JP2533376B2 - Skin cosmetics - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention comprises a mammalian synthesis whey having a collagen synthesis promoting action and a fractionation component thereof, and a collagen synthesis promoter and the same. Skin cosmetics (including quasi-drug medicated cosmetics. The same applies hereinafter).

[Conventional technology]

In Japan, which is facing an aging society, while medical interest in aging is increasing, research on skin aging mechanisms and the like is progressing in the field of dermatology as well, especially in the control of aging by administration of cell activating agents. Is attracting attention.

Conventionally, some plant and animal extracts and fungal extracts have been found to have a tissue-activating ability, and have been used in pharmaceuticals and cosmetics. However, among these, few have been evaluated as substances having physiological activity at the cellular level, and for example, the increase in oxygen consumption due to the addition of these extracts to tissue sections was examined using a Warburg pressure gauge or the like. Attempts have been made to perform a measurement method or a quantitative method using an enzyme that catalyzes oxidative phosphorylation of ATP and the like. However, in these means, for example, according to the oxygen consumption test method using a tissue section, water also shows an activating ability, and in extreme cases, many having a carbon or nitrogen source have an appropriate amount. It shows an increase in oxygen consumption depending on the concentration. At least these measuring methods are effective for evaluation as a nutritional supplement, but when assessing the sample by observing the skin function at the cell level, it was often impossible to capture the cell activation ability by these means. .

[Problems to be solved by the invention]

The skin fully fulfills its physiological function by viscoelasticity that protects appendages and blood vessels from various movements in daily life, and strong resistance that prevents destruction of itself.

Histologically, the skin is roughly divided into epidermis, dermis, and subcutaneous tissue. Particularly, the dermis plays an important role for maintaining homeostasis of the skin as a supporting tissue of the skin. The main constituent cells of the dermis are fibroblasts, which produce proteins such as collagen and glycosaminoglycans such as hyaluronic acid, and form a structured structure in these connective tissue components.

As the morphological changes of the skin with aging, firstly, it is known that the thickness and atrophy of the skin tissue are reduced (Marks, R., Bio
chemistry and Physiology of the Skin, Oxford Univer
sity Press, 1983). Also, the skin's torsional stretchability is reduced, its elastic recovery is reduced, and its tarmi is increased, eventually leading to the formation of wrinkles, which is the most characteristic change observed in the skin of aged people.

On the other hand, the greatest biochemical changes that occur with aging of the dermis are found in collagen, which is the main component of extracellular matrix. That is, the amount of skin collagen decreases by 65% between the ages of 20 and 80, and the thickness of the dermis decreases (Shuste
r, S., British Journal of Dermatology, 93 , 639,197
Five). In addition, the turnover rate of collagen also decreases with age and stays in the body for a long period of time, so unregulated intermolecular cross-linking and glycation proceed in collagen fibers, and insoluble collagen increases (Legrand, Y., Pathological Biology, 17,99
1,1969), causing hardening of the extracellular matrix.

From the results of these studies, if it was possible to specifically enhance collagen synthesis in dermal fibroblasts, the collagen content of the dermis increased, the dermis thickness increased, and the turnover was activated and The increase in collagen may improve the flexibility and extensibility of the extracellular matrix, and as a result, it may be possible to suppress the morphological changes of aged skin represented by wrinkles.

An object of the present invention is to provide a novel skin cosmetic having a aging control function by specifically enhancing collagen synthesis of dermal fibroblasts.

[Means for solving problems]

The present inventors first established a collagen synthesis ability test method using cultured fibroblasts in order to evaluate a specimen at a cell level.

That is, the present test method, the human fibroblasts cultured up to just before forming a monolayer on the entire culture surface (subconfluent), the sample at the same time as radioisotope-labeled proline - a ^([3 H] proline) After adding and further culturing to take up into cells, from the culture supernatant and the cell fraction, on the one hand, the amount of [ ³ H] -proline as the total protein amount, and on the other hand, by collagenase treatment, were obtained. The amount of [ ³ H] -proline in the obtained trichloroacetic acid (TCA) -soluble fraction is measured to quantify the amount of synthetic collagen.

The present inventors screened various substances arbitrarily selected from animals and plants and fungi for their collagen synthesis enhancing action by the above-described test method, and as a result, high collagen synthesis enhancement in mammalian whey and its fractional components. The activity was recognized.

Whey is the fraction that remains after removing casein and fat from milk and is obtained from milk as a by-product of cheese and casein production. Also, in ancient Greece, whey was used as a panacea food by Hypocrates as one of the natural medicines for body washing, diuresis, detoxification, function enhancement, recovery of physical strength, treatment of constipation, etc. Even today, whey is used for beverages and the like, and its significance is recognized particularly in nutritional physiology and preventive medicine. Further, the protein obtained from whey (whey protein concentrate; WPC) is in demand for processed food such as ham and sausage because of its excellent functional properties such as gel forming property and water retention property.

