research article -mangostin from cratoxylum arborescens...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 450840 10 pageshttpdxdoiorg1011552013450840

Research Article120572-Mangostin from Cratoxylum arborescens (Vahl) BlumeDemonstrates Anti-Ulcerogenic Property A Mechanistic Study

Heyam M A Sidahmed1 Siddig Ibrahim Abdelwahab2 Syam Mohan1

Mahmood Ameen Abdulla3 Manal Mohamed Elhassan Taha2

Najihah Mohd Hashim1 A Hamid A Hadi4 Jamunarani Vadivelu5 Mun Loke Fai5

Mawardi Rahmani6 and Maizatulakmal Yahayu6

1 Department of Pharmacy Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia2Medical Research Centre Jazan University PO Box 114 Jazan Saudi Arabia3 Department of Molecular Medicine Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia4Department of Chemistry Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia5 Department of Medical Microbiology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia6Department of Chemistry Faculty of Science Universiti Putra Malaya 43400 Serdang Malaysia

Correspondence should be addressed to Siddig Ibrahim Abdelwahab siddigroayahoocom

Received 26 October 2012 Accepted 28 January 2013

Academic Editor Evan Paul Cherniack

Copyright copy 2013 HeyamM A Sidahmed et alThis is an open access article distributed under theCreative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Cratoxylum arborescens (Vahl) Blume is an Asian herbal medicine with versatile ethnobiological properties including treatmentof gastric ulcer This study evaluated the antiulcerogenic mechanism(s) of 120572-mangostin (AM) in a rat model of ulcer AM is aprenylated xanthone derived through biologically guided fractionation of C arborescens Rats were orally pretreated with AM andsubsequently exposed to acute gastric lesions induced by ethanol Following treatment ulcer index gastric juice acidity mucuscontent histological and immunohistochemical analyses glutathione (GSH) malondialdehyde (MDA) nitric oxide (NO) andnonprotein sulfhydryl groups (NP-SH) were evaluated The anti-Helicobacter pylori cyclooxygenase-2 (COX-2) inhibitory effectand antioxidant activity of AM were also investigated in vitro AM (10 and 30mgkg) inhibited significantly (119875 lt 005) ethanol-induced gastric lesions by 6604 and 7439 respectively The compound induces the expression of Hsp70 restores GSH levelsdecreases lipid peroxidation and inhibits COX-2 activityTheminimum inhibitory concentration (MIC) of AM showed an effectivein vitro anti-H pylori activityThe efficacy of the AMwas accomplished safely without presenting any toxicological parametersTheresults of the present study indicate that the antioxidant properties and the potent anti-H pylori in addition to activation of Hsp70protein may contribute to the gastroprotective activity of 120572-mangostin

1 Introduction

Peptic ulcer disease (PUD) is the most common gastroin-testinal tract aliment of multiple causes which hinders a lotof people worldwide PUD occurs when the offensive factorsovercome the defensive ones [1 2] There are many noxiousagents that attack the stomach resulting inmucosal ulcerationsuch as Helicobacter pylori infection excessive ingestion ofnonsteroidal anti-inflammatory drugs (NSAIDs) or alcoholas well as psychological stress and cigarette smoking On theother hand the stomach protects itself by many defensive

mechanisms mainly the entire mucosal layer which acts asa barrier against inflammatory and cytotoxic agents [3] Itis reported that accumulation of reactive oxygen species isin charge of peptic ulcer aetiology in various gastric ulcermodels suggesting the participation of antioxidant enzymesand therefore there is a trend to explore antioxidant drugsfrom natural resources [2] The reciprocal antiulcer medica-tion to treat the peptic ulcer is either by inhibiting gastric acidsecretion or by enhancing mucus layer protection Howevermajority of them possess adverse effects that may limit theirusage [4] For that there is need to discover new antiulcer

2 Evidence-Based Complementary and Alternative Medicine

O

O

12

345

6

7

89

10

1112

13

14 15

19

16

1718

20

H2CO

OH

OHHO

9a

4a

8a

10a

Figure 1 Chemical structure of 120572-mangostin from Cratoxylum arborescens (Vahl) Blume

drugs Recent studies have proven a variety of antiulcercompounds of botanical origin which mainly are alkaloidssaponins xanthones triterpenes and tannins compounds [1]

The chemical constituents of Cratoxylum species is notwell studied so far regardless of their popular traditional usesHowever recent studies on this genus have led to the isolationand identification of oxygenated and prenylated xanthonesand some anthraquinones Cratoxylum arborescens is a well-known tropical tree The bark roots and leaves of thisplant have been used in folk medicine to treat fever coughsdiarrhea itches ulcers and abdominal complaintsThe plantwas reported to possess cytotoxic effect against human lungcancer cell line (NCI-H187) with IC

50values ranging from

065 to 52 120583gmL [5 6] Upon these potential activities Carborescens was subjected to several experimental tests toevaluate its antiulcerogenic property and to identify possiblegastroprotective mechanism(s) of its major bioactive com-pound against ethanol ulcer models in experimental animals

2 Material and Methods

21 Extraction and Isolation of 120572-Mangostin (AM) from Carborescens The stem bark of C arborescens was collectedfrom wild trees growing in Sarawak Malaysia in 2009 Avoucher specimen has been deposited at the HerbariumDepartment of Biology Faculty of Medicine UniversitiPutra Malaysia The finely ground air-dried stem bark of Carborescens (10 kg) was extracted consecutively with hexanechloroform and methanol to give 612 2818 and 4027 grespectively of dark viscous semisolid material on solventremoval The hexane extract was chromatographed over vac-uum column and eluted with solvent of gradually increasingpolarity to give 26 fractions of 200mL each After extensivefractionation and purification of fractions 14ndash20 yielded 120572ndashmangostin (AM Figure 1)

