JP3292869B2 - Hyaluronidase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel and highly safe hyaluronidase inhibitor comprising a plant extract.

[0002]

BACKGROUND ART Hyaluronidase is widely distributed in the human body,
It has been shown to be involved in the development of inflammation and is also called a inflammatory enzyme. Therefore, inhibiting this enzyme in the body is considered to be effective in preventing and treating inflammation and allergy caused by hyaluronidase.

[0003] Already, it has been found to inhibit hyaluronidase in extracts of jujube, gennoshoko, peonies, aegis, aseniak, pinlouge, black tea and green tea,
A patent application has been filed (JP-A-2-11520). Also, a hyaluronidase inhibitor (JP-A-2-221229) containing an organic solvent extract of a root of a licorice or other congener plant as an active ingredient, peonies, oren, psyllium, botanpi, genoshoko, tea, kujin, shibotuturu, odoranthus, A hyaluronidase inhibitor obtained from Salvia, Western murine, Hamamelis, and birch (Japanese Patent Application Laid-Open No. 1-128933) is known.

[0004]

Results obtained in the plant extracts [0004] The present inventors have new hyaluronidase inhibitor further with inhibitory effect, extracts of Lo Han Guo is high hyaluronic
Heading that you have a Nidaze inhibitory activity, which resulted in the completion of the present invention.

[0005]

Active ingredient of the hyaluronidase inhibitor of the present invention SUMMARY OF] is an extract of Luo Kanhate may be used alone or in appropriate combination. The crude drug can be extracted by appropriately mixing one or more of water, a lower primary alcohol such as methyl alcohol, ethyl alcohol or butanol, or an organic solvent such as ethyl acetate. The extraction temperature may be any of a high temperature, a room temperature, and a low temperature, but is preferably about 40 to 70 ° C.

[0006] Luo Kanhate has also been used as a food and drink as a full Kukara herbal medicine, the safety of the human body is extremely high. The extract of the present invention is used as a hyaluronidase inhibitor as it is, or in a diluted or concentrated state, or in the form of a concentrated dried powder, granules, tablets or the like.

[0007]

EXAMPLES Hereinafter, we specifically described <br/> based on the present invention embodiment. (1) Preparation of Hyaluronidase Inhibitor by Arkanka Extract Extract A 30-g portion of dried and crushed Arkanka herb was added with twice the amount of methanol, extracted by shaking, and then separated from supernatant and residue. The same operation was repeated, and the supernatant was combined and concentrated to dryness under reduced pressure. This extract was suspended in water and extracted with water-saturated butanol. Concentrate the butanol layer under reduced pressure to dryness and extract the plant
1.2 g was obtained and adjusted to 2 mg / ml with a 0.1 M phosphate buffer solution, and used as a sample .

(2) Method of Measuring Hyaluronidase Inhibitory Activity The measurement of hyaluronidase inhibitory activity was performed according to a method applying the Morgan-Elson method (J.
Biol. Chem., 217, 959 (1955).). Enzyme solution (Note 1)
Adding specimen 0.2ml obtained in the above manner 1 ml, 37
It was left at ℃ for 20 minutes. Next, 0.2m of enzyme activation solution (Note 2)
l was added and left at 37 ° C. for 20 minutes. After standing, 0.5 ml of a substrate solution (Note 3) was added, the mixture was allowed to stand at 37 ° C. for 40 minutes, and 0.2 ml of a 0.4N aqueous sodium hydroxide solution was added. Add 0.2 ml of boric acid solution (Note 4), boil for 3 minutes, allow to cool, p-DAB
6 ml of a reagent (Note 5) was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes to develop color, and the absorbance at 585 nm was measured. Control used as subjects, the same operation was carried out by adding a phosphoric acid buffer solution 0.2ml of 0.1M in place of specimen.

(Note 1) Enzyme solution: Bovine carcinoma hyaluronidase (manufactured by Sigma, type IV-S) is added to a 0.1 M phosphate buffer solution (pH
4.0) and adjusted to 1.9mg / ml. (Note 2) Enzyme activation solution: compound48 / 80 (manufactured by Sigma)
Was dissolved in 0.1 M phosphate buffer solution (pH 4.0) to adjust to 0.5 mg / ml. (Note 3) Substrate solution: Dissolve potassium hyaluronate (manufactured by Wako Pure Chemical Industries, Ltd.) in 0.1 M phosphate buffer solution (pH 4.0),
Prepared at g / ml. (Note 4) Boric acid solution: 4.59 g of boron and 50 ml of water are added.
PH adjusted to 9.1 with N aqueous sodium hydroxide solution and adjusted to 100 ml with water. (Note 5) p-DAB reagent: 12.5 ml of 10N hydrochloric acid and 87.5 glacial acetic acid
10 g of p-methylaminobenzaldehyde is dissolved in the mixed solution of ml and stored refrigerated. Dilute 10-fold with glacial acetic acid before use.

The inhibition rate was calculated by the following equation.

[0011]

(Equation 1)

[0012] As a result , the hyaluronida of the extract of arhat fruit
The enzyme inhibition rate was 96.2%.

[0013]

As described in detail above,LuoChinese fruit extractionGift
Indicates high hyaluronidase inhibition as can be seen from the examples.
Has action. Used as a crude drug and food and drink since ancient times
It is a highly safe substance and can be used for a long time
Is. LuoThe figures by Han Gu are described in the above-mentioned conventional technology.
It is a high value that is not in the literature, and a very large effect
Is shown.

────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI A61P 43/00 111 A61P 43/00 111 (56) References Chemical Abstracts, United States, October 25, 1959, Vol. 53, No. 16, 53: 15356 de, AN 53: 85104 Journal of Pharmaceutical Science, 1991, Vol. 45, No. 1, pp. 52-56 Sanitary Chemistry, 1991, Vol. 37, No. 3, pp. 205-210 Chemical Abstracts, Vol. 53, No. 16, 15256 de, 25, August (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/78 A61K 7/00 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (1)

(57) [Claims] 1. A hyaluronidase inhibitor comprising an extract of Arhat fruit as an active ingredient.