Universidade Estadual Paulista Júlio de Mesquita Filho 
Instituto de Biociências 

Programa de Pós-graduação em Ciências Biológicas 
Área de Concentração Farmacologia 

Curso de Doutorado 
 

 

 

 

EFEITO DO (-)-MENTOL SOBRE A ÚLCERA GÁSTRICA:  
ESTUDO DO MECANISMO GASTROPROTETOR 

 
 
 
 

 
ARIANE LEITE ROZZA 

 
Orientadora: Profa. Dra. Cláudia Helena Pellizzon 

 

 

 

 

 

Botucatu/SP 
- 2013 – 



 1 

Universidade Estadual Paulista Júlio de Mesquita Filho 
Instituto de Biociências 

Programa de Pós-graduação em Ciências Biológicas 
Área de concentração Farmacologia 

Curso de Doutorado 
 
 
 
 

EFEITO DO (-)-MENTOL SOBRE A ÚLCERA GÁSTRICA:  
ESTUDO DO MECANISMO GASTROPROTETOR 

 
 
 
 
 

ARIANE LEITE ROZZA 
 

Orientadora: Profa. Dra. Cláudia Helena Pellizzon 
 
 
 
 
 
 
 
 
 
 
 
 

Botucatu/SP 
- 2013 – 

 



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FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉCNICA DE AQUISIÇÃO E TRATAMENTO DA INFORMAÇÃO 
DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CAMPUS DE BOTUCATU - UNESP 

BIBLIOTECÁRIA RESPONSÁVEL: ROSEMEIRE APARECIDA VICENTE 
Rozza, Ariane Leite. 
     Efeito do (-)-mentol sobre a úlcera gástrica : estudo do mecanismo 
gastroprotetor / Ariane Leite Rozza. – Botucatu : [s.n.], 2013 
      
     Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências 
de Botucatu 
     Orientador: Cláudia Helena Pellizzon 
     Capes: 21001006 
      

1. Sistema gastrointestinal – Úlceras.  2.  Antioxidantes.  3. Intestinos – 
Doenças.  4. Diarréia.  5. Menta (Planta) – Uso terapêutico.                                                                                                                                
                                               
 
                    
Palavras-chave: Anti-inflamatório; Antioxidante; Antissecretório; Mentol; 
Úlcera gástrica. 

 
 
 
 



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                                                                               Apoio Financeiro 
 

 
 

 
 

Auxílio 2010/08536-9 
 

 
 
 
 
 
 
 
 
 



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Epígrafe 

 

 

 

 

 

 

 

 

 

 

 

“Tenho a impressão de ser uma criança brincando à beira-mar, divertindo-me em descobrir 

uma pedrinha mais lisa ou uma concha mais bonita que as outras, enquanto o imenso oceano 

da verdade continua misterioso diante dos meus olhos...” 

 

                                                                                                        Isaac Newton 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 5 

Dedicatória 

 

 

 

 

Aos meus pais, Remigio e Hilda, e minha irmã Mirela, 

 

A minha orientadora Profa. Dra. Cláudia Helena Pellizzon. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

"Só o que sonhamos é o que verdadeiramente somos, 

porque o mais, por estar realizado, 

pertence ao mundo e a toda a gente."  

 

 



 6 

Agradecimentos 

 

A minha orientadora Profa. Dra. Cláudia Helena Pellizzon, que me ensinou grande 

parte do que aprendi durante a iniciação científica e a pós-graduação, e também pela amizade, 

paciência, ajuda em todas as horas, 

 

Aos membros da banca do exame de qualificação, Dr. Celso Acácio Rodrigues de 

Almeida Costa, Prof. Dr. Luis Fernando Barbisan, Profa. Dra. Clélia Akiko Hiruma-Lima, 

Prof. Dr. Carlos Alan Cândido Dias Júnior e Prof. Dr. André Sampaio Pupo, pela disposição 

em colaborar, pelas valiosas contribuições e, posso dizer, pela amizade, 

 

 Aos membros titulares e suplentes da banca de defesa da tese, Profa. Dra. 

Clélia Akiko Hiruma-Lima, Prof. Dr. Carlos Alan Cândido Dias Júnior, Prof. Dr, Jairo 

Kenupp Bastos, Profa. Dra. Caden Soucar, Prof. Dr. Luis Fernando Barbisan, Prof. Dr. Sérgio 

Brito Garcia e Profa. Dra. Elfriede Marianne Bacchi, por aceitarem nosso convite e pelas 

muitas contribuições que certamente virão,  

 

A Profa. Dra. Alba Regina Monteiro Souza-Brito e seus alunos Felipe Meira de-Faria 

e Eduardo Augusto Rabelo Socca, pelas colaborações e muitas risadas, 

 

Aos amigos Flávia Bonamin e Thiago de Mello Moraes, sempre fomos uma equipe e 

continuamos sendo, 

 

Aos amigos do laboratório, Daniela e Lucas, e os que por aqui passaram, Gustavo, 

Bianca, Jean e Paulinho, 

 

Aos professores do departamento de Morfologia, em especial ao Prof. Dr. Robson 

Francisco de Carvalho, com quem aprendi muito à época em que fui bolsista didática, 

 

Aos professores do departamento de Farmacologia, que também me ensinaram muito 

durante as disciplinas e espero que continuem ensinando,  

 

Aos funcionários do departamento de Morfologia, Luciana, Ricardo e José Eduardo, 

sempre dispostos a ajudar e com um bom humor que contagia, 



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Aos funcionários da seção de pós-graduação, pela ajuda e paciência de sempre,  

 

A CAPES, pela bolsa, e FAPESP, pelo auxílio financeiro. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

"Confiança - o senhor sabe - não se tira das coisas feitas ou perfeitas:  

ela rodeia é o quente da pessoa." Guimarães Rosa 

 



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Agradecimentos Pessoais 

 

Aos meus pais, minha irmã Mirela, vó Teresinha, pelo amor e pelo apoio de sempre, e 

também a minha pequena Paquita, remédio para o estresse em forma de bolinha de pelo, 

 

Aos meus tios e primos, e os priminhos também, 

 

A amiga Larissa Aku, companhia desde 2006, a irmã que eu escolhi, que agora foi 

montar uma filial da nossa casa (vila do chaves) no Reino Unido, 

 

Aos amigos que fazem a vida mais divertida, Mariana Fragoso, Rodrigo Aero, Flavia 

Kalose, Celso Saks, Hélio Ponpon, Monize, Ana Júlia, Caroline Iaiz, Alessandra Carneiro, 

Felipe de-Faria, Marília Carneiro, Bruno Põe, Juliana Akinaga, Fernanda Nojimoto, Vanessa, 

Leandro Chico, Fabrício Dalva, Daniela Maracutaia, Lucas Zaraga, Jean Monje, entre todos 

os outros, 

 

Aos que foram meus alunos e hoje são meus amigos, e são muitos,  

 

Aos demais amigos da pós-graduação e do departamento, pelas ajudas nas técnicas, 

pela companhia e pelas risadas, 

 

A todos os amigos que podem não estar com o nome escrito nessa página, mas estão 

com o nome no meu coração e na minha história, 

 

Aos amigos do centro cristão espírita Caminho da verdade, 

 

A Deus, mestre, professor doutor. 

 

 

 

 

"Em um mundo de peregrinos, quando os caminhos se cruzam, o mundo parece um lar 

durante algum tempo." Herman Hesse 

 



 9 

ÍNDICE 

 

1. PRÓLOGO……………………………………………………………………1 

2. RESUMO……………………………………………………………………...8 

3. INTRODUÇÃO……………………………………………………………….9 

4. OBJETIVOS………………………………………………………………….33 

5. CAPÍTULO  1  

Effect of menthol in experimentally induced ulcers: pathways of 

gastroprotection……………………………………………………………….35 

6. CAPÍTULO 2  

The gastroprotective effect of menthol: involvement of anti-apoptotic, antioxidant and 

anti-inflammatory activities………………………………….64 

7. CONCLUSÕES……………………………………………………………….87 

 

 

 

 

 

 

 

 
 

 
 



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Prólogo 

 

 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



 11 

Atividades realizadas durante o curso de Doutorado (2009-2013) em Ciências Biológicas, 

área de concentração Farmacologia 

 

Disciplinas cursadas 

1. Estratégias digitais para ensino/aprendizagem 

2. Matriz extracelular animal 

3. Tópicos de atualização em Ciências 

4. Farmacologia do sistema nervoso autônomo simpático 

5. Empreendedorismo 

6. Estresse térmico e reprodução animal 

7. Farmacologia avançada 

8. Classificação de receptores farmacológicos 

9. Mecanismos de controle e adaptação do trato gastrointestinal 

10. Métodos de validação de novos fármacos e tópicos de atualização em plantas 

medicinais 

 

Atividades de Ensino 

02/2013 – 07/2013      Professora Bolsista da disciplina Farmacologia 

02/2011 - 07/2011 Professora Bolsista da disciplina Histologia nos cursos de Biologia e 

Medicina 

08/2010 - 12/2010 Estágio didático na disciplina Histologia no curso de Biomedicina 

02/2010 - 07/2010 Estágio docência na disciplina Biologia Celular e Tecidual no curso de 

Física Médica 

 

Artigos completos publicados em periódicos 

1. Rozza AL, Hiruma-Lima CA, Tanimoto A, Pellizzon CH. Morphologic and 

pharmacological investigations in the epicatechin gastroprotective effect. Evidence-Based 

Complementary and Alternative Medicine, v. 2012, p. 1 - 8, 2012. 

 



 12 

2. de-Faria FM, Almeida ACA, Luiz-Ferreira A, Dunder RJ, Takayama C, da Silva MS, 

Silva MA, Vilegas W, Rozza AL, Pellizzon CH, Toma W, Souza-Brito ARM. Mechanisms 

of action underlying the gastric antiulcer activity of the Rhizophora mangle L. Journal of 

Ethnopharmacology, v. 139, p. 234 - 243, 2012. 

3. de Faria FM, Luiz-Ferreira A, Socca EAR, de Almeida ACA, Dunder RJ, Manzo LP, 

Silva MA, Vilegas W, Rozza AL, Pellizzon CH, dos Santos LC, Souza Brito ARM. Effects 

of Rhizophora mangle on experimental colitis induced by TNBS in rats. Evidence-Based 

Complementary and Alternative Medicine, v. 2012, p. 1 - 11, 2012. 

