JPH06189780A - Hyaluronic acid production promoting agent - Google Patents

Abstract

PURPOSE:To provide the subject agent containing N-methyl-L-serine, ethanolamine, N-methylethanolamine or their salts, etc., acting on human- originated fibroblast cell and promoting the depressed hyaluronic acid productivity. CONSTITUTION:The objective hyaluronic acid production promoting agent having the form of cosmetic, bathing agent, pharmaceuticals, etc., acting on human- originated fibroblast cell and capable of promoting the hyaluronic acid productivity is produced by compounding one or more compounds selected from N-methyl- L-serine, ethanolamine, N-methylethanolamine and their salts in an amount of 0.001-3W/W% for N-methyl-L-serine or its salt, 0.001-1W/W% for ethanolamine or its salt and 0.00001-0.05 W/W% for N-methylethanolamine or its salt to an excipient or assistant such as petrolatum, squalane, higher alcohol, higher fatty acid lower alkyl ester and polyhydric alcohol, a surfactant such as polyethylene alkyl ether phosphate, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a hyaluronic acid production promoter which promotes hyaluronic acid production in cultured cells or in vivo.
Further, the present invention relates to a hyaluronic acid production promoter which can be added to cosmetics, medicines and the like to enhance the hyaluronic acid production ability of the skin.

[0002]

BACKGROUND OF THE INVENTION Hyaluronic acid retains water in intercellular spaces, retains cells based on the formation of a jelly-like matrix in tissues, retains lubricity and flexibility of tissues, and exerts external forces such as mechanical obstacles. It has many functions such as resistance to bacteria and prevention of bacterial infection (BIO INDUSTR
Y, vol. 8, p. 346, 1991).

[0003] For example, it is said that hyaluronic acid in the skin decreases with age, resulting in aging such as wrinkles and roughness.

As an agent for improving such aged skin,
Many cosmetics containing collagen or hyaluronic acid have been proposed, but they only improve the moisturizing effect on the surface and do not essentially improve aged skin. Other,
Although vitamins and crude drugs are used as skin cell activating agents, the current situation is that they have not yet reached the treatment of aged skin.

Hyaluronic acid in the synovial fluid covers the surface of the articular cartilage and contributes to the smooth operation of the joint function. Hyaluronic acid concentration in normal human joint fluid is approximately 2.3 mg
However, in the case of rheumatoid arthritis, the concentration of hyaluronic acid in the synovial fluid decreases to about 1.2 mg / ml, and at the same time, the viscosity of the synovial fluid decreases significantly (Arthriti
s Rheumatism, vol. 10, p. 357, 196
7 years).

[0006] In addition, it is known that a decrease in hyaluronic acid content occurs in purulent arthritis and gouty arthritis as in rheumatoid arthritis [Connective tissue (Kanehara Publishing Co., Ltd., 481, 1984)].

In the above diseases, it is considered that the amount of hyaluronic acid in the synovial fluid is increased in order to improve the lubrication function, cover and protect the articular cartilage, suppress pain, and improve the properties of pathological synovial fluid. For example, when rheumatoid arthritis patients are given joint injection therapy with sodium hyaluronate, the above improvement has been observed (inflammation, vol. 11, p. 16, 1
991).

Similarly, in traumatic arthrosis, osteoarthritis and osteoarthritis, the above-mentioned improving effect by the joint injection therapy of hyaluronic acid has been reported [Connective tissue and disease (Kodansha), 246, 1980. Year〕.

However, the treatment of the above diseases is long-term,
Moreover, it requires a doctor's prescription. Therefore, an ointment or gel containing a hyaluronic acid production promoter which can be easily treated in daily life has been desired.

Further, during the healing process after burn injury, hyaluronic acid is remarkably increased in the granulation during the period from the initial stage of the granulation tissue growing from below the necrotic tissue to the time when the whole tissue is replaced with the granulation tissue. Is known [connective tissue and disease (Kodansha), p. 153, 1980], and a hyaluronic acid production promoter is expected as an early therapeutic drug for burns.

