JPH0320206A - Skin cosmetic - Google Patents

Abstract

PURPOSE:To obtain a skin cosmetic, containing mammalian whey having collagen synthesis accelerating action and fractionated ingredients thereof, capable of specifically accelerating the collagen synthesis of dermal fibroblasts and having senescence controlling function. CONSTITUTION:A skin cosmetic, obtained by blending whey of a mammal having collagen synthesis accelerating action, preferably human, cattle, goat, sheep or pig, especially in initial whey within 5 days after delivery and fractionated ingredients thereof, preferably an acid- and a alcohol-soluble fraction prepared by treating the whey with a solution at pH1-5 in 10-50% (vol./vol.) alcohol concentration, especially pH2.0-4.5 in 30-40% (vol./vol.) alcohol concentration and then removing insoluble substances as an essential ingredient in a skin cosmetic, such as toilet water, cream, milky lotion or pack and having senescence controlling function so as to accelerate skin function, prevent wrinkles or the skin and provide fine and moist skin with high safety.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a skin cosmetic (non-medicinal) characterized by containing whey of a mammalian animal and its fractionated components, which have a collagen synthesis enhancing effect. (including high-quality medicated cosmetics (the same applies hereinafter)). [Prior art] In Japan, which is facing an aging society, medical interest in aging is increasing, and research into the mechanism of skin aging is progressing in the field of dermatology, with particular emphasis on the administration of cell activators. The control of aging is attracting attention at home and abroad. Extracts from several plants, animals, and fungi have been found to have tissue activating ability and have been used in pharmaceuticals and cosmetics. However, few of these substances have been evaluated as physiologically active substances at the cellular level. Attempts have also been made to measure oxidative phosphorylation using enzymes that catalyze oxidative phosphorylation of ATP, etc. However, with these means, e.g.
According to the oxygen consumption test method using tissue sections, water also shows activating ability, and to put it in an extreme case, many carbon and nitrogen sources have low oxygen consumption at appropriate concentrations. shows an increase in These measurement methods are effective at least for evaluation as nutritional supplements, but when assessing skin functions at the cellular level and evaluating samples, there are many points where it is impossible to use these methods to capture cell activation scent. ．． [Problems to be solved by the invention] By having a strong resistance, it satisfactorily fulfills its physiological functions. Histologically, the skin can be roughly divided into the epidermis, dermis, and subcutaneous tissue, and the dermis in particular plays an important role in maintaining skin homeostasis as a support tissue for 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 structural structures among these connective tissue components. The first known morphological changes in the skin associated with aging include a decrease in skin tissue thickness and atrophy (Marks
．． R. ．． Biochemistry and Ph
ysiology of theSkin. Oxford
rd University Press. 1983
In addition, the torsional extensibility of the skin decreases, the elastic recovery power decreases, and the walnuts further increase, eventually leading to the formation of wrinkles, which are the most characteristic changes seen in the skin of the elderly.

On the other hand, the most significant biochemical changes that occur with aging in the dermis are observed in collagen, the main component of the extracellular matrix. In other words, the amount of collagen in the skin decreases to 80% from the age of 20.
By age, the thickness of the dermis decreases by 65%, and the thickness of the dermis becomes thinner (
Shuster. S. * British Journal
al of Dermatolology. 93,639.
1975). In addition, the turnover rate of collagen decreases with age and remains in the body for a long time, so collagen S
Disordered intermolecular cross-linking and saccharification progress in the fibers, and insoluble collagen increases (Lagland. Y. Pa.
thological Biology. 17. 99
1. (1969), which causes hardening of the extracellular matrix. From the results of these studies, dermis 1! If it were possible to specifically accelerate collagen synthesis in miscellaneous blast cells, the collagen content in the dermis would increase and the thickness of the dermis would increase, along with activation of turnover and increase in soluble collagen, which would lead to cell growth. It improves the flexibility and extensibility of the outer matrix, and as a result, it may be possible to suppress morphological changes in aging skin, such as wrinkles. The present invention aims to improve the collagen synthesis of dermal miscellaneous blast cells [Means for solving the problem] The present inventors first conducted a collagen synthesis ability test using cultured fibroblasts to evaluate the specimen at the cellular level. Established the method. In other words, in this test method, human fibroblasts that have been cultured to a state just before forming a monolayer on the entire culture surface (subconfluent) are injected with radioisotope-labeled proline (('H)-proline) at the same time as the specimen. After adding and further culturing to incorporate into the cells, the culture medium and cell fraction were treated with ◆ and ('H) monoproline as the total protein amount on the one hand, and collagenase treatment on the other hand. [1H]- of the trichloroacetic acid (TCA) soluble fraction obtained by
The amount of proline is measured and the amount of synthetic collagen is quantified.

