Effect of Anthocyanin-Rich Extract from Black Rice (Oryza sativa L. Japonica) on Chronically Alcohol-Induced Liver Damage in Rats

Abstract

The study evaluated the protective effect of anthocyanin-rich extract from black rice (AEBR) on chronic ethanol-induced biochemical changes in male Wistar rats. Administration of ethanol (3.7 g/kg/day) to Wistar rats for 45 days induced liver damage with a significant increase (P < 0.05) of aspartate transaminase (AST), alanine transaminase (ALT), gamma glutamyl transferase (GGT) in the serum and the hepatic malondialdehyde (MDA) level. In contrast, administration of AEBR (500 mg/kg) along with alcohol significantly (P < 0.01) decreased the activities of liver enzymes (AST, ALT and GGT) in serum, the MDA levels and the concentrations of serum and hepatic triglyceride (TG) and total cholesterol (TCH). Rats treated with AEBR showed a better profile of the antioxidant system with normal glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and glutathione S-transferase (GST) activities. All these results were accompanied by histological observations in liver. The results demonstrate that AEBR has a beneficial effect in reducing the adverse effect of alcohol.

Full text

pubs.acs.org/JAFCPublished on Web 02/09/2010©2010 American Chemical Society
J. Agric. Food Chem. 2010, 58, 3191–3196 3191
DOI:10.1021/jf904407x
Effect of Anthocyanin-Rich Extract from Black Rice
(Oryza sativa L. Japonica)on Chronically Alcohol-Induced
Liver Damage in Rats
Z
HAOHUA
H
OU
,P
EIYOU
Q
IN
,
AND
G
UIXING
R
EN
*
Institute of Crop Science, Chinese Academy of Agricultural Sciences No. 80 South Xueyuan Road,
Haidian District, Beijing100081, P. R. China
The study evaluated the protective effect of anthocyanin-rich extract from black rice (AEBR)on chronic
ethanol-induced biochemical changes in male Wistar rats. Administration of ethanol (3.7 g/kg/day)to
Wistar rats for 45 days induced liver damage with a significant increase (P<0.05)of aspartate trans-
aminase (AST), alanine transaminase (ALT), gamma glutamyl transferase (GGT)in the serum and the
hepatic malondialdehyde (MDA)level. In contrast, administration of AEBR (500 mg/kg)along with
alcohol significantly (P<0.01)decreased the activities of liver enzymes (AST, ALT and GGT)in serum,
the MDA levels and the concentrations of serum and hepatic triglyceride (TG)and total cholesterol
(TCH). Rats treated with AEBR showed a better profile of the antioxidant system with normal
glutathione peroxidase (GSH-Px),superoxidedismutase(SOD)and glutathione S-transferase (GST)
activities. All these results were accompanied by histological observations in liver. The results demons-
trate that AEBR has a beneficial effect in reducing the adverse effect of alcohol.
KEYWORDS: Black rice; anthocyanin-rich extract; liver damage; rats; ethanol
INTRODUCTION
Anthocyanins are a group of plant pigments that are widely
distributed in flowers, fruits and cereals, and contribute to the
bright colors (green, red and blue) of these plant components (1).
As a group of flavonoid pigments, anthocyanins have not only
their colorant potential but also significant health implications,
because of their nontoxicity and nonmutagenic, antioxidant
activity (2). As a result, anthocyanins have been widely used in
the food industry as well as in human health (1).
The “health-promoting” activity of anthocyanins has drawn
attention in recent years (3,4). As an abundant component in the
human diet (fresh fruit, juices, wine, cereals), anthocyanins
have demonstrated anti-inflammatory (5) and antioxidant acti-
vities (6). Systemic antioxidative effects by circulating antho-
cyanins in body fluids are expected to reduce the body’s load of
oxidants and ultimately the risk for developing of diseases (7).
Black rice (Oryza sativa L. Japonica), as a special anthocyanin-
rich cultivarof rice, has been regarded as a health-promoting food
and widely consumed in Eastern Asia. Previous studies showed
that the supplementation of black rice pigment fraction markedly
reduced oxidative stress, improved lipid profile and modulated
atherosclerotic lesions in two different animal models (8).
The antioxidant nature of anthocyanin-rich extract from black
rice might be helpful to alleviate the pathological changes caused by
alcohol in liver. The liver is subject to acute and potentially lethal
injury by several substances including phalloidin, carbon tetra-
chloride (CCl
4
), galactosamine, ethanol, and other compounds (9).