Insulin-like growth factor-1 and epidermal growth factor (Biochimica Biophysica Ac) are used as agents for promoting the production of hyaluronic acid in human cells.
ta, 1014, p. 305, 1989) and interleukin-1 (Japanese Society of Obstetrics and Gynecology, 41, 1.
943, 1989), or cytokines such as phorbol ester (Experimental C)
ell Research, 148, 377, 19
1983) and the like are known, but none of them can be safely used as cosmetics, bath salts and pharmaceuticals.

[0012]

Therefore, the object of the present invention is to promote the hyaluronic acid-producing ability of cells to prevent aging of the skin or to treat diseases associated with abnormal decomposition of hyaluronic acid, Moreover, it is to provide a safe hyaluronic acid production promoter which has little effect on the human body.

[0013]

The above object comprises one or more compounds selected from the group consisting of N-methyl-L-serine, ethanolamine, N-methylethanolamine, or salts thereof. This is achieved by a hyaluronic acid production promoter characterized in that

N-methyl-L used in the present invention
-Serine, ethanolamine, N-methylethanolamine, or salts thereof are small molecules, and therefore, when applied to the skin, pass through the epidermal layer and basement membrane, and the dermal layer (connective tissue) where fibroblasts are present. ) Can be reached, which is advantageous in promoting the hyaluronic acid-producing ability of fibroblasts (see Test Example 7 below).

N-methyl-L-serine, ethanolamine, N-methylethanolamine, or salts thereof are known compounds, and the production method thereof is not particularly limited, and a commonly used method is used. Good. In the present invention, these compounds may be used alone or in combination of two or more.

The hyaluronic acid production promoter of the present invention can be applied to cultured cells or living cells by itself to promote the production of hyaluronic acid, but it is blended in cosmetics, pharmaceutical compositions and the like. You may use.

The hyaluronic acid production promoter and composition of the present invention may be in the form of liquid, solid or semi-solid preparation, preferably ointment, gel, cream, spray, patch, lotion, packs, Examples include emulsions, powders, bath salts and the like.

The excipients or auxiliaries used for producing these compositions may be appropriately selected from those usually used for the same purpose according to the dosage form, and are not particularly limited. However, for example, hydrocarbons such as petrolatum and squalane, higher alcohols such as stearyl alcohol,
Higher fatty acid lower alkyl esters such as isopropyl myristate, animal oils and fats such as lanolinic acid, glycerin,
Polyhydric alcohols such as propylene glycol, glycerin fatty acid esters, polyethylene glycol monostearate, surfactants such as polyethylene alkyl ether phosphate, methyl paraoxybenzoate, preservatives such as butyl paraoxybenzoate, waxes, resins, various flavors, Examples include various dyes, various inorganic salts such as sodium citrate, sodium carbonate, lactic acid, various acids, water, ethanol, and the like, and examples of the obtained composition include cosmetics, bath salts, pharmaceuticals, and the like.

The content of N-methyl-L-serine, ethanolamine, N-methylethanolamine, or a salt thereof in the hyaluronic acid production promoter varies depending on its form and cannot be specified unconditionally, but the composition of application In the case of N-methyl-L-serine or its salt, 0.001 to 3% (W / W) is preferable, and 0.01 to 0.1% is more preferable, assuming that the whole product is 100% (W / W). (W /
W), ethanolamine or a salt thereof, 0.00
1 to 1% (W / W) is preferable, and more preferably 0.
005-0.1% (W / W), in the case of N-methylethanolamine or its salt, 0.00001-0.05%
(W / W) is preferable, and more preferably 0.001 to
It is 0.01% (W / W). However, in the case of a bath additive that is diluted at the time of use, the content can be further increased.

