201124135 六、發明說明: 【發明所屬之技術領域】 本發明關於一種硫化氫產生酶抑制劑,係以兒茶素類 作爲有效成分。 【先前技術】 硫化氫是具有刺激性味道的氣體,已知具有刺激皮膚 黏膜,還有引起呼吸麻痺等的毒性。其毒性在社會層面亦 被視爲問題’而希望能夠進行硫化氫的除去。硫化氫在自 然界是被含於火山氣體或溫泉等之中,人爲生產則是藉由 石油化學工業等方式。另外,也會有因爲厭氣性細菌等的 作用使硫化物被還原而產生硫化氫的情形,例如厭氣性細 菌在污物或排水等之中繁殖而產生硫化氫。另外,在產生 硫化氫的細菌之中,也有口腔內細菌或腸內細菌在生物體 內生存。該等細菌係以食物殘渣或生物體內的蛋白質作爲 原料而生產硫化氫。甚至還已知在生物體內會產生內因性 硫化氫。 已知硫化氫毒性的作用機制,係硫化氫的高反應性造 成對皮膚黏膜的刺激,以及對細胞色素C氧化酶的抑制。 由於細胞色素C氧化酶的抑制作用會急速發生,因此暴露 於高濃度硫化氫的情況,肺中的氧分壓急速降低以至於昏 倒。另外還有文獻指出硫化氫在腦內會增強N M D A受體的 活性(非專利文獻1 )。 如上所述’硫化氫也會由口腔內細菌或腸內細菌產生 -5- 201124135 。由口腔內細菌或腸內細菌所產生的硫化氫,與自然界或 工業級生產的硫化氫相比規模較小。然而,如果在生物體 內產生硫化氫,該個體會變成在非常封閉的狀態下暴露於 硫化氫,其影響爲直接性的。另外,所產生的硫化氫,會 導致被稱爲口臭或腸內臭氣這些令人厭惡、不愉快的氣味 。因此,正殷切期望有辦法阻礙或抑制由口腔內細菌或腸 內細菌所產生的硫化氫。另外,在呼出的氣體中,揮發性 硫化物(VSC )之內,硫化氫、甲硫醇及二甲基硫占了約 9 0%,硫化氫可說是口臭的主要原因之一。 在口腔內,脫落的上皮細胞或白血球、食物殘渣等, 會被來自於細菌的蛋白酶分解成半胱氨酸或甲硫胺酸等含 硫胺基酸,其後因爲硫化氫產生酶或甲硫氨酸酶的作用而 產生硫化氫。已有文獻指出在口腔內的硫化氫產生酶爲半 胱氨酸脫毓基酶或胱硫醚合成酶等。 專利文獻1揭示了由假單胞菌屬細菌或大腸菌精製的 揮發性硫化合物生合成抑制劑。然而,該抑制劑係以特定 的醛、十一烯醛、脂環式酮等香料作爲有效成分,目的爲 使用於家庭用品,並不能安全地對人體等使用。 專利文獻2揭示了 一種腸內硫化氫減低組成物,其中 含有水溶性難消化性糖質作爲有效成分。然而,專利文獻 2的腸內硫化氫減低效果只限於腸內,關於其減低的機制 仍然不明。 專利文獻3揭示了一種鎭痛用組成物,能夠阻礙、抑 制生物體內硫化氫的產生。但是,專利文獻3含DL-块丙基 201124135 甘胺酸及θ -氰基丙胺酸的鎭痛用組成物,係抑制胱硫醚 r -裂解酶’以抑制生物體內產生內因性硫化氫。 非專利文獻4的兒茶素衍生物,對於來自牙齦卟啉單 胞菌的蛋白酶表現出抑制。但是關於硫化氫產生酶的抑制 ,並無揭示或者提示。 非專利文獻5的綠茶兒茶素,對於金屬蛋白酶表現出 抑制。 但是關於硫化氫產生酶的抑制,亦並無揭示或者提示 [先前技術文獻] [專利文獻] [專利文獻1]日本特開2008-173441號公報 [專利文獻2]日本特開2007-99672號公報 [專利文獻3]日本特開2007- 1 1 27 3 5號公報 [非專利文獻] [非專利文獻 1] Abe, K,Kimura H,J. Neurosci. 16, 1 066-7 1 , 1 996 [非專利文獻 2] Persson S, Edlund Μ-Β, Claesson R, Carlsson J :Oral Microbiol Immunol. 