JPH0940552A - Human collagenase activity inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a human collagenase activity inhibitor having high safety, useful for prevention and curing of periodontitis and suitable for foods such as candy, drink or gum, etc., an oral cavity composition such as a mouth rinsing solution or toothpaste. SOLUTION: This activity inhibitor contains polyporenic acid C, a mushroom extract containing polyporenic acid C or its dried extract powder, or an extract of Daedalea dickinsi as an active component. A mixing concentration is preferably 0.001-10wt.% reduced to a concentration of polyporenic acid C, or 0.005-20wt.% reduced to dried weight of the extract in a case of mixing the extract of the mushroom or its dried extract powder. The activity inhibitor has excellent inhibition activity against human collagenase as a cause of collagen absorption of gingiva and alveolar bone in periodontitis and specifically effects to human collagenase, then prevention and curing of periodontitis can be performed without giving any effect to other enzymes.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention is suitable for candy, beverages, foods such as gums, mouthwashes, oral compositions such as toothpaste, and can be expected to have preventive and therapeutic effects on periodontal disease and is safe. It relates to a highly active human 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 chromogenic Porphyromonas (Porphiromonas) bacterial group pathogenic theory is promising (e.g., Journal of Clinical Periodontolog
y, vol. 13, p. 912, 1986). It is considered that direct action factors (enzymes, endotoxins, etc.) derived from bacteria and indirect action factors (through the immune response of the host) are involved in the periodontal tissue destruction action. It is common that collagen of gingiva and alveolar bone is decomposed and absorbed (American Journal of Pathology,
92, p. 509, 1978).

As collagen degrading enzymes involved in periodontal disease, those derived from Porphyromonas and those derived from gingival fibroblasts have been attracting attention. The former is a recently partially purified rare enzyme that simultaneously requires a metal and a thiol, but there are still many unclear points (Journal of Periodontal Res
earch, 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, 16: 417, 1981), and that collagen has been reduced since the early stages of gingivitis (Archieves
of Oral Biology, 18, 899, 1973), it is considered that gingival collagenase is 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. It has been reported that minocycline has not only antibacterial activity but also activity of inhibiting porphyromonas- and neutrophil-derived collagenase in vitro (Journal of the Japanese
Association of Periodontology, 30, 182, 1988
See year).

[0009] The above known methods are based on physical, surgical or pharmaceutical treatment according to the instructions of the physician. However, periodontal disease is a daily disease with a high morbidity, and considering that the condition tends to worsen before treatment by a doctor, foods such as gum, candy, and beverages,
As an oral composition such as a dentifrice or a mouthwash, it is desired to use a highly safe substance that is useful for the prevention and treatment of periodontal disease by removing the above-mentioned etiological factors.

On the other hand, polyporenic acid C (Polypo
Renic acid C) has been used as a compound having a triterpene skeleton until now.
Page, 1970) and Polyporus betulinus (Journal o
f Chemical Society, page 2548, 1953, Journal of Che
It has been reported that it was extracted and purified from Mical Society, page 3070, 1954), but it has not been reported to have an action of inhibiting human collagenase activity.

[0011]

In view of the above circumstances, the present inventors have diligently studied the search for a highly safe human collagenase activity inhibitor useful for the prevention and treatment of periodontal disease by eliminating the etiology, and Polenic Acid C (Polyporeni
acid C), polyporenic acid C (Polyporenic
The present invention has been completed based on the finding that a mushroom extract containing acid C) or an extract of Ganoderma lucidum has an action of specifically inhibiting human collagenase activity. Therefore, an object of the present invention is to provide a human collagenase activity inhibitor which can be expected to have preventive and therapeutic effects on periodontal disease and which is highly safe.

[0012]

[Means for Solving the Problems] The above-mentioned object is to extract polyporenic acid C (Polyporenic acid C), a mushroom extract containing polyporenic acid C (Polyporenic acid C) or a dried extract powder thereof, or garlic mushroom. This is achieved by a human collagenase activity inhibitor characterized by comprising an extract or a dried extract powder thereof as an active ingredient.

[0013]

DETAILED DESCRIPTION OF THE INVENTION The constitution of the present invention will be described in detail below. Polyporenic acid C used in the present invention, polyporenic acid C
The mushroom extract containing (Polyporenic acid C) or the dried extract powder thereof, or the spinach extract or the dried extract powder thereof can be produced from mushroom fruit bodies and the like as follows.