When N-methyl-L-serine (salt) is used for producing hyaluronic acid in cultured cells, it is preferable to add 1 mM or more, more preferably 1 to 10 mM in the culture medium of the cells. In the case of (0.012 to 0.12% by weight) and ethanolamine (salt), 0.005% by weight or more is preferable, and 0.005 to 5% is more preferable.
In the case of 0.1% by weight and N-methylethanolamine (salt), 0.001% by weight or more is preferable, and 0.001 to 0.01% by weight is more preferable.

[0021]

When the hyaluronic acid production promoter of the present invention is added to a human dermal fibroblast culture system, the hyaluronic acid production is promoted in a concentration-dependent manner (see Test Examples 1 and 2 below). This hyaluronic acid production promoter had low toxicity (see Test Example 3 below) and low skin irritation (see Test Examples 4 to 6 below). When the hyaluronic acid production promoter (cream, lotion) of the present invention is applied to the skin, it passes through the epidermis layer and the basement membrane and reaches the dermis layer containing fibroblasts (see Test Example 7 described later). Furthermore, the hyaluronic acid production promoter of the present invention can also increase the amount of hyaluronic acid in the living body (see Test Example 8 below). Therefore, the hyaluronic acid production promoter of the present invention is highly safe and can act on fibroblasts to promote the hyaluronic acid production of connective tissues such as skin, which are pathologically or physiologically decreased.

[0022]

EXAMPLES Prior to the examples, test examples showing the effect of the present invention will be described. The methods for preparing and measuring the reagents used in each test are as follows.

(A) Method for preparing MEM medium Minimum Essential Medium
(Dainippon Pharmaceutical Co., Ltd., 10-101) 10.6 g each has a final concentration of 1% (V / V) Non Essential
l Amino Acid (Dainippon Pharmaceutical Co., 16-81
0), 0.1% lactalbumin hydrolyzate (manufactured by Sigma, L
-9010), 1 mM sodium pyruvate (manufactured by Dainippon Pharmaceutical Co., Ltd., 16-820), 1.2% (W / V) sodium hydrogen carbonate, 50 mg / l streptomycin sulfate were added, and distilled water was added to make 1 l. Then, carbon dioxide gas was blown in to adjust the pH to about 7 (hereinafter abbreviated as MEM medium).

(B) Inactivation of fetal bovine serum (FBS) FBS (manufactured by Irvine Scientific)
Heat treatment was performed at 6 ° C. for 30 minutes.

(C) Preparation of PBS 8 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate and 0.2 g of potassium dihydrogen phosphate were dissolved in 1 liter of purified water, and Phosphat
e Buffered Saline (hereinafter abbreviated as PBS).

(D) Buffer H 0.5 M 2- (N-morpholine) ethanesulfonic acid-sodium hydroxide buffer containing 0.1 M sodium acetate and 0.02% (W / V) sodium azide buffer (p
H6.0).

(E) Buffer C containing 0.15 M sodium chloride, 0.02% (W / V) sodium azide and 0.05% Bridge-35 50
mM Tris-HCl buffer (pH 7.5).

(F) Pronase solution 200 μg / ml pronase (manufactured by Calbiochem-Beering Corporation, Streptomyces
griseus ), 0.5 M Tris-HCl buffer (pH 8.0) containing 0.15 M sodium chloride and 0.02% sodium azide.

(G) Hyaluronidase solution 6 TRU / ml hyaluronidase (EC 4.2.2.
1, made by Seikagaku Kogyo, Streptomyces hya
Buffer H containing ( from lurolyticus ).

(H) Trypsin solution PBS containing 0.1% trypsin (manufactured by Sigma)

(I) Fractionation method of high molecular weight fraction using PD-10 column When a sample solution is applied to a PD-10 column (Pharmacia, gel filtration desalting column packed with Sephadex G-25) , 2.5 for high molecular weight components with a molecular weight of 5000 or more
In a fraction of ~ 4 ml, low molecular weight components with a molecular weight of less than 5000 are recovered in a fraction of 4-12 ml.

