Drug and Chemical Toxicology
ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: http://www.tandfonline.com/loi/idct20
Gastroprotective effects of oleuropein and thymol
on indomethacin-induced gastric ulcer in SpragueDawley rats
Kubra Koc, Salim Cerig, Serap Ucar, Suat Colak, Murat Bakir, Huseyin Serkan
Erol, Serkan Yildirim, Mirkhalil Hosseinigouzdagani, Nihal Simsek Ozek,
Ferhunde Aysin, Enver Fehim Kocpinar, Harun Budak & Fatime Geyikoglu
To cite this article: Kubra Koc, Salim Cerig, Serap Ucar, Suat Colak, Murat Bakir, Huseyin Serkan
Erol, Serkan Yildirim, Mirkhalil Hosseinigouzdagani, Nihal Simsek Ozek, Ferhunde Aysin, Enver
Fehim Kocpinar, Harun Budak & Fatime Geyikoglu (2018): Gastroprotective effects of oleuropein
and thymol on indomethacin-induced gastric ulcer in Sprague-Dawley rats, Drug and Chemical
Toxicology, DOI: 10.1080/01480545.2018.1530261
To link to this article: https://doi.org/10.1080/01480545.2018.1530261
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DRUG AND CHEMICAL TOXICOLOGY
https://doi.org/10.1080/01480545.2018.1530261
RESEARCH ARTICLE
Gastroprotective effects of oleuropein and thymol on indomethacin-induced
gastric ulcer in Sprague-Dawley rats
Kubra Koca , Salim Ceriga, Serap Ucara, Suat Colakb, Murat Bakira, Huseyin Serkan Erolc, Serkan Yildirimd,
Mirkhalil Hosseinigouzdagania, Nihal Simsek Ozeka, Ferhunde Aysina,e, Enver Fehim Kocpinarf, Harun Budakg
and Fatime Geyikoglua
a
Department of Biology, Faculty of Science, Ataturk University, Erzurum, Turkey; bDepartment of Biology, Erzincan University, Erzincan,
Turkey; cDepartment of Biochemistry, Faculty of Veterinary, Ataturk University, Erzurum, Turkey; dDepartment of Pathology, Faculty of
Veterinary, Ataturk University, Erzurum, Turkey; eEast Anatolian High Technology Research and Application Center (DAYTAM), Ataturk
University, Erzurum, Turkey; fDepartment of Medical Laboratory, Techniques Vocational School of Health Services, Alparslan University, Mus,
Turkey; gDepartment of Molecular Biology and Genetics, Faculty of Science, Ataturk University, Erzurum, Turkey
ABSTRACT
ARTICLE HISTORY
Ethnopharmacological studies demonstrated that thymol (Thym) and oleuropein (Ole) have therapeutic
potential for gastric ulcers. The molecular mechanism underlying the gastroprotective effects of these
compounds have not been elucidated yet especially for their individual and combination use at high
dose. Therefore, this study was conducted to explore their gastroprotective mechanisms on indomethacin (Indo)-induced gastric ulcer model. Ole (50,100, 250, and 500 mg/kg) and Thym (50,100, 200, and
500 mg/kg) were orally administered to the rats 10 min before the induction of ulcer with Indo. The
combination of 500 mg/kg doses of Ole and Thym were applied. The gastric mucosa was evaluated
histopathologically. Moreover, TAC/TOS, tumor necrosis factor-alpha (TNF-a), prostaglandin E2 (PGE2),
endothelial nitric oxide synthase (eNOS), and caspase-3 levels were assessed by ELISA and the caspase3 and TNF-a expressions were quantified by qRT-PCR. Indo-induced histopathological changes while
Ole and Thym pretreatment prevented these effects. Unlike the 500 mg/kg dose of Ole treatment, the
500 mg/kg dose of Thym administration enhanced these damages. The decreased TAC, PGE2 levels and
increased TOS, eNOS, TNF-a, caspase-3 levels were obtained in Indo group. However, these changes
were reversed by Ole and Thym groups except the 500 mg/kg dose of Thym and the combination
treatment groups. Similar trends were observed in the caspase-3 and TNF-a expression levels. These
results demonstrated that enhanced inflammation, oxidant/antioxidant imbalance, and apoptotic activities were occurred in Indo, 500 mg/kg dose of Thym and the combination treatment groups while not
in the other groups. The findings demonstrated the gastroprotective ability of Ole and low doses of
Thym in gastric ulcer models.
Received 7 April 2018
Revised 8 September 2018
Accepted 23 September 2018
Introduction
Gastric ulcer disease is one of the most common disorders of
the gastrointestinal tract with a global prevalence, affecting
about 4.6 million people annually and having a mortality of
one death per 10 000 cases (Oluwole 2015). Since this disease has several complications; it negatively affects the overall quality of life. It has been well known that several
etiological factors like alcohol consumption, the use of nonsteroidal anti-inflammatory drugs, stress, and Helicobacter
pylori infection increase the chances of gastric ulcer development (Verma and Kumar 2016). This development is related
to the imbalance between mucosal aggressive and defensive
factors caused by the oxidative stress and inflammation.
