Food Research International xxx (xxxx) xxx–xxx

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Ethyl oleate food-grade O/W emulsions loaded with apigenin: Insights to

their formulation characteristics and physico-chemical stability
⁎
Imen Abchaa,b, Safa Souilema,c, Marcos A. Nevesa, , Zheng Wanga, Mohamed Nefattib,
Hiroko Isodaa, Mitsutoshi Nakajimaa
a
Alliance for Research on North Africa (ARENA), University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan
b
Pastoral Ecology Laboratory, Institute of Arid Land (IRA), Medenine 4119, Tunisia
c
Laboratory of Environmental Bioprocess, Center of Biotechnology of Sfax (CBS), B.P. 1177, Sfax 3018, Tunisia

A R T I C LE I N FO A B S T R A C T

Keywords: Apigenin has attracted a great interest in the food industry due to the wide range of its biological activities
Apigenin including antioxidant and anti-inflammatory. The encapsulation of apigenin in oil-in-water (O/W) emulsions
O/W emulsion could overcome its low solubility and lead to the development of new functional food products. The aim of this
Ethyl oleate study is to formulate food-grade O/W submicron emulsions loaded with apigenin using high-pressure homo-
Heating pretreatment
genization. Supersaturated solutions of 0.1 wt% apigenin in ethyl oleate were heated at 100 °C for 30, 60, or
High-pressure homogenization
120 min and the supernant after centrifugation were used as to-be-dispersed phases. An aqueous solution con-
Retention
taining 1 wt% tween 20 was used as the continuous phase. We examined the effect of heating process of the ethyl
oleate prior to emulsification and the homogenization-pressure (60–150 MPa) on the physico-chemical char-
acteristics of the O/W emulsions immediately after formulation and during storage. Submicron O/W emulsions
were formulated and the lowest average droplet diameter (dav) was 169 ± 2.082 nm with a polydispersity index
(PDI) of 0.06 ± 0.002. After 30 days of storage at 4 °C, the O/W emulsion formulated remained physically stable
with little change in their dav and PDI values. The preheat treatment of ethyl oleate, affected the initial loaded
apigenin concentration but hardly affected the physico-chemical stability of O/W emulsions. However, HPLC
analysis demonstrated that the emulsification pressure was a relevant parameter affecting apigenin retention
during the storage of O/W emulsions. Apigenin degradation in ethyl oleate O/W emulsions followed zero order
kinetics and about 91.5–93.5% of apigenin could be retained in O/W emulsions after 30 days of storage.

1. Introduction such as parsley (Petroselinum crispum) (Kaiser, Carle, & Kammerer

The functional food development and increasing consumer aware-
ness of the link between food and health has lead to growing demand
for foods that achieve optimal health status by promoting the state of
well-being and possibly reducing the risk of disease (Siro, Kapoina,
Kapoina, & Lugasi 2008). Among the growing functional foods cate-
gories, are foods that are fortified with flavonoids. Flavonoids are sec-
ondary metabolites of plants, present mainly in epiderms of leaves and
in the skin of fruits and confer protection to plant at various levels i.e.
UV protection, pigmentation, nitrogen fixing and diseases resistance
(Pierpoint 2000).
Apigenin (AP), 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzo-
pyran- 4-one (Fig. 1) is a common bioactive belonging to a less toxic
and non-mutagenic flavone subclass of flavonoids (Ding, Chen, Wang,
Zhai, & Zhai 2013). It can be found in several plants and vegetables,
2013), celery (Apium graveolens L.) (Yan, Yu, Xu, Gu, & Zhu 2014) Yan
et al. 2014), olive (Olea europaea) and derivatives (Aliakbarian,
Casazza, & Perego 2011), pigeonpeas (Cajanus cajan) or chamomile (Fu
et al. 2008), while plant-derived beverages containing apigenin include
tea and wine (Janssen et al. 1998). Advanced scientific research re-
vealed that apigenin exhibits a protective effect against various dis-
eases. Recent studies have demonstrated that apigenin could play an
important role in the treatment of skin inflammation (Begum & Prasad
2012; Duarte, Quirino, Patrocínio, & Anbinder 2011; Rithidech,
Tungjai, Reungpatthanaphong, Honikel, & Simon 2012), diabetes (Choi
et al. 2014), Alzheimer (Choi et al. 2014), platelets aggregation, cor-
onary heart disease, endothelial function (Guerrero et al. 2005;
Olszanecki, Gebska, Kozlovski, & Gryglewski 2002; Woodman & Chan
2004; Zhang et al. 2000) and cancer. Apigenin is considered. Apigenin
is considered very safe and even at high doses no toxicity features were

⁎
Corresponding author at: Alliance for Research on North Africa (ARENA), University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan.
E-mail addresses: safasouilem1@gmail.com (S. Souilem), marcos.neves.ga@u.tsukuba.ac.jp (M.A. Neves).

https://doi.org/10.1016/j.foodres.2018.09.032
Received 10 March 2018; Received in revised form 28 June 2018; Accepted 9 September 2018
0963-9969/ © 2018 Published by Elsevier Ltd.

