Efeito cicatrizante do (-)-borneol incorporado ao filme bioativo de quitosana em roedores

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(1)0. UNIVERSIDADE FEDERAL DE SERGIPE PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE. ROSANA DE SOUZA SIQUEIRA BARRETO. EFEITO CICATRIZANTE DO (-)-BORNEOL INCORPORADO AO FILME BIOATIVO DE QUITOSANA EM ROEDORES. ARACAJU – SE 2013.

(2) 1. ROSANA DE SOUZA SIQUEIRA BARRETO. EFEITO CICATRIZANTE DO (-)-BORNEOL INCORPORADO AO FILME BIOATIVO DE QUITOSANA EM ROEDORES Tese apresentada ao Programa de Pós-Graduação em Ciências da Saúde do Núcleo de Pós-Graduação em Medicina da Universidade Federal de Sergipe para obtenção do título de Doutor em Ciências da Saúde. Área de concentração: Farmacologia.. Orientador: Prof. Dr. Lucindo José Quintans Júnior Co-orientador: Profa. Dra. Josimari Melo de Santana. ARACAJU – SE 2013.

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(4) 3. ROSANA DE SOUZA SIQUEIRA BARRETO. EFEITO CICATRIZANTE DO (-)-BORNEOL INCORPORADO AO FILME BIOATIVO DE QUITOSANA EM ROEDORES Tese apresentada ao Programa de Pós-Graduação em Ciências da Saúde do Núcleo de Pós-Graduação em Medicina da Universidade Federal de Sergipe para obtenção do título de Doutor em Ciências da Saúde. Área de concentração: Farmacologia.. Aprovada em: _____/_____/________ _______________________________________________ Orientador: Prof. Dr. Lucindo José Quintans Júnior _______________________________________________ 1º Examinador: Profa. Dra. Rosilene Calazans Soares _______________________________________________ 2º Examinador: Prof. Dr. Ricardo Luiz Cavalcanti Albuquerque Júnior _______________________________________________ 3º Examinador: Prof. Dr. Enilton Aparecido Camargo _______________________________________________ 4º Examinador: Prof. Dr. Adriano Antunes de Souza Araújo PARECER __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________.

(5) 4. Dedico o fruto deste trabalho ao meu príncipe Samuel e à minha querida princesa e companheirinha Giovana, ao meu amado esposo e amigo André e aos meus pais Osmar e Vanda sinônimos de amor incondicional, subsídio e paciência. Vocês são minha fonte de inspiração e alegria..

(6) 5. AGRADECIMENTOS. À Deus, o princípio e o fim de tudo, a quem entrego os meus planos e perfeitamente estabelece e desembaraça os meus caminhos. A Ele toda a honra, toda a glória e todo o louvor. Ao meu querido esposo André, amor que é confiança e cumplicidade, obrigada por me guiar e estar sempre ao meu lado. À meu príncipe Samuel e à minha princesa e companheirinha Giovana, meus filhos e a mais doce expressão do amor, os seus sorrisos inocentes alegram a minha vida e perdoam a minha ausência a cada dia. A mamãe ama vocês! Aos meus pais Osmar e Vanda pelo amor incondicional, subsídio, paciência, pelo exemplo de dedicação e sabedoria. Vocês são minha motivação e o meu pensamento de que tudo posso alcançar. Às minhas irmãs Polly e Jully, amigas eternas e verdadeiras. Meus bens querer, amores inseparáveis, sou a fã n. 1 de vocês! Ao meu orientador Prof. Dr. Lucindo J. Quintans Júnior, por mais essa grande oportunidade, pela confiança depositada e pelos ensinamentos transmitidos. Obrigada por mais uma vez ter acreditado em meu potencial e possibilitado meu crescimento profissional. A você o meu afeto e minha eterna gratidão. Ao Prof. Dr. Waldecy de Lucca Júnior que me recebeu em seu laboratório e clutou comigo e por mim, pela infraestrutura imprescindível e pelas valiosas contribuições à condução desta pesquisa sempre cooperando para o meu crescimento profissional e pessoal. Você foi peça fundamental para a finalização deste estudo..

(7) 6. Ao Prof. Dr. Ricardo Luiz C. Albuquerque Júnior que novamente e gentilmente me recebeu em seu laboratório e concedeu valiosas orientações na condução desta pesquisa contribuindo para o meu crescimento intelectual e acadêmico. Aos professores Dr. Enilton Camargo, Dra. Rosilene Calazans e Dra. Rogéria Nunes pelo fornecimento de precioso tempo e subsídios indispensávelis à realização desta pesquisa. À Rosinely, técnica do laboratório LMBE/ITP/UNIT, pelas dicas preciosas e imprescindíveis para a confecção das lâminas histolágicas. Aos professores Drs. Adriano Antunes, Waldecy de Lucca Jr. e Márcio Viana pelas valiosas contribuições no exame de qualificação Aos companheiros do LAPEC, pela agradável convivência durante todos estes anos e, em especial à Douglas Prado e Roberta Cardoso, meus alunos de PIBIC,pela grande ajuda no desenvolvimento dessa pesquisa. Ao Sr. Oswaldo, técnico do Biotério Setorial, e ao Biotério Central da UFS pelo atendimento gentil, profissional e cuidadoso e por sempre me ajudar a resolver os problemas do dia-a-dia. A UFS pela oportunidade e suporte à qualificação docente. À CAPES/CNPq, pelo auxílio financeiro durante grande parte do período de estudo. Aos amigos sempre presentes, muito obrigada a todos, o meu carinho especial. A todos que de alguma forma, direta ou indiretamente, contribuíram para a realização de mais essa etapa o meu MUITO OBRIGADA!. Rosana de Souza Siqueira Barreto.

(8) 7. Efeito cicatrizante do (-)-borneol incorporado ao filme bioativo de quitosana em roedores. BARRETO, Rosana de Souza Siqueira. Universidade Federal de Sergipe, Aracaju, 2013.. RESUMO Plantas medicinais e seus constituintes tem se mostrado como uma grande fonte de novas biomoléculas. O (-)-borneol é um monoterpeno alcoólico que tem apresentado diversas atividades biológicas com potencial para acelerar o processo de cicatrização. O presente estudo buscou realizar uma revisão sistemática de monoterpenos e derivados iridóides com efeito cicatrizante. Além de, avaliar a atividade cicatrizante do (-)-borneol incorporado ao filme bioativo de quitosana em modelo de ferida cutânea aberta em ratos. Revisão Sistemática: foram pesquisados artigos publicados na LILACS, PubMed e EMBASE. Sete. artigos foram encontrados sobre o efeito cicatrizante de 3 monoterpenos e 2 derivados iridóides. Ferida cutânea aberta: Foram utilizados 90 ratos, Wistar , machos, pesando entre 250-300 g. Os animais foram submetidos a uma excisão cutânea e tratados com filme de quitosana inerte (QUIN), filmes de quitosana contendo (-)-borneol a 0,5% (QUIBO05) ou filmes de quitosana contendo (-)-borneol a 1% (QUIBO1), por 3, 7, 14 e 21 dias. As feridas residuais foram fotografadas e avaliadas quanto à área, presença de edema, crosta, secreção, necrose e cicatriz patológica. O tecido foi retirado e devidamente tratado com técnica histoquímica. Grupos adicionais foram submetidos à excisão cutânea, tratados por 3 dias e tiveram as feridas retiradas para a realização da medida da mieloperoxidase. Medida da mieloperoxidase (MPO): amostras do tecido de granulação das feridas foram submetidas à. análise da atividade da MPO. Todas as concentrações do QUIBO reduziram a atividade da enzima de MPO ao 3º dia pós-lesão. As secções foram analisadas quanto à presença de células mononucleares e polimorfonucleares, proliferação de fibroblastos, deposição de colágeno e re-epitelização. O QUIBO foi capaz de reduzir significativamente a área da ferida, acelerar a resposta inflamatória e aumentar significativamente o índice de epitelização quando comparado ao QUIN ao 7º dia pós-lesão. Além disso, todas as concentrações do QUIBO estimularam a deposição de fibras colágenas durante todos os estágios do reparo quando comparado ao QUIN. Os resultados sugerem que o QUIBO possui efeito cicatrizante. Palavras-chave: cicatrização; monoterpenos; quitosana; técnicas de fechamento de ferimentos; terpenos..

