RENAN APARECIDO FERNANDES Fitossíntese de nanopartículas de prata a partir de extrato de cascas de romã e desenvolvimento de formulação para tratamento de feridas: avaliação antimicrobiana, citotóxica e potencial cicatrizante em um modelo in vivo. Araçatuba – SP 2017 RENAN APARECIDO FERNANDES Fitossíntese de nanopartículas de prata a partir de extrato de cascas de romã e desenvolvimento de formulação para tratamento de feridas: avaliação antimicrobiana, citotóxica e potencial cicatrizante em um modelo in vivo. Tese apresentada à Faculdade de Odontologia do Campus de Araçatuba – Universidade Estadual Paulista “Júlio de Mesquita Filho”- UNESP, para obtenção do Título de DOUTOR EM CIÊNCIA ODONTOLÓGICA (Área de concentração em Biomateriais). Orientadora: Profª. Ass. Drª. Debora de Barros Barbosa Co-Orientadora: Drª Andresa Aparecida Berretta e Silva Araçatuba – SP 2017 Catalogação na Publicação (CIP) Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Fernandes, Renan Aparecido. F363f Fitossíntese de nanopartículas de prata a partir de extrato de cascas de romã e desenvolvimento de formulação para tratamento de feridas: avaliação antimicrobiana, citotóxica e potencial cicatrizante em um modelo in vivo. / Renan Aparecido Fernandes. - Araçatuba, 2017 120 f. : il. ; tab. Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientadora: Profa. Debora de Barros Barbosa Coorientadora: Profa. Andresa Aparecida Berretta e Silva 1. Biofilmes 2. Candida albicans 3. Staphylococcus aureus 4. Prata 5. Nanotecnologia I. T. Black D15 CDD 617.695 DADOS CURRICULARES RENAN APARECIDO FERNANDES NASCIMENTO 02/02/1991 – JOSÉ BONIFÁCIO - SP FILIAÇÃO José Fernandes Sobrinho Maria Aparecida Paulo Fernandes 2009/2013 Curso de Graduação em Odontologia Faculdade de Odontologia de Araçatuba - Universidade Estadual Paulista “Júlio de Mesquita Filho”. 2013/2013 Curso de Aperfeiçoamento em Prótese Parcial Fixa Faculdade de Odontologia de Araçatuba - Universidade Estadual Paulista “Júlio de Mesquita Filho”. 2013/2013 Aperfeiçoamento em Endodontia automatizada. Faculdade de Odontologia de Araçatuba - Universidade Estadual Paulista “Júlio de Mesquita Filho”. 2013/2015 Obtenção do título de Mestre em Ciência Odontológica. Faculdade de Odontologia de Araçatuba - Universidade Estadual Paulista “Júlio de Mesquita Filho”. DEDICATÓRIA Aos meus amados pais, Maria Aparecida Paulo Fernandes e José Fernandes Sobrinho. São minha base, sem vocês nada do que almejo seria realidade. Agradeço por toda a educação, amor, carinho a mim dispensados, honrarei o nome de vocês por toda a minha vida. Obrigado por tudo o que fazem por mim, com certeza se pudéssemos voltar em uma outra vida pediria para ser filho de vocês novamente! Em meio a simplicidade me ensinaram tudo o que eu precisava saber sobre a vida, princípios como a honestidade, respeito, amor e a lutar sempre pelo que almejamos em nossas vidas. Amo vocês! Ao meu irmão Lucas José Fernandes. Que sempre foi meu confidente, sempre me apoiou, obrigado por me dar forças, mesmo diante das dificuldades. Tenho muito orgulho de ver o homem que se tornou! A minha avó Clarice (in memorian). Que sempre foi meu orgulho, pessoa de maior coração que já conheci em toda minha vida. Sempre esta em minhas orações e lembranças. Mesmo distante sei que estás em nosso meio, por mais que a saudade doa sei que estás me observando em todos os momentos. Amo você vó. A minha esposa Ana Sara Muriana Mendonça. Talvez a escolha mais sábia da minha vida até o presente momento foi ter me casado com você! Minha companheira, amiga, guerreira! Me espelho muito em sua determinação pelo trabalho, em seu amor pela família. Deus não poderia ter me dado presente maior do que você. Espero ter a oportunidade de dividir muitos e muitos momentos importantes da minha vida com você. Obrigado por tudo o que faz por mim e por tudo o que representa em minha vida! Te amo. AGRADECIMENTOS ESPECIAIS A Deus Agradeço a Deus por ser tão bom comigo, por me dar uma família linda e amigos verdadeiros, por permitir que minha caminhada na Terra seja tão suave, mesmo diante de todas as dificuldades já vividas, que só serviram para me fortalecer ainda mais. Obrigado Pai por todas as bênçãos. À minha orientadora, Profª. Ass. Drª. Débora Barros Barbosa Professora, agradeço primeiramente a Deus por ter me dado a oportunidade de conhecer você. Através de sua forma incrível de ser aprendi muito, não somente no âmbito profissional, como também em minha formação pessoal. Sou tão feliz por ter você como orientadora, pois sei que além de ser minha guia és minha amiga! Com toda certeza posso dizer que você faz parte da minha família! Te agradeço imensamente por tudo o que fez e faz por mim, com certeza transformou a minha vida! Muito obrigado por tudo! Ao minha co – orientadora Drª. Andresa Aparecida Berretta e Silva. Agradeço imensamente pela recepção na cidade de Ribeirão Preto e na empresa Apis flora. Seus ensinamentos foram de grande valia para minha formação acadêmica e pessoal. Muito obrigado por toda a contribuição. À Fundação de Ampara à Pesquisa do Estado de São Paulo (FAPESP) Pelo grande incentivo na forma de Bolsa de Doutorado (FAPESP, Processo 2016/04230-9) AGRADECIMENTOS À Faculdade de Odontologia de Araçatuba - UNESP, na pessoa de seu diretor Prof. Tit. Wilson Roberto Poi, pela oportunidade de aprendizado e crescimento pessoal e profissional. À Universidade Federal de São Carlos - UFSCar, nas dependências do Laboratório Interdisciplinar de Eletroquímica e Cerâmica do Departamento de Química, onde foi possível realizar todas às sínteses propostas no trabalho. À APIS FLORA INDUSTRIAL E COMERCIAL LTDA, onde parte fundamental deste trabalho foi realizado com a ajuda de extrema importância de alguns dos alunos e funcionários em especial Andrei, Elina, Rebeca e Andréia. À Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP, na pessoa da Profª Drª. Maria José Vieira Fonseca e Profº Dr. Eduardo Barbosa Coelho, por abrir as portas de seus laboratórios para o desenvolvimento de parte do trabalho, muito obrigada. Ao atual coordenador do Programa de Pós-graduação em Ciência odontologica, da Faculdade de Odontologia de Araçatuba - UNESP, Prof. Adj. Luciano Tavares Angelo Cintra, pela competência e acessibilidade. Ao Departamento de Materiais Odontológicos e Prótese da Faculdade de Odontologia de Araçatuba - UNESP, representado por todos os seus Funcionários, pelo convívio agradável. Ao Departamento de Odontopediatria da Faculdade de Odontologia de Araçatuba - UNESP, representado por todos os seus Professores e Funcionários, pela oportunidade de realizar meu mestrado e conviver com pessoas tão agradáveis. Ao Dr Luiz Gorup, Dr Francisco Nunes e Profº Dr Emerson Camargo por toda acessibilidade disposição e ajuda. Ao Prof. Adj. Alberto Carlos Botazzo Delbem, por toda contribuição não só material, mas também intelectual que me proporcionou nesses últimos anos, com toda certeza aprendi muito com o senhor. Obrigado. A Prof. Drª Elaine Conti, pela disponibilidade e aceite do convite para compor a banca examinadora de meu trabalho e toda a contribuição oferecida. A Prof Drª Denise Pedrini Ostini por toda atenção e ensinamentos a mim oferecidos ao longo de minha formação. Às excelentes funcionárias da seção de Pós-graduação da Faculdade de Odontologia de Araçatuba - UNESP, Valéria, Cristiane e Lilian, pelo apoio, suporte e ajuda a mim dispensados. Aos queridos Pós-graduandos do Departamento de Odontopediatria da Faculdade de Odontologia de Araçatuba - UNESP, pelo convívio maravilhoso, pela amizade e por toda a ajuda. Graças a vocês me sinto em casa neste Departamento. A minha amiga Gabriela Lopes Fernandes por todo companheirismo desde a nossa graduação, obrigado pelo apoio, incentivo, trabalhos divididos, momentos de lazer, com certeza Deus sempre sabe quem colocar em nossos caminhos, admiro muito você, obrigado por deixar minha caminhada mais doce e serena, espero poder estar ao seu lado por muitos e muitos anos. Obrigado por toda ajuda durante o desenvolvimento de meu trabalho, você foi essencial para o desenvolvimento do mesmo. Obrigado por tudo! A minha amiga Luciene Pereira de Castro por todo companheirismo, apoio, ensinamentos e toda amizade depositada. Obrigado por tudo. Ao meu amigo (irmão) e companheiro de república Diego Felipe Mardegan Gonçalves por todos os ensinamentos, amizade e incentivo. Meu muito obrigado. Aos pais de minha esposa Ana Cláudia Muriana Mendonça e João Costa Mendonça, por todo o apoio, carinho e por permitir que eu faça parte da família de vocês! Tenho um carinho e admiração muito grande por vocês, são como um espelho para minha vida pessoal e profissional. Muito obrigado por tudo! “A ciência nunca resolve um problema sem criar pelo menos outros dez”. (George Bernard Shaw) Fernandes RA. Fitossíntese de nanopartículas de prata e desenvolvimento de formulação para tratamento de feridas: avaliação antimicrobiana, citotóxica e potencial cicatrizante em um modelo in vivo [Tese]. Araçatuba: Universidade Estadual Paulista; 2017. RESUMO GERAL O objetivo deste estudo foi avaliar a capacidade de produção de nanopartículas de prata através do extrato da casca de romã, e produzir formulações contendo estas nanopartículas para uso em feridas. Elas foram testadas quanto à ação antimicrobiana, citotóxica e potencial cicatrizante. Para a produção das nanopartículas de prata propôs-se uma síntese utilizando-se como base duas metodologias já estabelecidas na literatura, utilizando-se para reação carboximetilcelulose, propilenoglicol, nitrato de prata, água e extrato da casca de romã como agente redutor. O extrato da casca de romã foi caracterizado em parâmetros como pH, massa seca e quantidade de taninos bioativos (ácido elágico e totais fenólicos expressos em ácido gálico). Os totais fenólicos do extrato foram também dosados após seu aquecimento nas diferentes condições de tempo e temperatura propostos para as sínteses das nanopartículas de prata (12 minutos, 1 hora e 2 horas, à 50ºC e 100ºC). As nanopartículas de prata produzidas foram, então, adicionadas a uma solução contendo compostos para produção de formulações para serem utilizadas no tratamento de feridas. Elas foram caracterizadas através de espectroscopia UV-Visível, microscopia eletrônica de varredura (MEV), potencial zeta e dosagem de íons remanescentes após as reações. A atividade antimicrobiana tanto das nanopartículas como de suas formulações contra Candida albicans SC 5314 e Staphylococcus aureus ATCC 25923 foi avaliada por meio do método da microdiluição. Na avaliação dos extratos, apesar de ocorrer um aumento na concentração