Journal of Cereal Science 64 (2015) 92e99

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Journal of Cereal Science

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Identification of anthocyanins in black rice (Oryza sativa L.) by UPLC/Q-

TOF-MS and their in vitro and in vivo antioxidant activities
Jie Hao, Hui Zhu, Zhenqing Zhang, Shilin Yang, Heran Li*
College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, People's Republic of China

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

Article history: Anthocyanins in different types of black rice are different and their in vivo antioxidant activity is poorly
Received 1 December 2014 studied. In this study, nine anthocyanins were detected by high performance liquid chromatography
Received in revised form (HPLC) and eight of them were identified by ultra performance liquid chromatography/time-of-flight
30 April 2015
mass spectrometry (UPLC/Q-TOF-MS) in black rice. The in vivo antioxidant activity of the black rice
Accepted 5 May 2015
extract containing 41.69% of anthocyanins was determined by the model of KBrO3-induced renal injury in
Available online 14 May 2015
mice. KBrO3 caused serious renal injury, with a significant increase in serum creatinine (14.37 ± 0.27 mg/
mL versus 8.80 ± 0.38 mg/mL) and blood urea nitrogen (BUN, 625.56 ± 42.62 mg/L versus
Keywords:
Black rice
348.49 ± 14.90 mg/L) levels. KBrO3 could also cause oxidative stress in the kidney, with a significant
Anthocyanin increase in renal xanthine oxidase (XOD), malondialdehyde (MDA) and nitric oxide (NO) levels. Pre-
UPLC/Q-TOF-MS treatment with the anthocyanin-rich black rice extract remarkably reduced the levels of serum creatinine
Antioxidant and BUN and the levels of renal XOD, MDA and NO. The protective effects were due to the free radical
scavenging capacity of the anthocyanins.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction components in different types of black rice is important to develop

Black rice (Oryza sativa L.), a special type of rice with color
pigments, has been widely cultivated and consumed in South-
eastern Asian countries for a long time (Hu et al., 2003). Black rice is
rich in fiber, minerals and several important amino acids (Zhang
et al., 2004, 2005). It was also reported that the content of pro-
tein was higher than that in white rice (Jiang et al., 1999).
Furthermore, black rice has been considered as an important
health-promoting crop as high content of anthocyanin was
observed in the aleurone layer and introduced to many other areas
such as North America (Lee, 2010; Hou et al., 2013; Hiemori et al.,
2009). Anthocyanin which belongs to flavonoids, is responsible
for the attractive colors of flowers, fruits and grains in nature (Kong
et al., 2003). It has been widely reported that anthocyanin plays an
important role in reducing risk of oxidative damage and is a kind of
the potential drug candidates to treat cancer and cardiovascular
diseases (Chen et al., 2012). Different types of anthocyanin are
observed in various types of black rice (Hou et al., 2013; Hiemori
et al., 2009; Chen et al., 2012). Thus, identifying the anthocyanin
* Corresponding author.
E-mail address: heranli@suda.edu.cn (H. Li).
an accurate database and expand the applications of black rice.
Potassium bromate (KBrO3) is widely used as a food additive in
the bread making process for the maturation of flour because of its
oxidation (Ahmad et al., 2012a). KBrO3 is also observed in many
drinks as a by-product of ozone disinfection (Kurokawa et al., 1982).
Recently, it is believed that KBrO3 can cause serious nephrotoxicity
(Ahmad et al., 2012b; Bao et al., 2008; Khan and Sultana, 2004).
More and more researchers have focused on the mechanism of
KBrO3-induced nephrotoxicity. It is widely accepted so far that the
oxidative stress caused by KBrO3 increases the reactive oxygen
species (ROS), ONOO
 and 8-hydroxydeoxyguanosine levels of
renal tissue of rats, which is subsequently injured (Ahmad et al.,
2012b; Giri et al., 1999; Kasai et al., 1987). Since anthocyanins
exhibit protective effects in various pathophysiological conditions
via their antioxidant properties, we speculate that the anthocyanin-
rich black rice extract may inhibit KBrO3-induced renal injury
caused by oxidative stress.
The objective of the present study is to firstly characterise an-
thocyanins in the black rice extract and investigate the protective
effects of the extract against KBrO3-induced renal injury in mice.
The research results will promote the nutritional and medicinal
values of black rice.