(9) 8. Wound healing effects of (-)-borneol incorporated in quitosan-based films in rodents. BARRETO, Rosana de Souza Siqueira. Universidade Federal de Sergipe, Aracaju, 2013.. ABSTRACT Medicinal plants and their constituents have been shown to be a major source of new biomolecules. The (-)-borneol is an alcohol monoterpene which shows a series of biological activities with potential to accelerate the wound healing process. The present study was to conduct a systematic review that provides an overview of the characteristics of monoterpenes and derivatives iridoids with wound healing effect and its mechanisms of action. Besides, evaluate the wound healing activity of (-)-borneol incorporated to chitosan film in excisional wound model in rats. Systematic Review: articles published in LILACS, EMBASE and PubMed were examined. Seven articles were found on the wound healing effect of 3 monoterpenes and 2 derivatives iridoids. Excisional wound model: A total of 90 Wistar male rats, weighing between 250-300 g. The animals underwent fullthickness wound were treated with placebo (inert chitosan film - QUIN), chitosan films containing (-)-borneol 0.5% (QUIBO05) or chitosan films containing (-)-borneol 1% (QUIBO1) by 3, 7, 14, and 21 days. The residual wounds were photographed and evaluated by wound area, presence of edema, crust, secretion and necrosis. The tissue was removed and properly treated with histochemistry. Additional groups were submitted to excision of the skin, treated for 3 days, and the wounds were taken for performing the measurement of myeloperoxidase. Mieloperoxidase (MPO) assays: Granulation tissue samples of the wounds were submitted to. MPO activity. Both QUIBO concentrations decrease the myeloperoxidase activity at 3rd day after injury. The histological sections were analyzed by the presence of polymorphonuclear and mononuclear cells, fibroblast proliferation, collagen deposition and epithelialization rates. The QUIBO05 was able to significantly reduces the wound area, accelerates the inflammatory response and it was able to increase significantly the epithelization rates at 7th day. Finally, QUIBO provided collagen proliferation when compared to QUIN group. The results suggest that QUIBO showed wound healing effect. Keywords: chitosan; monoterpenes; terpenes; wound closure techniques; wound healing..

(10) 9. LISTA DE FIGURAS Figura 1: Estrutura molecular plana do (-)-borneol .......................................................... 18 A systematic review of wound healing effects from monoterpenes and derivatives iridoids: Figure 1: Flowchart of included studies ............................................................................ 52 Figure 2: Chemical structure of (-)-borneol (a), thymol (b), α-terpineol (c), genipin (d) and aucubin (e) .......................................................................................................................... 54 Wound-healing activity of chitosan films containing (-)-borneol, a bicyclic monoterpene alcohol, in rats: Figure 1: Chemical structure of (-)-borneol ...................................................................... 77 Figure 2: Effect of the borneol (BOR) on wound area in rats. Groups of rats were treated with inert chitosan-based film (QUIN); chitosan-based containing BOR 0.5% film (QUIBO05) or chitosan-based containing BOR 1% film (QUIBO1). The wound area was measurement through craniocaudal and latero-lateral axis measure by digital caliper. Value represents the mean ± S.E.M. *p < 0.05 compared to control group. ANOVA followed by Dunnett`s test n = 6, per group) …...................................................................................... 78 Figure 3: Effect of the borneol (BOR) on clinical features of wounds in rats. Groups of rats were treated with inert chitosan-based film (QUIN) (a, d, g and j); chitosan-based containing BOR 0.5% film (QUIBO05) (b, e, h and k) or chitosan-based containing BOR 1% film (QUIBO1) (c, f, i and l). The wounds were monitored regarding to the presence of edema, crust, secretion and necrosis ……………………...……………………………………..……………………….….…. 79. Figure 4: Effect of the borneol (BOR) on myeloperoxidase (MPO) activity at 3rd day. Groups of rats were treated with inert chitosan-based film (QUIN); chitosan-based containing BOR 0.5% film (QUIBO05) or. chitosan-based containing BOR 1% film. (QUIBO1). The MPO activity was assessed as follows. The granulation tissue was collected and placed in presence of potassium phosphate buffer. Each tissue sample was homogenized, and aliquots were centrifuged. The supernatants were collected and MPO.

(11) 10. activity was assessed. Value represents the mean ± S.E.M. ***p < 0.001 when compared to control group. ANOVA followed by Dunnett`s test (n = 6, per group) ............................ 80 Figure 5: Effect of (-)-borneol (BOR) on histological analysis of sections stained HE of the wounds in rats. Acute inflammatory reaction seen in absolutely all the cases of inert chitosan-based film (QUIN); chitosan-based containing BOR 0.5% film (QUIBO05) and chitosan-based containing BOR 1% film (QUIBO1) at the 3rd day (a, b and c). Acute inflammation seen in QUIN (d) and chronic inflammation seen in QUIBO05 (e) and QUIBO1 (f) at the 7th day. In addition, development of a delicate network of slit capillary vessels forming an immature granulation tissue was observed in QUIBO05 and QUIBO1 (e and f). Moderate inflammatory response with lymphocytes and plasma cells the most abundant leukocyte infiltrate in all groups at the 14th day (g, h and i). Remarkable content of large spindle-shaped cells interpreted as active fibroblasts, mainly in the bottom of the healing area and formation of epithelial buddings interpreted as cutaneous appendages rudiments in the epithelial lining in QUIBO05 (h) and QUIBO1 (i). Mild or absent inflammatory response in all the cases of QUIN, QUIBO05 and QUIBO1 at the 21st day (j, k and l). Also, well-developed sebaceous glands were observed in QUIBO05 and QUIBO1 groups (k and l) ................................................................................................................... 83 Figure 6: Effect of (-)-borneol (BOR) in epithelization rates (ER) of excision wounds of rats. Groups of rats were treated with inert chitosan-based film (QUIN); chitosan-based containing BOR 0.5% film (QUIBO05) or. chitosan-based containing BOR 1% film. (QUIBO1). The ER were evaluated by measuring the lengths of the epidermal tongues from both wound edges in a microscope with a ruler, and data were expressed as percentage wound closure (distance of migrated keratinocytes from the wound edge/total wound width x 100). Value represents the mean ± S.E.M. ***p<0.001 when compared to control group. ANOVA followed by Dunnett`s test (n = 6, per group) ............................ 85 Figure 7: Effect of (-)-borneol on histological analysis of sections stained Sirius red of wounds in rats. Scant deposition of thin delicate reticular arranged fibrils exhibiting greenish and yellow-greenish birefringence (type-III collagen) in all groups inert chitosanbased film (QUIN); chitosan-based containing BOR 0.5% film (QUIBO05) or chitosanbased containing BOR 1% film (QUIBO1) at the 3rd day (a, b and c). Fibers appeared thicker and longer in QUIBO05 and QUIBO1 (b and c). Remarkable improvement in the collagen fibers, with clear reduction of the interfibrillary spaces, in QUIBO05 and QUIBO1 at the 7th day (d, e and f). The newly formed fibrils sustained the yellow-.

(12) 11. greenish birefringence (type-III collagen molecules) and the reticular arrangement. Clear replacement of the delicate type-III collagen fibrils for gross and denser disposed type-I collagen fibers exhibiting orange birefringence at the 14th day (g, h and i). High levels of collagenization with pattern of the fibers in parallel arrangement in QUIBO05 (i) and interlaced in QUIBO1 (h). Intense deposition of gross thick parallel-arranged of both typeI and –III collagen bundles, less densely deposited in top of scars in QUIN at the 21st day (j). Same mix of wavy and highly interlaced type I and type III collagen fibers, with the fibers less thick and exhibited a dense interlaced arrangement that resembled the normal dermis in QUIBO05 and QUIBO1 (k and l) ...................................................................... 86.

(13) 12. LISTA DE TABELAS A systematic review of wound healing effects from monoterpenes and derivatives iridoids: Table 1: Characteristics of included studies....................................................................... 53 Wound-healing activity of chitosan films containing (-)-borneol, a bicyclic monoterpene alcohol, in rats: Table 1: Assessment of the profile of the inflammatory infiltrate in the inert quitosanbased film (QUIN) and chitonan-based containing 0.5% or 1% (-)-borneol (QUIBO05 or QUIBO1, respectivelly) films groups after 3 days of the surgical procedures …………………………………………………………………………………................. 81 Table 2: Assessment of the profile of the inflammatory infiltrate in the inert quitosanbased film (QUIN) and chitonan-based containing 0.5% or 1% (-)-borneol (QUIBO05 or QUIBO1, respectivelly) films groups after 7 days of the surgical procedures ………………………………………………………………………………..…............... 81 Table 3: Assessment of the profile of the inflammatory infiltrate in the inert quitosanbased film (QUIN) and chitonan-based containing 0.5% or 1% (-)-borneol (QUIBO05 or QUIBO1, respectivelly) films groups after 14 days of the surgical procedures ……………………………………………...….………………………………................. 82 Table 4: Assessment of the profile of the inflammatory infiltrate in the inert quitosanbased film (QUIN) and chitonan-based containing 0.5% or 1% (-)-borneol (QUIBO05 or QUIBO1, respectivelly) films groups after 21 days of the surgical procedures ………………………………………………...….……………………………................. 82.

