AlMotwaa et al
Tropical Journal of Pharmaceutical Research June 2019; 18 (6): 1205-1211
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
Available online at http://www.tjpr.org
http://dx.doi.org/10.4314/tjpr.v18i6.9
Original Research Article
Hepatotoxic and hematotoxic effects of sage oil-loaded
ifosfamide nanoemulsion in Ehrlich ascites carcinomabearing mice
Sahar M AlMotwaa1,2, Mayson H Alkhatib1,3*, Huda M Alkreathy4
1
2
Department of Biochemistry, Faculty of Science, Department of Chemistry, College of Science and Humanities, Shaqra
3
4
University, Shagra, Regenerative Medicine Unit, King Fahd Centre for Medical Research, Department of Pharmacology,
Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
*For correspondence: Email: mhalkhatib@kau.edu.sa; Tel: +966-599240526; Fax: +966-26400376
Sent for review: 2 February 2019
Revised accepted: 20 May 2019
Abstract
Purpose: To investigate the hepatotoxic and hematotoxic effects of sage oil-loaded ifosfamide (IFO)
nanoemulsion (NE) in Ehrlich ascites carcinoma (EAC)-bearing mice.
Methods: Ifosfamide (IFO) was loaded into a NE containing sage oil, and its hepatotoxic and
hematotoxic effects were assessed in EAC-bearing mice. Female Swiss albino mice (n = 50) weighing
25 - 30 g (mean weight = 27.5 ± 2.50 g) were randomly assigned to five groups of ten mice each. With
6
the exception of group 1, the mice were inoculated intraperitoneally (i.p.) with 2.5 × 10 EAC/mouse for
48 h. Group I served as negative control, C (-); group II served as positive control, C (+); while groups
III - V were treated i.p. with 60 mg/kg IFO in 0.3mL water (free-IFO); 0.3 mL NE (SAGE-NANO), and 60
mg/kg IFO in 0.3 mL SAGE-NANO (SAGE-IFO), respectively. The treatments were administered for
three days.
Results: Treatment with 60 mg/kg bwt IFO (free-IFO) significantly elevated the activities of aspartate
aminotransferase (AST) and alanine aminotransferase (ALT, p < 0.05). However, subsequent treatment
with SAGE-IFO significantly reduced the activity of these liver enzymes (p < 0.05). The concentration of
reduced glutathione (GSH) as well as the activities of catalase and glutathione reductase (GR)
significantly increased, while malondialdehyde (MDA) level decreased significantly in SAGE-IFO group,
when compared with free-IFO group (p < 0.05). Treatment with SAGE-IFO significantly restored white
blood cell (WBC) count and platelet levels which were altered by free-IFO (p < 0.05).
Conclusion: The results obtained in this study suggest that loading IFO in sage oil-NE greatly reduces
its hepatotoxicity and hematotoxicity.
Keywords: Ehrlich ascites carcinoma, Nanoemulsion, Oxidative stress, Sage oil, Hepatotoxicity,
Hematotoxicity
This is an Open Access article that uses a fund-ing model which does not charge readers or their
institutions for access and distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0)
and
the
Budapest
Open
Access
Initiative
(http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited.
Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus,
International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African
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INTRODUCTION
Nanoemulsion (NE) consists of an emulsifying
agent, water and oil phases. Oil-in-water NEs are
colloidal systems in which the oil droplet is
dispersed in water and produces micelles with
small droplet sizes of 20 to 200 nm, which serve
as drug carriers [1]. Essential oils (EOs) exhibit
antioxidant,
antifungal, antibacterial, antiinflammatory, antitumor, and antiviral activities.
----------------------------------------------------------------------------------------------------------------------------------------------------Trop J Pharm Res, June 2019; 18(6): 1205
© 2019 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License
AlMotwaa et al
However, their volatility and hydrophobicity limit
their applications in food and pharmaceutical
industries. Incorporation of EOs into emulsions
increases their dispersibility and solubility in
aqueous phases [2]. The anticancer activity of
various EOs when incorporated into NEs in vitro
have been reported [3,4]. Studies have shown
that extracts of sage plant possess antiproliferative, cytotoxic and pro-apoptotic effects
on different cancer cell lines such as lung
carcinoma, melanoma, breast cancer, laryngeal
carcinoma, oral cavity squamous cell carcinoma
and colorectal cancer [5-7]. Ifosfamide (IFO), an
oxazaphosphorine alkylating cytotoxic agent, is
commonly used to treat solid tumors such as
sarcomas, breast cancer, lymphoma and lung
cancer, alone or in combination with other
anticancer drugs [8]. The toxicity of IFO is
attributed
to
its
reactive
metabolite,
chloroacetaldehyde (CAA). Only a few studies
have reported the hepatotoxic effect of IFO.
