JP2010070493A - Antibacterial agent, method for producing the same, cosmetic product and pharmaceutical product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibacterial agent developing synergistically enhanced antibacterial activity compared with lysozyme and triclosan, exhibiting wider antibacterial spectrum and having high solubility in water, a method for producing the antibacterial agent, and a cosmetic product and a pharmaceutical product developing high antibacterial activity against various bacteria causing infectious diseases, inflammation, underarm body odor, etc., suppressing the infectious diseases, inflammation, underarm body odor, etc., and having high antiseptic property. <P>SOLUTION: There are provided the antibacterial agent containing a dried material produced by drying a solution containing lysozyme and triclosan, the method for producing the antibacterial agent by drying the solution containing lysozyme and triclosan, and the cosmetic product and the pharmaceutical product containing the antibacterial agent. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an antibacterial agent. More particularly, the present invention relates to an antibacterial agent suitable for developing antibacterial activity in the field of cosmetics (including quasi-drugs) and pharmaceuticals, a method for producing the same, and a cosmetic and a pharmaceutical containing the antibacterial agent.

  Cosmetics and pharmaceuticals containing antibacterial agents that exhibit antibacterial activity against various bacteria, including the causative bacteria, are known as cosmetics and pharmaceuticals for controlling infections and inflammations in the skin, and odors. It has been. The antibacterial agent is also used as a preservative for cosmetics and pharmaceuticals. As such antibacterial agents, lysozyme and triclosan are known.

  As the cosmetics and pharmaceuticals, cosmetics and pharmaceuticals containing lysozyme or triclosan have been proposed (see, for example, Patent Documents 1 to 5). However, the cosmetics and pharmaceuticals have a drawback that the antibacterial spectrum is narrow and a sufficient antibacterial effect cannot be obtained depending on the application location or application case. Further, since the lysozyme or triclosan has a narrow antibacterial spectrum, cosmetics and pharmaceuticals containing such lysozyme or triclosan have a disadvantage that sufficient antiseptic properties cannot be obtained.

  Therefore, as an attempt to obtain a higher antibacterial effect, cosmetics and pharmaceuticals containing an antibacterial agent containing both lysozyme and triclosan have been proposed (see, for example, Patent Documents 6 to 8). However, lysozyme is a water-soluble protein, whereas triclosan is a hydrophobic compound. Therefore, when the cosmetic or pharmaceutical is formulated as an aqueous preparation, triclosan is It has the disadvantage that it does not dissolve in the preparation or triclosan precipitates over time.

JP 20032266652 A JP 2008-156268 A JP 2007-145749 A JP 2008-110992 A JP 2008-138027 A JP 2004-189633 A (paragraph numbers [0025], [0027]) JP 2007-145750 A (paragraph number [0037]) JP 2007-144771 A

  The present invention has been made in view of the above prior art, and exhibits synergistically enhanced antibacterial activity, exhibits a broader antibacterial spectrum, and has high solubility in water as compared with lysozyme and triclosan. An object is to provide an antibacterial agent. Moreover, an object of this invention is to provide the manufacturing method of the antibacterial agent which can manufacture the said antibacterial agent simply. Furthermore, the present invention expresses high antibacterial activity against various bacteria that cause infectious diseases, inflammation, odor, etc., and can suppress the infectious diseases, inflammation, odor, etc., and has high antiseptic properties. It aims at providing the cosmetics and pharmaceutical which have.

The present invention
(1) an antibacterial agent containing a dried product obtained by drying a solution containing lysozyme and triclosan,
(2) A method for producing an antibacterial agent, comprising drying a solution containing lysozyme and triclosan,
(3) It relates to the production method according to (2) above, wherein the solution is dried by freeze drying, spray drying or vacuum drying, and (4) a cosmetic or pharmaceutical product containing the antibacterial agent according to (1).

  Compared with lysozyme and triclosan, the antibacterial agent of the present invention exhibits synergistically enhanced antibacterial activity, exhibits a broader antibacterial spectrum, and has an excellent effect of having high solubility in water. Moreover, the method for producing an antibacterial agent of the present invention has an excellent effect that the antibacterial agent can be easily produced. Furthermore, the cosmetics and pharmaceuticals of the present invention express high antibacterial activity against various bacteria that cause infections, inflammation, odors, etc., and can suppress the infections, inflammations, odors, etc. It has an excellent effect of having high antiseptic properties.

  The antibacterial agent of the present invention contains a dried product obtained by drying a solution containing lysozyme and triclosan. In the dried product, a complex of lysozyme and triclosan is formed as shown in the examples described later, unlike a mixture that simply contains lysozyme and triclosan. The present inventors have not found a report example regarding the formation of such a complex of lysozyme and triclosan.

  Lysozyme is a polysaccharide hydrolase that is a constituent of bacterial cell membranes. Lysozyme has bactericidal action, anti-inflammatory action, hemostasis action, tissue repair action and the like. Examples of the lysozyme include human lysozyme, egg white lysozyme, fish body surface mucus lysozyme, microbial lysozyme, bacteriophage lysozyme and the like. Among these, egg white lysozyme is preferable because it can be prepared at low cost. Among egg white lysozymes, egg white lysozyme of chicken eggs is preferable because it can be prepared at low cost. The lysozyme may be a pharmaceutically acceptable salt. Examples of the salt of lysozyme include lysozyme hydrochloride.

  Triclosan is a fungicide with the chemical name 2 ', 4,4'-trichloro-2-hydroxydiphenyl ether. Triclosan can be easily obtained from Ciba Special Chemicals under the trade name: Irgasan DP-300, for example.

  The dried product can be obtained by drying a solution containing triclosan and lysozyme. Therefore, since the antibacterial agent of the present invention contains the dried product, it can be easily produced.

