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UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE ENGENHARIA DE ALIMENTOS
RAFAELA DA SILVA MARINELI CAMPOS
EVALUATION OF CHIA SEED AND OIL (SALVIA HISPANICA L.) POTENTIAL IN
THE PREVENTION AND TREATMENT OF OBESITY AND COMORBIDITIES
INDUCED BY HIGH FAT AND HIGH CARBOHYDRATE DIET IN VIVO
AVALIAÇÃO DO POTENCIAL DA SEMENTE E DO ÓLEO DE CHIA (SALVIA
HISPANICA L.) NA PREVENÇÃO E NO TRATAMENTO DA OBESIDADE E
COMORBIDADES INDUZIDAS POR DIETA HIPERLIPÍDICA E HIPERGLICÍDICA
IN VIVO
CAMPINAS
2016
RAFAELA DA SILVA MARINELI CAMPOS
EVALUATION OF CHIA SEED AND OIL (SALVIA HISPANICA L.) POTENTIAL IN
THE PREVENTION AND TREATMENT OF OBESITY AND COMORBIDITIES
INDUCED BY HIGH FAT AND HIGH CARBOHYDRATE DIET IN VIVO
AVALIAÇÃO DO POTENCIAL DA SEMENTE E DO ÓLEO DE CHIA (SALVIA
HISPANICA L.) NA PREVENÇÃO E NO TRATAMENTO DA OBESIDADE E
COMORBIDADES INDUZIDAS POR DIETA HIPERLIPÍDICA E HIPERGLICÍDICA
IN VIVO
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 Nutrition applied to
Food Technology.
Tese apresentada à Faculdade de Engenharia de Alimentos
da Universidade Estadual de Campinas como parte dos
requisitos exigidos para obtenção do título de Doutora em
Alimentos e Nutrição, na Área de Nutrição Experimental e
Aplicada à Tecnologia de Alimentos.
Prof. Dr. MÁRIO ROBERTO MARÓSTICA JUNIOR
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA POR RAFAELA DA SILVA MARINELI CAMPOS E ORIENTADA PELO PROF. DR. MÁRIO ROBERTO MARÓSTICA JUNIOR.
CAMPINAS
2016
Agência(s) de fomento e nº(s) de processo(s): FAPESP, 2012/23813-4
Ficha catalográficaUniversidade Estadual de Campinas
Biblioteca da Faculdade de Engenharia de AlimentosClaudia Aparecida Romano - CRB 8/5816
Campos, Rafaela da Silva Marineli, 1986- C157e CamEvaluation of chia seed and oil (Salvia Hispanica L.) potential in the
prevention and treatment of obesity and comorbidities induced by high fat andhigh carbohydrate diet in vivo / Rafaela da Silva Marineli Campos. – Campinas,SP : [s.n.], 2016.
CamOrientador: Mario Roberto Marostica Junior. CamTese (doutorado) – Universidade Estadual de Campinas, Faculdade de
Engenharia de Alimentos.
Cam1. Chia. 2. Ácidos graxos ômega-3. 3. Obesidade. 4. Estresse oxidativo. 5.
Resistência à insulina. I. Marostica Junior, Mario Roberto. II. UniversidadeEstadual de Campinas. Faculdade de Engenharia de Alimentos. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Avaliação do potencial da semente e do óleo de chia (SalviaHispanica L.) na prevenção e no tratamento da obesidade e comorbidades induzidas pordieta hiperlipídica e hiperglicídica in vivoPalavras-chave em inglês:ChiaOmega-3 fatty acidsObesityOxidative stressInsulin resistanceÁrea de concentração: Nutrição Experimental e Aplicada à Tecnologia de AlimentosTitulação: Doutora em Alimentos e NutriçãoBanca examinadora:Mario Roberto Marostica Junior [Orientador]Júlia Laura Delbue BernardiMariana Battaglin Villas Boas AlvaroRenato GrimaldiStanislau Bogusz JuniorData de defesa: 16-05-2016Programa de Pós-Graduação: Alimentos e Nutrição
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COMISSÃO EXAMINADORA
Prof. Dr. Mário Roberto Maróstica Junior (Orientador)
Faculdade de Engenharia de Alimentos – UNICAMP
Prof. Dr. Renato Grimaldi (Titular)
Faculdade de Engenharia de Alimentos – UNICAMP
Prof. Dr. Stanislau Bogusz Junior (Titular)
Instituto de Química de São Carlos – USP
Prof. Dra. Júlia Laura Delbue Bernardi (Titular)
Faculdade de Nutrição – PUC CAMPINAS
Prof. Dra. Mariana Battaglin Villas Boas Alvaro (Titular)
Faculdade de Nutrição - UNIP
Prof. Dr. Marcelo Alexandre Prado (Suplente)
Faculdade de Engenharia de Alimentos – UNICAMP
Prof. Dra. Elisa de Almeida Jackix (Suplente)
Faculdade de Nutrição – PUC CAMPINAS
Prof. Dra. Glaucia Maria Pastore (Suplente)
Faculdade de Engenharia de Alimentos – UNICAMP
A Ata de defesa com as respectivas assinaturas dos membros encontra-se no
processo de vida acadêmica do aluno
AGRADECIMENTOS
A Deus, pela vida e tudo que sou.
À minha Mãe, pelo amor, incentivo e apoio incondicional.
Ao meu pai pela educação e empenho à minha formação.
Ao meu marido, pelo amor, apoio e paciência.
À minha irmã, pelo apoio e admiração.
Ao Prof. Dr. Mário Roberto Maróstica Jr, pela orientação e por todo ensinamento e
estímulo ao desenvolvimento das minhas habilidades e competências para a
pesquisa.
Às minhas queridas amigas e parceiras nestes anos de trabalho, Érica Aguiar
Moraes e Sabrina Alves Lenquiste, por todos os momentos vividos, pelas alegrias
das conquistas, pelo ombro amigo nos momentos mais difíceis, pelas longas
conversas, viagens, e principalmente pela amizade construída. Sentirei saudades!
À Dra. Soely Maria Pissini Machado Reis, pelo apoio técnico, ajuda, profissionalismo
e amizade durantes todos esses anos.
À Maria Susana Corrêa Alves da Cunha por todo auxílio técnico indispensável
durante os ensaios biológicos.
À aluna de Iniciação Científica, Marcela Perez, pelo estágio prestado e ajuda
durante o desenvolvimento deste trabalho.
Ao Laboratório do Pâncreas Endócrino do Instituto de Biologia, em especial
Everardo Magalhães Carneiro e seus alunos pela assistência nas análises
desenvolvidas em parceria.
Ao Laboratório de Óleos e Gorduras, em especial ao Prof. Dr. Renato Grimaldi pelos
ensinamentos e assistência nas análises dos óleos.
Ao Prof. Dr. Stanislau Bogusz Junior, pelos ensinamentos, apoio, dedicação e por
todo auxílio técnico nas análises desenvolvidas em parceria.
À Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP pela
concessão do auxílio financeiro.
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pela
bolsa de estudos concedida.
Aos membros da Banca Examinadora pelas sugestões e críticas convenientes ao
aprimoramento do Trabalho.
Aos amigos do grupo de pesquisa ―Compostos Bioativos, Nutrição e Saúde‖, pelos
momentos alegres e difíceis convividos.
A todos os colegas e funcionários do Departamento de Alimentos e Nutrição que
contribuíram de forma importante para a realização desse Trabalho.
Aos amigos e família que de uma forma ou de outra estavam sempre presentes e
interessados, incentivando o meu crescimento profissional e torcendo pelo meu
sucesso.
À Universidade Estadual de Campinas (UNICAMP), pela oportunidade de realizar
este sonho.
A todos que contribuíram para a execução de mais esta etapa em minha vida.
― Feliz aquele que transfere o que sabe e aprende o que ensina ‖
(Cora Coralina)
― Não tenha medo do caminho, tenha medo de não caminhar ‖
(Augusto Cury)
" Ninguém é tão grande que não possa aprender, nem tão pequeno que não possa
ensinar "
(Esopo)
RESUMO
A diversidade de compostos bioativos presentes nos alimentos e sua ampla
aplicação terapêutica justificam a busca por conhecimentos relacionados às
propriedades fisiológicas ou funcionais dos mesmos. A chia (Salvia hispanica L.) é
uma antiga planta herbácea, com alto teor de óleo, principalmente ácido graxo α-
linolênico, proteína, fibra dietética e outros compostos bioativos. Ela tem sido
recentemente estudada por sua importante composição nutricional, possível
aplicação em alimentos, potencial funcional e por seus efeitos benéficos à saúde. O
objetivo deste trabalho foi avaliar os efeitos da ingestão da semente e do óleo de
chia sobre mecanismos fisiológicos e bioquímicos na prevenção e no tratamento da
obesidade e comorbidades, induzida por dieta em modelo animal. Foi realizado um
ensaio biológico, com 60 ratos machos Wistar, divididos em 6 grupos (n=10): Grupo
1, controle magro alimentado com dieta padrão AIN-93M; grupo 2, controle obeso
alimentado com dieta hiperlipídica (35%) e adicionada de frutose (20%) (HFF); grupo
3 e 4: animais alimentados com dieta HFF adicionada de semente de chia em longo
(12 semanas) e curto (6 semanas) tempo; grupo 5 e 6: animais alimentados com
dieta HFF adicionada de óleo de chia em longo (12 semanas) e curto (6 semanas)
tempo. Foram avaliados: ingestão alimentar, ganho de peso, peso de tecidos
adiposos e órgãos, perfil lipídico e hormonal séricos, perfil de ácidos graxos
plasmático, marcadores inflamatórios séricos, conteúdo de lipídeo hepático e fecal,
resistência à insulina, tolerância à glicose, estresse oxidativo, peroxidação lipídica,
capacidade antioxidante plasmática e hepática; assim como a caracterização
química e a capacidade antioxidante das matérias-primas. Os dados foram
submetidos à análise de variância ANOVA e as médias comparadas pelo teste T
e/ou Tukey, com limite de significância de P ≤ 0,05. A semente de chia apresentou
alta concentração de óleo, proteína, fibra dietética, ácidos graxos ômega-3,
compostos bioativos e importante capacidade antioxidante in vitro. A ingestão da
dieta obesogênica (HFF) induziu a obesidade, resistência à insulina, intolerância à
glicose, dislipidemia, inflamação, estresse oxidativo e peroxidação lipídica nos
animais. A adição da semente e do óleo de chia, como substituição do óleo de soja
nas dietas experimentais, não reverteu a obesidade, mas normalizou o estado
inflamatório sérico, a dislipidemia, a intolerância à glicose, a resistência à insulina,
aumentou a concentração plasmática dos ácidos graxos da família ômega-3, α-
linolênico (ALA), eicosapentaenoico (EPA) e docosahexaenoico (DHA), reduziu a
razão dos ácidos graxos n-6/n-3, reduziu a peroxidação lipídica e o estresse
oxidativo, e aumentou a capacidade antioxidante no plasma e no fígado de animais
obesos induzidos por dieta. Os efeitos benéficos foram encontrados tanto no período
de prevenção (12 semanas) quanto de tratamento (6 semanas). Visto que os
produtos estudados têm comprovado efeito biológico e potencial funcional e por isso
podem beneficiar o estado de saúde da população, seja via suplementação ou por
enriquecimento de alimentos, o trabalho de pesquisa em questão forneceu subsídios
à introdução destes compostos na dieta habitual da população, como coadjuvante
na prevenção e no controle de desordens metabólicas crônicas, incentivando
pesquisas clínicas a este respeito.
Palavras-chave: Chia, Salvia hispanica, ácidos graxos ômega-3, obesidade,
estresse oxidativo, resistência à insulina, peroxidação lipídica, inflamação.
ABSTRACT
The diversity of bioactive compounds present in foods and its therapeutic application
justify the search for knowledge related to its physiological or functional properties.
Chia (Salvia hispanica L.) is an ancient herbal plant with high oil content, specially α-
linolenic fatty acid, protein, dietary fiber and bioactive compounds. It has been
recently studied for its important nutritional composition, possible application in food,
functional potential and for its beneficial effects on health. The aim of this study was
to evaluate the effects of chia seed and oil intake on physiological and biochemical
mechanisms in the prevention and treatment of obesity and comorbidities in diet-
induced animal model. A biological assay was done using 60 male Wistar rats, which
were divided into 6 groups (n = 10): Group 1, lean control fed with AIN-93M diet;
Group 2, obese control fed with high-fat (35%) and high-fructose diet (20%) (HFF);
Group 3 and 4: HFF diet added with chia seed in short (6-weeks) and long (12-
weeks) treatments; Group 5 and 6: HFF diet added with chia oil in short (6-weeks)
and long (12-weeks) treatments. Food intake, weight gain, adipose tissues and
organs weight, hormonal and lipid profile, plasma fatty acid profile, serum
inflammatory markers, hepatic and fecal lipids content, insulin resistance, glucose
tolerance, oxidative stress, lipid peroxidation, plasma and hepatic antioxidant
capacity, as well as the chemical and antioxidant capacity of raw materials were
evaluated. Data was submitted to analysis of variance ANOVA and means compared
by Tukey and/or T test. The limit of significance was set at P ≤ 0.05. Chia seed
showed a high content of oil, protein, dietary fiber, omega-3 fatty acids, bioactive
compounds and important antioxidant capacity in vitro. HFF diet intake induced
obesity, insulin resistance, glucose intolerance, dyslipidemia, inflammation, oxidative
stress and lipid peroxidation in animals. Addition of chia seed and oil, as a
replacement for soybean oil on the experimental diets did not reverse obesity, but
normalized serum inflammatory state, dyslipidemia, glucose intolerance, insulin
resistance, increased plasma omega-3 fatty acid concentration, α-linolenic acid
(ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduced n-
6/n-3 ratio, reduced lipid peroxidation and oxidative stress, and increased plasma
and hepatic antioxidant capacity in diet-induced obese rats. Beneficial effects were
found in both prevention (12 weeks) and treatment (6 weeks) periods. Since the
products studied have proven biological effect and functional potential, and therefore
can benefit population health status, either through supplementation or food
fortification, the present research provided subsidies to the introduction of these
compounds in the usual diet of the population as an aid in the prevention and control
of chronic metabolic disorders, encouraging clinical research in this regard.
Key words: Chia, Salvia hispanica, omega-3 fatty acids, obesity, oxidative stress,
insulin resistance, lipid peroxidation, inflammation.
LISTA DE ILUSTRAÇÕES
INTRODUÇÃO
Fluxograma resumido das atividades do trabalho .................................................... 22
REVISÃO BIBLIOGRÁFICA
Figura 1. Esquema relacionando obesidade, alterações na secreção de adipocinas e
desenvolvimento da resistência à insulina ............................................................... 26
Figura 2. Vias metabólicas da biossíntese de PUFA a partir de ácidos graxos
insaturados da dieta ................................................................................................. 33
Figura 3. Esquema dos efeitos benéficos e mecanismos moleculares em diferentes
tecidos a partir da ingestão da semente de chia (Salvia hispanica L.) in vivo ......... 47
ARTIGO I
Figure 1. GC chromatogram (fatty acid profile) of chia oil (Salvia hispanica L.) ….. 84
Figure 2. TAG fingerprints obtained by EASI(+)-MS of chia oil (Salvia hispanica L.)
………………………………………………………………………………………….....… 85
Figure 3. Polyphenols fingerprints obtained by EASI(-)-MS of chia seed extract and
chia oil (Salvia hispanica L.) ………………………………....….................................. 88
ARTIGO II
Figure 1. Total food intake, total energy intake and total weight gain of rats fed on
different diets …………………………………………….…………………………...... 109
Figure 2. Plasma antioxidant enzymes: catalase (CAT) activity, superoxide
dismutase (SOD) activity, glutathione peroxidase (GPx), glutathione reductase
(GRd), and Thiol groups (GSH) in plasma of rats fed on different diets ………... 111
Figure 3. Liver antioxidant enzymes: catalase (CAT) activity, superoxide dismutase
(SOD) activity, glutathione peroxidase (GPx), glutathione reductase (GRd) and Thiol
groups (GSH) in liver of rats fed on different diets …………………………..……... 113
Figure 4. Lipid peroxidation by 8-isoprostane assay in plasma and TBARS assay in
plasma and liver of rats fed on different diets ……………………………………..... 115
Figure 5. Ferric Reducing Antioxidant Power (FRAP) assay in plasma and liver of
rats fed on different diets ………………………………………………………..…….. 116
ARTIGO III
Figure 1. Glucose AUC during intraperitoneal glucose tolerance test (iGTTAUC),
Mean blood glucose levels after intraperitoneal infusion of glucose solution, KITT
during intraperitoneal insulin tolerance test, Mean blood glucose levels after
intraperitoneal infusion of insulin in rats fed on different diets ……………………... 141
Figure 2. Pancreatic islet glucose-stimulated insulin secretion at 2.8 mmol/L and 16.7
mmol/L glucose in rats fed on different diets …………………………..……………...142
Figure 3. Total cholesterol (TC), HDL-c, LDL-c and Triacylglycerol (TAG) in serum of
rats fed on different diets ………………………………………………….....................143
Figure 4. Leptin, Insulin, Ghrelin, Adiponectin, TNF-α, MCP-1 and C-reactive protein
in serum of rats fed on different diets …………………….........................................145
LISTA DE TABELAS
REVISÃO BIBLIOGRÁFICA
Tabela 1. Estudos realizados em animais com a semente e óleo de chia (Salvia
hispanica L.) ............................................................................................................. 49
Tabela 2. Estudos realizados em humanos com a semente de chia (Salvia hispanica
L.) ............................................................................................................................. 55
ARTIGO I
Table 1. Proximate composition of Chilean chia seed (Salvia hispanica L.) (g/100 g)
…............................................................................................................................... 81
Table 2. Fatty acid profiles of Chilean chia oil (Salvia hispanica L.) ....................... 83
Table 3. Main TAG ions detected by EASI-MS in chia oil (Salvia hispanica L.) ….. 85
Table 4. Values of peroxide index and malondialdehyde of chia seed and chia oil
(Salvia hispanica L.) ………………………………………………………………….….. 86
Table 5. Identified phenolic compounds in chia seed and oil (Salvia hispanica L.)
analyzed by EASI-MS …………………………………………….……………………… 88
Table 6. Antioxidant activity of Chilean chia seed (Salvia hispanica L.) …..……….. 89
ARTIGO II
Table 1. Ingredients and fatty acid composition of experimental diets ……….…. 104
ARTIGO III
Table 1. Experimental diets ingredients and fatty acid composition .......................134
Table 2. Food and energy intake, weight gain, organs weight and metabolic
parameters of rats fed on different diets ..................................................................139
Table 3. Plasma fatty acid composition (%) of rats fed on different diets .…………147
SUMÁRIO
1. INTRODUÇÃO GERAL .........................................................................................19
2. REVISÃO BIBLIOGRÁFICA ..................................................................................24
2.1 OBESIDADE: ASPECTOS GERAIS ...............................................................24
2.1.1 Obesidade: tecido adiposo, inflamação e resistência à insulina ..............25
2.1.2 Obesidade e estresse oxidativo ...............................................................29
2.2 ÁCIDOS GRAXOS ÔMEGA-3..........................................................................31
2.3 CHIA (SALVIA HISPANICA L.) ........................................................................35
2.3.1 Origem, histórico e produção ...................................................................35
2.3.2 Composição química e valor nutricional ..................................................37
2.3.3 Influência da adição de chia em alimentos ..............................................41
2.3.4 Efeitos biológicos da semente e do óleo de chia .....................................44
REFERÊNCIAS .........................................................................................................57
CAPÍTULO I ...............................................................................................................71
Artigo I: Chemical characterization and antioxidant potential of Chilean chia seeds
and oil (Salvia hispanica L.) ………………………………………………….…...………72
Abstract ..............................................................................................................73
Introduction .........................................................................................................74
Materials and Methods .......................................................................................75
Results and Discussion ......................................................................................80
Conclusions ........................................................................................................90
References .........................................................................................................91
CAPÍTULO II ..............................................................................................................97
Artigo II: Antioxidant potential of dietary chia seed and oil (Salvia hispanica L.) in diet-
induced obese rats ……………..................................................................................98
Abstract ..............................................................................................................99
Introduction …...................................................................................................100
Materials and Methods .....................................................................................101
Results ..............................................................................................................108
Discussion ........................................................................................................117
References .......................................................................................................122
CAPÍTULO III ...........................................................................................................128
Artigo III: Dietary intake of chia seed and oil (Salvia hispanica L.) normalizes
dyslipidemia, inflammation and insulin resistance in diet-induced obese rats ….…129
Abstract ............................................................................................................130
Introduction .......................................................................................................131
Materials and Methods .....................................................................................132
Results ..............................................................................................................138
Discussion ........................................................................................................149
Conclusions ......................................................................................................154
References …....................................................................................................155
DISCUSSÃO GERAL ..............................................................................................160
CONCLUSÃO GERAL .............................................................................................168
REFERÊNCIAS........................................................................................................170
ANEXOS...................................................................................................................176
Anexo A.........................................................................................................176
Anexo B.........................................................................................................177
Anexo C.........................................................................................................178
18
INTRODUÇÃO GERAL
19
1. INTRODUÇÃO GERAL
O aumento da expectativa de vida aliado às mudanças dos hábitos
alimentares da população e à incidência cada vez maior de doenças crônicas, como
obesidade, hipertensão, dislipidemias, doenças cardiovasculares, diabetes e câncer,
levou a maior preocupação por parte da população e dos órgãos públicos de saúde
na promoção de práticas alimentares saudáveis. O papel da alimentação equilibrada
em prol da saúde vem despertando o interesse da comunidade científica, que tem
produzido diversos estudos com o intuito de comprovar a influência de determinados
alimentos na redução do risco de doenças crônicas não transmissíveis (DCNT)
(SWINBURN et al., 2011; GORTMAKER et al., 2011).
Os alimentos funcionais têm sido, nos últimos anos, discutidos em
eventos científicos de âmbito nacional e internacional, sendo de interesse crescente
para os consumidores e indústrias de alimentos. A diversidade de compostos
bioativos presentes nos alimentos e a ampla aplicação terapêutica destes, justificam
a busca por conhecimentos relativos às suas propriedades fisiológicas ou funcionais
(TRIGUEROS et al., 2013).
A Salvia hispanica L., cujo nome comum é chia, é uma antiga planta
herbácea, que pertence à família Lamiaceae, nativa do sul do México e norte do
Guatemala (COATES; AYERZA, 1996) que tem sido recentemente comercializada
como uma cultura na América do Sul (AYERZA; COATES, 2011). Seu consumo
pode ocorrer na forma de sementes inteiras, farinha, mucilagem e óleo de sementes.
O renascimento do interesse na semente de chia é devido a sua composição
nutricional, principalmente ao seu alto teor de óleo (25-39%), sendo uma fonte rica
em ácidos graxos poli-insaturados (PUFA) da família ômega-3, com a maior
concentração de α-linolênico (ALA) dentre as fontes botânicas conhecidas
(AYERZA, 1995). As sementes também são ótimas fontes de proteína e fibras
dietéticas, solúveis e insolúveis (WEBER et al, 1991; REYES-CAUDILLO et al.,
2008; VÁZQUEZ-OVANDO et al., 2009; CAPITANI et al., 2012). Além disso, também
tem sido identificada a atividade antioxidante da chia, possivelmente devido à
20
presença de compostos fenólicos, fitosteróis e tocoferóis (REYES-CAUDILLO et al.,
2008; ÁLVAREZ-CHÁVEZ et al., 2008; VÁZQUEZ-OVANDO et al., 2009; IXTAINA et
al., 2011; CAPITANI et al., 2012; MARTÍNEZ-CRUZ; PAREDES-LÓPEZ, 2014).
Estudos mostram que é possível utilizar o óleo de chia, ou seus
derivados, para a alimentação de animais, com o objetivo de obtenção de produtos
(como ovos, leite e carnes) enriquecidos em PUFA e consequente melhor valor
nutricional (AYERZA; COATES, 2002; AYERZA et al. 2002; COATES, AYERZA,
2009). Além disso, pesquisas em modelos animais (AYERZA; COATES, 2005, 2007;
ESPADA et al., 2007; CHICCO et al., 2009; POUDYAL et al., 2012) e em humanos
(VUKSAN et al. 2007, 2010) têm demonstrado os efeitos fisiológicos benéficos a
partir da ingestão das sementes ou óleo de chia, principalmente relacionadas à ação
deste composto na melhora de marcadores relacionados à dislipidemia, inflamação,
doenças cardiovasculares, homeostase da glicose, resistência à insulina, esteatose
hepática e composição corporal, sem a presença de efeitos adversos.
Dessa forma, as propriedades físico-químicas e funcionais da fração
lipídica das sementes de chia, assim como de seus subprodutos, motivam o uso
destes como ingredientes na indústria de alimentos. Considerando a atual tendência
para um aumento do consumo de alimentos funcionais e o alto potencial nutricional
e tecnológico da semente de chia e seus subprodutos, estes também podem ser
utilizados para produção de ingredientes funcionais com aplicações comerciais em
alimentos (REYES-CAUDILLO et al., 2008; ÁLVAREZ-CHÁVEZ et al., 2008;
SEGURA-CAMPOS et al., 2013).
Visto que o alimento citado tem comprovado efeito biológico e por isso
pode beneficiar o estado de saúde da população, seja via suplementação ou por
enriquecimento de alimentos, o objetivo desta tese de doutorado foi investigar os
possíveis efeitos causados pela ingestão da semente e do óleo de chia (Salvia
hispanica L.) na prevenção e no tratamento da obesidade e comorbidades induzidas
por dieta hiperlipídica e adicionada de frutose em modelo animal. A partir disso,
foram enfocados neste trabalho, os seguintes objetivos específicos:
Determinou-se a composição centesimal das sementes de chia e das
dietas experimentais;
21
Determinou-se a composição de ácidos graxos da semente, do óleo de
chia e das dietas experimentais;
Avaliou-se o potencial antioxidante das sementes e do óleo de chia por
métodos in vitro.
Identificou-se os compostos fenólicos da semente e óleo de chia;
Avaliou-se e monitorar a ingestão dietética e ganho de peso dos animais;
Determinou-se o perfil lipídico sérico (colesterol total, lipoproteína de alta
densidade (HDL), lipoproteína de baixa densidade (LDL), triglicérides e ácidos
graxos livres) e hormonal sérico (insulina, leptina, adiponectina e grelina) dos
animais;
Quantificou-se os marcadores inflamatórios séricos (proteína
quimioatrativa de macrófagos-1 (MCP-1), fator de necrose tumoral α (TNF-α),
proteína C-reativa (PCR)) nos animais;
Determinou-se o perfil de ácidos graxos no plasma dos animais;
Determinou-se o grau de resistência à insulina e intolerância à glicose pela
glicemia, Teste de Tolerância à Glicose intraperitoneal (ipGTT) e Teste de
Tolerância à Insulina intraperitoneal (ipITT), nos animais;
Avaliou-se a capacidade secretória de insulina de ilhotas isoladas in vitro
do pâncreas dos animais;
Determinou-se o perfil oxidativo e a capacidade antioxidante no plasma e
no fígado dos animais a partir dos marcadores de oxidação lipídica, da
atividade de enzimas antioxidantes e do ensaio de poder antioxidante redutor
de ferro (FRAP, do inglês ―Ferric Reducing Antioxidant Power‖).
22
Segue abaixo, um Fluxograma resumido das atividades do trabalho.
23
REVISÃO BIBLIOGRÁFICA
24
2 REVISÃO BIBLIOGRÁFICA
2.1 OBESIDADE: ASPECTOS GERAIS
A obesidade é uma doença crônica de origem multifatorial, que pode ser
definida como o acúmulo excessivo de gordura corporal, desenvolvida pela interação
entre fatores comportamentais, sociais, fisiológicos, psicológicos e genéticos
(ROMERO; ZANESCO, 2006; FERNÁNDEZ-SÁNCHEZ et al., 2011). O excesso de
peso está associado a efeitos negativos sobre a longevidade e a qualidade de vida,
além de favorecer o desenvolvimento de outras complicações, incluindo a Diabetes
Mellitus tipo 2 (DM2), doenças cardiovasculares, dislipidemias, alguns tipos de
câncer e outros problemas de saúde que estão relacionados às altas taxas de
morbidade e mortalidade (FERNÁNDEZ-SÁNCHEZ et al., 2011; SWINBURN et al.,
2011).
A prevalência da obesidade vem aumentando consideravelmente nos últimos
anos e tornou-se um dos maiores problemas de saúde pública mundial. Dados das
Estatísticas Mundiais de Saúde de 2012 demonstraram que a obesidade é a causa
de morte de 2,8 milhões de pessoas por ano (WHO, 2012). Baseado em dados de
diversos países, a Organização Mundial de Saúde relata que a prevalência de
obesidade mais do que duplicou entre 1980 e 2014, sendo que no ano de 2014, 39%
da população adulta estava em sobrepeso e 13% da população mundial foi
considerada obesa (WHO, 2015). A epidemia da obesidade representa não somente
uma ameaça para a saúde da população, mas também um fardo substancial para
muitos sistemas de saúde referente às doenças associadas, que impõem elevados
custos médicos de tratamento e perdas de produtividade (WANG et al., 2011).
As mudanças, demográficas, culturais e socioeconômicas ocorridas
permitiram alterações nos padrões alimentares. Por isso, nas últimas décadas,
verifica-se um processo de transição nutricional no Brasil, caracterizado pela
diminuição progressiva da desnutrição e aumento da obesidade (SCHMIDT et al.,
2011). O aumento do consumo energético, principalmente de alimentos
processados, com elevado teor de açúcares simples e gorduras saturadas,
25
associado ao menor gasto energético atribuído à redução da atividade física,
explicam a tendência crescente de sobrepeso e obesidade na população brasileira,
e das DCNT associadas (SCHMIDT et al., 2011; SWINBURN et al., 2011).
