KR101677460B1 - Cosmetic composition comprising allergen removed water soluble royal jelly for skin whitening and anti-aging activity - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin whitening and anti-aging comprising water-soluble royal jelly in which an allergen-induced protein is removed. The cosmetic composition comprising the water-soluble royal jelly having the allergen-induced protein of the present invention exhibits TRP-1, TRP-2, tyrosinase activity inhibitory activity and melanin formation inhibitory activity, and thus can be used as a cosmetic composition for skin whitening and skin aging prevention Can be usefully used.

Description

Technical Field [0001] The present invention relates to cosmetic compositions for skin whitening and anti-aging comprising water-soluble royal jelly with reduced allergenicity,

The present invention relates to a cosmetic composition for skin whitening and anti-aging comprising water-soluble royal jelly in which an allergen-induced protein is removed.

Royal jelly is a milky white secretion secreted from the external glands of worker bees and accumulated in the royal bee (queen bee room) of the honey bee hive. It is a jelly-like substance that is fed to larvae to be queen bees. The chemical composition is somewhat different depending on the mountain area and the season, but it becomes 65 to 75 mass% of water, 15 to 20 mass% of protein, 10 to 15 mass% of carbohydrate, 1.7 to 6 mass% of fat and 0.7 to 2 mass% of ash It also contains organic acids, various vitamins and various minerals. Royal jelly has been used for a long time as a human health food and recently it has been reported that it has a useful physiological activity for human body such as antibacterial activity, immunity enhancing action, antitumor action, antiinflammatory action, There are many.

However, since royal jelly contains allergen-inducing substances, there are individual differences but allergic symptoms may occur due to ingestion, and in some cases, severe anaphylaxis shock may also occur. For the purpose of solving the problem of such royal jelly, Japanese Patent Application Laid-Open No. 2002-112715 discloses a method of lowering allergy by treating the royal jelly with a saccharide-degrading enzyme treatment and a proteolytic enzyme treatment. In addition, International Publication WO2004 / 21803 discloses a royal jelly in which the amount of water soluble protein is reduced to less than 50% based on the total protein amount. However, the royal jelly has allergen-inducing water-soluble proteins. In the royal jelly which has been subjected to glycosidase treatment and proteolytic enzyme treatment, degradation of allergen inducing substances is insufficient, or the added enzyme itself causes a new allergen And there is still a problem in that it is not preferable as royal jelly for the purpose of health maintenance.

On the other hand, the skin pigmentation mechanism is related to melanin synthesis. Melanin synthesis is mediated through the action of tyrosinase, which is present in melanosomes. This tyrosinase acts as a tyrosine hydroxylase that oxidizes L-tyrosine to form L-DOPA and also acts as a DOPA oxidant that oxidizes DOPA to form DOPA quinone, which is known to be an important enzyme for synthesizing melanin. Therefore, it is essential to inhibit the activity of tyrosinase in the development of a skin whitening agent that inhibits melanin production.

TRQ-1 (tyrosinase related protein-1) oxidizes DHICA (5,6-dihydroxy indole-2-carboxylic acid) produced by TRP-2 to produce IQCA (indole-2-carboxylic acid) Dark brown. Therefore, whitening effect can be expected by inhibiting TRP-1 expression.

TRP-2 is an enzyme known as DOPA chromium tauterae, which produces DHICA (5,6-dihydroxy indole-2-carboxylic acid) using DOPA chromium. DHICA is oxidized by TRP-1 to produce IQCA (indole-2-carboxylic acid), which shows a dark brown color. Therefore, when the expression of TRP-2 is inhibited, a whitening effect can be expected.

(Japanese Patent Application Laid-open No. Hei 4-9320), hydroquinone (Japanese Patent Laid-Open No. 6-192062), kojic acid (Japanese Patent Laid-open No. 56-7710), arbutin ) And some compounds have a tyrosinase inhibitory activity and are used as a raw material for whitening cosmetics. However, since they are not stable in the prescription system, they are decomposed to be colored, and the occurrence of odor, efficacy at a living body level, And the use thereof is limited due to problems. And the inhibitory effect of tyrosinase has been proven, but the effect is lower in experiments similar to actual bio-levels. Therefore, an inhibitory effect by melanocyte cell culture similar to a living body level is required. In addition, hydroquinone is prescribed as a carcinogenic substance and its use is prohibited. Kojic acid and ascorbic acid are very unstable substances, and browning occurs when a cosmetic containing a small amount is stored at room temperature for several weeks.

