UNIVERSIDADE ESTADUAL DE CAMPINAS

FACULDADE DE ODONTOLOGIA DE PIRACICABA

DIEGO ROMARIO DA SILVA

ATIVIDADE FARMACOLÓGICA DE MEL ORGÂNICO DA MATA ATLÂNTICA BRASILEIRA NOS COMPONENTES MICROBIANO E INFLAMATÓRIO DA DOENÇA PERIODONTAL

PHARMACOLOGICAL ACTIVITY OF ORGANIC HONEY FROM THE BRAZILIAN ATLANTIC FOREST IN THE MICROBIAL AND INFLAMMATORY COMPONENTS OF PERIODONTAL DISEASE

Piracicaba 2021

DIEGO ROMARIO DA SILVA

ATIVIDADE FARMACOLÓGICA DE MEL ORGÂNICO DA MATA ATLÂNTICA BRASILEIRA NOS COMPONENTES MICROBIANO E INFLAMATÓRIO DA DOENÇA PERIODONTAL

PHARMACOLOGICAL ACTIVITY OF ORGANIC HONEY FROM THE BRAZILIAN ATLANTIC FOREST IN THE MICROBIAL AND INFLAMMATORY COMPONENTS OF PERIODONTAL DISEASE

Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor em Odontologia, na área de Farmacologia, Anestesiologia e Terapêutica.

Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dentistry, in Pharmacology, Anesthesiology and Therapeutics area.

Orientador: Prof. Dr. Pedro Luiz Rosalen

ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELO ALUNO DIEGO ROMARIO DA SILVA E ORIENTADA PELO PROF. DR PEDRO LUIZ ROSALEN.

PIRACICABA

2021 Ficha catalográfica Universidade Estadual de Campinas Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159

Romario-Silva, Diego, 1993- Si38a RomAtividade farmacológica de mel orgânico da Mata Atlântica brasileira nos componentes microbiano e inflamatório da doença periodontal / Diego Romario da Silva. – Piracicaba, SP : [s.n.], 2021.

RomOrientador: Pedro Luiz Rosalen. RomTese (doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.

Rom1. Mel. 2. Atividade antimicrobiana. 3. Atividade antiinflamatória. 4. Doenças periodontais. I. Rosalen, Pedro Luiz, 1960-. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.

Informações para Biblioteca Digital

Título em outro idioma: Pharmacological activity of brazilian honey from the brazilian Atlantic Forest in the microbial and inflammatory components of periodontal disease Palavras-chave em inglês: Honey Antimicrobial activity Anti-inflammatory activity Periodontal diseases Área de concentração: Farmacologia, Anestesiologia e Terapêutica Titulação: Doutor em Odontologia Banca examinadora: Pedro Luiz Rosalen [Orientador] Janaína de Cássia Orlandi Sardi Masaharu Ikegaki Andréa Cristina Barbosa da Silva Josy Goldoni Lazarini Data de defesa: 26-02-2021 Programa de Pós-Graduação: Odontologia

Identificação e informações acadêmicas do(a) aluno(a) - ORCID do autor: http://orcid.org/0000-0001-9724-0147 - Currículo Lattes do autor: http://lattes.cnpq.br/0522585351371155

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UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Odontologia de Piracicaba

A Comissão Julgadora dos trabalhos de Defesa de Tese de Doutorado, em sessão pública realizada em 26 de fevereiro de 2021, considerou o candidato DIEGO ROMARIO DA SILVA aprovado.

PROF. DR. PEDRO LUIZ ROSALEN

PROF. DR. MASAHARU IKEGAKI

PROFª. DRª. ANDRÉA CRISTINA BARBOSA DA SILVA

PROFª. DRª. JANAINA DE CASSIA ORLANDI SARDI

PROFª. DRª. JOSY GOLDONI LAZARINI

A Ata da defesa, assinada pelos membros da Comissão Examinadora, consta no SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da Unidade. DEDICATÓRIA

Dedico este trabalho aos meus pais, Arilânia e José, por terem acreditado nos meus sonhos, investido e me apoiado nessa fabulosa jornada científica e à Alexandra Elbakyan, pelo seu comprometimento em tornar o conhecimento um bem livre e gratuito. AGRADECIMENTOS

A Deus e ao universo por tudo que existe e por tornar possível a realização deste sonho do Diego de 8 anos, da 4ª série, que sempre quis ser cientista.

Aos meus amados pais, Arilânia e José, pelo carinho, amor, dedicação e confiança de sempre. Obrigado por sempre colocarem meus sonhos e minha educação como uma prioridade, apesar de todas as dificuldades que enfrentamos. Tudo que conquistei e tudo que eu ainda conquistar está diretamente ligado ao esforço vocês.

Ao meu orientador, Prof. Dr. Pedro Rosalen por todo o ensinamento, carinho e dedicação durante esses anos. Obrigado por ter acreditado no meu potencial e por ter me permitido participar desse fantástico grupo de pesquisa que sempre admirei. És um exemplo de pesquisador e de ser humano por quem carrego grande admiração. Obrigado também pelos incríveis encontros de estudo bíblico, cafés da manhã e incentivo na busca de emprego. Nessa jornada ganhei além de um grande mestre um amigo.

O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001.

O presente trabalho foi realizado com apoio do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - processo nº 141129/2017-4.

À empresa Breyer produtos das abelhas, nas pessoas dos senhores Henrique Breyer e Daniel Breyer, e toda sua equipe, pelo fornecimento dos meis e melatos usados nesta pesquisa e por agregarem conhecimento ao nosso grupo de pesquisa sobre os produtos apícolas.

Às minhas queridas irmãs, Amanda e Vitória, duas verdadeiras amigas que sempre estiveram dispostas a me ajudar nessa caminhada. Obrigado por todo carinho, amor e dedicação.

A Danilo Bérgamo, por todo apoio, carinho e dedicação, por acreditar nos meus sonhos e me ajudar em todos os desafios. Sua presença foi essencial para reenergização, crescimento pessoal, amadurecimento e reencontrar o ânimo e dedicação aos planos e sonhos. Sou muito grato por tê-lo em minha vida.

Ao ex-presidente Luiz Inácio Lula da Silva, pelos incríveis investimentos em educação e criação de novas universidade, bem como pelos discursos motivadores onde mencionava que o filho do pobre poderia sim se tornar doutor. Ver o presidente da república falando isso quando era criança me deu muita motivação e hoje sou a prova de que isso é possível.

Aos amigos/irmãos Matheus Souza, Sarah Amstalden, e Lincoln Pires, pelo carinho, conselhos, acolhimento e vivências. Vocês foram essenciais para que eu conseguisse chegar até aqui e eu sou muito grato por tê-los em minha vida. Obrigado por iluminarem minha vida e deixa-la mais feliz. Levarei todos vocês no meu coração por toda a vida.

À amiga/irmã, parceira de laboratório, de anaeróbios, de Mendeley, de músicas e cafés com Paulas Me. Rafaela Carvalho. Não consigo imaginar esses 4 anos sem sua presença

para enfrentarmos os desafios juntos. Obrigado por ter sido luz tantas vezes no meu caminho. Tenho certeza que é uma amizade para a vida toda.

Às professoras e amigas Dra. Janaína Sardi e Dra. Josy Lazarini, pelo acolhimento que recebi em Piracicaba, carinho e ensinamentos. Vocês me ajudaram a evoluir como pessoa e como pesquisador. Obrigado pelas boas risadas, almoços e sorrisos. Tenho certeza que é uma amizade para a vida toda.

À minha querida orientadora de Iniciação Científica e, hoje, grande amiga Profª. Dra. Andréa Silva por todo o incentivo, por ter me convencido a ir em busca desse doutorado, pelo carinho, amor, dedicação e parceria de sempre. Você foi a primeira pessoa que acreditou em no meu potencial nessa jornada acadêmica e onde quer que eu vá serei eternamente grato.

À minha querida orientadora de mestrado Profª. Dra. Edja Costa por ser um ser de luz na minha vida, por ter me acolhido com tanto amor e dedicação, por todos os ensinamentos e por ter me impulsionado até a UNICAMP para concretizar este sonho. Obrigado por cada palavra, força e sorriso.

Ao amigo Dr. Irlan Freires, pelo acolhimento, carinho, conselhos e ensinamentos. Sempre foi uma inspiração como pessoa e como pesquisador.

Aos professores Dr. Marcelo Franchin e Dr. Bruno Bueno pelos ensinamentos essenciais para a realização desta pesquisa.

Ao Prof. Dr. Severino Alencar, pelo apoio, ensinamentos e contribuição essencial para exploração das atividades biológicas dos méis orgânicos brasileiros.

Ao Prof. Dr. Masaharu Ikegaki pelos ensinamentos durante as reuniões de grupo de pesquisa e por ter aceitado participar da minha banca de defesa de doutorado.

À Maria Elisa, secretária da Farmacologia, pessoa de sorriso puro e dona do melhor café. Obrigado pelo acolhimento, por todas as conversas, todos os cafés e toda a boa energia que você traz para nossa farmacologia.

Aos professores da área de Farmacologia, Anestesiologia e Terapêutica da FOP/UNICAMP pelo aprendizado, apoio e convivência, especialmente aos queridos mestres Prof. Dr. Pedro Luiz Rosalen, Profª. Karina Kogo, Profª. Drª. Maria Cristina Volpato e Prof. Dr. Francisco Carlos Groppo.

À técnica de laboratório Eliane Melo pelo acolhimento e por apresentar o laboratório e seu funcionamento.

À Dona Estela e Dona Vanda, minhas queridas vizinhas que me acolheram de forma muito carinhosa quando morei na rua Edu Chaves.

Aos amigos Rodrigo Lins e Jairo Cordeiro, pelo acolhimento em Piracicaba, conversas, troca de aprendizados e divertimentos durante esses anos.

Aos amigos Renan Broggio e Natalia Furlan, pelo acolhimento em Piracicaba, carinho e troca de aprendizados sobre a vida.

Ao meu psicólogo Carlos Peixe, por todo apoio e conselhos que me ajudaram a evoluir como pessoa e como profissional.

Aos colegas de laboratório, Bruno Nani, Sindy Magri, Iago Cortês, Aylla Pestana e Klinger Amorim, pelas conversas, cafés e divertimentos durante esses anos.

Resumo

O objetivo deste estudo foi avaliar, in vitro e in vivo, o efeito de méis orgânicos brasileiros nos componentes microbiano e inflamatório da doença periodontal. Oito méis produzidos de forma orgânica (OH-1 a OH-8) foram georreferenciados, esterilizados por filtração e diluídos em diferentes concentrações (% - p/v). Nos ensaios in vitro, a atividade antimicrobiana foi avaliada por microdiluição para determinação da CIM contra Streptococcus mutans ATCC 700610, Streptococcus mitis NCTC 12261, Streptococcus oralis ATCC 10557, Streptococcus salivarus ATCC 7073, Streptococcus gordonii Challis, and Streptococcus sanguinis 3K36 e Porphyromonas gingivalis W83. A atividade antibiofilme foi realizada em biofilme monoespécie de S. mutans e subgengival multiespécie contendo 32 espécies. A atividade anti-inflamatória foi avaliada por inibição da ativação de NF-κB e liberação de TNF-α em macrófagos Raw 264.7 (ATCC TIB-71). Foram conduzidos dois experimentos in vivo em camundongos C57BL/6J, (CEUA/UNICAMP aprovação n. 5348-1/2019), sendo o primeiro experimento no modelo de doença periodontal induzida por ligadura inoculada com Porphyromonas gingivalis W83 e o segundo uma peritonite aguda induzida por carragenina. Os dois experimentos foram conduzidos com 4 grupos, sendo eles: G1: sem desafio microbiano ou inflamatório e sem tratamento; G2: com desafio microbiano ou inflamatório e sem tratamento; G3: com desafio microbiano ou inflamatório e tratamento de OH-7 40%; e G4: com desafio microbiano ou inflamatório e tratamento com OH-7 in natura. Os grupos submetidos ao tratamento receberam por via oral 100 µL de mel orgânico (OH-7, georreferenciado) 5x ao dia, por 5 dias. Na análise estatística foi usado ANOVA one-way seguido do pós teste Tukey, e Kruskal Wallis seguido o pós teste Dunnett’s. Os OHs apresentaram atividade antimicrobiana contra o grupo dos Streptococcus e contra P. gingivalis com CIM variando entre 10% (OH-3) e 40% (OH-6) e 2% (OH-2) e 7% (OH- 6), respectivamente. Todas as amostras apresentaram atividade antibiofilme em biofilme multiespécie (p<0,05), com destaque para OH-7 a 8% e 40%. Na concentração 4% todas as amostras reduziram a ativação do NF-κB (p<0,05). OH-2, 3, 4 e 7 diminuíram os níveis de TNF-α em mais de 50%. Nos ensaios in vivo, o tratamento com OH-7 a in natura diminuiu a migração de neutrófilos na cavidade peritoneal e a liberação de TNF-α em 49% e 72%, respectivamente, em relação ao grupo controle (G2 - p<0,05). Já o ensaio de periodontite, demonstrou diminuição da reabsorção óssea em 26% (p<0,05) com o tratamento com OH-7 na concentração 40%. Portanto, os méis orgânicos interferem de forma benéfica nos componentes microbiano e inflamatório da doença periodontal, sendo a variedade OH-7 promissora para indicação a substituto do açúcar comum na dieta a fim de propiciar saúde diminuindo doenças orais inflamatórias e biofilme-dependentes.

Palavras-chaves: Mel, Atividade antimicrobiana, Atividade Anti-inflamatória, Doença periodontal. Abstract

The aim of this study was to evaluate, in vitro and in vivo, the effect of Brazilian organic honeys on the microbial and inflammatory components of periodontal disease. Eight samples of honeys produced organically (OH-1 to OH-8), were georeferenced, sterilized by filtration and diluted in different concentrations (w/v). In in vitro assays, antimicrobial activity was assessed by microdilution to determine MIC against Streptococcus mutans ATCC 700610, Streptococcus mitis NCTC 12261, Streptococcus oralis ATCC 10557, Streptococcus salivarus ATCC 7073, Streptococcus gordonii Challis and Streptococcus sanguinis 3K36. The antibiofilm activity was performed on S. mutans monospecie biofilm and subgingival biofilms containing 32 species. Anti- inflammatory activity was assessed by inhibiting NF-κB activation and TNF-α release in Raw 264.7 macrophages (ATCC TIB-71). Two in vivo experiments were carried out on C57BL / 6J mice, n = 6 (CEUA / UNICAMP approval n. 5348-1 / 2019), the first experiment in the ligature-induced periodontal disease model plus Porphyromonas gingivalis W83 and the second a peritonitis acute carrageenan-induced. The two assays were conducted with 4 groups, namely: G1: without microbial or inflammatory challenge and without treatment; G2: with microbial or inflammatory challenge and without treatment; G3: with microbial or inflammatory challenge and 40% OH-7 treatment; and G4: with microbial or inflammatory challenge and treatment with natural OH-7. The groups submitted to treatment received orally 100 µL of organic honey (OH-7, georeferenced) 5x a day, for 5 days. In the statistical analysis used one-way ANOVA with Tukey post-hoc test and Kruskal Wallis followed by Dunnett’s post-hoc test. OHs showed antimicrobial activity against P. gingivalis with MIC ranging between 2% (OH-2) and 7% (OH-6). All samples showed antibiofilm activity (p <0.05), with emphasis on OH-7 at 8% and 40%. At 4% concentration, all samples reduced NF-κB activation (p <0.05). OH-2, 3, 4 and 7 decreased TNF-α levels by more than 50%. In in vivo tests, treatment with OH-7 in natura decreased the migration of neutrophils in the peritoneal cavity and the release of TNF-α by 49% and 72%, respectively, in relation to the control group (G2 - p <0, 05). The periodontitis trial, on the other hand, demonstrated a 26% reduction in bone resorption (p <0.05) with treatment with OH-7 at 40% concentration. Therefore, organic honeys have a promising influence on the microbial and inflammatory components of periodontal disease, with the OH-7 variety being promising for indication as a substitute for common dietary sugar in order to promote health by reducing inflammatory and biofilm-dependent oral diseases.

Keywords: Honey, Antimicrobian activity, Anti-inflammatory activity, Periodontal disease. SUMÁRIO

1. INTRODUÇÃO 12 2. ARTIGOS 15 2.1 Artigo: Antimicrobial activity of honey against oral : current reality and future directions 15 2.2 Artigo: Antimicrobial and antibiofilm activities of organic honeys against oral microorganisms. 41 2.3 Artigo: Brazilian organic honey act as antimicrobial and decrease the bone resorption in periodontal disease 53 2.4 Artigo: Anti-inflammatory activity of organic honey from Brazilian Atlantic rainforest 80 3. DISCUSSÃO 98 4. CONCLUSÃO 109 REFERÊNCIAS 110 ANEXOS 116 Anexo 1: Certificado de aprovação do Comitê de Ética no Uso de Animais 116 Anexo 2 – Comprovante autorização do Conselho de Gestão do Patrimônio Genético (CGEN) 118 Anexo 3 – Comprovante de submissão do artigo à revista científica internacional 120 Anexo 4 – Comprovantes de premiação em congressos científicos 121 Anexo 5 – Relatório de originalidade do sistema Turnitin 122

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1. INTRODUÇÃO

Produtos naturais, como mel e plantas medicinais, contém moléculas bioativas capazes de desempenhar atividades biológicas no organismo (Crane & Gademann, 2016). Desde a antiguidade o mel é utilizado não só como um alimento prazeroso, mas também pelas suas propriedades biológicas no restabelecimento da saúde em casos de doenças envolvendo pequenas lesões, com efeito na cicatrização e em processos infecciosos (Cooper, 2016). A utilização do mel para o tratamento de feridas, escaras e outras lesões é uma prática comum que perdurou nos hospitais da Europa até a década de 1970 e a sua funcionalidade tem sido atribuída por suas propriedades biológicas como: atividade antimicrobiana, anti-inflamatória, estimulação de crescimento tecidual, ação desodorizante e de debridamento de ferimentos (Simon et al., 2009). Atualmente, a medicina moderna tem redescoberto as utilidades terapêuticas do mel no tratamento de feridas, escaras e outras condições em pacientes hospitalizados (Molan, 2015; Abd El-Malek, 2017; Dreyfus, 2018). Por estas propriedades o mel é um alimento que está presente e se sustenta desde a antiguidade na dieta, no convívio e bem-estar da humanidade (Cooper, 2016). A dieta desempenha um importante papel na prevenção de doenças crônicas, visto que grande parte destas podem estar relacionadas à nutrição. Desse modo, o mel além de ser importante para a nutrição, como fonte de energia e sais minerais, também é considerado um alimento functional. Alimentos funcionais são aqueles que além de suprir as necessidades nutricionais, funcionam como fonte de bem-estar físico e mental, contribuindo para a prevenção e redução de fatores de risco para várias doenças ou para aprimorar/sustentar funções biológicas (Ozen, Pons & Tur, 2012). O mel é um alimento natural produzido por abelhas, principalmente da espécie Apis mellífera, que tem em sua composição açúcares, enzimas, aminoácidos, vitaminas, ácidos orgânicos, carotenoides, substâncias aromáticas e minerais, abundante quantidade de flavonoides e ácidos fenólicos, o que reflete a diversidade do bioma visitado pela abelha e pode explicar sua variedade em desempenhar atividades biológicas (Alqarni, Awayss, & Mahmoud, 2012). Em consequência disto, o mel tem perfil químico complexo que depende da região geográfica, da biodiversidade da flora local, e também das condições climáticas e sazonalidade (Escuredo et al., 2014). O mel pode ser considerado um mel floral, quando as abelhas coletam nectar das flores, ou mel de melato, quando as abelhas coletam melato extraído do floema das 13

plantas por determinados insetos parasitas. Os estudos com méis de melato são ainda mais escassos e devem ser incentivados (Ng et al., 2020). Desse modo, diferentes tipos de méis podem apresentar propriedades farmacológicas diferentes e em maior ou menor intensidade (Oryan, Alemzadeh & Moshiri, 2016), incluindo propriedades que possam ser usufruídas para doenças bucais como a doença periodontal. A doença periodontal é uma das condições da cavidade oral que tem alta prevalência mundialmente (Kassebaum et al., 2014); e semelhante a cárie dental (Sicca et al., 2016), é biofilme dependente. Bactérias que crescem dentro de um biofilme frequentemente exibem fenótipos alterados, como o aumento da resistência aos agentes antimicrobianos. As propriedades estruturais estáveis e a proximidade das células bacterianas dentro do biofilme representam, além de uma barreira física, um excelente ambiente para a transferência horizontal de genes, o que pode levar à disseminação da resistência aos antibióticos (Roberts & Mullany, 2010). Além da colonização microbiana por bactérias periodontopatogênicas, como Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum e Prevotella spp, outro componente importante é o processo inflamatório, que o organismo do hospedeiro executa em resposta à disbiose do biofilme subgengival (Agossa, 2016). Apesar de essencial para o estabelecimento das doenças periodontais, o biofilme por si só não causa a destruição severa dos tecidos, esta ocorre em associação com essa resposta imuno-inflamatória exacerbada do hospedeiro mediada por células de defesa (Tatakis, 2005). Na doença periodontal, as células do sistema immune produzem metabólitos que geram efeitos citotóxicos e levam a instalação de um processo inflamatório no periodonto, que causa perda no tecido de inserção e proteção. A inflamação é uma reação complexa a agentes nocivos e inclui respostas vasculares, migração e ativação de leucócitos, especialmente neutrófilos. A migração dessas células é muito importante, pois quando o tecido é injuriado, os neutrófilos tornam-se ativos e cruciais no combate a agentes infecciosos, por reconhecimento ou ataque direto (Wright et al., 2010). O estímulo é iniciado pela ativação de fatores de transcrição nuclear, como o NF-κB, que está relacionado com liberação de mediadores químicos, como o fator de necrose tumoral alfa (TNF-α) e as quimiocinas CXCL1/KC e CXCL2/MIP-2, liberados pelas células locais. Esses mediadores químicos promovem a exposição de receptores inflamatórios pré-formados em suas membranas que aumentam sua função, vida útil e sua afinidade por células endoteliais, que permite a diapedese e migração para o sítio da inflamação (Lawrence, 2009). 14

O controle associado do componente microbiano e da resposta imune do hospedeiro na doença periodontal representa uma abordagem vantajosa e importante para a diminuição da agressão e severidade desta doença. Assim, os méis e seus componentes podem ser uma abordagem alternativa, uma vez que possuem propriedades anti-inflamatórias e antimicrobianas promissoras (Bayron et al., 2019), porém escassamente estudada em relação a doenças bucais. Para a atividade antimicrobiana, alguns méis foram classificados como antimicrobianos contra como S. mutans e Lactobacillus, (Ahmadi et al., 2013) e P. gingivalis (Atwa et al., 2014; Eick et al., 2014). No contexto anti-inflamatório, experimentos in vivo com ratos demonstraram promissora atividade anti-inflamatória de méis no tratamento de inflamação crônica e aguda em modelo de edema de pata (Owoyele et al., 2011; Almasaudi et al., 2017), porém sem informação sobre a bioprospecção dos compostos ativos ou como se dá o mecanismo da ação anti-inflamatória em nível molecular. Apesar dos efeitos antimicrobiano e anti-inflamatório de mel já descritos na literatura, não há informações de atividade antibiofilme de mel contra biofilmes subgengivais complexos nem estudos in vivo que avaliem a ação desse alimento funcional na doença periodontal. Acresce-se a isso o fato de que méis produzidos em matas nativas e de forma orgânica podem apresentar composição distinta de outros méis e seu estudo ainda é incipiente (Young et al., 2005). Portanto, o objetivo desta pesquisa foi avaliar, in vitro e in vivo, as atividades farmacológicas de méis orgânicos da Mata Atlântica brasileira nos componentes microbiano e inflamatório da doença periodontal.