The polyporenic acid used in the present invention
Examples of mushrooms containing C (Polyporenic acid C)
For exampleDaedalea dickinsi(Porcelain),Fomitopsis
 pinicola(Tsugasarunokeshi),Fomitopsis rose
a(Balairo Sarnos moss),Fomitopsis nigra
(Black moss),Polyporus betulinusEquality
Mushroom mushroom,Ganoderma valesiacum(Tsugano
Ganoderma lucidum) and other mushrooms of the Ganoderma lucidum
It is.

As a method for producing the mushroom extract of the present invention, for example, an extraction solvent is added in a weight ratio of 5 to 30 times to the freeze-dried substance of mushroom fruiting body, and usually 15 to 50 ° C.
And a method of obtaining an extract of each mushroom by infiltration for 24 hours to 1 week. Alternatively, the extract can be filtered or centrifuged to remove insoluble matter, and then can be obtained as a concentrated extract by subjecting it to usual concentration means such as vacuum concentration.

Examples of the extraction solvent for producing the mushroom extract of the present invention include water, a water-soluble organic solvent such as ethanol, or a mixed solvent thereof.

Examples of the method for producing the dried mushroom extract powder of the present invention include a method for obtaining the above-mentioned extracted extract as a dried extract powder by an ordinary drying means such as vacuum drying, spray drying or freeze drying.

The polyporenic acid C (Poly
As a method for isolating porenic acid C), the extracted extract or dried extract powder is extracted with an organic solvent and purified by a separation means such as a silica gel column to isolate polyporenic acid C (Polyporenic acid C). Methods and the like.

The human collagenase activity inhibitor of the present invention includes the above-mentioned polyporenic acid C (Polyporenic ac).
In addition to id C), the extracted extract or its dried extract powder, known components usually used in oral compositions can be optionally mixed.

The composition form to which the human collagenase activity inhibitor of the present invention is applied may be any of a liquid agent, a solid agent, a semi-solid agent, and the like, particularly foods such as chewing gum, candy, beverages, and toothpaste. Agent, mouthwash, troche,
Oral compositions such as coating solutions are preferred.

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. However, for example, lactose, magnesium stearate, sorbit, mannitol, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, saccharin, sodium lauryl sulfate, sodium lauroyl sarcosine, glycerin, polyethylene glycol, polyvinyl alcohol, carrageenan, gum arabic, ethanol, Menthol, fatty acid, citric acid, silicic acid anhydride, dibasic calcium phosphate, hydroxyapatite, calcium carbonate, titanium dioxide and the like are used.

Further, sweeteners, colorants, flavors, preservatives and the like which are usually used in foods can be appropriately used,
A bactericide such as chlorhexidine and an antibiotic such as minocycline or ampicillin can be mixed to prevent or improve periodontal disease.

The composition concentration of the human collagenase activity inhibitor of the present invention is such that the content of polyporenic acid C (Polyporenic acid C) or the mushroom containing polyporenic acid C (Polyporenic acid C) in the composition is high. The ratio of the extracted extract or its dry extract powder varies depending on the applied composition and cannot be specified in general, but when the total amount of the composition is 100% by weight, Polyporenic Acid C is used.
The concentration of (Polyporenic acid C) is preferably 0.001 to 10.0% by weight, and particularly preferably 0.005 to 1.0% by weight.
In addition, when a human collagenase activity inhibitor containing an extract of mushrooms or the powdered extract thereof is added,
The dry weight of the extract is preferably 0.005 to 20.0% by weight, more preferably 0.05 to 5.0% by weight.

[0024]

EFFECTS OF THE INVENTION According to the present invention, it has an excellent inhibitory activity against human collagenase, which is the cause of collagen absorption in gingiva and alveolar bone in periodontal disease, and is useful for the prevention and treatment of periodontal disease and the compounding amount. It is possible to provide a collagenase activity inhibitor which is extremely safe in use regardless of the amount of the drug.
Moreover, since the human collagenase activity inhibitor of the present invention acts specifically on human collagenase, it is suitable for preventing and treating periodontal disease without affecting other enzyme groups.

