JPH11335274A - Argingipain inhibitor, inhibitor for growth of reriodontisis bacterial and antiperiodontisis agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent capable of specifically inhibiting argingipain and growth of Porphyromonas gingivalis, having high safety without adverse effects and useful as antiperidontitis agent by bringing the subject agent to contain malabaricon C as an effective ingredient. SOLUTION: This argingipain inhibitor is brought to contain malabaricon C expressed by the formula as an effective ingredient. The compound of the formula is obtained by extracting, e.g. mace which is aril of Myristicaceae plant or nutmeg which is a nucleolus in seeds using a solvent such as chloroform, methanol or the like. When the agent is used as a medicine, the dose of the effective ingredient is preferably 1-100 mg/dg/day and can be administered in several times a day.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an alginipain-specific inhibitor suitable for prevention and treatment of periodontal disease,
It relates to a periodontal disease growth inhibitor and an anti-periodontal agent.

[0002]

2. Description of the Related Art Periodontal disease is an infection mainly caused by destruction of the gingival epithelium and periodontal connective tissue and subsequent resorption of alveolar bone. It is said that Gram-negative anaerobic bacteria present in subgingival plaque are deeply involved in its onset and progression. Among them, Porphyromonas gingivalis (Porphyromonas gingivalis) is frequently detected in deep periodontal pockets and is considered to be the most important bacterium in adult periodontitis and rapidly progressive periodontitis (Holt, SC et al .: Crit. Rev. OralBiol. M
ed., 2, 177, 1991). Porphyromonas gingivalis has pili and lipopolysaccharide and produces hemagglutinin and many proteases, which are thought to be involved in the development and progression of periodontal disease. In particular, pili are necessary for the bacterium to adhere to gingival tissue and plaque formed thereon, and are deeply involved in the infectivity of the bacterium.

A cysteine protease that specifically cleaves the C-terminal side of arginine from Porphyromonas gingivalis, argingipain (gingipain-1 or gingipain R).
pain R)) was isolated and purified. It has been reported that this enzyme degrades the complement components C3 and C5 in vitro, and in particular the C5a fragment derived from C5 acts as a potent chemotactic factor and promotes neutrophil activation (Chen, Z. et al .: J. Biol. Chem. 267,18896, 1
992). In addition, it has been reported that alginguipain suppresses the bactericidal activity of polymorphonuclear leukocytes, but the bactericidal activity is released from the suppression by the addition of anti-argingipain antibody, leupeptin or EDTA that inhibits alginguipain. (Kadowaki, T. et al .: J. Biol. Chem. 269, 2
1371, 1994).

The arginipain-deficient strain of Porphyromonas gingivalis lacks almost the ciliary structure of the cell surface and accumulates precursors of major outer membrane proteins. It is expected to play important physiological functions as processing enzymes such as cell surface proteins (Nakayama, K. et al .: J. Bac
teriol. 178, 2818, 1996), it is considered that the infectivity of the bacterium can be suppressed by suppressing the function of arginipain. In addition, the culture supernatant of the Arginipain-deficient strain did not show any activity to suppress the bactericidal activity of polymorphonuclear leukocytes, and the hemagglutinin activity was significantly lower than that of the wild-type (Nakayam
a, K. et al .: J. Biol. Chem. 270, 23619, 1995).

[0005] As described above, argingipain plays an important role in the degradation of biological proteins, the suppression of bactericidal activity of polymorphonuclear leukocytes, and the growth of Porphyromonas gingivalis. Therefore, the arginipain-specific inhibitor is effective for the prevention and treatment of periodontal disease. Cysteine protease inhibitors such as leupeptin and E-64 are known to inhibit arginipain,
These compounds have no specificity for arginguipain and high toxicity, and thus have a problem that they cannot be used for prevention and treatment of periodontal disease. Currently, Streptococcus mutans (Streptococcus mutans)
s) and glucosyltransferase (glucos) produced by Streptococcus mutans
yltransferase). However, Streptococcus mutans is a bacterium involved in supragingival plaque formation, and these drugs have no effect on subgingival periodontal disease. Therefore, an anti-periodontal agent which has a high effect on subgingival periodontal disease bacteria and has few side effects is desired.

