MINISTÉRIO DA EDUCAÇÃO

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

AVALIAÇÃO DA ATIVIDADE TRIPANOSSOMICIDA DE NANOPARTÍCULAS DE

PRATA E O POLISSACARÍDEO XILANA CONTRA FORMAS EPIMASTIGOTAS DE

Trypanosoma cruzi

TALITA KATIANE DE BRITO PINTO

NATAL/RN

2019

ii

TALITA KATIANE DE BRITO PINTO

AVALIAÇÃO DA ATIVIDADE TRIPANOSSOMICIDA DE NANOPARTÍCULAS DE

PRATA E O POLISSACARÍDEO XILANA CONTRA FORMAS EPIMASTIGOTAS DE

Trypanosoma cruzi

Tese apresentada ao Programa de

Pós-Graduação em Ciências da Saúde

da Universidade Federal do Rio

Grande do Norte como requisito para a

obtenção do título de Doutor em

Ciências da Saúde.

Orientador: Dr. Hugo Alexandre de Oliveira Rocha

NATAL/RN

2019

iii

Universidade Federal do Rio Grande do Norte - UFRN

Sistema de Bibliotecas - SISBI

Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde - CCS

Pinto, Talita Katiane de Brito.

Avaliação da atividade tripanossomicida de

nanopartículas de prata e o polissacarídeo xilana contra formas epimastigotas de Trypanosoma cruzi / Talita

Katiane de Brito Pinto. - 2019.

74.: il.

Tese (Doutorado) - Programa de Pós-Graduação em

Ciências da Saúde, Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte. Natal,

RN, 2019.

Orientador: Hugo Alexandre de Oliveira Rocha.

1. Trypanosoma cruzi - Tese. 2. Nanopartículas de prata - Tese. 3. Benzonidazol - Tese. 4. Xilana -

Tese. I. Rocha, Hugo Alexandre de Oliveira. II. Título.

RN/UF/BS-CCS CDU

616.937

Elaborado por ANA CRISTINA DA SILVA LOPES - CRB-15/263

iv

MINISTÉRIO DA EDUCAÇÃO

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

Coordenadora do Programa de Pós-graduação em Ciências da Saúde:

Prof. Dra Ana Katherine da Silveira Gonçalves de Oliveira

v

TALITA KATIANE DE BRITO PINTO

AVALIAÇÃO DA ATIVIDADE TRIPANOSSOMICIDA DE NANOPARTÍCULAS DE

PRATA E O POLISSACARÍDEO XILANA CONTRA FORMAS EPIMASTIGOTAS DE

Trypanosoma cruzi

Aprovada em: 30/08/2019

Banca Examinadora:

Presidente da Banca:

Prof. Dr. Hugo Alexandre de Oliveira Rocha – Departamento de Bioquímica, Centro

de Biociências, Universidade Federal do Rio Grande do Norte.

Membros da Banca:

Prof. Dra. Ariane Ferreira Lacerda (membro externo) - Instituto Federal de Educação,

Ciência e Tecnologia do Rio Grande do Norte (IFRN) - Campus Macau.

Prof. Dra. Nathalia Kelly de Araújo (membro externo) – Instituto Federal de Educação,

Ciência e Tecnologia do Rio Grande do Norte (IFRN) - Campus Pau dos Ferros.

Prof. Dra. Ana Katarina Menezes da Cruz Soares (membro interno) – Departamento

de Bioquímica, Universidade Federal do Rio Grande do Norte (UFRN).

Prof. Dr. Valter Ferreira Andrade Neto (membro interno) – Departamento de

Microbiologia e Parasitologia da Universidade Federal do Rio Grande do Norte

(UFRN).

vi

Dedicatória

À Deus...

Autor e Senhor da minha vida!

Aos meus pais Francisca Brito e Reinaldo Brito (In Memoriam).

A eles todos os meus sonhos e projetos.

Aos meus filhos, Gabriel (Bi) e Miguel (Mi)

Todos os meus sorrisos e gestos de amor.

Ao meu eterno amor, Rafael Pinto. Eterna gratidão e amor daqui até a eternidade....

vii

Agradecimentos

Ao meu orientador, Prof. Dr. Hugo Rocha.

Minha sincera gratidão pelo amigo, professor e orientador que se deixou ser.

Muito obrigada por ter acreditado em mim!

Aos meus irmãos, cunhadas e sobrinhos minha eterna gratidão por todo

apoio, incentivo e torcida. Juntos seremos sempre mais fortes! Aos demais

familiares e amigos que sempre me acompanharam nesta jornada.

À grande família Biopol. Meus sinceros agradecimentos por todos os

momentos de aprendizado e alegria passados juntos.

Ao meu grande amigo Jefferson Barbosa (Jefildo). Minha eterna gratidão

por todo apoio, ensinamentos, amizade e companheirismo. Você é o cara!

Jailma Almeida (Jaja). Palavras são insuficientes para demonstrar todo meu

apreço, todo meu respeito e gratidão. A você minha amiga todas as bênçãos

dos céus sejam em forma de retribuição. Amo você!

Maíra Menezes (minha linda enfermeira) meus sinceros agradecimentos

por tudo que passamos juntos e pela linda amizade que fizemos. Você é

especial!

Dayanne Gomes (Day). Muito obrigada pelo companheirismo nessa

caminhada. Obrigada por sua alegria que enfeitava meus dias.

viii

Mônica Amorim (Monquinha) a você todo meu afeto e agradecimento por

tantos momentos preciosos passados juntas. Você é fantástica!

Rony Lucas (Ronildo) eterna gratidão pela amizade e apoio durante todo o

processo do doutorado. Que Jesus te abençoe sempre!

Ao grande amigo Diego Sabri (Popó) o chatolino da vida diária. O cara da

Ressonância e tantas outras técnicas que domina. Obrigada por tudo

amigo.

Adriana Oliveira (Adri) a você meus sinceros agradecimentos por ter

andado ao meu lado nesse final de projeto. Você tem um futuro brilhante

pela frente.

A Profa. e amiga Monique Gabriela (Moniquete) pela disponibilidade em

ajudar e aconselhar. Seu incentivo fez toda diferença.

Ao meu amigo Wesley pelo seu bom humor sempre que alegrava os meus

dias chuvosos, o meu eterno obrigada.

Ao laboratório de Imunoparasitologia do Departamento de Análises Clínicas

e Toxicológicas, em especial ao professor Marcelo Silva meus eternos

agradecimentos por todo apoio, amizade e ensinamentos. Você é um

profissional muito fácil de admirar! A minha grande amiga Cláudia por ter

se tornado muito mais que uma parceira de doutorado, e sim, uma amiga

pra vida toda. Você é muito especial!! Ao amigo Jhonny por todo apoio nos

experimentos e pela alegria da sua presença.

ix

Ao amigo Raniere, muito obrigada por todo carinho e ensinamentos

transmitidos a mim no início dessa jornada.

Aos demais colegas do laboratório: Marília Negreiros, Luciana Fentanes

(Lu), Verônica Aquino, Éder Galinari (MacGyver), Vinícius Campelo (Vini),

Moacir (Momo) , Flávia (Flavoca), Joana de Ângelis (Jojo), Larisse (Lala)

Leonardo Nobre (Leozito), Leonardo Aquino (Leo), Gabriel Fidelis, Jayane

Mayara, Ivan, Karoline Rachel (Carolzinha), Cynthia Haynara, Raynara,

Pablo Castro (Pablito), Mariana Santana (Mari) Lucas Alighieri

(Luquetes), Carla (Carlota), Cinthia Beatrice, Thiago Laurentino os meus

eternos agradecimentos pelo adorável convívio durante esses 4 anos.

Aos amigos do Departamento de Farmácia, em especial a Stella Barreto e

Gabriel Azevedo pela forte amizade e companheirismo na “saga da coluna”.

Vocês são fantásticos!

Aos professores Leandro Costa e Rafael Câmara pela amizade e

ensinamentos.

Aos professores do Departamento de Bioquímica, em especial a Profa.

Luciana Guimarães, Prof. Eliseu Santos, Profa. Suely Chavantes, o meu

muito obrigada!

Aos membros da banca de qualificação, prof. Marcelo Silva e Profa. Mariana

Santana. Muito obrigada por todo apoio e sugestões que enriqueceram meu

trabalho.

Aos membros da banca de defesa sinceros agradecimentos.

x

Aos técnicos do Departamento de Bioquímica Ana Katarina Menezes por

todo apoio, a Francisco Freire pelas incontáveis análises no citômetro de

fluxo e a Eliene por todo carisma e disposição em ajudar.

Ao Laboratório de Química de Coordenação e Polímeros, em especial ao

prof. Daniel Pontes, Mayara Jane e Francimar Júnior, por todo empenho

nas análises e auxílio dos dados.

Aos colegas da secretaria, em especial ao Seu Rogério, Ricardo e Samara

muito obrigada!

Ao colega Matheus por me fazer companhia nas primeiras horas do dia no

laboratório, tornando sempre o nosso ambiente de trabalho um lugar

agradável para se estar.

Ao Cnpq e a Capes, pelo fomento necessário a realização dessa pesquisa.

xi

Epígrafe

“Eu queria ter mais que uma voz,

Mais que um amor e uma vida para Te oferecer,

Pois, Tu és muito mais do que eu possa ter em meu ser.

Tu és o autor, aquele que pintou com perfeição a vida.

Tu és o Senhor, aquele que me amou e és o meu Deus, meu Senhor,

minha vida é para Teu louvor”.

(Marcelo Manhãs)

xii

RESUMO

A doença de Chagas ou Tripanossomíase americana é causada pelo

protozoário Trypanosoma cruzi. O tratamento atual compreende o uso de pelo menos

dois fármacos o benzonidazol (BNZ) e o nifurtimox, que apresentam elevada

toxicidade e ineficácia na fase crônica da doença, o que induz a busca de alternativas

terapêuticas mais eficazes. Estudos têm demonstrado que a síntese verde de

nanopartículas de prata com polissacarídeos tem potencializado a atividade biológica

tanto dos polissacarídeos como da prata. Portanto, o polissacarídeo xilana foi extraído

do sabugo de milho, utilizando um método amigável ao meio ambiente, e foi usado

para a síntese de nanopartículas de prata (nanoxilanas - NX). Estudos de microscopia

de força atômica verificaram que a NX apresentou formato esférico e tamanho médio

(~ 40 nm). Espectroscopias de infravermelho e Raman e de Espectrometria de

emissão atômica por plasma acoplado comprovaram a presença de prata (~19%) e

xilana na NX. Parasitas T. cruzi perderam 95% da sua capacidade de reduzir o MTT

(3- brometo (4,5- dimetiltiazol-2-il) -2,5- difeniltetrazólio) quando incubados com

nanoxilana (100 µg/mL). Análise de citometria de fluxo mostrou que 98% dos parasitas

estavam positivos para iodeto de propídeo quando incubados por 24 h com NX (100

µg/mL). Os dados mostram que a nanoxilana induz necrose dos parasitas e que é um

forte candidato para estudos pré-clínicos com animais infectados com Trypanosoma

cruzi.

Palavras-chaves: Benzonidazol; Nanopartículas de prata; Trypanosoma cruzi;

Xilana.

xiii

ABSTRACT

Chagas disease or American trypanosomiasis is caused by the protozoan

Trypanosoma cruzi. The current treatment comprises the use of at least two drugs,

benzonidazole (BNZ) and nifurtimox, which present high toxicity and inefficiency in the

chronic phase of the disease, which induces the search for more effective therapeutic

alternatives. Studies have shown that the green synthesis of silver nanoparticles with

polysaccharides has potentiated the biological activity of both polysaccharides and

silver. Therefore, the xylan polysaccharide was extracted from corn cob using an

environmentally friendly method and was used for the synthesis of silver nanoparticles

(NX). NX-verified atomic force microscopy studies showing spherical shape and mean

size (~ 40 nm). Infrared and Raman spectroscopy and coupled plasma atomic

emission spectrometry confirmed the presence of silver (~ 19%) and xylan in the NX.

T. cruzi parasites lost 95% of their ability to reduce MTT (3-bromide (4,5-

dimethylthiazol-2-yl) -2,5-diphenyltetrazolium) when incubated with nano xylan (100

µg/mL). Flow cytometric analysis showed that 98% of the parasites were positive for

propidium iodide when incubated for 24 h with NX (100 µg/mL). The data show that

nano xylan induces parasite necrosis and is a strong candidate for preclinical studies

with animals infected with Trypanosoma cruzi.

