Protective effect of chamomile (Matricaria recutita L.) decoction extract against alcohol-induced injury in rat gastric mucosa

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Protective effect of chamomile (Matricaria recutita L.) decoction extract against alcohol-induced injury in rat gastric mucosa Mohamed-Amine Jabri a,b,∗ , Nadhem Aissani b , Haifa Tounsi c , Mohsen Sakly a , Lamjed Marzouki b , Hichem Sebai a,b a

Laboratoire de Physiologie Intégrée, Faculté des Sciences de Bizerte, 7021 Zarzouna, Université de Carthage, Tunisie Laboratoire de Physiologie Fonctionnelle et Valorisation des Bio-Ressources, Institut Supérieur de Biotechnologie de Béja, Université de Jendouba, Avenue Habib Bourguiba, B.P. 382, 9000 Béja, Tunisie c Laboratoire d’anatomie pathologique humaine et expérimentale, Institut Pasteur de Tunis, 13, Place Pasteur, Tunis 1002, BP-74, Tunisie b

a r t i c l e

i n f o

Article history: Received 21 August 2016 Received in revised form 5 November 2016 Accepted 18 November 2016 Available online xxx Keywords: Oxidative stress Lipid peroxidation calcium Free iron

a b s t r a c t Background: Matricaria recutita L. (Asteraceae), German chamomile, has been widely used in the traditional Tunisian medicine because of having the powerful health benefits. the current study was conducted to determine the protective effect of chamomile (Matricaria recutita L.) decoction extract (CDE) in ethanolinduced ulcer and oxidative stress on gastric mucosa in rat. Methods: Adult male wistar rats were used and divided into seven groups: Control, EtOH, EtOH + various doses of CDE (25, 50 and 100 mg/kg, b.w.), EtOH + famotidine (FAM) and EtOH + ascorbic acid (AA). Gastric ulceration was induced by EtOH (4 g/kg, b.w. p.o.). Results: Firsly, we found that acute alcohol administration leads to mark macroscopic and histologic changes in gastric mucosa. EtOH also induced lipoperoxidation (486.99%), thiol (-SH) groups decrease (40.98%) as well as antioxidant enzyme activity depletion such as superoxide dismutase (SOD) (49.05%), catalase (CAT) (46.80%) and glutathione peroxidase (GPx) (38.20%). Our results also demonstrated that alcohol intoxication increased tissue and plasmatic hydrogen peroxide, calcium and free iron levels. More importantly, CDE reversed all macroscopic, histologic and biochemical changes induced by EtOH administration. Conclusion: A potential gastropreotective effect of CDE against EtOH-induced ulcer and oxidative stress might be partially to its antioxidant properties as well as to various gastric mucosal defense mechanisms, including protection of gastric sulfhydryls and its opposite effect on some intracellular mediators such as free iron, hydrogen peroxide and calcium. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Peptic ulcer disease (PUD) is a problem of the gastrointestinal tract characterized by mucosal damage secondary to pepsin and gastric acid secretion [1]. PUD has been considered the principal leading cause of death in both advanced and developing countries [2]. It is well known that ethanol is mainly metabolized via alcohol dehydrogenases to form acetaldehyde and acetate, which present prominent toxic effects on the gastrointestinal tract [3]. The administration of ethanol generates reactive oxygen species (ROS), including superoxide anion, hydroxyl radical and hydro-

∗ Corresponding author at: Laboratoire de Physiologie Intégrée Département des Sciences de la Vie, Faculté des Sciences de Bizerte, 7021 Zarzouna, Tunisia. E-mail address: [email protected] (M.-A. Jabri).

