As frações cromatográficas que exibiram atividade antioxidante nos ensaios realizados foram analisadas em espetrômetro de massas do tipo MALDI-TOF/TOF (do ingles, Matrix- assisted laser desorption/ionization-Time of flight) modelo AB SCIEX TOF/TOF™ 5800 (AB Sciex, Framingham, MA, EUA). As frações foram ressuspendidas em água contendo TFA 0,1% (v/v) e 1 μL de cada fração cromatográfica foi aplicada em placa de aço inoxidável juntamente com 1 μL de matriz ácido α-ciano-4-hidroxicinâmico (HCCA 20 µg/µL). O procedimento foi realizado no modo refletido positivo, sendo a faixa m/z analisada de 200 a 4000 Da.
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Compounds of tucum-do-cerrado (Bactris setosa) fruit with antioxidant activity
Marcela Berckmans Viégas Costa Dantas1,3, Osmindo Rodrigues Pires Júnior2, Luane Tomé de Paula Campos3, Élida Geralda Campos1,3
1
Programa de Pós-Graduação em Nutrição Humana, Faculdade de Ciências da Saúde, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, Brasília, DF, Brazil, CEP 70910-900.
2
Laboratório de Toxinologia, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, Brasília, DF, Brazil, CEP 70910-900.
3
Laboratório de Biologia Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, Brasília, DF, Brazil, CEP 70910-900.
Marcela Berckmans Viégas Costa Dantas - 55+61-98112-7999 Email[email protected]
Osmindo Rodrigues Pires Júnior 55-61-3107-3101 Email[email protected]
Luane Tomé de Paula Campos – 55-61-99326-8771 Email[email protected] Élida Geralda Campos – 55-61-3107-2950 and 61-996074859 Email
[email protected][email protected]
*Corresponding author: Dr. Élida Geralda Campos
Departamento de Biologia Celular Universidade de Brasília
Brasília - DF - Brazil CEP 70910-900
Tel: +55-61-3107-2950
Abstract
Among the edible fruits of the Brazilian savanna, tucum-do-cerrado (Bactris setosa Mart.) stands out due to its high antioxidant potential and polyphenol content. We investigated the aqueous extract of tucum-do-cerrado regarding its mechanism of action and the antioxidant potential of its fractions. The aqueous extract was fractionated by HPLC and the fractions analyzed in terms of their antioxidant activity by means of the oxidative degradation of 2- deoxyribose. Five fractions were selected based on the antioxidant activity observed in vitro and components of three of them were identified as cyanidin, peonidin and quercetin. The selected HPLC samples, on the other hand, showed a detrimental effect on yeast cell growth in the presence of H2O2. Thus our results advance in identifying tucum-do-cerrado
compounds with antioxidant activity. The results show dichotomous actions of the tucum-do- cerrado compounds that should be investigated thoroughly to uncover the real possibilities of tucum-do-cerrado in contributing to good human health.
1.Introduction
Several studies have shown an inverse association between the intake of fruits and vegetables and the risk of developing certain diseases, such as heart disease, stroke, and cancers of the lung, colon, stomach and esophagus (Hu et al. 2014, Lock et al. 2005). This protective effect is mainly related to the fact that fruits and vegetables are sources of many bioactive compounds with antioxidant properties (Gomes-Rochette et al. 2016). Such compounds minimize oxidation of biological constituents and therefore protect cells from oxidative stress damage, while playing a role in signal transduction as well (Murphy et al. 2011).
