JP2004143127A - Pathogenic cysteine protease activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new agent for inhibiting the activity of a pathogenic cysteine protease, effectively inhibiting the activity of the pathogenic cysteine protease, especially arginine-specific cysteine protease (Rgp) and lysine-specific cysteine protease (Kgp). <P>SOLUTION: The agent for inhibiting the pathogenic cysteine protease activity contains catechin or a catechin mixture as an active component. Especially, (-)-gallocatechin (GC) and (-)-gallocatechin gallate (GCg) have strong activity inhibiting effect on Rgp and (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCg), (-)-gallocatechin (GC) and (-)-gallocatechin gallate (GCg) have strong activity inhibiting effect on Kgp. <P>COPYRIGHT: (C)2004,JPO

Description

【００３５】
２）ＥＧＣｇのアルカリホスファターゼに及ぼす影響
ＥＧＣｇはＰ．ｉｎｔｅｒｍｅｄｉａの産生するアルカリホスファターゼを強く阻害し、酵素活性を５０％阻害する濃度は約５μｇ／ｍＬであった（図２）。
Ｐ．ｉｎｔｅｒｍｅｄｉａの培養上清にも本酵素は検出され、ＥＧＣｇの５０％阻害する濃度はＰ．ｎｉｇｒｅｓｃｅｎｓのアルカリホスファターゼとほぼ同程度で約５０μｇ／ｍＬであった。
しかし、Ｐ．ｇｉｎｇｉｖａｌｉｓの産生するアルカリホスファターゼ活性に対しては阻害効果を示さなかった。
【００３６】
３）ＥＧＣｇのＰ．ｉｎｔｅｒｍｅｄｉａの溶血活性および血球凝集活性に及ぼす影響
図３は、最終濃度０−１００μｇ／ｍＬのＥＧＣｇを反応系に加え、２、４および６時間後の溶血活性を示したものである。
ＥＧＣｇの濃度が増加するにつれＰ．ｉｎｔｅｒｍｅｄｉａの溶血活性は阻害され、１００μｇ／ｍＬ、６時間の反応では対照の溶血活性の２３％に減少した。
【００３７】
【表２】

【００３８】
表２は、ＥＧＣｇの黒色コロニー形成嫌気性菌の血球凝集活性に及ぼす影響を調べたものである。
３．６μｇ／ｍＬのＥＧＣｇを反応系に加え、細菌と血球のみ加えた対照と比較した。その結果、Ｐ．ｉｎｔｅｒｍｅｄｉａは対照の２分の１、Ｐ．ｎｉｇｒｅｓｃｅｎｓでは対照の８分の１に凝集活性を減少した。一方、ＥＧＣｇはＰ．ｇｉｎｇｉｖａｌｉｓの血球凝集活性には影響を及ぼさなかった。
黒色コロニー形成嫌気性菌は溶血活性、血球凝集活性を持ち、これらの細菌の育成に必要な鉄イオンを血球凝集、溶血という形で補給していることが示唆されている。ＥＧＣｇがこれらの活性を阻害することは、これらの細菌の育成を抑制し、種々の有害な代謝産物産生を抑制する可能性が示唆される。
【００３９】
４）ＥＧＣｇのＰ．ｇｉｎｇｉｖａｌｉｓの産生するＲｇｐ活性に及ぼす影響
ＥＧＣｇはＰ．ｇｉｎｇｉｖａｌｉｓの産生するＲｇｐ活性を強く阻害し、酵素活性を５０％阻害する濃度は約１０μｇ／ｍＬであった（図４）。
生体蛋白質の分解や、多形核白血球の殺菌活性の抑制、さらには菌の生長などにＰ．ｇｉｎｇｉｖａｌｉｓのＲｇｐやリジンジンジパイン（Ｋｇｐ）らの酵素が重要な役割を演じていることから、ＥＧＣｇはこれらの病原性効果を阻害できることが示唆される。
【００４０】
また、ＥＧＣｇ以外のカテキンすなわちＥＣ、ＥＧＣ、ＥＣｇ、Ｃ、ＧＣ、Ｃｇ　、ＧＣｇ及びＧＡ（表ではｇ）、さらにカテキン混合物としてのテアフラン３５（商品名；伊藤園社製）及びテアフラン９０Ｓ（商品名；伊藤園社製）についても、ＥＧＣｇと同様にＲｇｐ活性に及ぼす影響について試験し、各カテキンのＲｇｐ活性阻害率に関する結果を図５〜図７に示し、各カテキンの５０％阻害濃度を下記表３に示した。
