TW201124135A - Hydrogen sulfide-producing enzyme inhibitor - Google Patents

Abstract

To inhibit the generation of hydrogen sulfide, especially to provide an inhibitor of a hydrogen sulfide-producing enzyme. It is discovered that catechins have activity for inhibiting the hydrogen sulfide-producing enzyme as a result of performing the inhibition test against the production of the hydrogen sulfide by bacteria by paying attention to a green tea-derived components having no side effects, exhibiting high safety and utilized from old times. The catechins are components derived from the green tea, and exhibit the high safety and good image. As a result, the inhibitor of the hydrogen sulfide-producing enzyme contains the catechins as active ingredients.

Description

[Technical Field] The present invention relates to a hydrogen sulfide generating enzyme inhibitor which uses catechins as an active ingredient. [Prior Art] Hydrogen sulfide is a gas having an irritating taste and is known to have irritating skin mucous membranes and toxicity such as respiratory paralysis. Its toxicity is also considered a problem at the social level, and it is desirable to be able to remove hydrogen sulfide. Hydrogen sulfide is contained in volcanic gases or hot springs in nature, and is produced by the petrochemical industry. In addition, there is a case where hydrogen sulfide is produced by reduction of sulfide by the action of anaerobic bacteria or the like, and for example, anaerobic bacteria are propagated in dirt or drainage to generate hydrogen sulfide. In addition, among the bacteria producing hydrogen sulfide, there are also intraoral bacteria or intestinal bacteria that survive in the living body. These bacteria produce hydrogen sulfide from food residues or proteins in living organisms. It is even known that endogenous hydrogen sulfide is produced in living organisms. It is known that the mechanism of action of hydrogen sulfide toxicity is that the high reactivity of hydrogen sulfide causes stimulation of the skin mucosa and inhibition of cytochrome c oxidase. Since the inhibition of cytochrome c oxidase occurs rapidly, exposure to high concentrations of hydrogen sulfide rapidly reduces the partial pressure of oxygen in the lungs to faint. Further, it has been pointed out that hydrogen sulfide enhances the activity of the N M D A receptor in the brain (Non-Patent Document 1). As mentioned above, 'hydrogen sulfide will also be produced by bacteria in the mouth or bacteria in the intestines -5 - 201124135 . Hydrogen sulfide produced by bacteria in the mouth or bacteria in the intestine is relatively small compared to hydrogen sulfide produced in nature or industrial grade. However, if hydrogen sulfide is produced in the organism, the individual will become exposed to hydrogen sulfide in a very closed state, the effect of which is direct. In addition, the hydrogen sulfide produced can cause a disgusting, unpleasant odor known as bad breath or intestinal odor. Therefore, it is highly desirable to have a means to hinder or inhibit hydrogen sulfide produced by bacteria in the oral cavity or bacteria in the intestine. In addition, among the exhaled gases, within the volatile sulfide (VSC), hydrogen sulfide, methyl mercaptan and dimethyl sulfide account for about 90%, and hydrogen sulfide is one of the main causes of bad breath. In the oral cavity, exfoliated epithelial cells, white blood cells, food debris, etc., are decomposed by bacterial proteases into thiol-containing acids such as cysteine or methionine, followed by hydrogen sulfide to produce enzymes or methyl sulfide. The action of lysin produces hydrogen sulfide. It has been reported in the literature that the hydrogen sulfide producing