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Elaboration of curcumin-loaded rice bran albumin nanoparticles formulation

with increased in vitro bioactivity and in vivo bioavailability

Article  in  Food Hydrocolloids · November 2017

DOI: 10.1016/j.foodhyd.2017.11.027

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 Food Hydrocolloids 77 (2018) 834e842

Contents lists available at ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Elaboration of curcumin-loaded rice bran albumin nanoparticles

formulation with increased in vitro bioactivity and in vivo
bioavailability
Chun Liu a, Xiaoquan Yang b, Wei Wu a, Zhao Long a, Huaxi Xiao a, Feijun Luo a,
Yingbin Shen c, Qinlu Lin a, *
a
National Engineering Laboratory for Rice and By-product Deep Processing, College of Food Science and Engineering, Center South University of Forestry
and Technology, Changsha 410004, China
b
Research and Development Center of Food Proteins, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640,
China
c
Department of Food Science and Engineering, School of Science and Engineering, Jinan University, Guangzhou 510632, China

a r t i c l e i n f o a b s t r a c t

Article history: Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with various
Received 18 September 2017 bioactivities, but its optimum potential is limited by its lack of dispersibility in aqueous solvents and poor
Received in revised form oral bioavailability. Here, we employed a protein-based nanoparticle approach to improve bioactivity and
16 November 2017
bioavailability. Curcumin was encapsulated with 95.94% efficiency in biodegradable nanoparticulate
Accepted 17 November 2017
formulation based on rice bran albumin (RBA). The mean particle diameter and z-potential of curcumin-
Available online 20 November 2017
loaded RBA nanoparticles (Cur-RBA-NPs) were 120 nm and
36.3 mV, respectively. The in vitro bioac-
tivity and in vivo bioavailability of Cur-RBA-NPs were evaluated. The results indicated that the in vitro
Keywords:
Rice bran albumin
bioactivities (antioxidant activity, anti-inflammatory activity, and anti-proliferative activity on tumor
Curcumin cells) of Cur-RBA-NPs were superior to those of free curcumin, respectively. Moreover, Cur-RBA-NPs
Nanoparticles significantly enhanced the bioavailability of curcumin in rats as compared with free curcumin. Be-
Bioactivity sides, the results clearly indicated the promise of RBA-based nanoparticles for oral delivery of poorly
Bioavailability bioavailable molecules like curcumin.
© 2017 Elsevier Ltd. All rights reserved.
Chemical compounds studied in this article:
Ethanol (PubChem CID: 702)
Curcumin (PubChem CID: 969516)

1. Introduction trials have demonstrated that although a high dose of curcumin

(3600e12000 mg/day) in patients with colorectal cancer could
It has been well documented that many active compounds in achieve efficient chemopreventive effect (Garcea et al., 2004;
natural foods, such as fruits and vegetables, possess bioactivities Sharma et al., 2001; Steward & Gescher, 2008), the large dose of
that can help prevent and cure diseases (Yen, Wu, Tzeng, Lin, & Lin, curcumin and frequent administrations involved showed an in-
2010). Curcumin, a yellow pigment present in the turmeric (Cur- crease of side effects and lower compliance from the users (Yen
cuma longa), is commonly used as food spice in curry as well as a et al., 2010).
medicine for the treatment of various diseases. It has been exten- Delivery systems have been used to enhance the effectiveness of
sively studied for its pharmacological activities that include anti- drug and food materials and to decrease the dosage required.
oxidant, anti-inflammatory, anticancer, antiulcer, Nanonization is one of the drug/food delivery processes that can
immunomodulatory, wound healing, neuroprotective and anti- help overcome a material’s poor aqueous solubility, dissolution,
aging effects (Pari, Tewas, & Eckel, 2008). Despite a series of bio- and/or bioavailability (Yen et al., 2010). Nanoparticles are stable
activities that curcumin possesses, it has low bioavailability due to colloidal particles with a size ranges from 10 to 1000 nm. Many
its poor aqueous dispersibility (Yen et al., 2010). Several clinical studies have demonstrated that reduction in particle size of the
active ingredient to nanoparticle size can improve its efficacy, sol-
* Corresponding author.
ubility and bioavailability (Kesisoglou, Panmai, & Wu, 2007).
E-mail address: linqinlu@hotmail.com (Q. Lin). Recently, the application of protein-based delivery systems for the

https://doi.org/10.1016/j.foodhyd.2017.11.027
0268-005X/© 2017 Elsevier Ltd. All rights reserved.
 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842 835