(14) 13. LISTA DE ABREVIATURAS BOR. (-)-Borneol. CF. Collagen Fibers. COX. Ciclooxigenase. DHA. Docosahexaenoic Acid. EGF. Endothelial Growth Factor. EMBASE. Excerpta Medical Database by Elsevier. ER. Epithelization Rates. ERK. Extracellular Signal-Regulated Kinase. FDA. Food and Drug Administrotion. FGF. Fibroblasts Growth Factor. fMLP. N-Formyl-Methionyl-Leucyl-Phenylalanine. Gr. Gram. HE. Hematoxilina-Eosina. HTAB. Hexadecyltrimethylammonium Bromide. ICAM. Intercellular Adhesion Molecule. IL. Interleucina. iNOS. Inducible Nitric Oxide Synthase. IR. Inflammatory Response. KGF. Keratinocyte Growth Factor. LILACS. Latin American and Caribbean Health Sciences. LT. Leucotrieno. MCP. Monocite Chemiotatic Protein. MEDLINE-PUBMED. National Library of Medicine MEDLINE-PubMed. MIF. Migration Inhibitory Factor. MMP. Matrix metalloproteinases. MPO. Mieloperoxidase. mRNA. Messenger Ribonucleic Acid. NF. Nuclear Factor. NGF. Nerve Growth Factor. NO. Nitric Oxide. PAF. Platelet Activating Factor.

(15) 14. PAI. Plasminogen activator inhibitor. PDGF. Platelet Derivate Growth Factor. PG. Prostaglandina. PGI. Prostaciclinas. PMN. Leucócitos Polimorfonucleares. QUIBO05. Filme de Quintosana contendo 0.5% de (-)-borneol. QUIBO1. Filme de Quintosana contendo 1% de (-)-borneol. QUIN. Filme de Quintosana Inerte. RNA. Ribonucleic Acid. ROS. Reactive Oxygen Species. TCM. Traditional Chinese Medicines. TGF. Transforming Growth Factor. TIMP. Tissue inhibitor of Metalloproteinases. TNF. Tumoral Necrosis Factor. TX. Tromboxano. UMPO. Unidade de Mieloperoxidase. UNEP. United Nations Environment Program. UV. Ultravioleta. VEGF. Vascular Endothelial Growth Factor. WCMC. World Conservation Monitoring Centre. WHO. World Health Organization.

(16) 15. SUMÁRIO 1 INTRODUÇÃO ............................................................................................................. 16. 2 OBJETIVOS .................................................................................................................. 21. 2.1 OBJETIVO GERAL .................................................................................................... 21. 2.2 OBJETIVOS ESPECÍFICOS ...................................................................................... 21. 3 RESULTADOS ............................................................................................................. 23. 3.1 A SYSTEMATIC REVIEW OF WOUND HEALING EFFECTS FROM MONOTERPENES AND DERIVATIVES IRIDOIDS ................................................... 3.2 WOUND HEALING ACTIVITY OF CHITOSAN FILMS CONTAINING (-)BORNEOL, A BICYCLIC MONOTERPENE ALCOHOL, IN RATS ............................ 23 55. 4 CONCLUSÃO ............................................................................................................... 89. 5 PERSPECTIVAS .......................................................................................................... 91. REFERÊNCIAS ............................................................................................................... 93. ANEXOS ........................................................................................................................... 98. ANEXO A – Fotografias do material e métodos utilizados para avaliação cicatrizante do (-)-borneol em ratos ..................................................................................................... ANEXO B – Pedido de Patente requerida junto ao INPI – “PRODUTO BIOTECNOLÓGICO COM PROPRIEDADE CICATRIZANTE” .................................. 98 99. ANEXO C – Artigo publicado no The Science World Journal “Borneol, a bicyclic monoterpene alcohol, reduces nociceptive behavior and inflammatory response in. 102. mice” ................................................................................................................................. ANEXO D – Orientação de TCC concluída – Artigo “Avaliação da atividade cicatrizante da tens em roedores” ..................................................................................... ANEXO E – Declaração de aprovação do projeto de pesquisa pelo Comitê de Ética em Pesquisa com Animais da UFS........................................................................................... 107 125. ANEXO F – Normas para publicação de artigos da Phytotherapy Research ................... 126. ANEXO G – Normas para publicação de artigos da Phytomedicine ................................ 130.

(17) 16. INTRODUÇÃO.

(18) 17. 1 INTRODUÇÃO. No mundo, as feridas, principalmente crônicas, afetam um grande número de pacientes e reduzem seriamente a qualidade de vida dos mesmos. Devido a isso tem sido consideradas como uma das principais complicações e preocupações entre profissionais de saúde e pacientes. As estimativas indicam que nos Estados Unidos cerca de 6,5 milhões de pacientes hospitalizados sofrem de feridas crônicas (SINGER; CLARK, 1999; CROVETTI et al., 2004; KUMAR et al., 2007). A ferida pode ser definida como um defeito ou ruptura da integridade da pele que pode ou não estar acompanhada da lesão da estrutura e função dos tecidos subjacentes, sendo, portanto, resultado de danos físicos, químicos ou térmicos, ou mesmo de condições médicas ou fisiológicas. Desta forma, esta continuidade da pele deve ser restaurada, pois exerce um papel fundamental na manutenção da homeostase (ENOCH; LEAPER, 2007). O processo natural de reparo tecidual (processo de cicatrização) é um fenômeno complexo de interações entre os diferentes tipos de células, mediadores inflamatórios e matriz extracelular, que se seguem à lesão da pele e de outros tecidos moles (DIEGELMANN; EVANS, 2004). É caracterizado por uma sequência de eventos independentes e parcialmente sobrepostos, os quais são descritos como: fase de hemostasia e/ou coagulação; fase exsudativa ou inflamatória; fase proliferativa (formação de tecido de granulação e a síntese de colágeno) e fase de maturação e remodelação (DASH; MURTHY, 2011). Apesar do processo de cicatrização ocorrer naturalmente e sem intercorrências, algumas feridas não cicatrizam de maneira oportuna e ordenada. A falha na cura de uma lesão resulta em feridas crônicas, as quais exigem um tratamento contínuo. Por outro lado, distúrbios em certas etapas do processo podem levar às cicatrizes patológicas (cicatrizes hipertróficas e quelóides) (ENOCH; LEAPER, 2007). Ainda no passado, alguns estudos demonstraram que o processo de cicatrização pode ser acelerado e potencializado pelo uso de técnicas e produtos no cuidado à ferida (COOPER, 1990; HANNA; GIACOPELLI, 1997). Atualmente tem sido evidenciado que a cicatrização de uma ferida se torna mais rápida e bem sucedida quando se alcança um ambiente quente e úmido em torno da mesma (BOATENG et al., 2012). Devido aos avanços biotecnológicos, os curativos convencionais, tais como ataduras, naturais ou sintéticas, algodão e gaze, que passivamente fornecem diferentes graus de absorção e proteção à ferida, tem sido substituídos por curativos especiais,.

(19) 18. tais como pomadas, filmes, espumas, géis etc. que são capazes de fornecer esse adequado ambiente ao redor da ferida. Além disso, esses podem permear ingredientes ativos ou interagir diretamente com as células no local da ferida facilitando a cicatrização (OVINGTON, 2007; YUDANOVA; RESHETOV, 2006). Dentre os curativos especiais, os biológicos ou bioativos, feitos de biomateriais desempenham um papel ativo no processo de cicatrização de feridas e geralmente combinam polímeros, como o colágeno (RAMSHAW et al., 1995) e a quitosana (ISHIHARA et al., 2002). A grande vantagem dos curativos feitos de biomateriais está em fazer parte da matriz do tecido natural ou ser biocompatível do ponto de vista toxicológico, biodegradável, além de desempenhar um papel ativo na cicatrização e formação de novo tecido. Ao mesmo tempo, podem ser incorporados compostos ativos, como por exemplo, agentes antimicrobianos e fatores de crescimento disponibilizando-os diretamente no local da ferida (BOATENG et al., 2012). Neste contexto, estudos demonstram que a quitosana é biodegradável, biofuncional, biocompatível com baixa toxidade e tem características antimicrobianas (PRANOTO et al., 2005; PAUL, 2000; MUZZARELLI; MUZZARELLI, 2005; KUMAR et al., 2004; DASH, 2011). Além disso, apresenta propriedades favoráveis ao processo de reparo das feridas devido à sua característica de proporcionar substratos para o crescimento de muitas células, incluindo células endoteliais e adicionais efeitos bacteriostáticos (SECHRIEST et al., 2000; SHI et al., 2006; BERGER et al., 2004). Outros estudos mostram que a quitosana também acelera a cicatrização (KWEON et al., 2003; WITTAYA-AREEKU; PRAHSARN, 2006; ISHIHARA et al., 2002). Além do mais, a quitosana tem se mostrado como um material ideal para a engenharia de tecidos. Isso por possuir a característica de fornecer um reservatório para a liberação de substâncias bioativas e agir como um apoio estrutural (scaffold), podendo influenciar diretamente em células colonizadoras, levando a uma vantagem na adaptação do tecido (VANDEVORD et al., 2002). Recentemente, os filmes de quitosana incorporados com princípios ativos estão emergindo como promissores sistemas de liberação controlada de fármacos (DASH et al., 2011). É importante ressaltar que no mercado mudial vários são os produtos, aprovados pela Food and Drug Administrotion (FDA), à base de quitosana utilizados para o manejo de. feridas como o ChitoFlex® (DEVLIN et al., 2011), SurgiLux® (FOSTER; KARSTEN, 2012) entre outros (PORETTI et al., 2005; MILLNER et al., 2009)..

