An overview of essential oil-based nanoemulsion and their biological activities against some microbial pathogenic

The essential oils are constantly increasing due to their biological activities utilization in several fields, from agricultures to pharmaceuticals. However, their extensively use are still limited due to their disreputable physico-chemical properties. Nowadays, the most appropriate to overcome such restrictions is to develop of the proper formulation strategies. One of the oncoming prompted to achieve this goal is through nanoemulsions. Nanoemulsion are heterogeneous dispersions or a colloidal particulate system in the submicron size range acting as carriers of drug molecules. Nanoemulsion is most studied in encapsulation process due to inexpensive in their manufacturing, ease of formulation and easy waste handling. In this review, intends to offer an overview of the formulation, preparation of essential oil-based nanoemulsion and their biological activities against some microbial pathogenic.


Introduction
Essential oils (EOs) are hydrophobic fluids containing volatile compounds, which are produced by plant metabolism or as byproducts.EOs are generally derived from aromatic plants, and commonly from family Myrtaceae, Rutaceae, Lamiaceae, Lauraceae, Verbenaceae, and Asteraceae [1].EOs generally stored in the secretory glands in the form of liquid droplets.EOs are also abundant in some parts of plants such as roots, stems, leaves, and flowers.EOs are generally produced by means of hydrodistillation, steam distilling, dry distillation or mechanical pressing procedures [2; 3].
EOs has long been used as an aromatherapy and in medical applications [4].EOs are easily soluble in various organic solvents such as acetone, hexane, benzene, methanol, toluene and others.Some literature reports that EOs have antibacterial, antidiabetic, anti-carcinogenic, and anti-inflammatory activity.Due to its extensive activity, the EOs is also widely used in various industries, such as food, beverage, and pharmaceutical industries [8].Thyme essential oil produced by Thymus vulgaris contains a thymol compound known to have an inhibitory activity against some bacteria and fungi [9].Thyme essential oil is traditionally used as an antiseptic, antispasmodic, carminative, anesthetic, stimulant, and 1297 (2024) 012083 IOP Publishing doi:10.1088/1755-1315/1297/1/012083 2 analgesic [10; 11].Rosemary essential oil derived from Rosmarinus officinalis is known to have antioxidant, antibacterial, anti-cancer [12; 13], and anti-inflammatory activities [7; 14].Clove essential oil, containing eugenol, a phenolic compound, is known to have high antioxidant activity [15].Thymoquinone is a bioactive compound produced from black ginger oil known to have excellent antifungal activity [16; 17].
EOs have very low physico-chemical properties, solubility, and bioavilability.EOs are lipophilic and have high volatility properties.Therefore, it is necessary to take an approach to enhance their properties.One of the best approach to increase their properties is through nanoemulsion technology (essential oil-nanoemulsion).In addition, essential oil-nanoemulsion were known to increase the biological activity, due to their ability to penetrate into tissue cells, and easier cellular uptake.Essential oil-nanoemulsion is also capable of controlling and modulating the release of active substances to target cells [18; 19].Essential oil-nanoemulsion is also able to enhance the physico-chemical properties and stability of the oils by increasing their solubility in water, reducing their volatility properties, and protecting them from their interaction with the environment.Another advantage of this technology is that it is easy to formulate, it's easy to handle, and it doesn't cost much.Nanoemulsion technology is perfectly suitable for compounds that are lipophilic or soluble in low water.Another advantages are high stability properties, so it is very well used in the food industry, as a delivery agent in the pharmaceutical industry and excellent as a drug delivery system [20; 21].This article aims to review the manufacture, composition, antimicrobial activity and their mechanism of action of essential oil-nanoemulsion against some microbial pathogenic.

Fabrication of essential oil nanoemulsion
There are two approaches to fabrication of essential oil-nanoemulsion, "top-down" and the "bottom up" approaches.In the "top-down", the mechanical style is used to break oil particles into nanometer shapes, while the "bottom-up" method is used based on the thermodynamic properties of a molecule to produce an emulsion with a very fine particle size.In the food industry, the "top-down" method is generally done using high energy such as high-pressure homogenization, micro fluidization, sonication, and microporous membranes.While the "top-down" using low energy such as phase inversion temperature (PIT), phase inverse composition (PIC), and anti-solvent precipitation method (ASP).In fabrication of the essential oil-nanoemulsion, "bottom-up" with lower energy is preferred because it produces ultra-fine particle droplet [22; 23].
High energy methods "top-down" require mechanical energy to change or reduce the shape of a droplet to a ball shape (spherical shapes).This reduction process depends heavily on the type of homogenizer, temperature conditions, duration of the homogenization process, energy intensity, and composition as well as its physicochemical properties.Unlike the high energy nanoemulsion method, the low energy method does not require mechanical energy for the nanoemulsification process.This method also requires simple equipment with low energy consumption.However, in this method relative use of surfactants for the process of forming nanoemulsions [24].
However, essential oil-nanoemulsion can be made using several methods, namely nanoprecipitation, inclusion complexation, solvent evaporation-emulsification, coacervation, and supercritical fluid [25]; a. Nanoprecipitation Nanoprecipitation method is the most commonly used nanoemulsion method for nanoemulsification of lipophilic components such as essential oils into semi-polar solvents.This method is very easy to do, simple, reproducible, and the energy used is minimal.This method generally uses polymers as matrices (such as lactic-co-glycolic acid, alkylcyanoacrylate, polylactic acid, and ε-caprolactone to encapsulate lipophilic components).

