Industrial Crops & Products 126 (2018) 287–301

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Industrial Crops & Products

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Double emulsion solvent evaporation approach as a novel eugenol delivery T

system – Optimization by response surface methodology
Filipa Paulo, Lúcia Santos
⁎

LEPABE – Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465,
Porto, Portugal

ARTICLE INFO ABSTRACT

Keywords: Eugenol, a substance found in plant essential oils, exhibits significant health benefits due to its antioxidant,
Eugenol antibacterial, anti-inflammatory, anesthetic, and even anticancerogenic properties. Nevertheless, eugenol is
Double emulsion light, heat and oxygen sensitive which limits its applications at an industrial scale and for practical uses.
Optimization Considering these drawbacks, this study aimed to microencapsulate eugenol by double emulsion solvent eva-
Ethyl cellulose
poration technique. A response surface methodology was applied to obtain eugenol-loaded ethyl cellulose mi-
Response surface methodology
Essential oils
croparticles. Optimal encapsulation conditions were found considering a polymer concentration of 32.1 g/L and
a surfactant concentration of 1.1% w/w. The microparticles obtained under the optimized encapsulation con-
ditions provided the maximization of both eugenol encapsulation efficiency and the product yield to
94.7 ± 1.0% and 82.0 ± 11.2%, respectively. The obtained powder was characterized regarding wettability,
thermogravimetric stability, thermal transitions, morphology and particle size distribution. In vitro release
studies were performed using simulated gastrointestinal fluids. Microparticles obtained under optimized con-
ditions showed to be spherical, presenting smooth but porous surfaces. The particle size distribution was narrow,
and the mean particle size was 11.4 ± 1.1 μm. The release stimulation of eugenol in in vitro gastrointestinal
transit revealed that this compound was favorably protected in the simulated salivary and gastric fluids. The
highest release of eugenol was observed in the simulated intestinal fluid and was much higher than the minimum
amount of eugenol that exhibits positive biological effects. This work demonstrates the promising possibility of
incorporation of eugenol in ethyl cellulose polymer-based microparticles to potentiate added-value properties of
functional foods and nutraceuticals.

1. Introduction
In the recent years, is acknowledged an increasing interest in the
beneficial properties of essential oils, mainly to obtain natural products
with added-value properties as improved food-flavor, prolonged food-
preservation, and extended fragrance effect. Essential oils are volatile,
strong-odor compounds mainly found in aromatic plants’ parts as buds,
flowers, herbs, roots, seeds, leaves, twigs, fruits, barks and woods (Burt,
2004). They were generally recognized as safe (GRAS) by Food and
Drug Administration in the 21 Code of Federal a Regulations 182.20
(Burt, 2004) being eugenol one of the most commercially important
component of the majority of essential oils (Kaufman, 2015).
Eugenol (4-allyl-2-methoxyphenol), a water miscible compound, is
the major phenolic component present in clove oil (Eugenia car-
yophyllata, Myrtaceae) also existing in the basil, cinnamon, and nutmeg
essential oils (Phunpee et al., 2016). Considering the phenolic nature of
eugenol, many studies have been carried out to investigate the multiple
bioactive properties of this phenylpropene compound such as anti-
oxidant (Gu, 2011; Nam and Kim, 2013; Ou et al., 2006), antifungical
(Mihai and Popa, 2015; Ribes et al., 2017), antibacterial (Devi et al.,
2010; Kalemba and Kunicka, 2003) and anti-inflammatory properties.
Eugenol also exhibits local anesthetic properties (Chaieb et al., 2007;
Markowitz et al., 1992; Ohkubo and Shibata, 1997). Additionally, eu-
genol has been gaining increasing attention due to its anti-cancerogenic
properties (Aggarwal and Shishodia, 2006).
Regarding the fungi development in foods, chemical preservatives
have been used as food additives worldwide. However, recently, the
consumer’s negative perception of chemical preservatives has driven
research for the incorporation of natural preservatives in foods. Due to
its antifungical properties, eugenol has been widely studied to control
fungi decay in foods. Eugenol acts against fungi by promoting the dis-
ruption of the cellular membrane of these type of microorganisms

⁎
Corresponding author.
E-mail address: lsantos@fe.up.pt (L. Santos).

https://doi.org/10.1016/j.indcrop.2018.10.027
Received 14 May 2018; Received in revised form 6 October 2018; Accepted 8 October 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
F. Paulo, L. Santos
(Ribes et al., 2017). Nevertheless, the concentration of eugenol required
to be effective in the fungi decay control may give food strong flavor
and therefore modify the sensory profile of food products. Moreover,
this bioactive compound is light, heat and oxygen sensitive which may
limit its applications at an industrial scale and for practical uses (Kayaci
et al., 2013; Phunpee et al., 2016; Sajomsang et al., 2012; Yang and
Song, 2005).
Therefore, microencapsulation arises a technological approach to
reduce the sensorial impact of eugenol when incorporated in food
products as well as protect it from light, oxygen and heat degradation.
Furthermore, microencapsulation may enhance the bioavailability of
eugenol in the human body.
Microencapsulation can be defined as a technological strategy in
which tiny droplets and even particles are coated using an en-
capsulating agent. The active ingredient is embedded in a homogeneous
or heterogeneous polymer matrix (Aguiar et al., 2017; Casanova et al.,
2016; Paulo and Santos, 2017). The most reported technique for the
microencapsulation of bioactive compounds is the spray-drying tech-
nique. However, during the spray-drying procedure, the droplets con-
taining the bioactive compound are exposed to a stream of hot air
which may reduce the retention of essential oils in the polymer matrix
by volatilization, resulting in a significant loss of ingredients during the
process (Jafari et al., 2008, 2007;).
The double emulsion solvent evaporation technique is a feasible
approach for the microencapsulation of volatile compounds as eugenol
as no increase in the temperature is required. In the water-in-oil-in-
water (w/o/w) double emulsion solvent evaporation technique, the
active ingredient, soluble in an aqueous phase (internal aqueous phase)
is dispersed in an organic phase, forming the primary emulsion.
Afterward, this emulsion is re-emulsified in another aqueous phase
(external aqueous phase) containing a hydrophilic stabilizer (Gaitzsch
and Kraume, 2011; Paulo and Santos, 2018; Schuch et al., 2013). This
technique has been used by food (Garti and Bisperink, 1998; Rodrı,
2008), pharmaceutical (Florence et al., 1976; Jeffery et al., 1993; Ma,
2014) and cosmetic (Taylor et al., 2010) industries.
Several biocompatible polymers have been studied as encapsulating
agents which can be classified as natural polymers (e.g., gelatin, casein,
acacia gum, whey protein, xanthan gum), semi-synthetic polymers (e.g.,
ethyl cellulose and carboxymethylcellulose) and synthetic polymers
(e.g., polylactide, polycaprolactone, polyglycolide) (Aguiar et al., 2017;
Dickinson, 2011; Park et al., 2005). Ethyl cellulose is a biocompatible
cellulose-derived polymer, an appealing carrier for microencapsulation
purposes. Depending on the degree of ethylation, some of the hydroxyl
groups of cellulose were replaced by ethyl ether groups. This compound
is soluble in many organic solvents as ketones, alcohols, esters, and
ethers which make it attractive for the organic phase formulation when
is considered the double emulsion solvent evaporation technique.
Moreover, it is resistant to mechanical stress, and also it is oxygen, heat
and light stable (Kamel et al., 2008; Murtaza, 2012).
Among the most important characterization parameters of micro-
encapsulation processes, the encapsulation efficiency and the product
yield can be highlighted. The main factors that may affect these para-
meters are the polymer and the surfactant concentration in the external
aqueous phase as they may reduce or improve the entrapment of water-
miscible compounds in the polymer matrix during solvent evaporation.
The product yield is a viable indicator if the microencapsulation pro-
cedure is suited for scale-up and industrial applications.
Many studies described the influence of selected parameters on
spray-drying microencapsulation process variables of oils as orange oil
(Kim et al., 1996), flaxseed oil (Tonon et al., 2011), seed oil (Ahn et al.,
2008), coffee oil (Frascareli et al., 2012), lemon myrtle oil (Huynh
et al., 2008), fish oil (Aghbashlo et al., 2012) among others. To the best
of authors knowledge, studies regarding the optimization of micro-
encapsulation processes of water-miscible oils are not reported in the
literature. Moreover, reports regarding the micro-inclusion of eugenol
are limited to its inclusion in β-cyclodextrin complexes (Choi et al.,
288
Industrial Crops & Products 126 (2018) 287–301
2009; Hill et al., 2013; Wang et al., 2011) and in mesoporous silica-
based systems. Other studies focused on eugenol incorporation in na-
noemulsions (Sharif et al., 2017; Woranuch and Yoksan, 2013) and
nanodispersions (Shah et al., 2013). To the authors best knowledge is
this the first report regarding the microencapsulation of eugenol by w/
o/w double emulsion solvent evaporation technique.
Regarding the optimization of formulation conditions that may af-
fect selected characterization parameters as the encapsulation effi-
ciency and the product yield, a design of experiments may be useful.
This toolbox allows to establish and evaluate the influence of selected
input variables on responses. This approach also allows to reduce de-
sign costs, labor complexity, time and also raw materials (Paulo and
Santos, 2017).
Therefore, this study aimed to microencapsulate eugenol by w/o/w
double emulsion solvent evaporation technique using ethyl cellulose as
encapsulating material. The optimal formulation conditions were ob-
tained applying a response surface methodology with a central com-
posite design. In a two-level full factorial design, the effect of the
polymer and the surfactant concentrations in the external aqueous
phase concentration on the eugenol encapsulation efficiency and the
product yield were evaluated. The powder was characterized regarding
its physicochemical behavior. In vitro release studies were performed
and monitored under simulated gastrointestinal conditions.
2. Experimental
2.1. Materials
2.1.1. Chemicals
The bio-active ingredient, eugenol standard (Ref: E-5504, C10H12O2,
CAS 97-53-0, purity ≥ 99%), ethyl cellulose (Ref: 433837-250 G,
viscosity of 46 cP, CAS 9004-57-3) and polyvinyl alcohol (Ref: P8136-
250 G, 87–90% hydrolyzed, average molecular weight of
30,000–70,000, CAS 9002-89-5) were purchased from Sigma Aldrich
Chemical (St. Louis, MO, USA). Ethyl acetate (Ref: ACRO423680010,
C4H8O2, CAS 141-78-6) used as a solvent was obtained from VWR
International (Fontenay-sous-Bois, France). The salts potassium
chloride (Ref: 1049380050, KCl, CAS 7447-40-7), potassium dihy-
drogen phosphate (Ref: 104871001, KH2PO4, CAS 7778-77-0), sodium
chloride (Ref: 1370170001, NaCl, 7647-14-5), magnesium chloride
hexahydrate (Ref: 1023675000, MgCl2.6H2O, CAS 7791-18-6), sodium
hydrogen carbonate (Ref: 1063290500, NaHCO3, CAS 144-55-8), and
ammonium carbonate (Ref: 1011369051, (NH4)2CO3, CAS 10361-29-2)
were used for electrolyte solutions formulation and obtained from
Merck kGaA (Darmstadt, Germany). Water was deionized and double-
distilled in the laboratory using a Millipore™ water purification system
(Massachusetts, USA). All the reagents were either chromatographic or
analytical grade and used as received.
2.2. Methods
2.2.1. Analytical methods validation
The UV–vis spectrophotometer method was chosen as the in-
strumentation for the analytical methods validation, using a UV–vis V-
530 (Jasco, OK, USA) spectrophotometer at 279 nm for detection and
quantification of eugenol in ultra-pure water (UPW) and in the simu-
lated gastric fluid (SGF) and at 280 nm for the detection and quantifi-
cation of eugenol in a simulated salivary and intestinal fluids (SSF and
SIF) using quartz cells of 10 mm of light path. The SPECTA MANAGER
software was used for all the absorbance measurements.
2.2.2. Preparation of eugenol-loaded microparticles
The w/o/w double emulsion solvent evaporation technique was
selected for the microencapsulation of eugenol into ethyl cellulose
microparticles. In a two-step emulsification process, the w/o primary
emulsion was prepared adding 1 mL of the aqueous solution containing
F. Paulo, L. Santos Industrial Crops & Products 126 (2018) 287–301