2.1.1 Obesidade: tecido adiposo, inflamação e resistência à insulina
A obesidade está associada a um estado crônico de inflamação de baixo
grau, caracterizado pela produção anormal de citocinas e a ativação de vias de
sinalização inflamatórias no tecido adiposo branco (HOTAMISLIGIL, 2006). O tecido
adiposo é um órgão endócrino que tem papel fundamental na regulação da
homeostase metabólica através da secreção de várias adipocinas biologicamente
ativas com diferentes funções e efeitos em tecidos periféricos e centrais
(FERNÁNDEZ-SÁNCHEZ et al., 2011).
A expansão do tecido adiposo na obesidade leva ao aumento da infiltração de
macrófagos e marcadores biológicos da inflamação, com aumento da produção de
citocinas pró-inflamatórias, como o fator de necrose tumoral α (TNF-α), interleucina-6
(IL-6), inibidor de ativador do plasminogênio-1 (PAI-1), proteína quimioatrativa de
macrófagos-1 (MCP-1), acompanhado por um aumento na liberação de ácidos
graxos livres (AGL) e secreção desregulada de leptina, adiponectina, resistina e
proteína 4 ligadora de retinol (RBP4). Em conjunto, estas substâncias derivadas de
adipócitos e macrófagos podem atuar de forma autócrina ou parácrina para
exacerbar a inflamação no tecido adiposo. Em nível sistêmico, a secreção alterada
de adipocinas pode levar ao aumento da ingestão de alimentos e redução do gasto
energético por meio de ações no hipotálamo e decréscimo da sensibilidade à
insulina no músculo e no fígado através do aumento da deposição de lipídeos e
inflamação (GALIC, OAKHILL, STEINBERG, 2010) (Figura 1).
26
Figura 1. Esquema relacionando obesidade, alterações na secreção de
adipocinas e desenvolvimento da resistência à insulina. Adaptado de Galic et al.
(2010).
A adiponectina é uma proteína de 247 aminoácidos, secretada pelo tecido
adiposo. Esse hormônio desempenha, em seres humanos e animais, um papel
importante na regulação da obesidade, homeostase energética, modulação do
metabolismo de glicose e lipídios em tecidos sensíveis à insulina, protegendo contra
desordens metabólicas e inflamação, e também aumenta a ação da insulina,
promovendo proteção à resistência deste hormônio. Ao contrário da maioria das
adipocinas, a expressão e os níveis séricos de adiponectina são negativamente
correlacionados com a gordura corporal e circunferência da cintura, estando
diminuídos em indivíduos obesos (LAGO et al., 2009; VÁZQUEZ-VELA; TORRES;
TOVAR, 2008).
27
A leptina é um hormônio sintetizado pelos adipócitos e secretado no plasma,
que regula o apetite e o gasto de energia, via sistema hipotalâmico. A quantidade de
leptina produzida e sua concentração plasmática são dependentes do conteúdo de
gordura corporal e da ingestão de alimentos, estando aumentada no período pós-
prandial. Seus efeitos também se estendem ao metabolismo lipídico, com aumento
da lipólise e inibição da lipogênese, além do seu papel inflamatório. Os níveis de
leptina têm sido associados positivamente com a quantidade de gordura corporal,
sendo que uma redução no peso corporal pode conduzir a uma redução nos níveis
desse hormônio, porém no estado clínico da obesidade, a resistência à este
hormônio também tem sido relatada (ROMERO; ZANESCO, 2006; VÁZQUEZ-VELA;
TORRES; TOVAR, 2008; LAGO et al., 2009).
A grelina, outra adipocina composta por 28 aminoácidos, sintetizada e
secretada principalmente pelas células oxínticas da mucosa do estômago, tem sido
estudada quanto a sua ação reguladora sobre a secreção de insulina e metabolismo
de glicose, além das funções biológicas relacionadas ao controle do apetite e da
fome. Estudos em modelos animais indicam que esse hormônio orexígeno,
desempenha importante papel na sinalização dos centros hipotalâmicos que
regulam a ingestão alimentar e o balanço energético (ERDMANN et al., 2004;
NAKAZATO et al., 2001).
O TNF-α é uma potente citocina que induz a produção de IL-6 e está
envolvido na resposta inflamatória sistêmica, além de ter efeitos sobre o transporte
de glicose, metabolismo de lipídeos, e ação da insulina. Tem sido relatado que, em
indivíduos obesos e em modelos animais, os níveis de TNF-α e IL-6 estão
persistentemente elevados, e uma redução da massa adiposa leva a uma redução
nos níveis de expressão desses marcadores que são relacionados com o
desenvolvimento da resistência à insulina, obesidade e diabetes (KERN et al., 2001;
GALIC, OAKHILL, STEINBERG, 2010). Além disso, o TNF-α pode também modular
a secreção de leptina, aumentando a expressão de genes e os níveis circulantes, da
mesma forma que o TNF-α e IL-6 são potentes inibidores da expressão e secreção
de adiponectina (LAGO et al., 2009; GALIC, OAKHILL, STEINBERG, 2010).
A insulina é um hormônio polipeptídico anabólico produzido pelas células β
pancreáticas, cuja síntese e secreção são ativadas pelo aumento dos níveis
28
circulantes de glicose e aminoácidos após as refeições. Este hormônio é
responsável por diversos efeitos metabólicos relacionados aos carboidratos, lipídeos
e proteínas e age diretamente em vários tecidos periféricos, incluindo músculo,
fígado, tecido adiposo e sistema nervoso central. No hipotálamo, este hormônio
desempenha função destacada no controle da ingestão alimentar, atuando de
maneira anorexigênica (XU et al, 2005; TILG; MOSCHEN, 2006).
Durante a obesidade, também há um aumento de AGL circulantes,
principalmente saturados, que são liberados pelo tecido adiposo e favorecem o
desenvolvimento da esteatose hepática não alcoólica, inflamação e lipotoxicidade
(FERNÁNDEZ-SÁNCHEZ et al., 2011; HELLMANN et al., 2013). Os AGL também
são tóxicos para as células pancreáticas que são sensíveis à oxidação e induzem
alterações na liberação de insulina, podendo levar à resistência à insulina e
consequentemente ao desenvolvimento da DM2 (FERNÁNDEZ-SÁNCHEZ et al.,
2011).
A resistência à insulina pode ser definida como um fenômeno biológico no
qual há uma diminuição da capacidade da insulina endógena ou exógena em
estimular a utilização celular de glicose, e assim manter as respostas metabólicas a
este hormônio (DIAS; SANTOS; COUTINHO, 2009). Entretanto, a resistência à
insulina tem como mecanismo compensador a hiperinsulinemia, resultante da
capacidade adaptativa das células β, que mantêm a homeostase glicêmica. Porém,
quando há falência nestas células, surge então a intolerância à glicose e,
posteriormente, o estabelecimento da DM2 (OLEFSKY; GLASS, 2010). Esse quadro
pode ser um fator agravante ou causal na obesidade.
Os receptores ativados por proliferadores de peroxissoma (PPAR) são
receptores de ácidos graxos que regulam a expressão dos genes, que por sua vez,
afetam a homeostase energética e as funções imunes. Foram identificadas 3
isoformas desse receptor nuclear, PPAR α, PPAR β e PPAR γ1 e γ2, as quais são
codificadas por genes diferentes e expressas de forma distinta em diversos tecidos,
mas todas envolvidas na regulação do metabolismo de lipídeos e glicose, na
diferenciação celular, bem como no desenvolvimento do câncer e no controle da
resposta inflamatória. Basicamente, os PPARs α e β estão envolvidos no
metabolismo de lipídeos e glicose e o PPAR γ está envolvido na diferenciação de
29
adipócitos, sendo considerado o principal ativador de genes lipogênicos e importante
fator de transcrição envolvido na adipogênese (ATAROD; KEHRER, 2004;
VÁZQUEZ-VELA, TORRES, TOVAR, 2008).
Dessa forma, a produção e secreção de adipocinas em excesso ou
deficiência, influenciam significativamente a sensibilidade à insulina, metabolismo da
glicose e lipídeos, inflamação e arteriosclerose, podendo fornecer uma ligação
molecular entre o aumento da adiposidade e o desenvolvimento de DM2, resistência
à insulina, e doenças cardiovasculares (GALIC, OAKHILL, STEINBERG, 2010).
2.1.2 Obesidade e estresse oxidativo
Os radicais livres são moléculas altamente reativas com elétrons
desemparelhados em sua última camada, que se ligam rapidamente a outras
moléculas e desempenham importante papel na defesa e na regulação da
homeostase de oxirredução do organismo (HALLIWELL; GUTTERIDGE, 2007).
Espécies reativas de oxigênio (EROs) são encontradas em todos os sistemas
biológicos, formadas a partir do metabolismo celular aeróbio. Dentre as principais,
podemos destacar: ânion-radical superóxido (O2•–), peróxido de hidrogênio (H2O2),
radicais peroxila (RO2•), alcoxila (RO•), oxigênio singlete (1O2) e radical hidroxila
(•OH), sendo esse último reconhecido por ser um dos mais reativos e mais lesivos
dentre os radicais livres conhecidos (VASCONCELOS et al., 2007).
Neste contexto, inserem-se as espécies reativas de nitrogênio (ERNs) que
também são radicais livres de destaque. Baixas concentrações de radicais livres
(EROs e ERNs) são importantes para manter o estado redox normal das células,
função tecidual e o processo de sinalização intracelular. Em contrapartida, o excesso
de radicais livres pode danificar as macromoléculas, como lipídeos, proteínas e
DNA, e comprometer a função celular (YU, 1994). Dentre os prejuízos causados
pelas ERONs, destaca-se a peroxidação lipídica, que ocorre geralmente a partir do
ataque do radical livre a ácidos graxos poli-insaturados, que são parte estrutural das
membranas celulares. A peroxidação lipídica leva a uma modificação das
características físico-químicas das membranas celulares, com alteração de sua
fluidez e permeabilidade, provocando risco de ruptura e consequente morte celular
(VASCONCELOS et al., 2007; HALLIWELL, 1994).
30
O combate ao dano oxidativo é mediado endogenamente pelo sistema
antioxidante enzimático, composto basicamente pelas enzimas superóxido
dismutase (SOD), glutationa redutase (GRd), glutationa peroxidase (GPx) e catalase,
que são responsáveis pela remoção ou inativação das ERONs produzidas em
condições fisiológicas ou anormais, como doenças. Além da proteção endógena, o
organismo conta com os antioxidantes alimentares, como as vitaminas C e E, os
carotenóides, os polifenóis e alguns minerais que possuem a propriedade de
prevenir e amenizar os danos celulares causados pelos radicais livres (HALLIWELL,
1994; YU, 1994; SIES, 1991).
O desequilíbrio entre agentes pró-oxidantes e antioxidantes é chamado de
estresse oxidativo, que se caracteriza pela capacidade insuficiente dos sistemas
biológicos em neutralizar a produção excessiva de radicais livres, que pode ser
induzido principalmente por fatores ambientais. O estresse oxidativo tem sido
implicado na etiologia de diversas doenças crônicas, tais como a aterosclerose,
doenças cardiovasculares, diversos tipos de câncer, obesidade, diabetes, resistência
à insulina e doenças neurodegenerativas (KULKARNI; KUPPUSAMY; PARINANDI,
2007; SIES, 1991; PANDEY; RIZV, 2010).
Tem sido relatado que a obesidade pode induzir o estresse oxidativo
sistêmico que, por sua vez, está associado com uma produção irregular de
adipocinas e citocinas pró-inflamatórias, contribuindo para o desenvolvimento da
síndrome metabólica (FERNÁNDEZ-SÁNCHEZ et al., 2011). A alteração de
citocinas junto ao estado de hiperglicemia e elevação dos níveis de AGL são
estimuladores potentes da produção de oxigênio e nitrogênio reativos, sendo,
portanto, responsáveis pelo aumento do estresse oxidativo, resistência à insulina e
inflamação no tecido adiposo (KING; LOEKEN, 2004).
Patel e colaboradores (2007) demonstraram que uma dieta com alta
densidade de gordura saturada e carboidratos induz um aumento significativo no
estresse oxidativo e inflamação em indivíduos com obesidade. Estudos têm
demonstrado que o consumo de dietas com alta densidade de açúcares afeta
negativamente o balanço entre a defesa antioxidante e a produção de radicais livres
em animais, levando ao aumento da suscetibilidade à peroxidação lipídica
(BUSSEROLLES et al., 2002; REDDY et al., 2009). Segundo Feillet-Coudray e
31
colaboradores (2009), em animais obesos há uma elevada produção de espécies
reativas de oxigênio, e esse dano oxidativo é um importante iniciador da patogênese
da diabetes e outras complicações.
A obesidade também compromete o sistema de defesa antioxidante a partir
da redução das enzimas antioxidantes, como SOD, GPx e catalase, além de afetar o
sistema antioxidante não enzimático, como vitaminas, minerais, polifenois, entre
outros. A sensibilidade dos biomarcadores do dano oxidativo, assim como o nível de
peroxidação lipídica estão aumentados em indivíduos obesos e se correlacionam
diretamente com o índice de massa corporal (IMC), percentual de gordura corporal,
oxidação do LDL-colesterol, concentrações sanguíneas de triacilgliceróis (TG) e
colesterol. Em contraste, os marcadores de defesa antioxidante estão reduzidos de
acordo com a quantidade de gordura corporal e de obesidade central (FERNÁNDEZ-
SÁNCHEZ et al., 2011).
2.2 ÁCIDOS GRAXOS ÔMEGA-3
Os lipídeos dietéticos estão entre os compostos bioativos que têm recebido
atenção especial, em termos quantitativos e qualitativos, como modulador importante
da morbidade e mortalidade de doenças relacionadas com o estilo de vida. Os
ácidos graxos com duas ou mais duplas ligações são denominados ácidos graxos
poli-insaturados (PUFA), os quais têm sido reportados por fornecer benefícios
adicionais para a saúde humana, além de suas funções básicas, como produção de
energia, produção hormonal, sinalização, e parte da estrutura da membrana celular
(TRIGUEROS et al., 2013; GANESAN; BROTHERSEN; MCMAHON, 2014).
Estudos epidemiológicos descrevem correlações entre o tipo de lipídeo
consumido na dieta, a composição de ácidos graxos no tecido adiposo, a
concentração destes no sangue e a incidência de certas doenças metabólicas
(WARENSJÖ et al., 2006). Dietas ricas em ácidos graxos saturados aumentam a
resistência à insulina e a incidência de doenças cardiovasculares, principalmente
devido a sua implicação na inflamação, pela indução de citocinas pró-inflamatórias,
estresse oxidativo, liberação de AGL, alteração de lipídeos circulantes, dislipidemia,
entre outros. Por sua vez, aquelas ricas em alguns ácidos graxos mono e poli-
32
insaturados protegem contra o desenvolvimento dessas doenças e complicações
(QUEIROZ et al., 2009; GANESAN; BROTHERSEN; MCMAHON, 2014).
Os seres humanos podem sintetizar os ácidos graxos saturados e
monoinsaturados, mas não podem sintetizar os PUFA da família ômega-3 (também
designado por ω-3 ou n-3) e ômega-6 (também designado por ω-6 ou n-6). Isto
porque os seres humanos como outros animais, não possuem as enzimas
necessárias para produzir os ácidos graxos α-linolênico (ALA) e linoleico (LA), sendo
assim, considerados essenciais, devendo ser fornecidos pela dieta (TAPIERO et al.,
2002).
O ácido linoleico (18:2 n-6, 9,12-octadecadienoico, LA) é metabolicamente
convertido em ácido araquidônico (20:4 n-6, 5,8,11,14-eicosatetraenoico, AA). Já o
ácido α-linolênico (18:3 n-3, cis-9,12,15-octadecatrienoico, ALA), é precursor de
importantes ácidos graxos da família n-3, como o eicosapentaenoico (20:5 n-3, cis-
5,8,11,14,17-eicosapentaenoico, EPA) e docosahexaenoico (22:6 n-3, cis-
4,7,10,13,16,19-docosahexaenoico, DHA), os quais são formados no organismo a
partir da ação de enzimas dessaturases e alongases (ALABDULKARIM et al., 2012)
(Figura 2). Porém, a eficiência da conversão de ALA em EPA e de ALA em DHA é
baixa e estimada em cerca de 8% a 21% e de 0% a 9%, respectivamente, sendo
bastante variável e modulada por diversos processos, incluindo estado fisiológico,
dieta, idade e sexo. Tem sido observado um nível maior de conversão em mulheres,
e esta diferença atribuída à possível influência do estrogênio sobre a atividade das
enzimas dessaturases (BURDGE, 2004; GANESAN; BROTHERSEN; MCMAHON,
2014).
O AA, da família n-6, é precursor de eicosanoides pró-inflamatórios, como
prostaglandina E2, tromboxano A2 e leucotrienos da série 3 e 4. Enquanto, os ácidos
graxos n-3 são responsáveis pela formação de precursores eicosanoides anti-
inflamatórios, como tromboxano A3, prostaciclina I3, prostaglandinas 3 e leucotrienos
da série 5, os três primeiros via cicloxigenase e os últimos via lipoxigenase
(ALABDULKARIM et al., 2012; MARTIN et al., 2006). Tanto os prostanóides como os
leucotrienos agem de forma autócrina e parácrina, influenciando inúmeras funções
celulares que controlam mecanismos fisiológicos e patológicos no organismo
(SMITH, 1992). O LA e ALA competem pelas mesmas enzimas de conversão para
33
formação de AA e EPA, respectivamente. Da mesma forma, o EPA compete com o
AA via cicloxigenase na formação dos eicosanoides, e produz inibição da agregação
plaquetária, estimula a vasodilatação e apresenta propriedade anti-inflamatória
(ALABDULKARIM et al., 2012) (Figura 2). Embora o ALA seja o substrato preferido
pela delta-6 dessaturase, o excesso de LA típico de dietas dos seres humanos e
animais biológicos conduz a uma maior conversão de LA em comparação ao ALA,
assim como, de seus principais derivados (AA e EPA) (BURDGE, 2004).
Figura 2. Vias metabólicas da biossíntese de PUFA a partir de ácidos graxos
insaturados da dieta. Setas contínuas indicam reações enzimáticas individuais, setas
tracejadas indicam múltiplos processos enzimáticos, e setas cheias apontam para a
ação biológica dos compostos. PG = prostaglandinas, LT = leucotrienos. Adaptado
de Ganesan et al. (2014).
34
Em função dessas diferenças fisiológicas tem-se proposto que a produção
excessiva de prostanóides da série 2, advindos de ácidos graxos da família n-6, está
relacionada com a ocorrência de desordens imunológicas, doenças cardiovasculares
e inflamatórias, sendo recomendado aumentar a ingestão de ácidos graxos n-3 para
elevar a produção de prostanóides da série 3 (SIMOPOULOS, 2004).
Os ácidos graxos n-3 são essenciais para o crescimento e desenvolvimento
normal do organismo, e podem desempenhar função importante no tratamento
adjuvante de doenças cardíacas, hipertensão, dislipidemia, e DM2 (SIMOPOULOS,
1999; WU et al., 2012; GANESAN; BROTHERSEN; MCMAHON, 2014). Esses
ácidos graxos são reguladores da expressão de genes envolvidos no metabolismo
de lipídeos e glicose e na adipogênese, atuando via PPARs (JUMP, 2002).
O DHA é necessário em altos níveis no cérebro e na retina como um nutriente
fisiologicamente essencial para garantir ótimas funções neurais e visuais, já que
possui efeito benéfico para acuidade visual e cognição, assim como para o
desenvolvimento cerebral e neural, principalmente durante a gestação e os primeiros
anos de vida (GANESAN; BROTHERSEN; MCMAHON, 2014). Também têm sido
relatados, por diversos estudos epidemiológicos, os benefícios da ingestão de DHA
e EPA na prevenção e no tratamento de doenças cardiovasculares, fatores de risco
associados, bem como de outras doenças crônicas. Os efeitos cardiovasculares
protetores têm sido atribuídos à capacidade de afetar favoravelmente vários fatores
de risco para doença cardiovascular, incluindo propriedades anti-inflamatória,
antitrombótica, antiarrítmica, vasodilatadora, e de redução de lipídeos sanguíneos
(SIMOPOULOS, 1999; QUEIROZ et al., 2009).
Evidências epidemiológicas e clínicas sugerem que um maior consumo de
ALA também está associado com a redução do risco de doenças cardiovasculares,
dislipidemia, inflamação, resistência à insulina e diabetes (DE LORGERIL et al.,
1994; ALBERT et al., 2005; Wu et al., 2012), bem como com a redução da
lipogênese e aumento da β-oxidação de ácidos graxos (IDE, 2000; QUEIROZ et al.,
2009).
O ALA é essencialmente de origem vegetal, presente em diversas sementes,
nozes, e em óleos vegetais, como linhaça, chia, canola, soja, linho e vegetais
folhosos verdes escuros; enquanto o EPA e DHA são encontrados em algas,
35
animais marinhos, óleo de peixes e peixes, devido à ingestão de algas marinhas
(ALABDULKARIM et al., 2012). O LA está presente em alimentos de origem animal,
como leite, carne e ovos, e em óleos vegetais, como cártamo, girassol e soja
(MARTIN et al., 2006). Nos países industrializados, a dieta da maior parte da
população apresenta baixo teor de PUFA n-3, principalmente devido ao baixo
consumo de pescados e ao uso excessivo de óleo vegetal rico em LA, estando a
razão dos ácidos graxos n-6:n-3 em torno de 20-25:1. O equilíbrio entre os ácidos
graxos n-6 e n-3 é alterado no caso de doenças crônicas, sugerindo que as
alterações neste equilíbrio se correlacionam com maior risco de doenças. Dessa
forma, a proporção recomendada dos ácidos graxos n-6:n-3 na dieta humana é de 5-
1:1 (SIMOPOULOS, 2004; 2011; GANESAN; BROTHERSEN; MCMAHON, 2014).
Diante disso, faz-se necessário encontrar alternativas nutricionais capazes de
evitar o surgimento e progressão da obesidade e de DCNT, tais como os compostos
bioativos presentes naturalmente em alimentos e que possuem importante ação
biológica.
2.3 CHIA (SALVIA HISPANICA L.)
2.3.1 Origem, histórico e produção
A Salvia hispanica L., cujo nome comum é chia, é uma planta herbácea anual,
que pertence à família Lamiaceae, nativa do sul do México e norte do Guatemala
(COATES; AYERZA, 1996). A chia, juntamente com o milho, o feijão e o amaranto,
era um componente essencial na dieta de civilizações pré-colombianas da América
Central, incluindo as populações maias e astecas (SAHAGUN, 1989).
Além da importância como fonte de alimento básico, o óleo extraído das
sementes possuía vários usos artísticos e todas as partes da planta eram utilizadas
como ingredientes em várias receitas astecas medicinais. Relatam-se também
algumas propriedades curativas das sementes, como por exemplo, para o
tratamento de obstruções dos olhos, infecções, problemas de pele, doenças
respiratórias e gastrointestinais. O uso culinário da chia se destinava as sementes
inteiras, farinha, mucilagem e óleo de sementes. As sementes embebidas em água
36
ou suco de frutas foram e ainda são consumidas em algumas regiões como uma
bebida refrescante (CAHILL, 2003).
Nos últimos anos a chia vem sendo cultivada comercialmente no México,
Bolívia, Argentina, Equador, Guatemala, Peru e Colômbia, e o período e época do
seu cultivo variam de acordo com a região (AYERZA; COATES, 2005; MUÑOZ et al.,
2013). Embora as plantas sejam pouco tolerantes a geadas, podem ser cultivadas
em estufas em algumas partes da Europa. A chia tem sido, recentemente,
comercializada na América do Sul, onde é considerada como uma cultura alternativa
para ajudar na diversificação e estabilização da economia local, em regiões áridas e
semi-áridas, onde a disponibilidade de água é a principal limitação para a produção
de culturas e plantio de oleaginosas (COATES; AYERZA, 1996). Apesar de ser uma
cultura antiga, a chia tem sido investigada como uma "nova" cultura devido aos
séculos de seleção e adaptação de cultivo a que foi submetida. Além disso, nos
últimos anos, a chia e seus derivados estão disponíveis comercialmente nas
Américas, Austrália, Europa e Sudeste asiático (MOHD ALI et al., 2012).
No Brasil, as regiões do oeste Paranaense e noroeste do Rio Grande do Sul
começaram a investir no cultivo de chia nas últimas safras, apresentando bons
resultados, apesar de ser uma cultura pouco explorada no país (MIGLIAVACCA et
al., 2014).
A planta produz pequenas sementes brancas e escuras que amadurecem no
outono. A sua forma é oval, medindo 2,0 mm x 1,5 mm. A maioria dos cultivares de
chia atualmente contém uma baixa porcentagem de sementes brancas. Em geral,
existe uma pequena diferença de tamanho entre estas sementes, sendo as brancas
um pouco maiores do que as negras (AYERZA; COATES, 2005). Além disso,
existem algumas diferenças no teor de proteína e de ácidos graxos entre as
sementes escuras e brancas (IXTAINA et al., 2008).
Além disso, os resultados do cultivo comercial de sementes de chia
proveniente de três países (Bolívia, Equador e Argentina) também indicam que se
deve ter cuidado antes de introduzi-la como uma cultura em uma nova área, uma
vez que a localização pode ter um impacto significativo sobre a composição das
sementes (AYERZA; COATES, 2011). Cahill (2004) sugere uma ligeira perda de
diversidade acompanhando a domesticação das variedades comerciais modernas.
37
Os fatores predominantes que influenciam a diversidade genética da Salvia
hispanica L. são os atributos biológicos das espécies e sua distribuição geográfica,
sendo que a seleção humana atua como um terceiro fator que influencia padrões de
diversidade.
2.3.2 Composição química e valor nutricional
A investigação sobre a chia tem-se centrado na composição química das
sementes (WEBER et al., 1991; AYERZA; COATES, 2004a; REYES-CAUDILLO et
al., 2008; VÁZQUEZ-OVANDO et al., 2009; CAPITANI et al., 2012), óleo da semente
(AYERZA, 1995; AYERZA; COATES, 2011; ÁLVAREZ-CHÁVEZ et al., 2008;
IXTAINA et al., 2011; 2012), mucilagem da semente (LIN et al., 1994; MUÑOZ et al.,
2012) e óleo essencial da folha (TING et al . 1996).
As sementes têm sido investigadas e recomendadas devido ao seu teor de
óleo, proteínas, antioxidantes, e teor de fibra dietética. A semente possui,
aproximadamente, 25 a 39% de óleo, e contém uma das maiores concentrações
conhecidas do ácido graxo α-linolênico (até 67,8%) (AYERZA, 1995; COATES;
AYERZA, 1996). A composição em ácidos graxos do óleo de chia apresenta cerca
de 90% de ácidos graxos insaturados, principalmente o ácido graxo α-linolênico
(ALA, C18: 3 n-3, 54-68%), linoleico (LA, C18: 2 n-6; 17-26%) e oleico (C18:1 n-9, 6-
7,3%), com baixas concentrações de ácidos graxos saturados, como o palmítico
(C16:0) e esteárico (C18:0) (9-11%), além de apresentar uma boa proporção entre
os ácidos graxos n-6/n-3 (AYERZA, 1995, COATES; AYERZA, 1996; AYERZA;
COATES, 2004a). O óleo da semente de chia é obtido por prensagem a frio, e é
comercializado como óleo bruto sob os nomes comerciais e marcas específicas em
vários países da América Latina e América do Norte (IXTAINA et al., 2010).
O teor de proteínas (19-26%) das sementes de chia é mais elevado quando
comparado a outras culturas tradicionais, tais como trigo, (Triticum aestivum L.),
milho (Zea mays L.), arroz (Oryza sativa L.), aveia (Avena sativa L.) e cevada
(Hordeum vulgare L.) (WEBER et al., 1991; IXTAINA et al., 2008). A semente de chia
foi caracterizada recentemente como uma potencial fonte de peptídeos
biologicamente ativos (bioativos) (SEGURA-CAMPOS et al., 2013) e contém todos
os aminoácidos essenciais, além de apresentar um bom balanço destes, em
38
particular, de metionina e cisteína (SANDOVAL-OLIVEROS; PEREDES-LÓPEZ,
2013).
A chia também possui um alto teor de fibra dietética total que varia entre 47,1
e 59,8% (WEBER et al., 1991), mas não contém elementos tóxicos ou compostos
antinutricionais (BUSHWAY et al., 1981, WEBER et al., 1991, LIN et al., 1994).
Pesquisadores avaliaram o teor de fibras de sementes de chia de duas regiões
diferentes do México e reportaram valores de 37-41% para fibra dietética total, sendo
que desta 33-35% foi composta por fibra insolúvel, e 6-7% por fibra solúvel (REYES-
CAUDILLO et al., 2008). Em estudo subsequente, uma fração rica em fibras obtida
por secagem da farinha desengordurada de sementes de chia mexicana apresentou
cerca de 56% de fibra dietética total, das quais 53% de fibra dietética insolúvel e 3%
de fibra dietética solúvel; a fração fibrosa teve alta capacidade de retenção e
absorção de água (VÁZQUEZ-OVANDO et al., 2009). Da mesma forma, Capitani e
colabroradores (2012) avaliaram amostras da fração fibrosa e resíduos obtidos após
a extração do óleo da semente de chia argentina por prensagem ou solvente (n-
hexano) e encontraram um alto teor de fibra dietética total (52%), composta de fibra
insolúvel (47,5%) e fibra solúvel (4,5%), sendo o teor de celulose, hemicelulose e
lignina em torno de 25-28%, 30%, e 5,5-8%, respectivamente. Ambas as frações
fibrosas apresentaram alta capacidade de retenção e absorção de água, sugerindo
seu uso como possível agente emulsionante e estabilizante em alimentos.