http://dx.doi.org/10.1016/j.jcs.2015.05.003
0733-5210/© 2015 Elsevier Ltd. All rights reserved.
 J. Hao et al. / Journal of Cereal Science 64 (2015) 92e99
2. Materials and methods
2.1. Materials and chemicals
The medium black rice having vitreous endosperm was supplied
by Baoji Embellish Wood Agricultural Development Co., LTD (Baoji,
China). The whole grain was dehulled by using a rice huller (QKT-
400B, Dali Machinery Works, Weihui, China). The yield of black rice
kernels is almost 90%. Authentic standard of cyanidin-3-glucoside
chloride was purchased from MUST Bio-Tech Co. (Chengdu,
China). Authentic standards of cyanidin-3-sambubioside chloride
and cyanidin-3,5-diglucoside chloride were obtained from Hui-
cheng Bio-Tech Co. (Shanghai, China). Authentic standard of
creatinine was purchased from National Institute for Food and Drug
Control (Beijing, China). Ascorbic acid was purchased from China-
sun Specialty Products Co. (Changshu, China). Blood urea nitrogen
(BUN) kit, malondialdehyde (MDA) kit, xanthine oxidase (XOD) kit
and nitric oxide (NO) kit were purchased from Jian Cheng Bioen-
gineering Institute (Nanjing, China). 1,1-diphenyl-2-picrylhydrazyl
(DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS) were purchased from TCI Chemical Co.
(Tokyo, Japan).
2.2. Preparation of the anthocyanin-rich black rice extract
The unpolished black rice kernels were dried at 40  C, ground
with a high speed mill (DFT-200, Linda Machinery Co. LTD., Wen-
ling, China) and passed through an 80 mesh screen sieve. Antho-
cyanins in black rice were extracted according to the modified
method described by Prior et al. (2008). The dried black rice flour
was extracted twice with ethanol/water/hydrochloric acid
(50:50:0.5, v/v/v) by solideliquid ratio (1:10) at 30  C for 2 h. All
extracts were combined and filtered through a Buchner funnel. The
collected filtrates were subjected to vacuum evaporation at 35  C to
remove ethanol. The concentrated extracts were loaded on an AB-8
resin column to remove major impurities. The anthocyanins were
washed with distilled water containing 1% hydrochloric acid and
subsequently recovered with 70% ethanol containing 1% hydro-
chloric acid. The ethanol eluent was evaporated under vacuum at
35  C to yield the anthocyanin-rich black rice extract. The yield of
the extract from dried black rice flour is ~1% (w/w).
2.3. Identification and quantification of anthocyanins in the black
rice extract
Cyanidin-3-glucoside chloride was used as the reference to
quantify anthocyanins in the black rice extract (Hao et al., 2014).
Cyanidin-3-glucoside stock solution was diluted to a series of
concentrations (1, 2, 5, 10, 20, 50, 125 mg/mL) with 10% formic acid.
The standard solutions and extract in proper concentration were
analyzed with an Agilent 1260 HPLC system (Agilent, USA). The
diode array detector was set at 520 nm. Anthocyanins were sepa-
rated through a Cosmosil 5C18-MS-II column (4.6  250 mm,
Nacalai Tesque, Japan) with average particle size of 3 mm at 1 mL/
min at room temperature. Mobile phase consisted of methanol (A)
and 10% formic acid (B) with the following gradient elution:
0e5 min, 10% A; 5e20 min, 10e60% A; 20e30 min, 60% A. The in-
jection volume was 20 mL. The detector responses were plotted as a
function of concentration of standard solutions. The limit of
detection (LOD, S/N ¼ 3) calculated with cyanidin-3-glucoside was
22.2 ng/mL and the limit of quantification (LOQ, S/N ¼ 10) of cya-
nidin-3-glucoside was 100.0 ng/mL. The average recovery calcu-
lated with cyanidin-3-glucoside was 98.3%. Each anthocyanin
separated was quantified against the standard curve.
Anthocyanins in the black rice extract were identified using
93
ultra-performance liquid chromatography-quadrupole-time of
flight mass spectrometry (UPLC/Q-TOF-MS) analysis. UPLC analysis
was carried out on an ACQUITY™ ultra-performance liquid chro-
matography system (Waters Corporation, USA). Chromatographic
separation was performed using a Cosmosil 5C18-MS-II column
(4.6  250 mm, Nacalai Tesque, Japan) at a column temperature of
20  C. The mobile phases consisted of methanol (A) and 10% formic
acid (B) with the following gradients: 0e5 min, 10% A; 5e20 min,
10e60% A; 20e30 min, 60% A. The injection volume was 20 mL.
520 nm was selected as the preferred wavelength. The flow rate
was 0.8 mL/min and the injection volume was 5 mL. Mass analysis
was performed on a Xevo G2 Q-Tof mass spectrometer (Waters
Corporation, USA). Spectra were recorded in positive ion mode
between m/z 100 and m/z 1000. First of all, the quadrupole mass
analyzer was set as “TTI” mode and the collision cell didn't apply
energy to acquire the MS spectra. Then, the quadrupole mass
analyzer was changed to “SIM” mode for the targeted ions and the
collision cell applied energy to the targeted ions to get the MS/MS
spectra. The major MS/MS parameters were as follows: capillary
voltage, 2.50 kV; sampling cone voltage 28 V; extraction cone
voltage, 4.0 V; source temperature, 100  C; desolvation tempera-
ture, 600  C; cone gas flow, 50 L/h; desolvation gas flow, 1200 L/h;
collision energy ramp: 15 Ve25 V.
2.4. Evaluation of in vitro antioxidant activities of the anthocyanin-
rich black rice extract by DPPH and ABTS assays
DPPH
scavenging assay was performed according to the pre-
vious method with minor modification (Hao et al., 2014). The
samples of anthocyanin-rich black rice extract, ascorbic acid and
DPPH were dissolved in methanol, respectively. The anthocyanin-
rich black rice extract and ascorbic acid solutions were respec-