Please cite this article as: Abcha, I., Food Research International, https://doi.org/10.1016/j.foodres.2018.09.032
I. Abcha et al.
Fig. 1. Structure of apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1- ben-
zopyran-4- one).
observed. However, apigenin may cause muscle relaxation and sedation
at high doses (Venigalla, Gyengesi, & Münch 2015).The chemopre-
ventive effect of apigenin was explored in several studies, which tested
different doses, possible administration routes, and its treatment fre-
quencies. The oral administration of apigenin (20 and 50 μg/mice) for
20 weeks reduced tumor volumes and induced complete abolishment of
distant organ metastasis in the transgenic adenocarcinoma of a mouse
prostate (TRAMP) mode (Sung, Chung, & Kim 2016). The anticancer
activity of apigenin was correlated to the inhibition of both, nuclear
transcription factor NFkB, inducible cyclooxygenase (COX-2) and to the
suppression of Id1 (DNA binding protein1) through activating tran-
scription factor 3 (ATF3) (Li, Hu, Wang, & Fang 2009; Srivastava,
Pandey, & Gupta 2009). Recently, the pharmacological effects of api-
genin as a dietary supplement are under evaluation in a phase 2 clinical
study by Technische Universität Dresden. The 20 mg apigenin was
served as a daily nutritional supplement to patients with resected col-
orectal carcinomas to evaluate the prevention of the recurrence of
neoplasia (Yan, Qi, Li, Zhan, & Shao 2017).
Despite the promising nutraceutical and health benefits of apigenin,
its low solubility in both polar and nonpolar solvents and its low
bioavailability in the gastrointestinal tract limit its therapeutic value. In
fact, apigenin was recently classified as a BCS (Biopharmaceutical
Classification System) class II compound which indicates its low solu-
bility. Its solubility in water is only 2.16 μg/mL Zhang, Liu, Huang, Gao,
& Qian 2012) and 0.001–1.63 mg/mL in high hydrophilic or non-polar
solvents (Block 2000), which explain its poor absorption in gastro-
intestinal tract (Chen, Li, Lu, Jiang, & Zeng 2007). The low oral bioa-
vailability of apigenin was clinically proven (Madunic, Madunic, Gajski,
Popic, & Garaj-Vrhovac 2017; Zhang et al. 2012).
The solubility of flavonoids depends on several parameters i.e.,
temperature, the nature of the solvents and pH conditions, as well as
the thermodynamic properties of the compounds which allow forma-
tion of hydrogen bonds with the surrounding solvent (Saidman,
Yurquina, Rudyk, Molina, & Ferret 2002; Tommasini et al. 2004; Xiao,
Shao, Yana, & Zhang 2011). Micro/nanoencapsulation has been in-
troduced as an effective tool to improve the solubility of flavonoids, to
confer their protection, to avoid their unpleasant taste (astringency,
bitterness) and to extend their chemical stability until consumption
(Braithwaite, Tyagi, Tomar, Kumar, & Choonara 2014; McClements
2010). Several techniques have been attempted to encapsulate apigenin
such as nanocrystals (Al Shaal, Shegokar, & Müller 2011), poly (lactic-
co-glycolide) (PLGA) nanoparticles (Das, Das, Samadder, Paul, &
Khuda-Bukhsh 2013), polymeric micelles (Zhaia et al. 2013), etho-
somes (Shena, Zhanga, Wang, Xub, & Fenga 2014), liposomes (Arsić
et al. 2011; Banerjee, Banerjee, Das, & Mandal 2015) and W/O/W
emulsion (Kim, Cho, & Park 2016). Most of these systems were useful to
increase the water solubility of apigenin, while few research have fo-
cused on increasing the solubility of apigenin in more hydrophobic
media such as oils. So far, to the best of the authors knowledge, the
encapsulation of apigenin in O/W emulsions has not been reported yet.
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Food Research International xxx (xxxx) xxx–xxx
O/W nanoemulsions are promising delivery systems used in food,
cosmetic and pharmaceutical industries (Benjemaa et al. 2018; Calva-
Estrada, García, Mendoza, & Jiménez 2017) which offer the potential to
significantly improve the solubility and bioavailability of several lipo-
philic functional ingredients including flavonoids, carotenoids and
several other bioactive compounds (lipophilic bioactive lipids, drugs,
flavors, antioxidants, and antimicrobial agents) (Chen & Wagner 2004;
Mao et al. 2009; Moreau, Kim, Decker, & McClements 2003; Skiff,
Baaklini, & Vlad 2007; Wang, Wang, & Huang 2009). O/W emulsions
have wide applications in the formulation of conventional food pro-
ducts (sauces, soups, desserts, fruit beverages, ice cream, soft drinks),
but also for the development of functional food products (sugar beet
pectin, and corn fiber) by the incorporation of bioactive compounds
(Chen et al. 2017; Liu et al. 2017; McClements, Decker, Park, & Weiss
2009). O/W emulsion based delivery systems are highly depended upon
the composition of different phases, the emulsifier selection and the
physicochemical properties of dispersed and continuous phases (Ozturk
& McClements 2016; Raikos & Ranawana 2017). The stability, physi-
cochemical properties and functional performance of O/W nano-
emulsions can be controlled by altering their composition and pre-
paration methods (McClements 2010). Therefore, their production,
with different droplet concentrations, compositions, particle size dis-
tributions and/or interfacial properties, requires the addition of me-
chanical energy typically provided using a shear flow, as well as the use
of surface-active agents (surfactants, or amphiphilic molecules) to help
reducing the energy of the interface area (Payet & Terentjev 2008).
O/W nanoemulsions are produced either by high energy or low
energy processes (Acosta 2009; Leong 2009; Tadros, Izquierdo,
Esquena, & Solans 2004). High-energy approaches utilize mechanical
devices capable of generating intense disruptive forces that breakup the
oil and water phases and lead to the formation of tiny oil droplets, e.g.,
high-pressure valve homogenizers, microfluidizers and sonication
methods (Gutierrez et al. 2008; Velikov & Pelan 2008; Wooster,
Golding, & Sanguansri 2008). High-pressure homogenization generally
produces emulsions with submicron droplets; therefore, it is one of the
most frequently used methods for producing emulsions for food and
pharmaceutical applications (El Kinawy, Petersen, & Ulrich 2012; Gao,
Zhang, & Chen 2008; Pandolfe 1995). Nowadays, O/W nanoemulsions
loaded with antioxidant compounds are typically produced to serve as
functional food supplements which improved bioavailability and de-
livery of bioactive compounds. Moreover, O/W nanoemulsions loaded
with bioactive compounds could be added as preservative into food
formulations in order to improve oxidative, microbiological stability
and sensory attributes of food products. For instance, O/W nanoemul-
sions were added to meat and dairy products and were efficient to
extend their shelf life and sensory profiles (Artiga-Artigas et al. 2017;
Joe et al. 2012).
The aim of this work was to prepare submicron O/W emulsions
loaded with apigenin using high pressure homogenization. We first
investigated the effect of processing parameters (heating pretreatment
time of dispersed phase and homogenization pressure) on the droplet
size, droplet size distribution and apigenin retention of these O/W
emulsions. Next, we evaluated their physico-chemical stability.
2. Materials and methods
2.1. Chemicals
Apigenin (purity, 97.0%) was purchased from Tokyo Chemical
Industry Co. (Tokyo, Japan). Ethyl oleate, polyoxyethylene (20) sor-
bitan monolaurate (Tween 20), ethanol (99.5%), n-hexane, and acet-
onitrile were purchased from Wako Pure Chemical Industries (Osaka,
Japan). The water used was of ultrapure water (Millipore).
I. Abcha et al. Food Research International xxx (xxxx) xxx–xxx

