UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Engenharia de Alimentos
MÔNICA CRISTINA LOPES DO CARMO
MODELO DE INFLAMAÇÃO INTESTINAL IN VITRO E AVALIAÇÃO DO POTENCIAL DO EXTRATO FENÓLICO DE FOLHAS DE PASSIFLORA EDULIS
IN VITRO MODEL FOR INTESTINAL INFLAMMATION AND EVALUATION OF THE EFFECTS OF THE PHENOLIC EXTRACT OF PASSIFLORA
EDULIS
CAMPINAS 2020
MÔNICA CRISTINA LOPES DO CARMO
MODELO DE INFLAMAÇÃO INTESTINAL IN VITRO E AVALIAÇÃO DO POTENCIAL DO EXTRATO FENÓLICO DE FOLHAS DE PASSIFLORA EDULIS
IN VITRO MODEL FOR INTESTINAL INFLAMMATION AND EVALUATION OF THE EFFECTS OF THE PHENOLIC EXTRACT OF PASSIFLORA
EDULIS
Tese apresentada à Faculdade de Engenharia de Alimentos da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Alimentos e Nutrição, na área de Nutrição Experimental e Aplicada à Tecnologia de Alimentos.
Thesis presented to the Faculty of Food Engineering of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Food and Nutrition, in the area of Experimental and Applied to Food Technology Nutrition
Orientadora: Profa. Dra. Juliana Alves Macedo
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA MÔNICA CRISTINA LOPES DO CARMO, ORIENTADA PELA PROFa. DRa. JULIANA
ALVES MACEDO
CAMPINAS 2020
Claudia Aparecida Romano - CRB 8/5816
Carmo, Mônica Cristina Lopes do,
C213i CarIn vitro model for intestinal inflammation and evaluation of the effects of the phenolic extract of Passiflora edulis / Mônica Cristina Lopes do Carmo. – Campinas, SP : [s.n.], 2020.
CarOrientador: Juliana Alves Macedo.
CarTese (doutorado) – Universidade Estadual de Campinas, Faculdade de
Engenharia de Alimentos.
Car1. Passiflora edulis. 2. Células Caco-2. 3. Atividade anti-inflamatória. 4. Intestinos – Doenças inflamatórias. I. Macedo, Juliana Alves. II. Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Modelo de inflamação intestinal in vitro e avaliação do potencial do
extrato fenólico de folhas de Passiflora edulis
Palavras-chave em inglês: Passiflora edulis
Caco-2 cells
Anti-inflammatory activity Inflammatory bowel diseases
Área de concentração: Nutrição Experimental e Aplicada à Tecnologia de Alimentos Titulação: Doutora em Alimentos e Nutrição
Banca examinadora:
Juliana Alves Macedo [Orientador] Flavia Maria Netto
Juliano Lemos Bicas João Felipe Mota
Spencer Luiz Marques Payao
Data de defesa: 11-09-2020
Programa de Pós-Graduação: Alimentos e Nutrição
Identificação e informações acadêmicas do(a) aluno(a)
- ORCID do autor: https://orcid.org/0000-0002-8567-1641 - Currículo Lattes do autor: http://lattes.cnpq.br/4898503281316969
BANCA EXAMINADORA Profa. Dra. Juliana Alves Macedo
Orientadora
Faculdade de Engenharia de Alimentos / UNICAMP
Profa. Dra. Flavia Maria Netto
Membro Titular
Faculdade de Engenharia de Alimentos / UNICAMP
Prof. Dr. Juliano Lemos Bicas Membro Titular
Faculdade de Engenharia de Alimentos / UNICAMP
Prof. Dr. João Felipe Mota Membro Titular
Faculdade de Nutrição / UFG
Prof. Dr. Spencer Luiz Marques Payão Membro Titular
Faculdade de Medicina de Marília / FAMEMA
A ata da defesa com as respectivas assinaturas dos membros encontra-se no processo de vida acadêmico do aluno.
DEDICATÓRIA
Dedico esse trabalho à Larissa Lima Bona (in memorian). Jamais esquecerei seu abraço apertado, seu sorriso meigo, seu encanto, e é com uma luta diária que enfrento sua precoce despedida. Todos os dias farei meu luto, e noites também, em um constante status de luta, para compreender as decisões da vida e da morte. Até um dia.
AGRADECIMENTOS
Ao Departamento de Alimentos e Nutrição da Faculdade de Engenharia de Alimentos da Universidade Estadual de Campinas (UNICAMP), pela oportunidade de realização deste trabalho e pela oportunidade de conhecer professores, alunos, funcionários e pesquisadores brilhantes.
À professora orientadora Juliana Alves Macedo, pela orientação, pelo carinho e amor que sempre tratou a mim, e a todos seus orientados, pela atenção e paciência, e por tudo que me ensinou e motivou na vida acadêmica durante esta jornada. Não tenho palavras para demonstrar a gratidão e inspiração que a senhora representa para mim. Obrigada por sempre me estender as mãos para me ajudar a levantar a cada tombo.
À professora Gabriela Alves Macedo, por todo apoio para a execução deste trabalho.
Aos meus pais e meu irmão, sem vocês eu nada seria. Agradeço pelas palavras de acalento, pelos silêncios necessários, pelos abraços apertados, choros emocionados, pela vibração com cada conquista, e principalmente, por tanto compreender sem dizer ou ouvir uma só palavra.
Ao meu marido, meu amor, agradeço a paciência e ao companheirismo, e por me apoiar em todos os momentos.
À nossa técnica Carla Greghi (in memorian) pelo auxílio no laboratório de Compostos Bioativos, e por todo apoio.
À Ana Elisa Magalhães, por compartilhar comigo a execução desta pesquisa, sua dedicação e apoio foram fundamentais para nossas conquistas.
À amigas de laboratório Vânia, Taís, Amanda, Paula, Isa, Camila, Ana, Érika, Renata, Lívia, por estarem sempre disponíveis para ajudar, pela troca de ensinamentos, e pelos momentos de descontração que levarei sempre na memória com muita saudade.
Aos meus médicos que me deram suporte e confiança, em especial ao doutor João Soares por sempre zelar da minha saúde com tanto carinho e proporcionar, cuidando de meu intestino, que indiretamente eu pudesse retribuir com pequenas contribuições no meio científico pela saúde intestinal alheia. Agradeço por me ensinar que enfrentar a Doença Inflamatória Intestinal seria uma luta comigo mesma, e que nem todo remédio para doenças autoimunes vêm em pílulas. Pelos seus incentivos eu escolhi não Desistir, e Resistir. Gratidão.
Aos professores Mário Maróstica e Cíntia Cazarin, pela colaboração, ajuda, e doação das folhas de maracujá.
Aos Professores da banca examinadora pelo tempo despendido para correções e pelas valiosas contribuições ao aprimoramento do trabalho.