【００４１】
８種類全てのカテキン及びカテキン混合物のいずれにもＲｇｐに対する活性阻害効果を認めることができた。ただし、各カテキンの活性阻害能には大きな差が見られ、効果の大きい順に
ＧＣ、ＧＣｇ＞ＥＧＣｇ、ＥＧＣ＞ＧＡ＞Ｃｇ、ＥＣｇ＞ＥＣ、Ｃ
であった（なお、ＧＣとＧＣｇは同程度という意である）。
また、テアフラン３５及びテアフラン９０ＳのＲｇｐ活性の５０％阻害濃度（ＩＣ５０）は、テアフラン３５が４０μｇ／ｍＬ、テアフラン９０Ｓが４０μｇ／ｍＬであった。
【００４２】
さらにまた、ＥＧＣｇ以外のカテキンすなわちＥＣ、ＥＧＣ、ＥＣｇ、Ｃ、ＧＣ、Ｃｇ　、ＧＣｇ及びＧＡ（表ではｇ）、さらにカテキン混合物としてのテアフラン３５（商品名；伊藤園社製）及びテアフラン９０Ｓ（商品名；伊藤園社製）についても、ＥＧＣｇと同様にＫｇｐ活性に及ぼす影響について試験し、各カテキンのＫｇｐ活性阻害率に関する結果を図８〜図１０に示し、各カテキンの５０％阻害濃度を下記表３に示した。
【００４３】
８種類全てのカテキン及びカテキン混合物のいずれにもＫｇｐに対する活性阻害効果を認めることができた。ただし、各カテキンの活性阻害能には大きな差が見られ、Ｋｇｐに対する活性阻害効果は、効果の大きい順に
ＧＣ、ＧＣｇ、ＥＧＣｇ、ＥＧＣ＞ＧＡ＞Ｃｇ、ＥＣｇ＞ＥＣ、Ｃ
であった（なお、ＧＣ、ＧＣｇ、ＥＧＣｇ及びＥＧＣは同程度という意である）。
また、テアフラン３５及びテアフラン９０ＳのＫｇｐ活性の５０％阻害濃度（ＩＣ５０）は、テアフラン３５が５０μｇ／ｍＬ、テアフラン９０Ｓが１５０μｇ／ｍＬであった。これより、Ｋｇｐに対する活性阻害については、テアフラン９０Ｓよりもテアフラン３５の方が強いことが分かった。
【００４４】
【表３】

【図面の簡単な説明】
【図１】黒色コロニー形成嫌気性菌の産生するアルカリホスファターゼのザイモグラフである。
【図２】ＥＧＣｇのＰ．ｉｎｔｅｒｍｅｄｉａの産生するアルカリホスファターゼに対する阻害効果を示したグラフである。
【図３】最終濃度０−１００μｇ／ｍＬの各ＥＧＣｇを反応系に加えた場合における、溶血活性の変化を経時的に示したグラフである。
【図４】各種濃度におけるＥＧＣｇのＰ．ｇｉｎｇｉｖａｌｉｓの産生するＲｇｐに対する活性阻害を示したグラフである。
【図５】各種濃度におけるカテキン類のＰ．ｇｉｎｇｉｖａｌｉｓの産生するＲｇｐに対する活性阻害を示したグラフである。
【図６】同じく、各種濃度におけるカテキン類のＰ．ｇｉｎｇｉｖａｌｉｓの産生するＲｇｐに対する活性阻害を示したグラフである。
【図７】各種濃度における緑茶抽出物のＰ．ｇｉｎｇｉｖａｌｉｓの産生するＲｇｐに対する活性阻害を示したグラフである。
【図８】各種濃度におけるカテキン類のＫｇｐに対する活性阻害を示したグラフである。
【図９】同じく、各種濃度におけるカテキン類のＫｇｐに対する活性阻害を示したグラフである。
【図１０】各種濃度における緑茶抽出物のＫｇｐに対する活性阻害を示したグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a pathogenic cysteine protease activity inhibitor, which effectively inhibits the activity of arginine-specific cysteine protease (Rgp) and lysine-specific cysteine protease (Kgp).
[0002]
BACKGROUND OF THE INVENTION
Protease is a generic term for enzymes that degrade proteins, and breaks peptide bonds of proteins by hydrolysis to break them down into smaller peptide fractions and amino acids. These proteases are classified into 1) serine protease, 2) cysteine protease, 3) aspartic protease, 4) metalloprotease and the like according to the active primordium.