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2. ARTIGOS

2.1 Artigo 1: Antimicrobial activity of honey against oral microorganisms: current reality and future directions

Scientific article to be submitted to The Journal of Nutritional Biochemistry (IF: 4.873). Comprovante disónível no Anexo 3 deste documento.

Diego Romario da Silva, Janaína Orlandi Sardi, Bruno Bueno Silva, Rafaela Parolina Carvalho and Pedro Luiz Rosalen.

* Corresponding author e-mail: [email protected]

Abstract Honey has been shown to have antimicrobial activity against different microorganisms, but its effects on oral biofilms is largely unknown. In this review, we analyzed the literature currently available on the antimicrobial activity of honey against oral biofilms, in order to determine its potential as a functional food in the treatment and/or prevention of oral diseases. Here, we compare studies reporting on the antimicrobial activity of honey against systemic and oral , discuss methodological strategies and point out current gaps in the literature. To date, there are no consistent studies supporting the use of honey as a therapy for oral diseases of bacterial origin, but current evidence in the field is promising. The lack of studies examining the antibiofilm activity of honey against oral microorganisms reveals a need for additional research to better define aspects such as chemical composition, mechanism(s) of action and antimicrobial action.

Keywords: Honey, Antimicrobial Activity, Antibiofilm Activity, Functional Food.

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Introduction Honey is a natural food produced by bees, mainly Apis melífera, composed mainly of sugars, water, enzymes, organic acids, vitamins, minerals, pigments, phenolic compounds, volatile compounds, solid particles derived from harvest, and an abundant amount and variety of phytochemicals [1,2] Some honey constituents may play important biological roles in humans, since the diversity of the biome(s) reached by the insects reflects in a direct fashion the wide bioactivity of the honey sample [3]. Honey has been used by mankind since ancient times, not only to meet nutritional needs, but also as a medicine, which characterizes it as a functional food [4]. The most common reports of the medicinal use of honey were to treat small lesions, with an effect on healing and infectious processes [5]. The use of honey to treat wounds, bedsores and other injuries was once a common practice that persisted in hospitals across Europe until the 1970s. The effectiveness of honey has been attributed to its biological properties, such as antimicrobial, anti- inflammatory, tissue repairing, deodorizing, and for debridement of wounds. In recent years, modern medicine has rediscovered the therapeutic benefits of honey in the treatment of wounds, bedsores, and other conditions in hospitalized patients [6–8]. This has encouraged the exploration of the biological properties of honey against several medical conditions, such as biofilm-dependent oral diseases. Several infectious diseases affecting humans are caused by biofilm-forming microorganisms, which include oral infections such as dental caries, periodontal diseases, and endodontic and fungal infections [9]. Bacteria that grow within a biofilm usually exhibit altered phenotypes, such as increased resistance to antimicrobial agents. Stable structural properties and proximity to bacterial cells in the biofilm favor horizontal transfer of resistance genes, which can ultimately increase antibiotic resistance rates [10]. This may render antimicrobial agents ineffective, because in addition to providing a facilitated environment for gene mutation, the biofilm scaffold forms a physical barrier against antimicrobial agents [10], which seems to be a crucial factor for the establishment and progression of microbial diseases [11] While the antimicrobial and antibiofilm activity of different types of honey have been previously determined against several systemic pathogens [5], little is known about its use as a functional food for the prevention and/or treatment of biofilm-dependent oral diseases. Evidence suggests that honey is not only an important source of energy and minerals, but also a potential functional food that may contribute to physical and mental well-being, preventing and mitigating risk factors for various diseases and improving/maintaining bodily functions [12]. 17

Thus, the purpose of this review is to discuss the antimicrobial properties of honey as a functional food, with a special focus on biofilm-dependent oral diseases and the use of analytical prospecting methods.

Oral Microbiome

The oral microbiome is a complex and diverse community with more than 700 microbial species, which may be embedded in self-produced extracellular polysaccharides. The most common bacterial genera of the oral microbiome are Veillonella, Actinomyces and Streptococcus [13,14]. These microorganisms can develop pathogenic biofilms on the surface of the teeth and cause oral diseases, such as caries and periodontal disease [15]. Dental biofilm can be classified into supragingival – located at or above the gingival margin – or subgingival – below the gingival margin, between the tooth and the gingival sulcular tissue [9]. Primary colonizers of supragingival dental plaque are predominantly facultative anaerobes (Streptococcus and Actinomyces), while subgingival areas are populated by strict anaerobes (Bacteroidaceae spp. and spirochetes) as a result of reduced oxygen availability [16]. Early biofilm formation and maturation take place through an interplay between bacteria-host, bacteria-bacteria, and bacteria-other microorganisms by means of various types of chemical bonds [17]. The wide range of chemical interactions between microbial cells, which are embedded in an extracellular polysaccharide matrix, is a complex and communicable network that provides a physical barrier and enables the expression and sharing of genes encoding for virulence factors. These conditions render biofilm pathogens approximately 1,000-fold more resistant than their planktonic form. Thus, biofilm control represents the biggest challenge for the prevention and/or treatment of biofilm-dependent diseases [18]. Previously, mature subgingival biofilms were cataloged into different color-based complexes (purple, yellow, green, orange and red), according to the frequency and quantity or abundance with which the microbial species are recovered from healthy patients and from those with periodontal disease [19,20] (Figure 1). The Socransky diagram was based on the results of several clustering and sorting analyses of the microbiome with a large size of biofilm samples (n = 185 individuals) by different clustering and sorting techniques [19]. Purple, yellow and green complex microorganisms are associated with periodontal health, whereas orange complex bacteria are associated with the health-disease dysbiosis and red complex species are strongly associated with the onset of periodontal disease. 18

Currently, the most common periodontal treatment includes scaling and root planing (SRP). However, this procedure alone may be insufficient to eradicate oral pathogens from the subgingival environment. Thus, the use of antimicrobial therapy as an adjunct to SRP is frequently needed. Antimicrobials can be administered systemically or through a local delivery system. Several well-documented randomized controlled trials and systematic reviews have shown that the systemic use of amoxicillin plus metronidazole (in combination with SRP) considerably produces clinical benefits when compared to SRP alone. However, the risk of selecting resistant microorganisms with monodrug therapy should be considered. Alternatively, natural products containing several bioactive molecules and systems acting synergistically could be considered an interesting antimicrobial treatment [21].

Figure 1. Schematics of the anatomical location of supragingival and subgingival biofilms, and the main bacterial species in each color-based complex.

Natural resources are a promising source of antibiofilm agents. In recent years, plants and natural foods have been increasingly arousing attention of the scientific community because of their health benefits. While most studies on the antibiofilm activity of natural products have focused on oral pathogens [22], the effectiveness of honey has been examined mostly against systemic microorganisms [5]. Biofilms are complex and dynamic microbial communities whose control can be challenging [22]. Subgingival [19] and supragingival [20] biofilms are formed by different 19

microbial complexes that are essential for the progression of biofilm-dependent oral diseases. To be effective against these conditions, an antimicrobial or functional food should ideally act against most of these microorganisms, especially those in the form of biofilm. Considering that periodontal disease depends on a key factor, i.e., development of complex multispecies biofilms [23], and that honey has antimicrobial and antibiofilm activity against different bacteria [5], this functional food can be promising in the control of microbial colonization and oral biofilm formation.

What explains the antimicrobial activity of honey?

The broad-spectrum antibacterial activity of honey has previously been reported in the literature. However, the early studies in the field failed to explore the characteristics of honey to its full potential [24]. Since honey has long been used in folk medicine [25] and more recently in the hospital setting for the treatment of skin wounds [26], most studies have investigated its antibacterial activity against microorganisms that may cause skin or systemic infections [5]. Despite this, the broad antimicrobial activity of most honey varieties remains unclear and some aspects, such as the elucidation of the bioactive components and mechanism(s) of action, are yet to be determined. Thus far, the available evidence shows there is no specific component responsible for the antimicrobial action of honey. Instead, its different compounds and characteristics seem to act synergistically, among which are: low pH, osmotic effect, presence of hydrogen peroxide, phenolic compounds (mainly phenolic acids and flavonoids), methyl glyoxal and bee peptides [27]. Overall, the antimicrobial mechanism of honey is mostly related to the production of peroxides by the enzymatic complex, particularly the enzyme glucose oxidase [28]. A brief description of each of these components and their relationship to the antimicrobial activity of honey can be found in Table 1. In addition, induction of cytokine release in the host can also be an indirect antimicrobial mechanism of honey [29].

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Table 01. Factors related to antimicrobial activity of honey.

Component / Physical Considerations about the antimicrobial activity of honey Reference characteristic Low pH Due to the high concentration of organic acids (mainly gluconic [30] acid), most honey samples have a pH ranging between 3.4 and 6.0 which, in combination with high osmotic pressure, can eliminate and / or prevent microbial colonization. Osmotic effect High osmolarity, one of the colligative properties of honey, has [31] been shown to play a role in its antimicrobial properties. Despite this, high concentrations of glucose alone showed no inhibitory effect on bacterial growth. Thus, sugar-induced osmolarity in honey is only considered as an adjuvant that provides an unfavorable environment for pathogens. Hydrogen peroxide Hydrogen peroxide (H2O2) levels directly proportional to the [30,32,33] (H2O2) antibacterial activity of honey. Therefore, H2O2 is considered a predictive biomarker of its antibacterial activity. Glucose oxidase is an enzyme present in honey that is activated when the honey is diluted. This enzyme acts on endogenous glucose to produce hydrogen peroxide. H2O2 is involved with oxidative damage, causing inhibition of bacterial growth and DNA degradation. However, these effects are synergistically modulated by other components present in honey, since higher concentrations of pure H2O2 are needed to obtain similar effects as compared to the low H2O2 levels present in honey samples. Phenolic compounds The therapeutic effect of honey is attributed to the presence of [2,34] several antioxidants, including phenolic compounds, such as flavonoids and phenolic acids. Some phenolic compounds, such as pinocembrine and serum acid, have been strongly associated with the antimicrobial activity of honey. Nevertheless, information on the mechanism of action and effective doses of these compounds is yet to be determined. Methylglioxal MGO is an organic compound derived from dihydroxyacetone. [35] (MGO) The presence of MGO in honey contributes to its antimicrobial activity, even in honeys with low levels of peroxide (e.g., Manuka honey). MGO causes loss of membrane integrity and changes the structure of bacterial fimbriae and flagella, which impairs microbial adhesion and motility. Bee peptides (Defesin- Defensin-1 is a peptide secreted by the hypopharyngeal glands [36] 1) of bees. This peptide is active against Gram-positive bacteria, including Bacillus subtilis and . Although bees produce other peptides, only defensin-1 has been detected in honey and it is was found to have antimicrobial activity.

The biological properties of honey, including its antimicrobial activity, have been associated with the diverse chemical composition of this functional food. The phytochemical profile of honeys is complex and can be influenced by different factors, such as geographic location, biodiversity of the local flora, as well as climatic conditions and seasonality [37]. While the phytoconstituents contained in the plant material collected by bees are also considered determinants of the antibacterial activity of honeys [24], they remain poorly characterized [5]. Currently, several active components have already been identified in honeys, 21

but their inhibitory activity can no longer be attributed only to the common characteristics mentioned earlier [5], as other bioactive components are also likely to be involved. Thus, studies examining the antimicrobial activity of honey should be mostly focused on determining its underlying mechanism(s) of action. According to antimicrobial mechanisms, honeys can be divided into two main groups, namely: peroxide and non-peroxide honeys [38]. Overall, most honeys with proven antimicrobial activity are classified as peroxide honeys because their antimicrobial activity is linked to the production of hydrogen peroxide [38]. The most important non-peroxide honey representative is a New Zealand honey known as Manuka honey. It was designated as non-peroxide because even with the inactivation of these compounds, this honey exhibited significant antimicrobial activity. The hydrogen peroxide production in Manuka honey is relatively low. Nevertheless, Manuka honey has a potent antimicrobial activity, even after inactivating the peroxides with the enzyme catalase. Thus, the term "non-peroxide activity" (NPA) was proposed for that honey category. Thus, different samples of manuka honey have been classified through the NPA unit [39–41]. Manuka honey properties are related to the presence of methylglioxal [42]. Some types of honeys are more potent than others, but all of them contain the same antibacterial substance (H2O2), except for Manuka honey. H2O2 was believed to be produced mainly by the enzyme glucose oxidase by converting glucose into gluconic acid under aerobic conditions in diluted honey [43]. However, recent evidence suggests that H2O2 present in honey is also produced via an alternative non-enzymatic pathway. There is no correlation between the content of glucose oxidase and H2O2 levels. In addition, a similarity has been demonstrated between MIC values of diluted honeys untreated and treated with proteinase-K (protease). These findings suggest that the antimicrobial activity attributed to peroxides produced by the enzyme complex and defensin-1 is virtually negligible [33,44,45]. The answers to many questions about the antimicrobial mechanisms of honeys remain unclear. Studies have reported that for H2O2 killing to occur, compound concentrations greater than 50 mM are required [46]. However, the H2O2 content in honey is 900 times less than that used in medical disinfectants [47]. A study demonstrated that when present in honey at a concentration of 1 mM, H2O2 was able to inhibit the growth of and Staphylococcus aureus [44]. Furthermore, the authors suggested that the plant-derived polyphenolic compounds present in honey may be related to H2O2 production and, consequently, to the increase of the antibacterial potency of honey samples. A significant correlation between the concentration of total polyphenols and the antibacterial activity of honey samples was observed. The authors pointed out that the antimicrobial activity causally 22

related to phenolic compounds is minimal and practically negligible, since the concentration of polyphenols and flavonoids dissolved in honey are relatively low. In summary, the study suggested that the presence of phenolic compounds may be related to the antimicrobial activity of honey in two ways, namely: H2O2 production and reduction of Fe (III) into Fe (II), triggering the Fenton reaction and producing more potent reactive oxygen species. Other studies examining Polish honeys reported the importance of phenolic compounds for their antimicrobial activity [48–50]. The authors observed a small amount of these compounds in honey and suggested that a synergism between them and the H2O2- producing system is likely. The conditions of matured honeys could resemble the macromolecular agglomeration in the living cell and affect the concentration, reactivity and conformation of the macromolecules. Thus, a previous study evaluated the structure and distribution of honey components. [51]. The authors found that a high concentration of macromolecules promoted self- assembly of micron-sized superstructures. These structures were visible under a Scanning Electron Microscope (SEM) as a biphasic system composed of dense globules dispersed in sugar. After diluted, these particles showed greater conformational stability. Moreover, at the threshold concentration, the system went through phase transition with concomitant fragmentation of large micron-sized particles to nanoparticles in a hierarchical order. The authors concluded that the biphasic conformation of honey was needed to produce H2O2 and, consequently, it determined the antibacterial activity of the sample. These properties disappeared beyond the phase transition point. This study suggests that the arrangement of active macromolecules with colloidal properties, organized in compact and stable multicomponent sets, is an important discovery about the overall structure of honey and is essential to understand the complexity of the biological activities of such a functional food. Thus far, mounting evidence indicates that honey has a multifactorial antimicrobial activity consisting of the synergism between physical characteristics (reactivity and conformation of macromolecules) and chemical composition. It is unlikely that a specific isolated component of honey has an antimicrobial activity as good as that of its originating fresh honey. Hence, future research in the field should be focused on understanding the mechanisms of action of whole honey, particularly its molecular targets. Detailed studies, including transcriptome analysis, can contribute to elucidate the global effects of honey on microbial cells [52].

Analysis of the antimicrobial activity of honey 23

The antimicrobial activity of honey has been determined by two methods, namely: agar diffusion and broth microdilution. However, the agar diffusion method has not been commonly recommended for most antibacterial substances since growth inhibition does not necessarily mean microbial death. Therefore, this method does not distinguish between bactericidal and bacteriostatic effects. In addition, it is not possible to measure the amount of substance that diffuses through the agar nor the viscosity-related variability issues. These are some of the reasons why this method has not been indicated for determining the Minimum Inhibitory Concentration (MIC) [53]. Apart from the general considerations on the use of agar diffusion to determine the antimicrobial activity of any substance, there are particularities to consider. For instance, high viscosity and volume issues when placing honey samples into agar wells may render the results even more imprecise. Another important point to consider is that the high molecular weight active constituents present in honey may not diffuse properly through the agar (e.g., defensin- 1 and especially glucose oxidase) [36]. Therefore, we recommend and discuss here only the use of both microdilution to determine the antimicrobial activity of honey. Dilution methods are the most suitable for determining MIC values. The MIC is defined as the lowest concentration of an antimicrobial that inhibits visible microbial growth. There are many approved guidelines for testing antimicrobial susceptibility using dilution methods. The most recognized standards are provided by the Clinical & Laboratory Standards Institute (CLSI) and the European Committee of Antimicrobial Susceptibility Testing (EUCAST). While the development of these standards does not guarantee the clinical relevance of the tests, it allows for the standardization and reproducibility of in vitro assays [54]. Broth microdilution involves preparing 1:2 dilutions of the antimicrobial agent in a liquid in a 96-well plate, i.e., 1000, 500, 250 and 125 µg/mL. This procedure is called serial dilution. The most problematic step in this test is to prepare a work solution with the honey sample. The first difference in relation to the preparation of honey or lyophilized solutions (e.g., monodrug or extract) is that the dilution of honey is expressed as a percentage (%) instead of mg/mL or µg/mL. In this case, it is possible to determine this percentage by making proportions of honey and water or culture medium, as follows: weight/volume (w/v) or volume/volume (v/v). Although some authors prefer to use the v/v ratio [55,56], most of them perform the dilution based on the w/v ratio [28,57–59]. We recommend the use of w/v ratio due to the viscosity of the sample, which may cause substantial material loss during pipetting and errors. 24

Honeys from different botanical and geographic origins exhibit varying antimicrobial potency. Thus, they can be classified into different categories, of which monofloral honey seems to be the most promising and interesting type as a natural remedy. Manuka honey, a monofloral honey derived from Manuka tree (Leptospermum scoparium) stands out for its antimicrobial and antioxidant properties. Manuka honey has been used as a parameter to classify comparatively the antimicrobial potency of other honey [60]. In general, dark colored honeys exhibit more expressive antimicrobial activity. The most potent honeys, such as Manuka, dark buckwheat, Heather, or molasses, have MIC values ranging from 1% to 12.5% (w/v). Nevertheless, light colored honeys, such as clover honey (pasture honey) and acacia or rapeseed honey, were found to be less potent, with MIC values ranging between 25 and 50% (w/v) [30]. These well-known honeys can be used as parameters to define whether a sample has strong, moderate, or weak antimicrobial activity. Thus, in Table 2, we propose some parameters for determination of the antimicrobial potency of honeys, according to Albaridi’s review (2019).

Table 2. Parameters for determination of the antimicrobial potency of honey samples. MIC (%, w/v) Antimicrobial activity 1.0 to 12.5% Strong 12.5 to 50.0% Moderate > 50.0% Weak (Adapted from Albaridi, 2019)

We suggest the preparation of an initial solution which should be serially diluted at a 1:2 ratio, since it would be the maximum concentration (50% w/v) to determine a discrete antimicrobial activity that is not due to lack of culture medium [30]. We do not recommend 1:2 dilutions in the wells of the microplate since the concentration drops very sharply from one well to the other. Instead, we suggest preparing individual solutions to be added together with the inoculum. This way, the concentration drops by half from the initial solutions and will have a difference of 5% between from one well to the other in the 96-well plate, ranging from 50% to 1.25% (Figure 2).

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Figure 2. Schematic representation of the serial dilution of honey for antimicrobial testing. The honey sample should be diluted separately by preparing a solution with twice the concentration expected to be in the well of the microplate. After adding the diluted honey solution to the same amount of inoculum, the concentration will drop by half in the well. To obtain these concentrations, it is necessary to use the microplate horizontally, as demonstrated in the figure.

In addition to determining the antimicrobial activity of honey against planktonic bacteria, it is essential to assess its potential against biofilms. Biofilms are dense communities that grow on inert surfaces and are embedded in self-secreting high molecular weight polymers. When organisms form a biofilm, they can adapt to environmental changes by altering their gene expression and becoming highly resistant to antimicrobials [13]. As mentioned in the section 2.0 of this review, there is an association between the presence of oral biofilms and the onset and progression of oral diseases, such as caries and periodontal disease [61,62]. Thus, a drug or product with antimicrobial activity should necessarily have a positive antibiofilm activity against mature biofilms to be considered effective for the prevention and/or treatment of oral diseases. An ideal and standardized in vitro assay to assess the effectiveness of antibiofilm agents has not been established yet, although different testing methods are available. Approaches such as the modified Robbins device, Calgary biofilm device, disc reactor, Centers for Disease Control (CDC) biofilm reactor, perfused biofilm fermenter and model bladder have 26

been considered promising and representative of in vivo conditions [63]. In addition, multispecies biofilm models better mimic what occurs in the oral cavity than single-species biofilm models. There is a wide range of tools available for biofilm analysis, from colony counting to more modern techniques, such as fluorescent biofilm labeling in conjunction with mathematical predictive modeling, such as COMSTAT [64]. For a quick and low-cost analysis, we commonly use Calgary device to determine the antibiofilm activity of propolis samples [21] and suggest that it should be performed to assess the antibiofilm activity of honey. The Calgary device allows biofilms to grow on rods suspended from a lid that fits into a 96-well plate. Once the device is suspended in the culture medium with inoculum, it allows for actual biofilm formation without deposition of gravity [65]. Thus, for testing the antibiofilm activity of honey, we suggest diluting the sample as shown in Figure 1 and treating multispecies biofilms at concentrations greater than the MIC. Treatment intervals should be selected to simulate the daily consumption of honey by a person who substitutes the common sugar of the diet by this functional food.

