research article gastroprotective effect of geopropolis ...research article gastroprotective effect...

Research ArticleGastroprotective Effect of Geopropolis fromMelipona scutellaris Is Dependent on Productionof Nitric Oxide and Prostaglandin

Jerocircnimo Aparecido Ribeiro-Junior1 Marcelo Franchin1 Miriam Elias Cavallini1

Carina Denny1 Severino Matias de Alencar2 Masaharu Ikegaki3 and Pedro Luiz Rosalen1

1Department of Physiological Sciences School of Dentistry of Piracicaba University of Campinas Avenue Limeira 90113414 903 Piracicaba SP Brazil2Department of Agro-Food Industry Food and Nutrition ldquoLuiz de Queirozrdquo College of Agriculture University of Sao PauloAvenue Padua Dias 11 13418-900 Piracicaba SP Brazil3Faculty of Pharmaceutical Sciences Federal University of Alfenas Rua Gabriel Monteiro da Silva 700 37130-000 Alfenas MG Brazil

Correspondence should be addressed to Pedro Luiz Rosalen rosalenfopunicampbr

Received 15 December 2014 Revised 18 March 2015 Accepted 18 March 2015

Academic Editor Yoshiji Ohta

Copyright copy 2015 Jeronimo Aparecido Ribeiro-Junior et alThis 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

The aim of this study was to evaluate the gastroprotective activity of ethanolic extract of geopropolis (EEGP) from Meliponascutellaris and to investigate the possible mechanisms of action The gastroprotective activity of the EEGP was evaluated usingmodel ulcer induced by ethanol To elucidate the possible mechanisms of action we investigated the involvement of the nonproteinsulfhydryl (NP-SH) groups nitric oxide and prostaglandins In addition the antisecretory activity of EEGP was also evaluated bypylorus ligatedmodelTheEEGPorally administrated (300mgkg) reduced the ulcerative lesions induced by the ethanol (119875 lt 005)Regarding the mechanism of action the prior administration of nitric oxide and prostaglandins antagonists suppressed the activityof gastroprotective EEGP (119875 lt 005) On the other hand the gastroprotective activity of EEGPwas kept in the group pretreated withthe antagonist of the NP-SH groups furthermore the antisecretory activity was not significant (119875 gt 005) These results supportthe alternative medicine use of geopropolis as gastroprotective and the activities observed show to be related to nitric oxide andprostaglandins production

1 Introduction

Peptic ulcers are the imbalance between the aggressive agents(Helicobacter pylori and anti-inflammatory drugs amongothers) and the protective agents (prostaglandins and nitricoxide among others) [1 2] Despite the widespread use ofdifferent classes of monodrugs for the treatment of differenttypes of ulcers a large part of the worldrsquos population stillbenefits from the use of natural products [3]

The propolis is a nontoxic natural product collected bybees from different plant parts [4] Propolis has increased inpopularity as an alternative medicine or dietary supplementfor improving health and preventing disease in various partsof the world [5] Among the several biological activities

of propolis reported in the literature antimicrobial anti-inflammatory anticancer laxative and antiulcer can be found[6ndash10]

The geopropolis a mixture of resin wax and soil is apropolis collected by a native stingless bee of the Meliponinitribe [11 12] and also widely used in folk medicine for varioustherapeutic purposes [5]

The geopropolis from Melipona scutellaris bee speciespopularly known as ldquourucurdquo and found in northeasternBrazil has been the focus of our research Studies have shownthat geopropolis has antinociceptive and anti-inflammatoryproperties besides antimicrobial activity against differenttypes of bacteria In addition studies on the chemical profileof geopropolis revealed absence of flavonoid and phenolic

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 459846 5 pageshttpdxdoiorg1011552015459846

2 Evidence-Based Complementary and Alternative Medicine

acids commonly found in propolis from Apis mellifera andpresence of benzophenones [13ndash16]