enzyme in the oral cavity is cysteine deacetylase or cystathionine synthase. Patent Document 1 discloses a volatile sulfur compound biosynthesis inhibitor purified from Pseudomonas bacteria or Escherichia coli. However, the inhibitor is a specific aldehyde, an undecenal or an alicyclic ketone as an active ingredient, and is intended to be used in household products and cannot be safely used for a human body or the like. Patent Document 2 discloses an intestinal hydrogen sulfide reducing composition containing a water-soluble indigestible saccharide as an active ingredient. However, the effect of reducing the hydrogen sulfide in the intestine of Patent Document 2 is limited to the intestine, and the mechanism for its reduction is still unknown. Patent Document 3 discloses a composition for pain, which can inhibit and suppress the production of hydrogen sulfide in a living body. However, Patent Document 3 contains a composition for pain relief of DL-block propyl 201124135 glycine and θ-cyanoalanine, which inhibits cystathionine r-lyase </ RTI> to inhibit the production of endogenous hydrogen sulfide in the living body. The catechin derivative of Non-Patent Document 4 exhibits inhibition of protease derived from Porphyromonas gingivalis. However, there is no disclosure or suggestion regarding the inhibition of hydrogen sulfide generating enzyme. The green tea catechin of Non-Patent Document 5 exhibits inhibition of metalloproteinase. However, the suppression of the hydrogen sulfide generating enzyme is not disclosed or suggested. [Prior Art Document] [Patent Document] [Patent Document 1] JP-A-2008-173441 [Patent Document 2] JP-A-2007-99672 [Patent Document 3] Japanese Laid-Open Patent Publication No. 2007- 1 1 27 3 5 [Non-Patent Document] [Non-Patent Document 1] Abe, K, Kimura H, J. Neurosci. 16, 1 066-7 1 , 1 996 [ Non-Patent Document 2] Persson S, Edlund Μ-Β, Claesson R, Carlsson J: Oral Microbiol Immunol. 1 9 9 0; 5:1 95-20 1 [Non-Patent Document 3] Tanaka M, Yamamoto Y, Kuboniwa M, Nonaka A, Nishida N, Maeda K, Kataoka K, Nagata H, Shizukuishi S : Microbes and Infection 2007; 6: 1 078- 1 08 3 [Non-Patent Document 4] Okamoto M, Sugimoto A, Leung KP, 201124135

Nakayama K, Kamaguchi A, Maeda N: Oral Microbiol. Immunol. 2004; 19: 118-120 [Non-Patent Document 5] Demeule M, Brossard M, Page M, Gingras D, Beliveau R: Biochim Biophys Acta. 2000; 1478: [Problems to be Solved by the Invention] The present invention has an object of providing hydrogen sulfide generating enzyme inhibitors for the purpose of suppressing the generation of hydrogen sulfide. [Means for Solving the Problems] In order to solve the above problems, the present inventors have focused on green tea which has been used without any side effects and has high safety and has been used since ancient times. The components derived therefrom inhibit the generation of hydrogen sulfide by bacteria, and the results of the test are carried out. The catechins were found to have hydrogen sulfide generating enzyme inhibitory activity, and the present invention was completed. [Effect of the Invention] According to the present invention, by using catechins as an active ingredient, it is possible to effectively suppress the hydrogen sulfide generating enzyme and to inhibit or suppress the generation of hydrogen sulfide. In addition, catechins are ingredients derived from green tea, which are highly safe and have good prints. Therefore, a hydrogen sulfide generating enzyme inhibitor using catechins as an active ingredient is easily accepted by consumers. [Embodiment] The present invention provides a hydrogen sulfide generating enzyme inhibitor, wherein catechins are used as an active ingredient. In the narrow sense, catechin