Antimicrobial activity of honey against oral pathogens

Some literature reports show that honey is active against a wide range of pathogens associated with systemic infections, such as Pseudomonas aeruginosa [22,66], Staphylococcus aureus (MRSA) [67], , Proteus mirabilis, Staphylococcus aureus, Shigella flexneri, and Staphylococcus epidermidis [68]. Testing the effectiveness of honey against planktonic bacteria is relevant, but the use of biofilm cultures in antimicrobial assays is highly recommended. Biofilm communities are embedded in self-producing polysaccharides, which creates a barrier that provides resistance to antimicrobials [18]. Some authors showed that multifloral honey has promising antibiofilm activity against systemic pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus and Escherichia coli [44,69–71]. Honey samples should be ideally examined for their antimicrobial mechanism(s) of action, e.g., downregulation of virulence genes, disruption of the bacterial cell wall, interference in the formation or size of cells and/or accessory components (fimbriae and flagella) and damage to nucleic acids. The mechanisms of action of honey against systemic bacteria are well documented [5], but there are still gaps concerning the analysis of antibiofilm properties against oral bacteria. Thus, this comprehensive review brings an overview of all the evidence on the in vitro antimicrobial activity of honey against oral microorganisms. Bibliographical searches were carried out in the databases Medline via 27

Pubmed, SciVerse Scopus, Web of Science, SciELO, LILACS, Cochrane Library and Google Scholar using the following descriptors: honey, antimicrobial activity, and oral bacterium (Table 2).

Table 2. In vitro studies of antimicrobial activity of honey against oral microorganisms.

Type of Strain Method Results Analysis of Chemical Reference Honey mechanism analysis? (s) of action? Honey from Streptococcus Antimicrobial The three honey No No [72] central gordonii, activity (MIC samples inhibited Switzerland, Streptococcus determination) the specific honey from sanguinis, and antiadherent growth of oral the German Streptococcus activity by bacterial strains. plain and mutans, counting Colony- When using a Manuka Streptococcus forming Units multispecies honey. sobrinus, per mL biofilm model, Lactobacillus (CFU/mL). none of the acidophilus, samples was Actinomyces significantly naeslundii. effective. Kerala Streptococcus Antimicrobial A discrete zone of No No [73] commercial mutans activity inhibition of the honey, India. determined by growth of the agar diffusion Streptococcus method. mutans was observed. Manuka Staphylococcus Antimicrobial Both honeys No No [74] honey and aureus, activity by broth inhibited most of white clover Escherichia coli, microdilution for the tested stains, honey. Streptococcus determination of with similar MIC / mutans, MIC and MBC MBC values, but Streptococcus values. Streptococcus mutans, mutans was Streptococcus comparatively sobrinus, resistant. Honeys Streptococcus with neutral pH sanguinis, had little Streptococcus antimicrobial gordonii, activity. Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia

Swiss Aggregatibacter Initial screening for Manuka honey No No [75] multifloral actinomycetemco antimicrobial activity below an NPA honey, mitans, by the agar diffusion value of 15 Manuka Porphyromonas method. The most showed the least Honey NPA gingivalis active samples were potential to inhibit 5+, Manuka and Streptococcus selected for bacterial growth, Honey NPA mutans determination of MIC even less - 15+, Manuka and MBC values. although not honey Label significantly - than “MGO 400+”, Swiss multifloral equivalent to honey. Manuka NPA 20+, honey above an Manuka NPA value of 15 honey NPA showed a 25+, significantly 28

MediHoneyT greater M medicinal antibacterial effect honey, compared to the MediHoneyT other honeys M gel sheet. tested. All Manuka honey preparations were more effective in inhibiting the growth of P. gingivalis and A. actinomycetemco mitans as compared to S. mutans.

Manuka Streptococcus Antimicrobial activity The antibacterial No No [76] honey 1 and 2. mutans, (MIC determination) activity of Streptococcus and antiadherent Manuka 1 was the sobrinus, activity by counting most important. Lactobacillus Colony-forming Units The two honeys rhamnosus, per mL (CFU/mL). tested showed a Actinomyces weak ability to viscosus, inhibit the Porphyromonas adhesion of S. gingivalis, and mutans cells onto Fusobacterium a glass surface at nucleatum. sub-MIC concentrations. Manuka 1 completely inhibited multispecies biofilm formation at a concentration of 200 μg/ml. Manuka 2 inhibited biofilm formation weakly at a concentration of 200 μg/ml, but strongly at a concentration of 500 μg/ml. Eucalyptus Steptococcus Antimicrobial activity Eucalyptus honey No No [77] honey mutans, by broth microdilution had a MIC of 25% Steptococcus for determination of (v/v) on the tested sobrinus, MIC values. strains, except for Steptococcus Streptococcus gordonii, anginosus and Steptococcus Streptococcus salivarius, oralis, whose MIC Steptococcus values were 17% sanguinis, (v/v) and 12% Steptococcus (v/v), respectively. anginosus, Hypertonic sugar Steptococcus control had a MIC oralis, and of 25% (vol / vol) Escherichia coli. on all bacterial strains. Manuka Streptococcus Antimicrobial activity C. albicans No No [78] honey, mutans, by broth microdilution (MIC of 40%) was eucalyptus Streptococcus for determination of more resistant to honey, pin salivarius, MIC values. the tested honeys cushion honey Streptococcus than were the and Erica sanguis, bacterial strains. S. honey. Streptococcus anginosus (MIC 29

anginosus, of 17%) and S. Streptococcus oralis (MIC of gordonii, 12.5%) were more Streptococcus sensitive to honey oralis, than the other Streptococcus strains. The honey sobrinus, samples showed a albicans, MIC of 25% Escherichia coli against other oral and streptococci. Staphylococcus aureus. Azarian honey Staphylococcus Antimicrobial activity The lowest MIC No No [79] aureus, by broth microdilution value of the test Staphylococcus for determination of honeys was found epidermidis, MIC values. for Streptococcus Staphylococcus mutans, MRSA aureus. The MIC and Enterococcus of honey for faecalis Escherichia coli was higher than that for the other strains. The mean MIC for Staphylococcus epidermidis was similar to that of S. aureus. The MBC values for S. mutans, MRSA, S. aureus, S. epidermidis and Enterococcus faecalis were 7.81, 8.52, 7.55, 12.03, and 7.81 % (v/v), respectively. The combination of propolis and honey reduced the MIC for all bacterial strains. Commercial Streptococcus Antimicrobial activity Natural honey No No [80] honey from mutans (microdilution reduced S. mutans Saudi Arabia method) and growth more (Langnese inhibition of biofilm effectively than Honig, formation. artificial honey Germany) (control) at the concentrations of 25% and 12.5%. At 50% and 25%, both honeys significantly reduced bacterial growth and biofilm formation as compared to the TSB control. Natural honey was also able to decrease the maximum growth rate of S. mutans compared to artificial honey. 30

Ramadan Streptococcus Antimicrobial activity Significant No No [57] natural honey mutans by the agar diffusion antibacterial method. activity was detected against S. mutans at concentrations greater than 20% and against Lactobacillus at the concentration of 100%.

Manuka Porphyromonas Antimicrobial activity Manuka honey Yes Yes [81] honey and gingivalis by the microdilution and commercial commercial method honey inhibited honey from (determination of the 50% of P. Germany MIC) and antibiofilm gingivalis growth activity. at concentrations of 2% and 5%, respectively. Manuka honey contained 1.87 mg / kg of hydrogen peroxide whereas the commercial honey had 3.74 mg / kg. The amount of methylglioxal was 2 mg / kg in the domestic honey and 982 mg / kg in Manuka honey. At 10%, both types of honey inhibited P. gingivalis biofilm formation and reduced the number of viable bacteria in 42-h old biofilms.

Most of the studies shown in Table 2 used the broth microdilution method for determining the antimicrobial activity of honey samples [72,74–80] and only a few studies used the agar diffusion method [57,73]. These findings are consistent with what was discussed in the section 4.0 of this review. Of all the studies presented in Table 2, only one examined aspects such as the mechanism of action, chemical composition, the relationship between chemical profile and bioactivity, as well as the antibiofilm activity of honey in mature biofilms [81]. However, none of the studies showed data indicating that honey can cause cell damage in bacteria or that it has antibiofilm activity against multispecies biofilms. Although the antimicrobial activity of honey against microorganisms present in wounds is well documented [5], its effectiveness against oral microorganisms remains to be determined. Further research should determine the antimicrobial activity of honey against mature multispecies oral biofilms [18]. 31

In addition to the in vitro studies summarized in Table 2, the literature presents some clinical trials that have evaluated the antimicrobial effectiveness of honey against oral diseases. Because it is food and not a drug, clinical trials with honey can be ethically less complex. However, this does not diminish the importance of carrying out in vitro studies for classification, characterization, and definition of the antimicrobial mechanisms of action. Despite the advances, the selected clinical trials have important limitations to consider, as further discussed herein. A comparative in vitro and clinical study was performed to determine the antimicrobial activity of 0.2% chlorhexidine and a honey-containing mouthwash. The in vitro phase of the study was carried out using the agar diffusion method, whereas the second phase consisted of a randomized blind clinical trial, with a total sample size of 66 individuals. The in vitro data showed that the honey mouthwash effectively inhibited the growth of Eubacterium nodatum, Campylobacter rectus, Streptococcus mutans, Agrobacter actinomycetemcomitans, Porphyromonas gingivalis and Streptococcus sanguinis, although 0.2% chlorhexidine was more effective. In the clinical trial, the intergroup analysis between chlorhexidine and honey showed that both formulations significantly reduced plaque formation (P < 0.001). Although chlorhexidine was more effective than the honey-containing mouthwash, there was no significant difference between them (P = 0.670) [82]. This study showed the in vitro and clinical effectiveness of a honey formulation in reducing the growth of oral microorganisms, supporting the hypothesis that honey can be used as a protective food against biofilm-dependent oral diseases. However, the in vitro data lack important pharmacological information, such as the definition of the mechanisms of action and the chemical profile of the sample. Furthermore, the clinical trial lacks important indices for periodontal disease assessment, such as clinical parameters and/or salivary cytokine quantification. Another study investigated the effectiveness of Manuka honey in reducing clinical levels of biofilm and gingivitis. The authors developed a "honey strip" chewable formulation. The study was randomized and had a sample size of 30 participants, who were allocated into two groups, as follows: one group that chewed the Manuka product and other that chewed a sugar-free gum. The chewing time was 10 minutes, three times a day, after each meal. Plaque and gingivitis scores were recorded before and after 21 days. In the Manuka honey group, there were statistically significant reductions in mean plaque scores (from 10.99 to 0.65, P = 0.001) and in the number of bleeding sites (from 48% to 17%, P = 0.001). In contrast, no significant differences were observed in the control group [83]. This showed that Manuka honey is as effective against oral microorganisms as it is against systemic bacteria, as demonstrated in 32

previous clinical studies. The authors also examined the pattern of gingival bleeding, which is an important diagnostic index for periodontal diseases. While these findings correspond to a pilot study, they provide important evidence for the use of honey to prevent biofilm-dependent oral diseases or even as an alternative therapy. However, aspects such as mechanism of action, dosage and therapeutic indications need to be studied before honey formulations can be indicated as a therapy in dentistry. The antibacterial activity of commercial honey and green tea solutions was previously assessed based on salivary S. mutans counts. Thirty healthy individuals were randomly allocated into two groups of 15, one for each solution. Saliva samples were collected before and after rinsing the test solutions in both groups. The results showed that, after a single mouth rising, the count of colony-forming units (CFU/mL) decreased from 2.28 x 108 to 5.64 x 107 CFU/mL in the honey group and from 1.95 x 109 to 2.9 x 108 in the green tea group. There was a statistically significant difference for both groups in relation to baseline CFU counts [84]. A similar study assessed the effects of Manuka honey on the count of salivary streptococci [85]. Although the study showed positive results, the authors did not measure the antibiofilm activity of Manuka honey nor included any clinical parameters in the analysis. However, collectively, the study shows that honey significantly modulates the microaerophilic microbiota of the mouth. English et al., [83] carried out a pilot study with patients who were asked to chew a honey-containing gum for 21 days. At the end of the study, the authors observed a reduction in the biofilm score and in the number of gingival bleeding sites. Biofilm formation is a key virulence factor for a wide range of microorganisms that cause chronic infections. The multifactorial nature and drug resistance of biofilms pose great challenges for the use of conventional antimicrobials and indicate a need for novel drugs or even a combination of existing drug regimens. Taken together, the literature indicates that honey is a functional food with antimicrobial activity. However, the biological activity of honey against oral biofilms remains poorly understood and underexplored. Thus, further in vitro and in vivo studies are needed to support the use of honey as an antimicrobial and / or as adjuvant therapy in dental practice.

Concluding Remarks

Mounting evidence has shown that honey has promising antimicrobial activity. This functional food has been studied and used in folk medicine for many centuries to treat 33

infections, particularly those associated with wounds and bedsores. Nevertheless, only a few scientific studies have investigated the use of honey to prevent and/or treat biofilm-dependent oral diseases such as periodontitis. The antimicrobial activity of honey against oral microorganisms, as well as its antibiofilm activity against mature biofilms, mechanism of action, chemical composition, and clinical indications, remain largely unknown. Honey has a great potential to be used as an alternative therapy for biofilm- dependent oral diseases and/or as an auxiliary functional food for maintaining health. Yet, further research is needed to demonstrate this activity in mature oral biofilms, as well as the associated chemical pathways and the molecular targets in the bacterial cell. Filling these gaps may provide evidence to recommend the rational consumption of honey instead of common sugar for oral and systemic health benefits. Moreover, the promising antibiofilm properties of honey may encourage the development of clinical trials aimed at using honey products as a complementary therapy in dental care.

References

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2.2 Artigo 2: Antimicrobial and antibiofilm activities of organic honeys against oral microorganisms. Scientific article to be submitted to journal Biofouling (IF: 2.351). Diego Romario da Silva, Camila Furtunato Silva, Severino Matias de Alencar and Pedro Luiz Rosalen. * Corresponding author e-mail: [email protected] Abstract

This study aimed to evaluate the antimicrobial and antibiofilm activity of Brazilian organic honeys against oral microorganisms. Eight samples of organic honeys (OH-1 to OH-8) were georeferenced, collected, diluted (% - w/v), and sterilized by filtration. Antimicrobial activity was defined by determining MIC and CBM against six oral Streptococcus. The component responsible for the antimicrobial action was defined by a catalase assay. The antibiofilm activity was evaluated against the monospecies biofilm of Streptococcus mutans. All honeys showed antimicrobial activity against the tested bacterial strains, with emphasis on OH-1, OH-2, OH- 3, and OH-7 with MIC values ranging between 10 and 25%. The mechanism of action of antimicrobial activity occurs mainly by hydrogen peroxide produced by honey enzymes. All honey samples showed an antibiofilm activity. OH-1, OH-2, and OH-7 showed total biofilm inhibition at concentrations below 50%. Brazilian honeys have promising antimicrobial and antibiofilm activity. Thus, by replacing quotidian sugar in the diet, this functional food can control the oral microbiota and decrease the risk of biofilm-dependent oral diseases.

Key-words: Honey; Antimicrobial activity, Antibiofilm activity, Oral , Antioxidant activity.

42

Introduction Honey is a natural food produced by bees, mainly of the species Apis mellifera. This food has in its composition sugars, enzymes, amino acids, vitamins, organic acids, carotenoids, aromatic substances and minerals, an abundant amount of flavonoids and phenolic acids, which reflects the diversity of the biome and may explain its variety in biological activities [1]. Thus, honey is not only an important component of nutrition but can also be considered a functional food, that is, in addition to meeting nutritional needs, it functions as a source of physical and mental well-being, contributing to the prevention and reduction of risk factors for various diseases or to improve/sustain biological functions [2]. Thus honey can be an excellent natural source for bioprospecting biological activities related to injuries and diseases that affect the human being. Antimicrobial activity, antibiofilm, anti-inflammatory, tissue growth stimulation, deodorant, and wound debridement have been reported for some honey [3]. Of the bacterial diseases that affect the oral cavity, we can highlight dental caries as the most prevalent. This disease is related to the colonization of oral bacteria, such Streptococcus mutans (S. mutans) and other Streptococcus of the viridans group, as well to Lactobacillus spp., among others, and, consequently, the formation of dental biofilm [4]. Bacteria that grow within a biofilm often exhibit altered phenotypes, such as increased resistance to antimicrobial agents. The stable structural properties and proximity of bacterial cells within the biofilm represent an excellent environment for the horizontal transfer of resistance genes, which can lead to the spread of antibiotic resistance [5]. Besides, the polysaccharide component of the biofilm matrix itself can provide adhering, protection against stresses and immune cells of the host, allow stratification of the microbial community, and establishment of nutrient gradients and residues to maintain the complex structure [5]. This complexity hinders the action of antimicrobial agents because, in addition to providing a facilitating environment for mutation, biofilm forms a physical barrier against these agents [5]. Despite being a food rich in sugars, some honey has been reported as an antimicrobial against cariogenic bacteria, such as S. mutans, and Lactobacillus, [6] and gum disease [7]. However, there is no record of an investigation of antimicrobial activity against other cariogenic bacteria or antibiofilm in mature biofilms, as well as the mechanism by which this action occurs is not described. Furthermore, the fact that honey produced in native forests and organically may present distinct composition from other honey and their study is still incipient. Thus, this study aimed to evaluate the antimicrobial activity of eight honeys, produced 43

organically, against cariogenic microorganisms and antibiofilm activity in biofilms of S. mutans.

Material and methods

Obtaining, georeferencing and sample preparation Eight samples of honeys produced in a certified organic way were used, collected, and georeferenced in the Atlantic Forest region preserved in two municipalities in the south of Paraná, General Carneiro (26 ° 25 '44 ”South, 51 ° 19' 2” West) and Turvo (25 ° 2 '47 ”South, 51º 32' 32” West), from December 2015 to February 2016. Table 01 shows the division and naming of honeys, their collection sites, and their floral variations. The honeys were diluted in a culture medium to reach the necessary concentrations and filtered through 0.22 µm syringe filters. Research registered in the National Management System for Genetic Heritage and Associated Traditional Knowledge (SISGEN) register: AE0DBB2.

Table 1. Sample collection of organic honeys in southern Brazil.

Honey Collection place (city / state) Type sample OH-1 Turvo-PR Polyfloral honey OH-2 Turvo-PR Polyfloral honey OH-3 General Carneiro-PR Honeydew honey (Mimosa scabrella) OH-4 General Carneiro-PR Polyfloral honey OH-5 General Carneiro-PR Honeydew honey (Mimosa scabrella) OH-6 Turvo-PR Polyfloral honey OH-7 General Carneiro-PR Honeydew honey (Mimosa scabrella) OH-8 Turvo-PR Polyfloral honey Adapted from Silva et al., 2019 [8].

Microorganisms and microbial susceptibility Microorganisms: Streptococcus mutans ATCC 700610, Streptococcus mitis NCTC 12261, Streptococcus oralis ATCC 10557, Streptococcus salivarus ATCC 7073, Streptococcus gordonii Challis, and Streptococcus sanguinis 3K36. Antimicrobial activity was performed using the microdilution technique in 96-well plate broth, to determine the Minimum Inhibitory Concentration (MIC) using Mueller-Hinton culture medium (CLSI, 2012) [9]. The Minimum Bactericidal Concentration was defined by seeding aliquots from the MIC in Mueller-Hinton agar culture medium. The honey was diluted in a Mueller-Hinton culture medium so that in the 44

wells it reached concentrations between 60 and 5% (weight/volume). The bacterial inoculum was standardized by spectrophotometry to obtain a concentration of 5x105 cells in the wells. The results were analyzed using the visual method. The experiments were carried out in triplicate and in three independent experiments.

Antibiofilm activity The formation of S. mutans biofilm was induced in 96-well plates. Inoculum: 100 µL of inoculum of approximately 8x1011, standardized by optical density at 0.8 to 625 nm, was added to each well in brain heart infusion (BHI) broth supplemented with 1% sucrose as previously described by Cai et al., 2016 [10], with changes. The plates were incubated in an oven with an atmosphere of 5% CO2. After 24 h the culture medium was renewed. After 48 h the culture medium was discarded and a new culture medium was added with the concentrations of honey in the MIC concentrations, twice the MIC and 50% of that honey. The positive control was done with 0.12% chlorhexidine and the negative control with BHI broth without the addition of sucrose. After 24 h of treatment, the contents of the wells were removed, diluted, and plated on Mitis Salivarius Agar, for later counting of Colony Forming Units per mL (CFU/mL).

Catalase treatment To verify the mechanism of antimicrobial activity, the honeys were treated with catalase. The solutions were diluted in a similar way to the MIC test and then they were treated for two hours with a catalase solution (1/1 ratio). The catalase solution was prepared by adding 40 mg of catalase (Sigma, C-10: 4000 mg-I units) to 20 ml of culture medium [11].

Scanning Electron Microscopy (SEM) Biofilms were formed in coverslips (Lab-Tek, Nunc, Naperville, IL, USA) using the method described in item 2.3. Biofilms were treated with samples that show the most promising antibiofilm activity, in CIM and 2x CIM concentrations. The cells were fixed in 3% glutaraldehyde at room temperature for 12 hours. The biofilm was then dehydrated in increasing concentrations of ethanol, metalized with gold, and examined using Scanning Electron Microscopy (SEM) (WITHOUT JEOL JSM 5600LV, JEOL Tokyo, Japan). Statistical analysis 45

The data were checked for normality and submitted to one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. The results were considered significant at p < 0.05 and α: 5%.

Results All samples of organic honeys, in natura, (OH-1 to OH-8) showed the ability to inhibit the growth of oral Streptococcus in planktonic form. The ones that stood out the most, with lower MIC values, were the OH-1, OH-2, OH-3, and OH-07 honeys (Table 2). Table 2. Antimicrobial activity showing MIC and CBM of fresh organic honeys against cariogenic microorganisms.

MIC (%, w/v) | MBC (%, w/v) Sample S. mutans S. gordonii S. salivarius S. oralis S. mitis NCTC S. sanguinis ATCC 700610 Challis ATCC 7073 ATCC 10557 12261 3K36

OH-1 15|20 15|20 15|20 15|20 20|25 15|20 OH-2 20|25 15|25 15|20 15|15 15|20 15|15 OH-3 10|20 20|25 20|25 15|15 15|15 15|15 OH-4 25|25 15|20 20|25 20|25 20|25 15|25 OH-5 30|30 25|30 25|30 20|25 20|20 25|30 OH-6 40|50 20|45 40|45 30|55 30|55 25|50 OH-7 20|25 20|25 25|30 15|30 25|25 25|25 OH-8 30|35 35|40 40|40 35|45 35|40 25|40

Legend: OH - organically produced honey plus sample number. Values expressed as a percentage of honey (w/v) diluted in culture medium.

After catalase treatment, the MIC of all eight honey samples for the six bacteria tested increased considerably. S. mutans was the bacteria less sensitive to honey with inactivated peroxide, being affected only by OH-5, OH-7, and OH-8, in high concentrations.

