Pharmacological and Antioxidant Actions of Garlic and - or Onion in Non Alcoholic Fatty Liver Disease ( Nafld ) in Rats

Abstract

Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of fat-induced liver injury, ranging from mild steatosis to cirrhosis and liver failure. This study investigates the hepatoprotective properties of garlic and onion in NAFLD rat model. Ninety male Sprague-Dawley rats were randomly divided into 9 groups; normal (I), NAFLD induced with high fat diet (HFD; II), NAFLD switched to regular diet (RD; III), NAFLD-HFD or NAFLD-RD treated with garlic (IV, V), onion (VI, VII) or the combined garlic+onion (VIII, IX) respectively. A NAFLD rat model was established by feeding the animals with a high-fat diet for 12 wk. These animals were then treated with garlic or/and onion or vehicle for 8 wk (weeks 13-20) and then killed to obtain serum samples and liver tissues. Liver histology, lipids, parameters of oxidative stress, TNF-α and TGF-β were measured. The liver in NAFLD-HFD showed typical steatosis, accompanied with mild to moderate lobular inflammatory cell infiltration. Serum levels of ALT, AST, ALP, leptin, cholesterol, triglycerides, TNF-α, TGF-β and hepatic MDA' were significantly increased (P < 0.05) compared with normal group. This was accompanied with reduction of hepatic GSH, GR, GPx, GST, SOD and serum adiponectin. These changes were to a less degree in NAFLD-RD group. Combined administration of garlic+onion produced a better and significant decrease in liver steatosis, serum liver enzymes, oxidative markers and lipid peroxidation versus each one alone. In the same time, NAFLD-induced inflammation was also mitigated via reduction of TNF-α and TGF-β. In addition, these results were better in the group IX versus group VIII.

Full text

PHARMACOLOGICAL AND ANTIOXIDANT ACTIONS OF GARLIC AND/OR ONION
IN NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) IN RATS
By
SAYED H. SEIF EL-DIN
1
, ABDEL-NASSER A. SABRA
1
, OLFAT A. HAMMAM
2
,
FATMA A. EBEID
1
AND NAGLAA M. EL-LAKKANY*
1
.
Departments of
1
Pharmacology and
2
Pathology, Theodor Bilharz Research Institute, Warak El-
Hadar, Imbaba, P.O. Box 30, Giza 12411, Egypt.
*Corresponding author: E-mail: naglaaellakkany@yahoo.com
Abstract
Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of fat-induced liver injury,
ranging from mild steatosis to cirrhosis and liver failure. This study investigates the
hepatoprotective properties of garlic and onion in NAFLD rat model. Ninety male Sprague-
Dawley rats were randomly divided into 9 groups; normal (I), NAFLD induced with high fat diet
(HFD; II), NAFLD switched to regular diet (RD; III), NAFLD-HFD or NAFLD-RD treated with
garlic (IV, V), onion (VI, VII) or the combined garlic+onion (VIII, IX) respectively. A NAFLD
rat model was established by feeding the animals with a high-fat diet for 12 wk. These animals
were then treated with garlic or/and onion or vehicle for 8 wk (weeks 13-20) and then killed to
obtain serum samples and liver tissues. Liver histology, lipids, parameters of oxidative stress,
TNF-α and TGF-β were measured. The liver in NAFLD-HFD showed typical steatosis,
accompanied with mild to moderate lobular inflammatory cell infiltration. Serum levels of ALT,
AST, ALP, leptin, cholesterol, triglycerides, TNF-α, TGF-β and hepatic MDA were significantly
increased (P<0.05) compared with normal group. This was accompanied with reduction of
hepatic GSH, GR, GPx, GST, SOD and serum adiponectin. These changes were to a less degree
in NAFLD-RD group. Combined administration of garlic+onion produced a better and significant
decrease in liver steatosis, serum liver enzymes, oxidative markers and lipid peroxidation versus
each one alone. In the same time, NAFLD-induced inflammation was also mitigated via reduction
of TNF-α and TGF-β. In addition, these results were better in the group IX versus group VIII.
Conclusions; Garlic or onion alone could be considered as a potent food supplement in the
prevention of fatty liver disease. Combined administration of both supplements gave better results
than each one alone.
Keywords: Fatty liver; garlic; onion, leptin; adiponectin; TGF-β; TNF-α; liver enzymes and
antioxidants.
Introduction
Non-alcoholic fatty liver disease (NAFLD), deposition of fat in the liver due to causes other
than alcohol, includes non-alcoholic steatohepatitis (NASH). NASH is a more severe form of
NAFLD in which fatty infiltration of the liver is accompanied by necroinflammatory activity, and
is now recognized as one of the most common causes of chronic liver disease (Shifflet and Wu,
2009). NAFLD has also the potential to progress to hepatocellular carcinoma (HCC) or liver
failure. NAFLD is strongly linked to caloric overconsumption, physical inactivity, insulin
resistance and genetic factors. Although significant progress in understanding the pathogenesis of
NAFLD has been achieved in years, the primary metabolic abnormalities leading to lipid
accumulation within hepatocytes has remained poorly understood (Wei et al, 2008). Given the
increasing prevalence of obesity worldwide, the deleterious effects of NAFLD, and more
particularly NASH, are becoming of increasing concern for physicians (Shifflet and Wu, 2009).
The pathophysiology of NAFLD is described as a “two hit model”. The first hit is supposed
to be the increase of free fatty acids in hepatocytes, which results in a decrease of β-oxidation.
Down regulation of β-oxidation further aggravates accumulation of fatty acids and insulin