encapsulation of hydrophobic bioactives have gained increasing Biological Technology Co., Ltd. (Liyungang, China). Curcumin was
interest (Z.-L. Wan, Guo, & Yang, 2015). So far, drug or hydrophobic purchased from Sigma-Aldrich Co. (Shanghai, China). Ethanol was
bioactives-loaded nanoparticles have been synthesized success- purchased from Nanjing Chemical Industry (Nanjing, China). All
fully from various proteins, including both water-soluble (bovine or other chemicals used were of analytical grade.
human serum albumin, BSA (Fang et al., 2011) or HSA (Wartlick,
Sp€ankuchschmitt, Strebhardt, Kreuter, & Langer, 2004), b-lacto- 2.2. Isolation of albumin from rice bran
globulin (bLG) (Teng, Li, & Wang, 2014) and insoluble proteins (zein
(Zhong & Jin, 2009), gliadin (Ezpeleta et al., 1996) and barley pro- Rice bran albumin (RBA) extraction was carried out at room
tein (J. Yang, Zhou, & Chen, 2013) etc.). These nanoscaled systems temperature (25  C) by adapting the method of Adebiyi et al. (2009)
exhibited various advantages, such as improved solubility, with some modification. Defatted rice bran (DRB) (100 g) was
controlled release property, and enhanced bioavailability of extracted by using Ultra homogenizer (IKA, Germany) with 400 mL
encapsulated nutraceuticals (Z.-L. Wan et al., 2015). Additionally, of distilled water for 4 h and centrifuged at 8000g for 15 min to
they exhibited low toxicity due to superior biocompatibility and obtain supernatant (albumin extract). Then the albumin extract
nutritional value (Teng et al., 2014). was dialysed against Millipore pure water (15 MU) for three days in
Rice bran protein has been found to be of high quality and of refrigerator (4  C) and freeze-dried in a freeze-dryer (DELTA 1e24
great importance for food and pharmaceutical applications. It is a LSC, Christ, Germany). The yield of RBA is about 3.18%. The RBA was
plant protein that can be derived from rice bran, an abundant and analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-
cheap agricultural byproduct. The protein content in rice bran is phoresis (SDS-PAGE) according to Laemmli (Laemmli, 1970). The
approximately 10e15% and it consists of 37% water-soluble, 31% protein content of RBA was determined by the micro-Kjeldahl
salt-soluble, 2% alcohol-soluble, and 27% alkali-soluble storage method, the result indicated that the protein content of RBA was
proteins. Its unique properties as being hypoallergenic and having 95.16% (w/w).
anti-cancer activity make it a superior cereal protein that may find a
wide range of applications (Fabian & Ju, 2011). 2.3. Dynamic interfacial tension measurements
Albumin from rice accounts for about 2e6% of the total seed
proteins and about 35% of the rice bran (Adebiyi, Adebiyi, The dynamic interfacial tension of RBA solutions [0.1% (w/v), pH
Hasegawa, Ogawa, & Muramoto, 2009). Like other albumins, they 7.0, 25  C] at the oil-water interface (purified corn germ oil was
are readily soluble in water due to the presence of sufficient net used) was determined by an optical contact angle meter as
charge and the lack of any extensive disulfide cross-linking or ag- described elsewhere (Z. L. Wan et al., 2014).
gregation (Hamada, 1997). Among the storage proteins in rice, al-
bumins are reported to have the highest biological value being 2.4. Formulation and characterization of curcumin-loaded RBA
most readily absorbed and utilized by the body (Mawal, Mawal, & nanoparticles (Cur-RBA-NPs)
Ranjekar, 1987). In addition, Wei et al. stated that a 16-kDa rice
albumin exhibited antioxidant activity and rice albumin was more 2.4.1. Formulation of Cur-RBA-NPs
potent than other rice proteins in preventing Cu2þ induced low- To obtain curcumin-loaded RBA, 0.1 mL stock solution of cur-
density lipoprotein (LDL) oxidation similar to serum albumins cumin (4 mg mL
1 in ethanol) was added into 2.9 mL of RBA so-
(Wei, Nguyen, Kim, & Sok, 2007). Similar antioxidant activity to lutions (1, 2, 3, 4, 5 mg mL
1) in successive titrations with magnetic
that of serum albumin was observed with rice albumin because stirring. The mixtures were centrifuged at 10000g, 25  C for 20 min
their N-terminal amino acid sequences are homologous (Masayuki to pellet the unbound curcumin, and the supernatants containing
Nakase et al., 1996). Recently, Ina et al. reported that rice albumin curcumin nanocomplexes were preserved in a light-resistant
suppressed the elevation of blood glucose and plasma insulin levels container at 4  C for determination. As contrasts, RBA without
after oral glucose loading (Ina et al., 2016). curcumin and curcumin without RBA in the same PBS solution with
In our previous work, we have successfully fabricated two homologous concentration were also prepared.
nanoparticles by using two soybean albumins [Kunitz trypsin in-
hibitor (KTI) and Bowman-Birk inhibitor (BBI)] to improve bio- 2.4.2. Encapsulation efficiency (EE) and loading capacity (LC)
accessibility and bioavailability of curcumin (Liu, Cheng, & Yang, The EE (%) of curcumin in the curcumin-loaded RBA was esti-
2017; Liu et al., 2016). However, KTI, as a major antinutritional mated as the percentage of curcumin encapsulated in the proteins
factor in soybean, must be treated by heating in the presence of by the following equation: EE (%) ¼ 100 e [amount of free curcumin
reducing agents before being used to prepare nanodelivery carrier. (mg)/total amount of added curcumin (mg)]  100, where the
In addition, although BBI is an excellent carrier protein for curcu- amount of free curcumin is determined from the precipitate ob-
min, only small quantities of it can be found in soybean, and it is tained by centrifugation. The precipitate was extracted in 5 mL of
costly to isolate and purify. Therefore, in this work, efforts have ethanol with mild stirring for 5 min under magnetically stirred
been made from the following aspects: first, rice bran albumin conditions and then centrifuged at 10 000g for 15 min at 25  C to
(RBA) was efficiently isolated from defatted rice bran by using an remove the protein aggregates. The supernatant was subjected to
improved method based on Adebiyi et al. (2009); second, a nano- spectrophotometric analysis at 426 nm with a GENESYS 10S
particulate delivery carrier has been developed and characterized UVeVis spectrophotometer (Thermo Scientific, USA), and the cur-
by the use of RBA; third, the in vitro bioactivity (antioxidant activity, cumin concentration was determined by using an established
anti-inflammatory activity and anti-proliferative activity on tumor standard curve of curcumin (R2 ¼ 0.9965). The LC of the samples
cells) and the in vivo bioavailability of nanoparticulate curcumin in was calculated with the following equation: LC (%) ¼ mass of
rats have been investigated. encapsulated curcumin/total mass of RBA.

