ORIGINAL ARTICLE
Thermal cooking changes the profile of phenolic compounds, but
does not attenuate the anti-inflammatory activities of black rice
Sassy Bhawamai
1
, Shyh-Hsiang Lin
1
, Yuan-Yu Hou
2
and Yue-Hwa Chen
1,3
*
1
School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan;
2
Department of Food and Beverage
Management, Mackay Junior College of Medicine, Nursing and Management, Taipei, Taiwan;
3
Cancer Research Center,
Taipei Medical University Hospital, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan
Abstract
Background: Evidence on biological activities of cooked black rice is limited. This study examined the effects of
washing and cooking on the bioactive ingredients and biological activities of black rice.
Methods: Cooked rice was prepared by washing 03 times followed by cooking in a rice cooker. The acidic
methanol extracts of raw and cooked rice were used for the analyses.
Results: Raw black rice, both washed and unwashed, had higher contents of polyphenols, anthocyanins, and
cyanidin-3-glucoside (C3G), but lower protocatechuic acid (PA), than did cooked samples. Similarly, raw rice
extracts were higher in ferric-reducing antioxidant power (FRAP) activities than extracts of cooked samples.
Nonetheless, extracts of raw and cooked rice showed similar inhibitory potencies on nitric oxide, tumor
necrosis factor-a, and interleukin-6 productions in lipopolysaccharide-activated macrophages, whereas
equivalent amounts of C3G and PA did not possess such inhibitory effects.
Conclusions: Thermal cooking decreased total anthocyanin and C3G contents and the FRAP antioxidative
capacity, but did not affect anti-inflammatory activities of black rice. Neither C3G nor PA contributed to the
anti-inflammatory activity of black rice.
Keywords: black rice; anthocyanin; cyanidin-3-glucoside; protocatechuic acid; antioxidation; anti-inflammation
To access the supplementary material to this article, please see
Supplementary files under ‘Article Tools’.
Received: 21 July 2016; Revised: 20 August 2016; Accepted: 21 August 2016; Published: 20 September 2016
R
ice is a staple food for nearly half of the world’s
population, and pigmented cultivars have been in-
dicated to have health-promoting effects because
of their high contents of nutrients and phytochemicals.
Therefore, replacing white rice with pigmented rice would
be expected to have protective effects against various
diseases (1). Black rice (Oryza sativa L. indica) is a cultivar
widely consumed in Asian countries and is rich in antho-
cyanins, especially cyanidin-3-glucoside (C3G) and peonidin
(27). Raw black rice and its bioactive components were
indicated to possess antioxidative, anti-inflammatory, and
antiallergic activities (3, 4, 6, 8), which are closely asso-
ciated with various diseases. However, the evidence on
biological activities of cooked black rice is limited.
Because of the phenolic structures and substituted
groups, anthocyanins are water-soluble and labile to dif-
ferent conditions, including high temperatures. Therefore,
washing and heating, key events in cooking processes,
would likely influence anthocyanin contents, thus affect-
ing the biological activities of cooked rice. Studies showed
that thermal cooking, including boiling, frying, steaming,
roasting, and pan-frying, decreases the total anthocyanin
and C3G contents of black rice, but increases proto-
catechuic acid (PA), a major degradation product of
anthocyanins (912). Surh and Koh (10) indicated that
presoaking black rice for 3 h prior to cooking did not
affect the total anthocyanin contents (TACs) or total
polyphenol contents (TPCs), but the authors did not
explain whether the water was discarded. Black rice is
consumed after cooking, for which washing and heating
processes are required. Although studies have reported the
polyphenol and anthocyanin contents and antioxidative
and anti-inflammatory activities of raw black rice, knowl-
edge of the biological activities of cooked black rice is
limited. This study thus examined the effects of the
number of times black rice was washed and thermal
research
food & nutrition
æ
Food & Nutrition Research 2016. # 2016 Sassy Bhawamai et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even
commercially, provided the original work is properly cited and states its license.
1
Citation: Food & Nutrition Research 2016, 60: 32941 - http://dx.doi.org/10.3402/fnr.v60.32941
(page number not for citation purpose)
cooking on the contents of phenolic compounds, includ-
ing polyphenols, anthocyanins, C3G, and PA, as well as
the antioxidative and anti-inflammatory activities of raw
and cooked black rice. In addition, the potential roles of
C3G and PA in the anti-inflammatory activities of raw and
cooked rice were also evaluated. Results obtained from
this study can promote understanding of the optimal
cooking processes for preserving polyphenols and antho-
cyanins in black rice, and provide scientific evidence of the
health-promoting activities of cooked black rice.