Aos meus grandes amigos que estiveram ao meu lado, Agatha Louise, Angelo Zadra, Thais Mercuri, Sara Bueno, minhas primas Bruna e Maria, minha mãe do coração Sandra.
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível superior – BRASIL (CAPES) – Finance Code 001.
À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo apoio ao projeto (Processo 2015/50333-1).
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pela bolsa de doutorado concedida.
Agradeço a Deus por me conceder, a cada dia, uma página de vida nova no livro do tempo. De todas as vezes que eu ajoelhei Senhor, nenhuma delas foi para te pedir coragem.
A todos que, contribuíram para a realização deste trabalho.
EPÍGRAFE
“Se eu vi mais longe, foi porque me apoiei nos ombros de gigantes”.
RESUMO
O extrato de folhas de Passiflora edulis é rico em compostos fenólicos, com propriedades bioativas, incluindo atividades antioxidantes, antivirais, anticâncer e anti-inflamatórias. Este estudo teve como objetivos desenvolver um modelo in vitro para estudar a inflamação intestinal e avaliar os efeitos protetores do extrato de folhas de P. edulis (PELE), na integridade de monocamadas epiteliais intestinais, nos efeitos nas respostas inflamatórias produzidas por um sistema de co-cultura de células epiteliais e macrófagos, e investigar os efeitos deste extrato na adesão de linhagens de bactérias probióticas e patogênicas em modelo in vitro com monocamada de Caco-2. No modelo in vitro de monocamada de Caco-2 as células foram desafiadas com o estímulo pró-inflamatório composto por IL-1β e LPS. Após estímulo inflamatório, observou-se um aumento significativo na permeabilidade média das monocamadas. Após 48 horas de tratamento com PELE (10 mg mL-1), as monocamadas
apresentaram aumento nos parâmetros de integridade avaliados. O tratamento também suprimiu a produção de IL-8 pelas células intestinais. Já no modelo de co-cultura de células epiteliais com macrófagos, o tratamento com o extrato PELE, após o estímulo pró-inflamatório das células, promoveu redução na expressão de IL-8 e IL-6, reduziu também a produção de óxido nítrico no cultivo celular de macrófagos e melhorou a disfunção da barreira epitelial diminuindo a permeabilidade da monocamada. Em relação aos testes de adesão das bactérias probióticas e patogênicas na monocamada intestinal epitelial, PELE (5 e 10 mg mL-1) aumentou a adesão de todas as cepas probióticas testadas (L. casei, L.
rhamnosus, Bifidobacterium animalis subsp. Lactis). Para as cepas patogênicas (Listeria monocytogenes, Escherichia coli, Salmonella Typhimurium e Salmonella Enteritidis), entretanto, o extrato diminuiu a adesão celular em taxas significativas para todos os microrganismos testados. O extrato avaliado neste estudo mostrou potencial atividade anti-inflamatória em monocamada de células Caco-2, podendo contribuir para minimizar os eventos inflamatórios e danos decorrentes do aumento de permeabilidade intestinal, recuperando a integridade da barreira epitelial; a amostra também se mostrou atuante na redução da sinalização inflamatória em co-cultura com células Caco-2 e RAW264.7, tanto por parte das próprias células epitelias intestinais, quanto das citocinas produzidas por macrófagos. O extrato mostrou ser um potencial produto modulador da microbiota, estimulando a adesão de bactérias probióticas e inibindo a adesão de cepas de patógenos, sendo interessante para o desenvolvimento de um suplemento simbiótico. Além disso, os modelos com células intestinais humanas demonstraram respostas inflamatórias
fisiopatológicas observadas in vivo, fortalecendo a hipótese deste modelo como aplicável na prospecção de diversos extratos com potencial bioativo em diferentes doses.
Palavras-chave: Passiflora edulis, células Caco-2, atividade anti-inflamatória, intestinos – doenças inflamatórias, adesão bacteriana
ABSTRACT
Passiflora edulis leaf extract is rich in phenolic compounds, with bioactive properties, including antioxidant, antiviral, anticancer and anti-inflammatory activities. This study aimed to develop an in vitro model to study intestinal inflammation and evaluate the protective effects of P. edulis leaf extract (PELE), on the integrity of intestinal epithelial monolayers, on the effects on inflammatory responses produced by a co-system culture of epithelial cells and macrophages, and investigate the effects of this extract on the adhesion of strains of probiotic and pathogenic bacteria in an in vitro model with Caco-2 monolayer. In the Caco-2 monolayer in vitro model, cells were challenged with the pro-inflammatory stimulus composed of IL-1β and LPS. After inflammatory stimulation, a significant increase in the average permeability of the monolayers was observed. After 48 hours of treatment with PELE (10 mg mL-1), the
monolayers showed an increase in the evaluated integrity parameters. The treatment also suppressed the production of IL-8 by intestinal cells. In the model of co-culture of epithelial cells with macrophages, the treatment with the PELE extract, after the pro-inflammatory stimulus of the cells, promoted a reduction in the expression of IL-8 and IL-6, also reduced the production of nitric oxide in the macrophage cell culture and improved epithelial barrier dysfunction by decreasing the permeability of the monolayer. Regarding the adhesion tests of probiotic and pathogenic bacteria in the epithelial intestinal monolayer, PELE (5 and 10 mg mL-1) increased the adhesion of all tested probiotic strains (L. casei, L. rhamnosus,
Bifidobacterium animalis subsp. Lactis). For the pathogenic strains (Listeria monocytogenes, Escherichia coli, Salmonella Typhimurium and Salmonella Enteritidis), however, the extract decreased cell adhesion at significant rates for all tested microorganisms. The extract evaluated in this study showed potential anti-inflammatory activity in monolayer of Caco-2 cells, which may contribute to minimize inflammatory events and damage resulting from increased intestinal permeability, recovering the integrity of the epithelial barrier; the sample was also shown to be active in reducing inflammatory signaling in co-culture with Caco-2 and RAW264.7 cells, both by the intestinal epithelial cells themselves, and by the cytokines produced by macrophages. The extract proved to be a potential modulator product of the microbiota, stimulating the adhesion of probiotic bacteria and inhibiting the adhesion of strains of pathogens, being interesting for the development of a symbiotic supplement. In addition, the models with human intestinal cells demonstrated pathophysiological inflammatory responses observed in vivo, strengthening the hypothesis of this model as applicable in the exploration of different extracts with bioactive potential in different doses.