4. Rozza AL, Pellizzon CH. Essential oils from medicinal and aromatic plants: a review 

of the gastroprotective and ulcer-healing activities. Fundamental & Clinical Pharmacology, v. 

27(1), p. 51 – 63, 2012. 

5. Rozza AL, Moraes TM, Kushima H, Tanimoto A, Marques MOM, Bauab TM, 

Hiruma-Lima CA, Pellizzon CH. Gastroprotective mechanisms of Citrus lemon (Rutaceae) 

essential oil and its majority compounds limonene and ß-pinene: Involvement of heat-shock 

protein-70, vasoactive intestinal peptide, glutathione, sulfhydryl compounds, nitric oxide and 

prostaglandin E2. Chemico-Biological Interactions, v. 189, p. 82 - 89, 2011. 

6. Takayama C, de-Faria FM, de Almeida ACA, Valim-Araújo DAO, Rehen CS, Dunder 

RJ, Socca EAR, Manzo LP, Rozza AL, Salvador MJ, Pellizzon CH, Hiruma-Lima CA, Luiz-

Ferreira A, Souza-Brito ARM. Gastroprotective and ulcer healing effects of essential oil from 

Hyptis spicigera Lam. (Lamiaceae). Journal of Ethnopharmacology, v. 135, p. 147 - 155, 

2011. 

7. Rozza AL, Moraes TM, Kushima H, Nunes DS, Hiruma-Lima CA, Pellizzon CH. 

Involvement of glutathione, sulfhydryl compounds, nitric oxide, vasoactive intestinal peptide, 

and heat-shock protein-70 in the gastroprotective mechanism of Croton cajucara Benth. 

(Euphorbiaceae) essential oil. Journal of Medicinal Food, v. 14, p. 1011 - 1017, 2011. 

8. Bonamin F, Moraes TM, Kushima H, Silva MA, Rozza AL, Pellizzon CH, Bauab 

TM, Rocha LRM, Vilegas W, Hiruma-Lima CA. Can a Strychnos species be used as antiulcer 

agent? Ulcer healing action from alkaloid fraction of Strychnos pseudoquina St. Hil. 

(Loganiaceae). Journal of Ethnopharmacology, v. 138(1), p. 47 - 52, 2011. 

9. Lima ZP, Bonamin F, Calvo TR, Vilegas W, Santos LC, Rozza AL, Pellizzon CH, 

Rocha LRM, Hiruma-Lima CA. Effects of the ethyl acetate fraction of Alchornea triplinervia 

on healing gastric ulcer in rats. Pharmaceuticals, v. 4, p. 1423 - 1433, 2011. 



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Capítulos de livros publicados 

1. Rozza AL, Ortiz MOM, Hiruma-Lima CA, Pellizzon CH. Antiulcerogenic activity of 

Citrus lemon essential oil on acetic acid-induced gastric ulcer in rats. In: Medicinal Plants - 

Phytochemistry, Pharmacology and Therapeutics, New Delhi, India: Daya Publishing House, 

v. 2, p. 53-67, 2012. 

2. Rozza AL, Pellizzon CH. Pharmacological effects of menthol in the gastrointestinal 

diseases in clinical and experimental assays. In: Recent Progress in Medicinal Plants, 

Houston, Texas: Studium Press LCC, v. 34, p. 1-13, 2012. 

3. Pellizzon CH, Rozza AL, Vasconcelos PCP, Andreo MA, Vilegas W, Hiruma-Lima 

CA. Non-commercial plants of medicinal purposes from the Brazilian biomes for the 

treatment of gastrointestinal diseases. In: Recent Progress in Medicinal Plants - Drug Plants, 

Houston, Texas: Studium Press LLC, v. 29, p. 217-236, 2010. 

 

Trabalhos publicados em anais de eventos  

1.        Rozza AL, De-Faria FM, Souza-Brito ARM, Pellizzon CH. Role of PGE2 in the (-)-

menthol gastroprotective activity In: 5th International Conference in Drug Discovery and 

Therapy, 2013, Dubai, Emirados Árabes Unidos. 

2.  Cesar DAS, Rozza AL, De-Faria FM, Souza-Brito ARM, Saldanha LL, Dokkedal 

AL, Pellizzon CH. Avaliação das atividades gastroprotetora, antioxidante e antidiarreica do 

extrato hidroalcoólico de Myrcia bella In: 15o Encontro Nacional de Biomedicina, 2012, 

Botucatu/SP. 

3.  Cesar DAS, Rozza AL, Pellizzon CH. Avaliação do efeito gastroprotetor e toxicidade 

do extrato bruto hidroalcoólico de Bauhinia holophylla In: XXXIV CIC - Congresso de 

Iniciação Científica da UNESP, 2012, Botucatu/SP. 

4.  Rozza AL, Cesar DAS, Pellizzon CH. Gastroprotective and anti-diarrheic activities of 

menthol In: 6th European Congress of Pharmacology EPHAR2012, 2012, Granada, Espanha. 

5.  Rozza AL, Pellizzon CH. Menthol: elucidating its gastroprotective mechanisms of 

action In: 6th European Congress of Pharmacology EPHAR2012, 2012, Granada, Espanha. 



 14 

6.  Rozza AL, Pellizzon CH. Evaluation of gastric juice parameters and mucus 

production in the menthol gastroprotective effect In: XX Congresso Italo-Latinoamericano de 

Etnomedicina 'Prof Dr Francisco de Abreu Matos', 2011, Fortaleza-CE. 

7.  Rozza AL, Tanimoto A, Pellizzon CH. Evaluation of gastric juice parameters and 

role of alpha-2 receptors, K+
ATP channels, nitric oxide and sulfhydryl compounds in the 

gastroprotection of epicatechin In: XX Congresso Italo-Latinoamericano de Etnomedicina 

'Prof Dr Francisco de Abreu Matos', 2011, Fortaleza-CE. 

8.  Tanimoto A, Rozza AL, Gregório BB, Moraes TM, Kushima H, Hiruma-Lima CA, 

Pellizzon CH. Role of alpha-2 adrenoceptors and sulfhydryl groupments on gastroprotective 

and antiulcerogenic action of epicatechin In: XXVI Reunião Anual da Federação de 

Sociedades de Biologia Experimental - FeSBE 2011, 2011, Rio de Janeiro. 

9.  Rozza AL, Pellizzon CH. The gastroprotective mechanism of menthol is involved 

with sulfhydryl compounds and K+
ATP channels In: 8th International Congress of 

Pharmaceutical Sciences CIFARP, 2011, Ribeirão Preto. 

10.  Tanimoto A, Pellizzon CH, Gregório BB, Rozza AL, Seito LN, Di Stasi LC, Hiruma-

Lima CA. Atividade antioxidante da epicatequina na prevenção de peroxidação lipídica em 

membrana de cérebro de ratos In: XIV Semana da Bio, 2010, Botucatu. 

11.  Tanimoto A, Rozza AL, Gregório BB, Moraes TM, Kushima H, Hiruma-Lima CA, 

Pellizzon CH. Epicatechin stimulates gastric mucus production and protects the stomach 

against ethanol and indomethacin In: 11th Congress of the International Society of 

Ethnopharmacology - ISE 2010, 2010, Albacete, Espanha. 

12. Tanimoto A, Rozza AL, Gregório BB, Moraes TM, Kushima H, Hiruma-Lima CA, 

Pellizzon CH. Gastroprotective effect of epicatechin in ethanol-induced gastric ulcer by 

stimulation of mucus secretion and nitric oxide production In: XXV Reunião Anual da 

FeSBE, 2010, Águas de Lindóia. 

13.  Rozza AL, Tanimoto A, Floriano Junior PR, Hiruma-Lima CA, Pellizzon CH. 

Participação dos compostos sulfidrílicos e do óxido nítrico no efeito gastroprotetor do mentol 

frente a úlceras gástricas induzidas por etanol absoluto In: XXV Reunião Anual da FeSBE, 

2010, Águas de Lindóia. 

14.  Marques JF, Rozza AL, Pellizzon CH. Análise morfológica de colite ulcerativa 

experimental In: XIII Semana da Bio, 2009, Botucatu. 



 15 

15.  Rozza AL, Cardoso FF, Moraes TM, Nunes DS, Hiruma-Lima CA, Pellizzon CH. 

Avaliação da participação dos compostos sulfidrílicos e do óxido nítrico no efeito 

gastroprotetor do óleo essencial de Croton cajucara In: XXIV Reunião Anual da Federação 

de Sociedades de Biologia Experimental - FeSBE 2009, 2009, Águas de Lindóia. 

16.  Tanimoto A, Gregório BB, Rozza AL, Hiruma-Lima CA, Pellizzon CH. 

Caracterização microscópica de ulcerações gástricas causadas por etanol absoluto e efeito 

gastroprotetor da epicatequina contra este agente lesivo In: XIII Semana da Bio, 2009, 

Botucatu. 

17.  Tanimoto A, Rozza AL, Gregório BB, Hiruma-Lima CA, Pellizzon CH. 

Epicatequina: ação gastroprotetora frente a lesão gástrica por etanol absoluto em ratos In: 

XXIV Reunião Anual da Federação de Sociedades de Biologia Experimental - FeSBE 2009, 

2009, Águas de Lindóia. 

18.  Tanimoto A, Gregório BB, Rozza AL, Hiruma-Lima CA, Pellizzon CH. Role of 

epicatechin in gastroprotection against absolute ethanol in rats pretreated with L-NAME or 

NEM In: V Encontro da Pós Graduação da FMB, 2009, Botucatu. 

19.  De-Faria FM, Luiz-Ferreira A, Almeida ACA, Barbastefano V, Silva MA, Vilegas W, 

Rozza AL, Pellizzon CH, Souza-Brito ARM. Role of SOD in the protection of Rhizophora 

mangle on gastric injury induced by ethanol, ischemia-reperfusion and acetic acid in rats In: 

57th International Congress and Annual Meeting of the Society for Medicinal Plant and 

Natural Product Research, 2009, Genebra, Suíça. 

 

 

 

 

Prêmios e títulos 

2011 Honra ao Mérito, XXVI Reunião Anual da FeSBE  

2010 Honra ao Mérito, XXV Reunião Anual da FeSBE  

 

Participação em Eventos 

1. 6th European Congress of Pharmacology EPHAR2012.  



