)JOEBXJ1VCMJTIJOH$PSQPSBUJPO &WJEFODF …downloads.hindawi.com/journals/ecam/2015/706782.pdf ·...

Research ArticleThe Preventive Effect on Ethanol-Induced Gastric Lesions ofthe Medicinal Plant Plumeria rubra Involvement of the LatexProteins in the NOcGMPKATP Signaling Pathway

Nylane Maria Nunes de Alencar1 Rachel Sindeaux Paiva Pinheiro1

Ingrid Samantha Tavares de Figueiredo2 Patriacutecia Bastos Luz1

Lyara Barbosa Nogueira Freitas1 Tamiris de Faacutetima Goebel de Souza1

Luana David do Carmo1 Larisse Mota Marques1 and Marcio Viana Ramos3

1Departamento de Fisiologia e Farmacologia UFC Coronel Nunes de Melo 1127 Rodolfo Teofilo 60430-270 Fortaleza CE Brazil2Centro Universitario Estacio do Ceara Via Corpvs Rua Eliseu Uchoa Becco No 600 Bairro Agua Fria60810-270 Fortaleza CE Brazil3Departamento de Bioquımica e Biologia Molecular UFC Campus do Pici Caixa Postal 6033 60451-970 Fortaleza CE Brazil

Correspondence should be addressed to Nylane Maria Nunes de Alencar nylanegmailcom

Received 3 July 2015 Revised 26 October 2015 Accepted 26 November 2015

Academic Editor Guillermo Schmeda-Hirschmann

Copyright copy 2015 Nylane Maria Nunes de Alencar et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Plumeria rubra (Apocynaceae) is frequently used in folk medicine for the treatment of gastrointestinal disorders hepatitis andtracheitis among other infirmities The aim of this study was to investigate the gastroprotective potential of a protein fractionisolated from the latex of Plumeria rubra (PrLP) against ethanol-induced gastric lesions and describe the underlying mechanismsIn a dose-dependentmanner the pretreatment with PrLP prevented ethanol-induced gastric lesions inmice after single intravenousadministration The gastroprotective mechanism of PrLP was associated with the involvement of prostaglandins and balance ofoxidantantioxidant factors Secondarily the NOcGMPKATP pathway and activation of capsaicin-sensitive primary afferents werealso demonstrated as part of the mechanism This study shows that proteins extracted from the latex of P rubra prevent gastriclesions induced in experimental animals Also the results support the use of the plant in folk medicine

1 Introduction

Peptic ulcer is a multifactorial disease that affects an increas-ing number of people worldwide Etiological factors includeemotional stress improper diet excessive ethanol ingestiongenetic factors continuous or indiscriminate use of NSAIDsand infection by Helicobacter pylori [1 2] Antagonists ofhistamine H2ndash6 receptors and inhibitors of the proton pumpare currently the main classes of drugs used in the clinicfor the treatment of peptic ulcer [3] However some adverseeffects are associated with their long-term use such ashypergastrinemia and an increased risk ofHelicobacter pyloriand Clostridium difficile infections [4 5] Furthermore thehigh costs of these drugs are still concerns to be addressed

These inconsistencies stimulate the search for alternative orcomplementary strategies to improve the prevention andhealing of ulcers

Latex fluids have been reported to display numerouspharmacological properties This is in good agreement withthe traditional and folk medicinal use of latex-bearing plantsworldwide [6 7] Plumeria rubra is a laticifer plant com-monly known as the frangipani or temple tree and distributedmainly in tropical and subtropical regions [8] It is usedin folk medicinal purposes to treat or relieve fever coldcutaneous infections tracheitis gastrointestinal disordersureterolithiasis and hepatitis and to induce coughing upThe traditional uses have been further certified by scientificdocumentation [9 10] Active phytoconstituents (plumericin

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 706782 10 pageshttpdxdoiorg1011552015706782

2 Evidence-Based Complementary and Alternative Medicine

and isoplumericin) isolated from P rubra showed antial-gal antifungal antibacterial and molluscicidal effects andcytotoxic properties [11ndash13] The n-hexane fraction of crudemethanolic extract stem bark showed in vitro antimicrobialactivity [14] The flavone glycoside isolated from the flowersexhibited a significant reduction in serum triglycerides ofanimals [15] More recently the involvement of proteinsin pharmacological activities of P rubra was reported Aprotease Plumerin-R was isolated from the latex by acetoneprecipitation and displayed anti-inflammatory activity [16]In our previous findings the soluble proteins extracted fromthe latex (PrLP) showed antioxidant and proteolytic activity[17] and endothelial relaxation of rat thoracic aortic rings [18]Furthermore studies with the proteins extracted from latexof other plants have suggested that the proteins are stronglyassociated with the pharmacological properties claimed bythe popular medicine [6 7] In the present study the aqueousprotein fraction extracted from the latex of P rubra wasexamined in vivo to determine its gastroprotective potential

2 Materials and Methods

21 Latex Extraction and Protein Recovered The fresh latexfrom Plumeria rubra L (Jasmine) was collected in specimensgrowing in the Garden of Medicinal Plants UniversidadeFederal do Ceara (UFC) Brazil The plant material wasidentified by a taxonomist and a voucher specimen (number15018) was deposited at Prisco Bezerra Herbarium of theUFC Briefly terminal branches were used to extract freshlatex The fluid was taken into plastic tubes containingdistilled water to yield a dilution ratio of 1 2 (vv) Thesamples were centrifuged at 5000timesg at 10∘C for 10 minutesThe precipitated rubber-like material was discarded andthe supernatant was submitted to dialysis against distilledwater using a membrane with a cutoff of 8000Da After48 h the nondialyzable material (PrLP) was obtained aftercentrifugation under the same conditions described aboveThe supernatant lyophilized was stored at 25∘C until useSoluble proteins from the latex of Plumeria rubra (PrLP) usedin this studywere previously characterized [17]The summaryof their properties will be presented later in this paper

22 Animals Adult male Swiss mice weighing 25plusmn30 g wereobtained from the Central AnimalHouse of theUniversidadeFederal do Ceara Brazil The animals were kept in plasticcages under controlled environmental conditions (1212 hlightdark cycles temperature 25∘C and humidity 55 plusmn 10)with free access to water and fed with a commercial feed(Purina Paulınia SP Brazil) until 16 hours before the experi-ments All experimental procedures were handled accordingto the current Guide for the Care and Use of LaboratoryAnimals of the National Research Council after approval bythe ldquoEthical Committee for Animal Userdquo of the UniversidadeFederal do Ceara (protocol number 572010)

23 Chemicals Capsaicin capsazepine indomethacinglibenclamide diazoxide L-arginine N120596-nitro-L-arginine

methyl ester (L-NAME) 1H-[124]oxadiazolo[43-a]quinox-alin-1-one (ODQ) and absolute ethanol were purchasedfrom Sigma-Aldrich (St Louis MO USA) Prostaglandinanalog 1616-dimethyl PGE2 (misoprostol) was purchasedfrom Continental Pharma (Cytotec Italy) N-Acetylcysteinewas purchased from Uniao Quımica (Sao Paulo Brazil) Allother chemicals were of analytical grade unless otherwisespecified