A camada externa da semente de chia contém mucilagem que incha e a
envolve na forma de uma camada espessa quando embebida em água. Nesta
mucilagem gelatinosa, a xilose, glicose e ácido glucurônico metílico foram
identificados formando um polissacarídeo ramificado de alto peso molecular (LIN et
al., 1994). Essa característica seria responsável pela saciedade atribuída ao
consumo das sementes em humanos (VUKSAN et al., 2010), indicando um grande
potencial como um ingrediente funcional para ser utilizado como agente espessante
em alimentos (MUÑOZ et al., 2012).
A semente de chia também possui teores significativos de alguns minerais,
como por exemplo, potássio (653 mg/100g), cálcio (631 mg/100g), fósforo (770
mg/100g), ferro (7,72 mg/100g), zinco (5,68 mg/100g), magnésio (334,50 mg/100g),
manganês (3,28 mg/100g), cobre (1,66 mg/100g) e selênio (55,2 μg/100g)
39
(COATES, 2012; MUÑOZ et al., 2013). Além disso, a composição nutricional da chia
também se destaca pela presença de vitaminas, como a vitamina A (53,86 IU/100g),
vitamina C-ácido ascórbico (1,61 mg/100g), vitamina B1 (0,62 mg/100g), vitamina B2
(0,17 mg/100g), vitamina B3 (8,83 mg/100g) e folato (49,0 μg/100g) (COATES, 2012;
MUÑOZ et al., 2013).
Também tem sido identificada a atividade antioxidante da chia, tanto do óleo
quanto da fração desengordurada das sementes, sendo principalmente devida à
presença de polifenóis, como ácidos caféico e clorogênico, mericetina, quercitina,
kaempferol entre outros (TAGA et al., 1984; REYES-CAUDILLO et al., 2008;
VÁZQUEZ-OVANDO et al., 2009; IXTAINA et al., 2011; CAPITANI et al., 2012,
MARTÍNEZ-CRUZ; PAREDES-LÓPEZ, 2014). Porém, o conteúdo de polifenóis pode
variar dependendo dos métodos de extração utilizados e locais de cultivo das
sementes.
Um importante estudo avaliou a atividade antioxidante e o conteúdo de
fenólicos de extratos brutos (com etanol) e hidrolisados (com ácido clorídrico e
etanol) de sementes de chia de duas regiões diferentes do México. Os principais
compostos identificados foram quercetina e kaempferol, enquanto que os ácidos
caféico e clorogênico estavam presentes em baixas concentrações. O conteúdo
médio de fenólicos encontrado foi em torno de 0,9 mg/g dos extratos de semente de
chia, expresso como equivalente de ácido gálico (GAE) (REYES-CAUDILLO et al.,
2008). Os resultados deste estudo demonstram atividade antioxidante consistente e
comparável ao Trolox em todos os modelos de ensaio analisados, sugerindo que o
isolamento e a preparação de compostos bioativos a partir de sementes de chia
poderiam ser utilizados para produzir potentes antioxidantes naturais ou ingredientes
funcionais com aplicações comerciais.
Em sequência, Vázquez-Ovando e colaboradores (2009) avaliaram a
atividade antioxidante de uma fração rica em fibras obtida por secagem da farinha
desengordurada das sementes de chia de diferentes regiões do México e verificaram
que a atividade antioxidante avaliada foi de 489 umol de Trolox equivalentes
(TEAC)/g, sendo considerada elevada em relação à muitos cereais e bebidas tais
como vinho, chá, café e suco de laranja. Da mesma forma, Capitani e colaboradores
(2012) encontraram uma alta atividade antioxidante nas amostras de fração fibrosa e
40
resíduo obtidos após a extração do óleo da semente de chia argentina por
prensagem ou solvente (n-hexano). As amostras apresentaram uma maior atividade
antioxidante do que a encontrada no farelo de trigo, grãos de sorgo e cevada,
possivelmente devido aos compostos polifenólicos e à presença de tocoferóis totais,
com aproximadamente 470-545 mg/kg de óleo, onde γ-tocoferol foi o componente
principal (95%), seguido por α- e δ-tocoferol, mas o β-tocoferol não foi detectado.
Ixtaina e colaboradores (2011) também identificaram a concentração de
tocoferóis (238-427 mg/kg) no óleo de chia da Argentina e Guatemala extraído por
prensagem ou solvente (n-hexano), sendo composto principalmente por γ-tocoferol
(>85%), com δ-tocoferol e α-tocoferol encontrado em concentrações variáveis,
enquanto o β-tocoferol também não foi detectado. Os métodos de extração
influenciaram significativamente o rendimento do óleo, obtendo-se cerca de 30%
mais óleo por solvente do que por prensagem. A relação dos ácidos graxos n-3/n-6
variou entre 3,18-4,18, sendo significativamente maior do que o relatado para outros
óleos vegetais. Os principais compostos fenólicos identificados e quantificados no
óleo de chia foram os ácidos clorogênico e caféico, mericetina, quercetina, e
kaempferol, estando suas concentrações significativamente maiores quando o óleo
foi extraído por prensagem em relação ao método por solvente.
Álvarez-Chávez e colaboradores (2008) identificaram o teor de fitosteróis no
óleo de chia de variedades mexicanas, que variou entre 12,6 e 8,15 g/kg, sendo o β-
sitosterol responsável por até 74% da fração total insaponificável. O teor de esteróis
individuais, β-sitosterol, estigmasterol e estigmastanol foram superiores à de colza,
amendoim, cártamo, sésamo, girassol e óleos não refinados (PHILLIPS et al., 2002),
caracterizando a chia como uma fonte atrativa também de fitosteróis.
Apesar do teor considerável de componentes bioativos, como polifenóis,
tocoferóis, esteróis, carotenóides e fosfolipídios, o alto nível de insaturação do óleo
de chia determina a sua baixa estabilidade oxidativa (IXTAINA et al., 2011).
Entretanto, Ixtaina e colaboradores (2012) verificaram que o óleo de chia apresentou
boa estabilidade oxidativa após 225 dias quando armazenado a 4 °C, e entre 60 e
120 dias quando armazenado a 20 °C. Além disso, a estabilidade oxidativa do óleo
de chia pode ser considerada maior quando são comparados os valores de índice de
peróxido de amostras do óleo de chia (IXTAINA et al., 2012) e óleo de linhaça
41
quando submetidos a diferentes métodos de extração (KHATTAB; ZEITOUN, 2013)
ou quando adicionados ou não de antioxidantes (RUBILAR et al., 2012).
Recentemente, Martínez-Cruz e Paredes-López (2014) identificaram, em
sementes de chia do México, diferentes compostos fenólicos como, ácidos
rosmarínico, protocatecuico, caféico, gálico, ferúlico, entre outros, estando o ácido
rosmarínico em maior concentração. Além disso, isoflavonas como a genisteína,
daidzeína e gliciteína também foram identificadas.
As variações na composição nutricional das sementes e óleo de chia são,
provavelmente, devido às condições ambientais. A produtividade da chia, como
muitas culturas, é sensível ao tempo e à data de plantio. Dessa forma, alguns
estudos relatam que o teor de óleo, a composição de ácidos graxos, bem como a
produção de sementes são afetados pelo clima, localização e práticas de produção
no noroeste da Argentina (COATES; AYERZA, 1996; 1998). O mesmo ocorreu em
estudos realizados no México (ÁLVAREZ-CHÁVEZ et al., 2008) e Equador
(AYERZA, 2010).
A avaliação da composição da chia cultivada em seis ecossistemas tropicais e
subtropicais da América do Sul indicou que a localização do plantio afetou a
composição das sementes, com uma diferença significativa no conteúdo de proteína
e óleo, índice de peróxido e composição de ácidos graxos, presumivelmente devido
a um ou mais fatores ambientais, como a temperatura, luz, condições climáticas, tipo
de solo e os nutrientes disponíveis (AYERZA; COATES, 2004a; 2004b).
2.3.3 Influência da adição de chia em alimentos
As propriedades físico-químicas e funcionais da fração lipídica das sementes
de chia, assim como de seus subprodutos, motivam o uso deste alimento com
potencial nutricional e tecnológico, considerando a atual tendência no aumento do
consumo de alimentos funcionais, tornando-os importantes ingredientes para uso
industrial na produção de sobremesas, bebidas, pães, geleias, emulsões, biscoitos,
alimentos cozidos e fritos, entre outros (CAPITANI et al., 2012; VÁZQUEZ-OVANDO
et al., 2009; ÁLVAREZ-CHÁVEZ et al., 2008).
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Dessa forma, diversos estudos têm relatado a vantagem nutricional da
utilização da chia ou de seus derivados, na alimentação animal, em relação às
outras fontes de PUFA. A adição de chia em dietas de aves para produção de ovos
(AYERZA; COATES, 1999, 2000, 2001, 2002) e produção de carne (AYERZA et al.,
2002), na dieta de vacas para a produção de leite (AYERZA; COATES, 2006), e nas
dietas de coelhos (PEIRETTI; MEINERI, 2008) e porcos para produção de carnes
(COATES; AYERZA, 2009) trazem consequentes benefícios aos consumidores
quanto a qualidade nutricional desses produtos, caracterizando a chia como uma
das fontes mais eficientes de PUFA n-3 para o enriquecimento de alimentos.
Além do potencial como fonte de nutrientes viável para alimentação animal,
as sementes de chia e seus derivados têm crescente interesse para indústrias
alimentícias. As sementes também são usadas como suplementos nutricionais, bem
como na fabricação de barras de cereais, cereais matinais e cookies, nos EUA,
América Latina e Austrália com mercado promissor (DUNN, 2010). Em 2009, foi
aprovado como novo alimento pelo Parlamento Europeu e pelo Conselho Europeu
(COMMISSION, E.U., 2009). Não há evidência de efeitos adversos ou de
alergenicidade causada pela ingestão de sementes de chia inteiras ou moídas
(EFSA, 2005; 2009).
Ainda que não haja evidências sobre efeitos adversos da chia, vale ressaltar
que o US Dietary Guidelines recomenda que o consumo de chia não exceda 48
g/dia e que o Conselho Europeu aprova o uso de semente de chia em produtos de
panificação até o limite de 5%. Embora os produtos de panificação sejam os mais
comuns e viáveis tecnologicamente para o enriquecimento, a chia é amplamente
utilizada nas indústrias alimentícias de diversos países, até mesmo em produtos
aparentemente improváveis de serem enriquecidos como sucos de fruta e iogurte
(BORNEO et al., 2010; MOHD ALI et al., 2012).
Olivos-Lugo e colaboradores (2010) estudaram as principais frações proteicas
da chia e demonstraram excelentes propriedades tecnológicas como capacidade de
retenção de água e capacidade de retenção de óleo constituindo ótimas alternativas
para produtos de panificação e emulsões. Um estudo demonstrou que a inclusão de
hidrolisados enzimáticos de frações ricas em proteína de chia no pão branco e
creme de cenoura aumentou o potencial biológico sem afetar a qualidade dos
43
produtos (SEGURA-CAMPOS et al., 2013). Outro estudo determinou a aceitação
geral, as características sensoriais, propriedades funcionais e quantidade de
nutrientes de bolos formulados utilizando gel de chia (mucilagem gelatinosa, ou seja,
produto obtido quando sementes de chia são imersas em água) como um substituto
para óleo ou ovos, nas concentrações de 25%, 50% e 75%. Os resultados indicaram
que a substituição com 25% de gel de chia nos bolos não foi significativamente
diferente do controle para aspectos como cor, sabor, textura e aceitabilidade geral. A
substituição do óleo com 50% do gel de chia, em comparação com o controle,
promoveu a redução de quilocalorias e gordura nas formulações, demonstrando que
a utilização dessa substituição em preparações comuns pode gerar um produto mais
nutritivo mantendo suas características funcionais e sensoriais (BORNEO et al.,
2010).
Recentemente, Coelho e Salas-Mellado (2015) adicionaram semente e
farinha de chia em pães de farinha de trigo reduzindo o teor de gordura vegetal
hidrogenada nas formulações. Os autores encontraram, nas formulações
modificadas, redução de gordura saturada, aumento dos teores de gordura poli-
insaturada, principalmente ômega-3, além do aumento dos teores de fibras
apresentando características de produtos funcionais. Porém, a qualidade tecnológica
dos pães foi afetada pela adição de chia nas formulações, levando a uma diminuição
no volume específico. Na avaliação sensorial, os pães apresentaram elevados níveis
de aceitabilidade e planos de compra, demonstrando a viabilidade comercial desses
produtos.
A farinha de chia tem sido utilizada também como alternativa para produtos
isentos de glúten. Costantini e colaboradores (2014) demonstraram que a adição de
10% de farinha de chia em formulações de pães com trigo sarraceno melhorou o
valor nutricional dos pães sem afetar os parâmetros tecnológicos. Dessa forma, os
pães elaborados com farinha de chia e trigo sarraceno podem ser indicados para
pacientes celíacos, diabéticos e obesos devido à isenção de glúten, ao baixo teor de
carboidratos, alto teor de proteínas, fibras insolúveis e ácidos graxos ômega-3.
Porém, a aceitabilidade sensorial das formulações não foi avaliada nesse estudo.
Embora a chia esteja em ascensão quanto à sua utilização como ingrediente
funcional na indústria alimentícia, a sua incorporação na dieta habitual ainda é um
44
desafio por alguns motivos, a citar o custo final do produto e o hábito alimentar da
população. Uma forma interessante e bastante recente de apresentação da chia é a
composição com outros alimentos funcionais, na forma de grãos/sementes ou
farinhas. Encontra-se disponível no mercado opções de composição da chia com
linhaça, amaranto e quinoa, constituindo mais uma alternativa de inclusão desta
semente na alimentação habitual. Ademais, as expectativas de aumento do uso da
chia e dos produtos enriquecidos com ela pela população são muito positivas, visto
que o seu cultivo é de fácil manejo e rentável até mesmo para pequenos produtores.
2.3.4 Efeitos biológicos da semente e do óleo de chia
Pesquisas recentes têm mostrado os efeitos fisiológicos benéficos do
consumo das sementes e/ou óleo de chia em modelos animais (AYERZA; COATES,
2005, 2007; ESPADA et al., 2007; CHICCO et al., 2009; POUDYAL et al., 2012;
GONZÁLEZ-MAÑÁN et al., 2012; ROSSI et al., 2013; OLIVA et al., 2013;
VALENZUELA et al., 2014; SIERRA et al., 2015) (Tabela 1) e em humanos
(VUKSAN et al., 2007, 2010; NIEMAN et al., 2009, 2012; JIN et al., 2012) (Tabela 2).
Ayerza e Coates (2005) iniciaram as pesquisas relacionadas aos efeitos
biológicos da suplementação com produtos de chia in vivo. Animais alimentados com
óleo e/ou semente de chia apresentaram uma redução significativa nos níveis
séricos de triacilglicerois (TG) e colesterol total, um aumento nos níveis de HDL-
colesterol, sem alteração na concentração de LDL-colesterol sérico. Também houve
um aumento nas concentrações séricas de ALA e DHA, e uma melhor proporção dos
ácidos graxos n-6/n-3 a partir da suplementação com os produtos de chia em
relação ao controle, sem causar efeito adverso sobre o crescimento dos animais.
Posteriormente, Ayerza e Coates (2007) seguindo o mesmo delineamento
experimental, também observaram uma melhora no perfil lipídico sérico e na
composição de ácidos graxos plasmáticos após a ingestão de semente ou óleo de
chia durante 4 semanas.
Os resultados do estudo de Espada e colaboradores (2007) mostraram que
uma dieta rica em ácidos graxos poli-insaturados n-3, a partir da semente de chia, foi
capaz de inibir o crescimento e formação do tumor de glândula mamária e o número
de metástases, ligado a um aumento da apoptose e uma redução da mitose
45
encontradas em tecidos tumorais. As células neoplásicas apresentaram baixos
níveis de AA e ambos eicosanoides derivados da via lipoxigenase (ácido 12-
hidroxieicosatetraenoico, 12-HETE) e cicloxigenase (ácido 12-
hidroxiheptadecatrienoico, 12-HHT), em comparação com os controles. A infiltração
de linfócitos T também foi mais elevada em relação às outras dietas.
Chicco e colaboradores (2009) investigaram os benefícios da ingestão da
semente de chia sobre a dislipidemia e resistência à insulina (RI), induzida pela
ingestão de uma dieta rica em sacarose (62,5%) (SRD) em ratos Wistar. Os autores
observaram que a suplementação com as sementes de chia (36,2%) preveniu o
aparecimento de dislipidemia e RI em ratos alimentados com a dieta SRD durante 3
semanas, sem alteração da glicemia. Em longo prazo (5 meses) houve normalização
da dislipidemia e RI e redução da adiposidade visceral quando a semente de chia
substituiu a gordura dietética durante os últimos dois meses do período
experimental. Além disso, o presente estudo forneceu dados sobre o efeito benéfico
da semente de chia sobre o perfil lipídico e homeostase da glicose em um modelo
experimental de dislipidemia e RI.
Posteriormente, Poudyal e colaboradores (2012) avaliaram a suplementação
com as sementes de chia (5%) por 8 semanas, após 56 dias de indução da
obesidade a partir de uma dieta hiperlipídica (24%) e hiperglicídica (52%), com 25%
de frutose na água de beber, em ratos Wistar. Os autores observaram melhora da
sensibilidade à insulina e da tolerância à glicose, redução da adiposidade visceral e
da esteatose hepática, diminuição da inflamação e fibrose cardíaca e hepática, sem
alterações na concentração dos lipídeos plasmáticos e na pressão sanguínea. Além
disso, houve melhora na razão n-3/n-6 no plasma, coração, fígado e tecido adiposo
dos animais, depleção do ácido graxo linoleico e seus produtos em todos os tecidos,
sugerindo aumento da oxidação mitocondrial, inibição da estearoil-CoA dessaturase
(enzima chave que catalisa a produção de ácido graxo insaturado a partir dos
saturados) pelo ácido graxo ALA, induzindo uma redistribuição lipídica associada
com cardioproteção e hepatoproteção.
Outro estudo demonstrou que a adição de 10% de óleo de chia nas dietas de
ratos Sprague-Dawley saudáveis aumentou a concentração de ALA, EPA e DHA no
plasma, fígado e tecido adiposo, melhorou a proporção dos ácidos graxos n-6/n-3,
46
além de aumentar a expressão do PPAR-α e enzimas lipolíticas no fígado,
responsáveis por aumentar a b-oxidação de ácidos graxos, o que pode ser uma
possível explicação para os efeitos hipolipidêmicos da chia (GONZÁLEZ-MAÑÁN et
al., 2012).
Recentemente, Rossi et al (2013) e Oliva et al (2013) têm buscado elucidar
alguns dos mecanismos responsáveis pelas alterações metabólicas e pela
prevenção, melhora e/ou reversão da dislipidemia, esteatose hepática e resistência à
insulina em ratos Wistar induzidos por dieta. Dentre os seus achados destacam-se:
(i) no fígado, aumento da expressão gênica e da atividade de enzimas chave na
oxidação de ácidos graxos, promovendo o deslocamento do equilíbrio do
metabolismo de ácido graxo a favor da oxidação ao invés do armazenamento;
redução da expressão e da atividade de enzimas lipogênicas hepáticas; (ii) no tecido
adiposo, redução da atividade de enzimas lipogênicas, redução da lipólise basal e
da hipertrofia celular no tecido adiposo epididimal, aumento da sensibilidade do
adipócito à ação antilipolítica da insulina; (iii) no músculo, redução do
armazenamento de lipídeo e aumento da fosforilação e oxidação da glicose e da
sensibilidade à insulina. Tais mecanismos contribuem para a melhora/normalização
da disfunção do tecido adiposo, homeostase da glicose, esteatose hepática,
dislipidemia e resistência à insulina periférica, além da redução da lipotoxicidade.
Estes efeitos podem estar relacionados às alterações subsequentes no conteúdo de
ácidos graxos em fosfolipídios de membrana de tecidos alvo da insulina, devido às
interações competitivas no metabolismo dos ácidos graxos poli-insaturados da
família n-6 e n-3, o que poderia, por sua vez, melhorar a sensibilidade à insulina.
Valenzuela e colaboradores (2014) demonstraram que a ingestão de
diferentes óleos vegetais (ricos em ALA), incluindo o óleo de chia, alterou a
composição de ácidos graxos em diversos tecidos de ratos Wistar saudáveis. O óleo
de chia foi capaz de aumentar as concentrações de EPA nos eritrócitos, fígado, rins,
intestino delgado, coração e músculo, mas não no cérebro. Os níveis de DHA foram
aumentados no fígado, intestino e cérebro. Dessa forma, os autores sugerem que a
conversão de ALA em ácidos graxos poli-insaturados da família ômega-3 é efetiva,
mas bastante seletiva dependendo do tecido estudado.
47
A Figura 3 ilustra os efeitos metabólicos e os mecanismos moleculares em
diferentes tecidos a partir da ingestão da semente ou óleo de chia em animais
experimentais (AYERZA; COATES, 2005, 2007; CHICCO et al., 2009; POUDYAL et
al., 2012; GONZÁLEZ-MAÑÁN et al., 2012; ROSSI et al., 2013; OLIVA et al., 2013;
VALENZUELA et al., 2014).
Figura 3. Esquema dos efeitos benéficos e mecanismos moleculares em diferentes
tecidos a partir da ingestão da semente de chia (Salvia hispanica L.) in vivo. AA:
ácido graxo araquidônico; ACC: acetil CoA carboxilase; ALA: ácido graxo α-
linolênico; CPT: carnitina palmitoil transferase; CT: colesterol total; DAG:
diacilglicerol; DHA: ácido graxo docosahexaenoico; DPA: ácido graxo
docosapentaenoico; EPA: ácido graxo eicosapentaenoico; FAO: ácido graxo
oxidase; FAS: ácido graxo sintase; FFA: ácido graxo livre; G6-P: glicose 6-fosfato; G-
6-PDH: glicose-6-fosfato desidrogenase; GLUT-4: transportador de glicose
48
dependente de insulina; HDL-c: lipoproteína de alta densidade; LCA-CoA: acil-
Coenzima A de cadeia longa; n-6/n-3: razão entre os ácidos graxos n-6 e n-3; PCR:
proteína C-reativa; PDHc: complexo piruvato desidrogenase; PPARα: receptor
ativado por proliferadores de peroxissoma; SAT: ácido graxo saturado; SREBP-1c:
proteína 1c ligadora do elemento regulatório de esterol; TG: triacilglicerol. Adaptado
de MARINELI et al., (2015).
49
Tabela 1. Estudos realizados em animais com a semente e óleo de chia (Salvia hispanica L.).
Modelo
experimental N Tratamento Duração Resultados Referência
Ratos Wistar
saudáveis 24
Dietas isocalóricas derivadas do
óleo de milho (controle); 15% de
semente de chia; e 5% de óleo de
chia
4 semanas
Redução dos níveis séricos de TG e CT,
aumento nos níveis de HDL-c. Aumento
na concentração sérica de ALA e melhor
proporção dos ácidos graxos n-6/n-3
Ayerza e
Coates (2005)
Ratos Wistar
saudáveis 32
Dietas isocalóricas derivadas do
óleo de milho (controle); 16% de
semente de chia inteira; 16% de
semente de chia moída; e 5% de
óleo de chia
4 semanas
Aumento nos níveis séricos de HDL-c e
redução dos níveis de TG. Aumento da
concentração plasmática de ácidos graxos
n-3 (ALA, EPA e DHA) e redução dos
ácidos graxos n-6 (LA e AA), com melhor
proporção dos ácidos graxos n-6/n-3
Ayerza e
Coates (2007)
Camundongos
BALB/c inoculados
com adenocarcinoma
de glândula mamária
60
Dieta comercial (controle); 6% óleo
de chia; e 6% óleo de cártamo. Após
3 meses os animais foram
transplantados com adenocarcinoma
mamário com capacidade
metastática moderada
3 meses
(dieta) e
45-50 dias
(tumor)
Inibição do crescimento, número de
metástases e formação do tumor de
glândula mamária
Espada et al.
(2007)
50
Modelo
experimental N Tratamento Duração Resultados Referência
Ratos Wistar
dislipidêmicos e
resistentes à insulina
induzidos por dieta
72
96
Dieta comercial (controle); dieta rica
em sacarose (62,5%); dieta com
semente de chia (36,2%)
3 semanas
5 meses
Prevenção da dislipidemia e resistência à
insulina
Normalização da dislipidemia, resistência
à insulina e redução da adiposidade
visceral
Chicco et al.
(2009)
Ratos Wistar obesos
induzidos por dieta
48
Dieta a base de amido de milho
(controle); dieta controle com
semente de chia (5%); dieta
hiperlipídica (24%) e hiperglicídica
(52%), com 25% de frutose na água
de beber (obesogênica); dieta
obesogênica com sementes de chia
(5%)
8 semanas
Melhora da sensibilidade à insulina e da
tolerância à glicose, redução da
adiposidade visceral e da esteatose
hepática, diminuição da inflamação e
fibrose cardíaca e hepática. Melhor
proporção dos ácidos graxos n-3/n-6 no
plasma, coração, fígado e tecido adiposo
Poudyal et al.
(2012)
Ratos Sprague-
Dawley saudáveis 27
Dieta experimental com óleo de
girassol (10%) (controle), óleo de
rosa mosqueta (10%) e óleo de chia
(10%)
21 dias
Aumento da concentração de ALA, EPA e
DHA no plasma, fígado e tecido adiposo
com melhor proporção dos ácidos graxos
n-6/n-3. Aumento da expressão de genes
lipolíticos
González-
Mañán et al.
(2012)
51
Modelo
experimental N Tratamento Duração Resultados Referência
Ratos Wistar
dislipidêmicos e
resistentes à insulina
induzidos por dieta
24
40
Dieta comercial (controle); dieta rica
em sacarose (62,5%); dieta com
semente de chia (36,2%)
3 semanas
5 meses
Prevenção (3 semanas) ou
melhora/normalização (5 meses) da
dislipidemia, esteatose hepática e
homeostase da glicose a partir de
alterações no metabolismo de ácidos
graxos hepáticos (lipogênese/oxidação)
Rossi et al.
(2013)
Ratos Wistar
dislipidêmicos e
resistentes à insulina
induzidos por dieta
18
Dieta comercial (controle); dieta rica
em sacarose (62,5%); dieta com
semente de chia (36,2%)
6 meses
Redução da hipertrofia de adipócitos,
melhora na atividade de enzimas
lipogênicas. Normalização do
armazenamento e oxidação de lipídeos no
músculo esquelético. Reversão da
resistência à insulina e dislipidemia
Oliva et al.
(2013)
Ratos Wistar
saudáveis 60
Dietas experimentais suplementadas
com 10% de óleo de girassol,
canola, Rosa canina, sacha inchi e
óleo de chia
21 dias
Melhora do perfil de ácidos graxos em
diversos tecidos. Aumento da
concentração de EPA nos eritrócitos,
fígado, rins, intestino delgado, coração e
músculo; e de DHA no fígado, intestino
delgado e cérebro. A concentração de
ALA foi maior em todos os tecidos
estudados, exceto no cérebro
Valenzuela et
al. (2014)
52
Modelo
experimental N Tratamento Duração Resultados Referência
Coelhos saudáveis e
dislipidêmicos
induzidos por dieta
32
Dieta comercial (controle); dieta com
óleo de chia (10%); dieta com
colesterol (1%); dieta com colesterol
(1%) e óleo de chia (10%)
5-6
semanas
Melhora da função vascular contra efeitos
deletérios da hipercolesterolemia e
aumento da concentração de ALA no
plasma
Sierra et al.
(2015)
Ácido graxo α-linolênico (ALA); Ácido graxo araquidônico (AA); Ácido graxo linoleico (LA); Ácido graxo eicosapentaenoico (EPA); Ácido graxo
docosahexaenoico (DHA); Colesterol total (CT); HDL-colesterol (HDL-c); Triacilglicerol (TG). Adaptado de MARINELI et al. (2015).
53
Alguns grupos de pesquisa também têm avaliado os efeitos fisiológicos da
ingestão das sementes de chia em humanos. Em 2007, Vuksan e colaboradores
utilizaram o ―grão integral‖ Salba - uma marca utilizada para descrever duas
variedades brancas registradas, Sahi Alba 911 e 912, que são o resultado de
cruzamentos seletivos a partir do grão preto da chia (Salvia hispanica L.) - como
ingrediente na formulação de pães. Vinte indivíduos com DM2 consumiram cerca de
37g por dia de Salba ou farelo de trigo (controle) durante 12 semanas, mantendo
suas terapias convencionais do diabetes. Comparado ao controle, a Salba reduziu a
pressão arterial sistólica, as concentrações de proteína C-reativa, com reduções
significativas na hemoglobina glicosilada e fibrinogênio em relação aos valores
basais (inicial) no grupo Salba, mas não em relação ao controle. Não houve
mudanças nos parâmetros de segurança, incluindo fígado, rins e função
hemostática, ou peso corporal. As concentrações plasmáticas dos ácidos graxos n-3,
ALA e EPA foram aumentadas com o consumo da Salba. Dessa forma, os autores
concluíram que a suplementação com os grãos de Salba, em longo prazo, atenuou
importante fator de risco cardiovascular e fatores emergentes, mantendo adequado
controle lipídico e glicêmico em indivíduos com DM2 controlada.