eugenol (50.0 mg/L of eugenol in ultra-pure water) to a 10 mL of Table 1

17.9–32.1 mg/L of ethyl cellulose in ethyl acetate. The w/o primary Experimental design for the microencapsulation tests.
emulsion was vigorously emulsified using a vortex shaker ((IKA Run no.a Process variables Coded variables
VORTEX GENIUS 3, Staufen, Germany) for 3 min. Afterward, the w/o
primary emulsion was mixed with 100 mL of different aqueous solu- X1 - Polymer Conc. X2 - Surfactant Conc. (% X1 X2
tions of polyvinyl alcohol used corresponding to different polyvinyl (g/L) w/w)

alcohol concentrations (0.8–2.2% w/w). The polyvinyl alcohol was 1 20.0 1.0 −1 −1
used as a surfactant in the external aqueous phase. The w/o/w double 2 20.0 2.0 −1 +1
emulsion was emulsified using a high-performance liquid homogenizer 3 30.0 1.0 +1 −1
(IKA T18 Digital ULTRA-TURRAX®, Staufen, Germany) at 5 000 rpm for 4 30.0 2.0 +1 +1
5 17.9 1.5 −1.41 0
5 min. The evaporation of the solvent and the hardening of micro-
6 32.7 1.5 +1.41 0
particles were performed by continuously stirring the w/o/w double 7 25.0 0.8 0 −1.41
emulsion in a stirring plate (AREX Digital, VELP Scientifica, Monza, 8 25.0 2.2 0 +1.41
Italy) at 650 rpm for 3 h in the fume hood at room temperature. 9 25.0 1.5 0 0
10 25.0 1.5 0 0
Eugenol-loaded ethyl cellulose microparticles were recovered by fil-
11 25.0 1.5 0 0
tration using a 0.2 μm quantitative paper filter and washed with 500 mL 12 25.0 1.5 0 0
of distilled water to remove polyvinyl alcohol residues. The obtained 13 25.0 1.5 0 0
microparticles were frozen for 24 h at −4 °C and freeze-dried for 72 h in
a bench top freeze-dryer (SP Scientific, NY, USA). The weight mea- Conc. – Concentration.
a
surements were performed on an analytical scale Mettler Toledo AG245 Each run was performed in triplicate.
balance (Columbus, OH, USA).
1.5 mL of the w/o/w double emulsion using a 0.2 μm syringe filter (Ref:
2.3. Experimental design 514-0070, VWR International, Fontenay-sous-Bois, France). A volume
of 1 mL of the aliquot was transferred to a quartz cell and the weight of
2.3.1. The experimental design factors polymer and surfactant eugenol non-microencapsulated was calculated considering the absor-
concentrations bance measurement at 279 nm on the UV–vis spectrophotometer, using
In this work, a response surface methodology was used as a math- ultra-pure water as a blank.
ematical and statistical design to optimize the eugenol encapsulation The product yield (PY) was calculated according to the Eq. 3.
efficiency and the product yield. The effect of the polymer concentra- Qf Qf
tion ( X1: 20–30 g/L) and the surfactant concentration in the external PY (%) = × 100 = × 100
Qi Qp + Qu (3)
aqueous phase ( X2 : 1–2% w/w) on two responses (Y1 - eugenol en-
capsulation efficiency; Y2 - product yield) were assessed using a central where Qf corresponds to the amount of the freeze-dried powder and Qi
composite design. The two variables were coded in five levels (−1.414, the initial amount used for the w/o/w preparation (the mass of the
−1,0,1,1.414) and integrated in a design including a base run of 13 polymer - Qp and the mass of eugenol - Qu ).
experiments, all in triplicate, corresponding to a total number of runs of Considering that parametric tests as the linear regression or even
39. ANOVA are not quite sensitive to the deviations of the assumptions and
Data relative to the effects of the variables X1 and X2 on the response data may not assume a normal and homogeneous distribution, a arcsine
Yn were evaluated using a second-order polynomial regression (Eq. (1)): of square roots of proportions was applied before data analysis by a
2 2
design of experiments as described by McDonald (2014).
Yn = 0 + 1 X1 + 2 X2 + 12 X1 X2 + 11 X1 + 22 X2 (1)
where, Yn corresponds to the response (Y1 - eugenol encapsulation effi- 2.4. Physicochemical characterization of the optimized eugenol-loaded ethyl
ciency or Y2 - product yield), 0 is a constant, 1 and 2 are the coeffi- cellulose microparticles
cients of the linear effects, 12 is the coefficient of the interaction be-
tween the two factors and the coefficients 11 and 22 represent the 2.4.1. Wettability of the optimized powder
quadratic effect. The coefficients of the fitted polynomial regression The wettability of the optimized powder was evaluated as described
equations as well as the surface response and contour plots were ob- by de Barros Fernandes et al. (2014). For that, 1 g of optimized powder
tained using MINITAB 18 (Minitab Inc., State College, PA, USA) sta- was sprinkled over the surface of distilled water without agitation at
tistical computer software. The analysis of variance (ANOVA) was ap- 20 °C. The time required for the microparticles to disappear from the
plied to evaluate significant differences between the independent water’s surface, to sink, to be submersed or to become a sediment was
variables as described by (de Barros Fernandes et al., 2014). In Table 1 registered.
is presented the main formulation conditions of each run (process
variables) as well as the corresponding coded variables. 2.4.2. Differential Scanning Calorimetry (DSC)
The technique Differential Scanning Calorimetry (DSC) was em-
2.3.2. The experimental design responses eugenol encapsulation efficiency ployed to evaluate the thermal behavior of eugenol (purity ≥ 99%),
and product yield ethyl cellulose, polymer-only microparticles and eugenol-loaded ethyl
The obtained microparticles were evaluated using the statistical cellulose microparticles obtained under optimized conditions as de-
response surface methodology regarding the eugenol encapsulation scribed by Piletti et al. (2017). Slight modifications to the procedure
efficiency (EEE) and the product yield (PY). described by Piletti et al. (2017) were implemented. The analyses were
The EEE (%) was obtained considering the Eq. (2). performed using a NETZSCH DSC 214 Polyma calorimeter (NETZSCH
DSC 214 Polyma, Selb, Germany) with a temperature range of
Qu Qd
EEE (%) = × 100 30 °C–270 °C at a heating rate of 10 K min−1, under a nitrogen atmo-
Qu (2)
sphere (flow rate of 40 mL min−1).
where the terms Qu and Qd correspond to the initial amount of eugenol
used in the w/o/w double emulsion formulation and the amount of 2.4.3. Thermogravimetric analysis (TGA)
eugenol detected in the external aqueous phase after the microparticles The thermogravimetric analysis (TGA) was used to study the
recovering, respectively. The Qd was evaluated after the filtration of thermal stability of eugenol (purity ≥ 99%), ethyl cellulose, polymer-