However, until now, no study has been conducted on the action of mammalian whey and its fractional components to promote collagen synthesis, or their usefulness in blending into skin cosmetics, and it was first revealed by the present inventors. It is a thing.

That is, the action of mammalian whey to enhance collagen synthesis is
It was found to be particularly high in colostral whey within 5 days after delivery, and it was revealed that its active ingredient was transferred to the acid / alcohol-soluble fraction of whey. Furthermore, the present inventors have prepared a skin cosmetic containing a mammalian whey having a collagen synthesis promoting action and its fractional component and applied it to the skin, which enhances the skin function and prevents wrinkles on the skin. However, the present invention has been completed by discovering that the skin has a delicate and moist skin and by confirming its high safety. The skin cosmetic containing the mammalian whey and its fractionated component having the collagen synthesis-enhancing action of the present invention exhibits its effect by specifically enhancing the collagen synthesis of dermal fibroblasts. Was thought to be.

Further, the skin cosmetic of the present invention acts on granulation tissue formed by fibroblasts that have migrated to the wound bed during tissue damage,
It is also expected to have a wound healing effect by enhancing the collagen synthesis and promoting the reconstruction of the dermis.

That is, the present invention provides a collagen synthesis promoter characterized by containing a mammalian whey having a collagen synthesis promoting action and a fractionated component thereof, and a skin cosmetic containing the same.

 Hereinafter, the present invention will be described in detail.

The above-mentioned collagen synthesizing ability test method used in the process leading to the present invention does not limit the collagen synthesizing promoting action of the present invention, and other test methods capable of detecting collagen synthesizing ability at the cell level, for example, radioisotope In vitro, in vivo, i) methods such as a method using hydroxyproline labeled with, an immunological method using an antibody of collagen, a method of detecting collagen mRNA by hybridizing with a nucleic acid probe, etc.
It can be used in situ.

The whey used in the present invention includes human, bovine, goat,
Fresh milk collected from mammals such as sheep and pigs,
Alternatively, the milk is used after being dried and powdered in advance and then dissolved in water. Usually, the casein fraction can be obtained by various methods after degreasing by removing the fat content of the uppermost layer by centrifugation. The method for removing the casein fraction is generally divided into two methods. The first is the addition of acids such as lactic acid, hydrochloric acid and sulfuric acid to make casein isoelectrically precipitated at an acidity of about pH 4.4 to 4.6, and the other is the action of enzymes such as chymosin to coagulate casein, making both insoluble. Filtered the casein fraction,
The casein fraction obtained by a method such as centrifugation or decantation is called acid casein, and the latter is called rennet casein.

In addition, it was revealed that the colostral whey obtained from colostrum collected within 5 days after delivery of mammals has a particularly high collagen synthesis-enhancing action, and that colostrum contains a large amount of globulin in the past. From what is known, physiologically,
It is considered to have important meaning.

Furthermore, as a result of research on a fractionation method of the action of promoting collagen synthesis in whey, the present inventors have found that the whey is efficiently transferred to a soluble fraction by treatment with acid / alcohol.
The obtained acid / alcohol soluble fraction is a clear and stable solution, which is an advantageous property as a raw material in the formulation of skin cosmetics, and the concentration of high molecular substances such as proteins that may be an allergen is significantly reduced. Therefore, safety is also improved.

Hereinafter, the acid / alcohol treatment conditions will be described in detail. Essentially, the collagen synthesis-enhancing component present in mammalian whey can be subjected to various methods, for example, lactose, desalting,
Fractionation, purification or concentration can also be carried out by solvent fractionation, salting out, ultrafiltration, gel filtration, ion exchange method and the like.

In the preparation of the acid-alcohol-soluble fraction of whey of the present invention, first, the whey prepared as described above, or a whey prepared by preliminarily dry-powdering it and then dissolving it in water is used. Use, acid and alcohol are added separately or as an acid / alcohol mixture, final pH 1-5, alcohol concentration 10-50% (V / V) preferably pH 2.0-4.5, alcohol concentration 30-40% ( V / V). When the pH is less than 1 or the alcohol concentration is 50% (V / V) or more, the active ingredient for promoting collagen synthesis is inactivated or insolubilized. Also, pH
Treatment with 5 or more or with an alcohol concentration of 10% (V / V) or less, or with only one of acid or alcohol cannot remove the unnecessary fraction, and the fractionation efficiency is significantly reduced.
The alcohol used is methanol, ethanol,
Any of isopropanol, n-butanol and the like can be used, but ethanol is preferably used. Also,
The acid used is not particularly limited to hydrochloric acid, acetic acid, lactic acid, sulfuric acid, citric acid, phosphoric acid and the like. The insoluble matter generated as a result of the acid / alcohol treatment is removed by filtration, centrifugation, decantation or the like to obtain a soluble fraction rich in collagen synthesis promoting components. Furthermore, the acid / alcohol soluble fraction may be adjusted to neutral pH if necessary and concentrated by a method such as vacuum concentration, ultrafiltration or freeze concentration. It is also possible to dry powder by a method such as drying.