de totais fenólicos com o aumento da temperatura, a concentração inibitória mínima (CIM) manteve-se estável com valores de 391 µg/ml e 781 µg/ml para S. aureus e C. albicans. A formação das nanopartículas de prata foi confirmada com a formação de picos característicos na espectroscopia UV-Visível e pelas imagens de MEV, onde verificou-se que a síntese com tempo de reação de 12 minutos e aquecimento a 50ºC gerou nanopartículas mais uniformes e melhor distribuídas na formulação. As sínteses propostas promoveram a redução iônica da prata de aproximadamente 100%, independente do tempo temperatura utilizados na reação. Valores de CIM para as nanopartículas de prata foram de 67,50 µg/ml e 68,75 µg/ml respectivamente para S. aureus e C. albicans independente das variações das condições de síntese. Após a seleção da síntese das nanopartículas de prata por 12 min à 50ºC, estas partículas foram também caracterizadas por difração de raios-X e microscopia eletrônica de transmissão (TEM), e as respectivas formulações por meio de espalhamento de luz dinâmica, dosagem de íons livres, potencial zeta, MEV e TEM. Para essas formulações foi também realizado um teste de estabilidade variando-se umidade e temperatura. Além da atividade antimicrobiana contra Candida albicans SC 5314 e Staphylococcus aureus ATCC 25923, a citotoxicidade em fibroblastos (L929) das nanopartículas de prata e das formulações destas partículas e do extrato da casca de romã foram também avaliadas. Para os extratos foram observados valores de 3,13, 86,39±0,96% m/m, 3,64±0,03 mg/g, 392,0±9 mg/g respectivamente para pH, massa seca, ácido elágico e totais fenólicos. Como controle, foram produzidas nanopartículas de prata sintetizadas convencionalmente (AgNP química), e observou-se potencial redutor de 99,89% e 99,51% para as sínteses utilizando-se extrato de romã (AgNP green) e um agente redutor químico convencional (AgNP química). Verificou-se a formação de partículas com tamanhos médios de 89 e 19 nm para nanopartículas green e química. A formulação contendo as nanopartículas de prata apresentaram um potencial antimicrobiano expressivamente maior do que o princípio ativo isolado, sendo 255 e 4 vezes mais efetiva contra S. aureus e C. albicans, respectivamente. Os valores de citotoxicidade foram consideravelmente menores para as nanopartículas de prata sintetizadas pelo extrato de romã quando comparadas as produzidas convencionalmente. De acordo com os valores de CIM e da citotoxicidade, a concentração das formulações foi ajustada, gerando assim três formulações: i) AgNP green, ii) AgNP química e iii) extrato de romã, nas concentrações de 337,5 µg/ml, 5,55 µg/ml e 94 µg/ml respectivamente. Para o estudo in vivo, utilizou-se como controle um medicamento comercial contendo prata indicado para tratamento de feridas (Sulfadiazina de prata). Foram selecionados noventa ratos Wistar machos com peso médio de 180 gramas. Foi induzida diabetes nos ratos, e, em seguida, realizou-se duas incisões no dorso dos animais e as lesões foram imediatamente infectadas com S. aureus (ATCC 25923) e C. albicans (SC 5314). Após 24 h, os animais foram divididos em grupos de acordo com as formulações propostas em cada tratamento, seguindo-se um protocolo de duas vezes ao dia por 2, 7 e 14 dias. Após o período de tratamento os animais foram eutanasiados e verificado o potencial reparador através do índice de fechamento de ferida, avaliação do infiltrado inflamatório, angiogenese, mieloperoxidase e hidroxiprolina. Ainda foi verificado o potencial antimicrobiano das formulações através da contagem de células viáveis de cada microrganismo infectado nas feridas. De forma geral, as formulações contendo nanopartículas de prata mostraram os melhores resultados para o fechamento das feridas, apresentando ainda uma atividade anti-inflamatória maior que a do extrato de casca de romã, o qual apresentou atividade pró inflamatória. Todos os tratamentos não foram capazes de reduzir de forma significativa o número de células de C. albicans, enquanto para S. aureus todos os tratamentos apresentaram redução significativa após quatorze dias de tratamento. Independente das nanopartículas de prata serem produzidas quimicamente ou por meio do extrato da casca de romã, ambas apresentaram considerável potencial de reparo de feridas infectadas em modelos in vivo com ratos. Os achados das presentes pesquisas reforçam e estimulam o uso potencial das nanopartículas de prata no tratamento de feridas, com destaque para a síntese green por gerar menos danos ao meio ambiente e às pessoas envolvidas tanto em sua produção como aos pacientes, e por apresentar, ainda, custo inferior quando comparada às sínteses utilizando reagentes químicos e processos convencionais. Palavras-chave: Biofilmes, Candida albicans, Staphylococcus aureus, prata, nanotecnologia. Fernandes RA. Phytosynthesis of silver nanoparticles using pomegranate peel extract and development of formulation for wound healing: antimicrobial and cytotoxicity evaluation and repair potential in a in vivo model. [thesis]. Araçatuba: UNESP - São Paulo State University; 2017. GENERAL ABSTRACT The aim of this study was to investigate the production of silver nanoparticles through peel extract of pomegranate, and produce formulations containing these particles to be used in wound healings. Its antimicrobial action, cytotoxicity and healing potential were tested. The synthesis of silver nanoparticles were based on two methods proposed in the literature with some modifications, which were used carboxymethylcellulose, propylene glycol, silver nitrate, water and peel extract of pomegranate as reducing agent. The peel extract was characterized by pH, dry mass and bioactive tannins (elagic acid and total phenols expressed as galic acid). The total phenols were also quantified after being heated at 50ºC and 100ºC for 12 minutes, 1 hour and 2 hours. Then, silver nanoparticles were added in a solution containing products to develop a formulation to be tested in wound healing. They were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM), zeta potential and the quantification of remaining silver ions after the synthesis reaction. The antimicrobial activity of the nanoparticles and formulations were tested against Candida albicans (SC 5314) e Staphylococcus aureus (ATCC 25923) by microdilution method. After submitting the peel extract to different conditions of temperature and times (50ºC and 100ºC for 12 minutes, 1 hour and 2 hours), it was noted that the values of the minimum inhibitory concentration was not affected and were 391 µg/ml and 781 µg/ml for S. aureus and C. albicans. The formation of silver nanoparticles was confirmed through the formation of characteristic peaks in the UV-Vis spectroscopy and SEM images, and it was observed that the reaction at 50ºC for 12 min produced silver nanoparticles with regular forms and better dispersed in the formulation. The synthesis proposed promoted the reduction of silver ions at about 100%, regardless of the time and temperature used in the reaction, which also did not interfere in antimicrobial activity against C. albicans (68,75 µg/ml) and S. aureus (67,50 µg/ml). After selecting the reaction at 50ºC for 12 min, the silver nanoparticles produced ere also characterized by X-ray diffraction and transmission electron microscopy (TEM), and the respective formulations through dynamic light scattering, free ion dosage, zeta potential, SEM and TEM. The stability test varying humidity and temperature was also performed for those formulations. Besides antimicrobial assays, the cytotoxicity (L929 fibroblasts) of the silver nanoparticles and the formulations of these particles and of the pomegranate peel extract were evaluated. It was observed in the peel extract the values of 3,13, 86,39±0,96% m/m, 3,64±0,03 mg/g and 392,0±9 mg/g respectively for pH, dry mass, elagic acid and total phenols concentrations. Silver nanoparticles produced by conventional chemical method was prepared and used as controls, and it was noted the reduction potential of 99,89% and 99,51% for the synthesis using pomegranate peel extract (AgNP green) and chemical reducing agent (AgNP chemical). The averages sizes of green and chemical AgNP were 89 and 19 nm. The formulation containing silver nanoparticles presented an antimicrobial potential expressively higher than the active input, being 254 and 5- fold more effective against S. aureus and C. albicans. Also, the cytotoxicity was notable reduced when silver nanoparticles were produced using pomegranate peel extract. Based on the MIC values and the cytotoxicity findings, the concentration of the formulations were determined: i) AgNP green at 337.5 µg/ml, ii) AgNP chemical at 5.55 µg/ml, and iii) pomegranate peel extract at 94 µg/ml. In the in vivo study, a commercial form of silver (Sulfadizsine) to the wound healing was used as control. Ninety Wistar male rats were selected, and, after inducing diabetes, two incisions on the dorsum of the animals were made and followed infected with S. aureus (ATCC 25923) and C. albicans (SC 5314). After the infection, the animals were treated with the formulations twice a day for 2, 7 and 14 days. Then, the animals were euthanized and the repair potential was verified through wound closure index, inflammatory infiltrate evaluation, angiogenesis, myeloperoxidase and hydroxyproline. It was also determined the antimicrobial potential by counting the viable cells of each microorganism used to infect the wounds. In general, the formulations containing silver nanoparticles promoted a better closure of the wounds, and a higher anti-inflamatory activity than the peel extract formulation which otherwise presented a pro-inflamatory effect. All formulations could not significantly reduce the viable cells of C. albicans, while for S. aureus they reduced significantly the cells after 14 days of treatment. Silver nanoparticles produced by both green and conventional chemical process present notable potential in repairing infected wounds in in vivo rat model. The findings of the present research strengthen and stimulate the potential application of silver nanoparticles in wound healings, highlighting the green production of these particles which apart from being lower costly, it is ecofriendly and less detrimental to people involved in its production and use. Keywords: Biofilms, Candida albicans, Staphylococcus aureus, silver, virulence nanotechnology. LISTA DE FIGURAS Chapter 1 Silver nanoparticles: an in vitro investigation of phytosynthesis process and insights into antimicrobial formulations Figure 1 Spectral UV/Visible of silver nanoparticles produced under different conditions, controls without reducing agent (pomegranate peel extract) (A), 50°C for 12 min (B), 50ºC for 1 h (C), 100ºC for 2 h (D).