KR101776696B1 - Isosorbide derivatives having anti-microbial activity and anti-microbial composition composition the same - Google Patents

Abstract

The present invention relates to isosorbide derivatives having anti-microbial activity and an anti-microbial composition comprising the same as an active ingredient. The isosorbide derivatives having a structure represented by chemical formula 1 have no cytotoxicity, are dissolved in an aqueous solution, and have excellent antibiotic sensitivity and anti-microbial activity against resistant strains. Also, diffusivity of anti-microbial activity is improved by 27 to 66% compared to norfloxacin which has been used as an existing antibiotic. Accordingly, an anti-microbial composition comprising the isosorbide derivatives can be used as a medicine, a cosmetic additive or a food preservative for preventing or treating bacterial infectious diseases.

Description

[0001] The present invention relates to an isosorbide derivative exhibiting antibacterial activity and an antimicrobial composition comprising the same as an active ingredient.

The present invention relates to an isosorbide derivative exhibiting an antimicrobial activity and an antimicrobial composition containing the same as an active ingredient. More particularly, the present invention relates to an isosorbide derivative exhibiting an antimicrobial activity and an antimicrobial composition containing the isosorbide derivative as an active ingredient A pharmaceutical composition, a cosmetic additive composition, or a food preservative additive composition.

Antibiotics are substances that have antimicrobial activity against bacteria, in particular, substances that have excellent antimicrobial activity by inhibiting the system of bacterial cell wall or protein synthesis, or those produced from such substances.

These antibiotics are widely used to treat diseases caused by bacterial infection. The most common antibiotic is penicillin manufactured by the British physician Alexander Fleming in 1928. Penicillin was the first antibiotic manufactured by mankind to respond to bacteria in earnest. This led to the development of penicillin-based or better antibiotics. In particular, recently, antibiotic-resistant bacteria that are resistant to antibiotics are emerging due to resistance to existing antibiotics, and new diseases such as acquired immunodeficiency syndrome (AIDS) are increasing, and new drugs capable of replacing existing antibiotics have been developed In fact.

Although many antibiotics have been separated from natural products or synthesized, they have been able to treat many diseases and infectious diseases. However, due to excessive abuse of antibiotics worldwide, the incidence and resistance rate of resistant bacteria to existing antibiotics are increasing, and in addition, Infectious pathways have accelerated the spread of resistant strains and have become a serious social problem (Hiramatsu K, et al. , 1997, Lancet, 350: 1670-1673; Glod H. et al. , 1996, N. Engl. Med 335: 1445-1453.).

In order to solve the above problems, antibiotics such as penicillin and methicillin antibiotics resistant strains such as MRSA (methicillin resistant staphylococcus ) and MRSE (methicillin resistant Staphylococcus epidermidis ) The use of TEICOPLANIN has been continuously increasing. However, due to the excessive use of the above-mentioned antibiotics, enterococci , a pathogenic bacterium resistant to vancomycin, which is called the last antibiotic, has been found, and Enterococcus faecalis ( Enterococcus faecalis ) Mycobacterium tuberculosis and Pseudomonas aeruginosa are resistant to all known antibiotics (Stuart B. Levy, Scientific American, 46-53, 1998). In addition, the antibiotics are not used as injections because they have poor solubility in an aqueous solution, and since they are low in persistence, resistant bacteria can not be removed only by a single dose, and thus they are cytotoxic to normal cells as they are administered for a long time.

Accordingly, the present inventors have been studying an antimicrobial composition having no cytotoxicity against normal cells and improved solubility and antibiotic activity in an aqueous solution, and it is possible to produce economical and environmentally friendly mass production using an enzymatic catalyst The isosorbide derivatives exhibited strong antimicrobial activity against antibiotic resistant and resistant bacteria as well as improved solubility and antimicrobial activity diffusivity in aqueous solution. Thereby completing the present invention.

It is an object of the present invention to provide an isosorbide derivative which is free from cytotoxicity and exhibits excellent antimicrobial activity.