Keywords: Passiflora edulis, Caco-2 cells, anti-inflammatory activity, inflammatory bowel diseases, bacterial adhesion
LIST OF ILLUSTRATIONS
Figure 1.1 Intestinal barrier in physiological state. ... 28 Figure 1.2 Intestinal barrier in a pathological state, with loss of the intestinal barrier, absence of the mucus layer, irregular distribution and loss of function of the tigh junctions and dysbiosis ... 29 Figure 1.3 General chemical structure of a flavonoid: two aromatic rings (A and B) and an intermediate ring (C) ... 33
Figure 2.1 Schematic representation of the in vitro experimental system for epithelial barrier dysfunction and inflammation. ... 65 Figure 2.2 Viability of Caco-2 cells after treatments with different concentrations of P. edulis leaf extract for 24 h. ... 68 Figure 2.3 Effects of 3-h inflammatory stimuli on TER values (A), Papp (B), and IL-8 production (C) by Caco-2 cells ... 69 Figure 2.4 Effects of inflammatory stimuli and treatments on TER values (A), Papp (B), and IL-8 secretion in Caco-2 monolayers (C). ... 72
Figure 3.1 Design of cell culture experiments to evaluate the effect of aqueous extract of P. edulis on LPS-induced inflammation in Caco-2, RAW264.7, and co-culture. ... 84 Figure 3.2 Co-culture system constructed with Caco-2 cells and RAW264.7 cells ... 86 Figure 3.3 Cell viability (% of control cells) of Caco-2 (A) and RAW264.7 (B) cell lines after treatment with different concentrations of PELE. ... 87 Figure 3.4 Effect of PELE on cytokines and NO concentration in RAW264.7 cells. Levels of IL-6 (A), IL-8 (B), NO (C) ... 89 Figure 3.5 PELE reduces the expression of inflammatory mediators in Caco-2 monolayer. Levels of IL-6 (A), IL-8 (B), and NO (C). ... 91 Figure 3.6 PELE reduces the expression of inflammatory mediators in the co-culture system of Caco-2 and RAW264.7 cells. Levels of IL-6 (A), IL-8 (B), and NO (C).. ... 92
Figure 4.1 Adhesion of Lactobacillus rhamnosus - MB154 (A), Lactobacillus casei - MB151 (B), and Bifidobacterium lactis A. - BLC 1 (C) to the cultured Caco-2 cells monolayer after 1 h in the presence of 10 and 5 mg/mL of PELE. ... 113 Figure 4.2 Adhesion of Escherichia coli 11229 (A), Listeria monocytogenes (B), Salmonella enteritidis (C), and Salmonella typhimurium (D) to the cultured Caco-2 cells monolayer after 1 h in the presence of 10 and 5 mg/mL of PELE. ... 115
LIST OF TABLES
Table 1.1 Total phenolic content and antioxidant capacity of Passiflora edulis leaf extract...67
Table 2.1 Viability of Caco-2 cells after treatments with different concentrations of P. edulis leaf extract for 24 h. ... 111
ABBREVIATIONS AND ACRONYMS LIST
AAPH 2,2’-azobis(2-methylpropionamidine
ABS Absorbance
ANOVA Analysis of variance
ATCC American type culture collection AUC Area under curve
BUDE Budesonide
BSA Bovine serum albumin
CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico
COX Cyclooxygenase
COX-2 Cyclooxygenase-2
DMEM Dulbecco's Modified Eagle's medium DMSO Dimethyl sulphoxyde
DNA Deoxyribonucleic acid DPPH 2,2-difenil-1-picrilhidrazil DSS Dextran sulfate sodium
ELISA Enzyme Linked Immune Sorbent Assay ERO Espécies reativas de oxigênio
FBS Fetal bovine sérum
FL Fluorescein
FRAP Ferric-reducing antioxidant power
GA Gallic acid
GAE Gallic acid equivalente
HBSS Hank's Balanced Salt Solution HCl Hydrochloric acid
HEPES -(2-hydroxyethyl)-1-piperazineethanesulfonic acid IBDs Inflammatory bowel diseases
IFN-ϒ Interferon-ϒ
IKb Kinase
IKK Kinase complex
IL-6 Interleukin-6 IL-8 Interleukin-8 IL-10 Interleukin-10 IL-1β Interleukin-1β
iNOS Inducible nitric oxide synthase LPS Lipopolysaccharide
LY Lucifer yellow
MAPK Mitogen-activated protein kinase mRNA Messenger ribonucleic acid MRS Man-Rogosa-Sharpe broth
MRS* Man-Rogosa-Sharpe broth without glucose
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NC Cells with no inflammatory induction
NB Nutrient broth
NF-κB Fator nuclear kappa B
NO Nitric oxide
Papp Apparent permeability PBS Phosphate saline buffer
PC Cells with inflammatory induction PELE Passiflora edulis leaves extract
PELE 5 Passiflora edulis leaves extract at 5 mg mL-1
PELE 10 Passiflora edulis leaves extract at 10 mg mL-1
PGE2 Prostaglandin E2 pH Hydrogen potential RNA Ribonucleic acid
ROS Reactive oxigen species SD Standard deviation SDS Sodium dodecyl sulfate
TBARS Thiobarbituric acid reactive substances TE Trolox equivalente
TER Transepithelial electrical resistance
Th1 Lymphocyte Th1
Th17 Lymphocyte Th17
TLR Tool like receptor TLR-4 Toll like receptor 4 TNF-α Tumor necrosis fator TPTZ 4,6-tripryridyl-s-triazine
Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid UV/VIS Ultraviolet/visible
SUMARY
GENERAL INTRODUCTION ... 22
CHAPTER 1. LITERATURE REVIEW ... 25
1. Inflammatory Bowel Disease ... 25
1.1 Epidemiology ... 26
1.2 Etiology and Pathophysiology of Inflammatory Bowel Disease ... 26
1.3 Changes in Mucosa Permeability ... 27
1.4 Intestinal Microbiota ... 29
1.5 Intestinal Immune System ... 30
1.6 Free Radicals ... 30
1.7 Traditional therapy and new trends ... 31
1.8 Antioxidants in IBD ... 32
1.9 Phenolic compounds and their effects in IBD ... 33
1.10 Passion fruit (Passiflora edulis) and the role of its phenolics in IBD ... 34
1.11 Evaluation of phenolic extracts by in vitro IBD models ... 36
2. Conclusion ... 37
3. References ... 37
CHAPTER 2. PASSION FRUIT (PASSIFLORA EDULIS) LEAF AQUEOUS EXTRACT AMELIORATES INTESTINAL EPITHELIAL BARRIER DYSFUNCTION AND REVERTS INFLAMMATORY PARAMETERS IN CACO-2 CELLS MONOLAYER ... 59
1. Introduction ... 60
2. Materials and methods ... 61
2.1. Sample preparation ... 61
2.2. Sample characterization ... 62
2.2.1. Total phenolic content ... 62
2.2.2. DPPH radical-scavenging activity ... 62
2.2.4. Orac ... 63
2.3. Intestinal inflammation cell model ... 63
2.3.1. Cell culture ... 63
2.3.2. Determination of cell viability ... 63
2.3.3. Development of Caco-2 monolayer in transwell inserts ... 64
2.3.4. Transepithelial electrical resistance (TER) measurement ... 64
2.3.5. Determination of barrier integrity by transport of paracellular marker ... 64
2.3.6. Inflammatory stimuli and anti-inflammatory effect of P. edulis leaf extract ... 64
2.3.7. Determination of IL-8 secretion ... 66
2.4. Statistical analyses ... 66
3. Results and discussion ... 66
