Author’s Accepted Manuscript

Essential oils in nanostructured systems: challenges

in preparation and analytical methods

Sheila Porto de Matos, Leticia G. Lucca, Letícia S.

Koester

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PII: S0039-9140(18)31180-9
DOI: https://doi.org/10.1016/j.talanta.2018.11.029
Reference: TAL19262
To appear in: Talanta
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Revised date: 8 November 2018
Accepted date: 9 November 2018
Cite this article as: Sheila Porto de Matos, Leticia G. Lucca and Letícia S.
Koester, Essential oils in nanostructured systems: challenges in preparation and
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 Essential oils in nanostructured systems: challenges in preparation and analytical

methods

Sheila Porto de Matos, Leticia G. Lucca, Letícia S. Koester*.

Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia,

Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752, 90610-000, Porto

Alegre, RS, Brazil.

*
Corresponding author: Letícia Scherer Koester, Postal address: Avenida Ipiranga, 2752

– Santa Cecília, Porto Alegre/RS – Brazil 90610-000. Tel.: + 55 51 3308 5278. E-mail

address: leticia.koester@ufrgs.br

ABSTRACT

Essential oils are natural products extracted from plants that present volatile and

thermolabile characteristics. Essential oils have become products of interest in many

fields, including the pharmaceutical, due to their medicinal properties. In recent years,

the interest in the encapsulation of essential oils in nanometric systems for therapeutic

approaches has risen and a number of studies have been published. This review

intended to set a panorama on the research within this field through a data survey and

identify the organic nanostructured systems, the preparation techniques and analytical

quantification methods employed. Many techniques used to prepare nanosystems loaded

with essential oils involve heating or solvent evaporation steps that may damage their

composition. In this context, the quantification of essential oil on the final nanosystems

is impaired. However, in more than half of the research papers, the quantification is

1
ignored or an indirect quantification is performed, assuming no volatilisation upon

formulation processes. Analytical methods used to assess essential oil encapsulation

efficiency were discussed regarding their suitability.

Graphical abstract

Abbreviations

EO, Essential Oil; NE, Nanoemulsion; HSS, High Shear Stirring; HPH, High Pressure
Homogenization; US, Ultrasound; SNE, Spontaneous Nanoemulsification; HLB, Hydrophilic-
lipophilic balance; PI, Phase inversion; PIT, Phase inversion Temperature; PIC, Phase inversion
Composition; SLN, Solid Lipid Nanoparticles; LS, Liposomes; MLV, Multilamellar Vesicles; LUV,
Large Unilamellar Vesicles; SUV, Small Unilamellar Vesicles; TFH, Thin Film Hydration; EIT,
Ethanol Injection Technique; SNEDDS, Self-nanoemulsifiyng Drug Delivery Systems; SEDDS,
Self-emulsifiyng Drug Delivery Systems; NC, Nanocapsules; PLA - Poly(Lactic Acid); PLG -
Poly(Glycolic Acid); PLGA - Poly(Lactide-co-Glycolide); NPP, Nanoprecipitation; EDM, Emulsion-
diffusion Method; ES, Electrospray; NG, Nanogel; SD, Spray Dryer; IG, Ionic Gelation; TPP,
Tripolyphosphate; GC, Gas Chromatography; FID, Flame Ionization Detector; MS, Mass
Spectrometer; HPLC, High Performance Liquid Chromatography; UV, Ultraviolet; PDA,
Photodiode Array;

Keywords: Essential oils; encapsulation; nanotechnology; association efficiency;

quantification; Gas Chromatography.

1. Introduction

2
Essential oils (EO) are hydrophobic and aromatic fluids extracted from plants, which

generally, as for the majority of products obtained from biological matrices, present a

complex chemical composition [1-3]. These oils are composed of volatile molecules,

such as monoterpenes, sesquiterpenes and phenylpropenes [4]. EO components are

biosynthesised by plants as secondary metabolites and play an important role in

protection against pathogens and predators [5]. Such a complex composition of EOs

carries a wide spectrum of possible applications in many fields, for instance, food,

agricultural and pharmaceutical [6].

Recently, EO gained significant interest in the pharmaceutical field. Edris [1] reviewed

the literature concerning EO use in therapeutics and listed studies concerning several

potential pharmacological applications of EO, such as in chemotherapy, the treatment of

cardiovascular diseases, as an anti-diabetic and many others. However, the chemical

composition of EO can be influenced by environmental, harvest, processing and storage

conditions of the plant material, with terpenoids and other EO constituents in general

being volatile, thermolabile and easily oxidised and hydrolysed [6]. Recent advances in

the extraction of EO using innovative and green technologies include solvent-free

microwave extraction, supercritical fluid extraction, controlled pressure drop process

and ultrasound extraction [7, 8]. Périno-Issartier et al. [9] compared the essential oil of

lavandin extracted from different techniques, either classical or green/sustainable

alternatives and identified that among the eight techniques studied, the optimal

extraction was achieved with microwave hydrodiffusion and gravity. Likewise, Asfaw

et al. [10] compared the extraction of Artemisia afra by low energy techniques

(ultrasound, supercritical fluid and microwave assisted), and suggested that microwave-

assisted EO extraction was the “greener” alternative, due to the lower time consumed

and the absence of the need for solvents to be used in the process.

