Egypt. J. Comp. Path &Clinic Path. Vol. 29 No.1, 2016 ; 121-136 ISSN 1110-7537
121
Potential Apopreventive Effects of Garlic Oil And
Silymarin on Skin Papilloma Induced in Mouse
Mohamed F. Abou Elazab1, Nagwan M. El- Habashi
2 , Mohamed Abdou
3
and Nora F. Ghanem3
1
Department of Clinical Pathology, Faculty of Veterinary Medicine, Kafr Elsheikh
University, Egypt. 2
Department of Pathology, Faculty of Veterinary Medicine, Kafr Elsheikh University,
Egypt. 3
Department of Animal Biology, Faculty of Science, Kafr Elsheikh University, Kafr
Elsheikh, Egypt.
ABSTRACT— The potential apopreventive effects of garlic oil and silymarin on two-
papilloma in mice initiated by 7-12 dimethyl benz [a] anthracin (DMBA) and promoted with
croton oil were investigated. The highest tumor incidence (100%) and the shortest tumor latency
period (5 weeks) were observed in tumor induced group. In contrast, significant reduction in
tumor incidence to 0 and 20% as well as increase of latency period of papilloma formation (week
10) were observed in garlic oil and silymarin treated groups, respectively. Moreover, significant
increase in reduced glutathione and glutathione peroxidase activities as well as decrease in
malondialdehyde levels were also observed in garlic oil and silymarin treated groups in
comparison with the papilloma induced group. Hematological analysis revealed a significant
decrease in RBCs, Hb, PCV, MCH, and MCHC mean values as well as increase in the total
leukocytic count of papilloma induced group. But, there were no significance differences
between garlic oil and silymarin treated mice and those of vehicle control groups. Also,
histopathological examination revealed significant reduction in papilloma formation and
epidermal hyperplasia in garlic oil and silymarin treated mice when compared with papilloma
induced group.
Keywords: Apoprevention, Garlic oil, Silymarin, DMBA induced skin papilloma, Mice ——————— ——————————
INTRODUCTION
Skin cancer, the most frequently diagnosed
cancer, represents an important public health
problem due to its high incidence and
medical costs (Hara-Chikuma and
Verkman 2008). The incidence of both non-
melanoma and melanoma skin cancers has
been increasing over the past decades.
Currently, between 2 and 3 million non-
melanoma skin cancers and 132,000
melanoma skin cancers occur globally each
year. One in every three cancers diagnosed is
a skin cancer (World Health Organization
2013). Although there are available
treatments such as surgery, radiation therapy
and topical chemotherapy, up to date, still
there is no effective chemopreventive agent
against the development and/or progression
of skin cancer. Increase incidence of skin
cancer due to constant exposure of skin to
environmental carcinogens, provides a strong
basis for chemoprevention with both
synthetic and natural remedies (Gupta and
Mukhtar 2002). Polycyclic aromatic
hydrocarbons (PAHs), organic pollutants, are
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released into the environment in large
quantities mainly due to human activities and
the exposure to such environmental
pollutants is associated with the development
of numerous cancers in human (Neef 1985;
Nebert et al. 2004). Enzymatic activation of
PAHs leads to the generation of active
oxygen species such as peroxides and
superoxide anion radicals, which induce
oxidative stress in the form of lipid
peroxidation. Amongst many PAHs that are
used in induction of skin cancer is 7, 12-
dimethylbenz[a]anthracene (DMBA). It is the
most potent initiator that has ability to
damage DNA and produce cancer in short
time (Slaga 1983). The potent carcinogenic
properties of DMBA is related to its ability to
generate various reactive metabolic
intermediates leading to oxidative stress and
tissue damage (Bishayee et al. 2000).
Moreover, the repeated active oxidative
damage has a great link to all stages of tumor
development including initiation, promotion
and progression (Scandalios et al. 2005;
Visconti and Grieco 2009). A commonly
used two-stage model of skin carcinogenesis
in mice involves initiation by single topical
treatment of the skin with DMBA followed
by promotion through several weeks of
repetitive applications of an effective tumor
promoter such as TPA (Abel et al. 2009).