Long-term ethanol consumption induces oxidative stress in the
liver due to the imbalance between the prooxidant and antioxi-
dant systems (10). The susceptibility of liver to ethanol toxicity can
promote alcoholic liver disease (11), while the efficacy of any
protective drug essentially depends on its capacity of either redu-
cing the harmful effects or in maintaining the normal physiology of
cells and tissues. In recent years, a wide variety of antioxidants and
diverse diets have been tested to alleviate the oxidative stress
induced by ethanol abuse (12,13 ). The aim of the present study
was to investigate the effects of anthocyanin-rich extract from
black rice on ethanol-induced liver damage in rats.
MATERIALS AND METHODS
Animals.
Male Wistar rats (150 g (20 g) were used in the experiment.
The animals were kept under the standard conditions of animal house with
12 h light-dark cycle (light 7:00-19:00) at a temperature 22 °C(2°Cand
humidity 70% (4%, and had free access to food and water. The
experiment was performed following the European Community Guide-
lines for the Use of Experimental Animals and approved by the Peking
University Committee on Animal Care and Use.
Preparation of Anthocyanin-Rich Extract from Black Rice
(AEBR).
Black rice was purchased from a local market in Jilin province,
China. The procedure for preparing AEBR was described previously (14).
In brief, whole black rice was ground with a laboratory mill and passed
through a 60 mesh screen sieve. Black rice powder was extracted twice with
ethanol/water/hydrochloric acid (50:50:0.5, v/v/v) of solid-liquid ratio
1:10 for 2 h at 50 °C. The filtrates were combined and subjected to vacuum
evaporation (RotavaporR 210; B€
uchi, Flawil,Sweden) to remove ethanol.
The concentrated extracts were loaded onto an AB-8 resin. The AB-8 resin
was washed with distilled water, and subsequently the absorbed antho-
cyanins were eluted with 80% ethanol. The ethanol eluent was sprayed to
yield anthocyanin-rich powders.
*Author to whom correspondence should be addressed. Tel: þ86-10-
62-11-5596. Fax: þ86-10-62-15-6596. E-mail renguixing@caas.net.cn.

3192 J. Agric. Food Chem., Vol. 58, No. 5, 2010 Hou et al.
Identification and Quantification of Anthocyanins.
Anthocyanins
in AEBR were identified using HPLC-MS method by comparing their
retention time and MS data with standards and published data. Cyanidin-
3-glucoside (HPLC grade) was obtained from Polyphenols Laboratories
(Sandnes, Norway). Electrospray mass spectrometry was performed with
an Esquire-LC mass spectrometer (MS) (Bruker Daltoniks, Billerica,
MA), an ion trap instrument equipment with an electrospray interface.
Spectra were recorded in positive ion mode between m/z50 and 1500.
Major MS parameters were as follows: capillary exit, 4000 V; capillary
offset, 500 V; skim 1, 38.3 V; nebulizer gas, nitrogen, 40 psi; dry gas,
nitrogen, 12 L/min; dry temperature, 300 °C. The experimental conditions
were described previously (2,15).
Four anthocyanins were identified as cyanidin-3-glucoside (91.01%),
peonidin-3-glucoside (7.13%), cyanidin-3, 5-diglucoside (0.92%), cyani-
din-3-rutinoside (0.94%) (Figure 1). Analyses were performed on a
Shimadzu LC-20A HPLC. Figure 1 shows the HPLC anthocyanin profile
of black rice. Quantification of anthocyanin of AEBR were performed on
an Shimadzu LC-20A HPLC and expressed as cyanidin-3-glucoside. The
total anthocyanin content is up to 22.5% of AEBR.
In addition to anthocyanins, AEBR also contained moisture (6.96%),
crude protein (8.78%), crude fat (4.35%), total carbohydrate and other
ingredients (57.41%).
Experimental Design.
The experimental animals were randomly
divided into five groups with eight rats in each group. Alcohol and
AEBR dissolved in water (16) was administered using an intragastric
tube for 45 days, according to preliminary experiments and previous
literature (16,17). The experimental design was as follows. Group 1:
Control rats treated with distilled water (3.7 g/kg body weight). Group 2:
Normal rats orally received ethanol (3.7 g/kg BW) (17). Group 3: Normal
rats orally received ethanol (3.7 g/kg BW) with AEBR (500 mg/kg BW).
Group 4: Normal rats orally received ethanol (3.7 g/kg BW) with AEBR
(250 mg/kg BW). Group 5: Normal rats orally received ethanol (3.7 g/kg
BW) with AEBR (125 mg/kg BW). At the end of the experimental period,
animals were sacrificed by decapitation. Livers were excised immediately,
washed with ice-cold physiologic saline solution (0.9%), blotted dry, and
weighed.
Preparation of Subcellular Fractionation and Blood.
Blood was
taken from the neck vessels and stood for 30 min at room temperature.
Serum was obtained by centrifugation at 3000gfor 10 min and stored
at -80 °C in aliquots until the analysis.