3
After proper plant extraction, as discussed above, the application of EO in the

pharmaceutical field requires its protection from environmental factors that may lead to

degradation and a loss of bioactive compounds by volatilisation [11]. Although the

encapsulation in micrometric systems is already well described as a way to overcome

degradation and volatilisation, the nanometric systems also present the advantage of

increasing the bioefficacy of the EO due to its capacity to be absorbed by cells and

permeate through membranes and biological barriers [12].

On the other hand, encapsulation in nanometric systems may represent a challenging

approach to develop drug delivery systems containing EO as active compounds. In

many cases, the preparation techniques of organic nanostructured systems contain one

or more steps, such as heating, solvent evaporation and high pressure homogenisation,

in which EO components are subjected to conditions that can cause degradation and loss

of active content by volatilisation [13].

In face of the growing interest in the encapsulation of EO in nanometric systems for

pharmaceutical and medical purposes, there is a need to discuss the stability of EO

during the encapsulation processes and the assurance of the final product. This review

aims to identify studies concerning EO encapsulated in organic nanometric systems for

therapeutic purposes, the different nanosystems and preparation techniques used to

achieve them, as well as to address the quantification of the EO in the nanostructures,

with focus on the analytical methods employed and their suitability.

2. Literature survey concerning nanoencapsulation of EO

A literature survey was carried out in two databases, Scopus and Web of Science

(considering the results found until December 31st 2017, but not limiting the period

before that), due to the recent increase in the use of nanotechnology to develop new

4
feasible and efficient delivery systems for EO [11]. The expressions “nano*” and

“essential oil” were used as keywords in the search and all results containing the

expressions “nanotube*”, “nanocomposite*”, “nanofilm*” and “nanofiber*” were

dismissed. Among the obtained search results for the aforementioned entries, a

screening was performed looking for the studies concerning the nanotechnological

encapsulation of EO focused exclusively on pharmaceutical and medical purposes.

Initially, papers were sorted by publication year (Figure 1) in order to set a time profile

for the research into this field of application. It was found that the topic of EO

encapsulation in organic nanometric systems for pharmaceutical uses has been studied

in the last decade, with the oldest report dating back to 2007. In addition, besides the

relatively low and constant number of publications in the period between 2007 and

2014, an important increase in 2015 was noticed, which may suggest that

nanotechnology represents a promising strategy to improve the use of EO as a

therapeutic agent.

Additionally, a data survey was undertaken on the selected studies gathering

information such as the EO used, the nanometric system developed and the preparation

technique, as well as whether the EO is quantified on the final formulation and the

analytical methods employed for the dosing of EO components. Data were arranged in

Table 1 in order to easily assess the obtained information and set a panorama of the

research within the subject of this review.

3. Nanostructured systems and preparation techniques

During the last decade, the encapsulation of EO in nanometric organic systems has

come to light in drug delivery systems design. Nanotechnology has been shown to be an

5
important tool to improve the stability of EO in front of possible degradation by light,

heat and other environmental factors [5, 11]. In addition, this approach has been used to

achieve better bioavailability, improve permeation through the skin and other biological

barriers and reach the controlled delivery of active compounds [12, 14].

Data survey in the literature found a variety of different nanostructured systems

containing EO, which were arranged, in this review, into two categories: lipid-based

nanostructured systems and polymer-based nanostructured systems (Figure 2). Lipid-

based nanostructured systems, such as nanoemulsions, solid lipid nanoparticles,

liposomes and self-nanoemulsifying drug delivery systems, are formed by lipidic

components, which are usually biodegradable and considered safe for pharmaceutical

uses [15]. Polymer-based nanostructured systems, such as nanocapsules and nanogels,

are composed of polymers that can be either natural, semisynthetic or synthetic [14].

Special attention has been paid to the development of drug delivery systems containing

biodegradable polymers.

The choice of a nanostructured system and the preparation technique encompass

physical-chemical variables of the compound intended to be encapsulated as well as the

characteristics of the nanostructured system. When it comes to EO, their volatile and

thermolabile features require a preparation technique that avoids heating and solvent

evaporation as a means to achieve a more efficient process. Additionally, the loading

capacity of each nanocarrier has to be considered when formulating a drug delivery

system. In theory, nanoemulsions are capable of encapsulating an amount of EO up to

30–40% of its total composition [16]. Probably due to this capacity to incorporate high

doses of EO, nanoemulsions are the more deeply studied nanostructure systems with

which to encapsulate EO, representing nearly half of the studies selected for this review

6
(Figure 3), followed by nanocapsules, solid lipid nanoparticles, nanogels and other

structures.

3.1 Lipid-based nanostructured systems

3.1.1 Nanoemulsions

Nanoemulsions (NE) are colloidal systems, generally consisting of two distinct

immiscible phases, one hydrophilic and one hydrophobic, and also called aqueous and

oily (or organic) phases, where one of the phases is dispersed into the other as

nanometric-sized droplets. However, these systems are thermodynamically unstable due

to the high surface tension formed at the interface between the two phases. In order to

overcome the instability, surfactants are added to the NE formulation because of their

ability to lower the surface tension and stabilise the system [17, 18].

The NE preparation techniques employed in EO encapsulation for therapeutic uses can

be divided into two groups: low energy and high energy techniques. High energy

techniques (high-shear stirring, high pressure homogenisation and ultrasonication)

employ a mechanical device that provides the system with energy to form nanometric

droplets. In contrast, the low energy techniques (phase inversion temperature, emulsion

phase inversion and spontaneous nanoemulsification) are based on a change in the

process parameters such as temperature and composition of the phases to achieve a

nanoemulsion [17-19].

