KR101959994B1 - Antimicrobial isosorbide derivatives and antimicrobial composition comprising the same - Google Patents

Abstract

The present invention relates to an isosorbide derivative exhibiting an antibacterial activity and an antimicrobial composition comprising the same as an active ingredient. The isosorbide derivative of the present invention is free from cytotoxicity, is soluble in an aqueous solution, and has antibiotic sensitivity and resistance to bacteria and fungi Therefore, the antimicrobial composition containing the isosorbide derivative of the present invention is excellent in antimicrobial activity against a strain. Therefore, the antimicrobial composition of the present invention is useful as a medicament for preventing or treating a bacterial infectious disease, a sterilizing and disinfecting agent, a cosmetic additive, a feed additive, Can be usefully used.

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 ester synthesis product of isosorbide and aminolauric acid exhibiting antibacterial activity and an antimicrobial composition comprising the same as an active ingredient. More particularly, the present invention relates to an ester synthesis of isosorbide and aminolauric acid A cosmetic additive composition, or a food preservative additive composition containing the isosorbide derivative and the isosorbide derivative as an active ingredient.

Sorbitol is a natural component widely distributed in the natural world. Especially, it exists in fruits and seaweeds such as pears, apples and the like. However, it is a hexahydric alcohol which is industrially produced by reducing glucose. Isosorbide, which is converted by dehydration in sorbitol, has a rigid chemical skeleton in the form of a double ring and can be used as a monomer in various polymeric materials via two hydroxyl (-OH) functional groups. Recently, Bisphenol-A (Bisphenol-A) is recognized as an environmental pollutant and cancer-causing substance, and it is expected that a new market with high added value will be created if demand of isosorbide is rapidly increased to replace it.

Isosorbide can be converted into monomers or substances having diverse functions depending on the substance or functional group substituted with hydroxyl group (-OH) as a diol, and isosorbide It is also used as a diuretic for treating glaucoma and hydrocephalus in raw materials of beads and as a vasodilator for angina pectoris by adding nitrate to one of the hydroxyl groups of isosorbide. In addition, the US FDA has approved a product called Bydil, a combination of isosorbide dinitrate and hydralazine vasodilators, to treat race-specific heart failure, targeting only blacks. As a phthalate free plasticizer with no added phthalate, the isosorbide plasticizer is a typical eco-friendly material that can be used in food wraps, children's products (toys, stationery, etc.) Is being recognized. (-OH) of isosorbide is used as a corrosion inhibitor and the like, and it is possible to prepare an environmentally friendly solvent (solvent) having no volatility through an ether reaction and is used as a cosmetic and a paint additive. Methylation compounds have been used as cosmetic additives because of increased transdermal delivery efficiency. In addition, we are developing polymeric materials (bioplastics, contact lenses, films, etc.) by adding various functional groups or sharing with other materials, and we are developing products of isosorbide as a raw material in Korea, It is a next-generation material that is highly likely to be used in various fields.

