capacidade antioxidante de bebidas aromatizadas: … · contrebalancé par des mécanismes de...
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Maria de Fátima de Sá Barroso
Mestre em Engenharia do Ambiente
Capacidade antioxidante de bebidas aromatizadas:
águas e chás
junho, 2011
III
Tese de Doutoramento
Capacidade antioxidante de bebidas aromatizadas:
águas e chás
Maria de Fátima de Sá Barroso
Dissertação de candidatura ao grau de Doutor em
Ciências Farmacêuticas – Química Analítica,
apresentada à Faculdade de Farmácia da Universidad e do Porto
Orientação
Professora Doutora Maria Beatriz Prior Pinto Oliveira
Porto
junho, 2011
IV
© Autorizada a reprodução parcial desta dissertação (condicionada à autorização
das editoras das revistas onde os artigos foram pub licados) apenas para efeitos de
investigação, mediante declaração escrita do intere ssado, que a tal se
compromete.
V
A realização deste trabalho foi possível graças à c oncessão de uma Bolsa de
Doutoramento (SFRH/BD/29440/2006) pela Fundação par a a Ciência e a Tecnologia
(FCT), financiada pelo Programa Operacinal Potencia l Humano (POPH) - Quadro de
Referência Estratégico Nacional (QREN) - Tipologia 4.1 - Formação Avançada,
comparticipado pelo Fundo Social Europeu (FSE) e po r Fundos Nacionais do
Ministério da Ciência, Tecnologia e Ensino Superior (MCTES). Em associação à
Bolsa de Doutoramento, a candidata contou ainda com subsídios para a realização
de trabalho no estrangeiro, para deslocamento a con gressos internacionais e para
a execução gráfica desta tese.
VII
Os estudos apresentados nesta dissertação foram re alizados no Laboratório
Grupo de Reacção e Análise Química (GRAQ) do Instit uto Superior de Engenharia
do Porto do Instituto Politécnico do Porto, no Serv iço de Bromatologia da
Faculdade de Farmácia da Universidade do Porto, no Laboratório de Química
Orgânica Física/Química Radicalar do Departamento d e Química da Faculdade de
Ciências e Tecnologia da Universidade Nova de Lisbo a e no Grupo de
Electroanálisis da Faculdade de Química da Universi dade de Oviedo.
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AGRADECIMENTOS
.“…Eu não posso brincar contigo, disse a raposa. Não me cativaram ainda. Ah! desculpa, disse o principezinho. Após uma reflexão, acrescentou:
- Que quer dizer "cativar"? ……
- É uma coisa muito esquecida, disse a raposa. Significa "criar laços. - Criar laços?..”
Antoine de Saint-Exupéry em O Principezinho
Tal como Saint-Exupéry também a autora desta dissertação cativou, e deixou-se
cativar pelas inúmeras pessoas com quem se cruzou a nível profissional e pessoal nestes
4 anos de trabalho. Sem o apoio de muitas pessoas teria sido muito difícil chegar à reta
final deste trabalho e atracar num porto seguro. Por isso ficam os meus agradecimentos à:
Fundação para a Ciência e Tecnologia pela concessão de uma bolsa de doutoramento
(SFRH/BD/29440/2006), sem a qual seria impossível realizar este trabalho.
Ao diretor da Faculdade de Farmácia da Universidade do Porto Professor Doutor José
Luís da Costa, ao ex-diretor Professor Doutor José Manuel Sousa Lobo e à Doutora
Isabel Guimarães pelo apoio prestado nas questões burocáticas associadas ao processo
de doutoramento.
À minha orientadora Professora Doutora Beatriz Oliveira por me ter acolhido como
aluna, pela ajuda prestada no decorrer do trabalho, pelo apoio incondicional e pela
amizade que foi crescendo nestes 4 anos, e também à Professora Doutora Cristina
Delerue-Matos, com quem tenho o previlégio de ter criado laços à mais de 10 anos, pelo
seu apoio incondicional, pelos seus conselhos e por me ajudar a crescer a nível pessoal
e científico.
À Professora Teresa Teles, à Professora Sandra Ramos à Engenheira Aurora Silva e
à Engenheira Elisa Soares pelo apoio prestado nas análises mineralógicas e estudos
estatísticos.
Ao Professor João Paulo Noronha, pela simpatia e entusiasmo com que me recebeu
no seu laboratório, pels constante preocupação e permanente disponibilidade e por todo
o apoio científico prestado que permitiu valorizar este trabalho.
X
À Professora Rosa Fireman Dutra e ao Doutor Joilson Jesus, que me receberam no
outro lado do Atlântico de braços abertos, por estarem sempres disponiveis, pelo apoio e
por todo o ensinamento. Ao Professor Lauro Kubota pelo valioso contributo na análise
dos resultados.
À Professora Noemí de-los-Santos Alvarez, e ao Professor Paulino Tuñón Blanco, por
me terem acolhido num magnífico laboratório, por me terem oferecidas condições ótimas
de trabalho, e por todo apoio científico e aprendizagem adquiridos no decorrer do
trabalho.
Aos meus colegas do laboratório por me ouvirem e me apoiarem em inúmeras
situações, e em especial à Doutora Marta Neves pela simpatia e ajuda aquando na
estadia no estrangeiro.
À minha família, país, irmãos, sobrinhos, marido e filhos, mais do que agradecer a
paciência, apoio e conselhos seguem as minhas desculpas pela ausência e pelo apoio
que não foram prestados.
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RESUMO
A produção contínua de radicais livres, durante os processos metabólicos, é
compensada pelos mecanismos de defesa antioxidante. Estes visam eliminar ou reduzir
os níveis destes radicais nas células e assim proteger o organismo dos seus efeitos
pejorativos.
Os alimentos e as bebidas são uma boa fonte exógena de antioxidantes. As águas
com sabores, sendo constituídas por aromas naturais, extratos de vegetais (chá) e sumos
de fruta, devem apresentar alguma capacidade antioxidante. Este tipo de refrigerante
(águas com sabores) foi desenvolvido recentemente e, por isso, não se encontrava
caraterizado no que concerne aos fatores com efeito benéfico na saúde humana,
nomeadamente a capacidade antioxidante.
O objetivo principal desta dissertação consistiu em melhorar o conhecimento acerca da
composição química e antioxidante das diversas águas com sabores disponíveis no
mercado português.
Numa primeira fase efetuou-se a caraterização mineralógica destas águas, tendo-se
avaliado um total de 18 minerais: 4 macrominerais (Ca, Mg, K e Na), 3 microminerais (Fe,
Cu e Zn) e 11 elementos vestigiais (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se e Si). Os
teores determinados destes minerais estavam dentro dos limites estipulados por lei e
eram variáveis de marca para marca, o que está relacionado com a sua origem.
Seguiu-se a avaliação de parâmetros relacionados com a ação antioxidante, através
de métodos óticos convencionais, nomeadamente, o teor fenólico total, o teor de
flavonoídes total, o poder redutor e a atividade anti-radicalar das águas e dos aromas
usados na sua formulação. Para um melhor conhecimento da composição química dos
aromas foi avaliado o perfil de terpenóides por HS-SPME/GC-MS.
De acordo com os resultados, as amostras de águas e aromas apresentam capacidade
antioxidante e atividade anti-radicar. No entanto, não foram detetados flavonóides. A
análise dos aromas indicou a presença de monoterpenos e sesquiterpenos.
Na sequência do trabalho desenvolveram-se metodologias alternativas para a
quantificação da capacidade antioxidante de águas com sabores. Procedeu-se à
construção de biossensores de ADN, usando bases púricas (adenina ou guanina) ou
cadeias simples de ADN imobilizadas na superfície de elétrodos de carbono vítreo ou de
pasta de carbono, respetivamente.
O princípio de funcionamento destes biossensores baseou-se na avaliação do dano
provocado por radicais livres (hidroxilo, superóxido e sulfato) e da proteção de
antioxidantes (ácido ascórbico, ácido gálico, ácido cafeico, ácido cumárico e resveratrol),
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usando-se a voltametria de onda quadrada, voltametria cíclica ou voltametria de impulso
diferencial, como técnicas de deteção
Verificou-se que os radicais livres hidroxilo (OH•), superóxido (O2•-) e sulfato (SO4
•-)
provocam danos oxidativos na adenina e na guanina imobilizada na superfície do
elétrodo de carbono vítreo. Por outro lado, os antioxidantes, ácido ascórbico, ácido gálico,
ácido cafeíco, ácido cumárico e o resveratrol protegem a adenina e a guanina dos danos
provocados pelos radicais.
Com os biossensores de cadeia simples de ADN os radicais livres usados foram o
hidróxilo (OH•) e o superóxido (O2•-). Neste caso o dano oxidativo e a proteção produzida
pelo ácido ascórbico foram avaliados através da medição da corrente eletrocatalítica do
NADH.
Estes biossensores foram usados para determinar a capacidade antioxidante total das
águas aromatizadas, tendo-se verificado capacidade de proteção ao dano causado por
radicais livres.
Palavras-chave : águas com sabores, antioxidante, radicais livres, ADN, biossensores
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ABSTRACT
The continuous production of free radicals during the metabolic process is balanced by
antioxidant defense mechanisms. These mechanisms aim to eliminate or reduce the
levels of these radicals in the cells and thus protect the organism from its deleterious
effects.
Food and beverages are good exogenous sources of antioxidants. Flavoured water,
which has natural flavours, vegetable extracts (like tea) and fruit juices, should have some
antioxidant capability. This type of beverage (flavoured water) is quite recent and so, its
beneficial health factors, namely antioxidant capability, are not yet assessed.
The main goal of this dissertation consisted in improving the knowledge concerning the
chemical and antioxidant composition of the several flavoured waters available in the
Portuguese consumer market.
In a first stage, a minerologic caracterization of the waters was done; a total of 18
mineral were assessed: 4 macrominerals (Ca, Mg, K and Na), 3 microminerals (Fe, Cu
and Zn), and 11 trace elements (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se and Si). The levels
of all these minerals were within legal limits but varied from brand to brand, due to its
different sources.
Then, an assessment of antioxidant capability parameters was done. Conventional
optical methods were used, namely, total phenolic content, total flavonoid content,
reducing power and DPPH radical scavenging activity of the waters and flavours used. To
better understand the chemical composition of flavours, the terpenoid profile was
assessed, using HP-SPME/GC-MS.
According to the results, the water samples and flavours presented antioxidant capacity
and radical scavenging activity. However, no flavonoids were found. The analysis of
flavour detected the presence of monoterpenes and sesquiterpenes.
In the work flow, alternative methods were developed to quantify the antioxidant
capacity of flavoured waters. DNA biosensors were assembled, using purine bases
(adenine and guanine) or single strainded DNA, immobilized on the glassy carbon
electrode surface or carbon paste electrode surface, respectively.
The operating principle of these biosensors was based in assessing damage promoted
by free radicals (hydroxyl, superoxide, sulfate) and the protection made by the
antioxidants (ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol), using
square wave voltammetry, cyclic voltammetry and differential pulse voltammetry, as the
detection technique.
It was verified that the free radicals hydroxyl (OH•), superoxide (O2•-) and sulfate (SO4
•-)
induce oxidative damage in adenine and in the guanine immobilized in the electrode
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surface. On the other hand, antioxidants, ascorbic acid, gallic acid, caffeic acid, coumaric
acid and resveratrol protect adenine and guanine from free radical damage.
With DNA-based biosensors, hydroxyl and superoxide were the free radicals used. In
this case, the oxidative damage and the protection granted by ascorbic acid were
assessed through measuring electrocatalytic current of NADH.
These biosensors were used to determine the total antioxidant capability of flavoured
waters, and it was verified that these waters present antioxidant capacity.
Keywords : flavoured waters, antioxidant, free radical, biosensor
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RÉSUMÉ
La production continue de radicaux libres au cours du processus métabolique est
contrebalancé par des mécanismes de défense antioxydante. Ces mécanismes visent à
éliminer ou à réduire les niveaux de ces radicaux dans les cellules et donc de protéger
l'organisme contre ses effets délétères.
Les aliments et les boissons sont de bonnes sources d'antioxydants exogènes. Les
eaux aromatisées, qui ont des arômes naturels, des extraits de végétaux (comme le thé)
et des jus de fruits, devraient avoir une certaine capacité antioxydante. Ce type de
boissons (eaux aromatisées) sont assez récente et donc, ses effets bénéfiques pour la
santé, à savoir la capacité antioxydante, ne sont pas encore évalués. L'objectif principal
de cette thèse a consisté à améliorer les connaissances concernant la composition
chimique et antioxydantes de diverses eaux aromatisées disponibles sur le marché des
consommateurs portugais.
En premier, une caractérisation minéralogique des eaux a été faite; un total de 18
minéraux ont été évalués: 4 macrominéraux (Ca, Mg, K et Na), 3 microminéraux (Fe, Cu
et Zn), et 11 éléments résiduels (Al , As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se et Si). Les
niveaux de tous ces minéraux étaient dans les limites légales, mais variaient d'une
marque à l'autre, en raison de ses différentes sources.
Ensuite, une évaluation des paramètres de la capacité antioxydante a été faite. Les
méthodes classiques optiques ont été utilisés, à savoir, le contenu totale en phénoliques,
le contenu totale en flavonoïdes, le pouvoir réducteur et l'activité antioxydant mesurée par
le radical DPPH des eaux et arômes utilisés. Pour mieux comprendre la composition
chimique des saveurs, le profil de terpénoïdes a été évaluée, en utilisant HP-SPME/GC-
MS.
Selon les résultats, les échantillons d'eaux et de saveurs présentent des capacités
antioxydantes et une activité anti-radicale. Toutefois, aucune flavonoïdes n’ont été
trouvées. L'analyse des aromes détecte la présence de monoterpènes et sesquiterpènes.
Dans le deroulement des travaux, d'autres méthodes ont été développées pour
quantifier la capacité antioxydante des eaux aromatisées. Des biocapteurs d'ADN ont été
assemblés, en utilisant des bases puriques (adénine et la guanine) ou brin simple l'ADN
immobilisé sur la surface de l'électrode de carbone vitreux ou sur la surface de l’électrode
de pâte de carbone, respectivement.
Le principe de fonctionnement de ces biocapteurs a été fondé dans l'évaluation des
dommages provoqués par les radicaux libres (hydroxyle, superoxyde, sulfate) et la
protection faites par les antioxydants (l’acide ascorbique, l’acide gallique, l’acide caféique,
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l'acide coumarique et le resvératrol), en utilisant la voltamétrie à onde carrée, voltamétrie
cyclique et la voltamétrie à impulsion différentielle, ainsi que la technique de détection.
Il a été vérifié que les radicaux libres hydroxyles (OH•), superoxyde (O2• -) et de sulfate
(SO4• -) induisent des lésions oxydatives dans l'adénine et la guanine qui a été
immobilisée dans la surface de l'électrode. D’ une autre part, les antioxydants, l’acide
ascorbique, l’acide gallique, l’acide caféique, l'acide coumarique et le resvératrol
protégent l’adénine et la guanine des dommages provoqués par les radicaux libres. Avec
les biocapteurs basés sur l'ADN, les radicaux libres qui ont été utilisées ont été l’hydroxyle
et le superoxyde. Dans ce cas, les dommages oxydatifs et la protection accordée par
l'acide ascorbique ont été évalués par le mesure de courant électrocatalytique de NADH.
Ces biocapteurs ont été utilisés pour déterminer la capacité antioxydante totale des
eaux aromatisées, et l’on observe que ces eaux présentent des capacités antioxydantes.
Mots-clés : eaux aromatisées, antioxydant, radicaux libres, biocapteur
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RESUMEN
La continua producción de radicales libres en los procesos metabólicos es
contrarrestada por los mecanismos antioxidantes de defensa. El objetivo de dichos
mecanismos es eliminar o reducir los niveles de estos radicales en las células y, por lo
tanto, proteger el organismo de sus efectos nocivos.
Los alimentos y las bebidas son una buena fuente de antioxidantes. Las aguas con
sabores, las cuales contienen aromas naturales, extractos vegetales (como el te) y zumos
de frutas, deberían tener actividad antioxidante. Este tipo de bebidas (aguas de sabores)
es bastante reciente y, por tanto, su efecto beneficioso sobre la salud en relación con su
capacidad antioxidante aún no ha sido estudiada.
El principal objetivo de esta Tesis Doctoral consiste en mejorar el conocimiento
relacionado con la composición química y de antioxidantes de varias agua de sabores
disponibles en el mercado portugués.
En una primera etapa, se llevó a cabo la caracterización mineralógica. Un total de 18
minerales fueron estudiados: 4 macrominerales (Ca, Mg, K y Na), 3 microminerales (Fe,
Cu y Zn) y 11 elementos traza (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se y Si). El nivel
encontrado de estos minerales está dentro de los límites legales aunque varía entre
marcas debido a su diferente origen.
Posteriormente, se estudiaron los parámetros relacionados con la capacidad
antioxidante. Para ello se emplearon métodos ópticos convencionales como el contenido
fenólico total, contenido de flavonoides total, el poder reductor y la actividad de
eliminación del radical DPPH. Para comprender mejor la composición química de los
aromas, el perfil de terpenoides fue estudiado por HP-SPME/GC-MS.
Los resultados obtenidos permiten afirmar que las muestras de agua y aromas
presentan capacidad antioxidante y de eliminación de radicales. Sin embargo, no se
encontraron flavonoides. El análisis de aroma detectó la presencia de monoterpenos y
sesquiterpenos.
En el curso del trabajo también se desarrollaron métodos alternativos para cuantificar
la capacidad antioxidante de aguas de sabores. Se prepararon sensores usando las
bases púricas (adenina y guanina) o ADN monocatenario inmovilizado sobre electrodos
de carbono vítreo o de pasta de carbono, respectivamente.
El principio de operación de estos biosensores está basado en la detección del daño
ocasionado por los radicales libres (hidroxilo, superóxido, sulfato) y la protección ejercida
por los antioxidantes (ácido ascórbico, ácido gálico, ácido cafeico, ácido cumárico y
resveratrol) usando voltametría de onda cuadrada, voltametría cíclica y voltametría de
pulso diferencial como técnicas de detección.
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Se comprobó que los radicales libres hidroxilo(OH•), superóxido (O2•-) y sulfato (SO4
•-)
inducen daño oxidativo en adenina y guanina inmovilizada sobre la superficie electródica.
Por otra parte, los antioxidantes ácido ascórbico, ácido gálico, ácido cafeico, ácido
cumárico y resveratrol protegen a la adenina y a la guanina del daño de los radicales
libres.
Con los biosensores basados en ADN, los radicales libres usados fueron el hidroxilo y
el superóxido. En estos casos, el daño oxidativo y la protección ejercida por el ácido
ascórbico fue estudiada a través de la medida de la corriente electrocatalítica de NADH.
Estos biosensores fueron usados para determinar la capacidad antioxidante total de
aguas con sabores comprobándose que, en efecto, estas aguas presentan capacidad
antioxidante.
Palabras clave : aguas de sabores, antioxidantes, radical libre, biosensor.
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TRABALHOS (PUBLICAÇÕES E COMUNICAÇÕES) DESENVOLVIDO S NO ÂMBITO
DO PROJETO DE DOUTORAMENTO
Publicações de artigos em revistas de circulação in ternacional com arbitragem
científica referenciadas no Journal Citation Reports da ISI Web of Knowledge :
1. Flavoured versus natural waters: Macromineral (Ca, Mg, K, Na) and micromineral (Fe,
Cu, Zn) contents
M. Fátima Barroso, Aurora Silva, Sandra Ramos, M. T. Oliva-Teles, Cristina Delerue-
Matos, M. Goreti F. Sales, M.B.P.P. Oliveira
Food Chemistry, 2009, 116 (2), 580-589
2. Survey of trace elements (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se, and Si) in retail
samples of flavoured and bottled waters
M. F. Barroso, S. Ramos, M. T. Oliva-Teles, C. Delerue-Matos, M. G. F. Sales, M. B.
P. P. Oliveira
Food Additives and Contaminants: Part B – Surveillance, 2009, 2 (2), 121-130
3. Flavored waters: Influence of ingredients on antioxidant capacity and terpenoid profile
by HS-SPME/GC-MS
M. Fátima Barroso, J. P. Noronha, Cristina Delerue-Matos, M. B. P. P. Oliveira
Journal of Food and Agricultural Chemistry, 2011, 59 (9), 5062-5072
4. DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in
beverages
M. F. Barroso, N. de-los-Santos-Álvarez, M.J. Lobo-Castañón, A. J. Miranda-Ordieres,
C. Delerue-Matos, M. B. P. P. Oliveira, P. Tuñón-Blanco
Biosensors and Bioelectronics, 2011, 26 (5), 2396-2401
5. Electrochemical DNA-sensor for evaluation of total antioxidant capacity of flavours and
flavoured waters using superoxide radical damage
M. F. Barroso, C. Delerue-Matos, M. B. P. P. Oliveira
Biosensors and Bioelectronics, 2011, 26 (9), 3748-3754
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6. Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant
capacity assessment
M. F. Barroso, N. de-los-Santos-Álvarez, M. J. Lobo-Castañón, A. J. Miranda-
Ordieres, C. Delerue-Matos, M. B. P. P. Oliveira, P. Tuñón-Blanco
Journal of Electroanalytical Chemistry, 2011, em publicação
doi:10.1016/j.jelechem.2011.04.022
7. Electrochemical evaluation of total antioxidant capacity of beverages using a purine-
biosensor
M. F. Barroso, C. Delerue-Matos, M. B. P. P. Oliveira
Food Chemistry (submetido)
8. Evaluation of total antioxidant capacity of flavoured waters using sulfate radical
damage of purine-based sensors
M. F. Barroso, C. Delerue-Matos, M. B. P. P. Oliveira
Electrochimica Acta (submetido)
9. Towards a reliable technology for antioxidant capacity and oxidative damage
evaluation: electrochemical (bio)sensors
M. Fátima Barroso, N. de-los-Santos-Álvarez, C. Delerue-Matos, M. B. P. P. Oliveira
Biosensor and Bioelectronics (submetido)
Publicações de artigos ou resumos alargados em atas de encontros científicos
1. Águas naturais versus aromatizadas: Influência dos ingredientes adicionados na
composição mineral
M. F. Barroso, C. Delerue-Matos, M. G. Sales, M. B. P. P. Oliveira
Atas do 9º Encontro de Química dos Alimentos, 29 Abril-2 Maio, 2009, Angra do
Heroísmo, Açores
Comunicações em poster em encontros científicos internacionais:
1. Evaluation of trace elements in flavoured waters: a case study
M. F. Barroso, M. T. Oliva-Teles, C. Delerue-Matos, M. G. Ferreira Sales, M. B. P. P.
Oliveira
Rapid Methods Europe 2008 for Food and Feed Safety and Quality, P34, 21-23
Janeiro, 2008, Noordwijkerhout, Holanda, 2008
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2. Macrominerals in flavoured waters
M. Fátima Barroso, M.T. Oliva-Teles, Cristina Delerue-Matos, M. Goreti F. Sales,
M. B. P. P. Oliveira
AOAC Europe Section International Workshop e II encontro Nacional de
Bromatologia, Hidrologia e Toxicologia, P3FNFA, 17-18 Abril, 2008, Lisboa, Portugal
3. Antioxidant capacity evaluation in flavour and flavoured waters by total phenolic and
flavonoid contents, reuducing power and DPPH scavenging assays
Maria Fátima Barroso, Cristina Delerue-Matos, João Paulo Noronha, Maria Beatriz
Oliveira, Maria Goreti Sales
1st European Food Congress, P208, 4-9 Novembro, 2008, Ljubljana, Slovenia
4. Investigations on the electrocatalytic assessment of antioxidant capacity using a DNA-
Modified carbon paste electrode
M. F. Barroso, N. de-los-Santos-Álvarez, M. J. Lobo-Castanon, A. J. Miranda-
Ordieres, M. G. Ferreira Sales, M. B. P. P. Oliveira, C. Delerue-Matos
Euro Analysis 2009, P081-B2, 6-10 Setembro, 2009, Innsbruck, Áustria
5. Development of a DNA-modified sensor to evaluate the total antioxidant capacity of
flavoured waters
M. Fátima Barroso, J. Paulo Noronha, M. Goreti F. Sales, Cristina Delerue-Matos, M.
Beatriz P. P. Oliveira
4th International Symposium on Recent Advances in Food Analysis, N-23, 4-6
Novembro, 2009, Prague, Czech Republic
6. Antioxidant capacity of flavoured waters by electrochemical DNA-Biosensor
M. Fátima Barroso, M. Goreti Sales, Cristina Delerue-Matos, M. B. P. P. Oliveira
13th International Conference on Electroanalysis, Pin-40, 20-24 Junho, 2010, Gigon,
Spain
7. DNA damage generated by a sulphate radical and the protective effect of dietary
antioxidants using an electrochemical DNA biosensors
M. Fátima Barroso, M. Goreti Sales, Cristina Delerue-Matos, M. B. P. P. Oliveira
13th International Conference on Electroanalysis, Pin-41, 20-24 Junho, 2010, Gigon,
Spain
XXII
Comunicações em poster em encontros científicos nacionais:
1. Aguas naturais versus aromatizadas: Influência dos ingredientes adicionados na
composição mineral
M. F. Barroso, S. Ramos, C. Delerue-Matos, M. G. Sales, M. B. P. P. Oliveira
9º Encontro de Química dos Alimentos, P42, 29 Abril-2 Maio, 2009, Angra do
Heroísmo, Açores.
XXIII
ÍNDICE
Agradecimentos IX
Resumo XI
Abstract XIII
Résumé XV
Resumen XVII
Trabalhos desenvolvidos no âmbito do projeto de dou toramento XIX
Índice XXIII
Lista de Abreviaturas e Símbolos XXV
I. REVISÃO DO ESTADO DA ARTE 1
Organização e estrutura da dissertação 3
Objetivos 5
Introdução geral 7
Referências 12
CAPÍTULO 1. Métodos eletroquímicos 17
Towards a reliable technology for antioxidant capacity and oxidative damage evaluation: electrochemical (bio)sensors 19
II. INVESTIGAÇÃO E DESENVOLVIMENTO 51
CAPÍTULO 2. Composição mineralógica 53
2.1. Flavoured versus natural waters: Macromineral (Ca, Mg, K, Na) and
micromineral (Fe, Cu, Zn) contents 55
2.2. Survey of trace elements (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se, and
Si) in retail samples of flavoured and bottled waters 77
CAPÍTULO 3. Perfil antioxidante – métodos convenc ionais 99
Flavored Waters: Influence of Ingredients on Antioxidant Capacity and
Terpenoid Profile by HS-SPME/GC-MS
101
CAPÍTULO 4. Construção de biossensores de bases púricas
127
4.1. Electrochemical evaluation of total antioxidant capacity of beverages
using a purine biosensor 129
XXIV
4.2. Electrochemical DNA-sensor for evaluation of total antioxidant
capacity of flavours and flavoured waters using superoxide radical
damage
149
4.3. Evaluation of total antioxidant capacity of flavoured waters using
sulfate radical damage of purine-based sensors
167
CAPÍTULO 5. Construção de biossensores de ADN 187
5.1. DNA-based biosensor for the electrocatalytic determination of
antioxidant capacity in beverages
189
5.2. Electrocatalytic evaluation of DNA damage by superoxide radical for
antioxidant capacity
207
CONSIDERAÇÕES FINAIS 225
XXV
LISTA DE ABREVIATURAS E SÍMBOLOS
Na lista apresentada constam termos em português e em inglês, consoante a língua
em que são utilizados ao longo da dissertação.
A• reactive radical
AA ascorbic acid
AAS atomic absorption spectrophotometry
ACu ácido cumárico
AdSV Adsorptive stripping voltammetry
AH phenolic antioxidant
AOC antioxidant capacity
APHA American Public Health Association
AuE gold electrode
BHA tert-butylhydroxy anisole
BHT butylated hydroxytolurene
CA caffeic acid
CF carbon fiber
CMEs chemically modified electrodes
CNTECE carbon nanotube epoxy composite electrode
CPE carbon paste electrode
CV cyclic voltammetry
DA dopamine
dA21 deoxyadenylic acid oligonucleotide
DCTMACl docosyltrimethylammonium chloride
DNA deoxyribonucleic acid
DPPH 2,2-diphenyl-1-picrylhydrazyl
DPV differential pulse voltammetry
dsDNA double strainded DNA
E potential
EFSA European Food Safety Authority
EI electronic impact
Eº´ formal potential
Ep peak potential
EPA Environmental Protection Agency
FAD flavin adenine dinucleotide
XXVI
FAO Food and Agriculture Organisation
FC Folin-Ciocalteu
FDA Food and Drug Administration
FIA flow injection analysis
FNB/IOM Food and Nutrition Board of the Institute of Medicine
FRAP ferric reducing antioxidant power
GA gallic acid
GCE glassy carbon electrode
GC-MS gas chromatography-mass spectrometry
GPES general purpose electrochemical system
H2O2 hydrogen peroxide
HAT hydrogen atom transfer
HOCl hypochorous acid
HOPC highly oriented pyrolytic graphite
HRP horseradish peroxidase
HS-SPME Headspace-solid-phase microextraction
i current
Ia electrocatalytic current in the presence of an antioxidant
ICP–AES inductively coupled plasma-atomic emission spectroscopy
ICP–MS inductively coupled plasma-mass spectrometry
Id electrocatalytic current in the absence of an antioxidant
ip peak current
JECFA Joint Expert Committee on Food Additives
LDL low-density lipoprotein
LOD limit of detection
LOQ limit of quantification
LSV linear sweep voltammetry
LSW linear sweep voltammetry
M metal
m/m massa/massa
MAE microwave-assisted extraction
MWCNs multi-walled carbon nanotubes
NADH nicotinamide adenine dinucleotide disodium salt, reduced form
NiHCF nickel hexacyanoferrate
NO• nitric oxide radical
NPs nanoparticules
XXVII
O2•- Superoxide radical, radical superóxido
OH• hydroxyl radical, radical hidroxilo
ORAC oxygen radical absorbance capacity
PAMAM poly(amidoamine)
PBS phosphate buffer saline
PCL photochemiluminescence
PDMS/DVB polydimethylsiloxane/divinylbenzene
PGE pyrolytic graphite electrode
PGEs pencil graphite electrodes
PPO polyphenol oxidase
PRTC peroxyl radical trapping capacity
PS polystyrene
PTDI provisional tolerable daily intake
PtE platinium electrode
PTWI provisional tolerable weekly intake
RDA recommended dietary allowance
RE relative error
REC recovery
RES resveratrol
RfD reference dose
RNS reactive nitrogen species
RO• alkoxyl radical
ROO• peroxyl radical
ROS reactive oxygen species
RSA radical-scavenging activity
RSD relative standard deviations
SAM self assembled monolayer
SAMe s-adenosyl-L-methionine
SD standard deviation
SET single electron transfer
SFE supercritical fluid extraction
SO4•– sulfate radical, radical sulfato
SOD superoxide dismutase
SPE screen printed electrode
SPE-Au gold-screen printed electrode
SPME solid-phase microextraction
XXVIII
ssDNA single strainded DNA
SSPE concentrated saline sodium phosphate EDTA
SV stripping voltammetry
SWCNs single-walled carbon nanotubes
SWV square wave voltammetry
TAC total antioxidant capacity
TEAC trolox equivalent antioxidant capacity assay
TFC total flavonoid content
TOSC total oxidant scavenging capacity
TPC total phenol content or total phenolic content
TRAP total radical-trapping antioxidant parameter
Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
UV / VIS ultraviolet-visible
v/v volume/volume
WHO World Health Organization
XOD xanthine oxidase
Γ surface coverage
~ approximately
1
I.
REVISÃO DO ESTADO DA ARTE
Organização e estrutura da dissertação
3
ORGANIZAÇÃO E ESTRUTURA DA DISSERTAÇÃO
A presente dissertação inclui todos os artigos científicos (6 publicados e 3 submetidos)
resultantes do projeto de doutoramento, pretendendo-se assim que todos os trabalhos
desenvolvidos tivessem já sido objeto de análise crítica por parte de diferentes revisores
internacionais, especialistas na área de investigação em que se inserem, e selecionados
de acordo com os critérios de cada revista em que o respetivo artigo foi publicado.
Todos os artigos estão escritos em inglês. Optou-se por manter a formatação original,
adaptada ao corpo da tese, com a qual os textos foram submetidos ou publicados, de
acordo com as normas específicas de cada revista. Assim, encontram-se variações na
estrutura dos diferentes artigos apresentados ao longo dos capítulos e no modo como
são indicadas as referências. Toda a bibliografia que não está integrada nas publicações,
é apresentada de acordo com a norma de Vancouver aconselhado pelas “normas de
formatação das dissertações de mestrado e teses de doutoramento” da Faculdade de
Farmácia da Universidade do Porto.
Os textos que se apresentam em português foram elaborados de acordo com o novo
acordo ortográfico.
A dissertação está organizada em duas partes: a Parte I corresponde à Revisão do
Estado da Arte ; a Parte II é designada por Investigação e Desenvolvimento , onde se
inclui toda a investigação desenvolvida na componente experimental.
Assim, na Parte I , com uma abordagem mais teórica, faz-se inicialmente uma
introdução geral ao tema “águas com sabores”, com o objetivo de contextualizar o
trabalho desenvolvido, abordando questões como o consumo desta bebida em Portugal e
fazendo referência à legislação nacional sobre sobre águas de consumo. A esta breve
introdução, segue-se a descrição dos objetivos do trabalho. Ainda dentro desta primeira
parte, é apresentado o Capítulo 1 constituído por um artigo, em que se faz a Revisão do
Estado da Arte no que se refere a todos os métodos eletroquímicos utilizados para a
determinação da capacidade antioxidante de diversas matrizes.
A Parte II da dissertação é constituída por 4 capítulos (capítulos 2 a 5). No geral, cada
capítulo é constituído por um ou mais artigos. O Capítulo 2 integra 2 artigos científicos
correspondentes à determinação mineralógica das águas com sabores e respetivas
águas naturais. O primeiro artigo refere-se à determinação de macrominerais (Ca, Mg, K
e Na) e microminerais (Fe, Cu e Zn) enquanto que o segundo aborda a determinação dos
minerais vestigiais (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se e Si). O Capítulo 3 (1 artigo)
corresponde à determinação do perfil antioxidante das águas com sabores e seus
aromas através de métodos óticos convencionais. O perfil antioxidante foi feito através da
determinação do teor fenólico total, teor total de flavonóides, poder redutor e atividade
I. Revisão do estado da arte
4
anti-radicalar. O trabalho apresentado neste capítulo foi elaborado em colaboração com o
Laboratório de Cromatografia da Linha de Química Orgânica Física/Química Radicalar do
departamento de Química da FCT-UNL. O Capítulo 4 (3 artigos) e Capítulo 5 (2 artigos)
correspondem ao desenvolvimento das metodologias analíticas alternativas, para a
determinação da capacidade antioxidante, baseadas em técnicas eletroquímicas e na
construção de biossensores. No Capítulo 4 refere-se a construção de biossensores de
adenina e de guanina. Estes biossensores foram sujeitos ao ataque oxidativo induzido
por 3 radicais livres (hidroxilo, superóxido e sulfato) e avalia-se o efeito protetor
promovido por 5 antioxidantes (ácido ascórbico, ácido gálico, ácido cafeico, ácido
cumárico e resveratrol). Cada artigo corresponde a um dos radicais. No Capítulo 5 , os
artigos descrevem a construção de biossensores de cadeia simples de ADN em que se
usaram o radical hidroxilo e o superóxido para a promoção do dano oxidativo. O trabalho
experimental associado a este capítulo foi desenvolvido no Grupo de Electroanálisis da
Faculdade de Química da Universidade de Oviedo.
No final da dissertação apresentam-se as Considerações Finais . Optou-se por esta
designação, uma vez que se foram apresentando conclusões parciais ao longo dos
diferentes capítulos. Nesta medida, o final desta dissertação não é necessariamente um
culminar de um projeto, mas pretende apenas apresentar um conjunto de reflexões sobre
o trabalho realizado, perspetivando projetos e atividades de investigação futuras.
Objetivos
5
OBJETIVOS
Considerando a ausência de estudos científicos na matriz água aromatizada,
considerou-se importante avaliar algumas propriedades desta nova bebida lançada no
mercado. Por isso os objetivos desta dissertação foram:
• Caraterizar quimicamente, no que diz respeito ao teor de macrominerais,
microminerais e minerais vestigiais, as águas com sabores;
• Analisar os aromas adicionados a estas águas por HS-SPME/GC-MS;
• Avaliar o perfil antioxidante destas águas e dos aromas usados na sua formulação,
através da avaliação do teor fenólico total, do teor de flavonóides total, poder redutor e
atividade anti-radicalar usando os métodos óticos convencionais;
• Desenvolver metodologias analíticas alternativas para a determinação da capacidade
antioxidante das águas com sabores:
• Construir biossensores eletroquímicos de cadeia simples de ADN ou de bases
púricas (adenina ou guanina);
• Estudar o dano oxidativo induzido por radicais livres (hidroxilo, superóxido e
sulfato) na superfície modificada do elétrodo.
• Estudar a proteção promovida por antioxidantes na superfície modificada do
elétrodo;
• Avaliar a capacidade antioxidante total das águas com sabores utilizando estes
biossensores.
Introdução geral
7
INTRODUÇÃO GERAL
É do conhecimento geral que a água é indispensável à vida sendo um fator primordial
na qualidade de vida e imprescindível a todos os aspetos da existência humana. A água
representa cerca de 60 a 70 % do peso corporal e é responsável pelo controlo da
temperatura corporal, pela manutenção do volume vascular, por possibilitar as reações
enzimáticas envolvidas na digestão, e pela absorção e metabolismo. Além de transportar
substâncias como nutrientes e oxigénio para as células, promove a excreção de toxinas
através dos rins. Uma hidratação correta contribui para o bem-estar e para a manutenção
da saúde.
A ingestão diária de água deve compensar as perdas de fluidos que ocorrem através
da excreção urinária, transpiração e respiração (1,2). Dependendo do género e da idade
do indivíduo, as quantidades diárias recomendadas para a ingestão de água podem
variar entre 0,7 L (bebés entre os 0-6 meses) 2,7 L (mulheres com idade superior a 19
anos) e 3,7 L (homens com idade superior a 19 anos). As quantidades de água
recomendadas incluem a água presente nos alimentos sólidos e líquidos (como por
exemplo a sopa) e bebidas. De acordo com Ershow and Cantor (1989), 28 % da água
ingerida pelos adultos corresponde à água dos alimentos, 28 % à água potável, enquanto
44 % corresponde a outro tipo de bebidas (3,4).
A qualidade e quantidade de água disponível para consumo humano tem sido uma
preocupação ao longo da história. As principais fontes deste bem essencial são as
massas de água superficial e subterrânea. A população atual, preocupada com a
qualidade da água, tem aumentado o consumo de água engarrafada (5).
Estão disponíveis no mercado vários tipos de água engarrafada: água mineral natural,
água mineral gasosa, água mineral natural gaseificada e água de nascente. A água
mineral natural é uma água de circulação subterrânea, considerada bacteriologicamente
própria, com caraterísticas físico-químicas estáveis na origem, caraterizada por um teor
de substâncias minerais, oligoelementos ou outros constituintes, de que podem
eventualmente resultar efeitos favoráveis à saúde e que se distingue da água de
consumo. Este tipo de água pode conter gás natural (água mineral natural gasosa), ser
reforçada com gás (água mineral natural reforçada com gás carbónico) ou conter
somente gás adicionado (água mineral natural gaseificada). Por outro lado, a água de
nascente é uma água subterrânea, considerada bacteriologicamente própria, com
caraterísticas físico-químicas que a tornam adequada para consumo humano no seu
estado natural (5,6). Estas águas encontram-se regulamentas pelo Decreto-Lei nº 156/98
que define e carateriza as águas minerais naturais e as águas de nascente e estabelece
as regras relativas à exploração, acondicionamento e comercialização (6).
I. Revisão do estado da arte
8
No entanto, em determinadas águas minerais naturais, devido à sua origem
hidrogeológica, podem estar presentes, no estado natural, elementos químicos que, a
partir de uma certa concentração, podem representar um risco para a saúde pública (7).
Assim, o decreto-Lei nº 72/2004 estabelece uma lista com os limites máximos de
constituintes que se encontram, naturalmente, nas águas minerais naturais. Os
constituintes presentes nessa lista são o antimónio, arsénio, bário, boro, cádmio, cromo,
cobre, cianeto, fluoretos, chumbo, manganês, mercúrio, níquel, nitratos, nitritos e o
selénio (7).
Embora a água seja muito importante para o ser humano, muitos indivíduos não
consomem água, preferindo ingerir outro tipo de bebidas (sumos, refrigerantes ou
chás/infusões). Em resposta às necessidades atuais do consumidor e conhecedores das
suas preferências, os industriais de bebidas desenvolvem com frequência novos tipos de
bebidas. É o caso das águas com sabores, baseadas na adição de um ou mais aromas
(naturais ou sintéticos) à água natural. Estas águas podem conter, para além do aroma,
sumos de fruta, conservantes, reguladores de acidez e edulcorantes. De acordo com a
Portaria nº 703/96, as águas com sabores podem, legalmente, ter diferentes
designações. No caso de estas águas conterem entre 6 % e 16 % (m/m) de sumo de
fruta, a designação será “refrigerante de sumo de frutos”; e refrigerante aromatizado no
caso desta bebida resultar da diluição de aromatizantes. Se estas bebidas não
contiveram açúcares nem edulcorantes a designação será “água aromatizada” (8). Neste
trabalho optou-se pela designação de águas com sabores.
O consumo de água com sabores é um mercado em ascensão, tendo sido vendidos
em Portugal e no 1º semestre de 2010 cerca de 6,07 milhões de litros (9). No mercado
português existe uma grande oferta destas bebidas. As mais usuais e frequentes são as
águas com aroma de limão, mas também podem ser encontradas bebidas com aroma de
laranja, ananás, pêssego, melão, morango, maçã e goiaba. Há marcas comerciais que
apostaram no desenvolvimento de águas com dois aromas em simultâneo
(laranja/framboesa, pêssego/ananás, maçã/chá, framboesa/ginseng, pêssego/chá,
manga/biloba, melão/hortelã). Sendo estas bebidas relativamente recentes, não havia
publicações científicas abordando o estudo deste tipo de águas quando se iniciou este
trabalho.
Analisando os rótulos das garrafas de águas com sabores das diferentes marcas
comerciais disponíveis no mercado português, verifica-se uma grande diversidade nos
ingredientes adicionados à água. Para além dos aromas (naturais ou sintéticos) algumas
marcas referem a adição de fibras alimentares (ex, dextrina de trigo), sumos de fruta
(concentração máxima de 2,3 %) compostos bioativos (ginseng, L-carnitina, chá verde e
branco, ginkgo biloba), vitaminas (vitamina C e complexos da vitamina B) e ingredientes
Introdução geral
9
que, não tendo uma relação positiva com o bem-estar e saúde do consumidor, são
necessários para garantir a qualidade desejada, tais como, agentes acidificantes (ácido
cítrico e citrato de sódio), edulcorantes (acessulfame-K, aspartamo e sucralose) e
conservantes (sorbato de potássio e benzoato de sódio).
Em termos energéticos, as águas com sabores que contêm edulcorantes apresentam
um valor energético inferior (0,4 a 1,3 kcal/ 100 mL) ao de águas com sabores que não
contêm edulcorantes (9 a 13 kcal/ 100 mL). Comparativamente com os refrigerantes,
néctares e sumos de fruta, as águas com sabores um baixo valor calórico, apresentando
os primeiros um valor calórico que oscila entre os 21 kcal/ 100 mL e os 46 kcal/ 100 mL.
Naturalmente que as águas com sabores não devem substituir a água potável, mas
podem ser uma alternativa interessante aos refrigerantes. Estes possuem na sua
composição ingredientes com efeito negativo na saúde dos consumidores, especialmente
crianças. O consumo moderado de águas com sabores pode ser feito com prazer e sem
grandes preocupações de saúde. No entanto, será importante referir que são mais caras
(20-40 %) do que as águas naturais engarrafadas (2, 10-13).
Considerando que as águas com sabores podem conter, além da sua composição
normal, sumos de fruta e aromas naturais, extraídos de frutas ou vegetais (exemplo do
chá), é esperado que estas águas possam apresentar valores de capacidade antioxidante
superiores.
De acordo com Laguerre e colaboradores (2010), os antioxidantes são substâncias
que, mesmo quando presentes em baixas concentrações, comparativamente com um
substrato oxidante, protegem (por si mesmo ou através dos seus produtos de oxidação) o
substrato dos danos provocados pela oxidação (14). Os alimentos (fruta, vegetais,
legumes e cereais) e bebidas (sumos, chá, café e vinho) são boas fontes externas de
antioxidantes (15-20). Os compostos que vulgarmente conferem capacidade antioxidante
aos alimentos são o ácido ascórbico, os ácidos fenólicos, os tocoferóis, os terpenos,
entre outros (21). Por isso, aumentar a ingestão de antioxidantes na dieta alimentar
(incluindo as águas com sabores) pode ajudar a fortalecer os mecanismos de defesa
antioxidante do organismo humano (21). Conscientes da importância que estes alimentos
e bebidas têm na defesa do organismo, contra os radicais livres produzidos durante o
metabolismo celular, têm sido efetuados muitos estudos para caraterizar o perfil
antioxidante de uma diversidade de alimentos e bebidas, bem como no desenvolvimento
de novos produtos alimentares fortificados com antioxidantes.
Embora não existam estudos (nacionais ou internacionais) publicados sobre a
capacidade antioxidante de águas com sabores (somente existem os trabalhos realizadas
nesta dissertação) alguns grupos portugueses têm-se dedicado ao estudo e valorização,
quer de recursos naturais quer de produtos locais, a fim de desenvolver sistemas
I. Revisão do estado da arte
10
económicos, social e ambientalmente sustentáveis. Uma das formas de valorizar esses
produtos é conhecer o seu perfil antioxidante.
Há vários grupos de investigação portugueses que têm contribuído para o
conhecimento do perfil antioxidante destes produtos. Das diversas matrizes estudadas
salienta-se o estudo da capacidade antioxidante de cogumelos (20,22,23), ervas
aromaticas (15), verduras (24), castanha e amêndoa (25), plantas aromáticas (26), frutos
e sumos de fruta (18) e vinho (27). Sendo o vinho um produto muito produzido em
Portugal e valorizado nacional e internacionalmente, alguns grupos de investigação
traçaram o perfil antioxidante de vários tipos de vinhos produzidos no nosso país (17, 28-
31). O café é uma das bebidas mais consumidas em Portugal e nesta área o Laboratório
de Bromatologia da Faculdade de Farmácia da Universidade do Porto tem desenvolvido
um amplo trabalho de investigação incluindo a avaliação da capacidade antioxidante
desta bebida (32,33).
Outro tipo de matrizes, como por exemplo chá, frutos vermelhos (romã, mirtilos e
morangos) e óleos têm sido avaliados por grupos de investigação da Universidade do
Minho (34) e da Universidade do Porto (35,36).
A nível internacional há um grande número de publicações visando a avaliação da
capacidade antioxidante de diferentes tipos de alimentos e produtos processados. Dada a
extensão da bibliografia serão apenas citados alguns artigos mais recentes (37-39).
A metodologia analítica mais vulgarmente usada para quantificar a capacidade
antioxidante de produtos é a espetrofotometria de UV-Vis (40). Estes métodos
convencionais têm a desvantagem de ter tempos de reação e análise longos; as
amostras com cor precisarem de um pré-tratamento; e, sendo o alimento uma matriz
complexa, conter muitos interferentes (40). Considerando ser importante encontrar
alternativas aos métodos convencionais, foram desenvolvidos e construídos, ao longo
deste projeto, biossensores para a quantificação da capacidade antioxidante total de
águas com sabores. A nível da literatura, encontram-se algumas publicações envolvendo
a utilização de biossensores de ADN para a avaliação da capacidade antioxidante de
extratos vegetais e chás (41,42).
Esta dissertação surge no seguimento da investigação científica desenvolvida sobre
sensores e biossensores eletroquímicos no Grupo de Reacção e Análise Química
(GRAQ) do Instituto Superior de Engenharia do Instituto Politécnico do Porto e sobre
antioxidantes realizada no Laboratório de Bromatologia da Faculdade de Farmácia da
Universidade do Porto. Pretende-se assim dar mais um passo num processo cumulativo
em relação ao estado da arte.
Alguns dos trabalhos apresentados foram também desenvolvidos no Laboratório de
Cromatografia da Linha de Química Orgânica Física/Química Radicalar do Departamento
Introdução geral
11
de Química da Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa sob
supervisão do Professor Doutor João Paulo Noranha e no Laboratório de Electroanálisis
do Departamemto de Química Física e Analítica da Faculdade de Química da
Universidade de Oviedo, sob orientação da Professora Doutora Noemí de los Santos
Álvarez.
I. Revisão do estado da arte
12
REFERÊNCIAS
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3. Ershow AG, Cantor KP. Total Water and Tapwater Intake in the United States:
Population-based estimates of quantities and sources. Bethesda, MD: Life Sciences
Research Office, 1989.
4. Institute of Medicine of the National Academies. Dietary reference intakes for water,
potassium, sodium, chloride, and sulfate. Food and Nutrition Board. The National
Academies Press. Washington, D.C. 2005.
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17. Feliciano RP, Bravo MN, Pires MM, Serra AT, Duarte CM, Boas LV, Bronze MR.
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610-16.
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praticantes de hidroginástica em idade sénior. Motri 2007 Apr; (3) 2: 7-8.
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antioxidant properties of Vitis vinifera red grape malvidin-3-glucoside. Int J Food Sci
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Portuguese wine aged brandies. J Food Compos Anal 2008 Dec; 21 (8): 626-33.
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four varieties of Portuguese red grape skins determined by reverse-phase high-
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416-20.
17
Capítulo 1
Métodos eletroquímicos
Towards a reliable technology for antioxidant capacity and oxidative damage evaluation:
electrochemical (bio)sensors
M. Fátima Barroso, N. de-los-Santos-Álvarez, C. Delerue-Matos, M. B. P. P. Oliveira
Biosensors and Bioelectronics (submetido)
1. Métodos eletroquímicos
19
Towards a reliable technology for antioxidant capac ity and
oxidative damage evaluation: electrochemical (bio)s ensors
M. Fátima Barrosoa,b, N. de-los-Santos-Álvarezc, C. Delerue-Matosa, M. B. P. P. Oliveirab aREQUIMTE/Instituto Superior de Engenharia do Porto, Dr. Bernardino de Almeida 431,
4200-072 Porto, Portugal bRequimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, R.
Aníbal Cunha n. 164, 4050-047 Porto, Portugal cDepartamento de Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8,
33006 Oviedo, Spain
Abstract
To counteract and prevent the deleterious effect of free radicals the living organisms
have developed complex endogenous and exogenous antioxidant systems. Several
analytical methodologies have been proposed in order to quantify antioxidants in food,
beverages and biological fluids. This paper revises the electroanalytical approaches
developed for the assessment of the total or individual antioxidant capacity. Four
electrochemical sensing approaches have been identified, based on the direct
electrochemical detection of antioxidant at bare or chemically modified electrodes, and
using enzymatic and DNA-based biosensors.
Keywords: Antioxidants; free radicals; electrochemistry; chemically modified electrode;
enzymatic biosensors; DNA-based biosensor.
Disponível online em ww.sciencedirect.com
Bionsensor and Bioelectronics submitted
1. Métodos eletroquímicos
21
1. Introduction
The field of antioxidants has grown over the past decades invading a large number of
areas that affect food and health such as nutrition, biochemistry, pharmacology,
physiology, food processing and analytical chemistry. In general, researchers have been
focused on the i) study of the antioxidant pathway on free radical scavenging activity to
stop radical chain reactions; ii) study of the antioxidant protective role on proteins, lipids
and DNA against the free radical generated in vivo; iii) evaluation of the antioxidant profile
of natural foodstuffs and beverages because of their increasing appreciation; iv) synthesis
of antioxidants and development of novel artificially antioxidant-enriched food products; v)
study of the antioxidant efficacy on the preservation of foodstuff against deterioration
carried out by free radicals produced by food; vi) development of analytical methods for
the evaluation of the antioxidant content in food, beverages and biological samples.
The definition of antioxidant has been adjusted over the time (Halliwell and Gutteridge,
1990; Krinkly, 1992) and recently Laguerre et al. (2010) defined a biological antioxidant as
a substance that, when present at low concentrations compared to an oxidizable
substrate, protects (by itself and through its oxidation products) that substrate from
oxidation, and ultimately protects the organism from harmful effects of oxidative stress. An
imbalance between the generation of oxidants, either free radical or non-free radicals, and
the antioxidant system is known to cause oxidative stress, which is frequently associated
with many complex diseases (cardiovascular diseases, inflammatory disorders and
cancer) (Laguerre et al., 2010). Oxidation of key biological molecules (e.g. proteins,
carbohydrates, lipids and nucleic acids) is usually the mechanism that triggers these
pathologies along with modulation of gene expression and the inflammatory response
(Laguerre et al., 2007). Fortunately, nature has developed complex endogenous and
exogenous antioxidant systems to counteract and prevent the deleterious effect of
oxidants and minimize the oxidative stress in most living beings. Endogenous antioxidants
include enzymes that have the ability to promote an efficient repair of oxidative damaged
sites on macromolecules such as DNA. In general, hydrophilic antioxidants react with
oxidant compounds in the cell and blood plasma while hydrophobic antioxidants protect
cell membranes from lipid peroxidation (Barroso et al., 2011a). Fig. 1 sumarizes the
antioxidant defence system.
I. Revisão do estado da arte
22
Fig. 1. Antioxidant defence system
Foodstuffs constitute an excellent exogenous source of natural antioxidants. Although
further and more conclusive studies must be done to establish the precise correlation
between the intake of antioxidants and the reduction in the oxidative stress level, it seems
reasonable that increasing intake of dietary antioxidants may help to maintain an
adequate antioxidant status and, so, the normal physiological functions of a living system.
Some functional foods, vegetables, fruits, whole-grain cereals, juice, tea, wine are good
sources of exogenous antioxidants (Almajano et al. 2008; Frankel, 2007; Huang et al.
2009; Lee et al. 2009).
Oxidant compounds (radicals and non-radicals) can be generated as a consequence of
normal aerobic metabolism, and are able to induce damage to the cells by reacting with
biomolecules (proteins, lipids, among others) and causing serious lesions on DNA.
Radical species such as nitric oxide (NO•), superoxide (O2•-), hydroxyl (OH•), peroxyl
(ROO•) and alkoxyl (RO•) radicals, are known as reactive oxygen species (ROS) and
reactive nitrogen species (RNS) produced during the mitochondrial respiratory chain
(electron transport) or during the lipid oxidation chain reaction. Hypochlorous acid (HClO)
obtained by the enzymatic system myeloperoxidase/H2O2/Cl- is another free radicals
involved. All of them tend to donate or take another electron to attain stability, which
confers their characteristic high reactivity (Halliwell and Gutteridge, 1999; Rice-Evans C.A.
antioxidant defence system
endogenous exogenous(dietary source)ascorbic acid phenolic acidtocopherolcarotenesterpenes
enzymaticsuperoxide dismutase (SOD)catalaseperoxidasemyeloperoxidaseCofactorCu/Zn SOD (cytoplasm) Mn/SOD (mitochondria)
non-enzymaticglutathionecarnosinehistidineuric acid
repair mechanism
DNA-repair enzymesprevention mechanism
inhibition of radical generation (scavenger activity)stop radical chain reaction
stabilization of biological sites
antioxidant defence system
endogenous exogenous(dietary source)ascorbic acid phenolic acidtocopherolcarotenesterpenes
enzymaticsuperoxide dismutase (SOD)catalaseperoxidasemyeloperoxidaseCofactorCu/Zn SOD (cytoplasm) Mn/SOD (mitochondria)
non-enzymaticglutathionecarnosinehistidineuric acid
repair mechanism
DNA-repair enzymesprevention mechanism
inhibition of radical generation (scavenger activity)stop radical chain reaction
stabilization of biological sites
1. Métodos eletroquímicos
23
and Burdon, 1994). The group of non-radical compounds contains a large variety of
substances, some of which are extremely reactive. Among these compounds produced in
high concentration in the living cell are the hypochlorous acid (HClO) and the hydrogen
peroxide (H2O2) (Kohen and Nyska, 2002).
Several analytical methods have been developed in order to measure the total or
individual antioxidant capacity of foodstuff, beverages, processed food and biological
samples. The quantification of the individual antioxidants present in samples can be
performed by chromatographic techniques and using several extraction techniques, like
solid-phase microextraction (SPME), supercritical fluid extraction (SFE) and microwave-
assisted extraction (MAE) (Abidi, 2000; El-Agamey et al., 2004; Valls et al., 2009).
However, considering the complexity of the composition of foods and biological samples
separating each antioxidant compound and studying it individually is costly and inefficient,
notwithstanding the possible synergistic interactions among the antioxidant compounds in
a sample. This is the case of the formation of highly active antioxidant compounds from
less active promoters or positive interactions between two antioxidants with different
action pathway. Therefore, it is very appealing for researchers to have methods for the
quick quantification of antioxidant effectiveness (Huang et al., 2005).
The conventional analytical methods used for the quantification of the total antioxidant
capacity can be divided in two major mechanisms, based on the chemical reaction
involved: Hydrogen atom transfer (HAT) and single electron transfer (SET). HAT-based
methods measure the ability of an antioxidant to quench radicals by hydrogen donation,
while SET-based methods measure the ability of an antioxidant to transfer one electron to
reduce any compound, including metals, carbonyls and radicals (Huang et al., 2005; Prior
et al., 2005). SET and HAT mechanisms almost always occur together, with the balance
determined by antioxidant structure and pH (Prior et al., 2005). The assessment of
antioxidant capacity using the HAT reactions mechanisms can be performed by several
techniques, such as the oxygen radical absorbance capacity (ORAC), total radical-
trapping antioxidant parameter (TRAP), total oxidant scavenging capacity (TOSC), β-
carotene bleaching by ROO•, and low-density lipoprotein (LDL) oxidation. On the other
hand, the SET reaction mechanisms can be used in various analytical methods, like ferric
reducing antioxidant power (FRAP), trolox equivalent antioxidant capacity assay (TEAC),
DPPH-based assay (2,2-diphenyl-1-picrylhydrazyl), total phenolic assay by Folin-
Ciocalteu (FC). These methods present some disadvantages such as long analysis time,
pre-treatment of coloured samples, expensive equipment requirements, and correction for
interfering substances. These analytical methods and the reactions behind them have
been very well described previously (Sánchez-Moreno, (2002) and Prior et al., (2005)).