24 Ethanol-Induced Gastric Lesions The animals were ran-domly distributed into six groups (119899 = 8 per group) and pre-treated intravenously (iv) with vehicle (09NaCl 10mLkgpositive control group) or PrLP (005 05 5 or 50mgkg) ororally (po) with N-acetylcysteine the standard antioxidantdrug (NAC 750mgkg) After 30 minutes a single intra-gastric dose of absolute ethanol (02mL) was administeredto positive control (vehicle) or PrLP groups and after 60minutes to NAC group One hour after ethanol treatmentsthe animals were sacrificed with anesthetic overdose Thestomachs were excised opened along the greater curvatureand rinsed with saline (09) according to the methoddescribed by Robert et al [19] The stomachs were scannedand the extension of the ulcerated area () was estimatedusing a computer planimetry program (ImageJ NationalInstitutes of Health USA) [20] After the statistical analysisof the data the lower effective dose of the 4 doses tested wasused for all other assays Subsequently gastric corpus sampleswere then weighed frozen and stored at minus70∘C until beingassayed to determine glutathione (GSH) levels [21]

25 Microscopic Analyses Samples of the gastric mucosawere fixed in formaldehyde (10 vv) and prepared in 001MPBS pH 72 over 24 h for histopathology procedures Sec-tions (5 120583m)were stained with hematoxylin-eosin to evaluategastric mucosal injury according to the criteria of Laine andWeinstein [22] as follows edema in the upper mucosa (0ndash4) hemorrhagic damage (0ndash4) epithelial cell loss (0ndash3) andthe presence of inflammatory cells (0ndash3) Photomicrographsof sections were obtained with a Leica DM microscopeequippedwith a LeicaDFC 280 camera (200xmagnification)

26 Role of Prostaglandins in Gastroprotective Effect ofthe PrLP The influence of endogenous prostaglandins wasinvestigated as previously described by Morais et al [23]The animals (119899 = 8 per group) were pretreated withthe cyclooxygenase inhibitor (indomethacin 10mgkg po)60min before the PrLP (05mgkg iv) or a syntheticprostaglandin E1 analog (misoprostol 50 120583gkg po) Othergroups of animals were treated only with vehicle (09NaCl 10mLkg iv) PrLP (05mgkg iv) or misoprostol(50 120583gkg po) After 30 minutes a single intragastric doseof absolute ethanol (02mL) was administered to positivecontrol (vehicle) or PrLP groups and after 60 minutes tomisoprostol groups One hour after ethanol treatments theanimals were sacrificed with anesthetic overdose The stom-achs were excised and opened along the greater curvature[19] The extension of the ulcerated area () was estimatedusing ImageJ software [20]

Evidence-Based Complementary and Alternative Medicine 3

27 Role of Nitric Oxide in Gastroprotective Effect of thePrLP The involvement of nitric oxide was investigated aspreviously described [23] The animals (119899 = 8 per group)were pretreated with inhibitor of the nitric oxide synthase(L-NAME 20mgkg) by intraperitoneal (ip) administra-tion 30min prior to the treatment with PrLP (05mgkgiv) or a substrate for nitric oxide synthase (L-arginine600mgkg ip) Other groups of animals were treated onlywith vehicle (09 NaCl 10mLkg iv) PrLP (05mgkgiv) or L-arginine (600mgkg ip) After 30 minutesall animals received a single intragastric dose of absoluteethanol (02mL) One hour later the animals were sacrificedwith anesthetic overdose The stomachs were excised andopened along the greater curvature [19] The extension ofthe ulcerated area () was estimated using ImageJ software[20] Subsequently samples from gastric mucosa were thenweighed frozen and stored at minus70∘C until being assayed forNO3NO2production [24]

28 Evaluation of Transient Receptor Potential Vanilloid Type1 (TRPV1) Triggers The activation of capsaicin-sensitiveprimary afferents was investigated using a TRPV1 antagonistcapsazepine [25] The animals (119899 = 8 per group) werepretreated with capsazepine (5mgkg ip) 30min priorto the treatment with PrLP (05mgkg iv) or a vanilloidagonist (capsaicin 03mgkg po) Other groups of animalswere treated only with vehicle (09 NaCl 10mLkg iv)PrLP (05mgkg iv) or capsaicin (03mgkg po) After30 minutes a single intragastric dose of absolute ethanol(02mL) was administered to positive control (vehicle) orPrLP groups and after 60 minutes to capsaicin groups Onehour after ethanol treatments the animals were sacrificedwith anesthetic overdose The stomachs were excised andopened along the greater curvature [19] The extension of theulcerated area () was estimated using ImageJ software [20]

29 Evaluation of Soluble Guanylate Cyclase Activation Asoluble guanylate cyclase inhibitor (ODQ) was used toinvestigate the involvement of cGMP [26]The animals (119899 = 8per group) were pretreatedwithODQ (10mgkg ip) 30minprior to the treatment with PrLP (05mgkg iv) Othergroups of animals were treated only with vehicle (09 NaCl10mLkg iv) or PrLP (05mgkg iv) After 30 minutesall animals received a single intragastric dose of absoluteethanol (02mL) One hour later the animals were sacrificedwith anesthetic overdose The stomachs were excised andopened along the greater curvature [19] The extension of theulcerated area () was estimated using ImageJ software [20]

210 Role of ATP-Sensitive PotassiumChannels (119870119860119879119875

) in Gas-troprotective Effect of PrLP Pharmacological modulation ofKATP with diazoxide (agonist) or glibenclamide (antagonist)was used to investigate the involvement of these channels inthe gastroprotective effect of PrLP [22] The animals (119899 =8 per group) were pretreated with glibenclamide (5mgkgip) 30min prior to the treatment with PrLP (05mgkgiv) or diazoxide (3mgkg ip) Other groups of animalswere treated only with vehicle (09 NaCl 10mLkg iv)

PrLP (05mgkg iv) or diazoxide (3mgkg ip) After30 minutes all animals received a single intragastric doseof absolute ethanol (02mL) One hour later the animalswere sacrificed with anesthetic overdose The stomachs wereexcised and opened along the greater curvature [19] Theextension of the ulcerated area () was estimated usingImageJ software [20]

211 Measurement of NitrateNitrite Levels in the GastricMucosa Nitrite levels in biopsy lysates from the gastricmucosa were determined indirectly as the total content ofnitrite and nitrate (NO

3

minus

NO2

minus) by a spectrophotometricmethod based on the Griess reaction [24] Samples of thegastric mucosa were homogenized in 50mM potassiumphosphate buffer (pH 78) and centrifuged at 11000timesg for15 minutes at 4∘C An aliquot of each sample (80 120583L) wasincubated in a microplate with nitrate reductase for 12 h toconvert NO

3into NO

2 At room temperature (25∘C) 100 120583L

of Griess reagent (1 sulphanilamide in 1 phosphoric acidand 01 naphthalene diamine dihydrochloride in water) wasadded and incubated for 10 minutes The optical densitieswere measured at 540 nm in a microplate reader Nitriteconcentrations in the samples were determined from a stan-dard curve generated by different concentrations of sodiumnitrite (01ndash100mM) The data are expressed as micromolesof nitrite All analyses were performed in triplicate and werereproduced without significant differences

212 Glutathione (GSH) Levels in the Gastric Mucosa Sam-ples of the gastric mucosa were homogenized in a solutionthat contained 1mL of a 002M EDTA cooled solution320 120583L of distilled water and 400120583L of trichloroacetic acid(TCA) 50 (wv) The homogenates were centrifuged at3000timesg for 15 minutes The supernatants (400 120583L) weremixed with 800120583L of Tris buffer (40mM pH 89) and 551015840-dithiobis(2-nitrobenzoic acid) (DTNB 10mM) was addedThe absorbance was measured within 3 minutes after addi-tion of DTNB at 412 nm against a blank reagent withouthomogenate [21] The absorbance values were extrapolatedfrom a reduced glutathione standard curve and expressed asNP-SHg of stomach tissue