TPA, the most active phorbol ester present in
croton oil, acts as strong promoter through an
oxygen -mediated mechanism; oxygen
components are the critical components of
the tumor promotion process (Huachen and
Krystyna 1991). Therefore, reducing
intracellular oxidative stress or blocking ROS
generation may represent an effective
strategy for preventing skin carcinogenesis.
Human body is equipped with various
antioxidants such as superoxide dismutase
(SOD), glutathione peroxidase (GPx),
catalase (CAT), glutathione (GSH), ascorbic
acid (Vitamin C), α- tocopherol (Vitamin E),
etc., which can counteract the deleterious
effects of ROS and consequently protect the
body from cellular and molecular damage
(Cotgreave et al. 1988). Antioxidants act as
radical scavengers inhibiting free radical-
mediated processes thereby protecting the
human body from various diseases (Jagetia
and Rao 2006). Amongst various molecules
which can inhibit the formation of free
radicals associated with carcinogenesis are
the bioactive compounds extracted from plant
which have the potential to subside the
biochemical imbalances induced by free
radicals.
Silymarin (SIL), a standardized extract from
the milk thistle Silybum marianum (L), is
composed of many polyphenolic flavonoids,
including silibinin (the major one), isosilybin,
silychristin and silidianin (Polyak et al.
2013). Previously, several studies reported
that consumption of silymarin is safe and
non-toxic in animals and humans
(Wellington and Jarvis 2001; Singh et al.
2002) and there is no known LD50 for
silymarin (Gazak et al. 2007). In addition,
several recent studies have shown the
potential cancer preventive and therapeutic
efficacy of Silybinin in different animal
models and cell culture systems (Singh et al.
2004; Tyagi et al. 2007).
Garlic (Allium sativum), has a long history of
being used as a medicinal plant for over 4000
years for a variety of ailments including
headache, bites, intestinal worms and tumors
(Block 1985). Garlic also possesses many
biological properties including antimicrobial,
antioxidant, anticarcinogenic, antimutagenic,
antiasthmatic, immunomodulatory and
prebiotic activities. Previous report regarded
its anticancer effect to its ability to inhibit
free radical and mutations-mediated DNA
damage (Borek 1997). Dietary intake of
Allium vegetables, such as garlic, may play a
role in reducing the risk of certain cancers
(Kim and Kwon 2009). Allyl sulfides, the
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important organosulfur components of garlic
oil, have been reported to suppress the
growth of multiple cancer cells in culture and
in vivo models (Wu et al. 2004; Powolny
and Singh 2008; Yang et al. 2009). Diallyl
sulfide (DAS), diallyl disulfide (DADS), and
diallyl trisulfide (DATS) are most abundant
in garlic oil (Lawson 1991). DATS has been
reported to be the most potent in inhibiting
carcinogen-induced tumor promotion
(Shrotriya 2010).
Therefore, the aim of the present study was to
investigate the potential chemopreventive
effects of garlic oil and silymarin on DMBA
induced skin papilloma. Tumor incidence
(percentage of papilloma bearing
mice/group), average latent period of tumor
occurrence, clinico-pathological examination
and histopathological examination were used
to achieve the aim of the study.
MATERIALS AND METHODS
1. Chemicals
7, 12-dimethylbenz[a]anthracene and croton
oil were purchased from Sigma-Aldrich Co
(USA). Silymarin powder was purchased
from local pharmaceutical company and was
dissolved in corn oil at a concentration of
10mg/200µl. Garlic oil and corn oil were
purchased from local herbal market. DMBA,
as tumor initiator was dissolved in acetone at
a concentration of 200µg/200µl. Croton oil
which served as tumor promoter was
dissolved in acetone to give 1% croton oil
solution.
2. Animals
Seventy five male Swiss albino mice (6
weeks old) were used in the present study.