(J) Method for measuring the number of viable cells After removing the culture supernatant of a cell line cultured in a 6-well plate by suction, each well was washed 3 times with 1 ml / well of PBS.

After washing, 0.2 ml of trypsin solution was added to each well to release the attached cells, and then MEM medium containing 10% inactivated FBS was added.

20 μl of trypan blue solution was mixed with 20 μl of MEM medium containing cells, and the number of viable cells was measured.

Test Example-1 Hyaluronic acid production promoting action 1. Test compound: N-methyl-L-serine

2. Test method: Cell culture normal human fibroblast cell line [Detroit 551 strain (ATCC
CCL 110)] was adjusted to 1 × 10 ⁵ cells / ml with MEM medium containing 10% (V / V) inactivated FBS, and 2 ml each was seeded on a 6-well plate (Falcon) to obtain 95% ( V / V) Air-5% (V / V) Carbon dioxide gas atmosphere, static culture was carried out at 37 ° C for 2 days.

The culture supernatant was removed by suction and washed twice with PBS and once with MEM medium at 1 ml / well, respectively,
Add 2 ml of MEM medium to each well, 95% (V / V)
Air-5% (V / V) 1 at 37 ° C in an atmosphere of carbon dioxide
The culture was allowed to stand for a day.

The culture supernatant was removed by suction and washed once with PBS and once with MEM medium at 1 ml / well each.

Next, the final concentration is 0 mM, 0.1 mM, 1 m
N-methyl-L to give M, 3 mM and 10 mM
- 1 [mu] Ci was added serine / ml glucosamine hydrochloride D- [1,6- ³ H (N)] (Du Pont made, NET-
ME containing 557A, hereinafter referred to as [ ³ H] glucosamine)
Add 2 ml of M medium to each well (all n = 3),
It was used to measure the amount of hyaluronic acid produced.

Further, a medium having the same composition as above except that it did not contain [ ³ H] glucosamine was added, and used for measuring the number of viable cells.

95% (V / V) of the above two types of plates
2 at 37 ° C in an atmosphere of air-5% (V / V) carbon dioxide gas
The culture was allowed to stand for a day.

Measurement of production amount of hyaluronic acid The culture supernatant was collected from each well for measurement of production amount of hyaluronic acid, heated at 100 ° C. for 10 minutes, and then 0.7
0.08 ml of pronase solution was added to 2 ml to decompose proteins bound to hyaluronic acid and other proteins produced in the culture solution. The same operation was performed twice on the culture supernatant obtained from each well.

After standing at 37 ° C. for 18 hours, heat treatment was performed at 100 ° C. for 10 minutes to inactivate pronase.

Next, 0.3 ml each of 0.8 ml of the above reaction mixture was placed in two tubes, 0.3 ml of buffer H was added to one tube, and 0.1 ml of hyaluronidase solution was added to the other tube.
Add 3 ml and leave it at 37 ℃ for 18 hours, then at 100 ℃
The mixture was heat-treated for 10 minutes to inactivate hyaluronidase.

0.5 ml each of 0.6 ml of the above reaction mixture without hyaluronidase and with addition of hyaluronidase was added with 0.15 M sodium chloride, 0.02
The mixture was applied to a PD-10 column equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 50% sodium azide and 0.05% Bridge-35, and 2.5 to 4.0 ml of high molecular weight fraction was collected. .

1 ml of 1.5 ml of each high molecular weight fraction
10 ml of scintisole EX-H (manufactured by Dojindo Laboratories) was added to and the [ ³ H] radioactivity incorporated into the high molecular weight fraction was measured by a liquid scintillation counter (manufactured by Aloka).

[ ³ H] radioactivity (DPM / well) incorporated into the high molecular weight fraction without the addition of hyaluronidase was incorporated into the high molecular weight fraction when hyaluronic acid was specifically decomposed by hyaluronidase [ ³ H] The value obtained by subtracting the radioactivity value (base) is the amount of hyaluronic acid produced (D
PM / well).