213 Statistical Analysis Values are expressed as the mean plusmnstandard errors mean (SEM) or median Analysis of Variance(ANOVA) followed by Student-Newman-Keuls test was usedto compare means and Kruskal-Wallis nonparametric testfollowed by Dunnrsquos test to compare medians 119875 lt 005 wasdefined as statistically significant

3 Results and Discussion

PrLP comprises the water soluble protein fraction extractedfrom the whole latex of Plumeria rubra The antioxidativeenzymes ascorbate peroxidase and superoxide dismutasewere detected in PrLP while catalase was absent Chitinaseswere also found Proteolytic enzymes that were best inhibitedby E-64 were reported [17]These authors reported that PrLPrepresents nearly 033mg of protein in 1mL of crude latex


Vehicle NAC 005 05 5 50

EthanolPrLP

0

5

10

15

20

Ulc

erat

ed ar

ea (

)

lowast

lowast

lowastlowast

Figure 1 Ethanol-induced gastric lesions Animals were sacrificedand their stomachs were immediately excised to evaluate theextension of the ulcerated area Data are expressed as the mean plusmnstandard error mean (SEM) of the number of lesions (8 animalsper group) expressed in percent lowast119875 lt 005 indicates a significantdifference compared with the positive control group (vehicle) 119875 lt005 indicates a significant difference comparedwith animals treatedwith NAC (ANOVA Newman-Keuls test)

PrLP is free of other metabolites mainly those producedby the secondary metabolism Water insoluble compoundsare lost by precipitation along dialyses in water and thewater soluble ones are lost through the dialyses membraneChemical assays for measurement of saponins flavonoidsphenols tannins triterpenes and alkaloids on PrLP havefailed (data not shown) At least the overall profile of proteinspresent in PrLP can be found in the studies of de Freitas et al[17] cited above

Experimental studies to highlight gastroprotective sub-stances have been performed using a model of ethanol-induced gastric lesions [27ndash29] Here we investigated thegastroprotective potential of PrLP since the properties oflatex proteins in different pharmacological models have beensuccessfully confirmedThevenous route for sample adminis-tration was selected as the first approach in order to avoid thephysiochemical instability and enzymatic barrier of proteinsMoreover the influence of enterohepatic recirculation couldreduce the bioavailability of the soluble proteins We haveinitiated approaches to determine the potential subchronictoxicity of PrLP in animals Serum level of urea and the enzy-matic activities of alanine aminotransferase (ALT or TGP)and aspartate aminotransferase (AST or TGO) determinedwith standardized diagnostic kits were normal in animalsgiven PrLP at tested doses (unpublished data)

In a dose-dependentmanner the pretreatment with PrLPprevented the appearance of gastric mucosal ulceration whencompared with positive control group (vehicle) (119875 lt 005)The effect was similar to that observed in theN-acetylcysteine(NAC) group The highest gastroprotective effect of PrLPoccurred at a dose of 05mgkg (Figure 1)

Macro- andmicroscopic aspects of the gastricmucosa areshown in Figures 2(a)ndash2(d) and Table 1 No type of lesionwas observed in the samples of stomach from the negativecontrol group (saline) as seen by the gastric epithelia integrity

Table 1 Semi-quantitative evaluation of gastric lesions

Groups Microscopic scores

Hemorrhage Loss epithelialcell

Inflammatoryinfiltrate

Negative control group 0 (0-0) 0 (0-0) 0 (0-0)Positive control group 4 (4-4)lowast 3 (2-3)lowast 1 (0-1)PrLP 05 (0-1) 1 (1-1) 0 (0-1)NAC 0 (0-0) 1 (1-1) 0 (0-1)Data represent the median and range of scores from two separate exper-iments (0) absent (1) mild (2) moderate (3) intense (4) edema in theupper mucosa hemorrhagic damage epithelial cell loss and the presence ofinflammatory cells lowast119875 lt 005 indicates a significant difference comparedwith the negative control group 119875 lt 005 indicates a significant differencecompared with the positive control group (vehicle) (119899 = 8 animalsgroupKruskal-Wallis test followed by Dunnrsquos test)

(Figures 2(a1) and 2(a2)) Animals pretreated with vehicle(positive control group) and subjected to intragastric absoluteethanol presented a significant number of lesions on thegastric mucosa with intense signs of hemorrhage and loss ofepithelial cells when compared with negative control group(Figures 2(b1) and 2(b2)) The groups of animals pretreatedwith PrLP or NAC were protected from alterations inducedby ethanol (Figures 2(c1) 2(c2) 2(d1) and 2(d2))

The genesis of ethanol-induced gastric lesions ismultifac-torial and is associated with a decrease in the intrinsic gastricmucosal defense mechanisms or an increase in aggressivefactors mainly related to changes in the microcirculationand oxidative stress [26] The existence of cytoprotectiveeffect associated with a significant reduction of the ulcerindex besides free radical scavenging activity was previouslypointed to ethanol and chloroform extract of Plumeria rubra[30] However our data shown in this work suggest thatthe gastroprotective effect observed on animals submittedto induced gastric ulcer was mediated by the latex proteinfraction instead of other metabolites

It is well known that prostaglandin E2 (PGE2) aneicosanoid has gastroprotective effects through stimulatingbicarbonate and mucus release also provoking vasodilationand increased blood flow [31ndash33] The effects of PGE2 onethanol-induced gastric lesions are due to an increase inintracellular cGMP mediated by an increase in intracellu-lar calcium concentration and nitric oxide production [3435] The involvement of prostaglandins in the gastropro-tective mechanism of PrLP was investigated through phar-macological modulation with indomethacin (an inhibitorof prostaglandin synthesis) A significant extension of thegastric mucosal ulceration was observed in animals pre-treated with vehicle (positive control group) and subjectedto intragastric absolute ethanol (Figure 3) The appearanceof injuries was significantly prevented by the pretreatmentwith PrLP or misoprostol when compared with the pos-itive control group (vehicle) (119875 lt 005) However thepretreatment of animals with indomethacin reversed thegastroprotective effects promoted by PrLP or misoprostolsignificantly increasing the ethanol-induced gastric mucosallesions (119875 lt 005)


(a1) (a2)

(b1) (b2)

(c1) (c2)

(d1) (d2)

Figure 2 Gastric mucosal lesions macro- and microscopic aspects Animals were sacrificed and their stomachs were opened along thegreater curvature For macroscopic evaluation images were selected from animals belonging to the corresponding experimental groups(a1ndashd1) Samples of the gastric mucosa were removed to perform histological analyses (a2ndashd2) Hematoxylin-eosin stained sections wereemployed to obtain photomicrographs and to estimate hemorrhage loss of epithelial cells and inflammatory infiltrates (arrow) among thedifferent experimental groups (a1 a2) negative control group (b1 b2) positive control group (c1 c2) PrLP 05 mgkg iv and (d1 d2) NAC(200x magnification)

These results strongly indicate that PrLP acts through theinvolvement of prostaglandins possibly stimulating the pro-duction of this mediator Similarly the protective responseof chloroform and ethanolic extract of leaves from P rubraagainst ethanol-induced gastric lesions was also a suggestionof its effect on prostaglandin synthesis [22]