The skin cosmetic of the present invention may be any externally applicable dosage form such as lotion, cream, milky lotion, pack, etc., and the blending amount of mammalian whey having a collagen synthesis promoting action and its fractional component, It is appropriately changed depending on the degree of symptoms, dosage form, and the like. Also, as the base, any liquid and solid cosmetic bases which are acceptable for skin application can be widely used, and further, if desired, additives such as preservatives, fragrances and colorants can be contained. .

[Action]

The results of testing the effectiveness and safety of the thus obtained skin cosmetic of the present invention are as follows.

Test Example 1 Collagen Synthetic Ability Test of Cultured Fibroblasts WI-38 cells (human fetal lung normal diploid cells, PDL40) that had been maintained for passage were added to a new MEM medium (containing 10% fetal bovine serum) on the day before the test. Change culture medium and culture for 24 hours (preculture)
After that, a sample was added to the culture supernatant, and [ ³ H] -proline was added to a final concentration of 2 μCi for 6 hours, 5% CO ₂ + 95% Air, 37.
Incubated at ℃. The sample-free area was used as a control. Then remove the culture supernatant, cell fraction to collagenase type III (manufactured by Worthington Corp.) 5 units / ml to 37 ° C., 18 hours to act performs deproteinization with trichloroacetic acid solution, ^[3 H in the soluble fraction ] -The amount of proline was counted with a scintillator to obtain the amount of collagen.

The amount of non-collagen protein was expressed as a value obtained by subtracting the amount of collagen from the total amount of [ ³ H] -proline which was not treated with collagenase.

 The results are shown in Table 1.

 The sample preparation method and the amount of sample added to the medium are described below.

Sample A Bovine whey having an action of promoting collagen synthesis 570 g of skim milk powder (normal milk) of cow was mixed with 4.5 of deionized water, 700 ml of 1M acetic acid was added to adjust the pH to 4.4, and the mixture was stirred for 2 hours.
After leaving it at 5 ℃ overnight, centrifuge (700g × 30min),
The supernatant was filtered through Celite to obtain a clear whey.

Sample B Bovine colostral milk having an action of promoting collagen synthesis Colostrum 10 collected 2 to 3 days after calving of cattle was centrifuged (15000 g × 30 min) to remove the uppermost fat, and then 30 ° C. After heating and adding an appropriate amount of chymosin to the mixture, the mixture was stirred for 2 hours and then centrifuged (720 g × 30 min), and the supernatant was filtered through a membrane filter to obtain a clear colostrum.

Sample C Fractional component of bovine whey having a collagen synthesis promoting action Bovine whey 5 obtained in Sample A was concentrated 10 times under reduced pressure, and after cooling to 4 ° C., lactose precipitated was removed and water was removed. In addition, it returned to 5. Add ethanol 2.8 and concentrated hydrochloric acid 6
A mixed solution with 0 ml was added and mixed with a homomixer for 2 hours. After that, centrifuge (700g x 30min) and the supernatant 6
1N sodium hydroxide 0.2 to pH 7.0 and under reduced pressure,
It concentrated to 2 and filtered, and the clear whey fraction component was obtained.

Sample D Fraction component of bovine colostrum having an action of promoting collagen synthesis Bovine colostrum 5 obtained in Sample B was mixed with methanol 3, adjusted to pH 3.0 with acetic acid, and further mixed with a homomixer for 2 hours. After that, the supernatant is collected by decantation,
It was twice concentrated with an ultrafiltration membrane having a molecular weight cut-off of 10,000, and filtered through Celite to obtain a clear colostrum fraction component.

Sample E Fractional component of goat whey with collagen synthesis enhancing action Milk 10 collected from goat was centrifuged (15000 g × 30
min), remove the top fat and add lactic acid to adjust the pH.
The mixture was adjusted to 4.5, stirred for 2 hours, left overnight at 5 ° C., centrifuged (700 g × 30 min), and the supernatant was filtered through Celite to obtain a clear whey. N-butanol 3.4 in this whey 6
And a mixed solution of 70 ml of acetic acid are added, followed by centrifugation (700 g × 30
min) and add 1N sodium hydroxide to the supernatant 4 to adjust the pH.
It was adjusted to 6.5, concentrated to 3 under reduced pressure, and filtered to obtain a clear whey fraction component.