(20) 19. Durante séculos, os produtos naturais, tais como plantas medicinais e seus metabólitos secundários, tem sido utilizados para tratar várias doenças em todo o mundo, despertando interesses comerciais e científicos e ainda desempenhando um papel importante nos sistemas de saúde em muitos países desenvolvidos e em desenvolvimento, como o Brasil, os Estados Unidos e países da Europa (SÜNTAR et al., 2012; LI; VEDERAS, 2009). Estudos demonstram que os óleos essenciais de várias espécies de plantas medicinais apresentam classes de compostos, tais como os terpenóides, responsáveis por uma grande variedade de atividades farmacológicas (BHALLA et al., 2013; LANGEVELD et al., 2013; PAZYAR et al., 2013; WOOLLARD et al., 2007). Os compostos terpênicos mais frequentes nos óleos voláteis são os monoterpenos e os sesquiterpenos (SIMÕES; SPITZER, 2004). O borneol (endo-(1S)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-ol) é um monoterpeno alcoólico isolado de óleos essenciais de plantas, incluindo espécies do gênero Lavandula e Valerian que são largamente utilizados como aromatizante em cosméticos e pela indústria. farmacêutica (HUSNU; BASER, 2008). Preparações farmacêuticas contendo o (-)-borneol (Figura 1), tem sido utilizados como um agente terapêutico na China por mais de 1500 anos (HATTORI, 2000; HORVÁTHOVÁ et al, 2009). Na medicina popular, o mesmo tem sido empregado para diversos fins, tais como tratamento da dor abdominal (estômago), tratamento de feridas, queimaduras, alívio de dores reumáticas e hemorróidas (WANG et al., 2006; HORVÁTHOVÁ et al., 2009).. Figura 1: Estrutura molecular plana do (-)-borneol. Evidências científicas tem demonstrado que o (-)-borneol tem sido associado à atividade analgésica, anti-inflamatória, antioxidante, antiantibacteriana, antifibrótica e.

(21) 20. cicatrizante (CANDAN et al, 2003; HORVÁTHOVÁ et al., 2009;. JUHAS et al., 2008;. KORDALI et al., 2005; SLAMENOVÁ et al., 2009; DAI et al., 2009; LI et al., 2008; ALMEIDA et al., 2013; MAI et al. 2003; POTHULA et al., 2013; CHEN et al., 2013). Tratase, portanto, de um produto natural com potencial para ser estudado na cicatrização de feridas de segunda intenção e facilmente incorporado em filmes bioativos de quitosana. A correlação do (-)-borneol com atividades anti-inflamatória, antioxidante e antinociceptiva sugerem que esta substância possa participar modulando mediadores próinflamatórios do processo de cicatrização (ALMEIDA et al., 2013; LI et al., 2008). Além disso, a possibilidade de incorporação do (-)-borneol a uma matriz biotativa, como a quitosana, provavelmente potencializaria tais efeitos. No que se refere à atividade cicatrizante de feridas abertas não existe nenhum relato na literatura científica sobre os efeitos do borneol (nenhum dos enandiômeros) quando incorporado em filmes de quitosana no reparo tecidual (levantamento realizado nas bases PUBMED, LILACS e EMBASE, no periodo até 20 de maio de 2013). Desta forma, a hipotése de nosso trabalho se baseia na investigação da atividade cicatrizante do (-)-borneol incorporado aos filmes de quitosana em modelo animal..

(22) 21. OBJETIVOS.

(23) 22. 2 OBJETIVOS 2.1 OBJETIVO GERAL . Avaliar a atividade cicatrizante do (-) borneol incorporado aos filmes de. quitosana em modelo animal de feridas cutâneas de segunda intenção.. 2.2 OBJETIVOS ESPECÍFICOS . Realizar um levantamento bibliográfico (revisão sistemática) acerca dos monoterpenos e derivados iridóides com efeito cicatrizante em modelo. . tecidual de feridas; Investigar a possível atividade cicatrizante do (-)-borneol incorporado aos filmes bioativos de quitosana em modelo tecidual de ferida. . excisional em ratos; Verificar possíveis alterações no processo de cicatrização de feridas cutâneas induzidas pelo borneol incorporado aos filmes bioativos de quitosana;.

(24) 23. RESULTADOS.

(25) 24. 3 RESULTADOS 3.1 A SYSTEMATIC REVIEW OF WOUND HEALING EFFECTS FROM MONOTERPENES AND DERIVATIVES IRIDOIDS. Artigo escrito para:. Phytotherapy Research Edited By: Elizabeth M. Williamson, Angelo A Izzo, Sung Hoon-Kim and Rajan Radhakrishnan. Impact Factor - 2012: 2,068. ISI Journal Citation Reports © Ranking: 2012: 33/59 (Chemistry Medicinal); 145/260 (Pharmacology & Pharmacy). Online ISSN: 1099-1573.

(26) 25. A systematic review of wound healing effects from Monoterpenes and derivatives iridoids. Running Title: Wound healing effects from monoterpenes. Rosana S.S. Barretoa, Ricardo L.C. Albuquerque-Júniorb, André S. Barretoa, Lucindo J. Quintans-Júniora*. a. Department of Physiology, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil.. b. Institute of Technology and Research, Tiradentes University, Aracaju, SE, Brazil. Corresponding address: Lucindo J. Quintans Júnior, PhD, Msc, Pharmacist Universidade Federal de Sergipe. Departamento de Fisiologia, Laboratório de Farmacologia Pré-Clínica - LAPEC Av. Tancredo Neves, S/N, Bairro: Rosa Elza, CEP: 49.000-100, São Cristóvão-SE, Brazil Phone: 55 (79) 2105-6645, E-mail: lucindojr@gmail.com; lucindo@pq.cnpq.br.

(27) 26. Abstract The search for therapeutic approaches to wound healing that are more effective and low cost remains a challenge for modern medicine. In the search for new therapeutic options, plants and its metabolites are a great source of novel biomolecules. Among its constituints, the monoterpenes represent 90% of essential oils, and have a variety of structures with several activities such as antimicrobial, anti-inflammatory, antioxidant and wound healing. Given this, and also due to the absence of reviews about wound healing activity of monoterpenes, we performed this systematic review that provides an overview of their caracteristics and mechanisms of action. In this search the terms “terpenes”, “monoterpenes”, “wound healing” and “wound closure techniques” were used to retrieve published articles in LILACS, PUBMED and EMBASE until May, 2013. Seven papers were found concerning the potential wound healing effect of 5 monoterpenes. Among the products used for the wound care, the solutions followed by films were the most studied pharmaceutical forms. Amazingly, monoterpenes are a class of compounds of great diversity of biological activities and therapeutic potential. The data reviewed here suggest that monoterpenes, although poorly studied, are a promising compound for the treatment of chronic wound conditions.. Keywords: terpene, monoterpene, wound healing, wound closure technique. ..

(28) 27. INTRODUCTION. Wounds are physical, chemical or thermal injuries that result in an opening or breaking in the integrity of the skin. This continuity of the skin should be restored, and an appropriate methods for wound healing are essential for the restoration of disrupted anatomical continuity and disturbed fuction status of the skin (Meenakshi et al., 2006). The acute wound healing process is a complex series of interrelated events that are mediated over the phases by a wide range of chemically co-ordinate cellular processes as well as hormonal influences. It is characterized by a sequence of independent and/or overlapping events (Diegelmann and Evans, 2004; Chan et al., 2008). This process can be broadly categorized into three or four stages: coagulating phase, inflammatory phase, proliferative phase (formation of granulation tissue and collagen synthesis), and finally the remodeling phase, wich ultimately determines the strength and appearance of the healed tissue (Sumitra et al., 2005; Ayyanar and Ignacimuthu, 2009; Dash and Murthy, 2011; Reinke and Sorg, 2012; Rawat et al., 2012). For centuries, natural products, such as medicinal plants, has been used to treat a lot of illnesses in the worldwide, raising scientific and commercial interests and still playing an important role in health systems in many developed and developing countries, such as United States and Brazil, respectively (Süntar et al., 2012; Li and Vederas, 2009). Studies have shown that essential oils from many plant species have medicinal classes of compounds, such as terpenes, that are responsible for a wide variety of pharmacological activities (Bhalla et al., 2013; Langeveld et al., 2013). The terpene compounds most frequently found in volatile oils are monoterpenes and sesquiterpenes (Simões and Spitzer, 2004; Santos et al., 2011)..