The present inventors screened various substances arbitrarily selected from animals, plants, and fungi for their collagen synthesis stimulatory effects using the above-mentioned test method. Synthetic redundant activity was recognized.

Whey is the fraction that remains after casein and fat are removed from milk, and is obtained from milk as a by-product during the production of cheese and casein. In ancient Greece, Hibocrates used it as a natural medicine to cleanse the body, diuretic, detoxify, and improve function.
Whey was used as a panacea for improving physical strength and treating constipation. Even today, whey is used in beverages and other products, and its significance is particularly recognized in terms of nutritional physiology and preventive medicine. In addition, the protein obtained from whey (whey protein concentrate.wpc) is in demand for processed foods such as ham and sausages due to its excellent functional properties such as gel-forming and water-retaining properties. However, until now, no research has been conducted on the collagen synthesis enhancing effect of whey from chewing mammals and its fractionated components, or on its usefulness in blending into skin cosmetics. This is what was done. In other words, the collagen synthesis enhancing effect of mammalian whey is particularly high in colostrum produced within 5 days after delivery.A skin cosmetic containing animal whey and its fractionated components is prepared and applied to the skin. They discovered that it enhances skin function, prevents wrinkles, and makes skin smooth and moisturized.
Furthermore, we have confirmed its high safety, which led us to complete the present invention. The skin cosmetics of the present invention containing mammalian whey and its fractionated components that have collagen synthesis-enhancing effects specifically enhance the collagen synthesis of fibroblasts in the dermis. This was thought to indicate an effect. In addition, the skin cosmetic of the present invention acts on the granulation tissue formed by amblasts that have migrated to the wound bed at the time of tissue damage, advances collagen synthesis, and promotes dermal reorganization, thereby exerting a wound healing effect. It is also expected that That is, the present invention provides a novel skin cosmetic product characterized by containing mammalian whey and its fractionated components, which have a collagen synthesis enhancing effect. The present invention will be explained in detail below. The above-mentioned collagen synthesis ability test method used in the process leading to the present invention does not limit the collagen synthesis redundancy effect of the present invention, and other test methods that can detect collagen synthesis ability at the cellular level, such as radioisotope In vitro, methods using hydroxybroline labeled with elements, immunological methods using collagen antibodies, and detection methods by hybridizing collagen mRNA with nucleic acid probes, etc.
It can be used in vivo and in situ. The whey used in the present invention is first of all human, bovine,
Fresh milk collected from mammals such as goats, sheep, and pigs, or milk that has been previously dried and powdered and then dissolved in water, is used, and is usually defatted by removing the fat in the top layer by centrifugation. After that, the casein fraction can be removed using various methods. Methods for removing casein fractions are generally divided into two main methods. One is for adding acids such as lactic acid, hydrochloric acid, and sulfuric acid. J:') Casein is isoelectrically precipitated with acidic pH of about 4.4 to 4.6, and casein is coagulated by the action of enzymes such as chymosin, and the insolubilized casein fraction is filtered and centrifuged. It is removed by separation or decantation, and the resulting casein fractions are called acid casein and rennet casein. In addition, it has been revealed that colostrum obtained from colostrum collected within 5 days after parturition of chewing mammals has a particularly high collagen synthesis enhancing effect, and conventionally, colostrum contains a high globulin content. Since this is known, it is considered to have important physiological significance. Furthermore, as a result of research into a fractionation method for collagen synthesis redundancy in whey, the present inventors discovered that whey is efficiently transferred to a soluble fraction by acid/alcohol treatment. In addition, the obtained acid/alcohol soluble fraction is a clear and stable solution, which is advantageous as a raw material for skin cosmetics, and the concentration of polymeric substances such as proteins that can be allergens is significantly reduced. This also improves safety.