It is another object of the present invention to provide an antimicrobial composition comprising the isosorbide derivative as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for antimicrobial use, a cosmetic additive composition, or a food preservative additive composition containing the composition as an active ingredient.

In one embodiment, the present invention provides an isosorbide derivative free from cytotoxicity and exhibiting excellent antimicrobial activity.

In the present invention, the isosorbide derivative is a compound in which norfloxacin, which is a conventional antibiotic, is bonded to both hydroxyl groups of isosorbide and has a structure represented by the following formula (1).

[Chemical Formula 1]

In the present invention, the compound of formula (1) is hereinafter referred to as isosorbide norfloxacin (ISDN).

In the present invention, the isosorbide dinoflopsaccharide (ISDN) is not limited thereto, but may include, for example, isosorbide represented by Chemical Formula 2 and NO 2 represented by Chemical Formula 3 as shown in Reaction Scheme 1 below. Can be prepared by esterification reaction of norfloxacin. At this time, it is preferable to use an enzymatic catalyst for the esterification reaction. This results in the production of isosorbide dinoflouroxyne (ISDN) by eliminating only the enzyme catalyst without further purification during the purification process since it does not produce any byproducts during the esterification reaction.

[Reaction Scheme 1]

In one specific example, the isosorbide dinoflopsacin (ISDN) of the present invention is prepared by dissolving isosorbide and norfloxacin in an organic solvent, adding an enzymatic catalyst, stirring and removing the solvent Can be obtained.

The organic solvent may be any organic solvent that does not inhibit the esterification reaction. Examples of the organic solvent include 1,4-dioxane, toluene, N-methyl-2-pyrrolidone, DMF, dimethylacetamide) and the like can be used.

The enzymatic catalyst may be any of those used for the esterification reaction between compounds. For example, Novozyme 435 (lipase B, EC 3.1.1.3, from Candida antarctica ) may be used.

The isosorbide dinofloxacin (ISDN) of the present invention is free from cytotoxicity, is soluble in an aqueous solution, has excellent antimicrobial activity against pathogenic strains and antibiotic resistant strains, and is also effective against norfloxacin The drug diffusivity is improved by about 7 to 89%.

At this time, the pathogenic strain is selected from the group consisting of Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Staphylococcus aureus , Staphylococcus epidermidis , and Salmonella typhimurium . However, the present invention is not limited thereto.

The antibiotic-resistant strain may be any one or more selected from the group consisting of drug-resistant Escherichia coli and antibiotic-resistant Pseudomonas aeruginosa , It is not.

Accordingly, the isosorbide dinoflopsaccharide (ISDN) of the present invention is useful for preventing or treating a bacterial infectious disease, for adding a cosmetic additive for cleaning hands, feet, face or the like, Can be usefully used.

In another aspect, the present invention provides an antimicrobial composition comprising isosorbide dinoflopoaxin (ISDN) as an active ingredient.

The antimicrobial composition of the present invention may further contain, as an active ingredient, isosorbide dinofloparacin (ISDN) together with an optional component exhibiting an antibacterial activity.

Isosorbide dinofloparacin (ISDN) as an active ingredient in the composition of the present invention is contained in an amount of 0.01 to 50% by weight, preferably 0.01 to 30% by weight, more preferably 0.01 to 10% by weight, % ≪ / RTI > by weight. When the content of ISDN is less than 0.001% by weight, the antibacterial activity is insufficient. When the content of ISDN is more than 50% by weight, the effect of increasing the content is not proportionally increased, There is a problem that stability is not ensured.

As one specific use of the present invention, the antimicrobial composition of the present invention can be used as a pharmaceutical composition for preventing or treating a bacterial infectious disease.

In the present invention, the bacterial infectious disease is a disease that occurs when a bacterium enters a blood, body fluids, or tissues and starts to live there. The diseases include laryngitis, prostatitis, dermatitis, food poisoning, keratitis, osteomyelitis, meningitis, , Influenza, typhoid, heterozygous, or tuberculosis, but are not particularly limited thereto.