Fig. 2. Schematic diagram for preparing O/W emulsions loaded with apigenin.

further filtrated using a hydrophobic PTFE membrane with a pore size

Fig. 3. Effect of heating ethyl oleate at 100 °C at different times (30, 60 or
120 min) on apigenin solubility.
2.2. Emulsions preparation
The detailed process for emulsions preparation is described in Fig. 2,
An excess apigenin concentration (0.1 wt%) was dissolved in 10 mL of
ethyl oleate using a magnetic stirrer before being heated at 100 °C for
different times (30 min, 60 min or 120 min). Solutions were subse-
quently centrifuged at 10,000 rpm (13.000 g) using a high-speed cen-
trifuge (MX-307 centrifuge, Tomy Digital Biology, Co., Ltd., Tokyo,
Japan) for 10 min and the obtained clear phase of ethyl oleate were
of 0. 45 μm. The resulting clear phases of ethyl oleate solutions at dif-
ferent heating pretreatment times were used as to-be- dispersed phases
to prepare apigenin loaded O/W emulsions (Fig. 2).
Apigenin-loaded O/W emulsions were prepared using a two-step
homogenization process according to the method of Tan and Nakajima
(2005). Each clear ethyl oleate phase containing apigenin was mixed
with the aqueous Tween 20 solution (1 g/100 g) using a magnetic
stirrer at room temperature (about 25 °C) for 30 min. The choice of
Tween 20 was based on its common use in food and pharmaceutical
industries (Sapei, Sandy, Suputra, & Ray 2017), its low toxicity, and its
low sensivity to the destabilizing effects of formulation additives
(O'Hagan & Podda 2008).
Firstly, coarse emulsions containing the oil-water mixture with a
10% dispersed-phase volume fraction were obtained with a rotor-stator
homogenizer (Polytron PT 3100, Kinematica AG, Switzerland) at
5000 rpm for 5 min. Subsequently, the coarse emulsions were im-
mediately passed through a high-pressure homogenizer (NanoVater
NV200, Yoshida Kikai, Co., Ltd., Japan) at different pressure levels of
60, 100 and 150 MPa under a moderate temperature of 10 °C. Such a
low temperature was applied to avoid apigenin degradation due to heat
produced inside the homogenizer. Control O/W emulsion samples
without apigenin were also prepared following the same procedure and
conditions mentioned above. All the O/W emulsion samples prepared
were stored in the dark at 4 °C for 30 days.

