Gastroprotection Studies of Schiff Base Zinc (II)Derivative Complex against Acute SuperficialHemorrhagic Mucosal Lesions in RatsShahram Golbabapour1,3, Nura Suleiman Gwaram2, Pouya Hassandarvish1, Maryam Hajrezaie1,3,
Behnam Kamalidehghan4, Mahmood Ameen Abdulla1, Hapipah Mohd Ali2, A. Hamid A Hadi2, Nazia
Abdul Majid3*
1 Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 2 Department of Chemistry, University of Malaya, Kuala Lumpur,
Malaysia, 3 Institute of Biological Science, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia, 4 Department of Pharmacy, Faculty of Medicine, University of
Malaya, Kuala Lumpur, Malaysia
Abstract
Background: The study was carried out to assess the gastroprotective effect of the zinc (II) complex against ethanol-inducedacute hemorrhagic lesions in rats.
Methodology/Principal Finding: The animals received their respective pre-treatments dissolved in tween 20 (5% v/v),orally. Ethanol (95% v/v) was orally administrated to induce superficial hemorrhagic mucosal lesions. Omeprazole(5.79061025 M/kg) was used as a reference medicine. The pre-treatment with the zinc (II) complex (2.18161025 and4.36261025 M/kg) protected the gastric mucosa similar to the reference control. They significantly increased the activitylevels of nitric oxide, catalase, superoxide dismutase, glutathione and prostaglandin E2, and decreased the level ofmalondialdehyde. The histology assessments confirmed the protection through remarkable reduction of mucosal lesionsand increased the production of gastric mucosa. Immunohistochemistry and western blot analysis indicated that thecomplex might induced Hsp70 up-regulation and Bax down-regulation. The complex moderately increased thegastroprotectiveness in fine fettle. The acute toxicity approved the non-toxic characteristic of the complex(,87.24161025 M/kg).
Conclusion/Significance: The gastroprotective effect of the zinc (II) complex was mainly through its antioxidant activity,enzymatic stimulation of prostaglandins E2, and up-regulation of Hsp70. The gastric wall mucus was also a remarkableprotective mechanism.
Citation: Golbabapour S, Gwaram NS, Hassandarvish P, Hajrezaie M, Kamalidehghan B, et al. (2013) Gastroprotection Studies of Schiff Base Zinc (II) DerivativeComplex against Acute Superficial Hemorrhagic Mucosal Lesions in Rats. PLoS ONE 8(9): e75036. doi:10.1371/journal.pone.0075036
Editor: David D. Roberts, Center for Cancer Research, National Cancer Institute, United States of America
Received May 1, 2013; Accepted August 8, 2013; Published September 13, 2013
Copyright: � 2013 Golbabapour et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded and supported by the University of Malaya (HIR Grant UM.C/625/1/HIR/151 and PG016/2012B and RG057/11BIO). The fundershad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Zinc, the second most abundant transition metal, is an essential
trace element with a variety of biological roles in organisms [1–3].
It stabilizes macromolecules [4] and is critical in storage routines,
transcription factors, and replication proteins [5,6]. Involved in
various metabolisms of genome [7–10] and proteins [11–13], zinc
is a vital biological element (for a review, see [14]). Variation in
structural configuration of zinc proteins introduced zinc as the
only metal which appears in all six fundamental enzyme classes;
oxidoreductases, lyases, hydrolases, transferases, ligases, and
isomerases [15]. Zn2+ possesses lewis acid properties [16] and
redox activity [17]. Zinc based compounds potentially may have a
variety of therapeutic activities which makes it an attractive
element in drug therapy. Analogous zinc compound has anti-
bacterial activity against gram-positive bacteria [1]. Zinc controls
bacterial gene expression for instance, bacterial proteins such as
the iron responsive regulator fur, alcohol dehydrogenases,
hydrolases, lyases, and Cu/Zn superoxide dismutases utilize zinc
[18–20]. The effectiveness of the zinc (II) complex in preventing
mucosal damage might inhibit pathogenesis activity of bacteria in
the gastrointestinal (GI) tract.
Inflammatory reactions are governed by histamine, bradykinin,
serotonin, prostaglandins, the blood clotting system, and T cells
(lymphokines) [21]. Essential for T-cell proliferation, activation of
extracellular signal regulated kinase 2 in response to IL-2 is
dependent on zinc [21]. Zinc signals in neutrophil granulocytes
are required for the formation of neutrophil extracellular traps
[22]. The presence of bromine atoms coordinated to the zinc
metal ion seemed a possible active site for the complex and this
might be ascribed to the electron donating properties of the
halogens by resonance, making the lone pair electrons more
available to a plausible electron transfer (for a review, see [23,24]).
Similarly, bromine substituted copper complex showed amazing
PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e75036
gastroprotective activity [25]. Analogous zinc coordinated Schiff
base compounds demonstrated potential urease inhibition [26–
28]. This mechanism might be performed by this complex as a
potential treatment for eradicate helicobacter pylori and prevent
further recurrence of ulcer after therapy. Studies showed that
antioxidant activity is important in gastroprotection. Several
natural/synthetic agents introduced with protective effects against
acute hemorrhagic gastric lesions possessed free-radical scavenging
activity [29–33]. The antioxidant activity of several ingredients
might augment the total antioxidant activity of the tissue [30,34].
