WO2022035309A1

WO2022035309A1 - Nano emulsion composition having antimicrobial properties

Info

Publication number: WO2022035309A1
Application number: PCT/MY2021/050066
Authority: WO
Inventors: Nur Kartinee KASSIM; Rukman AWANG HAMAT; Norazlinaliza SALIM
Original assignee: Universiti Putra Malaysia
Priority date: 2020-08-14
Filing date: 2021-08-12
Publication date: 2022-02-17

Abstract

The present invention discloses an antimicrobial composition and methods for decreasing the infectivity, morbidity and rate of mortality associated with a variety of microorganisms, particularly pathogenic bacteria. The composition is an oil-in-water nano emulsion comprising of: a) a plant extract consisting of a lemon myrtle extract; b) vegetable oil-based fatty acids; c) a surfactant; d) water; and e) an organic solvent. In one embodiment, the method of the present invention includes decontaminating an area or a surface colonized or infected by pathogenic microorganisms with the oil-in-water nano emulsion disclosed herein. In another embodiment, the method of the present invention includes topically applying to a subject's part with the oil-in-water nano emulsion composition disclosed herein.

Description

NANO EMULSION COMPOSITION HAVING ANTIMICROBIAL PROPERTIES

FIELD OF THE INVENTION

The present invention relates generally to the field of antimicrobial compositions. More particularly, the present invention relates to a nano emulsion composition and methods for decreasing the infectivity, morbidity and rate of mortality associated with a variety of microorganisms, particularly pathogenic bacteria.

BACKGROUND OF THE INVENTION

Microorganism pathogens such as bacteria, bacteria spores, fungi and viruses are responsible for a number of human and animal health problems and diseases, as well as contamination of food, biological and environmental samples. The first step of microbial infections is generally attachment or colonization of skin or mucus membranes, followed by invasion and dissemination of the infectious microorganisms.

In the field of bacteriology, one of the biggest concerns is antibiotic resistant bacteria, for example Staphylococcus aureus and Acinetobacter baumannii. As microbes are exposed to more antibiotics, their ability to build resistance also increase. Acinetobacter baumannii is a gram-negative, non-motile and non-fermentative bacterium which is commonly associated with nosocomial or hospital-acquired infections namely urinary tract infections, surgical site infections, bacteraemia and others with high morbidity and mortality. Acinetobacter species are usually found in in the hospital environment as they can grow in various pH and temperature conditions. They are known to survive in harsh conditions (dry and moist) and can be present on the inanimate objects, such as medical instruments including ventilators and catheters. They commonly infect patients with prolonged hospital stay as well. Emergence of multi-drug resistant Acinetobacter baumannii has created heightened concerns worldwide. Several outbreaks have been reported, including in hospitals. In the latest Malaysia Antibiotic Guideline 2019, approximately 41% of Acinetobacter baumannii isolates from clinical samples collected from many Malaysian hospitals were resistant to carbapenem. More worryingly, about 0.5% were resistant to colistin, the last resort antibiotic for the therapy, which has complicated the treatment and management of patients with Acinetobacter baumannii infections. Nonetheless, several attempts have been developed to search for potential antibiotics from plants that might have active ingredients to kill this bacterium.

Effective disease prevention is key in maintaining healthy human and animal population. Over the years, the improvement in and availability of vaccines has greatly assisted in the prevention of many diseases. However, vaccinated humans and animals may succumb to severe challenges. In view of this and considering that vaccines are not available for all diseases, effective bio-security program, coupled with an effective disinfection and vaccination program, are essential for maintaining the health of these populations.

Bacteria, including spores, can be inactivated by heat, pressure and the use of chemical agents often referred to as bacteriocides. For example, corrosive compositions, e.g., formaldehyde and sodium hypochlorite (bleach), have been used to inactivate spores. Unfortunately, such compositions are toxic or irritating to skin and mucus membranes. It would therefore be desirable to provide compositions and methods for inactivating bacteria including bacterial spores, which are non-toxic to skin and mucus membranes. It would also be desirable to provide compositions and methods for inactivating bacteria and bacterial spores which are effective in vivo.

Antimicrobial formulations play a big role in any bio-security system, both in the process of terminal disinfection and hygiene maintenance. Most of the available antimicrobial formulations, including sanitizers and disinfectants, contain antimicrobial agents that are not naturally occurring.