Therefore in order to aggregate scientific value to thegeopropolis this study aims at evaluating the gastroprotectiveactivity of the ethanolic extract of geopropolis (EEGP) fromMelipona scutellaris and at investigating the possible mecha-nisms of action

2 Material and Methods

21 Obtaining the EEGP The geopropolis samples werecollected on municipality of Entre Rios (11∘571015840 S 38∘051015840 W)state of Bahia northeast of Brazil The geopropolis samples(100 g) were extracted in 80 ethanol in water (wv) at a 17dilution rate at 70∘C for 30min followed by filtration therebyobtaining the EEGPThe same process was repeated twice Atthe end the EEGP was concentrated in a rotary evaporator at40∘C [13]TheEEGPdissolutionwas carried out in PBS 1mMThe EEGP was administered to the animals by pathway oral(po)

22 Animals MaleWistar rats SPF (specific-pathogen-free)weighing 200ndash250 g were provided by CEMIBUNICAMP(Multidisciplinary Center for Biological Research SP Brazil)and kept in controlled temperature chambers (20 plusmn 2∘C)in light-dark 12 hours cycles relative humidity of 40 and60 with filtered water ad libitum The animals fasted for24 hours before the experiments The procedures describedwere reviewed and approved by the local Animal EthicsCommittee (CEUA Unicamp process number 2560-1)

23 Drugs and Reagents The drugs were purchased fromSigma Chemical Co St Louis MO USA (N-ethylmaleimideomeprazole N120596-nitro-L-arginine methyl ester hydrochlo-ride and ethanol) MP Biomedicals (indomethacin) andMerck (organic solvents)

24 Gastric Lesion Induced by Ethanol The rats were pre-treated with EEGP at dose of 100 200 or 300mgkg (po)The positive control group received omeprazole 30mgkg(po) and the negative control group received dissolvingvehicle of EEGP (po) One hour after the treatments 1mLof absolute ethanol was administered by po and one hourlater the animals were killed by anesthesia overload andthe stomach was removed and opened along the greatercurvature [17] The ulcerative lesions from each animal werecalculated according to the Gamberini et al [18] method asdescribed below

25 Role of the Nonprotein Sulfhydryl (NP-SH) Groups onGastroprotection Effect of the EEGP The rats were pretreatedwith inhibitor of the NP-SH groups (N-ethylmaleimide10mgkg sc) 30min prior to the treatment with EEGP(300mgkg) by po [19] The negative control group receiveddissolution vehicle of EEGP (po) One hour after the treat-ments 1mLof absolute ethanolwas administered by po andone hour later the animals were killed by anesthesia overloadand the stomach was removed and opened along the greater

curvature The ulcerative lesions from each animal werecalculated according to the Gamberini et al [18] method

26 Role of the Nitric Oxide on Gastroprotective Effect of theEEGP Therats were pretreatedwith inhibitor from the nitricoxide synthase (L-NAME 5mgkg) by intraperitoneal (ip)administration 30min prior to the treatment with EEGP(300mgkg po) The negative control group received disso-lution vehicle of EEGP (po) One hour after the treatments1mL of absolute ethanol was administered by po and onehour later the animals were killed by anesthesia overloadand the stomach was removed and opened along the greatercurvature [19] The ulcerative lesions from each animal werecalculated according to the Gamberini et al [18] method

27 Role of Prostaglandins on Gastroprotective Effect of theEEGP The rats were pretreated with the cyclooxygenaseinhibitor (indomethacin 5mgkg ip) 30min before theEEGP treatment (300mgkg po) The negative controlgroup received dissolution vehicle of EEGP (po) One hourafter the treatments 1mL of absolute ethanol was adminis-tered by po and one hour later the animals were killedby anesthesia overload and the stomach was removed andopened along the greater curvature [19]Theulcerative lesionsfromeach animalwere calculated according to theGamberiniet al [18] method

