Method for preparing an emulsion containing an essential oil as active ingredient, pickering emulsion and use of a pickering emulsion

Abstract

The present invention relates to a method for preparing antimicrobial Pickering emulsions stabilised by solid chitosan particles adsorbed on the oil / water interface composed of natural and non-toxic raw materials. The emulsions have a Melaleuca alternifolia essential oil (MaEO) phase dispersed in an aqueous phase, stabilised with micro and nanoparticles of chitosan (Qs). Moreover, the emulsions exhibit anti-bacterial, anti-fungal and anti-viral antibiotic activity.

Description

“METHOD FOR PREPARING AN EMULSION CONTAINING AN ESSENTIAL OIL AS AN ACTIVE INGREDIENT, PICKERING EMULSION AND USE OF A PICKERING EMULSION” FIELD OF INVENTION

[0001] The present invention describes the development of antimicrobial Pickering emulsions stabilized by solid chitosan particles adsorbed at the oil / water interface, composed of natural and non-toxic raw materials. The emulsions present a Melaleuca alternifolia essential oil (OEMa) phase dispersed in an aqueous phase, stabilized with micro and nanoparticles of chitosan (Qs). BACKGROUND OF THE INVENTION

[0002] Following the declaration of an international public health emergency by the World Health Organization (WHO) in January 2020 due to Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), several efforts around the world were made by industries and scientists to develop new solutions with increasingly effective antimicrobial characteristics to prevent the transmission of diseases caused by pathogenic microorganisms (GORDON et al., 2020).

[0003] Among the main sanitizing agents, quaternary ammonium compounds and hydrogen peroxide, among other synthetic compounds, stand out. However, growing environmental and health concerns have driven the search for new, environmentally friendly products. In this regard, essential oils have emerged as promising antibacterial, antifungal, and antiviral agents for a wide range of applications. This is due to their composition, which involves a a diverse mixture of organic compounds with antimicrobial properties that often exhibit a synergistic antimicrobial effect when combined.

[0004] Essential oils are a complex mixture of hydrocarbons, terpenes, terpenoids, and tertiary alcohols. The mechanism of action of essential oils depends on their composition. In general, they contain active ingredients with hydrophilic and hydrophobic characteristics, capable of permeating and damaging the cytoplasmic membrane (phospholipid layer) and the cell wall, leaking the cellular contents of microorganisms (AZIZ et al., 2018; MONDELLO et al., 2022; SÁNCHEZ-GONZÁLEZ et al., 2010; WANI et al., 2021; YADALAM et al., 2021).

[0005] Previous studies indicate that many essential oils have been effective in inhibiting a wide range of microorganisms, including RNA and DNA viruses such as avian influenza A virus, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), dengue virus type 2, Junin virus, adenovirus influenza virus type 3, poliovirus, and Coxsackievirus B1 (POURGHANBARI et al., 2016; TARIQ et al., 2019).

[0006] Despite their excellent bioactive properties, essential oils have some limitations such as high volatility, low solubility, high tendency to degradation and oxidation, requiring the use of different approaches to stabilize and protect these compounds, such as encapsulation and emulsification (REIS, 2020), increasing their shelf life and preserving biological activities, this fact is essential for several applications (MEYERS et al., 2021).

[0007] The practical use of OEMa is often limited by several drawbacks that may occur during handling and storage due to the high susceptibility of its components to undergo chemical reactions or degradation, such as oxidation, isomerization, polymerization and rearrangement, which mainly depend on parameters environmental factors such as temperature, light, and atmospheric oxygen (TUREK; STINTZING, 2013). These instability problems can result in a reduction or loss of effectiveness of the active components of OEMa, posing a serious problem in terms of technological application. Furthermore, OEMa is insoluble in water, exhibiting high volatility and a short half-life, making it difficult to handle (SANCHEZ-GONZALEZ et al., 2010).

[0008] There are different types of emulsions, the most common being oil-in-water (O / W) emulsions and water-in-oil (W / O) emulsions. O / W emulsions have a continuous water phase and a dispersed oil phase, while W / O emulsions have a continuous oil phase and a dispersed water phase. Emulsions must be stabilized with emulsifying agents to ensure that these multiphase systems remain thermodynamically stable (PEREIRA; GARCIA-ROJAS, 2014).

[0009] Emulsification is a stabilization technology defined as a mixture of an aqueous phase and an oil phase. In this system, the droplets are considered the dispersed phase, and the liquid in which they are found corresponds to the continuous phase. The dispersion of one phase into another, aiming to obtain a homogeneous system, occurs through amphiphilic molecules capable of reducing the interfacial tension between the phases, resulting in high thermodynamic stability (TADROS 2016, FRANZOL; REZENDE, 2015). To ensure the stability of an emulsion, emulsifying additives, such as synthetic surfactants, are added. However, these compounds increase toxicity and cause environmental problems due to their low biodegradability (SOUZA et al., 2020).

[0010] Unlike normal emulsions, Pickering emulsions are emulsions stabilized with solid particles instead of classic surfactants (E. DICKINSON, 2012). [Oi l] Strong repulsive forces, such as steric and electrostatic forces generated by the layer of emulsifier molecules at the oil-water interface, inhibit droplet aggregation. In this system, absorbed molecular emulsifiers are in equilibrium with unabsorbed ones. Conversely, in a Pickering emulsion, particles that are partially wetted by the oil and water phases are irreversibly absorbed at the interface without significantly decreasing the interfacial tension. These particles form a mechanical barrier against droplet coalescence (Cahyana, Y. et al, 2022).

