LWT - Food Science and Technology 162 (2022) 113474

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LWT
journal homepage: www.elsevier.com/locate/lwt

Digestion stability of curcumin-loaded nanostructured lipid carrier

Ji Eun Hyun a, Hye-Yoon Yi a, Geun-Pyo Hong b, Ji-Yeon Chun a, *
a
Department of Food Bioengineering, Jeju National University, Jeju, 63243, South Korea
b
Department of Food Science and Biotechnology, Sejong University, Seoul, 05006, South Korea

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

Keywords: Curcumin is known as a functional substance with various effects such as antioxidant, anti-inflammatory, and
Nanostructured lipid carrier anticancer. However, it is not easily utilized due to low water solubility and bioavailability. Therefore, this study
Curcumin encapsulated into a nanostructured lipid carrier (NLC) with MCT oil of coconut to improve the solubility of
In vitro digestion
insoluble curcumin. The physicochemical properties and stability of curcumin NLCs were observed, and the in
MCT oil
vitro gastrointestinal stability of curcumin NLCs was also studied. The produced curcumin NLCs had a hydro­
dynamic diameter of 126.9 nm, zeta potential of − 25.9 mV, PDI of 0.246, and encapsulation efficiency of 94.5%.
TGA and DSC analysis were performed to confirm the thermal properties (pyrolysis temperature (◦ C), weight loss
(%), melting temperature (◦ C), and crystallinity, etc.) of the components constituting the curcumin NLCs.
Through this, it was confirmed that curcumin was completely entrapped in the NLC structure. In the digestive
imitation system, NLCs released approximately 31% of curcumin in the imitation saliva step, and curcumin
release gradually increased. This study showed that NLCs were an effective carrier to apply in various fields such
as the functional food industry, because they increased the water solubility of curcumin and could also improve
bioavailability and curcumin storage stability.

1. Introduction intermediate carbon-length saturated fats are bound to the glycerol

Curcumin is a major component of yellow spices contained in the
roots of turmeric (Curcumac longae rhizoma) and curcuma roots (Cur­
cuma Longa Radix) (Jo, Kim, & Kwon, 2016). Curcumin is a type of
polyphenol and has a molecular weight of 368.37 g/mol and a melting
point of 183 ◦ C (Araiza-Calahorra, Akhtar, & Sarkar, 2018). It is widely
used as a natural pigment, spice, and medicine in India and other Asian
countries (Araiza-Calahorra et al., 2018). In addition, curcumin has
anti-inflammatory (Selvam, Jachak, Thilagavathi, & Chakraborti,
2005), anticancer (Fujisawa, Atsumi, Ishihara, & Kadoma, 2004), anti­
oxidant (Ak & Gulcin, 2008), neuropathic pain suppression (Kim, Park,
& Jeon, 2008), skin sclerosis relief, and psoriasis relief, to prevent skin
cancer (Aggarwal, Surh, & Shishodia, 2007; Balasubramanian & Eckert,
2006) and antialcoholism functions (Nanji et al., 2003). Despite having
various health functionalities, curcumin has poor water solubility (11
ng/mol, at ambient temperature), low oral bioavailability, chemical
instability, and photolysis. Furthermore, it has low membrane perme­
ability and has difficulties in rapid metabolism and excretion (Hussain
et al., 2017).
Medium chain triglycerides (MCTs) are lipids in which three
backbone (Saarela, 2011b), and they are between six and twelve carbons
in length. The absorption and transport of MCT differ from long chain
triglycerides (LCTs), which are over twelve carbons in length. They
present different metabolic pathways of absorption and
gastric-intestinal digestion. MCT has been applied and studied as an
effective lipid type for various studies on weight control (Henry, 2007),
obesity (Saarela, 2011b), functional fat (Saarela, 2011a), functional
health benefits (Rastall, 2007), etc. MCTs are a thinning fluid,
light-yellow color, clearness, non-flavor and they are usually made from
coconut or palm kernel.
Solid lipid nanoparticles (SLNs), developed in the 1990’s are
colloidal particles that maintain the solid-state at room temperature by
replacing the liquid lipid of the emulsion with a solid lipid. SLNs have
excellent physical stability to protect sensitive drugs (Madane &
Mahajan, 2014), high bioavailability, good biocompatibility and low
toxicity (Yuan et al., 2007). However, SLNs have low encapsulation ef­
ficiency due to lack of space to capture drug materials and encapsulated
drug is easily released because the crystallinity of solid lipids increases
during long-term storage (Behbahani, Ghaedi, Abbaspour, Rostamiza­
deh, & Dashtian, 2019; Muller, Radtke, & Wissing, 2002).