Keywords: Benzonidazole; Silver nanoparticles; Trypanosoma cruzi; Xylan.

xiv

LISTA DE ABREVIATURAS E SIGLAS

AFM Microscopia de força atômica

AgNO3 Nitrato de prata

ANOVA Análise de Variância

Ara Arabinose

BERENICE Benzonidazol e Triazol para Nanomedicina e

Inovação na Doença de Chagas

BIOPOL Laboratório de biotecnologia de polímeros

naturais

BNZ Benzonidazol

BOD Demanda bioquímica de oxigênio

BSA Albumina do soro bovino

CN Controle negativo

CP Controle positivo

º C Grau Celsius

DC Doença de Chagas

FBS Soro fetal bovino

FTIR Espectroscopia de infravermelho com

transformada de Fourier

g Aceleração da gravidade

Gal Galactose

Glc Glicose

GlcA Ácido glucurônico

h Hora

HCl Ácido clorídrico

HMF Hidroximetilfurfural

HPLC Cromatografia em camada delgada de alta

eficiência

H2SO4 Ácido sulfúrico

ICP-OES Espectrometria de emissão óptica com plasma

acoplado

KBr Brometo de potássio

KCl Cloreto de potássio

xv

LABMEV Laboratório de Microscopia Eletrônica de

Varredura

LIT Infusão hepática triptose

LQCPol Laboratório de Química de Coordenação e

Polímeros

Man Manose

MBHA3 (3-Hidroxi-2-metileno-3-(4-

nitrofenilpropanonitrilo)

min Minutos

mg Miligrama

mL Mililitro

MTT Brometo de 3-(4,5-dimetil- tiazol-2-il)-2,5-

difeniltetrazólio

NaCl Cloreto de sódio

NaOH Hidróxido de sódio

NH2 Amina

Na2HPO4 Fosfato dissódico

NX Nanoxilana

PI Iodeto de propídeo

PBS Tampão fosfato salino

RAW 264.7 Linhagem celular de macrófago murino

SDS Dodecil sulfato de sódio

Xyl Xilose

XYL Xilana

µg Micrograma

µL Microlitro

nm Nanômetro

xvi

LISTA DE FIGURAS

Figura 1: Distribuição de casos da Doença de Chagas no mundo de acordo com

países endêmicos e não endêmicos ....................................................................... 17

Figura 2: Ciclo de vida do Trypanosoma cruzi ....................................................... 19

Figura 3: Síntese de nanopartícula de prata com o polissacarídeo xilana ............ 22

xvii

SUMÁRIO

1. INTRODUÇÃO ............................................................................................. 18

1.1 Doença de Chagas ..................................................................................... 18

1.2 Polissacarídeos com atividades biológicas ................................................. 21

1.3 Nanopartículas de prata e método de síntese verde .................................. 22

2. JUSTIFICATIVA ........................................................................................... 24

3. OBJETIVOS ................................................................................................. 25

3.1 Geral: .......................................................................................................... 25

3.2 Específicos: ................................................................................................ 25

4. MATERIAIS E MÉTODOS ........................................................................... 26

4.1 Avaliação da composição química dos polissacarídeos ............................. 26

4.1.1. Dosagem de proteínas ........................................................................ 26

4.1.2. Dosagem de compostos fenólicos ....................................................... 26

4.1.3. Dosagem de açúcares ......................................................................... 26

4.2 Caracterização das nanopartículas ............................................................ 27

4.2.1. Espectroscopia de UV-visível .............................................................. 27

5. ARTIGO PRODUZIDO ................................................................................. 28

6. CONCLUSÕES ............................................................................................ 67

7. COMENTÁRIOS, CRÍTICAS E SUGESTÕES ............................................. 68

REFERÊNCIAS ................................................................................................... 69

18

1. INTRODUÇÃO

1.1 Doença de Chagas

A doença de Chagas (DC) ou tripanossomíase americana, descoberta a mais

de um século 1, é reconhecida como uma das doenças parasitárias tropicais mais

negligenciadas no mundo, atingindo cerca de 8 milhões de pessoas, principalmente

na América Latina, além de estar entre as mais importantes infecções parasitárias,

segundo a Organização Mundial de Saúde 2. Possui fatores de risco fortemente

relacionados com baixas condições socioeconômicas. Por muitos anos foi uma

doença restrita às Américas, principalmente a América Latina, e predominantemente

rural. No entanto, a partir da década de 40 devido a fatores socioeconômicos e

migração da população do campo para os centros urbanos, esse perfil foi sendo

alterado 3 levando a expansão da doença a outros locais, inclusive não endêmicos,

como Europa, EUA e Canadá (Figura 1), neste caso, devido a migração de pessoas

infectadas 4,5. No Brasil o número de infectados entre 2012 e 2016 chegou a quase 2

mil pessoas, sendo 1.190 casos confirmados de acordo com o Ministério da Saúde

(2019) 6.

Figura 1: Distribuição de casos da doença de Chagas no mundo de acordo com

países endêmicos e não endêmicos

Fonte: Adaptado de https://www.dndi.org/diseases-projects/chagas/

19

Uma das principais vias de transmissão é a vetorial em que o inseto vetor é um

triatomíneo, da subfamília Triatominae; várias espécies desta subfamília atuam

como vetores na transmissão da doença de Chagas. No Brasil são conhecidas mais

de 68 espécies, 13 delas de importância epidemiológica 6. O triatomíneo é conhecido

popularmente como “barbeiro ou bicudo”, e transporta em seu intestino o protozoário

flagelado Trypanosoma cruzi, agente etiológico da doença de Chagas. No momento

da picada em áreas como pele ou mucosas, o barbeiro além de sugar o sangue

também elimina através das fezes as formas tripomastigotas metacíclicas de

Trypanosoma cruzi (transmissão clássica). No entanto, outras vias de transmissão

têm emergido nos últimos anos e servido de alerta para as agências de saúde, são

elas: a via transfusional, via vertical e a via oral, esta última em maior quantidade 7.

O ciclo evolutivo do T. cruzi é composto por formas evolutivas distintas –

epimastigota, tripomastigota sanguínea, tripomastigota metacíclicas e amastigota.

Diferem entre si principalmente por sua morfologia e posição do cinetoplasto em

relação ao núcleo. Inicia-se o ciclo ainda dentro do intestino médio do inseto, quando

o sangue contaminado com formas tripomastigotas é ingerido pelo triatomíneo. Tais

formas diferenciam-se em epimastigotas que migram para a porção intestinal posterior

e reto, e lá se diferenciam novamente em uma forma potencialmente infecciosa,

tripomastigota metacíclico (metaciclogênese), que será eliminada nas fezes do vetor

no momento da picada. Essas formas entram em contato com o hospedeiro através

de ferimentos na pele ou mucosas e invadem diversas células nucleadas 8. Vias de

sinalização são ativadas após o reconhecimento celular, que culminam com a

internalização deste parasita pela célula hospedeira, formando um “vacúolo

parasitóforo” que abriga o parasita 9. As formas tripomastigotas, através de um

mecanismo enzimático, rompem a membrana deste vacúolo e alcançam o citoplasma

da célula, local este em que se diferenciam na forma amastigota. Uma vez que o

citoplasma celular está preenchido com inúmeras formas amastigotas, há o processo

de nova diferenciação em forma tripomastigota. Essas formas flageladas livres no

meio intercelular irão infectar células vizinhas ou migrar através da corrente sanguínea

para infectar novas células em diversos tecidos (músculo cardíaco, intestino, esôfago)

ou serem novamente ingeridas pelo inseto vetor 9,10. Na Figura 2 pode-se observar o

ciclo de vida deste parasita.

20

Figura 2: Ciclo de vida do Trypanosoma cruzi

Fonte: Adaptado de https://www.chagasfound.org/chagas-disease/life-cycle/

A doença apresenta duas fases clínicas, a aguda ou inicial, caracterizada por

sintomas geralmente leves (quando presentes), apesar do paciente apresentar altos

níveis de parasitemia nesta etapa 11. Alguns estudos demonstraram que a doença é

curável se o paciente for submetido ao tratamento logo após a infecção 12,13. Caso não

haja cura nesse período o paciente passa para a fase seguinte, a crônica, a qual

requer uma maior atenção, pois o mesmo pode apresentar quadros de miocardite e/ou

distúrbios do sistema gastrointestinal e nervoso periférico 8. Nesta fase o paciente

pode manter-se assintomático por anos ou apresentar sintomas graves decorrentes

da lesão da área afetada pelo parasita, podendo chegar a óbito 14–16.

O principal fármaco disponível atualmente no Brasil para o tratamento da

doença de Chagas é o benzonidazol (BNZ), que objetiva eliminar o parasita sanguíneo

na fase aguda da doença. No entanto, este composto apresenta acentuada toxicidade

e baixa eficácia na fase crônica, além de fortes efeitos adversos 17. Alguns estudos

realizados em camundongos verificaram a eficácia deste fármaco durante a infecção

crônica, no entanto, concluíram que o BNZ não erradica completamente o parasita 18–

21. Segura e colaboradores 20 não evidenciaram diminuição da miocardite em

camundongos tratados com o benzonidazol. Este fármaco, além disso, apresenta

limitações dentre elas a proibição do uso por mulheres grávidas ou por pessoas com

insuficiência renal ou hepática.

21

Diante de tantas desvantagens, surge a necessidade urgente de se identificar

melhores fármacos para o tratamento de pacientes chagásicos 22,23. Dentro deste

contexto, pesquisas voltadas à prospecção de moléculas bioativas a partir de fontes

naturais têm aumentado significativamente ao longo dos anos impulsionando os

estudos para um avanço no quadro terapêutico atual desta morbidade.

1.2 Polissacarídeos com atividades biológicas

Estudos diversos têm demonstrado que compostos isolados de produtos

naturais são ferramentas importantes utilizadas em muitas áreas que podem inclusive

auxiliar no tratamento de doenças. Em particular os polissacarídeos que se

apresentam como moléculas funcionais com vasta aplicabilidade nos campos da

medicina, farmácia, indústria de alimentos e coméstica 24 e já são relatadas as suas

atividades anticoagulante e antioxidante 25–27; antitumoral 28 anti-inflamatória 29,

hipoglicemiante 30,31; antitrombótica 32.

Os polissacarídeos são polímeros naturais, unidos por ligações glicosídicas.

Estes polímeros podem ser classificados de acordo com a sua função, podendo ser

de reserva energética (amido) nas plantas e (glicogênio) em fungos e animais, ou

estruturais como por exemplo a celulose. No universo desses compostos existem as

xilanas que são heteropolissacarídeos estruturais que compõem a parede celular de

plantas. A xilana é um composto linear com ligação β-(1-4)-D-xilopiranosil, que

apresenta diversos substituintes ligados à sua cadeia principal, como por exemplo,

resíduos de arabinose, ácido glucourônico, ácido metil-glucourônico e acetil. O arranjo

estrutural da xilana pode diferir dependendo da fonte e devido a fatores abióticos e

bióticos 33,34. As xilanas têm potencial de uso em vários processos biotecnológicos 35.

O sabugo de milho, um dos resíduos agrícolas, apesar de ter uma parcela

significativa descartada pela indústria, desponta como fonte promissora de xilanas

bioativas com elevado potencial farmacológico. Ebringerová e colaboradores 36

relataram em seus estudos propriedades imunomodulatórias das xilanas. Nagashima

e seu grupo de pesquisa 37 também demonstraram a utilização das xilanas do sabugo

de milho na indústria farmacêutica como micro e nanopartículas para liberação

controlada no tratamento de doenças de cólon. Atividades antioxidante, antitumoral,

antimicrobiana e anticoagulante de xilanas de sabugo de milho foram evidenciadas

por Melo-Silveira et al. 26. Além disso, devido as suas propriedades naturais, elas

22

também estão sendo empregadas em formulações para o tratamento de feridas 38.

Bourantane et al. 39 demonstraram a atividade antitumoral das xilanas através de

testes que evidenciaram morte de linhagens celulares de câncer colorretal.

Dada a sua importância, sua diversidade estrutural e suas propriedades

biológicas, as xilanas são candidatas promissoras para o desenvolvimento de

produtos com aplicações biotecnológicas e biomédicas. Estes dados geram

interesse para busca de novos fármacos mais eficazes e com menores efeitos

adversos envolvendo este composto e oportunidade para disponibilizar uma entrega

mais efetiva do fármaco ao tecido lesado, através de nanoformulações com a xilana,

principalmente a extraída do sabugo de milho 40,41.

1.3 Nanopartículas de prata e método de síntese verde

Uma série de vantagens aparecem no campo das nanopartículas feitas com

metais como a baixa polidispersidade, inércia química, biocompatibilidade e fácil

funcionalização, para que ofereçam grande potencial em processos biológicos,

bioquímicos e biomédicos, tanto in vitro como in vivo 42. E devido ao crescente

interesse na área da nanotecnologia cada vez mais pesquisadores procuram utilizar

métodos que agridam minimamente o meio ambiente, empregando métodos

ecologicamente corretos. Estes métodos, utilizados na síntese de nanopartículas são

conhecidos como método de síntese verde e objetivam utilizar reagentes não tóxicos

ao meio ambiente, como por exemplo a água 43,44.

