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Protective effect of Crataegus monogyna Jacq. ethanolic extracts in oxidant-induced DNA damage evaluated through comet assay with human peripheral lymphocytes

Publication . Barreira, João C.M.; Costa, Carla Sofia; Teixeira, João Paulo Fernandes; Ferreira, Isabel C.F.R.; Oliveira, Beatriz

Much attention of preventive medicine research is focused on natural antioxidants. This interest refers not only to isolation and identification of new biologically active molecules for the pharmaceutical industry, but also because of the emergent public interest in using crude plant extracts, such as infusions for self-medication (Krishnaiah et al., 2011). The use of antioxidants, such as the well-known polyphenolic compounds, to prevent genetic damage induced by physical or chemical agents is of considerable interest. This bioactivity might be related to their anticlastogenic effect, due to the presence of specific functional groups. Other antioxidant compounds, such as vitamins C and D, were reported for their DNA-damage decreasing effect, suggesting that reactive oxygen species may be involved in this activity (Benavente-García et al., 2004). Evaluating the antioxidant activity of natural matrices represents one of our primary research challenges (Ferreira et al., 2009). Among hundreds of studied species, Crataegus monogyna Jacq. stood out as being one of the most promising plants due to its high bioactivity. Besides the antioxidant activity, C. monogyna was also studied for the human tumour cells growth inhibitory capacity of its phenolic extracts; furthermore, individual phenolic compounds were fully characterized by high performance liquid chromatography-photo diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI/MS), revealing high levels of flavonols, flavones, hydroxycinnamic acid derivatives and anthocyanins (Rodrigues et al., 2012). However, there is a high limitation in examining if the detected bioactivity is actually transferred from in vitro to in vivo systems (Carocho and Ferreira, 2013). Nevertheless, there have been major advances to accurately measure end products of oxidative damage to proteins, lipids, and DNA. Short-term in vitro tests are commonly used to identify genotoxicants and antigenotoxicants. Comet assay, in particular, has been considered to be a useful biomarker to this purpose as it can easily establish a link between natural extracts and DNA repair damage (Cemeli et al., 2009). Although this assay might be carried out virtually with any cell type, lymphocytes are often used as these are in contact with xenobiotics after absorption and have proved to be good surrogate cells (Cemeli et al., 2009). Herein, the potential genotoxic and antigenotoxic effect of ethanolic extracts from different botanical parts of C. monogyna was evaluated by the alkaline comet assay. Extracts were assayed to match the previously determined effective concentrations (EC) corresponding to 25% (EC25), 37.5% (EC37.5), 50% (EC50), 62.5% (EC62.5) and 75% (EC75) of antioxidant activity. Blood samples were obtained from healthy non-smoking donors in EDTA tubes and lymphocytes were isolated by Ficoll density gradient. A pool of lymphocytes was exposed to extract in different concentrations in addition to a positive and negative control for 1h to test for extracts’ genotoxicity. To test for extracts antigenotoxicity, two concentrations were selected and cells were exposed to a mutagenic compound (MMS) and extract concentrations simultaneously for 1 h. In one experiment, comet assay was immediately performed after the exposure period while in the second experiment, cell media were renewed and the comet assay was performed after a recovery-period of 1 h. All experiments were carried out with and without metabolic activation by liver fraction (S9). The results showed significant differences within the assessed botanical parts and also among the assayed concentrations. The performed investigation might be considered as representing a step further in the evaluation of the in vivo bioactive potential of this highly promising species. Furthermore, it has established some practical bases for the evaluation of additional natural matrices with high scoring in bioactivity screening studies.

2015Conference object

Open access

A influência da radiação gama na composição nutricional de flores comestíveis de Bauhinia variegata L. provenientes do Brasil

Publication . Heleno, Sandrina A.; Villavicencio, Anna L.C.H.; Barros, Lillian; Ferreira, Isabel C.F.R.

