Food Chemistry 232 (2017) 67–78

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Phytochemical profiles and antioxidant activity of processed brown rice

products
Er Sheng Gong a,b, Shunjing Luo a, Tong Li b, Chengmei Liu a,⇑, Guowen Zhang a, Jun Chen a,
Zicong Zeng a, Rui Hai Liu b,⇑
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China
b
Department of Food Science, Cornell University, Ithaca, NY 14853, United States

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

Article history: The phytochemical profiles and antioxidant activity of free, soluble-conjugated, and bound fractions of
Received 23 October 2016 brown rice and its processed products (textured rice, cooked rice and rice noodle) were studied.
Received in revised form 10 March 2017 Nineteen phenolic acids were identified. Trans-ferulic acid was the most abundant monomeric phenolic
Accepted 28 March 2017
acid with trans-trans-8-O-40 diferulic acid being most abundant diferulic acid. Processing increased the
Available online 30 March 2017
content of free phenolic acids, but decreased the content of soluble-conjugated phenolic acids. The con-
tent of bound phenolic acids was increased by improved extrusion cooking technology and cooking, but
Chemical compounds studied in this article:
not affected by rice noodle extrusion. The total phenolic contents and antioxidant activities of free and
Trans-ferulic acid (PubChem CID: 445858)
Trans-p-coumaric acid (PubChem CID:
soluble-conjugated fractions were decreased after processing, whereas those of bound fraction were
637542) increased by improved extrusion cooking technology and cooking, but not affected by rice noodle extru-
Cis-ferulic acid (PubChem CID: 1548883) sion. Results indicated that whole foods designed for reducing chronic disease risk need to consider the
Trans-trans-8-O-40 diferulic acid (PubChem effects of processing on phytochemical profiles and antioxidant activity of whole grains.
CID: 101052653) Ó 2017 Elsevier Ltd. All rights reserved.
Cis-p-coumaric acid (PubChem CID:
1549106)
P-hydroxybenzoic acid (PubChem CID: 135)
Trans-sinapic acid (PubChem CID: 637775)
Chlorogenic acid (PubChem CID: 1794427)
Trans-trans-5-50 diferulic acid (PubChem
CID: 5281770)
Trans-trans-8-50 benzofuran diferulic acid
(PubChem CID: 10385446)

Keywords:
Phenolics
Whole grains
Antioxidant activity
Processing
Phytochemicals

1. Introduction reduced risk of chronic diseases, including cardiovascular disease,

Epidemiological studies suggest that increased consumption of
whole grains and whole grain products has been associated with
⇑ Corresponding authors at: State Key Laboratory of Food Science and Technol-
ogy, No. 235 Nanjing East Road, Nanchang University, Nanchang 330047, China
(C. Liu). Department of Food Science, 245 Stocking Hall, Cornell University, Ithaca,
NY 14853-7201, USA (R.H. Liu).
E-mail addresses: gongersheng@163.com (E.S. Gong), luoshunjing@aliyun.com
(S. Luo), tl24@cornell.edu (T. Li), liuchengmei@aliyun.com (C. Liu), gwzhang@ncu.
edu.cn (G. Zhang), chen-jun1986@hotmail.com (J. Chen), katzeng@163.com
(Z. Zeng), RL23@cornell.edu (R.H. Liu).
type II diabetes, obesity and cancer (Jiang, Li, & Liu, 2016;
Okarter & Liu, 2010; Xi & Liu, 2016). The phytochemicals in whole
grains are proposed to be partly responsible for the health benefits
of whole grains and whole grain products consumption (Liu, 2007;
Okarter & Liu, 2010). Rice (Oryza sativa L.) is the most important
grain crop in Asia, and its processed products, such as cooked rice,
rice noodle and textured rice, are staple foods in most populations.
Whole grain rice is a rich source of phytochemicals with antioxi-
dant activity (AOA), including phenolic acids (PAs), flavonoids,
anthocyanins, proanthocyanidins, tocopherols, tocotrienols and
c-oryzanol (Okarter & Liu, 2010). PAs are the most common

