JP2993614B2 - New cosmetics, glucosaminoglycan formation promoter and whitening agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel cosmetic and pharmaceutical containing a umbilical cord fibroblast culture solution.

(Prior art) As the skin ages, the skin changes into a dry wrinkle-free skin with wrinkles, and this change is closely related to the age-related disappearance of glucosaminoglycan and collagen. Is believed to be something. The function of glucosaminoglycan in vivo is considered to have various important functions in addition to the retention of intercellular water, the formation of an intercellular matrix by binding to collagen, and the like. Until now, focusing on the moisturizing effect of glucosaminoglycan, moisturizing skin,
To provide smoothness, cosmetics and pharmaceuticals containing glucosaminoglycans such as hyaluronic acid and chondroitin sulfate and collagen as moisturizers have been used.

(Problems to be Solved by the Invention) However, the above cosmetics and pharmaceuticals contain glucosaminoglycan or collagen as a humectant, but are administered from the outside. It is not clear whether glucosaminoglycan increases in E. coli. Therefore, development of a component that promotes the production of glucosaminoglycan in skin fibroblasts to increase the amount of glucosaminoglycan in the skin and consequently provides moisture and smoothness to the skin has been desired.

Thus, the present inventors have conducted intensive studies to solve these problems, and as a result, the umbilical cord fibroblast culture solution significantly promotes glucosaminoglycan production of skin fibroblasts,
In addition, they have found that they also have a skin conditioning effect and a whitening effect, and have completed the present invention.

(Means for Solving the Problems) That is, the present invention provides a novel cosmetic and pharmaceutical containing a umbilical cord fibroblast culture solution.

Umbilical cord fibroblasts are fibroblasts isolated from cords connecting the fetus and the placenta, and can be purchased from RIKEN Genebank. The umbilical cord fibroblast culture solution is a culture solution collected after culturing umbilical cord fibroblasts in a liquid medium. Umbilical cord fibroblasts can be cultured by the usual method of culturing animal cells. The umbilical cord fibroblast culture solution can be prepared, for example, as follows. Eagle's MEM containing 1-20% fetal bovine serum containing umbilical cord fibroblasts, RITC80-7, Dulbecco's modified Ea
The cells are cultured at 37 ° C., 5% CO ₂ in a medium capable of culturing umbilical cord fibroblasts, such as gle's MEM, Ham F12, M199, and ASF301. When the growth of the umbilical cord fibroblasts reached a steady state, the medium containing serum was discarded, replaced with a new medium without serum, cultured for several days, and the culture was collected. It can be used as a blast cell culture solution. It is preferable that the umbilical cord fibroblast culture solution is collected and filtered before use. In addition, the umbilical cord fibroblast culture solution may be used as a powder, for example, as a liquid or by freeze-drying.

The culture thus obtained is blended with ordinary skin cosmetics and baths, or external preparations for the skin. As the compounding amount, 0.
It is 001 to 100% by weight, preferably 10 to 50% by weight. The cosmetics and pharmaceuticals of the present invention are applied to the skin several times a day. The amount of the coating varies depending on the age and sex, but it is about 0.01 to 100 ml, preferably 1 to 10 ml per day for an adult umbilical cord fibroblast culture solution.

The cosmetics and pharmaceuticals of the present invention may contain, in addition to the umbilical cord fibroblast culture solution, raw materials used in cosmetics and pharmaceuticals as long as their effects are not impaired. Examples of the cosmetics of the present invention produced from these raw materials include lotions, emulsions, foundation cosmetics such as creams, foundations, makeup cosmetics such as lipsticks, hair liquids, hair tonics, hair cosmetics such as hair creams, Examples include washing agents such as shampoo and face wash, and bath agents. Also,
Examples of the medicament of the present invention include plasters, ointments, suppositories, tinctures, poultices, liniments, lotions, spirits and the like.

(Examples) Next, typical examples of the present invention will be described, but the present invention is not limited thereto. In addition, the part of the compounding amount shown in an Example shows a weight part.

Example 1 Human umbilical cord fibroblasts (cell number RCB197, cell name HUC-Fm) purchased from RIKEN GeneBank were cultured at 37 ° C. in 5% CO _{2 in} a modified RITC80-7 medium containing 10% fetal bovine serum. And cultured. After 10 days of culture, the serum-containing medium was discarded, replaced with a new serum-free modified RITC80-7 medium, cultured for 2 days, and the culture solution was collected. A cell culture solution was used. The modified RITC80-7 medium used here is RITC proposed by Yamane et al.
Bovine serum albumin, fibronectin, epidermal growth factor, insulin, 3,3 ', 5-triiodothyronine, copper sulfate, manganese sulfate, nickel chloride, ammonium molybdate, ammonium vanadate, ammonium suboxide from 80-7D medium ⁽¹⁾ Excluding selenic acid.