I. Revisão do estado da arte
24
The use of electrochemical devices for the assessment of the total antioxidant capacity
in samples is a good alternative to the optical methods. In general, they do not require
sophisticated and expensive equipment, are very sensitive and easy to miniaturize, which
pave the way to portability. In the particular case of antioxidant assessment, some
antioxidant capacity assays are based on electron transfer reactions that occur in food
samples, which can be easily monitored by electrochemical techniques. Additionally,
natural antioxidants included in the diet usually exhibit a non negligible native
electroactivity that can be exploited for detection. In that sense, the electrochemical
detection of the antioxidant is a direct measure of the total reducing power of the
antioxidant compounds present in samples, without the use of reactive species (Blasco et
al., 2007).
This paper highlights the most recent electroanalytical methodologies (since 2007) for
detection of individual antioxidants or the total antioxidant capacity in several kinds of
matrices. Four electrochemical approaches for this purpose are described. The simplest
one is based on the direct electrochemical evaluation of antioxidant on bare electrode
surfaces. The other three strategies rely on modification of the electrode surfaces. A
distinction between chemically and biologically modified electrodes is herein established
mainly because the difference in the principle of measurement. Among the latter group,
enzyme-based and DNA-based sensors are revised separately.
2. Direct electrochemical detection of antioxidants
In the last twenty years, a great number of electrochemical approaches have been
developed for the direct determination of the total or individual antioxidants present in
beverages/food. For the total antioxidant content, cyclic voltammetry (CV), is the most
widely used electrochemical technique although differential pulse voltammetry (DPV) and
square wave voltammetry (SWV) are advantageous in terms of sensitivity for individual
detection.
As reducing agents, antioxidants tend to be easily oxidized at bare electrodes such as
glassy carbon (GCE) (Intarakamhang et al., 2011) and Pt (Pisoschi et al., 2009)
electrodes (Fig. 3A). In general, this technique allows the evaluation of the antioxidant
capacity by measuring the charge associated to the oxidation process of the antioxidants
present in the sample, that is, measuring the area under the corresponding anodic waves.
It is worth noting that this method of estimating the antioxidant capacity assumes that the
reducing power is an absolute measure of the antioxidant capacity, which is open to
discussion (Cheng and Li, 2004). The antioxidant capacity is actually a wider concept
including radical scavenging efficiency, metal chelating capacity and oxidative enzyme
1. Métodos eletroquímicos
25
inhibition capacity (Huang et al, 2005). In spite of the complexity of food samples that
frequently contain more than one antioxidant compound, the potential at which the
oxidation takes place enables the identification of the type of antioxidant involved
(oxidizable functional group). Additionally, the peak potential (Ep) is an indication of the
reducing power because the higher the potential the more difficult the oxidation is
indicating a less reducing power. Total phenolic content can also be electrochemically
evaluated.
It has been suggested that isoflavonoids are more advantageous for human health than
flavonoids because of their cardiovascular protective properties as well as antioxidant
activity. Consistenly with this statement isoflavonoid genistein presented a lower Ep than
flavonoid apigenin. This higher antioxidant activity was confirmed by TEAC assay (Han et
al., 2009).
As mentioned in the introduction, the antioxidant capacity can be estimated by a large
number of methods. However, the values obtained only reflect the chemical reactivity
under the experimental conditions of the assay. For this reason it is not unexpected that
polyphenolic compounds showed different antioxidant capacities depending on the
spectrophotometric assay used. CV provides helpful information to understand this
behaviour. Two flavonols, kaempferol and morin that differ in a single hydroxyl group
underwent a first oxidation at 4′-OH in their B-ring. However, the Ep of kaemperol was 80
mV less positive than the morin one, which clearly explains its higher antioxidant capacity
when exposed to oxidants unable to oxidize the additional 2’-OH that is only present in
morin. On the contrary, under stronger oxidants conditions, morin exhibited almost twice
antioxidant capacity because two oxidation processes took place (He et al., 2009).
Piljac-Žegarac (2010) used CV to study the electrochemical properties of antioxidants
present in fruit tea infusions as well as to estimate the antioxidant capacity. Three
compounds were identified through their redox processes. The easiest oxidized
compound (Ep=130 mV) was ascribed to the oxidation of the ene-diol of ascorbic acid.
The low magnitude of its current indicated a very low concentration in tea infusions, which
maybe a consequence of its thermal instability in the boiling water used for preparation. A
quasi-reversible redox process (Eº’=395 mV) was attributed to the oxidation of the ortho-
dihydroxy-phenol and gallate group of low formal potential phenolics because the resulting
product is a stable quinone, which was further reduced in the backward scan. The less
oxidizable compound presented an irreversible oxidation process between 670 and 700
mV, which was ascribed to the oxidation of the monophenol group or the meta-diphenols
on the A-ring of flavonoids that led to a phenoxy radical or a phenoxonium ion undergoing
successively secondary reactions. The antioxidant capacity of these fruit teas was
determined by estimating the integrated area under the peak up to 600 mV.
I. Revisão do estado da arte
26
Fig. 3 . Principle of operation for the four electrochemical sensing approaches described for
antioxidant quantification
The CV profile of flavonoids (quercetin and its glucosides) in onions was also studied
(Zielinska et al., 2008). The registered cyclic voltammogram showed up to four anodic
waves that were associated to quercetin, quercetin-3,4’-diO-β-glucoside, quercetin-3-O-β-
glucoside and quercetin-4’-O-β-glucoside, from most to less oxidizable compounds. The
data provided by CV were confirmed by HPLC-UV-MS. The quantitative analysis in terms
of total antioxidant capacity was carried out by measuring the total charge (area under the
anodic waves). This value was referred to the charge corresponding to the anodic wave of
increasing concentrations of trolox standards. This method can be considered an
alternative method for the quercetin glucosides quality control without sample
pretreatment. Comparison with other spectrophotometric methods was also discussed.
The CV method was also applied to the evaluation of the antioxidant capacity of dark- and
light-grown buckwheat (Zielinska et al., 2007). The broad anodic wave observed indicated
the presence of several antioxidants such as flavonoids, phenolic acids and water-soluble
vitamins (B1, B6, C).
Native DNA signal
Damaged DNA signal
DNA signal (antioxidant andfree radical)
e-
O2Antioxidant
(Antioxidant)ox
TyrLac
H2O
e-
O2Antioxidant
(Antioxidant)ox
TyrLac
H2O(Med)rd
(Med)ox
e-Antioxidant
(Antioxidant)ox
Direct oxidation on bare electrodes
Modified electrodes
CME
enzymatic
DNA
(A)
(B)
(C)
(D)
(E)
e-
(Med)rd
(Med)oxAntioxidant
(Antioxidant)ox
Native DNA signal
Damaged DNA signal
DNA signal (antioxidant andfree radical)
Native DNA signal
Damaged DNA signal
DNA signal (antioxidant andfree radical)
Native DNA signal
Damaged DNA signal
DNA signal (antioxidant andfree radical)
e-
O2Antioxidant
(Antioxidant)ox
TyrLac
H2O
e-
O2Antioxidant
(Antioxidant)ox
TyrLac
H2O(Med)rd
(Med)ox
e-Antioxidant
(Antioxidant)ox
Direct oxidation on bare electrodes
Modified electrodes
CME
enzymatic
DNA
(A)
(B)
(C)
(D)
(E)
e-
(Med)rd
(Med)oxAntioxidant
(Antioxidant)ox
e-
O2Antioxidant
(Antioxidant)ox
TyrLac
H2O
e-
O2Antioxidant
(Antioxidant)ox
TyrLacTyrLac
H2O
e-
O2Antioxidant
(Antioxidant)ox
TyrLac
H2O(Med)rd
(Med)ox
e-
O2Antioxidant
(Antioxidant)ox
TyrLacTyrLac
H2O(Med)rd
(Med)ox
e-Antioxidant
(Antioxidant)ox
e-Antioxidant
(Antioxidant)ox
Direct oxidation on bare electrodes
Modified electrodes
CME
enzymatic
DNA
(A)
(B)
(C)
(D)
(E)
e-
(Med)rd
(Med)oxAntioxidant
(Antioxidant)ox
e-
(Med)rd
(Med)oxAntioxidant
(Antioxidant)ox
1. Métodos eletroquímicos
27
(Makhotkina and Kilmartin, 2010) proposed several analysis in wines. It is known that
polyphenol catalyzed the oxidation of sulfur dioxide. Cyclic voltammograms of SO2-
containing wines showed a catalytic current due to the polyphenol-mediated oxidation of
SO2. Addition. of acetaldehyde suppressed this current because actetaldehyde interacts
with SO2 in solution, reducing its availability to be catalytically oxidized. So, the content of
free sulfur dioxide can be estimated. The simplicity and quickness of this approach make
it a promising alternative method to the classic titration procedure. The total phenolic
content and the flavonol level was estimated by measuring the area under the CV up to
1.2 V and the peak current at 1.12 V, respectively. The current intensity of the anodic
wave at about 450 mV allowed the estimation of catechol and galloyl-containing
compounds content. In all cases much better correlations were obtained for white wines
than for red ones. This was attributed to the more complex composition of red wines that
can not be compared to a mixture of a limited number of model compounds as the
standards used for calibration.
Burratti et al., (2008) developed a renewable, low-cost flow injection analysis (FIA)
system composed of a pencil (graphite) working electrode and a adapted PVC Pasteur
pipette as a wall-jet flow cell, for the determination of reducing power and total phenolic
content of several types of tea infusions. These inexpensive electrodes have been
previously used for other purposes and showed excellent electrochemical features such
as high sensitivity, low background current, wide potential window and chemical inertness.
The reducing power and the total phenolic content were quantified by measuring the
oxidation current at +0.5 V (from the most easily oxidized compounds) and +0.8 V (all
polyphenols), respectively. The total phenolic content was correlated to FC phenolic index
and the reducing power to the DPPH assay.
Pisoschi et al. developed a chronobiamperometric method for the detection of natural
juices. The radical DPPH• can interact with antioxidants leading to DPPH. The analytical
signal was the differential current measured at two Pt electrodes polarized at a small
potential difference in the presence of the reversible redox couple DPPH•/DPPH. While
the reduction took place in a Pt electrode the oxidation is monitored in the other one. In
the presence of antioxidants the differential current increased as a consequence of the
consumption of radical (Pisoschi et al., 2009).
All these direct electrochemical methods described are rapid, reliable, easy to carry out
and in general, potentials below 700 mV are applied, which improves the selectivity. This
is consistent with the low oxidation potential commonly found in food and biological
samples informing about their high antioxidant capacity (Blasco et al., 2007). Table 1
summarizes the approaches reported for the direct electrochemical evaluation of the
antioxidant status.
I. R
evis
ão d
o es
tado
da
arte
28
Tab
le 1
. M
ain
anal
ytic
al fe
atur
es o
f dire
ct e
lect
roch
emic
al d
etec
tion
of a
ntio
xida
nts
Ant
ioxi
dant
S
ampl
e V
olta
mm
etric
tech
niqu
e (e
lect
rode
) Li
near
ran
ge
Lim
it of
det
ectio
n R
efer
ence
Asc
orbi
c ac
id
Tab
lets
, fru
it ju
ice,
he
rbal
tea
extr
act
DP
V (
GC
E)
0.1-
8.0
mM
50
µM
In
tara
kam
hang
et a
l., 2
011
Cat
echi
n, q
uerc
etin
, rut
in
- C
V, D
PV
, SW
V (
GC
E)
- -
Med
vido
vić-
Kos
anov
ić e
t al
., 20
10
Isor
ham
netin
T
able
ts
CV
, DP
V (
GC
E)
10 -
100
nM
5.0
nM
Liu
et a
l., 2
008
Ant
ioxi
dant
cap
acity
F
ruit
tea
infu
sion
C
V (
GC
E)
- -
Pilj
ac-Ž
egar
ac e
t al,
2010
T
otal
pol
yphe
nols
F
ree
SO
2 W
hite
and
red
win
e C
V (
GC
E)
- -
Mak
hotk
ina
and
Kilm
artin
20
10
Tot
al o
xida
nt-s
cave
ngin
g ca
paci
ties
B
lood
pla
sma
CV
(G
CE
) -
- K
oren
et a
l., 2
009
Tot
al a
ntio
xida
nt c
apac
ity
Fru
it ju
ice,
sof
t drin
k C
V (
Pt)
-
76.8
nM
P
isos
chi e
t al.,
200
9 2-
Sty
rylc
hrom
ones
(f
lavo
ne)
- C
V (
GC
E)
- -
Gom
es e
t al.,
200
8
Que
rcet
in
Oni
ons
CV
(G
CE
) -
- Z
ieliń
ska
et a
l., 2
008
Toc
ophe
rol
- C
V (
GC
E)
- -
Yao
et a
l., 2
008
Pom
iferin
, Is
opom
iferin
, O
sajin
C
atal
posi
de
- S
WV
(C
PE
) 0.
1-10
0 ng
mL-1
1–12
ng
mL-1
0.
1-1
ng m
L-1
0.1-
1 ng
mL-1
50 p
g m
l-1
800
pg m
l-1
40 p
g m
l-1
10 n
g m
l-1
Dio
pan
e ta
l., 2
008
Pol
yphe
nols
W
ines
(G
CE
) -
- F
ell e
t al.,
200
7 ph
enol
ic a
cids
-
CV
(G
CE
) -
- S
imić
et a
l., 2
007
α-Li
poic
aci
d D
ieta
ry s
uppl
emen
ts
CV
; DP
V; S
WV
2.
5-75
µM
1.
8 µ
M
Cor
dune
anu
et a
l., 2
007
1. Métodos eletroquímicos
29
3. Chemically modified electrodes for detection of antioxidants
The finding that rational modification of a conductive substrate can lead to electrode
surfaces possessing not only the properties of the substrate but also those of the
immobilized compound has paved the way to the development of chemically modified
electrodes (CMEs). Since then, the field of CMEs is an area of growing interest because
of their convenience for a great variety of fundamental studies and applications.
IUPAC defines a CME as an electrode made of a conducting or semiconducting material
that is coated with a selected monomolecular, multimolecular, ionic, or polymeric film of a
chemical modifier and that by means of faradaic (charge-transfer) reactions or interfacial
potential differences (no net charge transfer) exhibits chemical, electrochemical, and/or
optical properties of the film (Durst et al., 1997). The substrate is the platform on the
modifier is assembled and it is selected among the electrode materials available according
to the desired properties, e.g., mechanical and chemical stability, resistance to fouling etc.
The modifying layer can be fixed at the electrode surface by different techniques: i)
physical adsorption; ii) formation of organized monolayers (self-assembly) iii)
electropolymerization; iv) covalent attachment; v) entrapment in a polymer or inorganic
film; and vi) incorporation into an electrode matrix. The discussion about the preparation
and properties of the different layers is out of the scope of this revision but the general aim
is to obtain enhanced properties such as faster electron transfer, increased current and
elimination of interferences. The detection of antioxidants can also benefit from these
modifications as discussed below.
Ascorbic acid (AA) is electroactive and can be directly detected on bare electrodes.
However, the presence of interfering compounds that oxidize at similar potentials such as
dopamine (DA), is an important drawback. Immobilization of cationic surfactants on the
electrode surface made more difficult the oxidation of positively charged DA because of
electrostatic repulsion. Therefore, the Ep shifted to more positive potentials improving the
peak separation up to 200 mV using docosyltrimethylammonium chloride adsorbed on
GCE (Luo et al., 2010).
Adsorption is the simplest immobilization method for carbon nanotubes. This twenty-
year old material is an excellent promoter of the electron transfer and electrocatalytic
activity, so it is not rare that has been tested for the detection of uncountable molecules
including antioxidants. Not only single-walled carbon nanotubes (SWCNTs) increased the
electrochemical area of the GCE twice but also the electron transfer was accelerated and
the current of catechin increased by a factor of 10 due to their high adsorption capacity.
Consequently, subnM concentrations were detected in combination with DPV, which is
more sensitive than CV (Yang et al., 2009). Other carbon materials such as graphene
I. Revisão do estado da arte
30
nanosheets (Du et al., 2010) and acetylene black nanoparticles (Song et al., 2010) were
used for rutin determination taking advantage of their high number of adsorption active
sites. A technique directly based on the enhanced sensitivity associated to accumulation
processes is adsorptive stripping voltammetry (AdSV). Using a lead film plated on GCE,
rutin was accumulated and further oxidized by SWV allowing the detection of subnM
concentrations. The main drawback was the toxicity of the electrode material (Tyszczuk,
2009).
In many cases the molecule immobilized is a redox mediator that shuttles electrons from
the antioxidant to the electrode surface reducing the potential at which the oxidation of the
antioxidant occurs. This is advantageous in order to eliminate interferences. The mode of
action is depicted in Fig. 3B. The oxidized form of the immobilized mediator is able to
oxidize the antioxidant chemically in the electrode-solution interface. The reduced
mediator is then electrochemically oxidized on the electrode surface, which generates
more oxidized mediator available for oxidizing the antioxidant (catalytic cycle). As a result
an increased anodic current is observed (catalytic current) increasing the sensitivity of the
determination.
Films of mediators can be electrogenerated on the electrode substrate. This is the case
of o-aminophenol that was electrografted on GCE and effectively catalyzed the oxidation
of AA in the presence of organic hydroxyacids and sugars commonly found in fruit juices.
The sensor was successfully applied to AA detection in commercial fruit juices (Civit et al.,
2008). Electropolymerization of aspartic acid (Wang et al. 2010) or glutamic acid (Santos
et al., 2007b) also resulted in higher oxidation currents of catechin and rutin, respectively
because of increased adsorption and kinetics, which allowed the analysis of catechin in
several types of teas with good recoveries and rutin in pharmaceutical formulations. Poly
(3-(3-Pyridyl) acrylic acid (Zhang et al., 2007b), polycaffeic acid (Li et al., 2008) and p-
aminobenzene sulfonic acid doped polyaniline (Zhang et al., 2008b) films also promote
the peak separation of AA and DA.
Carbon pastes are easily modified by incorporation of modifiers into the own material
(graphite powder and a binder, e.g. parafilm oil, nujol, silicon). This method of
immobilization is simple, fast and the resulting electrode surface is easily renewable by
polishing or discarding after measurement and immediately replacing by a new one from
the large amount previously prepared.
Ionic liquids can totally or partially replace the classical binders in carbon pastes
because they exhibit high ionic conductivity and a wide potential window. In spite of the
dramatic increase in capacitive current, the concomitant increase in faradaic current
allowed the determination of rutin, although the detectability was highly dependent on the
ionic liquid selected (Sun et al., 2008; Zhang and Zheng, 2008).
1. Métodos eletroquímicos
31
Cobalt 5-nitrosalophen, a well known mediator of the oxidation of AA, was introduced
into carbon paste along with a cationic surfactant to minimize the interference of DA by
peak separation (about 374 mV) (Shahrokhian and Zare-Mehrjardi, 2007b). The limit of
detection was improved in comparison with the docosyltrimethylammonium chloride GCE
(Luo et al., 2010) probably due to the presence of the mediator. A better peak resolution
(395 mV) and an order of magnitude lower limit of detection were achieved by preparing a
MWCNTs-thionine-nafion carbon composite (Shahrokhian and Zare-Mehrjardi, 2007a).
This material without nafion also exhibited a good discrimination ratio towards other
interfering drugs, a very useful feature for AA determination in human blood plasma
(Shahrokhian and Asadian, 2010).
Micromolar concentrations of rutin cannot be detected on bare CPE. However, after
addition of poly(vinylpyrrolidone) to the carbon matrix, a significant peak was observed.
This is due to the strong adsorption of phenolic compounds on this polymer. In spite of the
high oxidation potential, the CME did not show interferences in pharmaceutical
formulations (Franzoi et al., 2008).
Fatibello-Filho’s group has exploited the catalytic properties of copper immobilized on
polyester resin incorporated into carbon matrix to detect catechin in teas (Freitas and
Fatibello, 2010a), rutin in pharmaceutical preparations (Freitas et al., 2009) and the
synthetic phenolic antioxidants butylated hydroxyanisole (BHA) and butylated
hydroxytoluene (BHT) (Freitas and Fatibello, 2010b). These phenolic compounds have to
be controlled in food samples because it is believed to cause nutrient loss and have
potential toxic effects. In spite of the high detection potential, the method showed good
recoveries in mayonnaise samples and compared well with the most commonly used
HPLC method. BHA was also monitored on films of nickel hexacyanoferrate (NiHCF)
generated on p-phenylendiamine modified carbon paste. The electrocatalytic effect of
NiHCF allowed the detection of BHA at relatively low potentials, 400 mV, in a flow
injection system with excellent reproducibility and stability. The interference of more easily
oxidized water-soluble compounds such as AA was eliminated during the sample
pretreatment but the signal from propyl gallate could not be prevented (Prabakar and
Narayanan, 2010).
Molecular recognition is another strategy to improve the selectivity of the detection by
preconcentration on specific receptors. An example of this approach is the ability of
cyclodextrins to form inclusion complexes with organic and inorganic compounds
depending on the size of their cavity that was applied to the selectively detection of
catechin in different beverages. Slightly higher sensitivities were achieved by SWV than
DPV (El-Hady, 2007; El-Hady and El-Maali, 2008).
I. Revisão do estado da arte
32
A completely different approach is based on the correlation between the antioxidant
properties of flavonoids and other phenolic compounds and their ability to promote the
enlargement of AuNPs (Wang et al., 2007) or Au nanoshells (Ma and Qian, 2010). The
Au seeds were immobilized on SAMs of cysteamine or on aminopropyltriethoxysilane-
modified ITO electrodes in the formed of SiO2-Au nanocomposite, respectively. Equimolar
concentrations of ferrycianide/ferrocyanide or ferricyanide alone were used as probes in
solution to test the growth of AuNPs. In the presence of flavonoids the redox process of
the probe clearly diminished indicating that its access to the electrode surface is blocked.
This was attributed to the growth of the Au seeds induced by reducing activity of
flavonoids or phenolic compounds. This resistance to the electron transfer linearly
correlated with the concentration of antioxidant in different ranges, which allowed
comparing the antioxidant capacity of several pure compounds. Since this methodology
cannot distinguish the origin of the antioxidant power in mixtures, it was used for the
assessment of the total antioxidant capacity of herbal extracts (Wang et al., 2007).
Self-assembly of thiolated compounds is a spontaneous process on Au surfaces that has
become very common for immobilization of molecules. Recently, a nickel (II) complex was
prepared on the surface of a mercaptopropanoic acid monolayer. This complex was able
to oxidize catechin on the interface, which was subsequently reduced on the electrode
surface. Results from SWV compared well with those from capillary electrophoresis
(Moccelini et al., 2009). In table 2 the main characteristics of the CMEs applied to
antioxidant detection is summarized.
4. Enzymatic biosensors for antioxidant evaluation
Electrochemical biosensors, a subclass of chemical sensors, combine the excellent
detectability of electrochemical transducers with the high selectivity of biological
recognition elements such as enzymes, proteins, antibodies, nucleic acids, etc. Since the
principle of measurement is different, in this section, biosensors employing enzymes for
antioxidant detection are revised while the DNA-based biosensors are discussed in
section 5.
33
Tab
le 2
. C
hem
ical
ly m
odifi
ed e
lect
rode
dev
ices
for
the
antio
xida
nt m
easu
rem
ent.
Ant
ioxi
dant
S
ampl
e Im
mob
iliza
tion
proc
edur
e (e
lect
rode
) T
echn
ique
Li
near
ran
ge
Lim
it of
det
ectio
n R
efer
ence
Asc
orbi
c ac
id
Inje
ctio
n D
ocos
yltr
imet
hyla
mm
oniu
m c
hlor
ide
(GC
E)
DP
V
0.01
-1.0
mM
4.
0 µ
M
Luo
et a
l, 20
10
Cat
echi
n -
SW
CN
Ts-
CT
AB
(G
CE
) D
PV
0.
372–
2.38
nM
0.
112
nM
Yan
g et
al.,
200
9 R
utin
T
able
ts
Gra
phen
e na
nosh
eets
(G
CE
)
CV
0.
1-10
µM
21
nM
D
u et
al.,
201
0 R
utin
ch
ines
e m
edic
ines
ac
etyl
ene
blac
k na
nopa
rtic
les
(GC
E)
DP
V
20-5
0 µ
g L-1
10
µg
L-1
Son
g et
al.,
201
0 R
utin
T
able
ts
Pb
film
(G
CE
) A
dSV
0.
5 -1
0 nM
0.
25 n
M
Tys
zczu
k, 2
009
AA
F
ruit
juic
es
o-am
inop
heno
l film
(G
CE
) am
pero
met
ry
2-20
µM
0.
86 µ
M
Civ
it et
al.,
200
8 C
atec
hin
Tea
bev
erag
e P
oly-
aspa
rtic
aci
d fil
m (
GC
E)
D
PV
0.
25-3
0 µ
M
72 n
M
Wan
g an
d F
an, 2
010
Rut
in
Pha
rmac
eutic
al
form
ulat
ion
Pol
yglu
tam
ic a
cid
(GC
E)
SW
V
0.7-
10 µ
M
- S
anto
s et
al.
2007
Asc
orbi
c ac
id
- P
oly
(3-(
3-P
yrid
yl)
Acr
ylic
(G
CE
) C
V
10-4
00 µ
M
0.8
µM
Z
hang
, et a
l., 2
007b
A
scor
bic
acid
P
harm
aceu
tical
s P
olyc
affe
ic a
cid
(G
CE
) C
V
0.2-
1.2
mM
9.
0 µ
M
Li e
t al.,
200
8 A
scor
bic
acid
T
able
ts, u
rine
p-am
inob
enze
ne s
ulfo
nic
acid
dop
ed p
olya
nilin
e (G
CE
) D
PV
35
-175
µM
7.
5 µM
Z
hang
et a
l., 2
008b
Rut
in
Tab
lets
io
nic
liqui
d (C
PE
) C
V
0.5-
100
µM
0.
35 µ
M
Sun
et a
l., 2
008
Rut
in
Tab
lets
, urin
e H
ydro
phili
c io
nic
liqui
d (C
PE
) S
WV
0.
04-1
0 µ
M
10 n
M
Zha
ng a
nd Z
heng
, 200
8 A
scor
bic
acid
-
MW
CN
TS
/Naf
ion-
coba
lt(II)
-nitr
osal
ophe
n (C
PE
) D
PV
0.
5-10
0 µ
M
0.1
µM
S
hahr
okhi
an a
nd Z
are-
Meh
rjard
i, 20
07b
Asc
orbi
c ac
id
- T
hion
ine-
nafio
n-M
WC
NT
s (C
PE
)
DP
V
0.1-
80.0
µM
0.
08 µ
M
Sha
hrok
hian
and
Zar
e-M
ehrja
rdi,
2007
a
Asc
orbi
c ac
id
Hum
an b
lood
se
rum
T
hion
ine-
nafio
n-M
WC
NT
s (C
PE
)
DP
V
1-10
0 µ
M
0.8
µM
S
hahr
okhi
ana
and
Asa
dian
a 20
10
Asc
orbi
c ac
id
Pha
rmac
eutic
al
prep
arat
ion
Cob
alt(
II)-n
itros
alop
hen-
tetr
aoct
ylam
mon
ium
bro
mid
e (C
PE
) D
PV
1-
100
µM
0.7
µM
S
hahr
okhi
an a
nd Z
are-
Meh
rjard
i, 20
07c
Rut
in
Pha
rmac
eutic
al
form
ulat
ions
po
ly(v
inyl
pyrr
olid
one
(CP
E)
LSV
0.
39 -
13.0
µM
0.15
µM
F
ranz
oi e
t al.,
200
8
Cat
echi
n T
eas
Cu
(II)
- po
lyes
ter
resi
n (C
PE
)
SW
V
0.09
9-1.
2 µ
M
58 n
M
Fre
itas
and
Fat
ibel
lo-
Filh
o 20
10a
BH
A/B
HT
F
ood
sam
ples
C
u (I
I) p
olye
ster
res
in (
CP
E)
S
WV
0.
34-4
1.0
µM
(bot
h)
72 n
M, 9
3 nM
F
reita
s F
atib
ello
-Filh
o,
2010
b
BH
A
Spi
ked
pota
to c
hips
N
iHC
F-
(gra
phite
com
posi
te)
FIA
, DP
V
1.2-
1070
µM
0.
6 µ
M
Pra
baka
r et
al.
2010
C
atec
hin
T
ea
Bev
erag
es
Inco
rpor
atio
n of
β-
cycl
odex
trin
(C
PE
)
SW
V
SW
V
DP
V
Up
to 7
0 µ
g m
L-1
0.00
1-7.
2 µ
g m
L-1
0.00
2-4.
2 µ
g m
L-1
1.35
µg
mL-1
0.12
ng
mL-1
0.
30 n
g m
L-1
El-H
ady
and
El-M
aali
2008
E
l-Had
y 20
07
34
Ant
ioxi
dant
S
ampl
e Im
mob
iliza
tion
proc
edur
e (e
lect
rode
) T
echn
ique
Li
near
ran
ge
Lim
it of
det
ectio
n R
efer
ence
Que
rcet
in
daiz
eol
puer
arin
Her
bal e
xtra
cts
Au/
cyst
eam
ine/
AuN
Ps
C
V
10-1
00 µ
M
0.1-
10 µ
M
0.5-
1 µ
M
1 nM
10
nM
10
0 nM
Wan
g et
al.,
200
7a
Siri
ngic
aci
d -
Gol
d na
nosh
ells
(IT
O)
CV
5-
100
µM
-
Ma
and
Qia
n, 2
010
Cat
echi
n G
reen
tea
Ni(I
I) c
ompl
ex o
n S
AM
S
WV
3.
31-2
5.3
µM
0.
826
µM
M
occe
lin e
t al.,
200
9 A
scor
bic
acid
F
ood
Film
of b
inuc
lear
Cu
com
plex
(G
CE
) D
PV
5.
0-16
0.0
µM
2.
8 µ
M
Wan
g et
al.,
200
7b
Rut
in
Tab
lets
P
oly(
p-am
inob
enze
ne s
ulfo
nic
acid
) (
GC
E)
DP
V
0.25
- 10
.0 µ
M
0.1 µ
M
Che
n et
al.,
201
0
LSV
- lin
ear
swee
p vo
ltam
met
ry.
1. Métodos eletroquímicos
35
The enzyme is the most critical component of the enzyme electrode since it provides the
selectivity for the sensor and catalyzes the formation of an electroactive product for
detection. Recovery of enzymes from solution is, in general, very expensive, so their
immobilization on the transductor allows their reusability, which contributes to a reduction
in the analysis cost. This fact along with the feasibility of obtaining enzyme preparations of
high purity at a reasonable price has fuelled the construction of enzyme electrodes. It is
expected that this trend will continue because of their relative simplicity and rapid
analysis. For antioxidant detection oxidase enzymes such as polyphenol oxidase (PPO)
(Tan et al., 2011), tyrosinase (Cortina-Puig et al., 2010; El Kaoutit et al., 2007; Singh,
2011; Wang and Hasebe, 2011), laccase (El Kaoutit et al., 2008, Di fusco et al., 2010;
Ibarra-Escutia et al., 2010; Tan et al. 2009), horseradish peroxidise (HRP) (Santos et al.,
2007), ascorbate oxidase (Chauhan et al., 2010; Pisoschi, et al., 2010; Vig et al., 2010;
Wang et al., 2008) or uricase (Moraes et al., 2007; Zhang et al., 2007) are used. These
redox enzymes are very convenient for electrochemical detection because the product
monitored usually exhibits a redox process at low potentials, where the electrochemical
interferences are minimized.
Immobilization of enzymes on the electrode surface is a critical step for the successful
design of a biosensor. A great variety of approaches have been reported as it is summed
up in table 3. All of them try to obtain the highest enzyme loading without losing activity,
minimizing its leaching from the electrode surface to the bulk solution and inactivation of
the layer by radicals generated in the enzymatic reactions or polymerization of the product
formed as in the case of polyphenols.
Antioxidant detection by means of enzymes mostly relies on the measurement of the
reduction current of the enzymatically oxidized antioxidant as schematically depicted in
Fig. 3C. Tyrosinase and laccase are Cu containing oxidases that catalyses the reduction
of oxygen to water in the presence of phenolic compounds. Most methods for phenolic
compounds cited in table 3 measures the reduction current of the corresponding quinone
formed. The use of a redox mediator along with the enzyme (Fig. 3D) was also proposed
for the detection of catechol on Pt at +0.65 (Akyilmaz et al., 2010). Gallic acid (Di Fusco et
al., 2010), caffeic acid (Cortina-Puig et al., 2010), catechol (Zejli et al., 2008) or rosmarinic
acid (Diaconu et al., 2010) among others have been selected as standards for the
assesment of phenolic content in food and environmental samples. HRP can also catalyze
the conversion of some phenolic compounds. This enzyme along with an adsorbed
mediator (methylene blue) improved the reduction current of catechol (Santos et al.,
2007a).
Measurement of oxygen consumption after enzymatic reaction with Clark electrodes is
another possibility reported for the detection of ascorbic (Pisoschi et al. 2010) or uric acids
I. Revisão do estado da arte
36
(Zhang et al., 2007a). A combination of two enzymes, uricase and microperoxidase-11
allowed the detection of uric acid by monitoring the formation of H2O2 (Behera and Raj,
2007). To eliminate interferences, a flow injection system with differential measurement
was reported for AA. An enzyme cell was constructed with ascorbate oxidase immobilized
in a membrane and the electrochemical cell was placed separately. The sample was
injected twice, after the enzyme cell to measure the total oxidation current, and before the
enzyme cell to measure the decreased oxidation current due to the enzymatic depletion of
AA (Vig et al., 2010).
A different strategy is based on the scavenging activity of antioxidant against ROS.
Superoxide radical is enzymatically generated by xanthine oxidase (XOD) in the presence
of (hipo)xanthine. Superoxide dismutase causes the dismutation of the radical forming
H2O2, which can be reoxidized on the electrode surface. However, the high potential
needed, 0.65V at Pt electrodes (Campanella et al., 2009), limits the selectivity and practical
applicability. To solve this drawback, immobilization of cytochrome c that is oxidized by the
radical is advantageous. This compound is further reduced at the electrode surface. The
presence of antioxidants diminished the reduction current of cytochrome c by scavenging
the radical formed, resulting in signal off approaches (Cortina-Puig et al., 2009a; Cortina-
Puig et al., 2009b). Table 3 summarizes the more relevant analytical features of successful
applications of enzymatic biosensors for the evaluation of antioxidant status.
5. DNA biosensors for quantification of antioxidant s
Not only enzymes but also nucleic acids can be immobilized on electrochemical
transducers. DNA layers can act as biomolecular recognition elements for diagnostics of
genetic or infection diseases as well as the detection of pathogens in food and
environmental samples taking advantage of one of the most specific reactions known:
hybridization. Likewise the so-called aptamers (synthetic single stranded oligonucleotides)
can act as high affinity receptors similarly to antibodies for a great variety of ligands
(Miranda-Castro et al., 2009). However, the usefulness of DNA layers is not restricted to
these important applications. On the contrary, they can be the target for the antioxidant
assessment by mimicking the damage caused in vivo by ROS. Nucleobases are the main
targets of ROS leading to oxidation of bases and, ultimately to their release but sugar are
also weak points which may result in strand breaking. From this fact, it seems reasonable
that the protective role of antioxidants at a cellular level could be properly studied by
monitoring the DNA integrity. Several DNA-based electrochemical sensors have been
developed for the measurement of the antioxidant capacity of different compounds.
37
Tab
le 3
. E
nzym
atic
bio
sens
ors
for
antio
xida
nt q
uant
ifica
tion.
A
ntio
xida
nt
S
ampl
e
Enz
yme
Im
mob
iliza
tion
proc
edur
e (e
lect
rode
)
Line
ar r
ange
Det
ectio
n lim
it
refe
renc
e
Phe
nolic
su
bstr
ates
P
lant
s La
ccas
e
Ent
rapm
ent i
n ch
itosa
n-M
WC
NT
s co
mpo
site
on
Au
0.91
- 12
µM
(ro
smar
inic
)
0.23
3 µM
D
iaco
nu e
t al.,
201
0
Pol
yphe
nol
Win
es
Lacc
ase
Cro
sslin
ked
with
pol
ymer
on
SW
CN
T-S
PE
or
MW
CN
T-S
PE
0.
1- 1
8.0
mg
L-1 (
galli
c ac
id)
0.3
mg
L- (ga
llic
acid
) D
i fus
co e
t al.,
201
0
Pol
yphe
nolic
co
mpo
unds
W
ines
La
ccas
e M
embr
ane
on P
t ele
ctro
de
5 -3
5 µM
(ca
ffeic
aci
d)
0.88
µM
(ca
ffeic
ac
id)
Júni
or a
nd R
ebel
o 20
08,
Gil
and
Reb
elo
2010
C
atec
hol
- La
ccas
e C
ossl
inke
d to
chi
tosa
n on
MW
CN
T/ G
CE
0.
1-50
µM
20
nM
T
an e
t al.
2009
C
atec
hin
Gre
en te
a
Hum
an u
rine
La
ccas
e co
vale
ntly
imm
obili
zed
on d
endr
imer
-en
caps
ulat
ed A
uNP
s 0.
1-10
µM
0.
05 µ
M
Rah
man
et a
l., 2
008
Ros
mar
inic
aci
d P
lant
ext
ract
La
ccas
e In
corp
orat
ed to
ioni
c liq
uid
carb
on p
aste
0.
99-6
5.4
µM
0.
188
µM
F
ranz
oi e
t al.,
200
9 P
olyp
heno
ls
Bee
r La
ccas
e C
ossl
inki
ng o
nto
sono
gel-c
arbo
n el
ectr
ode
0.04
-2 µ
M (
caffe
ic a
cid)
0.
04-2
µM
(fe
rulic
aci
d)
0.1-
22 µ
M (
galli
c ac
id)
0.04
-3 µ
M (
cate
chin
) 0.
04-8
µM
(ep
icat
echi
n)
0.06
µM
0.
16 µ
M
0.41
µM
0.
10 µ
M
0.16
µM
El K
aout
it et
al.,
200
8
Caf
feic
aci
d C
atec
hol
Hyd
roqu
inon
e R
esor
cino
l
Tea
infu
sion
s La
ccas
e E
trap
men
t in
a m
embr
ane
on S
PE
0.
5-13
0 µ
M
0.5-
175
µM
1.
1-13
0 µ
M
50-2
50 µ
M
0.52
4 µ
M
0.55
8 µ
M
1.07
1 µ
M
5.43
2 µ
M
Ibar
ra-E
scut
ia e
t al.,
201
0
Tot
al p
heno
lic
cont
ent
Pla
nt e
xtra
cts
Lacc
ase
Ads
orbe
d on
a S
PE
7x
10-7
–1.5
x10-6
M
11.9
9x10
-7M
Li
tesc
u et
al.,
201
0
Phe
nolic
co
mpo
unds
-
Lacc
ase
Phy
sica
l ads
orpt
ion
on c
arbo
n ce
ram
ic
elec
trod
e 0.
1-10
µM
(ca
tech
ol)
0.06
µM
H
aghi
ghi e
t al.,
200
7
Phe
nols
and
po
lyph
enol
s B
eers
and
In
dust
rial
was
tew
ater
s
Tyr
osin
ase
Naf
ion
coat
ed o
n so
noge
l-car
bon
elec
trod
e -
64 n
M c
atec
hol
85 µ
M g
allic
aci
d 1.
25 µ
M c
atec
hin
96 n
M p
heno
l 30
nM
4-c
hlor
o-3-
met
hylp
heno
l
El K
aout
it et
al.,
200
7
Cat
echo
l C
affe
ic a
cid
Chl
orog
enic
ac
id
Spi
ked
river
wat
er
Tyr
osin
ase
Alu
min
a so
l-gel
on
sono
gel-c
arbo
n el
ectr
ode
0.1-
30 µ
M
5-30
µM
5-
30 µ
M
30 n
M
0.62
µM
0.
61 µ
M
Zej
li et
al.
2008
Cat
echo
l W
ater
sam
ples
T
yros
inas
e O
n A
uNP
s en
caps
ulat
ed-d
endr
imer
lin
kded
to a
con
duct
ing
poly
mer
on
GC
E
0.00
5- 1
20 µ
M
2 nM
S
ingh
, 201
1
p-ch
loro
phen
ol
p-cr
esol
, P
heno
l,
Cat
echo
l
- T
yros
inas
e C
oval
ent t
o ac
tivat
ed c
arbo
n fe
lt
2.6
-300
0 nM
2.7
-100
0 nM
0019
-10
µM
2.3-
3000
nM
2.6
nM
2.7
nM
18.7
nM
2.
3 nM
Wan
g an
d H
aseb
e, 2
011
38
Ant
ioxi
dant
Sam
ple
E
nzym
e
Imm
obili
zatio
n pr
oced
ure
(ele
ctro
de)
Li
near
ran
ge
D
etec
tion
limit
re
fere
nce
Cat
echo
l A
queo
us a
nd
orga
nic
med
ia
Tyr
osin
ase
Ent
rapm
ent i
nto
laye
red
doub
le h
ydro
xide
-al
gina
te h
ybrid
nan
ocom
posi
te o
n G
CE
2
nM–3
0 µ
M (
aq)
0.01
-3 µ
M (
orga
nic)
0.
5 nM
0.
01 µ
M
Lópe
z et
al.,
201
0
Cat
echo
l,
Cat
echi
n,
Caf
feic
aci
d
Gal
lic a
cid
Tea
sam
ples
T
yros
inas
e O
n di
azon
ium
-fun
ctio
naliz
ed S
PA
uE
0.1-
22 µ
M
2.8-
29 µ
M
0.3-
83 µ
M
2.5-
65 µ
M
0.1
µM
2.8
µM
0.2
µM
2.1
µM
Cor
tina-
Pui
g et
al.,
201
0
Phe
nolic
co
mpo
unds
-
Tyr
osin
ase
Ads
orpt
ion
on n
anoc
ompo
site
(A
u)
0.01
-7 µ
M
5 µ
M
Lu e
t al.,
201
0
Cat
echo
l -
Tyr
osin
ase
Inco
rpor
atio
n to
CP
E
- 0.
008 µ
M
Ard
uini
et a
l., 2
010
Phe
nolic
co
mpo
unds
M
icro
caps
ules
T
yros
inas
e D
rop-
coat
ing
on e
lect
rosp
inne
d m
embr
ane
GC
E
1–10
0 µ
M (
pyro
cate
chol
) 0.
05 µ
M
Are
cchi
et a
l., 2
010
p-cr
esol
P
heno
l 4-
chlo
roph
enol
- T
yros
inas
e A
dsor
bed
on Z
nO n
anor
od c
lust
ers
at
nano
crys
talli
ne d
iam
ond
elec
trod
es
1-21
0 µ
M
1-19
0 µ
M
1-25
0 µ
M
0.2 µ
M
0.5 µ
M
0.4 µ
M
Zha
o et
al.,
200
9a
p-cr
esol
4-
clor
ophe
nol
phen
ol
- T
yros
inas
e C
oval
ently
on
ZnO
nan
orod
s at
na
nocr
ysta
lline
dia
mon
d el
ectr
ode
1-17
5 µ
M
1-15
0 µ
M
1-15
0 µ
M
0.1
µM
0.
2 µ
M
0.25
0M
Zha
o el
al 2
009b
Phe
nolic
co
mpu
nds
Land
fill l
each
ate
T
yros
inas
e O
n m
etha
cryl
ic-a
cryl
ic m
embr
anes
on
SP
CE
6.
2-54
.2 µ
M
0.13
µM
H
anifa
h et
al.,
200
9
cate
chol
-
Tyr
osin
ase
Inco
rpor
ated
to M
WC
NT
-epo
xy c
ompo
site
U
p to
0.1
5 m
M
0.01
mM
Lo
pez
et a
l., 2
009
Tot
al p
heno
lic
com
poun
ds
Red
win
es
Tyr
osin
ase
Ioni
c liq
uid
mod
ified
MW
CN
Ts
on IT
O
0.01
-0.0
8 m
M
- K
im e
l al.,
200
9
Cat
echo
l In
dust
rial
was
tew
ater
P
olyp
heno
l ox
idas
e
Inco
rpor
ated
to p
olya
nilin
e fil
m o
n P
t
2.5-
140
µM
0.
05 µ
M
Tan
et a
l., 2
011
Pol
yphe
nols
T
ea le
aves
, al
coho
lic b
ever
ages
, w
ater
Pol
yphe
nol
oxid
ase
B
SA
-glu
tara
ldeh
yde
cros
slin
king
on
PV
C
mem
bran
e pl
aced
on
Pt
1.25
–10 µ
M
7.5 µ
M
Cha
wla
et a
l., 2
010
Cat
echo
l -
Pol
yphe
nol
oxid
ase
Ele
ctro
poly
mer
izat
ion
on S
PP
t 5-
100
µM
0.
318
µM
A
kyilm
az e
t al.,
201
0
Phe
nolic
co
mpo
unds
S
pike
d riv
er w
ater
P
olyp
heno
l ox
idas
e C
ross
linki
ng a
fter
entr
apm
ent i
n B
iOx
film
on
GC
E
4 nM
-15 µ
M (
cate
chol
) 1
nM
Sha
n et
al.,
200
9
Asc
orbi
c ac
id
Mul
tivita
min
ef
ferv
esce
nt ta
blet
w
hite
win
es
Asc
orba
te
oxid
ase
Flo
w s
yste
m. O
n m
embr
ane
cell
not
dire
ctly
on
the
elec
trod
e su
rfac
e 25
-400
µM
5
µM
V
ig e
t al.,
201
0
Asc
orbi
c ac
id
Ser
um, f
ruit
juic
e,
vita
min
c ta
blet
s A
scor
bate
ox
idas
e C
oval
ent t
o eg
g sh
ell m
embr
ane
on A
uE
10 –
100
µM
1.0 µ
M
Cha
uhan
et a
l., 2
010
Asc
orbi
c ac
id
Fru
it ju
ice
Asc
orba
te
oxid
ase
On
nylo
n m
embr
ane
with
glu
atar
alde
hyde
on
Cla
rk e
lect
rode
0.
10-0
.55
mM
0.
023
mM
P
isos
chi,
et a
l., 2
010
L-as
corb
ic a
cid
Fru
it ju
ice
Asc
orba
te
oxid
ase
Mic
elle
s on
pol
ysty
rene
coa
ted
GC
E
5 -4
00 µ
M
4 µ
M
Wan
g et
al.,
200
8
39
Ant
ioxi
dant
Sam
ple
E
nzym
e
Imm
obili
zatio
n pr
oced
ure
(ele
ctro
de)
Li
near
ran
ge
D
etec
tion
limit
re
fere
nce
Uric
aci
d H
uman
ser
um
Urin
e sa
mpl
es
Uric
ase
On
eggs
hell
mem
bran
e on
a C
lark
sen
sor
4.0-
640
µM
2.
0 µ
M
Zha
ng e
t al.,
200
7
Uric
aci
d C
linic
al te
sts
Uric
ase
Laye
r by
laye
r on
Pru
ssia
n bl
ue-I
TO
0.
1-0.
6 µ
M
0.15
µM
M
orae
s et
al.,
200
7 U
ric a
cid
- m
icro
pero
xida
se-
11
Uric
ase
MxP
-11
was
cov
alen
tly b
ound
to S
AM
U
ricas
e in
to c
hito
san
on M
xP-1
1 el
ectr
ode
5–15
0 µ
M
2 µ
M
Beh
era
and
Raj
. 200
7
Phe
nolic
co
mpu
nds
- H
RP
O
n m
ethy
lene
blu
e-M
WC
NT
- C
PE
1-
150 µ
M
0.5 µ
M
San
tos
et a
l., 2
007
Ant
ioxi
dant
ca
paci
ty
Gar
lic b
ulbs
O
rang
e ju
ices
X
OD
C
oval
ently
atta
ched
to m
ixed
SA
Ms
Alli
n st
anda
rd
Asc
orbi
c ac
id s
tand
ard
- C
ortin
a-P
uig
et a
l., 2
009,
20
09b
antio
xida
nt
capa
city
P
apay
a fr
uit a
nd
papa
ya-b
ased
food
S
uper
oxid
e di
smut
ase
On
kapp
a-ca
rrag
eena
n ge
l bet
wee
n ce
llulo
se a
ceta
te a
nd a
dya
lisis
mem
bran
e on
Pt
- -
Cam
pane
lla e
t al.,
200
9
I. Revisão do estado da arte
40
The simplest immobilization strategy is physical adsorption of double stranded DNA
(dsDNA) (Diopan et al., 2008) or single stranded DNA (ssDNA) (Barroso et al., 2011b;
Barroso et al., 2011c) on CPE. Covalent attachment of dsDNA on PAMAM-encapsulated
Au-Pd/chitosan composite was also proposed (Qian et al., 2010). Guanine or adenine free
bases were also used as oxidant layers adsorbed on GCE (Barroso et al., 2011d; 2011e;
2012f). Although the resulting modified electrodes cannot be considered as DNA-based
biosensors, they are included in this section because the principle of measurement is also
based on the electroactivity of purine bases, the most easily oxidized ones, after radical
attack. The oxidation current of guanine or adenine dramatically decreases in the
presence of radicals when comparing with their native electroactivity. When an antioxidant
is added to the radical-containing solution, the current is partially recovered, which is
attributed to the scavenging activity of these compounds. CV, SWV and DPV were the
electrochemical techniques most widely used for these studies (Fig. 3E).
Since the antioxidant capacity is highly dependent on the source of ROS, that is, a
single antioxidant exhibits different scavenging efficiency against different radicals, the
development of methods based on several source of ROS is needed. Most methods rely
on the generation of hydroxyl radical through a Fenton-type reaction. Recently sulfate
radical (Barroso et al., 2011e) and superoxide radical enzymatically generated (Barroso et
al, 2011 d, Barroso et al. 2011b) were also reported.
An indirect electrocatalytic voltammetric method to assess total antioxidant capacity
using a DNA-modified CPE was developed (Barroso et al., 2011a, b). It was reported that
the electrochemical oxidation of both adenine and guanine homopolynucleotides in neutral
or alkaline conditions led to the formation of a common oxidized product that catalyzed the
oxidation of NADH (de-los-Santos-Alvarez et al., 2007). Therefore, the oxidative lesions
generated after immersion of the DNA-CPE in free radicals (hydroxyl or superoxide
radical) can be indirectly quantified after the electrochemical oxidation of the adenines that
remained unoxidized on the electrode surface. Under the conditions tested both radicals
produce around 80% of damage on DNA. In the presence of antioxidants (ascorbic acid,
gallic acid, caffeic acid and resveratrol) an increase in the electrocatalytic current of NADH
was obtained, which probed the capacity of these compounds as free radical scavengers.
The efficiency varied from 19 to 63% and a different trend was observed depending on the
source of radicals used suggesting a different mode of action against both radicals.
Zhang et al. observed an increase in the electroactivity of DNA after damage because of
the strand breaking that leaves the purine bases more exposed to the electrode surface
facilitating the electron transfer. In addition to this, it was also found that the role of AA
was prooxidant at concentrations below 1.5 mM (increasing damage) but antioxidant at
higher concentrations (Zhang et al., 2008a).
1. Métodos eletroquímicos
41
Ziyatdinova et al observed that the degraded DNA is less effective in blocking the
electron transfer from anionic redox probes in solution. So, the current recovery due to the
protection of rutin allowed its detection by CV (Ziyatdinova et al., 2008).
Finally, it is worth mentioning that DNA layers were also used as simple modifiers
without being subjected to radical damage, similarly to approaches explained in section 3
(Liu et al., 2011; Wang et al., 2011).
Table 4 shows the main features of the DNA-based biosensors reported for the
evaluation of antioxidant status.
42
Tab
le 4
. D
NA
-bas
ed b
iose
nsor
app
lied
for
the
antio
xida
nt s
tatu
s ev
alua
tion.
Ant
ioxi
dant
Sam
ple
Tar
get l
ayer
Im
mob
iliza
tion
proc
edur
e F
ree
radi
cal
Line
ar r
ange
Det
ectio
n lim
it re
fere
nce
pom
iferin
, is
opom
iferin
, os
ajin
and
ca
talp
osid
e
- ss
DN
A
Ads
orpt
ion
afte
r da
mag
e on
CP
E
Hyd
roxy
l rad
ical
-
- D
iopa
n e
tal.,
200
8
TA
C
Bev
erag
es
dA21
A
dsor
ptio
n on
CP
E
Hyd
roxy
l rad
ical
0.
05-1
.00 µ
M
50 n
M
Bar
roso
et a
l., 2
011c
TA
C
Bev
erag
es
Fla
vour
ed w
ater
s dA
21
Ads
orpt
ion
on C
PE
S
uper
oxid
e ra
dica
l 10
-100
µM
10
µM
B
arro
so e
t al.,
201
1b
TA
C
Bev
erag
es
Fla
vour
ed w
ater
s G
uani
ne
aden
ine
Ads
orpt
ion
on G
CE
S
uper
oxid
e ra
dica
l 1.
00–5
.00
mg
l-1
0.50
–4.0
0 m
g l-1
0.
77 m
g l-1
0.
50 m
g l-1
B
arro
so e
t al.,
201
1e
TA
C
Bev
erag
es
Fla
vour
ed w
ater
s G
uani
ne
aden
ine
Ads
orpt
ion
on G
CE
S
ulfa
te r
adic
al
0.50
– 4
.00
mg
l-1
0.50
– 4
.00
mg
l-1
0.47
mg
l-1
0.50
mg
l-1
Bar
roso
et a
l., 2
011f
TA
C
Bev
erag
es
Fla
vour
ed w
ater
s G
uani
ne
aden
ine
Ads
orpt
ion
on G
CE
H
ydro
xyl r
adic
al
0.50
– 2
.50
mg
l-1
2.00
– 6
.00
mg
l-1
0.29
mg
l-1
0.99
mg
l-1
Bar
roso
et a
l., 2
011
Ser
icin
-
dsD
NA
C
oval
ently
to P
AM
AM
den
drim
er
enca
psul
ated
Au-
PdN
Ps/
GC
E
Hyd
roxy
l rad
ical
-
- Q
ian
et a
l., 2
010
Asc
orbi
c ac
id
Man
nito
l
dsD
NA
A
dsor
ptio
n af
ter
dam
age
on G
CE
H
ydro
xyl r
adic
al
- -
Zha
ng e
t al.,
200
8
Rut
in
Tea
ext
ract
s ds
DN
A
Ads
oprt
ion
on M
WN
T/S
PC
E
Hyd
roxy
l rad
ical
-
- Z
iyat
dino
va e
t al.,
200
8
Uric
aci
d
- ds
DN
A
PA
MA
M-M
WC
NT
-chi
tosa
n A
uE
- 0.
5-10
0 µ
M
0.07
µM
Li
u et
al.,
201
1
baic
alei
n M
edic
inal
tabl
ets
Spi
ked
hum
an b
lood
ds
-DN
A
Lagm
uir-
Blo
dget
t film
-
0.01
-2 µ
M
6.0
nM
Wan
g et
al.,
201
1
1. Métodos eletroquímicos
43
6. Conclusions
Measurement of the individual antioxidant or total antioxidant capacity can be performed
by several techniques such as spectrophotometry, chromatography and electrochemistry.
Considering the complexity of food composition and the possibility of synergistic
interactions among the antioxidant compounds in the sample, the evaluation of total
antioxidant capacity is desired instead the individual antioxidant measurement.
Electrochemistry is well suited approach for the antioxidant evaluation because
antioxidant mechanism in vitro systems is based on the transference of charge involved in
electron transfer during the redox reactions. The use of electrochemical devices presents
several advantages such as short detection time, small sample volume, high accuracy,
simplicity and lacks interferences from coloured samples avoiding time-consuming pre-
treatments.
Four different electrochemical approaches for the antioxidant evaluation were identified
in this review. Direct electrochemical detection on bare and CMEs, enzymatic biosensors
and DNA-based biosensors were described in detail. Direct electrochemical detection
presents the advantage of simplicity. In order to increase the rate of the electrode reaction
as well the sensitivity and selectivity, chemical modification of electrode surface can be
successfully carried out. The use of enzymes on electrodes for antioxidant sensing
combines the high selectivity of these biomolecules with a significant amplification of the
analytical signal. Besides, enzymatic biosensors are reusable, relatively simple, rapid and
inexpensive. The potential window at which they operate minimize the electrochemical
interferences in complex real samples. Nevertheless, the use of DNA-based biosensor is
preferred because the principle of measurement is closer to the activity of antioxidant in
biological systems. dsDNA, ssDNA or nucleobases immobilized on the electrode are
exposed to radical attack similarly to what occurs within the cell, which may generate
replication errors and subsequent misleading protein synthesis. Therefore, DNA-based
biosensors are considered promising tools for rapid screening of TAC in different kind of
matrices.
Acknowledgements
M. Fátima Barroso is grateful to Fundação para a Ciência e a Tecnologia for a Ph.D.
grant (SFRH/BD/ 29440/2006). N.S.Á thanks to Ministerio de Ciencia y Tecnología for a
Ramón y Cajal contract and for financial support (Project CTQ2008-02429 granted to
“Grupos Consolidados”).
I. Revisão do estado da arte
44
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51
II.
INVESTIGAÇÃO E DESENVOLVIMENTO
53
Capítulo 2
Composição mineralógica
2.1.
Flavoured versus natural waters: Macromineral (Ca, Mg, K, Na) and micromineral (Fe, Cu,
Zn) contents
M. Fátima Barroso, Aurora Silva, Sandra Ramos, M.T. Oliva-Teles, Cristina Delerue-Matos, M.
Goreti F. Sales, M.B.P.P. Oliveira
Food Chemistry, 2009, 116 (2), 580–589
2.2.
Survey of trace elements (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se, and Si) in retail samples
of flavoured and bottled waters
M. F. Barroso, S. Ramos, M. T. Oliva-Teles, C. Delerue-Matos, M. G. F. Sales, M. B. P. P.
Oliveira
Food Additives and Contaminants: Part B – Surveillance, 2009, 2 (2), 121-130
2.1. Macrominerais e microminerais
55
Flavoured versus natural waters: Macromineral (Ca, Mg, K, Na)
and micromineral (Fe, Cu, Zn) contents
M. Fátima Barrosoa,b, Aurora Silvaa, Sandra Ramosc, M. T. Oliva-Telesa, Cristina Delerue-
Matosa, M. Goreti F. Salesa, M. B. P.P. Oliveirab aRequimte/Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de
Almeida 431, 4200-072, Porto, Portugal. bRequimte/Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, Rua
Aníbal Cunha, 164, 4099-030 Porto, Portugal. cInstituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida 431,
4200-072, Porto, Portugal.
