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Protective role of ellagitannins from Eucalyptus citriodora against ethanol-induced gastric ulcer in rats: impact on oxidative stress, inflammation and calcitonin-gene related peptide.

ABSTRACT

The gastroprotective activity of an ellagitannin-rich fraction obtained from Eucalyptus citriodora (ECF) was investigated against ethanol-induced gastric ulceration in rats. The rats were pretreated with ECF (25,50 and 100 mg/kg) 1 h before the administration of absolute ethanol to induce acute gastric ulceration. The gastric lesions were significantly reduced by all doses of ECF. Notably, pre-treatment with ECF (100 mg/kg) conferred 99.6% gastroprotection, which is significantly higher than that produced by omeprazole. Moreover, ECF administration markedly increased the mucin content in a dose-dependent manner. The potent gastroprotective effect of ECF could be partly mediated by attenuating ethanol-induced oxidative stress. ECF-pre-treatment markedly increased the depleted GSH and SOD levels in a dose-dependent manner. Moreover, ECF significantly decreased the elevated MDA tissue levels induced by ethanol administration. The results demonstrated that ECF administration exerted a powerful anti-inflammatory activity as evidenced by the reduction in the pro-inflammatory markers; IL-1[beta], TNF-[alpha], 5-LO and COX-2. Additionally, the caspase-3 tissue levels were significantly reduced in the groups pre-treated with ECF. These results suggest that ECF could exert a beneficial gastroprotective effect through their antioxidant, anti-inflammatory and anti-apoptotic properties. Furthermore, ECF pre-treatment significantly attenuated the ethanol-induced decrease in CGRP expression, which has a protective role against gastric ulceration. Histopathological examination revealed intact mucosal layer, absence of hemorrhage and necrosis in groups treated with ECF. Ellagitannins were identified as the major active constituents responsible for the marked antioxidant and gastroprotective properties of ECF. The HPLC-PDA-ESI/MS/MS technique was employed to identify the ellagitannins of E. citriodora.

Keywords:

Ellagitannins

Eucalyptus citriodora

HPLC-PDA-ESI/MS/MS

Gastroprotection

Anti-inflammatory

Antioxidant

Caspase-3

Introduction

Peptic ulcer is one of the major gastrointestinal disorders and is induced by many factors including stress, smoking, ethanol abuse and frequent use of non-steroidal anti-inflammatory drugs. The pathophysiology of peptic ulcer involves an imbalance between offensive and protective factors in the gastric mucosa. There are two approaches for treating peptic ulcer; the first deals with reducing the production of gastric juice and the second with enhancing the protective mechanisms of gastric mucosa (Arun and Asha, 2008; Viana et al., 2013). Several drugs have been used for the treatment of gastric ulcer; however, most of these drugs do not cure the ulcers are produce several adverse effects (Abdelwahab et al., 2013; Viana et al., 2013). Development of tolerance and relapses is also common after using these drugs (Arun and Asha, 2008). In view of the limited therapeutic options available for the treatment of peptic ulcer, the need for more effective and safer gastroprotective agents is quite apparent. Natural products represent a new therapeutic strategy in the search for new and effective gastroprotective agents, especially when the current drugs cannot be used for long periods (Sumbul et al., 2011). A growing body of evidence indicates that medicinal plants can assist in ulcer healing and in preventing relapse (Abdelwahab et al., 2013).

Plants rich in tannins have been traditionally used for the treatment of gastric ulcer primarily because of their astringent effects (Khennouf et al., 2003). In gastric ulcers, tannins react with tissue proteins to form a protective layer over the injured epithelial tissues of the stomach. The protective layer confers protection from offensive factors and promotes the healing process (de Jesus et al., 2012). The presence of tannins explains the anti-ulcer effects of many medicinal plants, such as Phyllanthus amarus, Quercus species (de Jesus et al., 2012; Khennouf et al., 2003), Rubus berries and pomegranate (Sangiovanni et al., 2013).

Eucalyptus citriodora Hook. (Myrtaceae), or lemon Eucalyptus, has been widely used in folk medicine for the treatment of various ailments (Gbenou et al., 2013). Several pharmacological activities have been reported for E. citriodora, including cytotoxic activity and anti-tumor activity against Ehrlich ascites carcinoma (Bhagat et al., 2012). The essential oil of E. citriodora possesses a wide spectrum of biological activities such as anti-inflammatory, gastroprotective (Gbenou et al., 2013) and antifungal effects (Javed et al., 2012). Despite the various medicinal properties of E. citriodora, very little is known about the chemical composition and pharmacological properties of the nonvolatile constituents of E. citriodora. Most studies have mainly focused on the chemical composition of the essential oil of E. citriodora. To the best of our knowledge, no studies have so far been reported on the gastroprotective activity of E. citriodora. Therefore, this study was undertaken to determine the gastroprotective activity of an ellagitannin-rich fraction obtained from E. citriodora (ECF) against ethanol-induced peptic ulcer in rats, with the aim to develop a safe effective gastroprotective agent. The mechanisms underlying the antiulcer effect were explored and a histopathological examination of gastric tissues was performed. Identification of the ellagitannins of E. citriodora was conducted for the first time utilizing the HPLC-PDA-ESI/MS/MS technique (high-performance liquid chromatography coupled with diode array detection-electrospray ionization mass spectrometry).

