Journal of Molecular Liquids 394 (2024) 123720

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Journal of Molecular Liquids

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

Stable nanoemulsions for poorly soluble curcumin: From production to

digestion response in vitro
Qianyu Ye , Sophie Kwon , Zi Gu , Cordelia Selomulya *
School of Chemical Engineering, UNSW Sydney, NSW 2052, Australia

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

Keywords: Curcumin, a polyphenol, can induce anticancer activity depending on dose. However, oral curcumin adminis­
Curcumin tration is limited by its low bioavailability due to aqueous insolubility and instability against physiological
Nanoemulsion conditions. This study aims at formulating nanoemulsions by phase inversion temperature to enhance curcumin
Stability
loading, stability, antioxidant performance, bioaccessibility, and in vitro absorption. The selection mechanisms
Antioxidant activity
In vitro digestion
for oil phase (coconut oil), surfactant (polyoxyl 40 hydrogenated castor oil), co-surfactant (soy phospholipid),
and aqueous phase (2 % wt citrate buffer at pH 4.5) are established. The nanoemulsions show tunable mean
droplet size (26–129 nm), high curcumin loading (9.53 ± 0.49 mg/mL), polydispersity < 0.3, and ζ-potential <
-26 mV. Nanoencapsulation increases curcumin photostability by 36–42 % and the antioxidant activity after 24-
hour UV radiation is unchanged (P > 0.05). The curcumin nanoemulsions show ~ 11 %, 24 %, and 57 % higher
retention and ~ 10 %, 12 %, and 17 % higher antioxidant activity than raw curcumin after 3-hour simulated
gastric, intestinal, and physiological incubations, respectively. During in vitro digestion and absorption, the
encapsulated curcumin shows higher bioaccessibility and absorption than free curcumin (P < 0.05). The samples
are stable during 4-week storage at 4˚C and room temperature without preservatives. These findings suggest the
potential to develop a nanoencapsulation strategy, particularly for an oral delivery system of oil-soluble drugs.

1. Introduction
Despite advances in cancer treatment, breast cancer remains one of
the leading causes of death for women, with over 2 million women
diagnosed with breast cancer and about 700,000 deaths globally in 2020
[1]. It has been demonstrated that several dietary components like
curcumin from turmeric spice enable to induce anticancer activity
against breast cancer cell lines by increasing reactive oxygen species
production and promoting apoptosis in cancer cells but not in normal
cells [2,3]. Curcumin has been classified as a Generally Regarded as Safe
(GRAS) compound by the US Food and Drug Administration and has
been established an adequate daily intake value of 3 mg/kg body weight
per day by Joint FAO/WHO Expert Committee on Food Additives and
European Food Safety Authority [4]. However, the anticancer activity of
curcumin is dose-dependent [5] and restrained by its poor aqueous
solubility (0.11 ± 0.01 µg/ml in Milli-Q water) [6], low photostability
[7], instability under neutral and alkaline conditions [8], low bio­
accessibility, and extensive first-pass metabolism [9,10]. To solve these
issues, various encapsulation strategies have been developed, including
liposomes [11], complex powders [6], nanoemulsions [12], and others.
Nanoemulsions are a nano-sized colloidal particulate system acting
as carriers for improving the delivery of active drug molecules. In oil-in-
water (o/w) nanoemulsions, oil droplets (d < 100 nm) are encapsulated
within a protective coating of surfactant and co-surfactant molecules
and are dispersed in an aqueous continuous phase [13]. The composition
and preparation conditions affect the physicochemical and functional
properties of nanoemulsions such as droplet size distribution, droplet
concentration, interfacial tension, storage stability, and transparency
[12]. When the dimension of oil droplets is significantly smaller than the
wavelength of light, the resulting weak scattering will make the nano­
emulsions optically transparent [14]. Compared with conventional
emulsions with a droplet size of 1–20 µm, nanoemulsions display some
advantages e.g., transparent or translucent appearance, longer shelf-life,
low viscosity and interfacial tension, higher surface area and cellular
uptake, and potential to enhance bioavailability and absorption [14,15].
Therefore, nanoemulsions have been considered as potential carriers for
entrapping, stabilising, and incorporating water-insoluble drugs like
curcumin into transparent aqueous-based medical [16], food [17],

* Corresponding author.
E-mail address: cordelia.selomulya@unsw.edu.au (C. Selomulya).

