JPS6251425B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a halitosis detecting agent, and more particularly to a halitosis detecting agent containing a substance that changes color by acting on a substance having an --SH group in saliva that causes bad breath. Generally, bad breath is considered to be composed of molecules having an unpleasant odor produced by the decay of proteins in the oral cavity, except for special cases caused by internal diseases. In other words, the oral cavity contains food proteins, exfoliated epithelium,
Various protein components such as blood cell components and periodontal tissue cells are present in the exudate. These proteins are decomposed into various amino acids by the action of proteolytic enzymes derived from oral bacteria, and further into amines and ammonia by the action of decarboxylases and deaminases. Hydrogen sulfide and methyl mercaptan are also produced from sulfur-containing amino acids. In recent years, the relationship between bad breath and sulfides has attracted particular attention, and flame photometric detectors (hereinafter referred to as "flame photometric detectors") that can selectively and highly sensitively detect sulfur compounds have been attracting attention.
Analysis of oral volatile sulfide (hereinafter referred to as VSC) using FPD (abbreviated as FPD) revealed that halitosis and VSC
Many reports have shown that there is a close relationship between the two. In other words, a high concentration of
VSC has been detected, and it has been confirmed that methyl mercaptan exhibits the closest correlation with the intensity of bad breath due to its aversive nature and its threshold concentration. The mechanism of development of VSCs has also been investigated. The production of VSCs is caused by the release of disulfides by the breakdown of cellular components such as leukocytes and exfoliated epithelial cells in whole saliva, which are further reduced to thiols. It is believed that VSCs are then produced. On the other hand, conventional methods for detecting and evaluating bad breath include a sensory evaluation method using the human sense of smell and a gas chromatography method that measures the amount of VSC using an FPD. In clinical practice, sensory evaluation methods are mainly used, and in fact, it is said that the human sense of smell is superior in its detection ability to that of any analytical instrument. Judgment is possible. However, the sense of smell is easily fatigued, its sensitivity varies greatly from person to person, and it is easily influenced by factors other than the oral cavity (e.g., diet). It is difficult to objectively and quantitatively evaluate the intensity of bad breath, and there is also a drawback that those who have bad breath cannot evaluate the same bad breath. Also
Although objective evaluation is possible using gas chromatography using FPD, it requires special analytical equipment, requires incubation (keeping warm for a certain period of time) when saliva is used, and takes time, and experimental operations. There are problems such as being complicated.
Therefore, it can be said that there is currently no simple and objective method for evaluating halitosis. Under the current circumstances, the present inventors conducted intensive research to develop a method that can easily and objectively detect and evaluate bad breath. As a result, the inventors measured VSC or (and) thiols, which are its precursors, in saliva. Then, the measurement results show an extremely good correlation with the halitosis measured by sensory evaluation or gas chromatography, and the VSC and thiols are expressed by the following formula (), 4,4'-bisdimethylaminodiphenylcarbitol (hereinafter sometimes abbreviated as BDC-OH), represented by We have completed the present invention by discovering that when BDC-OH is used in combination with a certain type of surfactant, the detection sensitivity can be improved by more than 10 times. That is, the present invention comprises a first invention which is a bad breath detection agent containing 4,4'-bisdimethylaminodiphenylcarbitol, and a first invention which is a bad breath detection agent containing 4,4'-bisdimethylaminodiphenylcarbitol and an anionic surfactant or The present invention provides a second invention, which is a bad breath detection agent containing an amphoteric surfactant. The principle of the present invention is that BDC-OH becomes carbonium-immonium ion (BDC ⁺ ) on the acidic side and exhibits a strong blue color, but when the -SH compound reacts with this, this blue color quickly disappears. It takes advantage of this fact. In the second invention, the surfactants that increase the sensitivity of BDC-OH are anionic surfactants and amphoteric surfactants, and other surfactants have little effect or only slightly increase the sensitivity. Examples of anionic surfactants include higher fatty acid salts (soaps), alkanesulfonates, hydroxyalkanesulfonates, alkylbenzenesulfonates, N-alkylsulfosuccinic acid monoamide salts, alkyl sulfate ester salts, polyoxyethylene alkyl Examples include ether sulfate ester salts, fatty acid monoglyceride sulfate ester salts, alkyl phosphate ester salts, and polyoxyethylene alkyl ether phosphate ester salts. Examples of amphoteric surfactants include N,N-dimethyl-N-alkyl-N-carboxyalkylammonium (alkylbetaine), N,N-dialkylaminoalkylcarboxylate, N,N-dimethyl-N-alkyl- N-sulfoalkyl ammonium (sulfobetaine), N,-alkyl-N,
Examples include N-bispolyoxyethylene sulfate salts and imidazoline type amphoteric surfactants. These surfactants have 8 to 20 carbon atoms as hydrophobic groups.
The salt usually has a hydrocarbon group, and the salt preferably has an alkali metal, alkaline earth metal, ammonium, soluble amine, etc. as a counter ion. The bad breath detection agent of the present invention is BDC-OH or BDC
-OH and the surfactant are preferably in the form of a solution or test strip. When preparing a solution, BDC-OH or a surfactant thereof is preferably dissolved in water or a mixed solvent of a water-soluble organic solvent (eg, acetone, methanol, ethanol, tetrahydrofuran, etc.) and water. The pH of the solution may be neutral to acidic (PH2-7), but preferably weakly acidic (PH4-6.5), and the pH can be adjusted by a conventional method using a buffer. Although the concentration of BDC-OH in the solution is not particularly limited, it is preferable that the final concentration is about 0.005 to 1%. Also, although the amount of surfactant can be varied over a wide range, it is important to note that the concentration in solution is 0.001.
It is preferable to adjust the amount to ~10%, especially 0.05 to 0.5%. In addition, the test paper was prepared as described above.
Solution containing BDC-OH (hereinafter referred to as BDC solution)
Alternatively, it can be produced by impregnating a suitable paper such as paper with a solution containing BDC-OH and a surfactant (hereinafter referred to as BDC-S solution) and drying it at room temperature while shielding from light. In this case, it is preferable to use a nonvolatile acid as a buffer to prevent volatilization during storage. To measure halitosis using the halitosis detection agent of the present invention, in the case of a solution, add one to several drops of BDC solution or BDC-S solution to a certain amount of saliva, mix immediately, and check the degree of discoloration. Judgment is made with the naked eye or using a colorimeter, or in the case of a test paper, by soaking saliva on it and observing the degree of discoloration. Although it is possible to detect bad breath by directly measuring the amount of -SH groups in saliva using the method described above, it is also possible to use this method to measure the amount of VSC, which is conventionally measured by gas chromatography. I understand. When measuring the amount of VSC, incubate saliva at 37°C and introduce volatile sulfides with SH groups such as hydrogen sulfide and methyl mercaptan into the BDC solution or BDC-S solution and observe the color change of the solution. do. In this case, since a volatile substance is handled, a special sealed container is required, but any commercially available trace measuring device such as a Conway dish or an alternative sealed container can be used. However, the BDC solution, etc. and the saliva sample must not come into direct contact, and only the gas generated must come into contact with the BDC solution, etc. Gas chromatography has disadvantages such as requiring a special detector called FPD, not being able to perform multiple evaluations at once, and requiring complicated and time-consuming experimental operations. However, when measuring the amount of VSC using this method, has many advantages such as easy operation and short measurement time. As mentioned above, the halitosis detection agent of the present invention can detect bad breath without relying on sensory evaluation, which has large individual differences, or without using special equipment such as FPD, so it is extremely simple and can detect bad breath. It has the advantage that even people who are not aware that they have bad breath can evaluate whether they have bad breath or not. The present invention will be explained below with reference to Examples, but the present invention is not limited thereto. Example 1 Measurement of SH group amount in saliva (without surfactant) <Measurement method> 6.5 mg of BDC-OH was dissolved in 10 ml of acetone.
A BDC solution was prepared by mixing 1 ml of BDC-OH solution (stored in the dark) and 9 ml of 0.5M acetate buffer (PH5.1). 0.5 ml of whole saliva and 0.5 ml of BDC solution were mixed, left at room temperature for 25 minutes, centrifuged at 1500 g for 5 minutes, and the absorbance of the supernatant was measured at a wavelength of 610 nm. <Results> (1) Preparation of calibration curve By the above method, dithiothreitol [HS-CH ₂ -
(CHOH) ₂ --CH ₂ --SH] was used to prepare a calibration curve, and as shown in FIG. 1, a calibration curve with good linearity passing through the origin was obtained. (2) Correlation between the amount of volatile sulfide (VSC) and the amount of SH groups in saliva The amount of VSC produced by incubating whole saliva at 37℃ for about 20 hours and the SH group in whole saliva before incubation When we examined the correlation with the amount, we found a very high correlation (correlation coefficient 0.81), as shown in Figure 2. This means that saliva
This shows that bad breath can be evaluated by measuring the amount of SH groups. Example 2 Measurement of SH group amount in saliva (combined with surfactant) <Measurement method> 1.0 mg of BDC-OH was dissolved in 10 ml of acetone.
1ml of BDC-OH solution (stored protected from light), 9ml of 0.5M citrate buffer (PH5.1) and 100μ of 20% aqueous solution of sodium dodecyl sulfate (SDS) (final concentration 0.2
%) to prepare a BDC-S solution. Mix 25 μ or 50 μ of whole saliva with 0.5 ml of BDC-S solution, add water to make a total volume of 1 ml, leave at room temperature for 25 minutes, centrifuge at 1500 g for 5 minutes, and measure the absorbance of the supernatant (wavelength: 610 nm). ) was measured. <Results> (1) Preparation of a calibration curve A calibration curve was prepared using dithiothreitol according to the above method, and as shown in FIG. 3, a calibration curve with good linearity passing through the origin was obtained. The detection sensitivity was approximately 10 times higher than when no surfactant was used. (2) Correlation between the sensory evaluation method and the amount of SH groups in saliva The results of investigating the correlation between the value evaluated by the sensory evaluation method for the intensity of bad breath and the amount of SH groups in saliva measured by the above method are shown in Figure 4. As shown, a good correlation (correlation coefficient 0.79) was shown. In addition, the sensory evaluation was performed based on the following criteria.