Hepatic subcellular fractions (microsomes, cytosol, and mitochondria)
were isolated as previously described (18,19). A portion of each liver
sample was homogenized in 0.1 M Tris-HCl, 0.25 M sucrose, 0.1 mM
ethylenediaminetetraacetic acid(EDTA, pH7.4), and centrifuged at 2000g
for 15 min at 4 °C, to obtain the postnuclearsupernatant. The postnuclear
supernatant was further centrifuged at 10000gfor 30 min at 4 °C; the pellet
was mitochondria. The postmitochondrial supernatant was then centri-
fuged at 100000gfor 1 h at 4 °C to obtain microsomes (pellet) and cytosol
(supernatant). Both mitochondrial and microsomal pellets were suspended
in 0.1 M Tris-HCl (pH7.4) containing 10% glycerol, 0.1 mM EDTA and
were quickly frozen in liquid nitrogen.
To prepare a 10% liver homogenate, liver tissue was homogenized with
an ice-cold 0.9% solution of NaCl (1:10).
Biochemical Parameters of Liver Function.
Activities of aspartate
transaminase (AST), alanine transaminase (ALT), γ-glutamyltransferase
(GGT), and glutathione S-transferase (GST) in serum were measured by
using kits [Nanjing Jiancheng Bioengineering Institute (NJBI), China].
Measurement of TC, TG and MDA in Liver Homogenate and
Serum.
Total cholesterol (TC), triglycerides (TG) and malondialdehyde
(MDA) were measured by colorimetric method. MDA was assayed by the
measurement of thiobarbituric acid-reactive substance (TBARS) levels
spectrophotometrically at 532 nm. The results were expressed as nmol/mg
protein (20).
Biochemical Assays.
Superoxide peroxidation (SOD) in microsomal
and mitochondrial fractions, glutathione S-transferase (GST) in postmi-
tochondrial fraction, GSH-peroxidase in serum and mitochondrial frac-
tion were determined by kits obtained from NJBI (China).
Histopathology.
The caudal portion of the left lobe of the liver was
removed and fixed in 10% neutral-buffered formaldehyde solution. Fixed
tissues were embedded in paraffin, cut into 5-6μm thick sections and
placed on microscope slides. Slides were stained with hematoxylin and
eosin (H&E), which mounted in neutral distyrene-dibutylphthalate-xylene
(DPX) medium for microscopic observations.
Statistical Analysis.
All data are expressed as the mean (standard
deviation.Data were analyzed statistically by one-way analysis of variance
(ANOVA), using SPSS Statistical program (version 13.0 software, SPSS
Inc. Chicago, USA). A value of P< 0.05 was considered as statistically
significant.
RESULTS AND DISCUSSION
Growth Performance and Liver Index.
Table 1 shows the initial
and final body weights in control and experimental animals. The
ethanol-treated rats gained significantly less weight than controls
(P<0.01). Liver index in the ethanol-fed group increased signi-
ficantly (P< 0.01) compared with the control group, while it
increased insignificantly in the AEBR (500 mg/kg BW) group.
The rats treated with ethanol showed a significantly smaller body
weight (Table 1), in comparison with the control group, which is
similar to the previous report (21); the reason may be due to food
intake loss and malabsorption (21).
Effect of AEBR on AST, ALT and GGT Activities.
Ethanol also
induced pathologic changes in the liver including hepatomegaly
and serologic changes along with the increase of the activities of
aspartate transaminase (AST), alanine transaminase (ALT) and
gamma glutamyl transferase (GGT). Table 2 shows the activities
of serum AST, ALT and GGT in control and experimental
rats. Ethanol administration significantly (P< 0.05) increased
the activities of AST, ALT and GGT. Administration of AEBR
(500 mg/kg) along with alcohol significantly (P<0.01) reversed
these functional markers toward to near normal. AEBR at a dose
of 500 mg/kg body weight was more effective when compared
with two other doses (250 and 125 mg/kg body weight).
It is well-known that ethanol ingestion causes liver damage
with the leakage of cellular enzymes into plasma being a sign of
hepatic injury (22). Alcohol-induced oxidative stress in the liver
cells plays a major role in the development of alcoholic liver
disease. ALT and AST are the reliable makers for liver function.
Figure 1.
Anthocyanin profile in black rice extract analyzed using
HPLC. Detection was performed at 520 nm. Peak 1, cyanidin-3,5-digluco-
side; peak 2, cyanidin-3-glucoside; peak 3, cyanidin-3-rutinoside; peak 4,
peonidin-3-glucoside.
