Antimicrobial insect repellent composition

Abstract

An antimicrobial (anticandidal, antibacterial, antifungal) and antiviral composition which repels insects such as flies, mosquitoes and ticks. The composition includes poloxamer, boron compound, ethyl butyl acetylaminopropionate and preferably hydrogen peroxide. The present composition is a composition which can be applied in different forms via oral, nasal, ophthalmic, otic, local, ventricle, vaginal, rectal, dermal, intravenous, intramuscular, subcutaneous and intradermal route.

Claims

1. An insect repellent composition, comprising poloxamer, a boron compound, ethyl butyl acetylaminopropionate and 1% (w/w) hydrogen peroxide, wherein the poloxamer is 5-15% (w/w), the boron compound is 1-5% (w/w), the ethyl butyl acetylaminopropionate is 15-25% (w/w), and the boron compound is sodium borate, wherein, the insect repellant composition is antimicrobial and antiviral, and the insect repellant composition exhibits antifungal activity on the fungi Aspergillus niger, Botrytis cinerea, Fusarium oxysporum and Penicillium vinaceum, and antimicrobial activity on VRE.

2. The insect repellent composition according to claim 1, wherein, the insect repellant composition exhibits antimicrobial activity on the bacteria Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Acinetobacter baumannii, and MRSA.

3. The insect repellent composition according to claim 1, wherein, the insect repellant composition exhibits anticandidal activity on the yeast Candida albicans.

4. The insect repellent composition according to claim 1, wherein, the insect repellant composition has antiviral activity against enveloped and non-enveloped DNA and RNA viruses.

5. The insect repellent composition according to claim 4, wherein, the insect repellant composition is in a form of tablets, capsules, pastilles, drops, syrups, suppositories, gels, lotions, ampoules, or tubes.

6. The insect repellent composition according to claim 5, wherein, the insect repellant composition is configured to be applied via oral, nasal, ophthalmic, otic, local, ventricle, vaginal, rectal, dermal, intravenous, intramuscular, subcutaneous or intradermal route.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) “An Insect Repellent Composition” developed to fulfill the objectives of the present invention is shown in the accompanying figures wherein

(2) FIG. 1 shows a view of the antifungal activity of the EBAAP (Ethyl Butyl Acetylaminopropionate) and Sodium borate containing composition comprising 5% poloxamer (reference “a”) and 3% poloxamer (reference “b”) respectively against Aspergillus niger.

(3) FIG. 2 shows a view of the anticandidal activity of the composition containing EBAAP and Sodium borate containing composition comprising 5% poloxamer (reference “a”) and 3% poloxamer (reference “b”) respectively against Candida albicans.

(4) FIG. 3 shows a view of the anticandidal activity of the EBAAP and Sodium borate containing composition comprising 5% poloxamer (reference “a”) and 3% poloxamer (reference “b”) respectively against Staphylococcus aureus.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(5) Experimental Studies

(6) By means of the present invention, compositions of different concentrations and combinations which contain boron compounds, EBAAP (Ethyl Butylacetyl Aminopropionate), hydrogen peroxide and poloxamer separately or together.

(7) In the present invention; one of sodium pentaborate pentahydrate, zinc borate, disodium octaborate tetrahydrate, sodium borate, sodium perborate tetrahydrate, borax pentahydrate, and preferably sodium borate is selected as the boron compound.

(8) Preparation of the Combination

(9) Composition-1

(10) The gel, which is a carrier for the active molecules, was prepared by using 1-15% (w/w) poloxamer. In preparation of the gel, first of all 15-25% (w/w) EBAAP was added to an amount of distilled water that will be used in the formulation, then 1-10% (w/w) poloxamer was added to this mixture and the resulting mixture was allowed to rest at +4° C. until poloxamer precipitated (approximately 24 hours). After poloxamer precipitated, the mixture was homogenized by the help of a homogenizer or a stirrer, and then upon adding 1-5% (w/w) sodium pentaborate pentahydrate, it was allowed to hydrate at +4° C. After jelling is completed, if considered necessary, sodium carbonate or citric acid can be used for neutralizing pH. The mixture became ready for use upon being allowed to rest at +4° C. for 24 hours. After formation of gelling, the composition of the invention is in gel form.

