Embed Size (px)
Citation preview
FABÍOLA FONSECA LAGE
CASCA DE JABUTICABA: INIBIÇÃO DE ENZIMAS DIGESTIVAS, ANTIOXIDANTE,
EFEITOS BIOLÓGICOS SOBRE O FÍGADO E PERFIL LIPÍDICO
LAVRAS-MG
2014
FABÍOLA FONSECA LAGE
CASCA DE JABUTICABA: INIBIÇÃO DE ENZIMAS DIGESTIVAS , ANTIOXIDANTE, EFEITOS BIOLÓGICOS SOBRE O FÍGADO E
PERFIL LIPÍDICO
Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós-Graduação em Agroquímica, para a obtenção do título de Doutor.
Orientadora
Dra. Angelita Duarte Corrêa
Coorientador
Dr. Raimundo Vicente de Souza
LAVRAS-MG
2014
Lage, Fabíola Fonseca. Casca de jabuticaba : inibição de enzimas digestivas, antioxidante, efeitos biológicos sobre o fígado e perfil lipídico / Fabíola Fonseca Lage. – Lavras : UFLA, 2014.
140 p. : il. Tese (doutorado) – Universidade Federal de Lavras, 2014. Orientador: Angelita Duarte Corrêa. Bibliografia. 1. Plinia jaboticaba. 2. α -amilase. 3. Propriedades funcionais
tecnológicas. 4. HDL-colesterol. 5. Efeito hepatoprotetor. I. Universidade Federal de Lavras. II. Título.
CDD – 574.192
Ficha Catalográfica Elaborada pela Coordenadoria de Produtos e Serviços da Biblioteca Universitária da UFLA
FABÍOLA FONSECA LAGE
CASCA DE JABUTICABA: INIBIÇÃO DE ENZIMAS DIGESTIVAS , ANTIOXIDANTE, EFEITOS BIOLÓGICOS SOBRE O FÍGADO E
PERFIL LIPÍDICO
Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós-Graduação em Agroquímica, para a obtenção do título de Doutor.
APROVADA em 27 de fevereiro de 2014.
Dra. Nísia Andrade Villela Dessimoni Pinto UFVJM Dr. Michel Cardoso de Angelis Pereira UFLA Dra. Celeste Maria Patto Abreu UFLA Dra. Luciana Lopes Silva Pereira UFLA Dra. Denise Alvarenga Rocha UFLA
Dra. Angelita Duarte Corrêa
Orientadora
Dr. Raimundo Vicente de Souza Coorientador
LAVRAS – MG
2014
DEDICO
À minha mãe, por todo o carinho e amor, por estar a meu lado sempre e acreditar em mim
Aos meus sobrinhos Alencar Henrique, Clarissa e
a pequena Alícia, por serem a grande alegria da minha vida
Às minhas irmãs Flaviane, Flávia e Fernanda, pelo carinho e presença constante.
AGRADECIMENTOS
E em tudo isso glorifica o Senhor que te criou (Eclo 32, 17a)
À Deus, por ser meu sustento, minha alegria e fortaleza. Porque foi por
sua misericórdia eu cheguei até aqui.
À professora Angelita Duarte Corrêa, por sua paciência, atenção, apoio e
carinho. Por tudo que me ensinou e ajudou.
Ao professor Raimundo Vicente de Souza, pela coorientação e
ensinamentos valiosos.
Aos amigos do Laboratório de Bioquímica e de Pós-graduação, que
fizeram com que meus dias no laboratório fossem felizes e agradáveis. Pela
ajuda certa, na hora certa.
Aos meu amigos na fé, profissionais do reino que fazem parte da família
GPP São Benedito, por me ouvirem e apoiarem em todos os momentos.
A Xulita, pelos momentos compartilhados durante a realização deste
trabalho.
Ao amigo Willian Cezar Cortez, funcionário do Laboratório de
Fisiologia e Farmacologia (DMV), pela essencial contribuição na realização do
ensaio biológico.
À Universidade Federal de Lavras, especialmente ao Programa
Agroquímica, pela oportunidade e acolhida.
Para tudo há um tempo,
para cada coisa há um momento debaixo dos céus:
tempo para nascer e tempo para morrer;
tempo para plantar e tempo para colher;
tempo para chorar e tempo para rir;
tempo para dar abraços e tempo para apartar-se;
tempo para calar e tempo para falar;
tempo para amar e tempo para odiar;
tempo para a guerra e tempo para paz.
Todas as coisas que Deus fez são boas a seu tempo.
(Eclesiastes 2)
RESUMO
A jabuticaba é uma fruta tipicamente brasileira. Sua casca possui alto teor de fibras e de compostos fenólicos, entre eles as antocianinas responsáveis por sua cor característica. Os compostos fenólicos possuem comprovado potencial antioxidante e estudos têm sugerido que podem inibir algumas enzimas digestivas in vitro e in vivo. Objetivou-se, neste trabalho, estudar a farinha de casca de jabuticaba (FCJ) investigando a capacidade inibitória sobre enzimas digestivas, seu perfil fenólico, o potencial antioxidante in vitro e in vivo, seu efeito sobre os níveis séricos de lipídios em ratas e a ação hepatoprotetora, visando uma melhor utilização do ponto de vista tecnológico e nutricional. Foi analisado o potencial inibitório in vitro da FCJ sobre as enzimas α-amilase, α-glicosidase, lipase e tripsina na presença e na ausência de fluido gástrico. O extrato utilizado para quantificação e identificação dos compostos fenólicos por cromatografia liquida de alta eficiência foi preparado com metanol 50% na razão 1:25 (m/v). A atividade antioxidante foi determinada pelo método ABTS. Foram analisadas as propriedades tecnológicas absorção de água e óleo, solubilidade de nitrogênio, volume de espuma e estabilidade de emulsão. Para o estudo do efeito da FCJ sobre a peroxidação lipídica e o perfil lipídico plasmático e hepático de ratas, os animais foram divididos em 4 grupos de 8 animais. Os grupos receberam 0 (controle); 0,50; 1,50 e 3,00 g de FCJ por 100 g dieta.O extrato de farinha de casca de jabuticaba (EFCJ) inibiu significativamente a enzimas α-amilase. A inibição da α-amilase pelo EFCJ foi de 585,60 µmol min-1 (unidades de enzima inibida - UEI) e com o fluido gástrico 560,00 UEI. Foram identificados na FCJ os ácidos gálico, elágico e salicílico; galocatequina e a epicatequina. O composto fenólico marjoritário foi a epicatequina. A FCJ apresentou potencial antioxidante e possui boa absorção de água e estabilidade de emulsão. O ensaio biológico demonstrou que a dieta com 3,00% de FCJ aumentou em 20,23% o nível de HDL. Os grupos que receberam FCJ mostraram atividade de aspartato aminotransferase e alanina aminotransferase inferior ao grupo controle. Houve diminuição significativa da esteatose macrovesicular no fígado dos animais que receberam dieta suplementada com 3,00% de FCJ. As dietas contendo 1,50% e 3,00% de FCJ reduziram em cerca de 50,00% a peroxidação lipídica no fígado. A capacidade inibitória do EFCJ sobre a α-amilase e a tripsina pode estar relacionada com a ocorrência dos compostos fenólicos.A FCJ mostra-se como uma alternativa promissora para ser utilizada como antioxidante natural e como aditivo em formulações como sopas, molhos, salsichas, massas, queijos, bolos, produtos de padaria.A FCJ foi eficiente na proteção cardiovascular por aumentar o nível sérico de HDL, possuir boa
atividade antioxidante e demonstrou possuir efeito hepatoprotetor. A utilização da FCJ na prevenção e controle do diabetes e obesidade pode ser viável. Palavras chave: Plinia jaboticaba. α – amilase. HPLC. Propriedade funcional tecnológica. HDL-colesterol. Efeito hepatoprotetor.
ABSTRACT
Jabuticaba is a typical Brazilian fruit. Its skin is high in soluble and insoluble fiber and has a high content of phenolic compounds, especially anthocyanins, responsible for its characteristic color. Phenolic compounds have an antioxidant potential and studies have suggested that they may inhibit some digestive enzymes in vitro and in vivo. The objective of this study was to evaluate jabuticaba skin flour (JSF), investigating the inhibitory capacity on digestive enzymes, its phenolic profile, in vitro and in vivo antioxidant potential, and its effect on the serum lipid levels in female rats and hepatoprotective effect, aiming for a better use, from a technological and nutritional point of view. The inhibitory potential in vitro of JSF on the enzymes α-amylase, α-glucosidase, lipase and trypsin was evaluated in the presence and absence of gastric fluid. The extract used for the quantification and identification of phenolic compounds by high performance liquid chromatography was prepared with 50% methanol in the ratio 1:25 (w/v). The antioxidant activity was determined by the ABTS method. The technological properties: absorption of water and oil, nitrogen solubility, foam volume and emulsion stability were analyzed. For the study of the effect of JSF on lipid peroxidation and plasma and hepatic lipid profile in rats, the animals were divided into 4 groups of 8 animals. The groups received 0 (control); 0.50; 1.50 and 3.00 g JSF per 100 g diet. The jabuticaba skin flour extract (JSFE) significantly inhibited α-amylase. The inhibition of α-amylase by JSFE was 585.60 µmol min-1 (units of inhibited enzyme - UIE) and, with gastric fluid, it was 560.00 UIE. Gallic, ellagic and salicylic acids were identified in JSF, as well as gallocatechin and epicatechin. The phenolic compound marjoritario was epicatechin. JSF showed an antioxidant potential, and has a good water absorption and emulsion stability. The biological assay showed that the diet with 3.00% JSF increased in 20.23% the level of HDL, compared to the control. The groups that received JSF showed that the activity of aspartate aminotransferase and alanine aminotransferase was inferior. There was a significant decrease in macrovesicular steatosis in the liver of the animals fed the diet supplemented with 3.00% JSF. The diets containing 1.50% and 3.00% JSF reduced lipid peroxidation in the liver by about 50.00%. The inhibitory capacity of JSFE on α-amylase and trypsin may be related to the occurrence of phenolic compounds . JSF is shown as a promising alternative to be used as a natural antioxidant and as an additive in formulations such as soups, sauces, sausages, pasta, cheeses, cakes, bakery products. JSF was effective in protecting cardiovascular, since it increases HDL serum levels, has a good antioxidant activity and an hepatoprotective effect. The use of JSF in the prevention and control of diabetes and obesity may be viable.
Keywords: Plinia jaboticaba. α-amylase. HPLC. Functional technological property. HDL-cholesterol. Hepatoprotective effect.
SUMÁRIO
PRIMEIRA PARTE
1 INTRODUÇÃO......................................................................... 13
2 REFERENCIAL TEÓRICO..................................................... 16
2.1 A jabuticabeira........................................................................... 16
2.2 Radicais livres e dano oxidativo................................................ 17
2.3 Antioxidantes.............................................................................. 19
2.4 Compostos fenólicos................................................................... 20
2.4.1 Ácidos fenólicos.......................................................................... 23
2.4.2 Flavonoides................................................................................. 25
2.4.2.1 Taninos........................................................................................ 27
2.4.2.2 Antocianinas............................................................................... 30
2.5 Metabolismo de colesterol, lipoproteínas e suas relações com doenças cardiovasculares ehepáticas......................................
32
REFERENCIAS.................................................................... 39
SEGUNDA PARTE – ARTIGOS
ARTIGO 1: Inhibitory potential of Plinia Jaboticaba skin on the digestive enzymes…………………………………………
45
ARTIGO 2: Jabuticaba skin flour as a functional food ingredient: phenolic profile, antioxidant potential and functional-technological properties………………………….
73
ARTIGO 3: Jabuticaba skins decrease lipid peroxidation and have hepatoprotective and antihyperlipidemic effects……………………………………………………………
102
APÊNDICES………………………………………………….. 130
12
PRIMEIRA PARTE
APRESENTAÇÃO
As referências bibliográficas correspondem somente às citações que
aparecem nos itens introdução e referencial teórico.
Os resultados que fazem parte desta tese estão apresentados sob a forma
de artigos, os quais se encontram no item artigos.
Cada artigo está estruturado de acordo com as normas das revistas
científicas escolhidas para a submissão ou publicação do mesmo.
13
1 INTRODUÇÃO
As recentes pesquisas desenvolvidas evidenciam a importância do
estudo dos compostos orgânicos existentes em plantas para várias áreas do
conhecimento.Entre essas substâncias, os compostos fenólicos têm despertado
grande interesse na comunidade científica por apresentarem efetiva atividade
antioxidante.
Antioxidantes fenólicos são os mais ativos estão presentes nos vegetais e
funcionam como sequestradores de radicais e algumas vezes como quelantes de
metais, agindo tanto na etapa de iniciação como na propagação do processo
oxidativo, sendo eficazes para previnir a oxidação lipídica. Esses compostos
atuam na inativação dos radicais livres nos compartimentos celulares lipofílico e
hidrofílico e inibem reações em cadeia provocadas pelos radicais livres doando
hidrogênio. Os compostos fenólicos bloqueiam as estruturas radicalares devido à
sua estrutura química, formada por, pelo menos, um anel aromático com
grupamentos hidroxila, sendo que a ressonância do anel aromático confere
relativa estabilidade aos intermediários formados por sua ação antioxidante.
A inclusão de alimentos ricos em antioxidantes na dieta é importante
devido à interceptação dos radicais livres, que diminui o risco do
desenvolvimento de patologias associadas ao acúmulo desses radicais como as
doenças neurodegenerativas como o Alzheimer, doenças cardiovasculares e
câncer. A peroxidação e o estresse oxidativo, por exemplo, são agravantes nas
alterações glicêmicas e nas dislipidêmicas. Dislipidemias são distúrbios do
metabolismo lipídico, com repercussões sobre os níveis de lipoproteínas na
circulação sanguínea e sobre as concentrações de seus diferentes componentes.
Estudos sobre antioxidantes têm ressaltado o uso de compostos isolados, como
os taninos condensados e as antocianinas, no entanto, conhecer e compreender a
14
forma como estes nutrientes atuam sinergicamente, potencializando a ação
antioxidante do alimento pesquisado pode agregar valor aos produtos “in
natura”.
Estudos sugerem que os compostos fenólicos também são capazes de
inibir enzimas digestivas como a α-amilase e as glicosidases. A inibição dessas
enzimas mostra-se uma estratégia interessante para o controle da glicemia,
diabetes do tipo II e obesidade. Devido a esses efeitos biológicos, vários
benefícios à saúde têm sido atribuídos aos compostos fenólicos presentes em
frutas, vegetais, chás e vinhos.
A jabuticaba é uma fruta tipicamente brasileira. As cascas de jabuticaba
são descartadas como resíduo quando o fruto é consumido, no entanto, devido ao
seu alto valor nutritivo, sua utilização na indústria alimentícia pode ser bastante
promissora. As cascas são ricas em minerais, compostos fenólicos, fibra
alimentar, além de conterem vitamina C. Entre os compostos fenólicos , podem-
se citar as antocianinas, que são responsáveis por sua cor característica.
Antocianinas são glicosídeos de antocianidinas e pertencem ao grupo dos
flavonoides. São pigmentos de coloração púrpura, vermelha ou violeta,
dependendo do pH do meio e atuam como antioxidantes endógenos. Análises
utilizando HPLC realizadas por Lima et al. (2011a) mostraram a ocorrência das
antocianinas cianidina-3-glicosídeo e delfinidina-3-glicosídeo nas cascas de
jabuticaba. Neste estudo,as cascas de jabuticaba apresentaram significativa
atividade antioxidante. Análises de compostos bioativos como saponina, inibidor
de tripsina e lectina confirmam que as cascas de jabuticaba apresentam níveis
inferiores ou semelhantes aos de alimentos convencionais (LIMA et al., 2008).
O teor elevado de fibras e compostos fenólicos, bem como o bom
potencial antioxidante indicam que a utilização da farinha de casca de jabuticaba
(FCJ) como componente na alimentação humana é bastante promissora. As
fibras atuam de forma benéfica sobre a mobilidade intestinal. Antioxidantes
15
naturais, ao serem adicionados aos alimentos protegem não somente o produto
alimentício, mas também o organismo animal visto que, ao serem ingeridos,
combatem radicais livres, contribuindo para a redução do risco de desenvolver
várias doenças crônicas não transmissíveis. Os compostos fenólicos presentes
nas cascas de jabuticaba podem atuar como bons inibidores de enzimas
digestivas. Além disso, o elevado teor de fibras associado a um bom potencial
antioxidante pode induzir a uma diminuição da concentração de colesterol
plasmático nos indivíduos.
Objetivo geral
Estudar a FCJ investigando a capacidade inibitória sobre enzimas
digestivas, o perfil fenólico, o potencial antioxidante in vitro e in vivo, as
propriedades funcionais tecnológicas, o efeito biológico sobre o fígado e sua
atuação sobre o nível sérico de colesterol em ratos, visando uma melhor
utilização das cascas de jabuticaba, do ponto de vista tecnológico e nutricional.
16
12 REFERENCIAL TEÓRICO
2.1 A jabuticabeira
A jabuticabeira é uma árvore pertencente à família Myrtaceae do tipo
perene, de grande rusticidade e longevidade. Os tupis chamavam seu fruto de
“iapoti´kaba”, que significa fruto em botão, devido a sua forma arredondada.
Sua madeira apresenta elevada dureza, suas folhas são opostas, lanceoladas e
vermelhas quando novas, as flores são brancas e sésseis. Seus frutos são do tipo
baga globosa de até 3 cm de diâmetro, com casca preta-avermelhada, ou verde-
claras e verde-bronzeadas ou com listras roxas ou vermelhas. A polpa, macia,
esbranquiçada, suculenta, mucilaginosa, agridoce, é circundada por um epicarpo
fino que apresenta comumente uma única semente, podendo apresentar até
quatro sementes (DANNER et al., 2006).
A casca e a semente são ricas em fibras insolúves e juntas representam
mais de 50% do peso do fruto. O teor de fibras insolúveis,determinado nas
cascas é de 27,03 g 100 g-1 de matéria seca (MS).As cascas também possuem um
elevado teor de fibras solúveis (6,77 g 100 g-1MS) e é rica em minerais como o
potássio, magnésio, cálcio e cobre. Os teores de proteína bruta e extrato etéreo
da jabuticaba são baixos, mesmo nas sementes. O fruto inteiro apresenta 48,33 g
100 g-1 MS de açúcar total, 40,21 g 100 g-1 MS de açúcares redutores e 7,70 g
100 g-1 MS de açúcares não redutores. Foram detectados, por HPLC, os
açúcares glicose, frutose e sacarose na polpa, antocianinas cianidina-3-
glicosídeo e delfinidina-3-glicosídeos na casca, e os ácidos orgânicos oxálico,
cítrico, málico, succínico e acético na polpa e na casca. (LIMA et al., 2008,
2011a, 2011b). Pesquisas recentes também demonstram que a jabuticaba é uma
17
boa fonte de ácido elágico e vitamina C (ABE; LAJOLO; GENOVESE, 2012;
LIMA et al., 2011a).
A casca de jabuticaba apresenta elevado teor de compostos fenólicos
totais, sendo de 9, 79 g 100 g-1 MS (ALVES et al., 2013). Entre os fenólicos
destacam-se as antocianinas com 2,06 g 100 g-1 MS, e o melhor método para a
extração desses compostos é a maceração com 50% de etanol acidificado com
HCl 1,5 mol L-1 (85:15). O baixo pH do extrato faz com que o pigmento
permaneça estável por um período de 185 dias (LIMA et al., 2011a).
Os compostos fenólicos são potentes antioxidantes naturais e,
provavelmente, são responsáveis pela alta atividade antioxidante das cascas da
jabuticaba. Em estudo utilizando-se os métodos ABTS, fosfomolibdênio e β-
caroteno/ácido linoleico para medir a atividade antioxidante em FCJ, o do ABTS
acarretou melhores respostas. Os valores obtidos paraa atividade antioxidante
foram de 1,56 mmol L-1 g-1 equivalente ao trolox e 99,99 mg g-1 equivalente à
vitamina C e de 159 mg g-1 equivalente à vitamina C, pelo método do
fosfomolibdênio (LIMA et al., 2011a).
Estudos in vitro sobre a atividade antiproliferativa das casca de
jabuticaba sobre células tumorais demonstram efeitos antiproliferativos contra
leucemia (K-562) e contra células cancerosas da próstata (PC-3). Estudos sobre
os efeitos do extrato de casca de jabuticaba sobre as células da medula óssea de
ratos, usando o teste do micronúcleo, demonstraram que o extrato não induziu
danos ao DNA, nem apresentou propriedades citotóxicas sobre as células
analisadas sendo considerado não mutagênicos (LEITE-LEGGATI et al., 2012).