Material and methods

Chemicals

Reduced glutathione (GSH), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent), bovine serum albumin and thiobarbituric acid (TBA), Folin-Ciocalteu reagent, 2,2-diphenyl-l-picrylhydrazyl (DPPH*) and sodium ascorbate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Orcinol, dipotassium hydrogen phosphate ([K.sub.2]HP[O.sub.4]), n-butanol, potassium dihydrogen phosphate (K[H.sub.2]P[O.sub.4]) and trichloroacetic acid (TCA) were purchased from El-Nasr Chemical Co. (Egypt). Sephadex LH-20 was obtained from Amersham Biosciences, Sweden.

Plant material

The leaves of E. citriodora were collected in July 2013 from the botanical garden of the Faculty of Agriculture, Cairo University, Cairo, Egypt. The plant was botanically identified by Eng. Therese Labib, the taxonomy specialist at the herbarium of El-Orman Botanical Garden, Giza, Egypt. A voucher specimen of E. citriodora was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU ECM2013).

Extract preparation

Air-dried powdered leaves of E. citriodora (500 g) were extracted three times with 70% acetone. The total extract was concentrated and freeze-dried to obtain a dry powder, which was dissolved in absolute MeOH. The MeOH-soluble portion was concentrated and freeze-dried to obtain a dry powder (40 g). Column fractionation was performed with a portion of the extract (20 g) using a Sephadex LH-20 column (5 cm x 50 cm). Elution was performed with [H.sub.2]O followed by [H.sub.2]O-MeOH mixtures of decreasing polarities to yield four fractions (I-IV). The ellagitannin-rich fraction eluted with 60% MeOH (fr 111) was concentrated and freeze-dried to obtain a dry powder of ECF (3.4 g).

HPLC-PDA-ESI/MS/MS method

Part of ECF was dissolved in 20% MeOH (20 mg/ml), and the solution was filtered through 0.45 [micro]m membranes. LC-HRESIMS was performed on a Bruker micrOTOF-Q Daltonics (API) Time-of-Flight mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) coupled to a 1200 series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with Starlight diode-array detector (PDA). Chromatographic separation was performed on a Superspher 100 RP-18 (75 mm x 4 mm i.d.; 4 [micro]m) column (Merck, Darmstadt, Germany) according to the method described by Al-Sayed et al. (2012). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B). The elution profile was 0-3 min, 100% B (isocratic); 3-30 min, 0-30% A in B; 30-35 min, 30-70% A in B; 35-37 min, 70% A in B (isocratic) with constant flow rate of 0.5 ml/min. The HPLC system was controlled by Hystar software (version 3.2; Bruker BioSpin GmbH, Rheinstetten, Germany). The mass spectrometer was controlled by the Compass 1.3 for micrOTOF software package (Bruker Daltonics GmbH, Rheinstetten, Germany). The ionization technique was a pneumatically assisted electrospray. The mass spectrometer was operated in the negative mode, and mass detection was performed in the full scan mode in the range of 50-2000 m/z. The following settings were applied to the instrument: capillary voltage, 4000 V; end plate offset -500 V. The drying gas ([N.sub.2]) flow rate was 8.4 l/min, and the drying gas temperature was 200[degrees]C. For collision-induced dissociation (CID) MS/MS measurements, the voltage over the collision cell varied from 20 to 70 eV. Argon was used as collision gas. The data were analyzed using Compass Data Analysis Software (version 4.0 SP5; Bruker Daltonics GmbH, Rheinstetten, Germany).

DPPH* radical-scavenging assay

The assay was carried out according to the methods of Al-Sayed et al. (2012) and Orhan et al. (2012). Briefly, 20 [micro]l of ECF solution at different concentrations, positive control, or solvent was pipetted into each well of a 96-well plate, followed by 280 [micro]l of 0.25 mM methanolic solution of DPPH*. The mixture was incubated at room temperature in dark for 30 min, and the absorbance was measured at 520 nm with a Multiskan Ascent V1.24 microplate reader. The inhibition percentage of the radical scavenging activity was calculated using the equation: Inhibition (%) = 100 - 100 ([A.sub.Sample]/[A.sub.Blank]). The antioxidant activity was expressed in terms of the [IC.sub.50]. The data is expressed as the mean [+ or -] SEM of three independent experiments. Sodium ascorbate were used as the positive control and the antioxidant was also expressed as ascorbic acid equivalents (Pozharitskaya et al., 2008).

Determination of total phenols

ECF methanol solution (50 [micro]l) was added to 100 [micro]l of methanol and mixed with 100 [micro]l of Folin-Ciocalteu reagent. The mixture was shaken and allowed to stand at room temperature for 3 min before the addition of 500 [micro]l of 20% [Na.sub.2]C[O.sub.3]. The solution was mixed thoroughly and the absorbance was measured at 730 nm after 2 h according to the method of Velioglu et al. (1998). The assay was conducted in triplicates. Results were expressed as gallic acid equivalents per g dry weight of ECF from a calibration curve of gallic acid (0-500 [micro]g/ml). The data is expressed as the mean [+ or -] SEM of three independent experiments.

Animals

Male albino rats (150-200 g) were obtained from the Nile Company for Pharmaceutical and Chemical industries, Egypt. The rats were housed in an air-conditioned atmosphere, at a temperature of 25[degrees]C with alternatively 12 h light and dark cycles. The animals were acclimated two weeks before experimentation and were kept on a standard diet and water, ad libitum. Standard diet pellets (El-Nasr Chemical Company, Abu-Zaabal, Egypt) contained not less than 20% protein, 3.5% fat, 6.5% ash and a vitamin mixture. All the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH 1985), and were approved by the ethical committee of the Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU 2014Research Article 4).