The high-shear stirring (HSS) technique uses a rotor-stator mixer system to subject the

formulation to high shear forces, leading to disruption of the two immiscible phases in

order to obtain dispersed droplets. A multi-pass regimen can be applied to yield smaller

particles and more dispersed systems [19]. Even though HSS has been used since the

beginning of nanoemulsion research, it does not present very efficient results in terms of

7
polydispersity and particle size as most of the energy applied to the system is spent as

heat generation and viscous friction phenomena [20]. Adhavan et al. [21] and Rossi et

al. [22] describe the use of HSS to obtain nanoemulsions containing Pogostemon cablin

and Cymbopogon flexuous EO, respectively.

High Pressure Homogenisation (HPH) applies high pressure to a coarse emulsion,

forcing it throughout tight spaces at high speed, causing the breakout of the emulsion’s

dispersed phase into finer droplets [20]. This process subjects the nanoemulsion

simultaneously to shear force, turbulence and cavitation. However, the efficiency of

system disruption can be affected by the viscosity and content of the oily phase [19]. In

Table 1, it is possible to observe that, of the techniques used in the preparation of NE,

the HPH technique is the most commonly selected.

NE formation using Ultrasonication (US) is attributed to two phenomena. First, the

acoustic field generated destabilises the interface between the phases of the system,

leading to emulsion formation, which is subsequently submitted to low frequency

ultrasound, leading to acoustic cavitation. Acoustic cavitation consists of microbubble

formation, followed by its collapse, which causes localised turbulence, breaking up the

system into a nanoemulsion [23, 24]. The US technique has shown to be an economical

method with the efficient use of energy to provide fairly stable nanoemulsions [25],

thereby justifying why this technique is one of the most frequently employed in NE

preparation (Table 1), being described in the nanoemulsification of Cinnamomum

zeylanicum [26], Cuminum cyminum [27], Eucalyptus globulus [28], Foeniculum

vulgare [29], Nigella sativa [30], Syzygium aromaticum [31] Rosmarinus officinalis and

Lavandula augustifolia [32] and Thymus daenensis [33] EOs.

Spontaneous Nanoemulsification (SNE) is the most widely used low energy technique

in the preparation of NE (Table 1). It consists of the preparation of two homogeneous

8
phases: one aqueous phase containing water, hydrophilic surfactants and other

hydrophilic components and an organic phase containing the lipids, hydrophobic

surfactants, other hydrophobic components and a water-soluble solvent. Then, the

organic phase is added to the aqueous phase by continuous flow under agitation. This

will make it possible for water-miscible components on the organic phase to diffuse

towards the aqueous phase and form nanometric droplets. After nanoemulsion

formation, the organic solvent needs to be evaporated. This may lead to an evaporation

of the volatile fraction of the oil during the evaporation step, as observed by Flores et al.

[34] during the preparation of NE containing the essential oils obtained from leaves of

Melaleuca alternifolia. Dias et al. [35] compared the preparation of copaiba oil NE

prepared by HAP and SNE and found that HAP led to more stable and more

monodisperse nanodroplets as well as a lower loss of the volatile component β-

caryophyllene, the main component of the oil.

The quality of the final nanoemulsion can be influenced by parameters such as

surfactant hydrophilic-lipophilic balance (HLB), lipid viscosity and solvent solubility

[36, 37].

Phase Inversion (PI) techniques are based on release of energy stored in the system

during phase transitions. The process of nanoemulsification can be performed by

disturbing the system temperature (Phase Inversion Temperature, PIT) or the

composition by adding oil or water (Phase Inversion Composition, PIC). In this context,

understanding phase behaviour of the surfactant used is fundamental to obtaining the

nanoemulsion. The PIT technique can only be applied in cases where the surfactant used

is sensitive to temperature variation; on the other hand, the PIC technique has a wider

feasibility [37].

9
 3.1.2 Solid Lipid Nanoparticles

Solid lipid nanoparticles (SLN), also named nanostructured lipid carriers, are

suspensions of solid lipid particles in the nanometric size range dispersed in an aqueous

media. Aside from being quite similar to NE in a formulation point of view, the lipid

phase is formed using lipids that are solid at room temperature, which results in solid-

dispersed particles instead of oil droplets. As a result of the solid nature of SLN, they

have an enhanced physical stability over nanoemulsions [38].

As can be observed in Table 1, the preparation techniques of SLN are the same as those

described for NE preparations; in particular, HSH is used in most cases. During the

preparation, the solid lipid content is melted and kept at around 5-10ºC above melting

temperature and the pre-emulsion is prepared under heat [24]. Following this, the

formulation is submitted to high or cold high pressure homogenisation. In the case of

hot homogenisation, the most popular approach is to use a temperature above the lipid

melting point during the process, which is not suitable for thermolabile drugs or active

ingredients [39, 40]. Solid lipids such as cetyl palmitate [41-43], glyceryl behenate [44-

46] and SOFTISAN® 154 [47] have been employed in the preparation of SLN

containing Artemisia arborescens [46], Frankincence and myrrh [45], Laurus nobilis

[48], Melaleuca alternifolia [41, 42, 49] , Nigella sativa [47], Rosmarinus officinalis

[43], Yuxingcao [44] and Zataria multiflora [50] oils.

