ATIVIDADE ANTIOXIDANTE DA FOLHA DE Smilax Fluminensis E DA CASCA DO CAULE DE Caesalpinia Ferrea EM CAMUNDONGOS Swiss

UNIVERSIDADE FEDERAL DE MATO GROSSO CÂMPUS UNIVERSITÁRIO DE SINOP INSTITUTO DE CIÊNCIAS NATURAIS, HUMANAS E SOCIAIS PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS AMBIENTAIS

Ana Paula Simões da Cunha

ATIVIDADE ANTIOXIDANTE DA FOLHA DE Smilax fluminensis E DA CASCA DO CAULE DE Caesalpinia ferrea EM CAMUNDONGOS Swiss

SINOP MATO GROSSO - BRASIL 2021 i

UNIVERSIDADE FEDERAL DE MATO GROSSO CÂMPUS UNIVERSITÁRIO DE SINOP INSTITUTO DE CIÊNCIAS NATURAIS, HUMANAS E SOCIAIS PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS AMBIENTAIS

Ana Paula Simões da Cunha

ATIVIDADE ANTIOXIDANTE DA FOLHA DE Smilax fluminensis E DA CASCA DO CAULE DE Caesalpinia ferrea EM CAMUNDONGOS Swiss

Orientador(a): Profa. Dra. Valéria Dornelles Gindri Sinhorin Co-orientador(a): Prof. Dr. Adilson Paulo Sinhorin

Dissertação apresentada ao Programa de Pós-Graduação em Ciências Ambientais da Universidade Federal de Mato Grosso, Câmpus Universitário de Sinop, na área de concentração Biodiversidade e Bioprospecção, como parte das exigências para a obtenção do título de Mestre em Ciências Ambientais.

Linha de pesquisa: Produtos Naturais

SINOP MATO GROSSO - BRASIL 2021 ii

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Sinopse:

Estudou-se a atividade antioxidante da folha de Smilax fluminensis e da casca do caule de

Caesalpinia ferrea em camundongos Swiss após uma overdose do medicamento paracetamol. Nesta investigação antioxidante dos extratos e frações obtidas das plantas foram analisadas as atividades das enzimas antioxidantes catalase e a glutationa-S-transferase, dos antioxidantes não enzimáticos glutationa reduzida e a vitamina C, marcadores do estresse oxidativo como a carbonilação proteica e a peroxidação lipídica nos tecidos hepático, renal e cerebral. Foram dosados no plasma o colesterol, triglicérides, glicose e as enzimas alanina aminotransferase e aspartato aminotransferase.

Palavras-chave: Estresse Oxidativo, Smilax fluminensis, Caesalpinia ferrea

DEDICATÓRIA

À pessoa mais importante da minha vida, minha mãe Magda. Sem ela não sou nada! Uma pessoa que está em todos os momentos de minha vida, torcendo e aplaudindo por cada vitória e conquista, e esta não é só minha e sim, NOSSA VITÓRIA! Te amo! vi

AGRADECIMENTOS

Agradeço primeiramente à minha mãe. Magda, por todo cuidado e amor que tenho e recebi por toda a minha vida. Um exemplo de ser humano a ser seguido por sua educação, carisma, amizade, amor e tantos outros adjetivos que não caberia neste simples agradecimento. Só tenho a agredecer pelo grande exemplo que és e por ser dedicada a cada conquista minha (e dos meus irmãos). Te amo! À minha família, meus irmãos Carlos Eduardo e Bruna e minha cunhada Léia por cada palavra de incentivo e por estar ao meu lado apoiando em tudo que faço. Amo vocês! À minha querida orientadora Profa. Dr(a). Valéria D. G. Sinhorin pelos ensinamentos nesta longa trajetória acadêmica, na graduação e no mestrado, por plantar a sementinha do conhecimento e fazer com que eu não pare de estudar e sempre busque aprender cada dia mais. Por ter me tornado uma parte de sua família, e a recíproca é a mesma vindo da minha família, e acima de tudo por ter se tornado uma grande amiga e um segundo exemplo de vida a ser seguido. Te admiro muito! Ao meu co-orientador, Prof. Dr. Adilson P. Sinhorin, pelo conhecimento, ajuda e suporte ao logo da jornada acadêmica. Aos colegas de laboratório, que são muitos, porém cada um sabe o quão sou grata pelo companheirismo, ajuda e amizade que conquistei durante esta jornada. À Ariana Érica, por longas horas conversando e apoiando nas minhas decisões e pelos conselhos e incentivos recebidos. À um amigo que se tornou uma pessoa especial em tão pouco tempo e que não imagina o quão importante se tonou nesta trajetória do mestrado. Obrigada, Michael Monteiro por tudo! Aos meus vizinhos Diana e Luiz pela companhia e ajuda nesta jornada. À todos os colegas que conheci ao longo do curso e aos professores pelos ensinamentos e conhecimentos adquiridos. Agradeço à Universidade Federal de Mato Grosso – UFMT e ao Programa de Pós Graduação em Ciências Ambientais – PPGCAM pela oportunidade de realizar o mestrado e pelas instalações utilizadas. À Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT), à Fundação CAPES pela disponibilização da bolsa e ao CNPq pelo apoio financeiro.

Obrigada a todos! vii

“A menos que modifiquemos à nossa maneira de pensar, não seremos capazes de resolver os problemas causados pela forma como nos acostumamos a ver o mundo”. (Albert Einstein)

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RESUMO

O paracetamol (PCM) é um medicamento antipirético e analgésico amplamente utilizado pela população, que em doses aceitáveis e recomendadas pode ser seguro, porém, se administrado em altas doses pode causar dano hepático, levando a hepatoxicidade, devido a formação do metabólito tóxico, N-acetil-p-benzoquinona imina (NAPQI). Smilax fluminensis é conhecida popularmente como “salsa-parrilha” ou “japecanga” e suas espécies são utilizadas na medicina popular para tratamento de diversas enfermidades, sendo que seus estudos vêm demonstrando potencial antioxidante decorrente da presença de alguns metabólitos secundários. A Caesalpinia ferrea, conhecida popularmente como “pau-ferro” ou “jucá” sofreu uma reclassificação e hoje é conhecida como Libidibia ferrea e é utilizada popularmente com a finalidade anti-inflamatória e analgésica. O presente trabalho investigou se o extrato bruto (EB) e as frações F1 e F2 das folhas de Smilax fluminensis e o extrato etanólico da casca do caule da Caesalpinia ferrea promoveu alterações bioquímicas num modelo de estresse oxidativo, através de uma alta dose de PCM, em camundongos Swiss. Após a aclimatação, os camundongos foram divididos em grupos e administrados o tratamento por via gavagem. Para a S. fluminensis foram divididos em CONTROLE (água filtrada); PCM (paracetamol 250 mg kg-1 + água filtrada); EB (extrato bruto - 250 mg kg-1), F1 (fração 1 - 250 mg kg-1), F2 (fração 2 - 250 mg kg-1), PCM + EB, PCM + F1, PCM + F2. Para a administração do extrato etanólico da C. ferrea os camundongos foram divididos em 4 grupos: CONTROLE (água filtrada + 0,1% Tween 80), PCM (250 mg kg-1 de PCM em dose única + 0,1% Tween 80), PCM + EXT (250 mg kg-1 de PCM em dose única + 100 mg kg-1 de EXT) e EXT (água filtrada + 100 mg kg-1 de EXT). Inicialmente foram administrados o indutor do estresse oxidativo (PCM) ou água filtrada ou 0,1% de Tween 80 e após três horas iniciou-se o tratamento com as frações ou os extratos ou 0,1% de Tween 80 ou água filtrada. Depois de 24 horas da última dose dos tratamentos, os animais foram anestesiados via i.p. (intraperitoneal), foi realizada a punção cardíaca para retirada do sangue, e após, eutanasiados através de deslocamento cervical para a retirada dos órgãos (fígado, cérebro e rins) e as amostras foram congeladas a -85 ºC. Os dados foram avaliados por Anova seguida pelo post hoc teste de Tukey (p<0,05). Foram verificados se houve alterações nos parâmetros bioquímicos como os antioxidantes enzimáticos catalase (CAT) e glutationa-S-transferase (GST), nos antioxidantes não enzimáticos glutationa reduzida (GSH) e vitamina C, na carbonilação proteica (CARBONIL) e na peroxidação lipídica (TBARS) nos tecidos hepáticos, cerebral e renal. No plasma foram avaliadas as transaminases aspartato aminotransferase (AST) e a alanina aminotransferase (ALT), glicose, triglicerídeos e colesterol. O melhor efeito antioxidante encontrado nas folhas da S. fluminensis foi encontrado na fração 1 da planta onde houve diminuição da atividade de AST e ALT plasmática, aumento no fígado da atividade das enzimas CAT e GST, do antioxidante GSH, embora tenha aumentado TBARS, somados a diminuição da CARBONIL renal e cerebral e apresentou ter ação hipoglicêmica e hipocolesterolêmica. Já o extrato de C. ferrea foi capaz de reverter o dano lipídico e proteico causado pelo fármaco no tecido hepático, causando o mesmo efeito nos tecidos renais e cerebral na CARBONIL, diminuição da atividade da GST hepática e aumento na atividade da CAT e GST cerebral, além de diminuir a glicose e o colesterol. Dessa forma, acredita-se que a boa atividade antioxidante observada no extrato da S. fluminensis possa ser devida à presença de compostos flavonoides, neste caso específico, a quercetina e para o extrato da C. ferrea seja, provavelmente, pela presença de taninos. Novas pesquisas são necessárias a fim de explorar ainda mais o potencial biológico destas plantas para se obter mais conhecimentos sobre seus possíveis benefícios.

Palavras-chave: Estresse Oxidativo, Smilax fluminensis, Caesalpinia ferrea. ix

ABSTRACT

Paracetamol (PCM) is an antipyretic and analgesic medication widely used by the population, which in acceptable and recommended doses can be safe, however, if administered in high doses it can cause liver damage, leading to hepatoxicity, due to the formation of the toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Smilax fluminensis is popularly known as “parsley-parrilla” or “japecanga” and its species are used in folk medicine to treat various diseases, and its studies have shown antioxidant potential due to the presence of some secondary metabolites. Caesalpinia ferrea, popularly known as “pau-ferro” or “jucá” has undergone a reclassification and is now known as Libidibia ferrea and is popularly used for anti-inflammatory and analgesic purposes. The present work investigated whether the crude extract (EB) and the F1 and F2 fractions of the leaves of Smilax fluminensis and the ethanolic extract of the stem bark of Caesalpinia ferrea promoted biochemical changes in a model of oxidative stress, through a high dose of PCM, in Swiss mice. After acclimatization, the mice were divided into groups and the treatment was administered via gavage. For S. fluminensis they were divided into CONTROL (filtered water); PCM (paracetamol 250 mg kg-1 + filtered water); EB (crude extract - 250 mg kg-1), F1 (fraction 1 - 250 mg kg-1), F2 (fraction 2 - 250 mg kg-1), PCM + EB, PCM + F1, PCM + F2. For the administration of the ethanolic extract of C. ferrea, the mice were divided into 4 groups: CONTROL (filtered water + 0.1% Tween 80), PCM (250 mg kg-1 of PCM in a single dose + 0.1% Tween 80), PCM + EXT (250 mg kg-1 of PCM in a single dose + 100 mg kg-1 of EXT) and EXT (filtered water + 100 mg kg-1 of EXT). Initially, the oxidative stress inducer (PCM) or filtered water or 0.1% tween 80 was administered and after three hours treatment with the fractions or extracts or 0.1% tween 80 or filtered water was started. After 24 hours of the last dose of treatments, the animals were anesthetized via i.p. (intraperitoneal), cardiac puncture was performed to remove blood, and then euthanized through cervical dislocation to remove organs (liver, brain and kidneys) and the samples were frozen at -85 ºC. The data were evaluated by Anova followed by the Tukey post hoc test (p <0.05). We checked for changes in biochemical parameters such as enzyme antioxidants catalase (CAT) and glutathione-S-transferase (GST), non-enzymatic antioxidants reduced glutathione (GSH) and vitamin C, protein carbonylation (CARBONYL) and lipid peroxidation (TBARS) in liver, brain and kidney tissues. In plasma, the transaminases aspartate aminotransferase (AST) and alanine aminotransferase (ALT), glucose, triglycerides and cholesterol were evaluated. The best antioxidant effect found in the leaves of S. fluminensis was found in fraction 1 of the plant where there was a decrease in the activity of plasma AST and ALT, an increase in the activity of the enzymes CAT and GST, of the antioxidant GSH, although it increased TBARS, added decreased renal and cerebral CARBONYL and showed hypoglycemic and hypocholesterolemic action. The C. ferrea extract was able to reverse the lipid and protein damage caused by the drug in the liver tissue, causing the same effect on the renal and brain tissues in CARBONYL, decreased hepatic GST activity and increased CAT and GST brain activity, in addition to lowering glucose and cholesterol. Thus, it is believed that the good antioxidant activity observed in the extract of S. fluminensis may be due to the presence of flavonoid compounds, in this specific case, quercetin and for the extract of C. ferrea, it is probably due to the presence of tannins. Further research is needed in order to further explore the biological potential of these plants to gain more insight into their possible benefits.