The Brazilian Cerrado is composed of three biomes: tropical grassland (campo limpo), savanna (campo sujo, campo cerrado, and cerrado sensu stricto), and seasonal forest (cerradão) (Batalha 2011), and is the second largest Brazilian ecosystem (IBGE 2016). Its biodiversity is exemplified by thousands of different plant species (Mendonça et al. 1988).The Cerrado region has a rainy season (October to March) followed by a dry season (April to September), with average annual rainfall of 1,500 mm and mild temperatures throughout the year (22C to 27C). In addition, its vegetation develops in acidic and nutrient-poor soils, with high aluminum concentration (Klink and Machado 2005). Cerrado plants must have developed specific mechanisms to survive the significant biotic and abiotic stresses (drought, fire in the dry season, lack of nutrients, excess aluminum for example) present throughout the year. Environmental stresses, such as drought, can induce an increase in plant antioxidants as a mean to counteract increased generation of reactive oxygen species (ROS) (Yang et al. 2015). Following this reasoning, the edible fruits of the Cerrado may be considered a distinct quantitative and/or qualitative source of antioxidants when compared to fruits produced in regions of temperate climate and fertile soil.
Tucum-do-cerrado (Bactris setosa Mart.) fruits are produced as clusters by medium- sized (4-6 meters high) palms with trunks covered with very fine thorns. Each fruit is 1.1 to 2.1 cm long and 1.3 to 2.2 cm wide (Figure 1) (Lima and Soares 2003). The tucum-do-cerrado palm tree bears fruits from January to April each year. The pulp of the ripe fruit is edible and can be consumed fresh. Tucum-do-cerrado fruits are used to make juices, liqueurs, ice cream, jellies, wines and vinegars (Silva et al. 2001). Fruits are also used in infusions of cachaça, a distilled alcoholic beverage.
A study of twelve native edible Cerrado fruits showed that tucum-do-cerrado contains a higher content of total phenolics (12-fold), total flavonoids (19-fold), yellow flavonoids
(8-fold), anthocyanins (35-fold) and vitamin c (3-fold) when compared to the apple Red Delicious (Malus domestica Borkh.) which is considered to have significant levels of antioxidants (Boyer and Liu 2004, Siqueira et al. 2013).Analysis of aqueous, methanolic and ethanolic tucum-do-cerrado peel extracts by high perfomance liquid chromatography coupled with a photodiode detector (HPLC-DAD) identified the major classes of phenolics as flavanols, anthocyanins, flavones phenolic acids and silbenes (Rosa et al. 2016). Measures of the antioxidants and antioxidant activity of tucum-do-cerrado fruit extracts showed that most activity is in the peel, instead of the pulp, and in aqueous instead of other types (hexanic, ethanolic or methanolic) of extracts (Rosa et al. 2016, Souza 2013). Regarding the in vivo effects of tucum-do-cerrado, rats fed an iron-enriched diet containing tucum-do-cerrado pulp and peel showed diminished liver malondialdehyde levels when compared to animals that received an iron-enriched diet (Fustinoni-Reis et al. 2016). Such results demonstrate the in vivo antioxidant protection of tucum-do-cerrado fruit.
Although the specific classes of phenolic compounds present in tucum-do-cerrado became recently known (Rosa et al. 2016), to our knowledge, there are no studies relating specific tucum-do-cerrado compounds to the antioxidant activity presented by the total peel aqueous extract or describing probable antioxidant mechanisms presented by the extract. Therefore, the objective of the present study was to relate specific compounds of tucum-do- cerrado peel to antioxidant activities measured by the 2-deoxyribose degradation assay (Genaro-Mattos et al. 2009) and by investigating Saccharomyces cerevisiae growth exposed to oxidants.
2. Results and discussion
Initially we performed the FRAP assay with the aim to investigate the best starting material and extraction method of bioactive compounds of tucum-do-cerrado (Table 1). The aqueous extract from lyophilized peel (EAqL) showed the highest activity among the other forms of extractionmaterial. Therefore, EAqL was chosen to be used in this study. In another study on the antioxidant potential of fresh and lyophilized samples of 18 non-traditional tropical fruits from Brazil, the lyophilized samples also had the highest antioxidant capacity of all fruit samples and this result was associated with higher total phenol contents (Rufino et al. 2010). Furthermore, the lyophilization process produced less variability of the antioxidant activity. A study about drying processes and extraction procedures pointed lower variability of the phenolic content in lyophilized rosemary, when compared to fresh sample, and it was
suggested that lyophilization is the best method for storing the sample before analysis because minimizes waste and oxidation of phenolic compounds (Mulinacci et al. 2011).