  The solution containing lysozyme and triclosan is, for example, a lysozyme solution in which lysozyme is dissolved in a solvent suitable for maintaining the physiological activity of lysozyme, and a triclosan solution in which triclosan is dissolved in a solvent suitable for dissolving triclosan. And can be obtained by mixing.

  Suitable solvents for maintaining the physiological activity of lysozyme include, for example, water, ethanol and the like, and these can be used alone or in combination, respectively. Among these, water is preferable from the viewpoint of maintaining lysozyme in a good state. When the solvent is water, the water preferably has weak alkalinity from the viewpoint of favorably forming a complex of lysozyme and triclosan. The pH of the water is preferably 7.5 or more, more preferably 8 or more, from the viewpoint of satisfactorily forming a complex of lysozyme and triclosan and sufficiently expressing the physiological activity of lysozyme. From the viewpoints of favorably forming a complex of lysozyme and triclosan and maintaining the stability of the three-dimensional structure of lysozyme, it is preferably 11 or less, more preferably 10 or less.

  The content of lysozyme in the lysozyme solution may be an amount sufficient to express the antibacterial activity by the dried product, and cannot be determined unconditionally, but usually from the viewpoint of increasing the productivity of the antibacterial agent, Preferably it is 0.01 mass% or more, More preferably, it is 0.1 mass% or more, From a viewpoint of improving the solubility to a solvent, Preferably it is 10 mass% or less, More preferably, it is 5 mass% or less.

  Suitable solvents for dissolving triclosan include, for example, lower alcohols having 1 to 3 carbon atoms such as ethanol and isopropanol, polyhydric alcohols having 2 to 4 carbon atoms such as ethylene glycol and 1,3-butylene glycol. Can be mentioned. Among these, from the viewpoint of sufficiently dissolving triclosan and maintaining the lysozyme in a good state when the lysozyme solution and the triclosan solution are mixed, and from the viewpoint of easily drying the solution containing lysozyme and triclosan. Ethanol is preferred.

  The content of triclosan in the triclosan solution may be an amount sufficient to express the antibacterial activity by the dried product and cannot be determined in general, but is usually preferable from the viewpoint of increasing the productivity of the antibacterial agent. Is 5% by mass or more, more preferably 10% by mass or more, and preferably 50% by mass or less, more preferably 30% by mass or less, from the viewpoint of enhancing the solubility in a solvent.

  In mixing the triclosan solution and the lysozyme solution, the molar ratio of triclosan to lysozyme (triclosan / lysozyme) is preferably 5/1 or more, more preferably 10/1 or more, from the viewpoint of developing high antibacterial activity. From the viewpoint of increasing the compatibility between the triclosan solution and the lysozyme solution, it is preferably 50/1 or less, more preferably 30/1 or less.

  The temperature at which the triclosan solution and the lysozyme solution are mixed is preferably 0 ° C. or more, more preferably 5 ° C. or more from the viewpoint of sufficiently dissolving the triclosan and lysozyme in the solution, and the lysozyme is maintained in a good state. In view of the above, it is preferably 70 ° C. or lower, more preferably 60 ° C. or lower, and further preferably 40 ° C. or lower.

  The mixing of the triclosan solution and the lysozyme solution is preferably performed with stirring. The time required for mixing and stirring the triclosan solution and the lysozyme solution may be a time sufficient for complexing the triclosan and lysozyme, and cannot be determined unconditionally. It is about -48 hours, More preferably, it is about 5 to 30 hours.

  The method for drying the solution containing lysozyme and triclosan is not particularly limited as long as the method can maintain the physiological activity of lysozyme. The solution is preferably dried by freeze-drying (freeze-drying), spray-drying (spray-drying), or reduced-pressure drying from the viewpoint of drying the solution at a temperature that can maintain the physiological activity of lysozyme.

  The solution is preferably dried until, for example, the amount of residual solvent in the dried product is about 1% by mass or less. By drying the solution, for example, a dry product can be obtained in the form of a powder.

  The lyophilization of the solution can be performed, for example, by freezing the solution and then drying a frozen product of the solution under reduced pressure. The solution can be lyophilized using, for example, a vacuum freeze dryer.

  In the freeze-drying, the temperature at which the solution is frozen may be a temperature below the freezing point of the mixture of the solvent used in the triclosan solution and the solvent used in the lysozyme solution, and the type of solvent used It is determined accordingly. For example, when the solvent used for the triclosan solution is ethanol and the solvent used for the lysozyme solution is water, the temperature at which the solution is frozen depends on the ethanol concentration in the solution, but is cooled to the freezing point of ethanol. Then, since the mixture of ethanol and water can be sufficiently frozen, it is sufficient to cool to about −115 ° C. even at the lowest temperature.

  The degree of vacuum when drying the frozen product is preferably 10000 Pa or less, more preferably 5000 Pa or less, from the viewpoint of quickly drying the solution. The lower limit of the degree of vacuum is the performance of the apparatus used. Is not particularly limited.

  The solution can be spray-dried, for example, by spraying the solution into a space maintained at a temperature capable of maintaining the physiological activity of lysozyme and drying the solution. Spray drying of the solution can be performed using, for example, a spray dryer.

  In the spray drying, the temperature of the atmosphere when spraying the solution is preferably 70 ° C. or less, more preferably 60 ° C. or less from the viewpoint of the stability of lysozyme to heat, and from the viewpoint of performing spray drying in a short time. The temperature is preferably 20 ° C. or higher, more preferably 30 ° C. or higher.

  Moreover, in the said spray drying, the spray rate of a solution can be suitably set according to the particle size of the dried material obtained.

