Gastroprotection Studies of Schiff Base Zinc (II) Derivative Complex against Acute Superficial...

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

Schiff Base Zinc (II) Complex- In Vivo Study


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


95% ethanol


95% ethanol

doi:10.1371/journal.pone.0075036.t001



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



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



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



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)



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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