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Phytomedicine 20 (2013) 834– 843

Contents lists available at SciVerse ScienceDirect

Phytomedicine

jou rn al h om e page: www.elsev ier .de /phymed

yranocycloartobiloxanthone A, a novel gastroprotective compound fromrtocarpus obtusus Jarret, against ethanol-induced acute gastric ulcer in vivo

eyam M.A. Sidahmeda, Najihah Mohd Hashima, Junaidah Amira, Mahmood Ameen Abdullab,. Hamid A. Hadic, Siddig Ibrahim Abdelwahabd, Manal Mohamed Elhassan Tahad,ouya Hassandarvishb, Xinsheng Tehe, Mun Fai Lokee, Jamuna Vadivelue, Mawardi Rahmani f,yam Mohana,∗

Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaFaculty of Science, University of Malaya, 50603 Kuala Lumpur, MalaysiaMedical Research Centre, Jazan University, P.O. Box 114 Jazan, Saudi ArabiaDepartment of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Chemistry, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia

r t i c l e i n f o

eywords:rtocarpus obtususyranocycloartobiloxanthone AastroprotectionSP70ntioxidantelicobacter pylori

a b s t r a c t

Pyranocycloartobiloxanthone A (PA), a xanthone derived from the Artocarpus obtusus Jarret, belongs tothe Moraceae family which is native to the tropical forest of Malaysia. In this study, the efficacy of PA asa gastroprotective compound was examined against ethanol-induced ulcer model in rats. The rats werepretreated with PA and subsequently exposed to acute gastric lesions induced by absolute ethanol. Theulcer index, gastric juice acidity, mucus content, histological analysis, glutathione (GSH) levels, malondial-dehyde level (MDA), nitric oxide (NO) and non-protein sulfhydryl group (NP-SH) contents were evaluatedin vivo. The activities of PA as anti-Helicobacter pylori, cyclooxygenase-2 (COX-2) inhibitor and free radicalscavenger were also investigated in vitro. The results showed that the oral administration of PA protectsgastric mucosa from ethanol-induced gastric lesions. PA pretreatment significantly (p < 0.05) restoredthe depleted GSH, NP-SH and NO levels in the gastric homogenate. Moreover, PA significantly (p < 0.05)reduced the elevated MDA level due to ethanol administration. The gastroprotective effect of PA was

associated with an over expression of HSP70 and suppression of Bax proteins in the ulcerated tissue. Inaddition, PA exhibited a potent FRAP value and significant COX-2 inhibition. It also showed a significantminimum inhibitory concentration (MIC) against H. pylori bacterium. The efficacy of PA was accomplishedsafely without the presence of any toxicological parameters. The results of the present study indicate thatthe gastroprotective effect of PA might contribute to the antioxidant and anti-inflammatory propertiesas well as the anti-apoptotic mechanism and antibacterial action against Helicobacter pylori.

ntroduction

Peptic ulcer disease (PUD) is the most common multifactorastrointestinal disease affecting a lot of people worldwide. Theisease occurs when the aggressive factors overcome the protectivene. Several factors are claimed to be involved in the developmentf gastric and duodenal mucosal injury; among them are the over

ngestion of non-steroidal anti-inflammatory drugs (NSAIDS) andacterial infection with Helicobacter pylori (Rao et al., 2004).

∗ Corresponding author. Tel.: +603 7967 7520; fax: +603 7967 4964.E-mail addresses: syammohanm@um.edu.my, syammohanm@yahoo.com

S. Mohan).

944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.phymed.2013.03.002

© 2013 Elsevier GmbH. All rights reserved.

It is known that ethanol is one of the necrotizing agents thatcauses vascular damage and gastric cell necrosis, resulting in gastriculcer formation (Mahmood et al., 2005). Previous studies reportedthat oxygen free radical species produced by ethanol adminis-tration were responsible for gastric damage. Nowadays, there isglobal interest in searching for alternative medicines from natu-ral resources, mainly those derived from the plant kingdom. Plantsremain as the most attractive source of new drugs that has demon-strated anti-ulcerogenic activities (Falcão et al., 2008). Severalcompounds from natural flora were found to exhibit antiulcer activ-ity, mainly alkaloids, saponins, xanthones, triterpenes and tannins

(de Souza Almeida et al., 2011).