[0012] Pickering emulsions are currently the subject of significant research interest (Asma Sharkawy, et al, 2022). This renewed interest is attributed to the growing awareness of the environmental risks and adverse health problems associated with low molecular weight surfactants, as well as to continued advances in nanoparticle production and characterization techniques (M. Rayner, et al, 2014).

[0013] In this sense, the Pickering emulsion system is a solution for hydrophobic essential oils prone to oxidation. This method allows essential oils to provide a better effect in the resulting product and maintain the benefits of their bioactive components.

[0014] Chitosan is a renewable biopolymer capable of acting as an emulsifying agent. This polymer can be used in solid particle form as a stabilizer for Pickering emulsions. This class of emulsions is characterized by being stabilized by solid particles as surfactant substitutes. Compared to conventional emulsions, which are stabilized by organic surfactants (Zhai et al., 2018).

[0015] Chitosan is a natural polymer obtained by partial deacetylation of chitin (ES de Alvarenga, et al., 2010). This compound consists of O-(l-4)-linked D-glucosamine units and N-acetyl-D-glucosamine units (D. Wu, et al, 2020).

[0016] Chitosan has been selected to prepare nanoparticles to stabilize Pickering emulsions, since it has excellent adhesion properties and many other positive effects for wound healing, such as stimulating hemostasis, accelerating tissue regeneration and promoting fibroblast proliferation (L. Fan, et al. 2018).

[0017] Furthermore, chitosan nanoparticles also have good biocompatibility, bioadhesiveness and biodegradability (MH Asfour, et al., 2017).

[0018] Melaleuca altemifolia essential oil (Melaleuca altemifolia) is composed of terpene hydrocarbons, primarily monoterpenes, sesquiterpenes, and their associated alcohols. Terpenes are volatile and aromatic hydrocarbons and can be considered polymers of isoprene, which has the formula C5H8 (CF Carson, et al. 2006).

[0019] Known as tea tree oil, it is extracted from a plant native to Australia and has gained increasing interest in Europe, Japan and the United States due to its antimicrobial (antifungal, antiviral, antibacterial), antitumor, antioxidant, antiseptic and anti-inflammatory properties (GIORDANI et al., 2006; KAUSHIK et al., 2020; MERONI et al., 2020; PUVA et al., 2020).

[0020] For these reasons, OEMa has been used in products for skin and wound care, respiratory infections, homeopathic medicine manufacturing, treatment of fungal mucosa and skin infections (GIORDANI et al., 2006; PUVA" et al., 2020; VETERE et al., 2020).

[0021] This essential oil is composed of hydrocarbons terpenes, in particular monoterpenes, sesquiterpenes and associated alcohols, consisting of a mixture of approximately 100 compounds including terpene aldehydes, esters, alcohols, 1,8-cineole, terpinolene and monoterpenoids. However, despite the excellent antimicrobial properties already well described in the literature, it has some limitations to preserve its properties (GAROZZO et al., 2011; OLIVA et al., 2018; ROANA et al., 2021, ROANA et al., 2021, OLIVA et al., 2018 and LI et al., 2016).

[0022] Bao et al., 2020, reveal that Pickering emulsions are those stabilized by solid particles. The authors developed a Pickering emulsion by emulsifying a mixture of curcumin (10 μg / mL) dissolved in various oils, including Melaleuca alternifolia oil. After this step, the oils containing curcumin were mixed with the chitosan nanoparticle suspension obtained using NaOH (sodium hydroxide).

[0023] The Pickering emulsions of these authors are intended to promote the wound healing process through spraying. For this reason, stabilization through network formation is favorable, as it causes an increase in viscosity, leading to an improvement in adhesion properties.

[0024] In this invention, Melaleuca alternifolia oil is used in its pure form, without additives or curcumin. The combination of Melaleuca alternifolia oil and nanoparticles aims to achieve a synergistic and enhanced effect. Furthermore, using the oil in its pure form allows for a simpler preparation process and eliminates the need for additional steps, which would involve greater energy and financial expenditure.

[0025] Furthermore, the aforementioned work used sodium hydroxide, a substance known for its high pH and corrosive properties. In our work, we did not use NaOH, but yes, just a small amount of acetic acid, highlighting the process differences at this point, in addition to having employed an environmentally friendly process.

[0026] Finally, another important aspect is particle size. In our proposal, we used micrometric particles, as shown in the image below, while the authors of this paper used nanometric particles. This difference in scale impacts variations in surface area and physicochemical properties known in the literature. This variation changes the particles' availability in the medium, their interaction with the essential oil, their stabilization capacity, among other important properties. Furthermore, while nanoparticles have health safety concerns due to possible bioabsorption by cells, microparticles have no known unsafe properties.

[0027] Cahyana, Y. et al, 2022 reveal Pickering emulsions as carriers for encapsulated tea tree oil result in a sustained release effect and increase antibacterial effects against Escherichia coli and Staphylococcus aureus.