[0003]
It is known that cysteine protease has an increased activity involved in various diseases, and if it can inhibit the cysteine protease enhanced in these diseases, it may be effective for treatment and prevention of diseases (modern age Biological functions of proteases, Tokyo Kagaku Dojin 1993; proteases and their inhibitors).
[0004]
Among them, arginine-specific cysteine prosthesis (hereinafter referred to as “Rgp”) and lysine-specific cysteine prosthesis (hereinafter referred to as “Kgp”) are gram-negative anaerobic bacteria that are both periodontopathogenic bacteria. It is known as a major enzyme produced by Porphyromonas gingivalis (gingivalis), but has not only a function as a virulence factor of periodontal disease but also various pathogenic functions. For example, Kgp and Rgp cooperate with each other to have a pathogenic function of directly destroying periodontal tissue and destroying the biological defense system, and Kgp independently exerts a pathogenic function such as enhancement of the fibrinolytic system. It is known that it independently exerts pathogenic functions such as enhancement of the blood coagulation system (Chemistry and Biology, vol. 40, No. 2, 72-74, 2002). Therefore, if these pathogenic functions can be inhibited, treatment and prevention of various diseases can be expected.
[0005]
Rgp is Arg-gingipain that specifically recognizes arginine residues and cleaves at the C-terminal side, and Kgp is Lys-specifically recognizes lysine residues and cleaves at the C-terminal side. Zingipine.
[0006]
[Prior art]
As a substance that inhibits the activity of a pathogenic cysteine protease, especially Kgp or Rgp, KYT-1 has a structure of Cbz-Lys-Arg-Co-Lys-NMe2 (Cbz: carbobenzyloxy, Me: methyl), It specifically inhibits Rgp, and KYT-36 has a structure of Cbz-Glu (NHNMePh) -Lys-CO-NHCH2Ph, and is known to specifically inhibit Kgp (Non-patent Document 1). reference).
[0007]
In addition, the invention described in Patent Document 1 examines protease inhibitory effects of extracts of various natural products including a green tea extract. In Example 1, protease is produced in an aqueous solution of the extracts of various natural products. The anaerobic bacterium Porphyromonas gingivalis cell suspension was added, stirred, incubated at 35 ° C. for 5 minutes, a casein solution was added thereto, and incubated at 35 ° C. for 10 minutes, and then a trichloroacetic acid solution was added thereto for reaction. Was stopped, and a Folin reagent was added to develop a color. The absorbance at 660 nm was measured, and the protease titer was determined from a calibration curve prepared in advance. As a result, the protease inhibition rate of the green tea extract was 100%. On the other hand, Example 2 shows the result that the protease inhibition rate of catechin is 0%.
[0008]
As a substance inhibiting RGP, Patent Document 2 reports a high molecular weight polyphenol. In addition, although not considered to be related to the present invention, examples of the cysteine protease inhibitor include JP-A-2001-247475, JP-A-11-228526, JP-A-09-295996, JP-A-09-165360, JP-A-2002-515902, JP 2002-515411, JP 2002-514192, JP 2002-512621, JP 2001-525809, JP 2001-525804, JP 2001-518513, JP 2001-514506, JP 2001-506596, JP 2001-501178, JP-T-11-509543, JP-T-11-509231 and the like.
[0009]
[Non-patent document 1]
Chemistry and biology, vol. 40, no. 2, 73-74, 2002
[Patent Document 1]
JP-A-2002-3353
[Patent Document 2]
JP-A-8-81380
[0010]
[Problems to be solved by the invention]
The present invention has been studied diligently on the pathogenic cysteine protease inhibitory activity of the tea extract component, which is a natural product, particularly the activity inhibitory effect on Kgp and Rgp, and inhibits the activity of Kgp and Rgp derived from the tea extract component. It is intended to provide a pathogenic cysteine protease activity inhibitor excellent in action.
[0011]
[Means for Solving the Problems]
The present invention proposes a pathogenic cysteine protease activity inhibitor comprising catechin or a catechin mixture as an active ingredient.
[0012]
As the catechin, (-) epicatechin (EC), (-) epigallocatechin (EGC), (-) epicatechin gallate (ECg), (-) epigallocatechin gallate (EGCg), (-) catechin (C ), (-) Gallocatechin (GC), (-) catechin gallate (Cg) or (-) gallocatechin gallate (GCg), and as a catechin mixture, two or more of the above catechins are used. Can be used.
