WO2024076330A1 - A bee exosome-based composition with antibiotic properties against biofilm formation and the preparation method of this composition - Google Patents
A bee exosome-based composition with antibiotic properties against biofilm formation and the preparation method of this composition Download PDFInfo
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- WO2024076330A1 WO2024076330A1 PCT/TR2023/051073 TR2023051073W WO2024076330A1 WO 2024076330 A1 WO2024076330 A1 WO 2024076330A1 TR 2023051073 W TR2023051073 W TR 2023051073W WO 2024076330 A1 WO2024076330 A1 WO 2024076330A1
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- bee
- exosome
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- 230000032770 biofilm formation Effects 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title description 5
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
Definitions
- the invention relates to a composition with antibacterial and antibiotic properties that can be used as a washing solution in wound, burn or infection areas or as a tooth washing solution in dental operations, which prevents the production of biofilm on the wound, and a method for the preparation of this composition.
- Biofilms are bacterial communities structured within a protective extracellular polymerised substance (EPS) matrix that adheres to living or non-living surfaces.
- This gel-like layer is a polysaccharide-based network structure produced by bacterial cells and is called “exopolysaccharide” or “exopolymer (EPS)” in the terminology.
- Biofilm formation by microorganisms is a complex phenomenon that allows them to survive in an adverse environment.
- Biofilm infections account for 65% or more of all infections, especially tool-related infections, skin and soft tissue infections, and chronic infections.
- Bacteria within the biofilm are quite heterogeneous, with a small group being slow growers known as persisters and some being fast growers. Due to this heterogeneity and EPS matrix, bacteria in the biofilm are resistant to host defense mechanisms and antibiotic effects. Therefore, biofilms delay the healing process of damaged tissues or wounds [1 ],
- Biofilm formation can occur in living cells or on inanimate surfaces and is affected by factors such as the presence of other bacteria, amount of nutrients, temperature, amount of oxygen and pH.
- Biofilms can be formed by a single type of microorganism or can contain more than one type. In biofilms consisting of different species, each species forms its own microcolony, and these microcolonies are separated from each other by water channels. The ongoing water flow in water channels ensures the diffusion of nutrients and oxygen. 98% of the biofilm composition consists of water and the other substances in its composition are held together by the extracellular matrix structure.
- EPSs are responsible for the morphological, structural and physicochemical properties of biofilms.
- the composition of EPS consists of polysaccharide, protein, nucleic acid, teichoic acid, phospholipid and other polymeric substances and constitutes 75-90% of biofilms.
- the water in the biofilm structure binds to the EPS created by the bacteria.
- EPSs form biofilms and help microorganisms form colonies and attach to the surface.
- Multiple layers of cells and EPS can form a complex structure that makes it difficult for antimicrobials to penetrate and reach the inner layers. Thus, they can protect the organism against osmotic stress, phage contamination, toxic compounds, disinfectants and antibiotics.
- Microorganisms in biofilms are 10-1000 times more resistant to antimicrobial compounds than their planktonic forms [2],
- Antibiotics or washing solutions with antibiotic properties have been developed against different bacterial infections around the world.
- Antibiotics and surfactants which are antimicrobial chemical agents, are the conventional methods for controlling biofilms by using them in as small amounts as possible when necessary, although they have the risk of being harmful to the environment.
- Current methods that fail to remove biofilms are also not financially advantageous [3],
- antibiotics or exosomes which are vesicles obtained from cells and have antibacterial properties, used in washing solutions with antibiotic properties, are used against bacterial infections.
- a vesicle is a relatively small intracellular sac consisting of a closed membrane that stores or transports substances. Exosomes are vesicles with a diameter of 30-150 nm. These structures carry bioactive substances. By means of their antibiofilm properties, exosomes reduce bacterial infection and prevent biofilm formation. In addition, exosomes contribute to strengthening the immune system and play an active role in reducing the bacterial population.
- Apitoxin also known as bee venom
- the active substance of apitoxin poison consists of proteins, and this substance can be neutralised by ethanol. It is an acidic chemical substance and its pH value is between 4.5 and 5.5.
- Bee venom is rich in hyaluronic acid, phospholipase A2 molecule, dopamine, seretamine, hitamin and noradrenaline.
- Hyaluronic acid a substance contained in the venom and which is very important especially for skin health, connects two healthy cells, delays the signs of aging, rejuvenates and beautifies the skin.