b. Inclusion complexation
This method involves molecular linkage between the core material and the matrix polymer.Molecular linkage during the encapsulation process includes Van der Waal bonds, hydrogen bonds and hydrophobic interactions.In this method, it generally uses β-cyclodextrin and β-lactoglobin as nanocarriers for the encapsulation of lipophilic components.c.Solvent evaporation-emulsification This method involves polymers for the process of nanoparticles emulsification.Ethyl cellulose, polycaprolactone, polylactic-co-glycolic acid (PLGA), and polylactic acid (PLA) are the most commonly used polymers for the process of forming nanoparticles.In this method, usually highspeed homogenization and ultrasonication were used.

d. Coacervation
In this method, the process of encircling depends heavily on the enzymatic reaction, ionic density, pH, polymer type, concentration and complex properties of the matrix formed.e. Supercritical fluid This method is used to encapsulate a thermal-sensitive bioactive compound, which is subsequently solvented by compressing, then proceeds with a process of precipitation against the dissolved particles.

The composition of nanoemulsion
In the fabrication of a nanomeulsion, it usually consists of two phases, namely the water phase and the oil phase, but in the preparation of nanoemulsion it always requires a surfactant or co-surfactant.
Surfactant is a molecule capable of lowering surface pressure.The molecular structure of the surfactant is hydrophilic and lipophilic.During the emulsification process, the surfactant easily absorbs the wateroil interface so it can prevent aggregation.Based on the electrical charge, surfactants can be classified into four classes, namely (1) non-ionic, (2) zwitteronic, (3) cationic or (4) anionic.Based on its electrical charge properties, the surfactant has an enormous effect on the formation and stability of a preparation.This aspect affects, influences the mechanism of stabilization of the polar end of a surfactant in aqueous medium.Among all classes, non-ionic surfactants is most preferred due to safe, low toxicity effects, and safe to be taken orally (oral ingestion).Currently, the most widely used surfactants are sucrose esters, fatty acid esters sorbitants, glycerol fatty acids esters (polyglycerols), polyoxyethylene sorbitant fattyacid ester (polysorbates) and polyoxyetylene ether surfactant.Meanwhile, the most widely used surfactants for essential oil-based nanoemulsions are Tween 80 and Tween 20.Both of these surfactant have excellent effects on droplet size, size distribution and PDI (polydispersiveindex) value.
Co-surfactant is a supporting component of the surfactant.Co-surfactant are generally amphiphilic molecules with an active side on their surfaces i.e. have polar ends (usually hydroxyl groups, -OH) and non-polar ends (hydrocarbon).The co-surface has a smaller size at the polar end, so it is unable to stabilize itself, but it is significantly capable of reducing the interfacial tension so that the fluidity at the non-polar end will increase, resulting in an increase in the entropy system [3; 26].There are several types of co-surfactant, but the most commonly used are short-chain alcohols, there are medium-chain long alcohols.Co-surfactant are usually used in the oil phase and the water phase.In its mechanism, the co-surfactant molecule is able to intercalate with the surfactant molecule so that it weakens each other at the polar and non-polar ends.This causes the interfacial film to be more flexible, thus affecting the droplet.Because of the partition between the two phases, alcohol also affects the solubility properties of the oil and water phases [27].Geraniol, terpinen-4-ol and α-terpineol are alcoholic compounds contained in nutmeg oil and tree tea oil that can enhance nanoemulsion formation and can act as cosurfacts.

