JPS63152309A - External preparation of skin - Google Patents

Abstract

PURPOSE:To obtain an external preparation for skin useful as a skin cosmetic or a wound remedy, etc., having preventing effects on skin aging, containing a specific coenzyme selected from 5'-deoxyadenosylcobalamin, flavin-adenine dinucleotid, etc. CONSTITUTION:An external preparation for skin containing 0.01-2wt% at least one compound selected from 5'-deoxyadenosylcobalamin (coenzyme B12), flavin-adenine dinucleotide (coenzyme of oxidoreductase), beta-nicotinamide adenine dinucleotide (coenzyme of dehydrogenase), beta-nicotinamide adenine dinucleotide phosphate (coenzyme of dehydrogenase), a reduction type compound thereof, coenzyme A, pyrroloquinoline quinone and a salt thereof. The external preparation for skin has activating action on skin cell, metabolic promotion, curing effects on wound, etc., and use of the external preparation provides smooth, wet and young skin free from wrinkles.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention has effects such as activating skin cells, promoting metabolism, and wound healing, and when used, prevents skin aging and makes the skin wrinkle-free and smooth. The present invention relates to skin cosmetics such as cosmetic creams and ointments that give moist and youthful skin, and external skin preparations such as wound treatment agents.

[Conventional technology]

Conventionally, animal and plant extracts, vitamins, amino acids, nucleic acids, etc. extracted from natural sources have been added to cosmetics and other external skin preparations to prevent skin aging, but their effects are limited to supplementary ones. Because of this, no significant anti-aging effect on the skin could be expected even when incorporated in relatively large amounts.

On the other hand, prosufuglandin (Japanese Patent Application Laid-open No. 18436/1983), which is a physiologically active substance that exists in small amounts in living organisms, and EGF
(J: Dermal cell growth factor) and the use of urogastrone in cosmetics (Japanese Patent Application Laid-open No. 61-5006) have been proposed. However, these methods require (1) low permeability to cells because the compound is a high molecular weight substance;
(ii) Since EGF and urogastrone are protein-based substances, they are easily degraded by proteases within cells;
It is not practical due to the following reasons: (iii) On the other hand, it may cause side effects and there are safety problems.

[Problem that the invention seeks to solve]

Therefore, an object of the present invention is to provide a practical skin preparation for external use that has an excellent anti-aging effect on the skin, has no side effects, and is highly safe.

[Means for solving problems]

The present invention effectively solves the above problems by incorporating specific coenzymes, which exist in small amounts in each tissue and cell in the body and act as essential components of various enzymatic reactions responsible for the metabolism of various substances, into a skin external preparation. This was done based on the knowledge that it could be done.

That is, the present invention provides 5'-deoxyadenosylcobalamin or a salt thereof, flavin adenine dinucleotide or a salt thereof, β-nicotinamide adenine dinucleotide, a reduced compound thereof or a salt thereof, β-nicotinamide adenine dinucleotide phosphate The present invention provides an external preparation for skin, characterized in that it contains at least one compound selected from the group consisting of , a reduced compound thereof or a salt thereof, coenzyme A or a salt thereof, and pyrroloquinoline quinone or a salt thereof.

5'-deoxyadenosylcobalamin used in the present invention is coenzyme B12 (hereinafter abbreviated as CoB 1 2 ).

), and is a different substance from dianocobalamin, hydroxocobalamin, and methylcobalamin, which are commonly used as vitamin L2 in medicines, cosmetics, etc. COB + 2 is widely present in trace amounts in tissues and cells of living organisms, and is known to be an essential component as a coenzyme for enzymes important in substance metabolism, such as methylmalonyl-CoA mutase. At present, CO[l, □ is only commercially available as a biochemical reagent, but it can be produced from vitamin B12 (dianocobalamin) by organic chemical methods or biochemical methods. (Biochemistry Experiment Course, Volume 13, Vitamins and Coenzymes, p. 55-458).

Note that salts of C0B12 can also be used in the present invention, such as COB+2 sodium salt (CoB, 2.Na
) COB+2 potassium salt (Cod, .・K) is exemplified.