Specifically, there is a need in the art for highly effective antibacterial agents that can eradicate bacterial infections. The widespread resistance to antibiotics has been reaching a critical level. Intolerable adverse effects and development of multi-drug resistant strains against conventional anti-microbial agents are among the major drawbacks. Urgent approaches need to be taken to overcome this, particularly with new antibacterial nanomaterial. It is desirable to develop a nano emulsion with multiple potential targets, complex mechanisms and effective biological, as well as physiochemical properties. The characteristic of the bacterial cell membrane which contains charged lipids with high binding efficiency and high potential for electrostatic attraction coupled with nano emulsions in the form of antibacterial nano droplets with lipids as the outer layers will ensure better delivery of the bioactive compounds to the targeted sites

Nano emulsions have a unique potential to solubilize high amounts of hydrophilic or hydrophobic compounds and therefore increase bioavailability of such compounds. Nano emulsions are emulsion systems that have three main phases: i.) water, ii.) oil, and iii.) interface. Despite their potential, few food grade nano emulsions have been developed for commercial use. The lack of development relating to food matrices and in part stability of food grade nano emulsions is blamed on limited choices for food grade surfactants, low oil solubilization capacity and stability of soluble compounds (Garti, Spernath, Aserin, & Lutz, 2005).

Thyme oil-based hand sanitizer products can be applied on human skin for disinfecting hands. Although this is a thyme oil-based antimicrobial for topical application on human skin, it does not include an antimicrobial isolated or synthetic phenolic compound of natural origin in in combination with a surfactant, a solvent for dissolving the phenolic compound and an aqueous carrier.

Prior teachings related to nano emulsions are described in U.S. Patent No. 6,015,832, which is directed to methods of inactivating a Gram-positive bacteria, a bacterial spore, or a Gram-negative bacteria. The methods comprise contacting the Gram-positive bacteria, bacterial spore, or Gram-negative bacteria with a bacteria-inactivating (or bacterial-spore inactivating) emulsion.

U.S. Patent No. 6,506,803 is directed to methods of killing or neutralizing microbial agents (e.g., bacteria, virus, spores, fungus, on or in humans using an emulsion. U.S. Patent No. 6,559,189 is directed to methods for decontaminating a sample (human, animal, food, medical device, etc.) comprising contacting the sample with a nano emulsion. The nano emulsion, when contacted with bacterial, virus, fungi, protozoa, or spores, kills or disables the pathogens. The antimicrobial nano emulsion generally comprises, in an aqueous medium, a quaternary ammonium compound, one of ethanol/glycero l/PEG, an oil, and a surfactant.

There is a need in the art for improved and highly effective nano emulsion composition having antimicrobial agent and methods for decreasing the infectivity, morbidity and rate of mortality associated with a variety of microorganisms, particularly pathogenic bacteria.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, there is provided an antimicrobial composition, characterized in that the composition is an oil-in-water nano emulsion comprising of: a) a plant extract consisting of a lemon myrtle extract; b) vegetable oil-based fatty acids; c) a surfactant; d) water; and e) an organic solvent.

The nano emulsion composition of the present invention has the following physicochemical characteristics:

• composed of droplets having a mean particle size ranging from 20 to 200 nm

• pH ranging from 3-5

• viscosity ranging from 1 -5 mPAS^-1

• polydispersity index of less than 0.5

• zeta potential in the range of -5 to -45.0 mV.

The nano emulsion composition comprises from about 0.09% to about 5.99% of the lemon myrtle extract, which is in oil form. The lemon myrtle oil is composed of 60% to 99% monoterpenoids. The monoterpenoids are citral A and citral B.

The vegetable oil-based fatty acids include a mixture of oleic acid, hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid, in appropriate amount of 0.5% to 3.9%.

The percentage of oleic acid, n-hexadecanoic acid, octadecanoic acid and 9,12- octadecadienoic acid are as follows:

The vegetable oil is preferably sunflower oil.

The nano emulsion composition comprises from 9.0% to 18.0% of the surfactant. The surfactant is a non-ionic surfactant consisting a mixture of polysorbate 80 and polysorbate 60.

Preferably the mixture of polysorbate 80 and polysorbate 60 is in a ratio of 1 :1 .

The nano emulsion composition comprises from 60% to 85% of water.

The organic solvent includes hexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol and ethanol, in appropriate amount of 10% to 90%.

According to another aspect of the present invention, there is provided a method of decreasing the infectivity, morbidity and rate of mortality associated with a variety of pathogenic microorganisms that can cause a disease or infection. A first embodiment according to this aspect of the present invention involves decontaminating an area or surface area colonized or infected by pathogenic microorganisms with an oil-in-water nano emulsion composition comprising of: a plant extract consisting of a lemon myrtle extract; vegetable oil-based fatty acids; a surfactant; water; an organic solvent; and a sufficient aqueous carrier to make if 100%.

The area or surface area that is subject to decontamination by applying the nano emulsion composition described herein may be, for example, vehicles, equipments, instruments, furniture, etc., may thus be decontaminated by applying the emulsions described herein to the area or surface area.

The nano emulsion composition for use in decontaminating an area or surface area according to the present invention has the following physico-chemical characteristics:

• composed of droplets having a mean particle size ranging from 20 to 200 nm

• pH ranging from 3 to 5

• viscosity ranging from 1 to 5 mPAS^-1

• polydispersity index of less than 0.5

• zeta potential in the range of -5 to -45.0 mV.