2.2 Radicais livres e dano oxidativo
Radicais livres são moléculas altamente instáveis, que contêm um ou
mais elétrons não pareados e com existência independente. Possuem meia vida
18
muito curta e são quimicamente muito reativos, sendo capazes de reagir com
qualquer composto situado próximo à sua órbita externa, passando a ter uma
função oxidante ou redutora. Radicais livres in vivo são formados via ação
catalítica de enzimas, durante os processos de transferência de elétrons que
ocorrem no metabolismo celular e pela exposição à fatores como o uso de
cigarros e uma dieta inadequada (BIANCHI; ANTUNES, 1999).
A geração de radicais livres constitui uma ação contínua e fisiológica
cumprindo funções biológicas essenciais. São formados em um cenário de
reações de óxido-redução, provocando essas reações ou delas resultando. Podem
ceder o elétron desemparelhado e serem oxidados ou podem receber outro
elétron e serem reduzidos. Essas reações ocorrem no citoplasma, nas
mitocôndrias ou na membrana, e o seu alvo celular (proteínas, vitaminas,
lipídeos, carboidratos e moléculas de DNA) está relacionado com seu local de
formação (MANACH et al., 2004).
O peróxido de hidrogênio, os peróxidos orgânicos, os ânions
superóxidos e o radical hidroxila são espécies reativas de oxigênio produzidas
pelo metabolismo aeróbio dos organismos vivos e possivelmente estão
envolvidas em vários tipos de doenças. Estima-se que os principais efeitos
negativos são causados pelo radical hidroxila (OH), gerado a partir do
superóxido (O2-) e peróxido de hidrogênio (H2O2). A proteção contra essas
espécies reativas é fornecida por compostos antioxidantes presentes na dieta
humana e por antioxidantes enzimáticos, como a catalase e a glutationa
peroxidase (TEDESCO et al., 2001).
Devido à produção contínua de radicais livres, durante os processos
metabólicos, ocorrem mecanismos de defesa antioxidante para limitar os níveis
intracelulares e impedir a indução de danos.
19
2.3 Antioxidantes
Antioxidantes são substâncias que atrasam ou inibem a oxidação de um
substrato de maneira eficaz. Geralmente, estão presentes em baixas
concentrações em relação à concentração do substrato. Podem ser classificados
em antioxidantes enzimáticos, que são capazes de interagir com as enzimas que
removem as espécies reativas ao oxigênio e não enzimáticos, que interagem com
as espécies radicalares e são consumidos durante a reação (MOREIRA;
MANCINI-FILHO, 2004).
Os antioxidantes impedem a formação de radicais livres pela inibição
das reações em cadeia com o ferro e o cobre; são capazes de interceptar os
radicais livres gerados pelo metabolismo celular ou por fontes exógenas;
impedem o ataque sobre lipídios, aminoácidos, a dupla ligação dos ácidos
graxos poliinsaturados e as bases do DNA, evitando a formação de lesões e
perda da integridade celular; reparam lesões causadas pelos radicais, removendo
danos das moléculas de DNA e reconstituem membranas celulares danificadas
(HEIM; TAGLIAFERRO; BOBILYA, 2002).
As características de um bom antioxidante são: presença de substituintes
doadores de elétrons ou de hidrogênio ao radical, em função de seu potencial de
redução; capacidade de deslocamento do radical formado em sua estrutura,
capacidade de quelar metais de transição implicados no processo oxidativo; e
acesso ao local de ação, dependendo de sua hidrofilia ou lipofilia e de seu
coeficiente de partição (MANACH et al., 2004).
Na indústria alimentícia, o uso de antioxidantes sintéticos é muito
importante para manter a integridade dos produtos, preservando características
sensoriais e nutricionais e prolongando a vida de prateleira dos produtos. O
20
oxigênio, a luz, o calor, a atividade de água, pH e os íons metálicos com mais de
um estado de valência agem como iniciadores e catalisadores das reações de
oxidação. Essas reações originam hidroperóxidos que se decompõem em
hidrocarbonetos, álcoois, aldeídos, cetonas e ácidos. Os compostos originados
conferem alterações no sabor, odor e textura dos alimentos, degradam os ácidos
graxos essenciais e vitaminas lipossolúveis e ainda ocorre eventual perda de suas
características funcionais. Essas alterações reduzem a qualidade dos alimentos e
seu valor nutricional (ALMEIDA-DORIA; REGITANO-DÁRCE, 2000;
PÉREZ-GALVÉS; MÍNGUEZ-MOSQUERA, 2002).
Os antioxidantes sintéticos mais utilizados são o butil hidroxitolueno
(BHT), o butil hidroxianisol (BHA) e o terc butil hidroquinona (TBHQ) por
apresentarem alta estabilidade e baixo custo. No entanto, seu uso tem sido
gradativamente reduzido devido à suspeitas de que esses compostos promovam
efeitos deletérios ao organismo (TSAKNIS; STRAVOS, 2005). Os
consumidores têm preferido adquirir produtos que utilizem aditivos naturais.
Assim, muitos estudos, visando a identificação de antioxidantes de fontes
naturais para a aplicação em produtos alimentícios, têm sido feitos. Ao serem
adicionados aos alimentos, os antioxidantes naturais protegem, não somente o
produto alimentício, mas também o organismo animal visto que, ao serem
ingeridos, os antioxidantes combatem radicais livres, contribuindo para a
redução do risco de desenvolvimento de várias patologias.
2.4 Compostos fenólicos
Compostos fenólicos são metabólitos secundários importantes na
formação dos pigmentos das plantas. Possuem um ou mais grupos hidroxilas,
ligados ao anel aromático. Apresentam estruturas variadas sendo encontrados
como ácidos fenólicos, antocianinas e taninos. Estruturas fenólicas ligam-se a
21
proteínas e açúcares e fazem parte de alcaloides e terpenoides. Suas massas
molares podem ser baixas, como a dos ácidos cafeico e gálico ou altas, como a
dos taninos, que apresentam alto grau de polimerização. Em sua maioria, são de
grande polaridade e muito reativos, além de serem suscetíveis à ação de enzimas
(KING; YOUNG, 1999). Os compostos fenólicos geralmente encontrados em
alimentos são ácidos fenólicos, flavonoides, estilbenos e taninos.
Os compostos fenólicos são antioxidantes de ocorrência natural. A
capacidade antioxidante deles é devida, principalmente, às suas propriedades
redutoras e depende do número e posição das hidroxilas presentes na molécula,
assim como da concentração desses compostos no alimento (BROINIZI et al.,
2007; MELO et al., 2008). A eficiência do antioxidante é determinada pelos
grupos funcionais presentes, pela posição no anel aromático e pelo tamanho da
cadeia. Compostos fenólicos são incluídos na categoria de interruptores de
radicais livres e são eficientes na prevenção da autoxidação. A ação antioxidante
desses compostos tem importante papel na redução da oxidação lipídica em
tecidos vegetal e animal. Quando os compostos fenólicos reagem com os
radicais livres, os radicais formados são estáveis, o que impede a oxidação de
vários componentes dos alimentos, particularmente ácidos graxos e óleos. Esse
poder de neutralização das estruturas radicalares é devido à sua estrutura
química formada por pelo menos um anel aromático com grupamentos
hidroxilas (ANGELO; JORGE, 2007; SOARES et al., 2008).
As frutas são, geralmente, mais ricas em compostos fenólicos que outros
vegetais. Frequentemente, contêm altas taxas de antocianidinas e antocianinas.
As principais fontes de compostos fenólicos são as frutas cítricas e frutas como
cereja, uva, ameixa e jabuticaba. Na jabuticaba são encontrados em maior
quantidade na casca. Em geral, os compostos fenólicos são multifuncionais
como antioxidantes, pois atuam combatendo radicais livres; doando um átomo
de hidrogênio de um grupo hidroxila (OH) da sua estrutura aromática. A
22
estrutura aromática é capaz de suportar um elétron desemparelhado através do
deslocamento desse ao redor de todo o sistema de elétrons da molécula; quelam
metais de transição como o Fe2+ e o Cu+; interrompem a reação de propagação
dos radicais livres na oxidação lipídica; modificam o potencial redox do meio e
reparam as lesões de células atacadas por radicais livres (PODSEDEK, 2007).
Os compostos fenólicos também bloqueiam a ação de enzimas específicas que
causam inflamação; modificam as rotas metabólicas das prostaglandinas;
protegem a agregação plaquetária; e inibem a ativação de carcinógenos
(VALKO et al., 2006).
Desta forma, os compostos fenólicos atuam por mecanismos variados,
dependendo da sua concentração e do tipo de composto presente no alimento,
podendo existir sinergismo ou antagonismo entre os diferentes compostos
(HASSIMOTTO; GENOVESE; LAJOLO, 2005; PEDRIELLI; SKIBSTED,
2002).
Os flavonoides atuam na inativação dos radicais livres nos
compartimentos celulares lipofílico e hidrofílico e possuem a capacidade de doar
átomos de hidrogênio, inibindo as reações em cadeia provocadas pelos radicais
livres (DEGÁSPARI; WASZCZYNSKYJ, 2004).
O potencial oxidante dos flavonoides depende do número e da posição
dos grupos hidroxilas e sua conjugação, assim como da presença de doadores
nos anéis aromáticos. Como a eficaz atividade antioxidante dos flavonoides está
relacionada ao grau de hidroxilação, ela também diminui com a substituição por
açúcares, apresentando os glicosídeos menor atividade antioxidante do que suas
agliconas correspondentes (KUSKOSKI et al., 2004). A capacidade de
sequestrar radicais livres dos flavonoides é devido principalmene à alta
reatividade da hidroxila. Hidroxilas presentes no anel B dos flavonoides doam
hidrogênios e um elétron para radicais hidroxila, peroxila e peroxinitrito
23
estabilizando-os e originando uma relativa estabilidade ao radical flavonoide
(HEIM; TAGLIAFERRO; BOBILYA, 2002).
Compostos fenólicos de vegetais são divididos em dois grupos: os
flavonoides e os não flavonoides. Os não flavonoides são os ácidos fenólicos.
2.4.1 Ácidos fenólicos
Os ácidos fenólicos têm como característica um anel benzênico, um ou
mais grupamentos carboxílico, hidroxila e/ou metoxila e outros substituintes na
molécula. Podem se ligar entre si ou com outros compostos.São divididos em
três grupos: o primeiro é composto pelos ácidos benzoicos, que possuem sete
átomos de carbono (C6-C1) e são os ácidos mais simples encontrados na
natureza (Figura 1) (LUZIA; JORGE, 2010; SOARES, 2002).
Figura 1 Estrutura química dos ácidos benzoicos Fonte: Ramalho e Jorge (2006).
24
O segundo grupo é formado pelos ácidos cinâmicos (Figura 2), que são
compostos aromáticos que formam uma cadeia lateral (C6-C3), sendo sete os
mais comumente encontrados no reino vegetal (ácidos cinâmico, o-cumárico, p-
cumárico, m-cumárico, cafeico, ferúlico e sinápico). A dupla ligação, presente
na molécula dos derivados do ácido cinâmico, participa da estabilidade do
radical por ressonância de deslocamento do elétron desemparelhado, tornando
ácidos como o sinápico, ferúlico e p-cumárico antioxidantes mais ativos do que
os derivados do ácido benzoico. Apresentam funções biológicas como ação anti-
inflamatória, anticarcinogênica e antimicrobiana (LUZIA; JORGE, 2010).
Figura 2 Estrutura química dos ácidos cinâmicos Fonte: Ramalho e Jorge (2006).
25
Os ácidos hidroxicinâmicos normalmente ocorrem como ésteres de
ácidos orgânicos ou glicosídeos. Afetam a estabilidade, a cor e o sabor de
alimentos. Seu maior representante é o ácido cafeico, que ocorre em alimentos
na forma esterificada como ácido clorogênico. Tanto o ácido cafeico como o
ácido clorogênico demonstraram atividade antioxidante in vitro. O ácido
sinápico atua como sequestrador do radical peroxinitrito, apresentando ação
antioxidante, o que o torna importante na proteção de células e na proteção
contra doenças oxidativas. Esse composto também apresenta propriedades
ansiolíticas e anti-inflamatórias (GASPAR et al., 2010; HAGHI; HATAMI,
2010; LUZIA; JORGE, 2010; SOARES, 2002).
Como terceiro grupo têm-se as cumarinas que compõem uma classe de
metabólitos secundários derivados do ácido cinâmico por ciclização da cadeia
lateral do ácido o-cumárico (LUZIA; JORGE, 2010; SOARES, 2002).
2.4.2 Flavonoides
Os flavonoides apresentam baixo peso molecular, sendo constituídos de
15 átomos de carbono, organizados na configuração C6 – C3 – C6 chamada
difenilpropano (Figura 3).
Figura 3 Difenilpropano
26
Fonte: Paganini-Costa e Silva (2011). O difenilpropano consiste em dois anéis aromáticos, denominados A e
B, ligados por três átomos de carbono que formam um heterociclo oxigenado,
denominado C. O anel A deriva do ciclo acetato/malonato, o B deriva da
fenilalanina. Dependendo do grau de hidrogenação e da substituição do anel
heterociclo, diferenciam-se em flavonoides, flavonas, flavonóis, flavanonas,
antocianidinas e isoflavonoides. São provenientes da via metabólica do ácido
chiquímico a partir de carboidratos, ou pela via do acetato-polimalato que se
inicia com acetil-coenzima A e malonil-coenzima A. Quando polimerizados, os
flavonoides formam os taninos e ligninas (ÂNGELO; JORGE, 2007).
A formação dos flavonoides é acelerada pela luz, sendo sua distribuição
dependente do grau de acesso à luminosidade, especialmente os raios
ultravioleta B. Englobam uma classe importante de pigmentos naturais
encontrados unicamente em vegetais. Geralmente ocorrem em plantas na forma
de glicosídeos e são uma das substâncias responsáveis pela atribuição sensorial
de frutas (KARAKAYA, 2004; SIMÕES et al., 2007).
Além das frutas, os flavonoides ocorrem em legumes, nozes, sementes,
flores e cascas. Em alimentos, os flavonoides existem principalmente como 3 -
O – glicosídeos e polímeros. Os polímeros são uma importante fração de
flavonoides consumidos na dieta e constituem as proantocianidinas e os taninos
hidrolisáveis. São encontrados principalmente nas frutas e bebidas (suco de
fruta, vinho, chá, café, chocolate e cerveja) e, em menor extensão, em vegetais,
legumes e cereais (HEIM; TAGLIAFERRO; BOBILYA, 2002).
Estima-se que a ingestão diária de flavonoides por humanos deve ser de
1g. Tem sido documentado que o consumo dessas substâncias reduz a
mortalidade por insuficiência cardíaca coronariana. Relata-se também que os
flavonoides têm ação antibacteriana, antiviral, antiinflamatória, vasodilatadora e
são inibidores da oxidação de lipoproteína de baixa densidade livre (LDL-c)
(ZHANG et al., 2001). A capacidade de transferir elétrons de radicais livres,
27
quelar catalisadores metálicos, ativar enzimas antioxidantes, reduzir os radicais
α-tocoferol e inibir oxidases dos flavonoides é que lhes confere o efeito protetor
em sistemas biológicos (HEIM; TAGLIAFERRO; BOBILYA, 2002).
2.4.2.1 Taninos
Os taninos possuem grupos hidroxila fenólicos que permitem a
formação de ligações estáveis com proteínas, o que lhes confere a habilidade de
complexar e precipitar esses compostos. Devido a esta habilidade são
considerados inibidores de enzimas. As reações com as proteínas ocorrem
através de ligações de hidrogênio e/ou ligações hidrofóbicas, quando os taninos
estão na forma não oxidada, formando complexos reversíveis, podendo ser
solúveis ou insolúveis, dependendo da proporção tanino/proteína, do pH e da
força iônica.
As ligações de hidrogênio provavelmente são formadas entre as
hidroxilas fenólicas dos taninos e a funções carbonílicas das ligações peptídicas
das proteínas. As interações hidrofóbicas ocorrem entre os núcleos aromáticos
dos taninos e as cadeias laterais alifáticas ou aromáticas dos aminoácidos
proteicos. Essas interações hidrofóbicas atuam como força de atração inicial na
complexação em meio aquoso entre o tanino e a proteína. Quando oxidados se
transformam em quinonas, que formam ligações covalentes com alguns grupos
funcionais de proteínas, como os grupos sulfidrilos da cisteína e ε – amino da
lisina formando complexos irreversíveis. Esses complexos ocorrem na planta
quando seus tecidos são danificados, por auto-oxidação ou oxidação catalisada
por enzimas (SIMÕES et al., 2007).
Os taninos são solúveis em água e apresentam alto peso molecular (500-
3.000 daltons). Os taninos podem ser classificados como hidrolisáveis ou
proantocianidinas (taninos condensados). Os hidrolisáveis contêm um núcleo
28
central de glicose ou álcool poliídrico, esterificados com ácido gálico ou elágico,
que são hidrolisáveis com ácidos, bases ou enzimas (Figura 4); os taninos
condensados são polímeros de flavonoides formados predominantemente por
unidades de flavan-3-ols (catequina) e flavan-3,4-diols (leucoantocianidinas)
(Figura 5).
Figura 4 Hidrólise de taninos hidrolisáveis Fonte: Queiroz, Morais e Nascimento (2002).
29
FIGURA 5 Taninos condensados. Fonte: Queiroz, Morais e Nascimento
(2002)
Em meio ácido, alcoólico e a quente, ocorre ruptura das ligações entre as
unidades monoméricas das proantocianidinas liberando antocianidinas como a
propelargonidina, a procianidina ou a prodelfinidina.
Os taninos condensados ocorrem em maior quantidade nos alimentos e,
como estão presentes na fração da fibra alimentar podem ser considerados pouco
digeríveis. Em frutos, quando em pequenas quantidades, confere-lhes
características sensoriais desejáveis (“corpo da fruta”), em quantidades maiores
conferem características adstringentes por complexarem com as proteínas da
saliva. Atuam como captadores de radicais interceptando o oxigênio ativo e
formando radicais estáveis (MONTEIRO et al., 2005).
Os taninos apresentam propriedades antimicrobianas e ação como
sequestradores de radicais livres. Suas propriedades antioxidantes são
30
importantes na prevenção de danos oxidativos celulares, incluindo a peroxidação
lipídica. Essas propriedades também podem estar relacionadas com seu potencial
anticarcinogênico e antimutagênico. Às proantocianidinas são atribuídas
propriedades nutricionais e farmacológicas como ação vasoprotetora e
antiagregante plaquetária, ação hepatoprotetora, atividade antiviral e proteção
em relação à artereosclerose (DEGÁSPARI; WASZCZYNSKYJ, 2004;
SOARES, 2002; SANCHES et al., 2005).
Como os taninos são altamente polimerizados e possuem muitos grupos
hidroxila fenólicos, possivelmente são antioxidantes mais eficientes do que
outros compostos fenólicos que apresentam menor peso molecular. Pesquisa
feita por Ariga e Hamano (1990) revelou que antioxidantes de baixa massa
molecular reagiram com um ou dois radicais peroxil por molécula, enquanto as
procianidinas diméricas reagiram com oito radicais peroxil por molécula.
2.4.2.2 Antocianinas
Antocianinas são glicosídeos de antocianidinas. Sua estrutura básica é o
cátion flavílio/2-fenilbenzopirilium. São pigmentos solúveis em água,
pertencentes ao grupo dos flavonoides, responsáveis pela coloração entre laranja
e vermelho de frutos e raízes e a cor azul em flores. A coloração das
antocianinas é influenciada pelo número de hidroxilas, de grupos metóxi e de
glicosídicos existentes na estrutura do composto. A intensidade da cor vermelha
está relacionada à quantidade de grupos metoxila e da cor azul está relacionada
aos grupos hidroxila e glicosídicos. Esses pigmentos são instáveis e sofrem
degradação pela ação da vitamina C, oxigênio, temperatura e pH do meio
(LIMA et al., 2011a).
As antocianinas possuem uma molécula de açúcar ligado ao carbono da
posição 3 da antocianidina, exceto no caso das desoxiantocianinas, quando
31
geralmente está ligado na posição 5. Poucas antocianidinas são glicosiladas na
posição 7. Quando são glicosiladas nas posições 5 e 7, os açúcares são na
maioria glicose. São muito sensíveis à mudança de pH. Em solução podem
existir em 4 formas estruturais: o cátion flavínium vermelho, a base azul
quinoidal, o carbinol incolor e a chalcona incolor. O grande número de
compostos pertecentes à família das antocianinas é devido às diferenças que
ocorrem entre as estruturas desses compostos. Essas diferenças se devem ao
número de grupos hidroxila em cada molécula; à natureza,ao número e à
localização dos açúcares e ao número e à natureza dos hidrocarbonetos alifáticos
ou ácidos aromáticos ligados aos açúcares (GALVANO et al., 2004).
Presumia-se que as antocianinas fossem pouco absorvidas durante a
digestão por não serem conhecidas enzimas específicas que hidrolisam
seletivamente suas ligações glicosídicas. No entanto, estudos relatam a
ocorrência de absorção in vivo de flavonoides glicosídicos (MATSUMOTO et
al., 2001; YOUNDIM et al., 2000).