[0028] However, the article shows that Pickering Emulsions had a zeta potential of +24 mV. The present proposed invention features fewer process steps, uses less material, and has a higher zeta potential (+71 mV), meaning high stability over time. Another important aspect is that these charges are associated with intrinsic characteristics of the emulsions, and since our system presented three times the available charges, the properties of both systems are not comparable. This variation impacts antimicrobial, antifungal, and antiviral properties differently due to the mechanism of action.

[0029] Another important point to highlight is that in these Pickering emulsions described in the article, the authors describe applications of various essential oils and different particle stabilizers in Pickering Emulsion systems, but several forms of stabilization are shown, including the use of curcumin, pH control, the use of corrosive substances such as sodium hydroxide, and relatively expensive equipment for particle dispersion, among others. The present invention relies on an innovative way of mixing the stabilizers with minimal assistance from a less aggressive reagent (Acetic Acid) to the environment, while still achieving a high standard of stability as already described.

[0030] Ferreira, GS, et al. (2023) state that tea tree oil is highly susceptible to oxidation due to weathering. Therefore, stabilizing the components of this oil is essential for the preparation of efficient emulsions. In this regard, Pickering emulsions using cellulose nanofibrils (CNF) as a stabilizing agent were tested.

[0031] The method for preparing the stabilizer is completely different from that of the present invention. The CNF used in the stabilization process is dispersed in an aqueous solution using an ultrasound tip. No reagents are added to this mixture. It is then used as an emulsifier in the preparation of the Pickering emulsion.

[0032] The present invention uses chitosan particles as a stabilizer and this emulsifier does not need to be dispersed, which makes the technology economically viable on an industrial scale as it does not require mechanical force to assist in the droplet coating process. On the contrary, the chitosan microparticle has a simple process with the addition of a small amount of Acetic Acid (0.5%) to contribute to the Pickering Emulsion stabilization process.

[0033] The CNF used in the article stabilizes the Pickering emulsion through steric hindrance. The cellulose nanofibrils form a 3D network leading to sterile obstacles, so the OEMa droplets continue to be prevented from coalescing by the CNF that are adsorbed and surrounded on their surfaces.

[0034] The stabilization of the Pickering emulsion stabilized by chitosan particles of the present invention occurs through an electrostatic mechanism, evidenced by the differences in chitosan charges when interacting with the active ingredients of Melaleuca altemifolia oil. The high stability of the emulsions is attributed to the amine (+) group of Cs, which can be partially neutralized through interaction with some negatively charged groups of Melaleuca altemifolia during adsorption at the droplet / oil interface (SÁNCHEZ-GONZÁLEZ et al., 2010).

[0035] Asma, S. et. Al. (2022) is aimed at preparing a Pickering emulsion comprising CBD stabilized in chitosan / gum arabic, with the stabilizer dissolved only in acetic acid (0.1N) through magnetic stirring. Gum arabic is shown to be a natural, low-cost, and sustainable stabilizer.

[0036] In relation to the present invention, the work used the stabilizer chitosan with the aid of gum arabic. In the present invention, the emulsifier was chitosan with different characteristics and without the aid of gum arabic.

[0037] Asma, S. et. Al. (2022) presents a chemical modification through complexes with the aid of Gum Arabic. The present invention opted for stabilization without the use of complex formation, obtaining a solution suitable for the manufacture of Pickering. In addition, the amount of weak acid (acetic acid) is much lower, precisely because we do not use chemical modification techniques. This peculiarity promotes a sustainability effect with less environmental damage by using fewer resources.

[0038] Still on this article, the objective of deacetylation of Chitin is the form of chitosan. The degrees of deacetylation, such as molar mass distribution and impurity content, depend on the natural raw material sources and the preparation method, leading to a variation between 40 and 95% in the degree of deacetylation (above 50%, the polymer becomes soluble in aqueous solutions).

[0039] Several factors are analyzed for obtaining chitosan from chitin, including alkaline solution concentration, reaction temperature, reaction time, chitin particle size, and impurities. The conditions used during the chitin deacetylation reaction influence the molar mass of the chitosan polymer and, consequently, its degree of deacetylation (the percentage of free amino groups present in the structure after the acetyl group has been removed).

[0040] These factors demonstrate the difficulty of obtaining chitosan with a high degree of deacetylation. The aforementioned document used a chitosan with 96% (high DDA) in its process. This DDA is important for analyzing whether chitosan has the potential to be used as a raw material for research studies and as inputs for the manufacture of products dependent on it.

[0041] The higher the percentage of (DDA%), the greater the ease of modification to a nanometric scale, thus increasing the surface area, in addition, there is the presence of a greater number of amino groups in the structure, therefore the INCREASE in the level of deacetylation is an important factor, as it allows greater adsorption of particles at the interfaces, leading to the formation of small spherical drops (morphology) improving the stability of the system, because of this, high rates of (DDA%) are obtained through more rigorous processes and consequently the monetary value of chitosan tends to increase, when it comes to executing the process, these data are relevant since affect the value of the final product.

[0042] One of the differences between the aforementioned article and the present application is the difference in deacetylation. The author of Document D7 uses deacetylation of around 96%, while we use chitosan with (DDA%) of around (75-80%). As already mentioned, the higher the degree of deacetylation, the easier the manipulation of chitosan, and the greater the direct influence on particle properties. However, the present invention used a much lower degree of deacetylation and obtained excellent results, reaching a level of +71 mV according to the zeta potential.