Thus, the present inventors confirmed that the allergen-reduced water-soluble royal jelly of the present invention has an effect of inhibiting melanin formation, inhibiting tyrosinase activity, and inhibiting tyrosinase, TRP-1 and TRP-2 expression, Completed.

Japanese Patent Application Laid-Open No. 2002-112715 International Publication WO2004 / 21803 Japanese Patent Application Laid-Open No. 4-9320 Japanese Patent Application Laid-Open No. 6-192062 Japanese Patent Application Laid-Open No. 56-7710 Japanese Patent Laid-Open No. 4-9315

It is an object of the present invention to provide a cosmetic composition for skin whitening and anti-aging comprising water-soluble royal jelly in which an allergen-inducing protein has been removed.

In order to achieve the above object, the present invention provides a cosmetic composition for skin whitening and anti-aging comprising water-soluble royal jelly in which an allergen-induced protein is removed.

As used herein, the term " allergen-induced protein has been removed " refers to the removal of allergenic substances contained in royal jelly. Specifically, in the royal jelly, the protein comprises 27 to 41%, of which more than 80% is soluble protein (Major Royal Jelly Protein, MRJPs), and MRJPs 1 to 9 among MRJPs are known to be more than 30%. The MRJP1 (55 kDa) has a weakly acidic pI and the MRJP2 to 5 represent a basic pI. Of the proteins in royal jelly, 47 and 55 Kd are known as major antigens (Tropical Biomedicine 25 (3): 243-251). The water-soluble royal jelly in which the allergen-induced protein of the present invention has been removed may be one in which the allergen-inducing protein of 40 to 60 kDa has been removed, and preferably the protein of 47 kDa and 55 kDa in size has been significantly reduced.

As used herein, the term " water soluble royal jelly " refers to the removal of the water-insoluble portion of the royal jelly, and in the embodiment of the present invention refers to the extract extracted with ethanol to remove the water-insoluble portion.

The water-soluble royal jelly of the present invention may have an allergen-induced protein of 40 to 60 kDa removed, and the water-soluble royal jelly may contain a 10-HDA content of 4.1% or more.

As used herein, the term " 10-HAD " refers to a physiological activity indicator material of royal jelly. 10-hydroxy-decanoic acid (10-hydroxy-decenoic acid) and 10-hydroxy-decanoic acid have various physiological activities in the alkali-soluble fraction of royal jelly. In the embodiment of the present invention, 10-HDA is only 1.95% in the bio-royal jelly, while the water-soluble royal jelly in which the allergen-induced protein of the present invention is removed has a 10-HDA content of 4.1% or more.

As used herein, the term " raw royal jelly " is used as a raw material for preparing the water-soluble royal jelly of the present invention, and includes the kind of bee securing the raw royal jelly, Is not particularly limited. The species of bees that secrete the raw royal jelly include Apis mellifera, Apis cerana, Apis dorsata, Apis florea and the like. Mountain regions include Japan, South America, North America, Australia, China, Asia, and Europe. These royal jelly can be advantageously used as a raw material of the royal jelly fraction of the present invention, and it is more preferable to use raw royal jelly which is preserved as fresh or low temperature as possible. In other words, unprocessed royal jelly, which has been collected from the queen of bees, is referred to simply as "raw royal jelly" in the present specification.

The water-soluble royal jelly of the present invention may be a neutralization solution of ethanol extract of freeze-dried raw royal jelly powder. In an embodiment of the present invention, the allergy-free water-soluble royal jelly can be prepared in the following manner:

1) freeze-drying the raw royal jelly;

2) extracting the royal jelly powder produced by lyophilization in step 1) above with ethanol and concentrating to obtain royal jelly extract; And

3) neutralizing the royal jelly extract of step 2).