22 Identification of 120572-Mangostin Melting point of AM was178ndash180∘C ([7] mp 181-182∘C) UV MeOH 120582max nm (log 120576)390 (241) 358 (399) 316 (399) and 238 (265) IR 120584max cm

minus1

(KBr) 3369 (OH) 2934 (CH) 1608 (C=C) 1462 and 1286EIMSmz (intensity) 410 (4306) 395 (614) 379 (161) 354(2577) 339 (10000) 311 (3257) 296 (1289) 285 (1890) 257(646) and 162 (1416) 1H-NMR (500MHz acetone-119889

6) 120575

1379 (OH-1) 962 (OH-6) 952 (OH-3) 681 (s 1H H-5) 638

(s 1H H-4) 526 (t J = 685Hz 2H H-12 and H-17) 412(d J = 685Hz 2H H-11) 378 (OMe-7) 335 (d J = 800Hz2H H-16) 182 (s 3H Me-14) 171 (s 3H Me-19) and 164(s 6HMe-15 andMe-20) 13C-NMR (125MHz acetone-119889

6)

120575 1820 (C-9) 1621 (C-4a) 1609 (C-1) 1566 (C-10a) 1554(C-6) 1549 (C-3) 1436 (C-7) 1373 (C-8) 1306 (C-18 and C-13) 1239 (C-12) 1226 (C-17) 1112 (C-8a) 1102 (C-2) 1028(C-9a) 1019 (C-5) 923 (C-4) 625 (OMe-7) 261 (C-11) 251(C-15 andC-20) 211 (C-16) 175 (C-14) and 171 (C-19) Purityof AMwas checked and confirmed using HPLC and LCMS

23 Drugs and Reagents Griess reagent omeprazoleDTNB(221015840-dinitro-551015840-dithiodibenzoic acid) and TPTZ (2 46-tri (2-pyridyl)-135-triazine) were purchased from Sigma-Aldrich (Singapore) All other used chemicals and reagentswere of analytical grade

24 Acute Toxicity Study Adult male and female ICR mice(6ndash8 weeks old 20ndash30 g) were obtained from the Experimen-tal Animal House (Ethic No PM 07052008 MAA (a)(R))Faculty of Medicine University of Malaya Thirty animalswere assigned equally into three groups Animals were fasted(24 h) prior to oral administration of AM at doses of 30and 300mgkg bw respectively Third group of mice wereadministeredwith 5Tween80 and served as a control groupAnimals were observed for two weeks for any mortality orchanges particularly changes in skin eyesmucusmembraneand also autonomic and central nervous system Food andwater were provided throughout the experiment ad libitumOn day 15th animals were sacrificed the blood collectedfor biochemical parameters analysis and the organs (heartlungs spleen liver and the kidney) were excised for organweight variation and histology study All animals receivedhuman care according to the criteria outlined in the ldquoGuidefor the Care and Use of Laboratory Animalsrdquo prepared by theNational Academy of Sciences and published by the NationalInstitute of Health Malaysia

25 Induction of Acute Gastric Lesion Sprague Dawley malerats (220ndash250 g) were obtained from the Experimental Ani-mal House (Ethic No PM 07052008 MAA (a)(R)) Facultyof Medicine University of Malaya To avoid coprophagyrats were kept individually in cages with raised floors ofwide mish and divided randomly into four groups (119899 = 6)

Evidence-Based Complementary and Alternative Medicine 3

The animals were fasted for 48 h prior to oral dosing Oneh before intragastric administration of ethanol (5mLkg)animals were orally pre-treated as follows group (1) servedas normal control without treatment group (2) treated withvehicle (5 Tween80 vv 5mLkgbsdotw) group (3) omepra-zole (20mgkg) and group (4 and 5) with AM (10 and30mgkg resp) One h after ethanol dosing all animalswere sacrificed under anesthesia (ketamine and xylazine)and their blood was collected [2]

251 Measurement of Gastric Juice Acidity Mucus Contentand the Biochemical Parameters Stomach contents of eachanimal were collected and centrifuged to measure the con-centration of the hydrogen ion concentration (pH) from thesupernatant using the pH meter expressed as meql and theweight of the gastric mucosa from the sedimentation usingprecise balance Animal Blood samples were analyzed atUniversity Malaya Medical Centre Kuala Lumpur Malaysiato evaluate changes in serum biomarkers [8]

252 Gastroprotective Assessments Gastric ulcer appears aselongated bands of hemorrhagic lesions The length (mm)and the width (mm) of each band were measured usingplanimeter ((10 times 10 mm 2 = ulcer area) under dissectingmicroscope (18times)) The area of each ulcer lesion was mea-sured by counting the number of small squares (2mm times2mm) covering the length and width of each ulcer bandThesum of the areas of all lesions for each stomach was appliedin the calculation of the ulcer area (UA) where in the sum ofsmall squares times 4 times 18 = UAmm2 The inhibition percentage(I) was calculated by the following formula described in [9]with slight modifications

Inhibition percentage (I)

= [

(UAcontrol minus UA treated)UAcontrol

] times 100(1)

253 Histopathological Evaluation Using Hematoxylin andEosin Periodic Acid-Schiff and Immunohistochemistry Forhistopathological evaluation a small fragment of each animalgastric ulcer was fixed with 10 buffered formalin solutionFormalin-fixed tissues were dehydrated with alcohol andxylene and finally embedded in paraffin wax Tissue sectionsof 5 120583m were stained with hematoxylin and eosin (H andE) for light microscopy [10] Slides were also stained withPeriodic Acid-Schiff (PAS) Base [11] for the evaluation ofmucus production following the manufacturerrsquos instructions(Sigma-Aldrich Singapore) A primary antibody of HSP-70(dilution 1 500) was used to investigate the mechanism ofAM as a potential antiulcerogenic agent Immunohistochem-ical detection for these antibodies was done using AnimalResearch Kit (ARK DakoCytomation)