Table 1
Effect of ethyl oleate heating pretreatment time at 100 °C and apigenin addition on measured interfacial tension and oil viscosity.
Oil composition Interfacial tension (mN/m) (Milli-Q water containing 1 wt% Tween 20) Oil viscosity (mPa s) at 25 °C

Unheated ethyl oleate 3,7a ± 0,2 5,15a ± 0.02

Heated ethyl oleate for 30 min 4b ± 0.1 5,20a ± 0.01
Heated ethyl oleate for 60 min 4,1 b ± 0.2 5,26 b ± 0.1
Heated ethyl oleate for 120 min 4,4 b ± 0.3 5,6c ± 0.05
Heated ethyl oleate for 30 min containing apigenin(0.005 wt%) 4,3 b ± 0.1 5,27 b ± 0.00
Heated ethyl oleate for 60 min containing apigenin (0.006 wt%) 4,2 b ± 0.1 5,3 b ± 0.03
Heated ethyl oleate for 120 min containing apigenin (0.007 wt%) 4,0 b ± 0.2 5,27 b ± 0.02

Different letters in the same column indicate significant differences (p < 0.05).

3
I. Abcha et al.
Fig. 4. (A) Effect of homogenization pressure on the average droplet size (d av)
of the fresh O/W emulsions. (B) Effect of homogenization pressure (MPa)
produced in10 wt% ethyl oleate containing 0.005 wt% apigenin-in-water
emulsions. (C) Droplet size distribution of the fresh O/W emulsions loaded with
apigenin.
2.3. Determination of the fluid properties
A density meter (DA- 130 N, Kyoto Electronics Manufacturing Co.
Ltd., Japan) was used to determine the density of liquid phases which is
required for interfacial tension measurements. The viscosity of ethyl
oleate in the presence and absence of apigenin was measured using a
sine-wave vibro viscometer (SV-10, A&D Company, Tokyo, Japan). The
interfacial tension between the liquid phases was measured using an
interfacial tensiometer (PD-W, Kyowa Interface Science Co. Ltd., Japan)
that adopts a pendant drop method (Souilem et al. 2013). Interfacial
tension data were averaged from at least 10 determinations. All the
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Fig. 5. Effect of heating pretreatment time at 100 °C of dispersed phase on: (A)
the d av. (B) Droplet size distribution of the fresh O/W emulsions prepared at
100 MPa.
measurements were performed at 25 °C.
2.4. Droplet size measurements
The average droplet size (dav) and size distribution of the for-
mulated O/W emulsions were determined at 25 °C using a dynamic
light scattering particle size analyzer having a measuring range of
0.6 nm to 6 μm (Zetasizer Nano-ZS, Malvern Instruments, UK). The re-
fractive index values used here were 1.332 for water and 1.451 for ethyl
oleate. A 0.5 mL aliquot of emulsion was diluted with 9.5 mL of Milli-Q
water and stirred for 10 min before size determination at room tem-
perature (about 25 °C) using a plastic cuvette (1 cm path length) to
break up any aggregated particles. The mean droplet size of the O/W
emulsions is described by the Sauter mean diameter (d3,2), as indicated
in Eq. 1:
d3,2 = Volume/Surface Area = ∑ ni di3/ ∑ ni di2 (1)
All samples were measured in triplicate. Polydispersity index (PDI)
is a dimensionless number that measures the width of the size dis-
tribution. Its maximum value is arbitrarily limited to 1.0.
2.5. Determination of apigenin content
The apigenin content in ethyl oleate prior to emulsification (or in O/
W emulsion) was determined according to a modified procedure, in-
itially described by Treesuwan et al. (2013). Briefly, ethyl oleate solu-
tion (2 g) containing apigenin was mixed with 90% ethanol (5 g) and
I. Abcha et al.
Fig. 6. Apigenin retention of fresh O/W emulsions: (A) Effect of high pressure.
(B) Effect of heating pretreatment time at 100 °C.
then added to hexane. Two phases consisting of an upper clear hexane
layer and a lower ethanolic layer were obtained. The mixed sample was
subsequently centrifuged at 13,000 g for 20 min at room temperature
(about 25 °C). After centrifugation, the hexane layer was removed, and
an eluent (5 g) of acetonitrile/water (6:4, v/v) was added into the
ethanolic layer. The final mixture was filtrated through a hydrophobic
PTFE membrane (0.45 μm) prior to determining apigenin concentration
using HPLC analysis. Each extraction experiment was performed in
triplicate.
2.5.1. Chromatographic conditions
The HPLC system (JASCO International Co., Tokyo, Japan) was
composed by a degasser (DGU-4A), solvent delivery module (LC-10AT),
UV-970 UV–Vis spectrophotometric detector, a PU-980 pump system,
and an AS-2055 autosampler. The chromatographic analyse were per-
formed on a ODS C18 column ((150 × 4.6) mm, 5 μm), mobile phase
composed of acetonitrile and water in a volume ratio of 35 to 65 at a
flow rate of 1.0 mL· min−1 and detection wavelength at 275 nm. Each
measurement was run for 10 min at 25 °C. Apigenin concentration in
ethyl oleate was determined using a linear calibration curve
(R2 = 0.9998) using the concentration range of 0.001–0.1 mg/mL.
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2.6. Storage stability
The formulated O/W emulsions at different heating pretreatment
times of the to-be-dispersed phase ethyl oleate and at different pres-
sures were stored at 4 °C during 30 days to examine their droplet size
evolution and their retention of apigenin. The retention of apigenin (R)
in samples was calculated using Eq. 2, as follows:
R (%) = Ct/C0 × 100 (2)
where Ct is the concentration of apigenin (mg/g) at a specific time,
while C0 is the initial concentration of apigenin (mg/g) loaded in the
nanoemulsions.
2.7. Statistical analysis
All samples were analyzed in triplicate. Data were expressed as the
mean ± standard deviation. Statistical analyses were based on differ-
ences between means using the Student's t-test. Statistical significance
was judged at a p level of 0.05.
3. Results and discussion
3.1. Physico-chemical properties of the liquid phases
3.1.1. Effect of ethyl oleate heating pretreatment time on the solubility of
apigenin
Supersaturated solutions of ethyl oleate containing 0.1 wt% of api-
genin were used to test the effect of heating pretreatment time at 100 °C
for 30, 60, or 120 min on the solubility of apigenin. The safety of the use
of ethyl oleate in food is supported by metabolism data in rats and
clinical safety data in humans: there were no clinically significant ne-
gative effects from consuming ethyl oleate at levels up to 16 g/day
(approximately 0.2 g/kg) (Bookstaff et al. 2003).We chose a moderate