http://dx.doi.org/10.1016/j.foodchem.2017.03.148
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
68 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78
phenolic compounds found in rice (Liu, 2007). Phenolics exist as
free, soluble-conjugated, and bound forms, with bound form being
predominant (Adom & Liu, 2002). The bound phenolics are linked
to cell wall structural components, such as cellulose, lignin and
proteins (Acosta-Estrada, Gutierrez-Uribe, & Serna-Saldivar, 2014;
Liu, 2007). Some monomeric and dimeric phenolics have been
detected and structurally identified in whole grains (Guo & Beta,
2013; Qiu, Liu, & Beta, 2010). High concentrations of these pheno-
lics are present in the germ and bran layers (Adom, Sorrells, & Liu,
2005). However, these bioactive phenolics are removed by polish-
ing or milling. Whole grain rice with abundant phytochemicals
exhibit several unique bioactivities, such as antioxidant (Adom &
Liu, 2002), antimicrobial (Zhang, Ali, & Khan, 2014), antiviral
(Ray et al., 2013), anti-inflammatory (Niu et al., 2013), anticancer
and immunomodulatory activities (Hudson, Dinh, Kokubun,
Simmonds, & Gescher, 2000; Verschoyle et al., 2007), thus promot-
ing overall human health.
Since whole grains are thermally processed before consuming,
it is important to understand the effect of processing on bioactive
phenolics. A few researchers have done this work. In case of total
phenolic contents (TPCs) and AOAs of free fractions, most studies
found decreases after processing (Massaretto, Madureira Alves,
Mussi de Mira, Carmona, & Lanfer Marquez, 2011; N’Dri et al.,
2013; Scaglioni, de Souza, Schmidt, & Badiale-Furlong, 2014;
Walter et al., 2013; Zaupa, Calani, Del Rio, Brighenti, & Pellegrini,
2015), Dewanto, Wu, and Liu (2002) reported increases in sweet
corns, whereas de la Parra, Saldivar, & Liu (2007) observed both
decreases and increases in corns. When it came to TPCs and AOAs
of bound fractions, a few studies found decrements after process-
ing (de la Parra et al., 2007; Dewanto et al., 2002; N’Dri et al.,
2013; Scaglioni et al., 2014), Massaretto et al. (2011) observed
increments, whereas Zaupa et al. (2015) reported decrements,
increments, and no significant effects in different grain varieties.
These processing effects depended on the processing methods
applied, the type, and variety of whole grains analyzed. There is
no related report on the effect of processing on the TPCs and AOAs
of soluble-conjugated fractions in these studies. Processing may
cause depolymerisation of oligomers and high polymers into
dimers and trimers, concomitant polymerisation reactions, and
formation of strong complexes of the soluble phenolics with
macromolecules in a food matrix, which may reduce their solubil-
ity (Massaretto et al., 2011), thus influencing health benefits of
rice. Acosta-Estrada et al. (2014) reviewed that different forms
(free, soluble-conjugated, and bound) of phytochemicals are
released and absorbed at different sites of the human gastrointesti-
nal tract, thus exhibiting different health benefits. To conclude,
however, limited data are available to understand the effect of pro-
cessing on the phytochemical profiles and AOA in free, soluble-
conjugated and bound fractions of brown rice (BR).
The objectives of the present study were (1) to identify phenolic
compounds in free, soluble-conjugated and bound fractions of BR;
(2) to determine the effects of three processing methods of the sta-
ple foods, i.e. improved extrusion cooking technology (IECT), cook-
ing, and rice noodle extrusion (RNE), on TPC and AOA; and (3) to
determine the effect of processing on phenolic profiles of BR.
2. Materials and methods
2.1. Materials
Chlorogenic acid (CHA), p-hydroxybenzoic acid (p-HA), vanillic
acid (VA), trans-caffeic acid (trans-CFA), syringic acid (SYA), trans-
p-coumaric acid (trans-p-COA), trans-ferulic acid (trans-FA), trans-
sinapic acid (trans-SIA), gallic acid, Folin-Ciocalteu reagent, 2,20 -a
zino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 6-hydrox
y-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri
(2-pyridyl)-s-triazine (TPTZ), ascorbic acid, and 20 ,70 -dichlorofluor
escein-diacetate (DCFH-DA) were purchased from Sigma–Aldrich
(St. Louis, MO, USA). Acetone, ethyl acetate, methanol, hexane,
potassium hydroxide (KOH), potassium phosphate monobasic,
potassium phosphate dibasic, sodium carbonate, sodium hydrox-
ide (NaOH), potassium persulfate, hydrochloric acid (HCl), EDTA
disodium salt (Na2EDTA), ferric chloride, ferrous sulfate, high per-
formance liquid chromatography (HPLC) grade acetonitrile and
acetic acid were purchased from Xilong Chemical Co., Ltd. (Guang-
dong, China). 2,20 -Azobis (2-amidinopropane) dihydrochloride
(ABAP) was purchased from Wako Chemicals (Richmond, VA,
USA). Mass spectrometry (MS) grade acetonitrile and formic acid
were purchased from Merck (Darmstadt, Germany).
2.2. Brown rice sample and sample preparation
A BR (Oryza sativa L.) cultivar (Sonjing 16) was used in this
study. It was grown in Wuchang (Heilongjiang Province, China)
during the 2014 growing season, and harvested in October. After
dehulled, BR was stored in sealed polyethylene containers at
20 °C until use. For chemical determination, BR was ground by
a DFY-200 mill (Linda Machinery, Zhejiang, China) and sieved
(100 mesh) to a uniform size, then stored in sealed polyethylene
containers at 20 °C.
Cooked rice. BR grains were cooked using 400 ml of tap water to
200 g of rice. BR was cooked for 30 min in an electric pressure coo-
ker (Medea MY-12SS509A, Guangdong, China). After cooling,
cooked rice was dried overnight in an oven at 45 °C, then ground
to a fine powder by a DFY-200 mill and sieved (100 mesh) to uni-
form size. The cooking treatment was performed in triplicate. Sam-
ples were stored in sealed polyethylene containers at 20 °C.
Texturized rice. Texturized rice was made using a single-screw
extruder based on the method reported previously (Liu et al.,
2011). BR grains were soaked in excess tap water for 2 h, drained
and then ground to a fine powder to pass through a 0.25 mm mesh
screen in a hammer mill (Hongxing Machinery Limited Liability
Company, Jiangxi, China). The moisture content of the rice flour
was measured by a halogen moisture analyzer HR83 (Mettler-
Toledo International Inc., Greifensee, Switzerland). In order to
obtain a total feed moisture content of 40%, 8 kg of the BR flour
was blended with 2226 ml tap water in a mixer (Jinan Saixin
Machinery Ltd., Shandong, China) for 20 min at a high speed
(365 rpm) to ensure homogeneity of the feeding material before
IECT. The temperature profiles in the feed, mix, screw conveyor,
shearing compression metering, and die head zones were kept con-
stant at 50, 65, 85, 120, and 95 °C, respectively. The feed and screw
speeds were 30 and 37.5 rpm, respectively. The rotary cut fre-
quency was set as 35.5 Hz. After cooling, texturized rice was dried
overnight in an oven at 45 °C, then ground to a fine powder by a
DFY-200 mill and sieved (100 mesh) to uniform size. The IECT
was performed in triplicate. Samples were stored in sealed poly-
ethylene containers at 20 °C until analysis.
Rice noodle. Rice noodle was made by a traditional rice noodle
machine. Briefly, 8 kg of BR grains were soaked in excess tap water
overnight. The soaked rice grains and proper water were passed
through an instant rice noodle machine (Shengdi Machinery Fac-
tory, Zhejing, China). The BR grains were extruded and milled at
high speed (800 rpm) in the noodle machine, resulting in heat gen-
eration and flour steaming rapidly. Then the gelatinized BR flour
was passed through a 72-hole die to produce uniform strips. Long
strips obtained from the cutting rolls of the rice noodle machine
were dried at room temperature for 24 h. The rice noodles were
ground to a fine powder by a DFY-200 mill and sieved (100 mesh)
to uniform size. The RNE treatment was performed in triplicate.
Samples were stored in sealed polyethylene containers at 20 °C.
 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78
2.3. Extraction of free phenolics
Free phenolics were extracted using the method reported previ-
ously by our laboratory (Adom & Liu, 2002; Zhang & Liu, 2015;
Zhang, Zhang, Zhang, & Liu, 2010). Briefly, 5 g of sample flour were
blended with 30 ml of 80% chilled acetone for 10 min. After cen-
trifugation at 2500g for 10 min, the supernatant was removed
and extraction was repeated two times. Supernatant was evapo-
rated in a rotary evaporator to dryness at 45 °C and reconstituted
in 10 ml of deionized water to obtain soluble phenolics. Soluble
phenolic extract was acidized with concentrated HCl to pH 2. The
solution was extracted with hexane to remove lipids and then
extracted with ethyl acetate for five times. The ethyl acetate frac-
tion was evaporated to dryness at 45 °C. Phenolic compounds were
reconstituted in 5 ml of 80% methanol and labeled as free pheno-
lics, then stored at 40 °C until use. All analyses were performed
in triplicate.
2.4. Extraction of soluble-conjugated phenolics
Soluble-conjugated phenolics were extracted using the method
previously reported by our laboratory (Adom & Liu, 2002, 2005).
Soluble-conjugated phenolics in the aqueous phase after free phe-
nolics extraction were digested with 10 ml of 2 M NaOH for 1 h
under nitrogen at room temperature. Then the solution was neu-
tralized with concentrated HCl to pH 2. The mixture was extracted
with hexane to remove lipids and then extracted with ethyl acetate
for five times. The ethyl acetate fraction was evaporated to dryness
at 45 °C. Phenolic compounds were reconstituted in 5 ml of 80%
methanol and stored at 40 °C until use. All analyses were per-
formed in triplicate.
2.5. Extraction of bound phenolics
Bound phenolics were extracted according to the method
reported previously by our laboratory (Adom & Liu, 2002;
Kremer Faller, Fialho, & Liu, 2012). Briefly, 0.5 g of sample were
extracted thrice with 10 ml of 80% chilled acetone and the super-
natant was discarded. The residue was digested with 10 ml of
2 M NaOH under the same conditions as the soluble-conjugated
phenolics. After acidification and centrifugation, the mixture was
extracted with hexane to remove lipids and then with ethyl acet-
ate. The ethyl acetate fraction was evaporated at 45 °C to dryness.
Phenolic compounds were reconstituted in 5 ml of 80% methanol
and stored at 40 °C until use. All analyses were performed in
triplicate.
2.6. Determination of total phenolic content
TPC was measured using the Folin-Ciocalteu colorimetric
method reported by Singleton, Orthofer, and Lamuela-Raventos
(1999) and modified in our laboratory (Adom & Liu, 2002; Zhu
et al., 2015). Sample extracts or standard solutions were treated
with Folin-Ciocalteu reagent for 6 min. Then the mixture was alka-
linized with Na2CO3. The blue colour of the solution was developed
for 90 min, and the absorbance was measured at 760 nm using a
UV–VIS spectrophotometer (Purkinje General Instrument Co.,
Ltd., Beijing, China). The measurement was compared to standard
curve of gallic acid and expressed as mg of gallic acid equiv.
(GAE)/100 g dry weight (DW). Data were reported as mean ± SD
for three replications.
2.7. Determination of trolox equivalent antioxidant capacity (TEAC)
TEAC was determined using the ABTS radical cation decoloriza-
tion assay reported by Re et al. (1999). ABTS+ was prepared by
69
reaction of 192 mg ABTS and 33 mg potassium persulfate in
50 ml of water. The mixture was stirred overnight in the dark,
afterwards, the solution was diluted with water up to a final absor-
bance of 0.70 ± 0.02 at 734 nm. 3920 ll of diluted ABTS+ solution
was vortexed with 20 ll of sample extracts, then incubated at
30 °C, and the absorbance was recorded at 734 nm after 10 min.
Trolox was used to obtain the standard curve. The results were
expressed as mg of Trolox equiv. (TE)/100 g DW. Data were
reported as mean ± SD for three replications.
2.8. Determination of ferric reducing antioxidant power (FRAP)
FRAP was estimated following the method described by Benzie
and Strain (1996). Briefly, the FRAP reagent was prepared by blend-
ing 2.5 ml of 10 mM TPTZ solution (in 40 mM HCl) plus 2.5 ml of
12.024 mM ferric chloride and 25 ml of 300 mM acetate buffer
(pH 3.6) at 37 °C. Then, 2.4 ml of sample extract or standard solu-
tion was mixed with 0.1 ml of FRAP reagent. The mixture was incu-
bated at 37 °C for 10 min and the absorbance was measured at
593 nm. Ferrous sulfate solutions were used to perform the cali-
bration curve. The results were expressed as mg of ferrous sulfate
equiv. (FE)/100 g DW. Data were reported as mean ± SD for three
replications.
2.9. Determination of peroxyl radical scavenging capacity (PSC)
PSC was determined using the assay developed by our labora-
tory (Adom & Liu, 2005) with modification (Wang, Chen, Guo,
Abbasi, & Liu, 2016). Briefly, 100 ll of vitamin C standard solutions
or sample extracts were mixed with 100 ll of DCFH, which was
prepared by hydrolyzing 2.48 mM DCFH-DA with 1.0 mM KOH to
remove the diacetate moiety. Then 50 ll of ABAP was added to ini-
tiate the reaction at 37 °C. The fluorescence at 485 nm excitation
was measured every 2 min in a run of 40 min after emission at
538 nm in a Fluoroskan Ascent fluorescent spectrophotometer
(Thermo Labsystems, MA, USA). Results were expressed as mg of
vitamin C equiv. (VCE)/100 g DW. Data are reported as mean ± SD
for three replications.
2.10. Identification of phenolics in brown rice by ultra-high
performance liquid chromatography coupled with electrospray
ionization quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-
MS)
The chromatographic separation was carried out on an Agilent
1290 infinity series UPLC system (Agilent Technologies, Santa
Clara, CA, USA) as reported previously (Li et al., 2016). The reversed
phase column was Eclipse XDB-C18 (4.6  150 mm, 5 lm). The
mobile phase was made up of 0.1% formic acid solution (A) and
acetonitrile (B). The solvent gradient elution was as follows:
0–3 min, 10% B; 3–15 min, 10–15% B; 15–20 min, 15–22% B;
20–30 min, 22–35%; 30–50 min, 35–45% B; 50–60 min, 45–100%
B; 60–65 min, 100–10% B. 10 ll of sample extract was injected
and eluted at a flow rate of 0.6 ml/min. MS was performed on an
Agilent 6538 Q-TOF Micromass (Agilent Technologies, Santa Clara,
CA, USA) equipped with an orthogonal electrospray ionization
source operating in negative mode. The optimal values of the
source parameters were as follows: ion source temperature,
150 °C; desolvation temperature, 325 °C; capillary voltage, 3500
V; cone voltage, 35 V; cone gas (N2) flow rate, 50 L/h; desolvation
gas (N2) flow rate, 900 L/h. The collision energies were set at 10, 15,
20 and 25 V for monomeric PAs, 15, 20, 25, and 30 V for diferulic
acids (DFAs), and the fragmentor voltage was set at 120 V.
70 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