Table 3. Antimicrobial activity showing MIC and CBM of fresh organic honeys (treated with catalase) against cariogenic microorganisms.

MIC (%, w/v) | MBC (%, w/v) Amostra S. mutans S. gordonii S. salivarius S. oralis S. mitis NCTC S. sanguinis ATCC 700610 Challis ATCC 7073 ATCC 10557 12261 3K36

OH-1 >60|>60 55|60 60|>60 45|50 50|50 >60|>60 OH-2 >60|>60 >60|>60 60|>60 45|50 50|55 45|50 OH-3 >60|>60 >60|>60 45|50 60|>60 45|50 50|55 OH-4 >60|>60 60|>60 55|60 >60|>60 >60|>60 >60|>60 OH-5 45|50 45|55 55|60 >60|>60 50|55 55|60 OH-6 >60|>60 >60|>60 45|50 45|50 >60|>60 >60|>60 OH-7 45|50 45|50 55|60 >60|>60 55|60 55|60 OH-8 45|50 60|>60 >60|>60 >60|>60 45|50 55|60

46

The assay for antibiofilm activity, with an S. mutans biofilm with 48 h old, showed that all honeys can significantly reduce the number of viable cells in the biofilm (figure 1). The highlights are for the samples OH-1 (figure 1A), OH-2 (figure 1B), and OH-7 (figure 1G), which in the 2x CIM concentration were able to reduce 10 logs of biofilm, which means totally kill compared the control without treatment (p> 0.05). All other honeys, except OH-6 (figure 1F), also reduced 10 logs of biofilm to a concentration of 50% (w/v).

A B C D

15 15 15 15

a a a 3 10 10 10 a a a 10 10 a 10 10 a 10 logs c

5 5 5 5 CFU/mL - Log CFU/mL - Log

CFU/mL - Log 10 CFU/mL - Log 10 10 10 10 10 logs logs logs logs logs logs b b b b b c 0 0 0 0 C- 15 30 50 C- 15 30 50 C- 10 20 50 C- 25 50 OH-1 (% - p/v) OH-2 (% - p/v) OH-3 (% - p/v) OH-4 (% - p/v) E F G H

15 15 15 15

a 3 a

a a 10 10 10 10 3 logs a 10 10 10 10 -4 logs 4 b log b logs b b 5 5 5 5

CFU/mL - Log 10 CFU/mL - Log 10 CFU/mL - Log CFU/mL - Log 10 10 10 10 10 logs logs logs logs logs logs logs b b c c c c c 0 0 0 0 C- 30 50 60 C- 40 50 80 C- 20 40 50 C- 30 50 60 OH-5 (% - p/v) OH-6 (% - p/v) OH-7 (% - p/v) OH-8 (% - p/v)

Figure 1. Antibiofilm activity of eight samples of organic honey (OH-1 to OH-8) in MIC, 2x MIC and 50% (w/v) concentrations S. mutans ATCC 700610 monospecies biofilm. Different letters represent statistical difference. Antibiofilm activity of organic honeys against. SEM, in an increase of 5000 times, visually reproduces the results found in the UFC / mL count. It is possible to observe in the control without treatment (figure 2 A) a dense biofilm of S. mutans and extracellular matrix. The treatment of biofilm with honeys with better antibiofilm activity (OH-1, OH2, and OH-7) shows severe destruction in the biofilm structure, showing cell debris and empty spaces (figures 2B, 2C, and 2D). 47

Figure 2. Scanning electron microscopy showing the biofilm of S. mutans. Without treatment (A), treated with 2xCIM of OH-1 (B), treated with 2xCIM of OH-2 (C), and treated with 2xCIM of OH-7 (D). Arrows indicate some cellular deformations and destruction of the matrix. Discussion Honey is an ancient food, which has been part of the human's diet since its inception and for many years was used as a therapy for the treatment of wounds [3]. Currently, modern medicine has rediscovered the therapeutic potential of honey and inserted it even for therapies at the hospital level [12]. This space gain is linked, in large part, to the antimicrobial activity of honey [3]. Thus, honey has exhibited antimicrobial activity against bacteria, yeasts, and fungi, including strains resistant to different antibiotics [13]. This antimicrobial action can be bacteriostatic or bactericidal and has been reported as concentration-dependent [14,15]. Here we evaluate the antimicrobial activity of eight samples of Brazilian organic honeys on six oral microorganisms of the genus Streptococcus. All samples showed antimicrobial activity against the strains tested. The samples OH-1, OH-2, OH-3, and OH-7 were the ones that stood out the most, with MIC values of 15%, 10%, 20%, and 20% (w/v), respectively. A previous study evaluated the antimicrobial activity of local honey from India 48

against S. mutans MTCC, using the agar diffusion method. The results showed that 5%, 10%, 20%, and 40% showed no zone of inhibition. Only at the concentration of 60% of the honey solution, an average inhibition zone of 10.0 mm was observed [16]. Another study evaluated the antimicrobial activity of Hamadan honey, by diffusion on agar and found that honey was able to inhibit the growth of S. mutans in concentrations above 20% (w/v) and of Lactobacillus at a concentration of 100% [6]. Although we have not evaluated the action of our honeys against Lactobacillus, all honeys tested, with the exception of honeys 5, 6, and 8, presented MIC less than 20% for one or more analyzed cariogenic bacteria. The divergence in the results of the aforementioned studies may be related to both the composition of the honeys and the difference in the method of analysis. In the present study, the analysis of antimicrobial activity was determined by broth microdilution [9] to determine MIC. Despite being a fast and easy performance test, the diffusion in agar to define the antimicrobial activity of honey has several limitations, such as difficulty in diffusing the active components of honey due to high viscosity, low reproducibility, and inability to distinguish bactericidal from bacteriostatic activity [17]. Using the broth microdilution technique, a study evaluated the antimicrobial activity of Saudi Arabian honey against S. mutans [18]. The work found that the wells that contained honey in concentrations of 50, 25, 12.5, and 6.25% showed significantly less growth than the control with TSB medium. A spectrophotometer was used for reading and a specific MIC was not defined, nor was the CBM defined. In our study, we did a visual reading and from the well that did not show growth (turbidity), we considered the MIC. From this concentration, aliquots were plated on BHI agar to determine CBM (Table 2). The antimicrobial activity of honey, generally, is related to two main components: hydrogen peroxide produced by enzymes in honey, such as glucose oxidase, and/or phenolic compounds [3]. One way to assess which component present in honey is primarily responsible for antimicrobial action is to treat honey samples with catalase [11]. This enzyme degrades the sample's peroxide but not the other components. Comparing the results of MIC and CBM in tables 2 and 3 we can see the difference in the values. Table 3 shows the results of honeys previously treated with catalase before exposure with MIC and CBM values mostly higher than those in Table 2, which has the MIC and CBM values of the honeys without any interference. However, it is possible to observe in Table 3 that some samples for some bacteria showed inhibition capacity at concentrations close to 60% (w/v). These data suggest that the antimicrobial action of Brazilian organic honeys is mainly related to the peroxide-producing enzyme complex. Nevertheless, in higher concentrations, the other components of the honeys, 49

especially phenolic compounds, may have a discrete action. Besides, the high osmolarity of honey can also be linked to the persistence of antimicrobial action [17]. In a previous study, our research group showed significant antioxidant activity of the phenolic extracts from these honey samples analyzed in this study [8]. Furthermore, we identified the chemical profile of these honeys. The content of phenolic components in honey extracts in mg (GAE/g) was: 73.15 (OH-1), 59.79 (OH-2), 49.79 (OH-3), 52.20 (OH-4), 117.68 (OH-5), 84.08 (OH-6), 83.19 (OH-7) and 53.03 (OH-8). Four phenolic compounds were identified and quantified: ferulic acid, caffeic acid, rutin, and hesperidin in the samples [8]. Previous studies have shown the antimicrobial activity of these molecules against oral microorganisms, which may partly explain the antimicrobial activity of honeys after the inactivation of peroxides [19, 20, 21]. Few studies have evaluated the antibiofilm activity of honeys in a mature biofilm of an oral microorganism. Concerning the exploration of this activity, showing the action of peroxides as responsible for it, this is a pioneering study. All the organic honeys evaluated here showed a capacity to destroy the biofilm of 48 h of S. mutans. According to Albaridi, 2019 [13] honeys with MIC between 12.5 50% (w/v) have moderate antimicrobial potency. Therefore, we consider the most promising results those in which honeys at 2x the MIC and below 50% were able to destroy the biofilm. In this case, the most prominent honeys were OH-1 (30%), OH-2, and OH-7 (40%). Except for OH-6, all other honeys were able to cause total biofilm death in higher concentrations. There are divergences between studies due to variations in the method of analysis and in the units of concentration of honey. One study evaluated the antimicrobial activity of two samples of Manuka honey against S. mutans. The two varieties tested weakly inhibited the adhesion of S. mutans cells to a glass surface in sub-MIC concentration. One of the samples weakly inhibited biofilm formation at a concentration of 200 μg/mL, but more effectively at a concentration of 500 μg/mL. Although the results are interesting, the unit of measurement used in the study makes it difficult to compare it with other studies of honey antibiofilm activity. The standardized form of honey concentrations indicated for analysis is the percentage, either volume/volume or weight/volume. Concentrations in μg or mg per ml can make reproducibility difficult, due to the viscosity of this food [17]. For visualization purposes, we analyzed by SEM the antibiofilm activity of the most promising honeys (OH-1, OH-2, and OH-7) against S. mutans. SEM illustrates the results of the graphs of antibiofilm activity. Figure 2A show the robust biofilm without treatment or any structural changes. Figures 2B, 2C, and 2D show the biofilm treated with OH-1 (30%), OH-2 50

(40%), and OH-7 (40%), respectively. These percentage values represent twice the MIC concentration. It is possible to observe extensive destruction of the biofilm, with some cellular deformations and destruction of the matrix. The evidence that Brazilian organic honeys have antimicrobial activity and antibiofilm against oral microorganisms demonstrate that honey has the potential to be used as a substitute for quotidian sugar controlling the microbiota, besides having other benefits already known for this functional food for the organism.

Conclusion Brazilian organic honeys show promising antimicrobial activity against oral microorganisms and significant antibiofilm activity against mono-species biofilm of S. mutans. The amount of sugar in honey precludes the claim that honey is not a cariogenic food. However, compared to common dietary sugar, honey has great benefits. In addition to the advantages already known in the literature, such as increased physical and mental well-being, and the improvement of important biological functions, Brazilian organic honey can act to control oral microorganisms with the potential to reduce the risk of biofilm-dependent diseases.

References

[1]. Alqarni AS, Owayss AA, Mahmoud AA, Hannan MA. Mineral content and physical properties of local and imported honeys in Saudi Arabia. J Saudi Chem Soc [Internet]. 2014;18(5):618–25. Available from: http://dx.doi.org/10.1016/j.jscs.2012.11.009 [2]. Ozen AE, Pons A, Tur JA. Worldwide consumption of functional foods: A systematic review. Nutr Rev. 2012;70(8):472–81. [3]. Cooper R. Honey for wound care in the 21st century. J Wound Care. 2016;25(9):544– 52. [4]. Halpin RM, O’Connor MM, McMahon A, Boughton C, O’Riordan ED, O’Sullivan M, et al. Role of Streptococcus mutans in human dental decay. Eur food Res Technol. 2008;227(5):353–80. [5]. Roberts AP, Mullany P. Oral biofilms: A reservoir of transferable, bacterial, antimicrobial resistance. Expert Rev Anti Infect Ther. 2010;8(12):1441–50. [6]. Ahmadi-Motamayel F, Hendi SS, Alikhani MY, Khamverdi Z. Antibacterial activity of honey on cariogenic bacteria. J Dent (Tehran) [Internet]. 2013;10(1):10–5. Available from: 51

http://www.ncbi.nlm.nih.gov/pubmed/23724198%0Ahttp://www.pubmedcentral.nih.gov/artic lerender.fcgi?artid=PMC3666059 [7]. Atwa ADA, AbuShahba RY, Mostafa M, Hashem MI. Effect of honey in preventing gingivitis and dental caries in patients undergoing orthodontic treatment. Saudi Dent J [Internet]. 2014;26(3):108–14. Available from: http://dx.doi.org/10.1016/j.sdentj.2014.03.001 [8]. Silva CF, Rosalen PL, Soares JC, Massarioli AP, Campestrini LH, Semarini RA, et al. Polyphenols in Brazilian organic honey and their scavenging capacity against reactive oxygen and nitrogen species. J Apic Res [Internet]. 2020;59(2):136–45. Available from: https://doi.org/10.1080/00218839.2019.1686573 [9]. M. P. Weinstein BLZFRCMAWJAMNDGMEMJFDJHDWHJAHJBPMPJMSRBTMMTJDT. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically ; Approved Standard — Ninth Edition. Vol. 32, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standar- Ninth Edition. 2012. 18 p. [10]. Cai JN, Jung JE, Dang MH, Kim MA, Yi HK, Jeon JG. Functional relationship between sucrose and a cariogenic biofilm formation. PLoS One. 2016;11(6):1–12. [11]. Allen KL, Molan PC, Reid GM. A Survey of the Antibacterial Activity of Some New Zealand Honeys. J Pharm Pharmacol. 1991;43(12):817–22. [12]. Krishnakumar GS, Mahendiran B, Gopalakrishnan S, Muthusamy S, Malarkodi Elangovan S. Honey based treatment strategies for infected wounds and burns: A systematic review of recent pre-clinical research. Wound Med [Internet]. 2020;30:100188. Available from: https://doi.org/10.1016/j.wndm.2020.100188 [13]. Albaridi NA. Antibacterial Potency of Honey. Int J Microbiol. 2019;2019. [14]. Israili ZH. Antimicrobial properties of honey. Am J Ther. 2014;21(4):304–23. [15]. Nweze JA, Okafor JI, Nweze EI, Nweze JE. Comparison of Antimicrobial Potential of Honey Samples from Apis mellifera and Two Stingless Bees from Nsukka, Nigeria. J Pharmacogn Nat Prod. 2016;02(04). [16]. Patel HR, Ajith Krishnan CG, Thanveer K. Antimicrobial effect of honey on Streptococcus mutans – An in vitro study. Int J Dent Sci Res [Internet]. 2013;1(2):46–9. Available from: http://dx.doi.org/10.1016/j.ijdsr.2013.11.004 [17]. Piotr Szweda (March 15th 2017). Antimicrobial Activity of Honey, Honey Analysis, Vagner de Alencar Arnaut de Toledo, IntechOpen, DOI: 10.5772/67117. Available from: https://www.intechopen.com/books/honey-analysis/antimicrobial-activity-of-honey 52

[18]. Nassar HM, Li M, Gregory RL. Effect of honey on Streptococcus mutans growth and biofilm formation. Appl Environ Microbiol. 2012;78(2):536–40. [19]. Slobodníková L, Fialová S, Rendeková K, Kováč J, Mučaji P. Antibiofilm activity of plant polyphenols. Molecules. 2016;21(12):1–15. [20]. Nakamura K, Shirato M, Kanno T, Lingström P, Örtengren U, Niwano Y. Photo- irradiated caffeic acid exhibits antimicrobial activity against Streptococcus mutans biofilms via hydroxyl radical formation. Sci Rep. 2017;7(1):1–13. [21]. Gutiérrez-Venegas G, Gómez-Mora JA, Meraz-Rodríguez MA, Flores-Sánchez MA, Ortiz-Miranda LF. Effect of flavonoids on antimicrobial activity of microorganisms present in dental plaque. Heliyon. 2019;5(12).

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2.3 Artigo 3: Brazilian organic honey act as antimicrobial and decrease the bone resorption in periodontal disease

Scientific article to be submitted to the Food Chemistry (IF: 6.306).

Diego Romario da Silva, Bruno Bueno-Silva, Marcelo Franchin, João Marcos Spessoto Pingueiro, Severino Mathias Alencar and Pedro Luiz Rosalen.

Abstract Honey is a functional food that besides supplies nutritional needs, moreover, can prevent the occurrence of diseases and providing human health. It is well-known the antimicrobial activity of honey, however, there are few reports about the microbiota periodontal disease-related. Thus, we evaluated in vitro and in vivo the effect of the Brazilian organic honey in microbial components and the bone resorption in periodontal disease model. The microdilution method was used to define the MIC of all organic honey (OH) samples against Porphyromonas gingivalis. The in vitro antibiofilm activity was carried out using a 7- days subgingival biofilm of 32 species growing in the Calgary device. Biofilms were treated with OH twice a day for 1 minute, and from day 3 the biofilms received 2x and 10x MIC. The chlorhexidine (0.12%) and the vehicle was used as a positive and negative control, respectively. A chronic periodontal disease model induced by ligature and P. gingivalis using mice C57BL. All results were evaluated by definition of bone loss and biofilms microbial art the ligature by DNA-DNA hybridization. All OH samples showed antimicrobial activity against P. gingivalis, with MIC ranging from 2% and 7% (w/v) and CBM ranging from 3% and 11% (w/v), as well as antibiofilm activity. The OH-7 was selected due to exhibit the most promising antibiofilm activity. The OH-7 showed a reduction in microbial species growth in in vitro and in vivo models ranging color from the yellow, orange, and red complex as well as it was able to decrease bone loss in periodontal disease. The organic honey, OH-7, from Brazilian Atlantic Forest, has a promising antimicrobial activity, in vitro and in vivo, as well as on the reduction of bone loss triggered by this periodontal disease.

Keywords: Honey, periodontal disease, antibiofilm

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Introduction Natural products have been used since ancient times to treat human diseases, and the popular knowledge in traditional medicine provides information for the discovery of novel medicines and therapies from products of animal, plant, and mineral origin [1]. The honey is a food produced by bees, especially by Apis melífera, that is extensity used due to its several biological activities [2] The honey usage to treat wound and tissue lesions was a common medical practice in hospitals across Europe until the1970s, due to its antimicrobial [3] and anti-inflammatory activities [4], stimulation of tissue repair, deodorant action, and injury tissue debris [5]. Nowadays, modern medicine has rediscovered the honey therapeutic properties in the infection treatments in hospitalized patients, as an adjuvant and alternative therapy against resistant microorganisms [6-8]. The microorganisms-resistance to antibiotics has been increased and considered a public health problem. The pharmaceutical industry invests in research and development in novel medicines with an alternative mechanism of action, however, the microorganisms increasingly possess an arsenal to dribbles all obstacles by the medicine [9]. Thus, the search for alternative therapies, and sources to bioprospection of novel medicines, it must be stimulated [10]. Several studies have shown honey as a promisor antimicrobial in different microorganisms such as protozoa [11], fungi [12], and bacteria [13], causing human being disease. Periodontal disease is one of the most prevalent pathology in the oral cavity [14] and similar the dental caries-dependent, both are biofilm dependent. The consequences of periodontal disease are including tooth loss, inflammation, bad breath and difficulty to chew [16]. The tooth, supragingival and subgingival biofilm formation is crucial factor to establish and subsequently progression of periodontal disease [17]. The microbial colonization by the periodontal such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella spp; and together with the inflammatory process, is directly related to the he periodontal disease progression [18]. The orientation of prevention to periodontal disease include the toothbrush, dental floss, mouthwash, and frequent visit to the dentistry [16]. However, until this moment, there is no report to use functional foods as an alternative therapy to prevent or treatment to periodontal disease. Despite honey antimicrobial and anti-inflammatory properties has already been related to the scientific literature, the research of these biological activities regarding periodontal disease is incipient primarily about the antimicrobial activities against oral biofilms. 55

Thus, we evaluated in vitro and in vivo the effect of the Brazilian organic honey in microbial components and the bone resorption in periodontal disease model.

Material and methods Organic honey georeferencing, collection and extraction. Eight samples of certified honey produced organically (OH-1 to OH-8) were collected, certified1 and geographically referenced in a preserved Atlantic rainforest region from South Paraná State, in the city of General Carneiro (S 26° 25’, W 51° 19’) and the city Turvo (S 25° 2’, W 51º 32’) between the months of December 2015 and February 2016. The collection was carried out under the permission of the Brazilian Ministry of Environment (Council for the Administration and Management of Genetic Heritage–CGEN/ CNPq, # AE0DBB2). All organic honey (OH) samples were diluted in several concentrations using saline or culture medium and sterilized by filtration.

Polyphenol honey extraction

The polyphenol fraction was extracted follow the Ferreres, Tomaas-Barberaan, Gil and Tomaas-Lorente (1991) method with modifications [19]. The organic honey (OH-1 to OH- 8) was separated weighing 60g and dissolved in 300 mL of water. Later, the pH of the solution was adjusted at pH 2, and mixed then with 90 g of AmberliteVR XAD-2 resin (pore size 9 nm; particle size 0.3-1.2 nm; Sigma -Aldrich, St. Louis, MO, USA) for 15 min using a magnetic stirrer. Then, the mixture was packed in a glass column (40 cm 2 cm). The solution on the column was then eluted with 500 ml of acid water (pH 2.0) and then with 500 ml of neutral distilled water to remove the sugars. The phenolic compounds adsorbed on the solid phase were eluted with 500 mL of methanol and then concentrated on a rotary evaporator under low pressure at 50 C. Finally, all the organic honey extracts (OHE) obtained were lyophilized and stored at 22 °C until analyzes.

Microbial susceptibility

1Bee products produced and organically certified for the national market by the IMO of Brazil (Case No. 11- 0030.11) and for the export market NOP (USDA) and EEC (European) (Case No. 23511). 56

For the microbial susceptibility assay, the Porphyromonas gingivalis W83 strain was used as a periodontal pathogen. The organic honey was diluted in culture medium BHI plus TSB and yeast supplemented with heminm and menadione. The metronidazole, a mono drug, was used as a positive control. The final concentration in the wells were 60% to 10% (w/v). The microorganisms susceptibility was carried out by the microdilution technique, and to determine the Minimum Inhibitory Concentration (MIC) [20] and Minimum Bactericidal Concentration (MBC). These assays were conducted in 96-wells microplates.

Evaluation of antibiofilm activity

In vitro model of subgingival biofilm multispecies The in vitro model of subgingival biofilm multispecies was conducted and based on Soares et al. (2015) [21] e Miranda et al. (2019) [22]. All bacterial strains that consist of the multispecies biofilms are listed in table 1 above.

Table 1. List of microorganisms used to grow multispecies biofilms, organized by Socransky complexes.