Another protective mechanism is the suppression of acid secretion
which have been considered a preventive strategy against gastric
superficial hemorrhagic mucosal lesions. For instance, proton
pump inhibitors (PPIs) are effective agents in inhibiting gastric acid
secretion [35]. Previous studies on Zn (II) [benzenesulfanohydra-
zide] [36] and Zn (II) [piperazine] [37] showed remarkable
gastroprotection. In this study, synthesized Schiff base zinc
(II){Dichlorido-2-morpholino-N-[1-(2-pyridyl)ethylidene] ethana-
mine k3 N,N’,N’’} was evaluated for its gastroprotective activity
against acute hemorrhagic gastric mucosal lesions in normal rats.
Materials and Methods
Synthesis of the ComplexIn this study, the chemicals were obtained from Fluka and
Aldrich, and used without further purification. Zinc (II) complex
(Figure 1) was synthesized by condensation reaction of 2-
acetylpyridine and 4-(2-aminoethyl)morpholine followed by com-
plexation of the ligand with zinc (II) acetate dihydrate in the
presence of potassium bromide [38]. Briefly, the product was
collected by filtration, washed several times with ethanol until a
milky coloured precipitate was obtained. The precipitate was dried
in a vacuum desiccator. Recrystallization was performed in a
mixture of methanol and dichloromethane. The x-ray crystal
structure of the zinc (II) complex was previously published [39].
Infrared spectra were obtained using KBr discs (4000–400 cm21)
on Perkin –Elmer FT-IR spectrometer. 1H and 13C NMR spectra
were recorded on Jeol JNM-LA400 FT-NMR system (Figures
S1and S2). TMS was used as an internal standard and deuteriated
DMSO-d6 as a solvent. Elemental analysis (C, H, N) were
performed using a Flash EA 1112 Series elemental analyser in the
University of Technology Malaysia. Elemental analysis and
spectral characterization for the ligand and its metal complex
was previously published by Gwaram et al. [38].
AnimalsSprague Dawley and ICR mice rats were attained from Animal
House, Faculty of Medicine, University of Malaya, Kuala
Lumpur. The animals were housed in an isolated cabin
maintained at ,24uC in a relative humidity of 80% using an
automated ventilation system. An artificial lighting system was
used for a daily ratio of 1:1. Animals had access to standard rat
pellets and tap water ad libitum. In our preliminary study
(unpublished data) and on the basis of acute toxicity results,
different doses of the zinc (II) complex were examined in rats to
find effective doses for gastroprotection.
Ethical IssuesAll procedures were performed in compliance with the National
Institutes of Health Guide for the care and use of Laboratory
Animals [40] and approved by the committee for animal
experimentation- Faculty of Medicine, University of Malaya
[University of Malaya- Ethic No. (ISB/30/05/2012/SG (R)].
Throughout the experiments, all of the animals received humane
care according to the ‘‘Guide for the Care and Use of Laboratory
Animals’’ prepared by the National Academy of Sciences [41].
Drugs and ChemicalsIn this study, a dilution (5% v/v) of tween 20 (Merck, Germany)
was used as the vehicle. Omeprazole, the reference drug for
prevention of superficial hemorrhagic mucosal lesions, was
obtained from UMMC and dissolved in the vehicle (5% tween 20).
Toxicity evaluation. The acute toxicity study was performed
in accordance with the OECD protocol [40]. The acute toxicity
study was to determine a non-toxic range of doses for the zinc (II)
complex. Thirty six mice (18 males and 18 females, 6–8 weeks old)
were assigned randomly into three groups (for each gender) and
were administrated orally with the vehicle (5% tween 20),
43.62161025 M/kg or 87.24161025 M/kg of the zinc (II)
complex (5% tween 20), accordingly. Prior to the dosing, the
animals were fasted for 24 h (water was accessible but the last 2 h).
Water and food was suspended for another 1 and 3 h after dosing,
respectively. During the first 48 h, animals were monitored for any
sign of abnormality. Onwards, they were examined for their health
condition, twice per day. The Animals were under assessment for a
period of 14-day to record any sign of toxicity or mortality. The
animals were euthanized on day 15 for histology and hematology
evaluations.
Ethanol-induced LesionPreventive effect of the zinc (II) complex against superficial
hemorrhagic mucosal lesions were assessed in the normal rats. 48
rats were randomly divided into 8 groups of 6 individuals; the
normal control group, the complex control group
(8.72461025 M/kg of the zinc (II) complex), the lesion control
group, the reference control group (5.79061025 M/kg omepra-
zole) and 4 experimental groups (1.09161025, 2.18161025,
4.36261025 and 8.72461025 M/kg of the zinc (II) complex).
Table 1 shows specifications for each group. The vehicle was
orally administrated (5 mL/kg) to the normal control and the
lesion control as a pre-treatment. The vehicle also was given orally
to the normal control and complex control groups as a treatment.