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2. II Simpósio de Farmacologia da UNESP, 2012 

3. 8th International Congress of Pharmaceutical Sciences CIFARP, 2011  

4. XX Congresso Italo-Latinoamericano de Etnomedicina 'Prof Dr Francisco de Abreu 

Matos' - SILAE 2011 

5. I Simpósio de Farmacologia da UNESP, 2011 

6. 14o Encontro Nacional de Biomedicina, 2011  

7. XXV Reunião Anual da FeSBE, 2010 

 

Curso de curta duração ministrado 

1.  Rozza AL, Marques JF. Dificuldades e soluções para a técnica de Ácido Periódico - 

Schiff (PAS), 2010. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



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Introdução 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



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1. ÚLCERA GÁSTRICA 

O estômago é um reservatório ativo que armazena, tritura e dispensa o alimento 

parcialmente digerido para o intestino para posterior digestão e absorção. Uma de suas 

principais funções é a secreção do ácido clorídrico (HCl), que tem a função de facilitar a 

digestão de proteínas através da conversão de pepsinogênio na enzima proteolítica pepsina. O 

ácido também facilita a absorção de ferro, cálcio, vitamina B12 e de alguns medicamentos, 

além de prevenir crescimento bacteriano e infecções entéricas (Choi et al., 2011; Pali-Scholl 

& Jensen-Jarolim, 2011).  

O estômago é dividido em quatro regiões anatômicas (cárdia, fundo, corpo e piloro). 

Sua unidade básica funcional é a glândula gástrica, que se divide em três regiões: istmo, colo 

e base. Células-tronco e células mucosas em diferenciação estão localizadas no istmo, a partir 

de onde tais células migram e se diferenciam em célula parietal, mucosa do colo, zimogênica 

ou enteroendócrina. As células dominantes são as células parietais (secretoras de ácido) e 

células enteroendócrinas (secretoras de histamina). Entre os outros tipos celulares, encontram-

se as células enterocromafins (produtoras de peptídeo natriurético atrial, serotonina e 

adrenomedulina) e células D (produtoras de somatostatina). A característica distintiva da 

mucosa pilórica é a célula G (produtora de gastrina) (Chu & Schuber, 2012). 

A mucosa da região do corpo gástrico e, em menor extensão, do fundo e antro 

gástricos, contem as células parietais. A principal função das células parietais é produzir o 

acido clorídrico (HCl), que ativa a pepsina a partir do pepsinogênio, atua na digestão de 

proteínas e reduz a colonização bacteriana no estômago e intestino. As células parietais 

contém a bomba de prótons, ou H+/K+ATPase, que transporta H+ para o lúmen gástrico e K+ 

do lúmen para o interior da célula. Por causa da grande quantidade de energia necessária para 

a manutenção da bomba de prótons, a célula parietal contém a maior quantidade de 

mitocôndrias entre todas as células do corpo humano (Mercer & Robinson, 2008). Na célula 

parietal em descanso, as bombas estão contidas em vesículas intracelulares (Urushidani & 

Forte, 1997). Há um nível basal constante da produção de ácido clorídrico, mesmo quando a 

célula está em repouso, que corresponde a 10% da produção ácida da célula estimulada. No 

estado estimulado, as vesículas se fundem com a membrana apical da célula parietal, 

relocando as bombas protônicas para a superfície que está em contato com o lúmen gástrico 

(Urushidani & Forte, 1997; Mercer & Robinson, 2008). A representação esquemática da 

secreção ácida pela célula parietal está contida na figura 1. 



 19 

 
Figura 1: Ilustração esquemática da secreção do ácido clorídrico (HCl) pela célula 

parietal gástrica. A secreção envolve uma bomba de prótons (P), um carregador de simporte 

para K+ e Cl- e um antiporte para íons Cl- e HCO3- (modificado de Rang et al., 2011). 

 

Há três tipos de receptores que, quando ativados, estimulam a secreção acida nas 

células parietais: o receptor muscarínico (M3), o receptor de colecistoquinina do tipo B 

(CCKB) e o receptor de histamina (H2) (Urushidani & Forte, 1997). Os três receptores estão 

localizados na membrana basolateral da célula parietal. O receptor M3 é estimulado pela 

acetilcolina, que é liberada através do estímulo da inervação parassimpática (Mercer & 

Robinson, 2008). O receptor CCKB é estimulado pela gastrina, produzida pelas células G, 

localizadas na região do antro gástrico (Mulholland, 2006; Mercer & Robinson, 2008). O 

receptor H2 é estimulado pela histamina, que é produzida pelas células enteroendócrinas, e 

sua liberação é estimulada pela gastrina e acetilcolina. O resultado final da ativação desses 

três receptores é a ativação da bomba protônica. (Mercer & Robinson, 2008). 

O efeito inibitório na secreção de ácido gástrico é ativado pela somatostatina, que é 

produzida pelas células D presentes na mucosa fúndica e antral. A acetilcolina tem efeito 

inibitório sobre a liberação de somatostatina. A somatostatina inibe a secreção ácida atuando 

diretamente sobre a célula parietal, mas também indiretamente através de inibir a liberação de 

gastrina e histamina (Mercer & Robinson, 2008). A célula parietal contém outro receptor 



 20 

inibitório da secreção para a PGE2, que inibe a produção ácida através da inibição da secreção 

de gastrina e estimula a secreção de somatostatina (Mulholland, 2006). 

O trato gastrointestinal é um ambiente dinâmico que permite a entrada de nutrientes 

enquanto restringe a entrada de antígenos, toxinas e microrganismos, e responde a agentes 

luminais potencialmente prejudiciais, num processo de defesa da mucosa. A mucosa gástrica 

está sob constante ataque por fatores endógenos ou exógenos que induzem dano ao tecido 

gástrico, mas tais fatores são contrabalanceados por fatores gastroprotetores endógenos que 

mantém a integridade da mucosa (Wallace & Devchand, 2005). 

Úlcera gástrica é uma erosão profunda na parede gástrica que penetra em toda a 

espessura da mucosa (Tarnawski, 2005). É uma doença comum de ocorrência global, com 

crescente incidência e prevalência, apesar de recentes avanços no campo médico. 

Anualmente, 14 milhões de pessoas apresentam úlceras, no mundo todo (Maity & 

Chattopadhyay, 2008). 

O desenvolvimento das úlceras gástricas decorre do desequilíbrio entre fatores 

agressores à mucosa e a habilidade da mucosa em defender-se e curar-se (Repetto & Llesuy, 

2002; Lu & Graham, 2006). Entre os fatores potencialmente agressores, incluem-se fatores 

endógenos (como secreção elevada de histamina e ácido clorídrico, bile, isquemia, 

leucotrienos, citocinas pró-inflamatórias, ativação de neutrófilos, espécies reativas de 

oxigênio e proteínas pró-apoptóticas) e fatores exógenos, como estresse (Cho et al., 1992), 

uso de drogas anti-inflamatórias não esteroidais (DAINEs) (Hawkey, 2000), álcool e 

colonização pela bactéria gram-negativa Helicobacter pylori (Peckenpaugh, 1997). 

 

2. GASTROPROTEÇÃO 

O estômago pode se defender de injúrias causadas por uma variedade de agentes 

irritantes e nocivos devido à ativação de várias linhas de defesa, no processo de defesa da 

mucosa (Brzozowski et al., 2005). Vários fatores permitem a mucosa permanecer intacta 

apesar de sua freqüente exposição à substâncias com larga variação de temperatura, pH e 

osmolaridade, assim como substâncias com ação citotóxica e produtos bacterianos capazes de 

causar reações inflamatórias (Wallace & Granger, 1996). Esse processo de defesa inclui 

mecanismos locais e neurohormonais. Entre os mecanismos locais, os mais importantes 

incluem a barreira de muco e bicarbonato, as células epiteliais de superfície e sua alta taxa de 

regeneração, adequada microcirculação no estômago, o fluxo alcalino, a geração contínua de 

prostaglandinas E2 e I2 (PGE2 e PGI2), a liberação de óxido nítrico (NO), a manutenção dos 

compostos sulfidrílicos não-proteicos (NP-SH), a atuação de enzimas antioxidantes e a 



 21 

supressão do processo inflamatório (Tarnawski et al., 2001; Brzozowski et al., 2005; Laine et 

al., 2008). 

A camada formada por muco, bicarbonato e fosfolipídeos surfactantes constitui a 

primeira linha de defesa da mucosa (Allen & Flemströn, 2005). O muco é secretado pelas 

células epiteliais secretoras de muco e células mucosas do colo e seus principais constituintes 

são glicoproteínas, mucinas e água (Phillipson et al., 2008). Sua secreção é estimulada pelos 

hormônios gastrointestinais, incluindo gastrina e secretina, acetilcolina e também pela PGE2, 

que aumenta a viscosidade e o conteúdo de glicoproteínas no muco (Allen & Flemströn, 

2005).  

Substâncias ulcerogênicas dissipam o muco e a camada de fosfolipídeos, levando a 

redifusão de ácido clorídrico, causando injúria na mucosa (Darling et al., 2004). A camada de 

muco retém os íons bicarbonato (HCO3-) secretados pelas células epiteliais, mantendo um 

microambiente de pH neutro na superfície das células e prevenindo a penetração da enzima 

pepsina e consequente proteólise do epitélio (Tarnawski et al., 2001; Valcheva-Kuzmanova et 

al., 2005). O muco também atua como lubrificante para reduzir o dano físico ao epitélio 

causado pelos materiais ingeridos e diminui a possibilidade de que bactérias tenham contato 

com o epitélio. A camada de muco é a única barreira pré-epitelial entre o lúmen e o epitélio. 

Quando ela está prejudicada, a próxima série de mecanismos protetores entra em ação 

(Wallace, 2008). 

A próxima linha de defesa é formada por uma camada contínua de células epiteliais 

que, além da secreção de muco e bicarbonato, produzem prostaglandinas e proteínas heat-

shock (HSPs). Devido à presença dos fosfolipídios na superfície, essas células são 

hidrofóbicas, repelindo agentes danosos solúveis em ácido ou água. Interconectadas por 

junções celulares, formam uma barreira que previne a redifusão de ácido e pepsina 

(Tarnawski, 1998; Allen & Flemströn, 2005). As células parietais, que secretam ácido 

clorídrico no lúmen da glândula gástrica, simultaneamente secretam íons bicarbonato no 

interstício e lúmen dos capilares sanguíneos adjacentes através de sua membrana basolateral. 