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F. Paulo, L. Santos Industrial Crops & Products 126 (2018) 287–301

only microparticles and eugenol-loaded ethyl cellulose microparticles Table 2

obtained under optimized conditions as described by Piletti et al. Concentrations of electrolytes of each simulated gastrointestinal fluid.
(2017). Minor modifications were employed. The analyses were per- Salt Stock Conc. Conc. (mmol/L) Conc. (mmol/L) Conc. (mmol/L)
formed using a Netzsch STA449 F3 Jupiter thermogravimetric analyzer (g/L) in SSF in SGF in SIF
(Netzsch STA449 F3 Jupiter, Selb, Germany) with a temperature range
KCl 37.3 15.1 6.9 6.8
of 30 °C–810 °C at a heating rate of 10 K min−1 under inert atmosphere
KH2PO4 68 3.7 0.9 0.8
(N2, flow rate of 30 mL.min−1). NaHCO3 84 13.6 25 85
NaCl 117 – 47.2 38.4
2.4.4. Fourier transform infrared spectroscopy (FTIR) studies MgCl2(H2O)6 30.5 0.15 0.1 0.33
(NH4)2CO3 48 0.06 0.5 –
The infrared spectra of eugenol, ethyl cellulose and eugenol-loaded
ethyl cellulose microparticles obtained under optimized formulation Conc. – Concentration; SGF – Simulated Gastric Fluid; SIF – Simulated Intestinal
conditions were recorded using an VERTEX 70 FTIR spectrometer Fluid; SSF – Simulated Salivary Fluid.
(BRUKER) in transmittance mode with a high sensitivity DLaTGS de-
tector at room temperature. Samples were measured in attenuated total
reflection (ATR) mode, with a A225/Q PLATINUM ATR Diamond Table 3
crystal with single reflection accessory. The spectra were recorded from Experimental procedure for the in vitro release studies.
4000 to 500 cm−1 with a resolution of 4 cm−1. Electrolyte Procedure Enzymes Duration of the pH
solution release

2.4.5. Assessment of the morphology of the optimized powder SSF SSF and enzymes Salivary amylase 2 minutes 7
The external morphology and polydispersity of eugenol-loaded 1:1 (v:v) (75 U/mL)
ethyl cellulose microparticles obtained under optimized conditions SGF SGF and enzymes Pepsin 2 hours 3
were evaluated using a PHENOM XL scanning light microscope 1:1 (v:v) (2 000 U/mL)
SIF SIF and enzymes Pancreatina (100 2 minutes 7
(Eindhoven, The Netherlands) at an accelerating voltage of 10 kV. 1:1 (v:v) U/mL)
Freeze-dried microparticles were placed on an aluminum stub with a Bile salts (10 mM)
carbon double-sided adhesive tape. Samples of the optimized powder
were sputter-coated with gold for 20 s using a vacuum-sputtering coater SGF – Simulated Gastric Fluid; SIF – Simulated Intestinal Fluid; SSF – Simulated
(Leica, EM SCD 500, Wetzlar, Germany) as described by Negrão- Salivary Fluid.
a
Murakami et al. (2017). based on trypsin activity.

2.4.6. Assessment of the particle size distribution of the optimized powder

The particle size distribution of eugenol-loaded ethyl cellulose mi-
croparticles obtained under optimized conditions was evaluated by
laser granulometry using a Coulter Counter-LS 230 Particle Size
Analyzer (Miami, FL, USA) as described by Aguiar et al. (2017). The
particle size distribution was recorded considering the differential vo-
lume distribution. A powder sample of 100 mg was suspended in dis-
tilled water under agitation, and the particle size distribution was re-
corded (3 times, 60 s each). Average values were considered in data
analysis. The polydispersity of particle size distribution was analyzed
considering the SPAN equation (Eq. 5).
Dv,90 Dv,10
SPAN =
Dv,50 (5)
where, Dv,90 , Dv,50 and Dv,10 are respectively the characteristic volume
diameters at 90%, 50% and 10% of the cumulative volume (Jinapong
et al., 2008).
2.4.7. In vitro eugenol release studies in simulated gastrointestinal fluids
The in vitro release studies of eugenol from eugenol-loaded ethyl
cellulose microparticles obtained under optimized conditions at dif-
ferent moments were performed in a simulated salivary fluid (SSF),
simulated gastric fluid (SGF) and in simulated intestinal fluid (SIF). To
all simulated gastrointestinal fluids were added enzymatic solutions.
In brief, about 10 mg of the optimized powder was suspended in
Table 2 are presented the composition and the concentration of each
salt used in each electrolyte solution as described by Minekus et al.
(2014). Further considerations are presented in Table 3.
3. Results and discussion
3.1. Analytical methods validation
The UV–vis spectrophotometer method was chosen for the detection
and quantification of eugenol in ultra-pure water as well as in the si-
mulated gastrointestinal fluids due its cost-efficiency, availability,
simplicity, and versatility as described by many authors (Dehghani
Mohammad Abadi et al., 2012; Mendez et al., 2003; Shokoufi et al.,
2007)
The maximum absorption wavelength of eugenol in ultra-pure
water and in the SGF was 279 nm and 280 nm in the case of the SSF and
SIF. These wavelengths were used in all measurements. The pH of the
SSF and the SIF was about 7 and the pH of ultra-pure water, and the
SGF was 6 and 3, respectively. An increase in the maximum absorption
wavelength with the increase of the pH was observed. The UV–vis
spectrophotometry is an electron-based phenomenon and therefore, the
UV–vis absorption spectrum changes as the pH of the solvent changes.
A bathochromic shift was observed. The observed maximum wave-
lengths on UV–vis absorption spectra are attributed to * transi-

1.5 mL of the selected simulated fluid in a flask. Each suspension was
placed in the incubator at 37 °C and stirred in a horizontal shaker
(Orbital IKA KS 130 basic, Germany) at 170 rpm. The amount of eu-
genol released was measured after filtration of each sample with a
0.2 μm syringe filter (Ref: 514-0070, VWR International, Fontenay-
sous-Bois, France). From each sample, a volume of 500 μL was trans-
ferred to a flask, and 1000 μL of the respective simulated fluid was
added to the flask.
The amount released was evaluated considering the absorbance
read of the sample using the respective simulated fluid as a blank at 279
or 280 nm of wavelength depending on the electrolyte solution. In
tion, typical of unsaturated functional groups (Monteiro et al., 2011).
To the authors best knowledge, this is the first time that is reported
the maximum absorption wavelength of eugenol in simulated gastro-
intestinal fluids. Woranuch and Yoksan (2013) used the UV–vis spec-
trophotometer method for the detection and quantification of eugenol
after its inclusion in nanoparticles however studies regarding the de-
tection and quantification of eugenol in simulated gastrointestinal
fluids were not found in the literature.
Reports regarding the use of UV–vis spectrophotometer methods for
the detection and quantification of eugenol are still restricted. High
performance liquid chromatographic methods (Fischer and Dengler,

