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January 2014, Vol.12, No.

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59
Journal of Integrative Medicine
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Available also online at www.sciencedirect.com.
Copyright 2014, Journal of Integrative Medicine Editorial offce.
E-edition published by Elsevier (Singapore) Pte Ltd. All rights reserved.
1 Introduction
Excessive consumption of alcohol causes the liver to become
fatty. Fatty liver is the accumulation of triglyceride (TG) and
other fats in the liver cells. This condition makes the liver
vulnerable to infammation and varieties of disorders such
as alcoholic hepatitis, fibrosis, cirrhosis, and even hepa-
tocellular carcinoma and its associated complications. In
recent years, there has been an escalation in alcohol abuse,
and inevitably alcohol-related disorders are becoming an
increasingly important cause of morbidity and mortality
[1]
.

Research Article
Anti-fatty liver effects of oils from Zingiber officinale
and Curcuma longa on ethanol-induced fatty liver
in rats
Sarah Onyenibe Nwozo
1
, Damilola Adeola Osunmadewa
1
, Babatunji Emmanuel Oyinloye
1,2
1. Nutritional and Industrial Research Laboratories, Department of Biochemistry, Faculty of Basic Medical
Sciences, College of Medicine, University of Ibadan, Ibadan 200002, Nigeria
2. Department of Biochemistry, College of Sciences, Afe Babalola University, Ado Ekiti 360001, Nigeria
OBJECTIVE: The present study is aimed at evaluating the protective effects of oils from
Zingiber offcinale (ginger) and Curcuma longa (turmeric) on acute ethanol-induced fatty liver in
male Wistar rats.
METHODS: Ferric reducing antioxidant power activity and oxygen radical absorbance capacity
of the oils were evaluated ex vivo. Rats were pretreated for 28 d with standard drug (Livolin
Forte) and oils from Z. offcinale and C. longa before they were exposed to 45% ethanol (4.8 g/kg)
to induce acute fatty liver. Histological changes were observed and the degree of protection
was measured by using biochemical parameters such as alanine aminotransferase, aspartate
aminotransferase and alkaline phosphatase activities. Serum triglyceride (TG) level, total
cholesterol (TC) level and the effects of both oils on reduced gluthatione (GSH), glutathione-S-
transferase (GST), superoxide dismutase (SOD) and hepatic malondialdehyde (MDA) levels
were estimated.
RESULTS: Oils from Z. offcinale and C. longa at a dose of 200 mg/kg showed hepatoprotection
by decreasing the activities of serum enzymes, serum TG, serum TC and hepatic MDA, while they
signifcantly restored the level of GSH as well as GST and SOD activities. Histological examination
of rats tissues was related to the obtained results.
CONCLUSION: From the results it may be concluded that oils from Z. offcinale and C. longa
(200 mg/kg) exhibited hepatoprotective activity in acute ethanol-induced fatty liver and Z. offcinale
oil was identifed to have better effects than C. longa oil.
KEYWORDS: Curcuma longa; Zingiber offcinale; plant extracts; fatty liver; in vivo
http://dx.doi.org/10.1016/S2095-4964(14)60006-6
Nwozo SO, Osunmadewa DA, Oyinloye BE. Anti-fatty liver effects of oils from Zingiber officinale and
Curcuma longa on ethanol-induced fatty liver in rats. J Integr Med. 2014; 12(1): 59-65.
Received October 31, 2013; accepted December 20, 2013.