- Phospholipase A2 molecule supports cell formation and destroys bacteria.
- Bee venom is a powerful anti-inflammatory. Reduces inflammation in the body; therefore, eliminates the factors that predispose to autoimmune diseases. Additionally, bee venom is effective against skin disorders and heals scars in a short time. It also soothes the skin in cases of psoriasis and eczema [5].
- Patent application numbered LIS2012088671 A1 in the state of the art relates to polyphenolic compounds derived from a plant that prevent biofilm formation. Also provided are combinations comprising at least one phenolic phytochemical and at least one antimicrobial agent that inhibits the growth of an established biofilm. Patent application number LIS2012088671 A1 provides methods for inhibiting the formation and growth of biofilms. In one embodiment, a polyphenolic composition is provided.
- Production method of the polyphenolic compound comprises the process steps of “(a) partitioning an alcoholic extract of a plant with a mixture of water and hexane to form a first water portion and a hexane portion; (b) dividing the first water portion with a mixture of water and ethyl acetate to form a second water portion and an ethyl acetate portion; (c) splitting the second water compartment with a mixture of water and butanol to form a third water compartment and a butanol compartment; and (d) fractionating the butanol fraction by column chromatography with a mobile phase containing a mixture of methanol and dichloromethane, wherein the polyphenolic composition is eluted by the mobile phase having a volume ratio of methanol to dichloromethane of about 40:60.”
- said composition does not comprise any components that would prevent the formation of biofilm, it does not provide an effective treatment for the healing of open or closed wounds.
- the invention relates to a composition that can be used as a washing solution in wounds, bums or infection areas or as a tooth washing solution in dental operations, is easily sprayable, comprises bee exosome obtained by isolating exosomes, prevents biofilm formation on the wound, and has antibacterial and antibiotic properties, and a method for the preparation of this composition.
- the composition of the invention comprises at least one antibiotic for use against bacteria, bee exosome and isotonic liquid (serum physiology) that facilitates spraying.
- the most important aim of the invention is to provide an antibacterial composition that can be used as a washing solution in wounds, burns or infection areas and prevents biofilm formation.
- the composition of the invention comprises at least one antibiotic used against bacteria and bee exosome obtained from bee venom.
- Bee venom which is rich in hyaluronic acid, phospholipase A2 molecule, dopamine, serethamine, hitamine and noradrenaline, supports cell formation and destroys bacteria.
- Bee venom is a powerful anti-inflammatory. Additionally, bee venom is effective against skin disorders and heals scars in a short time.
- an antibacterial composition that prevents biofilm formation is provided by using bee exosome obtained from bee venom.
- Another aim of the invention is to provide a composition that can be used as a washing solution in wounds, bums or infection areas and contributes to strengthening the immune system.
- the composition of the invention comprises an exosome obtained from bee venom. Bee venom contributes to reducing inflammation in the body; therefore, it eliminates the factors that predispose to autoimmune diseases.
- the invention provides a composition that contributes to strengthening the immune system by using bee exosome obtained from bee venom.
- Another aim of the invention is to provide a composition that reduces the bacterial population in wounds, burns or infection areas or prevents the reproduction of these bacteria.
- the composition of the invention comprises at least one antibiotic used against bacteria. Said antibiotic effectively kills bacteria and even prevents them from reproducing. Thus, a composition is provided to reduce the bacterial population in wound or infection areas and prevent the reproduction of these bacteria.
- Another aim of the invention is to provide a composition that prevents bacteria in wounds, bums or infection areas from developing antibacterial resistance.
- the synergistic effect of the antibiotic contained in the composition of the invention and the bee exosome obtained from bee venom bacteria in wounds, burns or infection areas are prevented from developing antibacterial resistance.
- Another aim of the invention is to provide an easily sprayable composition that can be used as a washing solution in wounds, burns or infection areas.
- the composition of the invention comprises isotonic liquid. This isotonic liquid enables easy spraying of said composition.
- composition which can be used as a washing solution in wounds, bums or infection areas and prevents biofilm formation, is easily sprayable, contributes to strengthening the immune system, reduces the bacterial population in wound, bum or infection areas or prevents the reproduction of these bacteria, and prevents the bacteria in wound, bum or infection areas from developing antibacterial resistance, is provided
- FIG. 1 Scanning electron microscope (SEM) image of bee exosome obtained from bee venom.