  The solution can be dried under reduced pressure, for example, by drying the solution at a temperature at which the physiological activity of lysozyme can be maintained and under reduced pressure. The solution can be dried under reduced pressure using, for example, a vacuum dryer.

  In the drying under reduced pressure, the temperature when drying the solution may be the same as the temperature of the atmosphere when spraying the solution in the spray drying.

  In the vacuum drying, the degree of vacuum when the solution is dried may be the same as the degree of vacuum in the freeze drying.

  The antibacterial agent of the present invention contains the dried product thus obtained, that is, a complex of lysozyme and triclosan.

  The content of the dried product in the antibacterial agent of the present invention is preferably 10% by mass or more, more preferably 30% by mass or more, from the viewpoint of developing the function as an antibacterial agent. The antibacterial agent of the present invention may consist only of a dried product. Further, the antibacterial agent of the present invention is, for example, a solvent such as water, ethanol, 1,3-butylene glycol, a surfactant, an antioxidant, and a sequestering agent, as long as the object of the present invention is not impaired. , And may contain auxiliary agents such as pH adjusters.

  The antibacterial agent of the present invention exhibits synergistically enhanced antibacterial activity, exhibits a broader antibacterial spectrum, and has a high solubility in water as compared with lysozyme and triclosan. Therefore, the antibacterial agent of the present invention can be used by being blended in various types of cosmetics such as aqueous cosmetics and various pharmaceutical types such as aqueous pharmaceuticals.

  Specific examples of cosmetics include deodorants, lotions, perfumes, facial cleansing creams, facial cleansing foams, disinfecting cleansers, massage creams, moisturizing creams, sunscreens, hair nourishing agents, hair creams, hair tonics, set lotions, shampoos, rinses , Permanent wave solution, hand cream, lipstick, foundation, mouthwash and the like.

  The content of the antibacterial agent in the cosmetic of the present invention is preferably 0.01% by mass or more, more preferably 0.1% by mass or more from the viewpoint of sufficiently expressing antibacterial activity, From the viewpoint of suppression, it is preferably 10% by mass or less, more preferably 5% by mass or less.

  If the cosmetic of the present invention is within the range not inhibiting the purpose of the present invention, for example, a stabilizer, a thickener, a moisturizer, a surfactant, powder, vitamins and other drugs, animal extracts, plants Extracts, pH adjusters, colorants, other bactericidal components, antiperspirant components, deodorant components, fats and oils, lower alcohols, polyhydric alcohols, water and the like may be contained as appropriate.

  Examples of pharmaceuticals include acne treatment drugs, gargles, and external creams, but the present invention is not limited to such examples.

  The content of the antibacterial agent in the pharmaceutical product of the present invention is preferably 0.01% by mass or more, more preferably 0.1% by mass or more from the viewpoint of sufficiently expressing antibacterial activity, and irritation to the application site. From the viewpoint of suppressing the above, it is preferably 10% by mass or less, more preferably 5% by mass or less.

  The pharmaceutical product of the present invention appropriately contains pharmaceutically acceptable auxiliaries, excipients, binders, stabilizers, buffering agents, isotonic agents, and the like as long as the purpose of the present invention is not impaired. May be.

  Next, the present invention will be described in more detail based on examples, but the present invention is not limited to such examples.

Examples 1-3
Egg white lysozyme of chicken egg (manufactured by Innovatech Labs, Inc.) was dissolved in distilled water having a pH of 9.0 to obtain a 1% by mass lysozyme aqueous solution. An ethanol solution of 20% by mass triclosan so that the amount of triclosan per 1 mol of lysozyme is 10 mol (Example 1), 15 mol (Example 2) or 30 mol (Example 3) in the obtained lysozyme aqueous solution. (Hereinafter referred to as “triclosan ethanol solution”) was added to obtain a solution containing lysozyme and triclosan.

  Each solution obtained was stirred at 29 ° C. for 24 hours, and then subjected to a freeze-dryer (manufactured by Tokyo Rika Kikai Co., Ltd., trade name: FDU-540), and freeze-dried to obtain a freeze-dried product. . The obtained freeze-dried product was used as the antibacterial agent of Examples 1 to 3.

Examples 4-6
0.1 g of each antibacterial agent obtained in Examples 1 to 3 was dissolved in 100 mL of distilled water. Each aqueous solution obtained was dialyzed against 1 L of water at 4 ° C. for 72 hours, and then freeze-dried again to obtain a freeze-dried product. The obtained freeze-dried products were used as antibacterial agents of Examples 4 to 6, respectively.

Example 7
Egg white lysozyme of chicken egg (manufactured by Innovatech Labs, Inc.) was dissolved in distilled water having a pH of 9.0 to obtain a 1% by mass lysozyme aqueous solution. The triclosan ethanol solution was added to the obtained lysozyme aqueous solution so that the amount of triclosan per 1 mol of lysozyme was 10 mol to obtain a solution containing lysozyme and triclosan.

  The obtained solution was stirred at 29 ° C. for 24 hours, and then subjected to a spray drying apparatus and spray-dried under an atmosphere at 60 ° C. to obtain a spray-dried product. The obtained spray-dried product was used as the antibacterial agent of Example 7.

Example 8
Egg white lysozyme of chicken egg (manufactured by Innovatech Labs, Inc.) was dissolved in distilled water having a pH of 9.0 to obtain a 1% by mass lysozyme aqueous solution. The triclosan ethanol solution was added to the obtained lysozyme aqueous solution so that the amount of triclosan per 1 mol of lysozyme was 10 mol to obtain a solution. The obtained solution was stirred at 29 ° C. for 24 hours, and then subjected to a vacuum drying apparatus and dried under reduced pressure in an atmosphere of 40 ° C. to obtain a vacuum dried product. The obtained dried product under reduced pressure was used as the antibacterial agent of Example 8.