Therefore, these factors are considered as an important contributor to the pathogenesis of gastric ulcers (Guruprasad
et al. 2015). Up to now, there are many studies performed to
reveal the mechanism of this disease development. However,
the pathogenesis of gastric ulcer has not been clarified yet
CONTACT Kubra Koc
kubrakc@hotmail.com
Apoptosis; cytokine; gastric
ulcer; oleuropein; oxidative
stress; thymol
since it is a multifactorial disease. To elucidate the etiopathogenesis, indomethacin (Indo) and ethanol-induced gastric
ulcer models in rats are commonly used as a model of differentiated-type human stomach diseases (Chen et al. 2016,
Yodoi et al. 2016). These models produce a unified size of
ulcer, which is very useful for the assessment of different
drugs’ healing effects.
The treatment strategy of the ulcer is generally based on
the antisecretory therapy and proton pump inhibitors.
However, these therapeutic strategies do not have a complete protective/curative effect of this disease and they have
side effects. Therefore, alternative therapies based on medicinal plants are considered as better alternatives and main
source of new drugs for the treatment of peptic ulcer since
histological studies revealed that these medicinal plants did
not show any acute toxicity. Due to their better compatibility
with the human body and lesser adverse effects as compared
to the current anti-ulcer agents, there is an extensive
Department of Biology, Faculty of Science, Ataturk University, Erzurum, Turkey
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
2
K. KOC ET AL.
research to find out plant derived antiulcer agents (Dharmani
et al. 2005, Kumar 2011). Among this agent, olive leaves,
thyme oil, and their extracts are taken into consideration
since they include valuable bioactive substances such as
oleuropein (Ole) and thymol (Thym) (Tuck and Hayball 2002).
Previous studies indicated that Ole was generally obtained
from an edible food Olea europaea with various medicinal
uses (El and Karakaya 2009). On the other hand, Thym is
mostly derived from medicinal plants and it was extracted
from Origanum species (Origanum onites and Origanum vulgare) and Thymus vulgaris (Chorianopoulos et al. 2004). Olive
leaf extracts itself had a potential to prevent gastritis induced
by HCl/ethanol via alleviating inflammation and oxidative
imbalance (Al-Quraishy et al. 2017). Especially, the extracts of
Origanum vulgare protected the intestinal mucosa from injuries (Azari et al. 2016) and it showed a possible anti-cancer
activity against colon carcinogenesis (Srihari et al. 2008). Also,
the extracts of T. vulgaris had potential antiulcer effects
(Oliveira et al. 2014).
Ole is a polyphenolic constituent of olive leaves with various biological benefits (Barbaro et al. 2014) (Figure 1(a)). The
potential antioxidant activity of Ole is mainly due to the presence of hydroxyl groups in its chemical structure. These
hydroxyl groups could donate hydrogen to prevent oxidation
and it protects from the possible side effects of the oxidative
stress (Hassanzadeh et al. 2014). In addition, Thym is a monoterpene phenolic compound found in the oils of thyme with
valuable biological characteristics (Basch et al. 2004) (Figure
1(b)). The pharmacophore feature of the phenolic hydroxyl
group in Thym’s chemical structure confer protection against
the deleterious effects of free radicals both by absorbing or
neutralizing free radicals and by augmenting endogenous
antioxidants (Wojdylo et al. 2007).
The therapeutic/protective efficacy of the Ole and Thym
on different diseases has been indicated in traditional medicine and clinical studies (Sumbul et al. 2011, Meeran et al.
2017). Our unpublished studies on the protective effects of
both Ole and Thym in Indo-induced liver and renal damages
demonstrated that pretreatment with these natural compounds attenuated the adverse effects of this drug. In addition, gastroprotective effects of Thym have been
demonstrated in ulcer models including Indo-induced ulcer
model (Chauhan and Kang 2015, Ribeiro et al. 2016). Ribeiro
et al. (2016) revealed that 100 mg/kg dose of Thym treatment
reduce acute and chronic ulcers induced by ethanol, Indo,
and acetic acid through the increased mucus secretion, prostaglandins, and ATP-sensitive K þ channels. In addition to
Thym, the anti-ulcer activity of Ole has been indicated only
in ulcer models except Indo-induced disease model (Alirezaei
et al. 2012). Alirezaei et al. indicated that 12 mg/kg dose of
Ole pretreatment significantly lowered gastric ulcers induced
by ethanol through the elevation of glutathione peroxidase,
superoxide dismutase, and catalase activities. However, in
these studies, only low doses of both compounds with a
range of 10–100 mg/kg were used and just biochemical and
physiological parameters such as enzyme activity and mucus
secretion were investigated. Up to now, there is no comprehensive study including histological, biochemical, and genetic
expression examinations on the anti-ulcer activities of Ole
and Thym with their low and moderate doses. Therefore, the
first aim of this study was to elucidate the protective potential via histopathological, genetic, and biochemical analysis.
In addition, the clinical results remained unclear for the availability and reliability of these natural compounds at higher
doses. For this reason, the second aim of this study was to
determine the protective efficacy of high doses of Thym and
Ole with their individual and combination use on gastric ulceration.
