REDE NORDESTE DE BIOTECNOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA

MELYSSA LIMA DE MEDEIROS

AVALIAÇÃO DO EFEITO CICATRIZANTE DO ÓLEO DE COPAÍBA VEICULADO EM SISTEMAS SNEDDS E PROCESSOS TERAPÊUTICOS EM MODELO EXPERIMENTAL IN VIVO DE LESÕES CUTÂNEAS

NATAL/RN 2019

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE REDE NORDESTE DE BIOTECNOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA

MELYSSA LIMA DE MEDEIROS

AVALIAÇÃO DO EFEITO CICATRIZANTE DO ÓLEO DE COPAÍBA VEICULADO EM SISTEMAS SNEDDS E PROCESSOS TERAPÊUTICOS EM MODELO EXPERIMENTAL IN VIVO DE LESÕES CUTÂNEAS

Tese apresentada ao programa de Pós-graduação em Biotecnologia da Rede Nordeste de Biotecnologia, RENORBIO, como requisito para a obtenção do título de Doutor em Biotecnologia.

Áreas de Concentração: Biotecnologia em Saúde

Orientadora: Profa. Dra. Maria Aparecida Medeiros Maciel

NATAL/RN 2019

MELYSSA LIMA DE MEDEIROS

AVALIAÇÃO DO EFEITO CICATRIZANTE DO ÓLEO DE COPAÍBA VEICULADO EM SISTEMAS SNEDDS E PROCESSOS TERAPÊUTICOS EM MODELO EXPERIMENTAL IN VIVO DE LESÕES CUTÂNEAS

Tese apresentada ao programa de Pós-graduação em Biotecnologia da Rede Nordeste de Biotecnologia, RENORBIO, como requisito para obtenção da aprovação do Exame de Qualificação da referida candidata, no Programa de Biotecnologia da Rede Renorbio.

Aprovada em 29 de julho de 2019, por:

______Profa. Dra. Maria Aparecida Medeiros Maciel Examinador Interno e Presidente

______Prof. Dr. Valdir Florêncio da Veiga Júnior Examinador Externo

______Prof. Dr. José Heriberto Oliveira do Nascimento Examinador Externo

______Prof. Dr. Ricardo Luiz Cavalcanti de Albuquerque Júnior Examinador Interno

______Prof. Dra. Caroline Addison Carvalho Xavier de Medeiros Examinador Interno

DEDICATÓRIA

A Deus, pelo dom da vida e bênçãos em minha caminhada. Aos meus pais, Danny Moura e Terezinha Lima, pelo incentivo, confiança e incontestável exemplo de sabedoria. A meu esposo e grande amor, Jung Dantas, por dividir comigo o nosso bem mais precioso, a família. Aos meus filhos, Fabrício, Enzo, Benício e Maria Luiza, a razão para todo esse esforço e minha fonte de inspiração. As minhas irmãs e familiares, o alicerce para concretização desse sonho.

AGRADECIMENTOS

A Profa. Dra. Maria Aparecida Medeiros Maciel, pela oportunidade de trilhar um caminho em busca de aprimoramento intelectual e também, pela contribuição na concretização deste sonho. Agradeço o incentivo e suporte em todos os momentos que pensei em desistir, ficando ao meu lado e acompanhando cada passo do meu desenvolvimento. Sem dúvida é minha “mãe científica”, que na maioria das vezes acreditou mais em mim do que eu mesma. Uma das pessoas mais incríveis que tenho ao meu lado! Ao Prof. Dr. Irami Araújo Filho, um honrado pesquisador e um “grande ser humano”, comprometido com sua profissão e que vivencia os princípios do método científico de uma forma encantadora. Muito obrigada pela colaboração nos estudos em animais e pelos conhecimentos compartilhados durante as discussões técnico-científicas. À Profa. Dra. Amália Cinthia Meneses do Rêgo, pela parceria, cordialidade e contribuições nos estudos em animais. Agradeço, especialmente, a Profa. Dra. Maria das Dores Melo, por compartilhar comigo parte de seu imenso saber no campo da elaboração dos projetos de uso em animais, e pelo cordial acolhimento, apoio e amizade ao longo do tempo. Ao Prof. Dr. Valdir Florêncio da Veiga Júnior , pela atenção e colaboração na parte das caracterizações químicas da amostra de óleo de copaíba. Ao Prof. Magnaldo Tavares, doutorando do PPGB-Renorbio, pelo apoio, confiança, suporte e troca de conhecimentos, em algumas das disciplinas cursadas na etapa final do meu doutorado, bem como em alguns dos experimentos realizados com animais. À Profa. Dra. Rejane Carvalho e, especialmente, ao Dr. Ciro Soares, pela inestimável colaboração e oportunas contribuições com as análises histopatológicas e estatísticas. Aos parceiros de pesquisa Profa. Dra. Denise Porfirio Emerenciano, bem como ao doutorando da Rede Renorbio, Joherbson Deivid dos Santos Pereira e ao aluno de IC, Bernardo Bruno Dias Baracho pela parceria na pesquisa dos bioformulados, desenvolvidos a base de óleo de copaíba.

Aos meus queridos alunos de iniciação científica, Everardo Lucena e Alana Guiotto que são exemplos de dedicação e comprometimento com o desenvolvimento da ciência. Gratidão pelo cuidado e empenho em algumas etapas desta pesquisa. Aos professores do Programa do Programa de Pós-Graduação em Biotecnologia da Rede Renorbio, pelo comprometimento com a ciência. Deixo meu agradecimento especial às professoras Dra. Lucymara F. Agnez Lima, Dra. Silvia Batistuzzo e Dra. Katia Scortecci, pelo apoio e oportunidades de desenvolvimento. Aos professores Dra. Sílvia Regina Batistuzzo de Medeiros, Dr. Hugo Alexandre de Oliveira Rocha e José Heriberto Oliveira do Nascimento, pela participação no meu Exame de Qualificação desta tese, e contribuições. A Rede RENORBIO, em especial a Universidade Federal do Rio Grande do Norte, por possibilitarem juntamente com s instituição parceira UnP, a realização do presente estudo. Aos meus colegas de doutorado pelos vários momentos compartilhados, convívio e cumplicidade. Aos meus colegas de trabalho da Rede Laureate e da Universidade Potiguar (UnP), que me apoiaram e contribuíram para a conclusão de mais uma etapa da minha trajetória acadêmica. Especialmente à Profa. Patrícia Klahr pelo apoio, incentivo e parceria, que foram indispensáveis para o fechamento desta etapa profissional que parecia uma missão impossível. Conciliar o meu trabalho na Rede Laureate e na UnP, e ainda me dedicar ao doutorado, foram, de fato, experiências fantásticas que exigem comprometimento e dedicação exaustivos. Portanto, sem o seu apoio eu não teria conseguido gerenciar a questão tempo. Muito obrigada! Finalmente, aos meus pais e minha família pelo companheirismo e compreensão nas minhas ausências em várias ocasiões.

“Ninguém vence sozinho, Nem no campo, Nem na vida”. Papa Francisco

RESUMO

O manejo da ferida crônica compreende um importante aspecto da prática médica e requer tratamento específico para que o tratamento terapêutico seja eficaz. Registros históricos mostram que o óleo de copaíba é utilizado preferencialmente para promover a cicatrização de feridas e tratar doenças inflamatórias e infecciosas. No entanto, sua baixa solubilidade em água limita seu uso farmacológico. Neste sentido, na presente pesquisa, sistemas nanoemulsionados do tipo SNEDDS desenvolvidos à base de óleo de copaíba (OC) em baixa concentração (0,5%), foram preparados na presença de Tween® 80 e meio aquoso (neutro ou salino). O encapsulamento deste óleo bioativo em sistema de liberação de fármacos SNEDDS (self-nanoemulsion drug delivery system) possibilita sua liberação lenta e prolongada com aplicação tópica em procedimento experimental de cicatrização de feridas diabéticas e não diabéticas. Análises físico-químicas foram realizadas para caracterizar o OC nas formas in natura e encapsulado em sistemas SNEDDS. As bionanoformulações de OC foram preparadas sem o uso de co-surfactante o que justifica sua maior importância nas aplicações farmacológicas, em função da redução de ingredientes carreadores que possam causar reações adversas. Especificamente, os sistemas SNEDDS possuem OC e óleo de girassol em mistura (1:1), percentuais variáveis de surfactante (da classe dos Tweens) e água (pH neutro ou salino). Os sistemas SNEDDS-OC foram avaliados em procedimentos terapêuticos de cicatrização. Os ensaios in vivo para cicatrização de feridas (não infectada e infectada) foram realizados com ratos Wistar ( Rattus novergicus albinus ). Os animais foram divididos em grupos de 5 animais (n = 5), submetidos a cirurgia para retirada de retalho cutâneo na região dorsal e receberam tratamento tópico diário, com os sistemas SNEDDS-OC, durante o período pós-operatório. Análises histopatológicas e imunohistoquímicas foram realizadas após 7 e 14 dias de tratamento para ambos os grupos. Análises histomorfométricas revelaram um menor número de neutrófilos e linfócitos nos grupos tratados (7 dias), bem como aumento da colagênese, proliferação de fibroblastos e maior espessamento epitelial, que por comparação, foram mais evidentes nos grupos tratados (14 dias). Nos grupos controle, o atraso no reparo tecidual foi relacionado à presença de queratinócitos em função do aumento da imunorreatividade para IL-1β. O maior número de células imunocoradas com FGF-2 foi associado à melhora da colagênese e do processo de reparo tecidual cutâneo. Além disso, foi realizada uma abordagem comparativa in vivo dos processos terapêuticos, em modelo experimental de lesões cutâneas, na qual foi avaliado o efeito da terapia com laser de baixa intensidade (LLLT) na angiogênese e imunoexpressão da metaloproteinase-2 de matriz, na ausência e/ou presença dos sistemas SNEDDS-OC.

Palavras-Chave: Óleo de copaíba; Sistemas carreadores SNEDDS; Lesões cutâneas; Processos terapêuticos de cicatrização; Análises histológicas.

ABSTRACT

Chronic wound management comprises an important aspect of medical practice and requires specific treatment for effective therapeutic treatment. Historical records show that copaiba oil is used preferentially to promote wound healing and treat inflammatory and infectious diseases. However, its low solubility in water limits its pharmacological use. In this sense, in the present research, self- nanoemulsion drug delivery systems (SNEDDS) developed by using low- concentration (0.5%) of copaiba oil (CO) were prepared in the presence of Tween® 80 and aqueous (neutral or saline) medium. The encapsulation of this bioactive oil in a SNEDDS-type system allows its slow and prolonged release with topical application in an experimental procedure for the healing of diabetic and non-diabetic wounds. Physical-chemical analyzes were performed to characterize the in natura CO and also encapsulated in SNEDDS formulations. The CO-nanoformulations were prepared without the use of co-surfactant, which justifies its foremost importance in pharmacological applications, due to the reduction of carrier ingredients that may cause adverse reactions. Specifically, the SNEDDS contain copaiba oil and sunflower oil (1:1) as oil phase, variable percentages of surfactant (Tween class) and water on neutral or saline pH. The SNEDDS-CO systems were evaluated in therapeutic healing procedures, in which the in vivo assays for wound healing (uninfected and infected) were performed on Wistar rats ( Rattus novergicus albinus ). The animals were divided into groups of 5 animals (n = 5), submitted to surgery to remove cutaneous flap in the dorsal region and received daily topical treatment with SNEDDS-CO systems during the postoperative period. Histopathological and immunohistochemical analysis were performed after 7 and 14 days of treatment for both groups. Histomorphometric analysis revealed a lower number of neutrophils and lymphocytes in the treated groups (7 days), as well as increased collagenesis, proliferation of fibroblasts and increased epithelial thickening, which by comparison were more evident in the treated groups (14 days). In the control groups, the delay in tissue repair was related to the presence of keratinocytes as a function of the increased immunoreactivity for IL-1β. The highest number of cells immunocornized with FGF-2 was associated with the improvement of collagenase and the tissue repair process. In addition, an in vivo comparative approach of therapeutic procedures was performed in an experimental model of cutaneous lesions, in which the effect of low intensity laser therapy (LILT) on angiogenesis and immunoexpression of matrix metalloproteinase-2 was evaluated in the absence and/or presence of SNEDDS-CO systems.

Key-words: Copaiba oil; SNEDDS-type nanoemulsion systems; Wound healing therapeutic processes; Histological analysis.

LISTA DE ABREVIATURAS, SÍMBOLOS E SIGLAS

ACP Água de coco em pó ANVISA Agencia Nacional de Vigilância Sanitária ATP Adenosina trifosfato A/O Água em óleo CE Células endoteliais cm Centímetro COX Ciclooxigenase ºC Graus Celsius IL-1β Interleucina um beta IL-6 Interleucina seis J Joule J/cm² Joule por centímetro quadrado LLLT Low level laser therapy/ terapia com laser de baixa intensidade m² Metro quadrdo mL Mililitro mm Milímetro MMP Metaloproteinase de matriz MMP-2 Metaloproteinase dois NEOCACP Nanoemulsão com óleo de copaíba em meio salinizado com água de coco em pó nm Nanômetro O/A Óleo em água OC Óleo de copaíba OG Óleo de girassol OMS Organização Mundial de Saúde pH Potencial de hidrogênio de uma solução SNEDDS Self-nanoemulsifying drug delivery system TNF-α Fator de necrose tumoral alfa VEGF Fator de crescimento do endotélio vascular W Watt

SUMÁRIO Dedicatória Agradecimentos Resumo Abstract Lista de Abreviaturas, Símbolos e Siglas

1. INTRODUÇÃO ...... 12 2. OBJETIVOS ...... 15 2.1 Objetivo Geral ...... 15 2.2 Objetivos Específicos ...... 15 3. JUSTIFICATIVA ...... 16 4. REFERENCIAL TEÓRICO ...... 18 4.1 Tecido Cutâneo e o Processo de Reparo Tecidual ...... 18 4.2 Efeito da LLLT no Reparo Tecidual Cutâneo ...... 22 4.3 O Gênero Copaifera e o Óleo Resina de Copaíba ...... 26 4.4 Biodisponibilidade de Fármacos via Sistemas Coloidais ...... 32 5. RESULTADOS E DISCUSSÃO ...... 35

5.1 Copaiba oil as a natural product challenge in the chemistry, pharmacological and biotecnological fields ...... 35 5.2. SNEDDS drug delivery system based on copaiba oil improves wound healing by promoting angiogenesis, neocollagenesis and reducing inflammatory reaction ...... 70 5.3 Effect of low-intensity laser therapy on angiogenesis and MMP-2 immunoexpression in wound repair ...... 96 5.4 Improvement of wound healing process by combining SNEDDS copaiba oil system and low level laser therapy on the inflammatory phase of wound healing ...... 110 5.5. Patente Número de Registro: BR102017014800-9 ...... 137 6. CONCLUSÕES GERAIS ...... 138 7. PRODUÇÃO EXTENSIVA AO PROJETO DE TESE ...... 140 8. REFERÊNCIAS ...... 141 9. ANEXO 1-Comprobatório das Publicações ...... 190 10. ANEXO 2-Comprobatório dos Projetos Aprovados no CEUA ...... 196

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1. INTRODUÇÃO Feridas provenientes de complicações vasculares, traumáticas, metabólicas, imobilismo prolongado, diabetes, dentre outros fatores, requerem assistência especializada. O manejo de feridas crônicas, mesmo com todos os avanços científicos que consistem no avanço da compreensão celular e molecular dos processos de cicatrização, juntamente com esforços colaborativos em diferentes áreas de pesquisa, como a biotecnologia por exemplo, ainda é considerado complexo e oneroso, repercutindo diretamente nas condições de saúde e qualidade de vida do paciente. Os gastos estimados em tratamentos de feridas crônicas com beneficiários do Medicare, sistema de seguro de saúde gerido pelo governo americano, atingem mais de US$32 bilhões. O agravamento dos índices estatísticos de lesões cutâneas crônicas consiste no envelhecimento da população mundial (BARAKAT-JOHNSON et al., 2019; EMING et al., 2014; GURTNER et al., 2008; NUSSBAUM et al., 2018; MANDELBAUM et al., 2003; MARTIN; NUNAN, 2015; SEN et al., 2009, 2019). No processo de reparo tecidual, a resposta inflamatória é considerada um fator determinante e as falhas mais relevantes ocorrem nos estágios iniciais, produzindo edema acentuado, reduzindo a proliferação vascular e os elementos celulares, como leucócitos, macrófagos e fibroblastos. Um dos desafios da abordagem terapêutica das lesões crônicas tem sido desenvolver estratégias eficazes para a modulação da resposta inflamatória. Desta forma, novas abordagens genéticas, moleculares e químicas estão sendo investigadas (DEBONE et al., 2019; HAWKINS et al., 2005; KRZYSZCZYK et al., 2018; LAROUCHE et al., 2018; MARTIN; NUNAN, 2015; SATISH, 2015; ROUSSELLE et al., 2018; WONG et al., 2015; WONG; CHOI, 2015 ). Neste contexto, destaca-se o interesse por fitomedicamentos advindos da biodiversidade em que se objetiva a melhoria de tratamentos com base no uso de recursos naturais. Dentre os recursos naturais advindos da biodiversidade brasileira, destaca-se o óleo de copaíba que por ser um agente anti-inflamatório, antimicrobiano, analgésico e cicatrizante, dentre outros usos terapêuticos, vem sendo amplamente extraído de diferentes espécies do gênero Copaifera , para diversos usos, em que se incluem as pesquisas científicas desenvolvidas na área da saúde. Neste sentido, apesar do efeito cicatrizante do óleo de copaíba ser amplamente difundido na área científica, existem relatos de reação adversa pelo 14 uso deste óleo na sua forma in natura (BRITO et al., 1998, 1999; CAVALCANTI NETO et al., 2005; VIEIRA et al., 2008; YASOJIMA et al., 2013). No entanto, em muitos outros estudos, a redução da concentração do óleo de copaíba, possibilitou avanços na terapêutica de cicatrização, tendo sido observado modulação da resposta inflamatória, aumento significativo dos fenômenos de proliferação de fibroblastos e deposição de colágeno em feridas (DE LIMA SILVA et al., 2009; ESTEVÃO et al., 2013; GUSHIKEN et al., 2017; LUCCA et al., 2015, 2018; MASSON-MEYERS et al., 2013; PAIVA et al., 2002). Apesar das vantagens acima mencionadas, a biodisponibilidade do óleo de copaíba é limitada devido à sua baixa solubilidade em água. Neste sentido, foram desenvolvidos sistemas de entrega de fármaco a base do óleo de copaíba, objetivando empreender seu uso em diferentes modelos farmacológicos (CAMPOS et al., 2017; DE MEDEIROS et al., 2019; DI SOTTO et al., 2018; LUCCA et al., 2018; OTAGUIRI et al., 2017). Em busca de novas estratégias para favorecer o processo de reparo tecidual cutâneo, o método LLLT de Terapia com Laser de Baixa Intensidade (Low-Level Laser Therapy) tem sido utilizada como uma opção terapêutica segura para acelerar os processos de cicatrização. Especificamente, os efeitos da LLLT no tratamento de feridas cutâneas estão sendo associados a modulação da resposta inflamatória e incremento da neoangiogênese, colagênese, contração da ferida e proliferação epitelial e de fibroblastos (FORTUNA et al., 2018; LI et al., 2007; OTTERÇO et al., 2018a). No presente estudo, preferencialmente, avaliou-se a ação cicatrizante do óleo de copaíba, veiculado em sistemas carreadores de fármaco, do tipo SNEDDS (self-nanoemulsifying drug delivery system), com ênfase no controle da resposta inflamatória de feridas cutâneas, tendo sido produzido evidências a cerca de uma possível intervenção para o tratamento de lesões cutâneas. Comparativamente, realizou-se estudo in vivo em modelo experimental de lesões cutâneas para dois processos terapêuticos, em que se utilizou laser de baixa intensidade para avaliação da angiogênese e imunoexpressão da metaloproteinase-2 de matriz, na ausência e/ou presença dos sistemas SNEDDS contendo o óleo de copaíba (SNEDDS-OC). O contexto descritivo deste documento de tese consiste em i) Introdução Geral; ii) Revisão da literatura, que abrange levantamentos bibliográficos 15 pertinentes aos processos de reparo tecidual de lesões cutâneas, em que se incluem o uso do método LLLT (Low-Level Laser Therapy); bem como aspectos histórico e farmacológico que destacam a importância do óleo de copaíba; iii) Resultados e Discussão, que se encontra organizado em forma de artigos científicos, cujos descritivos experimentais encontram-se inclusos de forma expandida; iv) Considerações Finais que equivale as conclusões gerais da tese; v) Referências que contém todas as citações presentes neste manuscrito de tese. Na parte dos Resultados e Discussão, no artigo 1 encontra-se uma revisão da literatura em que são destacados aspectos fitoquímicos e fitomedicinais em que se destacam formulações biotecnológicas desenvolvidas à base de óleo de copaíba. Este artigo foi intitulado “Copaiba oil as a natural product challenge in the chemistry, pharmacological and biotecnological fields” e será submetido ao periódico Current Medicinal Chemistry (JCR 3.469). No artigo 2, se incluem as caracterizações físico-químicas de um sistema nanoemulsionado (SNEDDS-type system) contendo 0.5% de óleo de copaíba e sua aplicação in vivo em cicatrização. Este artigo foi intitulado “SNEDDS drug delivery system based on copaiba oil improves wound healing by promoting angiogenesis, neocollagenesis and reducing inflammatory reaction” e será submetido ao periódico Industrial Crops and Products (JCR 3.849). No artigo 3, encontram-se descritos os resultados do método LLLT aplicado em modelo in vivo em cicatrização. Este artigo foi publicado no periódico Lasers in Medical Science (JCR 1.949), com título “Effect of low-intensity laser therapy on angiogenesis and MMP-2 immunoexpression in wound repair” (DOI: 10.1007/s10103-016-2080-y). O artigo 4 correlaciona o uso tópico de formulados SNEDDS (0,5% de óleo de copaíba) seguido de aplicação LLLT, objetivando-se a otimização do efeito terapêutico do óleo de copaíba. Este estudo possibilitou o depósito de uma patente (BR102017014800-9, Instituto Nacional de Propriedade Intelectual), que foi avaliada e publicada na RPI 2468. A referida patente foi intitulada “Formulados a base de óleo de copaíba biodisponibilizados para uso oral e tópico associado com aplicações terapêuticas na analgesia, inflamação e cicatrização”. Juntamente com esta patente, encontram-se em anexo ao manuscrito desta tese, os comprobatórios de outras produções acadêmicas correlatas aos sistemas SNEDDS desenvolvidos na presente pesquisa. 16

2. OBJETIVOS

2.1 Objetivo Geral

Avaliação do efeito cicatrizante do óleo de copaíba veiculado em sistemas SNEDDS em modelo experimental in vivo de lesões cutâneas.

2.2 Objetivos Específicos

Utilização do óleo de copaíba in natura obtido da espécie Copaifera multijuga Hayne e de uma amostra comercial como biomassas de formulações SNEDDS. Realização de análises físico-químicas para caracterização de amostras in natura e comercial de óleo de copaíba, via experimentos cromatográficos (HRGC-MS). Preparo de sistemas nanoemulsionados do tipo SNEDDS isentos de co- tensoativo, desenvolvidos a base de óleo de copaíba obtido de amostras in natura (SNEDDS-OC) e comercial (SNEDDS-OCC), e suas caracterizações físico-químicas. Avaliação do efeito cicatrizante dos bioprodutos SNEDDS-OC e SNEDDS-OCC em modelo experimental de ferida cutânea em ratos Wistar, após 7 e 14 dias de tratamento. Avaliação tecidual dos efeitos dos bioprodutos SNEDDS-OC e SNEDDS-OCC no processo de reparo tecidual cutâneo a partir da resposta inflamatória, reação de granulação, colagênese, neoangiogênese, repitelização, proliferação fibroblástica e imunoexpressão de anticorpos monoclonais para os antígenos CD3, CD105, α-SMA e IL-1β, em modelo experimental de ferida cutânea em ratos Wistar. Avaliação da terapia com laser de baixa intensidade (LLLT) na angiogênese e imunoexpressão da metaloproteinase-2 de matriz na ausência dos bioprodutos SNEDDS-OC e SNEDDS-OCC , em modelo experimental de ferida cutânea em ratos Wistar. Disponibilização de um protocolo científico que possibilite o uso de bioprodutos SNEDDS preparados a base de óleo de copaíba, associado à terapia com laser de baixa intensidade, que otimize o processo de reparo tecidual de lesões cutâneas.

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3. Justificativa

O Brasil possui uma das maiores biodiversidades do mundo, no entanto, o número de fitoterápicos registrados na Agência Nacional de Vigilância Sanitária (ANVISA) ainda é inexpressivo. Neste sentido, com a intenção de fomentar as pesquisas voltadas para tratamentos medicinais alternativos, o governo do Brasil criou estratégias para assegurar o uso racional de plantas medicinais e fitoterápicos, pela implementação do PPNPMF (Política e Programa Nacional de Plantas Medicinais e Fitoterápicos, aprovado por meio do Decreto nº 5.813, de 22 de junho de 2006), que compreende o Programa Nacional de Plantas Medicinais e Fitoterápicos (PNPMF), a Política Nacional de Práticas Integrativas e Complementares no SUS (PNPIC) e a Relação Nacional de Plantas Medicinais de Interesse do Sistema Único de Saúde (RENISUS). Desde então, 71 plantas medicinais têm aprovação da ANVISA para serem prescritas no Sistema Único de Saúde (SUS). Dentre as quais, encontram-se as copaíferas, em função do óleo de copaíba ter um rico histórico medicinal. A Organização Mundial de Saúde (OMS) reconhece a relevância da medicina tradicional na atenção primária à saúde, principalmente nos países em desenvolvimento, já que 80% da população faz uso de plantas medicinais que são utilizadas desde a antiguidade, até os dias atuais, como uma alternativa para prevenção e tratamento de doenças infeciosas, inflamatórias, oncológicas, parasitárias, dentre outras enfermidades (BLACKWELL et al., 2014; FABRICANT; FARNSWORTH, 2001). O PPNPMF promove o uso sustentável da biodiversidade, o desenvolvimento da cadeia produtiva e da indústria nacional e atua juntamente com as políticas PNPIC e RENISUS que estabelecem diretrizes para os avanços na área da medicina fitoterápica. Especificamente, busca-se: i) ampliar as opções terapêuticas aos usuários, com garantia de acesso a plantas medicinais, fitoterápicos e serviços relacionados à fitoterapia, com segurança, eficácia e qualidade, na perspectiva da integralidade da atenção à saúde, considerando o conhecimento tradicional sobre plantas medicinais; ii) construir o marco regulatório para produção, distribuição e uso de plantas medicinais e fitoterápicos a partir dos modelos e das experiências existentes no Brasil e em outros países; iii) promover pesquisa, desenvolvimento de tecnologias e 18 inovações em plantas medicinais e fitoterápicos, nas diversas fases da cadeia produtiva; iv) promover o desenvolvimento sustentável das cadeias produtivas de plantas medicinais e fitoterápicos e o fortalecimento da indústria farmacêutica nacional neste campo; v) promover o uso sustentável da biodiversidade e a repartição dos benefícios decorrentes do acesso aos recursos genéticos de plantas medicinais e ao conhecimento tradicional associado. O elevado valor do mercado farmacêutico mundial, estimado em mais de 800 bilhões de dólares, reforça a importância econômica do setor industrial farmacêutico e estimula a ampliação do desenvolvimento de novos formulados farmacológicos de origem natural que possam ser utilizados como meios biológicos (DIMASI et al., 2012). Nos últimos anos, a biotecnologia possibilitou avanços importantes para a medicina, incorporando um novo conceito na área da saúde. Neste contexto, destaca-se a nanomedicina, que objetiva conciliar o desenvolvimento tecnológico e a medicina moderna de forma a desenvolver novas terapêuticas ou otimizar as já existentes. A nanotecnologia é um ramo da ciência que engloba processos e suas tecnologias em que se incluem manipulação ou exploração de materiais em nano e microescala, permitindo o aprimoramento de materiais. As nanopartículas são materiais com dimensão inferior a 100 nm e seus estudos tornaram-se destaque na investigação científica e desenvolvimento tecnológico (CHHAYANI et al., 2013; DAS; BAKER; 2016; FIGUEIRAS et al., 2014; FORNAGUERA; GARCÍA-CELMA, 2017; JHA et al., 2011; SAINZ et al.; 2015). O óleo de copaíba é um dos produtos naturais mais utilizados pela população da Amazônia brasileira (RICARDO et al., 2018), o uso mais frequente é como agente cicatrizante de lesões cutâneas e mucosas. Com relação à biodisponibilidade e doseamento do óleo de copaíba, pesquisas biotecnológicas estão sendo desenvolvidas objetivando-se ação eficaz e redução da dose administrada e reações adversas. Em função da importância terapêutica do óleo de copaíba estima-se uma expansão significativa da sua exploração em função de novos estudos bionanotecnológicos, que poderão ampliar sua comercialização em escala mundial.

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4. REFERENCIAL TEÓRICO

4.1 Tecido Cutâneo e o Processo de Reparo Tecidual

A pele é um órgão extenso e complexo, que desempenha distintas funções e apresenta alterações estruturais de acordo com seu sítio anatômico. Histologicamente, está organizada em três camadas, epiderme, derme e tecido subcutâneo. O sistema tegumentar é constituído pela pele e seus anexos e funciona como uma barreira para o corpo protegendo-o contra diversos tipos de injúria, tais como: atrito, radiação ultravioleta, invasão de microrganismos, perda transepidérmica de água, dentre outros fatores. Além da função de barreira física, química e imunológica, o tecido cutâneo é capaz de responder a alterações do ambiente interno e externo, atuando na síntese de vitamina D, termorregulação, equilíbrio hidroeletrolítico e sensibilidade cutânea (EMING et al., 2014; McLAFFERTY; et al., 2012; PROKSCH et al., 2008; TAKEO et al., 2015). A cicatrização de feridas acontece a partir de um processo biológico complexo, requerendo esforços colaborativos e coordenados de muitos tecidos e linhagens celulares, como as dos sistemas imunológico e nervoso, a cascata de coagulação sanguínea e as vias inflamatórias. Quando a resposta fisiológica ao dano celular é bem-sucedida, após a restauração da homeostase e integridade cutânea, o processo de reparo será encerrado (EMING et al., 2014; GURTNER et al., 2008; MARTIN, 1997; SEN et al., 2009). Neste contexto, o reparo tecidual tem início após a perda de continuidade da pele e pode ser decorrente de estímulos internos ou externos, que podem ser físicos, químicos, elétricos e térmicos (GONZALEZ et al., 2016). Em humanos, a maior parte do fechamento das feridas acontece por re- epitelização, por se tratarem de lesões superficiais que envolvem apenas a epiderme e parte da derme, devolvendo as características estruturais e funcionais do tecido. No entanto, nos casos de feridas de espessura total, com destruição total da epiderme e derme, o processo de reparo do tecido tem início com a formação do tecido de granulação e posteriormente, acontecerá a re- epitelização da lesão (SORG et al., 2017).