A single treatment with ethanol (95% v/v), was orally (5 mL/kg)
administrated to the lesion control group, reference control group
and the experimental groups. Prior to the pre-treatment, the rats
were fasted for 24 h (water was accessible but the last 2 h). The
interval between the pre-treatment and the treatment was 60 min.
The animals were euthanized 60 min after the treatment with an
over-dose of xylazine and ketamine anesthesia and their stomachs
were immediately excised.Figure 1. Chemical structure of the zinc (II) complex [38].doi:10.1371/journal.pone.0075036.g001
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Macroscopic Appearance of LesionsAcute hemorrhagic gastric lesions were characterized grossly. In
accordance with several studies, the superficial mucosal lesions
were petechial and hemorrhagic in different bund sizes, parallel to
the long axis of the stomach [25,31,33,37,42,43]. Luminal surface
of each stomach was assessed for the hemorrhagic damage. To
calculate the protection percentage (P%) for each pre-treatment,
the lesion area (LA) was calculated using a dissecting microscope
(1.86) and a planimeter (10610 mm2) where LE and LG were
lesion area of the lesion control and lesion area of a given group,
respectively.
(p%)~LE{LG
LE|100%:
Evaluation of Mucosal Protective FactorsPrevious studies showed that several gastroprotective mecha-
nisms were involved in the protection of gastric tissue against
aggressive conditions [25,30,34]. The acidity of gastric juice,
gastric wall mucosa, antioxidant and enzymatic activities of the
stomach were assessed to identify protective mechanisms of zinc
(II) complex in the ethanol-induced gastric lesions in rats.
Measurement of gastric juice acid content. In order to
measure the acidity of gastric juice, after dissecting the stomach, its
contents drained into a falcon tubes and centrifuged at 4000 rpm
for 10 min. The supernatant pH was recorded with a digital pH
meter.
Gastric mucus production. Gastric wall mucus production
was measured for each group [44]. Briefly, after removing the
glandular segments, the stomach tissue was immersed in 1%
Alcian blue solution (in sucrose solution, buffered with sodium
acetate at pH 5) and was rinsed with sucrose solution to remove
the excess dye. Magnesium chloride solution (500 mM) extracted
the dye from the mucus-dye complex. The extract mixed with
diethyl ether was centrifuged at 3000 rpm for 10 min and the
absorbance of supernatant was measured at 580 nm to calculate
the content of alcian blue extracted (mg of alcian Blue) per gram of
glandular tissue.
Enzymatic activities of stomach tissue homogenate. For
each rat, the gastric tissue homogenate was prepared in
phosphate-buffered saline (PBS) at ,4uC (125 mg tissue/mL
PBS). The tissues were homogenized with a teflon homogenizer
(Polytron, Germany). After centrifugation at 4,500 rpm for 15 min
at 4uC, the supernatant was used for the enzymatic and protein
assays.
Antioxidant activities and formation of prostaglandins E2
of stomach homogenate. The nitric oxide (NO), catalase
(CAT), glutathione (GSH) and superoxide dismutase (SOD) levels
of the gastric tissues were measured using commercial kits
(Cayman, USA). The gastric tissue supernatant of each sample
was subjected to the assays according to the manufacturer
protocols. The levels of prostaglandin E2 (PGE-2) and malondial-
dehyde (MDA) in the gastric tissue supernatant were measured
using commercial kits (Cayman, USA). Protein concentrations
were determined through Biuret reaction [45].
Histological AnalysisSpecimens of the gastric tissue were fixed using 10% buffered
formalin, were processed in the paraffin tissue-processing machine
(Leica, Germany), and were embedded in paraffin blocks. Sections
of 5 mm were subjected to hematoxylin and eosin (H&E) staining
and periodic acid schiff (PAS) staining (Sigma Aldrich, Malaysia).
H&E staining was to evaluate the tissue architecture. Periodic acid
schiff (PAS) staining was to evaluate changes in glycoproteins
(acidic and basic) and to observe the produced mucus. The gastric
sections were observed and photographed under a light micro-
scope (Nikon, Japan).
Immunohistochemistry AnalysisSpecimens of the gastric tissue were fixed (10% buffered
formalin) and processed in the paraffin tissue-processing machine
(Leica, Germany). Sections of 5 mm were placed on 3-aminopro-
pyltrimethoxysilane (APES)-treated glass slides and were subjected
to the immunochemical staining Hsp70 (Abcam, USA) and Bax
(Abcam, USA), using a streptavidin peroxidase (Abcam, USA).
Western Blot AnalysisFor western blot analysis, proteins were extracted from the same
gastric mucosa samples using protein extraction buffer (Pierce,
USA), the gastric tissue supernatant of each sample was subjected
to the western blot assays according to the previously published
procedure [46,47], with some modifications. Proteins (30 mg) were
separated by 12% SDS-PAGE (25 mA, for 2 h). Proteins were
transferred to PVDF membranes (Pierce, USA) using a Trans-Blot
SD semi-dry transfer cell (Bio-Rad, USA) at 15 V, 95 mA, for 1 h.