(11) Composition-2

(12) The gel, which is a carrier for the active molecules, was prepared by using 1-15% (w/w) poloxamer. In preparation of the gel, first of all 1% (w/w) hydrogen peroxide (H.sub.2O.sub.2) was added to distilled water in an amount that will be used in the formulation, then 15-25% (w/w) EBAAP was added to the mixture, and upon adding 1-10% (w/w) poloxamer to the same mixture, the resulting mixture was allowed to rest at +4° C. until poloxamer precipitated (approximately 24 hours). After poloxamer precipitated, the mixture was homogenized by the help of a homogenizer or a stirrer, and then upon adding 1-5% (w/w) sodium pentaborate pentahydrate, it was allowed to hydrate at +4° C. After jelling is completed, if considered necessary, sodium carbonate or citric acid can be used for neutralizing pH. The mixture became ready for use upon being allowed to rest at +4° C. for 24 hours. After formation of gelling, the composition of the invention is in gel form.

(13) In the preparation of the gel of the present invention, sodium borate (Na[B.sub.5O.sub.6(OH).sub.4].Math.3H.sub.2O) was especially preferred as the boron compound. Apart from this compound; borax, boric acid, alkaline and alkaline earth metal borates and all hydrate forms of these borates, ammonium borates, boric acid esters can also be used.

(14) Antimicrobial Tests

(15) Modified Disc Diffusion Method

(16) Standard NCCLS disc diffusion method was used by being modified in order to determine the antimicrobial activity of boron compounds, EBAAP and the developed gel formulation on each microorganism that is being tested. The 100 μl solution including 10.sup.8 cfu/ml bacteria, 10.sup.6 cfu/ml yeast and 10.sup.4 spores/ml fungi was prepared with new cultures and inoculated with spreading method on Nutrient Agar (NA), Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA), respectively. 20 μl of sterile water was dropped on the empty discs and it was separately immersed into pulverized zinc borate, sodium borate, sodium perborate tetrahydrate, borax pentahydrate, disodium octaborate tetrahydrate. The discs coded with zinc borate, sodium borate, sodium perborate tetrahydrate, borax pentahydrate, disodium octaborate tetrahydrate were placed in inoculated petri dishes. Empty discs with 20 μl drop of sterile water were used as negative control. Furthermore, 19 μl empty discs were impregnated with EBAAP and antimicrobial activity tests were performed. Ofloxacin (10 μg/disc) and nystatin (30 μg/disc) were used as positive control for bacteria and fungi, respectively. The petri dishes, which were inoculated and on which modified disc diffusion method was applied, were incubated for 24 hours for bacteria and 48 hours for yeasts at 36±1° C., and for 72 hours for fungi at 25±1° C. Antimicrobial activity against microorganisms tested with modified disc diffusion method was assessed by measuring the inhibition zone (area where microorganisms do not grow). Antimicrobial activity test results of the tested boron compounds are summarized in Table 1 and 2. All tests were repeated at least twice.

(17) Antiviral Tests

(18) EBAAP Antiviral Activity Tests

(19) In order to produce Human adenovirus type 5 Adenoid 75 strain and Poliovirus type 1 Chat strain virus and to carry out the experiment, a complete layer of HEp-2 cells (ATCC CCL-23), which are human monolayer tumor cells, were used. For determining virus titration, reference Human adenovirus type 5 Adenoid 75 strain and Poliovirus type 1 Chat strain were inoculated by making serial dilutions to HEp-2 cells, and by taking as basis the virus dilution that produces a cytopathic effect visible in invert microscope, virus titration was computed by using Spearman-Karber method. In order to determine sub-cytotoxic concentration of EBAAP, EBAAP was 10-fold serially diluted with Eagle's minimum essential medium (MEM) and non-toxic concentrations were detected in the cell medium and these concentrations were used in the experiment. For the controls, MEM inoculated HEp-2 cells, full layer HEp-2 cells wherein EBAAP was not added, 10-fold diluted reference virus titration control, formaldehyde control and controls containing toxic concentrations of EBAAP were used as negative control instead of the virus.

