Colloids and Surfaces B: Biointerfaces 199 (2021) 111481

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Colloids and Surfaces B: Biointerfaces

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

Improving the oxidative stability of fish oil nanoemulsions by

co-encapsulation with curcumin and resveratrol
Qayyum Shehzad a, b, Abdur Rehman a, c, Seid Mahdi Jafari d, Min Zuo b,
Muhammad Aslam Khan a, Ahmad Ali a, f, Sohail Khan a, Aiman Karim a, Muhammad Usman e,
Arif Hussain a, Wenshui Xia a, *
a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China
b
National Engineering Laboratory for Agri-Product Quality Traceability, Beijing Technology and Business University, Beijing, China
c
Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, Jiangsu, 214122, China
d
Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural Science and Natural Resources, Gorgan, Iran
e
Beijing Engineering and Technology Research Center of Food Additives, School of Food and Chemical Engineering, Beijing Technology and Business University, Beijing,
100048, China
f
College of Biosystems Engineering and Food Science, Zhejiang University, Hanzhou, 310058, China

A R T I C L E I N F O A B S T R A C T

Keywords: Tuna fish oil (TFO), is a rich source of omega-3 fatty acids comprising particularly docosahexaenoic acid (DHA)
Tuna fish oil and eicosapentaenoic acid (EPA), which are essential for the human’s health enhancement. However, their
Modified starch foremost problems are poor solubility, low bioavailability, and easy oxidization, which limit their wide range of
Curcumin
applications in food and pharmaceutical products. The aim of this research is to develop TFO nanoemulsions
Resveratrol
encapsulating curcumin (CUR) and resveratrol (RES) as co-antioxidants, stabilized with different concentrations
Nanoemulsions
Oxidative stability of two modified starches including Purity Gum 2000 (PG) and Purity Gum Ultra (PGU). The effect of diverse
Antioxidant activity quantities of modified starches on droplet size, droplet charges, viscosity, and oxidative stability of produced
nanoemulsions was assessed at different storage temperatures (4, 25, and 40 ◦ C). Furthermore, to assess the
antioxidant activity and capacity of loaded nanoemulsions, DPPH, and ABTS assays were used, respectively.
Among various samples, PGU2.5 and PG9 emulsified by PGU and PG2000 had showed the premium results of
stability during storage at all temperatures over other formulations. Even at 40 ◦ C, 199.10 μL/mL of PGU2.5 and
258.59 μL/mL of PG9 were required to attain the level of DPPH IC50, which were the lowest concentration as
compare to other nanoemulsions. Taken together, it was accredited that co-encapsulation of CUR and RES inside
the TFO nanoemulsion-based delivery systems can be efficient for the production of functional foods.

1. Introduction and susceptibility to oxidation as well as low bioavailability and unfa­

Tuna fish oil (TFO) is abundantly comprised of polyunsaturated fatty
acids particularly DHA (Docosahexaenoic acid) and EPA (Eicosa­
pentaenoic acid). These fatty acids are very important and recognized as
essential nutrients for human’s diet [1]; because they can improve the
human health by preventing from several serious disorders, such as
cardiovascular disease, cancer, inflammation, arthritis, diabetes, al­
lergies and Alzheimer disease [2–6]. Also, they assist in improving the
infant’s nervous system and visionary development. Despite its
health-promoting benefits and disorders prevention attributes, TFO
cannot be used directly into the food systems, due to its lower solubility
vorable sensory properties [7]. For this purpose, the encapsulation
strategy could be an effective tactic for enriching food matrices based on
TFO nanoemulsions encapsulating DHA and EPA [8].
Nanostructured delivery assemblies, such as nanoemulsions are
beneficial and efficient approaches that can protect, transport, and
resolve the insolubility problems of bioactive [9]; alternatively, they can
improve the bioaccessibility/bioavailability of lipophilic bioactives and
their controlled delivery in aqueous suspensions [10,11]. Nano­
emulsions are defined as colloidal delivery systems having droplet sizes
< 500 nm; the reduced size of nanoemulsions provides a greater surface
area, which improves the interaction of entrapped bioactive compounds

* Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Jiangsu, Wuxi, 214122, China.
E-mail address: xiaws@jiangnan.edu.cn (W. Xia).