The PVDF membrane was blocked using BlockerTM Casein
(Pierce, USA) for 1 h at room temperature and washed twice using
TBST. The membranes were then incubated at 4uC overnight
with primary antibodies; Hsp70 mouse monoclonal antibody
(1:1000; Santa Cruz Biotechnology, USA), Bax mouse monoclonal
antibody (1:1000; Santa Cruz Biotechnology, USA) and b-actin
mouse monoclonal antibody (1:1000; Santa Cruz Biotechnology,
USA). The membranes were then incubated for 1 h at room
temperature with goat anti-mouse and goat anti-rabbit secondary
antibodies conjugated with alkaline phosphatase (i-DNA, USA) at
a ratio of 1:1000, then washed twice with TBST for 10 min. The
blotting were developed using the BCIP/NBT (Santa Cruz
Biotechnology, USA) solution for a period of 5–30 min to detect
the target protein band as a precipitated dark blue colour.
Statistical AnalysisThe data was analysed using analysis of variance by ANOVA
analysis followed by post-hoc analysis. A value of p,0.05 was
Table 1. The experimental design and specifications.
Groups Description Pre-treatment Treatment
Group 1 Normal control 5% tween 20 5% tween 20
Group 2 Complex control Complex8.72461025 M/kg
5% tween 20
Group 3 Lesion control 5% tween 20 95% ethanol
Group 4 Reference control omeprazole5.79061025 M/kg
95% ethanol
Group 5 Experimental group1 Complex1.09161025 M/kg
95% ethanol
Group 6 Experimental group 2 Complex2.18161025 M/kg
95% ethanol
Group 7 Experimental group 3 Complex4.36261025 M/kg
95% ethanol
Group 8 Experimental group 4 Complex8.72461025 M/kg
95% ethanol
doi:10.1371/journal.pone.0075036.t001
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considered significant. The data was analysed using the IBM SPSS
version 20 (IBM Corporation, USA) statistical software [48]. The
data is expressed as means 6 standard error.
Results
Acute Toxicity StudyFor duration of 14 days, none of the individuals in the acute
toxicity test showed any sign of abnormality or toxicity.
Histological assessment did not show any sign of nephrotoxicity
and/or hepatotoxicity. Hematological and serum biochemical
parameters were reported normal (Figure S3 and Table S1). The
lethal dose, 50% (LD50) for male and female mice were
1352.23 M/kg and 1169.02M/kg, respectively.
Macroscopic Evaluation of Gastric LesionsAs a pre-treatment, four doses of the zinc (II) complex
(1.09161025, 2.18161025, 4.36261025 and 8.72461025 M/kg)
were examined against the ethanol-induced gastric lesions in the
normal rats. Macroscopic evaluation of the lesions and the
comparisons among different groups showed that the doses of
1.09161025 M/kg and 2.18161025 M/kg of the zinc (II)
complex had the most remarkable protective effects (p,0.05) after
the reference group (5.79061025 M/kg omeprazole). Table 2
presents the inhibition percentage among the groups. Ethanol
caused extensive and remarkable hemorrhagic lesions on the
gastric epithelium. The pre-treatment with the omeprazole or the
zinc (II) complex significantly protected the gastric mucosa against
the damage (Table 2).
Evaluation of Mucosal Protective FactorsMeasurement of gastric juice acid content (pH). Table 2
represents the acidity of the gastric juice of the rats. The highest
pH was recorded in the reference control group (p,0.05).
Gastric mucus production. Alcian-blue-binding capacity
for each group was compared with the lesion control group
(Group 3) and the reference control group (Group 4). Table 2
shows the differences in the capacity for each group. The normal
control group and the complex control group showed a similar
binding capacity (p,0.05). In comparison, the lesion control
group possessed the lowest capacity. The pre-treatment with the
omeprazole or with the complex in the experimental groups
significantly compensated the lost capacity imposed by ethanol.
Among the experimental groups, the pre-treatment with
2.18161025 M/kg and 4.36261025 M/kg were relatively the
highest and close to that of the reference control group.
Protein concentration. Protein concentration for the com-
plex control group was the highest and the reference control group
showed non-significant differences to the experimental groups but
Group 5 (Table 3).
Antioxidant activities and formation of prostaglandins E2
of stomach homogenate. Table 3 shows antioxidant and
enzymatic activities of stomach tissue homogenates of the groups.
The lesion control group showed a major reduction of the level of
each antioxidant component (NO, CAT, GSH and SOD). In
contrast, the pre-treatment with either the omeprazole or the
complex in majority of the antioxidant assays compensated those
reductions to maintained the levels. The pre-treatment with
2.18161025, 4.36261025 and 8.72461025 M/kg of the zinc (II)
complex showed high level of activity for NO, but the reference
control group showed the highest activity. The activity of CAT
was significant in the rats pre-treated with the omeprazole
(5.79061025 M/kg) or with the zinc (II) complex (1.09161025
and 2.18161025 M/kg). The levels of SOD and GSH in those
rats pre-treated with omeprazole (5.79061025 M/kg) or the
complex (4.36261025 M/kg) were significantly high. The normal
control group and the complex control group did not show notable
differences in the antioxidant assays.
