JPH01128933A - Hyaluronidase inhibitor - Google Patents

Abstract

PURPOSE:To obtain a hyaluronidase inhibitor, containing a crude drug extract essence as an active ingredient and having high safety without any irritancy or hormonal side effects on the skin. CONSTITUTION:A hyaluronidase inhibitor obtained by containing a crude drug selected from the group consisting of Paeonia Radix, Coptidis Rhizoma, Phellodendri Cortex, Mountan Cortex, Geranium Herba, Thea sinensis L., Sophorae Radix, Clematis brevicaudata DC., Lamium album L. var. barbatum Franch. et Savat., Salvia officinalis L., Juniperus communis L., Hamamelis virginiana L. and birch or a solvent extract essence (any of extract solution, diluted solution or concentrated extract or dried powder thereof) of a plant selected from the afore-mentioned group as an active ingredient. The above- mentioned inhibitor is useful for prophylaxis and prevention of diseases caused by hyaluronidase of suppuration, inflammation and I-type allergy.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel hyaluronidase inhibitor. More specifically, the present invention relates to a novel and highly safe hyaluronidase inhibitor for preventing and preventing diseases caused by hyaluronidase, such as suppuration, inflammation, and type II allergies.

[Conventional technology]

Conventionally, hyaluronidase in the human body is produced by Propionibacterium acnes (Propio
nibacterium acnes) and Staphylococcus aureus (St.
aphyrococcus aureus) and others are known to have this activity (Appl,
Microbiol.

gy q, 1434-1436.196B). Staphylococcus aureus is known as the causative bacterium when suppuration occurs, and there is a report that when this occurs, the bacterium releases hyaluronidase and decomposes the hyaluronidase in the skin tissue, preparing it for proliferation (Brit).

J, Exp, Path, 26.124.1945).

On the other hand, human hyaluronitase other than bacterial cell-derived hyaluronidase normally exists in an inactive state in various tissues within the body.
It is activated by in-vivo metal ions such as Na+, K+, Ca+, Mg2+, and in-vivo organic bases such as spermine and spermidine.

The physiological significance of hyaluronidase in the human body is still unclear, but it is an inflammatory enzyme that increases the permeability of capillaries (J, Dental Re5t, 33゜1
14.1958), it is said to be an enzyme closely related to inflammation (Arch, Pathol, 8).
5,272°1968). In addition, since type I antiallergic agents such as sodium cromoglycate inhibit this enzyme, the involvement of this enzyme in type II allergies has been suggested (Immunopharm, 2.139.1
980).

As described above, it has been suggested that hyaluronidase is involved in exacerbation during suppuration, inflammation, and type I allergy, and it is thought that inhibiting hyaluronidase will alleviate these diseases.

To date, there are anti-inflammatory drugs that inhibit hyaluronidase (indomethacin, aspirin, mefenamic acid, etc.) and anti-allergic drugs (sodium cromoglycate, tranilast, traxanox, baicalein phosphate, ketotifen, azelastine, cyanidanol, oxatride, etc.). has been reported (Physiol, Re
v, 27.436.1947゜Inflammation and anti-inflammatory therapy, Ishiyaku Publishing, 7.411.1982).

Regarding herbal medicines, strong hyaluronitase inhibition has been observed in licorice ingredients such as Noriquiritigenin and Isoliquiritigenin, as well as picalin and baicalein, which are licorice ingredients.

However, these substances may not be able to exhibit hyaluronidase inhibitory effects due to their relationship with other ingredients, or may not always be satisfactory in terms of safety and effectiveness when applied topically.

[Problem that the invention seeks to solve]

The present invention was developed in consideration of the unsatisfactory points of existing hyaluronitase inhibitors, and as a result of intensive research to obtain a drug that is highly safe and truly has an excellent hyaluronitase inhibitory effect.
We have discovered a completely new fact that many extracts of herbal medicines currently in use have harmful effects on hyaluronitase, and based on this knowledge we have completed the present invention.

[Means to solve the problem]

That is, the present invention can be applied to peonies, orensis, orientalis,
A herbal medicine selected from one or more of the group consisting of Botanical herb, blackberry, tea, Japanese vine, white vine, dead nettle, salvia, juniper, Hamamelis, and birch, or a plant selected from one or more of the above group. The present invention provides a hyaluronidase inhibitor containing a solvent extracted extract of .

The present invention will be explained in detail below.

In the present invention, the herbal medicine is a dried and crushed plant, and the solvent-extracted extract means the above-mentioned plant or herbal medicine extract obtained by the following method, unless otherwise specified.
It shall mean its diluted solution, its concentrated extract or its drying bed.