The disinfectant produced by the present invention can be used for cleaning vegetables and fruits, cleaning agents for dishes and cookware, automatic dishwashers, hand washing machines, baby bottles, cleaning products for baby toys, gas stoves, As an important raw material component, there are many anionic and nonionic surfactants such as higher alcohol (anion), higher amine (nonion), and alpha olefin (anion). The main features of the active ingredients of the sterilization disinfectant used in Korea are as follows. The alcohol sterilizing disinfectant is used next to the chlorine system in the food factory for the purpose of improving the preservation of the food and the hygiene maintenance of the food processing environment by inhibiting microbial growth As a product, sanitizing disinfectants such as appliances have been continuously maintained as a mixing agent for food additives before they are newly established. Since most of the products contain more than 50% ethanol because of the strongest sterilizing power when the active ingredient, 50-70% ethanol, is added, organic acids, lower fatty acids or amino acids are added to lower the pH, And the like. The addition of amino acids (lysine, alanine or glycine) with organic acid (citric acid), low fatty acid (monoglyceride) and antimicrobial activity can show disinfection power even with 20 ~ 30% ethanol content. Chlorine-based disinfectant is the most commonly used disinfectant in domestic food factories and has been used as a food additive. The active ingredient, chlorine, is divided into inorganic, organic chlorine and chlorine dioxide, mostly inorganic and organic chlorine products. In particular, the disinfecting power of a wide range of disinfecting power is rapidly exhibited. Due to the function of discoloration and deodorization and economic advantages, the disinfecting power of the chlorine-based disinfectant is preferred in the food industry. However, And sulfates and chlorides present disadvantages such as corrosion on food-contact surfaces and tubes. Particularly, chlorine disinfection disinfectant is highly affected by organic matter, and degradation proceeds quickly, resulting in poor stability. Iodine, which is an effective ingredient of disinfectant for iodine, could be formulated without iodine, polyvinylpyrolidone, or indophors, a complex of iodine and surfactant, It has a disadvantage that it has a disinfecting power against bacteria forming spores with a wide range of disinfecting power and accompanies washing power by using a nonionic surfactant and the like, have. Quaternary ammonium disinfection disinfectants have excellent physico-chemical stability, cleaning power, and are not affected by pH, temperature and organic matter. It is colorless, odorless and noncorrosive. It has the advantage of maintaining the bacteriostatic effect continuously after use. However, when it is slightly weak for Gram negative bacteria and diluted with light water, sterilization disinfection power is low and sterilization persistence is low. Therefore, an ideal disinfectant must be easy to use on food contact surfaces, have a wide range of disinfecting power, and quickly remove microorganisms. It must also be stable in some form and should not be affected by the external environment. The toxicity to the human body should be low and the usage cost should be low. However, few sterilization disinfectants can meet all of the above conditions. Therefore, it is important to understand the characteristics of sterilization disinfectants under specific use conditions and to use them considering their advantages and disadvantages.

On the other hand, many antibiotics have been separated or synthesized from natural materials to treat many diseases or infectious diseases. However, due to excessive abuse of antibiotics worldwide, the appearance and resistance rate of resistant bacteria to existing antibiotics are increasing, The infectious pathway is accelerating the spread of resistant bacteria and is becoming a serious social problem. The occurrence of these resistant strains and the abuse of antibiotics in hospitals, which are the cultivation sites, caused the generation of these resistant strains more frequently, and these "Super-bacteria" were no longer available for treatment. Multinational pharmaceutical companies say that they can treat with new antibiotics. However, since antibiotic resistance appears in the intensive care unit of a hospital with weakened immunity, antibiotics cause septic shock due to the endotoxin secreted during the bacterial killing process It may be raised to death. Scientists around the world are studying and developing new natural or synthetic antibiotics.

Accordingly, the present inventors have found that isosorbide derivatives synthesized by ester bonding through dehydration using aminolauric acid as a catalyst in isosorbide can be mass-produced, but they are not toxic to normal cells and can not destroy cell membranes of bacteria and fungi And exhibit a strong bactericidal activity. Thus, the present invention has been completed.

Korean Patent Publication No. 10-2014-0072100 (published on June 12, 2014) Korea Patent No. 10-0943366 (Announcement of Feb. 18, 2010) Korean Patent Publication No. 10-2017-0029553 (published on Jul. Korean Patent Publication No. 10-2013-0061199 (published on June 10, 2013) Korean Patent Publication No. 10-2004-0035864 (published on April 29, 2004) U.S. Published patent application US 2014/0315996 (published on October 23, 2014)

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 aminolauric acid is bonded to one hydroxyl group or both hydroxyl groups of isosorbide and has a structure represented by the following formula (1), (2), or I have.

The compound of the formula 1 is 1,4: 3,6-dianhydro-5-o-aminododecanoyl-D-glucitol. glucoside, and isosorbide 5-aminolaurate (IS-5AL).

The compound of formula 2 is prepared by reacting 1,4: 3,6-dianhydro-2-o-aminododecanoyl-D- glucosyl-glucitol, hereinafter referred to simply as isosorbide 2-aminolaurate (IS-2AL).

The compounds of the above formulas (1) and (2) are all isosorbide monoaminolaurate. Since the compound of formula (1) is the main product by stereochemical reaction in the actual reaction, Isosorbide 5-aminolaurate (IS-5AL) may also be referred to as isosorbide monoaminolaurate (IS-mAL).

The compound of formula (3) is prepared by reacting 1,4: 3,6-dianhydro-2,5-di-o-aminododecanoyl-di- di-O-aminododecanoyl-D-glucitol, hereinafter referred to as isosorbide 2,5-diaminolaurate (IS-dAL).