https://doi.org/10.1016/j.colsurfb.2020.111481
Received 1 August 2020; Received in revised form 11 October 2020; Accepted 15 November 2020
Available online 10 December 2020
0927-7765/© 2020 Published by Elsevier B.V.
Q. Shehzad et al.
with emulsifier-based suspensions leading to an increase in oxidation
phenomenon [12]. Therefore, numerous kind of secondary oxidative
products, including ketones and aldehydes, not only decrease the
nutritional profile and sensory characteristics of food items but also may
leave negative impacts on human health [13].
Oxidative stability of bioactive-loaded nanoemulsions can be
improved via adding antioxidants to the system that are able to retard
the production of oxidative products through chelating the metal ions or
radical scavenging activities. In this context, there are many synthetic
and natural antioxidants available in the market; because of the concern
of safety issues and human’s demand for healthier food, synthetic
antoxidants are not appreciated by the consumers. Therefore, the use of
natural plant-based antioxidants such as phenolic compounds have
gained much attention owing to their health encouraging declarations as
well as accepted stimulating taste [14]. Curcumin (CUR) and resveratrol
(RES) (lipophilic polyphenols) are known for their wide range of po­
tential biological activities, such as antioxidant activity,
anti-inflammatory, anti-cancer, anti-diabetic and anti-aging effects as
well as a strong ability to protect us from cardiovascular and central
nervous diseases [15–17]. Therefore, they have successfully gained
great fame in the arena of food and pharmaceutical industries. However,
because of their poor solubility, lower bioavailability, and chemical
instability, these have limited application in the food systems [18,19].
Thanks to nanotechnology which has overwhelmed these barriers, such
bioactive compounds (CUR and RES) can be added into the food and
pharmaceuticals, with greater biocompatibility and bioavailability [18].
Recently, many studies have concluded that CUR and RES together have
better antioxidation effects than the individual ones [20]. In earlier
studies, CUR and RES have been successfully incorporated into the
nanoemulsion-based delivery systems [21,22]; while the mutual role of
CUR and RES as antioxidant agents to protect the TFO nanoemulsions
has not been investigated.
Therefore, in the current investigation, we have proposed the use of
two different OSA-MS to develop the TFO-based nanoemulsions encap­
sulating CUR and RES; the leading goal of this study was to analyze the
influence of MS types and concentrations along with storage tempera­
ture on the physiochemical characteristics and oxidative stability of TFO
nanoemulsions. Moreover, to improve the stability of TFO nano­
emulsions, co-encapsulation with CUR and RES was considered.
2. Materials and methods
2.1. Materials
TFO was provided by Novosana (Taicang) Ltd. (Suzhou, Jiangsu,
China). RES (≥98 %) was a gift from Sangon Biotech Co. Ltd. (Shanghai,
China). OSA-modified starches; PGU and PG2000 were purchased from
National Starch, (USA). CUR was kindly gifted by Zelang Medical
Technology Co. Ltd. (Nanjing, Jiangsu, China). All aqueous suspensions
were equipped by using double distilled water, and other chemicals used
in this study were of analytical grade.
2.2. Fatty acid profile analysis of TFO
Characterization of fatty acid profile in TFO was carried out
following the procedure projected by [25] with slight adjustments, using
an Agilent 7820A gas chromatograph (CA, USA) armed with aflame
ionization detector (GC-FID). Oil samples were investigated on a
TRACER TR-FAME column (60 m ×0.25 mm i.d., film thickness 0.25 μm,
Thermo Fisher, USA), where nitrogen gas with 1.5 mL/min flow rate was
filled as a carrier gas. 1.5 μL from each sample was manually injected