2. Material and methods 2.4.3. Particle size and z-potential measurements

The mean particle size and z-potential of the nanocomplexes
2.1. Materials were determined according to our previous publication (Z.-L. Wan,
Wang, Yang, Wang, & Wang, 2016) by using a Nanosizer ZS in-
Defatted rice bran was kindly provided by Liyungang Jinhong strument (Malvern Instruments Ltd, Worcestershire, UK).
836 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842

2.4.4. Transmission electron microscopy (TEM) CO2 incubator. The NO level was measured by reacting 100 mL of the
TEM was used to observe the surface morphology of nano- Griess reagent with 100 mL of the culture supernatant for 30 min at
particles and to further confirm particle diameter by dynamic light room temperature (25  C) in the dark. The absorbance was
scattering (DLS). A drop of diluted sample was deposited onto a measured at 540 nm by using an enzyme-linked immunosorbent
carbon-coated copper grid, and excess of sample was removed after assay (ELISA) microplate reader.
5 min with a filter paper. Then, a droplet of phosphotungstic acid
(1%, w/v) was put onto the grid and removed after 5 min. Obser- 2.5.2.3. Prostaglandin E2 (PGE2) scavenging activity. The PGE2
vations were made with a JEM-2100F transmission electron mi- concentration in the culture media was determined using an EIA kit
croscope operating at 200 kV (JEOL, Japan) (Liu et al., 2017). (R&D Systems, Minneapolis, MN) according to the manufacturer’s
protocol. RAW264.7 cells were seeded at a density of 5  104 cells
2.4.5. X-ray diffraction (XRD) per well in 96-well plates, allowed to adhere for 2 h, and then
The XRD patterns of the samples were characterized by a Bruker treated with KTI at the indicated concentrations for 24 h. The cells
AXS (Karlsruhe, Germany) D8 Advance diffractometer. The instru- were stimulated with 1 mg mL
1 LPS for 24 h at 37  C in a 5% CO2
ment was equipped with a copper anode that produced Cu Ka X- incubator. The culture supernatants (100 mL) were collected, and
rays (l ¼ 0.15418 nm) with an accelerating voltage 40 kV and a tube the PGE2 concentrations were determined using an ELISA micro-
current 40 mA. The diffractogram was collected with a monocap plate reader.
collimator of 0.3 mm during 300 s. XY amplitude of 2 mm resulted
in a 2q between 3 and 40 after the separate recordings were 2.5.2.4. Measurement of inflammatory cytokines. The
merged. Rocking and amplitude oscillation were used to obtain an RAW264.7 cells were plated at a density of 5  104 cells per mL on a
average diffractogram of the sample and minimize a preferred 96 well plate and incubated overnight. Then, cells were treated
orientation of crystals (Liu et al., 2017). with 1 mg mL
1 LPS, alone or with free curcumin or Cur-RBA-NPs at
the indicated concentration. After 24 h of treatment, the concen-
2.4.6. Stability measurement tration of the pro-inflammatory cytokine, TNF-a, interleukin (IL)-6
The free curcumin and the freshly prepared nanocomplex dis- and IL-1b were measured in the culture supernatants using
persions containing sodium azide (0.002%, w/v) were settled under commercially available kits (Invitrogen, Carlsbad, USA) according to
room temperature (25  C). At the specified time points, samples the manufacturer’s instructions. The concentrations of TNF-a, IL-6
(200 mL) were taken out and added to 1800 mL of ethanol for and IL-1b were calculated based on the standard curve prepared
quantitative analysis of curcumin by spectrophotometer, as using each recombinant cytokine in the kit.
described above. The results were represented by a retained ratio of
curcumin, which was calculated as the percentage of the retained 2.5.3. In vitro anti-proliferative activity on tumor cells assay
curcumin at a certain time point with respect to the initial value The anti-proliferative effects of Cur-RBA-NPs on tumor cells
(Liu et al., 2016). were analyzed by the MTT assay (K. Pan, Luo, Gan, Baek, & Zhong,
2014). Du145, HCT-116, HepG2, and MCF-7 cell lines were seeded
2.5. In vitro bioactivity measurement at 5000 cells per well density in 96-well plates. After 24 h, the cells
were treated with a medium containing DMSO-dissolved or
2.5.1. In vitro antioxidant activity different concentration (0, 5, 10, 15, 20, 25 and 30 mM) of Cur-RBA-
The in vitro antioxidant activities of RBA, Cur-RBA-NPs, curcu- NPs. Other cells were untreated (negative controls, NC) or treated
min in water and curcumin in ethanol were evaluated by DPPH only with DMSO or RBA at the concentrations as in the dispersions
radical scavenging activity (Yen et al., 2010), Trolox equivalent with encapsulated curcumin (positive controls, PC). Cells were
antioxidant capacity (TEAC) (Wang, Chen, Luo, & Yan, 2016) and incubated for 48 h for assessing the toxicity of samples. A standard
ferric-reducing ability of plasma (FRAP) assays (Wang et al., 2016). MTT based colorimetric assay was used to determine cell viability.
Vitamin C (Vc) was used as an antioxidant reference. Details of the Relative cell viability was expressed as the absorbance normalized
operation conditions and methods as described elsewhere (Wang by that of the control cells treated with same amounts of DMSO and
et al., 2016). RBA as in DMSO-dissolved and encapsulated curcumin samples.
The mean and standard deviation from six-well replicates were
2.5.2. In vitro anti-inflammatory activity assay calculated. The normalized cell viability was obtained after
2.5.2.1. Reactive oxygen species (ROS) scavenging activity. The normalizing the viability of a treatment by the viability of NC and
intracellular production of ROS in the RAW264.7 cells was deter- PC.
mined by measuring the oxidation of DCFH-DA to DCF. The
RAW264.7 cells were pre-incubated at a density of 5  104 cells per Cell viability (%) ¼ [(Atreated
APC)/(ANC
APC)]  100
mL in 96-well plates for 4 h, and then treated with 1 mg mL
1 LPS
for 24 h. This was followed by treatment with free curcumin or Cur- where Atreated, APC and ANC are the absorbance of the wells with cells
RBA-NPs at the indicated concentration for 24 h. The cells were treated by curcumin, the positive and the negative control,
exposed to 80 mM DCFH-DA for 30 min at 37  C, and the fluores- respectively (Liu et al., 2016).
cence was measured by using a microplate reader at excitation and
emission wavelengths of 400 and 505 nm, respectively (Choi, Ji, Lee, 2.6. In vivo bioavailability study
Lee, & Cho, 2015).
For the in vivo bioavailability study, 12 male Sprague-Dawley
2.5.2.2. Nitrite (NO) scavenging activity. The NO concentration in (SD) rats weighing 260e300 g were used. The protocol was
the medium was measured as an indicator of NO production ac- approved by the University Animal Ethics Committee and per-
cording to the Griess reaction (Kim, de Vera, Watkins, & Billiar, formed according to the guiding principles for the use and care of
1997) The RAW264.7 cells were seeded in 96-well plates at experimental animals at the University of South China. The animals
5  104 cells per mL and incubated overnight. Then, cells were were divided into two groups (n ¼ 6). Group 1 was administered
stimulated with LPS (1 mg mL
1), and treated with free curcumin or 454 (454 mg NPs  0.0441 (LE) ¼ 20 mg curcumin) mg kg
1 body
Cur-RBA-NPs at the indicated concentration for 24 h at 37  C in a 5% weight (bw) Cur-BBI-NPs, and group 2 was administered
 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842 837