………………………………………………. 50 Figure 2 Images of scanning electron microscopy of silver nanoparticles produced under different conditions, 50°C for 12 min (A), 50ºC for 1 h (B), 100ºC for 2 h (C) and formulations prepared from them (D, E and H). …………………………………………………… 51 Chapter 2 Antimicrobial potential and toxicity of silver nanoparticles phytosynthesized by pomegranate peel extract. Figure 1 XDR of green and silver nanoparticles...………......................... 80 Figure 2 Images of scanning electron microscopy (MEV) (1) and transmission electronic microscopy (TEM) (2). A- Green silver nanoparticles, B- Green sprat formulation, C- Chemical silver nanoparticles and D- Chemical spray formulation..……………………………………… 81 Figure 3 Total phenols concentration for green-spray-formulation and pomegranate-spray formulation in different periods...………………………………………………… 82 Figure 4 Cytotoxicity evaluation of respective active input and their formulations....…………………………………… 83 Chapter 3 Green and chemical silver nanoparticles and pomegranate formulations as potential wound healing and antimicrobial medicines. Figure 1 Images and wound healing rate of different treatments for 14 days. C: control (without treatment); SQ: chemical spray; SG: green spray; Ps: spray of pomegranate peel extract; Sulf: silver sulfadiazine..………........................................................ 107 Figure 2 Mean values of the logarithm of colony forming units per gram (log10 CFU/g) for mixed C. albicans and S. aureus biofilms treated for different treatments for 14 days. C: control (without treatment); SQ: chemical spray; SG: green spray; Ps: spray of pomegranate peel extract; Sulf: silver sulfadiazine..……………………………………………. 108 Figure 3 Results of inflammatory infiltrate and fibroblasts cells for 14 days of treatment. C: control (without treatment); SQ: chemical spray; SG: green spray; Ps: spray of pomegranate peel extract; Sulf: silver sulfadiazine…….. 109 Figure 4 Results of blood vessels count for 14 days of treatment. C: control (without treatment); SQ: chemical spray; SG: green spray; Ps: spray of pomegranate peel extract; Sulf: silver sulfadiazine……………………………………… 110 Figure 5 Results of myeloperoxidase for 14 days of treatment. C: control (without treatment); SQ: chemical spray; SG: green spray; Ps: spray of pomegranate peel extract; Sulf: silver sulfadiazine………………………………………. 111 Figure 6 Results of hydroxyproline for 14 days of treatment. C: control (without treatment); SQ: chemical spray; SG: green spray; Ps: spray of pomegranate peel extract; Sulf: silver sulfadiazine………………………………………. 112 LISTA DE TABELAS Chapter 1 Silver nanoparticles: an in vitro investigation of phytosynthesis process and insights into antimicrobial formulations. Table 1 Values of total phenols and minimum inhibitory concentration (MIC) for pomegranate peel extract submitted under different conditions.………………………………………………. 48 Table 2 Values of ion reduction and minimum inhibitory concentration (MIC) for pomegranate peel extract submitted under different conditions..……………………………………………… 49 Chapter 2 Antimicrobial potential and toxicity of silver nanoparticles phytosynthesized by pomegranate peel extract. Table 1 Values of silver ionic reduction and zeta potential for green and chemical spray formulations in different periods. ………………..................................................... 78 Table 2 Ion concentration and values of minimum inhibitory concentration (MIC) for S. aureus and C. albicans …………………………………………………………... 79 SUMÁRIO 1. Introdução Geral............................................................................. 26 2. Capítulo 1 - Silver nanoparticles: an in vitro investigation of phytosynthesis process and insights into antimicrobial formulations. 2.1. Abstract…………………………………………………….. 2.2. Introduction………………………………………………… 2.3. Materials and methods……………………………………... 2.4. Results……………………………………………………… 2.5. Discussion………………………………………………….. 2.7. References.............................................................................. 36 37 38 41 42 44 3. Capítulo 2 - Antimicrobial potential and toxicity of silver nanoparticles phytosynthesized by pomegranate peel extract. 3.1. Abstract.................................................................................. 3.2. Introduction............................................................................ 3.3. Materials and methods........................................................... 3.4. Results.................................................................................... 3.5. Discussion.............................................................................. 3.6. References.............................................................................. 53 54 56 62 65 70 4. Capítulo 3 - Green and chemical silver nanoparticles and pomegranate formulations as potential wound healing and antimicrobial medicines. 4.1. Abstract...................................................................... 4.2. Introduction................................................................ 4.3. Materials and methods............................................... 4.4. Results........................................................................ 4.5. Discussion.................................................................. 4.6. References.................................................................. Anexo................................................................................................. 85 87 89 94 97 100 113 INTRODUÇÃO GERAL 26 1. INTRODUÇÃO GERAL Muitas das doenças que acometem os seres vivos são caracterizadas por infecções que estão intimamente relacionadas com a capacidade de bactérias e/ou fungos em formar biofilmes. Os biofilmes são comunidades organizadas de microrganismos aderidos a uma superfície podendo ser biótica ou abiótica, rodeado por uma matriz extracelular em um ambiente aquoso (Costerton, Stewart et al 1999). Eles são geralmente compostos por mais de uma espécie de microrganismos, formando assim biofilmes mistos. No entanto biofilmes simples são encontrados em infecções especificas como nos casos de infecções de dispositivos e equipamentos médicos, recebendo destaque na literatura para os biofilmes de Pseudomonas aeuraginosa, Pseudomonas fluorescens, Escherichia coli, Vibrio cholerae e Staphylococcus aureus (O’Toole, Kaplan et al 2000). Os biofilmes além de serem capazes de colonizar as superfícies abióticas como mencionado anteriormente são capazes e se desenvolverem em diversos tecidos dos seres humanos como no trato gênito urinário, pele, aparelho respiratório, trato gastrintestinal dentre outros (De, Raj et al. 2017). Feridas crônicas são encontradas devido à presença de biofilmes persistentes e que podem ser localizados em diversos locais, como por exemplo, em bolsas periodontais na boca, úlceras venosas, infecções em dispositivos como implantes e infecções adquiridas em âmbito hospitalar (Ammons and Copie 2013). Assim estes biofilmes são caracterizados como um problema nos casos de úlceras, pois as feridas crônicas não cicatrizam se devido à persistência de infecções e inflamações (Chen and Wen 2011). 27 Diversos microrganismos estão relacionados ás úlceras crônicas, e dois em específico necessitam receber atenção especial neste processo, pois são abundantemente encontrados em diversas regiões do corpo humano, além de estarem presentes em biofilmes de úlceras crônicas, entre eles a bactéria Gram positiva Staphylococcus aureus e o fungo Candida albicans. Os biofilmes envolvendo Candida albicans e Staphylococcus aureus estão diretamente relacionados com patologias como a candidíase e queilite angular (Na and Gijzen 2016). O fungo Candida albicans é um microrganismo pleomórfico, oportunista, que pode apresentar diferentes formas de acordo com o meio e o estímulo recebido (Shepherd. 1990). As leveduras do gênero Candida constituem uma menor parte da flora microbiana oral (Kulak, Arikan et al. 1997) além de colonizar a pele, órgãos genitais e trato gastrintestinal, são consideradas comensais em indivíduos adultos saudáveis (Thein, Samaranayake et al. 2006). As espécies de Candida produzem enzimas hidrolíticas (exemplo: fosfolipases e proteinases) que colaboram para a invasão dos tecidos hospedeiros através da digestão ou destruição das membranas celulares (Ghannoum 2000); (Naglik, Challacombe et al. 2003) (Schaller, Borelli et al. 2005), este processo inicia se quando o sistema imunológico do hospedeiro encontra se debilitado (Bassetti, Merelli et al. 2013). O fungo C. albicans necessita de muita atenção, pois possui a capacidade de aderir-se firmemente em tecidos e dispositivos médicos (Ramage and Lopez-Ribot 2005), além de células em biofilmes mostrarem-se resistentes a maioria dos antifúngicos convencionais como os azoles e a anfotericina B que podem causar diversos efeitos colaterais no hospedeiro (Laniado-Laborin and Cabrales-Vargas 2009). 