JP3207912B2 - Collagenase activity 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 collagenase activity inhibitor useful for the prevention and treatment of periodontal disease, and more particularly to a composition suitable for oral compositions such as gums, mouthwashes and dentifrices. It relates to a collagenase activity inhibitor.

[0002]

2. Description of the Related Art Periodontal disease is a general term for inflammatory diseases including various pathological conditions in periodontal tissue. Generally, gingivitis, in which inflammation is limited to the gingival part, and teeth that reach the alveolar bone and become chronic. It is roughly divided into peritonitis. Periodontitis was called alveolar pyorrhea, but with the chronicity, gingiva and alveolar bone were destroyed, leading to tooth loss. Periodontal disease accounts for 50% of the causes of tooth loss, and affects about 80% of older people.

[0003] as the cause of periodontal disease, certain bacteria in the plaque of periodontal pockets, among them black dye-producing Bacteroides (Bacteroides) bacterial group pathogenic theory it is promising (e.g., Journal of Clinical Periodontology, 13
Vol. 912, 1986). The periodontal tissue destruction is believed to involve bacterial direct agents (such as enzymes and endotoxins) and indirect agents (mediated by the host immune response). Gingival and alveolar bone collagen is commonly degraded and absorbed (American Journal of Pathology, Vol. 92,
509, 1978).

As collagen-degrading enzymes (collagenase) related to periodontal disease, those derived from Bacteroides and those derived from gingival fibroblasts have attracted attention. The former is a rare enzyme that has recently been partially purified and requires both metal and thiol, but there are still many unclear points (Journal of Per.
iodontal Research, 23, 258, 1988).

On the other hand, stromal collagenase derived from fibroblasts (hereinafter referred to as collagenase unless otherwise noted) has been elucidated in detail, and its primary structure has been elucidated (The Journa).
l of Biological Chemistry, 261: 6600, 1986).

[0006] Collagenase is a rate-limiting enzyme in degrading interstitial collagen (type I, type II, and type III collagen) in connective tissue, and plays an important role in collagen metabolism. Increased collagenase activity in inflamed gingiva (Journal of Periodontal Re
search, vol. 16, p. 417, 1981), and that collagen is reduced from the early stages of gingivitis (Archieves
Considering the Oral Biology, 18, 899, 1973), gingival collagenase is considered to be deeply involved in the progression of periodontal disease.

Conventionally, for prevention and treatment of periodontal disease, removal of plaque and tartar in periodontal pockets by scaling and root planing, removal of periodontal pockets (gingival resection), and the like have been used.

[0008] Recently, a therapeutic agent containing an antibacterial agent minocycline has been developed as a drug therapy. Minocycline has been reported to have not only antibacterial activity but also activity to inhibit bacteroides and neutrophil-derived collagenase in vitro (Journal of the Japanese As
sociation of Periodontology, 30, 182, 1988).

The above known methods are based on physical, surgical or pharmaceutical treatments according to the instructions of the physician. However, considering that periodontal disease is an everyday disease with a high morbidity, and that the condition tends to worsen before being treated by a physician, foods such as gum, candy, and beverages,
It is desired to use a highly safe food-derived material which is useful for the prevention and treatment of periodontal disease by removing the above-mentioned etiology in oral materials such as dentifrices and mouthwashes.

[0010]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a collagenase activity inhibitor which can be expected to have a preventive and / or therapeutic effect on periodontal disease and which is highly safe.

[0011]

Means for Solving the Problems The above object is achieved and co Hi acid or a salt thereof, by collagenase activity inhibitor characterized by containing the ascorbic acid or erythorbic acid or salts thereof.

[0012] The caffeic acid used in the present invention is
It is a compound that exists as a glycoside, ester or free form in higher plants such as coffee beans and tea leaves.

The method for producing caffeic acid used in the present invention comprises:
There is no particular limitation, and a commonly used method may be used. It can also be obtained as a caffeic acid-containing extract from plants such as tea leaves, coffee beans, fruits, or the like, or by extracting as chlorogenic acid and then hydrolyzing.

Further, the obtained caffeic acid can be neutralized with a commonly available base and used as a salt such as sodium caffeate.

In the present invention, caffeic acid or a salt thereof may be used in combination.

The content of caffeic acid (salt) in the applied composition varies depending on the object to be applied and cannot be specified unconditionally, but 0.001% by weight of the whole applied composition as 100% by weight.
About 5.0% by weight, more preferably 0.01 to 3.0% by weight.
0% by weight.

However, when used in combination with ascorbic acid, erythorbic acid or salts thereof described below, 0.0001 to 5.0.
% By weight, more preferably 0.002 to 3.0% by weight.
It is.