3.1. Total phenolic content and antioxidant potential of PELE ... 66
3.2. Effects of PELE on Caco-2 cells viability ... 67
3.3. Development of an inflamed Caco-2 epithelial barrier ... 68
3.4. Anti-inflammatory effect of PELE on IL-8 secretion and epithelial barrier integrity 70 4. Conclusions ... 72
5. References ... 73
CHAPTER 3. PASSION FRUIT (PASSIFLORA EDULIS) LEAF EXTRACT INHIBITS INFLAMMATORY RESPONSE IN IN-VITRO CELLULAR MODEL (INTESTINAL EPITHELIUM AND MACROPHAGES) ... 79
1. Introduction ... 80
2. Materials and methods ... 82
2.1. Chemicals ... 82 2.2. Sample preparation ... 82 2.3. Sample characterization ... 83 2.4. Cellular assay ... 83 2.4.1. Cell culture ... 83 2.4.2. Inflammatory assay ... 83
2.4.3.1. Caco-2 inflammatory assay ... 84
2.4.3.2. RAW264.7 inflammatory assay ... 85
2.4.3.3. Inflammatory assay of co-culture ... 85
2.4.4. Inflammatory parameters analysis ... 86
2.4.4.1. Determination of IL-8 and IL-6 sobrenadant secretion ... 86
2.4.4.2. Transepithelial electrical resistance (TER) measurement ... 86
2.4.4.3. Determination of NO levels ... 86
2.5. Statistical analyses ... 87
3. Results and discussion ... 87
5. Conclusion ... 95
6. References ... 95
CHAPTER 4. PASSIFLORA EDULIS EXTRACT EFFECTS ON PROBIOTIC AND PATHOGENIC MODULATION FOR HEALTHIER MICROBIOTA ... 105
1. Introduction ... 106
2. Materials and methods ... 108
2.1. Bacteria strains and growth condition ... 108
2.2. Sample preparation ... 108
2.3. Cell culture ... 108
2.4. Citoxicity of Passiflora edulis extract in Caco-2 cells ... 109
2.5. Bacterial adhesion assay... 109
2.6. Statistical analyses ... 110
3. Results ... 110
3.1. Effects of PELE on Caco-2 cells viability ... 110
3.2. Effects of PELE and on probiotic bacteria adhesion... 111
3.3. Effects of PELE and on pathogen bacteria adhesion ... 113
4. Discussion ... 115
5. Conclusions ... 118
GENERAL DISCUSSION ... 126
1. Sample characterization ... 126
2.1 Caco-2 monolayer inflammated permeability assay ... 126
2.2 Inflammatory assay with co-cultures of Caco-2 and RAW264.7 interactions ... 127
2.3 Effects of PELE and on probiotic bacteria adhesion in Caco-2 monolayers ... 129
GENERAL CONCLUSION ... 130
REFERENCES ... 132
GENERAL INTRODUCTION
Inflammatory Bowel Disease (IBD) includes, fundamentally, Crohn's Disease (CD) and Ulcerative Colitis (UC), both are chronic inflammatory diseases that affect the gastrointestinal tract, with periods of exacerbation followed by prolonged intervals of symptom remission. (LENNARD-JONES, 1989). They are diseases marked by ulceration and infiltration of neutrophils in the mucosa, discomfort or abdominal pain with altered bowel habits, such as diarrhea, constipation and weight loss. Inflammatory bowel disease is considered a major problem in the modern population, as it also affects people's quality of life in its social, psychological and professional aspects (GEBOES; ECTORS; D'HAENS; RUTGEERTS, 1998).
Although IBD has been the subject of research for several decades, its etiology is not fully known and a single agent or mechanism alone does not seem to be sufficient to produce or trigger the disease. The interaction of genetic and environmental factors (stress, dietary factors, use of contraceptive drugs and non-steroidal anti-inflammatory drugs, among others), in combination with the intestinal microbiota, trigger a mechanism that activates cells of immune and non-immune origin that make up the defense system of the intestinal mucosa, so that its etiology is considered multifactorial (MOLLER et al., 2015). The microorganisms present in the intestine play a key role in the regulation of the immune system, which is why they are considered very important in the development of IBD (ANDERSON, 1985).
Current treatments for IBD are designed to induce or keep the patient in remission and to mitigate the side effects of the disease. Among the drugs used are aminosalicylates, glucocorticoids, immunosuppressants and biological therapy, which is based on the association of corticosteroids, immunomodulators or anti-TNF-α (YAMAMOTO-FURUSHO, 2007; MORRISON et al., 2009; PITHADIA et al., 2011). The available therapy does not represent a cure for IBD and the use of available drugs is associated with several problems, such as serious side effects, such as glucocorticoids, high cost and low patient response to different treatments (JANI et al., 2002).
For these reasons, studies that seek plant extracts rich in bioactive compounds with positive effects in the control of the inflammatory process and represent hope in the prevention and as adjuvants in the treatment of the disease in a less aggressive way to the patient (CRESPO et al., 1999; CRUZ et al., 2001; DI STASI et al., 2004; BASTOS et al., 2008; FRUET et al., 2012).
The phenolic compounds present in certain foods are excellent natural antioxidants, which can improve the antioxidant status in vivo (KARP et al., 2006). In addition, these compounds may have, among other effects, anti-inflammatory action (LUCHINI et al., 2008). In this sense, the evaluation of different phenolic extracts, could assist in the identification of the best samples to promote the inhibition of inflammatory activity through the modulation of inflammatory mediators present in ulcerative colitis, as well as modulation of the microbiota, which would be another mechanism for the control and remediation of disease. Experimental models that make it possible to test a large number of plant extracts, with good correlation with the responses obtained in humans and without the use of animal experimentation, would be an important part of the search strategy for alternative therapies for the treatment of IBD.
Since 2010, our researchers in the area of Food Science and Nutrition at UNICAMP, have carried out studies that evaluate the antioxidant and anti-inflammatory activity of Passiflora edulis leaf extracts, in a model of colitis induced by trinitrobenzene sulfonic acid (TNBS) in rats. These studies demonstrated the therapeutic potential of the passion fruit leaf (Passiflora edulis), controlling oxidative stress and inflammation seen in inflammatory bowel disease (CAZARIN; DA SILVA; COLOMEU; BATISTA et al., 2014; CAZARIN; DA SILVA; COLOMEU; BATISTA et al., 2015).