[0043] It is highlighted that the referred article achieved a stability of +68.5 ± 4.9 mV and +46.7 ± 1.7 mV, however, the authors achieved this zeta potential with the use of a chitosan with a high degree of deacetylation (DDA%) as already described, the (DDA%) positively affects the properties of the chitosan particles, and the higher the degree of deacetylation, the easier it is to achieve high stability. According to the article, the degree of deacetylation of 96% (DDA%) achieved a Zeta potential of +68.5 + 4.9, while 78% (DDA%) obtained a Zeta potential of 46.7 + 1.7 mV, in this sense, it is observed how important it is to use a chitosan with a high degree of deacetylation to achieve high stability in Pickering emulsions.

[0044] The present invention consists of a chitosan with a low degree of deacetylation (75-85%) and without the use of gum arabic to aid in Pickering stabilization. The aforementioned document employed gum arabic in the stabilization process. The literature shows the use of chitosan with gum arabic, as chitosan particles have been reported to be highly hydrophilic and positively charged. Therefore, the use of gum arabic is important for the Pickering stabilization process because it has the opposite charge to chitosan, increasing the reactivity with the polymer chain, thus resulting in greater polymer interconnection (complexation). This fact underlies the authors' reason for achieving good Pickering stability.

[0045] In contrast, the stabilization process of the present invention uses only chitosan (1 mL of Acetic Acid for 1 g of chitosan), and with this, a high Zeta potential +71 mV is obtained, handling fewer reagents during the procedure and consequently reducing the number of steps involved in the manufacture of the Pickering emulsion.

[0046] This makes clear the advances brought by the present invention, namely, a simple process, using fewer reagents, and without the need for a lot of equipment and with good results.

[0047] Still on Asma, S. et. Al. (2022), the Pickering stabilization process of the aforementioned article shows that the particles were prepared through the complexation of polyelectrolytes between chitosan and gum arabic following a methodology completely different from that proposed by the present invention.

[0048] The methodology employed in this work consists of a modification in the structure of chitosan, in short, lg of chitosan was dissolved in 50mL of acetic acid (0.1 N), followed by the addition of a gum arabic solution (consisting of lg of gum arabic dissolved in 50mL of deionized water) drop by drop with constant magnetic stirring (800 rpm) for 30 min, controlling the pH D7[4, 5],

[0049] The methodology of the present invention presents a simpler stabilization process with a chitosan with (DDA%) between (75-80%), that is, the present invention presents a chitosan with a lower degree of deacetylation when compared to the chitosan used in the article, in addition to using an infinitely smaller amount of acetic acid, (Asma, S. et. Al. (2022)-50mL for 1g / present application ImL for 1g), the present application presents only a simple process, where chitosan was dissolved in a solution of ImL of acetic acid in distilled water.

[0050] The aforementioned article presents a polyelectrolyte complexation between chitosan and gum arabic, this stage occurring before the preparation of the Pickering emulsion stabilization. The present invention presents fewer processes involved in obtaining a Pickering emulsion with a high degree of stability. The aforementioned document presents stability of +68.5 ± 4.9 mV and +46.7 ± 1.7 mV, but uses a chitosan with a high degree of (DDA%) in addition to additional steps shown in Table 1 below: Table 1: Comparison between the present invention and the state of the art Asma, S. et. Al. (2022) Present invention

[0051] It is possible to observe that the aforementioned article, despite achieving good stability, makes use of many resources, both equipment and reagents, and still presents a complex methodology when compared with the present invention, considering that we have a proposal for Pickering stabilization with chitosan using only a minimum amount of a weak acid (Acetic Acid), without agitation, and only using distilled water (24 h period), in addition, This simplicity in execution leads to a reduction in resources and less environmental damage associated with the process.

[0052] It is evident that in the polyelectrolyte process used by the author of the article, two types of water are consumed (deionized water and distilled water). Considering a direct application, it would be an additional cost to implement this methodology in an industrial process, since the production of deionized water has a high processing cost, as it is water with a higher degree of purity, since ions are removed by means of electrodes.

[0053] The present invention features the use of only distilled water throughout the entire process, both in the preparation of the chitosan solution and in the manufacture of the Pickering emulsion. This feature contributes to an innovative approach, in addition to, of course, reducing energy costs, contributing to a sustainable economy.

[0054] The use of Pickering Emulsions of essential oils is quite widespread, however, what the present invention highlights is that several ways are used to achieve the greatest possible stability, and for this reason, the use of equipment and additives that are harmful to the environment has become very common and widespread.

[0055] The challenge was to achieve high stability in a Pickering emulsion using few additional raw materials, making the process simpler and less costly, thus increasing both the economic viability of the process and the issues of sustainability and environmental friendliness.

[0056] Furthermore, the present invention optimized the active compounds of Melaleuca altemifolia oil through Pickering's emulsion, which demonstrated high stability and antimicrobial action, promising for a vehicle with diverse applications, especially against the SARS-CoV-2 virus. The active ingredients of Melaleuca altemifolia oil were able to immediately inactivate SARS-CoV-2, showing that stabilization was effective and the process is highly reproducible.

[0057] This fact is relevant, as it demonstrates that this system has an efficient action against new microorganisms that are emerging, being potential for diverse areas, especially cleaning highly contaminated surfaces, such as public transport and hospitals.