1 9 9 0; 5:1 95-20 1 [非專利文獻 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 [非專利文獻 4] Okamoto M,Sugimoto A,Leung K-P, 201124135[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 [非專利文獻 5] Demeule M,Brossard M,Page M, Gingras D, Beliveau R: Biochim Biophys Acta. 2000; 1478: 51-60. 【發明內容】 [發明所欲解決之課題] 本發明以抑制硫化氫的產生爲目的,特別是提供硫化 氫產生酶抑制劑作爲課題。 [用於解決課題之方法] 本發明人等爲了解決上述課題,注目於無副作用、安 全性高、自古以來一直被使用的綠茶,來自其中的成分會 抑制細菌的硫化氫產生,實施測試的結果,發現兒茶素類 具有硫化氫產生酶抑制活性,而完成本發明。 [發明之效果] 依據本發明’藉由使用兒茶素類作爲有效成分,能夠 有效地抑制硫化氫產生酶,可阻礙或抑制硫化氫的產生。 另外,兒茶素類爲來自於綠茶的成分,安全性高、印 象良好。因此,以兒茶素類作爲有效成分的硫化氫產生酶 抑制劑容易被消費者接受。 201124135 【實施方式】 本發明提供一種硫化氫產生酶抑制劑’係以兒茶素類 作爲有效成分。 兒茶素(catechin)狹義而言’是指Ci5H1406、分子 量2 9 0.2 7,木本植物的木芯所富含的水溶性多價酚,廣義 而言包含其衍生物的多酚。在本說明書中’爲了使定義明 確,兒茶素是指狹義的兒茶素,在兒茶素類這樣的情況, 是指兒茶素及其衍生物一系列的多酚。兒茶素類,尤其以 茶中所含的茶兒茶素爲所周知。兒茶素類已知有(+ )-兒茶素、(_)·表兒茶素、(―)-表掊兒茶素、(一)· 表兒茶素掊酸酯、(一)-表掊兒茶素掊酸酯、(一)-掊 兒茶素掊酸酯、(一)-兒茶素掊酸酯等。 第二,本發明提供一種硫化氫抑制劑,其中兒茶素類 係(—)-表兒茶素掊酸酯、(-)-表掊兒茶素掊酸酯、 (一)-掊兒茶素掊酸酯、(一)·兒茶素掊酸酯或該等混 合物作爲有效成分。 第三,本發明提供一種硫化氫產生酶抑制劑,係以( 一)-表兒茶素掊酸酯、(一)-表掊兒茶素掊酸酯、 )-掊兒茶素掊酸酯、(一)-兒茶素掊酸酯之任一者、或 該等混合物作爲有效成分,並抑制來自於具核梭桿菌( Fusobacterium nucleatum )、牙銀卩卜啉單胞菌( Porphyromonasg gingivalis )、或齒垢密螺旋體( Treponema denticola)的硫化氫產生酶。 已知具核梭桿菌、牙齦卟啉單胞菌、齒垢密螺旋體任 -9- 201124135 一者皆爲口腔內細菌,硫化氫或甲硫醇的生產能力高,另 外還已知其爲牙周病菌(非專利文獻2、非專利文獻3 )。 本發明另外還提供一種飲食品,係含有前述硫化氫產 生酶抑制劑。飮食品是指包含生鮮食品、肉、魚等動物性 食品、榖物、蔬菜等植物性食品、乳製品、麵包、即時食 品等加工食品、點心類等嗜好食品、甘味料、調味料等烹 飪調味用材料、健康食品,特殊用途食品、水、清涼飲用 水、酒精飲料、茶等飲料、食品加工材料、食品添加物等 〇 本發明進一步還提供一種含有前述硫化氫產生酶抑制 劑之口腔用組成物。口腔用組成物,可採用牙膏、液體牙 膏、液狀牙膏、濕潤牙膏等牙膏類、漱口劑、口腔洗劑、 喉錠劑、口香糖等形態。 以下對本發明舉例說明,而本發明之範圍並不受以下 範例侷限。 [實施例1 ] 進行關於兒茶素類對硫化氫產生酶及甲硫氨酸酶酶抑 制活性的實驗。