In an embodiment of the present invention, it is preferable, but not limited, to further include a step of lyophilizing and pulverizing the royal jelly extract of step 2) and then dissolving it in distilled water. In an embodiment of the present invention, it is further preferred that the method further comprises the step of freeze-drying the neutralized royal jelly solution in the step 3), but is not limited thereto. In an embodiment of the present invention, the royal jelly is water soluble by the ethanol extraction and concentration step of step 2), but is not limited thereto. In an embodiment of the present invention, the pH of the royal jelly is adjusted to 7 to 8 by the neutralization step in step 3), but is not limited thereto. In the embodiment of the present invention, the neutralization in step 3) is preferably performed by adding any one selected from the group consisting of sodium hydroxide, sodium carbonate and ammonia, but sodium hydroxide which does not destroy the components of royal jelly is most preferable But is not limited thereto.

In an embodiment of the present invention, the water-soluble royal jelly having the water-insoluble portion removed by the ethanol extraction can be obtained, and the allergenicity of the royal jelly is removed by the neutralization process.

The water-soluble royal jelly of the present invention from which the allergen has been removed can be obtained through the above-described production method. The water-soluble royal jelly of the present invention can be a neutralization solution of the ethanol extract of freeze-dried raw royal jelly powder . The neutralization solution may be a solution containing any one selected from the group consisting of sodium hydroxide, sodium carbonate and ammonia, preferably a solution neutralized with sodium hydroxide. In embodiments of the present invention, the water-soluble royal jelly may be a lyophilized powder of the neutralization solution.

In the embodiment of the present invention, the pH of the proteins in the royal jelly is kept constant in a basic state to minimize changes in the charge, thereby uniformizing the characteristics of the water-soluble royal jelly. In order to increase the activity of 10-HDA, The royal jelly was alkalized (pH before neutralization: pH 3.8 to 4.0, pH after neutralization: pH 7.4).

The water-soluble royal jelly of the present invention may contain 0.001 to 90% by weight of the allergen-free water-soluble royal jelly based on the total weight of the cosmetic composition for skin whitening and antiaging.

The cosmetic composition of the present invention can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, a foundation, , Cleansing foams, cleansing lotions, cleansing creams, lotions and body cleansers. When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component In the case of the formulations of the present invention as a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl Benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan. When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may be selected from aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyltaurates, sarcosinates, fatty acids Amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative or ethoxylated glycerol fatty acid ester.

The cosmetic composition of the present invention may comprise a composition selected from the group consisting of water-soluble vitamins, useful vitamins, high molecular weight peptides, polymeric polysaccharides and sphingogly lipids. In addition to the above components, Antimicrobial agents, antioxidants, plant extracts, pH adjusters, alcohols, coloring matters, perfumes, blood flow promoters, cold agents, antiperspirants, antioxidants, antioxidants, antioxidants, emollients, surfactants, organic and inorganic pigments, And other components such as water, purified water, and the like. In addition, the cosmetic composition of the present invention can be prepared into any formulation in which the cosmetic composition is usually prepared, and can be, for example, an emulsion, a cream, a lotion, a pack, a foundation, a lotion, a serum, a cleanser and a hair cosmetic.

In an embodiment of the present invention, the inventors of the present invention made the raw royal jelly into a powder by lyophilization and then extracted and concentrated with ethanol to make it water-soluble. The extract was powdered by lyophilization, dissolved in distilled water, Neutralized with sodium at pH 7.4 and powdered by lyophilization. In the thus-prepared royal jelly, the 25-55 kDa protein reacted with IgG, an allergic antibody, was found to be decreased. The expression of β-Hexosamindase and TNF-α, which are secreted in RBL- It was confirmed that the allergen reduced water soluble royal jelly of the present invention was remarkably reduced than that of the raw royal jelly, and thus allergen induction was reduced. In addition, it was confirmed that the royal jelly of the present invention maintains the effective effect of the royal jelly itself by confirming the increase of the 10-HDA content and the skin cell viability. As a result, it was confirmed that the water-soluble royal jelly having reduced allergenicity of the present invention has enhanced useful activity and reduced allergen-inducing properties, so that it can be applied to various consumer groups and can be easily absorbed and formulated.

In addition, the cosmetic composition comprising the water-soluble royal jelly with reduced allergicity of the present invention is characterized by having TRP-1, TRP-2, tyrosinase activity inhibiting activity and melanin formation inhibiting activity.