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1990; Yun et al., 2010), as well as gas chromatographic methods (;
Mehta et al., 1996), are more commonly employed for the detection
and quantification of this compound. However, due to the advantages
mentioned above of UV–vis spectrophotometry method, this method
was chosen for the detection and quantification of eugenol.
Calibration curves were drawn for eugenol detection in ultra-pure
water and in each gastrointestinal fluid. The linearity range for the
quantification of eugenol in ultra-pure water and in the SSF ranged
between 0.5–20.0 mg/L. In the case of the SGF and the SIF, the cali-
bration curves were constructed in the range of 0.125–20.0 mg/L.
Different linearity ranges were obtained so that it could be verified the
limit of quantification (LOQ) was lower than the lowest concentration
value obtained during the in vitro release studies. During these studies,
the minimum amount released in SSF, SGF and in the SIF, was
2.299 mg/L, 5.163 mg/L and 33.479 mg/L, respectively and the LOQ
values obtained respectively were 1.894 mg/L, 0.557 mg/L, and
0.799 mg/L.
The values obtained for the LOQ and the limit of detection (LOD)
were appropriate regarding the analytical methods validated, and
therefore they are suitable for the quantification of eugenol during the
in vitro release studies in simulated gastrointestinal fluids. The results of
the regression analysis of eugenol quantification by UV–vis spectro-
photometry are presented in Table 4. The regression equations are
presented considering a confidence interval of 95%.
3.2. Optimization process by response surface methodology
The bio-active compound of this study, eugenol, was successfully
entrapped in ethyl cellulose polymer-based matrix. Eugenol-loaded
ethyl cellulose microparticles were obtained by w/o/w double emulsion
solvent evaporation technique. A design of experiments was considered
to optimize characterization parameters of the final powder as the eu-
genol encapsulation efficiency and the product yield. For that, a re-
sponse surface methodology was employed. In a two-level full factorial
central composite design, five coded levels of two independent vari-
ables (factors) were tested resulting in thirteen experiments (in tripli-
cate each) with five replicates at the centre of the design.
A preliminary screening was performed to define the experimental
domain. Regarding the polymer concentration ( X1), concentration le-
vels below 15 g/L provoked an undesired instability of the double
microencapsulated a peptide in low molecular weight and hydrophilic
poly(D,L-lactide-co-glicolide) polymer matrix. Polymer concentrations
above 30 g/L resulted in high viscous polymer solutions which led to
the formation of clusters and microparticles with irregular shapes. This
type of observation was also noticed by Sutaphanit and Chitprasert
(2014) when they used gelatin as wall material for the optimization of
the microencapsulation process of holy basil essential oil. Regarding the
surfactant concentration ( X2 ), is acknowledged that an increase of
surfactant concentration is associated to an increase in the encapsula-
tion efficiency as described by Krishnamachari et al., (2007) in a report
when they were designing a budesonide delivery system to ileum and
colon. According to these authors, the encapsulation efficiency in-
creases linearly to the surfactant concentration. Nevertheless, other
studies report the possibility to formulate microparticles in a surfactant-
free formulation (Carrio et al., 1995; Sivakumar et al., 2009). Even
though some studies are available, a limited number of reports were
found regarding the influence of the surfactant concentration in the
formulation of microparticles by w/o/w double emulsion solvent eva-
poration. Considering this, the range of surfactant concentration was
defined to range between 1% w/w and 2% w/w. All the other for-
mulation parameters and operation settings remained constant during
this study.
Results of the experimental design demonstrate differences on the
eugenol encapsulation efficiency and the product yield, varying from
79.4 ± 0.4% (1.2 ± 0.0° considering a confidence interval of 95%) to
86.5 ± 1.6% (0.9 ± 0.1°; confidence interval of 95%), in the case of
the eugenol product yield and from 67.7 ± 1.8% (1.3 ± 0.1°; con-
fidence interval of 95%), to 96.1 ± 0.4% (1.3 ± 0.0°; confidence
interval of 95%), in the case of the product yield as depicted in
Table 5.
For the second-order equations, the regression coefficients and the P
value for the lack-of-fit are presented in Table 6. Considering a statis-
tical significance at 5% of probability, the obtained equations were
tested regarding adequacy and fitness by the ANOVA. The fitted second-
order models were acceptable as they demonstrate significant regres-
sion, no relevant lack of fit and low residual values.
The mathematical and statistical relationship between the process
variables and the responses are presented in coded variables on Eqs. (6)
and (7).
EEE (%) = 0.042 + 6.990 × 10 3X + 0.571 X2 8.000 × 10 6 X2
1 1

emulsion and consequently the coalescence of the microparticles. 6.740 × 10 2 X2

2 0.171 × 10 3X X
1 2 (6)
Additionally, a large amount of eugenol was not expected to be en-
trapped in the polymer matrix as the encapsulation efficiency is ex- PY (%) = 0.331 6.000 × 10 5X
1 + 0.899 X2 2 × 10 6 X12 0.233 X22
pected to increase with the increase of the polymer concentration
6.4 × 10 4X X
1 2 (7)
(Jyothi et al., 2010; Li et al., 1999; Mehta et al., 1996; Rafati et al.,
1997). Mehta et al. (1996) concluded that an increase of polymer Considering the presented coefficients and P-values (Table 6) and a
concentration from 20.0% to 32.5% resulted in an increase of the en- significance at P < 0.05, can be concluded that eugenol encapsulation
capsulation efficiency from 53.1% to 70.9% when they efficiency is mainly affected by the linear term of the surfactant

Table 4
Results from the regression analysis for eugenol quantification in ultrapure water and in a simulated salivary fluid, simulated gastric fluid and in a simulated
intestinal fluid.
Detection of Wavelength pH of the Regression equationa,b Correlation Relative standard Conc. range Limit of Limit of
EUG in (nm) analysis coefficient error of slope (%) (mg/L) detection (mg/L) quantification (mg/L)

UPW 279 6 Abs = (0.017 ± 0.009) C + 0.995 2.326 0.5–20 0.791 2.636
(0.002 ± 0.010)
SSF 280 7 Abs = (0.016 ± 0.001) C + 0.996 1.932 0.5–20 0.568 1.894
(0.079 ± 0.007)
SGF 279 3 Abs = (0.0160 ± 0.0002) C + 0.999 0.530 0.125-20 0.167 0.557
(-0.0004 ± 0.0019)
SIF 280 7 Abs = (0.0165 ± 0.003) C + 0.999 0.761 0.125-20 0.240 0.799
(0.0007 ± 0.0029)

Conc. – Concentration; EUG – Eugenol; SGF – Simulated Gastric Fluid; SIF – Simulated Intestinal Fluid; SSF – Simulated Salivary Fluid; UPW – Ultra-pure water.
a
Abs is the absorbance measured using a UV–vis method and C is the EUG concentration (mg/L).
b
Calibration curves are based on three absorbance reads.

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F. Paulo, L. Santos Industrial Crops & Products 126 (2018) 287–301