254 Determination of Glutathione (GSH) Levels Total GSHcontent was estimated using the following method [12] Inbrief small fragments of each stomach were homogenized inice-cooled trichloroacetic acid (5 wv) The homogenates

were centrifuged at 7000 times g for 15min at 4∘C and thesupernatants were used to quantify GSH content throughreactionwithDTNBAbsorbancewas read in 412 nm and theresults were expressed in nmol GSHg tissue

255 Determination of Thiobarbituric Acid Reactive Sub-stance and Nitric Oxide Thiobarbituric acid reactive sub-stance (TBARS) assay was used to estimate malondialdehyde(MDA) content according to the method of [13] Briefly05mL of the 10 stomach homogenate in 01molL PBS wasadded to a solution containing 15mL acetic acid (20 wv)20mL of 20 sodium dodecyl sulphate and SDS (pH 35adjusted with NaOH) The mixture was incubated in a waterbath at 95∘C for 1 h After cooling 3mL of the mixture wasvortex and centrifuged at 3000 g for 10min The absorbanceof the supernatant was determined in a spectrophotometerat 532 nm and the values of MDA in the gastric tissue areexpressed in 120583molg tissue malondialdehyde Nitric oxide(nmolg tissue) was quantified in gastric homogenates usingthe Griess assay [14]

256 Estimation of Nonprotein Sulfhydryls Gastric mucosalnonprotein sulfhydryls (NP-SH) (120583molg of tissue) weremeasured [15] In brief an aliquot of 5mL of the stomachhomogenate was mixed with a solution containing 4mL ofdistilled water and 1mL of 50 trichloroacetic acid Themixture was vortex for 15min and centrifuged at 3000 times gTwo mL of the supernatant were mixed with 4mL of 04MTris buffer (pH 89) One hundred microliter of DTNB [55dithiobis-(2-nitrobenzoic acid)] was added The absorbancewas recorded within 5min of addition of DTNB at 412 nmagainst a reagent blank with no homogenate

26 In Vitro Evaluation of Cyclooxygenase-2 Inhibitory Activ-ity 120572-Mangostin was tested for its COX-2 inhibitory activityusing a COX-inhibitor screening kit according to the man-ufacturerrsquos instructions (Cayman Chemical USA) COX-2 enzyme catalyzes the biosynthesis of prostaglandin-H-synthase-2 from arachidonic acid This assay measures theproduction of PGF2120572 generated by SnCl

2 using enzyme

immunoassay (acetylcholine esterase (ACE) competitiveEIA) The stock solution of AM was dissolved in dimethylsulfoxide (DMSO) and the final concentration was obtainedfrom 0 to 100 120583gmL using the reaction buffer Inhibition wascalculated by the comparison to control incubations

27 In Vitro Antioxidant Assays The Ferric-reducing antiox-idant power (FRAP) assay was accomplished followingthe procedure described earlier [16] with slight modifica-tions Briefly the FRAP reagent was prepared freshly fromacetate buffer (pH 36) 10mM TPTZ [246-Tri(2-pyridyl)-s-triazine] solution in 40mM HCl and 20mM iron (III)chloride solution in proportions of 10 1 1 (vv) respectivelyFifty 120583L of the compound were added to 15mL of the FRAPreagent in the dark 4min later the absorbance was thenrecorded at 593 nmThe standard curve was constructed lin-early (1198772=0996) using iron (II) sulfate solution (0ndash1000120583M)and the results were expressed as 120583M of Fe(II)g dry weight


Table 1 Observed ulcer area inhibition percentage pH and mucus content stomach

Animal group Pre-treatment (5mLkg dose) pH of gastric content Mucus content Ulcer index (mm)2(Mean plusmn SEM) Inhibition ()

1 Tween80 (normal control) 306aplusmn 009 195

aplusmn 006

2 Ethanol 95 (ulcer control) 167bplusmn 015 099

bplusmn 004 4824

aplusmn 3089

3 Omeprazole (20mgkg) 37aplusmn 114 45

cplusmn 007 108

bplusmn 960 7842

4 120572-mangostin (10 gkg) 464dplusmn 01 1875

dplusmn 004 16992

cplusmn 7505 6604

5 120572-mangostin (30mgkg) 320eplusmn 005 31

eplusmn 001 12816

dplusmn 8475 7439

All values are expressed as mean plusmn standard error of the mean Means with different superscripts are significantly different The mean difference is significantat the 005 level Groups with different superscript alphabets are statistically significant

Table 2 Effects of 120572-mangostin (AM) of liver function tests (ALT and AST) gastric MDA GSH NP-SH and nitric oxide

Animal groups Pre-treatment(5mLkg dose) AST ALT GSH MDA

(120583molg tissue)NP-SH

(120583molg tissue)Nitric oxide[NO2

minus] 120583M

1 Tween80(normal control) 6106

aplusmn 11 14506

aplusmn 18 16

aplusmn 003 14

bplusmn 031 06

aplusmn 009 92

aplusmn 019

2 Ethanol 95(ulcer control) 97

bplusmn 015 2922

bplusmn 192 07

bplusmn 010 28

aplusmn 014 72

bplusmn 05 332

bplusmn 021

3 Omeprazole(20mgkg) 63

aplusmn 076 176

aplusmn 41 17

aplusmn 003 15

aplusmn 021 171

aplusmn 14 77

aplusmn 112

4 120572-mangostin(10 gkg) 64

dplusmn 15 173

dplusmn 36 141

dplusmn 03 14

dplusmn 012 26

dplusmn 019 63

dplusmn 014

5 120572-mangostin(30mgkg) 59

eplusmn 62 164

eplusmn 50 17

eplusmn 002 14

eplusmn 011 140

eplusmn 093 81

eplusmn 053

Values are presented asmeanplusmn SEMof five rats in each groupGlutathione (GSH) levelsmalondialdehyde level (MDA) nitric oxide (NO) alanine transaminase(ALT) aspartame transaminase (AST) and non-protein sulfhydryl groups (NP-SH)weremeasured in all groups Groupswith different alphabets are statisticallydifferent

of the compound DPPH was conducted according to themethod described by Mahmood et al [23]