Cases of liver failure after treatment with a
combination of IFO and docetaxel for advanced
breast cancer treatment or sarcoma have been
reported [9,10]. In a previous study,
hepatotoxicity combined with Fanconi syndrome
was reported in female rats [11]. The aim of this
study was to investigate the hepatotoxic and
hematotoxic effects of sage oil- loaded IFO NE in
EAC-bearing mice.
EXPERIMENTAL
Chemicals
Ifosfamide (IFO) was purchased from Baxter
(USA); Span 20 and Tween 80 were obtained
from Sigma (USA), while sage oil was purchased
from Sokar Nabat for Natural Oils (Saudi Arabia).
Phosphate buffered saline (PBS) was a product
of Bio-Diagnostics Company (Egypt). Automated
cell counter was obtained from Beckman (USA).
Inverted light microscope was a product of
Olympus Optical Co., Ltd. (Japan).
Preparation of sage oil-based NE
Sage oil-based NE was prepared by adding, in
drops, 5.5 % (v/v) surfactant mixture (Tween 80
and Span 20, 2:1, v:v); 1.8 % sage oil, and 92.7
% water until a clear and transparent solution
was obtained after thorough mixing and heating
at 70 °C.
Experimental rats
Female Swiss albino mice (n = 50) weighing 25 30 g (mean weight = 27.5 ± 2.50 g) were
obtained from King Fahd Center for Medical
Research, King Abdulaziz University (Saudi
Arabia), and were randomly assigned to five
groups of 10 rats each. They were maintained
under standard laboratory condition: 25 ◦C, 12 h
light/dark cycle and 60 - 65 % humidity. The rats
were allowed free access to standard rat feed
and clean water. The study protocol was
approved by the Institutional Research Ethics
Committee of the Faculty of Medicine, King
Abdulaziz University (Approval no. 1-17-01-0090068). The guidelines for the proper use and
care of animals, prepared by the National
Academy of Sciences, National Institute of
Health, were followed to provide humane care to
all the mice [12].
Experimental design
With the exception of group 1, the mice were
6
inoculated intraperitoneally (i.p.) with 2.5 × 10
EAC/mouse for 48 h. Group I served as negative
control, C (-); group II served as positive control,
C (+), while groups III - V were i.p. treated with
60 mg/kg bwt IFO in 0.3mL water (free-IFO), 0.3
mL NE (SAGE-NANO), and 60 mg/kg bwt IFO in
0.3 mL SAGE-NANO (SAGE-IFO), respectively.
The treatments lasted three days [13].
Blood and tissue sample collection
On the fifth day of treatment, the mice were
fasted overnight and sacrificed via cervical
dislocation. They were anesthetized with diethylether. Blood sample was collected from the
venous sinus in EDTA or plain tubes for
biochemical analysis. The blood was centrifuged
at 3000 rpm for 15 min to obtain plasma or
serum sample which was refrigerated at -80 oC
prior to use. Mice liver and spleen were excised,
washed
with
PBS
and
subjected
to
histopathological examination. Portion of the liver
was used to prepare 20 % tissue homogenate
which was used for determination of oxidative
status of the mice.
Determination of relative liver weight
Following sacrifice, the liver was excised, blotted
dry and directly weighted after sacrifice to
calculate the organ weight ratio for each mouse
by dividing the weight of liver with pre-sacrifice
body weight of the same mouse.
Assessment of liver function
Serum activities of AST, ALT and alkaline
phosphatase (ALP), and concentrations of total
protein (TP), albumin, total bilirubin (T.Bil) and
direct bilirubin (D.Bil) were determined using their
respective kits.
Trop J Pharm Res, June 2019; 18(6): 1206
AlMotwaa et al
Determination
of
activities
and
concentrations of antioxidant enzymes and
molecules
Catalase activity was determined using the
method described by Aebi [14]. The
concentration of MDA was measured based on
a
previously
reported
method
[15].
Concentration of GSH was estimated according
to the method of Beutler et al [16], while GR
activity was determined using the method
described by Goldberg et al [17].
Hematological investigation
Complete blood count (CBC) was carried out
using automated cell counter.
Histopathological examination
Sections of excised liver and spleen were
prepared for histopathological examinations. The
tissues were cut into slices (5 µm) using a
microtome, fixed in 10 % formaldehyde,
dehydrated through gradient of alcohol,
embedded in paraffin blocks, and sectioned.
They were thereafter stained with hematoxylin
and eosin (H & E), and viewed under an inverted
light microscope.