The composition of essential oil-nanoemulsion
Ethnomedicinally, EOs are widely used health applications, especially as aromatherapy.Olive oil is highly volatile, transparent, and fat-soluble, has been proven used in health medical applications as antibacterial, antidiabetic, anti-inflammatory, and as anti-carcinogenic [8; 32].For these reasons, EOs are widely used in various applications both in the food industry and in the pharmaceutical industry [32].Due to their physicochemical properties and bioavailability are also very low, the application of EOs in foods, cosmetics, and pharmaceutical industries is very limited.Therefore, manufacturing of essential oil-nanoemulsion is very important to increase their physicochemical and bioavailability properties through dropping their needed levels of dosage [33].The essential oil-nanoemulsion can be prepared using lipid-based formulation.Several compositions of essential oil-nanoemulsion fabrication are presented in Table 2.

Microbial activity of essential oil-nanoemulsion
The use of essential oils in the manufacture of essential oil-nanoemulsions in the pharmaceutical industry aims to improve oral bioavailability and the solubility of water-soluble drugs.In addition, the production of essential oil-nanoemulsions also serves to enhance antimicrobial activity such as antibacterial and antifungal.Some antimicrobial activity of the formula essential oil-nanoemulsion can be seen in Table 3.

Tyme essential oilnanoemulsion
Culex tritaeniorhynchus LC50 22.58 ppm [45] Note: a : percentage reduction of mycelial growth; b: methicillin resistance Staphylococcus aureus (MRSA) The results showed that garlic oil-nanoemulsion produces good antibacterial activity against Staphylococcus aureus and Escherichia coli.Oksh et al also showed that garlic oil-nanoemulsion can act as an alternative antibiotic, as it significantly reduced the gene expression levels of MDR Pseudomonas aeruginosa in broiler farms [34].Pon et al mentioned that patchouli oil-nanoemulsion produced stable emulsions on the first day and on the 45th day.Pon also mentioned that patchouli oil-nanoemulsion produced the best antibacterial activity against the bacteria Shigella flexneri, Staphylococcus aureus and Streptococcus mutans with the MIC 50 of 12.5 mg/ml, 12.5 ng/ml and 25 mg/mL respectively [35].
IOP Publishing doi:10.1088/1755-1315/1297/1/0120839 Meanwhile, thyme oil-nanoemulsion preparation was effective activity against Escherichia coli bacteria than rosemary oil-nanomulsion with the MIC 50 value of 0.45 μl/ml [7].Sayed et al showed that a combination of clove seed oil with blake seed oil-nanoemulsion in a ratio of 2:1 was able to reduce the formation of sclerotial Botrytis cinerea.In addition, the combination of essential oilnanoemulsion in can also suppress seed rot and seedling mortality of cucumber [10].Ullah et al also showed that essential oil-nanoemulsions (coriander, clove, cinnamon, and cardamom essential oils) are capable of inhibiting the growth of bacteria Staphylococcus aureus and Staphylococcus epidermidis with a barrier zone diameter of about 9 mm [37].Mina et al also mentioned that cuminum oil-nanoemulsion has excellent antioxidant activity.Mina also showed that cuminum oilnanoemulsion has an effect on bacteria contained in fish Acipenser stellatus [38].
Abayomi was reported that sesame oil-nanoemulsion was able to increase zone inhibition on Klebsiella pneumoniae and Bacillus subtilis [39].Meanwhile, Aji et al was stated that cinnamon bark essential oil-nanomeulsion could be applied as poultry feed additive [40].Liu was mentioned that garlic oil-nanoemulsion significantly suppressed the cell proliferation of MRSA, which was mainly achieved by damaging the cell membrane as evidenced by membrane depolarization and considerable leakage of intracellular nucleic acids and protein [41].Meanwhile, Yuan reported that garlic oil-nanoemulsion was able to change the structure and the morphology of P. italicum.Garlic oil-nanoemulsion is also able to disrupt the structure of proteins, amino acids and lipids of P. italicum.Garlic oilnanoemulsion is also able to increase the bioavailability of garlic oil so that it can be recommended as a promising alternative to inhibit P. italicum in vegetables and fruits [42].Joyce et al mentioned that the formula celery seed oil-nanoemulsion can be used for cancer therapeutics and can also be used as an alternative to antibiotic therapy especially against S. aureus [43].Doan et al noted that orange peel essential oil-nanoemulsions supported by nanosilver have the superior antibacterial ability against E.coli [44].Flavia was reported that andiroba seed oil-nanoemulsion allowed achievement of a potential bioactive oil in water nanoemulsion that may be a promising controlled release system [57].Parisa et al also mentioned that Thymus vulgaris (L.) essential oil nanoemulsion had larvasida activity against C. tritaeniorhynchus with its LC 50 value of 22.58 ppm.Parisa also show that Thymus vulgars (L) essential oil nanomulsion encapsulated with chitosan was capable of killing A. stephensi larvae with a LC 50 of 18.88 ppm [45].