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Classicamente o processo de reparo tecidual pode ser dividido em três distintas fases, a hemostasia e inflamação, proliferação e remodelação tecidual, que dura em torno de duas ou três semanas (TEPOLE; KUHL, 2013; SINGER; CLARK, 1999; SINNO; PRAKASH, 2013). Vale ressaltar que a resposta de reparação do paciente também pode interferir no controle e regulação de processos multicelulares e moleculares da cicatrização de feridas, comprometendo ou otimizando a inflamação, angiogênese, deposição de matriz extracelular e recrutamento celular. Desta forma, os mecanismos regulatórios do processo de reparo tecidual podem estar comprometidos com enfermidades associadas como o diabetes, câncer, desnutrição, imunossupressão, quadros infecciosos e outras doenças crônicas e vasculares, que juntos ou isoladamente, podem estender o período de cura da ferida por semanas, meses e anos. O atraso no processo de reparo tecidual poderá acontecer em qualquer lesão, no entanto, é mais frequente nos casos de insuficiência vascular, diabetes, exposição à radiação ou tempo prolongado de imobilismo no leito (EMING et al., 2014; GURTNER et al., 2008; MARTIN, 1997; SEN et al., 2009). A primeira fase ocorre logo após o dano tecidual é caracterizada por hemostasia e inflamação, tendo como objetivo limitar a hemorragia e a perda de fluidos, a remoção de tecidos desvitalizados e a prevenção de infecções. A exposição do colágeno ativa a cascata de coagulação sanguínea e ainda nesta fase, haverá vasoconstrição e adesão e agregação plaquetária, formando o coágulo ou trombo sanguíneo. Em seguida haverá a infiltração da fibrina, formando o arcabouço fibroso para sustentação das células que irão iniciar o processo de reparo tecidual. A degranulação das plaquetas ativa a cascata de complemento (C5), libera mediadores vasoativos e quimiocinas, que desempenham um papel fundamental na quimiotaxia para neutrófilos. Concluída a fase de hemostasia, há liberação de histamina pelos mastócitos, que irá favorecer a vasodilatação, aumentando o fluxo sanguíneo e a permeabilidade vascular e, consequentemente, a migração de células inflamatórias. Os neutrófilos são as primeiras células de defesa a migrarem para o local da lesão e logo em seguida chegam os monócitos, que se diferenciam em macrófagos, continuam o processo de desbridamento da ferida iniciado pelos neutrófilos e liberam citocinas, colagenases e fatores de crescimento que estimulam os 21 fibroblastos a iniciarem a angiogênese (EMING et al., 2014; GONZALEZ et al.,

2016; GURTNER et al., 2008; SORG et al., 2017; TAKEO et al., 2015). A segunda fase do processo de reparo tecidual é a proliferativa, que se caracteriza pela formação do tecido de granulação, epitelização, angiogênese e deposição de colágeno. Nesta etapa haverá a substituição da matriz extracelular por um tecido conjuntivo mais resistente e elástico, produzido pelos fibroblastos, formando um novo substrato para migração subsequente de queratinócitos. Os macrófagos e as células endoteliais continuam liberando fatores de crescimento, tais como: Fator de Crescimento de Fibroblasto (FGF), Fator de Crescimento do Endotélio Vascular (VEGF) e o Fator de Necrose Tumoral Alfa (TNF-α), promovendo, desta forma, a fibroplasia, colagênese e angiogênese. A partir da formação de novos vasos e reparação dos capilares lesionados, estarão garantidos a nutrição celular e a manutenção do tecido de granulação. Ainda nesta fase, ocorre a re-epitelização, quando os queratinócitos basais proliferam para assegurar o número de células para para a re-estruturação do tecido e consequentemente, migram das bordas da ferida para o leito da lesão, restabelecendo a função de barreira do epitélio. Além disso, como células contráteis, os miofibroblastos depositados nas bordas da lesão irão contribuir para a contração da ferida (DRISKELL et al., 2013; GONZALEZ et al., 2016; GURTNER et al., 2008; LI et al., 2007; SINNO; PRAKASH, 2013). A terceira fase do processo de reparo é a de maturação, que tem início duas ou três semanas após a lesão, podendo estender-se por anos. A principal característica dessa etapa é a formação da cicatriz e a contração da lesão. Além da deposição do colágeno pelos fibroblastos, também ocorrerá a degradação e o alinhamento das fibras, aumentando a resistência do tecido às forças mecânicas. Inicialmente, o colágeno é depositado aleatoriamente no tecido, mas ao longo do processo de remodelamento as fibras serão reorganizadas, tornando-se mais espessas e firmes. O colágeno tipo III será substituído pelo tipo I, a partir da atividade das metaloproteinases de matriz, secretadas pelos fibroblastos, garantindo resistência à tração, mas não alcançará a força equivalente a pele não lesionada (cerca de 70%). Durante esta fase, a maioria das células endoteliais, macrófagos e miofibroblastos afastam-se da área da lesão por fenômeno de emigração ou sofrem apoptose. O número de capilares também reduz, deixando a cicatriz menos vermelha (BAUM; ARPEY, 2005; 22

GURTNER et al., 2008; LEVENSON et al., 1965; LOVVORN et al., 1999; MONACO; LAWRENCE, 2003; SINNO; PRAKASH, 2013). Apesar da maior parte das lesões cutâneas cicatrizarem eficazmente, em algumas situações pode ocorrer excesso ou atraso no processo de cicatrização. A deposição excessiva de matriz extracelular e alterações na vascularização e proliferação celular podem causar o aparecimento de cicatrizes hipertróficas e queloides, muito comuns nos pacientes com queimaduras, nas cicatrizes decorrentes de procedimentos cirúrgicos ou em casos de alterações genéticas (AARABI et al., 2007; GURTNER et al., 2008). A cicatrização de uma determinada lesão cutânea envolve, portanto, diversas fases que se sobrepõem e depende de muitas vias de sinalização, bem como de múltiplos processos celulares e moleculares que ocorrem simultaneamente, modulando fatores de crescimento, citocinas, metaloproteinases de matriz, receptores celulares e componentes da matriz extracelular. De forma geral, as falhas mais importantes no processo de reparo ocorrem nos primeiros estágios, produzindo edema acentuado, reduzindo a proliferação vascular e diminuindo os elementos celulares, como leucócitos, macrófagos e fibroblastos. No entanto, nas feridas crônicas a incapacidade de re-epitelizar é um indicador de que as etapas normais do processo de cicatrização não estão acontecendo de forma coordenada. Entre os aspectos celulares que atrasam os processos de epitelização e cicatrização, destacam-se a quantidade reduzida de macrófagos e a desorganização ou o excesso do tecido de granulação (EMING et al., 2017; HAWKINS et al., 2005; LAROUCHE et al., 2018; SATISH, 2015; ROUSSELLE et al., 2018; 2019). Pesquisas relacionadas a compreensão dos efeitos fotobiológicos do método LLLT (Low-Level Laser Therapy) nos tecidos lesionados, mostram que os resultados de cicatrização vinculados a este método, dependem diretamente do comprimento de onda do laser e da absorção da energia pelos cromóforos, via absorção dos fótons pela enzima citocromo-c-oxidase (DE FREITAS; HAMBLIN, 2016). Os efeitos estimulantes de baixo nível de luz e inibitório em nível mais alto, no tratamento de feridas cutâneas encontram-se descritos em diversos estudos, como detalhados a seguir.

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4.2 Efeitos da Laser Terapia no Reparo Tecidual Cutâneo

O fenômeno físico da emissão estimulada foi proposto pela primeira vez em 1916, por Albert Einstein, tendo se tornado objeto de estudo de muitos grupos de pesquisa. Com relação ao efeito bioestimulador em feridas cutâneas, destaca-se, originalmente, o estudo realizado por Kovács et al. (1974) por terem identificado o efeito bioestimulador do laser HeNe (632,8 nm) em feridas cutâneas de pele de ratos. Atualmente, apesar dos efeitos terapêuticos não estarem completamente elucidados, a terapia pelo uso da amplificação de luz por emissão estimulada de radiação, tem sido utilizada e reconhecida como uma opção segura para acelerar o processo de cicatrização tecidual cutâneo. Os efeitos terapêuticos deste método consistem na modulação da resposta inflamatória, colagênese, contração da ferida, bem como pela proliferação epitelial e de fibroblastos (DA SILVA et al., 2013; FARIVAR et al., 2014; GOUNDER; GOUNDER, 2016; PARKER, 2007; VALADAS et al., 2019; XU et al., 2018). De acordo com a potência e o comprimento de onda (determinante da profundidade de penetração) que atinge os tecidos biológicos, os lasers terapêuticos são classificados como de alta intensidade (High-Intensity Laser Therapy - HILT) e baixa intensidade (Low-Level Laser Therapy - LLLT). O laser HILT, por exemplo, tem efeito de ablação e vem sendo utilizado em procedimentos cirúrgicos como cortes, coagulação e cauterização; enquanto os de baixa intensidade são atérmicos, e apresentam efeitos analgésico, anti- inflamatório e de bioestimulação (ENWEMEKA, 2005; KARU, 1999). Com relação ao comprimento de onda, os lasers são classificados em visíveis (400 a 700 nm) e invisíveis (700 a 1.000 nm), no entanto, ambos apresentam efeitos terapêuticos decorrentes da fotobiomodulação, diferindo apenas, pelas propriedades fotoquímica e fotofísica (DE FREITAS; HAMBLIN, 2016; KARU, 1987; 1988; 1989a; 1989b; SMITH, 1991, 2005). As pesquisas relacionadas a compreensão dos efeitos fotobiológicos da LLLT mostram que inicialmente ocorre a fotoativação da molécula fotorreceptora, principalmente a Citocromo C Oxidase da cadeia respiratória, em seguida a absorção da luz, o que leva a tradução e amplificação do sinal, e por fim, o desencadeamento das respostas bioativas. Após a absorção dos fótons ocorre 24 a ativação de diferentes vias de sinalização envolvendo espécies reativas de oxigênio (ROS), AMP cíclico, oxido nítrico e cálcio (Ca 2+ ), culminando com a ativação de fatores de transcrição e posteriormente, com o aumento da expressão de genes envolvidos na síntese de proteínas, migração e proliferação celular, sinalização da resposta anti-inflamatória, ativação de enzimas antioxidantes e proteínas antiapoptóticas (DE FREITAS; HAMBLIN, 2016; KARU, 1999; 2008; MÁRQUEZ-MARTÍNEZ et al., 2008; SMITH, 1991). A comprovação dos efeitos terapêuticos da terapia LLLT é ampla e se estende para outras aplicações, como descrito na Figura 1.

FIGURA 1. Efeitos terapêuticos decorrentes do uso da amplificação de luz por emissão estimulada de radiação. Fonte: autoria própria

Apesar da amplitude de estudos que avaliam os efeitos da LLLT em tecidos biológicos, de acordo com alguns autores, foram observadas imprecisões nas descrições de parâmetros de irradiação, como comprimento de onda, potência, tempo de irradiação, área do feixe, parâmetros de pulso, intervalo entre tratamentos, número de sessões, dentre outros aspectos. Portanto, ainda não há consenso e diretrizes claras para a determinação da dosimetria ideal bem como definição dos mecanismos de ação envolvidos nos efeitos biológicos (DA SILVA et al., 2010; HADIS et al., 2016; MUSSTTAF et al.; 2019; POSTEN et al., 2005; RODRIGUES et al., 2015; WHINFIELD; AITKENHEAD, 2009). A seguir encontram-se destacadas algumas pesquisas decorrentes do uso da amplificação de luz por emissão estimulada de radiação. 25

Fortuna et al. (2018), aplicaram a LLLT, utilizando o laser de gálio- alumínio-arsênio (GaAlAs), com comprimento de onda de 670 nm e densidade de energia 4 J/cm 2, em feridas cutâneas de ratos Wistar, tendo sido observado neoangiogênese e colagênese no remodelamento tecidual. Sousa e Batista (2016) avaliando diversos estudos realizados com a laser terapia para o tratamento de feridas diabéticas, observaram que o laser He-Ne é o mais utilizado. Os procedimentos realizados com densidade de energia na faixa entre 3 a 5 J/cm 2, com emissão contínua, possibilitaram a otimização do processo de cicatrização de lesões cutâneas, como destacado a seguir. Byrnes et al. (2004) utilizaram a laser terapia He-Ne, comprovaram que a dosagem 4 J/cm 2 melhorou de forma significativa, a cicatrização de feridas cutâneas em ratos diabéticos. O efeito terapêutico foi associado ao aumento da liberação de fatores de crescimento, citocinas, proliferação fibroblástica e colagênese. Maiya et al. (2009), comprovaram a importância do efeito bioestimulador do laser He-Ne (632,8 nm) em modelo experimental de feridas cutâneas excionais em ratos Wistar, com diabetes, expostos a doses diárias entre 3 a 9 J/cm², cinco vezes por semana, até o fechamento completo das lesões. Foram observados aumento na deposição de colágeno e redução significativos, no tempo médio de cicatrização nos grupos tratados com doses entre 3 e 6 J/cm². No entanto, a dose entre 4 e 5 J/cm 2 foi a mais eficaz, podendo ser considerada como “janela terapêutica” no processo de cicatrização de feridas. Vale destacar que nos animais que receberam doses entre 7 e 9 J/cm² o processo de reparo foi desacelerado, sugerindo um efeito inibitório da fotobiomodulação. De forma abrangente, em muitas das publicações pertinentes ao uso do laser, os autores não especificam o tipo utilizado, como exemplificado a seguir. Houreld et al. (2014), comprovaram o efeito da fotobiomodulação na cicatrização, pela irradiação de fibroblastos humanos, com laser de comprimento de onda de 660 nm. Neste estudo, a LLLT modulou a expressão de genes envolvidos na colagênese, adesão celular, remodelamento da matriz extracelular; citocinas e quimiocinas inflamatórias; fatores de crescimento, dentre outras moléculas. Mathur et al. (2017), avaliaram o efeito da LLLT como coadjuvane ao tratamento convencional de feridas, em 30 pacientes diabéticos com lesões 26 crônicas nos pés. Nos pacientes que foram submetidos a terapia convencional associada a LLLT (660 ± 20 nm, 3 J/cm 2), tendo sido observado redução significativa na área da ferida já nas primeiras duas semanas, 37 ± 9% no grupo LLLT e 15 ± 5,4% no grupo controle. Além da redução da área, as lesões do grupo tratado com LLLT, apresentaram maior quantidade de tecido de granulação. De Alencar et al. (2018), realizaram ensaio clínico com 18 pacientes diabéticos, com idade variando entre 30 e 59 anos, que apresentavam feridas crônicas nos pés. A eficácia da LLLT foi avaliada em relação a dosimetria, em que foi utilizado um laser com comprimento de onda de 660 nm, 30 mW de potência, com densidade de energia 6 J/cm 2, emissão em modo contínuo, intervalo de 48h entre as sessões, durante 4 semanas. No grupo submetido a LLLT observou-se um aumento significativo (p <0,013) do índice de reparo tecidual quando comparado ao grupo controle. Szezerbaty et al. (2018), analisaram os mecanismos moleculares envolvidos nos efeitos da LLLT (660 nm), com densidade de energia ente 1 e 5 J/cm 2 na viabilidade celular e expressão dos genes do fator de crescimento do endotélio vascular (VEGF) e interleucina 6 (IL6) em células de fibroblastos L929. Em 72 h, foi observado que para densidade de energia 5 J/cm 2, houve aumento da atividade reticular, melhoria da organização e distribuição do citoesqueleto no citoplasma e promoção da redução da cromatina. Além disso, houve aumento da expressão do VEGF e redução da transcrição do gene da IL6, fornecendo evidências de que a expressão do RNAm dos genes da IL6 e VEGF foi modulada pela LLLT. Amaroli et al. (2019) comprovaram o aumento da produção de espécies reativas de oxigênio (ROS), a mudança do metabolismo anaeróbio para o aeróbio a nível celular e molecular, induzidos pela fotobiomodulação. A irradiação de culturas de células endoteliais humanas com laser infravermelho (808 nm) aumentou o consumo de oxigênio mitocondrial, a síntese de ATP e a produção de ROS. No entanto, não houve aumento significativo no estresse oxidativo e nem dos processos decorrentes de sua ativação. Portanto, o aumento da produção de ROS foi associado a melhoria do metabolismo aeróbio e a uma possível proteção da disfunção endotelial induzida pela inflamação.

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4.3 O Gênero Copaifera e o Óleo Resina de Copaíba

O gênero Copaifera , classificado na família Leguminosae, subfamília Caesalpinoideae, grupo Detarieae, é distribuído em toda a África, América Central, América do Sul e Ásia. No Brasil, a árvore Copaifera é comumente conhecida como copaibeira, pau-de-óleo, copaúva, copa copaíbana, copaibo, copal, marimari e bálsamo dos jesuítas, kupa'iwa, kupa'ü (tupi) e cupay (guarani). A origem do nome copaíba relaciona-se a “cupa-yba”, que em um dos idiomas indígena significa árvore do depósito, em alusão ao óleo que é extraído do tronco de espécies do gênero As copaibeiras são árvores de crescimento lento, atingindo de 25 a 40 metros de altura e podem viver até 400 anos. O tronco é áspero, de coloração escura, medindo de 0,4 a 4,0 m de diâmetro, as folhas são alternadas, pecioladas e penuladas. Os frutos contêm uma semente ovóide e as flores são pequenas, apétalas, hermafroditas e arranjadas em panículos axilares (MARTINS-DA-SILVA et al., 2008; PIERI et al., 2009; SALVADOR, 1975; VEIGA JUNIOR; PINTO, 2002). De acordo com o Index Kewensis (1996), existem 72 espécies de Copaifera e apenas dezessete foram quimicamente estudadas com análises limitadas ao oleorresina de copaíba. De acordo com o International Plant Name Index (IPNI, 2013) 106 espécies são citadas para o gênero Copaifera , das quais apenas 49 foram validadas, 49 são sinônimos, 2 espécies não são copaíferas e 6 não possuem identificação resolvida (ARRUDA et al., 2019). Na região amazônica brasileira nove espécies de Copaifera tiveram sua identificação botânica devidamente validadas, tais como: C. reticulata Ducke, C. duckei Dwyer, C. glycycarpa Ducke, C. martti Hayne, C. guianensis Desf., C. multijuga Hayne, C. piresii Ducke, C. publiflora Benth e C. paupera Herzog. Das quais, as copaíferas mais utilizadas para obtenção de óleo são a C. reticulata (que detém 70% da produção), C. guianensis (10%) e C. multijuga (10%). Em todo o mundo, dentre as mais abundantes espécies de copaíferas, destacam-se: i) as que são originárias do Brasil: C. cearensis Huber ex Ducke (nativa do estado do Ceará); C. confertiflora B (nativa do estado do Piauí); C. coriacea Mart. (nativa do estado da Bahia); C. langsdorffii Desf. (nativa de vários estados do país); C. multijuga , C. officinalis L. e C. reticulata (nativa da região Amazônica); ii) C. officinalis L. (nativa dos países Colômbia, Venezuela e San Salvador); C. 28 langsdorffii Desf. (nativa dos países Argentina e Paraguai); C. guianensis (nativa da Guiana) (ARRUDA et al., 2019; LEANDRO et al., 2012; VEIGA JÚNIOR; PINTO, 2002). O óleo de copaíba é um líquido transparente, constituído principalmente por diterpenos e sesquiterpenos, cuja composição química, viscosidade e coloração variam de acordo com a espécie, com características sazonais e a localização geográfica. Sua coloração varia do amarelo claro ao marrom, mas pelo conhecimento tradicional, aos óleos mais escuros e viscosos são atribuídas as melhores ações terapêuticas. A coleta deste óleo é realizada por extração, a partir de um orifício de 1 a 2 cm feito no tronco da árvore até o cerne da planta. Com o auxílio de um tubo de policloreto de vinila (PVC), o óleo é drenado por cerca de 24 horas e aproximadamente 20 a 30 litros são coletados. Após coleta, os orifícios são fechados com batoques de madeira da mesma árvore e argila, permitindo a regeneração e a produção continuada de oleoresina. Por outro lado, a identificação botânica dos óleos é desafiadora, porque na maioria das vezes, a coleta é feita em várias árvores e o óleo é armazenado em um mesmo recipiente (ARRUDA et al., 2019; BIAVATTI, 2006; BRUNETON, 1993; LEANDRO et al., 2012; PIERI et al., 2009; SANDNA et al., 2018; VEIGA JUNIOR; PINTO, 2002; WAGNER et al., 2017). No Brasil, o óleo de copaíba na forma in natura, tem sido comercializado em diferentes tipos de formulações, tais como: cápsulas, pomadas, cremes, loções e produtos capilares, espumas de banho, sabonetes, xampus, condicionadores. O óleo in natura é amplamente exportado para a Inglaterra, França, Alemanha e os Estados Unidos (DEL NUNZIO, 1985; FLEURY, 1997; VEIGA JÚNIOR; PINTO, 2002). Dentre as muitas aplicações farmacológicas do óleo de copaíba (Figura 2) destacam-se: as atividades anti-inflamatória, analgésica e cicatrizante, bem como o amplo uso nos tratamentos de enfermidades cutâneas, microbiológicas e infecciosas (AMORIM et al., 2017; DA COSTA et al., 2017; DIAS et al., 2014a; DIEFENBACH et al., 2018; DAMASCENO et al., 2019; LUCAS et al., 2017; SANDNA et al., 2018). A Figura 3 mostra a localização geográfica das publicações na área da saúde, reportadas para espécies do gênero Copaifera . 29

FIGURA 2. Descritivo das aplicações farmacológicas do óleo de copaíba. Fonte: autoria própria

FIGURA 3. Localização geográfica das publicações na área da saúde, reportadas para espécies do gênero Copaifera .

Fonte: autoria própria 30

Historicamente, o óleo de copaíba é um dos produtos naturais mais utilizados pela população da Amazônia brasileira. Em uma linha do tempo de 500 anos da história do Brasil, proposta por Ricardo et al. (2018), foi apresentado que o uso mais frequente deste óleo é como agente cicatrizante de lesões cutâneas e mucosas. Originalmente, relata-se que os índios observaram o comportamento dos animais, que esfregavam suas feridas nos troncos das copaibeiras e tinham suas lesões curadas. Os indígenas aplicavam o óleo de copaíba nos ferimentos dos guerreiros após as batalhas, e também no umbigo de recém-nascidos, sendo este o histórico original do óleo de copaíba que justifica seu uso como agente cicatrizante. A seguir, destacam-se alguns descritivos de uso do óleo de copaíba aplicado em processos de cicatrização. Estevão et al. (2013) investigaram os efeitos da pomada com óleo de Copaifera langsdorffii a 10%, em ferimentos cutâneos dorsais em ratos e constataram que o óleo de copaíba melhorou o processo de reparo tecidual e a viabilidade dos retalhos cutâneos, reduzindo a área de necrose e favorecendo a proliferação de vasos sanguíneos, possibilitando um melhor processo de reparo tecidual. Em outro estudo, o tratamento tópico com uma pomada de óleo de C. langsdorffii também a 10%, foi avaliado em feridas de coelhos. Os resultados confirmam o efeito cicatrizante deste óleo sobre a modulação da resposta inflamatória, proliferação de fibroblastos, re-epitelização dos tecidos, síntese de colágeno e remodelamento da matriz extracelular (MASSON-MEYERS et al., 2013). Martini et al. (2016) avaliaram o efeito do óleo de Copaifera multijuga em lesões cutâneas de ratos da linhagem Wistar e também observaram em todos os grupos, níveis semelhantes de inflamação e neovascularização. No entanto, a quantidade de fibras colágenas e elásticas foi maior nos grupos tratados com o óleo de C. multijuga e com nitrofurazona (controle positivo). O óleo de Copaifera paupera favoreceu o processo de reparação cutânea em ratos diabéticos, facilitando o processo regenerativo e diminuindo o tempo de cicatrização. Nos grupos tratados, observou-se redução nos níveis de IL-1β e de TNF-α na pele, menor duração da fase inflamatória, rápida evolução para a proliferativa e contração da lesão (AMORIM et al., 2017). 31

O efeito cicatrizante de uma pomada contendo 10% de óleo de Copaifera langsdorffii , aplicado no tratamento de feridas cutâneas em ratos Wistar, possibilitou o aumento da angiogênese, re-epitelização, retração da ferida e remodelamento precoce da matriz extracelular, com redução significativa da resposta inflamatória e maior expressão de FGF-2, comprovando sua eficácia na proliferação de fibroblastos e síntese de colágeno (GUSHIKEN et al., 2017). Paiva et al. (2002) observaram que a aplicação tópica do óleo de Copaifera langsdorffii, a 4%, acelera a contração de feridas cutâneas e aumenta a resistência à tração. Em outro estudo, o efeito protetor de C. langsdorffii também foi avaliado em um modelo experimental de retalhos cutâneos em dorso de ratos Wistar. Os animais foram tratados com 200 mg/kg e 400 mg/kg deste óleo, por gavagem, tendo sido evidenciado intensa atividade antioxidante e anti-inflamatória durante a isquemia e reperfusão de retalhos cutâneos (DE LIMA SILVA, 2009). Tratamento tópico com uso do óleo de copaíba na forma in natura, mostraram respostas adversas. Brito et al. (1998; 1999) administraram o óleo de Copaifera reticulata na forma in natura, por via tópica, em lesões cutâneas induzidas experimentalmente em ratos e observaram redução do número de fibras colágenas, aumento da resposta inflamatória e tamanho das crostas das lesões cutâneas, resultando em um tempo maior de cicatrização. Resultados semelhantes foram observados no reparo tecidual na presença de implante de uma lamínula de vidro no tecido subcutâneo dos animais. Neste caso, o tratamento tópico com óleo de C. langsdorffii aumentou o edema, hiperemia e o tempo de permanência das crostas, comprometendo a cicatrização cutânea na presença de corpo estranho (VIEIRA et al., 2008). Yasojima et al. (2013) investigaram os efeitos do óleo de copaíba na correção do defeito abdominal de ratos tratados com o uso de tela de polipropileno/poliglecaprone para correção de um defeito induzido na parede abdominal. A administração do óleo foi feita por gavagem ou por imersão do óleo na tela. No grupo tratado por gavagem constatou-se redução na quantidade de aderências abdominais e deposição precoce de fibras colágenas, além da modulação da fase inflamatória. No entanto, nos animais que receberam a tela imersa no óleo, observou-se danos ao tecido cutâneo, como a intensa presença 32 de zonas microscópicas de necrose de liquefação próximas às malhas, comprometendo o reparo da parede abdominal. Lucas et al. (2017) utilizando óleo de copaíba observaram que a redução da concentração do óleo possibilita a cicatrização de forma mais eficaz, tendo sido evidenciado modulação da resposta inflamatória, aumento significativo da proliferação de fibroblastos e deposição de colágeno nas feridas. O efeito cicatrizante do óleo de Copaifera reticulata , administrado diariamente (2 vezes/dia) durante 15 dias, foi investigado na terapêutica de lesões cutâneas em ratos Wistar. Neste estudo, três lesões foram realizadas no mesmo animal e cada uma delas recebeu tratamento diferenciado, tais como: soro fisiológico, clorexidina e óleo de C. reticulata . Ao final do estudo, observou- se que o grupo tratado com C. reticulata ainda apresentava tecido de granulação e ausência de anexos cutâneos, evidenciando o atraso no processo de maturação do tecido conjuntivo (CAVALCANTI NETO et al., 2005). Apesar da comprovada ação terapêutica do óleo de copaíba, sua biodisponibilidade em larga escala é limitada devido à sua baixa solubilidade em água e controle de doseamento (DIAS et al., 2012; DI SOTTO et al., 2018). A limitação de uso pela baixa solubilidade está ainda, associado à identificação botânica de algumas espécies, já que existem dados que são controversos. Em função da importância terapêutica do óleo de copaíba estima-se uma expansão significativa da sua exploração em função de novos estudos bionanotecnológicos, que poderão ampliar sua comercialização em escala mundial (DA TRINDADE et al., 2018; DIAS et al., 2014b; 2015; DI SOTTO et al., 2018). Com relação à biodisponibilidade e doseamento do óleo de copaíba, pesquisas biotecnológicas estão sendo desenvolvidas objetivando-se ação eficaz e redução da dose administrada e reações adversas. Neste sentido, dentre os sistemas carreadores do óleo de copaíba destacam-se os sistemas coloidais (micro e nanoemulsão) e nanosistemas sólidos, aplicados em diferentes modelos farmacológicos (ALENCAR et al., 2015; CAMPOS et al., 2017; BONAN et al., 2015; LUCCA et al., 2015; 2018; MAZUR et al, 2019; OLIVEIRA NEVES et al., 2018). Este tema encontra-se abordado no artigo intitulado “Copaiba oil as a natural product challenge in the chemistry, pharmacological and biotecnological fields” (item 5.1, página 35). 33

4.4 Biodisponibilidade de Fármacos via Sistemas Coloidais Dentre os sistemas farmacêuticos que promovam liberação lenta e controlada de fármacos, se destacam os sistemas coloidais micro e nanoemulsionados. Os sistemas coloidais conferem melhoria de solubilização de fármacos, evitam hidrólises enzimáticas digestivas, proporcionando o aumento do potencial de absorção em função da presença de um tensoativo, ampliam a eficácia terapêutica do fármaco e permitem que a dose administrada seja reduzida, com consequente diminuição de efeitos adversos (CAVALCANTI; BÜTTOW, 1999; DATE et al., 2010; DAMASCENO et al., 2011; DJORDJEVIC et al., 2005; FORMARIZ et al., 2005; ZHANG et al., 2015). No preparo de formulações do tipo micro ou nanoemulsão envolve a realização de estudos experimentais de vários óleos (girassol, soja, azeite, dentre outros), surfactantes (Tween 80, 40 ou 20; derivados de Acrysol, dentre outros), cotensoativos (labrafil M 2125, captex 300 ou 200, dentre outros) e cosolventes (etanol, PEG 400, propileno glicol, dentre outros) de modo que seja possível biodisponibilizar compostos de difícil solubilidade em solventes orgânicos ou em meio aquoso, ou ainda, biológico (CALLENDER et al., 2017; CAMPOS et al., 2017; CHRISTIANSEN et al., 2016; DATE et al., 2010; McCLEMENTS, 2016; OLIVEIRA NEVES et al., 2018; OLIVEIRA et al., 2004; SONI et al., 2014; ROSSI et al., 2007). Neste sentido, o maior desafio consiste em se obter uma combinação crítica eficaz entre os componentes, com solubilização máxima da fase dispersa. Neste contexto, o diagrama de fases descreve a condição experimental exata que possibilita avaliar as regiões limites de transição entre emulsões, fases separadas e microemulsão. Desta forma, facilita a escolha mais apropriada para a incorporação do fármaco em um determinado sistema coloidal (CHAUDHARY et al., 2018; DAMASCENO et al., 2011; DANTAS et al., 2010; GOMES et al., 2006; KANDAV; SINGH, 2018; MANDAL; MANDAL, 2011). Originalmente, Griffin (1954) objetivando selecionar qual seria o tensoativo ideal de uma determinada emulsão, desenvolveu um estudo denominado de “balanço hidrofílico-lipofílico” (BHL), que possibilitou quantificar as contribuições das partes polar e apolar de moléculas de tensoativos não-iônicos. Desta forma, foi possível observar a predominância do caráter hidrofílico ou lipofílico dos tensoativos avaliados. Griffin concluiu que: i) para que se tenha sistemas 34 espumantes o valor teórico de BHL deve estar na faixa entre 7,0 - 9,0; ii) sistema antiespumante, apresentam BHL na faixa entre 1,5 - 3,0; iii) emulsões do tipo A/O apresentam BHL na faixa entre 3,0 - 6,0 e BHL 8,0 - 18,0 para emulsões polares (sistema O/A); iv) dedetergentes apresentam BHL na faixa entre 13,0 - 15,0 e componentes solubilizantes têm BHL na faixa entre 15,0 - 20,0; v) no caso dos tensoativos os valores de BHL variam de acordo com sua polaridade e encontram-se na faixa entre 1 - 50; vi) tensoativos hidrofílicos apresentam BHL 10 e tensoativos lipofílicos encontram-se na faixa entre 1 - 10 (BOUCHEMAL et al., 2004). A formação de agregados micelares é um processo que acontece com dimunuição da entropia do sistema. Na concentração micelar crítica (CMC) ocorre uma transição de fase monômeros de tensoativos para micelas, nesta concentração soluções de tensoativos sofrem mudanças bruscas de propriedades físicas (condutividade elétrica, tensão superficial, densidade, pressão osmótica, espalhamento de luz, dentre outras). Através de dados físico- químicos é possível confirmar a formação de sistemas coloidais (micro e nanoemulsões), bem como efetuar modificações no seu comportamento para fins específicos. Dentre as propriedades mais comuns utilizadas destacam-se: reologia, condutividade elétrica, viscosidade, difusão da luz, birrefringência elétrica, sedimentação, difração de Raios-X a baixo ângulo (SAXS), difração de nêutrons, microscopia eletrônica de transmissão (TEM), dentre outros (ABOOFAZELI et al., 2000; CALLENDER et al., 2017; DJORDJEVIC et al., 2005; FAHMY et al., 2015; SINTOV; SHAPIRO, 2004). Micronanoemulsão são sistemas interfaciais sofisticados produzidos espontaneamente pela auto-organização de moléculas tensoativas nas interfaces óleo-água, formando microestruturas dispersas em um meio contínuo constituído por três ou mais constituintes (tensoativo, fase óleo e fase água). Os sistemas dispersos (micronanoemulsão ou nanoemulsão) possuem microgotículas dispersas e dinâmicas, com diâmetro variando entre 1 nm e 100 nm), apresentam uma camada mononuclear de moléculas anfifílicas (tensoativo) que envolve as microgotículas como uma membrana (FORMARIZ et al., 2005; KREILGAARD et al., 2000; LAWRENCE; REES, 2000; OLIVEIRA et al., 2004). As microemulsões são sistemas monofásicos, termodinamicamente estáveis, transparentes ou translúcidos, com baixa tensão interfacial que 35 possuem a capacidade de combinar grandes quantidades de dois líquidos imiscíveis em uma única fase homogênea. Os sistemas nanoemulsionados possuem propriedades semelhantes as observadas para as microemulsões e diferem na estabilidade por serem cineticamente estáveis. Ambos sistemas apresentam boas propriedades condutoras, em geral superiores as observadas para os solventes orgânicos, e podem dissolver simultaneamente substâncias hidrofóbicas e hidrofílicas. Conceitualmente, nanoemulsões do tipo O/A (sistema formado por micelas diretas) são heterogêneas e contém gotículas de óleo dispersas em um meio aquoso, estabilizadas por um tensoativo (ABOOFAZELI et al., 2000; KRAUEL et al., 2005; KREILGAARD et al., 2000; LI et al., 2017; SANDHU et al., 2015). Neste contexto, destacam-se ainda, os sistemas SNEDDS (self- nanoemulsifying drug delivery system) por serem caracteristicamente resistentes a elevadas diluições em água. Estes sistemas são polares (do tipo óleo em água, O/A); contém uma fase óleo, um (ou mais) surfactante e um cotensoativo (se necessário); são cineticamente estáveis; são misturas isotrópicas que emulsionam espontaneamente quando expostos a fluidos gastrointestinais para formarem óleo disperso em água com tamanho de gotícula manométrico; são viáveis para uso oral e tópico. No caso do uso oral, a motilidade digestiva do estômago e do intestino fornece a agitação necessária para o processo de autoemulsificação-dispersão. Considerando-se as condições fisiológicas hostis do trato gastrointestinal (estômago e intestino delgado), onde ocorrem ataques enzimáticos (enzimas digestivas), sobre os princípios bioativos, os sistemas SNEDDS aumentam a biodisponibilidade do fármaco promovendo a melhoria da permeação através da membrana intestinal, mantém concentrações plasmáticas do fármaco em níveis terapêuticos. Portanto, inibem a elevada atividade enzimática digestiva, associada as extremas flutuações das faixas de pH, além da presença dos tensoativos fisiológicos (sais biliares), que atuam na destruição ou complexação de várias moléculas de fármacos (CAVALCANTI; BÜTTOW, 1999; CHENG et al., 2015; DATE et al., 2010; MAHMOUD et al., 2013; MRSNY, 1992; REDDY et al., 2015; SONI et al., 2014; ZHANG et al., 2015).

