JPH08239404A - Hyaluronic acid production accelerator - Google Patents

Abstract

PURPOSE: To obtain a hyaluronic acid production accelerator which is useful for preventing the aging of the human skin and joints and safe for the human body by fractionating bovine serum by ultrafiltration, etc., and incorporating the resulting low-mol.-wt. fraction having a mol.wt. of 5,000 or lower into the accelerator. CONSTITUTION: A hyaluronic acid production accelerator for preventing the aging and degradation in the lubrication function of the human skin and joints due to decrease in hyaluronic acid content is obtd. by fractionating a low-mol.- wt. fraction having a mol.wt. of 5,000 or lower from bovine serum with an ultrafilter. Pref. bovine fetus serum or bovine neonate serum is used. The low.- mol.-wt. fraction, when applied to culture cells, accelerates the production of hyaluronic acid, and, according to the type of the fraction, can be compounded with known cosmetics or medicine components to give ointments, creams, plasters, lotions, bathing cosmetics, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the prevention of aging of the skin or treatment of diseases associated with abnormal decomposition of hyaluronic acid by promoting the hyaluronic acid-producing ability of human dermal fibroblasts, and the human body. The present invention relates to a hyaluronic acid production promoter that is safe against.

[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).

Among the hyaluronic acids, for example, the hyaluronic acid in the skin is said to decrease with age, resulting in aging such as wrinkles and lumps.

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] It is known that, in the case of rheumatoid arthritis, a decrease in hyaluronic acid content occurs in purulent arthritis, gouty arthritis and the like [connective tissue (Kanehara Shuppan), item 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.

[0012] Examples of agents that promote the production of hyaluronic acid in human cells include insulin-like growth factor-1 and epidermal growth factor (Biochimica Biophysica A).
Cta, 1014, p. 305, 1989) and interleukin-1 (Japanese Society of Obstetrics and Gynecology, Vol. 41,
1943, 1989) or other cytokines, or 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 human skin fibroblasts to prevent skin aging or diseases accompanied by abnormal decomposition of hyaluronic acid. For example, it is intended to provide a hyaluronic acid production promoter which can be used for treating rheumatism, osteoarthritis, gingivitis, etc. and is safe for the human body.

[0013]

The above object is achieved by a hyaluronic acid production promoter characterized by containing a low molecular weight fraction of bovine serum having a molecular weight of 5000 or less.

Examples of the bovine serum used in the present invention include fetal bovine serum and newborn bovine serum.

The molecular weight of bovine serum used in the present invention is 50.
As a means for fractionating a low molecular weight fraction of 00 or less, for example, an ultrafiltration membrane for molecular sieve can be used. Examples of the material of the ultrafiltration membrane include cellulose, cellulose acetate, polyacrylonitrile, and polyether sulfone.

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. However, the low molecular weight fraction of bovine serum having a molecular weight of 5,000 or less is used. Depending on the type, known ingredients used in ordinary cosmetics and medicines can be arbitrarily combined and added, and it is also possible to add them in the composition form of ordinary cosmetics and medicines.

The hyaluronic acid production-promoting agent and composition of the present invention may be in the form of liquid, solid or semi-solid agent, for example, ointment, gel, cream, spray, patch, lotion, packs. , Emulsions, powders and bath salts.

The excipients or auxiliaries used in the production of these compositions may be appropriately selected from those usually used for the same purpose depending on the dosage form, and are not particularly limited. However, for example, petroleum jelly, hydrocarbons such as squalane, higher alcohols such as stearyl alcohol, higher fatty acid lower alkyl esters such as isopropyl myristate, animal fats and oils such as lanolinic acid, glycerin, polyhydric alcohols such as propylene glycol, Glycerin fatty acid ester, polyethylene glycol monostearate, surfactants such as polyethylene alkyl ether phosphate, preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate, waxes, resins, various flavors, various pigments, sodium citrate, carbonic acid Various inorganic salts such as sodium and lactic acid and various acids Water, and ethanol and the like.