As nanopartículas de prata vêm demonstrando atividades biológicas

importantes, sendo as mais significativas as antivirais 45,46, anti-inflamatória 47,

antifúngica 48 e antiangiogênica 49,50. No entanto, não há relatos na literatura dessas

nanopartículas sendo elas de prata conjugadas ao polissacarídeo xilana com atividade

frente a formas evolutivas do Trypanosoma cruzi, o que gera uma oportunidade de

análises desta como proposta a candidatas no tratamento da doença de Chagas.

O grupo de pesquisa no qual está enserida esta tese relatou através de alguns

trabalhos o efeito sinérgico obtido pela união de um polissacarídeo a prata, na forma

de nanopartículas: Amorim et al. 51 em seus estudos demonstraram a atividade

citotóxica de polissacarídeos sulfatados (PS) da alga marinha Spatoglossum

schröederi potencializada frente às células do adenocarcinoma renal 786-0.

23

Fernandes-Negreiros et al. 52 também evidenciaram um aumento da intensidade nas

atividades antitumoral, imunomodulatória e antibacteriana dos polissacarídeos

sulfatados da alga Dictyota mertensii.

Embora estudos já tenham evidenciado que xilana de sabugo de milho exibe várias

atividades biológicas, particularmente, atividade antiproliferativa contra células de

adenocarcinoma uterino 53 e atividade antioxidante 26, a atividade tripanossomicida

dessa molécula quando unida a nanopartícula de prata ainda é desconhecida.

Em nosso grupo de pesquisa a síntese da nanopartícula de prata ligada a xilana e

a reação de redução da prata foram evidenciados através de uma mudança na

coloração da solução de nitrato de prata e extrato bruto de xilana, dados estes

monitorados através da Espectroscopia de UV-visível, conforme item 2.5.1. de

resultados do artigo produzido, conforme observa-se na Figura 3.

Figura 3: Síntese de nanopartícula de prata com o polissacarídeo xilana. A:

Solução de nitrato de prata; B: Solução de extrato bruto de xilana e nanopartículas de

prata e C: Solução de nanoxilana

Fonte: Imagens cedidas por Souza, A. O (2019).

24

2. JUSTIFICATIVA

A doença de Chagas está em ampla expansão, tanto com aumento do número de

casos, bem como, aumento do número de países que estão sendo atingidos por ela.

Seu tratamento, inclusive farmacológico, não é eficiente, o que tem impulsionado a

busca por alternativas mais eficientes e menos danosas ao paciente. Dentro deste

contexto, tem-se a busca por novas moléculas bioativas ou mesmo, a busca por

nanomateriais que possam potencializar a ação das moléculas, tanto novas, como dos

fármacos já existentes. Como é o caso da prata, um antiparasita já conhecido, mas

que não pode ser administrado em pacientes, pois apresenta toxicidade nas

concentrações em que é ativa. Polissacarídeos estão no cerne dessa linha de

pensamento, pois muitos são bioativos e/ou podem ser utilizados como matéria prima

para o desenvolvimento de nanomateriais. O Brasil é o terceiro maior produtor de

milho e esta grande produção gera um volume acentuado de resíduos os quais, se

não utilizados de forma correta, podem causar um problema ambiental significativo.

Um desses resíduos é o sabugo de milho que aparece como uma rica fonte natural

da xilana. A xilana é um polissacarídeo que não teve avaliação do seu potencial como

componente de nanomateriais feitos com prata como agente tripanossomicida. Fato

que não se justifica, pois a xilana é obtida facilmente por métodos não agressivos ao

meio ambiente; é obtida em grandes quantidades do sabugo de milho; e já foi

demonstrada como ser um composto com propriedades farmacológicas. Testes

realizados pelo nosso grupo de pesquisa evidenciaram que nanopartículas de prata

ligadas ao polissacarídeo xilana não possuem citotoxicidade frente a células da

linhagem 3T3, este é outro fato que justifica o nosso interesse pela união de uma

nanopartícula de prata ao polissacarídeo xilana extraído do sabugo de milho. Portanto,

os dados aqui apresentados nesta Tese visam começar a preencher esta lacuna de

conhecimento referente a xilana no tocante a temática da doença de Chagas.

25

3. OBJETIVOS

3.1 Geral:

Sintetizar nanopartículas de prata contendo xilana do sabugo de milho e avaliar

sua a atividade contra formas epimastigotas de Trypanosoma cruzi.

3.2 Específicos:

Extrair a xilana do sabugo de milho e caracterizá-la quimicamente;

Avaliar a composição monossacarídica da xilana;

Sintetizar a nanopartícula de prata unida a xilana (nanoxilana) e avaliá-la

morfologicamente e físico-quimicamente.

Avaliar a atividade antiparasitária contra Trypanosoma cruzi e identificar o

mecanismo de morte causado pela NX.

26

4. MATERIAIS E MÉTODOS

Todos os materiais e a maioria dos métodos encontrados nesta tese são

descritos no Tópico 2 do artigo 1. Devido a alguns fatores, aqueles métodos que

porventura, estejam muito resumidos no Tópico 2 são descritos com mais detalhe a

seguir.

4.1 Avaliação da composição química dos polissacarídeos

4.1.1. Dosagem de proteínas

Para analisar a contaminação da amostra por proteínas foi utilizado o reagente

de Bradford e a albumina bovina como padrão. As leituras foram feitas a 595 nm. Essa

técnica envolve a ligação do corante Comassie brilliant blue à proteína e um aumento

da absorção de luz (de 465 nm para 595 nm) pelo corante é evidenciado. O processo

de ligação completa do corante à proteína é rápido, dura em torno de 2 minutos e a

coloração é estável por 1 hora 54.

4.1.2. Dosagem de compostos fenólicos

A contaminação da xilana por compostos fenólicos foi avaliada utilizando o

método colorimétrico de Folin-Ciocalteau. O reagente de Folin-Ciocalteau é composto

pelos ácidos fosfomolibídico e fosfotunguístico, nos quais o molibdênio e o tungstênio

encontram-se no estado de oxidação 6+. No entanto quando estes estão em presença

de substâncias redutoras, formam-se compostos de molibdênio e tungstênio, ambos

de cor azul. A coloração permite a determinação da concentração das substâncias

redutoras. As leituras foram realizadas em 760 nm e o conteúdo desses compostos

foi calculado a partir de uma curva padrão de ácido gálico 55,56.

4.1.3. Dosagem de açúcares

O teor de açúcares totais foi determinado por espectrofotometria a um

comprimento de onda de 490 nm usando D-xilose como padrão. Como os

polissacarídeos são ricos em grupos hidroxila (-OH), eles podem ser facilmente

desidratados. Para isso utilizou-se um ácido forte como ácido sulfúrico que rompe

facilmente as ligações glicosídicas presentes nessas moléculas, liberando os

monossacarídeos presentes. Uma vez liberados esses monossacarídeos

27

desidratados geram um composto incolor conhecido furfural, quando o

monossacarídeo desidratado for uma pentose, e o hidroximetilfurfural (HMF), quando

for uma hexose. Após esse processo, adiciona-se fenol a 80%, que gera uma

coloração amarelo-alaranjado. A mudança de coloração da solução foi medida na

região do visível e é proporcional à quantidade de açúcares presentes na amostra.

Esse método é conhecido como Fenol-H2SO4 ou Método de Dubois 57.

4.2 Caracterização das nanopartículas

4.2.1. Espectroscopia de UV-visível

A formação das nanoxilanas (NX) e redução da prata foram analisadas

observando-se uma mudança na coloração da solução. A solução de coloração

amarelo claro representou o tempo zero (0 h); a intermediária o tempo 12 h e a de

coloração intensa (marrom escuro) o tempo total da síntese de 24 h. Esse aspecto foi

monitorado por espectroscopia eletrônica na região UV-visível com leituras realizadas

de 350 a 600 nm. Foi utilizado o Espectrofotômetro UV/Visível modelo DR 5000 Hach

Lange GmbH (Düsseldorf, Alemanha).

28

5. ARTIGO PRODUZIDO

O artigo “Synthesis of trypanocidal active silver nanoparticle employing

corn cob xylan as a reducing agent” foi aceito no periódico International Journal of

Nanomedicine que possui fator de impacto 4.471 (ano 2018) e Qualis A1 da CAPES

para área Medicina II.

29

Synthesis of trypanocidal active silver nanoparticle employing corn cob xylan as

a reducing agent.

Talita Katiane Brito1,2, Cláudia Jassica Gonçalves Moreno3,5, Rony Lucas Silva

Viana2,3, Jefferson da Silva Barbosa1,2,4, Francimar Lopes de Sousa Júnior6, Mayara

Jane Campos de Medeiros6, Raniere Fagundes Melo-Silveira2,3, Jailma Almeida-

Lima1,2,3, Daniel de Lima Pontes6, Marcelo Sousa Silva3,5,7, Hugo Alexandre Oliveira

Rocha 1,2,3 *

1Postgraduate Program in Health Sciences, Federal University of Rio Grande do Norte

(UFRN), Natal-RN 59012-570, Brazil; talitakatiane@hotmail.com (T.K.B);

biolottus23@yahoo.com.br (J.A.L).

2Laboratory of Biotechnology of Natural Polymers (BIOPOL), Department of

Biochemistry, Center of Biosciences, Federal University of Rio Grande do Norte

(UFRN), Natal-RN, 59078-970, Brazil; rony_lucas@hotmail.com (R.L.S.V);

ranieremelosilveira@gmail.com (R.F.M.S).

3Postgraduate Program in Biochemistry, Federal University of Rio Grande do Norte

UFRN), Natal-RN, 59.078-970, Brazil.

4Federal Institute of Education, Science and Technology of Rio Grande do Norte

(IFRN), Rio Grande do Norte 59500-000, Brazil; jefferson.barbosa@ifrn.edu.br (J.S.B);

5Laboratory of Immunoparasitology, Department of Clinical and Toxicological

Analysis, Health Sciences Center, Federal University of Rio Grande do Norte, Natal-

30

RN, 59012-570, Brazil; claudia.mrn1@gmail.com (C.J.G.M); mssilva.ufrn@gmail.com

(M.S.S).

6Laboratory of Chemistry of Coordination and Polymers (LQCPol), Institute of

Chemistry, Federal University of Rio Grande do Norte (UFRN), Natal - RN 59078-970,

Brazil; francimar_junnior@hotmail.com (F.L.S.J); mayarajane20049@hotmail.com

(M.J.C.M); pontesdl@yahoo.com (D.L.P).

7Global Health and Tropical Medicine, Institute of Hygiene and Tropical Medicine,

New University of Lisbon, 1349-008, Lisboa, Portugal.

Correspondence: Hugo Alexandre Oliveira Rocha, Laboratory of Biotechnology of

Natural Polymers (BIOPOL), Department of Biochemistry, Center of Biosciences,

Federal University of Rio Grande do Norte (UFRN), Natal-RN, 59078-970, Brazil;

hugo@cb.ufrn.br

Background: Chagas disease, also known as American Trypanosomiasis, is caused

by the protozoan Trypanosoma cruzi. It is occurring in Americas, including USA and

Canada, and Europe and its current treatment involves the use of two drugs as

follows: benznidazole (BNZ) and nifurtimox, which present high toxicity and low

efficacy during the chronic phase of the disease, thus promoting the search for more

effective therapeutic alternatives. Xylan, a bioactive polysaccharide, was extracted

from corn cob using an environmentally friendly method.

31

Methods: Xylan from corn cob was characterized by FTIR (Fourier-transform

infrared spectroscopy) and monosaccharide composition. MTT (3-bromo(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium).

Result: Data from atomic force microscopy, FTIR, UV-Visive, Raman spectrocopy,

and coupled plasma atomic emission showed that NX was composed by xylan and

silver (19%), with rounded shapes and average sizes (~ 40 nm). MTT and Flow

cytometry analyses showed that NX (100 µg/mL) induces almost 100% death of the

parasites (~95% necrosis), whereas xylan and silver were not effective.

Conclusion: This is the first time silver nanoparticles are presented as a trypanocidal

agent and the data point to the potential application of NX to new preclinical studies

in vitro and in vivo.

Keywords: Xylan; Silver nanoparticles; Trypanosoma cruzi; Benznidazole.