20 mg kg
1 bw free curcumin by oral gavage. Details of the 2.71% (i.e., decreased from 67.63 to 27.08 mg mg
1 of RBA). When
collection of blood samples and determination of serum curcumin the concentration of RBA was over 3 mg mL
1, the EE is comparable
concentration as described in our previous work (Liu et al., 2017). to that (>95%) of curcumin encapsulated in b-lactoglobulin nano-
particles prepared by the same method (Sneharani, Karakkat,
2.7. Statistical analysis Singh, & Rao, 2010), but reasonably higher than that of curcumin
encapsulated in zein-based colloidal particles produced by the
Results were expressed as mean values ± standard deviations. antisolvent precipitation method (71e87%) (Patel, Hu, Tiwari, &
Sample comparison, by multivariate analysis of variance (ANOVA) Velikov, 2010). The LC of curcumin is much greater than that
and followed by Duncan’s comparison test, was used to assess the (1.743e1.784 mg mg
1 of SPI) in SPI-curcumin nanocomplexes
differences. The level of significance used was p < 0.05. (Chen, Li, & Tang, 2015) and that (19 mg mg
1 of casein) in the
curcumin encapsulated in casein nanocapsules (Kang Pan, Zhong, &
3. Results and discussion Baek, 2013).
Because the increase of EE took very little change with RBA
3.1. SDS-PAGE and surface activity of RBA concentration increasing from 3 mg mL
1 to 5 mg mL
1, 3 mg mL
1
of RBA concentration was selected as the optimum concentration
The SDS-PAGE pattern of RBA isolated from rice bran by our for preparing RBA-curcumin complexes. The appearances of cur-
method is shown in the inset of Fig. 1. Several main bands of pro- cumin in pure PBS and different concentration RBA solution can be
teins on SDS-PAGE gel were detected, the molecular weight (MW) observed in the inset in Fig. 2A. The free curcumin in PBS was very
of these protein bands ranges from 10 to 100 kDa, which is in good turbid due to its poor water solubility. However, the dispersibility of
agreement to the literature data (Fabian & Ju, 2011). Proteins are curcumin was increased with the increase of the RBA solution
natural amphiphilic molecules with surface activity, which can be concentration, and the RBA-curcumin mixture solutions exhibited
determined by dynamic interfacial tension of proteins at the oil- yellow and highly transparent appearances when the concentra-
water interface. The time evolution of the interfacial tension (g) tion of RBA solution was over 2 mg mL
1. The water dispersibility of
of RBA at the oil-water interface is presented in Fig. 1. Generally, the curcumin could reach up to 127.92 mg mL
1
interfacial tension decreased with adsorption time, a phenomenon ((0.1 mL  4 mg mL
1)  95.94%/3 mL) when the RBA concentration
that can be associated with the surface-active substances adsorp- was 3 mg mL
1, which increased ca. 44 times compared with that of
tion at the interface. As can be seen from Fig. 1, the interfacial free curcumin in PBS (2.9 mg mL
1). It is necessary to note that
tension value of RBA was 6.94 mN m
1 after 180 min, which was curcumin dispersibility also has a great increase in PBS after the
lower than that (8.0 mN m
1) of pure soybean protein isolate (SPI) antisolvent process. Nevertheless, curcumin dispersibility is
at the same conditions in our previous work (Z. L. Wan et al., 2014). increased 11629-fold by this antisolvent process in RBA solutions
This data revealed that RBA possessed good surface activity. The according to previously reported results (11 mg L
1) (Kaminaga
favorable surface activity of RBA would provide driving force for the et al., 2003).
intermolecular self-assembly of RBA during preparation of Cur- The mean particle size and z-potential of Cur-RBA-NPs are
RBA-NPs by anti-solvent precipitation method. presented in Fig. 2B. As can be seen, the mean particle size of Cur-
RBA-NPs was decreased with the increase of the RBA solution
3.2. Formulation and characterization of cur-RBA-NPs concentration, but the decrease of particle size was insignificant
when the concentration of RBA solution was more than 2 mg mL
1.
The encapsulation efficiency (EE) and loading capacity (LC) of Oppositely, the absolute value of z-potential of Cur-RBA-NPs was
curcumin in Cur-RBA-NPs are shown in Fig. 2A. As can be seen from increased with the increase of the RBA solution concentration,
Fig. 2A, as the concentration of RBA increased from 1 mg mL
1 to while the increase of the absolute value of z-potential of Cur-RBA-
5 mg mL
1, the EE of curcumin progressively increased from 49.03% NPs took very little change when the concentration of RBA solution
to 98.18%, while the LC of curcumin linearly decreased from 6.76% to was over 3 mg mL
1. The mean particle size and z-potential of Cur-
RBA-NPs were 120 nm and
36.3 mV respectively when the con-
centration of RBA solution was 3 mg mL
1. The mean particle size of
Cur-RBA-NPs (fresh curcumin stabilized by 3 mg mL
1 RBA) was
25
also evidenced by TEM (the inset in Fig. 2B).
1
XRD was carried out to investigate the crystallinity of curcumin
after its complexation with RBA and the results are presented in
20 Fig. 2C. For the XRD patterns of free curcumin showed intense
diffraction peaks between 5 and 30 , suggesting its highly crys-
tallized structure. On the contrary, the classic amorphous XRD
(mN/m)