Materials and methods
Animals
Sixty Sprague-Dawley female rats, whose weight range
250–300 g were used in the study. Constant environmental
conditions were maintained with a temperature of 22 ± 1 C,
humidity of 55% and a 12-h light/dark cycle. Experiments
were performed according to the Guide for the Care and Use
of Laboratory Animals published by the US National
Institutes of Health (NIH publication No. 85–23, revised 1996).
All experimental procedures in this study were approved by
€rk University Local Ethics Committee for Animal
the Atatu
Experiments (No. 104, 02.06.2015).
Chemicals
Indo was purchased from DEVA Holding A.S. (Istanbul,
Turkey). Certified chemical standard of Thym (>99.5%), Ole
(HPLC grade 98%) and all the other chemicals were purchased from Sigma-Aldrich International (St. Louis, MO).
Experimental design
Figure 1. (a)The chemical structures of Oleopein and (b) Thymol.
The design of this study related to the application of the
tested agents was performed based on the previous studies
about protective effects of plant-based products such as Ole
and Thym (Manna et al. 2004, Sumbul et al. 2011, Alirezaei
et al. 2012). In these studies, natural compounds/plant-based
products were given with drugs which have tissue toxicities.
One reason of this experimental design is that both compounds administered alone did not cause any biochemical
and histological changes in animals (Alam et al. 1999, Kumral
et al. 2015, Achour et al. 2016, El-Sayed et al. 2016). Based on
DRUG AND CHEMICAL TOXICOLOGY
these studies, due to ethical considerations in animal experiments especially to keep sample number at minimum, we
have performed both natural products in only Indo-induced
gastric ulcer models.
The rats were divided into twelve groups: normal control,
ulcer (25 mg/kg dose Indo only), Indo þ reference standard
(50 mg/kg dose Rantidine hydrochloride [Ran]), Indo þ Ole
(50, 100, 250, and 500 mg/kg doses), Indo þ Thym (50, 100,
200, and 500 mg/kg doses), Indo þ Thym (500 mg/kg dose) þ
Ole (500 mg/kg dose). Doses of Ole and Thym were determined according to the previous studies (Miller et al. 2015,
Halici et al. 2016) and our dose-based preliminary studies.
The animals were fasted 24 h prior to administration of each
of control, standard, and tested compounds. All substances
used were dissolved in distilled water. First, Ole and Thym
solution (1 ml/rat) were administered orally to the abovementioned groups. After 10 min of this treatment, Indo solution (0.5 ml) was given orally at the specified doses. Six hours
after treatment, the rats were anesthetized using sevoflurane
(3.5 vol% and 30% oxygen: 70% N2O) for 30 min using a ratadapted mask. The stomachs were dissected, opened, and
rinsed with phosphate-buffered saline (PBS) to remove the
gastric contents.
Histopathological examinations and assessments
Stomach sections were fixed in 10% formalin embedded in
paraffin and then serially-sectioned (5-lm thick) by using a
Leica RM2135 microtome (Leica, Berlin, Germany), mounted
on glass slides and stained using with hematoxylin and eosin
(H&E) solution. To localize the gastric mucus production,
some slides were stained by periodic acid–Schiff (PAS) base
(Sigma-Aldrich, PAS kit). The extent of mucosal fibrosis and
definition of an amyloid fibril protein were detected
using Masson and Congo red-staining kits, respectively. Then
sections were examined under a light microscope by an
experienced pathologist who was blind for the treatment
(Yildirim et al. 2018). The high-resolution pictures of samples
(200 and 400) were taken under bright field using an
Olympus BX60 microscope.
Determination of TAC/TOS, PGE2, eNOS, TNF-a levels,
and caspase-3 activity
All activity measurements were obtained from tissue homogenates. Fresh tissues were rinsed with ice-cold saline and
immediately stored at –80 C. The tissues were weighed and
homogenized in 50-mM phosphate buffered saline (PBS) at
pH 7.0. Then centrifuged at 10 000 rpm at 4 C for 15 min to
collect the supernatant for subsequent analysis. The automated Trolox equivalent total antioxidant capacity (TAC) and
total oxidant status (TOS) assays were carried out in tissue
homogenates using commercially available kits (Rel Assay
DiagnosticsV, Gaziantep, Turkey). The tumor necrosis factoralpha (TNF-a), prostaglandin E2 (PGE2), endothelial nitric oxide
synthase (eNOS) levels, and caspase-3 activity were acquired
via the commercially available enzyme-linked immunosorbent
assay (ELISA) kits. Procedures were performed according to
R
3
the manufacturers’ instructions in each kit (rat TNF-a kit,
Biolegend, San Diego, CA; PGE2 kit, Cayman Chemical
Company, Ann Arbor, MI; eNOS kit, Korain Biotech, Junjiang
Internatioanl Bldg., SH. China; caspase-3 activity kit, Beyotime
Institute of Biotechnology, Haimen, China).
Caspase-3 and TNF-a expressions
Total RNA was isolated from frozen rat gastric tissues using
Pure Link RNA Mini Kit according to manufacturer’s instruction (Thermo Fisher Scientific, ambion life technologies Cat.
No. 12183018A). RNA concentrations and quality were verified by spectrophotometer (Thermo Scientific, Multiskan GO)
and cDNA was synthesized using ProtoScript First Strand
cDNA Synthesis Kit (BioLabs, New England, Cat. No.
