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32 Scripta Scientifica Medica, vol. 45, suppl. 3, 2013, pp. 32-38
Copyright © Medical University of Varna
ORIGINAL ARTICLES
EFFECT OF AQUEOUS INFUSION FROM COTINUS
COGGYGRIA LEAVES ON INDOMETHACIN-INDUCED
GASTRIC MUCOSAL DAMAGE AND OXIDATIVE STRESS
IN RATS
Danail Pavlov1, Diana Ivanova1, Miroslav Eftimov2, Maria Tzaneva3, Milka Nashar1,
Ina Kobakova4, Stefka Valcheva-Kuzmanova2
1Department of Biochemistry, Molecular Medicine and Nutrigenomics, 2Department of
Preclinical and Clinical Pharmacology, 3Department of Preclinical and Clinical Sciences,
and 4Department of General and Clinical Pathology, Forensic Medicine and Deontology,
Medical University of Varna
Address for correspondence:
Danail Pavlov
Dept. of Biochemistry, Molecular Medicine and
Nutrigenomics, Medical University of Varna,
55 Marin Drinov Str., 9002 Varna, Bulgaria
e-mail: danailpavlov@gmail.com
ABSTRACT
PURPOSE: The Smoke tree (Cotinus coggygria) is well known medicinal plant that is used mainly external-
ly by the Balkan folk medicine for its antiseptic and anti-inflammatory properties. There are scarce reports
about the internal usage of decoctions from C. coggygria leaves against gastric ulcers. Our study was aimed
to explore the effect of aqueous infusion from Cotinus coggygria leaves (AICCL) on indomethacin-induced
gastric mucosal damage in Wistar rats and its possible effect on the gastric oxidative status.
MATERIAL AND METHODS: Three AICCL (1/100, 2/100 and 4/100) were applied by gastric gavage (volume:
10 ml/kg) as a pretreatment 3 days before a single intragastric administration of indomethacin (dose: 100
mg/kg). Gastric ulcer formation was estimated morphometrically and histopathologically 4 h after the in-
domethacin administration. Malondialdehyde (MDA) in blood serum and stomach was measured as a bio-
chemical marker of lipid oxidation. Gastric necrosis was also evaluated by alkaline phosphatase (ALP) and
uric acid (UA) assays.
RE SULTS: Morphometrical examinations of stomachs showed that the 2/100 AICCL significantly decreased
the ulcer number and area. Histopathological studies demonstrated that AICCL induced a reduction of the
depth and severity of indomethacin-induced mucosal lesions. AICCL reduced the elevated by indomethacin
gastric MDA, ALP and UA levels.
CONCLUSION: Indomethacin-induced gastric mucosal damage was accompanied by the development of ox-
idative stress. AICCL-pretreatment alleviated the gastric lesions, and reduced the indomethacin-induced el-
evation of ALP and UA. It could be suggested that the gastroprotective effect of AICCL was due to its antiox-
idant properties as evidenced by the decreased gastric MDA levels.
Key words: Cotinus coggygria, Indomethacin, Gastric mucosal damage, Oxidative stress, Lipid peroxida-
tion, Antioxidant activity
Received: September 5, 2013
Accepted: October 7, 2013
INTRODUCTION
Gastric ulcer is a recurrent chronic illness that
affects approximately 10% of the world population
(1). It is induced by several factors, including infection
by Helicobacter pylori, emotional stress, smoking,
nutritional deficiencies, alcohol consumption and
treatment with non-steroidal anti-inf lammatory
Scripta Scientifica Medica, vol. 45, suppl. 3, 2013, pp. 32-38
Copyright © Medical University of Varna 33
Danail Pavlov, Diana Ivanova, Miroslav Eftimov et al.
drugs (NSAIDs) (2, 3). Ulceration occurs when there
is an imbalance between protective (mucus secretion,
blood flow, prostaglandins, enzymatic and non-
enzymatic antioxidants) and aggressive mechanisms
in the stomach (acid-pepsin, leukotrienes and react ive
oxygen species, ROS) (4). It is well known that ROS,
especially the superoxide anion and the hydroxyl
radical, play an important role in the pathogenesis
of acute experimental gastric lesions induced by
NSAIDs (5).
Indomethacin, like other NSAIDs, is known to
induce gastric mucosal damage in humans (6) and is
considered as an appropriate agent for development
of animal models of NSAIDs-induced ulcerogenesis
(7). The inhibition of cyclooxygenase (COX) and
deficiency of endogenous prostaglandins (PGs)
is accepted as a main mechanism implicated in
indomethacin-induced gastropathy (8).