3.1.3 Liposomes

Liposomes (LS) are vesicles built by amphiphilic lipids. The lipids organise themselves

in bilayers surrounding an aqueous core, where water and hydrophilic compounds can

be entrapped while hydrophobic compounds can be encapsulated on the bilayer. As a

10
result of this structure, liposomes are very versatile drug delivery systems since they can

incorporate either hydrophilic, hydrophobic or amphiphilic drugs [51, 52].

The US technique already described for nanoemulsion preparation is used for the

production of LS when multilamellar vesicles (MLV) or large unilamellar vesicles

(LUV) systems are converted to small unilamellar vesicles (SUV) [52, 53]. Aside from

being a fast technique to reduce size of LS, US can cause degradation by oxidation and

hydrolysis of the lipid content [53].

Thin film hydration (TFH) or thin membrane hydration, first described by Bangham

[54], is an easy technique for the preparation of LS. It consists of the hydration of a thin

lipid film using organic solvent, followed by solvent evaporation, resulting in a solid

lipid deposited on the surface. The lipid is then hydrated with an aqueous solution,

which leads to the spontaneous formation of liposomes. Such a simple technique has

some drawbacks though, as the technique often generates MLVs, with large and wide

size distribution [55]. TFH was described by Ge & Ge [56] in order to encapsulate

Melaleuca alternifolia EO; along with the TFH technique, the study employed US,

which was probably necessary to obtain nanometric-sized LS.

Ethanol injection technique (EIT) follows the principle of phase inversion. First, a

solution containing lipids and ethanol is prepared, which is injected rapidly, in

sequence, into the aqueous phase containing water and hydrophilic components. The

final step is the purification of the liposomal suspension by discarding ethanol, which

may be a difficult task as the ethanol forms an azeotropic mixture with water and

impairs their separation [55, 57, 58].

3.1.4 Self-Nanoemulsifying Drug Delivery Systems

11
Developing delivery systems for hydrophobic drugs has always been a challenge.

Among the many technological approaches to improve oral bioavailability, self-

emulsifying drug delivery system (SEDDS) have shown to be a promising strategy [59].

SEDDS is described in the literature as an isotropic mixture of lipids, surfactants, co-

solvents and active compounds that form emulsions spontaneously when agitated gently

in an aqueous media, such as the gastrointestinal tract. SEDDS that form emulsion

droplets under 100 nm are denominated SNEDDS [60, 61]. It is important to point out

that SNEDDS preparation does not involve any step of heating, solvent evaporation or

any procedure that may harm the molecular structure of EO components or which may

cause a loss of volatile content. Usually, the only purpose of this type of formulation is

to enhance oral bioavailability [62]. In other words, SNEDDS will not provide any

protection from environmental factors, since the encapsulation of the active compound

only occurs in situ. In fact, only 3 out of 73 studies reveal the development of

SNEDDS-containing essential oils.

Many of the techniques cited above can be employed in the preparation of different

nanostructures. As it can be noticed in Table 1, the most commonly used technique was

HPH, followed by US, SNE, phase inversion techniques, HSS and finally EIT.

3.2 Polymer-based nanostructured systems

3.2.1 Nanocapsules

Nanocapsules (NC) are vesicular systems of “core-shell” type of structures, which

consist of a hydrophobic or oily cavity surrounded by a polymeric layer. Drug or active

compounds can be loaded either inside the core or embedded on the polymeric shell [63,

64]. In general, most of the polymers used to prepare NC are synthetic, especially

poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and poly(lactide-co-glycolide)

12
(PLGA), due to their biocompatibility and the possibility of controlling drug release

upon variation of polymer composition [65].

The most widely used technique in the encapsulation of EO in NC is the

Nanoprecipitation (NPP) technique (Table 1), also called deposition of preformed

polymer, interfacial deposition or the solvent displacement technique. In this technique,

an organic phase, containing an organic solvent (usually acetone), a film-forming

polymer, a hydrophobic surfactant and oil, is poured into an aqueous phase containing

water and hydrophilic surfactant under agitation; this instantly forms the NC

suspension. The organic solvent diffuses towards the aqueous phase and is subsequently

removed by solvent evaporation, along with the excess of water as well, until the

suspension reaches the desired concentration [66].

Emulsion-Diffusion Method (EDM) is a technique that allows the high loading of

lipophilic active components and was first described by Quintanar-Guerrero et al.,

1998[67]. It consists in the preparation of an oil-in-water emulsion where the oily phase

contains polymer, oil, and organic solvent partially miscible in water and the aqueous

phase contains water and a stabiliser. After preparation of the emulsion, water is added

to the system, causing the organic solvent in the dispersed phase to migrate towards the

continuous phase, leading to a decrease in the droplets’ diameter and the precipitation of

the polymer [66, 68].

Besides the classically described techniques, Ghayempour and Mortazavi [69] describe a

new technique for the preparation of NC using electrospray (ES). In this method, two

needles of different gauge are placed in coaxial positions so that the core material, an

oil-in-water emulsion, and the shell material, an alginate solution, could exit the

apparatus through the same nozzle. A ring electrode is placed near the nozzle and

positive voltages are applied in order to build an electric field in the nozzle capable of

13
overcoming the surface tension of the alginate solution, creating the micro-nano

droplets. Then, the droplets are released into a calcium chloride solution which enables

the gelification of the particles coating. This technique was employed by Ghayempour

and Mortazavi in order to obtain nanocapsules containing Mentha piperitha EO [70]

3.2.2 Nanogels

Nanogels (NG) have been described recently in the literature as promising drug delivery

systems due to their high loading capacity, stability and the possibility of

biocompatibility and low toxicity, relying on polymer choice. They are defined as

dispersed hydrogel nanometric particles; in other words, they are nanometric networks

formed by cross-linked polymers, which can be natural, synthetic or semisynthetic.