The level of PGE-2 in the normal control group and the
complex control group appeared the highest level among the
groups. While the lesion control group showed the minimum
formation of PGE-2, the pre-treatment with omeprazole
(5.79061025 M/kg) or the complex (2.18161025 and
4.36261025 M/kg) recompensed the activity level, significantly.
The lesion control group showed increase in the tissue level of
MDA accompanied by impairment of anti-oxidative defence
mechanisms. Unsurprisingly, the MDA levels were well main-
tained in the pre-treatment with either omeprazole
(5.79061025 M/kg) or the zinc (II) complex (especially doses of
2.18161025 M/kg and 4.36261025 M/kg) in comparison to that
of the lesion control group.
Histological EvaluationIn compliance with the macroscopic appearance, histological
evaluation of the gastric tissues showed different microscopic
features as shown in Figure 2. An extensive superficial damage
induced by ethanol was observed in the gastric mucosa of the
lesion control groups. They showed extensive edema and
leukocyte infiltration of the submucosal layer (Figure 2C).
Histological examination indicated that the oral pre-treatment
with omeprazole (5.79061025 M/kg) prevented the gastric tissue
from hemorrhagic lesions (Figure 2D). Similarly, the pre-treatment
with the zinc (II) complex showed the reduction of the lesion area,
submucosal edema and leukocyte infiltration (Figures 2E–2H).
The PAS staining was performed to assess the production of
glycogen in the gastric epithelium. The gastric mucosa in the rats
pre-treated with omeprazole (5.79061025 M/kg) or the complex
(Groups 4–8) showed increase in PAS staining intensity comparing
with the lesion control group in which the PAS staining not
profusely noticeable. The glycoprotein content of gastric mucosa
appeared almost similar in all groups but the lesion control group.
Figure 3A shows the PAS staining of the gastric tissue received
4.36261025 M/kg of the complex.
Table 2. Measurement of the lesion area, inhibitionpercentage, alcian blue binding capacity and pH.
Groups Ulcer area (mm)2 Inhibition pH GWM
Group 1 0*# 60 0 4.60*# 60.02 696.23*# 63.41
Group 2 0*# 60 0 4.70*# 60.02 716.59*# 61.54
Group 3 970.13# 621.12 0 3.71*# 60.03 117.30# 65.12
Group 4 124.60* 65.38 87% 5.95*# 60.06 613.77* 66.66
Group 5 201.10*# 68.16 79% 4.19*# 60.03 552.16*# 66.13
Group 6 136.92* 67.41 86% 5.22*# 60.04 588.51*# 64.51
Group 7 159.06* 62.79 84% 5.38*# 60.09 570.35*# 64.35
Group 8 200.62*# 65.19 79% 5.52*# 60.06 546.74*# 67.18
The experiment consisted of the negative control group (Group 1), the complexcontrol group (Group 2), the lesion control group (Group 3), the referencegroup pre-treated with 20 of omeprazole (Group 4) and the experimentalgroups (Groups 5–8) which received 1.09161025, 2.18161025, 4.36261025 and8.72461025 M/kg of the zinc (II) complex as a pre-treatment. All values areexpressed as mean 6 standard error mean. Mean difference is significant at thep,0.05 level (one-way between groups ANOVA with post-hoc analysis).*significant when compared to the ulcer control group (Group 3).#significant when compared to the reference control group (Group 4).doi:10.1371/journal.pone.0075036.t002
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Immunohistochemistry EvaluationImmunohistochemical evaluation of the gastric tissues showed
up-regulation of Hsp70 protein in gastric mucosa in the rats pre-
treated with omeprazole (5.79061025 M/kg) or the complex-
especially in the dose of 4.36261025 M/kg (Figure 3B)- when
compared with the lesion control group. The normal control
group and the complex control group also showed more
expression of Hsp70 in comparison to the lesion control group.
Immunohistochemical staining of Bax protein of gastric mucosa
showed down-regulation of Bax protein in those groups pre-
treated with the omeprazole (5.79061025 M/kg) or
4.36261025 M/kg of the zinc (II) complex (Figure 3C) while the
Table 3. Measurement of the total protein concentration, antioxidant activity, lipid peroxidation and formation of prostaglandinsE2 of the tissue homogenates.