Dependendo do pH do meio apresentam cores diferentes, sendo usadas
como indicadores. Também atuam como antioxidantes, captando radicais livres,
o que lhes confere a capacidade de prevenir problemas cardiovasculares,
circulatórios e carcinogênicos, diabetes e o mal de Alzheimer (KUSKOSKI et
al., 2006; LIMA et al., 2008). Apresentam ação anti-inflamatória e
antimutagênica. O consumo de antocianinas na dieta promove efeito protetor
contra danos hepáticos, gástricos e degradação do colágeno, além de aumentar o
desempenho cognitivo (DEGÁSPARI; WASZCZYNSKYJ, 2004; SANTOS;
VEGGI; MEIRELES, 2010). A promoção da saúde que ocorre pelo consumo de
antocianinas deve-se à sua estrutura química e ao fato de serem muito reativas
com espécies reativas de oxigênio devido à sua deficiência eletrônica
(GALVANO et al., 2004).
32
A capacidade antioxidante das antocianinas está relacionada com sua
estrutura química sendo influenciada pelas posições e tipos de grupos químicos
ligados aos anéis aromáticos e pela variada capacidade de aceitar elétrons
desemparelhados (GALVANO et al., 2004). A eficiência das antocianinas em
eliminar radicais livres e cessar a reação em cadeia, responsável pelo dano
oxidativo, é comprovada na literatura, sendo esses compostos potentes
antioxidantes in vitro (HE; GIUSTI, 2010). Utilizando o método ORAC de
absorção de oxigênio radical, Wang, Cao e Prior (1997) determinaram a
atividade antioxidante de 14 antocianinas e seus derivados glicosilados em
solução aquosa e pH neutro. Nesse estudo, a antocianina cianidina-3-glicosídeo
apresentou um valor de atividade antioxidante 3,5 vezes maior que a vitamina E.
Estudos sobre a atividade antioxidante das antocianinas, em análises in vivo,
mostram-se promissores e de grande interesse médico-científico.
2.5 Metabolismo de colesterol, lipoproteínas e suas relações com doenças cardiovasculares e hepáticas
O consumo de alimentos para fins profiláticos e terapêuticos é uma
prática comum entre várias populações. Existe um crescente interesse em
diversas farinhas obtidas de vegetais como a farinha de casca de maracujá e
farinha de linhaça, por causa de seus efeitos medicinais. Atualmente, aumenta
cada vez mais a procura por alimentos que possuem substâncias ou componentes
bioativos capazes de produzir benefícios específicos à saúde, prevenindo
doenças e mantendo o bem- estar do indivíduo.
Entre as doenças que mais preocupam a população, citam-se- as que
estão relacionadas com lipídeos ou compostos que contenham lipídeos na
corrente sanguínea, como a hipercolesterolemia, importante no desenvolvimento
de doenças cardiovasculares. O desenvolvimento da arterosclerose está
relacionado com concentrações séricas elevadas de colesterol total,
33
principalmente quando ocorre aumento de lipoproteína de baixa densidade
(LDL-c), na forma de colesterol e redução de lipoproteínas de alta densidade
(HDL-c), na forma de colesterol.
As dislipidemias e o excessivo de acúmulo de lipídeos no fígado
também têm sido relacionados com a ocorrência de esteatose hepática não
alcoólica. Fígados com esteatose hepática são vulneráveis ao extress oxidativo,
havendo indução de peroxidação lipídica, o que pode acelerar a progressão da
doença. A esteatose hepática promove elevações nas aminotransferases séricas,
sendo a análise dessas enzimas uma das formas utilizadas para auxiliar no
diagnóstico da doença. Dietas antioxidantes têm sido utilizadas para a prevenção
e como estratégia para limitar o acúmulo de lipídios e a lesão hepática (BRUNO
et al., 2008; PARK et al., 2011).
O colesterol é adquirido através da dieta ou pode ser sintetizado pelo
organismo humano. O colesterol hepático é o resultado do balanço entre o
colesterol ingerido, o colesterol sintetizado pelo organismo e o colesterol
metabolizado pelo fígado. O colesterol possui uma estrutura em anel que o
organismo humano não consegue metabolizar a CO2 e H2O, sendo eliminado
pelo fígado como colesterol inalterado na bile, que o transporta até o intestino
para eliminação; como componente das lipoproteínas do plasma; ou como sais
biliares que são excretados nas fezes (CHAMPE; HARVEY; FERRIER, 2009).
O transporte do colesterol de um tecido para outro é realizado
utilizando-se lipoproteínas plasmáticas, que são agregados de moléculas, que
apresentam forma aproximadamente esférica, em que ocorre a associação de
lipídeos e proteínas. As apoliproteínas ou apoproteínas correspondem à fração
proteica das lipoproteínas. As diversas combinações entre lipídeos e proteínas
dão origem a partículas com densidades diferentes, caracterizando as principais
classes de lipoproteínas humanas. Entre estas classes estão os quilomícrons, as
lipoproteínas com densidade muito baixa (VLDL), lipoproteínas de baixa
34
densidade (LDL), lipoproteínas de densidade intermediária (IDL) e lipoproteínas
de alta densidade (HDL). As VLDL e LDL contém cerca de 10 e 25% de
proteínas. As HDL possuem cerca de 50% de proteínas, 20% de colesterol em
sua maioria esterificado, 30% de fosfolipídeos e traços de triacilgliceróis. Cada
lipoproteína possui uma função específica, que é determinada por seu lugar de
síntese, composição lipídica e conteúdo de apoproteína (BACHORICK;
RIFIKIND; KWITEROVICH JUNIOR, 2000; LEHNINGER; NELSON; COX,
1995).
Os quilomícrons, partículas formadas no intestino, são a principal via
pela qual os lipídeos provenientes da dieta entram na circulação sanguínea, onde
realizam trocas com as HDLs, adquirindo colesterol livre, colesterol esterificado,
fosfolipídeos e as apoliproteínas C-II, C-III e C-E. Devido à aquisição da apo C-
II tornam-se capazes de ativar a lipase proteica, responsável pela hidrólise dos
triacilgliceróis nos quilomícrons, resultando partículas menores, chamadas
quilomícrons remanescentes, que podem ser capturados pelo fígado, oxidados ou
podem ficar retidos nas células “scavenger”, que são receptores de lipoproteínas
de baixa densidade (SALES; PELUZIO; COSTA, 2003).
Os lipídeos de origem endógena têm seu metabolismo iniciado com a
síntese hepática da VLDL. As partículas de VLDL interagem com enzimas
lipase lipoproteica do endotélio capilar, liberando ácidos graxos. A VLDL
transfere as apoproteínas apo C e apo E para a HDL, sofre alterações na sua
composição que ocorrem com a metabolização de seus constituintes e passam a
ser classificadas como IDL. As IDL podem ser captadas no fígado e degradados
ou sofrem ação da lipase hepática, formando LDL (SALES; PELUZIO; COSTA,
2003).
As HDL são as frações lipoproteicas plasmáticas menores e mais densas.
São formadas quando apoliproteinas plasmáticas ligam-se a lipídeos. A apoA-I e
a apoA-II são as principais lipoproteínas presentes na HDL, sendo a apoA-I a
35
lipoproteína estrutural que representa o maior componente proteico. Ambas são
sintetizadas principalmente no fígado, no entanto, uma proporção da ApoA-I
pode ser formada no intestino. As HDL contêm, principalmente, ésteres de
colesterol, uma pequena quantidade de triacilgliceróis e colesterol não
esterificado formando um núcleo lipídico hidrofóbico, que é circundado por uma
camada monofásica de fosfolipídeos, colesterol não esterificado e
apolipoproteínas. Apenas uma fração da HDL é metabolizada como partícula
intacta, sendo a maioria dos constituintes removida separadamente através de
um processo bastante complexo (LIMA; COUTO, 2006).
A HDL tem sido subdividida em subclasses nomeadas segundo sua
densidade, tamanho e mobilidade eletroforética; ou constituição. A concentração
plasmática de HDL, apoA-1 e o metabolismo entre a subclasses de HDL
determinam parcialmente o transporte reverso de colesterol. Esse processo é
uma via de transporte que remove o colesterol das células extra-hepáticas para o
fígado e talvez para o intestino, para excreção; o que reduz o acúmulo de
colesterol na parede das artérias, prevenindo o desenvolvimento de
arterosclerose. No transporte reverso, o colesterol é retirado das células extra-
hepáticas por aceptores específicos, ocorre esterificação do colesterol dentro da
HDL pela ação da enzima lecitina-colesterol-acil-transferase (LCAT), o
colesterol é transferido para lipoproteínas que contêm a apoB, a HDL é
remodelada e capturada pelo fígado, rim e, possivelmente, intestino delgado
(SALES; PELUZIO; COSTA, 2003).
Compostos antioxidantes, como os compostos fenólicos e as fibras são
componentes reconhecidamente importantes na prevenção e no tratamento de
doenças cardiovasculares. Os produtos derivados da fermentação
microbiológica das fibras são capazes de reduzir os teores de colesterol total e da
lipoproteína de baixa densidade (LDL). A dose diária recomendada para se obter
o efeito benéfico da ingestão das fibras é entre 25 g (ORGANIZAÇÃO
36
MUNDIAL DA SAÚDE - OMS, 2004). Estudos sugerem que alimentos como o
chá verde e mirtilo, ricos em compostos fenólicos e que apresentam comprovado
potencial antioxidante, exibem efeito inibitório sobre a esteatose hepática
(BRUNO et al., 2008; LIU et al., 2011; PARK et al., 2011). Orientações
nutricionais enfatizando a importância dos compostos fenólicos e das fibras na
dieta têm aumentado a procura por produtos ricos em fibras e antioxidantes.
Modelos de animais têm sido usados para compreender melhor a ação dos
antioxidantes fenólicos e das fibras sobre as disfunções do metabolismo do
colesterol. Roedores são frequentemente utilizados para esse tipo de pesquisa.
Esses animais são resistentes ao colesterol da dieta e hiperlipidemias podem ser
induzidas somente por uma dieta rica em colesterol/gordura, contendo ácido
cólico e tiouracil (ROSSONI JÚNIOR, 2008). Os parâmetros bioquímicos
normais de colesterol, colesterol HDL (HDL-c), triacilgliceróis, aspartato
aminotransferase (AST) e alanina aminotransferase (ALT) para animais da
linhagem Fisher estão apresentados na Tabela 1. Alterações nesses parâmetros
confirmam indução de doença nos animais. Estudos sobre a atuação dos
antioxidantes fenólicos sobre disfunções no metabolismo do colesterol,
provocadas em animais, têm mostrado resultados bastante positivos.
37
Tabela 1 Intervalo de referência de parâmetros bioquímicos no sangue de animais da linhagem Fisher.
Parâmetros bioquímicos Macho Fêmea
Colesterol (mg dL-1) 57,18 - 92,00 64,56 - 95,60
HDL-c1(mg dL-1) 34,68 - 67,74 43,22 - 73,30
Triacilgliceróis (mg dL -1) 41,51 - 95,05 37,42 - 85,84
AST2 (U L1) 28,20 - 67,20 22,98 - 60,84
ALT 3 (U L1) 9,13 - 37,17 6,00 - 48,30 1HDL – c: lipoproteína de alta densidade 2AST – aspartato amino transferase 3ALT – alanina amino transferase Fonte: Gonçalves et al. (2011) e Oliveira et al. (2011).
Pesquisas desenvolvidas por Chen et al. (2005)demonstram que os
flavonoides presentes na casca da amêndoa atuam sinergicamente com as
vitaminas C e E para proteger a oxidação da LDL, em hamsters. Compostos
fenólicos presentes no pó liofilizado de uvas frescas afetaram diretamente a
aterogenicidade de macrófagos, reduzindo a oxidação mediada por macrófagos
de LDL e a absorção de LDL oxidada em ratos (FUHRMAN et al., 2005).
A oxidação das partículas de LDL ocorre quando os compostos
oxidantes fornecem radicais livres com um número de elétrons maior que a
capacidade do sistema antioxidante endógeno de prevenir a oxidação
espontânea. As LDL oxidadas (LDLox) apresentam propriedades aterogênicas
físico-químicas e biológicas que desempenham papéis importantes na
patogênese da arterosclerose, infarto do miocárdio e AVC isquêmico. A LDLox
consiste em uma classe heterogênea de lipídeos modificados e moléculas
protéicas que possuem lipídeos, ácidos graxos e antioxidantes cuja composição
difere da LDL nativa (RICCIONI et al., 2012).
38
Antioxidantes provenientes da dieta podem ser capazes de inibir a
peroxidação de LDL e mostram-se como uma estratégia terapêutica interessante
para a prevenção de aterosclerose e doença cardíaca coronária. Dessa forma, a
investigação de antioxidantes naturais capazes de atuar sobre as LDL ox mostra-
se bastante promissora.
39
REFERÊNCIAS
ABE, L. T.; LAJOLO, F. M. L.; GENOVESE, M. I. Potential dietary sources of ellagic acid and other antioxidants among fruits consumed in Brazil: jabuticaba (Myrciaria jaboticaba (Vell.) Berg).Journal of the Science of Food and Agriculture , London, v. 92, n. 8, p. 1679-1687, June 2012. ALMEIDA-DORIA, R. F.; REGITANO-D'ARCE, M. A. B. Antioxidant activity of rosemary and oregano ethanol extracts in soybean oil under thermal oxidation.Ciência e Tecnologia de Alimentos, Campinas, v. 20, n. 2, p. 197-203, 2000. ALVES, A. P. C. et al. Flour andanthocyanin extracts of jaboticaba skins used as a natural dye in yogurt. International Journal of Food Science and Technology, Oxford, v. 48, n. 10, p. 2007-2013, Oct. 2013. ANGELO, P. M.; JORGE, N. Compostos fenólicos em alimentos:uma breve revisão. Revista do Instituto Adolfo Lutz, São Paulo, v. 66, n. 1, p. 232-240, 2007. ARIGA, T.; HAMANO, M. Radical scavenging action and its mode in procyanidins B-1 and B-3 from adzuki beans to peroxyl radicals.Agricultural and Biological Chemistry, Tokyo, v. 54, n. 10, p. 2499-2504, Oct. 1990. BACHORICK, P. S.; RIFIKIND, B. M.; KWITEROVICH JUNIOR, P. O. Lypidis and dyslipoproteinemia.Clinical Chemistry,Baltimore,v. 46, n. 2,p. 208-236, Feb. 2000. BIANCHI, M. L. P.; ANTUNES, L. M. G. Radicais livres e os principais antioxidants da dieta. Revista de Nutrição, Campinas, v. 12, n. 2, p. 123-130, 1999. BROINIZI, P. R. B. et al. Avaliação da atividade antioxidante dos compostos fenólicos naturalmente presentes em subprodutos do pseudofruto de caju (Anacardium occidentale L.). Ciência e Tecnologia de Alimentos, Campinas, v. 27, n. 4, p. 902-908, 2007. BRUNO, R. S. et al. Green tea extract protects leptin-deficient, spontaneously obese mice from hepatic steatosis and injury. Journal of Nutrition , Philadelphia, v. 138, n. 2, p. 323-331, Feb. 2008.
40
CHAMPE, P. C.; HARVEY, R. A.; FERRIER, D. R. Bioquímica ilustrada.Porto Alegre: Artmed, 2009. 519 p. CHEN, C. Y. et al. Flavonoids from almond skins are bioavailable and act synergistically with vitamins c and e to enhance hamster and human LDL resistance to oxidation. Journal of Nutrition , Philadelphia, v. 135, n. 6, p. 1366-1373, 2005. DANNER, M. A. et al. Enraizamento de jabuticabeira 1(Plinia trunciflora) por mergulhia aérea.Revista Brasileira de Fruticultura, Jaboticabal, v. 28, n. 3, p. 530-532, 2006. DEGÁSPARI, C. H.; WASZCZYNSCYJ, N. Propriedades antioxidantes de compostos fenólicos. Visão Acadêmica, Curitiba, v. 5, n. 1, p. 33-40, 2004. FUHRMAN, B. et al. Grape powder polyphenols attenuate atherosclerosis development in apolipoprotein E deficient (E0) mice and reduce macrophage atherogenicity. Journal of Nutrition , Philadelphia, v. 135, n. 4, p. 722-728, 2005. GALVANO, F. et al. Cyanidins: metabolism and biological properties. Journal of Nutritional Biochemistry , Stoneham, v. 15, n. 1, p. 2-11, 2004. GASPAR, A. et al. Dietary phenolic acids and derivatives.evaluation of the antioxidant activity of sinapic acid and its alkyl esters. Journal of Agricultural and Food Chemistry, Washington, v. 58, n. 21, p. 11273-11280, 2010. GONÇALVES, S. C. et al. Valores de referência de parâmetros séricos de ratos Fischer do laboratório de nutrição experimental. In: CONGRESSO NACIONAL DE ALIMENTAÇÃO E NUTRIÇÃO, 1.; CONGRESSO MINEIRO DE ALIMENTAÇÃO E NUTRIÇÃO, 4., 2011, Ouro Preto.Anais... Ouro Preto: UFOP, 2011. p. 1346-1351. HAGHI, G.; HATAMI, A. Simultaneous quantification of flavonoids and phenolic acids in plant materials by a newly developed isocratic high-performance liquid chromatography approach.Journal of Agricultural and Food Chemistry, Washington, v. 58, n. 20, p. 10812-10816, 2010. HASSIMOTTO, N. M. A.; GENOVESE, M. I.; LAJOLO, F. M. Antioxidant activity of dietary fruits, vegetables, and commercial frozen fruit pulps.Journal of Agricultural and Food Chemistry, Washington, v. 53, n. 8, p. 2928-2935, Mar. 2005.
41
HE, J. A.; GIUSTI, M. M. Anthocyanins: natural colorants with health-promoting properties. Annual Review of Food Science and Technology, Palo Alto, v. 1, p. 163-186, 2010. HEIM, K. E.; TAGLIAFERRO, A. R.; BOBILYA, D. J. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. Journal of Nutritional Biochemistry , Stoneham, v. 13, n. 10, p. 572-584, 2002. KARAKAYA, S. Bioavailability of phenolic compounds. Critical Reviews in Food Science and Nutrition, Boca Raton, v. 44, n. 6, p. 453-464, 2004. KING, A.; YOUNG, G. Characteristics and occurrence of phenolic phytochemicals.Journal of the American Dietetic Association, Chicago, v. 99, n. 2, p. 213-218, 1999. KUSKOSKI, E. M. et al. Atividade antioxidante de pigmentos antociânicos.Ciência e Tecnologia de Alimentos, Campinas, v. 24, n. 4, p. 691-693, 2004. KUSKOSKI, E. M. et al. Frutos tropicais silvestres e polpas de frutas congeladas: atividade antioxidante, polifenóis e antocianinas. Ciência Rural, Santa Maria, v. 36, n. 4, p. 1283-1287, jul./ago. 2006. LEHNINGER, A. L.; NELSON, D.L.; COX, M.M. Princípios de bioquímica.2. ed. São Paulo: Sarvier, 1995. 839 p. LEITE-LEGATTI, A. V. et al. Jaboticaba peel: antioxidant compounds,antiproliferative and antimutagenic activities. Food Research International , Barking, v. 49, n. 1, p. 596-503, Nov. 2012. LIMA, A. J. B. et al. Caracterização química do fruto jabuticaba (Myrciaria cauliflora Berg) e de suas frações. Archivos Latinoamericanos de Nutrición, Caracas, v. 58, n. 4, p. 416-421, 2008. LIMA, A. J. B. et al. Anthocyanins, pigment stability and antioxidant activity in jabuticaba [Myrciaria cauliflora (Mart.) O. Berg]. Revista Brasileira de Fruticultura , Jaboticabal, v. 33, n. 3, p. 877-887, 2011a. LIMA, A. J. B. et al. Sugars, organic acids, minerals and lipids in jabuticaba. Revista Brasileira de Fruticultura, Jaboticabal, v. 33, n. 2, p. 540-550, 2011b.