28 In Vitro Anti-Helicobacter Pylori Activity Two H pyloristrains NCTC 11637 (ATCC43504) and J99 (ATCC700824)were cultured with brain heart infusion broth supplementedwith 10 horse serum (Invitrogen Malaysia) incubated at37∘C in a humidified CO

2incubator for 3 days Minimum

inhibitory concentration (MIC) was determined by a mod-ified microtiter broth dilution method on sterile 96-wellpolypropylene microtitre plates with round-bottom wells(Eppendorf Germany) BrieflyAMwas dissolved anddilutedin 5 DMSO to give a 10x working stock solution H pyloriwas diluted to a final concentration of 2 times 106 CFUmL inculturemediumAliquots of 10120583L ofAMwere added to 90 120583Lof H pylori in a well of the microtitre plate Concentrationof AM ranged from 0 to 250 120583gmL The microtiter platewas incubated for 3 days in a CO

2incubator The plate was

examined visually and measured using a microplate reader(Varioskan Flash) at 600 nm to determine the lowest con-centration showing complete growth inhibition which wasrecorded as the MIC Minimum bactericidal concentration(MBC) as the lowest concentration without growth on achocolate agar plate supplementedwith 7 lysed horse bloodWells containing H pylori with 10 120583L of 5 DMSO and BHImedium containing 250 120583gmL AMwere used as control andblank respectively The result was recorded in accordancewith the Clinical and Laboratory Standards Institute [17]

29 Statistical Analysis All values were reported as mean plusmnSEM Statistical significant differences between groups wereassessed using one-way ANOVA followed by Tukeyrsquos post hocmultiple comparison test A value of 119875 lt 005 or lower wasconsidered as a significant difference

3 Results

31 Toxicity Study There were no abnormal physiologicalbehavioural changes or body weight alterations at any timepoint of observation at the doses used during the twoweeks Histological examination to the liver and kidney andserum biochemical analysis did not show any differencein comparison to the control group (data not shown butavailable upon request)

32 Antiulcer Study

321 Gross andMacromorphometric Evaluation Results sho-wed that animals pretreated with AM and omeprazoleconsiderably reduced ulcer area formation compared toanimal group pretreated with only 5 Tween80 (Table 1) 120572-Mangostin (AM) at doses of 10 and 30mgkg bw significantly(119875 lt 005) inhibited ulcer formation by 6604 and 7439respectively as shown in Table 1 and Figure 2

322 Gastric Mucus Content Acidity and Biochemical Anal-ysis As shown in Table 1 ulcer control group produced


(a) (b)

(c) (d)

(e)

Figure 2 Gross evaluation of stomachs from various animal groups Results showed that rats pretreated with 120572-mangostin (AM) at doses of10 and 30mgkg ((d) and (e) resp) and omeprazole ((c) 20mgkg) had considerably reduced areas of gastric ulcer formation compared torats pretreated with only Tween80 (ulcer control group (a)) (magnification 18x)

the lowest mucus content of gastric mucosa while 10 and30mgkg of AM increased significantly (119875lt005) the mucusproduction On the other hand animal groups pretreatedwith AM showed increase in the pH of the gastric contentsSerum analysis showed that rats induced with ethanol ulcer-ation had increased levels of liver enzymes (AST and ALT)compared to normal and AM-treated groups as shown inTable 2

323 Morphological Findings Histological observation ofthe ulcer control group pretreated only with 5 Tween80showed highly extensive gastric lesion submucosal edemaand leucocytes infiltration PretreatmentwithAM (10 30mg)and omeprazole has relatively better protection as seen bydecreasing ulcer area reduction or complete absence ofedema and leucocytes infiltration as shown in Figure 3Treatment of animals with AM resulted in the expansion of a


(a) (b)

(c) (d)

(e)

Figure 3 Histopathological evaluation Results showed that 120572-mangostin (AM) at doses of 10 and 30mgkg ((d) and (e) resp) andomeprazole ((c) 20mgkg) improved the histopathology of ratsrsquo stomach compared to those pretreated with only Tween80 (ulcer controlgroup (b)) Black arrow indicates severe disruption to the epithelium surface and deep mucosa while white arrow indicates leukocyteinfiltration and edema in the submucosa layer (H and E stain magnification 10x)

substantial continuous PAS-positive mucous layer that liningthe entire gastric mucosal surface noted as a bright-purple-stained area lining the mucosa as shown in Figure 4

324 Protein Expression (HSP-70) To understand themech-anism of AM as an antiulcerogenic agent gastric tissues fromvarious experimental groups were stained with immunohis-tochemistry as shown in Figure 5 The gastric tissues wereexamined for immunohistochemical localization of Hsp70

protein Microscopic examination reveals high numbers ofproliferated cell as well as modulation of Hsp70These resultsindicate an effective role of this antioxidant protein in thegastroprotection mechanism of AM

325 Involvement of Oxidative Stress Markers and COX-2Glutathione levels were significantly (119875 lt 001) lower in theulcer control group than the normal group The treatmentof animals with the AM restored significantly (119875 lt 001) the