The animals were kept in Animal House of
Faculty of Sciences, University of
Kafrelshiekh. They were housed in cages
(with 7-8 mice per cage) under normal
laboratory conditions of humidity,
temperature (25±2ºC) and light (12/12 hour
light dark cycle). The mice were allowed for
2 week adaptation period with free access of
water and food ad libitum. The ethical
clearance had been approved by the ethical
committee of Kafr Elshiekh University.
Three days before the experiment, the dorsal
hair of all the animals was shaved by using
electrical hair clipper (2x2cm) and only the
mice showing no hair growth were chosen for
the study. The inhibition of tumor incidence
by garlic oil and silymarin was evaluated on
two-stage skin carcinogenesis, induced by a
single application of DMBA (as initiator),
and 2 weeks later, promoted by repeated
application of croton oil (as promoter) twice
per week, following the protocol for 12
weeks.
3. Experimental Design
Animals were randomly divided into five
groups each comprising 15 mice and treated
as described below: Group I (papilloma
induced group); mice were received a single
topical application of DMBA in acetone
(200µg/200µl/mouse) over the shaved dorsal
skin area, followed by topical application of
croton oil in acetone (200µl of 1% of croton
oil/mouse) twice a week for 12 weeks. Group
II (treatment group 1); All animal received
the same treatment as Group I and garlic oil
(200µl/mouse) was topically applied on the
shaved dorsal skin area for 7 days prior to 7
days after DMBA (anti-initiation)
application, and further applied 30 minutes
prior to croton oil treatment (twice weekly)
until the end of 12 weeks of promotion
period (anti-promotion). Group III (treatment
group 2); All animal received the same
treatment as Group I, in addition, silymarin
(10mg/200µl corn oil/mouse) applied on the
shaved dorsal skin area for 7 days prior to
7days after DMBA application, and further
applied 30 minutes prior to croton oil
treatment (twice weekly) until the end of 12
weeks of promotion period. Group IV
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(vehicle control 1); All animals were
received topical application of acetone
(200µl/mouse) on the shaved dorsal skin area
throughout the experiment. Group V (vehicle
control 2); All animals were received topical
application of corn oil (200µl/mouse) on the
shaved dorsal skin area throughout the
experiment. Body weight of mice, mortality
rate and tumor incidence were observed at
weekly interval.
4. Blood collection
Blood samples were collected from retro-
orbital venous plexus at the end of the
experiment. Nearly 2 ml of blood were
collected per mouse. Blood samples were
collected on an anticoagulant for
hematological profile and collection of
plasma. Plasma was separated from blood by
centrifugation at 3,000 x g for 15 minutes and
stored at -20 ºC until use.
5. Clinico-pathological examination
5.1. Hematological examination
Red blood cell count (RBC), hemoglobin
(Hb), packed cell volume (PCV), mean
corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), mean
corpuscular hemoglobin concentration
(MCHC), and total leukocyte count (TLC)
were measured by using an automated
hematologic analyzer for veterinary use and
calibrated for mice.
5.2. Biochemical analysis
5.2.1. Measurement of Malondialdehyde
Malondialdehyde (MDA) was
calorimetrically determined in plasma
according to the method adapted by
Esterbauer et al. (1982). This method is based
on the measurement of MDA as one of the
main end products of lipid peroxidation by
the thiobarbituric acid test. Thiobarbituric
acid reacts with MDA in acidic medium at
95oC for 30 minute to form thiobarbituric
acid reactive product. The absorbance of the
resultant color product measured at 534 nm.
5.2.2. Measurement of Reduced
glutathione
Reduced glutathione (GSH) was determined
in plasma according to the methods of
Beutler et al. (1963). This method is based on
spectrophotometrically measurement of the
yellow color of 2-nitro-5-thiobenzoic acid
which was produced from the following
reaction: Glutathione + 5,5'-dithiobis(2-
nitrobenzoic acid) (DTNB)→2-nitro-5-
thiobenzoic acid + glutathione disulfide
(GSSG).