42
LIMA, E. S.; COUTO, R. D. Estrutura, metabolismo e funções fisiológicasda lipoproteína de alta densidade. Jornal Brasileiro de Medicina e PatologiaLaboratorial, Rio de Janeiro, v. 42, n. 3, p. 169-178, 2006. LIU, Y. et al. Inhibitory effect of blueberry polyphenolic compounds on oleic acid-induced hepatic steatosis in vitro. Journal of Agricultural and Food Chemistry, Easton, v. 59, n. 22, p. 1254-1263, Oct. 2011. LUZIA, D. M. M.; JORGE, N. Antioxidant potential of lemon seed extracts (Citrus limon). Ciência e Tecnologia de Alimentos, Campinas, v. 30, n. 2, p. 489-493, 2010. MANACH, C. et al. Polyphenols: food sources and bioavailability. The American Journal of Clinical Nutrition , Bethesda, v. 79, n. 5, p. 727-747, 2004. MATSUMOTO, H. et al. Orally administered delphinidin 3-rutinoside and cyanidin 3-rutinoside are directly absorbed in rats and humans and appear in the blood as the intact forms. Journal of Agricultural and Food Chemistry, Washington, v. 49, n. 3, p. 1546-1551, 2001. MELO, E. A. et al. Capacidade antioxidante de frutas. Revista Brasileira de Ciências Farmacêuticas, São Paulo, v. 44, n. 2, p. 193-201, 2008. MONTEIRO, J. M. et al. Taninos: uma abordagem da química à ecologia. Química Nova, São Paulo, v. 28, n. 5, p. 892-896, set./out. 2005. MOREIRA, A. V. B.; MANCINI-FILHO, J. Influência dos compostos fenólicos de especiarias sobre a lipoperoxidação e o perfillipídico de tecidos de ratos1. Revista de Nutrição, Campinas, v. 17, n. 4, p. 411-424, 2004. OLIVEIRA, E. C. et al. Valores de referência para atividade enzimática sérica de ratos Fischer do laboratório de nutrição experimental. In: CONGRESSO NACIONAL DE ALIMENTAÇÃO E NUTRIÇÃO, 1.; CONGRESSO MINEIRO DE ALIMENTAÇÃO E NUTRIÇÃO, 4., 2011, Ouro Preto. Anais...Ouro Preto: UFOP, 2011. p. 1352-1357. ORGANIZAÇÃO MUNDIAL DE SAÚDE. Conferência internacional sobre cuidados primários de saúde: declaração deAlma-Ata, 1978. Brasília: Ministério da Saúde, 2004. 15 p.
43
PAGNINI-COSTA, P.; SILVA, D. C. Uma xícara (chá) de química. Revista Virtual de Química,Niterói, v. 3, n. 1, p. 27-36, 2011. PARK, H. J. et al. Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice. The Journal of Nutritional Biochemistry , Stoneham, v. 2, n. 4, p. 393-400, Apr. 2011. PEDRIELLI, P.; SKIBSTED, L. H. Anti-oxidant synergy and regeneration effect of quercetin, (-)-epicatechin, and (+)-catechin on alpha-tocopherol in homogeneous solutions of peroxidating methyl linoleate. Journal of Agriculture and Food Chemistry, Washington, v. 50, n. 24, p. 7138-7144, Nov. 2002. PÉREZ-GÁLVEZ, A.; MÍNGUEZ-MOSQUERA, M.I. Degradation of non-esterified and esterified xanthophylls by free radicals.Biochimica et Biophysica Acta, Amsterdam, v. 1569, n. 1/3, p. 31-34, Jan. 2002. PODSEDEK, A. Natural antioxidants and antioxidant capacity of Brassica vegetables: a review. Food Science and Technology, Trivandrum, v. 40, n. 1, p. 1-11, Jan. 2007. QUEIROZ, C. R. A. A.; MORAIS, S. A. L.; NASCIMENTO, E. A. Caracterização dos taninos da aroeira preta (Myracrodruon urundeuva). Revista Árvore ,Viçosa,MG,v. 26, n. 4, p. 485-492, jul./ago. 2002. RAMALHO, V. C.; JORGE, N. Antioxidantes utilizados em óleos, gorduras e alimentos gordurosos. Química Nova,São Paulo, v. 29, n. 4, p. 755-760, jul./ago. 2006. RICCIONI, G. et al. Novel phytonutrient contributors to antioxidant protection against cardiovascular disease. Nutrition , London, v.28, n. 6, p. 605-610, June 2012. ROSSONI JÚNIOR, J. V. Pefil lipídico, defesas antioxidantes e marcadores de função hepática e renal em hamsteres tratados com extratos de semente de urucum.2008. 97 p. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Ouro Preto, Ouro Preto, 2008. SALES, R.L.; PELUZIO, M.C.G.; COSTA, N.M.B. Lipoproteínas: uma revisão do seu metabolismo e envolvimento com o desenvolvimento de doenças cardiovasculares. Nutrire , São Paulo, v. 25, p. 71-86, 2003.
44
SANCHES, A. C. C. et al. Antioxidant and antifungal activities of extracts and condensed tannins from Stryphnodendron obovatum Benth. Brazilian Journal of Pharmaceutical Sciences, São Paulo, v. 41, n. 1, p.101-107, 2005. SANTOS, D. T.; VEGGI, P. C.; MEIRELES, M. A. A. Extraction of antioxidant compounds from Jabuticaba (Myrciaria cauliflora) skins: yield, composition and economical evaluation. Journal of Food Engineering, New York, v. 101, n. 1, p. 23-31, 2010. SIMÕES, C. M. O. et al. Farmacognosia, da planta ao medicamento. 6. ed. Porto Alegre: UFGRS, 2007. 1104 p. SOARES, M. et al. Avaliação da atividade antioxidante e identificação dos ácidos fenólicos presentes no bagaço de maçã cv. Gala. Ciência e Tecnologia de Alimentos, Campinas, v. 28, n. 3, p. 727-732, 2008. SOARES, S. E. Ácidos fenólicos como antioxidantes. Revista Nutrição, Campinas, v. 15, n. 1, p. 71-81, 2002. TEDESCO, I. et al. Antioxidant effect of red wine anthocyanins in normal and catalase-inactive human erythrocytes.Journal of Nutritional Biochemistry , Stoneham, v. 12, n. 9, p. 505-511, 2001. TSAKNIS, J.; STAVROS, L. Extraction and identification of natural antioxidant fromSideritis euboea (Mountain Tea). Journal of Agricultural and Food Chemistry,Washington, v. 53, p. 6375-6381, June 2005. VALKO, M. et al. Free radicals and antioxidants in normal physiological functions and human disease.International Journal of Biochemistry & Cell Biology, Oxford, v. 32, n. 3, p. 3-41, 2006. WANG, H.; CAO, G.; PRIOR, R. L. Oxygen radical absorbing capacity of anthocyanins.Journal of Agricultural and Food Chemistry, Washington, v. 45, n. 2, p. 304-309, Feb. 1997. YOUDIM, K. A. et al. Polyphenolics enhance red blood cell resistance to oxidative stress: in vitro and in vivo. Biochimica et Biophysica Acta-General Subjects, Alberta, v. 1523, n. 1, p. 117-122, 2000. ZHANG, Z. et al. Characterization of antioxidants present in hawthorn fruits. Journal of Nutritional Biochemistry , Stoneham, v. 12, n. 3, p. 144-152, 2001.
45
SEGUNDA PARTE – ARTIGOS
ARTIGO 1 Submetido para a Revista International Journal of Food Science Properties
Formato conforme normas da revista
INHIBITORY POTENTIAL OF PLINIA JABOTICABA SKIN ON THE
DIGESTIVE ENZYMES
Fabíola Fonseca Lage1, Angelita Duarte Corrêa1, Anderson Assaid Simão1,
and Flávia Cíntia Oliveira1
1Chemistry Department, Biochemistry Laboratory, Universidade Federal de
Lavras – UFLA, PO Box 3037, Zip Code 37200.000, Lavras, MG, Brazil
_______________________________
*Corresponding author: Telephone number: +55 (35) 38291272; Fax: +55 (35) 38291271 Email:[email protected] (F.F. Lage) and [email protected] (A.D. Corrêa).
46
INHIBITORY POTENTIAL OF PLINIA JABOTICABA SKIN ON
THE DIGESTIVE ENZYMES
Fabíola Fonseca Lage1, Angelita Duarte Corrêa1, Anderson Assaid
Simão1, andFlávia Cíntia Oliveira1
1Chemistry Department, Biochemistry Laboratory, Universidade Federal
de Lavras – UFLA, PO Box 3037, Zip Code 37200.000, Lavras, MG,
Brazil
ABSTRACT
The inhibitory potential of the jabuticaba skin flour aqueous extract
(JSFE) on the digestive enzymes α-amylase, α-glucosidase, lipase, and
trypsin in the absence and presence of simulated gastric fluid was
studied. The JSFE significantly inhibited α-amylase. The inhibition of
α-amylase by JSFE was 585.60 µmol min-1 (units of inhibited enzyme -
UIE) and, with gastric fluid, it was 560.00 UIE. The phenolic
compounds present in jabuticaba skins are probably responsible for the
inhibitory potential of JSFE.Jabuticaba skin flour might potentially
47
serve as a good alternative for the control of diseases, such as type 2
diabetesand obesity.
Keywords: Plinia jaboticaba, Inhibitory potential, phenolic compounds, Flavonoids, Anthocyanin.
INTRODUCTION
The inhibition of the enzymes α-amylase and glucosidase may be a
strategy for the control of hyperglycemia and type 2 diabetes, mainly
through the reduction in starch metabolism and in the absorption of
glucose in the intestines. Studies have suggested that phenolic compounds
may inhibit specific digestive enzymes in vitro and in vivo.
Phenolic compounds from almond seed shell (Prunus dulcis) were
purified by Tsujita et al.[1], and the ability of the purified fraction to
inhibit α-amylase was analyzed. This fraction strongly inhibited α-
amylase. In this study, extraction tests with water and aqueous organic
solvent in the ratio of 70% were performed. The solvents used were
methanol, ethanol, acetone and acetonitrile. The results showed that water
was the most efficient solvent for the extraction of phenolic compounds.
Water also has the advantage of not being toxic, in case there is the
48
possibility to test the performance of the extract to inhibit α-amylase in
humans.
The Brazilian jabuticaba tree (Plinia jaboticaba) produces jabuticaba,
a common Brazilian fruit rich in sugar and minerals, such as iron,
potassium, magnesium, and manganese[2]. This fruit, especially the skin,
also contains high amounts of phenolic compounds, including
anthocyanins, which are responsible for its characteristic reddish-black
color. Lima et al.[3] have analyzed the anthocyanin content of jabuticaba
skins and reported a total anthocyanin content of 2.057 mg 100 g-1 dry
matter (DM). It is a very high level, since the literature reports that grape
skins, considered rich in anthocyanins, present an anthocyanin content of
206 mg 100 g-1 DM, which is approximately 10 times lower[4].
Lima et al.[3] and Leite-Legatti et al.[5] investigated the anthocyanins
found in jabuticaba skins, and identified cyanidin-3-glucoside and
delphinidin-3-glucoside. Abe et al.[6] identified quercetin and ellagic acid
in ripe jabuticaba. The ellagitannins iso-oenothein and oenothein C and
the depside jaboticabin were isolated and identified in jabuticaba fruits
(Myrciaria cauliflora), lyophilized by Wu et al.[7] Depsides are
49
derivatives of phenolic compounds consisting of two or more units of
monocyclic aromatic nuclei, linked by an ester bond[8].
There are few studies in the literature that investigated the in vitro
inhibitory potential of the jabuticaba skin flour aqueous extract (JSFE) on
the digestive enzymes α-amylase, α-glucosidase, lipase and trypsin.
Alezandro et al.[9] analyzed the inhibitory potential of lyophilized
jabuticaba skins in various ripening stages, and observed the inhibition of
the enzyme α-amylase. The skin from the ripe fruit was the fraction that
showed the best inhibition, and the authors suggest that it was due to the
higher content of anthocyanins in this ripening stage.
Due to the high content of phenolic compounds in its skin, mainly
anthocyanins, the jabuticaba skin flour aqueous extract (JSFE) may
present a promising inhibitory effect on digestive enzymes, especially on
the key enzymes of carbohydrate metabolism, and may present a potential
antidiabetic effect. Thus, the objective of this study was to evaluate the
inhibitory potential of the JSFE on the digestive enzymes α-amylase, α-
glucosidase, lipase, and trypsin, in the absence and presence of simulated
gastric fluid, in order to find enzyme inhibitors.
50
MATERIALS AND METHODS
Chemicals and reagents
3,5-dinitrosalicylic acid (DNS), p-nitrophenyl-α-D-glucopyranoside,
citric acid, p-nitrophenyl palmitate, Nα-Benzoyl-DL-arginine p-
nitroanilide (BApNA), Triton X-100 were purchased from Sigma (St.
Louis, MO, USA). Tri(hydroxymethyl)aminomethane (Tris) was obtained
from Fisher Scientific (Fair Lawn, NJ). Sodium hypochlorite, sodium
hydroxide, acetic acid, ethanol, hydrochloric acid, sodium chloride,
calcium chloride, and sodium phosphate monobasic were obtained from
Vetec (RJ, Brazil).
Sample preparation
Ripe jabuticaba fruits (Plinia jaboticaba [Vell.] Berg, Sabara
genotype) were hand-picked on the morning of October, 2010, from a
farm in São José do Ismeril, in the municipality of Coqueiral (MG,
Brazil). The fruits for the experiment were selected, washed with tap
water, immersed in a sodium hypochlorite solution (200 mg kg-1) for 10
min, and crushed. The skins were weighed and separated into 3 fractions
of approximately 2.9 kg each. They were then placed in mesh metallic
51
material baskets and dried in a food dehydrator for 36 h at 45°C with a 1
m s-1airflow. The skins were then ground into flour, packaged in
hermetically sealed flasks, and wrapped in aluminum foil. In this flour,
the particle size was determined using a sieve shaker, and most of the
flour particles were retained on the sieves between 32 mesh (0.5 mm) and
60 mesh (0.25 mm). According to Zanotto and Bellaver[9], the uniformity
index indicates the relative proportion between coarse, medium and fine
particles, which are defined according to diameters larger than 2 mm,
between 2 and 0.60 mm and smaller than 0.60 mm, respectively.
Therefore, JSF can be classified as fine.
Moisture
Moisture analyses were carried out in triplicate, based on the
methodology described by the Association of Official Analytical
Chemists – AOAC[10].
Preparation of extracts
Jabuticaba skin flour (JSF) was mixed with distilled water in the
proportion 1:5 (w/v). The solutions were mixed for 1h using a horizontal
52
shaker set at room temperature and then centrifuged for 15 min at 10,000
× g at 4°C. The precipitate was discarded, and the resulting supernatant
was used for the enzymatic analyses as inhibitors in the enzymatic
analyses.
Analyzed enzymes
Type VI pancreatic porcine α-amylase (EC 3.2.1.1) (Sigma), porcine
pancreatic trypsin (EC 3.4.21.4), and type II porcine lipase (EC 3.1.1.3)
(Merck) were used in this study. α-glucosidase (EC 3.2.1.20) was
obtained from fresh pig duodenum. The tissue was triturated in a blender
with 0.5 mol L-1 Tris-HCl buffer, pH 8.0, at 4°C until a homogeneous
solution was obtained. The homogenate was filtered through a nylon
mesh and centrifuged at 2,500 × g for 10 min at 4°C. The supernatant was
collected and used as enzyme extract[11].
Activity of α-amylase
The determination of α-amylase activity was conducted according to
the methodology proposed by Noelting and Bernfeld[12]. 50 µL JSFE and
50 µL α-amylase were pre-incubated in a 37°C water bath for 30 min. A
53
1% starch substrate was prepared in 0.05 mol L-1 Tris, pH 7.0, 0.038 mol
L-1 NaCl, and 0.1 mmol L-1 CaCl2. After the addition of 100 µL of the
substrate, the mixture was incubated for 4 periods of time. The reaction
was stopped by adding 200 µL DNS, and absorbance was measured in a
spectrophotometer at a wavelength of 540 nm.Results are relative to a
blank control.
Activity of α-glucosidase
The activity of α-glucosidase was measured according to the protocol
of Pereira et al.[13] using 5 mmol L-1p-nitrophenyl-α-D-glucopyranoside in
0.1 mol L-1 citrate-phosphate buffer, pH 7.0. For this assay, 50 µL JSFE
and 100 µL enzyme were incubated in a 37°C water bath for 4 periods of
time. The reaction was stopped by adding 1,000 µL of 0.05 mol L-1
NaOH, and the absorbances of the product were measured using a
spectrophotometer at a wavelength of 410 nm. Results are relative to a
blank control.
Activity of lipase
For the determination of lipase, a mixture of 100 µL lipase, 50 µL
JSFE and 50 µL of 8 mmol L-1p-nitrophenylpalmitate in 0.05 mmol L-1
54
Tris-HCl, pH 8.0, with 0.5% Triton X-100, was incubated for 4 periods of
time after the addition of the substrate. The reaction was stopped by
transferring the tubes to an ice bath and adding 1,000 µL of 0.05 mmol L-
1 Tris-HCl, pH 8.0. The absorbance of the yellow p-nitrophenol product
was measured using a spectrophotometer at a wavelength of 410 nm[11].
Results are relative to a blank control.
Activity of trypsin
Trypsin activity was determined according to the method of Erlanger
et al.[14] Briefly, 200 µL JSFE and 200 µL enzyme were incubated in a
37°C water bath for 4 periods of time, after the addition of 800 µL Nα-
Benzoyl-DL-arginine p-nitroanilide (BApNA) prepared in 0.05 mol L-
1Tris, pH 8.2. (tri(hydroxymethyl)aminomethane) buffer with 20 mmol L-
1CaCl2. The reaction was stopped by adding 200 µL of 30% acetic acid,
and the absorbance was measured using a spectrophotometer at a
wavelength of 410 nm. Results are relative to a blank control.
Determination of enzyme inhibition
The inhibition of the enzymes was calculated according Pereira et
al.[13] from the determination of the slopes of the straight lines
55
(absorbance x time) of the control enzyme (without plant extract) and
enzymes + inhibitor (with plant extracts) activity assays.The slope of the
line was determined by the velocity of the reaction and product formation
per minute, in which the presence of the inhibitor caused a decrease in
that slope. From this slope, the absorbance values were converted into
µmol of product through a standard curve of glucose for amylase, and of
p-nitrophenol for glycosidase and lipase, whereas for trypsin, the molar
extinction coefficient of BApNA was determined using the protocol of
Erlanger et al[14]. The result of inhibition was expressed in µmol min g-1
sample, called UEI (unit of enzyme inhibited).
Preparation of simulated gastric fluid
To simulate the natural digestion process in the stomach, in vitro
assays of enzymatic activities were also performed in the presence of a
simulated gastric fluid. The extracts were incubated with simulated
gastric fluid, prepared according to the United States Pharmacopeia[15]
and kept in a 37°C water bath for 1 h. The extracts were then neutralized
with sodium bicarbonate salt until they reached physiological pH; activity
assays were then performed.
56
RESULTS AND DISCUSSION
Analyses of enzyme inhibition, where the extract is incubated with
simulated gastric fluid prior to the enzyme assay, try to simulate the
environment that the extract finds in the stomach before reaching the
intestine, aiming to observe if a low pH interferes with the interaction
between the enzyme and the components of the sample. Table 1 shows an
inhibition of 585.60 UEI and 560.00 UEI on the activity of α-amylase in
the absence and in the presence of simulated gastric fluid, respectively.
This shows that the inhibitor still acted on the gastric fluid, suggesting
that even in very acidic pH conditions, JSFE was able to maintain
practically the same inhibition of this enzyme.
Inhibition of α-amylase by JSFE may have occurred due to the
presence of phenolic compounds, especially anthocyanins, in JSF. A
previous study using the HPLC technique identified the phenolic acids
gallic acid, ellagic acid and salicylic acid in jabuticaba skins, as well as
the compounds gallocatechin and epicatechin, which was the major
one[17]. The anthocyanins cyanidin-3-glycoside and delphinidin-3-
glucoside were also identified[3]. Among the phenolic compounds that
57
occur in jabuticaba skins, condensed tannins and anthocyanins are
probably the compounds that act more effectively in the inhibition of α-
amylase.
Table I. Inhibition of digestive enzymes by jabuticaba skin aqueous
extract, in UEI1 without and after exposure to simulated gastric fluid
Enzyme2
Without gastric fluid
After gastric fluid
α-Amylase 585.60 ± 0.00 560.00 ± 0.00 α-Glycosidase 0.31 ± 0.01 nd2
Lipase 0.14 ± 0.00 nd Trypsin 0.26 ± 0.01 0.03 ± 0.01 1Unit of enzyme inhibited, in µmol min-1g-1
Moisture contentof JSF in g 100 g-1: 21.69 Results from 3 assays ± standard deviation
2nd = no inhibition detected
Gallocatechin and epicatechin are flavonoids that comprise condensed
tannins. Studies show that jabuticaba skins have a high content of total
condensed tannins[6]. α-amylase was inhibited by condensed tannins,
extracted from Vitis vinífera grape seeds. This extract was fractionated
according to the molecular weight of procyanidins, and the activity of α-
amylase was reduced with the increase in the concentration of
procyanidins for all tested fractions. The fluorescence quenching method
demonstrated that inhibition occurs through the formation of a stable
interaction between procyanidin and the enzyme[18].