[0025]

EXAMPLES The present invention will be described in more detail by way of test examples and examples. The human collagenase activity inhibitor of the present invention shows that it does not act as a chelator for all metalloproteases but specifically inhibits human collagenase, and therefore bacterial collagenase and angiotensin that are the same metalloproteases as human collagenase. The inhibitory activity against the converting enzyme was investigated.

(Test Example-1) Polyporenic Acid C
Collagenase Inhibitory Action of (Polyporenic acid C) and Ganoderma lucidum Extract

1. Test substance Fruit bodies of spinach mushrooms are freeze-dried and then 7.35k
g was extracted with 15 l of 85% ethanol to give a spinach extract. The extract was concentrated, extracted with ethyl acetate, applied to a silica gel column (BW-127ZH), and eluted with chloroform: acetone (8: 2). Furthermore, the silica gel column (Klesselgel 6
0) and eluted with chloroform: acetone (82:18) were applied to an ODS column for HPLC and eluted with methanol: water (86:14) to obtain 6.0 mg of polyporenic acid C. It was

2. Human Collagenase As human collagenase, human fibrosarcoma cell-derived anchorage-independent cells were prepared by adding human procollagenase produced in a serum-free protein-free medium to CM Sepharose ^™ (Pharmacia) and zinc. chelating Sepharose ^TM
Purified by (Pharmacia) and dissolved in buffer,
Trypsin (Activated by Sigma, Type)
e12) was added and incubated at 35 ° C. for 5 minutes, and then soybean trypsin inhibitor (manufactured by Merck) was added to inactivate trypsin.

3. Measurement of human collagenase inhibitory activity Polyporenic acid C against human collagenase
The measurement of the inhibitory activity of (Polyporenic acid C) and spinach extract was performed as follows.

First, polypore acid C (Polypore
In the case of nic acid C), 4.0 mg of polyporenic acid C (Polyporenic acid C) was dissolved in 100 μl of dimethylsulfoxide (DMSO) to prepare a stock solution, and a measurement buffer solution [0.2 M sodium chloride, 5 mM calcium chloride, 0.05% by volume Br
ij-35 (polyoxyethylene (23) lauryl ether manufactured by ICI), 50 mM Tris-HCl buffer containing 0.02% by volume sodium azide, pH 7.5] and diluted 100-fold. Furthermore, the solution was diluted 3 to 100 times with the measurement buffer.

Equal amounts of each diluted solution and a known amount (0.4 unit; 1 unit represents the amount of enzyme degrading 1 μg of type I collagen per minute at 35 ° C.) were mixed in equal amounts, Using the type I collagen (manufactured by Cosmo Bio Inc.) labeled with fluorescein isothiocyanate as a substrate, the method of Nagai et al. (Japanese Journal of Inflamatio
n, Volume 4, page 123, 1984), and the inhibition curve was determined by measuring the collagenase activity.
The amount of test drug required for 0% inhibition was read as the IC ₅₀ value.

In the case of spinach extract, it was dried to dryness with a centrifugal concentrator, weighed, and then 5.0 mg was dissolved in 100 μl of dimethyl sulfoxide (DMSO) to prepare a stock solution, which was diluted 25 times with a measurement buffer.

An equivalent amount of the diluted solution and a known amount (0.4 unit; 1 unit represents the amount of enzyme degrading 1 μg of type I collagen per minute at 35 ° C.) were mixed in equal amounts to prepare bovine I. Using type collagen (manufactured by Cosmo Bio Inc.) as a substrate, reaction was carried out at 35 ° C. for 16 hours. An equal amount of sample buffer [3.2% by volume sodium n-dodecyl sulfate was added to this solution.
(SDS), 8% by volume β-mercaptoethanol, 16% by volume BPB solution (0.05% by volume bromphenol blue, 70% by volume glycerin, 0.0625M Tris hydrochloric acid solution, pH 6.8) containing 0.1M Tris hydrochloric acid solution, pH 6 .8] was added and boiled for 3 minutes to prepare a sample.

This sample was prepared by the method of Laemmli (Natur
e, 227, 680, 1970), and after 1 hour staining with CBB staining solution (0.25 vol% Coomassie Brilliant Blue R-250, 45 vol% methanol, 10 vol% acetic acid) Decolorization was carried out with a decolorizing solution (25% by volume methanol, 8% by volume acetic acid).

After the electrophoretic gel was dried, the cut bands of collagen in the electrophoretic pattern were analyzed by an image analyzer [Maci
ntosh] (manufactured by Apple Computer), and the intensity of the band was used as an index of inhibition of human collagenase activity.

