JPH0344331A - Collagenase inhibitor - Google Patents

Abstract

PURPOSE:To provide a collagenase inhibitor comprising an extract obtained from cacao husks, effective to prevent and treat periodental diseases. CONSTITUTION:The cacao husks of cacao beans are extracted with an organic solvent (e.g. hexane, aretone or alcohol) to remove hydrophobic substances, and the residues are extracted with water at room temperature, followed by drying of lyophilizing the obtained extract to provide a collagenase inhibitor. The collagenase inhibitor has a low taste and is substantially tasteless and odorless to be a raw material highly suitable for the addition thereof into foods, thereby permitting to be compounded with chewing gums, candies, drinks, etc., to prepare compositions having a periodental disease-perventing function and used for oral cavities.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a collagenase inhibitor obtained by extraction from a predetermined part of a cacao plant, and more specifically, the present invention relates to a collagenase inhibitor obtained by extracting from a predetermined part of a cacao plant. Bactero
The present invention relates to a collagenase inhibitor for preventing and treating periodontal disease by inhibiting collagenase produced by Bacteroides gingivalis.

[Conventional technology] Periodontal disease is an inflammatory disease of bacterial origin.

Adult periodontitis is the most common type of periodontal disease, and the leading theory is that collagen fibers in the periodontal tissue are destroyed by collagenase, which is released from the patient's periodontal pocket. It is thought to be produced by Bacteroides gingivalis, which has been isolated in large numbers.

Bacteroides gingivalis is a non-motile Durham-negative individual anaerobic bacillus that forms black colonies on blood plates, and progress is being made in elucidating its virulence factors.

Bacteroides gingivalis, which is considered to be the most likely causative agent of periodontal disease, produces various proteolytic enzymes, but among these, we can specifically inhibit collagenase, which is a direct factor that leads to the destruction of periodontal tissues. If possible,
It is believed that prevention and treatment of periodontal disease can be effectively achieved.

Collagenase inhibitors intended for use in the prevention and treatment of periodontal disease are intended for use in the oral cavity, so from a safety point of view natural extracts are preferred over synthetic ones, ideally.
It is desirable that the material is extracted from an edible material that has been used by humans since ancient times and is recognized as being harmless to the human body.Furthermore, in order to be used for the oral cavity, it must have a good taste or taste. It is also necessary that it itself be tasteless and odorless. In addition, even if the product is extracted from a natural product, the natural product used as the raw material may be rare and difficult to obtain, or the extraction process may be complicated.
Those that require a manufacturing process are problematic in terms of cost and are inappropriate for industrial use.

Several synthetic drugs such as tetracycline have been reported as collagenase inhibitors, but there are almost no reports on natural products, and only soybean extracts (Japanese Patent Application Laid-Open Nos. 61-275224 and 62-28692) have been reported.
No. 6).

[Problems to be Solved by the Invention] The present invention provides an effective collagenase that is safe, has good taste, is tasteless and odorless, is easily available, and is easy to extract, and is made from an extract extracted from inexpensive raw materials. The purpose is to provide inhibitors.

[Means for Solving the Problems] According to the present invention, there is provided a collagenase inhibitor characterized by comprising an extract extracted from cacao husk, which is the hull of cacao beans.

This collagenase inhibitor is produced by extracting from cacao husk, which is the hull of cacao beans, and preferably by removing hydrophobic substances from cacao husk with an organic solvent and then extracting with water. Extraction efficiency can be increased.

The collagenase inhibitor does not contain hydrophobic substances that are extracted with organic solvents from cacao husk, which is the skin of cacao beans.
It is suitable if it does not contain extraction residues that cannot be extracted with water.

It is preferred if the organic solvent is selected from the group consisting of hexane, acetone and alcohol.

It is preferable that the cacao husk, which is the bean skin of cacao beans, is a bean skin that is separated after roasting the cacao beans.

The collagenase inhibitor according to the present invention can be prepared, for example, by crushing 100 g of cacao husk, adding 1000 to 5000 g of hexane, and extracting at room temperature for 10 to 30 hours to defatte the cacao husk.
Add 0-5000III ethanol and heat at room temperature for 10-5000
Extract for 30 hours to remove hydrophobic substances that cannot be removed by hexane.
00011 and extracted at room temperature for 10 to 30 hours to obtain an extract, and drying or freeze-drying this aqueous extract, whereby 10 to 15 g
A collagenase inhibitor according to the present invention can be obtained.

Cacao Husk
This is the seed coat (bean shell) of cacao, L, ), and can be obtained from cocoa powder or chocolate manufacturing process, or from ordinary cacao beans. Cocoa husk is MI fiber and is thought to contain lignins, tannins, polysaccharides, etc., but the details are not clear.However, by extracting cacao husk with water using the prescribed method disclosed in the present invention, Collagenase inhibitors can be obtained.

In addition, the collagenase inhibitor according to the present invention has a low taste and is substantially tasteless and odorless, and is a material that is very suitable for addition to foods. Therefore, the collagenase inhibitor according to the present invention can be used in chewing gum, candy, Prevent periodontal disease by adding it to drinks, etc. l! It is possible to provide an oral composition having the following properties. In this case, the collagenase inhibitor according to the invention is preferably used from 0.01 to 1% by weight.
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[Action] As a result of repeated research to develop a substance that inhibits collagenase activity produced by Bacteroides gingivalis 6. The present invention was completed based on this finding, which revealed that a water extract of cacao husk has a strong inhibitory effect on collagenase activity.