into the instrument with a split ratio of 1:100, while column, injector,
and detector temperatures were stored at 190, 230, and 290 ◦ C, sepa­
rately. Furthermore, the fatty acids were determined on the base of
retention times of standard fatty acid methyl esters under the same
conditions. All measurements were made in triplicate at each interval,
2
Colloids and Surfaces B: Biointerfaces 199 (2021) 111481
and the final values were presented in percentage.
2.3. Nanoemulsions preparation
Aqueous suspensions were developed by dissolving different con­
centrations of PGU and PG2000. PGU was used in less concentration
than PG2000 due to the difference in their structure, molecular weight,
and z-average radius of gyration. PGU (2, 2.5, and 3% w/w) and PG2000
(3, 6, 9, and 12 % w/w) were dispersed into distilled water, on a stirrer
for 6− 8 h to ensure the complete hydration. In the next stage, the oil
phase was prepared by dissolving 0.6 % (w/w) CUR into TFO by stirring
overnight at room temperature in a dark place. Then, 0.4 % (w/w) RES
was diluted into ethanol and poured into the oil phase prepared in the
previous stage. Nanoemulsions were fabricated via the procedure pro­
posed by [10], with small modifications. Firstly, the oil phase was added
to aqueous phase at a ratio of 10:90 and then homogenized by
Ultra-Turrax for 3− 4 min at 18,000 rpm to obtain the coarse emulsions.
Secondly, the coarse emulsions were microfluidized (Model 101,
Microfluidics, Newton, MA) at 110 MPa pressure with 4 processing cy­
cles in order to obtain the final nano-sized emulsions.
2.4. Measurement of mean particle size (MPS), polydispersity index
(PDI) and zeta-potential (ZP)
MPS, PDI, and ZP of nanoemulsions were examined through Zeta­
sizer (Nano-ZS90, Malvern Instruments, UK) according to the dynamic
light scattering (DLS) technique at 25 ◦ C. All formulations were diluted
200-times with distilled water to minimize scattering prior to the mea­
surements and stirred to make sure the consistency among samples.
Each analysis was repeated three times. The surface charge on all
nanoemulsion particles was obtained by 100-times dilution.
2.5. Viscosity analysis of nanoemulsions
The viscosity of all nanoemulsion formulations was determined by
the Discovery Hybrid Rheometer (DHR-2, USA) through the procedure
adopted by [26]. In short, DHR equipped with cone angle 2◦ and 40 mm
cone plate diameter was employed for all nanoemulsions. About 1 mL of
each sample was poured on the plate with 0.1 s− 1 to 1000 s-1 shear rate
under room temperature. The rheological behavior of nanoemulsions
was fitted to the Herschel-Bulkley model (following equation) [27]:
Ƞ = Kγn− 1
(1)
n
where ƞ is the viscosity (Pa. s), K is the consistency index (Pa.s ), γ is the
shear rate (s− 1), and n is the flow index behavior.
2.6. Microscopic observation of nanoemulsions
Confocal laser scanning microscopy (CLSM) technique was used to
demonstrate the structural variations and morphology of freshly pre­
pared nanoemulsions as well as all stored formulations at 40 ◦ C for 30
days. In brief, 2.5 μL of the Nile Red reagent was poured into the 300 μL
of each sample. Approximately, 5 μL of the stained sample was loaded on
the upper surface of the microscopic glass side, protected with a
coverslip and processed to capture images for all samples by fixing the
excitation wavelength (543 nm) of the dye. The images of all formula­
tions were taken by CLSM (Zeiss LSM 710, Leica, Heidelberg, Germany)
and further processed with the help of instrument LSM 710 ZEN
software.
2.7. Oxidative stability of nanoemulsions
The freshly prepared nanoemulsions and one-month stored nano­
emulsions at 4, 25, and 40 ◦ C were analyzed on the basis of oxidative
stability according to the procedure proposed by [28], with slight
Q. Shehzad et al. Colloids and Surfaces B: Biointerfaces 199 (2021) 111481