Flavin adenine dinucleotide (hereinafter abbreviated as FAD) is known as an important substance that is involved in the general oxidation of organic compounds in vivo as a coenzyme of redox enzymes. FAD is used as a pharmaceutical raw material to treat dermatitis,
It is widely used in the treatment of allergic diseases, stomatitis, and beriberi, and as an active ingredient in eye drops.

Currently, FAD is produced industrially by fermentation or synthesis, and can be easily produced manually. Furthermore, F.A.D.
Salts of FAD can also be used in the present invention, specifically FAD sodium salt (FAD・2Na), FΔD potassium salt (F
AD・2K> is exemplified.

β-nicotinamide adenine dinucleotide (hereinafter referred to as N
It is abbreviated as AD. ) and β-nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) are known as important substances that act as coenzymes for various dehydrogenases in redox reactions in living organisms.

These coenzymes are currently used as diagnostic agents and biochemical reagents, and those produced mainly by fermentation are commercially available. In the present invention, reduced compounds of NAD and NADP (abbreviated as NADH and NADPH, respectively) are used.
or these salts can also be used. Specifically, N.A.
D sodium salt (NAD・Na), NΔD lithium salt (
NAD-Li), NΔD potassium salt (NAD-K)
, NADP sodium salt (NADP-Na), N
ΔDP potassium salt (NADP・K), NADP) squirrel salt, NADH sodium salt, (NADH・2Na>,
NADH potassium salt (NADII ・2K), NADH
Cyclohexylamine salt, N〇H tris salt, NADP
H sodium salt (NADPH・4Na), NADP)
I potassium salt (NADPH・, IIK), NΔDP
Examples include H-cyclohexylamine salt, NADPH) lithium salt.

Coenzyme A (hereinafter abbreviated as CoA) is involved in acyl group transfer reactions in vivo, and is involved in fatty acid oxidation, fatty acid synthesis, steroid synthesis, pyruvic acid oxidation, amino acid metabolism, and biological It is known as an important substance that serves as a carrier for acyl groups in various enzymatic reactions such as acetylation.

CoA is used as a raw material for pharmaceuticals in Europe, and in Japan, CoA produced by fermentation is commercially available as diagnostic agents and reagents. In addition, in the present invention, CoA salts can also be used, such as C08 hetrium salt (CoA・
Examples include NaXCoA .3Na), CoA lithium salt (CoA -Li, CoA .3S1) and C08 helium salt (CoA .K, CoA .3K).

Pyrroloquinoline quinone (hereinafter abbreviated as PQQ) was discovered in 1979 as a coenzyme of methanol dehydrogenase in methanol-assimilating bacteria, and has since been extensively researched, and has been shown to be widely distributed in living organisms. became known.

It has been reported that PQQ exhibits coenzyme action of dehydrogenase or oxidase and cell growth promoting action (Japanese Patent Application Laid-open No. 61-58584), but other physiological actions are unknown. , how many ships have not been reported.

Currently, PQQ is produced by fermentation using methanol-assimilating bacteria.
is commercially available as a biochemical reagent.

In addition, in the present invention, a salt of PQQ can also be used, and as the salt, PQQ can also be used.
Examples include QQ potassium salt (PQQ.2K) and PQQ sodium salt (PQQ.2Na).

The above-mentioned coenzymes used in the present invention are usually included in various skin and external preparations in amounts of 0.001 to 5, depending on the product form and frequency of use.
Weight% (hereinafter abbreviated as %), preferably 0.01
It is preferable to contain up to 2%.

