JPH0967251A - Hyaluronidase inhibitor containing rosmarinic acid as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hyaluronidase inhibitor having excellent hyaluronidase- inhibitory activity, preferably a hyaluronidase inhibitor prepared in the cosmetic form. SOLUTION: This inhibitor contains rosmarinic acid obtained by extraction of perilla plants in an amount of 0.001-5wt.%, preferably 0.005-1wt.%. In addition, usual excipient, auxiliary agents and additives are appropriately formulated to give face-washing cream, cosmetic lotion, emulsion and other cosmetics. The rosmarinic acid is obtained by extracting perilla plants such as beefsteak plant, green perilla, perilla frutescens Britton var. crispa Decaisnef. crispa Makino, rosemary or the like or by chemical synthesis. Extraction is convenient and water or ethanol is used as a solvent to effect the extraction at room temperature to 100 deg.C. This cosmetic prevents skin roughening and fine wrinkles and can keep moistening feeling.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hyaluronidase inhibitor containing rosmarinic acid as an active ingredient, preferably a hyaluronidase inhibitor prepared in a cosmetic form.

[0002]

BACKGROUND OF THE INVENTION Hyaluronidase is an enzyme that is widely distributed in the living body and is also present in the skin. As its name implies, it decomposes hyaluronic acid. Hyaluronic acid is β-D-N-
It is a linear polymer polysaccharide in which acetylglucosaminoglycan and β-D-glucuronic acid are alternately linked, and is a kind of glucosaminoglycan widely present in connective tissues of mammals together with chondroitin sulfate and the like. The function of hyaluronic acid in connective tissue is thought to be to retain water in the intercellular spaces, maintain lubricity and flexibility of the skin, and prevent external force (mechanical damage) and bacterial infection. It is said that hyaluronic acid in the skin decreases with age, resulting in aging such as wrinkles and lumps. Therefore,
Suppressing the activity of hyaluronidase, which decomposes hyaluronic acid, contributes to the stability of hyaluronic acid used in formulations and to the stability of hyaluronic acid in formulations after application to the skin and hyaluronic acid present in the skin. It is supposed to do.

At present, as a hyaluronidase inhibitor,
Natural and synthetic are known. As natural products, it has already been found that extracts of jiuyu, geno-shoko, peony, agets, acacia yam, areca, black tea, and green tea inhibit hyaluronidase, and a patent application has been filed (Japanese Patent Laid-Open No. 11520/1990). Further, a hyaluronidase inhibitor (Japanese Patent Laid-Open No. 1-128933) obtained from peony, oren, psyllium, button pipi, genoderma lucidum, tea, kujin, shibototsuru, odorikosou, salvia, western mouse, hamamelis, and birch is known. On the other hand, sodium cromoglycate is known as a synthetic product having a strong hyaluronidase inhibitory action, but this is a therapeutic drug for bronchial asthma and its use as a cosmetic product is not preferable.

[0004]

An object of the present invention is to provide a hyaluronidase inhibitor having a hyaluronidase inhibitory activity superior to conventional products. Further, it is to provide the hyaluronidase inhibitor prepared in the form of a cosmetic product for the purpose of preventing rough skin and wrinkles and maintaining a moist feeling.

[0005]

Means for Solving the Problems As a result of various investigations for solving the above problems, the present inventors have found that rosmarinic acid contained in Lamiaceae plants or rosmarinic acid obtained by synthesis has excellent hyaluronidase inhibitory activity. I found a thing. That is, the present invention provides a hyaluronidase inhibitor containing rosmarinic acid as an active ingredient. The hyaluronidase inhibitor of the present invention is preferably prepared in a cosmetic form. Rosmarinic acid, which is a caffeine tannin, has a beautiful skin- and skin-conditioning action (Japanese Patent Laid-Open No. 63-162611) and 5-
Although a pharmacological action such as a lipoxygenase inhibitory action (JP-A-1-121217) has been known, it has not hitherto been known to have a hyaluronidase inhibitory activity.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Rosmarinic acid, which is an active ingredient of the hyaluronidase inhibitor of the present invention, can be obtained by extraction from Lamiaceae plants such as perilla, Aojiso, Chirimendiso, and Rosemary (mannen wax), or by synthesis. It is convenient to obtain rosmarinic acid by extraction of Lamiaceae plants. In this case, one or more kinds of water, lower alcohols such as methyl alcohol, ethyl alcohol or butanol, and organic solvents such as ethyl acetate can be appropriately mixed and used as the extraction solvent.
The extraction temperature may be any of low temperature to high temperature, but is preferably room temperature to about 100 ° C. The extract of the Lamiaceae plant thus obtained can be used as it is, or rosmarinic acid separated and purified from the extract can be used as an active ingredient of the hyaluronidase inhibitor of the present invention.