heating temperature as ethyl oleate might degrade at about 150–155 °C
(Cataldo 2013). The data expressed in Fig. 3 revealed that apigenin
solubility varied between 0.55 ± 0.02 and 0.66 ± 0.05 mg/g. In-
creasing the heating pretreatment time led to a slight increase of api-
genin solubility in ethyl oleate (p > 0.05). Recent reports suggest that
the solubility of apigenin is dependent on the solvent type (Xiao et al.
2011). Based on our results, ethyl oleate, with moderate viscosity
(Table 1) demonstrated a better dissolving power for apigenin as
compared to isopropyl myristate and labrafil M1944 CS (Zhao, Zhang,
Meng, Wang, & Zhai 2013). Ethyl oleate was also shown to have the
highest dissolving power for similar hydrophobic compounds i.e. cur-
cumin (Cui et al. 2009) and trans-ferulic acid (Zhang et al. 2016), as
compared to long chain oils. The clear ethyl oleate solutions heated at
100 °C for different times were cooled to room temperature (about
25 °C) and used as dispersed phases for O/W emulsions preparation.
3.1.2. Effect of heating pretreatment time on the viscosity and interfacial
tension
The O/W interfacial tension and the viscosity of the two phases
properties influences the efficiency of droplet disruption, and therefore
the minimum size attainable using high-pressure homogenization
(Walstra 1993; Cheng Qian, and David Julian McClements 2010). The
variation of the interfacial tension and oil viscosity with heating pre-
treatment time are presented in Table 1. In the absence of apigenin, the
viscosity of ethyl oleate increased significantly (p < 0.05) as the
heating pretreatment time of dispersed phase increases. But, in the
presence of apigenin, the oil viscosity did not exhibit distinct changes,
regardless of the heating pretreatment time (p > 0.05). These results
suggest that the ethyl oleate viscosity in the presence of apigenin was
unaffected by the heating process. Interfacial tension results showed
similar trends. In the absence of apigenin, interfacial tension increased
from 3.7 to 4.4 mN/m after heating ethyl oleate at 100 °C for 120 min.
These results are in agreement with (Mosayebia, Angaji, & Khadiv-Parsi
I. Abcha et al.
Fig. 7. Apigenin retention of fresh O/W emulsions: (A) Effect of high pressure. (B) Effect of heating pretreatment time at 100 °C.
2016; Ye et al. 2008) showing that increasing the heating pretreatment
time of oil phase can shorten the time needed for an interfacial tension
to reach equilibrium and therefore lead to an increase of the interfacial
tension values (Mosayebia et al. 2016; Ye et al. 2008). The heating
pretreatment may be led to increase mutual solubility of the solvents
(crude oil and water), lipophilicity of surfactants and diffusion velocity
of surfactant molecules onto the interface, adsorption velocity of sur-
factant molecules at the interface. Furthermore, it affected the time that
was needed to reach equilibrium interfacial tension to decrease. How-
ever, for the ethyl oleate systems containing apigenin, there was no
remarkable variation of the interfacial tension (p > 0.05) which sug-
gests that the presence of apigenin in ethyl oleate might suppress the
effect on oil caused by the heating pretreatment.
3.2. Preparation characteristics of apigenin-loaded O/W emulsions
3.2.1. Droplet size and size distribution of the formulated O/W emulsions
3.2.1.1. Effect of homogenization pressure. Fig. 4A and Fig. 4B depict the
droplet size distribution and the average droplet diameter (dav) of the
O/W emulsions obtained by high-pressure homogenization in the
presence and in the absence of apigenin at different operating
pressures ranging from 60 MPa to 150 MPa. Overall, we observed a
decrease in droplet diameter with increasing the pressure of
homogenization, which is in agreement with previous studies (Mei,
Che, Weng, Yang, & Yang 2003; Treesuwan et al. 2013). No significant
difference was obtained in the dav between formulated O/W emulsions
in the absence and in the presence of apigenin (p > 0.05). As for O/W
emulsions loaded with apigenin, their dav and PDI were
190 ± 1.567 nm and 0.086 ± 0.002 at 60 MPa, 174 ± 1.568 nm
and 0.070 ± 0.001 at 100 MPa, and 169 ± 2.082 nm and
0.06 ± 0.002 at 150 MPa. Thus, the addition of apigenin into the
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Food Research International xxx (xxxx) xxx–xxx
ethyl oleate to-be-dispersed phase hardly affects the dav of the
formulated O/W emulsions, which is an agreement with previous
studies about the formulation of O/W emulsions loaded with other
flavonoids using high-pressure homogenization (Treesuwan et al.
2013).
When plotting log (dav) = f (log (P)), we found a linear dependence
with a slope of about −0.6 (Fig. 4C). This result confirmed previous
mathematical and computer simulations findings that the breakup of
droplets under high-pressure homogenization associated with a slope of
log(d) versus log(P) of around −0.6 under primarily turbulent-inertial
flow conditions (Hakansson, Tragardh, & Bergenstahl 2009; Walstra
2003).
Fig. 4B plots the droplet size distributions of fresh O/W emulsions
loaded with apigenin at different homogenization pressures. Increasing
the pressure from 60 MPa to 100 MPa decreased significantly
(p < 0.05) the polydispersity by clearly comprising the droplet sizes
into narrower distribution, but droplet distributions at 100 and
150 MPa had almost similar width (Fig. 4B). The minimum average
droplet size dav attainable for fresh O/W emulsions was around 169 nm,
which is much smaller than the baicalein-loaded O/W emulsions (about
300 nm) formulated in similar homogenization conditions and using
soybean oil as to-be- dispersed phase (Treesuwan et al. 2013). Ethyl
oleate has lower viscosity (5.1 mPa s), as compared to the soybean oil
(49.3 mPa s), which contains long chain fatty acids. More viscous oil
droplets are more difficult to breakup by a high-pressure homogenizer,
because the coarse droplets may leave the disruption zone before they
have time to deform and disrupt, and therefore larger droplets still
remain (Shah, Carvajal, Patel, Infeld, & Malick 1994).
3.2.1.2. Effect of heating pretreatment time. Fig. 5A shows the effect of
the heating pretreatment time at 100 °C on the average droplet
I. Abcha et al. Food Research International xxx (xxxx) xxx–xxx