- the invention relates to a composition that can be used as a washing solution in wounds, bums or infection areas or as a tooth washing solution in dental operations, is easily sprayable, comprises bee exosome, prevents biofilm formation on the wound, and has antibacterial and antibiotic properties, and a method for the preparation of this composition.
- the bee exosome mentioned here is obtained from bee venom in liquid form.
- the composition of the invention comprises at least one antibiotic or antibiotic mixture to be used against bacteria, bee exosome obtained from bee venom, and isotonic liquid (serum physiology) that facilitates spraying.
- Said antibiotic may be gentamicin or amoxicillin, or it is preferred as a mixture of gentamicin and amoxicillin, which is a mixture with bacteriocide or bacteriostatic effects.
- the antibiotic mixture with ancidal or bacteriostatic effects is used in the composition of the invention at a rate of 1 -5% by weight.
- the composition comprises 0.1 -2% bee exosome by weight.
- Bee exosome is a component of bee venom in liquid form.
- the bee exosome is separated from the bee's venom using the centrifuge method or a kit.
- the minimum inhibition concentration (MIC) value of bee exosome obtained from bee venom is 400 ngr.
- the composition of the invention which comprises bee exosome, prevents biofilm formation on the wound, and has antibacterial and antibiotic properties, contains OS- OS.8% isotonic liquid by weight.
- Isotonic liquid prevents the bee exosome contained in the composition from breaking down and ensures easy spraying of the composition.
- isotonic water balances the pressure of the bee exosome in the composition, thus preventing the exosome from deteriorating.
- Isotonic liquid is a simple salt solution. 1 ml of isotonic liquid contains 0.9 milligrams of sodium chloride (NaCI). The pH of the isotonic liquid should be 7.2 and the temperature should be 20-40°C.
- isototic liquid in the composition of the invention, the internal pressure of the vesicle and the exosome is stabilised, preventing the exosome from bursting or shrinking.
- Isototic fluid is used for storage of exosomes.
- the production method of the composition that is the subject of the invention comprises the process steps of
- the antibiotic mixture used in the production of the composition whose process details are given above is obtained from gentamicin and amoxicillin.
- a mixture of 1 -5% by weight of gentamicin and amoxicillin is dissolved in 93-98.8% by weight of isotonic liquid, and 0.1 -2% by weight of an exosome is added to this solution.
- the resulting solution is subjected to ultrasonic vibration for 30-45 minutes at 20-40°C and as a result of rotation at 100000-200000 rpm for 1 hour, an antibacterial and antibiotic composition comprising bee exosome, which prevents the formation of biofilm on the wound, is obtained.
- Production method of the composition that is the subject of the invention comprises the process steps of
- the shaker of the ultrasonic vibration device operates at 1000 rpm, thus ensuring that the device vibrates at the desired level.
- the production method of said composition uses the ultracentrifuge method to separate the bee exosome from the bee venom
- the bee exosome is separated from the bee venom by rotating the bee venom in the ultracentrifuge at 100000-200000 rpm for 1 hour.
- the production method of said composition uses a commercial kit to separate bee exosome from bee venom; said kit disrupts the structure of liquid bee venom and ensures the collapse of exosomes, without the need for high centrifuge speeds in the separation of bee exosomes.
- the kit used disrupts the layers of water molecules in the bee venom and holds the surface of the exosome, allowing the bee exosome to decompose with lower centrifugal speed and time (5 minutes at 10000 rpm).
- the purity level of the bee exosome obtained using the kit is lower than the bee exosome obtained by high-speed centrifugation in the ultracentrifuge (it may also retain other proteins and residues). For this reason, the ultracentrifuge method is preferred over the kit.
- MIC Minimum inhibitory concentration
- MRSA Staphylococcus aureus
- the expression minimum inhibitory concentration is used to determine the minimum amount of antibiotic that stops the growth of bacteria.
- the MIC is the suppressive dose and the lower the MIC value, the stronger the effect.
- the concentration of the antibiotic reaches the MIC value and cures, the microorganism is considered to be sensitive to this antibiotic.
- the MIC values of bee venom and exosome components against the determined pathogens were determined using the microdilution method. Dosage ranges of bee venom exosome and antibiotic are determined as 1028-32 pg/ml.
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Abstract
The invention relates to a composition with antibacterial and antibiotic properties that can be used as a washing solution in wounds or infection areas, can be easily sprayed, prevents biofilm formation on the wound, and to the production method of this composition. The composition of the invention comprises at least one antibiotic to be used against bacteria, bee exosome obtained from bee venom and isotonic liquid that facilitates spraying.