pattern was observed for RBA. Nevertheless, the diffraction spec-

15 trum of Cur-RBA-NPs exhibited complete disappearance of all the
characteristic crystalline peaks of curcumin, indicating the forma-
tion of amorphous curcumin. This observation should be attributed
to the inhibition of its crystallization in the nanoscale confinement
10
and the formation of an amorphous complex with RBA within the
particle matrix.
Curcumin in aqueous solution is readily susceptible to hydro-
5 lysis or degradation, even at physiological Ph (Liu et al., 2016).
0 2000 4000 6000 8000 10000 12000 Therefore, a short-time storage stability of free curcumin and Cur-
RBA-NPs solutions was evaluated (Fig. 2D). The result revealed
Time (s) that free curcumin in PBS was very unstable, and only 3.6% cur-
Fig. 1. Dynamic interfacial tension of rice bran albumin (RBA) at the oil-water inter- cumin was retained after 24 h storage under ambient conditions. As
face. Inset: SDS-PAGE pattern of RBA. Lane M: Molecular weight markers, Lane 1: RBA. expected, the stability of curcumin in the Cur-RBA-NPs was
838 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842

A 110
Encapsulation efficiency
8
B 180
-33
100 Loading efficiency 170
Encapsulation efficiency (%)

160 -34

Loading capacity (%)

90
6

-potential (mV)
150
80 -35

Size (nm)
200 nm
140
70 -36
130
4
60
120 -37
Size
50 110 -potential
-38
40 2 100
1 2 3 4 5 1 2 3 4 5
RBA concentration (mg/mL) RBA concentration (mg/mL)

3000
C D 100
2500 Free curcumin
RBA
Cur-RBA-NPs 80

Residual ratio (%)

Intensity (A.U.)