3. Test results: various concentrations of N-methyl-L
-The number of viable cells and the amount of hyaluronic acid produced in the obtained culture supernatant when serine was added were measured (Table 1). As a result, it was found that the addition of N-methyl-L-serine did not affect the viable cell number and that the amount of hyaluronic acid produced by the Detroit 551 strain increased in a dose-dependent manner.

[0049]

[Table 1]

Test Example 2 Hyaluronic Acid Production Promoting Action 1. Test compound: ethanolamine, N-methylethanolamine

2. Test method: in the same manner as in Test Example-1,
The amount of hyaluronic acid produced by ethanolamine and N-methylethanolamine was measured (Table 2).

3. Test results: ethanolamine and N
-Methylethanolamine was found to promote hyaluronic acid production in a dose-dependent manner.

[0053]

[Table 2]

(Test Example 3) Acute toxicity test (JP-A-4-
(1130 publication)

1. Test compound: N-methyl-L-serine

2. Test method: water and N-methyl-L-
An aqueous solution of serine (prepared to be 5 g / kg body weight as a sample) was orally administered to male ICR mice (5 weeks old, weight 24-28 g, 5 mice per group) at a ratio of 0.2 ml / kg body weight, and then The mice were observed for 7 days.

3. Test results: In the N-methyl-L-serine administration group, as in the control group (administering only water), no death was observed at all.

(Test Example-4) Skin cumulative irritation test

1. Test compound: N-methyl-L-serine, ethanolamine, N-methylethanolamine

2. Test method: Using a Japanese domestic male rabbit (body weight of about 3 kg), the Draize method (Appraisal method)
of the safety of chemicala
lsin foods, drags and cosm
etics, p. 46, 1959, Edited an
d published by the editor
ial committee, association
of food and drug office
ls of U. S. A. ).

That is, the back of the rabbit (3x
4 cm), 0.1 ml of a 0.1% (W / V) aqueous solution of the test compound adjusted to pH 7 with hydrochloric acid was applied by open application to 1 cm.
It was applied once a day for 4 days. 24 hours later, erythema on the skin,
The edema, crust and crack states were observed, and the erythema, edema, crust and crack scores were assigned according to the evaluation criteria in Table 3, respectively.

[0062]

[Table 3]

The erythema score, edema score, scab score and fissure score listed in Table 3 were summed to give a cumulative irritation score.

Next, the degree of stimulation of N-methyl-L-serine, ethanolamine and N-methylethanolamine was determined from the cumulative stimulation scores based on the criteria shown in Table 4 below.

[0065]

[Table 4]

3. Test results: It was shown that N-methyl-L-serine, ethanolamine and N-methylethanolamine had low cumulative irritation to the skin (Table 5).

[0067]

[Table 5]

(Test Example-5) Primary skin irritation test (see JP-A-4-1130)

1. Test compound: N-methyl-L-serine, ethanolamine, N-methylethanolamine

2. Test method: Using a Japanese domestic male rabbit (body weight: about 3 kg), the drate method (Appraisal)
of the safety of chemical
s in foods, drags and cos
metrics, p. 46, 1959, Edited a
nd published by the edito
Rial committee, associatio
no of food and drug offici
als of U. S. A. ).

That is, a scratched site (damaged skin) was created on the back of a shaved rabbit, and 0.1 ml of water or 1% (W) of N-methyl-L-serine was added to the damaged skin and normal skin, respectively. / V) 0.1 ml of an aqueous solution or 0.1 ml of a 1% (W / V) aqueous solution of ethanolamine and N-methylethanolamine adjusted to pH 7 with hydrochloric acid is applied to a patch test plaster (1.2 x 1.6 cm, ribbon). Aid (registered trademark, manufactured by River Tape Pharmaceutical Co., Ltd.) was soaked and applied. Twenty-four hours later, the adhesive bandage was peeled off, and the erythema and edema state of the skin was observed. Further, the same observation was made 48 hours after the adhesive bandage was peeled off. Then, the erythema and edema scores were assigned according to the evaluation criteria of Table 6, respectively.