5.2.3. Measurement of Glutathione
peroxidase
Glutathione peroxidase (GPx) activity was
assayed in plasma according to the method of
Gross et al. (1967). The activity of GPx was
measured directly by determining the amount
of unconsumed GSH remaining at specific
time intervals in the presence of small
amounts of peroxide.
6. Histopathological examination
Animals were sacrificed at the end of the
experiment. Tissue specimen of papilloma, as
well as skin of normal mice were collected in
10% neutral buffered formalin for 24h,
routinely processed and embedded in paraffin
blocks. The 5 m thick sections were stained
with haematoxylin and eosin (Bancroft and
Gamble 2007). The morphological evaluation
of the skin sections was performed under the
light microscope.
7. Statistical analysis
Statistical analysis was carried out by using
the SPSS for windows software, version16
(SPSS Inc., Chicago, IL, USA). Groups data
were compared by one-way analysis of
variance (ANOVA), followed by LSD test.
The statistical significance was accepted at a
level of p<0.05.
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RESULTS 1- Effect of garlic oil and silymarin topical
application on body weight and mortality
rate of DMBA- induced skin papilloma
mouse:
The average initial and final body weight and
mortality rate of mice from all treatment
groups were measured (Table 1). There was a
significant decrease in the average body
weight and increase in the mortality rate of
carcinogenic control group in comparison
with vehicle control groups (p<0.05).
However, there were no significant
differences in the average body weight
between garlic oil and silymarin treated
groups and vehicle control groups (p>0.05).
2- Effect of silymarin and garlic oil topical
application on hemogram of DMBA-
induced skin papilloma mouse:
Some hematological parameters (RBCs, Hb,
PCV, MCV, MCH, MCHC and WBCs) were
measured in all experimental groups (Table
2). The mean values of RBCs, Hb and PCV
were significantly decreased (p<0.05) in
papilloma induced group in comparison with
the vehicle-control groups. However, there
were no significant difference between garlic
oil and silymarin treated groups and vehicle-
control groups (p>0.05). RBC indices (MCH,
MCHC) were also significantly decreased
(p<0.05) in papilloma induced group in
comparison with the vehicle-control groups.
But, the mean values of MCV were
significantly increased in papilloma induced
group in comparison with the vehicle-control
groups. However, there were no significant
difference between garlic oil and silymarin
treated groups and vehicle-control groups
(p>0.05). Total leukocytic counts were
significantly increased in papilloma induced
group in comparison with the vehicle-control
groups (p<0.05). However, there were no
significant difference between garlic oil and
silymarin treated groups and vehicle-control
groups (p>0.05).
3- Effect of silymarin and garlic oil topical
application on GSH and GPx activities and
MDA concentrations in DMBA- induced
skin papilloma mouse:
The activities of GSH and GPx, as well as
MDA concentrations were measured in all
experimental groups (Table 3). There was a
significant decrease in the activities of GSH
and GPx as well as increase in MDA
concentrations in papilloma induced group in
comparison with the vehicle-control groups
(p<0.05). Topical application of garlic oil and
silymarin before and after DMBA application
caused significant increase in GSH and GPx
activities as well as decrease in MDA
concentrations when compared with the
carcinogenic group (p<0.05).
4- Effect of silymarin and garlic oil topical
application on histopathological changes in
DMBA- induced skin papilloma mouse:
Normal skin, uniformly arranged epidermal
and dermal layers with normal layer of
keratin over the epidermis, (Figs. 1) were
observed in all vehicle control groups. Skin
lesions in form of papillomas (Figs. 2),
abnormally thickened epidermis due to an
increase of the layers number: acanthosis,
hypergranulosis and hyperkeratosis with
fibrovascular core were observed, however,
the basement membrane is intact (Fig. 2, 3)
in all DMBA treated animals (100%,
incidence of skin lesions /group). The extent
of lesion was much less in garlic oil and
silymarin treated groups (Fig. 3, 4) when
compared with the papilloma induced group.