4. Test Results Tables 1 and 2 show the results. Polyporenic acid C has a dose-dependent human collagenase inhibitory activity, and its IC ₅₀ value is 7.3 μg / ml.
(15.1 μM). Also 85% of spinach
The ethanol extract completely inhibited human collagenase.

[0037]

[Table 1]

[0038]

[Table 2] ++ Completely inhibited human collagenase activity.

Test Example-2 Human Collagenase Inhibitory Action of Various Mushroom Extracts

1. Test substance Fomitopsis pinicola , Fomito
psis rosea , Fomitopsis
nigra , Ganodermavalesiacu
The fruiting body of m (Tsuganomannentake) was freeze-dried, and 2.5 g of the fruiting body was extracted with 75 ml of 85% ethanol to obtain a mushroom extract.

2. Method The method for measuring the human collagenase and the human collagenase inhibitory activity used was the same as the method for the spinach extract of Test Example-1.

3. Test results Table 3 shows the results. The four types of mushroom extracts examined are
Both inhibited human collagenase.

[0043]

[Table 3] +: Inhibited human collagenase.

(Test Example-3) Polyporenic Acid C
(Polyporenic acid C) Specificity of inhibition

1. Test substance Polyporenic acid C (Po
lyporenic acid C) was used.

2. Bacterial collagenase and its activity inhibition assay method As bacterial collagenase, Clostridium hist
Collagenase derived from olyticum (manufactured by Sigma, Type
I) was used.

Polyporenic Acid C
acid C) 400 μg / ml is diluted 2-fold to 12.5 μg / ml with measurement buffer, 0.1 unit (1 unit indicates the amount of enzyme that decomposes 1 μmole of substrate per minute at 25 ° C)
The inhibition of the activity of Escherichia coli on bacterial collagenase was investigated.
The method for measuring the bacterial collagenase inhibitory activity is Test Example-
The same procedure as in the case of No. 1 spinach extract was carried out. At the same time, the activity inhibition against 0.4 unit of human collagenase was also examined.

3. Angiotensin-converting enzyme inhibition assay (a) Reaction buffer containing 50 mM Tris-HCl (p
H7.8)

(B) Angiotensin-converting enzyme solution Angiotensin-converting enzyme (derived from rabbit, manufactured by Sigma)
100 mM Tris-HCl containing 60 mM NaCl
It was dissolved in (pH 7.8) at 0.15 milliunit / ml. However, 1 unit means the amount decomposed from the substrate hippuryl-histidyl-leucine to 1 μmol hippuric acid in 1 minute at 37 ° C. and pH 8.3.

(C) Angiotensin I solution Angiotensin I (manufactured by Peptide Institute) was dissolved in 25% / ml with 10% acetonitrile solution (V / V) and then diluted with distilled water to 1.2 mg / ml.

(D) Method for measuring angiotensin converting enzyme inhibitory activity Polyporenic acid C (Polyporenic acid C) was dissolved in dimethyl sulfoxide to a concentration of 4.0 mg / ml, and then diluted appropriately with a reaction buffer. And

First, after mixing 20 μl of the above sample with 20 μl of angiotensin converting enzyme solution, 20 μl of substrate angiotensin I solution was added and reacted at 37 ° C. for 10 minutes.
The reaction was stopped by adding 60 μl of a 50% acetonitrile solution (V / V) containing 0.2% trifluoroacetic acid (V / V).

The amount of angiotensin II in the solution obtained by the above operation was measured by high performance liquid chromatography.

The elution condition of angiotensin II by high performance liquid chromatography is as follows: flow rate 1 ml, eluent 0.1%
A 23% acetonitrile (V / V) solution containing trifluoroacetic acid (V / V) was used as a column YMC A-312 (manufactured by Yamamura Chemical Laboratory, 6 × 150 mm). Moreover, the amount of angiotensin II was measured by the absorbance at 280 nm.
(This value is X.)

On the other hand, as a control, a group was prepared in which 20 μl of only the reaction buffer solution was added instead of polyporenic acid C (Polyporenic acid C), and the angiotensin converting enzyme inhibitory activity was measured by the same procedure as above. The amount of angiotensin II in the angiotensin converting enzyme reaction solution was defined as Y.