refers to Ci5H1406, a molecular weight of 2 9 0.2 7, a water-soluble polyvalent phenol rich in the wood core of a woody plant, and a polyphenol containing a derivative thereof in a broad sense. In the present specification, 'in order to clarify the definition, catechin refers to a narrowly defined catechin, and in the case of catechins, it refers to a series of polyphenols of catechin and its derivatives. The catechins, especially the tea catechins contained in tea, are well known. The catechins are known to have (+)-catechin, (_), epicatechin, (-)-epicatechin, (a) epicatechin, and (a)- Epigal catechin phthalate, (a)-quinone catechin phthalate, (a)-catechin phthalate and the like. Secondly, the present invention provides a hydrogen sulfide inhibitor, wherein the catechins are (-)-epicatechin phthalate, (-)-epium catechin phthalate, (1)-掊 catechin A sulfonate, (a) catechin phthalate or such a mixture is used as an active ingredient. Thirdly, the present invention provides a hydrogen sulfide generating enzyme inhibitor which is characterized by (a)-epicatechin phthalate, (a)-ep. catechin phthalate, and - catechin phthalate Any one of (a)-catechin phthalate, or a mixture thereof, as an active ingredient, and inhibiting Fusobacterium nucleatum, Porphyromonas g gingivalis, Or hydrogen sulfide producing enzyme of Treponema denticola. It is known that Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema pallidum are all -9-201124135. All of them are oral bacteria, hydrogen sulfide or methyl mercaptan have high productivity, and are also known as periodontal. Pathogens (Non-Patent Document 2, Non-Patent Document 3). The present invention further provides a food or drink comprising the aforementioned hydrogen sulfide generating enzyme inhibitor.飮Food refers to cooking foods such as raw foods, meat, fish and other animal foods, sorghum, vegetables and other plant foods, dairy products, bread, instant foods, processed foods, snacks, hobby foods, sweeteners, seasonings, etc. The present invention further provides a composition for oral cavity containing the aforementioned hydrogen sulfide generating enzyme inhibitor, using materials, health foods, special purpose foods, water, refreshing drinking water, alcoholic beverages, tea and the like, food processing materials, food additives, and the like. Things. As the oral composition, a toothpaste such as a toothpaste, a liquid toothpaste, a liquid toothpaste, a moist toothpaste, a mouthwash, an oral lotion, a throat lozenge, or a chewing gum may be used. The invention is exemplified below, and the scope of the invention is not limited by the following examples. [Example 1] An experiment was conducted on the inhibitory activity of catechins on hydrogen sulfide producing enzyme and methioninase. That is, using cysteine and methionine as a matrix, adding a crude enzyme solution from Fusobacterium nucleatum, P. gingivalis, and Treponema pallidum, mixing the respective reagents, and quantifying the methyl sulfide The product of α-ketobutyric acid and methyl mercaptan or a hydrogen sulfide generating enzyme: pyruvic acid and hydrogen sulfide, thereby measuring the inhibitory activity against methioninase and hydrogen sulfide generating enzyme. -10 - 201124135 Materials and methods 1 -1 Materials 1 ·1 2 3 -1 Sulfide gas production enzyme inhibitor candidate The following reagents were used as candidates for hydrogen sulfide production enzyme inhibitors. That is, catechins use (+)-catechin, (a) epicatechin, (-)-epicate catechin, (a)-epicatechin citrate, (a) ) - epigallocatechin phthalate, (a) - catechin phthalate, (a) - catechin phthalate (catechins from Nagara Science), and gallic acid, chlorine Zinc. These inhibitor candidates have the