tively diluted to a series of concentrations (25, 50, 100, 200, 400 mg/
mL). 0.5 mL of the samples diluted were added to 2.5 mL of 0.2 mM
DPPH
solutions. The mixture was vigorously shaken and incubated
in the dark at 25  C for 30 min. The absorbance at 517 nm of the
mixture was measured by using a UV-2600 spectrophotometer
(SHIMADZU, Japan). The scavenging rate was calculated as DPPH
-
scavenging rate (%) ¼ [1  (A  B)/A0]  100, where A ¼ absorbance
of test sample, B ¼ absorbance of 0.5 mL of test sample þ 2.5 mL of
methanol, and A0 ¼ absorbance of 0.5 mL of methanol þ 2.5 mL of
DPPH
solution. The experiment was conducted in triplicate.
ABTS was dissolved in water to acquire a concentration of
7.4 mM. The ABTS
þ was generated by adding equal ABTS stock
solution to potassium persulfate solution that was at a concentra-
tion of 2.6 mM. The reaction was incubated for 15 h at 25  C in the
dark to assure that the reaction was complete and the absorbance
was stable. The anthocyanin-rich black rice extract and ascorbic
acid solutions were also respectively diluted to a series of con-
centrations (5, 10, 15, 25, 50, 100 mg/mL). 0.5 mL of the samples
diluted were added to 2.5 mL of the ABTS
þ solutions. The mixture
was vigorously shaken and incubated in the dark at 25  C for 2 h.
The absorbance at 734 nm of the mixture was detected with a UV-
2600 spectrophotometer (SHIMADZU, Japan). The scavenging rate
was calculated as ABTS
þ-scavenging rate (%) ¼ [1  (A  B)/
A0]  100, where A ¼ absorbance of sample, B ¼ absorbance of
0.5 mL of sample þ 2.5 mL of water, and A0 ¼ absorbance of 0.5 mL
of water þ 2.5 mL of ABTS
þ solution. The experiment was con-
ducted in triplicate.
2.5. Animals and experimental design
Five-week-old male KM mice (18e22 g) were purchased from
Slac Laboratory Animal Limited Liability Company (Shanghai,
China) and maintained in Soochow University Laboratory Animal
94 J. Hao et al. / Journal of Cereal Science 64 (2015) 92e99
Center. All mice were kept in a specific germfree animal room under
controlled conditions of temperature (20e22  C) and lighting (12 h
darkelight cycle). All mice were provided with standard laboratory
diet and tap water. The animals were acclimatized to laboratory
conditions for 7 days before commencement of the experiment.
The study protocol was approved by the Ethical Committee of
Soochow University, Suzhou, China.
In this study, mice were randomly divided into six groups with
10 animals for each group. Group 1 and 2 received distilled water
once daily for 7 days at a dose of 0.1 mL/10 g by body weight. Group
3 received pretreatment with ascorbic acid by gavage once daily for
7 days at a dose of 200 mg/kg of body weight. Groups 4, 5 and 6
received pretreatment with the anthocyanin-rich black rice extract
by gavage once daily for 7 days at dosages of 50, 100, and 200 mg/
kg, respectively. After the last treatment, the animals of groups 2e6
received an intraperitoneal injection of KBrO3 at a dose of 200 mg/
kg, whereas group 1 received an intraperitoneal injection of saline
at a dose of 0.1 mL/10 g. After 9 h, the animals were anesthetized
with anesthetic ether.
2.6. Sample preparation
Under anesthesia, blood samples were collected in test tubes
without anticoagulative agents by cardiac puncture. Then, animals
were sacrificed by cervical dislocation method. Kidneys were
immediately removed and washed with ice-cold saline (0.9%). After
40 min of standing, blood samples were centrifuged at 5000 rpm
for 10 min at 4  C by high speed refrigerated centrifuge (Xiangyi Co.,
Changsha, China) to obtain serum. Next, the serum was acidified
with 6% perchloric acid to precipitate proteins. The samples were
centrifuged at 12,000 rpm for 15 min at 4  C and then the super-
natants were collected for further study. The 10% homogenate was
prepared in ice-cold saline (1:9, m/v) and centrifuged at
10,000 rpm for 10 min at 4  C. The supernatants were appropriately
diluted for further assays.
2.7. Determination of serum creatinine level
Serum creatinine concentration was determined according to
the method described by Jen et al. (2002) using an Agilent HPLC
(Agilent, USA). The HPLC conditions were as follows: a flow rate of
1.0 mL/min; a Cosmosil 5C18 column (4.6  150 mm; Nacalai Tes-
que, Japan) at room temperature (25  C); mobile phase, potassium
phosphate buffer solution (20 mM, pH 6.5); detection, 235 nm; and
injection volume, 20 mL.
2.8. Determination of blood urea nitrogen level
BUN concentration was determined using a BUN kit in accor-
dance with the diacetyl monoxime method. Under acidic and
boiling conditions, urea nitrogen in serum reacted with diacetyl to
produce pink-colored diazine that was measured at 520 nm with a
BioTek microplate reader (BioTek Co., USA).
2.9. Estimation of renal protein level
A 2% kidney homogenate was used to determine the concen-
tration of protein using a Coomassie (Bradford) protein kit with
bovine serum albumin as a standard. Homogenate combined with
Bradford staining to produce blue adduct that was measured at
595 nm with a BioTek microplate reader (BioTek Co., USA).
2.10. Estimation of renal xanthine oxidase level
A 5% kidney homogenate was used to determine the XOD level
with a commercial XOD kit. XOD in the homogenate catalyzed the
oxidation of hypoxanthine to xanthine with a coproduct of super-
oxide anion radical which ultimately led to the generation of pink-
colored adduct that was measured at 530 nm with a BioTek
microplate reader (BioTek Co., USA).
2.11. Estimation of renal malondialdehyde level