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5. RESULTADOS E DISCUSSÃO

5.1 Copaiba oil as a natural product challenge in the chemistry, pharmacological and biotecnological fields

Abstract As an attempt to update the copaiba oil research improvement regarding to pharmaceutical industry advancements the present review of published information on copaiba oil, contributes with a new compilation data focusing on its traditional uses, phytochemical and pharmacological updating and also highlight its ongoing biotechnological advances. Nowadays biotechnology has largely increased due to the large success involving the development of drug delivery systems in micro and nanometric scales, leading to lower bioactive doses, toxicity and side effects, as well as site-specific delivery, biocompatibility, stability, and optimize drug release rate. So, the findings of copaiba oil showed promising results and point towards the safe bioavailability of this important natural product which is herein discussed along with its medicinal properties.

Keywords: Copaiba oil; phytochemistry; pharmacology; biotechnological advances.

Introduction Copaiba oil is obtained from trees of the Copaifera genus, and become an important medicinal source particularly concerning to Amazonian region of Brazil wherein since the 16th century has been largely used in Brazilian folk medicine. Among the 106 predict species of the genus Copaifera (Leguminosae), only 49 have been validated, 49 showed to be synonyms and the remaining species are unresolved. Particularly in Brazil, 17 species can be found but from which there are only 9 main species, such as: C. duckei Dwyer , C. glycycarpa Ducke , C. guyanensis Desf. , C. martti Hayne , C. multijuga Hayne , C. paupera Herzog, C. piresii Ducke , C. publiflora Benth., and C. reticulata Ducke. Among those species the greatest natural sources of copaiba oil are C. reticulate (70% of oil production), C. multijuga (10%) and C. guianensis (10%) (ARRUDA et al., 2019; LEANDRO et al., 2012; VEIGA JÚNIOR; PINTO, 2002). 37

Copaiba oil is a transparent liquid ranging from yellow to light brown color, and is excreted as a plant defense against animals, fungi and bacteria. Its production could occasionally produce 30 L/tree in a single collection but the lower amounts (0.3 to 4 L/tree) are usually expected. In this sense, copaiba oil extraction for the specimens C. officinalis Jacq. L. and C. reticulate , seems optimal for dry season harvest. Meanwhile, from C. venezuelana and C. pubiflora Benth this oil yield may peak with a great fluidity, in the rainy season (ALMEIDA et al., 2012; PAIVA et al., 2002, SANDNA et al., 2018; VEIGA JÚNIOR et al., 1997; VEIGA JÚNIOR; PINTO, 2002). Generally, copaiba oleoresin is largely used in cosmetics and perfume industries as an important raw material as fixer, with fresh and acres notes which are compatible with traditional floral products. Along with its emollient property, this oil shows additional benefits such as antibacterial and anti-inflammatory therapeutics in the manufacture of soaps, creams and bath foams. Aiming at to soften hair, shampoos, conditioners, creams, lotions and capillaries have been largely commercialized (ALMEIDA et al., 2012; DEL NUNZIO, 1985; MACIEL et al., 2002; VEIGA JÚNIOR et al., 2005). Copaiba oil is also used as a protective agent applied on metal surfaces (EMERENCIANO et al., 2017) and among other uses, act as a drying product in the varnish industry, replacing the use of linseed oil (ALBUQUERQUE et al., 2017; ARRUDA et al., 2019; DIEFENBACH et al., 2018; FERRO et al., 2018; TOBOUTI et al., 2017). The present updating of copaiba oil brings a new compilation data focusing on its ongoing biotechnological advances and point towards the safe bioavailability of this important natural product, herein discussed along with its foremost importance on phytochemical and medicinal properties. Also, point out its therapeutic potential on wound healing and highlight in a general vision the drug delivery field progress in nanomedicine. Finally, associate the challenges of copaiba oil as a traditional floral product in the transition of nanomedicine products into modern commercial products and beyond.

Material and Methods The searched literature for this review was collected from different scientific sources such as Web of Science, PubMed, Google Scholar, and the brazilian virtual library CAPES´ Periodics Portal which includes ScienceDirect 38 and others scientific resources. Original studies published for copaiba oil in English or Portuguese without predefined period of time and for which a complete manuscript or abstracts available in those cited database, were taking in account. Expanded to the progress in nanomedicine on the therapeutic potencial of natural products and its dominant market.

Phytochemical and medicinal properties of copaiba oil The chemical composition, color, and viscosity of copaiba oil vary according to each species and regions, but usually are complex mixtures of chemicals. Because of that, copaiba oil become a huge challenge to the chemist of natural products and pharmacologist researchers. According to previously reports of copaiba oleoresin composition, there are described 72 sesquiterpenes and 28 diterpenes. However, only a few species of Copaifera have a full studied chemical composition wherein both oleoresin and volatile fractions were analyzed. Table 1 to 6 shows for some Copaiferas species their major components identified in their copaiba oil samples by applying phytochemical studies, as well as the plant natural occurrence and the country of the developed study. Among them C. multijuga Hayne is the most studied species, followed by C. reticulata Ducke, C. langsdorffii Desf., C. officinalis (Jacq.) L., C. cearensis Huber ex Ducke, C. guianensis Desf., C. lucens Dwyer, C. martii Hayne, C. paupera (Herzog) Dwyer, C. piresii Ducke, C. publiflora Benth., and C. trapezifolia Hayne. By comparing the results data, it is well known that presence and concentration of copaiba oil components often shows conflicting data. Despite this chemical variation the substances detected are basically the same, with different concentrations, but it still controversial that extraction according to day hour cause significant variations. In that studies β-caryophyllene is usually the major constituent. Since the sesquiterpene caryophyllene has been shown to be the main component of Copaifera specimens, it become the most used biomarker to authenticate copaiba oil. From the phytochemical studies it was realized that chromatography modifications procedures improved the isolations and purification of the bioactive copaiba oil constituents some examples are described forward for some copaiba oil characterized by GC/MS. The findings confirmed the occurrence of sesquiterpenes volatile compounds such as: β- 39

caryophyllene, caryophyllene oxide, α-copaene, α-humulene, τ-muurolene, β- bisabolene and β-bisabolol (VEIGA JÚNIOR; PINTO, 2002).

Table 1. Copaifera multijuga Hayne species occurrence and major components.

Country Reference and Major Components Occurrence Country Study

β-caryophyllene (5.1 -64.0%) α-copaene (2.0-15.0%) copalic acid (1.7-7.1%) SOUZA-BARBOSA et al., caryophyllene oxide (0.2-31.5%) Brazil 2012. (Brazil) α-humulene (0-8.9%) germacrene D (0-16.7%) δ-cadinene (0–5.4%) β-caryophyllene (10.6 -62.7%) α-copaene (2.5-14.9%) α-humulene (2.4-8.7%) SOUZA-BARBOSA et al., Brazil copalic acid (1.1-5.2%) 2013. (Brazil) germacrene D (0-18.9%) caryophyllene oxide (0.2-32.5%) β-caryophyllene (42.9 -60.3%) trans -β-bergamotene (2.0-7.0%) caryophyllene oxide (tr-8.8%) CASCON; GILBERT, Brazil α-copaene (2.1-5.2%) 2000. (Brazil) copalic acid (1.9-11.0%) 3-acetoxycopalic acid (0.8-6.2%) β-caryophyllene (60.2%) copalic acid (9.5%) SANT’ANNA et al., Brazil α-humulene (8.6%) 2007. (Brazil) trans -α-bergamotene (6.4%) β-caryophyllene (57.5%) VEIGA JUNIOR et al., α-humulene (8.3%) Brazil 2007. (Brazil) copalic acid (6.2%) β-caryophyllene (57.5%) SANTOS et al., Brazil copalic acid (6.2%) 2008. (Brazil) β-caryophyllene (57.5%) LIMA et al., α-humulene (8.3%) Brazil 2003. (Brazil) copalic acid (6.2%) β-caryophyllene (57.1%) TRINDADE et al., α-humulene (10.2%) Brazil 2013. (Brazil) β-sesquiphellandrene (9.9%) β-caryophyllene (36.0%) α-copaene (18.8%) KOBAYASHI et al., β-bisabolene (8.5%) Brazil 2011. (Brazil) trans -α-bergamotene (7.0%) δ-cadinene (6.1%) Tr = traces. Source: by author

40

Table 2. Copaifera reticulata Ducke species occurrence and major components.

Country Country Study Major Components Occurrence and Reference β-caryophyllene (1.4 -68.0%) trans -α-bergamotene (2.4-29.6%) β-bisabolene (3.7-42.4%) caryophyllene oxide (0.1-15.2%) ZOGHBI et al., Brazil 2009. (Brazil) β-elemene (0.5-5.6%)

α-humulene (1.1-9.7%) β-selinene (0-20.6%) α-selinene (0-13.2%) β-caryophyllene (25.1 -50.2%) trans -α-bergamotene (6.4-12.0%) SACHETTI et al., β-bisabolene (5.2-17.4%) Brazil 2011. (Brazil) α-humulene (4.1-5.8%) β-selinene (1.8-6.7%) β-caryophyllene (0 -43.4%) trans -α-bergamotene (12.0- 32.8%) β-bisabolene (24.2-50.3%) β-elemene (0-6.0%) HERRERO-JÁUREGUI et al., Brazil α-guaiene (0-9.5%) 2011. (Spain) * trans -β-guaiene (0-5.8%) α-humulene (0-7.0%) β-selinene (0-17.1%) α-selinene (0-10.4%) β-caryophyllene (40.9%) VEIGA JUNIOR et al., α-humulene (6.0%) Brazil 2007. (Brazil) germacrene D (5.0%) β-caryophyllene (37.3%) trans -α-bergamotene (9.0%) TEIXEIRA et al., β-bisabolene (14.5%) Brazil 2017. (Brazil) α-humulene + (E)-β-farnesene (5.4%) β-caryophyllene (37.3%) trans -α-bergamotene (9.0%) GUIMARÃES-SANTOS et al., Brazil β-bisabolene (14.5%) 2012. (Brazil) α-humulene (5.4%) β-caryophyllene (7.7%) trans -α-bergamotene (22.0%) BARDAJÍ et al., β-bisabolene (24.9%) Brazil 2016. (Brazil) β-selinene (12.2%) α-selinene (11.4%) *copaiba oil collected from Brazil.

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Table 3. Copaifera langsdorffii Desf . species occurence and major components.

Major* Reference and Major Components Country Country Study Occurrence

β-caryophyllene (5.5%) trans -α-bergamotene (48.4%) cyclosativene (5.0%) GELMINI et al., Brazil β-elemene (5.1%) 2013. (Italy) ** α-himachalene (11.2%) β-selinene (5.0%) β-caryophyllene (32.8%) copalic acid (5.6%) SANTOS et al., Brazil hardwickiic acid (8.2%) 2008. (Brazil) kaurenoic acid (44.3%) β-caryophyllene (31.4%) eremophilone (6.8%) ESTEVÃO et al., kaurene (6.8%) Brazil 2013. (Brazil) methyl oleate (26.5%) γ-muurolene (22.7%) trans-α-bergamotene (10.2%) ZIMMERMAM-FRANCO et al., β-elemene (8.0%) Brazil 2013. (Brazil) γ-muurolene (16.1%) β-caryophyllene (1.1 -9.0%) α-cadinol (3.2-7.9%) caryophyllene oxide (7.4-16.6%) DE ALMEIDA et al., Brazil spathulenol (12.6-35.7%), 2016. (Brazil) bicycle-germacrene (1.5-5.7%) germacrene D (4.0-18.0%) *other country occurrences: Argentina and Paraguay. ** copaiba oil collected from Brazil. Source: by author

Table 4. Copaifera cearensis Huber ex Ducke species occurrence and major components. Reference and Country Major Components Country of Study Occurrence

β-caryophyllene (19.7%) clorechinic acid (11.3%) α-copaene (8.2%) VEIGA JUNIOR et al., Brazil β-bisabolol (8.2%) 2007. (Brazil) δ-cadinene (7.2%) hardwickiic acid (6.2%) β-caryophyllene (19.7%) SANTOS et al., α-copaene (8.2%) Brazil 2008. (Brazil) hardwickiic acid (6.2%) β-caryophyllene (0.7 -6.2%) trans -α-bergamotene (3.4-7.9%) β-bisabolene (8.9-12.1%) CASCON; GILBERT, hardwickiic acid (0-24.3%) Brazil 2000. (Brazil) kaur-16-en-19-oic acid (19.8-24.5%) polyalthic acid (17.1-27.7%) β-selinene (5.5-7.3%) Source: by author 42

Table 5. Copaifera duckei Dwyer species occurrence and major components. Reference and Country Major Components Country of Study Occurrence

β-caryophyllene (25.1 -50.2%) trans -α-bergamotene (6.4-12.0%) LAMEIRA et al., β-bisabolene (5.2–33.6%) Brazil 2009. (Brazil) (E)-β-farnesene (2.9-5.8%) β-selinene (1.8-6.7%) β-caryophyllene (13.0 -15.5%) trans -α-bergamotene (8.3-10.6%) β-bisabolene (15.7-17.6%) LAMEIRA et al., Brazil β-elemene (8.3-9.4%) 2009. (Brazil) β-selinene (13.8-15.4%) α-selinene (8.8-9.9%) β-caryophyllene (0.7 -6.2%) trans -α-bergamotene (3.4-7.9%) β-bisabolene (8.9-12.1%) CASCON; GILBERT, hardwickiic acid (0-24.3%)kaur- Brazil 2000. (Brazil) 16-en-19-oic acid (19.8-24.5%), polyalthic acid (17.1-27.7%) β-selinene (5.5-7.3%) Source: by author

Table 6. Copaifera officinalis (Jacq.) L . species occurrence and major components. Major* Reference and Major Components Country Country of Study Occurence β-caryophyllene (24.9%) allo -aromadendrene (7.5%) β-bisabolene (6.3%) DIAS et al., 2014a. Brazil δ-cadinene (15.3%) (Brazil) α-cadinene (5.6%) germacrene B (5.1%) β-caryophyllene (8.5%) SANTOS et al., hardwickiic acid (30.7%) Brazil 2008. (Brazil) copalic acid (13.9%) *other country occurrences: Colombia, Venezuela and San Salvador.

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Table 7. Other Copaifera species from Brazil occurrence and major components. Reference Copaifera species Major Components and Country of Study β-caryophyllene (65.9%) Copaifera β-selinene (10.2%) ZOGHBI et al., publflora Benth α-humulene (7.3%) 2009. (Brazil) α-selinene (5.5%) β-caryophyllene (33.5%) Copaifera germacrene D (11.0%) VEIGA JUNIOR et al., trapezifolia Hayne spathulenol (7.6%) 2006b. (Brazil) α-humulene (6.2%) β-caryophyllene (14.1%) α- Copaifera copaene (42.5%) ZOGHBI et al., paupera (Herzog) δ-cadinene (10.4%) 2009. (Brazil) Dwyer α-cubebene (5.5%) β-Caryophyllene (11.48%) RIBEIRO et al., Copaifera spp. α-bergamotene (7.04%) 2019b. (Brazil) β-caryophyllene (10.3%) Copaifera ZOGHBI et al., α-copaene (45.5%) piresii Ducke 2009. (Brazil) δ-cadinene (13.7%) caryophyllene oxide (19.1%) kaur-16-en-19-oic acid (17.5%) Copaifera CASCON; GILBERT, hardwickiic acid (11.0%) guianensis Desf. 2000. (Brazil) polyalthic acid (10.6%) trans -α-bergamotene (7.2%) Copaifera polyalthic acid (69.8%) SANTOS et al., lucens Dwyer copalic acid (11.1%) 2008. (Brazil) β-bisabolene (10.7%) Copaifera kovalenic acid (29.0%) SANTOS et al., martii Hayne kaurenoic acid (7.9%) 2008. (Brazil) zingiberene (7.2%) β-bisabolene (20.2%) Copaifera zingiberene (19.4%) SANTOS et al., paupera (Herzog) kaurenoic acid (13.3%) 2008. (Brazil) Dwyer copalic acid (6.1%) Source: by author

Barreto Júnior et al. (2005), Leandro et al. (2012), Dos Santos et al. (2013) and Veiga Júnior et al. (2005) have been shown that specific phytoconstituents could be obtained by a specific chromatography approach with ionic resins who retain carboxylic acids and elute sesquiterpenes and then, sequentially diterpenic acids. Highlighting two examples: i) the ion exchange chromatography was applied to the fractionation of the Copaifera multijuga Hayne, in non-aqueous medium, for separation of basic or acidic fractions from copaiba oil as an important unit operation in preparative scale for commercial purpose. In that study an anionic macroporous resin was successful used for separation of the 44 acid fraction of C. multijuga rich in labdanic diterpenes (BARRETO JÚNIOR et al, 2005; LEANDRO et al, 2012; VEIGA JÚNIOR; PINTO, 2002); ii) silica modified with KOH was used to separate diterpenoic acids from a C. multijuga sample, aiming at analysing their biological activity (VEIGA JÚNIOR; PINTO, 2002). Volatile constituents of Copaifera langsdorffii Desf. sample collected from Brazil, were studied by using GC/MS analysis of the hydrodistilled essential oils obtained from leaves, root bark, fruit peel, trunk bark, trunk wood, root wood and fruits, allowing the identification of 40 different constituents The major compounds of those samples were: β-caryophyllene (53.3% from copaiba balsam oil; 16.6% from leaf oil of and 14.8% from fruit oil); caryophyllene oxide (47.3% fruit peel oil; 40.5% root wood oil; 31.0% trunk wood oil; 30.7% root bark oil); γ-muurolene (29.8% fruit oil; 25.2% leaf oil; 8.3% trunk wood oil); kaurene (30.2% trunk wood oil; 16.7% trunk bark oil; 8.2% root bark oil); 4-α-copaenol (17.6% root wood oil); β-bisabolol (30.5%) and kaurenal (31.9%) from trunk bark oil (GRAMOSA; SILVEIRA, 2005). From hydrodistillation chemical procedure of copaiba oilresin applied to C. langsdorffi and C. martii , the sesquiterpenes β-caryophyllene, α-calacorene gleenol and seline-3,7(11)-diene were identified. Indeed, from several samples from C. langsdorffi , C. duckei and C. reticulata collected in Brazil, β-caryophyllene is usually the major constituent. Meanwhile, α-copaene is the major constituent of samples from C. martii , C. paupera and C. piresii also collected in Brazil (ARRUDA et al., 2019; FERRO et al., 2018; LEANDRO et al, 2012; MACIEL et al, 2002; SOUZA et al., 2011a; TINCUSI et al., 2002; VARGAS et al., 2015; VEIGA JÚNIOR et al., 2002). Regarding to the complex chemical composition of Copaifera species oleoresin, several factors can influence the compounds presence and amount, such as genetics, climate and harvesting conditions. Considering these aspects and also the pharmacological properties of copaiba oil, validated analytical methods for the quality control of samples are important for quantitative and qualitative control (ARRUDA et al., 2019). In this sense, their identifications by using HRGC-FID and HRGC-MS analysis have been performed by comparing the obtained data with those stored in Espectoteca Wiley as well as by substances pattern. Specifically, the chromatography analysis of fractions obtained from copaiba oil (after suffering 45 esterification) are performed following specific conditions such as: i) a gas chromatography equipment (Hewlett Packard-5890 model), S4-54 column with 20 m length, 0.25 mm internal diameter and 0.25 μm thick phase; ii) hydrogen gas carrier gas at a flow rate of 2 mL/min and flow division (split 1:20); iii) initial temperature set at 120 °C with heating rate of 2 °C/min until reach 160 °C, this temperature is selected heating rate 10 °C/min up to 270 °C, and the final temperature is held constant for 5 min. This applied phytochemical methodology was standardized for copaiba oil commercialization (CASCON; GILBERT, 2000; LEANDRO et al., 2012; VEIGA JÚNIOR; PINTO, 2002). Xavier Júnior et al. (2017) developed a precise method for quantification of the main compounds of copaiba oil ( Copaifera langsdorffii Desf.) by using gas chromatography mass spectroscopy (GC/MS) method. In that work it was possible to identify diterpenes compounds form both the copaiba resin and its essential oil. Then the GC/MS method was transposed to be used with a flame ionization detector (FID) and validated as a quantitative method. A good correlation between GC/MS and GC/FID was obtained favoring method transposition. This chemical approach showed satisfactory sensitivity, specificity, linearity, precision, accuracy, limit of detection and limit of quantitation for β- caryophyllene, α-humulene and caryophyllene oxide. Specifically, the main compounds identified in copaiba essential oil were β-bisabolene (23.6%), β- caryophyllene (21.7%) and α-bergamotene (20.5%). Meanwhile, from the derivatized copaiba resin the diterpenes identified were copalic acid methyl ester (15.6%), β-bisabolene (12.3%), β-caryophyllene (7.9%), α-bergamotene (7.1%) and labd-8(20)-ene-15,18-dioic acid methyl ester (6.7%). The main non-volatile components belong to the diterpenes class are caurano, labdanum and clerodane skeletons, including kaurenol, kaurenoic acid, copalic acid, agathic acid, and hardwiickic acid (SOUZA et al., 2011a; 2011b). Some copaiba oils such as C. cearensis and C. langsdorfii could present a high content of kaurenoic acid as it can naturally precipitate forming crystals. For this reason, this diterpene is one of the most studied substance from copaiba oils (LEANDRO et al, 2012). Sesquiterpenes from copaiba oil of samples colleted from C. duckei Dwyer, C. pauper (Herzog) Dwyer, C. piresii Ducke, C. publiflora Benth., and C. reticulate Ducke, were identified such as: cis -α-bergamotene, trans-α- 46 bergamotene, (Z)-α-bisabolene, α-bulnesene, (E)-γ-bisabolene, epi-β-bisabolol, (Z)-γ-bisabolene, trans -cadina-1(6),4-diene, trans -cadina-1(2),4-diene, β- chamigrene, cubenol, epi-cubenol, β-curcumene, γ-curcumene, cyclosativene, cyperene, 4,5-diepiaristolochene, (E)-β-farnesene, (E,E)-α-farnesene, (Z)-β- farnesene, germacrene A, globulol, guaia-6,9-diene, cis -β-guaiene, trans -β- guaiene, γ-gurjunene, humulene epoxide II, epi-α-muurolol, epi-β-santalene, 7- epi-α-selinene, 7-epi-sesquithujene, sesquisabinene, valencene and viridiflorene. The main sesquiterpenes identified in copaiba oil samples are β- caryophyllene, caryophyllene oxide, α-copaene, α-humulene, τ-muurolene, β- bisabolene and β-bisabolol. Some such as α-curcumene, δ-cadinene, β- bisabolene, β-elemen, β-caryophyllene and bisabolol (Figure 1) have its bioactivities reported wherein α-curcumene and β-bisabolene are antiulcerongenic and antiviral agents; β-bisabolene also shows anti-inflammatory and analgesic proprieties; β-caryophyllene is also described as anticancer, anti- inflammatory and antimicrobial agent (SOUZA et al., 2011a; 2011b; VEIGA JÚNIOR; PINTO, 2002).

Figure 1. Some chemical constituent structures from Copaifera specimens.

Source: by author 47

Other biochemicals were identified from copaiba oil samples such as xyloglucans oligosaccharides (40.0%), linoleic (35.7%), palmitic (24.9%), oleic (35.3%), behenic (3.00%), araquidinic (1.1%) acids and coumarins (0.15%) (IZUMI et al., 2012; LEANDRO et al., 2012; DOS SANTOS et al., 2013; VEIGA JÚNIOR et al., 2002; 2005; 2006a; 2006b; 2007). Although Copaifera species have its traditional uses largely described, a restrict biological studies are available for C. cearensis Huber ex Ducke, C. duckei Dwyer, C. langsdorffii Desf., C. langsdorffii Desf., C. lucens Dwyer, C. martii Hayne, C. multijuga Hayne, C. officinalis (Jacq.) L., C. paupera (Herzog) Dwyer, C. reticulata Ducke and C. sp. (commercial copaiba oleoresins). For many reported studies from Copaifera species it was not discriminate which comes from commercial copaiba oil or specific plant species (LEANDRO et al, 2012; MACIEL et al, 2002; SOARES et al, 2013; SOUZA et al., 2011a; VEIGA JÚNIOR; PINTO, et al, 2002). Considering the whole copaiba oil bioactive constituents, the great amount comes from Brazilian Copaifera species, and some of them, showed anticancer, antileishmanial, microbiological, antiparasitic, antipsoriatic, among other properties. So, Table 8 shows the propose of some studies focusing on copaiba oil chemical composition associated with its biological effects and original country of collected samples. Regarding to the pharmacological improvement, the antileishmanial activity of several diterpenes isolated from copaiba oil were analyzed in which the 3-hydroxy-copalic acid was observed to be highly bioactive (DOS SANTOS et al., 2013). Similarly, diterpenic acids from copaiba oils had their synergistic effect together with caryophyllene analyzed to chagas disease. The activity was observed to copalic acid, 3-hydroxy-copalic acid and caryophyllene, but also, it was potentialyzed 20 times when copalic acid was put together with caryophyllene (IZUMI et al., 2012). For a general vision Figure 2 shows the lower researches statistics for copaiba oil isolated compounds and its pharmacological applications, in the years 2002 to 2019, which ranges between 2 and 3 published papers by year. Recent statistics reported for both chemical and pharmacological researches performed by using copaiba oil isolated compounds for the years 2016 to 2019, almost doubled by year showing a progressive tendency. 48

Table 8. Studies developed with bioactive constituents isolated from copaiba oil applied in healthcare.