The content of the low molecular weight fraction of bovine serum having a molecular weight of 5000 or less in the applied composition varies depending on the object to be applied and cannot be unconditionally specified, but 0.005 to 3.
0% (W / W) is preferable, and 0.01 is more preferable.
~ 2.0% (W / W). However, in the case of a bath additive that is diluted at the time of use, the content can be appropriately increased.

When hyaluronic acid is produced in cultured cells by a low molecular weight fraction of bovine serum having a molecular weight of 5000 or less, 0.01% (W / V) or more is preferably contained in the cell culture solution, Particularly preferably 0.1 to 2.0
% (W / V).

[0021]

The hyaluronic acid production-promoting agent of the present invention promotes the hyaluronic acid production ability of human skin fibroblasts (test example described below). Therefore, it can be used for the prevention of skin aging or diseases accompanied by abnormal decomposition of hyaluronic acid. It can be used for treatment and is safe for the human body.

[0022]

EXAMPLES The present invention will be described in detail below with reference to examples and comparative examples.

Example 1 (Preparation of low molecular weight fraction of fetal bovine serum (FBS) having a molecular weight of 5000 or less) 10 ml of FBS was added to a cellulose ultrafiltration membrane (Millipore,
It is subjected to a molecular weight cut-off of 5000) and the filtrate is 9.2 m under pressure.
1 was obtained and freeze-dried to obtain 148.4 mg of a low molecular weight fraction.

Example 2 (Preparation of low molecular weight fraction of bovine neonatal serum (NBS) having a molecular weight of 5000 or less) 10 ml of NBS was added to a cellulose ultrafiltration membrane (Millipore,
It is subjected to a molecular weight cut-off of 5000) and the filtrate is 9.5 m under pressure.
1 was obtained and freeze-dried to obtain 155.8 mg of a low molecular weight fraction.

Next, a test example 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.

(1) Method for preparing MEM medium Minimum Essential Medium
(Dainippon Pharmaceutical Co., Ltd., 10-101) 10.6 g each with a final concentration of 1% (V / V) Non Essentia
l Amino Acid (Dainippon Pharmaceutical Co., 16-81
0) 1 mM sodium pyruvate (manufactured by Dainippon Pharmaceutical Co., Ltd., 1
6-820), 1.2% (W / V) sodium hydrogen carbonate was added, and distilled water was added to make 1 liter, and then carbon dioxide gas was blown to adjust the pH to about 7 (hereinafter abbreviated as MEM medium). .

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

(3) Preparation method 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).

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

(5) 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).

(6) 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).

(7) 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.

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

Test Example 1 (Hyaluronan production promoting action of low molecular weight fraction of bovine serum having a molecular weight of 5000 or less) (1) Cell culture Normal human fibroblast cell line [Detroit 551 strain (ATCC
CCL 110)] was prepared at 1 × 10 ⁵ cells / ml in MEM medium containing 10% (V / V) inactivated FBS, and seeded in a 12-well plate (manufactured by Falcon) at 1 ml each, and 95% ( V / V) Air-5% (V / V) Carbon dioxide gas atmosphere, static culture was carried out at 37 ° C for 3 days.

The culture supernatant is removed by suction, and the cells are washed with MEM medium 1
Wash once with 1 ml / well, add 1 ml / well of MEM medium, 95% (V / V) air-5% (V / V)
V) Incubation was carried out for 1 day at 37 ° C. in an atmosphere of carbon dioxide.

The culture supernatant was removed by suction, and the cells were washed twice with PBS and once with MEM medium at 1 ml / well.

Next, the final concentration is 0.5%, 1.0%, 2.
Molecular weight of bovine serum 5000 to be 0% (W / V)
The following low molecular weight fractions (Examples 1 and 2) were added to 10 μ
Ci / ml glucosamine hydrochloride D- [1,6-3H
(N)] (manufactured by Du Pont, NET-557A, hereinafter referred to as [ ³ H] glucosamine) in 1 ml of MEM medium
Each was added to each well and used for the measurement of hyaluronic acid (n = 4). As Comparative Example 1, bovine serum has a molecular weight of 50.
A MEM medium was added instead of the low molecular weight fraction of 00 or less to serve as a control.