The ascorbic acid, erythorbic acid, and salts thereof used in the present invention are known compounds, and the production method thereof is not particularly limited, and may be a commonly used method.

In the present invention, ascorbic acid, erythorbic acid or salts thereof can be used in combination.

The content of ascorbic acid, erythorbic acid, or a salt thereof in the applied composition varies depending on the object to be applied and cannot be specified unconditionally, but 0.001% by weight of the entire applied composition as 100% by weight. ~ 5.0% by weight
The degree is preferably, more preferably 0.01 to 1.0% by weight.

[0021] Collagenase activity inhibitor of the present invention, A scan <br/> Colvin acid, by combination with erythorbic acid, or salts thereof, is intended to achieve the synergistic effect.

The subject to which the collagenase activity inhibitor of the present invention is applied may be any of liquid preparations, solid preparations and semi-solid preparations. Preferred compositions are dentifrices, mouthwashes, chewing gums, troches and coating solutions. And the like, or foods such as candies and beverages.

The excipients or auxiliaries used for producing these compositions may be appropriately selected from those usually used for the same purpose according to the dosage form, and are not particularly limited. For example, lactose, magnesium stearate, sorbitol, mannitol, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, saccharin, sodium lauryl sulfate, sodium lauroyl sarcosine, glycerin, polyethylene glycol, polyvinyl alcohol, carrageenan, gum arabic, ethanol, Menthol, fatty acids, citric acid, silicic anhydride, dibasic calcium phosphate, hydroxyapatite, calcium carbonate, titanium dioxide and the like are used.

Further, sweeteners, coloring agents, flavors, preservatives and the like usually used for foods can be appropriately used,
Sandalwood, Cascia, which inhibit the growth of periodontal disease-causing bacteria
Essential oil components such as cinnamon, guaiac and patchouli (Japanese Patent Application No. 2-126331), a bactericide such as chlorhexidine, and an antibiotic such as ampicillin can be added to enhance the effect of preventing and improving periodontal disease.

[0025]

Industrial Applicability The collagenase activity inhibitor of the present invention has an excellent inhibitory activity against collagenase, which is a cause of collagen absorption of gingiva and alveolar bone in periodontal disease, and is useful for prevention and treatment of periodontal disease. In addition, the safety in use is extremely high regardless of the blending amount.

[0026]

【Example】

(Test Example-1) Collagenase inhibitory activity of caffeic acid

1. Test drug Caffeic acid (manufactured by Wako Pure Chemical Industries, Ltd.)

2. Collagenase As collagenase, human procollagenase produced in anchorage-independent cells derived from human fibrosarcoma cells in a serum-free and protein-free medium was obtained by CM Sepharose ^™ (Pharmacia) and zinc chelating. Purified by Sepharose ^™ (manufactured by Pharmacia) and dissolved in a buffer, and trypsin (manufactured by Sigma, Type 1) was dissolved in the buffer.
After adding 2) and incubating at 35 ° C. for 5 minutes, soybean trypsin inhibitor (manufactured by Merck) was added to inactivate trypsin.
1-238941).

3. Measurement of Collagenase Inhibitory Activity The measurement of the inhibitory activity of caffeic acid on collagenase was performed by
It went as follows. First, the test drug was dissolved in dimethyl sulfoxide to obtain an 8% by weight solution, and then a measurement buffer [0.2M salt, 5mM calcium chloride, 0.05% by volume Brij-35
(Polyoxyethylene (23) lauryl ether manufactured by ICI) and 0.02% by volume of sodium azide.
mM Tris-HCl buffer, pH 7.5]
Dilute two times.

An equal amount of each of the diluents and a known amount (0.7 units; 1 unit represents the amount of the enzyme that degrades 1 μg of type I collagen per minute at 35 ° C.) is mixed in an equal amount, and Using type I collagen (manufactured by Cosmo Bio) labeled with olescein isothiocyanate as a substrate,
The method of Nagai et al. (Japanese Journal of Inflamation, 4
Volume, p. 123, 1984), the inhibition curve was determined by measuring the collagenase activity, and the amount of the test drug required for 50% inhibition was read as the IC ₅₀ value.

4. Test Results Table 1 shows the results. Caffeic acid showed a collagenase inhibitory activity in a dose-dependent manner, and had an IC ₅₀ value of about 50 μg / ml.

[0032]

[Table 1]

(Test Example 2) Human collagenase inhibitory action of ascorbic acid or erythorbic acid

1. Test drug L (+)-ascorbic acid (manufactured by Kanto Chemical Co., Ltd.) Sodium erythorbate (manufactured by Wako Pure Chemical Industries, Ltd.)