Table 1. Effects of AEBR on Body Weight and Liver Index in Ethanol-Treated
Rats
a
body wt (g)
parameters initial final liver wt (g)liver/wt (%)
control 157.92 (8.24 348.75 (15.56 8.26 (0.81 2.48 (0.12
ethanol 159.11 (6.22 316.70 (12.41** 8.63(0.59 2.74 (0.19**
ethanol þAEBR
125 mg/kg 155.72 (10.6 321.44(16.41 8.64 (0.73 2.58 (0.06
#
250 mg/kg 158.82 (7.02 335.75 (19.26 8.42 (0.58 2.55 (0.07
##
500 mg/kg 153.73 (5.37 343.57 (23.86
#
8.53 (0.97 2.52 (0.11
##
a
Values are mean (dev for 8 rats in each group. Compared with control group:
*P< 0.05, **P< 0.01. Compared with ethanol group:
#
P< 0.05,
##
P< 0.01.

Article J. Agric. Food Chem., Vol. 58, No. 5, 2010 3193
The increased levels of serum enzyme such as AST and ALT
indicate the increased permeability and damage and/or necrosis
of hepatocytes (23). The membrane bound enzyme GGT is
released into the bloodstream depending on the pathological
phenomenon (24). In our study, chronic ethanol consumption
caused a significant increase in the activities of AST, ALT and
GGT (Table 2), which could cause severe damage to tissue
membrane. Obi et al. (25) examined that anthocyanin obtained
from the petals of Hibiscus rosainensis significantly decreased the
levels of serum aspartate and alanine aminotransferase activities
(25). In this study, the decreased activities of these enzymes on
AEBR administrated rats indicate the hepatoprotective effect.
Effect of AEBR on TCH and TG in Liver and Serum.
Levels of
triglycerides (TG) and total cholesterol (TCH) in 10% liver
homogenate and serum are presented in Table 3. Ethanol intake
led to the increase of serum and hepatic TG and TCH levels (P<
0.05). AEBR administration improved these adverse effects.
Compared with the ethanol group, the serum and hepatic TG
levels decreased significantly in ABER group.
Hepatic steatosis has been defined as either more than 5% of
hepatocytes containing fat droplets or total lipid exceeding 5% of
liver weight (26). Accumulation of fat is the earliest and most
common response to heavy alcohol intake. Alcoholic fatty liver is
usually characterized by the enlargement of the liver, the increase
of the serum and hepatic TG levels, together with a lot of fat
droplets in the liver sections (27). In the current study, ethanol
administration resulted in the considerable increase of liver index,
the elevation of the serum and hepatic TG and TCH levels
(Table 3), suggesting that ethanol administration induced typical
fatty liver. Parallel to these changes, histological examination
showed a lot of droplets in ethanol-treated rat livers.
Effect of AEBR on GSH and MDA Concentrations and SOD,
GST and GSH-Px Activities.
The levels of MDA and GSH in
serum and liver of control and experimental rats are shown in
Table 4. Ethanol administration caused a severe increase of serum
and liver MDA concentrations (P<0.01). Pretreatment of rats
with AEBR reduced the formation of MDA of serum and liver.
Table 4 represents the levels of nonenzymatic antioxidant (GSH)
in tissues. The levels of GSH were significantly (P<0.05) reduced
in alcohol-treated rats when compared with control rats. Admin-
istration of AEBR (500 mg/kg) significantly (P<0.05) restored
the levels of nonenzymatic antioxidants in tissues.
Ethanol-induced liver injury associated with increased oxida-
tive stress and free radical-mediated tissue damage is widely
demonstrated in rats and humans (11,28,29 ). Free radicals or
reactive oxygen species (ROS) are responsible for ethanol induced
oxidative stress (10,30). Free radicals formed from the ethanol-
mediated process have a great potential to react rapidly with
lipids, which in turn leads to lipid peroxidation (LPO). The level
of malondialdehyde (MDA) has been widely used as a biomarker
of LPO for many years (31).
Chiang et al. (32) reported that black rice anthocyanin extract
(cyanidin-3-glucoside and peonidin-3-glucoside) may attenuate
oxidative stress by reducing ROS and increasing antioxidant
enzyme activities both in vitro and in vivo(32). Tsuda et al.
(1999) found that cyanidin-3-glucoside (C3G), a potent anti-
oxidant in vivo, can lower the serum thiobarbituric acid-reactive
substance (TBARS) concentration and increase the oxida-
tion resistance of serum to lipid peroxidation in rats (1). In
the current study, AEBR was found dramatically inhibiting
the elevation of MDA levels caused by ethanol, indicating that
the protective effects of AEBR may be associated with anti-
oxidant activities.
In antioxidant system, nonenzymatic antioxidant such as GSH
plays an important role in protecting the cell from lipid peroxida-
tion in biological system. Supplementation of AEBR to alcohol-
treated rats restored the nonenzymatic antioxidants levels in liver
and serum. We observed near-normal levels of these antioxi-
dants in alcohol-administered rats (Table 4). AEBR acts as an
oxygen-radical scavenger and chain-breaking antioxidant, which
minimizes the consumption of endogenous antioxidants and
improves the levels of those nonenzymic antioxidant in circu-
lation of alcohol-treated rats.