Artocarpus obtusus Jarrett (Moraceae) is an endemic speciesof Borneo and locally known as ‘pala tupai’. The species mostlygrow in lowland, peat swamp and freshwater swamp forests in

H.M.A. Sidahmed et al. / Phytome

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Fig. 1. Chemical structure of pyranocycloartobiloxanthone A (PA).

he tropical and subtropical regions. Recent studies on its mainioactive compound, pyranocycloartobiloxanthone A (PA) (Fig. 1),xhibited significant antioxidant and antibacterial activities againstelected assays (Hashim et al., 2012), in addition to the cytotoxicnd antiproliferative effects in MCF7 cells (Hashim et al., 2011)hrough NF-kB and Bcl2/Bax signaling pathways (Mohan et al.,012). Several studies suggest that oxidative stress is involved inhe pathology of gastric ulcer. Therefore, based on the potentialctivities of the PA, mainly the antioxidant property, the com-ound was subjected to several experimental tests to evaluate itsastroprotective activity for the first time and to identify possi-le mechanism (s) underlying this effect against the ethanol ulcerodel in experimental animals.

aterial and methods

lant materials

The stem bark of Artocarpus obtusus Jarret was collected fromarawak, identified by Dr. Rusea Go, and a voucher specimenS94402) has been deposited at the Herbarium, Department of Biol-gy, Faculty of Science, Universiti Putra Malaysia.

xtraction and isolation of pyranocycloartobiloxanthone A (PA)

Pyranocycloartobiloxanthone A as yellow needle-shaped crys-als was purified from the dried and ground stem bark in our lab.heir chemical and physical data as obtained in our work were ingreement with those reported previously (Hashim et al., 2010)Fig. 1).

cute toxicity study

The acute toxicity study was used to determine a safe dose of theompound. Healthy ICR mice of about 6–8 weeks old and 20–30 geight were obtained from the Experimental Animal House, Fac-lty of Medicine, University of Malaya. The animals were fedtandard pellets and free access to water ad libitum. Thirty six mice18 male and 18 female) were assigned equally into three groups.vernight fasted animals were orally administered with PA at lownd high dose (30 and 300 mg/kg of body weight); whereas theontrol group animals were received 5% Tween 80. The food wasithheld for further 3–4 h after dosing. During 14 days, the ani-als were observed for any mortality or physiological changes.n day 15, body weight variation was determined; blood sampleas collected for biochemical parameters analysis. Animals were

acrificed under anesthesia (ketamine & xylazil), and the liversnd kidneys were excised for organ weight variation and histol-gy study. This study was performed based on Organization for

conomic Co-operation and Development (OECD) Guideline 420rotocol year 1992, and was approved by Institutional Animalare and Use Committee, University of Malaya (UM ICUCA) [Ethico. FAR/29/06/2012/HMAS(R)], Faculty of Medicine, University of

dicine 20 (2013) 834– 843 835

Malaya, Malaysia. All animals were received human care accordingto the criteria outlined in the “Guide for the Care and use of Labo-ratory Animals” prepared by the National Academy of Sciences andpublished by the National Institute of Health (USA, VA).

Induction of acute gastric lesion

Sprague Dawley rats (200–220 g) were obtained from theExperimental Animal House [Ethic No FAR/29/06/2012/HMAS(R)],Faculty of Medicine, University of Malaya. To avoid coprophagy,the rats were kept individually in cages with raised floors of widemesh and divided randomly into six groups (n = 6). Overnight fastedanimals were treated as follows: group (A, B) treated with vehicle(5% Tween 80 v/v 5 ml/kg b.w.), group (C) 20 mg/kg omeprazolein vehicle (5 ml/kg) as positive group, group (D, E, F) were treatedwith PA at three doses 5, 10 and 20 mg/kg (5 ml/kg) in vehicle. After1 h all groups except group (A) received absolute ethanol (5 ml/kg)(Potrich et al., 2010). 1 h later, all animals were anesthetized usingketamine & xylazil, blood samples were collected from their jugularvein then euthanized by cervical decapitation and their stomachswere immediately removed.

Measurement of gastric juice acidity, mucus content and thebiochemical parameters

Each experimental stomach was opened along the greater cur-vature. Gastric contents were collected and centrifuged to measurethe pH of gastric juice from the supernatant by pH metric titrationwith 0.1 N NaOH solutions using digital pH meter. Stomachs werewashed with normal saline to keep the mucus layer on the mucosasurface. The gastric mucosa of each animal was rubbed off usinga glass slide and the weight of the collected mucus was measuredusing precise electronic balance (Tan et al., 2002). Animal bloodsamples were analyzed by clinical laboratory diagnostics (CLD)automated machine to evaluate changes in biochemical parame-ters, assessed at University Malaya Medical Centre, Kuala Lumpur,Malaysia.

Gastroprotective assessments

Gastric ulcer appeared as elongated bands of hemorrhagiclesions in which the elongated lesions are the pathological fea-tures that related with ethanol induced ulcer (Alvarez-Suarez et al.,2011). The length (mm) and the width (mm) of each band wasmeasured using planimeter [(10 mm × 10 mm = ulcer area) underdissecting microscope (1.8×)]. The area of each ulcer lesion wasmeasured by counting the number of small squares, 2 mm × 2 mm,covering the length and width of each ulcer band. The sum ofthe areas of all lesions for each stomach was applied in thecalculation of the ulcer area (UA) wherein the sum of smallsquares × 4 × 1.8 = UA mm2. The inhibition percentage (I %) was cal-culated by the following formula described in Njar et al. (1995) withslight modifications:

Inhibition percentage (I%)

= [(UAcontrol − UAtreated)/UAcontrol] × 100%.