A wide variety of signaling molecules are involved in the melanin biosynthetic signaling system. Melanin is synthesized through several signal transduction mechanisms. Among them, cAMP / PKA pathway is the main pathway of melanin synthesis. When skin is exposed to UV, melanin cell cAMP is increased and PKA, a downstream signaling substance, is activated. Increases expression of MITF via CREB, thereby promoting synthesis of tyrosinase, TRP-1, and TRP-2, thereby increasing melanin synthesis. In order to confirm skin whitening and anti-aging effects of the allergen-free water-soluble royal jelly in the embodiment of the present invention, experiments concerning inhibition of tyrosinase, TRP-1 and TRP-2 activities related to melanin synthesis were conducted .

As a result, the water-soluble royal jelly (WSRJ) with a 10-HDA content of 40 to 60 kDa allergen-induced protein was more effective than the conventional royal jelly (TRP-1, TRP-2 and Tyrosinase) RJ). In addition, in the embodiment of the present invention, it was confirmed that the allergen-free water-soluble royal jelly of the present invention has an effect of inhibiting melanin production. Therefore, it has been confirmed that the water-soluble royal jelly of the present invention having the allergen-free properties of the present invention has the above-mentioned TRP-1, TRP-2, tyrosinase activity inhibiting activity and melanin formation inhibiting activity, thus being effective for skin whitening and anti-aging.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The cosmetic composition comprising the water-soluble royal jelly having the allergen-induced protein of the present invention exhibits TRP-1, TRP-2, tyrosinase activity inhibitory activity and melanin formation inhibitory activity, and thus can be used as a cosmetic composition for skin whitening and skin aging prevention Can be usefully used.

FIG. 1 is a schematic diagram showing the process of removing allergens and water-solubilization of water-soluble royal jelly with reduced allergy according to an embodiment of the present invention.
FIG. 2 is a gel photograph showing that allergen-induced proteins of royal jelly have been removed according to an embodiment of the present invention (Root: royal royal jelly: bottom royal royal jelly of the present invention) J: Pearl).
FIG. 3 is a graph showing cytotoxicity of reduced allergenic water-soluble royal jelly according to an embodiment of the present invention (RJ: freeze-dried royal jelly; WSRJ: reduced allergenic water-soluble royal jelly of the present invention).
FIG. 4 is a graph showing melanin production inhibitory effects of water-soluble royal jelly and general royal jelly with reduced allergies according to an embodiment of the present invention (A: inhibition of melanin formation in B16F1 cells by royal jelly treatment Of the present invention at a concentration of 1: 1 ug / ml; 10: 10 ug / ml of the melanin content in the group treated with royal jelly; Royal jelly treated group)
Figure 5 shows the inhibitory effect of tyrosinase (A), TRP-1 (B) and TRP-2 (C) mRNA expression of generalized freeze-dried royal jelly and water-soluble royal jelly with reduced allergenicity according to an embodiment of the present invention FIG.
FIG. 6 is a graph showing the inhibitory effects of tyrosinase (A), TRP-1 (B) and TRP-2 (C) proteins on the reduction of allergenicity of water-soluble royal jelly and general lyophilized royal jelly FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Preparation of water-soluble royal jelly to remove allergenic protein

Example  1-1. Removal of royal jelly allergy and water absorption

The present inventors removed water-insoluble portions of royal jelly using ethanol and made it water-soluble (Fig. 1).

Specifically, 100 g of domestic raw royal jelly purchased directly from the farms of Anseong, Jinju and Cheolwon, which represent the central, southern and northern parts of Korea, were lyophilized to obtain 35 g of freeze-dried royal jelly powder. The royal jelly powder was extracted with 70% ethanol and concentrated twice to obtain an ethanol royal jelly extract. The extract was lyophilized and powdered, and the obtained lyophilized powder was dissolved in tertiary distilled water to confirm that it was water-soluble.

The resulting royal jelly aqueous solution was then neutralized with sodium hydroxide to inactivate allergen-inducing proteins and activate the active substance.

Specifically, the water-soluble royal jelly powder extracted with ethanol and lyophilized was dissolved in distilled water (pH 3.8 to 4.0), and 2N sodium hydroxide (NaOH) was added at a ratio of 0.5 to 1 ml per 100 g of royal jelly, followed by neutralization (pH 7.4). The neutralized royal jelly liquid was lyophilized to obtain 19.6 g of a water-soluble royal jelly powder with reduced allergenicity.