Abstract
Macro (Ca, Mg, K, Na) and micromineral (Fe, Zn, Cu) composition of 39 waters was
analysed. Determinations were made by atomic flame spectrophotometry for
macrominerals and electrothermic atomization in graphite furnace for microminerals.
Mineral contents of still or sparkling natural waters (without flavours) changed from
brand to brand. Mann–Whitney test was used to search for significant differences between
flavoured and natural waters. For that, the concentration of each mineral was compared to
the presence of flavours, preservatives, acidifying agents, fruit juice and/or sweeteners,
according to the labelled composition.
The statistical study demonstrated that flavoured waters generally have increased
contents of K, Na, Fe and Cu. The added preservatives also led to significant differences
in the mineral composition. Acidifying agents and fruit juice can also be correlated to the
increase of Mg, K, Na, Fe and Cu. Sweeteners do not provide any significant difference in
Ca, Mg, Fe and Zn contents.
Keywords: Macrominerals; Microminerals; Atomic spectrophotometry; Flavoured waters;
Health benefits
Available online at www.sciencedirect.com
Food Chemistry 2009, 116 (2), 580-589
2.1. Macrominerais e microminerais
57
1. INTRODUCTION
Water makes up more than two thirds of the human body, and it is the most consumed
drink in the world. To answer to consumer´s preferences, industries have applied several
technical improvements to plain water. Today, a significant part of marketed water is
flavoured. It consists in the addition of flavours, juices, bioactive compounds,
preservatives and/or sweeteners that provide singular tastes and smells appreciated by
consumers.
In the case of flavoured waters, either mineral or spring sources are used, both having
important mineral contents. According to Food and Drug Administration (FDA, 2002),
mineral water arises from a geologically and physically protected underground source,
characterised by constant levels and relative proportions of minerals and trace elements
at the source. Spring water is derived from an underground formation from which water
flows naturally to the surface at an identified location.
Minerals are necessary for human life and play important roles in metabolic functions
(Biziuk & Kuczynska, 2007) such as, maintenance of pH, osmotic pressure, nerve
conductance, muscle contraction, energy production, and in almost all other aspects of
life. Depending on the amounts needed, minerals can be divided into macro (g or mg/day)
and microminerals (few mg or µg/day). Physiologically, the most important macrominerals
are Ca, K, Na and Mg, and the same for Fe, Cu and Zn as microminerals (Silvera &
Rohan, 2007).
Bioavailability of minerals is affected by several factors. Host factors can be defined as
any attribute that can influence the amount of metal exposure, uptake, absorption,
biokinetics and susceptibility of an individual. Such factors include age, gender, size and
weight, nutritional status, genetics and some behaviours (Robson, 2003).
Although minerals are essential to normal health and development, they can become
toxic in higher amounts. Risk assessments of chemical elements show high intakes that
result in toxicity or nutritional problems related to low or no intakes (Goldhaber, 2003). So,
it is important to establish an adequate intake of certain substance to avoid adverse health
effects (Nasreddine & Parent-Massin, 2002). To answer this goal, the Joint Expert
Committee on Food Additives (JECFA) of the Food and Agriculture Organisation of the
United Nations and the World Health Organisation (FAO/WHO, 2000) established
acceptable or tolerable intakes for substances that exhibit thresholds of toxicity.
Provisional Tolerable Daily Intake (PTDI), calculated on a daily basis for certain
substances that do not accumulate in the human body, is the reference value that
indicates the safe level of intake. US Environmental Protection Agency (EPA) has also
carried out the Reference Dose (RfD), being the amount of a daily human exposure
II. Investigação e desenvolvimento
58
(including sensitive subgroups) to a certain compound, without an appreciable risk during
a lifetime (EPA, 1993). RfD is generally expressed in mg/kgbodyweight/day. US Food and
Nutrition Board of the Institute of Medicine (FNB/IOM) set forward the Recommended
Dietary Allowance (RDA) as the average daily intake that meets the nutrient requirements
of nearly all healthy individuals in a particular life stage and gender (Institute of Medicine
from United States, 2007). Table 1 presents the different values established for the
aforementioned minerals.
Table 1 Recommended dietary allowances (RDA), provisional tolerable intakes (PTDI)
and reference dose (RfD) for the studied minerals.
Mineral RDA (mg/day) PTDI (mg/kgbodyWeight/day) RfD (mg/kg/day)
Ca 1000a - -
Mg 420male;a
320female;a - -
K 4700b - -
Na 1500b - -
Fe 8male;a
18female;a 0.8c -
Zn 11male;a
8female;a 1.0d 0.3f
Cu 0.9a 0.5e -
Adult Values (male or female) with 31-50 years old.
- Not available. aInstitute of Medicine from United States (2001). bInstitute of Medicine from United States (2004). cWHO/FAO (1983). dWHO/FAO (1982a). eWHO/FAO (1982b). fEnvironmental Protection Agency of United States, EPA (2005)
Several analytical methods have been developed to determine the mineral contents in
biological, food and environmental samples. The most commonly employed techniques
are described below. Inductively coupled plasma mass spectrometry (ICP–MS) (Liu,
Chen, Yang, Chiang, & Hsu, 2007), inductively coupled plasma atomic emission
spectroscopy (ICP–AES) (Mehra & Baker, 2007), these techniques allow a multielement
analysis; however the equipment used is very expensive and also have high operation
coasts. Atomic absorption spectrophotometry (AAS) with flame or electrothermic
atomisation in graphite furnace (Galani-Nikolakaki,Kallithrakas-Kontos, & Katsanos, 2002;
Tamasi & Cini, 2004). The AAS based technique is robust, well establish, easy to use,
and presents good detection and quantification levels, mg/L and µg/L for flame or graphite
2.1. Macrominerais e microminerais
59
furnace technique, respectively. The use of voltammetry (Melucci, Torsi, & Locatelli, 2007)
to quantify metals is an inexpensive and fast technique but normally associated with the
use of mercury electrode considered as toxic and environmental unfriendly.
The present study aims to evaluate the contents of Ca, Mg, Na, K, Cu, Fe and Zn in 39
mineral and spring water samples, with and without flavours. Atomic absorption
spectrophotometry with flame or electrothermic atomisation in graphite furnace was the
implemented methodology. A nutritional and statistic study was carried out to compare
these water kinds.
There are no known reports, or any type of evaluation, concerning the mineral contents
of these flavoured waters. So, the presented research work is crucial to consumer’s
information about the advantages/disadvantages of the consumption of these beverages.
2. Materials and methods
2.1. Reagents and equipment
The water used had ultrapure quality (18.2MΩcm-1) and was obtained from a Millipore
Simplicity 185 system.
All reagents and solvents used were suprapure grade and acquired from Merck, except
CsCl that was from Sigma. Standard solutions of each element (Ca, Mg, Na, K, Cu, Fe
and Zn) were daily prepared by dilution of the corresponding stock solutions (1000 mg/L),
with water and 0.1% (v/v) nitric acid, and stored in polyethylene bottles.
Mg(NO3)2 (0.1%; v/v) was used as matrix modifier for the determination of Fe and Zn
and CsCl (0.1%; v/v) to evaluate Na and K contents.
All glassware and polyethylene vessels were soaked with 10% HNO3, at least overnight,
and then rinsed with ultrapure water prior to use.
Macrominerals were quantified in a Perkin Elmer AAnalystTM 200 spectrophotometer
with an air–acetylene flame. Ca, Mg, Na and K were analysed at wavelengths of 422.7,
285.2, 589.6 and 766.5 nm, respectively.
Microminerals (Fe, Zn and Cu) were quantified in an Analytik Jena Zeenit 650
spectrophotometer with electrothermic atomisation in graphite furnace (wavelengths of
248.4, 213.9 and 324.8 nm, respectively) equipped with an Analytik Jena MPE60
autosampler. Pyrolytically coated graphite tubes with integrated pin platform (Analytik
Jena AG) were used. Specific interferences from the matrix were not observed in all
samples and the Zeeman background correction was sufficient. Hollow cathode lamps
II. Investigação e desenvolvimento
60
were used (Varian). A stream of ultrapure argon at 5.5 bar was used in the electrothermic
determinations, except in the Auto-zero and atomisation step.
2.2. Samples and sample preparation
Thirty-nine water samples (flavoured and the natural ones) corresponding to 10 different
brands (mineral and spring) were collected in several supermarkets in the North of
Portugal. Each brand (still or sparkling) had different flavours and aromas.
Table 2 summarises the labelled information, namely the presence of vitamins,
sweeteners and preservatives.
All samples were acidified with suprapure HNO3 (1 mL/L) and stored in sealed
polyethylene bottles maintained at 4 ºC. The gas of sparkling water was removed by
sonication, before HNO3 treatment or acidification.
2.3. Validation of the methodology
Calibration standards were daily prepared (all samples were determined in triplicate).
The proposed methods were validated by linear range, limit of detection (LOD), limit of
quantification (LOQ), precision and accuracy. LOD and LOQ were defined, respectively,
as three and 10 times the standard deviation of 10 blank signals divided by the slope of
the calibration plot (Miller & Miller, 2000). The precision was investigated considering the
intra-day and inter-day determinations of standard solutions and expressed by relative
standard deviations (RSD). For intra-day evaluation each concentration was assessed by
three measurements, at three times along a working-day. The inter-day precision
measurements were performed over a period of one week. Accuracy and reproducibility
were checked by the recovery (REC), the relative error (RE) and the RSD.
2.4. Data analysis
All results were expressed as mean ± standard deviation. In the statistical analysis, data
were presented as median (1st quartile– 3rd quartile). The significance of the differences
between natural waters with and without natural gas was tested by Mann–Whitney test.
Comparisons between natural water group and the respective flavoured water group were
carried out by Wilcoxon test (dependent samples). All statistical analysis was performed
using the Statistica7 software, p < 0.05 was considered statistically significant.
2.1. Macrominerais e microminerais
61
Table 2 Label information in bottled flavoured waters evaluated.
Brand Sample Flavour Composition (mg/L) Other ingredients
Still water A (Mineral) 1 Lemon
2 Mango
3 Strawberry
Energy value 1.3 kcal/100 mL, Na+ (<200), Proteins (<1000); carbohydrate (1000), lipids (<1000)
Fibres (1%), Wheat dextrin (0.1%) citric acid, potassium sorbate, sodium benzoate, sodium citrate, acesulfame-K
4 Natural
Total dissolved solids (47), SiO2
(12.7), Ca2+ (0.75), F- (<0.08), NO3
- (1.7), Na+ (6.9), Mg2+ (1.7), Cl- (9.4), HCO3
- (11.6), pH 5.7
B (Spring)
5
Pineapple /orange
Apple juice concentrate, calcium lactate, citric acid, potassium sorbate, sodium benzoate, acesulfame-K, aspartame
6 Lemon
Apple juice concentrate, citric acid, potassium sorbate, sodium benzoate, vitamins: niacin, pantothenic acid, B6, folic acid, biotin, B12, acesulfame-K, aspartame
7 Natural Total dissolved solids (52), SiO2
(21.5), Na+ (<5.5), Ca2+ (<4.3), HCO3
- (<24), Cl- (<30); pH 5.8-6.9
C (Mineral)
8
Lemon/ magnesium
Energy value 13 kcal/100 mL, proteins (<1000), sugar (23000), Sat. fatty acids (<500), fibres (<1000), Na+ (12), Mg2+ (450)
Fruit juice concentrate, citric acid, potassium sorbate, dimethyl dicarbonate, magnesium carbonate, ginseng, vitamins (mg/100 mL): B3 (2.7), B5 (0.9), B6 (0.3), B8 (0.022), B9
(0.03), B12 (1.5x10-4)
9
Apple/ white tea
Energy value 17 kcal/100 mL, proteins (<1000), Sat. fatty acids (<500), Sugar (43000), fibres (<1000), Na+ (14), Ca2+ (1200)
Fruit juice concentrate, calcium lactate, citric acid, malic acid, potassium sorbate, vitamins (mg/100 mL): B3 (2.7), B5 (0.9), B6 (0.3), B8 (0.022), B9 (0.03) and B12 (1.5x10-4)
10
Pineapple /fibre
Energy value 9 kcal/100 mL, proteins (<1000), sugar (23000), Sat. fatty acid (<1000), fibres (9000), Na+ (14)
Fruit juice concentrate, wheat dextrin (0.9%), citric acid, potassium sorbate, dimethyl dicarbonate, L-carnitine (200 mg/L)
11 Natural
Total dissolved solid (45), SiO2
(18), HCO3- (5.1), Cl- (7.4), NO3
-
(2.1), Ca2+ (0.8), Na+ (5.8), Mg2+ (0.5), pH 5.7
D (Mineral) 12 Apple
13 Orange
/peach 14 Lemon
Energy value 0.9 kcal/100 mL
Citric acid, dimethyl dicarbonate, sodium benzoate, sucralose, acesulfame-K
15 Natural
Conductivity (515 µS/cm), HCO3
- (315), SO4
2- (25), Cl- (10), Ca2+ (83), Mg2+ (24), Na+ (4.7)
Sparkling water
E (Mneral) 16 lemon
Added gas 17 orange/ raspberry
18 peach/ pineapple
19 guava
Energy value 1.4 kcal/100 mL, proteins (<1000), carbohydrates, (<100) lipids (<1000)
Lemon juice, carbon dioxide, citric acid, sodium citrate, potassium sorbate, sodium benzoate, acesulfame-K, aspartame, vitamins (mg/100 mL): B3 (2.7), B12 (0.15)
20 natural Total dissolved solids (47), SiO2
(12.7), Ca2+ (0.75), F- (<0.08),
II. Investigação e desenvolvimento
62
Brand Sample Flavour Composition (mg/L) Other ingredients NO3
- (1.7), Na+ (6.9), Mg2+ (1.7), Cl- (9.4), HCO3
- (11.6)
F (Mineral) 21
lemon/ green tea
Natural gas 22 raspberry/ ginseng
23 peach/ white tea
24 mango/ ginkgo biloba
25 melon/ mint
Energy value 19 kcal/100 mL, proteins (1000), sugar (43000), lipids (<500), Na+ (600)
Lemon, apple and pear juice, fibres (<1000), citric acid
26 natural
Total dissolved solids (3011), SiO2
(62), HCO3- (2125), Cl- (31), NO3
- (0.3), Ca2+ (103), Na+ (622), Mg2+ (28), pH 6.1
G (Mineral) 27 lemon
Citric acid, vitamin C (12 mg/250 mL), potassium sorbate, acesulfame-K, sucralose
Added gas 28 lime
29 apple
30 peach
Energy value 0.4 kcal/100 mL
Citric acid, vitamin C (12 mg/250 mL), potassium sorbate, acesulfame-K, sucralose
31 natural
Total dissolved solids, 180ºC (497), Cl- (78), SO4
2- (22), HCO3-
(373), Na+ (37), Ca2+ (105), Mg2+ (29), pH 5.43
H (mineral) 32 lemon Energy value 4 kcal/100 mL proteins (<1000), carbohydrates (10000), lipids (<1000)
Lemon and apple juice, citric acid, vitamin C (30 mg/100 mL), sodium benzoate, potassium sorbate, aspartame
Natural gas
33 natural
Total dissolved solids (2776), SiO2 (37), HCO3
- (1954), Cl- (34), NO3-
(0.3), Ca2+ (75), Na+ (607), Mg2+ (14), pH 6.1
I (Mineral) 34 lemon Energy value 2 kcal/100 mL, carbohydrates (5000)
Lemon and apple juice, citric acid, aspartame, sodium benzoate
Natural gas 35 green apple
Energy value 4 kcal/100 mL, proteins (500), carbohydrates (9000)
Apple juice, citric acid, sodium benzoate, sucralose
36 strawberry
Energy value 2 kcal/100 mL, carbohydrates (5000)
Apple and strawberry juice, citric acid, sodium benzoate, aspartame
37 natural
CO2 free (2260), Total dissolved solids (3535), SIO2 (26.7), Cl- (145), HCO3
- (2393), Na+ (741), Mg2+ (35.2), Ca2+ (116)
J (Spring) 38 lemon
Energy value 4 kcal/100 mL, proteins (<1000), carbohydrates (<1000), lipids (<1000)
Lemon juice, citric acid, sodium citrate, potassium sorbate, sodium benzoate, aspartame, acesulfame-K
Added gas 39 natural
Total dissolved solids (180ºC) (21), SiO2 (8.5), HCO3
- (5.8), Cl- (3.4), Ca2+ (1.2), Mg2+ (0.3), K+ (0.2), Na+ (2.5)
2.1. Macrominerais e microminerais
63
3. Results and discussion
Macro (Ca, Mg, K, Na) and microminerals (Fe, Cu, Zn) are essential, in different
amounts, to normal human development. So, an adequate intake from dietary sources is
very important to avoid deleterious effects on human health and general well-being.
Before the evaluation of the mineral content in the samples it was necessary to
implement and validate the employed methodologies.
3.1. Minerals quantification
Table 3 summarises the data from calibration curves and the performance
characteristics for the seven minerals in study. Linearity ranges were from 0.10 to 5.00
mg/L in macrominerals and from 1.0 to 20.0 µg/L in microminerals. The calculated LOD
values ranged from 4.6 to 30.2 µg/L for macrominerals, and 0.21 to 1.7 µg/L for
microminerals. LOQ values range from 15.2 to 100.2 µg/L and 0.7 to 5.5 µg/L for
macrominerals and microminerals, respectively.
Precision and accuracy values are shown in Table 3. No significant differences were
found between intra-day and inter-day experiments. RSD values ranged from 1.0% to
4.3%, and confirmed the high precision of the method. REC and RE values assessed the
accuracy of the results. RE were always <10.0% and recovery trials ranged from 99% to
110%, confirming the accuracy of the implemented method.
64
Tab
le 3
Cal
ibra
tion
curv
es, l
imit
valu
es, p
reci
sion
and
acc
urac
y ob
tain
ed fo
r th
e m
iner
als
stud
ied.
R
EC
(%
) =
[met
al] fo
und/
[met
al] a
dded
x 1
00; R
E (
%)
= ([
met
al] fo
und
– [m
etal
] add
ed)/
[met
al] a
dded
x 1
00; R
SD
(%
) =
σ/[m
etal
] mea
n fo
und
x 10
0
a)A
vera
ge o
f thr
ee m
easu
rem
ents
thre
e tim
es a
long
a d
ay; b)
RE
C, r
ecov
ery;
c)R
E, r
elat
ive
erro
r; d)
RS
D, r
elat
ive
stan
dard
dev
iatio
n
e)A
vera
ge o
f thr
ee m
easu
rem
ents
ove
r a
wee
k.
Par
amet
ers
Ca
Mg
K
Na
Fe
Zn
Cu
Line
ar c
once
ntra
tion
(µg/
L)
100
0.0-
5000
.0
100
.0–4
00.0
4
00.0
–150
0.0
400
.0–1
500.
0 6
.0-2
0.0
2.5
0-20
.0
1.0
-10.
0
Slo
pe (
Abs
µg-1
L)
5.2
±0.2
(x1
0-5
) 9
.3±0
.5 (
x10
-4)
1.9
±0.8
(x1
0-4
) 2
.7±0
.2 (
x10
-4)
0.0
118±
0.00
02
11.
08±0
.04
0.0
14±0
.001
Inte
rcep
t (A
bs)
0.0
10±0
.006
2
.4±0
.1 (
x10
.2)
1.9
±0.7
(x1
0-3
) -
0.02
±0.0
2 1
.18x
10-2
± 0
.02x
10-2
0
.9±0
.4
0.0
03±0
.002
Cor
rela
tion
coef
ficie
nt (
n =
3)
0.9
97
0.9
95
0.9
97
0.9
92
0.9
994
1.0
0 0
.998
1
LOD
(µ
g/L)
30
.2
4.6
2
7.3
17.1
1.
7 0
.59
0.2
1
LOQ
(µ
g/L)
10
0.2
15.
2 9
1.0
57.1
5.
5 1
.90
0.7
Intr
a-da
y st
udie
sa)
Add
ed (
µg/
L):
foun
d (µ
g/L)
1000
.0
1020
.0
300.
0
305.
0
120
0.0
119
0.2
1000
.0
1090
.0
12.0
12.5
20.0
19.7
5.0
5.6
RE
Cb)
(%
) 10
2 10
1.7
99.
2 10
9.0
104.
2 98
.5
112.
0
RE
c) (%
) 2.
0 1.
7 -
0.8
9.0
4.2
-1.5
12
.0
RS
Dd)
(%
) 4.
0 3.
8 1
.0
2.3
3.4
1.8
4.3
Inte
r-da
y st
udie
se)
Add
ed (
µg/
L)
foun
d (µ
g/L)
1000
.0
1015
.0
300.
0
298.
0
1200
.0
1210
.0
1000
.0
1100
.0
12.0
12.3
20.0
20.4
5.0
5.3
RE
C (
%)
101.
5 99
.3
100.
8 11
0.0
102.
5 10
2.0
106.
0
RE
(%
) 1.
5 -0
.7
0.8
10.0
2.
5 2.
0 5.
7
RS
D (
%)
4.3
2.9
3.7
2.8
1.7
2.5
3.8
2.1. Macrominerais e microminerais
65
3.2. Global discussion
Table 4 shows the minerals contents of still and sparkling waters, respectively. The
mineral composition of the natural waters (without flavours) changed from brand to brand.
This was due to their different natural origins, from different geological structures.
According to the values described in the label (Table 2) it is possible to discriminate
three groups in natural waters, attending to the total dissolved solids values:
- one with reduced values ranging from 21 to 47 mg/L corresponding to natural waters
4, 7, 11, 20 and 39 (50% of the samples considering the flavoured ones). This group
included all spring waters studied (samples 7 and 39); sample 39 had added gas. The
other samples included in this group were mineral waters and only sample 20 had added
gas;
- another group included two samples with intermediate values (samples 15 and 31)
with total dissolved solids ranging 500 mg/L. This group corresponds to 25% of the total
samples analysed;
- the third group included mineral waters with natural gas and their total dissolved solids
ranged from 2776 to 3535 mg/L (samples 26, 33 and 37).
From all the evaluated samples, the excessive consumption of the mineral waters,
pertaining to this group, with or without flavours, can contribute to the development of
health problems namely kidneys disorders. Considering the values of Table 1, three
bottles of this water (about 1 L) per day correspond to an ingestion of a 1/3 of the RDA for
Na.
Taking into account the macromineral values presented in the label and the determined
ones (Tables 2 and 4), in general, there are in agreement. All label samples described the
contents of Ca, Mg and Na and only one (sample 39) presented K contents. None of them
expressed micromineral contents.
It is important to stress that the samples with high total dissolved solids (the integrated
measure of the concentrations of common ions, Na, K, Ca, Mg, Cl) have a high
contribution of Na, with contents ranging 600 mg/L. This fact reinforces the care needed in
the consumption of this foodstuff, with special attention to children and adults with renal
disorders. Nevertheless, chloride contents are not very high, decreasing the possible
influence in the development or contribution to hypertension.
II. Investigação e desenvolvimento
66
Table 4 Mineral contents in bottled waters.
Macrominerals, mg/L (%) Microminerals, µg/L (%) Brand Sample No. Ca Mg K Na Fe Zn Cu Still A 1 9.1 ± 0.5 3.4 ± 1.0 117.5 ± 0.1 209.1 ± 0.4 18.9 ± 3.4 24.7 ± 5.7 13.3 ± 8.5 2 8.2 ± 0.3 4.0 ± 0.8 107.5 ± 0.5 190.1 ± 2.1 16.5 ± 11.6 16.3 ± 11.0 12.5 ± 0.6 3 8.4 ± 0.5 4.4 ± 2.1 113.7 ± 0.9 151.0 ± 8.6 42.6 ± 3.9 15.3 ± 3.9 12.3 ± 5.8 4 0.8 ± 3.0 1.5 ± 4.0 1.0 ± 0.6 6.7 ± 0.4 5.1 ± 3.2 11.9 ± 8.4 9.1 ± 7.0
B 5 212.8 ± 0.8 0.2 ± 1.2 65.7 ± 0.7 46.1 ± 10.2 108.4 ± 10.2 8.3 ± 0.7 0.7 ± 5.7 6 3.1 ± 0.7 0.3 ± 2.8 137.4 ± 0.4 57.7 ± 6.9 262.9 ± 4.3 7.8 ± 1.3 2.3 ± 1.8 7 1.5 ± 1.4 0.2 ± 1.7 0.5 ± 0.5 4.2 ± 2.2 2.3 ± 15.2 8.6 ± 0.3 -
C 8 1.6 ± 0.5 28.0 ± 2.6 111.8 ± 0.1 16.6 ± 7.3 83.7 ± 2.0 29.4 ± 2.6 1.2 ± 4.1 9 238.8 ± 6.7 33.2 ± 3.3 105.7 ± 0.2 16.8 ± 2.4 87.0 ± 13.9 28.5 ± 2.8 2.3 ± 2.6 10 2.4 ± 1.4 0.4 ± 0.5 94.3 ± 1.2 10.6 ± 5.3 1 9.5 ± 7.7 30.9 ± 2.8 0.5 ± 0.6 11 0.2 ± 0.3 0.4 ± 1.3 2.8 ± 1.0 8.1 ± 0.6 1.8 ± 14.2 22. 5 ± 4.4 -
D 12 69.7 ± 0.6 12.2 ± 0.5 21.5 ± 0.9 35.4 ± 1.3 2.9 ± 33.6 5.8 ± 17.2 - 13 55.1 ± 0.5 10.7 ± 0.8 17.3 ± 0.8 31.9 ± 1.0 4.1 ± 6.9 6.9 ± 1.7 - 14 53.3 ± 1.4 14.3 ± 1.4 15.9 ± 1.6 31.9 ± 2.3 - 9.2 ± 0.6 - 15 69.9 ± 2.9 20.1 ± 1.6 1.1 ± 1.3 4.8 ± 1.3 - 6.5 ± 21.5 -
Sparkling E 16 1.1 ± 2.9 2.1 ± 2.2 246.9 ± 1.0 619.4 ± 1.1 6.3 ± 14.4 11.1 ± 7.2 9.0 ± 0.9 17 2.3 ± 0.7 2.3 ± 4.2 86.26 ± 3.0 245.2 ± 0.8 13.9 ± 12.7 20.4 ± 13.2 7.9 ± 1.9 18 1.1 ± 1.3 2.2 ± 4.2 92.7 ± 0.6 221.7 ± 2.3 35.4 ± 11.8 12.8 ± 10.9 9.6 ± 2.2 19 1.3 ± 1.6 2.2 ± 3.9 96.2 ± 0.2 290.9 ± 0.8 8.9 ± 1.0 13.4 ± 14.9 9.4 ± 1.2 20 0.2 ± 5.2 1.7 ± 1.7 0.5 ± 0.5 8.2 ± 1.8 83. 6 ± 2.5 31.2 ± 10.6 8.7 ± 0.3
F 21 82.5 ± 0.6 20.4 ± 1.5 36.7 ± 0.5 655.6 ± 1. 9 29.2 ± 7.3 12.2 ± 11.5 - 22 82.5 ± 1.4 19.4 ± 2.7 31.8 ± 2.0 618.2 ± 1.5 122.8 ± 1.5 8.3 ± 16.9 1.5 ± 6.9 23 79.5 ± 0.6 20.2 ± 1.2 41.9 ± 1.1 641.6 ± 2.4 9.4 ± 6.4 12.4 ± 25.8 1.4 ± 3.5 24 77.8 ± 1.6 21.3 ± 1.3 25.8 ± 1.1 496.6 ± 2.3 196.8 ± 11.7 10.3 ± 18.4 5.1 ± 3.2 25 89.6 ± 1.2 21.4 ± 2.6 41.8 ± 0.2 667.3 ± 1.8 27.7 ± 3.2 65.9 ± 2.1 1.5 ± 3.1 26 87.8 ± 0.6 20.0 ± 4.0 42.7 ± 1.5 560.1 ± 3.6 4.5 ± 11.5 4.8 ± 25.0 -
G 27 110.8 ± 1.7 15.0 ± 0.5 122.4 ± 1.8 56.9 ± 1.2 20.3 ± 6.4 15.4 ± 4.5 - 28 108.4 ± 0.2 14.4 ± 1.1 6.2 ± 0.5 43.4 ± 0.7 49.2 ± 4.4 6.8 ± 1.5 0.7 ± 2.7 29 112.1 ± 0.4 23.8 ± 0.9 88.7 ± 2.3 45.7 ± 1.4 67.0 ± 4.0 27. 6 ± 15.6 2.0 ± 4.3 30 98.8 ± 1.5 25.9 ± 5.5 78.6 ± 0.2 47.1 ± 1.8 41.0 ± 0.5 5.6 ± 3.6 - 31 108.0 ± 1.1 33.7 ± 7.3 2.2 ± 0.5 43.1 ± 2.4 58.0 ± 4.3 2.8 ± 0.1 -
H 32 58.2 ± 0.3 10.8 ± 12.3 37.1 ± 1.0 603.0 ± 0.9 35.1 ± 5.6 11.4 ± 14.0 -
33 81.6 ± 1.8 10.9 ± 2.0 24.8 ± 2.2 596.8 ± 0.5 25.8 ± 2.2 11.2 ± 21.4 -
I 34 123.0 ± 1.6 28.7 ± 21.7 36.2 ± 4.5 671.3 ± 0.1 39.7 ± 2.5 3.0 ± 0.3 - 35 118.6 ± 0.2 30.1 ± 11.4 39.6 ± 0.4 593.2 ± 1.9 104.4 ± 0.8 6.4 ± 10.9 - 36 127.4 ± 1.1 29.2 ± 3.2 34.5 ± 1.3 555.1 ± 1.3 36.4 ± 2.8 8.3 ± 1.1 0.9 ± 5.3 37 123.0 ± 1.5 24.9 ± 5.2 38.3 ± 0.6 535.1 ± 1.2 8.7 ± 1.2 3.5 ± 0.8 - J 38 3.6 ± 3.6 1.3 ± 1.2 69.2 ± 0.8 359.7 ± 1.4 10.9 ± 2.1 21.7 ± 3.0 7.2 ± 0.7 39 1.1 ± 1.4 0.2 ± 2.6 0.37 ± 1.0 3.7 ± 1.6 20 .7 ± 6.3 24.9 ± 1.4 1.0 ± 4.6
- Not detected.
In what concerns microminerals, several natural water samples have important levels of
Fe; it is the case of samples 20 and 31 (83.6 and 58.0 µg/L) as well as samples 33 and 39
(25.8 and 20.7 µg/L). Samples 4 and 20 presented 9 µg/L of Cu and sample 39 had 1
µg/L. Only these three natural water samples presented detectable Cu values.
2.1. Macrominerais e microminerais
67
From Table 2, it was possible to obtain more information, namely about the added
ingredients in the flavoured waters. Amongst them can be cited:
- fibres, that are listed in 11 samples from brands A, C and F;
- fruit juices or concentrates in about 50% of the samples. Only flavoured brands A, D
and G do not refer the addition of this type of ingredient;
- vitamins: 11 samples refer the presence of vitamins of B complex (seven samples) and
C (four samples). According to the label, the added amounts are very different in several
brands, some of them only refer its presence;
- other bioactive compounds, namely ginseng, L-carnitine, white and green tea and
ginkgo biloba; they are present in some flavoured waters from different brands.
Inevitably, these waters also need other ingredients, without positive relation with well-
being and health, but necessary to assure the desired quality for the producer and
consumers, and the safety of the product, such as, acidifying agents, sweeteners and
preservatives.
About 50% of the flavoured samples contained sweeteners as ingredients. There are
samples with only one (acesulfame-K, sucralose or aspartame) and with blends of two
sweeteners (acesulfame- K and aspartame; acesulfame-K and sucralose). The most used
were acesulfame-K (present in 14 samples) and aspartame in 10 samples. It is interesting
to note that, in general, the samples from the same brand have the same sweetener, with
exception of brand I that use different sweeteners for different flavours.
Brands C and F do not use sweeteners, providing more energetic products (9–13 and
19 kcal/100 mL), respectively. In the case of sweetened samples its energy value ranged
from 0.4 to 4 kcal/100 mL.
In what concerns to preservatives and the information contained in the label, each
sample can contain one (potassium sorbate or sodium benzoate) or two preservatives
(potassium sorbate and sodium benzoate; potassium sorbate and dimethyl dicarbonate;
sodium benzoate and dimethyl dicarbonate) simultaneously.
Flavoured waters would not ideally replace natural water, but can be an interesting
alternative to soft drinks which are products with more ingredients with negative influence
in health, namely obesity and generally in children health. Its moderate consumption can
be made with pleasure and without major concerns. It is also important to refer that
flavoured waters are more expensive (20–40%) than natural ones.
Confirming the referred above, except for Fe (p = 0.215), there were statistical
differences in mineral levels between natural waters (with and without natural gas or
added gas) (p < 0.05 for Ca, Mg, K, Na, Zn and Cu).
II. Investigação e desenvolvimento
68
After the presented discussion it is consensual that the minerals contents in flavoured
waters are higher than in natural ones. One justification for that is the use of several
ingredients in the salt form.
3.2.1. Flavour factor analysis
Considering only the flavour factor, the contents of K, Na, Fe and Cu are higher in the
flavoured waters than in the natural ones. For Mg and Ca median concentrations were
slightly higher in the natural waters. However, the difference observed between the
median concentration of the two groups (natural and flavoured) are statistically significant
only for K, Na, Cu (p < 0.001) and Fe (p = 0.045). Fig. 1 shows the median concentration
of the minerals studied in flavoured and natural waters.
Natural water Flavoured water0
20
40
60
80
100
120
Cal
cium
(m
g/L
)
Natural water Flavoured water
0
2
4
6
8
10
12
14
16
18
20
22
24
Mag
nesi
um (
mg/
L)
Natural water Flavoured water
0
20
40
60
80
100
120
Pota
ssiu
m (
mg/
L)
Natural water Flavoured water
0
100
200
300
400
500
600
700
Sodi
um (
mg/
L)
Natural water Flavoured water0
10
20
30
40
50
60
70
Iron
(m
cg/L
)
Natural water Flavoured water
0
1
2
3
4
5
6
7
8
Cop
per
(mcg
/L)
Natural water Flavoured water
0
2
4
6
8
10
12
14
16
18
20
22
24
Zin
c (m
cg/L
)
Fig. 1. Concentrations of Ca (p = 0.767), Mg (p = 0.456), K (p < 0.001), Na (p < 0.001), Fe (p =
0.045), Cu (p < 0.001) and Zn (p = 0.114) in flavoured waters and correspondent natural water.
The bars represent the median and the whisker represents the inter-quartile range.
3.3. Individual mineral composition
Calcium is the most important macromineral, with amounts ranging from 0.2 to 213
mg/L. Analysing Table 4 it was possible to verify that Ca contents are higher in sparkling
2.1. Macrominerais e microminerais
69
waters (except in brands E and J, that have added gas) than in still waters. In the former
group included the samples with the highest content in total dissolved solids and the one
with the lowest contents (brand J). In flavoured still waters, Ca levels increased, except in
brand D. This brand only has preservatives, sweeteners and acidifying agents.
Samples 5 and 9 presented the highest Ca contents (140 and 600 times higher than the
corresponding natural water). In this case, the addition of calcium lactate (Table 2) as
acidifying agent can justify the increased contents. Nevertheless the detected values are
lower than the claimed in the label (Ca 1200).
In sparkling waters Ca levels were kept fairly constant in all samples. As an exception,
in brand H, the flavoured water had lower contents than the natural one. This situation
could not be explained with the available information in Table 2.
Magnesium is a cofactor in almost all phosphorylation reactions involving ATP and is an
indirect antioxidant, being important for the control of the pro-oxidant and antioxidant
status (Lukaski, 2004). Its concentration ranged from 0.2 to 33.2 and 0.2 to 33.7 mg/L in
still and sparkling waters, respectively. Sample 9 had the highest Mg contents (when
compared with the corresponding natural sample). This increment was higher than the
determined in sample 8 with magnesium carbonate incorporation. According to Table 2
this sample should contain 450 mg/L. This claim does not correspond to the actual water
content, and therefore, the consumer is misled when looking for a good source of Mg. Mg
contents have little variation in the water samples evaluated. There are samples with an
increase in the contents and in others a decrease occurs.
Potassium is the most abundant positively charged electrolyte inside cells, being very
important for the muscle contractility, including cardiac muscle (European Food Safety
Authority (EFSA), 2006).
K concentration ranged from 0.5 to 137.4 mg/L and 0.4 to 246.9 mg/L in still and
sparkling waters, respectively. In general, water samples (still and sparkling) presented an
increment in K levels in the flavoured waters that can be explained by the addition of
ingredients in a K salt form, which is the case of some preservatives.
Sodium is the major extracellular electrolyte with functions in nerve conduction, active
transport and formation of the mineral apatite of the bone (WHO/FAO, 2003).
Table 4 points out high Na contents in some water samples, as referred above. In
flavoured waters, both still and sparkling, a significant increase in the Na contents was
verified, comparing to the natural ones. One of the major influential factors is the addition
of sodium benzoate and sodium citrate as preservatives.
Iron is essential for the haemoglobin (oxygen transport), myoglobin, fatty acid, DNA and
neurotransmitters synthesis, in peroxide conversions, in purine metabolism and in the
nitric oxide production (Lukaski, 2004).
II. Investigação e desenvolvimento
70
Fe contents of samples 14 and 15 (Table 4) were lower than the LOD value. Samples 4,
7, 11, 12, 13 and 26 (Table 4) presented levels between LOD and LOQ values.
In the other samples, Fe contents ranged from 16.5 to 262.9 µg/L and 6.3 to 196.8 µg/L
in still and sparkling water, respectively. In samples with added gas, iron contents are
lower than in the natural ones, which is an interesting factor.
Zinc is essential to enzymes function, acting as catalyst or stabilizing protein structure
(Silvera & Rohan, 2007; Zuliani, Kralj, Stibilj, & Milačič, 2005). Zn in excess competes with
the absorption of Cu and Fe. Zn was detected in all samples, in levels ranging from 5.8 to
30.9 µg/L and 2.8 to 65.9 µg/L in still and sparkling waters, respectively. The behaviour
presented is not constant, with increased levels in some samples and decreased in
others, comparing flavoured and natural waters. Sample 25 (melon/mint) presented the
highest contents of Zn and sample 10 (pineapple/fibre) the second higher content. A
possible justification can be the flavour used in the case of sample 25 (brand F). In brand
C all samples had similar contents regardless of flavour contribution.
Copper is an essential cofactor for a variety of enzymes and, like Zn and Fe, is involved
in the regulation of the expression of the genes for the metal-binding proteins (Zuliani et
al., 2005). Deficient intakes can promote breast cancer and cardiovascular diseases. Cu
was not detected in all samples of brands D and H, neither in some natural water
(samples 7, 11, 26, 31 and 37). As shown in Table 4, the presence of flavours can
increase Cu levels in water samples. In brand I, sample 36 (strawberry flavour) is the only
one with a detectable Cu level. In brand G only samples 28 and 29 (lime and apple) had a
detectable Cu level.
3.4. Effects of same labelled compounds in mineral composition
Table 5 shows the results of the statistical analysis considering the following factors:
preservatives, acidifying agents, fruit juice and sweeteners. These ingredients are added
to natural waters and this study aimed to verify its influence in the contents of the macro
and microminerals evaluated. Their influence in each mineral will be appreciated
individually.
2.1. Macrominerais e microminerais
71
Table 5 Statistical analysis of minerals content results obtained.
Natural water Flavoured water p Ca (mg/L) Preservatives Potassium sorbate+sodium benzoate (n=12) Potassium sorbate (n=7) Sodium benzoate (n=6) Acidifying agents Citric acid+sodium citrate (n=9) Citric acid+natural flavour (n=8) Citric acid (n=12) Fruit juice Yes (n=19) No (n=10) Sweeteners Yes (n=20) No (n=9)
0.22 (0.22-0.83) 108.00 (0.99-110.25) 96.45 (69.90-123.00) 0.83 (0.22-0.95) 87.80 (87.80-123.00) 69.90 (0.52-108.00) 1.47 (0.22-87.80) 69.90 (0.83-108.00) 35.68 (0.83-108.00) 87.80 (0.21-87.80)
2.25 (1.12-8.17) 103.55 (4.41-116.98) 94.17 (54.62-124.09) 3.07 (1.19-8.31) 86.03 (80.24-121.90) 84.25 (53.73-111.76) 77.75 (2.25-118.60) 62.40 (8.93-108.95) 54.17 (3.21-111.76) 82.45 (40.06-98.98)
0.018* 0.722 0.138 0.008* 0.063 0.754 0.845 0.959 0.494 0.678
Mg (mg/L) Preservatives Potassium sorbate+sodium benzoate (n=12) Potassium sorbate (n=7) Sodium benzoate (n=6) Acidifying agents Citric acid+sodium citrate (n=9) Citric acid+natural flavour (n=8) Citric acid (n=12) Fruit juice Yes (n=19) No (n=10) Sweeteners Yes (n=20) No (n=9)
1.72 (1.52-1.72) 24.87 (0.65-33.70) 22.48 (20.09-24.87) 1.52 (0.88-1.72) 19.99 (19.99-28.87) 20.09 (0.36-33.70) 1.72 (0.36-19.99) 20.09 (1.52-33.70) 6.32 (1.52-24.87) 19.99 (0.36-19.99)
2.20 (2.06-3.37) 19.41 (6.03-28.52) 21.50 (11.82-29.43) 2.20 (1.69-3.08) 21.35 (20.25-29.09) 14.33 (10.69-25.35) 19.41 (2.06-27.95) 13.25 (4.32-17.20) 7.54 (2.17-21.61) 20.42 (16.89-24.69)
0.019* 0.875 0.345 0.007* 0.036* 0.272 0.001* 0.028* 0.881 0.173
K (mg/L) Preservatives Potassium sorbate+sodium benzoate (n=12) Potassium sorbate (n=7) Sodium benzoate (n=6) Acidifying agents Citric acid+sodium citrate (n=9) Citric acid+natural flavour (n=8) Citric acid (n=12) Fruit juice Yes (n=19) No (n=10) Sweeteners Yes (n=20) No (n=9)
0.45 (0.45-1.03) 2.51 (2.21-34.96) 19.72 (1.10-38.34) 0.45 (0.45-1.03) 42.74 (38.34-42.74) 2.21 (1.10-2.81) 2.81 (0.45-42.70) 1.10 (1.03-2.21) 1.07 (0.45-2.21) 42.74 (2.81-42.74)
96.18 (86.26-117.50) 83.65 (36.46-113.24) 28.04 (16.98-37.04) 107.47 (89.47-127.45) 36.45 (32.45-41.24) 72.11 (18.39-102.82) 65.65 (36.70-96.18) 83.65 (16.98-114.68) 82.42 (36.42-112.17) 41.79 (28.77-99.99)
0.018* 0.008* 0.249 0.008* 0.030* 0.002* 0.014* 0.005* <0.001* 0.594
Na (mg/L)
0.017* 0.002* 0.029* 0.007* 0.050* 0.002*
Preservatives Potassium sorbate+sodium benzoate (n=12) Potassium sorbate (n=7) Sodium benzoate (n=6) Acidifying agents Citric acid+sodium citrate (n=9) Citric acid+natural flavour (n=8) Citric acid (n=12) Fruit juice Yes (n=19) No (n=10) Sweeteners Yes (n=20) No (n=9)
8.20 (6.70-8.20) 43.10 (8.10-535.10) 269.95 (4.80-535.10) 6.70 (5.45-8.20) 560.10 (535.10-560.10) 8.10 (4.80-43.10) 8.20 (8.10-560.10) 6.70 (4.80-43.10) 8.20 (4.80-43.10) 560.10 (8.1-560.10)
245.20 (209.00-359.70) 57.30 (43.98-583.68 295.25 (31.90-612.73) 221.70 (170.55-325.30) 629.90 (564.63-664.38) 39.40 (20.58-46.85) 496.60 (57.70-619.40) 46.40 (34.53-160.76) 199.60 (46.35-506.25) 496.60 (16.7-648.60)
0.001* 0.005* <0.001* 0.051
II. Investigação e desenvolvimento
72
Natural water Flavoured water p Fe (µg/L)
Preservatives Potassium sorbate+sodium benzoate (n=12) Potassium sorbate (n=7) Sodium benzoate (n=6) Acidifying agents Citric acid+sodium citrate (n=9) Citric acid+natural flavour (n=8) Citric acid (n=12) Fruit juice Yes (n=19) No (n=10) Sweeteners Yes (n=20) No (n=9)
83.60 (5.10-83.60) 8.60 (3.00-58.00) 4.70 (0.81-8.60) 20.70 (5.10-83.60) 4.50 (4.50-8.60) 2.05 (1.05-58.00) 4.50 (2.30-25.80) 5.10 (0.80-58.00) 8.60 (3.00-58.00) 4.50 (1.80-4.50)
13.90 (8.90-18.90) 41.80 (35.43-79.45) 20.25 (2.30-55.85) 16.50 (9.90-39.00) 38.05 (28.00-118.18) 38.05 (7.95-79.45) 35.40 (13.90-104.30) 19.60 (3.83-44.25) 27.70 (9.40-42.20) 49.2 (23.55-104.90)
0.128 0.050* 0.046* 0.594 0.012* 0.158 0.044* 0.646 0.601 0.015*
Zn (µg/L) Preservatives Potassium sorbate+sodium benzoate (n=12) Potassium sorbate (n=7) Sodium benzoate (n=6) Acidifying agents Citric acid+sodium citrate (n=9) Citric acid+natural flavour (n=8) Citric acid (n=12) Fruit juice Yes (n=19) No (n=10) Sweeteners Yes (n=20) No (n=9)
31.20 (11.90-31.20) 7.35 (2.80-11.73) 6.65 (5.10-8.23) 24.90 (11.90-31.20) 4.80 (4.80-6.00) 7.75 (2.80-19.68) 8.60 (4.80-24.90) 6.35 (2.80-11.90) 8.90 (5.95-21.65) 4.80 (4.80-22.50)
16.30 (13.80-21.70) 9.85 (6.50-24.55) 6.65 (5.00-8.79) 15.30 (11.95-21.05) 9.30 (12.35-6.88) 10.30 (6.83-28.28) 12.20 (8.30-21.70) 12.25 (6.55-18.40 ) 11.25 (7.13-16.08) 12.40 (23.55-30.15)
0.176 0.015* 0.991 0.260 0.043* 0.015* 0.796 0.018* 0.925 0.008*
Cu (µg/L) Preservatives Potassium sorbate+sodium benzoate (n=12) Potassium sorbate (n=7) Sodium benzoate (n=6) Acidifying agents Citric acid+sodium citrate (n=9) Citric acid+natural flavour (n=8) Citric acid (n=12) Fruit juice Yes (n=19) No (n=10) Sweeteners Yes (n=20) No (n=9)
8.65 (8.65-9.08) 0.10 (0.07-0.15) 0.08 (0.06-0.10) 8.65 (4.85-8.08) 6.10 (0.06-0.10) 0.10 (0.10-0.15) 0.10 (0.10-1.04) 0.15 (0.10-9.08) 0.15 (0.10-8.65) 0.10 (0.10-0.10)
9.35 (7.95-12.45) 0.61 (0.10-1.80) 0.10 (0.09-0.29) 9.35 (7.56-12.35) 1.13 (0.10-1.51) 0.33 (0.10-1.07) 1.46 (0.50-7.17) 0.41 (0.10-12.30) 1.43 (0.10-9.27) 1.39 (0.61-1.93)
0.051 0.018* 0.285 0.018* 0.026* 0.028* 0.001* 0.063 0.005* 0.012*
*Statistically significant p<0.05. Data are presented was median (1st quartile–3rd quartile).
3.4.1. Calcium
Taking into account the preservatives added, it was observed that the blend of
potassium sorbate and sodium benzoate lead to significant statistical differences (p =
0.018). For the remaining preservatives the differences observed are not significant (p >
0.05).
In what concerns acidifying agents, only the waters with, simultaneously, citric acid and
sodium citrate presented statistically significant differences from its natural corresponding
2.1. Macrominerais e microminerais
73
water (p = 0.008). The addition of fruit juice (p = 0.845) as well as sweeteners does not
influence Ca concentration.
3.4.2. Magnesium
In the case of Mg the statistical study showed that the addition of preservatives
(potassium sorbate and sodium benzoate) (p = 0.019) and acidifying agents increased
significantly Mg concentration. Regarding fruit juice, the statistical values are p = 0.001
and 0.028 for flavoured waters with and without fruit juice, respectively.
No other factor affected Mg concentration in a significant way.
3.4.3. Potassium
The addition of preservatives, only one (potassium sorbate) or in combination
(potassium sorbate and sodium benzoate) as well as all acidifying agents, increased
significantly K levels (p = 0.008 and 0.018, respectively) (Table 5).
Regarding the presence or absence of juice, there is also a significant statistical
difference in K concentration between flavoured and natural waters. The addition of
sweeteners also increased significantly K concentration (p < 0.001).
3.4.4. Sodium
Statistical analysis showed that the influence of numerous factors led to significant
differences in the results. This can be seen in Table 5.
3.4.5. Iron
The presence of potassium sorbate or sodium benzoate, as preservatives, induced
significant differences in Fe contents, as well as the presence of citric acid and natural
flavours. Also, significant differences, in Fe content, can be verified amongst waters
without fruit juice or sweeteners.
3.4.6. Zinc
The statistical analysis for the influence in Zn concentration showed that the major
influence came from potassium sorbate and citric acid (Table 5). As referred for Fe,
II. Investigação e desenvolvimento
74
significant differences amongst waters without fruit juice and sweeteners could also be
noticed.
3.4.7. Copper
Regarding the influence of the considered factors in Cu concentration, the ones that
caused statistical significant differences were: potassium sorbate (in the preservative
group), all acidifying agents and the presence of fruit juice. The presence of sweeteners
influenced the levels of this micromineral in all samples.
4. Conclusion
This study leads to conclude that flavoured waters can be an adequate alternative to
consumers that do not like natural water. The different ingredients added to natural waters
hardly influence its mineral composition. All consumers are advised to read the label
content, in order to avoid some health problems that can occur with some mineral waters
and some specific groups of consumers. Also, flavoured waters could represent
advantages due to the presence of certain minerals, some vitamins, antioxidants and
bioactive compounds. Some preservatives, acidifying agents and sweeteners are not
hazardous if consumed with moderation.
Acknowledgement
M. Fatima Barroso is grateful to Fundacao para a Ciencia e a Tecnologia for the Ph.D.
Grant (SFRH/BD/29440/2006).
2.1. Macrominerais e microminerais
75
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<http://www.iom.edu/Object.File/Master/20/004/Electrolytes%20Table%20for%20web.p
df>.
Institute of Medicine from United States. (2007). DRI values definitions. Food and Nutrition
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Liu, C. L., Chen, Y. S., Yang, J. H., Chiang, B. H., & Hsu, C. K. (2007). Trace element
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Miller, J. N., & Miller, J. C. (2000). Statistics and chemometrics for analytical chemistry.
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Nasreddine, L., Parent-Massin, D. (2002). Food contamination by metals and pesticides in
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Tamasi, G., & Cini, R. (2004). Heavy metals in drinking waters from Mount Amiata
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contaminants. 26th report of the joint FAO/WHO expert committee on food additives,
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<http://www.inchem.org/documents/jecfa/jecmono/v18je18.htm>.
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Zuliani, T., Kralj, B. L., Stibilj, V., Milačič, R. (2005). Minerals and trace elements in food
commonly consumed in Slovenia. Italian Journal of Food Science, 17(2), 155-166.
2.2. Minerais vestigiais
77
Survey of trace elements (Al, As, Cd, Cr, Co, Hg, M n, Ni, Pb, Se,
and Si) in retail samples of flavoured and bottled waters
M.F. Barrosoab, S. Ramosc, M.T. Oliva-Telesa, C. Delerue-Matosa, M.G.F. Salesa and
M.B.P.P. Oliveirab aRequimte/Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de
Almeida 431, 4200-072, Porto, Portugal. bRequimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto,
Rua Aníbal Cunha, 164, P-4099-030, Porto, Portugal. cInstituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida 431,
4200-072, Porto, Portugal.
Abstract
Concentrations of eleven trace elements (Al, As, Cd, Cr, Co, Hg, Mn, Ni, Pb, Se, and
Si) were measured in 39 (natural and flavoured) water samples. Determinations were
performed using graphite furnace electrothermetry for almost all elements (Al, As, Cd, Cr,
Co, Mn, Ni, Pb, and Si). For Se determination hydride generation was used, and cold
vapour generation for Hg. These techniques were coupled to atomic absorption
spectrophotometry. The trace element content of still or sparkling natural waters changed
from brand to brand. Significant differences between natural still and natural sparkling
waters (p < 0.001) were only apparent for Mn. The Mann–Whitney U-test was used to
search for significant differences between flavoured and natural waters. The concentration
of each element was compared with the presence of flavours, preservatives, acidifying
agents, fruit juice and/or sweeteners, according to the labelled composition. It was shown
that flavoured waters generally increase the trace element content. The addition of
preservatives and acidifying regulators had a significant influence on Mn, Co, As and Si
contents (p < 0.05). Fruit juice can also be correlated to the increase of Co and As.
Sweeteners did not provide any significant difference in Mn, Co, Se and Si content.
Keywords : health significance; metals analysis – atomic absorption spectrometry (AAS);
method validation; statistical analysis; heavy metals – arsenic; heavy metals – cadmium;
heavy metals – mercury; metals – nutritional; beverages; drinking water; water
Available online at www.inforworld.com
Food additives and Contaminants
2009, (2) 2, 121-130
2.2. Minerais vestigiais
79
Introduction
Trace elements are required in small quantities (µg kg-1 body weight) in the human diet
to maintain normal physiological functions (Goldhaber 2003; Silvera and Rohan 2007).
They are found naturally in the environment, and human exposure is derived from a
variety of sources, including the air, food and drinking water (Silvera and Rohan 2007).
Some trace elements have no known beneficial biological function in humans, but
generally these elements have an impact on human health in many ways (Santos et al.
2004). In several cases, slightly high exposure may be harmful to human health (Ysart et
al. 2000). Some trace elements, such as mercury (Hg), lead (Pb), arsenic (As), cadmium
(Cd), aluminium (Al), chromium (Cr) and nickel (Ni), produce a variety of toxic effects
when intake exceeds the established limit (World Health Organization (WHO) 1996;
Berdanier 1998; Chou and De Rosa 2003; Aziz et al. 2006; Diawara et al. 2006; Mehra
and Baker 2007; Barton 2008; Jorhem et al. 2008; Mor and Ceylan 2008). On the other
hand, some nutritional problems are related to low or no intake of some elements, like
selenium (Se), cobalt (Co) and manganese (Mn) (Magnuson et al. 1997; Barceloux 1999;
Institute of Medicine (IOM) of National Academies 2001; Goldhaber 2003; Alinnor 2005).
Almost all trace elements can be found in bones and teeth (Berdanier 1998).
The intake of trace elements by consumers depends on their content in foodstuffs, the
consumer’s drink/beverage eating habits, and of course the kind of drinking water. The
risks to health of the presence of certain trace elements in water can be assessed by
comparing estimates of dietary exposures with the provisional tolerable weekly intake
(PTWI) recommended by the Joint Expert Committee on Food Additives (JECFA) of the
Food and Agricultural Organization of the United Nations and the World Health
Organization (FAO/WHO). PTWI values range from 0.005 to 0.025 mg kg-1 body weight
week-1 for As, Cd, Hg and Pb. Consumers can also use the recommended dietary
allowance (RDA) from the US Food and Nutrition Board of the Institute of Medicine
(FNB/IOM). The RDA ranges between 25 and 1800 µg day-1 for the trace elements Cr, Se
and Mn (IOM 2001; WHO 2008).
The results presented in this paper focus on the determination of eleven trace elements
(Al, As, Pb, Cd, Co, Cr, Hg, Mn, Ni, Se and Si) in 39 mineral or spring bottled water
samples (with and without flavours) from the market in Portugal. Trace element
concentrations in water depend on soil characteristics, such as organic matter content,
pH, clay mineralogy, and physical and chemical forms in which they are dispersed, which
can affect the bioavailability of these elements (Santos et al. 2004). Flavoured water
consists of water with the addition of flavours, juices, bioactive compounds, preservatives
and/or sweeteners that provide a characteristic taste and odour appreciated by
II. Investigação e desenvolvimento
80
consumers. Mineral and spring waters with important mineral contents are used in the
case of flavoured waters. According to the US Food Drug and Administration (USFDA)
(2002), mineral water arises from a geologically and physically protected underground
source, characterized by constant levels and relative proportions of minerals and trace
elements at the source. Spring water is derived from an underground formation from
which water flows naturally to the surface at an identified location.
In this study atomic absorption spectrometry (AAS) was used with electrothermic
atomization in graphite furnace and hydride generation for the quantification of the
selected trace elements. A nutritional and statistical study was carried out to compare
these different kinds of water. The knowledge of trace element concentrations in waters
can provide important information on the impact of the use of these beverages. Until now,
in this respect no published studies were found.
Material and methods
Apparatus
For the quantification of almost all trace elements (Al, As, Pb, Cd, Co, Cr, Mn, Ni and Si)
an Analytik Jena Zeenit 650 spetrophotometer was used with electrothermic atomization
in a graphite furnace equipped with an Analytik Jena MPE60 autosampler. Pyrolytically
coated graphite tubes with integrated pin platform were used. Specific interferences from
the matrix were not observed in all samples and the Zeeman background correction was
sufficient. Hollow cathode lamps were used (Varian). A stream of ultrapure Ar at 5.5 bar
was used in the electrothermic determinations.
Hydride generation atomic absorption spectroscopy (HS60; Analityk Jena) was used for
Se and Hg quantifications. In order to determine the total content of Se, a preliminary
reduction of Se(VI) to Se(IV) is needed because only Se(IV) is hydride active (Magnuson
et al. 1997). The efficiency of the reduction depends on the temperature, reduction time
and HCl concentration. The resulting Se hydride was drawn off under inert atmosphere
(Ar) into a quartz T-tube at 960ºC and the determinations were performed at 196.0 nm. Hg
was determined by cold vapour generation at room temperature at 253.7 nm.
The optimization of analytical conditions for the quantification of trace elements was
based on the Standard Methods for the Examination of Water and Wastewater (American
Public Health Association (APHA), 1995).
