JPH0625000A - Protease inhibitor - Google Patents

Abstract

PURPOSE:To provide a protease inhibitor useful for preventing or improving periodontosis and capable of inhibiting the activity of protease. CONSTITUTION:This invented protease inhibitor contains one or more kinds of plants selected from a group of Allium, Citrus reticulate Blanco, Glycyrrhiza glabra L., Matricaria chamomilla L., Plantago psyllium L., Platycodon grandiflorum, cinnamon, Foeniculum vulgare Mill., hop, Valerianae faurier Brig. radix, Scutellaria baicalensis, Lithospermum erythrorhizon, Salvia miltiorrhiza Bunge, ginkgo, bamboo and Morus alba or solvent extracts thereof as the active components.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to novel protease inhibitors. More specifically, the present invention relates to a novel protease inhibitor that is useful for the prevention or treatment of periodontal diseases such as gingivitis and periodontal pyorrhea and that inhibits the activity of protease produced by periodontopathic bacteria.

[0002]

2. Description of the Related Art Recently, periodontal disease has been revealed to be caused by a certain Gram-negative anaerobic bacterium in the gingival sulcus, and Porphyromonas gingivalis ( Formerly known as Bacteroides gingivalis) and Treponema denticola are listed. It is said that these causative bacteria inhabit the periodontal tissues and produce proteases specific to these pathogens such as collagenase and trypsin-like enzyme, and these proteases are closely related to the development of periodontal disease such as the destruction of periodontal tissues. [Journal of Dental Research (J.Dent.Res.)
63, 412-421, 1984 and Journal of Dental Research 65, 1335-1340,
1986]. Therefore, it is considered that inhibiting the action of these proteases is a very effective method as a means for preventing or treating periodontal disease.

However, up to now, no proposal has been found to prevent or treat periodontal disease by paying attention to the activity of such protease and inhibiting the enzyme activity. In addition, conventionally, protease inhibitors such as leupeptin and antipain have been known, but there is a problem in terms of safety, and at present, they are not put into practical use as pharmaceuticals, quasi drugs or cosmetics.

[0004]

Means for Solving the Problems The present invention has been made as a result of earnest studies to obtain a drug having a high safety and a truly excellent protease inhibitory effect in consideration of the unsatisfactory point of known protease inhibitors. The present inventors have found that a plant extract of a seed has a novel activity called a protease inhibitory action, and completed the present invention.

That is, the present invention relates to garlic, chimpi,
One or more kinds of plants selected from the group consisting of licorice, chamomile, chamomile, zelkova, cinnamon, fennel, hops, valerian, staghorn, sikhon, sage, ginkgo, bamboo and sakuhaku, or a solvent-extracted extract of the plant. The present invention provides a protease inhibitor characterized by being an active ingredient.

The plant used in the present invention is not particularly limited in its part or form, but it is usually preferable to use a dry powder form obtained by drying and crushing the plant itself. In addition, the plant solvent extraction extract may be prepared by subjecting the plant or its dry powder to water or an organic solvent such as methanol, ethanol, n-propanol, butanol, acetone, pyridine, polyethylene glycol or propylene glycol, according to an extraction operation known per se. ,
It can be obtained by extraction with chloroform, ethyl acetate, ether, benzene, toluene, cyclohexane, carbon tetrachloride, etc., concentration, and drying.

The protease inhibitor of the present invention is a composition such as toothpaste, powder toothpaste, liquid toothpaste, mouthwash, pasta, cream, mouthwash, troche, candy, chewing gum, ointment, patch, tablet, etc., according to a conventional method. Can be In the present invention, the plant dry powder or the solvent-extracted extract can be used alone or in combination of two or more kinds, and the dry weight is 0.0001 to 30% by weight, preferably 0.001 with respect to the total amount of the protease inhibitor. A desired effect can be obtained by blending 5% by weight. The other compounding ingredients are not particularly limited, and any ingredient usually used in this type of composition can be compounded.

[0008] Furthermore, by further adding other medicinal components, such as chlorhexidine, triclosan, cetylpyridinium chloride, and other fungicides and antibacterial plant components, to these compositions, the effect of preventing or treating periodontal disease is further enhanced. Can be increased.

[0009]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples. Example 1 Preparation of plant extract Extract 10 g of each dried and pulverized plant powder was added with water, methanol or a mixed solution thereof, and extracted under heating and refluxing for 2 hours. The extract was concentrated under reduced pressure and then dried to obtain the desired plant extract.