Acute toxicity study

Acute oral toxicity was conducted according to the Guidlines for Testing of Chemicals adopted by the Organization for Economic Cooperation and Development (OECD, 2001). Three doses of ECF were used (100, 500, 1000 mg/kg body weight p.o.). Eight overnight fasting rats were used for each dose. Control group animals were given 8% Tween 80 in water. The animals were observed for any behavioral or physiological changes for fourteen days. The mortality percentage was recorded. On the fifteenth day, the body weight was recorded. Blood samples were collected from the retro-orbital plexus for the assessment of kidney and liver function tests.

Experimental design

The animals were divided randomly into six groups (six animals per group). Group (I) was given vehicle only (8% Tween 80 in water) and served as the normal control. Group (II) served as the ulcer control and was given the vehicle orally before giving absolute ethanol. Groups (III-V) were treated orally with 25, 50 and 100 mg/kg of ECF, respectively. Group (VI) was given standard omeprazole at a dose of 20 mg/kg orally. After 1 h, all groups except group (I) received absolute ethanol (5 ml/kg orally) to induce gastric ulcer. One hour after ethanol administration, the animals were anesthetized, sacrificed and their stomachs were dissected. The gastric contents were collected and centrifuged at 5000 rpm for 10 min. Gastric juices were used to measure titrable acidity and mucin content. Each stomach was opened along the greater curvature and washed with ice-cold saline. Stomach tissues from each group were homogenized in saline then the homogenate was used to assess oxidative stress markers: reduced glutathione (GSH), lipid peroxides and superoxide dismutase (SOD), inflammatory markers (IL-1[beta], TNF-[alpha], COX-2 and 5-LO), apoptotic marker (caspase-3) as well as calcitonin-gene related peptide (CGRP). Additionally, representative stomach specimens were taken from the treated groups for histopathological examination.

Assessment of gastroprotection

Ethanol-induced gastric ulcers appeared as elongated bands of hemorrhagic lesions (Alvarez-Suarez et al., 2011; Sidahmed et al., 2013). The areas of ulcerated lesions were determined using image analysis software (Image J, 1.46a, NIH, USA) and the percentages of the ulcerated area relative to the total stomach area were calculated. The percentage of inhibition was determined according to the following formula:

Percentage of inhibition = [Ulcerated area(ulcer control) - Ulcerated area(treated)/ Ulcerated area(ulcer control)] x 100%

Assessment of titrable acidity in gastric juice

Acid concentration (mequiv./l) was determined by titrating 200 [micro]l of gastric juice against 0.01 N NaOH solution, using phenol red as an indicator, till the first color changes from yellow to red (Grossman, 1963; Reitman, 1970).

Assessment of mucin content in gastric juice

The gastric mucin content was determined as described by Winzler (2006) and was expressed in terms of mg % hexoses. Gastric juice was diluted with distilled water (1:10, v/v) and 250 [micro]l was mixed with equal volume of orcinol solution (1.6%) and 2 ml sulfuric acid (60%, v/v). Samples were then placed in boiling water bath for 10 min, allowed to cool in ice-cold water and the developed color was measured at 425 nm.

Assessment of oxidative stress markers

GSH, MDA and SOD levels were assessed in the stomach homogenate of the different experimental groups. GSH was measured according to the method of Ellman (1959). Lipid peroxidation was determined by estimating the level of thiobarbituric acid reactive substances (TBARS) and the results were expressed as MDA equivalents (Uchiyama and Mihara, 1978). The SOD activity in tissue homogenates was assessed using SOD assay kit (Trevigen, Inc., Gaithersburg, MD, USA). The manufacturer's instructions were followed, where the homogenate was mixed with xanthine solution, NBT solution as well as the reaction buffer. The mixture was read spectrophotometrically at 550 nm by ELISA microplate reader (Chromate[R] microplate reader, Awareness Technology, Inc., Palm City, USA).

Assessment of protein content

The protein content in gastric tissue homogenate was determined using bovine serum albumin as a standard according to the method adopted by Bradford (1976).

Assessment of inflammatory markers

The involvement of inflammation in ethanol-induced gastric ulcer was assessed by measuring the levels of IL-1[beta], TNF-[alpha], COX-2 and 5-LO in the stomach homogenate of the different groups. Both IL-1 and TNF-[alpha] levels were assessed using RayBio[R] Rat IL-1[beta] and RayBio[R] Rat TNF-[alpha] ELISA Kits (RayBiotech, Inc., Norcross, GA, USA), respectively. COX-2 and 5-LO tissue levels were estimated using Rat COX-2 Assay Kit (Immuno-Biological Laboratories Co., Ltd, Minneapolis, USA) and Rat 5-LO ELISA kits (EIAab, Science Co., Ltd., Wuhan, China), respectively. The steps were carried out according to the manufacturer's instructions.

Assessment of caspase-3

Caspase-3 levels in gastric tissue homogenates were measured according the manufacturer's instructions of caspase-3 ELISA kit (USCN Life Science Inc, Wuhan, China).

Assessment of calcitonin-gene related peptide (CGRP)

Rat CGRP Elisa Kit (EIAab, Science Co., Ltd., Wuhan, China) was used to assess the gastric CGRP levels in experimental groups.