NEB #E6300S).
Quantitative real-time PCR (qPCR) was performed by using
SYBR Green method. Both target and reference genes were
purchased from metabion international (Martinsried,
Germany). Their gene symbols were: caspase-3, TNF-a, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as
a reference gene since it was not affected by any of the
treatments. The sequences of the specific primers of rat
GAPDH (NM_017008.4) were as follows: GAPDH forward primer 50 -TGGACCTCATGGCCTACATG-30 ; GAPDH reverse primer
50 -AGGGAGATGCTCAGTGTTGG-30 . Thermal cycler real-time
PCR (Rotor Gene Q, Qiagen) machine was used to perform
gene expression experiments. The 25 ml PCR reaction volume
contained 10 ng/ml template DNA, 0.2 pmol of forward and
reverse primers, 12.5 ml of SYBR Green Master Mix. The PCR
conditions were as follows: 50 C for 2 min, 95 C for 10 min,
45 cycles of 95 C for 10 s, annealing, and extension at 60 C
for 1 min. GAPDH expression was used for normalization and
relative quantification (DDCT method).
Statistical analysis
Results are expressed as the mean ± SD. The differences in
variance were analyzed statistically using one-way analysis of
variance (ANOVA) test by Graphpad Prism version 5.0 statistics software (GraphPad, La Jolla, CA). Tukey’s test was used
as a post hoc and p < 0.05 was considered as statistically significant. The superscripts of a and b were used to compare
the control and Indo groups with other studied groups.
Results
The macroscopic examination
Representative stomach of each group is shown in Figure 2.
Stomach of control rats showed a normal anatomical structure with an intact corpus (Figure 2(a)), whereas the oral
administration of Indo produced ulcers with well-defined
hemorrhagic streaks (Figure 2(b)). Macroscopic examinations
revealed that the Ran afforded the protection on the mucosa
(Figure 2(c)). However, the low doses (50 and 100 mg/kg) of
Ole provided a poor improvement on Indo-induced gastric
ulceration (Figure 2(d,e)). On the other hand, increasing doses
4
K. KOC ET AL.
Figure 2. Macroscopic findings of Ole and Thym treatments on Indo-induced gastric mucosal lesions. (a): Control (Normal), (b): Indo, (c): Indo þ Ran, (d): Indo
þ50 mg/kg Ole, (e): Indo þ100 mg/kg Ole, (f): Indo þ250 mg/kg Ole, (g): Indo þ500 mg/kg Ole, (h): Indo þ50 mg/kg Thym, (i): Indo þ100 mg/kg Thym, (j): Indo
þ200 mg/kg Thym, (k): Indo þ500 mg/kg Thym, (l): Indo þ500 mg/kg Thym þ500 mg/kg Ole. Abbreviations: Indo, Indomethacin; Ran, Ranitidine; Ole, Oleuropein;
Thym, Thymol.
(250 and 500 mg/kg) of Ole showed a remarkable protective
effect in comparison with Ran-treated rats (Figure 2(f,g)).
Similar effects were also detected in the Thym-treated groups
(Figure 2(h–j)). However, the gastric damage in the Indo
þ500 mg/kg Thym group was more profound than those of
the Indo group (Figure 2(k)). Although the protective effect
of Ole with 500 mg/kg was observed, this effect was found to
be insufficient for the gastric injury induced by 500 mg/kg
dose of Thym and Indo treatment (Figure 2(l)).
Histopathological studies and assessments
To evaluate the morphology of gastric mucosa, H&E, PAS,
Congo Red, and Masson trichrome staining were carried out.
The results of each dye are demonstrated in Figures 3–6. It
was found that there was a strong correlation between the
macroscopic and microscopic findings. As can be seen in
Figure 3(a–h), no gastric damage was observed in control
group (Figure 3(a1,a2)) whereas Indo group presented
remarkably distinctive injuries to the gastric mucosa, which
deeply penetrated into the gastric epithelium. Moreover, congestion, leukocyte aggregation, severe alterations in the
architecture of glandular parts, hemorrhages, the depletion
of the gastric mucus layer and amyloidosis were observed in
Indo group (Figure 3(b1–b4)). Although, a decrease in Indoinduced gastric lesions was obtained with Ran treatment,
congestion, hemorrhages, and cellular infiltration were
observed in this group (Figure 3(c)). In addition, the low
doses (50 and 100 mg/kg) of Ole and Thym-treatment could
not return stomach tissue into its normal architecture (data
not shown) (Figure 3(d–f)). This protective effect was
observed only in the 200 mg/kg dose of Thmy treatment.