https://doi.org/10.1016/j.molliq.2023.123720
Received 11 October 2023; Received in revised form 15 November 2023; Accepted 30 November 2023
Available online 2 December 2023
0167-7322/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Q. Ye et al.
beverage [18], and cosmetic products [19]. To produce nanoemulsions
with smaller oil droplets, high-energy emulsification methods are nor­
mally applied e.g., high-pressure homogenisation [13] and ultra­
sonication [20]. The high energy consumption during processing may
incur costs and degradation of active ingredients, making these methods
unfavourable for scaling up production. By contrast, the phase inversion
temperature (PIT) method is a low-energy emulsification method that
utilises the chemical energy of the surfactant i.e., the temperature-
dependent solubility and molecular geometry of nonionic surfactants
[21]. At the PIT, nonionic surfactants show the minimum of interfacial
tension and the same affinity for the aqueous and oil phases, caused by
the surfactant spontaneous curvature passing zero [22], which favour­
ably forms kinetically stable actives-loaded nanoemulsions.
Although there are several reports on the preparation of curcumin-
fortified nanoemulsions by the PIT method [12,23,24], to the best of
our knowledge, the selection criteria for oil phase, surfactant, co-
surfactant and aqueous phase, and the mechanisms underlying the
process have never been systematically studied. This study also aims to
formulate curcumin-fortified nanoemulsions by the PIT method to
enhance the loading capacity, storage, pH and UV stability, antioxidant
performance, bioaccessibility, and in vitro intestinal absorption of
curcumin.
2. Materials and methods
2.1. Materials
Curcumin from Curcuma longa (Turmeric) (≥65 %), soy phospho­
lipids, polyoxyl 40 hydrogenated castor oil (RH 40), octanoic acid, oleic
acid, linoleic acid, poly(ethylene glycol) 200 (PEG 200), PEG 400, PEG
600, propylene glycol, pepsin from porcine gastric mucosa powder
(≥3,200 units/mg protein), and bile salts were obtained from Sigma-
Aldrich, St Louis, Mo, USA. Liquid coconut oil was purchased from
Natural Raw C Pty Ltd, Australia. Sunflower oil was from Crisco Pre­
mium Oil, Australia. Canola oil was from Farmers Harvest, Australia.
Pancreatin from porcine pancreas (8 x USP specifications) was pur­
chased from MP Biomedicals. Other chemicals used were from Chem
Supply, Australia. Milli-Q water from a Millipore system (18.2 MΩ cm− 1
at 25 ◦ C). All chemicals were used without further purification.
Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF)
were prepared following the standard INFOGEST protocol [25]. Elec­
trolyte stock solutions containing 0.5 mol/L potassium chloride, 0.5
mol/L potassium dihydrogen phosphate, 1 mol/L sodium bicarbonate, 2
mol/L sodium chloride, 0.15 mol/L magnesium chloride, and 0.5 mol/L
ammonium carbonate were prepared. 100 mL SGF (adjusted to pH 1.6
using 6 M hydrochloric acid) was composed of 80 mL of the electrolyte
stock solutions, 0.05 mL 0.3 M calcium chloride solution, 19.95 mL
Milli-Q water, and porcine pepsin with an enzyme activity of 2000
units/mL in the final digestion mixtures. 200 mL SIF (adjusted to pH 7.0
with 6 M hydrochloric acid) was prepared with 160 mL of the electrolyte
Amount of curcumin encapsulated
Loading efficiency(LE)(%) =
Amount of curcumin added in preparing nanoemulsion
stock solutions, 0.4 mL 0.3 M calcium chloride solution, 39.6 mL Milli-Q
water, 10 mM bile salts, and pancreatin with a pancreatic lipase activity
of 16 or 400 units/mL in the final digestion samples.
2.2. Nanoemulsion preparation
2.2.1. Curcumin solubility in various vehicles
The solubility of curcumin in oil phases (canola oil, sunflower oil,
2
Journal of Molecular Liquids 394 (2024) 123720
liquid coconut oil, octanoic acid, oleic acid, and linoleic acid) and
nonionic surfactants (Tween 40, Tween 80, PEG 200, PEG 400, PEG 600,
and RH 40), was determined following a modified method from Mandal,
Mandal [16]. An excess of curcumin (~200 mg) was added into a glass
vial containing 2 g of each vehicle. After sealing, the mixtures were
heated at 60 ◦ C for 1 h in a water bath to accelerate the solubilisation of
curcumin and then were shaken at 200 rpm and 40 ◦ C for 24 h to reach
equilibrium. After centrifugation at 18,000g for 10 min using a Micro­
fuge® 22R centrifuge (Beckman Coulter, USA), the supernatants were
diluted with absolute ethanol for quantification using a microplate
reader (Infinite M Nano+, TECAN, Switzerland) at 425 nm based on
calibrations made with known amounts of curcumin dissolved in
ethanol.
2.2.2. Cloud point of nonionic surfactant solutions
Citrate buffer was selected as the aqueous phase of the o/w nano­
emulsions to dissolve surfactants, whose selection criteria and mecha­
nisms will be discussed in detail in section 3.4. The cloud point (CP) of
surfactant solutions was measured based on the procedure adopted by
Hasan, Leanpolchareanchai [26] with minor modifications. Nonionic
surfactants were dissolved in 0–2 % wt citrate buffer at surfactant con­
tents of 10 %, 20 %, 30 %, 40 %, and 50 % wt, and then were gradually
heated to 100 ◦ C under magnetic stirring, followed by three temperature
cycles (100–60-100–60-100–60 ◦ C). When a clear surfactant solution
reversed into a cloudy system, the temperature range was recorded as
the CP temperature of the surfactant solutions i.e., phase inversion
temperature (PIT). The three temperature cycles were used to check the
reproducibility of PIT.
2.2.3. Nanoemulsion preparation and pseudo-ternary phase diagram
construction
Blank and curcumin-loaded nanoemulsions were prepared by the PIT
method using a modified method from Jintapattanakit, Hasan [12]. The
selected components were mixed at room temperature (20–22 ◦ C) and
gradually heated to 90 ◦ C under magnetic stirring at 200 rpm, followed
by three temperature cycles (90–60-90–60-90–75 ◦ C) to achieve the
phase inversion process. Then, twice the amount of cold milli-Q water
was added into the system for fast cooling down from 75 ◦ C, followed by
20-min magnetic stirring at 200 rpm at room temperature. Formulations
containing different ratios of the oil phase, surfactant, co-surfactant, and
aqueous phase were examined accordingly to find out the nanoemulsion
existing zone in the pseudo-ternary phase diagrams.
2.3. Nanoemulsion characterisation
2.3.1. Loading efficiency of curcumin nanoemulsions
The loading efficiency of freshly prepared curcumin nanoemulsions
was measured to determine the appropriate curcumin incorporation,
using Eq. (1):
× 100 (1)
The amount of curcumin encapsulated was determined by centrifuging
the nanoemulsions at 18,000g for 20 min at 20 ◦ C, collecting an aliquot
of the supernatants, diluting the supernatants with absolute ethanol, and
measuring the curcumin absorbance at 425 nm with the microplate
reader. No clear phase separation and curcumin precipitation were
observed after centrifugation due to the small droplet size of nano­
emulsions (d < 100 nm). The supernatants collected were akin to the
original nanoemulsions.
Q. Ye et al.
2.3.2. Physical and chemical stability of nanoemulsions during storage
The nanoemulsions were stored in the darkness at room temperature
and 4 ◦ C for 4 weeks without additional preservatives. The physical
stability of nanoemulsions was examined by the change in droplet size
