Ultrasonics - Sonochemistry 64 (2020) 104995

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Ultrasonics - Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Ultrasound-assisted production of palm oil-based isotonic W/O/W multiple T

nanoemulsion encapsulating both hydrophobic tocotrienols and hydrophilic
caffeic acid with enhanced stability using oil-based Sucragel
⁎
Revathi Raviadarana,b, Mei Han Ngb, Sivakumar Manickama, , Davannendran Chandranc
a
Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Jalan Broga, 43500 Semenyih, Selangor
Darul Ehsan, Malaysia
b
Malaysian Palm Oil Board, No. 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia
c
Sunway University, No. 5, Jalan Universiti, Bandar Sunway, 47500 Subang Jaya, Selangor, Malaysia

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

Keywords: In this work, the effects of thickeners and tonicity towards producing stable palm oil-based water-in-oil-in-water
Ultrasound (W/O/W) multiple nanoemulsion using ultrasound and microfluidizer were investigated. Palm oil, Sucragel,
Nanoemulsion polyglycerol polyricinoleate, Tween 80, Xanthan gum, and NaCl were used. W/O/W was formed under the
Multiple optimized conditions of ultrasound at 40% amplitude and for 180 s of irradiation time, whereas for the mi-
Microfluidizer
crofluidizer, the optimized conditions were 350 bar and 8 cycles. This is the first work that successfully utilized
Palm-oil
Sucragel
Sucragel (oil-based thickener) in imparting enhanced stability in W/O/W. W/O/W with isotonic stabilization
produced the lowest change in the mean droplet diameter (MDD), NaCl concentration, and water content by
1.5%, 2.6%, and 0.4%, respectively, due to reduced water movement. The final optimized W/O/W possessed
MDD and dispersity index of 175.5 ± 9.8 and 0.232 ± 0.012, respectively. The future direction of formulating
stable W/O/W would be by employing oil phase thickeners and isotonicity. The observed ~12 times lesser
energy consumed by ultrasound than microfluidizer to generate a comparable droplet size of ~235 nm, further
confirms its potential in generating the droplets energy-efficiently.

1. Introduction
Water-in-oil-in-water (W/O/W) multiple emulsion is a dispersed
internal simple water-in-oil (W/O) emulsion within a continuous ex-
ternal water phase. It possesses several beneficial applications for the
cosmetic, food, nutraceutical, and pharmaceutical industries due to its
ability to encapsulate natural active compounds [1–3]. Majorly, W/O/
W multiple emulsion was studied to exploit its potential for sustained
release as active compounds could pass through several compartments
before their release [4,5]. However, it’s potential for encapsulating both
hydrophilic and hydrophobic active compounds simultaneously had
been scarcely studied.
Many active compounds are either hydrophobic or hydrophilic,
which could be encapsulated both simultaneously in W/O/W multiple
emulsion, especially for pharmaceutical applications. This could ensure
protection during processing and exposure to heat and light, delivery
with improved bioavailability, enhanced therapeutic potential due to
synergistic and reduced side effects [6–8]. Two specific compounds
with potential health benefits that can be encapsulated are hydrophobic
tocotrienol and hydrophilic caffeic acid.
Tocotrienol is vitamin E with three double bonds at positions 3′, 7′,
and 11′ in the alkyl side chain that has a composition of γ-tocotrienols
(46 wt%), α-tocopherol (22 wt%), α-tocotrienols (20 wt%), and δ-to-
cotrienols [9,10]. It is reported to possess antioxidant [11,12], anti-
inflammatory [13,14], anti-proliferative [15,16] and anti-cancer
[17,18] properties. Whereas, caffeic acid (3,4-dihydroxycinnamic acid)
is a phenolic compound which possesses an aromatic ring bearing two
hydroxyl groups. It is reported to have anti-oxidant [19,20], anti-cancer
[21], anti-tumour [22] and anti-proliferative [23,24] properties. Both
compounds have the potential to be used for cancer treatment. The best
drug delivery tool which could be used to encapsulate both hydro-
phobic and hydrophilic natural active compounds would be the W/O/
W multiple emulsion.
However, since 2015, there has been little progress on W/O/W

Abbreviations: DI, dispersity index; MDD, mean droplet diameter; min, minute; PGPR, polyglycerol polyricinoleate; rpm, revolution per minute; W/O, water-in-oil;
W/O/W, water-in-oil-in-water; wt, weight
⁎
Corresponding author.
E-mail address: Sivakumar.Manickam@nottingham.edu.my (S. Manickam).