Microorganisms of biofilm multispecies Actinomyces complex Actinomyces naeslundii ATCC 12104 Actinomyces oris ATCC 43146 Actinomyces gerencseriae ATCC 23840 Actinomyces israelii ATCC 12102

Purple complex Veillonella parvula ATCC 10790 Actinomyces odontolyticus ATCC 17929

Yellow complex

Streptococcus sanguinis ATCC 10556

Streptococcus oralis ATCC 35037

Streptococcus intermedius ATCC 27335

Streptococcus gordonii ATCC 10558

Streptococcus mitis ATCC 49456 57

Green complex

Aggregatibacter actinomycetemcomitans ATCC 29523

Capnocytophaga ochracea ATCC 33596

Capnocytophaga gingivalis ATCC 33624

Eikenella corrodens ATCC 23834

Capnocytophaga sputigena ATCC 33612

Orange complex

Streptococcus constellatus ATCC 27823

Eubacterium nodatum ATCC 33099

Fusobacterium nucleatum vincentii ATCC 49256

Parvimonas micra ATCC 33270

Fusobacterium nucleatum polymorphum ATCC 10953

Campylobacter showae ATCC 51146

Fusobacterium periodonticum ATCC 33693

Prevotella intermedia ATCC 25611

Red complex

Porphyromonas gingivalis ATCC 33277

Tannerella forsythia ATCC 43037 Other complexes

Eubacterium saburreum ATCC 33271

Streptococcus anginosus ATCC 33397

Selenomonas noxia ATCC 43541

Propionibacterium acnes ATCC 11827

G. morbillorum ATCC 27824

S. mutans ATCC 25175 58

All microorganisms were grown in specific solid culture medium and appropriate conditions for each strain. After 24 h, the bacterial strain was mixed with BHI (Becton Dickinson, Sparks, MD, USA) supplemented with 1% heminm for 24 h. The single microorganisms suspensions at 108 CFU/mL of cells (standardized by spectrophotometer) were diluted and 100 µl of the inoculum containing 106 cells were mixed with 11.8 ml of BHI broth supplemented with 1% heminm and 5% of sheep blood. The biofilm inoculum consisted of 15 ml with 106 cells of each bacterial strain. The multispecies biofilm formation was induced in a Calgary biofilm device (CBD) in 96-well microplate (Nunc; Thermo Scientific, Roskilde, Denmark). Aliquots of 150 µL of each inoculum were added to the wells, which corresponds to 104 cells of each bacterial strain. A top with polystyrene pins was used to seal the 96-well microplate (Nunc TSP system; Thermo Scientific, Roskilde, Denmark). The microplates were incubated at 37 °C under anaerobic conditions. After 3 days, medium consumed by the bacteria was replaced and the biofilms were maintained at 37 °C in anaerobic conditions for four days to obtain mature biofilms of seven days [21,22]. Organic honey treatment on biofilm

The mature biofilm was treated with organic honey diluted in BHI 3 days after the medium replacement. Also, after these 3 days, the mature biofilm received organic honey twice a day for more 4 consecutive days. The biofilm-coated pins were transferred to 96-well microplates containing diluted honey at 2x and 10x CIM, the dilution vehicle (negative control), and 0.12% chlorhexidine (Periogard, Colgate, positive control). The mature biofilm remained in contact with the treatments for 1 min, twice a day. After this procedure, the biofilm-coated pins were washed with PBS and returned to the same culture medium. The three independent experiments were carried out in triplicate. Metabolic biofilm activity and DNA-DNA hybridization (Checkerboard assay)

The metabolic activity of multispecies biofilm treated with honey, extracts, and controls were measured using 2,3,5-triphenyltetrazolium chloride (catalog number 17779; Fluka Analytical, Darmstadt, Germany) in a spectrophotometric. To measure the metabolic activity of biofilms, the coated pins were transferred to 96-well microplates with 200 µl of BHI medium supplemented with 1% heminm and 10% of TTC solution (1%). The microplates were incubated in anaerobic conditions for 24 hours at 37 °C. The reduction of TTC to red formazan was read at 485 nm using a spectrophotometer. Three biofilm-coated pins from each group were added in tubes, washed in PBS and mixed to 100 µL of TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 7.6)], followed by the addition of 100 µL of NaOH at 0.5 M. The solution above 59

was sonicated for 10 minutes, and then the solution was neutralized with 0.8 ml of ammonium at 5 M. The samples were individually analyzed in terms of the presence and quantity of 32 species bacteria, using the DNA-DNA hybridization technique [21]. Evaluation of organic honey potential in periodontal disease induced by ligature and P. gingivalis in mice.

Animals

Male SPF (specific-pathogen free) C57BL/6JUnib mice, purchased from CEMIB/UNICAMP (Multidisciplinary Center for Biological Research, SP, Brazil), weighing between 22 and 25 g. All animals were housed in vivarium under humidity (40–60%) and temperature (22 ± 2 ˚C) control in 12 h light-dark cycle, with access to food and water ad libitum. For the experiment, all animals were deprived of food for 8 h before oral administration. This study complied with the National Council for Animal Experimentation Control guidelines for the care and use of animals in scientific experimentation. The study protocol was previously approved by the Institutional Ethics Committee on Animal Research at the University of Campinas (CEUA/UNICAMP, Protocol Number 5348-1). Periodontal disease inducted by ligature model plus P. gingivalis W83

P. gingivalis W83 strain was cultivated in brain heart infusion (BHI, BD-Difco, Sparks, MD, USA), supplemented with 1 mg/ml of heminm, and 0.5 mg/ml of menadione, and incubated in anaerobiosis at 37º C for two days. To induce bone loss, a ligature 5-0 silk suture thread (Roboz Surgical Instrument Co., MD, USA) was previously contaminated with P. gingivalis (107 CFU/mL diluted in carboxymethylcellulose and PBS). This ligature-induced periodontitis was tied around the left upper second molar in mice. The ligature was gently tied avoiding damage to the periodontal tissue [23]. Subsequently, mice were inoculated with P. gingivalis (100 µL - 107 CFU/mL diluted in carboxymethylcellulose and PBS) 3 times a day until day 4 counting since the beginning of the treatment. Organic honey (OH-7) treatment

The experimental design was conducted as follow: 6 mice experimental group was divided: group 1 (no ligature and treated with NaCl); group 2 (with ligature and treated with NaCl); group 3 (with ligature and treated with 100 µL of OH-7 40% (w/v), orally 5x/day); and group 4 (with ligature and treated with 100 µL of OH-7 in natura 5 x/day). All mice were treated until day 7 and subsequently, they were euthanized.

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Bone loss measure

Mice jaws were added with 3% hydrogen peroxide and mechanically dissected. To distinguish the cement-enamel junction, the jaws were stained with 3% methylene blue. The face-palatal were photographed using 2.5 x extension using a stereoscopic microscope (ZEISS CL1500 ECO) and a digital camera (Canon E0S 1000D). The images were obtained and analyzed by the software Image J. For quantitative analysis, it was measured the distomesial area (mm2) between the cementoenamel junction and the alveolar bone crest on the palatal side of the first and second molars, as described by Prates et al., 2014 [24]. Evaluation of the tooth caries presence

The tooth caries decay on smooth and grooved surfaces and their severity (Ds, exposed dentin; Dm, 3/4 of the affected dentin; Dx, all affected dentin) were evaluated according to the Keyes system, modified by Larson (Larson 1981) [25]. The determination of the caries score was performed by a calibrated examiner. Statistical analysis The data were checked for normality and submitted to one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. The results shown not normality were submitted to Kruskal Wallis followed by Dunnett’s post-hoc test. The results are expressed as mean ± standard deviation (SD). The results were considered significant at p < 0.05 and α: 5%. Results

All organic honey sample shown antimicrobial activity against P. gingivalis with MIC values ranged from 2 to 7% (w/v) and MBC ranged from 3 and 11% (w/v). Considering the MIC and CBM values, OH-2 was the most promising sample with antimicrobial activity against P. gingivalis, whereas OH-6 and OH-8 were less effective (MIC and MBC high value) as shown in Table 1.

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Table 1. Antimicrobial activity (MIC and CBM) from eight Brazilian organic honey samples against P. gingivalis W83.

Honey samples and internal P. gingivalis W83 control MIC (% – µg/mL) | MBC (% – µg/mL)

OH-1 3.0 – 30000 |3.0 - 30000

OH-2 2.0 – 20000 |2.0 - 20000

OH-3 3.0 – 30000 |5.0 - 50000

OH-4 3.0 – 30000 |4.0 - 40000

OH-5 4.0 – 40000 |8.0 - 80000

OH-6 7.0 – 70000 |11.0 - 110000

OH-7 4.0 – 40000 |6.0 - 60000

OH-8 6.0 – 60000 |7.0 - 60000

Metronidazole 0.0000039 – 0.039 |0.0000078 – 0.078

The organic honey extracts (OHE) also exhibited antimicrobial activity against P. gingivalis, with MIC values ranged from 31.25 to 62.5 µg/mL, and MBC values ranged from 62.5 e 250 µg/mL, as seen in Table 2. Metronidazole, a gold standard effective against anaerobic microorganisms, was used as an internal positive control shown MIC and MBC values ranged from 0.039 and 0.078 µg/mL, respectively.

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Table 2. Antimicrobial activity (MIC and MBC) of Brazilian organic honey extracts (OHE) against P. gingivalis W83.

Honey extracts P. gingivalis W83 samples and MIC (% - µg/mL) | MBC (% - µg/mL) internal control

OHE-1 0.003125 – 31.25|0.00625 – 62.5

OHE-2 0.003125 – 31.25|0.00625 – 62.5

OHE-3 0.00625 – 62.5|0.0125 – 125

OHE-4 0.00625 – 62.5|0.0125 – 125

OHE-5 0.00625 – 62.5|0.025 – 250

OHE-6 0.003125 – 31.25|0.00625 – 62.5

OHE-7 0.00625 – 62.5|0.0125 – 125

OHE-8 0.003125 – 31.25|0.003125 – 31.25

Metronidazole 0.0000039 – 0.039|0.0000078 – 0.078

Regarding antibiofilm results, as seen in Figure 1, all organic honey samples showed in vitro antibiofilm activity against P. gingivalis when treated at 10x MIC. Also, at 10x MIC all organic honey samples decreased significantly the biofilm metabolic activity compared to the negative control. Interestingly, there was no statistical difference in terms of biofilm metabolic activity between the organic honey samples and those treated with the positive control, gold standard chlorhexidine (p>0.05). OH-7 at 8% (2x MIC) did not differ statistically when compared with chlorhexidine that shown the equivalent potency, however in 10x MIC.

63

150

a 100 b b b b b b 74,6% 50 c c c c 82,1% c c c c c c c Metabolic activity (%)

0 C- 6 30 4 20 6 30 6 30 8 40 14 70 8 40 12 60 0,12 OH-1 OH-2 OH-3 OH-4 OH-5 OH-6 OH-7 OH-8 CLX (%) Organic honey (OH) - 2 e 10xMIC (% - w/v)

Figure 1. Antibiofilm activity of eight organic honey samples in subgingival multispecies biofilm (32 microorganisms). Organic honey OH-1 (2xCIM 6% - 10xCIM 30%), OH-2 (2xCIM 4% - 10xCIM 20%), OH-3 (2xCIM 6% - 10xCIM 30%), OH-4 (2xCIM 6% - 10xCIM 30%), OH-5 (2xCIM 8% - 10xCIM 40%), OH-6 (2xCIM 14% - 10xCIM 70%), OH-7 (2xCIM 8% - 10xCIM 40%) e OH-8 (2xCIM 12% - 10xCIM 60%). The results are expressed in media ± DP in three different assays. Different letters indicate statistical difference and the symbol % indicates a decrease in metabolic activity. One-way ANOVA followed by Tukey’s post-hoc test, P < 0.05.

Since verified the antibiofilm activity of all organic honey samples, it was carried out the antibiofilm activity (10 and 20x MIC) with organic honey extracts sample. As seen in Figure 2, OHE-1, OHE-2, and OHE-7 at 20x CIM decreased significantly the biofilm growth, inhibiting by 24%, 25%, and 25% the biofilm viability, respectively (p<0.05).

150

a a a a a 100 a a a a a a a a 24% 24% 24% 25% b b b b 50 c Metabolicactivity (%)

0 C- 312,5 625 312,5 625 625 1250 625 1250 625 1250 312,5 625 625 1250 312,5 625 0,12 OHE-1 OHE-2 OHE-3 OHE-4 OHE-5 OHE-6 OHE-7 OHE-8 CLX (%)

Organic honey extract (OHE) (g/mL) - 10 e 20xMIC

Figure 2. Antibiofilm activity of eight organic honey extracts samples in subgingival multispecies biofilm (32 microorganisms). Organic honey extract OHE-1 (2xCIM 6% - 10xCIM 30%), OHE-2 (2xCIM 4% - 10xCIM 20%), OHE-3 (2xCIM 6% - 10xCIM 30%), OHE-4 (2xCIM 6% - 10xCIM 30%), OHE-5 (2xCIM 8% - 10xCIM 40%), OHE-6 (2xCIM 14% - 10xCIM 70%), OHE-7 (2xCIM 8% - 10xCIM 40%) and OHE-8 (2xCIM 12% - 10xCIM 60%). The results are expressed in media ± DP in three different assays. Different letters indicate 64

statistical difference and the symbol % indicates a decrease in metabolic activity. One-way ANOVA followed by Tukey’s post-hoc test, P < 0.05.

The results regarding biofilm metabolic activity shown the OH-7 as a most promising sample and then the OH-7 was selected for checkerboard analysis. As seen in Figure 3, the biofilm multispecies treated with OH-7 (40%) and chlorhexidine (0.12%) had a decrease of biofilm viability by 89% and 86%, respectively as compared to the negative control (p>0.05).

250 a )

5 200

150

100 of bacteria of

Total counts (x10 counts Total 50 b b 0 C- 40 CHX OH-7 (% - w/v)

Figure 3. Total bacteria count. Total bactéria count (105) in biofilm treated with negative control (vehicle; C-), OH-7 at 40% and chlorhexidine at 0.12%. The results are expressed in media ± DP in three different assays (N¼ 9). Different letters indicate statistical difference and the symbol % indicates a decrease in Total bacteria count. (Kruskal Wallis followed by Dunnett’s post hoc test, P < 0.05).

Since verified the biofilm viability, it was carried out the DNA-DNA hybridization (Checkerboard assay). As seen in Figure 4, the OH-7 at 40% reduced in 89% the total biofilm bacteria count and did not show statistical difference in relation to the positive control chlorhexidine (p>0.05) Figure 4 represents the media count of each bacteria strain based on the DNA-DNA hybridization (Checkerboard assay). The treatment with OH-7 (40%) decreased by 89% the total bacteria count on biofilm (p<0.05) and did not show statistical difference in relation to positive control chlorhexidine (p>0.05). In addition, the treatment with OH-7 (40%) decreased the levels of 13 bacterial species as follow: S. gordonii, S. mitis, V. parvula, C. showae, F. n. Vinc, P. micro, P. intermetia, S. costellatus, P. gingivalis, P. forsythia, S. anginosus, S.mutans and S. noxta compared to the vehicle group. Chlorhexidine positive control group reduced levels of 15 bacterial species as follow: S. intermedius, S. gordonii, S. mitis, V. parvula, C. achracea, C. showae, F. n. Vinc, P. micro, P. intermetia, S. costellatus, P. gingivalis, P. forsythia, S. anginosus, S.mutans and S. noxta. The bacterial species market in red such as P. 65

gingivalis e T. forsythia, showed the lower levels after the OH-7 treatments, as seen in Figure 4.

Figure 4. Media count of each bacteria strain (105) based on the Socransky's bacterial complexes. Bacterial complexes colored in yellow, purple, Actinomyces, green, orange, red and others present in the ligature. Mice were treated with the vehicle, OH-7 (40%) and chlorhexidine (0.12%). The data were analyzed by Kruskal Wallis followed by Dunnett’s post hoc test for each bacterial species separately. The letter (a) indicates statistical difference between biofilms treated with OH-7 and biofilms treated with vehicle. The letter (b) indicates statistical difference between CHX-treated biofilms and vehicle-treated biofilms. 66

Given the results obtained in vitro concerning antimicrobial activity and biofilm multispecies formation, we further investigated the in vivo activity of OH-7 in a periodontal disease model. As seen in Figure 5B, the image represents the differences at the mice hemimandibles in a periodontal disease induced by ligature and P. gingivalis inoculum. As seen in figure 5A, the ligature-induced periodontitis plus inoculum of P. gingivalis in mice showed a reduction of bone absorption when treated orally with OH-7 at 40% (p<0.05). The treatment OH-7 in natura given orally did not affect statistically compared to the control group.

Figure 5 AB. A) General measurement of distance CEJ-ABC to determine the bone loss. Mice treated with negative control (vehicle; C-), OH-7 at 40% and in natura (100%). The results are expressed in media ± DP and different letters indicate statistical difference (ANOVA One-away - pos teste Tukey’s post hoc test, p<0,05). B) Mice hemimandibles photograph in a periodontal disease induced by ligature and P. gingivalis inoculum. The image shown a control group with absence the disease and untreated (I), control group with presence of the disease and untreated (II), group with presence of the disease and treated with OH-7 at 40% (III), and group with presence of the disease and treated with OH-7 in natura (IV).

As seen in Figure 6, shows the total of microorganisms counted present in the ligature of each group in the periodontal disease model. The mice treated with OH-7 at 40% decreased the total of microorganisms counted as compared to the control group (p<0.05). In contrast, the mice group treated with OH-7 in natura showed no significant difference compared to the control group or the group treated with OH-7 at 40% (p> 0.05). 67

40 a 30

20 of bacteria of 10 ab Total counts (x105) counts Total b

0 Ligature 40 100 OH-7 (% - w/v)

Figure 6. Total bacteria count (105) at the ligature-induced periodontitis. Mice untreated (ligature group), treated with OH-7 at 40%, OH-7 in natura (100%). The results are expressed in media ± DP and different letters indicate statistical difference (Kruskal Wallis followed by Dunnett’s post hoc test, p<0,05).

As seen in Figure 7, the total bacteria counted separated by Socransky complexes was reduced after the treatment with OH-7 at 40% compared to the control (p<0.05). Interestingly, there was a significant reduction of the total bacteria counted in the red complex in bacteria treated with OH-7 at 40% and also in the group treated with OH-7 in natura (p <0.05). In the caries scores analysis, as described by Larson, 1981, none mice of the groups were affected by caries lesion and/or white spot.

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DNA-DNA hybridization bacterial counts

COUNTS OF EACH ESPECIE (X105)

0,0 0,1 1,0 10,0 100,0 A. gerencseriae A. israelli A. naeslundii A. oris V. parvula A. odontolyticus S. gordonii S. intermedius S. mitis S. oralis S. sanguinis A. actinomycetencomytans C. gingivalis C. ochracea C. sputigena E. corrodens C. gracilis C. rectus C. showae E. nodatum F.nucleatum (sp. nucleatum) F.nucleatum (sp. polymorphum) F.nucleatum (sp. Vincentii) F. periodonticum P. micra P. nigrescens S. constellatus P. intermedia T. forsythia P. gingivalis T. denticola E. saburreum G. morbillorum L. buccalis P. acnes P. melaninogenica N. mucosa S. anginosus S. noxia T. Socransky

Ligature OH-40% OH-100%

Figure 7. Media count of each bacteria strain (105), present in the ligature, based on the Socransky's bacterial complexes. Bacterial complexes colored in yellow, purple, Actinomyces, green, orange, red and others present in the ligature. Mice were treated with the vehicle, OH-7 (40% w/v) and OH-7 in natura (100%). The data were 69

analyzed by Kruskal Wallis followed by Dunnett’s post hoc test for each bacterial species separately. The letter (a) indicates statistical difference between group treated with OH-7 at 40% and vehicle-treated group. The letter (b) indicates statistical difference between OH-7 in natura and vehicle-treated group.

Discussion

Honey properties are described and use since the beginning of civilization [2]. Several studies have described the antibacterial activity of this functional food against important bacteria causing the systemic infection, such as Staphylococcus aureus (MRSA) [26], Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Shigella flexneri, and Staphylococcus epidermidis [27]. However, there is a lack of studies in the scientific literature regarding honey's antimicrobial activity against oral microorganisms and, specifically, pathogens of the periodontal region. Herein, we carried out a bio-guided screening with eight Brazilian organic honey samples from the Atlantic rainforest, and their extracts against P. gingivalis W83 strain. All samples analyzed, honey and extract, have shown antimicrobial activity against P. gingivalis. The best MIC values were for OH-2 (2%), OH-1, OH-3 and OH- 4 (3%), OH-5 and OH-7 (4%). These results suggested the organic honey as a promising functional food against pathogens of the periodontal region, as P. gingivalis, which justify the investment for research of this food against bacterial-components involved in periodontal disease etiology. In a study, the authors evaluated the Manuka honey antimicrobial activity against different strains of P. gingivalis (ATCC 33277, M5-1-2, MaRL e J361-1) [28] and the study revealed MIC value 2% for all bacteria strain. In another study using Manuka and white clover honey, the authors evaluated the antimicrobial against P. gingivalis (ATCC 33277) and shown MIC value 12.5% for both honey samples [29]. The Manuka honey is originally from New Zealand that has been considered a reference due to its antimicrobial activity no-related to peroxides and used in therapy due to medical degree [30,31]. Herein, Brazilian organic honey studied showed MIC values can be considered promising against P. gingivalis, since they are equal until better than values founded on Manuka honey against this microorganism. In two other studies using Egyptian honey [32], Manuka and a poly floral honey variety [33] against oral cavity microorganisms, including P. gingivalis. The authors founded positive results; however, agar diffusion method was utilized becoming difficult to compare results. It was already reported that the agar diffusion method is not recommended to evaluate honey antimicrobial activity that due to its high viscosity, there are 70

problems such as corrected volume into the wells and diffusion of the actives components, which become the results uncertain [34]. Honey antimicrobial activity has been known a long time ago, nowadays there is no consensus toward scientists regarding which methodology must be followed for describing this activity [34]. The antimicrobial activity of a novel medicine is just one of the initial steps of the bio-guided studies to signaling the discovery of novel substances and compounds with the potential for antimicrobial use [35]. Nonetheless, with the antimicrobial resistance increasing and the aggressively of biofilm-dependent disease, the evaluation of the antibiofilm activity for the medicines candidates to antimicrobial therapy is a need [36] since the microorganism in biofilm form is organized in terms of architecture and integrity, leading to became until 1000 times more resistant [37]. Biofilms are dense microorganisms community that grows on a surface and encapsulates with high weight molecular polymer release by them. Since the bacteria develop their biofilm, they are capable to adapt to certain environmental changes creating its gene expression patterns becoming highly resistant to antimicrobials [38]. Thus, the evaluation of the antibiofilm activity from antimicrobial honey is essential to characterize this functional food as a protector agent or therapeutic against biofilm-dependent disease, as a periodontal disease [39]. The honey antimicrobial activity evaluation against oral mature biofilms is scarce in the scientific literature. Nowadays, there is one published study regarding the antibiofilm activity of Manuka honey and poly floral honey from Germany against mature biofilm P. gingivalis [28]. The authors have shown that at 10% both honey inhibited P. gingivalis biofilm formation and decrease the bacteria viability into the biofilm for 42 h. However, there is no total prevention on biofilm formation and neither complete elimination of the biofilm [28]. In another study, the authors evaluates the S. mutans formation biofilm by honey acquired in supermarket from Saudi Arabia [40]. The results showed that the honey at 25 and 50% significantly decreased the growth of S. mutans biofilm in polystyrene plate [40]. In this sense, is important to emphasize that there are no reports on antimicrobial activity against multispecies biofilm or subgingival, and then it seems uncertain to state that the honey intake can act as protecting from the biofilm-dependent oral disease. Herein, we evaluate the antibiofilm of eight samples of Brazilian organic honey (OH-1 to OH-8). This is the first study investigating the antibiofilm activity of this functional food and providing important information regarding their activity against multispecies subgingival biofilm (32 species). The multispecies biofilm model, performed on a Calgary 71

device, was previously tested by our research group to analyze the antibiofilm potential of bee products and red propolis samples [22]. In our assays, we considered the concentration to treat the biofilm based on the MIC value of each OH their respective extracts for P. gengivalis (2 and 10x MIC for honey, and 10 and 20x MIC for the extract for each honey). The treatments were carried out twice a day, 1 minute for four days, respecting six hours between the treatments. According to our results, all honey samples at 10x MIC value significantly diminished the overall metabolic biofilm activity and there was no statistical difference with the positive control (CLX). Thus, given the strong antimicrobial activity that in lower concentration (2x MIC 8%) decreased the biofilm viability no differing with the gold standard, we selected the OH-7 to continue the subsequent assays. Regarding organic honey extracts, the OHE-1, 2, 5, and 7 showed a slight reduction of the biofilm viability as compared to the organic honey. These different results obtained are explained by how the extract can be obtained as described by Ferreres et al., 1991 [19]. This technique removes excess sugar and the protein component, such as the enzyme complex that produces peroxides. Thus, considering that there is a discrete antimicrobial activity by organic honey extracts compared to the organic honey, we hypothesize that the organic honey showed antimicrobial activity due to the presence of peroxide produced by the enzymatic complex in honey chemical composition. Also is highly likely that the biological activity can be due to a synergic effect among the enzymatic complex and the presence of the phenolic components (the activity of the extracts). Previously, our research group has studied the antioxidant activity of these eight organic honey samples (OH-1 to OH-8) and also identify the main chemical compounds of honey crude extracts. The total phenolic content founded at honey extracts (mg - GAE/g) was 73.15 (OH-1), 59.79 (OH-2), 49.79 (OH-3), 52.20 (OH-4), 117.68 (OH-5), 84.08 (OH-6), 83.19 (OH-7) e 53.03 (OH-8). In total, four phenolic compounds were identified in the samples such as ferulic acid, caffeic acid, rutin, and hesperidin. Besides that, we identified considerable amounts of ascorbic acid ranged 2.75 to 6.22 mg/100g at OH-3, OH-5, and OH-7 [41]. An expressive antimicrobial activity has previously been demonstrated for the compounds identified in organic honey samples [42–45]. Thus, these components may act as antimicrobial and the relation of the honey in natura components seems to be synergistically between peroxides and phenolic compounds. This synergism gives this Brazilian food an expressive antibiofilm activity in subgingival biofilm, different from phenolic extracts. 72

