JPH04299998A - Halitosis bacterium-examining chemical - Google Patents

Abstract

PURPOSE:To obtain a halitosis causative-examining chemical consisting of S group-substituted derivative of L-cysteine or L-methionine, and capable of simply and specifically detecting a halitosis bacterium such as Fusobacterium nucleatum or halitosis in high sensitivity. CONSTITUTION:The objective halitosis causative-examining chemical consisting of a L-cysteine or L-methionine derivative (salt) expressed by formula I or formula II (Y is functional group capable of measuring an amount of Y-SH produced by metabolism of halitosis bacteria by a visible light, ultraviolet ray, infrared ray or fluorescence). The compound expressed by formula I includes e.g. S-(p-nitrophenyl)-L-cysteine.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a drug for testing bad breath bacteria, which is used to test for bad breath.

0002

[Prior art and problems to be solved by the invention] Conventionally,
Methods for measuring halitosis include methods that directly or indirectly measure halitosis, methods that measure halitosis substitute substances, and methods that measure periodontal pathogenic substances based on the relationship between halitosis bacteria and periodontal pathogenic bacteria. are known, for example, the following (1) to (3)
The following method has been proposed. (1) Method of measuring and evaluating bad breath directly sensually or using equipment such as gas chromatography (Kenki Umezu, Japanese Periodontal Journal, 18, 1-22, (1976)), bad breath sensor (gas detection element) A method for measuring and evaluating bad breath (Japanese Unexamined Patent Publication No. 58-208651). (2) Method for measuring SH compounds (reducing substances) and nitrite ions in saliva as a substitute for halitosis bacteria (JP-A-57-1
48252, 62-115364, 62-1153
65, No. 59-33826, No. 62-294943). (3) Method for investigating the dynamics of Bacteroides gingivalis in lesions of periodontal disease using monoclonal antibodies against Bacteroides gingivalis (Japanese Patent Application Laid-Open No. 60-7
3463), a method for measuring periodontopathogenic bacterial antigens (JP-A-61-162753), and a method for specifically measuring aminopeptidase-like enzyme activity produced by periodontal pathogenic bacteria using a specific substrate. Periodontal disease test drug to measure
-36800).

[0003] However, methods for measuring halitosis require equipment, methods using halitosis sensors have problems with specificity as they react with components other than halitosis bacteria, and methods for measuring halitosis substitutes have poor measurement accuracy. There are issues that need to be improved, and periodontal disease testing agents have problems such as insufficient sensitivity.

0004

[Means for Solving the Problems and Effects] The present inventor has conducted intensive studies on a new method for testing bad breath bacteria, which is different from the above-mentioned conventional techniques. That is, sulfur-containing amino acids such as L-cysteine and L-methionine, which are substrates that cause bad breath, are
Focusing on the fact that it is metabolized by the enzymatic reaction of oral bacteria such as Polyphomonas (Bacteroides), Bionella, and Spirochete (Treponema) to become malodorous components H2S and CH3SH (see Dentistry Abstracts 29, 199 (1987)). As a result of further investigation, it was found that by using the above-mentioned sulfur-containing amino acid derivative (S-substituted derivative of L-cysteine or L-methionine (homocysteine) of formula (1) or (2) below, or a salt thereof), bad breath can be reduced. It has become possible to measure bacteria, and it has been discovered that bad breath can be easily tested.

YS-CH2-CH(NH)-CO
OH…(1) Y
-S-CH2-CH2-CH(NH2)-COOH
...(2) (However, Y represents a functional group that makes it possible to measure the amount of Y-SH produced by metabolism by halitosis bacteria in the visible region, ultraviolet region, infrared region, or fluorescence.) Here, the above L - The reason why halitosis bacteria can be measured and halitosis testing becomes possible by using derivatives of cysteine or L-methionine is presumed as follows.

That is, as shown in reaction formula A below, the metabolic enzyme L-cysteine desulfidolase, methionine γ
-It is thought that malodorous components (hydrogen sulfide H2S, methyl mercaptan CH3SH) are produced when lyase metabolizes L-cysteine and L-methionine, which are the substrates of bad breath-causing bacteria, but instead of the sulfur-containing amino acids mentioned above, For example, by using a sulfur-containing amino acid derivative as shown in reaction formula B below, α-
Keto acid or p-nitrophenyl mercaptan (yellow) is produced, and by measuring this, it is possible to measure halitosis bacteria or bad breath.

[0007]

[Chemical formula 1]

[0008] Therefore, L- in the above formula (1) or (2)
By using cysteine or L-methionine derivatives or their salts as halitosis bacteria testing agents, halitosis bacteria, particularly Fusobacterium nucleum, Fusobacterium necrophorm, Polyphomonas gingivalis, Bacteroides denticola, Bionella disper, Bionella・Detection of Parvula, Treponema denticola, etc. or bad breath test can be easily, highly sensitive, and specific, and by selecting the above-mentioned L-cysteine or L-methionine derivatives or their salts, it can be directly detected by color development in the visible part. It also makes it possible to recognize bad breath.

[0009] The present invention will be explained in more detail below. The halitosis bacteria testing agent of the present invention uses
- By using derivatives of cysteine, L-methionine, or salts thereof, halitosis bacteria or bad breath can be determined using the same metabolic (enzyme) system as used for the metabolism of L-cysteine and L-methionine. The following formula (1) or (2) is used as a derivative of cysteine or L-methionine.

Y-S-CH2-CH(NH2)-C
OOH…(1)
Y-S-CH2-CH2-CH(NH2)-COOH
...(2) (However, Y represents a functional group that allows the amount of Y-SH produced by metabolism by halitosis bacteria to be measured using visible light, ultraviolet light, infrared light, or fluorescence.)