Keywords: Oxidative Stress, Smilax fluminensis, Caesalpinea ferrea. x

LISTA DE ABREVIAÇÕES

ALT – Alanina Aminotransferase AST – Aspartato Aminotransferase CAT – Catalase CARBONIL – Proteínas Carboniladas / Carbonilação Proteica DP – Desvio Padrão EB – Extrato Bruto EO – Estresse Oxidativo EROs – Espécies Reativas de Oxigênio F1 – Fração 1 F2 – Fração 2 GSH – Glutationa Reduzida GST – Glutationa-S-transferase I.p. – Intraperitoneal NAPQI - N-acetil-p-benzoquinona imina PCM - Paracetamol TBARS – Substâncias Reativas ao Ácido Tiobarbitúrico UFMT – Universidade Federal de Mato Grosso

SUMÁRIO

1. INTRODUÇÃO ...... 13 2. REFERÊNCIA BIBLIOGRÁFICA ...... 16 CAPÍTULO I ...... 20 INVESTIGAÇÃO DO POSSÍVEL EFEITO PROTETOR DA FOLHA DE Smilax fluminensis STEUD. EM CAMUNDONGOS SUBMETIDOS AO ESTRESSE OXIDATIVO PELO PARACETAMOL ABSTRACT ...... 21 INTRODUCTION ...... 21 MATERIAL AND METHODS ...... 22 Extract preparation ...... 22 Animals and treatment ...... 22 Biochemical analysis ...... 23 Tissue ...... 23 Plasma ...... 23 Statistical analysis ...... 23 RESULT AND DISCUSSION ...... 23 CONCLUSION ...... 28 ACKNOWLEDGMENTS ...... 28 RESUMO ...... 28 REFERENCES ...... 29 CAPÍTULO II ...... 35 ATIVIDADE ANTIOXIDANTE DO EXTRATO ETANÓLICO DA CASCA DO CAULE DE Caesalpinia ferrea EM CAMUNDONGOS Swiss ABSTRACT ...... 36 INTRODUCTION ...... 36 MATERIAL AND METHODS ...... 37 Extract preparation ...... 37 Experimental Model ...... 37 Biochemical analysis ...... 37 Tissue ...... 37 Plasma ...... 38 Statistical analysis ...... 38 xii

RESULT AND DISCUSSION ...... 38 CONCLUSION ...... 42 ACKNOWLEDGMENTS ...... 42 REFERENCES ...... 42 3. CONCLUSÃO GERAL ...... 48 ANEXO I ...... 49 Certificado do Comitê de Ética em Pesquisa Animal ANEXO II ...... 50 Normas da Revista Bioscience Journal ANEXO III ...... 58 E-mail de aceite do artigo na Revista Bioscience Journal ANEXO IV ...... 59 Normas da Revista The Natural Products Journal

1. INTRODUÇÃO As espécies reativas de oxigênio (EROs) são moléculas formadas durante o metabolismo e desempenham papéis importantes na sinalização celular (SOSA-TORRES; SAUCEDO-VÁZQUEZ; KRONECK, 2015). As EROs possuem elétrons desemparelhados, tornando-se moléculas altamente reativas, podendo interagir com componentes celulares produzindo diversos danos oxidativos, que podem culminar na indução de morte celular (TELES et al., 2015). Seu excesso produz o chamado estresse oxidativo (EO), um processo decorrente do desequilíbrio entre a produção e o consumo de radicais livres, que se correlaciona com numerosas doenças (POHANKA, 2014; VYSAKH et al., 2014). Um radical livre é um átomo ou molécula com um ou mais elétrons desemparelhados no último orbital, sendo capaz de reagir com muitas biomoléculas através de sua oxidação (SÁNCHEZ- VALLE; MÉNDEZ-SÁNCHEZ, 2013). Estes são liberados durante o metabolismo humano (fonte endógena) ou adquiridos de uma fonte exógena através dos poluentes ambientais (atmosférico, aquático, solo), radiação (ultravioleta, gama) e podem estar relacionados ao consumo ou uso de substâncias tóxicas como álcool, tabaco e drogas ou devido a nutrição inadequada, exposição a fertilizantes ou pesticidas (LEÓN et al., 2015). Os radicais livres podem ser eliminados por antioxidantes e enzimas que transformam as espécies reativas em compostos estáveis (CERVELLATI et al., 2014). Existem os antioxidantes não enzimáticos – obtidos da dieta como as vitaminas A, E e C, pró- betacarotenos e os produzidos endogenamente como a glutationa reduzida (GSH) e ácido úrico – e os antioxidantes enzimáticos, protagonizados pelas enzimas superóxido dismutase, catalase e glutationa peroxidase (GAUTAM et al., 2012; MIROŃCZUK-CHODAKOWSKA; WITKOWSKA; ZUJKO, 2018; TELES et al., 2015). Eles têm a capacidade de neutralizar os efeitos nocivos dos radicais livres e peróxidos lipídicos, e sua suplementação pode ser útil na prevenção e tratamento de doenças mediadas pelos radicais livres (JAKUBCZYK et al., 2020). Com isso, podem agir de diferentes maneiras no processo oxidativo, tais como: reação com os radicais livres, ligação competitiva com o oxigênio molecular, sequestro de íons metálicos ou estabilização de hidroperóxidos (REGITANO-D’ARCE, 2006). O acetaminofeno, também conhecido como paracetamol, N-acetil-p-aminofenol e PCM, é um dos medicamentos mais comumente usados por suas propriedades analgésicas e antipiréticas (LANCASTER; HIATT; ZARRINPAR, 2015). Uma alta ingestão é considerada tóxica, podendo causar insuficiência hepática, quando exceder uma dose de 150 mg kg-1, porém um número crescente de notificações sugere que em doses mais baixas de PCM podem 14

conferir lesão hepática aguda e insuficiência hepática (BUNCHORNTAVAKUL; REDDY, 2013). O mecanismo de toxicidade ocorre através da ativação metabólica do PCM, catalisada pelo citocromo P450, que gera o metabólito reativo N-acetil-p-benzoquinona imina (NAPQI) e inicia a toxicidade em roedores e humanos (JAESCHKE; MCGILL; RAMACHANDRAN, 2012). Em condições normais, o NAPQI é imediatamente desintoxicado pela conjugação com a glutationa e este conjugado é metabolizado à cisteína e ácido mercaptúrico, que são eliminados na urina (BENSON; KOFF; TOLMAN, 2005; FLINT et al., 2017; MIAN et al., 2020). Já em altas doses do medicamento, ocorre o excesso de NAPQI que acaba esgotando as reservas de GSH e começa a formar adutos proteicos através da ligação a grupos cisteína nas proteínas celulares (MAZALEUSKAYA et al., 2015). Com este aumento de adutos de proteínas formados pelos NAPQI ocorre o estresse oxidativo, dano mitocondrial e necrose centro lobular e, eventualmente, levam a danos no fígado (BIAN et al., 2018) (Figura 1).

Figura 1. Farmacocinética do Paracetamol

Fonte. Casarett & Doull`S (2008).

A presença de antioxidantes em plantas, tais como, compostos fenólicos, flavonoides, taninos e proantocianidinas pode fornecer proteção contra várias doenças (GULCIN, 2012). O 15

Brasil se situa em posição privilegiada para a ciência de produtos naturais, em decorrência de ser um dos países com uma grande biodiversidade (BERLINCK, 2012). Assim, a bioprospecção designa a utilização comercial de recursos derivados de material genético de modo sustentável, visando o emprego de recursos naturais sem comprometer sua essência ou existência (JÚNIOR, 2014). Dessa forma, na investigação de plantas medicinais com potencial terapêutico, a abordagem geralmente utilizada envolve processos de extração, fracionamento e purificação, que resultam no isolamento e identificação de uma ou mais substâncias bioativas (CARMONA; PEREIRA, 2013; JUNIO et al., 2011; NIEMEYER; BELL; KOITHAN, 2013). A ação antioxidante da maioria dessas substâncias naturais se deve a presença de hidroxilas fenólicas e as suas propriedades de oxirredução, pois eles agem como agentes redutores, doadores de hidrogênio (FONSECA et al., 2009; REBELO et al., 2009). Por conta disso, as plantas medicinais são utilizadas desde a antiguidade pelas populações no tratamento de enfermidades, sendo uma prática atual deste uso através da fitoterapia, pois além de ser aceita, é acessível economicamente (VERRI; MOURA; MOURA, 2017). Smilax fluminensis é conhecida popularmente como “salsa-parrilha” ou “japecanga” (SOUZA; LORENZI, 2008). O gênero Smilax (família Liliaceae) compreende mais de 30 espécies de arbustos (XIE et al., 2018). Algumas espécies são encontradas em todo o território brasileiro como a Smilax brasiliensis Spreng., a S. campestris Griseb. e a S. fluminensis Steud. Estas produzem metabólitos secundários como os compostos fenólicos, terpenos e componentes contendo nitrogênio (LORENZI; MATOS, 2008; MARQUES, 2016). Petrica e colaboradores (2014) estudaram alguns extratos e frações obtidos das folhas de Smilax fluminensis, sendo que identificaram vários compostos fitoquímicos e obtiviveram como resultado final o isolamento e a elucidação de dois compostos contendo quercetina. A quercetina é um flavonoide que pode atuar como antioxidante e, com isso, têm efeitos benéficos para a saúde humana, agindo com propriedades farmacológicas tais como antitumoral, anticancerígena, anti-inflamatória e hepatoprotetora (GHOSH et al., 2011). Algumas espécies têm sido utilizadas na medicina popular desde o século XVI (MEDEIROS; SENNA-VALLE; ANDREATA, 2007), como é o caso do uso de extratos feitos a partir de folhas e raízes que são empregados no tratamento de sífilis, brucelose, nefrite, gota, reumatismo, afecções cutâneas, asma, dor de dente, ferimentos, tumores, diabetes, demência e também como depurativo do sangue e diurético (CHEN et al., 2011; WUNGSINTAWEEKUL et al., 2011). 16

Caesalpinia ferrea Martius (Leguminosae), popularmente conhecida como “pau- ferro” ou “jucá” sofreu uma reclassificação e hoje é conhecida como Libidibia ferrea e é uma grande árvore encontrada principalmente no norte e nordeste do Brasil (DOMINGUEZ- ROBLES et al., 2015). Numerosas análises realizadas com espécies do gênero Libidibia corroboraram sua eficácia como fonte natural de novas moléculas químicas e aplicações terapêuticas, além de demonstrar propriedades úteis para o desenvolvimento de medicamentos tradicionais e conhecimentos científicos para o uso desta planta como medicação (HUSSEIN et al., 2016). Com isso, há estudos utilizando extratos desta planta que demonstram ter propriedades anti-inflamatórias, antinociceptiva, antioxidante (FALCÃO et al., 2019 (A); FALCÃO et al., 2019 (B)), antimicrobiana (SAMPAIO et al., 2009) e atividade antiproliferativa (GUERRA et al., 2017). Wyrepkowski et al. (2014) caracterizou e quantificou compostos antioxidantes no extrato etanólico da casca do caule de C. ferrea, identificando vinte e seis moléculas, nas quais predominaram taninos hidrolisáveis derivados do ácido gálico e do ácido elágico. Já Vasconcelos et al. (2011) constataram a presença de flavonoides (catequina e epicatequina), cumarinas, saponinas, esteroides, taninos hidrolisáveis (ácido gálico e ácido elágico) e compostos fenólicos na casca do caule C. ferrea. Diante do exposto, o presente estudo teve por finalidade investigar a ação antioxidante do extrato bruto e de frações da folha da Smilax fluminensis e do extrato etanólico da casca do caule da Caesalpinia ferrea após um estresse oxidativo induzido por PCM em camundongos Swiss.

2. REFERÊNCIAS BIBLIOGRÁFICAS

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CHEN, L. et al. Anti-hyperuricemic and nephroprotective effects of Smilax china L. Journal of Ethnopharmacology, v. 135, p. 399–405, 2011.

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FALCÃO, T. R. et al. Libidibia ferrea Fruit Crude Extract and Fractions Show Anti- Inflammatory, Antioxidant, and Antinociceptive Effect In Vivo and Increase Cell Viability In Vitro. Evidence-Based Complementary and Alternative Medicine, 2019 (A).

FALCÃO, T. R. et al. Crude extract from Libidibia ferrea (Mart. ex. Tul.) L.P. Queiroz leaves decreased intra articular inflammation induced by zymosan in rats. BMC Complementary and Alternative Medicine, v. 19, n. 47, 2019 (B).

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FONSECA, A. M. et al. Constituents and antioxidant activity of two varieties of coconut water (Cocos nucifera L.). Revista Brasileira de Farmacognosia, v. 19, p. 193-198, 2009.

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Capítulo I

INVESTIGAÇÃO DO POSSÍVEL EFEITO PROTETOR DA FOLHA DE Smilax fluminensis STEUD. EM CAMUNDONGOS SUBMETIDOS AO ESTRESSE OXIDATIVO PELO PARACETAMOL

O artigo foi elaborado segundo as normas da revista Bioscience Journal

INVESTIGATION OF THE POSSIBLE PROTECTIVE EFFECT OF Smilax fluminensis STEUD. LEAF IN MICE SUBJECTED TO OXIDATIVE STRESS BY PARACETAMOL

ABSTRACT Paracetamol (PCM), a drug widely used by the population as an antipyretic and analgesic. If administered in high doses it can cause liver damage, leading to hepatoxicity. The genus Smilax, found in temperate and tropical regions, is traditionally used by the population through the extract of leaves and roots for several conditions, such as in the treatment of syphilis, diabetes, asthma and as a diuretic action. Through this, Smilax fluminensis leaf extracts were used to evaluate the protective effect against oxidative stress induced by a high dose of PCM in mice that received the drug and after receiving treatment with crude extract and fractions. Plasma analysis was performed using as partate aminotransferase (AST), alanine aminotransferase (ALT), glucose, triglycerides and cholesterol, in addition to biochemical techniques such as catalase (CAT), glutathione-S-transferase (GST), reduced glutathione (GSH), ascorbic acid (ASA), substances reactive to thiobarbituric acid (TBARS) and carbonylated proteins (CARBONYL) of liver, brain and kidneys. Fraction 1 of the extract was the most promising, decreasing the plasma levels of AST and ALT, the levels of CAT and GST of the liver, together with GSH and in the renal and brain tissue there was a decrease in carbonylated proteins (PCM + F1 versus PCM). Besides, fraction 1 proved to be hypoglycemic and hypocholesterolemic. It is concluded that fraction 1 of Smilax fluminensis leaves has good antioxidant activity in the face of the damage caused by the high dose of paracetamol.

KEYWORDS: Smilax fluminensis. Acetaminophen. Hepatotoxicity. Hypoglycemic agents. Free radicals.