GroupsProtein(mg/ml tissue) NO (mM) CAT nM/min/ml
SOD(U/mg protein)
GSH(mM/mg protein)
MDA(mM/g protein)
PGE-2(ng/mg protein)
Group 1 14.72*# 60.23 9.15*# 60.08 125.88*# 63.43 15.96*# 60.41 16.81*# 60.20 99.41*# 63.63 3.56*# 60.03
Group 2 15.53*# 60.39 8.79*# 60.14 129.35*# 61.03 16.40*# 60.19 17.32*# 60.17 87.14*# 64.71 3.65*# 60.02
Group 3 9.18# 60.23 3.64# 60.11 66.56*# 62.33 11.42# 60.29 9.87# 60.39 206.43# 63.48 1.16# 60.006
Group 4 13.39* 60.15 7.99* 60.19 151.79*# 64.39 27.43* 60.26 14.49* 60.18 128.80* 65.11 3.29* 60.02
Group 5 11.99*# 60.26 5.33*# 60.05 145.30* 61.28 19.56*# 60.19 11.13*# 60.20 101.59*# 63.68 2.18*# 60.006
Group 6 12.67* 60.41 7.19*# 60.08 149.56* 60.80 26.77* 60.12 14.21* 60.48 99.24*# 63.64 3.28* 60.014
Group 7 13.50* 60.31 6.90*# 60.09 140.86*# 60.77 25.30*# 60.38 13.11*# 60.19 127.72* 66.00 3.26* 60.002
Group 8 13.21* 60.19 6.12*# 60.09 142.13* 60.94 24.96*# 60.57 11.43*# 60.26 113.30* 63.98 3.15*# 60.005
This experiment consists of the negative control group (Group 1), the complex control group (Group 2), the lesion control group (Group 3), the reference group pre-treated with 5.79061025 M/kg of omeprazole (Group 4) and the experimental groups (Groups 5–8) which received 1.09161025, 2.18161025, 4.36261025 and8.72461025 M/kg of the zinc (II) complex as a pre-treatment. All values are expressed as mean 6 standard error mean. Mean difference is significant at the p,0.05 level(one-way between groups ANOVA with post-hoc analysis).*significant when compared to the ulcer control group (Group 3).#significant when compared to the reference control group (Group 4). NO, nitric oxide; CAT, catalase; SOD, superoxide dismutase; GSH, glutathione; MDA,Malondialdehyde; PGE-2, prostaglandins E2.doi:10.1371/journal.pone.0075036.t003
Figure 2. Hematoxylin and eosin staining evaluation of the gastric mucosa. The negative control and the complex control group have notany disruption to the gastric epithelium, submucosal edema or leucocyte infiltration (A). The lesion control group has extensive edema in thesubmucosal layer (B). Moreover acute hemorrhagic gastric lesions with severe disruption to the epithelium penetrated deeply into the mucosa (C)along with leucocyte infiltration are also noticeable (D and E). The pre-treatments with the complex (4.36261025 M/kg) show mild superficialdisruption to the gastric epithelium (F).doi:10.1371/journal.pone.0075036.g002
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lesion control groups showed up-regulation of the protein. In the
normal control group, the immunohistochemistry of Hsp70 and
Bax proteins was similar to the complex control group. Figure 3 (B
and C) shows immunohistochemistry for the expression of Hsp70
and Bax proteins of the gastric tissues in the rat received
4.36261025 M/kg of the complex.
Western Blot AssayWestern blot analysis confirmed the immunohistochemistry
results. The complex (2.18161025 and 4.36261025 M/kg) and
omeprazole (5.79061025 M/kg) caused up-regulation of Hsp70
protein in the pre-treated rats when compared with the normal
control group (Figure 4A). The complex control group by itself
caused mild up-regulation of the expression of Hsp70. In the lesion
control group, the expression of this protein was remarkably
down-regulated as compared with the normal control group
(Figure 4A).
The expression of Bax protein showed that the protein was up-
regulated in the lesion control group as a strong sign of apoptosis
while those rats pre-treated with omeprazole (5.79061025 M/kg)
or the complex showed remarkable down-regulation of the
expression of Bax protein (Figure 4A).
Discussion
In recent years, the awareness of the implications of cycloox-
ygenase 2 on prevention and maintenance of gastric mucosal
integrity and ulcer healing [49,50] persuade several studies to find
alternative therapeutics for preventing and treating superficial
hemorrhagic mucosal lesions. Several studies have promoted
ethanol-induced model of hemorrhagic gastric lesions [34,51–53].