However, the highest dose (500 mg/kg) of Thym and Indo
treatment enhanced the pathological structure in the gastric
DRUG AND CHEMICAL TOXICOLOGY
5
Figure 3. Rat stomach (H&E). (a): Control (a1): Normal structure of stomach (a2): Normal structure of gastric glands, (b): Indo group (b1): Focal necrosis of gastric
mucosa (white arrow) (b2): Congestion (black arrow), infiltration (I), Dilatation of glands (b3): Extensive congestion in glands (black arrow) (b4): Severe hemorrhagic
bands in the gastric mucosa (H), (c): Indo þ Ran group; Congestion (black arrows), hemorrhage (H), infiltration (I), (d): Indo þ250 mg/kg Ole; normalized appearances
of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in Indo þ200 mg/kg Thym, (g): The damaged gastric tract in Indo
þ500 mg/kg Thym, (g1): Hemorrhage (H), (g2): Congestion (black arrow), (h): Ulcer in Indo þ500 mg/kg Thym þ500 mg/kg Ole, Hemorrhage (H). For abbreviations
see legend Figure 2.
mucosa when compared with those of the Indo group
(Figure 3(g1,g2)). Moreover, the 500 mg/kg dose of Ole treatment indicated a slight protective effect on Indo þ500 mg/kg
Thym and the observed pathological findings were similar to
those of Indo group (Figure 3(h)). Congo Red staining findings indicated that no amyloid deposit was present in control, Indo þ250 mg/kg Ole, Indo þ500 mg/kg Ole, and Indo
þ200 mg/kg Thym groups. On the contrary, this deposit was
present in the Indo, Indo þ Ran, Indo þ500 mg/kg Thym, and
Indo þ500 mg/kg Thym þ500 mg/kg Ole groups (Figure
4(a–h)). Based on PAS staining results (Figure 5(a–h)), control
group had a gastric mucus whereas Indo induced mucus
depletion. Although less mucus content was observed in
Indo þ Ran treated group, an increased mucus secretion was
observed with the 250, 500 mg/kg doses Ole, and 200 mg/kg
dose of Thym treatment. In addition to these results, 500 mg/
kg dose of Thym treatment led to mucus depletion. The
fibrotic content in stomach was evaluated based on Masson
staining results, which revealed that this content was not
changed control and Indo treated groups. Similarly, any
fibrotic content was not observed in all of the doses of Ole
and Thym (Figure 6(a–h)).
6
K. KOC ET AL.
Figure 4. Rat stomach (Congo Red). (a): Stomach without amyloid in control, (b): Amyloidosis after Indo (A), (c): Indo þ Ran group; Amyloidosis (A), (d): Indo
þ250 mg/kg Ole; normalized appearances of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in Indo þ200 mg/kg
Thym, (g): The damaged gastric tract in Indo þ500 mg/kg Thym; Amyloidosis (A), (h): Ulcer in Indo þ500 mg/kg Thym þ500 mg/kg Ole; Amyloidosis. For abbreviations see legend Figure 2.
Biochemical studies
Indo treatment significantly reduced TAC and PGE2 levels
while it increased TOS, eNOS, TNF-a levels, and caspase-3
activity with respect to the control group (Figure 7). On the
other hand, the low doses (50 and 100 mg/kg) of Ole and
Thym-treatment reversed the amount of the measured biochemical parameters induced by Indo application. However,
the most profound changes in these parameters were determined in the 250 mg/kg dose of Ole and 200 mg/kg dose of
Thym treated group. Ole and Thym have better protective
potential against Indo-induced ulcer compared to Ran. In
addition, 500 mg/kg dose of Ole provided a similar protective
effect on these parameters. However, the highest dose
(500 mg/kg) of Thym caused a significant increase in TOS,
eNOS, TNF-a, and caspase-3 levels when compared with Indo
group. However, 500 mg/kg dose of Ole treatment did not
alter caspase-3 activity compared to the Indo group, implying
that it could not prevent apoptotic activity induced by Indo
þ500 mg/kg dose of Thym treated group.
Molecular biology studies
Indo administration induced a remarkable increase in mRNA
levels of TNF-a and caspase-3 which were 28.5- and 5-fold
higher than those levels of the control group. Both Ole and
Thym treatments significantly decreased TNF-a and caspase-3
expression levels with respect to the Indo group (p and 5-fold
higher than those levels of the control group. Both Ole and
Thym treatments significantly de 32 and 26% of the Indo
group’s values. A significant decrease in caspase-3 levels was
observed which corresponds to 43 and 35% of the Indo
groups’s values (p values (of the Indo group’s values. A significant decrease in caspase-3 levels was observed which co
mRNA expression levels of TNF-a and caspase-3 by 35.7 and
42.4% with respect to the ulcer control group. No significant
differences were also observed between Indoþ 500 mg/kg
Thym þ500 mg/kg Ole and Indo-treated rats for these levels.
Discussion
Several studies on the pathogenesis of gastric ulcer proved
that oxidative stress is the main cause of this disease.
Therefore, elimination of oxidative stress by antioxidant
agents, such as Ole and Thym can be used as a protective
approach. The protective efficacy of these compounds with
low doses between 10 and 100 mg/kg in gastric ulcer was
previously demonstrated (Alirezaei et al. 2012, Chauhan and
Kang 2015, Ribeiro et al. 2016). However, this ability of both
molecules at moderate and high doses in gastric ulcers has
not been shown yet. Acute toxicity study on Thym in rats
DRUG AND CHEMICAL TOXICOLOGY
7
Figure 5. Rat stomach (PAS). (a): Gastric-mucus in controls (asterisk), (b): Indo-induced mucus depletion, (c): Indo þ Ran grup, Slight mucus content in gastric
mucosa, (d): Indo þ250 mg/kg Ole; normalized appearances of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in
Indo þ200 mg/kg Thym, (g): The damaged gastric tract in Indo þ500 mg/kg Thym (g1): mucus depletion (g2): Extensive hemorrhage, (H), (h): Ulcer in Indo
þ500 mg/kg Thym þ500 mg/kg Ole; unregulated mucus discharge. For abbreviations see legend Figure 2.