Histopathological examination

Stomach specimens from all experimental groups were fixed in 10% formol saline for 24 h. Washing was done by tap water then serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56[degrees]C in a hot air oven for 24 h. Sections were embedded in paraffin and sliced into 4 [micro]m thick sections by a sledge microtome. The tissue sections were collected on glass slides, deparaffinized, stained with hematoxylin and eosin dye then examined by light electric microscope (Banchroft et al., 1996).

Statistical analysis

Data are presented as means [+ or -] SEM. Multiple comparisons were performed using one-way ANOVA test followed by Tukey-Kramer test for post hoc analysis. p-Values < 0.05 were considered statistically significant. All statistical analyses were performed using Instat version 3 software package. Graphs were sketched using GraphPad Prism (ISI[R], San Diego, CA, USA) version 5 software.

Results

Identification of ECF ellagitannins by HPLC-PDA-ESI/MS/MS

The individual constituents of ECF were identified using the HPLC instrument coupled to a PDA detector and a mass spectrometer. The combination of the PDA and mass spectrometry (MS/MS) data provided a sensitive method for the characterization of the constituents of ECF. Ten compounds were identified based on their molecular ions [[M-H].sup.-], MS/MS fragment ions and distinctive PDA data, which were consistent with the data reported in the literature (Table 1 and Fig. 1A and B). Quantitative analysis of ellagitannins was also determined; the identified ellagitannins make up 91% of the purified fraction.

DPPH* radical-scavenging and total phenolic content

ECF exhibited considerable inhibitory activity in the DPPH* radical-scavenging assay, with [IC.sub.50] value of 18.88 [+ or -]0.41 [micro]g/ml. The [IC.sub.50] value of sodium ascorbate (positive control) was 60.55 [+ or -] 0.03 [micro]M. Ascorbic acid equivalents of the antioxidant activity = 3.2 (Pozharitskaya et al., 2008). The strong radical-scavenging activity was consistent with the results obtained from the determination of the total phenol content. ECF had considerably high phenol content (504.31 [+ or -] 3.50 mg gallic acid-equivalents per g dry weight of ECF).

Acute toxicity study

During the 14 days, no mortality, physiological or behavioral changes were observed in rats treated with 100, 500 and 1000 mg/kg of ECF. In addition, the different treated groups did not show a significant change in body weight. The serum biochemical analyses were not significantly different from the normal control group. Taken together, these findings indicate that ECF has a low toxicity profile and that the lethal dose 50% ([LD.sub.50]) value of ECF is greater than 1000 mg/kg.

Effect of ECF on ethanol-induced gastric lesions

The percentage of ulcerated area in ulcer control rats reached 35.5% of total stomach area. However, pre-treatment with omeprazole (20 mg/kg) or ECF at doses of 25 and 50 mg/kg produced a significant reduction in the percentage of ulcerated areas (by 71.7, 50.4 and 69.2%, respectively) when compared to the ulcer control group. Notably, the ulcerated area was markedly reduced by 99.6% in the rats pre-treated with 100 mg/kg of ECF (Table 2 and Fig. 2).

Effect of ECF on mucin content and total acidity in gastric juice

Administration of absolute ethanol in the ulcer control group produced the lowest content of gastric mucin content (0.6 mg % hexose). In contrast, the mucin content was significantly increased, in a dose dependent manner, in the groups pre-treated with ECF (25, 50 and 100 mg/kg) or omeprazole reaching 7, 51.7, 238.3 and 21.5-fold, respectively, compared with the ulcer control group (Table 2). Pre-treatment with omeprazole and ECF (25 mg/kg) significantly reduced ethanol-induced acidity by 49.6 and 43%, respectively. However; ECF pre-treatment (100 mg/kg) increased gastric juice acidity by 55.8% compared to the positive ulcer group. All doses of ECF significantly decreased the gastric juice volume in a dose-dependent manner (Table 2).

Effect of ECF on oxidative stress markers

Table 3 shows the effects of ethanol and ECF on oxidative stress markers; GSH, MDA and SOD. Absolute ethanol administration significantly reduced GSH and SOD levels by 78.8 and 76.2%, respectively compared to normal control values. In contrast, the groups pre-treated with ECF (25 and 50 mg/kg) showed a marked increase in the level of GSH, in a dose-dependent manner by 154.5 and 436.4%, respectively, compared to the ulcer group. Moreover, the SOD levels were significantly elevated in ECF pre-treated groups (25 and 50 mg/kg) by 126.7 and 183.3%, respectively when compared to the positive ulcer group. Interestingly, gastric SOD levels were markedly higher in all groups treated with ECF compared with the omeprazole-treated group. Notably, pre-treatment with ECF at a dose of 100 mg/kg induced a marked increase in GSH and SOD levels even more than their normal values. In contrast, gastric MDA level was significantly increased in the ulcer group by 4.19 fold compared to the normal values. However, pre-treatment with ECF (25, 50 and 100 mg/kg) significantly attenuated the ethanol-induced MDA increase by 44.5, 67.3 and 79.8%, respectively. Administration of omeprazole, significantly increased GSH and SOD tissue levels by 245.5 and 70%, respectively. In contrast, the MDA levels were decreased by 51.7% compared to the ulcer control group. These results clearly indicated the strong in vivo antioxidant activity provided by ECF.