Comparative Examples 1-3
Egg white lysozyme of chicken egg (manufactured by Innovatech Labs, Inc.) was dissolved in distilled water having a pH of 9.0 to obtain a 1% by mass lysozyme aqueous solution. To the obtained lysozyme aqueous solution, the triclosan ethanol solution was added so that the amount of triclosan per mole of lysozyme was 10 mol (Comparative Example 1), 15 mol (Comparative Example 2) or 30 mol (Comparative Example 3). A solution was obtained.

  Each obtained solution was dialyzed against distilled water at 4 ° C. for 72 hours to obtain the dialyzed products of Comparative Examples 1 to 3.

Comparative Example 4
Egg white lysozyme of chicken egg (manufactured by Innovatech Labs, Inc.) was dissolved in distilled water having a pH of 9.0 to obtain a 1% by mass lysozyme aqueous solution. The triclosan ethanol solution was added to the resulting aqueous lysozyme solution so that the amount of triclosan per 1 mol of lysozyme was 10 mol to obtain a solution. The obtained solution was used as the antibacterial agent of Comparative Example 4.

Test Example 1 (Measurement of absorbance and turbidity)
(1) Preparation of sample for evaluation Each antibacterial agent obtained in Examples 1 to 6 or each dialysis product obtained in Comparative Examples 1 to 3 has a protein (lysozyme) concentration of 0.5 mg / mL. Then, it was dissolved in ultrapure water (manufactured by Millipore, ultrapure water produced by an ultrapure water production apparatus “MilliQ”) to obtain a sample for evaluation. The protein concentration was quantified using a Bradford protein assay (manufactured by Bio-Rad, protein assay kit).

  Further, in Examples 1 to 6 and Comparative Examples 1 to 3, except that ethanol was added to the lysozyme aqueous solution instead of the triclosan ethanol solution, the same operation as in each Example and each Comparative Example was performed, and a control lysozyme sample [ Triclosan / lysozyme (molar ratio) was 0/1].

  Triclosan concentration in the sample for evaluation of each antibacterial agent obtained in Examples 1 to 3 (Example 1: 0.35 μmol / mL, Example 2: 0.53 μmol / mL or Example 3: 1.05 μmol / mL) A sample was prepared by dissolving triclosan in ethanol so as to have the same concentration. The obtained samples are collectively referred to as “TCS (EtOH)”.

  Triclosan concentration in the sample for evaluation of each antibacterial agent obtained in Examples 1 to 3 (Example 1: 0.35 μmol / mL, Example 2: 0.53 μmol / mL or Example 3: 1.05 μmol / mL) A sample was prepared by adding triclosan to water so as to have the same concentration. The obtained samples are collectively referred to as “TCS (H 2 O)”.

(2) Measurement of absorbance Samples for evaluation of antibacterial agents obtained in Examples 1 to 6, samples for evaluation of dialysis products obtained in Comparative Examples 1 to 3, TCS (EtOH) and TCS (H2O), respectively The absorbance at a wavelength of 280 nm was measured, and the interaction between triclosan and lysozyme was evaluated using the fact that triclosan absorbs light in the ultraviolet wavelength region. Specifically, it is as follows.

  Using a spectrophotometer (trade name: SmartSpec-3000, manufactured by Bio-Rad, Inc.), samples for evaluation of the antibacterial agents obtained in Examples 1 to 6, obtained in Comparative Examples 1 to 3 The absorbance at a wavelength of 280 nm was measured for each of the dialysis product evaluation samples, TCS (EtOH) and TCS (H2O). The absorbance of triclosan in each of the antibacterial agents obtained in Examples 1 to 6 and each of the dialysis products obtained in Comparative Examples 1 to 3 is a sample for evaluation of each antibacterial agent of Examples 1 to 6 at a wavelength of 280 nm and It calculated | required by subtracting the measured value of the light absorbency of the said control lysozyme sample from the measured value of the light absorbency of each sample for evaluation of each dialysis product of Comparative Examples 1-3.

  About the sample for evaluation of each antibacterial agent obtained in Examples 1 to 6, the sample for evaluation of each dialysis product obtained in Comparative Examples 1 to 3, TCS (EtOH) and TCS (H2O), respectively, at a wavelength of 280 nm The results of measuring the absorbance are shown in Table 1 and FIG.

  In Table 1, TCS-LZ is a general term for samples for evaluation of antibacterial agents obtained in Examples 1 to 3, TCS-LZ-cd is a general term for samples for evaluation of antibacterial agents obtained in Examples 4 to 6, TCS-LZ-d is a generic name for samples for evaluation of dialysis products obtained in Comparative Examples 1 to 3.

  In FIG. 1A, the rectangle is the absorbance of TCS (EtOH), the black square is the absorbance of TCS (H 2 O), the black circle is the absorbance of TCS-LZ, the white square is the absorbance of TCS-LZ-cd, and the white triangle is The absorbance of TCS-LZ-d is shown. In Table 1 and FIG. 1A, the absorbance when triclosan / lysozyme (molar ratio) is 0/1 indicates the absorbance of the control lysozyme sample.

  Since each antibacterial agent obtained in Examples 1 to 3 contains the same amount of triclosan as TCS (H 2 O) and TCS (EtOH), the absorbance of TCS-LZ is usually TCS (H 2 O) and TCS ( It is considered that the absorbance is comparable to each of EtOH). However, from the results shown in Table 1 and FIG. 1 (A), it can be seen that the absorbance of TCS-LZ is lower than the respective absorbances of TCS (H 2 O) and TCS (EtOH). Therefore, it is suggested that a complex of triclosan and lysozyme is formed in the lyophilized product that is each antibacterial agent obtained in Examples 1 to 3.