[0058] The advantages of the invention will be evident from the description of the invention provided in this document. SUMMARY OF THE INVENTION

[0059] The present invention aims to develop antimicrobial Pickering emulsions stabilized by solid chitosan particles adsorbed at the oil / water interface.

[0060] In particular, the emulsions present a phase of Melaleuca altemifolia essential oil (OEMa) dispersed in an aqueous phase, stabilized with micro and nanoparticles of chitosan (Qs).

[0061] In a first embodiment, the present invention discloses a method for preparing an emulsion containing an essential oil as an active ingredient, characterized by the fact that it comprises the following steps: (i) prepare a chitosan dispersion in an acidic solution with acetic acid; (ii) mix the chitosan suspension and essential oil; (iii) homogenize the emulsion.

[0062] In a second embodiment, the present invention further discloses that the essential oil is an essential oil of Melaleuca altemifolia.

[0063] In a third embodiment, the present invention further discloses that the acetic acid used in step (i) is in a concentration of 0.1% to 10%, preferably 0.5% to 5%, even more preferably 1%.

[0064] In a fourth embodiment, the present invention further reveals that the chitosan dispersion used in step (i) has a degree of deacetylation (DDA%) of 70% to 85%, preferably 75% to 80%.

[0065] In a fifth embodiment, the present invention further reveals that the chitosan suspension used in step (ii) has from 0.1% to 1.0% in w / v, preferably from 0.2% to 0.7% in w / v, more preferably 0.5% in w / v.

[0066] In a sixth embodiment, the present invention discloses a Pickering emulsion prepared by a method as defined above.

[0067] In a seventh embodiment, the present invention discloses a Pickering emulsion comprising Melaleuca altemifolia essential oil as the active ingredient and stabilizers, wherein the stabilizer is chitosan microparticles.

[0068] In an eighth embodiment, the present invention further discloses that Pickering Emulsion can be used as an antibiotic against bacteria, fungi and viruses.

[0069] In an eighth embodiment, the present invention discloses the use of a Pickering emulsion prepared by the method as defined above as an antibiotic against bacteria, fungi and viruses. BRIEF DESCRIPTION OF THE FIGURES

[0070] Figure 1: Describes the emulsions analyzed by Optical Microscopy (OM). The emulsions of Melaleuca altemifolia essential oil in water stabilized with chitosan micro and nanoparticles do not undergo creaming or sedimentation during 30 days of storage. The average diameters were 16.66 ± 5.82 (emulsion 1) and 18.08 ± 6.56 (emulsion 2). 2). The diameters are statistically similar, so the amount of water did not affect droplet size, demonstrating that the emulsion preparation methods were suitable for the formation of stable microdroplets. Therefore, the droplets in the emulsions are attributed to the emulsification mechanism characterized by the adsorption of chitosan particles at the oil-water interface, leading to surface coverage of the droplets, preventing coalescence and thus contributing to the high stability of the emulsions.

[0071] Figure 2: Shows the result of the instability phenomenon. The emulsions showed transparency, indicating high stability and easy applicability.

[0072] Figure 3: Describes the FT-Raman analysis - MultiRaman (Bruker Optics, USA) with a wavelength of 1064 nm and a laser power of 150 W.

[0073] Figure 4: Depicts Fourier transform infrared (FTIR) spectroscopy with attenuated total reflectance (ATR) diamond attachment.

[0074] Figure 5: Describes the antimicrobial analysis of Gram-Positive and Gram-Negative Strains (E. coli). The inhibition zone against E. coli bacteria was 2.6 ± 0.2 cm and 2.5 ± 0.1 cm (Melaleuca altemifolia Emulsions 1 and 2) and Melaleuca altemifolia oil with 4.4 ± 0.2 cm, respectively. These values ​​are higher than the results observed for the antimicrobial agents sodium hypochlorite and Lysoform®, with zones of 2.3 ± 0.2 cm, respectively, currently marketed and widely used.

[0075] Figure 6: Describes the Antimicrobial Analysis of Gram-Positive and Gram-Negative Strains (S. Aureus). The inhibition zone against S. aureus bacteria was 3.0 ± 0.2 cm and 2.7 ± 0.1 cm (Melaleuca altemifolia Emulsions 1 and 2) and Melaleuca altemifolia oil was 4.5 ± 0.2 cm. cm. These values ​​are higher than the results observed for the antimicrobial agents sodium hypochlorite and Lysoform® with halos of 2.1 ± 0.2 cm and 2.7 ± 0.1 cm, respectively.

[0076] Figure 7: Describes the fungal test (Penicillium sp). Inhibition halo against fungi (Penicillium sp) was detected with diameters of 5.1 ± 0.2 cm and 4.5 ± 0.1 cm. Values ​​much higher than the results observed for other antimicrobial emulsions.

[0077] Figure 8: Describes the dispersivity analysis with Pickering emulsions with chitosan particles.

[0078] Figure 9: Optical fluorescence microscopy of Melaleuca altemifolia emulsion stabilized with chitosan microparticles. Scale bar = 100 pm.

[0079] Figure 10: Zeta potential of Pickering emulsions stabilized by chitosan particles.