In the present invention, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) and isosorbide 2- Sorbide 2,5-diaminouracil (IS-dAL) is not necessarily limited to the esterification reaction of isosorbide of the following formula (4) with 12-aminolauric acid of the formula (5) Can be manufactured.

A catalyst may be used to induce the esterification reaction. Examples of the catalyst include sulfuric acid (H ₂ SO ₄ ), hydrochloric acid (HCl), nitric acid (HNO ₃ ), p-toluenesulfonic acid (PTSA), phosphoric acid (H ₃ PO ₄ ) And Novozyme 435 (lipase B, EC 3.1.1.3, from Candida antarctica ). Amberlyst polymeric catalysts are used as the ion exchange resin catalysts, and zinc oxide (ZnO), aluminum oxide Al ₂ O ₃ ), zinc aluminate (ZnAl ₂ O ₃ ), and the like.

In addition, since water is generated in the esterification reaction, it is preferable to conduct the reaction while dehydrating under vacuum.

As one specific example, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) And isosorbide 2,5-diaminolaurate (IS-dAL) are prepared by dissolving isosorbide and aminolauric acid in a sulfuric acid catalyst and reacting under a reduced pressure at 140-150 ° C to dehydrate Followed by elimination of the sulfuric acid with an anion exchange resin and solid phase extraction with C ₁₈ .

Isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) and isosorbide 2 , 5-diaminolaurate (IS-dAL) is dissolved in an aqueous solution and has excellent antimicrobial activity against pathogenic bacteria and fungus strains and antibiotic resistant bacteria and fungi strains, There is no toxicity.

The pathogenic strain may be selected from the group consisting of Bacillus cereus , Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Staphylococcus aureus , Philo locus epidermidis (Staphylococcus epidermidis), Salmonella typhimurium (Salmonella typhimurium), such as a bacteria group and Candida albicans (Candida albicans), Candida para Shiro sheath (Candida parapsilosis), Candida krusei (Candida krusei), Fusarium Solar Fungi such as Fusarium solani , Fusarium moniliforme , Aspergillus flavus , and the like, but the present invention is not limited thereto.

The antibiotic-resistant strain may be selected from the group consisting of antibiotic-resistant Staphylococcus aureus , drug-resistant Escherichia coli , and antibiotic-resistant drug-resistant Pseudomonas aeruginosa . , But the present invention is not limited thereto.

Thus, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) and isosorbide 2- The bead 2,5-diaminolaurate (IS-dAL) can be used to prevent or treat bacterial infectious diseases or to provide sterilization disinfectants and cosmetic additives for cleaning hands, feet or face, It can be usefully used for antibacterial use.

In another embodiment, the present invention provides a pharmaceutical composition comprising isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL ) And isosorbide 2,5-diaminolaurate (IS-dAL) as an active ingredient.

The antimicrobial composition of the present invention contains, as an active ingredient, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) Bead 2,5-diaminolaurate (IS-dAL), and an isosorbide derivative selected from the group consisting of isosorbide derivatives.

Isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) and isosorbide 5-aminolaurate Beads 2,5-diaminolaurate (IS-dAL) is present in an amount of 0.01 to 50% by weight, preferably 0.01 to 30% by weight, based on the total weight of the total composition, Preferably in an amount of 0.01 to 10% by weight. When the content of the isosorbide derivative is less than 0.001% by weight, the antibacterial activity is insufficient. When the content of the isosorbide derivative is more than 50% by weight, the effect of increasing the content is not proportionally increased, there is a problem.

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.

The pharmaceutical composition of the present invention may be prepared by incorporating one or more pharmaceutically acceptable solvents in addition to the above-described effective ingredients for administration. Pharmaceutically acceptable solvents include saline, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. In addition, other conventional additives such as antioxidants, buffers, bacteriostats, and the like may be added, and they may be formulated into tablets, capsules, suspensions, emulsions, syrups, aerosols, oral preparations, external preparations, suppositories, Solution or the like. In addition, the polymer can be made into a hydrogel or a film using a biomolecule.