A 5% kidney homogenate was used to determine the MDA level
using a commercial MDA kit with 1,1,3,3-tetraethoxypropane as a
standard. Under acidic and boiling conditions, MDA reacted with
thiobarbituric acid (TBA) to produce the pink-colored adduct that
was measured at 532 nm with a BioTek microplate reader (BioTek
Co., USA).
2.12. Estimation of renal nitric oxide level
A 10% kidney homogenate was used to determine the NO level
using a commercial NO kit with sodium nitrite as a standard. NO
combined with water and oxygen leading to the generation of ni-
trite. The nitrite reacted with the Griess reagent to produce purple
azo-dye product that was detected at 550 nm with a BioTek
microplate reader (BioTek Co., USA).
2.13. Data analysis
The results were expressed as mean ± standard error of mean
(SEM). Differences between groups were evaluated by one-way
analysis of variance (ANOVA) using the Statistical Package for So-
cial Science (SPSS) software. Post hoc testing was used for inter-
group comparisons using the Dunnett's test. Values corresponding
to p < 0.05 were considered as statistically different.
3. Results
3.1. Identities and contents of anthocyanins in the black rice extract
In the present study, nine principal anthocyanins were detected
in the black rice extract by HPLC analysis at 520 nm (Fig. 1A). UPLC/
Q-TOF-MS was used to identify the anthocyanins. The MS/MS
spectra are shown in Fig. 2.
As seen in Fig. 2, both peak 1 (RT ¼ 13.126 min) and peak 2
(RT ¼ 13.424 min) showed a molecular ion [M þ H] þ at m/z 611 and
a fragment ion at m/z 287 ([M þ H-162-162] þ loss of two glucose
moieties). By reference to previous reports (Hou et al., 2013;
Tamura et al., 2010), the identities of the two peaks were cyani-
din-3,5-diglucoside and cyanidin-3-gentiobioside. The data of
cyanidin-3,5-diglucoside standard (absorption maxima, retention
time and MS in Table 1) were consistent with peak 1. Hence, peak 1
was identified as cyanidin-3,5-diglucoside and peak 2 as cyanidin-
3-gentiobioside. Peak 3 (RT ¼ 14.301 min) possessed a molecular
ion [M þ H] þ at m/z 449 and a fragment ion at m/z 287 ([M þ H-
162] þ loss of a glucose moiety). Peak 3 was identified as cyanidin-
3-glucoside by comparing with the standard. Peak 4
(RT ¼ 14.848 min) possessed a molecular ion [M þ H] þ at m/z 581
and a fragment ion at m/z 287 ([M þ H-162-132] þ loss of one
glucose moiety and one xylose moiety). The data (absorption
maxima, retention time and MS in Table 1) of cyanidin-3-
sambubioside standard were consistent with peak 4. Therefore,
peak 4 was identified as cyanidin-3-sambubioside. This anthocy-
anin has been identified in berries (Mullen et al., 2010; Wei et al.,
2011). However, it seems this is the first time that this anthocy-
anin is being reported in black rice. Peak 5 (RT ¼ 15.199 min)
showed a molecular ion [M þ H] þ at m/z 595 and a fragment ion at
m/z 287 ([M þ H-162-146] þ loss of one glucose moiety and one
 J. Hao et al. / Journal of Cereal Science 64 (2015) 92e99
Fig. 1. HPLC chromatograms of the anthocyanin-rich black rice extract at 520 nm (A), 254 nm (B) and 280 nm (C). Peak 1, cyanidin-3,5-diglucoside; peak 2, cyanidin-3-
gentiobioside; peak 3, cyanidin-3-glucoside, peak 4, cyanidin-3-sambubioside; peak 5, cyanidin-3-rutinoside; peak 6, peonidin-3-glucoside; peak 7, cyanidin-derivative; peak 8,
cyanidin; peak 9, peonidin.
rhamnose moiety). Peak 5 was identified as cyanidin-3-rutinoside
(Hou et al., 2013). Peak 6 (RT ¼ 16.085 min) showed a molecular
ion [M þ H] þ at m/z 463 and a fragment ion at m/z 301 ([M þ H-
162] þ loss of one glucose moiety). Peak 6 was identified as peo-
nidin-3-glucoside (Hou et al., 2013; Hiemori et al., 2009; Chen et al.,
2012; Lee, 2010). Peak 8 (RT ¼ 18.742 min) showed a molecular ion
[M þ H] þ at m/z 287. Peak 8 was identified as cyanidin (Kaneda
et al., 2006). Peak 9 (RT ¼ 20.672 min) possessed a molecular ion
[M þ H]þ at m/z 301. Peak 9 was identified as peonidin. The MS
profile of peak 7 (RT ¼ 17.924 min) presented a molecular ion
[M þ H]þ at m/z 477 and a fragment ion at m/z 287 which corre-
sponded to the cyanidin moiety. Peak 7 was not identified and it
was named cyanidin-derivative.
In this study, quantitative determination of anthocyanins in the
black rice extract was based on the area under the peaks from HPLC
analysis at 520 nm. The linear equation (y ¼ 40.751x  15.524,
R2 ¼ 0.9998, linear range 1e125 mg/mL, n ¼ 7) prepared using
cyanidin-3-glucoside standard, was used to calculate the contents
of anthocyanins in the black rice extract. The results are listed in
Table 2. Each gram of the black rice extract contained
416.92 ± 0.63 mg of total anthocyanins. In addition, the content of
cyanidin-3-glucoside was 11 times higher than that of peonidin-3-
glucoside, which was consistent with previous reports (Hou et al.,
2013; Lee, 2010).
3.2. DPPH
and ABTS
þ radical scavenging activities of the
anthocyanin-rich black rice extract
Previous findings indicated that different black rice varieties
also contained different contents of anthocyanins and exhibited
different antioxidant activities (Chen et al., 2012; Lee, 2010; Zhang
et al., 2010). There was a positive correlation between total
anthocyanin content and antioxidant activity (Yao et al., 2010). So,
DPPH
and ABTS
þ radical scavenging assays were used to evaluate
the antioxidant activities of the black rice extract containing 41.69%
of total anthocyanins. Ascorbic acid, a common antioxidant com-
pound, was used as positive control. Fig. 3A shows the DPPH