INTRODUCTION Oxidative stress is a result of the imbalance between reactive oxygen species (ROS), nitrogen (ERN) and the antioxidant defense system present in the body (SAHU et al., 2010). When such a balance tends towards an excessive production of these compounds or a deficiency of the antioxidant systems, the oxidative stress condition arises, which is harmful to the cellular components and to the individuals as a whole (CAROCHO, FERREIRA, 2013). Through this, several pathologies have been identified such as arthritis, atherosclerosis, diabetes, cataracts, multiple sclerosis, chronic inflammation, brain dysfunction, heart disease, emphysema, aging, cancer, among others. (NASCIMENTO, 2010). The presence of antioxidants in plants, such as phenolic compounds, flavonoids, tannins and proanthocyanidins can provide protection against various diseases (GULCIN, 2012), since they interact with free radicals before they can react with biological molecules, preventing the occurrence of chain reactions or preventing oxygen activation to highly reactive products (RATNAM et al., 2006). Paracetamol (PCM) is a medicine with analgesic and antipyretic activity most used by the population (SALWE et al., 2017). The administration of paracetamol is safe when the therapeutic dose is not exceeded, however, it can cause hepatic necrosis, nephrotoxicity, extrahepatic lesions and even death in humans and experimental animals when subjected to overdose (MOHANRAJ et al., 2013). PCM-induced hepatotoxicity involves the formation of a chemically reactive intermediate, namely N- acetyl-p-benzo-quinone-imine (NAPQI), which is formed by cytochrome P450 enzymes (CYP450s) and normally detoxified through the conjugation of glutathione (HINSON et al., 2004). Thus, paracetamol-induced toxicity (N-acetyl-p-aminophenol, acetaminophen) in rats is one of the experimental models widely used to assess the hepatoprotective activity of plant extracts (HUSSAIN et al., 2014). Medicinal plants have always been associated with cultural behavior and traditional knowledge. Many studies have shown that medicinal plants contain several bioactive compounds with antioxidant activity, which are responsible for their beneficial effects on health (DAI, MUMPER, 2010). Through this, there is a great interest in foods of plant origin with antioxidant properties due to their health benefits and the ability to prevent chronic diseases caused by oxidative stress (DESANMI et al., 2009). 22

Smilax fluminensis is commonly called “sarsaparilla” or “japecanga”, belongs to genus Smilax, comprises approximately 370 species that are widely distributed in tropical and temperate zones (SOUZA, LORENZI, 2008; JUDD et al., 2009; WU et al., 2010; ZHOU et al., 2017). It has a vast occurrence in the states of Roraima, Pará, Bahia, Minas Gerais, Rio de Janeiro, São Paulo and states of the Midwest Region, extending to Bolivia, Paraguay and Argentina. Smilax species, such as the leaves of S. excelsa, are used as food or pharmaceutical materials in many countries, such as in some parts of Turkey (OZSOY et al., 2008). In addition, the use in folk medicine of plants of this genus is quite common, as is the case of extracts of leaves and roots that are used in the treatment of syphilis, brucellosis, nephritis, gout, rheumatism, skin disorders, asthma, toothache, wounds, tumors, diabetes, dementia and also as a blood purifier and diuretic (WU et al., 2010; ANDRADE-CETTO, 2011; CHEN et al., 2011; WUNGSINTAWEEKUL et al., 2011; KHALIGH et al., 2016). Recently, studies performed with different Smilax species have demonstrated hypoglycemic and hypotensive activities (AMARO et al., 2014; ROMO-PÉREZ, 2019); antioxidant and antifungal potential (MORAIS et al., 2014; Wang et al., 2019) and cytoprotective effects against oxidative stress in pulmonary cells and in the liver (RAJESH, PERUMAL, 2014). Petrica and collaborators (2014) identified phytochemicals in extracts obtained from the leaf of Smilax fluminensis Steud. (S. fluminensis), obtaining as a final result the isolation and elucidation of two compounds containing Quercetin. Quercetin is a flavonoid found in vegetables and fruits, in which it has significant antioxidant and anti-inflammatory effects (SERBAN et al. 2016). Despite different biological activities and bioactive compounds that are well documented for some Smilax species and for not having biological studies with this plant, this is the first work that aimed to investigate the antioxidant action of the crude extract and leaf fractions of S. fluminensis using as a model oxidative stress induced by paracetamol in Swiss mice.

MATERIAL AND METHODS

Extract preparation The leaves of the plant were collected in the municipality of Nova Canaã do Norte - MT and an exsiccata of this species was deposited at the Herbarium of the Universidade Federal de Mato Grosso (UFMT), campus Sinop-MT / Brazil, under nº 1606, 1607 and 1608. After collection, the leaves were cleaned, dried and crushed. The crushed material was subjected to exhaustive maceration with ethanol (6 L) for one week. After filtration, the crude ethanolic extract (EB) (480 g) was obtained by evaporating the solvent (50 °C) under reduced pressure (700 mmHz). Part of the EB was partitioned with 1.5 L of methanol-water (1:1, V / V) resulting in two extracts, a suspension with chlorophyll (180.0 g) and another ethanolic extract (EE) without chlorophyll (280.0 g). Part of the EE was fractionated in a chromatographic column with silica gel as a stationary phase and elution by polarity gradient producing the fractions: hexane (FH), dichloromethane (FD), ethyl acetate (FAE), methanol (FM), methanol-water (9:1, V / V) (FMA) and methanol / water / acetic acid (7:3:1, V / V) (FMAA). Part of the EB and the FAE (F1) and FM (F2) fractions, which showed a higher concentration of flavonoids, were used in the experimental model.

Animals and treatment Male Swiss mice (average of 30 g), obtained from the Vivarium of the Universidade Federal de Mato Grosso, Campus in Cuiabá, were kept under conditions with temperature (22 ± 2 °C), light (12 hours light / dark) and humidity control (51 ± 2%), with water and pellets fed ad libitum. The animals remained for ten days under a period of acclimation. Afterwards, the animals received the treatment by gavage, initially being administered paracetamol (inducing liver damage) or filtered water. After three hours, treatment with the extract, fractions or filtered water was started, which lasted for 7 days. The animals were divided according to the following groups: CONTROL (filtered water), PCM (paracetamol 250 mg kg-1 + filtered water), EB (crude extract - 250 mg kg-1), F1 (fraction 1- 250 mg kg-1), F2 (fraction 2 - 250 mg kg-1), PCM + EB, PCM + F1, PCM + F2. Twenty-four hours after the last dose, the animals were anesthetized intraperitoneally with ketamine 50 mg/kg, xilaxine 2 mg/kg and acepromazine 2 mg/kg. Cardiac puncture was performed to collect blood with heparinized syringes and, after this stage, the animals were euthanized to remove 23

the liver, kidneys and brain, in which they were stored in the ultra-freezer at -85 °C. This study was approved by the Ethics Committee on the Use of Animals (CEUA), UFMT under nº 23108.781869/12-0.

Biochemical analysis

Tissue In the liver tissue, enzymatic antioxidants such as catalase (CAT) were analyzed according to the method of Nelson and Kiesow (1972) and the results expressed as µmol min-1 mg of protein-1 and glutathione-S-transferase (GST) by the method described by Hagib et al. (1974) and the results expressed in µmol GS-DNB min-1 mg protein-1. The dosed non-enzymatic antioxidants were reduced glutathione (GSH) determined according to the method of Sedlack and Lindsay (1968) and the data expressed as µmol GSH mg protein-1 and ascorbic acid (ASA) which followed Roe's method (1954) and the results presented in µmol of ASA g-1 tissue. The oxidative stress markers evaluated were carbonylated proteins (Carbonyl, protein damage marker) according to Yan et al. (1995) and the results expressed in nmol carbonyl mg protein-1 and substances reactive to thiobarbituric acid (TBARS, marker of lipid damage), which were evaluated according to Buege and Aust (1978), and the data presented in nmol MDA mg protein-1. In addition to liver tissue, CAT and protein damage in renal and brain tissue and the non-enzymatic antioxidant, GSH, in renal tissue were analyzed. The amount of protein in the tissues was determined by the method of Bradford (1976) and compared to a standard curve of bovine albumin.

Plasma The plasma measurements of aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, as well as the concentration of glucose, triglycerides and cholesterol were performed with commercial kits (Labtest®).

Statistical analysis Data were analyzed using the mean and standard deviation (SD) according to the unidirectional ANOVA, followed by the Tukey post hoc test, considering the differences between the groups when p < 0.05.

RESULTS AND DISCUSSION After carrying out this first biological study with the extract and fractions of the leaves of Smilax fluminensis, in the PCM-induced oxidative stress model in mice, we can observe that at the end of seven days of treatment, EB reduced the oxidative stress markers (TBARS and CARBONYL) in the liver tissue. Both fractions F1 and F2 showed similar results for CARBONYL, depending on the tissue (renal or brain). In addition, we found that F1 answered the objectives of the study, showing good results when assessing antioxidants in liver tissue (CAT, GST, GSH). Besides, with respect to liver damage markers, EB was toxic as well as F1 and F2, although these have partially reduced the damage caused by PCM. On the other hand, a hypoglycemic effect was also observed for most treatments, reduction of triglycerides by F2 and cholesterol by F1 (figure 1).

Figure 1. Effect of the ethanolic crude extract and leaf fractions of Smilax fluminensis, under oxidative stress induced by PCM in the tissues and plasma.

Protein oxidation can be an effect caused by high levels of ROS, resulting in protein carbonylation and oxidative damage (LI et al., 2010). Increased levels of hepatic MDA, an important reactive aldehyde that results from the peroxidation of polyunsaturated fatty acids in the cell membrane, are indicative of a causal role of lipid peroxidation resulting from liver damage induced by PCM (ADAM et al., 2016). According to figures 2. A and 2. B, analyzes of CARBONYL and hepatic TBARS showed an increase in the PCM group and EB was able to restore the levels of these markers at the control level, showing that treatment with this extract was able to inhibit the damage to proteins and lipids and the same occurred with F2 in protein carbonylation, but in lipid peroxidation these fraction didn’t present the same effect. The oxidative stress model is already well characterized, as in the work developed by Ajiboye et al. (2017) using the administration of PCM in dose of 300 mg kg-1 in mice, which showed the same protective effect, regarding the reductions in the dosages of TBARS and carbonylated proteins, although they administered the aqueous extract of the leaves of Phyllanthus muellarianus (100, 200 and 400 mg kg-1).

A B

6 0.20 -1

-1 * * ** 0.15 4 * * ** 0.10 ** ** 2

0.05 nmol MDA mg protein mg MDA nmol

nmol carbonyl mg protein mg carbonyl nmol 0 0.00 EB F1 F2 EB F1 F2 PCM PCM PCM+EBPCM+F1PCM+F2 PCM+EBPCM+F1PCM+F2 CONTROL CONTROL

Figure 2. Effect of the ethanolic crude extract and leaf fractions of Smilax fluminensis, under oxidative stress induced by PCM in the liver tissue, (A) CARBONYL and (B) TBARS (n = 8). * p <0.05 compared with the CONTROL group, ** p <0.05 compared with the PCM group. ANOVA followed by Tukey test.

High doses of PCM result in saturation of the metabolism pathways by glucuronidation and sulfation, thus, the PCM metabolism is diverted to the cytochrome P450 system generating the toxic and highly reactive N-acetyl-p-benzoquinone metabolite (KHANDELWAL et al., 2011). NAPQI depletes glutathione and initiates a covalent bond to cellular proteins leading to disruption of calcium homeostasis, mitochondrial dysfunction and oxidative stress, which can culminate in cell damage and cell death (HURKADALE et al., 2012). Depletion of glutathione causes severe liver damage by increasing the susceptibility of cells to oxidative stress (HINSON et al., 2010). The metabolism associated with glutathione is an important mechanism of cellular protection against agents that generate oxidative stress, eliminating cytotoxic products of lipid peroxidation, in addition, changes in GST activity can cause disturbances in the concentration of GSH (FERNÁNDEZ- IGLESIAS et al., 2014). Thus, the main function of glutathione-S-transferase (GST) is the cellular defense mechanism against electrophilic xenobiotics and their metabolites (GOPIA et al., 2017). Besides, this enzyme also plays a vital role in the detoxification of lipid and electrophilic hydroperoxides toxic by catalyzing their conjugation with GSH, which contributing to the protection of cell integrity (TOWNSEND, TEW, 2003). In our studies, GST activity in the liver tissue showed a decrease in the PCM group and the F1 was able to reverse the cellular damage caused by the drug (PCM + F1 versus PCM; figure 3. A). On the other hand, the GSH plays an important role in the detoxification of NAPQI, because when the therapeutic dose of PCM is within limits, NAPQI is promptly detoxified forming a conjugate with GSH, but when there is an overdose, NAPQI is excessively formed and depleted the content of GSH and, from there, binds to liver cell proteins, causing mitochondrial dysfunction (RADOSAVLJEVI´C et al., 2010). Similarly to GST, F1 of Smilax fluminensis promoted an increase in the concentration of GSH, important non-enzymatic antioxidant (PCM + F1 versus PCM; figure 3 B). These two results demonstrate the protective effect of this fraction against damage caused by PCM.

A B

-1 2.5 100 -1 2.0 80

** ** mg protein mg -1 1.5 * 60 *

1.0 40

0.5 20

mol GSH mg protein mg GSH mol 

0.0 0 µmol GS-DNB min GS-DNB µmol EB F1 F2 EB F1 F2 PCM PCM PCM+EBPCM+F1PCM+F2 PCM+EBPCM+F1PCM+F2 CONTROL CONTROL

Figure 3. Effect of the ethanolic crude extract and leaf fractions of Smilax fluminensis, under oxidative stress induced by PCM in the liver tissue, (A) GST and (B) GSH (n = 8). * p <0.05 compared with the CONTROL group, ** p <0.05 compared with the PCM group. ANOVA followed by Tukey test.

Free radical scavenging enzymes, such as SOD, CAT and GPx, are the first lines of defense against oxidative damage, so the dosage of these antioxidant enzymes is an indirect way to assess the 26

antioxidant status in tissues (SABIR et al., 2012). SOD converts superoxide radicals into hydrogen peroxide and this is toxic to the body, being subsequently metabolized by CAT resulting in water and oxygen. In our study, there was a decrease in hepatic CAT activity in the PCM group and F1 managed to reverse this damage (figure 4. A). In the study by Xia et al. (2010), using a plant from the same family, it was found that CAT activity in the liver of Wistar rats that received the ethanolic extract of Smilax glabra was restored after the stress caused by lead acetate, which was used as a damage inducer. On the other hand, the non-enzymatic antioxidant ASA did not present any significant difference between the groups (figure 4. B).

A B

24 1.5 -1 18 1.0 ** Weight * -1

mg protein mg 12 -1 0.5

mol ASA g ASA mol

 µmol min µmol 0 0.0 EB F1 F2 EB F1 F2 PCM PCM PCM+EBPCM+F1PCM+F2 PCM+EBPCM+F1PCM+F2 CONTROL CONTROL

Figure 4. Effect of the ethanolic crude extract and leaf fractions of Smilax fluminensis, under oxidative stress induced by PCM in the liver tissue, (A) CAT and (B) ASA (n = 8). * p <0.05 compared with the CONTROL group, ** p <0.05 compared with the PCM group. ANOVA followed by Tukey test.

