JP2001240529A - Cosmetics for skin care - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique which matures the keratinocyte and improves the skin barrier function. SOLUTION: A royal jelly characterized by containing a protein having the following properties, which constitutes 9 weight % or more with respect to the all proteins in the jelly, is contained in cosmetics; (1) a single band is formed in unmodified polyacrylamide gel electrophoresis of protein in royal jelly; (2) a molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kDa; (3) a amino acid sequence of amino acids n: 1-8 in sequence 1 (refer to the specification) is contained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to cosmetics, and more particularly, to cosmetics useful for preserving and enhancing the skin barrier function.

[0002]

2. Description of the Related Art Cosmetics are external preparations for skin used to keep the skin clean and healthy. The main focus so far has been on improving the function of retaining moisture in the skin. Was. However, in recent years, skin troubles, which cannot be controlled only by such a water retention function, have come to occur. Examples of such skin troubles include a decrease in the skin barrier function resulting from anti-stress load, that is, a rapid increase in the amount of dissipated water, and a permeation of a chemical substance into the skin. Such a phenomenon is
It is thought that due to stress or the like, keratinocytes cannot be sufficiently matured and are lifted to the stratum corneum. Therefore, there is a need for a technique that matures keratinocytes and improves the skin barrier function.
No. 3 compounds have been found, but their effects are of a degree that is not practical.

[0003] On the other hand, a technique for containing royal jelly in cosmetics for the purpose of moisturizing has been known for a long time. However, the fact that royal jelly has an action of improving the skin barrier function, and that the active substance of such action has the following protein In addition, it has not been known at all that royal jelly containing the protein in an amount of 9% by weight or more based on the total protein in royal jelly has a remarkable effect of maintaining or enhancing the skin barrier function. 1) Form a single band in native polyacrylamide gel electrophoresis of proteins in royal jelly. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodalton. 3) Includes the amino acid sequence of amino acid numbers 1 to 8 of SEQ ID NO: 1.

[0004]

SUMMARY OF THE INVENTION The present invention has been made under such circumstances, and it is an object of the present invention to provide a technique for maturing keratinocytes and improving a skin barrier function.

[0005]

In view of such circumstances, the present inventors have made intensive research efforts for a technique to mature keratinocytes and improve the skin barrier function, and as a result, royal jelly contained such components. Found that it was included. As a result of further studies, it has been found that a protein having the following properties in royal jelly is the active substance thereof.
In addition, the inventors have found that a cosmetic containing royal jelly containing 9% by weight of such a protein based on the total protein in royal jelly has an excellent preserving action and enhancing action of the skin barrier function, and has completed the invention. Was. That is, the present invention relates to the following technology. (1) A cosmetic containing royal jelly, wherein the royal jelly contains 9% by weight or more of a protein having the following properties with respect to all proteins in the royal jelly. 1) Form a single band in native polyacrylamide gel electrophoresis of proteins in royal jelly. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodalton. 3) Includes the amino acid sequence of amino acid numbers 1 to 8 of SEQ ID NO: 1. (2) The cosmetic according to (1), wherein the cosmetic is for maintaining or enhancing the barrier function of the skin. (3) The cosmetic according to (2), wherein the skin barrier function is suppression of the amount of dissipated moisture. (4) It further comprises a water-retaining saccharide,
(1) The cosmetic according to any one of (4) to (4). Hereinafter, the present invention will be described in detail focusing on embodiments.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Royal jelly which is an essential component of the cosmetic of the present invention The cosmetic of the present invention is characterized by containing royal jelly, and the royal jelly comprises a protein having the following properties. 9% by weight based on total protein
It is characterized by containing above. 1) Form a single band in native polyacrylamide gel electrophoresis of proteins in royal jelly. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodalton. 3) Includes the amino acid sequence of amino acid numbers 1 to 8 of SEQ ID NO: 1.