The above plate was placed in 95% (V / V) air-
Static culture was carried out at 37 ° C. for 3 days in an atmosphere of 5% (V / V) carbon dioxide gas.

(2) Measurement of hyaluronic acid production (method of [ ³ H] glucosamine uptake activity) The culture supernatant was recovered from the hyaluronic acid production measurement well and heated at 100 ° C. for 10 minutes. 0.72
0.08 ml of pronase solution was added to ml to decompose the protein bound to hyaluronic acid.

After leaving it at 37 ° C. for 18 hours, it was heated at 100 ° C. for 10 minutes to inactivate the pronase.

Next, 0.3 out of 0.8 ml of the above reaction solution
Put 2 ml each into two tubes, and put buffer H in one
0.3 ml, on the other hand 0.3 m hyaluronidase solution
1 and then left standing at 37 ° C for 18 hours and then at 100 ° C for 10 hours.
Heat treatment was carried out for a minute to inactivate hyaluronidase.

From 0.6 ml of each reaction mixture without hyaluronidase and with addition of hyaluronidase, 0.5 ml was added to 0.15 M sodium chloride, 0.02
Sephadex G-50 column (Pharmacia) equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 50% sodium azide and 0.05% bridge-35
The high molecular weight fraction of 2.5 to 3.5 ml was collected.

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

[ 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).

Low molecular weight fractions of bovine serum having a molecular weight of 5000 or less (Examples 1 and 2) were used to give final concentrations of 0.5% and 1.0%, respectively.
The amount of hyaluronic acid produced in the culture supernatant when added at 2.0% (W / V) was measured (Table 1). As a result, it was found that the amount of hyaluronic acid produced by the Detroit 551 strain increased compared to Comparative Example 1 at any of the concentrations of Examples 1 and 2.

[0046]

[Table 1]

The formulation examples of the present invention will be given below. The values in the table show% by weight. Further, the hyaluronic acid production promoter is referred to as HA production promoter.

Formulation Examples 1 to 3 (Cream) Creams were prepared with the compositions shown below.

[0049]

[Table 2]

Preparation method: The component (A) was uniformly mixed and dissolved at 80 ° C., and then the component (B) was mixed and dissolved (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.

Formulation Examples 4 to 6 (lotion) Lotions were prepared with the compositions shown below.

[0052]

[Table 3]

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

Formulation Examples 7 to 8 (Bathing Agent) Bathing agents were prepared with the compositions shown below.

[0055]

[Table 4]

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.

Formulation Examples 9 to 11 (Ointment) Ointments were prepared with the compositions shown below.

[0058]

[Table 5]

Preparation method: Each component of the above (B) was placed 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.

Formulation Examples 12 to 14 (gel) Gels were prepared with the compositions shown below.

[0061]

[Table 6]

Preparation method: (A) was swollen with a part of water (D), the 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.

Formulation Examples 15 to 17 (Hair Tonic) Hair tonics were prepared with the compositions shown below.

[0064]

[Table 7]

Preparation method: After the perfume was dissolved with the perfume-solubilizing agent, it was added to ethanol while stirring at room temperature and dissolved, 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.

Front page continued (72) Inventor Shintaro Inoue 5-328, Kotobuki-cho, Odawara-shi, Kanagawa Inside Kanebo Co., Ltd. Biochemistry Research Institute (72) Inventor Tadashi Matsui 5-28-3, Kotobuki, Odawara-shi, Kanagawa Kanebo Co., Ltd. Cosmetic Research Center

Claims (1)

[Claims] 1. A hyaluronic acid production promoter comprising a low molecular weight fraction of bovine serum having a molecular weight of 5000 or less.