28 Evaluation of the Antisecretory Activity Theanimals wereanesthetized their abdomen was dissected and the pyloruswas connected Immediately after this procedure the EEGP300mgkg cimetidine 100mgkg or dissolution vehicle ofEEGP (negative control) was administered by po to therespective group of animals The abdomens were suturedand four hours later the animals were killed The stomachswere removed and the gastric content was collected andcentrifuged The volume and the pH of the gastric juice weremeasured [20]

29 Statistical Analysis Data were expressed as means plusmnstandard error of the mean (SEM) and statistical comparisonbetween groups was made utilizing analysis of variance(ANOVA) followed by test of Dunnett or Tukey Significancewas accepted when 119875 lt 005

3 Results and Discussion

This study evaluated the gastroprotective activity of EEGPfromMelipona scutellaris as well as the possible actionmech-anisms involved

The geopropolis used in this work presents the samechemical composition as described in our previous publica-tions where absence of flavonoids and phenolic acids andpresence of benzophenone were found [15 16]

Ethanol is a potent gastric ulcer inducer agent and itis widely applied to evaluate the gastroprotective activityof plant extracts as well as new pharmaceutical drugsin animal models Its effect causes an imbalance betweenthe oxidizing and the antioxidants agents in the gastric

Evidence-Based Complementary and Alternative Medicine 3

C Omep 30 100 200 3000

10

20

30

40

50

(mgkg po)Treatments

EEGP

Ulc

erat

ive l

esio

n in

dex

lowastlowast

Figure 1 Effect of the po administration of ethanolic extract ofgeopropolis (EEGP) on the ethanol induced ulcers Control (C)treated with vehicle omeprazole 30mgkg (Omep 30) and EEGPwith doses of 100 200 and 300mgkg The results are expressedas means plusmn SEM 119899 = 5 Symbols indicate statistical difference(ANOVA followed by Dunnett test 119875 lt 005) lowast compared to thecontrol group

mucosa This imbalance causes bleeding resulting from theruptured blood vessels [21 22] In the present study weevaluated the EEGP gastroprotective activity on the ethanol-induced ulcermodel (Figure 1)TheEEGP (300mgkg) orallyadministrated decreased the ulcerative lesions where an 89reduction was detected compared to the negative controlgroup (119875 lt 005) Regarding the positive control group(omeprazole) there was an 85 reduction of the ulcerativelesions compared to the negative control group (119875 lt 005)

To elucidate the possible mechanisms of action involvedwith the gastroprotective activity of EEGP we investigatedthe participation of the NP-SH groups nitric oxide andprostaglandins In addition the antisecretory activity ofEEGP was also evaluated

TheNP-SHgroups are protective of the gastricmucosa Inthis case the role of thesemediators is associatedwith the freeradicals blockage in the gastric mucosa [23] Thus in orderto evaluate the EEGP association with these substances theanimals underwent a pretreatment with a blocker agent (N-ethylmaleimide 10mgkg) of the NP-SH groups The resultsshowed that the administration of the N-ethylmaleimide(Figure 2) did not suppress the gastroprotective activity of theEEGP (119875 gt 005) Thus suggesting that the EEGP activity isnot associated with NP-SH groups

Nitric oxide plays a vital role in gastric gastroprotectionalso Among its actions are regulation of gastric secretion andgastric stimulation of mucus secretion [24 25] Furthermorestudies have shown thatNO is able to decrease the adhesion ofneutrophils to endothelial cells during the inflammatory pro-cess [26] The pretreatment with L-NAME (inhibitor of syn-thesis of nitric oxide) suppressed the gastroprotective activityof EEGP (Figure 3 119875 lt 005) These results corroboratethe study of Franchin et al [14] where it was observed thatthe administration of inhibitors of nitric oxide productionsuppressed the anti-inflammatory activity of the EEGP In thesame study it was found that the activity of EEGP on theinflammatory processwas related to the increased production