(a) (b)

(c) (d)

(e)

Figure 4 PAS staining for the evaluation of mucus production Results showed that rats pretreated with 120572-mangostin (AM) at doses of 10and 30mgkg ((c) and (d) resp) and omeprazole ((e) 20mgkg) showed more PAS-positive mucus as compared to those pretreated withonly Tween80 (ulcer control group (b)) (Magnification 10x)

GSH levels depletion after ethanol administration The ulcercontrol group showed the highest level of malondialdehyde(MDA) an indicator of lipid peroxidation than the othergroups Gastric MDA level significantly (119875 lt 005) droppedafter AM administration Results forMDA NO and GSH areshown in Table 2

To investigate the gastroprotective mechanism NP-SHlevels were estimated The compound alleviated NP-SH levelwhich was dropped due to ethanol administrationMoreoverit was observed that AM in two concentrations 250 ngmLand 500 ngmL inhibited COX-2 activity in vitro by 277and 588 in comparison to the standard COX-2 inhibitorindomethacin (726)

33 Antioxidant Evaluation of 120572-Mangostin 120572-Mangostinexhibited FRAP value of 1128 plusmn 023 while the positivecontrols used in this study exhibited 214061plusmn0061 77039plusmn0003 246067 plusmn 0027 and 42194 plusmn 0003 for troloxquercetin gallic acid and ascorbic acid respectively Thisnatural compound did not show strong radical scavengingactivity with no value against DPPH

34 In Vitro Anti-Helicobacter Pylori Activity 120572-Mangostinrepresents minimum inhibitory concentration (MIC) of24120583gmL and minimum bactericidal concentration (MBC)of 246120583gmL against H pylori (Strain no NCTC11637) and


(a) (b)

(c) (d)

Figure 5 Immunohistochemical staining of rat gastric tissues with Hsp70 primary antibodies (a) Gastric tissues from AM (10mgkg)pretreated animals (b) gastric tissues from AM (30mgkg) pretreated animals (c) gastric tissues from ethanol-induced animals (d) gastrictissues from omeprazole (20mgkg) pretreated animals (magnification 10x)

MIC 19 120583gmL and MBC of 124120583gmL against H pylori(Strain no J99)

4 Discussion

Cratoxylum arborescens is well-known ethnomedicine usedin Asia for the treatment and prevention of gastric ulcer120572-Mangostin (AM) as a natural product is a prenylatedxanthone isolated by our group through biologically guidedfractionation from C arborescens Therefore this study eval-uated the antiulcerogenicmechanism(s) of AM in a ratmodelof ulcer This natural compound was reported earlier to pos-sess many biological properties such as anti-inflammatoryantioxidative damage and antioxidant activities [18]

Gastric ulcer is caused by different etiologies [19] Itis generally believed that this disease is the consequenceof an imbalance between destructive factors and defensivemechanisms of the mucosal integrity [20] Ethanol as apotential gastroulcerogenic agent is known to be involved

in the depression of these defensive mechanisms [21]The mechanism of this potential gastrotoxic agent includesmicrovascular injury increased vascular permeability edemaformation and epithelial lifting Ethanol produces necroticlesions in the gastric mucosa increased flow of Na+ and K+increased pepsin secretion and loss of H+ ions and histamineinto the lumen [22] Ethanol is commonly used for inducingulcer in experimental rats and leads to intense gastricmucosaldamage through oxidative injury Ethanol produces amarkedcontraction of the circular muscles of rat fundic strip [23]Alterations in the gastric motility and relaxation of circularmuscles may protect the gastric mucosa Such contractioncan lead to mucosal compression at the site of the greatestmechanical stress at the crests of mucosal folds leading tonecrosis and ulceration [24] Flattening of the gastric foldswill increase the mucosal area exposed to ulcerogenic factorsand reduce the volume of the gastric irritants on rugal crestIn the present study we observed the flattening of mucosalfolds (Figure 2) which suggests that antiulcerogenic propertyof AMmight be due to a reduction in gastric motility


Microscopic evaluation revealed protection of gastricmucosa and decrease in leucocytes infiltration of gastrictissues from AM-pretreated animals as shown in Figure 3Reduction of neutrophil infiltration into ulcerated gastricendothelia is known to encourage the healing of gastric ulcersin rats Infiltrated neutrophils produce oxygen free radicalswhich have inhibitory effect on gastric ulcers healing in ratsThese reactive oxygen species are also highly cytotoxic andcan provoke tissue damage Furthermore neutrophil accu-mulation in gastric mucosa has been shown to induce micro-circulatory abnormalities Cheng and Koo [25] reported thatplant extracts were able to protect gastric mucosa againstethanol induced ulceration through the decrease in neu-trophil infiltration These morphological results were in linewith biochemical indicators which revealed that AM has ananti-inflammatory activities To further confirm this cytopro-tective effect we have evaluated AM against the activity ofgastric NO and in vitro COX-2 This natural compound wasobserved to affect the activities of these cellular mediatorsNO was reported to inhibit neutrophil infiltration The roleof NO in the protective effect of phytochemicals in ethanol-induced gastric ulcer mice was reported earlier [26] 120572-Mangostin (AM) was reported to have anti-COX-2 and anti-NO activities [18]

The gastric tissues were also examined for immune-localization of Hsp70 protein to understand the mechanismof AM as an antiulcerogenic agent (Figure 5) Histologicalexamination reveals high numbers of proliferated cell as wellas modulation of Hsp70 expression These results indicatean effective role of this protein in gastroprotection of AMHsp70 is the most preserved and richly produced proteinin reaction to harmful effects and oxidative stress Heatshock proteins protect cellular homeostasis from injuries byprotecting the structure of normal proteins and repairingor removing damaged proteins Hsp70 proteins defend cellsfrom oxidative stress or heat shock Ethanol-generated ROSnormally acts to inhibit the expression of Hsp70 [27 28]