The activities of antioxidant enzymes such as superoxide
dismutase (SOD), glutathione peroxide (GPx) and glutathione
S-transferase (GST) are given in Table 5. A significant increase in
the activities of enzymes was observed in alcohol-treated rats.
Administration of AEBR (500 mg/kg) to alcohol-treated rats
significantly (P<0.01) decreased the activities.
Long-term ethanol treatment resulted in hepatic oxidative
stress by exacerbated lipid peroxidation and decreased anti-
oxidant enzyme activity. Free radical scavenging enzymes such
as SOD, GST and GPx are the first line of defense against oxidative
injury. The inhibition of antioxidant system may cause the
accumulation of ROS or products of its decomposition (32,33).
The GSTs are a multigene family of isozymes that catalyze the
conjugation of GSH to a variety of electrophilic compounds,
and thereby exert a critical role in cellular protection against
Table 2. Effect of AEBR on Hepatic Markers in the Serum of Control and
Ethanol-Administered Rats
a
parameters AST (IU/L)ALT (IU/L)GGT (IU/L)
control 48.58 (19.88 20.93 (3.99 4.65 (2.28
ethanol 75.79 (8.33** 28.76(2.03** 6.99 (1.57*
ethanol þAEBR
125 mg/kg 61.19 (11.16 25.83 (7.08 5.10 (1.66
250 mg/kg 49.71 (9.15
##
23.71 (5.97 4.77 (1.12
#
500 mg/kg 47.51 (11.94
##
20.85 (3.34
##
4.21 (1.52
##
a
Values are mean (dev for 8 rats in each group. Compared with control group:
*P< 0.05, **P< 0.01. Compared with ethanol group:
#
P< 0.05,
##
P< 0.01.
Table 3. Effect of AEBR on Hepatic and Serum TCH and TG
a
liver serum
parameters TCH (mmol/L)TG (mmol/L)TCH (mmol/L)TG (mmol/L)
control 1.24 (0.12 1.45(0.11 2.92 (0.09 1.48 (0.19
ethanol 1.54 (0.16** 1.73 (0.22** 3.26 (0.62 1.76 (0.08*
ethanol þAEBR
125 mg/kg 1.43 (0.14 1.59(0.06 2.71 (0.57
#
1.58 (0.21
250 mg/kg 1.43 (0.08 1.57(0.099
#
2.61 (0.33
#
1.46 (0.31
#
500 mg/kg 1.34 (0.10
##
1.51 (0.10
##
2.48 (0.39
##
1.57 (0.09
a
Values are mean (dev for 8 rats in each group. Compared with control group:
*P< 0.05, **P< 0.01. Compared with ethanol group:
#
P< 0.05,
##
P< 0.01.
Table 4. Effect of AEBR on Hepatic and Serum MDA and GSH Concen-
trations
a
MDA GSH
parameters
serum
(nmol/L)
liver
(nmol/mg protein)
serum
(mgl/L)
liver
(mg/g protein)
control 5.18 (0.29 3.61 (0.69 9.15 (0.48 5.31 (0.63
ethanol 5.92 (0.34** 4.79 (0.79** 7.92 (0.95* 3.67 (0.42**
ethanol þ
AEBR
125 mg/kg 5.43 (0.55 4.19(0.65 8.55 (0.55 4.32 (0.66
#
250 mg/kg 5.18 (0.36
##
3.83 (0.57
#
8.69 (1.01 4.45 (0.32
#
500 mg/kg 5.13 (0.63
##
3.85 (0.79
#
9.48 (0.93
##
4.47 (0.43
#
a
Values are mean (dev for 8 rats in each group. Compared with control group:
*P< 0.05, **P< 0.01. Compared with ethanol group:
#
P< 0.05,
##
P< 0.01.

3194 J. Agric. Food Chem., Vol. 58, No. 5, 2010 Hou et al.
ROS (34,35). Ethanol or its metabolic products might specifically
target GST isoenzymes, and the reduction in enzyme activity
or expression may contribute to ethanol hepatoxicity (36).
In consistent with these reports, our results showed that oral
supplementation of AEBR to rats treated with ethanol chronically
restored the activities of GSH-Px, SOD and GST in liver (Table 5).