and zeta potential of nanoemulsions using a Zetasizer Nano ZS (Malvern
Instruments, UK), during this storage. The chemical stability was
determined by the variation in the curcumin concentration of the
nanoemulsions during the same storage. The curcumin concentration
was evaluated using the same method in section 2.3.1.
2.3.3. IR spectra collection
IR spectra of nanoemulsions and various components were recorded
and analysed using an IR Alpha (Bruker) instrument with platinum ATR
(Bruker Optics GmbH, Germany) and OPUS software version 7.5 (Bruker
Optics GmbH, Germany).
2.3.4. Photostability of curcumin in nanoemulsions
2 g of nanoemulsions were loaded into an open glass vial, separately,
covered by a single layer of cling film to avoid moisture evaporation and
allow light to pass through [27]. As a control, a certain amount of raw
curcumin was dissolved in 2 g of absolute ethanol at the same concen­
tration as that of nanoemulsions. Soft ultraviolet (UV) radiation (18 W,
350–400 nm) was performed using a fluorescent blacklight blue lamp
(F18T8 BLB 1709, Crompton Lighting, Australia) with an 8-cm distance
at room temperature for 24 h to accelerate the photodegradation of
curcumin. The curcumin content as a function of radiation time was
tested using the method in section 2.3.1.
2.3.5. Curcumin stability under different physiological conditions
As curcumin is unstable at neutral and alkaline pH, the stability of
nanoemulsions was evaluated under simulated gastric, intestinal, and
physiological conditions [6]. Curcumin ethanolic solution with the same
curcumin concentration as that of nanoemulsions was used as a control.
The samples were dispersed and incubated in the SGF at pH 3.0, the SIF
with 16 units/mL pancreatic lipase at pH 7.0, and phosphate buffered
saline (PBS) at pH 7.4, separately, at 37 ◦ C for 3 h. At incubation time 0,
the curcumin content in all final mixtures was 16 µg/mL. The curcumin
concentration retained with time was determined with the microplate
reader at 425 nm.
2.3.6. DPPH scavenging activity of curcumin nanoemulsions
Before and after photodegradation: DPPH (2,2-diphenyl-1-picrylhy­
drazyl) radical scavenging activity was measured to evaluate the anti­
oxidant activity of curcumin in nanoemulsions following a modified
method [6]. The samples before and after 24-hour UV radiation in sec­
tion 2.3.4 were diluted with milli-Q water at a fixed ratio, separately.
Afterwards, 0.5 mL of the diluted samples was mixed with 0.5 mL of 0.2
mM DPPH ethanolic solution, followed by vigorously mixing and 30-min
incubation in darkness. Then, the final mixtures were centrifuged at
18,000g for 5 min and the absorbance of the supernatants was measured
at 527 nm using the microplate reader. The mixtures of 0.5 mL of the
diluted samples and 0.5 mL of ethanol were considered a blank. The
mixture of 0.5 mL of milli-Q water and 0.5 mL of DPPH ethanolic so­
lution was used as the control. The DPPH scavenging activity was
calculated using Eq. (2):
(
OD of sample − OD of blank
) content inside and outside the dialysis tube, cm.
Radical scavenging activity (%) = 1 −
OD of control
× 100
(2)
Before and after physiological incubation: In section 2.3.5, the samples
freshly mixed with the SGF, SIF, and PBS and the samples mixed and
incubated for 3 h were added to 0.2 mM DPPH ethanolic solution at 1: 1
v/v, separately. Other steps for the DPPH scavenging activity mea­
surement were the same as those above.
3
Journal of Molecular Liquids 394 (2024) 123720
2.3.7. In vitro gastrointestinal digestion and intestinal absorption
For standardised in vitro gastric digestion [25], 5 mL of the curcumin-
loaded nanoemulsions were mixed with 5 mL of the SGF, followed by
rapid pH adjustment to 3.0 with 1 M sodium hydroxide solution. A
certain amount of raw curcumin was dispersed into milli-Q water to
achieve the same curcumin addition of nanoemulsions containing 2 mg/
mL curcumin, as a control. The gastric digestion samples were incubated
in an orbital shaker incubator (Bioline, Edwards Instrument, Australia)
at 200 rpm and 37 ◦ C for 2 h. 200 µL of gastric digestion samples were
collected every 30 min and centrifuged at 18,000g and 4 ◦ C for 10 min,
while no curcumin precipitation and phase separation were found. 100
µL of the supernatants were diluted with ethanol and the absorbance of
curcumin was tested at 425 nm using the microplate reader. After each
sample collection, 200 µL of the SGF were added to the system to make
sure the final volume was unchanged.
After 2-hour in vitro gastric incubation, the 10 mL digestion samples
were mixed with 10 mL of the SIF, adjusted to pH 7.0 using 6 M hy­
drochloric acid and 1 M sodium hydroxide solution, with a pancreatic
lipase activity of 400 units/mL in the final mixtures [25]. The intestinal
digestion samples were incubated at 37 ◦ C and 200 rpm for 2 h. 200 µL of
the chyme were collected every 30 min and centrifuged at 18,000g and
4 ◦ C for 10 min. The curcumin concentration was determined using the
same method above. 200 µL of the SIF were added to the system after
each collection.
2.3.8. In vitro intestinal absorption using a dialysis model
The in vitro intestinal absorption of lipophilic curcumin was simu­
lated with a dialysis model (MWCO 12,000–14,000 Da) following a
modified method from Hu, Wu [28]. The gastrointestinal (GI) digestion
samples were dialysed against PBS-T buffer (PBS buffer containing 0.05
% wt Tween 80 outside of the dialysis bag) at 1:5 v/v, incubated at 37 ◦ C
and 130 rpm for 3 h. Tween 80 was added to the PBS buffer as an
emulsifier to dissolve the water-insoluble curcumin [6]. As curcumin in
this study was encapsulated in lipid nanodroplets, this would facilitate
drug-loaded droplets to pass through the intestinal lymphatic transport
[29]. The intestinal lymphatic fluid contains large amounts of albumin
up to 0.012–0.036 g/mL [30], which enables to solubilise curcumin via
binding [31]. Therefore, the addition of 0.05 % wt Tween 80 to PBS
buffer was regarded as an alternative way to simulate the solubilisation
and transport of curcumin in the lymphatic fluid.
The first approximation of the rate of a drug’s ability to cross the
intestinal barrier is expressed as the apparent permeability [32]. In this
study, the apparent permeability coefficient (Papp) of perfusate transport
across the dialysis model was obtained from the steady-state level of the
perfusate diffusing out of the dialysis membrane using Eq (3) as follows
[33]:
( ) dQnormalised dQ
Papp cm min− 1 = = (3)
dt A × (Ci − C0 )dt
where Q represents the amount of drug tested in the container outside of
the intestinal barrier (mimicked by the dialysis tube), mg; A means the
effective surface area of the dialysis membrane available for diffusion,
cm2; Ci and C0 are the drug content inside and outside the dialysis tube at
t = 0 min, mg/mL; and Qnormalised is the normalised Q given the differ­
ences in the surface area of the dialysis membrane and the initial drug
The overall ability of a drug to cross the intestinal barrier can be
expressed as the absorption ratio, using Eq (4) as follows [28]:
Q0 − Qt
Absorption ratio (%) = × 100% (4)
Q0
where Q0 represents the dose of the drug added to the dialysis tube at t =
0 min, mg; and Qt means the amount of the drug still in the dialysis tube
at t minutes, mg.
Q. Ye et al. Journal of Molecular Liquids 394 (2024) 123720