indicated that the lethal dose (LD50) of thymol was
2462.23 mg/kg (Gad 2012). Moreover, 1000 mg/kg dose of
Ole was found to be nontoxic for mice (Petkov and Manolov
1972). Therefore, this study was conducted to investigate and
determine for the first time, the protective mechanism of Ole
and Thym at moderate and high doses in the gastric injury
induced by Indo ingestion. In addition, we investigated the
gastroprotective efficiency of Ole and Thym in combination
especially for high dose (500 mg/kg). Indo-induced gastric
ulcer model was preferred since this drug often causes
gastrointestinal side-effects in patients and is widely used to
induce gastrointestinal ulcers in animal models (Park et al.
2014, Yodoi et al. 2016).
A number of studies suggested that Indo had common
severe effects on the gastric mucosa, such as ulceration,
deep erosions with shedding of the superficial epithelial cell,
and cellular infiltration (Xie et al. 2013, Farzaei et al. 2015).
Therefore, to assess the severity of gastric damage induced
by Indo, a detailed microscopic analysis was performed on
H&E stained sections. In agreement with the previous studies,
we detected a disarrangement of epithelial barrier integrity
and serious congestion of gastric vessels with Indo treatment.
On the contrary, the Ole (250 and 500 mg/kg) and Thym
(200 mg/kg) pretreatment prevented the formation of these
damages. It has been known that any disruption of the epithelial barrier causes gastric ulceration (Saeki et al. 2000).
Therefore, Indo-induced gastric ulcer may be arised from the
disruption of mucosal barrier. In addition, this drug-induced
congestion may be due to the constriction of the small
arteries and venules and the restriction of mucosal blood
flow (Freudenthaler et al. 2016). Based on these findings it
can be inferred that, the curative potential of Ole and Thym
may be stem from the strengthened mucosal barrier, maintenance of mucosal integrity, and accurate blood flow
(Chauhan and Kang 2015). In consistence with our findings,
the protective of Ole and Thym pretreatments in gastric ulcer
were also demonstrated in the histological structure of stomach in other studies (Dekanski et al. 2009, Chauhan and
Kang 2015).
In addition to H&E staining, to better understand the
architectural alterations in the gastric mucosa cells, we carried out Congo red, PAS, and Masson staining. Congo red
staining results indicated that an interstitial amyloid deposit
was found with Indo administration. However, these deposits
were not seen in the 250, 500 mg/kg doses of Ole and
200 mg/kg dose of Thym treated group. This deposit formation may be due to Indo-enhanced oxidative stress (Alirezaei
et al. 2014). However, the elimination of these amyloid
8
K. KOC ET AL.
Figure 6. Rat stomach (Masson). (a): Control stomach, (b): Unchanged fibrotic content after Indo (A), (c): Indo þ Ran grup; Unchanged fibrotic content, (d): Indo
þ250 Ole mg/kg; normalized appearances of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in Indo þ200 mg/kg
Thym, (g): The damaged gastric tract in Indo þ500 mg/kg Thym; unchanged fibrotic content, (h): Ulcer in Indo þ500 mg/kg Thym þ500 mg/kg Ole; Normal fibrotic
content. For abbreviations see legend Figure 2.
deposits may be associated the reduced oxidative stress by
Ole and Thym treatments. A previous study stated that this
deposit was responsible for the formation of hemorrhages.
Therefore, Indo-induced gastric hemorrhages may be arisen
from the formation of amyloid deposits (Miller et al. 2015). It
has been reported that mucus secretion had an important
role in the gastroprotection since the mucus lipids and glycoproteins had an antioxidant property and protected the
mucus against oxygen radicals (Gong et al. 1990, Tarique
et al. 2016). Therefore to evaluate the mucus content, PAS
staining was performed, indicating a decrease in the mucus
secretion by Indo treatment. This decrease may be due to
the Indo-enhanced free radical production (Naito and
Yoshikawa 2016). However, enhanced mucus secretion was
observed in the 250, 500 mg/kg doses of Ole and 200 mg/kg
dose of Thym treated group, which may be arisen from the
decreased free radical amount by these compounds. In consistence with our results, the enhanced effect of Thym on the
mucus secretion was previously demonstrated in Indoinduced gastric ulcer rat model (Ribeiro et al. 2016).
In addition to histopathological examinations, the determination of biochemical changes is an important factor for
revealing tissue damage caused by oxidative stress.
Numerous antioxidant enzymes are involved in the protection of gastric mucosa from oxidative stress. The imbalance
between oxidant status and antioxidant capacity is a
substantial determinant for demonstration of the oxidative
damage. Therefore, TAC and TOS levels were determined in
Indo, as well as in Ole and Thym pretreated tissue homogenates to assess alterations in oxidative stress levels of stomach. According to our study, Indo treatment inhibited the
production of antioxidants, whereas Ole (p According to our
study, Indo treatment inhibited the production of antioxidants, whereas Ole (hym nd Thym d to standard drug (ranitidine). Moreover, it has been reported that the use of
antioxidants as dietary supplements in combination with conventional treatment could play a pivotal part in the management of gastric and duodenal ulcers (Lee et al. 2016).