Topical application of garlic oil and
silymarin, on skin areas exposed to DMBA,
altered the incidence of skin lesions /group to
0% and 20 % of animals respectively, in
comparison with papilloma induced group
(100% of animals). Microscopically, 80% of
animals co-treated with silymarin showed
normal skin, however, 20% of animals
showed papillomas. Moreover, 60% of
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animals co-treated with garlic showed normal
skin, however, 40% of animals showed focal
thickening of epidermis as well as focal
hyperkeratosis mild congestion and
inflammatory cells infiltration in the dermis
(Fig.4).
DISCUSSION
Skin cancer is the most common malignancy
in the world and also one of the major causes
of death worldwide. The development of skin
cancer is a stepwise process, consisting of
initiation, promotion and progression in
experimental animals and possibly in human
cancer (Meeran et al. 2009). Murine skin
carcinogenesis, an excellent in vivo model
used for screening of new cancer protective
agent, is a classical design for the study of
biological changes during carcinogenesis
(Saha and Hait 2012). A commonly used
two-stage model of skin carcinogenesis in
mice involves initiation by treatment of the
skin with 7, 12-dimethylbenz[a]anthracene
(DMBA) followed by promotion through
treatment with croton oil or its active
principle 12-O-tetradecanoylphorbol-13-
acetate (TPA). DMBA is a polycyclic
aromatic hydrocarbon, considered to be one
of the etiological factors for the mice cancers
as well as human cancers (Das and
Bhattacharya 2004). The croton oil is the
most widely used distinguished promoting
agent to understand the cellular and
molecular alterations associated with
promotion stage and also a well-known
model to understand the role of
inflammation, generation of reactive oxygen
species (ROS), and hyperplasia in cancer
promotion (Yaar 1995; Sharma and
Sultana 2004; Ha et al. 2006).
Currently, apoprevention is an important
strategy for controlling the process of cancer
induction. Therefore, there is a need to
explore medicinal plants or other natural
agents that can work as chemopreventive
agents. Chemoprevention with food
phytochemicals offers a new promise for
reducing the incidence and mortality of
cancer. Phytochemicals have the potential of
chemoprevention through several cellular
mechanisms; oxidative or electrophilic
stresses that can trigger a wide variety of
cellular events from which the increasing in
the expression of detoxifying enzymes and/or
antioxidant enzymes (Surh 2003; Chen and
Kong 2005). Therefore, the present study
was designed to investigate the potential
apopreventive effects of garlic oil and
silymarin on DMBA/croton oil-induced
initial and promotional changes in mouse
skin. For testing this possibility, garlic oil and
silymarin were applied topically one week
before and after the topical application of
DMBA and continued 12 weeks thereafter
prior to croton oil treatment.
Firstly, effect of garlic oil and silymarin
topical application on body weight and
mortality rate of DMBA- induced skin
papilloma mouse were measured. There was
a significant decrease in the average body
weight and increase in the mortality rate of
carcinogenic control group in comparison
with vehicle control groups. However, there
were no significant differences in the average
body weight between garlic oil and silymarin
treated groups and vehicle control groups.
The anti-carcinogenic, antibacterial, anti-
inflammatory, antithrombotic, fibrinolytic,
antioxidant, and wound-healing properties of
garlic have been well-documented in
previous studies (Saifzadeh et al. 2006;
Vazquez-Prieto et al. 2011; Santhosha et
al. 2013). Furthermore, it has been reported
that silymarin having multiple
pharmacological activities including
antioxidants, hepatoprotectant, and anti-
infilammatory agent, antibacterial, anti-
allergic, antiviral and anti-neoplastic
(Pradeep et al. 2007; Toklu et al. 2007).
Hepatoprotection and Enhancement of liver
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functions may be could improve the
metabolic rate, body weight and decrease the
mortality rate.