amendments. Briefly, about 1 mL of each nanoemulsion sample was Table 1

combined with isooctane 2-propanol (2:1 v/v) and vortexed 3-times (10
s each time). Next, centrifuged at 1300 g for 3 min and the solvent phase
was collected; which was then added to 3.2 mL of 2:1 v/v
methanol/1-butanol mixture along with the addition of 3.94 M ferrous
iron reagent/ammonium thiocyanate/ (1:1 v/v; 30 mL). After 20 min in
the dark, it was analyzed at 510 nm wavelength in a UV–vis spectro­
photometer (Bckman Coulter Inc., USA). Each sample was analyzed in
triplicate.
2.8. Antioxidant activity of nanoemulsions
Radical scavenging activity of CUR and RES-loaded TFO nano­
emulsions were evaluated through DPPH (2,2 diphenyl-1-
picrylhydrazyl) and ABTS (2,2-Azino-bis (3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt) assays by the procedure of [29], with
slight modifications. For this purpose, a 50 μM DPPH solution was made
in methanol. About 0.1 mL of each sample was added into a 3.5 mL
DPPH solution. It was then placed in dark for 30 min at 25 ◦ C. A spec­
trophotometer (Bckman Coulter Inc., USA) was used to measure the
absorbance of each sample at 517 nm and Eq. (2) was applied to
calculate the %DPPH inhibition; the final values are indicated as DPPH
IC50 (μL/mL sample).
%DPPH inhibition = [(ABSBlank - ABSSample) / ABSBlank] × 100 (2)
ABTS assay was exploited to record the antioxidant capacity of
nanoemulsions [29]. For this, around 0.1 mL of each sample was mixed
with 3.5 mL ABTS solution. The samples were then placed in the dark for
10 min and analyzed in a spectrophotometer (Quimis, Brazil) at 734 nm.
The final results were noted as μg Trolox per mL of each sample.
2.9. Encapsulation efficiency (EE)
EE is one of the most important parameter for the development of
nanoemulsions during the process. EE of OSA-MS stabilized nano­
emulsions were analyzed by the solvent extraction technique. In short,
100 μL of each nanoemulsion sample was added in a test tube with 9 mL
hexane and stirred at 60 rpm for 15 s, followed by some extra hand-
shaking; the determined oil separated into hexane was considered as
the free oil. Absorbance wavelength was set as per the maximum peaks
perceived for each formulation. EE (%) was measured for each formu­
lation by following the method of [30].
2.10. Statistical analysis
Statistical analysis of the data was investigated using one-way
ANOVA and Minitab 17 (Minitab Inc., State College PA USA). The sig­
nificant differences among all formulations were examined using Dun­
can’s multiple range test on a 95 % confidence level ((p ≤ 0.05). Each
analysis was performed in triplicate.
3. Results and discussions
Fatty acids composition of raw tuna fish oil.
Fatty acids Content%
Docosahexaenoic acid, 25.263 ± 0.54
Hexadecanoic acid, methyl ester 20.303 ± 0.22
9-Octadecenoic acid, methyl ester, (E)- 12.085 ± 0.07
Methyl stearate 6.483 ± 0.35
Eicosapentaenoic acid, meth 6.12 ± 0.13
9-Hexadecenoic acid, methyl ester, (Z)- 5.752 ± 0.19
Methyl tetradecanoate 4.511 ± 0.01
Trans-13-Octadecenoic acid, methyl ester 2.298 ± 0.03
6-Hexadecenoic acid, 7-methyl,methyl est 1.528 ± 0.01
5,8,11,14-Eicosatetraenoic acid, methyl 1.337 ± 0.02
Methyl 4,7,10,13,16-docosapentaenoate 1.289 ± 0.05
Heptadecanoic acid, methyl ester (CAS) 1.133 ± 0.04
Octadeca-9,12-dienoic acid methyl ester 1.048 ± 0.01
Pentadecanoic acid, methyl ester 1.031 ± 0.03
Methyl stearidonate 0.912 ± 0.01
Others 8.93 ± 0.67
in the development of nanoemulsified systems [23]. The concern about
the consumer’s health and growing demands for natural foods have led
the food industries to design, develop, and increase the utilization of
natural emulsifiers rather than the synthetic ones. Earlier researchers
have explored the potential of proteins, polysaccharides, and phospho­
lipids as emulsifiers to fabricate the nanoemulsions [12]. Octenyl suc­
cinic anhydride-modified starches (OSA-MS) are efficient food-grade
biopolymers with emulsifying, gel-forming, and film-forming proper­
ties. They are cheap, easy to use, and GRAS (generally regarded as safe)
materials [24]. Therefore, different concentrations of two OSA-MS were
used and their influence on zeta potential and size are deliberated.
Zeta potential of the nanoemulsions provides the information about
their stability, because of the repulsion phenomenon among similarly
charged droplets [10]. All nanoemulsions showed negative ZP values
indicating the presence of carboxylic groups on the outer layer of
droplets resulted from the coated modified starch. PGU2 and PG3
showed the highest values of ZP than other formulations. Our findings in
terms of ZP are in between to the previous studies published on
PGU-stabilized nanoemulsions with ZP -42 to -43 and -2 to − 3 mV and
showed superior stability [33,34]. However, the nanoemulsions stabi­
lized by PG2000 exhibited slightly lower ZP values than the previous
findings (-29 to -30) of [34] (Table 2).
The PDI is an important parameter that governs the distribution of
the droplets in the nanoemulsion system; a narrow range of PDI exhibits
higher stability of the system. PDI values < 0.3 are considered as an
indicator of a uniform system, good for better stability. The PDI values
decreased as the concentration of PGU increased from PGU2 to PGU2.5,
whereas PGU2.5 showed the minimum PDI (0.13 ± 0.03). However,
upon further increase in PGU concentration, the increase in PDI was
observed. Moreover, in case of PG2000; the increase in its concentration
resulted in gradual decrease in PDI, thereby PG9 showed the lowest PDI
of 0.16 ± 0.01. The decrease in PDI probably because of the more
adsorption of modified starch at the interface which prevents the coa­
lescence of emulsion droplets. However, at higher concentrations of
both starches i.e., PGU3 and PG12, an increase in PDI was observed. The