95
radical scavenging activity of the anthocyanin-rich black rice
extract in the concentration range of 0e400 mg/mL. As seen in
Fig. 3A, the anthocyanin-rich black rice extract exhibited strong
DPPH
radical scavenging activity. The DPPH
radical scavenging
activity of the anthocyanin-rich black rice extract was comparable
to that of ascorbic acid (IC50, 53.51 ± 0.87 mg/mL versus IC50,
45.60 ± 0.31 mg/mL). Fig. 3B shows the ABTS
þ radical scavenging
activity of the anthocyanin-rich black rice extract in the concen-
tration range of 0e100 mg/mL. As shown in Fig. 3B, the
anthocyanin-rich black rice extract also exhibited strong ABTS
þ
radical scavenging activity. The ABTS
þ radical scavenging activity
of the anthocyanin-rich black rice extract was also comparable to
that of ascorbic acid (IC50, 13.09 ± 0.11 mg/mL versus IC50,
12.83 ± 0.03 mg/mL).
3.3. Effects of the anthocyanin-rich black rice extract on serum
creatinine and blood urea nitrogen levels
Serum creatinine and BUN levels are usually used as markers of
renal function. The changes in the creatinine and BUN levels in the
serum samples from all of the control and experimental groups are
shown in Table 3. Creatinine in serum, increased by 63.3% following
treatment with KBrO3. The creatinine level was significantly
(p < 0.05) reduced with the treatment of the anthocyanin-rich
black rice extract (100, 200 mg/kg) when compared with the
KBrO3-treated group. Ascorbic acid also reduced the creatinine
level significantly (p < 0.05) when compared with the KBrO3-
treated group. The effects of 100 and 200 mg/kg groups were
similar to that of ascorbic acid group.
Blood urea nitrogen increased by 79.5% following treatment
with KBrO3. The BUN level was significantly (p < 0.001) reduced
with the treatment of the anthocyanin-rich black rice extract (100,
200 mg/kg) when compared with the KBrO3-treated group.
Ascorbic acid also reduced the BUN level significantly (p < 0.001)
when compared with the KBrO3-treated group. Furthermore, the
BUN level of 200 mg/kg group was lower than that of 100 mg/kg
group.
96 J. Hao et al. / Journal of Cereal Science 64 (2015) 92e99