Materials and methods
Chemicals and reagents
C3G, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (TROLOX), dimethyl sulfoxide (DMSO),
2,4,6-tripyridyl-s-triazine, trifluoroacetic acid, potassium
persulfate, sodium hydroxide, FolinCiocalteu’s reagent,
acetonitrile, and methanol were purchased from Sigma-
Aldrich (St. Louis, MO). PA was from Cayman (Ann
Arbor, MI). Dulbecco’s modified Eagle medium (DMEM)
and fetal bovine serum (FBS) were obtained from GIBCO
(Grand Island, NY). All chemicals and solvents used in
the study were of reagent grade.
Preparation of black rice extracts
Black rice was purchased from a local market in Taipei,
Taiwan. The rice was washed 03 times followed by
immediate cooking with an electronic rice cooker for
25 min. Washing was performed by soaking the rice in
cold water for 5 min, and discarding the water. Ground
dry raw and cooked rice samples were then extracted with
water, methanol, ethanol, acidic methanol (methanol:
1 N HCl99:1, v/v), or acidic ethanol (ethanol: 1 N
HCl99:1, v/v) for 24 h, and the respective extracts were
obtained after centrifugation. All experiments were per-
formed at least three times, and the results are expressed
on a dry matter basis.
Determination of total polyphenols, total anthocyanins,
C3G, and PA
TPCs in black rice extracts were determined spectropho-
tometrically at 755 nm after adding the FolinCiocalteu
reagent (13), and the value is expressed as milligrams of
gallic acid equivalents (GAE) per gram of dry rice. TACs
were determined by directly measuring the absorbance of
the extracts at 530 nm (14), and are expressed as milligrams
of C3G equivalents per gram of dry rice.
Being one of the major anthocyanins in black rice,
C3G in the extracts was detected and quantitated by
high-performance liquid chromatography (HPLC; Hitachi,
Tokyo, Japan) with a C
18
column (Inertsil, ODS-2,
Phenomenex, Torrance, CA) under a visible wavelength
of 520 nm (Hitachi) using an authentic C3G standard.
The mobile phase consisted of solvent A (4.5% formic
acid in water) and solvent B (50% acetonitrile with 4.5%
formic acid in water) with a gradient of solvent B of 10%
(0 min), 25% (30 min), 33% (34 min), 90% (42 min), and
10% (4550 min) (14, 15). PA, one of the major degra-
dation products anthocyanins, was also analyzed and
quantitated under the same conditions, except that a
wavelength of 280 nm and a PA standard were used.
Determination of the in vitro antioxidative activity
To understand whether the TPCs or TACs were associated
with antioxidant activities, the antioxidant power of the
extracts was measured by a ferric-reducing antioxidant
power (FRAP) assay using TROLOX as a standard (16).
Cell culture, cell proliferation, and anti-inflammatory
activities
Murine RAW 264.7 macrophages were used to study the
anti-inflammatory activities of black rice. Cells were
obtained from the Culture Collection and Research Center
(CRCC6001; Hsinchu, Taiwan), and were grown in
DMEM supplemented with 10% FBS at 378C under a
5% CO
2
environment. Cells (1510
5
) were treated with
lipopolysaccharide (LPS at 100 ng/ml) in the presence of
different treatments for 16 h, and the medium and cells
were collected for analysis.
Cell proliferation was evaluated by measuring the absor-
bance of the formazan product of [3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2
H-tetrazolium (MTS) produced by live cells at 490-nm
wavelength. The production of inflammatory mediators,
including nitric oxide (NO), tumor necrosis factor (TNF)-
a, and interleukin (IL)-6, secreted into the medium was
determined. NO, as quantitated by nitrite, was determined
by the Griess reaction, and TNF-a and IL-6 were analyzed
by commercial enzyme-linked immunosorbent assay kits
(R&D Systems, Minneapolis, MN).
Statistical analysis
Values are expressed as the mean9standard deviation
(SD). A two-way analysis of variance (ANOVA) with
repeated measures was carried out to compare effects of
the cooking and washing processes. A Pearson correlation
analysis was performed to express the relationships of
total polyphenols, total anthocyanins, C3G, and PA with
antioxidant activities. Student’s t-test was used to compare
means between the LPS and control groups. A one-way
ANOVA followed by the least significance difference test
was used to compare means between groups of LPS-treated
macrophages. All statistical analyses were performed with
the aid of SPSS software version 19 (IBM, Armonk, NY).
Differences between groups were considered significant at
pB0.05.
Results
Phenolic compounds and FRAP antioxidation activity
Polyphenols, particularly anthocyanins, are major con-
tributors to the dark-purple color of black rice, but the
contents may vary with different cooking processes and
Sassy Bhawamai et al.