following abbreviations in this specification: (+)-catechin: C, (a)-epicatechin: EC, (_)-epicatechin: EGC , (-)- epicatechin phthalate: ECg, (a) - epigallocatechin phthalate: EGCg, (-)-掊 catechin phthalate: GCg, (a) - catechu Sulfate: Cg, gallic acid: G, zinc chloride: ZnCl2. Gallic acid is known to have bactericidal activity, and derivatives thereof are used as an oral composition. Zinc chloride is known to have a bad breath preventing activity and is used as a mouthwash. -11 - 1 -1 - 2 Use strain 2 Hydrogen sulfide to produce bacteria, using Fusobacterium nucleatum (in the present specification, abbreviated as F. η), and gingivalis (abbreviated as Pg in this specification) Case), Treponema pallidum (in the present specification, abbreviated as T · d). The strain C. nucleus was used for the strain C C 8 8 3 2, the strain W83 was used for P. gingivalis, and the strain of ATCC accession number 3 5405 was used for Treponema pallidum. 201124135 1 - 2 Test method 1-2-1 Preparation of crude enzyme solution from each strain F.n was inoculated into Trypticase Soy Broth (TSB) 15ml, and anaerobic culture was carried out until the growth reached a fixed period. In addition, Trypticase Soy Broth (TSB) was prepared by dissolving Trypticase Soy Broth ( DIFCO) 3g and Yeast Extract ( DIFCO ) 0.3g in 100ml of water, then adding hemin (5mg/ml in 0.1N NaOH) 0.1ml with menadione (0.5mg/ Ml in 50% EtOH) 0·1 ml and prepared. The above culture was used as the preculture, and then further cultured in 200 ml of TSB, and anaerobic culture was carried out at 37 °C from the beginning of the growth period to 24 hours (2 days after the culture period). Next, the culture solution was centrifuged at 3,000 x g, 10 min, and 4 ° C to remove the medium components, and the cells were collected. Thereafter, 50 mM Tris-HCl buffer (ρΗ7·5 ) was added (the trishydroxymethylaminomethane (molecular weight 121) 6. 〇 5 g was dissolved in water, and adjusted to pH 7.5 with HC1, and the amount was adjusted to 1 L. After further centrifuging and washing the cells, the buffer was removed, and the cells were frozen at -80 °C. The cells were further suspended in 20 to 25 ml of Tris-HCl buffer, and the cell membrane was disrupted by ultrasonic waves (20 W, 30 sec x 15 hours, 〇 ° C ). It was centrifuged at 20,000 x g, 30 min, and 4 ° C, and the above supernatant was used as a crude enzyme solution. The crude enzyme solution was quantified using a DC Protein Assay kit (Pierce) and diluted to a final concentration of 0.3 mg protein/ml in the test system. P.g was cultured in TSB medium in the same manner as F.n. In addition, T.d was cultured in -12-201124135 TYGVS medium. The cultivation time is 7 days. The modulation of the P.g and T.d crude enzyme solutions was carried out in the same manner as F.n. In addition, the modulation of the TYGVS medium is as follows. The following I liquid ~ 111 liquid is further mixed with the inactivated rabbit serum 10 〇 m 1 to prepare the liquid I: heart infusion broth 5g, tryptone 1 〇g, yeast extract lOg, gelatin lOg, ammonium sulfate 〇. 5g K2HP04 〇_5g, MgS04 O.lg was made into a 750 ml aqueous solution, adjusted to pH 7.0, and autoclaved at 121 t for 15 minutes. Liquid II: L-cysteine HC1 lg, glucose lg, sodium pyruvate 〇. 25 g, VFA solution (1.7 ml of acetic acid, 0.6 ml of propionic acid, ml. 4 ml of ortho-tyrosine, 0.1 ml of valeric acid, Isocetic acid 0.1ml, isobutyric acid 0.1ml, DL-metylbutyric acid 0.1ml and distilled water 27_9ml force to mix) 5ml, TPP solution (containing 0.25g of thiamine pyrophosphate and adding 100ml of distilled water and mixing) 5ml, 1 -N KOH 1 6 ml was mixed, further added with water to prepare a 50 ml aqueous solution, adjusted to pH 7.2, and sterilized by filtration. -13- 201124135 III Solution: Dissolve the pectin lg in 100 ml of distilled water and autoclave at 121 °C for 15 minutes. 