Fresh Garlic Extract Protects The Liver Against Acetaminophen-Induced Toxicity

Abstract

Acetaminophen toxicity is a major course of acute liver failure. Many plants have been reported to show hepatoprotective activities. This study was to determine the protective potential of fresh garlic extract on acetaminophen toxicity and to demonstrate its dose dependence. Sixty Swiss mice were divided into six groups of ten. Group I served as negative control and were treated with physiological saline. Group II served as the positive control and received 250 mg/kg body weight of acetaminophen only. Groups III, IV, and V were pretreated with daily administration of 250 mg/kg, 500 mg/kg, and 750 mg/kg of garlic respectively, for five days followed by 250 mg/kg of acetaminophen. Group VI were pretreated with 25 mg/kg silymarin prior to 250 mg/kg acetaminophen. Blood samples were collected after six hours of acetaminophen and used for biochemical studies, while liver was excised from each mouse and used to prepare hematoxylin / eosin sections. The alterations in AST, ALT, alkaline phosphatase, and serum albumin were significantly prevented by prior administration of garlic extract. Also the histological changes induced by acetaminophen overdose were prevented by garlic. We conclude that fresh garlic extract protected the liver against toxic doses of acetaminophen and suggest that its use could protect against hepatitis.

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ISPUB.COM The Internet Journal of Nutrition and Wellness
Volume 7 Number 1
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Fresh Garlic Extract Protects The Liver Against
Acetaminophen-Induced Toxicity
C Ezeala, I Nweke, P Unekwe, I El-Safty, E Nwaegerue
Citation
C Ezeala, I Nweke, P Unekwe, I El-Safty, E Nwaegerue. Fresh Garlic Extract Protects The Liver Against Acetaminophen-
Induced Toxicity. The Internet Journal of Nutrition and Wellness. 2008 Volume 7 Number 1.
Abstract
Acetaminophen toxicity is a major course of acute liver failure. Many plants have been reported to show hepatoprotective
activities. This study was to determine the protective potential of fresh garlic extract on acetaminophen toxicity and to
demonstrate its dose dependence. Sixty Swiss mice were divided into six groups of ten. Group I served as negative control and
were treated with physiological saline. Group II served as the positive control and received 250 mg/kg body weight of
acetaminophen only. Groups III, IV, and V were pretreated with daily administration of 250 mg/kg, 500 mg/kg, and 750 mg/kg of
garlic respectively, for five days followed by 250 mg/kg of acetaminophen. Group VI were pretreated with 25 mg/kg silymarin
prior to 250 mg/kg acetaminophen. Blood samples were collected after six hours of acetaminophen and used for biochemical
studies, while liver was excised from each mouse and used to prepare hematoxylin / eosin sections. The alterations in AST,
ALT, alkaline phosphatase, and serum albumin were significantly prevented by prior administration of garlic extract. Also the
histological changes induced by acetaminophen overdose were prevented by garlic. We conclude that fresh garlic extract
protected the liver against toxic doses of acetaminophen and suggest that its use could protect against hepatitis.
INTRODUCTION
Hepatitis is a major public health problem in the world today
and acetaminophen overdose contributes significantly to
cases of drug induced hepatitis. 1,2 Acetaminophen is quite
safe and well tolerated in therapeutic doses. However, at
toxic doses, acetaminophen produces acute liver failure
characterized by centrilobular necrosis in both man and
experimental animals. 3 This has been attributed to the
metabolic activation of acetaminophen to a toxic metabolite,
N-acetyl-p-benzoquinone imine (NAPQI) in the liver by
cytochrome p450 isoenzymes especially CYP2E1. 4 NAPQI
reportedly depletes liver glutathione thereby inducing
oxidative stress. It also binds to vital cellular and
mitochondrial proteins leading to cellular necrosis, and it
could activate cells of the immune system leading to the
release of pro-inflammatory cytokines. 5,6
Many natural products have been reported to possess
antioxidant and hepatoprotective effects in animal models of
hepatotoxicity and quite a good number are employed in folk
medicine for the treatment of liver diseases. Currently, many
plant extracts have been studied, and some have received
approval or are undergoing clinical trial for use in liver
related conditions. 7,8,9,10
Garlic is widely consumed in many cultures as spice and as
condiment in many dishes. Several cultures use garlic for
medicinal purposes. A plethora of publications are also
available on the pharmacological properties of garlic and
their beneficial health effects. 11,12,13,14,15 Although many
studies have focused on the antioxidant and anti-lipid
peroxidative properties of garlic extracts, many of these
studies employed aged garlic extract (AGE) that is known to
have a different phytochemical composition from fresh
garlic, and just a few reports investigated the actions of
garlic oil. 16,17 To our knowledge, no study has investigated
the effect of fresh extract of local Ugandan cultivars of garlic
on acetaminophen hepatotoxicity. In other to provide crucial
information that is needed for the possible development of a
prophylactic strategy for drug induced hepatitis, this study
was designed to determine the usefulness of ethanolic extract
of fresh garlic bulbs in preventing acetaminophen induced
hepatotoxicity in mice. Specifically, we investigated its
usefulness in preventing acetaminophen induced changes in
serum biochemistry and liver histology, and dose
dependence of these effects.
MATERIALS AND METHODS
A. PLANT COLLECTION, IDENTIFICATION, AND