In addition to the above-mentioned essential ingredients, the skin external preparation of the present invention contains oil,
Water, surfactants, humectants, lower alcohols, thickeners, antioxidants, chelating agents, pH adjusters, preservatives, fragrances, pigments, and other raw materials commonly used in external skin preparations such as cosmetics can be blended. . Specifically, oils include fats and oils such as olive oil, jojoba oil, and hydrogenated oils, waxes such as spermaceti wax, beeswax, and lanolin, hydrocarbons such as liquid paraffin, ceresin, and squalane, stearic acid, and oleic acid. Examples include fatty acids such as cetanopestearyl alcohol, alcohols such as lanolin alconopehexyldecanol, and esters such as isopropyl myristate and butyl stearate. Examples of surfactants include anionic surfactants such as sodium stearate, sodium cetyl sulfate, polyoxyethylene lauryl ether phosphate, and sodium N-acylglutamate, and cationic surfactants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride. Active agent, alkylaminoethylglycine hydrochloride solution, amphoteric surfactant such as lecithin, glycerin monostearate, sorbitan monostearate, sucrose fatty acid estenopemonostearate propylene glycol, polyoxyethylene oleyl ether, polyethylene monostearate Nonionic surfactants such as glycol, polyoxyethylene sorbicun monopalmitate, polyoxyethylene coconut fatty acid monoethanolamide, polyoxyethylene castor oil, polyoxyethylene lanolin, etc. are exemplified. be able to. Furthermore, as humectants, glycerin, 1,3-butylene glycol, propylene glycol, etc. are used, as lower alcohols, ethanol, isopropanol, etc. are used, and as thickeners, polyethylene glycol, carboxymethyl cellulose sodium, etc. are used, and oxidized As inhibitors, dibutyl hydroxytoluene, butyl hydroxyanisone propyl gallate, etc. are used. As chelating agents, disodium edetate, ethane hydroxy diphosphate, etc. are used. As pH adjusters, citric acid, sodium citrate, etc. , boric acid, borax, sodium hydrogen phosphate, etc., and examples of the preservative include methyl paraoxybenzoate, ethinobedehydroacetic acid paraoxybenzoate, salicylic acid, benzoic acid, etc., respectively. Note that the optional components are not limited to these. By appropriately blending the above essential components and optional components, for example, 0.00% of the essential components
1-5%, optional ingredients: oil 0-80%, surfactant 0.5-12%, humectant 2-15%, purified water 11-95%
%, it is possible to provide an external preparation for skin containing a trace amount of preservative. Specifically, it can be used in various product forms such as lotion, cream, pack, lotion, skin milk, milky lotion, and ointment.

When used as a skin cosmetic, preferred surfactants include sodium N-acylglutamate, glyceryl monostearate, sorbitan monostearate, sucrose fatty acid ester, polyoxyethylene oleyl ether, polyoxyethylene castor oil, and the like.

A specific example of use as a skin cosmetic is as a lotion: 0.01-2% of the essential ingredients of the present invention, 2-2% of lower alcohol.
10%, surfactant 0.5-1%, humectant 3-7%, p
A composition containing 0.05 to 0.2% of H regulator, 80 to 95% of purified water, a trace amount of preservative, a trace amount of pigment, and a trace amount of fragrance.As a skin cream: Essential ingredients 0.01 to 2%, Oil content 20-70%, surfactant 2-7%, humectant 5-10%, purified water 11-73%,
A composition containing a trace amount of preservative and a trace amount of fragrance, as a lotion; essential ingredients 0.01-2%, lower alcohol 5-10%,
Surfactant 0.5-2%, humectant 2-8%, antioxidant 0.01-0.05%, chelating agent 0.02-0.1%
, p++ adjuster 0.1-1%, purified water 77-92%, a trace amount of preservative, a composition containing a trace amount of fragrance, as a skin milk; essential ingredients 0.01-2%, oil content 20-40%, Surfactant 2-5%, purified water 53-78%, trace amount of preservative, fragrance
Compositions and emulsions containing trace amounts of: essential ingredients 0.01-2%, oil 10-30%, surfactants 1-5%, humectants 5-10%, purified water 53-84%,
Examples include compositions containing a trace amount of a preservative and a trace amount of a fragrance.

When used as a wound treatment agent, the ointment contains 0.01 to 2% of essential ingredients, 40 to 60% of oil, and 1 to 10% of surfactant.
12%, moisturizing agent 8-15%, purified water 12-51%, composition containing a trace amount of preservative (parent wood ointment), essential ingredients 0.01-2%, oil content 95-99%, purified water O
-5% (oil-based ointment).

Further, compositions similar to the above-mentioned lotions, skin creams, lotions, skin milks, and milky lotions can be mentioned.

Although the details of how the cosmetics of the present invention exhibit excellent anti-aging effects on the skin are unknown, the main cause of skin aging is the condition of the epidermis on the surface of the skin tissue. Rather, the fibrous proteins collagen and elastin that make up the dermis decrease with aging, and their structure changes.
It is presumed that this is due to weakening. Therefore, we extensively searched for substances that promote the growth of fibroblasts, which are cells that produce and secrete collagen and elastin in the dermis, based on the experiments described below. It has been revealed that it has dermal regeneration-promoting effects, collagen production-promoting effects, and fibrosis-promoting effects.
As shown in the examples, excellent anti-aging effects on the human body were also confirmed.