[0006]

SUMMARY OF THE INVENTION In order to meet the above demands, the present invention has a specific inhibitory action against arginipain, a specific antibacterial action against Porphyromonas gingivalis, and has no side effects. , Secure,
It is intended to provide an anti-periodontal agent.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and as a result, have found that malavaricon C contained in nutmeg or mace and represented by the following formula (1) is alginate. The present inventors have found that they specifically inhibit pine and also specifically inhibit the growth of Porphyromonas gingivalis, and thus have found that malavaricon C can effectively suppress periodontal disease, and completed the present invention. . That is, the present invention provides the following formula (1)

[0008]

Embedded image

[0009] The present invention provides an alginipain inhibitor and an anti-periodontal agent comprising malavaricon C as an active ingredient. Furthermore, the present invention provides an oral composition for preventing and ameliorating periodontal disease containing malavaricon C represented by the above formula (1), and prevention of periodontal disease containing malavaricon C represented by the above formula (1) And a functional food for improvement. Malabaricon C (Purushothaman, KK et al .:
J. Chem. Soc., Perkin Trans. 1, 587, 1977) has nematicidal activity (Nakamura, N. et al .: Chem. Pharm. Bull. 36,
2685, 1988), antitumor activity (Japanese Unexamined Patent Publication No.
No.), antibacterial activity (Orabi, KY et al .: J. Nat. Prod.
54, 856, 1991), an antioxidant effect and a collagen biosynthesis promoting effect (Japanese Patent Application Laid-Open No. 7-33651), but no inhibitory effect on argingipain. In addition, it is not known to specifically inhibit the growth of Porphyromonas gingivalis. Formula (1) compound represented by the tasteless, odorless, non-irritating, LD ₅₀ when administered orally to rats is 2000mg or more / kg. Malabaricon C represented by the above formula (1)
Is an Azumaceae plant (eg Myristica fragrans Houtt or
It can be obtained from a mace, which is a temporary seed coat of Myristica dactyloides, or nutmeg, which is a seed in seeds, by solvent extraction or the like (see Production Examples), or can be obtained from an appropriate starting material by organic synthesis.

[0010] In extracting the active ingredient of the present invention, there is no particular limitation as long as the method can effectively extract the active ingredient. The solvent used in this extraction is not particularly limited as long as the solvent can effectively extract the active ingredient. Examples thereof include water and acetone, or lower alcohols such as methanol and ethanol, and chlorine such as chloroform. Hydrocarbons, lower fatty acid esters such as ethyl acetate, or mixtures thereof can be used.

In order to produce the medicament according to the present invention, the compound of the formula (1) obtained as described above may be used as an active ingredient and formulated in accordance with a conventional method in combination with a known non-toxic pharmaceutical carrier. . For example, oral preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and lyophilized preparations. Parenteral preparations include injection preparations In addition, suppositories, sprays, transdermal absorbents and the like can be mentioned, and these preparations can be prepared by conventional means in preparation. Examples of the above non-toxic pharmaceutical carriers include, for example, glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, albumin, water And physiological saline. If necessary, conventional additives such as a stabilizer, a lubricant, a wetting agent, an emulsifier, and a binder can be appropriately added. In the medicament according to the present invention, the dose of the active ingredient is appropriately selected and determined according to the patient's age, body weight, symptoms, degree of disease, administration schedule, formulation, and the like. -10
It may be administered several times a day.

Further, the compound of the formula (1) can be blended as an active ingredient in an oral composition such as toothpaste and mouthwash. In this case, pure malavaricon C may be blended as it is, or an extract of nutmeg or mace may be blended.

The amount of the malavaricon C to be incorporated into the oral composition is preferably 0.0005 to 5%, particularly preferably 0.0025 to 1%. As other components, appropriate components according to the type of the oral composition are used. For example, in the case of toothpaste, dibasic calcium phosphate, calcium carbonate, insoluble sodium metaphosphate, amorphous silica, crystalline silica, abrasives such as aluminum oxide, carboxymethylcellulose, hydroxyethylcellulose, alginate, carrageenan, gum arabic , Binders such as polyvinyl alcohol, thickeners such as polyethylene glycol, sorbitol, glycerin, propylene glycol,
Sodium lauryl sulfate, sodium dodecylbenzenesulfonate, hydrogenated coconut fatty acid monoglyceride monosulfate sodium, sodium lauryl sulfoacetate,
A foaming agent such as sodium N-lauroyl sarcosinate and N-acyl glutamate, and components such as a sweetener, a flavor, and a preservative are mixed with water, and the mixture is produced according to a conventional method. Also, in mouthwashes such as mouthwashes, components according to the properties of the products are appropriately compounded.

In the present invention, in addition to the compound of the above formula (1), lysozyme chloride, dextranase, lytic enzyme, mutanase, chlorhexidine, sorbic acid,
Alexidine, hinokitiol, cetylpyridinium,
Alkyl glycine, alkyl diaminoethyl glycine salts, sodium monofluorophosphate, sodium fluoride, water-soluble primary or secondary phosphates, quaternary ammonium compounds, sodium chloride and the like can also be blended.