Em 2010, Vuksan e colaboradores avaliaram a ingestão de grãos integrais de
Salba na redução da glicemia pós-prandial em indivíduos saudáveis. No estudo
agudo, randomizado, duplo-cego e controlado, 11 indivíduos saudáveis receberam
0, 7, 15 ou 24 g de Salba no pão branco. A diminuição da glicemia pós-prandial foi
observada para todas as doses de Salba ingeridas, assim como o aumento da
saciedade. Estes achados sugerem que a ingestão de doses crescentes de Salba
melhorou a glicemia pós-prandial de maneira dose-dependente, fornecendo uma
possível explicação para as melhorias na pressão arterial, coagulação e marcadores
inflamatórios previamente observados após 12 semanas de suplementação com
Salba em diabéticos do tipo 2 (VUKSAN et al., 2007).
Apesar dos diversos efeitos benéficos relatados acima, Nieman e
colaboradores (2009) avaliaram a eficácia da ingestão de sementes de chia (50 g/dia
54
misturados com 250 mL de água, divididos em duas porções, antes das principais
refeições) na promoção da perda de peso e alteração de fatores de risco de doença
em 76 adultos com sobrepeso, durante 12 semanas e não verificaram melhora da
perda de peso, alteração da composição corporal, atenuação da inflamação
(proteina C-reativa, IL-6, MCP-1, TNF-α), aumento da capacidade antioxidante,
melhora do perfil lipídico sanguíneo ou da pressão arterial em homens e mulheres
com sobrepeso. Entretanto, a concentração plasmática de ALA aumentou 24,4% no
grupo suplementado em comparação com uma redução de 2,8% no controle.
No estudo de Jin e colaboradores (2012), dez mulheres na pós-menopausa
ingeriram cerca de 25 g/dia de semente de chia moída, durante 7 semanas, e
apresentaram aumento significativo da concentração plasmática de ALA, após uma
semana de suplementação, que permaneceu 138% acima dos níveis basais até o
final do estudo. A concentração plasmática de EPA aumentou 30% e foi
correlacionada ao longo do tempo com o ALA. Porém, não houve alteração
significativa na concentração de ácido docosapentaenoico (DPA), enquanto o nível
de DHA diminuiu ligeiramente até ao final do estudo e não foi relacionado ao
aumento de ALA. Pesquisadores do mesmo grupo de pesquisa avaliaram, em
estudo duplo-cego, a ingestão de 25 g/dia de semente de chia (inteira ou moída) ou
placebo (semente de papoula) em 56 mulheres na pós-menopausa com sobrepeso
durante 10 semanas e encontraram um aumento na concentração plasmática de
ALA e EPA em 58% e 39%, respectivamente, no grupo que recebeu semente de
chia moída em comparação aos grupos que receberam semente de chia inteira ou
placebo, sem alterações na inflamação ou fatores de risco de doenças avaliados por
diferentes parâmetros (NIEMAN et al., 2012).
Tendo em vista a importante composição química, o valor nutricional, o
potencial funcional e tecnológico, e os efeitos benéficos atribuídos a chia in vivo e in
vitro, são necessárias investigações mais detalhadas que avaliem os mecanismos
fisiológicos e moleculares, bem como o impacto biológico da sua suplementação, a
longo prazo, para que este composto possa ser utilizado como estratégia segura e
eficaz na prevenção e/ou tratamento de DCNT.
55
Tabela 2. Estudos realizados em humanos com a semente chia (Salvia hispanica L.).
Modelo N Tratamento Duração Resultados Referências
Indivíduos com
diabetes mellitus
tipo 2 (DM2)
20
Ingestão de 37 g/dia de
semente de chia ou
farelo de trigo (controle)
12
semanas
Redução da pressão arterial sistólica, da concentração de
proteína C-reativa, hemoglobina glicosilada e fibrinogênio.
Aumento das concentrações plasmáticas dos ácidos graxos n-3
(ALA e EPA). Atenuação de importante fator de risco
cardiovascular e fatores emergentes, mantendo adequado
controle lipídico e glicêmico
Vuksan et al.
(2007)
Indivíduos com
sobrepeso
76
Ingestão de sementes
de chia (50 g/dia com
250 mL de água) duas
vezes por dia
12
semanas
Aumento da concentração plasmática de ALA (24,4%). Não
apresentou alterações na composição corporal ou nos fatores
de risco de doença avaliados
Nieman et al.
(2009)
Indivíduos
saudáveis 11
Ingestão de 0, 7, 15 ou
24 g de semente de chia
no pão branco
5 dias Redução e melhora da glicemia pós-prandial de maneira dose-
dependente e aumento da saciedade
Vuksan et al.
(2010)
Mulheres na pós-
menopausa 10
Ingestão de 25 g/dia de
semente de chia moída
7
semanas
Aumento das concentrações plasmáticas de ALA (138%) e
EPA (30%), sem alteração de nos níveis de DPA e DHA
Jin et al.
(2012)
56
Modelo N Tratamento Duração Resultados Referências
Mulheres na pós-
menopausa 56
Ingestão de 25 g/dia de
semente de chia (inteira
ou moída) ou placebo
(semente de papoula)
10
semanas
Aumento das concentrações plasmáticas de ALA (58%) e EPA
(39%) somente no grupo que recebeu semente de chia moída.
Não apresentou alterações no perfil inflamatório ou nos fatores
de risco de doença avaliados.
Nieman et al.
(2012)
Ácido graxo α-linolênico (ALA); Ácido graxo eicosapentaenoico (EPA); Ácido graxo docosahexaenoico (DHA); Ácido graxo docosapentaenoico
(DPA) (MARINELI et al., 2015).
57
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CAPÍTULO I
72
ARTIGO I
Artigo publicado em revista internacional: LWT - Food Science and
Technology, 59, (2014), p. 1304-1310. http://dx.doi.org/10.1016/j.lwt.2014.04.014
Chemical characterization and antioxidant potential of Chilean chia seeds and
oil (Salvia hispanica L.)
Rafaela da Silva Marineli a, Érica Aguiar Moraes a, Sabrina Alves Lenquiste a,
Adriana Teixeira Godoy b, Marcos Nogueira Eberlin b, Mário Roberto Maróstica Jr a,*
a Department of Food and Nutrition, Faculty of Food Engineering, University of
Campinas – UNICAMP, Campinas, SP, Brazil.
b ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas – UNICAMP, Campinas, SP, Brazil.
*Corresponding author. Email address: [email protected] Tel.: +55 19 3521-
4059; fax: +55 19 3521-4060. Address: Monteiro Lobato st., 80, zip code 13083-862,
Campinas, São Paulo, Brazil.
1 Abbreviations
AOAC (Association of Official Analytical Chemists), ALA (α-Linolenic acid), EASI-MS (easy
ambient sonic-spray ionization mass spectrometry), FAME (fatty acids methyl esters), FID
(flame ionization detector), GC (Gas chromatography), IDF (Insoluble dietary fiber), MDA
(Malondialdehyde), PUFA (Polyunsaturated fatty acids), SDF (Soluble dietary fiber), TAG
(Triacylglycerols), TBARS (Thiobarbituric acid reactive substances), TDF (Total dietary
fiber), 3,4-DHPEA-EDA (3,4-dihydroxyphenylethanol-elenolic acid dialdehyde).
73
Abstract
The objective of this study was to chemically and nutritionally characterize the
commercial chia seeds and oil from Chile and investigate their antioxidant potential
by different in vitro methods. The chia seed presented a good source of protein
(25.32 g/100 g), oil (30.22 g/100 g) and total dietary fiber (37.50 g/100 g), with
predominant insoluble fiber (35.07 g/100 g). The main fatty acids, ranked order of
abundance, were α-linolenic acid > linoleic acid > palmitic acid ~ oleic acid > stearic
acid. The triacylglycerols (TAG) in chia oil were identified by direct mass
spectrometry using the easy ambient sonic-spray ionization (EASI-MS) technique
and linolenic acid (Ln) was present in the most TAG found. The oil also presented a
low peroxide index value (2.56 mEq peroxide/kg). The samples exhibited a high
antioxidant activity by the various in vitro methods evaluated; it is due to the
presence of phenolic compounds in the seed or oil, which were, mainly, myricetin,
quercetin, kaempferol, chlorogenic acid and 3,4-dihydroxyphenylethanol-elenolic acid
dialdehyde (3,4-DHPEA-EDA). Our results therefore suggest that Chilean chia seeds
and oil should be considered as functional ingredients with high antioxidant potential
in food products with commercial applications.
Key words: Chia, Salvia hispanica, Omega-3 fatty acid, Phenolic compounds,
Antioxidant activity
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1. Introduction
Chia (Salvia hispanica L.) is an annual herbaceous plant that belongs to the
Lamiaceae family. This plant is native from southern Mexico and northern Guatemala
(Ayerza, 1995), and has recently been marketed as a crop in South America (Ayerza
& Coates, 2011). The use of chia may be in the form of whole seeds, flour, mucilage
and oil seed. The chia seed has been described as a good source of oil, protein,
dietary fiber, minerals and polyphenolic compounds (Ayerza & Coates, 2004; Reyes-
Caudillo, Tecante & Valdivia-López, 2008; Capitani et al., 2012). The revival of
interest in chia seed is due to their oil content, which provides rich source of
polyunsaturated fatty acids (PUFA). The chia oil is unique since it contains the
highest proportion of omega-3-linolenic acid (ALA) of any known natural source
(Ayerza, 1995; Coates & Ayerza, 1996). ALA plays an important role in health and is
used in several foods and cosmetics. Many studies have provided evidence that
regular consumption or dietary supplementation with long chain n-3 PUFA brings
numerous health benefits, including the prevention of cardiovascular diseases,
hypertension and inflammatory diseases (Albert et al., 2005; Garg et al., 2006).
In addition, chia seed and oil contain a rich pool of natural antioxidants such
as tocopherols, phytosterols, carotenoids (Álvarez-Chávez et al., 2008; Ixtaina et al.,
2011) and phenolic compounds, including chlorogenic acid, caffeic acid, myricetin,
quercetin and kaempferol (Reyes-Caudillo et al., 2008; Capitani et al., 2012), which
protects consumers against many diseases and also promotes beneficial effects on
human health (Nijveldt et al., 2001).
Chia seed and oil are important raw materials to functional foods due to its
bioactive components, offering advantages over other available n-3 sources (Coates
& Ayerza, 1996). Previous studies have shown that the oil, or their by-products, can
be used for animal feed industries, to obtain PUFA enriched animal products and
consequent better nutritional value (Ayerza, Coates & Lauria, 2002; Coates &
Ayerza, 2009). The benefits of chia seed has recently been described for human
health and nutrition because their bioactive components were found to promote
health benefits (Vuksan et al. 2007, 2010), improving biological markers related to
75
dyslipidemia, inflammation, cardiovascular disease, glucose homeostasis, and insulin
resistance, without promote adverse effects.
The previous studies have characterized chia seed and oil mainly in Mexico
(Reyes-Caudillo et al., 2008; Vázquez-Ovando et al., 2009), Argentina (Ixtaina et al.,
2011; Capitani et al., 2012), and other countries in South America (Ayerza & Coates,
2011), but, to the best of our known, there are to date no reports on chia seed and oil
from Chile. Geographical origin is known to have significant impacts on seed
composition and concentration of bioactive compounds (Ayerza & Coates, 2011).
There is also little information available regarding the antioxidant potential of chia
seed and oil and few methods have been explored.
The purpose of this study was therefore to chemically and nutritionally
characterize the commercial chia seeds and oil from Chile and investigate their
antioxidant potential by different in vitro methods in order to assess their potential as
functional ingredients in food products.
2. Materials and methods
2.1. Seeds, oil and chemicals
Fresh chia seed (two different batches, 4.5 kg each) and chia oil (two different
batches, 5.0 L each) from FTP S.A. Santiago, Chile were purchased from R&S
Blumus Comercial de Produtos Alimentícios Ltda, Brazil. According to the producer,
chia oil was obtained by cold pressing and stored at 2-8 °C until use in amber glass
bottles without head space. All solvents were analytical grade from J.T. Baker
(Phillipsburg, NJ, USA). Total dietary fiber assay kit (TDF-100A), 6-hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid (Trolox), 2,2′-azobis(2-methylpropionamidine)
dihydrochloride (AAPH), 2,4,6- tris(2-pyridyl)-s-triazine (TPTZ), 2-thiobarbituric acid
(TBA), 2,2- diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu's reagent and gallic
acid were all obtained from Sigma-Aldrich (Sigma Co., St. Louis, MO, USA).
Fluorescein sodium salt was purchased from Vetec Química Fina (Sao Paulo, Brazil).
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2.2. Flours
The seeds were grounded in a laboratory impact mill (Marconi MA 630/1) and
passed through a 0.850 mm sieve. This flour was used to estimate the raw proximate
composition and malondialdehyde (MDA) assay. Defatting with n-hexane was carried
out in a Soxhlet apparatus. The defatted flour was stored in dark-tightly closed bottles
and stored at 2-8 °C until the analysis. The defatted flour was used for the analysis of
dietary fiber and for the preparation of the ethanolic extract.
2.3 Proximate composition
Protein was determined by the semi-micro Kjeldahl method and ashes were
quantified by means of sample incineration in a muffle furnace as described by
Association of Official Analytical Chemists (AOAC, 2000). Total lipids were
determined by the method of Bligh and Dyer (1959), and moisture in an oven at 105
± 1 °C. Carbohydrates were calculated by difference using Eq. (1). Total energy
value was estimated using the isoperibol automatic calorimeter (PARR 1261) with
oxygen pump (PARR 1108).
Carbohydrate = 100 - (Ash + Moisture + Protein + Lipids). Eq. (1).
2.4. Total, soluble and insoluble dietary fiber
Total dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber
(IDF) were determined using the enzymatic-gravimetric AOAC method (Prosky et al.,
1988).
2.5. Fatty acid profiles of chia oil
The fatty acid composition of the chia oil was determined by gas
chromatography (GC) (Kramer et al., 1997) using a capillary GC Agilent 6850 Series
GC System, capillary column DB-23 Agilent (50% cyanopropyl‐methylpolysiloxane,
60 m x 0.25 mm×0.25 μm), with flame ionization detector (FID). Oven temperature
was 110 °C for 5 min, 110–215 °C (5 °C/min), 215 °C for 24 min; detector
temperature: 280 °C; injector temperature: 250 °C; carrier gas: helium; split ratio
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1:50; injection volume: 1.0 uL. Oil samples were saponified in 0.5 mol/L methanolic
KOH. The fatty acids methyl esters (FAME) were prepared as described by Maia and
Rodriguez-Amaya (1993). FAMEs were identified by comparison of retention times
with FAME standard mixture under the same conditions (FAME Mix GLC-68A; Nu-
Chek-Prep, INC, Elysian, MN, USA) and quantified using area normalization.
2.6. Triacylglycerol composition of chia oil
Profiles of major TAG were obtained directly from the chia oil via easy ambient
sonic-spray ionization mass spectrometry work in positive ion mode (EASI(+)-MS)
(Simas et al., 2010). The EASI(+)-MS data were collected in the positive ion mode
using a unit mass resolution compact single-quadrupole mass spectrometer
(Shimadzu LCMS 2010, Japan) equipped with a homemade EASI source, which is
described in detail elsewhere (Haddad, Sparrapan & Eberlin, 2006). A tiny droplet of
the chia oil sample (2 μL) was dipped directly onto a paper surface (brown Kraft
envelope paper). Experimental EASI parameters were as follow: methanol flow rate
of 20 μL/min, N2 as the nebulizing gas at 3 L/min, and paper/source entrance angle
of ∼30°. Mass spectra were accumulated over 30 s and scanned over the m/z
100−1000 range.
2.7. Lipid autoxidation products
2.7.1. Peroxide Index
Peroxide index determination in chia oil was performed according to the official
Association of Official Analytical Chemists method (AOAC, 2000), which is based on
the oxidation of iodine in the presence of potassium iodide by the peroxide present in
the sample, assuming that all oxidizing substances in the sample are peroxides.
Results were expressed as mEq peroxide/kg sample.
2.7.2. Malondialdehyde
Determination of thiobarbituric acid reactive substances (TBARS) was done in
fresh chia seed samples (250 mg each) and chia oil samples (115 mg each) using
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the method proposed by Sinnhuber and Yu (1958). The method is based in the
formation of a pink-red pigment composed of 2 molecules of thiobarbituric acid (TBA)
and 1 molecule of MDA. Absorbance was read in visible light with a wavelength of
535 nm, using a 1% solution of 2-thiobarbituric acid for calibration. Results were
expressed as mg MDA/kg sample using Eq. (2):
Dilution factor = (100 (mL) / sample weight (g)) / 100 %
―TBA value‖ = [(Absorbance x Dilution factor) / bucket diameter (1 cm)] x 46 mg de
malondialdehyde
2.8. Extraction with ethanol
Extraction of phenolic compounds was carried out according to Capitani et al.
(2012), with some modifications. One gram of defatted flour was extracted with 10
mL of ethanol at room temperature for 3h under mechanical shaking. The mixture
was centrifuged at 3000g for 15 min. The supernatant was evaporated using a
rotavapor apparatus and dissolved in 1 mL ethanol. This extract was used for all
analyzes below.
2.9. Total phenolic content
The total phenolic content of chia seed extract was determined according to
Folin–Ciocalteu's method (Swain & Hillis, 1959), with some modifications. In a vial,
50 μL of extract, 800 μL distilled water and 25 μL (0.25 N) Folin–Ciocalteu's reagent
were mixed and incubated at room temperature for 3 min. Then, 100 μL sodium
carbonate solution (1 N) was added and further incubated for 2 h at room
temperature. The absorbance was read at 725 nm in a microplate reader SynergyHT,
Biotek (Winooski, USA); with Gen5™2.0 data analysis software spectrophotometer.
Gallic acid was used in a standard curve (16–500ug gallic acid/mL water) and the
results were expressed in terms of gallic acid equivalent (GAE/g).
79
2.10. Polyphenol analysis
Identification of the phenolic compounds in chia oil and in chia seed ethanolic
extract was carried out using EASI(-)-MS. The data was collected in the negative ion
mode and the equipment conditions were described in 2.6. section.
2.11. Antioxidant potential in chia seed extract
The readings of absorbance and fluorescence were done in a microplate
reader Synergy HT, Biotek (Winooski, USA); with Gen5™ 2.0 data analysis software
spectrophotometer.
2.11.1. DPPH free radical scavenging activity
The free radical scavenging activity of the extracts was determined based on
the DPPH method (Rufino et al., 2007). The extract (33 μL) was added in 1.3 mL
DPPH solution diluted in methanol (0.024 mg/mL), shaking and incubated for 30 min
in dark, and absorbance was measured at 515 nm. The Trolox standard curve was
made (5–600 μmol TE). Results were expressed in μmol Trolox equivalent (TE)/g.
2.11.2. FRAP assay (ferric reducing antioxidant power)
The ferric reducing ability was determined by FRAP method (Benzie & Strain,
1996), with adaptations. Under dark conditions, FRAP reagent was prepared with
300 mmol/L acetate buffer (pH 3.6), 10 mmol/L 2,4,6- tris(2-pyridyl)-s-triazine (TPTZ)
in a 40 mmol/L HCl solution and 20 mmol/L FeCl3. Sample or standard solutions,
ultrapure water and FRAP reagent were mixed and kept in a water bath for 30 min at
37 °C. After cooling to room temperature, samples and standard were read at 595
nm. The Trolox standard curve was made (5–600 μmol TE). Results were expressed
in μmol Trolox equivalent (TE)/g.
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2.11.3 Lipophilic and Hydrophilic ORAC assay (Oxygen Radical Absorbance
Capacity)
Lipophilic (L-ORAC) – chia oil – and hydrophilic (H-ORAC) – chia seed
ethanolic extract – ORAC assays were based on the method described previously
(Prior et al., 2003). Analyses were conducted in phosphate buffer pH 7.4 and the
peroxyl radical was generated by 2,2′-azobis(2-methylpropionamidine)
dihydrochloride (AAPH) solution, prepared just before the analysis and fluorescein
was used as the substrate. The microplate reader was adjusted (fluorescent filters,
excitation wavelength, 485 nm; emission wavelength, 520 nm) and Trolox was used
as standard. The fluorescence was read at every 1 min, for 80 min. The data were
calculated in the microplate reader, which calculated the area under the curve, the
linear regression and the dilution factor. ORAC values were expressed as µmol
Trolox equivalent (µmol TE) per gram of sample by using the standard curves (2.5 -
50.0 µmol TE) (Davalos, Gomez-Cordoves, & Bartolome, 2004). Total antioxidant
capacity (TAC) was calculated by summing the L-ORAC and H-ORAC (Wu et al.,
2004).
2.12. Statistical analysis
All determinations were done at least in triplicate. Statistical analysis was done
to determine the central tendency of the data and standard deviation using the
GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA) software.
3. Results and discussion
3.1. Proximate composition and total, soluble and insoluble dietary fiber of chia seeds
Proximate composition and dietary fiber analysis are presented in Table 1.
Chilean chia seeds contain an important nutritional source of protein (25.32 g/100 g)
and oil (30.22 g/100 g), and these results are similar with those found in chia seeds
of other countries of South America (Coates & Ayerza, 1998; Ayerza & Coates,
2004). Although chia seed is not commercially grown as a protein source, the protein
content is higher than those of traditional crops such as wheat, corn, rice and oats
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(Coates & Ayerza, 1996). A recent study also demonstrated that chia is a potential
source of biologically-active (bioactive) peptides, suggesting that this seed can be
incorporated into human diets to produce a more balanced protein source (Segura-
Campos et al., 2013).
TDF content in the seed was 37.50 g/100 g, with most of this content
represented by IDF (35.07 g/100 g) and the remainder by SDF (2.43 g/100 g) (Table
1), which is in accordance with other studies (Craig & Sons 2004; Reyes-Caudillo et
al., 2008) and markedly higher than wheat flours and whole grain cereals found by
Ragaee et al. (2006). Furthermore, dietary fiber has an essential role in intestinal
health, nutritional and physiological effects in consumers, and appears to be
significantly associated with a lower risk of developing coronary heart disease,
hypertension, diabetes and obesity (Willem van der Kamp et al., 2010).
Table 1. Proximate composition of Chilean chia seed (Salvia hispanica L.) (g/100 g)
Components Chia seed a
Moisture 5.82 ± 0.04
Ash 4.07 ± 0.02
Protein (N = 6.25) 25.32 ± 0.21
Lipids 30.22 ± 0.08
Carbohydrates b 34.57 ± 0.26
TDF 37.50 ± 1.07
IDF 35.07 ± 0.90
SDF 2.43 ± 0.30
Energy value c 576.50 ± 9.60
a Data are expressed as means values ± standard deviation (n = 4). b By difference. c
The energy value was expressed as Kcal/100 g. TDF: total dietary fiber, SDF: soluble
dietary fiber, IDF: insoluble dietary fiber.
3.2. Fatty acid profiles of chia oil
Table 2 shows the fatty acid composition of chia oil, which is found to contain
mainly α-linolenic, linoleic, oleic, palmitic and stearic acids, with a predominant
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amount of α-linolenic acid (62.8%) (Fig. 1). These findings are in agreement with
those reported in earlier studies (Ayerza, 1995; Coates & Ayerza, 1998, Ayerza &
Coates, 2004). Chia oil shows higher quantities of ALA compared with flaxseed oil
(up to 57%) (Khattab & Zeitoun, 2013).
The n-6/n-3 fatty acid ratio of Chilean chia oil (0.29) is comparable to chia
seed oil of Argentina (Ayerza, 2010) and markedly lower than that of most vegetable
oils, such as maize (76.57), rapeseed (2.26), soybean (6.68), sunflower (30.77) and
olive (17.86) (Tuberoso et al., 2007). The use of chia seed oil in human daily diet
seems indeed beneficial since the use of vegetable oils with high contents of PUFAs
have been shown to provide several health benefits (Ganesan, Brothersen &
McMahon, 2014). Low n-6/n-3 fatty acid ratio has also been associated with the
reduction of cardiovascular disease risk (Simopoulos, 2004).
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Table 2. Fatty acid profiles of Chilean chia oil (Salvia hispanica L.)
Fatty acids Chia oil (g/100 g) a
C14:0 0.07
C15:0 0.05
C16:0 7.07
C16:1 0.08
C17:0 0.07
C17:1 0.03
C18:0 3.36
C18:1 7.04
C18:2 trans 0.16
C18:2 18.23
C18:3 trans 0.40
C18:3 62.80
C20:0 0.29
C20:1 0.14
C22:0 0.09
C24:0 0.12
SFA 11.12
MUFA 7.29
PUFA 81.59
n-6/n-3 ratio 0.29
a Data are expressed as relative percentages of total methyl esters (n = 3). SFA:
saturated fatty acid, MUFA: monounsaturated fatty acid, PUFA: polyunsaturated fatty
acid.
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Figure 1. GC chromatogram (fatty acid profile) of chia oil (Salvia hispanica L.). Major
peaks and relative retention times: a palmitic acid (C16:0): 22.174 min; b stearic acid
(C18:0): 25.107 min; c,d oleic acid (C18:1): 25.557 and 25.668 min; e linoleic acid
(C18:2): 26.380 min; f α-linolenic acid (C18:3): 27.437 min.
3.3. Triacylglycerol composition of chia oil
Figure 2 shows a typical EASI(+)-MS TAG profile of the chia oil whereas Table
3 summarizes the observed TAG composition of the chia oil. Different TAG were
found in chia oil, in which the most abundant [TAG + Na]+ ions are those of m/z 895,
897, 899 corresponding to LnLnLn, LLnLn, LLLn, respectively, whereas the other
ions identified are also abundant. The same TAG identified as [TAG + Na]+ were also
identified as [TAG + K]+, however such ions displayed low abundance. Predominance
of [TAG + K]+ ions of LnLnLn, LLnLn, LLLn were also observed which corresponded
to those of m/z 911, 913, 915, respectively. Linolenic acid is present in the most TAG
found, which is in agreement with findings reported by Ixtaina et al. (2011) in chia oil
from Argentina and Guatemala. Furthermore, results obtained by EASI(+)-MS are in
agreement with the fatty acid composition of chia oil obtained by GC (Table 2). The
85
knowledge of the TAG profile of oil is useful to direct its proper use by the chemical,
food and pharmaceutical industries (Haddad et al., 2006).
Figure 2. TAG fingerprints obtained by EASI(+)-MS of chia oil (Salvia hispanica L.).
Table 3. Main TAG ions detected by EASI-MS in chia oil (Salvia hispanica L.)
TAG a Elemental
composition
[TAG + Na]+
m/z
[TAG + K]+
m/z
LnLnP C55H94O6 873 889
LLnP C55H96O6 875 891
LLP/LnOP C55H98O6 877 893
LnLnLn C57H92O6 895 911
LLnLn C57H94O6 897 913
LLLn/LnLnO C57H96O6 899 915
LLL/LLnO/LnLnO C57H98O6 901 917
LLO/LLnS/LnOO C57H100O6 903 919
a L = linoleic acid, Ln = linolenic acid, O = Oleic acid, P = palmitic acid and S = stearic
acid.
3.4. Lipid autoxidation products
Table 4 shows the values of peroxide index and MDA of chia seed and oil.
Peroxide index value of Chilean chia oil (2.56 mEq peroxide/kg) was similar to those
reported by Ayerza and Coates (2004) in chia oil of South America (2.7 – 3.8 mEq
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oxygen/kg) and by Ixtaina, Nolasco and Tomás (2012) in Argentina chia oil (1.0 mEq
peroxide/kg). Additionally, the samples from Chile presented lower values than
flaxseed oil (Khattab & Zeitoun, 2013). An important finding was to observe that none
of the samples exceeded the upper limit of peroxide index value (10.0 mEq active
oxygen /kg oil) established by the Codex Alimentarius (1999).
Chia oil contains bioactive components, such as tocopherols, polyphenols and
carotenoids (Ixtaina et al., 2011), which may be responsible for keeping low peroxide
values and consequently may present a good oxidative stability.
Regarding the secondary lipid autoxidation product, MDA value of Chilean
chia oil (17.46 mg MDA/kg) was higher than those reported by Martysiak-Żurowska
and Stołyhwo (2006) in soybean oil (9.17 mg MDA/kg) and in rapeseed oil (11.12 mg
MDA/kg). However, mean absorbance of chia oil at 535nm (0.044) was markedly
lower than canola oil (0.53) found by Hawrysh et al. (1988). Furthermore, the MDA
value (9.58 mg MDA/kg) or mean absorbance (0.052) found in Chilean chia seeds
were lower than those mentioned above.
Although the TBA test is one of the most commonly used chemical assays to
determine secondary oxidation products, this method has limitations, such as lack of
specificity and sensitivity. In addition to MDA, some other substances may react with
the TBA reagent and contribute to absorption, causing an overestimation of the
intensity of color complex (Pignitter & Somoza, 2012).
Table 4. Values of peroxide index and malondialdehyde of chia seed and chia oil
(Salvia hispanica L.) a
PI MDA
(mEq
peroxide/kg)
Absorbance
(535nm)
TBA
(mg MDA/kg)
Chia Seed - 0.052 ± 0.001 9.58 ± 0.19
Chia oil 2.56 ± 0.11 0.044 ± 0.002 17.46 ± 0.62
a Data are expressed as means values ± standard deviation (n = 4). PI: peroxide
index, MDA: malondialdehyde, TBA: 2-thiobarbituric acid.