It has been established that one of the most important detrimental
effects evoked by ethanol-induced gastric lesion is represented by
increments of gastric mucosal MDA levels, a marker for oxidative
stress [54]. ROS such as superoxide anions, hydrogen peroxide,
and hydroxyl radicals, is the main cause of oxidation of biological
constituents in gastric mucosal injury [55]. Ethanol, a necrotizing
agent, induces cell membrane injury through generating free
radicals [56–58] and lipid peroxidation [59,60]. Increase in the
permeability of the cell membrane causes extensive tissue
destruction [61] which macroscopically appears as hemorrhagic
erosions in the gastric mucosa. Moreover, oral administration of
ethanol imposes vascular permeability and, diffuse severe damage
to the capillaries of the gastric glandular mucosa [62] which in
turn appears as petechiation or hemorrhagic bundles. Results of
the several works demonstrated that, the effect of oral adminis-
tration of ethanol on gastric functions was to reduce the gastric
mucin content [31,34,37]. In the current study, the acute toxicity
test did not show any sign of toxicity or mortality in the given
dosages. In this study omeprazole was used as a reference
medicine in prevention of gastric lesion. The idea was to compare
the preventive activity of the zinc (II){Dichlorido-2-morpholino-N-
[1-(2-pyridyl)ethylidene] ethanamine k3 N,N’,N’’} complex with
that of the reference medicine, omeprazole, in ethanol-induced
gastric lesion rats. Omeprazole protected gastric mucosa signifi-
cantly against the induced aggressive factor, ethanol. Omeprazole,
a substituted benzimidazole derivative, is a proton pump inhibitor
that inhibits gastric acid secretion [63] and managing acute
hemorrhagic mucosal lesions. PPIs with antioxidant properties
[64] inhibit acid secretion, and promote gastric epithelial cell
migration [65]. However, its stimulating effect on mucus secretion
has remained controversy [66]. In addition to the effectiveness of
omeprazole on acid-dependent gastric lesion, it is also effective on
acid-independent gastric model [67–70]. Ethanol, as it was shown
in this study, causes severe macroscopic lesion with histological
changes such as extensive edema, leukocyte infiltration of the
submucosal layer and loss of integrity of gastroepithelium along
with the impairment of gastric mucosa [30,42]. In accordance with
previous studies, our result showed that omeprazole
(5.79061025 M/kg) prevented the reduction of protein concen-
tration along with increase in PGE-2 [25,30,34]. In addition to a
Figure 3. Glycoprotein-PAS staining and immunohistochemical evaluation for the expression of Hsp70 and Bax proteins of thegastric mucosa. Oral administration of the zinc (II) complex (2.18161025 M/kg) increased the glycoprotein content of the gastric tissue (A),enhanced the expression of Hsp70 protein (B) and suppress the expression of Bax protein (C). The arrows point to the respective proteinaccumulations.doi:10.1371/journal.pone.0075036.g003
Figure 4. Western blot analysis with Hsp70 and Bax mousemonoclonal antibody. Corresponding b-actin blots are shown as acontrol for sample loading. G1, normal control; G2, complex control; G3,lesion control; G4, reference control; G5, zinc (II) complex(2.18161025 M/kg); G6, zinc (II) complex (4.36261025 M/kg).doi:10.1371/journal.pone.0075036.g004
Schiff Base Zinc (II) Complex- In Vivo Study
PLOS ONE | www.plosone.org 6 September 2013 | Volume 8 | Issue 9 | e75036
newly published research on anti-ulcer effect of a synthesized
steroid [34], our study provided an evidence on gastroprotective
effect for PG through increasing the formation of PGE-2 [71].
The zinc (II) complex (as shown in the complex control group)
not only maintained the normal condition of the stomach but to
some extents enhanced the defensive efficacy of the tissue. The
antioxidant activity of the pre-treatment with the zinc (II) complex
was increased in the gastric homogenates similar to the antioxidant
activities of the reference medicine [72]. This study in consistence
with several studies showed that antioxidant property was one of
the main gastroprotective mechanisms [29,33,42,53]. The Zn (II)
complex appeared effective in the mid doses (2.18161025 and
4.36261025 M/kg). The highest dose (8.72461025 M/kg) ap-
peared comparatively less effective in the protection against acute
gastric lesions. Perhaps the main reason for such negative
protective feedback lied in pro-oxidant activity of the Zn (II)
complex in higher concentrations. Previous studies indicated that
some Schiff base compounds might possess pro-oxidant property
[73–76], however, a precise project should be conducted to show
the exact pro-oxidation activity of the Schiff base Zinc (II)
derivative complex.
The macroscopic and microscopic assessment among the
reference group and the pre-treated rats with the zinc (II) complex
(Groups 5–8) showed remarkable protection of gastric epithelium
from ethanol-induced hemorrhagic lesion in a similar pattern to
the reference control group. Consistent with previous studies
[25,34,37], the gastric mucosal content increased remarkably in
those groups that were pre-treated with the complex (Group 5–8),
when compared to the lesion control. In comparison with the
lesion control group, the reference control group among all the
groups showed the highest level of the mucus content, but
significant boost in mucosal content of Groups 5–8 (pre-treated
with the complex) was noticeable. The zinc (II) complex enhanced
the gastric mucosal content in the complex control group when
compared with the normal control. These findings showed that the
complex had the ability to stimuli the mucus secretion. In
agreement with several studies [33,34,69,70,77], our investigation
revealed that the exposure of gastric mucosa to oxidative stress was
restricted through the oral administration of omeprazole
(5.79061025 M/kg) or the complex as a pre-treatment. The
involvement of formation of PGE-2 in the gastroprotective
mechanisms was previously investigated with conflicting evidences
[25,29,33,34]. PGE-2, the most abundant GI prostaglandin, is
fundamentally important in the regulation of gastric mucus
secretion [78], gastric acid secretion [79] and gastric motility
[80,81]. In oral administration of ethanol, reduction in formation
of PGE-2 lessened acid secretion and gastric motility [78], and
decreased gastric mucus secretion [82]. As shown in the pre-
treatment with Zn (II) complex, there was the same expression
pattern (with a direct relation) between the level of secretion of
gastric mucosa and the level of PGE-2 in the homogenate [83]. On
the other hand, the inhibition percentage was almost in the same
pattern with the level of GWM. However, the protection was not
appeared in a clear dose-dependent manner. This finding could be
due to the possible anti-inflammatory effect of the complex, where
the pre-treatment in lower concentrations appeared more effective
(with relatively higher inhibition percentage, PGE2 and GWM
secretion). Increasing the pre-treatment dose showed more anti-
inflammatory effects, astonishingly. Several studies showed that
Schiff base derivative compounds possessed anti-inflammatory
activity [84–86]. The endogenous prostaglandins that contribute
to ulcer healing/protection are derived from COX-2 [87]. Some
of the Schiff base complexes are selectively COX-2 inhibitors in
relatively high concentrations [88,89] (For a review, see [87]).