3.1. Fatty acid composition of tuna fish oil

Table 2
Influence of different concentrations of modified starches on zeta potential and
The fatty acid profile of raw TFO is displayed in Table 1 including the
size of TFO nanoemulsions loaded with curcumin and resveratrol.
leading ones omega-3 DHA, palmitic acid, and oleic acid. It was
important to note that the collective percent of DHA and EPA in TFO was Emulsifier Concentration Sample Zeta Potential (mV) PDI
codes
31.383 %. Our results are in agreement with the findings reported by
[31,32]. To conclude, TFO could be used for the enrichment of food PGU 2% PGU2 − 33.6 ± 0.6 0.16 ± 0.04
PGU 2.5 % PGU2.5 31.3 ± 0.7 0.13 ± 0.03
products with DHA and EPA. −
PGU 3% PGU3 − 32.5 ± 0.4 0.17 ± 0.03
PG2000 3% PG3 − 24.3 ± 0.4 0.25 ± 0.02
3.2. Zeta Potential and size of nanoemulsions PG2000 6% PG6 − 22.5 ± 0.5 0.18 ± 0.01
PG2000 9% PG9 − 19.0 ± 0.2 0.16 ± 0.01
PG2000 12 % PG12 22.3 ± 0.3 0.22 ± 0.01
The selection of the most suitable emulsifier is an imperative aspect
−

3
Q. Shehzad et al.
increment in PDI is due to the formation of micelles with the increase in
the concertation of modified starch as reported earlier by [35].
The concentration of bioactive compounds (CUR and RES) were
constant in all the formulations, whereas the impact of different con­
centrations of modified starch (PGU) on droplet size is presented in the
Fig. 1A; as the amount of PGU increased from 2 to 2.5 % (w/v), the MPS
decreased significantly. This may be due to the more accessibility of an
emulsifier to adsorb at the interface which has the ability to prevent the
droplets from flocculation and coalescence. Whereas, a further increase
in the concentration of PGU from 2.5 % to 3% resulted in a higher MPS.
This increase is associated with the non-adsorbed molecules of PGU and
their engagement in the formation of free micelles [34,35]. Finally,
PGU2.5 nanoemulsion showed a lower droplet size than other formu­
lations. All formulations exhibited an increase in MPS after one-month
storage period; the highest increase in MPS was observed at 40 ◦ C,
higher temperatures cause to increase the motion of particles within the
system and more chances of coagulation. For instance, the increase in
MPS for PGU2.5 at 40 ◦ C was ⁓21 nm. In contrast, the rise in MPS for
PGU2 and PG3 at 40 ◦ C was ⁓28 nm and ⁓35 nm. In general, the rise in
MPS after one-month storage at all temperatures for PGU2.5 was lower
than PGU2 and PG3, thereby showing more stability. A similar rise in
MPS for nanoemulsions stabilized by OSA-starches after the storage
period was observed by [34]. Hence, our results are in accordance with
earlier studies conducted on PGU. All the nanoemulsions were stable
after storage which was credited to the higher density and molecular
weight of PGU as compared to the PG2000 embedded nanoemulsions.
However, the influence of different concentrations of PG2000 (3, 6,
9, and 12 %) on MPS was accessed for fresh nanoemulsions. Fig. 1 B is
clearly depicting that as the concentration of PG2000 increased from 3
to 9% there was a steady reduction in MPS. However, it was noteworthy
that upon a further increase in starch concentration from 9 to 12 %
caused an increment in MPS of nanoemulsion. The decrease in size was
possibly due to the more availability and binding of emulsifier molecules
at the interface which prevents coagulation. However, PG9 showed the
minimum MPS of 127.9 ± 3.1 nm which might be due to the formation
of micelles as discussed in the previous section. For checking the system
stability, all nanoemulsions were then stored at 4, 25, and 40 ◦ C for one