Gastric tolerability test

The gastric tolerability test was conducted to assess the effect

of the compound on the stomach. Each experimental stomachswere observed under an illuminated magnifier (3×) to evaluatethe severity of gastric lesions according to the modified gastricdamage scoring system of (Adami et al., 1964; Ðor –devic et al.,

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007) (0:no lesion; 0.5:slight hyperaemia or ≤5 petechiae; 1: ≤5rosions ≤5 mm in length;1.5: ≤5 erosions ≤5 mm in length andany petechiae; 2:6–10 erosions ≤5 mm in length; 2.5:1–5 ero-

ions > 55 mm in length; 3:5–10 erosions >5 mm in length; 3.5:10 erosions >5 mm in length; 4:1–3 erosions ≤ 5 mm in lengthnd 0.5–1 mm in width; 4.5:4–5 erosions ≤5 mm in length and.5–1 mm in width; 5:1–3 erosions > 5 mm in length and 0.5–1 mm

n width; 6:4 or 5 grade 5 lesions; 7: ≥6 grade 5 lesions; 8: completeesion of the mucosa with hemorrhage.

istological evaluation

A small specimen of each stomach was fixed in 10% bufferdormalin solution followed by tissue dehydrated with alcohol andylene. Then each sample was embedded in paraffin wax, sectionedt 5 �m in slides. Selected slides then stained with hematoxylin andosin (H & E) for light microscopy. Moreover, to evaluate mucusroduction, other slides also stained by periodic acid Schiff basePAS) following the manufacture instruction (Sigma Periodic Acid-chiff (PAS) Kit). For further analysis, study slides underwent formmunohistochemistry staining using ARKTM (Animal Researchit) to observe the immunhistochemical localization of HSP70

1:100) and Bax (1:50) proteins. Both proteins were purchase fromanta Cruz Biotechnology, Inc., California, USA.

reparation of gastric tissue homogenate

Glandular gastric tissue washed thoroughly with ice-cold saline.sing a homogenizer (Polytron, Heidolph RZR 1, Germany), Small

ragment of each stomach was homogenized (10% w/v) in ice coldBS (0.1 mol/l) containing mammalian protease inhibitor cocktail.he homogenates were centrifuged at 10000 g for 15 min at 4 ◦C.he pure supernatant was used to quantify the levels of GSH, MDA,O and NP-SH.

lutathione levels

Total GSH content was estimated by interaction with DTNB (5,5-itiobis-2-nitrobenzoic acid) and the absorbance was read in apectrophotometer (412 nm). The results were expressed in nmolSH/g tissue (Rahman et al., 2007).

hiobarbituric acid reactive substance assay

Thiobarbituric acid reactive substance (TBARS) assay was usedo estimate MDA content. Briefly, the stomach homogenate wasdded to a 0.126 ml solution containing 26 Mm thiobarbituric acid,.26 M HCl, 15% trichloroacetic acid and 0.02% butylated hydroxyl-oluene. The mixture was incubated in a water bath at 95 ◦C for 1 h.fter cooling, the mixture was centrifuged at 3000 × g for 10 min.he absorbance was read in a spectrophotometer at 532 nm and theesults were expressed in �mol/g tissue malondialdehyde. Tetram-thoxy propane was used as reference standard (Hodges et al.,999).

itric oxide level

Nitric oxide (NO) content was quantified by measuringitrite/nitrate concentration using Griess assay (Miranda et al.,001). In brief, stomach homogenates were deproteinated with

bsolute ethanol for 48 h at 4 ◦C, then centrifuged at 12000 g for5 min at 4 ◦C. To an aliquot of the supernatant, vanadium trichlo-ide 0.8% (w/v) in 1 M HCl was added for the reduction of nitrate toitrite, followed by rapid addition of Griess reagent (Sigma) and the

dicine 20 (2013) 834– 843

absorbance was measured at 540 nm. The results were expressedas �mol/g tissue. Sodium nitrite was used as reference standard.

Estimation of nonprotein sulfhydryls content

Gastric mucosal non-protein sulfhydryls content (�mol/g of tis-sue) was measured according to the method of (Sedlak and Lindsay,1968). Briefly, aliquot of 5 ml of the stomach homogenate wasmixed with a solution containing 4 ml of distilled water and 1 mlof 50% trichloroacetic acid. The mixture was vortex for 15 minand centrifuged at 3000 × g. Two milliliters of supernatant wasmixed with 4 ml of 0.4 M Tris Buffer at pH 8.9; 0.1 ml of DTNB [5,5dithiobis-(2-nitrobenzoic acid)] was added and the samples wereshaken. The absorbance was recorded within 5 min after DTNBaddition at 412 nm against a reagent blank with no homogenate.