Correspondence: Sarah Onyenibe Nwozo, PhD; Tel: +234-802-3658-268; E-mail: sonwozo@yahoo.com
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January 2014, Vol.12, No.1
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Journal of Integrative Medicine
Increased intake of alcohol damages mitochondria,
endoplasmic reticulum, and other cellular structures,
which contributes to the inhibition of fatty acid oxidation.
The amount of fatty acid in the liver depends on the balance
between the processes of delivery and removal
[2]
.
The liver is the principal site for alcohol metabolism.
Alcohol in the liver causes a shift in metabolic pathways
and could result in a buildup of fatty acids. Fatty acid
overload in hepatocytes acts as both a substrate and an
inducer of microsomal cytochrome P-450 (CYP) 2E1
and fatty acid oxidation systems that generate reactive
oxygen species resulting in oxidative stress
[3]
. Increasing
evidence indicates that multiple mechanisms contribute
to the development of the alcoholic liver diseases (ALD),
involving oxidative stress, inflammation, excess lipid
synthesis, as well as complex interactions between alcohol
metabolism, lipid metabolism and the immune system
[4,5]
.
Recently, researchers are working on therapeutic modalities
to halt or reverse the pathogenesis and progression of
ALD. Previously performed clinical and experimental
studies have demonstrated that oils from different plant
source works as a scavenger of various reactive oxygen
species, including superoxide anion and hydroxyl radicals.
The possible mechanism(s) of their action may be due to
free radical-scavenging potential caused by the presence
of antioxidant component(s) in the oils
[6-8]
.
With the current resurgence of medicinal herbs in the
world, some natural compounds isolated from plants,
seeds or fruits have been used to treat alcoholic fatty liver.
Zingiber offcinale and Curcuma longa are of such plants
having both medicinal and nutritive values, and popularly
used as herbal remedy against a wide range of ailments,
both in Nigeria and several other countries in the world
[9]
.
C. longa is a rhizomatous perennial herb that belongs
to family Zingiberaceae. It is a tropical plant and is the
source of the spice turmeric, which is derived from the
dried, ground rhizome. It is extensively grown and used as
dietary pigment and spices. C. longa possesses antioxidant,
antitumor, antimicrobial, anti-inflammatory, wound healing,
lipid-reducing, chemopreventive, immunomodulatory,
and gastroprotective activities and all these are well docu-
mented
[10-12]
.
Z. officinale, commonly known as ginger, belongs to
family Zingiberaceae. It is one of the most commonly
used spices in Nigeria and around the world; it is an
indispensable component of curry
[13]
. It has long been used
to treat gastrointestinal disorders and its constituents have
shown anti-infammatory, chemopreventive, antidiabetic,
antihepatotoxic and antioxidant properties
[14-17]
.
Though many documented reports have implicated the
rhizomes of Z. officinale and C. longa in folk medicine
and herbal preparations for treatment of liver disorders,
this informed our decision to extract the oils, evaluate
antioxidant properties of the oils and study the effect of
the oils in acute ethanol exposure in rats and we have compared
our results with commercial multivitamin standard drug
Livolin Forte