Table 5. Effect of AEBR on Activities of SOD, GST and GSH-Px
a
GST SOD GSH-Px
parameters serum (U/mL)
postmitochondria
(U/mg prot)serum (U/mL)
mitochondria
(U/mg prot)serum (U/mL)
microsome
(IU/L)
mitochondria
(IU/L)
control 32.31 (2.56 42.26 (2.61 144.75 (10.86 291.89 (44.32 1650.13 (156.59 62.44 (8.30 60.65 (6.48
ethanol 27.68 (2.88** 37.29 (2.63* 128.33 (13.14* 210.18 (48.23** 1343.69 (120.66** 45.18 (5.85** 53.31 (11.47*
ethanol þ
AEBR
125 mg/kg 28.49 (2.29 38.94 (6.36 139.39 (16.11 246.65 (23.43 1345.88 (95.20 46.57 (3.61 63.11 (7.04
250 mg/kg 29.34 (1.73 40.19 (5.38 142.79 (11.18 260.25 (41.99
#
1393.06 (168.26 49.95 (6.62 64.65 (7.31
#
500 mg/kg 31.48 (2.99
#
46.60 (1.96
##
175.76 (12.33
##
280.13 (29.51
##
1539.19 (137.17
#
52.99 (7.12
#
69.82 (9.75
##
a
Values are mean (dev for 8 rats in each group. Compared with control group: *P< 0.05, **P< 0.01. Compared with ethanol group:
#
P< 0.05,
##
P< 0.01.
Figure 2.
Representative photomicrographs of livers in control and experiment rats. The liver section of each rat from different groups was stained by
hematoxylin and eosin staining, and the images were examined by Olympus BX50 light microscope.

Article J. Agric. Food Chem., Vol. 58, No. 5, 2010 3195
Histopathological Examination of Rat Liver.
Ethanol can
induce severe liver damage. The liver samples of alcohol-treated
rats showed the focal hepatocytes’ damage and degeneration
(Figure 2B). It was found that administration of AEBR reversed
this liver damage. AEBR at a dose of 500 mg/kg body weight
(Figure 2C) was more effective when compared with two other
doses (250 and 125 mg/kg body weight) (Figure 2D,E). The
administration of ethanol along with AEBR (500 mg/kg body
weight) showed near normal appearance (Figure 2A). The liver
was almost normal in appearance with mild changes in hepato-
cytes of rats treated with AEBR (500 mg/kg BW) group. The
results of histological observations suggest that alcohol leads to
serious changes in histology of liver (Figure 2A). The present
study suggests that the focal hepatocytic necrosis with inflamma-
tory cell infiltration in alcohol-treated rats might be due to the
accumulation of lipids and its content in tissues, which could also
increase the LPO, as a basis for cellular damage. Administration
of AEBR (500 mg/kg) (Figure 2A) remarkably reduced the
histological alterations caused by alcohol, which may be due to
attenuation of the ethanol-mediated oxidative threat and reduc-
tion of the pathological changes with restoration of normal
physiological function.
AEBR contained four anthocyanins (cyanidin-3-glucoside,
peonidin-3-glucoside, cyanidin-3,5-diglucoside, and cyanidin-3-
rutinoside), and the anthocyanin structures were different. The
structure of anthocyanin modulated functionality of anthocya-
nins; absorption and metabolism of anthocyanins were different
in vivo. Delphinidin-3-glucoside was metabolized to 4
0
-methyl
delphinidin-3-glucoside (37), whereas cyanidin-3-glucoside pro-
duced both 3
0
-and4
0
-methyl cyanidin-3-glucoside (38). However,
synergistic effects of anthocyanins are not clear; it is worth being
researched.
In conclusion, our data indicate that AEBR has a protective
action against alcohol-induced toxicity as evidenced by the
lowered tissue lipid peroxidation and elevated levels of the
enzymic and nonenzymatic antioxidants in liver, which is prob-
ably due to its antioxidant properties, scavenging ethanol-in-
duced free radicals. AEBR plays a beneficial role in the treatment
of alcohol induced tissue damage, which indicates the therapeutic
values of black rice.
ABBREVIATIONS USED
AEBR, anthocyanin-richextract from black rice; ALT, alanine
transaminase; AST, aspartate transaminase; BW, body weight;
GGT, gamma glutamyl transferase; GST, glutathione S-transfer-
ase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde;
SOD, superoxide dismutase; TCH, total cholesterol; TG, trigly-
ceride; ROS, reactive oxygen species.
LITERATURE CITED
(1) Kong, J. M.; Chia, L. S.; Goh, N. K.; Chia, T. F.; Brouillard, R.
Analysis and biological activities of anthocyanins. Phytochemistry
2003,64, 923–933.
(2) De Pascual-Teresa, S.; Santo-Buelga, C.; Rivas-Gonzalo, J. LC-MS
analysis of anthocyanins from purple corn cob. J. Sci. Food . Agric.
2002,82, 1003–1006.
(3) Oszmianski, J. Aronia (Aronia melanocarpa Elliot) fruits and skull-
cap (Scutellaria baicalensis Georgi) roots are very high in biologically
active flavonoids. Farm. Pol. 2001,15, 726–730.