Description
A BEE EXOSOME-BASED COMPOSITION WITH ANTIBIOTIC PROPERTIES AGAINST BIOFILM FORMATION AND THE PREPARATION METHOD OF THIS COMPOSITION
Technical Field of the Invention
The invention relates to a composition with antibacterial and antibiotic properties that can be used as a washing solution in wound, burn or infection areas or as a tooth washing solution in dental operations, which prevents the production of biofilm on the wound, and a method for the preparation of this composition.
State of the Art
Biofilms are bacterial communities structured within a protective extracellular polymerised substance (EPS) matrix that adheres to living or non-living surfaces. This gel-like layer is a polysaccharide-based network structure produced by bacterial cells and is called “exopolysaccharide” or “exopolymer (EPS)” in the terminology. Biofilm formation by microorganisms is a complex phenomenon that allows them to survive in an adverse environment. Biofilm infections account for 65% or more of all infections, especially tool-related infections, skin and soft tissue infections, and chronic infections. Bacteria within the biofilm are quite heterogeneous, with a small group being slow growers known as persisters and some being fast growers. Due to this heterogeneity and EPS matrix, bacteria in the biofilm are resistant to host defense mechanisms and antibiotic effects. Therefore, biofilms delay the healing process of damaged tissues or wounds [1 ],
Biofilm formation can occur in living cells or on inanimate surfaces and is affected by factors such as the presence of other bacteria, amount of nutrients, temperature, amount of oxygen and pH. Biofilms can be formed by a single type of microorganism or can contain more than one type. In biofilms consisting of different species, each species forms its own microcolony, and these microcolonies are separated from each
other by water channels. The ongoing water flow in water channels ensures the diffusion of nutrients and oxygen. 98% of the biofilm composition consists of water and the other substances in its composition are held together by the extracellular matrix structure. EPSs are responsible for the morphological, structural and physicochemical properties of biofilms. The composition of EPS consists of polysaccharide, protein, nucleic acid, teichoic acid, phospholipid and other polymeric substances and constitutes 75-90% of biofilms. The water in the biofilm structure binds to the EPS created by the bacteria. EPSs form biofilms and help microorganisms form colonies and attach to the surface. Multiple layers of cells and EPS can form a complex structure that makes it difficult for antimicrobials to penetrate and reach the inner layers. Thus, they can protect the organism against osmotic stress, phage contamination, toxic compounds, disinfectants and antibiotics. Microorganisms in biofilms are 10-1000 times more resistant to antimicrobial compounds than their planktonic forms [2],
Antibiotics or washing solutions with antibiotic properties have been developed against different bacterial infections around the world. Antibiotics and surfactants, which are antimicrobial chemical agents, are the conventional methods for controlling biofilms by using them in as small amounts as possible when necessary, although they have the risk of being harmful to the environment. As the exopolysaccharide structure in the biofilm structure reduces the penetration of antibiotics and prevents them from reaching the bacteria, and the number of infections caused by antibiotic-resistant microorganisms increases, these traditional approaches are insufficient to destroy the biofilm structure and do not prevent biofilm formation. Current methods that fail to remove biofilms are also not financially advantageous [3],
In the state of the art, antibiotics or exosomes, which are vesicles obtained from cells and have antibacterial properties, used in washing solutions with antibiotic properties, are used against bacterial infections. A vesicle is a relatively small intracellular sac consisting of a closed membrane that stores or transports substances. Exosomes are vesicles with a diameter of 30-150 nm. These structures carry bioactive substances. By means of their antibiofilm properties, exosomes reduce bacterial infection and prevent biofilm formation. In addition, exosomes contribute to strengthening the immune system and play an active role in reducing the bacterial population. In addition, exosomes prevent the emergence of antibacterial resistance [4],
Apitoxin, also known as bee venom, is a bitter, colourless, liquid chemical. It is found inside the venom sacs of honey bees and is the substance that enters the human body when exposed to a bee sting. The active substance of apitoxin poison consists of proteins, and this substance can be neutralised by ethanol. It is an acidic chemical substance and its pH value is between 4.5 and 5.5. Bee venom is rich in hyaluronic acid, phospholipase A2 molecule, dopamine, seretamine, hitamin and noradrenaline. Hyaluronic acid, a substance contained in the venom and which is very important especially for skin health, connects two healthy cells, delays the signs of aging, rejuvenates and beautifies the skin. Phospholipase A2 molecule supports cell formation and destroys bacteria. Bee venom is a powerful anti-inflammatory. Reduces inflammation in the body; therefore, eliminates the factors that predispose to autoimmune diseases. Additionally, bee venom is effective against skin disorders and heals scars in a short time. It also soothes the skin in cases of psoriasis and eczema [5].