References (1) I. Yamane et al., Exp. Cell. Res., Vol.
981) Example-2 Human umbilical cord fibroblasts (cell number RCB197, cell name HUC-Fm) purchased from RIKEN GeneBank were incubated at 37 ° C in 5% CO _{2 in} Eagle's MEM medium containing 10% fetal calf serum. And cultured. After 10 days of culture, the serum-containing culture was discarded, replaced with a fresh serum-free Eagle's MEM, cultured for 2 days, and the culture broth was collected. And

Example 3 Lotion Formulation Formulation amount A) Umbilical cord fibroblast culture solution 30.0 parts 1,3-butylene glycol 8.0 glycerin 2.0 Xanthan gum 0.2 Purified water 43.1 B) Ethanol 5.0 P-methyl hydroxybenzoate 0.1 Polyoxyethylene (40) Hardened castor oil 0.1 Perfume qs Purified water 10.0 Production method: Dissolve component A and component B uniformly,
Mix to make the product.

Example-4 Cream Formulation Formulation amount A) 4.0 parts of stearic acid cetyl alcohol 3.0 stearyl alcohol 1.0 liquid paraffin 6.5 petrolatum 10.0 sorbitan monostearate 1.5 polyoxyethylene (25) monostearate 3.0 B) 1,3-butylene glycol 5.0 water Potassium oxide 0.1 Culture solution of umbilical cord fibroblasts 10.0 Methyl P-hydroxybenzoate 0.2 Purified water 54.7 C) Appropriate amount of fragrance Production method: 70-75 each of oil phase component A and aqueous phase component B
After heating and dissolving at ℃, component A was added to component A and emulsified,
During the cooling, Component C is added and mixed, and cooled to 30 ° C. to obtain a product.

Example-5 Emulsion Formulation Formulation amount A) Stearic acid 5.0 parts Cetyl alcohol 5.0 Glycerin monostearate 1.3 Liquid paraffin 2.0 Sorbitan monostearate 1.5 Polyoxyethylene (10) monostearate 3.0 B) Glycerin 6.0 Umbilical cord fibroblast culture solution 20.0 Methyl P-hydroxybenzoate 0.2 Purified water 57.4 C) Perfume qs Production method: Make a product in the same manner as in Example-2.

Example-6 Pack Prescription Formulation amount Umbilical cord fibroblast culture solution 10.0 parts 1,3-butylene glycol 10.0 glycerin 15.0 Methyl P-hydroxybenzoate 0.2 Polyoxyethylene (40) hydrogenated castor oil 0.5 citric acid 0.1 sodium citrate 0.3 flavor Appropriate amount Purified water 63.8 Production method: Dissolve each component uniformly to obtain a product.

Example-7 Foundation Prescription Formulation amount 1, umbilical cord fibroblast culture solution 10.0 parts 2, stearic acid 2.4 3, propylene glycol monostearate 3.0 4, setosteryl alcohol 0.25, liquid lanolin 2.0 6, liquid paraffin 3.0 7, myristin Isopropyl acid 8.58, butyl paraoxybenzoate 0.19, purified water 48.6 10, sodium carboxymethyl cellulose 0.211, bentonite 0.512, propylene glycol 4.013, triethanolamine 1.114, methyl paraoxybenzoate 0.215, titanium oxide 8.0 16, Talc 4.017, coloring pigment 5.018, perfume qs Production method: Heat components 1 and 9 to 70 ° C, add component 11 and swell well. The component 10 in which the component 12 is dispersed is added to and dissolved therein. Subsequently, the components 13 and 14 are dissolved to form an aqueous phase. Components 2 to 8 are heated and dissolved, and kept at 80 ° C. to form an oil phase. In the water phase,
Mix well, pass through a pulverizer, pulverize component 17 and stir with a homomixer to keep at 75 ° C. The oily phase is added to the aqueous phase with stirring, cooled, and the component 18 is added at 45 ° C. After stirring and cooling, the product is obtained.

Example-8 Bath Formulation Formulation Amount Sodium sulfate 28.0 parts Sodium bicarbonate 35.0 Umbilical cord fibroblast culture solution 30.0 Borax 2.0 Yellow 202 No. (1) Appropriate amount Fragrance Appropriate amount Production method: Mix well each component and produce product And

Example -9 Ointment Formulation Formulation amount Purified lanolin 5.0 parts Salivary beeswax 5.0 Umbilical cord fibroblast culture solution 10.0 White petrolatum 80.0 (Effect of the present invention) It has a skin conditioning effect and a whitening effect.