https://doi.org/10.1016/j.ultsonch.2020.104995
Received 20 November 2019; Received in revised form 29 January 2020; Accepted 30 January 2020
Available online 31 January 2020
1350-4177/ © 2020 Elsevier B.V. All rights reserved.
R. Raviadaran, et al.
multiple emulsion for oral-based pharmaceutical applications. One
primary reason that prevents the advancement of W/O/W is its stabi-
lity, mainly described by the unwanted loss of the internal water dro-
plets or rupture of oil globules [25,26]. Firstly, this could occur due to
coalescence in the internal water droplets and between the oil globules.
Raviadaran et al. [6] indicated that this could primarily be prevented
by utilizing an emulsifier that anchors deeply within the oil phase,
ensuring rigidity at the oil-water interphase during the generation of
primary W/O emulsion. Secondly, the diffusion or osmotic transfer
could also result in the loss of internal water droplets or rupture of the
oil globules [6,27]. The best method to control this is through thick-
eners and tonicity stabilization during the secondary formation of W/
O/W multiple emulsion.
In the past, most studies investigated the usage of thickeners in the
internal water phase of W/O/W multiple emulsion, emphasizing on the
sustained release of the active compound. Thickeners that were com-
monly used for the internal water phase were proteins such as sodium
caseinate [28,29], whey protein [2,30], gelatin [31,32], and poly-
saccharides such as starch [33,34] and xanthan gum [35,36]. However,
limited studies investigated the usage of thickeners for evaluating the
stability of W/O/W multiple emulsion. To the best of our knowledge,
the utilization of thickeners in the oil phase and its effect on the sta-
bility of W/O/W multiple nanoemulsion has not been reported to date.
Furthermore, not readily available oil-soluble thickeners and limited
choices for oral-based formulations present other challenges.
Addressing the above, a 100% natural oil-soluble thickener, i.e.,
Sucragel AOF Bio composed of Prunus Amygdalus Dulcis (Sweet
Almond) oil, glycerine, sucrose laurate, and Citrus Aurantium Dulcis
(Orange) fruit water was considered and the effect of Sucragel was
evaluated. This is the first work evaluating the stabilizing effect of
Sucragel, which acts as an oil phase thickener in palm oil-based W/O/W
multiple emulsion. Notably, Schmidt et al. [37] investigated the stabi-
lizing effect of thickener in the water globule of O/W/O multiple
emulsion. They have reported an increase in the stability against coa-
lescence of inner oil droplets owing to the reduced movement of dro-
plets. Hence, it is essential to evaluate the effect of thickener in the oil
globule for the stability of W/O/W multiple emulsions.
Concerning tonicity stabilization, the majority of the existing stu-
dies alters the concentration of solutes (NaCl, glucose) in the internal
and external water phases of W/O/W multiple emulsion to evaluate the
release rate and transport mechanism. Few studies [38–40] suggested
either isotonic or hypertonic or even both for attaining a sustained
release. Hence, there is a lack of study for evaluating the best tonicity
condition required for a stable formulation of W/O/W multiple emul-
sion. Therefore, a thorough investigation is necessary on the tonicity
condition required for a stable W/O/W multiple emulsion formulation.
Based on the above, the first objective of this study is to evaluate the
production of palm oil-based W/O/W multiple nanoemulsion utilizing
ultrasound and microfluidizer. Cavitation due to ultrasound forms and
collapses vapor cavities that lead to powerful shock waves throughout
the medium, breaking the dispersed phase into smaller droplets.
Whereas, the microfluidizer uses high pressure to direct the flow stream
through microchannels towards the impingement area, which creates a
very high shearing action into forming smaller droplets. The energy
consumption and oxidation of palm oil in the emulsion upon subjecting
to both the processing methods were also investigated. Also, the effect
of varying the concentration of hydrophilic emulsifier and the phase
volume ratio on the formation of W/O/W multiple nanoemulsion were
examined.
The second objective is to evaluate the effect of utilizing thickeners
and tonicity stabilization on the stable formulation of W/O/W multiple
emulsion. The effects of using oil-soluble thickener (Sucragel) and
water-soluble thickener (Xanthan gum) were studied for oil and in-
ternal water phases, respectively, on the stability of W/O/W multiple
emulsion. Furthermore, three conditions of tonicity, namely, hypotonic,
isotonic, and hypertonic, were simulated by modifying the
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Ultrasonics - Sonochemistry 64 (2020) 104995
concentration of NaCl (solute) in the internal and external water phases
to assess their effects on the stability of W/O/W multiple emulsion.
2. Materials and methods
2.1. Materials
Refined palm olein (palm oil), palm medium-chain triglyceride
(MCT) oil, and polyglycerol polyricinoleate (PGPR) were sourced from
Sime Darby (Malaysia), Kuala Lumpur Kepong Oleochemicals
(Malaysia) and Palsgaard (Denmark), respectively. The following che-
micals were also used in this investigation to produce the nanoemul-
sion: Tween 80 from Sigma Aldrich (Malaysia), Sucragel AOF Bio from
Alfa Chemicals (United Kingdom), and Xanthan gum from Sigma
Aldrich (Malaysia). Deionized water was produced from the Milli-Q®
Plus (USA) and was utilized for the generation of nanoemulsions. For
STEM analysis, a formvar-coated copper grid with 300 mesh procured
from Agar Scientific Ltd (UK) was utilized.
2.2. Comparison of processing W/O/W multiple nanoemulsion using
ultrasound and microfluidizer
2.2.1. Generation of W/O/W nanoemulsion
W/O coarse and nanoemulsion were prepared as reported by
Raviadaran et al. [6]. The obtained coarse emulsion was then subjected
to ultrasonication for 180 s at 40% amplitude. This led to the formation
of W/O nanoemulsion (~150 nm). The W/O nanoemulsion was then
added at 10 wt% into 89 wt% deionized water and 1 wt% Tween 80 to
form the W/O/W coarse multiple emulsion.
2.2.2. Generation of W/O/W nanoemulsion using ultrasound
Coarse W/O/W multiple emulsion was formed using VCX 750 W
Ultrasonic Horn Processor (USA) with 1⁄4′’ (6 mm) stepped microtip in
a pulse mode (60 s on and 60 s off) at 20 kHz. Amplitudes of 20, 30, 40,
and 50% were utilized to produce the W/O/W multiple nanoemulsion.
Irradiation times of 10, 30, 45, 60, 90, 120, 150, 180, 220, and 240 s
were utilized for sonication at each amplitude. The composition of W/
O/W multiple nanoemulsion constituted 90 wt% W/O nanoemulsion,
9 wt% deionized water, and 1 wt% Tween 80.
2.2.3. Generation of W/O/W nanoemulsion using microfluidizer
Microfluidizer (Model M-110Y, Microfluidics, USA) with the cooling
jacket was also used to form W/O/W multiple nanoemulsion. Operating
pressures at 150, 250, 350, and 450 bar were utilized. At each operating
pressure, the effect of the number of cycles of 2, 3, 4, 5, 6, 7, 8, 9, 10,
and 12 on the formation of W/O/W multiple nanoemulsion was eval-
uated. The composition of W/O/W multiple nanoemulsion constituted
of 90 wt% W/O nanoemulsion, 9 wt% deionized water, and 1 wt%
Tween 80.
2.3. Effect of phase volume ratio (water to W/O), the concentration of
hydrophilic emulsifier, thickeners and tonicities on the generation of W/O/
W multiple nanoemulsion
80:20 and 90:10 of phase volume ratio consisting of deionized
water:W/O nanoemulsion on W/O/W nanoemulsion was evaluated. For
this, 40% amplitude with a total irradiation time of 10, 30, 45, 60, 90,
120, 150, 180, 220, and 240 s was employed.
For the effect of concentration of hydrophilic emulsifier on the
formation of W/O/W multiple nanoemulsion, 0.5 wt%, 1.0 wt%, and
2.0 wt%, of Tween 80 were compared. 40% acoustic amplitude and 180
s of irradiation time were employed.
For the effect of thickener on the stability of W/O/W multiple na-
noemulsion, oil-based thickener (Sucragel), water-based thickener
(Xanthan gum), and without the addition of thickener were compared.
To thicken the oil phase, firstly, during the formation of W/O
R. Raviadaran, et al.
nanoemulsion, 1 wt% Sucragel was included in the oil phase. Whereas,
for the thickening of the water phase, Xanthan gum at 1 wt% was added
to the internal water phase during the formation of W/O nanoemulsion.
This was followed by the evaluation of the stability of W/O/W multiple
nanoemulsion with varying concentrations of oil-based Sucragel at
0.5 wt%, 1 wt%, and 2.0 wt%.
The effect of hypotonicity, isotonicity, and hypertonicity on the
stability of W/O/W multiple nanoemulsion was evaluated. W/O na-
noemulsion contains 1.0 M NaCl in the internal water phase. Hence, to
simulate hypotonicity, isotonicity, and hypertonicity, the external