[0080] Figure 11: Sars-Cov-2 test result

[0081] Figure 12: The hydrodynamic diameter of the microparticles and nanoparticles of the chitosan solution used in the manufacture of Pickering Emulsions. DETAILED DESCRIPTION OF THE INVENTION

[0082] As a preliminary point, it should be noted that the following description will be based on a preferred embodiment of the invention. As will be evident to anyone skilled in the art, however, the invention is not limited to that specific embodiment.

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one skilled in the art to which the invention belongs. The terminology used in the description of the invention is intended to describe particular embodiments only and is not intended to limit the scope of the teachings. Unless otherwise indicated, all numbers expressing quantities, percentages, and proportions, and other numerical values ​​used in the specification and claims, should be understood as being modified, in all cases, by the term "about." Therefore, unless otherwise indicated, the numerical parameters shown in the specification and claims are approximations that may vary depending on the properties to be obtained.

[0084] The present invention provides a method for preparing O / W Pickering emulsions containing essential oil droplets (MaEO) stabilized in aqueous media using chitosan particles without chemical modification and without the addition of reagents that are harmful to the environment.

[0085] The present invention relates to a new method for manufacturing oil-in-water Pickering emulsion with high stability and excellent biological activities against a series of pathogens.

[0086] The oil phase contains MaEO essential oil, composed primarily of terpenes and terpenoids. This Pickering emulsion can be used as a carrier for an active ingredient in cosmetics, pharmaceutical formulations, or cleaning agents, depending on the manufacturer's objectives.

[0087] The present invention provides a new method for preparing Pickering emulsions containing essential oil of Melaleuca altemifólia (Tea Tree).

[0088] Specifically, the present invention relates to a process for forming a Pickering emulsion composition with the oily phase containing essential oil using a suspension with chitosan (biopolymer) with a degree of deacetylation (DDA% 75-80%), with the use of a low toxicity acid in a small amount (1% Acetic Acid).

[0089] The new method revealed by the present invention comprises the following steps: (1) preparation of chitosan dispersion in acidic solution with acetic acid; (2) mixture of chitosan suspension (chitosan polymeric stabilizer (0.5% w / v)) and OEMa (30%); (3) homogenize the emulsion.

[0090] The acetic acid used in step (1) may be in a concentration of 0.1% to 10%, preferably 0.5% to 5%, even more preferably 1%.

[0091] Particles commonly used in Pickering emulsions include groups of polysaccharides, proteins, and other solid particles.

[0092] Particles used in polysaccharide-based Pickering emulsions include starch, starch nanocrystals, chitosan, cellulose nanocrystals, cellulose nanofibrils, and β-cyclodextrin, while those in protein-based Pickering emulsions include whey, zein, and others. Other particles commonly used as Pickering emulsion stabilizers include silica nanoparticles, synthetic hydroxyapatite (HAp) nanoparticles, and TiO2 nanoparticles.

[0093] The chitosan dispersion used in step (1) has a degree of deacetylation (DDA%) of 70% to 85%, preferably 75% to 80%.

[0094] In step (2), the chitosan suspension has 0.1% to 1.0% in w / v, preferably 0.2% to 0.7% in w / v, more preferably 0.5% in w / v.

[0095] Regarding the OEMa used in step (2), it is composed of terpinene-4-ol (41%), γ-terpinene (21%), α-terpinene (9%), α-terpineol (4%), terpinolene (3%), α-pinene (3%) and p-cymene (3%). It is presented in a concentration in the range of 10% to 50%, preferably 20% to 40%, more preferably 30%. Example 1 - Preparation of Pickering emulsions

[0096] 1 - Oil phase: OEMa (30%).

[0097] 2 - Aqueous Phase: Water (70%),

[0098] 3 - Preparation of the chitosan particle suspension: Chitosan (0.5% w / v) suspended in an aqueous solution of acetic acid (1% v / v) this suspension is stored for 24h and then used in the Pickering preparation process.

[0099] Process Parameters for the Preparation of Pickering Emulsions. Rotation: 12 RPM and Time: 5 Minutes

[0100] The aqueous chitosan suspension (0.5% w / v) is mixed with OEMa (30%) homogenized in an Ultra-Turrax Blender, model IKA T25 (IKA Werke, Staufen, Germany), rotation (12 rpm) was used for 5 minutes in order to cause a greater dispersion of the chitosan microparticles, leading to a homogeneous system. Example 2 - Optical Microscopy

[0101] Oil droplets in the emulsions (oil-in-water) were analyzed using a Leica DM KM optical microscope (Leica Microsystems GmbH, Wetzlar, Germany). A drop of emulsion was added to a glass slide, and representative images were obtained at days 0 and 30 (static storage). Droplet diameters were measured using ImageJ software as an average number of 120 droplets.

[0102] The results can be seen in figure 1. Example 3 - Emulsion Storage Stability

[0103] The samples were added to tubes and stored at room temperature. The stability of the emulsions was assessed through the instability phenomenon (creaming and sedimentation) evaluated in duplicates at 0–30 days using equations based on visual behavior.

[0104] The cremation index (CI), when present, is calculated using Equation (1), where H totai and H creme are the total heights of the emulsion and cream layer, respectively. The stability index is calculated according to equation 1: Cl = x 100. Total

[0105] The emulsions did not exhibit any instability, thus being 100% stable. The emulsions were transparent, indicating high stability and easy application (see Figure 2).