Table 5 OVAT methodology that an increase of polymer concentration from 1%

Results observed for the responses eugenol encapsulation efficiency and pro- w/v to 6% w/v, keeping the surfactant concentration constant at 2.5%
duct yield regarding the studied process variables polymer and surfactant w/v, the encapsulation efficiency of bovine serum albumin increased
concentration. from 6.5 ± 0.4% to 75.2 ± 3.0%. The results obtained in the present
Run no.a Process variables Coded variables Responses study are remarkable since it has been observed that increasing the
polymer concentration did not lead to an increase of encapsulation
X1 X2 X1 X2 Y1 Y2 efficiency while the surfactant concentration remained constant (Fig. 1-
Polymer Surfactant EEE PY
Conc. (g/L) Conc. V (CI)a V (CI)a
B).
(% w/w) The main effects and interaction plots of polymer and surfactant
concentrations for both eugenol encapsulation efficiency and product
1 20.0 1.0 −1 −1 1.1 (0.2) 1.0 (0.0) yield are depicted in Fig. 3 and Fig. 4. According to Fig. 3-A, there is an
2 20.0 2.0 −1 +1 1.2 (0.1) 1.0 (0.0)
increase, a maximum and a decrease in eugenol encapsulation effi-
3 30.0 1.0 +1 −1 1.3 (0.1) 1.0 (0.0)
4 30.0 2.0 +1 +1 1.1 (0.0) 1.0 (0.0) ciency as the polymer concentration increases. Still according to the
5 17.9 1.5 −1.41 0 1.2 (0.0) 1.0 (0.0) study of Rafati et al. (1997), was observed that an increase of surfactant
6 32.7 1.5 +1.41 0 1.2 (0.1) 0.9 (0.1) concentration led to a non-related set of values for the entrapment ef-
7 25.0 0.8 0 −1.41 1.3 (0.1) 0.8 (0.3)
ficiency. No increasing or decreasing trend was observed since the en-
8 25.0 2.2 0 +1.41 1.2 (0.0) 0.9 (0.1)
9 25.0 1.5 0 0 1.3 (0.1) 0.9 (0.0)
capsulation efficiency varied from 78.2 ± 5.2, 95.6 ± 3.0%,
10 25.0 1.5 0 0 1.3 (0.0) 1.0 (0.1) 75.2 ± 3.0 to 85.6 ± 9.0% with surfactant concentration variation of
11 25.0 1.5 0 0 1.3 (0.0) 0.9 (0.1) 10%, 5.0%, 2.5% and 1.0% w/v, respectively. These observations are in
12 25.0 1.5 0 0 1.2 (0.1) 1.2 (0.0) part in agreement with the observed in this study as it was observed an
13 25.0 1.5 0 0 1.2 (0.0) 1.0 (0.1)
increase, a maximum and a decrease in eugenol encapsulation effi-
Conc. – Concentration; EEE – Eugenol Encapsulation Efficiency; PY – Product ciency with an increase of surfactant concentration (Fig. 3-A and B). In
Yield. the studies presented by Krishnamachari et al. (2007), using an oil-in-
a
Data represent the mean value in degrees (V) in a confidence interval, CI at water o/w solvent evaporation technique is reported a linear correla-
95%; n = 3. tion between the surfactant concentration and the encapsulation effi-
ciency. However, the results of present study are not in agreement with
concentration and the interaction term of both polymer concentration the trend reported by these authors. The above-reported comparison
and surfactant concentration. The term that had the most significant should be considered with carefulness as both the active ingredient,
effect on eugenol encapsulation efficiency was the interaction term polymer, and emulsification type were different. Therefore, can be
between the polymer concentration and the surfactant concentration concluded that both the concentration of polymer and the surfactant,
and the linear term of the surfactant concentration. Regarding the re- are inter-dependent for the maximization of eugenol encapsulation ef-
sults obtained for the product yield, the term that exerted the greatest ficiency.
effect on this response was the quadratic term of the surfactant con- When considering the obtained results for the product yield (Fig. 4-
centration. Even though the constant terms of the response surface A and B), the highest values were obtained with the lowest values for
analysis performed for the eugenol encapsulation efficiency and the the polymer concentration and intermediate values of surfactant con-
product yield presented the highest values, they are not relevant in this centration. For a suited scale-up process, it is essential to study the
analysis as they only indicate that the starting-points are different from product yield. To the best knowledge of the authors, a limited number
zero and not really affect the responses (Zar, 2010). of reports are available regarding the study of this characterization
The response surface plots and their corresponding contour plots are parameter. Nevertheless, Li et al. (2015) recognized the importance of
presented in Fig. 1 and Fig. 2. The polymer and the surfactant con- the optimization of this parameter when they microencapsulated fish
centrations affected eugenol encapsulation efficiency as well as the oil using gum arabic, casein and β-cyclodextrin mixtures by spray-
product yield. The highest value for eugenol encapsulation efficiency drying. In an orthogonal design, they maximized the product yield to
appeared with the highest polymer concentration and the lowest sur- 55.6%, through the optimization of the selected process parameters. To
factant concentration (Fig. 1-A and B). To the authors best knowledge, the authors best knowledge, there are no reports available that de-
reports on microencapsulation of compounds from essential oils as monstrate the dependence of the product yield with both the polymer
eugenol by w/o/w double emulsion solvent evaporation technique are and the surfactant concentrations.
not available in the literature. Rafati et al. (1997) observed using a Results presented in Figs. 4-A and B are significative and

Table 6
Significant coefficients of second-order coded variables.
Term type Process variables Y1 – EEE Y2 PY

Coef. Significant level Coef. Significant level

(P value) (P value)

Constant 0 1.252a 0.000 1.000a 0.000

Linear 1 0.021b 0.272 −0.015b 0.651
2 −0.041a 0.039 0.029b 0.386
Quadratic 11 −0.041b 0.147 0.008b 0.873
22 −0.034b 0.234 −0.116a 0.026
Interaction 12 −0.086a 0.026 −0.032b 0.633
Lack of fit (P-value) – 0.052b – 0.110b –

X1 – Polymer Concentration; X2 - Surfactant Concentration.

Coef. – Coefficient; EEE – Eugenol Encapsulation Efficiency; PY – Product Yield.
a
Significant at 5% of probability.
b
non-significant considering P > 0.05.

292
F. Paulo, L. Santos
Fig. 1. Response surface (A) and contour (B) plots for the eugenol encapsulation efficiency (EEE – Eugenol Encapsulation Efficiency).
demonstrate that the product yield is dependent on both the polymer
and the surfactant concentrations. There was observed a decrease fol-
lowed by a minimum and a minor increase of the product yield with the
increase of the polymer concentration. Moreover, was observed an in-
crease, followed by a maximum and a decrease in the product yield
with the increase of the surfactant concentration. The effect of the in-
teraction of the polymer concentration and the surfactant concentration
in the product yield was not shown to be linear (Fig. 4-B).
The response was optimized using the Response Optimizer of
MINITAB 18, using a maximizing desirability function for both eugenol
encapsulation efficiency and the product yield, considering equal
weight and importance of these characterization parameters. The
variables polymer and surfactant concentrations were considered
without constraints and a two-sided confidential level of 95%. The
optimized obtained conditions corresponded to a polymer concentra-
tion of 32.1 g/L and a surfactant concentration of 1.1% w/w. The op-
timized conditions corresponded to the highest value defined for the
polymer concentration in the experimental design and to a relatively
low value of surfactant concentration. These optimized conditions are
predicted to provide satisfactory values for both the eugenol en-
capsulation efficiency and the product yield.
The fitted value of eugenol encapsulation efficiency was 96.4%.
Considering a 95% confidential interval is predicted that mean of assays
should be in the range of 96.8% and 99.1%. Considering a 95% pre-
diction interval is expected that a single assay should lay down in the
prediction interval ranging between 91.2% and 101.7%.
The fitted value for the product yield was 81.2% with a confidential
interval ranging between 72.3% and 90.1% and a prediction interval of
63.5% and 98.9%. In both cases was observed that the prediction in-
tervals were wider than the confidential intervals as the prediction in-
terval includes the uncertain involved in the prediction of a single
assay.
Fig. 2. Response surface (A) and contour (B) plots for the product yield (PY – Product Yield).
293
Industrial Crops & Products 126 (2018) 287–301
3.3. Physical and chemical characterization of eugenol-loaded ethyl
cellulose microparticles obtained under optimized formulation conditions
3.3.1. Evaluation of eugenol encapsulation efficiency and product yield
In this study, the optimized conditions obtained using a design of
experiments were tested to maximize both the eugenol encapsulation
efficiency and the product yield of eugenol-loaded ethyl cellulose mi-
croparticles using the w/o/w double emulsion solvent evaporation
technique. Considering the optimized conditions, three formulations
(OF-1, OF-2, and OF-3) were prepared under similar conditions as de-
scribed in the 2.2.2 section, considering a polymer concentration of
32.1 g/L and a surfactant concentration of 1.1% w/w. Eugenol en-
capsulation efficiency ranged from 93.6% to 95.5% (Fig. 5-A), and the
mean eugenol encapsulation efficiency was 94.7 ± 1.0%. The obtained
mean eugenol encapsulation efficiency is within the confidential in-
terval of 95% for the optimized conditions (confidential interval ranged
from 93.8% to 99.1%). Also, in accordance, all the obtained values of
eugenol encapsulation efficiency of each independent assay were
within the prediction interval of 95% for the optimized conditions
(prediction interval ranged from 91.9% to 101.7%).
The product yield varied from 69.1% to 89.0% (Fig. 5-B) corre-
sponding to a mean product yield of 82.0 ± 11.2%. The obtained mean
product yield and each obtained result of the independent assay were
within the confidence interval (the confidential interval ranged from
72.3% to 90.1%) and the prediction interval (the prediction interval
ranged from 63.5% to 98.9%), respectively.
The results of this study are fairly significant as the obtained results
of the mean eugenol encapsulation efficiency and product yield are
within the confidential interval, and the results of these parameters are
also within the prediction interval for this experimental design.
Sajomsang et al. (2012) studied the inclusion complex between water-
soluble β-cyclodextrin grafted with chitosan derivates and eugenol.
They found out that eugenol encapsulation efficiency ranged between
F. Paulo, L. Santos Industrial Crops & Products 126 (2018) 287–301

Fig. 3. Main effects (A) and interaction (B) plots for eugenol encapsulation efficiency (EEE – Eugenol Encapsulation Efficiency).