Fig. 9. Changes in apigenin concentration during storage of O/W emulsions

formulated using 100 MPa at different heating pretreatment time.

when heating ethyl oleate at 100 °C for 30 and 120 min, respectively.
Heating pretreatment time of oil phase might influence the dav of an
emulsion because it caused a slight increase of the oil viscosity
(Table 1). In fact, it was reported that the use of more viscous oils
caused the production of larger emulsions droplets (Souilem et al.
2014). On the other hand, following the heat treatment of the ethyl
oleate containing apigenin at 100 °C for 30, 60 and 120 min, all fresh
emulsions had similar dav (p > 0.05). Heating hardly affected the
droplet size distributions of apigenin-loaded O/W emulsions, as
confirmed by the combined size distributions at the different heating
pretreatment times of ethyl oleate (Fig. 5B).The above results suggested
that the effect of ethyl oleate heating pretreatment on the dav of fresh
O/W emulsions is attenuated by the incorporation of apigenin in the to-
be-dispersed phase.
Fig. 8. (A) Changes in apigenin concentration during storage of O/W emulsions
formulated at different pressures. (B) Linear correlation between apigenin
concentration during storage and the average droplet size (d av) of O/W
3.3. Apigenin retention in fresh O/W emulsions
emulsions formulated at different pressures. (C) Degradation kinetics of api-
genin during storage of O/W emulsions formulated at different pressures. The retention of apigenin in the formulated O/W emulsions was
quantitatively determined by HPLC analysis. The HPLC chromatogram
showed a sharp monomodal peak of apigenin eluted at 2.5 min. Fig. 6
diameter (dav) of the fresh O/W emulsions prepared at a fixed
illustrates the effect of homogenization pressure and heating pretreat-
homogenization pressure (100 MPa, 1 pass). In the absence of
ment of the oil phase on apigenin retention in the fresh O/W emulsions.
apigenin, the dav of fresh emulsions significantly increased
As seen in Fig. 6A, the apigenin retention was affected by homo-
(p < 0.05) with increasing heating pretreatment time of the ethyl
genization pressure, since 4–24% of apigenin was lost during the pro-
oleate. The dav increased from 181 ± 1.023 nm to 202 ± 0.503 nm
cess, depending on the applied pressure. The homogenization pressure

