JPH02221229A - Hyaluronidase inhibitor - Google Patents

Abstract

PURPOSE:To obtain a hyaluronidase inhibitor containing an extract obtained by extracting root of glycyrrhiza or a plant belonging to same genus as glycyrrhiza with an organic solvent as an active ingredient, having high safeness and capable of continuously using for a long period. CONSTITUTION:Root of glycyrrhiza or other plant belonging to same genus as glycyrrhiza is extracted with one or two or more kinds of an organic solvent such as ethyl alcohol, ethyl acetate, acetone or ethyl ether and glycols properly used in combination and the resultant extract is added as an active ingredient to provide the hyaluronidase inhibitor. The hyaluronidase inhibitor is useful to stabilization of hyaluronic acid salt having excellent water retaining effects and affinity to tissue of living body.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel and highly safe hyaluronidase inhibitor comprising an organic solvent extract of roots of licorice or other congenerous plants.

[Prior art and problems to be solved by the invention] Hyaluronate (hereinafter abbreviated as HA), which has an excellent water-retaining effect and affinity for living tissues, is used in pharmaceuticals such as eye drops, skin drugs, and arthritis treatment drugs. It is also widely used in basic cosmetics such as lotions, milky lotions, and creams. However, it is known that HA is decomposed by ultraviolet rays, enzymes, etc. in a water-containing system, and as the molecular weight decreases, the water retention effect also decreases and eventually disappears. Therefore, in order to rationally reflect the excellent properties of HA in products, the development of a method for stabilizing a hot solution of HA is an important issue, but at present it has hardly been studied.

The present inventors focused on enzymatic decomposition, which has the greatest effect among the causes of decomposition of HA in hot solutions, and focused on enzymatic decomposition by hyaluronidase (hereinafter referred to as Has
It is abbreviated as e. ) We started stabilizing HA warm solution by inhibiting its activity.

Other reasons include: (1) Ba5e is widely distributed in the human body and is known to be involved in the occurrence of inflammation, and is also called an inflammatory enzyme. Therefore, the discovery of an inhibitor of this enzyme is not only meaningful as a stabilizer for HAfl liquid, but also effective in preventing and treating inflammation and allergies caused by Iase, such as acne and gingivitis. (2) It was thought that it would be possible to prevent the effectiveness of HA from rapidly decreasing from the time of application due to decomposition by Hase present at the application site of the HA-containing preparation.

The inventors of the present invention discovered that vitamin B6 and plant extracts such as C. spp.
7, Japanese Patent Application No. 63-182205), but as a result of intensive research to find a new Hase inhibitor in plant extracts, it was discovered that an organic solvent extract of the roots of licorice or other congenerous plants inhibits Hase. The present invention has now been completed.

Incidentally, glycyrrhizin, glycyrrhetinic acid, and their salts, which are aqueous extracts of roots of licorice or other plants of the same genus, were not found to inhibit Hase activity.

[Means for Solving the Problems] The roots of the Hase inhibitor of the present invention are the roots of licorice or other congenerous plants, but they can be used regardless of their origin, and can be used alone or in appropriate mixtures. . In addition, for the extraction of the above plants, organic solvents such as ethyl alcohol, ethyl acetate, acetone, and ethyl ether, propylene glycol, 1,3-butylene glycol,
One type or a combination of two or more types of glycols such as polyethylene glycol can be used as appropriate.

Root powder and water extracts of licorice or other congenerous plants have been used as herbal medicines and sweeteners since ancient times, and are extremely safe for humans.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 (Extraction of Hasa inhibitor) Licorice (Glycyrrhiza glabra L, var.
, glanduliferaRcaALet, H! II
O-1 and below are abbreviated as licorice X. ) or licorice from northeast China to northern China.
ralensis F+5cox and below is abbreviated as licorice Y. ) to 1 kg of roots, add absolute ethyl alcohol at 10°C,
Extract while heating under reflux for about 5 hours.

The obtained extract was concentrated under reduced pressure, and ethyl acetate was added to it at 1°C.
In addition, the mixture is heated and extracted under reflux for about 5 hours.

The solvent is removed from the extract under reduced pressure, and the mixture is dried and ground.

10 g of powder is dissolved in 95 g of 1,3-butylene glycol and filtered.

The main components of the organic solvent extract of the root of licorice X are glabridin and graphrene, which have the effect of preventing the oxidation of fats and oils and inhibiting tyrosinase.On the other hand, those of licorice Y are cochalcone A1 and lycochalcone B, which prevent the oxidation of fats and oils. , is known to have an ultraviolet absorption effect.