Cocoa husks are bean husks separated from cacao beans during the chocolate manufacturing process, and theobromine and pigments have been extracted from them in the past, but they currently have no particularly useful use and most of them are disposed of. The reality is that there are. Therefore, the discovery of the new effect of cacao husk in inhibiting collagenase activity is extremely significant from the perspective of effective utilization of waste, and it is thought that it can be fully put to practical use as a material for the prevention and treatment of periodontal disease.

[Effects of the Invention] According to the present invention, an effective collagenase inhibitor is provided that is safe, substantially tasteless and odorless, easily available, and easy to extract, and is made of an extract extracted from inexpensive raw materials. Ru.

The collagenase inhibitor according to the present invention is very low cost because it is made from cacao husk, which is discarded during the chocolate manufacturing process, and has very high extraction efficiency because it is extracted from 10 to 15 g of IQOg cacao husk. Furthermore, it is a highly safe natural product and has a low taste and is virtually odorless, making it an extremely suitable material for addition to food, and has been put to practical use as a material for the prevention and treatment of periodontal disease. It's something you get.

[Examples] The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited only to the following Examples.

Collagenase inhibitors were extracted from cocoa husk by the following steps.

After crushing 100g of cacao husk, 10001'I1
Add hexane from step 1 and extract at room temperature for 24 hours to defatte the cacao husk. Add 100Q+ul of ethanol to the extraction residue and extract at room temperature for 24 hours to extract and remove hydrophobic substances that cannot be removed by hexane. Then, 1000 ml of water was added to the extraction residue and extracted for 24 hours at room temperature to obtain an extract, and this water extract was dried or freeze-dried to obtain 10 to 15 g of the collagenase inhibitor according to the present invention. Ta.

The collagenase inhibitory activity of the collagenase inhibitor of the present invention prepared as described above was measured.

For measurement of collagenase activity, collagenase) CLN-1
00 (registered trademark, CosmoBio Co., Ltd.) uses fluorescently labeled collagen as a substrate, extracts the decomposed product produced after the collagenase reaction, and measures the fluorescence intensity. It is used to quantify cell activity.

As a collagenase activity inhibition sample, the collagenase inhibitor according to the present invention and tetracycline hydrochloride, which has been confirmed to have a collagenase activity inhibition effect (for example,
Please refer to Vol. 0, No. 1, pp. 182-190) to compare their effects.

The Bacteroides gingivalis collagenase solution used in this example was prepared by the following method.

Bacteroides gingivalis strain FDC381, which had been cultured anaerobically for 4 days using blood plate medium, was cultured in Tributicase soy (Trypt 1case SOV) liquid medium 150 g.
On+I was inoculated and cultured anaerobically for 3 days. The culture solution was centrifuged (12,000 x G, 20 minutes, 4°C),
The supernatant was brought to 80% ammonium sulfate saturation and centrifuged (1
2,0OOx G, 20 minutes, 4°C) to 80%
The ammonium sulfate fraction was collected. This fraction was mixed with 0.05M Tris-H
Dissolved in 300 nl of Cl 8 solution (pH 7.5), 0
It was filtered through a 622 μm filter and used as a Bacteroides gingivalis collagenase solution.

The matrix collagen solution was 0.05M Tris-HCl 455 (p) containing 0.05% fluorescently labeled collagen.
H7,5), and the sample solution contained 0.05% of the collagenase inhibitor according to the present invention and tetracycline hydrochloride.
The concentration was adjusted to a predetermined concentration using M Tris-HCl 11 solution (pH 7.5).

The experimental system was a total of 500 μm consisting of 200 μm of Bacteroides gingivalis collagenase solution, 200 pi of matrix collagen solution, and 100 El of sample solution.
The reaction was carried out at 35°C for 2 hours, and the collagenase activity was measured based on the fluorescence intensity of the decomposed product. The results are shown below; the unit of activity, tU, is the amount of enzyme that decomposes 1 μg of collagen per minute.

Wave pattern 1±X± Activity stop-a bean-[2''-(n
u/n+I) and total amount 521 Lord Godanko Yotama Collagenase Utoyoda 0.1 324 0.05 503 0.01 706 0.005 1191 0.001 1492 Bearded 522 Eyu no 0.1 385 (1 ,05584 0,011088 00 5 8 2 0,005122380 0,001149298 From these results, the inhibitory activity of the collagenase inhibitor according to the present invention is stronger than that of tetracycline hydrochloride, which is known as an activity inhibitor, and the degree of inhibition is greater than that of tetracycline hydrochloride, which is known as an activity inhibitor. It can be seen that the inhibitory activity increases depending on the concentration, and the addition of 0.01% shows an inhibitory activity of 50% or more (the specific activity of collagenase is 46%).

Chewing gum was manufactured using the collagenase inhibitor according to the present invention according to the following formulation.

Placed directly gum base sugar glucose starch syrup fragrance According to the present invention Danshi (1!! Sumire) 20.0% 55.0 15.0 9.3 0.5 collagenase inhibitor total 00 %

Claims (4)

[Claims] (1) A collagenase inhibitor comprising an extract extracted from cacao husk, which is the skin of cacao beans. (2) The collagenase inhibitor according to claim 1, which does not contain a hydrophobic substance extracted with an organic solvent from cacao husk, which is the hull of cacao beans, and does not contain an extraction residue that cannot be extracted with water. (3) The collagenase inhibitor according to claim 2, wherein the organic solvent is selected from the group consisting of hexane, acetone, and alcohol. (4) The collagenase inhibitor according to any one of claims 1 to 3, wherein the cacao husk, which is the bean skin of cacao beans, is the bean skin separated after roasting the cacao beans.