[0011] Here, the above-mentioned functional group (chromogenic group) is a functional group that can be measured in the visible region, ultraviolet region, infrared region, and fluorescence, as described above, and such functional groups include phenyl group, Examples include groups having a benzyl group, benzoyl group, purine group, imidazole group, naphthyl group, etc., and specifically, o- or p-
-nitrophenyl group, o- or p-nitrobenzyl group,
o- or p-nitrobenzoyl group, α- or β-nitronaphthyl group, o- or p-bromophenyl group, o- or p-bromobenzyl group, o- or p-bromobenzoyl group, α- or β- Examples include bromonaphthyl group, o- or p-aminophenyl group, o- or p-aminobenzyl group, o- or p-aminobenzoyl group, α- or β-aminonaphthyl group, and furthermore, a nitro group in the above skeleton. , a bromo group, a group containing two or more amino groups, a benzothiazole group, and the like.

The test agent of the present invention can be prepared in various forms, for example, in the form of an aqueous solution, or by impregnating filter paper with this aqueous solution and drying it, or using dry chemistry techniques. This also includes those developed into various test papers by using .

[0013] Furthermore, the test reagent of the present invention can be made into a kit by combining other reagents, and by making it into a kit, the precision of quantitative determination can be further improved. Furthermore,
The halitosis bacteria test drug of the present invention is metabolized by a wide range of oral bacteria such as Fusobacterium nucleum, Polyphomonas gingivalis, and spirochetes, so it can also be applied to testing for periodontal disease-causing bacteria.

[0014]

[Example] Next, the effects of the present invention will be specifically explained with reference to experimental examples.

[Experiment example] First, various oral bacteria were
Anaerobic culture was performed in a medium at 37° C. for 48 to 72 hours, and the bacterial concentration was adjusted to 1.0 (absorbance at a wavelength of 660 nm). Next, add 2 ml of 1.0 mM substrate solution shown in Table 1 (pH 7.5, 0.5 M phosphate buffer) and 0.0 ml of bacterial solution.
．． 5 ml was reacted at 37°C for 30 minutes. After the reaction is complete,
The resulting α-keto acid (pyruvic acid or α-ketobutyric acid) was purified using the method of Soda et al. (Agric. Biol. Chem. 31
, 1054-1060 (1967)) and judged in four stages according to the following criteria. The results are also listed in Table 1. Judgment criteria -: α-keto acid cannot be detected +: Detection amount at the same level as α-keto acid produced from L-cysteine ++: 5 to 1 of α-keto acid produced from L-cysteine
Detection amount +++ at 0 times level: 20% of α-keto acid produced from L-cysteine
Detection amount more than double

[0016]

[Table 1]

From the results in Table 1, L-cysteine and L-
It has been found that derivatives having a methionine coloring group can easily and sensitively detect bacteria that cause bad breath.

The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Using gas chromatography, H2S and CH3SH (halitosis) in the exhaled breath of subjects were measured, and subgingival plaque samples were collected from 12 subjects whose measured values were relatively high. was suspended in 0.5 ml of physiological saline. S-(p-nitrophenyl)-L-cysteine was used as a test agent for bad breath bacteria, and the metabolic activities of bacteria were compared in the same manner as in Experiment 1. In this case, S-(p-nitrophenyl)-L-cysteine is metabolized to produce S-(p-nitrophenyl) mercaptan, so the absorbance of S-(p-nitrophenyl) mercaptan in the reaction solution is measured at the wavelength Measured at 405 nm, and H2S, CH3 in exhaled breath
The relationship with the SH concentration (VSC) was investigated. The results are shown in Figure 1.

[0020] From the results shown in Figure 1, H2S and CH3 in exhaled breath
SH concentration and S-(p-nitrophenyl)-L-cysteine produced using halitosis bacteria testing agent.
A high correlation was observed with the absorbance value of (nitrophenyl) mercaptan.

[Example 2] 2,4- as a halitosis bacteria test drug
Using dinitrophenyl-L-cysteine, its 2mM
A test paper was prepared by impregnating filter paper with the solution and drying it. Next, exudate from the periodontal pocket of the same subject as in Example 1 was 0.
Collect 01ml and soak it into the above test paper, 3
The test paper was left to stand for 5 to 30 minutes at 7°C, and changes in the color of the test paper were observed.

In this case, the test paper is initially white, but 2
, 4-dinitrophenyl-L-cysteine is metabolized by bacteria that cause bad breath to produce 2,4-dinitrophenyl mercaptan, which turns yellow, and the greater the amount, the darker the yellow becomes.

The results are shown in FIG. 2, and it was found that there was a high correlation between the concentration of H2S and CH3SH in exhaled breath and the change in color of the test paper.

[0024]

[Effects of the Invention] According to the halitosis bacteria test drug of the present invention, halitosis bacteria such as Fusobacterium nucleatum or halitosis can be detected simply, with high sensitivity, and specifically, and is useful for periodontal disease examinations and halitosis examinations. Used effectively.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the absorbance of S-(p-nitrophenyl)mercaptan and the concentrations of H2S and CH3SH in exhaled breath.

FIG. 2 is a graph showing the relationship between the color of a 2,4-dinitrophenyl-L-cysteine-containing test paper and the concentration of H2S and CH3SH in exhaled breath.

Claims (1)

[Claims] [Claim 1] The following formula (1) or (2)
Y-S-CH2-CH(NH2)-COOH
...(1) Y-S-CH
2-CH2-CH(NH2)-COOH...(2)(
However, Y represents a functional group that makes it possible to measure the amount of Y-SH metabolized by halitosis bacteria using visible light, ultraviolet light, infrared light, or fluorescence. ) A halitosis bacteria testing agent comprising an L-cysteine or L-methionine derivative or a salt thereof.