Next, the effects of the cosmetics and pharmaceuticals of the present invention will be described in more detail with reference to experimental examples.

Experimental Example-1 Glucosaminoglycan production promoting effect test of skin fibroblasts Using the umbilical cord fibroblast culture solution shown in Example 1, a glucosaminoglycan production promotion test was performed. Culture the skin fibroblasts to a confluent state, add the umbilical cord fibroblast culture solution to a final concentration of 0 to 100% (V / V), and culture at 37 ° C and 5% CO ₂ . did. After 7 days of culture, the culture solution is collected, and the final concentration is set to 1 to degrade the protein.
% (W / V) of pronase was added and left at 50 ° C. for 48 hours. Add trichloroacetic acid to 10% (W / V)
It was left at room temperature for 30 minutes with stirring. After removing insolubles by centrifugation, the mixture was dialyzed against water. The concentrated sample was used as a crude glucosaminoglycan fraction and quantified according to the Bitter-Muir method ⁽²⁾ .

As a result, as shown in Table 1, the umbilical cord fibroblast culture solution was found to have an effect of promoting glucosaminoglycan production by skin fibroblasts. When the addition amount was 10%, the maximum value was shown, and an effect 1.49 times that of the control was recognized.

A similar test was performed using the umbilical cord fibroblast culture solution shown in Example 2.

The results are shown in Table 2. Similarly, the umbilical cord fibroblast culture solution shown in Example 2 was found to have a glucosaminoglycan production promoting effect.

Reference (2) T. Bitter and H. Muir, anal. Biochem., VOL 4,330
(1962) Experimental Example-2 Use test (skin conditioning effect) The test sample was the lotion (A) shown in Example 3, and the control was the lotion (B) obtained by removing the umbilical cord fibroblast culture solution from the formulation of Example 3. Was. To 12 subjects, A was applied to the right cheek and B was applied to the left cheek for three consecutive days, twice in the morning and evening.
In the use test, a questionnaire survey was conducted on the moist feeling and smoothness of the skin after use. The determination of the effect was not changed because A was better (A> B) and B was better (A <B).

As a result, as shown in Table 3, both the moist feeling and the smoothness were recognized as effective.

In addition, when the same use test was performed also about the cosmetics and pharmaceutical products of Examples 4 to 9, the moist feeling and smoothness of the skin were improved.

Experimental Example-3 Inhibition test of melanin production using B16 mouse melanoma B16 using the umbilical cord fibroblast culture solution shown in Example 1
A melanin production inhibition test using mouse melanoma was performed. 2x melanoma in logarithmic growth phase into φ60mm dish
10 ⁴ cells were seeded, a umbilical cord fibroblast culture solution was added to a final concentration of 0 to 100% (V / V), and the cells were cultured at 37 ° C under 5% CO ₂ . After 3 days of culture, the culture solution was replaced. After 6 days, the cells were detached from the dish, the number of cells was counted, and the cells obtained by centrifugation were treated with 10% TCA, followed by centrifugation and containing 10% DMSO in the precipitate. 0.1NNaOH was added to 1 × 10 ⁶ cells / ml, and the measured value of OD ₄₇₅ was defined as the amount of melanin.

As a result, as shown in Table 4, the umbilical cord fibroblast culture solution
Suppressed melanin production in B16 mouse melanoma. When the amount of addition was 100%, the OD ₄₇₅ decreased the most,
It suppressed melanin production by 2.4%.

A similar test was performed using the umbilical cord fibroblast culture solution shown in Example 2. Table 5 shows the results. A similar effect was observed in the umbilical cord fibroblast culture solution shown in Example 2.

Experimental Example-4 Clinical Example The ointment shown in Example 9 was applied to the affected area once a day for four patients with post-inflammatory pigmentation and observed for up to three months. At the same time, the same test was performed on four patients with an ointment containing no umbilical cord fibroblast culture solution. Judgment of effect-:
Unchanged +: Lightened ++: Almost disappeared +++: Completely disappeared. No side effects were observed in all cases.

As a result, as shown in Table 4, the effect was observed in 75% (3 out of 4 cases). No effect was observed in patients using ointments without umbilical cord fibroblasts.

As described above, the cosmetics and pharmaceuticals of the present invention have remarkable glucosaminoglycan production accelerating effects, skin conditioning effects, and whitening effects.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 35/44 A61K 35/44

Claims (3)

(57) [Claims] 1. A novel cosmetic comprising a umbilical cord fibroblast culture solution. 2. A glucosaminoglycan production promoter comprising an umbilical cord fibroblast culture solution. 3. A whitening agent comprising an umbilical cord fibroblast culture solution.