Pharmacological Purpose of the study Reference activity and Country of Study Potential cytotoxic and genotoxic effects of the isolated kaurenoic acid and its semi-synthetic derivatives Antibacterial and CANO et al., methoxy kaurenoic acid antispasmodic 2017. (Brazil) (methyl ent-kaur-16(17)-en- 19-oate; MKA) and kaurenol (ent-kaur-16(17)-en-19-ol; KRN) in CHO-K1 cell lines. Copaiba oil chemoprevention assessment applied focusing on its some identified compounds, was investigated on DNA damage, pre- neoplastic lesions and mitotic SENEDESE et al., Anticancer frequencies induced by the 2019. (Brazil) 1,2-dimethylhydrazine (DMH; intraperitoneal injection) carcinogen by comet, aberrant crypt focus (ACF) and long- term assays, respectively. Antibacterial activity of Copaifera duckei Dwyer oleoresin and two isolated compounds [eperu-8(20)- Anticancer and ABRÃO et al.,

15,18-dioic acid and polyalthic antimicrobial 2018. (Brazil) acid] against bacteria involved in primary endodontic infections and dental caries. Synthetic ent-kaurenoic acid derivatives were obtained by microbial transformation DA COSTA et al., Anticancer methodologies and tested 2018.(Brazil) against breast cancer cell lines (MCF-7). Genotoxicity and the chemopreventive potential of ALVES et al., Anticancer Copaifera multijuga Hayne 2017. (Brazil) oleoresin and copalic acid. Effect of kaurenoic acid, obtained from copaiba oil CARDOSO et al., resin, in gastric cancer and a Anticancer 2017. (Brazil) normal mucosa of stomach (MNP01) cell lines. Antibacterial action of the Copaifera langsdorffii Desf. Anticancer and ABRÃO et al., oleoresin and (-)-copalic acid, antibacterial 2015. (Brazil) against a multiresistant 49 bacteria as well as their antiproliferative activity. New small chaperone inhibitors from copaiba oil LAMA et al., fractions (copalic acid, Anticancer 2014. (USA) hardwickiic acid and 3- (not identified sample origin) acetoxycopalic acid). Genotoxicity evaluation of copaiba oil, their volatile ALMEIDA et al., Anticancer compounds and also the 2012. (Brazil) resinous fractions. Evaluation of Copaifera multijuga Hayne fractions GOMES et al., Anticancer against ascitic and solid 2008. (Brazil) Ehrlich tumor. Genotoxicity evaluation of CAVALCANTI et al., Anticancer kaurenoic acid. 2006. (Brazil) Inhibition of lung metastasis and tumor growth induced by LIMA et al., melanoma cells using specific Anticancer 2003. (Brazil) compounds rich fractions from Copaifera multijuga Hayne. Effects of kaurenoic acid, a diterpene isolated from the oleo-resin of C. langsdorffii Desf. in developing sea urchin (Lytechinus variegatus ) COSTA-LOTUFO et al., Anticancer embryos, on tumor cell growth 2002. (Brazil) in microculture tetrazolium (MTT) test and on mouse and human erythrocytes in hemolysis assay. In vivo antiedematogenic activity of specific compounds VEIGA JÚNIOR et al., Antiedematogenic rich fractions obtained from 2006a. (Brazil) Copaifera multijuga Hayne. Antifungal activity of the copaiba oil and its isolated compounds caryophyllene oxide, copalic acid and NAKAMURA et al., acetoxycopalic acid against Antifungal 2017. (Brazil) Trichophyton rubrum , Trichophyton mentagrophytes and Microsporum gypseum strains. Protective effect of β- Anti ‐inflammatory and AMES ‐SIBIN et al., caryophyllene from copaiba antioxidant 2018. (Brazil) oils. Effects of L-arginine and kaurenoic acid of copaiba oil Anti ‐inflammatory and SILVA et al., against ischemia reperfusion antioxidant 2015. (Brazil) injury in a randomized skin flap model in rats. 50

In vitro cytotoxicity and anti- inflammatory effects of six diterpene acids: copalic, 3- hydroxy-copalic, 3-acetoxy- VARGAS et al., 2015. Anti-inflammatory copalic, hardwickiic, kolavic- (Brazil) 15-metyl ester, andkaurenoic, isolated from the oleoresins of Copaifera spp. Anti-inflammatory effect of PAIVA et al., kaurenoic acid from Copaifera Anti-inflammatory 2002. (Brazil) langsdorffi . Aantileishmanial activity of diterpene from C. officinales DOS SANTOS et al., (methyl copalate and agathic, Antileishmanial 2013. (Brazil) hydroxycopalic, kaurenoic, pinifolic and polyaltic acids). Caryophyllene from copaiba oils as an effective biomarker SOARES et al., in copaiba oils or specific Antileishmanial 2013. (Brazil) compounds rich fractions derived thereof. Investigation of leishmanicidal DOS SANTOS et al., activity of trans -β- Antileishmanial 2008. (Brazil) caryophyllene. Antimicrobial, Leishmanicidal, cytotoxic activities and Antileishmanial and TINCUSI et al., inhibitory aldose reductase of antimicrobial 2002. (Brazil) various constituents obtained from Copaifera pauper . Antimicrobial and cytotoxic properties of C. reticulata PFEIFER BARBOSA et al., Antimicrobial 2019. (Germany) oleoresin and also its specific (sample from Brazil) secondary metabolites. Antibacterial potential of ent- copalic acid against the SOUZA et al., bacterias Peptostreptococcus Antimicrobial 2018. (Brazil) anaerobius and Actinomyces naeslundii . Anticariogenic activity of nine terpenes and four SOUZA et al., Antimicrobial sesquiterpenes obtained from 2011a. (Brazil) Copaifera langsdorffii Desf. Antimicrobial activity of sclareol, manool, (−)-copalic acid, (−)-acetoxycopalic acid, SOUZA et al., (−)-hydroxycopalic acid, (−)- Antimicrobial 2011b. (Brazil) agathic acid isolated from Copaifera langsdorffii against periodontal bacteria. Antinociceptive effect of kaurenoic acid from Copaifera DALENOGARE et al., officinalis and its mechanism Antinociceptive 2019. (Brazil) of action, and possible adverse effects, in mice. 51

In vitro Schistosomicidal effects of Copaifera oleoresins BORGES et al., 2016. (C. duckei , C. langsdorffii , and Antiparasitic (Brazil) C. reticulata ) and its isolated terpenes from C. duckei . Antiparasitic and synergic activity of β-caryophylene methyl copalate and acids IZUMI et al., Antiparasitic (copalic, 3 β-hydroxycopalic, 2012. (Brazil) agathic, pinifolic, polyaltic and kaurenoic) from Copaifera. Anti-inflammatory mechanism and antipsoriatic effect of the GELMINI et al., volatile and non-volatile Antipsoriatic 2013. (Italy) compounds from Copaifera (sample from Brazil) langsdorffii Desf. Labdane diterpenes copalic acid, 3 β-acetoxy copalic acid, 3β-hydroxy copalic acid and ent -agathic acid from C. SILVA et al., Antitubercular Langsdorffii oleoresin in vitro 2017. (Brazil) assayed against Mycobacterium tuberculosis (H37Rv, ATCC 27294). Systemic immunomodulation potential of the trans - caryophyllene as possible CAMPOS et al., Immunomodulator prophylactic agent of 2015. (Brazil) leukopenia secondary in the chemotherapy. Larvicidal activity of diterpenoids (3-β- acetoxylabdan-8(17)-13-dien- 15-oic acid, alepterolic acid, 3- GERIS et al., Larvicidal β-hidroxylabdan- 8(17)-en-15- 2008. (Brazil) oic acid, andent-agatic acid) from C. reticulata Ducke against Aedes aegypti . In vitro effect of kaurenoic acid Relaxant DE ALENCAR CUNHA from C. langsdorffii , analyzed (smooth muscle) et al., 2003. (Brazil) on rat uterine muscle. Ability of copaiba oil and kaurenoic acid to eliminate Trypanosoma cruzi forms by infected macrophages through KIAN et al., Trypanocidal 2018. (Brazil) other mechanisms in addition to nitric oxide, reactive oxygen species, iron metabolism, and antioxidant defense.

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52

6

5

4

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Year

Figure 2. Statistics reported for chemical and pharmacological researches performed by using copaiba oil isolated compounds in the years 2002 to 2019. Source: by author

Previously to this review article, the chemical and pharmacological progress focusing on the medical potential of copaiba oil were largely documented assessed for i) occurrence, phytochemistry, pharmacology and analytical methods on Copaifera genus (ARRUDA et al., 2019; DA TRINDADE et al., 2018; LEANDRO et al., 2012; PIERI et al., 2009; VEIGA JUNIOR; PINTO, 2002; YAMAGUCHI et al., 2012); ii) evidences for the use of copaiba oil-resin in wound healing (MONTES et al., 2009); iii) chemical composition, biological activities; iv) antimicrobial activity of copaiba oil (Copaifera spp.) on oral pathogens (DIEFENBACH et al., 2018; TOBOUTI et al., 2017); v) incremental progress in the treatment of difficult to heal leishmaniasis wounds by using Copaifera oil (DE ALBUQUERQUE et al., 2017); vi) meta-analysis on copaiba oil: its functions in metabolismo and its properties as an anti-inflammatory agent (FERRO et al., 2018); vii) topical copaiba oil in treatments for inflammatory arthritis (DINI et al., 2019; HEBERT et al., 2017); viii) copalic acid analogs down- regulate androgen receptor and inhibit small chaperone protein (IDIPPILY et al., 2017) ; vx) matrix microparticles of Copaifera langsdorffii on renal physiology (HENRIQUES BRITO et al., 2017).

53

Copaiba oil toxicity

Concerning to copaiba oil dosage toxicity, a single dose of a volatile or resinous fractions obtained from this oil were administered by gavage in rats. The treatment with either one did not increase DNA damage, and there was no alteration in the incidence of micronucleated polychromatic erythrocytes (CHEN et al., 2009). In other study, it was demonstrated that the C. reticulata and C. multijuga oleoresin (500 mg/kg by oral route) did not show cytotoxicity in mammalian cells, or induced alterations such as lesions or bleeding in the stomach of treated mice (GOMES et al., 2007; VEIGA JÚNIOR; PINTO, 2002). Historicaly, in natura copaiba oil has been applied since the first colonizers of the Americas who reported its benefits on treatment of navel of newborns and wounded warriors (SILVA et al., 2012a; VEIGA JÚNIOR; PINTO, 2002). Indeed, this medicinal oil is used topically for a variety of painful and inflammatory conditions, including rashes, dermatitis, insect bites, and psoriasis in addition to join pain (HEBERT et al., 2017). The anti-inflammatory activity of copaiba oil was correlated to the high content of the sesquiterpenes β-caryophyllene and α-humulene, as well as to the diterpene kaurenoic acid. The anti-inflammatory activity and its mechanism are the most investigated for copaiba oils. This activity was related to the inhibition of the NF-B nuclear translocation, and consequently of proinflammatory cytokines secretion. Indeed, copaiba oil suppressed the proinflammatory cytokines interleukin (IL) 6, IL-8, and IL-1 β in LPS-exposed cells (AMILIA DESTRYANA et al., ARRUDA et al., 2019; BENTO et al., 2011; DIAS et al., 2012; 2014a; HEBERT et al., 2017; SILVA et al., 2002a; 2014; ROGERIO et al., 2009; SARPIETRO et al., 2015).

Silva et al. (2012a) performed a study with 10 patients affected by acne, which receive copaiba oil on a controlled double-blind trial. The findings showed good anti-inflammatory results since improvements occurred in the affected area, and no adverse reaction was reported. According to the medical popular tradition, they also reinforce the milenar practice of copaiba oil use on treatment of navel of newborns. So, this ethnopharmacological study carrying the responsibility for collect answer in sequential fashion critical questions, showed to be both efficient and successful support the medicinal importance of copaiba oil. 54

The antipsoriatic effect after oral intake and topical application was also investigated for copaiba oil. In a preliminary clinical trial three patients affected by chronic psoriasis, treated during 6 weeks with oral intake (two patients) and topical application (one patient), exhibited a significant improvement of the disease typical signs, e.g. erythema, skin thickness, and scaliness. Along with the findings, analysis of β-caryophyllene and β-caryophyllene oxide showed their bioavailabilities and absorption effectiveness through cell membranes (GELMINI et al., 2013).

According to Sachetti et al. (2011) higher copaiba oil doses (2 g/kg) did not show neurotoxic effect with a relative margin for safe use as an in natura therapeutic agent. Copaiba oleoresin does not pose a health risk to pregnant women when used according to the recommended doses which is up to five drops (730 mg), three times a day (about 2 g of copaiba oil). It seen that copaiba oil for a reduced period administered at control doses is healthy, but on a large amounts or even in the prolonged treatment periods, it may cause side effects such as gastrointestinal irritation, nausea, vomiting, salivation, diarrhea and depression of the central nervous system.

Reinforcing those results, Copaifera oils ( C. reticulate , C. officinalis and C. multijuga ) were applied focusing in wounds treating, such as ulcers scarring and Leishmanial wounds, as well as toxicological assays. The findings showed lower cytotoxicity and genotoxicity (DE ALBUQUERQUE et al., 2017).

Those studies along with the huge phytopharmacological results of copaiba oil brings out safety on practicability of the copaiba oil validation for therapeutic use in the modern medicinal market. Indeed, the strong use of copaiba oil is part of the Brazilian centuries-old folk medicine culture and the biochemical findings summarized in this article unite interdisciplinary scientific fields and largely encourage novel biotechnological approaches, resulting in new challenges for scientific advances of copaiba oil which has been loaded into nanostructured systems, as described in the following section.

55

Studies designed to bionanoformulations containing copaiba oil

Despite the pharmacological potential of some natural products such as copaiba oil, their poor water solubility remains a challenge on development of effective ecofriendly products. Nanotechnology has emerged as a promising area to solve this problem, especially oil-in-water (o/w) nano or microemulsion type systems. Nanoemulsions containing copaiba oil (Copaifera multijuga Hayne) were proposed as a delivery system for copaiba oil in view to treat locally inflamed skin. The SPME-GC method was performed with PDMS (polydimethylsiloxane) fiber (100 μm), by high pressure homogenization. The obtained nanoemulsions were exposed to acid hydrolysis, UV-A irradiation, oxidative (H2O2) and thermolitic (60 °C) conditions. Such reduction occurred in lower extent in the nanoemulsions, suggesting the β-caryophyllene protective effect. Since no degradation products were detected in the same retention time of β-caryophyllene, the specificity of the tested process was demonstrated. The method was linear in the range of 0.14-0.68 μg mL(-1) of β-caryophyllene (r(2)>0.999), and was also validated for precision (R.S.D. ≤5.0%), accuracy (97.85-101.87%) and robustness. This methodology was validated in the quantification of β-caryophyllene content in the developed formulations (DIAS et al., 2012). Polar nanoemulsions (o/w) developed by using copaiba oil (Copaifera duckei ) dispersed through a high internal phase were prepared and evaluated against Aedes aegypti larvae. Overall, 31 formulations were prepared, ranging from 11.5 ± 0.2 to 257.3 ± 4.1 nm. Some of them reached small mean droplet sizes (<200 nm) and allowed achievement of a nanoemulsion region. The formulation consisted of 5% (w/w) of oil phase (copaiba oil), 5% (w/w) of surfactant and 90% (w/w) of water, which presented mean droplet size of 145.2 ±0.9 nm and polidispersity of 0.378 ± 0.009. According to larvae mortality level (250 ppm - 93.3 after 48 h) the tested nanoemulsions are available as green ecofriendly larvicidal products (RODRIGUES et al., 2014). Nanoemulsions produced by high-pressure homogenization and spontaneous emulsification methodology were carried out to obtain stable copaiba oil formulations. The stability of the formulations stored at 4 °C and 25 °C was monitored for 90 days wherein the reduced loss of volatile fraction was 56 observed at 4 °C. Among the tested methods, high-pressure homogenization process proved to be the most efficient technique in which the most suitable nanoemulsion composition was achieved adding 20% of copaiba oil, 10% of medium chain triglycerides, 3% of Span 80® and 1% Tween 20® (a surfactant mixture). The use of medium chain triglycerides was shown to be a good strategy to fix copaiba oil volatile components incorporated into nanoemulsions during preparation and storage (DIAS et al., 2014b). A copaiba oil nanoemulsified carrier system (CopNEC) prepared by high- pressure homogenization method improved the oral delivery of amphotericin B (AmB) by increasing its oral bioavailability. The optimal CopNEC-AmB (AmB encapsulated CopNEC, d-α-tocopheryl polyethylene glycol 1000 succinate and phosphatidylcholine) had a small globule size, low polydispersity index, high ζ potential and encapsulation efficiency. The high resolution transmission electron microscopy illustrated spherical particle geometry with homogeny in their sizes and the stability of CopNEC-AmB was carried out in simulated gastric fluid and simulated intestinal fluid. CopNEC-AmB was found to be stable in gastrointestinal fluids showing insignificant changes in globule size and encapsulation efficiency. CopNEC-AmB and plain AmB were also compared regarding to the in vitro antileishmanial activity, pharmacokinetics, organ distribution and toxicity. CopNEC-AmB synergistically enhance copaiba oil antileishmanial activity. The AUC0-48 value of CopNEC-AmB in rats was significantly improved showing 7.2- fold higher oral bioavailability than free drug. This prototype CopNEC formulation showed improved bioavailability and cause drastic changes in the morphology of Leishmania parasite and rupturing its plasma membrane. Additionally, showed significantly less haemolytic toxicity and cytotoxicity, had a non-toxic synergistic effect on the antileishmanial activity of AmB, and did not change the histopathology of kidney tissues as compared with plain AmB. In conclusion, the synergistic enhancement of parasiticidal activity of amphotericin B using copaiba oil in nanoemulsified carrier for oral delivery could represent an important approach for non-toxic chemotherapy (GUPTA et al., 2015). Determination of β-caryophyllene (CAR) skin permeation/retention from crude copaiba oil ( Copaifera multijuga Hayne) and respective oil- based nanoemulsion using a novel HS-GC/MS method was used as a bioanalytic method gas chromatography in headspace mode coupled with mass 57 spectrometry. It was noted that nanoemulsification of copaiba oil convert this bioresource into a more acceptable hydrophilic formulation and may improve CAR penetration through the skin due to the small droplet size and also by the nanoemulsion higher contact surface. Copaiba oil nanoemulsion presented a better skin penetration compared to the crude oil, with CAR achieving the dermis, the most profound layer of the skin. In conclusion, according to authors, the finding results justify the validation of a novel, sensitive, practical and solvent free methodology, which demonstrate linearity (r(2)>0.99), specificity (no peaks co- eluting with CAR retention time), precision (RSD<15%) and accuracy (recovery>90%) within the accepted parameters and also reinforce β- caryophyllene studies since this compound is one of the major components of copaiba oil and its potent anti-inflammatory property has attracted large attention (LUCCA et al., 2015). The antimicrobial activity of nanostructured emulsions based on copaiba (Copaifera langsdorffii ) resin-oil and copaiba essential oil were investigated against fungi and bacteria related to skin diseases. The oils samples were characterized by gas chromatography combined with mass spectrometry (GC- MS). The antimicrobial susceptibility assay was performed followed by the Minimum Inhibitory Concentration (MIC) determination, the bioautography assay, and the antibiofilm determination. Strains of the genera Staphylococcus , Pseudomonas , and Candida were used. Copaiba resin-oil and essential oil nanostructured emulsions improved the antimicrobial activity of the pure oils, especially against Staphylococcus and Candida , resistant to azoles. The given results showed copaiba oil nanoemulsion samples as a promising candidates for the treatment of infections and also may be used to incorporate other antimicrobial drugs (ALENCAR et al., 2015). Copaiba oil emulsions (CO) and a suspension of ethanol extract obtained from propolis (EP) were applied on dentin cleaning in order to remove debris that may impair adaptation and marginal sealing. In that investigations through scanning electron microscopy (SEM) the morphology of the dentin surface, cut and treated with CO and EP were performed. The findings showed quantitatively reducing microorganisms.Twenty four upper pre-molars teeth, divided into eight groups (n=3), were used: G1: no cleaning, G2: air/water spray, G3: 10% CO, G4: 10% CO + A, G5: 30% CO, G6: 30% CO + A, G7: 1% EP, G8: 2% chlorhexidine. 58

The specimens were dentin discs (1 mm Ø). The SEM photomicrographs were classified and the results were: G1 - Debris dentin on the entire image/countless microorganisms, G2 and G7- 50-100 debris / countless microorganisms and G3, G4, G5, G6 and G8-0-50 debris/countable microorganisms (50-100 colonies). In conclusion, both products the copaiba oil emulsions and the suspension of ethanol extract of propolis quantitatively reducing microorganisms and showed feasibility to be used as bioactive dental cleaning agents (BANDEIRA et al., 2016). Copaiba oil has emerged as an alternative for the inhibition of microorganisms in dental biofilm. In this sense, the in vitro antibacterial activity of a gel formulation based on copaiba oil ( Copaifera multijuga ) was assayed against strains of Streptococcus sp present in dental biofilm. The oil emulsions were formulated and used with the Brain Heart Infusion agar diffusion method with strains of Streptococcus mitis , Streptococcus constellatus and Streptococcus salivarius isolated from patients as well as standard strains of S. mitis (ATCC903), S. mutans (ATCC10449), S. sanguinis (ATCC15300) and S. oralis (ATCC10557). The study groups were as follows: experimental copaiba oil gel, 1% chlorhexidine gel (positive control) and base gel (negative control). The seeded plates were incubated at 37 ºC for 12, 24 and 48 hours, respectively. The obtained results were analyzed by Shapiro-Wilk and Friedman Tests (p<0.05) for non parametric data and the Tukey test was used for pH values with 5% level of significance. The experimental copaiba oil gel and 1% chlorhexidine gel showed antibacterial activity against the tested microorganisms. The copaiba oil gel demonstrated antibacterial activity against all the tested strains of Streptococcus sp, suggesting that it can be used for dental biofilm control (SIMÕES et al., 2016). Copaiba oil (CO) was loaded on colloidal o/w microemulsions in the presence of low surfactant content as following: oil and water mixtures (15:85 and 25:75) were titrated with surfactant blends until a microemulsion formation. Microemulsions containing up to 19.6% and 13.7% of the selected surfactant blends afforded o/w microemulsions with a high volume of the oil phase (CO complex natural oil) in which a specific match of solubility parameters was developed between CO and surfactants aiming at forming colloidal formulations with a high dispersed volume of copaiba oil and low surfactant content. The obtained microemulsion systems were proposed as delivery systems for the oral 59 administration of poorly soluble drugs as well as CO pharmacological investigations (XAVIER JÚNIOR et al., 2016). Microemulsion systems based on CO aiming at its bioavailability, were used to loaded β-caryophyllene ( β-CP). The CO-carrier microemulsion systems (CO-MES) containing plurol oleique (8.5%), labrasol (33.8%), water (47.1%) and CO (10.6%) as well as plurol oleique (18.3%), labrasol (36.6%), water (39.0%) and CO (6.1%), behaved as Newtonian fluids and exhibited low viscosity. The applied pharmacological testes showed antimicrobial and anti-inflammatory activity for the CO-carrier systems containing the bioactive sesquiterpene β-CP. Comparatively, the CO-MES formulation prepared with 6.1% of CO, showed a stronger result against all target microorganisms (OLIVEIRA NEVES et al., 2018). A hydrogel formulation containing CO nanoemulsion prepared with carbopol and hydroxyeethylcellulose, presented a high retention in epidermis (9.76 ± 2.65 µg/cm 2 as higher result), followed by a smaller retention into dermis (2.43 ± 0.91 µg/cm 2 as higher result). Additionally, presented permeation to the receptor fluid (1.80 ± 0.85 µg/cm 2 as higher result and an anti-inflammatory effect was observed on edema inhibitions in mouse ear edema (67% of higher result) and in rat paw edema (72% of higher result). Histological cuts showed the decrease of infiltration, confirming its anti-inflammatory property (LUCCA et al., 2018). Solid nanoencapsulation containing copaiba oil as feasible and a promising alternative have been also described. In the earlier studies iron oxide nanoparticles dispersed in copaiba oil were developed with low and high velocity resolution by Mössbauer spectroscopy. The results demonstrated differences of Mössbauer parameters for iron oxide nanoparticles which was correlated to interactions of polar molecules of copaiba oil (kaurinic acid) with nanoparticles' surface (OSHTRAKH et al., 2013). The in vitro antimicrobial activity of solution blow spun poly(lactic acid)/polyvinylpyrrolidone nanofibers loaded with copaiba oil ( Copaifera sp.) were produced by solution blow spinning (SBS). All prepared compositions were able to produce continuous and smooth fibers by SBS. Neat PLA and four PLA/PVP blends containing 20% (wt.%) of copaiba oil were spun and characterized by scanning electron microscopy (SEM) and by studying the surface contact angle, in vitro release rate, and antimicrobial activity. The addition 60 of PVP increased fiber diameter, and decreased the surface contact angle. GC analyzes demonstrated that the main component of the copaiba oil was β- caryophyllene, a known antimicrobial agent. Results confirmed the potential of the fiber mats for use of in controlled drug release and could lead to promising applications in the copaiba oil biomedical field as well as other bioactives compounds (BONAN et al., 2015). Cutaneous nanoparticle formulation based on co-encapsulation of imiquimod (approved for the treatment of basal cell carcinoma) and copaiba oil were applied against skin carcinoma. The nanostructured capsule was prepared by high-pressure homogenization using the interfacial deposition method and characterized by average diameter (200 nm), zeta potential (-12mV), pH (6) and drug content of approximately 1 mg/mL, and exhibited homogeneity regarding particle size, high encapsulation efficiency and stability. The antitumor activity was considered satisfactory for human skin carcinoma treatment and was correlated with the applied copaiba-nanostructure system which maintain the skin drug release control (VENTURINI et al., 2015). Nanoencapsulation containing copaiba oil co-loaded with allantoin (NCOA) based on solid lipid nanoparticles were developed by using a high homogenisation technique and characterized by dynamic light scattering (126.06 ± 9.84 nm), laser diffraction (123 ± 1.73 nm), nanoparticle tracking analysis (homogeneous), multiple light scattering analysis (204 nm), high-pressure liquid chromatography, pH and rheology (Newtonian behaviour). The NCOA was in vitro evaluated against the emergent yeasts Candida krusei and Candida parapsilosis , and the fungal pathogens of human skin Trichophyton rubrum and Microsporum canis . Antifungal susceptibility showed a MIC90 as following: 7.8 μg/mL against C. parapsilosis , 250 μg/mL ( C. krusei ), 1.95 μg/mL ( T. rubrum ). Then, the nanoencapsulation of copaiba oil in the presence of allantoin could represent promising therapeutics for skin infections caused by yeasts and dermatophytes (SVETLICHNY et al., 2015). The in vitro pharmacological evaluation of nanocarriers composed of lamellar silicates and copaiba oil was investigated in order to endometriosis control. Intercalation was confirmed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analyzes (TGA) and differential scanning calorimetry (DSC). Pharmacological findings showed reduction in the 61 viability and proliferation of endometriotic cell cultures suggesting this nanocomposite system as a promising alternative therapy on oral treatment of endometriosis (BORGES et al., 2016). A study designed to test nanocapsules containing copaiba oil (400 mg/kg) applied to treat pulmonary arterial hypertension (PAH) was investigated on cardiovascular diseases. A single injection of MCT (60 mg/kg i.p.) was administered according to modulate monocrotaline (MCT) protocol, and measurements were performed after three weeks. MCT promoted a significant increase in pulmonary vascular resistance (PVR), right ventricle (RV) hypertrophy and RV oxidative stress and copaiba nanocapsules significantly reduced RV hypertrophy and oxidative stress. PVR was reduced by in natura copaiba oil+MCT but not by copaiba-nanocapsules+MCT. In conclusion, copaiba oil may be an important adjuvant treatment for pulmonary arterial hypertension (CAMPOS et al., 2017). The bactericidal effect of copaiba oil ( Copaifera multijuga Hayne) in natura or in combination with silver nanoparticles produced by green synthesis using Fusarium oxysporum (AgNPbio) were assayed against planktonic and sessile cells of GBS (group B Streptococcus agalactiae ) including those resistant to erythromycin and/or clindamycin. The combination of copaiba oil with AgNPbio resulted in a synergistic effect against planktonic cells and biofilm formation, reducing the minimal inhibitory concentration values of both compounds. No hemolytic activity was detected for both compounds. GBS remains a leading cause of neonatal infections and an important cause of invasive infections in adults with underlying conditions. Plain copaiba oil, or in combination with AgNPbio represent new strategies for controlling GBS infections (OTAGUIRI et al., 2016).

It is expected that Brazil become the fifth largest natural drug market. This fact attracted representatives of the pharmaceutical industry and leveraged discussions on the importance of patent protection to ensure the interests of inventors and society (ZUANAZZI; MAYORGA, 2010). For a general vision, Figures 3 show the copaiba oil formulations-type in the period 2009 to 2015 developed for therapeutic applications, and Figure 4 highlight the growth of 62 biotechnology studies developed with copaiba oil to be applied as phytomedicines

Ointment and Cream Nanoparticle (as solid formulations)

Nanofiber

Nanoemulsion

Microemulsion Formulation Gel

Endodontic pastes

Emulsion 0 5 10 15 20 Study representativeness

Figure 3. Copaiba oil applied into different formulations.

Source: by author

12

10

8

6

4

Study representativeness Study 2

0 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Year

Figure 4. Representative biotechnology studies growth of the copaiba oil focusing on formulations-type in the period 2009 to 2015, aiming at the healthcare. Source: by author 63

Comparatively, for the period 2016 to 2019, over than thirty papers have been published for copaiba oil and its isolated or derivatives compounds. This crescent improvement could be addressed to the new nanotechnologies, as well as the development of new analytical methods for analysis of Copaifera oleoresin, and also the difficult challenge to standardize the chemical composition of the main compounds (sesquiterpenes and diterpenes). So, recently there are a huge amounts of new publications concerning to copaiba oil focusing on its phytotherapeutic use. Table 9 summarizes its foremost medicinal importance, highlighting the main development of copaiba oil loaded into colloidal (gel, emulsion, microemulsion and nanoemulsion) formulations.

Table 9. Biotechnological and pharmacology results of copaiba oil studies.