58
Condensed tannins may be contributing to the inhibition of α-amylase
observed in this study. The stable interactions between the enzyme and
procyanidins, which occur at a high concentration in jabuticaba skins,
probably withstand the acidic conditions of the stomach, because the
inhibition in simulated gastric fluid was close to that observed in the
absence of gastric fluid.
Anthocyanins comprise approximately 36% of the total phenolic
compounds in jabuticaba skin flour[19].. Alezandro et al.[1] studied the
inhibitory potential of jabuticaba skins, and observed that the fully ripe
fruit successfully inhibited α-amylase, probably due to the higher
concentration of anthocyanins at this stage, and the same occurred in the
present study, which was also conducted with ripe jabuticaba skins. It has
been reported that anthocyanins measured glucose absorption via sodium
glucose transporter by intestinal membrane vesicles, and promote
upregulation of GLUT4 insulin-dependent glucose transport, through the
activation of adenosine monophosphate-activated protein kinase[19].
Analyses of the hypolipidemic effect of jabuticaba skins on rats show
that diets supplemented with 15% jabuticaba skin flour presented serum
glucose levels 8.8% lower than the control group[19]. The decrease in
59
glucose levels is a strong indicator of α-amylase inhibition, because this
enzyme reduces the metabolism of starch and glucose absorption in the
intestine, confirming the inhibitory power of JSFE.
The use of natural sources for α-amylase inhibitors has recently gained
interest, especially in terms of avoiding side effects that arise from the use
of commercial inhibitors. The treatment of diabetes using enzyme
inhibitors derived from fruits, such as strawberries, raspberries, and
blueberries have been examined elsewhere[20-22].
Studies on the specific activity of gallic acid, ellagic and salicylic acid
as inhibitors of α-amylase were not found in the literature.
The digestion of carbohydrates begins in the mouth, through the
mechanical action of chewing and the salivary action of α-amylase. The
function of α-amylase is to initiate starch digestion in the mouth,
catalyzing the hydrolysis of α-1,4 glycosidic bonds in starch; however,
the enzyme cannot hydrolyze branched α-1,6 glycosidic bonds. The α-
amylase secreted by the pancreas has the same specificity as the salivary
form, yet with a higher enzymatic activity. The action of salivary α-
amylase occurs until the food in the stomach is mixed with gastric acid,
60
which inactivates the enzyme; thus, no further processing of
carbohydrates occurs[23].
JSFE showed an inhibition of 0.31 and 0.14 UEI for α-glucosidase and
lipase, respectively, in the absence of gastric fluid (Tabela 1). Alezandro
et al.[9] found inhibition of ripe jabuticaba skins on lipase. The extraction
was carried out with extracted lyophilized powder in a solvent mixture,
comprising methanol/water/acetic acid in the ratio (70:30:0.5 v/v/v).
The disaccharides sucrose and lactose and the polysaccharide starch
are the main sources of carbohydrates in the normal human diet. The
glycosidic bond between monosaccharide residues in these compounds is
an α bond; thus, α-glucosidase inhibitors are more interesting to
investigate, when compared to β-glucosidase, which is of minor
importance in the energy metabolism of carbohydrates in humans. Thus,
the ability of JSFE to inhibit only α-glucosidase was examined.
α-glucosidase acts complementing the action of amylase during
carbohydrate digestion. Polysaccharides are hydrolyzed by α-amylase,
producing maltose and isomaltose, which can not be absorbed into the
bloodstream. Thus, it is necessary that these compounds are subsequently
hydrolyzed by intestinal α-glucosidase, in order to release glucose, which
61
will be absorbed in the intestinal epithelium and inserted into the
bloodstream[24].
Lipases are hydrolases acting on carboxylic ester bonds, hydrolyzing
os triacilgliceróis. Sergent et al.[25] analyzed the inhibitory potential of 20
phenolic compounds on lipase and, among these compounds, ellagic and
gallic acid were investigated, as well as epicatechin. Ellagic acid showed
only 30% inhibitory potential on lipase, while gallic acid and epicatechin
had no significant effect. Ellagic acid was indentified in JSF[16] and may
been contributing to low the lipase inhibition in this study.
The addition of JSFE resulted in an inhibition of 0.258 UEI in the
activity of trypsin. The inhibition of trypsin is usually associated with
procyanidins present in the sample, which interact with these compounds
and the proteins, forming tannin-protein aggregates[26].Several studies
have confirmed this hypothesis, showing that procyanidins are quite
efficient in inhibiting proteases such as trypsin[26-28].
Abe et al.[6] studied the effect of ripening on the concentration of
condensed tannins in jabuticaba, and found a concentration of 48 g tannic
acid kg-1 fresh weight in ripe jabuticaba skins. Condensed tannins are
flavonoid polymers formed mostly by flavan-3-ol units, and phenolic
62
hydroxyl groups that occur in their structure allow the formation of stable
bonds with proteins, allowing them to complex and precipitate these
compounds. Due to this ability, they are considered enzyme inhibitors.
When investigating the influence of carbohydrates on the interactions
of procyanidin B3 (catechin-(4β →8)-catechin) with trypsin, Gonçalves et
al.[29] used saturation transfer difference- nuclear magnetic resonance
(STD-NMR) to identify procyanidin B3 binding trypsin. The molar ratio
used was 1:30 (trypsin/procyanidin), due to the fact that preliminary
studies showed that, at this stoichiometry, procyanidin B3 is able to cause
a decrease in trypsin activity fluorescence. In this study, it was apparent
that trypsin saturation was transferred to the aromatic protons of
procyanidin. This ability of procyanidins to bind trypsin may be
responsible for the inhibitory activity JSFE presented on trypsin in this
study.
When in the presence of simulated gastric fluid, the inhibition of JSFE
decreased about 8.6 times, indicating that phenolic compounds present in
JSFE experienced a reduction in their stability. Studies suggest that
gastrointestinal conditions may adversely affect the bioavailability of
63
epigallocatechin-O-gallate[30], a condensed tannin whose bioavailability
has been widely analyzed[10,32].
Krokk and Hagerman[33] conducted studies on the stability of
epigallocatechin-O-gallate, a condensed tannin, when introduced in an in
vitro simulated digestive system, under a reduced oxygen concentration,
pH conditions, gastrointestinal enzymes, bile and food components. The
results found by these authors indicate that the stability of
epigallocatechin-O-gallate in solutions with a pH of 1.8 ± 0.1, similar to
the stomach, under a reduced oxygen concentration, remained stable;
however, in the absence of food or digestive components, it was lost by
90%. The authors suggest that the phenolic compounds have different
destinations and purposes, as they are ingested as compounds or as
constituents of foods or beverages. Thus, despite JSFE has its stability
greatly reduced, it is possible that as JSF its stability is preserved.
Research efforts focused on the inhibition of these enzymes are
important for the treatment of diseases, such as pancreatitis, cystic
fibrosis, emphysema, and asthma. However, because of the fact that
trypsin is also responsible for the degradation of proteins into peptides
64
and amino acids that are more easily absorbed through the intestinal
mucosa, its inhibition may sequentially lead to its lower absorption[26].
CONCLUSIONS
JSFE inhibited α-amylase and there was a little difference between the
inhibition in the presence and in the absence of simulated gastric fluid.
This inhibition is probably due to the phenolic compounds present in
jabuticaba skin flour, highlighting the anthocyanins and flavonoids
constituents of condensed tannins. This inhibition showed that jabuticaba
skin flour might potentially serve as a good alternative for the control of
diseases, such as type 2 diabetes and obesity, through the reduction in the
breakdown of ingested starch.
ACKNOWLEDGMENTS
The authors would like to thank CAPES, FAPEMIG, and CNPq for the
financial support.
65
REFERENCES
1. Alezandro, M.R.; Dubé, P.; Desjardins, Y.; Lajolo, F.M.;
Genovese, M.I. Comparative study of chemical and phenolic
compositions of two species of jaboticaba: Myrciaria jaboticaba
(Vell.) Berg and Myrciaria cauliflora (Mart.) O. Berg. Food
Research International 2013, 54, 468–477.
2. Lima, A.J.B.; Corrêa, A.D.; Dantas-Barros, A.M.; Nelson, D.L.;
Amorim, A.C.L. Sugars, organic acids, minerals and lipids in
jabuticaba. Revista Brasileira de Fruticultura 2011a, 33, 540–550.
3. Lima A.J.B.; Corrêa, A.D.; Saczk, A.A.; Martins, M.P.; Castilho,
R.O. Anthocyanins, pigment stability and antioxidant activity in
jabuticaba [Myrciaria cauliflora (Mart.) O. Berg] 2011b, Revista
Brasileira de Fruticultura 33, 877–887.
4. Bordiga, M.; Travaglia, F.; Locatelli, M.; Coissom, J.D.; Arlorio,
M. Characterisation of polymeric skin and seed proanthocyanidins
during ripeningin six Vitis vinifera L. cv. Food Chemistry
2011,127, 180–187.
5. Leite-Legatti, A.V.; Batista, A.G.; Dragano, N.R.V.; Marques,
A.C.; Malta, L.G.; Riccio, M.F.; Eberlin, M.N.; Machado, A.R.T.;
66
Carvalho-Silva, L.B.L.; Ruiz, A.L.T.G.; Carvalho, J.E.; Pastore,
G.M.; Júnior, M.R.M. Jaboticaba peel: antioxidant compounds,
antiproliferative and antimutagenic activities. Food Research
International 2012, 49, 596-503.
6. Abe, L.T.; Lajolo, F.M.L.; Genovese, M.I. Potential dietary
sources of ellagic acid and other antioxidants among fruits
consumed in Brazil: Jabuticaba (Myrciaria jaboticaba (Vell.)
Berg). Journal of the Science of Food and Agriculture 2012, 92,
1679–1687.
7. Wu, S-B.; Wu, J.; Yin, Z.; Zhang, J.; Long, C.; Kennely, E.J.;
Zheng, S. Bioactive and Marker Compounds from Two Edible
Dark-Colored Myrciaria Fruits and the Synthesis of Jaboticabin.
Journal of Agricultural and Food Chemistry 2013, 61: 4035−4043.
8. Reynertson, K.A.; Wallace, A.M.; Adachi, S.; Gil, R.R.; Yang, H.;
Basile, M.J.;D’Armiento, J.; Weinstein, I.B.; Kennelly, E.J.
Bioactive Depsides and Anthocyanins from Jaboticaba (Myrciaria
cauliflora) Journal of Natural Products2006, 69, 1228-1230.
67
9. Zanotto, D. L.; Bellaver, C. (1996). Método de determinação da
granulometria de ingredientes para uso em rações de suínos e
aves. Comunicado Técnico: Embrapa.
10. Association of Official Analytical Chemistry (2005).Official
Methods of Analysis of the association of the analytical chemists.
17th Ed. Washington.
11. Souza, S.P.; Pereira, L.L.S.; Souza, A.A.; Santos, C.D. Inhibition
of pancreatic lipase by extracts of Baccharis trimera: Evaluation
of anti-nutrients and effect on glycosidases. Revista Brasileira de
Farmacognosia 2010,21, 450–455.
12. Noelting, G.; Bernfeld, P. Sur les enzymes
amylolytiques-III. La β-amylase: dosage d’activité et contrôle de
l’absence d’ α-amylase. Helvetica Chimica Acta 1948, 31, 286–
290.
13. Pereira, L.S.; Santos, C.D.; Sátiro, L.C.; Marcussi, S.; Pereira,
C.A.; Souza, S.P. Ação inibitória e estabilidade do extrato de
farinha de feijão branco sobre enzimas digestivas na presença de
fluido gástrico simulado. Revista Brasileira de Farmacognosia
2011, 92, 367-372.
68
14. Erlanger, B.F.; Kokowsky, N.; Cohen, W. The preparation and
properties of two new chromogenic substrates of trypsin. Archives
of Biochemistry and Biophysics 1961, 95, 271–278.
15. The United States Pharmacopeia (2005). The national formulary
NF 18 (Pharmacopeial Convention Ing). Rockvile.
16. Lage, F.F.; Simão, A.A.; Guedes, M.N.S.; Ramos, V.O.; Sousa,
R.V.; Corrêa, A.D. Jabuticaba [Plinia jaboticaba (Vell) Berg]
skins decrease lipid peroxidation: hepatoprotective and
antihyperlipidemic effects. African Journal of Biotechnology
2014, 13, 1295-1302.
17. Gonçalves, R.; Mateus, N.M.; Freitas, V. Inhibition of a-amylase
activity by condensed tannins. Food Chemistry 2011a,125, 665-
672.
18. Araújo, C.R.; Esteves, E.A.; Dessimoni-Pinto, N.A.; Batista, A.G.
Myrciaria cauliflora Peel Flour Had a Hypolipidemic Effect in
Rats Fed a Moderately High-Fat Diet. Journal of Medicinal Food
2014,17, 262-267.
19. Flores, F.P.; Singh, R.K.; Kerr , W.L.; Pegg, R.; Kong, F.
Antioxidant and Enzyme Inhibitory Activities of Blueberry
69
Anthocyanins Prepared Using Different Solvents Journal of
Agricultural and Food Chemistry 2013,61, 4441−4447.
20. Cheplick, S.; Kwon, Y.; Bhowmik, P.; Shetty, M. Clonal variation
in raspberry fruit phenolics and relevance for diabetes and
hypertension management. Journal of Food Biochemistry
2007,31, 656–679.
21. Pinto, M.; Carvalho, J.; Lajolo, F.; Genovese, M.; Shetty, K.
Evaluation of anti-proliferative, anti-type 2 diabetes and anti-
hypertension potential of ellagitannins from strawberries (Fragaria
x ananassa Duch.) using in vitro models. Journal of Medicinal
Food 2010, 5, 1–9.
22. Stull, A.; Cash, K.; Johnson, W.; Champagne, C.; Cefalu, W.
Bioactives in blueberries improve insulin sensitivity in obese,
insulin-resistant men and women. Journal of Nutrition 2010,140,
1764–1768.
23. Moriel, P.; Madureira, H.L.; Uwagoya, A.K.Y.; Wlian, L.;
Pincinato, E.C. Influence of smoke in salivary amylase activity
and in the glycemic curve. Revista de Nutrição 2010, 23, 565–
572.
70
24. Zhang, L., Hogan, S.; Li, J.; Sun, S.; Canning, C.; Zheng, S.J.;
Zhou, K. Grape skin extract inhibits mammalian intestinal α-
glucosidase activity and suppresses postprandial glycemic
response in streptozocin-treated mice. Food Chemistry 2011, 126,
466–471.
25. Sergent, T.; Vanderstraeten, J.; Winand, J.; Beguin, P.; Schneider,
Y-J. Phenolic compounds and plant extracts as potential natural
anti-obesity substances. Food Chemistry 2012, 135, 68–73.
26. Gonçalves, R.; Soares, S.; Mateus, N.; Freitas, V. Inhibition of
trypsin by condensed tannins and wine. Journal of Agricultural
and Food Chemistry 2007, 55, 7596–7601.
27. Gonçalves, R.; Mateus, N.; Freitas, V. Biological relevance of the
interaction between procyanidins and trypsin: A multi-technique
approach. Journal of Agricultural and Food Chemistry 2010, 58,
11924–11931.
28. Zhang, L.; Li, J.; Hogan, S.; Chung, H.; Welbaum, G.E.; Zhou, K.
Inhibitory effect of raspberries on starch digestive enzyme and
their antioxidant properties and phenolic composition. Food
Chemistry 2010, 119, 592–599.
71
29. Gonçalves, R.; Mateus, N.M.; Freitas, V. Influence of
carbohydrates on the interaction of procyanidin b3 with trypsin.
Journal of Agricultural and Food Chemistry 2011b, 59, 11794–
11802.
30. Long, H.; Zhu, Y.X.; Gregor, M.; Tian, F.F.; Coury, L.; Kissinger,
C.B. et al. Liquid chromatography with multi-channel
electrochemical detection for the determination of
epigallocatechin gallate in rat plasma utilizing an automated blood
sampling device. Journal of Chromatography B 2001, 763, 47–51.
31. Dube, A.; Nicolazzo, J.A.; Larson, I. Assessment of plasma
concentrations of (−)-epigallocatechin gallate in mice following
administration of a dose reflecting consumption of a standard
green tea beverage. Food Chemistry 2011, 128, 7–13.
32. Williamson, G.; Dionisi, F.; Renouf, M. Flavanols from green tea
and phenolic acids from coffee: Critical quantitative evaluation of
the pharmacokinetic data in humans after consumption of single
doses of beverages. Molecular Nutrition & Food Research 2011,
55, 864–873.
72
33. Krook, M.A.; Hagerman, A.E. Stability of polyphenols
epigallocatechin gallate and pentagalloyl glucose in a simulated
digestive system. Food Research International 2012, 49, 112–116.
73
ARTIGO 2
Submetido para a Revista LWT – Food Science and Technology
Formato conforme normas da revista
Jabuticaba skin flour as a functional food ingredient: Phenolic profile, antioxidant potential and functional-technological properties F.F. Lagea1*, A.D. Corrêaa*, A.P.C. Alvesa, A.A. Saczka, F.C. Oliveiraa, M.M. Mendonçaa
Chemistry Department, Universidade Federal de Lavras, 37200-000, Lavras, MG, Brazil.
ABSTRACT
Jabuticaba skin flour (JSF) is especially rich in phenolic compounds with
antioxidant potential, but little is known about their characterization and
about the functional-technological properties of this flour. The objective
of this study was to identify and quantify phenolics, measure their
antioxidant capacity and investigate the functional-technological
properties of JSF, in order to verify the feasibility of its use in the food
industry. Gallic, ellagic and salicylic acids were identified in JSF, as well 1 * Corresponding authors: Biochemistry Laboratory, Chemistry Department, Universidade Federal de Lavras, 37200-000, Lavras, MG, Brazil. Tel: +55 (35) 38291272; fax number: +55 (35) 38291271. Email address:[email protected] (F.F. Lage) and [email protected] (A.D. Corrêa)
74
as monomers of condensed tannins, e.g. epicatechin and gallocatechin.
The phenolic compound which occurred in larger amounts was
epicatechin. Analyses of functional-technological properties indicated that
JSF has good water absorption and emulsion stability. JSF shows up as a
promising alternative to be used as a natural antioxidant and as an
additive in formulations, such as soups, sauces, sausages, pasta, cheese,
pastries and bakery products.
Keywords: Plinia jaboticaba, phenolic compounds,antioxidant activity,
HPLC, functional-technological properties.
1. Introduction
Jabuticaba is a fruit of the Myrtaceae family with a high incidence
in southern and southeastern Brazil. Jabuticaba trees are strong and
perennial trees, which remain in productive phase for a period of 30-50
years. The fruits are globose berry, mostly black with reddish skin,
reaching up to 3 cm in diameter, are consumed fresh, and as liqueurs and
jam. The residues produced by the consumption of this fruit are skins and
seeds (Danner et al., 2006; Lima, Corrêa, Dantas-Barros, Nelson, &
Amorim, 2011a).
75
Jabuticaba skins are rich in phenolic compounds, especially in
anthocyanins, which are responsible for their characteristic color, can be
highlighted (Lima, Corrêa, Saczk, Martins, & Castilho, 2011b). Phenolic
compounds are among the most active antioxidants present in plants, as
they act through various mechanisms, and their action depends on
concentration, compound structure, number of hydroxyl groups and their
distribution; there may be synergism or antagonism among the different
compounds (Palafox-Carlos, Yahi,a & González-Aguilar, 2012; Zhang,
Wang, & Mi, 2011).
Recent research shows that jabuticaba has a high antioxidant
potential (Abe, Lajolo, & Genovese, 2012; Lima, Corrêa, Saczk, Martins,
& Castilho, 2011b; Rufino, Alves, Fernandes, & Brito, 2011; Santos,
Veggi, & Meireles, 2010). Studies performed by Leite-Legatti et al.
(2012) with jabuticaba skins show that there is a high relation between
antioxidant activity and content of phenolic compounds. Determining the
composition and concentration of each phenolic compound can help in
understanding the observed antioxidant activity.
Ellagic acid was identified in the skin by HPLC, as well as the
following anthocyanins: cyanidin-3-glucoside, delphinidin-3-glucoside,
76
cyanidin 3-O-glucopyranoside and quercetin (Abe, Lajolo, & Genovese,
2012; Einbond, Reynertson, Luo, Basile, & Kennelly, 2004; Lima,
Corrêa, Saczk, Martins, & Castilho, 2011b). However, there are other
phenolic compounds which have not been identified.
Jabuticaba skins show up as a promising alternative for use as a
functional food by the food industry because, besides a high antioxidant
potential, they have a good nutritional value. According to Lima, Corrêa,
Alves, Abreu, & Dantas-Barros (2008), they present high contents of
soluble and insoluble fiber (27.03 and 6.77 g 100 g-1 dry matter (DM),
respectively), are rich in minerals, such as potassium (1,496.67 mg 100 g-
1 DM), magnesium (90.00 mg 100 g-1 DM), iron (1.68 mg 100 g-1 DM)
and manganese (1.71 mg 100 g-1 DM), as well as in vitamin C (0.025 g
100 g -1 DM).