Sample F Sheep whey with collagen synthesis enhancing action Milk 10 collected from sheep was centrifuged (15000 g x
30 min), and after removing the fat in the top layer, 1M hydrochloric acid 1.1
The mixture was adjusted to pH 4.5, stirred for 2 hours, left overnight at 5 ° C., centrifuged (700 g × 30 min), and the supernatant was filtered through Celite to obtain a clear whey.

Addition amount (V / V medium%) Sample A: 5% Sample B: 5% Sample C: 2% Sample D: 2% Sample E: 2% Sample F: 5% As is clear from Table 1, mammalian whey and its fractional components were found to significantly and specifically enhance collagen synthesis in fibroblasts.

Test Example 2 Panel Test For 50 females aged 31 to 52 (average age 40.1) having wrinkles, the following skin cosmetics of the present invention and control skin cosmetics were administered twice a day (in the morning, Evening) Table 2 shows the sensory evaluation of the results of continuous application for 3 months and use.

<Inventive Skin Cosmetics and Control Skin Cosmetics> Sample A of Test Example 1 (control skin cosmetics are purified water) 20% Glycerin 5% Ethanol 5% Methylparaben 0.1% Purified water 69.9% As apparent from Table 2, It was clarified that the skin cosmetic of the present invention exhibits high effectiveness.

Test Example 3 Patch Test A closed patch test was performed on the upper arm flexion side of the test subjects, which consisted of 30 males, 17 males and 13 females aged 21 to 64 years. The bovine whey of Example 1 was used as a sample.

Criteria for judgment −: No reaction, ±; slight erythema, +: clear erythema,
++; Erythema and swelling, papules As a result, it was-(totally non-reactive) in all subjects, and it was revealed that the possibility of stimulating reaction and allergic reaction is very low and the safety is high. It was

〔Example〕

Examples of the present invention are shown below, but the present invention is not limited thereto.

Example 1 <Lotion> Glycerin 5% Propylene glycol 4% Oleyl alcohol 0.1% Polyoxyethylene sorbitan monolaurate
1.5% Polyoxyethylene lauryl ether 0.5% Ethanol 10% Sample A of Test Example 1 20% Fragrance, dye, preservative, UV absorber Appropriate amount Purified water 58.9% Example 2 <Cream> Stearic acid 2% Stearyl alcohol 7% Reduction Lanolin 2% Squalane 5% Octyldodecanol 6% Polyoxyethylene cetyl ether 3% Lipophilic glyceryl monostearate 2% Propylene glycol 5% Sample B of Test Example 1 10% Fragrance, preservative, antioxidant Proper amount Purified water 58% Example 3 <Emulsion> Stearic acid 0.2% Cetanol 1.5% Vaseline 3% Lanolin alcohol 2% Liquid paraffin 10% Polyoxyethylene monooleate 2% Glycerin 3% Propylene glycol 5% Triethanolamine 1% Test Example 1 Sample C 15% fragrance, preservative , Antioxidant Proper amount Purified water 57.3% Example 4 <Pack> Polyvinyl alcohol 15% Sodium carboxymethyl cellulose 5% Propylene glycol 3% Ethanol 10% Sample E of Test Example 1 5% Fragrance, preservative, oxidizing agent Proper amount Purified water 62 % [Effect of the invention] As described in detail above, a collagen synthesis enhancer characterized by containing a mammalian whey having a collagen synthesis enhancing action of the present invention and a fraction component thereof, and a skin makeup containing the same The ingredient enhances skin function, prevents wrinkles on the skin,
Since it has a function of controlling aging that makes the skin delicate and moist, and has high safety, it is expected to be applied clinically. Therefore, the industrial significance of the present invention is very great.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masatsugu Yamashita 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. (72) Inventor Katsuya Nishimoto 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. In-house (72) Inventor Yoshinori Okuda 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. Inspector Mika Kagawa (56) Reference JP 63-269947 (JP, A) JP 57- 54108 (JP, A) JP-A-1-254610 (JP, A)

Claims (6)

(57) [Claims] 1. A collagen synthesis enhancer comprising a component derived from mammalian whey. 2. The collagen synthesis enhancer according to claim 1, wherein the whey is one or more selected from human, bovine, goat, sheep and porcine whey. 3. The collagen synthesis enhancer according to claim 1, wherein the whey is colostrum within 5 days after delivery. 4. The collagen synthesis enhancer according to any one of claims 1 to 3, wherein the whey-derived component is obtained by acid / alcohol treatment. 5. The collagen according to claim 4, which is obtained by acid / alcohol treatment under conditions of pH 1 to 5 and alcohol concentration 10 to 50% (V / V) to remove insoluble matter. Synthetic enhancer. 6. A skin cosmetic containing the collagen synthesis enhancer according to any one of claims 3 to 5.