(29) 28. Monoterpenes belong to a large and diverse group of chemical compounds named terpenes. They represent a group of naturally occurring organic compounds. They are the most representative molecules constituting 90% of the essential oils and have a great variety of structures (Bakkali et al., 2008), with relevant pharmacological properties, such as antimicrobial, anti-inflammatory, antioxidant, antipruritic, hypotensive and analgesic (Kordali et al., 2005; Bastos et al., 2010; Menezes et al., 2010; Guimarães et al., 2013). So, medicinal plants and related compounds traditionally have played an important role in drug discovery and were the basis of most early medicines (Butler, 2008). Additionally, the usage of techniques and products in wound care allied to substances with anti-inflammatory, antibacterial, and antioxidant properties are powerful on the treatment of skin lesions (Dias et al., 2011). Despite their importance, there are no reviews on the wound healing potential of monoterpenes. Accordingly, we conducted for the first time a systematic review of the literature to examine and synthesize the literature on monoterpenes, to identify and to evaluate those that assess healing effects in wound healing animal models.. METHODS. The present systematic review was conducted according to guidelines Transparent Reporting of Systematic Reviews and Meta-Analyses (PRISMA statement) (Liberati et al., 2009).. Search Strategy Three database (internet sources) were used to search for appropriate papers that satisfied the study purpose. These included the National Library of Medicine, Washington,.

(30) 29. D.C. (MEDLINE-PubMed), Excerpta Medical Database by Elsevier (EMBASE), and Latin American and Caribbean Health Sciences (LILACS), using different combinations of the following keywords: wound healing, wound closure techniques, monoterpenes and terpenes. The databases were searched for studies conducted in the period up to and including May, 2013. The structured search strategy was designed to include any published paper that evaluated a wound healing to identify those that shows potential therapeutic value. Citations were manually limited to animals studies. Additional papers were included in our study after analyses of all references from the selected articles. We did not contact investigators and did not attempt to identify unpublished data.. Study Selection All electronic search titles, select abstracts, and full-text articles were independently reviewed by a minimum of two reviewers (R.S.S.B., A.S.B. and L.J.Q.J.). Disagreements on study inclusion/exclusion were resolved with a consensus meeting. The following inclusion criteria were applied: wound healing studies, and the use of monoterpenes isolated or not isolated from medicinal plants (natural or synthetic product) for treatment. Studies were excluded according to the following exclusion criteria: studies in humans, studies of mixtures of substances or extracts from plants, review articles, meta-analyses, abstracts, conference proceedings, editorials/letters, case reports (Fig. 1).. INSERT FIGURE 1. Data Extraction Data were extracted by one reviewer using standardized forms and were checked by a second reviewer. Extracted information included data regarding the substance, animal models,.

(31) 30. dosages and concentrations, dosage form, evaluated parameters, results and proposed mechanisms of action.. RESULTS. A total of 1,895 abstracts/citations were identified from electronic and manual searches for preliminary review. The primary search identified 1,894 articles, with 1,116 from PUBMED, 722 from LILACS, 56 from EMBASE and 1 from manual search. After removal of duplicates and screening for relevant titles and abstracts, a total of 140 articles were submitted for a full-text review. Seven articles satisfied the inclusion and exclusion criteria established. A flow chart illustrating the progress of study selection and article number at each stage is shown (Fig. 1). From 7 final selected studies, most of those research were realized in China (43%), Korea (29%), Brazil (14%) and Peru (14%). Regarding the pharmaceutical form of the products used to wound healing presented in selected studies, the bioactives films (43%) were the most used following ointments (29%) and solutions (28%) (Table 1). In the articles selected, the wound models used to study the wound healing included excision and incision wound model. However the main used was the excision wound model (83%). In most of selected studies both macroscopic and microscopic features were evaluated. A total of 56% selected searches evaluated tissue morphology aspects involved in the wound healing process. Concerning the mechanisms of action involved in wound healing proposed for different monoterpenes were suggested: antimicrobian activity (inhibits RNA and protein biosynthesis of microorganisms); antiinflammatory activity (decreases the amount of IL-6 and.

(32) 31. TNF-α production in mast cells; inhibits the release of LTC4 and effect on release of TXB2); antioxidant activity (photoprotective effects and oxidative stress by inhibiting UVB-induced free radical production); low toxicity characteristic; macrophage migration inhibitory factor (MIF) and fibroblasts growth effects.. DISCUSSION. In this study, China was the country with the largest number of research on the healing effects of monoterpenes. The use of plants for medicinal purposes, to treat, cure and prevent diseases, is one of the oldest forms of medical practice of humanity (Viegas et al., 2006). In particular, Traditional Chinese Medicines (TCM) are composed of various combinations of medical plants and have been used as natural remedies for thousands of years (Xiao, 2002). Medicinal plants are the primary sources of many small molecule drugs and herbal products (Chen et al., 2010; Nalawade et al., 2003). Several recent publications reiterate the importance of natural products as a source of drugs (Oberlies and Kroll, 2004; Srivastava et al, 2005). In this context, the advent of modern technologies has boosted medicinal plants as a highly valuable commodity in patent market. Many developed and developing countries are actively engaged in biomining of medicinal plants for therapeutically precious and biologically active phytochemicals (Rajeswara et al., 2012). According to the World Conservation Monitoring Centre (WCMC) of United Nations Environment Program (UNEP), China was identified as one of the largest mega-biodiversity countries (Rajeswara et al., 2012). Accordance with Ravenhill (2006) China is one of the largest countries in Asia, which have the richest arrays of registered and relatively wellknown medicinal plants. In addition, medicinal plants have been used in developing countries for thousands of years. The World Health organization (WHO) estimated that 70-80% of the.

(33) 32. population living in developing nations depend on traditional healthcare systems for primary healthcare. Besides, in China about 40% of the total medicinal consumption is attributed to traditional tribal medicines (Hoareau and DaSilva, 1999). Brazil is one of the countries with the largest biodiversity in the planet and it is associates an extensive ethical and cultural diversity (Indigenous, African and European) that uses natural products as a tradition. It also presents social and economical characteristics that typify it as a developing country, where 80% of population depends on the use of plants for the primary health care (Schuster, 2001; Nodari and Guerra, 1999). Although the therapeutic potential of medicinal plants and their compounds, the great biodiversity and the ethnic and cultural aspects of developing countries such as China and Brazil, few studies were found regarding the wound healing effects of monoterpenes. For this study were included only isolated monoterpenes, due to these provide structural molds for obtaining synthetic substances and also are considered sources for drug development. Furthermore, they can be used as tools to identify mechanisms of action (McKernan, 1996). The healing process can be accelerated and enhanced by the use of techniques and products in the wound care (Cooper, 1990; Hanna e Giacopelli, 1997). In this review it was observed that, among the products used for the wound care, the solutions followed by films were the most studied pharmaceutical forms. The use of liquid dosage form provide the advantage of studying the action of the compounds isolated. However the major problem is the short residence times on the wound site, especially where there is a measurable degree of wound fluid exuding (Boateng et al., 2012). It might also be noted that the bioactive films were studied in the most current research. Currently, it has been shown that wound healing becomes rapid and successful when a warm moist environment around the wound provided. Unlike the solutions, recently the.

(34) 33. modern dressings have been developed with features to retain and create this great environment playing a active role in wound healing (Boateng et al., 2012). Moreover, the dressings-based biomaterials, for being part of the matrix of natural tissue, are biocompatible at the toxicological point, biodegradable and are able to permeate active ingredients such as antimicrobial agents or growth factors (Ovington, 2007; Yudanova et al., 2006). Wounds are heterogeneous, and the wound healing process is multifactorial, which is influenced by many extrinsic and intrinsic factors. In order to obtain new knowledge of the complexity of this process or substances effects, the use of an animal models are requested (Gottrup et al., 2000). More specific human chronic wound treatments are absent, in large part due to the lack of knowledge of the molecular abnormalities within the wound preventing the healing. Research is hindered by the absence of an easily reproducible animal model that mimics the human chronic wound state (Chen et al., 1999). In the present study, excisional wound model was the most used. This is an acute wound model that a great advantage is the rapid introduction of injury and a relatively rapid course, besides being a wound model of easy and inexpensive execution when compared to chronic wound models. Furthermore, the excisional wound model involves the removal of a significant volume of the target tissue, and the filling of the void created allows greater amount of material. The excision site can be harvested or biopsied to obtain cells, tissue, RNA, exudates, and histological specimens that have a wider cross-sectional area and volume when compared to incisional wound. This is suitable for in-situ techniques or biomechanical strength (tensile strength), (Gottrup et al., 2000)..