Formulation Pharmacological Reference Activity and Country of Study Cream CARVALHO et al., Antimicrobial (vaginal formulation) 2015. (Brazil)

Cream LIMA et al., Antimicrobial (vaginal formulation) 2011. (Brazil)

Cream Cutaneous wound MASSON-MEYERS et al., (cutaneous formulation) healing 2013. (Brazil)

DE BARI et al., Emulsion Antimicrobial 2016. (Brazil)

MARANGON et al., Emulsion Antimicrobial 2017. (Brazil)

XAVIER JÚNIOR et al., Emulsion - 2012. (Brazil)

DIAS et al., Endodontic pastes Antimicrobial 2015. (Brazil)

GARCIA et al., Endodontic pastes - 2011. (Brazil)

Gel SILVA et al., Antiacne (inflammation formulation) 2012a. (Brazil)

Gel LUCCA et al., Anti-inflammatory (hydrogel formulation) 2018. (Brazil)

Gel PEREIRA et al., Antimicrobial (dental formulation) 2010. (Brazil) 64

Antimicrobial and anti- OLIVEIRA NEVES et al., Microemulsion inflammatory 2018. (Brazil)

XAVIER JÚNIOR et al., Microemulsion - 2016. (Brazil)

DE ABREU et al., Nanoemulsion Anticancer 2018. (Brazil)

LUCCA et al., Nanoemulsion Anti-endometriosis 2015. (Brazil)

DIAS et al., Nanoemulsion Anti-inflammatory 2012. (Brazil)

RODRIGUES et al., Nanoemulsion Antileishmanial 2018. (Brazil)

ALENCAR et al., Nanoemulsion Antimicrobial 2015. (Brazil)

VAUCHER et al., Nanoemulsion Antimicrobial 2015. (Brazil)

EMERENCIANO et al., Nanoemulsion Antioxidant 2019. (Brazil)

DIAS et al., Nanoemulsion - 2014b. (Brazil)

Cutaneous anti- HENRIQUES DA SILVA Nanoemulsion inflammatory et al., 2015. (Brazil)

RODRIGUES et al., Nanoemulsion Larvicidal 2014. (Brazil)

GUPTA et al., Nanoemulsion Leishmanicidal 2015. (Brazil)

DE MORAES et al., Nanoemulsion Leishmanicidal 2018. (Brazil)

MAZUR et al., Nanoemulsion Leishmanicidal 2019. (Brazil)

Solid Nanoparticle SVETLICHNY et al., Antifungal (lipid formulations) 2015. (Brazil)

Solid Nanoparticle MITSUTAKE et al., Anti-inflammatory (cyclodextrin formulations) 2019. (Brazil)

Solid Nanoparticle PINHEIRO et al., Anti-inflammatory (cyclodextrin formulations) 2017. (Brazil) 65

Solid Nanoparticle BONAN et al., Antimicrobial (nanofiber formulations) 2015. (Brazil)

Solid Nanoparticle OTAGUIRI et al., Antimicrobial (silver formulations) 2016; 2017. (Brazil)

Solid Nanoparticle SIMÕES et al., Antimicrobial (from emulsion) 2016. (Brazil)

Solid Nanoparticle VENTURINI et al, Antineoplastic (lipid formulations) 2015. (Brazil)

Solid Nanoparticle CAMPOS et al., Cardioprotective (capsules formulations) 2017. (Brazil)

Solid Nanoparticle Cutaneous wound MILLAS et al., (nanofiber formulations) healing 2014. (Brazil)

Solid Nanoparticle Skin burns and other DEBONE et al., (chitosan formulations) chronic wounds 2019. (Brazil) Solid Nanoparticle QUIÑONES et al., (polyethylene glycol Transdermal delivery 2018. (Brazil) formulations) Solid Nanoparticle GASPAR et al., - 2017. (Portugal) Lipid carriers (NLC) (sample from Brazil)

Solid Nanoparticle REÁTEGUI et al., - (from emulsion) 2017; 2018. (Brazil)

Solid Nanoparticle XAVIER JÚNIOR et al., - (chitosan capsules) 2018. (Brazil)

Solid Nanoparticle DE ALMEIDA BORGES et al., - (silicate formulations) 2016. (Brazil)

Solid Nanoparticle GARRIDO et al., - (lipid formulations) 2010. (Brazil)

ESTEVÃO et al., Ointment Healing 2009; 2013 (Brazil)

GUSHIKEN et al., Ointment Healing 2017. (Brazil)

Source: by author

66

Socioeconomic development of copaiba oil Markets for products derived from plants (herbal, dietary supplements, cosmetics, insect repellents, dyes, among many other possibilities) are constantly expanding worldwide. It is known that 25% of the drugs currently used in the industrialized countries come directly or indirectly from natural products. So, countries with high biodiversity have the opportunity to go into billionaires markets such as pharmaceuticals and dietary supplements, which handle about 320 and 31 billion/year, respectively (SOUZA-BARBOSA et al., 2012). In the other hand, the preservation of biodiversity is of paramount importance and can be seen as a way to sustain life on the planet. In Brazil, changes in public health policy are being aligned with the World Health Organization (WHO) recommendations, seeking full and universal assistance to health services, without infringing right to preservation and rational use of biodiversity. So, the importance of plant species for humanity, studies for management, bioprospecting and conservation of the biodiversity are thoroughly carried out (KIM et al., 2012; NOGUEIRA et al., 2010; OLDHAM et al., 2013; WHO, 2014). Therefore, the appropriation of the biodiversity for industrial purposes is a powerful instrument for sustainable development, since organized and properly performed. In this sense, in the period 1974 to 1979, the state of Amazonas exported 101 tons for domestic market and 433 tons were exported to foreign. In 1992, the exports were about 24 tons of oil to the United States and Europe. During the last century this oil ranked the second place in Brazilian exports of medicinal drugs and represents approximately 95% of the entire oleoresin production country wise and its annual production is around 500 tons/year (ALENCAR, 1982; ALMEIDA et al., 2012; MEDEIROS; VIEIRA, 2008; SANT’ANNA et al., 2007; TAPPIN et al., 2004; VEIGA-JÚNIOR; PINTO, 2002). By the reason of copaiba oil widespread traditional importance its commercialization had become intense and Brazil became an important exported country to France, Germany and the United States. In fact, this oil was distributed to Europe about 50 tons per year with France responsible for consuming more than 6 tons/year. In this sense, Hamburg and Germany, in the period before the first World War became the main copaiba oil import center to Brazil commercialization. The largest global copaiba oil export period was in the post- war wherein values achieved 225 tons/year. In the very long past, French people 67 was the most dedicated to the study and exploration of copaiba oil. In 1972, the Food and Drug Administration approved the copaiba oil, after being subjected for sensitization and irritation tests on 25 volunteers, with negative results. Along with this application many studies have been performed in order to improve its pharmacological and industrial importance including wound healing assays (CALLENDER et al., 2017; EMING et al, 2014; GAJENDRAREDDY et al., 2013; GELMINI et al., 2013; MACIEL et al., 2014a; 2014b; ROSIQUE et al., 2015). The use of natural resources guided by WHO, has stimulate the economies of the developing countries and increased applications for pharmaceuticals and cosmetics patents arising out of the local biodiversity (OLDHAM et al., 2013; WHO, 2013; 2014). Patent is a form of protection of economic and personal interests, in which the state grants a temporary title to the creation (invention or utility model) to the authors, inventors or as individual or entity, regulating and promoting the technological innovation process. In fact, the current model of international intellectual property system favors patent holders, encourages the scientific production and technological innovation. Thus, the analysis of the patent documents is one strategy techniques for monitoring changes and advancements in technology, enabling identification of technological innovation trends over the years (MENELL et al., 2001; MUELLER; TAKETSUMA-COSTA et al., 2014). Regarding patents involving the copaiba oil, the oldest one is from 1898 (GB189803261) in which copaiba capsules was used in the inflammation treatment of the urethra (gonorrhea). One of the many companies using this oil is the Technico-flor S/A that obtained in France in December 1993 a patent registration (FR2692480) for a "new cosmetic or food compositions including copaiba". In June 1994 achieved the same record at WIPO (WO9400105) expanding it to patent world domination. In the United States, the Aveda Corp achieved in March 1999 a patent registration (US5888251) for a “Method of coloring hair or eyelashes with compositions which contain metal containing pigments and a copaiba resin". The Brazilian Pharmacopoeia describes an ointment containing copaiba oil, for external use, with anti-inflammatory, antiseptic and healing proprieties. The formulation is obtained by mixing 10 g of resin copaiba oil ( Copaifera langsdorffii Desf., C. multijuga H. Kuntze, C. 68 reticulata Ducke or C. paupera H. Dwyer ) and 100 g of lanolin and petrolatum ointment. In the dentistry field, an orthodontic cement containing Copaifera multijuga oil a developed product was subjected to laboratory analysis of its chemical and physical properties compared to other three commercial products. The results revealed that the experimental cement complies satisfactorily with the standards of the American Dental Association (GARRIDO et al., 2010). A considerable number of other patents for therapeutic applications can still be found in the current literature . In that, the number of patents containing copaiba oil for therapeutic purposes or cosmetics uses has increased and some examples are herein highlighted (BR8605738, GB637440, JP07-278001, MU8203234-3, PI1004276-8A). The patent register PI 0404266-2 shows the development of a gel containing copaiba oil to dental application. A patent process (WO2005110446) for preparations of copaiba oil extracts, fractions and isolated compounds from the Copaifera species was register for treatment of urinary lithiasis in human beings and animals. For a general vision, Figure 6 shows the number of patents per country that were requested in the period of 1950 to 2019 for copaiba oil aiming at health applications (human or veterinary needs) reported by INPI, EPO, USPTO and WIPO resources. From this amount 27 documents belonged to Brazil, 5 the United States, 6 Chine, 4 Japan, 4 Korea, 2 Spain, 2 Germany and 1 France. However, when considering the copaiba oil use the last 20 years based on the information of patents, they were found 17 documents and of these, fourteen were required by Japanese companies and only one was Brazilian (SOUZA- BARBOSA et al., 2012). In the other hand, the growth in the number of Brazilian patents highlights the interest in herbal market and could improve the national biotechnologic management. The Figure 7 shows the growth number of patents in the period of 1950 to 2019. Despite Brazil's leading position in relation to copaiba oil patent requests there was a high number of requests by countries where Copaifera is not part of their native flora. Among the patent applicants the participation of foreign companies is a frequent finding and may generate questions about the misappropriation (biopiracy) of natural resources as well as traditional knowledge (OLDHAM et al., 2013). 69

30

25

20

15

10 Number of patents of Number 5

0 Spain Korea China Brazil Germany France Japan United States Country

Figure 6. Number of patents per country requested in the period of 1950 to 2019 for copaiba oil aiming at healthcare.

Source: by author

9 8 7 6 5 4 3

Numbers of patents of Numbers 2 1 0

Year

Figure 7. Numerical growth of patents in the period of 1950 to 2019.

Source: by author

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Conclusion Copaiba oil is historically recognized as anti-inflammatory, antimicrobial, analgesic and healing agent, among other medicinal uses. In Brazil, it is largely administered to treat skin lesions, infectious, urogenital, respiratory, gastrointestinal, oncologic diseases, and many other folk indications. Copaiba oil pharmacological preparations are limited due to its lower water solubility. Nowadays, biotechnology enables the availability and therapeutic uses of some medicinal resources. This is the case of copaiba oil which has been loaded into colloid systems (emulsion, nanoemulsion, and microemulsion), as well as solid nanostructure systems for therapeutic applications. The pharmacological properties of copaiba oil were correlated with terpenoid compounds such as sesquiterpenes and diterpenes. The main diterpenes components are kaurenol, kaurenoic acid, copalic acid, agathic acid, and hardwiickic acid. The main sesquiterpenes identified are β-caryophyllene, caryophyllene oxide, α-copaene, α-humulene, τ-muurolene, β-bisabolene and β- bisabolol. It is known that β-caryophyllene is described as anticancer, anti- inflammatory and antimicrobial agent. Since this compound has been detected as the main component in several Copaifera species, become the most used biomarker to authenticate copaiba oil. Because of that, the anti-inflammatory activity of copaiba oils has been addressed to this compound. The foremost medicinal use of copaiba oil was designed to skin healing process in which is able to reduce inflammatory response at early stages with significant increase in the fibroblast proliferation phenomena and collagen deposition in wounds. The market trends investments in copaiba oil biotechnology approach have been justified in order to improve the therapeutic properties of copaiba oil, since it can be limited mainly by its insolubility in water. Therefore, the development of copaiba oil dispersed systems have been seen as a promising strategy, since they allow the delivery, topically, insoluble in water molecules, enhancing, including its therapeutic effect.

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5.2. SNEDDS drug delivery system based on copaiba oil improves wound healing by promoting angiogenesis, neocollagenesis and reducing inflammatory reaction

Abstract Copaiba oil (CO) from Copaifera species is largely administered as anti-inflammatory, antimicrobial and healing agent. Despite CO medicinal importance, generally, pharmacological CO-based preparations are limited due its lower water solubility. In this work a colloidal system based on copaiba oil ( Copaifera multijuga Hayne) containing a sorbitan surfactant was prepared and applied for wound healing. The CO-lower loaded content consisted in a self-nanoemulsion drug delivery system (SNEDDS) for CO- gradual and prolonged release to be evaluated on wound healing for non diabetic and diabetic rats. The C. multijuga characterization was performed using HRGC-MS analysis. The chemical approach (79% of water, 20% of surfactant, 1% of oil phase) afforded a colloidal SNEDDS-type system, so called SNEDDS-CO which was prepared without co- surfactant justifying its foremost importance on CO-pharmacologic applications. The ternary phase diagram was constructed by titration method to determine WIV region. Droplets diameters determined using the dynamic light-scattering technique. Wistar (Rattus novergicus albinus ) after dorsal cutaneous skin flap surgery, received a post- operative topically treatment with SNEDDS-CO formulation. Animals (n=6) were divided as non diabetic (G1, control and G2 treated group) and diabetic (G3, control and G4 treated group). Histopathological and immunohistochemical analyzes were performed after 7 and 14 days. Histomorphometric analysis revealed a smaller number of neutrophils and lymphocytes on treated group (7 days) as well as collagenesis, fibroblast proliferation and lager epithelial thickness (treated group, 14 days). Keratinocytes with increased immunoreactivity for IL-1β on control group contributed with repair delay on treated animals (G2 and G4). The greater number of FGF-2 immunostained cells were associated with positive collagenesis and accelerated the wound healing. This study shows an innovative approach for the use of copaiba oil loaded into SNEDDS colloidal- type system and highlighted the morphological and immunohistochemical characteristics of non diabetic and diabetic rats treated with a SNEDDS´s copaiba oil formulation.

Keywords: Copaiba oil; SNEDDS Drug Delivery Systems; Wound Healing; Immunohistochemistry.

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Introduction Copaiba oil (CO) has huge medicinal indications such as larvicide, antileishmanial, anticancer, antimicrobial, gastrointestinal, respiratory, urogenital, analgesic, anti-inflammatory, and wound healing properties. The anti- inflammatory property of copaiba oil has been reported since the first colonizers of the Americas who reported its use to treat navel of newborns and wounded warriors (ARRUDA et al., 2019; DAMASCENO et al., 2019; RICARDO et al., 2018; VEIGA JÚNIOR; PINTO, 2002). In respect of medicinal plants even with the large therapeutic record of copaiba specimens, this oil still remains with limited uses, justified by the fact that depending on its dosage, controversial results of in natura CO applied on healing process of skin diseases may be aggressive (BRITO et al., 1998; 1999; LEANDRO et al., 2012; VIEIRA et al., 2008). Due to its traditional and widespread medicinal importance the commercialization of CO or its common capsule formulations had become intense around the world (DE MEDEIROS et al., 2019; DIAS et al., 2014b). Nowadays, biotechnology enables availability and therapeutic uses of medicinal resources. This is the case of CO which has been loaded into nanoemulsions, microemulsions, and solid nanostructure systems aiming at some therapeutic applications (ALENCAR et al., 2015; BORGES et al., 2016; CAMPOS et al., 2017; BONAN et al., 2015; LUCCA et al., 2015; 2018; MAZUR et al, 2019; OLIVEIRA NEVES et al., 2018). Colloidal systems (nano and microemulsion) have been largely applied in pharmaceutical, cosmetic, and food domains, whose advantages enhanced solubility and bioavailability of bioactivities lipophilic and hydrophilic compounds, shows good chemicals dissolution content, rapid onset of action, pharmacokinetics improvement, enhancement of absorption phenomena and drug release kinetics, as well as reduction of side effects and decreased toxicity (CHRISTIANSEN et al., 2016; CALLENDER et al., 2017; DATE et al.; 2010; FAHMY et al., 2015; RASHID et al., 2015; SANDHU et al., 2015; SONI et al., 2014). As far as wound healing improvement is concerned several millenary natural compounds were analyzed and for most of them, tissue repair process of cutaneous ulcers has not yet been satisfactorily proven. Concerning to copaiba oil, controversial results were notified, depending on its dosage. Copaiba oil may 73 be pro-inflammatory, but at lower concentrations, reduces the inflammatory response at early stages with significant increase in the fibroblast proliferation phenomena and collagen deposition in wounds (AMES ‐SIBIN et al., 2018; ESTEVÃO et al., 2013; SILVA et al., 2015; PAIVA et al., 2002; MASSON- MEYERS et al., 2013). Generally, pharmacological CO-based preparations are limited due its lower water solubility, than the bioavailability of this therapeutic oil faces limitations such as the chemical variability of CO depending on its specimen origin, and also poor solubility on biologic solvents. In this work, copaiba oil was loaded, in a lower content (1% of CO-based oil mixture containing copaiba oil and sunflower oil on 1:1 of ratio) in order to improve its solubility and safe availability into a SNEDDS (self-nanoemulsifying drug delivery system) formulation which works with gradual and prolonged CO-release, to be applied as an efficient wound healing agent.

Material and Methods

Vegetal materials and chromatography analysis Copaiba oil ( Copaifera multijuga Hayne) was collected in Manaus, a voucher specimen (61212 INPA) is available at the Herbarium of National Institute for Amazonian Research. Characterization of copaiba oil after its chemical esterification, was performed using gas chromatography high resolution analyses with flame ionization detection (GC-FID) coupled with mass spectrometry (GC-MS, Hewlett Packard-5890 model) being in accordance with our previous reported method (SOUZA-BARBOSA et al., 2012; BARRETO- JÚNIOR et al., 2005; VEIGA JÚNIOR et al., 2006a; 2006b; 2007). Figure 1 shows the applied phytochemical methodology in order to obtain the chemical characterization of the copaiba in natura oil sample, and Figure 2 shows the chromatogram of the esterified copaiba oil in natura sample.

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Figure 1. Phytochemistry approach applied in the chemical characterization of the copaiba oil in natura sample (Copaifera multijuga Hayne).

Source: by author

75

Figure 2. Chromatogram of the esterified copaiba oil in natura sample (Copaifera multijuga Hayne). Source: by author

Colloidal SNEDDS system material and preparation approach The surfactant Tween® 80 (polyethylene glycol sorbitan monooleate) was acquired from Sigma-Aldrich (St. Louis, USA). Sunflower oil ( Helianthus annuus ) was obtained commercially as a food product. The chosen veterinary-use anesthetic and sedative agent, Zoletil 50 (tiletamine HCl 125 mg, zolazepam base 125 mg) acquired from Virbac (São Paulo, Brazil). All other used reagents were of analytical grade and purchased from Merck (Darmstadt, Germany). The temperature was measured using a stove 76 thermometer (HG-Brazil, #1876/11) and pH measurement was performed with Tecnopon MPA-210. Centrifugation was conducted at Centrifuge Clinic, Centribio, 80-2B-5ML-110). SensaDyne tensiometer was used for surface tension tests. Viscosity test was performed with the Thermo Scientific Haacke Mars Rheometer (Thermo Fischer Scientific, USA). Droplets diameters were determined using the dynamic light-scattering technique (Nanotrac Particle-Size Analyzer, Microtrac Incorporation, USA). A self-nanoemulsifying drug delivery system based on copaiba oil (named SNEDDS-CO) was obtained according to determination of the points of maximum solubility of active matter, through titration and mass fractions methodology (DANTAS et al., 2010; EMERENCIANO et al. 2019; FAHMY et al., 2015; FELIPE et al., 2013; MACIEL et al., 2014a; 2014b; 2014c; MANDAL; MANDAL, 2011; MEKJARUSKUL et al., 2013; SONI et al., 2014) by using 1% of the oil phase (copaiba oil mixed with sunflower oil on a ratio of 1:1), and 20% of Tween® 80 as surfactant, into aqueous neutral medium (79% of bidistilled water). The pH measurement was performed in a microprocessor-based pH bench meter, previously calibrated with buffer solutions of pH 4.0 and 7.0 at a temperature of 25 °C ± 0.5, in triplicate. Dynamic stability was evaluated from the centrifugation (Centrifuge Clinic, Centribio, 80-2B-5ML-110), at rate of two grams to 2000 RPM for 30 to 60 minutes. The acceptability criterion was the lack of occurrence of phase separation. From the maximum bubble pressure measurement method, surface tension using two capillary holes with different diameters, pumping nitrogen (N 2) at a constant pressure (200 kPa). The capillaries were immersed in the sample at a constant temperature (30 ± 2 °C) controlling gas bubble frequency. The viscosity evaluation was performed at 25 °C with shear rate range from 1 s -1 to 370 s -1. The droplets diameter was determined by using dynamic light scattering technique (Nanotrac Particle Size Analyzer, Microtrac Incorporation, USA), whose measurement was performed in triplicate with refractive index 1.4635.

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Wound healing assays Animals Forty-eight male Wistar rats (300 g), 120 days old, were provided by animal sector from Department of Biotechnology of the Potiguar University (UnP), located at Natal city (Rio Grande do Norte, Brazil). The number of animals per group (n=6) was recommended after application of the formula proposed by Charan and Kantharia (2013). This study was approved by the Ethics Committee on Animal Use (CEUA/UnP, 0019/240712). The animals were distributed in individual cages lined with shavings under satisfactory accommodation for an acclimation period and had a good overall health with free access to water and diet. The environment was under controlled temperature (automatically adjusted to 22 ± 2 °C), noise level below 60 dB, humidity around 45% ± 15% with light- dark cycles of 12 hours (DAMY et al., 2010; RÊGO et al., 2010).

Experimental groups and Streptozotocin (STZ) induced diabetes The animals were randomly divided in four-groups (n=6) for 7 and 14 experiment days as follow G1 (control group) and G2 (treated group) as non- diabetic rats, and also G3 (control group) and G4 (treated group) as diabetic rats. For diabetes induction the specimens of G3 and G4 groups received a single dose (65 mg/Kg) of Streptozotocin (Sigma Chemical, St. Louis, MO). STZ was diluted in 0.1 M, pH 4.5 solution of citrate. The glucose levels were daily monitored utilizing a glucometer (True Read™, HOME Diagnostics, EUA), and considered diabetics when showed glucose levels higher than 300 mg/dL.

Surgical procedures The animals were anesthetized with an intramuscular injection of Zoletil 50 (0.1 mL of solution/100 g) and then positioned in a fixed prone position on the operating table; underwent hair removal by manual traction in the dorsal region, in an area of about 4 cm 2. After that, were submitted to antisepsis with 2% chlorhexidine digluconate spraying and marking of the operative area with sterile fenestrated drape. In the center of the shaved area, an incision was made for the excision of a circular fragment of 1 cm 2 of skin, aiming at to expose the dorsal muscle fascia (ALVES et al., 2008). Hemostasis occurred with the assistance of 78 digital compression for two minutes with sterile gauze (COELHO et al., 2010). For pain control, a single dose of intraperitoneal dipyrone (50 mg/kg) diluted in saline solution, was administered (DAMY et al., 2010). 24h after the surgical procedure it was performed cleaning of the wound with saline solution (0.9% NaCl) and only for G2 and G4 groups, 1 mL of copaiba oil loaded into the SNEDDS system (so called SNECO) was topically administered, daily throughout the whole study period. Permanently, the animals were observed for the health and mobility, and submitted to macroscopic lesions evaluation (ARAÚJO-FILHO et al., 2010).

Euthanasia assays After the experimental period (7 and 14 days) the animals were submitted to new anesthesia, weighed and submitted to an excision biopsy including one centimeter of undamaged perilesional skin, reaching the dermal-epidermal region for dorsal muscle fascia exposure, and then sacrificed with an intracardiac anesthesia (thiopental sodium, 50 mg/kg).

Qualitative and quantitative histological analysis After biopsy, the skin specimens were fixed in 10% formalin for 48h and processed according to routine histological technique. Paraffin blocks with the specimens were sectioned with a thickness of 4-μm, mounted on slides and stained using the hematoxylin-eosin (HE) technique. For collagen tissue evaluation specimens were stained using the Masson’s trichrome protocol as described previously. Photomicrographs were obtained in optical microscope (OLYMPUS, CX31 model) attached to a digital camera (OLYMPUS, CX31), and saved in TIFF format, high resolution (300 dpi) for analysis with the image J 1:48 software (NIH, USA). Two calibrated examiners, treatment and subject-blinded, evaluated the images in a light microscope for qualitative and quantitative analyzes. Measurement of the epithelial thickness and wound area were performed in microphotographs with the ×40 magnification field, image J® open source software, and tools " Analyze->Measure ". Corresponding areas were designed and measured in μm and μm², respectively. For quantitative analyzes of 79 inflammatory cells, twenty fields were photographed per subject at ×400 magnification and images were used to count the total number of neutrophils and lymphocytes. The tool used was " Cell Count ". Data were tabulated and analyzed with proper statistics software.

Immunohistochemical staining On immunohistochemistry assays 3-μm sections of formalin-fixed and paraffin-embedded (FFPE) tissues were utilized. Expressions of FGF-2 (fibroblast growth factor) and IL-1β (interleukin 1 beta) in skin samples were analyzed using rabbit polyclonal antibody raised against an anti-FGF-2 recombinant protein (sc-79; Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:100 and rabbit polyclonal antibody to recombinant human anti-IL-1β (sc- 7884; Santa Cruz Biotechnology, Santa Cruz, CA) was also diluted at 1:100. FFPE sections of oral squamous carcinoma served as positive control for FGF-2 and IL-1β. Negative controls were performed by omission of the primary antibody. After deparaffinization in xylene, the tissue sections were washed twice in phosphate-buffered saline solution and treated with streptavidin-biotin- peroxidase (Dako, Carpinteria, CA, USA) at room temperature in order to bind the primary antibody. Tissue sections were stained with diaminobenzidine for visualization of peroxidase activity (D5637, Sigma Chemical, St. Louis, MO, USA) resulting in a brown-colored reaction product. The tissue sections were counterstained with Carazzi’s hematoxylin for 1 minute.

Immunohistochemical analysis The slides were photographed in ×40 magnification in the area central of wound, and digital images were loaded on image J® software (National Institutes of Health, Bethesda, Maryland, USA) to quantify immunostained cells. Immunoexpression of FGF-2 and IL-1β was evaluated on the epithelium and dermis of wound healing area. Two blinded and calibrated pathologists counted the total of positive and negative cells. Magnification fields at ×400 in twelve areas (six in epithelium and six in dermis) were selected. Cells with orange color, similar to positive control cells, were quantified and graded: 0 or no staining (<10% of positive immunostaining cells), 1 or weak staining (11% - 25%), 2 or moderate 80 staining (26% - 75%), and 3 or strong staining (>76%), as previously described (NONAKA et al., 2008).

Statistical analysis For the whole data set, Kolmogorov-Smirnov test was applied for data normality verification. Epithelial thickness, wound area and number of neutrophils and lymphocytes were evaluated using t-student test, two-tailed [data were analyzed for each tested non-diabetic (G1 and G2 groups) and diabetic (G3 and G4 groups) animals]. Comparative analysis of FGF-2 and IL-1β immunoexpression between the specimens was performed using the Mann– Whitney (two-tailed) non-parametric test. Significance level was set at 0.05 *(p<0.05). The tests were conducted in GraphPad Prism®, version 6.0 (GraphPad, La Jolla, CA, USA).

Results and Discussion

Copaiba oil chemical characterization The authenticity and quality of copaiba oil sample ( Copaifera multijuga Hayne) were confirmed by chromatography analysis using a recognized method which has been standardized for copaiba oil commercialization. Identification of its chemical components as well as comparing the obtained spectra with those stored in Wiley mass spectra library and also with substance pattern data, revealed the presence of CO-sesquiterpenes biomarkers being in accordance with previously reports (SOUZA-BARBOSA et al., 2012; VEIGA JÚNIOR et al., 2006a; 2006b; 2007). Figure 3 shows the chemical structures of the major sesquiterpenes biomarkers identified in the HRGC-MS analysis of copaiba oil in natura sample. Chromatographic and spectrometric analysis of the oil sample (Copaifera multijuga Hayne) allowed to identify sesquiterpenes and acidic diterpenes which were derivatized as their respective methyl esters and then, observed as their ester derivatives. The sesquiterpene fraction comprises about 82.35% of the oil and remain constituents such as β-caryophyllene (32.84%; retention index 1425.7) and germacrene D (18.78%; retention index 1480.5). Only 5.31% of the 81 oil sample correspond to diterpenes analyzed as their corresponding methyl esthers derivatives. In which, the main substance is copalic acid (4.44%).

CH3 CO2H

CH3 3HC CH CH2 2 H H CH3 CH3

HC CH CH3 3 3 CH3

β-caryophyllene Germacrene D Copalic acid

Figure 3. Major biomarkers identified on the copaiba oil sample by applying HRGC-MS analysis. Source: by author

Several studies attributed some of the copaiba oil activities to one or other terpene, in which the conjugation of sesquiterpenes and diterpenes are essential to promote the biological effect. An example of bioguided fractionation study of copaiba oil can be observed to the assessment of 3-β-copalic acid as the main responsible for the anti-leishmaniosis activity of copaiba oils (DOS SANTOS et al., 2013). Meanwhile, Lima et al. (2003) evidenced that copaiba oil loses its activity after its chemical fractionating, but the prompt regenerated was found when fraction were reunited, as observed in anticancer studies. Indeed, a very important study were performed with the sesquiterpene β-caryophyllene and several diterpenes isolated from copaiba oils on the inhibition of the Trypanossoma cruzi parasitc action. Isobolograms evidenced that, even though β-caryophyllene and copalic acid are very active, together they are 20 times more active at the same concentration, a potent synergistic effect of terpenes presented by the very first time (IZUMI et al., 2012). In fact, copalic acid pharmacological properties showed great findings such as anti-inflammatory (VARGAS et al., 2015), anticancer (LAMA et al., 2014), antiparasitic (IZUMI et al., 2012) and antimicrobial (SOUZA et al., 2011b). The sesquiterpene germacrene D as a biosynthetic precursor of some sesquiterpene and has large investigations such as acid catalyzed, 82 photochemically and thermally induced rearrangements agent (BARQUERA- LOZADA; CUEVAS , 2017; BÜLOW; KÖNIG, 2000). These previously findings about the sesquiterpenes β-caryophyllene and germacrene D and the diterpene copalic acid which act by synergistic effect along with the other minor compounds (Table 1) corroborate with this present study which brings results for copaiba oil ( Copaifera multijuga ) sample loaded into a colloidal SNEDDS-type formulation, instead its isolated compounds.

Table 1. HRGC-MS characterization of the oil sample ( Copaifera multijuga Hayne).

Constituents IR % NI 1214.2 0.45 NI 1340.6 0.24 α-cubebene 1352.2 0.80 α-copaene 1382.2 5.96 β-elemene 1393.4 1.84 α-gurjunene 1409.0 0.22 β-cary ophy llen e 1425 .7 32 .84 NI 1430.7 1.31 NI 1440.7 0.54 α-humulene 1453.2 4.47 allo-aromadendrene 1460.2 0.66 γ-muurulene 1472.8 4.19 Germacreno D 1480 .5 18 .78 NI 1490.2 0.86 Bicyclogermacrene 1495.2 3.36 NI 1508.6 1.49 δ-cadinene 1516.6 5.47 α-cadinene 1529.6 0.50 O. cariofilene 1567.8 0.22 NI 1572.4 0.21 Palustrol 1579.6 0.29 Ledol 1605.6 1.10 NI 1625.6 1.14 NI 1628.7 1.43 α-cadinol 1637.0 1.65 NI - 0.35

Copalic acid methyl ester - 4.44 NI - 0.17 Agatic acid methyl ester - 0.64 NI - 0.74 Agatoic acid methyl ester - 0.23 NI - 2.68 NI - 0.33 NI - 0.40 Non identified components 12 .34 % Sesquiterpen es 82 .35 % Diterpen es 5.31 % Total of identified components 87 .66 % NI = not identified; IR = retention index

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Copaiba oil loaded into colloidal formulation The ternary phase diagram (Figure 4) was constructed by titration method to determine Winsor IV nanoemulsion region (WIV). Applying the aqueous titration method under magnetic stirring at moderate temperature (50 oC to 60 oC), bidistilled water was titrated (drop by drop) to the solution of emulsifying agent (20% of Tween® 80) and oil phase (1% of CO-based oil mixture containing copaiba oil and sunflower oil on 1:1 of ratio). Under those conditions, the oil phase gets intimately dispersed throughout the aqueous continuous phase (79%) in the SNEDDS-type nanoemulsion system. During the preparations, after each water addition, the solution was examined in its appearance in which titration end-point consist of a transparent homogenous solution, affording the colloidal SNEDDS system (herein so called SNEDDS-CO). The surface tension (Figure 5) determines the cmc (8.62 x 10 -3 g/mL-1) value. This novel pharmacological copaiba oil formulation (SNEDDS-CO) presented a unique refractive index (1.490), none deviation of polarized light was observed and stability at 0 °C to 75 °C. The viscosity is 8.0 x 10 -3 cP and droplet diameter showed lower value (5.79 nm). The formulation stability in water was evaluated by successive dilutions and characterized this colloidal system as a SNEDDS-type formulation (self-nanoemulsifying drug delivery system) which stability analyzes allows to evaluate its storage time (breaks the WIV nanoemulsion region upon reaching 80 °C). After 60 minutes of centrifugation at 2 000 RPM, no change was observed. The pH data (6.46 ± 0.02) is compatible with dermatological products and with biological tolerance range (5.5 to 8.0), being suitable for human topical application (MAHMOOD; AKHTAR, 2013). Under the above conditions the SNEDDS-CO formulation is thermodynamically stable and remain stable without signs of precipitation or supernatant and can be reproduced on industrial scale.