  On the other hand, from the results shown in Table 1 and FIG. 1 (A), the absorbance of TCS-LZ-d is almost 0, and no absorption of light in the ultraviolet wavelength region based on triclosan is observed. By performing, it can be seen that triclosan is removed from the solution containing triclosan and lysozyme. These results suggest that the complexing of triclosan and lysozyme occurs by drying to remove the solvent.

  However, samples for evaluation of antibacterial agents obtained in Examples 4 to 6, which were freeze-dried lyophilized products after drying, were also obtained in Examples 1 to 3 as the triclosan / lysozyme (molar ratio) increased. Since the change in absorbance is similar to that of each antibacterial evaluation sample, once the complex of triclosan and lysozyme is formed, the formed complex of triclosan and lysozyme exhibits high stability. I understand that.

(3) Measurement of turbidity Samples for evaluation of antibacterial agents obtained in Examples 1 to 6, samples for evaluation of dialysis products obtained in Comparative Examples 1 to 3, TCS (EtOH) and TCS (H2O) About each, the sample for evaluation of each antibacterial agent obtained in Examples 1-6, the sample for evaluation of each dialysis product obtained in Comparative Examples 1 to 3, by measuring the absorbance at a wavelength of 500 nm, TCS ( EtOH) and TCS (H 2 O) were evaluated for water solubility. Specifically, it is as follows.

  Using a spectrophotometer (trade name: SmartSpec-3000, manufactured by Bio-Rad, Inc.), samples for evaluation of the antibacterial agents obtained in Examples 1 to 6, obtained in Comparative Examples 1 to 3 The absorbance at a wavelength of 500 nm was measured for each of the dialysis product evaluation samples, TCS (EtOH) and TCS (H2O). Samples for evaluation of each antibacterial agent obtained in Examples 1 to 6, samples for evaluation of each dialysis product obtained in Comparative Examples 1 to 3, turbidity of each of TCS (EtOH) and TCS (H2O) (wavelength 500 nm) Table 2 and FIG. 1 (B) show the results of measuring the absorbance at (1). In FIG. 1B, a rectangle, a black square, a black circle, a white square, and a white triangle are the same as those in FIG. In Table 2 and FIG. 1B, the absorbance when triclosan / lysozyme (molar ratio) is 0/1 indicates the absorbance of the control lysozyme sample.

  From the results shown in Table 2 and FIG. 1 (B), the turbidity of TCS (H 2 O) increases with an increase in triclosan concentration, but for evaluation of each antibacterial agent obtained in Examples 1-6. It can be seen that the turbidity of the sample does not change in the same manner as the turbidity of TCS (EtOH) even when triclosan / lysozyme (molar ratio) is increased. Therefore, it can be seen that a freeze-dried product obtained by freeze-drying a solution containing triclosan and lysozyme exhibits high solubility in water.

Test Example 2 (Measurement of fluorescence spectrum)
Each antibacterial agent obtained in Examples 1 to 6 or each dialysis product obtained in Comparative Examples 1 to 3 was 25 mM sodium phosphate buffer so that the protein (lysozyme) concentration was 0.1 mg / mL. A sample for measurement of each antibacterial agent obtained in Examples 1 to 6 or each dialysis product obtained in Comparative Examples 1 to 3 was obtained by dissolving in a liquid (pH 7.4). Further, lysozyme was dissolved in 25 mM sodium phosphate buffer (pH 7.4) so that the protein (lysozyme) concentration was 0.1 mg / mL to obtain a control lysozyme solution. Further, triclosan was added to 25 mM sodium phosphate buffer (pH 7.4) so that the triclosan concentration was the same as the triclosan concentration in each antibacterial agent obtained in Examples 1 to 3 in the measurement sample. And mixed to obtain a control triclosan sample.

  The obtained measurement sample was put in a 0.5 cm square cuvette and using a fluorescence spectrophotometer [manufactured by JASCO Corporation, trade name: JASCO FP-6600] at a rate of 200 mm / min at 25 ° C. The fluorescence spectrum of the measurement sample having an emission wavelength of 300 to 400 nm at an excitation wavelength of 285 nm (bandwidth 5 nm) at which the fluorescence of tryptophan residue is excited was measured.

  FIG. 2 (A) shows fluorescence spectra of the antibacterial agent measurement samples, control lysozyme solutions, and control triclosan samples obtained in Examples 1 to 3, respectively. FIG. 2B shows the fluorescence spectra of the antibacterial agent measurement samples and control lysozyme solutions obtained in Examples 4 to 6, respectively. FIG. 2 (C) shows the fluorescence spectra of the dialysis product measurement samples and control lysozyme solutions obtained in Comparative Examples 1 to 3, respectively. In the figure, cLZ indicates a control triclosan sample in which the control lysozyme solution, cTCS10, cTCS15 and cTCS30 have a triclosan concentration of 0.07 μmol / mL, 0.11 μmol / mL and 0.21 μmol / mL, respectively.

  Regarding the fluorescence of tryptophan residues, when the tryptophan residues are buried in a hydrophobic environment, the maximum fluorescence intensity (FImax) usually increases, and the wavelength of maximum absorption (λmax) shifts to the short wavelength side (blue shift). From the results shown in FIG. 2 (A), the fluorescence emission spectrum of the measurement sample of the control lysozyme solution shows a sharp peak with a maximum wavelength of 347 nm and a steep peak typical of tryptophan residues partially buried in a nonpolar environment. It can be seen that the wavelength end of the gradient is shown. However, the fluorescence spectra of the measurement samples for the antibacterial agents obtained in Examples 1 to 3 have peaks (blue shifts) at wavelengths of 346.4 nm, 346 nm, and 345 nm, respectively, and triclosan / lysozyme (molar ratio). It can be seen that the fluorescence intensity at the absorption maximum wavelength increases with an increase in.