These crosslinks can be separated between chemical or covalent crosslinks or physical

or non-covalent crosslinks, with the latter depending on intermolecular forces such as

Van der Waals forces, hydrogen bonding, electrostatic interactions and so on [71, 72].

Considering the aforementioned characteristics of NG, chitosan and alginate

nanoparticles were labelled as nanogels in order to simplify the particles’ classification

as they fulfil the feature of this system.

Spray drying (SD) technique is extensively employed in the preparation of micro- and

nanometric polymeric particles. It is based on the use of a spray dryer, built by an

atomiser, which can be a nozzle or a spinning disk from where the polymeric solution or

suspension is atomised, and a drying chamber, where the atomised droplets made

contact with hot air to evaporate the solvent [73, 74]. This technique was employed by

Paula et al. [75] and following studies [76, 77] in the attempt of encapsulating Lippia

sidoides EO in chitosan nanoparticles.

14
Another technique employed in the preparation of NG is Ionotropic Gelation (IG), in

which an ionic polymer is submitted to crosslinking through electrostatic interactions

when put into contact with a counter-ion. An example of ionotropic gelation applied in

NG preparation is between chitosan, a natural and positively charged polymer and

tripolyphosphate (TPP) [78, 79]. Esmaeili & Asgari [80] described the production of

NG containing Carum copticum EO using IG technique. The technique was also

employed by Hosseini et al. [81] in order to obtain Origanum vulgare EO NG.

As previously described in this review, the choice of nanostructured system and

preparation technique involves many factors. It is known that techniques using heating

or solvent evaporating steps can lead to degradation or the loss of the volatile content of

EO. However, many of the aforementioned techniques do perform such processes, for

instance, SNE, TFH, NPP and EDM represent an inefficient use of energy as HSS.

Furthermore, there is a growing concern on the development of green and sustainable

alternatives to techniques that employ heat, use of organic solvent, waste of energy and

generation of residues due to its environmental impact [82].

4. Quantification of EO in nanosystems

Nanometric systems containing encapsulated EO need to be characterised in order to

ensure the product quality as well as understand the system’s features. Physicochemical

parameters, such as particle size, surface charge, morphology, thermal stability and

association efficiency are general are investigated independently of the employed

system. Special care is needed with respect to the association efficiency, encapsulation

efficiency or loading capacity in reason of the considerable sensitivity of EO in front of

heating and solvent evaporation steps on the preparation processes [13, 83].

It can be noticed that the majority of the preparation techniques previously mentioned,

employed in the preparation of nanosystems containing EO, involve heating or solvent

15
evaporation steps, which may lead to volatilisation and/or degradation of components,

influencing the formulation final composition. At this juncture, it is crucial to perform

analytical methods in order to understand the EO behaviour during the nanostructure

preparation, especially the quantification of biologically active molecules when it is

intended for medicinal purposes, since it can interfere in the product efficacy and safety.

Techniques used in the analysis of EO are well described in the literature and good

reviews have been published, as illustrated by Smelcerovic, et al. [84], Rubiolo, et al.

[85], Jalali-Heravi and Parastar [3], Marriott, et al. [86] and many others, discussing

classical techniques and trends on sample preparation, analysis and quantification of

volatile components of EO. Also, in recent decades, special attention has been dedicated

to reducing environmental impacts and developing green chemistry [87]. Armenta, et al.

[88] discussed the fundamentals of green analytical chemistry and sustainable

alternatives to analytical methods and pre-analytical sample preparation. In a more

recent review, Gałuszka, et al. [89] suggest 12 principles to guide the development of

greener analytical methods.

In the present review, the studies were investigated concerning the quantification of EO

in the final formulations (Figure 4a). It was found that more than half of the selected

papers do not mention any quantification method to assess the marker content of EO in

the final formulation. Furthermore, 5 publications report the quantification only of the

non-encapsulated EO, which may not be reasonable, since the calculation of association

efficiency assumes a non-existent loss of EO content. Finally, the studies that performed

the quantification of EO were sorted according to the analytical method applied: indirect

quantification, spectrophotometric and chromatographic methods and further discussed

as follows (Figure 4b) (Table 2).

16
 4.1 Indirect Quantification

Indirect quantification is performed in two studies by assessing the mass balance of

essential oil on the formulation with the help of the Clevenger apparatus. First described

by Clevenger, 1928 [90], this allows the extraction of EO content from the formulation

avoiding the use of solvents. Upon heating of the material, the EO is carried by water

steam towards a condenser and falls into separator with a graduated tube [90]. Flores et

al.[34] and Ghayempour and Mortazavi [70] assessed the encapsulation efficiency of

EO in nanosystems by distillation of formulations in Clevenger apparatus. Flores et al.

[34] used the Clevenger apparatus to extract the EO from formulation after previous

extraction with acetonitrile and subsequentially weighing the EO extracted, obtaining an

encapsulation efficiency of 96% in NE prepared by SNE and 97.7% on NC prepared by

NPP. Ghayempour and Mortazavi [70] assessed the encapsulation efficiency of EO in

NC by the hydrodistillation of formulations in the Clevenger apparatus, performed by

distilling the nanoformulations for 2 hours at 70ºC, with an encapsulation efficiency in

the range of 72 to 96.4%. However, the hydrodistillation by Clevenger apparatus

involves heating the sample, which may lead to chemical conversions [91]. Also, the

Clevenger apparatus does not allow the identification of single components unless

associated with an additional analytical method.