resistance. The second step includes all mechanisms contributing to the development of
inflammation and fibrosis (Day and James, 1998). In detail, increase of fatty acids enhances the
expression of cytochrome peroxidase 2E1 (CYP2E1). CYP2E1 stimulates generation of
proinflammatory cytokines, oxidative species and thereby enhances lipid peroxidation of the
hepatocyte membrane (Esterbauer et al, 1991; Osmundsen et al, 1991). NAFLD represents a
spectrum of disease from benign fatty liver to NASH and even cirrhosis. The disease seems to
progress through a two- or even multiple-hit process, with successive liver insults leading from
fatty infiltration to inflammation and fibrosis. The interplay of various adipokines, the most
important of which are leptin, adiponectin, tumor necrosis factor-alpha (TNF-α), has a key role in
this process (Tsochatzis et al, 2009). Emerging data suggest that apoptosis plays a critical role in
NAFLD-induced liver injury and in the progression from steatosis to NASH and cirrhosis
(Alkhouri et al, 2011). Moreover, the degree of apoptosis is closely associated with the severity
of NASH and the stage of fibrosis (Feldstein et al, 2003).
The role of natural foods in disease prevention has been studied extensively in recent years.
Among these natural foods, garlic has attracted a great deal of attention. Garlic (Allium sativum
L.) has long been used both for flavoring and for the potential benefits of preventing and curing
ailments in many cultures (Rivlin, 2001). Preclinical and clinical studies reveal a close
relationship between dietary habits and the occurrence of diseases. Previous studies have found
that ingestion of garlic is inversely related to the incidence of hyperlipidemia, atherosclerosis and
thrombosis (Agarwal, 1996). Like other Allium vegetables, Welsh onion (Allium fistulosum L.)
can be expected as a good source of flavonoids and of many kinds of sulfur compounds. These
components have recently been suggested as having beneficial medicinal properties, including
those on arteriosclerosis (Yamamoto and Yasuoka, 2010). However, few reports on the
hypolipidemic activity of Welsh onion have been reported. In this study, we established a
NAFLD animal model through feeding a high-fat diet, and investigated the individual and
combined action of garlic and onion on serum liver markers, proinflammatory cytokines, hepatic
steatosis, apoptosis, and oxidative stress.
Materials and methods
Animals: Ninety male Sprague-Dawley rats, average weight 120g ± 20, were bred and maintained
at the Schistosome Biology Supply Center (SBSC) of Theodor Bilharz Research Institute (TBRI),
Giza, Egypt. Animals were housed in a controlled temperature and light environment, and were
given water and commercial chow ad libitum. The animal experiments were conducted at the
animal unit according to the international ethical guidelines for the care and use of animals for
research purposes.
Experimental design: Ninety male Sprague-Dawley rats were randomly divided into 9 groups (10
rats each); normal control (I), NAFLD-induced with high-fat diet (HFD; II), NAFLD switched to
regular diet (RD; III), NAFLD-HFD or NAFLD-RD treated with garlic (IV, V), onion (VI, VII)
or the combined garlic+onion (VIII, IX) respectively. Rats in the control group were maintained
on the standard chow diet for 20 weeks. A rat model of NAFLD was induced by a high-fat diet
(25 % fats + 1 % cholesterol + 0.25 % bile salts) for 12 weeks (Zulet et al, 1999). After 12 weeks,
rats in the garlic/onion-treated NAFLD groups were treated with garlic (Tomex plus®; Atos
Pharma; Egypt) at a dose of 500 mg/kg b.w. or onion (onion oil, CAP Pharm; Egypt) at a dose of
100 mg/kg body b.w. via oral gavage, for 8 weeks (starting from the 13
th
week to the 20
th
week).
Rats in the NAFLD-RD treated groups were switched and maintained on the standard chow diet
(weeks 13-20). At end of the study, all rats were weighted and then sacrificed by decapitation
after 12h of fasting. Blood samples were collected for biochemical assays. The liver was
immediately removed and weighted after rinsed with ice-cold saline, and sampled for assessment