The acid/alcohol ideal conditions will be explained in detail below, but the collagen synthesis-enhancing components essentially present in the whey of mammalian animals can be processed by various methods, such as delactose, desalt, and solvent fractionation. It can also be fractionated, purified, or concentrated by salting out, ultrafiltration, gel filtration, ion exchange methods, etc. In preparing the acid/alcohol soluble fraction of whey of the present invention, first, the whey prepared as described above, or the whey that has been previously dried and powdered and then dissolved in water, is prepared. The acid and alcohol are added separately or as a mixture of acid and alcohol, with a final pH of 1 to 5 and an alcohol concentration of 10 to 50% (V/V), preferably a pH of 2.0 to 4.
5,7 Adjust the 4-z-le concentration to 30-40% (V/V). pH less than 1 or alcohol concentration 50% (V
/v) or higher, the active ingredient for collagen synthesis redundancy becomes inactivated or insolubilized. In addition, in cases where the pH is 5 or more, the alcohol concentration is 10% (v/v) or less, or only one of citric acid or alcohol is used, concentration may be performed by an unequal method, and freeze-drying, spray-drying, or It is also possible to dry and powder it by methods such as plate drying. The skin cosmetic of the present invention may be in any form that can be used externally, such as lotion, cream, milky lotion, or pack, and contains the amount of whey from a mammalian animal that has a collagen synthesis enhancing effect and its fractionated components. The dosage can also be changed as appropriate depending on the severity of symptoms, dosage form, etc. Also, its groups ethanol, inpropanol, n
-Any substance such as butanol can be used, but ethanol is preferably used. Furthermore, the acids used are not particularly limited, such as hydrochloric acid, acetic acid, lactic acid, sulfuric acid, citric acid, and phosphoric acid. Insoluble matter generated as a result of the acid/alcohol treatment is removed by filtration, centrifugation, decantation, etc. A soluble fraction rich in components that enhance collagen synthesis can be obtained. Furthermore, the pH of the acid/alcohol soluble fraction can be adjusted to neutrality if necessary, and the fraction can be concentrated under reduced pressure, ultrafiltrated, or frozen. [Effect] The results of testing the effectiveness and safety of the skin cosmetics of the present invention thus obtained are as follows. 1. Collagen synthesis ability test of cultured w&fibroblasts W4-38 cells (human fetal lung normal diploid cells, PDL40), which have been maintained for generations, were added to fresh MEM medium (containing 10% fetal bovine serum) on the day before the test. ) and cultured for 24 hours (
precultura), the sample was added to the culture supernatant, [aH]-brolin was added to a final concentration of 2 μCi, and the mixture was incubated for 6 hours in 5% Co, +95% Air. Cultured at 37°C. In addition, the sample-free plot was used as a control. After that, the culture supernatant was removed, and the cell fraction was divided into furagenase type N (
Worthington) 5 units/ml
was allowed to act at 37°C for 18 hours, and the amount of collagen was determined by torn. The non-collagen protein amount was expressed as the value obtained by subtracting the collagen amount from the total [1H]-proline amount without fullagenase treatment. 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: 1 Bovine whey with lagen synthesis redundancy effect 570 g of bovine skim milk powder (conventional milk) was mixed with 4.5 g of deionized water.
Mix with IM vinegar @700mj! was added to adjust the pH to 4.4, stirred for 2 hours, left overnight at 5°C, and then centrifuged (
700g x 30min) L, and the supernatant was filtered through Celite to obtain clear whey.

Sample B: Bovine colostrum with collagen synthesis enhancing effect 10N of colostrum collected from cows 2 to 3 days after calving was centrifuged (15000g x 30min), after removing the fat in the uppermost layer, ℃, add an appropriate amount of chymosin, stir for 2 hours, and centrifuge XI (720 g
30 min), and the supernatant was filtered using a Membrane filter to obtain clear colostrum.

Sample C: Fractionation of bovine whey that has the effect of promoting collagen synthesis. 5 liters of the bovine whey obtained in Sample A was concentrated 10 times under reduced pressure, and after removing the lactose that precipitated when cooled to 4°C, Add water to bring the volume back up to 5 liters. A mixture of 2.82 ml of ethanol and 60 m2 of concentrated hydrochloric acid was added to this, and mixed for 2 hours using a homomixer. Then centrifugation (700g x 30min)
,, IN+, sodium oxide 0.21 was added to 6 liters of the supernatant to adjust the pH to 7.0, and the mixture was concentrated under reduced pressure to 22 and filtered to obtain a clear whey fraction. Sample D Fractionated component of bovine colostrum having collagen synthesis photostimulation activity Bovine colostrum 5Q obtained in sample B was mixed with methanol 32, adjusted to pH 3.0 with acetic acid, and further mixed for 2 hours in a homomixer. Thereafter, the supernatant was collected by decantation, concentrated twice using an ultrafiltration membrane with a molecular weight cutoff of 10,000, and filtered through Celite to obtain a clear colostrum fraction. Sample E Fractional component of goat whey that has a collagen synthesis enhancing effect Milk 102 collected from a goat was centrifuged with
00g x 30min) t, After removing the fat from the top layer, add lactic acid and adjust to pH 4. 5, stirred for 2 hours, left overnight at 5℃, and centrifuged (700g x 30℃).
min) L, the supernatant was filtered through Celite to obtain clear whey. A mixture of 3.42 ml of n-butanol and 70 ml of acetic acid was added to this whey 61, and then centrifuged (700 g
X30sin)L, its supernatant 42 was adjusted to pH 6.5 by adding IN sodium hydroxide, concentrated under reduced pressure to 31 and filtered to obtain a clear whey fraction.