When the antimicrobial composition of the present invention is used as a pharmaceutical composition, in addition to isosorbide dinofromosin (ISDN) as an active ingredient, a further pharmaceutically acceptable carrier may be further contained. The pharmaceutically acceptable carriers are those conventionally used in the formulation and include carbohydrate compounds such as lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose and the like, acacia rubber, calcium phosphate, (Such as corn oil, cottonseed oil, soybean oil, olive oil, coconut oil) such as, for example, water, syrup, salt solution, alcohol, gum arabic, , Polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be formulated in the form of powders, granules, capsules, suspensions, emulsions, oral preparations such as aerosols, external preparations, suppositories, and sterilized injection solutions according to a conventional method.

The pharmaceutical composition may prevent or treat a bacterial infectious disease, wherein the therapeutic method comprises administering the pharmaceutical composition in a pharmaceutically effective amount by various routes in a mammal such as a mouse, a mouse, a domestic animal, or a human. The above administration method can be carried out in any manner, for example, by oral, rectal or intravenous infusion, intraperitoneal administration, intramuscular injection, subcutaneous administration, or local administration by application or the like.

The dosage of the pharmaceutical composition may vary according to the formulation method, the administration method, the age, weight, sex, infection level, etc. of the patient. The daily dose of the pharmaceutical composition of the present invention is in the range of 0.01 to 10 mg / kg, preferably about 3 to 6 mg / kg on an adult basis. However, the dosage is not limited in any way to the scope of the present invention.

As another specific application, the antimicrobial composition of the present invention can be used as a cosmetic additive composition for maintaining cleanliness by inhibiting the growth of bacteria on the hands, feet, face or the like.

When the composition of the present invention is used as a cosmetic additive composition, in addition to isosorbide dinofloprolactin (ISDN) as an active ingredient, it may further include components commonly used in cosmetic compositions. For example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

The cosmetic additive composition of the present invention may be prepared in any formulation conventionally produced in the art and may be in the form of solutions, suspensions, emulsions, pastes, gels, creams, powders, soaps, surfactant- , A powder foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto. More specifically, it can be prepared as a nutritional cream, a convergent lotion, a soft lotion, a lotion, an essence, a nutritional gel or a massage cream.

When the formulation of the cosmetic additive composition is a paste, a cream or a gel, the carrier component may be an animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide Etc. may be used.

When the formulation of the cosmetic additive composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, a mixture of chlorofluorohydrocarbons , Propane / butane or dimethyl ether.

When the formulation of the cosmetic additive composition is a solution or an emulsion, a solvent, a solubilizer or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the cosmetic additive composition is in the form of a suspension, it may contain, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the cosmetic additive composition is an interface-active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

As another specific application of the present invention, the antimicrobial composition of the present invention can be used as a food preserving additive composition capable of preserving foods for a long time.

The food preservative additive composition of the present invention can be prepared by incorporating isosorbide dinoflopsacin (ISDN) as an active ingredient and one or more known food preservatives. Examples of the food preservative include sodium benzoate, potassium benzoate, calcium benzoate, dehydroacetic acid, potassium sorbate, calcium sorbate, methyl p-hydroxybenzoate, p-hydroxybenzoate. Sodium propionate, calcium propionate, and the like, but are not limited thereto.

There is no particular limitation on the kind of food using the food preserving additive composition of the present invention. Examples of food to which isosorbide dinofloprocyanate (ISDN) of the present invention can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and includes all the health foods in a conventional sense.

The isosorbide dinofloxacin (ISDN) of the present invention is free from cytotoxicity, is soluble in an aqueous solution, has excellent antimicrobial activity against pathogenic strains and antibiotic resistant strains, and is also effective against norfloxacin (ISDN) is effective for preventing or treating a bacterial infectious disease, a cosmetic additive, or a food additive for preventing or treating a bacterial infectious disease. Can be usefully used as a preservative.