Then, the effect of silymarin and garlic oil
topical application on hemogram of DMBA-
induced skin papilloma mouse were
measured. Significant decrease in RBCs, Hb,
PCV, MCH, and MCHC mean values as well
as increase in MCV and TLC of papilloma
induced group when compared with vehicle
control groups. But, there were no
significance differences between garlic oil
and silymarin treated mice and those of
vehicle control groups. These results indicate
presence of anemia in papilloma induced
group. Also, an increase in the number of
leukocytes (any or all type) above the normal
range usually part of an inflammatory
reaction. Generally, neoplastic lesions
usually associated with elevated numbers of
neutrophils and monocytes. As, macrophages
and neutrophils are both integrated in the
regulation of the innate and adaptive immune
responses in various inflammatory situations,
including cancer (Mantovani 2014).
Also, the effect of silymarin and garlic oil
topical application on some oxidative stress
markers in DMBA-induced skin papilloma
mouse were investigated. There was a
significant decrease in the activities of GSH
and GPx as well as increase in MDA
concentrations in carcinogenic group in
comparison with the vehicle-control groups.
Topical application with garlic oil and
silymarin before and after DMBA
application caused significant increase in
GSH and GPx activities as well as decrease
in MDA concentrations when compared with
the carcinogenic group. Oxidative stress has
long been implicated in cancer development
and progression (Hussain et al., 2003).
Repeated topical application of tumor
promoter croton oil on mouse skin involves
both oxidative burst as well as inflammation
by stimulating the generation of NO, reactive
nitric oxide species (RNS) and reactive
oxygen species (ROS) that plays an
important role in the process of mutagenesis
and carcinogenesis particularly tumor
promotion by increasing membrane lipid
peroxidation and decreasing cellular
antioxidant stores (Reed 2011; Das et al.
2012; Rauchová et al. 2012; Saha and Hait
2012). On the other hand, topical application
of garlic oil and silymarin confers
chemopreventive effects in terms of
reduction of lipid peroxidation and protection
against depletion of GSH and GPx. These
effects may be due to their potent antioxidant
properties.
Histopathological examination results
revealed that skin lesions in form of
papillomas, abnormally thickened epidermis
due to an increase of the layers number:
acanthosis, hypergranulosis and
hyperkeratosis with fibrovascular core were
observed, however, the basement membrane
is intact in all DMBA treated animals. During
the tumor promotion stage, repeated
application of TPA can trigger the production
of squamous papilloma and epidermal
hyperplasia, which is the pre-neoplastic
lesion of skin carcinoma (Shelton et al.
2000; Abel et al. 2009). These features have
been used routinely as quantitative markers
of pre-neoplastic lesions during
carcinogenesis via the measurement of the
level of epidermal thickness and the counting
of papilloma number (Cibin et al. 2011; Ko
et al. 2011). On the other hand, the extent of
lesion was much less in garlic oil and
silymarin treated groups when compared with
the papilloma induced group. Topical
application of garlic oil and silymarine, on
skin areas exposed to DMBA, altered the
incidence of skin lesions /group in
comparison with carcinogenic group. It has
been documented that garlic oil and silymarin
are sources of many anticarcinogenic agents
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and antioxidants, which may be useful for the
prevention of cancer. Lan et al. (2013)
documented that garlic oil has potent anti-
tumor property by inhibiting cells growth via
anti-proliferation and inducing apoptosis.
Also, Seki et al. (2008) reported that
phytochemical garlic-derived diallyl
trisulfide, a major constituent of the garlic
oil, has potent anticancer property. Moreover,
silymarin has a strong antioxidant capability
of scavenging free-radicals (Wellington and
Jarvis 2001) and several short-term studies
have suggested that silymarin may be a
potent anti-carcinogenic agent (Kren and
Walterova 2005).
In conclusion, the results of the present
study exhibits apopreventive potential of
garlic oil and silymarin on DMBA-induced
skin papilloma in male Swiss albino mice.
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Table 1. Effect of garlic oil and silymarin topical application on body weight and mortality
rate of DMBA- induced skin papilloma mouse.