Desde há muitos anos que a utilização de flores comestíveis na culinária se tornou uma prática comum, uma vez que conferem uma melhor qualidade sensorial e nutricional aos alimentos, para além do aspeto visual atrativo [1,2]. Bauhinia variegata L. é uma árvore muito comum no Brasil, sendo as suas flores grandes e de coloração lilás na variedade mais comum. Estas flores são vulgarmente conhecidas como “pata de vaca”, sendo comestíveis e muito utilizadas em saladas e na decoração de vários pratos nas cozinhas Gourmet. No presente estudo, as flores foram submetidas a radiação por feixe de eletrões em diferentes doses (0.5 e 0.8 KGy) para efeitos de descontaminação, e seguidamente analisadas em termos de parâmetros nutricionais nomeadamente, composição centesimal (humidade, proteínas, gordura, hidratos de carbono e cinzas), perfil em açúcares livres (determinados por HPLC-RI) e em ácidos gordos (analisados por GC-FID). As amostras não irradiadas (controlo) e irradiadas revelaram um perfil nutricional muito similar, sendo que os hidratos de carbono foram os nutrientes mais abundantes, seguidos das proteínas, cinzas e gorduras. Relativamente aos açúcares, as amostram mostraram também um perfil semelhante, tendo sido identificados: a frutose em maior quantidade, seguida da glucose e da sacarose. Os ácidos gordos: palmítico (C16:0), esteárico (C18:0), oleico (C18:1n9), linoleico (C18:2n6) e α-linolénico (C18:3n3) foram os mais abundantes nas amostras em estudo, tendo sido identificados mais 16 ácidos menos abundantes. Os ácidos polinsaturados (PUFA) foram maioritários, seguidos dos saturados (SFA) e monoinsaturados (MUFA). Também neste caso as amostras revelaram uma composição muito idêntica. Em suma, a técnica de irradiação pode ser utilizada como forma de descontaminação e preservação de flores comestíveis uma vez que as doses aplicadas não alteraram significativamente os parâmetros nutricionais das amostras em estudo.

2015Conference object

Open access

Crocin and β-Carotene bleaching assays as analytical tools in the optimization of the extraction of hydrophilic and lipophilic antioxidants from tomato

Publication . Pinela, José; Prieto Lage, Miguel A.; Barreiro, M.F.; Carvalho, Ana Maria; Oliveira, Beatriz; Vázquez, J.A.; Ferreira, Isabel C.F.R.

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop worldwide and a rich source of hydrophilic (H) and lipophilic (L) antioxidants. The H fraction is constituted mainly by ascorbic acid and soluble phenolic compounds, while the L fraction contains carotenoids (mostly lycopene), tocopherols, sterols and lipophilic phenolics [1,2]. To obtain these antioxidants it is necessary to follow appropriate extraction methods and processing conditions. In this regard, this study aimed at determining the optimal extraction conditions for H and L antioxidants from a tomato surplus. A 5-level full factorial design with 4 factors (extraction time (I, 0-20 min), temperature (T, 60-180 •c), ethanol percentage (Et, 0-100%) and solid/liquid ratio (S/L, 5-45 g!L)) was implemented and the response surface methodology used for analysis. Extractions were carried out in a Biotage Initiator Microwave apparatus. The concentration-time response methods of crocin and P-carotene bleaching were applied (using 96-well microplates), since they are suitable in vitro assays to evaluate the antioxidant activity of H and L matrices, respectively [3]. Measurements were carried out at intervals of 3, 5 and 10 min (initiation, propagation and asymptotic phases), during a time frame of 200 min. The parameters Pm (maximum protected substrate) and V m (amount of protected substrate per g of extract) and the so called IC50 were used to quantify the response. The optimum extraction conditions were as follows: r~2.25 min, 7'=149.2 •c, Et=99.1 %and SIL=l5.0 giL for H antioxidants; and t=l5.4 min, 7'=60.0 •c, Et=33.0% and S/L~l5.0 g/L for L antioxidants. The proposed model was validated based on the high values of the adjusted coefficient of determination (R2.wi>0.91) and on the non-siguificant differences between predicted and experimental values. It was also found that the antioxidant capacity of the H fraction was much higher than the L one.