* Corresponding author. Department of Food Bioengineering, Jeju National University, Jeju-si, 63243, South Korea.
E-mail address: chunjiyeon@jejunu.ac.kr (J.-Y. Chun).

https://doi.org/10.1016/j.lwt.2022.113474
Received 1 October 2021; Received in revised form 8 April 2022; Accepted 18 April 2022
Available online 21 April 2022
0023-6438/© 2022 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
J.E. Hyun et al.
Nanostructured lipid carriers (NLCs) were developed to compensate
for the deficiency of SLNs. NLCs form by containing liquid lipids (oil)
with solid lipids (fat) at room temperature, therefore, NLCs have a lower
melting point than SLNs (Jun, Lim, & Jin, 2015). NLCs are partially
crystallized and have a less ordered crystalline structure (Tamjidi,
Shahedi, Varshosaz, & Nasirpour, 2013). Therefore, the solubility of the
active ingredients and the drug loading capacity are increased minimize
the leakage of the active compound during storage due to the amorphous
solid structure of NLCs (Tamjidi et al., 2013; Varshosaz, Eskandari, &
Tabakhian, 2010). Therefore, NLCs may increase encapsulation effi­
ciency, drug loading and physical stability and may improve the
chemical stability, bioavailability, and controlled release of functional
lipophilic compounds in food. In addition, NLCs can include hydro­
phobic and hydrophilic active substances and various types of NLCs are
manufactured according to the mixing ratio of solid lipids and liquid
lipids in the oil phase of NLCs (Muller et al., 2002; Sezer, 2012).
In this study, insoluble curcumin was encapsulated into NLCs with
MCT oil from coconut to improve their solubility and availability. The
physicochemical properties and stability of curcumin NLCs were
observed, and the in vitro gastrointestinal stability of curcumin NLCs was
also studied.
2. Material and methods
2.1. Reagents and standard
The curcumin (≥65%), Tween 80, Span 80, glycerol tristearate, so­
dium hydroxide solution, pancreatin from porcine pancreas, pepsin
porcine gastric mucosa, monobasic potassium phosphate, uric acid so­
dium salt, sodium DL-lactate and Folin-Ciocalteu reagent were purchased
from Sigma-Aldrich Chemical Co., Ltd. MCT oil (Palm Kernel oil 99.9%,
canola oil 0.1%) was provided from Korea Medical Foods Co Ltd., and
Ethyl acetate purchased Junsei Chemical Co., Ltd (Japan). Hydrochloric
acid (HCl) solution was supplied by Yakuri Pure Chemicals Co., Ltd.
(Kyoto, Japan).
2.2. Preparation of nanostructured lipid carriers (NLCs)
Aqueous dispersion was Tween 80 300 mg in distilled water 100 mL.
Oil dispersion was glycerol tristearate (solid lipid) 600 mg/100 mL, MCT
oil (liquid lipid) 240 mg/100 mL, curcumin 3 mg/100 mL and Span 80
300 mg/100 mL. Each dispersion was mixed in magnetic stirring ho­
mogenization 80 ◦ C, 350 rpm for 10 min and then oil dispersion was
immediately dispersed dropwise into the aqueous dispersion. The
mixture was kept 80 ◦ C and mixed high speed homogenizer (T-25D, IKA,
Germany) at 8,000 rpm for 5 min. Finally, the mixture was homogenized
by high pressure homogenizer (Picomax MN400, Micronox, Korea) at
20,000 psi, 3 cycle and cooled to room temperature. After preparation,
each NLC dispersion was stored at 5 ◦ C, 21 ± 2 ◦ C, 40 ◦ C, 65 ◦ C for 28
days.
2.3. Nanostructured lipid carriers particle characteristic
The samples were diluted to distilled water (1:9 v/v). The hydro­
dynamic diameter (nm) and PDI were measured by dynamic light
scattering (DLS) on DelsaMax Pro (Beckman Coulter, USA) at 25 ◦ C. The
zeta potential (mV) was measured by electrophoretic light scattering
(ELS) on DelsaMax Pro. The samples were measured at least 3 times.
2.4. Encapsulation efficiency (EE%)
The encapsulation efficiency method (Riaz et al., 2019; Yu et al.,
2016) was modified. The standard calibration curve of curcumin was
obtained by measuring the absorbance of curcumin solution in con­