(35) 34. About the parameters evaluated, the analysis of the kinetics of biological events in response to pharmacological substances is crucial for the development of effective therapeutic products able to stimulate wound healing (Alborova et al., 2007). This review shows that no study prioritized the molecular biology assays. Monoterpenes or monoterpenoids are compounds with a core of 10 carbons. They are cyclized and oxidized in a variety of ways. Due to the low molecular weight many of them exist in the form of essential oils (Bakkali et al., 2008). A type of monoterpenes, the iridoids are derived from geraniol. They are different from the similarly named iridals (triterpenes). A subclass of iridoid, the iridoid glycosides and glucosides are compounds that include a glycoside or glucoside, respectively, moiety, usually found at the C-1 position. In present study were selected articles with the following monoterpenes, including types and their subclasses: borneol, thymol, α-terpineol, genipin and aucubin. According to the scientific literature these compounds possess a range of biological activities that may be directly or indirectly related to wound healing effects. Borneol is a bicyclic monoterpenoid alcohol (Fig. 2a). The borneol has shown effects such as. antithrombotic effects (Li et al., 2008), antibiotic activity (Unlu et al., 2002), wound healing activity (Mai et al., 2003), anti-inflammatory activity by reduce leukocyte migration (Almeida et al., 2013), anti-fibrosis activity by decrease the fibroblasts growth, inhibit collagen production, decrease MMP-2 activity and inhibit TIMP-1 production (Dai et al., 2009). It showed no cytotoxicity (Dai et al., 2009), radical scavenging properties (Candan et al., 2003; Kordali et al., 2005) and immunomodulatory effects (Juhás et al., 2008). This monoterpene was able to suppress the proinflammatory cytokine (IL-1β and IL-6) mRNA expression and act as bioactive material in the cellular signal transduction system (Park et al., 2003). It shows antibacterial activity and inhibitory effects on several Gr(-) and Gr(+) pathogenic microorganisms (Knobloch et al., 1989; Tabanca et al., 2001), antifungal activity (Knobloch.

(36) 35. et al., 1989; Kordali et al., 2005; Tuberoso et al., 2005; Tabanca et al., 2006; Wenqiang et al., 2006), antioxidant activity by reduce intracellular reactive oxygen species (ROS) generation and attenuate the elevation of nitric oxide (NO), the increase of inducible nitric oxide synthase (iNOS) enzymatic activity and the upregulation of iNOS expression (Liu et al., 2011). Borneol blocked NF-κB p65 nuclear translocation (Liu et al., 2011) and has been shown to be a mast cell membrane stabilizer (Watanabe et al., 1994). Finally, it was showed antiinflammatory property by fewer ICAM-1 positive vessels, IL-1β positive cells, TNF-α positive cells, and number of neutrophils (He et al., 2006). Thymol is a monoterpenoid phenol (Fig. 2b) which exhibits multiple biological activities.. Studies show that thymol modulates prostaglandin synthesis (Anamura et al., 1988), it has anti-inflammatory effect in human neutrophils incubated (Braga et al., 2006) and beneficial effects on the antioxidant status by the influenced on docosahexaenoic acid (DHA) concentration (Youdim and Deans, 2000). Thymol prevented autoxidation of lipids (Yanishlieva et al., 1999) and the formation of toxic products by the action of reactive nitrogen species (Prieto et al., 2007). It exhibits antimicrobial activity (Braga et al., 2006; Haeseler et al., 2002; Karpanen et al., 2008; Yanishlieva et al., 1999; Mastelic et al., 2008) and wound healing activity (Riella et al., 2012). Thymol is able to increase the levels of macrophage migration inhibitory factor (MIF) in central nervous system (Denkinger et al., 2012), enhance the fibroblasts growth in vitro (Khorshid et al., 2010) and interfere with elastase activity as evidenced by the reduced release of this proteinase by human neutrophils stimulated with the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) (Braga et al., 2006). It effectively inhibited COX-1 (Badr et al., 2011), inhibited inducible lymphocyte proliferation (Amirghofran et al., 2011) and showed anti-inflammatory effects by reduction of the edema, inhibition of MPO activity and decreased leukocyte influx (Riella et al., 2012)..

(37) 36. α-terpineol is a monoterpenoid alcohol (Fig. 2c) and is relatively non-toxic present in the essential oils of several species (Dagne et al., 2000). This monoterpene presented antiinflammatory activity by inhibits the COX enzyme and IL production (Khalil et al., 2004; Kawata et al., 2008). Also, α-terpineol is an NF-κB inhibitor and promotes down-regulation of IL-1β expression (Hassan et al., 2010) and IL-6 formation (Held et al., 2007). Futhermore, it was demonstrated the power in the reduction of TNF-α and NO production (Oliveira et al., 2012). In addition, α-terpineol showed selective inhibition of ovine COX-2 activity (Kawata et al., 2008), inhibited the neutrophil influx (Oliveira et al., 2012), exhibited strong antimicrobial activity (Park et al., 2012) and antifungal effects (Park et al., 2009). Genipin is an iridoid glucoside (Fig. 2d) and an alternative natural crosslinking agent (Jin et. al., 2004; González et al., 2011; Pujana et al., 2013). It has shown ability to form biocompatible and stable crosslinked products and showed low cytotoxicity (Sung et al., 1999). Moreover, it has been proved that genipin has anti-inflammatory (Nam et al., 2010), anti-angiogenesis (Tian et al., 2010) antithrombotic (Suzuki et al., 2001) and anti-oxidative effects (Wang et al., 2009) and abilities of inhibiting lipid peroxidation and production of nitrogen monoxide (NO) (Koo et al., 2004). Additionally, genipin can increase the mitochondrial membrane potential (Parton et al., 2007), increase the ATP levels and close KATP channels (Parton et al., 2007) and stimulate insulin secretion (Parton et al., 2007). Finally, studies showed that genipin suppress the alpha-TN4 lens epithelial cells and subconjunctival fibroblast migration induced by TGF-b (Kitano et al., 2006a and b). Aucubin is an iridoid glycoside (Fig. 2e) with a variety of pharmacological effects, such as. antimicrobial (Ishiguro et al., 1982; Davini et al., 1986; Yuan et al., 2003), anti-inflammatory (Maria et al., 1994; Recio et al., 1994; Park and Chang, 2004), dermal wound healing (Shim et al., 2007; Lee et al., 1999) and in vitro antioxidative capacity (Li et al., 2004). In addition, aucubin showed inhibition of RNA and protein biosyntheses (Chang et al., 1983; Davini et al.,.

(38) 37. 1986; Chang, 1998; Chang et al., 1989; Shim, 2007). Futher, aucubin inhibits TNF-α-induced secretion and mRNA synthesis including PAI-1, MCP-1, and IL-6 (Grundy et al., 2004). Furthermore investigation revealed that aucubin suppressed extracellular signal-regulated kinase (ERK) activation (Park, 2013), inhibitory kappa Bα (IκBα) degradation (Park, 2013), and subsequent nuclear factor kappa B (NF-κB) activation (Park, 2013). Finally, Aucubin was considered as a promising chemopreventive agent and was devoid of any cytotoxic activity (Hung et al., 2008; Cherng et al., 2007; Galvez et al., 2005).. INSERT FIGURE 2. CONCLUSION. For more than one decade, researchers have studied the wound healing potential of monoterpenes through in vivo and in vitro assays. Futher, as in nature there are about 20,000 different terpene metabolites known (Tholl, 2006), there is still a wide perspective to this superfamily, of which the monoterpenes are a part. Despite, this review described the study of only 5 monoterpenes or type of monoterpenes in models of wound healing in animals. In summary, it can be concluded that, although there are some studies about the wound healing effects of monoterpenes, a class of compounds of great diversity of biological activities and therapeutic potential, they have been little studied for the treatment of wounds. It occur especially in developing countries that have a wide biodiversity and tradition in use of natural products such as Brazil. Moreover, of those, every evaluated monoterpenes showed wound healing effects. The anti-inflammatory action of monoterpenes is often related e correlated to wound healing effect. However, further studies are required to better understand these mechanisms..

(39) 38. All these findings make the monoterpenes great compound for the development of new drugs for the treatment of various pathological processes that afflict humanity, including chronic wound conditions.. Declaration of interest: The authors report no conflicts of interest.. Acknowledgements. This work was supported by grants from National Council of Technological and Scientific Development (CNPq/Brazil) and the Research Supporting Foundation of State of Sergipe (FAPITEC-SE/Brazil)..

(40) 39. References Alborova A, Lademann J, Meye L, Kramer A, Patzelt A, Sterry W, Antoniou C. 2007. Application of laser scanning microscopy for the characterization of wound healing. GMS Krankenhaushyg Interdiszip 2(2): Doc37.. Almeida JRGS, Souza GR, Silva JG, Saraiva SRGL, Oliveira-Júnior RG, Quíntans JSS, Barreto RSS, Bonjardim LR, Cavalcanti SCH, Quíntans-Júnior, LJ. 2013. Borneol, a Bicyclic Monoterpene Alcohol, Reduces Nociceptive Behavior and inflammatory Response in Mice. The Scientific World Journal 2013: 1-5.. Amirghofran, Z, Hashemzadeh R, Javidnia K, Golmoghaddam H, Esmaeilbeig A. 2011. In vitro immunomodulatory effects of extracts from three plants of the Labiatae family and isolation of the active compound(s). J. Immunotoxicol 8: 265-273. Anamura S, Dohi T, Shirakawa M, Okamoto H, Tsujimoto A.1988. Effects of phenolic dental medicaments on prostaglandin synthesis by microsomes of bovine tooth pulp and rabbit kidney medulla. ArchOralBiol 33: 555-560. Ayyanar M, Ignacimuthu S. 2009. Herbal medicines for wound healing among tribal people in Southern India: Ethnobotanical and Scientific evidences. Int J Appl Res Nat Prod 2(3): 2942. Badr G, Alwasel S, Ebaid H, Mohany M, Alhazza I. 2011. Perinatal supplementation with thymoquinone improves diabetic complications and T cell immune responses in rat offspring. Cell Immunol 267: 133-140.. Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils – A Review. Food Chem Toxicol 46(2): 446-475..