2016Conference object

Open access

Spray-drying microencapsulation of synergistic antioxidant mushroom extracts and their use as functional food ingredients

Publication . Ribeiro, Andreia; Ruphuy, Gabriela; Lopes, José Carlos B.; Dias, Madalena M.; Barros, Lillian; Barreiro, M.F.; Ferreira, Isabel C.F.R.

In this work, hydroalcoholic extracts of two mushrooms species, Suillus luteus (L.: Fries) (Sl) and Coprinopsis atramentaria (Bull.) (Ca), were studied for their synergistic antioxidant effect and their viability as functional food ingredients tested by incorporation into a food matrix (cottage cheese). In a first step, the individual extracts and a combination of both, showing synergistic effects (Sl:Ca, 1:1), were microencapsulated by spray-drying using maltodextrin as the encapsulating material. The incorporation of free extracts resulted in products with a higher initial antioxidant activity (t0) but declining after 7 days (t7), which was associated with their degradation. However, the cottage cheese enriched with the microencapsulated extracts, that have revealed a lower activity at the initial time, showed an increase at t7. This improvement can be explained by an effective protection provided by the microspheres together with a sustained release. Analyses performed on the studied cottage cheese samples showed the maintenance of the nutritional properties and no colour modifications were noticed.

2015Journal article

Open access

Suplementos dietéticos à base de Cochlospermum angolensis Welw.: atividade antimicrobiana e compostos fenólicos

Publication . Barros, Lillian; Pereira, Carla; Alves, Maria José; Pereira, Liliana; Santos-Buelga, Celestino; Ferreira, Isabel C.F.R.

Cochlospermum angolensis Welw. (borututu) é uma árvore tropical pertencente à família das Cochlospermaceae e amplamente utilizada pelas suas propriedades medicinais, incluindo no tratamento da malária, da icterícia e de doenças hepáticas.1 Neste trabalho, foram caracterizados os compostos fenólicos presentes em três formulações (infusões, comprimidos e xarope) à base desta planta, utilizando cromatografia líquida de alta eficiência acoplada a detetores de díodos e de espetrometria de massa (HPLC-DAD-ESI/MS). Esses compostos foram relacionados com a atividade antimicrobiana das mesmas formulações contra isolados clínicos de bactérias multirresistentes (Escherichia coli, Escherichia coli produtora de β-lactamases de espectro estendido (ESBLs), Proteus mirabilis, Staphylococcus aureus resistentes à meticilina (MRSA) e Pseudomonas aeruginosa). As infusões e os comprimidos revelaram uma maior variedade de compostos fenólicos, com onze moléculas identificadas. O ácido protocatéquico foi encontrado apenas nas infusões, sendo o composto maioritário, enquanto a (epi)galocatequina-O-galato e a eucaglobulina/globulusina foram as moléculas mais abundantes nos comprimidos e no xarope, respetivamente. O ácido elágico e derivados metilados, a eucaglobulina/globulusina B e a (epi)galocatequina-O-galato foram compostos comuns a todas as formulações. As infusões apresentaram propriedades antimicrobianas contra todas as bactérias testadas, com exceção de P. mirabilis, ao passo que os comprimidos apenas revelaram atividade em E. coli ESBLs e MRSA. O xarope não apresentou atividade antimicrobiana, o que está em concordância com o seu baixo teor de compostos fenólicos. Nenhuma das formulações estudadas demonstrou capacidade de inibir o crescimento de P. mirabilis. Atendendo aos resultados obtidos neste estudo, as infusões de C. angolensis podem ser consideradas uma fonte de compostos fenólicos com boas propriedades antimicrobianas.

2015Conference object

Open access