Groups
Body weight (g)
Numbers of mouse
Mortality rate
(%) Initial
Final
Initial
Final
CAR
29.00±0.33
22.95±0.67*
15
5
66.67
CAR+SIL 29.00±0.37
28.35±0.29
15 12 20
CAR+GAR 28.81±0.25
28.91±0.23
15 12 20
CON1 27.55±0.29
29.50±.028
15 13 13.33
CON2 26.50±0.50
27.50±0.44 15 13 13.33
CAR means carcinogenic group (DMBA and croton oil); CAR+SIL means DMBA and croton oil plus silymarin;
CAR+GAR means DMBA and croton oil plus garlic; CON1 means vehicle control group number 1(acetone); CON2
means vehicle control group number 2 (corn oil). Data are expressed as means ± SD. * Indicate significant
difference between the treatments at p<0.05.
Table 2. Effect of silymarin and garlic oil topical application on hemogram of DMBA- induced skin papilloma mouse.
Groups
RBCs
(x106/µl)
Hb
(g/dl)
PCV (%)
MCV (%)
MCH (%)
MCHC
(%)
WBCs
(x103/µl)
CAR 6.35±0.25* 9.80±0.20*
29.03±0.51* 46.00±0.41* 9.70±0.83* 21.30±1.67* 5.40±1.56*
CAR+SIL 8.85±0.33 13.00±0.29
38.88±1.10 44.00±0.71 14.75±0.38 33.23±0.23 2.60±1.5
CAR+GAR 9.01±0.27 13.15±0.34
39.25±0.78 43.75±0.25 14.60±0.33 33.40±0.30 3.03±0.55
CON1 8.54±0.56 11.87±0.69
35.23±1.94 43.00±1.15 14.00±0.43 33.66±0.13 2.95±0.10
CON2 9.04±0.28
13.00±0.21
38.98±0.84 43.50±0.50 14.40±0.23 33.28±0.19 4.00±0.14
CAR means carcinogenic group (DMBA and croton oil); CAR+SIL means DMBA and croton oil plus silymarin;
CAR+GAR means DMBA and croton oil plus garlic; CON1 means vehicle control group number 1(acetone); CON2
means vehicle control group number 2 (corn oil). Data are expressed as means ± SD. * Indicate significant
difference between the treatments at p<0.05.
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Table 3. Effect of silymarin and garlic oil topical application on GSH and GPx activities and MDA concentrations in DMBA- induced skin papilloma mouse.
GPx (U/ml)
GSH (µmol/ml) MDA (nmol/ml) Groups
1.11±0.07* 6.73±0.53* 19.72±0.59* CAR
1.24±0.03* 8.46±0.54* 17.55±1.03* CAR+SIL
1.31±0.10* 10.13±0.83* 17.83±0.48* CAR+GAR
1.26±0.03 9.73±1.48 12.67±2.10 CON1
1.26±0.04 10.92±0.80 10.95±1.69 CON2
CAR means carcinogenic group (DMBA and croton oil); CAR+SIL means DMBA and croton oil plus silymarin;
CAR+GAR means DMBA and croton oil plus garlic; CON1 means vehicle control group number 1(acetone); CON2
means vehicle control group number 2 (corn oil). Data are expressed as means ± SD. * Indicate significant
difference between the treatments at p<0.05
Fig. 1. Skin section in group V (corn oil) showing normal histology with uniformly arranged
epidermal (E) and dermal layers (D) as well as normal layer of keratin (K) over the epidermis.
(H & E, X 100).
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Fig. 2. Skin section in DMBA treated mice (GI) showing papilloma, abnormally thickened
epidermis due to increase of the layers number: acanthosis, hypergranulosis and hyperkeratosis
with intact basement membrane. (H & E, X 100).
Fig. 3. Higher magnification of Fig. 2 showing papilloma, abnormally thickened epidermis,
acanthosis (A) as well as hyperkeratosis (H) along with fibrovascular core (arrow) with intact
basal cells layer (B). (H & E, X 200).
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Fig. 4. Skin section in group II (DMBA+ garlic) showing inflammatory cells infiltration in the
dermis and mild congestion. (H & E, X 100).
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