4.2 Spectrophotometric methods

Spectrophotometric techniques are based on the ability of certain molecules to absorb

light in the ultraviolet wavelength range. Absorbance, a dimensionless unit, represents

the light emitted by a light source in the spectrophotometer, which does not reach the

equipment’s detector since it has been absorbed or scattered in the optical pathway. In

general, the absorbance of a certain substance and its concentration can be correlated,

17
according to Beer’s Law. However, in some cases, such as highly concentrated samples

and presence of non-light absorbing molecules, the Beer Law may not be applicable.

Additionally, the quantification of complex samples, containing more than one analyte

requires, ideally, distinct maximum absorption wavelengths and avoid spectra overlaps

between analytes [92].

A significant number of studies described spectrophotometric assays to determine the

EO encapsulation efficiency or nanosystem loading capacity. In many cases, a sample

preparation step, such as centrifugation [81, 93, 94], solubilisation of the system [75-77,

95, 96], reflux [80, 81], and a colorimetric reaction [27] is performed in order to release

the EO encapsulated in the nanostructures, followed spectrophotometric measures,

using a calibration curve. This can be justified by the Tyndall effect, in which colloidal

dispersions scatter the light beams [97] and may interfere with the spectrophotometric

reading of the analyte’s absorbance. Among these studies, Paula et al., Abreu et al. and

Paula et al. [75-77] assessed the encapsulation efficiency of NG containing Lippia

sidoides EO with spectrophotometric methods, obtaining 15–62%, 45–60% and 16–

77.8%, respectively. Also preparing NG systems, Hosseini et al. [81] determined the

encapsulation efficiency of Origanum vulgare to be in the range from 5.45% to 24.72%

and Esmaeili and Asgari [80] measured the encapsulation efficiency of Carum copticum

in NG to be in the range of 7.6% to 26.9%. Castangia et al.[98] measured Santolina

insularis EO content in LS and found values up to 70% of the oil initially added in the

system. Natrajan et al. [93] evaluated the association of Tumeric EO and Lemongrass

EO in NC and detected encapsulation efficiencies of 71.7% for Tumeric EO and 86.9%

for Lemongrass EO. Kalita et al. [94] prepared NC containing Cymbopogon flexuous

EO and determined the EO content in the final formulation to be 86.9%. NE containing

Stenachaenium megapotamicum were prepared by Danielli et al. [95] and presented an

18
encapsulation efficiency of 77.6%. Spectrophotometry was also used by Bonferoni et al.

[96] to determine the loading capacity of Cymbopogon citratus in NE as 10% and by

Mostafa et al. [27] to assess the phenolic content of Cuminum cyminum in NE, also

subsequently calculating the encapsulation efficiency, found to be in the range from

1.49% to 2.80%.

On the other hand, Liakos et al [99] performed spectrophotometric assays in

nanoparticles dispersion, extrapolating the calibration curve of non-encapsulated EO,

obtaining a result of 0.9 µl of lemongrass EO in 50 µL of the NC formulation (1.8%)

when 5% of EO was initially added. Despite being a cheap and accessible option and

widely employed in the EO quantification, the spectrophotometry does not seem to be

an appropriate method. This technique lacks specificity, which is unfavourable in the

analysis of complex matrices such as EO. Also, the scattering of light by colloidal

suspensions such as polymers in dispersion and the nanoparticles demand sample

preparation processes.

4.3 Chromatographic methods

In analytical chemistry, chromatography is widely used as a separation technique. It is

often associated with other instrumental techniques in an attempt to accomplish the

identification and quantification of chemical mixtures. The separation occurs due to a

distribution equilibrium of chemical compounds between a mobile phase and a

stationary phase, where each different compound presents a different interaction with

the different phases, resulting in different migrations between compounds [92, 100].

4.3.1 Gas Chromatography (GC)

19
Gas chromatography is a technique where a gaseous or volatile sample is carried by a

gaseous mobile phase through a column coated by a solid or liquid stationary phase

[92]. EOs are composed mainly of apolar volatile components, which makes capillary

GC the most extensively used analytical method. Fused silica capillary columns bonded

to the stationary phase are usually employed in the separation of compounds. The

identification is achieved by coupling the gas chromatograph instrument to detectors

chosen according to the analytical needs. The Flame Ionisation Detector (FID) gives

information about retention time and peak intensity, while the Mass Spectrometer (MS)

provides the mass spectra [84, 101]. Furthermore, in many cases, the GC analysis

requires a sample preparation step, since the injection of non-volatile components may

damage the chromatographic column [92]. As described previously in this review,

nanostructured systems containing EO almost always include an aqueous fraction and

other non-volatile components that need to be extracted from the sample before

injection into the chromatographic system. Traditional extraction techniques such as

distillation and liquid phase extraction present disadvantages in sample preparation for

GC injection, since they may use large volumes of organic solvents and/or lead to the

thermal degradation of analytes. On the other hand, more recent techniques such as

headspace (HS), coupled or not with solid phase microextraction (SPME), are efficient

techniques that can be coupled to the chromatographic system, and allow the separation

of volatile fraction whilst avoiding the formation of degradation products, since these

preparation techniques do not require high temperature steps [102].