Gastric ulcer disease is the most common ulcer of anarea of the stomach and 80 of such ulcers are linked withHelicobacter pylori a spiral-shaped bacterium that lives inthe acidic environment of the stomach H pylori colonizesthe stomach and induces long-lasting inflammation (chronicgastritis) The bacterium persists for decades in the stomachAbout 10ndash20 of those colonized byH pylori will eventuallyexperience gastric and duodenal ulcers H pylori infection isalso associated with a 1-2 lifetime risk of stomach cancerand a less than 1 risk of gastric lymphoma 120572-Mangostinrepresents minimum inhibitory concentration (MIC) of25 120583gmL and minimum bactericidal concentration (MBC)of 250120583gmL against H pylori (Strain no NCTC11637) andMIC 19 120583gmL and MBC of 125120583gmL against H pylori(Strain no J99) 120572-Mangostin was reported before to showan antibacterial activity [29]

In conclusion AM could significantly and dose-dep-endently protect the gastric mucosa against ethanol-inducedinjury The antioxidant activity of this natural compoundthrough the induction of cellular antioxidant protection is apointer for scavenging the free radicals produced by ethanolregardless the weak in vitro antioxidant results obtained in

this study The compound also interferes with the naturalrelease of NO and inhibits COXs The current findingswarrant further research for the introduction of AM as apossible defensive and remedial agent for gastric ulcer thatis caused by different aetiologies

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The authors would like to express their utmost gratitude andappreciation to the University ofMalaya (HIRGrant F00009-21001) for providing Grant to conduct this study

References

[1] E S De Souza Almeida V C Filho R Niero B K Clasen SO Balogun and D T De Oliveira Martins ldquoPharmacologicalmechanisms underlying the anti-ulcer activity of methanolextract and canthin-6-one of Simaba ferruginea A St-Hil inanimal modelsrdquo Journal of Ethnopharmacology vol 134 no 3pp 630ndash636 2011

[2] F B Potrich A Allemand LM da Silva et al ldquoAntiulcerogenicactivity of hydroalcoholic extract of Achillea millefolium Linvolvement of the antioxidant systemrdquo Journal of Ethnophar-macology vol 130 no 1 pp 85ndash92 2010

[3] A L Rozza T D M Moraes H Kushima et al ldquoGastroprotec-tive mechanisms of Citrus lemon (Rutaceae) essential oil andits majority compounds limonene and 120573-pinene involvementof heat-shock protein-70 vasoactive intestinal peptide glu-tathione sulfhydryl compounds nitric oxide and prostaglandinE2rdquo Chemico-Biological Interactions vol 189 no 1-2 pp 82ndash892011

[4] M P D Barros M Lemos E L Maistro et al ldquoEvaluation ofantiulcer activity of the main phenolic acids found in BrazilianGreen Propolisrdquo Journal of Ethnopharmacology vol 120 no 3pp 372ndash377 2008

[5] P Pattanaprateeb N Ruangrungsi and G A Cordell ldquoCyto-toxic constituents fromCratoxylumarborescensrdquoPlantaMedicavol 71 no 2 pp 181ndash183 2005

[6] V Reutrakul W Chanakul M Pohmakotr et al ldquoAnti-HIV-1constituents from leaves and twigs of Cratoxylum arborescensrdquoPlanta Medica vol 72 no 15 pp 1433ndash1435 2006

[7] D Shankaranarayan C Gopalakrishnan and L KameswaranldquoPharmacological profile of mangostin and its derivativesrdquoArchives Internationales de Pharmacodynamie et de Therapievol 239 no 2 pp 257ndash269 1979

[8] P V Tan B Nyasse T Dimo and C Mezui ldquoGastric cytopro-tective anti-ulcer effects of the leaf methanol extract ofOcimumsuave (Lamiaceae) in ratsrdquo Journal of Ethnopharmacology vol82 no 2-3 pp 69ndash74 2002

[9] V C O Njar J K Adesanwo and Y Raji ldquoMethyl angolensatethe antiulcer agent of the stem bark of Entandrophragmaangolenserdquo Planta Medica vol 61 no 1 pp 91ndash92 1995

[10] O A Behmer Manual DE Tecnicas Para Histologia Normal EPatologica EDARTUSP 1976

[11] L L Vacca Laboratory Manual of Histochemistry Raven pressNew York NY USA 1985


[12] I Rahman A Kode and S K Biswas ldquoAssay for quantitativedetermination of glutathione and glutathione disulfide levelsusing enzymatic recycling methodrdquo Nature Protocols vol 1 no6 pp 3159ndash3165 2007

[13] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[14] K M Miranda M G Espey and D A Wink ldquoA rapid sim-ple spectrophotometric method for simultaneous detection ofnitrate and nitriterdquo Nitric Oxide vol 5 no 1 pp 62ndash71 2001

[15] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 no C pp 192ndash2051968

[16] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[17] J H Jorgensen and J F Hindler ldquoNew consensus guide-lines from the clinical and laboratory standards Institute forantimicrobial susceptibility testing of infrequently isolated orfastidious bacteriardquo Clinical Infectious Diseases vol 44 no 2pp 280ndash286 2007

[18] S H Liu L T Lee N YHu et al ldquoEffects of120572-mangostin on theexpression of anti-inflammatory genes in U937 cellsrdquo ChineseMedicine vol 7 p 19 2012