The nano emulsion composition for use in decontaminating an area or surface area comprises from about 0.09% to about 5.99% of the lemon myrtle extract, which is in oil form.

The lemon myrtle oil is composed of 60% to 99% monoterpenoids. The monoterpenoids are citral A and citral B. The vegetable oil-based fatty acids include a mixture of oleic acid, hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid in appropriate amount of 0.5% to 3.9%.

The vegetable oil is preferably sunflower oil.

The nano emulsion composition for use in decontaminating an area or surface area comprises from 9.0% to 18.0% of the surfactant. The surfactant is a non-ionic surfactant consisting a mixture of polysorbate 80 and polysorbate 60.

The nano emulsion composition for use in decontaminating an area or surface area comprises from 60% to 85% of water.

The aqueous carrier includes a thickener, essential oil and fragrance in appropriate amount of 1 - 2 wt.%, respectively. A second embodiment according to this aspect of the present invention involves topically applying to the subject’s part with an oil-in-water nano emulsion composition comprising of: a plant extract consisting of a lemon myrtle extract; vegetable oil-based fatty acids; a surfactant; water; an organic solvent; and a sufficient aqueous carrier to make if 100%,

The topical application prevents or treats microbial infection in a subject by applying the nano emulsion composition described herein to the skin or mucous membrane of the subject to inactivate the microorganisms, which include bacteria, bacteria spores, fungi and viruses. By inactivating the microorganisms before attachment or colonization, subsequent invasion and dissemination of the infectious pathogen may be prevented.

The treatment of microbial infection in a subject includes treating a subject by providing the emulsion preparation described herein suitable for pharmaceutical administration, which may also include a pharmaceutically acceptable carrier. The preparation can be applied topically to skin surface areas, mucus membranes, or oral surfaces, for example, as a cream, gel, spray, or mouthwash, to treat or prevent microbial infections. The preparation can also be applied to wounds caused by bacterial infection. Accordingly, the present invention further provides a method for inactivating a bacterium, including bacterial spores, by topical application of the emulsions described herein.

The subject’s part may be, for example, any one of a skin, a limb, a head, an ear, a nose, a hand, a foot, a mucosa, or any combinations thereof.

The subject is a mammal.

The nano emulsion composition for use in topical application according to the present invention has the following physico-chemical characteristics: • composed of droplets having a mean particle size ranging from 20 to 200 nm

• pH ranging from 3 to 5

• viscosity ranging from 1 to 5 mPAS^-1

• polydispersity index of less than 0.5

• zeta potential in the range of -5 to -45.0 mV.

The nano emulsion composition for use in topical application comprises from about 0.09% to about 5.99% of the lemon myrtle extract, which is in oil form.

The lemon myrtle oil is composed of 60% to 99% monoterpenoids. The monoterpenoids are citral A and citral B.

The vegetable oil-based fatty acids include a mixture of oleic acid, hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid in appropriate amount of 0.5% to 3.9%.

The vegetable oil is preferably sunflower oil.

The nano emulsion composition for use in topical application comprises from 9.0% to 18.0% of the surfactant. The surfactant is a non-ionic surfactant consisting a mixture of polysorbate 80 and polysorbate 60.

Preferably the mixture of polysorbate 80 and polysorbate 60 is in a ratio of 1 :1 . The nano emulsion composition for use in topical application comprises from 60% to 85% of water.

The aqueous carrier includes a thickener, essential oil and fragrance in appropriate amount of 1 - 2 wt.%, respectively.

It is an advantage of the present invention to provide an antimicrobial nano emulsion composition and a method that is effective in deactivating, killing or reducing the infectivity or presence of microorganisms in an area or surface area, article, sample or on a subject’s part.

The area or surface area may be for example, a room or solid surface that is potentially infected by pathogenic microorganisms, such as bacteria, bacteria spores, fungi and viruses that are responsible for a number of human and animal health problems and diseases.

The article may be for example, a medical equipment (e.g. catheter, ventilator, etc.) or a consumer article and surface area (e.g. writing tools, tables, stationery, door handles, etc.) that is potentially infected by pathogenic microorganisms, such as bacteria, bacteria spores, fungi and viruses that are responsible for a number of human and animal health problems and diseases.

The sample may be for example, food, biological or environmental samples that are potentially infected with microorganisms such as bacteria, bacteria spores, fungi and viruses that are responsible for a number of human and animal health problems and diseases. The subject is a mammal and the subject’s part may be, for example, any one of a skin, a limb, a head, an ear, a nose, a hand, a foot, a mucosa, or any combinations thereof.