The serum activities of AST and ALT are indicators of hepatotoxicity induced by acetaminophen (AJIBOYE, 2015). As a result, the induction of liver damage after a high dose of paracetamol (250 mg kg-1) caused an increase in blood transaminases (AST and ALT, table 1) in the PCM group, results that agree with the studies by Olaleye and Rocha (2008). However, EB, F1 and F2 showed toxicity, as they increased the activities of AST and ALT. When PCM was administered together with EB, the activity of these enzymes remained high. Although considered toxic by our findings, F1 managed to slightly reduce the increase caused by the drug, showing a significant difference in AST together with F2. Only the F1 fraction was able to decrease the plasma ALT activity increased by PCM (table 1). In this context, our findings are in agreement with Murali et al. (2012) who evaluated the methanol extract from Smilax zeylanica roots and rhizomes and observed a reduction in transaminases using also the experimental PCM model, but with a higher PCM dose (1 g kg-1). Due to their antioxidant and chelating properties, flavonoids inactivate ROS and thus neutralize the oxidation of LDL in the plasma and promote the reduction of inflammation of the blood vessel endothelium, in addition to acting by decreasing the activity of xanthine oxidase, NADPH oxidase and lipoxygenase. Under these circumstances, its anti-atherosclerotic actions are related to the reduction of inflammation in the blood vessel wall through the inhibition of leukocyte influx (MAJEWSKA-WIERZBICKA, CZECZOT, 2012; KOZŁOWSKA, SZOSTAK-WĘGIEREK, 2014). Added to the antioxidant study, we decided to complement this investigationy and evaluated the metabolic parameters in plasma and observed that the F1 fraction had a hypocholesterolemic effect and the F2 fraction promoted hypotriglyceridemia, which leads us to suggest that the presence of the glycosylated quercetin (PETRICA et al., 2014) may be inducing this hypolipidemic function. In our studies, except for the alone crude extract group, all the others groups had a hypoglycemic effect when compared to the control group (table 1). According to Rómo-Perez et al. (2019) who investigated and 27

demonstrated that the methanolic extract of roots of S. moranensis administered in hyperglycemic rats for a period of 42 days has hypoglycemic properties, corroborating with S. fluminensis, of the genus Smilax, which in this study presents this property. There are studies in the literature which presents mecanisms to glycosylated quercetin absortion (MUROTA, TERAO, 2003) and about the microbial catabolism process on the deglycosylation of quercetin glycosides (MUROTA et al., 2018, SERRA et al., 2012). BAHADORAN et al. (2013) explain that one of the main effects of this process is to decrease intestinal assimilation of carbohydrates in the diet, regulation of enzymes involved in glucose metabolism, alteration of β cell function, insulin activity and stimulation of insulin secretion, besides of the hypoglycemic effect. Therefore, we suggest that the hypolipidemic effect also observed in this work can be correlated to hypoglycemic effect presented by involvement of deglycosylation of quercetin glycosides.

Table 1. Effect of the ethanolic crude extract and leaf fractions of Smilax fluminensis, under oxidative stress induced by PCM in plasma. Grupos AST ALT Glucose Triglycerides Cholesterol CONTROL 75.67 ± 19.60 27.00 ± 5.27 314.60 ± 37.50 133.50 ± 26.22 63.38 ± 10.66 PCM 330.20 ± 37.36* 45.51 ± 3.89* 269.90 ± 40.64 128.40 ± 27.17 60.00 ± 7.07 EB 170.70 ± 29.72* 81.33 ± 11.28* 274.90 ± 61.79 115.40 ± 21.82 64.25 ± 14.64 F1 155.70 ± 34.27* 24.30 ± 3.30 227.50 ± 29.30* 110.50 ± 23.90 45.38 ± 10.84* F2 98.39 ± 21.70 41.32 ± 6.39* 225.80 ± 21.71* 87.29 ± 18.87* 48.29 ± 10.71 PCM + EB 288.50 ± 59.12* 41.03 ± 7.50 247.30 ± 39.69* 120.00 ± 27.32 54.50 ± 9.09 PCM + F1 164.50 ± 21.82*-** 29.63± 5.85** 189.40 ± 37.97* 104.80 ± 24.14 50.00 ± 6.26 PCM + F2 107.60 ± 20.48** 37.08 ± 8.89 229.00 ± 36.26* 90.13 ± 16.42* 60.57 ± 11.73 The results are expressed as mean ± SD; n = 7-8 animals. * p <0.05 compared with the CONTROL group, ** p <0.05 compared with the PCM group. ANOVA followed by Tukey test.

Drug-related nephrotoxicity is due to the role of the kidneys in the clearance of toxic substances (SHAHRBAF, ASSADI, 2015). Elevated levels of toxic liver metabolites, arising from high PCM intake, mainly NAPQI, quickly deplete GSH and covalently modify cellular proteins, leading to the generation of ROS and the depletion of ATP, this results in mitochondrial, hepatocyte and renal damage (HINSON et al. 2010; HODGMAN, GARRARD, 2012). In the renal tissue, there was no significant difference between the groups regarding the CAT activity and for the GSH levels, there was a decrease in the PCM and F1 groups compared to the CONTROL group (table 2). The oxidative stress marker, CARBONYL, showed an increase in the positive control group (PCM) and the extract and fractions were able to reverse the protein damage caused by the drug (PCM + EB, PCM + F1 and PCM + F2 versus PCM) (table 2). Studies realized by Pereira et al. (2018), who used the same experimental model of oxidative stress induction (dosage of 250 mg kg-1 PCM), showed the same pattern of kidney damage from the drug to GSH and CARBONYL. Some studies have shown that high doses of PCM can cause acute and chronic renal failure, and the mechanism involved included deficits in the antioxidant defense mechanisms and lipid peroxidation in the renal tissue (GHOSH et al., 2010). However, in our study, the crude extract or fractions could contribute positively to the improvement of the CARBONYL results in this tissue, in face of the damage caused by the PCM. On the other hand, Wang et al. (2019) showed that a flavonoid-rich fraction from rhizomes of S. glabra Roxb. ameliorates renal oxidative stress on uric acid nephropathy rats, restoring CAT activity and GSH levels besides reducing TBARS levels (lipid damage biomarker). Differently, in this study we investigate a protein damage biomarker and all treatments reduced this parameter although other parameters didn’t present similar results to the S. glabra in this tissue. According to Ghanem et al. (2016), there is the fact that acute liver failure occurs due to high ingestion of paracetamol and there is the secondary development of brain changes caused by the drug, with paracetamol crossing the blood-brain barrier and being distributed throughout the central nervous system (COURAD et al., 2001; KUMPULAINEN et al., 2007). Through this, we investigated the brain tissue that showed an increase only in the activity of the CAT enzyme in the EB group, and the increased protein damage caused by the PCM was reversed by F1 (table 2).

Table 2. Effect of the ethanolic crude extract and leaf fractions of Smilax fluminensis, under oxidative stress induced by PCM in renal and brain tissues Kidney Tissue Brain Tissue CAT GSH Carbonyl CAT Carbonyl CONTROL 18.72 ± 2.76 36.51 ± 8.38 4.40 ± 0.96 1.45 ± 0.31 4.52 ± 0.78 PCM 21.52 ± 2.88 22.27 ± 5.03* 6.55 ± 1.32* 1.44 ± 0.29 6.29 ± 1.02* EB 18.54 ± 2.30 27.75 ± 5.80 4.25 ± 0.99 2.50 ± 0.52* 5.33 ± 1.04 F1 15.61 ± 1.88 25.02 ± 5.99* 4.27 ± 0.95 1.54 ± 0.36 5.29 ± 1.32 F2 18.21 ± 3.35 32.60 ± 8.04 3.93 ± 0.92 1.60 ± 0.38 4.11 ± 0.83 PCM + EB 17.73 ± 3.69 21.33 ± 4.81* 3.40 ± 0.79** 1.84 ± 0.28 4.73 ± 1.15 PCM + F1 20.05 ± 2.91 25.77 ± 5.81 3.33 ± 0.68** 1.20 ± 0.29 3.94 ± 0.85** PCM + F2 18.64 ± 3.85 23.38 ± 3.23* 4.33 ± 0.92** 1.23 ± 0.29 4.97 ± 1.16 The results are expressed as mean ± SD; n = 8 animals. * p <0.05 compared with the CONTROL group, ** p <0.05 compared with the PCM group. ANOVA followed by Tukey test.

Studies by Posadas et al. (2010), found that PCM in the doses used (above 1 mM in vitro and 250 mg kg-1 in vivo), are neurotoxic to rat cortical neurons, thus, we can suggest that in this studied model, the EB and F1 were beneficial for this tissue. Therefore, according to Anand-David et al. (2016), flavonoids exert beneficial effects on the vascular system, which can lead to changes in cerebrovascular blood flow, being able to change neuronal morphology and can cause neurogenesis and angiogenesis, besides those also have the potential to protect neurons against neurotoxin-induced lesions. So, this first study suggest the involvement of these important molecules on the effects observed in mice treated with PCM.

CONCLUSION In this first study we identified that F1 reverses the effects caused by PCM in the enzymatic antioxidants CAT and GST and the non-enzymatic antioxidant GSH in the hepatic tissue and CARBONYL reduction in brain and kidney. F2 reverses protein damage in liver and kidney and EB presents the same effect in these tissues, besides reduces TBARS in the liver. In plasma biochemical analyses, EB was hepatotoxic, increasing ALT and AST activities, while F1 and F2 reduce the damage caused by PCM. The most of treatments presents hypoglycemic action and F1 being hypocholesterolemic and F2 reduces triglycerides. Therefore, it is possible that the good antioxidant activity which the extracts show may be due to the flavonoid compounds, in this specific case, glicosylated quercetins which were identified by our group of the study. However, more studies will be necessaries to explore the biological potential of Smilax fluminensis, in order to have more knowledge about the possible benefits of this plant.

ACKNOWLEDGMENTS To the Mato Grosso State Research Support Foundation (FAPEMAT) for scholarships (APSC and LSF). To CNPq for financial support process number 558225/2009-8.

INVESTIGAÇÃO DO POSSÍVEL EFEITO PROTETOR DA FOLHA DE Smilax fluminensis STEUD. EM CAMUNDONGOS SUBMETIDOS AO ESTRESSE OXIDATIVO PELO PARACETAMOL

Resumo Paracetamol (PCM), um fármaco amplamente utilizado pela população como antipirético e analgésico. Se administrado em altas doses pode causar dano hepático, levando a hepatoxicidade. O gênero Smilax, encontradas em regiões temperadas e tropicais, é tradicionalmente utilizado pela 29

população através do extrato das folhas e raízes para diversas afecções, como no tratamento da sífilis, diabetes, asma e com ação diurética. Através disto, utilizaram-se os extratos das folhas da Smilax fluminensis para avaliar o efeito protetor frente ao estresse oxidativo induzido através de uma alta dose de PCM em camundongos que receberam o medicamento e após receberam o tratamento com extrato bruto e frações. Foram realizadas análise do plasma através da aspartato aminotransferase (AST), alanina aminotransferase (ALT), glicose, triglicérides e colesterol, além de técnicas bioquímicas como, catalase (CAT), glutationa-S-transferase (GST), glutationa reduzida (GSH), ácido ascórbico (ASA), substâncias reativas ao ácido tiobarbitúrico (TBARS) e proteínas carboniladas (CARBONIL) em fígado, cérebro e rins. A fração 1 do extrato foi a mais promissora, diminuindo os níveis plasmáticos da AST e ALT, a atividade da CAT e GST do fígado, juntamente com a GSH e no tecido renal e cerebral houve uma diminuição das proteínas carboniladas (PCM+F1 versus PCM). Além disso, a fração 1 mostrou-se hipoglicêmica e hipocolesterolêmica. Conclui-se que a fração 1 das folhas da Smilax fluminensis possui boa atividade antioxidante frente aos danos causados pela alta dose de paracetamol.

Palavras chaves: Smilax fluminensis. Acetaminofeno. Hepatotoxicidade. Hipoglicemiante. Radicais livres.

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Capítulo II

ATIVIDADE ANTIOXIDANTE DO EXTRATO ETANÓLICO DA CASCA DO CAULE DE Caesalpinia ferrea EM CAMUNDONGOS Swiss

O artigo foi elaborado segundo as normas da revista The Natural Products Journal

1. INTRODUCTION [11], the presence of phenolic compounds, such as tannins and flavonoids present in the skin of C. Brazil has had a long history of using medicinal ferrea, can positively interfere in the effects plants to treat the health problems of the associated with wound healing and population, its use built on experience and antiinflammatory activity. In the phytochemical transmitted orally [1], with knowledge about analyses, several compounds in the genus medicinal plants often representing the only Caesalpinia were isolated, the main ones being therapeutic option for many communities [2]. flavonoids, diterpenes and steroids [12]. In studies According to the World Health Organization - carried out with the bark of the stem of C. ferrea WHO, a medicinal plant is defined as "any plant revealed the presence of flavonoids (catechins and that has, in one or more organs, substances that can epicatechins), coumarins, saponins, steroids, be used for therapeutic purposes or that are hydrolyzable tannins (gallic acid and ellagic acid) precursors of semi-synthetic drugs" [3]. and other phenolic compounds [10]. Caesalpinia ferrea Mart (C. ferrea) is a legume Oxidative stress is defined as a state of imbalance widely distributed in the North and Northeast between oxidizing radicals, free radicals, and regions of Brazil [4], popularly known as pau-ferro antioxidant systems as the body's internal defense or jucá, belonging to the Leguminosae- mechanism [13, 14]. The imbalance occurs as a Caesalpinoidae family [5]. Its bark is widely used result of the excessive level of reactive oxygen in folk medicine because it has antiinflammatory species (ROS) or the inadequate functioning of the [6], analgesic [7], antimutagenic [8] and antioxidant system [15]. ROS are molecules with an chemopreventive properties towards cancer [9]. unpaired electron, including peroxides, superoxide, Vasconcelos et al. [10] proved hypoglycemic hydroxyl radical and singlet oxygen, therefore activity and demonstrated molecular mechanisms of being very reactive and toxic to cells, as they affect C. ferrea bark extracts in streptozotocin-induced different cellular components in various ways [16]. diabetes in Wistar rats. According to Araújo et al. 37