The therapy used for treating gastric ulcers
includes the control of the H. pylori infection, the
control of the H+/K+-ATPase pump and the acid
secretion, as well as the damage and inf lammation
reversal to the mucosa (9). Medications prescribed
for the treatment of gastric ulcer are not completely
effective and exhibit many adverse reactions in
addition of high economic burden (10). This has been
the basis for screening of new sources of bioactive
compounds, such as herbal extracts.
The Eurasian smoke tree (Cotinus coggygria
Scop., Anacardiaceae) is a medicinal plant species
with wide distribution from southern Europe, the
Mediterranean, Moldova, and the Caucasus to
Central China and the Himalayas (11). According to
some authors (12, 13) the whole plant is poisonous
due to the large content of gallotannins (above 25%).
However, in the Balkan folk medicine, decoctions
from leaves of C. coggygr ia are applied to treat gingival
and throat inf lammations, stomach ache, gastric
ulcer, diarrhea, nephritis, anthrax, asthma, cardiac
and urinal diseases, and even diabetes mellitus, due
to their antiseptic, anti-inflammatory, antimicrobial,
anti-hemorrhagic, and wound-healing properties
(11, 13, 14, 15).
Studies on the chemical composition of leaves
infusion report the presence of gallic acid methyl ester
and anthocyanins (16), and gallotanins, gallic acid,
flavonic glycosides, myrcen, alpha-pinene, camphen,
linalool, and alpha-terpineol (11, 17). Numerous
polyphenolic compounds have been isolated,
including quercetin, fustin, and taxifolin (18).
The antioxidant activity of extracts from C.
coggygria leaves was demonstrated in several studies,
suggesting the role of high polyphenol content (19,
20, 21, 22). It is noteworthy that the ethanol and
aqueous extracts have shown the highest antioxidant
activity and the highest content of polyphenols
among dozens of investigated Bulgarian medicinal
plants (19). Although that the plant seems to be
extremely rich in biologically active compounds,
it has been somewhat ignored by pharmacological
studies because of the traditionally reported toxicity.
The aim of the present study was to investigate
the effect of aqueous infusion from Cotinus coggygria
leaves (AICCL) on indomethacin-induced gastric
mucosal damage in rats and its possible effect on the
gastric oxidative status.
MATERIAL AND METHODS
Experimental substances
Three aqueous infusions of Cotinus coggygria
leaves (AICCL) were prepared one hour before each
treatment: 1, 2 and 4 g dried material was scalded
in 100 ml boiling distilled water for 10 min. 1/100
AICCL is commensurable to a traditional Bulgarian
recipe for treatment of gastric ulcer (13).
Chemicals
Indomethacin (Indo) was obtained from Sig-
ma-Aldrich (Germany). It was prepared as a suspen-
sion in a vehicle (2 drops of Tween 80 per 5ml of dis-
tilled water). All chemicals used for the biochemical
analyses and histopathological examinations were
of analytical grade and were obtained from Merck
(Germa ny).
Experimental design
Male albino Wistar rats (2-2.5 months old; 220-
250g) were kept under the standard conditions of the
animal house with 12-h light-dark cycle (light 7:00-
19:00) at a temperature 23-25ºC. They were fasted 24
h before the indomethacin administration but had
free access to water. The cohort comprised of five
experimental groups each of eight rats: I. Control
(C), II. Indomethacin (Indo), III. 1/100 AICCL+Indo,
IV. 2/100 AICCL+Indo, V. 4/100 AICCL+Indo.
The rats were orally pretreated by direct stomach
34 Scripta Scientifica Medica, vol. 45, suppl. 3, 2013, pp. 32-38
Copyright © Medical University of Varna
Effect of aqueous infusion from Cotinus coggygria leaves on indomethacin-induced gastric mucosal damage ...
intubation (orogastric cannula) with water (groups I
and II, volume: 10 ml/kg) or AICCL (groups III, IV,
and V, volume: 10 ml/kg). Three days later, the rats
were treated by a single intragastric administration
of indomethacin (dose: 100 mg/kg). All procedures
concerning animal treatment and experimentation
were conducted in compliance with the national laws
and policies and in conformity with the international
guidelines (EEC Council Directive 86/609, IL 358, 1,
December 12, 1987).