gen peroxide [4]. Thus, over-production of ROS plays a key role in the pathophysiological changes in cell membrane fatty acid composition, resulting in the increase of lipid peroxidation and the depletion of endogenous antioxidant enzyme activities, such as SOD, CAT, and GPx [5]. However, there is evidence to suggest that the oxidative stress and depletion of antioxidants can be considered as a crucial part in ethanol-induced mucosal damage [6,7]. In this respect the medicinal plant extracts well known for their antioxidant properties are widely used to investigate their gastroprotective effects [8–10]. Chamomile [Matricaria recutita L. (Asteraceae)] is one of the most widely used and well-documented medicinal plants in the world [11]. It has been included for centuries in the pharmacopeia in many countries such as Tunisia. Owing to its antioxidant [12] and its anti-inflammatory [13] properties, chamomile extracts exhibit many beneficial health effects as anti-allergic [14], neuro-

http://dx.doi.org/10.1016/j.pathophys.2016.11.001 0928-4680/© 2016 Elsevier B.V. All rights reserved.

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protective [15], antimicrobial [16] and anticancer [17] activities. Recently we have shown that chamomile decoction extract protects against diarrhea and then gastrointestinal oxidative stress induced by acute oralcastor oil administration in rat [18]. Therefore, the present study was designed to evaluate the putative gastroprotective role of CDE treatment (10 days against both ethanol induced gastric ulcers ethanol-induced oxidative stress. We also studied the possible implication of sulfhydril groups and some intracellular mediators in such protection. 2. Materials and methods 2.1. Chemicals Epinephrine, bovine catalase, 2-thio-barbituric acid (TBA) and butylated hydroxytoluene (BHT) were from Sigma Chemicals Co (Germany). All other chemicals used were of analytical reagent grade. 2.2. Preparation of chamomile decoction extract Chamomile flowers were cultivated from the region of Béja (North-West of Tunisia) during March 2013 and identified by Mrs. Mouhiba Ben-Naceur, professor of taxonomy in the Higher Institute of Biotechnology of Béja-Tunisia. The voucher specimens (No. M121) have been deposited in the herbarium of the Higher Institute of Biotechnology of Béja and also in our laboratory of integrated physiology in the Faculty of Sciences of Bizerta. Plant material was thendried in an incubator at 50 ◦ C during 72 h and powdered in an electric blender. The decoction was made with double distilled water (1/5; w/v) at 100 ◦ C during 5 min under magnetic agitation and the homogenate was filtered through a colander (0.5 mm mesh size). Finally, the obtained extract (CDE) was lyophilized and stored at −80 ◦ C until used. 2.3. Animals and treatment Healthy adult male Wistar rats (200–220 g body weight; 15 weeks old) were purchased from the Pasteur Institute of Tunis and used in accordance with the local ethics committee of Tunis University for the use and care of animals in agreement with the NIH recommendations [19]. They were provided with standard diet (standard pellet diet- Badr Utique-TN) and water ad libitum and maintained in animal house under controlled temperature (22 ± 2 ◦ C) with a 12 h light-dark cycle. The animals were divided into seven groups of 10 rats each. Groups 1 and 2 served as controls and had a physiological solution (NaCl, 0.9%, p.o.). Groups 3, 4, and 5 were pre-treated with various doses of CDE (25, 50, and 100 mg/kg, b.w. p.o.) while groups 6 and 7 were pretreated respectively with famotidine (20 mg/kg, b.w. p.o.) and ascorbic acid (250 mg/kg, b.w. p.o.). A total of seventy rats were pretreated for 10 days. They were fasted for 24 h before the administration of CDE or reference molecules. After 2 h, each of them, except those of groups 1 and 2, was intoxicated by acute administration of EtOH (4 g/kg, b.w. p.o.). Sixty min later, animals were sacrificed. Blood sample was collected in heparinized tubes. After centrifugation at 3000g for 15 min, plasma was processed to determine the free-iron, H2 O2 and calcium level. 2.4. Evaluation of gastric mucosal damage The stomach was removed from each rat and opened along its greater curvature. The tissue was gently rinsed in NaCl 0.9%. The lesions in the gastric mucosa were macroscopically examined and the photographs of hemorrhagic erosions were acquired with a