2.11. Determination of phenolics in brown rice and processed brown trans-8-50 DFA, trans-trans-8-50 benzofuran DFA and trans-cis-8-50
rice products by reversed-phase high performance liquid benzofuran DFA displayed the same typical fragment ions at m/z
chromatography (RP-HPLC)

The chromatographic separation was carried out on an Agilent

1260 infinity series HPLC system (Agilent Technologies, Santa
Clara, CA, USA). The chromatographic column was a Waters Sun-
Fire C18 column (4.6  250 mm, 5 lm) (Massachusetts, USA). The
mobile phase for separation of monomeric PAs consisted of 1.3%
acetic acid solution (A) and acetonitrile (B). Separation of phenolic
monomers: 0–2 min, 10% B; 2–20 min, 10–13% B; 20–35 min,
13–20% B; 35–45 min, 20–35% B; 45–80 min, 35–45% B;
80–93 min, 45–100% B; 93–98 min, 100–8% B; 98–105 min, 8% B.
The mobile phase for separation of DFAs consisted of 0.1% acetic
acid solution (A) and acetonitrile (B). Separation of DFAs:
0–1 min, 10% B; 1–3 min, 10–11% B; 3–12 min, 11–15% B;
12–17 min, 15–18% B; 17–22 min, 18–20% B; 22–30 min, 20–30%
B; 30–55 min, 30–40% B; 55–58 min, 40–100% B; 58–65 min,
100–10% B. The injection volume of sample extract was 20 ll and
the flow rate was 0.8 ml/min. The determination of PAs was carried
out by area measurements at 280 nm for hydroxybenzoic acid
derivatives and 320 nm for hydroxycinnamic acid derivatives.
Calibration curves for the cis-isomers of hydroxycinnamic acid
derivatives were calculated using different fresh trans-
hydroxycinnamic acid derivatives solutions that were placed under
the UV lamp overnight to ensure different trans/cis transformation
ratios according to Hernanz et al. (2001). Trans-FA was used for
quantification of DFAs according to Chiremba, Taylor, Rooney,
and Beta (2012). The recoveries of PAs are shown in Supplemen-
tary Table 1.

2.12. Statistical analysis

Differences in mean values were determined by ANOVA fol-

lowed by Tukey’s tests at p < 0.05 significance level using SPSS ver-
sion 18.0 (SPSS Inc., USA). Multivariate analysis was performed
using the software XLSTAT 2016 (Addinsoft, NY, USA), where the
principal component analysis (PCA) was applied to the data set
after normalization by Pearson’s correlation matrix.

3. Results and discussion

3.1. Identification of phenolic compounds in brown rice

Twelve monomeric PAs were identified by UPLC-Q-TOF-MS

with authentic standards: CHA, p-HA, trans-CFA, VA, SYA, cis-CFA,
trans-p-COA, cis-p-COA, trans-SIA, trans-FA, cis-SIA, and cis-FA
(Supplementary Table 2).
By plotting the molecular ion at m/z 385 ([MH]) expected
from DFA, seven peaks (peaks 13–19) were detected. Their RP-
HPLC chromatogram are shown in Fig. 1E. After comparing RP-
HPLC elution sequences, MS spectra, MS/MS spectra, and UV spec-
tra (Supplementary Fig. 1) with the literature data (Dobberstein &
Bunzel, 2010; Guo & Beta, 2013), the seven peaks were identified
as: trans-trans-8-50 DFA, trans-trans-5-50 DFA, trans-cis-5-50 DFA,
trans-trans-8-O-40 DFA, trans-trans-8-50 benzofuran DFA, trans-cis-
8-50 benzofuran DFA, and trans-cis-8-O-40 DFA (Fig. 2). Trans-

"
Fig. 1. HPLC chromatogram (280 nm) of phenolic acids standards (A), free fraction
(B), soluble-conjugated fraction (C), bound fraction (D), and DFAs (E) of brown rice:
p-hydroxybenzoic acid (1), chlorogenic acid (2), vanillic acid (3), trans-caffeic acid
(4), syringic acid (5), cis-caffeic acid (6), trans-p-coumaric acid (7), cis-p-coumaric
acid (8), trans-ferulic acid (9), trans-sinapic acid (10), cis-ferulic acid (11), cis-sinapic
acid (12), trans-trans-8-50 DFA (13), trans-trans-5-50 DFA (14), trans-cis-5-50 DFA
(15), trans-trans-8-O-40 DFA (16), trans-trans-8-50 benzofuran DFA (17), trans-cis-8-
50 benzofuran DFA (18), and trans-cis-8-O-40 DFA (19).
 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 71