2.4. Statistical analysis nontoxic and nonionic surfactants with HLB values ranging from 11 to
18 suitable for o/w emulsions. To increase the loading capacity of cur­
All sample collections and measurements were conducted in tripli­ cumin in the nanoemulsions, RH 40 with the highest curcumin solubility
cate. The average and standard deviation were calculated using Micro­ was selected as the surfactant (Table 1). The difference in curcumin
soft Excel. The statistical differences between the groups were evaluated solubility in six surfactants was seemingly irrelevant to their HLB values,
using one-way analysis of variance (ANOVA) in IBM SPSS Statistics 26.0 as RH 40, Tween 40, and Tween 80 have similar HLB values ranging
(IBM Inc., USA) at a probability level of 0.05. Origin 2019 (OriginLab from 14 to 16 in Table 1 [36]. The higher curcumin solubility in RH 40
Corporation, USA) was applied to plot some figures. was presumably caused by its three hydrophobic tails in one molecule
which provided more lipophilic structures to interact with curcumin,
3. Results and discussion while other surfactants only had one hydrophobic tail in each molecule.
A similar phenomenon was observed in the lower curcumin solubility in
3.1. Selection criteria for the oil phase octanoic acid with one hydrophobic tail (C8) and the higher solubility in
coconut oil with three hydrophobic tails (mainly C8) in section 3.1.
As shown in Table 1, sunflower oil, canola oil, and coconut oil were Therefore, surfactants with HLB values ranging from 14 to 16 and more
tested as commonly used vegetable oils and exhibited statistically lipophilic structures can effectively stabilise nanoemulsions and in­
different solubilities for curcumin, probably due to their different fatty crease curcumin loading.
acid profiles. According to the nutrition information provided by the
manufacturers, canola oil, sunflower oil, and coconut oil contained 93.3 3.3. Selection criteria for co-surfactant
% wt of unsaturated fat, 88.2 % wt of unsaturated fat, and 98.6 % wt
saturated fat, respectively. The main fatty acids in canola oil were re­ Soy phospholipid, glycerol, and propylene glycol are three
ported to be 62.4 % wt of oleic acid and 20.1 % wt of linoleic acid, while commonly used co-surfactants with a short to medium hydrophobic
the principal fatty acids in sunflower oil were 15.3 % wt of oleic acid and chain, weak amphiphilicity, and terminal small hydrophilic group [37].
71.2 % wt of linoleic acid [34]. By contrast, the major fatty acids in These features allow them to disturb the packing and long-range order of
coconut oil were composed of 53.1 % wt of octanoic acid and 30.8 % wt surfactant molecules and improve droplet curvature and interfacial
of capric acid based on the manufacturer. Therefore, the solubility of fluidity in the formation of nanoemulsions. Additionally, combining
curcumin in three major fatty acids was measured as the controls: hydrophilic surfactants and hydrophobic co-surfactants can create
octanoic acid ≫ linoleic acid > oleic acid, which matches the curcumin smaller and more uniform oil droplets in nanoemulsions with improved
solubility in these oils: coconut oil (mainly containing octanoic acid) ≫ stability [38]. Thus, molecular structure, the ability to lower interfacial
sunflower oil (linoleic acid) > canola oil (oleic acid). This difference in tension, and HLB value are three key factors in the screening of co-
curcumin solubility in three fatty acids was probably associated with surfactants [38,39]. Soy phospholipid (HLB: 4–7 [40]) can lower the
their hydrophile-lipophile balance (HLB) values (Table 1). The HLB of a interfacial tension of its aqueous solutions more effectively [41] than
surfactant measures its balance of hydrophilic and lipophilic moieties to other co-surfactants such as glycerol [42] and propylene glycol [43].
indicate the type of emulsions (o/w or w/o) that the surfactant is suit­ Consequently, soy phospholipid was used as the co-surfactant in the
able for [22]. Compared to the other two fatty acids, the shorter carbon system.
chain in octanoic acid reduced its hydrophobicity and brought about a
higher HLB value of 5.8 to dissolve curcumin. The higher affinity of C– –C 3.4. Cloud point determination and selection criteria for the aqueous
bond for water than C–C bond may cause a higher HLB value and cur­ phase
cumin solubility in linoleic acid than in oleic acid [35], despite similar
estimated HLB values of these two fatty acids. This indicates that The dissolution of nonionic surfactants in water (e.g., RH 40 in this
selecting an oil with a suitable fatty acid profile and molecular structure study) is achieved through the hydrogen bonding between water mol­
can effectively increase the solubility of curcumin or other lipophilic ecules and the repeating oxyethylene units in RH 40 [44]. The hydrogen
drugs. Hence, coconut oil was selected as the oil phase in this study. bonding would be damaged with increasing temperature, causing RH 40
dehydration, the change in solubility behaviour of RH 40 from hydro­
philic to lipophilic, and then phase separation at a certain temperature,
3.2. Selection criteria for surfactant referred to as cloud point (CP). As seen in Fig. 1(A), the CP temperature
of 20 % wt RH 40 aqueous solution was higher than 100 ◦ C and thus salts
Tween 40, Tween 80, PEG 200, PEG 400, PEG 600, and RH 40 are should be introduced into the systems to lower the CP.
Screening of salts depends on their Hofmeister series or lyotropic
Table 1 sequence [45]. The kosmotropic anions (anti-solvents and salting out
Curcumin solubility in different oil phases and surfactants with HLB values. (a-f effect) in a sequence of Hofmeister series (citrate3- > tartrate2- > sul­
represent that under the same letter, there is statistical insignificances at P > phate2- > hydrogen phosphate2- > chromate- > carbonate2- > chloride-)
0.05, n = 3.).
can hydrate better with water molecules, which in turn causes the
Vehicle Component Curcumin solubility HLB oxyethylene units in RH 40 to be more hydrophobic to separate from the
type (mg/mL)
aqueous phase at lower temperatures (i.e., a lower CP). While the cha­
Oil Sunflower 1.340 ± 0.039c / otropic anions (co-solvents and salting in effect) in a sequence of hex­
oil
d
afluorophosphate- > thiocyanate- > perchlorate- > iodide- > nitrate- >
Canola oil 1.209 ± 0.049 /
Coconut oil 2.891 ± 0.032a /
bromide- show weak hydration ability and make the nonionic surfac­
Octanoic 1.506 ± 0.006b 5.8 (estimated by the tants more hydrophilic, which promotes the solubilisation of RH 40 even
acid method of Davies [65]) at higher temperatures (a higher CP). Therefore, citrate3- was selected as
Oleic acid 0.501 ± 0.046f 1.0 (estimated) the anion in the salt.
Linoleic acid 0.829 ± 0.041e 1.0 (estimated)
The anti-solvent cations in a sequence of Hofmeister series (tet­
Surfactant Tween 40 47.369 ± 2.245c 15.6
Tween 80 51.823 ± 1.924b 15.0 ramethylammonium+ > sodium ion+ > potassium ion+ > lithium ion+)
PEG 200 47.501 ± 1.482c 18.1 [66] salt out of nonionic surfactants, lowering the CP [45]. The co-solvent
PEG 400 43.285 ± 2.590d 11.3 [67] cations (guanidinium+ > barium ion2+ > calcium ion2+ > magnesium
PEG 600 42.658 ± 0.867d 12 [68] ion2+) function in the opposite way. Considering food safety and cost,
RH 40 81.142 ± 0.846a 14–16 [36]
sodium ion was chosen as the cation and thus sodium citrate was