Statistical analysis
Data are expressed as mean ± SEM. Statistical
analysis was performed using MegaStat Excel
(10.3). Groups were compared using Tukey’s
range tests. Values of p < 0.05 were considered
statistically significant.
RESULTS
Liver function
There were no significant differences in relative
weights of liver and concentrations of TP among
the groups (p > 0.05). The activity of AST was
significantly elevated in all the EAC-bearing
mice, when compared with C (-) group (p < 0.05).
However, the activities of AST in SAGE-NANO
and SAGE-IFO groups were significantly
reduced, relative to free-IFO group (p < 0.05).
The activity of ALT was significantly elevated in
C (+) and free-IFO groups, when compared with
C (-) group (p < 0.05). However, there were no
significant differences in ALT activities in SAGENANO and SAGE-IFO groups relative to C (-)
group (p > 0.05). There were no significant
differences in ALP activities among the groups,
except the SAGE-NANO group (p > 0.05).
In the SAGE-NANO group, ALP activity was
significantly elevated, when compared with the
other groups (p < 0.05). Albumin level was
significantly higher in EAC-treated groups than in
C (-) and C (+) groups (p < 0.05). The
concentrations of albumin in free-IFO and SAGEIFO groups were significantly higher than that in
SAGE-NANO (p < 0.05) group. The level T.Bil
was significantly increased in C (+) group,
relative to the other groups (p < 0.05). However,
D.Bil level was significantly reduced in C (-) and
C (+) groups, when compared with the other
groups (p < 0.05). These results are shown in
Table 1.
Oxidative status of mice
Catalase activity was significantly reduced in C
(+) and free-IFO groups, when compared with
SAGE-IFO, SAGE-NANO and C (-) groups (p <
0.05). Injection of free-IFO into the mice
significantly elevated the concentration of MDA
and significantly reduced the activities of the
antioxidant enzymes (p < 0.05). However,
SAGE-IFO treatment significantly ameliorated
the oxidative stress induced by free-IFO (p <
0.05).
The activities of catalase were significantly
reduced in C (+) and free-IFO groups relative to
C (-), SAGE-IFO and SAGE-NANO groups, while
MDA level was significantly increased in C (+)
and free-IFO groups, when compared with C (-),
Table 1: Relative liver weight and liver function indices
Parameter
C (-)
C (+)
free-IFO
SAGE-NANO
SAGE-IFO
-2
Liver weight (x 10 )
4.70 ± 0.90
5.50 ± 0.60
5.70 ± 0.80
5.80 ± 0.99
5.50 ± 0.30
a
a
ab
ab
AST (U/mL)
99.70 ± 1.30
117.90 ± 3.80
132.90 ± 3.50
126.15 ± 0.20
127.90 ± 0.30
75.30 ± 1.10
81.56 ± 2.60
83.60 ± 0.80
77.40 ± 4.60
74.70 ± 1.54
ALT (U/mL)
c
ALP (U/L)
142.10 ± 1.05
148.40 ± 8.07
138.40 ± 1.50
174.76 ± 8.00
149.60 ± 8.20
TP (g/dL)
0.56 ± 0.02
0.55 ± 0.03
0.57 ± 0.03
0.57 ± 0.04
0.55 ± 0.05
d
d
2.97 ± 0.05
3.04 ± 0.01
3.48 ± 0.05
3.23 ± 0.07
3.77 ± 0.02
Albumin (g/dL)
e
e
e
T.Bil (mg/dL)
0.13 ± 0.01
0.15 ± 0.00
0.12 ± 0.01
0.12 ± 0.01
0.12 ± 0.01
e
e
e
D.Bil (mg/dL)
0.01 ± 0.00
0.02 ± 0.01
0.05 ± 0.00
0.04 ± 0.01
0.06 ± 0.01
a
b
c
P < 0.05, when compared with C (-) group; p < 0.05, when compared with free-IFO; p < 0.05, when compared
with the other groups; dp < 0.05, when compared with SAGE-NANO group; and ep < 0.05, when compared with C
(+) group
Trop J Pharm Res, June 2019; 18(6): 1207
AlMotwaa et al
significantly reduced in free-IFO group, relative to
SAGE-IFO, SAGE-NANO and C (+) groups (p <
0.05). The level of GSH was significantly higher
in C (-) group than SAGE-NANO, C (+), SAGEIFO and free-IFO groups (p < 0.05). The order of
GSH concentrations was: C (-) > SAGE-NANO >
C (+) > SAGE-IFO > free-IFO. These results are
shown in Figure 1.