2. Collagenase Same as in Test Example 1.

3. Measurement of Collagenase Inhibitory Activity The same as in Test Example 1 except that the test drug was directly dissolved in a buffer for measurement.

4. Test Results Table 2 shows the results. Both test drugs showed a collagenase inhibitory activity in a dose-dependent manner, and showed almost the same activity as the caffeic acid IC ₅₀ value.

[0038]

[Table 2]

(Test Example 3) Effect of ascorbic acid and erythorbic acid on the inhibitory effect of caffeic acid on human collagenase.

1. Test drug Same as test examples 1 and 2.

2. Collagenase Same as Test Example-1.

3. Measurement of Collagenase Inhibitory Activity In order to observe the synergistic effect between ascorbic acid or erythorbic acid and caffeic acid, the presence of ascorbic acid or erythorbic acid at a final concentration of 0.01% by weight in the presence of ascorbic acid or erythorbic acid was confirmed. The inhibitory activity (IC ₅₀ value) was examined. The amount of purified collagenase used was about twice as large as that of Test Example-1 (1.2 units), and in the presence of 0.01% by weight of ascorbic acid or erythorbic acid, the amount of active collagenase was substantially 0%. 0.7 units.

4. Test Results Table 3 shows the results. In the presence of ascorbic acid and erythorbic acid, the IC of collagenase inhibition of caffeic acid
A decrease in ₅₀ values (increase in inhibitory activity) was observed.

[0044]

[Table 3]

Examples will be described below. The values in the table represent% by weight.

Reference Examples 1-2, Example 3 (Toothpaste)

[0047]

[Table 4]

It is produced according to a conventional method. That is, the prescribed amounts of water, glycerin, carrageenan, saccharin, butyl parahydroxybenzoate, chlorhexidine digluconate, fragrance, caffeic acid, and sodium erythorbate are measured and mixed to swell the binder, and then the calcium dibasic phosphate is added. And sodium lauryl sulfate were added, mixed well and defoamed, and then filled into a tube to obtain a toothpaste.

Reference Example 4, Example 5 (troche)

[0050]

[Table 5]

A troche having the above formulation was produced according to a conventional method.

Reference Example 6, Examples 7 to 8 (Mouthwash)

[0053]

[Table 6]

A mouthwash having the above formulation was produced according to a conventional method.

Reference Example 9, Examples 10 to 11 (chewing gum)

[0056]

[Table 7]

The whole amount of the chewing gum base and the whole amount of starch syrup kept at 40 ° C. were put into a kneader and kneaded for 10 minutes. 1/3 amount was added and kneaded for 5 minutes. Next, add caffeic acid, ascorbic acid and fragrance to the remaining 1/3
The mixture mixed with the amount of powdered sugar was added and kneaded for 5 minutes to obtain a gum mix.

Reference Example 12 and Example 13 (Nougat)

[0059]

[Table 8]

Mix and whisk. Boil down to 130 ° C. Add little by little and whip further. This was added, and the mixture was cooled to 90 ° C. while mixing. The mixture was added and mixed well, and then spread on a cooling board to form a nougat.

Reference Example 14, Comparative Example 1 (Coffee drink)

[0062]

[Table 9]

Roasted and ground coffee beans (particle size 20 mesh 9)
100 kg (0% on, medium roast) was put into a drip extractor. After heating 200 kg of water to 80 ° C. with a plate heater, it was allowed to flow down from the top of the extractor and extracted with hot water,
150 kg of coffee extract (Bx18) was obtained. To this extract, liquid sugar, water, caffeic acid and sodium bicarbonate having the above composition were added, and the prepared solution was Bx27.9, pH
It was adjusted to 5.65.

The obtained mixture was filled in a can and sealed.
At 25 ° C. for 25 minutes to obtain a coffee beverage, the amount of caffeic acid in the beverage was determined by high performance liquid chromatography [YMC-A312 reverse phase _C18 column manufactured by Yamamura Chemical Co., Ltd .; 15% by volume acetonitrile with trifluoroacetic acid by volume was used. Detection was measured at 350 nm].

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61K 31/341 A61K 31/341 A61P 43/00 111 A61P 43/00 111 C12N 9/99 C12N 9/99 (56) Reference Ciba Found. Symp. (1988) 141 (Metastasis) pp. 193-210 (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 31/191, 31/375, 31/341

Claims (1)

(57) [Claims] 1. A collagenase activity inhibitor comprising caffeic acid or a salt thereof and ascorbic acid or erythorbic acid or a salt thereof.