1 is a diagram illustrating the synthesis process of isosorbide dinorfloxacin (ISDN) using isosorbide and norfloxacin.
FIG. 2 is a graph showing the separation time of isosorbide dinofloparacin (ISDN), isosorbide, and norfloxacin according to the present invention.
FIG. 3 is a graph showing the molecular weights of isosorbide dinofloparacin (ISDN) and isosorbide mononofluorosaccharide (ISMN) synthesized according to the present invention.
FIG. 4 is a graph showing the structures of ISOS, ISOS, and norfloxacin according to the present invention using ¹ H-NMR spectroscopy. FIG.
FIG. 5 shows the results of measuring the solubility of isosorbide dinofro- oxacin (ISDN) and norfloxacin in an aqueous solution according to the present invention.
6 is isobutyl an isobutyl cyano sorbitan Escherichia coli (E.coli, ATCC 25922) or Escherichia coli O157 cyano antibacterial activity against (E.coli O157, ATCC 25922) of the bead flow Dino saksin (ISDN) according to the invention (ISMN), isosorbide, and norfloxacin. ≪ / RTI >
7 is a graph showing the antimicrobial activity against Pseudomonas aeruginosa (ATCC 15692) of isosorbide dinoflopsacin (ISDN) according to the present invention against isosorbide mononofluorosaccharide (ISMN), isosorbide ), And norfloxacin. ≪ / RTI >
8 is a graph showing cytotoxicity of isosorbide dinofloparacin (ISDN) to normal cells compared with norfloxacin according to the present invention.
9 is a graph showing the antimicrobial persistence against Pseudomonas aeruginosa (ATCC 15692) of isosorbide dinofloparacin (ISDN) according to the present invention versus isosorbide mononoproxacin (ISMN), isosorbide ), Norfloxacin, cefotaxime, and ciprofloxacin.
FIG. 10 is a graph showing the effect of isosorbide dinofloparacin (ISDN) on E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 15692), S. typhimurium , KCTC 1926), or S. epidermidis (KCTC 1917) in comparison to norfloxacin.
11 is observed with isosorbide Dino flow saksin (ISDN) treated Escherichia coli cyano (E.coli, ATCC 25922) of transmission electron microscope (Transmission electron microscope) according to the present invention.

Hereinafter, the present invention will be described in more detail by way of examples and the like. It will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples and the like according to the gist of the present invention. will be.

Example  1: Preparation of isosorbide derivatives

50 mM of isoorbide (IIC chemical) and 100 mM of norfloxacin (Sigma-Aldrich Co., St. Louis, Mo.) were dissolved in 30 ml of DMF solution, and 10% (Sigma-Aldrich Co., Saint Louis, Mo.) was added and mixed, followed by stirring at 37 ° C for 24 hours. Thereafter, the reaction product was filtered through a filter paper to remove the lipase-immobilized resin to prepare an isosorbide derivative. Subsequently, the reactant from which the lipase immobilized resin was removed was centrifuged by adding water, and then the supernatant was separated using a separation HPLC system at a retention time of 1 ml / min at a retention time of 26 minutes and at a retention time of 35 minutes (See Fig. 1).

The HPLC system consists of a Waters 600 pump and controller, and a Waters 717 autosampler and a Waters 966 photodiode-array (PDA) detector are controlled by Millennium chromatography manager. Quantitative analysis of the purified material was performed by comparing the peak area with the external standard under the same conditions for the same concentration. In addition, the molecular weight of the purified material was measured using MALDI-TOF, and the molecular structure of the purified material was confirmed using ¹ H-NMR spectrum. Isosorbide and norfloxacin were used as control for molecular structure analysis. The results are shown in FIG. 2 to FIG.

2, free isosorbide, a compound in which a hydroxyl group at one side of isosorbide is bonded to norfloxacin (isosorbide mononorphosporin, ISMN), and both hydroxyl groups of isosorbide Peaks were observed at 11, 26, and 35 minutes, respectively, in the form of a compound in which adenosine monophosphate (ISOSB) was coupled to isopropyl myristate (Isosorbide dinofloxacin, ISDN). HPLC analysis showed that both hydroxyl groups of isosorbide It was confirmed that the yield of the combined type compound was the highest at 50% or more.

A compound in which one of the hydroxyl groups of the isosorbide is bonded to norfloxacin is treated with isosorbide mononofluorosaccharide (ISMN) and a compound in which two hydroxyl groups of isosorbide are bonded to two hydroxyl groups Isosorbide dinofloxacin (ISDN).