Our project aims to establish models for evaluating aspects of IBD in vitro, with tests on mammalian cells, evaluating and comparing the responses obtained in this model to the action of the same leaf extract of Passiflora edulis, already successfully tested in models of IBD in animals. Cell models with human intestinal epithelial cells can be used to prospect for numerous extracts with bioactive potential, as well as adding important responses to those obtained in animal tests, strengthening the hypothesis that this extract is interesting in cases of intestinal inflammation.
The work presented in this thesis is organized in four chapters, the first chapter is based on a literature review, the second based on an article published in the Food Research International journal, entitled "Passion fruit (Passiflora edulis) leaf aqueous extract ameliorates intestinal epithelial barrier dysfunction and reverts inflammatory parameters in Caco-2 cells monolayer” (Appendix 1); the third based on the article: “Passion fruit (Passiflora edulis) leaf extract inhibits inflammatory response in in-vitro cellular model (intestinal epithelium and macrophages)", submitted to the Journal Toxicology in Vitro, which contains the results indicating the anti-inflammatory potential of the extract in the co-culture of Caco-2 and RAW264.7; and the fourth chapter, based on the article "Passiflora
edulis extract effects on probiotic and pathogenic modulation for healthier microbiota", published in the Nutrire Journal (Appendix 2), describing the potential effect on the adhesion of strains of pathogenic and probiotic bacteria from extract. Appendix 3 contains the register receipt on the SisGen Platform.
CHAPTER 1. LITERATURE REVIEW 1. Inflammatory Bowel Disease
Inflammatory Bowel Disease comprises two chronic and recurrent nosological entities: Crohn's Disease and Ulcerative Colitis (LENNARD-JONES, 1989). These two disorders have points of similarity, however, they differ in terms of different pathophysiological aspects and clinical manifestations (HENDRICKSON; GOKHALE; CHO, 2002).
Crohn's disease (CD) is a transmural inflammatory condition of the mucosa that can affect any region of the gastrointestinal tract (GIT), especially the terminal ileum and the perianal region. It typically presents in a discontinuous form, reaching several portions of the GIT and associated with complications, such as fistulas, strictures and abscesses (SHEPHERD, 1991). Unlike CD, Ulcerative Colitis is a disease characterized by diffuse inflammation of the colon mucosa. It affects the rectum (95% of cases) and the proximal portions of the colon, in a symmetrical and continuous manner, with no areas of normal mucosa between the affected parts (STROBER; JAMES, 1986).
Ulcerative Retocolitis (UC) can be classified according to its anatomical location and extension into proctitis (inflammation restricted to the rectum), distal colitis (inflammation encompasses the sigmoid colon with or without involvement of the descending colon) and pancolitis or total colitis, when inflammation involves the entire cervix (TRUELOVE; WITTS, 1955).
CD can be clinically described as mild, moderate and severe. Mild disease is characterized by the absence of severe abdominal pain, intestinal obstruction, weight loss and there are also no signs of systemic toxicity, which includes fever, tachycardia, anemia and an increase in the erythrocyte sedimentation rate. In moderate form there are symptoms of systemic toxicity, weight loss, pain and tenderness in the abdominal region, nausea and vomiting without intestinal obstruction or significant anemia. The severe form is characterized by high fever, cachexia, persistent vomiting, intestinal obstruction and abscess (HANAUER; SANDBORN, 2001).
Commonly, the symptoms of UC include hematoquey, rectal urgency and tenesmus, as well as CD can be characterized as mild (4 daily bowel movements with or without blood and absence of systemic toxicity), moderate (mixture of symptoms of mild and severe disease) and severe (bloody stools and more than 6 bowel movements per day,
abdominal tenderness and systemic toxicity). Patients with fulminating UCs typically evacuate more than 10 times a day, have anemia requiring transfusion, systemic toxicity, abdominal distension and tenderness (ROTHENBERGER; WONG; BULS; GOLDBERG et al., 1984).
1.1 Epidemiology
The epidemiology of inflammatory bowel disease is being analyzed in several studies and although trends for the onset of IBD are changing, certain demographic patterns appear to be similar. Incidence rates are higher in developed and more industrialized countries, such as Northeastern Europe, the United Kingdom and North America, indicating urbanization as a risk factor (MISRA; FAIZ; MUNKHOLM; BURISCH et al., 2018). However, in the last 20 years, this scenario is changing and developing countries are showing increasing rates of IBD incidence (ZUO; KAMM; COLOMBEL; NG, 2018).
Worldwide data indicate that the prevalence is in the range of 120- 200 cases of UC and 50-200 cases of CD for every 100,000 inhabitants; in the United States it is estimated that 1.4 million people have the disease (LICHTENSTEIN; SHAHABI; SEABURY; LAKDAWALLA et al., 2017).
In Brazil, few studies analyze the epidemiological aspects of IBD, with the majority only describing the clinical aspects and the frequency of admission to hospitals due to this disease, without any reference to the incidence or prevalence in the population. Although in Brazil these numbers are lower than the world rate, this disease is becoming increasingly important in our public and private health system (KARREMAN; LUIME; HAZES; WEEL, 2016).
Crohn's disease is more prevalent in women while Ulcerative Colitis is more common in men. In addition, CD and RCU are more frequent in Caucasian populations than in other ethnicities (COSNES; GOWER-ROUSSEAU; SEKSIK; CORTOT, 2011).
1.2 Etiology and Pathophysiology of Inflammatory Bowel Disease
Although IBD has been the subject of research for several decades, its etiology is not fully known and a single agent or isolated mechanism does not seem to be sufficient to produce or trigger the disease (NISHIDA; INOUE; INATOMI; BAMBA et al., 2018). The interaction of genetic and environmental factors (stress, dietary factors, use of contraceptive drugs and non-steroidal anti-inflammatory drugs, among others), in combination with the intestinal microbiota, triggers a mechanism that activates cells of immune and non-immune
origin that make up the defense system of the intestinal mucosa, so that its etiology is considered multifactorial (LODDO; ROMANO, 2015).
1.3 Changes in Mucosa Permeability
After many investigations about it, evidence supports that IBD is a multifactorial disorder of mucosal homeostasis, which leads to hyperresponsiveness of the innate and adaptive mucosal immune system (SUN; HE; WU; ZHOU et al., 2017). The first line of defense of the mucosal immune system is an epithelial barrier, which is protected by a layer of mucus, composed of glycoproteins (mucin), water, electrolytes, cellular macromolecules and microorganisms. Intestinal mucin is secreted by goblet cells, and any qualitative or quantitative change in this protective barrier can favor the attack of the epithelium, as well as important physiological changes (OKUMURA; TAKEDA, 2018).