.
2 Materials and methods
2.1 Experimental animals
Thirty male albino rats procured from the animal house
of the Biochemistry Department, College of Medicine,
University of Ibadan, Nigeria, weighing between 120-
200 g were randomly distributed into five groups of six
animals each. The animals were acclimatized to standard
laboratory conditions and were allowed free access to
standard rat chow (Ladokun Feed, Ibadan, Nigeria) and
water was given ad libitum for two weeks. Group I served
as normal control and was given corn oil (200 mg/kg).
Group II was a positive control given only 45% ethanol
(4.8 g/kg). Group III received Z. offcinale oil (200 mg/kg)
and 45% ethanol (4.8 g/kg). Group IV received C. longa
oil (200 mg/kg) and 45% ethanol (4.8 g/kg). Group V was
given standard drug (Livolin Forte) (20 mg/kg) and 45%
ethanol (4.8 g/kg). Rats were pretreated by oral gavage
for 28 d with standard drug (Livolin Forte) and oils from
Z. officinale and C. longa before they were exposed to
45% ethanol (4.8 g/kg) by oral gavage to induce acute
fatty liver. Animals were sacrifced after 24 h of ethanol
exposure. Animal experiments followed protocols established
by National Institute of Health for the Care and Use of
Laboratory Animals.
2.2 Plant collection and extraction
The rhizomes of Z. offcinale and C. longa, were purchased
from Ibode, Oja oba market, Ibadan, Nigeria. They were
identified and authenticated in the Botany Department,
University of Ibadan, Nigeria. One kilogram of each of
the plant materials was air-dried at room temperature,
grounded into powder and Soxhlet extracted using n-
hexane for 72 h. Oils were stored in sample bottles and
refrigerated until needed.
2.3 Ferric reducing antioxidant power activity
The ferric reducing antioxidant power (FRAP) assay
of oils from Z. officinale and C. longa was done on a
multiscan spectrum plate reader by using method developed
by Benzie and Strain
[18]
. This assay uses antioxidants as
reductants in a redox-linked colorimetric method which
measures the reduced oxidant at low pH (pH = 3.6). Ferric
tripyridyltriazine (Fe
III
-TPTZ) complex is reduced to the
ferrous form, forming an intense blue color with an absorption
maximum at 593 nm. The change in absorbance is directly
related to the reducing power of the electron-donating
antioxidants present in the reaction mixture.
2.4 Oxygen radical absorbance capacity
The oxygen radical absorbance capacity (ORAC) of Z.
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Journal of Integrative Medicine
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offcinale and C. longa oils was determined using fuorescein
C
20
H
10
Na
2
O
5
as the fuorescent probe and 2,2-azobis (2-meth-
ylpropionamidine) dihydrochloride (AAPH) as the peroxyl
radical generator based on the method of Ou et al
[19]
. This
was performed on a Fluoroskan plate reader with fourescense
flters for an excitation wavelength of 490 nm and emission
wavelength of 530 nm at 37 . Trolox caliberation solutions
were used as standard and ORAC values were calculated using
a regression equation. Data were expressed as a micromoles of
trolox equivalents (TE) per milligram of sample.
2.5 Chemicals and biochemical assays
Alkaline phosphatase (ALP), alanine aminotransferase
(ALT), aspartate aminotransferase (AST), total cholesterol
(TC) and TG assay kits were purchased from ABJ Chemicals,
Lagos (Nigeria). Adrenaline, thiobarbituric acid, Ellmans
reagent, glutathione and bovine serum albumin were
purchased from Sigma Chemical Company (St. Louis,
MO, USA). All other chemicals were of the highest purity
commercially available.
Lipid peroxidation (LPO) was assayed by measuring
thiobarbituric acid reactive substances as described by Varshney
and Kale
[20]
. Superoxide dismutase (SOD) activity was
determined by the method of Misra and Fridovich
[21]
. Reduced
glutathione (GSH) level was estimated using the method
described by Beutler et al
[22]
at 412 nm. Glutathione-S-
transferase (GST) activity was determined according to
Habig et al
[23]
. Serum ALP, ALT and AST activites and
serum TG and TC levels were quantified spectrophoto-
metrically using Randox commercial assay kits.
2.6 Preparation of tissues for biochemical analyses and
histological examination
Following the daily treatment for 28 d with standard
drug (Livolin Forte) and oils from Z. officinale and
C. longa, the animals were sacrificed 24 h after ethanol
exposure. Blood samples were collected by retro-orbital
puncture allowed to coagulate at room temperature
for half an hour and the serum was obtained by blood