The patent application numbered LIS2012088671 A1 in the state of the art relates to polyphenolic compounds derived from a plant that prevent biofilm formation. Also provided are combinations comprising at least one phenolic phytochemical and at least one antimicrobial agent that inhibits the growth of an established biofilm. Patent application number LIS2012088671 A1 provides methods for inhibiting the formation and growth of biofilms. In one embodiment, a polyphenolic composition is provided. Production method of the polyphenolic compound comprises the process steps of “(a) partitioning an alcoholic extract of a plant with a mixture of water and hexane to form a first water portion and a hexane portion; (b) dividing the first water portion with a mixture of water and ethyl acetate to form a second water portion and an ethyl acetate portion; (c) splitting the second water compartment with a mixture of water and butanol to form a third water compartment and a butanol compartment; and (d) fractionating the butanol fraction by column chromatography with a mobile phase containing a mixture of methanol and dichloromethane, wherein the polyphenolic composition is eluted by the mobile phase having a volume ratio of methanol to dichloromethane of about 40:60.” However, since said composition does not comprise any components that would prevent the formation of biofilm, it does not provide an effective treatment for the healing of open or closed wounds.
Due to negativities and disadvantages such as antibiotics, solutions or disinfectants in the state of the art not providing effective treatment in the treatment of bacterial infections or diabetic wounds, not preventing biofilm formation, not contributing to the immune system, are less effective in reducing the population of bacteria that form biofilms, and are insufficient to prevent the formation of antibacterial resistance, it has become necessary to make a development in the relevant field.
Brief Description and Aims of the Invention
The invention relates to a composition that can be used as a washing solution in wounds, bums or infection areas or as a tooth washing solution in dental operations, is easily sprayable, comprises bee exosome obtained by isolating exosomes, prevents biofilm formation on the wound, and has antibacterial and antibiotic properties, and a method for the preparation of this composition. The composition of the invention comprises at least one antibiotic for use against bacteria, bee exosome and isotonic liquid (serum physiology) that facilitates spraying.
The most important aim of the invention is to provide an antibacterial composition that can be used as a washing solution in wounds, burns or infection areas and prevents biofilm formation. The composition of the invention comprises at least one antibiotic used against bacteria and bee exosome obtained from bee venom. Bee venom, which is rich in hyaluronic acid, phospholipase A2 molecule, dopamine, serethamine, hitamine and noradrenaline, supports cell formation and destroys bacteria. Bee venom is a powerful anti-inflammatory. Additionally, bee venom is effective against skin disorders and heals scars in a short time. In the invention, an antibacterial composition that prevents biofilm formation is provided by using bee exosome obtained from bee venom.
Another aim of the invention is to provide a composition that can be used as a washing solution in wounds, bums or infection areas and contributes to strengthening the immune system. The composition of the invention comprises an exosome obtained from bee venom. Bee venom contributes to reducing inflammation in the body; therefore, it eliminates the factors that predispose to autoimmune diseases. Thus, the invention provides a composition that contributes to strengthening the immune system by using bee exosome obtained from bee venom.
Another aim of the invention is to provide a composition that reduces the bacterial population in wounds, burns or infection areas or prevents the reproduction of these bacteria. The composition of the invention comprises at least one antibiotic used against bacteria. Said antibiotic effectively kills bacteria and even prevents them from reproducing. Thus, a composition is provided to reduce the bacterial population in wound or infection areas and prevent the reproduction of these bacteria.
Another aim of the invention is to provide a composition that prevents bacteria in wounds, bums or infection areas from developing antibacterial resistance. With the synergistic effect of the antibiotic contained in the composition of the invention and the bee exosome obtained from bee venom, bacteria in wounds, burns or infection areas are prevented from developing antibacterial resistance.
Another aim of the invention is to provide an easily sprayable composition that can be used as a washing solution in wounds, burns or infection areas. The composition of the invention comprises isotonic liquid. This isotonic liquid enables easy spraying of said composition.