Therefore, when this cosmetic is applied to the skin, it has the effect of promoting the proliferation of dermal fibroblasts, promoting the production of collagen and elastin fibers, promoting the respiratory ability of skin cells, and activating the TCA cycle of skin cells through transdermal absorption. It is presumed that as a result of this, the above-mentioned excellent effects will be exhibited.

The above-mentioned coenzymes used in the present invention naturally exist widely in living organisms and are considered to be extremely safe. Practical use in terms of sensitization, etc. - No particular problems were observed and it was confirmed to be highly safe.

[Effects of the Invention] According to the present invention, by activating skin cells and preventing skin aging, it is possible to prevent wrinkles and give smooth, moist and youthful skin, which is much more effective than conventional products. Provided is a skin preparation for external use that has excellent effects and is extremely safe.

Therefore, the skin external preparation of the present invention includes various cosmetic creams,
Lotion, lotion, skin milk, emulsion, pack agent,
It can be widely used in various forms such as ointments.

Next, the present invention will be explained using test examples and examples.

[Test example]

Test Example 1 The fibroblast proliferation promoting effect of the coenzymes used in the present invention was evaluated as follows.

Human dermis-derived fibroblasts were cultured under serum-suppressed conditions in medium with and without the above compounds, and the effects on proliferation were investigated.

That is, the cells were cultured in a medium containing Eagle MEM medium supplemented with 10% fetal bovine serum (pH 7.6) for 24 hours at 37 degrees in a C02 incubator, and then replaced with a medium containing suppressed serum to 1%. The medium was then cultured for an additional 24 hours.The medium was then replaced with a medium containing to-5M of the test substance and 1% serum, and culture was continued.

Thereafter, the medium was replaced every other day. Table 1 shows the results.
Shown below. Here, the number of cells after 14 days in the case where no test substance was added (control) was set as 100, and the cell number when the test substance was added was expressed as a relative value.

From the results in Table 1, it can be seen that all of the active ingredients of the present invention exhibit a remarkable effect of promoting proliferation of human fibroblasts. Among them, 00ku, 2 and CoA.3Na showed particularly excellent effects.

Fibroblasts present in the dermis of the skin are cells that produce structural proteins such as collagen and elastin, which are responsible for maintaining skin flexibility and elasticity. Therefore, promoting the proliferation of fibroblasts is a necessary condition for promoting the production of collagen and elastin, and as a result, improving the softness and elasticity of the skin.

Test Example 2 The effect of promoting wound dermis regeneration and collagen production of the coenzymes used in the present invention was evaluated as follows.

One group consisted of six 6-week-old Wistar male rats. After hair was removed from the backs of the rats, the skin was cut into a circular shape (approximately 15 mm in diameter) and treated with
% ethanol solution or a 10% ethanol solution without these (control) (0.1 ml each) was applied twice daily for 6 consecutive days. After sacrifice on the 7th day, the surgical site was excised and the weight of the newly formed granulation, which is the dermis, and the amount of hydroxyproline, which is a constituent amino acid unique to collagen, were measured as indicators of the amount of regenerated collagen. The results are shown in Table-2. Here, the control granulation weight and hydroxyproline amount are each 100%.
, expressed as a relative value.

Table 2 From the results in Table 2, a clear increase in the weight of regenerated granulation and the amount of hydroxyproline was observed for all active ingredients of the present invention, indicating that the amount of collagen in the dermis increased. Among them, CoA.3Na and CoB,2 showed particularly excellent effects.

The above results indicate that the active ingredient of the present invention has an excellent wound healing promoting effect when applied externally. In other words, the proliferation of fibroblasts present in the skin dermis is promoted, resulting in an increase in granulation weight, which in turn promotes collagen production, which prevents and improves skin aging such as loss of elasticity and the appearance of fine wrinkles. This shows that it is possible to do so.

Test Example 3 The effect of the active ingredient of the present invention on the strength of collagen and elastin fibers in the dermis was evaluated as follows.

