WO 2009/057835 1 PCT/KR2007/005437 Description ACIDIC ANTIBIOTIC COMPOSITION CONTAINING PERACID AND ACETYL SALICYLIC ACID Technical Field [1] The present invention relates to an antibiotic composition, more particularly to an antibiotic composition useful for sterilizing and disinfecting medical equipments, such as tweezers, capillary tubes, silicone tubes, vinyl tubes, forceps, silicone caps, silicone stoppers, scalpels, scalpel grips, endoscopes, and so forth. Background Art [2] Proper use and management of medical equipments are demanded in order to improve the quality of medical cares provided to patients. Especially, cleaning, sterilizing and disinfecting of medical equipments are important to protect the patients from the risk of secondary infection. Normally, sterilizing and disinfecting of medical equipment are carried out using a diluted version of an antibiotic solution. [3] At present, chlorine-based, organic acid-based and aldehyde-based antibiotic agents are widely used in sterilizing and disinfecting of medical equipments. [4] The most typical example of chlorine-based antibiotic agents is sodium hypochlorite. Although sodium hypochlorite has a broad antibiotic spectrum, it may cause envi ronmental pollution. [5] Organic acid-based antibiotic agents provide only limited effect of sterilizing and dis infecting. [6] Examples of aldehyde-based antibiotic agents include glutaraldehyde, glyoxylic acid and formaldehyde. Among them, glutaraldehyde is the most widely used, at present. Although the agents containing glutaraldehyde as active ingredient have a broad antibiotic spectrum, they may cause offensive odor, toxicity to human body and allergic reactions. [7] Recently, use of antibiotic agents containing peracetic acid as active ingredient is increasing rapidly. Peracetic acid has an excellent antimicrobial activity and breaks down to safe and environment friendly residues (acetic acid, water and oxygen). [8] Normally, the peracetic acid contained in an antibiotic agent is provided in the form of a peracetic acid composition of a high concentration. [9] The highly concentrated peracetic acid composition is normally diluted to a peracetic acid concentration of 0.1 to 0.35 % when used to sterilize or disinfect medical equipments. [10] Normally, a peracetic acid composition is prepared and used in the form of a diluted solution. In general, such a diluted solution is for single use. It is because when the WO 2009/057835 2 PCT/KR2007/005437 conventional antibiotic composition, which comprises highly concentrated acetic acid and hydrogen peroxide as main components, is diluted with purified water, the antibiotic activity decreases with time. Typically, a diluted solution stored for 7 days or longer exhibits significantly reduced sterilizing and disinfecting performance. Hence, the conventional peracetic acid composition is disadvantageous in that it has to be used within a week or so when prepared into a diluted solution. Disclosure of Invention Technical Problem [11] The present invention has been made to solve the storage problem of a diluted peracetic acid solution. An object of the present invention is to provide an antibiotic composition which is capable of maintaining the concentration of peracetic acid in a diluted solution which is prepared by diluting a highly concentrated peracetic acid composition above a predetermined effective concentration level, even when stored for a long period of time of about 21 days, and a method for preparing the antibiotic composition. Technical Solution [12] In an embodiment of the present invention, an antibiotic composition containing acetic acid and hydrogen peroxide is provided. The antibiotic composition comprises acetic acid, hydrogen peroxide, peracetic acid and acetylsalicylic acid. [13] The antibiotic composition may further comprise 1,1 -diphosphonic acid, citric acid, sodium citrate, lower alcohol and purified water. [14] Further, the composition may comprise acetylsalicylic acid in an amount of 0.1 % to 10 % based on weight. [15] In another embodiment of the present invention, there is provided a preparation method of an antibiotic composition containing acetic acid and hydrogen peroxide. The antibiotic composition may be prepared by mixing a first solution comprising acetic acid, hydrogen peroxide and peracetic acid with a second solution comprising acetyl salicylic acid. [16] The first solution may further comprise 1,1 -diphosphonic acid, and the second solution may further