With the invention, a composition, which can be used as a washing solution in wounds, bums or infection areas and prevents biofilm formation, is easily sprayable, contributes to strengthening the immune system, reduces the bacterial population in wound, bum or infection areas or prevents the reproduction of these bacteria, and prevents the bacteria in wound, bum or infection areas from developing antibacterial resistance, is provided
Description of Drawings
Figure 1. Scanning electron microscope (SEM) image of bee exosome obtained from bee venom.
Detailed Description of the Invention
The invention relates to a composition that can be used as a washing solution in wounds, bums or infection areas or as a tooth washing solution in dental operations, is easily sprayable, comprises bee exosome, prevents biofilm formation on the wound,
and has antibacterial and antibiotic properties, and a method for the preparation of this composition. The bee exosome mentioned here is obtained from bee venom in liquid form.
The composition of the invention comprises at least one antibiotic or antibiotic mixture to be used against bacteria, bee exosome obtained from bee venom, and isotonic liquid (serum physiology) that facilitates spraying. Said antibiotic may be gentamicin or amoxicillin, or it is preferred as a mixture of gentamicin and amoxicillin, which is a mixture with bacteriocide or bacteriostatic effects. The antibiotic mixture with bacteriicidal or bacteriostatic effects is used in the composition of the invention at a rate of 1 -5% by weight. By using said antibiotic mixture in the composition of the invention, the antibacterial spectrum of the composition is expanded.
The composition comprises 0.1 -2% bee exosome by weight. Bee exosome is a component of bee venom in liquid form. The bee exosome is separated from the bee's venom using the centrifuge method or a kit. The minimum inhibition concentration (MIC) value of bee exosome obtained from bee venom is 400 ngr.
The composition of the invention, which comprises bee exosome, prevents biofilm formation on the wound, and has antibacterial and antibiotic properties, contains OS- OS.8% isotonic liquid by weight. Isotonic liquid prevents the bee exosome contained in the composition from breaking down and ensures easy spraying of the composition. In addition, isotonic water balances the pressure of the bee exosome in the composition, thus preventing the exosome from deteriorating. Isotonic liquid is a simple salt solution. 1 ml of isotonic liquid contains 0.9 milligrams of sodium chloride (NaCI). The pH of the isotonic liquid should be 7.2 and the temperature should be 20-40°C. By means of the use of 93-98.8% of isototic liquid in the composition of the invention, the internal pressure of the vesicle and the exosome is stabilised, preventing the exosome from bursting or shrinking. Isototic fluid is used for storage of exosomes.
The production method of the composition that is the subject of the invention comprises the process steps of
• separating bee exosome from bee venom by ultracentrifuge method or with a kit and obtaining bee exosome,
• dissolving at least one, and preferably at least two, antibiotics in isotonic liquid in a separate place and obtaining an antibiotic solution,
• adding the bee exosome separated from the bee venom to the antibiotic solution dissolved in isotonic liquid and obtaining an antibiotic solution containing the bee exosome, and
• subjecting the solution containing bee exosome and antibiotic to ultrasonic vibration and thus obtaining an antibacterial and antibiotic composition containing bee exosome, which prevents the formation of biofilm on the wound, sequentially or in a mixed order.
The antibiotic mixture used in the production of the composition whose process details are given above is obtained from gentamicin and amoxicillin. A mixture of 1 -5% by weight of gentamicin and amoxicillin is dissolved in 93-98.8% by weight of isotonic liquid, and 0.1 -2% by weight of an exosome is added to this solution. The resulting solution is subjected to ultrasonic vibration for 30-45 minutes at 20-40°C and as a result of rotation at 100000-200000 rpm for 1 hour, an antibacterial and antibiotic composition comprising bee exosome, which prevents the formation of biofilm on the wound, is obtained.
Production method of the composition that is the subject of the invention comprises the process steps of
• separating bee exosome from bee venom by ultracentrifuge method or with a kit,
• preparing the antibiotic mixture comprising gentamicin and amoxicillin in dry and powder form in a separate place and dissolving the mixture at a rate of 1 -5% by weight in isotonic liquid at a rate of 93-98.8% by weight,
• adding 0.1-2% by weight of bee exosome to the resulting solution,
• subjecting the solution comprising bee exosome to ultrasonic vibration at 20-
40°C for 30-45 minutes, and
• obtaining an antibacterial and antibiotic composition comprising bee exosome, which prevents the formation of biofilm on the wound, as a result of
rotating the solution comprising the bee exosome subjected to vibration for 1 hour at 100000-200000 rpm, sequentially or in a mixed order.