water phase with 0.5 M, 1.0 M, and 2.0 M NaCl, respectively, were used
when formulating W/O/W multiple nanoemulsion.
2.4. Analyses
Among the performed analyses for the generated W/O/W multiple
nanoemulsion were peroxide value, Fourier infrared transform spec-
troscopy (FTIR), fatty acid compositions, droplet size, dispersity index,
zeta potential, viscosity, conductivity, and water content. Peroxide
value and FTIR analyses were conducted to determine the formation of
primary oxidation products and secondary oxidation products, respec-
tively. Fatty acid composition analysis was performed to ascertain the
changes in fatty acid composition after being processed under ultra-
sound and microfluidizer. The droplet size and dispersity index were
performed to compare the sizes of the formed droplets using ultrasound
and microfluidizer. Viscosity, conductivity and water content analyses
were performed to evaluate the stability of the generated W/O/W
multiple nanoemulsion. For water content, the separated oil phase
(supernatant) after centrifuged at 15000 rpm for 15 min was used for
the analysis. The images and morphology of the generated W/O/W
multiple nanoemulsion were also analysed using a light microscope and
field emission scanning electron microscope, respectively. The detailed
explanations for these analyses and the utilized equipment can be ob-
tained from the previously published work [6].
3. Results and discussion
3.1. Comparison of processing W/O/W nanoemulsion using ultrasound and
microfluidizer
3.1.1. Generation of W/O/W nanoemulsion using ultrasound
As shown in Fig. 1, the MDD of W/OW multiple nanoemulsion
droplets decreased gradually with increased irradiation time and
acoustic amplitude. As the sonication intensity increases, the cavitation
activity is amplified, enhancing the droplet breakup [41]. Thus, the
obtained droplets with smaller MDD in W/O/W multiple nanoemulsion
agree with other reported studies [42,43].
Furthermore, a plateau in MDD at 243.3 ± 10.7 nm with a 40%
amplitude and 180 s of irradiation time was observed. This is the
Fig. 1. Effect of irradiation time towards droplet size at different amplitudes.
3
Ultrasonics - Sonochemistry 64 (2020) 104995
optimum power level required at 1% Tween 80 for achieving the
maximum droplet break-up. Fig. 2 shows the microscopic images ob-
tained for the W/O/W multiple nanoemulsion droplets. These images
demonstrate the reduction in MDD of W/O/W multiple nanoemulsion
with increased irradiation time and amplitude. The droplets of W/O/W
multiple nanoemulsion became more monodispersed with an increase
in irradiation time.
Nevertheless, a further increase in amplitude to 50% also produced
the smallest droplet size, i.e., MDD of ~240 nm. However, droplets with
MDD of ~ 240 nm were obtained much faster with an irradiation time
of 150 s. Also, at 50% amplitude, there was an increase in the MDD of
W/O/W multiple emulsion at a higher irradiation time of 240 s. Higher
power could lead to the break-up of internal water droplets, as shown in
Supplementary Fig. 1 [2,28]. As the cavitation intensity increases, the
power generated renders instability, especially at the interphase of the
internal water droplets. It is suggested that the instability leads to the
coalescence and droplet enlargement in the internal water droplets
during recoalescence and stabilization at the interphase during pro-
cessing [41]. This causes a rupture in the oil droplets and hence the loss
of internal water droplets, as illustrated in Supplementary Fig. 1. This is
also confirmed with the increase in DI by 89.8% at 50% amplitude as
the irradiation time increases. Thus, in this work, an optimal power at
40% amplitude is more favourable compared to the 50% amplitude for
the formation of W/O/W multiple emulsion. Droplet size of
243.3 ± 10.7 nm and DI of 0.248 ± 0.011 respectively, were
achieved by utilizing 1 wt% Tween 80 and optimized ultrasound at 40%
acoustic amplitude and a total irradiation time of 180 s respectively, as
tabulated in Supplementary Table 1.
3.1.2. Generation of W/O/W nanoemulsion using microfluidizer
The MDD of W/O/W droplets decreased gradually with the in-
creased number of cycles and operating pressure, as demonstrated in
Fig. 3. Higher pressure enhances the shearing rate of droplets, resulting
in the reduction of droplet sizes [41]. Similar findings were also ob-
tained in other studies [43,44].
At an operating pressure of 350 bar, a plateau was noticed after 8
cycles. This is the optimum power level required for a maximum dro-
plet breakup obtained with an MDD of 233.2 ± 7.3 nm using 1 wt%
hydrophilic surfactant (Tween 80). Fig. 4 shows the microscopic images
of W/O/W multiple emulsion droplets. The images exhibit reduced
droplet size with an increase in the number of cycles and operating
pressure. Droplets of multiple emulsions are seen to become more
monodispersed as the number of cycles increased.
Increased operating pressure beyond 450 bar gave an optimum
droplet breakup of ~230 nm, much uniform even with 6 cycles. The
higher power could have led to a reduction in the number of cycles
required. Similar to ultrasonication at a higher amplitude of 50%, mi-
crofluidization at 450 bar with the increasing number of cycles led to an
increase in MDD and DI by 11.1% and 45.2%, respectively. As men-
tioned previously in Section 3.1.1, the increasing operating pressure
and number of cycles render instability, especially at the interphase of
the internal water droplets. It is suggested that the instability leads to
coalescence and droplet enlargement in the internal water droplets
during recoalescence and stabilization at the interphase during pro-
cessing [41]. This causes a rupture in the oil droplets and hence the loss
of internal water droplets, as illustrated in Supplementary Fig. 1. This
phenomenon is suggested for the increase in MDD and DI. Thus, 350 bar
of operating pressure is advantageous over 450 bar. As shown in
Supplementary Table 2, the droplet sizes of 233.2 ± 7.3 nm and DI of
0.205 ± 0.008 were generated using microfluidizer at 350 bar of
operating pressure with 8 cycles.
3.1.3. Energy consumption
At optimum conditions, ultrasound and microfluidizer generated
W/O/W nanoemulsion with a final droplet size of 243.3 ± 10.7 nm
and 233.2.5 ± 7.3 nm, respectively. The ultrasound and microfluidizer
R. Raviadaran, et al.
Fig. 2. Images of W/O/W multiple nanoemulsion with increased irradiation time and amplitude.
Fig. 3. Effect of the number of cycles towards droplet size.
utilized 4.62 kJ and 53.71 kJ of energy consumption, respectively, to
obtain W/O/W multiple nanoemulsion with the droplet size of
~230 nm. Higher energy consumption by ~12 times was required by
microfluidizer as compared to ultrasound to generate a comparable
droplet size of ~235 nm. A similar outcome was reported by Tang et al.
[43], where ultrasound required lesser energy by 18 times than mi-
crofluidizer to produce W/O/W nanoemulsion. When comparing to this
study, 35% lower energy was required by the study reported by Tang
et al. [43] to formulate W/O/W multiple nanoemulsion using
4
Ultrasonics - Sonochemistry 64 (2020) 104995
ultrasound. The difference in the required energy could be attributed to
the utilization of less viscous Lauroglycol 90 oil as compared to the
palm oil used in this study. Less viscous oil would enable better pro-
pagation of acoustic waves and hence resulting in the reduction of the
required energy to obtain the optimum droplet diameter. For producing
W/O/W multiple nanoemulsion using microfluidizer, 5% higher energy
consumption was reported by Tang et al. [43] as compared to this
study. This could be associated with different emulsifiers used in these
studies. Tang et al. [43] utilized Cremophor EL, while PGPR was uti-
lized in this study. The utilization of PGPR emulsifier in the present
study, which has strong anchoring ability into the chains of fatty acid of
oil, ensures good elasticity at the interface that could resist the re-
coalescence of internal water droplets during microfluidization. This
could have led to a higher energy requirement for producing W/O/W
multiple nanoemulsion using microfluidizer as compared to the study
reported by Tang et al. [43].
3.1.4. Oxidation of W/O/W nanoemulsion
Three analyses were performed to determine the oxidation of W/O/
W nanoemulsion processed using microfluidizer and ultrasound. Firstly,
the peroxide value of the formed W/O/W nanoemulsion using ultra-
sonication and microfluidization remained unchanged in comparison to
the coarse W/O/W emulsion at ~15 meq/kg, as shown in
Supplementary Table 3. This shows that the primary oxidation products
were absent when W/O/W emulsion was processed using ultrasound
R. Raviadaran, et al. Ultrasonics - Sonochemistry 64 (2020) 104995