1_2-2 Hydrogen sulfide production enzyme (cysteine deacetylase) inhibition by colorimetric assay One of the enzymes for the production of hydrogen sulfide: a cysteine deaminase capable of producing pyruvate from a hemi-caloric acid , ammonia and hydrogen sulfide. In the hydrogen sulfide production enzyme inhibition test by colorimetric quantification, the amount of pyruvic acid produced by cysteine deaminase was used as an indicator to compare the production of hydrogen sulfide by each reagent. The inhibitory activity of the enzyme. Preparation of solution A 300 mM cysteine solution, an enzyme-coenzyme mixed solution, a sample solution, and a 3-methyl-2-benzothiazolinone oxime (MB TH) reaction solution were prepared in the following manner. 300 mM cysteine solution: The cysteine was diluted to 300 mM in Tris-HCl buffer. Enzyme-coenzyme mixed solution: The amount of protein in the crude enzyme solution in the reaction system was adjusted to 0.3 mg/ml and pyridoxal-5-phosphate to 0.05 mM in Tris-HCl buffer before the test was started. Sample solution: Dilute zinc chloride or catechins into various concentrations in Tris-HCl buffer. 3-Methyl-2·benzothiazolinone oxime (MBTH) reaction solution: dissolve -14- 201124135 MBTH 30mg In 50 ml of distilled water. Thereafter, 100 ml of a 1 M sodium acetate buffer solution (ρ Η 5 · 0) was added (82.03 g of sodium acetate was dissolved in water, and adjusted to pH 5.0 with acetic acid, and the amount was adjusted to 1 L). Quantification of pyruvate In 0.1 ml of 300 mM cysteine solution and 2 ml of sample solution, add enzyme-coenzyme mixed solution 〇 7 m 1, oscillate at 37 °C. After 1 hour, 0.5 ml of 6% perchloric acid was added to stop the reaction, and after centrifugation (3,000 x g, 1 minute, 4 °C), the supernatant was recovered. 1.2 ml of the MBTH reaction solution was added to 4 ml of the supernatant, and it was allowed to react at 50 ° C for 30 minutes. The reaction solution was diluted 3 times with a sodium acetate buffer, and the absorbance at 3 3 5 n m was measured. All tests were performed twice and averaged. In addition, the production of pyruvic acid calibration lines was also carried out. Inhibition rate The amount of pyruvic acid when the sample solution was added was determined as Pi, and the amount of pyruvic acid when Tris-HCl buffer was added instead of the inhibitor solution was determined as P(), and the inhibition rate was determined by the following method. Inhibition rate (%) = (P〇-Pi) /PqX1〇〇-15- 201124135 In the acidase inhibition test, the amount of α-ketobutyric acid produced by methioninase was used as an indicator to compare the test drug pairs. Inhibitory activity of methioninase. Preparation of reagents A 300 mM methionine solution was prepared as described below. The preparation of the enzyme-coenzyme mixed solution, the sample solution, and the 3-methyl-2-benzothiazolinone oxime (MBTH) reaction solution was carried out in the same manner as above. 3 00 mM methionine solution: The methionine was diluted to 300 mM in Tris-HCl buffer. Quantification of α-ketobutyric acid

0.1 ml of a 3.0 mM methionine solution and 0.2 ml of a sample solution were added to an enzyme-coenzyme mixed solution 77 ml, and shaken at 37 °C. After 1 hour, 0.5 ml of 6% perchloric acid was added to stop the reaction, and after centrifugation (3,000 x g, 10 minutes, 4 ° C), the supernatant was recovered. 1.2 ml of the MBTH reaction solution was added to 0.4 ml of the supernatant, and the mixture was reacted at 50 ° C for 30 minutes. The reaction solution was diluted 3 times with a sodium acetate buffer, and the absorbance at 3 35 nm was measured. All tests were performed twice and averaged. In addition, the production of 彳7 α-keto acid calibration line was also carried out. The inhibition rate was determined by adding the amount of α-ketobutyric acid to the sample solution to Ki, and the amount of α-ketobutyric acid when the Tris-HC1 buffer was added instead of the inhibitor solution was determined to be Kq, and the methioninase was determined by the following method. The inhibition rate. -16- 201124135