The use of the hyaluronidase inhibitor of the present invention is not particularly limited, and it can be used in any situation where inhibition of hyaluronidase is desired. Components other than rosmarinic acid, which is the active ingredient of the hyaluronidase inhibitor of the present invention, may be appropriately selected depending on the use of the inhibitor. These ingredients are conventional excipients (including bases, fillers, diluents, etc.), auxiliaries (including emulsifiers, dispersants, etc.) and additives (including antioxidants, preservatives, etc.). You may Although the content of the active ingredient rosmarinic acid in the hyaluronidase inhibitor of the present invention varies depending on the application, it is usually converted to purified rosmarinic acid to about 0.001 to 1% of the total amount of the inhibitor.
It is in the range of 5% by weight, preferably about 0.005 to 1% by weight.

The hyaluronidase inhibitor of the present invention is particularly preferably prepared in a cosmetic form. In this case, it can be prepared in any form such as facial cleansing cream, lotion, emulsion and the like, and ingredients other than the active ingredient are selected according to the form. When prepared in the form of cosmetics, the moisturizing effect can be further enhanced by appropriately mixing hyaluronic acid that is used externally as a moisturizing agent for cosmetics. It is desirable that the content of rosmarinic acid in the cosmetic is in the range of about 0.001 to 5% by weight, preferably about 0.005 to 1% by weight of the total amount of the cosmetic from the viewpoint of the effect.

[0009]

INDUSTRIAL APPLICABILITY As shown in Table 1 below, rosmarinic acid showed superior inhibitory activity to sodium cromoglycate known as a drug having a high hyaluronidase inhibitory effect. Therefore, the hyaluronidase inhibitor of the present invention containing rosmarinic acid as an active ingredient is useful for use in all situations where inhibition of hyaluronidase is desired.

[0010]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Preparation of perilla extract and measurement of hyaluronidase inhibitory activity (a) Preparation of perilla extract 50 g of Soba (Uchida Wanhan) with 80% ethanol (water content 20
%, V / v ratio) 600 ml, and the mixture was allowed to stand for 24 hours under heating at 40 ° C. for immersion extraction. After filtering the extract, 600 ml of 80% ethanol was further added to the residual soybean leaf, and the same operation was performed. Combine the two extracts to 1097 ml
Was obtained. This extract was concentrated under reduced pressure while heating at 50 ° C to make a volume of 5 ml, and then 100 ml of water was added to 100 ° C.
For 24 hours. The resulting insoluble precipitate was removed by filtration, 0.75% by weight of activated carbon was added, and the mixture was stirred for 1 hour. Then, the activated carbon was removed by filtration to obtain a perilla extract.

(B) Measurement of hyaluronidase inhibitory activity Hyaluronidase usually has an extremely low enzymatic activity. Compound 48/80 containing a metal ion is known as a substance that enhances the enzyme activity. The ability of a hyaluronidase inhibitor to inhibit the process of activation of enzyme activity in this compound was measured. Specifically, the inhibitory effect of compound 48/80 (purchased from Sigma) on the activation step of the inactive enzyme was measured using hyaluronidase derived from beef testis [purchased from Sigma, type IV]. . The enzyme activity was quantified by measuring the reducing power of the tetrasaccharide having N-acetylhexosamine as a reducing end produced by hydrolysis of hyaluronic acid by measuring the absorbance at a wavelength of 585 nm (Mieda Maeda et. , Volume 31, 233 ~
237, 1990). That is, an appropriate amount of the test sample was dissolved in 100 μl of 0.1 M acetate buffer (pH 4.0), and the whole amount was put into a test tube, to which 0.10 mg (10 mg of hyaluronidase enzyme was added.
Add 500 μl of the same buffer containing 0 NF unit)
Incubated at 20 ° C for 20 minutes. Then, 100 μl of the same buffer solution in which compound 48/80 (50 μg) was dissolved was added, and the mixture was further incubated at 37 ° C. for 20 minutes. Finally, sodium hyaluronate [Seikagaku Corporation] 20
Add 250 μl of the same buffer containing 0 μg, and add at 37 ° C for 4
Incubated for 0 minutes. Then 0.4N NaOH
After adding 100 μl and cooling on ice, borate buffer (pH 9.
1) Add 100 μl and boil for 3 minutes. After cooling with ice, 3 ml of a p-dimethylaminobenzaldehyde reagent solution was added, and the mixture was incubated at 37 ° C. for 20 minutes, and then the absorbance was measured at a wavelength of 585 nm. As a control, the acetate buffer was used instead of the sample solution. Further, as each blank, the above-mentioned acetate buffer was used instead of the enzyme solution. The inhibitory activity was expressed by the inhibition rate calculated from the following equation.