In the honey and extract chemical composition context, a study evaluated the presence and organization of the compounds present on this functional food. The authors conclude that there is a biphasic conformation on honey and this system is a requirement for hydrogen peroxide (H2O2) production and, consequently, the antibacterial activity [46]. The same research group has suggested that the honey arrangement of active macromolecules, organized jointly and stable with colloidal properties, is a remarkable discovery about the honey structure conformation and it is essential to understand the biological activities that this structural complexity can interfere [46]. Based on our results, the study was continued only using organic honey, since the antimicrobial and antibiofilm activities of the extracts were not significant. In the search for a better understanding concerning the honey anti-inflammatory activity against different species in subgingival biofilm, we carried out the hybridization DNA- DNA assay at biofilm in vivo samples aiming to verify the specific reduction based on Socransky complexes. It was analyzed biofilms treated with OH-7 at 40%, chlorhexidine at 0.12%, and the dilution vehicle. Studies have shown that periodontal disease, in the microbiological context, is initiated in a periodontal dysbiotic environment [47]. The development of dysbiosis occurs over time modifying gradually the semiotic relation microorganism-host to a pathogenic pattern. The orange complex is the first one related to the dysbiosis and disease establish, follow by the red complex that means the biofilm becoming mature and disease progression [48]. Also, it is well known that P. gingivalis stick to another pathogen from the yellow complex, as S. gordonii, leading to virulence increase and bone loss as a consequence [49]. Our results of hybridization DNA-DNA showed an expressive reduction of 14 species from bacteria biofilm from 34 species at the total when treated with OH-7 at 40% compared to the control. We highlighted the biofilm reduction from the red complex as follows T. forsythia, P. gengivalis, P. intermedia, P. micros, and F. nucleatum, and orange complex S. gordonii, which are closely related to environmental dysbiosis that is responsible to establish the disease began [47-50]. The reduction of total bacteria count of yellow, orange, and red complexes have indicated that honey consumption can control the growth of these microorganisms and, consequently, prevent the establishment and periodontal disease progression. However, despite the in vitro results be promising, it is necessary to prove the in vivo effectiveness. Then, we conducted the periodontal disease model in mice to verify the OH- 7 efficacy to prevent periodontal disease. 73

Periodontal disease assay was conducted as previously described by Abe and Hajishengallis 2013 [23], with modifications. The author used only the ligature silk suture thread on the mice' first molar. However, in our study, besides the ligature silk suture thread we expose the mice to a P. gingivalis inoculum of 107 for 48h under anaerobic conditions and for four consecutive days mice received 100 µL of P. gingivalis. In another study, the authors showed that the association between P. gingivalis and the ligature silk suture thread in the periodontal model induces in an exacerbated manner bone resorption dependent on RANKL protein (kappa B nuclear factor activator receptor ligand) [51]. We also verified that the daily treatment with OH-7 at 40% five times a day decreased 34% of bone loss compared to the untreated group (Figure 5B). However, the group that received honey in natura did not show statistical difference compared to the untreated group. The treatment frequency establish was based on a balanced diet to those who feed three regular meals and one light meal, totaling five times honey feed a day. In a representative image of each experimental group (Figure 5B) is possible to verify the anatomical preservation of the bone crest alveolar at the furcation area. The damage present in the furcation area with severity is an indicator of disease progression. Furthermore, in a general population who does not regularly periodontal treatment is increase the risk of molars loss involving the furcation area [52]. Also, after the conclusion of the periodontal assay, a dental caries score was determinate in a double-blind trial by a calibrated examiner, as described by Larson, 1981 [25]. None mice of the groups were affected by caries lesion and/or white spot. These findings indicate that despite the honey is considered a safe functional food rich in carbohydrates [2], the rational feed and for short time is safety. Ligature silk suture thread was carefully collected to perform the DNA-DNA hybridization assay. As shown in Figure 7, the group that received treatment with OH-7 at 40% had a significant decrease of S. gordonii, S. sanguinis, F. nucleatum, P. gingivalis and Prevotella melaninogenica corroborating with the effects observed in in vitro assay of multispecies subgingival biofilm. In the group treated with OH-7 in natura, P. gingivalis was the only strain with a significant reduction as compared to the control group. Several studies have related the presence and virulence of P. gingivalis to an exacerbated increase in bone loss in periodontal disease [53,54]. Thus, the reduction of bone loss in the OH-7 group may be related to a decrease of P. gingivalis biofilm viability and other pathogens of the periodontal region. Besides that, the presence of these microorganisms stimulates the immune system host to release inflammatory mediators that stimulate osteoclast activity [55]. 74

Surprisingly, the OH-7 at 40% showed more effect than honey in natura. This effect may be related for both production of H2O2 by the glucose oxidase enzyme and to the honey viscosity as well. The formation of H2O2 products depends on the honey dilution rate. The glucose oxidase enzyme is inactive in in natura honey [56] and its activation varies according to the honey dilution [57,58]. Previous studies have shown a strong correlation between the level of H2O2 products and the honey inhibition of the bacterial growth [56]. It has been shown that even maintain high dilutions, the presence of H2O2 continues in the solution and inhibits in different levels the microbial viability [59]. Thus, we hypothesize in natura honey did not show its glucose oxidase enzyme activated in enough level to produce peroxide in quantities capable to exert antimicrobial activity. Nevertheless, the honey viscosity may obstruct the biological compounds to reach the ligature region effectively. Another important point to consider is the mice self-cleaning movement behavior with their paws that can interfere with the honey reach to ligature region. Conversely, mice treated with

OH-7 at 40%, that there is the presence of glucose oxidase enzyme to produce H2O2, and is fluid honey to reach to ligature region, did not show the self-cleaning movement behavior. It was no possible to find in the scientific literature other studies that evaluated the honey biological activity specifically in the periodontal disease in vivo model. In our study, the honey treatment beginning after the fourth day of ligature silk suture thread installation, which is the period when bone resorption begins [23]. Take into consideration the honey is a food and not a medicine, it is possible that OH-7 can reduce the risk of onset periodontal diseases and be included in a rational way in the diet of healthy individuals. In a unique study [32], the authors verified the Egyptian honey activity on the microbial reduction species in orthodontic patients. Patients were asked to chew and ingest 10 g of Egyptian honey in natura for 2 minutes. The total bacteria count was significantly decreased after 30 minutes of exposition. Despite these promising results, there is no in vivo assay to show which microorganisms were affected by the honey consumption or the clinical consequences. Thus, until this moment, there is a lack of scientific to response the honey potential to prevent and/or treat the biofilm-dependent oral diseases. Altogether, clinical trials are necessary with healthy patients to verify the oral microbiota modifications and subsequently a study with patients affected by the periodontal disease to analyze their clinical, microbiological, and inflammatory conditions to then to able to indicate safety and active honey use.

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Conclusion

Collectively, our findings showed that OH-7, Brazilian organic honey from the Atlantic Forest, has a promising antimicrobial activity, in vitro and in vivo, against microbial components that trigger periodontal disease, as well as on the reduction of bone loss. Thus, the findings presented herein may open avenues for the rational consumption of this honey variety and the application as an alternative for the prevention and/or treatment of biofilm-dependent oral diseases.

References

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2.4 Artigo 4: Anti-inflammatory activity of organic honey from Brazilian Atlantic rainforest

Scientific article to be submitted to the Journal of Apicultural Research (IF: 1.8). Diego Romario da Silva, Josy Goldoni Lazarini, Marcelo Franchin, Severino Mathias Alencar and Pedro Luiz Rosalen. Abstract This study evaluated, in vitro and in vivo, the anti-inflammatory mechanism of action of 8 types of organic honey (OH-1, OH-2, OH-3, OH-4, OH-5, OH-6, OH-7, OH-8) from the Atlantic Rainforest. To determine the cell viability, macrophages RAW 264.7 were treated with several concentrations with OH-1 to OH-8. The anti-inflammatory activity was also determined by NF-kB activation and release of TNF-α levels in LPS-stimulated macrophages treated with OH-1 to OH-8 at 4, 2, 1 e 0.5% (w/v). It was selected the most promising OH and then the anti-inflammatory activity was evaluated in vivo through neutrophil migration and release of TNF-α levels. As result, the OH-1 to OH-8 did not interfere in macrophages viability up to 4% (w/v). All OH samples were capable to decrease the NF-kB activation and release of TNF-α levels in LPS-stimulated macrophages (p>0.05), with emphasis, the OH-7 was the most promising decreasing 93.8% and 59% the NF-kB activation and release of TNF-α levels (respectively; p<0.05). Regarding in vivo assays, mice pre-treated orally at 100% of OH-7 had a decrease of neutrophil migration (49%) and TNF-α levels (69%) compared to the carrageenan- challenge group (p<0.05). The Brazilian organic honey has shown a promising anti- inflammatory activity highlighting the variety OH-7. Therefore, this Brazilian functional food may lead to the development of new products for human health. Keywords: Honey; anti-inflammatory, neutrophil migration; cytokine. 81

Introdução During the Stone Age, several paintings record indicate the honey used in medicine for more than 8,000 years. Old documents such as parchment, bible, koran, Egyptian papyrus, and others, have described the honey treatment for diseases [1, 2]. The honey chemical composition varies according to local flora visited by the bees and also under the effect of seasonality. In general, honey is a mixture of sugar such as glucose, fructose and sucrose, minerals, vitamins, enzymes, amino acids, organic acids, and phenolic compounds. This complex composition of the honey food directly reflects upon the biological activities. For years, it is have been reported to honey relevant biological activities such as antimicrobial, anticancer, tissue repair, antioxidant and anti-inflammatory [3]. The inflammation is an immune system response to determinate agents such as pathogens, damaged cells, toxic compounds, irradiation, and others. The inflammation's main objective is to remove these damaged agents through cellular and molecular responses to restore the organism's homeostasis [4]. Nonetheless, the non-control acute inflammation may contribute to the establishment of the inflammatory disease, becoming chronicle [5]. Some diseases such as arthritis, asthma, cancer, neurodegenerative diseases, and inflammatory bowel disease are an example of pathologies related to inflammatory response uncontrolled and exacerbated [6, 7]. The inflammatory process is characterized by several mediators releases, including cytokines such as interleukin 6 (IL-6), IL-12, tumoral necrosis factor - α (TNF-α), interferon (INF-c), enzymes as cyclooxygenase-2 (COX-2) and inductive nitric oxide syntax (iNOS) [4,7]. In recent years, the nuclear factor kappa B (NF-kB) has been extensively studied due to perform an important role in the inflammatory response, acting on the regulation and inflammatory genes expression, e.g. cytokines [8]. Hence, the inhibition or a regulation of this nuclear factor or cytokines by a natural product e.g. honey, is an interesting strategy to develop new medicines and products prevent diseases inflammatory-related. In this context, it is need the development of new anti-inflammatory or therapy since the non-steroidal anti-inflammatory drugs (NSAID) and corticosteroids may promote certain adverse effects such as gastrointestinal, renal, and cardiovascular disorders [9,10]. The gastrointestinal injuries NSAID-induced can remain asymptomatic and the extensive use of NSAID to treat inflammatory diseases can lead to gastrointestinal bleeding (50%) or perforations (71%) [11]. In addition, the adverse effects of corticosteroids are also related to dermatological and ophthalmic alterations, Cushing’s syndrome, cardiovascular and gastrointestinal complications, as well as muscular bone pathologies [12]. Thus, natural products may be a novel source of compounds that can control inflammation and the anti- 82

inflammatory activity of honey has been considered as a promising source by our research group [13-16]. Several kinds of honey from different countries have been associated with a promising anti-inflammatory activity and this effect is associated with a significant amount of phenolic compounds [17–20]. In this regard, the honey has been related as a functional food with anti-inflammatory activity and due to be a natural product, is free of adverse effects compared with NSAID and corticosteroids [19]. However, there is no study elucidating the mechanism of action of the honey produced organically. Several studies have been related that products produced organically, shown a highest quantity of antioxidant compounds (vitamins, polyphenols, flavonoids), and minerals. These compounds which are present in organic products, may perform more significant biological activities [21,22]. The differences of bioactive compounds levels in a plant are attributed to adverse environmental conditions without the use of pesticides, resulting in greater production of secondary metabolites [23]. Thus, this study evaluated, in vitro and in vivo, the anti-inflammatory mechanism of action of 8 types of organic honey (OH-1, OH-2, OH-3, OH-4, OH-5, OH-6, OH-7, OH-8) from the Atlantic Rainforest

Material and methods

Organic honey georeferencing, collection and extraction. Eight samples of certified honey produced organically (OH-1 to OH-8) were collected and geographically referenced in a preserved Atlantic rainforest region from South Paraná State, in the city of General Carneiro (S 26° 25’, W 51° 19’) and the city Turvo (S 25° 2’, W 51º 32’) between the months of December 2015 and February 2016. The collection was carried out under the permission of the Brazilian Ministry of Environment (Council for the Administration and Management of Genetic Heritage–CGEN/ CNPq, # AE0DBB2). All organic honey (OH) samples were diluted in several concentrations using saline or culture medium and sterilized by filtration. Anti-inflammatory activity

Cell culture and viability (MTT)

RAW 264.7 macrophages (ATCC1 TIB-71™) transfected with the NF-κB-pLUC gene to express luciferase were cultured in endotoxin-free Roswell Park Memorial Institute 83

(RPMI1640) medium supplemented with 10% fetal bovine serum (FBS) plus 100 U/mL , 100 μg/mL streptomycin sulfate and L-glutamine (37 °C, 5 % CO2). To determine the cytotoxic effects of OH samples, macrophages were cultured in 96-well plates (5 × 105 cells/mL) and incubated for 24 h prior to treatment with OH-1 to OH-8 at 1, 2, 4 and 6 % (w/v) or culture media (negative control). After this period, the supernatant was removed and the MTT solution (0.3 mg/mL) was added to the wells. The plates remained incubated for 3 h (37 ˚C, 5% CO2). The supernatant was removed and DMSO was added. Absorbance was measured at 570 nm using an ELISA microplate reader [24]. NF-κB activation and TNF-α levels

Macrophage cells transfected were cultured in 24-well plates (3×105 cells/mL) overnight. The cells were treated with OH-1 to OH-8 at 0.5, 1, 2 and 4% (w/v) for 30 min before LPS stimulation (10 ng/mL) for 4 h. The culture media control was used as negative control. After 4 h, cell lysis buffer and 25 μL of luciferase reagent (luciferin at 0.5 mg/mL) were added to each well. Luminescence was measured using a white microplate reader (SpectraMax M3, Molecular Devices). For the TNF-α levels measure, the supernatant was recovered and determined according to the protocols provided by the manufacturers (R&D Systems, Minneapolis, MN, USA) and determined using an ELISA microplate reader. The results were expressed as pg/mL [25]. In vivo anti-inflammatory study

Animals To determine the OH capacity to decrease the neutrophil influx into the inflamed site, male SPF (specific-pathogen free) C57BL/6JUnib mice, purchased from CEMIB/UNICAMP (Multidisciplinary Center for Biological Research, SP, Brazil), weighing between 22 and 25 g. All animals were housed in vivarium under humidity (40–60%) and temperature (22 ± 2 ˚C) control in 12 h light-dark cycle, with access to food and water ad libitum. For the experiment, all animals were deprived of food for 8 h before oral administration. This study complied with the National Council for Animal Experimentation Control guidelines for the care and use of animals in scientific experimentation. The study protocol was previously approved by the Institutional Ethics Committee on Animal Research at the University of Campinas (CEUA/UNICAMP, Protocol Number 5348-1).

Neutrophil migration into the peritoneal cavity of mice and TNF-α levels 84

Mice were pre-treated orally (via gavage) five times a day during five days with 100 μL of OH-7 at 40% and 100 μL of OH-7 in natura (100%). The negative control group received oral administration of 0.9% saline (vehicle). After five days, all animals received an inflammatory challenge by intraperitoneal (i.p.) injection of the carrageenan (500 μg/cavity) in the peritoneum for 4 h, except the vehicle group. After 4 h upon challenge, mice were euthanized and their peritoneal cavity was washed and recovered to count for the total number of leukocytes and neutrophils. The volumes recovered were similar in all experimental groups and equated to approximately 95% of the injected volume. The results were expressed as number of neutrophils per cavity. For measure the release of TNF-α levels, the peritoneal fluid was recovered and determined according to the protocols provided by the manufacturers (R&D Systems, Minneapolis, MN, USA) and determined using an ELISA microplate reader. The results were expressed as pg/mL [26].

Statistical analysis The data were checked for normality and submitted to one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. The results were expressed as mean ± standard deviation (SD). The results were considered significant at p < 0.05 and α: 5%. Results

As seen in Figure 1, the macrophages were pre-treated with honey did not significantly affect cell viability up to 4 % (Fig 1A to 1H). However, all samples induced significantly cell cytotoxicity at 6 % as compared to the culture media control (p<0,05). 85

A B C 120 120 120 a a a a a a a a a a a a 80 49% 80 80 b 51% 61% b b 40 40 40 Cell viability (%) Cellviability (%) Cell viability (%)

0 0 0 M 6 4 2 1 M 6 4 2 1 OH-1 (% - w/v) M 6 4 2 1 OH-2 (% - w/v) OH-3 (% - w/v) D E F 120 120 a a a 120 a a a a a a a a a 80 37% 80 40% b b 80 34% b 40 40 40 Cell viability (%) Cell viability (%) Cell viability (%) 0 M 6 4 2 1 0 M 6 4 2 1 0 OH-4 (% - w/v) OH-5 (% - w/v) M 6 4 2 1 OH-6 (% - w/v) G H 120 120 a a a a a a a a

37% 80 80 b 47% b

40 40 Cell viability (%) Cell viability (%)

0 0 M 6 4 2 1 M 6 4 2 1 OH-7 (% - w/v) OH-8 (% - w/v)

Figure 1. Cell viability of macrophages RAW 264.7 treated with different concentrations of Brazilian organic honeys. A) OH-1, B) OH-2, C) OH-3, D) OH-4, E) OH-5, F) OH-6, G) OH-7, H) OH-8 (ANOVA One-way followed by the Tukey post-test, p <0.05). Different letters represent statistical difference. As seen in Figure 2, for the anti-inflammatory assay, macrophages were pre-treated with honey at 4% had a significant decrease in NF-κB activation as compared to the LPS group (p<0,05). OH-1 to OH-4 at 0.5%, 1% and 2% also decreased the NF-κB activation as compared to the LPS group (p < 0.05). OH-5 at 2%, OH-6 to OH-8 at 0.5%, 1% and 2% also decreased the NF-κB activation as compared to the LPS group (p < 0.05). 86

A B C 120 NF-B activation NF-B activation NF-B activation b 120 120 b b 80 d d 80 80 d c d c c c 40 c 40 40 95% 84% a 92% a a a a a Relative luminescence units (%) units luminescence Relative 0 0 V - 4 2 1 0.5 (%) units luminescence Relative 0

V - 4 2 1 0.5 (%) units luminescence Relative V - 4 2 1 0.5 OH-1 (% - w/v) OH-2 (% - w/v) OH-3 (% - w/v) LPS (10 ng/mL) LPS (10 ng/mL) LPS (10 ng/mL) D E F NF-B activation 120 NF-B activation 120 NF-B activation 120 b b b b b

80 80 80 c c d d c c

40 c 40 40 89% 90% 89% a a a a a a 0 0 0 Relativeluminescence units (%) Relative luminescence units (%) units luminescence Relative

V - 4 2 1 0.5 Relative luminescenceunits (%) V - 4 2 1 0.5 V - 4 2 1 0.5 OH-4 (% - w/v) OH-5 (% - w/v) OH-6 (% - w/v) LPS (10 ng/mL) LPS (10 ng/mL) LPS (10 ng/mL)

G H NF-B activation NF-B activation 120 b 120 b d d 80 d 80 c c 40 c 40 87% a a 93% a a 0 0 Relative luminescence units (%) luminescenceRelative units V - 4 2 1 0.5 (%) units luminescence Relative V - 4 2 1 0.5 OH-7 (% - w/v) OH-8 (% - w/v) LPS (10 ng/mL) LPS (10 ng/mL)