亦即,使用半胱氨酸及甲硫胺酸作爲基質 ,加入來自於具核梭桿菌、牙齦卟啉單胞菌、齒垢密螺旋 體的粗酵素液,將各試藥混合,並且定量甲硫氨酸酶的生 成物:α-酮酪酸及甲硫醇、或硫化氫產生酶的生成物: 丙酮酸及硫化氫,藉此測定對甲硫氨酸酶及硫化氫產生酶 的抑制活性。 -10 - 201124135 材料及方法 1 -1材料 1 ·1 2 3 -1硫化氣產生酶抑制劑候選 使用以下試藥作爲硫化氫產生酶抑制劑候選。亦即, 兒茶素類係使用( + )-兒茶素、(一)·表兒茶素、(-)-表掊兒茶素、(一)-表兒茶素掊酸酯、(一)-表掊兒 茶素掊酸酯、(一)-掊兒茶素掊酸酯、(一)-兒茶素掊 酸酯(兒茶素類由Nagara Science購得)、及没食子酸、 氯化鋅。該等抑制劑候選,在本說明書之中有採用以下簡 記的情形,(+ )-兒茶素:C、(一)-表兒茶素:EC、 (_ )-表掊兒茶素:EGC、(—)-表兒茶素掊酸酯: ECg、(一)-表掊兒茶素掊酸酯:EGCg、(—)-掊兒茶 素掊酸酯:GCg、(一)-兒茶素掊酸酯:Cg、没食子酸: G、氯化鋅:ZnCl2。没食子酸已知有殺菌活性,其衍生物 被使用作爲口腔用組成物。氯化鋅已知有口臭預防活性, 而被使用於漱口劑。 -11 - 1 -1 - 2使用菌株 2 硫化氫產生細菌,係使用具核梭桿菌(本說明書中有 簡記爲F . η的情形)、牙齦卟咐單胞菌(本說明書中有簡 記爲P.g的情形)、齒垢密螺旋體(本說明書中有簡記爲 T · d的情形)。具核梭桿菌使用j C μ 8 5 3 2菌株,牙齦卟啉單 胞菌使用W83菌株,齒垢密螺旋體使用ATCC寄存編號 3 5405之菌株。 201124135 1 - 2測試方法 1-2-1由各菌調製粗酵素液 將 F.n 植菌於 Trypticase Soy Broth ( TSB) 15ml,進行 厭氣培養直到生長達定常期。另外,Trypticase Soy Broth (TSB)係使 Trypticase Soy Broth ( DIFCO) 3g 與 Yeast Extract ( DIFCO ) 0.3g 溶於 100ml 水之後,加入 hemin ( 5mg/ml in 0.1N NaOH) 0.1ml 與 menadione ( 0.5mg/ml in 50%EtOH ) 0· 1ml而調製。 以上述培養作爲前培養,然後進一步植菌於TSB200ml ,從生長達定常期開始至24小時後(培養期間2days ), 在3 7 °C進行厭氣培養。 接下來,以3,000xg、lOmin、4°C將培養液離心分離 ,除去培養基成分,回收菌體。其後加入50mM Tris-HCl 緩衝液(ρΗ7·5 )(使三羥甲基胺基甲烷(分子量121 ) 6.〇5g溶於水,並以HC1調整成PH7.5後,定量爲1L而調製 ),進一步離心並將菌體洗淨後,除去緩衝液,將菌體冷 凍於-80 °C。進一步使菌體懸浮於20〜25ml的Tris-HCl緩 衝液中,以超音波將細胞膜打碎(20W,30secxl5times, 〇°C )。 以20,000xg、30min、4°C將其離心,以上清液作爲粗 酵素液。粗酵素液係使用DC Protein Assay kit ( Pierce) 進行蛋白質定量,稀釋成在測試系統中最終濃度爲0.3 mg protein/ml而使用。 與F.n相同地,以TSB培養基培養P.g。另外,T.d係以 -12- 201124135 TYGVS培養基進行培養。培養時間爲7天。關於P.g及T.d 粗酵素液之調製,係與F.n相同的方式進行。 