Inhibition rate (%) = (K〇-Ki) /KoxlOO 1 -2-4 Hydrogen sulfide production enzyme inhibition test by gas chromatography The hydrogen sulfide production enzyme is capable of producing hydrogen sulfide from cysteine. In the hydrogen sulfide production enzyme inhibition test by gas chromatography, cysteine was used as a substrate, and the amount of hydrogen sulfide produced by the hydrogen sulfide generating enzyme was used as an index to compare the inhibition of hydrogen sulfide generating enzyme by each reagent. active. Quantification of hydrogen sulfide Add L-cysteine solution to the test tube 0 · 1 m 1 , sample (inhibitor candidate) solution 〇 2 m 1 , enzyme-coenzyme mixed solution 0 · 7 m 1, sealed with rubber plug Stir in a water bath at 3 7 °C while keeping warm. After 60 minutes, a 3 Torr aqueous phosphoric acid solution was added to stop the reaction, and the mixture was further kept at 37 ° C and stirred for 10 minutes. The headspace gas was 15 yl in the case of F.n, 1 0 0 1 in P.g, and 50/z丨 in T·d, and the amount of hydrogen sulfide was measured by gas chromatography. In addition, the analysis conditions are as follows. Column: HP-PLOT/Q (ψ 0.53mmx30m) Temperature: 70°C (2.5min)—heating at 30°C/min to 190°C — 190 °C (3.5min)

Detector: FPD split ratio: 4 8 : 1 Injection volume: 15, 50 and 1 barium 1 1 - 2 - 5 methionin inhibition test by gas chromatography -17- 201124135 The enzyme inhibitory activity of methioninase serves as a control for hydrogen sulfide producing enzymes. The methioninase uses methionine as a substrate to produce methyl mercaptan, ammonia and hydrazine: ketobutyric acid. In the methioninase inhibition test by gas chromatography, the inhibitory activity of each reagent for methioninase was compared by using the amount of methyl mercaptan produced by methioninase as an indicator. Quantification of methyl mercaptan In the test tube, L·methionine solution 0·lml, sample solution 0.2 ml, and enzyme-coenzyme mixed solution 〇.7 ml were added to the test tube, sealed with a rubber plug, and stirred at 37 ° C in a water bath while keeping warm. After 60 minutes, a 3 M aqueous phosphoric acid solution was added, and after the reaction was stopped, the mixture was further kept at 37 ° C and stirred for 10 minutes. The top air gas was taken, and in the case of F.n, 40 μl, P.g and T.d were 200 //1, and the amount of methyl mercaptan was determined by gas chromatography. In addition, the analysis conditions are as described above. 2-3 results

2-3-1 Hydrogen sulfide generating enzyme (cysteine deacetylase) and methioninase inhibition test results of F. η produced by catechins by colorimetric quantification will be 1-2- The results of 2 are disclosed in Tables 1 and 2, and the results of 1-2-3 are disclosed in Table 3. The order of the inhibitory activity of the catechins on the hydrogen sulfide generating enzyme (cysteine deacetylase) of Fn is Cg&gt;GCg&gt;EGCg&gt;ECg&gt; gallic acid, (+)-C, especially in Cg, GCg , EGCg and ECg have high inhibitory activity. The inhibitory activity of catechins on F.n methioninase is EGCg &gt; ECg &gt; EGC > EC, gallic acid, C, and particularly high inhibitory activity in EGCg and ECg. The IC50 at this time is 0.4 mM -18- 201124135 