[0072]

[Table 6]

Each score listed in Table 7 was obtained, and the primary stimulation score was calculated based on the following formula.

[0074]

[Equation 1]

[0075]

[Table 7]

Next, the degree of stimulation of N-methyl-L-serine was determined from the primary stimulation score based on the criteria shown in Table 8 below.

[0077]

[Table 8]

3. Test result: N-methyl-L-serine,
It was shown that ethanolamine and N-methylethanolamine had low skin irritation (Table 9).

[0079]

[Table 9]

(Test Example 6) Skin irritation test (human patch test, see JP-A-3-227921)

1. Test compounds: N-methyl-L-serine, ethanolamine and N-methylethanolamine

2. Subjects: 19 healthy people

3. Test method: Closed patch test method (Journal of the Society o
f Cosmetic Chemist, Volume 31, 97
(1980), using a KI chamber on the forearm of the subject, 0.05 ml of a 1% (W / V) aqueous solution of the test compound adjusted to pH 7 with hydrochloric acid was occluded for 24 hours, and the patch was removed. The skin reaction was observed after 1 hour and 24 hours.

4. Test results: In any patch test of N-methyl-L-serine, ethanolamine and N-methylethanolamine, one subject had a slight erythema at both 1 hour and 24 hours after patch removal. However, it was judged that there was almost no skin irritation.

(Test Example-7) N-methyl-L-serine,
Skin permeability test of ethanolamine and N-methylethanolamine

1. Test drug Creams and lotions of Examples 3, 6, 9, 14, and 17 described below

2. Preparation of rat skin Wistar male rats shaved on the day before the test were sacrificed with ether, and the abdominal skin was immediately peeled off.

3. Diffusion Cell Experiment Method Using the vertical diffusion cell device (Kerkoso Engineering, effective area 8 cm ² ) shown in FIG. 1, the cell was placed in an air thermostat at 32 ° C., and an isotonic phosphate buffer solution ( 45 ml of 1.44% sodium hydrogen carbonate and 2.33% potassium dihydrogen phosphate) was added, and 1 g of the sample was applied to the rat abdominal peel skin on the donor side (n = 3), 1,
After 2, 4 and 6 hours, add 0.2% buffer to the receptor bath.
Each ml was collected and immediately frozen and stored at -20 ° C.

4. Quantitative method of N-methyl-L-serine, ethanolamine, and N-methylethanolamine After thawing a frozen sample, it was diluted with 10 mM hydrochloric acid to an appropriate concentration, and asparatylglycine (final concentration 40 μM) was added as an internal standard. Amino acid analysis standard solution (A
N-type), asparatylglycine and N-methyl-L-serine, ethanolamine, and N-methylethanolamine (final concentration 10 μM) were added at the same concentration and used. Quantification of these samples was performed by post-column amino acid analysis method (lithium method) using ο-phthalaldehyde method (Analy
mechanical Biochemistry, Volume 96, 29
8 pages, 1979).

5. Test Results The results are shown in FIG. 2 and Table 10. N-methyl-L-serine (Example 3) permeated rat skin, and the permeation rate was about 0.12 μmol / cm ² / hour for ^{2 to} 6 hours after application of the cream (FIG. 2). In addition, ethanolamine (Examples 6 and 14), N-methylethanolamine (Example 9,
17) was also found to penetrate the skin (Table 10).

[0091]

[Table 10]

(Test Example-8) Effect on the amount of hyaluronic acid in rat skin

1. Test compound: N-methyl-L-serine

2. Method for measuring the amount of hyaluronic acid in the skin Extraction of hyaluronic acid from rat skin After slaughtering hairless rats with ether, the back skin was immediately exfoliated.