  Moreover, from the results shown in FIG. 2B, the fluorescence spectra of the measurement samples for the antibacterial agents obtained in Examples 4 to 6 are the measurement samples for the antibacterial agents obtained in Examples 1 to 3. It can be seen that the same tendency as the fluorescence spectrum of is shown. From these results, it is suggested that the solution containing triclosan and lysozyme is dried to remove the solvent, whereby the tryptophan residue in lysozyme is further buried in a nonpolar environment.

  From the results shown in FIG. 2 (A), only a peak that can be ignored in the wavelength region of the protein was observed in the fluorescence spectrum of the control triclosan sample. Therefore, from these results, an increase in the fluorescence intensity at the absorption maximum wavelength in the fluorescence spectrum of the measurement sample of each antibacterial agent obtained in Examples 1 to 6 indicates the formation of a ground state complex, This suggests that during the lifetime, triclosan, which is a hydrophobic quencher, comes into contact with a fluorescent tryptophan residue. In addition, since the phenol compound causes a π-π interaction with an aromatic residue in a protein such as tryptophan residue, the increase in the fluorescence intensity at the absorption maximum wavelength is caused by triclosan via a non-covalent bond by a π-π bond. It is suggested that this is due to the interaction between the phenolic group of lysozyme and the tryptophan residue in lysozyme.

  Further, from the results shown in FIG. 2 (C), the fluorescence spectra of the samples for measurement of each dialysis product obtained in Comparative Examples 1 to 3 obtained by dialyzing a solution obtained by simply mixing lysozyme and triclosan were compared with each other. Compared to the fluorescence spectrum of the lysozyme solution, there is no increase in the fluorescence intensity or peak shift at the absorption maximum wavelength, and the fluorescence intensity at the absorption maximum wavelength disappears slightly, but it is the same as the fluorescence spectrum of the control lysozyme solution. I know that there is. From these results, the increase in the fluorescence intensity at the absorption maximum wavelength in the fluorescence spectra of the measurement samples of the antibacterial agents obtained in Examples 1 to 6 is not only the interaction between lysozyme and triclosan, but also the residual lysozyme tryptophan. It is suggested that the indole side chain of the group is due to being located in a more hydrophobic environment, ie lysozyme molecule.

Test example 3 (electrophoresis analysis)
In order to evaluate the influence of the interaction with triclosan on the three-dimensional structure of the lysozyme molecule, electrophoresis analysis of each antibacterial agent obtained in Examples 1 to 3 was performed using a denaturing gel and a non-denaturing gel.

  In accordance with the Laemmli method, each antibacterial agent obtained in Examples 1 to 3 was added in a non-reduced state or a reduced state with 4 to 15% by weight polydosyl containing 0.1% by weight sodium dodecyl sulfate (SDS). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter referred to as non-reducing SDS-PAGE or reducing SDS-PAGE) using acrylamide gel was performed. The protein band was stained with Coomassie Brilliant Blue R-250 (CBB).

  The result of reduced SDS-PAGE is shown in the drawing-substituting photograph of FIG. In addition, the result of non-reducing SDS-PAGE is shown in the drawing-substituting photograph of FIG. In the figure, “Mr” is a molecular weight marker, “C” is lysozyme alone, “10” is the antibacterial agent obtained in Example 1, “15” is the antibacterial agent obtained in Example 2, and “30” is The antibacterial agent obtained in Example 3 is shown.

  In addition, each antibacterial agent obtained in Examples 1 to 3 was subjected to acidic polyacrylamide gel electrophoresis (hereinafter referred to as acidic) on a 15% by mass native polyacrylamide gel (pH 4.5) according to the method of Lewis et al. (Referred to as PAGE). The protein band was stained with CBB. The result of acidic PAGE is shown in the drawing-substituting photograph of FIG. In the figure, “N” is natural lysozyme, “C” is lysozyme alone, “10” is the antibacterial agent obtained in Example 1, “15” is the antibacterial agent obtained in Example 2, and “30” is The antibacterial agent obtained in Example 3 is shown.

  From the results shown in FIG. 3 (A) and FIG. 3 (B), in both reduced SDS-PAGE and non-reduced SDS-PAGE, the mobility of the band corresponding to the electrophoresis pattern and the fraction containing lysozyme. It turns out that there is no difference. Further, from the result shown in FIG. 3C, it can be seen that even when the value of triclosan / lysozyme (molar ratio) is increased, the mobility of the protein band is not changed. Therefore, from these results, in each antibacterial agent obtained in Examples 1 to 3, polymerization or fragmentation of lysozyme molecules by triclosan did not occur, and triclosan did not affect the three-dimensional structure of lysozyme molecules. It is suggested.

Test Example 4 (Antimicrobial activity test 1)
By the liquid broth method, the antibacterial activity test against bacteria by each antibacterial agent obtained in Examples 1 to 3 was performed as follows. As test bacteria, Staphylococcus aureus (NBRC 14462), Staphylococcus epidermidis (ATCC 12228), and Streptococcus zooepidemicus negative (Streptococcus zooepidemicus) and wild-type bacteria of Streptococcus zooepidemicus Escherichia coli (NBRC 3301) and Pseudomonas aeruginosa (ATCC 27853) were used. Staphylococcus epidermidis and Pseudomonas aeruginosa were obtained from the American Type Culture Collection. Staphylococcus aureus and Escherichia coli were obtained from the National Institute of Product Evaluation Technology Biotechnology Headquarters and Biogenetic Resources Division. The wild strain of Streptococcus zooepidemicus was obtained from the Bacteriological Institute of Animal Hospital, Zurich, Switzerland.