of liver glutathione antioxidant-related enzymes, MDA, and for histological study. Liver weight
index (%) was calculated as liver weight/body weight × 100.
Biochemical analyses: Blood biochemical parameters including triglyceride (TG), total
cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline
phosphatase (ALP) were assayed spectrophotometrically using the commercially available kits.
Serum TNF- and adiponectin (Assaypro, St. Louis, MO, USA), leptin (RayBiotech Inc.,
Norcross, GA, USA) and TGF-β levels (IBL International GMBH, Hamburg, Germany) were
measured using mouse enzyme-linked immunosorbent assay (ELISA) kits.
Assay of lipid peroxidation and oxidative stress: Liver tissue was homogenized in 4 volumes
(w/v) of ice-cold 100 mM KH
2
PO
4
buffer containing 1 mM EDTA, pH 7.4 and centrifuged at
10,000 g for one hour at 4 °C. The supernatant was collected and kept at −80 °C for subsequent
analysis for determination of liver levels of glutathione-related antioxidant enzymes and lipid
peroxidation. GSH content was measured by the method of Ellman (1959). GST activity was
measured using chlorodinitrobenzene (CDNB) as a substrate (Habig et al, 1974). Glutathione
reductase (GR) activity was assayed by using oxidized glutathione as a substrate according to the
method described by Zanetti (1979). Glutathione peroxidase (GPx) catalyzes the oxidation of
glutathione and its activity was measured based on the method described by Paglia and Valentine
(1967). Superoxide dismutase (SOD) activity was assayed spectrophotometrically by the
procedure of Winterbourn et al (1975). Degree of lipid peroxidation in the liver tissue
homogenates was determined in terms of thiobarbituric acid reactive substances (TBARS) which
were expressed by malondialdehyde (MDA) formation (Ohkawa et al, 1979).
Histological studies: The sections of liver tissues were fixed in 10% buffered formaldehyde, and
then embedded in paraffin wax. A 5 µm-thick section cut from a paraffin-embedded block was
stained with H&E and Masson’s trichrome. Steatosis was assessed by a morphological semi-
quantitative approach and graded as follows: mild= 5-30%, moderate= 30-60%, and severe >
60% of hepatocytes affected. The specimens were also examined for histological features
including; ballooning degeneration, hepatocytes with cytoplasmic vacuolation, acidophilic
necrosis, sinusoidal fibrosis and polymorph nuclear infiltration.
Immunohistochemical staining: Survivin polyclonal antibody was obtained from Santa Cruz
(Santa Cruz, CA, USA). Immunohistochemical staining of 5 µm-thick paraffin-embedded liver
sections was performed according to its manufacturer’s protocol (Vectastain Elite ABC kit;
Vector, Burlingame, CA, USA). Briefly, liver sections were deparaffinized in xylene and
rehydrated in graded ethanol. After endogenous peroxidase and biotin were blocked, the tissues
were preincubated with 3% horse serum for 30 min to prevent non-specific reactions. The
sections were then incubated with primary antibodies diluted at 1:150 for 60 min. On negative
control sections, the step with primary antibodies was omitted. Polyclonal antibodies were
detected using a biotinylated anti-goat or rabbit IgG diluted at 1:200 in 5% bovine serum albumin
for 30 min. The sections were incubated with R.T.U. Vectastain Elite ABC reagent for 30 min,
stained with diaminobenzidine for 5 min and counter stained with hematoxylin before they were
mounted. The liver sections were examined using a Zeiss light microscope (Oberkochen,
Germany). The number of positively stained cells with the highest expression recorded within 10
successive fields (x400) was counted per section ⁄ animal in a semi-quantitative way; the final
value represented the mean of 80 readings per group. Zero percentage was given to unstained
sections. Survivin expression sites (Baba et al, 2009) were examined intralobularly, in the
periportal areas, in hepatocytes, bile duct epithelium and monocytes.