Gastric mucosal defense is also mediated physiologicallyby nitric oxide (NO) through blood flow regulation mucusrelease and inhibition of inflammatory infiltrates [36ndash38]Thus drugs that block the synthesis of NO exacerbate thelesions associated with ethanol Therefore an inhibitor ofNO synthesis (L-NAME) was used in order to observe the


Vehicle PrLP Misoprostol PrLP Misoprostol

Ethanol

Indometacin

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowast

lowast

Figure 3 Involvement of prostaglandins in ethanol-induced gastriclesions Animals were sacrificed and their stomachs were immedi-ately excised to evaluate the extension of the ulcerated area Dataare expressed as the mean plusmn standard error mean (SEM) of thenumber of lesions (8 animals per group) expressed in percent lowast119875 lt005 indicates a significant difference compared with the positivecontrol group (vehicle) 119875 lt 005 indicates a significant differencecompared with animals treated only with PrLP amp119875 lt 005 indicatesa significant difference compared with animals treated only withmisoprostol (ANOVA Newman-Keuls test)

Vehicle PrLP L-Arg PrLP L-Arg

EthanolL-NAME

amp

0

10

20

30

40

50

Ulc

erat

ed ar

ea (

)

lowastlowast

Figure 4 Involvement of nitric oxide in ethanol-induced gastricmucosal lesions Animals were sacrificed and their stomachs wereimmediately excised to evaluate the extension of the ulcerated areaData are expressed as the mean plusmn standard error mean (SEM) ofthe number of lesions (8 animals per group) expressed in percentlowast

119875 lt 005 indicates a significant difference compared with thepositive control group (vehicle)

119875 lt 005 indicates a significantdifference comparedwith animals treated only with PrLP amp119875 lt 005indicates a significant difference comparedwith animals treated onlywith L-arginine (ANOVA Newman-Keuls test)

involvement of NO in the gastroprotective effect of PrLP Asignificant extension of the gastric mucosal ulceration wasobserved in animals pretreated with vehicle (positive controlgroup) and subjected to intragastric absolute ethanol (Fig-ure 4) The pretreatment with PrLP (iv) or L-arginine (ip)significantly prevented the appearance of lesions comparedwith the positive control group (119875 lt 005) The gastroprotec-tive effects promoted by PrLP or L-arginine were reversed inthe group of animals pretreated with L-NAME (ip) which

Saline Vehicle L-Arg PrLP

Ethanol

lowast

0

10

20

30

[NO

3minus

NO

2minus

] (120583

M)

Figure 5 Nitrite levels in gastric mucosa Animals were sacrificedand their stomachs were immediately excised Samples of the gastricmucosa were removed to determine nitrite levels by the Griessreaction Data are the mean of three independent experimentsand are expressed as the mean plusmn standard error mean (SEM)of nitric oxide (NO

3

NO2

) levels (120583M) lowast119875 lt 005 indicates asignificant difference compared with the negative control group(saline) (ANOVA Newman-Keuls test)

significantly increased ethanol-induced gastric lesions (119875 lt005) As demonstrated nitric oxide was involved in thegastroprotective effects of PrLP To confirm this hypothesisstomach samples were used to measure nitritenitrate levelsAnimals pretreated with vehicle (positive control group) andsubmitted to intragastric absolute ethanol exhibited reducedlevels of nitrite in the gastric mucosa when compared withthe negative control group (119875 lt 005) (Figure 5) Thereduction in nitrite levels induced by ethanolwas significantlyprevented in the group of animals pretreated with PrLP or L-arginine (119875 lt 005)

These results confirm the involvement of nitric oxidein the gastroprotective effect of PrLP probably by con-stitutive overexpression of nitric oxide synthases (NOS)and their efficiency on catalytic actions According to thepublished literature nitric oxide increases vascular perme-ability and prostaglandin production in the gastric mucosaMoreover its effects on gastric microcirculation and thesynthesis of mucus also result from cooperative activity withprostaglandins [39 40]

It is well known that the vasodilator effect of NO ismediated by the stimulation of the enzyme soluble guanylylcyclase (sGS) and the consequent release of cGMP [41] Inexperimentalmodels a highly selective irreversible heme-siteinhibitor of soluble guanylyl cyclase known as ODQ (1H-[124]oxadiazolo[43-a]quinoxalin-1-one) has been used toidentify the correlation of gastroprotective substances withthe NO-sGS-cGMP signaling system [42 43] Accordingto this approach animals pretreated with vehicle (positivecontrol group) and subjected to intragastric absolute ethanolexhibited a significant extension of the gastric mucosal ulcer-ation (Figure 6) The appearance of lesions was significantlyprevented in the group of animals pretreated with PrLP (iv)Another group of animals was pretreated with ODQ in


Vehicle PrLP PrLP

Ethanol

ODQ

0

20

40

60

Ulc

erat

ed ar

ea (

)

lowast

Figure 6 Involvement of the NO-sGS-cGMP signaling system inethanol-induced gastric lesions Animals were sacrificed and theirstomachs were immediately excised to evaluate the extension ofthe ulcerated area Data are expressed as the mean plusmn standarderror mean (SEM) of the number of lesions (8 animals per group)expressed in percent lowast119875 lt 005 indicates a significant differencecompared with the positive control group (vehicle)

119875 lt 005

indicates a significant difference comparedwith animals treated onlywith PrLP (ANOVA Newman-Keuls test)

which the protective effects of PrLP against ethanol-inducedgastric mucosal injury were reversed (119875 lt 005) The sameinvolvement of the NO-sGS-cGMP signaling systemwas alsoused to explain the gastroprotective mechanism of sildenafil(a drug commercially used to treat erectile dysfunction) byMedeiros et al [42]

An ATP-dependent potassium channel (KATP) is associ-ated with the regulation of blood flow acid secretion andmuscle contractility of the gastric mucosa [44] Some com-pounds such as diazoxide inhibit ethanol-induced gastricmucosal damage through the opening of KATP channelswhile glibenclamide a blocker of these channels attenuatesgastric injuries [25 45 46] Thus to assess the contributionof KATP channels to the gastroprotective effects of PrLPpharmacological approaches were employed A significantextension of the gastric mucosal ulceration was observed inthe group of animals pretreated with vehicle (positive controlgroup) and subjected to intragastric absolute ethanol Thedamage in the gastric mucosa was significantly prevented bythe pretreatment with PrLP (iv) or diazoxide (ip) (119875 lt005) In another group of animals the prior administrationof glibenclamide (ip) reversed the gastroprotective effectspromoted by PrLP or diazoxide against ethanol-inducedgastric mucosal injury (119875 lt 005) (Figure 7)

The gastroprotective mechanisms involving prostaglan-dins and nitric oxide are related to the activation of the guany-lyl cyclase enzyme and release of the intracellular secondmessenger cyclic GMP (cGMP) Moreover the activation ofATP-dependent potassium channels may occur in responseto nitric oxide and cyclic GMP [26 47 48] Therefore thisset of results suggests that the NOcGMPKATP pathway is ofprimary importance in the gastroprotective effect of PrLP

Capsaicin-sensitive sensory nerves are involved as adefense system to protect against gastric damageThemecha-nism involves receptor stimulation at the plasma membrane