Esse processo, chamado fluxo alcalino, neutraliza qualquer íon H+ que atinja a membrana 

basolateral e possa causar injúria na mucosa (Silen, 1993).  

As HSPs são produzidas por essas células em resposta ao estresse celular, como 

aumento de temperatura, estresse oxidativo e agentes citotóxicos, prevenindo a denaturação 

de proteínas e consequentemente protegendo a célula de injúrias. As principais HSPs que 

atuam no estômago são a HSP-70 e a HSP-90 (Ishihara et al., 2011).  



 22 

A contínua geração de PGE2 e PGI2 também é crucial para manutenção da integridade 

da mucosa e proteção contra agentes ulcerogênicos. Quase todos os mecanismos de defesa são 

estimulados ou facilitados pelas prostaglandinas. Elas inibem a secreção ácida, estimulam 

produção de muco e bicarbonato, estimulam o fluxo sanguíneo, aumentam a concentração de 

compostos sulfidrílicos não-proteicos (NP-SH) na mucosa e aceleram a regeneração epitelial e 

cicatrização da mucosa através do estímulo à proliferação celular (Brzozowski et al., 2005). 

Robert et al. (1979) concluíram que a prostaglandina aplicada exogenamente previne danos a 

mucosa gástrica induzidos por diferentes agentes, como etanol, ácido (HCl), base (NaOH), 

solução hiperosmótica (NaCl hipertônico), bile concentrada e até água fervente. A partir dessa 

descoberta, Robert et al. criaram a expressão citoproteção, definida como a habilidade do 

agente farmacológico de prevenir injúria a mucosa gástrica ou duodenal causada por uma 

variedade de agentes ulcerogênicos (Robert et al., 1979; Robert et al., 1983). 

A adequada microcirculação na mucosa também é essencial para entrega de oxigênio e 

nutrientes e remoção de substâncias tóxicas. Quando a mucosa gástrica é exposta a um agente 

lesivo, há rápido aumento no fluxo sanguíneo, que permite remover ou diluir a concentração 

do agente lesivo. Essa resposta parece ser essencial para a defesa da mucosa, já que a restrição 

mecânica do fluxo sanguíneo leva a necrose hemorrágica (Laine et al., 2008). O aumento no 

fluxo sanguíneo na mucosa é mediado, pelo menos em parte, pelo NO, que é gerado pela 

enzima NO-sintase. A liberação de acetilcolina e de histamina, além de estimular a secreção 

ácida, tem efeitos vasodilatadores sobre a mucosa gástrica (Peskar, 2001; Holzer, 2006). 

Os compostos NP-SH estimulam a formação de muco, ligam-se as espécies reativas de 

oxigênio (que estão envolvidas no dano tecidual) e ainda participam da síntese de proteínas 

(Banerjee et al., 2008). Agentes ulcerogênicos, como por exemplo o etanol, levam à queda na 

concentração de compostos NP-SH, que são neutralizados quando se ligam aos radicais livres 

produzidos pelo agente lesivo (Reyes-Trejo et al., 2008). 

 



 23 

 

 
 

Figura 2: Representação esquemática de alguns dos processos de defesa da mucosa 

gástrica (modificado de Laine et al., 2008). 

 

O contato do agente ulcerogênico com a mucosa do estômago induz um processo 

inflamatório local. A ativação dos neutrófilos que decorre da inflamação é uma das principais 

fontes de espécies reativas de oxigênio (ROS) (Kwiecien et al., 2002). Além disso, os 

neutrófilos produzem mieloperoxidase (MPO), enzima que libera radicais livres que 

danificam a mucosa, contribuindo para o desenvolvimento e manutenção da úlcera gástrica 

(Potrich et al., 2010). Para proteger os tecidos de injúrias causadas pelos radicais livres, as 

células contem enzimas antioxidantes, incluindo a glutationa peroxidase (GSH-Px), catalase, 

superóxido dismutase (SOD), glutationa redutase (GR) e o composto sulfidrílico glutationa 

(GSH).  

A primeira enzima antioxidante a ser ativada na mucosa gástrica é a SOD, que catalisa 

a dismutação do ânion superóxido O2- em H2O2, que é menos nociva. O passo seguinte no 

metabolismo da H2O2 envolve a atividade da GSH-Px. A redução de H2O2 em H2O pela 

GSH-Px é acompanhada pela conversão da glutationa em sua forma reduzida (GSH) para sua 

forma oxidada (GSSH), que é convertida novamente a GSH pela atividade da enzima GR, 

usando NADPH como agente redutor (Kwicien et al., 2002). Compostos com ação 



 24 

antioxidante podem bloquear a apoptose que levaria à necrose do tecido na região da úlcera 

gástrica (Amira et al., 2012). 

 

  
Figura 3: Representação da formação de espécies reativas de oxigênio no organismo. 

SOD: superóxido dismutase, CAT: catalase, GPx: glutationa peroxidase, GSH: glutationa, 

GSSG: glutationa na forma oxidada (retirado de Kwicien et al., 2002). 

 

A defesa da mucosa gástrica é regulada também por fatores hormonais. Vários 

peptídeos, como gastrina, colecistoquinina e leptina tem função gastroprotetora através de 

variados mecanismo, como o estímulo da proliferação das células epiteliais (Peterson et al., 

2010; Tanigawa et al., 2010).  

Várias pesquisas tem mostrado o envolvimento da motilidade gastrointestinal no 

processo de defesa da mucosa. A motilidade normal ajuda a manter o fluxo sanguíneo regular, 

contribuindo no processo de gastroproteção (Tanaka et al., 2001). 

 

3. PRINCIPAIS FATORES CAUSADORES DE ÚLCERA GÁSTRICA 

O uso de DAINEs e a colonização por H. pylori no estômago são atualmente os 

principais fatores responsáveis pelo desenvolvimento de úlceras gástricas. DAINEs são os 

medicamentos mais utilizados nos Estados Unidos. Onze por cento da população faz uso 

regular de DAINE e um número muito maior faz uso intermitente dessa classe de 

medicamento (Paulose-Ram et al., 2005). Estudos comprovam que, após 10 minutos da 

ingestão de um comprimido contendo 650 mg de aspirina, 25% das células epiteliais 

apresentam danos quando observadas ao microscópio óptico e eletrônico (Baskin et al., 1976). 

Se analisado sob endoscopia, o estômago apresenta hemorragias subepiteliais dentro de 15 



 25 

minutos após a ingestão do comprimido em 100% dos pacientes (Laine, 1996). Cinco por 

cento desses pacientes apresentam complicações severas no trato gastrointestinal, como 

perfurações e hemorragias (Yuan et al., 2006). 

DAINEs são ácidos fracos, que se ligam a mucosa gástrica causando injúria local. O 

principal mecanismo de ação é a inibição sistêmica da ciclooxigenase (COX), uma enzima da 

via de produção das prostaglandinas a partir do ácido aracdônico. Existem duas isoformas, 

COX-1 e COX-2; DAINEs não seletivos bloqueiam as duas isoformas. A atuação dessas 

enzimas resulta na produção de PGE2, que protege a mucosa através da inibição da secreção 

ácida, estímulo na produção de muco e bicarbonato e aumento do fluxo sanguíneo (Ramsay & 

Carr, 2011), além de ter efeito trófico na mucosa, estimulando a proliferação celular (Pai et 

al., 2002). Estudos que utilizaram inibidores seletivos para COX-1 ou COX-2 evidenciaram 

que o efeito ulcerogênico das DAINEs decorre da inibição de ambas as isoformas (Takeuchi, 

2012). A sequência de eventos que induz a formação de úlcera após administração de DAINE 

é a inibição da síntese de prostaglandina, indução da hipermotilidade gástrica, seguida por 

distúrbios microvasculares e ativação de neutrófilos (Laine et al., 2008). 

Helicobacter pylori é uma bactéria gram-negativa flagelada que coloniza o estômago. 

A colônia de bactérias produz a enzima urease, que converte uréia em amônia (NH3) e 

dióxido de carbono (CO2), criando um microambiente que permite a sobrevivência das 

bactérias no baixo pH do estômago. A colonização pela bactéria H. pylori no estomago induz 

a formação de úlceras na mucosa através de diversos mecanismos. A colônia bacteriana 

produz enzimas que dissolvem a barreira de muco e também induz uma queda na população 

de células D, que baixa a produção de somatostatina e consequentemente aumenta a produção 

de gastrina e de ácido clorídrico (Ramsay & Carr, 2011). Mais de 50% da população mundial 

está infectada pela H. pylori, e essa infecção pode ser assintomática ou evoluir para gastrite, 

úlcera gástrica, úlcera duodenal, linfoma e adenocarcinoma. A erradicação da colônia de 

bactérias reestabelece a população de células D, resultando em queda na produção de ácido 

(Ramsay & Carr, 2011). O tratamento para erradicação consiste na chamada terapia tripla, que 

combina um inibidor de bomba protônica com os antibióticos claritromicina e amoxicilina. O 

sucesso dessa terapia está em torno de 80% (Calvet et al., 2000), mas ainda há a necessidade 

de uma terapia mais simples, que melhore o desconforto para o paciente, apresente custo 

reduzido e aumente a taxa de erradicação da bactéria (Yuan et al., 2006). À época de seu 

lançamento, tal terapia apresentava taxas de erradicação próximas a 90%, mas atualmente tem 

vivenciado um declínio, estando próxima a 70% (Paoluzi et al., 2010). Há várias razões que 



 26 

justificam tal perda de eficácia, mas a principal delas é a crescente resistência da H. pylori aos 

antibióticos (Selgrad & Malfertheiner, 2011). 

Antes da introdução do tratamento para erradicação da bactéria H. pylori no estômago, 

a úlcera gástrica afetava 10% da população mundial, com episódios cíclicos de cura e 

reincidência. O tratamento com erradicação da bactéria alterou esse ciclo da doença, 

entretanto, a crença „sem H. pylori, sem úlcera‟ está desatualizada (Arakawa et al., 2000). A 

prevalência de úlceras gástricas que não são induzidas por H. pylori nem por DAINEs, que 

podem ser chamadas idiopáticas, está próximo de 40% dos casos (Jyotheeswaran et al., 1998; 

Jang et al., 2008) e vem crescendo gradualmente nos últimos anos (Musumba et al., 2012). A 

taxa de reincidência da úlcera gástrica idiopática é maior que a reincidência das úlceras 

induzidas por H. pylori ou DAINE, e seu tratamento, por não combater uma causa específica, 

é mais difícil, mais longo e mais caro (Kang et al., 2012).  