Unexpectedly, a few publications have reported the use of GC in the direct

quantification of EO in nanosystems. Nantarat et al. [103] studied the stability of EO

blend after encapsulation, submitted to different storage conditions using GC coupled to

an FID, and found no significant changes in the EO markers. Nasseri et al. [50] used

20
GC coupled to MS detectors to assess the encapsulation efficiency of Zataria multiflora

in SLN and determined the encapsulation efficiency to be 84% EO. Stability of

Lavandula officinalis EO in NE and Mentha piperita EO in NC before and after

encapsulation in nanosystems was evaluated respectively by Rocha-Filho et al. [104]

and Ghayempour and Mortazavi [70] using GC/MS. Ghayempour and Mortazavi [70]

observed no significant changes in the composition of peppermint EO after

encapsulation in NC, while Rocha-Filho et al. [104] identified a loss of linalyl from

lavender EO and an increase in α-terpineol and geranyl isobutyrate content during the

storage of NE, which the author attributes to linalyl degradation reactions. Ghaderi et al.

[33] employed a GC coupled to a MS detector to quantify the EO in the formulations

upon storage. It was found out that the content of EO remained in relatively high

amounts (96.3% and 93.5% for two different formulations) after 6 months of storage.

Lucca et al. [105] described a solvent free sample preparation method with a Headspace

(HS) extraction system coupled to the GC/MS to assess the content of Copaifera

multijuga EO through its majoritarian compound (β- caryophyllene) in the NE

formulation and skin permeation studies with the formulation. In a previous study, Dias

et al. [35] developed an analytical method to assess β-caryophyllene by extracting the

volatile content from C. multijuga nanoemulsion using SPME (solid phase

microextraction) and analysing with a GC/FID system.

4.3.2 High Performance Liquid Chromatography (HPLC)

In contrast to GC, HPLC techniques apply liquid mobile phases in which the analyte is

solubilised. The mobile phase is forced to pass through a packed column by high

pressure. This system enables high-resolution separations to be achieved. However,

HPLC is generally more expensive than GC and produces chemical residues [92]. HPLC

21
is a versatile technique that can be used in the analysis of EO as an alternative to GC,

especially when dealing with less volatile components [84, 106].

Mostafa et al. [29] employed a HPLC coupled to UV (ultraviolet) detector in order to

assess the amount of fennel EO encapsulated in NE by measuring the marker trans-

anethole. Also using a HPLC/UV system, Li et al. [107] quantified terpinen-4-ol, the

major component of Tea Tree EO in the NE and related it to the final content of EO in

nanosystem, found to be 70mg/ml, which was equivalent to approximately 58% of the

marker content initially added to the system. Lai et al. [46] performed a quantitative

determination of encapsulated EO using HPLC coupled to a Photodiode array (PDA)

detector, obtaining a range of 87–92% of Artemisia arborescens EO encapsulated in the

SLN. In both cases, spectrophotometric detectors were employed [92].

5. Final Remarks

Due to the recent growing interest in nanotechnological approaches to encapsulate EO

for pharmaceutical purposes, this review aimed to conduct a literature survey and

identify the organic nanostructured systems, preparation techniques and analytical

methods employed in the encapsulation efficiency of formulations. It was found that

most of the identified preparation techniques involve heating or solvent evaporation

steps that could damage the chemical composition of EO and lead to a loss of content

by volatilisation. In light of this information, the appropriate quantification of EO in the

final formulation is crucial.

However, in the course of the survey, a lack of investigation concerning EO

encapsulation efficiency was observed. Most of the publications do not mention any

analytical assays, while a few perform indirect analysis, assuming that there is no EO

content loss in the production of nanostructures systems. The remaining studies that

22
perform quantification employ different analytical tools in order to assess the EO

chemical marker content; many times, the methods are not suitable for the purpose. GC

is broadly used and considered the gold standard for the analysis of EO, but few

researchers take advantage of this method.

In this context, it is essential to emphasise the importance of the quantification of EO in

developed formulations. Future studies need to focus on the development and

implementation of analytical methods on this concern.

Acknowledgments

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de

Nível Superior - Brasil (CAPES) - Finance Code 001. S.P.M., L.G.L. and L.S.K. thank

FAPERGS, CAPES and CNPq (grant nº 304655/2015-5) respectively for their

scholarships.

Declaration of interest

Authors declare no conflict of interest.

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33
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34
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35
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Figure 1. Annual distribution of publications concerning the nanoencapsulation of EO

for pharmaceutical and medical purposes.
Figure 2. Schematic representation of the different organic nanostructured systems.
Figure 3. Percentual distribution of nanostructured systems containing EO for
therapeutic uses.
Figure 4. Graphical representation of (a) EO quantification status of the analyzed
publications and (b) the analytical method employed in the cases that performed EO
quantification.

36
Table 1. Publications concerning EO encapsulation in nanometric systems found in data
survey with application in pharmaceutical field.