Photometrics Quantix digital camera. Ulcer indexes were calculated as the sum of the lengths of the whole gastric lesions (in mm2 ). A blind measurement of lesion lengths was performed by two independent observers. 2.5. Stomach mucosa preparation After the macroscopic analyses, the gastric mucosa was rapidly excised and homogenized in phosphate buffer saline (KH2 PO4 /K2 HPO4 , 50 mM, pH 7.4) with Potter–Elvehjem homogenizers. After centrifugation (10 000g for 10 min at 4 ◦ C), supernatant was used for biochemical determination of protein, free iron, H2 O2 , calcium, SH- groups, MDA and antioxidant enzyme activities. 2.6. Histopathological analysis Immediately after the acrifice, samples of stomach were harvested and washed with ice-cold saline. Tissue fragments were then fixed in a 10% neutral buffered formalin solution, embedded in paraffin and used for histopathological examination; 5 ␮m thick sections were cut, deparaffinized, hydrated and stained with hematoxylin and eosin (HE). The gastric sections were examined in a blind fashion in all treatments. 2.7. Lipid peroxidation measurement Gastric mucosa lipid peroxidation was determined by MDA measurement according to the double heating method [20]. Briefly, aliquots from gastric mucosa homogenates were mixed with BHTTCA solution containing 1% BHT (w/v) dissolved in 20% TCA (w/v) and centrifuged at 1000g for 5 min at 4 ◦ C. The supernatant was blended with 0.5 N HCl 120 mM TBA in 26 mM Tris and then heated at 80 ◦ C for 10 min. After cooling the absorbance of the resulting chromophore was determined at 532 nm by using a UV–vis spectrophotometer (Beckman DU 640B). MDA levels were determined by using an extinction coefficient for MDA-TBA complex of 1.56 105 M−1 cm−1 . 2.8. Antioxidant enzyme activity assays The activity of SOD was determined using modified epinephrine assays [21]. At alkaline pH, superoxide anion O2 − caused the autoxidation of epinephrine to adenochrome; while competing with this reaction, SOD decreased the adenochrome formation. One unit of SOD is defined as the amount of the extract that inhibits the rate of adenochrome formation by 50%. Enzyme extract was added in 2 mL reaction mixture containing 10 ␮L of bovine catalase (0.4 U/␮l), 20 ␮L epinephrine (5 mg/ml) and 62.5 mM sodium carbonate/bicarbonate buffer pH 10.2. Changes in absorbance were recorded at 480 nm. The activity of CAT was assessed by measuring the initial rate of H2 O2 disappearance at 240 nm [22]. The reaction mix contained 33 mm H2 O2 in 50 mm phosphate buffer pH 7.0 and the activity of CAT calculated using the extinction coefficient of 40 mM−1 cm−1 for H2 O2 . The activity of GPx was quantified by the procedure of Flohé and Günzler [23]. Briefly, 1 mL of reaction mixture containing 0.2 mL of gastric mucosa supernatant, 0.2 mL of phosphate buffer 0.1 M pH 7.4, 0.2 mL of GSH (4 mM) and 0.4 mL of H2 O2 (5 mM) was incubated at 37 ◦ C for 1 min and the reaction was stopped by the addition of 0.5 mL TCA (5%, w/v). After centrifugation at 1500g for 5 min, aliquot (0.2 mL) from supernatant was combined with 0.5 mL of phosphate buffer 0.1 M pH 7.4 and 0.5 mL DTNB (10 mM) and absorbance was read at 412 nm. The activity of GPx was expressed as nmol of GSH consumed/min/mg protein.

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Fig. 1. Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on macroscopic changes induced by ethanol (EtOH). Rats were pretreated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bi-distilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour.