In the process of subjecting the solution comprising the bee exosome to ultrasonic vibration, the shaker of the ultrasonic vibration device operates at 1000 rpm, thus ensuring that the device vibrates at the desired level.
In an embodiment of the invention, if the production method of said composition uses the ultracentrifuge method to separate the bee exosome from the bee venom, the bee exosome is separated from the bee venom by rotating the bee venom in the ultracentrifuge at 100000-200000 rpm for 1 hour.
In another embodiment of the invention, if the production method of said composition uses a commercial kit to separate bee exosome from bee venom; said kit disrupts the structure of liquid bee venom and ensures the collapse of exosomes, without the need for high centrifuge speeds in the separation of bee exosomes. The kit used disrupts the layers of water molecules in the bee venom and holds the surface of the exosome, allowing the bee exosome to decompose with lower centrifugal speed and time (5 minutes at 10000 rpm). The purity level of the bee exosome obtained using the kit is lower than the bee exosome obtained by high-speed centrifugation in the ultracentrifuge (it may also retain other proteins and residues). For this reason, the ultracentrifuge method is preferred over the kit.
Table 1. Minimum inhibitory concentration (MIC) values and antibacterial activities of bee venom, bee venom exosome, and gentamicin and amoxicillin antibiotic mixture.
Bacterial Strains Bee Venom Bee venom Antibiotics MIC
MIC g/ml exosome MIC mg/L pg/ml
Escherichia co// ATCC 25922 125 1 ,95 4
Klebsiella pneumoniae ATCC 1.95 1 ,95 16
700603
Methicillin resistant 250 7,81 16
Staphylococcus aureus (MRSA)
ATCC 43300
Pseudomonas aureginosa 3.9 1 ,95 3
ATCC 27853
Methicillin-resistant 1000 62,5 1
Staphylococcus aureus
(MRKNS) growing in blood cultures ATCC 29213
Staphylococcus heamolyticus 15.62 1 ,95 1
ATCC JCSC1435
Enterobacter aerogenes 1 ,95 1 ,95 No data
ATCC 13048
Enterococccus feacalis 250 31 ,25 2
ATCC 29212
Streptococcus mitis ATCC 1000 250 2
12261
As seen in Table 1 , the expression minimum inhibitory concentration (MIC) is used to determine the minimum amount of antibiotic that stops the growth of bacteria. The MIC is the suppressive dose and the lower the MIC value, the stronger the effect. After applying antibiotics for treatment purposes, if the concentration of the antibiotic reaches the MIC value and cures, the microorganism is considered to be sensitive to this antibiotic. For a composition containing the bee exosome of the invention, which prevents biofilm formation on the wound and has antibacterial and antibiotic properties, the MIC values of bee venom and exosome components against the determined pathogens were determined using the microdilution method. Dosage ranges of bee venom exosome and antibiotic are determined as 1028-32 pg/ml.
REFERENCES
[1 ] Gun, I. & Ekinci, F. Y. (2009). Biyofilmler: Yuzeylerdeki Mikrobiyal Ya§am. Gida,
34 (3), 165-173. Retrieved from https://dergipark.org.tr/tr/pub/gida/issue/6849/91833
[2] Unal Turhan, E., & Erginkaya, Z. (2019). Bakteriyel Biyofilmlerdeki Antimikrobiyel Direng Mekanizmasi. Akademik Gida, https://doi.Org/10.24323/akademik- gida.544978
[3] Nagela Bernadelli Sousa, S., Lara De Andrade, M., & Denise Von Dolinger De Brito, R. (2020). Antibiyofilm Activity of Natural Products: Promising Strategies for Combating Microbial Biyofilms. Annals of Public Health Reports, 4(1 ). https://doi.org/10.36959/856/502
[4] INANIR, C., & EKICI, L. (2020). Eksozomlar: Kompozisyonlan, Biyolojik Fonksiyonlan ve Biyoaktif Bilegiklerin Taginmasmdaki Potansiyelleri. Akademik Gida, 421-432. https://doi.org/10.24323/akademik-gida.850939
[5] Kubala, M. J. S. (2019, June 24). Bee Venom: Uses, Benefits, and Side Effects.
Healthline, https://www.healthline.com/nutrition/bee- venom#:%7E:text=May%20benefit%20skin%20health,antibacterial%20effects %2C%20and%20reducing%20wrinkles.