2.2. Minerais vestigiais
81
Reagents
The water used was ultrapure quality (18.2MΩ cm-1) and obtained from a Millipore
Simplicity 185 system.
All chemicals used for the analytical determinations were supra-pure grade and
acquired from Merck. Standard solutions of each element (Al, As, Pb, Cd, Co, Cr, Hg, Mn,
Ni, Se and Si) were prepared daily by dilution of the stock solutions (1000 mg l-1) with
water and 0.1% (v/v) nitric acid and stored in polyethylene bottles.
All glassware and polyethylene vessels were soaked with 10% HNO3 at least overnight
and then rinsed with ultrapure water before use.
Samples collection and pre-treatment
Thirty-nine water samples corresponding to ten different brands (mineral and spring)
were collected in several supermarkets in Portugal. Each brand (still or sparkling) had
different flavours and aromas. Table 1 summarizes the nutrient information on the labels,
namely the presence of vitamins, sweeteners and preservatives.
All samples were acidified with supra-pure HNO3 (1 ml l-1) and stored in sealed
polyethylene bottles maintained at 4ºC. In the sparkling waters, gas was removed by
sonication before HNO3 conservation and storage.
Matrix modifiers were used in order to eliminate the interference of the water sample
matrix. Mg(NO3)2 (0.1%; v/v) was used for the analysis of Al, Cr and Co. For the As
quantification Pd(NO3)2 (0.1%; v/v) was used NH4H2PO4 (0.1%; v/v) was used for Cd and
Pb analyses. A mixture of Mg(NO3)2 and Pd(NO3)2 was utilized for Si determinations.
For the evaluation of Se, water samples were acidified with HCl 6 mol l-1 and heated at
about 100ºC during 45 min. After this treatment the samples could be subjected to hydride
generation that was performed with a 0.3% sodium borohydride (NaBH4) in 0.1% NaOH
and acidic medium (HCl 3%).
To analyse Hg, all water samples were digested with HNO3 and HCl followed by
reduction to elementary mercury vapour by SnCl2 (Akagi et al. 2000; Ohno et al. 2007).
II. Investigação e desenvolvimento
82
Table 1. Label information in bottled flavoured waters evaluated.
Brand Sample Flavour Other ingredients Still water
A Mineral 1 Lemon 2 Mango
3 Strawberry
preservatives (potassium sorbate, sodium benzoate) acidity regulators ( acid citric, sodium citrate), sweeteners
4 Natural
B Spring 5 Pineapple /orange Apple juice concentrate, preservatives (potassium sorbate, sodium benzoate) acidity regulators (citric acid) and sweeteners
6 Lemon
Apple juice concentrate, preservatives (potassium sorbate, sodium benzoate) acidity regulators (citric acid), sweeteners, added vitamins
7 Natural
C Mineral 8 Lemon/magnesium
9 Apple/ white tea
10 Pineapple/fibre
Fruit juice concentrate, preservative (potassium sorbate), acidity regulator (citric acid), added vitamins
11 Natural
D Mineral 12 Apple
13 Orange/peach
14 Lemon
Preservatives (sodium benzoate), acidity regulator (citric acid), sweeteners
15 Natural
Sparkling water
E Mineral Added gas
16
Lemon
17 Orange/raspberry
18 Peach/pineapple
19 Guava
Lemon juice, preservatives (potassium sorbate, sodium benzoate), acidity regulator (citric acid, sodium citrate) and sweeteners
20 Natural
F Mineral Natural gas
21
Lemon/green tea
22 Raspberry/ginseng
23 Peach/white tea
24 Mango/ginkgo beloba
25 Melon/mint
Lemon, apple and pear juice, acidity regulator (citric acid)
26 Natural
2.2. Minerais vestigiais
83
Brand Sample Flavour Other ingredients
G Mineral Added gas
27
Lemon
Preservatives (potassium sorbate), regulator acidity (citric acid) added vitamin, sweeteners
28 Lime
29 Apple
30 Peach
Preservatives (potassium sorbate), regulator acidity (citric acid) added vitamin, sweeteners
31 Natural
H Mineral Natural gas
32
Lemon
Lemon and apple juice, preservatives (sodium benzoate, potassium sorbate) acidity regulator (citric acid), added vitamins, sweeteners
33 Natural
I Mineral Natural gás
34
Lemon
Lemon and apple juice, preservative (sodium benzoate), acidity regulator (citric acid), sweeteners
35
Green Apple
Apple juice, preservative (sodium benzoate), acidity regulator (citric acid), sweeteners
36
Strawberry
Apple and strawberry juice, preservative (sodium benzoate), acidity regulator (citric acid), sweeteners
37 Natural
J Spring Added gas
38
Lemon
Lemon juice, preservative (sodium benzoate, potassium sorbate), acidity regulator (citric acid, sodium citrate), sweeteners
39 Natural
Note: Natural waters have no added ingredients.
Analytical quality assurance
Triplicate determinations were made on all samples. Validation of the proposed methods
was evaluated by linearity range, limit of detection (LOD), limit of quantification (LOQ),
precision and accuracy. The LOD is defined as 3 σ s-1 and the LOQ 10 σ s-1, where σ is
the standard deviation of the blank signal (n = 20) and s is the slope of the calibration plot
(Miller and Miller 2000). The precision of the proposed methods was investigated by intra-
and inter-day determinations of standard solutions and expressed by relative standard
deviations (RSD). For intra-day studies, each concentration was assessed by performing
three repeated measurements three times during a working day. The inter-day
measurements studies were performed over 1 week. Accuracy and reproducibility of
methods were checked by recoveries.
Statistical analysis
All results are expressed as mean ± standard deviation (SD). In the statistical analysis
data were presented as median (first quartile – third quartile). The significance of the
II. Investigação e desenvolvimento
84
differences between natural still and natural sparkling waters was tested by a Mann–
Whitney U-test. Comparisons between natural water group and the respective flavoured
water group were carried out by a Wilcoxon test (dependent samples). All graphical and
inferential statistical analysis was performed using the Statistica7 software. p ≤ 0.05 was
considered as being statistically significant.
Results and discussion
Trace elements quantification
Table 2 summarizes the data obtained for calibration curves and the performance
characteristics of the methods optimized for all elements studied. The linearity range was
from 0.20 to 200.0 µg l-1.The calculated LOD values ranged from 0.005 to 3.70 µg l-1 and
LOQ values ranged from 0.018 to 12.34 µg l-1. No significant differences were found
between intra- and inter-day experiments. RSD values ranged from 0.5% to 9.6%,
showing a high degree of precision. Recoveries were determined and all values ranged
from 99% to 110%, showing good accuracy.
Table 2. linear range, limit values and precision obtained for the trace elements studied.
Notes: a)Average of three measurements, three times along a day. b)RSD: relative standard deviation.c)Average of three
measurements over a week.
Label information
Figure 1 shows the median concentration of the trace elements analysed in flavoured
and natural waters. The mineral composition of natural waters (without flavours) depends
of the brand, and the different natural waters origins, namely different geological
structures. Labelling only indicated the concentrations of macrominerals (Ca2+, Na+, Mg2+,
K+) and some anions (NO3-, Cl-, HCO3
-). No information is given concerning trace element
Parameters Al As Cd Cr Co Hg Mn Ni Pb Se Si
Linear concentration
(µg l-1)
16.00-
40.00
1.00–
10.00
0.20–
1.00
1.00–
10.00
2.00–
10.00
0.10–
10.00
1.00–
5.00
2.00–
10.00
2.00–
10.00
0.200–
1.00
20.00–
200.00
LOD ( µg l-1) 0.73 0.11 0.005 0.04 0.11 0.02 0.13 0.07 0.49 0.04 3.70
LOQ ( µg l-1) 2.45 0.40 0.018 0.14 0.35 0.05 0.45 0.23 1.63 0.15 12.34
Intra-day studiesa)
RSDb) (%) 9.60 3.94 0.56 2.02 3.66 0.48 2.63 2.61 3.28 2.41 2.41
Inter-day studiesc)
RSDb) (%) 3.80 1.24 4.05 2.99 2.50 5.20 1.87 3.76 4.27 5.64 5.64
2.2. Minerais vestigiais
85
contents, with the exception of some natural waters that reported Si contents. Si values
determined in this work were similar to those described in the labels.
Natural water Flavoured water0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Hg
(ug
l-1)
p = 0.310
Natural water Flavoured water0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
160,0
Mn
(ug
l-1)
p = 0.458
Natural water Flavoured water0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Hg
(ug
l-1)
p = 0.310
Natural water Flavoured water0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
160,0
Mn
(ug
l-1)
p = 0.458
Natural water Flavoured water0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
160,0
Al (
ug l-1
)
p = 0.024
Natural water Flavoured water0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
As
(ug
l-1)
p = 0.001
Natural water Flavoured water0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
160,0
Al (
ug l-1
)
p = 0.024
Natural water Flavoured water0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
As
(ug
l-1)
p = 0.001
Natural water Flavoured water0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
Cr
(ug
l-1)
p = 0.709
Natural water Flavoured water0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
Co
(ug
l-1)
p = 0.064
Natural water Flavoured water0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Hg
(ug
l-1)
p = 0.310
Natural water Flavoured water0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
160,0
Mn
(ug
l-1)
p = 0.458
Natural water Flavoured water0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Hg
(ug
l-1)
p = 0.310
Natural water Flavoured water0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
160,0
Mn
(ug
l-1)
p = 0.458
II. Investigação e desenvolvimento
86
Figure 1. Al, As, Cr,Co, Hg, Mn, Ni, Pb, Se and Si concentrations in flavoured and correspondent natural
eaters. Bars represent the median and whiskers represent the inter-quartile range.
Several natural water samples showed important levels of Al, being the case for
samples 33 and 37 (441 and 158 µg l-1). Ni, Pb and Se were only detected in one (sample
20), three (samples 4, 20 and 39) and three (samples 20, 21 and 31) natural water
samples, respectively. Hg was detected in 50% of natural waters (in five samples). Cd
was not detected in all natural waters.
Except for manganese, there were no statistical differences in trace element
concentrations between natural still and natural sparkling waters (p < 0.001). From the
bottle labels (Table 1) it was possible to obtain more information, namely about the added
Natural water Flavoured water0,0
0,1
0,2
0,3
0,4
0,5
Ni (
ug l-1
)
p = 0.197
Natural water Flavoured water0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
Pb (
ug l-1
)
p = 0.225
Natural water Flavoured water0,0
0,1
0,2
0,3
0,4
0,5
Ni (
ug l-1
)
p = 0.197
Natural water Flavoured water0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
Pb (
ug l-1
)
p = 0.225
Natural water Flavoured water0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
Se (
ug l-1
)
p = 0.237
Natural water Flavoured water0,0
10,0
20,0
30,0
40,0
50,0
Si (
ug l-1
)
p = 0.299
Natural water Flavoured water0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
Se (
ug l-1
)
p = 0.237
Natural water Flavoured water0,0
10,0
20,0
30,0
40,0
50,0
Si (
ug l-1
)
p = 0.299
2.2. Minerais vestigiais
87
ingredients to the flavoured waters. Among them can be cited fibres, fruit juices and
vitamins.
Inevitably, these waters also need ingredients, other than those added to provide health
and well-being claims, but necessary to assure the quality desired for the producer and
consumers and safety of the product such as acidifying agents, sweeteners and
preservatives.
About 50% of the flavoured water samples contained sweeteners as ingredients. There
were samples with only one (acesulfame-K, sucralose or aspartame) and with blends of
two sweeteners (acesulfame-K and aspartame; acesulfame-K and sucralose). The most
used was acesulfame-K (present in 14 samples) and aspartame (ten samples). It is
interesting to note that, in general, the samples from the same brand have the same
sweetener, with the exception of band I.
Concerning the preservatives, each sample contained one (potassium sorbate or
sodium benzoate) or two (potassium sorbate and sodium benzoate). Flavoured waters
would not ideally replace natural water, but can be an interesting alternative to soft drinks.
Flavour factor analysis
Considering only the flavour factor, the contents on Cr, Co and Al were higher in the
flavoured waters than in the natural ones. For Mn, As, Pb, Se, and Si the median
concentrations were slightly higher in the natural waters. Ni and Hg when present had
similar concentrations in both groups (Figure 1). However, the differences observed
between the median concentration of the two groups (natural and flavoured) were
statistically significant only for As (p = 0.001) and Al (p = 0.024).
Considering that Cd was not detected in any natural water samples, it was not possible
to perform any statistical analysis.
Discussion of individual trace element composition
Aluminium (Al) was detected in all 39 water samples. Its concentrations ranged from 5.8
to 143.5 µg l-1 (still waters) and from 7.5 to 441 µg l-1 (sparkling waters). In general,
flavoured waters contain Al concentration higher than natural ones, probably due to the
addition of the indicated ingredients. Nookabkaew et al. (2006) and Mehra and Baker
(2007) detected Al contents in tea samples ranging from 1878 to 16 520 µg l-1 and from
458 to 1307 mg kg-1, respectively. Theses values are much higher than those found in
flavoured waters. A high Al content in tea samples is not surprising, because the tea plant
is known to be an Al accumulator (Mehra and Baker 2007). Another paper reported Al
II. Investigação e desenvolvimento
88
concentrations in drinking waters ranging from 3.9 to 16.4 µg l-1 (Tamasi and Cini 2004),
values that are similar to those found in two samples analysed (samples 15 and 20). The
other samples had values several fold higher.
As occurs naturally in soil and in many kinds of rock, especially in minerals that contain
lead and copper (Chou and De Rosa 2003) and it is a natural constituent of water. No As
was detected in 20 samples analysed (five still and 15 sparkling). Some brands (D, F, H
and I) have no As detectable content in all samples analysed (natural and flavoured). In
other brands some flavoured samples have lost the As content detected in the
corresponding natural water. Six samples had values between the LOD and the LOQ and
13 samples had As levels between 0.45 and 2.63 µg l-1.
Drinking water is one of the most important sources of As exposure. There are many
reports of chronic arsenism resulting from drinking water containing high levels of As in
endemic areas worldwide (Chou and De Rosa 2003). Analysis of tea samples indicated
As levels ranging from 0.2 to 1.5 µg l-1 (Nookabkaew et al. 2006). These values are in
agreement with those found in the flavoured waters studied.
Cd is widely distributed in the environment being a contaminant of soil, air, water, plants,
and food supplies. Unless contamination has occurred, the levels of Cd in most foods are
normally very low. In this work only four water samples contained Cd (all flavoured) with
values around 0.1 µg l-1. Similar results were found in drinking water and teas (Tamasi
and Cini 2004; Nookabkaew et al. 2006).
Cr is often found in soil and groundwater of abandoned industrial plants. In the waters
studied no Cr was found in five samples (four still and one sparkling). Only one sample
(number 12) was between the LOD and the LOQ values. Cr values ranged from 0.13 to
5.42 µg l-1. The higher values of Cr were found in sparkling flavoured waters, samples 19
(5.42 µg l-1) and 39 (2.13 µg l-1). Cr levels increased in all still flavoured waters, probably
due to the addition of flavours and other ingredients. Almost all flavoured sparkling brand
samples had a lower Cr concentration than the natural waters. Brand H showed the same
Cr value for the two samples analysed and only in brand B the Cr content increased in
waters with flavour. Comparing these values with those obtained by Nookabkaew et al.
(2006), where Cr ranged from 3 to 14 µg l-1, it is easy to verify that flavoured waters had
lower values of Cr.
Co was not detected in ten samples (three still, seven sparkling) and the values from
nine samples were between the LOD and the LOQ values. The other samples showed a
Co concentration ranging from 0.17 to 2.2 µg l-1. In all still flavoured waters the Co
contents increased. In sparkling flavoured waters some samples had increased levels,
and in others a decrease occurred without any obvious explanation. These results are
2.2. Minerais vestigiais
89
similar to those obtained by other authors (Tamasi and Cini 2004; Nookabkaew et al.
2006) in drinking waters.
The persistence in the environment, bioaccumulation, transport in the aquatic chain and
presence in a variety of foods make Hg among the most dangerous of all metals in the
human food chain. The widespread use of Hg and its derivatives in industry and
agriculture (now banned in most countries) had resulted in serious environmental
pollution. Only six of 24 sparkling samples contained Hg (from 0.06 to 0.3 µg l-1). Still
waters had Hg levels higher than sparkling ones. Two natural waters had contents of 0.8
and 0.67 µg l-1. Similar values were also reported in other studies with herbal tea and
drinking water (Alinnor 2005; Nookabkaew et al. 2006).
Mn occurs in the environment and is a natural water component. In the samples studied
Mn was not detected in six still water samples. The other samples had Mn levels ranging
from 0.28 to 1.22 µg l-1 in still water and from 0.77 to 236 µg l-1 in sparkling waters. It was
shown that sparkling natural water with natural gas had a higher Mn content (about 100
time higher) than still/added gas natural water. In general, the addition of flavours and
other ingredients increased the Mn concentration. This behaviour may be due to different
physiological properties or structures of flavours/aromas, levels of phytochelating
phenolics and other mineral-binding components present in the final product. Similar
behaviour can be found with respect to other trace elements. Several tea samples had
higher values of Mn than the flavoured waters studied. In tea samples, Mn concentration
ranges from 483 to 2766 µg l-1 (Nookabkaew et al. 2006; Mehra and Baker 2007).
Ni is used in many industrial and consumer products, including stainless steel, magnets,
coinage, and special alloys. In the present study only one still water sample had a
detectable content of Ni. In sparking water samples only brand E contained Ni in all
samples. In the other brands only one or two samples had a detectable Ni content. The
values are in agreement with those in Tamasi and Cini (2004).
Pb has probably the longest history of environmental contamination and toxicity to
humans. Its presence in the human food chain continues to be a great health problem
worldwide. Pb values ranged from 0.79 to 2.22 µg l-1 and from 0.55 to 1.28 µg l-1 in still
and sparkling waters, respectively. It was detected in 50% of the analysed samples.
Generally, the addition of flavours and aromas increased Pb content. The values found
are similar to those obtained in drinking waters and herbal teas (Soylak et al. 2002;
Tamasi and Cini 2004; Nookabkaew et al. 2006; Obiri 2007).
Se is frequently found in combination with Pb, Cu, Hg, and Ag in the environment. Se
was not found in eleven still and ten sparkling waters. Se content values are low (from
0.05 to 0.44 µg l-1). These values are similar to others obtained by other authors in tea
samples (Nookabkaew et al. 2006).
II. Investigação e desenvolvimento
90
Si is the second most abundant element on Earth and is usually found in the form of
silicon dioxide (also known as silica) and silicate. Si was found in all water samples. Si
concentration values ranged from 4.8 to 541.1 µg l-1. The highest Si values were found in
sparkling waters. Considering still waters, only in brand D did Si concentration levels
increase in flavoured water samples. In other brands, Si levels decreased. In sparkling
waters, Si contents decreased in all flavoured waters compared with the values presented
in natural ones. Sripanyakorn et al. (2004) reported Si levels ranging from 9.6 to 22.5 µg l-1
in several kinds of beers. Dejneka and Łukasiak (2003) analysed fruit juice where the Si
concentration ranged from 3.66 to 14.38 µg l-1. These values are similar to the majority of
the samples analysed, but there was one sample with 541.1 mg l-1. According with the
label information this sample had raspberry/ginseng flavour with addition of lemon, apple
and pear juice, fibres and citric acid.
Effects of some labelled compounds in mineral compo sition
Considering the following factors – preservatives, acidifying agents, fruit juice and
sweeteners – a statistical study was made. These ingredients are added to natural waters
and this study aimed to verify their influence on the contents of the trace elements
evaluated. Their influence in each element will be appreciated individually.
Aluminium
Statistical analysis showed that almost all ingredients added to the water do not
influence Al content (p > 0.05). Only significant differences could be found in flavoured
waters without sweeteners (p < 0.05).
Arsenic
Taking into account the added preservatives, it was observed that potassium sorbate
and the blend of potassium sorbate and sodium benzoate led to significant differences (p
< 0.05). Concerning acidifying agents, only the sample with, simultaneously, citric acid
and sodium citrate had significant differences compared with its natural corresponding
water (p = 0.043). Regarding fruit juice, the statistical values were p = 0.018 and 0.028 for
flavoured waters with and without fruit juice, respectively. The addition of sweeteners (p =
0.008) also seemed to influence As concentration.
2.2. Minerais vestigiais
91
Chromium
In the case of Cr, the statistical study showed that the addition of acidifying regulators
(citric acid and natural flavour) (p = 0.012) increased Cr concentrations significantly.
Regarding the presence or absence of sweeteners, only flavoured waters without
sweeteners increased Cr contents significantly (p = 0.046). No other factor affected Cr
concentration in a significant way.
Cobalt
Regarding the influence of the considered factors on Co concentration, those that
caused significant differences were potassium sorbate and sodium benzoate (in the
preservatives group; p = 0.028); and citric acid and sodium citrate (in acidifying regulators;
p = 0.017). The presence of fruit juice also influenced the levels of this trace element in all
samples (p = 0.019).
Manganese
The blend of potassium sorbate and sodium benzoate as preservatives led to significant
differences in Mn contents, as well as the presence of citric acid and sodium citrate (p =
0.017 for both). Also, a significant difference was verified in flavoured waters without fruit
juice (p = 0.018).
Silicon
The addition of preservatives (potassium sorbate and sodium benzoate) (p = 0.008) and
acidifying regulators (citric acid and sodium citrate) (p = 0.008) increased Si levels
significantly. Regarding the presence of fruit juice or sweeteners, these groups do not
influence Si concentration (p = 0.070 and 0.765, respectively).
Cadmium, mercury, nickel, lead and selenium
Taking into account that only four water samples contained Cd, it was not possible to
perform the statistical analysis for this trace element.
For Hg, Ni, Pb and Se, it was difficult to carry out the statistical analysis because these
elements had several missing values. However, it was shown that the addition of
ingredients in flavoured waters does not affect Hg, Ni, Pb and Se concentrations.
II. Investigação e desenvolvimento
92
Conclusion
The different ingredients added to natural waters (flavours, preservatives, acidifying
regulators, fruit juice and sweeteners) usually influence the concentrations of trace
element. It was verified that preservatives provide a significant contribution to the contents
of As, Co, Mn and Si. The acidifying regulators also produce differences in the
concentrations of As, Cr, Co, Mn and Si. Fruit juice and sweeteners can be correlated to
the increase of Co and As levels.
Although there was an increase in concentrations of trace elements by the added
ingredients, it did not impact on human health because levels never exceeded the
established values recommended by the WHO and the IOM. It is important to note that Cd
was not detected in natural waters and that 50% of the analysed samples had detectable
levels of Hg and Pb. Mn levels were the only element where there were differences in
natural still and sparkling waters (100 times higher).
Acknowledgement
M. Fátima Barroso is grateful to the Fundação para a Ciência e a Tecnologia for a PhD
grant (Grant Number SFRH/BD/29440/2006).
93
Sup
lem
enta
r in
form
atio
n (D
atas
ete)
Tab
le 3
. Min
eral
con
tent
ana
lyse
d in
mar
ket b
ottle
d w
ater
s.
sam
ple
Al
As
Cd
Cr
Co
Hg
Mn
Ni
Pb
Se
Si
nº
µg
l-1 (
%)
mg
l-1 (
%)
1 77
.80
± 3.
00
0.26
± 3
.00
0.17
± 4
.41
0.48
± 1
.80
1.38
± 1
2.64
0.
38 ±
2.9
1 1.
08 ±
8.2
6 -
1.17
± 1
6.63
-
9.3
± 0.
8
2 14
3.53
± 2
.00
0.17
± 1
0.00
-
1.92
± 9
.30
1.48
± 9
.83
0.13
± 1
0.09
0.
90 ±
5.5
1 0.
08 ±
20.
00
1.18
± 7
.81
- 9.
9 ±
8.3
3 48
.12
± 1.
91
- 0.
05 ±
2.0
9 0.
60 ±
11.
24
1.49
± 2
.18
0.12
± 7
.99
0.52
± 2
.81
- 1.
23 ±
13.
83
- 10
.9 ±
3.2
4 11
1.11
± 1
0.90
0.
38 ±
19.
40
- 0.
26 ±
7.5
0 1.
26 ±
10.
20
0.67
± 9
.37
0.28
± 1
2.74
-
1.17
± 6
.10
- 14
.0 ±
1.5
5 11
2.29
± 9
.2
0.74
± 1
3.09
-
0.44
± 0
.76
- -
1.22
± 2
3.88
-
2.22
± 1
2.01
-
17.7
± 8
.1
6 20
.43
± 2.
3
1.05
± 6
.66
- 0.
64 ±
2.8
8 0.
54 ±
13.
00
0.06
± 6
.54
- -
- 0.
22 ±
5.0
8 24
.4 ±
3.9
7 41
.97
± 2.
68
2.14
± 1
.87
- -
0.14
± 8
.30
0.08
± 1
.57
- -
- -
25.7
± 2
.4
8 74
.67
± 9.
1
0.64
± 1
7.36
-
0.28
± 1
2.88
0.
60 ±
28.
20
0.05
± 4
.14
1.46
± 1
2.09
-
1.48
± 4
.45
0.24
± 1
5.46
17
.5 ±
8.4
9 97
.06
± 2.
30
0.45
± 1
0.94
-
0.32
± 4
.23
0.95
± 8
.71
0.07
± 1
7.97
1.
37 ±
11.
85
- 0.
79 ±
10.
12
- 15
.8 ±
5.6
10
87.2
4 ±
4.77
1.
09 ±
2.8
5 -
0.31
± 4
.29
0.94
± 6
2.7
5 -
0.85
± 2
.23
-
- -
17.0
± 4
.8
11
34.0
4 ±
4.60
1.
75 ±
4.2
7 -
- 0.
57 ±
20.
62
0.80
± 5
.15
0.82
± 1
4.69
-
- -
49.7
± 7
.9
12
51.7
9 ±
5.80
-
- 0.
13 ±
18.
17
- -
- -
0.79
± 9
.99
0.20
± 4
.11
7.4
± 1.
7
13
43.0
7 ±
6.0
- -
0.25
± 1
2.13
0.
29 ±
4.0
0 -
- -
0.89
± 1
5.61
0.
12 ±
11.
63
10.9
± 6
.0
14
57.8
0 ±
6.70
- -
0.81
± 2
3.67
-
0.34
± 5
.97
- -
- 10
.0 ±
4.9
15
5.80
± 2
.80
-
- -
- -
- -
- -
5.2
± 7.
4
16
21.0
0 ±
15.2
0
- -
0.50
± 5
.83
1.65
± 2
7.84
0.
30 ±
5.4
0 7.
40 ±
0.8
1 0.
37 ±
2.6
2
0.64
± 1
8.04
-
10.6
± 1
.3
17
51.8
0 ±
1.97
-
- 0.
30 ±
10.
19
1.59
± 5
.77
0.26
± 0
.48
18.9
1 ±
1.32
0.
35 ±
5.8
9
- -
12.3
± 0
.1
18
66.0
3 ±
1.30
-
- 0.
33 ±
2.8
9 1.
78 ±
14.
08
- 19
.11
± 5.
15
1.05
± 2
.58
0.78
± 1
6.37
-
16.7
± 2
.3
19
8.90
± 3
.05
0.
24 ±
7.9
5 -
5.42
± 8
.37
2.20
± 5
.82
0.
13 ±
6.2
0 12
.19
± 1.
07
0.27
± 6
.96
0.87
± 7
.56
- 11
.8 ±
3.8
20
7.50
± 8
.00
0.
49 ±
16.
63
- 0.
24 ±
4.3
0 1.
60 ±
10.
70
0.12
± 0
.45
7.45
± 1
0.90
0.
31 ±
6.0
9 0.
94 ±
8.9
8 0.
42 ±
11.
58
18.1
± 9
.6
94
sam
ple
Al
As
Cd
Cr
Co
Hg
Mn
Ni
Pb
Se
Si
nº
µg
l-1 (
%)
mg
l-1 (
%)
21
104.
91 ±
1.9
3
- -
0.33
± 1
0.19
0.
59 ±
12.
11
- 2
23.6
7 ±
1.53
-
- 0.
28 ±
17.
87
46.5
± 3
.5
22
221.
42 ±
1.4
0
- -
0.61
± 9
.13
0.48
± 1
0.05
-
118.
72 ±
1.8
9
0.15
± 8
.15
1.28
± 8
.99
0.05
± 9
.03
541.
1 ±
9.1
23
119.
22 ±
5.1
0
- -
0.75
± 5
.21
- -
176.
46 ±
1.3
7
0.08
± 1
5.00
0.
87 ±
15.
10
0.10
± 1
3.72
22
1.5
± 6.
0
24
323.
96 ±
1.1
0
- -
0.91
± 5
.09
- 0.
06 ±
21.
93
184.
86 ±
0.7
0 -
- -
44.2
± 0
.5
25
402.
22 ±
6.3
-
- 0.
35 ±
8.3
6 -
- 21
6.30
± 1
.52
- -
- 32
.0 ±
4.6
26
56.5
1 ±
1.80
-
- 1.
71 ±
2.1
3 0.
54 ±
6.0
0 -
185.
62 ±
2.5
0 -
- 0.
30 ±
3.0
5 79
.6 ±
5.8
27
68.1
0 ±
14.3
1
0.54
± 4
4.84
-
0.22
± 4
.66
0.32
± 3
.25
- 1.
83 ±
0.6
2
- 1.
22 ±
2.5
3 0.
44 ±
9.4
6 30
.0 ±
6.0
28
28.5
4 ±
0.20
0.
85 ±
3.4
9 -
- 0.
31 ±
1.2
4 -
3.58
± 2
.86
- -
0.12
± 1
6.57
27
.0 ±
9.2
29
253.
34 ±
1.8
0.
31 ±
12.
96
0.04
± 5
.98
0.34
± 6
.95
0.17
± 3
.89
- 4.
09 ±
3.3
1 0.
39 ±
2.6
6 1.
59 ±
3.4
3 0.
08 ±
16.
46
25.8
± 3
.9
30
95.8
2 ±
0.70
0.
35 ±
18.
32
- 0.
16 ±
18.
44
0.20
± 4
.05
- 1.
68 ±
7.1
1 -
0.55
± 3
.93
0.14
± 6
.09
26.2
± 8
.6
31
63.1
7 ±
0.70
0.
94 ±
0.9
3 -
0.24
± 1
3.49
0.
39 ±
5.0
0 -
1.50
± 3
.98
- -
0.17
± 7
.03
21.4
± 5
.0
32
221.
43 ±
4.7
0 -
- 0.
23 ±
8.0
5 -
- 22
9.40
± 0
.89
- -
- 47
.3 ±
10.
9
33
441.
26 ±
2.8
0
- -
0.22
± 1
.82
0.30
± 5
.96
- 23
6.6
2 ±
0.40
-
- -
35.5
± 5
.5
34
343.
66 ±
0.6
0
- -
0.33
± 4
.09
- -
79.7
1 ±
1.24
-
- 0.
03 ±
8.5
4 26
.5 ±
3.8
35
50.0
1 ±
0.50
-
- 0.
49 ±
1.6
1 0.
45 ±
6.0
5 -
58.9
0 ±
0.91
0.
12 ±
7.5
6 0.
85 ±
3.2
2 0.
13 ±
9.1
4 23
.9 ±
1.7
36
58.9
7 ±
0.70
-
- 0.
43 ±
8.8
7 0.
25 ±
5.0
6 -
72.9
1 ±
1.73
-
0.78
± 7
.29
0.03
± 1
2.27
25
.1 ±
8.5
37
158.
00 ±
4.5
0
- -
0.63
± 4
.71
0.26
± 3.
86
- 86
.55
± 0.
89
- -
- 25
.6 ±
1.7
3
38
106.
72 ±
8.7
0
1.10
± 8
.79
0.07
± 3
.36
0.82
± 4
.17
- -
1.55
± 0
.38
0.53
± 5
.43
- 0.
11 ±
20.
80
4.8
± 0.
8
39
99.0
6 ±
1.10
2.
63 ±
0.7
0 -.
2.
13 ±
1.1
7 -
0.1
± 4
.27
0.77
± 7
.20
-
0.88
± 1
1.89
-
9.2
± 0.
7
2.2. Minerais vestigiais
95
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Tamasi G, Cini R. 2004. Heavy metals in drinking waters from Mount Amiata (Tuscany,
Italy). Possible risks from arsenic from public health in the Province of Siena. Sci Total
Environ. 327:41-51.
US Food Drug and Administration (USFDA). 2002. Bottled Water Regulation and the FDA.
Washington DC: USFDA, Center for Food Safety and Applied Nutrition. Available from:
http://www.cfsan.fda.gov/~dms/botwatr.html
World Health Organization (WHO). 1996. Trace elements in human nutrition and health.
Geneva (Switzerland): WHO.
2.2. Minerais vestigiais
97
World Health Organization (WHO). 2008. homepage website available from: :
http://www.inchem.org/
Ysart G, Miller P, Croasdale M, Crews H, Robb P, Baxter M, L’Argy C, Harrison N. 2000.
1997 UK Total Diet Study – dietary exposures to aluminium, arsenic, cadmium,
chromium, copper, lead, mercury, nickel, selenium, tin and zinc. Food Addit Contam.
17:775-786.
99
Capítulo 3
Perfil antioxidante – métodos convencionais
Flavored waters: Influence of ingredients on antioxidant capacity and terpenoid profile by
HS-SPME/GC-MS
M. Fátima Barroso, J. P. Noronha, Cristina Delerue-Matos, M. B. P. P. Oliveira
Journal of Food and Agricultural Chemistry, 2011, 59 (9), 5062 - 5072
3. Perfil antioxidante
101
Flavored Waters: Influence of Ingredients on Antiox idant
Capacity and Terpenoid Profile by HS-SPME/GC-MS
M. Fátima Barroso,†,§ J. P. Noronha,*,# Cristina Delerue-Matos,§ and M. B. P. P. Oliveira†
†Requimte/Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4099-
030 Porto, Portugal §Requimte/Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de
Almeida 431, 4200-072 Porto, Portugal #Requimte/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
Abstract
The antioxidant profiles of 39 water samples (29 flavored waters based on 10 natural
waters) and 6 flavors used in their formulation (furnished by producers) were determined.
Total phenol and flavonoid contents, reducing power, and DPPH radical scavenging
activity were the optical techniques implemented and included in the referred profile.
Flavor extracts were analyzed by HS-SPME/GC-MS to obtain the qualitative and
quantitative profiles of the volatile fraction of essential oils. Results pointed out a higher
reducing power (0.14-11.8 mg of gallic acid/L) and radical scavenging activity (0.29-211.5
mg Trolox/L) of flavored waters compared with the corresponding natural ones, an
interesting fact concerning human health. Bioactive compounds, such as polyphenols,
were present in all samples (0.5-359 mg of gallic acid/L), whereas flavonoids were not
present either in flavored waters or in flavors. The major components of flavor extracts
were monoterpenes, such as citral, α-limonene, carveol, and α-terpineol.
KEYWORDS: total antioxidant capacity, flavored water, essential oils, total phenols and
flavonoids contents, radical scavenging activity, reducing power, HS-SPME/GC-MS
Available online at pubs.acs.org
Journal of Agricultural and Food Chemistry
2011, 59 (9), 5062-5072
3. Perfil antioxidante
103
INTRODUCTION
Reactive oxygen species (ROS) are continuously produced in all living beings,
especially in higher organisms, as a result of normal cellular metabolism, phagocytises,
inflammation, and exogenous factors such as ionizing radiations and xenobiotics.1 ROS
can induce cell damage by reacting with biomolecules (proteins, lipids) and cause serious
lesions in the DNA molecule,2 such as strand breaks, DNA-protein cross-linking, and
base-free sites.3 The mammalian body has certain endogenous antioxidant defense
mechanisms to combat and reduce oxidative damage such as enzymatic systems, and
exogenous antioxidant systems, such as as vitamins, minerals, and proteins. Antioxidants,
which can inhibit or delay the oxidation of a substrate in a chain reaction, therefore,
appear to be very important in the prevention of many diseases.4 Foodstuffs constitute an
excellent exogenous source of natural antioxidants. It is known that vegetables, fruits,
whole-grain, and some beverages (tea, juice, wine) are rich in antioxidants and bioactive
compounds. Examples of antioxidants present in food are vitamins (particularly C and E),
phenolic compounds (flavonoids, catechins, flavones, flavonols, anthocyanins), and
carotenoids including β-carotene.5 A healthy diet should provide an adequate and
continuous supply of these antioxidants. Other antioxidants, such as ubiquinol and thiol
compounds, produced in small amounts by the organism, can be obtained in higher
amounts by dietary supplements.6 Consequently, interest is increasing in new effective
natural antioxidants as well as in the chemical and biochemical characterization of
foodstuffs and beverages to evaluate them with regard to their antioxidant profiles.
To answer consumers’ preferences, the food industry has applied several technical
improvements to plain water. Today, a significant part of commercialized water is in
flavored formulation. Flavors, juices, bioactive compounds, preservatives, and/or
sweeteners are added to water, providing a product with singular tastes and smells
appreciated by consumers.
Flavors (or essential oils) from fruits contain 85-99% of volatile and 1-15% of nonvolatile
compounds. Volatile constituents are a mixture of monoterpenes and sesquiterpenes,7
being flavonoids present in the nonvolatile fraction.8 Terpenes and flavonoids present
antioxidant and antiradical properties9 and can be transferred to water samples if
flavors/aromas extracts are used. Therefore, drinking this type of beverage can improve
the daily intake of antioxidants, contributing to the exogenous protective system. However,
there are no reports concerning the antioxidant properties of these waters, although their
macro- and micromineral compositions are known.10,11 These properties will be a new
source of information for consumer’s about the advantages/disadvantages on the
consumption of these beverages.
II. Investigação e desenvolvimento
104
Antioxidant capacity determination is not an easy task to perform. Several factors
(substrates, conditions, analytical methods, and concentrations) can affect the estimated
values, and it is difficult to measure each antioxidant component separately and/or the
interactions among different antioxidant components in the samples.4 Total antioxidant
capacity measures can be classified in two groups: assays based on the inhibition of
human low density lipoprotein oxidation or those based on oxygen free radical scavenging
ability. Current In vitro methods for antioxidant efficacy evaluation have as a basic
principle the oxidation inhibition of a suitable substrate. After oxidation of the substrate,
under standard conditions, the extent of the reaction is determined at a fixed time point or
over the range that is characteristic of the generated free radical.3 UV-vis
spectrophotometric, chemiluminescence, fluorometric,4 and chromatographicmethods12
can be used to do that.
In the present study, four optical methods were applied to evaluate the antioxidant
profile of 39 mineral and spring, natural and flavored water samples, and 6 flavors/aromas
used in their formulation. This was carried out by means of the total phenol content (TPC),
total flavonoid content (TFC), reducing power, and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical-scavenging activity (RSA). The volatile fractions of the flavor extracts were isolated
by headspace solid-phase microextraction (SPME) and analyzed by gas chromatography-
mass spectrometry (GC-MS).
MATERIALS AND METHODS
Chemicals . Gallic acid, (-)-epicatechin, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (Trolox, a water-soluble analogue of vitamin E) standards were from
Sigma-Aldrich or Fluka. Folin-Ciocalteu reagent and DPPH were obtained from Sigma-
Aldrich. All of these chemicals, of the highest quality available (95-99%), were used
without purification. Other compounds of analytical grade, such as sodium carbonate,
sodium nitrite, aluminum chloride, sodium hydroxide, ethanol, and sodium acetate (0.1
mol/L, pH 4.3), were from Merck. All solvents used were of HPLC grade. Standard
antioxidant solutions were prepared daily and stored in the dark at 4 ºC Cwhen not in use.
Water used was ultrapure (18.2MΩ/cm), obtained from a Millipore Simplicity 185 system.
For spectrophotometric measurements a Shimadzu 160-A spectrophotometer was used.
Sample Preparation . Mineral water arises from a geologically and physically protected
underground source, characterized by constant levels and relative proportions of minerals
and trace elements at the source. Spring water derives from an underground formation
from which water flows naturally to the surface at an identified location.
3. Perfil antioxidante
105
Thirty-nine water samples, corresponding to 10 different brands, acquired in
supermarkets in northern Portugal and stored in the dark at 4 ºC were analyzed. Each
brand (still or sparkling, mineral or spring water) had different flavors and aromas. Natural
waters of each brand were used as control. Sonication was used to eliminate gas from
sparkling water samples.
Table 1 summarizes the nutrient information on the labels, taking into account its
different composition in gas, flavor, vitamins, preservatives, acidifying regulators, and
sweeteners.
Six flavors or concentrate extracts (lime, tangerine, strawberry, lemon, apple, and
gooseberry) used in the formulation of some water brands, and provided by producers,
were also analyzed. As expected, these flavors had no description about its chemical
composition.
TPC Determination . TPC values of flavors and flavored waters were determined by a
colorimetric assay based on procedures described by Singleton and Rossi13 with some
modification. Folin-Ciocalteu reagent and the reduced phenols produced a stable blue
product at the end of reaction. The reaction mixture (20 µL of sample, 1.58 mL of ultrapure
water, and 100 µL of Folin-Ciocalteu reagent) was sonicated for 30 s. After this, it was
added to 300 µL of 7% Na2CO3, and the mixture was incubated for 10 min at 50 ºC.
Factor dilutions of 10 times on the mother standard antioxidant gallic acid (GA) were
carried out to obtain a calibration curve ranging from 0 to 5.00 mg of GA/L of water.
Quantifications were carried out in triplicate, and the absorbance was measured at 760
nm.
TFC Determination . TFC was determined by a colorimetric assay based on the
formation of flavonoid-aluminum compound.14 One milliliter of flavored water was mixed
with 4 mL of ultrapure water and 300 µL of 5% NaNO2 solution. After 5 min, 300 µL of
10% AlCl3 solution was added. After 6 min, 2 mL of 1 mol/L NaOH and 2.4 mL of
ultrapure water were added. The solution was mixed well, and the absorbance of a pink
color was read at 510 nm. (-)-Epicatechin was used to plot the standard curve ranging
from 0 to 66.26 mg/L, and the results of TFC were expressed as milligrams of epicatechin
per liter of water. All measurements were carried out in triplicate.
10
6
Tab
le 1
. Lab
el In
form
atio
n in
the
Eva
luat
ed B
ottle
d F
lavo
red
Wat
ers
bran
d sa
mpl
e fla
vor
juic
e vi
tam
in
pres
erva
tives
ac
idify
ing
regu
lato
rs
swee
tene
rs
othe
r in
gred
ient
s
Stil
l Wat
er A
1 le
mon
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid,
sodi
um c
itrat
e
aces
ulfa
me-
K
fibre
s (1
%),
whe
at d
extr
in (
0.1%
) m
iner
al
2 m
ango
po
tass
ium
sor
bate
,
sodi
um b
enzo
ate
citr
ic a
cid,
sod
ium
citr
ate
aces
ulfa
me-
K
fibre
s (1
%),
whe
at d
extr
in (
0.1%
)
3
stra
wbe
rry
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid,
sodi
um c
itrat
e
aces
ulfa
me-
K
fibre
s (1
%),
whe
at d
extr
in (
0.1%
)
4
natu
ral
B
5 pi
neap
ple/
oran
ge
appl
e
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid
aces
ulfa
me-
K,
aspa
rtam
e
calc
ium
lact
ate
spr
ing
6 le
mon
ap
ple
niac
in, p
anto
then
ic a
cid,
B6,
folic
aci
d, b
iotin
, B12
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid
aces
ulfa
me-
K,
aspa
rtam
e
7
natu
ral
C
8 le
mon
/mag
nesi
um
frui
t (m
g/10
0 m
L): B
3 (2
.7),
B5
(0.9
),
B6
(0.3
), B
8 (0
.022
), B
9 (0
.03)
,
B12
(1.
5x10
-4)
pota
ssiu
m s
orba
te,
dim
ethy
l dic
arbo
nate
citr
ic a
cid
mag
nesi
um c
arbo
nate
,
gin
seng
min
eral
9
appl
e/w
hite
tea
frui
t (m
g/10
0 m
L): B
3 (2
.7),
B5
(0.9
),
B6
(0.3
), B
8 (0
.022
), B
9 (0
.03)
B12
(1.5
x10-4
)
pota
ssiu
m s
orba
te
citr
ic a
cid
m
alic
aci
d,
calc
ium
lact
ate
10
pi
neap
ple/
fibre
fr
uit
po
tass
ium
sor
bate
,
dim
ethy
l dic
arbo
nate
citr
ic a
cid
w
heat
dex
trin
(0.
9%)
L-ca
rniti
ne (
200
mg/
L)
11
na
tura
l
D
12
appl
e
di
met
hyl d
icar
bona
te,
sodi
um b
enzo
ate
citr
ic a
cid
sucr
alos
e,
aces
ulfa
me-
K
min
eral
13
or
ange
/pea
ch
dim
ethy
l dic
arbo
nate
, so
dium
ben
zoat
e ci
tric
aci
d su
cral
ose,
aces
ulfa
me-
K
10
7
bran
d sa
mpl
e fla
vor
juic
e vi
tam
in
pres
erva
tives
ac
idify
ing
regu
lato
rs
swee
tene
rs
othe
r in
gred
ient
s
14
le
mon
dim
ethy
l dic
arbo
nate
, so
dium
ben
zoat
e ci
tric
aci
d
sucr
alos
e,
aces
ulfa
me-
K
15
na
tura
l
E
16
lem
on
lem
on
(mg/
100
mL)
: B3
(2.7
), B
12 (
0.15
) po
tass
ium
sor
bate
,
sodi
um b
enzo
ate
citr
ic a
cid,
sodi
um c
itrat
e
aces
ulfa
me-
K,
aspa
rtam
e ca
rbon
dio
xide
min
eral
17
or
ange
/ras
pber
ry
oran
ge, r
aspb
erry
(m
g/10
0 m
L): B
3 (2
.7),
B12
(0.
15)
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid,
sodi
um c
itrat
e
aces
ulfa
me-
K,
aspa
rtam
e ca
rbon
dio
xide
add
ed g
as
18
peac
h/pi
neap
ple
peac
h, p
inea
pple
(m
g/10
0 m
L): B
3 (2
.7),
B12
(0.
15)
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid,
sodi
um c
itrat
e
aces
ulfa
me-
K,
aspa
rtam
e ca
rbon
dio
xide
19
gu
ava/
lime
guav
a/lim
e (m
g/10
0 m
L): B
3 (2
.7),
B12
(0.
15)
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid,
sod
ium
citr
ate
aces
ulfa
me-
K,
aspa
rtam
e ca
rbon
dio
xide
20
natu
ral
F
21
lem
on/g
reen
tea
frui
t, le
mon
, app
le
citr
ic a
cid
gr
een
tea
min
eral
22
ra
spbe
rry/
gins
eng
rasp
berr
y, a
pple
, pea
r
ci
tric
aci
d
gins
eng
nat
ural
gas
23
pe
ach/
whi
te te
a fr
uit,
peac
h, a
pple
, pe
ar
citr
ic a
cid
w
hite
tea
24
m
ango
/gin
kgo
belo
ba
man
go, a
pple
, pea
r
ci
tric
aci
d
Gin
kgo
bilo
ba
25
m
elon
/min
t fr
uit,
mel
on, a
pple
, pe
ar
citr
ic a
cid
m
int
26
na
tura
l
G
27
lem
on
C
(12
mg/
250
mL)
po
tass
ium
sor
bate
ci
tric
aci
d ac
esul
fam
- K
, su
cral
ose
min
eral
28
lim
e
add
ed g
as
29
appl
e
C (
12 m
g/25
0 m
L)
pota
ssiu
m s
orba
te
citr
ic a
cid
aces
ulfa
me-
K,
sucr
alos
e
30
pe
ach
C
(12
mg/
250
mL)
po
tass
ium
sor
bate
ci
tric
aci
d ac
esul
fam
e-K
, su
cral
ose
31
natu
ral
H
32
lem
on
lem
on, a
pple
C
(30
mg/
100
mL)
so
dium
ben
zoat
e,
pota
ssiu
m s
orba
te
citr
ic a
cid
aspa
rtam
e
min
eral
n
atur
al g
as
33
na
tura
l
I 34
le
mon
le
mon
, app
le
so
dium
ben
zoat
e ci
tric
aci
d as
part
ame
10
8
bran
d sa
mpl
e fla
vor
juic
e vi
tam
in
pres
erva
tives
ac
idify
ing
regu
lato
rs
swee
tene
rs
othe
r in
gred
ient
s
min
eral
35
gr
een
appl
e ap
ple
so
dium
ben
zoat
e ci
tric
aci
d su
cral
ose
nat
ural
gas
36
st
raw
berr
y ap
ple,
str
awbe
rry
so
dium
ben
zoat
e ci
tric
aci
d as
part
ame
37
natu
ral
J
38
lem
on
lem
on
pota
ssiu
m s
orba
te,
sodi
um b
enzo
ate
citr
ic a
cid
so
dium
citr
ate
aspa
rtam
e,
aces
ulfa
me-
K
spr
ing
add
ed g
as
39
natu
ral
3. Perfil antioxidante
109
Reducing Power Assay . Reducing power was determined according to the method of
Oyaizu.15 One milliliter of sample was mixed with 2.5 mL of 0.2 mol/L sodium phosphate
buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. This mixture was incubated for
20 min at 50 ºC, and then 2.5 mL of 10% trichloroacetic acid (w/v) was added and
centrifuged at 1000 rpm for 10 min. The upper layer of the solution (2.5 mL) was mixed
with distilled water (2.5 mL) and 0.5 mL of 0.1% ferric chloride, and the absorbance was
measured at 700 nm. The calibration curve was prepared with GA solutions ranging from
0 to 19.6 mg/L, and the results are given as milligrams of GA per liter of water.
DPPH Radical Scavenging Activity . RSA of samples against the stable nitrogen
radical DPPH• was determined spectrophotometrically at 517 nm.16 DPPH• free radical is
reduced to the corresponding hydrazine when it reacts with hydrogen donors, such as an
antioxidant. In this technique, samples (200 µL) were mixed with 2.80 mL of 1.86x10-4
mol/L ethanolic solution of DPPH•. The mixture, vigorously shaken, was left to stand for 15
min in the dark (until stable absorption values). Lower absorbance values of the reactive
mixture indicated higher free radical scavenging activity. The calibration curve was
prepared with Trolox solutions ranging from 0 to 19.6 mg/L, and the results are given as
milligrams of Trolox per liter of water.
Validation of the Optical Methodologies . Calibration standards were daily prepared,
and all samples were determined in triplicate. The methods were validated by linear
range, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. LOD
and LOQ were defined, respectively, as 3 and 10 times the standard deviation of 10 blank
signals divided by the slope of the calibration plot.17 Precision was calculated by intraday
and interday determinations of standard solutions and expressed by relative standard
deviations (RSD). For intraday evaluation, each concentration was assessed by five
measurements, three times during a working day. The interday precision measurements
were made over 1 week. Accuracy and reproducibility were checked by recovery (REC),
relative error (RE), and RSD. All results were expressed as the mean ( standard deviation.
Flavor/Fragrance Extraction by Headspace SPME and D etection by GC-MS .
Extraction of fragrances was carried out by SPME using a 65 µm
polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber (Supelco, Inc., Bellefonte, PA), for
all experiments. This fiber was selected according to the best results for the extraction of
fruit volatiles.18,19 Fibers were conditioned for 30 min at 250 ºC before use.
For each extraction 2 g of flavor and 0.5 g of NaCl (to inhibit enzymatic reactions and to
favor the transfer of the analytes from the aqueous solution to the headspace) or 50 µL of
extract were transferred into a 10 mL Teflon-lined septum cap vial equipped with a
Tefloncoated magnetic bar. To favor the transfer of the analytes from the aqueous
solution to the headspace, the solution was stirred (200 rpm) at 70 ºC. The PDMS/DVB
II. Investigação e desenvolvimento
110
fiber was used to extract the nonpolar volatile compounds (in the headspace). The fiber
was exposed to the sample headspace for 20 min at 70 ºC. The fiber was then removed
and introduced into the injector port of the GC-MS for desorption at 250 ºC for 3 min, in
the splitless mode.
The separation and detection of the analytes was achieved using a GC-MS system
(Agilent Technologies, USA) with a GC 6850 coupled to a 595C VL MSD mass selective
detector, with a silica capillary column (30 m x 0.32 mm i.d.; df, 0.25 µm) covered with 5%
phenyl/95% dimethylpolysiloxane (DB-5 ms, Agilent-J&W Scientific), kept at 30 ºC for 3
min, and then ramped to 300 ºC at 8 ºC/min and held at the final temperature for 4 min.
The splitless injection (3 min) was achieved with an injector temperature at 250 ºC.
Helium was the carrier gas used at flow of 1.0 mL/min. Ion source, quadrupole, and
transference line were kept at 230, 150, and 280 ºC, respectively. MS spectra were
obtained by electronic impact (EI) at 70 eV and collected at the rate of 1 scan/s over an
m/z range of 35-400, and using MSD ChemStation E.02.00493 software (Agilent
Technologies, USA). Identification of the individual components was performed by
comparing their mass spectra with the standards and spectral libraries of GC-MS (NIST
98 and Wiley 275), enabling the detection of some minor components and identification of
compounds that arise from incompletely resolved chromatographic peaks.
For each compound, quantitation was performed by measuring the corresponding peak
area of the total ion chromatogram and expressed as relative (percent) areas by
normalization.
RESULTS AND DISCUSSION
Descriptive Statistics . Table 1 represents the labeled nutrient information in flavored
waters. About 38% of water samples are still and 62% sparkling (11 water samples with
added gas). Labels indicate the presence of several compounds added for technological
purposes, with biological activity (flavors, juice fruit, and vitamins). Inevitably, these waters
also need other ingredients, without positive relationship with well-being and health, but
necessary to ensure the quality desired for producers and consumers and for the safety of
the product. This is the case of preservatives, acidifying regulators, and sweeteners.
Twelve different flavors were present in flavored waters: lemon (10 samples); mango,
strawberry, lime, and raspberry (2 samples each); pineapple, apple, and orange (3
samples each); peach (4 samples); guava, melon, and green apple (1 sample each).
Lemon is the predominant flavor, present in all water brands (A-J; 10 samples).
Seventeen flavored water samples had only one flavor, and 12 samples had a
3. Perfil antioxidante
111
combination of two flavors. About 50% of the samples have fruit juices or concentrates.
Only flavored brands A, D, and G do not report the addition of this type of ingredient.
Eleven samples, according to the label, have in their composition vitamins of the B
complex (7 samples) and C (4 samples). It is important to remember that vitamin C is an
antioxidant with protection capacity against oxidative stress, being also a cofactor in
several vital enzymatic reactions. Other bioactive compounds (ginseng, L-carnitine, white
and green tea, and Ginkgo biloba) are present in some samples from different brands.
Green tea contains numerous components with antioxidant activity, such as polyphenols
(catechins, epicatechin, epigallocatechin) and vitamins.20 Ginseng is an herbal medicine
with antioxidant and anti-inflammatory activities and G. biloba is rich in phenolic and
flavonoid compounds.
Forty-nine percent of samples contain sweeteners. There are water samples with only
one (acesulfame-K, sucralose, or aspartame) ad with two sweeteners in association
(acesulfame-K and aspartame; acesulfame-K and sucralose). The most used was
acesulfame-K (present in 14 samples), followed by aspartame (10 samples). It is
interesting to note that, in general, the samples from the same brand have the same
sweetener, the exception being brand I that uses different sweeteners for different flavors.
Brands C and F do not have sweeteners, providing more energetic products, of 9-13 and
19 kcal/100 mL, respectively (sweetened samples ranged from 0.4 to 4 kcal/100 mL).
Each sample contains a single preservative (potassium sorbate or sodium benzoate) or
the association of two (potassium sorbate and sodium benzoate; potassium sorbate and
dimethyl dicarbonate; sodium benzoate and dimethyl dicarbonate). From this discussion,
different behaviors and antioxidant values among the samples in the study are expected.
Method Validation . Table 2 presents the results obtained in the validation procedures
of the applied methodologies (TPC, TFC, reducing power, DPPH RSA). Linearity ranges
from 0 to 5.0 mg of GA/L in TPC, from 0 to 66.2 mg of epicatechin/L in TFC, and from 0 to
19.6 mg of GA/L and Trolox/L in reducing power and DPPH RSA methods, respectively.
LOD values ranged from 5.43x10-3 (reducing power) to 1.00x10-1 (TFC) mg of standard
antioxidant/L, and LOQ values ranged from 1.81x10-2 to 3.33x10-1 mg of standard
antioxidant/L.
Precision and accuracy values are also shown in Table 2. RSD values ranged from 2.1
(intraday studies) to 7.4 (interday studies) and confirmed the high precision of the
methods. REC and RE values assessed the accuracy of the results. RE were always
<11.0%, and recovery trials ranged from 93 to 111%, confirming the accuracy of the
implemented methods.
II. Investigação e desenvolvimento
112
Table 2. Calibration Curves, Limit Values, Precisio n, and Accuracy Obtained in the
Determination of Antioxidant Activity Assays
Determination of TPC and TFC . Recently, bottled flavored waters have become
popular, and the consumption of flavored waters is globally increasing, including in
Portugal. In the first half of 2010, 6.08 million liters of this kind of water was consumed by
the Portuguese population. Considering the emergent market of this kind of beverage, it is
important to deepen the knowledge of the antioxidant capacity of these beverages. The
method used to determine TPC has been extensively applied in plants and beverages.
Phenolic and flavonoid compounds, correlated with antioxidant activity, seem to have an
important role in stabilizing lipid oxidation. Generally, the antioxidant mechanism of
phenolic compounds is inactivating lipid free radicals and preventing decompositionof
hydroperoxides into free radicals. This is the case of fruits and beverages in relation to
their phenolic compounds.5 Therefore, in this research TPC and TFC were evaluated in 6
flavors used in flavored water formulation and in 39 water samples commercialized in
northern Portugal. However, TPC determination should always be considered as an
parameters
TPC
(mg Gallic acid/L)
TFC
(mg Epicatechin/L)
reducing Power
(mg Gallic acid/L)
DPPH scavenging activity
(mg Trolox/L)
linear concentration (µg/L) 0 - 5.0 0 - 66.2 0 - 19.6 0 - 19.6
slope (Abs mg/L) 7.34 ± 0.10 (x10-2) 3.52 ± 0.03 (x10-2) 2.74 ± 0.07 (x10-1) -6.76 ± 0.2 (x10-2)
intercept (Abs) -9.24 ± 0.40 (x10-4) 1.67 ± 0.80 (x10-2) -2.65 ± 0.5 (x10-2) 1.30 ± 0.02
correlation coefficient (n = 5) 0.999 0.999 0.998 0.997
LOD (mg standard/L) 3.22x10-2 1.00x10-1 5.43x10-3 2.84x10-2
LOQ (mg standard/L) 1.07x10-1 3.33x10-1 1.81x10-2 9.48x10-2
Intra-day studiesa
added (µg/L)
found (µg/L)
5.0
4.8
6.0
5.8
10.0
11.1
10.0
9.3
RECb (%) 95.0 96.7 111.0 93.0
REc (%) - 5.0 -3.3 11.0 -7.0
RSDd (%) 3.2 4.6 2.1 6.9
Inter-day studiese
added (µg/L)
found (µg/L)
5.0
5.2
6.0
5.6
10.0
9.5
10.0
9.8
REC (%) 104.0 93.3 95.0 98.0
RE (%) 4.0 -6.7 -5.0 -2.0
RSD (%) 4.0 5.8 6.3 7.4
aAverage of three measurements, three times during a day. bREC, recovery. cRE, relative error. dRSD, relative standard deviation. eAverage of five measurements over a week.