[0007] The above-mentioned protein is
The present inventors have confirmed that it is present in royal jelly, and have found that royal jelly is an active substance having an effect such as an anti-fatigue effect, a critical momentum enhancing effect, and a cell viability enhancing effect. As a means for separating such a protein from royal jelly, the following method can be preferably exemplified. That is, freeze-dried royal jelly was added to 0.1 ml.
7% by weight of 10 mM Tris-HCl buffer (pH 7.0)
And concentrated 6 times with UF100,000 (Miniplate 100; ultrafiltration) and desalted 7 times. The filtrate was further filtered with UF30,000 (Miniplate30; ultrafiltration) for 8 times.
Double concentration and desalting were performed once to obtain a fraction having a molecular weight of 100,000 to 30,000. The sample having a molecular weight of 100,000 to 30,000 can be fractionated by anion exchange chromatography or gel filtration chromatography to separate a protein having a molecular weight of 57 kDa. The anion exchange chromatography may be performed according to a generally known method,
For example, DEAE-Toyopearl manufactured by Tosoh Corporation
Using 650 M as a column, 20 mM Tris-HCl buffer (pH 7.0) was used as a developing solution A, 20 mM Tris-HCl buffer (pH 7.0) and 1 M NaCl were used as a developing solution B, and the flow rate was 5 ml / min at 280 nm.
Of the fraction No. which was detected by the absorbance of From 119 to 127, a protein fraction having a molecular weight of 57 kDa can be detected. Further, this fraction may be subjected to gel filtration chromatography according to a generally known method. Preferred examples of such a fraction include, for example, Himac manufactured by Pharmacia.
Using Load16 / 60 Superdex600 as a column, 50 mM potassium phosphate buffer pH 7.0 containing 0.15 M sodium chloride as a developing solution, a flow rate of 1.0 ml / min, and a column temperature of 35 ° C.,
Fraction No. was detected by absorbance at 280 nm and fractionated at 2.0 ml / tube. A protein having a molecular weight of 57 kDa can be detected in 35 to 43. (The same protein was confirmed by electrophoresis.) From the results of gel filtration analysis of known molecular weight, the protein was determined to be a 57-kilodalton monomer protein. In addition, the present inventors have found that this protein is a glycoprotein because it is digested by N-glucosidase F and has a molecular weight of about 48 kDa after digestion. The protein content was analyzed using a standard product separated and purified as described above, using an absolute value calibration curve method, and using HiLoad 16/60 Superdex600 manufactured by Pharmacia as a column, containing 0.15 M sodium chloride. 50m
M potassium phosphate buffer pH 7.0 as a developing solution,
The flow rate can be determined from the area value of the peak detected at an absorbance of 280 nm at a flow rate of 1.0 ml / min and a column temperature of 35 ° C.

[0008] The fresh royal jelly usually contains about 9 to 13% by weight of the total protein in the freshly collected royal jelly, and as the protein deteriorates, it decreases to the order of several percent. I do. To concentrate the fraction, for example, royal jelly is divided into a water-soluble fraction and a non-water-soluble fraction, and the water-soluble fraction passes through an ultrafiltration membrane having a molecular weight of 100,000 and a molecular weight of 10,000 An operation that collects a portion that does not pass through the ultrafiltration membrane and freeze-drys may be performed. In the cosmetic of the present invention, it can be embodied by including fresh royal jelly so as to satisfy the above conditions, or as described above, using a fraction obtained by concentrating the protein. Thus, the above condition can be satisfied. In the cosmetic of the present invention, the preferred content of royal jelly containing 9% by weight or more of the protein is 0.01 to 10% by weight, and more preferably 0.1 to 1% by weight. This is because if the amount is too small, the effect may not be exhibited, and if the amount is too large, the effect will reach a plateau. As described above, the cosmetic of the present invention containing the 57-kilodalton protein is excellent in the action of preserving and enhancing the skin barrier function, which is caused by stress and the like.