Vehicle PrLP Diazoxide PrLP Diazoxide

Ethanol

amp

Glibenclamide

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast

Figure 7 Involvement ofATP-dependent potassiumchannel (KATP)in ethanol-induced gastricmucosal lesions Animals were sacrificedand their stomachs were immediately excised to evaluate the exten-sion of the ulcerated area Data are expressed as themeanplusmn standarderror mean (SEM) of the number of lesions (8 animals per group)expressed in percent lowast119875 lt 005 indicates a significant differencecompared with the positive control group (vehicle)

119875 lt 005

indicates a significant difference comparedwith animals treated onlywith PrLP amp

119875 lt 005 indicates a significant difference comparedwith animals treated only with diazoxide (ANOVA Newman-Keulstest)

primarily of the transient receptor potential vanilloid type1 (TRPV1) [49 50] A vanilloid antagonist capsazepine hasbeen used to determine the involvement of these receptorsas part of the gastroprotective mechanism for differentsubstances [51 52] A significant extension of the gastricmucosal ulceration was observed in the group of animalspretreatedwith vehicle (positive control group) and subjectedto intragastric absolute ethanol As expected PrLP (iv)and capsaicin (po) significantly prevented the appearanceof injuries when compared with the positive control group(119875 lt 005) In the group of animals pretreated with vanilloidantagonist capsazepine the gastroprotective effect of PrLP(iv) and capsaicin (po) was significantly reduced (119875 lt005) (Figure 8) Our results indicate that the gastropro-tective effect of PrLP is also mediated by the activation ofcapsaicin-sensitive primary afferents A similar profile wasobserved in the gastroprotective effect of barbatusin and 3-beta-hydroxy-3-deoxibarbatusin diterpenes able to stimulatereceptor potential vanilloid type 1 (TRPV1) and promote anincrease in nitric oxide displaying mucosal defense throughboth of these mechanisms [22]

Ethanol-induced gastric mucosal injury is related tothe generation of free radicals and imbalance of oxi-dantantioxidant factors [53 54] Clinical and experimentalevidence suggest that antioxidant substances may promotegastroprotective effects [31 52]Therefore grounded by stud-ies that showed the antioxidative and proteolytic activitiesof laticifer cells of P rubra [17] the involvement of PrLPin the antioxidant defense mechanisms was investigatedthrough determination of glutathione (GSH) levels in thegastric mucosa Animals from the negative control group(saline) displayed glutathione (GSH) levels within normalrange according to the published literature [55] Animals


Vehicle PrLP Capsaicin PrLP Capsaicin

Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast

Figure 8 Involvement of transient receptor potential vanilloid 1 (TRPV1) in ethanol-induced gastric lesions Animals were sacrificed andtheir stomachs were immediately excised to evaluate the extension of the ulcerated area Data are expressed as the mean plusmn standard errormean (SEM) of the number of lesions (8 animals per group) expressed in percent lowast119875 lt 005 indicates a significant difference compared withthe positive control group (vehicle) 119875 lt 005 indicates a significant difference compared with animals treated only with PrLP amp119875 lt 005indicates a significant difference compared with animals treated only with capsaicin (ANOVA Newman-Keuls test)

Saline Vehicle PrLP NAC

amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)

Figure 9 Glutathione (GSH) levels in ethanol-induced gastric lesions Animals were sacrificed and their stomachs were immediately excisedSamples of the gastric mucosa from animals subjected to ethanol (a) or not (b) were removed to determine glutathione levels Data are themean of three independent experiments and are expressed as the mean plusmn standard error mean (SEM) of 120583g GSHg of tissue lowast119875 lt 005indicates a significant difference compared with the negative control group (saline) 119875 lt 005 indicates a significant difference comparedwith the positive control group (vehicle) amp119875 lt 005 indicates a significant difference comparedwith theNACgroup (ANOVANewman-Keulstest)

pretreatedwith vehicle (positive control group) and subjectedto intragastric absolute ethanol displayed significant mucosalGSH depletion when compared with the negative controlgroup (119875 lt 005) GSH levels were significantly (119875 lt005) restored in animals pretreated with NAC (po) or PrLP(iv) before administration of intragastric absolute ethanol(Figure 9(a)) A second approach was taken to verify whetherPrLP acts by increasing the levels of GSHwithout influencingethanol-induced gastric mucosal injury GSH levels were notaltered in animals pretreated with PrLP when compared withthe negative control group while NAC promoted an increaseregardless of the deleterious effects promoted by ethanol(Figure 9(b))

4 Conclusions

Soluble proteins from the latex of Plumeria rubra (PrLP) pre-vented ethanol-induced gastric lesions through the involve-ment of prostaglandins and balance of oxidantantioxidantfactors Secondarily the NOcGMPKATP pathway and acti-vation of capsaicin-sensitive primary afferents were alsodemonstrated as part of the mechanism This study alsosuggests that the use of the latex of P rubra in folk medicineis pertinent The diversity of manners in which people uselatex as alternative medicine (as topical application or oralingestion) encourages the scientific studies However it stillrepresents a challenge to understand the true potentialities of


latex compounds and determine the toxic potential and thusestablish the best practices for their use

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The study has been supported by grants from the followingBrazilian agencies Concelho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) and Fundacao Cearensede Apoio ao Desenvolvimento Cientıfico e Tecnologico(FUNCAP) This study is part of the consortium MolecularBiotechnology of Plant Latex

References

[1] K S Jain A K Shah J Bariwal et al ldquoRecent advancesin proton pump inhibitors and management of acid-pepticdisordersrdquo Bioorganic amp Medicinal Chemistry vol 15 no 3 pp1181ndash1205 2007

[2] C Musumba D M Pritchard and M Pirmohamed ldquoReviewarticle cellular and molecular mechanisms of NSAID-inducedpeptic ulcersrdquoAlimentary PharmacologyampTherapeutics vol 30no 6 pp 517ndash531 2009

[3] J L Wallace and J G P Ferraz ldquoNew pharmacologic therapiesin gastrointestinal diseaserdquo Gastroenterology Clinics of NorthAmerica vol 39 no 3 pp 709ndash720 2010

[4] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[5] E Palencia-Herrejon B Sanchez I Escobar andM L Gomez-Lus ldquoProton pump inhibitors and infection riskrdquo RevistaEspanola de Quimioterapia vol 24 no 1 pp 4ndash12 2011

[6] K C Mousinho C D C Oliveira J R D O Ferreira et alldquoAntitumor effect of laticifer proteins of Himatanthus drasticus(Mart) PlumelmdashApocynaceaerdquo Journal of Ethnopharmacologyvol 137 no 1 pp 421ndash426 2011

[7] P Chaudhary C D A Viana M V Ramos and V KumarldquoAntiedematogenic and antioxidant properties of high molec-ular weight protein sub-fraction of Calotropis procera latex inratrdquo Journal of Basic and Clinical Pharmacy vol 6 no 2 pp69ndash73 2015

[8] M E Endress and P V Bruyns ldquoA revised classification of theApocynaceae slrdquoThe Botanical Review vol 66 no 1 pp 1ndash562000

[9] L M Perry Medicinal Plants of East and Southeast AsiaAttributed Properties and Uses MIT Press Cambridge UK1980

[10] G Ye Z-X Li G-X Xia et al ldquoA new iridoid alkaloid from theflowers of Plumeria rubra L cv AcutifoliardquoMagnetic Resonancein Chemistry vol 46 pp 1195ndash1197 2009

[11] M O Hamburger G A Cordell and N Ruangrungsi ldquoTra-ditional medicinal plants of Thailand XVII Biologically activeconstituents of Plumeria rubrardquo Journal of Ethnopharmacologyvol 33 no 3 pp 289ndash292 1991