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EXTRACTION
Local cultivars of garlic (Allium sativum L.) were purchased
from a local market in Ishaka Town in Western Uganda, and
identified by a qualified taxonomist, Professor Samson
Bitawha of the Rukararwe Partnership Workshop for Rural
Development, Bushenyi. A voucher specimen number
KIU/PHM/0026 was deposited at the Herbarium of the
Pharmacy Department of the Kampala International
University, Western Campus.
Cold extraction of the garlic was carried out at room
temperature (18-22 ° C) as follows:
Fresh garlic bulbs were ground to a fine paste using a
mechanical grinder.
50 g of the paste was put in a 250 ml conical flask and
covered with about 100 ml of 80 % ethanol, stoppered with
cotton wool, and allowed to stand in the dark at room
temperature for 48 hours.
The ethanolic extract was filtered off into pre-weighed
evaporating dishes, while the residue in the flask was
washed with a further 100 ml of 80 % ethanol and added to
the extracts in the evaporating dishes.
The filtrates were then evaporated to a syrupy residue using
a rotary extractor at 40 ° C.
The dishes were then weighed again on a triple beam
balance and the percentage yield was calculated as follows:
Weight of extract = weight of evaporating dish after
evaporation – weight of dish before addition of extract;
Percentage yield = total weight of extract ÷ weight of paste
used (50 g) × 100.
the extracts were pooled together into an air-tight container
and stored refrigerated (-4 ° c) until required for use.
For use, a portion of the extract was weighed and dissolved
in normal saline solution. Fresh preparations were made on
each day of the experiment. The resulting solutions were
injected intraperitonially into the mice.
B. LABORATORY ANIMALS
Swiss mice 6-8 weeks old weighing 18-32 g were purchased
from the Pharmacology Department of the Mbarara
University of Science and Technology in Uganda. They
were maintained and habituated in plastic cages in the
animal house of the School of Health Sciences, Kampala
International University, Western Campus for one week
before use. The mice had free access to water and were fed
standard rodent pellets (purchased from a local commercial
supplier) ad libitum. Habituation conditions were 12 hr
dark/light cycles, and average environmental temperature of
20 ° C.
C. ACUTE TOXICITY TEST AND
DETERMINATION OF LD
The LD50 of each of the extracts was determined in the mice
by the procedure described by Bernas et al. (2004), 18 and the
confidence interval of the LD50 was estimated by the
Litchfield – Wilcoxon method (1949). 19
D. EXPERIMENTAL DESIGN
Sixty Swiss mice were grouped randomly into 6 groups of
10 each and administered with the drugs/extract as follows:
Group I (10 mice): given physiological saline i.p. only.
Group II (10 mice): given acetaminophen 250 mg/kg i.p.
single dose only.
Group III (10 mice): given garlic extract 250 mg/kg for 5
days before a single i.p dose of acetaminophen 250 mg/kg.
Group IV (10 mice): given garlic extract 500 mg/kg for 5
days before a single i.p dose of acetaminophen 250 mg/kg.
Group V (10 mice): given Garlic extract 750 mg/kg for 5
days before a single i.p dose of acetaminophen 250 mg/kg.
Group VI (10 mice): given Sylimarin 25 mg/kg for 5 days
before a single i.p dose of acetaminophen 250 mg/kg.
The extract was administered as a single once daily dose,
while acetaminophen was administered after 12 hours fast
after the last dose of garlic extract.
The study received approval from the ethics committee of
the Kampala International University, Western Campus,
Bushenyi Uganda, as well as the Postgraduate Committee of
the College of Medicine, Abia State University, Uturu,
Nigeria.
E. COLLECTION OF SAMPLES
a) Blood Samples: Blood samples were collected by cardiac
puncture into sterile plastic tubes under ether anaesthesia
exactly six hours after acetaminophen administration, and
used for biochemical studies. The blood samples were
allowed to clot and retract at room temperature, and the sera
were separated into plastic vials and stored in the freezer