centration range prepared from stock solutions in methanol at 423 nm in
triplicate. Calibration curve of curcumin was then plotted with
2
LWT 162 (2022) 113474
absorbance on y-axis and curcumin concentration on x-axis (R2 =
0.9998). To remove free curcumin NLCs dispersion was mixed with ethyl
acetate (1:1, v/v) and then vortexed for a few seconds. The mixture was
centrifuged (3,134×g, 10 min, 25 ◦ C) and the supernatant, which
included free curcumin was removed. The remained curcumin NLCs
dispersion was mixed with methanol and then centrifuged (3,134×g, 40
min, 4 ◦ C). The middle part of curcumin NLCs dispersion was filtered by
using a syringe filter (0.45 μm), and then the absorbance was deter­
mined by UV–Vis spectrophotometer (Epoch™, BioTek Instruments, Inc,
Winooski, USA) at λmax = 423 nm. Encapsulation efficiency of curcumin
was calculated as follows (Eq. (1)):
EC(g)
EE (%) = × 100 (1)
IC (g)
where EC is from the middle part of curcumin NLCs dispersion and IC is
the initial amount of curcumin at NLCs preparation.
2.5. Total phenolic content
This test was performed by modifying the Folin-Ciocalteu method of
Singleton and Rossi (1965). In order to evaluate only the curcumin
collected in NLCs, free curcumin was removed in the same way as in the
encapsulation efficiency (EE%) experiment and used as a sample.
Briefly, 0.2 mL of sample was mixed with a 0.9 mL distilled water, fol­
lowed by the addition of 0.1 mL of 2 M Folin-Ciocalteu’s phenol reagent
and incubated for 5 min, protected from light. Subsequently, 0.3 mL of
Na2CO3 (2 g/100 mL) and 0.5 mL of distilled water were added and the
mixture left at protected from light of room temperature for 1 h before
the absorbance was measured at 760 nm. The results were calculated by
using a standard curve (R2 = 0.9988) which was built by dissolving
gallic acid in distilled water at different concentrations (100, 200, 300,
400, 500 and 600 μg/mL) and expressed as μg of gallic acid equivalents
per mL sample (μg GAE/mL).
2.6. Thermogravimetric analysis (TGA) and differential scanning
calorimetry (DSC)
Blank NLCs and Curcumin NLCs were used after lyophilization for
analysis. The thermal degradation of glycerol tristearate, MCT oil, cur­
cumin, blank NLCs, and curcumin NLCs were analyzed by TGA Q500
(TA Instruments, USA). Samples were heated at a rate of 10 ◦ C/min from
room temperature to 500 ◦ C. Thermograms of the curcumin NLCs
components were obtained using a DSC Q20 (TA Instruments, USA).
Samples (2–3 mg) were heated from 0 to 200 ◦ C at the scan rate of 10 ◦ C/
min. TA Universal analysis software (v 4.5A, TA Instruments, USA) was
used to analyze data for both TGA and DSC.
2.7. Preparation of simulated gastrointestinal fluids (saliva, gastric, small
intestinal fluids)
The in vitro digestion model (Fig. 1) used was the modification of
those described previously (Mao & McClements, 2012). Simulated saliva
fluid (SSF, pH 6.8) consisted of mucin and various salts (Table 1) and pH
was adjusted by using 1 mol/L sodium hydroxide (NaOH). The SSF was
continuously shaken to heat at 105 rpm and 37 ◦ C for 15 min. The
simulated gastric fluid (SGF) conditions were defined by the ‘United
States Pharmacopeia and National Formulary 200’ method (United
States Pharmacopeial Convention, 2009). For the SGF condition, 0.7 mL
of 1 mol/L hydrochloric acid (HCl) was added to 90 mL of deionized
water, and 200 mg of sodium chloride (NaCl) was dissolved in the HCl
solution. Then, 302 mg of pepsin from porcine gastric mucosa (≥2,500
Units/mg) was added in the HCl–NaCl solution. The pH of SGF was
adjusted to 1.2. The simulated small intestinal fluid (SIF) conditions
were defined by the ‘United States Pharmacopeia and National Formu­
lary 200’ method. For the SIF condition, 680 mg of monobasic potassium
J.E. Hyun et al. LWT 162 (2022) 113474