The antioxidant action of an extract can be achieved by means of two main mechanisms: chelation of metals or scavenging of free radicals. The 2-deoxyribose (2-DR) degradation assay for determination of hydroxyl radicals (·OH) is a very popular assay to analyze the pro- or antioxidant activities of pure compounds or plant extracts and aid in description of mechanism of action (Genaro-Mattos, Dalvi, Oliveira, Ginani and Hermes- Lima 2009, Lopes et al. 1999). Figures 2a-b show that EAqL has a protective effect in vitro in different reaction times, except for the concentration of 0.5 mg/ml that did not provided protection to 2 -DR in any of the times tested. In addition, the oxidative damage to 2 -DR was inversely proportional to the concentration of EAqL, which features a dose-dependent antioxidant effect (Figure 2c-d). The results obtained by varying the concentration of 2-DR suggest that the mechanism of action of EAqL is of the scavenger type, because its performance was similar to mannitol, a classic scavenger (Figure 2e-f). When the concentration of 2-DR was increased, EAqL showed a decrease in antioxidant protection, which indicated the capacity to quench hydroxyl radicals generated in the in vitro oxidative system. This probably happened because the antioxidants present in EAqL and 2 –DR, the target molecule, compete for the hydroxyl radical in the reaction (Andrade et al. 2006).
The tucum-do-cerrado peel extract has high antioxidant activity when compared to other cerrado fruits and apple (Rosa, Arruda, Siqueira and Arruda 2016, Siqueira, Rosa, Fustinoni, de Sant'Ana and Arruda 2013). To be able to relate the antioxidant activity to specific bioactive compounds, we performed extract fractionation by HPLC (Figure S1) and antioxidant tests in vitro. Figure 3a shows that all extract fractions had antioxidant activity in the 2 -DR degradation assay. However fractions 06, 09, 15 and 17 standed out with respect to their antioxidant protection percentage (Table 2). From this result, we seek to identify these fractions and also test its effect on yeast cell growth in oxidative stress conditions.
Fractions 06 and 09 were identified by HPLC and through comparison of the retention time and UV-visible spectral data of standards, while fractions 15, 17 and 18 were identified by HPLC and mass spectrometry (MS) analysis in the positive ion mode (Table 3). The UV-spectral data of fractions 06 and 09 were similar with the UV-spectral data of catechin standard (Figure S2-3). The MS analysis of fraction 15 showed two peaks with m/z 449 and a fragment ion at m/z 287 (Figure S4), corresponding to cyanidin-3-glucoside and cyanidin, respectively (Liu, Guo and Sun 2012). The mass ion (m/z = 301) of fraction 17 corresponded to a peonidin fragment (Liu, Guo and Sun 2012) and fraction 18, with mass ion
(m/z = 303), was identified as a fragment of quercetin (Petrovic et al. 2010). A recent study on characterization of phenolic compounds in different extracts from tucum-do-cerrado peel detected catechin and cyanidin compounds in all extracts, the quercetin was found only in the aqueous extract with partition methanol/water (1:3) and peonidin not was identified (Rosa et al. 2016). These results suggest that lyophilization of the peel before extract preparation optimizes the extraction of phenolic compounds.
Finally, we tested the antioxidant activity of fractions 06, 09, 15 and 17 in yeasts cells exposed to hydrogen peroxide, an oxidant agent. Figure 3b shows that all fractions, when combined with H2O2 treatment, caused a delay in cell cycle progression. In addition,
fractions used in higher concentrations completely abolished cell growth (results not shown). Therefore, despite the fractions showing antioxidant activity in a chemical assay, the assay with yeast cells indicated an opposite activity (pro -oxidant or even toxic), depending on the concentration. This can be explained by the fact that polyphenol compounds are susceptible to oxidation in cell culture medium and as oxidation product can be generated reactive oxygen species (ROS) (Halliwell 2014). The oxidation of these molecules may present toxic effect,