in EGCg ' at E C g is _ _ 8 m Μ. [Table 1] Table 1 Various catechins for the inhibition of Fn hydrogen sulfide producing enzyme (cysteine depurination _____^ enzyme) _ ----- MH#· / HV J 1 inhibitor concentration Inhibition rate (%) UM) Gallic acid (+)-C EC EGC ECfi EGCg 1.50 4.6 1.6 0.1 0.0 40.3 53.2 [Table 2] Table 1 Inhibition rate of various catechins for F. __碁 enzyme) η hydrogen sulfide Enzyme production (cysteine depurination rate inhibitor concentration (mM) Cg inhibition rate (%) GCg EGCg 78.8 68.1 54 [Table 3] Inhibitory concentration of fish catechins on Fn methioninase Inhibition rate (%) (mM) Gallic acid (+)-C EC EGC ECg EGCg 2.00 10.7 8.1 11.0 25.3 83.8 82.1 1.00 9.9 5.7 8.2 17.9 63.8 75.1 0.44 9.6 2.7 5.1 12.3 29.3 50.5 0.22 9.2 2.1 4.4 6.6 15.5 26.4 _____〇 JJ 7.9 — 3.4 2.1 10.9 13.2 -19- 1 -2-2 Enzyme inhibition of En, Pg and Td by EGCg by colorimetry 2 Test 201124135 Test results of enzyme inhibition of 1-2-2 hydrogen sulfide production and 1 A comparison of EGCg with zinc chloride inhibitory activity in the 2-3 methioninase inhibition test is disclosed in Figure 1. Further, 1C 50 is disclosed in Table 4. In Fig. 1, the white point is EGCg, and the black spot is the result of zinc chloride. Further, it is known that zinc chloride has a bad breath preventing activity and is expected to inhibit hydrogen sulfide generating enzyme and methioninase. From the results of Fig. 1, it appears that, with respect to the inhibition of methioninase, the inhibitory activity by chlorination is higher than that of EGCg in any of the cases from Fn, Pg and Td. Regarding the production of hydrogen sulfide, in any of the cases derived from Fn, Pg and Td, the inhibitory activity by EGCg is higher than that of zinc chloride. The same results can be obtained from Table 4. 4] Table 4 IC50 (colorimetric analysis) of crude enzyme solution from F. nucleatum JCM8532, P.gingivalis W83 and T.denticola ATCC3 5405 methionine enzyme hydrogen sulfide generating enzyme IC50 (mM) zinc chloride EGCg chlorination Zinc EGCg Fn JCM8532 0.038 0.363 2.080 1.336 Pg W83 0.007 0.131 1.571 0.189 Td ATCC35405 0.009 0.259 2.223 0.199 2-3-3 EGCg by gas chromatography The enzyme inhibition test for Fn, Pg and Td will be 1-2-4. Hydrogen sulfide production enzyme inhibition test 1-2-5 inhibition of methionine results disclosed in FIG. In addition, the 1C 5 0 system is disclosed in Table 5. From the results of Fig. 2, the inhibition of methioninase 'in any case from Fn-20-201124135, Pg and Td' has an inhibitory activity by zinc chloride higher than that produced by EGC g. Inhibition activity. On the other hand, regarding the production of hydrogen sulfide, in any of the cases from Fn, Pg and Td, the inhibitory activity produced by EGCg is higher than that of the inhibitory activity produced by chloranilated zinc. Obtain the same knot # ° [Table 5] Table 5 IC50 of the crude enzyme solution from F. nucleatum JCM8532, P.gingivalis W83 and T.denticola ATCC3 5405 (gas chromatography analysis) methionine hydrogen sulfide generating enzyme IC50 ( mM ) zinc chloride EGCg zinc chloride EGCg Fn JCM8532 0.039 0.351 2.369 1.106 Pg W83 0.003 0.078 1.840 0.136 Td ATCC35405 0.009 0.175 1.812 0.133 2 - 4 Summary of Example 1 From the above, the GCg of the catechins, Cg, ECg and EGCg have hydrogen sulfide generating enzyme inhibitory activity. In particular, it is apparent that it has an inhibitory activity against a hydrogen sulfide-producing enzyme: cysteine deaminase. It is further known that the activity of E G C g is significantly higher than that of zinc chloride. It is known that hydrogen sulfide of e G C g produces an enzyme inhibitory activity, and is highly active in a crude enzyme solution derived from P.g and