In vitro evaluation of COX-2 inhibitory activity

PA was tested for COX-2 inhibitory activity using a COX-inhibitor screening kit (Cayman Chemical, USA) according to themanufacturer’s instructions. PA was dissolved in DMSO to a finalconcentration was 0–100 �g/ml. The inhibition was calculatedby the comparison of compound treated to control incubation.Indomethacin was used as a reference standard.

Ferric-reducing antioxidant power (FRAP) assay

The FRAP value of PA was estimated according to the rec-ommended method of (Benzie and Strain, 1999) with slightmodification. Briefly, the FRAP reagent was prepared freshlyfrom acetate buffer (pH 3.6), 10 mM TPTZ [2,4,6-Tri(2-pyridyl)-s-triazine] solution in 40 mM HCl and 20 mM iron (III) chloridesolution in proportions of 10:1:1 (v/v), respectively. Fifty micro-liters of the compound was added to 1.5 ml of the FRAP reagent inthe dark for 4 min, then the absorbance was recorded at 593 nm.The standard curve was constructed linear (R2 = 0.9723) using iron(II) sulfate solution (100–1000 �M), and the results were expressedas �M Fe (II)/g dry weight of the compound.

In vitro anti-Helicobacter pylori activity

Two Helicobacter pylori strains, NCTC 11637 (ATCC 43504) andJ99 (ATCC 700824) were cultured with brain heart infusion broth(BHI; Oxoid) supplemented with 10% horse serum (Invitrogen)incubated at 37 ◦C in a humidified CO2 incubator for 3 days. Mini-mum inhibitory concentration (MIC) was determined by a modifiedmicrotiter broth dilution method on sterile 96-well polypropyl-ene microtitre plates with round-bottom wells. Briefly, PA wasdissolved and diluted in 5% DMSO to give a 10× working stocksolution. H. pylori was diluted to a final concentration of 2 × 106

CFU/ml in culture medium. Aliquot of 10 �l of PA was added to90 �l of H. pylori in a well of the microtitre plate. Concentrationof PA ranged from 31.25 to 250 �g/ml. The microtiter plate wasincubated for 3 days in a CO2 incubator. The plate was exam-ined visually and measured using a microplate reader at 600 nmto determine the lowest concentration showing complete growthinhibition, which was recorded as the MIC. Minimum bactericidalconcentration (MBC) as the lowest concentration without growthon a chocolate agar plate supplemented with 7% lysed horse blood.

Wells containing H. pylori with 10 �l of 5% DMSO and BHI mediumcontaining 250 �g/ml PA were used as control and blank, respec-tively. The result was recorded in accordance with the Clinical andLaboratory Standards Institute (Jorgensen et al., 2007).

H.M.A. Sidahmed et al. / Phytomedicine 20 (2013) 834– 843 837

Table 1Protective effect of PA on ethanol-induced gastric ulcer in rats. Observed ulcer area, inhibition percentage and mucus stomach content.

Animal group Pretreatment (5 ml/kg) pH of gastric tissue Mucus weight (g) (mean + S.E.M) Ulcer area (mm2) (mean + S.E.M) Inhibition%

1 Normal control 7.06 ± 0.09 2.90 ± 0.06 0 02 Tween 80 (ulcer control) 2.67 ± 0.15 0.99 ± 0.04 557.28 ± 14.7 NA3 Omeprazole 20 mg/kg 6.72 ± 1.14* 1.75 ± 0.07* 108 ± 20.78* 79.074 PA 5 mg/kg 5.40 ± 0.2* 1.67 ± 0.05* 158.4 ± 18.36*,# 71.585 PA 10 mg/kg 6.00 ± 0.80* 2.05 ± 0.03* 128.16 ± 15.34*,# 77.006 PA 20 mg/kg 5.0 ± 0.16* 1.45 ± 0.08* 201.6 ± 47.60*,# 63.82

NA, not applicable.All value are expressed as mean ± standard error mean.

* Indicates significance at p < 0.05 compared to ulcer control.

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tatistical analysis

All values were reported as mean ± S.E.M. The Statistical signif-

cance of differences between groups was assessed according toPSS version 16 and Graph Pad prism 6.0. A Value of p < 0.05 wasonsidered significant.

ig. 2. Gross evaluation of gastric mucosa of the rats in different groups. Extensive visontrol animals (B). The PA protected against acute ethanol-induced erosion of gastric mumeprazole (20 mg/kg) treated groups respectively. White arrow indicates elongated baMagnification: 1.8×).