PCM is oxidized by cytochrome P450, which leads 2.2 Experimental Model to the formation of the toxic electrophilic Male Swiss mice with an average weight of 30 ± 5 g metabolite, N-acetyl-p-benzoquinone imine were obtained from the Vivarium of the Federal (NAPQI) [18]; overdosing of PCM leads to the accumulation of this metabolite, which is a highly University of Mato Grosso, Campus of Cuiabá. First, the animals were acclimated with food and reactive compound that acts as a precursor to the water ad libitum, room temperature (25 ± 1 °C), toxic side effects observed in the body [19]. This is because there is saturation of the glucuronidation relative humidity (51 ± 2%) and with a 12-hour and sulfation pathways, producing more NAPQI, light / dark period. which depletes GSH reserves and forms protein After acclimatization the animals were divided into adducts by binding to the cysteine groups in cellular 4 groups (n = 8) according to the following groups: proteins and forming N-acetyl-p-aminophenol- Control (filtered water + Tween 80), PCM (250 mg cysteine (APAP-Cys) adducts [20]. With the kg-1 PCM in a single dose + Tween 80), PCM + increase in these protein adducts formed by EXT (250 mg kg-1 of PCM in a single dose + 100 NAPQI, oxidative stress, mitochondrial damage mg kg-1 of EXT) and EXT (filtered water + 100 mg and centrilobular necrosis occurs, and eventually kg-1 of EXT). Subsequently, oxidative stress was leads to liver damage [21]. induced through acetaminophen acute intoxication (PCM) at a dose of 250 mg kg-1, which was based Considering the antiinflammatory properties on a study by Olaleye and Rocha [23]. The already described by C. ferrea and studies done by Wyrepkowski et al [8] who identified antioxidant administration of the treatment started after three hours of the administration of the PCM, in which it compounds such as ellagic acid and gallic acid occurred with filtered water or with the ethanolic found in the ethanolic extract of the stem bark of C. ferrea, in addition to that overdosage with PCM can extract of the C. ferrea stem bark (EXT) prepared in an aqueous solution containing 0.1% of Tween deplete glutathione and release proinflammatory 80 (vehicle) at the dosage of 100 mg kg-1. This dose agents, for instance, inflammatory cytokines [22], we hypothesized that the stem bark extract from C. was established according to the Malone Hippocratic test [23]. The animals were treated ferrea can exhibit antioxidant activity against PCM- orally by gavage (0.3 mL / day / per animal) for a induced oxidative stress in mice. For this, the animals will be intoxicated with a high dose of period of 7 days. PCM and treated for seven days with the ethanolic After 24 hours of the last dose and after 8 hours of extract of the plant. fasting, the animals were anesthetized (ketamine 50 mg kg-1, xylazine 2 mg kg-1 and acepromazine 2 mg

kg-1), blood was collected (with heparinized syringes) by cardiac puncture and centrifuged at 1000 g for 10 minutes to obtain plasma. *Address correspondence to this author at the: Instituto de Subsequently, the animals were euthanized by Ciências Naturais, Humanas e Sociais, Câmpus Universitário de Sinop, Universidade Federal de Mato Grosso, Avenida cervical dislocation, the liver, kidneys and brain Alexandre Ferronato, 1200. CEP 78557-267, Sinop - MT, Brazil; were removed and washed with saline solution Tel/Fax: +55-66-99622-6670; E-mail: (0.15 M NaCl) and then frozen at -85 °C. [email protected]

2. MATERIALS AND METHODS 2.3 Biochemical Analysis: 2.1 Extract Preparation 2.3.1 Tissues The bark of the stem of C. ferrea was collected in Enzymatic antioxidant activities were measured in the city of Juína / MT, Brazil (11 ° 22'40 "S and 58 liver, kidney and brain tissues. Catalase activity ° 44'27" W) and the botanical identification was (CAT) was determined according to Nelson and performed at the Federal University of Mato Grosso Kiesow [25]. The principle is based on the (UFMT), Sinop / MT campus, Brazil. An exsiccata decomposition of H2O2 and measured No. 3021 was deposited at the Herbarium Centro spectrophotometrically at 240 nm and expressed in -1 -1 Norte Mato-Grossense (CNMT). μmol H2O2 min mg protein . The activity of glutathione-S-transferase (GST) was determined According to the methodology of Wyrepkowski et according to Habig et al. [26] and the enzymatic al. [8] for the preparation of the extract, the stem activity was measured according to the formation of bark was dried and ground to powder (1610.0 g) of the GS-DNB adduct. The result was expressed in C. ferrea. After it was mashed at room temperature µmol GS-DNB min-1 mg protein-1. with ethanol (4 × 48 h). The solution was evaporated under vacuum resulting in 260.09 g The non-enzymatic antioxidant reduced glutathione (16.2%) of crude ethanolic extract from the stem (GSH) was measured by the colorimetric method bark. that consists of a reaction of the sulfhydryl groups developed by Sedlak and Lindsay [27] in the liver, kidneys and brain and quantified at 412 nm. The 38

result was expressed in µmol GSH mg protein-1 and ethanolic extract of the stem bark of C. ferrea as an compared to a standard GSH curve. antioxidant in the model of oxidative stress induced by paracetamol, whose hepatotoxicity in high doses The oxidative damage to lipids in the liver was generates reactive oxygen species and determined by spectrophotometric identification of inflammatory processes [23]. the levels of thiobarbituric acid reactive substances (TBARS), according to Buege and Aust [28]. The There is a great diversity of secondary metabolites amount of lipid peroxidation was expressed in nmol present in plants, making them a source of MDA mg protein-1. biomolecules for therapeutic purposes, such as phenolic compounds that contain antioxidant Oxidative damage to proteins in the liver, kidneys properties due to their chemical structure [35]. The and brain was measured by determining carbonyl ethanolic extract of the C. ferrea bark used in this groups according to Yan et al. [29]. The amount of experiment was studied and the phytochemical carbonylated proteins (CARBONYL) was -1 parts were analyzed by Wyrepkowski et al. [8] who expressed in nmol of carbonyl mg protein . characterized and quantified the compounds, The levels of ascorbic acid (ASA) in the liver and identifying twenty-six molecules, in which brain were determined according to Roe [30], using hydrolyzable tannins derived from gallic acid and the colorimetric method and read at 520 nm ellagic acid predominated. On the other hand, de absorbance. The result was expressed in μmol ASA Souza et al. [35], studying the leaves of the same g tissue-1 and compared with a standard curve of plant, and showed that the flavonoids rutin, ascorbic acid. amentoflavone, quercetin-3-β-D-glycoside, taxifolin, apigenin, quercitrin and luteolin were The protein content of the sample (except ASA) found in the crude ethanolic extract and all of these, was determined during the analysis by the method except quercitrin in the extract ethyl acetate, which of Bradford [3] at 595 nm, using bovine serum demonstrates that different parts of the plant have albumin as a standard for the construction of the different bioactive compounds. calibration curve. Tannins are polyphenolic compounds extracted from plants and are composed of different chemical 2.3.2 Plasma structures [36], in which hydrolyzable tannins can be found that include gallotannins and ellagitannins The enzymatic activities of alanine [37]. They consist of gallic acid esters and ellagic aminotransferase (ALT) and aspartate acid glycosides that are formed from chiquimic acid aminotransferase (AST) and the quantification of [38] or condensed tannins that are polymers of plasma glucose, total cholesterol and triglycerides proanthocyanidins [36]. They are secondary ® were performed using commercial kits (Labtest , metabolites whose characteristic are the ability to Diagnostic S.A, Minas Gerais, Brazil). complex with macro and micro molecules and can act in the sequestration of free radicals, allowing for a series of pharmacological applications [39]. 2.3.3 Statistical Analysis Gallic acid has the ability to eliminate free radicals, Results were presented as mean ± standard acting as an antioxidant and is responsible for some deviation (SD), analyzed according to one-way biological activities [40] as an antioxidant and ANOVA and followed by Tukey's post hoc test. The antiinflammatory agent [41], as well as a homogeneity of the variances was verified using the bactericidal and bacteriostatic agent [42], along Bartlett test. The level of significance established with anticancer and antiangiogenic properties [43]. for rejection of the null hypothesis was 5% (p Ellagic acid, on the other hand, has antioxidant <0.05). properties and the ability to capture free radicals that result in the prevention or reduction of oxidative stress, a condition involved in several disorders, in addition to having a neuroprotective 3. RESULTS AND DISCUSSION and anti-carcinogenic effect [44, 45]. On the same The use of medicinal plants for the treatment of note, flavonoids contain antioxidant, anti-cancer, diseases in humans has increased considerably antimicrobial, antiviral and anti-aging properties, in worldwide [32]. This is due to the difficulty in which they can promote various biological effects accessing health care by the population, who do not in different types of cells [46]. As an antioxidant, have their demands and needs met and therefore, flavonoids can suppress the formation of ROS by are partially provided by the use of alternative inhibiting enzymes that generate these compounds therapies and also by a personal alternative [33]. (such as microsomal monooxygenase and NADH One of the major problems faced is that the use of oxidase), scavenging, chelating metals involved in phytotherapies and medicinal plants currently used the generation of these molecules, or by increasing by self-medication or by medical prescription has the activities of antioxidant and detoxifying no known toxic profile [34]. Thus, in this study, we enzymes (such as glutathione peroxidase, decided to investigate the possible effect of the glutathione reductase and catalase) [47, 48]. 39

Catalase (CAT) is involved in blocking the chain of free radicals produced by living beings together with superoxide dismutase (SOD) and glutathione peroxidase (GPx), in which they prevent oxidative modifications of DNA, proteins and lipids [49]. Its action occurs in catalyzing the conversion of hydrogen peroxide (H2O2) into water and molecular oxygen [50]. In our study, the ethanolic extract of the C. ferrea stem bark was not able to recover the inhibition of this enzyme that was caused by the medication (figure 1.A).

Paracetamol (PCM, N-acetyl-p-aminophenol, acetaminophen) is a widely studied drug, as it has a hepatotoxic potential after an overdose in experimental animals and in humans [51]. The conventional dose of PCM is metabolized to non- toxic compounds that are excreted in the urine, while a small remaining part is metabolized by CYPs to a highly reactive intermediate metabolite, N-acetyl-p-benzoquinone imine (NAPQI) [52]. NAPQI binds to glutathione (GSH) and leads to the rapid depletion of hepatic GSH, consequently after GSH depletion; NAPQI binds to other cellular thiol proteins (especially mitochondrial proteins) and disrupts the mitochondrial respiratory chain, which causes the formation of mitochondrial reactive oxygen species (ROS) [53]. In our studies, we found that there was a decrease in hepatic GSH Figure 1. Effect of ethanolic extract of C. ferrea, levels after the administration of the oxidative under oxidative stress induced by PCM on liver stress inducer (PCM) and the extract was not able tissue, graphs of (A) CAT and (B) GST (n = 8). *p to reverse this damage (PCM+EXT versus PCM) <0.05 compared to the Control group. 1-way (figure 2.A). However, in pharmacological doses of ANOVA followed by Tukey test. PCM, NAPQI is rapidly conjugated with GSH spontaneously and by glutathione-S-transferases (GST) [54], which in this case, in high doses, is The depletion of GSH induced by NAPQI can depleted and therefore is not conjugated. As a aggravate oxidative stress and lipid peroxidation, consequence, there is a decrease in GST activity, as occurred in our study in which GST in the liver which ends up leading to the acceleration of necrosis and apoptosis in the liver tissue [55]. decreased in the PCM groups and the extract was According to Hasanein and Sharifi [56], oxidative not able to reverse this damage (PCM+EXT versus stress induced by a high dose of PCM significantly PCM) (figure 1.B). increased the concentration of malondialdehyde (MDA) in the liver of Wistar rats. This is in line with our study, in which there was an increase in TBARS in the liver of the PCM group (PCM versus Control) and the extract was able to reverse the damage caused by the drug, decreasing liver lipid peroxidation (PCM+EXT versus PCM), however when the extract was administered alone, it was found to be toxic, as it increased TBARS levels (EXT versus Control) (figure 2.B). Lipid peroxidation, under toxic conditions as in the case with this drug, can induce cells to necrosis or apoptosis because the extent of oxidative damage exceeds their repair capacity, and therefore, causes damage to the molecular components of cells, which can facilitate the development of various diseases and premature aging [57,58].

Protein carbonylation is irreversible and generally results in impairment or even loss of protein function [59]. Its oxidative modification can be initiated by the reaction with hydroxyl radicals (OH•), but it is even more dependent on the •- concentration of the superoxide radical (O2 ) and hydrogen peroxide (H2O2) in the medium [60]. In our studies, protein damage increased in the PCM group (PCM versus Control) and the plant extract was able to reverse this damage (PCM+EXT versus PCM) (figure 2.C). This fact may have happened due to ellagic acid, which is considered a polyphenolic compound, with protective effects that can be attributed to some factors, which include binding to DNA, inhibition of ROS production and its elimination, protection of DNA against injuries from alkylating agents, thereby ellagic acid neutralizes free radicals and inhibits lipid peroxidation and protein carbonylation [61]. For the levels of ascorbic acid (ASA), a good antioxidant, soluble in water and considered essential to the organism through its action as a redox agent in biological systems [62], it was found that this non- enzymatic antioxidant decreased in PCM group (PCM versus Control) and the extract did not interfere in this parameter (figure 2.D). The same results occurred in the assay by Magalhães et al. [63], in which they used the same oxidative stress model, but used the ethanolic extract and the ethyl acetate fraction extract from the leaves of Trattinnickia rhoifolia. Drug-induced liver damage, in this work using PCM as a model, can lead to increased concentrations of serum enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), in addition to increased serum bilirubin, glucose, triglycerides, urea and creatinine [64]. The permeability of hepatocyte cell membranes is impaired after cell damage, which in this case was caused by PCM. Because of this, the liver enzymes AST and ALT are released into the circulation, which causes a significant increase in their levels [65-67]. In our study there was an increase in the enzymes ALT and AST in the PCM group (PCM versus Control), however in ALT the extract was able to restore the values at the Control level (PCM + EXT versus PCM). In AST there was an increase in the activity of this enzyme in the PCM + EXT and EXT groups compared to the Control group (Table 1). The increases in the activities of these enzymes (PCM group) are in line with the model used in the studies by Olaleye and Rocha [23]. On the other hand, Figure 2. Effect of ethanolic extract of C. ferrea, although the extract had a good effect on ALT, the under oxidative stress induced by PCM on liver same cannot be seen in the mitochondrial enzyme tissue, graphs of (A) GSH, (B) TBARS, (C) AST. According to Krithika and Verna [68], Carbonyl and (D) ASA (n = 8). *p <0.05 compared flavonoids are able to reduce ALT and AST activity with the Control group, **p <0.05 compared with in the serum of the animal exposed to the liver the PCM group. 1-way ANOVA followed by Tukey injury model. This extract presents several test. compounds and we didn’t know the extent of effect each one can cause. Interestingly, GST reduction and an increase in TBARS, which measures lipid 41