The epithelial tissue of the gastrointestinal tract is an environment that is constantly attacked by these immunogens, which cause mild inflammation in healthy tissues. To maintain homeostasis, the gastrointestinal tract uses a colic epithelium with a mechanical barrier function, in addition to a commensal flora suppressing the inflammatory process and expression of antimicrobial peptides (BEVINS; SALZMAN, 2011).
The contact between potentially pathogenic organisms and the intestinal epithelium, results in the activation of mesenteric lymph nodes, lymphoid follicles in the inductive sites, epithelium and the lamina propria underlying the effector sites and release of cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-a). These cytokines induce the expression of adhesion molecules on the walls of vascular endothelial cells, which neutrophils, monocytes and lymphocytes will bind, allowing increased permeability. Other cytokines that participate in this process are interleukin-8 (IL-8) and interferon-g (IFNg), increasing chemotaxis for leukocytes and phagocytosis. All of these effects result in edema and accumulation of leukocyte cells in the affected areas (MCGEE; BAMBERG; VITKUS; MCGHEE, 1995).
The integrity of the protective barrier, which is exercised by the intestinal epithelium, favors the normal traffic of substances between the mucosa and the colic environment. The physical barrier formed by the intestinal epithelium is complemented by a well-evolved innate immune system, which has the function of defending against the invasion of pathogens, as well as controlling the inflammatory response inherent to the presence of the commensal microbiota (MALOY; POWRIE, 2011).
It is noted, through sigmoid colon biopsies of patients with mild stage CD, that there is a decrease in the structural capacity of this barrier, and a decrease in the number of intercellular junctions (tight junctions). Pro-inflammatory cytokines present in IBD, among other factors, can induce this type of defect in the intestinal barrier, as well as regulate gene expression and the processes of redistribution of intercellular junctions (LEE, 2015).
The intestinal barrier is established by a monolayer of polarized columnar epithelial cells, which are connected by intercellular junctions, which maintain the connection of cells and the separation of the luminal contents of the intestine with the intracellular medium (CAZARIN; DA SILVA; COLOMEU; BATISTA et al., 2015) (Figure 1.1).
Changes in this physical barrier can lead to changes in the immune response, observed in IBD, favoring the entry of substances present in the lumen, such as enteric bacteria and antigens, in the systemic circulation, promoting an overload of the immune system, which induces a response inflammatory (CAZARIN; DA SILVA; COLOMEU; BATISTA et al., 2015; CAZARIN; RODRIGUEZ-NOGALES; ALGIERI; UTRILLA et al., 2016) (Figure 1.2).
1.4 Intestinal Microbiota
The microorganisms present in the intestine play a key role in the regulation of the immune system, which is why they are considered very important in the development of IBD (RANGAN; CHOI; WEI; NAVARRETE et al., 2019). The mammalian gastrointestinal tract is colonized by a large number of microorganisms, and this microbiota is responsible for maintaining homeostasis and intestinal function. Experimental studies have shown that animals kept in pathogen-free conditions do not develop IBD, showing the importance of the intestinal microbiota in the pathogenesis of the disease (DANESE; FIOCCHI, 2006).
Microorganisms such as, for example, Listeria monocytogenes, Chlamydia tracomatis, Escherichia coli, Cytomegalovirus, Saccharomyces cerevisiae, and others are being studied as possible triggering agents of DII (LINSKENS; HUIJSDENS; SAVELKOUL; VANDENBROUCKE-GRAULS et al., 2001).
On the other hand, beneficial bacteria, such as Bifidobacterium longum, B. infantis, B. breve, Lactobacillus acidophilus, L. casei, L. delbrueckii spp. Bulgaricus, L. plantarum, Streptococcus salivarus spp. and S. thermophilus can normalize the physiological Figure 1.2 Intestinal barrier in a pathological state, with loss of the intestinal barrier, absence of the mucus layer, irregular distribution and loss of function of the tigh junctions and dysbiosis (Adapted from Cazarin et al., 2014).
function and the integrity of the intestinal barrier, in addition to increasing the integrity of the tight junctions between the epithelial cells. The balance between pathogenic and commensal bacteria in the intestine is a significant factor in the pathogenesis of IBD (CHAPMAN; GIBSON; ROWLAND, 2011).
1.5 Intestinal Immune System
Pattern recognition receptors (RRPs), such as Toll-like membrane receptors (TLR), recognize pathogens or pathogen-associated molecular patterns (PAMPs), and commensal microbiota by intestinal cells. It is possible that in chronic inflammation the expression of TLR is increased, mainly due to the presence of pro-inflammatory cytokines (PIERIK; JOOSSENS; VAN STEEN; VAN SCHUERBEEK et al., 2006).
One of the hypotheses raised is that pathogenic species cause damage to tissue through their extracellular matrix products, as well as cellular products such as nucleic acids, uric acid and mitochondrial components that are released and can be detected by means of various RRPs, thus activating an inflammatory response. This confirms the possibility that the blocking or deficiency of these receptors that recognize these cellular products may attenuate the inflammatory response (HENCKAERTS; PIERIK; JOOSSENS; FERRANTE et al., 2007).
1.6 Free Radicals
Oxidative stress is a potential etiological factor of both diseases, as the harmful effects of reactive oxygen (EROS) or nitrogen (ERN) molecules have been widely observed in inflammatory processes (SERRA; INCANI; SERRELI; PORRU et al., 2018). The imbalance between the production of free radicals and their removal by antioxidants is called oxidative stress (LOBO; PATIL; PHATAK; CHANDRA, 2010). The main free radicals observed in inflammatory bowel diseases are: nitric oxide (NO), superoxide (O2-), nitrite
peroxide (ONOO2), hydrogen peroxide (H2O2) and hypochlorite (OCl2), being neutrophils in
RCU and monocytes in CD, the main source for the production of these free radicals, especially after the disease is already installed (SIMMONDS; RAMPTON, 1993).
Studies indicate that free radicals may have immunomodulatory activity, such as O2- (superoxide) has been reported to mediate infiltration and accumulation of neutrophils at
the site of inflammation, in addition to being involved in the mobilization of arachidonic acid. H2O2 seems to be related to neutrophil chemo-attraction, leukocyte bearing, activation of T
lymphocytes, induction of angiogenesis and mobilization of arachidonic acid (GRISHAM; GRANGER, 1988).
In addition to the inflammatory process, it is possible to observe an increase in oxidative stress and a decrease in the endogenous antioxidant capacity of patients with IBD, especially among patients with active disease (KORDJAZY; MIRZAIAN; HAJ-MIRZAIAN; ROHANI et al., 2018; WANG; ZHOU; ZHANG; LEI et al., 2019).