[12] M Grignon-Dubois B Rezzonico A Usubillaga and L BVojas ldquoIsolation of plumieride from Plumeria inodorardquo Chem-istry of Natural Compounds vol 41 no 6 pp 730ndash731 2005

[13] G M Kuigoua S F Kouam B T Ngadjui et al ldquoMinorsecondary metabolic products from the stem bark of Plumeriarubra Linn displaying antimicrobial activitiesrdquo Planta Medicavol 76 no 6 pp 620ndash625 2010

[14] A Dey T Das and S Mukherjee ldquoIn vitro antibacterial activityof n-Hexane fraction of methanolic extract of Plumeria rubra L(Apocynaceae) stem barkrdquo Journal of Plant Sciences vol 6 no3 pp 135ndash142 2011

[15] A J Merina D Sivanesan V H Begum and N SulochanaldquoAntioxidant and hypolipidemic effect of Plumeria rubra L inalloxan induced hyperglycemic ratsrdquo E-Journal of Chemistryvol 7 no 1 pp 1ndash5 2010

[16] I Chanda U Sarma S K Basu M Lahkar and S K Dutta ldquoAprotease isolated from the latex of Plumeria rubra linn (apoc-ynaceae) 2 Anti-inflammatory and wound-healing activitiesrdquoTropical Journal of Pharmaceutical Research vol 10 no 6 pp755ndash760 2011

[17] C D T de Freitas D P de Souza E S AraujoMG CavalheiroL S Oliveira andM V Ramos ldquoAnti-oxidative and proteolyticactivities and protein profile of laticifer cells of Cryptostegiagrandiflora Plumeria rubra and Euphorbia tirucallirdquo BrazilianJournal of Plant Physiology vol 22 no 1 pp 11ndash22 2010

[18] S V F Gaban W S Costa C N Assis et al ldquoLatex extract ofPlumeria rubra induces antihypertensive and vasodilator effectson rat aortardquo International Journal of Indigenous MedicinalPlants vol 46 pp 2051ndash4263 2013

[19] A Robert J E Nezamis C Lancaster and A J HancharldquoCytoprotection by prostaglandins in rats Prevention of gastricnecrosis produced by alcohol HCl NaOH hypertonic NaCland thermal injuryrdquoGastroenterology vol 77 no 3 pp 433ndash4431979

[20] N RD Costa RO Silva L ADNicolau et al ldquoRole of solubleguanylate cyclase activation in the gastroprotective effect of theHO-1COpathway against alendronate-induced gastric damagein ratsrdquo European Journal of Pharmacology vol 700 no 1ndash3 pp51ndash59 2013

[21] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 pp 192ndash205 1968

[22] P D A Rodrigues S M de Morais C M de Souza et alldquoGastroprotective effect of barbatusin and 3-beta-hydroxy-3-deoxibarbatusin quinonoid diterpenes isolated from Plectran-thus grandis in ethanol-induced gastric lesions inmicerdquo Journalof Ethnopharmacology vol 127 no 3 pp 725ndash730 2010

[23] T CMorais N B Pinto KMM B Carvalho et al ldquoProtectiveeffect of anacardic acids from cashew (Anacardium occiden-tale) on ethanol-induced gastric damage in micerdquo Chemico-Biological Interactions vol 183 no 1 pp 264ndash269 2010

[24] J-C Chen H-M Chen M-H Shyr et al ldquoSelective inhibitionof inducible nitric oxide in ischemia-reperfusion of rat smallintestinerdquo Journal of the Formosan Medical Association vol 99no 3 pp 213ndash218 2000

[25] A P De Vasconcellos Abdon G C De Souza L N C De Souzaet al ldquoGastroprotective potential of frutalin a d-galactose bind-ing lectin against ethanol-induced gastric lesionsrdquo Fitoterapiavol 83 no 3 pp 604ndash608 2012

[26] HWDavenport ldquoGastricmucosal hemorrhage in dogs Effectsof acid aspirin and alcoholrdquoGastroenterology vol 56 no 3 pp439ndash449 1969


[27] D Wong and C W Ogle ldquoChronic parenterally administerednicotine and stress- or ethanol-induced gastricmucosal damagein ratsrdquo European Journal of Pharmacology EnvironmentalToxicology and vol 292 no 2 pp 157ndash162 1995

[28] K Sairam C V Rao M D Babu and R K Goel ldquoProphylacticand curative effects ofBacopamonniera in gastric ulcermodelsrdquoPhytomedicine vol 8 no 6 pp 423ndash430 2001

[29] F M De-Faria A C A Almeida A Luiz-Ferreira et alldquoAntioxidant action of mangrove polyphenols against gastricdamage induced by absolute ethanol and ischemia-reperfusionin the ratrdquo The Scientific World Journal vol 2012 Article ID327071 9 pages 2012

[30] M Vimlesh Y Garima U S Mubeen and S Vivek ldquoDetermi-nation of antiulcer activity of Plumeria rubra leaves extractsrdquoInternational Research Journal of Pharmacy vol 3 no 9 pp194ndash197 2012

[31] T Brzozowski P C Konturek D Drozdowicz et alldquoGrapefruit-seed extract attenuates ethanol and stress-inducedgastric lesions via activation of prostaglandin nitric oxide andsensory nerve pathwaysrdquo World Journal of Gastroenterologyvol 11 no 41 pp 6450ndash6458 2005

[32] L Laine K Takeuchi and A Tarnawski ldquoGastric mucosaldefense and cytoprotection bench to bedsiderdquo Gastroenterol-ogy vol 135 no 1 pp 41ndash60 2008

[33] K Higuchi E Umegaki Y Yoda T Takeuchi M Murano andS Tokioka ldquoThe role of prostaglandin derivatives in a treatmentand prevention for gastric ulcers in the aged patientsrdquo NipponRinsho vol 68 no 11 pp 2071ndash2075 2010

[34] G B Glavin S Szabo B R Johnson et al ldquoIsolated rat gastricmucosal cells optimal conditions for cell harvesting measuresof viability and direct cutoprotectionrdquo Journal of Pharmacologyand Experimental Therapy vol 276 pp 1174ndash1179 1986

[35] H Sakai E Kumano A Ikari and N Takeguchi ldquoA gastrichousekeeping Clminus channel activated via prostaglandin EP3receptor-mediated Ca2+nitric oxidecGMP pathwayrdquoThe Jour-nal of Biological Chemistry vol 270 no 32 pp 18781ndash187851995

[36] J F Brown A C Keates P J Hanson and B J R WhittleldquoNitric oxide generators and cGMP stimulate mucus secre-tion by rat gastric mucosal cellsrdquo The American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 265 no3 pp G418ndashG422 1993

[37] J L Wallace W McKnight T L Wilson P D Del Soldatoand G Cirino ldquoReduction of shock-induced gastric damage bya nitric oxide-releasing aspirin derivative role of neutrophilsrdquoAmerican Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 273 no 6 pp G1246ndashG1251 1997

[38] J L Wallace ldquoNitric oxide aspirin-triggered lipoxins and NO-aspirin in gastric protectionrdquo Inflammation amp AllergymdashDrugTargets vol 5 no 2 pp 133ndash137 2006

[39] M Ding Y Kinoshita K Kishi et al ldquoDistribution ofprostaglandin E receptors in the rat gastrointestinal tractrdquoProstaglandins vol 53 no 3 pp 199ndash216 1997