2.9. Thiol groups measurement

2.13. Protein determination

The total concentration of thiol groups (-SH) was performed according to Ellman’s method [24]. Briefly, aliquots from stomach mucosa were mixed with 100 ␮L of 10% SDS and 800 ␮L of 10 mM phosphate buffer (pH 8), and the optical density was measured at 412 nm (A0). Then, 100 ␮L of DTNB were added and then incubated at 37 ◦ C during 60 min. After incubation, the absorbance of the sample was quantified at 412 nm (A1). The thiol group concentration was calculated from A1 to A0 subtraction using a molar extinction coefficient of 13.6 × 103 M−1 × cm−1 . The results were expressed as nmol of thiol groups per milligrams of protein.

Protein concentration was defined according to Hartree [28] which is a slight change of the Lowry method. Serum albumin was used as standard. 2.14. Statistical analysis Statistical analyses were performed using one-way ANOVA and were expressed as means ± standard error of the mean (SEM). All statistical tests were two-tailed, and a p value of 0.05 or less was considered significant. 3. Results

2.10. H2 O2 determination The gastric mucosa H2 O2 level was performed according to Dingeon et al. [25]. Briefly, the hydrogen peroxide reacts with phydroxybenzoic acid and 4-aminoantipyrine in the presence of peroxidase leading to the formation of quinoneimine that has a pink color detected at 505 nm.

2.11. Iron measurement Gastric mucosa and plasma non-heme iron were measured colorimetrically using ferrozine as described by Leardi et al. [26]. Briefly, the iron dissociated from transferrin-iron complex by a solution of guanidine acetate was reduced by ascorbic acid and reacted with ferrozine leads to the formation of pink complex measured at 562 nm.

2.12. Calcium determination Gastric mucosa and plasma calcium were performed using a colorimetric method according to Stern and Lewis [27]. Briefly, at alkaline medium, calcium reacts with cresolphtalein leading to color complex measurable at 570 nm.

3.1. Macroscopic examination of the CDE effect on EtOH-induced gastric mucosa injuries Data from Fig. 1 shows the macroscopic examination of gastric mucosa after EtOH, CDE and reference molecules administration. As expected, EtOH (4 g/kg b.w.) administration induced a marked gastric ulceration. However, CDE (25, 50 and 100 mg/kg; b.w.) prehandling, significantly and does-dependently protected against gastric mucosal damage caused by alcohol administration (Fig. 1). On the other hand, quantitative analyses showed that pretreatment with CDE, at 25, 50 and 100 mg/kg, exhibited significant reduction of gastric lesions (ulcer index) with protection percentage of 46.77%, 63.30% and 90.95%, respectively when compared to EtOH group. However, sub-acute pretreatment with famotidine or ascorbic acid significantly protects against EtOH induced gastric mucosal damage (81.13% and 73.12%, respectively) (Table 1). 3.2. Histopathological examination of the CDE effect on EtOH-induced gastric mucosa injuries The stomach of the control section showed normal histologic features while following EtOH administration it revealed the prominent and severe gastric mucosal damage, hemorrhagic erosion, edema and leukocytes infiltration of the submucosal layer. pretreatment with CDE, FAM and AA greatly but only partially reduced EtOH-induced gastric structural changes. (Fig. 2).

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Fig. 2. Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on histologic changes induced by ethanol (EtOH). Animals were pre-treated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bi-distilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour.

Table 1 Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on macroscopic changes induced by EtOH in rats: ulcer index and inhibition percentage. Animals were pre-treated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bi-distilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour. Group

Ulcer index (mm2 )

Protection percentage (%)

Control EtOH EtOH + CDE-25 EtOH + CDE-50 EtOH + CDE-100 EtOH + FAM EtOH + AA

– 77.42 ± 4.22a 41.24 ± 2.02b 28.41 ± 1.61b,c 7.04 ± 1.5b,c,d 14.63 ± 2.02b 20.83 ± 2.42b

– 00 46.77 63.30 90.95 81.13 73.12

a b c d

p < 0.05 compared to control group. p < 0.05 compared to EtOH group. p < 0.05 compared to CDE-25 group. p < 0.05 compared to CDE-50.