Effect of ECF on inflammatory markers

The effect of ECF pre-treatment on the pro-inflammatory markers is shown in Table 4. IL-1[beta]), TNF-[alpha], 5-LO and COX-2 levels were markedly increased in the ulcer control group reaching 4.2-, 3.9-, 5.3- and 7.7-fold, respectively compared to the normal control group. Pre-treatment with ECF (25 mg/kg) significantly ameliorated the ethanol-induced increase in inflammatory mediators (IL-1[beta], TNF-[alpha], 5-LO and COX-2) by 60.7, 52.7, 51.9 and 58%, respectively. Furthermore, increasing ECF dose to 50 mg/kg induced a more significant decrease in IL-1[beta], TNF-[alpha], 5-LO and COX-2 levels by 65.6, 65.6, 70.5 and 75.4%, respectively, when compared to the ulcer control group. Notably, the levels of pro-inflammatory markers were returned to their normal levels in the group pretreated with 100 mg/kg of ECF. All doses of ECF were more effective in reducing the levels of pro-inflammatory markers compared to omeprazole.

Effect of ECF on caspase-3 expression

The normal level of caspase-3 activity, an apoptotic marker, was 12.08 nmol/mg protein. The gastric caspase-3 activity was significantly increased (4.9-fold) in absolute ethanol-ulcerated rats compared to the normal group. Pre-treatment with ECF (25, 50 and 100 mg/kg) significantly attenuated the ethanol-induced increase in caspase-3 in a dose-dependent manner, by 49.7, 68.2 and 80%, respectively (Fig. 3A). Based on these findings, all the tested doses of ECF were more effective than omeperazole in restoring the caspase-3 activity.

Effect of ECF on CCRP expression in stomach mucosa

The CGRP expression was significantly reduced by 26.5% in ulcer control group as compared to the normal group. Nevertheless, pretreatment with ECF (25,50 and 100 mg/kg) significantly elevated the CGRP levels by 1.7, 1.9 and 2.5-fold, respectively as compared to ulcer control values (Fig. 3B).

Histopathology

Fig. 4 and Table 5 show the histopathological alterations in stomach specimens of the different experimental groups. Stomach specimens from normal control rats showed normal histological structures of the mucosa, submucosa as well as the muscularis. The administration of absolute ethanol induced severe focal ulceration, necrosis and severe hemorrhage in a focal manner in the mucosal layer. Edema, inflammatory cells infiltration and congested blood vessels were observed in the submucosa of the ulcer group. The stomach specimen of omeprazole pre-treated group showed moderate focal ulceration and necrosis associated with mild congestion of blood vessels in the lamina propria, along with edema and inflammatory cells infiltration in the underlying submucosa. It was clear that the lower dose of ECF is as effective as omeprazole in reducing all the pathological changes induced by absolute ethanol. Notably, the pre-treatment of rats with 100 mg/kg of ECF conferred marked protection from ethanol-induced ulcer as evidenced by the intact mucosal layer, absence of hemorrhage and necrosis. Interestingly, treatment with ECF at a dose of 100 mg/kg conferred more gastroprotection than omeprazole.

Discussion

The present study aimed to investigate the potential protective effect of ECF against ethanol-induced stomach ulceration in rats. In addition, the possible mechanisms underlying the gastroprotective effect were explored including the anti-oxidant, anti-inflammatory and anti-apoptotic effects as well as the modulatory effect on CGRP expression. Absolute ethanol administration induced severe hemorrhagic lesions. This finding was supported by the histopathological examination, where severe focal ulceration, hemorrhage, degeneration and necrosis were detected in the stomach mucosa. Also, severe submucosal edema and inflammatory cells infiltration were observed. In contrast, the total ulcerated area was significantly decreased by ECF pre-treatment, in a dose-dependent manner. Interestingly, ECF at dose of 100 mg/kg provided 99.6% gastroprotection which is significantly higher than that provided by omeprazole. In addition, ECF ameliorated the aforementioned gastric histopathological changes induced by ethanol. The present study demonstrated that absolute ethanol administration significantly decreased the mucin content in gastric juice. Nevertheless, this effect was markedly ameliorated in all ECF pre-treated groups. It is well known that mucus represents the first defensive line of gastric mucosa that protects the stomach from necrotizing agents (Sidahmed et al., 2013; Wallace, 2008). Based on the results of this study, the gastroprotective effect of ECF could be partly attributed to its ability to increase the gastric mucin content.

Generation of reactive oxygen species (ROS) is critically involved in the pathogenesis of ethanol-induced gastric damage (Bonamin et al., 2014; Sidahmed et al., 2013). The results of the present study showed that the administration of ethanol induced a marked oxidative stress, as evidenced by increased lipid peroxidation in gastric mucosa. Furthermore, the anti-oxidant capacity was markedly reduced in the ulcer group, where GSH and SOD levels were significantly depleted as compared to normal control values. In contrast, pre-treatment with ECF significantly ameliorated ethanol-induced oxidative stress, in a dose-dependent manner. Interestingly, pretreatment with ECF, at a dose of 100 mg/kg, increased GSH and SOD levels even more than their normal values, which confirmed the strong antioxidant effect of ECF. Experimental evidence indicated that antioxidant compounds can be used to protect against stomach ulcer (Bonamin et al., 2014; Sidahmed et al., 2013). The results of the DPPH* assay clearly indicated the strong radical-scavenging activity of ECF. The potent scavenging of the DPPH* was closely correlated with the high phenol content of ECF. The remarkable antioxidant effect of ECF may be related to the presence of different ellagitannins, which may confer a synergistic action. Experimental evidence have reported that whole plant fractions usually possess much better pharmacological activities than single isolated components due to synergistic interactions between the individual ingredients (Wagner and Ulrich-Merzenich, 2009). Ellagitannins are a class of polyphenolic compounds that have attracted considerable attention for their marked pharmacological activities, including potent radical scavenging activity, inhibitory effect on lipid peroxidation and gastroprotective properties (de Jesus et al., 2012; Sangiovanni et al., 2013). Based on the results of this study, ECF pre-treatment returned the increased MDA to its normal level. Therefore, it can be concluded that the gastroprotective properties of ECF might be partly attributed to the inhibitory effect of its ellagitannins on lipid peroxidation and oxidative stress.