4
Q. Ye et al.
Fig. 1. Change in CP (A) with increasing concentration of citrate buffer at pH 4.5 and (B) with the addition of RH 40. (a-c mean that under the same letter, there is
statistical insignificances at P > 0.05, n = 3.).
selected as the salt added into the aqueous. On the other hand, curcumin
is sensitive to pH and shows better stability at acidic pH [6]. A pH of less
than 4.0 is potentially damaging to the teeth [46]. Therefore, instead of
sodium citrate, citrate buffer at pH 4.5 (containing citric acid and so­
dium citrate) would be selected as the aqueous phase candidate.
In Fig. 1(A), the CP of RH 40 decreased from over 100 ◦ C to 75–88 ◦ C
with increasing citrate buffer concentration from 0 % to 2 % wt (0.084
M). A similar reduction in CP was achieved by using 6 % wt NaCl so­
lution as an aqueous phase [26], demonstrating that citrate can lower
the CP of nonionic surfactants more efficiently than chloride ions due to
the salt kosmotrope and chaotrope explained above. With increasing RH
40 concentration, the CP reduced slightly and the time duration for
cloudy state completion increased, as shown in Fig. 1(B). This is pre­
sumably due to less available non-associated water molecules to hydrate
the oxyethylene units in RH 40 at higher RH 40 contents [44]. Curcumin
is sensitive to heat and starts partial degradation at around 50 ◦ C in
methanolic solutions and 150 ◦ C in oily solutions [47]. To reduce cur­
cumin thermal degradation and costs, the CP of RH 40 should be lower
than the boiling point of water but higher than the storage/shipping
temperature of nanoemulsions. Otherwise, the nanoemulsions with a CP
close to or lower than the storage/shipping temperature would undergo
phase separation during storage and shipping, causing nanoemulsions
destabilisation [48]. In this study, the CP of RH 40 was adjusted to
75–88 ◦ C and met the criteria above. Consequently, 2 % wt citrate buffer
at pH 4.5 would be used as the aqueous phase.
3.5. Pseudo-ternary phase diagrams and storage stability
As shown in Fig. 2(A), (B), and (C), blank samples without curcumin
were prepared first to understand the effect of component mass ratios on
samples after the PIT method and rapid cooling-dilution process. Each
number in Fig. 2(A-C) represents a component mass ratio and is used as a
formulation number. Smix represents the mixture of co-surfactants and
surfactants. Smix = 0.5 means that the molecular ratio of co-surfactant
(soy phospholipid) to surfactant (RH 40) was fixed at 0.5. The number
and component mass ratio of nanoemulsions (red dots) varied with
Smix. A larger nanoemulsion existing zone implies a more stable system
to support the formation of nanoemulsions [12], and thus 0.3 and 0.5
were considered the candidate Smix values.
The detailed information and statistical analyses of nanoemulsions
and transition-state emulsions are summarised in Table S1 in the Sup­
plementary Materials, in which Sample name 0.3–9 represents the
sample prepared with Smix 0.3 and formulation number 9. The samples
with droplet size < 100 nm and polydispersity (PDI) < 0.2 were regar­
ded as nanoemulsions. The samples with droplet size < 100 nm, 0.2 <
PDI < 0.3, or 100 nm < droplet size < 200 nm, PDI < 0.2 were
considered as transition-state emulsions. The samples with droplet size
5
Journal of Molecular Liquids 394 (2024) 123720
> 100 nm and PDI > 0.2 were referred to as non-nanoemulsions. As seen
in Table S1 and Fig. 2(D, E, F), the oil droplet size distribution of
samples at Smix 0.3 was in the range of 18 to 47 nm. With increasing
Smix, nanoemulsions with adjustable oil droplet size from ~ 17 to 103
nm can be produced. This should not be simply explained as the addition
of co-surfactants would cause an increase in droplet size. Instead, when
observing the samples with different Smix values but the same compo­
nent mass ratio (e.g., 0.3–15, 0.5–15, and 0.7–15), the droplet size
decreased from 19.56 ± 0.26 nm, 18.83 ± 0.23 nm, to 18.45 ± 0.22 nm,
respectively. The droplet size of formulation number 21 also reduced
from 23.64 ± 0.06 nm at Smix 0.3, 21.76 ± 0.36 nm at Smix 0.5, to
20.88 ± 0.21 nm at Smix at 0.7. For most formulations, the reduction in
droplet size with increasing Smix was because adding co-surfactants can
increase the fluidity of interfacial film, the rate of intermicellar ex­
change, and the curvature of oil droplets, resulting in the formation of
smaller oil droplets [49]. For some formulations such as formulation
numbers 13 and 14, the excess addition of co-surfactants may lead to the
formation of micelles and vesicles, causing nanoemulsion destabilisation
and the formation of transition-state emulsions and non-nanoemulsions.
For samples with the same Smix, oil droplet size increased with the oil
phase ratio (Table S1). The oil droplet size of samples was controlled by
the component mass ratio and Smix in a complex manner.
The effect of Smix on PDI was not as evident as that on droplet size,
as shown in Fig. 2(G, H, I) and Table S1. However, for some formula­
tions like 13 and 14, an excess of co-surfactants also increased the PDI of
samples, presumably due to the nanoemulsion destabilisation
mentioned above. Fig. 2(J, K, L) exhibit the negative zeta potential of
nonpolar oil droplets covered by soy phospholipids and nonionic RH 40,
which is presumably caused by the adsorption of hydroxyl ions at the o/
w interface via hydrogen bonding to the repeating oxyethylene units in
RH 40 [50]. The addition of soy phospholipids elevated the absolute
value of zeta potential, making the systems more stable. This is because
zeta potential measurement was performed on samples by diluting with
milli-Q water to reach an attenuator setting of 10. The pH values of the
original and diluted samples were 4.7–5.0 and around 5.0, respectively,
which were higher than the isoelectric point of phospholipids ~ 4.5
[51]. Hence, oil droplets became more negatively charged with
increasing Smix.
The nanoemulsions were then kept in darkness for 4 weeks at 4 ◦ C
and room temperature without additional pasteurisation to evaluate
their storage stability based on the oil droplet size variation (Fig. 3).
Droplet size hardly changed with time, whereas PDI was more sensitive
to long-term storage. The storage stability of nanoemulsions was
possibly due to the inhibition effect of citric acid in citrate buffer on the
growth of bacteria due to its Ca2+ chelating activity [52]. Considering
the nanoemulsion existing zone and the effects of Smix on droplet size,
PDI, electrostatic stability (zeta potential), and storage stability, 0.5 was
Q. Ye et al. Journal of Molecular Liquids 394 (2024) 123720