Fig. 2. MS/MS spectra of seven DFAs acquired at a collision energy of 20 eV: trans-trans-8-50 DFA (13), trans-trans-5-50 DFA (14), trans-cis-5-50 DFA (15), trans-trans-8-O-40
DFA (16), trans-trans-8-50 benzofuran DFA (17), trans-cis-8-50 benzofuran DFA (18), and trans-cis-8-O-40 DFA (19).
72 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78
326, 311, 297, 282, and 267. The further identification of these
three DFAs was achieved by comparing UV spectra and the reten-
tion times in RP-HPLC with literature data (Dobberstein & Bunzel,
2010; Guo & Beta, 2013). The identification of trans-trans-8-O-40
DFA was confirmed by the UV spectrum of 8-O-40 DFA standard
(Dobberstein & Bunzel, 2010) and its typical fragment ion at m/z
193 (Guo & Beta, 2013). The fragments at m/z 178, 149, and 134
of trans-trans-8-O-40 DFA were attributed to losses of –CH3, –
COOH, and –CH3 plus –COOH from the typical fragment ion at
m/z 193, respectively. Trans-cis-8-O-40 DFA, which is the cis-
isomer of trans-trans-8-O-40 DFA, exhibited the same fragment ions
as trans-trans-8-O-40 DFA. It eluted after trans-trans-8-O-40 DFA in
RP-HPLC (Fig. 1E). Trans-trans-5-50 DFA displayed typical fragment
ions at m/z 385, 370, 341, 326, 282, and 267, which were similar to
those reported by Guo and Beta (2013). What’s more, the UV spec-
trum of trans-trans-5-50 DFA in this study matched that of the
trans-trans-5-50 DFA standard reported by Dobberstein and
Bunzel (2010). Trans-cis-5-50 DFA, which is the cis-isomer of
trans-trans-5-50 DFA, exhibited the same fragment ions as trans-
trans-5-50 DFA. It eluted after trans-trans-5-50 DFA in RP-HPLC
(Fig. 1E).
Most of DFAs reported previously are trans-trans forms
(Dobberstein & Bunzel, 2010; Guo & Beta, 2013; Qiu et al., 2010),
and only the cis-isomers of 8-50 , 8-50 benzofuran and 5-50 DFA
had been identified by Bunzel and Steinhart (2004). Other DFA
cis-isomers have not been reported previously in whole grains. In
the present study, we successfully detected and identified another
DFA cis-isomer, i.e., trans-cis-8-O-40 DFA (Fig. 2, 19).
3.2. Contents of phenolic acids in brown rice and processed brown rice
products
The contents of PAs in BR and processed BR products are given
in Table 1. The total content of free-PAs of BR (4.29 lmol/100 g
DW) was significantly increased after processing (p < 0.05). The
values for processed BR products ranged from 5.17 (textured rice)
to 6.83 lmol/100 g (rice noodle). Processing significantly
decreased the total content of soluble-conjugated-PAs
(39.29 lmol/100 g). The values for processed BR products ranged
from 21.71 (textured rice) to 26.27 lmol/100 g (rice noodle). How-
ever, the total content of bound-PAs (316.52 lmol/100 g) was sig-
nificantly increased after IECT and cooking (p < 0.05), but not
significantly changed after RNE (p > 0.05). The values for processed
BR products ranged from 303.45 (rice noodle) to
407.59 lmol/100 g (textured rice). The total content of PAs
(360.10 lmol/100 g) was significantly increased after IECT and
cooking (p < 0.05), but not significantly changed after RNE
(p > 0.05). The values for processed BR products ranged from
336.55 (rice noodle) to 434.47 lmol/100 g (textured rice).
To date, information on the effects of IECT, cooking, and RNE on
the phytochemical profiles and AOA in free, soluble-conjugated
and bound fractions of BR was rare, only a few studies have inves-
tigated the effect of processing on the phenolic profiles of whole
grains. Zielinski, Kozlowska, and Lewczuk (2001) found increases
in the contents of free-PAs and bound-PAs of four grains (wheat,
barley, rye and oat) after processing. Dewanto et al. (2002) and
de la Parra, Saldivar, and Liu (2007) observed increases in the con-
tents of free and conjugated FA, and a decrease in the content of
bound FA in corn after thermal processing. Fares, Platani, Baiano,
and Menga (2010) analyzed the free and bound PAs in pasta, and
found that cooking increased the content of bound-PAs, but did
not affect the content of free-PAs. N’Dri et al. (2013) found that,
in sorghum, the content of free-PAs increased, whereas that of
bound-PAs decreased after cooking; in millet, cooking increased
the content of free-PAs without affecting that of bound-PAs;
whereas in fonio, a decrease in the content of free-PAs was
observed along with an increase in the content of bound-PAs after
cooking. Scaglioni et al. (2014) showed that the contents of both
free and bound PAs of rice were reduced after hydrothermal treat-
ment. These processing effects depended on the processing meth-
ods applied, the type, and variety of whole grains analyzed. To our
knowledge, there was no information on the effect of processing on
the soluble-conjugated-PAs profiles of whole grain rice. The reduc-
tions of soluble-conjugated-PAs are likely due to the fact that pro-
cessing promotes the formation of strong complexes of the
soluble-conjugated-PAs with macromolecules in the BR matrix,
which may reduce their solubility in extraction solvent, thus
decreased their determined contents (Walter et al., 2013).
In BR and processed BR products, bound-PAs were predominant
forms (88.4–94.1% of total), followed by soluble-conjugated
(4.9–10.5%) and free PAs (1.1–1.8%), which was consistent with
previous studies (Adom & Liu, 2002; Okarter, Liu, Sorrells, & Liu,
2010). As reported in whole grains previously (Okarter et al.,
2010), the most representative PAs in free and bound fractions
were FA isomers and trans-p-COA. However, in the soluble-
conjugated fraction, the most representative PAs were FA isomers
and trans-SIA, which was inconsistent with the previous results
of Okarter et al. (2010). In addition, other PAs, i.e., cis-p-COA,
CHA, cis-SIA, p-HA, SYA, VA, trans-CFA, and cis-CFA were also
detected in significant or small quantities in three fractions. In
most cases, long phenolic extraction procedures, exposure to air
and light, or application of hydrothermal treatment would result
in chemical isomerization of PAs. However, most previous studies
had failed to detect cis-isomers of PAs using HPLC (Chiremba et al.,
2012; Guo & Beta, 2013; Qiu et al., 2010). Therefore, PAs in whole
grains will be significantly underestimated without including the
cis-isomers. In the present study, a gradient elution procedure of
105 min for separation of PAs using RP-HPLC was developed in
our laboratory. After applying it, the cis-isomers of four hydrox-
ycinnamic acid derivatives (FA, p-COA, SIA, and CFA) were success-
fully separated (Fig. 1A–D). Cis-isomers of PAs were detected in
significant amounts when compared to their corresponding
trans-isomers (Table 1). The total contents of FA, p-COA, SIA, and
CFA in BR and processed BR products would be significantly under-
estimated by 17.2–28.2%, 17.9–22.4%, 24.4–35.5%, and 19.5% with-
out including the cis-isomers. In addition, the contents of bound
cis-FA, cis-COA, and cis-SIA in cooked rice were higher than those
in textured rice and rice noodle (p < 0.05) (Table 1). It suggests that
the cooking treatment may be more effective in promoting the iso-
merization of these bound hydroxycinnamic acid derivatives.
Since ultraviolet absorbance of trans-SIA was much lower than
that of trans-FA at the same concentration (data not shown), and
the polarity of trans-SIA was closer to that of trans-FA in RP-
HPLC, previous studies did not detect it in whole grains
(Chiremba Rooney, & Beta, 2012; Fares et al., 2010; Scaglioni
et al., 2014). In this study, we successfully separated it from
trans-FA, and separated its cis-isomer from cis-FA in RP-HPLC
(Fig. 1A–D). In addition, the content of bound trans-SIA was dra-
matically increased by 277.6–407.8% after processing, suggesting
that proper processing could be used to promote the release of
bound trans-SIA in whole grains.
DFAs were only detected in the bound fraction, with trans-
trans-8-O-40 , trans-trans-5-50 , trans-trans-8-50 benzofuran, and
trans-trans-8-50 DFA being abundant DFAs, which was consistent
with previous studies (Dobberstein & Bunzel, 2010; Guo & Beta,
2013; Qiu et al., 2010). However, the other three DFAs, trans-cis-
5-50 , trans-cis-8-O-40 and trans–cis 8-50 benzofuran DFA were not
reported in these studies. The most abundant DFA in BR and pro-
cessed BR products was trans-trans-8-O-40 DFA, followed by
trans-trans-5-50 , trans-trans-8-50 benzofuran, trans-cis-5-50 , trans-
cis-8-O-40 , trans-trans-8-50 and trans-cis-8-50 benzofuran DFA. The
sequence of the DFA contents was not the same in different whole
 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 73

Table 1
The composition of phenolic acids in brown rice and processed brown rice products (lg/g DW).a