Nanostruct Preparati Quantificat Quantificatio Rerefen

ure on ion of EO n method ce
techniqu in
Achyrocline NC NPP
e No
nanosyste NA [108-
satureioides m 110]
Artemisia SLN HPH Yes HPLC/PDA [46]
arborescens
Carapa NE SNE and No NA [111]
guaianensis and HPH
Casearia
Schinus molle NE PIT No NA [112]
sylvestris
Carum copticum NG IG Yes Spectrophotom [80]
etry
Cinnamomum NE US No NA [26]
Zeylanicum
Clove oil, SNEDDS NA NA NA [113]
Peppermint oil,
Cedar wood oil
Copaifera NE PIT No NA [114]
and Rose oil
langsdorffii
Copaifera NE HPH Yes HS-GC/FID [105]
multijuga
Cuminum NE US Yes Spectrophotom [27]
cyminum etry
Cuminum NG IG Only non- Spectrophotom [93]
cyminum encapsulate etry
Curcuma longa NE SNE No
d NA [115]

Curcuma SNEDDS NA No NA [116]

zedoaria
Curcuma NC NPP Yes Spectrophotom [93]
zedoaria and etry
Cymbopogon NE SNE Yes Spectrophotom [96]
citratus etry
Cymbopogon NC NPP Yes Spectrophotom [94]
flexuous etry
Cymbopogon NE HSS No NA [22]
flexuous
Cymbopogon NE HPH No NA [117]
nardus, Ocimum
Drimys
americanum and NE HPH No NA [118]
angustifolia
Vetiveria and
Essential
D. Oil
brasiliensis
zizanioides NE PIC Yes GC/FID [103]
Blend
Eucalyptus NE US No NA [28]
globulus
Eucalyptus NE SNE No NA [119]
staigeriana
Foeniculum NE HPH No NA [120]
vulgare
Foeniculum NE US Yes HPLC/UV [29]
vulgare

37
Frankincence and SLN HPH Only non- GC/FID [121]
myrrh encapsulate
Laurus nobilis SLN HPH ans No
d NA [48]
HSS
Lavandula NE PIC Yes GC-MS [104]
officinalis
Lemongrass NC NPP No NA [122]
essential oil
Lemongrass NC NPP Yes Spectrophotom [99]
essential oil etry
Lippia sidoides NC EDM Only non- HPLC/PDA [123]
encapsulate
Lippia sidoides NG IG No
d NA [124]

Lippia sidoides NG SD Yes Spectrophotom [76, 77]

etry
Lippia sidoides NG SD Yes Spectrophotom [75]
etry and
Melaleuca LS TFH and Only non- GC/FID [56]
alternifolia US encapsulate
Melaleuca NE SNE No
d NA [125]
alternifolia
Melaleuca NE PIT No NA [126]
alternifolia
Melaleuca NE HPH Yes HPLC/UV [107]
alternifolia
Melaleuca NE and NC SNE and No NA [127-
alternifolia NPP 129]
Melaleuca NE and NC SNE and Yes Clevenger [34]
alternifolia NPP
Melaleuca SLN HSS No NA [41]
alternifolia
Melaleuca SLN HPH No NA [42, 49]
alternifolia
Mentha piperita NC ES Yes Clevenger and [70]
GC/MS
Mentha piperita NG IG No NA [130]

Nigella Sativa SLN HPH No NA [47]

Nigella sativa L NE US No NA [30]

Origanum NG IG Yes Spectrophotom [81]

vulgare etry
Piper cubeba SNEDDS NA No NA [131]

Pogostemon NE HSS No NA [21]

cablin
Rosmarinus SLN PIT No NA [43]
officinalis
Rosmarinus NE PIC No NA [132]
officinalis
Rosmarinus NE US NO NA [32]
officinalis and
Lavandula
angustifolia 38
Santolina LS US Yes Spectrophotom [98]
insularis etry
Stenachaenium NE SNE Yes Spectrophotom [95]
megapotamicum etry
Synzygium NE SNE No NA [133]
aromaticum
Syzygium LS EIT Only non- HPLC/PDA [134]
aromaticum encapsulate
Syzygium NE SNE No
d NA [135]
aromaticum
Syzygium NE US No NA [31]
aromaticum
Sweet Fennel, NE HPH No NA [136]
Peppermint,
Orange, Lime,
Buriti, Avocado,
Thymus
Clove and NE US Yes GC [33]
daenensis
Copaiba essential
Thymus vulgaris NG IG Only non- Spectrophotom [137]
oils encapsulate etry
Urtica dioica NE HPH No
d NA [138]

Yuxingcao SLN HSS Only non- GC/FID [44]

encapsulate
Zataria SLN HPH and Yes
d GC/MS [50]
multiflora US

Table 2. Overview of the analytical methods employed in the assessment of

encapsulation efficiency of EO in nanosystems.

Method Sample Preparation Drawbacks

Indirect Quantification No sample preparation Non specific
required Use of heat and solvents

Spectrophotometry Need to dilute sample Lack of specificity

Analyte needs to be in Light scatter effects
solution
GC Need to displace water Difficult to analyze non-
content of formulation volatile fraction of EO

HPLC The sample needs to be More expansive

filtered and sonicated to Demands use of solvents
avoid air bubbles

39
40
Highlights

 A data survey was performed in order to set a panorama of the research

concerning the nanoencapsulation of essential oils for medicinal purposes;

 Encapsulation of essential oils in nanosystems has been studied in the last

decade. The number of publications on this concern has raised in 2015 and the

following years;

41
 Preparation techniques of nanoencapsulated essential oils many times involve

heating and solvent evaporation steps, which may cause volatilization

and/degradation of essential oils components.

 Many studies evaluated do not perform the quantification of chemical markers

on the essential oils after the nanoencapsulation. Among the one that quantify, a

few techniques are employed, but not always suitable for the purpose;

42