[Table] (3) Diurnal variation in salivary SH group amount Figure 5 shows the results of investigating the diurnal variation in salivary SH group amount in five people using the above method. The results are shown in Figure 6. Both figures show similar patterns, indicating that this method can evaluate bad breath. Example 3 Measurement of volatile sulfides (BDC-Conway method) <Measurement method> BDC-OH 10mg dissolved in acetone 10ml
-OH solution 50μ (kept out of light), 50mM Tris-Maleate buffer (PH6.5) 10ml
and 50μ of 20% sodium dodecyl sulfate aqueous solution
(final concentration 0.1%) to prepare a BDC-S solution. Place 1.2 ml of this BDC-S solution inside the Conway dish shown in Figure 7.
After adding methyl mercaptan (benzene solution) to the outside, it was immediately sealed, incubated at 37°C for 5 hours, and then left at room temperature for 1 hour.
Absorbance (610 nm) was measured. <Results> (1) Preparation of calibration curve A curve with good linearity as shown in FIG. 8 was obtained. (2) Correlation with GC method We investigated the usefulness of this method by examining the correlation between this method and gas chromatography (GC) method using FPD, which has been traditionally used as a method for measuring volatile sulfides. The results are shown in Figure 9.
A correlation coefficient of 0.72 (n = 14) showed a highly significant correlation (risk rate of 1% or less). This shows that volatile sulfides can be sufficiently evaluated by this method.