peroxidation, were observed in the EXT group. So, suggest that this beneficial effect against protein new studies are required to purify and identify the carbonylation may be related to gallic acid that has active compounds in this extract. On the other hand, antioxidant and antiinflammatory effects [76], and in our research, there was a decrease in glucose and its protective action consists of the ability to inhibit cholesterol in the PCM+EXT and EXT groups cell damage induced by ROS, in addition to compared to the Control group (Table 1). regulating positively the expression of glutathione peroxidase (GPx), although it was not investigated These data demonstrate that the extract had a in this study, and attenuate the presence of free beneficial action, as it decreased serum glucose and radicals [77]. cholesterol, demonstrating that the extract has a hypoglycemic and hypocholesterolemic action. Studies have shown that different hydrolyzable Table 2. Effect of ethanolic extract of C. ferrea, tannins contain biological properties such as under PCM-induced oxidative stress on renal antitumor, antimutagenic, antidiabetic and antibiotic [69], and in the ethanolic extract of the C. tissues. ferrea stem bark some compounds were found, CAT GST Carbonyl consisting of hydrolyzable tannins such as gallic Control 13.4 ± 2.1 1.3 ± 0.19 6.3 ± 1.4 acid and ellagic acid that were quantified according PCM 14.2 ± 2.7 1.0 ± 0.23* 15.0 ± 3.1* to Wyrepkowski et al. [8]. Still, the results by PCM + EXT 14.2 ± 1.6 1.1 ± 0.15 7.7 ± 1.9** Souza et al. [35] demonstrated that the extract of EXT 15.1 ± 1.3 1.2 ± 0.17 8.8 ± 1.8 the leaves of C. ferrea has a hypoglycemic action The results are expressed as mean ± SD; n = 8 and decreased triglyceride levels, which goes animals. *p <0.0001 compared with the Control against our findings. It is suggested that tannins group, **p <0.0001 compared with the PCM group. may have an insulin-like effect on tissues sensitive 1-way ANOVA followed by Tukey test. to their action, this demonstrates that they can act on cells by modifying or interacting with certain specific proteins found in important intracellular In brain tissue, oxidative stress occurs because this signaling pathways and, therefore, affect their role structure is more susceptible, it has high metabolic in the improvement of hyperglycemia [70, 71]. activity, a high density of oxidizable substrates and Furthermore, tannins are associated with the because it has a relatively low antioxidant defense inhibition of cholesterol biosynthesis, in which it [78]. Through this, the metabolism of PCM via the inhibits the enzyme HMG-CoA reductase, so that cytochrome P450-dependent pathway is associated its absorption can decrease [72]. with the development of harmful substances, such as N-acetyl-p-benzoquinone imine (NAPQI) in brain astrocytes and neurons [79, 80]. Our findings Table 1. Effect of ethanolic extract of C. ferrea, showed that the extract increased the activity of under oxidative stress induced by PCM in plasma antioxidant enzymes CAT and GST in brain tissue parameters. (EXT versus Control) and was able to decrease the Triglyc ALT AST damage caused by the drug to protein carbonylation Glucose Cholesterol erides (EXT+PCM versus PCM), while non-enzymatic Control 208.6±31 79.0±9 88.4±17 27.1±6 85.3±21 antioxidants GSH and ASA had no significant PCM 188.9± 7 70.3±14 76.4±16 47.0±10* 169.0±39* PCM + difference between groups (table 3). This damage 132.5±4* 46.2±9* 87.2±20 32.9±5** 155.2±23* EXT was also seen by Pereira et al. [81], in which they EXT 91.3± 9* 61.1±14* 82.8±14 32.6±7 362.6±8* used the same experimental model, but it was The results are expressed as mean ± SD; n = 8 reversed by the crude ethanolic extracts and ethyl animals. *p <0.0001 compared with the Control acetate from the stem bark of Copaifera multijuga group, **p <0.0001 compared with the PCM group. at a concentration of 250 mg kg-1. This action of the 1-way ANOVA followed by Tukey test. extract may be due to the fact that gallic acid has the ability to permeate the blood-brain barrier and act in a neuroprotective way [82] through its However, paracetamol overdose can cause antioxidant and free radical scavenging properties, hepatotoxicity and nephrotoxicity [73], through inactivating the enzymes responsible for production reactive oxygen species (ROS), which are one of of ROS or by the positive regulation of antioxidant the main mechanisms of renal pathogenesis and enzymes [83], which in this way, increased CAT which can lead to apoptosis and senescence of these and GST. cells, in addition to fibrosis in the kidney [74, 75]. In our studies, there was a depletion in GST in the PCM group and an increase in protein damage in the PCM group (PCM versus Control) and the extract was able to reverse this renal protein damage (PCM+EXT versus PCM) (table 2). A similar result was found by Souza et al. [35] who used the same oxidative stress model. We can 42

Table 3. Effect of ethanolic extract of C. ferrea, Nacional de Gestão do Patrimônio Genético under PCM-induced oxidative stress on brain brasileiro – SISGEN (Nº AA4F748). tissue.

CAT GST GSH ASA Carbonyl CONSENT FOR PUBLICATION Control 0.74±0.16 0.12±0.02 12.7±1.96 0.41±0.04 4.5 ± 0.8 PCM 0.79± 0.18 0.14±0.03 15.2±3.43 0.45±0.03 7.2±1.8* Not applicable. PCM+EXT 0.68±0.15 0.12±0.02 12.8±1.31 0.39±0.03 4.7±1.1**

EXT 1.29±0.19* 0.18±0.02* 14.5±2.43 0.40±0.02 5.5 ±1.3 The results are expressed as mean ± SD; n = 8 AVAILABILITY OF DATA AND MATERIALS animals. *p <0.0001 compared with the Control group, **p <0.0001 compared with the PCM group. Not applicable. 1-way ANOVA followed by Tukey test. FUNDING 4. CONCLUSION Not applicable. It is known through the literature that paracetamol overdose causes several damages to the organism, however, with the administration of the ethanolic CONFLICT OF INTEREST extract of C. ferrea it was possible to reduce liver The author declares no conflict of interest, damage through the reversal of lipid peroxidation financial or otherwise. (TBARS) and protein damage (Carbonyl) caused in the three tissues (liver, kidney and brain). The extract was shown to have a hypoglycemic and ACKNOWLEDGEMENTS hypocholesterolemic effect. The antioxidant effect of the extract can be demonstrated through its To the Research Support Foundation of the constituents, mainly tannins such as gallic acid and State of Mato Grosso (FAPEMAT), the CAPES ellagic acid. The study with this part of the plant is Foundation for the availability of a scholarship and unprecedented and, therefore, it is necessary to CNPq for financial support. To the teachers and investigate further to better elucidate the possible collaborators for the help received during the benefits of the stem bark of C. ferrea. research. Besides, the authors are grateful to Anna Scheffer Sinhorin that reviewed the manuscript.

C.C.W. collected and prepared the plant extract; LIST OF ABBREVIATIONS A.P.S. idealized and supervised the plant chemical Not applicable. study; A.P.S.C. performed all biochemical studies, performed data analyses and wrote this manuscript; D.R. participated of biochemical analyses and ETHICS APPROVAL AND CONSENT TO V.D.G.S. supervised the pharmacological study, PARTICIPATE performed data analyses and review the manuscript. The authors declare that this study was approved by the Ethics Committee on the Use of Animals REFERENCES (CEUA), UFMT under nº 23108.781869/12-00. [1] Bruning, M.C.R.; Mosegui, G.B.G.; Viana, C.M.M. A. Utilização da fitoterapia e de HUMAN AND ANIMAL RIGHTS plantas medicinais em unidades básicas de The authors declare that no patient data appear in saúde nos municípios de Cascavel e Foz do this article. The mice were treated according to the Iguaçu-Paraná: a visão dos profissionais de Ethical Guidelines for Animal Experimentation In saúde. Cien. Saúde Colet., 2012, 17(10), 2675- accordance with the standards of “Guide for the 2685. Care and Use of Laboratory Animals” (grants.nih.gov/grants/olaw/guide-for-the-care-and- [2] Oliveira, A.K.M.; Oliveira, N.A.; Resende, use-of-laboratory-animals_prepub.pdf published by U.M.; Martins, P.F.R.B. Ethnobotany and the National Academy of Sciences, The National Academies Press, Washington, D.C.). traditional medicine of the inhabitants of the Patanal Negro sub-region and the raizeiros of Miranda and Aquidauna, Mato Grosso do Sul, RESEARCH INVOLVING PLANTS Brazil. Braz. J. Biol. 2011, 719, 283-289.

The author declares that the C. ferrea leaves used [3] Costa, V.P; Mayworm, M.A.S. Plantas in this experiment does not violate to the Sistema medicinais utilizadas pela comunidade do 43

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3. CONCLUSÃO GERAL A alta dose de paracetamol demonstrou dano hepático, no qual diminuiu a atividade das enzimas CAT e GST e do antioxidante não enzimático GSH, as transaminases AST e ALT apresentaram um aumento nos seus parâmetros e também houve um aumento de proteínas carboniladas. Foi constatado um dano renal através da diminuição da GSH e um aumento da CARBONIL, além de um aumento da carbonilação proteica do cérebro. A administração da fração 1 da Smilax fluminensis resultou em um melhor efeito desta planta onde, no plasma houve uma diminuição da atividade da AST e da ALT, um aumento das enzimas hepáticas CAT e GST e do antioxidante não enzimático GSH e constatou uma diminuição da CARBONIL renal e cerebral, evidenciando uma proteção do extrato F1 frente ao estresse oxidativo causado pela alta dose de PCM. Além disso, a fração 1 evidenciou ser hipoglicemiante e hipocolesterolêmica. Já a administração do extrato etanólico da Caesalpinia ferrea conseguiu reverter os danos causados pelo medicamento na peroxidação lipídica (TBARS) e na oxidação de proteínas (CARBONIL) no tecido hepático. Ademais, houve uma diminuição da CARBONIL após a administração do extrato nos tecidos renal e cerebral. Além disto, no plasma houve uma diminuição da atividade das transaminases AST e ALT e, também, este extrato apresentou efeito hipoglicemiante e hipocolesterolêmico. O efeito antioxidante destas plantas pode ser demonstrado através de seus constituintes, no caso da S. fluminensis pode ser devido a compostos flavonoides, neste caso em específico, a quercetina e para a C. ferrea foram encontrados os taninos, como o ácido gálico e o ácido elágico. Novas pesquisas podem explorar mais o potencial fitoquímico e biológico destas plantas, a fim de desenvolver mais conhecimentos dos possíveis benefícios delas. Resaltando que este é o primeiro estudo com a casca do caule da C. ferrea.

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numbered. Papers should be submitted without the identification of the authors. The authors' names, title and address of work must be presented to metadata submission and in the cover letter. Figures and tables must be inserted in the text, as close as possible to where cited. The article will be sent to three (03) reviewers in the area in question, in the shortest possible time, without identifying the authors and will be considered as approved upon 02 favorable opinions. Only papers written in English will be accepted. Author must present a certificate of proofreading made by an expert in the English language. The journal reserves the right to make changes as to rules, spelling and grammar in the original, in order to maintain the standard patterns of the language, while respecting the style of the authors. The final proofs will be sent to the authors, together with the payment slip for publication. Papers which are published become the property of the Bioscience Journal, having their reprint, in whole or in part, subject to the express permission of the journal Editor. The original source of publication must be assigned. No reprints will be provided. The articles will be available for printing in PDF format on the journal website. A publication fee will be charged to the amount of R$ 40.00 (forty reais) per published page of the approved papers to national authors and $ 40 (forty US dollars) for foreign authors. (Form of payment will be informed later). Once the article has been reviewed and approved, the journal will categorize the contributions according to the following categories:

1. Original Articles - Articles that present a contribution which is entirely new to knowledge and allow other researchers, based on the written text, to judge the conclusions, check the accuracy of the analyzes and deductions of the author and repeat the investigation if they so wish. The articles must contain: Title, Abstract (200 to 400 words), Keywords, Introduction, Material and Methods, Results, Discussion (or Results and Discussion) and Conclusion (optional), Acknowledgements (if applicable). They must also contain: Title, Abstract (200 to 400 words) and key words in Portuguese and References. The papers must not exceed 20 pages (including text, references, figures, and annexes).

2. Review Articles - Articles that present comprehensive and updated review of a subject of interest from the scientific community and which offer significant contribution to the area of knowledge under discussion. The articles must contain: Title, Abstract (200 to 400 words), Keywords, Introduction, Development, Conclusion, Acknowledgements (if applicable). They must also contain: Title, Abstract (200 to 400 words) and keywords in Portuguese and References. The papers must not exceed 30 pages (including text, references, figures and any annexes). In this paper/work category only contributions made at the invitation of the editors (General or Associate) will be accepted for submission.

3. Communication - Non original article, demonstrating the experience of a group or a service, preferably covering teaching, research, health policy and professional practice. Or an article to report the results (partial or not) of work that offers relevant information to scientific knowledge, but which does not allow for firm conclusions. It must contain: Title, Abstract (200 to 400 words), Keywords, Introduction, Contents and Acknowledgements (if necessary). It must also contain: Title, Abstract (200 to 400 words) and Keywords in Portuguese and References. The papers must not exceed 10 pages, including attachments.

Presentation of Papers Format: All papers/collaborations must be submitted through the Electronic System for Journal Publishing - SEER, Address: http://www.seer.ufu.br/index.php/biosciencejournal/index The text must be saved in RTF (Rich Text Format) extension or Microsoft Word format. The metadata must be filled out with the Paper/Work title, name (s) of author (s), last academic degree, work institution, postal address, telephone and email. The text will be cordially written with intercalation of tables and figures, already inserted in the text, with the minimum amount required for its understanding. As a measure of secrecy the body of the paper must not include the authors' names, which must be sent separately, with personal data (title, mailing address, email address and institution to which he/she is connected).

Paper title: The title must be brief and sufficiently specific and descriptive, containing the keywords that represent the contents of the text separated by colon, both accompanied by their translation into Portuguese.

Abstract: An informative summary must be prepared with about 200 to 400 words, including objective, method, results, conclusion, accompanied by its translation into Portuguese. Both must have 800 words at most.

Keywords: The keywords must not repeat words in the title, the scientific name of the species studied must be included. Words should be separated by a colon and begin with a capital letter. Authors must submit 3-6 terms, taking into consideration that a term may be composed of two or more words.

Acknowledgements: Acknowledgements as to help received in the preparation of the paper must be mentioned at the end of the article, before the references.

Notes: The notes contained in the article must be indicated with an asterisk immediately after the sentence to which they refer. The notes must be at the bottom of the corresponding page. Exceptionally, numbers may be adopted for the notes together with asterisks on the same page. In which case, the notes with asterisks precede the notes with numbers, regardless of the order of these notes in the text.

Appendices: Appendices can be used in the case of extensive lists, statistics and other supporting elements.

Figures and Tables: Clear photos (black and white or in color), graphs and tables in black and white (strictly essential for clarity of the text) will be accepted, and must be marked in the text by their order number, in the places where they must be inserted. If the illustrations submitted have already been published, mention the source. (See rules for preparation of figures, in the next section). Manuscripts, even if they present scientific relevance and are methodologically correct, may be refused if they are not properly organization and if they are outside the norms of the Bioscience Journal.