Inflammation plays a prominent causative role in the pathogenesis of ethanol-induced ulceration and the use of anti-inflammatory agents to ameliorate the gastric mucosal damage was shown to be promising (Moezi et al., 2014; Warzecha et al., 2014). Previous studies indicated that the ellagitannins of blackberries and strawberries exhibited gastroprotection via their anti-inflammatory effect (Sangiovanni et al., 2013). In the present study, the positive ulcer group showed a marked increase in pro-inflammatory response as evidenced by a significant increase in gastric mucosal levels of IL-1[beta], TNF-[alpha], 5-LO and COX-2. On the other hand, the pre-treatment of rats with ECF exhibited marked anti-inflammatory effects, which was evident from the reduction of the mucosal contents of several pro-inflammatory markers in a dose-dependent manner.

Several lines of evidence showed that the induction of apoptosis is involved in the pathogenesis of ethanol-induced gastric ulcer. This is attributed to the up-regulation of caspases, a family of enzymes responsible for apoptosis execution (Luo et al., 2013; Yu et al., 2013). In this study, caspase-3 enzymatic activity was significantly increased in ulcer control group, an effect which was reversed by ECF pre-treatment. This suggests that ECF effectively ameliorates ethanol-induced gastric lesions via anti-apoptotic activity. CGRP is a member of the calcitonin peptides family (Rosenfeld et al., 1983). Recently, CGRP gene down-regulation was shown to be involved in gastric ulcer pathogenesis (Evangelista, 2009; Gyires et al., 2013). In this context, many agents exhibited an anti-ulcer activity via the up-regulation of CGRP (de Carvalho et al., 2014; Li et al., 2012). Notably, the pretreatment of rats with ECF significantly increased CGRP expression, in a dose dependent manner, which might contribute to the gastroprotective properties of ECF.

In conclusion, the present study is the first one to provide evidence that ECF has a potent gastroprotective effect against ethanol-induced gastric ulcer, which was demonstrated by reduced ulcerated area and increased mucin content. The remarkable gastroprotective effect of ECF could be partially mediated by attenuating ethanol-induced oxidative stress. ECF enhanced the cellular antioxidant defense status and reduced the elevated MDA tissue levels induced by ethanol administration. In addition, ECF exerted a powerful anti-inflammatory activity as demonstrated by the reduction in pro-inflammatory markers; IL-1[beta]), TNF-[alpha], 5-LO and COX-2. Also, the gastroprotective effect of ECF was attributed to its ability to down-regulate the caspase-3 activity as well as to markedly attenuate the ethanol-induced decrease in CGRP expression The gastroprotective effect of ECF was further supported by histopathological observations of stomach tissues. ECF conferred marked protection from ethanol-induced ulcer as evidenced by intact mucosal layer, absence of hemorrhage and necrosis. Ellagitannins were identified as the major active constituents responsible for the marked antioxidant, anti-inflammatory and gastroprotective properties of ECF. This study represents the first report that employed the HPLC-PDA-ESI/MS/MS technique for the identification of the ellagitannins of E. citriodora. The adopted HPLC-PDA-ESI/MS/MS method provided a useful tool to develop a characteristic chromatographic fingerprint for the authentication of ECF and for the identification of its composition.

Conflict of interest statement

The authors declare that there is no conflict of interest.

ARTICLE INFO

Article history: Received 23 June 2014

Revised 27 August 2014

Accepted 15 October 2014

Acknowledgements

Professor Juha-Pekka Salminen, department of chemistry, university of Turku, Finland is acknowledged for the use of the HPLC-MS instrument during this study.

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Eman Al-Sayed (a), *, Reem N. El-Naga (b)

(a) Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, 11566 Cairo, Egypt

(b) Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, 11566 Cairo, Egypt

* Corresponding author. Tel.: +20 2 01001050293: fax: +20 2 405 1106.

E-mail address: em_alsayed@pharma.asu.edu.eg, emanalsayedasu@gmail.com (E. Al-Sayed).

http://dx.doi.org/10.1016/j.phymed.2014.10.002

Table 1
LC-PDA-ESI/MS/MS identification of ECF ellagitannins.