Further, the compound of formula (1) can be taken as a functional food for the purpose of preventing and improving periodontal disease. In this case, pure malavaricon C may be blended as it is, or an extract of nutmeg or mace may be blended. For example, after adding the above-mentioned suitable auxiliaries to the compound of the formula (1) or the extract of nutmeg or mace, an edible form may be obtained by a conventional means,
For example, it can be obtained by forming a liquid, granule, granule, tablet, capsule, or paste. This functional food may be served as it is, or various foods (for example, ham, sausage, kamaboko, chikuwa, bread, butter, milk powder, gum,
Candy, etc.) or water, fruit juice, milk, soft drinks and other drinks. The amount of malavaricon C to be taken in such a food form is appropriately selected and determined according to age, body weight, symptoms, degree of disease, food form, etc., for example, per 1 kg of body weight per day.
It is about 1 to 1000 mg.

[0016]

【Example】

The present invention will be described in more detail with reference to the following Examples, which by no means limit the present invention.

(Production Example) Nutmeg powder (Myristi)
250 g of chloroform: methanol (2: 1) was added to 50 g of ca.
Extracted at room temperature for minutes. 250 ml of chloroform: methanol (2: 1) was again added to the extraction residue, and the mixture was extracted for 30 minutes at room temperature. The obtained extracts were combined and concentrated under reduced pressure. Next, 500 ml of water was added to this extract, and the mixture was extracted three times with 500 ml of ethyl acetate. The ethyl acetate layer was dried under reduced pressure to obtain 19.7 g of an oily substance. After dissolving this oily substance in chloroform: methanol (2: 1), 40 g of silica gel powder was added, mixed well with stirring, and the solvent was removed under reduced pressure. This silica gel powder was charged into a silica gel column (5 × 10 cm) previously filled with hexane. Hexane: acetone (100: 0), (9
0:10) The fraction eluted in 3 L each was removed. Next, elution was carried out with a mixed solvent of hexane: acetone (80:20), and 300 ml was fractionated. Fraction No. 3 to No. In FIG. 8, malavaricon C was eluted. These fractions were collected and dried under reduced pressure to obtain 272 mg of an oily substance.
This oily substance was suspended in 27.2 ml of methanol and then filtered to obtain a filtrate. This solution was used as a sample for high performance liquid chromatography performed under the following conditions (column size: 3
0 × 250 mm, carrier; CAPCELL PAK C ₁₈
UG120 (manufactured by Shiseido), solvent composition; 50% acetonitrile, 50% water, flow rate: 40 ml / min, temperature 2
3 ° C, detection wavelength: 210 nm, device: Tosoh HLC-
8070). A fraction having a retention time of 18 to 21 minutes was collected, the solvent was distilled off under reduced pressure, and then extracted with ethyl acetate.
g of purified product was obtained. The physical properties of this product are
Of the literature.

(Test Example 1) Enzyme Inhibitory Activity Test (1) Preparation of Enzyme Arginipain was obtained from Porphyromona according to a known method.
gingivalis ATCC 33277 (see, eg, Kadowaki, T. et al .: J. Biol. Chem. 269, 21371,
1994). 3% trypticase soy broth, 0.5% yeast extract, 0.05% cysteine, 0.5%
Porphyromonas gingivalis ATCC 33277 was anaerobically cultured in a medium containing 0.1% hemin and 0.1% menadione. The culture was centrifuged to obtain a supernatant. At 4 ° C.,
Ammonium sulfate was added to 75% saturation.
The precipitate was collected by centrifugation at 10,000 xg for 20 minutes,
Dissolve in 10 mM sodium phosphate buffer (pH 7.0) containing 5% Brij 35, and in the same buffer overnight at 4 ° C.
Dialysed. Insoluble matter was removed by centrifugation at 25,000 × g for 30 minutes, and the supernatant was used as crude algingingine.