3. Perfil antioxidante
113
indicative value instead of an accurate measure of phenolic compounds. This method
should be aware of possible interferences (reducing sugars and some amino acids) that
can overestimate evaluated amounts. On the other hand, it is difficult to measure all
phenolic molecules individually. Nevertheless, this assay is a simple, sensitive, and
precise technique.4 Table 3 presents TPC and TFC values obtained in samples.
With regard to TPC, only natural waters (without added ingredients) and two samples of
flavored waters (13 and 14) present “not detected” values. As an exception, sample 37
(natural water) presents trace TPC levels (0.07 mg of GA/L). TPC values ranged from 8.5
(gooseberry) to 380.2 (lemon) mg of GA/L in flavors and from 0.29 (sample 27) to 284 mg
of GA/L (sample 29) in flavored waters. Comparing flavor TPC values, the highest
contents are from citrus fruits such as tangerine, lime, and lemon, respectively, 117, 359,
and 380 mg of GA/L. These values are similar to those obtained by other authors in juices
of citrus fruits,5 but less than those found in other studies with beverages containing milk
and fruits of the same kind used in this work.21 Gooseberry, in contrast to what was
expected, presented the lowest levels. This flavor is different from Indian gooseberry,
described by Mayachiew and Devahastin,22 as rich in TPC (290 mg of GA/g extract).
Calixto and Goñi23 reported the TPC of beverages (coffee, tea, and red wine) and fruits.
The TPC values were higher than the TPC values obtained in this work and ranged from
76 mg of GA/100 mL in beverages to 538 mg of GA/100 g in dry fruit.
With regard to flavored waters and their TPC contents, the lowest value (0.29 mg of
GA/L, lemon flavor, sample 27) and the highest value (284 mg of GA/L, apple flavor,
sample 29) were determined in samples from the same brand (G). This information can be
important for consumers because they generally correlate brands with similar behaviors.
In this case, flavored waters from the same brand can be distinct. According to Table 3
and the values presented, brand G is unique, having significant differences.
The addition of bioactive compounds such as tea (samples 9, 21, and 23), ginseng
(sample 22), and G. biloba (sample 24) seems to increase TPC contents of the flavored
waters. These samples presented values ranging from 28.1 to 39.7 mg of GA/L, the
highest ones excluding samples 29 and 30. It is important to remember that according to
the label information, these samples (29 and 30) have vitamin C as an added ingredient
(12 mg/ 250 mL), a compound related with antioxidant properties. Nevertheless, the
presence of the vitamin, referred to on the label, does not always imply high TPC levels.
This is the case for samples 27 and 32, with added vitamin, which have very different TPC
values lower than those previously mentioned. Sometimes, the expectation that samples
with bioactive compounds have a dual phenolic protective effect is not true.
II. Investigação e desenvolvimento
114
Table 3. TPC, Reducing Power, and DPPH RSA Determin ed in Flavors and Flavored
and Natural Waters
brand sample TPC (mg GA/L) reducing power (mg GA/L) DPPH (mg trolox/L)
Flavours tangerine 116.70 ± 1.50 10.21 ± 0.09 51.21 ± 0.35 lime 359.30 ± 0.60 11.03 ± 0.07 78.51 ± 0.23
strawberry 15.34 ± 0.10 11.12 ± 0.04 213.53 ± 2.50
lemon 380.20 ± 0.09 10.71± 0.08 38.90 ± 0.42
gooseberry 8.53 ± 0.04 11.80 ± 0.04 211.53 ± 4.60
apple 37.15 ± 0.03 10.62 ± 0.03 54.43 ± 0.29
A 1, lemon 4.68 ± 0.05 2.61 ± 0.11 14.71 ± 0.04 2, mango 7.72 ± 0.10 3.68 ± 0.09 13.16 ± 0.05 3, strawberry 9.26 ± 0.03 3.91 ± 0.08 12.27 ± 0.04 4, natural nda nd 0.76 ± 0.03
B 5, pineapple/orange 18.30 ± 0.09 6.01 ± 0.42 13.49 ± 0.32
6 lemon 17.62 ± 0.020 5.45± 0.14 16.26 ± 0.15
7, natural nd nd 0.62 ± 0.03
C 8, lemon/magnesium 24.44 ± 0.20 8.48 ± 0.20 15.71 ± 0.09
9, apple/white tea 28.10 ± 0.03 8.20 ± 0.15 16.49 ± 0.03
10, pineapple/fibre 11.40 ± 0.05 4.56 ± 0.08 8.05 ± 0.45
11, natural nd nd 0.89 ± 0.04
D 12, apple 0.54 ±0.03 3.31 ± 0.03 46.55 ± 0.45
13, orange/peach nd 2.79 ± 0.06 44.11± 0.07
14, lemon nd 3.07 ± 0.07 44.56 ± 0.04
15, natural nd nd 0.41 ± 0.02
E 16, lemon 2.24 ± 0.11 0.28 ± 0.05 12.38 ± 0.25
17, orange/raspberry 6.18 ± 0.05 1.07 ± 0.02 16.49 ± 0.05
18, peach/pineapple 1.51 ± 0.02 0.14 ± 0.03 15.27 ± 0.10
19, guava/lime 8.57 ± 0.03 5.45 ± 0.04 14.71± 0.04
20, natural nd nd 0.76 ± 0.02
F 21, lemon/green tea 39.70 ± 0.10 9.64 ± 0.03 41.45 ± 0.27
22, raspberry/ginseng 37.90 ± 0.08 13.78 ± 0.05 48.66 ± 0.32
23, peach/white tea 29.20 ± 0.15 8.29 ± 0.02 42.45 ± 0.04
24, mango/ginkgo beloba 36.50 ± 0.02 10.52 ± 0.04 45.89 ± 0.37
25, melon/mint 19.70 ± 0.04 10.11 ± 0.04 41.56 ± 0.05
26, natural nd nd 0.29 ± 0.05
G 27, lemon 0.29 ± 0.02 nd 38.23 ± 0.06
28, lime 1.75 ± 0.04 nd 38.79 ± 0.17
29, apple 284.0 ± 2.3 154.04 ± 0.26 268.89 ± 2.45
30, peach 147.0 ± 1.3 nd 133.87 ± 1.35
31, natural nd nd 0.42 ± 0.06
H 32, lemon 7.23 ± 0.05 3.77 ± 0.03 44.78 ± 0.48
33, natural nd nd 0.31 ± 0.05
I 34, lemon 4.31 ± 0.03 5.91 ± 0.04 43.67 ± 0.28
35, green apple 4.92 ± 0.06 4.00 ± 0.07 54.21 ± 0.03
36, strawberry 5.89 ± 0.03 6.10 ± 0.38 42.67 ± 0.06
37, natural 0.07 ± 0.03 nd 0.27 ± 0.13
J 38, lemon 1.88 ± 0.02 4.33 ± 0.29 41.89 ± 0.04
39, natural nd nd 0.38 ± 0.08 and, not detected
3. Perfil antioxidante
115
Samples from brand D presented the lowest TPC values (from not detected to 0.54 mg
of GA/L). According to the label information, these samples have only flavors in its
formulation. It is possible to speculate whether this is a synthetic substance without the
complexity of vegetable/fruit extracts, namely, without phenolic compounds. Another
approach can be the use of trace amounts without influence in values of the parameters in
appreciation. All brands have water flavored with lemon. TPC values have extreme
discrepancies, ranging from not detected (sample 14) to 17.62 mg of GA/L (sample 6) and
higher in association with magnesium (24.44 mg ofGA/L, sample 8) or green tea (39.70
mg of GA/L, sample 21). It is also interesting to verify that waters flavored with lemon
generally presented the lowest TPC values compared with other flavors, the exception
being the examples referred to above with magnesium and green tea. This is especially
important taking into account that lemon flavor was the richest in TPC. The use of more
diluted extract, due to its strong taste, can be a possible explanation for the obtained
results.
In the case of lime flavor, the second most rich in TPC, is only present in sample 28.
However, this flavor, being slightly poorer in TPC compared with lemon, is present at
levels 6-fold higher in flavored waters from the same brand.
With regard to flavors and their TPC contents, it is important to note that TPC values
from red fruits (strawberry and gooseberry) were the lowest. Taking into account its
antioxidant power, it can be speculated that the mechanism does not involve phenolic
compounds. However, when used as ingredients, they provide water samples the highest
TPC values compared with other samples of the same brand. This is the case of sample 3
in brand A and sample 36 in brand I.
From the studied samples, labels do not reveal the presence of gooseberry. Two
samples (17 and 22) have raspberry as an ingredient, being the second richest
considering all samples of the brands.
Unfortunately, the labels of the samples evaluated do not declare tangerine flavor in
their compositions. Probably it is used in combination with other flavors, in minimal
amounts, and not declared in the final list of ingredients. The same explanation can be
proposed for gooseberry flavor.
With regard to TFC, all flavors and flavored water samples had no flavonoids, in
detectable amounts, in their composition. These results are consistent with those obtained
by Tabard and collaborators.24 Using the same optical technique used in this work, these
authors did not find flavonoids in apple, grape, or vegetable juices. However, those
authors found a high TFC level in red wine. On the other hand, flavors (essential oils)
contain about 1-15% of nonvolatile components when flavonoids are included.8 Therefore,
II. Investigação e desenvolvimento
116
flavonoids are present in small or not detected amounts in flavors. When quantified by a
colorimetric method, and after dilution in water, it is difficult to detect them.
Reducing Power Assay . In reducing power determination, the yellow color of the
solution changes to various shades of green and blue, depending on the compounds
present in the solution. The presence of antioxidants causes the reduction of the Fe3+
/ferricyanide complex to the ferrous form.
Table 3 presents the values of reducing power from flavors and flavored waters. As
expected, flavors presented higher values than flavored waters due to their higher
concentrations of bioactive compounds. Nevertheless, samples from brand F present
similar values, and one sample from brand G (sample 29) presents values 15-fold higher
than those in flavors.
With regard to flavors, reducing powers are very similar (from 10.2 to 11.8 mg of GA/L).
No correlation among the different evaluated parameters was verified. As referred to
above, flavors presented very different TPC values. It is interesting to remember that the
lowest TPC value (gooseberry) corresponds to the highest value in reducing power
determination. Some studies indicated a high reducing power activity in wild fruits.25
All natural waters and three flavored water samples from the same brand (27, 28, and
30) presented values of this parameter below the LOD. It should be noted that samples 27
and 30 have vitamin C as an added ingredient and sample 30 presented the second
highest content in TPC. Inversely, sample 29, with apple flavor, had the highest value in
TPC and reducing capacity and had also vitamin C as an added ingredient. The highest
reducing power values were obtained (like in TPC) in flavored waters with bioactive
compounds (tea, ginseng, and G. biloba) ranging from 8.3 to 13.8 mg of GA/L.
It is verified that samples from the same brand had similar values, except for brand C
(sample 10, without addition of bioactive compounds) and brand E (sample 19 with a
value 5-fold higher than the other samples). This behavior occurred also in TPC values,
sample 19 being also the richest in these compounds.
From a general point of view and except for brand F (with values similar to flavors) and
brand G, as referred to above, all brands can be grouped into two sets: A, D, E, H, and J
with lower values ranging from 3 to 4 mg/L of GA/L; and B, C, and I with relatively higher
values near 6-7mg of GA/L.
DPPH RSA. DPPH RSA is a technique based on the reduction of the DPPH radical in
the presence of a hydrogen-donating antioxidant. A DPPH solution, freshly prepared,
exhibits a deep purple color with maximum absorption at 517 nm. This color disappears in
the presence of an antioxidant, because antioxidant molecules can quench DPPH free
radicals and convert them into a colorless product. Hence, the more rapidly the
absorbance decreases, the more potent is the antioxidant. Table 2 presents RSA values
3. Perfil antioxidante
117
obtained with water samples and flavors. According to the previously discussed
parameters, flavors presented, in general, higher RSA values than flavored waters, except
some samples of brand G (samples 29 and 30) with higher values than some flavors.
Flavor RSA values ranged from 39 (lemon) to 214 (strawberry) mg of Trolox/L. The
highest RSA values were determined in strawberry and gooseberry flavors (214 and 212
mg of Trolox/L), which presented the lowest values of TPC (15 and 9 mg of GA/L). Choi
and collaborators26 reported the RSAs of 34 kinds of citrus essential oils and their
components by HPLC, showing that all essential oils have scavenging effects on DPPH
ranging from 5.4 to 172 mg of Trolox equiv/mL.
As with other parameters (TPC and reducing power), flavored waters with bioactive
compounds (tea, ginseng, and G. biloba) have increased RSA values, demonstrating the
dual effect of radical scavenging of these bioactive compounds. Further global
comparisons are difficult to establish due to the fact that different standards are used in
the several analytical methods described.
GC-MS Analysis of Flavors/Fragances . Six flavors were evaluated with regard to
antioxidant activity, but only citrus flavors (lime, lemon, and tangerine) were analyzed by
GC-MS. Flavors (essential oils) are volatile and complex natural mixtures characterized by
a strong odor, which can contain about 20-60 components at quite different
concentrations. Terpenes and terpenoids constituted the main group of compounds with
other aromatic and aliphatic constituents, all characterized by low molecular weight.7
Volatile compound profiles were obtained by HS-SPME using a PDMS/DVB fiber and
analyzed by GC-MS. Figure 1 shows the chromatograms of citrus flavors (lime, lemon,
and tangerine). The characterization of individual components was performed with mass
spectrometry (MS). Qualitative and quantitative composition of the citrus flavors (lime,
lemon, and tangerine), obtained by comparison of mass spectra data, and library data are
listed in Tables 4-6. A total of 28 terpenes were identified: 22 monoterpenes and 6
sesquiterpenes. Terpenes are a combination of several 5-carbon-base (C5) units called
isoprenes. The main terpenes are monoterpenes (C10) and sesquiterpenes (C15). A
terpene containing oxygen is called a terpenoid. The monoterpenes identified and present
in the flavors can be classified as (i) acyclic (β-myrcene) (ii) monocyclic (α-limonene, γ-
terpinene, o-cymene; β-cymene); (iii) bicyclic (6-isopropylidene-1-
methylbicyclo[3.1.0]hexane, (-)-β-pinene, 3-carene); (iv) terpenoid alcohol acyclic (cis-
geraniol); (v) terpenoid alcohol monocyclic (1,6-dihydrocarveol, 1-terpinen-4-ol, α-
terpineol, L-isopulegol, carveol, cis-p-menth-2,8-dienol, cis-β-terpineol); (vi) terpenoid
aldehyde (geranial, p-mentha-1,8-dien-7-al); (vii) terpenoid ester (linalyl butyrate); (viii)
terpenoid ether (1,4-cineole, 1,8-cineole); and (ix) terpenoid phenol (carvacrol).
II. Investigação e desenvolvimento
118
Figure 1. GC-MS chromatograms of flavor extracts: (a) lime; (b) lemon; (c) tangerine.
a)
b)
c)
11
9
Tab
le 4
. C
hem
ical
Com
posi
tion
of th
e V
olat
ile F
ract
ion
of L
ime
Fla
vor
peak
re
tent
ion
time
(min
) co
mpo
und
MW
m
/z
rela
tive
cont
enta (
%)
Mon
oter
pene
s
1 9
.53
(-)-β
-Pin
ene
13
6 93
; 41;
91
2.1
7 2
10.4
0 1,
4-C
ineo
l 15
4 11
1; 4
3; 7
1 1
.06
3 10
.60
o-cy
men
e 13
4 11
9; 1
34
0.6
7 4
10.6
9 α
-lim
onen
e 13
6 68
; 67;
93
7.4
5 5
10.7
4 eu
caly
ptol
(1,
8-C
ineo
l) 15
4 43
; 81;
108
1
.87
6 11
.33
γ-t
erpi
nene
13
6 93
; 91
1.4
3 7
12.1
7 lin
alyl
but
yrat
e 22
4 93
; 43;
41
1.2
7 8
12.8
7 3-
care
ne
136
93; 9
1; 7
9 0
.95
9 13
.07
p-m
enth
-8-e
n-2-
ol (
1,6-
dihy
droc
arve
ol)
154
93; 1
07; 1
21; 1
36
0.8
6 10
13
.65
n-oc
tana
l dim
ethy
l ace
tal
174
75; 7
1; 4
1 0
.38
11
13.7
0 1-
terp
inen
-4-o
l 15
4 71
; 93;
111
1
.90
12
13.9
8 α
-ter
pine
ol
154
59; 9
3; 1
21
19.3
4 13
14
.09
6-is
opro
pylid
ene-
1-m
ethy
l-bic
yclo
[3.1
.0]h
exan
e 13
6 12
1; 9
3; 1
36
2.4
3 14
14
.19
L-is
opul
egol
15
4 41
; 67;
69;
81;
55
1.0
6 15
14
.71
gera
nyl i
sova
lera
te
238
85; 4
3; 5
7; 4
1; 6
9 0
.52
16
14.8
7 ca
rveo
l (p-
men
tha-
1,8-
dien
-6-o
l) 15
2 11
9; 9
1; 1
34
17.1
3 17
15
.40
citr
al (
gera
nial
) 15
2 69
; 41;
84
27.1
2 18
15
.49
cis-
p-m
enth
-2,8
-die
nol
152
91; 1
34; 4
3; 1
19; 1
34
0.4
7 19
16
.94
cis-
gera
niol
15
4 93
; 41;
91
6.7
4 20
17
.25
β-m
yrce
ne
136
93; 4
1; 9
1 4
.19
Ses
quite
rpen
es
21
17.9
6 β
-car
yoph
ylle
ne
204
41; 6
9; 9
3; 1
33; 7
9 0
.30
22
18.1
5 α
-ber
gam
oten
e 20
4 93
; 41;
119
; 91
0.3
8 23
19
.27
β-b
isab
olen
e 20
4 69
; 41;
93
0.3
1 a R
elat
ive
cont
ent w
as c
alcu
late
d fr
om a
rea
ratio
.
12
0
Tab
le 5
. C
hem
ical
com
posi
tion
of th
e vo
latil
e fr
actio
n of
lem
on fl
avou
r.
peak
re
tent
ion
time
(min
) co
mpo
und
MW
m
/z
rela
tive
cont
enta
(%)
Mon
oter
pene
s 1
10.4
0 ci
s-β
-Ter
pine
ol
154
43; 7
1 2
.55
2 10
.69
α-L
imon
ene
(p-M
enth
a-1,
8-di
ene)
13
6 68
; 67;
93
8.5
7 3
10.7
5 eu
capl
ypto
l (1,
8-C
ineo
l) 15
4 43
; 81;
108
2
.95
4 13
.96
α-T
erpi
neol
(p-
Men
th-1
,8-d
ien-
6-ol
) 15
4 59
; 93;
121
2
.92
5 14
.85
carv
eol (
p-M
enth
a-1,
8-di
en-6
-ol)
152
119;
91;
134
27
.09
6 15
.37
citr
al (
Ger
ania
l) 15
2 69
; 41;
84
44.3
2 S
esqu
iterp
enes
7
17.9
5 β
-Car
yoph
ylle
n 20
4 41
; 69;
93;
133
; 79
11.5
9 a R
elat
ive
cont
ent w
as c
alcu
late
d fr
om a
rea
ratio
.
Tab
le 6
. C
hem
ical
com
posi
tion
of th
e vo
latil
e fr
actio
n of
tang
erin
e fla
vour
. pe
ak
rete
ntio
n tim
e (m
in)
com
poun
d M
W
m/z
a re
lativ
e co
nten
t (%
) M
onot
erpe
nes
1 10
.61
β-c
ymen
e 13
4 11
9; 9
1 0
.89
2 10
.69
α-li
mon
ene
(p-m
enth
a-1,
8-di
ene)
13
6 68
; 67;
93
11.7
8 3
11.3
3 γ-
terp
inen
e (p
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tha-
1,4-
dien
e)
136
93; 9
1 3
.76
4 12
.17
linal
yl b
utyr
ate
224
93; 4
3; 4
1 1
.01
5 13
.70
4-te
rpin
eol (
p-m
enth
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n-4-
ol; 1
-ter
pene
n-4-
ol)
154
71; 9
3; 1
11
4.7
4 6
13.9
5 α
-ter
pine
ol (
p-m
enth
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n-8-
ol)
154
59; 9
3; 1
21
11.3
8 7
14.1
9 n-
deca
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156
41; 4
3; 5
7 1
.01
8 15
.49
p-m
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(-)-
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lald
ehyd
e)
150
68; 7
9 1
.22
9 15
.73
carv
acro
l (p-
cym
en-2
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150
135;
150
2
.29
10
17.6
7 n-
dode
cana
l 18
4 41
; 57;
55
0.7
1 11
17
.76
met
hyla
min
oben
zoat
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5 16
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05
48.7
5 S
esqu
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12
17
.96
β-c
aryo
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len
204
41; 6
9; 9
3; 1
33; 7
9 3
.84
13
19.1
4 α
-sel
inen
e 20
4 10
8; 2
04; 9
3 1
.54
14
19.2
2 α
-far
nese
ne
204
41; 9
3 6
.65
15
19.5
3 β
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inen
e 20
4 16
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04; 1
34
0.4
4 a R
elat
ive
cont
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as c
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.
3. Perfil antioxidante
121
The sesquiterpenes are classified as (x) acyclic (α-farnesene); (xi) monocyclic (β-
bisabolene); and (xii) bicyclic (β-caryophyllene, α-bergamotene, α-selenene, and β-
cadinene).
For the lime flavor the mass spectral data revealed that monoterpenes represented
>99% of the volatile fraction. Citral (27.12%) was the major ingredient followed by α-
terpineol (19.34%), carveol (17.13%), and α-Limonene (7.45%). The sesquiterpenes β-
bisabolene (0.31%), β-caryophyllene (0.30%), and α-bergamotene (0.38%) were in minor
quantities.
In lemon flavor, the volatile fraction extracted was represented by 88.41% of
monoterpenes and 11.59% of sesquiterpenes. The major ingredients were the terpenes
citral (44.32%), carveol (27.09%), and α-limonene (8.57%) and the sesquiterpene β-
caryophyllene (11.59%). With regard to the tangerine flavor, methyl aminobonzoate
(48.75%), α-limonene (11.78%) and α-terpineol (11.38%) were the major compounds
followed by the sesquiterpene α-farnesene (6.65%). With regard to the tangerine flavor, α-
limonene (11.78%) and α-terpineol (11.38%) were the major compounds followed by the
sesquiterpene α-farnesene (6.65%).
By comparison of the obtained results with those of the literature, several analogies can
be pointed out. According to studies carried out by several authors, the essential oil
obtained from citrus fruit (orange, lemon, bergamot, grapefruit) had a similar composition
to that described in this study, considering only the analysis of the most volatile fraction of
the essence.9,27-29 Some authors reported that the major ingredients present in essential
oils from citrus fruit (orange and lemon) is limonene9,28 followed by α- and β-pinenes and
γ-terpinene.28 However, Caccioni and collaborators27 reported that lemon oil collected in
February showed the highest content of oxygenated compounds, two geraniol-geranial
and nerol-neral couples being the main compounds. Thus, the analysis and extraction of
the compounds in flavors can change in quality and quantity with seasonal variation,
ripeness, soil composition, and geographical region.7,8 Almost authors agree that
monoterpenes make up 97% of the citrus oil composition, with alcohol, aldehydes, and
esters being the lowest percentage components ranging from 1.8 to 2.2%.29 Flavonoids
are another group of components that are present in citrus flavors, making up the
nonvolatile part of the oils.8 Indeed, antimicrobial, antifungal, antioxidant, and radical
scavenging properties have been reported for flavors (essential oils) and fruits.9 Di Vaio
and collaborators9 reported that the peel ethanol extract from lemon presented antioxidant
activity and high radical scavenging power, suggesting that lemon essential oils and their
related flavor components may contribute to preventing oxidation in foods and inhibit lipid
oxidation. Other studies reported by Crowell30 revealed that terpenoids such as carveol
II. Investigação e desenvolvimento
122
and limonene present in plant essential oils are effective in treating breast, liver, and/or
other cancers.
Funding Sources
This research was supported by a Ph.D. grant from FCT (Fundação para a Ciência e
Tecnologia - SFRH/BD/29440/2006).
ACKNOWLEDGMENT
We acknowledge the Frize (Portuguese farmers) for providing flavor samples.
3. Perfil antioxidante
123
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127
Capítulo 4
Construção de biossensores de bases púricas
4.1.
Electrochemical evaluation of total antioxidant capacity of beverages using a purine-biosensor
M. F. Barroso, C. Delerue-Matos, M. B. P. P. Oliveira
Food Chemistry (submetido)
4.2.
Electrochemical DNA-sensor for evaluation of total antioxidant capacity of flavours and flavoured
waters using superoxide radical damage
M. F. Barroso, C. Delerue-Matos, M. B. P. P. Oliveira
Biosensors and Bioelectronics, 2011, 26 (9), 3748-3754
4.3.
Evaluation of total antioxidant capacity of flavoured waters using sulfate radical damage of purine-
based sensors
M. F. Barroso, C. Delerue-Matos, M. B. P. P. Oliveira
Electrochimica Acta (submetido)
4.1. Biossensores de bases púricas – radical hidroxilo
129
Electrochemical evaluation of total antioxidant cap acity of
beverages using a purine-biosensor
M. Fátima Barroso1,2, C. Delerue-Matos1, M. B. P. P. Oliveira2 1REQUIMTE/Instituto Superior de Engenharia do Porto.
Dr. Bernardino de Almeida 431, 4200-072 Porto. Portugal 2Requimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto.
R. Aníbal Cunha n.º 164, 4050-047 Porto. Portugal
Abstract
In this paper, it was evaluated the total antioxidant capacity (TAC) of beverages using
an electrochemical biosensor. The biosensor consisted on the purine base (guanine or
adenine) electro-immobilization on a glassy carbon electrode surface (GCE). Purine base
damage was induced by the hydroxyl radical generated by a Fenton-type reaction. Five
antioxidants were applied to counteract the deleterious effects of the hydroxyl radical. The
antioxidants used were ascorbic acid, gallic acid, caffeic acid, coumaric acid and
resveratrol. These antioxidants have the ability to scavenger the hydroxyl radical and
protect the guanine and adenine immobilized on the GCE surface. The interaction carried
out between the purine-base immobilized and the free radical in the absence and
presence of antioxidants was evaluated by means of changes in the guanine and adenine
anodic peak obtained by square wave voltammetry (SWV). The results demonstrated that
the purine-biosensors are suitable for rapid assessment of TAC in beverages.
Keywords: Purine bases; Total antioxidant capacity (TAC); Ascorbic acid; Phenolic acid;
Reactive oxygen species (ROS); Hydroxyl radical (OH•); Biosensor
Available online at www.sciencedirect.com
Food Chemistry submitted
4.1. Biossensores de bases púricas – radical hidroxilo
131
1. Introduction
In recent years, the interest for DNA-based diagnostic tests has been growing. The
development of systems allowing DNA detection is motivated by applications in many
fields: DNA diagnostics, fast detection of biological warfare agents and forensic
applications. Detection of genetic mutations at the molecular level opens up the possibility
of performing reliable diagnostics even before any symptom of a disease appears
(Sassolas, Leca-Bouvier, & Blum, 2008).
Reactive oxygen species (ROS) produced in living organisms by normal metabolism
and by exogenous sources such as carcinogenic compounds and ionizing radiations
induce oxidative DNA damage producing a variety of modifications at DNA level including
base and sugar lesions, strand breaks, DNA-protein cross-link and base-free sites
(Dizdaroglu, Jaruga, Birincioglu, & Rodriguez, 2002; Mello, Hernandez, Marrazza,
Mascini, & Kubota, 2006; Vertuani, Angusti, & Manfredini, 2004). However, the
mammalian cells have developed a complex endogenous defence system to repair the
damaged DNA through specific enzymes such as superoxide dismutase, catalase,
peroxidase, myeloperoxidase, that are involved in the base excision repair (Cadet, Douki,
Gasparutto, & Ravanat, 2003). Beyond this endogenous system, the living organisms also
use exogenous antioxidant compounds. An antioxidant is any substance that when
present at low concentration compared to those of an oxidizable substrate significantly
delays, inhibits or prevents oxidation of that substrate, in a chain reaction, therefore,
appears to be very important in the prevention of many diseases (Frankel, 2007; Halliwell,
Gutteridge, & Cross, 1992; Mello & Kubotta 2007). Antioxidants may delay or inhibit the
chain initiation, propagation and termination by reaction with a peroxyl radical (ROO•) or
alkoxyl radical (RO•) resulting in a lesser reactive radical (A•). In the inhibited oxidation,
termination occurs through the reaction of ROO• and RO• with a chain-breaking phenolic
antioxidant (AH), by interrupting the chain reaction by hydrogen transfer to produce a
phenoxy radical (A•) (Eq. 1 and Eq. 2) that is too stable to continue the chain by reaction.
The antioxidant radical can either react again with the ROO• (Eq. 3) and RO• (Eq. 4) to
form a stable peroxide or hydroxyl or react with another antioxidant radical to form a dimer
(Eq. 5) (Frankel, 2007).
II. Investigação e desenvolvimento
132
(5) A -A A A
(4) ROA A RO
(3) ROOA A ROO
(2) A ROH AH RO
(1) A ROOH AH ROO
→+
→+
→++→+
+→+
••
••
••
••
••
Increasing intake of dietary antioxidant may help to maintain an adequate antioxidant
status and, therefore, the normal physiological functions of a living system. Some
functional foods, vegetables, fruits, whole-grain cereals, wine and infusions are good
sources of exogenous antioxidants (Ignat, Volf & Popa, 2011). These foodstuff and
beverages include in its composition exogenous antioxidants such as vitamins (A, E, C),
phenolic compounds (gallic acid, caffeic acid, ferulic acid, p-coumaric acid, sinapic acid),
flavonoids (quertecin, rutin), minerals (selenium, zinc) or proteins (transferrin,
ceruloplasmin, albumin).
Ascorbic acid is a γ-lactone synthesized by plants and many animals (except primates).
This powerful exogenous antioxidant is a water-soluble vitamin, and plays a key role in the
protection against biological oxidation processes. Indeed, ascorbic acid is a good
scavenger of free radicals acting as a reducing agent by donation of a one electron
producing the semi-dehydroascorbate radical. It justifies its association to protection
against cancer agents by the prevention of formation of carcinogens precursors’
compound (Lee, Davis, Rettmer, & Lable, 1988; Mello & Kubotta, 2007; Smiroff, 2000).
Phenolic compounds (originated from vegetables) alsi present antioxidant activity. In
general, the antioxidant activity of the phenolics-derived compounds is determined by its
ideal chemical structure in terms of some properties such as free-radical scavengers or
chain breakers agents. It also, the fact of the resulting antioxidant-derived radical, namely
phenoxy radical is relatively stable due to the resonance delocalization and lack of
suitable sites for attack by molecular oxygen. The last property, the transition metal-
chelating potential, in special iron and copper supports the role of polyphenols as
preventive antioxidants in terms of inhibiting transition metal-catalysed free radical
formation (Soobrattee, Neergheen, Ramma, Aruoma, & Bahorun, 2005; Thavasi, Leong,
& Bettens, 2006).
Several methods have been proposed for the evaluation of the total antioxidant capacity
(TAC) in biological and food samples. These methodologies are based on UV-vis
spectrometry, chemiluminescence, fluorimetry (Sanchez-Moreno, 2002), chromatography
(Jaitz, Siegl, Eder, Rak, Abranko, Koellensperger, & Hann, 2010) and electrochemistry
techniques (Piljac-Žegarac, Valek, Stipčević, & Martinez, 2010).
4.1. Biossensores de bases púricas – radical hidroxilo
133
Electrochemical DNA-based have been developed in order to assess the antioxidant
capacity (Mello, & Kubota, 2007). These biosensors were based on the ds-DNA (double-
stranded DNA) (Mello, Hernandez, Marrazza, Mascini, & Kubota, & 2006), dA21
(deoxyadenylic acid) (Barroso, de-los-Santos-Álvarez, Lobo-Castañón, Miranda-Ordieres,
Delerue-Matos, Oliveira, & Tuñón-Blanco, 2011a) immobilization on the electrode surface,
as oxidation target and a Fenton-type reaction were used for (hydroxyl) OH• generation
(Eq. 6). Hydroxyl radicals interact with DNA bases inducing damage.
)6( OHOH Fe OH Fe 322
2 •−++ ++→+
In this work, the TAC of flavoured waters was evaluated using a purine-based
biosensor. This purine-based biosensor consisted on the electro-deposition of purine
bases (guanine and adenine) on a glassy carbon electrode (GCE) surface. The biosensor
was damaged by the hydroxyl radical according the procedure of Kamel and collaborators
(Kamel, Moreira, Delerue-Matos, & Sales, 2008). The influence of five antioxidants on the
scavenger free radical activity was studied. The antioxidants used were ascorbic acid, and
the following phenolic acids, gallic acid, caffeic acid, coumaric acid and resveratrol
(polyphenol). The protective effect of these five antioxidants on the purine bases was
observed. Square wave voltammetry (SWV) was the electroanalytical technique used to
relate the extent of oxidative damage carried out by the hydroxyl radical and the protective
role made by antioxidants.
2. Material and methods
2.1. Chemicals
Guanine (G-0381), adenine (A-8626), iron (II) sulphate heptahydrate, hydrogen peroxide
(100 % w/v), gallic acid, resveratrol were purchased from Sigma. Caffeic acid was from
Fluka, L(+) ascorbic acid and p-coumaric were acquired from Riedel-de-Haën. Other
chemicals were Merck pro-analysis grade and were used as received. (1 g L-1) Guanine
stock solution was prepared by dissolving an amount of this solid in 0.1 mol L-1 of NaOH
and dilution in phosphate buffered saline (PBS) at pH 7.4. Stock solution of 1 g L-1 of
adenine was prepared in PBS pH 7.4 and stored at +4 ºC.
Working standard solution (ascorbic acid, gallic acid, caffeic acid, coumaric acid) were
prepared daily and immediately before measurements by dissolving an amount of the
solid standard in water until the desired concentration. In order to dissolve the resveratrol
II. Investigação e desenvolvimento
134
antioxidant, an amount of this compound was dissolved in ethanol and then diluted with
water until to the desired concentration.
Hydroxyl radical was generated by mixing Fe2+:EDTA:H2O2 (0.20 mmol L-1: 0.40 mmol L-1
:8 mmol L-1) in the molar ratio of 1:2:40. Mello and collaborators (Mello, Hernandez,
Marrazza, Mascini, & Kubota, 2006) reported that when an excess of hydrogen peroxide
is added in the reaction a high DNA damage is obtained. EDTA was added for solubility
reasons. All solutions were prepared with water purified with a Direct-Q (Millipore) system.
2.2. Apparatus
Square wave voltammetry (SWV) was performed with an Autolab PSTAT 10
potentiostat controlled by GPES software (EcoChemie, The Netherlands). A conventional
three electrode cell was used, which includes glassy carbon electrode (GCE) (0.07 cm2)
as working electrode, a glassy carbon counter electrode and a Ag|AgCl|KClsat reference
electrode to which all potentials are referred. GCE was mechanically polished using a
polishing kit (Metrohm 6.2802.010) first with γ-Al2O3 (0.015 µm) until a shining surface
was obtained and after with only water. After this step the GCE was treated by applying a
fixed potential of +1.7 V for 30 s in PBS pH 4.8. This initial conditioning step improves the
resolution of the analytical signal because the application of high potentials in acidic
medium increases the hydrophilic properties of the electrode surface through the
introduction of oxygenated functionalities (Mello, Hernandez, Marrazza, Mascini, &
Kubota, 2006; Rice, Galus, & Adams, 1983).
2.3. Voltammetric procedure
Unless otherwise mentioned, all experiments consisted of three steps: i) guanine or
adenine electro-immobilization on the purine-based GCE, ii) damage of purine bases by
the immersion of DNA-GCE on the hydroxyl radical, and study the effect of the presence
of antioxidants in the reactive system; iii) detection and measurement of the peak current
of adenine or guanine in a PBS at pH 7.4.
Purine base (adenine or guanine) immobilization was performed by the application of an
adsorptive accumulation step. For that, the activated GCE was immersed in PBS pH 4.8
containing 10 mg L-1 of adenine or 3 mg L-1 of guanine and it was applied a positive
potential of +0.4 V for 180 s, after this the electrode was washed with water (Scheme 1).
For the purine bases biosensor preparation procedure (cleaning and immobilization step)
it was used the conditions optimized in previous works (Kamel, Moreira, Delerue-Matos, &
Sales, 2008; Mello, Hernandez, Marrazza, Mascini, & Kubota, 2006).
4.1. Biossensores de bases púricas – radical hidroxilo
135
Scheme 1. Electroimmobilization of the purine base on the CGE surface procedure and the SWV signal of the purine-based sensor in PBS (PH 4.8): a) blank signal (maximum peak current); after b) immersion in hydroxyl radical; c) immersion in hydroxyl radical with an antioxidant (ascorbic acid).
electroimmobilization step
detection stepSWV technique(frequency= 50 Hzstep potential = 4.12 mV amplitude= 0.09 V)
blank signal
immersionin hydroxyl radical
immersion in hydroxyl radicalwith an antioxidant (ascorbic acid)
N
N
NH2
N
HN
activated GCE surface+++++++
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
Ep = 0.82 V
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
a)
b)
c)
activated GCE surface+++++++
+ 0.4 V 180 s
N
N
NH2
N
HN
adenineadenine
electroimmobilization step
detection stepSWV technique(frequency= 50 Hzstep potential = 4.12 mV amplitude= 0.09 V)
blank signal
immersionin hydroxyl radical
immersion in hydroxyl radicalwith an antioxidant (ascorbic acid)
N
N
NH2
N
HN
activated GCE surface+++++++
N
N
NH2
N
HN
activated GCE surface+++++++
activated GCE surface+++++++
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
Ep = 0.82 V
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
Ep = 0.82 V
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
E / V15.0
25.0
35.0
45.0
0.50 0.70 0.90 1.10
i / µ
A
a)
b)
c)
activated GCE surface+++++++
+ 0.4 V 180 s
N
N
NH2
N
HN
adenineadenine
activated GCE surface+++++++
+ 0.4 V 180 s
activated GCE surface+++++++
+ 0.4 V 180 s
N
N
NH2
N
HN
adenineadenine
activated GCE surface+++++++
+ 0.4 V 180 s
NH
NNH2N
HN
O
guanine
electroimmobilization step
blank signal
25.0
35.0
45.0
55.0
0.20 0.40 0.6 0.80E / V
i / µ
A
Ep=0.55 V
25.0
35.0
45.0
55.0
0.20 0.40 0.60 0.80E / V
i / µ
A
25.0
35.0
45.0
55.0
0.20 0.40 0.60 0.80E / V
i / µ
A
HN N
N
NH2
HN
O
activated GCE surface+++++++
immersionin hydroxyl radical
immersion in hydroxyl radicalwith an antioxidant (ascorbic acid)
detection stepSWV technique(frequency= 50 Hzstep potential = 4.12 mV amplitude= 0.09 V)
guanine
activated GCE surface+++++++
+ 0.4 V 180 s
NH
NNH2N
HN
O
guanine
activated GCE surface+++++++
+ 0.4 V 180 s
activated GCE surface+++++++
+ 0.4 V 180 s
NH
NNH2N
HN
O
guanine
electroimmobilization step
blank signal
25.0
35.0
45.0
55.0
0.20 0.40 0.6 0.80E / V
i / µ
A
Ep=0.55 V
25.0
35.0
45.0
55.0
0.20 0.40 0.6 0.80E / V
i / µ
A
Ep=0.55 V
25.0
35.0
45.0
55.0
0.20 0.40 0.60 0.80E / V
i / µ
A
25.0
35.0
45.0
55.0
0.20 0.40 0.60 0.80E / V
i / µ
A
25.0
35.0
45.0
55.0
0.20 0.40 0.60 0.80E / V
i / µ
A25.0
35.0
45.0
55.0
0.20 0.40 0.60 0.80E / V
i / µ
A
HN N
N
NH2
HN
O
activated GCE surface+++++++
HN N
N
NH2
HN
O
activated GCE surface+++++++
activated GCE surface+++++++
immersionin hydroxyl radical
immersion in hydroxyl radicalwith an antioxidant (ascorbic acid)
detection stepSWV technique(frequency= 50 Hzstep potential = 4.12 mV amplitude= 0.09 V)
guanine
2
1
a)
b)
c)
II. Investigação e desenvolvimento
136
Purine base damage was carried out by immersing the biosensor in a freshly prepared
Fenton solution in the absence or in the presence of antioxidant in PBS pH 7.4. After a
fixed period of reaction time, the purine-based biosensor was rinsed with water and
immediately immersed in PBS (pH 4.8) to carry out the SWV between +0.2 V and +1.4 V.
(frequency 50 Hz, step potential 4.12 mV and amplitude 0.09 V). The peak current of
guanine and adenine obtained was used as a detection signal. For the electrochemical
studies it was considered that the maximum signal current obtained was for the purine
base signal without damage neither antioxidant effect (Scheme 1).
2.4. Samples
Thirty-nine water samples corresponding to 10 different brands were purchased in
several supermarkets in the North of Portugal and stored in the dark at +4 ºC. Each brand
(still or sparkling, mineral or spring water) had different flavours and aromas. The natural
water of each brand was also used as control. Sonication (30 min) was used to eliminate
gas from the sparkling water samples. The labels on the water bottles indicate the nutrient
information, namely the presence of fruit juice, vitamins, sweeteners and preservatives.
Six liquid flavours used in the formulation of some water brands, provided by a
producer, were also analysed. The flavours used corresponded to different fruit aromas,
such as lime, tangerine, strawberry, lemon, apple and gooseberry. These flavours had no
description about their chemical or aroma composition, but were known to be present in
the flavoured waters used in this study.
2.5. TAC measurement on beverages
The purine-based biosensor was applied to the determination of TAC on flavour and
flavoured waters. For the measurement of TAC in beverages, a volume of the flavoured
water or flavour were diluted in PBS to a final volume of 500 µl. Then, the purine-based
GCE was immersed in the solution and a freshly prepared hydroxyl radical was added for
120 s. After this period of time the biosensor was washed and immersed in PBS buffer to
measured the oxidation peak current of guanine and adenine. Ascorbic acid, gallic acid,
caffeic acid, coumaric acid and resveratrol were the working standard antioxidants used to
study the protective effect made by the antioxidant on the free-radical scavenging and to
carry out the linear calibrations studies. Measurements were made at least three times
and all results were expressed as mean ± standard deviation.
4.1. Biossensores de bases púricas – radical hidroxilo
137
3. Results and discussion
Previous studies reported in the literature indicate the oxidative damage of dsDNA
(Mello, Hernandez, Marrazza, Mascini, & Kubota, 2006), dA21 (Barroso, de-los-Santos-
Álvarez, Lobo-Castañón, Miranda-Ordieres, Delerue-Matos, Oliveira, & Tuñón-Blanco,
2011) and purine bases (Kamel, Moreira, Delerue-Matos, & Sales, 2008) induced by the
hydroxyl radical generated by the Fenton solution. Hydroxyl radical (OH•) is one of the
most reactive radical species that induce lesions in DNA. This ROS cause cell injury when
is generated in excess or the cellular antioxidant defence is impaired. When hydroxyl
radical is generated adjacent to DNA, it attacks both deoxyribose sugar and the purine
and pyrimidine bases resulting intermediates radicals, which are the immediate precursors
for DNA base damage (Jaruga & Dizdaroglu, 1996).
In order to study the protective effect promoted by antioxidants on the deactivation of
the hydroxyl radical and consequently protect the purine bases from the oxidative
damage, the purine-based biosensor was placed in a PBS pH 4.8 in presence of an
antioxidant and hydroxyl radical during 120 s. Next the biosensor was rinsed with water
and a SWV was made from +0.2 V to +1.4 V. Fig. 1 shows the performance of the purine-
based biosensor in the presence of antioxidants (0.5 mg L-1 of ascorbic acid, gallic acid,
caffeic acid, coumaric acid and resveratrol) and the hydroxyl radical.
Fig. 1. Effect of the antioxidants presence on the signal of guanine and adenine immobilized on the GCE: blank purine base signal (guanine 3 mg L-1 and adenine 10 mg L-1); after immersion in a hydroxyl radical (Fe2+: EDTA: H2O2; 0.1 mmol L-1: 0.2 mmol L-1: 4.0 mmol L-1 during 120 s); immersion in hydroxyl radical with five different antioxidants (0.50 mg L-1).
100value) expected (maximum done wasdamage no whenmeasured pi
t)antioxidan an of presence the in (or radical hydroxyl withdamage base purine after measured pi (%) signal base purine ×=
0
20
40
60
80
100
120
blank hydroxyl radical ascorbic acidhydroxyl radical
gallic acidhydroxyl radical
caffeic acidhydroxyl radical
coumaric acidhydroxyl radical
Resveratrolhydroxyl radical
guanine
adenine
pu
rin
eba
se s
ign
al (
%)
0
20
40
60
80
100
120
blank hydroxyl radical ascorbic acidhydroxyl radical
gallic acidhydroxyl radical
caffeic acidhydroxyl radical
coumaric acidhydroxyl radical
Resveratrolhydroxyl radical
guanine
adenine
pu
rin
eba
se s
ign
al (
%)
II. Investigação e desenvolvimento
138
To perform this electrochemical study all current peaks were compared with the signal
current obtained with the non damaged adenine and guanine bases (blank signal). Purine
bases of DNA measured in SWV presented two oxidation peaks at around +0.55 V and
+0.82 V corresponding, respectively, to guanine and adenine oxidation peak (Scheme 1a
and 1b). Hydroxyl radical had the ability to produce 61.4% and 55.2% of damage in
guanine and adenine base, respectively (Fig. 1). Other free radicals had also the capacity
to induce oxidative damage on the purine bases. It was verified that and superoxide
radical produce from about 64% of damage on the guanine-based biosensor (Barroso,
Delerue-Matos, & Oliveira, 2011a) and 85% on the dA21 (Barroso, de-los-Santos-Álvarez,
Lobo-Castañón, Miranda-Ordieres, Delerue-Matos, Oliveira, & Tuñón-Blanco, 2011b)
while the sulfate radical produced 61% of damage on guanine-based biosensor (Barroso,
Delerue-Matos, & Oliveira, 2011b).
When it was added an antioxidant (0.5 mg L-1) in the reactive system a less decrease of
the anodic current of guanine and adenine was recorded. It was observed a protective
effect on the purine base carried out by the antioxidants ranged from 47 to 79%. Using the
guanine-biosensor the lowest values were found for caffeic and coumaric acid, 47.6% and
49.1%, respectively. The highest values was obtained for resveratrol (74.6 %) followed by
gallic acid (72.0%) and ascorbic acid (62.8%). Using the adenine-biosensor the protective
effective of the antioxidants ranged from 60 to 79%. The highest values was observed for
the resveratrol antioxidant (79.1%) followed by gallic acid (77.7%) and caffeic acid
(73.6%). The lowest values were found for ascorbic acid (60.4%) and coumaric acid
(61.9%). Using a DNA-based biosensor, ascorbic acid (0.5 µmol L-1) presented a
protective role of 58 % against the hydroxyl radical, and a concentration of 10 µmol L-1 of
ascorbic acid presented a protective role of 53.8 % against the superoxide radical
(Barroso, de-los-Santos-Álvarez, Lobo-Castañón, Miranda-Ordieres, Delerue-Matos,
Oliveira, & Tuñón-Blanco, 2011a; Barroso, de-los-Santos-Álvarez, Lobo-Castañón,
Miranda-Ordieres, Delerue-Matos, Oliveira, & Tuñón-Blanco, 2011b).
The protection action mode of antioxidants may involve multiple mechanisms,
depending on the source material and, possible presence of synergists and antagonists.
In general, the antioxidant activity of ascorbic acid and phenolics-derived compounds is
related to reducing properties as hydrogen or electron-donating agents, which is
determined to its reduction potential (Buettner, 1993; Mello & Kubota, 2007; et al., 2007;
Rice-Evans, 2001).
4.1. Biossensores de bases púricas – radical hidroxilo
139
3.1. Optimization of the experimental conditions
In order to evaluate the TAC on beverages, some parameters concerning the damaging
reaction (iron concentration and reaction time between hydroxyl radical) at a fixed time
reaction were implemented in order to achieve the maximum purine base of DNA effect,
but without a complete damage (non-zero ip). The level of purine bases damage was
evaluated as function of the variation of the concentration of Fe2+ keeping constant the
molar ratio Fe2+: EDTA: H2O2 used (1:2:40) (Mello, Hernandez, Marrazza, Mascini, &
Kubota, 2006). Fe2+ concentration was studied between 5.0 µmol L-1 to 1.0 mmol L-1. A
range of 19% to 60% decrease in the ip of guanine and adenine immobilized on the GCE
surface was observed over the Fe2+ concentration studied. When it was used the adenine-
biosensor, a 52% decrease on the ip was recorded when the Fe2+ was increased from 50
µmol L-1 to 0.2 mmol L-1. At Fe2+ concentrations higher than 0.2 mmol L-1 the peak current
remained essentially unchanged so, this concentration was chosen for the next
experiments. At a guanine-biosensor, the increase of Fe2+ concentration promoted a
decrease of 20% to 58% in the ip. At Fe2+ concentration higher than 0.15 mmol L-1 ip was
achieved to remains unchanged, so this value was used for the next experiments.
Reaction time between the hydroxyl radical and the DNA bases immobilized on the GCE
surface depends on the half-life time of the generated free radical, so this parameter is an
important feature to optimize.
In this study the incubation time were ranged from 0 to 120 s. A 62% and a 53%
decrease on the ip of guanine and adenine, respectively was observed after an incubation
time of 120 s. Fig. 2 shows the correlation between the damage on the purine bases
measured (correlated with the anodic peak current) and the incubation time. The
incubation time of 120 s was chosen for both purine-based biosensors for all experiments.
Fig. 2. Influence on the peak current on the biosensor with the incubation time a) 10 mg L-1 adenine base; b) 3 mg L-1 guanine base.
Time (s)
4.0
6.0
8.0
10.0
12.0
14.0
0 20 40 60 80 100 120
a) guanine
b) adenine
i p(µ
A)
Time (s)
4.0
6.0
8.0
10.0
12.0
14.0
0 20 40 60 80 100 120
a) guanine
b) adenine
i p(µ
A)
II. Investigação e desenvolvimento
140
3.2. Determination of TAC
Beverages, such as juice and infusions are an excellent source of exogenous
antioxidants. The total phenolic (TPC) reducing power and DPPH radical scavenging
activity of these flavoured waters were determined using conventional optical methods.
The polyphenols compounds were present in all flavoured water samples (0.5 to 359 mg
of gallic acid L-1). The highest TPC levels were from citrus fruits (tangerine, lime and
lemon) and from waters with bioactive compounds, like tea, gingeng and Gingko biloba.
The reducing power values were ranged from (0.14 to 11.8 mg gallic acid L-1) and DPPH
radical scavenging activity (0.29-211.5 mg trolox L-1) (Barroso, Noronha, Delerue-Matos,
& Oliveira, 2011).
For the evaluation of the TAC of flavoured waters it was used the five antioxidants
referred before. These antioxidants can be found in fruit, grapes, wine and teas. As
expected, the anodic peak current of guanine and adenine immobilised on the GCE
surface increased when the concentration of the antioxidant increased. The analytical
parameters obtained in linearity studies between antioxidants concentration and peak
current of purine-based biosensor are presented in Table 1.
Table 1. Analytical feature obtained for the 5 antioxidants standards. Parameters Ascorbic acid Gallic acid Caffeic acid Coumaric acid Resveratrol
Guanine-GCE
Linear range (mg L-1) 0.50–2.50 0.10–0.50 0.40–0.80 0.31–0.73 0.10–0.50
Slope (µA mg-1L) 2.82 9.33 8.76 9.20 11.8
Intercept (µA) 1.88 4.31 1.27 1.69 3.76
Correlation coefficient (n=5) 0.990 0.986 0.992 0.990 0.986
RSD (%) (mg L-1 ) 3.43 (2.00) 4.87 (0.30) 2.58 (0.50) 4.63 (0.50) 3.25 (0.30)
LOD (mg L-1) 0.29 0.09 0.06 0.08 0.07
Adenine-GCE
Linear range (mg L-1) 2.00–6.00 0.11–0.44 0.10–0.50 0.10–1.00 0.10–0.50
Slope (µA mg-1L) 0.40 7.38 11.9 3.81 8.78
Intercept (µA) 5.08 5.30 2.08 4.35 3.81
Correlation coefficient (n=5) 0.983 0.986 0.990 0.972 0.972
RSD (%) (mg L-1) 2.45 (3.00) 5.35 (0.30) 4.86 (0.30) 7.56 (0.50) 6.35 (0.30)
LOD (mg L-1) 0.99 0.08 0.07 0.27 0.10
RSD (%) = σ/[antioxidant]mean found x 100
4.1. Biossensores de bases púricas – radical hidroxilo
141
Some authors reported the study of dsDNA (Liu, Roussel, Lagger, Tacchini, & Girault,
2005; Korbut, Buckova, Labuda, & Grundler, 2003; Mello, Hernandez, Marrazza, Mascini,
& Kubota, 2006), or ssDNA (Barroso, de-los-Santos-Álvarez, Lobo-Castañón, Miranda-
Ordieres, Delerue-Matos, Oliveira, & Tuñón-Blanco, 2011a) or purine bases (Kamel,
Moreira, Delerue-Matos, & Sales, 2008) damage induced by hydroxyl radical, generated
by the fenton system (Mello, Hernandez, Marrazza, Mascini, & Kubota, 2006) or UV
radical (Liu, Roussel, Lagger, Tacchini, & Girault, 2005) and its protection with the
ascorbic acid (Kamel, Moreira, Delerue-Matos, & Sales, 2008) gallic acid (Liu, Roussel,
Lagger, Tacchini, & Girault, 2005) and flavonoids (Korbut, Buckova, Labuda, & Grundler,
2003). Zhang et al. (2008) reported the study of DNA damage induced by Fenton system
on a GCE and its protection with the antioxidant ascorbic acid. Ascorbic acid promoted
protective effect on the DNA in a norrow concentration range (from 1.5 to 2.5 mmol l−1)
(Zhang, Wang, Li, Jia, Cui, & Wang, 2008). Nobushi and Uchikura, (2010) reported the
protective effects on the DNA by applying ascorbic acid as a scavenging antioxidant.
Enzyme-modified electrodes using ascorbate oxidase and peroxidase enzymes for the
detection of ascorbic acid showed linear ranges in the submM level (Mello and Kubota,
2007).
The purine-based biosensors were applied to the evaluation of TAC of flavours and
flavoured waters. Table 2 shows the TAC values expressed in mg L-1 of ascorbic acid,
gallic acid, caffeic acid, coumaric and resveratrol. It was verified that all flavours and
flavoured waters presented antioxidant capacity. Like it was expected the natural waters
not presented antioxidant capacity. Flavours presented the highest TAC values, as
demonstrated by results in Table 2. Indeed, flavours are fruit extract and have in its
composition several concentrated antioxidant compounds, so higher TAC values were
expected.
Using the guanine and adenine-biosensor the higher TAC values were found with the
antioxidant standard ascorbic acid. When it was used the guanine-based biosensor, the
flavour that presented the highest TAC value was apple, followed by tangerine,
strawberry, lemon, gooseberry and lime. At the adenine-GCE lime was the flavour that
presented the highest TAC value followed by lemon, apple, tangerine, strawberry and
gooseberry.
When the guanine-biosensor was applied to the quantification of TAC in flavoured
waters, TAC values ranged from 2.98 to 40.32 mg L-1; 0.11 to 2.27 mg L-1; 1.50 to 4.34 mg
L-1, 1.18 to 3.88 mg L-1, 0.10 to 2.05 mg L-1 with the antioxidant ascorbic acid, gallic acid,
caffeic acid, coumaric acid and resveratrol, respectively. Using ascorbic acid as standard
antioxidant, lemon flavoured waters presented the highest TAC values on all brands
(except in brand I).
14
2 Tab
le 2
. T
AC
val
ues
obta
ined
for
the
flavo
urs
and
flavo
ured
wat
ers
usin
g a
guan
ine-
base
d bi
osen
sor
and
aden
ine-
base
d bi
osen
sor.
Gua
nine
-bio
sens
or
Ade
nine
-bio
sens
or
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Bra
nd
Sam
ple
m
g L-1
Lem
on
120.
23 ±
11.
48
10.3
0 ±
3.47
45
.67
± 3.
69
38.9
2 ±
3.52
12
.81
± 2.
74
526.
45 ±
35.
62
32.5
2 ±
1.92
3
0.42
± 1
.19
35.4
3 ±3
.71
21.5
3 ±
1.62
Tan
gerin
e 17
3.56
± 2
7.61
26
.41
±8.3
4 62
.84
± 8.
89
55.
27 ±
8.4
6 25
.55
± 6.
60
375.
31 ±
25.
45
16.9
4 ±
2.9
3 25
.38
± 2.
57
19.6
9 ±
2.75
14
.69
± 3.
87
App
le
185.
64 ±
5.7
7 30
.06
± 1.
74
66.7
2 ±
1.86
58
.97
± 1
.77
28.4
3 ±
1.38
47
6.83
± 1
6.69
16
.30
± 1.
97
28.7
6 ±
1.22
30
.26
± 3.
82
19.2
8 ±
1.66
Str
awbe
rry
126.
26 ±
19.