After washing the cells in the logarithmic growth phase grown in Brain Heart Infusion Medium [manufactured by Nissui Pharmaceutical Co., Ltd.], 1% by weight of the bird so that it becomes 10 ⁵ to 10 ¹⁴ colony forming units (CFU) / mL. Resuspension broth of each bacterium was obtained by resuspension in petit case soy broth (TSB) [manufactured by Nissui Pharmaceutical Co., Ltd., pH 7.3].

  As the test substance, each antibacterial agent or lysozyme obtained in Examples 1 to 3 was added to the 1% by mass TSB so that the concentration was twice the protein concentration shown in Tables 3 to 7, and mixed. A solution was obtained. Next, each of the obtained mixed solutions was mixed with an equal amount of the resuspension broth to obtain bacterial mixtures having various protein concentrations shown in Tables 3-7. Each bacterial mixture obtained was incubated at 30 ° C. for 2 hours or 24 hours.

  In addition, as a test substance, triclosan alone was added to the 1% by mass TSB so as to be twice the concentration of triclosan in the bacterial mixture containing each antibacterial agent obtained in Examples 1 to 3, and a mixed solution was obtained. Obtained. Next, each mixed solution obtained was mixed with an equal amount of the resuspension broth to obtain a bacterial mixture. Each bacterial mixture obtained was incubated at 30 ° C. for 2 hours or 24 hours. The bacterial mixture having the same concentration as the triclosan concentration in the bacterial mixture containing the antimicrobial agent obtained in Example 1 is TCS10, and the bacterial mixture having the same concentration as the triclosan concentration in the bacterial mixture containing the antimicrobial agent obtained in Example 2 is TCS15. The bacterial mixture having the same concentration as the triclosan concentration in the bacterial mixture containing the antibacterial agent obtained in Example 3 is referred to as TCS30.

  In addition, incubation was performed in the same manner as described above without adding the test substance to obtain a control bacterial mixture having a protein concentration of “0”. Dilutions obtained by serially diluting 30 μL of each bacterial mixture after incubation or 30 μL of the control bacterial mixture with physiological saline were respectively prepared on a normal agar plate medium [trade name: Compact Dry, manufactured by Nissui Pharmaceutical Co., Ltd. "Nissui"] was smeared. After incubation of each bacterial mixture or control bacterial mixture smeared on normal agar plates for 18 hours at 37 ° C., colony forming units (CFU) were measured and the viable count of each bacterial mixture or control bacterial mixture (log CFU / mL medium). ) Was calculated. The antibacterial activity test was performed three times. Moreover, the viable count was the average value of the results of each test.

  The number of viable bacteria after incubation with each antibacterial agent, lysozyme alone or triclosan alone obtained in Examples 1 to 3 is shown in Tables 3 to 5. Table 3 shows the number of viable bacteria of Staphylococcus aureus after incubation with each antibacterial agent obtained in Examples 1 to 3, lysozyme alone or triclosan alone, and Table 4 shows each antibacterial agent obtained in Examples 1 to 3. Table 5 shows the number of living Staphylococcus epidermidis after incubation with lysozyme alone or triclosan alone. Table 5 shows the production of Streptococcus zooepidemicus after incubation with each antibacterial agent, lysozyme alone or triclosan alone obtained in Examples 1-3. The number of bacteria is shown respectively. In addition, Tables 6 to 7 show the results of examining the number of viable bacteria after incubation with each antibacterial agent obtained in Examples 1 to 3, lysozyme alone or triclosan alone. Table 6 shows the number of viable Escherichia coli after incubation with each antibacterial agent, lysozyme alone or triclosan alone obtained in Examples 1 to 3, and Table 7 shows each antibacterial agent or lysozyme obtained in Examples 1 to 3. The viable cell counts of Pseudomonas aeruginosa after incubation with simple substance or triclosan simple substance are shown. In Tables 3 to 7, the triclosan concentration in TCS10 and the triclosan concentration in the bacterial mixture containing the antimicrobial agent obtained in Example 1, the triclosan concentration in TCS15 and the bacterial mixture containing the antimicrobial agent obtained in Example 2 The viable cell counts in TCS10, TCS15, and TCS30 are displayed so that the triclosan concentration of TCS10 and the triclosan concentration in TCS30 and the triclosan concentration in the bacterial mixture containing the antibacterial agent obtained in Example 3 correspond to each other.

  From the results shown in Tables 3 to 5, each antibacterial agent obtained in Examples 1 to 3 has a stronger antibacterial activity against Staphylococcus aureus, which is a gram-positive bacterium, compared to lysozyme alone and triclosan alone. It can be seen that In particular, lysozyme alone and triclosan alone have low antibacterial activity against Staphylococcus epidermidis and Streptococcus zooepidemicus, but each antibacterial agent obtained in Examples 1 to 3 shows strong antibacterial activity. Further, from the results shown in Tables 6 to 7, the antibacterial agents obtained in Examples 1 to 3 were more effective against Escherichia coli and Pseudomonas aeruginosa, which are Gram-negative bacteria, compared to lysozyme alone and triclosan alone, respectively. It can be seen that it exhibits strong antibacterial activity. From these results, it can be seen that each of the antibacterial agents obtained in Examples 1 to 3 has a broad antibacterial spectrum that cannot be obtained with lysozyme alone and triclosan alone, and has strong antibacterial activity.

Test Example 5 (Antimicrobial activity test 2)
Instead of each antibacterial agent or lysozyme obtained in Examples 1 to 3, using the antibacterial agent obtained in Example 1 or the antibacterial agent obtained in Comparative Example 4, the protein concentration is 60 μg / mL. In the same manner as in Test Example 4 except that a bacterial mixture was obtained, Staphylococcus epidermidis and Gram negative, which are Gram-positive bacteria obtained by the antibacterial agent obtained in Example 1 and the antibacterial agent obtained in Comparative Example 4, respectively. Antibacterial activity test was carried out against each bacterium Escherichia coli.