1. Introduction

For several years, Chagas disease had assumed a predominantly rural profile,

but has now become part of the urban zone due to movement of the population from

country sides to cities, due to increased deforestation and socioeconomic changes. This

migration has also affected non-endemic areas such as Europe, USA and Canada.1–3

Also known as American Trypanosomiasis, it is caused by the protozoan Trypanosoma

cruzi and is transmitted among other forms by insects of the Triatominae subfamilys.4

It is estimated that around 8 million people worldwide, particularly those residing in

32

Latin America, are infected with Trypanosoma cruzi. More than 10 thousand people die

of the disease every year and more than 25 million are at a risk of acquiring it.5

Chagas disease has two successive phases, acute and chronic.2 There are no

vaccines to treat the patients affected by the disease and their treatment is

pharmacological. On the other hand, these drugs are used to treat the disease mainly

in its acute phase, reducing symptoms and parasitemia. The current treatment

involves the use of two drugs: benznidazole (BNZ) and nifurtimox. Both are far from

ideal due to their side effects, limited efficacy and little is known about their activity

in the chronic phase. These disadvantages justify the urgent need to identify better

drugs for the treatment of chagasic patients.6,7 Considering this scenario, researches

aimed at prospecting bioactive molecules from natural sources has increased

significantly over the years.8–11

In the secondary cell walls of Gramineae and dicotyledons, for example, the

main hemicellulose is a polymer composed of D-xylose units linked by β glycosidic (1

→ 4) bonds called xylans.12 Xylans from different sources have been used in the

biosynthesis of grafts, bandages and sutures, synthesis of micro and nanoparticles13,14

as well as for the controlled release of drugs.15–17 It has been reported in the literature

that corn cobs (Gramineae), underutilized in industry and often discarded, are the

most promising material for the extraction of this biopolymers.18

Xylans obtained from corn cobs had a chemical composition consisting of 4-O-

methyl-D-glucuronic acid, L-arabinose and D-xylose in a ratio of 2:7:19.19,20 It has been

also reported that the xylans from corn cobs demonstrate immunomodulatory21,

33

antioxidant, anticoagulant and antibacterial properties as well as cytotoxicity to tumor

cells.22

The synthesis of nanoparticles, including silver nanoparticles, often leads to the

production of several undesirable wastes. Therefore, the search for more effective and

environmentally friendly methods is ongoing. These methods are known as green

synthetic methods and are considered efficient since they do not use toxic reagents,

such as water, for the environment.23

Studies from our research group have shown that the green synthesis of silver

nanoparticles with polysaccharides has potentiated the biological activity of both

polysaccharides and silver. According to Amorim et al24, the cytotoxic activity of

sulfated polysaccharides (sPS) of the seaweed Spatoglossum schröederi was potentiated

against renal adenocarcinoma cells 786-0. Also, the antitumor, immunomodulatory

and antibacterial activities of the sPS of the seaweed Dictyota mertensii had intensified.25

In both cases, they composed the structure of silver nanoparticles.

Our group also demonstrated that xylan from corn cobs exhibits several

biological activities, particularly, antiproliferative activity against uterine

adenocarcinoma cells26 and antioxidant activity.22 However, the activities of this

molecule were not evaluated when they are part of the silver nanoparticle structure

regarding its antiparasitic activity against T. cruzi. Therefore, in the present study,

silver nanoparticles containing xylans of corn cobs were synthesized to evaluate their

effects against T. cruzi.

34

2. Materials and Methods

2.1. Materials

MTT (3-bromo-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), silver

nitrate (AgNO3), D-xylose, bovine serum albumin (BSA), cell culture media - liver

infusion tryptose (LIT), dextrose, hemina, tryptose and liver infusion broth were

purchased from the Sigma Chemical Company (St. Louis, MO, USA). NaCl, KCl and

Na2HPO4 from the INLAB (São Paulo, SP, Brazil). Sterile fetal bovine serum was

purchased from the Cultilab (Campinas, SP, Brazil). Benznidazole (Rochagan)

Hoffmann-La Roche (Basel, Switzerland) was provided by the consortium of the

BERENICE (BEnznidazole and Triazole REsearch group for Nanomedicine and

Innovation on Chagas diseasE - Barcelona, Spain) project. We also procured the

Bradford reagent from the Bio-Rad Laboratories, Inc. (Hercules, CA, USA) and the

Folin-Ciocalteau reagent from the Merck KGaA (Darmstadt, Hessen, Germany).

Penicillin and streptomycin were obtained from Thermo Fisher Scientific (Waltham,

Massachusetts, USA). Sodium hydroxide, sulfuric acid and methanol were purchased

from the CRQ (Diadema, SP, Brazil). Phenol was obtained from the Reagen Quimibrás

Indústrias Químicas S. A. (Rio de Janeiro, RJ, Brazil). Annexin V Apoptosis Detection

Kit for flow cytometry (Annexin-FITC, Propionate Iodide - PercP) was purchased from

BD Biosciences Inc. (San Diego, CA, USA).

35

2.2. Extraction of xylan from corn cobs

Fresh corn samples bought from a local market (Natal, RN - Brazil) were peeled,

cleaned and washed. They were then cut into small pieces, dried in a kiln at 60 °C and

ground until its flour was obtained. 160 mL of methanol was added to 10 g of flour

produced from the cob and the material was stirred for 24 h in the dark to remove

lipids and pigments. The precipitate was collected by centrifugation (10,000 × g, 20

min, 4 °C), dried and stored in hermetically sealed vials at 25 °C until use. For the

extraction of the polysaccharides, 25 mL of the solution of NaOH (1.8 mol/L) in the

ratio of 1:25 (w/v) was added to 1 g of corn cob flour. This solution was sonicated at

an ultrasound power of 200 W for 30 min at intervals of 5 min and a temperature of 60

°C. The solution was centrifuged to separate the extracted material from the insoluble

residue. Four volumes of chilled methanol was added to the supernatant under

conditions of gentle agitation and was held protected from light at 4 °C for 24 h. The

precipitate thus formed was collected by centrifugation (10,000 × g, 20 min, 4 °C), dried

and stored in flasks at 25 °C for further analysis.

2.3. Chemical analysis of xylan

The contamination of xylan by phenolic compounds was evaluated using the

Folin-Ciocalteau method and gallic acid was used as the standard, as described by

Nascimento et al.27 Protein contamination was assessed using the Bradford reagent

36

and bovine albumin was used as the standard.28 Total sugars were analysed by the

reaction of phenol-H2SO4 and D-xylose was used as the standard.29

2.3.1 Determination of the monosaccharide composition of xylan

An analysis of the monosaccharide composition was performed as described by

Melo-Silveira et al.22 The material after being hydrolysed was subjected to sugar

composition analysis using the LaChrom Elite® VWR-Hitachi high performance

liquid chromatography (HPLC) system with a L-2490 refractive index detector. The

column used was LichroCART® 250-4 Lichrospher® 100 NH2 (10 μM). The mobile

phase used in the test was with acetonitrile: water (80:20) in a flow rate of 1 mL/ min,

at 40 °C. The following sugars were analysed as references: arabinose, galactose,

glucose, glucuronic acid, fructose, fucose, mannose, N-acetyl-glucosamine,

galactosamine, rhamnose and xylose.

2.4. Synthesis of nano xylans silver

The nano xylans were obtained using the process of green synthesis described

by Dipankar et al.30 The corn cob xylan was used as a bioreductor in this experiment.

The synthesis of NX was performed by the addition of a solution of 1.0 mM silver

nitrate to a 10 mg/mL (1:9 w/v) solution of xylan. The solution was subjected to

continuous stirring for 24 h and was placed in conditions protected from the light.

After this period, it was centrifuged at 10,000 × g for 15 min at 25 °C. The precipitate

was collected and lyophilized.

37

2.5. Characterization of the nanoparticles

2.5.1. UV-Visible spectroscopy

The formation of NX and reduction of silver were analysed by observing a

change in the color of the solution. Electron spectroscopy in the UV-visible region with

readings in the 350 to 800 nm range was performed to monitor these reactions using

the UV/Visible spectrophotometer model DR 5000 Hach Lange GmbH (Düsseldorf,

Germany).

2.5.2. Atomic force microscopy (AFM) analysis

The samples were dried on coverslips in a desiccator. Images of the NX were

obtained using the scanning probe microscope SPM – 9700 (Shimadzu, Kyoto, Japan),

in the Scanning Electron Microscopy Laboratory (Laboratório de Microscopia

Eletrônica de Varredura – LABMEV – Department of Materials Engineering) of the

Federal University of Rio Grande do Norte (Universidade Federal do Rio Grande do

Norte - UFRN).

2.5.3 Quantitative determination of silver in nano xylans by coupled plasma optical emission

spectroscopy (ICP-OES)

2.5.3.1 Sample digestion

Analytical scales were used to weigh 100 mg of sample in Teflon cups (total 200

mg of sample; 100 mg of each cup), to which 7 mL of 65% nitric acid purified by sub-

38

boiling distillation (Berghof, Eningen, Germany) and 1mL of 30% hydrogen peroxide

(Merck, Darmstadt, Germany) were added. The digestion cups were subsequently

closed and digestion was performed in a microwave digester (Start D, Milestone, Italy)

using 6 stages and a power of 1100W:(1) 5 min at 70 °C; (2) 2min at 70 °C;(3) 3min at

120 °C; (4) 2 min at 120 °C;(5) 10min at 170 °C;(6) 15 min at 170 °C and lastly 30 min of

ventilation before there moval of the rotor from the microwave. The digested content

was quantitatively made up to 15 mL using deionized water and filtered through a

0.45 µm membrane. The analyses were conducted in duplicate and analytical blanks

were performed by conducting the procedure in the absence of sample.

2.5.3.2 Determination of silver content the digested sample

Silver content of digested sample (NX) was quantified using an inductively

coupled plasma emission spectrometer (ICP-OES 5100 VDV, Agilent Technologies,

Tokyo, Japan) in axial view, equipped with a radio frequency source (RF) of 27 MHz,

using a simultaneous optical detector, a peristaltic pump, a double pass cyclonic spray

chamber, a 1.8 mm quartz torch, and a spray glass nebulizer. The gas used in the

system was plasma liquid argon with a purity of 99.996% White Martins, SP, Brazil).

The ICP-OES equipment operated under the following conditions: power of the

plasma, 1.5 kW; argon flow, 12.0 L/min; auxiliary argon flow, 1.0 L/min; nebulization

flow, 0.70 L/min; number of replicates, 3; stabilization and reading time, 15 s,

wavelength, 328.068 nm. The analytical curve for silver was prepared by diluting 1000

mg/L of reference standard solution (Merck, Darmstadt, Germany) into a 2.5–100 g/L

range (r= 0.9999) in a solution of 5% (v/v) nitric acid prepared from 65% acid distilled

39

in a subboiling system (Distillacid, Berghof, Eningen, Germany). Serial dilutions were

prepared for sample measurement: i) 0.1 mL/10 mL and ii) 0.2 mL/10 mL.

2.5.4 Analysis using Fourier-transform infrared spectroscopy (FT-IR)

The samples (10 mg) were mixed with KBr, triturated, and pellets, made from

this material, were subjected to FT-IR. Spectra of the samples were obtained using a

spectrophotometer (IRAAffinity-1 Shimadzu Corp., Kyoto, Japan) equipped with the

IRsolution Shimadzu Corp. software (Kyoto, Japan) version 1.60 with scan number 32

and 4 cm-1 resolution. The frequency range for the analysis was 4000 to 400 cm-1. The

analyses were carried out at the analytical center of UFRN's Chemistry Institute and

the Coordination and Polymers Chemistry Laboratory (Laboratório de Química de

Coordenação e Polímeros - LQCPol - Department of Chemistry) of the Federal

University of Rio Grande do Norte (Universidade Federal do Rio Grande do Norte -

UFRN). Three independent analyses were performed.

2.5.5 Analysis using Raman spectroscopy

The Raman spectra were obtained from pre-crushed samples in the solid state.

The samples were analysed using the Raman confocal microscope, model LabRAM

HR Evolution, HORIBA Scientific with the CCD detector. For this purpose, the 633 nm

laser with an acquisition time of 20 s and 2 s of accumulation in the region of 30 to 4000

cm-1 was used. The analyses were carried out at the Coordination and Polymers

Chemistry Laboratory (Laboratório de Química de Coordenação e Polímeros –

40

LQCPol – Department of Chemistry) of the Federal University of Rio Grande do Norte

(Universidade Federal do Rio Grande do Norte - UFRN).