Fig. 4. Images of W/O/W multiple nanoemulsion with the increasing number of cycles and operating pressure.

and microfluidizer. stretching off C]O at 2850–2800 cm−1) were not detected. This shows
Secondly, from the fatty acid composition analysis of capric acid that the processed W/O/W multiple emulsion did not oxidize under
(C10:0), lauric acid (C12:0), myristic acid (C14:0), palmitic acid ultrasound and microfluidizer. Similar findings of the absence of oxi-
(C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), dation products were also reported in these studies [45–47].
and linolenic acid (C18:3), < 3.33% of maximum difference was ob-
served between before and after processing under both the equipment,
3.2. Effect of water to W/O phase volume ratio towards the generation of
independently as shown in Supplementary Table 4. The minimal
W/O/W nanoemulsion at 40% amplitude
change observed could have resulted from the instrumental measure-
ment errors. This result shows that the fatty acid compositions of palm
Fig. 6 shows the comparison between the phase volume ratio of
oil were not altered due to processing under ultrasound and micro-
water:W/O, at 90:10 and 80:20. A reduction in the MDD with an in-
fluidizer.
crease in the irradiation time in both the phase volume ratios has been
Thirdly, FTIR analysis was performed to ascertain any changes in
noted. However, for a phase volume ratio of 90:10, a plateau for the
terms of functional groups before and after the processing under ul-
MDD of W/O/W multiple nanoemulsion was observed at 180 s, whereas
trasound and microfluidizer, independently. From the performed three
for a phase volume ratio of 80:20, a plateau was not observed up to
analyses to determine the oxidation of processed W/O/W nanoemul-
240 s. At 180 s of irradiation time, the MDD obtained at 90:10 was
sion, FTIR analysis is the most important. This is because if oxidation
smaller by 49.8% as compared to 80:20. Also, the DI was larger by
has taken place, it will eventually lead to the formation of secondary
177.0% for the phase volume ratio of 80:20 compared to 90:10. The
oxidation products, which could be detected from the FTIR spectra. As
optimal power at 40% amplitude and 180 s of irradiation time were
shown in Fig. 5, the functional groups before and after processing under
insufficient for the phase volume ratio of 80:20 to attain plateau with
both equipment independently remained unchanged. Importantly, the
smaller MDD and DI due to the presence of a higher volume of W/O
key functional groups for secondary oxidation products such as car-
compared to the phase volume ratio of 90:10. In line with the above,
boxylic acid (hydrogen-bonded OeH stretching at 3400–2400 cm−1
Onuki et al. [48] reported that a decrease in the phase volume ratio of
and C]O stretching at 1730–1650 cm−1), ketones (C]O stretching at
water to W/O from 85.7 to 66.7% resulted in a larger droplet size by
1750–1625 cm−1) and aldehydes (CeH stretching off C]O at
43.7%. The authors suggested that this could be due to the agglom-
2750–2700 cm−1, C]O stretching at 1750–1625 cm−1 and CeH
eration of inner droplets. Similar findings were also observed in other