 

4. TERAPÊUTICA ATUAL E PERSPECTIVAS 

O conhecimento sobre a doença mudou drasticamente com a descoberta da relação 

entre colonização estomacal por H. pylori e o desenvolvimento de úlcera gástrica, por Warren 

& Marshall (1983). Essa descoberta alterou a ideia de uma doença não apenas causada pelo 

ácido gástrico, mas também seria uma doença infecciosa, abrindo campo para intensivas 

pesquisas sobre a patogênese da doença. Além disso, essa pesquisa recebeu pleno 

reconhecimento em 2005, quando os autores foram laureados com o Prêmio Nobel. 

Entretanto, esse reconhecimento não fechou o capítulo sobre as úlceras gástricas. O 

tratamento da úlcera e principalmente de suas complicações ainda são desafios no campo da 

gastroenterologia (Garcia Rodriguez & Barreales, 2007). 

As drogas atualmente usadas no tratamento de úlceras gástricas incluem antibióticos 

(nos casos de infecção por H. pylori), bloqueadores de receptores H2 (cimetidina, ranitidina), 

inibidores da bomba protônica (omeprazol, lansoprazol) e agentes citoprotetores (bismuto, 

sucralfato). A classe de drogas que obteve maior êxito é a que visa inibir a secreção ácida. Os 

antagonistas de receptores H2 revolucionaram a tratamento das úlceras, cicatrizando-as e 

mantendo-as em remissão durante o período em que eram administrados (Bianchi-Porro & 

Lazzaroni, 1992). Essa classe de drogas foi gradualmente substituída por uma classe de 

drogas mais potente na inibição da secreção ácida, os inibidores de bomba de prótons, que 

entraram no mercado em 1989. Tais medicamentos atuam bloqueando a bomba 

H+/K+ATPase nas células parietais (Fellenius et al., 1981). Já que a rapidez da cicatrização 

da úlcera estaria associada com o grau de supressão da produção ácida, os inibidores da 



 27 

bomba protônica tornaram-se uma marca no tratamento das úlceras. Entretanto, após a fase de 

cicatrização, as úlceras geralmente reincidiam e, por muitos anos, a pratica padrão era manter 

o tratamento de supressão ácida para o paciente (Malfertheiner et al., 2009). Essas drogas 

geraram declínio nas taxa de morbidade, mas produzem muitos efeitos adversos, não 

impedem a reincidência da úlcera (DeVault & Talley, 2009), induzem tolerância (Komazawa 

et al., 2003) e geralmente tem um custo que não é acessível a grande parte da população 

(Maity & Chattopadhyay, 2008).  

Assim, tem se tornado consenso que um fármaco antiulcerogênico deve atuar não 

apenas na supressão da secreção ácida, mas sim apresentar mecanismo de ação multifatorial. 

Um fármaco antiulcerogênico considerado ideal deve apresentar atividade antisecretória, 

antioxidante, antiapoptótica, anti-inflamatória e ainda estimular a proliferação celular e a 

angiogênese (Maity et al., 2009). A busca por esse composto ainda tem sido um desafio e tem 

impulsionado as pesquisas recentes da área (Maity & Chattopadhyay, 2008). Nesse sentido, 

extensivos trabalhos têm sido desenvolvidos com plantas e produtos naturais derivados de 

plantas. As plantas são fontes atrativas de novas biomoléculas, e elas têm mantido sua 

popularidade como tratamento para várias doenças por razões históricas e culturais. O 

interesse no uso de plantas para o tratamento de várias doenças é disseminado em várias 

partes do mundo. 

Entre as principais classes de compostos químicos derivados de plantas que vem 

mostrando resultados promissores para a atividade antiulcerogênica, estão os carotenóides, 

terpenóides (Favier et al., 2005; Pertino et al., 2006) e os flavonóides (Alanko et al., 1999; 

Maity & Chattopadhyay, 2008). 

O uso das espécies do gênero Mentha para fins medicinais é bastante disseminado. 

Suas folhas são utilizadas internamente para alívio dos sintomas de doenças dos tratos 

gastrointestinal e urinário, ou por via inalatória para alívio de sintomas de problemas 

respiratórios e tosse. De acordo com o conhecimento popular, seus benefícios incluem 

distúrbios biliares, dispepsia, enterite, flatulência, gastrite, úlcera péptica, cólica intestinal e 

síndrome do intestino irritável (Blumenthal et al.., 2000). 

Mentol é o composto majoritário dos óleos essenciais do gênero Mentha. Apresenta 

fórmula molecular C10H20O e peso molecular de 156 kDa. É um terpeno cíclico que dá as 

plantas desse gênero seu aroma e sabor característicos. Atualmente, o consumo de mentol 

excede 700 toneladas por ano, e o produto puro movimenta cerca de 300 milhões de dólares 

(Croteau et al.., 2005). Sua aplicação é versátil e inclui produtos de higiene oral, confeitos, 

medicamentos, cosméticos, aromatizantes, além de estar presente na composição de fórmulas 



 28 

antipruríticas, antissépticas, analgésicas e refrescantes (Eccles, 1994; Croteau et al., 2005). 

Ainda, o mentol apresenta comprovado efeito anti-nociceptivo, anestésico local (Haeseler et 

al., 2002), bactericida e fungicida (Işcan et al., 2002).  

   

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 34 

 

 

Objetivos 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



 35 

OBJETIVOS 

 

- Investigar e caracterizar o mecanismo de ação gastroprotetor do mentol contra 

diferentes agentes ulcerogênicos (etanol e indometacina),  

- Investigar a atividade do mentol sobre a diarreia e motilidade intestinal, 

- Investigar possível indução de efeito tóxico agudo do mentol em ratos. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



 36 

 

 

 

Capítulo 1 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



 37 

Artigo a ser submetido para  

Evidence-Based Complementary and Alternative Medicine (eCAM) 

 

EFFECT OF MENTHOL IN EXPERIMENTALLY INDUCED ULCERS: 

PATHWAYS OF GASTROPROTECTION 

 

ABSTRACT 

Based on ethnopharmacological indications that Mentha species may be used in the treatment 

of gastrointestinal diseases, this study aimed to characterize the gastroprotective mechanisms 

of menthol (ME), the major compound of the essential oil from species of the genus Mentha. 

The gastroprotective action of ME was analyzed in gastric ulcers that were induced by ethanol 

or indomethacin in Wistar male rats. The mechanisms responsible for the gastroprotective 

effect were assessed by analyzing the amount of mucus secreted, involvement of non-protein 

sulfhydryl (NP-SH) compounds, involvement of calcium ion channels and NO/cGMP/K+
ATP 

pathway, gastric antisecretory activity and the prostaglandin E2 (PGE2) production. The anti-

diarrheal activity and acute toxicity of ME were also evaluated. Oral treatment with ME (50 

mg/kg) offered 88.62% and 72.62% of gastroprotection against ethanol and indomethacin, 

respectively. There was an increased amount of mucus and PGE2 production. The 

gastroprotective activity of ME involved NP-SH compounds and the stimulation of K+
ATP 

channels, but not the activation of calcium ion channels or the production of NO. The oral 

administration of ME induced an antisecretory effect as it decreased the H+ concentration in 

gastric juices. ME displayed anti-diarrheal and antiperistaltic activity. There were no signs of 

toxicity in the biochemical analyses performed in the rats‟ serum. These results demonstrated 

that ME provides gastroprotective and anti-diarrheal activities with no toxicity in rats. 

Keywords: menthol, gastroprotection, gastric ulcer, diarrhea, acute toxicity 



 38 

 

1. INTRODUCTION 

 The gastric mucosa is continuously exposed to damaging agents that are involved in 

the pathogenesis of gastric ulcers. These damaging agents can be endogenous (e.g., 

hydrochloric acid, pepsin, refluxed bile, and reactive oxygen species) or exogenous (e.g., 

alcohol consumption, excessive coffee ingestion, and administration of non-steroidal anti-

inflammatory drugs). The basic physiopathology of gastric ulcers results from an imbalance 

between damaging factors and cytoprotective factors, which include an intact mucus barrier, 

prostaglandins, adequate mucosal blood flow, activity of antioxidant compounds, and other 

mediators (Laine et al., 2008). 

 There is a continuous search for a new bioactive compound derived from medicinal 

plants and natural products with gastroprotective and ulcer-healing properties. The new 

compound should be accessible, safe, and gastroprotective, and it should effectively heal the 

ulcer, thus avoiding its reoccurrence (Lallo et al., 2013). Plants have presented promising 

results in the treatment of gastric ulcers in several research projects worldwide (Schmeda-

Hirschmann & Yesilada, 2005). Among the natural compounds that have been studied in 

recent studies, several terpenes have presented gastroprotective effects, such as limonene 

(Rozza et al., 2011), suaveolol (Vera-Arzave et al., 2012) and carvacrol (Silva et al., 2012). 

Menthol (ME) is a cyclic terpene with molecular weight 156 kDa and the formula C10H20O. 

ME is the main compound of the essential oil from the species of the genus mentha and is 

responsible for giving mentha species their distinctive smell and flavor. Among the optical 

isomers, (-)-menthol occurs most widely in nature and is endowed with the peculiar property 

of being a fragrance and flavor compound (Eccles, 1994). This study aimed to characterize 

the mechanism of action of ME against ethanol- and indomethacin-induced gastric ulcers in 



 39 

rats. We also sought to evaluate the anti-diarrheal activity and the acute toxicity induced by 

ME.    

 

2. MATERIAL AND METHODS 

2.1. Menthol 

(-)-Menthol (catalog #63660) was purchased from Sigma Chemical Co. (St. Louis, MO, 

USA). The chemical structure of ME is presented in figure 1. 

 

 

Figure 1: Chemical structure of (-)-menthol.  
 

2.2. Animals 

Male Wistar rats (200–250 g) from the Central Animal House of UNESP were fed a 

certified diet with free access to tap water under standard light-dark cycles (12 h dark–12 h 

light), humidity (60 ± 1%) and temperature (21 ± 2°C). All rats were fasted for at least 16 

hours prior to each experiment because the treatments were orally administered. Rats were 

housed in cages with raised, wide-mesh floors to prevent coprophagy. All experimental 

protocols followed the recommendations of the Canadian Council on Animal Care and were 

approved by the UNESP Institutional Animal Care and Use Committee. 