N   [t.sub.R]   Compounds                   UV maximum
    (min)                                   (nm)

1   9.5         Pedunculagin                222,273 (sh)
2   10.2        Vescalagin/castalagin       222, 273 (sh)
3   11.4        Pedunculagin                222, 273 (sh)
4   12.0        Acutissimin A               218, 260
5   12.5        Pterocarinin A              215,270
6   12.9        Stynophyllanin A            215,273
7   13.6        Casuarinin                  215, 275
8   14.6        Guajavin A                  213, 275
9   15.4        Tellimagrandin 1:           214,273
                  2,3-di-0-galloyl-4,6-0-
                  HHDP-[beta]-d-
                  glucopyranose
10  19.2        Ellagic acid                260, 300 (sh).
                                              365

N   [(M-H).sup.-] m/z           Fragments (MS/MS) m/z      % area

1   783.07                      301. 00, 275.02, 257.01,    4
                                  229.0
2   933.07                      631.06, 425.02, 301.00      5
3   783.07                      301.00, 275.02, 257.01,     4
                                229
4   1205.64                     613.04, 467.03, 301.00,     2
      [(M-2H).sup.2-]: 602.56     289.07, 275.02, 249.04
5   1067.13                     301.00, 275.02, 249.04,    18
      [(M-2H).sup.2-]: 533.06     169.01
6   1207.16                     458.03, 301.00, 289.07,    19
      [(M-2H).sup.2-]:603.07      275.02, 249.04, 169.01
7   935.08 [(M-2H).sup.2-]:     783.08, 633.07, 481.06,    21
      467.03                      301.00, 275.02, 169.01
8   1223.15                     306.44, 301.00, 275.02,     7
      [(M-2H).sup.2-]: 611.07     249.04, 169.01
9   785.09                      301.00. 275.02, 249.04,    4
                                  169.01
10  301.00                                                 7

N   References

1   Al-Sayed et al. (2014)
2   Hager et al., 2008;
      Saucier et al. (2006)
3   Al-Sayed et al. (2014)
4   Saucier et al. (2006)
5   Nonaka et al. (1989)
6   Yoshida et al. (2008)
7   Al-Sayed et al. (2014)
8   Tanaka et al. (1992)
9   Al-Sayed et al. (2014)
10  Hager et al. (2008)

Table 2
Effect of ECF on gastric juice volume, total titrable acidity,
ulcerated area and mucin content in ethanol-induced gastric ulcer in
rats.

Treated groups          Gastric juice
                        volume (ml)

Negative control        NA
Ulcer control           2.99 [+ or -] 0.13 (b)
ECF (25 mg/kg)          2.03 [+ or -] 0.12 (a)
ECF (50 mg/kg)          1.80 [+ or -] 0.33 (a)
ECF (100 mg/kg)         1.59 [+ or -] 0.10 (a)
Omeprazole (20 mg/kg)   1.74 [+ or -] 0.12 (a)

Treated groups          Total titrable
                        acidity (mequiv./l)

Negative control        NA
Ulcer control           38.30 [+ or -] 0.80 (b)
ECF (25 mg/kg)          21.83 [+ or -] 1.76 (a, c)
ECF (50 mg/kg)          41.83 [+ or -] 7.63 (b)
ECF (100 mg/kg)         59.67 [+ or -] 4.77 (a, b)
Omeprazole (20 mg/kg)   19.30 [+ or -] 1.23 (a, c)

Treated groups          Mucin content
                        (mg % hexose)

Negative control        NA
Ulcer control             0.60 [+ or -] 0.22 (b)
ECF (25 mg/kg)            4.21 [+ or -] 0.32 (a, c)
ECF (50 mg/kg)           31.00 [+ or -] 7.61 (a)
ECF (100 mg/kg)         143.00 [+ or -] 24.10 (a, b, c)
Omeprazole (20 mg/kg)    12.90 [+ or -] 6.10 (a)

Treated groups          Observed ulcerated
                        area (%)

Negative control        0
Ulcer control           35.49 [+ or -] 5.16 (b)
ECF (25 mg/kg)          17.61 [+ or -] 1.82 (a, b, c)
ECF (50 mg/kg)          10.93 [+ or -] 0.52 (a)
ECF (100 mg/kg)          0.13 [+ or -] 0.09 (a, b, c)
Omeprazole (20 mg/kg)   10.02 [+ or -] 0.40 (a)

Treated groups          Inhibition percentage
                        (gastroprotection) (%)

Negative control        NA
Ulcer control           NA
ECF (25 mg/kg)          50.46 [+ or -] 5.14 (b, c)
ECF (50 mg/kg)          69.21 [+ or -] 1.50
ECF (100 mg/kg)         99.58 [+ or -] 0.28 (b, c)
Omeprazole (20 mg/kg)   71.74 [+ or -] 2.85

Data are expressed as means [+ or -] SEM (n = 6). NA, not applicable.

(a) Significantly different from the ulcer control group at p < 0.05.

(b) Significantly different from the omeprazole group at p < 0.05

(c) Significantly different from the corresponding group treated with
ECF 50 mg/kg at p < 0.05.

Table 3
Effect of ECF on oxidative stress markers in ethanol-induced gastric
ulcer in rats.