Inflammatory bowel diseases are characterized by the presence of neutrophils and macrophages, which, when activated, release free radicals, which cause mucin destruction which allows bacteria to enter the intestinal lamina. This fact promotes the migration of more phagocytic cells and, consequently, the release of more reactive oxygen molecules, in addition to the release of toxins, which together cause the degradation of collagenase and hyaluronic acid. The degradation of these enzymes added to lipid peroxidation, caused by free radicals, act as the main responsible for the development of ulceration of the intestinal mucosa (YAMADA; GRISHAM, 1991).
Based on the above, it is easy to see that excessive production or lack of control in the regulation of free radicals is related to inflammatory diseases. Therefore, it is essential that cells and tissues have a defense system to control the levels of free radicals and prevent these inflammatory processes. For this, the cells have an antioxidant defense system (KRUIDENIER; VERSPAGET, 2002).
There is a growing interest in the application of compounds with antioxidant activity in IBD, since there is a decrease in the endogenous antioxidant response of the intestinal mucosa of IBD patients, which can contribute to both the pathogenesis and the perpetuation of the inflammatory process (HABTEMARIAM; BELAI, 2018; MILLAR; RAMPTON; CHANDER; CLAXSON et al., 1996). Thus, the search for new products with antioxidant, anti-inflammatory and immunomodulatory activity could represent an effective option in the prevention and treatment of IBD.
1.7 Traditional therapy and new trends
According to Mayer (2010), the inflammatory process can be divided into three phases called: initiation, phase of increase and phase of perpetuation or regulation. Taking this statement into account, the drugs currently developed, which aim to interact with the intestinal microflora or the mediating agents of the immune response, focus on the first two stages of the process, stimulating both the innate immune response and the adaptive immune response.
Defects in the regulation phase are intrinsic reflexes to changes in the adaptive immune response and, therefore, few drugs are developed to act at this stage.
Until the 1990s, the current treatments for IBD focused only on the remission of the symptoms of the disease, not acting on reducing inflammation and healing of the mucosa (DE CHAMBRUN; COLOMBEL; POULAIN; DARFEUILLE-MICHAUD, 2008). Studies to obtain new drugs, aiming at both reducing the inflammatory process and healing the mucosa, have intensified since the 1990s. Since then, the use of corticosteroids, aminosalicylates and their derivatives, as well as antibiotics and immunomodulatory drugs in treatment of IBD has intensified. As a result, reduction of the inflammatory process and healing of the mucosa were achieved satisfactorily. Other therapies have emerged from these studies, such as preparations with monoclonal antibodies, anti-sense nucleic acid drugs and therapies with integrin antibodies. Many of these drugs are already being used in the treatment of IBD, but they are associated with severe side effects (VERSTOCKT; FERRANTE; VERMEIRE; VAN ASSCHE, 2018).
1.8 Antioxidants in IBD
An antioxidant is defined as a substance that, in low concentrations, slows or prevents oxidation of the substrate. When the mechanism of action of the antioxidant occurs through its reaction with the free radical, the new radical formed must be stable and unable to propagate the reaction (HALLIWELL, 1990).
The hypothesis that the diet affects oxidative damage in vivo is based on the fact that foods provide both antioxidant substances, nutrients or non-nutrients, with the ability to fight free radicals, as well as oxidizable substrates (polyunsaturated fatty acids - PUFA) and traces of metals with catalytic action (Fe2+, Cu+). Thus, food can have positive and negative
effects on the balance between oxidative damage and antioxidant action (KABEL, 2014). This statement proves to be very simple given the complexity of the human organism's functioning. In addition, there are some gaps with regard to antioxidants, such as: the lack of a recommendation for each antioxidant, the lack of standardization as to the real antioxidant value of foods, and the possible toxic effects of administering high doses of antioxidants.
The category of food antioxidants includes carotenoids and vitamins E and C, with recognized antioxidant properties among non-nutrients, polyphenolic compounds are mentioned (DIMITRIOS, 2006).
1.9 Phenolic compounds and their effects in IBD
Plant phenolic compounds fall into several categories such as: simple phenols, phenolic acids (derived from benzoic and cinnamic acids), coumarins, flavonoids, stilbenes, condensed and hydrolyzable tannins, lignans and lignins (VERMERRIS; NICHOLSON, 2008).
The basic structure of a flavonoid can be seen in Figure 1.3.
The antioxidant activity of phenolic compounds is mainly due to their reducing properties and chemical structure. These characteristics play an important role in neutralizing or scavenging free radicals and chelating transition metals, acting both in the initiation stage and in the propagation of the oxidative process. The intermediates formed by the action of phenolic antioxidants are relatively stable due to the resonance of the aromatic ring present in the structure of these substances (HEIM; TAGLIAFERRO; BOBILYA, 2002).
Researches have been directed to evaluate the effect of phenolic compounds in the inflammatory response, and in this study, we are interested in knowing the action potential of phenolic compounds in intestinal inflammation.
Varilek et al., (2001) observed that the ingestion of green tea polyphenols decreased the activity of inflammatory disease and the production of INF-ɣ and TNF-α in IL-2-/- mice, demonstrating the effects of the bioactive compounds present in green tea in the
experimental model of spontaneous development of IBD. Decreased oxidative stress and inflammation were also observed by Denis et al., (2013) using polyphenols extracted from apple peel in in vitro tests, using Caco-2 cells.
Another polyphenol, ellagic acid (10-20 mg/kg), found in several fruits such as pomegranate and raspberries, promoted a decrease in the severity and extent of intestinal Figure 1.3 General chemical structure of a flavonoid: two aromatic rings (A and B) and an intermediate ring (C) (Adapted from Rosa et al., 2007).
lesions, a decrease in cell infiltrate, an increase in mucus production, in addition to a protective effect by through the inhibition of the mitogen-activated protein kinase (MAPK) and NFκB signaling pathways in a TNBS-induced model of TNBS (trinitrobenzene sulfonic acid), (ROSILLO et al., 2011).
Polyphenols, such as the quercetin flavonoid (AMASHEH et al., 2008), kaempferol (SUZUKI et al., 2011), genistein (WELLS et al., 1999), naringenin (AZUMA et al., 2013), among others, demonstrated an effect in the restoration of the intestinal barrier, by increasing the expression of proteins from tight junctions.
1.10 Passion fruit (Passiflora edulis) and the role of its phenolics in IBD
Passion fruit is a popular name given to several species of the genus Passiflora, this being the largest and most important of the Passifloraceae family. There are 500 species distributed in regions of tropical and subtropical climate of the globe, Brazil being its biggest producer with more than 79 species (DHAWAN et al., 2004), with an annual production of 923 thousand tons of fruits, with emphasis on the regions northeast and southeast producers (IBGE, 2012). Passion fruit Passiflora edulis known as sour or yellow passion fruit is the most produced and commercialized (ZERAIK et al., 2010), representing the species, present in 95% of the orchards (BRUCKNER et al., 2001).