2
(page number not for citation purpose)
Citation: Food & Nutrition Research 2016, 60: 32941 - http://dx.doi.org/10.3402/fnr.v60.32941
extraction solvents, so TPCs and TACs in black rice
extracts from different preparations were analyzed. Results
showed that acidic methanol extracts had 285730% higher
TACs from raw black rice than other extracts, including
water, ethanol, and acidic ethanol (Supplementary Fig. 1),
so acidic methanol extracts were used in the following
experiments. Figure 1 shows that cooking significantly
decreased the polyphenol and anthocyanin contents of
black rice. Raw rice had higher TPCs (5.285.88 mg GAE/g
dry weight) and TACs (2.052.36 mg C3G/g dry weight),
which contribute to approximately 50% of TPCs, than did
cooked samples (3.094.35 mg GAE/g dry weight and 0.
961.20 mg C3G/g dry weight, respectively), indicating
that thermal cooking respectively reduced TPCs and
TACs to 6780% and 4554% of levels of raw black rice.
However, washing up to three times did not affect the total
contents of polyphenols and anthocyanins in raw or
cooked black rice.
C3G and PA, as one of the major anthocyanins and
anthocyanin degradation products in black rice (8, 17),
respectively, were identified and quantified by HPLC.
Figure 2a and b show the representative HPLC chromato-
grams and peak identifications of C3G and PA of acidic
methanol extracts of black rice, respectively. Raw rice
possessed a higher amount of C3G, ranging 650791 mg/g
dry weight (2934% of TACs), and a lower amount of PA
(1218 mg/g dry weight) compared with cooked samples, at
238296 mg C3G/g dry weight (24.727% of TACs) and
5667 mg PA/g dry weight, respectively (Fig. 2c and d).
Parallel to results for TPCs and TACs, washing did not
alter the contents of C3G or PA in either raw or cooked
black rice. Therefore, cooking significantly decreased C3G,
but increased PA (by 343540%), but washing produced no
effects on the C3G or PA contents of either raw or cooked
rice. Interestingly, a major peak, with a retention time of ca.
21 min, was found in the HPLC chromatogram of PA, and
the peak area decreased in cooked rice.
FRAP capacity of black rice
In order to understand the antioxidative activities of the
acidic methanol extracts of black rice, the FRAP anti-
oxidant capacity activity was determined. Results showed
that washing slightly decreased the FRAP antioxidant
capacity, but cooked rice had a lower FRAP capacity
than did raw samples (Fig. 3). In addition, the FRAP
antioxidant capacity was positively associated with the
contents of phenolic compounds, including polyphenols,
anthocyanins, C3G, and PA, in black rice (Table 1).
Fig. 1. Total polyphenol (a) and anthocyanin (b) contents in acidic methanol extracts of raw and cooked black rice washed
different number of times. Values are expressed as the mean9SD (n3). A two-way ANOVA with repeated measures was used
for analyses of the cooking effect (*pB0.05) and washing effect (p0.05).
Cooking does not attenuate anti-inflammatory activities of black rice
Citation: Food & Nutrition Research 2016, 60: 32941 - http://dx.doi.org/10.3402/fnr.v60.32941 3
(page number not for citation purpose)
Effects of black rice extracts, C3G, and PA on inflammatory
mediators in LPS-stimulated macrophages
To understand the biological effects of black rice, LPS-
stimulated RAW 264.7 cells were used to study the anti-
inflammatory activities of extracts of different black
rice preparations. Results indicated that extracts of black
rice (1,000 mg/ml) showed no cytotoxicity (data not
shown), but extracts from both raw and cooked rice
showed similar inhibitory effects on the production of
LPS-stimulated inflammatory mediators, including
NO (Fig. 4a), TNF-a (Fig. 4b), and IL-6 (Fig. 4c),
suggesting that thermal cooking did not attenuate the
anti-inflammatory activity of black rice. However,
equivalent amounts of C3G (0.240.96 mg/ml) and PA
(0.0380.077 mg/ml)inblackrice,whendissolved
in acidic methanol, did not show such suppressive
effects (Fig. 5), although higher concentrations of PA
(1.530.8 m g/ml) significantly suppressed these mediators
stimulated by LPS (Fig. 5b) with no cytotoxicity (Supple-
mentary Fig. 2). On the contrary, similar concentrations
of C3G when dissolved in DMSO significantly decreased
NO production by LPS-activated macrophages (Fig. 6).
Discussion
In this study, we demonstrated that thermal cooking
for 25 min with no presoaking significantly reduced
the TPCs, TACs, and C3G levels in black rice, and
such decreases were associated with decreased FRAP
Fig. 2. Representative HPLC chromatograms of cyanidin-3-glucoside (C3G) (a) and protocatechuic acid (PA) (b) in acidic
methanol extracts of raw (left panel) and cooked (right panel) black rice. Thumbnails inside indicated the authentic C3G and PA
standards. The contents of C3G (c) and PA (d) were calculated by standard curves from HPLC analyses. Values are expressed as
the mean9SD (n3). A two-way ANOVA with repeated measures was used for analysis of the cooking effect (*pB0.05) and
washing effect (p0.05).