Fig. 2. Pseudo-ternary phase diagrams with Smix at 0.3 (A), Smix 0.5 (B), and Smix 0.7 (C) for developing nanoemulsions. The Z-average of the nanoemulsions and
emulsions in the transition state with Smix 0.3 (D), Smix 0.5 (E), and Smix 0.7 (F). The PDI of the samples above with Smix 0.3 (G), Smix 0.5 (H), and Smix 0.7 (I). The zeta
potential of the samples above with Smix 0.3 (J), Smix 0.5 (K), and Smix at 0.7 (L). Non-nanoemulsions are excluded in (D-L).

selected as the optimal Smix.
Fig. 3(C) and (G) reflect that the appearance of blank nanoemulsions
was strongly affected by the oil droplet size. With increasing droplet size
from 18.83 ± 0.23 nm to 84.49 ± 1.00 nm, the blank nanoemulsions
changed from transparent to hazy and milky appearance, due to the
weak scattering explained in the Introduction. Considering the PDI
stability and appearance in Fig. 3(D) and (G), Samples 0.5–12, 0.5–13,
and 0.5–14 would be used in the following curcumin loading
experiments.

6
Q. Ye et al.
Fig. 3. Storage stability of blank nanoemulsions prepared at Smix 0.3 (A, B), 0.5 (C, D) and 0.7 (E, F) and kept at 4 ◦ C and room temperature in darkness for 4 weeks
without additional pasteurisation. (G) The appearance of freshly prepared blank nanoemulsions at Smix 0.5. (Room temperature is abbreviated as r.t.).
3.6. Curcumin loading capacity and location
In Table S2 in the Supplementary Materials, all curcumin-loaded
samples showed loading efficiency > 95 %, electrostatic stability (zeta
potential < -25 mV), monodispersed oil droplets (PDI < 0.3), adjustable
droplet size distribution from about 25 to 129 nm, and acceptable pH in
the range of 4.7–5.2. With increasing oil phase ratio from 6.7 % to 13.3
% wt, the maximum curcumin loading capacity of Sample 0.5–14,
0.5–13, and 0.5–12 reached 4.76, 6.20, and 9.53 mg/mL, respectively,
indicating the critical role of oil phase content in tuning curcumin
loading.
Fig. 4(A) shows the different trends of droplet size as a function of
curcumin loading for three samples. In the presence of an excess Smix
(Sample 0.5–14 with an oil: Smix ratio of 1: 2), non-adsorbed Smix (RH
40 and soy phospholipids) would exist as micelles in the aqueous phase.
Micelles are thermodynamically stable and enable to incorporate hy­
drophobic curcumin within their structures [12], and thus the droplet
size hardly increased with curcumin loading. For Sample 0.5–12 with an
7
Journal of Molecular Liquids 394 (2024) 123720
oil: Smix ratio of 2: 1, loading curcumin caused a statistically significant
increase in droplet size (P < 0.05), implying curcumin molecules
migrated into the oil droplets rather than micelles. Sample 0.5–13 was
somewhere in between. The impact of curcumin content on PDI followed
the similar trend above, in Fig. 4(B). It also implies curcumin loading
tended to elevate the polydispersity of nanoemulsions, no matter which
vehicle dominated the systems, micelles or oil droplets. In Fig. 4(C) and
(D), Sample name like 0.5–12-2C represents the sample with Smix 0.5,
Formulation number 12, and curcumin content 2 mg/mL. The appear­
ance of curcumin-loaded nanoemulsions became more orange with
higher curcumin loading. When curcumin loading was fixed at 2 mg/mL,
different formulations showed various transparency, due to the droplet
size-dependent scattering mentioned above.
To investigate the effect of curcumin content on the nanoemulsions,
the samples containing 2 and 4 mg/mL of curcumin were selected in the
following functional property measurements.
Q. Ye et al.
Fig. 4. Change in (A) droplet size and (B) PDI as a function of curcumin
loading. Changes in nanoemulsion appearance with (C) curcumin loading for
Sample 0.5–13, and (D) component mass ratio at the same curcumin loading, 2
mg/mL. (a-h represent statistical significances at P < 0.05, n = 3. The numbers
on top of the Saturated column mean the saturated curcumin contents loaded in
the samples.).
3.7. Storage stability and IR spectra of curcumin nanoemulsions
Storage stability of nanoemulsions includes the variations of droplet
size, PDI (physical stability), and curcumin retention (chemical stabil­
ity) with storage condition and time. As seen in Fig. 5(A) and (B), both
formulations containing 2 and 4 mg/mL curcumin showed physical
stability during 4-week storage at 4 ◦ C and room temperature in dark­
ness. In Fig. 5(C), the samples with low-dose curcumin (2 mg/mL)
retained most of the curcumin during storage at 4 ◦ C and room tem­
perature. Interestingly, storage temperature and delivery vehicle
affected the curcumin retention of samples with high-dose curcumin
simultaneously. The micelle-dominated formulation (Sample 0.5-14-4C)
8
Journal of Molecular Liquids 394 (2024) 123720
suffered curcumin degradation during room-temperature storage, from
4.2 mg/mL to ~ 3.6 mg/mL in the 1st week and 3.4 mg/mL in the 4th
week, a drop of ~ 19 % in total from initial to week 4. During storage at
4 ◦ C, the curcumin retention of the above samples reduced to ~ 3.7 mg/
mL in the 1st week and 3.6 mg/mL in the 4th week (around 14 %
decrease). For the oil droplet-dominated formulation (Sample 0.5-12-
4C), its curcumin retention decreased from ~ 4.2 mg/mL to 3.9 mg/
mL in the 1st week and 3.8 mg/mL in the 4th week (9.5 % drop),
regardless of storage temperature. The micelle-oil droplet balanced
samples (0.5-13-4C) were somewhere in between. This indicates that the
low-dose samples and the oil droplet-dominant high-dose ones can adapt
to a broader storage temperature range (i.e., better storage stability),
while the micelle-dominant high-dose samples preferred a lower storage
temperature. Compared to surfactant micelles, oil droplets stabilised by
surfactants showed better protection of curcumin from degradation,
probably due to the double isolation (oil phase plus surfactant layer).
The IR spectra of components and nanoemulsions are shown in
Fig. S1(A) and (B) in the Supplementary Materials, respectively. After
encapsulation by the PIT method and cooling-dilution process, the
nanoemulsions still exhibited the characteristic bands of curcumin,
including the band at 1634 cm− 1 attributed to the overlapping stretch­
ing vibrations of alkenes (C––C) and carbonyl (C– –O) groups, band at
1280 cm− 1 ascribed to the bending vibration of the ν(C-O) phenolic
group, and band at 1515 cm− 1 assigned to the in-plane bending vibra­
tions around aliphatic δ(CC-C) and δ(CC– –O), in-plane bending vibra­
tions around aromatic δ(CC-H) of keto and enol configurations, and
stretching vibrations around aromatic ν(C–C) bonds of keto and enolic
form of curcumin [53]. In Fig. S1(B), the bands of coconut oil, RH 40,
and soy phospholipids consisted of 1462 cm− 1 originating from the
–C–H deformation vibrations in CH2 and CH3 groups (bending vibra­
tions), 1745 cm− 1 ascribed to the stretching vibrations of the C– –O
groups in esters, and 2958, 2925, and 2854 cm− 1 attributed to the
–C–H stretching vibrations in –CH3 and CH2 groups in triglycerides
[54]. This indicates that each ingredient, especially curcumin, was well
retained in the nanoemulsions during storage.
3.8. Photostability of curcumin nanoemulsions
Curcumin degrades under light exposure and UV has a more signif­
icant effect on curcumin photodegradation than daylight [7]. Therefore,
the photostability of free curcumin and curcumin-loaded nanoemulsions
under UV radiation is shown in Fig. 6(A). 24-hour UV radiation reduced
the retention of curcumin to 66–72 % in nanoemulsions and ~ 30 % in
raw curcumin, suggesting that encapsulation may partially shield UV
radiation, causing lower degradation in the encapsulated samples. The
similar degradation percentages of micelle-dominant (Sample 0.5-14-
2C) and oil droplet-dominant formulations (0.5-12-2C) imply that the
delivery vehicle hardly affected the photostability of curcumin. Major
curcumin decomposition seemingly occurred in the first five hours in
Fig. 6(A). However, some curcumin photodegradation products present
a yellow colour and remain detected at 425 nm [27], which can be
considered as the limitation of absorbance spectra for curcumin
measurement.
Additionally, the DPPH inhibition activity of samples before and
after 24-hour radiation was tested to evaluate the effect of UV radiation
on curcumin antioxidant activity. In Fig. 6(B), the antioxidant activities
of curcumin-loaded nanoemulsions after 24-hour UV radiation were as
high as the fresh ones, while raw curcumin displayed much lower
antioxidant activity after radiation by half. When the DPPHCurcumin
sample-DPPHBlank sample, the antioxidant activities of curcumin nano­
emulsions were statistically similar. This suggests that after 24-hour
photodecomposition, the degradation products of curcumin still had
good radical scavenging activity. This is because the antioxidant
mechanisms of curcumin include phenoxide radicals formed via electron
transfer and carbon-centred radicals via direct hydrogen abstraction
[55]. The photodegradation products of curcumin probably lost the
Q. Ye et al. Journal of Molecular Liquids 394 (2024) 123720