Besides, TOS level was significantly lower in the control
group (p Besides, TOS level was significantly lower in
thIndo group. TOS levels were higher in Ole and Thymtreated groups than in Indo group. Our results were in
accordance with those of Chu et al. (2016) that evaluated the
oxidative stress using Indo. Along with the determination of
TAC and TOS levels, eNOS, TNF-a, PGE2, and Caspase-3
amounts were also detected as bioimportant markers of oxidative stress. eNOS causes loss of membrane fluidity, impairment of ion transport and membrane integrity, and
ultimately failure in cellular function (Bindu et al. 2013). eNOS
generated at the inflammatory site contributed to the onset
and progression of gastric ulcer (El-Ashmawy et al. 2016).
Previous studies stated that the first physiologic defense
DRUG AND CHEMICAL TOXICOLOGY
9
Figure 7. The effects of Thym and Ole on stomach (a) TAS (b) PGE2 (c) TOS (d) eNOS (e) TNF-a (f) Caspase-3 levels after treated with Indo. Control: TAS, PGE2, TOS,
eNOS, TNF-a, and Caspase-3 levels in normal rats. Indo: Indo induced a significant increase in TOS, eNOS, TNF-a levels and caspase-3 activity, while decrease TAC
and PGE2 levels in comparison to the control. Indo þ Ran: Reversal of TAS, PGE2, TOS, eNOS, TNF-a, and Caspase-3 levels in Ran treated rats in comparison to Indo.
Indo þ50 mg/kg Ole, Indo þ100 mg/kg Ole, Indo þ50 mg/kg Thym, Indo þ100 mg/kg Thym: Dose-dependent improvement of Ole and Thym treatment on the levels of biochemical parameters against Indo-induced ulcer. Indo þ200 mg/kg Thym, Indo þ250 mg/kg Ole, Indo þ500 mg/kg Ole: Ole and Thym treatment most profoundly improved the levels of biochemical parameters against Indo-induced ulcer. Indo þ500 mg/kg Thym: Thym caused a significant increase in TOS, eNOS, TNF-a
and caspase-3 levels when compared with Indo group. Indo þ500 mg/kg Thym þ500 mg/kg Ole: Worsening effects of Thym and Ole treatment on the levels of biochemical parameters against Indo-induced ulcer. Data are presented as mean ± SD (n ¼ 5). a denotes significant differences between other studied groups and control (a1:p < 0.05, a2:p < 0.01, a3:p < 0.001, a4:p < 0.0001), b denotes significant differences between other studied groups and Indo group (b1:p < 0.05, b2:p < 0.01,
b3:p < .0.001, b4:p < 0.0001) by Tukey’s multiple range tests. For abbreviations see legend Figure 2.
against cellular damage in any organ was the inflammation
emerging with rapid vascular changes (i.e., increased permeability and blood flow), followed by cellular alteration (e.g.,
infiltration by acute and chronic inflammatory cells). Indoinduced eNOS can cause serious early vascular injury resulting in blood flow stasis and necrosis of peripheral epithelial
and mesenchymal cells (Antonisamy et al. 2014). In order to
further confirmation of the NO production, TNF-a was measured as an indicator of NO production in endothelial cells
(Boss et al. 2016). Our data support a similar situation in
ulcerated animals where inducible TNF-a production
enhanced inflammation-associated cytokines such as eNOS
(Jagla and Pechanova 2015). In our experiments, we observed
that high doses of Ole (250 and 500 mg/kg) lead to the
changes in microcirculation modulated through eNOS and
anti-inflammatory activity. Olive polyphenols have been
found to play an important protective role in inflammationrelated diseases (Wedler et al. 2016). The polyphenols are
10
K. KOC ET AL.
Figure 8. The effects of Thym and OLE on stomach relative mRNA expression of (a) TNF-a (b) Caspase 3 after treated with Indo. Data are presented as mean ± SD
(n ¼ 5). a denotes significant differences between other studied groups and control (a4:p < 0.0001), b denotes significant differences between other studied groups
and Indo group (b4:p < 0.0001) by Tukey’s multiple range tests. For abbreviations see legend Figure 1. For abbreviations see legend Figure 2.
vasoprotective and the protection mechanisms may include
the upregulation of eNOS expression and NO production
(Bertin et al. 2016), reduction in TNF-a and superoxide anion
generation (Sinnett et al. 2016). In this study, however, antiinflammatory function of Ole may be due to its direct effect
on neutrophils, in which inhibited the production of proinflammatory cytokines, such as TNF-a and eNOS. The inflammation is aggravated by the recruitment of inflammatory
cells, such as neutrophils and mononuclear cells (Murch et al.