Fig. 1. Preparation of simulated gastrointestinal fluids and protocol of curcumin release in in vitro lipid digestion.

2.9. Statistical analysis

Table 1
Chemical composition of artificially simulated saliva fluid (Mao & McClements,
All experiments were carried out at least three times. The results are
2012).
reported as means ± standard deviation. Data were analyzed by one-
Chemicals Chemical formula Concentration (g/L) way ANOVA followed by Tukey’s multiple range test at p values of
Sodium chloride NaCl 1.594 <0.05 using Minitab ver. 17(PA, USA) statistical tests.
Ammonium nitrate NH4NO3 0.328
Potassium phosphate KHPO4 0.636
3. Results and discussion
Potassium chloride KCl 0.202
Potassium citrate K3C6H5O7•H2O 0.308
Uric acid sodium salt C5H3N4O3•Na 0.021 3.1. Effect of curcumin concentration on curcumin NLC properties
Urea H2NCONH2 0.198
Lactic acid sodium salt C3H5O3Na 0.146 Curcumin NLCs were produced after establishing the blank NLCs
Porcine gastric mucin (type II) – 30
optimal formulation in our previous study (Supplementary material).
Several conditions were confirmed, such as hydrophilic emulsifier con­
phosphate (KH₂PO₄) was dissolved in 75 mL of deionized water and then centration (Tween 80), lipophilic emulsifier concentration (Span 80), oil
mixed with 7.7 mL of 0.2 mol/L NaOH. One g of pancreatin from the type (MCT oil or soybean oil), oil concentration, and high-pressure ho­
porcine pancreas and 240 mg of bile extract was added in KH₂PO₄-NaOH mogenization cycles. To manufacture curcumin NLCs, Tween 80 300
solution. Finally, SIF was adjusted to pH to 6.8 ± 0.1. mg/100 mL, Span 80 300 mg/100 mL, MCT oil 240 mg/100 mL, and 3
cycles of high-pressure homogenization were applied (data not shown).
2.8. Curcumin release in in vitro lipid digestion Various concentrations (1–20 mg/100 mL) of curcumin were applied
to the blank NLCs, and then the appearance, particle properties, and
NLCs dispersion was applied into three simulated gastrointestinal encapsulation efficiency were observed. Particle properties of curcumin
fluids to observe curcumin release in in vitro lipid digestion. In the first NLC dispersion, such as hydrodynamic diameter, zeta potential, poly­
digestive step (mouth condition), each NLC was mixed with preheated dispersity index, and encapsulation efficiency, were estimated (Table 2
SSF (1:1 v/v) and then reacted for 10 min at 37 ◦ C with 105 rpm shaking. and Fig. 2). Hydrodynamic diameter was not significantly different ac­
In the second digestive step (stomach condition), digested NLCs cording to curcumin concentration, and it ranged from 115.2 to 141.4
dispersion was mixed with SGF (1:1 v/v) and then reacted in a shaking nm. Zeta potential was similar to hydrodynamic diameter, and the value
water bath (37 ◦ C, 105 rpm) for 2 h. The sample was collected at 5, 10,
30, 60, 90, and 120 min. In the third digestive step (small intestinal
condition), digested NLCs dispersion mixed with the SIF (1:1 v/v) and Table 2
adjusted pH of intestinal condition sample to pH 7.0 ± 0.1 using 1 mol/L Hydrodynamic diameter and zeta potential of curcumin-loaded nanostructured
NaOH. The mixture was then reacted in a shaking water bath (37 ◦ C, lipid carriers with different curcumin concentrations.
105 rpm) for 2 h. The sample was collected at 5, 10, 30, 60, 90, and 120 Curcumin (mg/100 mL) Hydrodynamic diameter (nm) Zeta potential (mV)
min. The release amount of curcumin was measured at each time of the 1 118.9 ± 1.1cde − 25.4 ± 0.4bc
digestive process, and the particle properties of curcumin NLCs were 2 118.4 ± 1.3de − 26.6 ± 0.4cd
also determined at the same time. The release amount of curcumin was 3 126.9 ± 1.6b − 25.9 ± 0.6bc
4 126.3 ± 2.3b 25.8 ± 0.3bc
measured through the encapsulation efficiency method and then −
5 125.3 ± 1.3bc − 25.3 ± 0.7b
calculated as follows (Eq. (2)): 10 139.8 ± 1.8a − 23.2 ± 0.4a
[ ] 12 122.8 ± 3.3bcd − 29.7 ± 1.1e
EC(g)
Curcumin release (%) = 100 − × 100 (2) 14 137.4 ± 3.0a − 25.9 ± 1.3bc
IC (g) 16 115.2 ± 2.9e − 27.5 ± 0.6d
18 118.2 ± 1.7de − 27.9 ± 0.6 d
where EC is from the middle part of curcumin NLCs dispersion and IC is 20 141.4 ± 8.7a − 25.7 ± 0.4bc
the initial amount of curcumin at NLCs preparation. Data are expressed as the mean ± standard deviation (n = 3).
a-e
The means followed by the same letter are not significant by Tukey’s multiple
range test at p > 0.05.