As shown in FIG. 3, it was confirmed that the molecular weights of purified isosorbide mononofluorosaccharide (ISMN) and isosorbide dinofloxacin (ISDN) were 447.42 and 748.75, respectively.

As shown in FIG. ^{4 1.5 (δH 3), 3.5} (δH 2), and 3.6 (δH ²⁾ the identified peaks in ppm are characteristic peaks of the furnace flow saksin (Figure 4C (a) and (b)), 4.5 It was confirmed that the peak at the (隆 H ¹ ) ppm shifts to the lower field is the peak indicated by the esterification result (Fig. 4C (a ')).

From the above results, it was found that isosorbide dinofloxacin (ISDN) was successfully synthesized and purified. In addition, since the by-product is not produced due to the use of the enzymatic catalyst, it is possible to purify the enzyme catalyst only by removing the enzyme catalyst, so that it is possible to mass-produce isosorbide dinofloxacin (ISDN) through an economical and eco-friendly process.

Test Example 1: Determination of solubility of isosorbide dinofloparacin (ISDN) in distilled water

0.1, 0.5, 1, 5, or 10 mg of isosorbide dinofloparacin (ISDN) purified in Example 1 was placed in an EP tube containing 1 ml of PBS (phosphate buffered saline), and the mixture was vortexed using a vortex mixer And the absorbance was measured at a wavelength of 620 nm using a spectrophotometer. As a negative control, PBS was used. As a positive control, 0.1 mg of norfloxacin was mixed with 1 ml of PBS. As a comparative group, 0.1, 0.5, 1, 5, or 10 mg of a substance mixed with 1 ml of PBS was used. The mean difference between the control group, the comparison group, and each experimental group was analyzed by Student's t-test. When the p-value was less than 0.05, the difference was statistically significant . The results are shown in Fig.

Experimental results show that norfloxacin is soluble in saline but not in distilled water. On the other hand, isosorbide dinofloxacin (ISDN) was found to have significantly improved solubility in distilled water compared to norfloxacin.

Test Example 2: Antimicrobial activity of isosorbide dinofloxacin (ISDN)

Micro-dilution analysis was carried out using seven pathogenic strains and eight antibiotic resistant strains in order to confirm whether ISOBS synthesized in the present invention can be used as an antimicrobial agent.

2-1. Microbial preparation

Escherichia coli (ATCC 25922), Escherichia coli O157, ATCC 25922, Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa ( Pseudomonas aeruginosa) aeruginosa , ATCC 15692) was purchased from the American Type Culture Collection and Listeria monocytogenes ( Listeria monocytogenes , KCTC 3710), were prepared to purchase from Salmonella typhimurium (Salmonella typhimurium, KCTC 1926), Philo and spa locus epidermidis (Staphylococcus epidermidis, KCTC 1917) Microbial Resources Center for South Korea (Korean Collection for Type Cultures).

In addition, drug-resistant E. coli DREC1, an antibiotic resistant strain, DREC1), DREC2 (drug-resistant E. coli DREC2), Pseudomonas aeruginosa PS-002 (drug-resistant P. aeruginosa PS-002), Pseudomonas Rouge industrial PS-003 (drug-resistant P. aeruginosa to the PS-003), Pseudomonas Rouge industrial PS-006 (drug-resistant P. aeruginosa to the PS-006), industrial PS-009 Rouge Pseudomonas drug-resistant P. aeruginosa PS-010, drug-resistant P. aeruginosa PS-009, drug-resistant P. aeruginosa PS-010 and drug-resistant P. aeruginosa 4891, The patient was directly separated from the patient and checked for drug resistance.