Nutritional guidelines emphasizing the importance of fiber and
antioxidants in the diet have increased the demand for products that are
rich in these components (Agboola, Mofolasayo, Watts, & Aluko, 2010).
Jabuticaba skins efficiently meet these requirements, which makes the use
of their flour as a promising functional food ingredient.Functional foods
can be naturally present or added in processed food products. The
77
commercial demand for functional foods has grown greatly in recent
years. Consumers have changed their attitudes when choosing food to
consume and are increasingly concerned about the potential health
benefits that food can provide. Therefore, knowing the functional-
technological properties of JSF can aid in their application for the
formulation of new products.
Thus, the objective of this study was to identify and quantify
phenolic compounds, measure their antioxidant capacity, and investigate
the functional-technological properties of JSF, in order to verify the
feasibility of its use in the food industry.
2. Material and methods
2.1 Reagents and standards
Sodium hypochlorite, ethanol, hydrochloric acid and acetone were
acquired from Vetec Química Fina (RJ - Brazil), methanol from JT Baker
Chemical Co. (Phillipsburg, NJ) and trolox from Sigma-Aldrich (St.
Louis, MO, USA). For the preparation of the mobile phase used in the
HPLC analyses, ultrapure Milli-Q water (Millipore, Billerica, MA, USA),
acetic acid and methanol (Merck, Darmstadt, Germany) were used. HPLC
78
grade standards of gallic, p-coumaric, ferulic, ellagic, 3,4-
dihydroxybenzoic, syringic and salicylic acids were obtained from Sigma-
Aldrich (St. Louis, MO, USA), as well as the condensed tannin
monomers gallocatechin, catechin, epigallocatechin gallate and
resveratrol. Vanillic acid and m-and o-cumaric acids were obtained from
Fluka (St. Louis, MO, USA). Stock standard solutions were prepared in
dimethylsulfoxide and/or methanol (Merck).
2.2 Sample preparation
The harvest of jabuticaba (Plinia jaboticaba (Vell.)Berg) ripe
fruits, genotype Sabará, was made in October 2010, in the morning, by
hand, on Fazenda São José do Ismeril, in the municipality of Coqueiral,
MG, Brazil.
The fruits were transported to the laboratory, where they were
selected, washed in tap water, sanitized with sodium hypochlorite
solution (200 mg kg-1), by a 10-minute immersion; they were then
squeezed and the skins were weighed and separated into three fractions of
approximately 2.9 kg.
79
The jabuticaba skins were dried in a food dehydrator, in mesh
metallic material baskets, at a temperature of 45°C, with a 1 m s-1 air flow
over a period of 36 hours. The skins were then ground and the resulting
jabuticaba skin flour (JSF) was packaged in hermetically sealed flasks in
three replicates, wrapped in aluminum foil, stored at room temperature
and subjected to analyses. In this flour, the particle size was determined
using a sieve shaker, and most of the flour particles were retained on the
sieves between 32 mesh (0.5 mm) and 60 mesh (0.25 mm). According to
Zanotto and Bellaver (1996), the uniformity index indicates the relative
proportion between coarse, medium and fine
particles, which are defined according to diameters larger than 2 mm,
between 2 and 0.60 mm and smaller than 0.60 mm, respectively.
Therefore, JSF can be classified as fine.
2.3 Extraction and quantification of phenolic compounds
The extraction of phenolic compounds (in three replicates) in JSF
was performed with 50% methanol under reflux for three consecutive
times, at 80°C, at a ratio of 1:25 (w/v). The extracts were collected and
evaporated to 25 mL. The content of phenolic compounds was measured
80
using the Folin-Denis method, with tannic acid as a standard (AOAC,
1995).
2.4 Determination of antioxidant activity
The antioxidant activity was determined with a method proposed by
Re et al. (1999), with modifications made by Rufino et al. (2007). Four
different dilutions were made to the assays, and subsequently a calibration
curve was constructed. For the analyses, 3.0 mL of the ABTS•+ radical
were placed in a test tube with a 30 µL aliquot of each extract dilution
and homogenized on a magnetic stirrer. The absorbance was measured at
734 nm after 6 minutes of reaction, using ethanol as a blank.
Calculations were made with the absorbances obtained from
different extract dilutions (four concentrations), and a graph was plotted
for each sample, with the absorbance on the y-axis and the concentration
for each dilution (mg L-1) on the x-axis, determining the line equation. In
this equation, the absorbance corresponding to 1.000 µmol L-1 trolox
standard was replaced; thus, it was possible to find the concentration of
the sample (mg L-1), equivalent to 1.000 µmol trolox L-1. The final result,
81
which was expressed in µmol L-1 g-1, was calculated by dividing 1.000
(µmol L-1) by the x value (g) and multiplying it by 1 g.
2.5 Chromatographic study of phenolic compounds
Chromatographic analyses were performed using an Agilent HPLC
equipment model 1100, and the best response was obtained at a
wavelength of 280 nm. The extracts of phenolic compounds and the
standards were injected into an Ascentis C18 column (25 cm x 4.6 mm x
5 mm), attached to a Supelguard Ascentis C18 pre-column (2 cm x 4.0
mm x 5 µm). The mobile phase was composed of the solutions: 2% acetic
acid (A) and methanol:water:acetic acid (70:28:2 v/v/v) (B). The flux
used in all analyses was 1.00 mL min-1; the injection volume was 20 µL.
Analyses were performed in a total time of 65 minutes at 15ºC in a
gradient-type system: 100% solvent A for 5 minutes, 70% solvent A for
20 minutes, 60% solvent A for 18 minutes , 55% solvent A for 7 minutes,
0% solvent A for 10 minutes. Until the end of the run, solvent A was
increased to 100%, in order to balance the column.
The phenolic compounds in JSF were identified by comparison
with retention times of standards, and confirmed by co-chromatography
82
of extracts and standards. For the quantification of the phenolic
compounds, the external standardization method was used. The analytical
curves of the compounds standard solutions found in the samples were
obtained by dilutions of the stock solutions (1,0 x 10-3 mol L-1), resulting
in a concentration range from 5,0 x 10-7 to 1,5 x 10-3 mol L-1. Calibration
curves were constructed by plotting the average detector response (n=3)
to the peak area as a function of concentration.
Addition of standards to the extracts was also used as an
identification parameter. Quantitation was performed using external
standardization with concentrations of standard stock solutions: gallic
acid (85.06 mg L-1), gallocatechin (153.13 mg L-1), 3,4 dihydroxybenzoic
acid (77.06 mg L-1), catechin (145.14 mg L-1), chlorogenic acid (177.15
mg L-1), epigallocatechin gallate (229.18 mg L-1), vanillic acid (84.07 mg
L-1) , epicatechin (145.13 mg L-1), para, meta, and ortho coumaric acids
(82.08 mg L-1), ferulic acid (97.09 mg L-1), resveratrol (114.12 mg L-1),
ellagic acid (151.09 mg L-1) and salicylic acid (69.06 mg L-1). Each
solution was injected three times on the HPLC system, with the purpose
of obtaining concentration means and retention times.
83
2.6 Functional-technological properties
2.6.1 Water and oil absorption
JSF was suspended in water or oil, mixed at high speed
(Robot Classic mixer - Mallory) and then centrifuged. The volume of the
supernatant was measured, and the amount of water or oil absorbed was
multiplied by their respective densities for conversion to grams (Okezie &
Bello, 1988).
2.6.2 Nitrogen solubility
JSF was mixed in distilled water, and the pH was adjusted to 2, 3,
4, 5, 6, 7, 8 and 9 with NaOH or HCl solution. Then, it was centrifuged
and the supernatant was analyzed according to the Kjeldahl method
(Beuchat, 1977).
2.6.3 Foam volume
JSF was suspended in distilled water and agitated for 3.5 minutes
(Robot Classic mixer - Mallory); the mixture was transferred to a
graduated cylinder, in which foam volumes were determined at different
times (0, 30, 60 and 120 minutes). The foam volume, expressed as a
84
percentage, was calculated considering the foam volume at time 0 as
100% (Wang, Caballero-Corboba & Sgarbieri, 1992).
2.6.4 Emulsion Stability
JSF was dispersed in distilled water and the oil was slowly added
under agitation for 30 seconds (Robot Classic mixer – Mallory); it was
then homogenized at high speed for another 60 seconds. The volumetric
change of foam, oil and aqueous phase was observed after 30 minutes, 2
hours and 6 hours (Okezie & Bello, 1988).
3. Results and discussion
The content of phenolic compounds in JSF was 8.45 ± 0.3 g tannic
acid 100 g-1 DM. Lima, Corrêa, Alves Abreu, & Dantas-Barros (2008)
determined a phenolic compounds content of 11.99 g tannic acid 100 g-1
DM in JSF using lyophilized jabuticaba skins, higher than that of husks at
45ºC. Environmental factors, such as harvest time, rainfall and soil
nutrition may have been decisive for the differences among the levels of
phenolic compounds in JSF, as well as dehydration process.
Lyophilization better preserves bioactive compounds; however, it is a
85
relatively expensive process. Food drying using a food dehydrator is a
cheaper and quite efficient alternative.
The phenolic extract of JSF showed good antioxidant potential
(866.39 µmol L-1 g-1) by the ABTS method. In a previous study, in which
the methods ABTS, phosphomolybdenum and β-carotene/linoleic acid
were used to measure the antioxidant activity in JSF, the ABTS method
resulted in better responses (Lima, Corrêa, Saczk, Martins, & Castilho,
2011b); for this reason, this was the method used in this study.
The phenolic profile of JSF was investigated and three phenolic
acids were identified, as well as two monomers of condensed tannins,
presenting the following quantitative order: epicatechin ˃ salicylic acid >
ellagic acid ˃ gallic acid > gallocatechin (Figure 1). The total content of
phenolic compounds was 2.68 g 100 g-1 DM (Table 1), lower than the one
determined by the colorimetric method. However, it is possible to observe
in Figure 1 that there are other non-identified peaks, which could lead to
higher contents.
86
0 10 20 30 40 50 60-5
0
5
10
15
20
25
30
Inte
nsity
(m
Vol
ts)
Time (min)
1
2
3
4
5
Fig. 1.Chromatogram of total phenolics extract. Peak identification: 1- gallic acid; 2- gallocatechin; 3 – epicatechin; 4- ellagic acid; 5- salicylic acid.
87
Table 1 Phenolics average (g 100 g-1 DM) in JSF, by HPLC Phenolic compound
Structure
Retention time (min)
Phenolic
content (g 100 g-1 DM)
Gallic acid
7.44 0.11
Gallocatechin
8.89 0.01
Epicatechin
21.95 1.9
Ellagic acid
51.39 0.12
Salicylic acid
52.14 0.54
Total 2.68
88
Using the HPLC method, Lima, Corrêa, Saczk, Martins, &
Castilho (2011b) identified the anthocyanins cyanidin-3-glucoside and
delphinidin-3-glucoside in jabuticaba Sabará skins, which were harvested
from the same propoerty and the same plants as the jabuticabas used in
this study, but in previous years.
There are no other studies in the literature that detected condensed
tannins in jabuticaba skins by HPLC. The amounts of gallocatechin and
epicatechin together represent 71.27% of the total content of phenolic
compounds quantified.
Gallocatechin, when polymerized, forms prodelphinidin, which is
a condensed tannin. Due to the occurrence of many hydroxyl groups in its
structure, gallocatechins have a high antioxidant effect (Cai, Xing, Sun,
Zhan, & Corke, 2005). The gallocatechin detected in JSF may contribute
to its good antioxidant potential.
Phenolic acids are reported as good antioxidants. This antioxidant
action is probably due to the occurrence of an easily ionizable carboxyl
group in its structure, which is an efficient hydrogen donor (Palafox-
Carlos, Yahia & González-Aguilar, 2012).
89
Studies with other jabuticaba skins in which salicylic acid has
been quantified for comparison were not found in the literature. Salicylic
acid is derived from hydrolysis of acetylsalicylic acid in living organisms,
and has significant anti-inflammatory properties. Prolonged exposure to
diets of silicates from fruits and vegetables may be beneficial for
inflammatory processes, as well as for some cardiovascular diseases
(Battezzati, Fiorillo, Spadafranca, Bertoli, & Testolin, 2006).
Gallic and ellagic acids presented similar concentrations. Abe,
Lajolo, & Genovese (2012), using the HPLC technique, found a content
of 2.25 g 100 g-1 in the fresh weight of the jabuticaba lyophilized fruit.
80% acetone was used as an extragent, and the extract underwent
subsequent acid hydrolysis. Extraction in fresh jabuticaba skins was
probably determinant for the higher content of ellagic acid in relation to
that quantified in this study, in addition to the influence of harvest year,
rainfall and soil nutrition, among other factors.
Ellagic acid has four OH groups attached to the benzofuran
structure. It has a minimum solubility in water; however, its solubility
increases in organic solvents, such as methanol and dimethyl sulfoxide
(DMSO). This feature implies that ellagic acid can act as a good
90
lipophilic antioxidant (Hayes, Allen, Brunton, Grady, & Kerry, 2011).
Several studies confirm the antioxidant power of ellagic acid (Cuartero,
Ortuno, Truchado, Garcia, Tomás-Barberán, & Albero, 2011; Hayes,
Allen, Brunton, Grady, & Kerry, 2011; Kumar, Raja, Vidhya, & Devaraj,
2012). The presence of ellagic acid confirms the high antioxidant power
of JSF, which makes it an interesting alternative as a substitute for
synthetic antioxidants used in the food industry.
Gallic acid has a carboxyl group and three hydroxyl groups in its
structure, which are available to donate hydrogens, and may then
contribute significantly to the antioxidant activity of JSF (Table 1).
Water absorption by JSF was 350% and oil absorption was
475.55%. JSF had a good water absorption, when compared to the water
absorption of proteins isolated from legumes and yam bean flour varieties
Country Ekona and Sosso Chad (Pastor-Cavada, Juan, Pastor, Alaiz &
Vioque, 2010; Njintang et al., 2007). This high water absorption can be
attributed to the presence of a considerable amount of fibers and
carbohydrates in the flour. Flours with a high water absorption capacity
can be used in products in which a good viscosity is required, such as
91
soups and sauces, sausage, pasta, processed cheese and bakery products
(Kaushal, Kumar, & Sharma, 2012).
At 30 minutes, there was a retention of only 50% of the initial
foam volume. At 60 minutes, there was no longer foam, indicating that
the foam formed by JSF is not very stable, and therefore has no good
foaming characteristics. Foam stability in good flours suggest that the
native proteins, which are soluble in the continuous phase (water), are
very active on the surface of these flours (Kaushal, Kumar, & Sharma,
2012). The protein content in JSF is low, corresponding to 1.16 g 100g-1
DM (Lima, Corrêa, Alves, Abreu, & Dantas-Barros, 2008), which may
explain the low stability of the foam formed.
Changes in pH resulted in variations in the sample nitrogen
solubility. The isoelectric point of vegetable proteins is between 3 and 5.
In general, nitrogen solubility is minimal at this point and increases as the
pH moves away (Naves, Corrêa, Abreu, & Santos, 2010). Nitrogen
solubility in JSF remained constant from pH 2.0 through 5.0 and
increased in pH 6.0, keeping constant again up to pH 9.0.
The average volumes of foam, oil and aqueous phase observed
when analyzing the emulsion stability in JSF are shown in Table 2. After
92
2 hours of agitation, a 25% reduction in foam was observed, as well as a
small increase in the oil volume. Thus, JSF has a considerable emulsion
stability. The results indicate that JSF can be used in the production of
sausage, soups and pastries.
Table 2 Emulsion stability: average volume of foam, oil and aqueous phase at times: 0.5, 2.0 and 6.0 hours
Average volume (mL) Time after
agitation (hours) Foam Oil Aqueous phase
0.5 2 10 9
2.0 1.5 10.5 9
6.0 1.5 10.5 9
The studies conducted show that JSF has a high potential to be
used as a natural antioxidant, in replacement of the synthetic antioxidants
used in the food industry. JSF was also demonstrated to have good water
absorption and emulsion stability, properties that indicate it as a
promising alternative to be used as an additive in formulations such as
soups, sauces, sausage, pasta, cheese, pastries and bakery products.
93
4. Conclusions
JSF has a high content of phenolic compounds, in which three
phenolic acids and two monomers of condensed tannins were identified,
presenting the following quantitative order: epicatechin ˃ salicylic acid >
ellagic acid ˃ gallic acid > gallocatechin, in addition to others that have
not been identified in this study.
JSF has a good antioxidant potential, probably due to phenolic
compounds. Therefore, the JSF extract shows up as a promising
alternative to be used as a natural antioxidant in food industry, in
replacement of synthetic antioxidants.
The functional-technological properties of JSF indicate that this
flour can be used in the formulation of foods, such as soups, sauces,
sausages, pasta, cheese, pastries and bakery products, enriching them not
only with nutrientes, but also with antioxidants.
REFERENCES
Abe, L. T., Lajolo, F. M. L., & Genovese, M. I.(2012). Potential dietary
sources of ellagic acid and other antioxidants among fruits consumed in
94
Brazil: Jabuticaba (Myrciaria jaboticaba (Vell.) Berg).Journal of the
Science of Food and Agriculture, 92, 1679–1687.
Agboola, S. O., Mofolasayo, O. A., Watts, B. M., & Aluko, R. E. (2010).
Functional properties of yellow field pea (Pisum sativum L.) seed flours
and the in vitro bioactive properties of their polyphenols. Food Research
International, 43, 582–588.
Agostini-Costa, T. S., Lima, A., & Lima, V. V. (2003). Determinação de
tanino em pedúnculo de caju: método da vanilina versus método do
butanol ácido. Química Nova, 26(5), 763-765.
AOAC.(1995). Official Methods of Analysis. (16th ed.). Washington, DC:
Association of Official Analytical Chemists.
Battezzati, A., Fiorillo, G., Spadafranca, A., Bertoli, S., & Testolin, G.
(2006).Measurement of salicylic acid in human serum using stable
isotope dilution and gas chromatography–mass spectrometry.Analytical
Biochemistry, 354, 274–278.
95
Beuchat, L.R. (1977). Functional and eletrophoretic characteristics of
succinylated peanut flour protein.Journal of Agricultural and Food
Chemistry, 25(2), 258-261.
Cai, Y-Z., Xing, J., Sun, M., Zhan, Z-Q., & Corke, H. (2005). Phenolic
antioxidants (hydrolyzable tannins, flavonols, and anthocyanins)
identified by lc-esi-ms and maldi-qit-tof ms from rosa chinensis flowers.
Journal of Agricultural and Food Chemistry, 53, 9940-9948.
Cuartero, M, Ortuno, J. A., Truchado, P., Garcia, M. S., Tomás-Barberán,
F. A., & Albero, M. I. (2011).Voltammetric behaviour and square-wave
voltammetric determination of the potent antioxidant and anticarcinogenic
agent ellagic acid in foodstuffs.Food Chemistry, 128, 549–554.
Danner, M. A., Citadin, I., Fernandes –Jr, A. A., Assmann, A. P., Mazaro,
S. M., Donazzolo, J., & Sasso, S. A. Z. (2006). Enraizamento de
jabuticabeira (Plinia trunciflora) por mergulhia aérea. Revista Brasileira
de Fruticultura, 28(3), 530-532.
96
Einbond, L. S., Reynertson, K. A., Luo, X-D, Basile, M. J., & Kennelly,
E. J. (2004). Anthocyanin antioxidants from edible fruits.Food Chemistry,
84(1), 23-28.
Hayes, J. E., Allen, P., Brunton, N., Grady, M. N. O, & Kerry, J. P.
(2011).Phenolic composition and in vitro antioxidant capacity of four
commercial phytochemical products: olive leaf extract (Olea europaea
L.), lutein, sesamol and ellagic acid. Food Chemistry, 126, 948–955.
Kaushal, P., Kumar, V., & Sharma, H. K. (2012). Comparative study of
physicochemical, functional, antinutritional and pasting properties of taro
(Colocasia esculenta), rice (Oryza sativa) flour, pigeonpea (Cajanus
cajan) flour and their blends. LWT – Food Science and Technology, 48,
59-68.
Kumar, K. N., Raja, S. B., Vidhya, N., & Devaraj, S. N. (2012). Ellagic
acid modulates antioxidant status, ornithine decarboxylase expression,
and aberrant crypt foci progression in 1,2- dimethylhydrazine-instigated
97
colon preneoplastic lesions in rats. Journal of Agricultural and Food
Chemistry, 60, 3665−3672.
Leite-Legatti, A. V., Batista, A. G., Dragano, N. R. V., Marques, A. C.,
Malta, L. G., Riccio, M. F., Eberlin, M. N., Machado, A. R. T., Carvalho-
Silva, L. B.L, Ruiz, A. L. T. G., Carvalho, J. E., Pastore, G. M., & Júnior,
M. R. M. (2012). Jaboticaba peel: antioxidant compounds,
antiproliferative and antimutagenic activities.Food Research
International, 49, 596-503.