[40] M N Muscara and J L Wallace ldquoNitric oxide therapeuticpotential of nitric oxide donors and inhibitorsrdquo AmericanJournal of Physiology vol 276 pp 1313ndash1316 1999

[41] J W Denninger J P M Schelvis P E Brandish Y ZhaoG T Babcock and M A Marletta ldquoInteraction of solubleguanylate cyclase with YC-1 kinetic and resonance Ramanstudiesrdquo Biochemistry vol 39 no 14 pp 4191ndash4198 2000

[42] J V R Medeiros G G Gadelha S J Lima et al ldquoRole of theNOcGMPKATP pathway in the protective effects of sildenafil

against ethanol-induced gastric damage in ratsrdquo British Journalof Pharmacology vol 153 no 4 pp 721ndash727 2008

[43] A E Chavez-Pina G R Tapia-Alvarez A Reyes-Ramınrezand A Navarrete ldquoCarbenoxolone gastroprotective mecha-nism participation of nitric oxidecGMPKATP pathway inethanol-induced gastric injury in the ratrdquo Fundamental ampClinical Pharmacology vol 25 no 6 pp 717ndash722 2011

[44] H P Toroudi M Rahgozar A Bakhtiarian and B DjahanguirildquoPotassium channel modulators and indomethacin-inducedgastric ulceration in ratsrdquo Scandinavian Journal of Gastroen-terology vol 34 no 10 pp 962ndash966 1999

[45] F F B P Freitas H B Fernandes C A Piauilino et alldquoGastroprotective activity of Zanthoxylum rhoifolium Lam inanimal modelsrdquo Journal of Ethnopharmacology vol 137 no 1pp 700ndash708 2011

[46] B Saxena and S Singh ldquoInvestigations on gastroprotectiveeffect of citalopram an antidepressant drug against stress andpyloric ligation induced ulcersrdquo Pharmacological Reports vol63 no 6 pp 1413ndash1426 2011

[47] BM Peskar K Ehrlich andBA Peskar ldquoRole ofATP-sensitivepotassium channels in prostaglandin-mediated gastroprotec-tion in the ratrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 301 no 3 pp 969ndash974 2002

[48] M I G Silva B A Moura M R D Q Neto et al ldquoGastropro-tective activity of isopulegol on experimentally induced gastriclesions inmice investigation of possiblemechanisms of actionrdquoNaunyn-Schmiedebergrsquos Archives of Pharmacology vol 380 no3 pp 233ndash245 2009

[49] G Mozsik J Szolcsanyi and A Domotor ldquoCapsaicin researchas a new tool to approach of the human gastrointestinal phys-iology pathology and pharmacologyrdquo Inflammopharmacologyvol 15 no 6 pp 232ndash245 2007

[50] A P C Castelo BNArruda RG Coelho et al ldquoGastroprotec-tive effect of Serjania erecta Radlk (Sapindaceae) involvementof sensory neurons endogenous nonprotein sulfhydryls andnitric oxiderdquo Journal of Medicinal Food vol 12 no 6 pp 1411ndash1415 2009

[51] J V R Medeiros V H Bezerra A S Gomes et al ldquoHydrogensulfide prevents ethanol-induced gastric damage in mice roleof ATP-sensitive potassium channels and capsaicin-sensitiveprimary afferent neuronsrdquo Journal of Pharmacology and Exper-imental Therapeutics vol 330 no 3 pp 764ndash770 2009

[52] G S Cerqueira G S Silva E R Vasconcelos et al ldquoEffectsof hecogenin and its possible mechanism of action on exper-imental models of gastric ulcer in micerdquo European Journal ofPharmacology vol 683 no 1ndash3 pp 260ndash269 2012

[53] M G Repetto and S F Llesuy ldquoAntioxidant properties ofnatural compounds used in popularmedicine for gastric ulcersrdquoBrazilian Journal of Medical and Biological Research vol 35 no5 pp 523ndash534 2002

[54] R Nassini E Andre D Gazzieri et al ldquoA bicarbonate-alkalinemineral water protects from ethanol-induced hemorrhagicgastric lesions in micerdquo Biological and Pharmaceutical Bulletinvol 33 no 8 pp 1319ndash1323 2010

[55] S R B Damasceno J C Rodrigues R O Silva et al ldquoRoleof the NoKATP pathway in the protective effect of a sulfated-polysaccharide fraction from the algae Hypnea musciformisagainst ethanol-induced gastric damage in micerdquo BrazilianJournal of Pharmacognosy vol 23 no 2 pp 320ndash328 2013

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


and isoplumericin) isolated from P rubra showed antial-gal antifungal antibacterial and molluscicidal effects andcytotoxic properties [11ndash13] The n-hexane fraction of crudemethanolic extract stem bark showed in vitro antimicrobialactivity [14] The flavone glycoside isolated from the flowersexhibited a significant reduction in serum triglycerides ofanimals [15] More recently the involvement of proteinsin pharmacological activities of P rubra was reported Aprotease Plumerin-R was isolated from the latex by acetoneprecipitation and displayed anti-inflammatory activity [16]In our previous findings the soluble proteins extracted fromthe latex (PrLP) showed antioxidant and proteolytic activity[17] and endothelial relaxation of rat thoracic aortic rings [18]Furthermore studies with the proteins extracted from latexof other plants have suggested that the proteins are stronglyassociated with the pharmacological properties claimed bythe popular medicine [6 7] In the present study the aqueousprotein fraction extracted from the latex of P rubra wasexamined in vivo to determine its gastroprotective potential

2 Materials and Methods

21 Latex Extraction and Protein Recovered The fresh latexfrom Plumeria rubra L (Jasmine) was collected in specimensgrowing in the Garden of Medicinal Plants UniversidadeFederal do Ceara (UFC) Brazil The plant material wasidentified by a taxonomist and a voucher specimen (number15018) was deposited at Prisco Bezerra Herbarium of theUFC Briefly terminal branches were used to extract freshlatex The fluid was taken into plastic tubes containingdistilled water to yield a dilution ratio of 1 2 (vv) Thesamples were centrifuged at 5000timesg at 10∘C for 10 minutesThe precipitated rubber-like material was discarded andthe supernatant was submitted to dialysis against distilledwater using a membrane with a cutoff of 8000Da After48 h the nondialyzable material (PrLP) was obtained aftercentrifugation under the same conditions described aboveThe supernatant lyophilized was stored at 25∘C until useSoluble proteins from the latex of Plumeria rubra (PrLP) usedin this studywere previously characterized [17]The summaryof their properties will be presented later in this paper

22 Animals Adult male Swiss mice weighing 25plusmn30 g wereobtained from the Central AnimalHouse of theUniversidadeFederal do Ceara Brazil The animals were kept in plasticcages under controlled environmental conditions (1212 hlightdark cycles temperature 25∘C and humidity 55 plusmn 10)with free access to water and fed with a commercial feed(Purina Paulınia SP Brazil) until 16 hours before the experi-ments All experimental procedures were handled accordingto the current Guide for the Care and Use of LaboratoryAnimals of the National Research Council after approval bythe ldquoEthical Committee for Animal Userdquo of the UniversidadeFederal do Ceara (protocol number 572010)