5
R. Raviadaran, et al. Ultrasonics - Sonochemistry 64 (2020) 104995

Fig. 5. FTIR spectra of W/O/W multiple nanoemulsion generated utilizing ultrasonication and microfluidization.

increase in MDD and DI.

Furthermore, after 1 week, at 1 wt%, 3 wt%, and 5 wt% of Tween
80, there was a change in MDD by 10.49%, 46.84%, 100.92%, and DI
by 19.48%, 51.48%, and 121.81% respectively. As the concentration of
Tween 80 increased, the MDD and DI increased steeply, which agree
with other studies [50,51]. When the concentration of hydrophilic
emulsifier used is higher than the critical micelle concentration, both
the internal hydrophobic emulsifier and the external hydrophilic
emulsifier migrate. This movement causes instability at the oil-water
interphases, hence, an increase in the MDD and DI. Jiao and Burgess
[50] also elaborated that the micelles may carry the hydrophobic sur-
factant into the external continuous water phase; hence, the rupturing
of the oil globules and loss of internal water droplets occur. Therefore,
1 wt% is the best concentration of hydrophilic surfactant (Tween 80)
Fig. 6. Effect of 40% acoustic amplitude and irradiation time towards droplet that can be used to formulate W/O/W multiple nanoemulsion.
size.

3.4. Effect of thickener on the formation of W/O/W multiple nanoemulsion

studies [5,49].
3.3. Effect of concentration of hydrophilic surfactant (Tween 80) on the
stability of W/O/W multiple nanoemulsion
The MDD and DI were larger with 0.5 wt% stabilized W/O/W
multiple nanoemulsion compared to 1 wt% stabilized W/O/W multiple
nanoemulsion, as shown in Table 1. The percentage change of MDD and
DI after 1 week, was higher for 0.5 wt% Tween 80 stabilized W/O/W
multiple nanoemulsion as compared to 1 wt% Tween 80 stabilized W/
O/W multiple emulsion. At an optimal ultrasonic amplitude of 40%,
and 180 s of irradiation time, 0.5 wt% Tween 80 was insufficient in
obtaining a smaller droplet size compared to 1 wt% Tween 80. There-
fore, coalescence prevailed at the water-oil interphase leading to an
3.4.1. Comparison of the stability of W/O/W multiple nanoemulsion
stabilized by oil-based thickener (Sucragel), water-based thickener
(Xanthan gum) and no thickener
For the formation of W/O nanoemulsion, as shown in Table 2, oil-
based thickened W/O nanoemulsion produced the smallest MDD in
comparison to W/O nanoemulsion with no thickener and water-based
thickened W/O nanoemulsion. Also, comparatively, the oil-based
thickened W/O nanoemulsion had the smallest change in MDD and DI
after 1 week. Majorly, this is suggested due to the presence of Sucragel
in the oil phase, which is composed of glycerine, sucrose laurate, Prunus
Amygdalus Dulcis (Sweet Almond) oil and Citrus Aurantium Dulcis fruit
water that enhanced hydrogen bonding and fatty acid-binding with
PGPR stabilized at the palm oil-water interphase. The sucragel

Table 1
Comparison of the concentration of hydrophilic emulsifier (Tween 80) on the formation of W/O/W multiple nanoemulsion.
Concentration of Tween 80 (wt%) W/O/W multiple nanoemulsion Initial 1 week Percentage change

0.5 Mean droplet diameter (nm) 355.06 ± 7.8 407.7 ± 12.9 14.85
Dispersity index 0.368 ± 0.037 0.426 ± 0.029 15.76
1 Mean droplet diameter (nm) 212.6 ± 2.6 234.9 ± 2.1 10.49
Dispersity index 0.231 ± 0.032 0.276 ± 0.041 10.82
3 Mean droplet diameter (nm) 166.5 ± 3.5 244.5 ± 3.4 46.84
Dispersity index 0.235 ± 0.016 0.356 ± 0.004 51.48
5 Mean droplet diameter (nm) 163.7 ± 2.5 328.9 ± 2.3 100.92
Dispersity index 0.188 ± 0.009 0.417 ± 0.015 121.81