[0009] In addition to the above essential components, the cosmetic of the present invention can contain optional components usually used in cosmetics. Such optional components include, for example, hydrocarbons such as squalane, petrolatum, microcrystalline wax, jojoba oil, carnauba wax,
Esters such as octyldodecyl oleate, triglycerides such as olive oil, tallow, coconut oil, fatty acids such as stearic acid, oleic acid and ritinoleic acid, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, sulfosuccinate esters and the like. Anionic surfactants such as sodium polyoxyethylene alkyl sulfate, amphoteric surfactants such as alkyl betaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, and polyoxyethylene adducts thereof , Nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyethylene fatty acid esters, polyhydric alcohols such as polyethylene glycol, glycerin and 1,3-butanediol, thickening Gelling agents, antioxidants, ultraviolet absorbers, may contain coloring material, preservatives, powders and the like. Of these, particularly preferred are water-retentive saccharides. This is because the water retention function that has been reduced due to stress or the like can be complemented. Here, the water-retentive saccharide means a generic name of polysaccharides having water retentivity and their acylated products, sulfated products or salts thereof, and specifically, trehalose, acylated trehalose, One or more selected from sulfated trehalose and / or salt, chondroitin sulfate and / or salt, hyaluronic acid and / or salt, heparin-like substance, glycerin and diglycerin can be preferably exemplified. The preferred content of these water-retaining saccharides is 0.001 to 10% by weight, and more preferably 0.01 to 5% by weight. The cosmetic of the present invention can be produced by treating these essential components and optional components in a conventional manner. In addition, the cosmetics referred to in the present invention are makeup cosmetics such as lotion, milky lotion, cream, foundation, under makeup, cheek color, lip color, eye color, eyeshadow, mascara. , Hair oil, hair tonic, shampoo, rinse, hair care cosmetics, soap, body shampoo and other cleaning cosmetics.

[0010]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to only these Examples.

Example 1 An animal experiment was set up to examine the relationship between overcrowding stress and skin function and the effect of the stress-reducing agent of the present invention on it. That is, S of 12 weeks old
kh-1 mice (male) were bred in a normal group with 5 mice per cage (95.2 cm 2 per mouse), and in an overcrowded stress group with 21 mice per cage (22.7 cm per mouse).
Reared in 2). The breeding period was one week. Thereafter, the back was shaved and then stripped. From the day after the stripping, one week after the stripping, the sample was applied to the back by 0.1 m on the back every day.
1 was applied. As a specimen, a protein of 57 kilodaltons was used for 11.6 of the total protein in royal jelly.
A 1% aqueous solution of fresh royal jelly containing 1% (sample 1) and a 1% aqueous solution of degraded royal jelly containing 57 kilodaltons of protein 5.1% of the total protein in royal jelly (sample 2), the control group was: Water alone was used. The normal group was bred by a normal breeding method during this stress load period. The amount of water dissipated from the skin was measured using a dissipative water meter. The measurement was performed 24 hours after the sample administration was completed. Table 1 shows the results. Thus, in the royal jelly containing 10% or more of the protein of 57 kilodaltons, which is an essential component of the present invention, the skin barrier function has been restored to the level of the normal group.
The deteriorated royal jelly group showed only a skin barrier function comparable to that of the control. That is, it is understood that the essential components of the cosmetic of the present invention have excellent effects of preserving and enhancing the skin barrier function.

[0012]

[Table 1]

Example 2 A lotion was prepared according to the following formulation. That is, the prescription components were solubilized with stirring to obtain lotion 1. Lotion 2 in which fresh royal jelly (57 kilodalton protein; 11.6%) is replaced with degraded royal jelly is replaced with water.
Was prepared and evaluated in the same manner as in Example 1. Table 2 shows the results. From this, it was confirmed that the cosmetic of the present invention was excellent in the action of preserving and enhancing the skin barrier function. It is also found that it is preferable to contain a water-retaining saccharide such as a heparin-like substance. Fresh royal jelly (57 kilodalton protein; 11.6%) 1 part by weight Heparin-like substance 0.1 part by weight 1,3 butanediol 5 parts by weight Methylparaben 0.1 parts by weight Water 94.8 parts by weight

[0014]

[Table 2]

Example 3 A cream was prepared according to the following recipe. That is, (a), (b) and (c) were each heated and dissolved at 80 ° C., (b) was gradually added to (a), and (c) was further added and emulsified. The homogenized particles were homogenized by a homomixer and cooled to obtain a cream. It was excellent in preserving and strengthening the skin barrier function, and was useful for recovering skin damaged by stress and the like. B) Squalane 10 parts by weight Cetanol 3 parts by weight Sorbitan sesquistearate 2 parts by weight POE (20) behenyl ether 2 parts by weight Vitamin A acid 1 part by weight b) 1,3-butanediol 5 parts by weight Royal jelly (57Kd; 11.6%) 0.1 part by weight Carboxyvinyl polymer 0.3 part by weight Water 40 parts by weight Sodium sulfated trehalose 0.1 part by weight c) Water 36.3 parts by weight Potassium hydroxide 0.2 parts by weight