7
I. Abcha et al.
of 100 MPa showed the highest apigenin retention of about 96% which
corresponds to an initial concentration of 5.40 mg/100 g present in the
emulsion sample (Fig. 8A). Our O/W emulsion system is fortified with a
relative high concentration of apigenin as compared with previously
formulated liposomes containing a range of concentration from
32.8–230 μg/mL (Paini et al. 2015; Pápay et al. 2016). The heating
pretreatment time at 100 °C of ethyl oleate prior to emulsification did
not affect apigenin retention, which ranges from 96.0–99.0%
(p > 0.05) (Fig. 6B). The heating pretreatment time affected rather the
solubility of apigenin and therefore the initially loaded concentration of
apigenin, which ranges from 5.36–6.58 mg/100 g of emulsion (Fig. 9).
The high apigenin retention might be due to its high hydrophobicity.
3.4. Storage stability of apigenin-loaded O/W emulsions
The emulsion stability is defined in terms of physical and chemical
stability of the emulsion. The stability of O/W emulsions formulated at
different heating pretreatment times of the to-be-dispersed phase ethyl
oleate and at different pressures was evaluated based on the changes in
mean droplet diameter, size distribution and apigenin retention over a
storage time of 30 days at 4 °C.
3.4.1. Physical stability of apigenin-loaded O/W emulsions
3.4.1.1. Effect of homogenization pressure. All emulsions remained
relatively stable without phase separation, creaming or flocculation
upon 30 days of storage. Fig. 7A and Fig. 7B show the stability of
apigenin loaded O/W emulsions at the different homogenization
pressures. Slight increases in dav were seen in all the emulsions upon
the different applied pressures. O/W emulsions formulated at 100 and
150 MPa followed similar increase rate of their dav upon storage
(p > 0.05) (Fig. 7A.). However, O/W emulsions formulated at
60 MPa exhibited higher rate of dav increase after 30 days of storage.
For instance the dav of apigenin loaded O/W emulsions and their PDI
values after 30 days were 300 ± 1.056 nm and 0.202,
237 nm ± 0.523 and 0.168, and 289 nm ± 2.368 and 0.179, at
60 MPa, 100 MPa and 150 MPa respectively. Fig. 7B showed
monomodal size distributions with a narrow width for O/W
emulsions formulated at 150 MPa even after 30 days of storage which
confirmed the high physical stability of these emulsions.
3.4.1.2. Effect of heating pretreatment time. Fig. 7C and Fig. 7D show the
effect of heating pretreatment of ethyl oleate on the dav of apigenin
loaded O/W emulsions upon 30 days of storage at 4 °C. There was little
variation in their droplet size over 30 days of storage, and their dav
remained in the range of 225–234 nm either when heating ethyl oleate
at 100 °C for 30, 60 or 120 min. The heating pretreatment time did not
affect the resultant droplet size after 30 days of storage (p > 0.05).
These results indicate that the formulated O/W emulsions were
considered physically stable during storage at 4 °C. In general, nano-
emulsions are kinetically and thermodynamically stable (Anton &
Vandamme 2011; Mason, Wilking, Meleson, Chang, & Graves 2006;
McClements 2010). McClements (2005) noted also that nano-sized O/W
droplets have a lesser tendency to cream but a greater tendency to
aggregate, because they are more numerous at a given phase ratio and
more susceptible to the influence of Brownian motion, both of which
would lead to greater chance of collision (McClements 2005). Apigenin
addition did not affect the trends in the dav values during storage at any
heating pretreatment time of dispersed phase.
3.4.2. Apigenin retention in O/W emulsions during storage
3.4.2.1. Effect of homogenization pressure. Fig. 8 shows the effect of
homogenization pressure on the apigenin retention during storage. For
all O/W emulsion samples, apigenin was gradually degraded and by the
end of the 30 days storage, about 12–48% of the apigenin was lost. The
total apigenin concentrations after 30 days of storage were 2.60 mg/
100 g, 4.70 mg/100 g, and 2.60 mg/100 g at 60, 100 and 150 MPa,
8
Food Research International xxx (xxxx) xxx–xxx
respectively. Emulsions prepared at 100 MPa depicted the highest
retention of apigenin upon 30 days of storage (Fig. 8C.). In order to
better compare the effect of homogenization pressure on apigenin
retention, degradation kinetics were monitored. Zero-order (Eq. 3) and
first-order (Eq. 4) kinetic models have been used to evaluate the
apigenin degradation kinetics in the prepared O/W emulsions
(Souilem, Debbabi, Demmargi, & Bellegha 2015).
Zero order:C = C0 + k 0. t (3)
First order:C = C0 . exp.(k1. t) (4)
where C is the apigenin concentration in the O/W emulsions during
storage, C0 is the initial concentration of apigenin; k is the rate constant
of degradation and t corresponds to storage time. Both zero and first
order reaction kinetic models were tested to predict the degradation of
apigenin in O/W emulsions during storage (Fig. 8A. and Fig. 8B.). The
rate constants were calculated by applying the regression function of
Microsoft Excel 2010, obtaining best-fit results. Regardless of the
applied pressure, the zero order equation was more plausible to
predict apigenin degradation since its corresponding R2 values
(Fig. 8A.) were higher than those of the first-order model k1
(correlations exceeded 0.9) (Fig. 8B.). O/W emulsions prepared at
100 MPa presented the lowest degradation rates followed by samples
prepared at 150 and 60 MPa, respectively. It was expected that
increasing the homogenization pressure from 100 to 150 MPa would
not affect apigenin retention in O/W emulsions since high-pressure
homogenization-induced degradation is not a serious problem for the
majority of active ingredients (Tan & Nakajima 2005). For instance,
Treesuwan et al. (2013) found that the degree of baicalein degradation
was similar in the nanoemulsions produced at 60, 100 and 150 MPa