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3.5. Total phenolic content
Regarding the total phenolic content, previous reports are also consistent with
our results. Chia seed from two different regions in Mexico showed values between
0.88 to 0.92 mg GAE/ g (Reyes-Caudillo et al., 2008), which is similar with that found
for our samples from Chile (0.94 mg GAE/ g) (Table 6).
3.6. Polyphenol analysis
Table 5 shows the major phenolic compounds identified in chia seed and chia
oil.
3.6.1 Chia seed
Chia seed extract evidenced the presence of chlorogenic acid, quercetin,
myricetin and decarboxymethyl elenolic acid linked to hydroxytyrosol (3,4-DHPEA-
EDA) by EASI(-)-MS (Table 5, Figure 3.A), which is in agreement with some findings
from other studies (Capitani et al., 2012; Reyes-Caudillo et al., 2008) despite the
presence of the 3,4-DHPEA-EDA and absence of caffeic acid and kaempferol in our
samples. These differences may occur due to different extraction and identification
techniques, losses during processing, in addition to the location variation of seeds,
which, in turn could have a significant impact on concentration of bioactive
compounds (Ayerza & Coates, 2011). Recently, Reyes-Caudillo et al. (2008) found
that the major phenolic components of Mexican chia seed crude extracts are
glycosides of quercetin and kaempferol, furthermore, hydrolyzed extracts showed
significant amounts of the same aglycon phenolic compounds.
3.6.2 Chia oil
Myricetin, quercetin, kaempferol and chlorogenic acid were identified in chia oil
(Table 5, Figure 3.B). These compounds are the same substances found by Ixtaina,
et al. (2011) and this makes the oil quite stable in spite of its high PUFA content
(Ixtaina et al., 2012). It is interesting to note that most of the phenolic compounds
found in chia oil are not present in other oilseeds (Tuberoso et al., 2007).
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Table 5. Identified phenolic compounds in chia seed and oil (Salvia hispanica L.)
analyzed by EASI-MS
Compounds Elemental
composition
[M - H]-
m/z Chia seed Chia oil
Quercetin C15H10O7 301.04 X X
Chlorogenic acid C16H18O9 353.09 X X
Kaempferol C15H10O6 285.05 X
Myricetin C15H10O8 317.04 X X
3,4-DHPEA-EDA C19H22O8 377.13 X
( X ) Means that the compound is present in sample.
Figure 3. Polyphenols fingerprints obtained by EASI(-)-MS of (A) chia seed extract
and (B) chia oil (Salvia hispanica L.).
3.7. Antioxidant potential of chia seeds
Table 6 displays antioxidant activity of chia seeds. All the methods evaluated
in this study provided quite similar results to the ones reported by Capitani et al.
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(2012) for Argentina chia meals (557.2 TEAC, µmol/g) and fibrous fractions (446.4
TEAC, µmol/g) obtained by pressing extraction; and by Vázquez-Ovando et al.
(2009) for a Mexican chia fibrous fraction (488.8 TEAC, µmol/g).
The samples also displayed higher antioxidant activity than that found in wheat
flour, barley and sorghum whole grain cereals (8.3, 14.9 and 51.7 TEAC, µmol/g)
(Ragaee et al., 2006) and were similar to that of sorghum bran with high tannin
content (512.0 TEAC, µmol/g) (Awika et al., 2003). Chia seeds might therefore have
the same health beneficial effects attributed to other grains and vegetables.
Table 6. Antioxidant activity of Chilean chia seed (Salvia hispanica L.)
Antioxidant activity Results a
Total phenolic content 0.94 ± 0.06 mg GAE/g
ORAC hydrophilic 517.30 ± 9.09 µmol TE/g
ORAC lipophilic 6.48 ± 0.47 µmol TE/g
TAC 523.78 µmol TE/g
DPPH 436.61 ± 9.67 µmol TE/g
FRAP 405.71 ± 6.55 µmol TE/g
a Data are expressed as means values ± standard deviation (n = 4). TAC: total
antioxidant capacity.
Results regarding inhibition of lipid peroxidation in crude extract of the Sinaloa
and Jalisco chia seeds suggest that the phenols in both extracts have an important
activity as oxygen singlet quenchers, which could explain the antioxidant potential
found in chia seeds extract (Reyes-Caudillo et al., 2008).
Wu et al. (2004) investigated lipophilic and hydrophilic antioxidant capacities
using ORAC assay in different nuts. For H-ORAC, the lowest value found was 4.43
µmol TE/g for pine nut and the highest value was 175.2 µmol TE/g for pecan. For L-
ORAC, the lowest value found was 1.72 µmol TE/g for almonds and the highest
value was 5.57 µmol TE/g for Brazil nuts. The present results show that Chilean chia
seeds (H-ORAC) and oil (L-ORAC) have a high antioxidant capacity compared to the
previous study that used the same in vitro methodologies (Table 6). This could be
90
due to a high content of polyphenolic compounds (Reyes-Caudillo et al., 2008) and
lipophilic compounds, such as tocopherols, polyphenols, carotenoids and
phospholipids already found in chia seed oils from Argentina and Guatemala (Ixtaina,
et al., 2011).
Total antioxidant capacity, as measured by ORAC or any other in vitro
methods, could provide no information on in vivo antioxidant effects. Many other
issues such as metabolism, absorption and physicochemical properties should also
be considered (Wu et al., 2004).
The antioxidant activity of a bioactive compound has been attributed to various
mechanisms, among which are prevention of chain initiation, binding of transition
metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen
abstraction and radical scavenging protecting against oxidative damage to DNA,
proteins, and lipids (Halliwell, 1994). In this way, the consumption of Chilean chia
seed and oil could be interesting for health improvement.
4. Conclusions
The Chilean chia seeds and oil are important sources of protein, dietary fiber,
omega-3 PUFA and bioactive compounds with nutritional and technological potential.
The main fatty acid was α-linolenic acid, which was present in the most TAG found in
chia oil. Chia seeds and oil also contains polyphenols, mainly, myricetin, quercetin,
kaempferol, chlorogenic acid and 3,4-DHPEA-EDA, that contributed to the high
antioxidant capacity and low levels of lipid autoxidation products found in the
samples, which could be used to produce functional ingredients in dietetic products
with commercial applications. The consumption of Chilean chia seed and oil can
therefore be an important alternative to improve consumer‘s health suggesting its use
as a functional food in human daily diet.
Conflicts of interest
This work does not present conflicts of interest.
All authors participated in protocol development, conception and design of the study,
result evaluation, writing, editing and have approved the final version of the
91
manuscript. None of the authors had any financial or personal interest in any
company or organization sponsoring the research.
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP, 2012/23813-4) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq 140386/2012-2 and 300533/2013-6).
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CAPÍTULO II
98
ARTIGO II
Artigo publicado em revista internacional: Food Research International, 76,
(2015), p. 666-674. http://dx.doi.org/10.1016/j.foodres.2015.07.039
Antioxidant potential of dietary chia seed and oil (Salvia hispanica L.) in diet-
induced obese rats
Rafaela da Silva Marineli a, Sabrina Alves Lenquiste a, Érica Aguiar Moraes a, Mário
Roberto Maróstica Jr a,*
a Department of Food and Nutrition, Faculty of Food Engineering, University of
Campinas – UNICAMP, Campinas, Sao Paulo, Brazil.
*Corresponding author. Email address: [email protected] Tel.: +55 19 3521-
4059; fax: +55 19 3521-4060. Address: Monteiro Lobato st., 80, zip code 13083-862,
Campinas, Sao Paulo, Brazil.
Abbreviations
AIN, American Institute of Nutrition; ALA, alpha-linolenic acid; CAT, catalase; FAME,
fatty acids methyl esters; FRAP, ferric reducing antioxidant power; GC, gas
chromatography; GPx, glutathione peroxidase; GRd, glutathione reductase; GSH,
reduced thiol; HFF, high-fat and high-fructose diet; LDL, low-density lipoprotein;
MDA, malondialdehyde; PB, phosphate buffer; ROS, reactive oxygen species; SOD,
superoxide dismutase; TBA, 2-thiobarbituric acid; TBARS, thiobarbituric acid reactive
substances; TCA, trichloroacetic acid.
99
Abstract
This study aimed to investigate the effects of dietary chia seed and oil on plasma and
liver oxidative status in diet-induced obese rats. Thirty-six Wistar rats were divided in
six groups (6 animals each): control group was fed the American Institute of Nutrition
(AIN)-93M diet; HFF group was fed a high-fat and high-fructose (HFF) diet; chia seed
short (6-weeks) and long (12-weeks) treatments received an HFF diet with chia
seed; chia oil short (6-weeks) and long (12-weeks) treatments received an HFF diet
with chia oil. Plasma and hepatic biomarkers of lipid peroxidation, endogenous
enzymatic and non-enzymatic antioxidant systems and antioxidant capacity were
determined. HFF diet induced weight gain, oxidative stress and lipid peroxidation in
plasma and liver of animals. Compared to HFF group chia seed and chia oil (12 and
6 weeks) intake increased plasma reduced thiol (GSH) levels, plasma catalase
(CAT) and glutathione peroxidase (GPx) activities. In the liver glutathione reductase
(GRd) activity was enhanced, while CAT and GPx activities did not change. There
were no differences in plasma and liver superoxide dismutase activity among chia
diets and HFF group. Chia (seed and oil) intake did not modify liver lipid
peroxidation, but was able to reduce plasma thiobarbituric acid reactive substances
(TBARS) and 8-isoprostane levels increased by HFF group. Plasma and hepatic
antioxidant capacity values were increased in chia seed and oil groups about 35%
and 47%, respectively, compared to HFF group. Chia groups presented similar
antioxidant potential, regardless of treatment time. Dietary chia seed and oil reduced
oxidative stress in vivo, since it improved antioxidant status and reduced lipid
peroxidation in diet-induced obese rats.
Keywords: Chia (Salvia hispanica), Omega-3 fatty acid, Oxidative stress, Lipid
peroxidation, Antioxidant enzymes, Rats.
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1 Introduction
Obesity has become a worldwide epidemic and may be a state of chronic
oxidative stress, which consists in an imbalance between protector endogenous
antioxidant systems and free radicals generation (Fernández-Sánchez et al., 2011).
The reactive oxygen species (ROS) excess can damage cell proteins, lipids and
DNA, which might result in loss of function and even cellular death. This mechanism
has been linked to many diseases, such as cardiovascular disease,
neurodegeneration, cancer and diabetes (Brambilla et al., 2008; Pandey & Rizv,
2010). Furthermore, obesity impairs the enzymatic antioxidant system, with reduction
of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and
glutathione reductase (GRd) activities, and also affects the non-enzymatic
antioxidant systems (reduced thiol, minerals, vitamins and polyphenols) which play
essential roles in many antioxidant mechanisms (Brambilla et al., 2008; Fernández-
Sánchez et al., 2011).
Many investigations have shown that consumption of natural dietary sources
(fruits, nuts, vegetables) with antioxidant bioactive compounds (polyphenols,
tocopherols, carotenoids, vitamins) may support the prevention of oxidative stress
and could be a natural alternative for preventing and controlling chronic diseases
(Bullo et al., 2011; Avignon et al., 2012; Landete, 2012). Several mechanisms have
been proposed for this health protection, such as the improvement of antioxidant
defense system which protects and reduces ROS damage in the organism
(Brambilla et al., 2008; Avignon et al., 2012). Therefore, the evaluation of potential
functional foods that may improve antioxidant systems efficacy or alleviate reactive
oxygen and nitrogen species formation could be a strategy to prevent obesity
complications.
In this context, chia (Salvia hispanica L.) is an herbaceous plant that belongs
to Lamiaceae family native from southern Mexico and northern Guatemala (Ayerza,
1995). Today it is commercially available for human consumption as whole seeds,
flour, and oil in the Americas, Australia, Europe and Southeast Asia (Mohd Ali et al.,
2012). Chia seed is the richest known botanical source of omega-3 α-linolenic acid
(C18:3, ALA, up to 68%) (Ayerza, 1995) compared to flaxseed (50.6%), rapeseed
(8.1%), soybean (7.6%) and sunflower (1.8%) oils (Tuberoso et al., 2007); and has
101
been also described as an important source of protein, dietary fiber, minerals and
bioactive compounds (Reyes-Caudillo, Tecante & Valdivia-López, 2008; Marineli et
al., 2014). Literature data indicates that chia seed and oil possess high antioxidant
potential confirmed by different in vitro assays (Reyes-Caudillo et al., 2008;
Martínez-Cruz & Paredes-López, 2014; Marineli et al., 2014). Chia seed and chia oil
are considered new sources of natural antioxidants, due to the content of
tocopherols, phytosterols, carotenoids (Álvarez-Chávez et al., 2008; Ixtaina et al.,
2011) and phenolic compounds (Reyes-Caudillo et al., 2008; Martínez-Cruz &
Paredes-López, 2014), which have the potential to protect consumers against many
diseases and also promotes beneficial effects on human health (Avignon et al., 2012;
Landete, 2012). Chia lipids could be an omega-3 alternative source to vegetarians
and people with fish allergies, since chia seed and oil have not shown problems
associated with other nutritional sources such as flaxseed and marine products,
including fish flavor, animal weight loss and digestive problems (Mohd Ali et al.,
2012).
Our previous study (Marineli et al., 2015) reported that chia ingestion induced
heat shock protein expression and restored SOD and GPx expression in skeletal
muscle, with a higher potential of oil relative to seed. However, chia seed and oil has
been little explored from a scientific point of view, especially in relation to its
antioxidant potential in vivo. To the best of our knowledge, this is the first report
about antioxidant effects in liver and plasma after chia seed and oil intake. We
hypothesized that dietary chia seed and chia oil could protect against oxidative
stress by improving antioxidant defenses and reducing lipid peroxidation in diet-
induced obese rats, due to the content of bioactive compounds. Therefore, the
present study aimed to evaluate the effects of dietary chia seed and chia oil on
oxidative stress and antioxidant biomarkers of plasma and liver in diet-induced obese
Wistar rats.
2 Materials and methods
2.1 Chemical, chia seed and oil
Solvents were all analytical grade from J.T. Baker (Phillipsburg, NJ, USA).
Metaphosphoric acid was purchased from Vetec Química Fina (Sao Paulo, Brazil). L-
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Glutathione reduced, glutathione reductase, glutathione oxidized form disodium salt,
β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate
(NADPH), 5′5′-dithio-bis-2-nitrobenzoic acid (DTNB), albumin from bovine serum
(BSA), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,4,6-
tris(2-pyridyl)-s-triazine (TPTZ), 2-thiobarbituric acid (TBA) were all obtained from
Sigma-Aldrich (St. Louis, MO, USA). Trichloroacetic acid (TCA) was purchased from
Synth Laboratorios (Sao Paulo, Brazil), Bradford reagent was purchased from
BioAgency (Sao Paulo, Brazil) and malondialdehyde (MDA) was purchased from
Cayman Chemical Company (Ann Arbor, MI, USA). Fresh chia seed and chia oil
from FTP S.A. Santiago, Chile were purchased from R&S Blumos Comercial de
Produtos Alimentícios Ltda, Brazil. According to the producer, chia oil was obtained
by cold pressing (< 40 °C) and stored at 2-8 °C until use in amber glass bottles
without head space. Seeds were ground in a laboratory impact mill (Marconi MA
630/1, Piracicaba – Sao Paulo, Brazil) and passed through a 0.850 mm sieve.
Chemical analysis and antioxidant activity of Chilean chia seed and oil samples used
in this study were previously determined by Marineli et al. (2014) in the same raw
material.
2.2 Animals, diets and interventions
This work was approved by the Ethics Commission on Animal Use (CEUA/
UNICAMP, protocol no. 2936-1) and followed the University guidelines for the use of
animals in experimental studies. Thirty-six male Wistar rats aged 21 to 23 days were
obtained from Multidisciplinary Center for Biological Investigation, University of
Campinas. Animals remained in individual cages for growth with free access to water
and chow diet for 4 weeks and were maintained under controlled conditions (22 ± 1
ºC, 60-70% humidity, 12 hours light/dark cycle). After growth, animals (234.08 g ±
19.12) were randomly divided in six experimental groups (n=6/group) for 12 weeks.
Diets were based on the American Institute of Nutrition (AIN)-93M diet
(Reeves, Nielsen & Fahey, 1993) with protein concentration of 12%. Control group
received the standard diet (AIN-93M); High-fat and high-fructose (HFF) group
received a diet containing 4% (w/w) soybean oil, 31% (w/w) lard and 20% fructose
(w/w) (Shapiro et al., 2011); chia seed short and long treatments received an HFF
103
diet with 13.3% (w/w) of chia seed; chia oil short and long treatments received an
HFF diet with 4% (w/w) of chia oil. There were a long (12 weeks) and a short (6
weeks) treatment with chia seed or chia oil. Animals from the long treatment were
fed only an HFF diet containing chia seed or chia oil for 12 weeks. Short groups were
initially fed only an HFF diet for the first 6 weeks, followed by an additional 6 weeks
with an HFF diet containing chia seed or chia oil. Soybean oil for both chia seed and
oil diet was replaced by the oil content of chia seed or chia oil. Chia diets were
formulated to provide equal quantities of oil and ALA from chia (Ayerza & Coates,
2007). The seeds contained 30.2% of oil (Marineli et al., 2014). Protein and dietary
fiber content in HFF-chia seed groups was balanced taking into account the amount
of such nutrients present in chia seed (Marineli et al., 2014). Diets composition is
presented in Table 1. Diets were prepared monthly and packed in dark polyethylene
bags and stored at -20 ºC to minimize the oxidation of fatty acids. Weight gain was
monitored weekly and food intake every 2 days.
2.2.1 Diet analyses
Calorie values of diets were determined using isoperibol automatic calorimeter
(PARR 1261) instrument equipped with oxygen bomb (PARR 1108). (Instrument Co,
Moline, IL, USA). Lipids were extracted according to the method of Bligh and Dyer
(1959) and samples were saponified in 0.5 mol/L methanolic KOH. Fatty acids
methyl esters (FAME) were prepared as previously described by Maia and
Rodriguez-Amaya (1993). The fatty acid composition of the experimental diets was
determined by gas chromatography (GC) (Kramer et al., 1997) using a capillary GC
Agilent 6850 Series GC System (Agilent Technologies, Wilmington, DE, USA),
capillary column DB-23 Agilent (50% cyanopropyl-methylpolysiloxane, 60 m × 0.25
mm × 0.25 mm) (Wilmington, DE, USA), with flame ionization detector (FID). Oven
temperature was 110 °C for 5 min, 110-215 °C (5 °C/min), 215 °C for 24 min;
detector temperature: 280 °C; injector temperature: 250 °C; carrier gas: helium; split
ratio 1:50; injection volume: 1.0 µL. FAME were identified by comparison of retention
times with FAME standard mixture under the same conditions (FAME Mix GLC-68A;
Nu-Chek-Prep, INC, Elysian, MN, USA) and quantified using area normalization.
104
Table 1. Ingredients and fatty acid composition of experimental diets
AIN-93M HFF Chia seed Chia oil
Ingredients (g/kg diet)
Casein a 139.53 139.53 106.53 139.53
Maltodextrin 155.0 45.40 45.40 45.40
Corn starch 465.69 136.44 123.44 136.44
Sucrose 100.0 29.32 29.32 29.32
Soybean oil 40 40 - -
Chia oil - - - 40
Chia seed - - 133 -
Lard - 310 310 310
Fructose - 200 200 200
Cellulose 50 50 3.6 50
Mineral mixture 35 35 35 35
Vitamin mixture 10 10 10 10
L-Cystine 1.8 1.8 1.8 1.8
Choline bitartrate 2.5 2.5 2.5 2.5
tert-Butyl hydroquinone 0.008 0.008 0.008 0.008
Energy density (kcal/g
diet) 3.74 5.89 5.84 5.88
Fatty acids b
SFA 7.38 127.86 125.35 125.99
MUFA 10.20 146.94 138.87 140.11
PUFA 22.42 71.10 81.11 79.54
Linoleic (C18:2n-6) 19.89 66.74 54.97 53.53
α-Linolenic (C18:3n-3) 2.20 4.06 25.88 25.73
n-6/n-3 ratio 9.04 16.44 2.12 2.08
a Amount was calculated based on protein content equal to 86% to provide 12 g of
protein/100 g of diet. b Fatty acids expressed as g/kg diet. AIN-93M group: control
standard diet; HFF group: high-fat and high-fructose diet; Chia seed groups: HFF diet
plus 13.3% (w/w) chia seed; Chia oil groups: HFF diet plus 4% (w/w) chia oil. SFA:
105
saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty
acids.
2.3 Blood and tissue collection
Animals were euthanized by decapitation preceded by 12-hour-fasting after 12
weeks of experimental period. Blood samples were collected in polyethylene tubes
with EDTA anticoagulant to obtain plasma. Tubes were centrifuged at 3000 g (15
min, 4 ºC). Plasma was separated and stored in polypropylene microtubes at -80 °C
until analysis. Liver was removed, washed, frozen in liquid nitrogen and stored at -
80ºC. Liver homogenates were prepared in a ratio of about 100 mg wet tissue per 1
mL of 50 mmol/L phosphate buffer (PB) (pH 7.4) or 5% trichloroacetic acid (TCA)
solution using a homogenizer (MA102/Mini, Marconi, Piracicaba – Sao Paulo, Brazil).
PB and TCA homogenates were used in the enzymatic antioxidant activities and
GSH assays, respectively (Batista et al., 2014). Liver was also freeze-dried,
manually ground with mortar and pistil and kept at -80 °C until analysis of lipid
peroxidation.
2.4 Biochemical analyses
Absorbance readings were done in a microplate reader Synergy HT, Biotek
(Winooski, VT, USA); with Gen5™ 2.0 data analysis software spectrophotometer.
2.4.1 Endogenous enzymatic and non-enzymatic antioxidant system
The protein concentration of tissue homogenates was determined by Bradford
method (Bradford, 1976).
2.4.1.1 Reduced thiol (GSH) content
GSH levels were determined in plasma and liver TCA homogenates by
Ellman's reaction using DTNB (Faure & Lafond, 1995). GSH solution (2.5–500 nmol
GSH/mL) was used as standard curve and absorbance was read at 412 nm. Results
were expressed as nmol GSH/µg protein or nmol GSH/mL plasma.
106
2.4.1.2 Glutathione reductase (GRd) activity
GRd activity was measured in plasma and liver PB homogenates by the
method described by Carlberg and Mannervik (1985). The decrease in absorbance
was monitored at 340 nm after induction with 1 mmol/L oxidized glutathione in the
presence of 0.1 mmol/L NADPH in PB. Results were expressed as nmol NADPH
consumed/min/mg protein or nmol NADPH consumed/min/mL plasma.
2.4.1.3 Glutathione peroxidase (GPx) activity
GPx activity was quantified in plasma and liver PB homogenates according to
Flohe and Gunzler (1984). This assay is based on the oxidation of 10 mmol/L
reduced glutathione by GPx coupled to the oxidation of 4 mmol/L NADPH by 1 unit
(U) enzymatic activity of GRd in the presence of 0.25 mmol H2O2. The rate of
NADPH oxidation was monitored by the decrease in absorbance at 365 nm. Results
were expressed as nmol NADPH consumed/min/mg protein or nmol NADPH
consumed/min/mL plasma.
2.4.1.4 Catalase (CAT) activity
CAT activity was analyzed in plasma and liver PB homogenates using
commercial assay kits (#707002, Cayman Chemical Company, Ann Arbor, MI, USA).
The method is based on reaction of the enzyme with methanol in the presence of an
optimal concentration of H2O2. The formaldehyde produced is measured
colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the
chromogen. Results were expressed as nmol CAT activity/min/g protein or nmol CAT
activity/min/mL plasma.
2.4.1.5 Superoxide dismutase (SOD) activity
SOD activity was analyzed in plasma and liver PB homogenates using
commercial assay kits (#706002, Cayman Chemical Company, Ann Arbor, MI, USA).
The method utilizes a tetrazolium salt for detection of superoxide radicals generated
by xanthine oxidase and hypoxanthine. SOD assay measures all three types of SOD
107
(Cu/Zn, Mn, and FeSOD). One unit of SOD is defined as the amount of enzyme
needed to exhibit 50% dismutation of the superoxide radical. Results were
expressed as U SOD activity/mg protein or U SOD activity/mL plasma.
2.4.2 Lipid peroxidation
2.4.2.1 TBARS (thiobarbituric acid reactive substances) assay
TBARS determinations were done in plasma and liver according to Ohkawa,
Ohishi, and Yagi (1979), with adaptations. Hepatic freeze-dried tissue (10 mg/mL)
was sonicated for 30 min in a 300 mmol/L acetate buffer solution (pH 3.6). Plasma
samples (100 µL) were used without previous preparation. Working solution was set
with 2-thiobarbituric acid (TBA), acetic acid (20%) and sodium hydroxide (5%) at
ratio of 1:100:100 (w:v:v). One hundred microliters of 8.1% sodium dodecyl sulfate
solution and 4 mL of work solution were homogenized with samples and standard
and heated at 95 °C for 60 min. Samples were cooled in an ice bath for 10 min and
then centrifuged at 10,000 g (10 min, 4 °C). The supernatant was read at 532 nm
using a 96-well microplate. Standard curve (0.625–85 nmol MDA/mL) was obtained
using malondialdehyde standard (MDA) (#10009202, Cayman Chemical Company,
Ann Arbor, MI, USA). Results were expressed as nmol MDA/mg tissue or nmol
MDA/mL plasma.
2.4.2.2 8-isoprostane assay
8-Isoprostane was determined in plasma samples by an enzyme
immunoassay (EIA) kit (#516351, Cayman Chemical Company, Ann Arbor, MI,
USA). The method is based on the competition between 8-isoprostane and an 8-
isoprostane-acetylcholinesterase (AChE) conjugate (marker) for a limited number of
8-isoprostane-specific rabbit antiserum binding sites. Results were expressed as pg
8-Isoprostane/mL plasma.
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2.4.3 Antioxidant capacity measured by ferric reducing antioxidant power (FRAP)
assay
Plasma samples and liver PB homogenates were treated with
ethanol:ultrapure water (2:1) and 0.75 mol/L metaphosphoric acid (Batista et al.,
2014). Samples were centrifuged at 14000 g for 5 min at 4 °C and the supernatant
removed. The ferric reducing ability of plasma and liver extracts was determined by
FRAP method (Benzie & Strain, 1996), with adaptations. Under dark conditions,
FRAP reagent was prepared with 10 mmol/L TPTZ, 20 mmol/L FeCl3 and 300
mmol/L acetate buffer (pH 3.6) following the 1:1:10 ratio. Samples or standard
solutions, ultrapure water and FRAP reagent were mixed in 1:3:30 ratios and kept in
a water bath for 30 min at 37 °C. After cooling to room temperature, samples and
standard were read at 595 nm. Trolox standard curve was made (5–400 μmol/L).
Results were expressed as μmol Trolox equivalent (TE)/g protein or μmol TE/L
plasma.
2.5 Statistical analysis
Data are expressed as mean values ± standard deviation. Differences
between AIN-93M and HFF group were tested by T test. Differences among HFF,
chia seed 12 weeks, chia seed 6 weeks, chia oil 12 weeks and chia oil 6 weeks
groups were tested by one-way analysis of variance (ANOVA) followed by Tukey's
test using the GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA)
software. P<0.05 was considered statistically significant.
3 Results
3.1 Food intake and weight gain
Total food intake was lower (Fig. 1A) and total energy intake was higher (Fig.
1B) in HFF-fed animals compared to AIN-93M group. These parameters did not differ
among the chia (seed or oil) and HFF groups. Animals fed on the HFF diet showed
an increase in weight gain compared to AIN-93M group (Fig. 1C). Chia (seed or oil)
intake did not attenuate rat‘s weight gain, regardless of treatment time, when
compared to HFF group.
109
Figure 1. Total food intake (A), Total energy intake (B) and Total weight gain (C) of
rats fed on different diets: control standard diet (AIN-93M); high-fat and high-fructose
(HFF) diet; HFF diet plus 13.3% chia seed during 12 weeks (chia seed 12-wk) or 6
weeks (chia seed 6-wk); HFF plus 4% chia oil during 12 weeks (chia oil 12-wk) or 6
weeks (chia oil 6-wk). Different capital letters (A-B) mean significant differences
110
between AIN-93M and HFF group according to T test (P<0.05). Different small letters
(a-b) mean significant differences between HFF, chia seed and chia oil groups by
one-way analysis of variance followed by Tukey‘s test (P<0.05). Data expressed as
mean ± standard deviation (n = 6/group).
3.2 Enzymatic antioxidant activities and GSH levels
Animals fed with HFF diet showed the lowest plasma and liver CAT activity
when compared to AIN-93M group (Fig. 2A and Fig. 3A, respectively). All chia seed
and chia oil groups presented higher plasma CAT activity and no changes in liver
CAT activity in relation to HFF group. Although plasma SOD activity did not differ
among the experimental groups (Fig. 2B), HFF group presented lower liver SOD
activity compared to AIN-93M group (Fig. 3B). However, the groups that consumed
chia seed or chia oil diet did not show differences in liver SOD activity compared to
HFF group.
Plasma and liver GSH values decreased in HFF-fed animals compared to
AIN-93M group (Fig. 2E and Fig. 3E, respectively). Chia groups (seed and oil)
showed an increase in plasma GSH level compared to HFF group. Moreover, the
group that received chia seed diet for 12 weeks showed a higher GSH level in liver
(28%) compared to HFF group and also compared to other chia groups.