3.3. Effect of CDE on EtOH-induced oxidative stress in gastric mucosa Bearing on the effect of EtOH and CDE on oxidative stress condition, we firstly studied the gastric mucosa lipoperoxidation. As expected EtOH alone dramatically increased the gastric mucosa MDA level while CDE pretreatment significantly and doesdependently reversed alcohol-induced lipid peroxidation (Table 2). In this respect, the effect of EtOH and CDE pretreatment on gastric mucosa antioxidant enzyme activities was also investigated and the results are presented in Table 2. EtOH per se significantly decreased gastric antioxidant enzyme activities including SOD (A), CAT (B), and GPx (C). More importantly, CDE pretreatment significantly reversed all EtOH-induced antioxidant enzyme depletion in a dose-dependent manner. Subacute treatment with both famotidine and ascorbic acid protected against alcohol-induced oxidative stress in gastric mucosa.

Fig. 3. Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on ethanol (EtOH)-induced changes in stomach mucosa SH- groups level. Animals were pre-treated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bidistilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour. Essays were carried out in triplicate. a: p < 0.05 compared to control group, b: p < 0.05 compared to EtOH group, c: p < 0.05 compared to CDE-25 group and d: p < 0.05 compared to CDE-50.

3.5. Effect of CDE on EtOH-induced changes in gastric mucosa and plasma H2 O2 , free iron and calcium levels In the present study, we also examined the effect of Ethanol and CDE on intracellular mediators including hydrogen peroxide (Fig. 4), free iron (Fig. 5) and calcium (Fig. 6) levels in the plasma and gastric mucosa tissue. Alcohol per se significantly increased H2 O2 , free iron and calcium levels in plasma and gastric mucosa. CDE pretreatment significantly and does-dependently protected against EtOH-induced intracellular mediator disturbances. Reference molecules reversed these parameters to near control levels too.

3.4. Effect of CDE against EtOH-induced decrease of sulfhydril groups in gastric mucosa

4. Discussion

We further looked at the effect of ethanol and CDE on gastric mucosa sulfhydril group contents. Our results indicated that alcohol treatment significantly decrease gastric thiol groups level. Interestingly, CDE, famotidine or ascorbic acid pre-treatments significantly protected against EtOH-induced sulfhydrils decrease in a dose-dependent manner (Fig. 3).

The main purpose of the present study was to investigate whether chamomile decoction extract (CDE) has a protective effect on ethanol-induced acute gastric mucosal damage in rat. We also investigated the implication of oxidative stress, sulfhydril groups and some intracellular mediators such as hydrogen peroxide, free iron and calcium in such gastroprotection.

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Table 2 Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on ethanol (EtOH)-induced changes in stomach mucosa MDA level and antioxidant enzyme activities (SOD, CAT and GPx). Animals were pre-treated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bi-distilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour. Essays were carried out in triplicate. Group

MDA (nmol/mg protein)

SOD activity (U/mg protein)

CAT activity (nmol H2 O2 /min/mg protein)

GPx activity (nmol GSH/min/mg protein)

Control EtOH EtOH + CDE-25 EtOH + CDE-50 EtOH + CDE-100 EtOH + FAM EtOH + AA

141.12 ± 12.88 687.25 ± 35.69a 358.75 ± 28.48b 288.83 ± 28.29b,c 162.41 ± 11.89b,c,d 147.5 ± 31.85b 202.27 ± 33.67b

0.53 ± 0.03 0.26 ± 0.01a 0.33 ± 0.03b 0.43 ± 0.02b,c 0.50 ± 0.02b,d 0.47 ± 0.03b 0.53 ± 0.03b

34.23 ± 2.19 16.02 ± 1.52a 20.11 ± 1.91b 27.23 ± 1.81b,c 32.70 ± 1.16b,d 31.41 ± 2.31b 29.07 ± 2.71b

3.90 ± 0,11 1.49 ± 0.10a 2.44 ± 0.29b 3.03 ± 0.25b,c 3.47 ± 0.17b,c,d 3.59 ± 0.15b 3.42 ± 0.27b

a b c d

p < 0.05 compared to control group. p < 0.05 compared to EtOH group. p < 0.05 compared to CDE-25 group. p < 0.05 compared to CDE-50.