C NEM EEGP0

20

40

60

80

Treatments

Ulc

erat

ive l

esio

n in

dex

lowast

lowast

EEGP + NEM

Figure 2 Effect of the po administration of ethanolic extract ofgeopropolis (EEGP) on the ethanol induced ulcers Rats pretreatedsubcutaneous (sc) with the N-ethylmaleimide 10mgkg (NEM)After 30min the rats were treated with vehicle (C and NEM) andwith 300mgkg doses of EEGP The results are expressed as meansplusmn SEM 119899 = 5 Symbols indicate statistical difference (ANOVAfollowed by Tukey test 119875 lt 005) compared to control (C) groupand lowast compared to control group

C L-NAME EEGP0

20

40

60

80

100

Treatments

EEGP + L-NAME

Ulc

erat

ive l

esio

n in

dex

lowast

lowastlowast

Figure 3 Effect of the po administration of ethanolic extract ofgeopropolis (EEGP) on the ethanol induced ulcers Rats pretreatedwith 5mgkg of the L-NAME (ip) After 30min the rats weretreatedwith vehicle (C and L-NAME) and 300mgkg doses of EEGPThe results are expressed as means plusmn SEM 119899 = 5 Symbols indicatestatistical difference (ANOVA followed by Tukey test 119875 lt 005) compared to control group lowast compared to control (C) group lowastlowastcompared to EEGP group

of nitric oxide (verified by quantifying nitrite) which resultedin decreased adhesion of neutrophils on endothelial cellsTherefore the EEGP increased the gastroprotective responseand reduced the ulcerogenic effect induced by ethanol in thegastric mucosa probably by increased production of NO

This study also evaluated the role of prostaglandins inthe EEGP gastroprotective activity Indomethacin a nons-teroidal anti-inflammatory drug is widely used for inductionof ulcers in animal research These drugs are known to


C INDO EEGP0

20

40

60

80

100

Treatments

EEGP + INDO

Ulc

erat

ive l

esio

n in

dex

lowast

lowastlowast

Figure 4 Effect of the po administration of ethanolic extract ofgeopropolis (EEGP) on the ethanol induced ulcers Rats pretreatedwith 5mgkg of the indomethacin-INDO (ip) After 30min therats were treated with vehicle (C and Indo) and 300mgkg doses ofEEGP The results are expressed as means plusmn SEM 119899 = 5 Symbolsindicate statistical difference (ANOVA followed by Tukey test 119875 lt005) compared to control group lowast compared to control (C)group lowastlowast compared to EEGP group

Table 1 Evaluation of the anti-secretory activity of the ethanolicextract of geopropolis (EEGP) using the pyloric ligation modelMeans plusmn SEM 119899 = 5

Treatments Dose (mgkg) Volume (mL) pHControl mdash 33 plusmn 06 34 plusmn 05

Cimetidine 100 19 plusmn 03lowast

64 plusmn 01lowast

EEGP 300 26 plusmn 05 34 plusmn 05

lowastCompared to control group (ANOVA followed by Dunnett 119875 lt 005)

inhibit the production of prostaglandins including thosethat have protective action in the gastric tissue [27 28] Theprostaglandins produced in the stomach exert gastroprotec-tive activity through the stimulatory action of the gastricmucus and of bicarbonate secretion [29] According to theresults (Figure 4) it was found that the prior administrationof indomethacin in ulcer model induced by ethanol revertedthe EEGP gastroprotective activity (119875 lt 005) These resultssuggest that the EEGP beyond develops its gastroprotectiveactivity through modulation of nitric oxide and also acts byincreasing the levels of prostaglandins present in the gastricmucosa

Finally we evaluated the antisecretory activity of theEEGP Acetylcholine histamine and gastrin are endogenoussubstances responsible for the regulation of acid secretion[30] Currently antiulcer drugs act by blocking the acidsecretion for example omeprazole (proton pump blockers)and cimetidine (H

2inhibitor) [31]