另外,TYGVS培養基之調製如以下所述。亦即將以下 I液〜111液進一步與不活化的兔血清1 0 〇m 1加以混合而調製 I液: 將 heart infusion broth 5g、 tryptone 1 〇g、 yeast extract lOg、明膠 lOg、硫酸銨 〇.5g、K2HP04 〇_5g、 MgS04 O.lg製成750ml之水溶液,調整成pH7.0後,在121 t高壓滅菌1 5分鐘。 II液: 將L-半胱氨酸HC1 lg、 葡萄糖lg、 丙酮酸鈉〇 . 2 5 g、 VFA液(將醋酸1.7ml、丙酸0.6ml、正酪酸〇.4ml、正 纈草酸0.1ml、異纈草酸0.1ml、異酪酸0.1ml、DL-metylbutyric acid 0.1ml及蒸餾水 27_9ml力□以混合)5ml、 TPP液(於硫胺素焦磷酸0.25 g添加蒸餾水100ml並混 合)5ml、 1 -N KOH 1 6ml 加以混合,進一步加水製成50ml水溶液,調整成 PH7.2後,過濾滅菌。 -13- 201124135 III 液: 使果膠lg溶於蒸餾水100ml,並在121 °c高壓滅菌15分 鐘。 1_2-2以比色定量進行的硫化氫產生酶(半胱氨酸脫毓基酶 )抑制測試 硫化氫產生酶的一種:半胱氨酸脫锍基酶,能夠由半 腕氣酸產生丙酮酸、氨及硫化氫。在以比色定量進行的硫 化氫產生酶抑制測試中,係以半胱氨酸爲基質,由半胱氨 酸脫毓基酶所產生的丙酮酸量作爲指標,比較各試藥對硫 化氫產生酶的抑制活性。 溶液之調製 如以下方式調製出300mM半胱氨酸溶液、酶-輔酶混 合溶液、試樣溶液、3-甲基-2-苯并噻唑啉酮腙(MB TH) 反應溶液。 300mM半胱氨酸溶液:以Tris-HCl緩衝液將半胱氨酸 稀釋成爲300mM。 酶-輔酶混合溶液:在正要開始測試之前,以Tris-HC1緩衝液將反應系統中粗酵素液之蛋白質量調製成爲 〇.3mg/ml、5·-磷酸吡哆醛成爲0.05mM。 試樣溶液:以Tris-HCl緩衝液將氯化鋅或兒茶素類稀 釋成各濃度》 3-甲基-2·苯并噻唑啉酮腙(MBTH )反應溶液:使 -14- 201124135 MBTH30mg溶於50ml蒸餾水。其後,添加1M醋酸鈉緩衝液 100ml ( ρΗ5·0)(使醋酸鈉82.03g溶於水,並以醋酸調整 成PH5.0之後,定量爲1L而調製)。 丙酮酸之定量 在3 00mM半胱氨酸溶液0.1 ml及試樣溶液〇.2ml加入酶 —輔酶混合溶液〇 . 7 m 1,在3 7 °C震盪。1小時後,加入6 %過 氯酸0.5ml,使反應停止,離心分離(3,000xg、1〇分鐘、4 °C )後,回收上清液。 於上清液〇.4ml添加MBTH反應溶液1.2ml,使其在50 °C反應3 0分鐘。以醋酸鈉緩衝液將反應液稀釋3倍,測定 3 3 5 n m的吸光度。全部的測試進行2次,求其平均値。另外 還進行丙酮酸檢量線的製作。 抑制率 將加入試樣溶液時的丙酮酸量定爲Pi,加入Tris-HCl 緩衝液代替抑制劑溶液時的丙酮酸量定爲P(),由以下方式 求得抑制率。 抑制率(% ) = ( P〇- Pi ) /PqX1〇〇 -15- 201124135 酸酶抑制測試中,係由甲硫氨酸酶產生的α -酮酪酸之量 作爲指標,比較各試藥對甲硫氨酸酶的抑制活性。 試藥之調製 如以下所述方式,調製出3 00mM甲硫胺酸溶液。關於 酶-輔酶混合溶液、試樣溶液、3-甲基-2-苯并噻唑啉酮腙 (MBTH )反應溶液之調製,係與上述相同的方式進行。 3 00mM甲硫胺酸溶液:以Tris-HCl緩衝液將甲硫胺酸 稀釋成300mM。 α -酮酪酸之定量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