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Figure 4. Ternary phase diagram of the SNEDDS-CO colloidal system.

Figure 5. Surface tension analysis of the SNEDDS-CO colloidal system.

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Figure 6. Viscosity analyzes of the SNEDDS-CO colloidal system.

The satisfactory dissolution of copaiba oil was possible in the SNEDDS- type formulation having Tween® 80 as surfactant which was selected for presenting a lower risk of toxic reaction and reducing effect on the particle diameter, and also aiming at to increase the thermodynamic stability of the system. The sunflower oil (mixconstituent) also increases the copaiba oil dissolution. Additionally, physiological skin acidity will not interfere in the stability of SNECO, since Tween® 80 as well as copaiba oil are biocompatible on acidic conditions. Since a co-surfactant was not necessary on SNEDDS-CO preparation this novel bioformulation represent a safe alternative for CO-cutaneous absorption with prevention and/or reduction of CO in natura side effects. Copaiba oil was chosen to be load into a self-nanoemulsifying carrier system due to the need of its bioavailability at lower concentration in order to some advantages such as i) micelles with reduced diameter contribute with the stability of colloidal systems and enhance drug carriers as they increase dissolution rates of poorly soluble in water; ii) SNEDDS systems are stable on water dilutions and maintain the gradual and prolonged release of biocompounds with pharmacokinetics improvement, iii) copaiba oil applied at lower concentration shows absence of toxicity and could to be successful applied as an efficient wound healing agent. 86

Previously we accessed the antioxidant in vitro property of some self- nanoemulsion drug delivery system based on copaiba oil, as a potential carrier to improve the solubility and bioavailability of this oil and other poor water soluble compounds. The greater inhibitory activity (84.11%) by using hydroxil radical scavenging test, was found for copaiba oil 0.5% blended on 1:1 ratio with coconut oil into a SNEDDS formulation. Meanwhile, the nanoemulsion with sunflower oil prepared at the same conditions, showed higher reduction power (52.46%). These finds suggest that copaiba oil co-encapsulated with other oils favor a more suitable oil phase which showed satisfactory antioxidant results (EMERENCIANO et al., 2019). So, from this approach by changing the oil phase composition and/or the surfactant content and even adding a cosurfactant agent or blend surfactants, the SNEDDS-CO target system may afford new biological colloidal systems and could be new carriers for synthetic and natural bioactive compounds. In fact, some of SNEDDS-CO derivative formulations were already applied (ALVES-NETO, et al., 2018; EMERENCIANO et al., 2019; MACIEL et al., 2014b; DE MEDEIROS et al., 2015; 2017). Hence, extensively application on therapeutics studies become possible, including medicinal investigations undertaken for the oral administration of poorly water soluble drugs.

Histomorphometric analysis The animals were clinically stable, in good general condition, monitored, with no evidence of side effects or infection in the surgical wound. On day 7 after the surgical procedure, for the control group G1 (normoglycemic subjects), wounds showed an exuberant granulation tissue with considerable number of neutrophils and lymphocytes (Fig. 7A, 7B, and 7C). Meanwhile, for group G2 (treated normoglycemic treated-subjects) wounds showed a moderate inflammatory infiltrate process (Fig. 7D, 7E and 7F) with decreased number of lymphocytes (p=0.0002) and neutrophils (p<0.0001) compared with G1. However, these events were more evident on diabetic animals with intense inflammatory infiltration on G3 control group (Fig. 7G, 7H, and 7I) and discreet inflammation on G4 treated group (Fig. 7J, 7K and 7L) revealed by a significant decreasing of lymphocytes (p=0.0004) and neutrophils (p=0.0048). Additionally, for G2 and G4 groups higher fibroblastic activity associated with prominent angiogenic process, benefiting repair, was observed, and been stronger on 87 treated diabetic animals (G4 group). These findings highlight the anti- inflammatory activity of CO-bioformulation (SNEDDS-CO) in the treated groups (G2 and G4), causing a reduced inflammatory infiltrate in treated-animals, mainly in diabetic subjects.

Figure 7. Microscopic events day 7 after SNEDDS-CO treatment: A) G1, intense inflammatory response, discreet fibroblast proliferation, and a cellular connective tissue; B) few blood vessels, thin and relatively sparse collagen fibers of G1; C) Graphic of neutrophils number of G1 and G2; D) G2, connective tissue granulation with intense fibroblast proliferation and reduced number of inflammatory cells; E) G2, collagenized connective tissue many blood vessels and thick collagen fibers; F) Graphic of lymphocytes number of G1 and G2; G) Fibroblasts and inflammatory cells more evident for diabetic control group (G3) indicating wound in inflammatory/proliferative phase transition; H) The main changes observed consists of short and dispersed collagen fibers, vascular endothelial cells form new blood vessels; I) Graphic of neutrophils number of diabetic G3 and G4 groups; J) G4 showed wound with evident collagen production, and fully formed blood vessels, in maturation phase of wound healing; K) Collagen showed thick fibers and more distributed in the treated diabetic specimen (G4); L) Graphic of lymphocytes number of G3 and G4 groups. 88

As illustrated in the Figure 8, fourteen days after the surgical procedure wounds showed predominance of proliferative and remodelation phases. Specifically, for G1 control group a persistent infiltrate inflammatory process (Fig. 8A, 8B and 8C) was observed and for both treated groups (G2 and G4) there was significant decrease on the number of inflammatory cells (p<0.0001 for neutrophils and p=0.0002 for lymphocytes), with greater collagen activity, and fibroblast proliferation increases (Fig. 8D, 8E and 9F). From which, an exuberant granulation tissue (inflammatory stage) with higher number of neutrophils and lymphocytes was evidenced on the G3 diabetic control group. Some fibroblasts and blood vessels were observed on G4 group (treated diabetic animals) with wounds in proliferative stage (Fig. 8J, 8K and 8L). This find showed that CO- formulation (SNEDDS-CO) was capable to reduce the number of inflammatory cells of the treated animals (G2 and G4 groups) confirming its modulated anti- inflammatory effect, being important because persistent inflammatory infiltrate by neutrophils process can delay neocollagenesis, and consequently wound contraction, since they play an important role in activation of metalloproteinase’s matrix (GAJENDRAREDDY et al., 2013; WETZLER at al., 2000). The number of neutrophils observed for G2 and G4 groups was significantly lower compared to the controls G1 and G3 groups (Fig. 8C, 8I, 8C and 8I). Since neutrophils cells are the first leukocytes that migrate for damaged area, followed by monocytes and macrophages, and also produce higher number of cytokines and proteases (GALLI at al., 2011), the results observed on treated G2 and G4 groups confirm that CO-bioformulation (SNEDDS-CO) present anti-inflammatory activity. In addition, in the 14 th day of the experimental period, the persistence of the inflammatory response on G1 control group and also bigger cellularity was compared to the smaller number of neutrophils (p=0.0002) and lymphocytes (p=0.0192) presented on G2 treated group which demonstrate greater collagenesis. On diabetic G3 control group, a moderate or intense inflammatory infiltrate and persistent neutrophils and lymphocytes process were evidenced. In the other hand, collagenesis and angiogenesis activities were identified on treated groups (G2 and G4), especially in day 14 of treatment.

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Figure 8. Microscopiceventsonday 14 after SNEDDS-CO treatment: A) G1, persistent inflammatory infiltrate and delayed fibroblastic proliferation; B) G1, cellular connective tissue with thin and relatively sparse collagen fibers; C) Graphic of neutrophils number of G1 and G2; D) G2, small number of mononuclear inflammatory cells; E) G1 with thick and organized collagen fibers of dermis, a few cells; F) Graphic of lymphocytes number of G1 and G2; G) G3, proliferative wound with moderate inflammatory infiltrate and fibroblasts and smaller collagen activity; H) Collagen with delicate fibers disperses in dermis and endothelial cells that formed immature blood vessels; I) Graphic of neutrophils number of G3 and G4; J) G4, wounds showed completely repair and complete reepithelization and thick collagen fibers; K) The dermis demonstrated collagen and blood vessels sparse; L) Graphic of lymphocytes number of G3 and G4 groups.

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The largest number of lymphocytes observed in the control groups (G1-7 days and G3-14 days) revealed occurrence of transition, from inflammatory to proliferative stage. Meanwhile, for treated groups (G2-7 days and G4-14 days) greater number of fibroblasts and re-epithelialization were confirmed and a few number of lymphocytes demonstrated a more advanced repair phase (maturation stage), indicating the healing efficiency of SNEDDS-CO. Epidermis coated the damaged area and, in all cases, the keratin layer formation was evidenced in the treated groups (G2 and G4). Moreover, this groups showed greater epithelium thickness (p<0.0001 Fig. 9C), and reduced wound area (p=0.0016 Fig. 9F). These events correspond to the final stage of healing process, favoring especially the treatment of the diabetic animals (G4 group). With respect to the angiogenesis, comparatively, control groups presented dispersed endothelial cells and immature blood vessels indicating a delayed and discrete angiogenic activity that reinforce the angiogenic property of SNEDDS-CO. This find is very important because angiogenesis supports and improves the repair process, modulating chemokines expression, and also improves the healing of chronic wounds, especially on diabetic subjects (BODNAR, 2015).

CO-bioformulation (SNEDDS-CO) effect on IL-1β expression As demonstrated in the Figure 9, treated wounds (G2 as non-diabetic and G4 as diabetic subjects) assessed for immunoexpression analyzes of interleukin 1 beta (IL-1β) in dermis and epidermis showed cytokine decreasing on dermis (Table 2) and epidermis (Table 3). It is kwon that IL-1β together with other proinflammatory chemokine play an important role in recruitment of inflammatory cells and regulate the neutrophilic function during repair process (ROSIQUE et al., 2015; MOLLOY et al., 2003). According to our findings on day 7, immunoreactivity of IL-1β (Fig. 9A and 9B) reinforce histomorphometric findings demonstrating reduction on lymphocytes and neutrophils in the tissues of the treated animals. For diabetic G3 control group the stronger expression of IL-1β in dermis (Fig. 9G and 9H) corroborate with the delay in wound repair, reinforcing that there is a persistent inflammatory response delaying the wound healing. On the other hand, no appreciable difference was observed in the expression of IL- 1β in epidermis of the treated groups (G3 and G4) probably due the incomplete reepithelization (Table 3). 91

Figure 9. Immunohistochemical analyzes on day 7 after SNEDDS-CO treatment: A) G1, immunoexpression of IL-1β in epidermis (moderate, yellow arrow) and dermis (moderate, green arrow); B) G2, immunoexpression of IL-1β in epidermis (absent, yellow arrow) and dermis (weak, green arrow) of; C) Graphic of epithelial thickness of G1 and G2; D) G1, immunoexpression of FGF-2 in epidermis (weak, green arrow) and dermis (moderate, yellow arrow); E) G2, immunoexpression of FGF-2 in epidermis (intense, green arrow) and dermis (intense, yellow arrow); F) Graphic of wound area of G1 and G2; G) G3, moderate immunoreactivity of IL- 1β in keratinocytes and inflammatory cells; H) G4, absence of immunoreactivity of IL-1β in dermis (yellow arrow); I) Graphic of epidermal thickness of G3 and G4; J) G3, weak immunoreactivity of FGF-2 on inflammatory cells (yellow arrow) and fibroblasts (green arrow) in dermis; K) G4, moderate immunoreactivity of FGF-2 in fibroblasts (green arrow) and inflammatory cells (yellow arrows); L) Graphic of wound area of G3 and G4 groups.

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Table 2. Immunoexpression of interleukin 1 beta in dermis.

Table 3. Immunoexpression of interleukin 1 beta in epidermis.

Meanwhile, on day 14 some differences were detected (Table 2) on diabetic G4 treated group which show lower IL-1β-immunoreactivity (Fig. 9J and 9K) than the control group. These findings suggest SNEDDS-CO as IL-1β inhibitor, contributing with repair of diabetic chronic wounds (Table 1). On the other hand, for non-diabetic G2 treated group, the immunoreactivity analysis (IL- 1β at day 14) the diminished IL-1β expression was more discrete (Table 1, dermis) and (Table 2, epidermis) (Fig. 10A and 10B). Comparatively, at day 14, control groups showed stronger immunoreactivity for IL-1β in epidermis and dermis (Fig. 4G and 4H; Table 1, and Table 2, for G1, non-diabetic group, and Fig. 4G and 4H; Table 1, Table 2, for G3 diabetic group). Meanwhile, IL-1β-immunostaining was absent or weak for diabetic G4 treated group, this event corroborates with the reduced number of inflammatory cells (Fig. 8J, 8K, 8I, and 8L, and Fig. 10J, 10K, 10I, and 10L) reinforcing SNEDDS-CO as IL-1β inhibitor contributing with repair of chronic wounds (Table 1). This finding is very important, once IL-1β is suggested as a complicate factor in wound repair, especially in diabetic patients (BǍDULESCU et al., 2013) and consequently its inhibition is associated with improved wound repair in type II diabetic subjects (MIRZA et al., 2013).

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Figure 10. Immunohistochemical analyzes on day 14 after SNEDDS-CO treatment: A) G1, immunoexpression of IL-1β in epidermis (intense, green arrow) and dermis (intense, yellow arrow); B) G2, immunoexpression of IL-1β in epidermis (weak, green arrow) and dermis (weak, yellow arrow); C) Graphic of epithelial thickness of G1 and G2; D) G1, immunoexpression of FGF-2 in epidermis (absent, green arrow) and dermis (absent, yellow arrow); E) G2, immunoexpressionof FGF-2 in epidermis (moderate, green arrow) and dermis (moderate, yellow arrow); F) Graphic of wound area of G1 and G2; G) Stronger expression of IL-1β in epidermis (green arrow), in fibroblasts and inflammatory cells of dermis (yellow arrow) for diabetic control group (G3); H) G4 (diabetic treated group), weak expression of IL-1β, in epidermis (green arrow) and dermis; I) Graphic of epidermal thickness of G3 and G4; J) G3, focal immunopositivity of FGF-2 in inflammatory cells (yellow arrow) in dermis; K) G4, stronger immunoreactivity of FGF-2 on kerathinocytes (green arrow) and fibroblasts (yellow arrow); L) Graphic of wound area of G3 and G4 groups.

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CO-bioformulation (SNEDDS-CO) effect on FGF-2 expression On treated G2 and G4 groups higher FGF-2 immunoreactivity in dermis (Table 3) and epidermis (Table 4) (Fig. 9C, 9D, 9G, and 9J), higher epithelial thickness and wound size reduction was also notified on treated groups than control groups (G1 and G3). On day 7, the number of cells immunostained with anti-FGF-2 (antibody) was higher (G2 and G4) than in the control groups (G1 and G3) (Table 4 and Table 5).

Table 4. Immunoexpression of FGF-2 in dermis.

Table 5. Immunoexpression of FGF-2 in epidermis.

Epidermis keratinocytes of the treated groups (G2 and G4) showed moderate immunoreactivity (Fig. 10E and 10K) as well as epithelial thickness and wound size reduction. On control groups (G1 and G3) the FGF-2 expression was weak or absent for both dermis and epidermis (Fig. 10D and 10L). Several previous studies demonstrated the correlation of FGF-2 overexpression with improvement in repair process (NUGENT et al., 2000; CROSS et al., 2001; DATAS-FILHO et al., 2007) by regulating important process such as angiogenesis and fibroblast proliferation, the higher FGF-2 expression on treated wounds (G2 and G4) was associated to repair improvement (Figure 9D and 9E; 10D and 10E; Tables 3 and 4). Previous results of in natura copaiba oil showed its anti-inflammatory by means of cytokines inhibition, which was attributed to the CO-phytochemicals compounds such as sesquiterpenes and diterpenes. Since β-bisabolene has anti- 95 inflammatory and analgesic proprieties and β-caryophyllene is described as anticancer, anti-inflammatory and antimicrobial agent (GOMES et al., 2010; VEIGA-JÚNIOR et al., 2007; PAIVA et al., 2003). Since the sesquiterpene β- caryophyllene has been detected as a main component in several Copaifera species, the present findings may be addressed to the high content of β- caryophyllene synergic acting with the other terpenoid constituents. Certainly, treatment with SNEDDS-CO up regulate FGF-2 expression which is correlated with fibroblast proliferation, neocollagenesis, tissue reepithelization, wound reduction area and angiogenic process, confirming the improved healing effect of CO. So, the present findings corroborate with the indication of copaiba oil for treatment of chronic wounds. Reinforcing the importance of the present study, Arruda et al. (2019) covering mostly the last two decades on the distribution, chemistry, pharmacology, quality control and safety of Copaifera species showed the lack of studies concerning different copaiba species wound healing effect. According to these authors, only after several additional research studies approaching these and other pharmacological findings, the traditional use of Copaifera as healing agent will be accordingly corroborated.

Conclusions

A colloidal SNEDDS-type system (self-nanoemulsifying drug delivery system) based on copaiba oil (CO) and Tween® 80 (surfactant) was prepared and applied on wound healing of non-diabetic and diabetic rats. CO was loaded at lower content aiming at CO-gradual and prolonged release as well as to avoid possible occurrence of CO in natura side-effects. The chemical approach afforded a SNEDDS formulation containing 1% of oil phase (CO and sunflower oil,1:1 ratio), 20% of surfactant and 79% of bidistilled water, which is stable at water dilutions conditions. Chromatographic HRGC-MS analysis of the oil sample ( Copaifera multijuga Hayne) allowed to identify sesquiterpenes (82.35%) and diterpenes (5.31%). The major sesquiterpenes are β-caryophyllene (32.84%) and germacrene D (18.78%). The major diterpene analyzed as its corresponding methyl esther derivative is copalic acid (4.44%). 96

The repairing process showed successful results for the copaiba oil non- toxic SNEDDS formulation, herein so called SNEDDS-CO, which in vivo treatment optimized CO-bioavailability, demonstrating wound healing improvement as well as anti-inflammatory effect. Sunflower oil was co-encapsulated with copaiba oil favoring a more suitable oil phase in this CO-colloidal formulation which increased fibroblast proliferation and neocollagenesis, and showed to be IL-1β inhibitor, and also activated FGF-2 production, associated to positive neocollagenesis. These therapeutic events support the use of CO-lower content loaded into a SNEDDS system aiming at CO-gradual and prolonged release applied on wound healing in normal and diabetic rats. Animals, Rattus novergicus albinus , were subjected to dorsal cutaneous skin flap surgery and received (topically) the copaiba oil loaded into the SNEDDS carrier. For control group saline solution was applied. The histomorphometric analysis revealed a smaller number of neutrophils and lymphocytes in the treated group as well as collagenesis, fibroblast proliferation and larger epithelial thickness. Copaiba oil was able to reduce the IL-1β expression and increase the FGF-2 expression and these molecular alterations were directly associated with were neocollagenesis which accelerated the wound healing process. Therefore, these molecules may represent possible targets to treat chronic wounds, and the mechanism of action of the copaiba oil. Taken together, the results of this study reinforce the healing potential of copaiba oil, and provide new insights about its nanoencapsulation by using the SNEDDS biotechnological approach, which improves the CO-resin bioavailability and consequently its healing activity. It is important to mention that our findings can help to develop new therapeutic options for the management of chronic wounds, particularly in diabetic subjects.

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5.3 Effect of low-intensity laser therapy on angiogenesis and MMP-2 immunoexpression in wound repair

Artigo publicado no periódico Lasers in Medical Science DOI: 10.1007/s10103-016-2080-y

Abstract Low-level Laser Therapy (LLLT) shows anti-inflammatory and angiogenic activities in wound healing. Several studies have demonstrated benefits of the Low-level laser therapy (LLLT) therapy including collagen production, synthesis of adenosine triphosphate (ATP) and fibroblasts growth, as proliferative activities. Despite of that, LLLT mechanisms of action in increased levels of growth factors, enzymes and extracellular matrix remodeling proteins (ECM) as well as stimulation of angiogenesis still not fully clarified. Thus, in this study the effect of LLLT on wound healing in rats, highlighting the MMP-2 immunoexpression. Additionally, we evaluated the epidermal and dermal MMP-2 expression and its correlation with angiogenesis during wound healing. Persistence crust and moderate number of inflammatory cells were evidenced in the control groups CG7 and CG14. In the LG14 treated group, wounds demonstrated completely re- epithelization on remodeling phase. Angiogenesis and MMP-2 expression were higher in LLLT treated groups (LG7 and GL14) especially in the 14th day, and they were correlated according to Spearman correlation test. LLLT results improve wound healing by enhancing the neocollagenesis and increasing the amount of new vessels formed in the tissue (neoangiogenesis) and modulates MMP-2 expression. Epidermal overexpression of MMP-2 was correlated to angiogenic process. Meanwhile, the mechanism of action and optimal parameters still need further clarification.

Keywords: Low-Level Laser Therapy; Wound Healing; Angiogenesis; MMP-2.

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Introduction Despite substantial progress in wound treatment, impaired wound healing is associated with morbidity and mortality, specifically in patients with deficient angiogenesis and neocollagenesis, such as diabetic patients. Additionally, it has been recognized that several cellular and molecular mechanisms in these patients culminate in persistent inflammatory cell infiltration, overexpression of cytokines, and low expression of growth factors (BARRIENTOS et al., 2014; REINKE; SORG, 2012). According to the National Institutes of Health, the prevalence of chronic ulcers in American patients is estimated to be 2%of all patients with wounds, and the costs for treatment are estimated to be US $50 billion per year (BLACKWELL et al., 2014; JUNG et al., 2016; MENKE et al., 2007; MENKE et al., 2008; SEARLE et al., 2008; SEN et al., 2009; SUN et al., 2014). In Australia, the health care system spends about US $2.85 billion per year for chronic wound treatments (NORMAN et al., 2016). Wound healing is a coordinated, complex, and dynamic biological process. Three phases have been described during wound healing, inflammation, proliferation, and maturation. During the first phase, inflammatory cells and keratinocytes produce cytokines and growth factors responsible for fibroblast proliferation and chemotaxis. A higher intensity or persistence of this step can delay healing and impair angiogenesis and neocollagenesis, resulting in amputation procedures (ENOCH; LEAPER, 2005; ENOCH; LEAPER, 2008; WILLIAMSON; HARDING, 2004). Several molecular mechanisms regulate wound healing, particularly overexpression of matrix metalloproteinases (MMPs), a family of calcium- dependent, zinc-containing endopeptidases involved in matrix remodeling. Additionally, low levels of tissue inhibitors of metalloproteinases (TIMPs) can affect skin wound healing without a fully clarified mechanism of action (BELLAYR; MU; LI, 2009; HAUBNER et al., 2015). During wound healing at inflammatory and maturation phases, MMP-2 is specifically expressed, resulting in degradation of the extracellular matrix, particularly in the maturation phase (FRANKOVA et al., 2013; GILLARD et al., 2004; KARIM et al., 2006). In addition to MMP-mediated collagen degradation, these proteinases play an important role in wound healing that is supported by 99 the ability of MMP-2 to enable endothelial cell (EC) migration during angiogenesis. Associated with this process, MMP-2 overexpression appears to facilitate reepithelization and fibroblast growth and reduce cellular adhesion by degradation of cadherins (junctional proteins), thereby improving the angiogenic process (GILL; PARKS, 2008; HAUBNER et al., 2015; MOTT; WERB, 2004; NEDEAU et al., 2011; PARKS et al., 2004). It has been recently demonstrated that MMP-2 plays a role in angiogenesis during tumorigenesis (ROJIANI et al., 2010). This effect would be advantageous in wound healing, but other studies have demonstrated that MMP-2 is responsible for impaired wound healing (KARIM et al., 2006; KREJNER; GRZELA, 2015). Furthermore, its mechanism of action and the correlation between MMP-2 immunoexpression and angiogenesis in wound healing remain unclear. Several studies have demonstrated the benefits of low-level laser therapy (LLLT), including collagen production, synthesis of adenosine triphosphate (ATP), and fibroblast growth. Lymphocyte activity has also been observed, exerting an antibacterial effect, as well as the formation of new blood vessels (neoangiogenesis) (CARVALHO et al., 2010; FATHABADIE et al., 2013; FERRARESI et al., 2015; FONSECA et al., 2012; GONÇALVES et al., 2013; HOURELD et al., 2014; SHARIFIAN et al., 2014; YAN et al., 2011). However, LLLT mechanisms of action in enhancing the levels of growth factors, enzymes, and extracellular matrix-remodeling proteins as well as stimulation of angiogenesis have not been clarified fully. Aparecida da Silva et al. (2013) evaluated the effect of LLLT on the mRNA expression levels of MMP-2 and MMP-9 in diabetic rats. They concluded that LLLT decreases MMP levels during repair processes, but optimal parameters and the cellular mechanisms remain to be clarified, and contradictory reports on the correlation betweenMMP-2 expression and angiogenic processes need clarification. Therefore, in this study, we examined the effect of LLLT on wound healing in rats, focusing on MMP-2 immunoexpression. In addition, we evaluated epidermal and dermalMMP-2 expression and its correlation with angiogenesis during wound healing.

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Material and Methods Animals and ethical considerations Twenty female Wistar rats were used in this study (average weight, 250 ± 50 g; 60 days of age). The animals were kept in individual cages under controlled temperature conditions (22 ± 2 °C) and lighting (12-h light/dark cycles) with minimal noise and food and water provided ad libitum. The study followed the guidelines of the Animal Experimentation Code of Ethics of the Brazilian College of Animal Experimentation and was approved by the Ethics Committee of Potiguar University Laureate International Universities (Protocol# 001/2013).

Surgical procedures After wound induction, the animals were randomly allocated into control groups (CG) and laser-treated groups (LG) at days 7 and 14. CG animals were treated daily with saline and LG animals received LLLT. Groups (n = 5 each) were designated as CG7 (day 7 control group), LG7 (day 7 LLLT-treated group), CG14 (day 14 control group), and LG14 (day 14 LLLT-treated group). Before wound induction, the back of each animal was sterilized with 2 % chlorhexidine gluconate. Animals were anesthetized via intramuscular injection of 100 mg/kg of an anesthetic solution including 10 % ketamine (Cetamin®; Syntec, São Paulo, Brazil) and 5 mg/kg of 2 % xylazine (Dopaser®; Syntec). An incision in the animal’s backmeasuring 2×2 cm was made to expose the muscle fascia dorsal, producing a square open wound. Pain at postoperative recovery (48 h) was controlled by a single dose of intraperitoneally injected dipyrone (50 mg/kg diluted in saline) followed by continuous monitoring.

Low-level laser therapy (LLLT) LLLT irradiation of wounds (LG7 and LG14 groups) was performed with an indium–gallium–aluminum–phosphide (GaAlInP) diode laser (PHOTON LASER III, DMC® equipment Ltda., São Carlos, Brazil) using parameters as follows: visible red, 4 J/cm 2 irradiation, 660-nm wavelength in continuous emission, 40mW, and punctual using the pen positioned vertically and observing a distance of 1 cm from the edge of the lesion and from one point to another. The first 101 application of LLLT was performed at 2h after wound induction and then every 48h until the end of the experimental period.

Tissue collection After each experimental period, the animals were anesthetized and we performed an excisional biopsy of the entire lesion including undamaged portions. For the biopsy, a 1-cm lesion of the normal skin margin adjacent to the wound or scar reaching the dermo-epidermal region was necessary. This material was collected and placed in a histological cassette. The specimens were fixed in 10 % formaldehyde and processed accordingly the routine histochemical procedures. For euthanasia, the animals were intracardially injected with 300 mg/kg of 10 % ketamine and 15 mg/kg of 2 % xylazine.

Histological and histomorphometric analysis For morphological analysis, the specimens were fixed in 10 % formalin for 48 h and then processed in a Leica® type TP1020 Tissue Processor, embedded in paraffin, and sectioned on a microtome to thicknesses of 5 μm. Sections were mounted and stained with hematoxylin and eosin (HE) and Masson’s trichrome. The sections were examined under a BX50 optical microscope (Olympus) coupled to camera (Sony) and ImageJ software (National Institutes of Health, Bethesda, USA). Photomicrographs were obtained at various magnifications (×40, ×100, and ×400). Analyses were performed by two blinded evaluators. In the qualitative analysis, microscopic images were analyzed for damage by inflammatory cell infiltration, neocollagenesis, and tissue repair. Quantitative analysis was performed using ImageJ software. Neutrophils and lymphocytes were counted in 12 fields at ×400 magnification for each specimen. The total area of the wound ( μm2) and epithelial wound thickness ( μm) were measured. Analysis of angiogenesis was conducted by previously reported methodology (YAMAKAWA et al., 2004; ZGHEIB et al., 2013), in which the evaluators quantified the number of blood vessels in 10 fields at ×100 magnification for each animal and calculated the mean number of blood vessels per field. The scores were (1) up to 20 vessels per field; (2) 21-50 blood vessels per field; (3) 51-80 blood vessels per field; and (4) more than 80 blood vessels per field. 102

Immunohistochemical staining Paraffin-embedded tissue samples were sectioned (3 μm thick) and mounted on silane-coated glass slides for immunohistochemistry. Briefly, the sections were dewaxed with xylene and then hydrated through a graded series and ethanol concentrations. Antigen retrieval was performed by autoclaving the sections for 15 min in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation in 10 % hydrogen peroxide for 15 min. The sections were then incubated overnight at 4 °C with a primary anti-MMP-2 antibody obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The sections were exposed to an avidin-biotin complex and then horseradish peroxidase reagents (Rat LSAB Kit; Dako Cytomation, Carpinteria, CA, USA) and diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA). Finally, the sections were counterstained with Carazzi hematoxylin.

Immunohistochemical analysis Semiquantitative analysis was performed by the following scoring system: (0) no immunostaining; (1) weak or moderate staining in 25 % of cells; (2) weak or moderate staining in 50 % of cells; and (3) immunostaining stronger in >50 % of cells. Data were tabulated for the dermis and epidermis.

Statistical analysis A statistical test was selected for each experimental period (7 and 14 days). Student’s t test was used for comparisons of neutrophil and lymphocyte numbers and the epithelial wound area and thickness. The Mann–Whitney test (nonparametric) was used for comparison of angiogenesis scores. The Spearman correlation test was performed to evaluate possible correlations between the number of immunopositive cells for MMP-2 and angiogenesis scores. All tests were performed in GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA). The significance level was set at 0.05 (p < 0.05).