The model of ligature of the pylorus of the stomach inmice is widely used to study the antisecretory activity ofnew drugs This fact is understandable due to the effectgenerated by ligation of the pylorus which is the acidshypersecretion on stomach [20] According to Table 1 it isobserved that the volume and pH of gastric juice remained

unaltered in the stomachs of animals submitted to bindingthe pylorus remained (119875 gt 005) after administration ofEEGP (300mgkg) On the other hand the positive control(cimetidine 100mgkg) not only decreased the gastric juicevolume but also increased the pH (119875 lt 005) These resultstherefore suggest that the gastroprotective activity of theEEGP is not related to the regulation of gastric secretion

4 Conclusion

The results of this study indicate that geopropolis exerts agastroprotective effect in rats with ethanol-induced gastricmucosal damage The results also suggest that the gastropro-tective effect of geopropolis could be related to the gastropro-tective mechanism in which nitric oxide and prostaglandinsare involved

Abbreviations

EEGP Ethanolic extract of geopropolisIndo IndomethacinNEM N-EthylmaleimideNP-SH Nonprotein sulfhydrylOMEP OmeprazoleL-NAME N120596-nitro-L-arginine methyl ester

hydrochloride

Conflict of Interests

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

Acknowledgments

The authors are grateful to Mr Jose Emıdio Borges de Souzafor providing the geopropolis samples This research wassupported by FAPESP (201020214-7)

References

[1] G Mozsik ldquoGastric cytoprotection 30 years after its discoveryby Andre Robert a personal perspectiverdquo Inflammopharmacol-ogy vol 18 no 5 pp 209ndash221 2010

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

[3] 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

[4] B B Silva P L Rosalen J A Cury et al ldquoChemical compositionand botanical origin of red propolis a new type of Brazil-ian propolisrdquo Evidence-Based Complementary and AlternativeMedicine vol 5 no 3 pp 313ndash316 2008

[5] S Castaldo and F Capasso ldquoPropolis an old remedy used inmodern medicinerdquo Fitoterapia vol 73 supplement 1 pp S1ndashS62002

[6] M K Choudhari S A Punekar R V Ranade and K MPaknikar ldquoAntimicrobial activity of stingless bee (Trigona sp)


propolis used in the folk medicine of Western MaharashtraIndiardquo Journal of Ethnopharmacology vol 141 no 1 pp 363ndash367 2012

[7] M C Bufalo I Ferreira G Costa et al ldquoPropolis and itsconstituent caffeic acid suppress LPS-stimulated pro-inflam-matory response by blocking NF-120581B and MAPK activation inmacrophagesrdquo Journal of Ethnopharmacology vol 149 no 1 pp84ndash92 2013

[8] R Markiewicz-Zukowska M H Borawska A FiedorowiczS K Naliwajko D Sawicka and H Car ldquoPropolis changesthe anticancer activity of temozolomide in U87MG humanglioblastoma cell linerdquo BMC Complementary amp AlternativeMedicine vol 13 article 50 9 pages 2013

[9] M Kakino H Izuta K Tsuruma et al ldquoLaxative effectsand mechanism of action of Brazilian green propolisrdquo BMCComplementary amp Alternative Medicine vol 12 article 192 8pages 2012

[10] M P de Barros J P B Sousa J K Bastos and S F deAndrade ldquoEffect of Brazilian green propolis on experimentalgastric ulcers in ratsrdquo Journal of Ethnopharmacology vol 110no 3 pp 567ndash571 2007

[11] G Nates-Parra ldquoLas Abejas sin aguijon (Hymenoptera ApidaeMeliponini) de Colombiardquo Biota Colombiana vol 2 pp 233ndash248 2001

[12] O M Barth ldquoPalynological analysis of geopropolis samplesobtained from six species of Meliponinae in the Campus of theUniversidade de Ribeirao Preto Brazilrdquo Apiacta vol 41 pp 71ndash85 2006