Figure 2. Effect of organic honeys (OH-1 to OH-8) on decreasing NF- ⱪB activation in macrophages RAW 264.7. A) OH-1, B) OH-2, C) OH-3, D) OH-4, E) OH-5, F) OH-6, G) OH-7, H) OH-8 (ANOVA One-way followed by the Tukey post-test, p <0.05). Different letters represent statistical difference. Besides, as seen in Figure 3, macrophages were pre-treated with honey at 4% had a significant decrease TNF-α level as compared to the LPS group. The OH-1 to OH-3 (1% and 2%); and OH-4 at 2% also decreased TNF-α level as compared to the LPS group (p< 0.05). Nevertheless, OH-4 at 1% did not reduced the cytokine level (p >0.05).OH-5 and OH-6 at 2% and OH-7 at 1% and 2% also decreased significantly the release of TNF-α. Lastly, the OH-5, OH-6 and OH-8 at 1% did not decreased TNF-α level as compared to the LPS group (p>0.05). 87

A TNF- level B TNF- level C TNF- level 6000 b 6000 6000 b b d d d 41% c c c 4000 d 4000 4000 54% 54% c c pg/mL pg/mL pg/mL 2000 2000 2000 a a a

0 0 0 V - 4 2 1 V - 4 2 1 V - 4 2 1 OH-1 (% - w/v) OH-2 (% - w/v) OH-3 (% - w/v) LPS (10 ng/mL) LPS (10 ng/mL) LPS (10 ng/mL) D E F TNF- level TNF- level TNF- level 6000 6000 b b b 6000 b b b c d 30% 35% c c 4000 4000 c 4000 52% c pg/mL pg/mL pg/mL 2000 2000 2000 a a a

0 0 0 V - 4 2 1 V - 4 2 1 V - 4 2 1 OH-4 (% - w/v) OH-5 (% - w/v) OH-6 (% - w/v) LPS (10 ng/mL) LPS (10 ng/mL) LPS (10 ng/mL)

G TNF- level H TNF- level 6000 b 6000 b b b d 27% d c 4000 4000 59% c L pg/m pg/mL 2000 2000 a a

0 0 V - 4 2 1 V - 4 2 1 OH-8 (% - w/v) OH-7 (% - w/v) LPS (10 ng/mL) LPS (10 ng/mL)

Figure 3. Effect of organic honeys (OH-1 to OH-8) in decreasing the release of TNF-α in macrophages RAW 264.7. A) OH-1, B) OH-2, C) OH-3, D) OH-4, E) OH-5, F) OH-6, G) OH-7, H) OH-8 (ANOVA One -way followed by the Tukey post-test, p <0.05). Different letters represent statistical difference. 88

Among all the OH-1 to OH-8 samples analyzed for their cytotoxicity and anti- inflammatory activities, the OH-7 was found to be the most bioactive one and then it was follow to pre-clinic anti-inflammatory assays using animal model. The inhibitory effects of OH-7 on neutrophil influx were determined in vivo. Mice pretreated with OH-7 at 100% (in natura) showed a significant decrease in neutrophil influx (49%) as compared to those treated with the control carrageenan (Fig 3A). However, OH-7 at 40% did not decreased the neutrophil migration (p>0.05). Regarding to release of TNF-α level, mice pretreated with OH-7 at 100% (in natura) showed a significant decrease TNF-α level (69%) as compared to those treated with the control carrageenan (Fig 3B). However, OH-7 at 40% did not decrease TNF-α level (p>0.05).

A B Neutrophil TNF- level 2.5 migration b 250 2.0 b b 200 /cavity 6 1.5 b 150 49% c

1.0 pg/mL 100 69% a a 0.5 50 Neutrophils x 10 x Neutrophils a 0.0 0 V - 40 100 C - 40 100 OH-7 (%) OH-7 (%) Carrageenan Carrageenan

Figure 4. Effects of OH-7 on neutrophil migration and TNF-α release in vivo. (A) Effect of vehicle (V), carrageenan (-), OH-7 (40% and 100%) on neutrophil migration into the peritoneal cavity of mice induced by i.p. administration of carrageenan (500 μg/cavity, -). (B) Effect of the OH-7 treatment on the release of TNF-α in mice. The results were expressed as mean ± SD, n = 5–6. Different letters indicate statistical difference and all groups were compared to each other. One-way ANOVA followed by Tukey’s post-test, P < 0.05.

Discussion

Chronic non-communicable diseases (NCDs), such as cardiovascular diseases, cancer, and diabetes, exhibit highly epidemiological rates and great potential for morbidity worldwide. The poor quality diet has been related to the appearance of NCDs on the worldwide population [27]. The low 89

intake of fruits and vegetables (poor quality diet) has contributed to emerging subclinical inflammation, which is a risk factor to arise the inflammatory diseases cited above [28]. The analysis of the inflammatory state include evaluation of biomarkers such as pro-inflammatory cytokines, and enzymes inflammation-related. In general, the inflammatory may be triggered to specific conditions such as obesity, active smoking and sedentary [29,30]. Inspired by the relation of diet, inflammation, and chronicle diseases, a research group have develop a dietetic tool named Dietary Inflammatory Index (DII®) that evaluate the consumption of biological actives, micronutrients and macronutrients in a diet [31]. This research group emphasize the importance to adopt a diet rich in polyphenols, as flavonoids preventing the appearance of NCDs [27]. In this regard, the Brazilian natural products, rich in bioactive molecules that act in different biological functions, are a promising to become a food function [25]. Honey is a natural product of animal origin produced by bees, especially the Apis melífera specie [32]. Despite being an animal product, the honey chemical composition varies according to the flora visited by bees as well as the seasonality. In general, honey is a mixture of sugar, carbohydrates, minerals, vitamins, enzymes, amino acids, organic acids, alkaloids, and phenolic compounds [33]. Besides that, honey is shown to have an enzymatic complex composed of diastase, invertases, glucose oxidase, catalase, and acid phosphatase [34]. The chemical honey complexity reflects several biological activities that may modulate biological functions. Thus, honey is a functional food that supplies nutritional needs, moreover, can prevent the occurrence of diseases and providing human health [35]. Despite all honey benefits [3], it was not found on scientific literature the anti-inflammatory activity from certified honey produced organically. Therefore, it was evaluate the anti-inflammatory mechanism of action of 8 types of organic honey (OH-1 – OH-8) from the Atlantic Rainforest. The anti-inflammatory assays of organic honey were conducted by macrophages cell culture evaluating the reduction of NF-κB activation and release of TNF-α level. Firstly, the cell viability assay was conducted to evaluate whether organic honey showed cytotoxicity; despite the honey be a non-toxic food to the human being, the in vitro methods shown some limitation [36]. None of the organic honey samples tested showed cell toxicity until 4% (w/v), however, at the highest concentration (6%) all samples induced toxicity, which can be explained by the osmosis change provided by honey becoming in a hypertonic solution to the macrophages. 90

Regarding anti-inflammatory activity, all samples of organic honey at 4% decreased the NF-κB activation and these preliminary results have indicated a promising anti- inflammatory activity since the NF-κB is involved in the different intracellular inflammatory pathway. The NF-κB represents a nuclear transcription family, which regulates a wide amount of genes involved in the immunological system, including the inflammatory process on the organism [37]. Since the immune system cells, as macrophages, are bound by a molecule, e.g. lipopolysaccharide (LPS), this nuclear transcription family is triggered and initiate a cascade regulating processes such as differentiation of T lymphocytes, the release of cytokines, pattern recognition receptors (PRRs), and others [38,39]. Thus, the deregulation of NF-κB is intrinsically related to the appearance and progression of inflammatory chronicle diseases; and the therapeutical approach that may interfere in a controlled manner on the exacerbated NF-κB activation represents important strategies that must be explored and developed [40]. Several cells of the immune system, including macrophages, have the NF-κB intracellular that can be activated by canonical and non-canonical (alternative) pathways. The canonical pathway is related to the signaling of TNF-α and IL-1, pro-inflammatory cytokines in the context of chronic inflammatory diseases [41]. Since the inflammatory cells released these mediators, it initiates the protein expression under endothelial cell surface, such as selectins and integrins, which will increase and direct for diapedesis, the neutrophils flow to the inflammatory focus (Soares et al., 2019). Some diseases, such as asthma [42], chronic obstructive pulmonary disease (COPD) [43], rheumatoid arthritis [44], and periodontal disease [45], have been related to disordered NF-κB activation. It is well-known the intrinsic relation between the NF-κB activation and the TNF-α level [46]. To verify whether the organic honey decreased the inflammatory in vitro parameters, it was carried out the NF-κB activation and the release of TNF-α level. After 4h with the honey treatment on macrophages, it was observed a decrease of NF-κB activation and release of TNF- α level by the macrophages treated with OH-7 showing a percentage of inhibition 85% and 59%, respectively. In a study conducted by [15], the authors evaluated the anti-inflammatory activity of Manuka honey, medical-grade honey, from New Zealand, in HL-60 cells treated with 0.5% and 3% (v/v). They concluded that Manuka honey at 0.5% reduced the release of TNF-α level and other inflammatory mediators, however, the cells treated at 3% had an increase of the TNF- α release level [15]. In contrast, the eight samples of Brazilian organic honeys decreased the release of TNF-α at 4% (w/v). This result may be explain due to variations related to chemical and enzymatic profile of the organic honey and the different cell lines used in both studies 91

compared as well. It is worth noting that in both studies the honey represents an important source to reduce inflammatory mediators in chronic diseases. Collectively, among all the samples analyzed, the OH-7 was the most promising sample to decrease inflammatory markers as NF-κB and TNF-α and then it was select to subsequent anti-inflammatory in vivo assay. Mice were pre-treated with OH-7 before the challenge of carrageenan-induced peritonitis and had a decrease of the neutrophil influx (49%) compared to the control. Similarly, the release of TNF-α level by mice decreased (69%). These findings indicates that OH-7 has a promising anti-inflammatory potential for acute inflammation Studies evaluating chronic inflammation must be conducted to select the honey as a functional food (adjuvant therapy) in chronic diseases. Furthermore, our findings validate the hypothesis that organic honey consumption decreased the leukocyte migration and, consequently, the release of inflammatory mediators. A research group evaluated the anti-inflammatory effect in vivo of Malaysian Gelam honey using carrageenan-induced paw edema model [47]. The authors found that the animals had decrease of paw-edema in a dose-dependent manner and shown reduction of TNF- α level in the plasma and paw tissue. In another study, the authors evaluated the anti-inflammatory potential of Manuka honey and concluded that at 0.5, 1, and 3% the sample reduced the phosphorylation of IκBα in a dose-dependent manner, also decreased the chemotaxis and consequently the reduction of neutrophil influx. Manuka honey is medical-grade known worldwide for its antimicrobial and anti-inflammatory potential. This honey has been used in the hospital environment in New Zealand to treat wounds inflamed and infected resistant to allopathic drugs [48,49]. Thus, OH- 7 showed an anti-inflammatory activity similar to Manuka honey and, therefore, it can be considered as a promising natural product and candidate to become Brazilian medical-grade honey. Besides the importance of honey as a functional food or therapeutic input, e.g. Manuka honey [50], studies that are guided biologically (bioguided) needs to be encouraged mainly in the matter of natural products [51]. Honey has a chemical profile that varies according to the local flora and reflects the secondary metabolites from different plants, increasing the chances of discovering promising molecules with biological activities for the development of medicines [50]. The honey anti-inflammatory activity has been reported due to its expressive quantity of phenolic composition [52]. The phenolic compounds have been related to decrease 92

the pro-inflammatory mediators' release by interfering in enzymes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) [53]. Previously, our research group has studied the antioxidant activity of these eight organic honey samples (OH-1 to OH-8) and also identify the main chemical compounds of honey crude extracts. The total phenolic content founded at honey extracts (mg - GAE/g) was 73.15 (OH-1), 59.79 (OH-2), 49.79 (OH-3), 52.20 (OH-4), 117.68 (OH-5), 84.08 (OH-6), 83.19 (OH-7) e 53.03 (OH-8). In total, four phenolic compounds were identified in the samples such as ferulic acid, caffeic acid, rutin, and hesperidin. Besides that, we identified considerable amounts of ascorbic acid ranged 2.75 to 6.22 mg/100g at OH-3, OH-5, and OH-7. The presence of these compounds can explain, in part, the in vitro anti-inflammatory activity exerted by all organic honey and in vivo by OH-7. Previous studies have shown the in vitro anti-inflammatory of cafeic acid. Macrophages LPS-stimulated treated by cafeic acid had a decrease of the inflammatory markers such as nitric oxide, prostaglandin E2, mRNA TNF-α levels from TNF-α and COX-2, and inducible nitric oxide synthase (iNOS). Another compound identified in organic honey was the ferulic acid that which is well known for its antioxidant activity [56]. In addition, ferulic acid also decreases the inflammation and oxidative stress in a model of respiratory distress syndrome in rats [57]. In another anti-inflammatory study, the authors carried out in vivo assays using acute and chronicle inflammatory model. The authors treated rats with the flavonoids rutin, quercetin, and the flavone hesperidin. The intraperitoneal injection of these compounds daily (80 mg/kg) reduced the acute and chronicle inflammation, and the rutin compound was the most effective in the chronicle phase. Thus, the organic honey anti-inflammatory activity can be related to the presence of these compounds [58]. The honey chemical composition also has short-chain fatty acids (AGCC) and this chemical structure can be responsible for the slow absorption promoting the release of immunomodulators molecules by the fermentation agents. Thus, these fermentable sugars produced from honey, as nigero-oligosaccharides, have the ability to induce an immune response [59,60]. Another important fact to consider is the presence of some proteins present in honey composition such as glycoproteins and glycopeptides. In Ziziphus honey composition it was verified that fractional proteins inhibited the reactive oxygen species in human neutrophils (IC50 = 6-14 ng/mL) and murine macrophages (IC50 = 2-9 ng/mL). In addition, honey proteins significantly suppressed the production of TNF-α in human monocytic cell lines [61]. 93

This is the first report elucidating the anti-inflammatory mechanism of action (in vitro and in vivo) from Brazilian honey produced organically in the Atlantic Rainforest. Although our results have shown the promising anti-inflammatory activity of the organic honey decreasing NF-κB activation, the release of TNF-α level, and the neutrophil influx into the inflammatory site; it is highly possible that this functional food acts by other pathways not elucidate yet. In addition, it seems that there is a synergism effect among the organic honey chemical composition resulting in the biological activities, as the anti-inflammatory. Considering that the organic honey is food and not medicine, and its use probably does not trigger adverse effect, the insertion of this food on diet it is a promising strategy to protect and decrease the exacerbated inflammatory process, and, consequently, chronicle diseases-related. Conclusion

Brazilian organic honeys reduced the inflammatory process, through the modulation of NF-κB activation and TNF-α level, as well as the OH-7 variation was the most promising one. This study adds value to the promising Brazilian organic honeys that have emerged as a source of bioactive compounds providing benefits for human health (functional foods), contributing to the development of agribusiness for many families, and also contributing to preservation of biodiversity, particularly in the Brazilian Atlantic Rainforest.

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3. DISCUSSÃO

Muitas evidências mostraram que mel apresenta atividade antimicrobiana promissora para muitos microrganismos (Cooper, 2016). Este alimento funcional tem sido estudado e usado na medicina popular por muitos séculos para tratar infecções, particularmente aquelas associadas com feridas e escaras. A atividade antibacteriana, com amplo espectro, já foi relatada para diferentes amostras de méis e em várias bactérias patogênicas. No entanto, muitos dos primeiros estudos exploraram mal as características do mel (Molan, 1996). Como o mel é usado há muito tempo na medicina popular (Eteraf-Oskouei e Najafi, 2013) e, mais recentemente, em hospitais (Goharshenasan et al., 2016), para o tratamento de feridas na pele, a maioria dos estudos de atividade antibacteriana é direcionada aos microrganismos que colonizam esta região, bem como bactérias que causam infecções sistêmicas (Cooper, 2016). Foi documentado para diferentes amostras de mel atividade antimicrobiana contra muitas bactérias, causadoras infecções sistêmicas de grande relevância clínica, como Pseudomonas aeruginosa (Lu et al., 2019), Staphylococcus aureus (MRSA) (Cremers et al., 2019), Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Shigella flexneri, and Staphylococcus epidermidis (Ghramh et al., 2019). Porém, a literatura é escassa de resultados com atividade antimicrobiana de mel contra microrganismos orais. Poucos estudos científicos têm investigado o uso de mel para prevenir e/ou tratar doenças bucais biofilme-dependentes. A atividade antimicrobiana do mel contra microrganismos orais, bem como sua atividade antibiofilme contra biofilmes maduros, mecanismo de ação, composição química e indicações clínicas, permanecem amplamente desconhecidas. No primeiro artigo que compõe esta tese, realizamos uma extensa busca na literatura acerca das propriedades antimicrobianas de mel contra microrganismos bucais. Existem poucos resultados sobre o assunto e diferentes lacunas científicas sem respostas. A maioria dos estudos que abordaram essa temátia utilizaram a microdiluição em caldo como método para determinação da atividade antimicrobiana de mel (Habluetzel et al., 2018, Safii et al., 2017, Schmidlin et al., 2014, Badet e, Quero, 2011, Badet e, Quero, 2011, Bassone e Grobler, 2008,, Nassar et al., 2012) e alguns poucos utilizaram o método de difusão em ágar (Mathai et al., 2017, Ahmadi – Motamayel et al., 2012). De todos os estudos encontrados, apenas um avaliou questões como mecanismo de ação, composição química do mel e essa relação química-atividade, bem como a atividade antibiofilme em biofilme maduro (Eick et al., 20014). Entretanto, nenhum dos estudos mostrou dados relacionados a danos celulares que este 99

alimento funcional possa causar nas bactérias ou atividade antibiofilme em biofilme multiespécie. Embora essas informações sejam bem difundidas em relação a atividade antimicrobiana de microrganismos presentes em feridas (Cooper et al., 2016), a literatura é escassa nesta questão para atividade antimicrobiana de mel contra microrganismos orais. Além disso, considerando que os biofilmes orais são complexos, multiespécie e multirresistentes (Marsh, 2004), é essencial que a atividade antimicrobiana de mel ou qualquer outro produto seja avaliada contra esse tipo de comunidade bacteriana reproduzindo de forma mais fiel possível as condições reais. Dessa forma, a revisão apresentada no primeiro capítulo fornece as justificativas necessárias para a realização dos artigos subsequentes. No segundo artigo avaliamos a atividade antimicrobiana das oito amostras de méis orgânicos da Mata Atlântica brasileira (OH-1 a OH-8) contra colonizadores primários e secundários do biofilme dental, bem como a atividade antibiofilme contra um biofilme monoespécie de S. mutans. Todas as amostras apresentaram atividade antimicrobiana contra as cepas testadas. Não encontramos na literature nenhum estudo que tenha mencionado ter utilizado amostras de mel produzidos de forma orgânica certificada. Alguns estudos demonstraram atividade antimicrobiana positiva de mel contra microrganismos orais (S. mutans e Lactobacillus) (Nassar et al., 2012; Ahmadi-Motamayel et al., 2013; Patel et al., 2017), porém com divergência nos resultados entre si e com os resultados do artigo 1. A diferença nos resultados dos estudos citados pode estar relacionada tanto à composição dos méis quanto à diferença no método de análise. No presente estudo, a análise da atividade antimicrobiana foi determinada pela microdiluição em caldo (CLSI, 2012) para determinação da CIM. Apesar de ser um ensaio rápido e de fácil desempenho, a difusão em ágar para definição da atividade antimicrobiana de mel tem várias limitações, como dificuldade de difusão dos componentes ativos do mel devido a alta viscosidade, baixa reprodutibilidade e impossibilidade de distinguir atividade bactericida da bacteriostática (Szweda, 2017). A atividade antimicrobiana do mel, em geral, está relacionada a dois componentes principais: o peróxido de hidrogênio produzido pelas enzimas existentes no mel, como a glicose oxidase, e/ou os compostos fenólicos (Cooper, 2016). Uma forma de avaliar qual componente presente no meu desencadeia sua ação antimicrobiana é tratar as amostras de mel com catalase (Allen et al., 1991). No artigo 1 demonstramos que ao tratar os méis com catalase a atividade antimicrobiana foi suprimida na maioria das amostras de mel. Porém, em outras a atividade persistiu em altas concentrações. Esses dados sugerem que a atividade 100

antimicrobiana está ligada majoritariamente aos peróxidos produzidos pelo complexo enzimático do mel, mas que os outros componentes, principalmente os compostos fenólicos, também desempenham uma ação discreta. Nenhum dos estudos mencionados na revisão do capítulo 1 desta tese definiu se a atividade antimicrobiana estava marjoritariamente ligada aos peróxidos produzidos pelo complexo enzimático, aos compostos fenólicos ou a ambos. Esse achado suporta a afirmaçao de que o artigo apresentado no capítulo 2 é um estudo pioneiro de atividade antimicrobiana para microrganismos orais com definição dos components responsáveis por essa ação. Em estudo anterior, nosso grupo de pesquisa mostrou uma expressiva atividade antioxidante dos extratos fenólicos dessas mesmas amostras analisadas neste estudo. Além disso, identificamos o perfil químico desses méis. O teor de componentes fenólicos nos extratos de mel em mg (GAE/g) foi: 73,15 (OH-1), 59,79 (OH-2), 49,79 (OH-3), 52,20 (OH-4), 117,68 (OH-5), 84,08 (OH-6), 83,19 (OH-7) e 53,03 (OH-8). Quatro compostos fenólicos foram identificados e quantificados: ácido ferúlico, ácido cafeico, rutina e hesperidina nas amostras. Além disso, foi detectada uma alta quantidade de ácido ascórbico (vitamina C) nas amostras OH-3, OH-5 e OH-7 que variou de 2,75 a 6,22 mg/100 g (Silva et al., 2019). Estudos anteriores mostraram atividade antimicrobiana dessas moléculas contra microrganismos orais, o que pode explicar em parte a atividade antimicrobiana dos méis após inativação dos peróxidos (Slobodníková et al., 2016; Nakamura et al., 2017; Gutierréz-Venegaz et al., 2019). Com relação a atividade antibiofilme, o artigo 2 também é um dos estudos pioneiros na avaliação da atividade antibiofilme de méis em biofilme maduro de um microrganismo oral. Todos os méis orgânicos avaliados aqui apresentaram capacidade de destruição do biofilme de 48 h de S. mutans. A escassez de estudos semelhantes dificulta a comparação dos nossos resultados. Entretanto, usando a atividade de inibição da formação do biofilme de P. gingivalis do mel de Manuka (Eick, 2014), um mel de grau medico da Nova Zelândia, podemos considererar os méis orgânicos brasileiros alimentos funcionais com atividade antibiofilme promissora. A comprovação de que os méis orgânicos brasileiros possuem atividade antimicrobiana e antibiofilme contra microrganismos orais demonstra que o mel tem potencial para ser usado em substituição ao açúcar comum controlando a microbiota, além de desempenhar outros benefícios já conhecidos desse alimento funcional para o organismo. Entretanto, apesar dos resultados promissores, muitas perguntas sobre a atividade antimicrobiana de mel contra biofilmes bucais permaneciam obscuras, o que motivou a estender a pesquisa para microrganismos aneróbios presentes no biofilme subgengival e relacionados a 101

doença periodontal. Assim, foi conduzido o artigo 3 que avaliou in vitro a atividade antibiofilme das oito amostras de méis e in vivo a capacidade da variedade OH-7, amostra mais promissora, em interferir no estabelecimento e progressão da doença periodontal. Desse modo, foi realizado um screening inicial para avaliar a atividade antimicrobiana de oito amostras de méis oriundos da Mata Atlântica brasileira, e seus respectivos extratos, contra P. gingivalis W83. Todas as amostras analisadas, méis e extratos, demonstraram atividade antimicrobiana contra P. gingivalis. Esses resultados sinalizaram os méis como alimentos promissores contra P. gingivalis, justificando maiores investigações desse alimento funcional contra componentes envolvidos na etiologia da doença periodontal. Apesar da atividade antimicrobiana de mel ser conhecida há muito tempo, atualmente não há um consenso entre os pesquisadores sobre quais caminhos metodológicos devem ser seguidos na exploração dessa atividade (Szweda, 2017). A determinação da atividade antimicrobiana de uma substância é apenas um dos passos iniciais dos estudos bioguiados para sinalização de novas moléculas com potencial de uso como antimicrobianos (Ramírez-Rueda et a., 2019). Entretanto, com a evolução da resistência antimicrobiana e a agressividade das doenças biofilme-dependente, a análise da atividade antibiofilme das substâncias candidatas à terapia antimicrobiana é essencial (Slobodníková et al., 2016), uma vez que microrganismos organizados na forma de biofilmes podem ser até 1000 vezes mais resistentes (Marsh, 2004). A análise da atividade antimicrobiana de mel contra biofilmes orais complexos e maduros é incipiente na literatura. Até o momento, apenas um único estudo que avaliou a atividade antibiofilme do mel Manuka e um mel multifloral da Alemanha contra biofilme monoespécie maduro de P. gingivalis (Eick et al., 2014). Os resultados de Eick et al., 2014 mostraram que na concentração 10% ambos os tipos de méis inibiram a formação de biofilmes de P. gingivalis e reduziram o número de bactérias viáveis em biofilmes de 42 horas. Entretanto, não houve uma prevenção total da formação de biofilme nem sua erradicação completa. Um outro estudo avaliou a inibição da formação de biofilme monoespécie de S. mutans por um mel natural adquirido em um supermercado da Arábia Saudita (Nassar et al., 2012). Os resultados de Nassar et al., 2012 demonstraram que o mel da Arábia Saudita, nas concentrações 25 e 50%, foi capaz de diminuir significativamente a formação de biofilme de S. mutans em placa de poliestireno. Há muitas variações metodológicas entre os estudos e não há relatos de atividade antimicrobiana contra biofilmes multiespécie e/ou subgengivais. Assim, até o momento a afirmação de que o mel pode ser uma alimento com potencial protetor para doenças orais biofilme-dependente permaneceu incerta. 102