Result

Acute toxicity study

There were no abnormal physiological or behavioral changes,body weight alteration at any time of observation at the doses used

ible hemorrhagic necrosis of gastric mucosa caused by absolute ethanol in ulcercosa at 5, 10 and 20 mg/kg dose (D–F). (A and C) represents the normal control and

nds of hemorrhagic lesions, while black arrow indicates flattening of mucosal fold

838 H.M.A. Sidahmed et al. / Phytomedicine 20 (2013) 834– 843

Table 2Effects of PA on liver function test renal function test and HDL level in ethanol-induced gastric ulcer in rats.

Animal group Treatment groups (mg/kg) AST (U/l) ALT (U/l) Creatinine mmol/l HDL mmol/l

1 Normal Control 207 ± 9.81 35.67 ± 1.67 45.33 ± 3.71 1.29 ± 0.272 Tween 80 (ulcer control) 292 ± 7.15 63 ± 1.47 49.67 ± 0.33 1.085 ± 0.203 Omeprazole (20 mg/kg) 174.67 ± 6.01* 34 ± 2.08* 43.33 ± 2.19* 1.30 ± 0.20*

4 PA 5 mg/kg 218 ± 4.01* 50.07 ± 1.3* 39 ± 2.20* 1.29 ± 0.10*

5 PA 10 mg/kg 185.4 ± 3.91* 42.2 ± 2.24* 32.8 ± 3.32* 1.31 ± 0.11*

6 PA 20 mg/kg 226.2 ± 5.36* 52 ± 5.54* 43.2 ± 6.70* 1.27 ± 0.21*

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ll values expressed as mean ± S.E.M.* Asterisk are statistically significant p < 0.05 compared to ulcer control.

uring the 14 days. Histological examination to the liver and kidneynd the serum biochemical analysis did not showed any significantifferences when compared to the control group. Thus, our resulthowed that oral 50% lethal dose (LD 50) value for both female andale mice is greater than 300 mg/kg.

ffect of PA on gastric lesions induced by ethanol

Ulcerated rats pretreated with omeprazole or PA showed signif-cantly reduced (p < 0.05) areas of gastric ulcer formation comparedo ethanol group. Omeprazole 20 mg/kg b.w and PA at doses of, 10, 20 mg/kg b.w, significantly (p < 0.05) has reduced the for-ation of ulcer by 79.07%, 71.58%, 77% and 63.82% respectively.

he results showed significant (p < 0.05) differences between thehree doses of PA (Table 1). Flattening of gastric mucosal folds andeduced mucosal damage were also observed in gross evaluationf PA treated rats (Fig. 2 D, E, F) or in omeprazole group (Fig. 2C)hen compared to ulcer control group (Fig. 2B).

astric mucus content, pH and biochemical analysis

PA or omeprazole treated groups produced statistically signifi-ant (p < 0.05) increase in mucus contents of gastric mucosa when

ig. 3. Histological evaluation of rats pretreated with Tween 80 (normal control), Tween

mproved histological appearance compared to ulcer control rats which have extensive dnto gastric mucosa (white arrow) and extensive edema and leucocytes infiltration of subs magenta color (yellow arrow). PA treatment had observed magenta color in the apicaMagnification at 2×). (For interpretation of the references to color in the artwork, the re

compared to ulcer control group (Table 1). Animal groups pre-treated with PA significantly (p < 0.05) increased the pH of thegastric contents (Table 1). Serum analysis showed that animals inulcer control group had increased levels of HDL, creatinine andliver enzymes (AST and ALT) compared to normal animal group.Meanwhile animals in PA-pretreated groups significantly (p < 0.05)reduced elevation of such parameters (Table 2).

Histological evaluation

H&E and PAS staining resultsAccording to H&E staining the histological observation of gastric

ulcer in the ulcer control group showed visible extensive damageof the gastric mucosa, and necrotic lesions penetrated deeply intothe mucosa. Moreover, extensive oedema and leucocytes infiltra-tions of the submucosal layer were also observed (Fig. 3). Animalspretreated with PA had better protection for the gastric mucosashowed reduction or absence of ulcer area, submucosal oedema,

and leucocytes infiltration (Fig. 3). PAS staining showed that PAincreased the glycoprotein content of gastric mucosa in pretreatedgroups, as evident in Fig. 3 (the magenta color), in comparison tothe ulcer control group, indicating that PA had the ability to reserve

80 (ulcer control) and PA 10 mg/kg. H&E staining shows that PA pre-treatment hadisruption to the surface epithelium and hemorrhagic necrosis penetrating deeply

mucosa (black arrow). PAS staining presented gastric tissue glycoprotein appearedl epithelial cells showed gradual increased in mucosal secretion of gastric glands

ader is referred to the web version of the article.)