(2) Measurement of Arginipain Inhibitory Activity The measurement of the alginipain inhibitory activity was carried out by partially modifying the method described in the above literature. 0.01 units (1 of enzyme activity)
The unit is 1 nmol of 7-amino-per minute at 37 ° C.
Arginipain (defined as 4-methylcoumarin release) and an inhibitor dissolved in 10 mM cysteine and dimethylsulfoxide in 20 mM sodium phosphate buffer (pH 7.5) after preincubation at 37 ° C. for 5 minutes, The substrate Z-Phe-Arg-MCA (10
μM) was added to initiate the reaction (where Z represents carbobenzoxy and MCA represents 4-methylcoumaryl-7-amide). After reacting at 37 ° C. for 10 minutes, a 100 mM sodium acetate buffer (pH 5.0) containing 10 mM iodoacetic acid was used.
0) was added to stop the reaction, and the fluorescence intensity (excitation wavelength: 365 nm, fluorescence wavelength: 450 nm) of the reaction solution was measured.
Table 1 shows the results. Here, the relative values are shown assuming that the alginipain activity of the control is 100.

[0021]

[Table 1] Table 1. Arginipain inhibitory activity ──────────────────────────── Sample concentration Arginipain activity (relative value) ─────── ───────────────────── Control １００ 100 Malabaricon C 3.8 × 10 ^-7 M 82 1.1 × 10 ^-6 M 70 3.4 × 10 ^-6 M 41 1.0 × 10 ^-5 M 0 ────────────────────────────

(3) Measurement of Cathepsin L Inhibitory Activity Cathepsin L inhibitory activity was determined by the method described in the literature (Barrett,
AJ et al .: Methods Enzymol. 80, 535, 1981). Inhibitors dissolved in human liver cathepsin L (0.01 units (the definition of units is similar to arginipain), Calbiochem) and dithiothreitol (2 mM) and dimethylsulfoxide were Brij 35 (0.01%) and EDTA-2Na (1 mM) in a 0.1 M sodium acetate buffer (pH 5.5) at 37 ° C.
After preincubation for 5 minutes in Z-Phe-Ar
The reaction was started by adding g-MCA (10 μM). 37
After reacting at 10 ° C. for 10 minutes, 100 mM acetate buffer (pH 4.3) containing 100 mM sodium monochloroacetate.
To stop the reaction, and the fluorescence intensity of the reaction solution (excitation wavelength:
365 nm, fluorescence wavelength: 450 nm). Table 2 shows the results. Here, the concentration (IC ₅₀ ) at which malavaricon C inhibits each enzyme activity by 50% is shown.

(4) Measurement of papain, ficin, bromelain, chymotrypsin and trypsin inhibitory activities The inhibitory activities of papain, ficin and bromelain were determined by methods described in the literature (Suda, H. et al .: J. Antibiotics, 25, 26).
3, 1972) with some modifications. Each enzyme solution (30
17.5 m) of the inhibitor dissolved in 280 nm at 280 nm in 0.4 to 0.5), cysteine (0.2 mg / ml) and dimethylsulfoxide.
M in borate buffer (pH 7.4)
For 5 minutes. Casein (0.5%) was added thereto and reacted at 37 ° C. for 30 minutes.
The reaction was stopped by adding a 1.7 M perchloric acid solution, and the mixture was allowed to stand at room temperature for 1 hour. Then, the absorbance of the centrifuged supernatant was measured at 280 nm. The inhibitory activity of chymotrypsin and trypsin was determined by the method described in the literature (Umezawa, H. et al .: J. Antibioti
cs, 23, 425, 1970). The procedure was the same as for papain, except that cysteine was changed to 2.5 mM calcium chloride. Table 2 shows the results. Here, the concentration at which malavaricon C inhibits each enzyme activity by 50% (IC
₅₀ ).

[0024]

[Table 2] Table 2. Enzyme inhibitory activity of malavaricon C 酵素 Enzyme inhibitory concentration (IC ₅₀ (M)) ───────────── ──────── Cathepsin L> 2.8 × 10 ^-4 papain 1.3 × 10 ^-4 ficin> 2.8 × 10 ^-4 bromelain> 2.8 × 10 ^-4 chymotrypsin 1.8 × 10 ^-4 trypsin 1.3 × 10 ^-4 ─────────────────────

From these results, it was found that malavaricon C was 2.8.
50% inhibition of argingine pine at × 10 ^-6 M, 1.0%
It was completely inhibited at × 10 ⁻⁵ M. It was found that it specifically inhibited Arginipain by 50 times or more as compared with other proteases.

(5) Measurement of Arginipain and Cathepsin L Inhibitory Activity of Nutmeg Extract To 1 g of nutmeg powder, 5 ml each of methanol, 50% aqueous methanol, acetone, 50% aqueous acetone, and chloroform: methanol (2: 1) was added. , Extracted for 30 minutes and filtered. The alginipain and cathepsin L inhibitory activities of these extracts were measured by the method described above. Table 3 shows the results
Shown in Here, the amount of the extract that inhibits each enzyme activity by 50% is shown by% (v / v).