Hematological parameters
As shown in Table 2, WBC level was significantly
higher in C (+) and SAGE-NANO groups, when
compared with the C (-), free-IFO and SAGE-IFO
groups (p < 0.05).
Platelets were also
significantly higher in C (+), free-IFO and SAGENANO groups than in C (-) and SAGE-IFO
groups (p < 0.05). There were no significant
differences in the levels of RBC and Hb among
the groups (p > 0.05).
Histopathological features of mouse liver
The results of histology are shown in Figure 2.
Photomicrographs of hepatic tissue sections of C
(-) group showed of central vein (CV) surrounded
by hepatocytes separated by vascular sinusoids.
Liver section of C (+) group revealed several
microscopic changes such as presence of
prominent Kupffer cells and leukocyte infiltration
around CV, and excessive dilated blood
sinusoids, when compared with C (-) group.
Vasodilatation, presence of abundant Kupffer
cells and leukocyte infiltration, in addition to
some enlarged hepatocytes were observed in the
liver section of free-IFO group. The leukocyte
infiltration around CV was significantly reduced,
and non-dilated blood sinusoids were seen in the
hepatic tissue of SAGE-NANO group relative to
C (+) group. Liver section of SAGE-IFO group
revealed less dilated blood sinusoids and
leukocyte infiltration than those of C (+) and freeIFO groups.
Figure 1: Some biochemical activities of mice
Histopathological characteristics of mouse
spleen
SAGE-IFO and SAGE-NANO groups (p < 0.05).
The activity of GR was significantly higher in C
(+) group than in the other groups, and it was
Normal spleen architecture was observed in C (-)
group as evidenced by the presence of red pulp
and lymphoid white pulp, which consisted of
Table 2: Levels of hematological parameters in the different groups
Parameter
C (-)
C (+)
free-IFO
SAGE-NANO
3
a
a
WBC (1 × 10 /µL)
1.70 ± 0.02
2.35 ± 0.05
1.20 ± 0.01
2.50 ± 0.01
Platelets (1 × 103/µL) x
7.15 ± 0.43
8.58 ± 0.30
8.36 ± 0.55b
10.54 ± 0.28b
2
10
6
8.91 ± 0.12
8.94 ± 0.19
8.71 ± 0.49
8.27 ± 0.05
RBC (1 × 10 /µL)
Hb (g/dL)
13.80 ± 0.80
14.50 ± 0.35
13.70 ± 0.30
13.85 ± 0.25
a
P < 0.05, when compared with C (+) group; bp < 0.05, when compared with C (-) group
SAGE-IFO
a
1.77 ± 0.10
6.87 ± 0.70
8.62 ± 0.28
13.85 ± 0.50
Trop J Pharm Res, June 2019; 18(6): 1208
AlMotwaa et al
periarteriolar lymphoid sheaths (PALS), adjacent
follicles and marginal zone. However, significant
reduction of lymphocytes with no clear border
between red pulp and white pulp was evident in
C (+) group, while the spleen section of SAGENANO group revealed decreased lymphocytes
with clear borders. Spleen sections of free-IFO
and SAGE-IFO groups revealed the presence
of lymphocytes (Figure 3).
a
b
investigated the hepatotoxic and hematotoxic
effects of sage oil-loaded IFO NE in EAC-bearing
mice.
a
b
c
d
e
c
d
e
Figure 2: Tissue sections of liver stained with H & E (x
400). (a): C (-) tissue showing normal liver histology
including CV, blood sinusoids (S), hepatic cells (H)
and Kupffer cells (K); (b): C (+) tissue showing
enlarged CV, excessive dilated blood sinusoids,
hepatocytes with prominent Kupffer cells, infiltration of
tumor cells, and leukocytes (LI); (c): Free-IFO tissue
showing excessive dilated blood sinusoids, leukocyte
infiltration and enlarged hepatocytes with abundant
Kupffer cells; (d): SAGE-NANO tissue showing minute
leukocyte infiltration around CV, blood sinusoids close
to normal structure; and (e): SAGE-IFO tissue
showing dilated blood sinusoids and leukocyte
infiltration less than that of C (+) and free-IFO tissues,
hepatocytes with prominent Kupffer cells
DISCUSSION
Nanoemulsions (NEs) are single-phase and
thermodynamically stable isotropic systems that
consist of emulsified oil, water and amphiphilic
molecules. They are sub-micrometre-sized
emulsions that are under extensive investigation
as drug carriers for improving the delivery of
therapeutic agents [1]. The present study
Figure 3: Histological sections of spleen stained with
H & E (x 400). (a): C (-) tissue showing normal
morphology of spleen characterized by red pulp (RP)
and lymphoid white pulp (WP); (b): C (+) tissue
revealing severe reduction of lymphocytes in the
periarteriolar lymphoid sheaths (PALS) of the WP with
undefined boundary between WP and RP; (c): FreeIFO tissue displaying WP with basically normal
numbers and arrangement of lymphocytes in the
PALS and follicles; (d): The WP in SAGE-NANO
tissue showing reduction of lymphocytes in PALS; and
(e): SAGE-IFO tissue presenting WP with basically
normal numbers and arrangement of lymphocytes in
the PALS and follicles
Elevation of serum activities of AST, ALT and
ALP are indicative of acute liver toxicity [10,11].