Example  1-2. Allergen-induced protein reduction and 10- HDA  Content check

The present inventors confirmed the content of the allergen-induced protein of the water-soluble royal jelly reduced in allergy obtained in the <Example 1-1>. Specifically, in the serum of 54 patients with allergic reactions to royal jelly, the reaction of IgE, known as an allergic antibody, with the proteins of the royal jelly was confirmed by SDS-PAGE protein separation. As a result, 25 to 55 Kd Were identified as antigens by reacting with IgE, and proteins of 47kd and 55kd were deduced as major antigens. In order to confirm the change of the content of the main antigen protein of the royal jelly according to the present invention, 0.1 g of the raw royal jelly and the royal jelly according to the present invention were dissolved in 1 ml of PBS, Was loaded onto an SDS-PAGE gel and electrophoresed. After electrophoresis, the gel was stained with Coomassie staining buffer for 1 hour and decolorized with destaining buffer for 2 hours. The decolorized gel was treated with gel drying buffer for 40 minutes, and then the gel was obtained using a dry system.

As a result, it was confirmed that the neutralized water-soluble royal jelly of the present invention had no change in the total protein content, but the protein of 40-60 kDa, which is an allergen-induced protein of royal jelly, was reduced as compared with the control royal jelly (FIG. 2 ).

In addition, the present inventors confirmed the 10-HDA content of the allergen reduced water-soluble royal jelly of the present invention. Specifically, the content of 10-HDA in the raw royal jelly and the royal jelly of the present invention was measured using a mobile phase 0.02M (NH4) 2HPO4 · EtOH (7: 3) mixture solution (pH 7.0) and a μ-Bondapak C18 column Respectively.

As a result, the royal jelly of the present invention has a 10-HDA content of at least 4.1%, and has a 10-HDA content of at least twice that of the raw royal jelly (Table 1).

Analysis (%) Raw royal jelly Invention 10-HDA 1.95 + 0.02 4.1 ± 0.06

* P <0.05, compared with raw royal jelly

Example  2. Identification of whitening and anti-aging effects of allergenic protein-free water-soluble royal jelly

Example  2-1. B16F1 cell culture and survival rate measurement

The present inventors cultured mouse black B16F1 cells to examine cytotoxicity by treating the allergen reduced water-soluble royal jelly of Example 1 above.

Specifically, mouse black B16F1 cell line was purchased from Korean cell line bank (Seoul, Korea). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco BRL, MD, USA), 1% penicillin / streptomycin , Gibco BRL, MD, USA) in DMEM (Dulbecco's modified Eagle's medium) at 37 ° C in a 5% CO2 incubator. To determine the survival rate of B16F1 cells to α-MSH (α-melanocyte stimulating hormone, Sigma, USA) used to induce melanin synthesis in freeze-dried royal jelly and water-soluble royal jelly, 3- (4,5-dimethythiazol- -yl) -2-5-diphenytetrazolium bromide (MTT) reduction method. B16F1 cells were seeded at a density of 5 × 10 ³ cells / ml in a 96-well plate, cultured for 24 hours, and then subjected to various concentrations of allergen-removing water-soluble royal jelly and α-MSH. After culturing for 24 hours, the medium was removed, and then 50 μl of MTT solution was added thereto, followed by culturing for 3 hours, treating 200 μl of DMSO, and then measuring the absorbance at 570 nM.

As a result, it was confirmed that there is no cytotoxicity against the allergen reduced water-soluble royal jelly (WSRJ) of the present invention (FIG. 3).

Example  2-2. Confirming melanin production inhibitory effect

The present inventors quantified melanin and examined the melanin production inhibitory effect by treating the water-soluble royal jelly with reduced allergenicity in Example 1.