58
12.1
2 ±
5.91
47
.61
± 6.
30
40.7
7 ±
6.00
14
.24
± 4.
68
309.
82 ±
7.8
7 20
.87
± 2.
68
23.1
9 ±
1.66
12
.86
±1.8
7 11
.73
± 2.
26
Goo
sebe
rry
100.
82 ±
10.
03
9.18
± 1
.03
39.1
6 ±
3.22
32
.72
± 3.
07
7.97
± 2
.40
211.
59 ±
17.
82
15.5
8 ±
0.9
6 19
.92
± 0.
59
10.5
0 ±
0.06
10
.71
± 0.
81
Fla
vour
Lim
e 10
2.62
± 1
1.33
10
.97
± 2.
42
40.0
0 ±
3.65
33
.52
± 3
.47
8.59
± 2
.71
571.
91 ±
13.
45
14.6
4 ±
2.29
31
.94
± 4.
52
40.1
7 ±
10.1
2 23
.58
± 6.
13
A
1 Le
mon
12
.27
± 0.
69
2.27
± 0
.29
4.34
± 0
.23
3.88
± 0
.22
2.05
± 0
.17
18.3
3 ±
2.59
1.
38 ±
0.1
6 1.
26 ±
0.0
9 4.
06 ±
0.3
1 0.
71 ±
0.1
2
2 M
ango
9.
53 ±
0.7
0 1.
44 ±
0.0
9 3.
45 ±
0.1
5 3.
04 ±
0.0
4 1.
40 ±
0.0
5 7.
42 ±
3.6
1 0.
51 ±
0.0
2 0.
87 ±
0.0
2 2.
36 ±
0.0
4 0.
21 ±
0.0
6
3 S
traw
berr
y 3.
46 ±
0.4
1 -
1.50
± 0
.13
1.18
± 0
.13
- 7.
21 ±
0.7
6 0.
43 ±
005
0.
93 ±
0.0
3 2.
21 ±
0.0
7 0.
20 ±
0.0
3
4 N
atur
al
- -
- -
- -
- -
- -
B
5 P
inea
pple
/ora
nge
5.42
± 0
.52
0.20
± 0
.02
2.13
± 0
.10
1.78
± 0
.13
0.42
± 0
.09
37.5
4 ±
1.97
0.
18 ±
0.0
6 1.
92 ±
0.0
7 1.
73 ±
0.1
1 1.
57 ±
0.0
9
6
Lem
on
5.13
± 0
.28
0.11
± 0
.08
2.04
± 0
.09
1.69
± 0
.08
0.35
± 0
.07
34.0
6 ±
5.69
0.
09 ±
0.0
3 1.
72 ±
0.1
9 1.
56 ±
0.0
5 1.
41 ±
0.2
6
7
Nat
ural
-
- -
- -
- -
- -
-
C
8 Le
mon
/Mag
nesi
um
8.26
± 0
.28
1.06
± 0
.06
3.04
± 0
.05
2.65
± 0
.09
1.09
± 0
.06
22.6
6 ±
1.19
0.
11 ±
0.0
5
1.49
± 0
.04
1.59
± 0
.10
0.90
± 0
.05
9
App
le/w
hite
tea
6.99
± 0
.14
0.67
± 0
.04
2.63
± 0
.04
2.26
± 0
.04
0.79
± 0
.03
4.16
± 0
.67
0.41
± 0
.05
0.86
± 0
.02
2.17
± 0
.02
0.60
± 0
.03
10
Pin
eapp
le/fi
bre
6.41
± 0
.15
0.50
± 0
.13
2.44
± 0
.17
2.08
± 0
.13
0.65
± 0
.02
13.7
1 ±
5.52
0.
21 ±
0.0
2 1.
30 ±
0.1
8 1.
78 ±
0.0
4 0.
49 ±
0.0
5
11
Nat
ural
-
-
-
-
-
-
-
-
-
-
D
12 A
pple
4.
64 ±
0.6
9 -
1.88
± 0
.22
1.54
± 0
.21
0.23
± 0
.06
5.43
± 0
.96
0.
84 ±
0.0
7 2.
25 ±
0.1
6 0.
12 ±
0.0
2
13
Ora
nge/
peac
h 4.
59 ±
0.3
5 -
1.86
± 0
.11
1.52
± 0
.11
0.22
± 0
.08
3.02
± 0
.09
- 0.
75 ±
0.0
8 1.
52 ±
0.0
5 -
14
Lem
on
4.74
± 0
.28
- 1.
91 ±
0.0
8 1.
57 ±
0.0
4 0.
25 ±
0.0
5 3.
34 ±
0.0
8 -
0.84
± 0
.03
1.81
± 0
.08
-
15
Nat
ural
-
-
-
-
-
-
-
-
-
-
E
16 L
emon
7.
57 ±
0.2
9 0.
85 ±
0.0
6 2.
82 ±
0.1
9 2.
44 ±
0.3
2 0.
93 ±
0.0
6 19
.14
± 2.
41
1.37
± 0
.13
1.29
± 0
.08
4.02
± 0
.26
0.74
± 0
.11
17
Ora
nge/
rasp
berr
y 2.
98 ±
0.4
1 -
1.50
± 0
.14
1.18
± 0
.13
- 4.
23 ±
0.2
5 0.
52 ±
0.1
0 0.
88 ±
0.0
4 2.
39 ±
0.2
0 0.
70 ±
0.0
6
18
Pea
ch/p
inea
pple
3.
86 ±
0.1
4 -
1.63
± 0
.04
1.30
± 0
.05
- 11
.78
± 6.
15
0.34
± 0
.05
1.25
± 0
.21
2.02
± 0
.10
0.41
± 0
.02
19
Gua
va/li
me
3.66
± 0
.70
- 1.
56 ±
0.0
7 1.
24 ±
0.3
2
- 3.
96 ±
0.1
8 0.
11 ±
0.0
5 1.
01 ±
0.2
4 1.
59 ±
0.1
0 -
20
Nat
ural
-
-
-
-
-
-
-
-
-
-
F
21 L
emon
/gre
en te
a 9.
83 ±
0.2
8 1.
53 ±
0.0
4 3.
55 ±
0.9
1 3.
13 ±
0.09
1.
47 ±
0.0
7 15
.94
± 0.
86
0.34
± 0
.05
1.
26 ±
0.0
3 2.
03 ±
0.1
4 0.
60 ±
0.0
4
22
Ras
pber
ry/g
inse
ng
8.90
± 0
.35
1.25
± 0
.10
3.25
± 0
.11
2.84
± 0
.10
1.25
± 0
.08
27.3
6 ±
1.28
0.
28 ±
0.0
8 1.
59 ±
0.0
4 1.
92 ±
0.1
5 1.
11 ±
0.0
6
14
3
Gua
nine
-bio
sens
or
Ade
nine
-bio
sens
or
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Bra
nd
Sam
ple
m
g L-1
23
Pea
ch/w
hite
tea
6.89
± 0
.28
0.64
± 0
.03
2.60
± 0
.09
2.23
± 0
.08
0.77
± 0
.06
28.4
9 ±
1.16
0.
39 ±
0.0
4 1.
63 ±
0.0
4 2.
14 ±
0.1
5 1.
16 ±
0.0
5
24
Man
go/g
inkg
o be
loba
7.
48 ±
0.0
8 0.
82 ±
0.0
5 2.
79 ±
0.0
5 2.
41 ±
0.0
8 0.
91 ±
0.0
2 19
.33
±1.9
7 0.
49 ±
0.0
2 1.
40 ±
0.0
7 2.
32 ±
0.0
4 0.
75 ±
0.0
9
25
Mel
on/m
int
8.58
± 0
.38
1.16
± 0
.11
3.15
± 0
.03
2.7
5 ±
0.12
1.
17 ±
0.1
1 24
.89
± 1.
97
0.41
± 0
.05
1.4
9 ±
0.07
2.
17 ±
0.0
7 1.
00 ±
0.0
8
26
Nat
ural
-
-
-
-
-
-
-
-
-
-
G
27 L
emon
7.
47 ±
0.2
0 0.
82 ±
0.0
6 2.
79 ±
0.0
6 2.
40 ±
0.0
6 0.
91 ±
0.0
5 29
.77
± 0.
96
2.02
± 0
.21
1.73
± 0
.03
5.29
± 0
.41
1.22
± 0
.04
28
Lim
e 4.
11 ±
0.0
6 -
1.71
± 0
.02
1.37
± 0
.02
0.10
± 0
.01
5.42
± 1
.98
- 0.
94 ±
0.0
7 0.
76 ±
0.0
3 0.
12 ±
0.0
8
29
App
le
7.48
± 0
.28
0.82
± 0
.09
2.79
± 0
.10
2.41
± 0
.08
0.91
± 0
.07
45.0
5 ±
2.95
1.
20 ±
0.2
1 2.
28 ±
0.0
9 3.
70 ±
0.0
5 1.
91 ±
0.1
3
30
Pea
ch
9.14
± 0
.57
1.33
± 0
.13
3.33
± 0
.15
2.92
± 0
.13
1.31
± 0
.10
14.2
5 ±
2.65
0.
79 ±
0.1
6 1.
12±
0.08
2.
90 ±
0.3
1 0.
51 ±
0.0
2
31
Nat
ural
-
-
-
-
-
-
-
-
-
-
H
32 L
emon
6.
38 ±
0.3
9 0.
49 ±
0.0
7 2.
44 ±
0.0
7 2.
07 ±
0.1
2 0.
65 ±
0.0
9 18
.43
± 1.
27
0.09
± 0
.01
1.35
± 0
.04
1.54
± 0
.03
0.71
± 0
.06
33
Nat
ural
-
- -
- -
- -
- -
-
I 34
Lem
on
34.6
2 ±
1.97
0.
63 ±
0.0
3 1.
91 ±
0.0
7 2.
59 ±
0.5
6
1.44
± 0
.09
34.6
2 ±
1.97
0.
63 ±
0.0
3 1.
91 ±
0.0
7 2.
59 ±
0.5
6
1.44
± 0
.09
35
Gre
en A
pple
37
.40
± 1.
69
0.92
± 0
.14
2.00
± 0
.07
3.
15 ±
0.2
6 1.
57 ±
0.0
2 37
.40
± 1.
69
0.92
± 0
.14
2.0
0 ±
0.07
3.
15 ±
0.2
6 1.
57 ±
0.0
2
36
Str
awbe
rry
40.3
2 ±
2.78
0.
14 ±
0.0
3 2.
07 ±
0.0
3 -
1.
70 ±
0.0
4 40
.32
± 2.
78
0.14
± 0
.03
2.07
± 0
.03
-
1.
70 ±
0.0
4
37
Nat
ural
-
-
-
-
-
-
-
-
-
-
J 38
Lem
on
5.71
± 0
.09
0.29
± 0
.04
2.22
± 0
.09
1.86
± 0
.05
0.48
± 0
.04
36.3
5 ±
2.47
0.
69 ±
0.1
3 1.
86 ±
0.1
5 2.
90 ±
0.2
5 1.
52 ±
0.1
1
39
Nat
ural
-
- -
- -
- -
- -
- -N
ot d
etet
ed.
II. Investigação e desenvolvimento
144
The highest TAC values was found in brand I (sample 34 to 36) followed by brand F
(sample 21 to sample to 25) brand G (sample 27 to 30) and sample C (sample 8 to 10).
Using the gallic acid as a standard antioxidant some flavoured waters not presented
antioxidant activity such as brand D (sample 12 to 14) brand E (sample 17 to 19) sample
3 (brand A), and sample 28 (brand G). The lowest TAC value was from the brand B and
the highest was from brand F. With the caffeic acid standard antioxidant all flavoured
waters presented antioxidant capacity. The highest TAC values were from brand F
following brand A and brand C. Using the coumaric acid and the resveratrol as standard
antioxidant some samples not presented antioxidant activity, such as, sample 36 (brand I)
with coumaric acid and sample 3 (brand A), sample 17, sample 18 and sample 19 (brand
E) with the resveratrol antioxidant. TAC values obtained with the four antioxidant, gallic
acid, caffeic acid, coumaric acid and resveratrol are narrower than the values obtained
with the standard ascorbic acid antioxidant. Theses differences obtained between the
ascorbic acid and the others antioxidants can be elucidated by the fact that ascorbic acid
presented a larger linear range (0.50 to 2.50 mg L-1).
When it was used the adenine-GCE, the highest TAC contents were found with the
ascorbic acid antioxidant. With this antioxidant, TAC values ranged between 3.02 to 45.05
mg L-1 of ascorbic acid. The highest TAC values were obtained in brand I, followed from
brand J, brand B, brand F and brand G. The lowest TAC value was obtained in brand D.
When it was use the gallic acid antioxidant, some flavoured waters not presented
antioxidant activity, such as brand D (sample 12 to 14), and sample 28 (brand G). TAC
values ranged from 0.09 to 2.02 mg L-1 of gallic acid. The highest TAC value was found on
brand G (samples 27 and 29) following brand E and brand A. The lowest TAC value was
found in brand H followed by brand B, brand C and brand F. With the caffeic acid
antioxidant all flavoured waters presented antioxidant activity and the TAC values ranged
from 0.75 to 2.28 mg L-1. When it was used the coumaric acid and the resveratrol as
standard antioxidant some flavoured waters not presented antioxidant activity, such as
sample 36 with the coumaric acid antioxidant and sample 13, sample 14 and sample 19
with the resveratrol antioxidant. TAC values ranged between 0.76 to 5.29 mg L-1 and 0.12
to 1.57 mg L-1 when it was used coumaric acid and resveratrol respectively. Like it was
happened with the guanine-biosensor, larger TAC values were obtained with the ascorbic
acid and the the other four antioxidants presented a narrow TAC range.
Analysing results from Table 2 it is possibly to confirm that the purine bases immobilized
on GCE can be used for the quantification of TAC in beverages, however using the
adenine-GCE and ascorbic acid as antioxidant standard it was obtained the highest TAC
values.
4.1. Biossensores de bases púricas – radical hidróxilo
145
4. Conclusion
A guanine-biosensor and adenine-biosensor for the TAC quantification of beverages was
used. The electroanalytical technique is based on the interaction of adenine or guanine
immobilized on the GCE surface with the hydroxyl radical. The hydroxyl radical had the
capacity to damage the purine base. Five antioxidants (ascorbic acid, gallic acid, caffeic
acid, coumaric acid and resveratrol) were tested as hydroxyl radicals scangers exihiting
efficiencies ranging from 47 to 79 %. The protective effect on the DNA bases performed
by the presence of these antioxidants allowed the evaluation of TAC in food samples.
Ascorbic acid presented the highest TAC values and seems to be the most sensitive
standard antioxidant. The purine-based biosensor developed is disposable, and requires a
very easy, rapid, reproducible preparation and also the advantage to combine with
portable equipment.
Acknowledgements
M. Fátima Barroso is grateful to Fundação para a Ciência e a Tecnologia for a Ph.D.
grant (SFRH/BD/ 29440/2006). The authors thank Frize for providing flavours samples.
II. Investigação e desenvolvimento
146
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4.2. Biossensores de bases púricas - radical superóxido
149
Electrochemical DNA-sensor for evaluation of total antioxidant
capacity of flavours and flavoured waters using sup eroxide
radical damage
M. F. Barroso1,2, C. Delerue-Matos1, M. B. P. P. Oliveira2 1REQUIMTE, Instituto Superior de Engenharia do Porto. R. Dr. Bernardino de Almeida
431, 4200-072 Porto, Portugal 2Requimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto. R.
Aníbal Cunha n.º 164, 4050-047 Porto, Portugal
Abstract
In this paper, a biosensor based on a glassy carbon electrode (GCE) was used for the
evaluation of the total antioxidant capacity (TAC) of flavours and flavoured waters. This
biosensor was constructed by immobilising purine bases, guanine and adenine, on a
GCE. Square wave voltammetry (SWV) was selected forthe development of this
methodology. Damage caused by the reactive oxygen species (ROS), superoxide radical
(O2• −), generated by the xanthine/xanthine oxidase (XOD) system on the DNA-biosensor
was evaluated. DNA-biosensor encountered with oxidative lesion when it was in contact
with the O2• −. There was less oxidative damage when reactive antioxidants were added.
The antioxidants used in this work were ascorbic acid, gallic acid, caffeic acid, coumaric
acid and resveratrol. These antioxidants are capable of scavenging the superoxide radical
and therefore protect the purine bases immobilized on the GCE surface. The results
demonstrated that the DNA-based biosensor is suitable for the rapid assess of TAC in
beverages.
Keywords: DNA biosensor; Total antioxidant capacity (TAC); Ascorbic acid; Phenolic
acid; Reactive oxygen species (ROS); Superoxide radical (O2• -).
Available online at www.sciencedirect.com
Biosensors & Bioelectronics 2011, 26 (9), 3748-3754
4.2. Biossensores de bases púricas - radical superóxido
151
1. Introduction
Recently, bottled flavoured waters are becoming popular, and the consumption of
flavoured waters is globally increasing including Portugal. In the first half of 2010, 6.08
million L of this kind of water were consumed by the Portuguese population (ANIRSF,
2010). Flavoured waters produced from mineral and spring waters consist of the addition
of flavours, juices and sugar or sweeteners that provide water with a particular taste and
aroma appreciated by consumers. Considering that flavours/aromas are fruit extracts, and
fruits are good sources of exogenous antioxidants, it is expected that the use of this fruit
extracts in beverages can introduce antioxidants to the water (Barroso et al., 2009, 2011).
Antioxidant defence mechanisms include the use of enzymes, vitamins, phenolic
compounds, minerals or proteins. Consequently, increasing intake of dietary antioxidants
may help maintain an adequate antioxidant status and, therefore, sustain normal
physiological functions of a living system. Antioxidants are very important in the
mammalian body because they have the ability to combat and reduce oxidative damage
caused by reactive oxygen species (ROS) (Halliwell et al., 1992). ROS are continuously
produced in all living beings as a result of normal cellular metabolism (Benherlal and
Arumughan, 2008).
The superoxide anion radical (O2• −) is the most abundant radical in biological systems
resulting from the univalent reduction of oxygen (Ge and Lisdat, 2002). This radical
species is enzymatically produced by xanthine oxidase (XOD). XOD is a mettalloenzyme
that catalyses the oxidation of hypoxanthine and xanthine to form O2• − that is generated
during the respiratory burst of phagocytic cells such as neutrophils (Gobi and Mizutani,
2000; Laranjinha, 2009).
Several analytical methods have been proposed for the quantification of the total
antioxidant capacity (TAC) in biological and food samples. These methodologies are
based on UV-vis spectrometry, chemiluminescence, fluorimetry, electrochemistry and
chromatography techniques (Sanchez-Moreno, 2002).
Recently, several electrochemical methods based on enzymatic biosensors have been
developed for the determination of superoxide radical and TAC. These biosensors are
based on the immobilization of Cytochrome c (this enzyme acts as an oxidant of
superoxide radical) or on the immobilization of the enzyme superoxide dismutase (SOD;
this enzyme has a protective scavenging function against the superoxide radical), on the
electrode surface (gold, platinium, glass, carbon paste or screen printed electrode (SPE),
SPE-Au) (Ge and Lisdat, 2002; Emregül, 2005). In this type of protein immobilised
biosensor, an electrochemical signal was found to be proportional to the superoxide
II. Investigação e desenvolvimento
152
concentration generated in aqueous solution by the xanthine and xanthine oxidase (Eq.
(1)).
(1) O 2H aciduric O OH xanthine -2
XOD 22
•+ ++ →++
For the immobilization of enzymes on an electrode surface, some strategies have been
demonstrated. The immobilization of the enzyme can be carried out via short-chain thiol
modified gold electrodes, long-chain thiol (mercaptoundecanoic acid), mixed-thiol, long-
chain mixed thiol (mercaptoundecanoic acid/mercaptoundecanol) and hemin modified
electrode (McNeil et al., 1995; Gobi and Mizutani, 2000; Ignatov et al., 2002). However,
the performance of many of these types of devices is interfered by hydrogen peroxide,
uric acid and some communication interference between the protein and the electrode
(Chen et al 2000; Beissenhirtz et al., 2004; Endo et al, 2002; Campanella et al 2004;
Emregül, 2005). The protective effect of antioxidants at a cellular level could only be
achieved by monitoring the DNA integrity (Barroso et al., 2011). For this purpose,
electrochemical DNA-based biosensors have been developed in order to assess the TAC
of foodstuff (Mello et al., 2006; Barroso et al., 2011). In many studies (Fojta et al., 2000;
Mello et al., 2006), the oxidative damage of double stranded DNA or of the nucleobases
(guanine or adenine) by the hydroxyl radical was evaluated. The oxidative damage
produces a significant decrease in the current intensity on the strand scission of DNA or
on the decreasing oxidation current after damage of the nucleobases (Liu et al., 2005;
2006; Mello et al., 2006; Qian et al., 2010). In this work, a DNA-sensor was used in order
to evaluate TAC in bottled flavoured waters. This DNA-biosensor consisted of
electrochemically deposited purine base (adenine or guanine) on a glassy carbon
electrode (GCE). All DNA bases (purine and pyrimidine) can be used for the
electrochemistry study. However, purine bases (adenine and guanine) are more sensitive
for detection and present lower potential peaks than the pyrimidine bases (+1.3 V for
thymine and +1.5 V for cytosine). Considering that purine bases have peaks more well-
defined and larger than those of the pyrimidines (Brett and Matysik, 1997), the purine
bases were used in this study. In experiments evaluating the oxidative damage of the
purine bases, the biosensor was firstly immersed in an aqueous superoxide radical
solution that was generated in the enzymatic reaction between XOD and xanthine (Eq.
(1)). Then, the decrease of the oxidation current of guanine and adenine recorded in
square wave voltammetry (SWV) was used to relate the extent of oxidative damage. The
influence/protection of five antioxidants, such as, ascorbic acid, gallic acid, caffeic acid,
reverastrol and p-coumaric acid was studied.
4.2. Biossensores de bases púricas - radical superóxido
153
2. Materials and methods
2.1. Chemicals
Guanine, adenine, xanthine oxidase (XOD, X1875) xanthine, gallic acid, resveratrol
were purchased from Sigma. Caffeic acid was from Fluka, L(+) ascorbic acid and
reveratrol was acquired from Riedeil-de-Haën. Other chemicals were Merck pro-analysis
grade and were used as received. Guanine stock solution (1 g L-1) was prepared by
dissolving an amount of this solid in 0.1 mol L-1 of NaOH and diluting in pH 7.4 phosphate
buffered saline (PBS). Stock solution of 1g L-1 of adenine was prepared in PBS pH 7.4
and stored at +4 ºC. For all voltammetric measurements, pH 4.8 PBS was used as the
supporting electrolyte. Superoxide radical was generated by adding XOD (0.0015 U mL-1)
to oxygen-satured PBS (pH 7.4) containing xanthine (10 µmol L-1). All solutions were
prepared with water purified with a Direct-Q (Millipore) system.
2.2. Instrumentation
SWV was performed with an Autolab PSTAT 10 potentiostat controlled by GPES
software (EcoChemie, The Netherlands). A conventional three electrode cell was used,
which includes a GCE (0.07 cm2) as working electrode, a glassy carbon counter electrode
and a Ag|AgCl|KClsat reference electrode to which all potentials were referred. The GCE
was mechanically polished using a polishing kit (Metrohm 6.2802.010) first with γ-Al2O3
(0.015 µm) until a shining surface was obtained and then rinsed with water. After this step
the GCE was treated by applying a fixed potential of +1.7 V for 30 s in PBS pH 4.8. This
initial conditioning step improves the resolution of the analytical signal because the
application of high potentials in acidic medium increases the hydrophilic properties of the
electrode surface through the introduction of oxygenated functionalities (Rice et al., 1983;
Mello et al., 2006).
2.3. Assay procedure
Unless otherwise mentioned, all experiments consisted of three steps: (i) Guanine or
adenine electro-immobilization on the GCE, (ii) damage of purine bases by the immersion
of DNA-GCE in the XOD/xanthine solution, and study of the effect of the presence of
antioxidants in the system, and (iii) detection and measurement of the peak current of
adenine or guanine in a PBS at pH 7.4.
II. Investigação e desenvolvimento
154
Purine bases (adenine or guanine) immobilization was performed by the application of
an adsorptive accumulation step. For that, the activated GCE was immersed in PBS pH
4.8 containing 10 mg L-1 of adenine or 3 mg L-1 of guanine and a potential of +0.4 V was
applied for 180 s. The electrode was next rinsed with water. A reported procedure
(Marrazza et al., 1999; Chiti et al., 2001; Mello et al., 2006) for cleaning and
immobilization step was adopted in this work. DNA damage was carried out by immersing
the biosensor in a freshly prepared XOD/xanthine mixture in the absence or in the
presence of antioxidant in PBS pH 7.4 for a fixed period of reaction time. Next, the
biosensor was immersed in pH 4.8 PBS. SWV was then conducted between +0.2 V and
+1.4 V and the oxidation peak current of guanine and adenine obtained was used as a
detection signal. For the electrochemical studies it was considered that the maximum
signal current obtained were for the purine base electrochemical signal without damage
neither antioxidant effect.
2.4. Samples
Thirty-nine water samples corresponding to 10 different brands were purchased in
several supermarkets in the North of Portugal and stored in the dark at +4 ºC. Each brand
(still or sparkling, mineral or spring water) had different flavours and aromas. The natural
water of each brand was also used as control. Sonication was used to eliminate gas from
the sparkling water samples. The labels on the water bottles indicate the nutrient
information, namely the presence of fruit juice, vitamins, sweeteners and preservatives
(Barroso et al., 2009).
Six liquid flavours used in the formulation of some water brands, provided by a
producer, were also analysed. The flavours used corresponded to different fruit aromas,
including lime, tangerine, strawberry, lemon, apple and gooseberry. These flavours had
no description about their chemical or aroma composition, but were known to be present
in the flavoured waters used in this study.
2.5. TAC measurement on beverages
The purine-based biosensor was applied to the determination of TAC on flavour and
flavoured waters. For the measurement of TAC in beverages, 100 µL of the flavoured
water or 5 µL of flavour were diluted in PBS to a final volume of 500 µL. Then, the DNA-
GCE was immersed in the solution and a freshly prepared superoxide radical was added.
After 120 s, the biosensor was rinsed and immersed in PBS buffer before SWV of guanine
and adenine was carried out.
4.2. Biossensores de bases púricas - radical superóxido
155
3. Results and discussion
The ease of oxidation of purine bases in DNA depends, predominantly, on the
secondary structure of the polynucleoside. Owing to the flexibility and better accessibility,
nucleobases in a ssDNA are readily oxidised than in a dsDNA, leading to a higher
oxidation current at an electrode surface (de-los-Santos-Álvarez et al., 2002). SWV was
used to observe the electrochemical response of the oxidation of guanine and adenine
immobilised on a GCE. Fig. 1 (curve a in (i) and (ii)) shows the anodic peak of guanine
and adenine bases. The less positive peak potential (+0.55 V) corresponds to the
oxidation of guanine, while the peak at more positive potential (+0.82 V) corresponds to
the electrooxidation of adenine. This results are in agreement with +0.55 V for guanine
and +0.82 V for adenine reported in the literature (Brett et al., 1994; Brett and Matysik,
1997), which focussed on the dependence of the oxidation peak of purine bases on pH,
buffer and ionic strength.
Fig. 1. SVW obtained in PBS pH 4.8: (i) guanine-biosensor and (ii) adenine-biosensor: after: (a) total oxidation of guanine and adenine signal (maximum peak current), (b) immersion of the biosensor in a superoxide radical solution and (c) immersion in superoxide radical solution with ascorbic acid.
Damage of DNA is the major endogenous type of pathogenesis that induces a variety of
diseases including cancer. ROS induced oxidative lesion in the DNA will cause
modifications at the DNA. Superoxide radical generated in situ by XOD can mediate the
direct strand scission of DNA and this can be attributed to hydrogen atom abstraction of
C5’ of the deoxyribose (Burrows and Muller, 1998). In order to verify if O2•- radicals
generated by xanthine/XOD reaction are able to damage purine base immobilized on the
GCE, the DNA-GCE was placed in a freshly prepared solution of xanthine/XOD in PBS pH
7.4 for 5 min. Next, the biosensor was rinsed with water and SWV at this biosensor was
repeated. A 61.4% and a 64.5% decrease in the anodic peak current (ip) of guanine and
adenine, respectively was observed after the biosensor was immersed on the superoxide
40.0
50.0
60.0
70.0
0.50 0.70 0.90 1.10 1.30
E (V) vs. AgCl/Ag
i / µ
A
a
b
c
40.0
50.0
60.0
70.0
0.50 0.70 0.90 1.10 1.30
E (V) vs. AgCl/Ag
i / µ
A
40.0
50.0
60.0
70.0
0.50 0.70 0.90 1.10 1.30
E (V) vs. AgCl/Ag
i / µ
A
a
b
c
10.0
20.0
30.0
40.0
50.0
0.10 0.30 0.50 0.70 0.90
E (V) vs. AgCl/Ag
i / µ
A
a
b
c
10.0
20.0
30.0
40.0
50.0
0.10 0.30 0.50 0.70 0.90
E (V) vs. AgCl/Ag
i / µ
A
10.0
20.0
30.0
40.0
50.0
0.10 0.30 0.50 0.70 0.90
10.0
20.0
30.0
40.0
50.0
0.10 0.30 0.50 0.70 0.90
E (V) vs. AgCl/Ag
i / µ
A
a
b
c
i) ii)
II. Investigação e desenvolvimento
156
radical solution (curve b in Fig. 1i and ii). This decrease in the peak current was used to
infer damage of the DNA bases after being oxidised by the O2• −radicals. According to the
literature (de-los-Santos-Álvarez et al., 2007; Freidman and Heller, 2004), guanine base is
the most easily oxidized of the nucleic acid base, yielding 8-oxoguanine (8-oxoG) and the
tautomer 8-hydroxyguanine. However, a common diimine structure was produced when
guanine and adenine were electrochemically oxidised at neutral or alkaline solution. As
shown by curve c in Fig. 1i and Fig. 1ii, when an antioxidant, in this case ascorbic acid,
was added to the superoxide radical solution a 43.86% and a 50.11% increase of ip of
guanine and adenine, respectively, compared to curve b of Fig. 1i and Fig. 1ii. Indeed, this
is indicative that the DNA was protected by the antioxidant presents in the solution.
Antioxidants are well-known to exhibit a protective effect with a scavenging effect of ROS
preventing DNA damage. Consequently, the number of lesions diminishes, yielding a
larger number of adenine and guanine for electrochemical oxidation (Barroso et al., 2011).
Indeed, ascorbic acid is considered a good scavenger of free radicals produced during the
metabolic pathways of detoxification. Ignatov et al. (2002) reported the development of a
methodology for the electrochemical detection of antioxidants based on a superoxide
radical measurement with a cytochrome c modified electrode. In this study the authors
have used several antioxidants such as ascorbic acid (standard antioxidant) and sub-
groups of the phenolic acid (flavanols, flavanones, isoflavones, flavones and flavonols).
The antioxidants used by these authors presented scavenger capacity of the superoxide
radical. Considering the good correlation between antioxidant concentration and the
protective effect on the DNA, an analytical procedure to evaluate TAC was developed.
3.1. Optimization of the experimental conditions
To measure the TAC of beverages, some parameters concerning the damage on the
purine base immobilized on the GCE (xanthine and XOD concentration, reaction time
between superoxide radical and the target molecule) were implemented in order to
achieve the maximum DNA effect, but without a complete damage (non-zero ip). XOD
concentration was studied between 0.0015 and 0.1 U mL-1. A range of 25%-66%
decrease in the ip of guanine and adenine was observed over the XOD concentration
studied. This is indicative of the effectiveness of XOD on the generation of the superoxide
radical. At an adenine-biosensor, a 62% decrease in ip was observed when the XOD
concentration was increased from 0.0015 U mL-1 to 0.07 U mL-1. At higher XOD
concentration, ip was observed to remains essentially unchanged. Considering that the
lowest XOD concentration was 0.0015 U mL-1, this XOD concentration was used for the
next optimisation steps for the adenine biosensor. Similar results were obtained with the
4.2. Biossensores de bases púricas - radical superóxido
157
guanine-biosensor. The increase of XOD concentration on the reactive system generates
high damage on the DNA as indicated by a decrease of 22 %-60 % in ip. At XOD
concentration higher than 0.008 U mL-1, ip was observed to remains similar, so this value
was used for the next experiments.
For both purine-based biosensors (guanine and adenine) xanthine concentration was
ranged between 10 and 800 µmol L-1. With the increase of xanthine concentration a
decrease between 57% and 66% in the ip of guanine and adenine was observed,
however, the decrease of the ip in all range of xanthine concentration studied was very
similar and remained essentially unchanged. Therefore, to be more cost effective, the
lowest xanthine concentration of 10 µmol L-1 was used in the next optimisation step.
Reaction time between the superoxide radical and the purine bases immobilized on the
GCE depends of the half-life time of the generated ROS, so this parameter is an important
feature to optimize. In this study the incubation time were ranged from 0 to 120 s. Fig. 2
shows the correlation between the damage on the purine base produced by the
superoxide radical (correlated with the ip values) and the incubation time. A more than
50% decrease in the ip was observed with an increase of the reaction time from 0 to 120 s.
However, there was no complete damage of DNA as indicated by the non-zero ip results
shown in the Fig. 2. The lower ip obtained at the adenine biosensor than the guanine
biosensor indicates more damage at the former. However, de-los-Santos Álvarez et al.
(2007) reported more damage of guanine than adenine at a pyrolic graphite electrode in a
neutral and alkaline aqueous solutions. The incubation time of 120 s was chosen for all
experiments.
Fig. 2. Influence on the peak current on the biosensor with the incubation time (a) 10 mg L−1 adenine base, (b)
3 mg L−1 guanine base.
a) adenine
b) guanine
0.0
5.0
10.0
15.0
0 20 40 60 80 100 120
t (sec)
i p/µ
A
a) adenine
b) guanine
0.0
5.0
10.0
15.0
0 20 40 60 80 100 120
t (sec)
i p/µ
A
II. Investigação e desenvolvimento
158
3.2. Determination of TAC
Foodstuff constitutes an excellent source of exogenous antioxidants to counteract the
alteration of lipids in cellular membranes, protein, enzymes, carbohydrates and DNA
promoted by ROS. Antioxidants, such as, ascorbic acid, and phenol-derived compounds
are natural components of fruits and beverages (tea and wine). For the evaluation of the
TAC of flavoured waters, five antioxidants including ascorbic acid, gallic acid, caffeic acid,
coumaric acid and resveratrol were used. Ascorbic acid is a water-soluble vitamin, is
considered a powerfull antioxidant and plays a key role in the protection against biological
oxidation processes participating in many metabolic reactions (Mello and Kubotta, 2007).
Gallic, caffeic and coumaric acid are phenolic acids with a large protective action.
Phenolic acids include several groups such as the hydroxybenzoic acid (gallic acid) and
the hydroxycinnamic acid (caffeic and coumaric acid). In general, the antioxidant activity
of the phenolic-derived compounds is determined by some properties, such as, free-
radical scanvengers (Thavasi, et al., 2006). Resveratrol is a polyphenolic natural product,
derived stilbene that exists in various foods and beverages, has attracted increasing
attention over the past decade because of its multiple beneficial properties, including
chemopreventive and antitumor activities (Fulda, 2010). Linearity studies between the five
antioxidants and ip of guanine and adenine oxidation were carried out. Fig. 3i and ii shows
the SWV of electrochemical current obtained after immersing the purine-biosensor on the
superoxide radical containing increasing concentration of ascorbic acid. As expected, the
oxidation current of guanine and adenine increased when the concentration of ascorbic
acid increased. Similar voltammograms were obtained when the other antioxidants
(resveratrol, gallic, caffeic and coumaric acid) were used with the both DNA-biosensors
(guanine and adenine).
Table 1 presents a summary of analytical parameters of the guanine and adenine
biosensors obtained after being immersed in the respective five antioxidants used. Among
them, ascorbic acid showed the widest linear range from 1.00 to 5.00 mg L-1 at guanine-
GCE and 0.50-4.00 mg L-1 at adenine-GCE. The other antioxidants presented a narrow
linear range, 0.10-1.00 mg L-1 of gallic acid or caffeic acid, 0.10-0.50 mg L-1 of resveratrol
and 0.50-1.00 mg L-1 of coumaric acid when the guanine biosensor was used. For the
adenine biosensor, the linear range was from 0.10 to 0.50 mg L-1 for the antioxidants
caffeic acid, coumaric acid and resveratrol, and from 0.50 to 0.90 mg L-1 for gallic acid.
RSD values were below 10% confirmed the high precision of the methods.
4.2. Biossensores de bases púricas - radical superóxido
159
Fig. 3. SWV obtained after immersion of (i) guanine-biosensor in superoxide radical containing a standard
solution of ascorbic acid: (a) 1.00, (b) 2.00, (c) 3.00, (d) 4.00 and (e) 5.00 mg L−1 and (ii) adenine-biosensor in
superoxide radical containing a standard solution of ascorbic acid: (a) 0.50, (b) 1.00, (c) 2.00, (d) 3.00 and (e)
4.00 mg L−1. Inset: relationship between ip and ascorbic acid concentration.
Table 1. Analytical feature obtained for the 5 antioxidants standards. Parameters Ascorbic acid Gallic acid Caffeic acid Coumaric acid Resveratrol
Guanine-GCE
Linear range (mg L-1) 1.00–5.00 0.10–1.00 0.1–1.00 0.50–1.00 0.10–0.50
Slope (A mg-1L) 1.05x10-6 5.38x10-6 5.23x10-6 7.33x10-6 1.27x10-5 Intercept (A) 4.11x10-6 4.66x10-6 4.25x10-6 2.09x10-6 1.92x10-6
Correlation coefficient (n=5) 0.990 0.980 0.987 0.993 0.998
RSD (%) (mg L-1 ) 3.43 (2.00) 2.36 (0.30) 2.96 (0.50) 1.05 (0.70) 3.86 (0.20)
LOD 0.77 0.10 0.10 0.08 0.06
Adenine-GCE Linear range (mg L-1) 0.50–4.00 0.50–0.90 0.10–0.50 0.10–0.50 0.10–0.50
Slope (A mg-1L) 5.02x10-7 9.40x10-6 1.30x10-5 6.49x10-6 1.11x10-5
Intercept (A) 4.26x10-6 8.00x10-8 1.74x10-6 2.99x10-6 3.02x10-6
Correlation coefficient (n=5) 0.985 0.993 0.995 0.998 0.994
RSD (%) 1.00 (2.00) 2.11 (0.70) 4.00 (0.30) 4.93 (0.20) 6.43 (0.20) LOD 0.50 0.06 0.05 0.02 0.10
Table 2 shows the TAC values expressed in mg L-1 of ascorbic acid, gallic acid, caffeic
acid, coumaric acid and resveratrol. All flavours and flavoured waters were observed to
show antioxidant capacity; except the natural waters. Flavours that showed the highest
TAC values are fruit extracts that contain several concentrated antioxidant compounds.
Using the adenine and guanine GCE the highest TAC values were found with the ascorbic
acid standard. At the adenine biosensor apple, fallowed by lemon, gooseberry strawberry,
tangerine and lime were the flavours that showed the highest TAC values. At the guanine-
10.0
20.0
30.0
40.0
50.0
0.30 0.40 0.50 0.60 0.70 0.80
E (V) AgCl/Ag
i/ µA
10.0
20.0
30.0
40.0
50.0
0.30 0.40 0.50 0.60 0.70 0.80
E (V) AgCl/Ag
i/ µA
i)
[AA] mg/L
I p/(µ
A
50.0
60.0
70.0
0.60 0.70 0.80 0.90 1.00 1.10 1.20
E (V) AgCl/Ag
i/ µ
A
i p/µ
A
a
e
ii)
[AA] mg/L
a
e
ii)
[AA] mg/L
a
e
4.0
6.0
8.0
10.0
1.0 2.0 3.0 4.0 5.04.0
6.0
8.0
10.0
1.0 2.0 3.0 4.0 5.0
4.0
5.0
6.0
7.0
0.0 1.0 2.0 3.0 4.04.0
5.0
6.0
7.0
0.0 1.0 2.0 3.0 4.0
II. Investigação e desenvolvimento
160
biosensor tangerine showed the highest TAC value, fallowed by strawberry, apple, lime,
lemon and gooseberry.
When the adenine-biosensor was applied to the analysis of flavoured waters, brand G
showed the highest TAC values (sample 27 and 30), maybe because this brand had in its
composition vitamin C (sample 28 has no vitamin and the TAC value was lower than the
other samples from the same brand). Brand A also presented higher TAC values and the
other commercial brands presented TAC values ranging between 0.33 mg L-1 and 7.31
mg L-1 with the standard ascorbic acid. Using the antioxidant ascorbic acid the lowest TAC
value was obtained from the Brand D (sample 12-14) and sample 35. Analysing TAC
results obtained using the water brands (brand A, B, C, D, E, F and I) it was verified that
the TAC values obtained within the same brand were similar, hence, the Adenine-GCE
might not discriminate the different flavours present in same brand. Using the gallic acid
standard the TAC values ranged from 37 to 57 mg L-1 for the flavours and 0.34-3.37 mg L-1
for the flavoured waters. The lowest TAC values were obtained in brand D (samples 12-
14) and the highest TAC contents were from brand A (samples 1-3). With the caffeic acid
antioxidant the TAC ranged from 13 to 27 mg L-1 and 0.72-1.74 mg L-1 in flavours and
flavoured waters respectively. Similar results were obtained with the other standard
antioxidants, coumaric acid and resveratrol. TAC values obtained with the ascorbic acid
were larger than the other four antioxidants (gallic acid, caffeic acid, coumaric acid and
resveratrol) that presented a narrow TAC levels. Theses differences obtained between the
ascorbic acid and the other antioxidants can be elucidated because the ascorbic acid is a
powerful antioxidant and in this study presented a larger linear range.
A similar behaviour was observed with the guanine-GCE and using the standard
ascorbic acid, brand G (samples 27) presented also the highest TAC values fallowed by
brand F, brand A and brand H. TAC values ranged between 0.68 and 18.7 mg L-1
equivalents of ascorbic acid. It was verified that TAC results obtained within the same
brand were similar (analogous to that at the adenine biosensor) with the exception of
brand C. Considering that sample 9 (from brand C) had two added ingredient; apple and
white tea a higher TAC value was expected compared with the other samples of brand C.
For other antioxidants, the TAC values ranged from 0.34 mg L-1 to 3.15 mg L-1 and 0.41
mg L-1-3.20 mg L-1 or between 0.01 mg L-1 and 4.71 mg L-1 and from 0.33 mg L-1 to 2.19
mg L-1 for the gallic acid, caffeic acid, coumaric acid and resveratrol, respectively. Larger
TAC values were obtained with the ascorbic acid antioxidant and the other four
antioxidants presented a narrow TAC range, a similar behaviour was obtained with the
adenine-GCE.
16
1
Tab
le 2
. T
AC
val
ues
obta
ined
for
the
flavo
urs
and
flavo
ured
wat
ers
usin
g a
guan
ine-
GC
E a
nd a
deni
ne-G
CE
(m
g L-1
) A
deni
ne-G
CE
G
uani
ne-G
CE
Bra
nd
Sam
ple
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Fla
vour
Le
mon
16
9.52
± 1
1.20
55
.22
± 3.
90
25.9
3 ±
2.82
32
.68
± 5.
64
25.
53 ±
2.9
3
93.1
4 ±
19.9
3 20
.96
± 3.
89
16.0
2 ±
4.00
47
.36
± 3.
31
17.0
1 ±
1.89
T
ange
rine
131.
6 ±
1.16
39
.14
± 0.
06
14.3
0 ±
0.04
9.
39 ±
0.0
9
13.4
3 ±
0.05
22
0 ±
26.4
7 32
.71
± 9.
07
41.4
9 ±
9.33
68
.40
± 7.
71
29.0
1 ±
4.40
A
pple
20
2.69
± 5
5.08
56
.99
± 2.
94
27.2
1 ±
2.13
35
.25
± 4.
26
26.8
6 ±
2.21
17
7.05
± 3
.10
24.3
3 ±
0.60
32
.87
± 0.
62
61.2
8 ±
0.51
24
.95
± 0.
29
S
traw
berr
y 16
3.75
± 4
.23
37.4
3 ±
3.43
13
.06
± 2.
48
6.90
± 0
.97
12.1
4 ±
2.58
18
6 ±
29.0
3 9.
86 ±
1.6
6 2.
30 ±
5.8
3
32.2
2 ±
4.81
8.
37 ±
2.7
5
G
oose
berr
y 16
9.42
± 5
9.30
55
.22
± 3.
17
25.9
3 ±
2.29
32
.67
± 4.
59
25.5
2 ±
2.38
74
.81
± 7.
18
4.38
± 0
.89
12
.34
± 1.
29
44.3
2 ±
1.81
15
.27
± 6.
73
Li
me
126.
00 ±
6.7
7 39
.44
± 4.
10
14.5
2 ±
2.96
9.
82 ±
0.3
2 13
.66
±3.0
8 13
3.52
± 3
4.21
15
.84
± 2.
68
24.1
3 ±
2.8
7 54
.06
± 5.
67
20.8
3 ±
3.24
A
1 Le
mon
13
.91
± 2.
78
3.23
± 0
.11
1.64
± 0
.08
2.32
± 0
.16
1.63
± 0
.08
15.0
5 ±
2.50
2.
43 ±
0.0
9 2.
13 ±
0.5
7 4.
09 ±
0.4
1 1.
83 ±
0.2
4
2 M
ango
13
.31
± 4.
43
3.16
± 0
.04
1.59
± 0
.03
2.
21 ±
0.5
1.
58 ±
0.0
3 9.
98 ±
5.3
5 1.
44 ±
0.0
4 0.
88 ±
0.3
2 3.
25 ±
0.8
9 1.
35 ±
0.5
1
3 S
traw
berr
y 16
.08
± 2.
06
3.33
± 0
.12
1.71
± 0
.09
2.4
6 ±
0.18
1.
71 ±
0.0
9 6.
58 ±
1.7
8 0.
77 ±
0.0
5 0.
62 ±
0.0
4 2.
69 ±
0.3
0 1.
03 ±
0.1
7
4 N
atur
al
- -
- -
- -
- -
0.01
± 0
.04
-
B
5 P
inea
pple
/ora
nge
1.03
± 0
.03
2.53
± 0
.20
1.13
± 0
.14
1.30
± 0
.28
1.10
± 0
.15
8.00
± 0
.48
1.05
± 0
.09
0.
94 ±
0.0
6 2.
92 ±
0.0
8 1.
17 ±
0.0
5
6
Lem
on
1.29
± 0
.06
2.39
± 0
.08
1.03
± 0
.06
1.09
± 0
.11
1.00
± 0
.06
4.39
± 0
.13
0.34
± 0
.03
0.45
± 0
.05
2.32
± 0
.02
0.82
± 0
.01
7
Nat
ural
-
- -
- -
- -
- 0.
05 ±
0.0
4 -
C
8 Le
mon
/Mag
nesi
um
4.22
± 0
.26
2.38
± 0
.06
1.02
± 0
.05
1.07
± 0
.09
0.99
± 0
.05
5.80
± 2
.02
0.48
± 0
.03
3.20
± 0
.27
1.
62 ±
0.3
4 0.
42 ±
0.0
9
9
App
le/w
hite
tea
3.69
± 0
.99
2.52
± 0
.25
1.12
± 0
.18
1.28
± 0
.36
1.09
± 0
.19
14.6
9 ±
5.21
2.
36 ±
0.0
2
2.54
± 1
.4
4.03
± 0
.86
1.80
± 0
.05
10
Pin
eapp
le/fi
bre
1.70
± 0
.25
2.45
± 0
.40
1.07
± 0
.29
1.18
± 0
.57
1.04
± 0
.30
4.86
± 0
.53
0.44
± 0
.10
1.
54 ±
0.5
3 2.
40 ±
0.0
9 0.
87 ±
0.0
5
11
Nat
ural
-
- -
- -
- -
0.
05 ±
0.0
3 -
D
12 A
pple
0.
33 ±
0.0
7 0.
77 ±
0.5
2 1.
30 ±
0.3
8 1.
65 ±
0.7
6 1.
28 ±
0.3
9 0.
95 ±
0.0
6 0.
67 ±
0.0
2 0.
70 ±
0.0
3 0.
60 ±
0.0
4 0.
15 ±
0.0
3
13
Ora
nge/
peac
h 0.
82 ±
0.0
4 0.
38 ±
0.2
1 -
1.09
± 0
.31
0.99
± 0
.16
0.68
± 0
.03
0.38
± 0
.07
0.41
± 0
.05
0.80
± 0
.02
0.33
± 0
.05
14
Lem
on
0.48
± 0
.09
0.34
± 0
.11
0.99
± 0
.08
1.02
± 0
.15
0.96
± 0
.08
0.81
± 0
.06
0.62
± 0
.09
0.60
± 0
.51
0.75
± 0
.08
0.96
± 0
.04
15
Nat
ural
-
- -
- -
- -
- -
-
E
16 L
emon
6.
12 ±
0.0
4 1.
96 ±
0.4
5 0.
72 ±
0.3
3 0.
47 ±
0.6
5 0.
67 ±
0.3
4 10
.71
± 0.
61
1.58
± 0
.09
1.76
± 0
.48
3.37
±0.
93
1.42
± 0
.53
17
Ora
nge/
rasp
berr
y 7.
31 ±
0.9
5 2.
68 ±
0.6
6 1.
24 ±
0.4
8 1.
52 ±
0.0
6 1.
22 ±
0.5
0 14
.09
± 2.
63
2.24
± 0
.51
1.91
± 0
.58
3.93
± 0
.44
1.74
± 0
.25
18
Pea
ch/p
inea
pple
6.
55 ±
0.7
7 2.
72 ±
0.2
8 1.
26 ±
0.2
0 1.
57 ±
0.2
1 1.
24 ±
0.2
1 14
.20
± 3.
42
2.26
± 0
.27
2.34
± 0
.22
3.95
± 0
.57
1.75
± 0
.32
19
Gua
va/li
me
6.56
± 0
.57
2.45
± 0
.12
1.07
± 0
.09
1.1
8 ±
0.18
1.
04 ±
0.0
9 6.
11±
0.40
1.
70 ±
0.0
3 1.
07 ±
0.7
9
1.58
± 3
.16
1.17
± 0
.04
20
Nat
ural
-
- -
- -
- -
- -
-
F
21 L
emon
/gre
en te
a 4.
14 ±
0.2
1 2.
28 ±
0.0
2 0.
95 ±
0.0
1 0.
94 ±
0.0
3 0.
91 ±
0.0
1 11
.10
± 1.
21
1.66
± 0
.21
1.82
± 0
.44
3.44
± 0
.86
1.46
± 0
.04
22
Ras
pber
ry/g
inse
ng
3.58
± 0
.26
2.39
± 0
.12
1.03
± 0
.09
1.10
± 0
.18
1.00
± 0
.09
12.0
1 ±
0.24
1.
83 ±
0.0
5 1.
67 ±
0.8
1 3.
59 ±
0.0
4 1.
55 ±
0.0
2
23
Pea
ch/w
hite
tea
2.66
± 0
.18
2.03
± 0
.09
0.77
± 0.
06
0.58
± 0
.13
0.73
± 0
.07
12.4
2 ±
0.47
1.
91 ±
0.4
2
1.61
± 0
.63
3.66
± 0
.36
1.58
± 0
.21
24
Man
go/g
inkg
o be
loba
1.
62 ±
0.3
0 2.
22 ±
0.1
2 0.
91 ±
0.0
9 0.
85 ±
0.1
8 1.
87 ±
0.0
9 15
.92
± 1.
51
2.60
± 0
.29
2.40
± 0
.64
4.24
± 0
.25
1.92
± 0
.04
25
Mel
on/m
int
2.10
± 0
.05
2.19
± 0
.15
0.88
± 0
.11
0.8
0 ±
0.21
1.
84 ±
0.1
1
8.17
± 1
.39
1.08
± 0
.06
1.2
6 ±
0.02
2.
95 ±
0.7
3 1.
18 ±
0.0
2
16
2
Ade
nine
-GC
E
Gua
nine
-GC
E
Bra
nd
Sam
ple
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
26
Nat
ural
-
- -
- -
- -
- -
-
G
27 L
emon
19
.28
± 1.
44
3.37
± 0
.24
1.74
± 0
.18
2.52
± 0
.35
1.74
± 0
.18
18.7
7 ±
1.61
3.
15 ±
0.3
1 3.
29 ±
0.8
1 4.
71 ±
0.2
7 2.
19 ±
0.o
5
28
Lim
e 5.
01 ±
0.2
0 2.
76 ±
0.0
8 1.
29 ±
0.0
6 1.
63 ±
0.1
2 1.
27 ±
0.0
6 10
.92
± 1.
41
1.62
± 0
.28
1.38
± 0
.68
3.41
± 0
.23
1.44
± 0
.13
29
App
le
11.2
3 ±
0.96
3.
17 ±
0.2
6
1.59
± 0
.19
2.22
± 0
.38
1.58
± 0
.20
15.9
8 ±
0.88
2.
61 ±
0.1
7 2.
46 ±
0.7
0 4.
25 ±
0.1
5 1.
92 ±
0.0
8
30
Pea
ch
21.2
5 ±
0.96
2.
45 ±
0.1
3 1.
07 ±
0.0
9 1.
18 ±
0.1
9 1.
04 ±
0.1
0 14
.87
± 0.
61
0.44
± 0
.51
0.67
± 0
.77
2.40
± 0
.43
0.87
± 0
.01
31
Nat
ural
-
- -
- -
- -
- -
0.04
± 0
.02
H
32 L
emon
4.
03 ±
0.4
0 2.
48 ±
0.4
4 1.
09 ±
0.3
2 1.
23 ±
0.6
3 1.
06 ±
0.3
3 8.
92 ±
0.7
5 1.
23 ±
0.5
4 0.
92 ±
0.4
9 3.
08 ±
0.4
6 1.
25 ±
0.0
6
33
Nat
ural
-
- -
- -
- -
- -
-
I 34
Lem
on
3.91
± 0
.73
2.55
± 0
.20
1.15
± 0
.14
1.33
± 0
.29
1.12
± 0
.15
9.48
± 0
.91
1.34
± 0
.18
1.
16 ±
0.0
9 3.
17 ±
0.1
5 1.
31 ±
0.0
9
35
Gre
en A
pple
0.
37 ±
0.2
1 2.
61 ±
0.3
9 1.
19 ±
0.2
8 1.
42 ±
0.5
6 1.
16 ±
0.2
9 14
.48
± 0.
57
2.31
± 0
.11
2.15
± 0
.07
4.00
± 0
.09
1.78
± 0
.05
36
Str
awbe
rry
1.25
± 0
.05
2.05
± 0
.11
0.78
± 0
.08
0.6
1 ±
0.16
0.
74 ±
0.0
8 15
.19
± 1.
09
2.45
± 0
.21
2.4
2 ±
0.25
4.
11 ±
0.1
8 1.
85 ±
0.1
0
37
Nat
ural
-
- -
- -
- -
- -
-
J 38
Lem
on
2.10
± 0
.21
2.06
± 0
.03
0.
79 ±
0.0
2 0.
62 ±
0.0
4 0.
75 ±
0.0
2 10
.49
± 0.
23
1.54
± 0
.04
1.41
± 0
.05
3.34
± 0
.04
1.40
± 0
.02
39
Nat
ural
-
- -
-
-
-
-
4.2. Biossensores de bases púricas - radical superóxido
163
By analysing the results in Table 2, the applications of adenine and guanine-immobilised
GCEs to the evaluation of TAC in beverages were demonstrated. Standards off all
antioxidants were available for use in the TAC determination in this study. Among them,
we recommend ascorbic acid should be used as a common standard in the determination
of TAC of foodstuff and beverages as it exhibited the widest linear calibration range at
both the guanine and adenine biosensors.
4. Conclusion
Adenine and guanine-immobilised GCEs for the evaluation of TAC in beverages was
developed. The methodology is based on the interaction of adenine or guanine with the
superoxide radical generated by the xanthine/xanthine oxidase system. Five standard
antioxidants (ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol) were
used in order to protect adenine and guanine base. Ascorbic acid presented the highest
TAC values and seems to be the most sensitive standard capable to discriminate the
several ingredients added to the waters.
The biosensors described in this study have some advantages over the conventional
methodologies such as a shorter detection time, a smaller sample volume, higher
accuracy and a high simplicity. In addition, coloured samples can be directly used for the
measurement without pretreatment. The use of these biosensors is closer to biological
systems, with a nucleotide being damaged by free radical.
Acknowledgements
M. Fátima Barroso is grateful to Fundação para a Ciência e a Tecnologia for a Ph.D.
grant (SFRH/BD/ 29440/2006). The authors thank Frize for providing flavours samples.
II. Investigação e desenvolvimento
164
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.
4.3. Biossensores de bases púricas - radical sulfato
167
Evaluation of total antioxidant capacity of flavour ed waters using
sulfate radical damage of purine-based sensors
M.F. Barrosoa,b, C. Delerue-Matosa,, M. B. P. P. Oliveirab aREQUIMTE/Instituto Superior de Engenharia do Porto R. Dr. Bernardino de Almeida 431,
4200-072 Porto. Portugal bRequimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto.
R. Aníbal Cunha n.º 164, 4050-047 Porto. Portugal
Abstract
In this study, a method for the electrochemical quantification of the total antioxidant
capacity (TAC) in beverages was developed. This method is based on the oxidative
damage of the purine bases, adenine or guanine, immobilized on glassy carbon electrode
(GCE) surface. The oxidative lesions on the DNA bases were promoted by the sulfate
radical generated by the persulfate/iron (II) system. The presence of antioxidants on the
reactive system promoted the protection of the DNA bases immobilized on the GCE by
scavenging the sulfate radical. Square wave voltammatry (SWV) was the electrochemical
technique used to perform this study. Five antioxidants (ascorbic acid, gallic acid, caffeic
acid, coumaric acid and resveratrol) were used to study its efficiencies on the scavenger
sulfate radical and consequently protect the purine bases immobilized on the GCE. The
results demonstrated that the purine-based biosensor is suitable for rapid assessment of
TAC in flavours and flavoured waters.
Keywords: Purine-based biosensor, Total antioxidant capacity (TAC), Ascorbic acid,
Phhenolic acids, Sulfate radical (SO4• −).