[13] M Franchin M G da Cunha C Denny et al ldquoGeopropolisfrom Melipona scutellaris decreases the mechanical inflamma-tory hypernociception by inhibiting the production of IL-1120573and TNF-120572rdquo Journal of Ethnopharmacology vol 143 no 2 pp709ndash715 2012

[14] M Franchin M G da Cunha C Denny et al ldquoBioactivefraction of geopropolis fromMelipona scutellaris decreases neu-trophils migration in the inflammatory process involvementof nitric oxide pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 907041 9 pages 2013

[15] M G da Cunha M Franchin L C C Galvao et al ldquoAntimi-crobial and antiproliferative activities of stingless beeMeliponascutellaris geopropolisrdquo BMC Complementary amp AlternativeMedicine vol 13 article 23 9 pages 2013

[16] M G da Cunha M Franchin L C C Galvao et al ldquoApolarbioactive fraction of melipona scutellaris geopropolis on Strep-tococcus mutans biofilmrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 256287 7 pages 2013

[17] Y Morimoto K Shimohara S Oshima and S TakayukildquoEffects of the new anti-ulcer agent KB-5492 on experimentalgastric mucosal lesions and gastric mucosal defensive factorsas compared to those of teprenone and cimetidinerdquo JapaneseJournal of Pharmacology vol 57 no 4 pp 495ndash505 1991

[18] M T Gamberini L A Skorupa C Souccar and A J LapaldquoInhibition of gastric secretion by a water extract from Baccha-ristriptera Martrdquo Memorias do Instituto Oswaldo Cruz vol 86supplement 2 pp 137ndash139 1991

[19] P C Dias M A Foglio A Possenti and J E de CarvalholdquoAntiulcerogenic activity of crude hydroalcoholic extract ofRosmarinus officinalis Lrdquo Journal of Ethnopharmacology vol69 no 1 pp 57ndash62 2000

[20] H Shay S A Komarov S S Fels D Meranze M Gruenteinand H Siplet ldquoA simple method for the uniform production of

gastric ulceration in the ratrdquo Gastroenterology vol 5 pp 43ndash611945

[21] C H Cho and C W Ogle ldquoThe pharmacological differencesand similarities between stress- and ethanol-induced gastricmucosal damagerdquo Life Sciences vol 51 no 24 pp 1833ndash18421992

[22] U N S Prakash and K Srinivasan ldquoGastrointestinal protec-tive effect of dietary spices during ethanol-induced oxidantstress in experimental ratsrdquo Applied Physiology Nutrition andMetabolism vol 35 no 2 pp 134ndash141 2010

[23] S Szabo J S Trier and P W Frankel ldquoSulfhydryl compoundsmay mediate gastric cytoprotectionrdquo Science vol 214 no 4517pp 200ndash202 1981

[24] S KatoMKitamura R P Korolkiewicz andK Takeuchi ldquoRoleof nitric oxide in regulation of gastric acid secretion in ratseffects of NOdonors andNO synthase inhibitorrdquoBritish Journalof Pharmacology vol 123 no 5 pp 839ndash846 1998

[25] C H Cho ldquoCurrent roles of nitric oxide in gastrointestinaldisordersrdquo Journal of Physiology vol 95 no 1ndash6 pp 253ndash2562001

[26] D Dal-Secco J A Paron S H P de Oliveira S H Ferreira J SSilva and F Q Cunha ldquoNeutrophil migration in inflammationnitric oxide inhibits rolling adhesion and induces apoptosisrdquoNitric Oxide Biology and Chemistry vol 9 no 3 pp 153ndash1642003

[27] F L Lanza ldquoEndoscopic studies of gastric and duodenal injuryafter the use of ibuprofen aspirin and other nonsteroidal anti-inflammatory agentsrdquoTheAmerican Journal ofMedicine vol 77no 1 pp 19ndash24 1984

[28] K Takeuchi ldquoPathogenesis of NSAID-induced gastric damageimportance of cyclooxygenase inhibition and gastric hyper-motilityrdquo World Journal of Gastroenterology vol 18 no 18 pp2147ndash2160 2012