 



 40 

2.3. Experimental assays 

2.3.1. Ethanol-induced gastric ulcers: determination of dose 

Male Wistar rats were distributed into five groups (n=7) and then orally dosed with 

vehicle (10 mL/kg), carbenoxolone (100 mg/kg) or ME (25, 50 or 100 mg/kg). After 1 hour, 

the animals received an oral dose of 1 mL of absolute ethanol. One hour after ethanol 

treatment, the rats were euthanized, and their stomachs were removed (Robert et al., 1979). 

The stomachs were then opened along the greater curvature and washed. The flattened 

stomach samples were scanned, and the ulcer area (mm2) was measured using the AVSoft 

BioView software. The lower effective dose from the 3 doses tested was adopted for all other 

assays. 

After scanning, the mucosa of each stomach was gently scraped using two glass slices, 

homogenized in phosphate-buffered saline (PBS, 0.1 M, pH 7.4), and frozen at −80°C until 

biochemical analyses. Stomach samples were collected for histological slide preparation and 

were either stained with hematoxylin and eosin (HE) or periodic acid-Schiff (PAS) or used for 

immunohistochemical analyses. A microscopic score (Jahovic et al., 2007) was determined 

for the following parameters: epithelial desquamation, hemorrhage, glandular damage and 

eosinophilic infiltration. A scale ranging from 0 to 3 (0: none, 1: mild, 2: moderate and 3: 

severe) was used for each criterion. The highest possible score was 12.  

 

2.3.2. Involvement of the NO/cGMP/K+ATP pathway in gastroprotection 

Rats were distributed into eight groups (n=7). Two groups of rats were subjected to 

intraperitoneal treatment with the following drugs: vehicle (8% tween 80, 10 mL/kg), L-

NAME (N-nitro-L-arginine methyl ester 70 mg/kg, a NO synthase inhibitor), ODQ 

(1H[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one 10 mg/kg, a guanylate cyclase inhibitor) or 

glibenclamide (K+
ATP channel blocker 3 mg/kg). One hour later, the vehicle (10 mL/kg) and 



 41 

ME (50 mg/kg) were orally administered to four groups each (Chávez-Piña et al., 2011). After 

60 minutes, all groups were orally treated with 1 mL of absolute ethanol for gastric ulcer 

induction. Animals were euthanized one hour after ethanol administration, and the stomachs 

were removed, opened along the greater curvature, scanned and the ulcer area (mm2) was 

determined using the AVSoft BioView software. 

 

2.3.3. Involvement of non-protein sulfhydryl compounds (NP-SH) or calcium ion 

channels in gastroprotection 

Rats were distributed into six groups (n=7). Two groups of rats were subjected to 

intraperitoneal treatment with the following drugs: vehicle (8% tween 80, 10 mL/kg), NEM 

(N-ethylmaleimide 5 mg/kg, a NP-SH compounds blocker) or verapamil (a calcium channel 

blocker, 5 mg/kg). One hour later, the vehicle (10 mL/kg) and ME (50 mg/kg) were orally 

administered to three groups each. After 60 minutes, all groups were orally treated with 1 mL 

of absolute ethanol for gastric ulcer induction. Animals were killed one hour after ethanol 

administration, and the stomachs were removed, opened along the greater curvature, scanned 

and the ulcer area (mm2) was determined using AVSoft BioView. 

  

2.3.4. Evaluation of gastric juice parameters 

Rats were randomly divided into six groups (n=7). Thirty minutes after oral treatment 

or immediately after the intra-duodenal administration of a single dose of vehicle (10 mL/kg), 

cimetidine (100 mg/kg) or ME (50 mg/kg), the rats were subjected to pyloric ligation (Shay et 

al., 1945). Four hours later, the animals were euthanized, the abdomen was opened and 

another ligature was placed around the esophagus, close to the diaphragm. The stomach was 

removed, and its contents were drained into a graduated centrifuge tube, which was then 

centrifuged at 2,000 g for 15 minutes. The total acid content of the gastric secretions was 



 42 

determined by titration to pH 7.0 with 0.01 N NaOH using a digital burette. The total 

concentration of acid was expressed as mEq/mL/4 h. 

 

2.3.5. Determination of mucus adherence to the gastric wall 

After 24 hours of fasting, anesthetized rats (n=7) were subjected to longitudinal 

incisions slightly below the xiphoid apophysis for the placement of a pyloric ligature. The 

oral administration of vehicle, carbenoxolone (200 mg/kg) or ME (50 mg/kg) was performed 

1 hour before the ligature. After 4 hours, the rats were euthanized, and the glandular portion 

of the stomach was weighed and immersed in Alcian Blue solution for the mucus 

quantification procedure. The absorbance was measured in a spectrophotometer at a 

wavelength of 598 nm, and the results were expressed as µg Alcian Blue/g tissue (Rafatullah 

et al., 1990). 

 

2.3.6. Non-steroidal anti-inflammatory drug (NSAID)-induced gastric ulcers  

Rats were distributed into three groups (n=7). Vehicle (10 mL/kg), cimetidine (100 

mg/kg) or ME (50 mg/kg) were orally administered 30 minutes prior to the induction of 

gastric lesions by the oral administration of the ulcerogenic agent indomethacin (100 mg/kg). 

The animals were euthanized 5 hours after treatment with indomethacin (Puscas et al., 1997). 

The stomachs were removed, opened along the greater curvature and then scanned. The ulcer 

area (mm2) was determined using AVSoft BioView. 

 

2.3.7. Prostaglandin E2 (PGE2) assay 

Rats were randomly divided into five groups (n=7): sham, vehicle, vehicle + 

indomethacin, ME (50 mg/kg) and ME (50 mg/kg) + indomethacin. The +indomethacin 

groups subcutaneosly received indomethacin (PGE2 inhibitor) at 30 mg/kg, and the other 



 43 

groups received vehicle. After 30 minutes, rats orally received vehicle or ME. The sham 

group received neither drug nor treatment. After 30 minutes, the rats were euthanized, and the 

stomachs were removed. Following stomach harvesting, the corpus was excised, weighed and 

suspended in 10 mM PBS at pH 7.4 (1 mL). The tissue was minced finely with scissors and 

incubated at 37°C for 20 minutes. After centrifugation at 9,000 g, the PGE2 levels in the 

supernatant were measured by ELISA and read in spectrophotometer (420 nm). The results 

were expressed as ng/mL (Curtis et al., 1995).  

 

2.3.9. Effect of menthol on castor oil-induced diarrhea 

Three groups of rats were orally treated with vehicle (10 mL/kg), loperamide 

hydrochloride (3 mg/kg) or ME (50 mg/kg). After 30 minutes, each rat received 1 mL of 

castor oil orally. Immediately after ingesting the castor oil, each rat was kept in an individual 

cage, the floor of which was lined with blotting paper, and the rats were observed for 5 hours. 

The following parameters were then observed: onset of diarrhea, number of solid, semi-solid 

and watery feces and total frequency of fecal outputs. Each rat received an evacuation index 

(EI) expressed according to the formula: EI = 1×(no. solid stool) + 2×(no. semi-solid stool) + 

3×(no. watery stool) (Crocci et al., 1994).  

 

2.3.10. Effect of menthol on gastrointestinal motility 

Rats were orally treated with vehicle (10 ml/kg), loperamide hydrochloride (4 mg/kg) 

or ME (50 mg/kg). After 20 minutes, each rat orally received 1 mL of charcoal meal (10% 

charcoal suspension in 5% aqueous gum Arabic). After 30 minutes, rats were euthanized, and 

the stomach and small intestine were removed. The distance between the charcoal meal and 

the pylorus was measured and correlated to the distance from the pylorus to the caecum 

(Calzada et al., 2010). 



 44 

2.3.11. Acute toxicity 

Male rats (n=10) orally received vehicle or a single acute dose of ME (500 mg/kg, 

corresponding to ten times the therapeutic dose) after 12 hours of fasting. Possible signs and 

symptoms associated with toxicity were observed at 0, 30, 60, 120, 180 and 240 minutes after 

the administration and then twice daily for the next 14 days. Body weights were noted daily. 

At the end of the period, the rats were euthanized, and the kidneys and liver were withdrawn, 

weighed and evaluated. Biochemical analyses were performed on the rats‟ serum to quantify 

AST (aspartate aminotransferase), ALT (alanine aminotransferase), -GT (gamma 

glutamyltransferase) and alkaline phosphatase to evaluate liver damage and creatinine and 

urea to evaluate kidney damage using the automated biochemical analyzer SBA-200, CELM, 

Brazil. 

 

2.4. Statistical analysis 

Parametric data were analyzed using an unpaired t-test or a one-way analysis of 

variance (ANOVA) followed by Dunnett‟s test and compared to the vehicle group or Tukey‟s 

test. The results were presented as the mean ± standard error of the mean (SEM). 

Nonparametric data (histology scoring) were analyzed using the Kruskal-Wallis 

(nonparametric ANOVA) test, followed by a Dunn multiple comparison test. The results were 

presented as the median (range). All analyses were performed using GraphPad InStat 

software. A value of p<0.05 was considered significant. 

 

 

 

 



 45 

3. RESULTS  

3.1. Ethanol-induced gastric ulcers 

3.1.1. Gastric ulcer area 

The vehicle group presented several hemorrhagic bands that were usually parallel to 

the long axis of the stomach, with an average ulcer area of 374.82 ± 12.75 mm2. They were 

located mostly in the gastric corpus, and no visible lesions developed in the non-secretory part 

of the stomach. The two highest ME doses tested (i.e., 50 and 100 mg/kg) exhibited 

gastroprotective effects (p<0.01) when compared to the vehicle group. ME presented a 

gastroprotective effect of 20.06% for the lower dose (25 mg/kg), 88.62% for 50 mg/kg (ulcer 

area 42.64 ± 15.64 mm2) and 98.42% for the highest dose, 100 mg/kg (ulcer area 5.91 ± 5.33 

mm2). However, according to Tukey‟s test, there was no difference between the ulcer areas of 

the groups treated with 50 mg/kg or 100 mg/kg; therefore, the dose of 50 mg/kg was used for 

all subsequent experiments. The ulcer areas (mm2) are represented in figure 2. 