[0106] Due to the absence of instability phenomena, the stability value was 100%. Example 4 - Fourier Transform Raman Spectroscopy (FT-Raman)

[0107] The emulsions were analyzed with a FT-Raman - MultiRaman (Bruker Optics, USA) with a wavelength of 1064 nm and a laser power of 150 W. Data acquisition was performed at 600-4000 cm' 1 , with 64 scans and a spectral resolution of 2 cm 1 (see figure 3)- Example 5 - Infrared Spectroscopy with Transform Fourier

[0108] Two emulsions called 4 and 5 with different process parameters and (FTIR) were used. The tea tree emulsion with chitosan (4) showed better results.

[0109] Fourier transform infrared (FTIR) spectroscopy with attenuated total reflectance (ATR) diamond attachment was performed on a Frontier 94942 (PerkinElmer Inc., Massachusetts, USA). Spectra were collected with a spectral resolution of 4 cm' 1, 64 scans, from 4000 to 400 cm' 1 (see figure 4). Example 6 - Bacteria Inhibition Zone

[0110] Bacterial stock cultures were grown in Mueller-Hinton broth with 15% glycerol and stored at -70 °C. Stock cultures were grown on Mueller-Hinton agar (Oxoid) and inoculated at 37 °C for 24 h. [H l] The inoculation culture (for 24 h with turbidity equivalent to 0.5 Mc Farland, concentration equivalent to 1.5 x 108 CFU mL-1) prepared the suspensions of the isolated and standard bacterial strains. Then, 30 mL of agar were melted and cooled to 48 ± 1 °C. Subsequently, 500 pL of the bacterial inoculum at 1.5 x 108 CFU mL-1 were mixed with the agar.

[0112] This mixture was then transferred to a Petri dish. After solidification, a 0.9 cm diameter hole was made in the middle of the plates and the emulsion (0.2 - 0.5 mL) was placed in this hole.

[0113] Finally, the inoculated plates were incubated for 24 h at 36 ± 1°C. The appearance of inhibition zones around the well containing the product was evaluated. These inhibition zones correspond to the diameter of a merged inhibition zone around the antimicrobial samples. Commercial sanitizers were used as controls. widely used common (hypochlorite and Lysoform®) (Figures 5 and 6). Example 7 - Bacterial Colony Formation Counting Assays

[0114] For bacterial counting, 100 pL of a previously adjusted stock bacterial inoculum (105 CFU.mL' 1in Nutrient Broth, HIMEDIA, India) was mixed with 9.9 mL of nutrient broth (HIMEDIA, India) and 100 pL of the essential oil or emulsion. The samples were incubated at 36 ± 1 °C for 24 h with 85% humidity. Then, 100 pL of the incubated samples were mixed with 9.9 mL of nutrient agar medium (HIMEDIA, India), poured into Petri dishes (90 mm in diameter) and incubated at 36 ± 1 °C for 24 h with 85% humidity.

[0115] The viability of the inocula was confirmed using a pour-plate method on nutrient agar as a positive control. The number of count-forming units was measured using ImageJ software. The tests were performed in triplicate.

[0116] Total bacterial count (in CFU.mL' 1 ) was determined by multiplying the number of bacterial colonies counted on the plates by the dilution factor (10-4) of the plated bacterial inoculum. Example 8 - Fungicide Tests

[0117] Fungal strains of the genus Penicillium sp were isolated in the laboratory after exposure of a petri dish containing potato destrose agar, followed by growth in an oven at 25°C for 5 days according to a modified methodology from ANVISA's RE09 and 2003.

[0118] The cultivated fungus was suspended in solution sterile physiological solution, 10 μL of this solution was used in the petri dishes containing potato destrose agar per spread plate, 100 μL of the emulsions was used as an inhibitory factor, immediately after the petri dishes (potato destrose agar) containing the emulsions and the strains were incubated in an oven at 25 °C for 5 days.

[0119] At the end of the fifth day, fungicidal inhibition halos with a diameter of 5.1 ± 0.2 cm and 4.5 ± 0.1 cm with a standard deviation of 0.8 cm were observed against Penicillium sp (Figure 7) Example 9 - Zeta Potential

[0120] The zeta potential was estimated by the Smoluchowski model from measurements of the electrophoretic mobility of the nanoparticles using Zetasizer Nano-ZS (Malvez Instruments). The reading time to obtain the zeta potential data was 10 s, and the measurements were performed in triplicate.

[0121] As previously stated, the present proposed invention presents fewer process steps, with the use of little material and with a higher Zeta potential (+71mV), that is, high stability over time (see figure 10). Example 10 - Dynamic Light Scattering (DLS)

[0122] The particle diameter distribution was recorded using the dynamic light scattering (DLS) technique with a scattering angle of 90°. The Zetasizer Nano-ZS (Malvern Panalytical Ltd., Malvern, UK) was the equipment used for DLS characterization. Aliquots of the suspensions (100 pL) were suspended in distilled water (2 mL), and each sample was analyzed in duplicate.