3
J.E. Hyun et al.
Fig. 2. Encapsulation efficiency and polydispersity index of curcumin-loaded
nanostructured lipid carriers with different curcumin concentrations; gray
square: encapsulation efficiency and black square: polydispersity index. a-cThe
means followed by the same letter in the encapsulation efficiency result are not
significant by Tukey’s multiple range test at p > 0.05. Error bars indicate
standard deviation.
was from − 23.2 to − 29.7 mV, which is usually described as unstable
stability in colloid suspensions. The stable state of colloid solution is
usually over ±30 mV (Aditya et al., 2014). Even after measuring the
hydrodynamic diameter and zeta potential, selecting an appropriate
curcumin NLC dispersion was not clear.
The results of the polydispersity index and encapsulation efficiency
showed significant differences among various samples. Fig. 2 shows
monodisperse curcumin NLC dispersions from 1 to 10 mg/100 mL cur­
cumin, and the value was below the 0.25 polydispersity index. However,
the polydispersity index increased depending on the curcumin concen­
tration, especially from 14 to 20 mg/100 mL. Furthermore, curcumin
NLC dispersions (14–20 mg/100 mL) were over the 0.3 polydispersity
index, which is polydisperse, and precipitates were also observed.
Encapsulation efficiency was also significantly different according to
concentration (p < 0.05). The encapsulation efficiency of the curcumin
NLCs according to the different curcumin concentrations was
80.9–95.9% (Fig. 2), and all samples had high efficiency, but it was
significantly decreased depending on the increase in curcumin concen­
tration (p < 0.05). When the curcumin was over 4 mg/100 mL, pre­
cipitation was observed after 24 h at room temperature (Fig. 3). The
yellow color derived from the original curcumin color increased up to 4
mg/100 mL curcumin NLC dispersion, but sedimentation was observed
from the 5 mg/100 mL curcumin NLC dispersion. This phenomenon did
not affect hydrodynamic diameter or zeta potential, but it seemed that
encapsulation efficiency influenced the polydispersity index of curcu­
min NLC dispersions (Fig. 2). From NLC with 4 mg/100 mL or more of
curcumin added, the encapsulation efficiency was significantly lower (p
< 0.05), and as the concentration of curcumin increased, a polydisperse
particle distribution was shown. These results show that at the con­
centrations of solid and liquid lipids and emulsifiers set in this experi­
ment, up to 3 mg/100 mL of curcumin can be stably entrapped in the
NLC structure. However, at concentrations above that, curcumin ap­
pears to precipitate without being completely dissolved in the lipid
structure. Therefore, in the characterization of particle properties, 3 mg/
Fig. 3. Pictorial views of nanostructured lipid carriers with different curcumin concentrations (% w/v). (a) 1 mg/100 mL, (b) 2 mg/100 mL, (c) 3 mg/100 mL, (d) 4
mg/100 mL, (e) 5 mg/100 mL, (f) 20 mg/100 mL.
4
LWT 162 (2022) 113474
100 mL curcumin was selected to manufacture the optimum curcumin
NLC dispersion with 300 mg/100 mL Tween 80, 300 mg/100 mL Span
80, and 240 mg/100 mL MCT oil. Moreover, the particle properties of
the optimum curcumin NLC dispersion were 126.9 nm in diameter,
− 25.9 mV zeta potential, 0.246 PDI (monodisperse), and 94.5%
encapsulation efficiency.
NLCs can be manufactured in various formulations and have
different particle properties. Dolatabadi et al. (2021) made curcuminoid
(79.4% curcumin) NLCs using various lipids (glycerol palmitostearate,
MCT oil) and emulsifiers (Na cholate, Span 80, Poloxamer 188). Some
materials were similar to our study, but they produced NLCs using high
shear homogenization and ultrasound. The hydrodynamic diameter was
148.8–225.5 nm, the zeta potential was − 16.7–24.9 mV, and the best
encapsulation efficiency was over 95%. In the Araujo et al. (2020) study,
curcumin NLCs were produced by using specific lipids (MCT oil, castor
oil) and emulsifiers (Tween 20, Tween 80), and they used only
high-speed homogenization to manufacture curcumin NLCs. The hy­
drodynamic diameter was 192.6 or 247.7 nm, the PDI was 0.164 or
0.161 according to oil type, and the encapsulation efficiency was 88.34
or 94.14%. Rapalli et al. (2020) also produced curcumin NLCs, and
probe sonication was applied to reduce size variation. The hydrody­
namic diameter was 96.2–298.0 nm, and the PDI was 0.257–0.690
depending on sonication time. The encapsulation efficiency was
46.3–70.5%, which was not sonication time-dependent. There are many
formulations to produce optimum NLC. NLC properties, such as hydro­
dynamic diameter, PDI, zeta potential, encapsulation efficiency, etc., are
influenced by solid lipids, liquid lipids, emulsifier type, emulsifier HLB,
emulsification method, etc.
In this study, curcumin NLCs were relatively smaller (nm), had a
higher zeta potential (mV), and had a higher encapsulation efficiency
(%) than other curcumin NLCs. The reason may be the emulsification
method among various conditions. High-pressure homogenization is a
high-energy emulsification. The intensifier pump makes 2,000–30,000
psi, and pressure generates power, such as high shear, impact, and
cavitation. Three energy effects on fluid, including oil in water emulsion,
solid in liquid suspension and the energy, break and disperse particles.
Therefore, this energy could reduce the hydrodynamic diameter and
polydispersity index of a particle in colloid solution. Moreover, the
number of process cycles usually improves particle properties.
3.2. Temperature-dependent storage stability
The appearance of the curcumin NLC dispersion according to storage
temperature is shown in Table 3. There was no phase separation at any
storage temperatures, but at 65 ◦ C, the color of the curcumin NLC
dispersion changed as if curcumin was decomposed by high temperature
(Table 3). Fig. 5 shows that the encapsulation efficiency of NLCs grad­
ually decreased; therefore, the released free curcumin might be influ­
enced by high temperature. Curcumin is labile to basic solvents, oxygen,
and light and decomposes, eventually yielding volatile phenolic com­
pounds, such as vanillin, guaiacol and isoeugenol, which are relatively
hydrophilic (Kharat, Du, Zhang, & McClements, 2017). It is thought that
during this storage period, the components are hydrolyzed and volatil­
ized (Esatbeyoglu, Ulbrich, Rehberg, Rohn, & Rimbach, 2015), and the
curcumin content decreases.
The hydrodynamic diameter and zeta potential during the 28-day
storage period obtained by varying the storage temperature of
J.E. Hyun et al. LWT 162 (2022) 113474