(4) Hertog, M. G.; Feskens, E. J.; Hollman, P. C.; Katan, M. B.;
Kromhout, D. Dietary antioxidant flavonoids and the risk of
coronary heart disease: the Zutphen Elderly Study. Lancet 1993,
342, 1007–1011.
(5) Subarnas, A.; Wagner, H. Analgesic and anti-inflammatory activity
of the proanthocyanidin shellegueain A from Polypodium feei
METT. Phytomedicine 2000,7, 401–405.
(6) Wolniak, M. Mechanisms of antioxidative action of anthocyanins.
Farm. Pol. 2002,20, 931–934.
(7) Pool-Zobel, B. L.; Bub, A.; Schr€
oder, N.; Rechkemmer, G. Antho-
cyanins are potent antioxidants in model systems but do not reduce
endogenous oxidative DNA damage in human colon cells. Eur. J.
Nutr. 1999,38, 227–234.
(8) Guo, H. H.; Ling, W. H.; Wang, Q.; Liu, C.; Hu, Y.; Xia, M.; Feng,
X.; Xia, X. D. Effect of anthocyanin-rich extract from black rice
(Oryza sativa L. india) on hyperlipidemia and insulin resistance in
fructose-fed rats. Plant Foods Hum. Nutr. 2007,62, 1–6.
(9) Valcheva-Kuzmanova, S.; Borisova, P.; Galunska, B.; Krasnaliev,
I.; Belcheva, A. Hepatoprotective effect of the natural fruit juice
from Aronia melanocarpa on carbon tetrachloride-induced acute
liver damage in rats. Exp. Toxicol. Pathol. 2004,56, 195–201.
(10) Nordmann, R.; Ribi
ere, C.; Rouach, H. Implication of free radical
mechanisms in ethanol induced cellular injury. Free Radical Biol.
Med. 1992,12, 219–240.
(11) Lieber, C. S. Alcohol and the liver: metabolism of alcohol and its
role in hepatic and extrahepatic diseases. Mt. Sinai J. Med. 2000,67,
84–94.
(12) Nanji, A. A.; Jokelainen, K.; Tipoe, G. L.; Rahemtulla, A.; Thomas,
P.; Dannenberg, A. J. Curcumin prevents alcohol-induced liver
disease in rats by inhibiting the expression of NF-kB-dependent
genes. Am. J. Physiol. 2003,284, 321–327.
(13) Ramirez-Farias, C.; Madrigal-Santillan, E.; Gutierrez-Salinas, J.;
Rodriguez-Sanchez, N.; Martinez-Cruz, M.; Valle-Jones, I.; Gramlich-
Martinez, I.; Hernadez-Ceruelos, A.; Morales-Gonzalez, J. A. Pro-
tective effect of some vitamins against the toxic action of ethanol on
liver regeneration induced by partial hepatectomy in rats. World J.
Gastroenterol. 2008,14, 899–907.
(14) Prior, R. L.; Wu, X. L.; Gu, L. W.; Hager, T. J.; Hager, A.; Howard,
L. R. Whole berries versus berry anthocyanins: interactions with
dietary fat levels in the C57BL/6J mouse model of obesity. J. Agric.
Food Chem. 2008,56, 647–653.
(15) Wu, X. L.; Gu, L. W.; Prior, R. L.; McKay, S. Characterization of
anthocyanins and proanthocyanidins in some cultivars of Ribes,
Aronia, and Sambucus and their antioxidant capacity. J. Agric. Food.
Chem. 2004,52, 7846–7856.
(16) Hassimotto, N. M. A.; Genovese, M. I.; Lajolo, F. M. Absorption
and metabolism of cyanidin-3-glucoside and cyanidin-3-rutinoside
extract from wild mulberry (Morus nigra L.) in rats. Nutr. Res. (N.Y.)
2008,28, 198–207.
(17) Kundu, R.; Dasgupta, S.; Biswas, A.; Bhattacharya, A.; Pal, B. C.;
Bandyopadhyay, D.; Bhattacharya, S.; Bhattacharya, S. Cajanus
cajan Linn. (Leguminosae) prevents alcohol-induced rat liver da-
mage and augments cytoprotective function. J. Ethnopharmacol.
2008,118, 440–447.
(18) Lieber, C. S.; DeCarli, L. M. Hepatic microsomal ethanol-oxidizing
system: in vitro characteristics and adaptive properties in vivo.J. Biol.
Chem. 1970,245, 2505–2512.
(19) Raza, H.; Prabu, S. K.; Robin, M. A.; Avadhani, N. G. Elevated
mitochondrial cytochrome P4502E1 and glutathione S-transferase
A4-4 in streptozotocin-induced diabetic rats tissue-specific varia-
tions and roles in oxidative stress. Diabetes 2004,53, 185–194.