[0013]
A pathogenic cysteine protease activity inhibitor containing catechin or a catechin mixture as an active ingredient effectively inhibits the activity of Rgp and Kgp among pathogenic cysteine proteases. In particular, any one of GC and GCg or a mixture thereof has a more excellent activity inhibitory effect on Rgp than other catechins, and any one of EGC, EGCg, GC, and GCg, or a mixture thereof. A mixture composed of a combination of more than two kinds has more excellent activity inhibitory effect on Kgp than other catechins.
Therefore, the present invention also provides an Rgp activity inhibitor containing any one of GC and GCg or a mixture thereof as an active ingredient, and any one of EGC, EGCg, GC, and GCg, or two or more of these. The present invention proposes a Kgp activity inhibitor containing a mixture composed of the combinations as an active ingredient.
[0014]
In the present invention, "a specific catechin (for example, (-) epicatechin (EC) or (-) epigallocatechin (EGC), or a limited catechin such as a mixture thereof, as an active ingredient)" As long as the specific catechin does not inhibit the pathogenic cysteine protease activity-inhibiting effect, it includes the meaning that other components such as catechins other than the specific catechin may be included.
In addition, a polymer of each catechin of EC, EGC, ECg, EGCg, C, GC, Cg, and GCg, and a copolymer of each catechin and another catechin, or a combination of two or more thereof The mixture is an equivalent of “catechin or a mixture of catechins”, “EC, EGC, ECg, EGCg, C, GC, Cg, or GCg, or a mixture of two or more of these”. Can be considered.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
The pathogenic cysteine protease activity inhibitor of the present invention can be produced by mixing catechin or a catechin mixture at an appropriate concentration. However, it is possible to appropriately mix other components or perform other treatments as long as the pharmacological activity of catechin or the catechin mixture is not inhibited.
[0016]
As the above catechin, any one of eight catechins of EC, EGC, ECg, and EGCg, and their epimers, C, GC, Cg, and GCg, can be used.
Further, as the catechin mixture, a mixture composed of a combination of two or more of the above eight catechins can be used.
All eight types of catechins have activity inhibitory effects on pathogenic cysteine proteases, especially Rgp and Kgp, but there is a large difference in the activity inhibitory ability of each catechin.
For example, the activity inhibitory effects on Rgp are in descending order of effect.
GC, GCg> EGCg, EGC> Cg, ECg> EC, C
And
The activity inhibitory effects on Kgp are in descending order of effect.
GC, GCg, EGCg, EGC> Cg, ECg> EC, C
It has been confirmed that
(Incidentally, the above-mentioned "GC, GCg> EGCg, EGC" indicates that the effects of GC and GCg are almost the same, and that the effects of GC and GCg are greater than the effects of EGCg and EGC. And similar designations in this specification have the same meaning.)
[0017]
Since EC, EGC, ECg and EGCg are contained in a large amount in natural products, particularly tea leaves, they can be obtained by extraction and purification by a conventionally known method or a known method in the future.
[0018]
On the other hand, these epimers, C, GC, Cg and GCg, are hardly present in natural plants including tea leaves, but are each a mixture of EC, EGC, ECg and EGCg or a combination of two or more thereof. Can be obtained by heat isomerization (epimerization) by heat treatment. For example, each of purified EC, EGC, ECg and EGCg or a mixture comprising a combination of two or more thereof, or a tea extract or leaching solution, preferably adjusted to pH 5 to 6, and then heated to 80 ° C. or higher. The heat treatment promotes the thermal isomerization of catechin to increase the concentration of C, GC, Cg and GCg. From such a heat-treated product, each of C, GC, Cg and GCg or a combination of two or more of these It can be obtained by separating and purifying a mixture comprising the combinations.
As the heat treatment for thermally isomerizing (epimerizing) catechin, a solution containing catechin (a catechin solution) may be heated to at least 80 ° C. or more. For example, heating at 100 ° C. for 15 minutes, heating at 115 ° C. for 20 minutes, heating at 120 ° C. for 3 to 30 minutes, heating at 123 ° C. for 10 minutes, heating at 131 ° C. for 30 seconds, and catechin at 133 ° C. for 45 seconds. Thermal isomerization has been observed.
It is preferred that the catechin solution be adjusted to pH 5 to 6 and then heat-treated because it has been reported that catechins hardly undergo thermal isomerization below pH 4.5 (Shinichi Suematsu et al .: Nisshin Jitsugaku Magazine, 39, 178 (1992)).
[0019]
The method for separating and purifying catechin may be a conventionally known method or a method known in the future. For example, it is known that the tea extract can be separated by subjecting it to reverse phase HPLC using, for example, a mixed solution of water-acetonitrile-phosphoric acid as a mobile phase, and applying a gradient with an acetonitrile concentration.