Table 3 curcumin NLCs are shown in Fig. 4. The hydrodynamic diameter

Pictorial views of curcumin nanostructured lipid carriers at
different temperatures during 28 days of storage.
Fig. 4. Hydrodynamic diameter and zeta potential of curcumin nanostructured
lipid carriers at different temperatures during 28 days of storage. Error bars
indicate standard deviation.
increased significantly to 112.5 nm, 100.8 nm and 118.8 nm when
stored at 5 ◦ C, 21 ◦ C and 65 ◦ C, respectively (p < 0.05). However, at
40 ◦ C, the hydrodynamic diameter was maintained at an average of
90–100 nm, and there was no difference during the storage period (p >
0.05). Hydrodynamic diameter might why the emulsifier and lipid that
were used for forming NLCs were structurally changed. NLC was pro­
duced by the hot high shear homogenization (HSH) method (Salvi &
Pawar, 2019). Solid lipids were mixed with liquid lipids and core ma­
terials, such as curcumin, and then emulsifiers and aqueous phases, were
added above the melting temperature of solid lipids. Generally, the
melting point of solid lipid glycerol tristearate is 54.0–72.5 ◦ C. Then,
while emulsification of the lipid phase and aqueous phase occurred by
passing through HPH (high-pressure homogenization) at varying pres­
sures for 3 cycles, the pre-emulsion cooled rapidly, and NLC was formed
(Salvi & Pawar, 2019). Namely, solid lipids easily melt above the
melting point, and the crystallin structure of NLCs breaks down. In
addition, NLCs are usually amorphous at high temperatures (Klucker,
Dalencon, Probeck, & Haensler, 2012); therefore, curcumin might be
easily released at 65 ◦ C. The absolute zeta potential was significantly
decreased at 21, 40, and 65 ◦ C and it was rapidly decreased to − 16.9 mV
after 7 days at 65 ◦ C. This result was due to the release and decompo­
sition of curcumin, and wall materials, such as lipids and emulsifiers,
were also unstable during storage under temperature and time stress.
The encapsulation efficiency (EE%) and total phenolic content were
measured for 28 days at certain times, as shown in Figs. 5 and 6. Storage
temperature affected the release rate of curcumin encapsulated in NLCs.
Significantly, EE% was dramatically reduced from 92% to 24% at 65 ◦ C.
Next, curcumin NLCs were stored at 40 ◦ C, which was a faster release
rate than other samples. Curcumin NLCs were relatively stable when
stored at 5 ◦ C and 21 ◦ C for 28 days after preparation. The content of
curcumin is reduced due to structural instability from high-temperature
exposure, exposure to the water phase of curcumin, oxidation and hy­
drolysis (Esatbeyoglu et al., 2015). The total phenolic content of NLC
also showed a trend similar to that of EE%. At the beginning of storage (7
days), gallic acid (μg/mL) rapidly decreased and then slowly decreased
in all samples (Fig. 6). However, significantly, gallic acid (μg/mL) was
dramatically reduced from 76% to 27% at 65 ◦ C. As the storage period of
the curcumin emulsion increases, the curcumin content gradually de­
creases, and the curcumin content in the emulsion is lower at 55 ◦ C than
at 20 ◦ C (Kharat et al., 2017). Therefore, as a result of this study, the
dramatic reduction in the total phenolic content of curcumin NLCs at
65 ◦ C appears to be the result of a large amount of curcumin being
released at relatively high temperatures, resulting in the loss of many
phenolic components. From this analysis, it was observed NLC could be
stable and maintain functionality at certain storage condition. As we
mentioned in introduction, NLC system is effective to improve entrap­
ment efficiency, loading efficiency, physico-chemical stability and
bioavailability compare with other carriers. Therefore, it is useful to
apply into encapsulation of functional component such as curcumin.
Especially, curcumin is one of health functional materials but it is not
appropriate to apply directly in food production because of its low sol­
ubility, low chemical stability, low bioavailability and so on. For the
food production, food processing efficiency could be improving by using
curcumin NLC instead of pure curcumin. For the human health, around
30 ppm of curcumin which is produced by using nano-encapsulation
technology also have function in cancer drug delivery system (Santad­
kha, Skolpap, & Thitapakorn, 2021). According to the report 30–50 μM
concentration of curcumin affected proliferation and apoptosis of breast
cancer cells (Kim, Yi, Lee, Cho, & Yoon, 2004; Park et al., 2013).
Thermogravimetric analysis (TGA) is a useful method for evaluation
the thermal stability of compounds by recording the weight loss of a
sample with increasing temperature (Soltanzadeh, Peighambardoust,
Ghanbarzadeh, Mohammadi, & Lorenzo, 2021). The TGA results for the
components constituting the curcumin NLCs are shown in Fig. 7 and
Table 4. The thermal decomposition temperature and weight loss rates

5
J.E. Hyun et al. LWT 162 (2022) 113474

a-d
Fig. 5. Encapsulation efficiency of curcumin nanostructured lipid carriers at different temperatures during 28 days of storage. The means followed by the same
letter are not significant by Tukey’s multiple range test at p > 0.05. Error bars indicate standard deviation.