Example 2 Toothpaste A toothpaste was produced according to a conventional method according to the following formulation. Ingredients Amount (% by weight) Silicic anhydride 15 Sodium lauryl sulphate 1 Polyacrylic acid 1 Sorbit (70% solution) 60 Carrageenan 0.2 Chalazion extract 0.01 Cayhi extract 0.01 Sodium saccharin 0.2 Preservatives and fragrances 1.2 Distilled water adjusted to 100%

Example 3 Mouthwash A mousewash was produced according to a conventional method according to the following formulation. Ingredients Amount (wt%) Ethanol 5 Solbit (70% solution) 15 Polyoxyethylene (60EO) hydrogenated castor oil 0.5 Polyoxyethylene monooleate (20EO) sorbitan 0.5 Fennel extract 0.05 Saccharin sodium 0.1 Preservative and fragrance 0.8 Adjusted to 100% distilled water

Example 4 Mouthwash A mousewash was produced by the following method according to a conventional method. Ingredients Amount (% by weight) Ethanol 15 Glycerin 15 Polyoxyethylene (60EO) hydrogenated castor oil 1 Valerian extract 0.05 Cetylpyridinium chloride 0.05 Saccharin sodium 0.1 Perfume 0.5 Distilled water 100% adjusted

Example 5 Oral pasta An oral pasta was produced according to a conventional method according to the following formulation. Ingredients Amount (wt%) White petrolatum 10.0 Stearic alcohol 8.0 Propylene glycol 5.6 Sodium lauryl sulphate 0.6 Ethyl paraoxybenzoate 0.01 Distilled water 16.1 Sohakuhi extract 1.0 Carboxymethylcellulose sodium 100% Adjusted to

Example 6 Hard Candy A hard candy was produced by the following method according to a conventional method. Ingredients Amount (% by weight) Granulated sugar 46.8 Starch syrup 52.37 Citric acid 0.3 Chimp extract 0.03 l-menthol 0.5 Total 100.0

Test Example 1 The protease inhibitory activity of each plant extract prepared according to the method of Example 1 was examined. (1) Preparation of Protease Porphyromonas gingivalis strain 381 strain
Anaerobically cultivated in heart infusion broth (0.5% yeast extract, 5 μg / ml hemin, 0.2 μg / ml menadione added) at 37 ° C. for 48 hours and centrifuged (7
The culture supernatant was subjected to the following test as a crude protease enzyme.

(2) Preparation of substrate solution N-carbobenzoxy-glycyl-glycyl-arginine-β-naphthylamide was treated with dimethyl sulfoxide to give 10
Adjust to obtain mM concentration, and use distilled water for 1m
What was diluted to M was used as the substrate solution.

(3) Measurement of protease activity In a test tube (inner diameter 12 mm, length 100 mm), 0.3 ml of 1 mM substrate solution and 0.1 M Tris-HCl buffer solution (p
H7.0) 0.9 ml and plant extract of various concentrations or 0.3 ml of distilled water was added to make a reaction solution, and 0.3 ml of the above protease crude enzyme was added thereto and incubated at 37 ° C. for 1 hour. At the end, 0.5 mg /
ml Fast Garnet GBC (1 M acetate buffer containing 10% Tween 20, dissolved in pH 4.2) 0.6
After adding 15 ml and leaving at room temperature for 15 minutes or more, 525 nm
Absorbance at a spectrophotometer (Perkin-Elmer, Mode
55B). When the enzyme activity (ΔOD ₅₂₅ ) when using the test plant extract is a and the enzyme activity when using distilled water is b, the protease inhibition rate (%) of each plant extract extract extract is calculated from the following formula. did.

[0018]

[Equation 1]

The results are shown in Table 1.

[Table 1]

As is clear from Table 1, each plant extract had a concentration of 61% to 99% at a concentration of 0.1% and 22% at a concentration of 0.01%.
It showed an inhibition rate of ˜98%. In particular, the inhibition rate of leupeptin used as a positive control (95% at 0.1% concentration, 0.
The plant extracts exhibiting an inhibition rate equal to or higher than 50% at a concentration of 01%) were extracts of Chinpi, Chazensou, Keihi, Kanokoso, Bamboo, and Sakuhaku.

Test Example 2 A strain of Treponema denticola isolated from the periodontal region of a patient with periodontal disease was anaerobically cultivated in TYGVS medium at 37 ° C. for 1 week (a turbidity is OD ₆₆₀ = 0.5). The enzyme inhibitory solution prepared by the same culture medium) was used as an enzyme solution, and the protease inhibitory activity of various concentrations of the Chazensou extract and the Sohakuhi extract was examined. The substrate solution was prepared and the protease activity was measured according to Test Example 1. The results are shown in Table 2.

[0022]

[Table 2]

As shown in Table 2, each plant extract had an inhibition rate of 10% or more at a concentration of 0.0001%, and
At a concentration of 1% or more, an inhibition rate of 50% or more was shown.

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, not only is it a strong and effective inhibitor of protease and is very useful for the prevention or treatment of periodontal disease, but also the active ingredient is a natural product, so that there are no side effects. And a protease inhibitor which is very excellent in safety can be obtained.

Claims (1)

[Claims] 1. One or more kinds of plants selected from the group consisting of garlic, chinpi, licorice, chamomile, chamomile, cypress, cinnamon, fennel, hops, valerian, corn, sage, sage, ginkgo, bamboo and sakuhaku. Or a protease inhibitor comprising a solvent-extracted extract of the plant as an active ingredient.