A combination of the lemon myrtle extract, vegetable oil-based fatty acids, surfactant and solvent in appropriate amounts that forms a nano emulsion composition has demonstrated an isotropic and nano-sized particle with good stability. The composition exhibits good antimicrobial and antioxidant properties despite low concentration of the lemon myrtle extract.

The desired properties of the nano emulsion composition of the present invention, particularly on good bioactivity and stability, are aided with the following physicochemical characteristics of the composition:

• composed of droplets having a mean particle size ranging from 20 to 200 nm

• pH ranging from 3 to 5

• viscosity ranging from 1 to 5 mPAS^-1

• polydispersity index of less than 0.5

• zeta potential in the range of -5 to -45.0 mV.

Particularly, the nano emulsion composition of the present invention exhibits antimicrobial and antioxidant properties at low concentration of the lemon myrtle. The antimicrobial and antioxidant activities of the terpene essential oil components (citral), which are compounds extracted from plants through distillation (via steam and/or water) or mechanical methods, such as cold pressing, are highly affected by their chemical nature and interactions.

Interaction of the terpene essential oil components (citral) with the fatty acids of the sunflower oil are effective and contribute towards the biological activities by producing a synergistic and additive effect on the pathogenic microorganisms. The nano-sized particles of the emulsion composition further enhance and contributes towards the bioactivities and stability of the composition. The antimicrobial property of the nano emulsion composition of the present invention is advantageous and aided by the following:

• nanometric size that permits drug delivery through impermeable barriers

• large surface area to volume ratios for improved efficacy, surface modification and backbone functionalization versatility that facilitates cellular membranes passage or enhancing stability and bioavailability

• virucidal activity against a series of viruses (e.g. HIV, HSV, HBV, etc.) due to biomimetic properties, increased specificity, improved antiviral delivery and controlled drug release to the target

• decrease the emergence of drug resistance

It is another advantage of the present invention to provide an antimicrobial nano emulsion composition that is categorized as “food grade”, which renders it non-toxic, non-irritating and safe for use.

It is also another advantage of the present invention to provide an antimicrobial nano emulsion composition that can be produced using low energy. With this low energy production method, the energy cost is reduced, since high pressure homogenizers in the production method will not be applied.

It is yet another advantage of the present invention to provide an antimicrobial nano emulsion composition that can be formulated for use in a variety of applications, including: i) as a functional food ii) as a food additive iii) as a medicament in pharmaceuticals, therapeutic products and nasal spray. iv) as a nutritional supplement in dietary, supplements and culinary. v) as a component ingredient in cosmeceuticals, for example in aromatherapy, hair-skin care, body cream, shower gel, etc. vi) as a disinfectant, for example in hand sanitizer, surface spray, body spray. vii) as anti-bacteria air purifier. Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a chromatogram that shows the chemical components in the lemon mrytle extract evaluated by gas chromatography mass spectrum (GCMS);

Figure 2 is a graph that shows the effect of a preferred nano emulsion formulation of the present invention on VERO cells after 72 hours exposure; and

Figure 3 shows a broth microdilution by resazurin assay to determine the minimum inhibitory concentration value of a preferred nano emulsion formulation, according to the present invention.

DETAILED DESCRIPTION

The present invention provides an antimicrobial composition, characterized in that the composition is an oil-in-water nano emulsion comprising of: a) a plant extract consisting of a lemon myrtle extract; b) vegetable oil-based fatty acids; c) a surfactant; d) water; and e) an organic solvent.

The nano emulsion composition of the present invention having the following physicochemical characteristics exhibits good bioactivity and stability:

• composed of droplets having a mean particle size ranging from 20 to 200 nm

• pH ranging from 3 to 5

• viscosity ranging from 1 to 5 mPAS^-1

• polydispersity index of less than 0.5 • zeta potential in the range of -5 to -45.0 mV.

The present invention also provides a method of decreasing the infectivity, morbidity and rate of mortality associated with a variety of pathogenic microorganisms that can cause a disease or infection.

A first embodiment according to the method of the present invention involves decontaminating an area or a surface area colonized or infected by pathogenic microorganisms with an oil-in-water nano emulsion comprising of: a plant extract consisting of a lemon myrtle extract; vegetable oil-based fatty acids; a surfactant; water; an organic solvent. a sufficient aqueous carrier to make if 100%, which includes thickener, essential oil and fragrance in appropriate amounts of 1 -2 wt.%.

The area or surface area that is subject to decontamination by applying the emulsion described herein, may be, for example vehicles, equipment, instruments, etc., may thus be decontaminated by applying the emulsions described herein to the surfaces.

A second embodiment according to the method of the present invention involves topically applying to the subject’s part with an oil-in-water nano emulsion comprising of: a plant extract consisting of a lemon myrtle extract; vegetable oil-based fatty acids; a surfactant; water; an organic solvent. a sufficient aqueous carrier to make if 100%, which includes thickener, essential oil and fragrance in appropriate amounts of 1 -2 wt.%. The topical application prevents or treats microbial infection in a subject by applying the emulsion described herein to the skin or mucous membrane of the subject to inactivate the microorganisms, which include bacteria or spores. By inactivating the microorganisms before attachment or colonization, subsequent invasion and dissemination of the infectious pathogen may be prevented.