Solvent extraction extracts are obtained by extracting the above plants or herbal medicines from a dry bed using water or organic solvents (petroleum ether, cyclohexane, carbon tetrachloride, toluene, benzene, dichloromethane, chloroform, ether, ethyl acetate, butanol, acetone, n-propanol, ethanol). , methanol, pyridine, polyethylene glycol, propylene glycol)
Alternatively, it can be obtained by extraction using a solvent in which they are mixed at a certain ratio, such as aqueous alcohol, usually at 3°C to 70°C. The above herbal medicines and solvent extraction can be used as hyaluronidase inhibitors as they are, or after being diluted, concentrated, or lyophilized, prepared into a powder or paste form, and formulated into an appropriate formulation as desired.

[Effects of the Invention] The hyaluronidase inhibitor of the present invention strongly and effectively inhibits hyaluronidase, as can be seen from the examples described below.Furthermore, it does not cause irritation to the skin or hormone-like side effects. It is extremely safe for the skin as it does not give any irritation to the skin.

(Example 1) (1) Preparation of hyaluronitase Hyaluronitase (derived from Ushibimaru, manufactured by Sigma) was added to a final concentration of 0.1 to give a final concentration of 340 NFunit/ml.
M acetate buffer PH3,5 was prepared.

(2) Preparation of solvent-extracted extract sample Add 10 times the amount of water, methanol, or a mixture thereof to 1 to 10 g of dried and ground herbal medicine, and extract with shaking or extract under heating and reflux, and then soak in the supernatant and precipitate. separated. The supernatant was concentrated to dryness under reduced pressure to obtain a crude drug extract, which was diluted with water or methanol as necessary to prepare a crude drug extract sample.

(3) Hyaluronitase activity measurement method Hyaluronitase activity measurement is carried out by Morgan-Ellson (Morgan-E
It was carried out according to the method of applying the method (ibid
, 242, 437.1967).

'To 0.1 ml of the enzyme buffer solution, 0.2 ml of the enzyme activator buffer was added, and the mixture was left at 37° C. for 20 minutes to activate the enzyme. Next, add 0.2 ml each of the buffer solution or sodium carbonate neutralized aqueous solution of the solvent extracted extract sample prepared in (2) Preparation of the solvent extracted extract sample, and
Leave it for a minute. The enzyme solution used as a target contains 0.2 ml of the sample buffer solution or sodium carbonate neutralized aqueous solution.
Instead, 0.2 ml each of buffer solution or water was added. Thereafter, 0.5 ml of potassium hyaluronate buffer was added and reacted at 37°C for 40 minutes.

After stopping the reaction by adding 0.2n+1 of 0.4N sodium hydroxide, color was developed by the Morgan-Elfn method.
Absorbance at 5 mm was measured.

O. Morgan-Elfn method Potassium borate solution was added to each reaction solution, heated in water for 3 minutes, cooled in running water, and 6.0 LIll of P-dimethylaminobenzaldehyde reagent was added.

After shaking immediately, the color was developed for 20 minutes at 37°C.
The absorbance at m was measured.

(Potassium boric acid solution: 0.8M boric acid aqueous solution 100+
2.24 g of potassium hydroxide was added to al and dissolved.

P-dimethylaminobenzaldehyde: 10 g of P-dimethylaminobenzaldehyde was dissolved in 5 ml of acetic acid 87 and 12.5 ml of ION hydrochloric acid. This reagent is usually stored refrigerated and diluted 10 times with acetic acid before use.

The inhibition rate was calculated using formula (1).

The final concentrations of compound 48/80 (manufactured by Sigma) were 2.5n+M, 0.15M and 0.5n+M, respectively.
A buffer solution prepared to have a concentration of 1 mg/ml was used.

The results are shown in Table-1.

(Comparative Example 1) (1) Preparation of hyaluronitase It was prepared in the same manner as (1) Preparation of hyaluronitase in Example 1.

(2) Preparation of hyaluronitase inhibitors Samples of tranilast, sodium chlormonoglyphate, and cyanidanol, which are known as drugs having a hyaluronitase inhibitory effect, were prepared.

(3) Example 1 of the method for measuring hyaluronidase activity,
(3) The procedure was performed according to the method for measuring hyaluronidase activity.

The results are shown in Table-1.

Table 1: Hyaluronitase inhibitory effect of various herbal medicine extracts and hyaluronidase As is clear from Table 1, the hyaluronidase inhibitor according to the present invention is effective when the crude medicine extract sample is present in the reaction system at 0.001 to 0.1%. ,20%
It can be seen that it exhibits ~85% hyaluronidase inhibitory effect.

Furthermore, even if tranilast, sodium chlormonoglyphate, and cyanidanol, which are known as hyaluronitase inhibitors, are present in the reaction system at 0.1%, they have a hyaluronitase inhibitory effect of less than 50%, except for thorium chlormonoglynate. I understand.

Patent applicant: Shiseido Co., Ltd.

Claims (1)

[Claims] Herbal medicines selected from one or more of the group consisting of peonies, oriental laurel, giant tabara, botanpi, genus shoko, tea, kujin, white vine, dead nettle, salvia, juniper, hamamelis, and birch, or one or two of the above groups. A hyaluronitase inhibitor whose active ingredient is a solvent-extracted extract of a plant selected from the above.