 

 

Figure 2: Gastric ulcer area (mm2) of rat stomachs with ethanol-induced gastric ulcers after 

treatment with vehicle, carbenoxolone (100 mg/kg) or menthol (25, 50 or 75 mg/kg). The 

results are reported as the mean ± SEM. ANOVA followed by Dunnett´s test, p<0.01. 



 46 

3.1.2. Microscopic analyses 

Microscopically, ME presented moderate epithelial desquamation, mild hemorrhage 

and glandular damage as well as an absence of eosinophilic infiltration, presenting a score of 

3(1-3). Carbenoxolone showed moderate epithelial desquamation and glandular damage as 

well as mild hemorrhage and eosiniphilic infiltration, presenting a score of 4(2-5). The score 

of the vehicle group was 12(11-12). The mucus polysaccharides were evidenced after the PAS 

staining, which conferred a purple barrier covering the gastric pits in the ME-treated rats. The 

HE and PAS staining of the ulcers are displayed in figure 3. 

 

3.2. Involvement of the NO/cGMP/K+
ATP pathway in gastroprotection 

In rats that were pre-treated with L-NAME (a NO-synthase inhibitor) or ODQ (a 

guanylate cyclase inhibitor), the gastroprotective effect of ME (50 mg/kg) was maintained 

(62.65% and 97.71% gastroprotection, respectively, compared to the vehicle-treated group, 

p<0.01), showing that the NO synthesis or cGMP were not involved in the protective 

mechanism of ME. However, in rats pretreated with glibenclamide (a K+
ATP channel blocker), 

the gastroprotective effect of ME (50 mg/kg) was reversed, indicating the involvement of 

K+
ATP channels in gastroprotection (table 1).  

 

3.3. Involvement of NP-SH compounds or calcium channels in gastroprotection 

In rats pretreated with NEM (an NP-SH compound reagent), the gastroprotective 

effect of ME (50 mg/kg) was reversed, indicating the involvement of SH compounds in 

gastroprotection. However, in rats that were pre-treated with verapamil (a calcium ion channel 

blocker), the gastroprotective effect of ME (50 mg/kg) was maintained (a protective effect of 

98.31% compared to the vehicle-treated group, p<0.001), indicating that the calcium ion 

channels are not involved in the gastroprotective mechanism of ME (table 2).  



 47 

 

Figure 3: Photomicrography of rat stomachs with ethanol-induced gastric ulcers after 

treatment with (A, D) vehicle, (B, E) carbenoxolone (100 mg/kg) or (C, F) menthol (50 

mg/kg). In the HE staining (A, B, C), * indicates epithelial desquamation and # indicates 

glandular damage. In the PAS staining (D, E, F), + indicates the mucus secretion in the gastric 

glands. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 48 

Table 1: Effect of menthol (50 mg/kg) on ethanol-induced gastric ulcer area (mm2) in rats 

that were pretreated with L-NAME (NO synthase inhibitor), ODQ (guanylate cyclase 

inhibitor) or glibenclamide (K+
ATP channels blocker) 

Pretreatment (i.p) Treatment (p.o) Ulcer area (mm2) Gastroprotection 

(%) 

Vehicle Vehicle 501.70 ± 35.00 - 

 Menthol 50.52 ± 10.99 *** 89.93 

L-NAME 70 mg/kg Vehicle 476.44 ± 89.17 - 

 Menthol 177.94 ± 25.29 ** 62.65 

ODQ 10 mg/kg Vehicle 652.54 ± 144.21 - 

 Menthol 14.94 ± 6.10 ** 97.71 

Glibenclamide 3 mg/kg Vehicle 134.13 ± 16.03 - 

 Menthol 154.41 ± 28.50 0 

The results are reported as the mean ± SEM. Unpaired t test, **p<0.01, ***p<0.001. 

 

Table 2: Effect of menthol (50 mg/kg) on ethanol-induced gastric ulcer area (mm2) in rats 

that were pretreated with NEM (SH blocker) or verapamil (calcium channel blocker) 

Pretreatment (i.p) Treatment (p.o) Ulcer area (mm2) Gastroprotection 

(%) 

Vehicle Vehicle 1825.62 ± 192.76 - 

 Menthol 17.86 ± 11.46 *** 99.02 

NEM 5 mg/kg Vehicle 1799.24 ± 250.73 - 

 Menthol 1574.03 ± 346.59  8.34 

Verapamil 5 mg/kg Vehicle 1155.17 ± 336.88 - 

 Menthol 19.53 ± 12.38 **  98.31 

The results are reported as the mean ± SEM. Unpaired t test, **p<0.01, ***p<0.001. 
 

 
 
 
 
 



 49 

3.4. Evaluation of the gastric juice parameters  

A comparison of the gastric juice parameters of rats treated with oral or intra-duodenal ME 

(50 mg/kg) demonstrated that the oral treatment was able to diminish the H+ concentration 

(p<0.01) in the gastric juice without modifying its volume. The intra-duodenal administration 

was not able to decrease the H+ concentration, but it did diminish the volume of the gastric 

juice (table 3).  

 

Table 3: Effect of menthol (50 mg/kg) on gastric juice parameters in rats with pyloric ligation 

The results are reported as the mean ± SEM. ANOVA followed by Dunnett‟s test, p<0.01. 

 

3.5. Determination of mucus adherence to the gastric wall  

There was a 2.2-fold increase in the amount of gastric mucus adhering to the stomach wall in 

the ME-treated (50 mg/kg) group (3632.00 ± 66.06 µg/g, p<0.01) compared to the vehicle-

treated group (1633.43 ± 43.73 µg/g) (figure 4). 

Route Treatment Gastric Juice Volume 

(mL) 

[H+] mequiv/mL/4h 

Oral Vehicle 

Cimetidine 100 mg/Kg 

Menthol 50 mg/Kg 

2.93 ± 0.36 

1.9 ± 0.13 

3.39 ± 0.63 

9.60 ± 0.95 

2.23 ± 0.18 ** 

5.90 ± 0.67 ** 

Intraduodenal Vehicle 

Cimetidine 100 mg/Kg 

Menthol 50 mg/Kg 

5.08 ± 0.21 

2.42 ± 0.17 ** 

2.95 ± 0.15 ** 

10.23 ± 0.44 

4.62 ± 0.50 ** 

9.05 ± 0.32 



 50 

 

Figure 4: Quantification of adherent mucus (µg/g of tissue) in the gastric mucosa of 

rats treated with vehicle, carbenoxolone (200 mg/kg) or menthol (50 mg/kg). The results are 

reported as the mean ± SEM. ANOVA followed by Dunnett´s test, p<0.01. 

 

3.6. Indomethacin-induced gastric ulcers  

The vehicle-treated group had a large quantity of small petechiae in the stomach and a 

mean ulcer area of 38.06 ± 8.02 mm2. In this model, ME presented a gastroprotective effect of 

72.62% (ulcer area 10.42 ± 2.71 mm2, p<0.01) in comparison to the vehicle group (figure 5).  

 

3.7. PGE2 assay 

The decreased level (p<0.01) of PGE2 in the vehicle + indomethacin group, compared 

to that of the sham group, proves that indomethacin induces decrease of PGE2 production. 

The ME groups maintained PGE2 levels near that of the sham group, even with indomethacin 

administration (figure 6). 

 

 



 51 

 
Figure 5: Gastric ulcer area (mm2) in rat stomachs with indomethacin-induced gastric ulcers 

after treatment with vehicle, carbenoxolone (100 mg/kg) or menthol (50 mg/kg). The results 

are reported as the mean ± SEM. ANOVA followed by Dunnett´s test, compared to vehicle 

group, p<0.01. 

 

 

 
Figure 6: PGE2 measurement (ng/mL) in the gastric mucosa of rats treated with vehicle or 

menthol (50 mg/kg) alone or in association with nonselective COX inhibitor (indomethacin). 

The results are reported as the mean ± SEM. ANOVA followed by Dunnett‟s test, compared 

to sham group, p<0.01. 



 52 

 

3.9. Effect of menthol on castor oil-induced diarrhea and gastrointestinal motility 

ME inhibited evacuation in 36.15% of the treated group compared to the vehicle group 

(p<0.01), indicating an antidiarrheal activity comparable to the effect of the standard drug 

loperamide hydrochloride (38.50%, table 4). The antidiarrheal effect of ME was connected to 

an antiperistaltic activity, decreasing the intestinal motility in 56.18% of the treated animals 

compared to the vehicle group (p<0.01, table 5).  

 

Table 4: Evacuation index and % of evacuation inhibition after oral treatment with vehicle, 

loperamide hydrochloride (3 mg/kg) or menthol (50 mg/kg) 

The results are reported as the mean ± SEM. ANOVA followed by Dunnett‟s test, p<0.01. 

 

Table 5: Evaluation of intestinal motility (cm1/2) after oral treatment with vehicle, loperamide 

hydrochloride (5 mg/kg) or menthol (50 mg/kg) 

The results are reported as the mean ± SEM. ANOVA followed by Dunnett‟s test, p<0.01. 

 

3.10. Acute toxicity 

Treatment Evacuation index % of evacuation inhibition 

Vehicle 

Loperamide 3 mg/Kg 

Menthol 50 mg/Kg 

13.0 ± 0.84 

8.0 ± 1.0 ** 

8.3 ± 0.42 ** 

- 

38.50 

36.15 

Treatment Intestinal motility % of motility inhibition 

Vehicle 

Loperamide 5 mg/Kg 

Menthol 50 mg/Kg 

4.15 ± 0.31 

1.91 ± 0.23 ** 

1.81 ± 0.32 ** 

- 

53.97 

56.38 



 53 

No signs or symptoms associated with toxicity were observed on the day of oral 

administration or over the subsequent 14 days. Body weights progressed normally. After the 

rats were euthanized, there were no alterations in the macroscopic appearance of the kidney or 

liver or in their weights. There were no alterations in the biochemical parameters analyzed in 

the rats‟ serum between the groups, indicating that there was no hepatotoxicity or 

nephrotoxicity associated with the ME oral treatment (table 6).   