Fig. 4. Main effects (A) and interaction (B) plots for product yield (PY – Product Yield).

59.3 ± 2.4% to 77.5 ± 7.4%. Phunpee et al. (2016) studied the en-
capsulation of eugenol in water-soluble quaternized β-cyclodextrin
grafted with chitosan. They observed a maximum encapsulation effi-
ciency below 80%. Hill et al. (2013) characterized microencapsulated
compounds of essential oils as eugenol β-cyclodextrin inclusion com-
plexes for antimicrobial delivery systems. They found out that the en-
capsulation efficiency was 90.15 ± 0.06%. Shinde and Nagarsenker
(2011) microencapsulated eugenol by gelatin-sodium alginate complex
coacervation and obtained a maximum encapsulation efficiency of
15.99 ± 0.55%. Comparing to the previous studies, the encapsulation
efficiency was remarkably improved in this study.
To the best authors knowledge, there are no available reports re-
garding the study of the product yield using this bio-active ingredient.
The applied response surface methodology is shown to be an ef-
fective approach to obtain eugenol-loaded ethyl cellulose micro-
particles with the maximization of both eugenol encapsulation effi-
ciency and product yield.
3.3.2. Microparticles wettability
In this study, the mean wettability value of eugenol-loaded ethyl
cellulose microparticles obtained under optimized formulation condi-
tions was 1652 ± 44 s. The time required for the powders become
entirely wet ranged between 1610s and 1698s.
According to Gaiani et al. (2007), the ability of powders to rehy-
drate in water is characterized regarding wettability and sinkability.
The wettability is defined as the capability of water or other liquid to
penetrate into the microparticles pore system. On the other hand, the
sinkability is related to the ability of microparticles to sink below the
liquid surface (Fäldt and Bergenståhl, 1996). These physicochemical
properties are related to the reconstitution of powders (Bae and Lee,
2008) and affect the molecular interaction between the solid and the
liquid (Cuq et al., 2011) and therefore are usually used inter-
changeably. The wettability of powders depends on several parameters
as the hydrophobicity and hydrophilicity of substances present in the
surface of the powder, the particle size, the density and the porosity of
the powder (de Barros Fernandes et al., 2014; Kim et al., 2002).
To the authors best knowledge this is the first time that the

Fig. 5. Observed results of eugenol encapsulation efficiency (A) and product yield (B) using the optimized conditions.