Specifically, melanin quantitation was measured by modifying Hosoi's method. B16F1 cells were cultured in 6-well plates at a concentration of 2 × 10 4 cells / ml and then replaced with medium containing 10 nM of MSH. After 48 hours, the cells were treated with a medium containing normal freeze-dried royal jelly (RJ) and allergen-free water-soluble royal jelly (WSRJ) for 24 hours, and the cells were collected and centrifuged at 1200 rpm for 10 minutes. After removing the supernatant, 500 μl of lysis buffer (50 mM sodium phosphate pH 6.5, 1% Triton X-100, 2 mM PMSF) was added to the pellet to dissolve and centrifuged at 14,000 rpm for 15 minutes to obtain pellet. After incubation at 80 ° C for 1 hour with 200 μl of 1 N NaOH containing 10% DMSO, the absorbance was measured at 405 nm. The amount of melanin was determined based on the value obtained from the melanin standard (Sigma, USA).

After that, melanin biosynthesis inhibitory activity was measured in B16F1 melanoma cell of water - soluble royal jelly which had allergy component removed. As shown in FIG. 4, the melanin synthesis in the cell was significantly inhibited compared to the α-MSH-treated group at 1 μg and 10 μg of the water-soluble royal jelly, and no difference in inhibition of melanin synthesis was observed between the freeze-dried common royal jelly. In order to visually confirm the inhibitory effect of melanin synthesis, the morphology of the cells before harvesting the cells was observed. As a result, as shown in FIG. 4, the α-MSH-treated group was able to confirm the degree of melanin formation in a very dark condition, but melanin production was hardly observed in the normal control group and 10 μg and 1 μg of water-soluble royal jelly treatment group. As a result, it was confirmed that the water-soluble royal jelly that eliminated allergy at a concentration of 1 ㎍ or more effectively inhibited melanin synthesis in B16F1 cells promoting melanin synthesis by α-MSH (FIG. 4).

Example  2-3. Tyrosinase, TRP -One, TRP -2 mRNA  Expression measurement

To examine the effect of the reduced allergenicity of the water-soluble royal jelly of Example 1 on expression of whitening-related tyrosinase, TRP-1 and TRP-2 mRNA, B16F1 cells were cultured in 100 mM tissue culture Dissociated to 2 x 10 ⁴ cells / ml and cultured for 24 hours. After the medium was removed, the medium was replaced with medium containing 10 nM of MSH, followed by incubation for 48 hours, followed by lyophilization and water-soluble royal jelly, respectively. After the treatment, the cells were cultured for 24 hours. Total RNA extraction was performed using the Total RNA Purification Kit (Invitrogen, USA). 1 μg of total RNA was used for cDNA synthesis and reverse transcriptase was inactivated by RT Premix kit (Oligo dT primer) (iNtRON Biotechnology, USA) for 1 hour at 45 ° C and 5 minutes at 95 ° C. The synthesized cDNA was subjected to PCR using PCR Premix kit (i-Taq) (iNtRON Biotechnology, USA). PCR conditions include denaturation at 94 ° C for 5 minutes; After 30 cycles of 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 1 minute; Gt; 72 C &lt; / RTI &gt; for 10 minutes.

The primer sequences used in the experiments are as follows:

Mouse tyrosinase, 5'-GGCCAGCTTTCAGGCAGAGGT-3 '(SEQ ID NO: 1, forward primer) and 5'-TGGTGCTTCATGGGCAAAATC-3' (SEQ ID NO: 2, reverse primer); Mouse TRP-1, 5'-GCTGCAGGAGCCTTCTTTCTC-3 '(SEQ ID NO: 3, forward primer) and 5'-AAGACGCTGCACTGCTGGTCT-3' (SEQ ID NO: 4, reverse primer); (SEQ ID NO: 5, forward primer) and 5'-CGGTTGTGACCAATGGGTGCC-3 '(SEQ ID NO: 6, reverse primer) and mouse GAPDH (glyceraldehyde- 3-phosphate dehydrogenase) internal trp-2, 5'- GGATGACCGTGAGCAATGGCC- 5'-ACCACAGTCCATGCCATCAC-3 '(SEQ ID NO: 7, forward primer) and 5'-TCCACCACCCTGTTGCTGTA-3' (SEQ ID NO: 8, reverse primer) as internal controls.

PCR products were analyzed by electrophoresis on 1.5% agarose gel. The intensity of each band was quantified by Quantity One (Bio-Rad, USA).