centrifugation at 3 000g for 10 min and kept at 20
until analyses were done. Liver samples were quickly
excised and washed in ice-cold 1.15% KCl solution, dried
using flter paper and weighed. They were then homogenized
in 4 volumes of 56 mmol/L Tris-HCl buffer (pH 7.4)
containing 1.15% KCl, and then centrifuged at 10 000g
for 15 min.
The supernatant was collected and stored until needed
for assays. Small pieces of liver sections were fixed in
10% formal saline. Sections were cut and stained with
haematoxylin and eosin. The stained tissue sections
were observed under a light microscope for histological
assessment.
2.7 Statistical analysis
All values were expressed as the mean standard error
of mean of six animals. Data were analyzed using one-
way analysis of variance followed by the post-hoc LSD-
t test for analysis of biochemical data using SPSS (10.0)
statistical software. P < 0.05 was considered statistically
signifcant.
3 Results
3.1 FRAP activity and ORAC
Prior to the commencement of the animal experiment,
FRAP and ORAC activities of the oils from Z. offcinale
and C. longa were determined. FRAP values obtained from
the result showed that Z. officinale oil ((976.911.32) mol
AA/g) possessed the highest reducing power while C. longa
oil possessed the lowest reducing power ((113.105.67) mol
AA/g). The ORAC value of the oils determined also
showed that Z. officinale oil ((2 649.000.91) mol
TE/g) possessed a higher ORAC value than C. longa oil
((587.000.78) mol TE/g).
3.2 Effects of Z. officinale and C. longa oils on the
marker of LPO in the liver, and serum TC and TG
levels
The effects of Z. offcinale and C. longa oils on the marker
of LPO in the liver are presented in Table 1. Malondialdehyde
(MDA) level of ethanol-treated rats (Group II) was
signifcantly (P<0.05) higher than those of the normal control
rats (Group I). The increase observed in the MDA level of
the ethanol-treated rats was signifcantly (P<0.05) reduced
as a result of pretreatment with the standard drug (Livolin
Forte) and oils from Z. offcinale and C. longa. In like manner,
signifcant (P<0.05) increases noted in serum TC and TG
levels of ethanol-treated rats (Group II) when compared
with Group I, were significantly (P<0.05) reduced as a
result of pretreatment with the standard drug (Livolin
Forte) and oils from Z. offcinale and C. longa.
3.3 Effects of Z. offcinale and C. longa oils on hepatic
GSH level, and SOD and GST activities
GSH level, and SOD and GST activities in liver homogenate
were significantly (P<0.05) decreased in ethanol-treated
rats (Group II) compared with the normal control (Group
I). Moreover, in the pretreated groups (Groups III, IV
and V), GSH level, and SOD and GST activities in liver
homogenate were significantly (P<0.05) higher than
that of the ethanol-treated rats (Group II). Group IV rats
showed signifcantly (P<0.05) higher GSH level, and SOD
and GST activities than corresponding GSH level, and
SOD and GST activities in Groups III and V rats (Table 2).
3.4 Effects of Z. officinale and C. longa oils on liver
marker enzymes
The effects of Z. officinale and C. longa oils on liver
marker enzymes are presented in Table 3. Exposure to
ethanol caused abnormal liver function in ethanal-treated
rats (Group II). The levels of serum hepatospecifc enzymes
such as ALP, ALT and AST were significantly (P<0.05)
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Journal of Integrative Medicine
increased when compared with the normal control (Group
I). It was observed that the elevated levels of ALP, ALT
and AST were signifcantly (P<0.05) lowered in the pre-
treated groups (Groups III, IV and V) when compared
with the ethanol-treated rats (Group II).
3.5 Histological analysis of liver sections
The results of the histological assessment of the liver section
are presented in Figure 1. Group I showed normal liver archi-
tecture, with no visible lesions seen. There were marked portal
congestion and cellular infiltration by mononuclear cells in
Group II. Group III was characterized by periportal hydropic
degeneration of hepatocytes with cellular infiltration by
mononuclear cells. Mild portal congestion and mononuclear
cellular infltration with mild hydropic degeneration were
Table 2 Effects of Z. offcinale and C. longa oils on hepatic GSH level, and SOD and GST activities in rats
(Mean standard error of mean)
Group n GSH (g/mL) SOD (U/mg protein) GST (mol/min/mg protein)
Group I (corn oil 200 mg/kg) 6 6.910.14 8.550.36 0.560.03
Group II (45% ethanol) 6 5.950.41
*
7.450.36
*
0.270.05
*
Group III (Z. offcinale oil 200 mg/kg + 45% ethanol) 6 6.750.23