23 Chemicals Capsaicin capsazepine indomethacinglibenclamide diazoxide L-arginine N120596-nitro-L-arginine

methyl ester (L-NAME) 1H-[124]oxadiazolo[43-a]quinox-alin-1-one (ODQ) and absolute ethanol were purchasedfrom Sigma-Aldrich (St Louis MO USA) Prostaglandinanalog 1616-dimethyl PGE2 (misoprostol) was purchasedfrom Continental Pharma (Cytotec Italy) N-Acetylcysteinewas purchased from Uniao Quımica (Sao Paulo Brazil) Allother chemicals were of analytical grade unless otherwisespecified

24 Ethanol-Induced Gastric Lesions The animals were ran-domly distributed into six groups (119899 = 8 per group) and pre-treated intravenously (iv) with vehicle (09NaCl 10mLkgpositive control group) or PrLP (005 05 5 or 50mgkg) ororally (po) with N-acetylcysteine the standard antioxidantdrug (NAC 750mgkg) After 30 minutes a single intra-gastric dose of absolute ethanol (02mL) was administeredto positive control (vehicle) or PrLP groups and after 60minutes to NAC group One hour after ethanol treatmentsthe animals were sacrificed with anesthetic overdose Thestomachs were excised opened along the greater curvatureand rinsed with saline (09) according to the methoddescribed by Robert et al [19] The stomachs were scannedand the extension of the ulcerated area () was estimatedusing a computer planimetry program (ImageJ NationalInstitutes of Health USA) [20] After the statistical analysisof the data the lower effective dose of the 4 doses tested wasused for all other assays Subsequently gastric corpus sampleswere then weighed frozen and stored at minus70∘C until beingassayed to determine glutathione (GSH) levels [21]

25 Microscopic Analyses Samples of the gastric mucosawere fixed in formaldehyde (10 vv) and prepared in 001MPBS pH 72 over 24 h for histopathology procedures Sec-tions (5 120583m)were stained with hematoxylin-eosin to evaluategastric mucosal injury according to the criteria of Laine andWeinstein [22] as follows edema in the upper mucosa (0ndash4) hemorrhagic damage (0ndash4) epithelial cell loss (0ndash3) andthe presence of inflammatory cells (0ndash3) Photomicrographsof sections were obtained with a Leica DM microscopeequippedwith a LeicaDFC 280 camera (200xmagnification)

26 Role of Prostaglandins in Gastroprotective Effect ofthe PrLP The influence of endogenous prostaglandins wasinvestigated as previously described by Morais et al [23]The animals (119899 = 8 per group) were pretreated withthe cyclooxygenase inhibitor (indomethacin 10mgkg po)60min before the PrLP (05mgkg iv) or a syntheticprostaglandin E1 analog (misoprostol 50 120583gkg po) Othergroups of animals were treated only with vehicle (09NaCl 10mLkg iv) PrLP (05mgkg iv) or misoprostol(50 120583gkg po) After 30 minutes a single intragastric doseof absolute ethanol (02mL) was administered to positivecontrol (vehicle) or PrLP groups and after 60 minutes tomisoprostol groups One hour after ethanol treatments theanimals were sacrificed with anesthetic overdose The stom-achs were excised and opened along the greater curvature[19] The extension of the ulcerated area () was estimatedusing ImageJ software [20]









3

minus

NO2


3into NO









EthanolPrLP

0

5

10

15

20

Ulc

erat

ed ar

ea (

)

lowast

lowast

lowastlowast















(a1) (a2)

(b1) (b2)

(c1) (c2)

(d1) (d2)






Ethanol

Indometacin

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowast

lowast



EthanolL-NAME

amp

0

10

20

30

40

50

Ulc

erat

ed ar

ea (

)

lowastlowast






Ethanol

lowast

0

10

20

30

[NO

3minus

NO

2minus

] (120583

M)


3

NO2






Vehicle PrLP PrLP

Ethanol

ODQ

0

20

40

60

Ulc

erat

ed ar

ea (

)

lowast


119875 lt 005







Ethanol

amp

Glibenclamide

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast


119875 lt 005







Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast



amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)



4 Conclusions






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3

minus

NO2


3into NO









EthanolPrLP

0

5

10

15

20

Ulc

erat

ed ar

ea (

)

lowast

lowast

lowastlowast















(a1) (a2)

(b1) (b2)

(c1) (c2)

(d1) (d2)






Ethanol

Indometacin

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowast

lowast



EthanolL-NAME

amp

0

10

20

30

40

50

Ulc

erat

ed ar

ea (

)

lowastlowast






Ethanol

lowast

0

10

20

30

[NO

3minus

NO

2minus

] (120583

M)


3

NO2






Vehicle PrLP PrLP

Ethanol

ODQ

0

20

40

60

Ulc

erat

ed ar

ea (

)

lowast


119875 lt 005







Ethanol

amp

Glibenclamide

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast


119875 lt 005







Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast



amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)



4 Conclusions






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















EthanolPrLP

0

5

10

15

20

Ulc

erat

ed ar

ea (

)

lowast

lowast

lowastlowast















(a1) (a2)

(b1) (b2)

(c1) (c2)

(d1) (d2)






Ethanol

Indometacin

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowast

lowast



EthanolL-NAME

amp

0

10

20

30

40

50

Ulc

erat

ed ar

ea (

)

lowastlowast






Ethanol

lowast

0

10

20

30

[NO

3minus

NO

2minus

] (120583

M)


3

NO2






Vehicle PrLP PrLP

Ethanol

ODQ

0

20

40

60

Ulc

erat

ed ar

ea (

)

lowast


119875 lt 005







Ethanol

amp

Glibenclamide

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast


119875 lt 005







Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast



amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)



4 Conclusions






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a1) (a2)

(b1) (b2)

(c1) (c2)

(d1) (d2)






Ethanol

Indometacin

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowast

lowast



EthanolL-NAME

amp

0

10

20

30

40

50

Ulc

erat

ed ar

ea (

)

lowastlowast






Ethanol

lowast

0

10

20

30

[NO

3minus

NO

2minus

] (120583

M)


3

NO2






Vehicle PrLP PrLP

Ethanol

ODQ

0

20

40

60

Ulc

erat

ed ar

ea (

)

lowast


119875 lt 005







Ethanol

amp

Glibenclamide

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast


119875 lt 005







Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast



amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)



4 Conclusions






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Ethanol

Indometacin

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowast

lowast



EthanolL-NAME

amp

0

10

20

30

40

50

Ulc

erat

ed ar

ea (

)

lowastlowast






Ethanol

lowast

0

10

20

30

[NO

3minus

NO

2minus

] (120583

M)


3

NO2






Vehicle PrLP PrLP

Ethanol

ODQ

0

20

40

60

Ulc

erat

ed ar

ea (

)

lowast


119875 lt 005







Ethanol

amp

Glibenclamide

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast


119875 lt 005







Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast



amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)



4 Conclusions






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Vehicle PrLP PrLP

Ethanol

ODQ

0

20

40

60

Ulc

erat

ed ar

ea (

)

lowast


119875 lt 005







Ethanol

amp

Glibenclamide

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast


119875 lt 005







Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast



amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)



4 Conclusions






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Ethanol

amp

Capsazepine

amp

0

10

20

30

40

Ulc

erat

ed ar

ea (

)

lowastlowast



amp

Ethanol

lowast

lowast

0

200

400

600

800

(120583g

GSH

g ti

ssue

)

(a)Saline PrLP NAC

amp

lowast

0

200

400

600

(120583g

GSH

g ti

ssue

)

(b)



4 Conclusions






Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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