Available online at www.sciencedirect.com
Electrochimica Acta submitted
4.3. Biossensores de bases púricas - radical sulfato
169
1. Introduction
Free radical generation is directly related with oxidation in food and biological systems. It
is well know that free radicals have the facility to promote alterations on the DNA inducing
oxidative damage and causing several diseases in humans.
Sulfate radical (SO4•–) is a free radical that can also damage DNA. Some studies indicate
that sulfate radical can interact with the nucleic acids producing 8-oxoGuo and induce
modifications on the DNA, such as strand breaks and DNA-protein cross-link. Guanine
base would be the preferential target to suffer these oxidative lesions due to its low
oxidation potential and to its ability to bind to transition metal ions that can catalyze
oxidative processes [1-3]. Some authors reported the oxidation and damage on the
adenine and adenosine produced by the sulfate radical generated by photooxidation of
peroxydisulfate and quantified by UV-Vis method [4,5], while Luke and collaborators [6]
indicated the deprotonation of the nitrogen at acidic medium from the 6-methyl uracil and
5,6-dimethyl uracil induced by the referred radical.
Sulfate radical can be generated by several ways, such as via scission of peroxide bond
by radiolytic, photolytic or thermal activation of the persulfate anion [7,8], or formed via
electron transfer by transition-metal activation of persulfate and via transition-metal
catalysis [9,10]. To counteract and prevent the deleterious effect of free radicals the living
organisms have developed complex endogenous and exogenous antioxidant systems.
The exogenous antioxidant system can be provided by functional foods, vegetables, fruit,
and beverages. These matrices are rich in several antioxidants, like, vitamins (A, E, C, β-
carotene), phenolic compounds, minerals (selenium, zinc) or proteins [11].
Several methods have been proposed for the quantification of total antioxidant capacity
(TAC) in food and biological system. These methodologies are based in photometric,
fluorimetric and chromatographic techniques [12]. Recently, several electrochemical
devices have been developed in order to measure the antioxidants in several types of
matrixes [13-15], however the protective effect of antioxidants at a cellular level could only
be achieved by monitoring the DNA integrity [16]. For this purpose, several
electrochemical DNA-based biosensors have been developed. As far as we know, all the
methodologies cited in the literature reported only the use of the hydroxyl radical
generated by the Fenton system or by the UV irradiation for the evaluation of the oxidative
damage on double stranded of DNA or on the nucleobases (guanine or adenine). In these
works the protective effect on the nucleic acid, produced by the free radical scavenger,
such as vitamins and phenolic acids were also studied [17-22].
In this paper, a purine-based biosensor had been developed in order to assess TAC in
flavoured waters. This biosensor consisted in the electro-deposition of a purine base
II. Investigação e desenvolvimento
170
(adenine or guanine) on a glassy carbon electrode (GCE). To evaluate the oxidative
lesions in the DNA bases, and for the first time, it was used the sulfate radical generated
by the persulfate activated with Fe2+ Eq. (1). For that the purine-based biosensor was
immersed in a freshly solution of sulfate radical for a fixed time period. Depending on the
pH values, the sulfate radical can interconvert in hydroxyl radical according to the
following emical reactions Eq. (2) and Eq. (3). It should be noted that in a general
persulfate oxidation system (acid condition, low [OH-] concentration) reactions of sulfate
radical with water and OH- can be negligible [23,24].
Fe2+ + S2O82- → Fe3+ + SO4
2- + SO4•– (1)
SO4•– + H2O → HO• + HSO4
- (2)
SO4•– + HO- → HO• + SO4
2- (3)
The ability of some antioxidants to scavenge oxidizing free radicals and protect the
integrity of purine bases has also studied. The antioxidants used were, ascorbic acid, and
phenolic acids, such as the hydroxybenzoic acid (gallic acid), hydroxycinnamic acid
(caffeic acid and coumaric acid) and the stilbene (resveratrol). All the electrochemical
studies were performed using the square wave voltammetry (SWV) technique. The
increase of the oxidative current of the purine-based biosensor recorded in SWV observed
when it was added to the reactive system antioxidants allows to the development of a
rapid and alternative electrochemical method to evaluate TAC in beverages, namely
flavoured waters.
2. Experimental
2.1. Chemical reagents
Guanine, adenine, iron (II) sulfate heptahydrate, potassium persulfate, gallic acid, were
purchased from Sigma. Caffeic acid was from Fluka, L(+) ascorbic acid and resveratrol
was acquired from (Riedeil-de-Haën). Other chemicals were Merck pro-analysis grade
and were used as received. Guanine stock solution (1 g L-1) was prepared by dissolving
an amount of this solid in 0.1 mol L-1 of NaOH and diluting it in phosphate buffer (PBS 0.2
mol L-1) at pH 7.4. Stock solution of 1 g L-1 of adenine were prepared in PBS pH 7.4 and
stored at 4ºC.
Working standard solution (ascorbic acid, gallic acid, caffeic acid, coumaric acid) were
prepared daily and immediately before measurements by dissolving an amount of the
solid standard in water until the desired concentration. In order to dissolve the resveratrol
4.3. Biossensores de bases púricas - radical sulfato
171
antioxidant, an amount of this compound was dissolved in ethanol and then diluted with
water until to the desired concentration.
Considering that free radicals have a very short-lived time, the sulfate radical generation
was prepared immediately before each assay by mixing Fe2+:EDTA: S2O82- (1x10-5: 2x10-5:
2.0x10-5 mol L-1). According Liang et al. [25] if the Fe2+ is in excess (Eq. 4) can destroy the
radical and produce the sulfate ions, so, in this study it was used a molar excess of
persulfate anions. All solutions were prepared with water purified with a Direct-Q
(Millipore) system.
Fe2+ + SO4•– → Fe3+ + SO4
2- (4)
2.2. Apparatus
Square wave voltammetry (SWV) was performed with an Autolab PGSTAT 10
potentiostat controlled by GPES software (EcoChemie, The Netherlands). A conventional
three electrode cell was used, which includes glassy carbon electrode (GCE) (0.07 cm2)
as working electrode, a glassy carbon counter electrode and a Ag|AgCl|KClsat reference
electrode to which all potentials are referred. GCE was mechanically polished using a
polishing kit (Metrohm 6.2802.010) first with γ-Al2O3 (0.015 µm) until a shining surface
was obtained and after with only water. After this step the GCE was treated by applying a
fixed potential of +1.7 V for 30 s in PBS pH 4.8. After this treatment a thin blue film can be
observed on the activated GCE surface. This procedure was made after each experiment.
This initial conditioning step improves the resolution of the analytical signal because the
application of high potentials in acidic medium increases the hydrophilic properties of the
electrode surface through the introduction of oxygenated functionalities [18,26].
2.3. Assay procedure
Unless otherwise mentioned, most experiments consisted of three steps: i) Guanine or
adenine electro-immobilization on the GCE, ii) damage of DNA bases by the immersion of
purine-GCE on the sulfate radical solution, and study of the effect of the presence of
antioxidants in the system; iii) detection and measurement of the peak height of adenine
or guanine in a PBS at pH 4.8.
Purine bases (adenine or guanine) immobilization was performed by the application of
an adsorptive accumulation step. For that, the activated GCE was immersed in 0.2 mol L-1
PBS pH 4.8 containing 10 mg L-1 of adenine or 3 mg L-1 of guanine and it was applied a
positive potential of +0.4 V for 180 s on permanent stirring, after this the electrode was
II. Investigação e desenvolvimento
172
washed with water. When this procedure was made the purine base was subsequently
immobilized onto activated GCE surface by adsorptive accumulation (controlled-potential
at +0.4 V) involving the application of positive electrode potential to achieve electrostatic
binding of negatively charged purine base. The purine-based biosensor (cleaning and
immobilization step) and the general analytical procedure (buffer composition, pH, ionic
strength) were optimized in previous works [18,27,28].
Purine base damage was carried out by immersing the biosensor in a freshly prepared
sulfate radical in the absence or the presence of antioxidant in PBS pH 4.8 for a fixed
period of reaction time. Damage to purine base layer was made through diffusion of the
radicals to the surface of the transducer. To study the effect of the antioxidant on the free-
radical scavenging, the standard antioxidant (ascorbic acid, caffeic acid, gallic acid,
coumaric acid and resveratrol) were added to the cleavage mixture. Protective effects of
antioxidants in flavours and flavoured waters were done by replacing the standard
antioxidant by samples.
For detection, the damaged purine-based biosensor was immersed in PBS solution pH
4.8. The peak height of the purine base was obtained by sweeping the potential between
+0.2 V and +1.4 V using the SWV technique with a frequency of 50 Hz, step potential of
4.12 mV and amplitude of 0.09 V. For these electrochemical studies it was considered
that the maximum intensity current obtained was for the purine base signal without
damage neither antioxidant effect.
2.4. Samples
Thirty-nine water samples corresponding to 10 different brands were purchased in
several supermarkets in the North of Portugal and stored in the dark at +4ºC. Each brand
(still or sparkling, mineral or spring water) had different flavours and aromas. The natural
water of each brand was also used as control. Sonication was used to eliminate gas from
the sparkling water samples. The labels on the water bottles indicate the nutrient
information, namely the presence of fruit juice, vitamins, sweeteners and preservatives
[29,30].
Six flavours in liquid state used in the formulation of some water brands, provided by a
producer, were also analysed. The flavours used corresponding to different fruit aromas,
such as lime, tangerine, strawberry, lemon, apple and gooseberry. These flavours had no
description about their chemical or aroma composition, only knowing that they are present
in the flavoured waters used in this study.
4.3. Biossensores de bases púricas - radical sulfato
173
2.5. TAC measurement in flavoured waters
The purine-based biosensor was applied to the determination of TAC on flavour and
flavoured waters. For the measurement of TAC in beverages, 100 µL of the flavoured
water or 5 µL of flavour were diluted in PBS to a final volume of 500 µL. Then, the purine-
GCE was immersed in the solution and a freshly prepared sulfate radical was added for
120 s and 60 s for guanine-GCE and adenine-GCE respectively. After this period of time
the biosensor was washed and immersed in PBS buffer pH 4.8 to measured the oxidation
current of guanine and adenine. Ascorbic acid, gallic acid, caffeic acid, coumaric acid and
resveratrol were the working standard antioxidants used, to study the protective effect
made by the antioxidant on the free-radical scavenging, and to carry out the linear
calibrations studies.
3. Results and discussion
To develop the purine-based biosensor for the quantification of TAC in beverages, the
first important analytical parameter optimized was the concentration of the purine base
immobilized on the GCE. As can be seen in Fig. 1, a saturation behaviour on the
electrochemical signal of the biosensor was observed for a concentration higher than 3
mg L-1 and 10 mg L-1 for guanine and adenine, respectively. Considering that with these
concentrations it was found the better analytical condition for the immobilization of the
purine bases on the GCE these concentration were chosen for further experiments.
In order to verify if the sulfate radical generated by the persulfate/iron (II) system have
the ability to induce oxidative lesion on the purine base (guanine and adenine)
immobilized on the GCE, the purine-based biosensor was placed in a freshly prepared
SO4•− in PBS pH 4.8 for 1 minute. After this process the purine-based biosensor was
washed with water and a SWV was made from +0.2 to +1.4 V. It was observed that the
sulfate radical induce damage on the purine bases, indeed when the purine bases
immobilized on the GCE interact with the sulfate radical a decrease on the oxidation peak
of guanine and adenine (Fig. 2) was observed.
Fig. 2 shows the performance of the purine-based biosensor in presence of the sulfate
radical and of ascorbic acid antioxidant on radical scavenging. Sulfate radicals had the
ability to produce 63% and 61% of damage in guanine and adenine base, respectively.
When ascorbic acid was added to the reactive system an increase of the anodic current of
guanine and adenine was registered using SWV and a protective effect of 69% and 73%
was observed in guanine-GCE and adenine-GCE, respectively. These results confirm the
ability of the antioxidant to deactivate the sulfate radical and consequently protect the
II. Investigação e desenvolvimento
174
purine bases from the oxidative damage. To perform this electrochemical study all current
peaks were compared with the signal current obtained with the non damaged adenine and
guanine bases (blank signal).
Fig. 1. Square wave voltammogram of a) guanine (3 mg L-1) and b) adenine (10 mg L-1); Influence of the
concentration of the purine base immobilized on the electrochemical current: a) guanine; b) adenine.
Conditions: potential range from to, frequency = 50 Hz, step potential= 4.12 mV and amplitude= 0.09 V.
20.0
30.0
40.0
50.0
60.0
0.20 0.40 0.60 0.80
E(V) vs. AgCl/Ag
i/ µA
20.0
30.0
40.0
50.0
60.0
0.20 0.40 0.60 0.8020.0
30.0
40.0
50.0
60.0
0.20 0.40 0.60 0.8020.0
30.0
40.0
50.0
60.0
0.20 0.40 0.60 0.80
E(V) vs. AgCl/Ag
i/ µA
45.0
50.0
55.0
60.0
65.0
70.0
0.60 0.80 1.00 1.20
E(V) vs. AgCl/Ag
i/ µA
45.0
50.0
55.0
60.0
65.0
70.0
0.60 0.80 1.00 1.20
E(V) vs. AgCl/Ag
i/ µA
4.0
8.0
12.0
16.0
20.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0
Purine base concentration / mg L -1
i p/ µ
A
a)b)
4.0
8.0
12.0
16.0
20.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0
Purine base concentration / mg L -1
i p/ µ
A
a)b)
guanine a)
adenine b)
4.3. Biossensores de bases púricas - radical sulfato
175
Fig. 2. Signal of the immobilized purine base on the GCE: blank purine base signal (guanine 3 mg L-1 and
adenine 10 mg L-1); after immersion in a sulfate radical (Fe2+ 2.0x10-5 mol L-1; S2O82- 4.0x10-5 mol L-1 for 120
s); immersion in sulfate radical with ascorbic acid antioxidant (4.0 mg L-1).
Purine bases of DNA measured in SWV presented two oxidation peaks at around +0.55
V and +0.82 V corresponding, respectively, to guanine and adenine oxidation peak (Fig.
1). With the analysis of the undamaged purin a maximum current peak was registered
because all adenine and guanine were available for electro-oxidation. But when the
biosensor interact with the sulfate radical, the radical attacks the purine base and promote
the oxidative damage of some amount of the guanine or adenine. So, after this process
the peak current decrease because only the undamaged DNA bases were available for
electro-oxidation in voltammetric techniques. With the addition of antioxidants to the
reactive system the number of lesions diminishes yielding a larger number of adenine or
guanine available for electrochemical oxidation. So, an increase of the peak current
occurs confirming the protective effect on the purine bases made by the antioxidants.
Swaraga and colaborators [4,5] reported the photooxidation of adenosine and adenine
induced by the sulfate radical generated by the photolysis of peroxydisulfate and the
protection of this DNA bases promoted by the antioxidant caffeic acid. Scheme 1 shows a
protective mechanism promoted by caffeic acid on the DNA bases against the sulfate
radical. In this purposed mechanism [5] caffeic acid act as sensitizers and as an efficient
scanvenger of SO4•− by receiving the free electron from the sulfate radical. Considering
the good correlation between antioxidant concentration and the protective effect on the
purine bases of DNA, an analytical procedure to evaluate TAC was developed.
guanine
guanine
guanine
adenine
adenine
adenine
0
25
50
75
100
125
blank sulphate radical ascorbic acid
DN
A s
igna
l / %
guanine
guanine
guanine
adenine
adenine
adenine
0
25
50
75
100
125
blank sulphate radical ascorbic acid
DN
A s
igna
l / %
100value) expected (maximum done wasdamage no whenmeasured i
radical sulfate withdamage base purine after measured i (%) signalDNA
p
p ×=
II. Investigação e desenvolvimento
176
Scheme 1 . Mechanism of adenine protection produced by caffeic acid against the sulfate radical (adapted
from Swaraga and collaborator [5]).
3.1. Optimization of the experimental conditions
In order to evaluate the TAC on beverages, some parameters concerning the damage
on the purine base immobilized on the GCE surface (persulfate and Fe2+ concentration,
and reaction time between sulfate radical and the target molecule) were optimized in order
to achieve the maximum DNA bases effect, but without a complete damage.
Persulfate (S2O82-) concentration was studied between 2.0x10-5 and 1.0x10-4 mol L-1. It
was observed a decrease on the peak current (ip) of guanine and adenine immobilized on
the GCE in all range of persulfate concentration studied. This is indicative of the powerful
effect of persulfate on the generation of the sulfate radical. However considering that the
persulfate anion caused some electronics problems on the GCE surface, for the next
optimization step, it was chosen the persulfate concentration of 2.0x10-5 and 4.0x10-5 mol
N
N
NH2
N
HN
+ SO4·-
N
N
NH2
N
HN
+ SO42- +
(adenine)
(caffeic acid radical)
·N
N
NH2
NH
N
H + + SO42-(damage)
(protection)
OH
CH=CH-COOH
O·CH=CH-COOH
(caffeic acid)
OH
OH
N
N
NH2
N
HN
+ SO4·-
N
N
NH2
N
HN
+ SO42- +
(adenine)
(caffeic acid radical)
·N
N
NH2
NH
N
H +
·N
N
NH2
NH
N
H + + SO42-(damage)
(protection)
OH
CH=CH-COOH
O·
OH
CH=CH-COOH
O·CH=CH-COOH
(caffeic acid)
OH
OH
CH=CH-COOH
(caffeic acid)
OH
OH
(caffeic acid)
OH
OH
4.3. Biossensores de bases púricas - radical sulfato
177
L-1 for the guanine and adenine biosensor, respectively. For the both purine-based
biosensors (guanine and adenine) Fe2+ was ranged from 1.0x10-5 and 1x10-4 mol L-1. With
the increases of Fe2+ concentration a decrease of the ip of guanine and adenine was
recorded on SWV. However, the decrease of the ip in all Fe2+ concentration range studied
remain very similar, so, for the next optimization step it was chosen a Fe2+ concentration
of 1.0x10-5 and 2.0x10-5 mol L-1 for guanine and adenine biosensor, respectively. Using
these optimized concentrations of Fe2+ and persulfate it was obtained a molar ratio S2O82-
/Fe2+ of 2 for both purine-based biosensor.
Reaction time between the sulfate radical and the bases immobilized on the GCE
surface depends on the half-life time on the generated free radical, so this parameter is an
important feature to optimize. In this study the incubation time were ranged from 0 to 120
s. Fig. 3 shows the correlation between the damage on the purine base produced by the
sulfate radical (interrelated with the ip values) and the incubation time. A decrease on the
ip was observed with the increase of the reaction time, however it was not observed a
complete damage of the purine bases. When it was used the adenine-biosensor it was
observed the lower ip indicating that adenine is more damaged than the guanine-
biosensor, but 2 minutes after, the damage was similar in the two purine bases. These
results are agreed with those obtained by other authors [31].The incubation time of 120 s
and 60 s was chosen for guanine-GCE and adenine-GCE respectively for all experiments.
Fig. 3. Influence on the peak current of the biosensor after immersion in a sulfate radical with the incubation
time a) 10 mg L-1 of adenine base; b) 3 mg L-1 of guanine base.
3.2. Determination of TAC in flavoured waters
Beverages (such as juice) are an excellent source of exogenous antioxidants. So,
drinking this type of foodstuff can help the human body to provide an adequate and
continuous supply of antioxidants. Antioxidants, such as, ascorbic acid and phenol-
0.00
4.00
8.00
12.00
0 20 40 60 80 100 120
Time / s
i p/ µ
A
a) guanine
b) adenine
0.00
4.00
8.00
12.00
0 20 40 60 80 100 120
Time / s
i p/ µ
A
0.00
4.00
8.00
12.00
0 20 40 60 80 100 1200.00
4.00
8.00
12.00
0 20 40 60 80 100 120
Time / s
i p/ µ
A
a) guanine
b) adenine
II. Investigação e desenvolvimento
178
derived compounds are natural components of fruits. Flavoured waters consist of the
addition of flavours/aromas, juices and considering that these added ingredients are fruit
extracts, they contain natural antioxidants, transferring them to the bottled water. So,
drinking this type of water can increase the daily intake of natural exogenous antioxidants
and may contribute to the protective system against free radicals. The consumption of
flavoured water is increasing over the world namely in Portugal where it was consumed
around 6.08 million L only in the first half of 2010 [32]. Phenolic compounds are correlated
with antioxidant activity and seem to have an important role in stabilizing lipid oxidation
[33]. The total phenolic contents (TPC) of this kind of waters was evaluated (data not
showed). It was observed that all flavoured waters presented phenolic compounds in its
composition. Like it was expected, natural waters did not have TPC. The highest TPC
levels were from citrus fruits flavours (tangerine, lime and lemon) and from waters with
bioactive compounds, like, tea, ginseng and gingko biloba [34]. For the evaluation of the
TAC of flavoured waters it was used five antioxidants compounds: ascorbic acid, gallic
acid, caffeic acid, coumaric acid and resveratrol. Ascorbic acid is a water-soluble vitamin,
considerated a powerfull antioxidant and plays a key role in the protection against
biological oxidation processes participating in many metabolic reactions [15]. Gallic,
caffeic and coumaric acids are phenolic acids with a large protective action. The phenolic
acids, can included several groups, such as the hydroxybenzoic acid (gallic acid) and the
hydroxycinnamic acid (caffeic and coumaric acid). In general, the antioxidant activity of
the phenolic-derived compounds is determined by some properties, such as free-radical
scavengers [35]. Resveratrol is a stilbene, can be found in grapes and wine and have
multiple beneficial properties, including chemopreventive and antitumor activities [36].
Linearity studies between the five antioxidants and ip of guanine and adenine oxidation
was carried out. Fig. 4 (i and ii) shows the SWV of electrochemical current obtained after
immersion the purine-biosensor on the sulfate radical containing increasing concentration
of gallic acid or coumaric acid. Like it was expected the oxidation current of DNA-bases
increased when the concentration of the antioxidant also increase. Similar
voltammograms were obtained when it was used the other antioxidants (ascorbic acid,
resveratrol, gallic, caffeic and coumaric acid) with the both purine-based biosensors
(guanine and adenine).
4.3. Biossensores de bases púricas - radical sulfato
179
Fig. 4. SWV obtained after immersion of i) guanine-biosensor in sulfate radical containing a standard solution
of gallic acid: a) 0.20; b) 0.30; c) 0.40; d) 0.50 and e) 0.60 mg L-1. Inset: relationship between ip and gallic acid
concentration and a SWV of a flavoured water sample and ii) Adenine-biosensor in sulfate radical containing a
standard solution of coumaric acid: a) 0.50; b) 0.60; c) 0.70; d) 0.80 and e) 1.00 mg L-1. Inset: relationship
between ip and coumaric acid concentration and a SWV of a flavoured water sample.
Table 1 presents the several analytical parameters obtained for the five antioxidants
standard used on the protection of the purine base (adenine or guanine) immobilized on
the GCE against the sulfate radical. Ascorbic acid presents the larger linear range ranging
from 0.50 to 4.00 mg L-1 in both purine-based biosensors. The other antioxidants
presented a narrow linear range, from 0.20 to 0.60 mg L-1 of gallic acid, 0.40 to 1.20 of
caffeic acid, 0.50 to 0.90 mg L-1 of coumaric acid and 0.10 to 0.50 mg L-1 of resveratrol
when it was used the guanine-GCE. For the adenine-GCE the linear ranges were from
20.0
40.0
60.0
0.10 0.30 0.50 0.70 0.90
4.0
6.0
8.0
10.0
0.20 0.40 0.60
i)
Blank signal
Sulfate radical
E(V) vs. AgCl/Ag
30.0
40.0
50.0
0.3 0.5 0.7i/µ
A
Flavoured water sample
i/ µ
A
E(V) vs. AgCl/Ag
i p/µ
A
gallic acid (mg L-1)
20.0
40.0
60.0
0.10 0.30 0.50 0.70 0.90
4.0
6.0
8.0
10.0
0.20 0.40 0.60
i)
Blank signal
Sulfate radical
E(V) vs. AgCl/Ag
30.0
40.0
50.0
0.3 0.5 0.7i/µ
A
Flavoured water sample
i/ µ
A
E(V) vs. AgCl/Ag
i p/µ
A
gallic acid (mg L-1)
E(V) vs. AgCl/Ag
60.0
0.50 0.70 0.90 1.10 1.30
80.0
4.0
6.0
8.0
10.0
0.50 0.60 0.70 0.80 0.90 1.00
ii)
Blank signal
Sulfate radical
40.0
50.0
60.0
70.0
0.6 0.8 1.0 1.2
i/ µ
A
E(V) vs. AgCl/Ag
Flavoured water sample
i/ µ
A
coumaric acid (mg L-1)
i p/µ
A
E(V) vs. AgCl/Ag
60.0
0.50 0.70 0.90 1.10 1.30
80.0
4.0
6.0
8.0
10.0
0.50 0.60 0.70 0.80 0.90 1.00
ii)
Blank signal
Sulfate radical
40.0
50.0
60.0
70.0
0.6 0.8 1.0 1.2
i/ µ
A
E(V) vs. AgCl/Ag
Flavoured water sample
i/ µ
A
coumaric acid (mg L-1)
i p/µ
A
II. Investigação e desenvolvimento
180
0.10 to 0.50 mg L-1 for the antioxidants gallic acid, 0.30 to 0.80 mg L-1 of caffeic acid, 0.50
to 1.00 mg L-1 of coumaric acid and 0.10 to 0.60 mg L-1 of resveratrol. RSD values were
below 10% confirmed the high precision of the methods.
Table 1. Analytical feature obtained for the 5 antioxidants standards. Parameters Ascorbic acid Gallic acid Caffeic acid Coumaric acid Resveratrol
Guanine-GCE Linear range (mg L-1) 0.50–4.00 0.20–0.60 0.40–1.20 0.50–0.90 0.10–0.50
Slope (A mg L-1L) 1.14x10-6 8.84x10-6 6.41x10-6 9.17x10-6 9.87x10-6
Intercept (A) 4.69x10-6 2.44x10-6 1.99x10-6 2.39x10-7 5.80x10-6
Correlation coefficient (n=5) 0.995 0.999 0.990 0.997 0.999
RSD (%) (mg L-1) 4.71 (3.00) 6.58 (0.50) 2.51 (1.00) 2.77 (0.60) 5.05 (0.10) LOD (mg L-1) 0.47 0.02 0.15 0.04 0.02
Adenine-GCE
Linear range (mg L-1) 0.50–4.00 0.10–0.50 0.30–0.80 0.50–1.00 0.10–0.60
Slope (A mg L-1) 5.58x10-7 1.00x10-5 5.02x10-6 8.96x10-6 9.21x10-6
Intercept (A) 6.28x10-6 5.07x10-6 6.52x10-6 4.05x10-7 3.74x10-6 Correlation coefficient (n=5) 0.990 0.975 0.990 0.998 0.990
RSD (%) (mg L-1) 0.54 (1.00) 4.45 (0.20) 3.86 (0.50) 7.26 (0.60) 5.44 (0.20)
LOD 0.50 0.10 0.10 0.50 0.10 The purine-based biosensor was applied for the determination of TAC on flavour and
flavoured waters. Table 2 shows the TAC values expressed in mg L-1 of ascorbic acid,
gallic acid, caffeic acid, coumaric acid and resveratrol.
It was verified that all flavours and flavoured waters presented antioxidant capacity; the
natural waters, like it was expected, not presented antioxidant capacity. Flavours
presented the highest values of TAC, indeed, flavours are fruit extract and have in its
composition several concentred antioxidant compounds, so these results was expected.
Using the adenine and guanine GCE the higher TAC values were found with the
standard ascorbic acid. The flavours that presented the highest TAC when it was used the
guanine-biosensor was the apple flavour fallowed by tangerine, strawberry, lemon, lime
and gooseberry. With the adenine-biosensor apple had also the highest TAC level
fallowed by lemon, gooseberry, lime, tangerine and strawberry.
When it was used, the guanine-biosensor applied to the analysis of TAC in flavoured
waters, TAC values ranged from 2 to 16 mg L-1, 0.2 to 3 mg L-1, 0.7 to 5 mg L-1, 1.2 to 4.5
mg L-1 and 0.7 to 2 mg L-1 with the antioxidant ascorbic acid, gallic acid, caffeic acid,
coumaric acid and resveratrol, respectively. Using the ascorbic acid as standard
antioxidant it was observed higher TAC values in sparkling flavoured waters (brand E to J)
than in still waters (brand A to D). The higher TAC content were from brand F (sample 21,
22, 24 and 25), brand G (sample 27, 29 and 30) and brand H (sample 32). Samples from
brand F have several added ingredients, such as tea, ginseng and ginkgo beloba, brand
G and H have also vitamin C.
.
18
1
Tab
le 2
. T
AC
val
ues
obta
ined
for
the
flavo
urs
and
flavo
ured
wat
ers
usin
g a
guan
ine-
GC
E a
nd a
deni
ne-G
CE
(m
g L-1
).
Gua
nine
-GC
E
Ade
nine
-G
CE
Bra
nd
Sam
ple
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Lem
on
154.
39 ±
5.3
2 40
.72
± 3.
78
69.5
8 ±
9.84
67
.73
± 6
.88
19.7
6 ±
2.40
23
5.35
± 4
.89
50.1
3 ±6
.86
235.
35 ±
10.
54
77.8
0 ±
9.30
36
.81
± 8.
64
Tan
gerin
e 20
9.12
± 1
0.12
47
.09
± 5.
31
79.3
1 ±
6.30
74
.54
± 11
.39
26.0
8 ±
3.62
91
.40
± 8.
04
27.8
8 ±
3.7
6 91
.40
± 5.
87
53.9
2 ±
8.46
14
.63
± 2.
86
App
le
281.
89 ±
7.3
2 55
.52
± 7.
41
92.2
5 ±
3.76
83
.58
± 2
.63
34.4
8 ±
2.44
23
9.12
± 2
1.36
50
.57
± 5.
95
239.
12 ±
15.
23
78.2
7 ±
6.38
37
.24
± 5.
93
Str
awbe
rry
184.
21 ±
15.
12
44.2
1 ±
1.60
74
.88
± 4.
67
71.4
4 ±
4.96
23
.20
± 4.
75
24.6
1 ±
2.44
25
.72
± 3.
76
24.6
1 ±
2.56
51
.60
± 4.
03
12.4
7 ±
3.75
Goo
sebe
rry
101.
75 ±
2.4
5 34
.65
± 2.
65
60.2
2 ±
5.78
61
.19
± 6.
23
13.6
8 ±
2.32
14
0.23
± 9
.15
7.16
± 1
.43
14
0.00
± 1
2.43
66
.05
± 6.
94
25.8
0 ±
2.97
Fla
vour
Lim
e 12
6.93
± 4
.32
37.5
7 ±
4.87
64
.70
± 8.
76
64.3
2 ±
9.42
16
.59
± 4.
32
110.
13 ±
4.4
0 35
.63
± 3.
50
110.
13 ±
4.4
0 62
.23
± 5.
47
22.3
5 ±
5.09
A
1 Le
mon
2.
57 ±
0.5
4 1.
44 ±
0.1
3 2.
56 ±
0.1
9 2.
75 ±
0.1
3 0.
39 ±
0.0
2 3.
14 ±
0.2
3 1.
24 ±
0.0
4 2.
26 ±
0.5
8 2.
53 ±
0.0
6 0.
84 ±
0.0
2
2 M
ango
6.
63 ±
0.7
2 1.
91 ±
0.4
0 3.
28 ±
0.1
9 3.
25 ±
0.4
3 1.
14 ±
0.0
8 2.
26 ±
0.0
7 1.
16 ±
0.0
3 2.
12 ±
0.0
4 2.
44 ±
0.0
2 0.
74 ±
0.0
7
3 S
traw
berr
y 5.
79 ±
0.6
2 1.
81 ±
0.7
1 3.
14 ±
0.0
9 3.
15 ±
0.0
6 1.
27 ±
0.0
2 2.
81±
0.04
1.
34 ±
0.0
4 2.
46 0
.28
2.64
± 0
.05
0.68
± 0
.03
4
Nat
ural
- -
- -
-
B
5 P
inea
pple
/ora
nge
5.87
± 0
.26
1.82
± 0
.06
3.15
± 0
.09
3.16
± 0
.07
0.77
± 0
.06
2.56
± 0
.04
1.24
± 0
.03
2.
25 ±
0.0
4 2.
53 ±
0.0
8 0.
78 ±
0.0
3
6
Lem
on
9.66
± 0
.41
2.26
± 0
.09
3.82
± 0
.15
3.63
± 0
.10
1.21
± 0
.09
2.11
± 0.
02
1.06
± 0
.04
1.97
± 0
.23
2.33
± 0
.01
0.72
± 0
.05
7
Nat
ural
-
- -
- -
C
8 Le
mon
/Mag
nesi
um
9.19
± 0
.43
2.21
± 0
.56
3.74
± 0
.87
3.57
± 0
.17
1.16
± 0
.07
4.22
± 0
.56
1.63
± 0
.07
2.86
± 0
.03
2.95
± 0
.02
0.97
± 0
.05
9
App
le/w
hite
tea
9.18
± 0
.45
2.21
± 0
.34
3.74
± 0
.52
3.57
± 0
.36
1.16
± 0
.03
5.23
± 0
.26
0.82
± 0
.02
1.61
± 0
.02
2.08
± 0
.05
0.30
± 0
.03
10
Pin
eapp
le/fi
bre
11.2
5 ±
1.58
2.
45 ±
0.3
7 4.
11 ±
0.0
6 3.
82 ±
0.3
9 1.
40 ±
0.0
7 3.
12 ±
0.7
2 1.
16 ±
0.0
5 2.
13 ±
0.0
6 2.
44 ±
0.0
6 0.
84 ±
0.0
7
11
Nat
ural
-
- -
- -
D
12 A
pple
3.
77 ±
0.4
7 0.
15 ±
0.1
1 2.
78 ±
0.1
6 2.
90 ±
0.1
2 0.
53 ±
0.0
2 -
0.54
± 0
.02
1.51
± 0
.03
1.89
± 0
.23
0.96
± 0
.07
13
Ora
nge/
peac
h 2.
43 ±
0.6
7 0.
23 ±
0.0
8 0.
71 ±
0.0
9
1.45
± 0
.23
0.66
± 0
.08
- 0.
95 ±
0.0
6 1.
34 ±
0.0
6 2.
06 ±
0.0
3 0.
28 ±
0.0
4
14
Lem
on
2.88
± 0
.30
0.28
± 0
.05
0.79
± 0
.07
1.51
± 0
.31
0.76
± 0
.04
1.82
± 0
.05
2.20
± 0.
10
2.73
± 0
.05
3.56
± 0
.05
1.53
± 0
.15
15
Nat
ural
- -
- -
E
16 L
emon
10
.82
± 0.
03
1.60
± 0
.13
2.80
± 0
.07
2.91
± 0
.21
1.35
± 0
.12
5.00
± 0
.34
2.12
± 0
.06
3.61
± 0.
04
3.48
± 0
.34
1.36
± 0
.05
17
Ora
nge/
rasp
berr
y 6.
36 ±
0.3
7 1.
88 ±
0.0
8 3.
24 ±
0.1
3 3.
22 ±
0.0
9 0.
83 ±
0.0
9 0.
83 ±
0.0
9 1.
59 ±
0.0
7 2.
79 ±
0.0
5 1.
39 ±
0.0
7 0.
54 ±
0.0
4
18
Pea
ch/p
inea
pple
4.
73 ±
0.3
0 1.
69 ±
0.0
3 2.
95 ±
0.0
2 3.
02 ±
0.0
5 1.
02 ±
0.0
3 7.
56 ±
0.8
4 1.
29 ±
0.0
1
2.32
± 0
.05
2.58
± 0
.05
0.62
± 0
.05
19
Gua
va/li
me
2.33
± 0
.19
1.41
± 0
.07
0.48
± 0
.08
1.2
9 ±
0.02
0.
90 ±
0.0
7 4.
23 ±
0.0
5 0.
72 ±
0.0
5 1.
46 ±
0.0
3 1.
98 ±
0.0
4 0.
14 ±
0.0
1
20
Nat
ural
-
- -
- -
F
21 L
emon
/gre
en te
a 11
.89
± 0.
09
2.52
± 0
.02
4.22
± 0
.04
3.91
± 0
.02
1.47
± 0
.02
1.31
± 0
.07
1.24
± 0
.01
2.25
± 0
.18
2.53
± 0
.03
0.58
± 0
.05
22
Ras
pber
ry/g
inse
ng
16.8
0 ±
0.58
3.
09 ±
0.0
6 5.
09 ±
0.3
2 4.
52 ±
0.1
4 2.
04 ±
0.0
6 2.
86 ±
0.0
4 0.
86 ±
0.0
3 1.
68 ±
0.0
9 2.
13 ±
0.3
5 0.
20 ±
0.0
3
18
2
Gua
nine
-GC
E
Ade
nine
-G
CE
Bra
nd
Sam
ple
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
Asc
orbi
c ac
id
Gal
lic a
cid
Caf
feic
aci
d C
oum
aric
aci
d R
esve
ratr
ol
23
Pea
ch/w
hite
tea
9.81
± 0
.10
2.27
± 0
.23
3.85
± 0
.36
3.65
± 0
.25
1.23
± 0
.03
0.98
± 0
.03
1.16
± 0
.06
2.
13 ±
0.0
5 2.
44 ±
0.0
4 0.
50 ±
0.0
4
24
Man
go/g
inkg
o be
loba
11
.78
± 0.
65
2.51
± 0
.15
4.20
± 0
.23
3.89
± 0
.16
1.45
± 0
.15
5.56
± 0
.07
1.13
± 0
.01
1.52
± 0
.04
2.02
± 0
.43
0.45
± 0
.01
25
Mel
on/m
int
14.0
5 ±
1.36
2.
77 ±
0.3
2 4.
61 ±
0.0
8 4.
17 ±
0.0
4 1.
72 ±
0.0
1 4.
00 ±
0.4
5 0.
98 ±
0.0
4
1.02
± 0
.07
1.67
± 0
.05
0.37
5 ±
0.0
26
Nat
ural
-
- -
- -
- -
- -
-
G
27 L
emon
10
.85
+ 2
.90
2.40
± 0
.07
4.04
± 0
.03
3.7
8 ±
0.0
8 1.
35 ±
0.0
7 4.
00 ±
0.0
5 1.
24 ±
0.0
3 2.
25 ±
0.0
9 2.
53 ±
0.0
4 0.
57 ±
0.0
4
28
Lim
e 12
.14
± 0.
80
2.55
± 0
.19
4.27
± 0
.28
3.94
± 0
.19
1.50
± 0
.09
1.00
± 0
.03
0.98
± 0
.01
1.65
± 0
.05
2.11
± 0
.04
0.15
± 0
.03
29
App
le
13.7
8 ±
2.72
2.
74 ±
0.0
2 4.
56 ±
0.0
7 4.
14
± 0
.07
1.69
± 0
.03
1.52
± 0
.027
1.
28 ±
0.0
4 2.
31
± 0.
24
2.57
± 0
.03
0.62
± 0
.04
30
Pea
ch
11.6
1 ±
2.11
2.
49 ±
0.0
9 4.
17 ±
0.0
5 3.
87
± 0
.03
1.44
± 0
.05
2.56
± 0
.15
1.18
± 0
.05
2.16
± 0
.06
2.46
± 0
.05
0.78
± 0
.05
31
Nat
ural
-
- -
- -
- -
- -
-
H
32 L
emon
10
.06
± 0.
72
2.31
± 0
.04
3.90
± 0
.07
3.6
8 ±
0.0
7 1.
26 ±
0.0
2 4.
29 ±
0.0
5 1.
23 ±
0.0
3 2.
24 ±
0.0
5 2.
52 ±
0.0
1 0.
98 ±
0.0
3
33
Nat
ural
-
- -
- -
- -
- -
-
I 34
Lem
on
12.8
3 ±
0.52
2.
63 ±
0.1
2 4.
39 ±
0.1
8 4.
02
± 0
.13
1.58
± 0
.11
8.01
± 1
.23
2.07
± 0
.17
2.54
± 0
.04
2.73
± 0
.05
1.4
± 0.
07
35
Gre
en A
pple
13
.72
± 0.
53
2.73
±0.
59
4.55
± 0
.06
4.13
± 0
.04
1.68
± 0
.09
0.75
± 0
.02
1.08
± 0
.02
2.
01 ±
0.0
3 2.
36 ±
0.0
6 0.
57 ±
0.0
4
36
Str
awbe
rry
13.6
0 ±
1.46
2.
72 ±
0.3
4 4.
52 ±
0.5
2 4.
12 ±
0.3
6 1.
66 ±
0.0
4 0.
98 ±
0.0
5 0.
79 ±
0.0
1
1.56
± 0
.06
2.05
± 0
.03
0.12
± 0
.06
37
Nat
ural
-
- -
- -
- -
- -
-
J 38
Lem
on
11.4
5 ±
1.86
2.
47 ±
0.4
3 4.
14 ±
0.6
7 3.
85
± 0
.47
1.42
± 0
.03
1.89
± 0
.04
0.69
± 0
.03
1.40
± 0
.05
1.94
± 0
.02
0.87
± 0
.09
39
Nat
ural
-
- -
-
-
-
-
4.3. Biossensores de bases púricas - radical sulfato
183
These ingredients are antioxidants, so it was expected an increase of the TAC values is
these samples due to the dual antioxidant effect on the protection of DNA. The lowest
TAC content was from Brand D. Using the gallic acid as a standard antioxidant the higher
TAC were from brand F (sample 22) fallowed by brand G (sample 27-29), and I (sample
34-36), the lowest TAC values was from brand D. With the others antioxidants (caffeic
acid, coumaric acid and resveratrol) it was obtained a similar TAC behaviour. TAC values
obtained with the four antioxidant, gallic acid, caffeic acid, coumaric acid and resveratrol
are narrower than the values obtained with the standard ascorbic acid antioxidant. Theses
differences obtained between the ascorbic acid and the others antioxidants can be
elucidated by the fact that ascorbic acid is a powerful antioxidant and in this study
presented a larger linear range.
Using the adenine-GCE all TAC values were very similar. Using the ascorbic acid as
standard, TAC contents ranged from 0.83 to 8 mg L-1. The highest TAC value was from
sample 34 (brand I) followed by sample 18 (brand E), sample 25 (brand F), and sample 9
(brand C). Sample 12 and 13 from brand D did not presented TAC activity. With the
standard gallic acid TAC values ranged from 0.5 to 2.2 mg L-1, 1.0 to 3.6 mg L-1 with the
caffeic acid, 1.6 to 3.5 mg L-1 with the coumaric acid and 0.20 to 1.41 mg L-1 with the
standard resveratrol. With the resveratrol standard it was observed a narrow range of TAC
values. Using the other antioxidants (gallic acid, caffeic acic and coumaric acid) the TAC
behaviour was similar.
Analysing results from Table 2 it is possibly to confirm that the purine bases immobilized
on GCE can be used for the quantification of TAC in beverages, however using the
guanine-GCE and ascorbic acid as antioxidant standard it was obtained the highest TAC
values.
The purine-based biosensors described in this study have some advantages over the
conventional methodologies previously reported in the literature, such as determination of
TAC based on the electrochemical properties do not require the use of reactive
compounds, since it is based on electrochemical behaviour and consequently on their
chemical-physical properties. Furthermore, the electrochemical method has some
advantages over the commonly used optical method, such as a shorter detection time, a
smaller sample volume, higher accuracy and a high simplicity. In addition, colored
samples can be directly used for the measurement without pretreatment. The use of the
purine-based biosensor are closer to biological systems, with a nucleotide being damaged
by free radical, in this case, the radical sulphate. These radical may develop oxidative
attack against DNA in biological systems which may generate replication errors and
subsequent misleading protein synthesis. These advantages indicate that a purine-based
II. Investigação e desenvolvimento
184
biosensors can be used as a useful tool for a rapid screening in the determination of TAC
in food matrices.
4. Conclusion
A purine-based biosensor for the evaluation of TAC in beverages was developed. The
adenine and guanine bases immobilized on the GCE surface were damage by the sulfate
radical generated by the persulfate /iron (II) system. The protective effect on the DNA
bases performed by the presence of five antioxidants was confirmed and allows to the
development of a methodology for the quantification of TAC in food samples. The purine-
based biosensor developed is disposable, and requires a very easy, rapid, reproducible
preparation and also the advantage to combine with portable equipment.
Acknowledgements
M. Fátima Barroso is grateful to Fundação para a Ciência e a Tecnologia for a Ph.D.
grant (SFRH/BD/29440/2006). The authors thank Frize for providing flavours samples.
4.3. Biossensores de bases púricas - radical sulfato
185
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Oliveira, Food Addit. Contam. Part B-Surveill. 2 (2009) 121.
[31] N. de-los-Santos-Álvarez, P. de-los-Santos-Álvarez, M.J. Lobo-Castañón, R. López,
A.J. Miranda-Ordieres, P. Tuñon-Blanco, Electrochem. Commun. 9 (2007) 1862.
[32] ANIRSF, 2010. Associação Nacional dos industriais de refrigerantes e sumos de
frutos, 2010. Informar nº29. from the website www.anirsf.pt (accessed 03.09.2010).
[33] M.L. Rodríguez-Méndez, C. Apetrei, J.A. de Saja, Electrochim. Acta 53 (2008) 5867.
[34] M. Fátima Barroso, J.P. Noronha, Cristina Delerue-Matos, M. B.P.P. Oliveira.
Flavoured waters: influence of ingredients in antioxidant capacity and terpenoid
profile by HS-SPME/GC-MS. J. Agric. Food Chem. 2011 (59) 5062.
[35] V. Thavasi, L.P. Leong, R.P.A. Bettens, J. phys.Chem. A 110 (2006) 4918.
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187
Capítulo 5
Construção de biossensores de ADN
5.1.
DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages
M.F. Barroso, N. de-los-Santos-Álvarez, M.J. Lobo-Castañón, A.J. Miranda-Ordieres,
C. Delerue-Matos, M.B.P.P. Oliveira, P. Tuñón-Blanco
Biosensors and Bioelectronics, 2011, 26 (5), 2396 - 2401
5.2.
Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity
M.F. Barroso, N. de-los-Santos-Álvarez, M.J. Lobo-Castañón, A.J. Miranda-Ordieres,
C. Delerue-Matos, M.B.P.P. Oliveira, P. Tuñón-Blanco
Journal of Electroanalytical Chemistry, 2011, em publicação
doi:10.1016/j.jelechem.2011.04.022
5.1. Biossensores de ADN – radical hidroxilo
189
DNA-based biosensor for the electrocatalytic determ ination of
antioxidant capacity in beverages
M.F. Barrosoa,b,c, N. de-los-Santos-Álvareza, M.J. Lobo-Castañóna, A.J. Miranda-
Ordieresa, C. Delerue-Matosc, M.B.P.P. Oliveirab, P. Tuñón-Blancoa aDepartamento de Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8,
33006 Oviedo, Spain bRequimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, R.
Aníbal Cunha n. 164, 4050-047 Porto, Portugal cREQUIMTE/Instituto Superior de Engenharia do Porto, Dr. Bernardino de Almeida 431,
4200-072 Porto, Portugal
Abstract
Reactive oxygen species (ROS) are produced as a consequence of normal aerobic
metabolism and are able to induce DNA oxidative damage. At the cellular level, the
evaluation of the protective effect of antioxidants can be achieved by examining the
integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an
adenine-rich oligonucleotide (dA21) adsorbed on carbon paste electrodes for the
assessment of the antioxidant capacity is proposed. The method was based on the partial
damage of a DNA layer adsorbed on the electrode surface by OH• − radicals generated by
Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases
to generate an oxidation product that was able to catalyze the oxidation of NADH. The
presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines
unoxidized, and thus, increasing the electrocatalytic current of NADH measured by
differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant
species, the detection of as low as 50 nM of AA in aqueous solution was possible. The
protection efficiency was evaluated for several antioxidant compounds. The biosensor
was applied to the determination of the total antioxidant capacity (TAC) in beverages.
Keywords : NADH; DNA biosensor; Electrocatalytic oxidation;Total antioxidant capacity
(TAC); Ascorbic acid; Reactive oxygen species (ROS).
Available online at www.sciencedirect.com
Biosensor and Bioelectronics 2011, 26 (5), 2396-2401
5.1. Biossensores de ADN – radical hidroxilo
191
1. Introduction
Oxidative lesions in DNA are the primary risk factor for gene mutations, which plays a
key role in carcinogenesis and aging (Freidman and Heller, 2004). Reactive oxygen
species (ROS) are continuously generated in living cells, such as, in the inner
mitochondrial membrane, outer membrane, and in several metabolic pathways in
mammalian cells, for instance, in the microsomal electron transport. Hydroxyl radical, OH•
is one of the most powerful oxidant known in a biological setting, and upon formation, it
oxidizes indiscriminately and site-specifically any biomolecule (Laranjinha, 2009). In living
systems, most of the hydroxyl radicals are generated from the metal (M) ion-dependent
breakdown of hydrogen peroxide. In the presence of reduced transition metal ions, e.g.
ferrous or cupric ions, hydrogen peroxide is turned into OH• and OH− through a one-
electron redox reaction commonly called Fenton reaction (Eq. (1)) (Mello et al., 2006). The
Fenton chemistry is important because it is involved in oxidative damage in vivo leading to
changes in DNA that induce mutagenesis, and eventually, carcinogenesis:
H2O2 +Fe2+→ OH• + OH− +Fe3+ (1)
Most living organisms have developed complex endogenous and exogenous antioxidant
systems to counteract and prevent the deleterious effects of ROS. Antioxidants act as
reductants agents (free radical terminators), metal chelating and singlet oxygen
quenchers (Vertuani et al., 2004). Endogenous antioxidant systems include enzymes,
such as, superoxide dismutase, glutathione peroxidase, glutathione reductase,
glutathione-S-transferase and catalase (Huang et al., 2005). An additional protection can
be provided by exogenous antioxidant compounds, such as, vitamins (A, E, C, β-
carotene), phenolic compounds, minerals (Se, Zn) or proteins (transferrin, ceruloplasmin,
albumin). Foodstuffs constitute an excellent exogenous source of natural antioxidants. It is
well-known that vegetables, fruits, whole-grain and some beverages (tea, juice, wine) are
rich in many antioxidant and bioactive compounds. Recently, to answer to consumer’s
preferences, flavoured waters produced from mineral and spring waters were developed
and commercialized. In the first semester of 2009, 6.23 million litters of this kind of water
were consumed by Portuguese population (ANIRSF, 2009). This kind of water consists of
the addition of flavours, juices and sugar or sweeteners that provide water with singular
tastes and aromas appreciated by consumers. Considering that flavours/aromas are fruit
extracts, they contain natural antioxidants, transferring them to the bottled water. So,
II. Investigação e desenvolvimento
192
drinking this type of water can increase the daily intake of natural exogenous antioxidants
or may contribute to the protective system against ROS.
Several methods have been reported to evaluate the total antioxidant capacity (TAC) in
biological and food samples, defined as the moles of a given free radical scavenged by a
sample solution disregarding the antioxidant present (Mello and Kubota, 2007). These
methods rely on the inhibition of the oxidation of a suitable substrate by the antioxidant
agent. After reaction, the extent of the oxidation is measured at a fixed time by UV–vis
spectrometry, chemiluminescence, fluorescence and after chromatographic separation
(Sanchez-Moreno, 2002).
The protective effect of antioxidants at a cellular level could only be achieved by
monitoring the DNA integrity. In recent years, several electrochemical DNA-based sensors
have been developed in order to assess the antioxidant capacity (Labuda et al., 2002,
2003; Mello and Kubota, 2007; Qian et al., 2010). UV irradiation (Liu et al., 2005) or most
commonly Fenton reaction were used for OH• generation. Recently, a ruthenium complex
was also used as an electrogenerated oxidant to cause the oxidation of DNA in the
presence of TiO2 nanoparticles (Liu et al., 2006).
Taking advantage of the two main lesions caused by ROS in DNA, two detection
estrategies have been proposed. On one hand, the use of redox active dsDNA
intercalators allowed evaluating the oxidative damage on dsDNA layers because of a
significant decrease in the current intensity of the intercalator upon strand scission
(Labuda et al., 2002, 2003; Liu et al., 2005). On the other hand, the intrinsic electroactivity
of DNA can be exploited. The oxidation of nucleobases on solid electrodes, mainly
guanine, and also adenine in a lesser extent, allowed the use of their decreasing oxidation
current after damage on carbon electrodes (Mello et al., 2006; Qian et al., 2010).
In this work, an electrocatalytic voltammetric method to assess TAC using DNA-
modified carbon paste electrodes (CPE) was developed. It has been reported that the
electrochemical oxidation of both adenine and guanine homopolynucleotides in neutral or
alkaline conditions led to the formation of a common oxidized product that catalyzed the
oxidation of NADH (de-los-Santos-Alvarez et al., 2007). Therefore, the oxidative lesions
generated after immersion of the DNA-CPE in the Fenton mixture were indirectly
quantified after the electrochemical oxidation of the adenines that remained unoxidized on
the electrode surface. The increase of this electrocatalytic current in the presence of
several antioxidant species was studied. The biosensor developed was applied to the
determination of TAC in several beverages and the results were compared with those
attained using other methodologies to obtain an overall picture of the antioxidant profile.
5.1. Biossensores de ADN – radical hidroxilo
193
2. Materials and methods
2.1. Chemicals
Deoxyadenylic acid oligonucleotide (dA21) was purchased as a desalted product from
Sigma-Genosys (London, UK). Concentrated saline sodium phosphate EDTA (20× SSPE;
0.2 mol l−1 sodium phosphate, 2 mol l−1 NaCl, 0.02 mol l−1 EDTA), tris–HCl pH 9.0,
phosphate buffer pH 9.0, iron (II) sulphate heptahydrate, hydrogen peroxide (30%, w/v),
gallic acid, resveratrol, nicotinamide adenine dinucleotide disodium salt, reduced form
(NADH), were acquired from Sigma–Aldrich (Spain). L(+)-Ascorbic acid was from Riedel-
de-Haën. Caffeic acid, and trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic
acid, a water-soluble derivative of vitamin E) were from Fluka (Spain). Other chemicals
employed were of analytical grade.
Stock solutions of 1 g l−1 dA21 were stored at 4 ºC and diluted with 2× SSPE buffer
solution (prepared by dilution of 20× SSPE solution) prior to use. Fenton solution
(generation of hydroxyl radical) was prepared by mixing Fe2+:EDTA:H2O2 (µmol l−1:2 µmol
l−1:40 µmol l−1) in the molar ratio of 1:2:40. Mello et al., 2006 reported that when an excess
of hydrogen peroxide was added in the reaction a high DNA damage was obtained. EDTA
was added for solubility reasons. All solutions were prepared with water purified with a
Direct-Q (Millipore) system.
2.2. Instrumentation
Cyclic voltammetry (CV) and differencial pulse voltammetry (DPV) were performed with
a µAutolab II controlled by GPES software, version 4.8 (EcoChemie, The Netherlands). A
conventional three-electrode cell was used, which included a home-made CPE (3 mm in
diameter) as a working electrode, a platinum wire counter electrode and a Ag|AgCl|KClsat
reference electrode to which all potentials were referred. The CPE was prepared by
mixing 1.8 g of paraffin oil as a pasting liquid with 5 g of spectroscopic grade graphite
powder (Ultracarbon, Dicoex, Spain). The unmodified carbon paste was introduced into
the well of a teflon electrode body provided by a stainless steel piston. The surface was
smoothed against a plain white paper while a slight manual pressure was applied to the
piston. Unless otherwise stated, after each experiment, the CP was discarded and a new
electrode surface was freshly prepared.
II. Investigação e desenvolvimento
194
2.3. Assay procedure
Unless otherwise mentioned, most experiments consisted of four steps: DNA
immobilization, damage of oligonucleotide by the immersion of DNA-CPE on the Fenton
mixture and study of the effect of the presence of antioxidants in the system;
electrooxidation of the remaining unoxidized adenines on the CPE, and detection in a
Ca2+ containing-NADH solution.
DNA immobilization was performed by dry adsorption placing a 5-µl droplet of dA21 (180
mg l−1) in 2× SSPE solution on the electrode surface and evaporating it to dryness under a
stream of warm air.
DNA damage was carried out by immersing the modified electrode in a freshly prepared
Fenton mixture in the absence or the presence of antioxidant in 2× SSPE buffer.
After a fixed period of reaction time, the DNA-CPE was washed with water and
immediately immersed in a 0.1 mol l−1 phosphate buffer (pH 9.0) to carry out the electro-
oxidation of the remaining unoxidized adenine bases. A hundred potential scans were
performed between −0.2 and +1.4 V at 500 mV s−1 to ensure a complete oxidation.
For detection, the DNA-CPE was placed in a NADH solution (0.5 mmol l−1 in 0.1 mol l−1
tris–HCl pH 9.0 containing 0.01 mol l−1 CaCl2). The electrocatalytic current of NADH was
obtained by sweeping the potential between −0.2V and +0.5 V at 50 mV s−1 when CV was
used as a detection technique. For DPV experiments, the potential was swept from −0.2
to +0.3 V. The step potential was 0.005 V and the modulation amplitude 0.05 V.
2.4. Samples and alternative methods description
Two lemon sparkling flavoured water samples corresponding to two different brands
were collected in a supermarket and stored in the dark at +4 ºC. Sonication was used to
eliminate gas from the sample. A lemon flavour used in the formulation of some water
brands, was also analysed. This flavour had no description about their chemical or aroma
composition, only knowing that they are present in some brands of flavoured water.
Label information from brand A indicated the presence of vitamin C, some
preservatives, such as, sodium benzoate, potassium sorbate and the acidifying regulator
citric acid. Label from brand B sample indicated the presence of green tea and citric acid.
In order to compare the results obtained with the DNA based sensor developed, the
total phenolic contents (TPC) of these samples was determined by a colorimetric assay
based on procedures previously described (Singleton and Rossi, 1965). Folin-Ciocalteu
reagent was used, and the reduced phenols produced a stable blue product at the end of
reaction and the results were given as milligram of gallic acid l−1.
5.1. Biossensores de ADN – radical hidroxilo
195
Other two methods were used to obtain a complete profile of the antioxidant capacity.
The radical scavenging ability of these samples was tested by DPPH (1,1-diphenyl-2-
picrylhydrazyl) stable radical assay (Hatano et al., 1988) and reducing power method
(Oyaizu, 1986). DPPH values were expressed in mg trolox l−1 and reducing power in mg
of gallic acid l−1. A comprehensive study on the chemistry behind these methods has been
reviewed (Huang et al., 2005).