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 of the present invention varies depending on the body weight, age, sex, health condition, diet, method of administration, excretion rate, severity of disease and the like. The daily dose of the pharmaceutical composition of the present invention is 0.1-1 mg / kg, preferably about 3-6 mg / kg on an adult basis, but may be increased or decreased according to clinical results. It is preferable to administer them separately.

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

When the composition of the present invention is used as a cosmetic additive composition, in addition to isosorbide monoaminauralate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) May additionally include components used as < RTI ID = 0.0 > For example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

The cosmetic additive composition of the present invention can be prepared in any formulations conventionally produced in the art. Examples of the cosmetic compositions of the emulsified form include nutritional lotion, cream, essence and the like, and the solubilized cosmetic composition has a softening longevity.

The cosmetic additive composition of the present invention can be prepared in the form of an adjuvant which can be applied topically or systemically, which is conventionally used in the field of dermatological science by containing an acceptable medium or base in addition to the isosorbide derivative.

The excipient used in the cosmetic additive composition of the present invention may further contain arbutin, kojic acid, rucinol, vitamin C and vitamin C derivatives, And may further contain retinol and its derivatives, indole acetic acid and its derivatives, adenosin, tocoperol and derivatives thereof, and carnitine for the purpose. In addition, the excipients include conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances, which are commonly used ingredients in cosmetic additives.

Suitable formulations of the cosmetic additive composition include solutions, gels, solid or kneaded anhydrous products to which at least one of the isosorbide derivatives are added, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, In the form of granules or ionic forms (liposomes), non-ionic follicular dispersions, creams, skins, lotions, powders, ointments, sprays or conical sticks. It can also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. Also, it can be formed into a form such as encapsulation and grafting by using chitosan, dextran, and hyaluronic acid, which are biocompatible polymers, as a carrier.

The cosmetic additive composition of the present invention preferably contains the isosorbide derivative of the present invention in an amount of 0.01 to 0.1% by weight based on the total weight of the cosmetic additive composition. However, the above content does not limit the addition amount.

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

The food additive composition of the present invention contains, as an active ingredient, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) Bead 2,5-diaminouracil (IS-dAL), and one or more of the 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 type of food using the above food additive composition, and examples thereof include drinks, meat, sausage, bread, biscuits, rice cakes, chocolates, candies, snacks, confections, pizza, ramen noodles, , Various soups, beverages, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The food additive composition preferably contains the isosorbide derivative in an amount of 0.01 to 0.1% by weight based on the total weight of the food additive composition. However, the addition amount is not limited.

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

The food additive composition of the present invention contains, as an active ingredient, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) Bead 2,5-diaminouracil (IS-dAL), together with at least one isosteric bead derivative selected from the group consisting of known animal feed preservatives.

The feed additive composition preferably comprises the isosorbide derivative in an amount of 0.01 to 0.1% by weight based on the total weight of the feed additive composition. However, the above content does not limit the addition amount.

As another specific application of the present invention, the antimicrobial composition of the present invention can be used as an antiseptic disinfectant composition for cleaning and disinfecting infant products, medical surgical tools, household products, industrial products and the like.

The disinfecting and disinfecting composition of the present invention contains, as an active ingredient, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) Bead 2,5-diaminouracil (IS-dAL), and one or more isosorbide derivatives selected from the group consisting of at least one of the known sterilizing disinfectants.

The sterilization disinfectant composition preferably comprises the isosorbide derivative in an amount of 0.01 to 0.1% by weight based on the total weight of the sterilization disinfectant composition. However, the above content does not limit the addition amount.

The isosorbide 5-aminolaurate (IS-5AL), the isosorbide 2-aminolaurate (IS-2AL) and the isosorbide 2,5- The diaminolaurate (IS-dAL) is useful as a safe antimicrobial composition since it has no irritation to the skin as a result of skin irritation test and has no toxicity to normal cells in the human body.

Thus, isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) and isosorbide 2, 5-diaminolaurate (IS-dAL) exhibits high antimicrobial and antifungal effects under physiological conditions, is not toxic to human cells, exhibits strong antibacterial activity against antibiotic resistant bacteria, and has excellent bioavailability. Thus, the isosorbide 5-aminolaurate (IS-5AL), isosorbide 2-aminolaurate (IS-2AL) and isosorbide 2,5 -Diminolaurate (IS-dAL) can be used as a pharmaceutical composition for antimicrobial use, a cosmetic composition and a food additive composition.