Treated groups          GSH ([micro]mol/g tissue)

Negative control        2.58 [+ or -] 0.16 (b, c)
Ulcer control           0.55 [+ or -] 0.04 (a, c)
ECF (25 mg/kg)          1.35 [+ or -] 0.03 (a, b, c, d)
ECF (50 mg/kg)          2.95 [+ or -] 0.05 (b, c)
ECF (100 mg/kg)         3.72 [+ or -] 0.10 (a, b, c, d)
Omeprazole (20 mg/kg)   1.86 [+ or -] 0.04 (a, b, d)

Treated groups          MDA (nmol/g tissue)

Negative control        15.65 [+ or -] 0.50 (b, c)
Ulcer control           65.42 [+ or -] 5.50 (a, c)
ECF (25 mg/kg)          36.28 [+ or -] 5.30 (a, b, d)
ECF (50 mg/kg)          21.40 [+ or -] 0.50 (a, b)
ECF (100 mg/kg)         13.20 [+ or -] 0.89 (b, c, d)
Omeprazole (20 mg/kg)   31.60 [+ or -] 0.68 (a, b, d)

Treated groups          SOD (U/g tissue)

Negative control        12.57 [+ or -] 0.38 (b, c)
Ulcer control            3.00 [+ or -] 0.14 (a, c)
ECF (25 mg/kg)           6.81 [+ or -] 0.22 (a, b, c, d)
ECF (50 mg/kg)           8.50 [+ or -] 0.21 (a, b, c)
ECF (100 mg/kg)         13.98 [+ or -] 0.33 (b, c, d)
Omeprazole (20 mg/kg)    5.07 [+ or -] 0.17 (a, b, d)

Data are expressed as means [+ or -] SEM (n = 6). GSH, reduced
glutathione; MDA, malondialdehyde; SOD, superoxide dismutase.

(a) Significantly different from the normal control group at p < 0.05.

(b) Significantly different from the ulcer control group at p < 0.05.

(c) Significantly different from the omeprazole group at p < 0.05.

(d) Significantly different from the corresponding group treated with
ECF 50 mg/kg at p < 0.05.

Table 4
Effects of ECF on inflammatory markers in ethanol-induced gastric
ulcer in rats.

Treated groups          IL-1[beta] (pg/mg protein)

Negative control        17.30 [+ or -] 0.80 (b, c)
Ulcer control           73.32 [+ or -] 4.84 (a, c)
ECF (25 mg/kg)          28.83 [+ or -] 0.79 (a, b, c)
ECF (50 mg/kg)          25.17 [+ or -] 0.29 (a, b, c)
ECF (100 mg/kg)         16.60 [+ or -] 0.76 (b, c, d)
Omeprazole (20 mg/kg)   43.00 [+ or -] 1.93 (a, b, d)

Treated groups          TNF-[alpha] (pg/mg protein)

Negative control        20.83 [+ or -] 0.82 (b, c)
Ulcer control           80.92 [+ or -] 4.74 (a, c)
ECF (25 mg/kg)          38.27 [+ or -] 0.94 (a, b, c, d)
ECF (50 mg/kg)          27.80 [+ or -] 0.54 (a, b, c)
ECF (100 mg/kg)         20.80 [+ or -] 0.82 (b, c, d)
Omeprazole (20 mg/kg)   50.32 [+ or -] 1.74 (a, b, d)

Treated groups          5-LO (ng/mg protein)

Negative control         5.35 [+ or -] 0.28 (b, c)
Ulcer control           28.48 [+ or -] 1.10 (a, c)
ECF (25 mg/kg)          13.70 [+ or -] 0.44 (a, b, c, d)
ECF (50 mg/kg)           8.38 [+ or -] 0.29 (a, b, c)
ECF (100 mg/kg)          4.90 [+ or -] 0.20 (b, c, d)
Omeprazole (20 mg/kg)   19.70 [+ or -] 0.41 (a, b, d)

Treated groups          COX-2 (pg/mg protein)

Negative control         2.72 [+ or -] 0.16 (b, c)
Ulcer control           20.73 [+ or -] 0.75 (a, c)
ECF (25 mg/kg)           8.68 [+ or -] 0.20 (a, b, c, d)
ECF (50 mg/kg)           5.05 [+ or -] 0.12 (a, b, c)
ECF (100 mg/kg)          2.30 [+ or -] 0.17 (b, c, d)
Omeprazole (20 mg/kg)   13.25 [+ or -] 0.32 (a, b, d)

Data are expressed as means [+ or -] SEM (n = 6). IL-1[beta],
interleukin-1 beta; TNF-[alpha], tumor necrosis factor-alpha; 5-LO,
5-lipoxygenase; COX-2, cyclooxygenase 2.

(a) Significantly different from the negative control group at
p < 0.05.

(b) Significantly different from the ulcer control group at p < 0.05.

(c) Significantly different from the omeprazole group at p < 0.05.

(d) Significantly different from the corresponding group treated with
ECF 50 mg/kg at p < 0.05.

Table 5
Histopathological alterations in stomach specimens of different
treated groups.

Histopathological alteration     Negative   Ulcer     ECF
                                 control    control   (25 mg/kg)
                                 group      group

Ulceration in the mucosal area   -          ++++      ++
Necrosis in the mucosal area     -          ++++      ++
Hemorrhage in the mucosal area   -          ++++      -
Edema and inflammatory cell      -          +++       +
  infiltration in the
  submucosa

Histopathological alteration     ECF          ECF           Omeprazole
                                 (50 mg/kg)   (100 mg/kg)   (20 mg/kg)

Ulceration in the mucosal area   +            -             ++
Necrosis in the mucosal area     ++           -             ++
Hemorrhage in the mucosal area   _            -             -
Edema and inflammatory cell      +            +             +
  infiltration in the
  submucosa

++++, very severe; +++, severe; ++, moderate; +, mild; -, nil.
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