在3 OOmM甲硫胺酸溶液0.1 ml及試樣溶液0.2ml加入酶 —輔酶混合溶液〇.7ml,在37°C震盪。1小時後,加入6%過 氯酸0.5ml使反應停止,離心分離( 3,000xg、10分鐘、4°C )後,回收上清液。 在上清液0.4ml添加MBTH反應溶液1.2ml,使其在50 °C反應30分鐘。以醋酸鈉緩衝液將反應液稀釋3倍,測定 在3 3 5nm的吸光度。全部的測試進行2次,求其平均値。另 外還進彳7 α-酮酷酸檢量線的製作。 抑制率 將加入試樣溶液時的α -酮酪酸量定爲Ki,加入Tris-HC1緩衝液以代替抑制劑溶液時的α -酮酪酸量定爲Kq,由 以下方式求得甲硫氨酸酶的抑制率。 -16- 2011241350.1 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
抑制率(%) = (K〇-Ki) /KoxlOO 1 -2-4藉由氣相層析進行的硫化氫產生酶抑制測試 硫化氫產生酶能夠由半胱氨酸產生硫化氫。在藉由氣 相層析進行的硫化氫產生酶抑制測試中,係以半胱氨酸作 爲基質,由硫化氫產生酶產生的硫化氫量作爲指標,比較 各試藥對硫化氫產生酶的抑制活性。 硫化氫的定量 在試管中加入L -半胱氨酸溶液0 · 1 m 1、試樣(抑制劑 候選)溶液〇 . 2 m 1、酶一輔酶混合溶液0 · 7 m 1,以橡膠栓密 封,在3 7 °C水浴攪拌同時保溫。6 0分鐘後,加入3 Μ磷酸水 溶液,使反應停止後,進一步保溫於3 7 °C並攪拌1 0分鐘。 抽取頂空氣體(headspace gas)在F.n的情況爲15yl、P.g 則爲1 0 0 1、T · d爲5 0 /z丨,並以氣相層析測定硫化氫量。 另外,分析條件如以下所述。 管柱:HP-PLOT/Q ( ψ 0.53mmx30m) 溫度:70°C (2.5min)—以 30°C/min升溫至 190°C — 190 °C (3.5min)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)
偵測器:FPD 分流比:4 8 : 1 注入量:15、50及1〇〇仁1 1 - 2 - 5藉由氣相層析進行的甲硫氨酸酶抑制測試 -17- 201124135 測定對甲硫氨酸酶的酶抑制活性,以作爲硫化氫產生 酶的對照。甲硫氨酸酶係以甲硫胺酸作爲基質,產生甲硫 醇、氨及〇:·酮酪酸。在藉由氣相層析進行的甲硫氨酸酶 抑制測試中,係以由甲硫氨酸酶產生的甲硫醇之量作爲指 標,比較各試藥對甲硫氨酸酶的抑制活性。 甲硫醇之定量 在試管中加入L-甲硫胺酸溶液0· lml、試樣溶液0.2ml 、酶—輔酶混合溶液〇.7ml,以橡膠栓密封,在37 °C水浴 攪拌同時保溫。60分鐘後,加入3M磷酸水溶液,使反應停 止後,進一步保溫於37 °C並攪拌10分鐘。抽取頂空氣體, 在F.n的情況爲40 μ 1、P.g及T.d爲200 // 1,以氣相層析測 定甲硫醇量。另外,分析條件如同上述。 2-3結果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藉由比色定量進行的兒茶素類產生的F.η的硫化氫產 生酶(半胱氨酸脫锍基酶)及甲硫氨酸酶抑制測試之結果 將1-2-2之結果揭示於表1、2,將1-2-3的結果揭示於 表3。兒茶素類對F.n的硫化氫產生酶(半胱氨酸脫毓基酶 )抑制活性的高低順序爲Cg> GCg> EGCg> ECg>没食子 酸、(+ ) -C,特別是在Cg、GCg、EGCg及ECg具有高抑 制活性。