(20) Preparation of Cell Culture Medium and the Chemicals

(21) MEM medium: 10% serum (FBS) containing enzymes, hormones and growth factors for the cells to be able to cling to the surfaces and proliferate; and 40 IU/ml penicillin, 0.04 mg/ml streptomycin, 0.5 mg/ml glutamine to prevent fungi and bacteria contamination; and 1% Sodium Bicarbonate as a buffer solution were added therein.

(22) FBS: Inactivated and mycoplasma-free

(23) Sodium bicarbonate: Sterile 7.5% solution

(24) Medium Used in Virus Inoculation: The medium included 1% antibiotic (Penicillin, Streptomycine, Amphotericin B) in order to prevent fungi and bacteria contamination, and 1% Sodium bicarbonate as a buffer solution. FBS serum is not added to this medium.

(25) Preparation of Clean and Contaminated Media

(26) Clean medium; 0.3 gr Bovine Serum Albumin Fraction V was dissolved in 100 ml sterile water. The solution that was obtained was sterilized by being passed through a filter with mesh size 0.22 μM.

(27) Contaminated medium; Sheep Erythrocyte and BSA were used for the contaminated medium. 3 g BSA was dissolved in 100 ml sterile water and filtered. 3 ml sheep erythrocyte was completed to 97 ml BSA.

(28) Erythrocyte; 8 ml fresh sheep blood was rotated at 800 G for 10 minutes and then its supernatant was removed. Upon adding 8 ml phosphate buffered saline (PBS) thereto, pipetting was performed and it was again rotated at 800 G for 10 minutes. This procedure was repeated 3 times.

(29) Analysis:

(30) Firstly, liquid EBAAP was solid serially diluted with the cell culture medium (MEM) and its non-toxic concentration in cell culture was calculated. 8 ml of the EBAAP that was to be tested was mixed with 2 ml hard water. The obtained solution was serially diluted (dilution step 1:10) with MEM. It was inoculated in 96-well monolayered cells. The microscopic changes that occurred were recorded. Concentrations that showed cytopathic effect (CPE) were determined. EBAAP and formaldehyde CPE values were compared. After determining non-toxic concentration of EBAAP on the cells, the effect of EBAAP on virus titration as a result of 5-60 minute separate application periods in clean and contaminated media was studied. For the controls, MEM inoculated HEp-2 cells, full layer HEp-2 cells wherein EBAAP was not added, 10-fold diluted reference virus titration control, formaldehyde control and controls containing toxic concentrations of EBAAP were used as negative control instead of the virus.

(31) Taking as basis the virus dilutions wherein cytopathic effect that is visible in invert microscope is formed, virus titration was calculated as TCID.sub.50 value by using Spearman-Karber method. Antiviral activity test results are summarized in Table 5, 6 and 7.

(32) Antimicrobial Activity Tests of the Prepared Formulations;

(33) Antimicrobial activity tests were performed for the compositions claimed to have antimicrobial and antiviral activity by means of the modified disc diffusion method. Modified disc diffusion experiments of the formulations were performed by providing wells on the medium and adding the developed formulation into the said wells. Antimicrobial activity test results are summarized in Table 3. Antiviral activity tests of the formulations were carried out by means of the above mentioned method. The test results are summarized in Table 6 and 7.

(34) Experimental Results

(35) Antimicrobial Test Results

(36) Antimicrobial activity test results of the tested boron compounds are summarized in Table 1. All tests were repeated at least twice.