28 Staphylococcus aureus é mundialmente conhecido por causar doenças na pele, tecidos moles, pneumonia, abcessos e osteomielite. (Chowers, Paitan et al. 2009). O Staphylococcus aureus é uma bactéria gran positiva encontrada em diversos locais do corpo humano e também em outros animais, relacionado a grande taxa de morbidade e mortalidade mundial (Van Hal et al. 2012). Relatos de pacientes portadores da Síndrome da Imunoficiencia Adquirida (AIDS) e infectados com este microrganismo chegaram ir a óbito, além de contribuir para o desenvolvimento da tuberculose, enfatizando assim a necessidade de se estudar tal microrganismo (van Hal, Jensen et al. 2012). Este também é conhecido por diversas infecções nasocomiais, além de algumas cepas mostrarem-se capazes de infectar pacientes saudáveis e ser responsável desde infecções brandas na pele, infecções de tecido mole até doenças fatais como a pneumonia necrotizante, osteomielite e septicemia (Saavedra-Lozano, J et al. 2015). Ainda há relatos da presença de Staphylococcus aureus em úlceras e osteomielite sendo frequente a presença de cepas resistentes a metacilina (MRSA) (Martini et al. 2015). Eles tem se tornado resistentes à maioria dos agentes antimicrobianos convencionais devido ao uso indiscriminado e incorreto de antibióticos pela população (Thurlow, L.R et al. 2012). Foi verificado também que a presença de Staphylococcus aureus em úlceras venosas é mais prevalente do que outras espécies de microrganismos (Ortiz et al. 2014). Para cicatrização de feridas o organismo precisa prevenir ou combater estes microrganismos além de passar por um processo de reconstrução de tecidos lesionados e estruturas celulares. Este processo normalmente está dividido em três passos, incluindo a inflamação, proliferação das células e remodelação tecidual 29 (Gurtner et al. 2008). A fim de melhorar a qualidade do tecido reparado e para aumentar a eficácia de materiais utilizados na cicatrização de feridas, medicamentos e dispositivos com diferentes tipos de nanopartículas têm sido desenvolvidos. Alternativas estudadas incluem algodões com nanopartículas de prata, que foram eficazes contra Escherichia coli, Staphylococcus aureus e Candida albicans em concentração de 250 ppm (Hebeish et al. 2014), também celulose bacteriana com deposição de nanopartículas de prata foi desenvolvida, apresentando atividade antibacteriana contra Staphylococcus aureus (Wu et al. 2014), assim como o desenvolvimento de nanopartículas encapsuladas com curcumina que também inibiram o crescimento de S. auereus MRSA e Pseudomonas aeruginosa (Krausz et al. 2015). Apesar das alternativas apresentadas até o presente momento, a busca de soluções efetivas para a cura de feridas infectadas ainda tem sido constante. Neste sentido é necessária a busca por alternativas de tratamentos que visem o combate de microrganismos patogênicos, além de auxiliar o organismo a controlar seu potencial inflamatório e estimular o reparo da lesão. A nanotecnologia associada a compostos naturais tem recebido intensa atenção na literatura atual. Dentre os compostos naturais, a romã (Punica granatum) se destaca por ser uma fonte rica de elagitaninos bioativos, e tem sido utilizada como vermífugo, adstringente, antibiótico, anti-inflamatório, em feridas, úlceras, vaginite e infecções do trato respiratório e urinário (Harde et al. 1970, Willianson et al. 2002; Duke, 4 Codwin & Cillier, 2002). O extrato da romã é composto principalmente de alcalóides e polifenóis. Ele tem demonstrado uma variedade de ações benéficas, incluindo efeito antioxidante e atividade antiviral. Dentre as 30 moléculas antioxidantes presentes nos extratos, existem uma grande quantidade de flavonoides, alcaloides, ácidos orgânicos, compostos polifenólicos, dentre outros. (Orgil et al. 2014). Atualmente, estudos tem focado na capacidade antioxidante e anti-inflamatória de extratos naturais, como investigado por Omar et al. 2015, onde o extrato de Punica granatum (romã) exerceu um grande potencial anti- inflamatório e antioxidante em ratos. A atividade antimicrobiana de extratos dessa planta tem sido relatada principalmente contra Staphylococcus aureus e Escherichia coli (Machado et al. 2003). Devido às suas propriedades biológicas, a casca da romã tem recebido atenção por parte de pesquisadores e consumidores (Reddy et al. 2007). O interesse de uso da romã tem se voltado também para o campo da nanotecnologia, onde já se tem comprovado a redução da prata iônica por meio de diferentes partes da romã (Barbosa et al, 2014) caracterizando a redução da prata iônica a nanopartículas de prata. Por se utilizar de plantas, a síntese de nanopartículas tem sido considerada sustentável ou “green”. Estudos prévios (não publicado ainda) dos mesmos autores indicam uma efetividade antifúngica semelhante às nanopartículas de prata (NPAg) obtidas convencionalmente por rota química (Monteiro et al 2014a; Monteiro et al 2014b). Assim, com a crescente tolerância dos microrganismos frente aos antimicrobianos convencionais e consequente aumento em sua dosagem para melhorar sua eficácia, há a necessidade pela busca de novas drogas que sejam efetivas não somente contra biofilmes patogênicos, mas também menos tóxicas as células do organismo hospedeiro auxiliando assim no processo anti-inflamatório e cicatrizante. Portanto, o objetivo geral do presente trabalho foi avaliar a 31 capacidade de produção de nanopartículas de prata através do extrato da casca de romã, e produzir formulações contendo estas nanopartículas para uso em feridas. Elas foram testadas quanto à ação antimicrobiana, citotóxica e potencial cicatrizante em um modelo in vivo em rato. Referências Ammons, M. C. and V. 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Lett Appl Microbiol 54(5):383-91. 35 CAPÍTULO 1 Silver nanoparticles: an in vitro investigation of phytosynthesis process and insights into antimicrobial formulations. *Artigo enviadopara o periódico Biofouling 36 Capítulo 1 Silver nanoparticles: an in vitro investigation of phytosynthesis process and insights into antimicrobial formulations. 2.1. Abstract In the present study, silver nanoparticles (AgNP) mediated by pomegranate peel extract were prepared under different times and temperatures of reaction, and the effectiveness of each synthesis process was determined by the quantification of silver ions remaining. It was also prepared spray formulations containing those AgNP, and the antimicrobial activity of the AgNP and AgNP formulations was tested against Candida albicans and Staphylococcus aureus. The phytosynthesis were carried out at 50ºC for 12 minutes (I) or 50ºC for 2 hours or 100ºC for 2 hours (III), and the samples were physicochemical characterized by spectroscopy Uv-Visible and scanning electron microscopy. The silver ions concentration was verified by a specific electrode and the rate of ion reduction was calculated. Microdilution method was used to determine the minimum inhibitory concentration (MIC) of the AgNP and AgNP formulations against the microorganisms. The AgNP formation was confirmed by Uv-Vis at wave-leght peaks ranging from 375 to 445 nm. SEM images showed regular forms and well- dispersed AgNP specially phytosynthesized at 50ºC for 12 minutes. The rate of reduction of the synthesis was I (99.89%) > II (96%) < II (98.6%). Time and temperature did not interfere in the antimicrobial activity of the AgNP samples, and surprisingly AgNP in the spray formulations increased 244-and 5-fold the effectiveness against S. aureus and C. albicans. The phytosynthesis of AgNP using pomegranate peel extract besides being quite effective and eco-friendly, it may be suitable for prophylaxis and treatment of infected wounds. 37 Capítulo 1 Keywords: Biosynthesis, silver, nanoparticles, Punica granatum, heating. 2.2. Introduction Due to its optical, magnetic, antimicrobial and electronic properties metallic nanoparticles has received a special attention in recent years. One of the most exploited metals in this sense is silver, more specifically the silver nanoparticles, being used in several areas such as biomedical, pharmaceutical, water treatment, energy, among others (Abbasi, Milani et al. 2016), (Liu and Astruc 2017), (Sai Saraswathi, Kamarudheen et al. 2017). For the production of silver nanoparticles several methodologies can be employed as by means of chemical reductions (Gurusamy 2017), electrochemical (Elemike, Fayemi et al. 2017), photochemical (Gabriel, Gonzaga et al. 2017) , sonochemical (Elsupikhe, Shameli et al. 2015), microwave (Francis, Joseph et al. 2017), γ-radiation (Mansour, Eid et al. 2017) and laser ablation (Delmee, Mertz et al. 2017). One of the most used techniques for obtaining silver nanoparticles is the chemical reduction, however the use of chemical reagents causes the production to become expensive, besides producing unwanted effects such as the production of toxic nanoparticles to human cells (Gurusamy 2017) and to the environment. Even so that the aggregation of the silver nanoparticles does not occur in stabilizing agents are added, which can thus generate even more unwanted effects (Khodashenas 2015), (Ren 2015). In this sense there is a need to search for cheaper alternatives, eco-friendly and biocompatible with the human organism. 38 Capítulo 1 Nowadays the use of green chemistry has been approached as an alternative to the previously reported techniques. Different natural processes are used to obtain metal nanoparticles such as bacteria, fungi (Wypij, Golinska et al. 2017), honey (Philip 2010) and plant extract (Francis, Joseph et al. 2017). Thinking of the benefits offered by plant extracts such as the possibility of availability in different regions of the world, low cost, possibility of large scale production, safe for the environment, besides the possible synergism of properties like anti-inflammatory, antioxidant and antimicrobial, this technique shows very promising. For synthesis of silver nanoparticles several plants have been used, time and temperatures of varied reactions. Therefore, the aim of this study was to produce silver nanoparticles mediated by pomegranate peel extract, and evaluate its potential of reduction according to different times and temperatures of reaction. 2.3. Materials and methods Collecting fruit and obtaining extracts The fruits were collected in May of 2015 from a farm in the city of Mirandópolis, São Paulo, Brazil. The peels were withdrawals from the pomegranates, dried at 50ºC (approximately 48 hours), grinding and siving, tracked by the extraction by maceration followed by percolation process (de Oliveira, de Castro et al. 2013). Phytosynthesis of silver nanoparticles For the development of this synthesis it was based on two methodologies proposed by Gorup et al. (2011) and Das Kumar et al. (2015) with modifications. 