[19] J K Grover G Adiga V Vats and S S Rathi ldquoExtracts ofBenincasa hispida prevent development of experimental ulcersrdquoJournal of Ethnopharmacology vol 78 no 2-3 pp 159ndash1642001

[20] D W Piper and D D Stiel ldquoPathogenesis of chronic pepticulcer current thinking and clinical implicationsrdquo Medical Pro-gress vol 2 pp 7ndash10 1986

[21] M Kinoshita T Noto and H Tamaki ldquoEffect of a combinationof ecabet sodium and cimetidine on experimentally induced(gastric lesions and gastric mucosal resistance to ulcerogenicagents in ratsrdquo Biological and Pharmaceutical Bulletin vol 18no 2 pp 223ndash226 1995

[22] S Szabo ldquoMechanisms of mucosal injury in the stomach andduodenum time-sequence analysis of morphologic functionalbiochemical and histochemical studiesrdquo Scandinavian Journalof Gastroenterology Supplement vol 22 no 127 pp 21ndash28 1987

[23] A Mahmood K Sidik I Salmah K Suzainor and K PhilipldquoAntiulcerogenic activity of Ageratum conyzoides leaf extractagainst ethanol-induced gastric ulcer in rats as animal modelrdquoInternational Journal of Molecular Medicine and Advance Sci-ences vol 1 pp 402ndash405 2005

[24] A Jamal K Javed M Aslam andM A Jafri ldquoGastroprotectiveeffect of cardamom Elettaria cardamomum Maton fruits inratsrdquo Journal of Ethnopharmacology vol 103 no 2 pp 149ndash1532006

[25] C L Cheng and M W L Koo ldquoEffects of Centella asiatica onethanol induced gastric mucosal lesions in ratsrdquo Life Sciencesvol 67 no 21 pp 2647ndash2653 2000

[26] V Sibilia G Rindi F Pagani et al ldquoGhrelin protects againstethanol-induced gastric ulcers in rats studies on the mecha-nisms of actionrdquo Endocrinology vol 144 no 1 pp 353ndash3592003

[27] K C Kregel ldquoInvited review heat shock proteins modifyingfactors in physiological stress responses and acquired thermo-tolerancerdquo Journal of Applied Physiology vol 92 no 5 pp 2177ndash2186 2002

[28] A Parcellier S Gurbuxani E Schmitt E Solary and C Gar-rido ldquoHeat shock proteins cellular chaperones that modulatemitochondrial cell death pathwaysrdquo Biochemical and Biophysi-cal Research Communications vol 304 no 3 pp 505ndash512 2003

[29] P T M Nguyen and R E Marquis ldquoAntimicrobial actions of120572-mangostin against oral streptococcirdquo Canadian Journal ofMicrobiology vol 57 no 3 pp 217ndash225 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


O

O

12

345

6

7

89

10

1112

13

14 15

19

16

1718

20

H2CO

OH

OHHO

9a

4a

8a

10a

Figure 1 Chemical structure of 120572-mangostin from Cratoxylum arborescens (Vahl) Blume

drugs Recent studies have proven a variety of antiulcercompounds of botanical origin which mainly are alkaloidssaponins xanthones triterpenes and tannins compounds [1]

The chemical constituents of Cratoxylum species is notwell studied so far regardless of their popular traditional usesHowever recent studies on this genus have led to the isolationand identification of oxygenated and prenylated xanthonesand some anthraquinones Cratoxylum arborescens is a well-known tropical tree The bark roots and leaves of thisplant have been used in folk medicine to treat fever coughsdiarrhea itches ulcers and abdominal complaintsThe plantwas reported to possess cytotoxic effect against human lungcancer cell line (NCI-H187) with IC

50values ranging from

065 to 52 120583gmL [5 6] Upon these potential activities Carborescens was subjected to several experimental tests toevaluate its antiulcerogenic property and to identify possiblegastroprotective mechanism(s) of its major bioactive com-pound against ethanol ulcer models in experimental animals

2 Material and Methods

21 Extraction and Isolation of 120572-Mangostin (AM) from Carborescens The stem bark of C arborescens was collectedfrom wild trees growing in Sarawak Malaysia in 2009 Avoucher specimen has been deposited at the HerbariumDepartment of Biology Faculty of Medicine UniversitiPutra Malaysia The finely ground air-dried stem bark of Carborescens (10 kg) was extracted consecutively with hexanechloroform and methanol to give 612 2818 and 4027 grespectively of dark viscous semisolid material on solventremoval The hexane extract was chromatographed over vac-uum column and eluted with solvent of gradually increasingpolarity to give 26 fractions of 200mL each After extensivefractionation and purification of fractions 14ndash20 yielded 120572ndashmangostin (AM Figure 1)

22 Identification of 120572-Mangostin Melting point of AM was178ndash180∘C ([7] mp 181-182∘C) UV MeOH 120582max nm (log 120576)390 (241) 358 (399) 316 (399) and 238 (265) IR 120584max cm

minus1

(KBr) 3369 (OH) 2934 (CH) 1608 (C=C) 1462 and 1286EIMSmz (intensity) 410 (4306) 395 (614) 379 (161) 354(2577) 339 (10000) 311 (3257) 296 (1289) 285 (1890) 257(646) and 162 (1416) 1H-NMR (500MHz acetone-119889

6) 120575

1379 (OH-1) 962 (OH-6) 952 (OH-3) 681 (s 1H H-5) 638

(s 1H H-4) 526 (t J = 685Hz 2H H-12 and H-17) 412(d J = 685Hz 2H H-11) 378 (OMe-7) 335 (d J = 800Hz2H H-16) 182 (s 3H Me-14) 171 (s 3H Me-19) and 164(s 6HMe-15 andMe-20) 13C-NMR (125MHz acetone-119889

6)