Fig. 4. Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on ethanol (EtOH)-induced changes in stomach mucosa (A) and plasma (B) hydrogen peroxide levels. Animals were pre-treated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bi-distilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour. Essays were carried out in triplicate. a: p < 0.05 compared to control group, b: p < 0.05 compared to EtOH group, c: p < 0.05 compared to CDE-25 group and d: p < 0.05 compared to CDE-50.

Fig. 5. Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on ethanol (EtOH)-induced changes in stomach mucosa (A) and plasma (B) free iron levels. Animals were pre-treated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bi-distilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour. Assays were carried out in triplicate. a: p < 0.05 compared to control group, b: p < 0.05 compared to EtOH group, c: p < 0.05 compared to CDE-25 group and d: p < 0.05 compared to CDE-50.

We firstly examined the protective effect of CDE on EtOHinduced macroscopic and microscopic changes in gastric mucosa. We showed that CDE pretreatment significantly protected against alcohol-induced marked morphological and structural changes in gastric mucosa. Based on previous data from rat models, acute ethanol intoxication induced significant lesions and damaging effects on the gastric mucosa severely including hemorrhagic erosion, oedema and leucocytes infiltration of the submucosal layer

[29,30]. In addition, ethanol-induced gastric mucosal injury is associated with over production of free radicals, which lead to oxidative stress status assessed by increased MDA level and depletion of antioxidant enzyme activities such as SOD, CAT and GPx. MDA, the end-product of lipid peroxidation, is used as a marker of tissue damage [31]. Moreover, lipid peroxidation and antioxidant enzyme activity depletion are considered as reliable biomarkers of the degree of oxidative stress [32]. In this respect, and as there

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Fig. 6. Subacute effect of chamomile decoction extract (CDE), famotidine (FAM) and ascorbic acid (AA) on ethanol (EtOH)-induced changes in stomach mucosa (A) and plasma (B) calcium levels. Animals were pre-treated with various doses of CDE (25, 50 and 100 mg/kg, b.w., p.o.), FAM (20 mg/kg, b.w., p.o.), AA (250 mg/kg, b.w., p.o.) or bi-distilled water, challenged with a single oral administration of EtOH (4 g/kg, b.w., p.o.) or NaCl 9‰ for one hour. Assays were carried out in triplicate. a: p < 0.05 compared to control group, b: p < 0.05 compared to EtOH group, c: p < 0.05 compared to CDE-25 group and d: p < 0.05 compared to CDE-50.

is a close relationship between ROS production and gastric inflammation, we will try in our future project to study the effect of CDE on the pro-inflammatory cytokines production. More importantly, our results showed that CDE significantly attenuated EtOH-induced oxidative stress in a dose-dependent manner. It indicates that can attenuate the process of lipid peroxidation and/or antioxidant enzymes depletion implicated in the pathogenesis of ethanol-induced gastric damage. EtOH-induced oxidative stress and gastric mucosa injuries have been attenuated by a number of medicinal plant extracts as Opuntia ficus [9], Momordica charantia [33], Brassica oleracea [34] Acacia nilotica [35], Acacia ferruginea [36], Piper aduncum [37] and Amukkara choornam [38]. The results of our studies of CDE revealed the presence of high concentrations of total polyphenols, total flavonoids, and condensed tannins. The use of HPLC-PDA-MS allowed to the identification of gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, cafeoylquinic acid, salicylic acid, quercetin, quinic acid derivative, hydroxybenzoic acid-O-hexoside, 5,7,4 -Trihydroxy6,3 -dimethoxyflavone [39]. These phytomolecules have been shown to be responsible for the antioxidant and bio-functional properties of many plant extracts [40]. The antioxidant mechanism of would be due mainly to their redox properties, by allowing them to act as reducing agents, hydrogen donors, free radical quenchers, metal chelators and decomposers of peroxides [41]. In order to combat and neutralize the deleterious effects of ROS, various antioxidant strategies have been proposed for our