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(-20 ° C) until required for use. For the biochemical assays,
the sera were allowed to thaw at room temperature and
mixed by repeated inversion of the vials.
b) Liver Samples: After sacrifice, livers were obtained from
the mice and after physical examination they were fixed in
10 % formal saline (1 part of tissue to 10 parts of fixative).
Tissue samples were processed by wax impregnation and
embedding, and 5 uM sections cut using a rotary microtome.
Staining was by Haematoxylin & Eosin method.
F. BIOCHEMICAL ASSAYS
Assays for serum aspartate transaminase (AST) and alanine
transaminase (ALT) was carried out with Randox kits
(Randox Laboratories UK) and based on the method of
Reitman and Frankel. 20 Serum Alkaline Phosphatase was
determined by the method of Armstrong and King. 21 Serum
albumin was determined by reaction with BCG using
Randox kits. Manufacturer's procedures in the kits were
diligently followed.
G. STATISTICAL ANALYSIS
Results were expressed as mean ± SE. Comparison of means
was by the student's t-test and the Mann-Whitney U test. A p
value ? 0.05 is considered significant.
RESULTS
Extraction of the fresh garlic bulbs yielded 22 % extract. The
LD50 of the extract was determined to be 1524 +/- 83
mg/Kg body weight. Administration of acetaminophen to the
untreated mice (group 2) resulted in a marked escalation of
serum transaminases and alkaline phosphatase levels and a
concomitant decrease in serum albumin values. However,
treatment of the mice with the fresh garlic extracts for five
days produced significant dose dependent prevention of
these biochemical changes that is similar to that produced by
silymarin, as shown in table 1 and figure 1.
Histological examination also showed characteristic
pathological changes in the group 2 mice that received only
acetaminophen. These include centrizonal necrosis, steatosis,
leukocyte infiltration, portal triaditis, and evidence of edema
as shown in figure 2 slides. Pretreatment of the mice in
groups 3 – 5 demonstrated dose dependent prevention of
these changes. Mice in groups 3 and 4 showed mild triaditis
and mild venous congestion, whereas those in group 5 and 6
showed no remarkable changes as shown in figures 4-6.
Figure 1
Table 1: Biochemical values from the mice in the six groups
Figure 2
Figure 1: Serum AST, ALT, Alkaline Phosphatase, and
albumin from the mice in the six study groups