Fig. 2. MS/MS spectra of anthocyanins detected in the black rice extract. Peak 1, cyanidin-3,5-diglucoside; peak 2, cyanidin-3-gentiobioside; peak 3, cyanidin-3-glucoside, peak 4,
cyanidin-3-sambubioside; peak 5, cyanidin-3-rutinoside; peak 6, peonidin-3-glucoside; peak 7, cyanidin-derivative; peak 8, cyanidin; peak 9, peonidin.

Table 1
Chromatographic and spectral properties of anthocyanins in the black rice extract.

Peak no. RTa (min) lvisb (nm) luvb (nm) [M þ H]þ (m/z) MS/MS (m/z) Identity

1 13.126 518 278 611 287 Cyanidin-3,5-diglucoside

2 13.424 518 280 611 287 Cyanidin-3-gentiobioside
3 14.301 518 280 449 287 Cyanidin-3-glucoside
4 14.848 520 280 581 287 Cyanidin-3-sambubioside
5 15.199 520 280 595 287 Cyanidin-3-rutinoside
6 16.085 518 278 463 301 Peonidin-3-glucoside
7 17.924 520 280 477 287 Cyanidin-derivative
8 18.742 520 280 287 e Cyanidin
9 20.672 530 274 301 e Peonidin

Peak numbering refers to the peaks of Fig. 1.

a
Retention time.
b
Maximum absorption wavelength obtained from online HPLC-UV-Vis spectra.
 J. Hao et al. / Journal of Cereal Science 64 (2015) 92e99 97