(20) Yuan, G. J.; Gong, Z. J.; Li, J. H.; Li, X. Ginkgo biloba extract
protects against alcohol-induced liver injury in rats. Phytother. Res.
2007,21, 234–238.
(21) Lieber, C. S. Alcohol and liver: 1994 update. Gastroenterology 1994,
106, 1085–1105.
(22) Baldi, E.; Burra, P.; Plebani, M.; Salvagnini, M. Serum malondial-
dehyde and mitochondrial aspartate aminotransferase activity as
markers of chronic alcohol intake and alcoholic liver disease. Ital. J.
Gastroenterol. 1993,25, 429–432.
(23) Goleberg, D. M.; Watts, C. Serum enzyme changes as evidence of
liver reaction to oral alcohol. Gastroenterology 1965,49, 256–261.
(24) Sillanaukee, P. Laboratory makers of alcohol abuse. Alcohol Alco-
holism 1996,31, 613–616.
(25) Obi, F. O.; Usenu, I. A.; Osayande, J. O. Prevention of carbon
tetrachloride-induced hepatotoxicity in the rat by H. Rosainensis
anthocyanin extract administered in ethanol. Toxicology 1998,131,
93–98.

3196 J. Agric. Food Chem., Vol. 58, No. 5, 2010 Hou et al.
(26) Salt, W. B. Nonalcoholic fatty liver disease(NAFLD): a compre-
hensive review. J. Insur. Med. 2004,36, 27–41.
(27) Zeng, T.; Guo, F. F.; Zhang, C. L.; Zhao, S.; Dou, D. D.; Gao, X. C.;
Xie, K. Q. The anti-fatty liver effects of garlic oil on acute ethanol-
exposed mice. Chem.-Biol. Interact. 2008,176, 234–242.
(28) Halliwell, B. Antioxidant defense mechanisms: from the beginning to
the end. Free Radical Res. 1999,31, 261–272.
(29) Minana, J. B.; Gomez-Gambronero, L.; Lloret, A.; Pallard
o, F. V.;
Olmo, J. D.; Escudero, A.; Rodrigo, J. M.; Pellı
´n, A.; Vi~
na, J. R.;
Vi~
na, J.; Sastre, J. Mitochondrial oxidative stress and CD95 ligand: a
dual mechanism for hepatocytes apoptosis in chronic alcoholism.
Hepatology 2002,35, 1205–1214.
(30) Hoek, J. B.; Pastorino, J. G. Ethanol, oxidative stress and cytokine
induced liver cell injury. Alcohol 2002,27, 63–68.
(31) Lykkesfeldt, J. Malondialdehyde as biomarker of oxidative damage
to lipids caused by smoking. Clin. Chim. Acta 2007,308, 50–58.
(32) Chiang, A. N.; Wu, H.L.; Yeh, H. I.; Chu, C. S.; Lin, H. C.; Lee, W. C.
Antioxidant effects of black rice extract through the induction of
superoxide dismutase and catalase activities. Lipids 2006,41, 797–803.
(33) Halliwell, B. Free radicals, antioxidants and human diseases-
curiosity, causes or consequence. Lancet 1994,344, 721–724.
(34) Wilce, M. C.; Parker, M. W. Structure and function of glutathione
S-transferases. Biochim. Biophys. Acta 1994,1205, 1–18.
(35) Hayes, J. D.; Pulford, D. J. The glutathione S-transferase supergene
family: regulation of GST and the contribution of the isoenzymes to
cancer chemoprotection and drug resistance. Crit. Rev. Biochem.
Mol. Biol. 1995,30, 445–600.
(36) Alin, P.; Danielson, U. H.; Mannervik, B. 4-Hydroxyalk-2-enals are
substrates for glutathione transferase. FEBS Lett. 1985,179, 267–
270.
(37) Ichiyanagi, T.; Rahman, M.; Kashiwada, K.; Ikeshiro, Y.; Shida, Y.;
Hatano, Y.; Matsumoto, H.; Hirayama, M.; Tsuda, T.; Konishi, T.
Absorption and Metabolism of delphinidin-3-O-β-
D
-glcopyranoside
in rats. Free Radical Biol. Med. 2004,36, 930–937.
(38) Ichiyanagi, T.; Shida, Y.; Rahman, M. M.; Hatano, Y.; Matsumoto,
H.; Hirayama, M.; Konishi, T. Metabolic pathway of cyanidin-
3-O-β-D-glucopyranoside in rats. J. Agric. Food Chem. 2005,53,
145–150.
Received for review December 14, 2009. Revised manuscript received
January 26, 2010. Accepted January 26, 2010. The present study was
supported by the Institute Fund (No. 2060302-15)from The Ministry of
Sciences and Technology, People’s Republic of China, and the Public
Service (Agriculture)Special Fund for Research (No.200803056)from
Ministry of Agriculture, People’s Republic of China.