[0020]
As the “catechin mixture” of the present invention, a tea extract having a catechin concentration of 20% by weight or more, preferably 25% or more, and especially 30% or more can be used.
The tea extract is obtained by extracting tea with water, hot water or hot water, preferably hot water at 40 ° C to 100 ° C, especially hot water at 90 to 100 ° C, or an organic solvent such as an aqueous ethanol solution or ethanol harmless to the human body. Preferably, the tea extract is obtained by purifying the tea extract in a direction to increase the catechin content by a purifying means such as a solvent extraction method, a resin adsorption method, and filtration such as ultrafiltration and reverse osmosis filtration. Can be. At the time of extraction, extraction may be performed under acidic conditions to which hydrochloric acid or the like is added in order to increase the catechin content. Also, a commercially available tea extract can be used. For example, for both Teafuran 35 and 30A (trade name; manufactured by ITO EN Co., Ltd.), green tea is subjected to a hot water extraction treatment, and the extract is dried to have a catechin concentration of about 30% (composition (% by weight)). : EGC7.62, EGCg 13.70, EC 2.13, ECg 2.61, GC 2.61, GCg 0.49, (+) C0.43, Cg0.28), and theafuran 90S (trade name; manufactured by ITO EN Co., Ltd.) Green tea extract obtained by subjecting green tea to hot water extraction and separation using an adsorption column using water and low- and high-concentration alcohols and drying to obtain a tea polyphenol concentration of about 85 to 99.5% (Composition (% by weight): EGCg 50.18, EC 0.57, ECg 17.11, GCg 3.96, Cg 0.91).
[0021]
In the pathogenic cysteine protease activity inhibitor of the present invention, catechin or a catechin mixture can be used alone as the active ingredient of the present invention. However, pathogenic cysteine proteases, particularly Rgp and Kgp, are already or in the future. It is also possible to mix it with a substance that has been shown to have an activity inhibiting effect on, and use it in combination with these active ingredients.
When formulated as the active ingredient of the present invention alone, for example, catechin or a catechin mixture is dissolved in purified water, physiological saline, or the like, and a pathogenic cysteine protease activity inhibitor (eg, an oral administration agent, an intraperitoneal administration agent, etc.) ) Is also possible.
[0022]
The pathogenic cysteine protease activity inhibitor of the present invention can be orally administered or parenterally administered (intramuscular injection, intravenous injection, subcutaneous administration, rectal administration, transdermal administration, nasal administration, etc.). It is preferred that the formulation and dosage form be suitable for administration.
With respect to the dosage form, for example, for oral administration, it can be prepared in the form of liquid, tablet, powder, granule, dragee, capsule, suspension, emulsion, pill, etc., and for parenteral administration. It can be prepared in the form of injection, ampoule, rectal administration, oily suppository, water-soluble suppository and the like.
As for the formulation (formulation), commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, dissolution aids, It can be produced by a conventional method using a preservative, a flavoring agent, a soothing agent, a stabilizer and the like. Also, for example, lactose, fructose, glucose, starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or a salt thereof, gum arabic, polyethylene glycol, syrup, petrolatum, glycerin, ethanol, propylene It is also possible to incorporate non-toxic additives such as glycol, citric acid, sodium chloride, sodium sulfite, and sodium phosphate.
[0023]
In addition, the pathogenic cysteine protease activity inhibitor of the present invention may be used in addition to drugs, quasi-drugs, health foods / healthy drinks / specified health foods / functional foods having pharmacological effects, food additives, and other non-human Can be provided as drugs, feeds, feed additives, etc. for animals.
For example, by preparing it as a quasi-drug and making it into a drinking form such as a bottled drink or a form such as tablets, capsules, granules, etc., it is possible to make it easier to ingest.
[0024]
Examples of health foods, health drinks, specified health foods, and functional foods having a pathogenic cysteine protease activity inhibitory effect include, for example, catechin or a catechin mixture, or a catechin solution containing the mixture (eg, catechin extract or leachate). A group of food and drink materials such as carbonic acid, excipients (including granulating agents), diluents, or sweeteners, flavors, flour, starch, sugars, oils and fats, various carbohydrate raw materials, vitamins and minerals Or mixed with one or more selected from, or currently known foods and drinks, such as sports drinks, fruit drinks, milk drinks, tea drinks, vegetable juices, milk drinks, alcoholic drinks, jelly, jelly drinks, Carbonated beverages, chewing gum, chocolate, candy, biscuits, snacks, bread, dairy products, fish paste products, animal meat products, frozen desserts Dry food, can be prepared by adding such as supplements.