(37) TABLE-US-00001 TABLE 1 Antimicrobial activity of Sodium Pentaborate Pentahydrate (SPP), Zinc borate (ZB), Borax (SB), Borax Pentahydrate (BP) and Disodium Octaborate Tetrahydrate (DOT) on the tested microorganisms Boron compounds Microorganisms SPP ZB SB BP DOT Bacteria Escherichia coli + + + + + Staphylococcus aureus + + + + + Pseudomonas aeruginosa + + + + + Klebsiella pneumoniae + + + + + Acinetobacter baumannii + + + + + Bacillus subtilis + + + + + Methicillin-resistant + + + + + Staphylococcus aureus (MRSA) Vancomycin-resistant + + + + + Enterococcus(VRE) Yeast Candida albicans + + + + + Fungi Aspergillus spp. + + + + + Fusarium oxysporum + + + + + Botrytis cinerea + + + + + Penicillium spp. + + + + +

(38) TABLE-US-00002 TABLE 2 Antimicrobial activity of EBAAP on the tested microorganisms Microorganisms EBAAP Bacteria Escherichia coli ATCC 25922 − Staphylococcus aureus ATCC 29213 + Pseudomonas aeruginosa ATCC 27853 + Klebsiella pneumoniae ATCC 13883 + Acinetobacter baumannii + Bacillus subtilis ATCC 6633 + Yeast Candida albicans + Fungi Aspergillus niger + Botrytis cinerae + Fusarium oxysporum + Penicillium vinaceum +

(39) TABLE-US-00003 TABLE 3 Antimicrobial activity of the developed formulations on the tested microorganisms Microorganisms F 1 F 2 Bacteria Escherichia coli ATCC 25922 + + Staphylococcus aureus ATCC 29213 + + Pseudomonas aeruginosa ATCC 27853 + + Klebsiella pneumoniae ATCC 13883 + + Bacillus subtilis ATCC 6633 + + Acinetobacter baumannii + + Methicillin-resistant Staphylococcus aureus (MRSA) + + Vancomycin-resistant Enterococcus(VRE) + + Yeast Candida albicans + + Fungi Aspergillus niger + + Botrytis cinerae + + Fusarium oxysporum + + Penicillium vinaceum + +

(40) Antimicrobial activities of the insect repellent composition of the present invention were tested by using bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Acinetobacter baumannii, MRSA and VRE), yeast (Candida albicans) and fungus (Aspergillus niger, Botrytis cinerea, Fusarium oxysporum and Penicillium vinaceum) isolates. According to the obtained results; it was observed that the products containing boron compounds, EBAAP and/or hydrogen peroxide had antimicrobial activity on all of the tested microorganisms.

(41) Antiviral Tests:

(42) It was observed in the calculations made as a result of the test that EBAAP caused 2 log reduction at the end of 5 minutes and 4 log reduction at the end of 60 minutes in virus titer (Table 4 and Table 5) as a result of application at a ratio of 1/1, at room temperature (20° C.), in clean and contaminated media and with 5 and 60 minute application periods.

(43) TABLE-US-00004 TABLE 4 Test results of antiviral activity of EBAAP against Adenovirus Type 5 (Av-5) AV-5 5 minutes 60 minutes Virus titer* 6.0 6.0 Clean Contm. Clean Contm. medi- medi- medi- medi- um um um um EBAAP EBAAP virüs titer** 4.0 4.0 2.0 2.0 Ratio of reduction 2.0 2.0 4.0 4.0 in virus titer*** *Logarithmic TCID.sub.50 value of the virus in ml. **Logarithmic TCID.sub.50 value of the virus treated with EBAAP at different periods and media. ***Logarithmic TCID50 ratio between the virus titer and the virus titer treated with EBAAP