A group of eight 6-week-old Wistar male rats were made. After hair was removed from the backs of the rats, a 4 cm sharp linear incision was made along the midline of the back using a scalpel, and three points were placed at equal intervals. It was sutured with a Mitsuhel needle. Misohel needle after surgery 4
I removed it the other day. Physiological saline containing 50mM of test substance or physiological saline without these (control)
Each dose of 0.1 mjl' was administered once daily to the incision site for 71 consecutive days. After slaughter on the 8th day, the skin at the incision site was cut out, and three skin pieces with a width of 1 cm perpendicular to the incision line were prepared for each animal. Ten5ile strength
, g/cm) was measured and used as an index of the strength of regenerated collagen/elastin fibers.

The results are shown in Table-3. Here, the tensile strength of the control is expressed as a relative value as 100.

Table-3 In this test, if the relative value is 110 or more,
It is generally judged that the tensile strength has been improved, but Table 3
The results show that the active ingredients of the present invention clearly increase the tensile strength of regenerated collagen and elastin fibers, indicating that wound repair is promoted.

Among them, CoA, 3Na and CoB. showed particularly excellent effects. In other words, it is possible that the elasticity and flexibility of the skin may be improved by increasing the tensile strength of the regenerated largen-elastin fibers.

〔Example〕

Example 1 After dissolving polyoxyethylene human oil, perfume, and methyl paraoxybenzoate in ethanol, the solution was added to an aqueous solution containing other components to make them solubilized, thereby preparing lotions I to ■. The ingredients used in each lotion are summarized in Table 4. Note that the numerical values in the table are mm% (the same applies hereinafter).

Table 4 Next, the effectiveness of the lotions ① to ② prepared in this way was evaluated as follows.

Two groups of 20 women (30 to 50 years old) who were statistically equivalent were selected, and one group of women was given lotion I and ■ twice a day (morning and night) for three consecutive months. For the other group of women, lotions ■ and ■ were used separately on the left and right sides of the face in a half-face method, and the degree of improvement in skin elasticity, skin luster, rough skin, and fine wrinkles was examined. . Tables 5 and 6 summarize the results.
Shown below.

From the results in Tables 5 and 6, it can be seen that the lotion (■) of the present invention containing C0B12 has better skin elasticity compared to the lotion (2) containing vitamin B12 (dianocobalamin) and the lotion (2) without these ingredients. It showed excellent effects in improving gloss, rough skin, and fine wrinkles.

On the other hand, a slight improvement was observed with the lotion (2) containing vitamin B12, but the effect was far inferior to that of the lotion (2) of the present invention.

From the above results, CoB. It is clear that it has excellent effects on skin activation and anti-aging.

In addition, no abnormality was observed in the condition of the skin during and after using the lotions 1 to 2 for 3 months. The results of the safety test of COB+2 used in lotion (1) of the present invention are shown below.

Acute oral toxicity (rat): LDso value; 5 g/kg or more Skin irritation (1) - Secondary irritation (guinea pig): 5%; no irritation (2)
Phototoxicity (guinea pig): Negative (3) Eye mucosal irritation (
Rabbits): 5%: Non-irritating skin Sensitization (1) Sensitivity (guinea pigs): Negative (2) Photosensitivity (guinea pigs): Negative Human patch test: 5%; No stimulation
From the second result, it was confirmed that the safety of C0B12 is extremely high.

Example 2 Components 1 to 6 and components 7 to 10 shown in Table-7 were separately prepared at 80%
After heating and dissolving at ℃, the mixture was mixed and emulsified, and while cooling, component 11 was added and uniformly dispersed to prepare the cream shown in Table 7.

Table 7 The effectiveness of the cream thus prepared was measured as follows.

Twice a day (morning and evening) to 20 women (30 to 50 years old)
After using the product of the present invention and a comparative example separately on the left and right faces of the face using the half-face method for three consecutive months, the degree of improvement in skin elasticity, skin gloss, rough skin, and fine wrinkles was examined. The results are summarized in Table-8.

From the results in Table 8, the cream of the present invention containing FAD/2Na showed superior effects in each evaluation item compared to the additive-free comparative example. In addition, no abnormality was observed in the condition of the skin during and after using the above cream for three months.