1 is a schematic diagram of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) using isosorbide and aminolauric acid. This is a schematic diagram of the synthesis process.
2 is a graph showing the time during which isosorbide monoaminauralate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) according to the present invention are separated in reversed phase HPLC.
3 is a graph showing the structure of isosorbide 2,5-diaminolaurate (IS-dAL) according to the present invention using ¹³ C-NMR spectrum.
FIG. 4 is a graph showing the results of a Fourier transform infrared spectroscopy of isosorbide, aminolauric acid, isosorbide monoaminauralate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) And the result of confirming the synthesis.
FIG. 5 is a graph showing the activity of L. monocytogenes , S. typhimurium , E. coli O157, and E. coli O157 of isosorbide monoaminolaurate (IS-mAL) Bacillus cereus , or S. aureus in the case of the present invention.
FIG. 6 is a graph showing the fluorescence intensity of Candida parapsilosis , Candida krusei , Fusarium solani , Fusarium monilomus, and the like of the isosorbide monoaminolaurate (IS-mAL) ( Fusarium moniliforme ), or Aspergillus flavus in a liquid culture.
Figure 7 is a graphical representation of the isosorbide monoaminolauate (IS-mAL) of the present invention and the isosorbide monoaminolaurate (IS-mAL) according to the present invention, Fusarium solani , Fusarium moniliforme ), or Aspergillus flavus in an agar culture medium.
FIG. 8 is a graph showing the results obtained by treating normal cells of isosorbide, melittin, isosorbide monaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) , And cell growth.
FIG. 9 is a graph showing the results obtained by treating the isosorbide monoaminolaurate (IS-mAL) of the present invention with Escherichia coli pretreated with SYTOX-Green by concentration, And the change in the green fluorescence due to the fluorescence of the sample.
10 is a graph showing the effect of isosorbide, melittin, isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) ( E. coli ) was observed with a scanning electron microscope.
Figure 11 is a graph showing the activity of Candida albicans treated with isosorbide, melittin, isosorbide monaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) C. albicans ) was observed with a scanning electron microscope.
FIG. 12 is a graph showing the effect of isosorbide, melittin, isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) ( E. coli ) was observed with a transmission electron microscope.
13 is a result of a skin irritation test after treatment of isosorbide monoaminolaurate (IS-mAL) according to the present invention on skin, such as female rabbits, with scratching and irritation.

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.

Preparation Example 1: Preparation of isosorbide derivatives

1 mol of isoorbide (IIC chemical) and 0.75 mol and 2 mol of 12-aminolauric acid (TCI) were completely dissolved in a sulfuric acid solution (0.85 mol and 2.2 mol), respectively, and a round flask reactor The first 30 minutes were elevated to 100-150 ° C, slowly to 140-150 ° C and stirred (see FIG. 1). Vacuum pressure reduction was carried out during the reaction to dehydrate to the amount of water generated by the ester reaction, and then passed three times through an anion exchange resin (1/2 of the volume of the reaction solution) to prepare an isosorbide derivative. Subsequently, the reactant with sulfuric acid removed was passed through a C18 solid-phase extraction resin, extracted with acetonitrile concentration, lyophilized, and analyzed using an analytical HPLC system at a flow rate of 1 ml / min for a retention time of 38 minutes (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) (see Fig. 2).

As a result of the above results, isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) were separated from each other using a separation HPLC system for subsequent experiments.

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 formation of the ester bond and the molecular structure of the purified substance were confirmed using ¹³ C-NMR spectrum. Isosorbide and aminolauric acid were used as a control for molecular structure analysis. The results are shown in Fig.

As shown in Fig. 3, free isosorbide, isosorbide, isosorbide, isosorbide, isosorbide monoaminolaurate (IS- It was confirmed that the carbon shifts to 172 and 175 ppm by the ester bond in the NMR peak of isosorbide 2,5-diaminolaurate (IS-dAL) and isosorbide (IS-dAL).

The results showed that isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) were successfully synthesized and purified.

Test Example 1: Antimicrobial activity of isosorbide derivatives

To confirm whether the isosorbide monoaminolaurate (IS-mAL) synthesized in Preparation Example 1 and the isosorbide 2,5-diaminolaurate (IS-dAL) can be used as an antibacterial or bactericide, And micro-dilution analysis using fungi, crop-related fungi and antibiotic-resistant strains.