GUIDELINES FOR THE PREPARATION OF FIGURES 1. Figures may be made in software depending on the authors' preference (Excel, Sigma Plot, etc.) They must be inserted and sent in TIFF or JPG format with a minimum resolution of 300 dpi. 2. The figures must have a maximum width of8.0 cmor16.0 cm. 3. The titles and the x and y axes scale must be in Times New Roman size 11. The axel lines and other lines (e.g., regression curves) must have a thickness of 0.3mm. All information contained inside the figure (e.g., equations, captions) must be in Times New Roman size 10 or at least 8. Right hand and top edges in graphs are not necessary. 4. All figures must be conveniently inserted into the text after being mentioned, consecutively and in Arabic numerals. The figures should be inserted in the text by means of the Insert Image/Figure File• command. 5. Figures may be made up of multiple graphs, both horizontal and vertical, respecting the maximum width of 16.0cm and 8.0cm, respectively. When dealing with figures of multiple graphs, the same must be identified by letters (A, B, C, D) in capital letters in brackets, source Times New Roman size 11. Papers that have been consulted and cited in the text are the responsibility of the author.

Clarification Note: The ISO 690: 2010 (E) standard was adopted as of this year, 2020, being required only for new submissions. Therefore, articles that were already approved will still be published using the ABNT standard.

References

References: ISO 690:2010(E) - Information and Documentation – Guidelines for bibliographic references and citations to information resources. 3nd ed. 2010. Ex.: https://feup.libguides.com/c.php?g=670877&p=4763581&preview=7cdb1f09 0b67eda50984ab80492405a8 The accuracy and appropriateness of the references to papers that have been consulted and cited in the text are the responsibility of the author. Information coming from personal communication, papers in progress and unpublished papers must not be included in the reference list, but indicated in a footnote on the page where they are cited. The references included at the end of each article must be written on separate pages from the main text, in alphabetical order according to the ISO 690:2010(E)

ISO 690: 2010 - International standard that establish the order of the elements in the elaboration of bibliographic references, as well as the conventions for the transcription and presentation of information from the most diverse sources of information. The consultation of the examples does not dispense with the consultation of the standards, which have more detailed information.

See examples below:

Printed documents

Books (technical term: monographs) SURNAME, Names. Title. Secondary liability. Edition. Place of publication: Publisher, Year of publication. ISBN.

Up to 3 authors: BERTONI, J. e LOMBARDI NETO, F. Conservação do solo. 7ª ed. São Paulo: Ícone, 2010. ISBN 85-274-0143-6.

More than 3 authors: DONAGEMA, G. K., et al. Manual de métodos de análise de solos. 2ª ed. Rio de Janeiro: Embrapa Solos, 2011. ISBN 85-85864-03-6.

Volumes and parts of books SURNAME, Names. Part title. Edition. Numbering. Secondary liability. Place of publication: Publisher, Year of publication. Part location. Example: HINKELMANN, Klaus. Design and analysis of experiments, 2th ed. Vol. 1. Hoboken: Wiley-Interscience, c2008. Section 4, Linear Model Theory, p. 73-140.

Book chapters SURNAME, Names. Title of contribution. In SURNAME, Names. Title of the monograph. Edition. Place of publication: Editor, Year of publication, Location in the monograph. Example: HARRIS, Robin F. and KARLEN, Douglas L. and MULLA, David J. A conceptual framework for assessment and management of soil quality and health. In: DORAN, J. W. and JONES, A. J. (Ed.). Methods for assessing soil quality. Madison: Soil Science Society of America, 1996, pp. 61-82.

Articles from magazines, newspapers, etc. SURNAME, Names. Title of the article. Secondary liability. Title of serial publication. Edition. Year of publication, Numbering, Location in publication. ISSN. Example: GEHRING, C. A. Growth responses to arbuscular mycorrhizae by rain forest seedlings vary with light intensity and tree species. Plant Ecology. July 2003, vol. 167, nº 1, pp. 127- 139. ISSN 1385-0237. or GEHRING, C. A. Growth responses to arbuscular mycorrhizae by rain forest seedlings vary with light intensity and tree species. Plant Ecology. 2003, 167 (1), 127-139. ISSN 1385- 0237.

Theses, dissertations and other academic evidence SURNAME, Names. Title. Supplementary note, Academic Institution, Year. Example: FERNANDES, J. C. Fontes e doses de nitrogênio na adubação do capim-Mombaça em cerrado de baixa altitude. São Paulo: Universidade de São Paulo, 2011. Dissertação de Mestrado.

Conference communications SURNAME, Names. Title of the communication. In Title of the proceedings of the congress, number, place, date. Place of publication: Editor. Location in the publication. ISBN. Example: 55

MILLARD, Frédérique e TOUPANCE, Gérard. Indicators concept applied to a european city: the ile de france area during ESQUIF Campaign. In: Conference on air pollution modelling and simulation, 2, Champs-sur-Marne, France, 2001. Berlin : Springer, c2002. ISBN 978-3-642-07637-4.

Standards Acronym and Number of the Standard: Year. Area - Title. Edition. Year of publication Example: ISO 690:2010(E). Information and Documentation – Guidelines for bibliographic references and citations to information resources. 3nd ed. 2010.

Patents RESPONSIBLE ENTITY. Title. Secondary liability. Country Document type Number. Publication date Example: SUN CHEMICAL CORP. Binder for fibers or fabrics. LINDEMANN, Martin K. and DEACON, Kim.US Patent 4 683 165. 1987-07-28.

Electronic documents

E-books SURNAME, Names. Title. [Type of support]. Secondary liability. Edition. Place of publication: Publisher, Year of publication. Update / revision date. [Consultation date]. Availability and access. ISBN. Example: PINTO, Luísa Janaina Lopes Barroso. Inovação e regulamentação ambiental no setor de alimentos sob a égide evolucionária [Online]. Ponta Grossa: Atena, 2019 [consultado 24 Janeiro de 2020]. Disponível em: https://www.atenaeditora.com.br/arquivos/ebooks/inovacao- e-regulamentacao-ambiental-no-setor-de-alimentos-sob-a-egide-evolucionaria. ISBN 978-85- 7247-820-5.

Book chapter (e-books) SURNAME, Names. Title of contribution. In Title of the monograph [Type of support]. Secondary Liability. Edition. Place of publication: Publisher, Year of publication. Update / revision date [Consultation date]. Location in the monograph. Availability and access. Example: ALMEIDA, Suise Carolina Carmelo de e GONÇALVES, Luciana Márcia. Indicadores de sustentabilidade urbana: panorama das principais ferramentas utilizadas para gestão do desenvolvimento sustentável. In: PACHECO, Juliana Thaisa Rodrigues e KAWANISHI, Juliana Yuri e NASCIMENTO, Rafaelly do (org.). Meio ambiente e desenvolvimento sustentável [online]. Ponta Grossa: Atena, 2019 [consultado em 25 Janeiro 2020]. pp. 113- 126. Disponível em: https://www.atenaeditora.com.br/arquivos/ebooks/meio-ambiente-e- desenvolvimento-sustentavel-2. ISBN 978-85-72477-54-3.

Online journals Title. [type of support]. Edition. Place of publication: Publisher, Date of publication [date of citation]. Series. Note. Availability and Access. ISSN. Example: 56

Journal of Technology Education. [online]. Blacksburg (Va): Virginia Polytechnic Institute and State University, 1989- [cited 15 March 1995]. Semi-annual. Disponível em: https://scholar.lib.vt.edu/ejournals/JTE/. ISSN 1045-1064.

Articles in electronic documents: magazines, newspapers, etc. SURNAME, Names. Title of the article. Title of publication [Type of support]. Edition. Numbering. Update / revision date [Consultation date], Location in publication. Availability and access. ISSN. Example in Portuguese: ALVAREZ-PARDO, V. M. e FERREIRA, A. G. e NUNES, V. F. Seed desinfestation methods for in vitro cultivation of epiphyte orchids from Southern Brazil. Horticultura Brasileira [online], junho 2006, vol. 24, nº 2, pp. 217-220 [consultado em 2020-01-24]. Disponível em: http://dx.doi.org/10.1590/S0102-05362006000200019. ISSN 1806-9991. Or ALVAREZ-PARDO, V. M. e FERREIRA, A. G. e NUNES, V. F. Seed desinfestation methods for in vitro cultivation of epiphyte orchids from Southern Brazil. Horticultura Brasileira [online], junho 2006, 24 (2), 217-220 [consultado em 2020-01-24]. Disponível em: http://dx.doi.org/10.1590/S0102-05362006000200019. ISSN 1806-9991. Example in English: DUTRA, D. e KANE, M. e RICHARDSON, L. Asymbiotic seed germination and in vitro seedling development of cyrtopodium punctatum : a propagation protocol for an endangered Florida native orchid. Plant Cell Tissue and Organ Culture [online]. March 2009, vol. 96, nº 3 [cited 2004-01-24], pp. 235-243. Available from Internet: http://dx.doi.org/10.1007/s11240-008-9480-z. ISSN 1573-5044.

Transfer of Copyright: All persons listed as authors must sign the Transfer of Copyright: I declare that, in the case of acceptance of the article, the Bioscience Journal shall be the owner of the copyrights relating to same, which will become the sole property of the Journal, prohibiting any reproduction, in whole or in part, in any other place or means of publication, printed or electronic, without the prior and required authorization being requested, and if obtained, will include an appropriate acknowledgment to the Journal.

Signature (s) of author (s) Date ___ / ___ / ____ Opinions expressed by authors are their exclusive responsibility.

Statement of Responsibility: All persons listed as authors must sign the responsibility statement in the following terms: I certify that I participated in the conception of the paper to take public my responsibility for its content, not omitting any affiliations or financial agreements between authors and companies that may be interested in publishing this article; - I certify that the manuscript is original and that the paper, in part or in whole, or any other paper with substantially similar content of my authorship, was not sent to another journal and will not be sent, while its publication is being considered by the Bioscience Journal, be it in printed or electronic format. Signature (s) of author (s) Date ___ / ___ / ____

ETHICAL PRINCIPLES Research involving human beings and animals 57

The Bioscience Journal believes it is important that all studies follow the principles incorporated in the Declaration of Helsinki and insists that all studies involving human beings, in case of publication in this journal, are performed according to these principles. In the case of experiments with animals, these ethical principles should also be followed. Studies in both animals and human beings require the presentation of an approval from the local Ethics Committee, providing the number of protocol approval. Thus, research involving both animals and human beings requires the presentation of an approval from the local Ethics Committee by means of a document submitted as supplementary material. The Editor-in-Chief and the Editorial Board reserve the right to decline manuscripts that do no show clear evidence that the methods used are not adequate to experiments in human beings and animals. Registration of Clinical Trials and Systematic Reviews The Bioscience Journal supports policies of registration of Clinical Trials and Systematic Reviews. The journal acknowledges the importance of such initiatives for the international registration and publication of open-access protocols. Therefore, the Bioscience Journal will publish only the clinical trials and systematic reviews that have previously received an identification number validated by the responsible agencies. We encourage the use of checklist (CONSORT, ARRIVE, STROBE, PRISMA, among others for the performance of studies).

Address for submission of papers: http://www.seer.ufu.br/index.php/biosciencejournal/submission/wizard Copyright Notice The copyright for articles published in this journal belong to the authors, with first publication rights granted to the journal. In virtue of their appearance in this journal of open access, the articles are free to use, with proper attribution, in educational and non- commercial applications. Authors who publish in this journal agree to the following terms: This work is licensed under a Creative Commons Attribution 4.0 International License.

The copyrights belong exclusively to the authors. Published content is licensed under Creative Commons Attribution 3.0 (CC BY 3.0) guidelines, which allows sharing (copy and distribution of the material in any medium or format) and adaptation (remix, transform, and build upon the material) for any purpose, even commercially, under the terms of attribution. Privacy Statement The names and email addresses entered in this journal site will be used exclusively for the stated purposes of this journal and will not be made available for any other purpose or to any other party.

ANEXO III E-mail de aceite do artigo na Revista Bioscience Journal.

ANEXO IV Normas da Revista The Natural Products Journal. Send Orders for Reprints to [email protected] Natural Products Journal, 2019, 9, Pagination 1 ARTICLE TYPE Title: (The Title of the Article should be Precise and Brief and Must Not be More Than 120 Characters. Authors should avoid the Use of Non-Standard Abbreviations. The Title Must be Written in Title Case Except for articles, conjunctions and prepositions.)

Principle Authora, Corresponding author*b, Co-author, Co-authora and Co-authorb

aDepartment Name, Faculty Name, University Name, City, Country; bDepartment Name, Faculty Name, University Name, City, Country

Abstract: The abstract of an article should be clear, concise and accurate summary, having no more than 250 words, and including the explicit sub-headings (as in-line or run-in headings in bold). Use of abbreviations should be avoided and the references should not be cited in the abstract. Ideally, each A R T I C L E H I S T O R Y abstract should include the following sub-headings, but these may vary according to requirements of the article. Received: Revised: Accepted: • Background

DOI: • Objective • Method • Results • Conclusion

Keywords: Provide 6 to 8 keywords.

1. INTRODUCTION manuscript submission should always have the The Introduction section should include the name of the corresponding author as part of the file background and aims of the research in a name, i.e., “Cilli MS text.doc”, “Cilli MS Figure 1” comprehensive manner. etc.