However, another study should be performed to evaluate the anti-
inflammatory effect of the Zn (II) complex and to highlight the
exact anti-inflammatory mechanisms in different doses.
Immunohistochemistry evaluation of the gastric tissue for each
group confirmed the gastroprotection effect of the pre-treatment,
either with the omeprazole (5.79061025 M/kg) or the zinc (II)
complex. Heat shock proteins is classified into four families
(HSP90, HSP70, HSP60, and Hsp70). The low molecular weight
chaperone, Hsp70 mediates a variety of post translational
modification of polypeptides [90]. Many studies have shown the
importance of Hsp70 as a cytoprotective protein under various
stress conditions [91–93]. In consistence with the previous studies
[25,30,34], the present study demonstrated that the mucosal
expression of Hsp70 in those rats pre-treated either with the
omeprazole (5.79061025 M/kg) or with the zinc (II) complex was
increased when compared with the lesion control group. In the
process of gastroprotection, balance between apoptosis and cell
proliferation should be maintain. Bcl-2 family consists of different
proteins such as Bax and Bcl-2 [94]. Bax protein promotes
apoptosis but Bcl-2 protein is an antagonist to the function of Bax
[95]. Immunohistochemistry evaluation for Bax protein in the
gastric tissue of each group showed that Bax protein was at its
highest level of expression in the pre-treatment with ethanol but
significantly reduced when the rats pre-treated with omeprazole or
the complex. Previous study demonstrated that a copper (II)
complex was able to reduce the expression of Bax protein,
significantly [25]. A newly published research showed effectiveness
of a new Schiff base derived copper (II) complex on gastropreven-
tion [25]. The complex showed the inhibition percentage of that
complex in a dose-dependent manner, in which the protection
with 80 mg/kg of the complex was the most effective dose. In
comparison, this study showed a remarkable protection in lower
administrated doses (2.18161025 and 4.36261025 M/kg). The
effectiveness of the zinc (II) complex appeared similar to
omeprazole (5.79061025 M/kg) but it could make the prevention
in significantly lower concentration in comparison to the copper
(II) complex.
Conclusions
The zinc (II){Dichlorido-2-morpholino-N-[1-(2-pyridyl)ethyli-
dene] ethanamine k3 N,N’,N’’} complex did not appear toxic in
administrated doses (43.62161025 or 87.24161025 M/kg) in
mice. The complex could significantly enhance the protective
mechanisms of mucosa against acute hemorrhagic mucosal lesions.
Antioxidant activities (NO, CAT, SOD and GSH) improved the
protection against free radicals and maintain the level of the
MDA. The zinc (II) complex, in some extent, stimulated the
release of PGE-2 in the gastric tissue homogenates similar to that
of omeprazole. The macroscopic and microscopic evaluations of
the gastric tissues confirmed the gastroprotective effect of the
complex through reduction of epithelial mucosal lesion, submu-
cosal edema and neutrophil infiltration and through increase in
glycoprotein content of the gastric homogenate and Hsp70
protein. This study introduced the complex as an efficient
gastroprotective agent against acute hemorrhagic gastric lesions
in rats.
Supporting Information
Figure S1 1H-NMR spectrum. DMSO-D6 record of the zinc
(II) complex [38].
(TIF)
Schiff Base Zinc (II) Complex- In Vivo Study
PLOS ONE | www.plosone.org 7 September 2013 | Volume 8 | Issue 9 | e75036
Figure S2 13C-NMR spectrum. DMSO-D6 record of the
zinc (II) complex [38].
(TIF)
Figure S3 Microscopic evaluation for the acute toxicitytest. H&E staining for liver (A) and kidney (B) do not show any
sign of toxicity in rats received 87.24161025 M/kg of the
complex. There is no significant difference in structures of liver
and kidney among the groups.
(TIF)
Table S1 Acute toxicity test.(DOCX)
Acknowledgments
The authors express gratitude to the staff of the Faculty of Medicine and
Faculty of Science for their support. The authors also are deeply grateful
for all supports from late Prof. Datuk Dr. A. Hamid A. Hadi throughout
this study.
Author Contributions
Conceived and designed the experiments: SG MAA NAM. Performed the
experiments: SG MH PH NSG BK. Analyzed the data: SG NSG NAM.
Contributed reagents/materials/analysis tools: SG NSG BK AHAH
HMA. Wrote the paper: SG NAM NSG.
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Schiff Base Zinc (II) Complex- In Vivo Study
PLOS ONE | www.plosone.org 9 September 2013 | Volume 8 | Issue 9 | e75036
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