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Figure 3
Figure 2: Section from mice in group 1. Normal hepatocytes
arranged in trabecular pattern.
Figure 4
Figure 3: Sections from mice in group 2 showing extensive
hepatocyte necrosis and centrizonal necrosis.
Figure 5
Figure 4:Mild portal triaditis in a section from group 4 mice.
Figure 6
Figure 5:A section from group 5 mice showing no
remarkable changes except for mild venous congestion.
Figure 7
Figure 6: A section from group 6 mice showing no
remarkable changes.
DISCUSSION
This study has shown that pretreatment of mice with fresh
ethanolic extract of garlic prevented the escalation of serum
liver enzymes such as AST, ALT, and alkaline phosphatase,
and the decrease in serum albumin that are usually
associated with acetaminophen hepatotoxicity. Similarly, the
characteristic histological changes associated with
acetaminophen toxicity such as centrizonal necrosis,
steatosis, and sinusoidal enlargement, were significantly
prevented by the garlic extract in a dose-dependent manner.
This adds to several reports on the pharmacological
usefulness of several plant extracts as liver protective agents.
7,8 There is currently a dart of articles on the

Fresh Garlic Extract Protects The Liver Against Acetaminophen-Induced Toxicity
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hepatoprotective action of garlic, with those available
reporting on aged garlic extract and garlic oil, 16,17 but here
we report that fresh ethanolic extract of garlic produced
dose-dependent hepatoprotection in acetaminophen toxicity.
The medical implication of this finding could be that
consumption of raw garlic might be a useful prophylactic
strategy against toxic hepatitis.
Although the mechanism by which garlic exerts its
hepatoprotective effect was not investigated in this study,
several reported pharmacological properties of garlic could
contribute to this. It is now a well recognized finding that
garlic possesses antioxidant and anti-lipid peroxidative
effects. 22,23 Oxidative stress and lipid peroxidation are key
processes in the toxicity of acetaminophen. Therefore
prevention of these processes could be central event through
which garlic exerts its salutary effects in acetaminophen
toxicity. Garlic contains several organosulfur compounds
such as allicin, diallyl sulfide, and diallyl disulfide which are
valuable precursors for glutathione biosynthesis. 24 It is
possible that garlic prevents glutathione depletion induced
by acetaminophen overdose by upregulating its biosynthesis
in the liver. Garlic contains high levels of selenium and other
constituents that could actively scavenge free radicals. 25
selenium also upregulates glutathione peroxidase activity in
the liver thereby contributing also to the antioxidant defense
of the organ. 26
The constituents of garlic are reported to inhibit phase I
metabolism through specific inhibition of cytochrome p450
enzymes, while they enhance the activities of phase II
enzymes that are involved in the conjugation and excretion
of drugs and their phase I metabolites. 27 limiting the
production of NAPQI production by the above mechanisms
would minimize the toxic effects of acetaminophen
overdose.
NAPQI produces cellular necrosis through the formation of
lethal protein adducts with important structural and
functional proteins. 28 Prevention of adduct formation could
also be one way through which garlic exerts its effect. More
so, garlic is known to inhibit the secretion of pro-
inflammatory cytokines such as tumor necrosis factor a,
interleukin-1, and interleukin -6; these cytokines play
significant roles in the toxicity of acetaminophen. 29 Finally,
many steroidal saponins and sapogenins are present in garlic,
30 and could play vital roles as anti-inflammatory agents, in
the induction of protein synthesis, and in tissue regeneration
and repair.
CONCLUSION
This study has shown that fresh extract from garlic exerts
significant protection of the liver against acetaminophen
toxicity. Garlic contains many secondary metabolites, trace
elements, amino acids, and proteins which could act in a
variety of ways to protect the hepatocytes from toxic assault.
Consumption of fresh garlic could therefore be a reasonable
antidote to the development of hepatitis.
CORRESPONDENCE TO
Christian Ezeala, MICR, CSci.
Dept of Medical Laboratory Sciences, School of Health
Sciences, Kampala International University, WC, Box 71
Bushenyi, Uganda. Email: christian40ezeala@yahoo.com;
Tel: +256782751614
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Author Information
Christian Chinyere Ezeala, PhD, MICR, CSci (UK)
School of Health Sciences, Kampala International University
Ifeoma Nneka Nweke, Ph.D.
Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, Abia State University
Prince C. Unekwe, Ph.D.
Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, Abia State University
Ibrahim Ahmed El-Safty, Ph.D.
School of Health Sciences, Kampala International University
Emenike Jacob Nwaegerue, MSc.
Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, Abia State University