1-1. 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), Salmonella typhimurium (Salmonella typhimurium, KCTC 1926), Bacillus serious (Bacillus cereus, KCTC 1012) and spa Philo locus epidermidis (Staphylococcus epidermidis, KCTC 1917) South Korea Microbial Resources Center (Korean Collection for Type Cultures) And prepared.

In addition, drug-resistant Pseudomonas aeruginosa DRPa-001 (drug-resistant P. aeruginosa 001), drug-resistant P. aeruginosa 002 (Pseudomonas aeruginosa 002), Pseudomonas aeruginosa DRPa-003 drug-resistant P. aeruginosa 003), Pseudomonas aeruginosa DRPa-004 (drug-resistant P. aeruginosa 004), and Pseudomonas aeruginosa DRPa-005 (drug-resistant P. aeruginosa 005) The drug resistance was confirmed.

In addition, the fungal strains include Candida parapsilosis , Candida krusei , Candida albicans , Fusarium solani , Fusarium moniliforme , Aspergillus flavus was purchased from the Korean Collection for Type Cultures.

1-2. Analysis of the antibacterial activity of isosorbide monoaminauralate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) against bacteria

Eight antibiotic susceptible strains and five antibiotic resistant strains prepared in 1-1 above were inoculated on nutrient medium and collected in the mid log phase. Then, the strain of the mid-log phase was diluted with PBS (phosphate buffered saline) containing 10% of culture medium to a concentration of 1 x 10 ⁶ cfu / mL. Thereafter, isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolane (IS-mAL) were continuously added to each well of a 96-well plate with the diluted strain in two- (IS-dAL) was added, followed by incubation at 37 占 폚 for 24 hours. The lowest isosorbide monoaminolaurate (IS-mAL), which is then visually identified by light microscopy and the absorbance of each well is measured at 600 nm to completely inhibit the growth of the microorganism, And isosorbide 2,5-diaminolaurate (IS-dAL) concentrations were determined as the minimal inhibitory concentration (MIC). Isosorbide and aminolauric acid were used as controls. 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 and 5.

As a result, isosorbide monoaminolaurate (IS-mAL) exhibited a minimum inhibitory concentration of 15.6 to 62.5 ug / mL for all tested bacteria, and isosorbide 2,5-diaminolaurate (IS- dAL) showed a minimum inhibitory concentration of 62.5 ~ 250 ug / mL. It has been found that the isosorbide 2,5-diaminolaurate (IS-dAL) has a somewhat lower antimicrobial activity than isosorbide monoaminolaurate (IS-mAL).

Pathogenic strain MIC (ug / mL) Isosorbide Aminolauric acid IS-mAL IS-dAL B.cereus > 2,000 > 500 62.5 125 E. coli > 2,000 > 500 15.6 62.5 E. coli O157 > 2,000 > 500 15.6 62.5 L.monocytogens > 2,000 > 500 62.5 125 P.aeruginosa > 2,000 > 500 15.6 62.5 S.aureus > 2,000 > 500 15.6 62.5 S. epidermidis > 2,000 > 500 62.5 125 S.typhimuirm > 2,000 > 500 62.5 250

Antibiotic resistant strain MIC (ug / mL) Norfloxacin IS-mAL IS-dAL P. aeruginosa 001 256 15.6 62.5 P. aeruginosa 002 64 15.6 62.5 P. aeruginosa 003 64 15.6 62.5 P. aeruginosa 004 256 15.6 62.5 P. aeruginosa 005 256 15.6 62.5

In addition, the antimicrobial activity of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) against antibiotic resistant strains was the same as that of antibiotic sensitive strains .

These results indicate that the antibacterial action mechanism of isosorbide monoaminauralate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) is different from that of norfloxacin, and antibiotic resistant bacteria Which can effectively be used as an antimicrobial agent.