[0025]
The content of the active ingredient in the present invention varies depending on the method of use, but in the case of a pharmaceutical, catechin or a mixture thereof is 0.001 to 1% by weight, particularly 0.01 to 0.5% by weight in terms of dry weight. %, Preferably 0.001 to 1% by weight, particularly 0.01 to 0.5% by weight, of catechin or a mixture thereof.
The amount of catechin or a mixture thereof is preferably 10 to 5000 mg, preferably 100 to 1500 mg, per day of an adult in terms of dry weight.
[0026]
<Test 1>
(Culture of cells)
The black colony-forming anaerobic bacterium was cultured at 37 ° C. in an anaerobic bacterium culturing device MIP-1025 (Forma Scientific) using Brucella HK agar medium (Far East) supplemented with 5% sheep defibrinated blood (Japan Biotest Research Institute). N ₂ 80%, CO ₂ 10%, H ₂ The strain was maintained by culturing at 10%. In the experiment, brain-heart infusion broth was cultured in a medium containing 0.5% yeast extract, 0.05% cysteine hydrochloride, 5 μg / mL hemin 1 μg / mL, and menadione under the above culture conditions. In this study, Prevotella intermedia ATCC 25611 ^T , Prevotella nigrescens ATCC 33563 ^T , ATCC 25261 and Porphyromonas gingivalis ATCC 33277 ^T Was used.
[0027]
(Preparation of cell extract)
The cultured bacteria are washed twice with a phosphate buffered saline (pH 7.2) after centrifugation, suspended in a Tris-HCL buffer (pH 7.4) containing 1% Triton-X, and mixed with glass beads (particles). (Diameter: 0.15 mm) and vigorously stirred to crush. After the crushed liquid was centrifuged at 12,000 rpm for 20 minutes, the supernatant was used for enzyme activity measurement.
[0028]
(Zymograph of alkaline phosphatase)
The cell extract was subjected to electrophoresis using a 7.5% polyacrylamide gel and a commonly used Tris-glycine electrophoresis buffer, and the electrophoresed gel was subjected to 1 mg / mL naphthol AS-BI phosphate, 0.1 mg / mL fast blue. BB was added to Tris-HCl buffer (0.1 M Tris-HCL buffer (pH 10.0), 5 μm g CL ₂ ), And the enzyme activity was detected.
[0029]
(Measurement of alkaline phosphatase activity)
The alkaline phosphatase activity was determined by reacting paranitrophosphate (pNP) as a substrate in a 96-well multiwell and measuring the absorbance at a wavelength of 405 nm. That is, alkaline phosphatase activity was measured using 0.1 mM MgCL ₂ , 8 mM pNP, 0.1 M Tris buffer (0.1 M Tris-HCL buffer (pH 10.0), 5 μm gCL ₂ 80 μL), 10 μL of EGCg of various concentrations, and 10 μL of crude enzyme extract were stirred and reacted at 37 ° C. for 20 minutes.
[0030]
(Measurement of Rgp activity)
The Rgp activity was measured by using Benzoyl arginine p-nitroanilide (BapNA) as a substrate in a 96-well multiwell and measuring the absorbance at a wavelength of 405 nm. That is, 10 mM Benzoyl arginine p-nitroanilide (HCL) is dissolved in DMSO to prepare a substrate stock solution. While 1 mM cysteine hydrochloride is dissolved in 0.1 M Tris-HCl buffer (pH 7.4), the above substrate stock solution is dissolved. After diluting and dissolving 10-fold with 1/10 buffer 2, the mixture was stirred to 80 μL of the substrate stock solution (final substrate concentration: 0.8 μM), 10 μL of EGCg of various concentrations, and 10 μL of the crude enzyme extract, 37 ° C., 10 minutes Reacted.
[0031]
(Measurement of Kgp activity)
The Kgp activity was measured by using Np-tosyl-Gly-Lys-p-nitroanilide as a substrate in a 96-well multiwell and measuring the absorbance at a wavelength of 405 nm. That is, 10 mM N-p-tosyl-Gly-Lys-p-nitroanilide (HCL) is dissolved in DMSO to prepare a substrate stock solution, and 1 mM cysteine hydrochloride is dissolved in a 0.1 M Tris-HCl buffer (pH 7.4). On the other hand, after diluting and dissolving the substrate stock solution 50-fold with 1/10 buffer 2, the mixture was stirred so that the substrate stock solution became 80 μL (final substrate concentration 0.16 μM), 10 μL of EGCg of various concentrations, and 10 μL of crude enzyme extract. Then, the mixture was reacted at 37 ° C. for 10 minutes.