Despite the economic and social importance for the country, the cultivation of passion fruit is still hampered due to phytosanitary problems that result in limited yields. The average yield is 22 to 28 tonnes/hectare, and can reach 50 tonnes/hectare, when producers make use of improved seeds, production technology and appropriate cultural treatments (AGRIANUAL, 2014).
The pulp and also the fruit by-products such as peel, leaves and seeds, although little studied, have characteristics of technological and biological interest (MARTÍNEZ et al., 2012). More than 75% of this waste can be transformed into an ingredient with bioactive properties for health promotion, based on the extraction of phenolic compounds (ARVANITOYANNIS, 2008). In addition, in recent years, awareness of environmental conservation and actions involving the fight against poverty and misery has grown worldwide, as well as in Brazil. Thus, seeking solutions to reduce food waste and the use of agro-industrial waste is an alternative for preserving the environment, as well as collaborating in the solution of some economic and social issues.
Passion fruit is known in popular medicine due to its pharmacological properties: antioxidants, sedatives, anti-inflammatory, antispasmodic, anxiolytic, antitumor and
antifungal properties attributed to compounds present in fruits, peels, seeds and leaves (CORRÊA et al., 2016; SCHUMACHER et al., 2015; SILVA, et al., 2015; ZERAIK et al., 2010)
Studies referring to the chemical composition of different Passiflora species show alkaloids, flavonoids, saponins, steroids, lignans, fatty acids, tannins, cyanogenic glycosides (SEIGLER, 1975), maltol (DHAWAN et al., 2001), and phenolic glycosides (CHASSAGNE et al., 1997).
In the review by Zeraik et al., (2010), the authors describe the main pharmacological studies and related to this fruit. The antioxidant activity of the fruits is attributed to polyphenols, mainly to flavonoids. The passion fruit peels, previously considered to be agro-industrial residues, are now used in animal and human food, as they are rich in soluble fibers, mainly in pectin. In addition, dried passion fruit peel flour is also used as a blood glucose reducer. Seeds, in turn, are good sources of essential fatty acids, used in the food and cosmetic industries, such as linoleic acid (ω-6) (55-66%), oleic acid (18-20%) and palmitic acid (10-14%) (ZERAIK et al., 2010). The toxicological aspects of passion fruit are associated with the presence of cyanogenic glycosides, mainly prunasin, substances that produce hydrocyanic acid (HCN) as a product of its hydrolysis. The authors also describe the nutritional properties of passion fruit juice attributed to sugars, fibers, organic acids, amino acids, carotenoids, vitamins, volatile substances, flavonoids and alkaloids (ZERAIK et al., 2010).
Cazarin et al., (2015) evaluated in their study the anti-inflammatory activity of the aqueous extract of P. edulis leaves in healthy mice induced by colitis by TNBS (trinitrobenzene sulfonic acid), observing that the oral ingestion of the leaf extract Passiflora edulis significantly improved the status of endogenous antioxidants and decreased lipid peroxidation in serum, liver and colon. The consumption of P. edulis extract decreased the level of pro-inflammatory cytokines in the colon tissue, especially through the reduction of IL-1β and TNF-α, compared to the control group induced to colitis and not supplemented with the extract. The authors therefore conclude that P. edulis leaf extract has therapeutic activity in rats and in TNBS-induced colitis, in particular, controlling oxidative stress and inflammation observed in this disease. Another research carried out by this same group of researchers showed that the ingestion of passion fruit tea leaves promoted a significant growth of colon bacteria of rats that consumed the tea in relation to the non-supplemented ones, the latter also presented higher levels of lipid peroxidation in the liver. In relation to the group that received the tea, the animals obtained increased levels of GSH in the kidneys, and
reduced activity of SOD in the liver and brain, when compared to controls. The same study also concludes that the aqueous extract of P. edulis leaf may be an option to increase the supply of antioxidants and protect against oxidative stress, which, as previously mentioned, may be involved in the etiology of IBD (DA SILVA et al., 2013).
1.11 Evaluation of phenolic extracts by in vitro IBD models
The co-culture system with Caco-2 and RAW 264.7 cells has been used successfully in studies of intestinal inflammation (MULLIN et al., 2005; TANOUE et al., 2008).
According to Tanoue et al., (2005), the in vitro model, standardized by the authors, is representative of inflammatory bowel disease in humans. In his model, a three-dimensional co-culture system with human intestinal epithelial cells, Caco-2, and murine macrophages, RAW264.7, was developed. To induce the inflammatory stimulus, RAW 264.7 cells were treated with lipopolysaccharide (LPS) from E. coli 0127, which responded to the stimulus by secreting TNF-α and IL-8. The mucosa integrity of the Caco-2 monolayer was assessed by measuring the transepithelial electrical resistance. The authors conclude that this co-culture system provides a model of relevant intestinal inflammation to evaluate the anti-inflammatory effects of different types of phenolic compounds, encouraging the refinement and replacement of models in vivo.
Leonard et al., (2010) established a cell model of intestinal inflammation using a co-culture of human intestinal epithelial cells (Caco-2, HT-29 and T84) and cells obtained from primary human cultures, such as macrophages and derived dendritic cells of human peripheral blood. Different combinations of proinflammatory stimuli were used (E. coli lipopolysaccharide, Salmonella typhimurium, interleukin-1β and interferon-γ). The results showed that Caco-2 cells were responsive to different pro-inflammatory stimuli with a positive regulation of these markers and showed a reduction in transepithelial electrical resistance, indicating changes in the properties of the epithelium barrier. The three-dimensional co-culture model using Caco-2 cells (apical membrane), and dendritic cells and macrophages (basolateral culture), showed a good pathophysiological response to the stimuli suffered, since the responses to the pro-inflammatory compounds were superior in this model when compared to the Caco-2 monolayer single culture model, demonstrating the stimulation of immunocompetent cells (apical membrane). This cellular interaction provides greater proximity to the model in vivo with patients.
The authors state that the three-dimensional cell model they present mimics the pathophysiological changes that affect patients with inflammatory bowel diseases, also claim that this co-culture model using different cell lines provides greater complexity and information compared to the basic cell model, in which a single cell type is stimulated. Thus, the model proposed by Leonard and collaborators represents an important tool in the development of formulations for the treatment of inflammatory bowel diseases (Leonard, et al., 2010).
2. Conclusion
Since current conventional therapies focus their effects on inducing remission, as they have several side effects that contraindicate many maintenance drugs, an auxiliary measure with improvement in the inflammatory condition and intestinal disorders of patients with IBD would be a good therapy strategy in the attempt to improve the quality of life of these individuals.
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