Phenolic acid Free Soluble-conjugated Bound Total

Trans-ferulic acid
Brown rice 3.04 ± 0.07c (0.9) 30.41 ± 0.75a (8.6) 320.70 ± 12.55bc (90.6) 354.15 ± 12.83bc
Textured rice 4.09 ± 0.07b (0.9) "34.6 13.19 ± 0.82c (3.0) ;56.6 417.59 ± 17.77a (96.0) "30.2 434.87 ± 18.56a "22.8
Cooked rice 2.38 ± 0.10d (0.7) ;21.7 12.25 ± 0.93c (3.4) ;59.7 346.74 ± 10.49b (96.0) "8.1 361.36 ± 9.82b "2.0
Rice noodle 5.22 ± 0.22a (1.6) "71.8 17.31 ± 0.58b (5.3) ;43.1 301.08 ± 10.36c (93.0) ;6.1 323.61 ± 9.88c ;8.6
Trans-p-coumaric acid
Brown rice 1.37 ± 0.12d (1.4) 7.29 ± 0.16a (7.5) 88.34 ± 5.15b (91.1) 97.01 ± 4.97b
Textured rice 1.78 ± 0.07c (1.6) "29.8 5.98 ± 0.30b (5.2) ;18.0 106.42 ± 6.14a (93.2) "20.5 114.18 ± 6.07a "17.7
Cooked rice 3.69 ± 0.13a (3.2) "168.8 7.40 ± 0.37a (6.3) "1.5 105.58 ± 4.76a (90.5) "19.5 116.67 ± 5.07a "20.3
Rice noodle 2.42 ± 0.17b (2.7) "76.4 7.14 ± 0.66a (7.8) ;2.0 81.60 ± 6.34b (89.5) ;7.6 91.16 ± 5.95b ;6.0
Cis-ferulic acid
Brown rice 0.97 ± 0.08b (1.1) 7.33 ± 0.27a (8.2) 80.66 ± 4.55b (90.7) 88.95 ± 4.69b
Textured rice 1.22 ± 0.09ab (1.3) "26.8 4.35 ± 0.28c (4.8) ;40.7 85.04 ± 8.07b (93.8) "5.4 90.62 ± 8.03b "1.9
Cooked rice 0.54 ± 0.07c (0.4) ;44.1 4.11 ± 0.20c (2.9) ;44.0 137.06 ± 11.20a (96.7) "69.9 141.70 ± 11.07a "59.3
Rice noodle 1.44 ± 0.23a (1.8) "48.8 5.23 ± 0.27b (6.6) ;28.6 72.04 ± 3.11b (91.5) ;10.7 78.71 ± 3.57b ;11.5
Trans-trans-8-O-40 DFAb
Brown rice NDc ND 33.68 ± 1.15b (100) 33.68 ± 1.15b
Textured rice ND ND 38.32 ± 1.93a (100) "13.8 38.32 ± 1.93a "13.8
Cooked rice ND ND 33.58 ± 1.21b (100) ;0.3 33.58 ± 1.21b ;0.3
Rice noodle ND ND 22.83 ± 1.25c (100) ;32.2 22.83 ± 1.25c ;32.2
Cis-p-coumaric acid
Brown rice 0.14 ± 0.02b (0.6) 1.08 ± 0.12b (4.6) 22.40 ± 1.17b (94.8) 23.62 ± 1.29b
Textured rice 0.15 ± 0.02b (0.6) "10.5 1.20 ± 0.06b (4.8) "10.7 23.60 ± 0.94b (94.6) "5.4 24.95 ± 1.01b "5.6
Cooked rice 0.37 ± 0.04a (1.1) "171.8 1.88 ± 0.11a (5.6) "73.7 31.40 ± 1.68a (93.3) "40.2 33.65 ± 1.58a "42.5
Rice noodle 0.19 ± 0.01b (0.8) "39.5 1.87 ± 0.09a (7.6) "73.3 22.54 ± 1.25b (91.6) "0.6 24.60 ± 1.21b "4.2
Trans-sinapic acid
Brown rice 0.33 ± 0.01c (1.5) 13.99 ± 0.28a (61.2) 8.55 ± 0.94c (37.4) 22.86 ± 0.74c
Textured rice 0.43 ± 0.03b (0.8) "30.3 8.92 ± 0.31b (16.9) ;36.2 43.40 ± 2.48a (82.3) "407.8 52.75 ± 2.69a "130.7
Cooked rice 0.41 ± 0.03b (1.0) "24.4 9.95 ± 0.63b (23.3) ;28.9 32.27 ± 1.69b (75.7) "277.6 42.63 ± 2.11b "86.5
Rice noodle 0.61 ± 0.03a (1.4) "83.3 9.29 ± 0.33b (21.8) ;33.6 32.61 ± 1.37b (76.7) "281.7 42.52 ± 1.70b "86.0
Chlorogenic acid
Brown rice ND 4.39 ± 0.36a (24.6) 13.45 ± 0.85b (75.4) 17.85 ± 1.03ab
Textured rice ND 2.97 ± 0.12b (15.9) ;32.3 15.70 ± 0.82a (84.1) "16.7 18.67 ± 0.72a "4.6
Cooked rice ND 2.62 ± 0.19b (13.5) ;40.3 16.77 ± 0.12a (86.5) "24.7 19.40 ± 0.26a "8.7
Rice noodle ND 2.79 ± 0.13b (17.1) ;36.5 13.57 ± 0.94b (82.9) "0.8 16.35 ± 0.81b ;8.4
Trans-trans-5-50 DFA
Brown rice ND ND 16.37 ± 0.23b (100) 16.37 ± 0.23b
Textured rice ND ND 19.58 ± 0.80a (100) "19.7 19.58 ± 0.80a "19.7
Cooked rice ND ND 15.07 ± 0.25c (100) ;7.9 15.07 ± 0.25c ;7.9
Rice noodle ND ND 12.54 ± 0.42d (100) ;23.4 12.54 ± 0.42d ;23.4
Trans-trans-8-50 benzofuran DFA
Brown rice ND ND 15.49 ± 0.77a (100) 15.49 ± 0.77a
Textured rice ND ND 17.13 ± 0.81a (100) "10.6 17.13 ± 0.81a "10.6
Cooked rice ND ND 8.76 ± 0.50c (100) ;43.4 8.76 ± 0.50c ;43.4
Rice noodle ND ND 10.67 ± 0.51b (100) ;31.1 10.67 ± 0.51b ;31.1
Cis-sinapic acid
Brown rice 0.19 ± 0.01b (1.5) 5.17 ± 0.19a (41.1) 7.23 ± 0.55c (57.4) 12.59 ± 0.70c
Textured rice 0.22 ± 0.03b (1.3) "14.5 3.57 ± 0.34c (21.0) ;31.1 13.22 ± 0.92b (77.8) "83.0 17.01 ± 0.97b "35.1
Cooked rice 0.24 ± 0.02b (1.1) "26.0 4.28 ± 0.30b (20.2) ;17.2 16.63 ± 1.04a (78.6) "130.1 21.15 ± 1.32a "68.0
Rice noodle 0.37 ± 0.02a (2.4) "93.3 3.42 ± 0.22c (22.1) ;33.9 11.68 ± 0.77b (75.5) "61.6 15.46 ± 0.87b "22.8
Trans-cis-5-50 DFA
Brown rice ND ND 11.09 ± 0.44a (100) 11.09 ± 0.44a
Textured rice ND ND 12.17 ± 0.91a (100) "9.7 12.17 ± 0.91a "9.7
Cooked rice ND ND 12.30 ± 0.71a (100) "10.9 12.30 ± 0.71a "10.9
Rice noodle ND ND 7.53 ± 0.52b (100) ;32.1 7.53 ± 0.52b ;32.1
Trans-cis-8-O-40 DFA
Brown rice ND ND 10.60 ± 0.89b (100) 10.60 ± 0.89b
Textured rice ND ND 14.44 ± 0.82a (100) "36.2 14.44 ± 0.82a "36.2
Cooked rice ND ND 5.70 ± 0.43c (100) ;46.2 5.70 ± 0.43c ;46.2
Rice noodle ND ND 2.21 ± 0.06d (100) ;79.1 2.21 ± 0.06d ;79.1
P-hydroxybenzoic acid
Brown rice 0.54 ± 0.05b (6.5) 4.85 ± 0.43a (57.5) 3.03 ± 0.16c (36.0) 8.43 ± 0.25a
Textured rice 0.53 ± 0.04b (5.5) ;2.9 1.57 ± 0.19c (16.4) ;67.6 7.51 ± 0.64a (78.1) "147.4 9.61 ± 0.85ab "14.0
Cooked rice 0.60 ± 0.06b (5.5) "10.3 2.04 ± 0.14bc (18.7) ;57.9 8.28 ± 0.77a (75.8) "173.0 10.92 ± 0.64a "29.6
Rice noodle 0.86 ± 0.07a (10.4) "58.5 2.39 ± 0.16b (28.9) ;50.6 5.03 ± 0.31b (60.7) "65.7 8.28 ± 0.51a ;1.7
Syringic acid
Brown rice 0.94 ± 0.02b (13.1) 1.85 ± 0.11a (25.8) 4.38 ± 0.30c (61.1) 7.17 ± 0.35b
Textured rice 0.64 ± 0.01d (8.8) ;31.3 0.78 ± 0.02d (10.6) ;57.8 5.92 ± 0.11b (80.6) "35.4 7.35 ± 0.11b "2.6