Fig. 5. Storage stability of curcumin-loaded nanoemulsions kept at 4 ◦ C and room temperature in darkness for 4 weeks without additional pasteurisation. (Room
temperature is abbreviated as r.t.).

9
Q. Ye et al. Journal of Molecular Liquids 394 (2024) 123720

Fig. 6. Photostability (A) and DPPH radical scavenging activity (B) of raw curcumin and curcumin-loaded nanoemulsions against 24-hour UV radiation. (Curcumin is
abbreviated as CUR. a-e represent that under the same letter, there is statistical insignificances at P > 0.05, n = 3.).

Fig. 7. Chemical stability of curcumin against gastric (A), intestinal (B) and PBS incubation (C). DPPH radical scavenging activity of curcumin before (D) and after 3-
hour gastric (E), intestinal (F), and PBS incubation (G). (Curcumin is abbreviated as CUR and normalised samples mean DPPHCurcumin sample-DPPHBlank sample. a-h
indicate that under the same letter, there is statistical insignificances at P > 0.05, n = 3.).

β-diketone moiety but retained the active hydroxyl methoxyphenyl
group after UV radiation, which is also crucial for antioxidant activity
[7,56]. This well-retained antioxidant activity of curcumin after radia­
tion indicates a great potential for nanoemulsions as an oral delivery
system with a longer shelf life.
3.9. Curcumin retention against various physiological conditions
Curcumin decomposes in neutral and alkaline environments i.e., in­
testinal and physiological conditions. [6]. As seen in Fig. 7(A), (B), and
(C), raw curcumin and curcumin nanoemulsions were dispersed in SGF