month as described in Fig. 1B. All nanoemulsions after one month were
subjected to MPS analysis. The MPS of PG3 and PG6 was increased
immensely and crossed the nanometric range. These formulations
turned bimodal due to the fusion of molecules [36]. The highest increase
was reported at 40 ◦ C. On the contrary, PG9 exhibited the lowest in­
crease in MPS for one month and remained monomodal. The minimum
increase in size for PG9 was 10 nm at 4 ◦ C, whereas the maximum was 21
nm at 40 ◦ C. The PG9 showed optimum results and maximum stability
among all the formulations upon storage.
Fig. 1. Effect of storage circumstances on MPS of TFO nanoemulsion enclosing curcumin and resveratrol. (A) Stabilized by PGU. (B) Stabilized by PG2000.
4
Colloids and Surfaces B: Biointerfaces 199 (2021) 111481
3.3. Viscosity characterization of nanoemulsions
Viscosity is an important parameter that is used to evaluate the
nanoemulsion’s stability. The viscosity attributes of nanoemulsion at
day 1 and after one-month storage (at 4, 25, and 40 ◦ C) were investi­
gated to assess the influence of diverse temperature and storage time on
their viscosity. Fig. 2A and B illustrate that all nanoemulsions had a
shear thinning behavior. In addition, for the fresh nanoemulsion sam­
ples, PG3 showed a higher viscosity which was because of the higher
concentration of modified starch in this sample.
The variation in the shear thinning behavior was because of the type
of modified starch and its concentration, oil composition, and storage
conditions. On storage, all nanoemulsions displayed a slight decline in
viscosity at all storing temperatures due to shear thinning behavior,
hence, signifying extra shear thinning behavior as presented in Fig. 2A
and 2B. According to previous studies, rheological behavior is influ­
enced by solution concentration and particle size and distribution [37].
The change in the rheological properties was due to the change in MPS
of nanoemulsions during storage. However, at a higher shear rate, there
were no substantial fluctuations detected for all nanoemulsions that
certainly due to the insensitivity of viscosity [38]. Our results were in
agreement with the earlier research directed on oil-in-water nano­
emulsions emulsified by modified starches [10].
3.4. Microstructure of nanoemulsions
CLSM was used to analyze the morphological changes in oil droplets
of freshly prepared and stored nanoemulsions at 40 ◦ C (Fig. 3A and B).
Freshly taken images of the formulations affirmed that all nano­
emulsions stabilized by both PGU and PG2000 starches had very fine
and evenly distributed oil particles; hence, micro-fluidization proved to
be an efficient approach to formulate nanoemulsions. Previous studies of
[10] and [34] on the OSA-MS stabilized O/W nanoemulsions also
revealed the clear and uniformly distributed oil droplets. Although,
more observation about droplet properties is difficult owing to their very
small sizes. After a one-month storage period at 40 ◦ C, all nanoemulsions
showed an increase in droplet size. The increase in MPS was similar to
the DLS data of these formulations. However, PGU2.5 and PG9 depicted
the smallest changes in droplet size. Though, the increase in MPS was
highest for the PGU3 and PG3 formulations. The increase in size
confirmed the coalescence phenomenon. The CLSM findings were in
accordance with DLS data of the nanoemulsions.
3.5. Oxidative stability (peroxide value) of nanoemulsions
Generally, the peroxide value (POV) is used to analyze the quality of
Q. Shehzad et al. Colloids and Surfaces B: Biointerfaces 199 (2021) 111481