The treatment of microbial infection in a subject include treating a subject by providing the emulsion preparation described herein suitable for pharmaceutical administration, which may also include a pharmaceutically acceptable carrier. The preparation can be applied topically to skin surface areas, mucus membranes, or oral surfaces, for example, as a cream, gel, spray, or mouthwash, to treat or prevent microbial infections. The preparation can also be applied to wounds caused by bacterial infection. Accordingly, the present invention further provides a method for inactivating a bacterium, including bacterial spores, by topical application of the emulsions described herein.

A description of every constituent of the antimicrobial nano emulsion of the present invention is provided below.

1 . Monoterpenes / Monoterpenoids

Monoterpenes/monoterpenoids are a class of terpenes that consist of two isoprene units and have the molecular formula C10H16. Monoterpenes may be linear (acyclic) or contain rings. Modified terpenes, such as those containing oxygen functionality or missing a methyl group, are called monoterpenoids.

The monoterpenes/monoterpenoids contained in the composition of the present invention is derived from about 0.09% to about 5.99% of the lemon myrtle extract, which is in oil form.

Referring to Figure 1 , the lemon myrtle oil is composed of 60% to 99% monoterpenes/monoterpenoids. The main monoterpene/monoterpenoid component is citral. Citral exists in an isomeric form, namely Citral A and Citral B. 2. Fatty acids

The oil phase of the inventive composition comprises one or more fatty acids. Preferably, these are selected from fatty acids having 6 to 22 carbon atoms, more preferably 13 to 21 carbon atoms, more preferably 16 to 20 carbon atoms, most preferably 18 carbon atoms. The fatty acids may be saturated, monounsaturated or polyunsaturated. Monounsaturated fatty acids are preferred.

The fatty acids are vegetable oil-based fatty acids. Preferably, the vegetable oil is sunflower oil.

The vegetable oil-based fatty acids may be, for example, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, hexadecanoic acid and octadecanoic acid. Oleic acid is especially preferred. However, mixtures of fatty acids are also preferred.

In a preferred embodiment, the vegetable oil-based fatty acids include a mixture of oleic acid, hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid, in appropriate amount of 0.5% to 3.9%.

Preferably, the percentage of oleic acid, n-hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid are as follows:

3. Surfactant

Surfactant, or surface-active agent, is a substance which when dissolved in water, or other aqueous systems, reduces the surface or interfacial tension between it and another substance or material.

The suitable surfactant used in conjunction with the oil-in-water nano emulsion composition according to the present invention comprises a non-ionic surfactant. In a preferred embodiment, the non-ionic surfactant that can be used in conjunction with the present invention includes, but not limited to, Polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), Polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) with Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) being most preferred.

Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate) and Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) are commercially available as Tween 60® and Tween 80®.

Preferably, the non-ionic surfactant used in conjunction with the nano emulsion according to the present invention comprises a mixture of Tween 60® and Tween 80® in a ratio of 1 :1 .

According to an embodiment, the nano emulsion composition comprises from 9.0% to 18.0% of the surfactant.

The surfactant aids in the dispersion or emulsification of the nano emulsion composition. In a further embodiment, the composition in accordance with the present invention can contain one or more functional aspects such as stabilizers, antioxidants, preservatives, absorption enhancers and pH adjusting agents in order to improve the physical and chemical stability of the composition. 4. Solvent

Water and organic solvents including hexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol and ethanol.

In a preferred embodiment, the composition comprises 60% to 85% by weight of water.

In another preferred embodiment, the organic solvent includes hexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol and ethanol, in appropriate amount of 10% to 90%.

6. Other ingredients

Other ingredients or constituents that can be used in conjunction with the nano emulsion composition of the present invention include a sufficient aqueous carrier to make it 100% (w/w).

In a preferred embodiment, the carriers used in conjunction with the composition of the present invention include a thickener, essential oil and fragrance in appropriate amounts of 1 -2 wt.%.

Method of Disinfection

According to an embodiment of the present invention, the antimicrobial nano emulsion formulation may be applied on a surface or topically onto a live subject’s part by way of a variety of techniques known in the art. The formulation may be applied or formulated into:

• a diffuser or blower

• aerosol formulation

• bath

• direct application

• a wipe • a cream

• an ointment

The live subject is preferably a mammal.

The area or surface area that is subject to disinfection or decontamination by applying the emulsion described herein may be, for example, vehicles, equipments, instruments, furniture, etc., may thus be disinfected or decontaminated by applying the emulsions described herein to the surfaces.

The present invention will now be described in further detail by way of non-limiting examples.