T.d, and also exhibits activity in a crude enzyme solution derived from F.n. [Example 2] A toothpaste, an oral spray, a throat lozenge, a chewing gum, a candy, and a soft drink containing a hydrogen sulfide-producing enzyme inhibitor composed of the catechins of the present invention were produced by a hanging method. These formulations are disclosed below. In addition, the scope of the invention is not limited by the scope of the invention. Toothpaste formula Calcium carbonate 50.0% by weight Glycerin 20.0 Carboxymethylcellulose 2.0 Sodium lauryl sulfate 2.0 Perfume 1.0 Saccharin 0.1 EGCg 1.0 Chlorhexidine 0.01 _ rest 100.0 Formulation of oral spray Ethanol 1 0.0% by weight Glycerol 5.0 Perfume 0.05 Colorant 0.00 1 ECg 1 .0 The rest of the water 100.0 -22- 201124135 Formulation of throat lozenges Glucose 7 3 . 3 wt % Lactose 16.0 Acacia 6.0 Perfume 1.0 Sodium monofluorophosphate 0.7 GCg 1.0 Lactose 2.0 100.0 Formula of chewing gum Gum base 2 0.0% by weight Sugar 54.7 Glucose 15.3 Water 9.3 EGCg 0.2 Perfume 0.5 100.0 Confectionery formula sugar 5 0.0% by weight Otter 34.0 Citric acid 2.0 EGCg 0.6 -23- 201124135 Perfume 0.2 The rest of the water 100.0 Formulation of soft candy gelatin 6 0.0wt% leeches 20.5 granulated sugar 8.5 vegetable oil 4.5 mannitol 3.0 malic acid 2.0 Cg 1.0 fragrance 0.5 100.0 beverage formula orange juice 3 0.0% by weight isomerized sugar 15.24 citric acid 0.10 vitamin C 0.04 fragrance 0.10 ECg 0.10 water remaining part 100.0 -24 - 201124135 [Simplified Schematic Description] Fig. 1A shows the results of inhibition activities of EGCg and zinc chloride on cysteine deacetylase and methioninase by colorimetric analysis. Figure 1 A is the result of F. η. Fig. 1A shows the results of inhibition activities of EGCg and zinc chloride on cysteine deacetylase and methionase by colorimetric analysis. Figure 1 B is the result of P . g. Fig. 1C shows the results of inhibition activities of EGCg and zinc chloride on cysteine deacetylase and methionase by colorimetric analysis. Figure 1 C is the result of T.d. Fig. 2A shows the results of the inhibitory activities of e g C g and zinc chloride on the hydrogen sulfide generating enzyme and methioninase by analysis by gas chromatography. Figure 2A is the result of F.n. Fig. 2B shows the results of the inhibitory activities of E G C g and zinc chloride on the hydrogen sulfide generating enzyme and methioninase by analysis by gas chromatography. Figure 2 B is the result of P.g. Fig. 2C shows the results of the inhibitory activities of E G C g and zinc chloride on the hydrogen sulfide generating enzyme and methioninase obtained by the analysis by gas chromatography. Figure 2 C is the result of T.d. -25-

Claims (1)

201124135 VII. Patent application scope: 1. A hydrogen sulfide generating enzyme inhibitor characterized by using catechins as an active ingredient. 2. The hydrogen sulfide generating enzyme inhibitor according to the first aspect of the patent application, wherein the catechins are (a)-epicatechin phthalate, (-)-epium catechin phthalate, a) - any of catechin phthalate, (i)-catechin phthalate or mixtures thereof. 3. A hydrogen sulfide producing enzyme inhibitor characterized by: (-)-epicatechin phthalate, (a)-ep. catechin phthalate, (_)-掊 catechin 掊Any one of the acid esters, catechins and the like, or the mixture is an active ingredient, and inhibits Fusobacterium nucleatum, Porphyromonas gingivalis, or tartar dense Hydrogen sulfide producing enzyme of Treponema denticola. A food or drink comprising the hydrogen sulfide generating enzyme inhibitor according to any one of claims 1 to 3. An oral composition comprising the hydrogen sulfide generating enzyme inhibitor according to any one of claims 1 to 3. -26-