(continued on next page)

74 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

Table 1 (continued)

Phenolic acid Free Soluble-conjugated Bound Total

Cooked rice 2.01 ± 0.04a (18.8) "114.6 1.48 ± 0.10b (13.9) ;20.0 7.19 ± 0.71a (67.3) "64.3 10.68 ± 0.77a "49.1
Rice noodle 0.80 ± 0.05c (12.9) ;14.7 1.06 ± 0.04c (17.0) ;43.0 4.35 ± 0.16c (70.1) ;0.6 6.21 ± 0.21b ;13.4
Trans-trans-8-50 DFA
Brown rice ND ND 6.90 ± 0.43bc (100) 6.90 ± 0.43bc
Textured rice ND ND 8.03 ± 0.60b (100) "16.4 8.03 ± 0.60b "16.4
Cooked rice ND ND 11.08 ± 1.03a (100) "60.7 11.08 ± 1.03a "60.7
Rice noodle ND ND 6.14 ± 0.15c (100) ;11.0 6.14 ± 0.15c ;11.0
Trans-cis-8-50 benzofuran DFA
Brown rice ND ND 5.42 ± 0.41ab (100) 5.42 ± 0.41ab
Textured rice ND ND 5.24 ± 0.45ab (100) ;3.4 5.24 ± 0.45ab ;3.4
Cooked rice ND ND 5.99 ± 0.55a (100) "10.6 5.99 ± 0.55a "10.6
Rice noodle ND ND 4.37 ± 0.27b (100) ;19.4 4.37 ± 0.27b ;19.4
Vanillic acid
Brown rice 0.35 ± 0.02b (12.9) 0.89 ± 0.09a (32.7) 1.48 ± 0.12c (54.4) 2.72 ± 0.16c
Textured rice 0.43 ± 0.04b (12.7) "22.9 0.62 ± 0.02c (18.1) ;30.4 2.36 ± 0.08b (69.2) "59.7 3.41 ± 0.04b "25.5
Cooked rice 0.44 ± 0.05b (10.1) "24.7 0.61 ± 0.04c (14.0) ;31.8 3.29 ± 0.20a (75.9) "123.0 4.34 ± 0.25a "59.7
Rice noodle 0.59 ± 0.06a (18.4) "67.0 0.75 ± 0.01b (23.7) ;15.1 1.84 ± 0.17c (57.9) "24.6 3.18 ± 0.21bc "17.1
Trans-caffeic acid
Brown rice ND ND 0.70 ± 0.05a (100) 0.70 ± 0.05a
Textured rice ND ND 0.51 ± 0.02b (100) ;27.9 0.51 ± 0.02b ;27.9
Cooked rice ND ND 0.31 ± 0.02c (100) ;56.4 0.31 ± 0.02c ;56.4
Rice noodle ND ND 0.35 ± 0.01c (100) ;50.6 0.35 ± 0.01c ;50.6
Cis-caffeic acid
Brown rice ND ND 0.17 ± 0.02 (100) 0.17 ± 0.02
Textured rice ND ND ND ND
Cooked rice ND ND ND ND
Rice noodle ND ND ND ND
Total phenolic acids (lmol/100 g DW)d
Brown rice 4.29 ± 0.22c (1.2) 39.29 ± 1.39a (10.9) 316.52 ± 14.87b (87.9) 360.10 ± 16.48b
Textured rice 5.17 ± 0.21b (1.2) "20.5 21.71 ± 1.19c (5.0) ;52.4 407.59 ± 18.83a (93.8) "28.8 434.47 ± 18.24a "20.7
Cooked rice 5.98 ± 0.30b (1.4) "39.3 23.74 ± 1.45bc (5.6) ;48.7 394.83 ± 18.05a (93.0) "24.7 424.55 ± 19.78a "17.9
Rice noodle 6.83 ± 0.45a (2.0) "59.2 26.27 ± 1.26b (7.8) ;28.5 303.45 ± 13.37b (90.2) ;4.1 336.55 ± 15.06b ;6.5
a
Results are expressed as mean ± SD (n = 3). Percent contribution to total phenolic acid content is in parentheses. Numbers with (") or (;) indicate percentage increase or
decrease when compared to the control. Values of each column with no letters in common are significantly different (p < 0.05). bDFA: diferulic acid. cND = not detected. dTotal
amounts of phenolic acids in each fraction are expressed as lmol/100 g DW due to the differences in the molecule weights of phenolic acids.

grains (Dobberstein & Bunzel, 2010; Guo & Beta, 2013). The total
content of DFAs was slightly increased by 15.4% after IECT
(p < 0.05), whereas the content was not changed after cooking
(p > 0.05), but significantly decreased by 33.4% after RNE
(p < 0.05). To conclude, limited data were available on the effect
of processing on the DFAs in whole grains. Zaupa et al. (2015)
found that two cooking techniques (boiling and risotto) decreased
the contents of DFAs in red, black, and white rice, except that boil-
ing did not affect those in white rice.
Processed BR products had high contents of PAs
(336.55–434.47 lmol/100 g DW) with bound-PAs being the
predominant forms (88.4–94.1% of total), followed by soluble-
conjugated (4.9–10.5%) and free PAs (1.1–1.8%). The potential
health benefits of PAs are mostly related to their antioxidant activ-
ity. Beyond the antioxidant activity, other biological activities of
PAs, such as prevention of cardiovascular diseases, anticancer
activity, protection against side effects of chemotherapy, antimi-
crobial activity, anti-osteoclast activity, anti-inflammatory and
analgesic activities had also been reported (El-Seedi et al., 2012).
3.3. Total phenolic content
The free, soluble-conjugated, bound TPCs of BR and processed
BR products are shown in Table 2. The free-TPC of BR (15.80 mg
GAE/100 g DW) significantly dropped after processing (p < 0.05).
The values for processed BR products ranged from 11.15 (rice
noodle) to 13.78 mg GAE/100 g (cooked rice). The soluble-
conjugated-TPC (31.25 mg GAE/100 g) was significantly
decreased after processing (p < 0.05). The values for processed
BR products ranged from 9.72 (rice noodle) to 13.61 mg
GAE/100 g (cooked rice). The bound-TPC (85.24 mg GAE/100 g)
was significantly increased after IECT and cooking (p < 0.05),
but not affected after RNE (p > 0.05). The values for processed
BR products ranged from 85.85 (rice noodle) to 106.41 mg
GAE/100 g (textured rice). The total-TPC (132.29 mg GAE/100 g)
was not significantly changed after IECT and cooking (p > 0.05),
but significantly decreased after RNE (p < 0.05). The values for
processed BR products ranged from 106.72 (rice noodle) to
129.65 mg GAE/100 g (textured rice).

Table 2
Total phenolic contents (TPC) of brown rice and processed brown rice products.a

Sample TPC (mg gallic acid equiv./100 g DW)

Free Soluble-conjugated Bound Total
Brown rice 15.80 ± 0.63a (11.9) 31.25 ± 1.53a (23.6) 85.24 ± 4.06b (64.4) 132.29 ± 6.19a
Textured rice 11.71 ± 0.45c (9.1) ;25.9 11.14 ± 0.60c (8.6) ;64.3 106.41 ± 6.51a (82.3) "24.8 129.26 ± 7.48a ;2.3
Cooked rice 13.78 ± 0.48b (10.6) ;12.8 13.61 ± 0.59b (10.5) ;56.4 102.26 ± 4.25a (78.9) "20.0 129.65 ± 5.29a ;2.0
Rice noodle 11.15 ± 0.34c (10.4) ;29.4 9.72 ± 0.51c (9.1) ;68.9 85.85 ± 2.63b (80.4) "0.7 106.72 ± 3.48b ;19.3
a
Results are expressed as mean ± SD (n = 3). Percent contribution of each fraction to the total value is in parentheses. Numbers with (") or (;) indicate percentage increase or
decrease when compared to the control in each column. Values of each column with no letters in common are significantly different (p < 0.05).
 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78 75