2000

60
1500 Cur-RBA-NPs
Free curcumin
40
1000

500 20

0 0
10 20 30 40 0 10 20 30 40 50 60 70 80
2 Time (h)
Fig. 2. (A) Encapsulation efficiency (EE) and loading capacity (LC) of curcumin in RBA solutions with different concentration. Inset: 0e5: fresh curcumin stabilized by the 0, 1, 2, 3, 4,
and 5 mg mL
1 RBA, respectively. (B) Size and z-potential of Cur-RBA-NPs (fresh curcumin stabilized by RBA with different concentration). Inset: TEM image of fresh curcumin
stabilized by 3 mg mL
1 RBA. (C) XRD diffractograms of free curcumin powder, RBA power and lyophilized Cur-RBA-NPs powder. (D) Residual ratios of curcumin as a function of
storage time under ambient conditions.

prominently improved. Curcumin was very stable in the initial 24 h, curcumin in complexes with serum albumins (Leung & Kee, 2009),
in spite of the stability slowly decreasing with further extension of soy Kunitz trypsin inhibitor nanoparticles (Liu et al., 2016), and milk
storage time. The final retained ratio of curcumin was still above proteins including as1-casein (Sneharani, Singh, & Appu Rao, 2009)
75% in RBA solution with a concentration of 3 mg mL
1. A similar and b-lactoglobulin (Sneharani et al., 2010).
improvement of storage stability has also been observed for

A 100 B 200
*
TEAC (mmol Trolox/g sample)

TEAC
Scavenging activity (%)

FRAP (mmol Fe /g sample)

80 FRAP *
150
60
2+

40 100

20 Vc
Cur-RBA-NPs 50
Free curcumin
0 RBA
0
0 50 100 150 200 RBA Cur-RBA-NPs Free curcumin
Concentration ( g/mL)
Fig. 3. In vitro antioxidant activities of Cur-RBA-NPs, Free curcumin (curcumin in 10 mM PBS), and RBA in terms of (A) scavenging capacity on 2,2-diphenyl-1-picrylhydrazyl radicals
(DPPH), and (B) Trolox equivalent antioxidant capacity (TEAC) and ferric-reducing ability of plasma (FRAP) assays. Each value is expressed as mean ± SD (n ¼ 3). * indicated the
significant difference between Cur-RBA-NPs and free curcumin (p < 0.05).
 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842 839

3.3. In vitro bioactivity positive reference are exhibited in Fig. 3A. Obviously, all samples
showed dose-dependent DPPH radical scavenging capacity in the
3.3.1. In vitro antioxidant activity concentration range of 0e200 mg mL
1. At 200 mg mL
1, the scav-
The potential antioxidant activity of curcumin after encapsula- enging activities of Vc, Cur-RBA-NPs, free curcumin (curcumin in
tion has been evaluated by DPPH scavenging, TEAC, and FRAP as- PBS), and RBA on DPPH radicals were 95.62%, 90.87%, 70.12%, and
says. The in vitro antioxidant activities of curcumin dissolved in PBS 45.65%, respectively. The DPPH radical scavenging capacity of the
and RBA were also evaluated and compared. The scavenging ca- Cur-RBA-NPs was close to that of Vc but was significantly stronger
pacities of the samples on the DPPH radical compared with Vc as a

A 400
450
5 M 10 M 20 M
A 130
120
5 M 10 M 20 M
*
350
* *
110 * *

TNF- (pg/mL)
300
100 *
250
ROS (%)

90
80 200

70 150
60 100
50
50
40
Blank LPS Cur-RBA-NPs Free curcumin RBA
Blank LPS Cur-RBA-NPs Free curcumin RBA

B 400
B 130
120
5 M 10 M 20 M
350
5 M 10 M 20 M

* 300 *
110
* *
* *
Nitric oxide (%)

100 250
IL-6 (pg/mL)

90 200
80
150
70
60 100

50 50
40
0
Blank LPS Cur-RBA-NPs Free curcumin RBA Blank LPS Cur-RBA-NPs Free curcumin RBA

C 130 5 M 10 M 20 M C 70 5 M 10 M 20 M

120
* 60
*
110
* *
*
IL-1 (pg/mL)

100 *
PGE2 (%)

50
90

80 40
70

60 30

50
20
Blank LPS Cur-RBA-NPs Free curcumin RBA Blank LPS Cur-RBA-NPs Free curcumin RBA