Statistical analysis: All data were presented as means ± SD. Differences among groups were
assessed using one-way analysis of variance (ANOVA) followed by post-hoc LSD test. P-values
of less than 0.05 were considered to be statistically significant. Calculations were performed with
the SPSS, Version 16.0 statistical software package.
Results
Effects on hepatic gross manifestations: The liver weight and liver index in NAFLD-HFD or
NAFLD-RD groups were significantly increased (P<0.05) over those of normal control group.
However, the body weight of rats in both groups decreased significantly (P<0.05). Compared
with NAFLD-HFD or NAFLD-RD groups, the liver weight and liver index were significantly
decreased (P<0.05) in the groups treated with either garlic or onion alone or in combination
(Table 1).
Effects on hepatic biochemistry: Compared with normal control group, the serum levels of ALT,
AST, ALP, cholesterol, triglycerides, TNF-α, TGF-β1 and leptin in NAFLD-HFD group
increased significantly (P<0.05). These elevations were to a less extent in NAFLD-RD group. At
the same time, the level of adiponectine was significantly decreased (P<0.05). Compared with
NAFLD-HFD group, ALT, AST, ALP triglycerides, TNF-α and TGF-β1 serum levels decreased
significantly (P<0.05) in the groups treated with either garlic or onion alone. The serum levels of
cholesterol, leptin and TGF-β1 (in onion treated-group) also had decreasing tendency, but there
was no significant difference (P>0.05). Compared with NAFLD-RD group, the serum levels of
ALT, AST, ALP, cholesterol, triglycerides and TGF-β1 (in garlic group) were significantly
increased in the groups treated with either garlic or onion alone (Table 2 & Fig. 1). On the other
hand, serum level of adiponectine was insignificantly changed in either treated groups compared
with NAFLD-HFD or NAFLD-RD groups.
Effects on oxidative stress and lipid peroxidation: Compared with normal control group, the
hepatic GSH content and GST, GR, GPx and SOD activities in NAFLD-HFD group were
significantly decreased (P<0.05), meanwhile, MDA level was significantly increased. These
changes were to a less extent in NAFLD-RD group. Compared with NAFLD-HFD or NAFLD-
RD groups, administration of either garlic or onion alone or both significantly decreased the
effects of the oxidative stress and lipid peroxidation in the hepatic tissue by restoring GSH
content, increasing GST, GR, GPx, SOD activities and decreasing of MDA levels (Table 3).
In the garlic+onion combined group, all the measured serum and hepatic markers gave better
results over those of each treated group, weather maintained on HFD or switched to regular diet.
Moreover, the best results were obtained in all groups switched to regular diet.
Histological evaluation: After 20 weeks, in normal control group (Fig. 2A), the liver lobules were
distinct (preserved lobular architecture), the liver cell cords arranged regularly, whereas, the liver
in NAFLD-HFD group (Fig. 2B) showed typical steatosis (65.0±2.69, micro and macrosteatotic
changes), lobular infiltration (22.5±2.01) by mononuclear cell and lymphocyte, hepatocytes with
cytoplasmic vacuolation (23.5±2.36), fat droplets accumulation, spotty necrosis and focal
necrosis. These findings suggest that the animal model of NAFLD was successfully established.
However, the degree of hepatic injury including steatosis, hepatocytes with cytoplasmic
vacuolation and lobular inflammation were to a less degree in NAFLD-RD group (30.1±6.54,
18.0±3.43 and 15.3±3.39 respectively; Table 4 & Fig. 2C). Treatment of NAFLD-induced rats
with either garlic or onion alone or both, maintained on HFD or RD respectively, markedly
attenuated steatosis (mean score: 20.0±3.57, 9.0±2.77, 42.0±6.96, 7.5±2.39, 12.5±3.59 and
2.5±0.83), hepatocytes with cytoplasmic vacuolation (mean score: 15.0±2.11, 4.0±1.45,

13.5±2.59, 7.0±2.49, 7.0±3.0 and 2.5±0.83) and inflammation (mean score: 9.5±1.89, 3.0±0.82,
8.5±0.76, 2.5±0.83, 3.5±1.07 and 1.5±0.76) 8 weeks post treatment (Fig. 2D-I). In the
garlic+onion combined group, steatosis and extension of lobular inflammation were relieved
significantly over those of each treated group, and in all NAFLD-RD groups over those groups
maintained on HFD (Table 4; Fig. 2H & I).
Immunohistochemical aspects: The control normal rats were negative for survivin polyclonal
antibody (Table 4 & Fig. 3A), meanwhile, NAFLD induced rats showed positively survivin-
stained hepatocyte cells indicating the presence of survivin cells (Fig. 3B). Switching to RD from
HFD reduced the percentage of hepatocytes expressing survivin (34.0±3.06 vs 43.0±1.86
respectively, Fig. 3C). Treatment of NAFLD-induced rats with either garlic or onion alone,
maintained on HFD or RD, markedly reduced the percentage of hepatocytes expressing survivin
(19.0±2.77, 7.5±2.39 for garlic, 26.0±3.93 and 8.5±3.17 for onion, respectively, Fig. 3 D-G). The
highest reduction in the expression of survivin was recorded in the group treated with both garlic
and onion, maintained on HFD or RD (11.0±3.48 and 5.5±1.57 respectively, Table 4 & Fig. 3H &
I).
Discussion
NAFLD represents a wide spectrum of disorders, the hallmark of which is hepatic steatosis.
NAFLD was considered a benign condition, but is now increasingly recognized as a major cause
of liver-related morbidity and mortality (Xiao et al, 2013). Insulin resistance is the basis for
accumulation of free fatty acids and triglyceride storage in hepatocytes or steatosis. Oxidative
stress from steatotic hepatocytes leads to lipid peroxidation, impaired mitochondrial and
peroxisomal oxidation of fatty acids, and cytokine release (Parekh and Anania, 2007). Endotoxins
and endotoxin-inducible cytokines, particularly TNF-α, are required for the pathogenesis of
NAFLD in experimental animals. Therefore, the TNF-α plays an important role in NAFLD (Day,
2006). Garlic (Allium sativum L.) has a wide range of pharmacological effects including
antimicrobial, cardiovascular, anti-inflammatory, anticancer, and immunomodulatory activity
among many other effects. Onions (Allium cepa L.) might be useful for preventing obesity-related
breast, colorectal, laryngeal, and ovarian cancers (Galeone et al, 2006; Wang et al, 2012). To our
knowledge, this is the first report investigating the pharmacological effects of garlic and onion
combination either maintained on HFD or switched to RD in a NAFLD-rat model.
In this study, a rat model of NAFLD induced by high fat diet was established. Decreased
body weight, increased liver weight and liver weight index, elevated serum liver enzyme levels
and altered liver histological conditions including steatosis, inflammation and fibrosis were
observed in the NAFLD-HFD rats, and to a less extent in NAFLD-RD rats. These abnormalities
were significantly improved 8 weeks after treatment with either garlic or onion or both. Body
mass significantly decreased with the high-fat diet and dietary restriction. This may be due to a
metabolic imbalance of carbohydrate, protein, and fat. However, hepatic index was higher in the
group with steatosis as compared to the garlic or onion-treated and control groups, which means
that garlic or onion acts by decreasing fat accumulation in the liver and fat weight, and therefore
decreases hepatic index. In our study, the elevated transaminase serum levels correlated strongly
with NAFLD, are the result of leakage from damaged cells, therefore, reflect hepatocyte damage
(Gholam et al, 2007). In agreement with previous reports (Assy et al, 2006), the present study
also showed that the levels of serum triglycerides, cholesterol, leptin as well as TNF-α and TGF-
β1 were significantly increased, whereas the levels of serum adiponectin remarkably decreased in
the NAFLD group. These abnormalities were significantly improved, and hepatic steatosis was
significantly decreased in all treated groups. Kuda et al (2004) mentioned that garlic has anti-
hyperlipidemic, hypocholesterolaemic and hypo triacylglyceride activities. Garlic may exert a