Example 2 (Measurement of Hasa activity inhibition effect by viscosity method) Based on the phenomenon that the viscosity of HA solution decreases due to enzymatic decomposition, the viscosity change in the system without and with the inhibitor added in Example 1 was measured using the Ostwald method. Tested by measuring over time using a viscometer.

(Test method) ■ In the inhibitor-added system of Example 1, the viscosity change at 37°C of a mixed solution consisting of the following substrate solution (7 ml), enzyme solution (1 company), and inhibitor solution (1 mM) was measured over time. was measured. On the other hand, (2) In a system in which no inhibitor was added (control), the change in viscosity of a three-component mixed solution in which 95'% ethyl alcohol (1d) was added instead of the inhibitor solution was measured in the same manner as in (2) above.

・Substrate solution: Sodium hyaluronate (manufactured by Kewpie Co., Ltd., molecular weight 1,000,000 to 1,200,000) in 0.1 molar phosphate buffer (pH
6,0) Dissolved and 0.4! A li solution was prepared.

- Enzyme solution: Ushigomaru hyaluronidase (manufactured by Sigma, type I-5) was dissolved in 0.1 molar phosphate buffer (p)I 6.0) to prepare an o,oot°4 solution.

- Inhibitor solution: Each inhibitor prepared in Example 1 was diluted 50 times and 100 times with 95% ethyl alcohol.

The relative viscosity of each solution at the start of measurement was set as 1, and the ratio of relative viscosity at each reaction time was calculated. The results are shown in the table. The numbers in parentheses in the table indicate the stabilization rate of HA, that is, H
This represents the effect of suppressing and stabilizing the decomposition of HA due to ase, and was calculated using the following formula based on the relative viscosity at each reaction time.

Example 3 (Measurement of Hase activity inhibition effect by reducing force method) Based on the phenomenon that N-acetylhexosamine with a reducing end is generated when HA is decomposed by Hase, the inhibitor-free system of Example 1 and the The change in reducing power in the added system was colored using the DMAB reagent.

(Test method) ■ In the system in which an inhibitor was added to Example 1, the following substrate solution (t, amffi) and enzyme solution (1mM) were used.
After reacting a mixed solution consisting of and inhibitor solution (o, 2 ml) at 37°C for 1 hour, a 0.8 molar potassium tetraborate solution (o
, 11111) and immerse in boiling water for 3 minutes.

After cooling with water, DMAB reagent (3 ml) was added and the mixture was allowed to develop color at 37° C. for 20 minutes, after which the absorbance at 544 nm (0,0,) was measured.

(2) In a system in which no inhibitor was added (control), the same operation as (1) above was carried out using a three-component mixture solution in which 95% ethyl alcohol was added instead of the inhibitor solution.

・Substrate solution: Sodium hyaluronate (manufactured by Kewpie Co., Ltd., molecular weight 1,000,000 to 1,200,000) in 0.1 molar phosphate buffer (pH
A, o) were dissolved to prepare a 0.2* solution.

- Enzyme solution: Gyugomaru hyaluronidase (manufactured by Sigma, type rs) was dissolved in 0.1 molar phosphate buffer (p)I 6.0). A solution was prepared.

- Inhibitor solution: Each inhibitor prepared in Example 1 was diluted 10 times with 9596 ethyl alcohol.

-DMAB reagent: Dissolve 10 g of p-dimethylaminobenzaldehyde in a mixture of 12.5 mfl of 10N hydrochloric acid and 87.5 mM of glacial acetic acid. Before use, it was diluted 10 times with glacial acetic acid.

The inhibition rate calculated by the following formula is 50 for Licorice X root extract, ON
, licorice Y root were 313.5, respectively.

The above is an example of the test results when an organic solvent extract of licorice root was used as an inhibitor.

[Effects of the Invention] As detailed above, organic solvent extracts of licorice or other plants of the same genus are highly safe substances that inhibit Hase activity, and the hyaluronidase inhibitor of the present invention containing these as active ingredients has the extremely useful feature of being able to be used continuously over a long period of time.

Therefore, the present invention is applicable to H, which has been widely used in various products in recent years.
This responds to the important need of stabilizing A.

4 others

Claims (1)

[Claims] 1. A hyaluronidase inhibitor whose active ingredient is an organic solvent extract of the roots of licorice or other congenerous plants.