Sample F: Milk 102 collected from a hedge whey sheep with collagen synthesis hyperactivity was centrifuged (1500
0g x 30min) I, After removing the fat from the top layer, add IM hydrochloric acid 1.12 to pH 4. Adjust to 5, then 2
After stirring for an hour and leaving it at 5℃ overnight, centrifugation (700g
x30min) The supernatant was filtered through Celite to obtain clear whey. Addition amount (V/V medium It has been revealed that whey from lying mammals and its fractions significantly and specifically enhance collagen synthesis in fibroblasts. Ward Team 1 Bunnell Test The following skin cosmetics of the present invention and control skin cosmetics were administered twice a day to 50 women aged 31 to 52 (average age: 40.1 years) suffering from fine wrinkles. Table 2 shows the sensory evaluation of the results of applying and using the product (morning and evening) for three consecutive months. <Skin cosmetics of the present invention and control skin cosmetics> Sample A of Test Example 1 (control skin cosmetics are purified water) 202 glycerin 5x ethanol
5z methylparaben
0.1x purified water
69.9X: There was a significant difference from the control with a confidence level of 99% (n = 4) As is clear from Table 2, it became clear that the skin cosmetics of the present invention showed high effectiveness. ．． Patch test 17 men and 13 women between the ages of 21 and 64, total 3
A closed patch test was performed on the flexor side of the upper arm of 0 subjects. The bovine whey from Example 1 was used as a sample. Judgment criteria -: No reaction at all, ±: Slight erythema, 10: Obvious erythema, ++: Erythema and swelling, papules As a result, in all subjects, 1 (all ← no reaction), irritation reaction It has been found that the possibility of causing an allergic reaction is extremely low, and that it is highly safe. [Examples] Examples of the present invention are shown below, but the present invention is not limited by these. Back collection lotion Glycerin 5x Probylene glycol 4x Layl alcohol 0.1x Polyoxyethylene sorbitan monolauryl 1.5x Polyoxyethylene lauryl ether 0.5x Ethanol 10x Test example 1
Sample A 20% fragrance, dye,
Preservatives, UV absorbers Appropriate amount of purified water
58.92 Dog Cover 1 <Cream> Stearic acid stearyl alcohol reduced lanolin squalane cutyl dodecanol polyoxyethylene cetyl ether lipophilic monostearic acid glycerin propylene glycol Sample B of Test Example 1 Fragrance, preservative, antioxidant Purified Sailor Boat Group 1 Milky lotion> Stearic acid cetanol Vaseline Lanolin Alcohol Liquid paraffin Polyethylene glycerol 0.2x 1.5x 3x 2X 10X Phosphate ester 2x 3x Propylene glycol 5x Triethanolamine 1x Sample of Test Example 1 C 15% fragrance, preservative,
# Antioxidant Appropriate amount of purified water
57.3X scarlet 1 pack> Polyvinyl alcohol 15X Sodium carboxymethylcellulose 5x Bropylene glycol 3x Ethanol
Sample E of 10% Test Example 1
5X fragrance, preservative, oxidizing agent
Appropriate amount of purified water
62% [Effect of the invention] As detailed above, the skin cosmetic product of the present invention, which is characterized by containing whey of a mammalian mammal and its fractionated components, which has an effect of promoting collagen synthesis, improves skin function. It has aging control functions that prevent wrinkles and make the skin smooth and moisturized, and it is also highly safe, so it has high expectations for clinical application. Therefore, the industrial significance of the present invention is extremely large.

Claims (5)

[Claims] (1) Skin cosmetics characterized by containing mammalian whey and fractionated components thereof that have a collagen synthesis enhancing effect (2) The skin cosmetic according to claim 1, wherein the mammal is a human, cow, goat, sheep, or pig. (3) The skin cosmetic according to claim 1, wherein the whey is colostrum produced within 5 days after delivery. (4) The skin cosmetic according to claim 1, wherein the fractionated component is an acid/alcohol soluble fraction. (5) The skin cosmetic according to claim 4, which is obtained by treating the acid/alcohol soluble fraction at a pH of 1 to 5 and an alcohol concentration of 10 to 50% (V/V) to remove insoluble matter.