As a result, the inhibition of melanin synthesis related protein tyrosinase expression of the allergen - free water - soluble royal jelly was confirmed, and the effect on tyrosinase mRNA expression was measured. As a result, it was confirmed that the expression of tyrosinase, TRP-1 and TRP-2 mRNA was greatly inhibited in the group treated with water-soluble royal jelly at a concentration of 1 μg and 10 μg (FIG. 5) , Which was more effective than general freeze-dried royal jelly (Fig. 5).

Example  2-4. Tyrosinase, TRP -One, TRP -2 protein expression measurement

The present inventors observed the effect of the reduced allergenicity of the water-soluble royal jelly of Example 1 on the expression of tyrosinase, TRP-1 and TRP-2 protein as whitening-related factors. First, B16F1 cells were subcultured to a density of 2 x 104 cells / ml in a 100 mm tissue culture dish and cultured for 24 hours. The medium was replaced with medium containing 10 nM of MSH, cultured for 48 hours, and treated with royal jelly. The cells were cultured for 24 hours after treatment with royal jelly. Proteins obtained by lysing cells using a cell lysis buffer containing protease inhibitor cocktail and centrifuging at 12,000 rpm for 20 minutes were analyzed using a Bio-rad protein assay kit Was used to quantify the protein. Electrophoresed using 10% SDS-PAGE gel and transferred to a PVDF membrane. Tyrosinase and β-actin (β-actin, abCam, USA) were diluted by adding 5% skim milk to a tris buffer saline containing 1% Tween 20 The reaction was carried out at 4 ° C. The next day, horseradish peroxidase-conjugated antibody was reacted for 1 hour and developed with ECL. The intensity of each band was measured by Quantity One (Bio-Rad, USA).

After that, the degree of expression of tyrosinase, TRP-1, and TRP-2, which are melanin synthesis-related factors, was examined to verify the melanin inhibitory effect of water-soluble royal jelly. 1ug and 10ug, respectively, it was confirmed that the expression of tyrosinase, TRP-1 and TRP-2 was greatly reduced in the water-soluble royal jelly-treated group. Which was similar to that of the normal control group. Especially, it was confirmed that the inhibitory effect was greater than that of the general royal jelly treatment group. It was confirmed that water-soluble royal jelly reduces melanin production through inhibition of tyrosinase, TRP-1 and TRP-2 expression.

The statistical significance of the results was analyzed using SPSS (18.0 version, USA) statistical program, and the results were expressed as mean ± standard error (mean ± SEM). The significance of the mean between the control and experimental groups was verified at p <0.05 using Duncan's multiple range test of ANOVA (analysis of variance).

Manufacturing example . Cosmetics  Preparation of composition

Manufacturing example  1-1. Preparation of oil-soluble (W / O formulation) ointment agent (% by weight)

Reduced allergic water soluble royal jelly 0.01-10.0%

Stearyl alcohol 7.0%

Stearic acid 2.2%

Squalene 3.4%

Octyl dodecanol 5.0%

Olive oil 6.0%

Glycerin monoauric fatty acid ester 10.0%

Isopropyl adipate 2.0%

Tego 0.3%

Carboxyl vinyl polymer 3.0%

Propylene glycol 5.0%

Kojic acid 0.05%

Alpha-keto glutamic acid 0.2%

Cysteine 0.2%

Parabens 0.3%

Sterile purified water or 0.02 M phosphate or buffer solution balance

After mixing the above components, a fat-soluble ointment was prepared according to a conventional method.

Manufacturing example  1-2. Preparation of hydrophilic (O / W type) ointment agent (% by weight)

Reduced allergic water soluble royal jelly 0.01-10.0%

White vaseline 25.0%

Stearyl alcohol 20.0%

Propylene glycol 12.0%

Propylene misstatin 0.1%

Sodium glycerin monolaurate 0.2%

Polyoxyethylene hardened castor oil 4.0%

Betaine-containing cationic emulsifier 0.2%

Glycerin monostearate 0.1%

0.1% paraoxybenzoic acid propylate

Purified water balance

After mixing the above components, a hydrophilic ointment was prepared according to a conventional method.