39 Capítulo 1 The base of the reaction was fixed in propylene glycol (PG) (Labsynth, Diadema, Brazil) (20%), carboxymethylcellulose (CMC) (Labsynth, Diadema, Brazil) (3.5%), and silver nitrate (100 mM), pomegranate peel extract at 30 mg/ml (based on previously data) was used as reducing agent and water to make up 100% of the samples. The variables were time and temperature of reaction. I- Synthesis at 50ºC for 12 minutes. II- Synthesis at 50ºC for 1 hours. III- Synthesis at 100ºC for 2 hours. Development of formulations For the development of formulations, it was used reagents compatible with the synthesis of silver nanoparticles, carboxymethylcellulose (Labsynth, Diadema, Brazil), propylene glycol (Labsynth, Diadema, Brazil) and methylparaben (Labsynth, Diadema, Brazil) in a proportion of 0.1%, 7% and 0.1%, respectively, and the active input (green silver nanoparticles synthesized under different conditions at 1 mg/ml). Determination of total phenolic Total phenolic contents in the extract submitted to different conditions were expressed based on a previously analytical curve of gallic acid (Sigma-Aldrich Chemical Co, St Louis, USA). The samples were homogenized in water and submitted to ultrasonic bath for 30 minutes. An aliquot was mixed with Folin- Denis reagent (Qhemis - High Purity, Hexis, São Paulo, Brazil) and sodium carbonate at 29% (Cinética, São Paulo, Brazil). The next step was the light 40 Capítulo 1 isolation for 30 minutes followed by the measurement at 760 nm (Zielinski and Kozlowska 2000). All samples were analyzed in triplicate. Determination of antimicrobial activity Evaluation of antimicrobial activity of silver nanoparticles and the extract prepared in the same conditions of each synthesis were verified by minimal inhibitory concentration (MIC) according to the Clinical Laboratory Standards Institute. The microorganisms selected were Staphylococcus aureus ATCC 25923 (S. aureus) and Candida albicans SC 5314 (wild type) (Gillum, Tsay et al. 1984) (C. albicans). The assay was performed in triplicate. Characterization of silver nanoparticles and formulations UV/Visible spectroscopy and rate of ion reduction The qualitative verification of the silver nanoparticle formation was verified through the UV/Visible spectroscopy with wavelength ranging from 300 to 800 nm (Spectrophotometer Shimadzu MultSpec-1501, Shimadzu Corporation, Tokyo, Japan). For determination of ion reduction dosages of free silver ions were done. The content of ions was determined using a specific electrode 9616 BNWP (Thermo Scientific, Beverly, MA, USA) coupled to an ion analyzer (Orion 720 A+, Thermo Scientific, Beverly, MA, USA). A previously curve was prepared at 1000 µg Ag/ml, and the samples diluted in water. A silver ionic strength adjuster solution (ISA, Cat. No. 940011) which provides a constant background ionic strength was used (1 ml of each sample/standard: 0.02 ml ISA). 41 Capítulo 1 SEM analyzes The morphology of silver nanoparticles was verified and characterized by scanning electron microscopy on a S-360 microscope, Leo, Cambridge, USA. Statistical analyses GraphPad Prism software (GraphPad Software, Inc, La Jolla, USA) was employed for the statistical analysis with a confidence level of 95 %. Parametric statistical analyses were conducted with one-way ANOVA followed by Tukey's Multiple Comparison test for total phenolic. 2.4. Results Determination of total phenolic The content of total phenolic are exposed on table 1 where interesting results were obtained for heating and time of reaction increasing the content of total phenolic in higher temperature and time. Moreover, the antimicrobial activity for the extract of peel pomegranate did not change with the altering time and temperature. UV/Visible analyzes and rate of ion reduction Figure 1 shows the qualitative formation of silver nanoparticles for all reactions developed. The rate of ion reduction are expressed in table 2 where it’s possible to note that the great reduction occurred in the reaction performed at 50ºC for 12 min, with a rate of reduction of 99.89%. Moreover, synthesis without pomegranate peel extract did not produced silver nanoparticles. 42 Capítulo 1 SEM analyzes SEM images (fig.2) shows different morphology for all synthesis, the synthesis performed at 50ºC for 12 minutes produced more regular and dispersed silver nanoparticles. Determination antimicrobial activity MIC values of silver nanoparticles obtained for S. aureus was 67.5 µg/ml and for C. albicans 68.75 µg/ml and interestingly the values did not change against the different conditions of synthesis. The MIC values obtained for pomegranate peel extract was 391 µg/ml for S.aureus and 781 µg/ml for C. albicans, even temperature and time of reaction did not interfere in this values. 2.5. Discussion Nanomaterials has been applied in several areas such as drug delivery, environment, biomedical, food packaging, among others. In this sense, the green synthesis of nanoparticles has been largely studied (Ahmed S 2016). Studies have reported that parameters for synthesis are very abstract and not very standardized for green synthesis. Factors such as pH, extract volume and ion concentration alter the final production of silver nanoparticles (Velu, Lee et al. 2017), so our research provides interesting results of the variation of parameters such as temperature and reaction time in the production of silver nanoparticles phytosynthesized using pomegranate peel extract. It is asserted that the phenolic compounds present in the extracts perform the reduction of the silver nanoparticles (El-Kassas and Ghobrial 2017). Thus, the 43 Capítulo 1 temperature ratio check and reaction time in the concentration of phenols and the activities of the extract as the antimicrobial activity are important. It can be observed that with increasing temperature and reaction time the total phenolic concentration increased significantly, however the antimicrobial activity remained stable, thus being able to attribute the increase concentration in relation to water evaporation, especially because the technique used to determine total phenols is colorimetric. However, we must take into account that the active compounds present in the extract with antimicrobial potential were not degraded, since MIC values were not altered. Profiles obtained in the UV/Visible analysis as the same way determined by S Agnithori et al. (2014) showed that the increase of time and temperature occurs the formation of two peaks and larger bands, suggesting the formation of particles of varied sizes. This fact can be confirmed by analyzing the SEM images obtained, observing that with the passage of time and increasing the temperature irregular and agglomerated silver nanoparticles are formed, thus confirming the spectral profiles obtained. Moreover, the silver nanoparticles produced by lower temperature and reaction time generated more homogeneous formulation. Regarding to antimicrobial activity values founded for S.aureus and C. albicans were very similarly. The values did not change for the different synthesis. One possible explanation would be the formation of different sizes of silver nanoparticles in the different conditions. As it is known different sizes and shapes of nanoparticles exert specific activities (Pal, Tak et al. 2007), thus given the variety of form occurred in the syntheses with higher temperature and time they also presented particles similar to the occurred synthesis in a shorter time and 44 Capítulo 1 temperature. According to the presented results, it can be concluded that the high reaction temperature and time can negatively favor the production of silver nanoparticles principally in the morphology and aggregation of nanoparticles. It is also worth noting that an average temperature of 50ºC and a short time of twelve minutes showed the highest efficiency both in the reduction of ions and in the production of regular and dispersed silver nanoparticles. As well as the preparation of formulations with possible uses in medical field. Funding This study was supported by São Paulo Research Foundation (FAPESP, process 2016/04230-9), Brazil. 2.7. References Abbasi, E., M. Milani, S. Fekri Aval, M. Kouhi, A. Akbarzadeh, H. Tayefi Nasrabadi, P. Nikasa, S. W. Joo, Y. Hanifehpour, K. Nejati-Koshki and M. Samiei (2016). "Silver nanoparticles: Synthesis methods, bio-applications and properties." Crit Rev Microbiol 42(2): 173-180. Ahmed S, S. A. M., Swami BL, Ikram S (2016). "Green synthesis of silver nanoparticles using Azadirachta indica aqueous leaf extract. ." J Radiat Res Appl Sci 9: 1-7. Das, A., A. Kumar, N. B. Patil, C. Viswanathan and D. Ghosh (2015). "Preparation and characterization of silver nanoparticle loaded amorphous hydrogel of carboxymethylcellulose for infected wounds." Carbohydr Polym 130: 254-261. de Oliveira, J. R., V. C. de Castro, P. das Gracas Figueiredo Vilela, S. E. Camargo, C. A. Carvalho, A. O. Jorge and L. D. de Oliveira (2013). "Cytotoxicity 45 Capítulo 1 of Brazilian plant extracts against oral microorganisms of interest to dentistry." BMC Complement Altern Med 13: 208. Delmee, M., G. Mertz, J. Bardon, A. Marguier, L. Ploux, V. Roucoules and D. Ruch (2017). "Laser Ablation of Silver in Liquid Organic Monomer: Influence of Experimental Parameters on the Synthesized Silver Nanoparticles/Graphite Colloids." J Phys Chem B. El-Kassas, H. Y. and M. G. Ghobrial (2017). "Biosynthesis of metal nanoparticles using three marine plant species: anti-algal efficiencies against "Oscillatoria simplicissima"." Environ Sci Pollut Res Int 24(8): 7837-7849. Elemike, E. E., O. E. Fayemi, A. C. Ekennia, D. C. Onwudiwe and E. E. Ebenso (2017). "Silver Nanoparticles Mediated by Costus afer Leaf Extract: Synthesis, Antibacterial, Antioxidant and Electrochemical Properties." Molecules 22(5). Elsupikhe, R. F., K. Shameli, M. B. Ahmad, N. A. Ibrahim and N. Zainudin (2015). "Green sonochemical synthesis of silver nanoparticles at varying concentrations of kappa-carrageenan." Nanoscale Res Lett 10(1): 916. Francis, S., S. Joseph, E. P. Koshy and B. Mathew (2017). "Green synthesis and characterization of gold and silver nanoparticles using Mussaenda glabrata leaf extract and their environmental applications to dye degradation." Environ Sci Pollut Res Int. Francis, S., S. Joseph, E. P. Koshy and B. Mathew (2017). "Microwave assisted green synthesis of silver nanoparticles using leaf extract of elephantopus scaber and its environmental and biological applications." Artif Cells Nanomed Biotechnol: 1-10. 46 Capítulo 1 Gabriel, J. S., V. A. M. Gonzaga, A. L. Poli and C. C. Schmitt (2017). "Photochemical synthesis of silver nanoparticles on chitosans/montmorillonite nanocomposite films and antibacterial activity." Carbohydr Polym 171: 202-210. Gillum, A. M., E. Y. Tsay and D. R. Kirsch (1984). "Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations." Mol Gen Genet 198(2): 179-182. Gorup, L. F., E. Longo, E. R. Leite and E. R. Camargo (2011). "Moderating effect of ammonia on particle growth and stability of quasi-monodisperse silver nanoparticles synthesized by the Turkevich method." J Colloid Interface Sci 360(2): 355-358. Gurusamy, V., Krishnamoorthy, R., Gopal, B., & Veeraravagan, V. (2017). "Systematic investigation on hydrazine hydrate assisted reduction of silver nanoparticles and its antibacterial properties." Inorganic and Nano-Metal Chemistry 47: 761–767. Khodashenas, B., & Ghorbani, H. R. (2015). "Synthesis of silver nanoparticles with different shapes. ." Arabian Journal of Chemistry. Liu, X. and D. Astruc (2017). "From Galvanic to Anti-Galvanic Synthesis of Bimetallic Nanoparticles and Applications in Catalysis, Sensing, and Materials Science." Adv Mater 29(16). Mansour, H. H., M. Eid and M. B. El-Arnaouty (2017). "Effect of silver nanoparticles synthesized by gamma radiation on the cytotoxicity of doxorubicin in human cancer cell lines and experimental animals." Hum Exp Toxicol: 960327116689717. 