2.6 Evaluation of the antiparasitic activity of nano xylan by the colorimetric MTT (3-

bromo(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) reduction assay

The epimastigote forms of the Y strain of T. cruzi were maintained in LIT

supplemented with 10% fetal bovine serum (FBS) from Thermo Fisher Scientific

(Waltham, MA - USA) at 28 ± 2 °C in a biochemical oxygen demand (BOD) kiln. The

strains were cultured until the exponential phase of growth (1 ×107 parasites/mL) was

reached. Samples were tested at concentrations of 2.5, 10 and 100 μg/mL and incubated

at 28 ± 2 °C for 24 h and 48 h. 90 μL of the culture (1 ×107 parasites) and 10 μL of

nanoparticles were added to a 96-well plate and incubated for 24 h. After incubation,

10 μL of MTT (5 mg) was added, and after 75 minutes of incubation in a BOD kiln, 100

μL of the solubilisation solution (0.01N HCl and 10% SDS) was added. Absorbance

was read at 570 nm using the Epoch Microplate Spectrophotometer obtained from

BioTek (Winooski, VT, USA). The assays were performed in triplicate and the data

obtained were processed with the GraphPad Prism software version 5.0, 2014 (La Jolla,

California, USA).

2.7 Flow cytometry

The mechanisms of cell death of the T. cruzi parasite through apoptosis/necrosis

were investigated by labeling with Annexin V/Propidium iodide by following the

manufacturer’s instructions from the Annexin V FITC Apoptosis Detection Kit -

41

Invitrogen. After treatment with nanoparticles for 24 hours, the parasites were

centrifuged at a speed of 2000 rpm at 4 °C for 5 minutes. The pellet, thus obtained, was

washed with phosphate buffered saline (PBS), resuspended in the binding buffer and

the Annexin V/PI labeling was performed for 10 min at 25 °C protected from the light.

The analysis was performed on the FACSCanto II flow cytometer (BD Biosciences,

Eugene, OR, USA) with the FACSDiva software, version 6.1.2 (Becton Dickson,

Franklin Lakes, NJ, USA). For each experimental condition, 20,000 events were

captured, and the percentage of labeled cells was calculated in the FlowJo Software

Version vX.0.7 1997-2014 (FlowJo, Ashland, OR, USA).

2.8 Statistical analysis

The experiments were performed at least twice and in triplicates. Data were

analysed using the ANOVA test, followed by the Tukey test. Statistical analyses were

conducted using the Prism 5 software (GraphPad software, version 5.0, 2014 La Jolla,

California, USA). Values of p < 0.05 were considered statistically significant. The

results were expressed as mean ± standard deviation.

3. Results and discussion

3.1 Extraction and chemical analysis of the corn cob xylan

Xylan was extracted as described in the methods section, and the content of its

total sugars, proteins and phenolic compounds was determined. It can be observed in

Table 1 that the sample consists majorly of carbohydrates (97.42%), and the obtained

42

xylan is composed mainly of xylose (50%), glucose (20%), arabinose (15%) and

galactose (10%) as well as some amounts of mannose (2.5%) and glucuronic acid (2.5%)

residues. Similar compositions of monosaccharides were observed in other studies that

evaluated xylans extracted from corn cob31 although their values differed from those

observed in our work. On the other hand, in the corn cob xylan studied by Melo-Silveira

et al22,26 the composition of monosaccharides as well as their proportions were similar

to those presented in Table 1. It is worth mentioning that these authors used the same

process described in this study.

Table 1. Chemical composition of xylan extracted from corn cob

axyl: xylose; bara: arabinose; cglc: glucose; dgal: galactose; eman: mannose; fGlcA:

glucuronic acid.

3.2. Synthesis of silver nanoparticles with xylan (nano xylan)

The UV-Vis spectroscopy was used as an essential tool to confirm the synthesis

of nanoparticles containing silver30 and assist in the analysis for the formation of

nanocomposites with reduced silver. Therefore, an analysis was performed using this

technique with a scanning range of 350-800 nm for the silver nanoparticle solution. In

the region highlighted by the rectangle (between 400-450 nm) in Figure 1, a change in

Molar Ratio (%)

Sample Polysaccharide

(%)

Protein

(%)

Phenolic

Compounds

(%)

aXyl bGlc cAra dGal eMan fGlcA

Xylan 97.42 2.18 0.40 50.0 20.0 15.0 10.0 2.5 2.5

43

the light absorption profile, evidenced by a small increase in the absorbance values, is

observed. From this region, a decrease in the absorbance is observed that almost

reaches zero during the sweep. When the silver nitrate or xylan solutions were

analysed separately in the 400-450 nm range, the same absorption profile was not

observed in this wavelength range (data not shown). This signal is characteristic of

reduced silver and indicates the formation of silver nanoparticles.32,33 Similar to the use

of xylan employed in this work, in other studies, the use of polysaccharides with

reducing agents to synthesize silver nanoparticles has already been described.24,25,34

Figure 1. UV-visible light absorption spectrum of the silver nanoparticle suspension. Scan performed

between 350 and 800 nm.

3.3. Determination of the composition of NX

Constituents of NX were determined as described in the methods section and

were found to consist of xylan (81%) and silver (19%). There are no reports in the

literature about studies that have evaluated the amount of silver in nanoparticles

44

synthesized with the xylan polysaccharide. However, some authors have verified the

amount of silver in the structure of nanoparticles containing other polysaccharides. In

studies conducted by Amorim et al24 and Chen et al35, 7% and 6.7% of silver were

found, respectively, in a fucan-coated nanoparticle composition and in nanoparticles

synthesized with fungal exopolysaccharide. These values are lower than those

obtained by us in our study using corn cob xylans. However, it is worth mentioning

that the efficiency of synthesis of silver nanoparticles and their characteristics depend

on certain factors such as temperature, concentration of plant extract used during the

process and reaction time36, which would explain the previously described variation

in values as well as the variation presented in this study.

3.3.1. Morphological analysis of NX by AFM

To evaluate the size of NX in the solution, their average size was determined using

AFM, which was approximately 40 nm, and had a rounded/spherical shape (Figure 2).

45

Figure 2. Atomic Force Microscopy of silver nanoparticles containing corn cob xylan. In A, the

nanoparticle can be seen in two dimensions. The arrow indicates the size of the nanoparticle (40.17 nm).

B shows the representation of image A in three dimensions. It is also possible to observe the rounded

shape of the particles.

It is possible to obtain nanoparticles larger or smaller than those found in this

research. However, silver nanoparticles with sizes ranging from 5 to 150 nm in

diameter (45-48) have been commonly observed in literature. Another essential

characteristic is the relation between the size of the nanoparticles and the response

they cause in biological systems, since the cytotoxicity of silver nanoparticles is

influenced by variations in their particle size.37

Studies report that smaller particles can induce higher cytotoxicity since they

can be easily internalized by the cells. To support this hypothesis, Carlson et al38 using

silver nanoparticles of sizes 15, 30 and 55 nm, found that the smaller nanoparticles (15

nm) generated more reactive oxygen species in murine macrophage lines. Liu et al39 in

turn, working with four human cell lines (A549, HepG2, MCF-7 and SGC-7901), also

found that silver nanoparticles with a diameter of 5 nm were more toxic than those

with 20 and 50 nm.

Morphology is another factor that can influence the activity of different types of

silver nanoparticles. The most commonly produced structures are as follows:

spherical, triangular, square, cubic, rectangular, oval and acicular. Considering their

toxicity, it is not yet known which form of the particle has the best effect on biological

systems and it will depend on several factors (concentration, pH, temperature, electric

46

charge) instead of a single factor. However, what has already been observed is that

spherical-shaped silver nanoparticles, such as the NX studied in this work, do not have

damaging effects on the cells.40,41

3.3.2 Analysis using FT-IR

FT-IR is an important and widely used technique to study the physicochemical

and conformational properties of compounds.42 Therefore, corn cob xylan and NX

were analysed using infrared spectroscopy and its spectra are presented in Figure 3.

There was no overlap in the spectra but there is a coincidence of bands found in both.

Figure 3. Infrared spectra of corn cob xylan and nano xylan in the 4000 to 500 cm-1 regions.

47

Bands in both spectra with absorption close to the 3430.70-3429.93 cm-1 regions

were attributed to the O-H stretch of the xylan monosaccharides. Oliveira et al20

reported that this absorption bandwidth occurs because of the stretching of the

monosaccharide hydroxyls associated with the water molecules by intra and

intermolecular hydrogen bonds.

The presence of arabinose side chains is characterized by a low-intensity band

in the 1161.41 cm-1 region. This band corresponds to the C–O–C vibrations in the

anomeric region of the xylan.43 Another prominent band observed in the 1046.59 cm-1

region of both spectra, which is a characteristic of xylans, was attributed to the

stretching of the C-C and C-O groups present in the sugar residues.44

The two bands that need to be highlighted are those observed at 896.90 cm-1 and

1039.63 cm-1. The first band was attributed to the anomeric carbon of the β-glycosidic

bonds of the monosaccharides, while the second band is characteristic of xylans

containing β (1→ 4) glycosidic bonds.45 Also, C, Li46 and Nacos et al47 have stated that

these bands are characteristics of polysaccharides with xylose units in their main chain.

In both spectra, bands of moderate intensity in the 2936.63 cm-1 region were also

observed which are characteristic of the C-H group stretches typical of

monosaccharides.48,49

It has been reported in the literature that bands near the 1400 cm-1 region of the

nanoparticle spectra correspond to those of reduced silver.50 With xylan, this band

appears in the spectrum of the silver nanoparticles around 1392 cm-1.

48

We also observed the presence of a band in the 1923 cm-1 region of the NX

spectrum and its absence in the xylan spectrum, which can be an indicator of the

presence of clusters during the formation of the nanoparticle or the superposition of

vibrational movements. However, we did not find data in literature reporting the

presence of this band in the spectra of the silver nanoparticle with xylan.

Some xylan bands appeared to be displaced in the NX spectra compared to those

of the free xylan spectrum, probably because of interaction between the silver and

polysaccharide, resulting in modifications in the electron density around the xylan

groups and displacing their vibrational modes.

Infrared analyzes suggest the interaction of polysaccharide (xylan) groups with

silver, confirming the formation of silver nanoparticles with the corn cob xylan.

3.3.3. Analysis of xylan and NX using Raman spectroscopy

Raman spectroscopy is a technique widely used to complement the study of

infrared spectroscopy.51 This analysis was carried out to confirm the structure of xylan

due to its complex nature52 and to analyse the formation of silver nanoparticles bound

to this compound.

There are few reports in literature on the analysis of the structure of xylan

using the Raman technique. Therefore, the assignment of bands in the present work

was based on interpretations of the spectra and performed using the data obtained

from the Raman analyses of other polysaccharides.

49

In Figure 4, the presence of β-type glycosidic bonds (1→4) is identified by a

band common to both spectra in the region around 840.18 cm-1.53 These authors also

evidenced the same band in the Raman spectra of wheat arabinoxylans, which have

xylans in its main chain.

Three bands around 1187 cm-1 (xylan) and 1201.80 cm-1 (NX) can be observed in

Figure 4. These bands are closely related to the C-O-H and C-H groups. Zeng et al52

reported that xylans from the cell walls, that are solubilized by enzymes, also showed

bands around the region between 1100 and 1220 cm-1.

Bands of glucopyranose rings were observed in the spectral range of 1100 to

900 cm-1, which are indicative of the C–C and C–O stretch modes.54

In the spectrum of NX, it can be noted that the main vibrational modes of xylan,

as discussed already, are present and the majority of them have coincident bands

except for an intense and wide band in the region of 2721.86 cm-1. However, after

analysing many studies that have used the Raman technique to evaluate nanoparticles

containing silver and polysaccharides, it was found that none presented an

interpretation of this band making it difficult to understand its significance in the NX

spectrum. Thus, it is necessary to conduct a study that goes beyond the scope of our

work to explain its meaning.

Moreover, this spectrum is clearer and contains bands that are better defined

than that of xylan. This corroborates with the report of Li et al,55 which asserts that the

quality of Raman signals is affected by the size of their metal cores.

50

Data presented in this paper confirm the interaction of xylan with silver and the

formation of silver nanoparticles. The synthesis of silver nanoparticles containing

polysaccharides aims to improve biological activities not only of the polysaccharides

themselves but also of the silver, since the presence of polysaccharides, such as xylan,

allows a better acceptance of the nanoparticles by the cells. For example, in the study

conducted by Akmaz et al,56 it was noted that the chitosan/silver nanoparticles had

significantly higher bacterial activity than that of pure chitosan.

Figure 4. Raman and infrared spectra of xylan and nano xylan.

The analyses were performed using a 633 nm laser with an acquisition time of 20 s and 2 s of

accumulation in the region of 500 to 4000 cm-1. All samples were obtained using this methodology.