[0032]
(Measurement of hemolytic activity and hemagglutination activity)
For the measurement of the hemolytic activity, 10% rabbit erythrocytes and cultured cells were mixed in a 96-well multiwell, reacted appropriately at 37 ° C., and 25 μL of the reaction solution was added to a 10-fold amount of NCN buffer (3 mM sodium chloride, 150 mM sodium chloride, pH 6). .8), and after centrifugation, the absorbance of the supernatant at a wavelength of 414 nm was measured to determine the amount of released hemoglobin.
The degree of hemolysis was expressed as [(a sample to which EGCg and P.intermedia were added)-(a sample to which only EGCg was added)] / [(a sample to which 100% hemolysis was performed)-(blank)] × 100 (%).
Hemagglutinating activity was measured by adding equal amounts of 2% rabbit erythrocytes, cultured cells and EGCg of each concentration to a V-bottom multiwell, stirring for 30 minutes, storing overnight in a refrigerator, and causing the highest dilution ratio to cause aggregation. Was used to determine the cohesion value.
[0033]
(Results and Discussion)
1) Zymograph of alkaline phosphatase
FIG. 1 shows a zymograph of alkaline phosphatase produced by black colony-forming anaerobic bacteria.
P. intermedia has the lowest mobility, followed by P.I. nigrescens is a P.C. gingivalis has the highest mobility, and was thought to produce different types of alkaline phosphatase depending on the bacterial species.
Table 1 shows the effect of the vanadate compound on each enzyme. Although vandic acid compounds strongly inhibit the alkaline phosphatase activity of Prevotella, gingivalis did not inhibit alkaline phosphatase activity.
[0034]
[Table 1]

[0035]
2) Effect of EGCg on alkaline phosphatase
EGCg is P.O. The concentration which strongly inhibited alkaline phosphatase produced by intermedia and inhibited enzyme activity by 50% was about 5 μg / mL (FIG. 2).
P. This enzyme is also detected in the culture supernatant of intermedia, and the concentration at which EGCg is inhibited by 50% is P. Nigrescens alkaline phosphatase was about 50 μg / mL, almost the same level.
However, P. gingivalis had no inhibitory effect on alkaline phosphatase activity.
[0036]
3) E.g. Effect of intermedia on hemolytic and hemagglutinating activities
FIG. 3 shows the hemolytic activity after 2, 4 and 6 hours when EGCg at a final concentration of 0-100 μg / mL was added to the reaction system.
As the concentration of EGCg increases, P.P. The hemolytic activity of intermedia was inhibited and decreased to 23% of the control hemolytic activity in a 100 μg / mL, 6 hour reaction.
[0037]
[Table 2]

[0038]
Table 2 shows the effect of EGCg on the hemagglutinating activity of black colony-forming anaerobic bacteria.
3.6 μg / mL EGCg was added to the reaction system and compared to a control containing only bacteria and blood cells. As a result, P.I. intermedia is one half of the control, P.I. nigrescens reduced agglutinating activity to one-eighth of control. On the other hand, EGCg gingivalis had no effect on the hemagglutinating activity.
It has been suggested that black colony-forming anaerobic bacteria have hemolytic activity and hemagglutinating activity, and supplement iron ions necessary for the growth of these bacteria in the form of hemagglutination and hemolysis. The fact that EGCg inhibits these activities suppresses the growth of these bacteria, suggesting the possibility of suppressing the production of various harmful metabolites.
[0039]
4) E.g. Effect of gingivalis on Rgp activity
EGCg is P.O. gingivalis strongly inhibited the Rgp activity, and the concentration at which the enzyme activity was inhibited by 50% was about 10 μg / mL (FIG. 4).
It is important for the degradation of biological proteins, suppression of bactericidal activity of polymorphonuclear leukocytes, and growth of bacteria. The role of enzymes such as gingivalis Rgp and lysine zindipain (Kgp) suggests that EGCg can inhibit these pathogenic effects.
[0040]
In addition, catechins other than EGCg, that is, EC, EGC, ECg, C, GC, Cg, GCg and GA (g in the table), and theafuran 35 (trade name; manufactured by ITO EN Co., Ltd.) and theafuran 90S (trade name) as a catechin mixture. (Itoen Co., Ltd.) was also tested for its effect on Rgp activity in the same manner as EGCg, and the results on the Rgp activity inhibition rate of each catechin are shown in FIGS. Indicated.