兒茶素類對F.n的甲硫氨酸酶的抑制活性高低順 序爲EGCg > ECg > EGC〉EC、没食子酸、C,特別是在 EGCg及ECg具有高抑制活性。此時的IC50在EGCg爲0.4mM -18- 201124135 '在 E C g 爲 〇 _ 8 m Μ。 [表1] 表1各種兒茶素類對於F.n硫化氫產生酶(半胱氨酸脫毓 一_____^酶)的抑制宅_ ----- —MH#· / HV J 1 抑制劑濃度 抑制率(%) UM) 没食子酸 ( + )-C EC EGC ECfi EGCg 1.50 4.6 1.6 0.1 0.0 40.3 53.2 [表2] 表1 各種兒茶素類對於F . __碁酶)的抑制率 η硫化氫 產生酶 (半胱氨酸脫锍 制率 抑制劑濃度 (mM) Cg 抑制率(%) GCg EGCg 78.8 68.1 54 [表3] 鱼種兒茶素類對於F.n 甲硫氨酸酶的抑 抑制劑濃度 抑制率(%) (mM) 没食子酸 ( + )-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藉由比色進行的EGCg對於En、P.g及T.d的酶抑制測 2 試 201124135 將1-2-2的硫化氫產生酶抑制測試結果及1-2-3的甲硫 氨酸酶抑制測試之中,EGCg與氯化鋅抑制活性的比較揭 示於圖1。另外將1C 50揭示於表4。圖1中,白點是EGCg, 黑點是指氯化鋅的結果。另外,已知氯化鋅具有口臭預防 活性,是被期待能夠抑制硫化氫產生酶及甲硫氨酸酶的物 質。由圖1的結果看來,關於甲硫氨酸酶的抑制,在來自 F.n、P.g及T.d的任一個情況中,由氯化辞產生的抑制活性 皆高於由EGCg產生的抑制活性。另一方面,關於硫化氫 的產生,在來自F.n、P.g及T.d的任一個情況中,由EGCg 產生的抑制活性皆高於由氯化鋅產生的抑制活性。由表4 也可得到同樣的結果。 [表4] 表 4 來自 F.nucleatum JCM8532、P.gingivalis W83 及 T.denticola ATCC3 5405的粗酵素液的IC50 (比色分析) 甲硫氨酸酶 硫化氫產生酶 IC50 (mM) 氯化鋅 EGCg 氯化鋅 EGCg F.n JCM8532 0.038 0.363 2.080 1.336 P.g W83 0.007 0.131 1.571 0.189 T.d ATCC35405 0.009 0.259 2.223 0.199 2-3-3藉由氣相層析進行的EGCg對於F.n、P.g及T.d的酶抑 制測試 將1-2-4之硫化氫產生酶抑制測試及1-2-5之甲硫氨酸 酶抑制測試結果揭示於圖2。另外,關於1C 5 0係揭示於表5 。由圖2之結果看來,關於甲硫氨酸酶的抑制’在來自F.n -20- 201124135 、P.g及T.d的任一個情況中’由氯化鋅產生的抑制活性皆 高於由E G C g產生的抑制活性。另一方面,關於硫化氫的 產生,在來自F.n、P.g及T.d的任一個情況中,由EGCg產 生的抑制活性皆高於'由氯彳匕鋅產生的抑1制活性°由表5也 可得到同樣的結# ° [表5] 表 5 來自 F.nucleatum JCM8532、P.gingivalis W83及 T.denticola ATCC3 5405的粗酵素液的IC50 (氣相層析分析) 甲硫氨酸酶 硫化氫產生酶 IC50 ( mM ) 氯化鋅 EGCg 氯化鋅 EGCg F.n JCM8532 0.039 0.351 2.369 1.106 P.g W83 0.003 0.078 1.840 0.136 T.d ATCC35405 0.009 0.175 1.812 0.133 2 - 4實施例1之總結 由以上可知’兒茶素類其中的GCg、Cg、ECg及EGCg 具有硫化氫產生酶抑制活性。