3.4 Evaluation of the antiparasitic activity of nano xylan by the colorimetric MTT (3-

bromo(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) reduction assay

51

Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin

America and manifests itself in two distinct phases, acute and chronic. BNZ is the drug

of choice for treatment in the acute phase. However, despite its effectiveness, it is

highly toxic. On the other hand, for the chronic phase of the disease, currently, no

effective treatment is available. BNZ has low solubility/bioavailability and high

toxicity and because of these factors may not affect an intracellular form the parasite.57

Therefore, it is crucial to search for new drugs that are effective. Some studies show

silver as a treatment of choice due to its antimicrobial properties, however, due to its

known cytotoxicity, its application is limited.58 One way around this problem is to use

silver in the form of nanoparticles, because in this formulation it is possible to use

smaller doses of the element, thus making it less toxic. Moreover, the use of silver

nanoparticles with polysaccharides becomes interesting because many of these

molecules can easily cross the cell membrane without causing damage to normal

cells.34,59

Figure 5 shows the antiparasitic activity of NX when incubated at

concentrations of 2.5, 10 and 100 μg/mL for 24 and 48 h. BNZ was used as a positive

control, as shown in Figure 5. In 24 h, only the highest concentration evaluated (100

μg/mL) was able to decrease the ability of the parasite to reduce MTT. However, at 48

h, its ability to reduce MTT was decreased when subjected to lower concentrations of

BNZ (2.5 and 10 μg/mL). On the other hand, the effect of BNZ at its highest

concentration on the ability of the parasite to reduce MTT was lower than that

52

observed at 24 h (Figure 5B). This decrease in the effect of BNZ on the parasites in

strain Y has also been reported by other authors.60

Under no conditions was the xylan able to inhibit the parasite-reducing action

against MTT, even with the highest concentration tested (100 μg/mL). A similar result

was observed when the effect of silver nitrate was evaluated, which showed that the

two NX compounds, when isolated, do not affect the parasites. It is noteworthy that

the amount of silver nitrate used contained 30 μg/mL of silver, which is 1.5 times

greater than that present in the 100 μg/mL of NX.

Figure 5. Antiparasitic activity of xylan and nano xylan on the epimastigote forms of T. cruzi after

incubation for 24 h (A) and 48 h (B). The negative control is represented by CN. The positive control

(BNZ) is represented by BNZ. Silver nitrate (AgNO3 - 30 μg/mL), nano xylan (NX and xylan (XYL).

Different letters (a, b, c) indicate a significant difference (p < 0.05) between various concentrations of the

same sample. Different numbers (1, 2, 3) indicate a significant difference (p < 0.05) between the same

concentration of different samples. Statistical analysis was performed using ANOVA followed by the

Student-Newman-Keuls post-test.

53

The reduction of MTT by the parasite decreased significantly (82% to 95%, 24

and 48 h respectively) in comparison with the negative control and the BNZ when

parasites were exposed to the highest concentration of NX used in the study (100

μg/mL).

For the first time a research group has demonstrated the antiparasitic activity of

silver nanoparticles with xylan against the T. cruzi protozoan. Although the

mechanism of silver nanoparticles has not been properly elucidated, several studies

have proposed that the nanoparticles can adhere to the surface of the cell membrane

and alter the transmembrane permeability,61 which may justify the significant decrease

in MTT reduction in T. cruzi. Therefore, it is believed that the mode of action of NX the

same, and this possibility can be confirmed in future studies.

3.5 Evaluation of the process of cell death by Annexin V-FITC and PI labeling

To examine whether data observed in the MTT test were correlated to cell death,

the Annexin-V-FITC and PI labeling assay was performed, which differentiates

apoptotic, necrotic and viable cells according to the labeling pattern. Strains of T. cruzi

were treated with NX at concentrations of 2.5, 10 and 100 μg/mL and analysed by flow

cytometry. In Figure 6, it can be seen that, for the control group, 95.1% of the cells are

negative for Annexin V-FITC and PI labeling.

No cell death was observed when lower concentrations of NX were used (2.5

and 10 μg/mL). However, a considerable increase in the percentage of PI-positive cells

54

was found in the treatment that used NX at a concentration of 100 μg/mL (98%), thus

demonstrating cell death by necrosis. This fluorochrome has been widely used to

determine the necrotic potential of different substances, since it has the characteristic of

marking the DNA of cells with membrane damage.62 Other compounds, such as

MBHA3 (3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile), kill T. cruzi by

inducing necrosis in which extensive damages to the mitochondrial and plasma

membranes are promoted.63

Figure 6. Evaluation of the process of cell death through flow cytometry using untreated parasites, and

those treated with (NX) at concentrations of 2.5, 10 and 100 μg/mL for 24 h. After this period, the T.

cruzi parasites were labeled with Annexin V-FITC and Propidium iodide (PI) and analysed using flow

cytometry.

4. Conclusions

The analyses confirmed that a xylan was obtained from the corn cobs. Using this

xylan, it was possible to synthesize spherical-shaped silver nanoparticles containing

81% xylan and 19% silver. This nanoparticle was able of causing death of the Y strain

of the trypanosome T. cruzi by the mechanism of necrosis, which was more powerful

55

than that of silver or xylan when evaluated independently. The data described here

point to the use of silver nanoparticles as a possible therapeutic alternative for the

treatment of Chagas disease, which can be proven by conducting in vivo studies in the

future. In future studies it is aimed to investigate the mechanism of action of

nanoxylan in the tripomastigote and intracellular (amastigote) forms of Trypanosoma

cruzi.

Acknowledgments: The authors wish to thank Conselho Nacional de

Desenvolvimento Científico e Tecnológico-CNPq, Coordenação de Aperfeiçoamento

Pessoal de Nível Superior-CAPES and Ministério de Ciência, Tecnologia, Informação e

Comércio – MCTIC for the financial support. Hugo Rocha is CNPq fellowship honored

researchers. To the Department of Biochemistry of the UFRN for providing the flow

cytometer where the experiments presented in this paper were performed. Rony Viana

and Mayara Medeiros received a MSc. scholarship from CAPES, and Claudia Moreno

had a Ph.D. scholarship from CAPES. This research was submitted to the Graduate

Program in Ciências da Saúde (Health Sciences – loose translation) at UFRN, as part of

the doctoral thesis of Talita Katiane de Brito.

Abbreviations

BNZ, Benznidazole; FTIR, Fourier-transform infrared spectroscopy; MTT, 3-bromo

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; NX, nano xylan; UV visible,

56

ultraviolet; T. cruzi, Trypanosoma cruzi; AFM, Atomic force microscopy; ICP-OES,

Quantitative determination of silver in nano xylans by coupled plasma optical

emission spectroscopy; Xyl, xylose; Glc, glucose; Ara, arabinose; Gal, galactose; Man,

mannose; GlcA, glucuronic acid. CN, negative control; XYL, xylan; PI, propidium

iodide.

57

References

1. Gascon J, Bern C, Pinazo MJ. Chagas disease in Spain, the United States and

other non-endemic countries. Acta Trop. 2010;115(1-2):22-27.

doi:10.1016/j.actatropica.2009.07.019

2. Moncayo Á́, Silveira AC. Current epidemiological trends for Chagas disease in

Latin America and future challenges in epidemiology, surveillance and health

policyfile. Mem Inst Oswaldo Cruz. 2009;104(SUPPL. 1):17-30. doi:10.1590/S0074-

02762009000900005

3. Schmunis GA, Yadon ZE. Chagas disease: A Latin American health problem

becoming a world health problem. Acta Trop. 2010;115(1-2):14-21.

doi:10.1016/j.actatropica.2009.11.003

4. Pan American Health Organization. Chagas Disease in the Americas: A Review

of the Current Public Health Situation and a Vision for the Future.

https://www.paho.org/hq/index.php?option=com_docman&view=download&

category_slug=technical-reports-3829&alias=45143-chagas-disease-americas-a-

review-current-public-health-situation-a-vision-for-future-report-conclusions-

recommendations-2018-143&Itemid=270&. Published 2018. Accessed March 18,

2019.

5. World Health Organization. Chagas Disease (American Trypanosomiasis).

https://www.who.int/chagas/disease/en/. Published 2018. Accessed January 23,

2019.

58

6. Salomao K, Menna-Barreto RFS, de Castro SL. Stairway to Heaven or Hell?

Perspectives and Limitations of Chagas Disease Chemotherapy. Curr Top Med

Chem. 2016;16(20):2266-2289. doi:10.2174/1568026616666160413125049

7. Salomão K, et al. Trypanosoma cruzi mitochondrial swelling and membrane

potential collapse as primary evidence of the mode of action of naphthoquinone

analogues. BMC Microbiol. 2013;13(1):196. doi:10.1186/1471-2180-13-196

8. Annang F, Pérez-Moreno G, García-Hernández R, et al. High-Throughput

Screening Platform for Natural Product–Based Drug Discovery Against

Neglected Tropical Diseases. J Biomol Screen. 2014;20(1):82-91.

doi:10.1177/1087057114555846

9. Izumi E, et al. Natural products and Chagas’ disease: A review of plant

compounds studied for activity against Trypanosoma cruzi. Nat Prod Rep.

2011;28(4):809-823. doi:10.1039/c0np00069h

10. Scotti L, Ferreira EI, Da Silva MS, Scotti MT. Chemometric studies on natural

products as potential inhibitors of the nadh oxidase from Trypanosoma cruzi

using the volsurf approach. Molecules. 2010;15(10):7363-7377.

doi:10.3390/molecules15107363

11. Uchiyama N. Antichagasic Activities of Natural Products against Trypanosoma

cruzi. J Heal Sci. 2009;55(1):31-39. doi:10.1248/jhs.55.31

12. Rennie EA, Scheller HV. Xylan biosynthesis. Curr Opin Biotechnol. 2014;26:100-

107. doi:10.1016/j.copbio.2013.11.013

59

13. Petzold-Welcke K, Schwikal K, Daus S, Heinze T. Xylan derivatives and their

application potential - Mini-review of own results. Carbohydr Polym. 2014;100:80-

88. doi:10.1016/j.carbpol.2012.11.052

14. Wang Y, et al. Fabrication of optically transparent and strong nanopaper from

cellulose nanofibril based on corn cob residues. Carbohydr Polym. 2019;214:159-

166. doi:10.1016/j.carbpol.2019.03.035

15. Bouramtane S, Bretin L, Pinon A, et al. Porphyrin-xylan-coated silica

nanoparticles for anticancer photodynamic therapy. Carbohydr Polym.

2019;213:168-175. doi:10.1016/j.carbpol.2019.02.070

16. Kayserilioǧlu BŞ, Bakir U, Yilmaz L, Akkaş N. Use of xylan, an agricultural by-

product, in wheat gluten based biodegradable films: Mechanical, solubility and

water vapor transfer rate properties. Bioresour Technol. 2003;87(3):239-246.

doi:10.1016/S0960-8524(02)00258-4

17. Nagashima T, Oliveira EE, da Silva AE, et al. Influence of the Lipophilic External

Phase Composition on the Preparation and Characterization of Xylan

Microcapsules—A Technical Note. AAPS PharmSciTech. 2008;9(3):814-817.

doi:10.1208/s12249-008-9115-z

18. Yang R, Xu S, Wang Z, Yang W. Aqueous extraction of corn cob xylan and

production of xylooligosaccharides. LWT - Food Sci Technol. 2005;38(6):677-682.

doi:10.1016/j.lwt.2004.07.023

19. Garcia RB, Ganter JLMS, Carvalho RR. D -xylans from corn cobs. Eur Polym J.

60

2000;36:783-787.

20. Oliveira EE, Silva AE, Júnior TN, et al. Xylan from corn cobs, a promising

polymer for drug delivery: Production and characterization. Bioresour Technol.

2010;101:5402-5406. doi:10.1016/j.biortech.2010.01.137

21. Ebringerová A, Hromádková Z, Hřibalová V. Structure and mitogenic activities

of corn cob heteroxylans. Int J Biol Macromol. 1995;17:327-331. doi:10.1016/0141-

8130(96)81840-X

22. Melo-Silveira RF, Fidelis GP, Pereira Costa MSS, et al. In vitro antioxidant,

anticoagulant and antimicrobial activity and in inhibition of cancer cell

proliferation by xylan extracted from corn cobs. Int J Mol Sci. 2012;13:409-426.

doi:10.3390/ijms13010409

23. Burdușel A-C, Gherasim O, et al. Biomedical Applications of Silver

Nanoparticles: An Up-to-Date Overview. Nanomaterials. 2018;8(9):681.

doi:10.3390/nano8090681

24. Amorim MOR, Lopes Gomes D, Dantas LA, et al. Fucan-coated silver

nanoparticles synthesized by a green method induce human renal

adenocarcinoma cell death. Int J Biol Macromol. 2016;93:57-65.

doi:10.1016/j.ijbiomac.2016.08.043

25. Fernandes-Negreiros M, Araújo Machado R, Bezerra F, et al. Antibacterial,

Antiproliferative, and Immunomodulatory Activity of Silver Nanoparticles

Synthesized with Fucans from the Alga Dictyota mertensii. Nanomaterials.