H.M.A. Sidahmed et al. / Phytomedicine 20 (2013) 834– 843 839

Fig. 4. Immunohistochemical evaluation of expression of HSP70 and Bax proteins in rats pretreated with Tween 80(normal control), Tween 80 (ulcer control) and PA 10 mg/kg.T proteia terprev

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he image showed over expression of HSP70 proteins and down expression of Bax

ntigen sites appeared as brown color (yellow arrow) (Magnification 20×). (For inersion of the article.)

he decrease in glycoprotein content induced by absolute ethanolFig. 3).

mmunohistochemical staining resultsImmunohistochemistry showed that the PA-pre-treatment

aused up regulation of HSP70 protein excepression in gastricissue. The expression of HSP70 protein in the ulcer controlroup underwent down regulation when compared to that ofxperimental groups (Fig. 4). Bax protein, on the other hand, inhe PA-pre-treated animals was down regulated. In contrast, thexpression of Bax protein in the ulcer control group was up regu-ated (Fig. 4). Antigen sites are characterized by the brown stainingn the cytoplasm of epithelial cells in the gastric glands.

ffects of PA on antioxidant system

GSH levels were significantly (p < 0.05) lower in ulcer con-rol group than the normal group. The treatment of animalsith the PA restored the GSH levels depletion after ethanol

dministration (Fig. 5 A). Together, the ulcer control group hadhowed the highest MDA level than the other groups, which isn indicator for lipid peroxidation. Gastric MDA level significantlyp < 0.05) dropped after PA administration (Fig. 5B). In vitro antiox-dant evaluation of PA revealed (p < 0.05) potent significant FRAPalue of 4197.94 ± 50.03 0.03, while the positive controls used inhis study exhibited 2140.61 ± 102.284 0.061, 770.39 ± 5.41 0.003,460.67 ± 45.80 0.027 and 421.94 ± 5.44 0.003 �M Fe (II)/g forrolox, quercetin, gallic acid, and ascorbic acid, respectively (Fig. 6).

ffect of PA on Non protein-Sulfhydryl compounds (NP-SH)

Pretreatment of rats with PA significantly (p < 0.05) restored theepletion of NP-SH caused by ethanol administration (Fig. 5C)

ns in the stomachs of rats pretreated with PA compared to ulcer control group. Thetation of the references to color in the artwork, the reader is referred to the web

Role of endogenous NO and COX-2 activity in gastroprotection

As demonstrated in (Fig. 5D), PA exhibited significant (p < 0.05)elevation of NO level at the three treatment doses, which wasdropped due to ethanol administration. Moreover, it was observedthat PA inhibited COX-2 activity by 69.4% and 28.6% at 500 and250 �g/ml, respectively compared to the standard COX-2 inhibitorusing EIA kit (Fig. 7).

In vitro anti-Helicobacter pylori activity

Pyranocycloartobiloxanthone A represented minimuminhibitory concentration MIC of 250 �g/ml and minimum bacteri-cidal concentration MBC >250 �g/ml against H. pylori NCTC11637strain and interesting MIC of 62.5 �g/ml and MBC of 125 �g/mlagainst H. pylori J99.

Discussion

Nowadays, there is global interest in traditionalmedicine/complementary and alternative medicine. Numer-ous medicinal plants have been evaluated as gastroprotectiveagents. Induction of gastric ulcer by ethanol administration isa common experimental model used to evaluate the antiulceractivity of tested compounds, since ethanol easily penetratesinto the gastric mucosa (Li et al., 2008; Mahmood et al., 2005).It was found that ethanol administration resulted in depressionof the stomach homogeneity, inhibited prostaglandin synthesis,disrupted the mucus layer that covers the inner surface of thestomach as well as decreased GSH levels and the protective factors

of the gastric mucosa. Moreover, it increased the microvascularpermeability as well as lipid peroxidation (Rozza et al., 2011). Inaddition, ethanol increased free radical production and bluntednitric oxide in the gastric mucosa, resulting in gastric mucosa

840 H.M.A. Sidahmed et al. / Phytomedicine 20 (2013) 834– 843

Fig. 5. Effect of PA on gastric tissue homogenate content of (A) glutathione (GSH), (B) malondialdehyde (MDA), (C) Non protein sulfhydryl compound (NP-SH) and (D)nitric oxide (NO) level. PA pre-treatment significantly increased GSH, NP-SH and NO level along with decreased in MDA level. * Indicate significance differences in animalp presset

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properties refer to properties of a compound that can delay orinhibit cellular oxidative stress and thus protect tissue damagethrough radical scavenging mechanism (Tachakittirungrod et al.,2007). To evaluate the antioxidant activity of PA, we demonstrated

FRAP va lue ( µM Fe (II)/g dry mass )

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re-treated with PA on 5, 10 and 20 mg/kg treatments at (p < 0.05). The results are exests to see the differences between groups using GraphPad Prism version 6.

amage (Salga et al., 2011). In the present study, we examinedhe gastroprotective activities of pyranocycloartobiloxanthone

against ethanol-induced stomach ulceration. This xanthoneompound derived from Artocarpus obtusus exhibited significantastroprotective effects.

