JPS61275224A - Collagenase-inhibiting agent - Google Patents

Abstract

PURPOSE:To obtain the titled inhibiting agent, by extracting a soybean raw material with water or a salt solution under heating at a specific temperature. CONSTITUTION:Soybean raw material (various soybeans or its processed product such as whole soybean with or without pod, defatted soybean, etc.) is crushed or pulverized, mixed with water or a salt solution (e.g. aqueous solution of NaCl, KCl, etc., having a concentration of about 0.01-1M), and extracted by heating at >=60 deg.C, preferably 80-121 deg.C. The amount of the solvent is 10-200 pts.vol. per 1pt.wt. of the soybean raw material. The heating is continued for 5-120min, preferably at 100 deg.C for about 30min. After the heat-treatment, the aqueous fraction containing said inhibiting agent is separated, salted out with 80% saturated ammonium sulfate and centrifuged. The obtained precipitate is dissolved in water, dialyzed sufficiently with water using a dialysis membrane and freeze-dried to obtain the objective collagenase inhibitor. The agent is useful for the prevention and remedy of periodontosis.

Description

[Detailed description of the invention] East! TECHNICAL FIELD The present invention relates to a novel collagenase inhibitor that inhibits the action of the enzyme collagenase. Collagenase is an enzyme that decomposes collagen, which is a type of hard protein, and inhibition of its action is useful in the fields of medicine and food. In particular, the collagenase inhibitor of the present invention is effective against Bacteroides gingivalis, which is one of the causative bacteria of periodontal diseases.
It strongly inhibits the action of collagenase produced by otdesgingivalis).

BACKGROUND OF THE INVENTION In recent years, it has been revealed that Durum-negative anaerobic bacteria in the gingival sulcus are involved in the cause of periodontal disease. It turns out that this effect is a contributing factor. Therefore, inhibition of this collagenase is useful for the prevention and treatment of periodontal diseases, but few inhibitors are known for this purpose.

On the other hand, while conducting various studies on the components of soybeans, the present inventors unexpectedly discovered that a fraction extracted by heat-treating soybeans in water at high temperatures strongly inhibits Bacteroides gingivalis collagenase.

The present invention provides a novel collagenase inhibitor extracted by heating soybean raw materials at a temperature of 60° C. or higher in water or a salt solution. As is known,
Soybeans contain trypsin inhibitor, which inhibits the action of the enzyme trypsin, but it does not inhibit collagenase and is different from the collagenase inhibitor of the present invention. In addition, although not limited to any particular theory, since the inhibitor cannot be obtained without heat treatment, there is a theory that heat treatment causes some change in the components of soybeans to produce the inhibitor. It is understood that

As the soybean raw material for obtaining the collagenase inhibitor described in the detailed description of the invention, various soybeans or processed products thereof such as whole soybeans with or without pods, pods, and defatted soybeans can be used. , used in crushed or powdered form.

Soybean raw materials are extracted using water or salt solution as an extraction solvent. Examples of the salts used include sodium chloride and potassium chloride, and generally an aqueous solution of about 0.01 to 1M is used. The pH of the solvent is not particularly limited, but is usually 2 to 12, preferably 5 to 9, more preferably 6.
~8. From the viewpoint of extraction efficiency, these solvents are generally used in a ratio of 10 to 200 parts by volume per 1 part by weight of the soybean raw material.

Extraction is performed by combining the soybean raw material and extraction solvent and heating the mixture to 60°C or higher. Although the upper limit of the heating temperature is not particularly limited, the desired collagenase inhibitor can usually be sufficiently extracted by heating at a temperature that can be reached by an autoclave, preferably 80 to +21°C. Note that the inhibitor is hardly extracted at temperatures lower than 60°C.

The heating time is not particularly limited, and is usually 5 to 12
Heating is performed in the range of 0 minutes, but the lower the temperature, the longer the heating time. Generally, heating at 100° C. for about 30 minutes is preferable. This heating extraction operation can be carried out using a known suitable heating extraction device.

After the heat treatment is completed, the inhibitor can be isolated by a known method. For example, the aqueous fraction containing the inhibitor is separated and 80%
Salt out with saturated ammonium sulfate and centrifuge. The desired collagenase inhibitor of the present invention can be obtained by dissolving the obtained precipitate in water, thoroughly dialyzing it against water using a dialysis membrane, and lyophilizing it.

This substance is generally a yellow powdery substance, and its main component is protein with a molecular weight of about 20,000 or more. According to polyacrylamide electrophoresis, one band at a molecular weight of about 20,000 and two bands at a molecular weight of about 30,000 are generated. This substance is easily soluble in water and slightly soluble in methanol and ethanol. Insoluble in chloroform and benzene. It has almost no hygroscopicity and its aqueous solution is stable.

The 50% inhibitory concentration (IC6°) of this yellow powder substance against Bacteroides gingivalis collagenase is 7
7 μg/ml.

The collagenase inhibitor of the present invention also includes those obtained by further purifying the powder obtained above. This purification can be carried out according to conventional methods such as ion exchange method, gel filtration method, and electrophoresis method. For example, after adsorption to a column such as DEAE-cellulose (manufactured by Whatman), concentration gradient elution with sodium chloride is performed. By collecting both fractions eluted with 0.25-0.3M sodium chloride, the activity was determined to be 5.
The purified collagenase inhibitor of the present invention in the form of a pale yellow powder is obtained which is enhanced more than twice as much.

The thus obtained collagenase inhibitor of the present invention can be used in fields where inhibition of collagenase is required, and in particular, it can be used for the prevention and treatment of periodontal diseases since it strongly inhibits collagenase produced by Bacteroides gingivalis. can do.