Blood serum and tissue preparation
The animals were anaesthetized with diethyl-
ether 4 h after the indomethacin treatment. Blood
was collected from the sublingual veins in heparin-
ized tubes. Samples were was centrifuged at 2000g
rpm for 10 min and serum was obtained and stored
at 20°C until biochemical analyses of uric acid (UA),
malondialdehyde (MDA) and sulphydryl groups
(SH-groups) concentrations. After the animals de-
capitation the stomachs were removed immediately,
opened along the great curvature, gently washed in
physiological salt solution, spread over the pad and
observed macroscopically for appearance of muco-
sal lesions. The length of each lesion was measured.
In the case of petechia, five of them were considered
as a 1 mm lesion. Mean ulcer number and area were
calculated. Stomachs were homogenized in 1:5 w/v
50mM phosphate buffer (pH 7.4) at 4000 rpm for 10
min. The homogenate was centrifuged at 800g rpm
for 15 min to discard the sediment and the super-
natant was taken for biochemical analyses: activi-
ty of alkaline phosphatase (ALP); concentrations of
UA and MDA. All manipulations were performed at
4-8ºC. MDA, ALP and UA were determined imme-
diately after thawing the samples.
Histopathological study
Pieces of the stomachs were fixed immediately
after morphometrical examination in 10% neutral
buffered formaldehyde solution. Fixed tissues were
embedded in paraffin, cut into sections and placed
on microscope slides. Staining of the slides with
hematoxylin-eosin was used for the histopathological
examination which was performed under light
microscopy and documented by microphotocamera.
Biochemical assays
Membrane lipid peroxidation was monitored by
MDA in blood serum and stomach homogenates us-
ing the method of Porter (23). Determination of SH-
groups was performed spectrophotometrically in se-
Fig. 1. Effect of AICCL-pretreatment on the number (A) and area (B) of gastric mucosal lesions in rats with indometha-
cin-induced ulceration.
***P<0.001 vs. water control; **P<0.01 vs. Indo; *P<0.05 vs. Indo
Scripta Scientifica Medica, vol. 45, suppl. 3, 2013, pp. 32-38
Copyright © Medical University of Varna 35
Danail Pavlov, Diana Ivanova, Miroslav Eftimov et al.
rum according to the method of Ellman (24). Activi-
ty of ALP in stomach homogenates was measured by
the standard kit of BioSystems S.A. (Spain). UA levels
in blood serum and homogenates were measured by
the standard kits of HUMAN liquicolor (Germany).
Statistical analyses
Data were analyzed by one-way analysis of
variance (ANOVA) followed by Dunnett’s multiple
comparison posttest. Each two independent groups
were compared also by Student’s t-test. A value of
P<0.05 was considered as statistically significant.
Data are expressed as mean ± SEM. GraphPad Prism
v 5.00 statistical software was used.
RESU LTS
Morphometric evaluation of gastric mucosal
damage
No mucosal lesions were detected in rats from
water control group. Indomethacin induced multiple
gastric mucosal lesions in the glandular part of the
stomach, most often 1-4 mm2 in size. The mean
ulcer number in the Indo-group was 11.58 (Fig.
1A), and the mean ulcer area was 22.39 mm2 (Fig.
1B). Pretreatment with 2/100 AICCL significantly
reduced the ulcer number by 87% (Fig. 1A), and the
ulcer area by 96% (Fig. 1B), respectively.
Biochemical
markers
Control
(Water)
Indomethacin
(Indo+Water)
1/100 AICCL
+ Indo
2/100 AICCL
+ Indo
4/100 AICCL
+ Indo
Stomach
МDА [μmol/L] 1.216± 0.10οοο3.285±0.32 **1.462±0.23 **1.369±0.16**1.660±0.11
UA [μmol/L] 225.7±37.9 οοο447.1±19.0 **210.7±38.6 **312 .5±39.5 **247.4±40.2
ALP [U/L] 19.17±1.53 οο47.28±5.68 **18.23±2.28 **18.31±2.53 **20.90±3.68
Blood
МDА [μmol/L] 0.507±0.02 0.541±0.03 0.483±0.02 0.530±0.02 0.441±0.02
UA [μmol/L] 105.9±4.50 94.13±5.32 89.10±4.22 83.91±4.82 83.17±3.60
SH-groups[μmol/L] 225.6±9.08 220.8±7.47 230.5±9.55 232.0±14.5 4 224.1±12.86
Table 1. Results from biochemical analyses of stomach homogenates and blood serum from rats, treated in a model of
indomethacin-induced ulcerogenesis
Legend: οοοP<0.001 vs. Water Control; οοP<0.01 vs. Water Control; **P<0.01 vs. Indomethacin
Fig. 2. Microscopic appearance (X100) of a rat stomach following the application of water (A), water plus indomethacin
(B), and 2/100 AICCL plus indomethacin (C)
36 Scripta Scientifica Medica, vol. 45, suppl. 3, 2013, pp. 32-38
Copyright © Medical University of Varna
Effect of aqueous infusion from Cotinus coggygria leaves on indomethacin-induced gastric mucosal damage ...