61

2017;8(1):6. doi:10.3390/nano8010006

26. Melo-Silveira RF, Viana RLS, Sabry DA, et al. Antiproliferative xylan from corn

cobs induces apoptosis in tumor cells. Carbohydr Polym. 2019;210(November

2018):245-253. doi:10.1016/j.carbpol.2019.01.073

27. Nascimento AKL, Melo-Silveira RF, Dantas-Santos N, et al. Antioxidant and

Antiproliferative Activities of Leaf Extracts from Plukenetia volubilis Linneo

(Euphorbiaceae). Evidence-Based Complement Altern Med. 2013;2013:1-10.

doi:10.1155/2013/950272

28. Bradford MM. Sistema séptico domiciliario | Rotomoldeo en Colombia Tanques

Plasticos En Colombia Rotoplast. Anal Biochem. 1976;72:248-254.

doi:10.1016/0003-2697(76)90527-3

29. Dubois M, Gilles KA, Hamilton JK, et al. Colorimetric Method for Determination

of Sugars and Related Substances. Anal Chem. 1956;28(3):350-356.

doi:10.1021/ac60111a017

30. Dipankar C, Murugan S. The green synthesis, characterization and evaluation of

the biological activities of silver nanoparticles synthesized from Iresine herbstii

leaf aqueous extracts. Colloids Surfaces B Biointerfaces. 2012;98:112-119.

doi:10.1016/j.colsurfb.2012.04.006

31. Kačuráková M, Belton PS, Wilson RH, et al. Hydration properties of xylan-type

structures: An FTIR study of xylooligosaccharides. J Sci Food Agric. 1998;77:38-

44. doi:10.1002/(SICI)1097-0010(199805)77:1<38::AID-JSFA999>3.0.CO;2-5

62

32. Martinez-Castanon GA, Niño-Martínez N, et al. Synthesis and antibacterial

activity of silver nanoparticles with different sizes. J Nanoparticle Res.

2008;10(8):1343-1348. doi:10.1007/s11051-008-9428-6

33. Song JY, Jang HK, Kim BS. Biological synthesis of gold nanoparticles using

Magnolia kobus and Diopyros kaki leaf extracts. Process Biochem. 2009;44(10):1133-

1138. doi:10.1016/j.procbio.2009.06.005

34. Pandey S, Goswami GK, Nanda KK. Green synthesis of biopolymer-silver

nanoparticle nanocomposite: An optical sensor for ammonia detection. Int J Biol

Macromol. 2012;51:583-589. doi:10.1016/j.ijbiomac.2012.06.033

35. Chen X, Yan JK, Wu JY. Characterization and antibacterial activity of silver

nanoparticles prepared with a fungal exopolysaccharide in water. Food

Hydrocoll. 2016;53:69-74. doi:10.1016/j.foodhyd.2014.12.032

36. Silva LP, Bonatto CC, Pereira FDES, et al. Nanotecnologia verde para síntese de

nanopartículas metálicas. Biotecnol Apl à Agro&Indústria - Vol 4. 2017:967-1012.

doi:10.5151/9788521211150-26

37. Nel A. Toxic Potential of Materials at the Nanolevel. Science (80- ).

2006;311(5761):622-627. doi:10.1126/science.1114397

38. Carlson C, Hussein SM, Schrand AM, et al. Unique cellular interaction of silver

nanoparticles: Size-dependent generation of reactive oxygen species. J Phys

Chem B. 2008;112(43):13608-13619. doi:10.1021/jp712087m

39. Liu W, Wu Y, Wang C, et al. Impact of silver nanoparticles on human cells: Effect

63

of particle size. Nanotoxicology. 2010;4(3):319-330.

doi:10.3109/17435390.2010.483745

40. Akter M, Sikder MT, Rahman MM, et al. A systematic review on silver

nanoparticles-induced cytotoxicity: Physicochemical properties and

perspectives. J Adv Res. 2018;9:1-16. doi:10.1016/j.jare.2017.10.008

41. Stoehr LC, Gonzalez E, Stampfl A, et al. Shape matters: Effects of silver

nanospheres and wires on human alveolar epithelial cells. Part Fibre Toxicol.

2011;8(1):36. doi:10.1186/1743-8977-8-36

42. Kumar S, Negi YS. Corn cob xylan-based nanoparticles: Ester prodrug of 5-

aminosalicylic acid for possible targeted delivery of drug. J Pharm Sci Res.

2012;4:1995-2003.

43. Sun JL, Sakka K, Karita S, et al. Adsorption of Clostridium stercorarium xylanase

A to insoluble xylan and the importance of the CBDs to xylan hydrolysis. J

Ferment Bioeng. 1998;85(1):63-68. doi:10.1016/S0922-338X(97)80355-8

44. Colom X, Carrillo F, Nogués F, Garriga P. Structural analysis of photodegraded

wood by means of FTIR spectroscopy. Polym Degrad Stab. 2003;80(3):543-549.

doi:10.1016/S0141-3910(03)00051-X

45. Sun XF, Xu F, Sun RC, et al. Characteristics of degraded hemicellulosic polymers

obtained from steam exploded wheat straw. Carbohydr Polym. 2005;60(1):15-26.

doi:10.1016/j.carbpol.2004.11.012

46. Li C. Exploring C - C Bond Formations beyond. Accounts. 2009;42(2):335-344.

64

47. Nacos MK, Katapodis P, Pappas C, et al. Kenaf xylan - A source of biologically

active acidic oligosaccharides. Carbohydr Polym. 2006;66(1):126-134.

doi:10.1016/j.carbpol.2006.02.032

48. Barroso EMA, Costa LS, Medeiros VP, et al. A non-anticoagulant heterofucan

has antithrombotic activity in vivo. Planta Med. 2008;74(7):712-718. doi:10.1055/s-

2008-1074522

49. Luo Y, Shen S, Luo J, et al. Green synthesis of silver nanoparticles in xylan

solution via Tollens reaction and their detection for Hg2+. Nanoscale. 2015;7:690-

700. doi:10.1039/c4nr05999a

50. He Y, Du, Tang, et al. Green synthesis of silver nanoparticles by Chrysanthemum

morifolium Ramat. extract and their application in clinical ultrasound gel. Int J

Nanomedicine. 2013;8:1809. doi:10.2147/IJN.S43289

51. Christophe F, Séné B, et al. Fourier-Transform Raman and Fourier-Transform

lnfrared Spectroscopy. Plant Physiol. 1994;106:1623-1631.

52. Zeng Y, Yarbrough JM, Mittal A, et al. In situ label-free imaging of hemicellulose

in plant cell walls using stimulated Raman scattering microscopy. Biotechnol

Biofuels. 2016;9:256. doi:10.1186/s13068-016-0669-9

53. Barron C, Robert P, Guillon F, et al. Structural heterogeneity of wheat

arabinoxylans revealed by Raman spectroscopy. Carbohydr Res. 2006.

doi:10.1016/j.carres.2006.03.025

54. Cao Y, Shen D, et al. A Raman-scattering study on the net orientation of

65

biomacromolecules in the outer epidermal walls of mature wheat stems

(Triticum aestivum). Handb Environ Chem Vol 5 Water Pollut. 2006;97:1091-1094.

doi:10.1093/aob/mcl059

55. Li JF, Zhang YJ, et al. Core-shell nanoparticle-enhanced raman spectroscopy.

Chem Rev. 2017;117(7):5002-5069. doi:10.1021/acs.chemrev.6b00596

56. Akmaz S., Adıgüzel E.D., et al. The Effect of Ag Content of the Chitosan-Silver

Nanoparticle Composite Material on the Structure and Antibacterial Activity.

Adv Mater Sci Eng. 2013:6. doi:http://dx.doi.org/10.1155/2013/690918

57. Lamas MC, Villaggi L, Nocito I, et al. Development of parenteral formulations

and evaluation of the biological activity of the trypanocide drug benznidazole.

Int J Pharm. 2006;307(2):239-243. doi:10.1016/j.ijpharm.2005.10.004

58. Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of

antimicrobials. Biotechnol Adv. 2009;27(1):76-83.

doi:10.1016/j.biotechadv.2008.09.002

59. Pal S, Tak YK, Song JM. Does the antibacterial activity of silver nanoparticles

depend on the shape of the nanoparticle? A study of the gram-negative

bacterium Escherichia coli. J Biol Chem. 2015. doi:10.1128/AEM.02218-06

60. Muelas-Serrano S, Nogal-Ruiz JJ, Gómez-Barrio A. Setting of a colorimetric

method to determine the viability of Trypanosoma cruzi epimastigotes. Parasitol

Res. 2000;86:999-1002. doi:10.1007/PL00008532

61. Sharma VK, Yngard RA, Lin Y. Silver nanoparticles: Green synthesis and their

66

antimicrobial activities. Adv Colloid Interface Sci. 2009;145:83-96.

doi:10.1016/j.cis.2008.09.002

62. Kumar SP, Birundha K, et al. Antioxidant studies of chitosan nanoparticles

containing naringenin and their cytotoxicity effects in lung cancer cells. Int J Biol

Macromol. 2015;78:87-95. doi:10.1016/j.ijbiomac.2015.03.045

63. Sandes JM, Borges AR, Junior CGL, et al. 3-Hydroxy-2-methylene-3-(4-

nitrophenylpropanenitrile): A new highly active compound against

epimastigote and trypomastigote form of Trypanosoma cruzi. Bioorg Chem.

2010;38(5):190-195. doi:10.1016/j.bioorg.2010.06.003

67

6. CONCLUSÕES

Os dados aqui descritos apontam para o potencial da NX como agente anti-

Tripanosoma cruzi e espera-se que estudos futuros indiquem o seu uso no tratamento

da doença de Chagas.

68

7. COMENTÁRIOS, CRÍTICAS E SUGESTÕES

Há décadas nosso grupo de pesquisa tem realizado uma extensiva busca de

atividades biológicas de polissacarídeos sulfatados obtidos de macroalgas marinhas.

Entretanto, uma nova linha de pesquisa vem ganhando destaque investigando a ação

do polissacarídeo, xilana, extraído de sabugo de milho, sendo este um produto

subutilizado pelo mercado agrícola e industrial. Atualmente trabalhos com xilana vêm

demonstrando que este polímero possui inúmeras atividades biológicas, tais como

antioxidante, antitumoral, antifúngica dentre outras. E para investigar mais sobre

essas atividades a inserção da nanotecnologia na nossa pesquisa também tem sido

testada.

No entanto, apesar de algumas atividades biológicas já terem sido descritas

para esse polímero, escassos são os trabalhos realizados sobre a atividade

antiparasitária, principalmente frente ao parasita T. cruzi. Este trabalho visa ajudar na

elucidação dos mecanismos de ação como também oferecer no futuro uma nova

alternativa terapêutica para a doença de Chagas.

Durante o desenvolvimento deste trabalho tive a oportunidade de aprimorar e

aprender novas técnicas nos mais diferentes campos de atuação (parasitologia,

farmacologia, bioquímica, cultura de célula). Além disto, esta experiência me propôs

conhecer novos laboratórios e a interagir com vários grupos de pesquisas localizados

aqui na UFRN.

Com relação à produção científica, ao longo do doutorado, participei de vários

congressos e encontros com submissão de resumos científicos, colaborei com a

orientação de alunos de iniciação científica e de mestrado. Além disso, também fiz

parte na colaboração de experimentos de Dissertações e Teses do próprio laboratório.

Após a conclusão dessa etapa acadêmica, tenho como projeto para o futuro,

continuar com os estudos em um futuro pós-doutorado aqui no Brasil ou no Exterior,

procurando sempre a possibilidade de aprovação em concursos docentes em

Instituições de Ensino ou Pesquisa na minha área de atuação, que é a Farmácia. Ao

longo desse trajeto houve vários percalços que poderiam promover a desistência de

chegar ao objetivo final deste trabalho, mas com a perseverança e apoio de familiares,

amigos e principalmente do meu orientador/amigo Dr. Hugo Rocha pude realizar o

cumprimento de mais esta meta.

69

REFERÊNCIAS

1. Lidani KCF, Andrade FA, Bavia L, Damasceno FS, Beltrame MH, Messias-

Reason IJ, et al. Chagas Disease: From Discovery to a Worldwide Health Problem.

Front Public Heal. 2019 Jul 2;7(July):1–13.

2. World Health Organization. Chagas disease (American trypanosomiasis)

[Internet]. 2019 [cited 2019 Apr 29]. Available from: https://www.who.int/news-

room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis)

3. Rodriguez Coura J, Albajar Viñas P. Chagas disease: a new worldwide

challenge. Nature. 2010;115(June):14–21.