 

Table 6: Effect of orally administered menthol (500 mg/kg) on rats‟ body weight, organ 

weight, and serum biochemical parameters 

 Vehicle Menthol 

Initial body weight (day 0) 196.70 ± 5.31 190.45 ± 4.54 

Final body weight (day 14) 290.60 ± 8.29 272.67 ± 8.11 

Kidneys weight 0.15 ± 0.00 0.15 ± 0.00 

Liver weight 0.72 ± 0.02 0.70 ± 0.02 

AST 105.80 ± 6.24 115.70 ± 7.44 

ALT 46.90 ± 0.99 47.90 ± 1.00 

Gama-GT 5.36 ± 0.46 5.09 ± 0.58 

Alkaline phosphatase 295.00 ± 10.71 277.37 ± 14.30 

Creatinine 0.28 ± 0.02 0.30 ± 0.03 

Urea 45.30 ± 1.51 42.44 ± 1.20 

Body weights are expressed in g. Organ weights are expressed in comparison to the body 

weight (g1/2). AST, ALT, γ-GT and alkaline phosphatase are expressed in U/L, creatinine and 

urea are expressed in mg/dL. The results are reported as the mean ± SEM. Unpaired t test. 

 

 



 54 

4. DISCUSSION 

Gastrointestinal diseases are major public health issues throughout the world and are 

estimated to affect 70% of the general population (Ouyang & Chen, 2004). Although the 

exact ulcer etiology is complex and multifactorial, it is generally accepted that an imbalance 

between acid production and mucosal integrity is one of the main causative factors (AlRashdi 

et al., 2012). The present study investigated the gastroprotective activity of (-)-menthol (ME) 

in experimental models of gastric ulcer and its possible mechanisms of action. 

The oral administration of ethanol is widely used to induce experimental gastric ulcer 

because it is easily reproducible and rapidly penetrates into the gastric mucosa. Ethanol 

causes necrotic lesions of the gastric mucosa in a multifactorial way. Ethanol is known to 

induce ulcers in the glandular part of stomach due to the secretion of mast cell secretory 

products and reactive oxygen species (Rao et al., 2004). Ethanol also acts by reducing the 

secretion of bicarbonate and the production of mucus, thus resulting in an increased flow of 

Na+ and K+, increased pepsin secretion, and a loss of H+ ions into the lumen (Szabo, 1987; 

Mahmood et al., 2005), which leads to cell necrosis and ulcer formation. HCl secretion further 

deepens necrosis and increases tissue injury. Because of these factors, ethanol-induced ulcers 

can be inhibited by agents that enhance mucosal defensive factors (Morimoto et al., 1991; 

Sener et al., 2004). In the ethanol-induced gastric ulcer model, ME exhibited a dose-

dependent effect and exerted substantial protective action on the gastric mucosa. Considering 

the multifactorial ways in which ethanol exerts its ulcerogenic action, the possible 

mechanisms of gastroprotection conferred by ME were investigated as follows.  

Several studies have shown the importance of mucus secretion in gastroprotection 

(Alrashdi et al., 2012; Laloo et al., 2013). The mucus barrier is considered the first line of 

mucosal defense because it decreases physical damage to the epithelium by ingested foods. It 

is an important barrier against self-digestion (Wallace, 2008) and acts as an antioxidant, 



 55 

scavenging free radicals in the mucosa (Repetto & Llesuy, 2002). Ulcerogenic substances 

cause disruptions in this barrier and allow contact between the gastric juice and epithelial 

cells, leading to mucosal injury (Darling et al., 2004). Thus, the increased stimulation of 

mucus production by 2.2-fold over the control group is a relevant part of the hypothesized 

mechanism of gastric mucosal protection by ME. The increase in mucus secretion was easily 

evidenced in the photomicrographies of the PAS-stained gastric ulcer. 

Endogenous NP-SH compounds help maintain the integrity of the mucus barrier by 

uniting its subunits by disulfide bridges, preventing the mucus from becoming soluble and 

easily withdrawn by ulcerogenic agents, including ethanol (Avila et al., 1996). NP-SH 

compounds also prevent the production of free radicals by ethanol and act as recycling 

antioxidants (Banerjee et al., 2008). The rats that were pretreated with an inhibitor of NP-SH 

compounds presented ulcer areas similar to those of the vehicle-treated rats, indicating the 

importance of an intact NP-SH barrier to the maintenance of the ME gastroprotective effect.  

Mucus secretion also can be regulated by nitric oxide (NO) (Brown et al., 1993), an 

endogenous gaseous mediator synthesized by the enzyme NO-synthase (NOs). NO also plays 

key roles in enhancing blood flow (Wallace & Miller, 2000), regulating acid secretion and 

inhibiting neutrophil aggregation (Wallace et al., 1997) and leukocyte adherence to the 

vascular endothelium (Zanardo et al., 2006). Our results showed that despite the inhibition of 

NO synthesis by the action of the NO-synthase inhibitor (L-NAME), ME exerted a 

gastroprotection similar to that observed in the group receiving ME treatment without NO 

blocking, indicating that maintaining NO synthesis is not crucial to gastroprotection by ME. 

In the gastric mucosa, NO interacts with neuropeptides and prostaglandins to maintain 

mucosal integrity. NO activates guanylyl cyclase to increase cyclic guanosine monophosphate 

(cGMP) levels and subsequently activates the ATP sensitive potassium channels (K+
ATP). The 

activation of this NO/cGMP/K+
ATP pathway leads to gastroprotection (Medeiros et al., 2008). 



 56 

K+
ATP channels mediate gastroprotection by enhancing gastric microcirculation and inhibiting 

neutrophil activation and the subsequent superoxide production (Campos et al., 2008). 

Despite this mediation, there was no loss of gastroprotection after the inhibition of NO 

synthesis or the cGMP blockade, but the blockade of the K+
ATP channels reversed the 

gastroprotective action of ME, thus indicating the involvement of these channels in the 

mechanism of action of ME. 

K+
ATP channels are not exclusively activated by the NO pathway but can also be 

activated by PGE2 activity (Lira et al., 2009). The oral treatment with ME was able to 

maintain PGE2 levels, even after the administration of the COX-inhibitor indomethacin, 

indicating the importance of PGE2 in the mechanism of action of ME. This result can explain 

the activation of the K+
ATP channels as well as the cytoprotective activity observed in the 

indomethacin-induced gastric ulcer. Indomethacin, as a non-steroidal anti-inflammatory drug 

(NSAID), induces gastric damage mainly by inhibiting prostaglandin production through 

inhibiting the activity of COX-1 and COX-2 isozymes (Wallace et al., 2000; Takeuchi et al., 

2010). 

The physiological functions of PGE2 in the gastrointestinal tract include stimulating 

bicarbonate and mucus secretion, maintaining mucosal integrity, and inducing a trophic effect 

in gastric and intestinal mucosa by triggering mitogenic signaling in mucosal cells (Pai et al., 

2002). It is also known that PGE2 presents a strong anti-secretory activity (Peskar & Maricic, 

1998), which can, at least in part, explain the anti-secretory effect observed after the oral 

administration of ME. The H+ concentration was decreased, but the gastric juice volume was 

not altered. This effect did not occur in the intra-duodenally treated rats, leading to the 

conclusion that ME acts via a local rather than a systemic mechanism. In addition to the 

increasing effect on PGE2 production, this antisecretory effect may also be due to increased 

mucus secretion, as the mucus barrier is able to neutralize secreted H+. 



 57 

The cellular perturbation caused by a damaging agent such as ethanol disrupts the 

normal Ca2+ homeostasis. Ethanol administration leads to an intracellular Ca2+ accumulation, 

providing an injurious action in the gastric mucosa (Kokoska et al., 1998; Miller et al., 2001). 

The oral administration of the calcium ion channel blocker verapamil was not able to reverse 

the ME gastroprotective effect, indicating that the blockade of the calcium ions efflux did not 

interfere with the protective mechanism and, therefore, that this gastroprotective pathway is 

not involved in ME activity.  

The incidence of gastric ulcers also can be influenced by changes in gastric motility. 

Natural compounds that affect gastrointestinal motility usually present effects on the ulcer 

formation (Abdulla et al., 2010). Constipation and diarrhea are common and prevailing 

disorders that affect the intestinal tract. Constipation is known to affect approximately 27% of 

the population and is more prevalent in women than in men (Cheng et al., 2009). Diarrhea is 

the passage of watery stool three or more times in a day or the passage of unformed bowel 

contents at a daily rate twice that of a person‟s usual rate (Feldman & Pickering, 1981). In 

developing countries, the death rate due to diarrhea is high (approximately 4 million 

deaths/year), and it is one of the leading causes of mortality (WHO, 2009). Antidiarrheal 

therapy is used to achieve increased resistance to flow (e.g., segmental contraction, decreased 

propulsion and peristalsis), increased mucosal absorption or decreased secretion (Burks, 

1991).   

Medicinal plants are commonly employed to treat constipation and diarrhea (Gilani & 

Rahman, 2005). This study also evaluated ME activity in castor oil-induced diarrhea and 

gastrointestinal motility. Experimentally, castor oil induces diarrhea by increasing the 

secretion of fluids and electrolytes in the intestinal lumen, thus resulting in fluid accumulation 

and in an aqueous content that flows rapidly in the small and large intestines (Burks, 1991). In 

this assay, ME displayed significant activity against the diarrhea induced by castor oil, an 



 58 

effect that can be compared to the standard drug loperamide. The antidiarrheal effect of ME 

was accompanied by an antiperistaltic effect. 

The acute toxicity test did not show any signs of toxicity or mortality. Behavioral 

changes such as irritation, restlessness, respiratory distress, abnormal locomotion, and 

catalepsy were not observed over a period of 14 days. There were no alterations in the dosage 

of renal and hepatic enzymes or in body weight evolution, revealing that ME has no toxicity 

when administered orally at a dose of 500 mg/kg, which is ten times higher than the 

therapeutic dose employed in this study. 

 

5. CONCLUSION 

The results described here suggest that menthol presents antiulcer activities against 

ethanol and indomethacin. The gastroprotective activity of menthol is associated mainly with 

mucus secretion, which is related to the maintenance of NP-SH compounds, PGE2 production 

and K+
ATP channel activation and to an anti-secretory effect. Menthol also presents an 

antidiarrheal and antiperistaltic effect. No signs of acute toxicity have been associated with 

the administration of a high dose of menthol. However, further clinical and toxicological 

studies must be conducted to support the use of menthol as a potential antiulcerogenic drug.  

 

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