6
R. Raviadaran, et al.
Table 2
Comparison of different thickeners on the formation of W/O nanoemulsion and W/O/W multiple nanoemulsion.
Type of thickener W/O nanoemulsion
Initial
No thickener Mean droplet diameter (nm) 143.1 ± 8.8
Dispersity index 0.102 ± 0.062
Oil thickener Mean droplet diameter (nm) 97.6 ± 3.23
Dispersity index 0.137 ± 0.025
Internal water thickener Mean droplet diameter (nm) 132.6 ± 1.1
Dispersity index 0.323 ± 0.014
solubilized well within palm oil, which improves the binding strength.
Besides, the water thickened W/O nanoemulsion had droplets with
larger MDD than the oil thickened W/O nanoemulsion, which could be
linked to the higher viscosity of the water thickened nanoemulsion in
comparison to the oil thickened W/O nanoemulsion, as shown in
Supplementary Table 5. The high propagation of acoustic waves in a
less viscous medium provides stronger cavitation-induced forces [43].
Furthermore, water thickener (Xanthan gum) could not improve the
stability at the oil-water interphase as compared to the oil thickener
(Sucragel) due to the absence of multiple OH groups and fatty acid
chains.
As shown in Table 2, both the water thickened and oil thickened W/
O nanoemulsion had a smaller change in MDD and DI after 1 week in
comparison to the non-thickened W/O nanoemulsion. The thickened
W/O nanoemulsion was more stable as it improves physicochemical
interaction by forming the complex stable networks [2,52,53].
However, it is to be noted that DI was smaller for the non-thickened
W/O nanoemulsion than both the oil and water thickened W/O na-
noemulsions. This is due to the influence of the viscosities in W/O
nanoemulsions in obtaining a more uniform MDD at the same optimal
power and irradiation time. At higher viscosity, cavitation-induced
disruptive forces are reduced. Regarding the formation of W/O/W
multiple nanoemulsion, from Supplementary Table 5, it is to be noted
that despite a lower viscosity, the droplet size of the non-thickened W/
O/W multiple nanoemulsion was much larger than both the water and
oil thickened W/O/W multiple nanoemulsion. Thus, it is suggested that
the internal water thickened phase and the oil thickened globule en-
abled greater strength at the inner water–oil interphase that assisted the
formation of W/O/W multiple nanoemulsion with smaller MDD and DI
especially during recoalescence and stabilization with high power
processing. The thickened entities imparted better strength that could
withstand high-power processing than the non-thickened.
This is the first study which utilized water content analysis to
evaluate the loss of entrapped internal water molecules, as shown in
Table 3. An increase in the internal water content was 25.57%, 7.56%,
and 12.47% for non-thickened, oil-thickened, and water-thickened W/
O/W multiple nanoemulsion, respectively after 1 week. The increase in
the internal water content was the smallest for the oil thickened W/O/
W multiple nanoemulsion. The thickened oil could retain and protect
the internal water droplets within the oil phase by forming a stable and
robust network within the oil globules and hence, better stability at the
oil-water interphase. Sucragel’s solubility in palm oil increased the
Table 3
Comparison of the water content in W/O/W multiple nanoemulsion with dif-
ferent thickeners.
W/O/W nanoemulsion Water content (%)
Initial After 1 week
No thickener 0.8252 ± 0.0023 1.0362 ± 0.0029
Oil thickener 0.8268 ± 0.0031 0.8893 ± 0.0046
Internal water thickener 0.8139 ± 0.0008 0.9208 ± 0.0071
7
Ultrasonics - Sonochemistry 64 (2020) 104995
W/O/W multiple nanoemulsion
1 week Initial 1 week
156.2 ± 6.5 242.9 ± 1.4 281.9 ± 3.6
0.283 ± 0.051 0.247 ± 0.025 0.318 ± 0.055
102.9 ± 1.4 175.4 ± 4.3 197.9 ± 1.4
0.219 ± 0.020 0.225 ± 0.005 0.263 ± 0.023
141.4 ± 1.9 182.7 ± 1.7 228.9 ± 2.0
0.467 ± 0.037 0.324 ± 0.037 0.467 ± 0.014
viscosity of the oil phase, and enhanced stability by co-interaction with
PGPR at water-oil interphase is the key to the formation of the smallest
MDD and DI. Many studies were reported with improved stability of W/
O/W multiple emulsion with the thickening of inner water; however, oil
phase thickening was hardly evaluated [28,31,32,54]. Inner water
thickening is more suitably used to fulfil the objective of improving
sustained release than improving the stability in W/O/W multiple na-
noemulsion.
Hence, the oil phase thickened W/O/W multiple nanoemulsion was
most stable. However, there was a change in MDD and an increase in
the water content by 12.82% and 7.56%, respectively. Therefore, an
optimal thickening concentration and tonicity stabilization would be
required to further improve the stability of W/O/W multiple nanoe-
mulsion, as elaborated in sections 3.4.2 and 3.5.
3.4.2. Effect of the concentration of oil-soluble thickener (Sucragel) towards
the generation of W/O/W multiple nanoemulsion
From Table 4, it could be noted that there is an increase in MDD by
3.2% in the W/O/W multiple nanoemulsion formulated with 0.5 wt%
Sucragel in comparison to W/O/W multiple nanoemulsion formulated
with 1.0 wt% Sucragel. Also, there is an increase in MDD by 17.03%
after 1 week in the case of W/O/W multiple nanoemulsion formulated
with 0.5 wt% Sucragel, whereas, with 1.0 wt% Sucragel, the increase is
12.8%. Furthermore, as shown in Table 4, the increase in water content
after 1 week in 0.5 wt% and 1.0 wt% Sucragel was 14.25% and 7.56%,
respectively. Sucragel at a concentration of 1.0 wt% had better re-
sistance to an increase in the water content and change in MDD. The
concentration of Sucragel at 0.5 wt% was insufficient in forming a
structured network at the water-oil interphase. The increase in visc-
osity, as shown in Supplementary Table 6, is small, hence, not able to
retain the internal water droplets within the oil globule due to the
absence of a rigid and packed network in the oil phase.
Whereas, W/O/W multiple nanoemulsion formulated with Sucragel
at 2.0 wt% produced a larger MDD by 21.09% compared to W/O/W
multiple nanoemulsion formulated with 1.0 wt% Sucragel. The DI was
also larger in W/O/W multiple emulsion formulated with 2.0 wt% in
comparison to the W/O/W multiple nanoemulsion formulated with
1.0 wt% Sucragel, as given in Table 4. Nevertheless, the increase in
water content is 7.56% and 2.51% in W/O/W multiple nanoemulsion
formulation using 1.0 wt% and 2.0 wt% Sucragel, respectively. The W/
O/W multiple nanoemulsion formulated with 2.0 wt% Sucragel had an
increase in MDD and DI by 14.03% and 34.81%, respectively, after
1 week. An increase in the viscosity of W/O/W multiple nanoemulsion,
as given in Supplementary Table 6, reduced the propagation of acoustic
waves resulting in larger MDD and DI. The higher DI promoted floc-
culation and coalescence in the W/O/W multiple nanoemulsion,
leading to an increase in MDD and DI after 1 week. However, as the
viscosity is the highest in W/O/W multiple nanoemulsion formulated
by 2 wt% Sucragel, the increase in water content was comparatively
minimal. The higher viscosity retained the water droplets and
strengthened the interaction at the water-oil interphase.
Therefore, 1 wt% Sucragel is the most suitable for the formation of
W/O/W multiple nanoemulsion as the increase in viscosity improved
R. Raviadaran, et al. Ultrasonics - Sonochemistry 64 (2020) 104995