Claims
1. An antibacterial and antibiotic composition that prevents biofilm formation on the wound, comprising 1-5% by weight of at least one antibiotic, 0.1-2% bee exosome obtained from bee venom and 93-98.8% isotonic water.
2. A composition according to Claim 1 , wherein said antibiotic comprises gentamicin and amoxicillin.
3. A composition according to Claim 2, wherein said antibiotic comprises gentamicin:amoxicillin in the ratio of 1 : 10.
4. A composition according to any one of claims 1 -3 for use as a rinsing solution in areas of wounds, bums or infections, or as a dental rinsing solution in dental operations.
5. Production method of a composition according to any one of claims 1 to 3, wherein the method comprises the process steps of:
• separating bee exosome from bee venom by ultracentrifuge method or with a kit and obtaining bee exosome,
• dissolving at least one, and preferably at least two, antibiotics in isotonic liquid in a separate place and obtaining an antibiotic solution,
• adding the bee exosome separated from the bee venom to the antibiotic solution dissolved in isotonic liquid and obtaining an antibiotic solution containing the bee exosome, and
• subjecting the solution containing bee exosome and antibiotic to ultrasonic vibration and thus obtaining an antibacterial and antibiotic composition containing bee exosome, which prevents the formation of biofilm on the wound.
6. A method according to claim 5, wherein the antibiotic mixture used is obtained from the antibiotic mixture gentamicin and amoxicillin.
7. A method according to claim 5, wherein a mixture of 1 -5% by weight of gentamicin and amoxicillin is dissolved in 93-98.8% by weight of isotonic liquid, and 0.1 -2% by weight of an exosome is added to this solution.
A method according to claim 5, wherein the isotonic solution comprising bee exosome and antibiotic mixture is subjected to ultrasonic vibration at 20-40°C for 30-45 minutes. A method according to claim 6, wherein, in the ultrasonic vibration process, the shaker of the ultrasonic vibration device operates at 100000-200000 rpm and rotates for 1 hour. Production method of a composition according to any one of claims 1 to 3, wherein the method comprises the process steps of:
• separating bee exosome from bee venom by ultracentrifuge method or with a kit,
• preparing the antibiotic mixture comprising gentamicin and amoxicillin in dry and powder form in a separate place and dissolving the mixture at a rate of 1-5% by weight in isotonic liquid at a rate of 93-98.8% by weight,
• adding 0.1-2% by weight of bee exosome to the resulting solution,
• subjecting the solution comprising bee exosome to ultrasonic vibration at 20-
40°C for 30-45 minutes, and
• obtaining an antibacterial and antibiotic composition comprising bee exosome, which prevents the formation of biofilm on the wound, as a result of rotating the solution comprising the bee exosome subjected to vibration for 1 hour at 100000-200000 rpm. A composition produced by a method according to any one of claims 5-10. A composition according to claim 11 with antibacterial and antibiotic properties that prevents biofilm formation on the wound. A composition according to claim 11 or 12 for use as a rinsing solution in areas of wounds, bums or infections or as a dental rinsing solution in dental operations.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101791327A (en) * | 2009-01-30 | 2010-08-04 | 农村振兴厅 | Natural antibiotics comprising bee-venoms and crude drug-extracts and preparing method thereof |
| WO2014094375A1 (en) * | 2012-12-20 | 2014-06-26 | 中国科学院南海海洋研究所 | Bee venom composition with acne removing effect |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101791327A (en) * | 2009-01-30 | 2010-08-04 | 农村振兴厅 | Natural antibiotics comprising bee-venoms and crude drug-extracts and preparing method thereof |
| WO2014094375A1 (en) * | 2012-12-20 | 2014-06-26 | 中国科学院南海海洋研究所 | Bee venom composition with acne removing effect |
Non-Patent Citations (1)
| Title |
|---|
| SCHUH CHRISTINA M. A. P., AGUAYO SEBASTIAN, ZAVALA GABRIELA, KHOURY MAROUN: "Exosome-like vesicles in Apis mellifera bee pollen, honey and royal jelly contribute to their antibacterial and pro-regenerative activity", JOURNAL OF EXPERIMENTAL BIOLOGY, COMPANY OF BIOLOGISTS CAMBRIDGE, 16 October 2019 (2019-10-16), XP093058868, ISSN: 0022-0949, DOI: 10.1242/jeb.208702 * |
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