294
F. Paulo, L. Santos
wettability of eugenol-loaded microparticles is studied. Therefore,
comparative studies were not found in the literature.
3.3.3. Differential scanning calorimetry (DSC) analysis
The DSC thermograms of pure eugenol, ethyl cellulose, polymer-
only microparticles and the powder obtained under optimized condi-
tions are depicted in Fig. 6.
The DSC thermograms allowed to confirm the inclusion of eugenol
in ethyl cellulose polymer-based matrix. Eugenol is liquid at room
temperature, therefore, the comparison of the thermal stability of pure
eugenol and the encapsulated pattern allowed to assess if the inclusion
of eugenol in ethyl cellulose polymer-based matrix occurred. The use of
this thermoanalytical technique for the detection of inclusion of
bioactive ingredients in microsystems is widely described in the lit-
erature (Ficarra et al., 2002; Gomes et al., 2011; Hill et al., 2013;
Karathanos et al., 2007).
The DSC thermogram exhibit a sharp endothermic peak at 254.0 °C
corresponding to its boiling point. The absence of this sharp en-
dothermic peak in the DSC thermogram of the powder obtained under
optimized conditions (OF MPs) confirms that eugenol was efficiently
embedded in ethyl cellulose polymeric matrices. In contrast to the re-
sults obtained by Piletti et al. (2017), a significant phase transition was
not observed at 44.3 °C. The authors described that at a temperature of
44.3 °C, eugenol experienced a significant phase transition due to eu-
genol volatilization. However, the DSC thermogram for pure eugenol
obtained in this study agreed with the presented DSC thermograms of
eugenol by other authors (Chaar et al., 2004; Monteiro et al., 2011;
Nuchuchua et al., 2009; Pramod et al., 2015; Santos et al., 2009). Ac-
cording to the results presented by these authors, eugenol presents only
one endothermic peak around 250 °C, attributed to its volatilization.
The DSC thermograms of polymer-only microparticles, eugenol-
loaded ethyl cellulose microparticles and ethyl cellulose exhibit a broad
endothermic peak around the temperature of 60 °C as shown in Fig. 7
(denoted as thermal event A). Many authors describe the presence of
these endotherms as the dehydration temperature (TDH ) related to the
evaporation and volatilization of the volatile portion of molecules or
bounded water (Luo et al., 2012; Müller et al., 2011; Neo et al., 2013).
The presence of these endotherms in ethyl cellulose and polymer-only
microparticles were also noted and described by Dubernet et al. (1990),
when they were performing a thermal analysis of ethyl cellulose and
ethyl cellulose microspheres prepared by solvent evaporation method.
The DSC thermogram of ethyl cellulose presents a subtle baseline
change starting around the temperature of 130 °C (Fig. 7, thermal event
B). This baseline change, attributed to the transition temperature (Tg ) of
ethyl cellulose, was also observed by Lai et al. (2010) when they were
295
Industrial Crops & Products 126 (2018) 287–301
Fig. 6. Differential Scanning Calorimetry
thermograms of different samples: eugenol
( ), ethyl cellulose ( ), polymer-only
microparticles ( ) and eugenol-loaded
ethyl cellulose microparticles ( ) (range
temperature from 30 °C to 270 °C).
studying the thermal properties of ethyl cellulose. Even though, the Tg
value of ethyl cellulose depends on the measurement method and
conditions used as well as the type of ethyl cellulose (e.g. the, ethylation
degree), its Tg is expected to be found around 130 °C and 150 °C
(Sakellariou et al., 1985). The results of this study revealed that ethyl
cellulose is a strong glass former and presents a limited heat capacity
change. The baseline change associated to the Tg of ethyl cellulose got
more predominant when ethyl cellulose was aggregated into micro-
particles. Therefore, polymer-only microparticles favored rheological
and heat capacity changes during the glass transition.
The mid-point of the of Tg of ethyl cellulose, polymer-only micro-
particles and eugenol-loaded microparticles was 127.9 °C, 153.6 °C and
143.9 °C, respectively. It was observed a shift of the Tg values higher
temperatures when eugenol was encapsulated into ethyl cellulose
polymer-based microsystem. The thermal profile of the eugenol-loaded
ethyl cellulose microparticles obtained under optimal conditions did
not shown the volatilization peak of eugenol (Fig. 6) suggesting that
eugenol was efficiently incorporated in ethyl cellulose polymeric ma-
trices. The incorporation of eugenol in the ethyl cellulose polymer-
based microsystem resulted in the formation of solid solution due to
anti-plasticizing effects. Additionally, the absence of the new peaks in
the DSC thermogram of eugenol-loaded ethyl cellulose microparticles
(Fig. 7) indicated that no chemical interactions occurred between the
wall material, ethyl cellulose and the encapsulated bio-active in-
gredient, eugenol.
The temperature and the peak shape of the first endothermic high-
temperature event (Fig. 7; thermal event C) is similar for both eugenol-
loaded ethyl cellulose microparticles and ethyl cellulose suggesting
again that eugenol was efficiently protected by ethyl cellulose polymer
matrix. According to Sakellariou et al. (1985), this endotherm may be
related to the melting of microcrystals present in ethyl cellulose poly-
meric matrix. The second-high temperature event (thermal event D) is
quite predominant the DSC thermogram of polymer-only microparticles
and is reported to be related to the degradation of ethyl cellulose
(Dubernet et al., 1990; Lai et al., 2010). This exotherm was also ob-
served in eugenol-loaded ethyl cellulose microparticles, even though is
not presented in the form of a sharp exothermic peak.
3.3.4. Thermogravimetric analysis (TGA)
The TGA thermograms and the derivative thermogravimetry (DTG)
thermograms from 30.0 °C to 810.0 °C of eugenol (purity ≥ 99%), ethyl
cellulose, polymer-only microparticles and eugenol-loaded ethyl cellu-
lose microparticles are presented in Fig. 8 and 9, respectively. The TGA
thermograms allow assessing the material weight change as a function
of temperature and DTG thermograms display the rate of weight loss as
F. Paulo, L. Santos
described by Yoksan et al. (2010). Still, according to these authors,
peaks in the DTG thermograms corresponds to the highest rate of
weight loss known as degradation temperature (Td ) of the components
present in the material analyzed.
According to Fig. 8, the weight of eugenol, ethyl cellulose, polymer-
only microparticles and eugenol-loaded ethyl cellulose microparticles
decreases as the temperature increase from 30 °C to 810 °C. The degree
of weight loss was similar for ethyl cellulose, polymer-only micro-
particles and the eugenol-loaded ethyl cellulose microparticles in the
temperature range of the study. All the samples analyzed exhibited only
one level of weight loss as it was only observed one peak in each DTG
sample thermogram (Fig. 9).
The weight loss change of eugenol (purity ≥ 99%) occurred in the
range of 92.9 °C–232.8 °C, whereas the weight loss change of ethyl cellu-
lose, polymer-only microparticles and eugenol-loaded ethyl cellulose mi-
croparticles occurred in the range of 265.4–411.9 °C, 270.8–427.1 °C,
286.2–403.3 °C, respectively (Fig. 9). The DTG thermograms exhibit Td
values at 214.1 °C, 352.4 °C, 356.2 °C and 365.3 °C, corresponding to the
degradation temperature of ethyl cellulose, polymer-only microparticles
and eugenol-loaded ethyl cellulose microparticles, respectively (Fig. 9).
Eugenol-loaded ethyl cellulose microparticles obtained under optimized
formulation conditions were relatively stable in a temperature range of
30.0 °C–286.2 °C (weight loss of 1.1%). Piletti et al. (2017) found out that
eugenol molecules entrapped in β-the cyclodextrin structure were de-
graded at 280 °C. In the present study, the Td value of eugenol-loaded ethyl
cellulose microparticles was found to be 365.3 °C. Therefore, it can be
concluded that the thermogravimetric stability was significantly improved
in this study.
3.3.5. Fourier transform infrared spectroscopy (FTIR) analysis
The Fourier transform infrared spectroscopy (FTIR) is a useful tool to
evaluate if the integration of eugenol in ethyl cellulose microparticles
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Industrial Crops & Products 126 (2018) 287–301
Fig. 7. Differential Scanning Calorimetry ther-
mograms of different samples: ethyl cellulose
( ), polymer-only microparticles ( )
and eugenol-loaded ethyl cellulose micro-
particles ( ) in a temperature range of
30 °C–270 °C. Letters A, B, C, and D denote the
thermal events under study: dehydration tem-
perature, glass transition, first endothermic
and the second endothermic high-temperature
events, respectively.
occurred, to verify the presence of specific functional groups in eugenol,
ethyl cellulose and eugenol-loaded ethyl cellulose microparticles obtained
under optimized conditions. Moreover, the analysis of FTIR spectra allows
evaluating if the incorporation of eugenol affected the chemical integrity
of the coating material, ethyl cellulose as described by Piletti et al. (2017).
The wave numbers (cm−1) and assignments for the bands observed in the
FTIR spectra of eugenol (EUG), ethyl cellulose (EC) and microparticles
obtained under optimized conditions (OF MPs) are presented in Table 7.
The FTIR spectra of EUG, EC and OF MPs are presented in the supple-
mentary information. Signature sharp peaks of eugenol were not observed
in the eugenol-loaded ethyl cellulose microparticles (OF MPs) FTIR spec-
trum. The aromatic C–H stretching vibration at 3076 cm−1, the aromatic
C–H out-of-plane bending vibration at 793 cm−1 and the C–H out-of-plane
bending vibration of tri-substituted benzenoid compounds at 1,2 and 4
positions at the wave numbers of 850 cm−1 and 797 cm−1 of eugenol
were not observed in the FTIR spectrum of eugenol-loaded ethyl cellulose
microparticles obtained under optimized formulation conditions. More-
over, the characteristic peaks of eugenol as follow: the C–H stretching
vibration of alkenes at 3058 cm−1, the C–H out-of-plane stretching vi-
bration of alkenes at the wave numbers of 995 cm−1 and 912 cm−1, the
C]C stretching vibration of alkenes at 1616 cm−1 and the C]C aromatic
stretching vibration at the wave numbers of 1607 cm−1 and 1510 cm−1
were not observed in the FTIR spectrum of eugenol-loaded ethyl cellulose
microparticles obtained under optimized formulation conditions. The
signature sharp peaks of ethyl cellulose (EC) were observed in eugenol-
loaded ethyl cellulose microparticles (OF MPs) FTIR spectrum whereas the
characteristic bands of eugenol were not observed in the OF MPs FTIR
spectrum.
Significant band shiftings were not observed in the FTIR spectrum of
eugenol-loaded ethyl cellulose microparticles when compared to the
FTIR spectrum of ethyl cellulose. These observations indicate that no
strong interactions between eugenol and the OeH and CeOeC groups
Fig. 8. Thermogravimetric analysis thermo-
grams of different samples: eugenol ( ),
ethyl cellulose ( ), polymer-only micro-
particles ( ) and eugenol-loaded ethyl
cellulose microparticles ( ).
F. Paulo, L. Santos Industrial Crops & Products 126 (2018) 287–301

Fig. 9. Derivative thermogravimetry thermo-

grams of different samples: eugenol ( ),
ethyl cellulose ( ), polymer-only micro-
particles ( ) and eugenol-loaded ethyl
cellulose microparticles ( ).

Table 7 3.3.6. Evaluation of the morphology

Wavenumbers (cm−1) and assignments for the bands observed in the FTIR The SEM microphotographs (Fig. 10) display the main morpholo-
spectra of eugenol (EUG), ethyl cellulose (EC) and microparticles obtained gical characteristics of eugenol-loaded ethyl cellulose microparticles
under optimized conditions (OF MPs). obtained under optimized conditions (polymer and surfactant con-
centration of 32.1 g/L and 1.1% w/w, respectively). The micro-
Bond type Chemical Vibration Wavenumber (cm−1)
functional group mode photographs demonstrate the presence of agglomerates (Fig. 10-A).
EUG EC OF MPs Additionally, clusters of bigger with smaller microparticles are widely
observed (Fig. 10-B).
OeH – Stretching 3508 3479 3479
Generically, microparticles showed to be spherical, presenting
3425
CeH Alkane Stretching 2971 2974 2976 smooth but porous surfaces (Fig. 10-C and D). The Fig. 10-C shows the
2935 2908 2910 presence of wrinkles and cracks in bigger microparticles. Similar con-
2902 2877 2885 clusions regarding the sphericity and the smoothness were observed by
2842 2870 2874 Hill et al. (2013) when they were using a transmission electron mi-
Alkene 3058 – –
Aromatic 3076 – –
croscope to evaluate particle morphology of β-cyclodextrin inclusion
-CH3 Bending 1464 1444 1442 complexes containing compounds of essential oils including eugenol by
1365 1373 1377 freeze-drying. However, rough and overlapped layers were observed by
-CH2 1431 Overlapped Choi et al. (2009) when they produced inclusion complexes of β-cy-
with -CH3
clodextrin and eugenol by molecular inclusion followed by freeze-
bending
Alkene Bending out- 995 – – drying. Additionally, these authors described the appearance of some
of-plane 912 smoother and rounder surfaces when they encapsulated eugenol into
Tri-Substituted 793 – – polycaprolactone polymer-based matrix by emulsion-diffusion method
Benzenoid at 850 followed by freeze-drying. Similar conclusions to those obtained by
1,2,4 positions 797
C]C Alkene Stretching 1616 – –
Choi et al. (2009) were drawn by Piletti et al. (2017). The SEM analyses
Aromatic 1607 – – of this study provide the evidence that the inclusion of eugenol in ethyl-
1510 cellulose-based microparticles by double emulsion solvent evaporation
CeO – Stretching 1265 1049 1054 technique significantly improved the morphological characteristics of
the obtained powder.