which is contradictory to our results. McClements (2012) reported that
the factors responsible for the chemical degradation of hydrophobic
bioactive components such as flavonoids vary from one system to
another, depending on the molecular structure of the bioactive
component and its environment. According to previous works (Tan &
Nakajima 2005), the influence of interfacial area is among the relevant
factors affecting the stability of bioactive compounds in
nanodispersions. In order to better explore this parameter, the
coefficients of correlation (R2) between the apigenin retention in the
O/W emulsions formulated at different pressures and dav during
30 days of storage were calculated (Fig. 8C).The results revealed that
apigenin degradation is very dependent on the droplet size of emulsions
during storage (R2 > 0.9). Results showed that by decreasing the
droplet size of emulsions, a higher apigenin retention was achieved
(Fig. 8C). Larger O/W droplets are less stable and might enhance the
diffusion of apigenin across the interface, its release and therefore
degradation. However, if we compare similar sized droplets formulated
at different pressures, we found different retention rates, which explain
that operating pressure also plays a significant effect on the emulsion
droplet stability of apigenin-loaded O/W emulsions. High shear stress
has been reported to be a major cause that promotes cavitation within
the high-pressure homogenizer, and evidence exists of free radical
formation (Lander et al. 2000). Moreover, Hebishy, Buffa, Guamis,
Blasco Moreno, and Trujillo (2015) reported that increasing the
homogenization pressure for > 100 MPa resulted in more oxidation in
the O/W emulsions produced by conventional and ultra-high-pressure
homogenization. Therefore, it is possible that the homogenization
process at 150 MPa might be a contributing factor to promote
oxidation and then could trigger higher degradation rate of apigenin
in the prepared O/W emulsions.
3.4.2.2. Effect of heating pretreatment time. Fig. 9 shows the effect of
heating pretreatment time of ethyl oleate prior to emulsification on the
apigenin retention in O/W emulsions during storage. Fig. 9A
demonstrates that apigenin degradation in the prepared O/W
emulsions exhibits similar tendency regardless of the heating
I. Abcha et al.
pretreatment time. For all O/W emulsion samples formulated at
100 MPa, the apigenin retention was high, regardless of the heating
pretreatment time (p > 0.05), and by the end of the 30 days storage
only around 6% of the apigenin was lost (Fig. 9B). The total apigenin
concentrations after 30 days of storage were 5.00 mg/100 g, 5.20 mg/
100 g and 6.20 mg/100 g of emulsion at 30, 60 and 120 min,
respectively.
During 30 days of storage, the heating of the dispersed phase hardly
affected the apigenin loaded O/W emulsions stability. The high phy-
sical stability of the formulated emulsions at different heating pre-
treatment times, predominantly contributed to the better apigenin
stability (Fig. 9). The retention of apigenin was almost similar following
the different heating pretreatments which also suggest the high stability
of ethyl oleate against oxidation. One possible consideration would be
that the association of colloid surfactants (Tween 80) may have retarted
ethyl oleate oxidation (Homma, Suzuki, Cui, McClements, & Decker
2015). Alternately, the type of oil used as dispersed phase represents an
important excipient in emulsions because they could produce remark-
able effects on emulsion area, drug solubility (Gershanik & Benita
2000). In fact, long-chain triglycerides seem to facilitate lymphatic
transport, while medium-chain oils were efficient in enlarging micro-
emulsion area and solubilizing drugs (Zhang et al. 2016).
Ethyl oleate used as dispersed phase in our formulations is capable
of enlarging emulsion region, solubilizing well apigenin and enabling
its high retention during storage. Recent research revealed that ethyl
oleate is a preferred oil candidate due to its integrated advantages of
high solubilizing capability, large microemulsion area and effective
lymphatic transport as compared to long-chain triglycerides and
medium-chain oils (Xing et al. 2016; Zhang et al. 2016).
4. Conclusion
The present study has demonstrated the successful formulation of
food-grade O/W emulsions loaded with apigenin using high-pressure
homogenization. The formulated O/W emulsions remained physically
stable with a slight decrease in their dav and PDI values during 30 days
of storage. Increasing the heating pretreatment time of ethyl oleate
prior to emulsification enhanced the apigenin solubility and therefore
its initial concentration in the dispersed phase but it hardly affected the
physico-chemical stability of the produced O/W emulsions. The O/W
emulsions also demonstrated relatively high retention of apigenin
during storage, and their retention kinetics followed a zero-order ki-
netics equation. The homogenization pressure affected the degradation
rate of apigenin in the prepared O/W emulsions. In particular, emul-
sions formulated at 100 MPa achieved the highest retention rate of
around 93%. Although the use of a high homogenization pressure at
150 MPa generated a physically stable O/W emulsion, it caused an in-
tense degradation of apigenin in fresh and stored O/W emulsions. Our
results provide a useful information on the preparation of stable food-
grade apigenin loaded O/W emulsions using high-pressure homo-
genization as promising system for nutraceutical formulation fortified
with an hydrophobic flavonoid.
Acknowledgments
The authors are grateful to the Science and Technology Research
Partnership for Sustainable Development (SATREPS) Project, finan-
cially supported by JICA and JST, Japan.
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