extract, either by enhancing the non-enzymatic defenses or by increasing the endogenous antioxidant enzyme defenses through dietary or pharmacological means. In fact, Tanigawa et al. reported that quercetin-induced ARE activity involves upregulation of Nrf2 through the regulation of both transcription and posttranscription sites and repression of Keap1 by affecting the posttranscription site in HepG2 cells [42]. In this respect, many studies strongly support the contribution of polyphenols to the prevention of digestive disorder [43]. However, it is well documented that phenolic compounds display a number of pharmacological properties in the GIT area, acting as antisecretory, cytoprotective, and antioxidant agents [9,18,44]. We further looked at the direct or indirect effec of EtOH and CDE on certain intracellular mediators related to oxidative stress status. Unsurprisingly, Alcohol administration significantly increased hydrogen peroxide, free iron and calcium levels in plasma and gastric mucosa tissue. Furthermore, both iron and/or H2 O2 accumulation catalyzed the highly toxic hydroxyl radical (OH. ) production via the Fenton reaction leading to membranes lipoperoxidation and enhancement of its permeability to calcium [45]. It seems that calcium homeostasis deregulation have a primal role in the injury induced by acute or chronic alcohol consumption [46,47]. However, it is generally reported that oxidants can cause a rapid increase of the cytoplasm calcium levels in various cell types [48,49]. More importantly, CDE pretreatment significantly attenuated EtOH-induced intracellular mediator deregulation in a dose-dependent manner. The phenolic compound extracts protection against oxidant-induced intracellular mediator deregulation has been previously described for myrtle berries seeds extract [50,51] and CDE [52]. Meanwhile, it’s tempting to speculate that CDE exerts its beneficial effect by chelating free iron and scavenging H2 O2 leading to calcium homeostasis. We next sought to determine the putative involvement of sulfhydril groups in the EtOH and CDE mechanism of action. We realized that CDE pretreatment significantly and dose- dependently protected against −SH groups decrease induced by alcohol administration in gastric mucosa. The implication of thiol groups in the loss of integrity of the gastric mucosa has been previously shown for several products capable of inducing ulceration such as absolute ethanol [53], indomethacin [54], aspirin [55] and ketoprofen [56]. However, it is well known that sulfhydryls are partly involved in gastric cytoprotection [57,58], by maintaining the integrity of the mucosal barrier and scavenging free radicals resulting from noxious agents [59]. Indeed, we can suggest that CDE exerts its beneficial effect by maintaining gastric sulfhydryls as previously described for silymarin [55].

5. Conclusion We have clearly demonstrated that chamomile decoction extract protects against ethanol-induced gastric mucosal damage. The gastroprotection offered by CDE may be related partly to gastric mucosa sulfhydryls safety as well as its antioxidant properties and opposite effect on some intracellular mediators such as free iron, hydrogen peroxide and calcium.

6. Declaration of interest The authors alone are responsible for the content of this paper.

7. Financial disclosures None declared.

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Acknowledgement Financial support of the Tunisian Ministry of Higher Education and Scientific Research is gratefully acknowledged.

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Please cite this article in press as: M.-A. Jabri, et al., Protective effect of chamomile (Matricaria recutita L.) decoction extract against alcohol-induced injury in rat gastric mucosa, Pathophysiology (2016), http://dx.doi.org/10.1016/j.pathophys.2016.11.001