[Equation 1] The same measurement was performed on sodium cromoglycate (obtained from Nippon Bulk Chemical Co., Ltd.), and the comparison results are shown in Table 1 below.

[Table 1] Test sample Hyaluronidase inhibitory activity IC ₅₀ (μg / ml) Sodium cromoglycate 102.5 Rosmarinic acid 72.0 Perilla extract 2530 As shown in Table 1, rosmarinic acid is known as a drug having a hyaluronidase inhibitory effect. The inhibitory activity was superior to that of sodium cromoglycate.

Example 2 Face Wash Cream The following face wash cream was formulated using rosmarinic acid as an active ingredient (hyaluronidase activity inhibiting ingredient) (total 100% by weight).
Used for.

[Table 2] Component A wt% myristin 14.0 Stearic acid 12.0 Lauric acid 3.5 Oleyl alcohol 1.5 Coconut oil fatty acid amidopropyl betaine 10.5 Component B wt% concentrated glycerin 18.0 Potassium hydroxide 7. 0 Purified water Residual preservative (paraoxybenzoic acid ester) Appropriate amount Component C wt% Rosmarinic acid 0.5 Perfume 0.2 Component A is dissolved by heating and kept at 80 ° C. Separately, the component B heated and dissolved at 80 ° C. is added to the component A and mixed well. The mixture was cooled while stirring, and the component C was added at 50 ° C. to obtain a face wash cream.

Example 3 Toner Lotion Rosmarinic acid was used as an active ingredient in the following lotion formulation (total 100% by weight).

[Table 3] Ingredients% by weight Purified water balance Concentrated glycerin 4.0 Sorbit solution (70% by weight aqueous solution) 4.0 Citric acid (pH adjusting agent) Appropriate amount Sodium citrate 0.3 Polyoxyethylene hydrogenated castor oil 0.5 Ethanol 15.0 Rosmarin Acid 1.0 Fragrance 0.05 All ingredients are stirred and mixed at room temperature to form a uniform solution,
The skin lotion was obtained by adjusting the pH to 5.5.

Example 4 Emulsion The following emulsion formulation was prepared using rosmarinic acid as an active ingredient (total 1
00% by weight).

[Table 4] Ingredient A wt% purified water balance Sucrose fatty acid ester (Daiichi Kogyo Seiyaku S-160) 1.0 Concentrated glycerin 6.0 Preservative (paraoxybenzoic acid ester) Appropriate amount Carboxyvinyl polymer 0.06 Potassium hydroxide 0.028 component B wt% olive oil 4.0 jojoba oil 4.0 myristyl lactate 2.0 self-emulsifying glyceryl monostearate 1.5 lipophilic glyceryl monostearate 1.5 component C wt% rosmarinic acid 0.5 Fragrance 0.2 Component A is melted by heating and kept at 80 ° C. Separately, the component B heated and dissolved at 80 ° C. is added to the component A and mixed well. The mixture was cooled while stirring, and the component C was added at 50 ° C. to obtain an emulsion.

Example 5 The face cleansing cream, lotion and emulsion obtained in Examples 2 to 4 were applied to humans. As a result, the sensory effect on the skin was extremely good.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 35/78 AED A61K 35/78 AEDQ C12N 9/99 C12N 9/99

Claims (2)

[Claims] 1. A hyaluronidase inhibitor containing rosmarinic acid as an active ingredient. 2. The hyaluronidase inhibitor according to claim 1, which is prepared in a cosmetic form.