Com o objetivo de preencher essas lacunas na literatura, analisamos a atividade antibiofilme de OH-7 em um biofilme subgengival multiespécie (32 espécies). O artigo 3 é o primeiro estudo que avaliou a atividade antibiofilme de um mel em biofilme oral com essas condições metodológicas. O modelo de biofilme, realizado em dispositivo Calgary, foi testado anteriormente com sucesso pelo nosso grupo de pesquisa para analisar o potencial antibiofilme de outro produto apícola, a própolis vermelha (Miranda et al., 2019). As concentrações usadas no tratamento do biofilme foram baseadas na CIM de cada OH (mel orgânico, do inglês Organic Honey) e seus respectivos extratos para P. gengivalis (2 e 10x CIM para mel, e 10 e 20x CIM para extrato de mel). Foram realizados dois tratamentos diários de 1 minuto durante quatro dias, respeitando-se o intervalo de 6 horas entre os tratamentos. Nossos resultados demonstraram que todas as amostras de mel na concentração 10x CIM reduziram intensamente a atividade metabólica geral do biofilme, não apresentando diferença estatística com o controle positivo (CLX). Nós consideramos OH-7 o mel mais promissor, uma vez que foi o que em menor concentração (2x CIM | 8%) apresentou essa redução promissora na viabilidade sem diferir da CLX. Com relação aos extratos dos méis os únicos que apresentaram uma discreta redução na viabilidade geral do biofilme foram os extratos dos méis 1, 2, 5 e 7. A forma de obtenção dos extratos anteriormente descrita por (Ferreres et al., 1991) remove o excesso de açúcar e o componente protéico, como o complexo enzimático produtor de peróxidos. Assim, considerando a discreta atividade antimicrobiana demonstrada pelos extratos dos méis em comparação com os méis in natura é possível observar que a atividade antimicrobiana se dá, em grande parte pela presença de peróxido produzido pelo complexo enzimático do mel. Esse achado foi sinalizado no artigo 2 e reproduzido posteriormente no artigo 3, confirmando também a hipótese de que uma pequena parte dessa atividade biológica está ligada aos componentes fenólicos (atividade dos extratos). Dessa forma, a relação entre os componentes do mel in natura é direcionada e sinérgica entre peróxidos e compostos fenólicos. Esse sinergismo conferem a este alimento brasileiro uma expressiva atividade antibiofilme em biofilme subgengival multiespécie. Baseado nesses achados iniciais foi dado seguimento na pesquisa apenas com a variedade OH-7 in natura, considerada a mais promissora. Assim, realizamos o ensaio de hibridização DNA-DNA nas amostras de biofilme in vitro tratados com OH-7. O objetivo foi verificar a redução específica de espécies bacterianas baseada nos complexos de Socransky. Os estudos mostram que a doença periodontal é desencadeada inicialmente devido a um ambiente periodontal disbiótico (Hajishengallis e Lamont, 2012). O desenvolvimento 103

dessa disbiose ocorre ao longo de um determinado período de tempo, modificando de forma gradual a relação simbiótica microrganismo-hospedeiro para um padrão patogênico. O primeiro complexo de Socransky (Socransky, 1998) que tem sido relacionado à disbiose e, consequentemente ao estabelecimento da doença é o complexo laranja. Em seguida, o amadurecimento do biofilme e progressão da doença tem sido relacionados com complexo vermelho (Nath e Raveendran, 2013). Também foi relatado que P. gingivalis demonstra aderência a um patógeno acessório do complexo amarelo, como S. gordonii, causando um aumento da virulência e consequentemente da perda óssea (Lamont e Hajishengallis, 2015). A redução na contagem de bactérias dos complexos laranja e vermelho, nos ensaios in vitro, indica que o consumo racional de mel pode controlar o crescimento desses microrganismos e, consequentemente, prevenir o estabelecimento e progressão da doença periodontal. Entretanto, apesar dos resultados serem promissores é necessária a comprovação da reprodutibilidade in vivo. Para isso, foi conduzido o ensaio de modelo de doença periodontal em camundongos para verificação da eficácia de OH-7 na prevenção da doença periodontal experimental in vivo. O ensaio de doença periodontal foi realizado como descrito anteriormente por Abe e Hajishengallis 2013, com modificações. O modelo proposto por esses autores utilizou apenas ligadura de fio de seda no primeiro molar dos animais. Aqui expomos previamente a ligadura a um inóculo inicial de 107 de P. gingivalis por um período de 48 h, sob condições de anaerobiose, e nos primeiros quatro dias de estímulo foi adicionado 100 µL de um inóculo de P. gingivalis na mesma concentração, diluído em carboximetilcelulose, para garantir a presença do periodontopatógeno no ambiente. Um outro estudo demonstrou que a associação de P. gingivalis ao modelo de periodontite induzida por ligadura exacerba a reabsorção óssea dependente da proteína RANKL (ligante do receptor ativador do fator nuclear kappa B) (Lin et al., 2014). Assim, a adição de P. gingivalis induziu uma estímulo mais intense. A ingestão de OH-7 na concentração 40%, 5 vezes ao dia, reduziu a perda óssea em 34% (p< 0,05). A frequência de tratamento foi baseada em uma dieta balanceada na qual fossem feitas três refeições principais com uma refeição auxiliar entre cada uma delas, totalizando a ingestão de mel 5 vezes ao dia. A figura 6 do artigo 3, que mostra uma imagem representativa de cada grupo, é possível perceber a preservação da crista óssea alveolar na região de furca. A presença de lesão de furca em graus severos é um indicador da progressão da doença. Foi demonstrado um risco aumentado de perda de molares com envolvimento da região de furca periodontal em uma população geral que não faz tratamento periodontal regular (Nibali et al., 2017). Ao final do experimento foi determinado escore de cárie de forma cega feita por um 104

examinador calibrado, como descrito por Larson, 1981. Nenhum dos animais de nenhum dos grupos apresentou lesões de cárie e/ou mancha branca. Esse achado indica que, apesar do mel ser um alimento rico em carboidratos (Cooper, 2016), o consumo racional é seguro e saudável. A variedade OH-7 é considerado um mel de melato ou simplismente melato. Esse tipo de mel, diferentemente do mel convencional, não provém do nectar das plantas mas sim de secreções de partes vivas de plantas ou excreções de insetos sugadores de floema de plantas (Pita-Calvo e Vásquez, 2017). Já foi reportado na literature que melatos em geral apresental propriedade antioxidante e antimicrobiana superior aos méis florais (Majtan et al., 2011). Apesar disso, a literatura é ainda mais escassa no que diz respeito a atividade antimicrobiana de melatos (Ng et al., 2020). Os meis de melato apresentam apresentam propriedades físico- químicas diferentes dos méis florais. Geralmente esses méis possuem maiores valores de pH, condutividade elétrica, absorbância líquida, maior teor de dissacarídeos, trissacarídeos e menor nível de monossacarídeos. Também caracterizados por terem uma cor mais escura e características sensoriais, como odor e sabor, mais expressivos (Seraglio et al., 2019). Outro diferencial de OH-7 é o seu alto teor de vitamina C. OH-7 apresentou 6,22 mg de vitamina C, muito superior a encontrada nos outros melatos (Silva et al., 2019). Um estudo suplementou méis florais com vitamina C e verificou um expressivo aumento nas propriedades antimicrobiana e antibiofilme desses méis contra S. aureus (Majtan et al., 2020). Os autores sugerem que a combinação de mel com vitamina C possa desencadear a produção de espécies reativas de oxigênio no interior das células bacterianas. Porém, estudos de mecanismo de ação precisam ser realizados. Acreditamos que o alto teor de vitamina C pode estar ligado a sua expressive atividade antimicrobiana in vitro e in vivo (análise das ligaduras). Os resultados para a análise de hibridização DNA-DNA das ligaduras foram semelhantes aos encontrados no estudo in vitro, reduzindo significativamente a quantidade de S. gordonii, S. sanguinis, F. nucleatum, e P. gingivalis e Prevotella melaninogenica. Surpreendentemente o mel diluído apresentou um efeito melhor que o mel in natura. Esse efeito parece estar relacionado tanto à produção de H2O2 pela enzima glicose oxidase quanto à viscosidade do mel e comportamento dos animais após administração. A formação de H2O2 depende da diluição do mel. A glicose oxidase encontra-se inativa no mel não diluído (White et al., 1963; Brudzynski, 2006) e sua ativação varia de acordo com a diluição do mel (Bang et al., 2003; Majtan et al., 2014). Estudos anteriores demonstraram uma forte correlação entre o nível de peróxido de hidrogênio endógeno e a extensão da inibição do crescimento bacteriano pelo mel (White et al., 1963; Brudzynski, 2006). 105

Foi demonstrado que mesmo com altas diluições a presença de H2O2 persiste na solução e inibe em diferentes níveis a viabilidade microbiana (Brudzynski et al., 2011). Desse modo, acreditamos que o mel in natura não teve sua glicose oxidase ativada em nível suficiente para produzir uma quantidade peróxido capaz de exercer atividade antimicrobiana. Não obstante, a viscosidade do mel pode ter impedido de chegar de forma efetiva na região de ligadura. Outro ponto importante é que os animais do grupo do mel in natura apresentaram comportamento de autolimpeza com as patas dianteiras, o que podeter dificultado a dissolução adequada do mel no ambiente oral. Em contrapartida, o mel diluído a 40% teria glucose oxidase ativada para produção de H2O2, estava fluído suficiente para chegar nas profundidades da ligadura e os animais não demonstraram reação de autolimpeza. Não encontramos na literatura outros estudos que avaliassem a atividade biológica de mel em modelo in vivo de doença periodontal. Em nosso estudo o tratamento com mel foi iniciado após o quarto dia de instalação da ligadura que é o período onde há um início da reabsorção óssea (Abe e Hajishengallis, 2013). Levando em consideração que o mel é um alimento e não um medicamento e que protegeu a reabsorção em um processo já iniciado, é possível que OH-7 possa reduzir o risco de doenças periodontais ao ser incluído de forma racional na alimentação de indivíduos saudáveis. Assim, até aqui a literatura permanecia carente de respostas sobre o potencial de mel na prevenção e/ou tratamento de doenças orais biofilme-dependente. Os artigos 2 e 3 fornecem evidências consistentes para explorar mais essa hipótese. Os próximos passos para que essa indicação seja seguramente feita são a realização de um ensaio clínico com pacientes saudáveis para verificar as modificações na microbiota oral e posteriormente um ensaio com pacientes com doenças orais biofilme-dependentes para análise das condições clínicas, microbiológicas e inflamatórias. Levando em consideração o componente inflamatório da doença periodontal, bem como em outras doenças crônicas de relevância clínica, conduzimos a realização do artigo 4 desta tese, que analisa as atividades anti-inflamatórias in vitro das oito amostras de meis, bem como os efeitos in vivo da variedade OH-7. De maneira geral, as doenças crônicas não transmissíveis, como as doenças cardiovasculares, o câncer e o diabetes, apresentam altas taxas epidemiológicas e grande potencial de morbidade em todo o mundo. A dieta de má qualidade tem sido considerada um fator que está intimamente relacionado ao aparecimento dessas patologias na população (Shivappa, 2019). Há indícios de que uma inflamação clinicamente silenciosa de baixo grau é um fator de risco em comum para o aparecimento dessas condições (Camps e Garcia-Heredia, 106

2014). A análise desse estado inflamatório inclui a avaliação de biomarcadores, como citocinas pró-inflamatórias e enzimas relacionadas. Em geral, o disparo inflamatório está ligado a condições específicas, como obesidade, tabagismo ativo e sedentarismo (Bluher, 2016; Tir, Labor e Plavec, 2017). Os produtos naturais brasileiros, ricos em moléculas bioativas que desempenham diferentes funções biológicas no organismo são destaques para compor um arsenal de alimentos funcionais que podem modular os componentes da inflamação (Lazarini et al., 2018). Desse modo, avaliamos no artigo 4 o potencial anti-inflamatório das oito amostras de méis (OH-1 a OH-8). Os resultados sinalizaram as oito amostras de mel orgânico brasileiro como potentes inativadores de NF-κB. Desregulações na ativação do NF-κB estão intimamente relacionada ao aparecimento e progressão de doenças inflamatórias crônicas. Assim, abordagens terapêuticas que interfiram de forma controlada na ativação exacerbada desse fator nuclear, representam estratégias terapêuticas que devem ser exploradas (Liu et al., 2017). Dessa forma, esses resultados iniciais mostraram indícios de que os méis orgânicos brasileiros poderiam ser alimentos funcionais com promissora atividade anti-inflamatória. A relação de ativação de NF-κB com a citocina TNF-α (Wong e Tergaonkar, 2009) motivou a condução do ensaio, in vitro, da liberação de TNF-α por macrófagos. Assim como a inativação de NF-κB pelos méis, a diminuição da liberação de TNF-α foi eficaz e diminuiu à medida que foi diminuída a concentração de mel. Esses resultados positivos também podem estar relacionados com a diminuição da perda óssea demonstrada no artigo 3, uma vez que TNF-α está relacionado a perda de fibroblastos e reabsorção óssea que ocorre durante a agressão por periodontopatógenos (Graves e Cochran, 2003). Tanto a inflamação aguda quanto a inflamação crônica apresenta concentrações aumentadas de TNF-α. Apesar da estratégia de bloqueio de TNF-α piorar o prognóstico em pacientes com abscessos e infecções granulomatosas, essa abordagem tem sido considerada benéfica para intervenções em inflamatórias crônicas, como artrite reumatoide (Popa et al., 2007). Os resultados dos estudos in vitro nos artigos 2, 3 e 4 sinalizaram OH-7 como a amostra mais promissora para condução de ensaios in vivo para prospecção de atividades biológicas. Assim, foi conduzido o ensaio de peritonite induzida por carragenina para analisar a capacidade de OH-7 em reduzir a migração de neutrófilos para a cavidade peritoneal, bem como a liberação de TNF-α. Os animais foram tratados com OH-7 in natura (100%) e diluído na concentração 40% (p/v). Surpreendentemente, o tratamento com OH-7 in natura 5 vezes ao 107

dia por 5 dias antes do estímulo reduziu em 49% a migração de neutrófilos para a cavidade peritoneal. De forma semelhante, a liberação de TNF-α foi diminuída em 69%. Essas reduções expressivas indicam que OH-7 têm um potencial anti-inflamatório promissor para inflamação aguda. Estudos conduzidos com modelos de inflamação crônicas são necessários para eleger esse alimento funcional como adjuvante em doenças crônicas inflamatórias. Entretanto, nossos resultados confirmam a hipótese de que o consumo racional de mel reduz a migração de leucócitos e, consequentemente, a liberação de mediadores inflamatórios. Tem sido relatado que o efeito anti-inflamatório do mel de abelha é devido a sua quantidade substancial de conteúdo fenólico (Al-Waili e Boni, 2003; Nweze, 2019). A diminuição dos eventos pró-inflamatórias de enzimas importantes no processo de inflamação, como, óxido nítrico sintase induzível (iNOS) e ciclooxigenase-1 e ciclooxigenase-2 (COX-1 e COX-2), acontece pela ação de compostos fenólicos e flavonóides (Viuda-Martos et al., 2008). Como já mencionado, em estudo anterior mostramos a abundância dos compostos fenólicos presentes nas amostras de mel aqui analisadas (Silva et al., 2019), o que pode explicar a expressiva interferência dos méis nos mediadores inflamatórios. Além disso, os méis egm geral possuem ácidos graxos de cadeia curta (AGCC). Acredita-se que a lenta absorção do mel resulte na produção desses agentes de fermentação que tem atividades imunomoduladoras comprovadas. Isso significa que esses açúcares fermentáveis produzidos a partir do mel, como os nigerooligossacarídeos, têm a capacidade de modular a resposta imune (Schley, 2002; Sanz et al., 2005). Outro fator anti-inflamatório importante já relatado para méis, são as glicoproteínas e glicopeptídeos. Proteínas fracionadas de mel de Ziziphus demonstraram uma potente inibição, concentração-dependente, da produção de espécies reativas de oxigênio em neutrófilos humanos (IC50 = 6-14 ng/mL) e macrófagos murinos (IC50 = 2-9 ng/mL). Além disso, as proteínas do mel suprimiram significativamente a produção de TNF-α em linhagem de células monocíticas humanas (Mesaik et al., 2015). Embora nossos resultados tenham mostrado um potencial anti-inflamatório promissor, por meio da inativação de NF-κB, diminuição de TNF-α e diminuição da migração de leucócitos para o foco inflamatório, é possível que esse alimento funcional atue também por outras vias ainda não decifradas. Parece haver um sinergismo finamente orquestrado entre todos os componentes do mel que resulta nas expressivas atividades biológicas desse alimento, como a atividade anti-inflamatória. Levando em consideração que os méis orgânicos brasileiros são alimentos e não medicamentos e que seu uso racional não resulta em efeitos adversos, a inclusão desse alimento funcional na dieta pode ser uma estratégia promissora para diminuir a exacerbação inflamatória e, consequentemente, doenças crônicas relacionadas. 108

Os resultados desta pesquisa mostram que os méis orgânicos da Mata Atlântica brasileira interferem nos componentes microbiano e inflamatório da doença periodontal de forma benéfica para o organismo. Esses achados além de estimularem hábitos alimentares mais saudáveis, dadas as outras propriedades biológicas de mel já relatadas na literatura (Samarghandian, Farkhondeh e Samini 2017), também podem agregar valor econômico e científico a esse produto nacional. Isso pode refletir de forma positiva no contexto econômico local. Além disso, a Mata Atlântica é um bioma ameaçado e que tem sido severamente agredido nas últimas décadas. A demonstração das riquezas naturais desse bioma poderá estimular ainda mais políticas de preservação dessa área. 109

4. CONCLUSÃO

Conclui-se que: ● Apesar de ser um alimento amplamente consumido e estudado, a literatura é incipiente no que diz respeito a atividades biológicas de mel para doenças bucais inflamatórias e/ou biofilme-dependentes; ● Os méis da Mata Atlântica brasileira, produzidos de forma orgânica, apresentam atividade antimicrobiana e antibiofilme in vitro contra microrganismos bucais; ● Os méis orgânicos interferem, in vitro, na modulação de fatores inflamatórios como o fator NF-κB e a citocina TNF-α. ● OH-7, a variedade de mel orgânico mais promissora nos estudos in vitro é capaz de interferir de forma benéfica na progressão da doença periodontal experimental em camundongos, diminuindo a microbiota local e reduzindo a reabsorção óssea alveolar. ● OH-7 diminui a migração de neutrófilos para o foco inflamatório em modelo experimental em camundongos, bem como reduz a liberação de TNF-α.

Os méis orgânicos da Mata Atlântica brasileira, especialmente a variedade OH-7, podem ser considerados alimentos funcionais promissores que modulam os componentes microbiano e inflamatório da doença periodontal. Esses achados podem agregar valor científico e econômico a esse alimento nacional, favorecendo setores como o agronegócio, indústria alimentícia e abordagens alternativas na saúde, proporcionando benefícios à saúde humana, além de contribuir para a preservação do bioma da Mata Atlântica. 110

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ANEXOS

Anexo 1: Certificado de aprovação do Comitê de Ética no Uso de Animais

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Anexo 2 – Comprovante autorização do Conselho de Gestão do Patrimônio

Genético (CGEN)

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Anexo 3 – Comprovante de submissão do artigo à revista científica internacional

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Anexo 4 – Comprovantes de premiação em congressos científicos

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Anexo 5 – Relatório de originalidade do sistema Turnitin