Table 2 level of 200 mg/kg group was similar to that of 100 mg/kg group.
Individual and total anthocyanin content in the black rice extract. MDA, lipid peroxidation product in kidney tissues, was found to
Peak no. Anthocyanin Amount (cyanidin-3-glucoside be significantly (p < 0.001) increased in KBrO3-treated group when
equivalent) compared with control group (25.54 ± 1.92 nmol/mg protein versus
mg/g 15.50 ± 0.38 nmol/mg protein). Treatment with the anthocyanin-
a rich black rice extract (100, 200 mg/kg) reduced the MDA level
1 Cyanidin-3,5-diglucoside 7.46 ± 0.04
2 Cyanidin-3-gentiobioside 7.55 ± 0.01 significantly (p < 0.001) when compared with KBrO3-treated group.
3 Cyanidin-3-glucoside 268.29 ± 0.36 Ascorbic acid also reduced the MDA level significantly (p < 0.01)
4 Cyanidin-3-sambubiosidea 7.61 ± 0.04 when compared with KBrO3-treated group. Moreover, the MDA
5 Cyanidin-3-rutinoside 7.91 ± 0.06
levels of 100 and 200 mg/kg groups were lower than that of the
6 Peonidin-3-glucoside 23.81 ± 0.06
7 Cyanidin-derivative 6.97 ± 0.01 ascorbic acid group.
8 Cyanidin 80.01 ± 0.82 The NO level in KBrO3-treated group was significantly
9 Peonidin 7.31 ± 0.09 (p < 0.001) increased when compared with control group
e Total anthocyanins 416.92 ± 0.36 (407.95 ± 34.26 nmol/g protein versus 168.51 ± 28.98 nmol/g pro-
Each anthocyanin was quantified by the cyanidin-3-glucoside standard curve. tein). Treatment with the anthocyanin-rich black rice extract
Results were the average of three analyses and expressed as mean ± SEM. reduced the NO level significantly (p < 0.001) in a dose-dependent
a
Respectively compared with the authentic standards.
manner. Ascorbic acid also reduced the NO level significantly
(p < 0.001) when compared with KBrO3-treated mice. The effects of
100 and 200 mg/kg groups were better than that of the ascorbic
3.4. Effects of the anthocyanin-rich black rice extract on renal
acid group. The NO levels of 100 and 200 mg/kg groups were even
xanthine oxidase, malondialdehyde and nitric oxide levels
lower than that of the control group.
The changes in the XOD, MDA and NO levels in the kidney tis-
4. Discussion
sues of all of the control and experimental groups are shown in
Table 3. As shown in Table 3, the XOD level of KBrO3-treated group
Anthocyanins in different black rice varieties vary greatly. Lee
was much higher than that of control group (14.62 ± 1.26 U/g
(2010) identified two anthocyanins (cyanidin-3-glucoside, peoni-
protein versus 6.21 ± 0.27 U/g protein). Seven-day oral adminis-
din-3-glucoside) whereas Hou et al. (2013) identified four antho-
tration of the anthocyanin-rich black rice extract (100, 200 mg/kg)
cyanins (cyanidin-3-glucoside, peonidin-3-glucoside, cyanidin-3-
and ascorbic acid reduced the XOD levels significantly (p < 0.001)
rutinoside, cyanidin-3,5-diglucoside) in black rice. Chen et al.
when compared with KBrO3-treated group. What's more, the XOD
(2012) identified another two anthocyanins (malvidin-3-
glucoside, petunidin-3-glucoside) from two different types of
black rice. Hiemori et al. (2009) detected six anthocyanins in black
rice. Two of them were identified as cyanidin-3-glucoside, peoni-
din-3-glucoside unambiguously; three of them were classified as
cyanidin-dihexoside; and one of them was a cyanidin-hexoside, in
which the hexosides were not confirmed. Besides types, the total
contents of anthocyanins in different black rice are also different.
The contents of anthocyanins in 10 Korean black rice varieties are
5.2e168.3 mg/100 g expressed as cyanidin-3-glucoside and peo-
nidin-3-glucoside (Lee, 2010) and in 6 Japanese pigmented rice
varieties are 7.9e473.3 mg/100 g (Chen et al., 2012). The differences
of above results may probably be attributed to the differences of
black rice varieties, extraction and identification methods. Very
minor contents of some anthocyanins in black rice also affected the
identification results. In the present study, UPLC/Q-TOF-MS method
was used to identify the anthocyanin compositions in the black rice
extract. Four minor anthocyanins (cyanidin-3,5-glucoside, cyani-
din-3-gentiobioside, cyanidin-3-rutinoside, cyanidin-3-
sambubioside) were identified simultaneously in black rice. Cya-
nidin-3-sambubioside was reported in black rice for the first time.
In addition, the total content of anthocyanins in this type of black
rice is 416.9 mg/100 g, which is very high either compared with
the10 Korean black rice varieties or compared with the 6 Japanese
pigmented rice varieties.
In vitro antioxidant properties of black rice are well documented
in scientific literature. In addition, a few studies had explored the
antioxidant activities of black rice in vivo. However, the activities
couldn't definitely be attributed to the anthocyanin composition, as
the content of anthocyanin in black rice was very low (Lee, 2010).
Hence, the black rice extract containing rich anthocyanins was
made to explore its in vivo antioxidant activity using the model of
KBrO3-induced renal injury in mice.
In this work, the serum creatinine and BUN levels were signif-
Fig. 3. (A) DPPH
radical-scavenging activity of the anthocyanin-rich black rice extract. icantly elevated in KBrO3-treated mice, which was reported pre-
(B) ABTS
þ radical-scavenging activity of the anthocyanin-rich black rice extract. viously (Giri et al., 1999; Nishioka et al., 2006). The elevation of
98 J. Hao et al. / Journal of Cereal Science 64 (2015) 92e99

Table 3
Effects of the anthocyanin-rich black rice extract on serum creatinine and BUN and renal XOD, MDA and NO levels of mice treated with KBrO3.

Group Creatinine (mg/mL) BUN (mg/L) XOD (U/g protein) MDA (nmol/mg protein) NO (nmol/g protein)

Control 8.80 ± 0.38 348.49 ± 14.90 6.21 ± 0.27 15.50 ± 0.38 168.51 ± 28.98
KBrO3 14.37 ± 0.27a 625.56 ± 42.62a 14.62 ± 1.26a 25.54 ± 1.92a 407.95 ± 34.26a
KBrO3 þ Vc (200 mg/kg) 11.02 ± 0.93b 386.37 ± 27.60c 6.44 ± 0.54d 19.67 ± 0.96e 197.04 ± 21.90d
KBrO3 þ ARBRE (200 mg/kg) 11.18 ± 0.26b 401.90 ± 38.75c 8.45 ± 0.55d 16.68 ± 0.67d 77.36 ± 11.05d
KBrO3 þ ARBRE (100 mg/kg) 11.40 ± 0.91b 415.48 ± 38.74c 8.57 ± 0.66d 16.85 ± 0.56d 125.78 ± 42.75d
KBrO3 þ ARBRE (50 mg/kg) 12.18 ± 1.03 607.25 ± 21.25 14.20 ± 1.22 24.93 ± 1.31 202.67 ± 17.43d

Vc refers to ascorbic acid.