Sassy Bhawamai et al.
4
(page number not for citation purpose)
Citation: Food & Nutrition Research 2016, 60: 32941 - http://dx.doi.org/10.3402/fnr.v60.32941
antioxidant activity, indicating that ordinarily consumed
cooked black rice possessed lower polyphenol contents
and antioxidative activities. On the contrary, washing up
to three times did not have significant effects on the
polyphenol contents, although it slightly decreased the
FRAP antioxidant capacity. We observed that 1 g of dry
raw and cooked black rice respectively contained 5.35.9
versus 3.14.4 mg polyphenols, 2.12.4 versus 1.0 1.2 mg
anthocyanins, and 650791 versus 238296 mg C3G,
which agreed well with results obtained by Sompong
et al. (8), who indicated that different species of raw black
rice contain 3.46.7 mg/g of TPCs, 1.12.6 mg/g of TACs,
and 1901,408 mg C3G. In addition, Abdel-Aal et al. (17)
showed that black rice had the highest amounts of TACs
(3.27 mg/g) and C3G (2,013 mg/g) among different grains,
including rice, corn, wheat, and barley. However, several
studies indicated different levels of TPCs or TACs in
black rice, ranging 14.773 mg/g and 1,4702,721 mg/g,
respectively (15, 18), and these differences may be ex-
plained by different sources of the black rice, extraction
solvents, and detection techniques used in these studies.
Polyphenols and anthocyanins are labile to heat treat-
ment and decreased anthocyanin contents after thermal
treatment were observed in various polyphenol-rich
foods, such as blueberry juice, pomegranate juice, and
black rice (19, 20). Results obtained by Hiemori et al. (9)
indicated that TACs in black rice cooked by different
methods, including a rice cooker, a pressure cooker, and
a gas range, were reduced to about one-third of the raw
sample, and those levels were lower than our results,
which showed about a 55% loss of anthocyanins and
a 67% loss of C3G after cooking in a rice cooker. The
higher TACs in the present study can possibly be
explained by a shorter cooking time (25 min) than the
90 min used by Hiemori’s group. On the contrary, a
significantly higher amount of PA, one of the major
anthocyanin degradation products, was found in cooked
rice, and this is similar to results obtained by Hiemori
et al. (9) who also observed 3- to 4-fold higher PA in rice
after cooking in different cookers. Unexpectedly, washing
prior to cooking did not affect the contents of phenolic
compounds of black rice. Anthocyanins are mainly loca-
lized in the aleurone layer and endosperm of grains, so
washing may only remove the superficial anthocyanins of
the rice, and interior pigments are not exposed until they
are powdered. Therefore, we did not observe a significant
loss of anthocyanins in black rice, because it was powdered
after washing and cooking. Because of the potent anti-
oxidative activities of the phenolic compounds, the
decreased polyphenols in cooked rice were also reflected
in the decreased FRAP antioxidative activities. Taken
together, thermal cooking reduced the TPCs, TACs, and
C3G levels, in association with decreasing FRAP acti-
vities in cooked black rice. On the contrary, washing up
to three times did not show significant effects on the
phenolic contents of black rice.
Although cooked rice had lower phenolic com-
pounds and antioxidative activity, it possessed similar
anti-inflammatory activities as raw rice, as determined by
assays of NO, TNF-a, and IL-6. Prolonged inflammation
evoked by over-activated macrophages is positively asso-
ciated with a variety of pathological conditions, including
Fig. 3. Ferric-reducing antioxidant power (FRAP) capacity of acidic methanol extracts of black rice. Values are expressed as the
mean9SD (n3). A two-way ANOVA with repeated measures was used for analysis of the cooking effect (*p B0.05) and
washing effect (abc, data not sharing the same letter within cooked or raw rice significantly differ at p B0.05).
Table 1. Correlation coefficients of total polyphenols, total antho-
cyanins, cyanidin-3-glucoside (C3G), protocatechuic acid (PA), and
the ferric-reducing antioxidant power (FRAP) antioxidant capacity
of black rice
TPC TAC C3G PA FRAP
TPC 0.800* 0.788* 0.773* 0.851*
TAC 0.800* 0.937* 0.914* 0.830*
C3G 0.788* 0.937* 0.933* 0.838*
PA 0.773* 0.914* 0.933* 0.801*
TPC, total phenolic contents; TAC, total anthocyanin contents; C3G,
cyanidin-3-glucoside; PA, protocatechuic acid. *
p
B0.01.
Cooking does not attenuate anti-inflammatory activities of black rice
Citation: Food & Nutrition Research 2016, 60: 32941 - http://dx.doi.org/10.3402/fnr.v60.32941 5
(page number not for citation purpose)