10
Q. Ye et al.
at pH 3.0, SIF at pH 7.0, and PBS at pH 7.4 for 3 h at 37 ◦ C. To fully
expose curcumin to the surrounding environments, all the samples
(including raw curcumin) were diluted 125 times with the media.
Compared to raw curcumin, the encapsulated curcumin showed higher
retention up to > 93 % and > 87 % during 3-hour gastric and PBS in­
cubations, respectively. Note that the pancreatic lipase activity was
fixed at 16 units/mL in the intestinal digestion samples to slow down the
lipid hydrolysis and observe curcumin decomposition. Fig. 7(B) implies
that lipid hydrolysis by lipase probably damaged the integrity of oil
droplets and RH 40 micelles, causing a continuous curcumin release and
exposure to neutral pH, and a gradual decrease in curcumin retention
during simulated intestinal digestion. By comparing the raw curcumin
retentions in the SIF at pH 7.0 to those in PBS at pH 7.4, we found in the
intestinal phase, bile salts might emulsify raw curcumin and form mi­
celles [57], which alleviated curcumin degradation in the first two
hours. Whereas a neutral pH led to the destabilisation of curcumin after
3 h.
The DPPH inhibition activity of samples before and after 3-hour in­
cubation was measured to understand the impact of various physiolog­
ical conditions on curcumin antioxidant activity, in Fig. 7(D-G). The
antioxidant activity of blank nanoemulsions presumably resulted from
the phenolic compounds of coconut oil [58]. Normalised samples mean
DPPHCurcumin sample-DPPHBlank sample. In water, the normalised DPPH
scavenging activities of encapsulated samples were slightly lower than
that of raw CUR, presumably because one phenoxy group in curcumin
with free radical scavenging activity formed molecularly hydrogen
bonding with the head group of soy phospholipids [59]. DPPH assay is
strongly pH-dependent and presents lower results under acidic pH [60],
which caused the lower scavenging activities of samples in SGF (pH 3.0)
than those in water. Under the unfavourable SIF and PBS conditions for
curcumin, the DPPH scavenging activity of 0.5-14-2C was lower than
those of 0.5-12-2C and 0.5-13-2C, as the curcumin in 0.5-14-2C tended
to stay in the aqueous surfactant micelles and thus became more
accessible to the physiological pH than other two samples. Compared to
PBS, lipolysis in SIF caused higher release, exposure, and degradation of
curcumin. However, the normalised DPPH scavenging activities were
similar in SIF and PBS (33–37 %). This is possibly because nano­
encapsulation alleviated curcumin degradation during in vitro intestinal
incubation, and the degradation products of curcumin at physiological
pH (bicyclopentadione, vanillin, ferulic acid, etc.) still had the active
hydroxyl methoxyphenyl group for antioxidant activity [61].
Fig. 8. The bioaccessibility of curcumin during in vitro GI digestion (green and yellow parts) and the normalised content of curcumin in PBS-T buffer diffusing from
the GI digestion samples of nanoemulsions or raw curcumin in the dialysis model (blue part) for 3 h (A) and for 20 h (B). The data were linearly fitted where the slope
of the straight line represents the apparent permeability coefficient (Papp). (Curcumin is abbreviated as CUR.). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
11
Journal of Molecular Liquids 394 (2024) 123720
3.10. In vitro GI digestion and intestinal absorption
The in vitro GI digestion and intestinal absorption of curcumin are
shown in Fig. 8(A) and (B). The release (bioaccessibility) curves of
nanoencapsulated curcumin showed an ultra-fast release in the initial
gastric phase followed by a relatively slow gastric release. This specific
“Γ” shape-like release profile is poorly fitted by zero-order, Higuchi or
first-order models, and is considered as a rapid and substantial release
drug delivery system. The slight reduction in the release curve in the
intestinal phase is attributed to the degradation of curcumin under
neutral pH.
In the gastric phase, raw and nanoencapsulated curcumin exhibited
completely different bioaccessibility of ~ 1.6 % and ~ 99 %, respec­
tively, due to the aqueous insolubility of raw curcumin [8]. Note that the
pancreatic lipase activity was set at 400 units/mL in the final intestinal
digestion samples, to simulate human lipolysis following a modified
INFOGEST protocol [25]. During 2-hour intestinal digestion, most
nanoemulsions showed good curcumin bioaccessibility up to 81 %,
except for Sample 0.5–14-2C which was a low-dose micelle-dominant
formulation with bioaccessibility of ~ 67 %. This further indicates the
advantage of oil droplet dominant formulations in curcumin stabilisa­
tion. The bioaccessibility of raw curcumin increased slightly to ~ 19 %,
presumably due to the emulsification and dissolution effect of bile salts
on lipophilic compounds like curcumin [57]. At the endpoint of intes­
tinal digestion, coconut oil droplets should be hydrolysed into a free
fatty acid-monoglyceride mixture (lipid digesta), while RH 40 was less
effectively hydrolysed [62]. Thus, curcumin was possibly loaded in the
lipid digesta droplets stabilised by partially hydrolysed RH 40
molecules.
The in vitro intestinal absorption was simulated with a dialysis model
[28], implying that the size of delivery vehicles would play a key role in
the amount of curcumin transported. During 3-hour intestinal absorp­
tion, the low-dose oil droplet-dominant formulations (Samples 0.5–12-
2C and 0.5–13-2C) exhibited less curcumin transport, probably owing to
their relatively large droplet size and low curcumin concentration gra­
dients during dialysis. Raw curcumin had significantly faster transport
because it was emulsified in small-size bile salt micelles. However, this
does not mean bile salt micelle was an ideal vehicle. The apparent
permeability coefficient (Papp) and absorption ratio of curcumin in
different samples are summarised in Fig. 8(B) and Table S3 in the
Supplementary Materials. All the nanoemulsions showed continuous
curcumin transport, indicating that the encapsulated curcumin
remained stable and accumulated after 4-hour digestion and 20-hour
Q. Ye et al.
intestinal transport. The raw curcumin decomposed in bile salt micelles
under unfavourable physiological pH. Sample 0.5–13-4C transported
more curcumin than others, owing to its high curcumin dosage and oil
droplet-dominant feature. Sample 0.5–14-2C had the second highest
curcumin in vitro absorption ratio, indicating that the small-size RH 40
micelles had a higher cross-membrane transport ability than oil droplets
and could stabilise curcumin better than bile salt micelles. Sample
0.5–13-6C had the saturated curcumin loading and oil droplet-dominant
formulation, whose less transport suggests that the saturated loading
might cause curcumin to distribute in the outside layer of oil droplets or
micelles, causing curcumin destabilisation. The absorption ratio of free
fatty acid was reported to be 26.62 ± 7.26 % using an Ex vivo rat small
intestine model [28]. In this study, the highest absorption ratio of oil-
soluble curcumin was found to be 3.36 ± 0.24 % in Sample 0.5–13-
4C. The difference in absorption ratios may be due to the complex
structure of intestinal epithelial cell layers which can generate
membrane-associated fatty acid binding proteins to promote the uptake
of fatty acids and oil-soluble nutrients [63]. Therefore, a Caco-2 cell
monolayer model will be applied in future to simulate the in vitro in­
testinal absorption of curcumin-loaded nanoemulsions [64].
4. Conclusion
The selection criteria for oil phase (coconut oil), surfactant (RH 40),
co-surfactant (soy phospholipid), aqueous phase (2 % wt citrate buffer at
pH 4.5), and Smix (0.5) were proposed via systematic studies to prepare
nanoemulsions by the PIT method. After curcumin was loaded, the
nanoemulsions had 4-week storage stability without the need for pre­
servatives and pasteurisation. The nanoemulsions exhibited curcumin
loading capacity of up to 9.53 mg/mL, adjustable transparency with
mean droplet size from 26 to 129 nm and low polydispersity < 0.29, and
electrostatic stability (zeta potential < -26 mV). Decreasing the mass
ratio of Smix to coconut oil shifted the nanoemulsions from micelle-
dominant formulation to oil droplet-dominant formulation and caused
the migration of curcumin from micelles to oil droplets. The use of
nanoemulsions also increased the photostability of curcumin by 36–42
% and kept the antioxidant activity unchanged after 24-hour UV radi­
ation, owing to the retained active phenolic groups in the encapsulated
samples. Compared to free curcumin, the nanoencapsulated curcumin
showed ~ 11 %, 24 %, and 57 % higher retention and 10 %, 12 %, and
17 % higher antioxidant activity after 3-hour simulated gastric, intes­
tinal, and physiological incubations, respectively. Nanoemulsions
exhibited significantly higher bioaccessibility than free curcumin during
in vitro GI digestion, while free curcumin displayed a faster apparent
permeability coefficient in the first three hours, due to size-dependent
cross-membrane transport in the dialysis model. However, the high-
dose oil droplet-dominant nanoemulsions had a higher curcumin ab­
sorption ratio than the other samples, due to the higher curcumin con­
centration gradient and protective effect of encapsulation. The proposed
formulation also had higher curcumin retention and antioxidant activity
during storage and various in vitro physiological incubations. The actual
intestinal absorption and lymphatic transport of the curcumin-fortified
nanoemulsions remained unknown under the current experimental
conditions and will be further studied with a Caco-2 cell monolayer
model in our next study. A three-dimensional tumour spheroid model
incorporating a typical breast cancer cell line (4 T1 cells) will be
leveraged to evaluate the anti-cancer activity and cytotoxicity of the
nanoemulsion digesta permeating from the Caco-2 cell model.
CRediT authorship contribution statement
Qianyu Ye: Conceptualization, Methodology, Investigation, Visual­
ization, Formal analysis, Writing – original draft. Sophie Kwon:
Investigation, Methodology. Zi Gu: Supervision, Writing – review &
editing. Cordelia Selomulya: Supervision, Writing – review & editing,
Project administration, Funding acquisition.
12
Journal of Molecular Liquids 394 (2024) 123720
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This project was supported by ARC Discovery grant (DP200100642).
Sophie Kwon was a Taste of Research scholarship recipient. The authors
would like to acknowledge Dr Nicholas Bedford for access to Malvern
Zetasizer Nano.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.molliq.2023.123720.
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