[Brief explanation of the drawing]

FIG. 1 shows a calibration curve obtained by measuring absorbance using dithiothreitol as a substance having an SH group and a BDC solution as a detection agent. Figure 2 shows the correlation between the amount of SH groups in saliva measured using a BDC solution and the amount of VSC produced by incubation. Figure 3 uses dithiothreitol as a substance with an SH group and as a detection agent.
A calibration curve obtained by measuring absorbance using a BDC-S solution is shown. Figure 4 shows the amount of SH groups in saliva.
The correlation between the values measured using the BDC-S solution and the strength of bad breath evaluated by the sensory evaluation method is shown.
Figure 5 shows the diurnal variation in the amount of SH groups in saliva measured using BDC-S solution. FIG. 6 shows the diurnal variation in halitosis investigated by the sensory evaluation method. FIG. 7 is an explanatory cross-sectional view of the Conway dish. FIG. 8 shows a calibration curve obtained by measuring absorbance using a Conway dish, using methyl mercaptan as the substance having an SH group, and using a BDC-S solution as the detection side. FIG. 9 shows the correlation between the amount of volatile sulfide measured using a Conway dish and the amount measured using a gas chromatograph.

Claims (1)

[Claims] 1 Formula (), A halitosis detection agent containing 4,4'-bisdimethylaminodiphenylcarbitol represented by: 2. The bad breath detection agent according to claim 1, which is in the form of a solution. 3. The bad breath detection agent according to claim 1, which is in the form of a test paper. 4 formula (), A halitosis detecting agent containing 4,4'-bisdimethylaminodiphenyl carbitol represented by: and a surfactant selected from the group consisting of anionic surfactants and amphoteric surfactants. 5. The bad breath detection agent according to claim 4, which contains 0.001 to 10% by weight of a surfactant in the total composition. 6 Hz is 4 to 6.5, the halitosis detecting agent according to claim 4 or 5. 7. The bad breath detection agent according to any one of claims 4 to 6, which is in the form of a solution. 8. The bad breath detection agent according to any one of claims 4 to 6, which is in the form of a test paper.