2-2. Micro-dilution analysis of pathogenic and antibiotic-resistant strains of isosorbide dinofloxacin (ISDN)

Seven antibiotic susceptible strains and eight antibiotic resistant strains prepared in 2-1 above were inoculated on a nutrient medium and collected in the mid log phase. Then, the strain of the mid-log phase was diluted to a concentration of ⁵ 10 ⁵ cfu / mL using PBS (phosphate buffered saline) containing 10% of culture medium. Then, isosorbide dinofloparacin (ISDN), which was serially diluted twice with the diluted strain, was added to each well of a 96-well plate and then cultured at 37 DEG C for 24 hours. The absorbance of each well was then measured at 600 nm using light microscopy to determine the lowest isosorbide dinofloxacin (ISDN) concentration that completely inhibited microbial growth to a minimum inhibitory concentration concentration, MIC). As a control group, norfloxacin was used instead of isosorbide dinofloparacin (ISDN). The MIC values were determined as the mean value of three independent experiments, three at a time. The results are shown in Tables 1, 2, 6, and 7.

As a result, isosorbide dinofloxacin (ISDN) showed an antibacterial effect similar to norfloxacin against E. coli O157 and P. aeruginosa , whereas isosorbide monoproloxacin (ISMN) The activity was somewhat decreased.

Pathogenic strain MIC (ug / ml) Norfloxacin ISDN E. coli <0.4 <0.4 E. coli O157 <0.4 <0.4 L.monocytogens <0.4 <0.4 P.aeruginosa <0.4 <0.4 S.aureus 4 4 S. epidermidis <0.4 <0.4 S.typhimuirm <0.4 <0.4

Antibiotic resistant strain MIC (ug / ml) Norfloxacin ISDN E.coli DREC1 16 4 E. coli DREC2 16 2 P. aeruginosa PS-002 64 16 P. aeruginosa PS-003 256 256 P. aeruginosa PS-006 32 8 P. aeruginosa PS-009 16 2 P. aeruginosa PS-010 64 8 P. aeruginosa 4891 516 128

As a result, the antimicrobial activity of isosorbide dinofloxacin (ISDN) was similar to that of norfloxacin, while the antimicrobial activity against antibiotic resistant strains was superior to that of norfloxacin Respectively.

These results suggest that the molecular weight of norfloxacin is increased by the binding of isosorbide, which prevents the release of drug through the efflux pump.

Test Example 3: Measurement of cytotoxicity of isosorbide dinoflopsacin (ISDN)

In order to examine the toxicity of isosorbide dinofloparacin (ISDN) of the present invention to normal cells, L929 cells treated with isosorbide dinoflopoaxan (ISDN) were subjected to MTT assay Respectively.

Specifically, L929 cells were inoculated into DMEM (Dulbecco's modified Eagle medium) containing antibiotics (100 U / mL penicillin, 100 ㎍ / mL streptomycin) and 10% FCS (fetal calf serum) They were cultured in a chamber that CO ₂ is maintained. Then, the cells were subcultured in a 96-well plate so that the concentration of the L929 cells was 4 × 10 ³ cells / well, followed by culturing for 24 hours. Each well was then treated with 0.5 to 100 μg / ml of isosorbide dinofloparacin (ISDN), which was serially diluted 2-fold with DMEM, and then cultured at 37 ° C. for 24 hours. Then, 10 쨉 l of 5 mg / ml MTT solution was added to each of the wells, followed by further incubation for 4 hours. Then, the supernatant was removed and the precipitate was dissolved in 50 쨉 l of DMSO. Then, the absorbance was measured using a microplate rederer having a wavelength of 490-620 nm to confirm the viability of the cells. As a comparative group, norfloxacin was used instead of isosorbide dinofloxacin (ISDN). Cell viability was determined as an average value obtained from four independent experiments, and viability (%) was expressed as a percentage based on the survival rate of cells treated with isosorbide dinofloparacin (ISDN). The results are shown in Fig.

As a result, L929 cells treated with norfloxacin had an IC ₅₀ of 16.5 μg / ml, whereas IS92 treated L929 cells were treated with 100 μg / mL of isosorbide dinofloxacin (ISDN) It was confirmed that it almost survived.

This is probably due to the decrease in the hydrophobic properties of the drug.