[0027]

[Table 3] Table 3. Enzyme inhibitory activity of nutmeg extract ─────────────────────────────────── Extraction solvent Inhibitory concentration (IC ₅₀ ( % (v / v))) Arginipaine cathepsin L ─────────────────────────────────── methanol 0 0.080> 1.0 50% aqueous methanol 0.23> 1.0 acetone 0.042> 1.0 50% aqueous acetone 0.065> 1.0 chloroform: methanol (2: 1) <0.012> 1 0.0 ─────────────────────────────────── From these results, the nutmeg extract is specific for Arginipain It was found that it inhibited.

Test Example 2 Measurement of Antibacterial Activity As a method for measuring the antibacterial activity, the minimum inhibitory concentration (MIC) for various microorganisms including periodontal disease bacteria was determined by a liquid medium dilution method. That is, the following microorganisms are cultured, and the number of bacteria is 2 × 1
It was prepared to be ⁵ cells / ml and used as a bacterial solution. 50 μl of the above bacterial solution was added to a U-bottomed 96-well plate containing 50 μl of a medium containing a test antimicrobial agent in which the concentration was changed stepwise. After culturing for 2 days under the following culture conditions, the presence or absence of growth of the bacteria was visually observed. The minimum concentration (μg / ml) at which no growth was observed was defined as the MIC. Table 4 shows the results
Shown in Similarly for erythromycin for comparison
The MIC was measured and the results are shown in Table 4. The strain used, the composition of the liquid medium and the culture conditions are as follows.

Porphyromonas gingivalis (Porphy
ATCC33277 and Porphyromonas gingivalis 381 are 30 g / L tryptic soy broth, 5 g / L yeast extract, 0.5 g / L cysteine, 5 mg / L hemin and 1 mg / L menadione at 37 ° C. The cells were cultured anaerobically (Aneropak (Mitsubishi Gas Chemical)). Fusobacterium nucleatu
m) ATCC25586 was anaerobically cultivated (Aneropak (Mitsubishi Gas Chemical)) at 37 ° C in brain heart infusion broth containing 0.05% cysteine. Streptococcus mutans IFO13955 and Actinomyces viscus
osus) ATCC15987 was cultured aerobically at 37 ° C in Brainheart Infusion Broth. Helicobacter
Helicobacter pylori ATCC 43526, 43579 and 43629 were cultured microaerobically (Campyropac (Mitsubishi Gas Chemical)) at 37 ° C in Muller Hinton broth containing 10% fetal calf serum. Escherichi
a.) ATCC 25922 and Staphylococcus aureus ATCC 25923 were cultured aerobically in Muller Hinton broth at 37 ° C. Aspergillus terreus was aerobically cultured at 25 ° C. in Sabouraud's medium.

[0030]

[Table 4] Table 4. Measurement results of antibacterial activity ────────────────────────────── Microorganisms Minimum inhibitory concentration MIC (μg / ml) Malavalicon C Erythromycin ─ ───────────────────────────── P. gingivalis 33277 0.39 0.078 P. gingivalis 381 0.098 0.020 F. nucleatum 3.1 10 S. mutans 50 0.039 A. viscosus 13 0.0098 H. pylori 43526 25 0.16 H. pylori 43579 13 0.078 H. pylori 43629 25 0.31 E. coli> 50> 10 S. aureus 13 0.31 A. terreus> 50> 10 ─────── ───────────────────────

As is apparent from the above results, Malavaricon C is specific and effective as compared to the oral flora Fusobacterium nucleatum, Streptococcus mutans, Actinomyces viscosus and other bacteria and fungi. It was found that it shows antibacterial activity against Porphyromonas gingivalis, which is a periodontal pathogen.

[0032]

Industrial Applicability The inhibitor of the present invention specifically inhibits arginipain and specifically inhibits the growth of Porphyromonas gingivalis. The anti-periodontal agent of the present invention can effectively prevent and treat periodontal disease.

Claims (5)

[Claims] 1. An alginguipain inhibitor comprising malavaricon C represented by the following formula (1) as an active ingredient. Embedded image 2. A growth inhibitor of Porphyromonas gingivalis comprising as an active ingredient malavaricon C represented by the above formula (1). 3. An anti-periodontal agent comprising malavaricon C represented by the above formula (1) as an active ingredient. 4. An oral composition for preventing and ameliorating periodontal disease, comprising malavaricon C represented by the above formula (1). 5. A functional food for preventing and ameliorating periodontal disease, comprising a malavaricon C represented by the above formula (1).