of ethyl cellulose occurred. Similar conclusions were drawn by Wang
et al. (2011) when they were analyzing the FTIR spectra of eugenol and
β-cyclodextrin complexes loaded with eugenol. According to these au-
thors, the non-significant shiftings of eugenol β-cyclodextrin complexes
compared with the FTIR bands of β-cyclodextrin complexes reveal that
no strong interaction occurred between eugenol and the OeH, CeC and
CeOeC groups of β-cyclodextrin complexes. Still, according to Wang
et al. (2011), a shift of OeH stretching vibration band of the polymer to
lower wavenumbers should be observed when eugenol is encapsulated.
This phenomenon is related to a change of hydration bonds structure
due to the reorganization of intramolecular hydrogen bonds between
the OeH groups and the polymer when eugenol is encapsulated. In this
study, was not observed a change in the wave number of OeH
stretching band from ethyl cellulose to eugenol-loaded ethyl cellulose
microparticles. Therefore, it can be concluded that significant change of
hydration bonds structure at the surface of ethyl cellulose polymer-
based system when eugenol was encapsulated did not occur.
The non-observation of significant chemical modifications on ethyl
cellulose when eugenol was loaded, confirm the incorporation of eu-
genol in ethyl cellulose polymer matrix. Similar conclusions regarding
the efficient incorporation of eugenol in β-cyclodextrin complexes
thought the study of the FTIR spectra were drawn by Piletti et al. (2017)
and Wang et al. (2011).
3.3.7. Evaluation of the particle size distribution
The mean differential volume versus particle size curve is presented
in Fig. 11 for the powders obtained using the optimized conditions
(polymer concentration of 32. 1 g/L and a surfactant concentration of
1.1% w/w). The particle size ranged from 10.8 μm to 12.6 μm, corre-
sponding to a mean particle size of 11.4 ± 1.1 μm. The SPAN values
ranged from 1.6 to 1.7, corresponding to a mean SPAN value of
1.6 ± 0.0.
The mean average diameters of the three triplicates of the optimized
powder were well-nigh similar. The variability in the SPAN value may
be attributed to the presence of some agglomerates with a mean particle
size around 40 μm and 60 μm.
Choi et al. (2009) presented a research focused on the micro-
encapsulation of eugenol in β-cyclodextrin inclusion complexes and in
polycaprolactone polymer-based particles. In this research article, the
authors reported a mean particle size ranging from 312.0 ± 5.3 nm to
321.3 ± 1.5 nm. Similar results were obtained by Hill et al., (2013) as
they obtained a mean particle size of 0.860 ± 0.007 μm. In the present
study, the mean particle size of eugenol-loaded ethyl cellulose micro-
particles is larger than the studies presented by Hill et al. (2013) and
Choi et al. (2009). Nevertheless, the results presented in this study for
the particle size distribution are reasonably satisfactory considering the
low complexity associated with this process and also considering that

297
F. Paulo, L. Santos
Fig. 10. Microphotographs of eugenol-loaded ethyl cellulose microparticles obtained under optimized conditions (amplification of 1 000 times is presented in (A), 2
000 times is presented in (B), 3 000 times is presented in (C) and 5 000 times is presented in (D); beam intensity of 10.00 kV).
Fig. 11. Mean particle size distribution of the microparticles obtained under
optimized formulation conditions.
was not proceed particle size sieving during the manufacturing process.
The mean particle size of the microencapsulation of eugenol in ethyl
cellulose polymer-based matrix obtained in this study was similar (in
the micrometer range) to the obtained values of the mean particle size
of eugenol immobilized on mesoporous silica microparticles (mean
particle size of 4.37 ± 0.12 μm) as presented by Ribes et al. (2017).
3.3.8. In vitro Release studies
The development of delivery systems to increase oral bioavailability
298
Industrial Crops & Products 126 (2018) 287–301
and retain the main biological properties of bioactive ingredient have
been widely investigated (Neves et al., 2016; Sanna et al., 2015; Sonaje
et al., 2007; Takahashi et al., 2009)
The in vitro release profiles of eugenol from eugenol-loaded ethyl
cellulose microparticles obtained under optimized conditions (polymer
concentration of 32. 1 g/L and a surfactant concentration of 1.1% w/w)
are presented in Fig. 12. The release studies were performed in tripli-
cate. The release profile of eugenol in the SSF during the 2 min of study
(Fig. 12-A) reveal that approximately 3% of the total amount of eugenol
entrapped was released from eugenol-loaded ethyl cellulose micro-
particles. Therefore, due to the slow release of eugenol in the SSF, can
be concluded that eugenol is favorably protected in the SSF, as in-
tended.
When the release of eugenol was simulated in the SGF (Fig. 12-B),
the total amount release was 10.0 ± 0.9% after 2 h of study. The
gastrointestinal environment (acidic pH) probably promoted the de-
gradation of the polymer shell though acidic hydrolysis of ethyl cellu-
lose. However, the amount released was low, indicating that eugenol
can be efficiently protected in the gastric environment. The metabolism
of eugenol occurs mostly in hepatocyte cells, the main tissue of the liver
(Thompson et al., 1991). Therefore, this study remarkably presents a
possibility to protect the active compound from oral and gastric de-
gradation. Therefore, the active compound can be delivered to hepa-
tocytes without significant transformation.
Regarding the release of eugenol from ethyl cellulose polymer-based
microparticles, the total amount released was 44.4 ± 4.4% after 2 h of
F. Paulo, L. Santos
Fig. 12. Release profiles of eugenol from the powder produced under optimized conditions during the release studies.
((A) – SSF; (B) – SGF; (C)-SIF).
the study. This result is remarkably relevant as for example, Fischer
et al., (1990) concluded that the presence of unchanged eugenol in
serum and bile after oral ingestion in a human trial was below of 10 ng/
mL. In the present study, the total amount released after 2 h in the SIF
corresponds to 47 037 ng/mL and therefore is 4 704 times higher than
the value reported by Fischer et al. (1990). Kim et al., (2003) presented
in their research paper that eugenol presents inhibitory effects on
prostaglandin E2 production in lipopolysaccharide-activated RAW264.7
cells corresponding to 98.3% of inhibition at a concentration level of
10 μg/mL. Prostaglandins are involved in multiple pathophysiological
processes like inflammation and carcinogenesis. In this study, the total
amount released (44.4 ± 4.4%) corresponds to a concentration level of
47.0 μg/mL, this is, 4.7 times higher than the minimum concentration
level for the inhibition of prostaglandin E2 production.
4. Conclusions
This study aimed to microencapsulate eugenol into ethyl cellulose
polymer-based matrix by double emulsion solvent evaporation tech-
nique and optimize encapsulation conditions providing high eugenol
encapsulation efficiency and product yield. The polymer concentration
of 32.1 g/L and the surfactant concentration of 1.1% w/w were ob-
tained as optimal encapsulation conditions using a response surface
methodology. Microparticles obtained for the selected optimal condi-
tions presented high eugenol encapsulation efficiency and product yield
(94.7 ± 1.0% and 82.0 ± 11.2%, respectively). Additionally, this
study provides the evidence that the inclusion of eugenol in ethyl-cel-
lulose-based microparticles by double emulsion solvent evaporation
significantly improve the morphological characteristics of eugenol en-
trapped in a polymer-based system. A release stimulation of eugenol
from eugenol-loaded ethyl cellulose microparticles obtained under op-
timized conditions was performed in simulated gastrointestinal fluids,
and the assessment results revealed that eugenol was favorably pro-
tected in the simulated salivary and gastric fluids. The highest amount
released of eugenol was observed in the simulated intestinal fluid and
was much higher than the minimum amount of eugenol that demon-
strates positive biological effects.
Declaration of interest
Authors declare no conflict of interest or any financial benefit from
direct or indirect applications of the research.
Acknowledgments
This work was financially supported by the projects POCI-01-0145-
FEDER-006939 (Laboratory for Process Engineering, Environment,
Biotechnology, and Energy – UID/EQU/00511/2013) funded by the
European Regional Development Fund (ERDF), through COMPETE2020
- Programa Operacional Competitividade e Internacionalização (POCI)
and by national funds, through FCT - Fundação para a Ciência e a
Tecnologia and NORTE‐01‐0145‐FEDER‐000005 – LEPABE-2-ECO-
INNOVATION, supported by North Portugal Regional Operational
Programme (NORTE 2020), under the Portugal 2020 Partnership
299
Industrial Crops & Products 126 (2018) 287–301
Agreement, through the European Regional Development Fund (ERDF).
This work was developed under the doctoral program in Chemical
and Biological Engineering (PDEQB) NORTE-08-5369-FSE-000028, co-
financed by the Northern Regional Operational Program (NORTE 2020)
through Portugal 2020 and the European Social Fund (ESF).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2018.10.027.
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