47 Capítulo 1 Pal, S., Y. K. Tak and J. M. Song (2007). "Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram- negative bacterium Escherichia coli." Appl Environ Microbiol 73(6): 1712-1720. Philip, D. (2010). "Honey mediated green synthesis of silver nanoparticles." Spectrochim Acta A Mol Biomol Spectrosc 75(3): 1078-1081. Ren, M., Jin, Y., Chen, W., & Huang, W. (2015). " Rich capping ligand–ag colloid interactions." The Journal of Physical Chemistry C 119: 27588–27593. S Agnihotri, S. M., S Mukherji (2014). "Size-controlled silver nanoparticles synthesized over the range 5–100 nm using the same protocol and their antibacterial efficacy." RSC Advances 8(4): 3974-3983. Sai Saraswathi, V., N. Kamarudheen, K. V. BhaskaraRao and K. Santhakumar (2017). "Phytoremediation of dyes using Lagerstroemia speciosa mediated silver nanoparticles and its biofilm activity against clinical strains Pseudomonas aeruginosa." J Photochem Photobiol B 168: 107-116. Velu, M., J. H. Lee, W. S. Chang, N. Lovanh, Y. J. Park, P. Jayanthi, V. Palanivel and B. T. Oh (2017). "Fabrication, optimization, and characterization of noble silver nanoparticles from sugarcane leaf (Saccharum officinarum) extract for antifungal application." 3 Biotech 7(2): 147. Wypij, M., P. Golinska, H. Dahm and M. Rai (2017). "Actinobacterial-mediated synthesis of silver nanoparticles and their activity against pathogenic bacteria." IET Nanobiotechnol 11(3): 336-342. Zielinski, H. and H. Kozlowska (2000). "Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions." J Agric Food Chem 48(6): 2008-2016. 48 Capítulo 1 Table 1. Values of total phenols and minimum inhibitory concentration (MIC) for pomegranate peel extract submitted under different conditions. *CMC= Carboximetilcelulose; PG= Propilenoglicol. Compounds Total phenols Mean ± SD (mg / g) MIC values S. aureus C. albicans CMC + PG -1.605 ± 0.1834 Absent Absent Extract at room temperature 10.628 ± 0.354 0.391 mg/ml 0.781 mg/ml Extract at 50°C/12 min 9.310 ± 0.7345 0.391 mg/ml 0.781 mg/ml Extract at50°C/2 h 9.915 ± 0.4777 0.391 mg/ml 0.781 mg/ml Extract at 100°C/2 h 13.451 ± 0.4805 0.391 mg/ml 0.781 mg/ml 49 Capítulo 1 Table 2. Values of ion reduction and minimum inhibitory concentration (MIC) for pomegranate peel extract submitted under different conditions. *Control= Carboximetilcelulose, propilenoglicol, nitrato de prata. Samples µg Ag+/mL % of reduction MIC (µg/ml) S. aureus C. albicans *Control at 50ºC/12 min 10303.26 4.59 4.13 0.53 *Control at 50ºC/2 h 9470.56 12 4.13 0.53 *Control at 100ºC/2 h 8447.06 22.14 4.13 0.53 Pomegranate peel extract - - 391 781 Phytosynthesis at 50ºC/12 min (I) 10.89 99.89 67.50 68.75 Phytosynthesis at 50ºC/2 h (II) 453.05 96 67.50 68.75 Phytosynthesis at 100ºC/2 h (III) 149.49 98.61 67.50 68.75 Formulation I - - 0.26 16.87 Formulation II - - 0.26 16.87 Formulation III - - 0.26 16.87 50 Capítulo 1 Figure 1. Spectral UV/Visible of silver nanoparticles produced under different conditions, controls without reducing agent (pomegranate peel extract) (A), 50°C for 12 min (B), 50ºC for 1 h (C), 100ºC for 2 h (D). 51 Capítulo 1 Figure 2. Images of scanning electron microscopy of silver nanoparticles produced under different conditions, 50°C for 12 min (A), 50ºC for 1 h (B), 100ºC for 2 h (C) and formulations prepared from them (D,E and H). 52 CAPÍTULO 2 Antimicrobial potential and toxicity of silver nanoparticles phytosynthesized by pomegranate peel extract * Artigo nas normas do periódico Biofouling. 53 Capítulo 2 Antimicrobial potential and citoxicity of silver nanoparticles phytosynthesized by pomegranate peel extract 3.1. Abstract Antimicrobial activity of colloidal and spray formulation of silver nanoparticles (AgNP) synthesized by pomegranate peel extract against Candida albicans and Staphylococcus aureus, and cytotoxicity in mammalian cells were tested. Dry mass, pH, total phenolics and ellagic acid in the extract were determined. Then AgNP were phytosynthesized and characterized by X-ray diffraction, electron scanning and transmission microscopies, dynamic light scattering, zeta potential, and Ag+ dosage. Spray formulations and respective chemical-AgNP controls were prepared and tested. Peel extract reduced more than 99% of Ag+, and produced nanoparticles with irregular forms and 89 nm mean size. All AgNP presented antimicrobial activity, and spray formulation of green-AgNP increased 255 and 4 times the effectiveness against S. aureus and C. albicans respectively. Cytotoxicity of colloidal and spray green-AgNP was expressively lower than the respective chemical controls. Pomegranate peel extract produced stable AgNP with antimicrobial action and low cytotoxicity, stimulating its use in the biomedical field. Keywords: Silver; nanoparticles, Candida albicans, Staphylococcus aureus, herbal medicine, Punicaceae. 54 Capítulo 2 3.2. Introduction Recently a state of alert on a topic that affects people all over the world, the antimicrobial resistance has received attention. This fact leads to the death of more than 700000 people a year around the world and this number has risen every year (Raut and Adhikari 2017). It is estimated that a world population will have a reduction of 11-444 million people in 2050 if the antimicrobial resistance was not bypassed (Raut and Adhikari 2017). As a contour form and an alternative to antimicrobial resistance, an approach of inorganic particles at nanoscale has become strong. The most prominent metals in the group of inorganic nanoparticles are copper, zinc, titanium, magnesium, gold and silver (Guzman, Dille et al. 2012, Jankun, Landeta et al. 2014, He, Qiao et al. 2017). In this context, silver nanoparticles are the most exploited because it have a wide range of toxicity against several microorganisms, such as Staphylococcus aureus, Escherichia coli, Candida albicans and others (Hebeish, El-Rafie et al. 2014). The incorporation and use of silver nanoparticles is observed in sundry sectors, for instance in the food industry as an attempt to produce packages with antimicrobial activity (Kuorwel, Cran et al. 2011). The area of cosmetics has also received prominence, using in housecleaning, antiseptics, sunscreens, soap and shampoo (Benn, Cavanagh et al. 2010, Bansod, Bawaskar et al. 2015, Tulve, Stefaniak et al. 2015), as well as in textile manufacturing (Velazquez-Velazquez, Santos-Flores et al. 2015). 55 Capítulo 2 Considering the synthesis of silver nanoparticles many routes have been presented, such as electrochemical (Treshchalov, Erikson et al. 2017), by radiation (Malkar, Mukherjee et al. 2014), photochemistry (Lombardo, Poli et al. 2016), and by biological methods (Rafique, Sadaf et al. 2016). Phytochemical synthesis has been noteworthy since the use of chemical compounds may result in undesirable toxic effects not only for the human organism as well as the environment. Quite effectiveness in the production of silver nanoparticles has been demonstrated when compounds of different plants are used in the ions reduction, being characterized as rapid, low cost and nature friendly synthesis (Roy, Gaur et al. 2013). Furthermore, green-silver nanoparticles are usually less cytotoxic when compared to those reduced by conventional chemical agents (Das and Brar 2013). Important aspects in green synthesis should be taken into account, including the choice of plant to be used, being the plants which grow in different regions of the world more eligible for (Das and Brar 2013). The previously known potential of the plant, including antioxidant, anti-inflammatory and antimicrobial, such as the case of Punica granatum (pomegranate), should be considered (Lansky and Newman 2007, Pande and Akoh 2009, Edison and Sethuraman 2013). Some studies have been used Punica granatum to reduce silver ions to silver nanoparticles (Naik and Chand 2003, Ahmad 2012, Edison and Sethuraman 2013). Thus, taking together the benefits of pomegranate and the antimicrobial applicability of silver nanoparticles, the present study aimed to synthesize silver nanoparticles using pomegranate peel extract, and produce spray formulations 56 Capítulo 2 containing the previously synthesized green-silver nanoparticles. Characterize and teste against strains of Staphylococcus aureus and Candida albicans, as well as their cytotoxicity against mammalian cells. 2. Materials and methods Plant material and preparation of pomegranate peel extract Pomegranate samples were collected from a plantation cultivated in Eixo (21º 08' 01" S, 51º 06' 06" W), Mirandópolis, São Paulo, Brazil, during May 2015. Pomegranate peels were separated and stove-dried at 50°C, ground and sieved to a granulometry lower than 2 mm. Peels were submitted to alcohol extraction using ethanol 70% by maceration followed by percolation process (de Oliveira, de Castro et al. 2013). The extract was characterized in relation of pH, dry mass, and total phenolics expressed as gallic acid. The chemical marker of pomegranate, ellagic acid, was also identified and quantified. Determination of total phenolic, pH and dry mass For determination of total phenolic an analytical curve of gallic acid (Sigma- Aldrich Chemical Co, St Louis, USA) was carried out (Waterman PG 1994). All extracts obtained and the plant drug were prepared in 50 ml volumetric flasks using water as solvent. The samples were homogenized and, the flasks were brought to the ultrasonic bath for 30 minutes. A 0.5 mL aliquot was transferred to another 50 mL flask where 2.5 mL of Folin-Denis reagent (Qhemis - High Purity, Hexis, São Paulo, Brasil) and 5.0 mL of 29% sodium carbonate (Cinética, São Paulo, Brasil) were added. The samples were protected from the light and the 57 Capítulo 2 readings were performed after 30 minutes in a UV-Vis spectrophotometer at 760 nm (Zielinski and Kozlowska 2000). The pH was measured direct of solution of 1% extract, using a kit of pH (Merck KGaA, Darmstadt, Germany) and dry mass was calculated after the sample stove drying at 105°C and it was expressed in percentage m/m. All data were analyzed in triplicate. Determination of the ellagic acid content A Shimadzu liquid chromatograph and a Shimpack ODS C18 (Shimadzu Corporation, Kyoto, Japan) reverse phase column (100 mm x 2.6 mm) were used to determine the ellagic acid content by high performance liquid chromatography (HPLC). Analytical conditions were optimized based on de Sousa et al 2007 (de Sousa, Bueno et al. 2007) with modifications. As the mobile phase, HPLC grade methanol and a 2% aqueous acetic acid solution with gradient elution (0-7 min, 20-72.5% v/v methanol, 7-7.5 min, 72.5-95 % v/v methanol, 7.5-8.5 min 95% v/v methanol, 8.5-9 min 95-20% v/v methanol, 9-10 min 20% v/v methanol) were used. The flow rate was 1.0 mL/min, and the separation was achieved at 25 °C. The injection volume was 5 μL and the wavelength used was 254 nm. Peaks were determined by comparison with authenticated ellagic acid standard. Briefly, the sample for the plant drug was transferred to a 20 mL volumetric flask which was quenched with HPLC grade methanol. Extraction was done using a vortex for 5 minutes and ultrasonic bath for 1 hour. For the extracts, samples were transferred to volumetric flasks of 10 mL, using methanol HPLC as solvent. All samples were vortexed for 5 minutes and sonicated for 30 minutes. Samples were filtered through 0.45 μm filter. All samples were prepared in triplicate. 