(41) 40. Bastos JF, Moreira IJ, Ribeiro TP, Medeiros IA, Antoniolli AR, De Souza DP, Santos MR. 2010. Hypotensive and vasorelaxant effects of citronellol, a monoterpene alcohol. Basic Clin Pharmacol Toxicol 106(4):331–337.. Bhalla Y, Gupta VK, Jaitak V. 2013. Anticancer activity of essential oils: A review. J Sci Food Agric. Jun 14. doi: 10.1002/jsfa.6267.. Boateng JS, Matthews KH, Stevens HN, Eccleston GM. 2012. Wound Healing Dressings and Drugs Delivery Sytems: A Review. Journal of Pharmaceutical Sciences 97(8), 2892-2923. Braga PC, dal Sasso M, Culici M, Bianchi T, Bordoni L, Marabini L. 2006. Antiinflammatory activity of thymol inhibitory effect on the release of human neutrophil elastase. Pharmacology 77: 130-136.. Butler MS. 2008. Natural products to drugs: natural product-derived compounds in clinical trials. Nat Prod Rep 25: 475-516. Candan F, Unlü M, Tepe B, Daferera D, Polissiou M, Sökmen A, Akpulat HA, 2003. Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp. Millefolium Afan. (Asteraceae). J Ethnopharmacol. 87: 215-220. Chan EWC, Lim YY, Wong LF, Lianto FS, Wong SK, Lim KK, Joe CE, Lim TY. 2008. Antioxidant and tyrosinase inhibition properties of leaves and rhizomes of ginger species. Food Chem 109: 477-483.. Chang IM, Ryu JC, Park YC, Yun HS, Yang KH. 1983. Protective activities of aucubin against carbon tetrachloride-induced liver damage in mice. Drug Chem Toxicol.6(5): 443-453..

(42) 41. Chang IM.1998. Liver-protective activities of aucubin derived from traditional oriental medicine. Res Commun Mol Pathol Pharmacol 102(2): 189-204. Chang KS, Yun HS and Chang IM. 1989. Effects of aucubin on DNA and RNA biosyntheses in sarcoma 180 cells. Korean Biochem J 22: 322-326. Chen C, Schultz GS, Bloch M, Edwards PD, Tebes S, Mast BA. 1999. Molecular and mechanistica validation of delayed healing rat wounds as a model for human chronic wounds. Wound Repair an Regenaration 7(6): 486-494.. Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C. 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One, 5(1): e8613. Cherng JM, Shieh DE, Chiang W, Chang MY, Chiang, LC. 2007. Aucubin has no effect on proliferation in cancer cells derived from nine human organs, including the lungs. Biosci Biotechnol Biochem 71: 1500-1504.. Cooper DM. Optimizing wound healing. A practice within nursing's domain. Nurs. Clin. North Am 25(1): 165-177, 1990.. Dagne E, Bisrat D, Alemayehu M, Worku T. 2000. Essential oils of twelve eucalyptus species from Ethiopia. J Essent Oil Res 12(4): 467-470. Dai JP, Chen J, Bei YF, Han BX, Wang S. 2009. Influence of borneol on primary mice oral fibroblasts: a penetration enhancer maybe used in oral submucous fibrosis. J Oral Pathol Med 38(3): 276-281..

(43) 42. Dash GZ, Murthy PN. 2011. Studies on Wound Healing Activity of Heliotropium indicum Linn. Leaves on Rats ISRN Pharmacol 2011: 847980. Davini E, Iavarone C, Trogolo C, Aureli P, Pasolini B. 1986. The quantitative isolation and antimicrobial activity of the aglycone of aucubin. Phytochemistry 25: 2420-2422. Denkinger MD, Nikolaus T, Denkinger C, Lukas A. 2012. Physical activity for the prevention of cognitive decline. Current evidence from observational and controlled studies. Z Gerontol Geriatr 45(1): 11-16.. Diegelmann, RF, Evans, MC. 2004. Wound healing: An overview of acute, fibrotic and delayed healing. Front Biosci 9: 283-289. Gálvez M, Martín-Cordero C, Ayuso MJ. 2005. Iridoids as DNA topoisomerase I poisons. J Enzyme Inhib Med Chem 20(4): 389-392.. González A, Strumia, MC, Alvarez Igarzabal CI. 2011. Cross-linked soy protein as material for biodegradable films: Synthesis, characterization and biodegradation. Journal of Food Engineering 106(4): 331-338.. Gottrup F, Agren MS, Karlsmark T. 2000. Models for use in wound healing research: A survey focusing on in vitro and in vivo adult soft tissue. Wound Repair and Regeneration 8(2): 83-96. Guimarães AG, Quintans JS, Quintans- Jr LJ. 2013. Monoterpenes with analgesic activity – a systematic review. Phytother Res 27(1): 1-15. Grundy SM. 2004. Obesity, metabolic syndrome and cardiovascular diseases. J Clin Endocrinol Metab 89: 2595-2600..

(44) 43. Haeseler G, Maue D, Grosskreutz J, Bufler J, Nentwig B, Piepenbrock S, Dengler R, Leuwer M. 2002. Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymo land menthol. European Journal of Anaesthesiology 19: 571-579. Hanna JR, Giacopelli JA. 1997. A review of Wound Healing and Wound Dressing Products. The Journal of Foot and Ankle Surgery 36(1): 2-14.. Hassan SB, Gali-Muhtasib H, Göransson H, Larsson R. 2010. Alpha terpineol: a potential anticancer agent which acts through suppressing NF-kappaB signalling. Anticancer Res. 30(6): 1911-9. He X, Lu Q, Liu Y. 2006. Effects of borneol injection on inflammation in focal cerebral ischemia reperfusion rats. Hua Xi Yao Xue Za Zhi 21: 523-526. He X, Lu Q, Liu Y. 2006. Effects of borneol injection on inflammation in focal cerebral ischemia reperfusion rats. Hua Xi Yao Xue Za Zhi 21: 523-526. Held S, Schieberle P, Somoza V. 2007. Identification of α-terpineol as an anti-inflammatory component of orange juice by in vitro and ex vivo studies. In Recent Highlights in Flavor Chemistry & Biology, 8th ed.; Proceedings of the Wartburg Symposium on Flavor Chemistry. and Biology, Eisenach, Germany, 27 February–2 March 2007; Deutsche Forschungsanstalt für Lebensmittelchemie: Garching, Germany, 239-244. Hoareau L, DaSilva E. 1999. Medicinal plants: a re-emerging health aid. Electronic Journal of Biotechnology 2: 56-71.. Hung,J.Y., Yang CJ, Tsai YM, Huang HW, Huang MS. 2008. Antiproliferative activity of aucubin is through cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells. Clin Exp Pharmacol Physiol 35: 995-1001;.

(45) 44. Ishiguro K, Yamaki M, Takagi S. 1982. Studies on the iridoid- related compound. I. On the antimicrobial activity of aucubigenin and certain iridoid aglycones. Journal of the pharmaceutical Society of Japan 102(8): 755-759.. Jin J, Song M, Hourston DJ. 2004. Novel chitosan based films cross-linked by genipin with improved physical properties. Biomacromolecules 5: 162-168. Juhás S, Cikos S, Czikková S, Veselá J, Il’ková G, Hájek T, Domaracká K, Domaracky M, Bujnáková D, Rehák P, Koppel J. 2008. Effects of borneol and thymo quinone on TNBSinduced colitis in mice. J FoliaBiol 54: 1-7. Karpanen TJ, Worthington T, Hendry ER, Conway BR, Lambert PA. 2008. Antimicrobial efficacy of chlorhexidine digluconate alone and in combination with eucalyptus oil, tea tree oil and thymol against planktonic and biofilm cultures of Staphylococcus epidermidis. Journal of Antimicrobial Chemotherapy 62: 1031-1036.. Kawata, J, Kameda M, Miyazawa M. 2008. Cyclooxygenase-2 inhibitory effects of monoterpenoids with a p-methane skeleton. Int J Essent Oil Ther . 2(4): 145-148. Khalil Z, Pearce AL, Satkunanathan N, Storer E, Finlay-Jones JJ, Hart PH. 2004. Regulation of wheal and flare by tea tree oil: complementary human and rodent studies. J invest Dermatol 123: 683-690.. Khorshid F, Ali SS, Alsofyani T, Albar H. 2010. Plectranthustenuiflorus (Shara) promoteswoundhealing: Invitro and invivo studies. International Journal of Botany 6(2): 6980..