Fig. 2. Viscosity attributes of fresh and stored TFO nanoemulsions encompassing CUR and RES stabilized by PGU (A) and PG2000 (B) kept for one-month. (a)
Nanoemulsions at day 1. (b) Nanoemulsions kept for 30 days at 4 ◦ C. (c) Nanoemulsions kept for 30 days at 25 ◦ C. (d) Nanoemulsions kept for 30 days at 40 ◦ C.

5
Q. Shehzad et al.
Fig. 3. A. CLSM images of nanoemulsions stabilized by PGU (A): (i) fresh, and (ii) one-month stored samples.
the oil. The aim of the current work was to estimate the oxidative sta­
bility of TFO nanoemulsions emulsified by different concentrations of
OSA-MS, and co-encapsulating two antioxidants (CUR and RES), as
shown in Fig. 4A and B. The first-day results of all the samples revealed
nearly the same POV. A slight increment in POV was reported due to the
emulsification method [39]. After one-month storage at 4, 25, and 40 ◦ C,
an identical effect of PGU and PG2000 concentration as well as storage
conditions on oxidation for all the nanoemulsions was observed as
highlighted in Fig. 4A and B. Lower POV was noticed for all the samples
kept at 4 ◦ C compared with the samples kept at 25 and 40 ◦ C. Concerning
emulsifier concentration, the lowest POV equal to 7.87 ± 0.24 meq/kg
oil and 9.84 ± 0.69 meq/kg oil were observed for PGU2.5 and PG9 at 4
◦
C, respectively. Temperature does affect POV, at lower temperature
POV was less as compared to higher temperatures.
However, the highest POV (41.75 ± 1.71 meq/kg oil and 49.76 ±
2.13 meq/kg oil) was observed for PG3 and PG12 at temperature 40 ◦ C,
respectively. Hence, high temperature causes higher POV. All the
Fig. 4. Effect of storage conditions on POV of TFO nanoemulsions encompassing curcumin and resveratrol. (A) Stabilized by PGU. (B) Stabilized by PG2000.
6
Colloids and Surfaces B: Biointerfaces 199 (2021) 111481
samples revealed a higher POV at 40 ◦ C which is clearly because of the
influence of higher temperature. After a one-month storage period,
PGU2.5 and PG9 formulations were found to be more stable even at a
higher temperature compared with other formulations. It is evident from
the results that starch concentration has an obvious effect on the
oxidation stability; PGU2.5 and PG9 proved better over other formula­
tions for a constant proportion of CUR and RES. Higher POV was
observed for PGU2 and PG6 at all temperatures which by reason of the
small droplet size offering greater exposure to the environment [24,40].
3.6. Antioxidant capacity of nanoemulsions
3.6.1. DPPH radical scavenging results
Curcumin and resveratrol are two natural anti-oxidants which have
been widely used by pharmacists and druggists for medicinal purposes.
However, in this series of experiments, the anti-oxidant potential of CUR
and RES-loaded nanoemulsions and the effect of different
Q. Shehzad et al.
concentrations of wall material on their anti-oxidant capacity was
assessed by DPPH• assay. The radical scavenging activity of all the
formulations stabilized by different concentrations of PGU and PG2000
were analyzed and results are presented in Fig. 5A (for PGU) and Fig. 5B
(for PG2000).
At day first all the formulations of PGU showed uncertain anti-
oxidant activity. As depicted in Fig. 5A, 106.04 ± 3.45 μL of PGU2,
91.08 ± 4.34 μL of PGU2.5 and 113.32 ± 2.95 μL of PGU3 were found
enough to reach the DPPH• IC50 level. After a one-month storage period
for all nanoemulsions at temperatures 4, 25, and 40 ◦ C, consequences
displayed a distinctive influence of starch concentration and storage
conditions on anti-oxidant activities. However, all samples showed
decreased DPPH• scavenging activity (p < 0.05). As compared to other
formulations, PGU2.5 was required in a little amount at all temperatures
for scavenging the DPPH•. However, samples were required in larger
quantities from those formulations that were stored at 25 and 40 ◦ C than
4 ◦ C for showing the scavenging activities.
Fig. 5B describes that PG2000 could hold a considerable quantity of
the scavenging activity of CUR and RES. At day first, 174.21 ± 5.78 μL of
PG3 and 154.34 ± 5.53 μL of PG12 were required to attain the DPPH
IC50. After storage at different temperatures for one month, all samples
displayed distinct results. PG3 and PG6 were required in the highest
amount at 40 ◦ C to achieve the required scavenging activity as compared
to other emulsions. This perhaps due to the instability of the emulsions
at high temperatures after one-month. However, the lowest volume of
PG9 was used at all temperatures for scavenging the DPPH• as compared
to other formulations (Fig. 5B).
It is evident from the results presented in Fig. 5 that there was a
noteworthy influence of temperature, storage time, and wall material
concentration on anti-oxidant activities of all nanoemulsions in
contradiction of DPPH•. Emulsifier concentration in CUR and RES
entrapped nanoemulsions have diverse kinds of phenol and hydroxyl
groups which impart disparate anti-oxidant activity to the nano­
emulsified system. Curcumin and resveratrol have various active sites
for free radical scavenging. However, the free phenolic and hydroxylic
groups are the main radically active groups that can undergo autoxi­
dation degeneration. A number of researches have described that poly­
saccharide may have efficient interactions with the above-mentioned
groups that can aid significantly in prevention from degradation.
Hereafter, CUR and RES can hold their optimal anti-oxidant capacity
when in conjugation with polysaccharides. Our anti-oxidant activity
findings of all nanoemulsions stabilized by PGU and PG2000 were in
agreement with the discoveries of Section 3.7, where we determined