30 ml of each skin (100-300 mg)
Washed 3 times with 0.5M sodium acetate and then 15M
L methanol / chloroform solution (7: 3, V / V)
Degreased three times. The skin is cut into small pieces, 2.4 ml of 0.15 M sodium chloride is added to 100 mg of skin, and 28,000 r is obtained using a histocol (manufactured by Nichine Medical Science Equipment Co., Ltd.)
After homogenizing twice at pm for 30 seconds, heat treatment was performed at 121 ° C. for 20 minutes.

After cooling the heat-treated skin sample, 1M containing 5 mg / ml Pronase E (manufactured by Merck) of 1/9 volume
Add Tris-HCl buffer (pH 7.9), and add 2 at 37 ° C.
Shaked for days. After heat treatment at 100 ° C. for 10 minutes to inactivate pronase, centrifugation was performed at 3000 rpm for 3 minutes to obtain a supernatant (hereinafter referred to as an extract).

Measurement of the amount of hyaluronic acid in the extract : The extract obtained above was divided into 0.5 ml aliquots, and the amount was adjusted to 0.5.
It is applied to a Sephadex G-50 column (1.5 x 5 cm) equilibrated with 2 M acetic acid, and hyaluronic acid is added at 2.0 to
Elution was performed in 3.5 ml of high molecular weight fraction, and high molecular weight fractions derived from the same extract were mixed. Each high molecular weight fraction mixture was lyophilized and then dissolved in 1.5 ml of buffer H.

From 1.5 ml of the above lysate, take 950 μl in one tube and add 50 μl of 200 TR to it.
Add U / ml hyaluronidase-containing buffer H (hyaluronidase treated sample) and add 4 to the other tube.
75 μl was taken and 525 μl of buffer H was added (untreated sample). After standing at 60 ℃ for 18 hours, 1
Heat treatment was performed at 00 ° C. for 10 minutes to inactivate hyaluronidase.

The hyaluronidase treated and untreated samples were divided into 0.5 ml aliquots and separated on a Sephadex G-50 column (1.5 x 5) equilibrated with 0.2 M acetic acid.
cm) to obtain 2.0 to 3.5 ml of high molecular weight fraction. Mix the high molecular weight fractions from each sample,
After lyophilization, 0.22 ml of 10 mM citric acid-sodium hydroxide containing 0.5 M sodium chloride (pH 3.
5, hereinafter referred to as buffer solution D).

Of the above 0.22 ml of each solution, 0.2
ml was applied to a DEAE-Sepharose column (column volume 0.1 ml) equilibrated with buffer solution D, and the non-adsorbed fraction 0.
2 ml was obtained. Further, the column was washed with 0.3 ml of buffer solution D, and the washed fraction and the non-adsorbed fraction were mixed.

Using the above mixture, the glucuronic acid, which is one of the two components of hyaluronic acid, was converted into Bitter acid.
Et al. Method (Analytical Biochemis
try, vol. 4, page 330, 1962), and the amount of hyaluronic acid in the skin was calculated by the following formula 2.

[0102]

[Equation 2]

A; glucuronic acid quantitative value (μg / ml) of hyaluronidase-untreated sample b; glucuronic acid quantitative value (μg / ml) of hyaluronidase-treated sample 194.14; glucuronic acid molecular weight 221.21; N-acetylglucosamine Molecular weight of 36.04; molecular weight of 2 water molecules

3. Test subject: W of 6 weeks or 14 weeks
istar male hairless rat, 6 per group

4. Test method: The amount of hyaluronic acid in the skin of each group of 6-week-old and 14-week-old hairless rats was calculated according to the above method. Also, from the age of 6 weeks, 50% (V
/ V) ethanol (control drug) or 10% (W / V) N
-Methyl-L-serine-containing 50% (V / V) ethanol (test drug) was applied once a day at a dose of 0.1 ml to the back for 60 days continuously, and two groups of rats that were 14 weeks old, The amount of hyaluronic acid in the skin was calculated according to the above method.