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Results Qualitative and quantitative histological analysis Morphological analysis of the inflammatory phase in CG7 group wounds revealed the presence of a crust, evident inflammatory cell infiltration, a small number of fibroblasts in the dermis, and a small number of blood vessels (Fig. 1). Evident angiogenesis with more blood vessels (p < 0.05) and fibroblast proliferation (the transition between inflammatory/proliferation phases) were observed in wounds of the LG7 group, but no differences were observed in inflammatory cell infiltration, epithelial thickness, or wound areas compared with the CG7 group (Figure 1). During this experimental period, inflammatory cells had infiltrated at the wound margins and a crust consisting of neutrophils and a few lymphocytes was observed. In the LG14 group, neocollagenesis and collagen deposition were the main findings, whereas a considerable number of inflammatory cells and fibroblasts were found in the corresponding control group (CG14) (Figure 2). Conversely, both LG14 and CG14 groups showed inflammatory cell infiltration with a predominate mononuclear component and few neutrophils, lymphocytes, and macrophages. The LG14 group exhibited re-epithelialization at the early stages, whereas the corresponding control group (CG14) exhibited wounds with incomplete re-epithelialization and more cellular granulation tissue with immature blood vessels. All animals in the LG14 group showed wavy, thin, short, and reticular collagen fibers, while the CG14 group showed poor collagen formation.

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Figure 1. Histological and immunohistochemistry at day 7. a) Presence of a crust and moderate inflammatory cell infiltration. Epithelialization was incomplete in CG7 (HE staining; scale bar - 50 μm); b) LG7 showed intense neovascularization and a crust (HE staining; scale bar - 50 μm); c) Neocollagenesis and vascularization were discrete in CG7 (Masson’s trichrome staining; scale bar - 100 μm); d) Neocollagenesis and neovascularization were more evident in LG7 (Masson’s trichrome staining; scale bar - 100 μm). Immunohistochemical staining of formalin- fixed scar tissues. MMP-2 demonstrated stronger staining in inflammatory cells in CG7 (e) and LG7 (f) (IHC stain, scale bar - 50 µm); g) Graphic representation of neoangiogenesis (Mann-Whitney test); h) epithelial thickness and (i) wound area (Student t test) in CG7 and LG7. 105

Figure 2. Histology and immunohistochemistry at day 14. a) In CG14, we observed a crust with strong neutrophilic infiltration and a small number of blood vessels and collagen fibers (HE staining, scale bar - 100 μm); b) LG14 specimens had a completely re-epithelialized wound with the presence of keratin (HE staining; scale bar - 100 μm); c) Evident neovascularization in CG14 and poor neocollagenesis (Masson’s trichrome staining; scale bar - 100 μm); d) Absence of inflammatory cell infiltration and complete re-epithelialization in LG14 (Masson’s trichrome staining; scale bar - 50 μm). Immunohistochemical staining of formalin-fixed scar tissues; e) MMP-2 was expressed in inflammatory cells and fibroblasts in CG14; f) Intense immunostaining of MMP-2 was observed in the dermis and epidermis of LG14 (immunohistochemical staining; scale bar - 50 μm); g) Graphic representations of neoangiogenesis (Mann–Whitney test); h) epithelial thickness; i) wound area (Student’s t test) in CG14 and LG14 groups.

In the quantitative analysis of inflammatory cell infiltration (Figure 3), the mean number of cells per field was 19.5 neutrophils and 18.0 lymphocytes in the CG7 group, 20.5 neutrophils and 24.0 lymphocytes in the LG7 group, 10.0 neutrophils and 7.5 lymphocytes in the CG14 group, and 1.5 neutrophils and 0.5 lymphocytes in the LG14 group. No difference was observed at 7 days (p > 0.05). 106

The LG14 group showed a smaller number of neutrophils and lymphocytes (p < 0.05).

Figure 3. Quantitative analysis of inflammatory cell infiltration. Graphs show the number of neutrophils and lymphocytes at day 7 (a) and (c) and day 14 (b) and (d) .

Angiogenesis scores were higher in LLLT-treated groups (Fig. 1, LG7, and Fig. 2, LG14, p = 0.0239 in the CG7 group and p = 0.267 in the CG7 group; Mann–Whitney test). Additionally, incomplete or immature blood vessels were predominante in the control groups, whereas the blood vessels had matured in LLLT-treated groups.

Immunohistochemical analysis Analysis of MMP-2 immunoreactivity in the epidermis of wounds revealed weak expression in CG7 and CG14 groups, moderate expression in the LG7 group, and strong expression in the LG14 group. In the dermis, similar immunoexpression of MMP-2 was found in CG7 and LG7 groups. However, theLG14 group demonstrated higher expression in the dermis compared with the CG14 group (p<0.05) (Tables 1 and 2). A correlation between angiogenesis scores and epidermal MMP-2 immunoexpression was only observed in LLLT- treated groups (LG7 and LG14, p < 0.05; Spearman ranks test, Table 3).

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Discussion In this present study, LLLT induced faster repair and a higher quality of newly formed tissue, mainly at 14 days after wound induction in animals (LG7 and LG14 groups) compared with untreated animals (CG7 and CG14 groups). These observations are in accordance with previous studies (DE LIMA et al., 2014; FERNANDES et al., 2015; KOO et al., 2015). Furthermore, we found a correlation between epidermal MMP-2 immunoexpression and angiogenesis. According to the LLLT mechanism of action in wound healing (FERRARESI et al., 2015; FONSECA et al., 2012; YAN et al., 2011), photobiomodulation occurs mainly in mitochondria, resulting in an increase of the respiratory chain and, consequently, ATP synthesis. These events favor the healing process through cellular proliferation, production of nucleic acids, 108 collagen synthesis, and a reduction of inflammation (GONÇALVES et al., 2013; HOURELD et al., 2014). Additional effects are associated with the cell membrane (ANWER et al., 2012). In the qualitative analysis of CG7 and LG7 groups, the wounds showed granulated tissue with abundant inflammatory responses of predominantly neutrophils and lymphocytes, and proliferation of blood vessels in the LG7 group as well as leukocytes associated with a fibrin crust. At day 7, no morphological differences were observed in both groups. Conversely, in the LG7 group, angiogenesis and neocollagenesis were more evident. LLLT irradiation (4 J/cm 2) stimulated fibroblasts for collagen formation and wound retraction. These events were more evident at 14 days after LLLT (LG14). This group also showed a reduction of inflammatory cells. According to Maiya, Kumar e Nayak, (2009), LLLT (He–Ne laser type) at 8–9 J/cm 2 induces less neocollagenesis activity and granulated tissue formation compared with 3–7 J/cm 2. This finding corroborates the present results in which LG14 (4 J/cm 2 irradiation) showed intense collagen deposition. However, the absence of granulated tissue in treated rats (LG14) was associated with a longer experimental period (14 days) compared with 5 days in the previous study (MAIYA et al., 2009). Wound healing is the biological process of repair and reestablishment of damaged areas. However, the biology is still poorly understood, including the expression of metalloproteinases during its diverse steps. Upregulation of MMP- 2 during wound healing has been described as a delaying factor (GILL; PARKS, 2008; KREJNER; GRZELA, 2015; LITWINIUK et al., 2012) but several studies describe the role of this proteinase in dynamic remodeling of the extracellular matrix as an important factor during the remodeling phase of wound healing (APARECIDA DA SILVA et al., 2013; DE VASCONCELOS CATÃO et al., 2015). Gill and Parks (2008) reported that MMPs and their inhibitors act during the stages of wound healing but highlight its role in regulating the inflammatory response and interfering with the activities of signaling molecules and cell adhesion, especially their direct interactions with chemokines and chemokine receptors. Another important aspect in wound healing is the balance between MMP and TIMP expressions. 109

In the present study, all groups demonstrated immunoreactivity for MMP- 2. This finding suggests stronger involvement of MMP-2 in the physiological processes of LLLT-treated groups (LG7 and LG14) compared with control groups (CG7 and CG14). Because upregulation of MMP-2 has been described in irradiated human skin fibroblasts (AYUK; HOURELD; ABRAHAMSE, 2014), the present findings corroborate the overexpression of MMP-2 in keratinocytes, dermal fibroblasts, and inflammatory cells. Additionally, in an in vivo LLLT study, Guerra et al. (2013) demonstrated improvements in tendon healing through an increase in MMP-2 activity. Furthermore, DA Silva et al. (2013) showed decreases in MMP-2 and MMP-9 expressions during wound healing in diabetic animals that were subjected to a single application of LLLT (50 mW, 660 nm, 4 J/cm 2, 80 s) in a short period (24 h). This finding contradicts our results in which a correlation was found between MMP-2 expression and angiogenesis, accelerating wound healing and contraction. This effect was more evident in the LG14 group compared with the other groups. Despite the contradictory results, probably due to methodological differences, collagen degradation by MMPs plays an important role in healing, because MMP-2 enables EC migration during angiogenesis and wound healing (NEDEAU et al., 2011). In this context, the present study corroborates the beneficial effects of laser applications, because immunohistochemical analysis demonstrated a positive correlation between epidermal MMP-2 expression and angiogenesis (Spearman rank correlation). This finding should be significantly considered because few studies have evaluated and correlated the effects of LLLT between MMP-2 expression and angiogenesis during wound healing (CORAZZA et al., 2007; CURY et al., 2013). Neocollagenesis and neoangiogenesis are predominant factors for complete wound repair, particularly in diabetic patients that have deficiencies in these processes. Activation of several genes has been found during healing in irradiated tissues (SHARIFIAN et al., 2014), including genes involved in cell adhesion and the extracellular matrix, which are responsible for regulation of the production of inflammatory cytokines and chemokines, growth factors, and signal transduction. Seventy-six genes are regulated by laser irradiation, including 43 upregulated genes and 33 downregulated genes. 110

The regulation of genes that activate inflammatory cytokine production supports the anti-inflammatory activity observed in our study. Additionally, most events in advanced healing (reepithelization, wound contraction, no inflammatory cell infiltration) occurred after 14 days. This finding corroborates related studies on the cumulative effects of LLLT, highlighting the need for further studies to analyze the expression of genes related to MMP-2 expression and angiogenesis.

Conclusions In this work it was demonstrated the possible utility of a GaAlInP laser with an appropriate energy density (4 J/cm 2) as an adjunctive modality for wound healing in clinical practice as well as a correlation between epidermal MMP-2 expression and angiogenesis. In fact, LLLT improved wound healing, especially at the 14 day, as evidenced by wound contraction, anti-inflammatory activity, neocollagenesis, and neoangiogenesis. These findings may be associated with increased mitochondrial activity with the consequential increase in ATP, vasodilation, protein synthesis, decreased levels of prostaglandins, mitosis, and the migration and proliferation of keratinocytes. Considering the limitations of immunohistochemical techniques, the correlation of MMP-2 overexpression with angiogenesis in wound healing warrants further studies to confirm MMP functions in wound healing and the correlation with angiogenesis.

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5.4 Improvement of wound healing process by combining SNEDDS copaiba oil system and low level laser therapy on the inflammatory and proliferative phase of wound healing

Abstract The low level laser therapy (LLLT) procedure was assessed along with the natural pharmacological formulation SNEDDS-CO (Self-nanoemulsifying drug delivery systems based in copaiba oil) as wound dressing, for employment of fibrin and collagen along with other benefits. Efforts to overcome the wound healing led to the design and optimization a bioavailability copaiba oil nanoformulation so called SNEDDS-CO. Indeed, the copaiba oil loaded into a SNEDDS cosurfactant-free formulations was prepared by using phase diagram performed on different concentration oil, surfactant and water constituents, such as 1%(w/w) of oil phase, 20%(w/w) of Tween 80®, and 79%(w/w) of double-distilled water. Copaiba oil sample ( Copaifera langsdorffii) was authenticated by gas chromatography equipped with a flame ionization detector and the nanoformulation SNEDDS-CO was characterized by droplet particle size, zeta potential, polarized light microscopy, pH, conductivity, refractive index, rheological and surface tension analysis. The pharmacological findings showed that both LLLT radiation (wavelength 660 nm) and SNEDDS-CO topical treatment of rat´s cutaneous lesions, strongly favored healing. LLLT treatment showed to be effective on neoangiogenic, collagenases, modulation of inflammatory response favoring the re-epithelialization of the assayed lesions. Meanwhile, combined SNEDDS-CO with LLLT significantly accelerated the healing process of experimentally induced cutaneous lesions in rats, especially in the initial phase (7 days), as evidenced by the early re-epithelization, high histologic grading regeneration repair, intense collagenases were observed at the 14 day, due to the appearance of cutaneous attachments and reduction of the lately inflammatory response. These results may be related to the modulation of the inflammatory response, observed especially from the decrease of lymphocytes in the proliferative phase.

Keywords : Copaiba oil; SNEDDS Drug Delivery Systems; Wound Healing; Angiogenesis, Neocolagenesis. 112

Introduction Low level laser therapy LLLT has been proposed for various treatment modalities of wound healing. Indeed, laser irradiation modulates biological activities in many tissues. On skin heal lasers act in its four stages (hemostatic, inflammatory, proliferative and remodelative) by emitting a highly concentrated, non-invasive, non-ionising radiation. Specifically, decreases inflammatory cell infiltration, accelerates wound healing processes with an increase of collagen synthesis. So, through photobiomodulation during the tissue-healing period, promote photobiochemical effect when in contact to different tissues, causing differentiation of fibroblasts into myofibroblasts, cell proliferation, angiogenesis and collagen synthesis and provides closure of the injured area in a short period (BABUCCU et al., 2014; CARVALHO et al., 2010; CATÃO et al., 2015; DANTAS et al., 2011; FATHABADIE et al., 2013; LOPES et al., 2010; FREITAS et al., 2013; GONÇALVES et al., 2013; HOURELD et al., 2014; PUGLIESE et al., 2003; REIS et al., 2008; SAITO et al., 2011; SHARIFIAN et al., 2014). Due to the complexity of the tissue repair process, several resources such as laser radiation, acupuncture, physiotherapeutic, and pharmacological products have been proposed on isolate treatment or in association, to accelerate the wound healing biological phenomena (AHMED et al., 2018; ARAGÃO-NETO et al., 2017; DANTAS et al., 2011; FIROUZI et al., 2018; OTTERÇO et al., 2018a; 2018b; SOLEIMANI et al., 2018). Colloidal systems (nano and microemulsion) have been largely applied in pharmaceutical, cosmetic, and food domains, whose advantages enhanced solubility and bioavailability of bioactivities lipophilic and hydrophilic compounds, shows good chemicals dissolution content, rapid onset of action, pharmacokinetics improvement, enhancement of absorption phenomena and drug release kinetics, as well as reduction of side effects and decreased toxicity. In this sense, self-nanoemulsion drug delivery system (SNEDDS) represent a resistant o/w nanoemulsion system which is stable at dilution (upon thirty dilutions) and thermal conditions, and became a potential carrier to improve solubility and bioavailability of health care products (CHRISTIANSEN et al., 2016; CALLENDER et al., 2017; DATE et al., 2010; FAHMY et al., 2015; RASHID et al., 2015; SANDHU et al., 2015; SONI et al., 2014). 113

Concerning to copaiba oil, controversial wound healing results were notified, depending on its dosage. Copaiba oil may be pro-inflammatory, but at lower concentrations, reduces the inflammatory response at early stages with significant increase in the fibroblast proliferation phenomena and collagen deposition in wounds (AMES ‐SIBIN et al., 2018; ESTEVÃO et al., 2013; SILVA et al., 2015; PAIVA et al., 2002; MASSON-MEYERS et al., 2013). In this sense, the present study shows an innovative approach for the use of copaiba oil loaded into a SNEDDS colloidal-type system applied along with LLLT radiation, during the tissue-healing period in rats, aiming at to improve the wound healing process. The findings highlight the morphological and immunohistochemical characteristics of the treated rats with the SNEDDS´s copaiba oil nanoformulation. Physicochemical analysis of the in natura copaiba oil sample and the SNEDDS-CO development and characterization are also herein presented.

Material and Methods

Copaiba oil sample and chemical characterization The copaiba ( Copaifera langsdorffii ) sample was commercially obtained, and the surfactant Tween 80 ® was purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). Copaiba oil was analyzed in a gas chromatography (Thermo Scientific - Trace 1310) equipped with a flame ionization detector (GC-FID). The sample (5.0 mg) was derivatized in situ using trimethylsilildiazomethane (TMSD), converting the diterpene carboxylic acids into the correspondent methyl esthers. Split injections (1:20) were performed in a DB-1 dimethylpolisiloxane column (25 m x 0.25 mm x 0.25 μm), using He as carrier gas at 2 mL.min -1. Oven temperature was programmed from 120 oC to 150 oC (at 3 oC.min -1), followed by another heating ramp until 290 oC, at 15 oC.min -1. Detector and injector temperatures were set at 300 oC and 270 oC, respectively. Two standard mixtures were injected at this same condition: a homologous series of linear hydrocarbons from tridecane to heptadecane (C13 to C17), and a mixture containing the sesquiterpenes caryophyllene, humulene and caryophyllene oxide. The homologous series of hydrocarbons were applied to obtain the Linear Retention Index (LRI) of the copaiba oil constituents, and then compared with literature. The 114 mixture of this three sesquiterpenes, very common constituents from copaiba oils, were used to correct the LRI obtained and compare them with literature data. Mass spectrometry (MS) experiments were useful to confirm the identification of the sesquiterpenes by comparing their mass spectra with automatic database (NIST) and also to obtain the diterpene methyl esthers mass spectra and compare all of them with mass spectra data from previously isolated substances from copaiba oils that were stored and compose a personal data library. Derivatized copaiba oil samples were injected into a gas chromatography (Trace CG Ultra, Thermo Scientific), coupled with mass spectrometry detector (DSQ II, Thermo Scientific) with quadrupolanalyser and auto-injetor (AI 3000, Thermo Scientific). Mass spectra were obtained by electron impact (70 eV), from 40 to 400 u.m.a. and using a similar oven program from GC-FID: 120 oC to 150 oC, at 2o C.min -1, followed by 150 oC to 260 oC, at 15 oC.min -1, and finally from 260 oC to 290 oC, at 14 oC.min -1. Samples were injected (split 1:40), using He as carrier gas at 2 mL.min -1 in a zebron ZB-5ms (Phenomenex-20 m x 0.18 mm x 0.18 μm) column. The applied methodology is accordance with our previous reported studies (BARRETO-JÚNIOR et al., 2005; EMERENCIANO et al., 2019; VEIGA- JUNIOR et al., 1997; 2006a; 2006b).

Preparation of the SNEDDS-CO colloidal nanoformulation SNEDDS-CO nanoformulation was prepared by using ternary phase diagram, constructed by using the surfactant mass titration methodology into the aqueous and oily phases in order to obtain a polar o/w microemulsion region (DATE et al., 2010; DANTAS et al., 2010; EMERENCIANO et al., 2019; McCLEMENTS, 2016). The mechanical stirring Vortex (IKA, Staufen, Germany) was applied (5 min.) to optimize homogenization under moderate temperature. Aiming at to confirm the colloidal homogenization the SNEDDS-CO (approximately 5 mL) was charged to the centrifugation tube, and then submitted to accelerate centrifugation (3500 rpm) for 15 minutes (Centribio 80-2B, Teknika, Brasil). The SNEDDS-CO system was chosen from the ternary phase diagram by fixing the emulsifier and water contents such as: 20% w/w of Tween 80 ®, 1% w/w of oil phase [copaiba oil mixed with sunflower oil ( Helianthus annuus ) in the ratio 1:1], and 79% w/w of doubly-distilled water, under moderate heating (55 oC 115 to 65 oC). After physicochemical characterizations it was clear that the applied process afforded a nanoemulsion system which was classified as SNEDDS colloidal-type system.

Physicochemical characterization of the SNEDDS-CO colloidal nanoformulation The characterization of SNEDDS-CO was conducted by using polarized light microscopy, pH, conductivity, refractive index, droplet size, zeta potential, rheological behavior and surface tension analysis. The pH was measured by using a pre-calibrated pH meter PG-2000 (Gehaka, São Paulo, SP, Brazil) at 25 ± 2 °C. The electrical conductivity of the samples was measured using a DM-32 conductivity meter (Digicrom Analytical, Campo Grande, SP, Brazil) with a cell constant of 0.11 cm -1. The measurements were performed at 25 ± 2 °C. The refractive index of microemulsion samples were determined using Abbe's refractometer (Bellingham plus Stanley Limited, England) at 25 ± 2 °C. The average droplet size and Zeta potential was measured by Zeta Potential Analyzer (ZetaPlus, Brookhaven Instruments Corporation, New York, USA) using a detector angle of 90°. Samples were analyzed in a polystyrene cell at 25 °C. Rheological property was determined using oscillatory Haake Mars rheometer (Thermo Fisher Scientific, Karlsruhe, Germany, cup Z43 DIN 53018 and rotor Z41 DIN 53018). The temperature was kept at 25.0 ± 0.1 °C using a thermostatic bath. The analysis was carried out by applying a shear rate sweep from 0 to 10 3 s−1. Surface tension was carried out using a SensaDyne tensiometer (model QC-6000, Research Corp., USA) employing the maximum pressure bubble technique, using nitrogen as gas phase. The results of the surface tension assays, expressed in mN m −1 (or dynes cm −1), were analyzed with the SensaDyne tensiometer software, version 1.21.

Experimental groups and animal accommodation conditions The in vivo experimental study was approved by the Ethics Committee on Animal Use (CEUA) of the Potiguar University (CEUA / UnP, 0019/240712). A total of 40 male Wistar rats weighing 300g and 120 days old were used. The animals were randomly divided into 4 groups (n = 10) and subdivided into two groups (n = 5), according to the experimental intervention time, being: control groups, 7 and 14 days (CG7 and CG14); pharmaceutical SNEDDS-CO groups, 116

7 and 14 days (FG7 and FG14); laser groups, 7 and 14 days (LG7 and LG14); and then the pharmaceutical SNEDDS-CO groups associated with laser, 7 and 14 days (FLG7 and FLG14). At the time of admission, the animals were evaluated the general health conditions and the acclimation period of three days was observed (GALVÃO et al., 2011). The rats were housed in individual cages lined with excelsior; with free access to water and diet; in an environment with temperature controlled by automatic adjustment (22 ºC ± 2 ºC); noise below 60 dB; humidity around 45% ± 15%; with a 12-h light-dark cycle (DAMY et al., 2010; RÊGO et al., 2010).

Surgical procedures and post-operative management All animals underwent two surgical procedures, one for induction of cutaneous lesion and the other, at the end of the study period, for excisional skin biopsy. On the day of surgery for induction of injury, the animals were weighed and then anesthetized with intramuscular injection of Zoletil® 50, with 0.1 mL / 100g, in the medial region of the right thigh. Under anesthesia, they were positioned and immobilized in the ventral decubitus at the surgical table, submitted to trichotomy in the dorsal region, in an area of approximately 4 cm2. The preoperative marking was performed with a permanent ink pen and a digital caliper. Then, cutaneous antisepsis was performed with chlorhexidine gluconate spray and incision was performed on the animal's back, with excision of a fragment of 1cm2 of total skin, until exposure of the dorsal muscular fascia (ALVES et al., 2008). Postoperative pain was treated with a single intraperitoneal dose of 50 mg / kg of dipyrone diluted in saline solution. After 24 hours of the surgical procedure the treatment of the skin lesions of the animals was started, according to the type of treatment provided for the group and experimental time. On a daily basis, 1mL of 0.9% NaCl solution (topical route) was administered in the control groups (CG7 and CG14) and in the groups treated with SNEDDS-CO (FG7 and FG14) 1 mL of the formulation was applied during the period of study. The animals treated with LLLT (LG7 and LG14) underwent a laser application on alternate days. The LLLT-SNEDDS-CO combination therapy groups (FLG7 and FLG14) received 1 mL of the formulation daily (topical) and one LLLT application on alternate days. 117

For wound irradiation, a diode laser of the gallium-aluminum-indium- phosphorus type (GaAlInP) (FOTON LASER III, DMC® Equipamentos Ltda, São Carlos, Brazil) was used. wavelength 660 nm, continuous emission, beam size 0.028 cm², power of 40mW, energy density of 156 j / cm², energy 4 J, for a minute, in a punctual way, using the pen positioned vertically and with interval of 48h. At the time of application, a cotton blanket was used to minimize stress (OTTERÇO et al., 2018). The total number of LLLT sessions varied according to the group, for the experimental period of 7 days there were 3 applications and the 14 days performed 7.

Biopsy and euthanasia assays On the day of euthanasia, all animals were weighed and anesthetized. An excisional biopsy was performed, including one centimeter of perilesional intact tissue, reaching the dermo-epidermal region, exposing the dorsal muscular fascia. For induction of death, thiopental sodium (50 mg / kg) was given via the intracardiac route.

Histological analysis procedures After the period of 48 hours for fixation, the pieces were sectioned transversely, dehydrated in alcohol, diaphanized in xylol and embedded in paraffin in the form of blocks. The blocks were mounted on the microtome and cut 5 μm thick and then subjected to hematoxylin and eosin (HE) staining. The general histological characteristics of each group were initially presented in a descriptive way. For histological grading of the repair process over the experimental period, 5 serial sections of each animal were used. The methodology of analysis followed the protocol proposed by Myers et al (1961) modified by Almeida (2016). To do so, the morphological parameters (Table 1) were analyzed semiquantitatively according to the following scores: 0 (absent), 1 (when isolated), 2 (when they appeared more frequently and dispersed in the optical fields), 3 ( when they appeared more frequently, constituting dense aggregates, but allowing to visualize free areas between the optical fields) and 4 (when they appeared with great frequency, constituting dense and juxtaposed aggregates, without free areas between the optical fields). To obtain the scores, 10 histological fields were 118 analyzed, in magnification of 400 x, selected by systematic randomization. Thus, the analysis always began in the upper (external) portion of the left margin of the wounds and, for each selected field, a despised histological field was followed until the right margin was reached. The procedure was repeated in the internal portion of the wound until all 10 fields were selected and representative of the entire extent of the lesion. The scores obtained in the analysis of each parameter were multiplied by a correction factor (Table 1), to better represent its relevance in the evolution of the repair, and to allow the determination of a more reliable final score of the evolutionary stage of the repair process.

Table 1. Morphological parameters considered for analysis of the histological gradation of dermal cicatricial repair process.

Criterion Score Correction factor Final score Abscess formation -10 Crust -1 Acute inflammation -4 Chronic Inflammation +2 Epithelial regeneration +5 Fibroblasts +5 Granulation reaction +5 Collagen +10 Eosinophils -4 Neovascularization +5 Total score

Immunohistochemical procedures Histological sections of 3 μm thickness were mounted on pre-flagellated glass slides and then submitted to immunohistochemical staining using the indirect streptoavidin-biotin method. Sections were dewaxed in xylol and washed in decreasing concentrations of ethyl alcohol (100%, 95%, 90%, 80%, and 70%). To potentiate the reaction, the antigenic recovery was carried out by immersing the sections in citrate solution, heated for 20 minutes in steamer. Antibody labeling was performed using rabbit anti-mouse monoclonal antibodies to the CD3, CD105, α-SMA and IL-1β (Sigma) antigens. Diaminobenzidine (DAB, Ventana Medical Systems, Tucson, AZ, USA) was used for the development of the reaction and Meyer's Hematoxylin for counterstaining. The tissue structures of the healthy margin of biopsied skin samples were considered as positive 119 controls. For the negative control of the reaction, the primary antibody was replaced with phosphate buffered saline. Histological analysis was performed under a light microscope (Olympus CX31), with an increase of 40/100 / 400X. In the evaluation of the immunohistochemical marking, any cytoplasmic and / or nuclear marking for the antibodies was considered as positive, represented by brownish color, regardless of the staining intensity. In addition, a quantitative analysis of the immunolabelled cells was performed following the following parameters: 10 photomicrographs histological sections of each case (ten histological fields) in the capture and scanning system of Olympus Imaging Corp. no. C-7070 wave zoom in magnification of 200x. The areas to be scanned were selected by systematic randomization (for each selected field, followed by two scorned fields), always from left to right, and from top to bottom. The photomicrographs were processed in ImageLab software (São Paulo-SP) and the immunomarker cells (highlighted in brownish color) were counted, regardless of the intensity of their labeling. The means of each group (experimental times) are expressed as the mean ± standard error of the mean.

Statistical procedures All data were submitted to analysis of normal distribution and homoscedasticity using the Shapito-Wilk and Bartlett tests, respectively. Data that obeyed normality and homoscedasticity assumptions were expressed as mean ± standard error of the mean and analyzed using the analysis of variance (ANOVA) followed by the post-test of Tukey multiple comparisons. Data that did not obey these assumptions, or data obtained by means of scores (considered non-Gaussian by definition) were expressed as median and interquartile range and analyzed using the Kruskal-Wallis test followed by the Dunn multiple-posttest test. The differences between means and medians were considered significant when the p value was lower than 0.05. For statistical analysis, GraphPad Prism software version 5.03 was used.

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Results and Discussion Chromatographic and spectrometric analysis of the copaiba in natura oil allowed to identify terpenes, sesquiterpenes and diterpene, as well as fatty acids which were derivatized and analyzed as their respective methyl esters observed as their ester derivatives. The sesquiterpene fraction (Figure 1) comprises about 73.1% of the oil and remain constituents such as β-caryophyllene (13.9%; 5.8 min.), β-bisabolene (13.1%; 7.4 min.), α-bergamotene (10.2%; 6.0 min.), β- selinene (5.6%) and α-selinene (5.3%) were also detected. About 16.9% of the oil sample correspond to diterpenes, including the kauranes kaur-16-en (0.7%) and kaurenoic acid (8.7%; 16.8 min.) and the labdane polyaltic acid (6.3%; 17.1 min.). About 10.5% of the oil sample correspond to fatty acids, analyzed as their corresponding methyl esthers derivatives. In which, the main substances are oleic acid (3.9%), linoleic acid (3.4%), palmitic acid (2.0%) and estearic acid (1.2%), with similar amounts of saturated (palmitic and estearic acids, 3.2%), monounsaturated (oleic acid, 3.9%) and polyunsaturated fatty acids (linoleic acids, 3.4%). Several studies attributed some of the copaiba oil activities to one or other terpene, in which the conjugation of sesquiterpenes and diterpenes are essential to promote the biological effect. An example of bioguided fractionation study of copaiba oil can be observed to the assessment of 3-β-copalic acid as the main responsible for the anti-leishmaniosis activity of copaiba oils (SANTOS et al., 2013). Meanwhile, Lima et al. (2003) evidenced that copaiba oil loses its activity after its chemical fractionating, but the prompt regenerated was found when fraction were reunited, as observed in anticancer studies. Indeed, a very important study were performed with caryophyllene and several diterpenes isolated from copaiba oils on the inhibition of the Trypanossoma cruzi parasitc action. Isobolograms showed that, even though caryophyllene and copalic acid are very active, together they are 20 times more active at the same concentration, a potent synergistic effect of terpenes presented by the very first time (IZUMI et al., 2012). These findings corroborate with the present study which brings results for the copaiba oil sample loaded into a colloidal SNEDDS-type formulation, instead its isolated compounds.

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β-caryophyllene β-bisabolene

α-bergamotene Figure 1. Sesquiterpenes as the major biomarkers identified on the copaiba oil sample by applying HRGC-MS analysis. Source: by author

SNEDDS-CO design and characterization The ternary phase diagrams of the SNEDDS-CO nanoformulation [20% of surfactant (Tween 80 ®), 1% of oil phase oil (0.5% of copaiba oil) and 79% of double-distilled water], was prepared based on the proposed model of colloidal system formation designed by Winsor. The procedure used to obtain the Winsor IV region (WIV) in the ternary phase diagram was based on determination of the maximum solubility of the active matter (surfactant) in aqueous and oil phase, by means of mass titrations. According to this methodology, the mixture of several compounds with different physicochemical properties can generate various colloidal formulations including spontaneous monophasic systems (DATE et al., 2010; DANTAS, et al., 2010; EMERENCIANO et al., 2019; McCLEMENTS, 2016; ROSSI et al., 2007; XAVIER-JUNIOR et al., 2016) (DANTAS et al., 2010; EMERENCIANO et al., 2019). The copaiba oil solubility and miscibility study was conducted by ternary phase diagram to obtain the maximum drug loading per formulation (target system SNEDDS-CO) and to provide large self-emulsifying areas. Specifically, precise amounts of oil phase and surfactant were mixed together using a 122 magnetic bar at moderate temperature and speed (on a magnetic stirring plate), followed by addition of water. Both phases were gradually heated separately (55 oC to 65 oC) and during the water addition the highest temperature was maintained. Figure 2 shows a representative phase diagram of these formulations focusing on WIV, and Table 1 shows the physicochemical characterization of the SNEDDS-CO cosurfactant-free colloidal nanoformulation.