120575 1820 (C-9) 1621 (C-4a) 1609 (C-1) 1566 (C-10a) 1554(C-6) 1549 (C-3) 1436 (C-7) 1373 (C-8) 1306 (C-18 and C-13) 1239 (C-12) 1226 (C-17) 1112 (C-8a) 1102 (C-2) 1028(C-9a) 1019 (C-5) 923 (C-4) 625 (OMe-7) 261 (C-11) 251(C-15 andC-20) 211 (C-16) 175 (C-14) and 171 (C-19) Purityof AMwas checked and confirmed using HPLC and LCMS

23 Drugs and Reagents Griess reagent omeprazoleDTNB(221015840-dinitro-551015840-dithiodibenzoic acid) and TPTZ (2 46-tri (2-pyridyl)-135-triazine) were purchased from Sigma-Aldrich (Singapore) All other used chemicals and reagentswere of analytical grade

24 Acute Toxicity Study Adult male and female ICR mice(6ndash8 weeks old 20ndash30 g) were obtained from the Experimen-tal Animal House (Ethic No PM 07052008 MAA (a)(R))Faculty of Medicine University of Malaya Thirty animalswere assigned equally into three groups Animals were fasted(24 h) prior to oral administration of AM at doses of 30and 300mgkg bw respectively Third group of mice wereadministeredwith 5Tween80 and served as a control groupAnimals were observed for two weeks for any mortality orchanges particularly changes in skin eyesmucusmembraneand also autonomic and central nervous system Food andwater were provided throughout the experiment ad libitumOn day 15th animals were sacrificed the blood collectedfor biochemical parameters analysis and the organs (heartlungs spleen liver and the kidney) were excised for organweight variation and histology study All animals receivedhuman care according to the criteria outlined in the ldquoGuidefor the Care and Use of Laboratory Animalsrdquo prepared by theNational Academy of Sciences and published by the NationalInstitute of Health Malaysia

25 Induction of Acute Gastric Lesion Sprague Dawley malerats (220ndash250 g) were obtained from the Experimental Ani-mal House (Ethic No PM 07052008 MAA (a)(R)) Facultyof Medicine University of Malaya To avoid coprophagyrats were kept individually in cages with raised floors ofwide mish and divided randomly into four groups (119899 = 6)






= [


] times 100(1)







2 using enzyme







aplusmn 006


bplusmn 004 4824

aplusmn 3089


cplusmn 007 108

bplusmn 960 7842


dplusmn 004 16992

cplusmn 7505 6604


eplusmn 001 12816

dplusmn 8475 7439






minus] 120583M


aplusmn 11 14506

aplusmn 18 16

aplusmn 003 14

bplusmn 031 06

aplusmn 009 92

aplusmn 019


bplusmn 015 2922

bplusmn 192 07

bplusmn 010 28

aplusmn 014 72

bplusmn 05 332

bplusmn 021


aplusmn 076 176

aplusmn 41 17

aplusmn 003 15

aplusmn 021 171

aplusmn 14 77

aplusmn 112


dplusmn 15 173

dplusmn 36 141

dplusmn 03 14

dplusmn 012 26

dplusmn 019 63

dplusmn 014


eplusmn 62 164

eplusmn 50 17

eplusmn 002 14

eplusmn 011 140

eplusmn 093 81

eplusmn 053









3 Results


32 Antiulcer Study




(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)



4 Discussion












Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















= [


] times 100(1)







2 using enzyme







aplusmn 006


bplusmn 004 4824

aplusmn 3089


cplusmn 007 108

bplusmn 960 7842


dplusmn 004 16992

cplusmn 7505 6604


eplusmn 001 12816

dplusmn 8475 7439






minus] 120583M


aplusmn 11 14506

aplusmn 18 16

aplusmn 003 14

bplusmn 031 06

aplusmn 009 92

aplusmn 019


bplusmn 015 2922

bplusmn 192 07

bplusmn 010 28

aplusmn 014 72

bplusmn 05 332

bplusmn 021


aplusmn 076 176

aplusmn 41 17

aplusmn 003 15

aplusmn 021 171

aplusmn 14 77

aplusmn 112


dplusmn 15 173

dplusmn 36 141

dplusmn 03 14

dplusmn 012 26

dplusmn 019 63

dplusmn 014


eplusmn 62 164

eplusmn 50 17

eplusmn 002 14

eplusmn 011 140

eplusmn 093 81

eplusmn 053









3 Results


32 Antiulcer Study




(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)



4 Discussion












Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















aplusmn 006


bplusmn 004 4824

aplusmn 3089


cplusmn 007 108

bplusmn 960 7842


dplusmn 004 16992

cplusmn 7505 6604


eplusmn 001 12816

dplusmn 8475 7439






minus] 120583M


aplusmn 11 14506

aplusmn 18 16

aplusmn 003 14

bplusmn 031 06

aplusmn 009 92

aplusmn 019


bplusmn 015 2922

bplusmn 192 07

bplusmn 010 28

aplusmn 014 72

bplusmn 05 332

bplusmn 021


aplusmn 076 176

aplusmn 41 17

aplusmn 003 15

aplusmn 021 171

aplusmn 14 77

aplusmn 112


dplusmn 15 173

dplusmn 36 141

dplusmn 03 14

dplusmn 012 26

dplusmn 019 63

dplusmn 014


eplusmn 62 164

eplusmn 50 17

eplusmn 002 14

eplusmn 011 140

eplusmn 093 81

eplusmn 053









3 Results


32 Antiulcer Study




(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)



4 Discussion












Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)



4 Discussion












Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)



4 Discussion












Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e)







(a) (b)

(c) (d)



4 Discussion












Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)



4 Discussion












Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























Acknowledgment


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article -mangostin from cratoxylum arborescens...

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