In this study, AST and ALT activities in free-IFO
group were significantly increased, while
administration of the combined formula, SAGEIFO, significantly reduced the activity of these
liver enzymes. On the other hand, activities of
ALP in free-IFO and SAGE-IFO groups were not
significantly different from that of C (-) group. The
concentration of direct bilirubin was significantly
increased in free-IFO and SAGE-IFO groups,
suggestive of hepatotoxicity induced by IFO [18].
There was induction of oxidative stress in freeIFO group, indicative of impairment of the
antioxidant defence mechanism in the mice. The
activities of GR and catalase, and concentration
of GSH were significantly reduced, and
concentration of MDA was significantly increased
in free-IFO mice. The activity of GR, and GSH
level were significantly increased, and the activity
of catalase and concentration of MDA were
Trop J Pharm Res, June 2019; 18(6): 1209
AlMotwaa et al
normalised in SAGE-IFO group, when compared
with
free-IFO
group.
The
results
of
histopathological examination revealed the
presence of inflammatory leukocyte infiltration in
the liver of free-IFO group. Degeneration of the
liver may have resulted from IFO toxic
metabolite, CAA, which might have promoted
GSH depletion and lipid peroxidation in the
hepatocyte [19].
CONCLUSION
The inclusion of sage oil in SAGE-IFO formula
may have contributed in diminishing IFO toxicity.
A number of studies have reported the antiproliferative, anti-migratory, anti-angiogenic and
antioxidant activities of sage oil [7,20]. Sage oil
plays increases the activity of glutathione
peroxidase, which in turn protects hepatocytes
from dimethoxy naphthoquinone and H2O2induced DNA damage. It has been reported that
oxidative stress in rat hepatocytes may be
improved by drinking water enriched with sage
extract [21]. Studies have also shown that the
sage extract, rosmarinic acid stimulates the
activities of various antioxidant enzymes such as
pancreatic catalase, glutathione peroxidase,
glutathione-S-transferase
and
superoxide
dismutase in streptozotocin-induced diabetic rats
[22].
The authors wish to sincerely thank King
Abdulaziz City for Science and Technology for
financial support for this work (project no. 1-1701-009-0068).
Hematological parameters are markedly affected
by the growth of tumor cells. Tumor cells induce
the release of vascular endothelial growth factor
(VEGF), which is an angiogenic factor stored in
platelet granules [22]. In this study, the levels of
WBCs were significantly higher in C (+) and
SAGE-NANO groups, when compared with C (-),
free-IFO and SAGE-IFO groups. Platelet levels
were significantly higher in C (+), free-IFO and
SAGE-NANO groups than in C (-) and SAGEIFO groups. There were no significant
differences in the levels of red blood cell (RBC)
and hemoglobin among the groups. These
results suggest that sage oil may possess
antiangiogenic properties. It has been reported
that ethanol extract of sage inhibits the
proliferation and migration of human umbilical
vein endothelial cells (HUVECs) and prevents
VEGF expression in breast cancer cells (MCF-7)
[23]. It has also been reported that EAC raises
the level of WBC, which reflects acute
inflammatory response or stress due to
metastasis of the cancer cells [24]. These results
also suggest that sage oil when used to
formulate NE may improve drug penetration
which leads to reduced tumor inflammation [25].
This is an Open Access article that uses a funding model which does not charge readers or their
institutions for access and distributed under the
terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/
4.0) and the Budapest Open Access Initiative
(http://www.budapestopenaccessinitiative.org/rea
d), which permit unrestricted use, distribution,
and reproduction in any medium, provided the
original work is properly credited.
The results obtained in this study suggest that
loading IFO in sage oil-NE greatly reduces its
hepatotoxicity and hematotoxicity.
DECLARATIONS
Acknowledgement
Conflict of interest
No conflict of interest is associated with this
work.
Contribution of authors
We declare that this work was done by the
authors named in this article and all liabilities
pertaining to claims relating to the content of this
article will be borne by the authors.
Open Access
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