that PG2 and PG9 were originated most stable even at higher temper­
atures because of their ability of minimum producing of POV instead of
others.
Fig. 5. Effect of storage conditions on DPPH of TFO nanoemulsions encompassing curcumin and resveratrol. (A) Stabilized by PGU. (B) Stabilized by PG2000.
7
Colloids and Surfaces B: Biointerfaces 199 (2021) 111481
3.6.2. ABTS scavenging activity
The purpose of this experiment was to assess the anti-oxidant ca­
pacity of CUR and RES entrapped inside the TFO nanoemulsions emul­
sified by different concentrations of PGU and PG2000, stored for one-
month. CUR and RES are bioactive compounds that are blessed with
numerous properties including resistance to oxidation and free radical
scavenging abilities. This analysis was also conducted to check the
suitability of the system encompassing CUR and RES and the system
developed should support their bioactivities and the effect of stabilizer
was also assessed.
All the formulations stabilized by PGU were examined at day first
and after one-month storage at 4, 25, and 40 ◦ C. With increasing tem­
perature and time, all samples exhibited a declining trend. The lowest
anti-oxidant capacity was 97.56 ± 4.2 displayed by PGU2 at 40 ◦ C.
PGU2 and PGU2.5 showed a significant decline after one-month storage
even at 4 ◦ C. PGU2.5 showed optimum values of 158.63 ± 5.68, 148.54
± 4.68, 128.22 ± 4.7 at 4, 25 and 40 ◦ C temperatures (Fig. 6A).
At day first, nanoemulsions prepared by PG2000 revealed an
increasing trend while PG9 was at the peak. A fresh sample of PG9
exhibited the highest anti-oxidant capacity of 173.98 ± 2.71. Upon
storage at different temperatures for one-month, all samples were
analyzed again and the results are presented in Fig. 6B. As depicted in
Fig. 6B that PG3 and PG6 declined deeply in anti-oxidant capacity and
the lowest values were 80.13 ± 4.34 and 89.35 ± 5.4 at 40 ◦ C. This can
be due to the creaming and flocculation of the emulsions after storage.
Though the anti-oxidant activity of PG9 was also declined, however, it
remained stable at all temperatures. After one month, the highest and
lowest values of PG9 at 4 ◦ C and 40 ◦ C were 156.44 ± 3.66 and 123.2 ±
5.53. Additionally, ABTS assay findings endorsed our POV and DPPH•
results.
3.7. Encapsulation efficiency (EE) of TFO within nanoemulsions
`The results of EE for each sample are described in Fig. 7A for PGU-
based nanoemulsions and Fig. 7B for PG2000-based nanoemulsions.
Among both kinds of OSA-MS, EE of PGU-based nanoemulsions ranged
from 83.17 ± 3–96.38 ± 1.78 and of PG2000 ranged from 80.74 ±
2.61–95.83 ± 1.74. Our findings concluded that EE is directly propor­
tional to the particle size of fabricated nanoemulsions; e.g., reduced
particle size provides superior EE. Our results were strongly agreed with
what proposed by [41] who extremely recommended that stable nano­
emulsions normally exhibit EE more than 80 %. Among all samples,
PGU2.5 and PG9 displayed optimum and satisfactory results of size,
anti-oxidant, and EE.
Q. Shehzad et al.
Fig. 6. Anti-oxidant capacity of fresh and stored TFO nanoemulsions encompassing curcumin.
and resveratrol measured using the ABTS method at (4, 25 and 40 ◦ C). (A) Stabilized by PGU.
(B) Stabilized by PG2000.
Fig. 7. Encapsulation efficiency of TFO nanoemulsions encompassing curcumin and resveratrol (A) Stabilized by PGU. (B) Stabilized by PG2000. Different letters in
the column show a significant difference (p ≤ 0.05).
4. Conclusion
This research exhibited that the OSA-MS concentration and type
positively affected the physiochemical properties of TFO nanoemulsions
entrapped with CUR and RES. Emulsions stabilized by PGU2.5 and PG9
exhibited optimum results even after one-month storage period. Coag­
ulation and flocculation were observed in PG3 and PG6. PG3 also
showed the highest instability after storage. Curcumin and resveratrol
served their great potent potential, being natural anti-oxidant agents.
The results of the present work revealed that PGU2.5 and PG9 provide
better protection to the enclosed substances; additionally, showed
incredible anti-oxidant activities that can be a valuable model for the
production TFO enriched nanoemulsions encompassing CUR and RES
into functional food matrixes, dairy and beverages products.
Referees
Iman Katouzian imaneternity@gmail.com.
Talita Comunian talita.comunian@gmail.com.
Amr M Bakry amr.bakry@agr.suez.edu.eg.
Gabriela I. Denoya denoya.gabriela@inta.gob.ar.
CRediT authorship contribution statement
Qayyum Shehzad: Conceptualization, Data curation, Investigation,
8
Colloids and Surfaces B: Biointerfaces 199 (2021) 111481
Writing an original draft. Abdur Rehman & Seid Mahdi Jafari:
Conceptualization, Visualization, Investigation. Wenshui Xia: Project
administration, Resources, Supervision, review & editing. Min Zuo &
Muhammad Aslam Khan: Formal analysis, writing, review & editing.
Ahmad Ali: Formal and statistical analysis. Sohail Khan: Formal
analysis. Aiman Karim: Writing, review & editing. Muhammad Usman
& Arif Hussain: Writing - review and editing.
Declaration of Competing Interest
None.
Acknowledgments
This research was financially supported by China Agriculture
Research System (grant no. CARS-45-27), Jiangsu Agricultural Industry
Technology System (grant no. JATS [2019] 467) and National first-class
discipline program of Food Science and Technology (grant no.
JUFSTR20180201).
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.colsurfb.2020.111481.
Q. Shehzad et al. Colloids and Surfaces B: Biointerfaces 199 (2021) 111481

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