HFF-fed animals decreased plasma and liver GPx and GRd activities
compared to AIN-93M group (Fig. 2C,D and Fig. 3C,D respectively). Chia groups
(seed and oil) showed an increase in plasma GPx and liver GRd activities, but not in
plasma GRd and liver GPx activities compared to HFF group.
111
Figure 2. Plasma antioxidant enzymes: catalase (CAT) activity (A), superoxide
dismutase (SOD) activity (B), glutathione peroxidase (GPx) (C), glutathione
reductase (GRd) (D), and Thiol groups (GSH) (E) in plasma of rats fed on different
diets: control standard diet (AIN-93M); high-fat and high-fructose (HFF) diet; HFF
diet plus 13.3% chia seed during 12 weeks (chia seed 12-wk) or 6 weeks (chia seed
112
6-wk); HFF plus 4% chia oil during 12 weeks (chia oil 12-wk) or 6 weeks (chia oil 6-
wk). Different capital letters (A-B) mean significant differences between AIN-93M and
HFF group according to T test (P<0.05). Different small letters (a-b) mean significant
differences between HFF, chia seed and chia oil groups by one-way analysis of
variance followed by Tukey‘s test (P<0.05). Data expressed as mean ± standard
deviation (n = 6/group).
113
Figure 3. Liver antioxidant enzymes: catalase (CAT) activity (A), superoxide
dismutase (SOD) activity (B), glutathione peroxidase (GPx) (C), glutathione
reductase (GRd) (D) and Thiol groups (GSH) (E) in liver of rats fed on different diets:
control standard diet (AIN-93M); high-fat and high-fructose (HFF) diet; HFF diet plus
13.3% chia seed during 12 weeks (chia seed 12-wk) or 6 weeks (chia seed 6-wk);
114
HFF plus 4% chia oil during 12 weeks (chia oil 12-wk) or 6 weeks (chia oil 6-wk).
Different capital letters (A-B) mean significant differences between AIN-93M and
HFF group according to T test (P<0.05). Different small letters (a-b) mean significant
differences between HFF, chia seed and chia oil groups by one-way analysis of
variance followed by Tukey‘s test (P<0.05). Data expressed as mean ± standard
deviation (n = 6/group).
3.3 Lipid peroxidation
HFF-fed animals increased plasma 8-isoprostane levels and plasma and liver
TBARS compared to AIN-93M group (Fig. 4A,B and C, respectively). Chia (seed or
oil) intake did not modify liver TBARS levels in comparison to HFF group. On the
other hand, chia diets were able to reduce plasma TBARS and 8-isoprostane levels
compared to HFF group.
3.4 Antioxidant capacity by FRAP assay
HFF diet promoted a decrease in plasma and liver antioxidant values
compared to AIN-93M group (Fig. 5A,B). The addition of chia (seed or oil) to the
diets reversed the effect caused by the HFF diet. Plasma FRAP values were
increased in chia groups about 35% and liver FRAP values were higher in chia
groups about 47% as compared to HFF group.
115
Figure 4. Lipid peroxidation by 8-isoprostane assay in plasma (A) and TBARS assay
in plasma (B) and liver (C) of rats fed on different diets: control standard diet (AIN-
93M); high-fat and high-fructose (HFF) diet; HFF diet plus 13.3% chia seed during 12
weeks (chia seed 12-wk) or 6 weeks (chia seed 6-wk); HFF plus 4% chia oil during
12 weeks (chia oil 12-wk) or 6 weeks (chia oil 6-wk). Different capital letters (A-B)
116
mean significant differences between AIN-93M and HFF group according to T test
(P<0.05). Different small letters (a-b) mean significant differences between HFF, chia
seed and chia oil groups by one-way analysis of variance followed by Tukey‘s test
(P<0.05). Data expressed as mean ± standard deviation (n = 6/group).
Figure 5. Ferric Reducing Antioxidant Power (FRAP) assay in plasma (A) and liver
(B) of rats fed on different diets: control standard diet (AIN-93M); high-fat and high-
fructose (HFF) diet; HFF diet plus 13.3% chia seed during 12 weeks (chia seed 12-
wk) or 6 weeks (chia seed 6-wk); HFF plus 4% chia oil during 12 weeks (chia oil 12-
wk) or 6 weeks (chia oil 6-wk). Different capital letters (A-B) mean significant
differences between AIN-93M and HFF group according to T test (P<0.05). Different
small letters (a-b) mean significant differences between HFF, chia seed and chia oil
groups by one-way analysis of variance followed by Tukey‘s test (P<0.05). Data
expressed as mean ± standard deviation (n = 6/group).
117
4 Discussion
Excessive energy intake from saturated fatty acids leads to an increase in
energy storage as fat, accompanied by an elevation in mitochondrial macronutrient
oxidation, favoring an excessive production of free radicals and oxidative stress
(Avignon et al., 2012). Obesity and more specifically visceral fat are associated with
oxidative stress in humans, which may contribute to metabolic syndrome
development (Fernández-Sánchez et al., 2011).
Our data show that a HFF diet intake induced lipid peroxidation, reduced
antioxidant capacity and enzyme activities involved in the antioxidant defense
system in plasma and liver of rats. These findings are in agreement with those
reported in earlier animals (Feillet-Coudray et al., 2009; Laissouf et al., 2013) and
humans studies (Patel et al., 2007), which demonstrated that high-fat and high-
carbohydrate diets are able to induce oxidative stress. Furthermore, previous studies
showed that high-fructose diet intake negatively affects the balance between
antioxidant defense and free radical production in rats, leading to increased lipid
susceptibility to peroxidation (Busserolles et al., 2002). The increasing of fructose
catabolism causes a reduction in total glutathione levels and the suppression of
hepatic antioxidant enzyme activities, such as CAT, SOD and GPx (Reddy et al.,
2009).
In the present study, a daily consumption of chia seed and oil diets enhanced
plasma antioxidant status through CAT and GPx activity and GSH levels reduction.
However, no effect was observed in SOD and GRd activities, regardless of treatment
time. It is important to consider that different nutrients such as natural antioxidants
and other substances should not necessarily affect all enzymes of redox system in
the same fashion and to the same extent. Antioxidant enzymes respond
independently to different inducing radicals and therefore we should not expect these
enzymes respond identically (Kohen & Nyska, 2002).
Although plasma and liver SOD activity did not differ statistically after chia
ingestion, our previous study showed a restored SOD expression in skeletal muscle
after chia seed and oil intake. These results suggest that tissues respond differently
to the same diet intervention. Furthermore, we should highlight that different
methodologies were used in both experiments (Marineli et al., 2015). A previous
118
study (Laissouf et al., 2013) found that flaxseed oil diet (2.5 and 5%) intake
attenuated lipid and protein oxidation in different tissues and improved antioxidant
status reducing CAT and GPx in obese aged rats.
With regard to effects of chia seed and oil intake on lipid peroxidation, we
found a significant reduction in plasma 8-isoprostane and TBARS levels, suggesting
that both seed and oil could act as antioxidant agents and counteract the pro-
oxidative effect caused by HFF consumption. 8-isoprostane is a specific lipid
peroxidation product formed via a non-enzymatic mechanism which involves free
radical-catalyzed peroxidation of arachidonic acid and is the most specific biological
indicator for the assessment of oxidative stress in vivo (Musiek et al., 2005). In this
way, the dietary intake of chia prevents plasma 8-isoprostane increase in rats fed
with HFF diet which endorse the beneficial effect of chia seed and oil in reducing this
non-enzymatic product of lipid oxidation in vivo.
Dietary contribution of antioxidant compounds affects the overall resistance of
lipoproteins to lipid peroxidation (Shinitzky, 1984). In this way, the reduction in
plasma lipid peroxidation may be involved to hypolipidemic effect of chia seed and
chia oil previous shown by Ayerza & Coates, 2007 and Poudyal, et al., 2012. This
effect is associated with reduced susceptibility of low-density lipoprotein (LDL) to
oxidation, apparently due to the reduction in plasma LDL-cholesterol levels.
Although lipid peroxidation reduction demonstrated in plasma through TBARS
levels, it was not reversed in hepatic tissue, since liver TBARS levels remained
unchanged in chia seed and oil groups in both 6 and 12weeks of treatment
compared to HFF group. Lipid peroxidation is a basic cellular deteriorating process
induced by oxidative stress and occurs readily in the tissues rich in highly oxidizable
polyunsaturated fatty acids (PUFA), as in the liver (Halliwell, 1994). Furthermore,
investigations have shown that different types of fatty acids intake affect fatty acid
composition in a tissue-specific manner (Valenzuela et al., 2014). It indicates that
metabolic rate may be slower in the liver than in the plasma, which the plasma may
respond more readily to metabolizing fatty acids (Kang & Choi, 2008).
Interestingly, despite the non-reversal of lipid peroxidation in rats liver, chia
seed and oil groups showed an increase in liver GRd activity and no change in liver
GPx activity. In addition, only the group that received chia seed diet for 12 weeks
119
increased liver GSH levels. An increase in liver GRd activity may occur to recover
reduced glutathione involved in glutathione cycle. However, there are several
interactions among enzymes and substrates variations in glutathione cycle which
could explain the change or not of the enzymes of this cycle (Kohen & Nyska, 2002).
Moreover, the potential effect of daily chia consumption on these biomarkers is still
recent and not enough studied.
Plasma and liver antioxidant capacity expressed by FRAP assay was
markedly higher in chia seed and oil-fed animals, which seemed to contribute to an
improvement in the endogenous antioxidant defenses. In addition to the assay
validity itself, special attention has to be paid to confounding factors from sample
matrix when biological samples are measured. Many other issues such as
metabolism, absorption and other physiological activities should also be considered
(Huang, Ou & Prior, 2005). These limitations represent difficulties to be overcome in
further studies about bioavailability of bioactive compounds and their antioxidant
capacity in vivo.
Furthermore, higher plasma and liver antioxidant status found in chia groups
could explain previous studies that showed increased cardio and hepatoprotective
parameters in obese rats fed with chia seed diet (Poudyal et al., 2012).
Literature data show that different types of fatty acids have variable effects on
free radical production and each fatty acid acts direct on ROS scavenging and
indirect on antioxidant enzymes induction (Bullo et al., 2011). Chia seed oil is a rich
source of ALA (Ayerza, 1995) and previous studies with the same fatty acid from
other source (Pal & Ghosh, 2012a, 2012b) corroborates to our present results on the
antioxidant status in vivo. These authors found that dietary intake of 0.5-1.0% ALA,
extracted from linseed oil, attenuated organic mercury-induced oxidative stress in
liver, kidney and blood of rats. ALA presented antioxidant effects by reducing lipid
peroxidation and restoring antioxidant enzymes such as CAT, SOD and GPx (Pal &
Ghosh, 2012a, 2012b). Thus, it may be possible that in in vivo conditions ALA might
reduce the hydroperoxides formation by scavenging free radicals and PUFA
peroxidation that occurs in lipids.
Our results seem to indicate that the protective effects against oxidative stress
caused by HFF diet might be due to either natural antioxidants present in chia
120
(Marineli et al., 2014; Martínez-Cruz & Paredes-López, 2014) or to PUFA action,
especially ALA, that give a greater mobility in cell membranes and therefore greater
membrane access to the antioxidants (Shinitzky, 1984). A recent study (Valenzuela
et al., 2014) demonstrated that dietary ALA intake from different vegetable oils,
including chia oil, caused important modifications in fatty acid composition of
phospholipids extracted from the various tissues of rats. However, the metabolites
absorption extension in plasma or tissues was not assessed in this present study.
The endogenous antioxidant system is supplemented by exogenous nutrients,
i.e. vitamins, minerals, polyphenols and others. Additionally, previous study
demonstrated that chia seed and oil could be a rich source of bioactive compounds,
due to an elevated content of polyphenolic compounds, including chlorogenic acid,
caffeic acid, myricetin, quercetin and kaempferol, and lipophilic compounds, such as
tocopherols, phytosterols, carotenoids and phospholipids (Reyes-Caudillo et al.,
2008; Ixtaina et al., 2011; Martínez-Cruz & Paredes-López, 2014). Therefore, these
compounds may be responsible for the high antioxidant potential in vitro as we
previously found in Chilean chia seed and oil (Marineli et al., 2014), which could also
explain our present findings. The antioxidant activity of a bioactive compound has
been attributed to several mechanisms, among which are chain initiation prevention,
binding of transition metal ion catalysts, peroxides decomposition, prevention of
continued hydrogen abstraction and radical scavenging (Halliwell, 1994). Dietary
antioxidants, mainly polyphenolic compounds, are exposed to an intensive in vivo
metabolism which affects their chemical structure, bioavailability, tissue retention and
subsequently bioactivity (Landete, 2012). A previous study (Gladine et al., 2007)
investigated the effect of plant extracts rich in polyphenols on different tissues of rats
fed omega-3 rich diets. These authors demonstrated that tissues are differently
affected by polyphenols and suggested that PUFA supplements did not sensibilize
tissues in a similar extension and that hepatic metabolism of polyphenols could have
reduced their activity. Nevertheless, it is necessary to evaluate the antioxidant
potential of chia seed and oil against oxidative injury including phenolic compounds
absorption mechanism and bioactivity in vitro and in vivo assays.
Another important finding was to observe the same antioxidant status
improvement and oxidative stress reversion after ingestion of both, chia seed and
chia oil, as well as the treatment time which the animals were submitted. The
121
absence of significant differences between both treatments time could suggest that 6
weeks is the enough time to observe significant changes in plasma and/or tissue
biomarkers. Furthermore, a possible explanation for the similar effects of oil and
seed intake may be related to different food matrices (seed vs. oil), since nutrients
interaction between compounds present in chia seed and the complexity of food
matrix synergism should be considered. The nutrient releasing from food matrix,
interactions with other food components, presence of cofactors and stable
compounds formation influence the nutrient bioavailability (Parada & Aguilera, 2007).
Therefore, the matrix state of natural foods may favor or hinder their nutritional
response in vivo, since the mode of action is more relevant than the total amount
present in the original food.
However, we previously found that dietary ingestion of chia seed and oil
showed different responses in heat shock protein and antioxidant enzymes
expression in skeletal muscle, depending on the treatment time and the ingredient
(seed or oil) added to the diet (Marineli et al., 2015). These findings lead to the
discussion that chia may influence the oxidative stress process in different ways, and
this influence is dependent on the biomarker used, including its tissue and
determination method. Moreover, considering the lack of studies assessing the
antioxidant potential of dietary chia seed and oil in vivo, determination of bioactive
compounds and their main in vivo metabolites in plasma, urine and tissues by
specific analytical techniques are absolutely crucial. The results presented in this
study are a good starting point for further investigations that should conduct further
analysis to clarify the bioactivity and mechanisms of action related to the beneficial
effects of chia intake.
In summary, the present study show that a daily consumption of chia seed
and oil diets enhanced plasma and liver antioxidant status, reduced plasma lipid
peroxidation and promote a protective effect against oxidative stress arising from
obesity. Chia seed and oil presented similar in vivo antioxidant properties,
independent of treatment time, probably due to the presence of polyphenols, ALA
and other active compounds. Thus, chia seed and oil intake could possibly improve
antioxidant defense system in diet-induced obese rats, protecting against cellular
oxidative damage and obesity related diseases. Caution is warranted before
extrapolating these results to humans, since the amount of chia tested in the present
122
study should be further evaluated in clinical studies for chia oil diet replacement or
introduction of chia seed in usual human diet.
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP, 2012/23813-4) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq 140386/2012-2 and 300533/2013-6).
Conflict of interest
This work does not present conflicts of interest.
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CAPÍTULO III
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ARTIGO III
Artigo em processo de submissão na revista Journal of Functional Foods
Chia seed and oil (Salvia hispanica L.) dietary intake normalizes dyslipidemia,
inflammation and insulin resistance in diet-induced obese rats
RUNNING HEAD: “Beneficial effects of chia in obese rats”
Rafaela da Silva Marineli a, Érica Aguiar Moraes a, Sabrina Alves Lenquiste a, Renato
Chaves Souto Branco b, Rafael Ludemann Camargo b, Everardo Magalhães Carneiro
b, Stanislau Bogusz Jr c, Mário Roberto Maróstica Jr a,*
a Department of Food and Nutrition, Faculty of Food Engineering, University of
Campinas (UNICAMP), Campinas, SP, Brazil
b Department of Structural and Functional Biology, Institute of Biology, University of
Campinas (UNICAMP), Campinas, SP, Brazil
c Institute of Chemistry of São Carlos (IQSC), University of São Paulo (USP), São
Carlos, SP, Brazil
*Corresponding author. Email address: [email protected] Tel.: +55 19 3521-
4059; fax: +55 19 3521-4060. Address: Monteiro Lobato st., 80, zip code 13083-862,
Campinas, São Paulo, Brazil.
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Abstract
Objective: This study investigated the effects of dietary chia seed and chia oil on
obesity parameters and associated complications in diet-induced obese rats.
Methods: Sixty Wistar rats were divided in six groups: control (AIN-93M), high-fat and
high-fructose (HFF) diet, HFF diet added with chia seed or chia oil in short (6-weeks)
and long (12-weeks) treatments. Biochemical markers, lipid and hormonal profile,
inflammatory markers, glucose and insulin tolerance, insulin secretion by pancreas
islet isolation as well as plasma FAME content were determined. Results: HFF diet
induced weight gain, dyslipidemia, inflammation, glucose intolerance, insulin
resistance and altered parameters related to obesity complications. Chia seed and
chia oil intake did not reduce weight gain, adipose tissues weight and hepatic fat
accumulation, but was able to reduce total cholesterol, LDL-cholesterol and
triacylglycerol and increase HDL-cholesterol levels as well as fecal lipids excretion.
Chia diets intake also reduced tumor necrosis factor alpha (TNF-α), monocyte
chemoattractant protein-1 (MCP-1) and C-reactive protein concentrations and
increased adiponectin levels; improved glucose and insulin tolerance and reduced
insulin secretion by the pancreatic islets. Additionally, chia seed and oil intake did not
reduce plasma saturated fatty acids content, but increased α-linolenic acid (ALA),
eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic
(DHA) plasma concentrations, and consequently reduced the n-6/n-3 ratio compared
to HFF group. Chia groups presented similar physiological effects, regardless of
treatment time. Conclusion: Dietary chia seed and chia oil were able to normalize
dyslipidemia, inflammation, glucose intolerance, insulin resistance and improve
plasma fatty acid profiles in diet-induced obese rats.
Key words: Chia, Salvia hispanica, Omega-3 fatty acid, obesity, insulin resistance,
glucose tolerance, inflammation.
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1. Introduction
Obesity is an epidemic condition and is considered the largest public health
problem worldwide. The global obesity rise has increased the prevalence of
cardiovascular diseases, type 2 diabetes mellitus, cancer and others health
problems, which can lead to further morbidity and mortality (Swinburn et al., 2011).
As a chronic disease of multifactorial origin, an excessive energy intake from
saturated fatty acids and refined carbohydrates leads to an increase in energy
storage as fat, accompanied by metabolic disorders, which may contribute to
metabolic syndrome development (Fernández-Sánchez et al., 2011). High-fat and
high fructose diet (HFF) consumption have been related with the increase in obesity,
glucose intolerance, insulin resistance, inflammation and nonalcoholic fatty liver
disease in animals (Poudyal et al., 2012; Shapiro et al., 2011).
Recent studies have been suggested that changes in the diets composition
and functional foods addition are important environmental determinants in the
prevention or improvement of obesity related diseases (Trigueros et al., 2013).
Health benefits of regular consumption or dietary supplementation with long chain n-
3 polyunsaturated fatty acids (PUFA) are widely acknowledged, especially in relation
to cardiovascular diseases, dyslipidemia, inflammation and diabetes (Baranowski et
al., 2012; Wu et al., 2012; Ganesan et al., 2014). Therefore, the evaluation of
potential functional foods rich in n-3 PUFA could be a strategy to obesity
complications management.
Chia (Salvia hispanica L.) is an herbaceous plant that belongs to Lamiaceae
family native from southern Mexico and northern Guatemala (Ayerza, 1995). Today it
is commercially available for human consumption as whole seeds, flour, and oil in the
Americas, Australia, Europe and Southeast Asia (Mohd Ali et al., 2012). Chia seed is
the richest known botanical source of omega-3 α-linolenic acid (C18:3 n-3, ALA, up
to 68%) (Ayerza, 1995) and has been also described as an important source of
protein, dietary fiber, minerals and polyphenolic compounds (Reyes-Caudillo,
Tecante & Valdivia-López, 2008; Marineli et al., 2014). Moreover, chia lipids could
also be an omega-3 alternative source to vegetarians and people with fish allergies
(Mohd Ali et al., 2012).
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Additionally, the effects of chia seed ingestion has been recently described
for human health and nutrition because their bioactive components were found to
promote health benefits (Vuksan et al., 2007; Chicco et al., 2009; Poudyal et al.,
2012) without promote adverse effects. Our previous studies reported that chia intake
increased tissue protection-associated proteins expression, restored antioxidant
system in skeletal muscle (Marineli et al., 2015a) and reduced oxidative stress and
lipid peroxidation in diet-induced obese rats (Marineli et al., 2015b). However, chia
seed and oil has been little explored from a scientific point of view, especially in
relation to its in vivo inflammatory potential. We hypothesized that dietary chia seed
and chia oil, due to the content of bioactive compounds and n-3 PUFA, could protect
against obesity related diseases by improving glucose and insulin tolerance,
inflammation and dyslipidemia in diet-induced obese rats. Moreover, there was an
interest in determining whether the oil alone would produce the same response as
the seed. Therefore, the present study aimed to evaluate the beneficial effects of
dietary chia seed and chia oil ingestion on obesity parameters and associated
complications in high-fat and high-fructose diet-induced obese Wistar rats.
2. Materials and methods
2.1. Chia seed and oil
Fresh chia seed and chia oil from FTP S.A. Santiago, Chile were purchased
from R&S Blumos Comercial de Produtos Alimentícios Ltda, Brazil. According to the
producer, chia oil was obtained by cold pressing and stored at 2-8 °C until use in
amber glass bottles without headspace. Seeds were ground in a laboratory impact
mill (Marconi MA 630/1, Piracicaba – São Paulo, Brazil) and passed through a 0.850
mm sieve. Chemical analysis and antioxidant activity of Chilean chia seed and oil
samples used in this study were previously determined by Marineli et al. (2014).
2.2 Animals, diets and interventions
This work was approved by the Ethics Commission on Animal Use (CEUA/
UNICAMP, protocol no. 2936-1) and followed the University guidelines for the use of
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animals in experimental studies. Sixty male Wistar rats, aged 21 to 23 days, were
obtained from Multidisciplinary Center for Biological Investigation, University of
Campinas. Animals remained in individual cages for growth with free access to water
and chow diet for 4 weeks and were maintained under controlled conditions (22 ± 1
ºC, 60-70% humidity, 12 hours light/dark cycle). After growth, animals (226.52 g ±
18.74) were randomly divided in six experimental groups (n=10/group) for 12 weeks.
Diets were based on the American Institute of Nutrition (AIN-93M) (Reeves,
Nielsen & Fahey, 1993) with protein concentration of 12%. Control group received
the standard diet (AIN-93M); high-fat and high-fructose (HFF) group received a diet
containing 4% (w/w) soybean oil, 31% (w/w) lard and 20% fructose (w/w) (Marineli et
al., 2015a); chia seed short and long treatments received an HFF diet with 13.3%
(w/w) of chia seed; chia oil short and long treatments received an HFF diet with 4%
(w/w) of chia oil. There were a long (12 weeks) and a short (6 weeks) treatment with
chia seed or chia oil. Animals from long treatment were fed only an HFF diet
containing chia seed or chia oil for 12 weeks. Short groups were initially fed only an
HFF diet for the first 6 weeks, followed by an additional 6 weeks with an HFF diet
containing chia seed or chia oil. Soybean oil for both chia seed and chia oil diet was
replaced by the oil content of chia seed or chia oil. Chia diets were formulated to
provide equal quantities of oil and ALA from chia (Ayerza & Coates, 2007). According
to Marineli et al., 2014 chia seeds contained 30.2% of lipids. Protein and dietary fiber
content in HFF-chia seed groups was balanced taking into account the amount of
such nutrients present in chia seed (Marineli et al., 2014). Diets composition is
presented in Table 1. Diets were prepared monthly and packed in dark polyethylene
bags and stored at -20 ºC to minimize the oxidation of fatty acids. Weight gain was
monitored weekly and food intake every 2 days.
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Table 1. Experimental diets ingredients and fatty acid composition
Ingredients (g/kg diet) AIN-93M HFF Chia seed Chia oil
Casein 1 139.53 139.53 106.53 139.53
Maltodextrin 155.0 45.40 45.40 45.40
Corn starch 465.69 136.44 123.44 136.44
Sucrose 100.0 29.32 29.32 29.32
Soybean oil 40 40 - -
Chia oil - - - 40
Chia seed - - 133 -
Lard - 310 310 310
Fructose - 200 200 200
Cellulose 50 50 3.6 50
Mineral mixture 35 35 35 35
Vitamin mixture 10 10 10 10
L-Cystine 1.8 1.8 1.8 1.8
Choline bitartrate 2.5 2.5 2.5 2.5
tert-Butyl hydroquinone 0.008 0.008 0.008 0.008
Energy density (kcal/g
diet) 3.75 5.89 5.85 5.88
Fatty acids 2
SFA 7.38 127.86 125.35 125.99
MUFA 10.20 146.94 138.87 140.11
PUFA 22.42 71.10 81.11 79.54
Linoleic (C18:2n-6) 19.89 66.74 54.97 53.53
α-Linolenic (C18:3n-3) 2.20 4.06 25.88 25.73
n-6/n-3 ratio 9.04 16.44 2.12 2.08
1 Amount was calculated based on protein content equal to 86% to provide 12 g of
protein/100 g of diet. 2 Fatty acids expressed in g/kg diet was determined by gas
chromatography according to Maia & Rodriguez-Amaya, 1993 and Kramer et al, 1997.
AIN-93M group: control standard diet; HFF group: high-fat and high-fructose diet; Chia
seed groups: HFF diet plus 13.3% (w/w) chia seed; Chia oil groups: HFF diet plus 4%
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(w/w) chia oil. SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA:
polyunsaturated fatty acids.
2.2 Intraperitoneal glucose tolerance test (iGTT) and insulin tolerance test (ITT)
An intraperitoneal glucose tolerance test (iGTT) and insulin tolerance test (ITT)
were performed on food-deprived (12 h) rats after 10 and 11 weeks of experiment,
respectively. Blood glucose levels were measured with a FreeStyle Lite® handheld
glucometer (Abbott Diabetes Care, Abbott Laboratorios do Brazil LTDA) using
appropriate test strips. For the iGTT, a solution of 50% D-glucose (2 g/kg body
weight) was administered into the peritoneal cavity. Blood samples were collected
from the tail vein at 30, 60, 90 and 120 min for the determination of glucose
concentrations. The AUC of glucose was calculated. For the ITT, glucose blood
levels were sampled at 5, 10, 15, 20, 25 and 30 min following intraperitoneal injection
of human insulin (0.75 U/kg body weight, Novolin R, Novo Nordisk® Farmacêutica do
Brazil LTDA). The constant rate for glucose disappearance during the ITT (KITT
%/min) was calculated using the formula [0.693 / (t1/2)]. The glucose t1/2 was
calculated from the slope of the least-square analysis of the glucose concentrations
during the linear phase (Bonora et al., 1987).
2.3 Blood and tissue collection
Animals were euthanized by decapitation preceded by 12-hour-fasting. Blood
samples were collected in polyethylene tubes with or without EDTA anticoagulant to
obtain plasma and serum, respectively. Tubes were centrifuged at 3000 g (15 min, 4
ºC). Serum and plasma were separated and stored in polypropylene microtubes at -
80 °C until analysis. Liver, heart, kidneys, spleen, epididymal, mesenteric and
retroperitoneal adipose tissues were removed and weighed. Liver was frozen in liquid
nitrogen and stored at -80 ºC for subsequent analysis. Relative organs weights were
calculated [RWL= (wet organ weight (g) x 100) / final body weight (g)]. Pancreatic
islet samples were isolated from the rats by the collagenase method, as described
previously (Rezende et al., 2007).
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2.4 Evaluation of pancreatic islet glucose-stimulated insulin secretion
Batches of five islets were pre-incubated for 1 h at 37 C in Krebs–Henseleit
buffer solution (KHBS) containing 0.5 g/L bovine serum albumin and 5.6 mM glucose
and equilibrated at 95% O2 and 5% CO2 (pH 7.4). The medium was discarded, and
the islets were incubated for an additional 1 h in 1 mL of KHBS containing 2.8 or 16.7
mM glucose. Subsequently, the supernatant fraction was collected to evaluate insulin
secretion which was measured by radioimmunoassay (RIA) (Rezende et al., 2007).