[0041]
All eight kinds of catechins and catechin mixtures were able to show an activity inhibitory effect on Rgp. However, there is a large difference in the activity inhibitory ability of each catechin, and
GC, GCg> EGCg, EGC>GA> Cg, ECg> EC, C
(Note that GC and GCg are almost the same).
In addition, the 50% inhibitory concentrations (IC50) of Rf activity of theafuran 35 and theafuran 90S were 40 μg / mL for theafuran 35 and 40 μg / mL for theafuran 90S.
[0042]
Furthermore, catechins other than EGCg, ie, EC, EGC, ECg, C, GC, Cg, GCg and GA (g in the table), and theafran 35 (trade name; manufactured by Itozoen) and theafran 90S (trade name) as a catechin mixture ; Manufactured by Itoen Co., Ltd.) as well as EGCg. The effect on Kgp activity was also tested, and the results on the Kgp activity inhibition rate of each catechin are shown in FIGS. It was shown to.
[0043]
All eight catechins and catechin mixtures were able to show an activity inhibitory effect on Kgp. However, there is a large difference in the activity inhibiting ability of each catechin, and the activity inhibiting effect on Kgp is in descending order of the effect.
GC, GCg, EGCg, EGC>GA> Cg, ECg> EC, C
(Note that GC, GCg, EGCg, and EGC mean the same degree).
Further, the 50% inhibitory concentrations (IC50) of the Kgp activity of theafran 35 and theafran 90S were 50 μg / mL for theafran 35 and 150 μg / mL for the theaflan 90S. From this, it was found that the theafuran 35 was stronger than the theafuran 90S in terms of the inhibition of the activity against Kgp.
[0044]
[Table 3]

[Brief description of the drawings]
FIG. 1 is a zymograph of alkaline phosphatase produced by a black colony-forming anaerobic bacterium.
FIG. 2 shows the P.C. It is the graph which showed the inhibitory effect with respect to the alkaline phosphatase which intermedia produces.
FIG. 3 is a graph showing changes in hemolytic activity over time when each EGCg at a final concentration of 0 to 100 μg / mL is added to a reaction system.
FIG. 4: P.C. of EGCg at various concentrations. 1 is a graph showing the inhibition of Rgp produced by Gingivalis.
FIG. 5. P. of catechins at various concentrations. 1 is a graph showing the inhibition of Rgp produced by Gingivalis.
FIG. 6 shows that P. catechins at various concentrations. 1 is a graph showing the inhibition of Rgp produced by Gingivalis.
FIG. 7. P. of green tea extract at various concentrations. 1 is a graph showing the inhibition of Rgp produced by Gingivalis.
FIG. 8 is a graph showing the inhibition of the activity of catechins against Kgp at various concentrations.
FIG. 9 is also a graph showing the inhibition of the activity of catechins against Kgp at various concentrations.
FIG. 10 is a graph showing the inhibition of Kgp activity of green tea extract at various concentrations.

Claims (6)

A pathogenic cysteine protease activity inhibitor comprising catechin as an active ingredient. A pathogenic cysteine protease activity inhibitor comprising a catechin mixture as an active ingredient. The above catechin or catechin mixture includes (-) epicatechin (EC), (-) epigallocatechin (EGC), (-) epicatechin gallate (ECg), (-) epigallocatechin gallate (EGCg), (-) -) Catechin (C), (-) gallocatechin (GC), (-) catechin gallate (Cg) and (-) gallocatechin gallate (GCg), or a combination of two or more of these. The pathogenic cysteine protease activity inhibitor according to claim 1 or 2, which is a mixture. The pathogenic cysteine protease activity inhibitor according to any one of claims 1 to 3, wherein the pathogenic cysteine protease is arginine-specific cysteine protease (Rgp) or lysine-specific cysteine protease (Kgp). . An arginine-specific cysteine prosthesis (Rgp) activity inhibitor comprising any one of (-) gallocatechin (GC) and (-) gallocatechin gallate (GCg) or a mixture thereof as an active ingredient. (-) Epigallocatechin (EGC), (-) epigallocatechin gallate (EGCg), (-) gallocatechin (GC), (-) gallocatechin gallate (GCg), or two or more of these A lysine-specific cysteine prosthesis (Kgp) activity inhibitor comprising, as an active ingredient, a mixture comprising a combination of:

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