*Address correspondence to this author at the Department of 1.1. Section Headings xxxy, Faculty of xxx, xxx University, P.O. Box: 0000-000, City, Section headings should be numbered sequentially, Country; Tel/Fax: ++0-000-000-0000, +0-000-000-0000; E- left aligned and have the first letter capitalized, mails: [email protected] starting with the introduction. Sub-section headings It is imperative that before submission, authors however, should be in lower-case and italicized with their initials capitalized. They should be should carefully proofread the files for special characters, mathematical symbols, Greek letters, numbered as 1.1, 1.2, etc. equations, tables, references and images, to ensure that they appear in proper format. The main text should begin on a separate page and 1.2. Text Organization should be divided into title page, abstract and the Please provide soft copies of all the materials (main main text. The text may be subdivided further text in MS Word or Tex/LaTeX), according to the areas to be discussed, which figures/illustrations in TIFF, PDF or JPEG, and should be followed by the Acknowledgements and chemical structures drawn in ChemDraw Reference sections. For Research papers, the (CDX)/ISISDraw (TGF) as separate files, while a manuscript should begin with the title page and PDF version of the entire manuscript must also be abstract followed by the main text, which must be included, embedded with all the structured into separate sections as Introduction, figures/illustrations/tables/chemical structures etc. Materials and Methods, Conclusion, Conflict of It is advisable that the document files related to a Interest, Acknowledgements and References. The 60

Review Article should mention any previous preferred file format TIFF, with color mode being important recent and old reviews in the field and RGB or Grayscale, in a resolution of 500-900 dpi. contain a comprehensive discussion starting with the general background of the field. It should then go on to discuss the salient features of recent 1.3.2. Formats developments. The authors should avoid presenting material which has already been published in a Illustrations may be submitted in the following previous review. The authors are advised to present file formats: and discuss their observations in brief. • Illustrator References, figures, tables, chemical structures etc. should be referred to in the text at the appropriate • EPS (preferred format for diagrams) place where they have been first discussed. Figure • PDF (also especially suitable for diagrams) legends/captions should also be provided. • PNG (preferred format for photos or images) 1.3. Figures/Illustrations • Microsoft Word (version 5 and above; All authors must strictly follow the guidelines figures must be a single page) below for preparing illustrations for publication. If • PowerPoint (figures must be a single page) the figures are found to be sub-standard, then the manuscripts will be rejected. • TIFF • JPEG (conversion should be done using The authors are expected to submit good quality the original file) figure(s) in PDF, PPT, MS Word, TIFF or JPEG versions, which, if required, should be improved • BMP yourself or by professional graphic designers of • CDX (ChemDraw) your organization/ country. You may even consider approaching our contracted service • TGF (ISISDraw) providers Eureka Science for Graphics Bentham Science does not process figures Enhancement Services. submitted in GIF format. The Graphics Designing team at Eureka Science For TIFF or EPS figures with considerably large can assist in improving the quality of your images file size restricting the file size in online at affordable rates. Eureka Science has contracted submissions is advisable. Authors may therefore special rates with us of US $125 for the convert to JPEG format before submission as this improvement of up to five figures, with any results in significantly reduced file size and upload additional figures being charged at US $20 each. time, while retaining acceptable quality. JPEG is a ‘lossy’ format, however. In order to maintain The quality of Graphic Enhancement Services acceptable image quality, it is recommended that offered by Eureka Science can be viewed JPEG files are saved at High or Maximum quality. at http://www.eureka- science.com/images/Binder1.pdf, along with Zipit or Stuffit tools should not be used to valuable feedback on their services compress files prior to submission as the resulting at http://www.eureka-science.com/testimonials.php. compression through these tools is always You may contact Eureka Science at info@eureka- negligible. science.com Please refrain from supplying: a) Graphics embedded in word processor 1.3.1. Scaling/Resolution (spreadsheet, presentation) document. Line Art image type is normally an image based b) Optimized files optimized for screen use on lines and text. It does not contain tonal or shaded (like GIF, BMP, PICT, WPG) because areas. The preferred file format should be TIFF or of the low resolution. EPS, with the color mode being Monochrome 1-bit c) Files with too low a resolution. or RGB, in a resolution of 900-1200 dpi. d) Graphics that are disproportionately Halftone image type is a continuous tone large for the content. photograph containing no text. It should have the preferred file format TIFF, with color mode being RGB or Grayscale, in a resolution of 300 dpi. 1.3.3. Image Conversion Tools

There are a number of software packages Combination image type is an image containing available, many of them freeware or shareware, halftone, text or line art elements. It should have the 61

capable of converting to and from different The µ in µg or µm should be in Roman. The symbol graphics formats, including PNG. for litre is L and that for minute is min. For temperature, please use only one of °C, °F or K in the General tools for image conversion include entire manuscript. As the Angström (1Å = 10-10m) is Graphic Converter on the Macintosh, Paint Shop not an SI unit, it should be replaced by the nanometre Pro, for Windows, and ImageMagick, available on Macintosh, Windows and UNIX platforms. (1nm = 10-9 m) or by the picometer (1pm = 10-12 m): 1Å = 0.1nm = 100 pm. Multiple units should be Bitmap images (e.g. screenshots) should not be written with negative superscripts (for example, converted to EPS as they result in a much larger file 25mgµL-1 µs-1). The list of notations should appear size than the equivalent JPEG, TIFF, PNG or BMP, just before the first paragraph of full text. and poor quality. EPS should only be used for images produced by vector-drawing applications A list of symbols and units should be provided if used such as Adobe Illustrator or CorelDraw. Most extensively throughout the text. vector-drawing applications can be saved in, or exported as, EPS format. If the images were originally prepared in an Office application, such as 1.5. Tables (if any) Word or PowerPoint, original Office files should be • Data Tables should be submitted in directly uploaded to the site, instead of being Microsoft Word table format. converted to JPEG or another format of low quality. • Each table should include a title/caption being explanatory in itself with respect to 1.3.4. Color Figures/Illustrations the details discussed in the table. Detailed legends may then follow. • The cost for color figures/plates/illustrations is US$ 540 per • Table number in bold font i.e. Table 1, article for up to 3 colour pages and should follow a title. The title should be in subsequently US$ 215.00 per page for any small case with the first letter in caps. A additional colour pages. full stop should be placed at the end of the title. • Color figures should be supplied in CMYK and not RGB colors. • Tables should be embedded in the text exactly according to their appropriate Note for authors To maintain publication quality, placement in the submitted manuscript. figures submitted in colour will be published in colour only. • Columns and rows of data should be made visibly distinct by ensuring that the borders of each cell are displayed as black 1.3.5. Chemical Structures lines. Chemical structures MUST be prepared in • Tables should be numbered in Arabic ChemDraw/ CDX and provided as a separate file. numerals sequentially in order of their citation in the body of the text.

• If a reference is cited in both the table and 1.4. Symbols and Units text, please insert a lettered footnote in the table to refer to the numbered reference in Greek symbols and special characters often undergo the text. formatting changes and get corrupted or lost during preparation of a manuscript for publication. To ensure • Tabular data provided as additional files that all special characters used are embedded in the can be submitted as an Excel spreadsheet. text, these special characters should be inserted as a symbol but should not be a result of any format styling (Symbol font face) otherwise they will be lost during 1.6. Construction of References the conversion to PDF/XML. All references should be numbered sequentially Authors are encouraged to consult reporting [in square brackets] in the text and listed in the guidelines. These guidelines provide a set of same numerical order in the reference section. The recommendations comprising a list of items relevant reference numbers must be finalized and the to their specific research design. bibliography must be fully formatted before submission. Only ISO symbols, written in italic, should be used for the various parameters. All kinds of measurements Sample references are provided at the end of this should be reported only in International System of template in the reference section. Correct reference Units (SI). SI units should always be written in Roman format and list must be provided in the article. and separated from the numerical value by a space (whatever the language). 62

2. MATERIALS AND METHOD (FOR For all manuscripts reporting data from studies RESEARCH ARTICLES ONLY) involving human participants, formal review and This section provides details of the approval by an appropriate institutional review methodology used along with information on any board or ethics committee is required. For research previous efforts with corresponding references. Any involving animals, the authors should indicate details for further modifications and research whether the procedures followed were in should be included. accordance with the standards set forth in the eighth edition of Guide for the Care and Use of Laboratory Animals (grants.nih.gov/grants/olaw/guide-for-the- EXPERIMENTAL (FOR RESEARCH care-and-use-of-laboratory- ARTICLES ONLY)- animals_prepub.pdf published by the National Academy of Sciences, The National Academies Repeated information should not be reported in Press, Washington, D.C.). the text of an article. A calculation section must include experimental data, facts and practical development from a theoretical perspective. HUMAN AND ANIMAL RIGHTS

Research work on animals should be carried out in 3. RESULTS (FOR RESEARCH ARTICLES accordance with the NC3Rs ARRIVE Guidelines. ONLY) For In Vivo Experiments, The Results and discussions may be presented visit https://www.nc3rs.org.uk/arrive-guidelines individually or combined in a single section with short and informative headings. Authors must clearly state the name of the approval committee, highlighting that legal and ethical approval was obtained prior to initiation of the 4. DISCUSSION research work carried out on animals, and that the experiments were performed in accordance with the This should explore the significance of the results relevant guidelines and regulations stated below. of the work, present a reproducible procedure and emphasis the importance of the article in the light of recent developments in the field. Extensive • US authors should cite compliance with citations and discussion of published literature the US National Research Council's should be avoided. "Guide for the Care and Use of Laboratory Animals" The Results and Discussion may be presented • The US Public Health Service's "Policy on together under one heading of “Results and Humane Care and Use of Laboratory Discussion”. Alternatively, they may be presented under two separate sections (“Results” section and Animals" and "Guide for the Care and Use “Discussion” Sections). Short sub- headings may be of Laboratory Animals" added in each section if required. • UK authors should conform to UK legislation under the Animals (Scientific Procedures) Act 1986 Amendment CONCLUSION Regulations (SI 2012/3039). • European authors outside the UK should A small paragraph summarizing the contents of conform to Directive 2010/63/EU. the article, presenting the final outcome of the research • Research in animals must adhere to ethical or proposing further study on the subject, may be guidelines of The Basel Declaration and given at the end of the article under the Conclusion the International Council for Laboratory section. Animal Science (ICLAS) has also published ethical guidelines. LIST OF ABBREVIATIONS • The manuscript must clearly include a declaration of compliance with relevant If abbreviations are used in the text either they guidelines (e.g. the revised Animals should be defined in the text where first used, or a (Scientific Procedures) Act 1986 in the list of abbreviations can be provided. UK and Directive 2010/63/EU in Europe) and/or relevant permissions or licences ETHICS APPROVAL AND CONSENT TO obtained by the IUCN Policy Statement on PARTICIPATE Research Involving Species at Risk of Extinction and the Convention on the All clinical investigations must be conducted Trade in Endangered Species of Wild according to the Declaration of Helsinki principles. Fauna and Flora. 63

RESEARCH INVOLVING PLANTS SUPPORTIVE/SUPPLEMENTARY MATERIAL All experimental research on plants (either Supportive/Supplementary material intended for cultivated or wild), must comply with international publication must be numbered and referred to in the guidelines. The manuscript must clearly include a manuscript but should not be a part of the submitted declaration of compliance of field studies with paper. In-text citations as well as a section with the relevant guidelines and/or relevant permissions or heading "Supportive/Supplementary Material" licences obtained by the IUCN Policy Statement on before the "References" section should be provided. Research Involving Species at Risk of Here, list all Supportive/Supplementary Material Extinction and the Convention on the Trade in and include a brief caption line for each file Endangered Species of Wild Fauna and Flora. describing its contents. Any additional files will be linked to the final published article in the form supplied by the author, CONSENT FOR PUBLICATION but will not be displayed within the paper. They If the manuscript has an individuals’ data, such as will be made available in exactly the same form as personal detail, audio-video material etc., consent originally provided only on our Web site. Please should be obtained from that individual. In case of also make sure that each additional file is a single children, consent should be obtained from the table, figure or movie (please do not upload linked parent or the legal guardian. worksheets or PDF files larger than one sheet). Supportive/ Supplementary material must be A specific declaration of such approval must be provided in a single zipped file not larger than 4 made in the copyright letter and in a stand-alone MB. paragraph at the end of the Methods section especially in the case of human studies where inclusion of a statement regarding obtaining the REFERENCES written informed consent from each subject or subject's guardian is a must. The original should be retained by the guarantor or corresponding author. References must be listed in the ACS Style Editors may request to provide the original forms only. All references should be numbered by fax or email. sequentially [in square brackets] in the text and All such case reports should be followed by a proper consent prior to publishing. listed in the same numerical order in the reference section. The reference numbers must be finalized CONFLICT OF INTEREST and the bibliography must be fully formatted before Financial contributions and any potential submission. conflict of interest must be clearly acknowledged under the heading ‘Conflict of Interest’. Authors must list the source(s) of funding for the study. This See below few examples of references should be done for each author. listed in the ACS Style

ACKNOWLEDGEMENTS Journal Reference All individuals listed as authors must have contributed substantially to the design, • Bard, M.; Woods, R.A.; Bartón, D.H.; performance, analysis, or reporting of the work and Corrie, J.E.; Widdowson, D.A. Sterol are required to indicate their specific contribution. mutants of Saccharomyces cerevisiae: Anyone (individual/company/institution) who has chromatographic analyses. Lipids, 1977, substantially contributed to the study for important 12(8), 645-654. intellectual content, or who was involved in the • Zhang, W.; Brombosz, S.M.; Mendoza, article’s drafting the manuscript or revising must J.L.; Moore, J.S. A high-yield, one-step also be acknowledged. synthesis of o-phenylene ethynylene cyclic Guest or honorary authorship based solely on trimer via precipitation-driven alkyne position (e.g. research supervisor, departmental metathesis. J. Org. Chem., 2005, 70, head) is discouraged. 10198-10201.

Book Reference Australian National University: Canberra, December 2004.

• Crabtree, R.H. The Organometallic E-citations Chemistry of the Transition Metals, 3rd ed.; Wiley & Sons: New York, 2001. • Citations for articles/material published Book Chapter Reference exclusively online or in open access (free- to-view), must contain the accurate Web addresses (URLs) at the end of the • Wheeler, D.M.S.; Wheeler, M.M. D. reference(s), except those posted on an Stereoselective Syntheses of Doxorubicin author’s Web site (unless editorially and Related Compounds In: Studies in essential), e.g. ‘Reference: Available from: Natural Products Chemistry; Atta-ur- URL’. Rahman, Ed.; Elsevier Science B. V: Amsterdam, 1994; Vol. 14, pp. 3-46. Some important points to remember:

Conference Proceedings • All references must be complete and accurate. • All authors must be cited and there should • Jakeman, D.L.; Withers, S.G.E. In: be no use of the phrase et al. Carbohydrate Bioengineering: • Date of access should be provided for Interdisciplinary Approaches. In: online citations. Proceedings of the 4th Carbohydrate • Journal names should be abbreviated Bioengineering Meeting, Stockholm, according to the Index Sweden, June 10-13, 2001; Teeri, T.T.; Medicus/MEDLINE. Svensson, B.; Gilbert, H.J.; Feizi, T., Eds.; • Punctuation should be properly applied as Royal Society of Chemistry: Cambridge, mentioned in the examples given above. UK, 2002; pp. 3-8. • Superscript in the in-text citations and reference section should be avoided. URL(WebPage) • Abstracts, unpublished data and personal communications (which can only be included if prior permission has been • National Library of Medicine. Specialized obtained) should not be given in the Information Services: Toxicology and references section. The details may Environmental Health. however appear in the footnotes. sis.nlm.nih.gov/Tox/ToxMain.html (Accessed May 23, 2004). • The authors are encouraged to use a recent version of EndNote (version 5 and above) or Reference Manager (version 10) when Patent formatting their reference list, as this allows references to be automatically • Hoch, J.A.; Huang, S. Screening methods extracted. for the identification of novel antibiotics. U.S. Patent 6,043,045, March 28, 2000.

Thesis

• Mackel, H. Capturing the Spectra of Silicon Solar Cells. PhD Thesis, The