  Table 8 shows the results of examining the viable cell count after incubation of Staphylococcus epidermidis and Escherichia coli with each antibacterial agent obtained in Example 1 or the antibacterial agent obtained in Comparative Example 4.

  From the results shown in Table 8, the antibacterial agent obtained in Example 1 is a gram-positive bacterium, Staphylococcus epidermidis, and the antibacterial agent obtained by simply mixing lysozyme and triclosan (Comparative Example 4). It can be seen that it exhibits strong antibacterial activity against any of the gram-negative bacteria Escherichia coli. From these results, it can be seen that the antibacterial agent obtained in Example 1 has strong antibacterial activity that cannot be obtained by simply mixing lysozyme and triclosan.

Formulation Example Hereinafter, a formulation example of the cosmetic or pharmaceutical of the present invention will be shown.

(Formulation Example 1: Liquid deodorant)
Ingredient Mass%
Zinc paraphenol sulfonate 2.0
Ethanol 30.0
1,3-butylene glycol 3.0
Antibacterial agent obtained in Example 1 0.4
Isopropylmethylphenol 0.1
Polyoxyethylene hydrogenated castor oil 0.5
(Oxyethylene group average addition mole number 50)
Perfume appropriate amount Purified water balance Total 100.0

(Formulation example 2: Deodorant spray)
Ingredient Mass%
Purified water 30.0
Ethanol 20.0
Antibacterial agent obtained in Example 2 0.3
Isopropylmethylphenol 0.1
Dimethyl ether 49.6
Total 100.0

(Formulation example 3: Roll-on deodorant)
Ingredient Mass%
Ethanol 30.0
Polyoxyethylene hydrogenated castor oil 1.0
(Oxyethylene group average addition mole number 40)
Chlorohydroxyaluminum 10.0
Hydroxypropyl methylcellulose 41.4
Antibacterial agent obtained in Example 3 0.3
Isopropylmethylphenol 0.1
Perfume appropriate amount Purified water balance Total 100.0

(Formulation 4: Acne cream)
Ingredient Mass%
Stearyl alcohol 8.0
Stearic acid 3.0
Purified lanolin 6.0
Antibacterial agent obtained in Example 1 0.4
Glycerin 3.0
Glyceryl monostearate 2.0
Polyoxyethylene cetyl ether 3.0
Perfume appropriate amount Purified water balance Total 100.0

(Formulation Example 5: Acne Lotion)
Ingredient Mass%
1,3-butylene glycol 4.0
Glycerin 3.0
Ethanol 10.0
Polyoxyethylene oleyl ether 1.0
(Oxyethylene group average addition mole number 20)
Antibacterial agent obtained in Example 7 0.3
Citric acid 0.01
Sodium citrate 0.1
Perfume appropriate amount Purified water balance Total 100.0

(Formulation example 6: solid white powder)
Ingredient Mass%
Sericite 15.0
Kaolin 10.0
Titanium dioxide 5.0
Zinc myristate 5.0
Magnesium carbonate 5.0
Squalane 3.0
Glycerin triisooctanoate 2.0
Antibacterial agent obtained in Example 3 0.3
Coloring pigment Appropriate amount Perfume Appropriate amount Talc Balance Total 100.0

(Prescription Example 7: Hair Tonic)
Ingredient Mass%
Pantothenyl ethyl ether 0.2
Polyoxyethylene polyoxypropylene tetradecyl ether 0.5
(Oxyethylene group average addition mole number 20, oxypropylene group average addition mole number 6)
Antibacterial agent obtained in Example 8 0.5
Ethanol 40.0
Perfume appropriate amount Purified water balance Total 100.0

(A) is a sample for evaluation of each antibacterial agent obtained in Examples 1 to 6, a sample for evaluation of each dialysis product obtained in Comparative Examples 1 to 3, TCS (EtOH), and TCS (H2O). It is a graph which shows the result of having investigated the light absorbency in wavelength 280nm. (B) is a sample for evaluation of each antibacterial agent obtained in Examples 1 to 6, a sample for evaluation of each dialysis product obtained in Comparative Examples 1 to 3, TCS (EtOH) and TCS (H2O). It is a graph which shows the result of having investigated turbidity (absorbance in wavelength 500nm). (A) is a figure which shows the fluorescence spectrum of each measurement sample of each antibacterial agent obtained in Examples 1-3, a control lysozyme solution, and a control triclosan sample. (B) is a figure which shows the fluorescence spectrum of each measurement sample of each antibacterial agent obtained in Examples 4 to 6 and a control lysozyme solution. (C) is a figure which shows the fluorescence spectrum of each sample for a measurement of each dialysis product obtained by Comparative Examples 1-3, and each control lysozyme solution. (A) is a drawing-substituting photograph showing the results of reduction SDS-PAGE of each antibacterial agent obtained in Examples 1 to 3. (B) is a drawing-substituting photograph showing the results of non-reducing SDS-PAGE of each antibacterial agent obtained in Examples 1 to 3. (C) is a drawing-substituting photograph showing the results of acidic PAGE of each antibacterial agent obtained in Examples 1 to 3.

Claims (4)

  An antibacterial agent containing a dried product obtained by drying a solution containing lysozyme and triclosan.   A method for producing an antibacterial agent, comprising drying a solution containing lysozyme and triclosan.   The production method according to claim 2, wherein the solution is dried by freeze drying, spray drying, or vacuum drying.   A cosmetic or pharmaceutical comprising the antibacterial agent according to claim 1.