Antimicrobial mechanism action of essential oil-nanoemulsion
EOs also known as aetheric oil, volatile oil, and aromatic oil, EOs is the basic ingredient of fragrances in cosmetic industry.The biologists consider that EOs to be a secondary metabolite that usually serves as self-defence to avoid being eaten by animals or as an agent to compete with other plants.Naturally, essential oils in nature are lipophilic and hydrophilic because they are composed of a mixture of volatile and non-volatile compounds, either stable or unstable.In general, essential oil in nature generally consists of terpenes, terpenoids, and phenylpropanoids [58; 59], some of them contain phenolic compounds.These secondary metabolites are known to be responsible for the biological properties of essential oils, especially their antimicrobial properties [60].On the other hand, the EOs also contains some minor compounds, which is also known to play an active role in the anti-microbial activity of essential oil.However, the mechanism of the action of essential oils as antimicrobials is still a matter of debate, due to their very low solubility and bioavailability properties [61].The mechanism of action of antimicrobial essential oil-nanoemulsion is still well understood, however several researchers stated that the secondary metabolites contains EOs play an important role in their mechanisms of action.Some studies mentioned that essential oil-nanoemulsion is more effective than essential oil itself as an antimicrobial agent.
The schematic of mechanism of action of essential oil-nanoemulsion as an antimicrobial agent can be seen in Figure 1 [62].In general, the mechanisms of the action of an essential oil-nanoemulsium (EOs-NE) as an antibacterial can be divided into several stages;   Essential oil-nanoemulsions (EOs-NE) also showed activity against pathogenic fungi.However, the mechanisms action of EOs-NE against the fungus were still unknown.Some studies have been reported that the mechanisms action of EOs-NE is shown in Figure 2. The Figure 2 shows that the action of EOS-NE against fungi is by interfering the cell wall, structure and permeability of the cell membrane.EOs-NE also reported damages cell integrity and membrane permeability by removing vital components inside cells such as Ca 2+ , K + , and Mg 2+ ions by inhibiting the proton pump with subsequent loss in the ATP pool, and eventually apoptosis [62].Other studies also reported that EOS-NE affects ergosterol by destabilization of membrane integrity and stability [63].

Conclusions
The essential oils (EOs) is constantly increasing due to their biological activities utilization in several fields, from agricultures to pharmaceuticals.However, their extensively use are still limited due to EOs have very low physico-chemical properties, solubility, and bioavailability.Essential oil-nanoemulsion (EOs-NE) able to enhance the physico-chemical properties and stability of the oils by increasing their solubility in water, reducing their volatility properties, and protecting them from their interaction with the environment.EOs-NE were known to increase the biological activity, due to their ability to penetrate into tissue cells, and easier cellular uptake.EOs-NE are also capable of controlling and modulating the release of active substances to target cells.EOs-NE is suitable for compounds that are lipophilic or soluble in low water.Another advantages are high stability properties, so it is very well used in the food industry, as a delivery agent in the pharmaceutical industry and excellent as a drug delivery system.For example, the Eos from Ocimum sanctum and Illicium verum can induce considerable impairment in ergosterol biosynthesis in Aspergillus flavus.Thus, Essential oil-nanoemulsions (EOs-NE) EOs can be utilized as possible natural antimicrobial agents against some microbial pathogenic.

10 a.
Essential oil-nanoemulsions (EOs-NE) provoke and disorganize the bilayer and mitochondrial phospholipids of bacterial membrane cells, resulting in damaged protein membrane structures.b.Protein membrane damage resulted in increased cell permeability, which then led to the cellular structure being unstable.c.The instability of the cellular structure leads to a decrease in the proton motive force, electron flow, and active cell transport.The presence of EOs-NE increases the leakage of vital cellular ions like Na + , Mg 2+ , and K + thus disrupting DNA, RNA, and protein synthesis.d.This leak causes the cell to change significantly, and eventually the cell dies (significant changes in the bacterial cell responsible for cell death).

Figure 1 .
Figure 1.Schematic representation of the possible antibacterial mechanisms of action of essential oilnanoemulsion.Adopted from [62].

Figure 2 .
Figure 2. Schematic representation of the possible antifungal mechanisms of action of essential oilnanoemulsion.Adopted from [62].

Table 1 .
Several surfactan and co-sufactan were commonly used in fabrication of essential oil-nanoemulsion.

Table 2 .
Several compositions of EOs-NE fabrication

Table 3 .
The antimicrobial activity of EOs-NE on some microbial pathogenic and other organisms.