(46) 45. Kitano A, Saika S, Yamanaka O, Ikeda K, Reinach OS, Nakajima Y, Okada Y, Shirai K, Ohnishi Y. 2006. Genipin suppresses subconjunctival fibroblast migration, proliferation and myofibroblast transdifferentiation. Ophthalmic Res 38: 355-360. Kitano A, Saika S, Yamanaka O, Reinach PS, Ikeda K, Okada Y, Shirai K, Ohnishi Y. 2006. Genipin suppression of fibrogenic behaviors of the alpha-TN4 lens epithelial cell line. J Cataract Refract Surg 32: 1727-1735.. Knobloch K, Pauli A, Iberl B, Weigand H, Weis N. 1989. Antibacterial and antifungal properties of essential oil components. J Essential Oil Res 1: 119-128. Koo HJ, Song YS, Kim HJ, Park EH. 2004. Antiinflammatory effects of genipin, an active principle of gardenia. Eur.J.Pharmacol.495: 201-208. Kordali S, Kotan R, Mavi A, Cakir A, Ala A, Yildirim A. 2005. Determination of the chemical composition and antioxidant activity of the essential oil of Artemisia dracunculus and of the antifungal and antibacterial activities of Turkish Artemisia absinthium, Artemisia dracunculus, Artemisia santonicum and Artemisia spicigera essential oils. J Agric Food Chem 53(24): 9452-9458. Langeveld WT, Veldhuizen EJ, Burt SA. 2013. Synergy between essential oil components and antibiotics: a review. Crit Rev Microbiol. Feb 28. Lee SW, Kho HS, Lee SG. 1999. The effects of iridoid compounds on wound healing. J Korean Acad Oral Med 24: 137-142.. Lei J, Xue HY, Jin LJ, Li SY, Xu YP. 2008. Antioxidant and pancreas-protective effect of aucubin on rats with streptozotocin-induced diabetes. European Journal of Pharmacology 582: 162-167..

(47) 46. Li FR, Yang JX, Shen XT, Li SJ. 2004. Antioxidative activities of aucubin invitro. J Shaanxi Norm Univ 32: 98-101.. Li JW, Vederas JC. 2009. Drug discovery and natural products: end of an era or an endless frontier? Science 325: 161-165. Li YH, Sunet XP, Zhang YQ, Wang NS. 2008. The Antithrombotic Effect of Borneol Related to Its Anticoagulant Property. The American Journal of Chinese Medicine 36(4): 719-727. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, Ioannidis JPA, Clarke M, Devereaux PJ, Kleijnen J, Moher D. 2009. The PRISMA Statement for Reporting Systematic Reviews and Meta-Analyses of Studies That Evaluate Health Care Interventions: Explanation and Elaboration. BMJ 339: b2700. Liu R, Zhang L, Lan X, Li L, Zhang TT, Sun JH, Du GH. 2011. Protection by borneol on cortical neurons against oxygen-glucose deprivation/reperfusion: Involvement of antioxidation and anti-inflammation through nuclear transcription factor κappaB signaling pathway. Neuroscience 176: 408-419. Mai LM, Lin CY, Chen CY, Tsai YC. 2003.. Synergistic effect of bismuth subgallate and. borneol, the major components of Sulbogins, on the healing of skin wound. Biomaterials 24: 3005-3012. Maria DCR, Rosa MG, Manez S and Rios JL. 1994. Structural considerations on the iridoids as anti-inflammatory agents. Plant Med 60: 232-234. Mastelic J, Jerkovic I, Blazevic I, Poljak-Blazi M, Borovi S, Ivancic-Bace I, Smrecki V, Zarkovic N, Brci-Kostic K, Vikic-Topic DZ, Müller N. 2008. Comparative study on the.

(48) 47. antioxidant and biological activities of carvacrol, thymol and eugenol derivatives. J Agric Food Chem 56: 3989-3996.. McKernan RM, Whiting PJ. 1996. Which GABAA-receptor subtypes really occurin the brain?.Trends Neurosci 19(4): 139-143. Meenakshi S, Raghavan G, Nath V, Ajay Kumar SR, Shanta M. 2006. Antimicrobial, wound healing and antioxidant activity of Plagiochasma appendiculatum Lenm et Lind. J Ethnopharmacol 107: 67-72.. Menezes IA, Barreto CM, Antoniolli AR, Santos MR, DeSousa DP. 2010. Hypotensive activity of terpenes found in essential oils. ZNaturforschC 65:562–566. Mentz PR, Petrovick. 1999. Farmacognosia da planta medicamento . Editora da Universidade Federal do Rio Grande do Sul, Porto Alegre. Nalawade SM, Sagare AP, Lee CY, Kao CL, Tsay HS. 2003. Studies on tissue culture of Chinese medicinal plant resources in Taiwan and their sustainable utilization. J Botanical Bulletin of Academia Sinica 44: 79-98.. Nam KN, Choi YS, Jung HJ, Park GH, Park JM, Moon SK, Cho KH, Kang C, Kang I, Myung SO, Eunjoo HL. 2010. Genipin inhibits the inflammatory response of rat brain microglial cells. Int Immunopharmacol 10: 493-499. Nodari RO, Guerra MP. 1999. Biodiversidade: Aspectos Biológicos, geográficos, legais e éticos. Apud: Simões OMC, Schenkel RP, Gosmann G, Mello PCJ, Mentz AL, Petrovick PR.. Farmacognosia da planta medicamento. Editora da Universidade Federal do Rio Grande do Sul. Porto Alegre..

(49) 48. Oberlies NH, Kroll DJ. 2004. Camptothecin and taxol: historic achievements in natural products research.J. Nat. Prod. 67: 129. Oliveira MG, Marques RB, de Santana MF, Santos AB, Brito FA, Barreto EO, De Sousa DP, Almeida FR, Badauê-Passos D Jr, Antoniolli AR, Quintans-Júnior LJ. 2012. α-terpineol reduces mechanical hypernociception and inflammatory response. Basic Clin Pharmacol Toxicol. 111(2): 120-125.. Ovington LG. 2007. Advances in wound dressings. Clin Dermatol 25: 33-8. Park KS, Chang IM. 2004. Anti-inflammatory activity of aucubin by inhibition of tumor necrosis factor-alpha production in RAW264.7cells. PlantaMed 70:778-779. Park KS. 2013. Aucubin, a naturally occurring iridoid glycoside inhibits TNF-α-induced inflammatory responses through suppression of NF-κB activation in 3T3-L1 adipocytes. Cytokine 62(3): 407-412.. Park MJ, Gwak KS, Yang I, Kim KW, Jeung EB, Chang JW, Choi IG. 2009. Effect of citral, eugenol, nerolidol and α-terpineol on the ultrastructural changes of Trichophyton mentagrophytes. Fitoterapia 80(5): 290-296. Park SN, Lim YK, Freire MO, Cho E, Jin D, Kook JK. 2012. Antimicrobial effect of linalool and α-terpineol against periodontopathic and cariogenic bacteria. Anaerobe 18(3): 369-372. Park TJ, Park YS, Lee TG, Ha H, Kim KT. 2003. Inhibition of acetylcholine- mediated effects by borneol. Biochem Pharmacol 65: 83-90. Parton LE, Ye CP, Coppari R. 2007. Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity. Nature 449: 228-233..

(50) 49. Prieto JM, Iacopini P, Cioni P, Chericoni S. 2007. In vitro activity of the essential oils of Origanum vulgare, Satureja montana and their main constituents in peroxynitrite-induced oxidative processes. Food Chem.104: 889-895. Pujana, MA, Pérez-Álvarez L, Iturbe LCC, Katime I. 2013. Biodegradable chitosan nanogels crosslinked with genipin. Carbohydrate Polymers 94: 836-842. Rajeswara RBR, Syamasundar KV, Rajput DK, Nagaraju G, Adinarayana G. 2012. Biodiversity, conservation and cultivation of medicinal plants. Journal of Pharmacognosy 3(2): 59-62. Rakotondramasy VC, Mouriès C, Cachet X, Neghra A, Mourabet ME, Tillequin F, Koch M, Deguin B. 2010. A novel series of cytotoxic iridoid glucosides derived from aucubin: Design, synthesis and structure–activity relationships. European Journal of Medicinal Chemistry 45(6): 2314-2320 Ravenhill J. 2006. Is China an economic threat to southeast Asia? Asian survey 46(5): 653674. Rawat S, Singh R, Thakur P, Kaur S, Semwal A. 2012. Wound healing Agents from Medicinal Plants: A Review. Asian Pacific Journal of Tropical Biomedicine S1910-S1917. Recio MC, Giner RM, Manez S, Rios JL. 1994. Structural considerations on the iridoids as anti-inflammatory agents. Planta Med 60:232-234. Reinke JM, Sorg H. 2012. Wound Repair and Regeneration. European Surgical Research 49: 35-43..