Manufacturing example  1-3. Preparation of nutritional lotion (% by weight)

Glycerin 8.0%

Butylene glycol 4.0%

Hyaluronic acid extract 5.0%

Beta-glucan 7.0%

Carbomer 0.1%

Reduced allergic water soluble royal jelly 0.05%

Capphilic / capric triglyceride 8.0%

Squalane 5.0%

Cetearyl glucoside 1.5%

Sorbitan stearate 0.4%

Cetearyl alcohol 1.0%

Triethanolamine 0.1%

Purified water balance

After mixing the above ingredients, a nutritional lotion was prepared according to a conventional method.

Manufacturing example  1-4. Preparation of nutritional cream (% by weight)

Glycerin 3.0%

Butylene glycol 3.0%

Liquid paraffin 7.0%

Beta-glucan 7.0%

Carbomer 0.1%

Reduced allergic water soluble royal jelly 3.0%

Cappylic / capric triglyceride 3.0%

Squalane 5.0%

Cetearyl glucoside 1.5%

Sorbitan stearate 0.4%

Polysorbate 60 1.2%

Triethanolamine 0.1%

Purified water balance

After mixing the above ingredients, a nutritional cream was prepared according to a conventional method.

Manufacturing example  1-5. Preparation of Massage Cream (% by weight)

Glycerin 8.0%

Butylene glycol 4.0%

Liquid paraffin 45.0%

Beta-glucan 7.0%

Carbomer 0.1%

Reduced allergic water soluble royal jelly 1.0%

Cappylic / capric triglyceride 3.0%

Waste 4.0%

Cetearyl glucoside 1.5%

0.9% sorbitan sesquioleate

Vaseline 3.0%

Paraffin 1.5%

Purified water balance

After mixing the above components, a massage cream was prepared according to a conventional method.

Manufacturing example  1-6. Preparation of pack (% by weight)

Glycerin 4.0%

Polyvinyl alcohol 15.0%

Hyaluronic acid extract 5.0%

Beta-glucan 7.0%

Allantoin 0.1%

Reduced allergic water soluble royal jelly 0.5%

Nonyl phenyl ether 0.4%

Polysorbate 60 1.2%

Ethanol 6.0%

Purified water balance

After mixing the above components, packs were prepared according to a conventional method.

<110> REPUBLIC OF KOREA <120> COSMETIC COMPOSITION COMPRISING ALLERGEN REMOVED WATER SOLUBLE          ROYAL JELLY FOR SKIN WHITENING AND ANTI-AGING ACTIVITY <130> P14R12D0367 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mouse tyrosinase forward primer <400> 1 ggccagcttt caggcagagg t 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse tyrosinase reverse primer <400> 2 tggtgcttca tgggcaaaat c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mouse TRP-1 forward primer <400> 3 gctgcaggag ccttctttct c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mouse TRP-1 reverse primer <400> 4 aagacgctgc actgctggtc t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mouse TRP-2 forward primer <400> 5 ggatgaccgt gagcaatggc c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mouse TRP-2 reverse primer <400> 6 cggttgtgac caatgggtgc c 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GAPDH forward primer <400> 7 accacagtcc atgccatcac 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mouse GAPDH reverse primer <400> 8 aagacgctgc actgctggtc t 21

Claims (8)

A cosmetic composition for skin whitening and anti-aging comprising water-soluble royal jelly having an allergen-induced protein of 40 to 60 kDa removed,
The water-soluble royal jelly is a freeze-dried powder of a neutralization solution which is prepared by freeze-drying an ethanol extract of lyophilized raw royal jelly powder, dissolving the powder in a distilled water, neutralizing it with sodium hydroxide and adjusting the pH to 7 to 8,
The non-water-soluble portion of the royal jelly is removed by ethanol extraction of the lyophilized raw royal jelly powder, and
Characterized in that the allergen-inducing protein of 40 to 60 kDa is removed by the neutralization.
delete The cosmetic composition for skin whitening and antiaging according to claim 1, wherein the water-soluble royal jelly has a 10-HDA content of 4.1% or more.
delete delete delete The cosmetic composition for skin whitening and anti-aging according to claim 1, wherein the water-soluble royal jelly is contained in an amount of 0.001 to 90% by weight based on the total weight of the composition.
The cosmetic composition for skin whitening and anti-aging according to claim 1, wherein the cosmetic composition has TRP-1, TRP-2, tyrosinase activity inhibiting activity and melanin formation inhibiting activity.

Images