5. Test results: Table 11 shows the results of measuring the amount of hyaluronic acid in the skin of 6-week-old and 14-week-old rats. As a result, the amount of hyaluronic acid per gram of skin of 14-week-old rats (216 ± 21 μg) was compared with the amount of hyaluronic acid per gram of skin of 6-week-old rats (333 ± 16 μg).
μg) was significantly lower (p <0.01). This indicates that the amount of hyaluronic acid in rat skin decreases with age.

Further, as is clear from Table 12, the group to which the control drug was applied (204 ± 14 μg) was about the same as the 14-week-old rat (216 ± 21 μg) shown in Table 11. To the group to which the test drug was applied (250 ± 18
μg) is significantly higher, and N-methyl-L-serine suppresses the decrease in the amount of hyaluronic acid due to aging.

[0108]

[Table 11]

[0109]

[Table 12]

Examples of the present invention will be given below. The values in the table show% by weight.

Examples 1 to 9 (Cream) Creams were prepared with the compositions shown below.

[0112]

[Table 13]

Preparation method: The component (A) was uniformly mixed and dissolved at 80 ° C., and then the component (B) was mixed and dissolved therein (mixed solution I). Separately from this, the component (D) was uniformly mixed and dissolved at 80 ° C., and then the component (C) was mixed and dissolved therein (mixed liquid II). Next, the mixed solution II was gradually added to the mixed solution I and cooled to 30 ° C. with sufficient stirring to obtain a cream.

Examples 10 to 17 (lotion) Lotions were prepared with the compositions shown below.

[0115]

[Table 14]

Preparation method: Each component was mixed and dissolved to prepare a lotion.

Examples 18 to 20 (Bathing agent)

[0118]

[Table 15]

Preparation method: Each component was mixed to prepare a bathing agent. In addition, this bathing agent is diluted about 3000 times at the time of use.

Examples 21 to 26 (ointment)

[0121]

[Table 16]

Preparation method: Each component of the above (B) was mixed in a hot water bath for 8 hours.
The mixture was mixed while being heated to 0 ° C., and this was gradually added to the mixture of each component of the above (A) which was heated to 80 ° C. with stirring. Next, a homogenizer (Tokusyukik)
a Vigorous stirring for 250 minutes (from Kogyou) (250
(0 rpm) to sufficiently emulsify and disperse each component, and then gradually cooled with stirring to obtain an ointment.

Examples 27-32 (gel)

[0124]

[Table 17]

Preparation method: (A) was swollen with a part of water (D), component (C) was dissolved with the remaining water (D), and then both were uniformly mixed (mixed solution I). The component (B) was uniformly mixed and dissolved (mixed liquid II). The mixed solution II was added to the mixed solution I and dispersed to obtain a gel.

Examples 33 to 38 (hair tonic)

[0127]

[Table 18]

Preparation method: After the perfume was dissolved with the perfume-solubilizing agent, it was dissolved by adding it to ethanol while stirring at room temperature, and the component (B) was sequentially added and dissolved (mixed solution I). The component (C) was dissolved and added to the mixed solution I with stirring to make the mixture uniform, and then filtered to obtain a hair tonic.

[Brief description of drawings]

FIG. 1 is a diagram showing a vertical diffusion cell device used in Test Example-7.

FIG. 2 is a diagram showing the results of a skin permeability test of N-methyl-L-serine in Test Example-7.

[Explanation of symbols]

 1 Teflon lid part 2 Sampling port 3 Drug sample 4 Skin 5 O-ring 6 Receptor phase 7 Stirrer

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number in the agency FI technical display location C12R 1:91) (72) Inventor Shingo Sakai 5-3 28, Kotobura-cho, Kanagawa Prefecture Bell Inside the Biochemistry Research Institute (72) Inventor Shintaro Inoue 5-3 28, Kotobuki-cho, Odawara-shi, Kanagawa

Claims (1)

[Claims] 1. A hyaluronic acid production promoter comprising one or more compounds selected from the group consisting of N-methyl-L-serine, ethanolamine, N-methylethanolamine, or salts thereof. Agent.