comprise citric acid, sodium citrate, lower alcohol and purified water. Advantageous Effects [17] The conventional antibiotic composition containing highly concentrated acetic acid and hydrogen peroxide as main components exhibited decreased antibiotic activity with time when diluted with purified water. In general, a diluted solution stored for 7 days or longer shows significantly reduced sterilizing and disinfecting performance. In contrast, the antibiotic composition of the present invention can maintain the properties WO 2009/057835 3 PCT/KR2007/005437 for over 21 days, since acetylsalicylic acid is consistently hydrated in diluted solution to provide acetic acid, and the hydrate salicylic acid also acts as another antibiotic agent. Best Mode for Carrying Out the Invention [18] The present invention relates to an antibiotic composition comprising a mixture of a first solution comprising acetic acid, hydrogen peroxide and peracetic acid and a second solution comprising acetylsalicylic acid at a predetermined proportion. The antibiotic composition is diluted with purified water before use. For actual use, the concentration of peracetic acid in the diluted solution may be adjusted to 0.05-0.5 %. [19] In the first solution, peracetic acid is produced from acetic acid and hydrogen peroxide, and a chemical equilibrium is reached by the following equation: [20] CH COOH+H 02 - CH COOOH+H O (1) [21] In the equilibrated state, peracetic acid and hydrogen peroxide react with water to generate nascent oxygen, which exhibits antibiotic effect through oxidizing property. Whereas peracetic acid exhibits superior antibiotic effect in the short term, hydrogen peroxide exhibits superior antibiotic effect in the long term. [22] When the first solution is diluted by purified water, the equilibrium in the equation (1) may move toward decreasing the concentration of peracetic acid. Accordingly, in order that the effective concentration of peracetic acid can be maintained for a long period of time even when the first solution is diluted by purified water, through inhibiting the reverse reaction in the equation (1), a second solution containing acetyl salicylic acid as main component is mixed with the first solution at a predetermined proportion. [23] In the diluted solution of the antibiotic composition prepared by mixing the first solution and the second solution, acetylsalicylic acid reacts with water to produce an acetate anion: [24] Acetylsalicylic acid + H 20 "+ Salicylic acid + CH COO (2) [25] The acetate anion reacts with proton to produce acetic acid: [26] CH COO + H* CH COOH (3) [27] As a result, in the diluted solution of the antibiotic composition obtained by diluting the mixture of the first solution and the second solution with purified water, the acetic acid produced from the acetylsalicylic acid moves the equilibrium in the equation (1) to the right side and, thereby, effectively prevents the decrease of the concentration of peracetic acid in the long term. In addition, the salicylic acid produced from the hydration of acetylsalicylic acid further strengthens the antibiotic activity of the diluted solution. [28] The antibiotic composition containing peracetic acid according to the present WO 2009/057835 4 PCT/KR2007/005437 invention is prepared as follows. Each of the first solution and the second solution is prepared. Subsequently, the first solution and the second solution are mixed at a pre determined proportion to obtain an antibiotic agent solution. The antibiotic agent solution is mixed with purified water to obtain a diluted solution. In the diluted solution, the concentration of peracetic acid may be from 0.05 % to 0.5 %. [29] For the preparation of the first solution, 84-25 % of purified water and 10-40 % of acetic acid are mixed well in a container. Subsequently, 5-30 % of hydrogen peroxide is added and mixed well. Then, acetic acid reacts with hydrogen peroxide to give peracetic acid at an equilibrium concentration. [30] Next, 1-5 % of 1,1-diphosphonic acid, which is a stabilizer, is added to stabilize the resultant product. The resulting mixture is warmed at 35-45 'C for 4-7 hours and aged at room temperature for 3 days to obtain the first solution. [31] As a consequence, by the equilibrium reaction of producing peracetic acid, the first solution contains 0.05-15 % of peracetic acid. [32] The second solution is prepared by mixing acetylsalicylic acid dissolved in lower alcohol with an aqueous buffer. The resulting mixture may