1-3. Analysis of the antimicrobial activity against fungal of isosorbide monoaminauralate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL)

Six species of the fungi prepared in 1-1 above were inoculated into a nutrient agar medium and cultured. The spores were obtained with a 0.08% Triton X-100 solution and diluted to a concentration of 2 × 10 ⁴ cells / mL. Thereafter, isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolane (IS-mAL) were continuously added to each well of a 96-well plate with the diluted strain in two- (IS-dAL) was added, followed by incubation at 28 DEG C for 24 hours. The isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS) which completely inhibited the growth of microorganisms were visually confirmed by light microscopy. -dAL) concentration was determined as the minimal inhibitory concentration (MIC). Isosorbide and aminolauric acid were used as controls. The MIC values were determined as the mean value of three independent experiments, three at a time. The minimum inhibitory concentrations are shown in Table 3, and cell growth inhibition is shown in Fig. The results of Fig. 6 are shown in Fig. 7 when 100 uL is added to the agar medium and cultured at 28 DEG C for 72 hours.

The results showed that isosorbide monoaminolaurate (IS-mAL) exhibited a minimum inhibitory concentration of 7.8 to 15.6 ug / mL for all fungi tested, and isosorbide 2,5-diaminolaurate (IS- dAL) showed a minimum inhibitory concentration of 31.3 to 62.5 ug / mL. It was found that the antibacterial activity of isosorbide 2,5-diaminolaurate (IS-dAL) was slightly lower than that of isosorbide monoaminolaurate (IS-mAL) in the same manner as the antibacterial agent.

mold MIC (ug / mL) Isosorbide Aminolauric acid IS-mAL IS-dAL C.albicans > 2,000 500 15.6 62.5 Candida krusei > 2,000 500 15.6 62.5 Candida parapsilosis > 2,000 500 15.6 62.5 Fusarium solani > 2,000 > 500 7.8 31.3 Fusarium moniliforme > 2,000 > 500 15.6 62.5 Aspergillus flavus > 2,000 > 500 7.8 31.3

Test Example 2: Measurement of cytotoxicity of isosorbide derivatives

To examine the toxicity of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) of Preparation Example 1 to normal cells, L929, a sarcoma cell line, was treated with isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) followed by MTT assay.

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 isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL), which were serially diluted 2-fold in DMEM, 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. Thereafter, the absorbance was measured using a microplate rederer having a wavelength of 490 nm to 620 nm to confirm the viability of the cells. Isosorbide and aminolauric acid were used as comparative groups. Cell viability was determined as an average value obtained from three independent experiments, and viability (%) was expressed as a percentage based on the survival rate of the untreated cells. The results are shown in Fig.

As a result, survival rate of L929 cells treated with isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) was significantly higher than that of L929 cells treated with aminolauric acid alone Respectively.

It was considered that the hydrophilic nature of aminoleic acid was reduced and hydrophobic properties were decreased.

Test Example  3: Identification of antimicrobial activity of isosorbide derivatives

In order to investigate the antimicrobial activity of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) in Preparation Example 1, Experiments were conducted to compare the intracellular inflow of the reagent SYTOX-Green and the morphological changes of bacteria and fungi.

Specifically, SYTOX-Green was pre-treated for 15 minutes in Escherichia coli , treated with isosorbide monoaminolaurate (IS-mAL) of the present invention at a concentration, FIG. 9 shows the result of measuring the change of green fluorescence due to the combination with time.

SYTOX-Green was not able to enter the cell unless the cell membrane was destroyed, and thus the nucleus could not be stained. As a result, green fluorescence in Escherichia coli bacteria treated with isosorbide monoaminolaurate (IS-mAL) increases with time, so that isosorbide monoaminolaurate (IS-mAL) I could see that it was destroyed.

In addition, Escherichia coli cyano (E.coli, ATCC 25922) and Candida albicans in (C. albicans) at a concentration of MIC melittin (Melittin), isosorbide laurate monoamino- (IS-mAL) and isosorbide (IS-dAL), cultured for 2 hours, fixed with 2% glutaraldehyde, dehydrated, coated with gold and observed with a scanning electron microscope. In the results of Escherichia coli cyano shown in Figure 11 relative to Figure 10 and Candida albicans (C. albicans) with respect to (E.coli, ATCC 25922).

In addition, cells stained with 2% Uranyl acetate were observed with a transmission electron microscope. Cells without samples were used as a control group and cells treated with melittin as a comparative group were used. The results are shown in Fig.