[0123] The Figure below obtained in DLS shows the result of the hydrodynamic diameter of the microparticles and nanoparticles of the chitosan solution used in the preparation of Pickering emulsions of tea tree stabilized with chitosan (Figure 12). Particle size measurements of Pickering emulsions

[0124] The particle diameter distribution was recorded using the Laser Particle Analysis technique. Using a laser diffraction particle analyzer (Bettersize ST, Instruments Ltd., China) with a range of 0.10–1000 pm, the emulsion samples were added to the measurement chamber (600 mL of water) until an obscuration level of 5–10% was reached. Each measurement was repeated in triplicate (see Figure 8). Example 11 - Fluorescence optical microscopy analysis

[0125] Two simple, low-cost procedures were performed in which the chitosan suspension, made with 0.5% acetic acid, is mixed with the essential oil. This results in electrostatic stabilization through physical and chemical effects due to chemical interactions between the components.

[0126] An optical fluorescence microscopy analysis was performed (Figure 9), in which we observed well dispersed and separated particles, where Congo red was adsorbed by chitosan, creating a difference in the red fluorescence signal detected by the microscope (MICHELS; BÜNTZOW, 2010).

[0127] The examples given below are merely illustrative and should be used only to better understand the developments contained in the present invention, not however, they should be used with the intention of limiting the objects described. Example 12 - SARS-CoV-2 Assay

[0128] Rapid antigen test kits were provided by PanbioTM COVID-19 Ag Rapid Test (Abbott, USA).

[0129] The Rapid Antigen Test methodology applied in this investigation detects protein denaturation or degradation through the absence of antigen-antibody interaction, an indicator of viral inviability. Tea tree essential oil and Pickering emulsions disrupt the viral capsid, rendering it inviable, producing viral fragments, leading to virus inactivation.

[0130] Initially, nasopharyngeal samples collected from patients were suspended in saline and tested for SARS-CoV-2 in the laboratory's routine diagnostics using the RT-qPCR (real-time reverse transcription-polymerase chain reaction) method. They were stored in the biorepository of the Adolfo Instituto Lutz - Santo André Regional Laboratory Center. To this end, 100 pL of a positive sample was diluted in 100 pL of saline and analyzed using the Rapid Antigen test. A swab was dipped into this dilution, transferred to the kit tube containing 300 pL of buffer, and five drops were added to the device, according to the manufacturer's instructions, to confirm the integrity of the viral protein in the sample.

[0131] After obtaining a positive result, a mixture of 100 pL of the same SARS-CoV-2-positive sample and 100 pL of tea tree essential oil or Pickering's emulsions was prepared, and the samples were tested at different contact times. The test times were (0, 5, 10, 15, 20). Tea tree essential oil and Pickering's emulsions presented satisfactory results for inactivation of the SARS-CoV-2 virus (Figure 11).

[0132] Ethics Statement: Antiviral tests against SARS-CoV-2 were performed following standard guidelines approved by the Research Ethics Committee of the Federal University of ABC and Adolfo Lutz Institute CAAE: 49573421.2.3001.0059, and by the Technical-Scientific Council of the Adolfo Lutz Institute, CTC 18-N / 2021. All procedures followed the biosafety standards of the Adolfo Lutz Institute of the Santo André Regional Laboratory Center. References ASMA Sharkawy, Filomena Barreiro, Alírio Rodrigues, Pickering emulsions stabilized with chitosan / gum Arabic particles: Effect of chitosan degree of deacetylation on the physicochemical properties and cannabidiol (CBD) topical delivery, Journal of Molecular Liquids, Volume 355, 2022 AZIZ, Z. A. A. et al. Essential Oils: Extraction Techniques, Pharmaceutical And Therapeutic Potential - A Review. Current Drug Metabolism, v. 19, n. 13, p. 1100-1110, 2018. Xieru Bao, Jie Wu, Guanghui Ma. 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Claims

CLAIMS 1. Method for preparing an emulsion containing an essential oil as an active ingredient, characterized by the fact that it comprises the following steps: (i) prepare a chitosan dispersion in an acidic solution with acetic acid; (ii) mix the chitosan suspension and essential oil; (iii) homogenize the emulsion.

2. Method according to claim 1, characterized in that the essential oil is an essential oil of Melaleuca altemifólia.

3. Method according to claim 1 or 2, characterized in that the acetic acid used in step (1) is in a concentration of 0.1% to 10%, preferably 0.5% to 5%, even more preferably 1%.

4. Method according to claim 1 or 2, characterized in that the chitosan dispersion used in step (1) has a degree of deacetylation (DDA%) of 70% to 85%, preferably 75% to 80%.

5. Method according to claim 1 or 2, characterized in that the chitosan suspension used in step (2) has from 0.1% to 1.0% in w / v, preferably from 0.2% to 0.7% in w / v, more preferably 0.5% in w / v.

6. Pickering emulsion characterized by the fact that it is prepared by a method as defined in any one of claims 1 to 5.

7. Pickering emulsion characterized by the fact that it comprises Melaleuca altemifólia essential oil as an active ingredient and stabilizers, in which the stabilizer is chitosan microparticles.

8. Pickering emulsion according to claim 6 or 7, characterized by the fact that it is for use as an antibiotic against bacteria, fungi and viruses.

9. Pickling emulsion according to claim 8, characterized in that it inactivates the SARS-CoV-2 virus.

10. Use of a picketing emulsion prepared by a method as defined in any one of claims 1 to 5, characterized in that it is an antibiotic against bacteria, fungi and viruses.

11. Use according to claim 10, characterized in that it inactivates the SARS-CoV-2 virus.

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