lipid-lowering effect partly through reducing intestinal microsomal triglyceride transfer protein
(MTP) gene expression, thus suppressing the assembly and secretion of chylomicrons from
intestine to the blood circulation (Lin et al, 2002) or due to an increase in cholesterol degradation
to bile acids and neutral sterols and mobilization of triacyl glycerols in treated rats (Kempaiah and
Srinivasan, 2006). Moreover, Rai et al (2009) and Lii et al (2012) mentioned that garlic's
organosulfur compounds (such as diallyl trisulfide) display hypolipidemic effects by inhibiting
fatty acids and cholesterol synthesis.
Oxidative stress is believed to play an important role in pathogenesis of NAFLD. It is likely
to be involved in disease progression from steatosis to steatohepatitis and potentially cirrhosis. It
has been shown that chronic oxidative stress, generated through oxidation of cytotoxic free fatty
acids, may lead to cytokine upregulation and depletion of hepatic antioxidant levels (Garcia-Ruiz
et al, 1995). In addition, enhanced lipid peroxidation leads to the generation of by-products, such
as MDA, which have been shown to further stimulate cytokine production. They are involved in
hepatic stellate cells activation, fibrogenesis, and enhanced extracellular matrix protein deposition
(Thong-Ngam et al, 2007). In this study, treatment of NAFLD-HFD or RD groups with garlic
and/or onion restored the depleted hepatic antioxidant activities as GSH, SOD, GR, GST and GPx
and decreased MDA levels in the liver. The lowered activities of hepatic antioxidant enzymes in
hypercholesterolemic rats were effectively countered by garlic (Kempaiah and Srinivasan, 2004).
Gorinstein et al (2006) reported that dietary garlic was effective in reducing the oxidant stress,
which was indicated by an increase of antioxidant activity and a decrease of lipids in the rats'
blood. Onion bulbs contain more than 20 flavonoids other than quercetin (Slimestad et al, 2007).
According to Jung et al (2011), Onion peel extract (OPE) was found to be composed of
polyphenols at the level 60%, of these, 16% was quercetin. Therefore, it was postulated that the
higher potency of OPE might be attributed to the additive or synergistic effect of an array of
phytochemicals. The antioxidant activity of Allium spp. has been attributed mainly to a variety of
sulphur-containing compounds and their precursors (Nishimura et al, 2004). Scientific evidence
shows that allicin, diallyl disulphide and diallyl trisulphide appeared to be the main antioxidative
compounds (Kim et al, 1997). In addition, the antioxidant activity is also related to other
bioactive compounds: dietary fibers, microelements (especially Selenium) and polyphenols
(Lanzotti, 2006). Nencini et al (2010) reported that that fresh Allium homogenates (leaves or
bulbs) possess antioxidant properties and provide protection against ethanol-induced liver injury.
Helen et al (2000) reported that onion oil is an effective antioxidant against the oxidative damage
caused by nicotine as compared to vitamin E. In previous studies, the authors reported that outer
dry layers of onion bulb have strong anti-inflammatory and antioxidant activities and proposed
quercetin as the major component responsible for this activity (Coskun et al, 2005; Park et al,
2007).
Apoptosis is important for the regulation of many physiological and pathological processes.
Apoptosis signaling pathway can be abnormally activated in various pathological processes in the
liver, including acute and chronic hepatitis, alcohol-induced liver disease, non-alcoholic fatty
liver disease, cholestatic liver disease, fibrogenesis, liver cirrhosis and liver carcinogenesis. In
NAFLD, Inflammatory response, oxidative stress and apoptosis serve as “following-hits” that
contribute to the ongoing inflammation (NASH). Emerging data suggest that apoptosis plays a
critical role in NAFLD-induced liver injury and in the progression from steatosis to NASH and
cirrhosis (Alkhouri et al, 2011). Liver regeneration is a complex process involving both
proliferation and apoptosis. Survivin is a fascinating little protein that acts as a component of the
chromosomal passenger complex, which is essential for cell division, and as an inhibitor of
apoptosis. With dual roles in promoting cell proliferation and preventing apoptosis, it is
considered a protein that interfaces life and death (Wheatley and McNeish, 2005). In this study,
different treatment regimens tested decreased the expression of survivin stained cells indicating