4. Dias JCP, Schofield CJ. Social and medical aspects on Chagas disease

management and control. In: American Trypanosomiasis Chagas Disease. Elsevier;

2017. p. 47–57.

5. Simões MV, Romano MMD, Schmidt A, Martins KSM, Marin-Neto JA. Chagas

Disease Cardiomyopathy. Int J Cardiovasc Sci. 2018;31(2):173–89.

6. Ministério da Saúde. Doença de Chagas Aguda e distribuição espacial dos

triatomíneos de importância epidemiológica, Brasil 2012 a 2016 [Internet]. 2019 [cited

2019 Feb 25]. Available from:

http://portalarquivos2.saude.gov.br/images/pdf/2019/janeiro/23/2018-025.pdf

7. Barroso Ferreira RT, Branquinho MR, Cardarelli-Leite P. Transmissão oral da

doença de Chagas pelo consumo de açaí: um desafio para a Vigilância Sanitária.

Vigilância Sanitária em Debate. 2014 Nov 25;2(4):4–11.

8. Tanowitz HB, Kirchhoff L V, Simon D, Morris SA, Weiss LM, Witfner M. Chagas

’ Disease. 1992;5(4):400–19.

9. Alves MJM, Colli W. Trypanosoma cruzi: Adhesion to the host cell and

intracellular survival. IUBMB Life. 2007;59(4–5):274–9.

10. Bern C, Kjos S, Yabsley MJ, Montgomery SP. Trypanosoma cruzi and chagas’

disease in the united states. Clin Microbiol Rev. 2011;24(4):655–81.

70

11. Giordanengo L, Gui N, Stempin C, Fretes R, Gea S. Cruzipain , a major

Trypanosoma cruzi antigen , conditions the host immune response in favor of parasite.

2002;1003–11.

12. Sosa-estani S, Colantonio L, Segura EL. Therapy of Chagas Disease :

Implications for Levels of Prevention. 2012;2012.

13. Meymandi S, Hernandez S, Park S, Sanchez DR, Forsyth C. Treatment of

Chagas Disease in the United States. 2018;373–88.

14. Sánchez-guillén MC, López-colombo A, Ordóñez-toquero G, Gomez-albino I,

Ramos-jimenez J, Torres-rasgado E, et al. Clinical forms of Trypanosoma cruzi

infected individuals in the chronic phase of Chagas disease in Puebla , Mexico.

2006;101(November):733–9.

15. Menezes, Cristiane; Costa, Germano Carneiro; Gollob, Kenneth J. and Dutra

WO. therapies. Drug Dev Res. 2012;72(6):471–9.

16. De la Rosa E, Paglini-Oliva P, Prato LB, Benizio E, Triquell MF, Muñoz SE, et

al. Early Detection of Chronic Asymptomatic Chagas Infection. Med Sci Monit. 2018

Jul 2;24:4567–71.

17. Cançado JR. Long Term Evaluation of etiological treatment of Chagas Disease

with. 2002;44(1):29–37.

18. Andrade, Sonia G; Magalhães, Juraci B. and Pontes AL. Terapêutica da fase

crônica da infecção experimental pelo Trypanosoma cruzi com o Benzonidazol e o

Nifurtimox. 1989;22(3):113–8.

19. Camandaroba, Edson L.P.; Reis, Eliana A.G.; Gonçalves, Marilda S.; Reis,

Mitermayer G. and Andrade SG. Trypanosoma cruzi : susceptibility to chemotherapy

with benznidazole of clones isolated from the highly resistant Colombian strain.

2003;36(2):201–9.

20. Segura MA, Raspi EM de, Basombrio MA. Reversibility of muscle and heart

lesions in chronic Trypanosoma cruzi infected mice after late trypanomicidal treatment.

Mem Inst Oswaldo Cruz. 1994 Jun;89(2):213–6.

21. Toledo MJO, Bahia MT, Veloso VM, Carneiro CM. Effects of specific treatment

on parasitological and histopathological parameters in mice infected with different

Trypanosoma cruzi clonal genotypes. 2004;53(6):1045–53.

71

22. Salomão K, De Santana NA, Molina MT, De Castro SL, Menna-Barreto RFS.

Trypanosoma cruzi mitochondrial swelling and membrane potential collapse as

primary evidence of the mode of action of naphthoquinone analogues. BMC Microbiol.

2013;13(1):196.

23. Salomao K, Menna-Barreto RFS, de Castro SL. Stairway to Heaven or Hell?

Perspectives and Limitations of Chagas Disease Chemotherapy. Curr Top Med Chem.

2016;16(20):2266–89.

24. Leila P, Cunha R, Célia R, Paula M De, Feitosa JPA. Quim. Nova,.

2009;32(3):649–60.

25. Santana M, Pereira S, Costa LS, Cordeiro SL. Evaluating the possible

anticoagulant and antioxidant effects of sulfated polysaccharides from the tropical

green alga Caulerpa cupressoides var . flabellata. 2012;1159–67.

26. Melo-Silveira RF, Fidelis GP, Pereira Costa MSS, Telles CBS, Dantas-Santos

N, de Oliveira Elias S, et al. In vitro antioxidant, anticoagulant and antimicrobial activity

and in inhibition of cancer cell proliferation by xylan extracted from corn cobs. Int J Mol

Sci. 2012;13:409–26.

27. Fidelis GP, Camara RBG, Queiroz MF, Costa MSSP, Santos PC, Rocha HAO,

et al. Proteolysis, NaOH and ultrasound-enhanced extraction of anticoagulant and

antioxidant sulfated polysaccharides from the edible seaweed, Gracilaria birdiae.

Molecules. 2014;19(11):18511–26.

28. Martínez-montemayor MM, Otero-franqui E. NIH Public Access.

2011;63(7):1085–94.

29. Dore, Celina Maria P Guerra Gabriela M, Faustino C, Sheyla L, Will EP, Costa

TG, Sabry DA, et al. A sulfated polysaccharide , fucans , isolated from brown algae

Sargassum vulgare with anticoagulant , antithrombotic , antioxidant and anti-

inflammatory effects. Carbohydr Polym. 2013;91(1):467–75.

30. Luo Q, Cai Y, Yan J, Sun M, Corke H. Hypoglycemic and hypolipidemic effects

and antioxidant activity of fruit extracts from Lycium barbarum. 2004;76:137–49.

31. Li SP, Zhang GH, Zeng Q, Huang ZG, Wang YT, Dong TTX, et al.

Hypoglycemic activity of polysaccharide , with antioxidation , isolated from cultured

Cordyceps mycelia. 2006;13:428–33.

72

32. Almeida-lima J, Costa S, Silva B, Melo-silveira RF, Silva V, Beatriz M, et al.

Evaluating the possible genotoxic , mutagenic and tumor cell proliferation-inhibition

effects of a non-anticoagulant , but antithrombotic algal heterofucan. 2010;(June):708–

15.

33. Jensen JK, Johnson N, Wilkerson CG, Great DOE, Bioenergy L, Lansing E.

Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a

heteroxylan containing mucilaginous tissue. 2013;4(June):1–15.

34. Rennie EA, Scheller HV. Xylan biosynthesis. Curr Opin Biotechnol.

2014;26:100–7.

35. Barakat A, Chuetor S, Monlau F, Solhy A, Rouau X. Eco-friendly dry chemo-

mechanical pretreatments of lignocellulosic biomass : Impact on energy and yield of

the enzymatic hydrolysis q. Appl Energy. 2014;113:97–105.

36. Ebringerová A, Hromádková Z, Hřibalová V. Structure and mitogenic activities

of corn cob heteroxylans. Int J Biol Macromol. 1995;17:327–31.

37. Nagashima T, Oliveira EE, da Silva AE, Marcelino HR, Gomes MCS, Aguiar LM,

et al. Influence of the Lipophilic External Phase Composition on the Preparation and

Characterization of Xylan Microcapsules—A Technical Note. AAPS PharmSciTech.

2008;9(3):814–7.

38. Petzold-Welcke K, Schwikal K, Daus S, Heinze T. Xylan derivatives and their

application potential - Mini-review of own results. Carbohydr Polym. 2014;100:80–8.

39. Bouramtane S, Bretin L, Pinon A, Leger D, Liagre B, Richard L, et al. Porphyrin-

xylan-coated silica nanoparticles for anticancer photodynamic therapy. Carbohydr

Polym. 2019;213:168–75.

40. Garcia RB, Nagashima T, Praxedes AKC, Raffin FN, Moura TFAL, Do Egito

EST. Preparation of micro and nanoparticles from corn cobs xylan. Polym Bull.

2001;46(5):371–9.

41. Kumar S, Negi YS, Upadhyaya JS. Studies on characterization of corn cob

based nanoparticles. 2010;(September 2015).

42. Reguera J, Langer J, Jime D, Liz-marza LM, Langer J. Chem Soc Rev

Anisotropic metal nanoparticles for surface enhanced Raman scattering. 2017;3866–

85.

73

43. Dipankar C, Murugan S. The green synthesis, characterization and evaluation

of the biological activities of silver nanoparticles synthesized from Iresine herbstii leaf

aqueous extracts. Colloids Surfaces B Biointerfaces. 2012;98:112–9.

44. Burdușel A-C, Gherasim O, Grumezescu AM, Mogoantă L, Ficai A, Andronescu

E. Biomedical Applications of Silver Nanoparticles: An Up-to-Date Overview.

Nanomaterials. 2018 Aug 31;8(9):681.

45. Orlowski P, Tomaszewska E, Gniadek M, Baska P, Nowakowska J, Sokolowska

J, et al. Tannic Acid Modified Silver Nanoparticles Show Antiviral Activity in Herpes

Simplex Virus Type 2 Infection. 2014;9(8):1–15.

46. Hang X, Peng H, Song H, Qi Z, Miao X, Xu W. Antiviral activity of cuprous oxide

nanoparticles against Hepatitis C Virus in vitro. J Virol Methods. 2015;222:150–7.

47. Uchiyama MK, Deda DK, Fernandes S, Rodrigues DP, Drewes CC, Bolonheis

SM, et al. In vivo and In vitro Toxicity and Anti-Inflammatory Properties of Gold

Nanoparticle Bioconjugates to the Vascular System. 2014;142(2):497–507.

48. He L, Liu Y, Mustapha A, Lin M. Antifungal activity of zinc oxide nanoparticles

against Botrytis cinerea and Penicillium expansum. Microbiol Res. 2011;166(3):207–

15.

49. Mukherjee P, Bhattacharya R, Wang P, Wang L, Basu S, Nagy JA, et al.

Antiangiogenic Properties of Gold Nanoparticles Cancer Therapy : Preclinical

Antiangiogenic Properties of Gold Nanoparticles. Clin Cancer Res. 2005;11(9):3530–

4.

50. Mukherjee S, Patra CR. Therapeutic application of anti-angiogenic

nanomaterials in cancers. Nanoscale. 2016;8(25):12444–70.

51. Amorim MOR, Lopes Gomes D, Dantas LA, Silva Viana RL, Chiquetti SC,

Almeida-Lima J, et al. Fucan-coated silver nanoparticles synthesized by a green

method induce human renal adenocarcinoma cell death. Int J Biol Macromol.

2016;93:57–65.

52. Fernandes-Negreiros M, Araújo Machado R, Bezerra F, Nunes Melo M, Alves

M, Alves Filgueira L, et al. Antibacterial, Antiproliferative, and Immunomodulatory

Activity of Silver Nanoparticles Synthesized with Fucans from the Alga Dictyota

mertensii. Nanomaterials. 2017;8(1):6.

74

53. Melo-Silveira RF, Viana RLS, Sabry DA, da Silva RA, Machado D, Nascimento

AKL, et al. Antiproliferative xylan from corn cobs induces apoptosis in tumor cells.

Carbohydr Polym. 2019;210(November 2018):245–53.

54. Bradford MM. Sistema séptico domiciliario | Rotomoldeo en Colombia Tanques

Plasticos En Colombia Rotoplast. Anal Biochem. 1976;72:248–54.

55. Sousa CMDM, Silva HRE, Vieira GM, Ayres MCC, Da Costa CLS, Araújo DS,

et al. Fenóis totais e atividade antioxidante de cinco plantas medicinais. Quim Nova.

2007;30(2):351–5.

56. Nascimento AKL, Melo-Silveira RF, Dantas-Santos N, Fernandes JM, Zucolotto

SM, Rocha HAO, et al. Antioxidant and Antiproliferative Activities of Leaf Extracts from

Plukenetia volubilis Linneo (Euphorbiaceae). Evidence-Based Complement Altern

Med. 2013;2013:1–10.

57. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric Method for

Determination of Sugars and Related Substances. Anal Chem. 1956;28(3):350–6.