Table 4
Comparison of the stability of W/O/W multiple nanoemulsion at different concentrations of Sucragel.
Concentration ofSucragel (wt%) W/O/W multiple nanoemulsion Initial After 1 week

0.5 Mean droplet diameter (nm) 181.2 ± 4.3 212.9 ± 8.7

Dispersity index 0.234 ± 0.072 0.322 ± 0.025
Water content (%) 0.8142 ± 0.0021 0.9302 ± 0.0018
1.0 Mean droplet diameter (nm) 175.4 ± 4.3 197.9 ± 1.4
Dispersity index 0.225 ± 0.005 0.263 ± 0.023
Water content (%) 0.8268 ± 0.0031 0.8893 ± 0.0046
2.0 Mean droplet diameter (nm) 212.4.1 ± 4.8 242.2 ± 2.3
Dispersity index 0.362 ± 0.008 0.488 ± 0.005
Water content (1%) 0.8314 ± 0.0012 0.8523 ± 0.0028

Table 5
Conductivity values of W/O/W multiple nanoemulsion at different tonicities.
Osmotic condition Conductivity values (µ/s)

Day

Initial 2 4 6 8 10

Hypotonic 15.44 ± 2.1 16.35 ± 1.8 17.78 ± 1.2 19.95 ± 2.1 20.31 ± 1.1 22.75 ± 1.1
Isotonic 27.10 ± 1.4 26.78 ± 1.1 26.88 ± 1.3 26.74 ± 1.2 26.42 ± 1.3 26.35 ± 1.0
Hypertonic 53.81 ± 1.6 50.22 ± 2.1 47.76 ± 1.1 44.23 ± 1.3 42.85 ± 1.2 41.90 ± 1.2

Table 6
Comparison of the concentrations of NaCl in W/O/W multiple nanoemulsion.
Osmotic condition Initial After 10 days

Concentration of NaCl (mol/dm3) Water content (%) Concentration of NaCl (mol/dm3) Water content (%)

Hypotonic 0.522 0.8368 ± 0.0044 0.782 0.8711 ± 0.0054

Isotonic 0.913 0.8235 ± 0.0058 0.937 0.8268 ± 0.0031
Hypertonic 1.890 0.8282 ± 0.0038 1.464 0.7703 ± 0.0087

Table 7
Effect of different tonicities towards the droplet size of W/O/W multiple nanoemulsion.
Osmotic condition W/O/W multiple nanoemulsion Initial After 1 week

Hypotonic Mean droplet diameter (nm) 175.3 ± 4.8 182.9 ± 7.2

Dispersity index 0.214 ± 0.013 0.308 ± 0.077
Isotonic Mean droplet diameter (nm) 172.2 ± 5.4 174.5 ± 9.8
Dispersity index 0.225 ± 0.008 0.232 ± 0.012
Hypertonic Mean droplet diameter (nm) 182.6 ± 3.8 172.4 ± 6.7
Dispersity index 0.281 ± 0.014 0.235 ± 0.022

water retention and produced the optimal MDD and DI. However, the external water phase. The isotonic condition has a balanced water po-
tonicity would need to be further controlled to improve the water tential with none/minimal water movement.
movement, as discussed in section 3.5. W/O/W multiple nanoemulsion simulating the hypotonic condition
showed an increasing trend in the conductivity values when analysed
for 10 days, as given in Table 5. The initial concentration of NaCl was
3.5. Effect of hypotonicity, isotonicity, and hypertonicity on the stability of
0.522 M, and it increased to 0.782 M after 10 days, as given in Table 6.
W/O/W multiple nanoemulsion
The concentration of NaCl was obtained based on Supplementary Fig. 3,
which showed the calibration of conductivity values for a concentration
Three conditions, i.e., hypotonic, isotonic, and hypertonic, in the
of NaCl solution between 0 and 2.5 M. The obtained best-fit line was
W/O/W multiple nanoemulsion were simulated, where hypotonic is a
used to calculate the concentration of NaCl in the external water phase
condition with the concentration of NaCl in the external water phase
using the obtained conductivity values of W/O/W multiple nanoemul-
(0.5 M) is lower than the internal water phase (1.0 M). Isotonic is a
sion. Also, as shown in Table 7, after 1 week, there was an increase in
condition where both the external and internal water phases have si-
MDD by 4.1% for the hypotonic condition. There was also an increase
milar concentrations of NaCl (1.0 M). Lastly, hypertonic is a condition
of 4.1% of water content in the internal water phase. Based on osmosis,
where the external water phase (2.0 M) has a concentration of NaCl
since the water potential is higher in the external phase than the in-
higher than the internal water phase (1.0 M). Supplementary Fig. 2
ternal phase, the water moves from the external to the internal phase
shows the movement of water in and out of both the internal and ex-
during the hypotonic condition. Thus, this explains the increase in
ternal water phases of a W/O/W multiple nanoemulsion at different
MDD, conductivity, and concentration of NaCl in the W/O/W multiple
tonicity conditions, where hypotonic promotes the movement of water
nanoemulsion.
from the external water phase to internal water phase. While hyper-
For the isotonic condition, a stable trend was observed with a
tonic promotes water movement from the internal water phase to the