Fig. 6. Total phenolic content of curcumin nanostructured lipid carriers at different temperatures during 28 days of storage. a-eThe means followed by the same letter
are not significant by Tukey’s multiple range test at p > 0.05. Error bars indicate standard deviation.

of glycerol tristearate and MCT oil, which were used as lipids for NLCs
preparation, were 250.6, 243.4 ◦ C and 94.1, 99.8%, respectively. Cur­
cumin showed the highest thermal decomposition temperature of
251.7 ◦ C and the lowest weight loss rate of 76.4% compared to other
materials composing NLC in this experiment, because phenolic com­
pounds have good thermal stability (Valencia et al., 2021). The thermal
decomposition temperatures of blank NLCs and curcumin NLCs did not
show a significant difference at 207.8 and 192.4 ◦ C, respectively. In
addition, both samples showed a stable state up to 180 ◦ C. These results
may suggest that curcumin is efficiently encapsulated in the amorphous
state. This showed a similar trend to the studies related to
carbamazepine-loaded solid lipid nanoparticles and nanostructured
lipid carriers (Montoto et al., 2018).
Differential scanning calorimetry (DSC) is a method to determine the
change in temperature and energy in a phase transition. Therefore,
through DSC analysis, the melting point of a compound and the crys­
tallization or amorphous state of a drug can be known (Madane &
Mahajan, 2014). The results of DSC analysis of glycerol tristearate, MCT
oil, curcumin, blank NLCs, and curcumin NLCs are shown in Fig. 7F and
Table 4. Glycerol tristearate, used as a solid lipid in the preparation of
NLCs showed a sharp melting peak around 55.2 ◦ C, suggesting that this
material has crystallinity. And curcumin exhibited the maximum
endothermic peak near about 177.7 ◦ C, which was found to have a value
similar to the results of several studies related to curcumin formulation
(Agrawal, Saraf, Pradhan, Patel, Singhvi, & Alexa et al., 2021; Behba­
hani et al., 2019; Puglia, Frasca, Musumeci, Rizza, Puglisi, & Bonina
et al., 2012). However, the maximum endothermic peak of curcumin
NLCs appeared around 56.2 ◦ C, and there was no peak near 177 ◦ C

6
J.E. Hyun et al. LWT 162 (2022) 113474

Fig. 7. Thermal characterization using TGA and DSC: TGA of glycerol tristearate (A), MCT oil (B), curcumin (C), blank NLCs (D), and curcumin NLCs (E); and DSC of
curcumin NLCs components.

3.3. In vitro digestion

Table 4
Parameter extracted from the TGA and DSC of the curcumin NLCs components.
The change in the curcumin NLC particle properties was observed
TGA DSC under in vitro digestion, as shown in Fig. 8A. There was no change in
Samples
Thermal Weight Melting ΔH curcumin NLC hydrodynamic diameter in in vitro model saliva. Hydro­
decomposition loss (%) temperature (J/g) dynamic diameter was increased to 165.3–180.8 nm in simulated gastric
temperature (◦ C) (◦ C)
fluid conditions, but there was no significant difference between the
Glycerol 250.6 94.1 55.2 179.3 original diameter and the hydrodynamic diameter after in vitro model
tristearate saliva (p > 0.05). Notably, there was no significant difference in hy­
MCT oil 243.4 99.8 ND ND
drodynamic diameter according to digestion time (p > 0.05). A nonionic
Curcumin 251.7 76.4 177.7 138.8
Blank NLCs 207.8 99.6 55.7 86.9 surfactant, Tween 80, which surrounded NLCs, was adequate to main­
Curcumin 192.4 99.7 56.2 77.5 tain the stability of NLCs from hydrolysis by pepsin and low pH (Van
NLCs Aken, Bomhof, Zoet, Verbeek, & Oosterveld, 2011). Similarly, the re­
ND: not detected. ported curcumin NLC maintained a hydrodynamic diameter of 200–300
nm, and there was no aggregation in simulated gastric fluid for 2 h
where the peak of curcumin appeared. These results indicate that cur­ (Behbahani et al., 2019). In the simulated small intestinal fluid, the
cumin is completely encapsulated inside the NLCs. In addition, the hydrodynamic diameter was increased by more than 100 nm after 5 min
enthalpy changes of blank NLCs (86.9 J/g) and curcumin NLCs (77.5 of digestion time and increased significantly to 445.1 nm at 120 min of
J/g) was decreased compared to glycerol tristearate (179.3 J/g) and reaction time. The enzymes, and bile extract might hydrolyze the
curcumin (138.8 J/g), indicating that blank NLCs and curcumin NLCs emulsifier and lipid layer as free micelles and monoacylglycerols,
have an amorphous structure. In the study of Behbahani et al. (2019), resulting in micelle formation (Aditya et al., 2014; Park, Garcia, Shin, &
curcumin-loaded NLCs were prepared using stearic acid as a solid lipid Kim, 2017). Simultaneously, NLCs were also treated in an in vitro
and Tween 80 and pluronic F167 as emulsifiers. At this time, the result digestion system without an enzyme. There was no hydrodynamic
of DSC analysis of curcumin NLC showed a small peak near 173 ◦ C, diameter change by adjusting the pH. According to one study related to a
which is the maximum endothermic peak of curcumin. This showed that phloretin-loaded nanostructured lipid carrier, an in vitro digestion model
some curcumin was not enclosed by NLCs, showing a different trend of phloretin NLC initially showed a hydrodynamic diameter of approx­
from the results of this study. imately 100 nm. However, it has been reported that NLC aggregation
occurred during the small intestine stage, which increased the hydro­
dynamic diameter to approximately 800 nm (Gu, Sun, Wang, & Xia,
2022).