2.5 Serum determinations
Serum total cholesterol (TC) (1220001), high-density lipoprotein cholesterol
(HDL-c) (1220223), triacylglycerol (TAG) (1780105), glucose (1400106) (Wiener lab.,
Argentina), uric acid (700320) and free fatty acid (FFA) (700310) (Cayman Chemical
Company, Ann Arbor, MI, USA) were determined using commercial enzymatic
colorimetric assays kits following manufacturer's protocol. Low-density lipoprotein
cholesterol (LDL-c) level was estimated using the Friedewald formula [LDL-c = (TC –
HDL-c) – (TAG/5)] (Friedewald et al., 1972). Serum adiponectin (EZRADP-62K),
ghrelin (EZRGRT-91K), insulin (EZRMI-13K), leptin (EZRL-83K) and C-Reactive
Protein (CRP) (CYT294) were analyzed using enzyme-linked immunosorbent assay
(ELISA) method (Millipore, MA, USA) following manufacturer's protocol. Absorbance
readings were done in a microplate reader Synergy HT, Biotek (Winooski, VT, USA);
with Gen5™ 2.0 data analysis software spectrophotometer. Tumor necrosis factor
alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were determined
using Rat Metabolic Magnetic Bead Panel (RMHMAG-84K) (Millipore, MA, USA)
following manufacturer's protocol.
2.6 Hepatic and fecal lipids content
Liver was freeze-dried, manually ground with mortar and pistil and kept at -80
°C until analysis. Feces were collected for 7 days during the last experimental week
to determine wet and dry weight by incubation for 24 hours at 60 C. Then, feces
were milled and stored at -20 C. Hepatic and fecal total lipids content were
determined by the method of Bligh and Dyer extraction (Bligh & Dyer, 1959).
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2.7 Plasma fatty acid methyl esters (FAME)
Fatty acids methyl esters (FAME) were prepared as previously described by
(Glaser et al., 2010) with adaptations. A total of 100 µL of plasma, 100 µL of internal
standard (Pentadecanoic methyl ester; Nu-Chek-Prep, INC, Elysian, MN, USA) and
600 µL methanol (precooled to 5 °C) were combined in glass tubes and shaken for
30 s. Precipitated proteins were separated from the methanolic phase by
centrifugation at 900 g for 5 min. Methanolic supernatant, which contained mainly
polar lipids, was transferred into another glass tube. Twenty-five µL sodium
methoxide solution (25% in methanol) were added to the supernatant, then the tubes
were shaken at room temperature. The reaction was stopped after 3 min by adding
75 µL methanolic HCl (3N). FAME were extracted by adding 300 µL hexane and
shaking for 30 s. Upper hexane phase was transferred into a 2 mL vial. The
extraction was repeated and combined extracts were dried under nitrogen flow at
room temperature. Dry residue was taken up in 500 µL hexane (containing 2 g/L
butylated hydroxytoluene) for gas chromatography analysis. All analyses were
carried out in triplicate.
Chromatographic conditions
FAME were analyzed by gas chromatography using an Agilent 6890N
equipment with an autosampler N10149 and flame ionization detector (Agilent, CA,
USA). The separation was performed in a 100 m × 0.25 mm i.d. × 0.20 µm, CP-Sil 88
capillary column (Agilent, DE, USA). The chromatographic conditions were: injector
and detector temperatures, 250 and 270 °C; injected volume, 1 µL in splitless mode;
carrier gas, hydrogen at 1.7 mL/min; oven, 78 °C for 10 min, with an increase of 8
°C/min to 180 °C, then 3 °C/min until 205 °C and finally 10 °C/min to 240 °C. FAME
were identified by comparison with authentic standards FAME Mix GLC-85 (Nu-
Chek-Prep, INC, Elysian, MN, USA), methyl cis-5,8,11,14,17 eicosapentaenoate
(EPA), methyl cis-7,10,13,16,19 docosapentaenoate (DPA) and cis-4,7,10,13,16,19
docosahexaenoic (DHA) (Supelco, Bellefonte, PA, USA). The quantification was
expressed as percentage of total fatty acid content.
2.8. Statistical analysis
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Data are expressed as mean values ± standard deviation. Differences
between AIN-93M and HFF group were tested by T test. Differences among HFF,
chia seed 12 wk, chia seed 6 wk, chia oil 12 wk and chia oil 6 wk groups were tested
by one-way analysis of variance (ANOVA) followed by Tukey's test using the
GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA) software. P<0.05
was considered statistically significant.
3. Results
Food intake, weight gain and organs weight
Food intake, energy intake, weight gain, and organs weight are shown in
Table 2. Food intake was lower and energy intake was higher in HFF-fed animals
compared to AIN-93M group. These parameters did not differ among chia (seed or
oil) and HFF groups. Animals fed on HFF diet showed an increase in weight gain
compared to AIN-93M group. Chia seed and oil intake did not attenuate rat‘s weight
gain when compared to HFF group. Weight of epididymal, mesenteric and
retroperitoneal adipose tissues in HFF-fed animals were higher than those in AIN-
93M group. Chia seed and chia oil diet intake did not show differences in adipose
tissues weight compared to HFF group. Kidneys of group that consumed chia seed
for 12 weeks were smaller than those in HFF group. There was no difference in heart
and spleen weight among the experimental groups. Animals fed on HFF diet showed
an increase in liver weight when compared to AIN-93M group. Only groups that
consumed chia seed (6 and 12 wk) showed a decrease in liver weight compared to
HFF and chia oil groups. Hepatic lipids content was higher in HFF-fed animals
compared to AIN-93M group. However, chia seed and chia oil intake did not reduce
hepatic fat accumulation in animals. HFF group showed an increase in fecal lipids
content when compared do AIN-93M group. Groups that consumed chia seed (6 and
12 wk) showed higher fecal lipids content compared to HFF group (Table 2). Uric
acid concentration was higher in HFF group compared to AIN-93M group. There was
no difference in uric acid concentration among HFF and chia groups. FFA levels
showed the same behavior among experimental groups. No alteration was observed
for serum glucose concentration (Table 2).
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Table 2. Food and energy intake, weight gain, organs weight and metabolic parameters of rats fed on different diets1
AIN-93M HFF Chia seed 12 wk Chia seed 6 wk Chia oil 12 wk Chia oil 6 wk
Food intake (g/day) 24.78 1.39A 20.47 0.95B a 19.37 0.51a 19.30 0.81a 19.69 0.84a 19.25 0.86a
Energy intake (kcal/day) 92.93 5.23B 120.50 5.63A a 113.30 3.04a 112.90 4.75a 115.80 4.98a 113.20 5.06a
Weight gain (g/day) 3.16 0.27B 3.89 0.25A a 3.91 0.19a 4.02 0.35a 3.97 0.38a 3.98 0.40a
Epididymal fat (g/100g) 2.90 0.55B 4.02 0.64A a 4.33 0.56a 4.06 0.32a 3.78 0.41a 3.97 0.91a
Retroperitoneal fat
(g/100g) 3.92 0.99B 5.56 0.71A a 6.09 1.01a 5.42 1.10a 5.76 0.60a
5.38 0.83a
Mesenteric fat (g/100g) 1.99 0.50B 3.15 0.50A a 3.58 0.38a 3.35 0.56a 3.09 0.60a 3.17 0.90a
Liver (g/100g) 2.73 0.23B 3.21 0.22A a 2.80 0.26b 2.87 0.15b 3.16 0.34a 3.31 0.28a
Kidneys (g/100g) 0.54 0.06A 0.54 0.07A a 0.46 0.03b 0.49 0.03ab 0.50 0.03ab 0.50 0.03ab
Spleen (g/100g) 0.19 0.02A 0.18 0.02A a 0.16 0.02a 0.18 0.02a 0.18 0.02a 0.19 0.03a
Heart (g/100g) 0.26 0.02A 0.25 0.03A a 0.24 0.02a 0.25 0.01a 0.25 0.02a 0.24 0.02a
Hepatic lipids (g) 2 26.82 3.83B 50.05 9.44A a 47.35 4.57a 47.03 5.27a 48.05 3.49a 51.44 9.68a
Fecal lipids (g) 2 2.52 0.31B 5.66 0.94A b 9.46 2.18a 9.13 2.74a 6.63 1.62ab 6.16 1.26ab
Glucose (mg/dL) 108.42 3.97A 114.93 7.0A a 119.69 10.17a 111.99 11.49a 115.82 4.08a 111.59 7.91a
Uric acid (µM/mL) 27.76 14.38B 64.86 19.61A a 55.50 23.02a 62.08 19.91a 49.65 13.46a 48.22 19.16a
FFA (µM/mL) 613.15 18.3B 679.55 7.82A a 645.05 21.2a 667.91 13.3a 644.64 32.4a 661.48 31.6a
1 Control standard diet (AIN-93M); high-fat and high-fructose (HFF) diet; HFF diet plus 13.3% chia seed during 12 weeks (chia seed
12-wk) or 6 weeks (chia seed 6-wk); HFF plus 4% chia oil during 12 weeks (chia oil 12-wk) or 6 weeks (chia oil 6-wk). Different
140
capital letters (A-B) mean significant differences between AIN-93M and HFF group according to T test (P<0.05). Different small
letters (a-b) mean significant differences between HFF, chia seed and chia oil groups by one-way analysis of variance followed by
Tukey‘s test (P<0.05). Data expressed as mean ± standard deviation (n = 8/group). 2 Values expressed in dry basis. FFA: free fatty
acid.
141
Glucose and Insulin Tolerance Tests
Glucose and insulin tolerance tests are shown in Figure 1. HFF diet intake
induced glucose and insulin intolerance compared to AIN-93M group (Figure 1). All
groups that consumed chia seed and chia oil diets (6 and 12 wk) improved glucose
and insulin tolerance compared to HFF group. Chia groups were more glucose
tolerant, as revealed by a glucose tolerance test (iGTT) (Fig. 1 B) and reflected by a
lower AUC of glucose during the iGTT (Fig. 1 A). Additionally, chia groups were more
insulin sensitive, as revealed by the lower blood glucose values observed during the
ITT (Fig. 1 D) and increased KITT (Fig. 1 C).
Figure 1. (A) Glucose AUC during intraperitoneal glucose tolerance test (iGTTAUC),
(B) Mean blood glucose levels after intraperitoneal infusion of glucose solution, (C)
KITT during intraperitoneal insulin tolerance test, (D) Mean blood glucose levels after
142
intraperitoneal infusion of insulin in rats fed on different diets: control standard diet
(AIN-93M); high-fat and high-fructose (HFF) diet; HFF diet plus chia seed during 12
weeks (chia seed 12-wk) or 6 weeks (chia seed 6-wk); HFF plus chia oil during 12
weeks (chia oil 12-wk) or 6 weeks (chia oil 6-wk). Different capital letters (A-B) mean
significant differences between AIN-93M and HFF group according to T test
(P<0.05). Different small letters (a-b) mean significant differences between HFF, chia
seed and chia oil groups by one-way analysis of variance followed by Tukey‘s test
(P<0.05). Data expressed as mean ± standard deviation (n = 8/group).
Static insulin secretion
Evaluation of pancreatic islet glucose-stimulated insulin secretion is shown in
Figure 2. Animals fed on HFF diet showed an increase in insulin secretion by the
pancreatic islets compared to AIN-93M group. Additionally, the pancreatic islets of
chia groups (seed and oil) secreted less insulin under both sub-stimulatory (2.8
mmol/L) and supra-stimulatory (16.7 mmol/L) glucose conditions (Fig. 2 A and B,
respectively).
Figure 2. Pancreatic islet glucose-stimulated insulin secretion at 2.8 mmol/L (A) and
16.7 mmol/L (B) glucose in rats fed on different diets: control standard diet (AIN-
93M); high-fat and high-fructose (HFF) diet; HFF diet plus chia seed during 12 weeks
(chia seed 12-wk) or 6 weeks (chia seed 6-wk); HFF plus chia oil during 12 weeks
143
(chia oil 12-wk) or 6 weeks (chia oil 6-wk). Different capital letters (A-B) mean
significant differences between AIN-93M and HFF group according to T test
(P<0.05). Different small letters (a-b) mean significant differences between HFF, chia
seed and chia oil groups by one-way analysis of variance followed by Tukey‘s test
(P<0.05). Data expressed as mean ± standard deviation (n = 8/group).
Lipid profile
Lipid profile results are shown in Figure 3. HFF diet intake reduced HDL-c
levels and increased LDL-c and TAG levels compared to AIN-93M animals. All
groups that consumed chia seed or chia oil showed a reduction in TC, LDL-c and
TAG levels and also showed an increase in HDL-c levels compared to HFF group
(Fig. 3).
Figure 3. (A) Total cholesterol (TC), (B) HDL-c, (C) LDL-c and (D) Triacylglycerol
(TAG) in serum of rats fed on different diets: control standard diet (AIN-93M); high-fat
and high-fructose (HFF) diet; HFF diet plus chia seed during 12 weeks (chia seed 12-
144
wk) or 6 weeks (chia seed 6-wk); HFF plus chia oil during 12 weeks (chia oil 12-wk)
or 6 weeks (chia oil 6-wk). Different capital letters (A-B) mean significant differences
between AIN-93M and HFF group according to T test (P<0.05). Different small letters
(a-b) mean significant differences between HFF, chia seed and chia oil groups by
one-way analysis of variance followed by Tukey‘s test (P<0.05). Data expressed as
mean ± standard deviation (n = 8/group).
Hormonal and Inflammatory markers
Inflammatory markers results are shown in Figure 4. Animals fed on HFF diet
showed an increase in serum leptin and insulin concentrations compared to AIN-93M
group (Fig. 4 A and B, respectively). Chia seed and chia oil groups were not able to
reduce HFF-induced hyperinsulinemia and hyperleptinemia. Ghrelin concentrations
were higher in HFF group in relation to AIN-93M group. There was no difference in
ghrelin concentrations among HFF and chia groups (Fig. 4 C). HFF diet intake
showed a reduction in adiponectin concentration compared to AIN-93M group. Chia
seed and chia oil groups displayed increased serum adiponectin concentration in
relation to HFF group (Fig. 4 D). Furthermore, TNF-α, MCP-1 and C-reactive protein
concentrations were increased in HFF-fed animals when compared to AIN-93M
group, whereas all chia groups were able to reduce these inflammatory markers
compared to HFF group (Fig. 4 E-G, respectively).
145
Figure 4. (A) Leptin, (B) Insulin, (C) Ghrelin, (D) Adiponectin, (E) TNF-α, (F) MCP-1
and (G) C-reactive protein in serum of rats fed on different diets: control standard diet
(AIN-93M); high-fat and high-fructose (HFF) diet; HFF diet plus chia seed during 12
weeks (chia seed 12-wk) or 6 weeks (chia seed 6-wk); HFF plus chia oil during 12
146
weeks (chia oil 12-wk) or 6 weeks (chia oil 6-wk). Different capital letters (A-B) mean
significant differences between AIN-93M and HFF group according to T test
(P<0.05). Different small letters (a-b) mean significant differences between HFF, chia
seed and chia oil groups by one-way analysis of variance followed by Tukey‘s test
(P<0.05). Data expressed as mean ± standard deviation (n = 8/group).
FAME contents
Results of plasma fatty acid composition, as a percentage of total fatty acid
content, are shown in Table 3. Animals fed with HFF diet showed a reduction in
C16:0 and C16:1 n-7 plasma concentrations compared to AIN-93M group and no
changes were found in chia groups when compared to HFF group. Linoleic acid
(C18:2 n-6, LA) plasma concentrations showed the same behavior among
experimental groups. No alteration was found for C11:0, C12:0, C13:0, C17:0, C18:1
n-9, C18:1 n-9T, C20:2 n-6, C20:4 n-6 and C22:1 n-9 among experimental groups.
HFF diet intake increased plasma concentration of C18:0, C20:0 and C20:3 n-6 in
relation to AIN-93M group, and no changes were found in chia groups when
compared to HFF group. C17:1 n-7 and C20:1 n-9 plasma concentrations were
higher in chia seed and oil groups compared to HFF group. Additionally, all chia
groups increased concentration of C18:3 n-3 (ALA), C20:5 n-3 (EPA) and C22:5 n-3
(DPA) in relation to HFF group. DHA (C22:6 n-3) concentration was lower in HFF-fed
animals compared to AIN-93M group. However, chia seed and chia oil intake
increased plasma DHA concentration compared to HFF group, as occurred with n-3
FAME contents. PUFA and n-6 FAME contents were lower in HFF-fed animals
compared to AIN-93M group and no changes were found in chia groups when
compared to HFF group. Groups that received chia seed or chia oil showed a
markedly reduction in n-6/n-3 ratio compared to HFF group. Furthermore, SFA
contents were higher in HFF group in relation to AIN-93M. The same occurred with
chia groups compared to HFF group. However, no alterations were found in MUFA
contents among experimental groups.
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Table 3. Plasma fatty acid composition (%) of rats fed on different diets1
FAME AIN-93M HFF Chia seed 12 wk Chia seed 6 wk Chia oil 12 wk Chia oil 6 wk
C11:0 0.19 ± 0.06A 0.22 ± 0.06A a 0.42 ± 0.11a 0.34 ± 0.09a 0.55 ± 0.20a 0.31± 0.15a
C12:0 0.08 ± 0.01A 0.12 ± 0.03A a 0.10 ± 0.004a 0.09 ± 0.004a 0.11 ± 0.02a 0.12 ± 0.01a
C13:0 0.18 ± 0.01 A 0.14 ± 0.04A a 0.20 ± 0.08a 0.15 ± 0.03a 0.15 ± 0.02a 0.22 ± 0.06a
C16:0 31.00 ± 0.68A 25.10 ± 0.48B a 25.00 ± 0.40a 25.29 ± 0.61a 26.05 ± 0.27a 26.60 ± 0.68a
C16:1 n-7 1.93 ± 0.32A 0.30 ± 0.03B a 0.31 ± 0.03a 0.30 ± 0.02a 0.30 ± 0.02a 0.27 ± 0.01a
C17:0 0.49 ± 0.02A 0.45 ± 0.02A a 0.43 ± 0.01a 0.43 ± 0.004a 0.44 ±0.004a 0.46 ±0.008a
C17:1 n-7 0.16 ± 0.05A 0.19 ± 0.02A b 0.52 ± 0.15a 0.45 ± 0.09a 0.66 ± 0.25a 0.55 ± 0.18a
C18:0 19.86 ± 1.09B 29.46 ± 1.25A a 30.43 ± 0.65a 29.92 ± 0.99a 28.78 ± 0.62a 29.37 ± 0.80a
C18:1 n-9 1.13 ± 0.15A 1.10 ± 0.04A a 0.80 ± 0.13a 0.73 ± 0.12a 0.74 ± 0.20a 0.97 ± 0.18a
C18:1 n-9T 7.56 ± 0.92A 8.51 ± 0.66A a 7.24 ± 0.54a 7.98 ± 0.67a 7.73 ± 0.65a 7.39 ± 0.57a
C18:2 n-6 (LA) 18.33 ± 0.64A 16.07 ± 0.19B a 15.32 ± 0.60a 14.83 ± 0.35a 15.84± 0.43a 14.95 ± 0.21a
C18:3 n-3 (ALA) 0.25 ± 0.02A 0.23 ± 0.04A b 0.58 ± 0.22a 0.36 ± 0.04 a 0.44 ± 0.04 a 0.38 ± 0.03 a
C20:0 0.10 ± 0.004B 0.14 ± 0.01A a 0.12 ± 0.04a 0.13 ± 0.04a 0.11 ± 0.02a 0.12 ± 0.02a
C20:1 n-9 0.33 ± 0.04A 0.29 ± 0.03A b 0.81 ± 0.09a 0.62 ± 0.08a 0.69 ± 0.08a 0.62 ± 0.19a
C20:2 n-6 0.18 ± 0.01A 0.18 ± 0.02A a 0.20 ± 0.01a 0.19 ± 0.01a 0.19 ± 0.02 a 0.20 ± 0.01a
C20:3 n-6 0.33 ± 0.02A 0.21 ± 0.03B a 0.25 ± 0.02a 0.24 ± 0.02a 0.24 ± 0.01a 0.24 ± 0.01a
C20:4 n-6 (AA) 16.00 ± 0.80A 15.62 ± 0.54A a 14.11 ± 0.48a 14.97 ± 0.42a 13.97 ± 0.66a 14.68 ± 0.68a
C20:5 n-3 (EPA) 0.22 ± 0.06A 0.36 ± 0.10A b 0.64 ± 0.21a 0.52 ± 0.04 a 0.54 ± 0.06 a 0.51 ± 0.04 a
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FAME AIN-93M HFF Chia seed 12 wk Chia seed 6 wk Chia oil 12 wk Chia oil 6 wk
C22:0 Nd 0.06 1.94 Nd 1.94 nd
C22:1 n-9 0.65 ± 0.11A 0.70 ± 0.07 A a 0.75 ± 0.06 a 0.71 ± 0.02 a 0.79 ± 0.06 a 0.82 ± 0.07 a
C22:5 n-3 (DPA) 0.28 ± 0.02A 0.26 ± 0.01A b 0.56 ± 0.01a 0.54 ± 0.04a 0.48 ± 0.03a 0.52 ± 0.04a
C22:6 n-3 (DHA) 1.54 ± 0.08A 1.08 ± 0.05B b 1.35 ± 0.07a 1.37 ± 0.09a 1.30 ± 0.08a 1.40 ± 0.10 a
SFA 51.76 ± 1.23B 55.50 ± 0.89A a 56.94 ± 0.75a 56.29 ± 0.41a 56.42 ± 0.87a 57.12 ± 0.30a
MUFA 11.29 ± 1.44A 10.65 ± 0.95A a 10.33 ± 0.55a 10.73 ± 0.85a 10.91 ± 0.80a 10.53 ± 0.97a
PUFA 36.82 ± 0.59A 33.86 ± 0.55B a 32.72 ± 0.57a 33.00 ± 0.71a 32.67 ± 0.59a 32.35 ± 0.91a
n-6 34.53 ± 0.64A 31.92 ± 0.59B a 29.72 ± 0.48a 30.23 ± 0.67a 30.03 ± 0.49a 29.92 ± 0.75a
n-3 2.28 ± 0.10A 1.94 ± 0.08B b 3.01 ± 0.66a 2.77 ± 0.13a 2.65 ± 0.15a 2.43 ± 0.19a
n-6/n-3 15.14 ± 0.78A 16.45 ± 0.81A a 9.87 ± 1.06b 10.91 ± 0.89b 11.33± 0.73b 12.31 ± 1.25b
1 Control standard diet (AIN-93M); high-fat and high-fructose (HFF) diet; HFF diet plus 13.3% chia seed during 12 weeks (chia seed
12-wk) or 6 weeks (chia seed 6-wk); HFF plus 4% chia oil during 12 weeks (chia oil 12-wk) or 6 weeks (chia oil 6-wk). Different
capital letters (A-B) mean significant differences between AIN-93M and HFF group according to T test (P<0.05). Different small
letters (a-b) mean significant differences between HFF, chia seed and chia oil groups by one-way analysis of variance followed by
Tukey‘s test (P<0.05). Data expressed as mean ± standard deviation (n = 8/group). nd: Not detected. SFA: saturated fatty acids,
MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids.
149
4. Discussion
Our data show that a HFF diet intake induced obesity, dyslipidemia, hepatic fat
accumulation, inflammation and impaired glucose and insulin tolerance in Wistar rats.
These findings are in agreement with those reported in earlier studies, which
demonstrated that high-fat and high-carbohydrate diets are able to mimic many signs
of human metabolic syndrome associated with obesity development (Poudyal et al.,
2012; Shapiro et al., 2011). In addition, fructose intake could influence carbohydrate
and lipid metabolism, which is associated with metabolic abnormalities in humans
and animals (Tappy et al., 2010).
In the present study, a daily consumption of chia seed and oil did not reduce
weight gain or adipose tissues weight after 6 or 12-weeks of experimental period.
These results are consistent with previous reports using chia seed (Chicco et al.,
2009 and Oliva et al., 2013) which demonstrated that chia intake did not affect body
weight gain in high sucrose-fed rats. However, these authors found a reduction in
adipose tissues weight and visceral adiposity in rats that consumed chia seed, which
was not found in the current study. These outcomes were related to improvement in
basal lipolysis and lipogenic enzyme activities such as a reduction in fatty acid
synthase (FAS) and acetyl-CoA carboxylase (ACC) in adipose tissue (Oliva et al.,
2013). Furthermore, it is needed to emphasize that different diets‘ compositions were
used in both studies, since chia seed was added to those diets almost 3 times more
(36.2%) than in the present study (13.3%), which may influence the results found.
Although chia seed showed a decrease in liver weight, chia seed and oil intake
did not reduce hepatic fat accumulation in animals. On the other hand, previous
studies found that chia seed ingestion reduced hepatic steatosis in diet-induced
obese rats (Poudyal et al., 2012; Rossi et al., 2013). Rossi et al (2013) explain this
result by the alterations in key enzymes activities involved in lipogenesis and
oxidative mitochondrial fatty acid oxidation, accompanied by changes in the sterol
regulatory element-binding protein-1 (SREBP-1) and peroxisome proliferator-
activated receptor-α (PPARα) expression in the liver. Moreover, Poudyal et al (2012)
also found a reduction in portal inflammatory cell infiltration and fibrosis in the liver of
150
obese rats fed with chia seed diet. However, liver histology or hepatic TAG
accumulation were not evaluated in the present research.
Current study demonstrates that chia seed and chia oil intake was able to
normalize dyslipidemia as well as increase fecal lipids excretion in obese rats. In this
regard, some animal studies have shown a reduction in serum or plasma total
cholesterol, TAG and an increase in HDL-cholesterol when chia was used as a fat
dietary source in healthy or dyslipidemic rats (Ayerza & Coates 2007; Chicco et al.,
2009), which corroborates with our findings. Moreover, hypolipidemic effects found in
chia groups could explain previous studies that showed an increase in
cardioprotective parameters in obese rats fed with chia seed diet (Poudyal et al.,
2012). Nevertheless, our results show that FFA concentrations were not altered with
chia diets intake. Poudyal et al (2012) found an increase in FFA concentrations in
obese rats when chia was added to the diets at 5%. On the other hand, other studies
demonstrated a reduction in this marker when chia seed was added to the diets at
36.2% in dyslipidemic rats (Oliva et al., 2013; Rossi et al., 2013).
High dietary fiber content in chia seed could play a role in lowering circulating
lipid levels since the hypocholesterolemic effect of dietary fiber is widely reported
(Willem van der Kamp et al., 2010). Additionally, ALA, the major fatty acid present in
the chia, was converted to long-chain n-3 PUFA, mainly to EPA and DHA in the liver
(González-Mañán et al., 2012). The effectiveness of this bioconversion can
determine its effect on plasma lipids. In this way, chia, as a precursor of EPA and
DHA, could modify fatty acid metabolism in the liver through PPARα activation and
SREBP-1c down-regulation, leading to a decrease in circulating lipids (Rossi et al.,
2013). Similar results were found by González-Mañán et al (2012) in relation to fatty
acid oxidation and biomarkers expression involved in lipogenesis after chia oil
ingestion in health rats. It could also explain the hypolipidemic effects of chia oil
intake in the present study. Furthermore, Sierra et al (2015) demonstrated that chia
oil can improve vascular function under hypercholesterolaemic conditions in rabbits.
Chia seed and oil groups improved glucose and insulin tolerance and reduced
insulin secretion by the pancreatic islets, although fasting plasma insulin
concentration was not affected. Similar results were reported by Chicco et al (2009)
when chia seed provided dietary fat during the last 2 months of feeding period in
151
dyslipidemic rats. They found an improvement in insulin resistance and glucose
homeostasis without changes in insulinemia. In addition, chia seed intake normalized
the decrease capacity of glucose phosphorylation and glucose oxidation during the
euglycemic–hyperinsulinemic clamp, as well as improved peripheral insulin
insensitivity in high sucrose-fed rats (Oliva et al., 2013). In this regard, long-chain n-3
PUFA have shown to prevent or normalize peripheral insulin resistance in both high-
carbohydrate and high-fat-fed animals (Lombardo & Chicco 2009). In addition, long-
chain n-3 PUFA affect the membrane lipid composition and this, in turn, moderates a
wide range of eicosanoid- and non-eicosanoid-mediated effects upon the metabolic
process and could directly affect insulin action (Chicco et al., 2009). Moreover, it has
been shown that dietary ALA or flaxseed oil improved insulin sensitivity and glycemic
responses in animal models and moderately improved fasting plasma glucose and
insulin resistance markers in humans (Wu et al., 2012).
Furthermore, in our previous study (Marineli et al., 2015a) we suggest that
chia seed and oil effect on increasing heat shock proteins (HSP) and restoring
antioxidant defense expression was an important factor in the improvement of
glucose tolerance, either coming from oxidative stress or from the mitochondrial
dysfunction that obesity may cause. Additionally, the antioxidant agents use, before
or after the onset of obesity, may ease glucose intolerance (Henriksen et al., 2011).
This could also explain our present findings, since chia seed and oil intake improved
antioxidant status in plasma and liver in diet-induced obese rats (Marineli et al.,
2015b). It is important highlight that insulin receptors and insulin signaling via should
be further addressed in vivo studies with chia to explain these outcomes in a
molecular way.
Chia seed and chia oil intake was able to normalize serum inflammation
caused by HFF-fed diet, since chia promote a decrease in pro-inflammatory markers
such as TNF-α, MCP-1 and C-reactive protein and an increase in adiponectin, an
anti-inflammatory marker. Adiponectin plays a protective role against insulin
resistance by regulating glucose and lipid metabolism and has potent angiogenic,
antiatherogenic and anti-inflammatory properties (Galic et al., 2010). However, there
are a few studies about chia effects on hormone and inflammatory markers linked to
obesity.