contain 0.1-10 % of acetyl salicylic acid based on weight. Specifically, the lower alcohol may be one selected from the group consisting of methanol, ethanol, propyl alcohol, isopropyl alcohol and a combination thereof. In the mixture, the weight proportion of the lower alcohol may be from 20 to 70 %. [33] The aqueous buffer may be prepared by mixing one selected from the group consisting of phosphoric acid, citric acid, phosphate, citrate and a combination thereof with purified water. In the second solution, the weight proportion of electrolytes, e.g., phosphoric acid, may be from 1 to 10 %, and that of purified water may be from 10 to 78.9 %. [34] Further, benzotriazole may be added to the second solution in order to prevent the surface damage of the medical equipments to be sterilized. In that case, the addition amount of benzotriazole may be adjusted within the range from 1 to 5 weight % of the second solution. Mode for the Invention [35] <Example 1> [36] In a mixture solution of the first solution and the second solution, the change of peracetic acid concentration with time was measured at various concentrations of acetylsalicylic acid. [37] For this purpose, the addition amount of acetylsalicylic acid to the second solution was adjusted, so that the final mixture of the first solution and the second solution contained 0.0 %, 0.4 %, 1.2 % and 2.0 % acetylsalicylic acid. The initial peracetic acid WO 2009/057835 5 PCT/KR2007/005437 concentration of the four solutions was adjusted to about 0.13 weight %. [38] Table 1 [Table 1] [Table ] Change of peracetic acid concentration with time was measured at various co ncentrations of acetylsalicylic acid (ASA) Days ASA 0.0 % ASA 0.4 % ASA 1.2 % ASA 2.0 % 0 0.134 0.137 0.137 0.137 6 0.134 0.138 0.141 0.142 12 0.134 0.141 0.143 0.146 20 0.133 0.140 0.143 0.154 30 0.131 0.138 0.142 0.154 [39] [40] As seen in Table 1, when the acetylsalicylic acid concentration was 0 %, the peracetic acid concentration decreased slightly 20 days later. In contrast, as the acetyl salicylic acid increased, the concentration of peracetic acid gradually increased with time. This indicates that the peracetic acid production reaction moved favorably for the forward reaction as acetic acid was supplied by the hydrolysis of acetylsalicylic acid. As a result, it can be predicted that the addition of acetylsalicylic acid at a pre determined concentration to the mixture of the first and second solutions diluted with purified water may effectively prevent the movement of equilibrium or decrease of the peracetic acid concentration caused by the reaction with organic materials or other con taminates, over a long period of time. [41] In Examples 2-4 described below, antibiotic activity of the diluted solution of the peracetic acid containing antibiotic compositions including acetylsalicylic acid was tested. The first solution and the second solution were prepared and mixed at a pre determined proportion to obtain an antibiotic agent solution. This antibiotic agent solution was diluted with purified water, so that the peracetic acid concentration in the diluted solution was 500 ppm, 750 ppm and 1000 ppm. [42] Staphylococcus aureus, Escherichia coli (E. coli) and Candida albicans were used to measure the antibiotic activity. The strains were obtained from the KFCC (Korean Federation of Culture Collections). Strains were cultured in commonly used media for 24 hours and stored at 4 0 C for test. [43] Specifically, each of the diluted antibiotic solutions was mixed with 0.1 mL of medium containing each strain under shaking. After a duration of reaction time, 1 mL of the solution was immediately taken into a test tube containing a neutralizing agent.
WO 2009/057835 6 PCT/KR2007/005437 After adequately diluting with physiological saline, living cells were counted after st reaking on a plate. For the neutralizing agent, 4.26 % sodium phosphate dibasic, 3 % Tween, and so forth were used. [44] The experiment was performed at 20±1 'C and the reaction times were 0, 5, and 10 minutes. [45] [46] <Example 2> [47] Three experiments were performed to evaluate antibiotic activity against Staphylococcus aureus. [48] Experiment 1: The initial number of Staphylococcus aureus cells was 1.9xI0 9 CFU/ mL. Experiment was performed under hygienic condition and the peracetic acid con centration of the diluted solution was set at 500 ppm. When the acetylsalicylic acid concentration was 0 % (control), the cell number decreased to 1.8x 10 3 CFU/mL after 5 