EXAMPLES

Example 1 : Preparation of oil-in-water emulsion

Lemon myrtle extract was dissolved in a mixture of fatty acids at ratio 1 :1 . The Tween 60 and Tween 80 was mixed well and added to lemon myrtle mixture. The mixture was centrifuged followed by addition of solvent. Then, the formulation was characterized used Zetasizer Nano ZS (Malvern Instrument Ltd., UK) to determine its physico-chemical characteristics, including the particle size, pH, viscosity, polydispersity index (PDI) and zeta potential.

Example 2: Cytoxicity Test

The cytotoxicity test of the formulation was done on Vero cells (green monkey kidney epithelial cells).

Method Cytotoxicity is the killing ability of synthesized chemicals, naturally occurring toxins or immune-mediator cells. One of the parameters to determine cytotoxicity is by using MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Cell culture with the concentration of 2 x 103 cells/ml was prepared and was plated (100 pl/well) onto 96-well plates. The diluted ranges of sample extracts were added to each well with identified concentrations; 500, 100, 50, 20, 10, 5 and 1 pg/ml further incubated for 72 hr. MTT solution was added by the end of incubation samples to the cells and continued for incubation in incubator for 3 hours. After solubilization of the purple formazan crystals using DMSO were completed, the Density (OD) of the plants extract was measured using an ELISA reader at a wavelength of 570 nm. The cytotoxicity was recorded as the drug concentration causing 50% growth inhibition of the tumour cells (IC50 value) using the formula given below:

Cell viability = Absorbance sample (mean) X 100 Absorbance control (mean)

After the determination of the percentage of cell viability, graphs were plotted with the percentage of cell viability against their respective concentrations.

Result (ICso in iq/ml): No IC50 up to 500 pg/ml (indication of non-cytotoxicity of the said formulation)

Acceptance Criteria of the Assay:

Absorbance values that are lower than the control cells indicate a reduction in the rate of cell proliferation. Conversely a higher absorbance rate indicates an increase in cell proliferation.

Table 1 : Optical Density value and percentage of cell viability in various concentration of the said formulation exposed to VERO cells

Results in Table 1 and Figure 2 suggest there were no cytotoxic effect of the formulation that have been observed when treated on VERO cells up to the identified concentrations. Hence, there was no IC50 determined.

Example 3: Characterization of the Selected Formulations

The samples were characterized by using Zetasizer Nano ZS to determine the polydispersity index (PDI) and particle size by diluting the samples with deionized water at 1 L/10mL. The instrument was using dynamic light scattering method to detect dispersion of particle moving under Brownian movement and the size converted to size distribution by using Stokes-Einstein relationship:

Where Dh is the thermodynamic diameter, Dt\s the translational diffusion coefficient, KB is Boltzmann’s constant, T is a thermodynamic temperature and q is dynamic constant. Zeta potential can be used to measure the electrokinetic potential of the particle. The electrical was applied across the samples and the movement was measured by using Zetasizer Nano ZS. By using Henry’s equation, zeta potential can be calculated:

Where Ue is the electrophoteric mobility, e is the dielectric constant, q is absolute zero shear viscosity of the medium, /(KO) is the Henry function, and (KO) is measure of the ratio of the particle radius of Deby length.

The particle size, polydispersity index (PDI) and zeta potential of the nano emulsion composition of the present invention were found to be at 20 to 200 nm, less than 0.5 and ranging from -5 to -45.0 mV, respectively.

Example 4: Antimicrobial Efficacy

Methodology for minimal inhibitory concentration (MIC) by resazurin assay.

Minimal inhibitory concentration (MIC) of lemon myrtle essential oil formulation, FWB, was determined by a microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). An overnight culture of methicillin-resistant Staphylococcus aureus (MRSA) was grown on Mueller-Hinton agar at 37°C. A total of 100 pL of 100% FWB formulation (the formulation as it is without any dilution) was pipetted into the first well of a sterile 96-well plate. The second up to the tenth wells were filled with 50 pL of Mueller-Hinton broth (MHB). 2-fold serial dilution was done by taking out 50 pL of FWB formulation from the first well and mixing it with the second well, this was repeated until the tenth well was reached, the last 50 pL of the mixture was discarded. 0.5 Me Farland of MRSA inoculum was prepared by comparing the turbidity with a standard, the inoculum was diluted in MHB with the ratio of 1 in 100 to achieve a bacterial suspension of 5 x 105 CFU/mL. 50 pL of the inoculum was pipetted into each well (at this point the formulation was diluted in half therefore, the new concentration of FWB ranges from 50% to 0.1 %). Sterility control, which is the FWB alone, growth control which is the inoculum alone as well as tetracycline as the quality control were included. The plate was incubated for 24 h at 37°C. The next day, 30 pL of 0.0015% resazurin dye were mixed into each well and the plate was further incubated for another 2 hours at the same temperature. The well with live bacteria will appear pink whereas the well with dead bacteria will remain blue, MIC was recorded from the well with the lowest FWB concentration that remains blue. This test was done in triplicate.