Fig. 4. In vitro anti-inflammatory activities of Cur-RBA-NPs, free curcumin (curcumin Fig. 5. Effects of anti-inflammatory of Cur-RBA-NPs, free curcumin (curcumin in
in 10 mM PBS), and RBA in LPS-stimulated RAW264.7 macrophage cells in terms of (A) 10 mM PBS), and RBA on the expression of pro-inflammatory cytokines in LPS-
ROS production, (B) NO production, and (C) PGE2 production. Each value is expressed stimulated RAW264.7 macrophage cells in terms of (A) Level of TNF-a, (B) Level of
as mean ± SD (n ¼ 3). * indicated the significant difference between Cur-RBA-NPs and IL-6, and (C) Level of IL-1b. Each value is expressed as mean ± SD (n ¼ 3). * indicated
free curcumin (p < 0.05). the significant difference between Cur-RBA-NPs and free curcumin (p < 0.05).
840 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842

than that of curcumin in PBS (p < 0.05). RBA also showed DPPH cells. As seen from Fig. 4A, treatment of the cells with Cur-RBA-NPs
radical scavenging capacity in a concentration-dependent manner in the concentration range of 5e20 mM markedly inhibited the LPS-
due to the potential antioxidant ability of some sequence in the stimulated ROS production, however, the inhibition effect of free
protein. curcumin on ROS production was weaker as compared with that of
The results of TEAC and FRAP assays indicated that the encap- Cur-RBA-NPs. In addition, RBA also showed slight dose-dependent
sulated curcumin in Cur-RBA-NPs exhibited higher antioxidant inhibition effect on ROS production. In the case of the inhibition
capacities (TEAC value of 190.09 ± 9.42 mmol Trolox/g sample and effects of the samples on NO and PGE2 production, similar results
FRAP value of 146.11 ± 7.26 mmol Fe2þ/g sample) than curcumin in were obtained (Fig. 4B and C).
PBS (TEAC value of 125.13 ± 6.20 mmol Trolox/g sample and FRAP Cytokines are low-molecular weight proteins that affect im-
value of 97.21 ± 4.82 mmol Fe2þ/g sample) (p < 0.05) (Fig. 3B). mune cell function, proliferation and activity, and play a key role in
These results indicated that encapsulated curcumin can achieve the inflammatory cascade. Hence, we further analyzed the effects of
efficient antioxidant activities in vitro. Similar to DPPH scavenging, the samples (Cur-RBA-NPs, free curcumin, and RBA) on the pro-
RBA also showed relatively high TEAC and FRAP values. duction of pro-inflammatory cytokines, including TNF-a, IL-6 and
IL-1b, in LPS-stimulated RAW264.7 macrophages. As shown in
3.3.2. In vitro anti-inflammatory activity Fig. 5A, a significant increase in TNF-a production was observed in
Macrophage activation by LPS can initiate an increase in oxygen the LPS-stimulated RAW264.7 macrophages. However, both Cur-
uptake that gives rise to a variety of reactive oxygen species (ROS), RBA-NPs and free curcumin showed dose-dependent inhibition
which are the major factors driving the oxidative stress-induced effects on TNF-a production in the concentration range of 5e20 mM,
inflammation in immune cells. Besides, activated macrophages and the inhibition effect of Cur-RBA-NPs was superior to that of free
produce a variety of pro-inflammatory molecules, including nitric curcumin. As can be seen from Fig. 5B and C, Cur-RBA-NPs relative
oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF- to free curcumin could suppress LPS-stimulated IL-6 and IL-1b
a), and interleukins, which are involved in the development and production more effectively. These results indicated that nano-
progression of inflammatory diseases and cancer (H.-L. Yang et al., particulate curcumin possessed favorable in vitro anti-
2015). As shown in Fig. 4, LPS treatment significantly increased ROS, inflammatory activity as compared with free curcumin.
NO, and PGE2 production compared with the untreated normal

Du145 HCT-116
A 100
B 100 *
* * *
80 * 80
*
Cell viability (%)
Cell viability (%)

* *
60 60
* *
RBA * RBA *
40 40
Free curcmin Free curcumin
Cur-RBA-NPs Cur-RBA-NPs
20 20

0 0
5 10 15 20 25 30 35 5 10 15 20 25 30 35
Concentration ( M) Concentration ( M)

HepG2 MCF-7
C 100 * D 100 *
*
* * *
80 80
Cell viability (%)
Cell viability (%)

* *
60 * 60
*
40 RBA * 40
RBA *
Free curcumin Free curcumin
Cur-RBA-NPs Cur-RBA-NPs
20 20

0 0
5 10 15 20 25 30 35 5 10 15 20 25 30 35
Concentration ( M) Concentration ( M)
Fig. 6. Dose dependent cytotoxicity of RBA, free curcumin, and cur-RBA-NPs in A549 (A), HCT-116 (B), HepG2 (C) and MCF-7 (D) cell lines. The extent of growth inhibition was
measured at 48 h by the MTT assay. The inhibition was calculated with respect to controls. Data as mean ± SD, n ¼ 6. (*) p < 0.05, free curcumin in solution versus Cur-RBA-NPs. RBA
toxicity was tested at the concentrations that are required to achieve the active concentrations of curcumin, i.e. the concentrations of RBA were 0.04, 0.08, 0.13, 0.17, 0.21, and
0.25 mg mL
1 corresponding to the concentrations of curcumin of 5, 10, 15, 20, 25, and 30 mM, respectively.
 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842 841