Test Example 4: Measurement of killing kinetics of isosorbide dinofluoracin (ISDN)

(ISMN), isosorbide dinofluorosuccinate (ISDN), and isosorbide dinofloxacin (ISDN) were added to the pathogenic strain Pseudomonas aeruginosa (ATCC 15692; OD ₆₀₀ 0.05) , Norfloxacin or isosorbide were added and mixed, and the mixture was diluted 20 times according to the incubation time. The resulting mixture was spread on LA (luria broth agar) medium, cultured for one day, The number of colonies was counted. As a positive control, cells treated with cefotaxime or ciprofloxacin were used. As a control (without sample), isosorbide monodifluorosaccharide (ISMN), isosorbide dinofloxacin (ISDN) , Norfloxacin, or a strain not treated with isosorbide was used. The results are shown in Fig.

Experimental results confirmed that the antimicrobial activity of isosorbide dinofloparacin (ISDN) remained constant for 24 hours in the same manner as norfloxacin, cefotaxime, or ciprofloxacin.

Test Example 5: Diffusivity measurement of isosorbide dinofloproscine (ISDN)

In order to verify the diffusion of isosorbide dinofloparacin (ISDN) of the present invention, agar medium diffusion analysis was performed.

Specifically, the 2-1 pathogenic strains of Escherichia coli cyano (E.coli, ATCC 25922) prepared by, Rouge Pseudomonas labor (P. aeruginosa, ATCC 15692), Salmonella typhimurium (S. typhimurium, KCTC 1926), Or S. pylori epidermidis (KCTC 1917) were preliminarily cultured and then mixed with 1% agarose gel added with 0.03% culture solution so that the final strain concentration was 5 × 10 ⁵ cfu / mL Pour 10 ml into a Petri dish and let stand for 5 minutes at room temperature. Thereafter, holes were drilled in the agarose gel so as to have a diameter of 4 mm to form wells. Isosorbide dinofluorosuccinate (ISDN) of 0.1 to 20 μg / mL was added to each of the wells, and the mixture was incubated for 30 minutes And cultured at 37 ° C. Thereafter, a mixture of 1% agarose gel containing 6% TSB was poured on the culture medium, and the mixture was incubated at room temperature for 24 hours to measure the diameter of the clearance zone formed around the wells . As a comparative group, norfloxacin was used instead of isosorbide dinofloxacin (ISDN). The results obtained in the above experiment were expressed as mean ㅁ standard error (mean SE SE), and the mean difference from each test group was analyzed by Student's t-test to determine statistically significant difference when p value was less than 0.05. The results are shown in Fig.

As a result of the experiment, it was confirmed that isosorbide dinofloxacin (ISDN) has about 7 to 89% enhanced diffusivity compared to norfloxacin.

Test Example 6: Identification of antimicrobial activity of isosorbide dinofloxacin (ISDN)

In order to investigate the antimicrobial activity of isosorbide dinoflopsacin (ISDN) of the present invention, experiments were conducted to compare bacterial morphological changes by treatment with actual bacteria.

Specifically, Escherichia coli, stained with a cyano (E.coli, ATCC 25922), 1 % uranyl acetate (Uranyl acetate) and incubated for one hour to process the isosorbide Dino flow saksin (ISDN) at a concentration of MIC in And observed with a transmission electron microscope. As a control group, Escherichia coli (ISDN) -free Escherichia coli was used. As a comparative group, norfloxacin was used instead of isosorbide dinofloparacin (ISDN) Treated Escherichia coli ( E. coli ) was used. The results are shown in Fig.

As a result of the experiment, it was observed that isosorbide dinofloxacin (ISDN), like norfloxacin, excreted intracellular components of Escherichia coli cells.

From these results, it was found that the isosorbide dinofloparacin (ISDN) of the present invention can kill bacteria by a mechanism similar to norfloxacin.

Formulation Example 1: Preparation of medicines

1-1. Manufacture of liquid agent

Isosorbide dinofloxacin (ISDN) 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to a conventional liquid preparation method, the lemon flavor was added in an appropriate amount, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 mL, And sterilized to prepare a liquid preparation.