The minimum inhibitory concentration (MIC) value of the said formula is determined as the lowest concentration of the said formula that inhibits the growth of the test bacterium. In resazurin assay, pink colour indicates growth while blue means inhibition of growth. The said formula has excellent activity against MRSA and Acinetobacter baumannii with MIC value of 1 .56 pg/ml.

Referring to Figure 3, a broth microdilution method by resazurin assay was used to determine the minimum inhibitory concentration value for the said formulation (pink colour indicates growth while blue means inhibition of growth). The said formulation in serial dilution in wells 1 -10. Well 12: the test bacterium. Row D: tetracycline as positive control in serial dilution. Row A-C: triplicate.

Referring to Table 2, the lowest MIC values of the said formulation by resazurin assay tested on MRSA and Acinetobacter baumannii in triplicate. The lowest concentration of the said formulation is 6.25 ug/ml, which has 0.125 % of LM. This concludes that the nano emulsion composition of the present invention exhibits good antibacterial activity on Methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii with minimum inhibitory concentration of (MIC) value of 1 .56 ug/ml for both.

Table 2

Example 5: Antioxidant test

First, 1 mg/mL of said formulation, Vitamin C and a-tocopherol were prepared in analytical grade methanol. Meanwhile 1 mg/mL of [3-carotene solution was prepared in chloroform. After that, 210 pL of [3-carotene solution (orange color) was pipetted into round bottomed flask which contains 5 pL of linoleic acids (one of fatty acid) and 42 pL of Tween 20. The chloroform from the mixture was then removed using rotary evaporator. Next, 10 mL distilled water was added to the dried mixture and shook to form emulsion. 200 pL of emulsion was pipetted into 96-well microplate which contains 50 pL of samples and a-tocopherol, respectively. Methanol (250 pL) was added around the samples and standard. The absorbance of mixtures at initial (t = 0) and after 2 h (t = 2) was read at 470 nm. Within 2 hours, the plate was incubated in oven at 40-50°C. The sample that has antioxidant properties will show decreasing in absorbance reading which were measured after every 30 minutes. The bleaching can be seen by observing the color changes from orange to white color. The percentage of antioxidant activity as illustrated in Table 3, was calculated as follows: Antioxidant activity, AA% =1 - [(At = 0 - At = 2)/ (Ac = 0 - Ac = 2)] xx 100%

At = formulation of the present invention

Ac = control.

Table 3

Referring to Table 3, the nano emulsion composition of the present invention demonstrated higher antioxidant activity than Vitamin C, and the antioxidant strength is comparable to Vitamin E. The formulation is also non toxic to normal cell lines when tested on Vero cells (green Monkey kidney epithelial cells).

Further, the presence of antioxidants in the nano emulsion composition of the present invention has been demonstrated to enhance the antiviral activity compared to formulations without antioxidants. This showed the synergistic effects of its components. With high antioxidant and antibacterial activities, it is likely that the formulation of the present invention also exhibits antiviral properties.

Claims

26 CLAIMS

1 . An antimicrobial composition, characterized in that the composition is an oil-in- water nano emulsion comprising of: i.) a plant extract consisting of a lemon myrtle extract; ii.) vegetable oil-based fatty acids; iii.) a surfactant; iv.) water; and v.) an organic solvent.

2. An antimicrobial composition according to claim 1 , wherein the oil-in-water nano emulsion having the following physico-chemical characteristics:

• composed of droplets having a mean particle size ranging from 20 to 200 nm

• pH ranging from 3-5

• viscosity ranging from 1 -5 mPAS^-1

• polydispersity index of less than 0.5

• zeta potential in the range of -5 to -45.0 mV.

3. An antimicrobial composition according to claim 1 , wherein the oil-in-water nano emulsion comprises from 0.09% to 5.99% of the lemon myrtle extract in oil form.

4. An antimicrobial composition according to claim 3, wherein lemon myrtle extract is composed of 60% to 99% monoterpenoids.

5. An antimicrobial composition according to claim 4, wherein the monoterpenoids are citral A and citral B.

6. An antimicrobial composition according to claim 1 , wherein the vegetable oilbased fatty acids include a mixture of oleic acid, hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid, in appropriate amount of 0.5% to 3.9%.

7. An antimicrobial composition according to claim 6, wherein the percentage of oleic acid, n-hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid are 60-85%, 3-10% and 1 -5%, respectively.

8. An antimicrobial composition according to claim 1 , wherein the vegetable oil is sunflower oil.

9. An antimicrobial composition according to claim 1 , wherein the oil-in-water nano emulsion comprises from 9.0% to 18.0% of the surfactant.