8
R. Raviadaran, et al.
Fig. 7. STEM image of W/O/W multiple nanoemulsion.
change of 0.75µ/s between the highest and lowest conductivity values
in 10 days, as given in Table 5. The concentration of NaCl initially and
after 10 days was 0.913 M and 0.937 M, respectively, as calculated
using the best fit line. Table 7 shows both the MDD and DI had an
increase of 1.3% and 3.0%, respectively, after 10 days. There was an
increase of 0.40% in the internal water content after 10 days, as given
in Table 6. Due to the similar concentration in NaCl at 1.0 M in the
internal and external phases, the water potential was similar; hence, it
reduced the movement of water in and out of both the internal and
external water phases.
Whereas, the hypertonic condition showed a decreasing trend in the
conductivity values. This reflected in the initial concentration of NaCl
at 1.890 M, decreasing to 1.464 M after 10 days, calculated using the
best fit line from Supplementary Fig. 3. There was a decrease of 5.58%
in the MDD of W/O/W multiple nanoemulsion for hypertonic condi-
tions, as given in Table 7. There was a decrease of 7% in the internal
water content, as shown in Table 6. Due to osmosis, the water potential
is higher in the inner water phase in comparison to the external water
phase. Hence, water moves from the internal to the external phase re-
sulting in the decrease in the conductivity and concentration of NaCl
and MDD in the W/O/W multiple nanoemulsion.
Thus, for the oil thickened W/O/W multiple nanoemulsion, the
isotonic condition provided the best stability. Zeta potential of
−29.9 ± 1.8 mV was obtained. Supplementary Table 7 shows the final
optimized parameters with oil thickener (Sucragel) at 1 wt% and iso-
tonicity used to generate stable W/O/W multiple nanoemulsion. Cor-
roborating with this work, Zhang et al. [55] formed W/O/W multiple
emulsion that was isotonically-stabilized with NaCl and they reported
achieving stability for at least 2 months.
3.6. Analysis of W/O/W multiple nanoemulsion using STEM
Fig. 7 shows the STEM image of W/O/W multiple nanoemulsion,
where there is an internal water droplet entrapped within the oil dro-
plet. The inner water droplet has an average diameter of ~100 nm,
while the oil globule has an average diameter of ~190 nm. The en-
trapped water droplet indicates the successful formulation that com-
plies with the MDD and DI obtained at 175.5 ± 9.8 nm and
0.232 ± 0.012, respectively, in the final optimized W/O/W multiple
nanoemulsion. However, the increase in MDD could be related to
sample preparation procedure involving staining using phosphotungstic
9
Ultrasonics - Sonochemistry 64 (2020) 104995
acid solution. Tang et al. [54] indicated this type of multiple emulsion
formation as the single-core W/O/W multiple emulsion.
4. Conclusions
Stable palm oil-based W/O/W multiple nanoemulsion was success-
fully formed with the optimized ultrasound conditions of 40% ampli-
tude and 180 s of irradiation time, whereas in the case of micro-
fluidizer, it was 350 bar and 8 cycles. Ultrasound was more energy
saving by 12 times as compared to microfluidizer. Palm oil in W/O/W
multiple nanoemulsion processed both by ultrasound, as well as mi-
crofluidizer, did not oxidize as there was no change in the peroxide
values and fatty acid composition, and FTIR indicated the absence of
primary and secondary oxidation products. The formulation with 90:10
of phase volume ratio was more stable with smaller MDD and DI as
compared to 80:20. A higher concentration of hydrophilic emulsifier
(Tween 80) led to instability in the W/O/W multiple nanoemulsion
with larger MDD and DI due to the enhanced migration of emulsifiers
between the internal and external water–oil interphases. This is the first
study that successfully utilized Sucragel in imparting enhanced stability
in the W/O/W multiple nanoemulsion as the thickened oil retained and
protected the internal water droplets within the oil phase by forming a
packed and stable network inside the oil globules and rigid stability at
the oil-water interphase. Its solubility in palm oil increased viscosity of
the oil phase, and the enhanced stability by co-interaction with PGPR at
the internal water–oil interphase were the keys to the formation of the
smallest MDD and DI. Isotonic stabilized W/O/W multiple nanoemul-
sion gave the lowest change in MDD, the concentration of NaCl and
water content by 1.5%, 2.6%, and 0.4%, respectively, due to the re-
duced movement of water between the internal and external water
phases. The final optimized formation of isotonic stabilized W/O/W
multiple emulsion with 1 wt% Sucragel had MDD and DI of
175.5 ± 9.8 and 0.232 ± 0.012, respectively. Based on the outcome,
future studies should be directed to improve the stabilization of W/O/
W multiple nanoemulsion by thickening the oil phase and isotonicity
modification. Sucragel is a natural oil-based thickener that interacts
well with palm oil in forming a stable W/O/W multiple nanoemulsion.
The encapsulation and loading efficiency of tocotrienol and caffeic acid
would be considered in future work.
Acknowledgements
The resources provided by The University of Nottingham (Malaysia
Campus), Malaysian Palm Oil Board, and Sunway University to facil-
itate this study are acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ultsonch.2020.104995.
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