7
J.E. Hyun et al.
Fig. 8. Changes in properties of curcumin nanostructured lipid carriers ac­
cording to simulated gastrointestinal conditions and pH: hydrodynamic diam­
eter (A) and curcumin release (B); Cur-NLC (curcumin nanostructured lipid
carrier), stomach condition (S), and small intestine condition (I). a-gThe means
followed by the same letter are not significant by Tukey’s multiple range test at
p > 0.05. Error bars indicate standard deviation.
Regarding curcumin release (Fig. 8B), approximately 31.7% of cur­
cumin was released in in vitro model saliva, and curcumin was released
up to 37.7% after 2 h in imitation gastric juice. There was no significant
difference in imitation saliva (p > 0.05). In imitation intestinal fluid,
curcumin release dramatically increased to 48.1% in 5 min, and up to
74.5% after 30 min, and the amount of curcumin released increased over
time and increased to 82% after 2 h, but after 30 min, there was no
significant difference (p > 0.05). In addition, when measuring the
amount of curcumin released by adjusting the pH and temperature,
excluding enzymes, curcumin was released when the pH was 6.8 or
higher, and there was no significant difference from the amount of
curcumin first released when the pH was acidic (p > 0.05). When the pH
was 8 or higher, the amount of curcumin released increased significantly
after 10 min to 30.9–53.6% (p < 0.05). Curcumin is highly affected by
pH. In an alkaline environment, oxidation and hydrolysis reactions
occur at the interface between the aqueous and oil phases, producing
reaction products such as bicyclopentadione, ferulic acid, and feruloyl
methane. Because most of these decomposition products are relatively
hydrophilic, they migrate to the water phase, which is unstable. How­
ever, to restore equilibrium, more curcumin molecules will migrate from
the oil phase to the interface, and the decomposition process will
continue (Kharat et al., 2017). At acidic pH, curcumin is predominantly
in an enolic structure, so the main decomposition mechanism is oxida­
tion rather than hydrolysis. Curcumin is also released under the
8
LWT 162 (2022) 113474
influence of pH, but considering the hydrodynamic diameter, it is
thought that the amount of curcumin release increased to 82% by
enzyme and pH as the hydrodynamic diameter of NLCs increased by an
enzyme (Fig. 8). Curcumin is rapidly decomposed under alkaline con­
ditions, so it is not easy to maintain curcumin from the digestive tract to
the intestine.
4. Conclusions
In this study, optimal conditions were established, and various
characteristics were observed to prepare nanostructured lipid carriers
(NLCs), which were included to increase the safety and utilization rate of
the poorly soluble material curcumin. Curcumin NLCs were micronized
into 3 mg/100 mL curcumin, 300 mg/100 mL Tween 80 and 300 mg/
100 mL Span 80. The produced curcumin NLCs had a hydrodynamic
diameter of 126.9 nm, zeta potential of − 25.9 mV, PDI of 0.246, and
encapsulation efficiency of 94.5%. When the curcumin NLC was stored
at various temperatures for 28 days, it was stably maintained at 5 ◦ C and
21 ◦ C, but at 40 ◦ C and 65 ◦ C, the encapsulation efficiency and total
phenolic content decreased dramatically. As a result of thermal char­
acterization, it was confirmed that curcumin NLCs did not show a peak
near 177 ◦ C, the maximum endothermic peak of curcumin, and was
completely captured in the NLC structure. In the digestive imitation
system, NLCs released approximately 31% of curcumin in imitation
saliva, and curcumin release gradually increased. This study showed
that NLC was an effective transporter for the solubilization of curcumin.
Furthermore, NLCs could apply to various fields by increasing water
solubility, bioavailability, and curcumin storage stability. Especially,
curcumin NLC could be applied into functional foods such as beverage,
jelly food, etc. in the food industry.
CRediT authorship contribution statement
Ji Eun Hyun: Investigation, Writing – original draft, Writing – re­
view & editing, Visualization. Hye-Yoon Yi: Validation, Investigation,
Writing – review & editing, Visualization, Software. Geun-Pyo Hong:
Conceptualization, Writing – original draft, Resources. Ji-Yeon Chun:
Conceptualization, Investigation, Writing – original draft, Writing – re­
view & editing, Supervision.
Declaration of competing interest
All authors declared that there is no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2022.113474.
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