When the safety of FAD/2Na was tested using the same method as in Example 1, no problems such as skin irritation were observed, and it was confirmed that it was extremely safe.

Example 3 Components 1 and 2 shown in Table 9 were mixed uniformly.

Component 7 was added to this while stirring, and after completely dissolving at around 60°C, Components 3.5 and 6, which had been mixed and dissolved in advance, and Component 4 were sequentially added and mixed. No. 9 pack agents were prepared.

Table 9 The effectiveness was investigated in the same manner as in Example 1, except that this pack was used on the face twice a week for six consecutive months. The results are shown in Table-10.

From the results in Table 10, the pack agent of the present invention containing NAD-Na showed remarkable effects in each evaluation item compared to the comparative example. In addition, no abnormality was observed in the condition of the skin during or after using the above pack for 6 months.

In addition, when we tested the safety of NAD-Na using the same method as in Example 1, no problems such as skin irritation were observed.
It was confirmed that the safety is extremely high.

Example 4 After separately mixing and dissolving components 1 to 6 and components 7 to 11 shown in Table 11, the solution of components 1 to 6 was added thereto while stirring the solution of components 7 to 11, and the solution of components 1 to 6 was added to the solution shown in Table 1. A lotion shown in No. 11 was prepared.

Table: The effectiveness of this lotion was investigated in the same manner as in Example 1. The results are shown in Table-12.

From the results in Table 12, it can be seen that the lotion of the present invention containing NADP-Na exhibits remarkable effects in each evaluation item compared to the comparative example. In addition, 3 of the above lotions
No abnormalities were observed in the condition of the skin during or after use.

Furthermore, when the safety of NADP-Na was tested using the same method as in Example 1, no problems such as skin irritation were observed, and it was confirmed that the safety was extremely high.

Example 5 Components 1 to 3 shown in Table 13 were stirred and mixed, and then heated and dissolved at 80°C. Next, components 4 to 6 and component 8 were sequentially added thereto and uniformly emulsified, and then component 7 was added and cooled to room temperature to prepare the homogeneous skin milk shown in Table 13.

Table 13 The effectiveness of the skin milk thus obtained was investigated in the same manner as in Example 1. The results are shown in Table-14.

From the results in Table 14, the skin milk of the present invention containing CoA/3Na showed remarkable effects in each evaluation item compared to the comparative example. In addition, no abnormality was observed in the condition of the skin during or after using the skin milk for three months.

Further, when the safety of CoA/3Na was tested using the same method as in Example 1, no problems such as skin irritation were observed, and it was confirmed that the safety was extremely high.

Example 6 Components 1 to 4 shown in Table 15 were heated and dissolved at 80°C. On the other hand, components 5 to 9 were dissolved by heating at 80°C, added to the oil phase solution (components 1 to 4), and emulsified. While cooling, component 10 was added midway through the process, and the mixture was cooled to room temperature. An emulsion shown in -15 was prepared.

Table 15 The effectiveness of the emulsion thus prepared was investigated in the same manner as in Example 1. The results are shown in Table-16.

From the results in Table 16, the emulsion of the present invention containing PQQ.2K showed remarkable effects in each evaluation item compared to the emulsion of the comparative example without additives. In addition, no abnormality was observed in the condition of the skin during or after using the above emulsion for three months.

Furthermore, when the safety of PQQ-2 was tested using the same method as in Example 1, no problems such as skin irritation were observed, and it was confirmed that the safety was extremely high.

Example 7 Ointments 1 to 3 shown in Table 17 were prepared as wound treatment agents.

Example 8 Solutions 1 to 2 shown in Table 18 were prepared as wound treatment agents.

Table-18

Claims (3)

[Claims] (1) 5'-deoxyadenosylcobalamin or its salt, flavin adenine dinucleotide or its salt, β-nicotinamide adenine dinucleotide, its reduced compound or salt thereof, β-nicotinamide adenine dinucleotide phosphate, its reduction 1. An external preparation for skin, comprising at least one compound selected from the group consisting of type compounds or salts thereof, coenzyme A or salts thereof, and pyrroloquinoline quinone or salts thereof. (2) The skin external preparation according to claim 1, which is used as a wound treatment agent. (3) The external skin preparation according to claim 1, which is used as a skin cosmetic.