10. An antimicrobial composition according to claim 9, wherein the surfactant is a non-ionic surfactant.

1 1. An antimicrobial composition according to claim 10, wherein the surfactant comprises a mixture of polysorbate 80 and polysorbate 60 in a ratio of 1 :1 .

12. An antimicrobial composition according to claim 1 , wherein the oil-in-water nano emulsion comprises from 60% to 85% of water.

13. An antimicrobial composition according to claim 1 , wherein the organic solvent in the oil-in-water emulsion includes hexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol and ethanol, in appropriate amount of 10% to 90%.

14. A method of decreasing the infectivity, morbidity and rate of mortality associated with pathogenic microorganisms, comprising decontaminating an area or a surface area colonized or infected by pathogenic microorganisms with an oil-in-water nano emulsion composition comprising of: i.) a plant extract consisting of 0.09 wt.% to 5.99 wt.% lemon myrtle extract in oil form; ii.) vegetable oil-based fatty acids comprising a mixture of oleic acid, hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid, in appropriate amount of 0.5 wt.% to 3.9 wt.%; iii.) an ionic surfactant ranging from 9.0 wt.% to 18.0 wt.%; iv.) water ranging from 60 wt.% to 85 wt.%; v.) an organic solvent ranging from 10 wt.% to 90 wt.%; vi.) a sufficient aqueous carrier to make it 100%, which includes thickener, essential oil and fragrance in appropriate amounts of 1 -2 wt.%.

15. A method according to claim 14, wherein the oil-in-water nano emulsion composition is having the following physico-chemical characteristics:

• composed of droplets having a mean particle size ranging from 20 to 200 nm.

• pH ranging from 3-5

• viscosity ranging from 1 -5 mPAS^-1

• polydispersity index of less than 0.5

• zeta potential in the range of -5 to -45.0 mV.

16. A method according to claim 14, wherein the lemon myrtle extract is composed of 60% to 99% monoterpenoids.

17. A method according to claim 16, wherein the monoterpenoids are citral A and citral B.

18. A method according to claim 14, wherein the percentage of oleic acid, n- hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid are 60-85%, 3- 10% and 1 -5%, respectively.

19. A method according to claim 14, wherein the vegetable oil is sunflower oil.

20. A method according to claim 14, wherein the ionic surfactant comprises a mixture of polysorbate 80 and polysorbate 60 in a ratio of 1 :1 .

21 . A method according to claim 14, wherein the organic solvent in the oil-in-water emulsion includes hexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol and ethanol. 29

22. A method of decreasing the infectivity, morbidity and rate of mortality associated with pathogenic microorganisms, comprising topically applying to a subject’s part with an oil-in-water nano emulsion composition comprising of: i.) a plant extract consisting of 0.09 wt.% to 5.99 wt.% lemon myrtle extract in oil form; ii.) vegetable oil-based fatty acids comprising a mixture of oleic acid, hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid, in appropriate amount of 0.5 wt.% to 3.9 wt.%; iii.) an ionic surfactant ranging from 9.0 wt.% to 18.0 wt.%; iv.) water ranging from 60 wt.% to 85 wt.%; v.) an organic solvent ranging from 10 wt.% to 90 wt.%; vi.) a sufficient aqueous carrier to make it 100%, which includes thickener, essential oil and fragrance in appropriate amounts of 1 -2 wt.%.

23. A method according to claim 22, wherein the oil-in-water nano emulsion composition is having the following physico-chemical characteristics:

• composed of droplets having a mean particle size ranging from 20 to 200 nm

• pH ranging from 3-5

• viscosity ranging from 1 -5 mPAS^-1

• polydispersity index of less than 0.5

• zeta potential in the range of -5 to -45.0 mV.

24. A method according to claim 22, wherein the lemon myrtle extract is composed of 60% to 99% monoterpenoids.

25. A method according to claim 24, wherein the monoterpenoids are citral A and citral B.

26. A method according to claim 22, wherein the percentage of oleic acid, n- hexadecanoic acid, octadecanoic acid and 9,12-octadecadienoic acid are 60-85%, 3- 10% and 1 -5%, respectively. 30

27. A method according to claim 22, wherein the vegetable oil is sunflower oil.

28. A method according to claim 22, wherein the ionic surfactant comprises a mixture of polysorbate 80 and polysorbate 60 in a ratio of 1 :1 .

29. A method according to claim 22, wherein the organic solvent in the oil-in-water nano emulsion composition includes hexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol and ethanol.

30. A method according to claim 22, wherein the subject’s part is one of a skin, a limb, a head, an ear, a nose, a hand, a foot, a mucosa, or any combinations thereof.

31 . A method according to claim 22, wherein the subject is a mammal.

32. A method according to claim 22, wherein the topical applying to is one of a spray, cream, gel or mouthwash.