The free-TPC of BR in the present study (13.77 mg GAE/100 g
FW) was lower than that of the rice (35.73 mg GAE/100 g FW)
reported by Adom and Liu (2002). It may be attributed to differ-
ences in the extraction method and varieties used. The Folin-
Ciocalteu reagent used for quantification of TPC can interact with
a number of reducing nonphenolic substances, such as amino
acids, certain reducing sugars, vitamin C, and other reducing com-
pounds in crude extracts, and thus may lead to overestimation of
TPC. Therefore, an additional ethyl acetate extraction (mainly for
purifying phenolics) was applied to the crude extracts in the pre-
sent study, but it was not included in their study. However, the
bound-TPC of BR (74.26 mg GAE/100 g FW) was higher than that
of rice (58.86 mg GAE/100 g FW) in their study due to varietal dif-
ference. Soluble-conjugated-TPC was not reported in their study.
The reductions in free-TPC of BR in the present study were congru-
ent with the results of N’Dri et al. (2013), Massaretto et al. (2011)
and Scaglioni et al. (2014). To our knowledge, there are no reports
on the effect of processing on the soluble-conjugated-TPC of rice.
The decreases in soluble-conjugated-TPC is likely attributed to
the fact that processing can promote the formation of strong com-
plexes of the soluble-conjugated phenolics with macromolecules
in the BR matrix. These decreases in soluble-conjugated-TPC were
consistent with the decreases in soluble-conjugated-PAs in BR. The
increases in bound-TPC of BR after IECT and cooking were in con-
sistence with the result of Massaretto et al. (2011). These changes
in bound-TPCs were consistent with those of bound-PAs in BR.
However, RNE did not affect the bound-TPC of BR. It is likely due
to that IECT and cooking might have allowed a more effective
extraction of bound phenolics under alkaline conditions through
destruction of the cell wall matrix, but RNE did not achieve that
because of its momentary treatment (10 s).
Several studies on whole grain rice phenolics did not include
the determination of bound forms (Huang & Ng, 2012; Shao,
Zhang, & Bao, 2011). Our laboratory developed a method to deter-
mine the complete phenolic profiles of whole grains, which
included both free and bound forms (Adom & Liu, 2002). Whole
grain rice phenolics will be significantly underestimated without
accounting for bound forms. In the present study, the free and
soluble-conjugated phenolics of BR and processed BR products
contributed 9.1–11.9% and 8.6–23.6% to total-TPCs, respectively,
while bound phenolics contributed 64.4–82.3%. Therefore, total-
TPCs of BR and processed BR products would be underestimated
by 64.4–82.3% without including bound phenolics. This is consis-
tence with the results reported by Adom and Liu (2002).
As suggested by Adom and Liu (2002) and Acosta-Estrada et al.
(2014), free phenolics are rapidly released and absorbed in the
stomach and small intestine, whereas soluble-conjugated pheno-
lics are released by mucosa cells cinnamoyl esterases and absorbed
in the small intestine, then these two forms would distribute
throughout the body with health benefits, such as inhibition activ-
ities against oxidation of LDL cholesterol and liposomes
(Chandrasekara & Shahidi, 2011). Bound phenolics would survive
stomach and intestinal digestion and reach the colon, thus exhibit-
ing health benefits in the prevention of colon cancer and other
digestive cancers (Adom & Liu, 2002). In the present study, pro-
cessed BR products contained significant amounts of free phenolics
(11.15–13.78 mg gallic acid equiv./100 g DW) and soluble-
conjugated phenolics (9.72–13.61 mg gallic acid equiv./100 g
DW), which may be released and absorbed in the stomach and
small intestine and exhibit inhibition activities against oxidation
of LDL cholesterol and liposomes. Processed BR products had large
amounts of bound phenolics (85.85–106.41 mg gallic acid
equiv./100 g DW), which may be released by colon fermentation
and exhibit health benefits in the prevention of cancer (Liu, 2007).
3.4. Antioxidant activity
Three antioxidant assays (TEAC, FRAP, and PSC) were used to
determine the AOA in the present study. The free, soluble-
conjugated, bound AOAs of BR and processed BR products are
presented in Table 3. The free-TEAC of BR (30.05 TE/100 g DW)
was significantly decreased after processing (p < 0.05). The values
for processed BR products ranged from 18.75 (rice noodle) to
23.53 mg TE/100 g (cooked rice). The soluble-conjugated-TEAC
(62.61 TE/100 g) was significantly decreased after processing
(p < 0.05). The values for processed BR products ranged from 35.36
(rice noodle) to 44.71 mg TE/100 g (cooked rice). However, bound-
TEAC (229.64 mg TE/100 g) was significantly increased after IECT
and cooking (p < 0.05), but not significantly affected after RNE
(p > 0.05). Bound-TEACs of processed BR products ranged from
241.77 (rice noodle) to 280.97 mg TE/100 g (textured rice). The
total-TEAC of BR (322.31 mg TE/100 g) was not significantly affected
after processing (p > 0.05). The values of processed BR products ran-
ged from 295.88 (rice noodle) to 340.87 mg TE/100 g (textured rice).
The free-FRAP of BR (25.78 mg FE/100 g DW) significantly
dropped after processing (p < 0.05). The values for processed BR
products ranged from 6.80 (rice noodle) to 9.24 mg FE/100 g
(cooked rice). The soluble-conjugated-FRAP (38.34 mg FE/100 g)
was also significantly decreased after processing (p < 0.05). The
values for processed BR products ranged from 7.77 (rice noodle)
to 14.59 mg FE/100 g (cooked rice). The bound-FRAP (99.97 mg
FE/100 g) was significantly increased after IECT and cooking

Table 3
Antioxidant activities of brown rice and processed brown rice products.a

Free Soluble-conjugated Bound Total

Trolox equivalent antioxidant capacity (TEAC) (mg Trolox equiv./100 g DW)
Brown rice 30.05 ± 1.34a (9.3) 62.61 ± 2.12a (19.4) 229.64 ± 7.43c (71.2) 322.31 ± 8.30ab
Textured rice 19.05 ± 0.94c (5.6) ;36.6 40.85 ± 1.59b (12.0) ;34.8 280.97 ± 13.91a (82.4) "22.4 340.87 ± 16.45a "5.8
Cooked rice 23.53 ± 0.76b (7.1) ;21.7 44.71 ± 1.41b (13.4) ;28.6 264.48 ± 8.51ab (79.5) "15.2 332.73 ± 10.68a "3.2
Rice noodle 18.75 ± 0.82c (6.3) ;37.6 35.36 ± 1.05c (11.9) ;43.5 241.77 ± 7.04bc (81.7) "5.3 295.88 ± 8.91b ;8.2
Ferric reducing antioxidant power (FRAP) (mg ferrous sulfate equiv./100 g DW)
Brown rice 25.78 ± 0.81a (15.7) 38.34 ± 1.49a (23.4) 99.97 ± 3.55b (60.9) 164.08 ± 5.83a
Textured rice 7.16 ± 0.14c (5.1) ;72.2 10.48 ± 0.31c (7.5) ;72.7 122.70 ± 5.94a (87.4) "22.7 140.35 ± 6.39b ;14.5
Cooked rice 9.24 ± 0.21b (6.8) ;64.2 14.59 ± 0.43b (10.7) ;61.9 112.80 ± 3.93a (82.6) "12.8 136.63 ± 4.57b ;16.7
Rice noodle 6.80 ± 0.13c (6.0) ;73.6 7.77 ± 0.21d (6.8) ;79.7 98.97 ± 3.03b (87.2) ;1.0 113.53 ± 3.37c ;30.8
Peroxyl radical scavenging capacity (PSC) (mg vitamin C equiv./100 g DW)
Brown rice 5.03 ± 0.17a (13.5) 8.76 ± 0.28a (23.5) 23.45 ± 0.67b (63.0) 37.24 ± 0.84a
Textured rice 3.61 ± 0.09c (10.0) ;28.2 3.20 ± 0.07c (8.9) ;63.4 29.01 ± 1.34a (80.5) "23.7 36.02 ± 1.50a ;3.3
Cooked rice 4.18 ± 0.09b (11.8) ;16.9 4.11 ± 0.12b (11.6) ;53.1 27.12 ± 1.01a (76.6) "15.6 35.41 ± 1.23a ;4.9
Rice noodle 3.38 ± 0.10c (11.3) ;32.7 2.90 ± 0.05d (9.7) ;66.9 23.58 ± 0.48b (79.0) "0.5 29.86 ± 0.62c ;19.8
a
Results are expressed as mean ± SD (n = 3). Percent contribution of each fraction to total value is in parentheses. Numbers with (") or (;) indicate percentage increase or
decrease when compared to the control. Values of each column with no letters in common are significantly different (p < 0.05).
76 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78