Kanjiki carp Next, the present invention will be explained in more detail with reference to Examples.

Example 1 10 g of defatted soybean flour was placed in a 50011IL bottle with a screw cap, 500 ml of water was added, and the mixture was heated at 100° C. for 30 minutes. After the heat treatment is completed, the mixture is cooled to room temperature, and the soybean flour is removed using a glass filter to obtain 480 ml of Shito liquid. Add 269g of ammonium sulfate to this and perform salting out.
, p, m for 15 minutes, and the resulting precipitate is suspended in 50 ml of water. Further, the mixture is dialyzed against water overnight and freeze-dried to obtain 2.5 g of the desired collagenase inhibitor in the form of a yellow powder.

The anti-collagenase activity of this inhibitor was measured as follows.

14c Acetylated collagen (prepared from rat skin collagen) was mixed with 0,0
Dissolved in 5M Tris-HCl buffer (pH 7,4), 4%
Make a solution. Add 0.1ml of 20mM dithiothreitol to 0.5J of this solution and 0.2ml of water or sample aqueous solution.
After heating at 35°C for 3 minutes, add 0.2111 fl of collagenase solution (flagenase obtained by fractionating 1 volume of Bacteroides gingivalis culture supernatant with 1.5 times the volume of acetone). Tris-HCl buffer (+) H7,
4) Add 100m (dissolved in M) and react at 35°C. After 60 minutes, dioxane IJ was added to stop the reaction.
3000r, p.

The undenatured collagen is precipitated by centrifugation for 20 minutes at 500 ml of supernatant, and the radioactivity of 1 ml of supernatant is measured. The inhibition rate (%) is calculated from the following formula, where a is the radioactivity when an aqueous sample solution is used and b is the radioactivity when water is used.

By this method, the 50% inhibitory concentration (ICio) is determined,
It is expressed as the concentration in the reaction 11mfj.

As a result, the IC6° of the collagenase inhibitor obtained was 7
It was 7.5 μg. This substance is a yellow powdery substance whose main component is protein with a molecular weight of about 20,000 or more. This substance is easily soluble in water, sparingly soluble in methanol and ethanol, and insoluble in chloroform and benzene. It has almost no hygroscopicity and its aqueous solution is stable.

Example 2 2.5 g of the collagenase inhibitor obtained in Example 1 was mixed with 0.0 g of the collagenase inhibitor obtained in Example 1.
A column (5 x 25 cm) filled with DEAE-cellulose (manufactured by Whatman), which had been equilibrated with the same solvent, was prepared by dissolving 01M Tris-HCl in 250 mQ of a slow solution (M7.4).
) of θ~0°5M aqueous solution of sodium chloride, total t
, 2, and the fractions eluted with 0.25M to 0.3M sodium chloride aqueous solution are collected, dialyzed against water, and lyophilized to obtain 375 mg of the desired partially purified collagenase inhibitor. This substance is a pale yellow powdery substance whose main component is protein with a molecular weight of about 20,000 or more. By polyacrylamide gel electrophoresis, two faint bands are confirmed, one at a molecular weight of about 20,000 and one at a molecular weight of about 30,000. This substance is easily soluble in water, sparingly soluble in methanol and ethanol, and insoluble in ether, chloroform, and benzene. It has almost no hygroscopicity and its aqueous solution is stable. The IC5a of this partially purified inhibitor was 15.6 μg.

Example 3 Put 100 mg each of charred soybeans with pods, whole soybeans without pods, coffee mill pulverized products of pods and defatted soybeans, and papain digested products of defatted soybeans into a bottle with a screw cap, and add 5 II of water.
Add IL one by one, heat and extract in the same manner as in Example 1, and extract an aqueous solution.
Get the liquid. The anti-collagenase activity of 0.05n of each Y solution was measured in the same manner as above, and the inhibition rate was calculated. The results are shown in Table 1.

As shown in Table 1, the desired collagenase inhibitor can be obtained from any soybean raw material.

Effects of the invention (1) Relationship between temperature and time of heating extraction Using 100 mg of defatted soybean flour, in the same manner as in Example 3,
Heated extraction was performed while varying the heating temperature and time, and the inhibition rate of 0.05Ili of each furnace liquid was calculated.

The results are shown in Table 2.

As shown in Table 2, collagenase inhibitors are extracted at temperatures of 60° C. or higher, preferably 80° C. or higher, and lower temperatures require longer heating times.

(2) Effect of the type of extraction solvent 100 mg of defatted soybean flour was extracted using 5 J of various extraction solvents in the same manner as in Example 3, and the inhibition rate of the extraction solvent was calculated. The results are shown in Table 3.

Table 3 As shown in Table 3, the collagenase inhibitor of the present invention is well extracted with water and salt solutions such as sodium chloride and potassium chloride. In particular, it is preferably extracted at pH 6 to 8.

(3) Antienzyme spectrum Similarly to Example 3, each 0.05 ml of aqueous I solution obtained from defatted soybean flour. Using 0.1 i of each enzyme solution, the inhibitory activity against infants was measured according to the measurement of anti-collagenase activity described above, and the inhibition rate (%) was calculated.

The results are shown in Table 4.

Table 4 As shown in Table 4, the inhibitor of the present invention exhibits a high specific inhibitory effect on collagenase derived from Bacteroides gingivalis.

Claims (3)

[Claims] (1) A collagenase inhibitor extracted by heating soybean raw materials at a temperature of 60° C. or higher in water or a salt solution. (2) Said step (1) in which the heat treatment is performed at 80 to 121°C.
Inhibitor of the term. (3) The inhibitor according to item (1) or item (2) above, which is heated for 5 to 120 minutes.