As a result, isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) were synthesized from E. coli , such as melittin, Destruction of cell membrane was observed. In addition, isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL), like melittin, produced pores in the surface of Candida albicans cells Respectively.

These results indicate that the isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) of the present invention can kill bacteria by a mechanism similar to melitin And it was found.

Test Example 4: Skin irritation test of isosorbide monoaminolaurate (IS-mAL)

In order to confirm the skin toxicity of the isosorbide monoaminolaurate of Preparation Example 1, a local skin irritation test was carried out in the condition of scratching and irritation.

Specifically, New Zealand White rabbits were shaved from the back of the New Zealand White rabbits. After 24 hours, the site of application was scratched and the skin (with the corrective knife damaged, but the dermis was not damaged and the blood was not spoiled) (Tegaderm ^TM 1626W, manufactured by Nippon Denshoku Kogyo Co., Ltd.) was applied to each of the two portions in a single application of isosorbide monoaminauralate (0.5 mL of 10 mg / mL) and sterile physiological saline (control, 3M HealthCare) was wrapped with a bandage and fixed with tape. Skin irritation was observed at 72 hours after appearance, feed and water intake, presence of deaths, weight, erythema formation, scarring and edema. The state of appearance is shown in Fig.

As a result of the test, there were no specific general symptoms of the animal or reflexive animal. In addition, no change in body weight was observed by the administration of isosorbide monoaminauralate as a test substance. After 72 hours, erythema, scurf and edema were not observed visually. The results of scoring according to the skin reaction evaluation criteria of the Food and Drug Safety Notification No. 2015-82 of the Toxicological Test Standards for Drugs and Medicines were as follows: Isosorbide monoaminauralate was identified as a non-irritant substance with a skin irritation index of 0.0.

≪ Formulation Example 1 > Preparation of pharmaceutical composition

1-1. Manufacture of liquid agent

Isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) Each 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 monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) Each 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 monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) Each 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 monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) Each 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 monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL) Each 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 additives

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

0.25 wt% each of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL)

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 each of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL)

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 each of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL)

Beta-1,3-glucan 5.0 wt%

Wax 10.0 wt%

Polysorbate 60 1.5 wt%

≪ tb > < tb > < tb >

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 each of isosorbide monoaminolaurate (IS-mAL) and isosorbide 2,5-diaminolaurate (IS-dAL)

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 (11)

A compound having a structure represented by the following formula (1) and exhibiting an antimicrobial activity.
Formula 1

A compound having a structure of the following formula (2) and exhibiting an antimicrobial activity.
(2)

A compound having a structure of the following formula (3) and exhibiting antimicrobial activity.
(3)

4. The method according to any one of claims 1 to 3,
Wherein said compound exhibits an antimicrobial activity against at least one microorganism selected from the group consisting of a pathogenic bacterium, a pathogenic fungal strain, an antibiotic resistant bacterium, and an antibiotic resistant fungal strain. 4. The method according to any one of claims 1 to 3,
The compounds are useful for the treatment and / or prevention of diseases such as Bacillus cereus , Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Staphylococcus aureus , locus epidermidis (Staphylococcus epidermidis), Salmonella typhimurium (Salmonella typhimurium), Candida albicans (Candida albicans), cis (Candida parapsilosis), Candida krusei (Candida krusei), Fusarium solani (Fusarium solani) para Shiro Candida Fusarium moniliforme , Aspergillus flavus , drug-resistant Staphylococcus aureus , drug-resistant Escherichia coli , antibiotic-resistant Staphylococcus aureus , And drug-resistant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ). Wherein the compound exhibits an antimicrobial activity against any one or more microorganisms selected from the group consisting of genus. 4. The method according to any one of claims 1 to 3,
Wherein said compound is prepared by the esterification reaction of isosorbide and 12-aminolauric acid. A pharmaceutical composition for antimicrobial use comprising the compound of any one of claims 1 to 3 as an active ingredient. An antimicrobial cosmetic additive composition comprising the compound of any one of claims 1 to 3 as an active ingredient. A food additive composition for antimicrobial use comprising the compound of any one of claims 1 to 3 as an active ingredient. A sterilizing disinfectant composition comprising the compound of any one of claims 1 to 3 as an active ingredient. A feed additive composition for antibiosis comprising the compound of any one of claims 1 to 3 as an active ingredient.

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