Figure 2. Ternary phase diagram of the SNEDDS-CO nanoformulation.

SNEDDS-CO remained isotopically stable after centrifuge procedure (3500 rpm) as well as by water dilution (upon thirty dilutions) ensuring no phase change. This likely occurs due to the spontaneous formation of such systems at low oil concentration, and high water/surfactant concentration. Even under variation of the oil phase there is no significant modification on the WIV regions formation. The coalescence phenomena, creaming, and phase separation were inhibited by using a biocompatible surfactant with copaiba oil sample, that lowered the interfacial tension favoring the interfaces formation, causing a strong and elastic physical barrier which is enough to prevent the coalescence of dispersed droplets and favored reduction of the interfacial tension. When the interface is saturated with the surfactant molecules self-assembling are formed and the surface tension reached its minimum value at the critical micellar 123

concentration (CMC) and remains constant above it (EMERENCIANO et al., 2019). A water diluted SNEDDS-CO dispersion gives rise to the Tyndall effect in which a laser beam passing through the colloidal solution leaves a discernible track as a result of light scattering. This phenomenon is the effect of light scattering in colloidal dispersion, while showing no light in a common solution (EVERETT, 1988).

Table 1. Physicochemical characterization of the SNEDDS-CO nanoformulation.

Zeta Surface Conductivity Refractive Droplet size Viscosity Formulation pH Potential tension (µS cm -1) index (nm) (cP) (ζmV) (Dynes cm -1) SNEDDS-CO 6.28 215.0 1.481 6.940 -2.460 7.4 47.490

The pH can influence significantly the stability of colloidal systems, due to the effect on the ionization and surface charges of droplets (XUE et al., 2018). Since the target SNEDDS-CO system shows slightly acid pH rationed at water (6.10 and 6.28), become appropriate for biological applications. At this point, it is important to highlight the phytochemical data of the copaiba in natura oil sample, which shows a significant acid composition (25.5%) such as: kaurenoic acid (8.7%), polyaltic acid (6.3%) and 10.5% of fatty acids, from which the main substances are oleic acid (3.9%), linoleic acid (3.4%), palmitic acid (2.0%) and estearic acid (1.2%). Hence, the finding slightly acid pH was just possible because the SNEDDS-CO system contain small content (1% w/w) of copaiba oil, on dispersed phase. Reinforcing this suggestion, Alencar et al. (2015) find strongly acidic pH on copaiba oil nanostructured colloidal formulations based on 5% (w/w) of copaiba oil ( Copaiba langsdorffii ) with a blend surfactant composition (Tween 20 ® and Span 80 ®) for a colloidal o/w nanostructured systems prepared by using high- energy approach (high-pressure homogenizers and ultrasonic devices). The prepared formulations showed pH with values ranging from 3.22 to 3.48, justified by the higher amounts of copaiba oil fatty acids. In the other hand, Oliveira Neves et al. (2018) observed pH data 5.25 and 5.74 for w/o microemulsion systems [concentrated of oil phase (6.1 % w/w) based on copaiba oil ( Copaifera multijuga 124

Hayne) comprising small amount (% w/w) 47.1 or 39.0 of water, and a surfactant blend containing higher amounts (% w/w) of plurol oleique (18.3) and labrasol (36.6), suggesting that the surfactant fixed around the oil droplet interfere on the acidity of copaiba components. Additionally, it is known that the charge of stabilizing molecules depends on the pH value of the solution. For example, pH value 6 of carboxy groups (COOH) compounds, become negatively charged (COO -), whereas they are neutral for pH 6 to 5. So, nanoparticles stabilized by the charge of carboxy groups are therefore only stable under neutral and alkaline pH conditions. In the other hand, steric repulsion also stabilizes nanoparticles formulations. The electrical conductivity represents an important parameter to characterize o/w type system formation, which characteristically shows high conductivity values. In fact, for the SNEDDS-CO target system the observed conductivity is 215.0 µS cm -1 and the refractive index is 47.49, confirming the external aqueous phase (o/w type system formation), which was quite close to the water tension value at the temperature of 25 °C. Surface tension (dynes cm -1) versus concentration (g mL -1) of the surfactant Tween 80® showed lower surface tension. The intersection point (Figure 3) resulted in the maximum dilution values of the micellar aggregates (16.701 g mL -1) from which dispersed monomers are found below these values, and above them the self-nanoemulsion system is formed. The negative Zeta potential -2.46 mV allow to predict the electrical repulsion and attractive van der Waals´ forces causing fast colloidal aggregation as a result of this attractive force-type (HONARY; ZAHIR, 2013). According to Oliveira Neves et al. (2018) the Zeta potential data of copaiba oil load in microemulsion for example, could be a modular small number very close to zero due the presence of nonionic surfactants (a blend containing plurol oleique and labrasol). Their findings also showed strong negative Zeta potential such as - 2.63 mV and -3.57 mV for two different w/o microemulsion systems, such effect was correlated to the solvation phenomena at the polar head group of the surfactant and also by the presence of carboxylic acid groups in the acidic fraction of copaiba oil sample that are stabilized through a steric rather instead stabilization by electrostatic effects (OLIVEIRA NEVES et al., 2018).

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Figure 3 . Representative graph of surface tension (Dynes cm-1) versus surfactant concentration (g mL -1) of the SNEDDS-CO formulation.

Comparatively, no matter the amount of water, similar Zeta potential (-2.46 mV) was found for the polar o/w target SNEDDS-CO system (79% of double- distilled water). According to Sharma et al. (2016), nonionic surfactant provides steric stabilization due to the presence of a dense hydrophobic tail and does not allow particles to come closer to one another preventing particle agglomeration. Considering all this results it is mandatory to suggest that the ordered organization of building micellar blocks on the SNEDDS-CO nanostructure relies on specific molecular recognition compromised by i) combination of nonelectrostatic and noncovalent interactions (London forces or even dipole- dipole hydrogen bonds, since the water contents do not influence the observed values of Zeta potential; ii) van der Waals forces (dipole permanent-dipole permanent) occurring between copaiba oil acidic components and the nonionic surfactant; iii) steric interactions (reduce repulsive forces between two particles), and iv) thermal forces. Concerning to higher Zeta potential values (>30 mV), due to electrostatic repulsion, particle aggregation did not occur for charged particles. However, this rule cannot strictly be applied for systems containing steric stabilizers, because its absorption will decrease the Zeta potential due to the shift in the shear plane of the particle (HONARY, S.; ZAHIR, 2013; HEURTAULT et al., 2003; YAN et al., 2010). 126

According to Yan et al. (2010), although individually these forces are relatively weak, when combined as a whole. Hence, they can govern the self- assemble of molecular building blocks into superior and ordered structures. Then, the individual forces are also not strong compared to the thermal forces which enable variations of structures and properties by a small variation of oil and surfactant parameters. Conventional emulsions have often nanoscale droplets in the ranging of 50 nm - 500 nm, which fall into the 5 nm to 200 nm in transparent or semi- transparent nanoemulsion systems, and become milky opalescent up to 500 nm (KAVIANI et al., 2016). In this sense, the SNEDDS-CO nanoformulation exhibited strongly small droplet size (6.94 nm) as well as similar lower viscosity (7.4 cP) (Figure 4) indicating that this specifically self-nanoemulsion system is a metastable dispersion of nanodroplets of one liquid (oil phase and Tween 80®) into water medium (MASON et al., 2006). Rheology behavior is a fundamental approach to provide useful information about the consistency or fluidity of colloidal systems, and indicate micellar aggregates performance over time (FORMARIZ et al., 2010). Figure 4 presents the rheology, as revealed by the flow curves, the CO-SNEDDS formulation showed a linear relationship between shear stress and shear rate, characteristic of Newtonian flow which is very important for convenient handling for commercial applications. The viscosity is very low (<7.4 cP) near of water value, owing to the external character of nanoemulsion system. Additionally, when an emulsion is characterized as a colloidal stable system, the flow curves usually exhibit a constant value of apparent viscosity at low shear rates (ALENCAR et al., 2015).

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Figure 4 . Representative graph of rheological behavior of the CO-SNEDDS formulations.

Descriptive morphological analysis In 7 days, the formation of an exuberant granulation reaction band of variable thickness was observed in all groups analyzed. However, the CG7 group showed focal areas of persistence of neutrophil infiltration especially in the more superficial areas of the wounds. The granulation reaction range was extensive, reaching the entire thickness of the dermis, and composed of a network of capillary vessels in the shape of a slit, generally disposed perpendicular to the long axis of the wound surface, interspersed with fusocellular proliferation (interpreted as endothelial cells and active fibroblasts). This stromal component is supported by a delicate mesh of thin collagen fibrils arranged parallel to the surface. From the inside, moderate to intense inflammatory infiltration was predominantly lymphocytic, with presence of possible macrophages, a morphological aspect consistent with a very immature granulation reaction. The re-epithelialization of the wound surface was very inconspicuous, being limited to the edges of the injured area. In the LG7 group, the granulation tissue exhibited a distinctly biphasic, exuberant, but still richly vascular characteristic in the central 1/3 of the wound, while in the periphery there was intense fusocellular proliferation arranged in long 128 bundles, organized parallel to the surface of the wound, and component less evident, suggesting a more mature (fibroblastic) phase. The granulation reaction range was narrow, occupying about 2/3 of the mean depth of the wound and showed inflammatory lymphocytic infiltrate remaining, with the presence of possible macrophages. In this area, the interface with the normal dermis, densely collagenized and less cellular, was visible. In the more central areas there was still no evident re-epithelization, but on the margins epithelial signs were evident, with a well-defined but relatively thin layer of Orth keratinized stratified squamous epithelium. The FG7 and FLG7 groups were characterized by a very cellularized granulation reaction and collagen deposition if well marked and composed of long beams of variable thickness. The thickness of the granulation reaction ranges also occupied about 2/3 of the total thickness of the injured dermis. The main difference between the groups was in the re-epithelialization, quite inconspicuous and marginal in FG7, but much more apparent, sometimes occupying almost 2/3 of the surface of the wound, in FLG7. In addition, the base (bed) of the FLG7 wounds was composed of rather dense fibrous connective tissue with clear demarcation of the more superficial granulation reaction zone. Figure 5 shows the main histological characteristics observed in the different groups analyzed. By the 14 day, all groups were characterized by the formation of a primary scar composed of proliferating spindle and ovoid cells (interpreted as fibroblasts) and deposition of denser bundles of variable thickness of collagens, which were disposed in a predominantly parallel orientation to the surface of the wound (Figure 6). It was also observed the persistence of narrow bands of reaction of remaining granulation, characterized by discrete mononuclear inflammatory infiltration and presence of small hyperemic capillaries in the middle of the fusocellular component, which was thicker and exuberant in the CG14 group than in all others. The re-epithelialization was well advanced in the groups analyzed and collected almost the entire surface of the wound. In the LG14 and FLG14 treated groups, bulbous formations appeared from the Orth keratinized stratified squamous epithelium of the dermal surface, interpreted as probable rudiments of developing skin attachments, but were clearly more abundant in FLG14.

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Figure 5. Photomicrographs of histological sections stained in HE of the most central areas of the wounds of the different groups studied in seven days. (A) CG7 group exhibiting broad immature granule (rgi) reaction (double black dotted arrow) occupying the entire dermis, with persistence of neutrophil polymorphonuclear (pmn) -inflammatory infiltration on the surface (100 ×). (B) LG7 group showing richly vascularized granulation reaction range (dotted black double arrow) occupying 2/3 superficial of the injured area of the dermis in the most central area of the wound; in the depth, a higher density of extracellular matrix is observed, at the expense of increased collagen deposition (dotted red double arrow) (100 ×). (C) FG7 group showing a highly cellularized granulation reaction band (dotted black double arrow) occupying 2/3 superficial of the injured area of the dermis in the most central area of the wound; in the depth, greater density of collagen deposition (dotted red double arrow) (100 ×) is observed. (D) FLG7 group demonstrating a wide range of mature (richly cellular) granulation reaction occupying slightly less than half the thickness of the damaged dermis throughout the wound and the presence of fairly advanced re-epithelialization (ep) in approximately 2/3 of the surface of the wound; in the depth, the collagen density was more marked and the line of separation between this area and the granulation reaction was very clear (dotted black line) (100 ×).density was more marked and the line of separation between this area and the granulation reaction was very clear (dotted black line) (100 ×). 130

Figure 6. Photomicrographs of histological sections stained in HE of the central areas of the wounds of the different groups studied in seven days. Observed re- epithelialization (Ep) advanced in all groups. (A) CG14 group thin-skinned epithelium and primary scar still with persistence of relatively abundant capillaries (100 ×). (B) LG14 group showing thick epithelium and peripheral bulbous formations compatible with rudiments of cutaneous appendages (arrows) (100 ×). (C) FG14 group showing richly cellularized primary scar (100 ×). (D) FLG14 group showing multiple bulbs budding, compatible with rudimentary cutaneous attachments, along the entire extension of the scar area (arrows) (100 ×).

Determination of the mean histological grading scores of the cicatricle repair The analysis of the histological grading of healing showed that all the treated groups (LG, FG and FLG) presented mean scores significantly higher than the CG control group (p <0.001) in the 7 and 14 days (p <0.001). Likewise, the FLG group had mean scores greater than FG and LG in seven (p <0.001) and 14 days 131

(p <0.05), but there was no significant difference between the two (FG and LG) in any of the times analyzed (p> 0.05) (Figure 7).

Figure 7 . Determination of the mean scores of the histological grading of healing in the different groups analyzed in 7 and 14 days. Data expressed as median and interquartile range. Significant differences with respect to the CG group are expressed as *** p <0.001. Significant differences regarding LG and FG groups are expressed as ### p <0.001 (Kruskall-Wallis and post-test of Dunn's multiple comparisons).

Quantitative analysis of immunohistochemical expression of CD3 antigens (T lymphocytes), α-SMA (myofibroblasts), CD105 (endothelial cells) and interleukin 1β (IL-1β) Positivity to all the antigens studied was identified by means of brownish cytoplasmic immunostaining. Figure 8 shows the immunohistochemical expression pattern and quantitative analysis of CD3 antigen-expressing cells expressed on T lymphocytes. Figure 9 shows the pattern of immunohistochemical expression and quantitative analysis of cells immunolabelled for CD105 antigen expressed on endothelial cells lining blood vessels. Figure 10 demonstrates the immunohistochemical expression pattern and quantitative analysis of immunolabelled cells for the α-SMA smooth-muscle antigen expressed in cells with myofibroblast / perictic differentiation. Figure 11 shows the pattern of immunohistochemical expression and quantitative analysis of cells immunolabelled for the interleukin 1 β antigen (IL- 1β).

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Figure 8 . Immunohistochemical expression (A and C) and quantitative analysis of CD3 + T lymphocytes (B and D) in the different groups analyzed in 7 and 14 days, respectively. Data expressed as mean ± standard error of the mean. Significant differences with respect to the CG group are expressed as *** p <0.001. Significant differences regarding LG and FLG groups are expressed as ### p <0.001. Significant differences regarding LG and FG groups are expressed as ФФ p <0.01 (ANOVA and post-test of Tukey multiple comparisons).

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Figure 9 . Immunohisistochemical expression (A and C) and quantitative analysis of blood vessels formed by CD105 + (B and D) endothelial cells in the different groups analyzed at 7 and 14 days, respectively. Data expressed as mean ± standard error of the mean. Significant differences with respect to the CG group are expressed as *** p <0.001. Significant differences regarding LG and FLG groups are expressed as ### p <0.001 (ANOVA and post-test of Tukey's multiple comparisons).

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Figure 10 . Immunohistochemical expression (A and C) and quantitative analysis of α-SMA + cells (B and D) in the different groups analyzed in 7 and 14 days, respectively. Data expressed as mean ± standard error of the mean. Significant differences with respect to the CG group are expressed as * p <0.05, ** p <0.01 and *** p <0.001. Significant differences regarding LG and FLG groups are expressed as ++ p <0.001 (ANOVA and post-test of Tukey multiple comparisons).

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Figure 11 . Immunohistochemical expression (A and C) and quantitative analysis of IL-1β + cells (B and D) in the different groups analyzed at 7 and 14 days, respectively. Data expressed as mean ± standard error of the mean. Significant differences with respect to the CG group are expressed as * p <0.05, ** p <0.01 and *** p <0.001 (ANOVA and post-test of Tukey multiple comparisons).

SNEDDS-CO colloidal formulation applied through biostimulation (LLLT) (item pendente: a ser elaborado) From the morphological analysis it was observed that the association of SNEDDS-OCC and LLLT favored collagenase, early re-epithelialization of the lesion and recovery of cutaneous attachments. From the histologic gradation score of the cicatricial repair it was observed that the association of SNEDDS-OCC with LLLT significantly favored cutaneous healing, especially in the initial phase of the repair process. From the quantitative analysis of the immunohistochemical expression of CD3 antigen expressed on T lymphocytes, it was observed that the association 136 of SNEDDS-OCC with LLLT significantly reduced the inflammatory response in the late repair phase. From the quantitative analysis of the immunohistochemical expression of the CD105 antigen, expressed in endothelial cells, it was observed that the association of SNEDDS-OCC to LLLT did not potentiate neoangiogenesis. In the initial phase LLLT significantly favored angiogenesis. From the quantitative analysis of the immunohistochemical expression of α-SMA antigen expressed in cells with myofibroblast differentiation, it was observed that the association of SNEDDS-OCC to LLLT did not potentiate cutaneous lesion contraction. LLLT significantly favored contraction of the wound in the early and late stages. From the quantitative analysis of the immunohistochemical expression of the interleukin 1 β antigen (IL-1β), it was observed that the association of SNEDDS-OCC to LLLT did not potentiate the reduction of the inflammatory response.

Conclusions Sesquiterpenes, diterpenes and fatty acids were identifying in the copaiba oil (CO) by using CG-MS analysis which shows 73.1% of sesquiterpene, 16.9% correspond to diterpenes, and 10.5% of the oil correspond to fatty acids. Non- toxic compounds were used to prepare the copaiba oil-based system SNEDDS- CO which was disign aiming at copaiba oil bioavailability. Sunflower ( Helianthus annuus ) oil (0.5%) was co-encapsulated with copaiba oil favoring a more suitable oil phase. Additionally, copaiba oil applied on lower content (0.5%) on the SNEDDS-CO non-toxic formulation justify its foremost importance as a promising target system for general therapeutic applications. Physicochemical characterization was performed by using polarized light microscopy, pH, conductivity, refractive index, droplet size, rheological behavior and surface tension analysis. which can afford new o/w nano or microemulsion systems by new changes in the oil phase composition. The observed negative zeta potential contributes for the SNEDDS-CO storage stability since the repulsive forces exceed attractive forces among droplets and such droplet-droplet repulsion prevents the formulation against 137 coagulation and/or coalescence phenomena on dispersed phase of the colloidal system. The association of the topical application of SNEDDS-CO with LLLT significantly accelerated the healing process of experimentally induced cutaneous lesions in rats, especially in the initial phase (7 days), as evidenced by early re-epithelization, high histologic grading cicatricial repair, intense collagenases and at 14 days, due to the appearance of cutaneous attachments and reduction of the late inflammatory response. These results may be related to the modulation of the inflammatory response, observed especially from the decrease of lymphocytes in the late phase. So, further studies will be needed to evaluate the influence of keratinocytes in the repair process. In isolation, both LLLT and the topical treatment of cutaneous lesions with SNEDDS, the copaiba oil base, favored healing. About LLLT, its effects on neoangiogenic, collagenases, modulation of inflammatory response and re- epithelialization of lesions were confirmed. The results of this study suggest that the association of LLLT to copaiba oil, carried in SNEDDS-like systems, may be a promising strategy for the treatment of cutaneous wounds. Considering the limitations of the histopathological and immunohistochemical analyzes, further studies are needed to elucidate the effects of the association of different types and doses of laser to the SNEDDS bioproducts, based on copaiba oil, in the tissue repair of cutaneous lesions.

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5.5. Patente Número de Registro: BR102017014800-9

Resumo No presente invento, amostras de óleo de copaíba (OCP) das espécies Copaifera reticulata Ducke e Copaifera multijuga Hayne, bem como de uma amostra comercial deste óleo, foram veiculadas (0,5% - 1,0%) em microemulsão ou nanoemulsão com características SMEDDS e SNEDDS, respectivamente. Especificamente, objetivou-se a otimização da ação terapêutica de OCP na administração por via oral e tópica associada, aplicada em processo de reparo tecidual cutâneo, com ação fitoterápica analgésica, anti-inflamatória e cicatrizante. Os bioformulados de OCP foram avaliados via protocolos experimentais in vivo em feridas cutâneas e pelo método de analgesia via inibições das contorções abdominais induzidas pelo ácido acético, tendo apresentado atividades anti-inflamatória, cicatrizante e analgésica satisfatórias. O efeito antisséptico, que inibe à proliferação de microorganismos, bem como aplicações extensivas na terapêutica dermatológica e musculoesquelética, também estão sendo reivindicadas para os bioformulados de OCP. Além do uso oral e tópico associado, a liberação lenta e controlada de OCP nas membranas celulares, via administração transdérmica, também é alvo de reivindicação.

Potencial de inovação: Produção de fitomedicamento preparado à base de óleo de copaíba biodisponibilizado para administração por via oral e tópica associada, com otimização da ação terapêutica em processo de reparo tecidual cutâneo.

MEDEIROS, M. L.; ARAÚJO FILHO, I.; RÊGO, A. C. M.; PEREIRA, J. D. S.; BARACHO, B. B. D.; LIMA, M. C. F.; VEIGA JÚNIOR, V. F.; MEDEIROS, M. I. T.; MACIEL, M. A. M. Formulados a base de óleo de copaíba biodisponibilizados para uso oral e tópico associado com aplicações terapêuticas na analgesia, inflamação e cicatrização. Data do Depósito: 09/07/2017. INPI – Instituto Nacional de Propriedade Intelectual. Data da Publicação (RPI/INPI): 24/04/2018 (RPI 2468).

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6. GENERAL CONCLUSIONS

The pharmacological properties of copaiba oil were correlated with terpenoid compounds such as sesquiterpenes and diterpenes. Among the main sesquiterpenes identified are β-caryophyllene, caryophyllene oxide, α-copaene, α-humulene, τ-muurolene, β-bisabolene and β-bisabolol. The sesquiterpene β-caryophyllene has been detected as a main component in several Copaifera species, and become the most used biomarker to authenticate copaiba oil, and the anti-inflammatory activity of copaiba oils may be addressed to the high content of this compound. Chromatographic HRGC-MS analysis of the copaiba oil sample ( Copaifera multijuga Hayne) allowed to identify sesquiterpenes (82.35%) and diterpenes (5.31%). The major sesquiterpenes are β-caryophyllene (32.84%) and germacrene D (18.78%). The major diterpene analyzed as its corresponding methyl esther derivative is copalic acid (4.44%). Sesquiterpenes, diterpenes and fatty acids were identifying in the commercial copaiba oil sample by using CG- MS analysis, which shows 73.1% of sesquiterpene, 16.9% correspond to diterpenes, and 10.5% of the oil correspond to fatty acids. This sample may correspond to the Copaifera langsdorffii Desf. species. Colloidal SNEDDS-type systems (self-nanoemulsifying drug delivery system) based on copaiba oil and Tween® 80 (surfactant) were prepared by using both copaiba oil samples (Copaifera multijuga Hayne and the commercial sample/Copaifera langsdorffii Desf.). The chemical approach afforded two SNEDDS formulations containing 1% of oil phase (CO and sunflower oil,1:1 ratio), 20% of surfactant and 79% of bidistilled water, which is stable at water dilutions conditions. Physicochemical characterizations of the SNEDDS-CO both formulations (Copaifera multijuga Hayne and the commercial sample/ Copaifera langsdorffii Desf.) were performed by using polarized light microscopy, pH, conductivity, refractive index, droplet size, rheological behavior and surface tension analysis. The observed negative zeta potential contributes for the SNEDDS-CO storage stability since the repulsive forces exceed attractive forces among droplets and such droplet-droplet repulsion prevents the formulation against coagulation and/or coalescence phenomena on dispersed phase of the colloidal system. 140

The repairing process showed successful results for the copaiba oil SNEDDS-CO both formulations (Copaifera multijuga Hayne and the commercial sample/ Copaifera langsdorffii Desf.), which in vivo treatment optimized its bioavailability, demonstrating wound healing improvement as well as anti- inflammatory effect. Since sunflower oil was co-encapsulated with copaiba oil favoring a more suitable oil phase in this CO-colloidal formulation which increased fibroblast proliferation and neocollagenesis, and showed to be IL-1β inhibitor, and also activated FGF-2 production, associated to positive neocollagenesis. Histomorphometric analysis revealed a smaller number of neutrophils and lymphocytes in the treated group as well as collagenesis, fibroblast proliferation and larger epithelial thickness. Copaiba oil was able to reduce the IL-1β expression and increase the FGF-2 expression and these molecular alterations were directly associated with were neocollagenesis which accelerated the wound healing process. Therefore, these molecules may represent possible targets to treat chronic wounds, and the mechanism of action of the copaiba oil. The association of the topical application of SNEDDS-CO with low level laser therapy (LLLT) significantly accelerated the healing process of experimentally induced cutaneous lesions in rats, especially in the initial phase (7 days), as evidenced by early re-epithelization, high histologic grading cicatricial repair, intense collagenases and at 14 days, due to the appearance of cutaneous attachments and reduction of the late inflammatory response. In the isolation process, both LLLT and the topical treatment of cutaneous lesions with SNEDDS-CO, favored healing. About LLLT, its effects on neoangiogenic, collagenases, modulation of inflammatory response and re- epithelialization of lesions were confirmed. The findings of the present study suggest that the association of LLLT to copaiba oil, carried in SNEDDS-CO type systems, may be a promising strategy for the treatment of cutaneous wounds. Considering the limitations of the histopathological and immunohistochemical analysis, further studies are needed to elucidate the effects of the association of different types and doses of laser to the SNEDDS bioproducts, based on copaiba oil, in the tissue repair of cutaneous lesions.

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7. PRODUÇÃO EXTENSIVA AO PROJETO DE TESE

A seguir encontram-se descritos as produções acadêmicas extensivas ao projeto de tese, que são correlatas aos sistemas SNEDDS-CO desenvolvidos na presente pesquisa.

7.1 Capítulo de Livro (CL4) EMERENCIANO, D. P.; ANDRADE, A. C. C.; MEDEIROS, M. L.; MOURA, M. F. V.; MACIEL, M. A. M. Effectiveness of copaiba oil loaded on microemulsion system as green corrosion inhibitor. In Corrosion Inhibitors, Editor: Esther Hart, Nova Science Publishers, Chapter 4, 2017.

7.2 Patente Número de Registro: BR102018010871-9 ALVES-NETO, E. L.; MACIEL, M. A. M.; PEREIRA, J. D. S.; MEDEIROS, M. L.; VEIGA-JUNIOR, V. F.; CARVALHO, R. A.; MARQUES, M. M. Óleo de copaíba (OCP) bioformulado em sistemas nanocarreadores de fármaco contendo fase oleosa mista (OCP + óleo de soja; OCP + óleo de girassol; OCP + óleo de coco) para uso odontológico em procedimento de implante dentário. Data do Depósito: 28/05/2018. Data da Publicação (RPI/INPI): 06/11/2018 (RPI 2496).

7.3 Artigo Científico (JCR 1,4) EMERENCIANO, D. P.; BARACHO, B. B. D.; MEDEIROS, M. L.; ROCHA, H. A. O.; XAVIER-JÚNIOR, F. H.; VEIGA-JUNIOR, V. F.; MACIEL, M. A. M. Physicochemical characterizations and antioxidant property of copaiba oil loaded into SNEDDS systems. Jornal of the Brazilian Chemical Society, v. 30, n. 2, p. 234-246, 2018.

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8. ANEXO 1 COMPROBATÓRIO DAS PUBLICAÇÕES

A1.1 CAPÍTULO DE LIVRO INTERNACIONAL (DE MEDEIROS et al., 2019)

192

A1.2 Artigo Internacional (IF 1.949; B1) (DE MEDEIROS et al., 2016)

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A1.3 Artigo Científico (FI 1,444; B2) EMERENCIANO, D. P.; BARACHO, B. B. D.; DE MEDEIROS, M. L.; ROCHA, H. A. O.; XAVIER-JÚNIOR, F. H.; VEIGA-JUNIOR, V. F.; MACIEL, M. A. M. Physicochemical Characterizations and Antioxidant Property of Copaiba Oil Loaded into SNEDDS Systems. Jornal of the Brazilian Chemical Society, v. 30, n. 2, p. 234-246, 2019.

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A1.4 CAPÍTULO DE LIVRO INTERNACIONAL (CL4; B2) EMERENCIANO, D. P.; ANDRADE, A. C. C.; DE MEDEIROS, M. L .; MOURA, M. F. V.; MACIEL, M. A. M. Effectiveness of copaiba oil loaded on microemulsion system as green corrosion inhibitor. In Corrosion Inhibitors, Editor: Esther Hart, Nova Science Publishers, Chapter 4, 2017.

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A1.5 Número de Registro: BR102017014800-9 (DE MEDEIROS et al., 2017)

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A1.6 Patente Número de Registro: BR102018010871-9 ALVES NETO, E. L.; MACIEL, M. A. M.; PEREIRA, J. D. D.; DE MEDEIROS, M. L. ; VEIGA JÚNIOR, V. F.; DE CARVALHO, R. A.; MARQUES, M. M. Óleo de copaíba (OCP) bioformulado em sistemas nanocarreadores de fármaco contendo fase oleosa mista (OCP + óleo de soja; OCP + óleo de girassol; OCP + óleo de coco) para uso odontológico em procedimento de implante dentário. INPI, 2018.

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9. ANEXO 2 COMPROBATÓRIO DOS PROJETOS APROVADOS NO CEUA A2.1 Título do projeto: Avaliação dos Efeitos de óleo de Copaíba em Processo Terapêutico em Modelo Experimental de Feridas Cutâneas Infectadas em ratos Diabéticos. Protocolo No. 017/2015; aprovado pela Comissão de Ética no Uso de Animais (CEUA/UnP).

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A2.2 Título do projeto: Avaliação dos Efeitos do Laser em Feridas Cutâneas Infectadas em Ratos Diabéticos. Protocolo No. 008/2016, aprovado pela Comissão de Ética no Uso de Animais (CEUA/UnP).

199

A2.3 Título do projeto: Efeitos Terapêuticos da Associação do Laser e Nanoemulsão com Óleo de Copaíba em Modelo Experimental de Feridas Cutâneas Infectadas em Ratos Diabéticos. Protocolo No. 006/2016, aprovado pela Comissão de Ética no Uso de Animais (CEUA/UnP).