1-2. Manufacture of Powder

Isosorbide dinofloxacin (ISDN) 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-3. Injection preparation

Isosorbide dinofluorosuccine (ISDN) 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ H ₂ O 26 mg

(2 mL) per 1 ampoule in accordance with the usual injection preparation method.

1-4. Manufacture of tablets

Isosorbide dinofluorosuccine (ISDN) 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-5. Preparation of capsules

Isosorbide dinofluorosuccine (ISDN) 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation Example 2: Preparation of cosmetic additive

2-1. Manufacture of softening longevity (skin lotion)

0.25 wt% of isosorbide dinofluorosuccinate (ISDN)

Beta-1,3-glucan 1.0 wt%

Butylene glycol 2.0 wt%

Propylene glycol 2.0 wt%

Carboxyvinyl polymer 0.1 wt%

0.2% by weight of phage-12 nonylphenyl ether

Polysorbate 80 0.4 wt%

Ethanol 10.0 wt%

0.1% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

2-2. Manufacture of nutrition lotion (milk lotion)

0.5% by weight of isosorbide dinofluorosuccinate (ISDN)

Beta-1,3-glucan 1.0 wt%

4.0 wt%

Polysorbate 60 1.5 wt%

1.5% by weight of sorbitan sesquioleate

0.5% by weight liquid paraffin

Caprylic / capric triglyceride 5.0 wt%

Glycerin 3.0 wt%

3.0% by weight of butylene glycol

3.0% by weight of propylene glycol

Carboxyvinyl polymer 0.1 wt%

0.2% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

2-3. Manufacture of nutrition cream

0.5% by weight of isosorbide dinofluorosuccinate (ISDN)

Beta-1,3-glucan 5.0 wt%

Wax 10.0 wt%

Polysorbate 60 1.5 wt%

&Lt; tb &gt; &lt; tb &gt; &lt; tb &gt;

0.5% by weight of sorbitan sesquioleate

Liquid paraffin 10.0 wt%

Squalane 5.0 wt%

Caprylic / capric triglyceride 5.0 wt%

Glycerin 5.0 wt%

3.0% by weight of butylene glycol

3.0% by weight of propylene glycol

0.2% by weight triethanolamine

Preservative, pigment, perfume

The amount to make the purified water 100% by weight

Formulation Example 3: Preparation of food preservative

0.5% by weight of isosorbide dinofluorosuccinate (ISDN)

0.1% by weight of dehydroacetic acid

0.1% by weight potassium sorbate

0.2% by weight calcium sorbate

0.5% by weight sodium benzoate

0.1% by weight potassium benzoate

Calcium benzoate 0.5 wt%

0.1% by weight of methyl paraoxybenzoate

0.1% by weight of p-hydroxybenzoic acid propylate

0.1% by weight sodium propionate

0.1% by weight calcium propionate

They are prepared separately according to a conventional preservative manufacturing method.

Claims (6)

An isosorbide derivative having a structure represented by the following formula (1) and exhibiting an antimicrobial activity.
[Chemical Formula 1]

The method of claim 1,
Wherein the isosorbide derivative is dissolved in an aqueous solution and the diffusibility of the antimicrobial activity is improved by 27 to 66% as compared to norfloxacin. The method according to claim 1,
Wherein the isosorbide derivative is selected from the group consisting of Escherichia coli , Listeria monocytogens , Pseudomonas aeruginosa , Staphylococcus aureus , (Staphylococcus epidermidis), Salmonella typhimurium (Salmonella typhimurium), drug-resistant Escherichia cyano coli (drug-resistant Escherichia coli) and a drug which at least one strain selected from the group consisting of labor (drug-resistant Pseudomonas aeruginosa) Rouge the resistance Pseudomonas Wherein the isosorbide derivative is an isosorbide derivative. A pharmaceutical composition for antimicrobial use comprising the isosorbide derivative of any one of claims 1 to 3 as an active ingredient. An antimicrobial cosmetic additive composition comprising an isosorbide derivative according to any one of claims 1 to 3 as an active ingredient. An antimicrobial food preservative additive composition comprising the isosorbide derivative of any one of claims 1 to 3 as an active ingredient.

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