As a comparative example, captopril known as an angiotensin-converting enzyme inhibitor was dissolved and diluted in a reaction buffer instead of polyporenic acid C, and the mixture was added. The angiotensin-converting enzyme inhibitory activity was measured by the above procedure.

The inhibitory rate of angiotensin converting enzyme was calculated from the amount of angiotensin II by the formula 1.

[0058]

[Equation 1]

4. Test results As shown in the results in Table 4, various concentrations of polyporenic acid C inhibited human collagenase in a concentration-dependent manner, but the inhibitory action against bacterial collagenase was observed at any concentration. Not shown.

Table 5 shows the results of determining the angiotensin converting enzyme inhibition rate. Comparative Example (Captopril 10n
M) showed 50%, but polyporenic acid C did not inhibit angiotensin converting enzyme as compared with the comparative example.

From these results, it is considered that the inhibitory action of polyporenic acid C on human collagenase is not an action as a chelator for metalloproteases but a specific one.

[0062]

[Table 4]

[0063]

[Table 5]

Example 1 (Toothpaste) Toothpaste containing 0.1% by weight of Polyporenic acid C (purified by the method of Test Example-1) according to a conventional method for the formulations shown in Table 6 The agent was manufactured.
That is, water, glycerin, carrageenan, saccharin, butyl paraoxybenzoate, chlorhexidine digluconate, fragrance, and polyporenic acid C (Polypore).
nic acid C) is weighed and mixed to swell the binder, then dibasic calcium phosphate and sodium lauryl sulfate are added, mixed well and defoamed, then filled into a tube and toothpaste Got

[0065]

[Table 6]

Example 2 (Toothpaste) Spinach extract (extracted by the method of Test Example-1) was used in place of Polyporenic acid C (Polyporenic acid C), and the addition amount was 1.0% by weight. A toothpaste was obtained in the same manner as in Example 1 except for the above.

Example 3 (lozenge) A lozenge containing 0.05% by weight of Polyporenic acid C (purified by the method of Test Example-1) according to a conventional method for the formulations shown in Table 7. Was manufactured.

[0068]

[Table 7]

Example 4 (Lozenge Agent) Spinach extract (extracted by the method of Test Example-1) was used in place of Polyporenic acid C (Polyporenic acid C), and its addition amount was 0.5% by weight. Except
A troche was obtained in the same manner as in Example 3.

Example 5 (Mouthwash) Mouthwash containing 0.1% by weight of Polyporenic acid C (purified by the method of Test Example 1) according to a conventional method for the formulations shown in Table 8 The agent was manufactured.

[0071]

[Table 8]

Example 6 (Mouthwash) Spinach extract (extracted by the method of Test Example 1) was used instead of Polyporenic acid C, and the addition amount was 1.0% by weight. A mouthwash was obtained in the same manner as in Example 5 except that

Example 7 (Chewing Gum) A chewing gum containing 1.0% by weight of spinach extract (extracted by the method of Test Example-1) was prepared according to a conventional method for the formulations shown in Table 9.

[0074]

[Table 9]

That is, the whole amount of chewing gum base and the whole amount of starch syrup kept at 40 ° C. were put into a kneader and kneaded for 10 minutes, and 1/3 amount of powdered sugar and the whole amount of glucose were added for 5 minutes and then powdered 1/3 amount of sugar was added and kneading was performed for 5 minutes. Next, a mixture of spinach extract and flavor in the remaining 1/3 amount of powdered sugar was added and kneaded for 5 minutes to obtain a gum mix.

Example 8 (Nougat) 0.2% by weight of spinach extract (extracted by the method of Test Example-1) was prepared according to a conventional method for the formulations shown in Table 10.
A nougat containing was produced.

[0077]

[Table 10]

That is, first, mix and foam,
Boiled down to ℃. Add little by little, and whisk again,
This was added to and mixed with, spread on a cooling plate and molded to obtain a nougat.

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Claims (3)

[Claims] 1. A polyporenic acid C (Polyporeni)
c acid C) as an active ingredient, a human collagenase activity inhibitor. 2. A polypore acid C (Polyporeni)
A human collagenase activity inhibitor, which comprises an extract derived from mushrooms containing c acid C) or a dried extract powder thereof as an active ingredient. 3. The mushrooms are spinach ( Daedalea di
ckinsi ), The human collagenase activity inhibitor according to claim 2.