1991). A number of studies indicated that Indo induced the
infiltration of neutrophils in gastric mucosa, which directly
correlated with the genesis of ulcerative lesions with the
amount of migrant neutrophils and the initiation of acute
inflammation process. In innate immune response, neutrophils represent the first step of basic defense. On the other
hand, there was also alternative ways of free radical generation and other forms of ROS, such as superoxide anions
(Wallace and Granger 1996). Finally, the inflammation of gastric mucosa induced the release of cytokines (Wallace et al.
2004). Our first explanation for this is that the Ole treatment
may prevent the inflammatory process and suppress inflammatory responses of inflammatory cells in Indo-induced
inflammation.
Prostaglandins play a key role in the defense mechanisms
of the gastric mucosa, and their production seems to be
dependent on the capacity for free-radical-neutralization and
the antioxidant potential following the damage by Indo (da
Silva Junior et al. 2016). Especially, PGE2 increases the resistance of microvascular endothelial cells and prevents thrombin-induced deterioration of endothelial junctions (Alirezaei
et al. 2014). Therefore, the deficiency in prostaglandin levels
may play a substantial role in the pathogenesis of the formation of ulcers induced by Indo, and this may be due to the
presence of oxidative damage (Boss et al. 2016). Thereby,
drugs that have an antioxidant effect can protect the gastric
mucosa against damage caused by ulcerogenic agents
(Bouaziz et al. 2005). Our second explanation encompasses
the antioxidant activity of agents like Ole. In this study, oral
administration of Ole with indicated an antioxidant activity
significantly prevents gastric injuries induced by Indo (Otani
et al. 2016). It has been reported that these agents contain a
phenolic hydroxyl group, which allows them to perform antioxidant activity by scavenging free radicals (Lin et al. 2016).
Thus, it can be said that Ole may be beneficial for the prevention of oxidative stress in the stomach and hemorrhage
can be controlled with PGE2 regulation. Apoptosis is another
important pathophysiological pathway activated during the
formation of Indo-induced ulcer (Yin et al. 2016). There are
two major apoptotic pathways (extrinsic and intrinsic).
Caspase-3 is a key factor particularly associated with the
DRUG AND CHEMICAL TOXICOLOGY
intrinsic pathway and its activation may take place either
following the activation of the mitochondrial-mediated
pathway or the death receptor pathway (Bakkali et al.
2008). In this study, the high doses of Ole (250 and
500 mg/kg) provided a significant reduction in pro-apoptotic marker (Caspase-3) in Indo-induced ulceration.
However, the therapeutic potential of 500 mg/kg Ole was
inadequate against apoptotic behavior of high dose Thym
(500 mg/kg) and Indo in which they used together in a
treatment. There have been no documented reports on
antiulcerogenic activity of increasing doses of Thym. As a
phenolic compound, Thym has a high potential to scavenge oxidative radicals by donating protons from its phenolic hydroxyl groups (Michiels et al. 2010). According to a
previous study, lower doses of Thym (30 and 100 mg/kg)
displayed gastroprotective actions on the acute ulcer
model (10 mg/kg Indo) through mechanisms involving the
increase in mucus secretion, prostaglandin levels, and activity of ATP-sensitive K þ channels (Slamenova et al. 2013,
Halici et al. 2016). A diet supplemented with high doses of
Thym (500 and 2000 mg/kg) in piglets could improve stomach and gut health as antimicrobial (Gao et al. 2016).
However, our in vivo study revealed a negative effect of
the highest dose of Thym (500 mg/kg) on Indo-induced
gastric ulcer in rats. This may be due to the irreversible
cell membrane damaging activity of Thym associated with
its antifungal activity (Thakre-Nighot and Blikslager 2016).
Hence the Ole could not prevent these damages in
Indo þ Thym group. Additionally, gene expression analysis
of caspase-3 and TNF-a supported the findings of biochemical assays (Figure 8). TOS, eNOS, and TNF-a measurements
indicated that they may have a potential to trigger the
apoptotic pathways, whereas PGE2 was able to inhibit
apoptosis and regulate the gene expressions of caspase-3
and TNF-a. Of 500 mg/kg dose of Thym-treatment caused a
significant increase in caspase-3 and TNF-a expression as
well as inflammatory mediators and oxidative stress
determinants.
Conclusion
The findings of this study showed that high doses (250 and
500 mg/kg) of Ole and 200 mg/kg dose of Thym have therapeutic potential against gastric mucosal damage caused by
Indo via anti-inflammatory, anti-oxidative, and anti-apoptotic
mechanisms. On the other hand, the high dose of Thym
treatment (500 mg/kg), showed serious side effects. The
administration of 500 mg/kg dose combination of Ole and
Thym revealed that this dose of Ole treatment is inadequate
for the prevention of apoptosis and gastric damages induced
by high dose of Thym administration. Since the current antiulcer agents as synthetic drugs are expensive and are likely
to produce more side effects when compared to herbal medicines, Ole and Thym may be more effective and less toxic
therapeutic potential for the treatment of peptic ulcers as
comparison to the synthetic anti-ulcer agents. However, to
able to be proved as anti-ulcer agents, comprehensive studies on high doses of these agents are required.
11
Disclosure statement
The authors declare that there are no conflicts of interest.
Funding
This work was supported by BAP (No. 2015/344) from Atat€
urk
€
Universitesi.
ORCID
Kubra Koc
http://orcid.org/0000-0001-6208-165X
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