[29] T Brzozowski P C Konturek S J Konturek I Brzozowskaand T Pawlik ldquoRole of prostaglandins in gastroprotection andgastric adaptationrdquo Journal of Physiology andPharmacology vol56 supplement 5 pp 33ndash55 2005

[30] M L Schubert ldquoGastric secretionrdquo Current Opinion in Gas-troenterology vol 27 no 6 pp 536ndash542 2011

[31] R la Corte M Caselli G Castellino G Bajocchi and F TrottaldquoProphylaxis and treatment of NSAID-induced gastroduodenaldisordersrdquo Drug Safety vol 20 no 6 pp 527ndash543 1999

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


acids commonly found in propolis from Apis mellifera andpresence of benzophenones [13ndash16]

Therefore in order to aggregate scientific value to thegeopropolis this study aims at evaluating the gastroprotectiveactivity of the ethanolic extract of geopropolis (EEGP) fromMelipona scutellaris and at investigating the possible mecha-nisms of action

2 Material and Methods

21 Obtaining the EEGP The geopropolis samples werecollected on municipality of Entre Rios (11∘571015840 S 38∘051015840 W)state of Bahia northeast of Brazil The geopropolis samples(100 g) were extracted in 80 ethanol in water (wv) at a 17dilution rate at 70∘C for 30min followed by filtration therebyobtaining the EEGPThe same process was repeated twice Atthe end the EEGP was concentrated in a rotary evaporator at40∘C [13]TheEEGPdissolutionwas carried out in PBS 1mMThe EEGP was administered to the animals by pathway oral(po)

22 Animals MaleWistar rats SPF (specific-pathogen-free)weighing 200ndash250 g were provided by CEMIBUNICAMP(Multidisciplinary Center for Biological Research SP Brazil)and kept in controlled temperature chambers (20 plusmn 2∘C)in light-dark 12 hours cycles relative humidity of 40 and60 with filtered water ad libitum The animals fasted for24 hours before the experiments The procedures describedwere reviewed and approved by the local Animal EthicsCommittee (CEUA Unicamp process number 2560-1)

23 Drugs and Reagents The drugs were purchased fromSigma Chemical Co St Louis MO USA (N-ethylmaleimideomeprazole N120596-nitro-L-arginine methyl ester hydrochlo-ride and ethanol) MP Biomedicals (indomethacin) andMerck (organic solvents)

24 Gastric Lesion Induced by Ethanol The rats were pre-treated with EEGP at dose of 100 200 or 300mgkg (po)The positive control group received omeprazole 30mgkg(po) and the negative control group received dissolvingvehicle of EEGP (po) One hour after the treatments 1mLof absolute ethanol was administered by po and one hourlater the animals were killed by anesthesia overload andthe stomach was removed and opened along the greatercurvature [17] The ulcerative lesions from each animal werecalculated according to the Gamberini et al [18] method asdescribed below

25 Role of the Nonprotein Sulfhydryl (NP-SH) Groups onGastroprotection Effect of the EEGP The rats were pretreatedwith inhibitor of the NP-SH groups (N-ethylmaleimide10mgkg sc) 30min prior to the treatment with EEGP(300mgkg) by po [19] The negative control group receiveddissolution vehicle of EEGP (po) One hour after the treat-ments 1mLof absolute ethanolwas administered by po andone hour later the animals were killed by anesthesia overloadand the stomach was removed and opened along the greater

curvature The ulcerative lesions from each animal werecalculated according to the Gamberini et al [18] method

26 Role of the Nitric Oxide on Gastroprotective Effect of theEEGP Therats were pretreatedwith inhibitor from the nitricoxide synthase (L-NAME 5mgkg) by intraperitoneal (ip)administration 30min prior to the treatment with EEGP(300mgkg po) The negative control group received disso-lution vehicle of EEGP (po) One hour after the treatments1mL of absolute ethanol was administered by po and onehour later the animals were killed by anesthesia overloadand the stomach was removed and opened along the greatercurvature [19] The ulcerative lesions from each animal werecalculated according to the Gamberini et al [18] method