Lima, A. J. B., Corrêa, A. D., Alves, A. P. C., Abreu, C. P. P., & Dantas-
Barros, A. M. (2008). Caracterização química do fruto jabuticaba
(Myrciaria cauliflora Berg) e de suas frações. Archivos Latinoamericanos
de Nutrición, 58(4), 416-421.
Lima, A. J. B., Corrêa, A. D., Dantas-Barros, A. M., Nelson, D. L., &
Amorim, A.C.L. (2011a). Sugars, organic acids, minerals and lipids in
jabuticaba. Revista Brasileira de Fruticultura, 33(2), 540-550.
98
Lima, A. J. B., Corrêa, A. D., Saczk, A. A., Martins, M. P., & Castilho,
R. O. (2011b). Anthocyanins, pigment stability and antioxidant activity in
jabuticaba [Myrciaria cauliflora (Mart.) O. Berg]. Revista Brasileira de
Fruticultura, 33(3), 877-887.
Naves, L. P., Corrêa, A. D., Abreu, C. M. P., & Santos, C. D. (2010).
Nutrientes e propriedades funcionais em sementes de abóbora (Cucurbita
maxima) submetidas a diferentes processamentos. Ciência e Tecnologia
de Alimentos, 30(1), 185-190.
Njintang, Y. N., Mbofung, C. M. F., Moates, G. K., Parker, M. L., Craig,
F., Smith, A. C., & Waldron, W. K. (2007). Functional properties of five
varieties of taro flour, and relationship to creep recovery and sensory
characteristics of achu (tare based paste). Journal of Food Engineering,
82, 114-120.
Okezie, B. O., & Bello, A. B. (1988). Physicochemical and functional
properties of winged beanflour and isolated compared with soy isolate.
Journal of Food Science, 53 (2), 450-454.
99
Palafox-Carlos, H., Yahia, E. M., & González-Aguilar, G. A.
(2012).Identification and quantification of major phenolic compounds
from mango (Mangifera indica, cv. Ataulfo) fruit by hplc–dad–ms/ms-esi
and their individual contribution to the antioxidant activity during
ripening. Food Chemistry, 135, 105–111.
Pastor-Cavada, E., Juan R. Pastor, J. E., Alaiz, M., & Vioque, J.
(2010).Protein isolates from two mediterranean legumes: Lathyrus
clymenum and Lathyrus annuus.chemical composition, functional
properties and protein characterization. Food Chemistry, 122, 533-538.
Porgali, E., & Büyüktuncel, E. (2012).Determination of phenolic
composition and antioxidant capacity of native red wines by high
performance liquid chromatography and spectrophotometric
methods.Food Research International, 45, 145–154.
Re, R., Pellegrini N., Proteggente A., Pannala A., Yang M., & Rice-Evans
C. (1999). Antioxidant activity applying an improved ABTS radical
100
cation decolorization assay. Free Radical Biology and Medicine, 26,
1231-1237.
Rufino, M. S. M., Alves, R. E., Fernandes, F. A. N., & Brito, E. S.
(2011).Free radical scavenging behavior of ten exotic tropical fruits
extracts. Food Research International, 44, 2072–2075.
Santos, D. T., Veggi, P. C., & Meireles, M. A. A. (2010).Extraction of
antioxidant compounds from jabuticaba (Myrciaria cauliflora) skins:
yield, composition and economical evaluation. Journal of Food
Engineering, 101, 23–31.
Wang, S. H., Caballero-Corboba, G. M.; & Sgarbieri, V. C.
(1992).Propriedades funcionais de misturas de farinhas de trigo e soja-
desengordurada, pré-tratada em microondas. Ciência e Tecnologia de
Alimentos, 12(1), 14-25.
101
Zanotto, D. L., & Bellaver, C. (1996). Método de determinação da
granulometria de ingredientes para uso em rações de suínos e aves.
Comunicado Técnico: Embrapa.
Zhang, J., Zheng-Wu Wang, Z-W., Mi, Q. (2011). Phenolic compounds
from Canna edulis Ker residue and their antioxidant activity. LWT - Food
Science and Technology 44, 2091-2096.
102
ARTIGO 3
Publicado pela revista African Journal of Biotechnology
Formato conforme normas da revista
Jabuticaba skins decrease lipid peroxidation and have hepatoprotective and
antihyperlipidemic effects
LAGE Fabíola Fonseca1*, SIMÃO, Anderson Assaid1, GUEDESMayara
Neves Santos1, RAMOS Vinicius de Oliveira1, SOUSA Raimundo Vicente
de2, CORRÊA Angelita Duarte1*
1Chemistry Department, Biochemistry Laboratory, Universidade Federal de
Lavras – UFLA, PO Box 3037, Zip Code 37200.000, Lavras, MG, Brazil.
2Veterinary Medicine Laboratory, Universidade Federal de Lavras – UFLA, Zip
Code 37200.000, Lavras, MG, Brazil
_______________________________
*Corresponding author: Telephone number: +55 (35) 38291272; Fax: +55 (35)
38291271 Email:[email protected] (F.F. Lage) and [email protected]
(A.D. Corrêa).
103
Jabuticaba skins decrease lipid peroxidation and have
hepatoprotective and antihyperlipidemic effects
ABSTRACT
The effect of Jabuticaba [Plinia jaboticaba (Vell.) Berg] Skin Flour (JSF)
was studied on peroxidation, plasma and hepatic lipid profiles of female
rats, as well as quantification and characterization of phenolic
compounds. The animals were divided into four groups of eight rats. The
groups received 0 (control); 0.5; 1.5 and 3.0 g JSF per 100 g diet. The diet
with 3.0% JSF increased the HDL level by 20.23% compared to the
control. The groups that received JSF had lower AST and ALT activities,
when compared to the control group. There was a decrease of macro
vesicular steatosis in the liver of animals fed the diet supplemented with
3.0% JSF. The diets containing 1.5% and 3.0% JSF reduced lipid
peroxidation in the liver by about 50%. JSF was effective in protecting
against dyslipidemia, because it increased the serum level of HDL
cholesterol, showed a good antioxidant activity and demonstrated
hepatoprotective effect.
104
Keywords: Plinia jaboticaba, phenolic compounds, HDL cholesterol,
antioxidant action, HPLC.
Abbreviations: ABTS - 2,2′-azinobis-(3-etilbenzotiazolin-6-sulphonic) acid ADC - average daily consumption ADG - average daily weight gain ALT - alanine aminotransferase AOAC - Association of Official Analytical Chemists AST - aspartate aminotransferase DM – dry matter FER - feed efficiency ratio GGT - gamma glutamyl transferase HDLc - high-density lipoprotein cholesterol HPLC - High-performance liquid chromatography JSF - jabuticaba skin flour LDLc - low-density lipoprotein cholesterol MDA - malondialdehyde acid NFE – Nitrogen-free extract TBARS - Thiobarbituric acid reactive substances
INTRODUCTION
Jabuticaba [Plinia jaboticaba (Vell.) Berg] is a typical Brazilian fruit
that features pleasant sensory characteristics, with its soft, juicy and
bittersweet pulp (Danner et al., 2006). When consumed fresh, the skin is
discarded as waste. The fruit has high water content and sugars, which
makes it highly perishable, with a short life after harvest. Aiming to
minimize significant economic losses, several studies have been
105
conducted for a better use of the fruits (Alves et al., 2013; Asquieri et al.,
2004). Jabuticaba skin contains bioactive compounds with potential to
promote health benefits. Research has shown that they have an
antiproliferative effect against leukemia and prostate cancer cells (Leite-
Legatti et al., 2012).
Jabuticaba Skin Flour (JSF) is rich in soluble and insoluble fiber
[27.03 and 6.77 g 100 g-1 dry matter (DM), respectively] and has a high
content of total phenolic compounds (11.99 g 100 g-1 DM), including
anthocyanins (2.06 g 100 g-1 DM), responsible for its characteristic color
(Lima et al., 2008, 2011b). High-performance liquid chromatography
(HPLC) analyses detected the anthocyanins cyanidin-3-glucoside and
delphinidin-3-glucoside in JSF (Leite-Legatti et al., 2012). It presents
antioxidant potential, probably due to the high content of phenolic
compounds (Lima et al., 2008, 2011a). The consumption of fiber and
phenolic compounds can beneficially affect the population health, and
are known to be important in the prevention and treatment of diseases.
Afonso et al. (2013) suggested that phenolic compounds attenuate
oxidative stress and reduce cholesterol levels in the blood of rats. Food
that is rich in phenolic compounds, such as green tea and blueberry,
106
show an inhibitory effect on hepatic steatosis (Park et al., 2011; Liu et
al., 2011). The occurrence of non-alcoholic hepatic steatosis has been
associated with the excess accumulation of lipids in the liver, liver injury
and dyslipidemia. Hepatic steatosis causes elevations in serum
aminotransferases, and the analysis of these enzymes is one of the forms
to diagnose its occurrence. Diets containing antioxidants have been used
for the prevention and as a strategy to limit the accumulation of lipids
and liver damage (Park et al., 2011; Bruno et al., 2008).
Jabuticaba skin have chemical characteristics that demonstrate
their potential as functional and/or nutraceutical food; however, studies
on possible applications in health promotion are scarce. In this context,
the objective of this study was to analyze the effect of JSF on
peroxidation, plasma and hepatic lipid profiles of female rats, and
quantification and characterization of its phenolic compounds.
MATERIAL AND METHODS
Preparation of the jabuticaba skin flour (JSF)
P. jaboticaba (Vell.) Berg fruits, Sabará genotype, were hand-picked on
São José do Ismeril Farm, in the municipality of Coqueiral, MG, Brazil,
107
transported to the laboratory, where they were selected, washed in tap
water, sanitized with sodium hypochlorite solution (200 mg kg-1), by a 10
min immersion; they were then squeezed and the skins were weighed and
separated into three lots of approximately 2.9 kg. The jabuticaba skins
were dried in a food dehydrator, in mesh metallic material baskets, at a
temperature of 45°C, with a 1 m s-1 air flow over a period of 36 h. The
skins were then ground and the resulting JSF was packaged in
hermetically sealed flasks in three replicates, wrapped in aluminum foil,
stored at room temperature and subjected to analysis. This JSF was
classified as fine grain.
Proximate composition
The proximate composition (moisture, ether extract, crude protein (N X
6.25), ash, dietary fiber and nitrogen-free extract) was performed, based
on the methodology described by the Association of Official Analytical
Chemists (AOAC, 2005).
108
Chromatographic study of phenolic compounds
The extraction of phenolic compounds was performed using 50%
methanol in the ratio 1:25 (w/v). Chromatographic analyses were
performed using an Agilent HPLC equipment model 1100, and the best
response was obtained at a wavelength of 280 nm. The extract of
phenolic compounds and the standards were injected, in three replicates,
into an Ascentis C18 column (250 mm × 4.6 mm × 5 µm), attached to a
Supelguard Ascentis C18 pre-column (20 mm × 4.0 mm × 5 µm). The
mobile phase was composed of the solutions: 2% acetic acid (A) and
methanol:water:acetic acid (70:28:2 v/v/v) (B). The flux used in all
analyses was 1.00 ml min-1; the injection volume was 20 µL. Analyses
were performed in a total time of 65 min at 15°C in a gradient-type
system: 100% solvent A for 5 min, 70% solvent A for 20 min, 60%
solvent A for 18 min, 55% solvent A for 7 min, 0% solvent A for 10 min.
Until the end of the run, solvent A was increased to 100%, in order to
balance the column. Addition of standards to the extracts was also used
as an identification parameter. Quantitation was performed using external
standardization with concentrations of standard stock solutions: Gallic, p-
coumaric, ferulic, ellagic, 3,4-dihydroxybenzoic, syringic and salicylic
109
acids, as well as the gallocatechin, catechin, epigallocatechin gallate and
resveratrol (Sigma-Aldrich - St. Louis, MO, USA). Vanillic acid and m-
and o-cumaric acids (Fluka - St. Louis, MO, USA). Stock standard
solutions were prepared in dimethylsulfoxide and/or methanol (Merck).
Each solution was injected three times on the HPLC system, with the
purpose of obtaining concentration means and retention times.
Animals and treatments
All procedures were performed in accordance with the ethical
principles in animal experimentation, adopted by the Ethics Committee
on Animal Use of the Universidade Federal de Lavras (Protocol 009/11,
approved on 09/01/2011).
Thirty two female Fischer rats were used, with a body weight of
approximately 140 g, divided into four groups with eight animals in each
group. The animals were kept in individual cages, in a room with a
temperature of 25 ± 3°C (light/dark cycle of 12 h) with access to water
and feed ad libitum for a period of 28 days. The experimental diets were
prepared according to AIN-93G (Reeves et al., 1993) modified by the
addition of crystalline cholesterol (0.5 g 100 g-1 diet) and sodium cholate
110
(0.25 g 100 g-1 diet). The four groups were divided according to the
amount of JSF added to the diet: 0 (control); 0.5, 1.5 and 3.0 g 100 g-1
diet (Table 1).
Feed consumption and animal weight were monitored weekly, in
order to calculate the average daily consumption (ADC), the average
daily weight gain (ADG) and the feed efficiency ratio (FER). At the end
of the experiment, the animals were fasted for about 12 h, and then were
anesthetized with thiopental sodium, intraperitoneally. Blood was
removed from the heart and then centrifuged at 2,500 x g for 5 min for the
collection of the plasma, which was stored at - 20°C. The liver was
removed by median laparotomy, washed with 0.9% saline solution,
weighed and stored at -25°C for further analyses.
111
Table 1 Composition of the experimental diets
Diets (g 100 g-1 diet)
Ingredients Control (0) 0.5 1.5 3.0
Starch 40 40 40 40
Casein 20 20 20 20
Sucrose 10 10 10 10
Oil 10 10 10 10
Cellulose 5 5 5 5
Mineral mixture 3.5 3.5 3.5 3.5
Vitamin mixture 1 1 1 1
Methionine 0.5 0.5 0.5 0.5
Cholesterol 1 1 1 1
Sodium cholate 0.25 0.25 0.25 0.25
JSF1 0 0.5 1.5 3.0
Kaolin 8.75 8.25 7.25 5.75
Caloric value (cal g-1) 3,022.32 2,975.31 2,912.32 3,019.97 1JSF – Jabuticaba skin flour.
Blood analyses
The analyses were performed with blood plasma. For all tests, Lab
Test kits were used. Analyses of total cholesterol and of the HDL-c
fraction were performed, as well as triacylglycerols, cholesterol in VLDL
+ LDL fractions, activities of aspartate aminotransferase (AST), alanine
aminotransferase (ALT) and gamma glutamyl transferase (GGT) were
also determined.
112
Analyses in the liver
Moisture, lipid and total cholesterol
The livers were lyophilized until constant weight and finely
ground. Moisture and lipid content were determined using the methods
proposed by AOAC (2005). The extraction of cholesterol was carried out
with isopropanol (Haug and Hostmark, 1987) and the dosage was
performed in the same way for the blood analyses.
Thiobarbituric acid reactive substances
The peroxidation of lipids isolated from the liver of animals was
determined by the formation of thiobarbituric acid reactive substances
(TBARS), according to Winterbourn et al. (1981). The pigment produced
by the colorimetric reaction was read in a spectrophotometer at 535 nm.
The TBARS concentration was calculated from the standard curve of
1,1,3,3 tetraethoxypropane. The results were expressed as nmoles of
malondialdehyde acid (MDA) g-1 protein.
Histopathological analysis
A liver fragment from each animal was fixed in 10% formalin.
The fragments were soaked in paraffin, sectioned (5 µm) and stained with
113
hematoxylin and eosin (HE method). The slides were evaluated under a
microscope and identified for the presence of hepatic steatosis,
considering mild (+), moderate (+ +) or severe (+ + +) lesion.
Statistical analysis
The experimental design was completely randomized with four
treatments, which were the control group (0%) and the groups containing
0.5, 1.5 and 3.0% JSF, with eight replicates, and each animal represented
an experimental plot. For the analyses of ADC, ADG and FER, split plots
in time were used. The software Sisvar (Ferreira, 2003) was used to
perform the analysis of variance and, when significant, the regression
analysis was performed, with p ≤ 0.05.
RESULTS AND DISCUSSION
The proximate composition of JSF is presented in Table 2. The
lipid content was low, but higher than that found by Leite et al. (2001)
and Lima et al. (Lima 2011b) in lyophilized jabuticaba skin samples (1.27
and 1.16 g 100 g-1 DM, respectively). Lima et al. (2011a) reported
114
contents of crude protein and ash (1.16 and 4.40 g 100 g-1 DM,
respectively) for Sabará skins, lower than those found in this study. On
the other hand, the contents of soluble (7.73 g 100 g-1 DM) and insoluble
fiber (35.13 g 100 g-1 DM) were higher than those reported by Lima et al.
(2011a) (6.80 g 100 g-1 DM for soluble fiber and 26.43 g 100 g-1 DM for
insoluble fiber). The component with the highest content was dietary
fiber. These differences are probably inherent in harvest, among other
factors.
Table 2 Proximate composition1 of the jabuticaba skin flour, in g 100 g-1
Constituents Contents Moisture 21.69 ± 0.20 Lipids 1.59 ± 0.41 Crude protein (N x 6,25) 5.53 ± 0.18 Ash 5.46 ± 0.57 Insoluble fiber Soluble fiber Total dietary fiber NFE2
27.51 ± 1.08 6.05 ± 0.78 33.56 ± 1.45 32.17 ± 1.30
1Data are the mean of triplicate ± standard deviation. 2NFE – Nitrogen-free
extract
These differences are probably inherent in harvest, among other
factors. In the chromatographic analyses of the JSF extract, phenolic acids
and flavonoids were identified, presenting the following quantitative
order: epicatechin ˃ salicylic acid ˃ ellagic acid ˃ gallic acid ˃
115
gallocatechin (Figure 1) and the total content of phenolic compounds was
2.68 g 100 g-1 DM (Table 3).
0 10 20 30 40 50 60-5
0
5
10
15
20
25
30
Inte
nsity
(m
Vol
ts)
Time (min)
1
2
3
4
5
Figure 1.Chromatogram of the JSF extract Peak identification: 1- gallic acid; 2- gallocatechin; 3 – epicatechin; 4- ellagic acid; 5- salicylic acid. Table 3.Phenolics average in jabuticaba skin flour, by HPLC
Phenolic compounds Phenolic content (g 100 g-1 DM) Gallic acid 0.11 Gallocatechin 0.01 Epicatechin 1.9 Ellagic acid 0.12 Salicylic acid 0.54 Total 2.68
The anthocyanins cyanidin-3-glucoside and delphinidin-3-
glucoside were identified in Sabará JSF by HPLC (Leite-Legatti et al.,
116
2012). JSF has proven antioxidant action (Leite-Legatti et al., 2012),
probably due to the high content of phenolic compounds. In Table 4, the
analyses of ADC, ADG and FER are shown
The analyses of variance for these variables using the split-plot
scheme in time showed significant difference at 1% by f test, just for the
time. Lenquiste et al. (2012) evaluated the effect of lyophilized jabuticaba
skin on rats, in the proportions 0, 1, 2 and 4% added to the diets rich in
fat, and did not observe significant statistical differences in the average
daily consumption (ADC) and average daily weight gain (ADG) of the
animals. These results suggest that the addition of JSF did not affect the
palatability of the diets.
Table 4.Averagedaily consumption (ADC), average daily weight gain (ADG) and feed efficiency ratio (FER) of animal during the experimental phase
Diets Control
(0%JSF1) 0.5% JSF 1.5% JSF 3.0% JSF
ADC (g) 17.35 17.11 18.55 17.94 ADG (g) 1.43 1.15 1.46 1.43 FER 0.09 0.07 0.08 0.09
1JSF – Jabuticaba skin flour.
The analyses of total cholesterol, triacylglycerols and GGT activity
carried in the blood of the animals showed no significant difference and
averages are shown in Table 5.
117
Table 5 Total cholesterol, triacylglycerols (TAG) and gama glutamyl transferase (GGT) of animal during the experimental phase
Diets Control
(0% JSF1) 0.5% JSF 1.5% JSF 3.0% JSF
Total cholesterol 193.14 165.99 147.56 166.37 TAG 29.72 25.3 26.73 26.10 GGT 4.87 4.46 3.72 3.82 1JSF – Jabuticaba skin flour.
The analyses of total cholesterol, triacylglycerols and GGT
activity carried in the blood of the animals showed no significant
difference and averages are shown in Table 5. The diet supplemented
with 3.0% JSF had the highest increase in the level of HDL-c, compared
to the control (Figure 2), that was, 20.23%. In a research conducted by
Lenquiste et al. (2012), using lyophilized jabuticaba skin, the authors also
reported that there was no significant difference for the levels of total
cholesterol and triacylglycerols between the diets supplemented with
jabuticaba skins and the control and that there were statistical differences
for the level of HDL-c. The diets supplemented with 2 and 4% jabuticaba
skins showed the lowest values of HDL-c.