8.610.38

0.490.03
*
Group IV (C. longa oil 200 mg/kg + 45% ethanol) 6 6.920.29

8.790.21

0.810.04
*
Group V (Livolin Forte 200 mg/kg + 45% ethanol) 6 6.391.10

8.090.16

0.350.03
*
*
P < 0.05, vs Group I;

P < 0.05, vs Group II;

P<0.05, vs Group IV.


GSH: gluthatione; SOD: superoxide dismutase; GST: glutathione-S-transferase.
Table 3 Effects of Z. offcinale and C. longa oils on liver marker enzymes in rats
(Mean standard error of mean, U/L)
Group n ALP ALT AST
Group I (corn oil 200 mg/kg) 6 30.030.70 37.870.23 4.350.17
Group II (45% ethanol) 6 50.003.26
*
65.801.71
*
7.100.55
*
Group III (Z. offcinale oil 200 mg/kg + 45% ethanol) 6 35.140.87
*
49.470.41
*
4.940.51

Group IV (C. longa oil 200 mg/kg + 45% ethanol) 6 31.161.87

45.801.74
*
4.640.77

Group V (Livolin Forte 200 mg/kg + 45% ethanol) 6 44.011.24


*
47.602.20
*
5.900.60
*
*
P < 0.05, vs Group I;

P < 0.05, vs Group II.


ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase.
observed in Group IV while in Group V, there were mild
portal congestion, mild hydropic degeneration and few
foci of cellular infltration by mononuclear cells.
4 Discussion
Many pathways have been suggested as playing a key
role in how alcohol induces oxidative stress. Toxic substances
generated during the metabolism of alcohol in the liver
may contribute greatly to the development of ALD.
Importantly, oxidation of alcohol through the CYP 2E1
generates superoxide anion radical and hydrogen peroxide.
These free radicals are capable of damaging many cellular
components such as DNA, protein, and lipid
[24-27]
.
Table 1 Effects of Z. offcinale and C. longa oils on the marker of lipid peroxidation in the liver, and serum TC and TG activities in rats
(Mean standard error of mean)
Group n
MDA in liver tissue
(nmol/g)
Serum TC
(mg/dL)
Serum TG
(mg/dL)
Group I (corn oil 200 mg/kg) 6 2.890.08 3.590.09 4.130.05
Group II (45% ethanol) 6 3.360.12
*
4.820.06
*
9.350.02
*
Group III (Z. offcinale oil 200 mg/kg + 45% ethanol) 6 2.390.10
*
3.940.07
*
3.880.04
*
Group IV (C. longa oil 200 mg/kg + 45% ethanol) 6 2.030.06
*
3.510.16

4.030.42

Group V (Livolin Forte 200 mg/kg + 45% ethanol) 6 2.330.02


*
4.090.02
*
4.860.12
*
*
P < 0.05, vs Group I;

P < 0.05, vs Group II.