the increase of apoptosis in hepatocyte cells. The maximum reduction was recorded in animals
receiving both garlic and onion in combination, either maintained on HFD or RD (11.0±3.48 and
5.5±1.57 respectively). These results are in agreement with those of Colín-González et al (2012)
and Wang et al (2012) who reported that garlic and onion stimulate inhibition of cell proliferation
and increase of apoptosis. The underlying mechanisms for this phenomenon require further
investigations.
In this study, combined administration of garlic and onion has an enhancement effect in
modulating the biochemical and histological markers related to NAFLD disease compared to
either administration alone. This may be due to their synergistic antioxidative, anti-inflammatory
and apoptotic effects. Organosulfur compounds present in garlic and onion are the most important
contents responsible for most of their pharmacological effects. It is evident that A. sativum may
exert toxicity only at high doses and that there have been few reports of intoxications following
the ingestion of garlic (Mikaili et al, 2013). In conclusion, the current study shows for the first
time that combined administration of garlic and onion has a modulating effect on the extent of
fatty infiltration and on lipid peroxidation compared with the effect of either drug alone. Inducing
apoptosis together with decreasing leptin, TGF-β1, TNF-α and reduction of oxidative stress
remains the best targeted treatment to date for NAFLD. Since garlic and onion are common food
supplements around the world, we believe that garlic and onion or their derivatives could be
considered as one of the preventive measures in the treatment strategy of NAFLD. Further studies
are needed to confirm the safety and quality of the plants to be used by clinicians as therapeutic
agents.
Acknowledgment
This work was supported by the internal research project 95/A for basic and applied research, a
grant from Theodor Bilharz Research Institute.
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b
a
a
a
b
a
a
0
10
20
30
40
50
60
70
Normal NAFLD
Control Garlic Onion Garlic &
Onion
Adiponectine (ng/ml).
HFD RD
b
a
a
a
b
a
a
0
1
2
3
4
5
6
Normal NAFLD
Control Garlic Onion Garlic &
Onion
Leptin (pg/ml).
HFD RD
b
ab
ab
a
a
0
2
4
6
8
10
12
Normal NAFLD
Control Garlic Onion Garlic &
Onion
TNF-alpha (pg/ml).
HFD RD
b
a
ab
a
b
a
b
a
0
20
40
60
80
100
120
Normal NAFLD
Control Garlic Onion Garlic &
Onion
TGF-beta (pg/ml).
HFD RD
Fig. 1: Effect of garlic and/or onion on the cytokines; adiponectine, leptin, TNF-α
αα
α and
TGF-β
ββ
β in NAFLD-induced rats.
Values are presented as mean±SD. Number of animals in each group= 10
a
Significant difference from normal at P<0.05.
b
Significant difference from the relative NAFLD control (HFD or RD) at P<0.05.