Example 4 A pack cosmetic was prepared according to the following formulation. That is, the ingredients are mixed well,
Packed. The pack was excellent at repairing damaged skin in the stratum corneum in winter. When the size of the keratinocytes before and after using this pack was compared by using an atomic force microscope for a paneler who had a particularly significant effect (use period: 1 month), the size was 162 mμ3 before use and after use. The size was 188 mμ3, which was clearly large, indicating that the cosmetics of the present invention greatly matured keratinocytes and improved the barrier ability. Vinyl acetate polymer emulsion 50 parts by weight 1,3-butanediol 5 parts by weight Glycerin 5 parts by weight Methyl paraben 0.3 parts by weight Royal jelly (57 kDa; 11.6%) 1 part by weight Water 37.7 parts by weight Sodium hyaluronate 1 part by weight

Example 5 An under-makeup cosmetic was prepared according to the following formulation. That is, a, b, and c are each heated and melted at 80 ° C, and b is gradually added to b, and further c is added and emulsified. Then, the emulsified particles are homogenized by a homomixer, cooled, and under-make-up is performed. Obtained. Since this product is excellent in the action of preserving and strengthening the skin barrier function, it was also excellent in the action of suppressing the generation of bulkiness due to the powder foundation even in winter. A) Squalane 5 parts by weight Cetanol 2 parts by weight Hydrogenated tallow 1 part by weight Sorbitan monostearate 2 parts by weight POE (20) behenyl ether 2 parts by weight b) 1,3-butanediol 5 parts by weight glycerin 3 parts by weight diglycerin 2 Parts by weight Royal jelly (57 Kd; 11.6%) 1.1 parts by weight Carboxyvinyl polymer 0.3 parts by weight Water 40 parts by weight Trehalose 0.1 parts by weight C) Water 36.3 parts by weight Potassium hydroxide 0.2 parts by weight

[0018]

[Sequence List] SEQUENCE LISTING &# 60110 &# 62 Polar Chemical Co., Ltd. &# 60120 &# 62 Cosmetic for beautiful skin &# 60130 &# 62 P20000281 &# 60140 &# 62 &# 60141 &# 622000-3-1- &# 60160 &# 62 1 &# 60170 &# 62 PatentIn Ver.2.0

&# 60210 &# 62 1 &# 60211 &# 62 25 &# 60212 &# 62 PRT &# 60213 &# 62 Apis mellifera &# 60220 &# 62 &# 60221 &# 62 UNSURE &# 60222 &# 62 (24) &# 60223 &# 62 Xaa = unknown &# 60BR &# 62 &# 60400 &# 62 1 Asp Ile Leu Arg Gly Glu Ser Leu Leu Lys Lys Leu Pro Ile Leu His 1 5 10 15 Glu Met Lys Phe Phe Asp Tyr Xaa Asp 20 25

[0020]

According to the present invention, keratinocytes are matured,
A technique for improving the skin barrier function can be provided.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 7/00 A61K 7/00 U // C07K 14/435 ZNA C07K 14/435 ZNA (72) Inventor Miyazaki Hirotaka 560, Kashio-cho, Totsuka-ku, Yokohama-shi, Kanagawa Prefecture F-term in the Totsuka Research Laboratory 4C083 AA071 AA072 AA082 AB032 AC022 AC072 AC122 AC182 AC442 AC482 AD042 AD092 AD191 AD212 AD312 AD332 AD411 AD412 AD622 CC01 CC03 CC04 CC05 CC07 4DD23 DD27 AA30 BA18 CA51 EA01 FA71 GA22 HA06

Claims (4)

[Claims] 1. A cosmetic containing royal jelly, wherein the royal jelly contains at least 9% by weight of a protein having the following properties, based on the total protein in the royal jelly. Fees. 1) Form a single band in native polyacrylamide gel electrophoresis of proteins in royal jelly. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodalton. 3) Includes the amino acid sequence of amino acid numbers 1 to 8 of SEQ ID NO: 1. 2. The cosmetic according to claim 1, wherein the cosmetic is for preserving or enhancing the barrier function of the skin. 3. The cosmetic according to claim 2, wherein the barrier function of the skin is suppression of the amount of dissipated moisture. 4. The cosmetic according to claim 1, further comprising a water-retaining saccharide.