58 Capítulo 2 Synthesis of green-silver nanoparticles The protocols described by Gorup et al. (2011) and Das K et al. (2015) with modifications were used to produce silver nanoparticles. Briefly, 3.5% of carboxymethylcellulose (CMC) (Labsynth, Diadema, Brazil), 20% of propylene glycol (PG) (Labsynth, Diadema, Brazil), 100 mM of silver nitrate (SN) (Merck KGaA, Darmstadt, Germany), pomegranate peel extract at 30 mg/ml, and water to make up 100% of the samples were used. The reaction was carried out at 50ºC for 12 minutes, and it was selected based on previous results (data not yet published). Synthesis of chemical-silver nanoparticles Chemical-silver nanoparticles were produced according to Gorup et al. (2011). AgNO3 (Merck KGaA, Darmstadt, Hesse, Germany) was dissolved in water, and brought to boiling at 90ºC. After 2 min of boiling an aqueous solution of sodium citrate (Na3C6H5O7)(Merck KGaA, Darmstadt, Hesse, Germany) was added, and kept boiling for more 6 min until the solution reaches yellow amber color. The stoichiometric ratio was 1:3, respectively for AgNO3 and Na3C6H5O7. Preparation of formulations The reagents used were CMC (Labsynth, Diadema, Brazil), PG (Labsynth, Diadema, Brazil), methylparaben (Labsynth, Diadema, Brazil) in a proportion of 0.1%, 7% and 0.1%, respectively. The active inputs (green- or chemical-silver nanoparticles and pomegranate peel extract) concentrations were based on the minimum inhibitory concentration and cytotoxicity. Therefore, the final 59 Capítulo 2 concentrations of active inputs in the spray formulations were: 337.5 µg/ml of green-silver nanoparticles, 5.55 µg/ml chemical-silver nanoparticles, and 94 µg/ml of crude peel extract dry mass. Characterization of silver nanoparticles and formulations X-ray diffraction (XRD), Dynamic Light Scattering (DLS) and Zeta Potential analysis Shimadzu XRD diffractometer with a Cu Kα radiation operating at 30 kV and 30 mA and 2θ range from 35 to 85° with step scan of 0.02° and scan speed 0.2°.min- 1 was used to perform XRD analysis. To collect silver nanoparticles patterns, the nanoparticles were deposited on the surface of a silicon substrate (Si) by dripping the aqueous colloidal dispersion on the substrate at a room temperature, and the solvent was evaporated. DLS experiments were performed at room temperature and at a fixed angle of 173° on a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, United Kingdom) equipped with 50 mW 533 nm laser and a digital auto correlator. The number-average values obtained were compared to the size distributions of silver nanoparticles. For Zeta Potential test a Zetasizer (Malvern instruments, Malvern, United Kingdom) with a MPT-2 titrator was used. Aliquots from each test suspension were obtained to conduct zeta potential, and mean values were obtained from three independent measurements. SEM and TEM analyzes 60 Capítulo 2 The nanocompounds morphology was characterized by scanning electron microscopy (SEM) on a S-360 microscope (Leo, Cambridge, USA), and Jeol JEM-100 CXII (JEOL USA Inc. Peabody, USA) microscope equipped with Hamamatsu ORCA-HR digital camera was used to obtain TEM images. Silver ions dosage The dosages of free silver ions (Ag+) present in the compounds and spray formulations were performed to observe if the total amount of Ag added in the synthesis reaction was successfully reduced. A specific electrode 9616 BNWP (Thermo Scientific, Beverly, MA, USA) coupled to an ion analyzer (Orion 720 A+, Thermo Scientific, Beverly, MA, USA) was used. A 1000 µg Ag/ml standard was prepared adding 1.57 g of AgNO3 in 1 L of deionized water. The combined electrode was calibrated with standards containing 6.25 to 100 µg Ag/ml to achieve equivalent silver concentrations in the compounds. A silver ionic strength adjuster solution (ISA, Cat. No. 940011) which provides a constant background ionic strength was used (1 ml of each sample/standard: 0.02 ml ISA). Stability test of the formulations The formulations were submitted to a stability test with controlled conditions of temperature and time. This test was based on Anvisa protocols (Cosmetics stability guide (ISBN 85-88233-15-0; Copyright© Anvisa, 2005) and guide to stability studies (Ordinance No. 593 of August 25, 2000). Briefly, samples of each spray formulations were submitted to alternating cycles of temperature daily ranging from 40 to -5°C for 28 days. The tests selected to evaluate the stability of 61 Capítulo 2 the samples were ion dosage, total phenolic content, zeta potential and microdilution broth. All tests were done in the same conditions as described before, and were carried out at 0, 7, 14 and 28 days. Antimicrobial activity of silver nanoparticles and formulations For determination of the minimal inhibitory concentration (MIC) the instructions of the Clinical Laboratory Standards Institute were followed with modifications. The samples were first diluted in water and subsequently in culture medium specific for each microorganism, Mueller Hinton broth (BD Difco, USA) for Staphylococcus aureus (ATCC 25923) and RPMI (Sigma-Aldrich, St Louis, USA) for Candida albicans SC 5314 (wild type) (Gillum, Tsay et al. 1984). The microorganisms were adjusted to 5 x 105 cells/ml for S. aureus and 5 x 103 cells/ml for C. albicans, and the plates were incubated for 24 hours and 48 hours in aerobiosis at 37°C respectively for S. aureus and C. albicans. After the incubation, the plates were visually read. The assays were performed in triplicate. Evaluation of cytotoxicity For evaluation of cytotoxicity fibroblast cells L929 lineage were used. Cells were cultured in DMEM culture supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/ml) (Gibco®), streptomycin (100 μg/ml), amphotericin B (25 μg/ml) and incubated in stove at 37 °C with 5% CO2. Cells were subcultured (5-7 days), using 0.9% saline to wash them and 0.25% trypsin to disintegrate them from the vial. After disruption, these cells were centrifuged at 1000 rpm for 10 62 Capítulo 2 minutes at 10 °C, resuspended in complete DMEM medium (supplemented with FBS), and cell counted in a Neubauer's chamber. The sub-cultured 3rd to 8th passage fibroblasts were inoculated into 96- well microplates at a density of 0.5x105 cells/well. They were then incubated at 37 ° C with 5% CO2. After 24 hours, 20 µl of different dilutions of each samples were added to the wells of the plate containing the cells in medium not supplemented with SBF (incomplete medium) and incubated. 24 hours post- treatment the medium was withdrawn, cells were washed with saline and 20 µl of resazurin (Sigma-Aldrich) 0.01% w/v in deionized H2O was added to each well containing 180 µl of DMEM medium supplemented with 10% Of SFB. The plates were then incubated for 4 hours at 37 ºC and fluorescence was measured at 540 and 590 nm for excitation and emission, respectively (Kuete, Wiench et al. 2013). Cell viability was expressed as percentage of viable cells compared to the control group without treatment. Statistical analysis GraphPad Prism software (GraphPad Software, Inc, La Jolla, USA) was employed for the statistical analysis with a confidence level of 95 %. Parametric statistical analyses were conducted with one-way ANOVA followed by Tukey's multiple comparison test for total phenols and zeta potential. For ion test the statistical analyses was Dunn's multiple comparison test. 3. Results Characterization of peel extract, silver nanoparticles and formulations 63 Capítulo 2 The pH and the dry mass of the peel extract obtained by maceration followed by percolation were 3.13 and 86.39 (±0.96) % m/m, and the total phenolic expressed in gallic acid and the ellagic acid were 392.0 (±9) and 3.64 (±0.03) mg/g respectively. The formation of silver nanoparticles was confirmed by comparing the XRD patterns and the corresponding standard patterns of cubic of silver nanoparticles (Fig. 1), according to the diffraction standard (JCPDS file No. 04- 0783). The reflection peak (2 2 2) is a characteristic of the substrate (Si), where silver particles were deposited as a thin film. MEV and TEM images (Fig 2) show different forms and sizes of silver nanoparticles fabricated by green and conventional chemical routes as well as in their respective formulations. In general, green synthesis produced particles with larger size than those obtained by conventional synthesis. DLS analyses of the formulations prepared with green or conventional silver nanoparticles demonstrated different sizes of the particles, being the mean values of 89 ± 21 and 19 ± 4 nm for green and conventional formulation respectively. The values of zeta potential of green- and conventional- silver nanoparticles were lower than -30 mV (-46.2 ± 6.06 mV green, and -67.5 ± 3.69 mV conventional), indicating the stability of both colloidal silver nanoparticles. Almost 100% of the Ag+ ions coming from AgNO3 were reduced by pomegranate peel extract (99.89%) and sodium citrate (99.51%). However, in the spray formulation containing chemical-silver nanoparticles, the percentage of reduction was diminished to 68.18%. Although it has happen, that formulation maintained stable regarding Ag+ ions concentration for 28 days (Table 2). Zeta 64 Capítulo 2 potential data confirmed the stability of spray formulations regardless of the method used to obtain the silver nanoparticles (Table 1). Total phenols in the spray formulations with or without silver nanoparticles were quantified at 0, 7, 14 and 28 days after having been prepared (Fig. 3), and it were significantly reduced in the green-silver nanoparticles formulation after 14 days with values ranging from 0.405 to 0.295 mg/g. Antimicrobial activity The antimicrobial activity expressed as MIC values (µg/ml) (Table 1) was, in general, considerably lower for spray formulations than active inputs regardless of the microorganisms tested. MIC values against C. albicans for active inputs and spray formulations were 781 and 0.18 for peel extract, 68.75 and 16.87 for green-, and 0.25 and 1.12 for chemical-silver nanoparticles. While for S. aureus the values were 391 and 0.37, 67.5 and 0.26, and 0.5 and 0.56, respectively for pomegranate peel extract, green- and chemical-silver nanoparticles in active inputs and in spray formualtions. In addition, different conditions of humidity and temperature did not affect the effectiveness of the spray formulations against both microorganisms. Cytotoxicity Figure 4 shows the fibroblast L929 cells viability in view of different concentrations of silver nanoparticles (green and conventional route). Green-silver nanoparticles presented lower cytotoxicity than conventional-ones. It was necessary a dosage of 50 µg/ml to initiate the toxicity, but the cell viability was 65 Capítulo 2 nearly 80%, while conventional-silver nanoparticles were quite toxic at very low concentration (6.25 µg/ml) and it was similar with the negative control (DMSO) with viability lower then 20%. Also, the addition of the reagents to prepare the formulations did not interfere in the toxicity of the conventional-silver nanoparticles, whereas the cytotoxicity for the green-silver nanoparticles formulation as well as for the extract formulation were considerable increased. 