Fig. 2 (A-I): Liver sections from; normal untreated rats (A) showing the liver lobules were distinct (preserved lobular architecture), the
liver cell cords arranged regularly, NAFLD-HFD rats (B) showing lost hepatic architecture, macro (black arrow) and micro (red arrow)
35% steatotic changes, many hepatocytes with cytoplasmic vacuoles (yellow arrow), and to a less extent in NAFLD-RD rats (C), garlic-
HFD-treated rats (D) showing preserved hepatic architecture, macro (black arrow) and micro (red arrow) 8% steatotic changes, scattered
lymphocytes between hepatocytes (yellow arrow), garlic-RD-treated rats (E) showing normal hepatic architecture, liver appear within
normal limit, onion-HFD-treated rats (F) showing preserved hepatic architecture, macro (black arrow) and micro (red arrow) 15%
steatotic changes, scattered lymphocytes between hepatocytes (yellow arrow), onion-RD-treated rats (G) showing preserved hepatic
architecture, micro (red arrow) 3% steatotic changes, scattered lymphocytes between hepatocytes (yellow arrow), garlic+onion-HFD-
treated rats (H) showing preserved hepatic architecture, micro (red arrow) 5% steatotic changes, scattered lymphocytes between
hepatocytes (yellow arrow) and garlic+onion-RD-treated rats (I) showing normal hepatic architecture, liver appear within normal limit (H
& E, x100).
A
B
D
E
F
G
H
I
C

Fig. 3 (A-I): Liver sections from; normal untreated rats (A) were negative for survivin polyclonal antibody, NAFLD-
HFD rats (B) showing moderate cytoplasmic expression of polyclonal antibody survivin (50%) as brownish stains
(arrows), and to a less extent in NAFLD-RD rats (C), garlic-HFD-treated rats (D) showing mild cytoplasmic expression
of polyclonal antibody survivin (20%) as brownish stains (arrow), garlic-RD-treated rats (E) showing hepatocytes
negative for polyclonal antibody surviving, onion-HFD-treated rats (F) showing moderate cytoplasmic expression of
polyclonal antibody survivin (50%) as brownish stains (arrow), onion-RD-treated rats (G) showing scattered
hepatocytes with cytoplasmic expression of polyclonal antibody survivin as brownish stains (arrow), garlic+onion-
HFD-treated rats (H) showing mild cytoplasmic expression of polyclonal antibody survivin (20%) as brownish stains
(arrow) and garlic+onion-RD-treated rats (I) showing faint scattered cytoplasmic expression of polyclonal antibody
survivin (arrow) (IHC, DAB, x400).
A
B
D
E
F
G
H
I
C

Table 1: Effect of garlic and/or onion on liver gross
manifestations in NAFLD-induced rats.
Body weight Liver weight Liver weight
index (%)
Normal
219.0±35.18 5.71±1.13 2.60±0.26
NAFLD Control
117.8±29.01
a
9.01±2.16
a
7.66±0.48
a
Garlic
150.1±34.51
ab
7.02±1.54
ab
4.74±0.72
ab
Onion
139.4±18.36
a
7.51±1.38
a
5.37±0.54
ab
HFD
Garlic & Onion
179.0±25.69
abcd
6.51±0.98
b
3.64±0.18
abcd
NAFLD Control
135.0±29.25
a
7.14±1.62
a
5.31±0.74
a
Garlic
161.5±24.73
ab
7.70±1.30
a
4.76±0.31
ab
Onion
149.8±37.21
a
6.91±1.26
a
4.72±0.65
a
RD
Garlic & Onion
195.5±21.79
bcd
6.03±1.12
c
3.08±0.39
abcd
Values are presented as mean±SD. Number of animals in each group= 10
a
Significant difference from normal at P<0.05.
b
Significant difference from NAFLD control at P<0.05.
c
Significant difference from garlic at P<0.05.
d
Significant difference from onion at P<0.05.
Table 2: Effect of garlic and/or onion on liver enzymes and lipid profile in NAFLD-
induced rats.
ALT
(U/L) AST
(U/L) ALP
(IU/L) Cholesterol
(mg/dl) Triglycerides
(mg/dl)
Normal
17.40±2.27 64.90±14.11 113.58±18.45 17.70±4.66 24.56±5.98
NAFLD Control 76.90±14.49
a
157.00±21.48
a
248.67±29.02
a
95.95±29.52
a
111.03±26.16
a
Garlic
44.20±9.84
ab
102.60±10.08
ab
166.00±20.79
ab
82.27±12.46
a
75.53±12.62
ab
Onion
47.70±8.11
ab
108.00±10.68
ab
165.94±41.94
ab
87.40±18.01
a
82.25±15.11
ab
HFD
Garlic & Onion
36.10±6.19
abd
89.40±9.00
abcd
156.55±21.25
ab
63.41±5.74
abcd
62.39±16.81
abd
NAFLD Control 61.40±16.89
a
119.30±13.32
a
204.40±19.15
a
83.82±22.12
a
90.83±19.59
a
Garlic
38.70±10.87
ab
96.80±10.77
ab
129.40±16.31
b
61.92±7.43
ab
65.40±12.10
ab
Onion
43.80±7.18
ab
102.10±9.73
ab
133.19±16.80
b
68.42±21.07
ab
78.61±13.42
a
RD
Garlic & Onion
27.50±5.19
abcd
78.60±8.51
abcd
117.70±10.26
b
53.13±7.77
abd
49.29±9.54
abcd
Values are presented as mean±SD. Number of animals in each group= 10
a
Significant difference from normal at P<0.05.
b
Significant difference from NAFLD control at P<0.05.
c
Significant difference from garlic at P<0.05.
d
Significant difference from onion at P<0.05.