3.3.3. In vitro anti-proliferative activity on tumor cells curcumin concentrations in the plasma after oral administration of
To further evaluate the bioactivity of nanoparticulate curcumin, Cur-RBA-NPs (454 (454 mg NPs  0.0441 (LE) ¼ 20 mg curcumin)
in vitro anti-proliferative activity on tumor cells of curcumin mg/kg bw) and free curcumin (20 mg/kg bw) at single dose in SD
(nanoparticulate and free curcumin) was studied in different cell rats are illustrated in Fig. 7. The relevant pharmacokinetic param-
lines by MTT assay. All the studied cell lines showed a typical dose eters including Cmax, Tmax and AUC0-∞ are listed in the inset in Fig. 7.
dependent anti-proliferative effect (Fig. 6). The in vitro half A sustained release of curcumin over 24 h was observed in the
maximal inhibitory concentration (IC50) is the quantitative mea- nanoparticle form, where as in the case of free curcumin, the level
surement for the cell toxicity induced by chemotherapeutic drug. was very low beyond 5 h. A wide variability in the absorption phase
These IC50 values were calculated from the obtained curves of all was observed between Cur-RBA-NPs and free curcumin. Free cur-
the studied cell lines and the results demonstrated nanoparticulate cumin formulation upon oral administration resulted in sharp Cmax
curcumin has higher anti-proliferative activity than free curcumin within 0.5 h, however, the plasma concentration of curcumin
(Table 1). Nanoparticulate curcumin is 1.60, 1.58, 1.42 and 1.66 times decreased rapidly, suggesting rapid metabolism of curcumin.
more effective than free curcumin as observed in D145, HCT-116, Nevertheless, relatively slow increase and sustained plasma con-
HepG2 and MCF-7 cell lines, respectively. The obtained results centration of curcumin for a longer time was observed after
confirmed comparable inhibition of cell proliferation, where administration of nanoparticulate curcumin, with delayed Cmax
nanoparticulate curcumin was more effective than free curcumin in occurring at 1 h, indicating an obvious sustained release of curcu-
solution by controlling the tumor cell growth. min from the nanoparticles. There was a prominent difference in
the AUC0-∞ between free curcumin and Cur-RBA-NPs. The AUC0-∞
3.4. In vivo bioavailability for curcumin was higher in the animals administered with nano-
particulate curcumin formulation, with a relative bioavailability of
Cur-RBA-NPs were devised to improve the oral bioavailability of 10.21 as compared to free curcumin. These results indicated that
curcumin. Blood levels after oral administration of nanoparticulate Cur-BBI-NPs formulation could improve the bioavailability of
formulation were compared with oral free curcumin. The mean curcumin.

4. Conclusion
Table 1
IC50 values of free curcumin and Cur-RBA-NPs in different tumor cells as assayed by
MTT cytotoxicity assay. In summary, Cur-RBA-NPs were prepared successfully by anti-
solvent precipitation approach. The nanoparticles were spherical
Tumor cells IC50 values (mM)
with a particle size of about 120 nm and z-potential ca.
36.3 mV.
Free curcumin Cur-RBA-NPs The Cur-RBA-NPs with high EE exhibited favorable water dis-
A549 26.11 16.27 persibility and storage stability. In addition, nanoparticulate cur-
HCT-116 26.39 16.67 cumin formulation showed improved in vitro antioxidant activity,
HepG2 26.72 18.89 anti-inflammatory activity and in vitro antiproliferative activity on
MCF-7 26.22 15.83
tumor cells of curcumin in aqueous solution as compared with free

300
Serum curcumin concentration (ng/mL)

250
256 + 19a 1 1715 + 97a

82 + 8b 0.5 168 + 9b
200

150
Cur-RBA-NPs
Free Curcumin
100

50

0
0 5 10 15 20 25
Time (h)
Fig. 7. Bioavailability of Cur-RBA-NPs and free curcumin. All values are presented as the mean ± SD, n ¼ 6. (Inset) Pharmacokinetics parameters of two curcumin formulations. AUC,
area under the plasma concentration-time curve from 0 h to ∞; Cmax, peak concentration; Tmax, time to reach peak concentration. Means of a column marked with different letters
indicate a significant difference between two groups (p < 0.05).
842 C. Liu et al. / Food Hydrocolloids 77 (2018) 834e842

curcumin. The in vivo bioavailability study revealed that Cur-RBA- 6(88), 85621e85633.
Liu, C., Cheng, F., & Yang, X. (2017). Fabrication of a Soybean Bowman-Birk inhibitor
NPs demonstrated more than 10-fold increase in oral bioavail-
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that RBA could be employed to develop a novel nano-delivery Masayuki Nakase, Takahiro Adachi, Atsuo Urisu, Takeya Miyashita, A. M. A., Satoru
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Acknowledgements characterization of rice albumin. Bioscience Reports, 7(1), 1e9.
Pan, K., Luo, Y., Gan, Y., Baek, S. J., & Zhong, Q. (2014). pH-driven encapsulation of
curcumin in self-assembled casein nanoparticles for enhanced dispersibility
This research was supported by grants from the National Natural and bioactivity. Soft Matter, 10(35), 6820e6830.
Science Foundation of China (No. 31571874), the Grain-oil Process Pan, K., Zhong, Q., & Baek, S. J. (2013). Enhanced dispersibility and bioactivity of
curcumin by encapsulation in casein nanocapsules. Journal of Agricultural and
and Quality Control 2011 Collaborative and Innovative Grant from Food Chemistry, 61(25), 6036e6043.
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