4. Discussion For future reproducibility of the experiment, the extract obtained by maceration followed by percolation was duly characterized in relation to dry mass, total phenols content, ellagic acid and pH. The correct selection of the plant and the standardization of the methods to obtain the extracts to be used as reducing or capping agent in nanosynthesis of metal particles should be preponderant when green process is elected the produce products in large scale. Also, a plethora of plants used in the phytosynthesis of metal nanoparticles (Ovais, Khalil et al. 2016, Soman and Ray 2016, Elemike, Onwudiwe et al. 2017) and the lack of information of the extraction techniques used in the articles hinder the comparison of the present results with those found in the literature. For instance, different values and methods of total phenol quantification can be observed in the literature as described by Kalaycioglu and Erim (2017). Similarly, other factors interfere in the quantification of the bioactive compounds in the extracts as the chemical and genotypic composition of the plant, the variety and the soil type, the place of the plant origin, the harvest season, maturation method, among others (Li, Yang et al. 2016). 66 Capítulo 2 MEV and TEM images showed the smallest particles obtained by conventional chemical synthesis (Fig 2). DLS data confirmed these findings, with mean sizes of 89 and 19 nm respectively for green and chemical nanoparticles. The fission of colloidal particles of different sizes and shapes may be related to additives (salts, polymers), solvent properties (boiling temperature, affinity with created surfaces), addition of nucleation, among others (Martins 2012). The reagents used in the chemical synthesis would produce particles with more predictable characteristics than the several substances and compounds present in the plant extract and used in the phytosynthesis route. It would interfere on the size and form of the nanoparticles and make the phytosynthesis a challenge in controlling the reaction process and the morphological aspects of the particles. Moreover, the presence of different bioactive substances in the extract would reduce only a fraction of the silver ions present in the solution. The remaining silver ions would form other nuclei and further the growth of the silver nanoparticles previously formed (Agnihotri 2014). This process is called Ostwald Ripening, where the largest particles consume the smaller ones and grow larger, where the dissolution of the smaller ones and deposition of ions on the surface of larger ones occur (Houk, Challa et al. 2009). Almost 100% of ions reduction was observed for both route of synthesis. However, there was an increase of about 30% in the Ag+ concentration after adding the conventional-silver nanoparticles in the formulation. This fact could be due to the presence of the components as carboxymethylcellulose and propylene glycol in the spray formulation which possibly favored the silver ions dissociation into the system (Prema, Thangapandiyan et al. 2017). This fact was not observed 67 Capítulo 2 when green synthesis was carried out. It could be related to the several compounds present in the extract which would readily react with the released silver ions, or even the encapsulation of the silver nanoparticles promoted by those phytocompounds may have avoided the silver ions dissociation from the silver nanoparticles and its release to the solution. Zeta potential test demonstrated the stability of the silver nanoparticles, notedly in the spray formulations. Electrical charges on the surface of the nanoparticles prevent agglomeration, and thus afford the stability of the nanoparticles (Sadowski 2008, Salem 2011). Indeed, silver nanoparticles and spray formulations presented a mean of 70 mv, which indicates their high stability of silver nanoparticles (Leite 2012). Antimicrobial results are promising for the silver nanoparticles as well as the pomegranate extract obtained. The formulations notable showed better results compared with the input active only. This fact could be explained for the proper dissolution of the actives inputs (silver nanoparticles and pomegranate peel extract) in the spray formulation. Also, a synergistic effect could be occurred between those active inputs and the metylparaben present in the formulation. In the literature studies with antimicrobial effect of pomegranate extract were conducted against Staphylococcus aureus, Enterobacter aerogene, Salmonella typhi, Klebsiella pneumonia (Malviya, Arvind et al. 2014). MIC values obtained in this study for pomegranate extract is in accordance with Bakkiyaraj et al. (2013) for both microorganisms studied, a difference was observed in C. albicans, but this fact may be explained by de difference between the C. albicans strains used in the studies. 68 Capítulo 2 Chemical-silver nanoparticles produced MIC values against S. aureus similar to those found by Prema et al.(2017) (60 µg/ml), whom also produced silver nanoparticles stabilized with CMC. Indeed, the antimicrobial activity of chemical silver nanoparticles was also determined by Monteiro et al. (2011) with MIC values for C. albicans (0.5 µg/ml) in accordance with this present study. Noteworthy is the difference found in the present study in respect of cytotoxicity between the chemical and green routes to obtain silver nanoparticles. Studies have shown that silver nanoparticles produced with Protium serratum and Nyctanthes arbortristis extract were biocompatible when tested in L929 fibroblasts (Gogoi, Babu et al. 2015, Mohanta, Panda et al. 2017). It is believed that what makes the silver nanoparticle toxic to human cells is the type of reducing agent used, such as sodium citrate or sodium borohydride (Asharani, Lian Wu et al. 2008). Even in the conventional syntheses of silver nanoparticles it is used reagents that prevent the aggregation of these nanoparticles (Ren 2015), which may further favor their cytotoxicity. In the case of phytosynthesis of metal nanoparticles, plant extracts besides acting as reducing agent it would act stabilizing the particles, and hence reducing the toxicity of the silver nanoparticles solution. Furthermore, it is possible that some compounds in the extracts may have a synergistic effect with the silver nanoparticles (Gengan, Anand et al. 2013), becoming them less toxic to human cells. Also, extracts of Punica granatum exhibits antioxidant (Delgado, Rouver et al. 2016) and anti-inflammatory (Houston, Bugert et al. 2017) activity, and it may have contributed in reducing the cytotoxicity of green- in comparison with chemical-silver nanoparticles. 69 Capítulo 2 In general, the stability assay (silver ions dosage, zeta potential and antimicrobial activity) showed a high stabilizing capacity of the formulations. However, the spray formulations of green silver nanoparticles and pomegranate peel extract showed a significant reduction in the content of total phenols in 14 and 28 days. The decrease in the content of phenolic total may have occurred due to the temperature variations inherent of stability test, as occurred in the study of (Mgaya-Kilima et al. (2015) which the temperature affected the total phenolic content in the roselle-mango juice blends. Moreover, in formulations containing green-silver nanoparticles, the components of the extract may have been degraded or associated with the nanoparticles, explaining the faster decrease of the total phenol content compared to the pomegranate extract formulation. Interestingly, ion dosage, zeta potential and antimicrobial activity were not affected by different conditions of temperature, time and humidity of the stability test. In view of the results obtained and the limitations of present study, it is concluded that the use of pomegranate peel extract shows as an efficient reducing agent for the production of silver nanoparticles. Moreover, the antimicrobial potential and the low cytotoxicity demonstrated by green-silver nanoparticles stimulate the search for improvements in the bio-nanotecnhology field. Also, anti- inflammatory and antioxidant properties of pomegranate encourage further researches to use nanosystems with future application in prophylaxis or treatment of biofilm-dependent diseases. 70 Capítulo 2 Acknowledgments The authors thank the company Apis Flora Indl. Coml. Ltda. for the facilities and for the production of the spray formulations containing pomegranate peel extract, and the Laboratory of photochemioprotection of the Faculty of Pharmaceutical Sciences of Ribeirão Preto for the facilities in performing some of the laboratory work. We thank the Brazilian Agricultural Research Corporation (EMBRAPA) to allow for some tests the research on its premises and also the Federal University of São Carlos for disposal of its dependencies and technologies. Funding This study was supported by São Paulo Research Foundation (FAPESP), Brazil, (Process nº 2016/04230-9). 3.6. References Agnihotri, S. M., S. & Mukerji, S (2014). 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