Histopathological study
The microscopic appearance of the gastric
mucosa of the control rats was normal (Fig. 2A). The
experimental lesions evoked by indomethacin were
manifested by multiple erosions comprising about
2/3 of the mucosal layer thickness. The defects were
filled with hemorrhages and epithelial cell necroses
(Fig. 2B). In the rats pretreated with 2/100 AICCL
before the indomethacin administration, the gastric
erosions were more superficial and in some cases only
bleeding and focal desquamation of the epithelium
were found (Fig. 2C).
Biochemical assays
All results from biochemical measurements are
presented in Table 1. The gastric MDA levels of Indo-
group were increased by 270% versus the control. In
the groups pretreated with AICCL before the indo-
methacin administration, the concentration of MDA
decreased significantly (P<0.01) in comparison with
the Indo-group. The same tendency was observed in
UA and ALP levels in the stomach. No significant
changes were found in blood serum levels of MDA,
UA and SH-groups.
DISCUSSION
The indomethacin-induced gastric lesions
could be caused by several mechanisms. It is gener-
ally accepted that the ulcerogenic activity of NSAIDs
is related to their ability to inhibit endogenous PGs
synthesis due to the non-selective inhibition of COX
(8). Some authors (25) concluded that the complete
inhibition of COX leading to decrease in PGE2 con-
tent probably consumed a much longer time and did
not occur 6 h after a single dose. In regard to these
findings, we might suppose that in the current ex-
perimental model the inhibition of the PGs secre-
tion is not the main pathobiochemical mechanism of
ulcerogenesis.
There are data that ROS are involved in the de-
velopment of mucosal damage by NSAIDs (5), and
that they increase lipid peroxidation, an important
cause for cellular membranes damage. In our study,
the involvement of extensive lipid peroxidation in in-
domethacin-induced gastric mucosal damage was
evidenced by the accumulation of MDA in gastric
mucosa (Table 1). Similar are the results obtained
by some other authors (26), who studied the perox-
idation of lipids in the same experimental model. In
their study, the amount of MDA in the gastric muco-
sa was significantly increased 4 h after indomethacin
administration. Presented results demonstrated that
the AICCL reduced the oxidative stress and the re-
lated histomorphological signs of indomethacin-in-
duced gastric mucosal damage. One possible mech-
anism of this effect is the antioxidant potential of
AICCL (19), due to the polyphenols in the infusion,
predominantly high gallic acid content (11, 16, 17).
Another possible mechanism of the gastroprotective
effect of AICCL might be the anti-histamine activity
demonstrated for some f lavonoids (27).
ALP activity has been reported as a biochemi-
cal marker in bone, liver and gastrointestinal lumen
diseases (28). The release of this enzyme is related to
the mechanisms of tissue necrosis (29). The results
from biochemical analysis (Table 1) showed that in-
domethacin increased significantly the levels of ALP
in gastric tissue. The pretreatment with AICCL de-
creased significantly gastric ALP levels, suggesting
a strong protective effect of Cotinus coggygria infu-
sions that was confirmed histopathologically (Fig. 2).
The absence of gastric lesions in AICCL-pretreated
groups was also confirmed by the significantly low-
er gastric UA concentrations. Higher UA levels in
Indo-group could be associated with the degrada-
tion of nucleotides due to elevated necrosis caused by
indomethacin.
CONCLUSIONS
Our study demonstrated a strong protective
effect of aqueous infusion from Cotinus coggygria
leaves against indomethacin-induced gastric
ulceration. Based on results discussed above, we
suggest that the most probable mechanism of this
beneficial effect is the significant decrease of lipid
peroxidation, due to antioxidant properties of plant
investigated.
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Acknowledgments
The financial support from the European Social
Fund within the Project BG051PO001-3.3.06-0028
“Enhancing of the research potential and opportu-
nities for career development in the fields of med-
icine, healthcare and biotechnology” is greatly
acknowledged.