27 Role of Prostaglandins on Gastroprotective Effect of theEEGP The rats were pretreated with the cyclooxygenaseinhibitor (indomethacin 5mgkg ip) 30min before theEEGP treatment (300mgkg po) The negative controlgroup received dissolution vehicle of EEGP (po) One hourafter the treatments 1mL of absolute ethanol was adminis-tered by po and one hour later the animals were killedby anesthesia overload and the stomach was removed andopened along the greater curvature [19]Theulcerative lesionsfromeach animalwere calculated according to theGamberiniet al [18] method

28 Evaluation of the Antisecretory Activity Theanimals wereanesthetized their abdomen was dissected and the pyloruswas connected Immediately after this procedure the EEGP300mgkg cimetidine 100mgkg or dissolution vehicle ofEEGP (negative control) was administered by po to therespective group of animals The abdomens were suturedand four hours later the animals were killed The stomachswere removed and the gastric content was collected andcentrifuged The volume and the pH of the gastric juice weremeasured [20]

29 Statistical Analysis Data were expressed as means plusmnstandard error of the mean (SEM) and statistical comparisonbetween groups was made utilizing analysis of variance(ANOVA) followed by test of Dunnett or Tukey Significancewas accepted when 119875 lt 005

3 Results and Discussion

This study evaluated the gastroprotective activity of EEGPfromMelipona scutellaris as well as the possible actionmech-anisms involved

The geopropolis used in this work presents the samechemical composition as described in our previous publica-tions where absence of flavonoids and phenolic acids andpresence of benzophenone were found [15 16]

Ethanol is a potent gastric ulcer inducer agent and itis widely applied to evaluate the gastroprotective activityof plant extracts as well as new pharmaceutical drugsin animal models Its effect causes an imbalance betweenthe oxidizing and the antioxidants agents in the gastric


C Omep 30 100 200 3000

10

20

30

40

50

(mgkg po)Treatments

EEGP

Ulc

erat

ive l

esio

n in

dex

lowastlowast






C NEM EEGP0

20

40

60

80

Treatments

Ulc

erat

ive l

esio

n in

dex

lowast

lowast

EEGP + NEM


C L-NAME EEGP0

20

40

60

80

100

Treatments

EEGP + L-NAME

Ulc

erat

ive l

esio

n in

dex

lowast

lowastlowast





C INDO EEGP0

20

40

60

80

100

Treatments

EEGP + INDO

Ulc

erat

ive l

esio

n in

dex

lowast

lowastlowast





64 plusmn 01lowast





2inhibitor) [31]



4 Conclusion


Abbreviations


hydrochloride



Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C Omep 30 100 200 3000

10

20

30

40

50

(mgkg po)Treatments

EEGP

Ulc

erat

ive l

esio

n in

dex

lowastlowast






C NEM EEGP0

20

40

60

80

Treatments

Ulc

erat

ive l

esio

n in

dex

lowast

lowast

EEGP + NEM


C L-NAME EEGP0

20

40

60

80

100

Treatments

EEGP + L-NAME

Ulc

erat

ive l

esio

n in

dex

lowast

lowastlowast





C INDO EEGP0

20

40

60

80

100

Treatments

EEGP + INDO

Ulc

erat

ive l

esio

n in

dex

lowast

lowastlowast





64 plusmn 01lowast





2inhibitor) [31]



4 Conclusion


Abbreviations


hydrochloride



Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C INDO EEGP0

20

40

60

80

100

Treatments

EEGP + INDO

Ulc

erat

ive l

esio

n in

dex

lowast

lowastlowast





64 plusmn 01lowast





2inhibitor) [31]



4 Conclusion


Abbreviations


hydrochloride



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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