3. Results and discussion
Previous studies reported by our group indicated that the electro-oxidation of different
adenine nucleosides and nucleotides, such as adenosine (de-los-Santos-Álvarez et al.,
2001), (dA)20 (delos- Santos-Álvarez et al., 2002), SAMe (de-los-Santos-Álvarez et al.,
2004), or FAD (de-los-Santos-Álvarez et al., 2005) on pyrolytic graphite electrodes (PGE)
or CPE (Alvarez-González et al., 2000) in phosphate buffer ranging from pH 7 to 12, led to
the formation of a diimine species, strongly adsorbed on the electrode surface. The
common oxidized compound exhibits a quasi-reversible redox process at low potentials
and is able to efficiently catalyze the oxidation of NADH reducing the overpotential by
more than 300 mV.
The oxidizability of purine bases in DNA depends, predominantly, on the secondary
structure of the polynucleotide. Accordingly, ssDNA leads to higher oxidation currents
than dsDNA because of their flexibility and better accessibility of nucleobases to the
electrode surface (de-los-Santos-Álvarez et al., 2002). Since polynucleotides strongly
adsorb on carbon electrodes, the oxidation of a layer of dA21 was carried out after physical
adsorption of the ssDNA on the electrode surface. The oxidation of adenine bases in DNA
takes place at about 1.2 V at CPE. In Fig. 1 (inset), the redox process associated to the
oxidation products arisen from dA21 after 100 potential scans at 500 mV s−1 from −0.2 to
+1.4 V on CPE is depicted. As anticipated, the redox process has a formal potential (Eo`)
of 0.035 V, which corresponds to the diimine species. After addition of NADH in the
presence of calcium ions, a great enhancement in the anodic current was observed at low
potentials associated to the mediated oxidation of NADH (Fig. 1, curve a). The onset of
the electrocatalytic wave appeared at potentials as low as 0.010 V with a plateau at 0.184
V. A further increase in the magnitude of the anodic current at more positive potentials
was adscribed to the direct uncatalyzed oxidation of NADH at CPE, as it is shown in curve
b, where the oxidation of NADH at a bare unmodified CPE (Ep = 0.70 V) is depicted. The
use of calcium ions has been reported to greatly improve the electrocatalytic current of
NADH (de-los-Santos-Alvarez et al., 2006; Mano and Kuhn, 1999; Toh et al., 2003). This
effect is not completely understood at the molecular level, although it is speculated that
II. Investigação e desenvolvimento
196
divalent cations can effectively counterbalance the negative charge of the catalyst
favouring the approach of the negatively charged NADH to form a complex between
NADH, Ca2+ and the catalyst. In addition to this, calcium ions can interact directly with
DNA contributing to DNA stabilization and conformation (de-los-Santos-Alvarez et al.,
2006).
Fig. 1. CVs obtained at 50 mV s−1 in tris–HCl pH 9.0 in the presence of 0.5 mmol l−1 NADH + 0.01 mol l−1
CaCl2 with (solid line) a DNA-CPE and (dotted line) a bare CPE after electrochemical oxidation up to 1.4 V at
500 mV s−1. Inset: CV obtained with a DNA-CPE at 50 mV s−1 in tris–HCl pH 9.0 in the absence of NADH+
CaCl2 solution after identical electrochemical oxidation.
It is well-known that hydroxyl radicals generated in the close proximity to DNA, can
attack both the deoxyribose sugar moiety and the nucleobases resulting intermediate
radicals, which are precursors of DNA base damage such as base oxidation, sugar
fragmentation and DNA strand structural changes (Jaruga and Dizdaroglu, 1996). In order
to verify that OH• generated by a Fenton-type reaction are able to oxidize dA21 on the
electrode surface, the DNA-CPE was placed in the Fenton mixture for 120 s. After
transferring to a phosphate buffer solution (pH 9), no redox process at low potentials was
observed. An extended voltammetric scan up to 1.4 V did not show any oxidation peak at
1.2 V indicating that, at least, part of the adenine bases were effectively oxidized by the
generated radicals. This result suggested that Fenton-generated hydroxyl radicals
induced oxidative damage on adenine bases following a pathway somehow different from
the electrochemical oxidation because the adsorbed compound was not formed or the
yield was so low that cannot be detected by CV. At this point it is important to remark that
the NADH catalyst generated on the electrode surface after oxidation of adenine residues
E / VE / V
- 0.20 0.20 0.60 1.00-- 0.50
0.75
2.00
3.25
4.50
- 0.20 0.20 0.60 1.00-- 0.50
0.75
2.00
3.25
4.50
I/ µ
A
-0.20 0.15 0.50-0.04
0
0.40
E / V
I / µ
A
a) b)
E / VE / V
- 0.20 0.20 0.60 1.00-- 0.50
0.75
2.00
3.25
4.50
- 0.20 0.20 0.60 1.00-- 0.50
0.75
2.00
3.25
4.50
I/ µ
A
-0.20 0.15 0.50-0.04
0
0.40
E / V
I / µ
A
- 0.20 0.20 0.60 1.00-- 0.50
0.75
2.00
3.25
4.50
- 0.20 0.20 0.60 1.00-- 0.50
0.75
2.00
3.25
4.50
I/ µ
A
-0.20 0.15 0.50-0.04
0
0.40
E / V
I / µ
A
a) b)
5.1. Biossensores de ADN – radical hidroxilo
197
is not the main oxidation product but only one of the several electrogenerated products
reported so far (Dryhurst and Elving, 1968; Goyal et al., 1991; Goyal and Sangal, 2002).
The damaged DNA-CPE was subjected to the above mentioned procedure to oxidize
the remaining undamaged adenine bases and immersed in a Ca2+-containing NADH
solution. An apparent electrocatalytic wave was observed with a plateau at about 0.18 V
(Fig. 2, curve a). This current was clearly smaller than that obtained when no Fenton
reaction was carried out (Fig. 2, curve b) indicating that only few adenine bases remained
unoxidized after exposure to Fenton mixture, confirming the ability of the generated OH• to
partially oxidized the DNA layer. When ascorbic acid was added to the Fenton mixture, an
electrocatalytic current higher than that obtained in its absence but smaller than when no
Fenton reaction was performed was observed (Fig. 2, curve c). This behaviour was in
good agreement with a scavenging effect of the antioxidant that prevented the DNA
damage to occur. As a consequence, the number of lesions diminished yielding a larger
number of adenine available for electrochemical oxidation. A positive correlation between
the partial oxidation of DNA and the concentration of antioxidant species in the tested
solution would allow the use of the electrocatalytic current of NADH to evaluate the TAC.
Fig. 2. CV obtained with a DNA-CPE at 50 mV s−1 in tris–HCl buffer pH 9.0 in 0.5 mmol l−1 NADH with 0.01
mol l−1 CaCl2 after: (a) immersion in Fenton solution and electrochemical oxidation (b) only electrochemical
oxidation; (c) immersion in Fenton solution with 10 µmol l−1 AA and electrochemical oxidation.
It is worth noting that a common oxidation product for the electrochemical oxidation of
both adenine and guanine bases was proposed (de-los-Santos-Alvarez et al., 2007).
Therefore, a layer of DNA containing guanine could be also used, in principle, for the
preparation of the biosensor layer. However, guanines are the primary nucleobase target
for hydroxyl radicals suggesting that the number of intact guanine bases for the
subsequent electrochemical oxidation would be smaller than in the case of adenine.
II. Investigação e desenvolvimento
198
Besides, the yield of the catalyst generated from guanine derivatives is much lower than in
the case of adenine derivatives (de-los-Santos- Alvarez et al., 2007). Thus, the selection
of adenine-rich oligos for the preparation of biosensors seems to be advantageous.
3.1. Optimization of the experimental conditions
Firstly, the influence of the number of potential scans to obtain a complete oxidation of
the dA21 was studied. It was observed that when increasing the number of potential scans
between −0.2 V and +1.4 V at 500 mV s−1, the amount of catalyst produced also
increased, and consequently, an enhancement in the electrocatalytic current was verified.
This increase in the peak current of the catalyst was observed until the 100th scan after
that, the amount of catalyst generated reached the highest value (surface coverage (Γ) of
1.1×10−11 mol cm−2) and remained constant, indicating the complete oxidation of dA21
adsorbed on CPE. Therefore, this number of potential scans was chosen for the next
optimization steps.
In order to evaluate the TAC on beverages, some parameters concerning the damaging
reaction (iron concentration, reaction time between hydroxyl radical and the target
molecule) at a fixed concentration of antioxidant compound has to be optimized in order to
achieve the maximum DNA effect, but without a complete damage.
The ratio between the electrocatalytic current obtained after exposing the DNA-CPE
electrode to the Fenton mixture in the presence of a fixed amount of antioxidant (ascorbic
acid as a model molecule) (Ia) and the electrocatalytic current obtained in the absence of
the antioxidant (Id) was selected as a criteria for optimization. Id is the minimum value of
the electrocatalytic current under each experimental condition because the absence of
antioxidants in the damaging solution precluded the protection of the nucleobases from
radical oxidation, leaving the lowest amount of adenine bases for further electrochemical
oxidation. Therefore, the maximum value for this ratio will be selected in each
optimization.
The level of DNA damage was evaluated as a function of the variation of the
concentration of Fe2+ keeping constant the molar ratio Fe2+:EDTA:H2O2 used (1:2:40)
(Mello et al., 2006). Fig. 3 shows the effect of Fe2+ concentration (from 1 to 100 µmol l−1)
on the amount of adenine lesions generated, indirectly evaluated using the electrocatalytic
current of the NADH. When increasing the Fe2+ concentration in the absence of
antioxidant compound, the electrocatalytic current decreased indicating an increased
damaging power attributable to the generation of a higher concentration of ROS.
However, when experiments were carried out in the presence of 10 µmol l−1 ascorbic acid,
the scavenging effect was apparent (much higher currents) especially at lower Fe2+
5.1. Biossensores de ADN – radical hidroxilo
199
concentrations. From this results it is clear that the protective role of ascorbic acid strongly
depended on the concentration of radicals generated, and thus, at high OH•
concentrations, ascorbic acid could not longer prevent DNA damage (Ia very similar to Id).
The maximum value for the Ia/Id ratio was obtained at a Fe2+ concentration of µmol l−1,
which was chosen for further experiments.
Fig. 3. Influence of Fe2+ concentration (ratio Fe:EDTA:H2O2; 1:2:40) on the electrocatalytic current intensity of
0.5 mmol l−1 NADH with 0.01 mol l−1 CaCl2 in tris–HCl buffer at pH 9.0.
Considering that the reaction time between hydroxyl radicals and DNA depends on the
half-time of the generated free radicals, this parameter was optimized. The reaction time
was studied from 10 to 120 s. In the absence of antioxidant, a decrease in the
electrocatalytic current was observed when increasing the incubation time from 10 to 30 s.
At longer reaction times the electrocatalytic signal remained almost constant. On the
contrary, a continuous increase in the analytical signal when increasing the reaction time
in the presence of ascorbic acid was observed. The maximum value for the Ia/Id ratio was
obtained after 120 s, so this time was used in subsequent experiments.
3.2. Determination of antioxidant capacity
Foodstuff constitutes an excellent exogenous source of natural antioxidants to
counteract and prevent the deleterious effects of ROS. This protective effect is analytically
defined as TAC. In this work, ascorbic acid was used as a model antioxidant compound.
Ascorbic acid is a potent reductant agent that can reduce metal transition ions, thus, a
potential pro-oxidant role in vivo was suggested (Podmore et al., 1998). However, most
evidences point out to a predominant antioxidant role of ascorbic acid (Carr and Frei,
1999; Evans and Halliwell, 2001). Owing to the fact that DPV is a technique more
sensitive than CV, it was used for calibration purposes. The concentration of ascorbic acid
0.0
0.1
0.2
0.3
0 25 50 75 100
[Fe2+] / µmol l-1
I / µ
A
0.0
0.1
0.2
0.3
0 25 50 75 100
[Fe2+] / µmol l-1
I / µ
A
II. Investigação e desenvolvimento
200
was varied from 0.05 to 1.00 µmol l−1. Fig. 4 shows the catalytic current obtained after
immersion of the DNA-CPE on Fenton mixtures containing increasing concentrations of
ascorbic acid.
Fig. 4. DPVs obtained after immersion of DNA-CPE in Fenton solution containing a standard solution of AA:
(a) 0.05, (b) 0.10, (c) 0.50, (d) 0.80 and (e) 1.00 µmol l−1. Inset: relationship between Ia and AA concentration.
As expected, the electrocatalytic current of NADH increased when the concentration of
ascorbic acid increased up to µmol l−1 because of the availability of a larger number of
undamaged adenines for electrochemical oxidation. In the inset, the I measured by DPV
was plotted against the concentration of AA. A linear range from 0.05 to 1.00 µmol l−1 of
AA was found (I (nA) = 9.34 [AA (µmol l−1)] + 5.72; r = 0.995, n = 5). A limit of detection of
50 nmol l−1 was estimated from the regression parameters (concentration at which the
analytical signal is equal to the y-intercept plus three times the standard deviation of the
regression). The RSD was 3.2% at 1 µmol l−1.
Zhang et al. (2008) reported the study of DNA damage induced by Fenton system on a
glassy carbon electrode (GCE) and its protection with the antioxidant ascorbic acid. These
authors verified that ascorbic acid promoted protective effect on the DNA in a narrow
concentration range (from 1.5 to 2.5 mmol l−1) while at lower concentrations, a pro-oxidant
role was observed. This behavior was not observed in our experiments carried out on the
electrode surface and not in solution as in that study. Other recent study also reported the
protective effects on the DNA by applying ascorbic acid as a scavenging antioxidant
(Nobushi and Uchikura, 2010). Enzyme-modified electrodes using ascorbate oxidase and
I / n
A
E / V
-0.05 0.10 0.25- -0.20
20
30
40
50
a
e
[AA] / µmol l-1
I / n
A
0
9
18
0.0 1.0 2.0 3.0
I / n
A
E / V
-0.05 0.10 0.25- -0.20
20
30
40
50
a
e
[AA] / µmol l-1
I / n
A
0
9
18
0.0 1.0 2.0 3.0[AA] / µmol l-1
I / n
A
0
9
18
0.0 1.0 2.0 3.0
5.1. Biossensores de ADN – radical hidroxilo
201
peroxidase enzymes for the detection of AA showed linear ranges in the submM level
(Mello and Kubota, 2007), several orders of magnitude higher than the exhibited by this
DNA-based sensor, which emphasizes the good analytical performance.
Fig. 5 shows a comparison of the efficiency of different antioxidant compounds on
hydroxyl radical scavenging.
Fig. 5. Efficiency of hydroxyl radical scavenging of several antioxidant compounds: AA – ascorbic acid; GA –
gallic acid; CA – caffeic acid; RES - resveratrol.
The efficiency was expressed as the percentage of the electrocatalytic current according
to the following expression: % efficiency = Ia/Ib ×100, where Ia is the current intensity
measured after DNA damage in the presence of the antioxidant compound and Ib is the
electrocatalytic current measured when no damage was done (maximum expected value).
The compounds used were ascorbic acid, gallic acid, trolox, caffeic acid, and resveratrol.
Using an antioxidant concentration of 0.5 µmol l−1, it was observed that AA presented the
highest protective role among all compounds tested (58.6%). Hydroxyl radicals had the
ability to produce 87.6% of damage on the dA21 layer. The protective effect of antioxidants
ranged from 19.3 to 58.6%. The lowest values were found for gallic acid and trolox, 19.3
and 20%, respectively. Caffeic acid and resveratrol presented a similar protective role of
34.1 and 37.9%, respectively. Using chemiluminescence detection, gallic acid and trolox
showed higher protection than AA (Nobushi and Uchikura, 2010).
Antioxidant compound
0
Blank fenton AA Trolox GA CA RESBlank fenton AA Trolox GA CA RES
20
40
60
80
100
20
40
80
120
An
tio
xid
an
t e
ffic
ien
cy
(%
)
Antioxidant compound
0
Blank fenton AA Trolox GA CA RESBlank fenton AA Trolox GA CA RES
20
40
60
80
100
20
40
80
120
0
Blank fenton AA Trolox GA CA RESBlank fenton AA Trolox GA CA RES
20
40
60
80
100
20
40
80
120
20
40
60
80
100
20
40
80
120
An
tio
xid
an
t e
ffic
ien
cy
(%
)
II. Investigação e desenvolvimento
202
3.3. Application to TAC assessment in beverages
The methodology developed was used to quantify TAC on a lemon flavour and two
different brands of lemon flavoured waters samples. A lemon flavour was chosen because
this fruit is an important source of antioxidants such as vitamin C, phenolic compounds
and it is the most commercialised flavour in the world (Orak, 2009; Xu et al., 2010). For
the quantification of TAC in beverages, 5 ml of the flavor water or 20 µl of flavour were
diluted in 2× SSPE to a final volume of 10 ml. Then, the DNA-CPE was immersed in the
solution and a freshly prepared Fenton solution was added for 120 s. After this period of
time the DNA-CPE was washed and immersed in phosphate buffer pH 9 to carry out the
electro-oxidation of the remaining unoxidized adenine bases. The detection was carried
out in a Ca2+-containing-NADH solution. In Table 1, TAC values are expressed in ascorbic
acid content, both µmol l−1 and mg l−1. It was observed that all samples exhibited
antioxidant activity. As expected, the highest level was found in the flavour because is a
concentrated product. Brand B lemon flavoured water presented an order of magnitud
higher TAC value than brand A. In fact, water sample from brand B has lemon flavor and
also green tea. Green tea is a bioactive compound and contains numerous components
with antioxidant activity, such as polyphenols (catechins, epicatechin, epigallocatechin)
and vitamins (Cabrera et al., 2006; Neyestani et al., 2009) so, this may explain the high
TAC value.
Table 1. TAC of flavoured waters
Samples TAC (expressed in ascorbic acid)
(µmol l-1) (mg l-1)
Lemon flavour 480 ± 20 85 ± 4
Lemaon flavoured water
Brand A
Brand B
0.19 ± 0.08
2.8 ± 0.3
0.03 ± 0.01
0.50 ± 0.05
The reducing capacity of samples was measured by the total phenolic content assay
(TPC). This parameter is commonly assumed to be equivalent to the antioxidant capacity,
so a good correlation is expected (Huang et al., 2005). The TPC level of the flavour and
water from brand A and B was 380, 7.2 and 39.7 g of gallic acid l−1, respectively. As it was
expected, the highest TPC values were from lemon flavour. The water sample from brand
5.1. Biossensores de ADN – radical hidroxilo
203
B exhibited a higher TPC value than water from brand A, in good agreement with TAC
results. Finally, the DPPH radical scavenging activity and the reducing power activity were
both evaluated. All samples presented DPPH scavenging activity and reducing power
activity. DPPH values were 38.9, 44.88 and 41.5 g trolox l−1 for lemon flavour and water
from brand A and B, respectively. Reducing power activity values for the flavour and water
samples from brand A and B were 10.7, 3.8 and 9.6 mg of gallic acid l−1, respectively.
These four different methodologies applied constituted a powerful tool to elucidate a full
profile of TAC in food samples.
4. Conclusions
The natural electrochemistry of adenine bases was exploited to develop a DNA-based
biosensor for the assessment of total antioxidant capacity in beverages. A layer of dA21
adsorbed on CPE was damaged by hydroxyl radicals generated in a Fenton-type reaction.
The remaining unoxidized adenine bases were electrochemically oxidized to give rise to
an adsorbed oxidation product that was able to catalyze the oxidation of NADH in the
presence of calcium ions. Several antioxidant compounds were tested as hydroxyl
radicals scavengers exhibiting efficiencies ranging from 19 to 59%. Ascorbic acid showed
the highest protective role, so the DNA-CPE biosensor was used for the detection of this
molecule. The biosensor developed was disposable and required a very easy, rapid and
reproducible preparation. In addition to this, the low detectability (50 nM) allowed its
advantageous used for TAC evaluation in foodstuffs as it was sucessfully shown in
several beverages.
Acknowledgements
N.S.A. thanks to MICINN for a Ramón y Cajal contract. M.F.B. is grateful to the
Fundação para a Ciência e a Tecnologia for a PhD grant (Grant Number
SFRH/BD/29440/2006). This work was cofinanced by Projects CTQ2008-02429 and
FICYT IB08-087 and the European Regional Development Fund.
II. Investigação e desenvolvimento
204
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5.2. Biossensores de ADN – radical superóxido
207
Electrocatalytic evaluation of DNA damage by supero xide radical
for antioxidant capacity assessment
M.F. Barrosoa,b,c, N. de-los-Santos-Álvareza, M.J. Lobo-Castañóna, A.J. Miranda-
Ordieresa, C. Delerue-Matosc, M.B.P.P. Oliveirab, P. Tuñón-Blancoa aDepartamento de Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8,
33006 Oviedo, Spain bRequimte, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, R.
Aníbal Cunha n. 164, 4050-047 Porto, Portugal cREQUIMTE/Instituto Superior de Engenharia do Porto, Dr. Bernardino de Almeida 431,
4200-072 Porto, Portugal
Abstract
The integrity of DNA purine bases was herein used to evaluate the antioxidant capacity.
Unlike other DNA-based antioxidant sensors reported so far, the damaging agent chosen
was the O2• - radical enzymatically generated by the xanthine/xanthine oxidase system. An
adenine-rich oligonucleotide was adsorbed on carbon paste electrodes and subjected to
radical damage in the presence/absence of several antioxidant compounds. As a result,
partial damage on DNA was observed. A minor product of the radical oxidation was
identified by cyclic voltammetry as a diimine adenine derivative also formed during the
electrochemical oxidation of adenine/guanine bases. The protective efficiency of several
antioxidant compounds was evaluated after electrochemical oxidation of the remaining
unoxidized adenine bases, by measuring the electrocatalytic current of NADH mediated
by the adsorbed catalyst species generated. A comparison between O2• −
and OH• radicals
as a source of DNA lesions and the scavenging efficiency of various antioxidant
compounds against both of them is discussed. Finally, the antioxidant capacity of
beverages was evaluated and compared with the results obtained with an optical method.
Keywords : NADH electrocatalysis; DNA damage; Antioxidant capacity; Ascorbic acid;
Reactive oxygen species; Superoxide radical
Available online at pubs.acs.org
Journal of Electroanalytical Chemistry In press doi:10.1016/j.jelechem.2011.04.022
5.2. Biossensores de ADN – radical superóxido
209
1. Introduction
Deleterious oxidative processes mediated by free radicals, such as ROS, are involved in
aging and in a vast array of diseases, including cancer, inflammation, cardiovascular and
neurodegenerative diseases [1]. Therefore, overproduction of ROS can be dangerous for
cells [2]. The superoxide anion radical (O2•-) is the primary component of ROS and the
most abundant radical in biological systems, resulting from the single electron reduction of
oxygen [3]. This cytotoxic species is enzymatically produced by xanthine oxidase (XOD),
a metalloenzyme that catalyzes the oxidation of hypoxanthine and xanthine to uric acid
generating O2•- during the respiratory burst of phagocytic cells (Eq. (1)) [1]. Under normal
physiological conditions, the highly reactive superoxide radical undergoes dismutation by
non-catalytic and enzymatic reactions, thus the physiological concentration is rather low
[4].
(1) O 2H aciduric OOHXanthine -2
XOD22
•+ ++ →++
The biological effects of highly reactive ROS are controlled in vivo by a variety of
nonenzymatic and enzymatic antioxidant mechanisms. Superoxide radical is easily
attacked by other active biomolecules and scavenged by enzymes and antioxidants [5].
The major scavenger of this radical in vivo is the superoxide dismutase enzyme (SOD)
that catalyzes its disproportionation to H2O2. Subsequently, catalase detoxifies H2O2, and
glutathione peroxidase detoxifies H2O2 and converts lipid hydroperoxides into non-toxic
alcohols [1]. An additional protection can be provided by exogenous antioxidant
compounds, such as low molecular weight molecules, vitamin (A, E, C, β-carotene), and
minerals (Se, Zn). This exogenous protective effect can be achieved by the intake of
foodstuff and beverages, like vegetables, fruit, whole-grain, tea, juice and wine.
Photometric, chemiluminescent, fluorimetric, chromatographic and electrochemical
methods have been proposed for in vitro quantification of the antioxidant capacity (AOC)
in biological and food samples [6]. Electrochemical biosensors use two main sources of
ROS: OH• and O2
•-. The former can be generated photocatalytically [7] or by Fenton
reaction in DNA-based antioxidant sensors [8, 9], and the latter is mostly enzymatically [2,
10, 11] but also chemically [3, 12, 13] or electrochemically [14] formed for the
determination of both superoxide radical and AOC. Sensors based on O2•- commonly rely
on the immobilization of cytochrome c, which is reduced by superoxide radical, on gold [2-
4], carbon [15] or screen printed-Au-electrode [16] surfaces, where it is reoxidized. To
enhance the electrical contact between cytochrome c and the electrode and to increase
II. Investigação e Desenvolvimento
210
the surface coverage of this compound, several immobilization strategies have been
proposed mostly based on SAMs of thiols of different length [2-4, 15] and hemin modified
electrodes [17]. However, these sensors present the interference of H2O2, uric acid and
also some electrical communication problems between the protein and the electrode.
Another strategy is the immobilization of SOD by physical adsorption or through SAM
[18-20] on the electrode surface in order to follow the disproportionation of superoxide
radical by measuring the O2 and H2O2 formed. These biosensors presented interferences
derived from the high potential at which the generated H2O2 is detected, limiting the
practical application of the sensor.
Nonetheless, the protective effect of antioxidants at a cellular level could only be
achieved by monitoring the DNA integrity. To the best of our knowledge, all
electrochemical DNA-based antioxidant sensors developed so far used the hydroxyl
radical as a damaging agent, which caused strand scission or oxidative lesions in
nucleobases (guanine or adenine). Superoxide radical has not been used for this purpose
probably because the mechanism of O2•- damage on DNA is not completely understood. It
is believed that its participation is limited to promote the production of OH• radicals [21-
23]. However, it is important to develop assays to study other radical sources active in
cells and tissues and the way antioxidants eliminate it preventing its deleterious effect.
Antioxidants can react by different mechanisms depending on the free radical/oxidant
source or by multiple pathways against a single oxidant [24]. This observation implies that
there is no a universal assay for the detection of all antioxidants. To obtain a full profile of
antioxidant capacity against various ROS, the development of methods specific for each
ROS is needed.
In this work, the effectiveness of superoxide radical generated by the enzymatic reaction
between XOD and xanthine to induce damage on a DNA-based sensor is studied. Based
on previous work on electrochemical oxidation of adenine and guanine derivatives [25-28],
a minor product of the radical oxidation was identified. The oxidative lesions were
indirectly quantified after electrochemical oxidation of the remaining intact adenine bases
to generate a well-known catalyst species that mediates the oxidation of NADH. CV was
used to measure the electrocatalytic current after the subsequent immersion of the
damaged DNA-modified CPE in a NADH-Ca2+ containing solution. A dependence of the
electrocatalytic current on the concentration of antioxidant in the damaging solution was
found, which allowed the development of a voltammetric method for the determination of
AOC in flavored waters.
5.2. Biossensores de ADN – radical superóxido
211
2. Material and methods
2.1. Chemicals
Deoxyadenylic acid oligonucleotide (dA21) purchased as a desalted product, xanthine
oxidase (XOD) and xanthine were from Sigma-Aldrich (Madrid, Spain). Concentrated
saline sodium phosphate EDTA (20 × SSPE; 0.2 M sodium phosphate, 2 M NaCl, 0.02 M
EDTA), tris-HCl pH 9.0, phosphate buffer pH 9.0, gallic acid (GA), resveratrol (RES),
nicotinamide adenine dinucleotide disodium salt, reduced form (NADH), were also
acquired from Sigma-Aldrich. L(+)-ascorbic acid (AA) was from Riedel-de-Haën
(Germany). Caffeic acid (CA), and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid, a water-soluble derivative of vitamin E) were from Fluka (Madrid, Spain).
Other chemicals employed were of analytical grade. Stock solutions of 1 g L-1 dA21 were
stored at 4 ºC and diluted with 2 × SSPE buffer solution (prepared by dilution of 20 ×
SSPE solution) prior to use. All solutions were prepared with water purified with a Direct-Q
(Millipore) system.
2.2. Instrumentation
Cyclic voltammetry was performed with a µAutolab II controlled by GPES software,
version 4.8 (EcoChemie, The Netherlands). A conventional three electrode cell was used,
which includes a home-made CPE (3 mm in diameter) as a working electrode, a platinum
wire counter electrode and a Ag|AgCl|KClsat reference electrode to which all potentials are
referred. The CPE was prepared by mixing 1.8 g of paraffin oil as pasting liquid with 5 g of
spectroscopic grade graphite powder (Ultracarbon, Dicoex, Spain). The unmodified
carbon paste was introduced into the well of a Teflon electrode body provided by a
stainless steel piston. The surface was smoothed against a plain white paper while a
slight manual pressure was applied to the piston. Unless otherwise stated, after each
experiment, the CP was discarded and a new electrode surface was freshly prepared.
For temperature-controlled experiments a circulating thermostat HAAKE DC1 (Thermo
Electron GmbH, Germany) was used.
2.3. Assay procedure
Unless otherwise mentioned, experiments were structured in four steps: DNA layer
preparation, damage of oligonucleotide by immersion of the DNA-CPE on a XOD/xanthine
solution in the absence/presence of several antioxidants; electro-oxidation of the
II. Investigação e Desenvolvimento
212
remaining unoxidized adenines on the CPE, and detection in a Ca2+-containing NADH
solution.
DNA immobilization was performed by dry adsorption placing a 5-µL droplet of dA21 (180
mg L-1) in 2 × SSPE solution on the electrode surface and evaporating it to dryness under
a stream of warm air.
DNA damage was carried out by immersing the dA21-CPE in a freshly prepared
XOD/xanthine mixture (superoxide radical generating solution) in the absence or the
presence of antioxidant under controlled temperature (27.0 ± 0.1 ºC). The superoxide
radical was generated by the addition of XOD (0.1 U mL-1) to oxygen-saturated 2 × SSPE
solutions at pH 7.4 containing xanthine (4.4 × 10-5 M).
After a fixed reaction time, the DNA-CPE was washed with water and immediately
immersed in a 0.1 M phosphate buffer (pH 9.0) to carry out the electro-oxidation of the
remaining unoxidized adenine bases. 100 potential scans were performed between -0.2
and +1.4 V at 500 mV s-1 to ensure a complete oxidation [29].
For detection, the DNA-CPE was placed in a NADH solution (5.0 × 10-4 M in 0.1 M tris-
HCl pH 9.0) containing 0.01 M CaCl2. The electrocatalytic current of NADH was obtained
by CV sweeping the potential between -0.2 V and 0.5 V at 50 mV s-1.
2.4. Samples and description of alternative methods
Two lemon sparkling flavored water samples corresponding to two different brands were
purchased in a supermarket and stored in the dark at +4 ºC. Sonication was used to
eliminate gas from the sample. Label information from brand A indicates the presence of
vitamin C, some preservatives, such as sodium benzoate, potassium sorbate and the
acidifying regulator citric acid. Label from brand B sample indicates the presence of green
tea and citric acid. A lemon flavor used in the formulation of some water brands was also
analyzed. This flavor had no description about its chemical or aroma composition.
For the measurement of AOC in beverages, 200 µl of the flavored water or 10 µl of
flavor were diluted in 2 x SSPE to a final volume of 500 µl. Then, the DNA-CPE was
immersed in the solution and a freshly prepared superoxide radical was added for 10 min.
After this period of time the DNA-CPE was washed and immersed in a phosphate buffer to
carry out the electro-oxidation of the remaining unoxidized adenine bases. The detection
was carried out in a Ca2+-containing NADH solution.
A colorimetric assay, based on a procedure previously reported [30], was used to
elucidate the antioxidant profile of the simples, expressed as the total phenolic content
(TPC). Folin-Ciocalteu reagent was used, and the reduced phenols produced a stable
blue product at 760 nm. The results were expressed as mg of GA L-1.
5.2. Biossensores de ADN – radical superóxido
213
3. Results and discussion
Oxygen and its reactive species are very important in oxidative metabolism. ROS induce
oxidative damage producing a variety of modifications at DNA level including base and
sugar lesions, strand breaks, DNA-protein cross-linking and base-free sites [31]. In order
to verify that O2•- generated by a xanthine/XOD reaction is able to oxidize dA21 on the
electrode surface, the DNA-CPE was placed in a freshly prepared xanthine/XOD solution
in 2 × SSPE buffer (pH 7.4) for 15 min. After transferring to a phosphate buffer solution
(pH 9), a small quasi-reversible redox process was observed at low potentials, Eº’ = 0.041
V (Fig. 1a). The amount of compound generated (surface coverage, Γ) was estimated to
be 1.2 × 10-11 mol cm-2 from the integrated change under the anodic wave. An extended
voltammetric scan up to 1.4 V did not show any oxidation peak at 1.2 V (oxidation
potential of adenine bases in DNA) but a gradual increase in the magnitude of the redox
process at low potentials was observed after several potential scans (Fig. 1b).
Fig. 1 . CVs obtained at 50 mV s-1 in tris–HCl pH 9.0 after: (a) immersion of dA21-CPE in a superoxide radical
generating solution ([XOD] = 0.3 U mL-1, [xanthine] = 4.4 x 10-5 M) for 15 min and (b) subsequent
electrochemical oxidation of the undamaged adenine bases adsorbed on the dA21-CPE.
Since the only oxidizable species was the oligonucleotide adsorbed on CPE, this
behavior is in good agreement with a partial oxidation of adenine bases by the superoxide
radical. Therefore, the intact adenines can be further electrochemically oxidized at the
electrode surface at 1.2 V leading to the product responsible for the redox couple at low
potentials. It is worth mentioning that the oxidation current of the remaining adenines was
not observed because it was overlapped by the rising background current at the high
potential at which it takes place. Additionally, the Eº’ of redox processes originated from
radical attack (Fig. 1a) and electrochemically (Fig. 1b) were virtually identical suggesting
II. Investigação e Desenvolvimento
214
that the same compound is formed in both types of oxidation. According to previous
studies on the electro-oxidation of adenine derivatives on carbon electrodes in phosphate
buffer [28, 32, 33], the compound responsible for redox process at +0.041 V is a diimine
species strongly adsorbed on the electrode surface. This compound was also identified
after oxidation of guanine derivatives [25-27]. Therefore, it can be concluded the existence
of a common lesion on DNA generated by O2•- generated by the xanthine/XOD system
and by electrochemical oxidation. However, from Fig. 1 it is apparent that the adsorbed
diimine species was a minor product of the radical oxidation and the yield was much lower
than in the electrochemical oxidation. This result indicated that the product profile and
compound distribution differed, thus, both oxidations are mechanistically different.The fact
that the O2•- attack on DNA led to the generation of this adsorbed compound is remarkable
because the oxidation of adenine bases through OH• radicals generated by Fenton-type
reaction was recently demonstrated not to occur via the formation of the diimine species,
at least, at levels detectable by CV [29]. Given that both radical attacks led to different
products, the reported primary OH• radical promoter role of O2•- remains uncertain.
The adsorbed species was shown to efficiently catalyze the oxidation of NADH reducing
the overpotential by more than 300 mV at pyrolytic graphite electrodes [27, 28, 32]. This
ability can be exploited, in principle, to detect the DNA damage. However, the low yield
achieved by radical oxidation did not allow observing an electrocatalytic current sufficiently
high to be used as an analytical signal. In fact, no significant current was observed at
potentials close to the redox process when NADH was added to the solution after DNA
damage by superoxide radicals (data not shown). To solve this problem an indirect
method was tested. The unoxidized adenine bases were electrochemically oxidized to
generate a larger amount of diimine (catalyst) species. Therefore, the higher the damage,
the lower the intact adenine available for further electrocatalytic measurement in the
presence of NADH. To electro-oxidize the remaining adenine adsorbed on the CPE,
several cyclic scans were carried out up to 1.4 V. After this step, the damaged DNA-CPE
was immersed in a NADH-Ca2+ solution. The use of calcium ions was reported to greatly
improve the electrocatalytic current of NADH [34, 35]. An apparent electrocatalytic wave
was observed at a potential as low as 0.011 V with a plateau at about 0.14 V (Fig. 2,
curve a). Given that the oxidation peak of the uncatalyzed oxidation of NADH at a bare
unmodified CPE is 0.70 V [29], a decreased of more than 550 mV is achieved. A low
potential is advantageous for analytical purposes because of the diminution of potential
oxidizable interferent compounds present in real food samples. Under these conditions,
this was the lowest electrocatalytic current possible because it arose from the maximum
damage. In the presence of antioxidant compounds a diminution in the damage was
expected along with an increase in the electrocatalytic current. When an antioxidant, AA
5.2. Biossensores de ADN – radical superóxido
215
(10 µM), was added to the superoxide radical generating solution, a high augment of the
electrocatalytic current was observed (Fig. 2, curve b).
Fig. 2 . CVs obtained with a dA21-CPE at 50 mV s-1 in tris–HCl pH 9.0 containing 0.5 mM NADH + 0.01 M
CaCl2 after; immersion in O2•- generating solution ([XOD] = 0.1 U mL-1, [xanthine] = 4.4 x 10-5 M) for 10 min (a)
in the absence of antioxidant (b) in the presence of 10 µM of AA; and further complete electrochemical
oxidation in both cases.
This anticipated behavior was related to the ability of antioxidant compounds to
scavenge or inactivate the ROS and prevent the damage on DNA. As a consequence, the
number of lesions diminished, yielding a larger number of adenine available for
electrochemical oxidation. A positive correlation between the partial oxidation of DNA by
O2•- and the concentration of antioxidant species in the tested solution would allow the use
of the electrocatalytic current of NADH to evaluate the AOC on flavored waters.
3.1. Selection of the experimental conditions for the damaging reaction
In order to determine AOC on beverages, some parameters concerning the damaging
reaction (xanthine and XOD concentration, reaction time between superoxide radical and
the target molecule) at a fixed concentration of antioxidant compound were varied in order
to achieve the maximum effect on DNA without a complete damage. For this reason, for
each experiment the ratio between the electrocatalytic current obtained after exposing the
DNA-CPE to the superoxide radical in the presence of a fixed amount of AA as antioxidant
(Ia) and the electrocatalytic current obtained in the absence of AA (Id, minimum value
expected) was estimated. The highest value for this ratio was always selected for further
experiments. The level of DNA damage was evaluated as a function of the amount of
radical formed through the variation of the concentration of XOD and xanthine. XOD
concentration was studied between 0.05 and 0.20 U mL-1. Fig. 3A shows the influence of
XOD concentration on the electrocatalytic current of NADH.
II. Investigação e Desenvolvimento
216
Fig. 3. Influence on the electrocatalytic current of 0.5 mM NADH + 0.01 M CaCl2 in tris–HCl at pH 9.0 of (A): XOD concentration: (B) xanthine concentration: and (C) time reaction: (o) without AA and (·) with 10 µM of AA to the O2
·−.
When increasing the XOD concentration in the absence of antioxidant compound (open
circles), the electrocatalytic current decreased until a XOD concentration of 0.10 U mL-1.
At higher concentrations the current remained constant. This behavior suggested that an
increase in the enzyme concentration implied a larger number of lesions attributed to the
5.2. Biossensores de ADN – radical superóxido
217
superoxide radical attack. The damage on the dA21 layer exhibited a maximum (minimum
electrocatalytic current) at a XOD concentration of 0.10 U mL-1. When the same
experiments were carried out in the presence of ascorbic acid (10 µM), the protective
effect on the DNA was apparent because the electrocatalytic currents were virtually
constant up to 0.10 U mL-1, within the experimental error (Fig. 3A, filled circles). Only at
higher concentrations of enzyme the analytical signal diminished suggesting that the
ascorbic acid concentration is not sufficient to compensate the increase in the amount of
superoxide radicals generated. In addition to this, it is worth noting that, with the addition
of this powerful antioxidant, consistently higher currents were measured at all XOD
concentrations (Fig. 3A). The highest value for the Ia/Id ratio was obtained at a XOD
concentration of 0.10 U mL-1, which was chosen for the next optimization steps.
Xanthine concentration was varied from 4.40 × 10-6 to 4.40 × 10-4 M. The influence of
this parameter within the range assayed was very limited. A slight decrease in the
electrocatalytic current was observed when increasing the xanthine concentration in the
absence of AA, which is not significant within the experimental error (Fig. 3B open circles).
In the presence of antioxidant species, all currents were clearly higher and a small but
relevant increase was apparent at 4.40 × 10-5 M (filled circles). At higher concentrations a
further diminution was observed. This behavior is in good agreement with a scavenging
activity of AA. The highest Ia/Id ratio was observed at a xanthine concentration of 4.40 ×
10-5 M, and this value was used for the next experiments.
The reaction time between the superoxide radical and dA21 layer depends on the
halftime on the generated ROS, so, this parameter is an important feature to select. The
reaction time between the free radical, the superoxide, and the DNA adsorbed on the CPE
was studied between 5 and 30 min. Increasing the incubation time, the electrocatalytic
current of NADH decreased during the first 10-15 min. (Fig. 3C, open circles). At longer
reaction time the current remained constant. With the introduction of ascorbic acid (10 µM)
on the reaction system, the electrocatalytic current measured was higher than in its
absence at all reaction times assayed in good agreement with the radical scavenging role
(Fig. 3C, filled circles). Nevertheless, a decrease is observed up to 15 min although the
remaining electrocatalytic current is significantly higher than in the absence of AA (Fig.
3C). This behavior indicated that, even at very long times, AA was able to partially protect
the integrity of DNA from O2•-
radical attack. The highest value of Ia/Id ratio was found
when an incubation time of 10 min. was used, so, this value was selected for further
studies.
II. Investigação e Desenvolvimento
218
3.2. Determination of AOC
In this work, the antioxidant ascorbic acid was used as a model for the study of the
behavior of antioxidants on the protection of DNA against O2•- radicals generated by
XOD/xanthine reaction. The feasibility of measuring the antioxidant concentration was
investigated varying the concentration of AA from 10 to 100 µM. A linear range wasfound
for the entire range (I (nA) = (0.85 ± 0.07) [AA (µM)] + (16 ± 5); r = 0.990 n = 5). The limit
of detection was estimated using the regression parameters obtaining a value of 10 µM.
The reproducibility expressed as RSD was 4.2% at 50 µM. Fig. 4 shows CVs obtained in a
Ca2+-containing NADH solution after immersing the DNA-CPE in a superoxide radical
solution with increasing concentrations of AA. The catalytic current of NADH increased up
to 100 µM due to the availability of a larger number of undamaged adenines for
electrochemical oxidation. At concentrations above this value the electrocatalytic current
remained constant indicating the saturation of the ability of AA to counterbalance the
radical attack.
Fig. 4. CVs at 50 mV s-1 obtained in tris–HCl at pH 9 containing 0.5 mM NADH + 0.01 M CaCl2 after
immersion of DNA-CPE in O2·−)generating solution ([XOD] = 0.1 U mL-1, [xanthine] = 4.4 x10-5 M) containing a
standard solution of AA: (a) 10, (b) 30, (c) 50, (d) 80 and (e) 100 µM for 10 min. Inset panel: relationship
between Ia and AA concentration.
Other authors have also used AA in order to study its protective effect on the DNA
(adsorbed at an electrode surface) against free radicals. However, all these reports only
described the scavenging role of AA towards hydroxyl radicals [29, 36, 37].
5.2. Biossensores de ADN – radical superóxido
219
As mentioned before, no DNA sensors for antioxidant assessment using other ROS,
such as superoxide radical, have been reported so far. Two reports described the use of
AA as a standard antioxidant against superoxide radical, but the biolayer on the electrode
was formed by cytochrome c or SOD [2]; or the electrochemically generated radical was
detected directly on a glassy carbon disk electrode [14]. From our previous work on
antioxidant activity against OH• on DNA-CPE, it can be concluded that AA seemed to be
less efficient as a scavenger of superoxide radical than hydroxyl radicals. In fact, the
minimum AA concentration able to show a protective action is more than two order of
magnitude lower in the case of OH• [29].
In order to compare the efficiency of radical scavenging, several antioxidants (AA, GA,
trolox, CA, and RES) were tested at a concentration of 10 µM under the same
experimental conditions and the results are shown in Fig. 5.
Fig. 5. O2
•- scavenging efficiency of several antioxidant compounds: AA – ascorbic acid, GA – gallic acid, CA –
caffeic acid, RES – resveratrol. Values are expressed as percentage of the electrocatalytic current obtained
with an intact (not damaged) dA21 layer that remained after exposure to a damaging solution containing 10 µM
of antioxidant species.
The efficiency was expressed as the percentage of the electrocatalytic current according
to the following expression: % efficiency = Ia/Ib x 100, where Ia is the current intensity
measured after DNA damage in the presence of the antioxidant compound, and Ib is the
electrocatalytic current measured when no damage was done (maximum expected value).
It was found that the superoxide radical generated 85% of damage on the dA21 layer, that
is, in the absence of a scavenging molecule. The protective effect of antioxidants ranged
from 33% to 63%. The lowest values were found for trolox and CA, 33% respectively.
0
20
40
60
80
100
120
Blank superoxideradical
AA Trolox GA CA Res
Antioxidant compound
An
tio
xid
an
t e
ffic
ien
cy
(%
)
0
20
40
60
80
100
120
Blank superoxideradical
AA Trolox GA CA Res
Antioxidant compound
An
tio
xid
an
t e
ffic
ien
cy
(%
)
II. Investigação e Desenvolvimento
220
RES presented the highest protective effect (62.5%). AA and GA presented a protective
role of 53.8% and 53.2 % respectively.
At this point, it is interesting to note that superoxide radicals caused a similar degree of
damage on DNA adsorbed on CPE to hydroxyl radical [29]. Although efficiency values
were similar or much higher than those obtained with OH•, the antioxidant concentration
employed is much higher, which is in good agreement with the lower scavenging activity
above found. This result was not unexpected because it is commonly accepted that not all
antioxidants behaves equally against different radicals [24]. It was clear that the efficiency
order differed from that obtained against. Whereas AA and CA exhibited similar protecting
roles against both radicals (about 55% and 33% respectively), the effectiveness of RES
dramatically increased from 38% for OH• to 62.5% for O2•-. In any case both compounds
were the most effective antioxidants assayed. Similarly, GA was much more active for O2•-
than for OH• shifting from 19.3% (the worst one) to 53.2% virtually identical to AA within
the experimental error.
Once the analytical features of the electrocatalytic voltammetric method were
characterized in aqueous solution, it was applied to the determination of AOC in real
samples. A lemon flavor and two different brands of lemon flavored water samples were
chosen because this citrus fruit is used and commercialized all over the world and is rich
in antioxidants such as vitamin C and phenolic compounds. As it is shown in Table 1, all
samples presented antioxidant capacity. Lemon flavor exhibited the highest level of AOC
expressed in mg L-1 of AA. This finding was expected because this flavor is extracted from
the fruit along with essential oils, and has several substances at high concentration in its
composition. Lemon water from brands A and B had a similar AOC value. However, the
composition of both samples was different because brand B had green tea in addition to
vitamin C.
Table 1. AOC values of flavored waters obtained using the electrochemical and optical methods
Samples DNA-CPE (mg l-1 AA) TPC (mg l-1 GA)
Lemon flavor 124 ± 13 380
Lemon flavored water
Brand A
Brand B
30.2 ± 0.7
31.0 ± 7.5
7.2
39.7
5.2. Biossensores de ADN – radical superóxido
221
Among the methods used for antioxidant capacity assessment, the Folin-Ciocalteu
method for the quantification of the phenolic content is widely used because its
robustness, simplicity and cost-effectiveness [24]. In general, phenolic compounds
content correlates with antioxidant activity and seems to have an important role in
stabilizing lipid oxidation. Therefore, the TPC of these samples was evaluated and
expressed in mg L-1 of GA. As expected, the highest TPC value was found in the lemon
flavor (Table 1). The water sample from brand B exhibited a significantly higher TPC value
than water from brand A. This difference on the TPC values was attributed to the
presence of green tea in brand B. Green tea contains polyphenols (catechins, epicatechin,
epigallocatechin) in addition to vitamins [38, 39]. Some phenols react with TPC reagents
although they may not necessarily be efficient radical scavengers [40]. The presence of
this type of phenolic compounds might explain the discrepancy between values obtained
for both voltammetric and TPC methods.
4. Conclusions
A DNA-CPE antioxidant biosensor for the assessment of AOC in beverages was
developed. For the first time in this type of devices, the effectiveness against damage of
superoxide radical on DNA was evaluated testing different antioxidant compounds.
Although the damage in terms of adenine oxidative lesions was similar to that found using
hydroxyl radicals, the scavenging activity of the antioxidant tested was lower because a
much higher concentration was needed to obtained similar efficiencies. The order of
protective efficacy was also different and as follows, RES> AA> GA> trolox ~ CA. A minor
product of the radical oxidation was identified by CV as a diimine compound that did not
appear when the oxidant source was the OH• radical. This result suggested that the
mechanism of O2•- attack on DNA is more complex that the reported promotion / source of
OH• radicals. In spite of the lower efficiency of AA as O2•- scavenger, the indirect
electrocatalytic method described allowed the quantification of ascorbic acid from 10 µM
and AOC determination in flavored waters and extracts.
Acknowledgements
N.S.A thanks to MICINN for a Ramón y Cajal contract. M. Fátima Barroso is grateful to
the Fundação para a Ciência e a Tecnologia for a PhD grant (Grant Number
SFRH/BD/29440/2006). This work was co-financed by Projects CTQ2008-02429 and
FICYT IB08-087 and the European Regional Development Fund.
II. Investigação e Desenvolvimento
222
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225
CONSIDERAÇÕES FINAIS
Considerações finais
227
Considerações gerais
Com muita frequência são lançados no mercado novos produtos alimentares e bebidas
com o objetivo de proporcionar ao consumidor uma alimentação diversificada e saudável.
Geralmente, no momento de distribuição, os consumidores não têm informações relativas
à completa composição destes novos produtos e aos seus benefícios para a saúde.
Ainda recentemente, assistimos ao aparecimento no mercado de uma grande diversidade
de águas com sabores. Quase todas as marcas de águas engarrafadas, prepararam as
respetivas águas de sabores os refrigerantes.
Este projeto de doutoramento centrou-se essencialmente no estudo de águas com
sabores designadamente (i) na avaliação do teor de macrominerais, microminerais, e
elementos vestigiais, (ii) na análise dos aromas usados nas formulações, identificando a
presença de alguns compostos recorrendo à cromatografia gasosa com deteção MS; (iii)
na determinação da capacidade antioxidante por métodos convencionais; e (iv) no
desenvolvimento de metodologias alternativas com recurso a biossensores
eletroquímicos para a avaliação da capacidade antioxidante total. Este último
procedimento foi o mais moroso mas ao mesmo tempo o mais inovador por envolver
pesquisa, desenvolvimento, otimização e validação das metodologias.
Inicialmente fez-se uma prospeção de mercado de modo a saber quantas marcas
comercializavam estas bebidas e quais os sabores disponíveis. Foram encontradas 10
marcas comerciais diferentes e 12 sabores diferentes. Ao longo do trabalho foram
surgindo novos sabores enquanto, outros aromas iam sendo retirados do mercado.
Considerando que os rótulos existentes nas garrafas destas águas com sabores não
contêm informações sobre o seu conteúdo mineralógico, fez-se uma análise química às
águas tendo-se analisado 18 minerais (4 macrominerais, 3 microminerais e 11 elementos
vestigiais). O critério de escolha destes minerais teve ainda em consideração o fato de os
minerais poderem ser interferentes (a nível do sinal elétrico) na análise eletroquímica da
capacidade antioxidante das amostras. Os resultados comprovam que os teores destes
minerais encontram-se dentro do limite estipulado por lei variando contudo de marca para
marca, o que está relacionado com a litologia e geoquímica do local de captação da
água. De um modo geral verificou-se que as águas minerais com gás natural (água
mineral natural gasosa) têm um teor mais elevado em minerais do que as águas de
nascente ou as águas minerais com gás adicionado (água mineral natural gaseificada).
Na avaliação da capacidade antioxidante das águas com sabores usaram-se
metodologias óticas convencionais (UV-vis). O perfil antioxidante foi obtido através da
Considerações finais
228
determinação do teor fenólico e de flavonóides total, poder redutor e atividade anti-
radicalar. A tabela 1 apresenta os valores mínimos e máximos referentes ao perfil
antioxidante.
Tabela 1 . Quadro resumo do perfil antioxidante das águas com sabores e aromas por métodos
convencionais.
amostras Teor fenólico total
(mg GA L-1)
Poder redutor
(mg GA L-1)
DPPH
(mg trolox L-1)
Aromas 8,53-380,20 10,72-11,80 38,90-213,53
Agua com 1 aroma 0,29-17,62 0,28-6,10 8,05-54,21
Água com 2 aromas 1,51-39,70 0,14-13,78 13,49-48,66
Agua com vitamina C 147,0-284,0 3,77-154,04 44,78-268,89
GA – gallic acid
Como se pode verificar cada método conduz ao seu resultado pois embora todos eles
meçam a capacidade antioxidante, cada um reporta-se a uma família de compostos. Por
exemplo, o teor fenólico total e o poder redutor medem a capacidade antioxidante
equivalente ao ácido gálico, e a atividade antiradicalar (radical DPPH) usando o
antioxidante trolox. Genericamente, os aromas apresentam um elevado teor fenólico,
poder redutor e atividade antiradicalar. Relativamente às águas com sabores, de um
modo geral os valores mais elevados obtiveram-se com amostras de águas contendo 2
sabores mas em que o segundo aroma é um composto bioativo (chá verde, ginseng,
Ginkgo biloba, menta). Algumas amostras contendo vitamina C também apresentam
composição com altos valores de capacidade antioxidante. Em nenhuma das amostras
foram detetados flavonóides.
Considerando que o laboratório onde decorreram os trabalhos tem uma larga
experiência no domínio da eletroquímica, neste trabalho pretendeu-se também
desenvolver métodos alternativos para a avaliação da capacidade antioxidante das águas
com sabores, tendo-se construído um biossensor e usado técnicas voltamétricas. De
acordo com o que se encontra publicado, os radicais livres provocam danos oxidativos no
ADN, mas, como mecanismo de defesa, a célula usa os antioxidantes que conseguem
desativar os radicais livres e evitar as reações de propagação destes e assim proteger as
macromoléculas. De acordo com este princípio, a construção dos biossensores baseou-
se na imobilização de bases púricas ou de cadeia simples de ADN na superfície do
elétrodo, na danificação deste biossensor com radicais livres e na proteção do material
imobilizado no elétrodo com antioxidantes. O sinal eletroquímico obtido na voltametria de
onda quadrada foi usado para avaliar a eficácia dos biossensores.
Considerações finais
229
Na tabela 2 apresenta-se um quadro resumo da capacidade antioxidante obtida
(valores mínimos e máximos) com os biossensores de adenina e guanina, e de cadeia
simples de ADN usando-se como danificador o radical hidroxilo, superóxido e sulfato e
como antioxidante padrão o ácido ascórbico (AA), o ácido gálico (AG), o ácido cafeico
(AC), o ácido cumárico (ACu) e o resveratrol (RES).
Tabela 2. Quadro resumo da utilização de biossensores para a determinação da capacidade
antioxidante de aromas e águas com sabores.
Amostras Biossensor Radical Antioxidante
Aromas Águas
AA (mg L-1) 100,8-185,6 2,9-40,3
GA (mg L-1) 9,2-30,1 0,1-1,4
CA (mg L-1) 39,2-66,7 1,5-4,3
Acu (mg L-1) 32,7-59,0 1,2-3,9
Hidroxilo
RES (mg L-1) 7,8-28,4 0,4-1,7
AA (mg L-1) 74,8-220 0,7-15,9
GA (mg L-1) 4,4-32,7 0,4-3,2
CA (mg L-1) 2,3-41,5 0,5-3,2
Acu (mg L-1) 32,2-68,4 0,6-4,7
Superóxido
RES (mg L-1) 15,3-29,0 0,3-2,1
AA (mg L-1) 101,8-281,9 3,8-16,8
GA (mg L-1) 34,7-55,5 0,2-2,8
CA (mg L-1) 60,2-92,3 0,5-5,1
Acu (mg L-1) 61,2-83,6 1,3-4,5
Guanina
Sulfato
RES (mg L-1) 13,7-34,5 0,4-1,7
AA (mg L-1) 211,6-571,9 3,3-37,5
GA (mg L-1) 14,6-32,5 0,1-2,0
CA (mg L-1) 19,9-31,9 0,9-2,3
Acu (mg L-1) 12,9-35,4 0,8-5,3
Hidroxilo
RES (mg L-1) 10.7-23,6 0,1-1,7
AA (mg L-1) 126,0-202,7 0,3-19,3
GA (mg L-1) 37,4-55,2 0,3-3,4
CA (mg L-1) 13,1-25,9 0,7-1,7
Acu (mg L-1) 6,4-35,3 0,6-2,5
Superóxido
RES (mg L-1) 12,1-26,9 0,7-1,9
ÁA (mg L-1) 24,6-239 0,8-5,6
GA (mg L-1) 7,2-50,6 0,5-2,1
CA (mg L-1) 24,6-235,4 1,3-3,6
Acu (mg L-1) 51,6-77,8 1,4-3,5
Adenina
Sulfato
RES (mg L-1) 12,5-37,2 0,2-1,5
Hidroxilo AA (mg L-1) 85 0,5 dA21
Superóxido AA (mg L-1) 124 31,0
Considerações finais
230
Estes valores, não são comparáveis com os obtidos pelos métodos convencionais já
que a unidade de medida é diferente, mas a metodologia desenvolvida, à semelhança
dos métodos convencionais permite, comparar a capacidade antioxidante relativa nas
diferentes amostras. Como se pode verificar, as águas com sabores apresentam alguma
capacidade antioxidante sendo que os maiores teores foram encontrados nos aromas
utilizados nas formulações.
Assim sendo, traçou-se o perfil antioxidante das águas com sabores, e constatou-se
que em princípio terão algum efeito positivo na saúde.
A matriz chá encontra-se em estudo, estando-se a efetuar o mesmo procedimento que
foi realizado às águas.
Este trabalho permitiu um avanço significativo na área da eletroquímica,
concretamente na construção de biossensores, no grupo onde foi desenvolvido. Como
trabalho futuro seria interessante otimizar a utilização dos biossensores, nomeadamente
reduzir o tempo de construção recorrendo-se para isso a elétrodos descartáveis (screen-
printed) e pré-fabricados com o material biológico imobilizado e assim usar estes
biossensores para análises de rotina, da capacidade antioxidantes de alimentos e
bebidas.