特別是明顯具有對硫化氫產 生酶的一種:半胱氨酸脫巯基酶的抑制活性。進一步可知 E G C g之活性大幅高於氯化鋅之活性。可知e G C g之硫化氫 產生酶抑制活性,在來自P.g及T.d的粗酵素液中活性特別 高’而且在來自F.n的粗酵素液之中也會表現出活性。 [實施例2] 以吊法製造含有本發明由兒茶素類所構成之硫化氫產 生酶抑制劑的牙膏、口腔噴劑、喉錠、口香糖、糖果、軟 -21 - 201124135 糖、飲料。於以下揭示該等配方。另外,本發明品之範圍 不會受該等所限制。 牙膏之配方 碳酸鈣 50.0重量% 甘油 20.0 羧甲基纖維素 2.0 月桂基硫酸鈉 2.0 香料 1.0 糖精 0.1 EGCg 1.0 洗必泰(chlorhexidine) 0.01 _其餘部分 100.0 口腔噴劑之配方 乙醇 1 0.0重量% 甘油 5.0 香料 0.05 著色劑 0.00 1 ECg 1 .0 水 其餘部分 100.0 -22- 201124135 喉錠之配方 葡萄糖 7 3 . 3重量% 乳糖 16.0 ***膠 6.0 香料 1.0 單氟磷酸鈉 0.7 GCg 1.0 乳糖 2.0 100.0 口香糖之配方 口香膠基材 2 0.0重量% 砂糖 54.7 葡萄糖 15.3 水飴 9.3 EGCg 0.2 香料 0.5 100.0 糖果之配方 砂糖 5 0.0重量% 水飴 34.0 檸檬酸 2.0 EGCg 0.6 -23- 201124135 香料 0.2 水 其餘部分 100.0 軟糖之配方 明膠 6 0.0重量% 水飴 20.5 砂糖 8.5 植物油脂 4.5 甘露醇 3.0 蘋果酸 2.0 Cg 1.0 香料 0.5 100.0 飲料之配方 柳橙汁 3 0.0重量% 異構糖 15.24 檸檬酸 0.10 維生素C 0.04 香料 0.10 ECg 0.10 水 其餘部分 100.0 -24- 201124135 【圖式簡單說明】 圖1 A表示由比色分析所得到EGCg及氯化鋅對於半胱 氨酸脫锍基酶及甲硫氨酸酶的抑制活性之結果。圖1 A爲 F . η之結果。 圖1Β表示由比色分析所得到EGCg及氯化鋅對於半胱 氨酸脫锍基酶及甲硫氨酸酶的抑制活性之結果。圖1 B爲 P . g之結果。 圖1C表示由比色分析所得到EGCg及氯化鋅對於半胱 氨酸脫巯基酶及甲硫氨酸酶的抑制活性之結果。圖1 C爲 T . d之結果。 圖2 A表示藉由氣相層析進行的分析所得到e g C g及氯 化鋅對於硫化氫產生酶及甲硫氨酸酶的抑制活性之結果。 圖2A爲F.n之結果。 圖2 B表示藉由氣相層析進行的分析所得到E G C g及氯 化鋅對於硫化氫產生酶及甲硫氨酸酶的抑制活性之結果。 圖2 B爲P . g之結果。 圖2 C表示藉由氣相層析進行的分析所得到E G C g及氯 化鋅對於硫化氫產生酶及甲硫氨酸酶的抑制活性之結果。 圖2 C爲T . d之結果。 -25-2-3-1 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>GCg>EGCg>ECg> 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 > ECg > 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-