MDA: malondialdehyde; TC: total cholesterol; TG: triglyceride.
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In recent years, researchers have shown that a way of
preventing or delaying the pathogenesis of free radical-
mediated cellular injuries is to augment the oxidative
defense capacity of the cell through intake of antioxidants.
Sequel to this, much attention has been focused on the
health benefcial role of naturally occurring antioxidants
in biological systems. Phytochemicals derived from plants
are being considered to play an important role as nutraceuticals
and are being utilized for treatment and prevention of
metabolic diseases related to oxidative stress, even though
their modes of action may still not be fully elucidated
[28]
.
The results of the present study indicate that ethanol
treatment caused signifcant morphological abnormalities
in the liver, as were demonstrated by the appearance and
observed changes in the histoarchitecture of the liver sections.
This was confirmed by the noticeable elevation in the
serum transaminases and ALP activities in Group II rats
following administration of single dose of 45% ethanol
(4.8 g/kg) compared to the control rats (Group I). The
elevated levels of these biochemical parameters are a
direct reflection of alterations in the hepatic structural
integrity, possibly, a necrosis of hepatocytes that results in
the leakage of transaminases and the elevation of serum
ALP. The signifcantly decreased serum transaminases and
ALP activities in the groups pretreated with standard drug
(Livolin Forte) and oils from Z. offcinale and C. longa prior
to ethanol administration demonstrated their hepatopro-
tective ability
[29-31]
.
We noticed that ethanol administration resulted in a
signifcant elevation in the hepatic MDA level. Oxidative
injury induced by ethanol can be monitored in animal
models by measuring the extent of LPO. Ethanol administra-
tion results in excessive generation of free radicals such
as hydroxyethyl radical, superoxide radical, hydroxyl
radical, peroxyl radical and hydrogen peroxide. All these
radicals formed from the ethanol-mediated process have
a great potential to react rapidly with lipids which in turn
leads to LPO. It is generally accepted that the enhanced
LPO is one of the toxic manifestations of ethanol ingestion.
However, the signifcant decrease in the MDA level of the
treated groups, indicated the anti-fatty liver effects of oils
from Z. offcinale and C. longa
[32-34]
.
Disturbance observed in lipid metabolism in ALD is
often important in determination of the disease status. The
increase in serum concentration of TG may be attributed
to inhibition of cholesterol catabolism or mobilizations
of fatty acids from adipose tissues by lipolysis while
the increase in serum TG level may be due to increased
biosynthesis or dismissed clearance from the blood. Previous
works reported that chronic ethanol ingestion results
in hypercholesterolemia and hypertriglyceridemia and
increased concentration of lipids in liver. The increased
reduced form of nicotinamide adenine dinucleotid/
nicotinamide adenine dinucleotide ratio during alcohol
intake enhances the concentration of glycerophosphate,
which favors hepatic TG accumulation by trapping fatty
acids. However, these were significantly reduced in the
serum of pretreated rats
[35,36]
.
The reduction observed in hepatic antioxidant (GSH,
SOD and GST) status of ethanol-treated rats compared
with the control is related to oxidative stress and elevation
of LPO that resulted in the leakage of hepatic enzymes to
serum in the ethanol-treated rats. The endogenous GSH,
synthesized mainly in the liver, plays an important role in
Figure 1 Representative histological section of liver in acute toxicity (Light microscopy, 400; haematoxylin and eosin staining)
A: Group I, no visible lesions seen. B: Group II, there is marked portal congestion and cellular infltration by mononuclear cells. C: Group III, there
are periportal hydropic degeneration of hepatocytes with cellular infltration by mononuclear cells. D: Group IV, there are mild portal congestion and
mononuclear cellular infltration, and mild hydropic degeneration. E: Group V, there are mild portal congestion, mild hydropic degeneration and few
foci of cellular infltration by mononuclear cells.
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Journal of Integrative Medicine
the system of cell defence. It is involved in detoxifcation
of many xenobiotics through conjugation of toxic metabolites
in the second phase of biotransformation. The substrate of
SOD is the superoxide radical anion (O
2

) which is generated
by the transfer of one electron to molecular oxygen. This
is responsible both for the direct damage of biological
macromolecules and for generating other reactive oxygen
species. SOD keeps the concentration of superoxide radicals
at low levels and therefore plays an important role in the
defense against oxidative stress. Taken together, the pre-
treated groups generally show a level of protection and
restoration in hepatic antioxidant (GSH, SOD and GST)
status. Excessive generation of free radicals, caused by
ethanol metabolism, can be suppressed by enzymatic and
molecular antioxidants such as catalase, glutathione per-
oxidase, SOD, GST, and GSH
[37-40]
.
ORAC and FRAP are two of the most popular total
antioxidant capacity assays. Based on the results obtained
from this study, significant correlations exist between
ORAC and FRAP values obtained which may be responsible
for the ameliorative and antioxidant activities observed in
other biochemical parameters estimated.
5 Conclusions
The present study demonstrates that pretreatment of rats
for 28 d with oils from Z. offcinale and C. longa (200 mg/kg)
exhibits a signifcant hepatoprotective effect in acute ethanol-
induced fatty liver. This was ascertained by a comparative
analysis of the results obtained in rats pretreated with
oils from Z. officinale and C. longa and Livolin Forte

.
Histopathological findings further confirmed the
protective effect of the oils, with Z. offcinale oil identifed
to have better effects than C. longa oil.
6 Acknowledgements
We are grateful to the Antioxidant Research Laboratory,
Cape Peninsula University, Cape Town, Nigeria for the
FRAP and ORAC analysis of the oils.
7 Competing interests
The authors declare that they have no competing interests.
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