(44) TABLE-US-00005 TABLE 5 Test results of antiviral activity of EBAAP against Poliovirus Type 1 (PV-1) PV-1 5 minutes 60 minutes Virus titer* 5.0 5.0 Clean Contm. Clean Contm. medi- medi- medi- medi- um um um um EBAAP EBAAP virus titer** 3.0 3.0 1.0 1.0 Ratio of reduction 2.0 2.0 4.0 4.0 in virus titer*** *Logarithmic TCID.sub.50 value of the virus in ml. **Logarithmic TCID.sub.50 value of the virus treated with EBAAP at different periods and media. ***Logarithmic TCID50 ratio between the virus titer and the virus titer treated with EBAAP

(45) As a conclusion; these experiment results show that EBAAP is 99.9% active against AV-5 and PV-1 viruses when used directly without being diluted at room temperature (20° C.) as a result of a 60 minute application.

(46) TABLE-US-00006 TABLE 6 Test results of antiviral activity of the composition with 3% poloxamer against Adenovirus Type 5 (Av-5) AV-5 5 minutes 60 minutes Virus titer* 5.0 5.0 Clean Contm. Clean Contm. medi- medi- medi- medi- um um um um 3% Jelli virus titer** 2 2 1 1 poloxamer Ratio of reduction 3 3 4 4 gel in virus titer*** *Logarithmic TCID.sub.50 value of the virus in ml. **Logarithmic TCID.sub.50 value of the virus treated with the gel at different periods and media. ***Logarithmic TCID50 ratio between the virus titer and the virus titer treated with the gel

(47) TABLE-US-00007 TABLE 7 Test results of antiviral activity of the composition with 5% poloxamer against Adenovirus Type 5 (Av-5) AV-5 5 minutes 60 minutes Virus titer* 5.0 5.0 Clean Contm. Clean Contm. medi- medi- medi- medi- um um um um 5% Jelli virus titer** 1 1 1 1 poloxamer Ratio of reduction 4 4 4 4 gel in virus titer*** *Logarithmic TCID.sub.50 value of the virus in ml. **Logarithmic TCID.sub.50 value of the virus treated with the gel at different periods and media. ***Logarithmic TCID50 ratio between the virus titer and the virus titer treated with the gel

(48) These experimental results show that when the gel containing 3% poloxamer (composition 1) was applied directly at room temperature (20° C.), it was not active against AV-5 in 5 minutes while it was 99.9% active against AV-5 in an application period of 60 minutes. The results further show that when the gel containing 5% poloxamer (composition 1) was applied directly at room temperature (20° C.), it was 99.9% active against AV-5 in application periods of both 5 and 60 minutes.

(49) Application of the Invention

(50) The compositions of the present invention can be obtained as antimicrobial (antibacterial, anticandidal, antifungal) and antiviral lotions, creams, emulsions, sprays, foams, gelatins, pastes, powders or plasters, skin plates and wound dressing textile products in different therapeutic ratios.

(51) All kinds of perfumes, moisturizers and surfactants, which will not chemically interact with the products, can be added to the said composition at suitable concentrations such that they will not reduce the antimicrobial and antiviral properties thereof.

(52) By application of the present invention, antiviral activity prevents viral replication and prevents proliferation of the virus.

(53) The composition of the present invention is suitable for use in many pharmacological areas such as applications of tablets, capsules, pastilles, drops, syrups, suppositories, gels, lotions, ampoules, tubes.

(54) The prepared composition can be administered by all kinds of ways that enable body absorption such as by oral, nasal, ophthalmic, otic, local, ventricle, vaginal, rectal, dermal, intravenous, intramuscular, subcutaneous and intradermal route.

(55) The product of the present invention can be used in all medical products, personal care products, cosmetic applications, drug formulations and medical applications upon being optimized.

(56) The said invention can find use in all kinds of products in textile, electronic goods, automotive industry, medical sector, construction materials, agriculture, biomedical science, packaging, hygiene, food, industrial design, sports goods, energy industry, defense industry, and in all sectors wherein antimicrobial and antiviral activities are desired and biodegradation is desired to be controlled; and thus antimicrobial and antiviral products with a very broad spectrum can be obtained.