To evaluate possible side effects of PA on other organs, a liverunction test was also conducted. In our study, the levels of ALTnd AST liver enzymes were increased in the ulcer control groups opposed to normal and PA-treated animals. Elevation of livernzymes can be attributed to alcoholic hepatitis or inflammationf the liver due to ethanol administration (Salga et al., 2011). Thereatment with PA was found to be significantly protective againsthis chemically-triggered tissue damage. In addition, the elevatedreatinine and HDL levels in the ulcer groups were found to beormalized in rats that were pre-treated with PA.

Relaxation of circular muscles result in the flattening of mucosalolds which increases the mucosal area exposed to necrotizinggents and reduces the volume of gastric irritant on the rugal crest.thanol increased circular muscle contraction of the rat fundic stripJamal et al., 2006). In this study, the observed flattening of mucosalolds suggests that the gastroprotective effect of PA might be due

o a decrease in gastric motility.

The involvement of reactive oxygen species (ROS) in the patho-enesis of gastric ulcer was well documented (Graziani et al., 2005).esides, the scavenging of free radicals protects the gastric mucosa

d as mean ± SEM by ordinary one way ANOVA with Dunnett’s multiple comparison

from oxidative stress damage (Hahm et al., 1996). Antioxidant

as

Fig. 6. Ferric reducing antioxidant power assay of PA in compare with trolox,quercetin, galic acid and ascorbic acid. Results are expressed in �M Fe (II)/g drymass.

H.M.A. Sidahmed et al. / Phytome

Fig. 7. Inhibition of COX-2 enzyme. PA was evaluated in a COX-2 catalyzedpw(

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p

rostaglandin biosynthesis assay. PA was observed to inhibit COX-2 enzymeshereby, the two final dilutions for PA 500 ng/ml and 250 ng/ml inhibit significantly

*p < 0.05). Data represent mean ± SEM.

he FRAP assay and the results were compared with the controlroup. A positive FRAP result indicates that the tested compounds an electron donor that terminates the oxidation chain reac-ion by reducing the oxidized intermediates into the stable formTachakittirungrod et al., 2007). Thus, the results of the FRAP assaynderline the antioxidant capacity of PA. In agreement with theseesults, a recent study reported that PA possesses a strong free rad-cal scavenging effect against DPPH free radicals (Hashim et al.,012). Thus, it could be suggested that PA mediates its gastropro-ective activity through scavenging mechanisms.

It is known that lipid peroxidation is the result of ROS interactionith the cell membrane, subsequently producing highly reactive

ipid-derived free radicals such as MDA to cause oxidative gastricamage (Kwiecien et al., 2002). In our current study, ethanol sig-ificantly increased the MDA level of the stomach homogenate;he TBRAS assay revealed that PA was able to lower the elevated

DA level as an indicator of radical scavenging activity. Apart fromDA, lowered GSH level in the gastric homogenate also indicate the

everity of oxidative stress damage caused by ethanol administra-ion (Bafna and Balaraman, 2004). GSH is an important intracellularntioxidant which protects the gastric mucosa from free radical-nduced tissue damage (Hiraishi et al., 1994; Li et al., 2008). Weound that the potential antioxidant activity of PA was proved by

reverse in the lowered GSH level in contrast to the ulcer con-rol group. Thus, it could be said that the ability of PA in reducinghe MDA level and restoring the GSH level might contribute to itsastroprotective effect by inhibiting oxidative gastric damage.

Heat shock protein (HSP70) is a protein of 70 kDa moleculareight; it functions by maintaining normal protein structures and

etting rid of the damaged ones induced by various external andellular stresses such as heat intolerance, immune response orxidative stress. The expression of HSP70 plays an important rolen the intracellular mechanism of gastric protection against ethanolHahm et al., 1996). Hence, the over expression of HSPs is con-idered as a direct indicator of the protective function of manyompounds against oxidative damage (Mikami et al., 2006). In ordero evaluate this effect, we observed the localization of HSP70 in thelcerative tissues. In our experiments, the gastric tissues pretreatedith PA in the ethanol ulcer model exhibited an up regulation ofSP70 proteins. This evidence led us to hypothesize that the induc-

ion of HSP70 protein synthesis might play an essential role in

romoting the gastroprotective effect of PA, possibly by attenuatingOS-mediated gastric oxidative stress.