(p < 0.05), but not significantly affected after RNE (p > 0.05). The
values for processed BR products ranged from 98.97 (rice noodle)
to 122.70 mg FE/100 g (textured rice). The total-FRAP (164.08 mg
FE/100 g) was significantly decreased after processing (p < 0.05).
The values for processed BR products ranged from 113.53 (rice
noodle) to 140.35 mg FE/100 g (textured rice).
The free-PSC of BR (5.03 mg VCE/100 g DW) was significantly
decreased after processing (p < 0.05). The values for processed BR
products ranged from 3.38 (rice noodle) to 4.18 mg VCE/100 g
(cooked rice). The soluble-conjugated-PSC (8.76 mg VCE/100 g)
was also significantly decreased after processing (p < 0.05). The
values for processed BR products ranged from 2.90 (rice noodle)
to 4.11 mg VCE/100 g (cooked rice). The bound-PSC (23.45 mg
VCE/100 g) was significantly increased after IECT and cooking
(p < 0.05), but not significantly affected after RNE (p > 0.05). The
values for processed BR products ranged from 23.58 (rice noodle)
to 29.01 mg VCE/100 g (textured rice). The total-PSC (37.24 mg
VCE/100 g) was not significantly affected after IECT and cooking
(p > 0.05), but decreased after RNE (p < 0.05). The values for pro-
cessed BR products ranged from 29.86 (rice noodle) to 36.02 mg
VCE/100 g (textured rice).
Processing significantly affected AOA of BR, which was similar to
the effects on TPC. There are significant correlations between TPC and
TEAC (r = 0.995, p < 0.01), PA and TEAC (r = 0.995, p < 0.01), TPC and
FRAP (r = 0.996, p < 0.01), PA and FRAP (r = 0.984, p < 0.01), TPC and
PSC (r = 0.999, p < 0.01), PA and PSC (r = 0.992, p < 0.01), indicating
that phenolic compounds were the main components responsible
for the AOAs of BR and processed BR products. To date, limited
reports have studied the processing effects on AOA of rice. Previous
studies showed that processing can increase or decrease the AOA of
foods depending on the nature and molecular structure of the antiox-
idant compound, processing methods, food matrix, and the interac-
tion of antioxidants with other components (Fares et al., 2010;
Walter et al., 2013; Wu et al., 2004). The decreases of free-AOAs in
the present study were in agreement with the results of N’Dri et al.
(2013), who found that the free-AOAs (using TEAC assay) of three
African cereals (fonio, millet, and sorghum) were significantly
reduced by 90, 68, and 76% after cooking, respectively. However,
the increases in bound-AOAs were not congruent with those in their
study, where bound-AOAs were significantly decreased after cooking.
The decreases were mainly caused by the decreases of bound-TPCs in
their study. The changes in AOAs after processing in our study were
inconsistent with those reported by de la Parra et al. (2007), who
reported that free-AOAs (using PSC assay) of corns were increased,
and bound-AOAs were decreased after processing (thermal-alkaline
treatment). They suggested that it was probably attributed to the
increased soluble phenolics from bound phenolics. The inconsistency
is mainly caused by differences in the composition of whole grain and Fig. 3. Scores plot (A) and loading plot (B) of PCA of the three fractions of BR and
processing methods. To date, there is no report on the effect of pro- processed BR products. The labels of the samples BR, TR, CR, and RN refer to brown
rice, textured rice, cooked rice, and rice noodle, respectively; the labels of the
cessing on the soluble-conjugated-AOA of rice. The reductions in
fractions f, sc, and b refer to free, soluble-conjugated, and bound fractions,
soluble-conjugated-AOAs after processing were related to the respectively. The labels of evaluated parameters refer to Table 2, Table 3 and Fig. 1.
decreases in corresponding TPCs.
Oxidative stress is involved in aging and the pathology of a wide
range of chronic diseases (Ames & Gold, 1991). Dietary antioxi- noodle and textured rice, could be valuable as functional foods to
dants may combat oxidative stress in the body to maintain a bal- promote consumer health.
ance between oxidants and antioxidants, thus protecting the In rice milling, the bran layers and germ fraction removed dur-
body’s cells from damage caused by reactive oxygen species and ing polishing are high in fibres, vitamins, minerals, phenolics, and
free radicals. A diet rich in natural antioxidants, such as PAs, can other bioactive compounds, which are of a lot of health benefits
thus significantly increase the reactive antioxidant potential of (Okarter & Liu, 2010). Consuming whole grain rice, instead of pol-
the organism and thereby decrease the risk of oxidative stress- ished rice in diet, could maximally preserve the health benefits of
related diseases (Liu, 2007). In addition to antioxidant activity, a rice. Processing had significant effects on health benefits of rice. It
number of other bioactivities of rice have also been reported, such was found that some processing methods, such as germination
as antimicrobial, antiviral, anti-inflammatory, anticancer and (Patil & Khan, 2011) and fermentation (Liu et al., 2017), had poten-
immunomodulatory activities (Hudson et al., 2000; Niu et al., tial to enhance the health benefits of rice. The present study
2013; Ray et al., 2013; Verschoyle et al., 2007; Zhang et al., revealed that cooking and IECT could significantly increase the
2014). Therefore, brown rice products, such as cooked rice, rice content of bound phenolics of BR, thus may enhance health
 E.S. Gong et al. / Food Chemistry 232 (2017) 67–78
benefits of rice in the prevention of colon cancer and other diges-
tive cancers (Adom & Liu, 2002).
3.5. Principal component analysis
PCA was carried out to evaluate the influence of tested parameters
(TPC, TEAC, FRAP, PSC, and nineteen PAs) in the classification and dif-
ferentiation of the free, soluble-conjugated and bound fractions of BR
and processed BR products. PCA reduces the dimensionality of data
and enables the visualisation of data structure and relationships
between data and samples. The PCA results for three fractions of
BR and processed BR products are shown in Fig. 3. A total of
96.27% of difference was explained by relation between principal
component 1 vs. principal component 2 (PC1 vs. PC2). PC1 was
responsible for 88.71% while PC2 was responsible for 7.56% of the dif-
ference. PCA allowed differentiation among the three fractions (free,
soluble-conjugated, and bound). In addition, a significant effect of
processing on the bound fraction of BR was observed, shown by
the quadrant change of the samples (Fig. 3A). Regarding the distribu-
tion of the evaluated parameters, the PA composition (except for cis-
caffeic acid), TPC and AOA (TEAC, FRAP, and PSC) were responsible for
the separation in the PC1 (Fig. 3B). The evaluated parameters were
most likely concentrated in the bound fractions, which was indicated
by the vectors (representing evaluated parameters) directed to the
same quadrants of bound fractions of the samples in Fig. 3A.
4. Conclusions
Twelve monomeric PAs and seven DFAs were identified in BR
and its processed BR products. Trans-FA was found to be the most
abundant PA in the free and bound fractions, followed by trans-p-
COA and cis-FA. However, in the soluble-conjugated fractions, the
most representative PAs were trans-FA, trans-p-COA and trans-
SIA. Processing methods of IECT, cooking and RNE significantly
affect the phytochemical profiles and AOAs of free, soluble-
conjugated, and bound fractions of BR. IECT, cooking and RNE
increased the total content of free-PAs, but decreased the total con-
tent of soluble-conjugated-PAs. The content of bound-PAs was
increased after processing with IECT and cooking, but not affected
by RNE. Free and soluble-conjugated TPCs and AOAs were
adversely affected by processing of IECT, cooking and RNE, whereas
bound TPC and AOA were increased by IECT and cooking, but not
affected by RNE. PCA allowed differentiation among the three frac-
tions (free, soluble-conjugated, and bound), and had shown that
the evaluated parameters were most likely concentrated in the
bound fractions. Therefore, the phytochemicals and AOAs in free,
soluble-conjugated, and bound fractions of BR and processed BR
products would help consumers understand the health benefits
of whole grain products consumption.
Notes
The authors declare no competing financial interest.
Acknowledgments
This study was supported by the Ganpo Outstanding Talents
555 Projects (18000007) and the State Key Laboratory of Food
Science and Technology Projects (SKLF-ZZA-201608).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.
03.148.
77
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