118
Figure 2.Analyses in the blood (A) and liver (B) of animals, after four weeks with the experimental diets (p ≤ 0.05).
Phenolic compounds, which are antioxidants, may be responsible
for the increase in HDL-c. The accumulation of cholesterol in
erythrocytes, leukocytes, platelets and endothelial cells can lead to a
reduction in the antioxidant defense systems and cause an increase in the
concentration of reactive species (Afonso et al., 2013). Flavonoids act to
inactivate free radicals in hydrophilic and lipophilic cellular
119
compartments and have the ability to donate hydrogen atoms, inhibiting
chain reactions caused by free radicals (Degáspari and Waszczynscyj,
2004). The flavonoids identified in JSF may have acted as lipophilic
antioxidants, attenuating the oxidative stress associated with
cardiovascular diseases, which may have increased HDL levels.
One possible mechanism proposed for the reduction of plasma
cholesterol levels is the formation of insoluble complexes with bile acids,
increasing their fecal excretion, therefore there is no reabsorption of bile
acids; cholesterol is then used to synthesize new bile acids, thus
decreasing the level of cholesterol (Mäkynena et al., 2013). Studies
indicate that phenolic compounds can induce an increase in the fecal
excretion of bile acids (Lee et al., 2010). The high content of these
compounds in JSF may have increased the fecal excretion of sterols, bile
acids and non-fecal cholesterol, contributing to the occurrence of the
antihyperlipidemic action of the flour. Although, not significant, a
decrease in total cholesterol was observed in the animals. Analyses of the
enzymes AST and ALT are used to identify changes in the function of the
liver and of the biliary tract, and the enzyme GGT identifies biliary
lesions. There was no statistical difference between the control group and
120
the treatments in relation to the enzyme GGT (Table 5). It was observed
in Figure 2 that the groups that received JSF showed activities of AST
and ALT significantly lower than the control group (p ≤ 0.05). JSF is
probably acting as a hepatoprotective, since all groups received 1.0 g
cholesterol, therefore with an accumulation of fat in the liver. The results
suggest that this accumulation of fat was less harmful to the animals that
received the treatment with JSF.
Total flavonoids extracted from the fruit Rosa laevigata Michx
showed a significant hepatoprotective effect on mice that suffered liver
damage, caused by the ingestion of paracetamol. The results were based
on the determination of liver enzymes and histopathological tests (Liu et
al., 2001). The authors suggest that this effect is due to the antioxidant
potential of flavonoids. The decrease in the levels of AST and ALT,
caused by the addition of JSF in the diets, may be due to its high content
of phenolic compounds. Confirming these results, the histopathological
study revealed a significant decrease in macrovesicular steatosis in the
liver of the animals fed the diet containing 3.0% JSF (Figure 3).
121
Figure 3.Distribution of macrovesicular steatosis observed in the histopathological analysis of the liver of the animals after four weeks of experiment. JSF – Jabuticaba skin flour.
The increase in cholesterol intake has been associated with lipid
peroxidation processes. Analysis of the lipid peroxidation index in the
plasma of hamsters treated with hypercholesterolemic diets show an
increase in the occurrence of thiobarbituric acid reactive substances
(TBARS) (Sánchez-Muniz, 2012). Diets containing 1.5 and 3.0% JSF
reduced the production of TBARS by about 50% in the liver of animals
with an average weight of 140 g, indicating that JSF conferred protection
against oxidative attack (Figure 2). Lenquiste et al. (2012) analyzed the
antioxidant potential, by the 2,2′-azinobis-(3-etilbenzotiazolin-6-
122
sulphonic) acid (ABTS) method, of the plasma of rats treated with a diet
containing 0, 1, 2 and 4% lyophilized jabuticaba skin, and observed that
there was an increase in the levels of ABTS with the diet with up to 2%
jabuticaba skin for animals with an average weight of 250 g, also
protecting against oxidative attack. The colonic microbiota causes the
fermentation of phenolic compounds that occur in matrices rich in fibers,
and releases absorbable compounds (Liu et al., 2001).
Regarding flavonoids, colonic bacteria share the heterocyclic ring
and degrade flavonoids into phenyl acids that can be absorbed. After
absorption, they are conjugated in the liver by glucuronidation, sulfation,
methylation, or are metabolized into smaller phenolic compounds. They
are able to inhibit cell proliferation and oxidative stress, as well as induce
enzyme detoxification, apoptosis and activate the immune system (Guida-
Cardoso et al., 2004). The phenolic acids and flavonoids present in JSF,
after being metabolized and deposited in the liver of the animals, may be
acting to inhibit oxidative stress and cause body detoxification. There was
no significant difference for liver moisture, and the average moisture
content was 52.79 g 100 g-1. There was a reduction in the level of
cholesterol and lipids in the liver of the animals, compared to the control.
123
The high content of fiber and phenolic compounds in JSF may be
responsible for these results. Hepatic cholesterol is the result of the
balance between the cholesterol acquired through food, the cholesterol
synthesized by the body and the cholesterol eliminated by the liver.
Cholesterol has a ring structure, which the human body is unable to
metabolize into CO2 and H2O, and is then eliminated by the liver as
unchanged cholesterol into the bile, whichtransports it to the intestine for
elimination, either as a component of plasma lipoproteins, or as bile salts,
which are excreted in the feaces (Pérez-Jimenéz, 2009). The animals fed
diets supplemented with 3.0% JSF showed a decrease in the cholesterol
level of 37.39% in the liver, when compared to the animals fed the control
diet (Figure 2). The phenolic compounds in JSF may be acting to cause
the decrease in cholesterol synthesis.
CONCLUSION
The JSF was effective in the protection against dyslipidemia, because
it increases the serum level of cholesterol in HDL. The JSF has a high
content of phenolic compounds and these components may be responsible
for the occurrence of the hepatoprotective effect observed. Furthermore,
124
the JSF has an antioxidant activity, since it protected the liver against
lipoperoxidation. The phenolic acids and flavonoids identified
epicatechin, salicylic acid, ellagic acid, gallic acid and gallocatechin,
probably are responsible for this antioxidant activity.
CONFLICT OF INTERESTS
The author(s) have not declared any conflict of interests.
ACKNOWLEDGMENTS
The authors thank the development agencies CAPES, FAPEMIG, and
CNPq for financial support for the completion of this study.
REFERENCES
Afonso MA, Silva AM, Carvalho EBT, Rivelli DP, Barros ABM, Rogero
MM, Lottenberg AM, Torres RP, Mancini-Filho J (2013). Phenolic
compounds from Rosemary (Rosmarinus officinalis L.) attenuate
oxidative stress and reduce blood cholesterol concentrations in diet-
induced hypercholesterolemic rats. Nutr. Metab. 10: 19-28.
125
Alves APC, Corrêa AD, Pinheiro ACM, Oliveira FC (2013). Flour
andanthocyanin extracts of jaboticaba skins used as a natural dye in
yogurt. Int. J. Food Sci. Technol. 48: 2007-2013.
Asquieri ER, Candido MA, Damiani C, Assis EM (2004). Fabricación de
vinoblanco y tinto de jabuticaba (Myrciaria jaboticaba Berg) utilizando
la pulpa y la cáscara respectivamente. Alimentaria 355: 97-109.
Association of Official Analytical Chemistry (2005). Official Methods of
Analysis of the association of the analytical chemists.17th Ed.
Washington.
Bruno RS, Dugan CE, Smyth JA, DiNatale DA, Koo SI (2008). Green tea
extract protects leptin-leficient, spontaneously obese mice from hepatic
steatosis and injury. J. Nutr. 138: 323-331.
Danner MA, Citadin I, Fernandes-Jr AA, Assmann AP, Mazaro SM,
Donazzolo J, Sasso SAZ (2006). Enraizamento de jabuticabeira (Plinia
trunciflora) por mergulhia aérea. Rev. Bras. Frutic. 28: 530-5321.
Degáspari CH, Waszczynscyj N (2004). Propriedades antioxidantes de
compostos fenólicos. Visão Acadêmica 5: 33-40.
126
Guida-Cardoso SM, Pinto WJ, Ogo SH, Reyes FGR, Areas MA (2004).
Dietary fiber reduces lipid peroxidation and mean arterial blood pressure
in hypercholesterolemic hamsters. Alimentaria 359: 31-34.
Haug A, Hostmark AT (1987). Lipoprotein lipases, lipoproteins and
tissue lipids in rats fed fish oil or coconut oil. J. Nutr. 117: 1011-1017.
Ferreira DF (2003). SISVAR: versão 4.6 (build 61) software. Lavras:
UFLA. Disponível em: <htpp://www.dex.ufla.br/danielff/dff02.htm>
Acesso em: 27 setembro 2012.
Lee J-S, Bok S-H, Jeon S-M, Kim H-J, Do K-M, Park Y-B, Choi M-S
(2010). Antihyperlipidemic effects of buckwheat leaf and flower in rats
fed a high-fat diet. Food Chem. 119: 235-240.
Leite AV, Malta LG, Riccio MF, Eberlin MN, Pastore GM, Júnior MRM
(2011).Antioxidant potential of rat plasma by administration of freeze-
dried jaboticaba peel (Myrciaria jaboticabaVell Berg). J. Agric. Food
Chem. 59: 2277-2283.
Leite-Legatti AV, Batista AG, Dragano NRV, Marques AC, Malta LG,
Riccio MF, Eberlin MN, Machado ART, Carvalho-Silva LBL, Ruiz
ALTG, Carvalho JE, Pastore G M, Júnior MRM (2012). Jaboticaba peel:
127
antioxidant compounds, antiproliferative and antimutagenic activities.
Food Res. Int. 49: 596-503.
Lenquiste SA, Batista AG, Marineli RS, Dragano NRV, Maróstica Jr. MR
(2012). Freeze-dried jaboticaba peel added to high-fat diet increases
HDL-cholesterol and improves insulin resistance in obese rats. Food Res.
Int. 49:153–160.
Lima AJB, Corrêa AD, Alves APC, Abreu CPP, Dantas-Barros AM
(2008). Caracterização química do fruto jabuticaba (Myrciaria cauliflora
Berg) e de suas frações. ALAN. 58: 416-421.
Lima AJB, Corrêa AD, Dantas-Barros AM, Nelson DL, Amorim ACL
(2011a).Sugars, organic acids, minerals and lipids in jabuticaba.Rev.
Bras. Frutic. 33:540-550.
Lima AJB, Corrêa AD, Saczk AA, Martins MP, Castilho RO (2011b).
Anthocyanins, pigment stability and antioxidant activity in jabuticaba
[Myrciaria cauliflora (Mart.) O. Berg]. Rev. Bras. Frutic. 33: 877-887.
Liu YT, Lu BN, Peng JY (2001). Hepatoprotective activity of the total
flavonoids from Rosa laevigataMichx fruit in mice treated by
paracetamol. Food Chem. 125: 719-725.
128
Liu Y, Wang D, Zhang D, Lv Y, Wei Y, Wu W, Zhou F, Tang M, Mao T,
Li M, Ji B (2011). Inhibitory effect of blueberry polyphenolic compounds
on oleic acid-induced hepatic steatosis in vitro. J. Agric. Food Chem. 59:
1254-1263.
Mäkynena K, Jitsaardkul S, Tachasamranc P, Sakai N, Puranachoti S,
Nirojsinlapachai N, Chattapat V, Caengprasatha N, Ngamukotea S,
Adisakwattanaa S (2013). Cultivar variations in antioxidant and
antihyperlipidemic properties of pomelo pulp (Citrus grandis [L.]
Osbeck) in Thailand. Food Chem. 139: 735-743.
Park HJ, DiNatale DA, Chung M-Y, Park Y-K, Lee J-Y, Koo S-I,
O´Connor M., Manautou JE, Bruno RS (2011). Green tea extract
attenuates hepatic steatosis by decreasing adipose lipogenesis and
enhancing hepatic antioxidant defenses in ob/ob mice. J. Nutr. Biochem.
22: 393-400.
Pérez-Jiménez J, Serrano J, Tabernero M, Arranz S, Díaz-Rubio ME,
García-Diz L, Goñi I, Saura-Calixto F (2009). Bioavailability of phenolic
antioxidants associated with dietary fiber: plasma antioxidant capacity
after acute and long-term intake in humans. Plant Foods Hum.Nutr. 64:
102–107.
129
Reeves PG, Forrest HN, Fahey-Jr GC (1993). AIN 93 Purified diets for
laboratory rodents: final report of the American Institute of Nutrition Ad
Hoc Writing Committee on the Reformulation of the AIN-76 A Rodent
Diet. J. Nutr. 123: 1939-1951.
Sánchez-Muniz FJ (2012). Dietary fibre and cardiovascular health.Nutr.
Hosp. 27:31-45.
Winterbourn CC, Gutteridge JM, Halliwel B (1981). Doxorubicin-
dependet lipid peroxidation at low partial pressures of O2. Free Radical
Biol. Med. 2: 1119-1122.
130
APÊNDICES
APÊNDICE A– ARTIGO 2 Tabela 1A Parâmetrosdas curvas de calibraçãoconstruídas para a
quantificação dos compostos fenólicos presentes nos
extratos de casca de jabuticaba.........................................
130
Figura 1A Cromatograma da solução de ácidos fenólicos com detecção
espectrofotométrica em 280 nm........................................
131
Gráfico 1A Curva analítica obtida a partir das soluções de ácido gálico 131
Gráfico 2A Curva analítica obtida a partir das soluções de
galocatequina......................................................................
132
Gráfico 3A Curva analítica obtida a partir das soluções de epicatequina 132
Gráfico 4A Curva analítica obtida a partir das soluções de ácido elágico 133
Gráfico 5A Curva analítica obtida a partir das soluções de ácido
salicílico............................................................................
134
Tabela 1A Parâmetros das curvas de calibração construídas para a quantificação dos compostos fenólicos presentes nos extratos de FCJ.
Compostos fenólicos Equação da regressão
linear1 Linearidade
(mg L-1) R2 LD
(mg L-1)2 LQ
(mg L-1)3 tR
(min) Ácido gálico y= 63,44x -80,22 0,34-42,53 0,998 2,7 8,18 7,51
Galocatequina y= 8732,46x – 2388,24 1,53-39,47 0,999 0,8 2,3 8,65
Epicatequina y=20,02x – 109,81 0,46-26,22 0,964 2,68 8,13 20,37
Ácido elágico y = 23,13x – 61,73 0,48–25,98 0,995 0,27 0,54 51,63
Ácido salicílico y=7,06x - 36,95 7,28-118,78 0,997 9,68 29,32 53,49 1 y: concentração, mg L-1; x: área do pico. 2Limite de detecção. 3Limite de quantificação.
130
131
Figura 1A Cromatograma da solução de ácidos fenólicos com detecção
espectrofotométrica em 280 nm. Identificação dos picos: 1 = ácido gálico; 2 = Galocatequina; 3 = 3,4 dihidroxifenólico 4 = Catequina; 5 = ácido clorogênico; 6 = epigalocatequina; 7 = ác. vanilico; 8 = epicatequina; 9 = ácido siríngico; 10 = ácido p-cumárico; 11 = ácido ferúlico; 12 = ácido m-cumárico; 13 = ácido o-cumárico; 14 = resveratrol; 15 = ácido elágico e 16 = ácido salicílico
Gráfico 1A Curva analítica obtida a partir das soluções de ácido gálico Equação da reta: y= 63,44x -80,224 Coeficiente de correlação linear = 0,998
132
0 2 0 4 0 6 0 8 0 1 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
Áre
a (
x103 )
/u.a
.
C o n c e n t r a ç ã o /m g L-1
Gráfico 2A Curva analítica obtida a partir das soluções de galocatequina Equação da reta: y = 8732,46x- 2388,24 Coeficiente de correlação linear = 0,999
Gráfico 3A Curva analítica obtida a partir das soluções de epicatequina Equação da reta: y = -20,02x - 109,8 Coeficiente de correlação linear = 0,964
133
Gráfico 4A Curva analítica obtida a partir das soluções de ácido elágico Equação da reta: y = 23, 13x – 61,74 Coeficiente de correlação linear = 0,995
134
Gráfico 5A Curva analítica obtida a partir das soluções de ácido salicílico Equação da reta: y = 7,07x – 36,95 Coeficiente de correlação linear = 0,997
135
APÊNDICE B – ARTIGO 3
Tabela 1B Resumo da análise de variância do consumo diário, ganho
de peso diário e coeficiente de eficiência alimentar de ratas
Fisher submetidas aos tratamentos, durante quatro semanas
136
Tabela 2B Resumo da análise de variância das análises de colesterol
total, fração HDL-c, triacilglicerois, atividade de aspartato
aminotransferase (AST) e alanina aminotransferas (ALT) e
gama gt do sangue de ratas Fisher submetidas ao
tratamento durante quatro semanas..................................
137
Tabela 3B Resumo da análise de variância das análises de umidade,
teor de lipídeos, colesterol total e substâncias reativas ao
ácido tiobarbitúrico (TBARS) do fígado de ratas Fisher
submetidas ao tratamento durante quatro semanas ...............
138
136
Tabela 1B Resumo da análise de variância do consumo diário, ganho de peso
diário e coeficiente de eficiência alimentar de ratas Fisher submetidas aos tratamentos1, durante quatro semanas.
FV GL QM CV(%)
Consumo Diário
Tratamentos 3 16,44
Resíduo1 28 11,46
Tempo 4 3268,88*
Tratamentos*Tempo 12 3,40
Resíduo 2 112 3,55
CV1 = 19,09 CV2 = 10,63
Ganho de peso Tratamentos 3 2,50
Resíduo1 28 16,00
Tempo 4 179,78*
Tratamentos*Tempo 12 9,94
Resíduo 2 112 137,18
CV1 = 55,19
CV2 = 80,78
Coeficiente de eficiência alimentar Tratamentos 3 0,004
Resíduo1 28 0,004
Tempo 4 0,073*
Tratamentos*Tempo 12 0,005
Resíduo 2 112 0,004
CV1 = 80,55
CV2 = 80,98
* Teste de regressão significativo a 1% de probabilidade. 1Controle – dieta hipercolesterolêmica; FCJ 0,5% - dieta hipercolesterolêmica contendo 0,5% de FCJ; FCJ 1,5% - dieta hipercolesterolêmica contendo 1,5% de FCJ e FCJ 3,0% - dieta hipercolesterolêmica contendo 3,0% de FCJ.
137
Tabela 2B Resumo da análise de variância das análises de colesterol total, fração HDL-c, triacilglicerois, atividade de aspartato aminotransferase (AST) e alanina aminotransferas (ALT) e gama gt do sangue de ratas Fisher submetidas ao tratamento durante quatro semanas.
Parâmetro FV GL QM CV (%)
Tratamentos 3 2816,67 22,99 Colesterol total
Resíduo 28 1497,04
Tratamentos 3 1340,27* 15,18 Fração HDL-c
Resíduo 28 273,35
Tratamentos 3 29,73 14,18 Triacilglicerois
Resíduo 28 14,61
Tratamentos 3 11953,64* 15,26 AST
Resíduo 28 223,96
Tratamentos 3 270,74* 9,19 ALT
Resíduo 28 6,62
Tratamentos 3 2,39 35,13 Gama GT
Resíduo 28 2,20
* Teste de regressão significativo a 1% de probabilidade. 1Controle – dieta hipercolesterolêmica; FCJ 0,5% - dieta hipercolesterolêmica contendo 0,5% de FCJ; FCJ 1,5% - dieta hipercolesterolêmica contendo 1,5% de FCJ e FCJ 3,0% - dieta hipercolesterolêmica contendo 3,0% de FCJ.
138
Tabela 3B Resumo da análise de variância das análises de umidade, teor de lipídeos, colesterol total e substâncias reativas ao ácido tiobarbitúrico (TBARS) do fígado de ratas Fisher submetidas ao tratamento durante quatro semanas.
Parâmetro FV GL QM CV (%)
Tratamentos 3 7,02 5,30 Umidade
Resíduo 28 7,83
Tratamentos 3 51,80* 5,03 Teor de lipídeos
Resíduo 28 12,42
Tratamentos 3 3520,93* 10,27 Colesterol total
Resíduo 28 60,29
Tratamentos 3 22,59* 20,35 TBARS
Resíduo 28 27,37
* Teste de regressão significativo a 1% de probabilidade. 1Controle – dieta hipercolesterolêmica; FCJ 0,5% - dieta hipercolesterolêmica contendo 0,5% de FCJ; FCJ 1,5% - dieta hipercolesterolêmica contendo 1,5% de FCJ e FCJ 3,0% - dieta hipercolesterolêmica contendo 3,0% de FCJ.