Earlier, apoptosis (programmed cell death) was reported to takelace in gastric ulceration (Konturek et al., 1999). The normal

dicine 20 (2013) 834– 843 841

physiologic state of gastric mucosa is always under equilibriumbetween the cell death and cell renewal process, and gastric lesionis developed when there is an increase in apoptosis and/or inhi-bition of cell proliferation (Maity et al., 2008). It was proved thatethanol induced gastric lesion formation through accelerating thecell death process (Piotrowski et al., 2008). The Bcl-2 family pro-teins are the main organizers of apoptotic cell death, where Bcl-2protein inhibits apoptosis. Inversely, the Bax proteins promote theapoptosis process (Konturek et al., 1999). In the present study,the effect of PA on gastric epithelial apoptosis was determined bymeans of Bax expression in the gastric mucosa following ethanol-induced gastric ulcer. Our results showed that as opposed to theulcer control group, treatment with PA exhibited a down regulationof the Bax protein expression in gastric mucosal tissue as shown byimmunohistochemical staining. This result suggests that PA effec-tively inhibits ethanol-induced gastric ulceration via anti-apoptoticproperty.

Endogenous NP-SH compounds are thought to be involved inprotecting the gastric mucosa against various chemicals by reg-ulating the production and nature of mucus (Chen et al., 2005;Salim, 1992). It is known that ethanol exerts its aggressive effectson gastric mucosa through diminishing endogenous NP-SH content(Loguercio et al., 1993). Our study indicates that PA-treated groupshave effectively elevated the gastric NP-SH content in ulcerated ani-mals when compared to the ulcer control group. Hence, it could besuggested that the replenishment of the endogenous NP-SH mightcontribute in the gastroprotective action of PA.

It was reported earlier that mucus is an important and thefirst defensive line of gastric mucosa, protecting the stomach fromexternal and internal necrotizing agents (Wallace, 2008). Ethanoladministration causes dissipation of the mucosal layer, leading toacid diffusion and lesion formation (Kryger et al., 2000). Our resultsshowed that the gastric mucus content markedly increased in PA-treated groups as well as the positive control group as opposed tothe ulcer control group. Thus, maintenance of the gastric mucusintegrity might contribute to the PA gastroprotective effect. Wepostulated that there is an involvement of NP-SH effect in maintain-ing mucus layer integrity as part of the hypothesized mechanismof gastric mucosal protection by PA.

The roles of NO and COX-2 enzymes in the gastric ulcer are con-troversial. NO is a free radical gas that mitigates gastric lesionsby regulating acid alkaline secretion, mucus secretion and gas-tric mucosal blood (Chandranath et al., 2002). On the other hand,NO regulates microvascular contraction and cellular neurolog-ical and immunological functions that are implicated in manyphysiologic conditions such as inflammation, where NO in tissuecontrols the release of proinflammatory cytokines. Besides its COX-2 induction effect, an enzyme activates prostaglandins production,which mediates cellular inflammatory and tissue damage (Tianet al., 2008). Suppression of inflammatory mediators enhances thehealing of stomach ulceration (Abdelwahab et al., 2011b) and sev-eral natural compounds have been reported to mediate their antiinflammatory effects through COX -2 inhibition as well as NO pro-duction (Abdelwahab et al., 2011a). The current study showed thatPA had significantly increased the NO level at all doses of treatment.In addition, there was a significant inhibitory activity on the COX-2compared to the standard COX-2 inhibitor. Based on these results,it could be hypothesized that PA have anti-inflammatory activitythrough the inhibition of COX-2-enzyme, but no involvement ofNO; which might be involved in its gastroprotective activity.

H. pylori is the most prevalent bacteria worldwide, proliferatingspecifically in the gastric epithelium and considered as the main eti-

ologic agent of gastric ulcer as well as consuming time and moneydue to its prolonged therapy (Marshall, 2002). There is a tendencyto discover new antiulcer remedies through anti H. pylori activities(Bonamin et al., 2011). A recent study considered that a compound

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ith an MIC value of less than or equal to 250 will exhibit strongerntibacterial activity (Moraes et al., 2008). In this study, PA showednteresting MIC values against two strains of H. pylori, providedhat its anti H. pylori action might be one of the mechanisms thatnderlies its gastroprotective effect.

In conclusion, pyranocycloartobiloxanthone A significantlynd dose-dependently showed gastroprotective effects againstthanol-induced gastric lesions in the animal model. The antiul-er effect of PA could be associated with the potent direct radicalcavenging activity and enhancement of the cellular antioxidantechanism by replenishing gastric GSH and NP-SH levels alongith reducing lipid peroxidation in addition to the activation

f HSP70. Moreover, PA showed a significant involvement ofhe anti-inflammatory and anti-apoptotic mechanisms togetherith acceptable MIC against H. pylori activity. The present studyarrants further research on pyranocycloartobiloxanthone A as aromising gastroprotective agent.

cknowledgment

The authors would like to express their utmost gratitude andppreciation to University of Malaya (PG109-2012B) and Ministryf Higher education (HIR F00009- 21001) for providing grant toonduct this study.

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