ARBRE refers to the Anthocyanin-Rich Black Rice Extract.
The results represent the mean ± SEM obtained from 10 animals in each group.
a
Indicates significant difference from the control group at p < 0.001.
b
Indicates significant difference from the KBrO3 group at p < 0.05.
c
Indicates significant difference from KBrO3 group at p < 0.001.
d
Indicates significant difference from the KBrO3 group at p < 0.001.
e
Indicates significant difference from the KBrO3 group at p < 0.01.

above two markers in serum resulted from the injuries to glomeruli
and tubules. KBrO3 also caused oxidative stress with an increase in
renal XOD, MDA and NO levels, which was also reported previously
(Bao et al., 2008; Khan and Sultana, 2004). Excessive XOD in kidney
catalyzed the oxidation of hypoxanthine to xanthine with a
coproduct of superoxide anion radical (O2
). Excessive NO reacted
with O2
 radical to produce cytotoxic radical ONOO
 (Watanabe
et al., 2004). The accumulation of O2
 and ONOO
 caused the
lipid peroxidation of cytomembrane of renal tissues and damaged
the normal physiological function of kidney. MDA, the final product
of lipid peroxidation, had been regarded as an available marker of
oxidative damage. MDA caused oxidative damage via converting
ROS into active chemicals and magnifying the effect of ROS through
the chain reaction of cellular metabolism and functional impair-
ment (Cheeseman, 1993). The present study revealed that appro-
priate dose of the anthocyanin-rich black rice extract inhibited
KBrO3-induced renal injury with a reduction in serum creatinine
and BUN levels. The anthocyanin-rich black rice extract also
ameliorated the oxidative stress markers including renal XOD, MDA
and NO levels.
Previous studies showed that some phytochemicals were
effective in ameliorating nephrotoxicity caused by KBrO3. The
protective effects were probably attributed to their antioxidant
activities (Khan and Sultana, 2004; Nishioka et al., 2006). Although
the detailed mechanism of KBrO3-induced renal injury is still un-
known, previous studies found that the production of ROS and
antioxidant defenses was approximately balanced in vivo. When
this balance tended to the production of ROS, oxidative stress
occured. Oxidative stress could lead to cellular dysfunctioning and
then tissue lesioning by damaging DNA, protein, lipids and other
biomolecules (Nishioka et al., 2006). The in vitro antioxidant assays
showed that the anthocyanin-rich black rice extract exhibited
remarkable DPPH
and ABTS
þ radical scavenging activities com-
parable to ascorbic acid. Due to its free radical scavenging activities,
we concluded that the anthocyanin-rich black rice extract exhibited
protective effects against KBrO3-induced renal injury in vivo by
means of rebalancing the levels of ROS and antioxidant defenses.
Our analysis results showed that the black rice extract contained
41.69% of total anthocyanins. In addition, 254 nm and 280 nm were
also used during the HPLC analysis. As seen in Fig. 1B and C, except
for anthocyanin peaks, few other potential phenolic compounds
with antioxidant activities were observed. So, the excellent in vitro
and in vivo antioxidant activities of the black rice extract were due
to its high content of anthocyanins.
5. Conclusions
In this study, we detected 9 different anthocyanins by HPLC and
identified 8 anthocyanins by UPLC/Q-TOF-MS in black rice. Cyani-
din-3-sambubioside was reported in black rice for the first time.
The analysis results indicated that the total anthocyanin content
was 416.92 ± 0.63 mg/g black rice extract. Afterwards, we esti-
mated the in vitro and in vivo antioxidant activities of the
anthocyanin-rich black rice extract. As a result, the anthocyanin-
rich black rice extract showed remarkable DPPH
and ABTS
þ
radical-scavenging activities in vitro. In addition, the anthocyanin-
rich black rice extract exhibited protective effects against KBrO3-
induced renal injury in vivo. The antioxidant activities of the black
rice extract were due to its high content of anthocyanins. Our re-
sults indicated that black rice, diet, might be a novel therapeutic
and preventive agent for oxidative stress-related diseases.
Acknowledgements
The authors are grateful to Baoji embellish wood agricultural
development Co., LTD for providing black rice. The fiancial support
from the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD) was appreciated.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jcs.2015.05.003.
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