152
Obesity has been linked with a low-grade, chronic inflammatory response,
characterized by altered production of adipokines and increases in biological
inflammation markers, such as TNF-α, MCP-1 and interleukin-6 among other (Galic
et al., 2010; Fernández-Sánchez et al., 2011). TNF-α is involved in the systemic
inflammatory response and it has also been linked with development of insulin
resistance, obesity and diabetes (Galic et al., 2010). C-reactive protein is the most
extensively studied systemic inflammation marker in humans and its elevated levels
has been associated with cardiovascular risk factors, including insulin resistance,
diabetes, metabolic syndrome, hypertension and dyslipidemia (Fernández-Sánchez
et al., 2011). Long-term supplementation with chia seed (37g/day) reduced C-
reactive protein levels, while maintained a good glycemic and lipid control in type 2
diabetes individuals (Vuksan et al., 2007). Poudyal et al (2012) also found a
reduction in plasma C-reactive protein in obese rats fed with chia seed diet, which
corroborates with our findings.
In addition, an increased fat mass and larger adipocytes lead to elevated
circulating leptin concentrations and leptin, in turn, exerts pro-inflammatory actions by
activating various immune cells (Fernandez-Riejos et al., 2010). It is consistent with
our findings related to metabolic damage caused by HFF diet. However, chia groups
did not reduce serum hyperleptinemia, although the reduction in serum inflammatory
markers. Moreover, the potential effect of daily chia consumption on these
biomarkers is still recent and not enough studied.
Based on animal experimental studies, n-3 PUFA can improve several
metabolic abnormalities and such effects include insulin-sensitizing improvement
through increasing adipocytokines production and secretion; and potential prevention
of insulin resistance via anti-inflammatory effects (Wu et al., 2012).
Baranowski et al (2012) demonstrated that dietary interventions with ALA-rich
flaxseed oil promote a reduction in the adipose tissue protein levels of several
inflammatory markers in obese Zucker rats. Additionally, the cardioprotective effects
of ALA are mediated in part by a reduction in inflammatory cytokines production
(Zhao et al., 2007). The mechanism whereby n-3 fatty acids alter gene expression is
multi-fold. It has been postulated that fatty acids alter inflammatory responses by
inhibiting nuclear factor kappaB (NF-kB) activity/signaling, which regulates the
153
induction of cytokine gene expression (Zhao et al., 2007). In this way, Jangale et al
(2013) demonstrated that flaxseed oil ingestion (rich in ALA) down-regulated the
expression of hepatic inflammatory genes in diabetic rats including transcription
factors such as NF-kB. However, inflammatory markers modulation in adipose and
hepatic tissues by chia ingestion has not yet been studied.
In the present research, chia seed and chia oil intake increased plasma n-3
PUFA, ALA, EPA, DPA and DHA concentrations and also markedly reduced n-6/n-3
ratio in animals. Similar results were found in serum when chia seed was added at
16% (Ayerza & Coates 2007) or 36.2% (Chicco et al., 2009) to the diets in healthy
and dyslipemic rats. The same occurred in plasma, liver and adipose tissue of
healthy rats when chia oil was added at 10% to the diets (González-Mañán et al.,
2012). These outcomes indicate that chia seed or oil potential benefit occurred
independently of supplemented chia amount used in previous studies. In this way, it
is important to further assess the minimum amount of chia enough to alter the fatty
acid composition in vivo.
Low n-6/n-3 fatty acid ratio has also been associated with the reduction of
cardiovascular disease risk (Simopoulos, 2004). In addition, a cardioprotective effect
of long-term ALA intake has been suggested by a number of epidemiological studies,
and an additive effect of ALA with n-3 long-chain PUFA from fish and fish oil has
been observed (Vedtofte et al., 2011). EPA and DHA, which are products of
elongation and desaturation of ALA, are also involved in biological mechanisms
related to fatty acids cardioprotective potential. It could also explain the hypolipidemic
effects found in chia groups in the current study. However, chia groups did not
reduce plasma SFA concentration increased by HFF diet, which is in agreement with
previous reports using chia seed (Chicco et al., 2009; Poudyal et al., 2012) and chia
oil (González-Mañán et al., 2012).
A recent study (Valenzuela et al., 2014) demonstrated that dietary ALA intake
from different vegetable oils, including chia oil (10%), caused important modifications
in fatty acid composition extracted from the various tissues of healthy rats. These
authors showed that EPA concentrations were increased in erythrocytes, liver,
kidney, small intestine, heart and quadriceps, but not in the brain. Furthermore, DHA
was increased in the liver, small intestine and brain tissues. These results leading to
154
the suggestion that ALA metabolism and its conversion into n-3 long-chain PUFA is
effective but markedly selective depending on the tissues studied (Valenzuela et al.,
2014). However, in the present study we did not evaluate the hepatic bioconversion
of ALA to EPA and DHA, and their accumulation in tissues.
In this regard, it has been reported that chia ingestion also modifies plasma
fatty acid profile in humans. Vuksan et al (2007) demonstrated that a long-term
supplementation with chia seed (37 g/day) increased plasma ALA and EPA
concentrations in people with well-controlled type 2 diabetes. Jin et al (2012) also
show that an ingestion of 25 g/day milled chia seed by postmenopausal women for
seven weeks increased plasma ALA and EPA concentrations, but not DPA or DHA.
Moreover, it should be highlighted that the conversion rate of ALA to EPA, especially
to DHA, in humans is generally low and it is modulated by several processes
including physiological status, diet, gender and age (Ganesan et al., 2014). In this
way, caution is warranted before extrapolating our results to humans.
In the current research, we observed the same effects and metabolic
improvement after ingestion of both, chia seed and chia oil, as well as in treatment
time which the animals were submitted. The absence of significant differences
between both treatments time could suggest that 6 week of chia intake is enough
time to observe significant changes in biomarkers related to obesity complications.
We previously found the same behavior in antioxidant status improvement and
oxidative stress reversion after chia seed and oil intake in diet-induced obese rats
(Marineli et al., 2015b). The results presented in this study are a good starting point
for further investigations that should conduct further analysis to clarify the bioactivity
and mechanisms of action related to the beneficial effects of chia intake.
5. Conclusion
In summary, the present study shows that a daily consumption of chia seed
and oil did not reduce weight gain or adiposity but was able to normalize
dyslipidemia, inflammation, glucose intolerance and insulin resistance caused by
HFF-fed diet. Chia diets also improved plasma n-3 PUFA levels and lowered the n-
6/n-3 ratio. In addition, similar physiological effects could be observed regardless of
treatment time. Therefore, our results suggest that chia seed and oil could be used
155
as a functional food and may be a dietary strategy to prevent or control obesity
related diseases. Moreover, the present findings warrant further research on the use
of chia seed and oil as a complimentary therapy for treating some signs of metabolic
syndrome in humans.
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP, 2012/23813-4) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq 140386/2012-2 and 300533/2013-6).
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DISCUSSÃO GERAL
Levando em consideração os resultados obtidos a partir da caracterização
química e nutricional das sementes e óleo de chia podemos destacar que as
amostras apresentaram um conteúdo significativo de proteína, óleo, fibra dietética
(principalmente fibra insolúvel) e compostos bioativos, assim como reportado em
estudos anteriores (AYERZA; COATES, 2004; REYES-CAUDILLO et al., 2008).
Deve-se destacar o teor de ácido graxo α-linolênico (ALA, C18:3 n-3) presente no
óleo de chia, seguido por ordem decrescente, os ácidos graxos linoleico, oleico,
palmítico e esteárico. O óleo da semente de chia tem sido amplamente estudado por
apresentar a maior concentração de ALA dentre as fontes botânicas estudadas
(AYERZA, 1995). Além disso, a razão de ácidos graxos n-6/n-3 do óleo de chia é
expressivamente menor em relação a outros óleos vegetais (TUBEROSO et al.,
2007), uma importante característica do alimento, já que a baixa razão de ácidos
graxos n-6/n-3 está relacionada à redução do risco de desenvolvimento de doenças
cardiovasculares (SIMOPOULOS, 2004).
A dieta da maior parte da população, nos países industrializados, apresenta
baixo teor de ácidos graxos poli-insaturados (PUFA) ômega-3, principalmente devido
ao baixo consumo de pescados e alimentos vegetais fontes de ALA e ao uso
excessivo de óleo vegetal rico em ácido graxo linoleico (LA) (SIMOPOULOS, 2004;
GANESAN et al., 2014). A Sociedade Brasileira de Cardiologia estimula o consumo
de ácidos graxos ômega-3 de origem vegetal, como parte de uma dieta saudável,
sendo recomendado para reduzir o risco cardiovascular (SANTOS et al., 2013).
Dessa forma, a semente de chia pode ser considerada um alimento com uma
proporção interessante de ácidos graxos conforme recomendado pela referida
Sociedade para pacientes que apresentam síndrome metabólica, já que o óleo de
chia possui baixa concentração de ácidos graxos saturados e monoinsaturados, e
alta concentração de ácidos graxos poli-insaturados, sendo principalmente da família
n-3 (ALA) e pouco atribuído à família n-6 (LA) (SBC, 2005; SANTOS et al., 2013).
Estudos têm relatado a vantagem nutricional da utilização da semente de
chia ou de seus derivados, na alimentação animal, em relação às outras fontes de
PUFA (COATES; AYERZA, 2009; AYERZA; COATES, 2002, 2006; AYERZA et al.,
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2002). Além do potencial como fonte de nutrientes viável para alimentação animal,
as sementes de chia e seus derivados têm crescente interesse para indústrias
alimentícias devido a sua importante composição nutricional e potencial funcional.
As amostras de semente e óleo de chia também apresentaram alta atividade
antioxidante dentre os métodos in vitro avaliados, o que pode ser devido
principalmente à presença de compostos fenólicos como, mericetina, quercitina,
kaempferol e ácido clorogênico nas amostras estudadas. Vale ressaltar que seria
importante, também, a quantificação destes compostos na semente e óleo de chia,
uma vez que, no presente trabalho, os compostos fenólicos foram somente
identificados nas amostras de chia. Estudos anteriores também reportaram a alta
capacidade antioxidante da chia (REYES-CAUDILLO et al., 2008; IXTAINA, et al.,
2011; CAPITANI et al., 2012) e justificam esta propriedade não somente aos
compostos fenólicos, mas também a presença de compostos lipofílicos, como,
tocoferóis, carotenoides e fosfolipídeos encontrados no óleo de chia. A presença
desses compostos contribuem para a estabilidade oxidativa do óleo, apesar do alto
conteúdo de PUFA deste alimento, já que o valor de peróxido do óleo de chia (2.56
mEq/kg) foi considerado significativamente baixo quando comparado com outros
óleos vegetais (KHATTAB; ZEITOUN, 2013) e também com o recomendado pela
resolução n. 270 da Agência Nacional de Vigilância Sanitária (ANVISA) para óleos
brutos extraídos a frio, em que o máximo é de 15 mEq/kg (BRASIL, 2005). Além
disso, é importante ressaltar que os compostos fenólicos encontrados no óleo de
chia não estão presentes em outros óleos de sementes (TUBEROSO et al., 2007).
Dessa forma, a semente de chia pode ter os mesmos efeitos benéficos à
saúde atribuídos a outros grãos e vegetais, já que o teor de fibra dietética, e sua
atividade antioxidante são muitas vezes maior ou semelhante a outros grãos e
farinhas (RAGAEE et al., 2006). A importante composição nutricional e propriedades
funcionais da semente de chia, assim como de sua fração lipídica, motivam o uso
destes como uma importante fonte de alimento, considerando a atual tendência para
um aumento do consumo de alimentos funcionais e seu alto potencial nutricional e
tecnológico, já que também podem ser utilizados para produção de ingredientes
funcionais com aplicações comerciais em alimentos (REYES-CAUDILLO et al., 2008;
ÁLVAREZ-CHÁVEZ et al., 2008; SEGURA-CAMPOS et al., 2013).
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A partir disso, avaliamos a ingestão da semente e óleo de chia na prevenção
e no tratamento da obesidade e comorbidades induzida por dieta em modelo animal.
Em relação aos resultados do ensaio in vivo podemos destacar que a ingestão da
dieta HFF (hiperlipídica e adicionada de frutose) induziu a obesidade e promoveu
diversas alterações metabólicas relacionadas a esta, como resistência à insulina,
intolerância à glicose, dislipidemia, inflamação, acúmulo de lipídeo hepático,
estresse oxidativo e peroxidação lipídica nos animais.
O excessivo consumo energético, principalmente a partir de carboidratos
simples e ácidos graxos saturados leva ao aumento do armazenamento de energia
na forma de gordura, acompanhado por desordens metabólicas que contribuem para
o desenvolvimento da síndrome metabólica associada à obesidade (FERNÁNDEZ-
SÁNCHEZ et al., 2011). Tem sido relatado que o consumo de dietas ricas em
gorduras e açúcares está relacionado com o aumento da obesidade, intolerância à
glicose, resistência à insulina, inflamação, estresse oxidativo e esteatose hepática
em modelos animais (FEILLET-COUDRAY et al., 2009; LAISSOUF et al., 2013;
POUDYAL et al., 2012; SHAPIRO et al., 2011). Devido às suas propriedades
lipogênicas, o excesso de frutose na dieta pode causar má absorção de glicose,
maiores elevações de triglicerídeos, colesterol e ácidos graxos livres em
comparação a outros carboidratos. Estes distúrbios metabólicos estão relacionados
à indução da resistência à insulina e leptina geralmente observada a partir da
ingestão de alta concentração de frutose em seres humanos e modelos animais
(JOHNSON et al., 2007; SAMUEL, 2011). O estado de resistência à insulina induzida
pela frutose é normalmente caracterizado por uma dislipidemia metabólica profunda,
que parece resultar da superprodução hepática e intestinal de partículas de
lipoproteínas aterogênicas responsáveis pelo desenvolvimento da doença hepática
gordurosa não alcoólica (esteatose hepática) (BASCIANO et al., 2005).
Em contrapartida, nossos resultados demonstraram que a adição da semente
e do óleo de chia, como substituição do óleo de soja nas dietas experimentais, não
reverteu a obesidade, mas foi capaz de promover a normalização de quase todas as
alterações metabólicas ocasionadas pela ingestão da dieta HFF.
Em relação ao perfil oxidativo, a ingestão da semente e óleo de chia
melhorou o sistema de defesa antioxidante no plasma e no fígado, reduziu a
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peroxidação lipídica plasmática, promovendo, dessa forma, um efeito protetor contra
o estresse oxidativo advindo da obesidade. Desse modo, a redução da peroxidação
lipídica plasmática pode estar envolvida com o efeito hipolipidêmico observado após
a ingestão da semente e óleo de chia, que está associada à redução da
suscetibilidade do LDL-colesterol a oxidação, aparentemente devido à redução
plasmática desta lipoproteína. Além disso, a melhora do status antioxidante
encontrada nos grupos que receberam chia podem explicar estudos anteriores que
demonstraram efeito hepato e cardioprotetor em ratos obesos alimentados com
semente de chia durante 8 semanas (POUDYAL et al., 2012).
Dados da literatura indicam que diferentes tipos de ácidos graxos têm efeitos
variáveis na produção de radicais livres podendo agir diretamente na remoção das
espécies reativas de oxigênio e indiretamente na indução de enzimas antioxidantes
(BULLO et al., 2011). Conforme demonstrado, a semente de chia é rica em ALA, e
estudos anteriores que utilizaram o mesmo ácido graxo de outras fontes vegetais
(PAL; GHOSH, 2012a, 2012b) corroboram com nossos resultados sobre a melhora
do status antioxidante in vivo. É possível que, em condições in vivo, o ALA pode
reduzir a formação de hidroperóxidos pela eliminação de radicais livres e redução da
peroxidação de PUFA.
Tendo em vista que o sistema antioxidante endógeno é suplementado por
nutrientes exógenos, por exemplo, vitaminas, minerais, polifenóis, dentre outros, o
efeito protetor da chia contra o estresse oxidativo pode ser devido não somente a
ação do ALA, mas também aos antioxidantes naturais presentes na semente e óleo
de chia, conforme demonstrado pelos resultados da capacidade antioxidante in vitro
do presente estudo e por outros estudos que também reportaram o importante
conteúdo de compostos bioativos e potencial antioxidante da semente e óleo de chia
(REYES-CAUDILLO et al, 2008; IXTAINA et al, 2011; MARTÍNEZ-CRUZ;
PAREDES-LÓPEZ, 2014). No entanto, é necessário avaliar o potencial antioxidante
da semente e óleo de chia contra o estresse oxidativo incluindo mecanismos de
absorção de compostos fenólicos e bioatividade em ensaios in vitro e in vivo, já que
antioxidantes dietéticos, principalmente os polifenóis, são expostos a um intenso
metabolismo in vivo que afeta a sua estrutura química, biodisponibilidade e
bioatividade (LANDETE, 2012).
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Nossos resultados também demonstraram que a ingestão da semente e óleo
de chia foi capaz de normalizar a dislipidemia, bem como aumentar a excreção de
lipídios fecais nos animais. A este respeito, alguns estudos em animais têm
mostrado a redução sérica ou plasmática de colesterol total, triacilgliceróis e
aumento nos níveis de HDL-colesterol quando a chia foi utilizada como fonte
dietética de gordura em ratos saudáveis ou dislipidêmicos (AYERZA; COATES
2007; CHICCO et al., 2009), o que corrobora com nossos achados. Além disso,
Sierra e colaboradores (2015) demonstraram que o óleo de chia pode melhorar a
função vascular em condições hipercolesterolêmicas em coelhos.
O alto teor de fibra alimentar das sementes de chia pode desempenhar um
papel na redução dos níveis de lipídios circulantes, já que o efeito
hipocolesterolêmico da fibra dietética é amplamente relatado (WILLEM VAN DER
KAMP et al., 2010). Além disso, o ALA, principal ácido graxo presente na chia, é
convertido principalmente em eicosapentaenoico (EPA) e docosahexaenoico (DHA)
no fígado (GONZÁLEZ-MAÑÁN et al., 2012). A eficácia dessa bioconversão pode
determinar o seu efeito sobre os lipídios plasmáticos. Desta forma, a chia, como um
precursor de EPA e DHA, pode modificar o metabolismo dos ácidos graxos no
fígado através da ativação do receptor ativado por proliferadores de peroxissoma
alpha (PPARα) e da baixa regulação da proteína de ligação ao elemento de
resposta a esterol (SREBP-1c), levando a uma diminuição da circulação de lipídios
(ROSSI et al., 2013). Resultados semelhantes foram encontrados por González-
Mañán e colaboradores (2012), em relação à oxidação de ácidos graxos e
expressão de biomarcadores envolvidos na lipogênese após a ingestão de óleo de
chia em ratos saudáveis.
No presente estudo, os animais que receberam semente ou óleo de chia
também apresentaram melhora da tolerância à glicose e à insulina, segundo os
testes de GTT e ITT, além da redução da secreção de insulina pelas ilhotas
pancreáticas. Porém, a concentração de insulina plasmática não foi afetada.
Resultados semelhantes foram relatados por Chicco e colaboradores (2009) quando
ratos dislipidêmicos receberam semente de chia nos últimos 2 meses do
experimento. Eles encontraram uma melhora na resistência à insulina e homeostase
da glicose, sem alterações na insulinemia. Além disso, a ingestão da semente de
chia normalizou a diminuição da capacidade de fosforilação e oxidação da glicose,
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bem como melhorou a insensibilidade à insulina periférica em ratos alimentados
com dieta rica em sacarose (OLIVA et al., 2013). A este respeito, estudos têm
demonstrado que ácidos graxos poli-insaturados da família ômega-3 podem prevenir
ou normalizar a resistência à insulina periférica em animais alimentados com dieta
rica em gordura ou carboidratos (LOMBARDO; CHICCO, 2009). Também tem sido
relatado que a ingestão de ALA ou de alimentos ricos neste ácido graxo melhora a
sensibilidade à insulina e respostas glicêmicas em modelos animais e humanos (WU
et al., 2012). No entanto, é importante destacar a necessidade de estudos que
avaliem a ação da chia ou de seus compostos nos receptores de insulina, assim
como na via de sinalização da insulina, para elucidar os mecanismos relacionados à
melhora da resistência à insulina.
Os resultados do presente estudo também demonstraram que a ingestão da
semente ou óleo de chia foi capaz de normalizar a inflamação sérica causada pela
dieta HFF, uma vez que a chia promoveu a redução de marcadores pró-
inflamatórios, como TNF-α, MCP-1 e proteína C-reativa, além do aumento nos níveis
de adiponectina, um marcador anti-inflamatório. A adiponectina desempenha um
papel protetor contra a resistência à insulina através da regulação do metabolismo
da glicose e lipídeos, apresentando propriedades angiogênicas, antiaterogênicas e
anti-inflamatórias (GALIC et al., 2010). No entanto, existem poucos estudos sobre os
efeitos da chia em hormônios e marcadores inflamatórios relacionados à obesidade.
A proteína C-reativa está associada à fatores de risco cardiovascular,
incluindo resistência à insulina, diabetes, síndrome metabólica, hipertensão e
dislipidemia (FERNÁNDEZ-SÁNCHEZ et al., 2011). A suplementação com a
semente de chia (37 g/dia) em indivíduos diabéticos tipo 2 reduziu os níveis de
proteína C-reativa, enquanto manteve um bom controle glicêmico e lipídico
(VUKSAN et al., 2007). Poudyal e colaboradores (2012) também encontraram uma
redução nos níveis de proteína C-reativa no plasma de ratos obesos alimentados
com sementes de chia, o que corrobora com nossos achados.
Segundo estudos experimentais realizados em animais, ácidos graxos poli-
insaturados da família ômega-3 podem melhorar diversas anormalidades
metabólicas e esses efeitos incluem a melhora da sensibilidade à insulina através do
aumento da produção e secreção de adipocitocinas e potencial prevenção da
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resistência à insulina através dos efeitos anti-inflamatórios (WU et al., 2012).
Baranowski e colaboradores (2012) demonstraram que intervenções dietéticas com
óleo de linhaça (rico em ALA) promoveram uma redução em diversos marcadores
inflamatórios no tecido adiposo de ratos obesos. Além disso, os efeitos
cardioprotetores do ALA são mediados, em parte, por uma redução na produção de
citocinas inflamatórias (ZHAO et al., 2007). Têm sido relatado que os ácidos graxos
podem alterar as respostas inflamatórias através da inibição da atividade do fator
nuclear kappa B (NF-kB), o qual regula a indução da expressão de citocinas (ZHAO
et al., 2007). Desta forma, Jangale e colaboradores (2013) demonstraram que a
ingestão do óleo de linhaça regulou a expressão de genes inflamatórios hepáticos
em ratos diabéticos, incluindo o NF-kB. No entanto, a modulação de marcadores
inflamatórios no tecido adiposo e hepático após a ingestão de chia ainda não foi
estudada. Dessa forma, mais estudos que avaliem a ingestão da semente de chia
precisam ser realizados com o objetivo de elucidar os mecanismos de ação pelos
quais a chia exerce tal efeito.
Levando em consideração que grande parte das alterações metabólicas e
efeitos benéficos observados a partir da ingestão da semente e óleo de chia podem
ser explicados pela presença do ALA ou da sua conversão em outros ácidos graxos
da família ômega-3, é importante ressaltar que a ingestão da semente e do óleo de
chia aumentou as concentrações plasmáticas dos ácidos graxos ALA, EPA e DHA
nos animais, além de reduzir a razão n-6/n-3. Resultados semelhantes foram
encontrados quando a semente de chia foi adicionada a 16% (AYERZA; COATES
2007) ou 36,2% (CHICCO et al., 2009) nas dietas de ratos saudáveis e
dislipidêmicos. O mesmo ocorreu em diversos tecidos de ratos saudáveis quando o
óleo de chia foi adicionado a 10% nas dietas experimentais (GONZÁLEZ-MAÑÁN et
al., 2012; VALENZUELA et al., 2014). Estes resultados indicam que a semente ou
óleo de chia podem alterar a composição de ácidos graxos independentemente da
quantidade ingerida. Deste modo, é importante avaliar a quantidade mínima de chia
suficiente para alterar o perfil de ácidos graxos in vivo.
Além disso, deve-se destacar que em humanos a taxa de conversão de ALA
em EPA, e especialmente em DHA, é geralmente baixa e pode ser modulada pelo
estado fisiológico, dieta, sexo e idade (GANESAN et al., 2014). Jin e colaboradores
(2012) demonstraram que a ingestão de chia (25 g/dia) durante 7 semanas
167
aumentou as concentrações plasmáticas de ALA e EPA, mas não de DHA em
mulheres na pós menopausa. Dessa forma, é preciso cuidado antes de extrapolar os
resultados para humanos.
No presente estudo, foi possível observar efeitos benéficos similares a partir
da ingestão tanto do óleo quanto da semente de chia, bem como do tempo de
tratamento em que os animais foram submetidos. A ausência de diferença
significativa entre o tempo de tratamento (6 ou 12 semanas) sugere que 6 semanas
de ingestão de chia é tempo suficiente para observar mudanças significativas nos
biomarcadores relacionados às complicações da obesidade. Além disso, uma
possível explicação para os efeitos similares do óleo e da semente de chia pode ser
devido as concentrações similares de ALA que estes forneceram às dietas, ou
também pode estar relacionada às diferentes matrizes alimentares (semente x óleo),
uma vez que a interação de nutrientes entre os compostos presentes na semente
chia e a complexidade do sinergismo da matriz alimentar deve ser considerada. A
liberação de nutrientes a partir da matriz alimentar, as interações com outros
componentes alimentares, assim como a presença de cofatores, podem influenciar a
biodisponibilidade de nutrientes (PARADA; AGUILERA, 2007). Portanto, o estado da
matriz alimentar pode favorecer ou dificultar a sua resposta in vivo, uma vez que o
modo de ação pode ser mais relevante do que a quantidade total presente no
alimento. O que também pode explicar o fato da semente de chia não ter
apresentado melhores resultados em relação ao óleo de chia.
Os resultados apresentados nessa pesquisa são um bom ponto de partida
para investigações futuras que devem realizar uma análise mais aprofundada para
esclarecer os mecanismos de ação relacionados aos efeitos benéficos da ingestão
de chia. É necessária precaução antes de extrapolar estes resultados para
humanos, uma vez que a quantidade de chia testada no presente estudo deve ser
avaliada em estudos clínicos como substituição do óleo ou introdução da semente
como alimento funcional na dieta da população.
168
CONCLUSÃO GERAL
Como conclusão geral do trabalho, podemos destacar que a ingestão da dieta
obesogênica (HFF) induziu a obesidade, resistência à insulina, intolerância à glicose,
dislipidemia, inflamação, estresse oxidativo e peroxidação lipídica nos animais,
sendo que tais alterações podem indicar a influência da frutose e do alto teor de
gordura sobre o estado metabólico destes animais. A adição da semente e do óleo
de chia, como substituição do óleo de soja nas dietas experimentais, não reverteu a
obesidade, mas normalizou o estado inflamatório sérico, a dislipidemia, a
intolerância à glicose e a resistência à insulina, aumentou a concentração plasmática
de ácidos graxos da família ômega-3, reduzindo, dessa forma, a razão dos ácidos
graxos n-6/n-3, também reduziu a peroxidação lipídica e o estresse oxidativo, e
aumentou a capacidade antioxidante no plasma e no fígado de animais obesos
induzidos por dieta. Os efeitos benéficos foram encontrados tanto no período de
prevenção (12 semanas) quanto no de tratamento (6 semanas). Tais efeitos podem
ser explicados pelos compostos bioativos e ácido graxo ômega-3 presentes na
semente e no óleo de chia, assim como pelo potencial antioxidante in vitro
encontrado nesses alimentos.
Visto que os produtos estudados tem comprovado efeito biológico e potencial
funcional e por isso podem beneficiar o estado de saúde da população, seja via
suplementação ou por enriquecimento de alimentos, o trabalho de pesquisa em
questão forneceu subsídios à introdução destes compostos na dieta habitual da
população, como coadjuvante na prevenção e no controle de desordens metabólicas
crônicas. Porém, é preciso precaução antes de extrapolar os resultados para
humanos ou estipular quantidades de recomendação de ingestão. Dessa forma,
estimula-se a realização de estudos em humanos baseados em diferentes ensaios
clínicos para confirmação dos resultados.
Após a experiência adquirida com a elaboração deste trabalho, é possível
destacar algumas ideias ou sugestões a serem observados em futuras pesquisas:
- Avaliar os efeitos da semente e do óleo de chia em estudos clínicos como
substituição do óleo da dieta ou introdução da semente como alimento funcional.
169
- Avaliar, com base em estudos clínicos, a quantidade mínima de óleo e/ou
semente de chia consumida que seja necessária para promover efeitos benéficos.
- Avaliar o potencial antioxidante da semente e do óleo de chia contra o
estresse oxidativo incluindo mecanismos de absorção de compostos fenólicos e
bioatividade em ensaios in vitro e in vivo.
- Avaliar a ação da chia ou de seus compostos nos receptores de insulina,
assim como na via de sinalização da insulina, para elucidar os mecanismos
relacionados à melhora da resistência à insulina.
- Avaliar os efeitos da chia e seus compostos em hormônios e marcadores
inflamatórios relacionados à obesidade e seu mecanismo de ação.
- Estudar a viabilidade tecnológica e elaboração de produtos adicionados com
o óleo ou a semente de chia, como iogurtes, sucos, pães, bolos, barras de cerais,
biscoitos, cereais em geral, sobremesas, entre outros.
170
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ANEXOS
ANEXO A: ESQUEMA DO DELINEAMENTO EXPERIMENTAL DO ENSAIO
BIOLÓGICO
177
ANEXO B: PARECER DA COMISSÃO DE ÉTICA NA EXPERIMENTAÇÃO
ANIMAL
178
ANEXO C: AUTORIZAÇÃO DA EDITORA PARA INCLUSÃO DOS ARTIGOS
PUBLICADOS
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