minutes, and no microorganism was detected after 10 minutes. In contrast, when the acetylsalicylic acid concentration was 1.2 %, no microorganism was observed both after 5 and 10 minutes, under the same condition. [49] Experiment 2: The initial number of Staphylococcus aureus cells was 1.9xI0 9 CFU/ mL. Experiment was performed under contamination condition (1 % yeast) and the peracetic acid concentration was set at 750 ppm. When the acetylsalicylic acid con centration was 1.2 %, the number of Staphylococcus aureus cells decreased to 4.9x10 4 CFU/mL after 5 minutes, and 8.Ox 10' CFU/mL after 10 minutes. [50] Experiment 3: The initial number of Staphylococcus aureus cells was 1.9xI09 CFU/ mL. Experiment was performed under contamination condition (1 % yeast) and the peracetic acid concentration was set at 1000 ppm. When the acetylsalicylic acid con centration was 0 % (control), the cell number decreased to 2.2x10 4 CFU/mL after 5 minutes, and 1.1xI0 4 CFU/mL after 10 minutes. The rate of the decrease of the mi croorganism was slow. In contrast, when the acetylsalicylic acid concentration was 1.2 %, no microorganism was observed both after 5 and 10 minutes, under the same condition. [51] Comparing the result of the three antibiotic activity experiments for Staphylococcus aureus, the diluted antibiotic solution containing acetylsalicylic acid consistently exhibited better antibiotic activity than the control sample containing no acetylsalicylic acid. Further, when the antibiotic activity of the sample containing acetylsalicylic acid was adjusted similarly to that of the control sample after 5 minutes by reducing the peracetic acid concentration than that of the control sample, however, the antibiotic effect of the control sample decreased considerably compared to that of the sample containing acetylsalicylic acid after 10 minutes. [52] WO 2009/057835 7 PCT/KR2007/005437 [53] <Example 3> [54] An experiment was performed to evaluate antibiotic activity against E. coli. The initial number of E. coli cells was 1.9x10 9 CFU/mL. Experiment was performed under contamination condition (1 % yeast) and the peracetic acid concentration was set at 500 ppm. When the acetylsalicylic acid concentration was 0 % (control), the cell number decreased to 1.6x 104 CFU/mL after 5 minutes, and no microorganism was detected after 10 minutes. In contrast, when the acetylsalicylic acid concentration was 1.2 %, no microorganism was observed both after 5 and 10 minutes, under the same condition. [55] [56] <Example 4> [57] Two experiments were performed to evaluate antibiotic activity against Candida albicans. [58] Experiment 4: The initial number of Candida albicans cells was 4.4x10' CFU/mL. Experiment was performed under hygienic condition and the peracetic acid con centration in the diluted solution was set at 750 ppm. When the acetylsalicylic acid concentration was 0 % (control), the cell number decreased to 4.9x 10 3 CFU/mL after 5 minutes, and no microorganism was detected after 10 minutes. In contrast, when the acetylsalicylic acid concentration was 1.2 %, no microorganism was observed both after 5 and 10 minutes, under the same condition. [59] [60] Experiment 5: The initial number of Candida albicans cells was 4.4x10' CFU/mL. Experiment was performed under hygienic condition and the peracetic acid con centration in the diluted solution was set at 1000 ppm. When the acetylsalicylic acid concentration was 0 % (control), the cell number decreased to 1.4x102 CFU/mL, and no microorganism was detected after 10 minutes. In contrast, when the acetylsalicylic acid concentration was 1.2 %, no microorganism was observed both after 5 and 10 minutes, under the same condition. Industrial Applicability [61] The antibiotic composition according to the present invention is an acidic antibiotic composition comprising acetic acid, hydrogen peroxide, peracetic acid and acetyl salicylic acid as main components. In the composition, peracetic acid is produced from the reaction of acetic acid and hydrogen peroxide, and remains in equilibrium state. Normally, the antibiotic composition is diluted with purified water, so that the peracetic acid concentration is from 0.1 to 0.35 %, and is used for sterilizing and dis infecting of medical equipments. [62] Although the present invention has been described in some detail by way of il- WO 2009/057835 8 PCT/KR2007/005437 lustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of the invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.