Table 3: Effect of garlic and/or onion on glutathione-related antioxidant enzymes and
lipid peroxidation in NAFLD-induced rats.
GSH
(umole/g
liver)
GR
(umole/min/g
liver)
GST
(umole/min/g
liver)
GPx
(umole/min/g
liver)
SOD
(umole/min/g
liver)
MDA
(nmol/g liver)
Normal
5.35±0.52 2.51±0.70 55.26±13.59 3.42±0.90 676.00±51.31 5.67±0.28
NAFLD Control
3.40±0.27
a
1.32±0.58
a
23.60±10.95
a
1.77±0.58
a
351.70±25.27
a
28.06±4.09
a
Garlic
3.89±0.39
ab
1.84±0.49
ab
38.55±10.46
ab
2.67±0.50
ab
452.40±89.49
ab
18.70±2.06
ab
Onion
3.73±0.30
a
1.79±0.47
ab
34.66±7.46
ab
2.71±0.49
ab
430.00±75.66
ab
18.89±2.06
ab
HFD
Garlic & Onion
4.19±0.24
abd
2.12±0.47
b
44.23±11.34
abd
2.91±0.62
b
544.10±63.52
abcd
11.98±2.68
abcd
NAFLD Control
3.68±0.44
a
1.63±0.56
a
32.79±7.22
a
2.56±0.54
a
434.70±64.52
a
16.17±1.67
a
Garlic
4.19±0.25
ab
1.94±0.47
a
41.75±11.62
a
2.77±0.60
a
473.30±85.56
a
13.44±2.52
ab
Onion
4.28±0.35
ab
1.77±0.58
a
37.21±8.26
a
2.82±0.60
a
457.30±84.79
a
14.11±2.66
a
RD
Garlic & Onion
4.71±0.47
abcd
2.29±0.46
bd
48.67±10.03
bd
3.11±0.48
b
581.30±69.53
abcd
9.68±1.63
abcd
Values are presented as mean±SD. Number of animals in each group= 10
a
Significant difference from normal at P<0.05.
b
Significant difference from NAFLD control at P<0.05.
c
Significant difference from garlic at P<0.05.
d
Significant difference from onion at P<0.05.

Table 4: Effect of garlic and/or onion on histological parameters in NAFLD-induced rats.
Hepatic architecture
Steatosis Type of steatosis Inflammation Hepat
ocytes with
cytoplasmic
vacuolation Fibrosis Survivin expression
Percentage of positive
stained with survivin
group
Preserved Lost % of cells
Microvesicular
Macrovesicular
% of inflamed
cells in 10
microscopic
fields
% of cells in 10
microscopic
fields None Present
% of positive
cells
Intensity of
stain in 10
HMF
Control-HFD 0/10 10/10 65.0±2.69 4/10 6/10 22.5±2.01 23.5±2.36 0/10 10/10 43.0±1.86 50%
moderate
50% marked
Control-
HFD/RD 6/10 4/10 30.1±6.54 5/10 5/10 15.3±3.39 18.0±3.43 4/10 6/10 34.0±3.06 60%
moderate
40% marked
Garlic-HFD 6/10 4/10 20.0±3.57
a
5/10 5/10 9.5±1.89
a
15.0±2.11
a
4/10 6/10 19.0±2.77
a
30%
moderate
70% mild
Garlic-
HFD/RD 10/10 0/10 9.0±2.77b
7/10 3/10 3.0±0.82
b
4.0±1.45
b
8/10 2/10 7.5±2.39
b
100% mild
Onion-HFD 4/10 6/10 42.0±6.96
a
5/10 5/10 8.5±0.76
a
13.5±2.59
a
2/10 8/10 26.0±3.93
a
80%
moderate
20% marked
Onion-
HFD/RD 8/10 2/10 7.5±2.39
b
6/10 4/10 2.5±0.83
b
7.0±2.49
b
6/10 4/10 8.5±3.17b 90% mild
10%
moderate
Garlic+Onion-
HFD 10/10 0/10 12.5±3.59
ce
5/10 5/10 3.5±1.07
ce
7.0±3.0
c
10/10 0/10 11.0±3.48
e
70% mild
30%
moderate
Garlic+Onion-
HFD/RD 10/10 0/10 2.5±0.83
d
5/10 5/10 1.5±0.76 2.5±0.83 10/10 0/10 5.5±1.57 50% mild
50%
moderate
Data are presented as mean ± SEM. Number of rats in each group= 10.
a
significant difference from NAFLD-HFD at P<0.05.
b
significant difference from NAFLD-RD at P<0.05.
c
significant difference from Garlic-HFD at P<0.05.
d
significant difference from Garlic-HFD/RD at P<0.05.
e
significant difference from Onion-HFD at P<0.05.