Compounds of tucum-do-cerrado (Bactris setosa) fruit with antioxidant activity

Abstract ‘Tucum-do-cerrado’ (Bactris setosa) is an edible fruit from the Brazilian ‘Cerrado’ biome marked by a high antioxidant potential and polyphenol content when compared to other fruits from the same biome. Its antioxidant activity is higher in the peel than in the pulp. Ethanolic and aqueous peel extracts were analyzed by the ferric reducing antioxidant power (FRAP) assay. We also investigated the aqueous peel extract for its antioxidant mechanism and isolated some of its compounds using high-performance liquid chromatography. Among the extracts tested, the aqueous peel extract exhibited the highest FRAP values, with a predominant free radical scavenger activity. The isolated compounds were identified as two catechins, a cyanidin, a peonidin, and a quercetin. Testing the antioxidant potential of the isolated compounds using the 2-deoxyribose degradation assay revealed that catechin and quercetin showed the highest antioxidant activity. Thus, our results advance the identification of ‘tucum-do-cerrado’ compounds with antioxidant activity. Graphical Abstract


Introduction
The Cerrado biome plant 'tucum-do-cerrado' (Bactris setosa Mart.) or simply 'tucum' are medium-sized palm trees (4-6 meters high) with trunks covered with very fine thorns. The 'tucum' palm produces its fruit in clusters. Each fruit is 1.1 to 2.1 cm long and 1.3 to 2.2 cm wide (Lima and Soares 2003). The 'tucum' palm bears fruit from January to April of each year. The ripe fruit pulp and peel are edible and can be eaten fresh. The 'tucum' fruits are used to make juices, liqueurs, ice creams, jellies, wines, and vinegars (Silva et al. 2001). The fruits are also used in infusions of 00 cachaça 00 , a distilled alcoholic beverage.
A study with edible fruits from 12 native species from the 'Cerrado' biome showed that 'tucum' contains a higher content of total phenolic compounds (12 times), total flavonoids (19 times), yellow flavonoids (8 times), anthocyanins (35 times) and vitamin C (3 times) when compared to Red Delicious apple (Malus domestica Borkh.), which is considered to have significant levels of antioxidants (Boyer andLiu 2004, Siqueira et al. 2013). The analysis of aqueous, methanolic, and ethanolic extracts of 'tucum' peel by high performance liquid chromatography coupled to a photodiode detector (HPLC-DAD) identified the main classes of phenolic compounds such as flavanols, anthocyanins, flavones, phenolic acids and stilbenes (Rosa et al. 2016).
Carotenoids have also been identified in 'tucum' extract (Boeing et al. 2017). Measures of the antioxidant activity of 'tucum' fruit extracts showed that the greatest activity is in the peel rather than the pulp, and in aqueous rather than other types (hexane, ethanolic or methanolic) of extracts (Rosa et al. 2016;Souza 2013). Regarding the in vivo effects of 'tucum', rats fed an iron-enriched diet containing 'tucum' pulp and peel showed decreased levels of hepatic malondialdehyde, an indicator of lipid peroxidation, when compared to animals fed an iron-enriched diet (Fustinoni-Reis et al. 2016). In rats with azomethane-induced premalignant colon lesions, consumption of 'tucum' causes decreased liver and colon lipid peroxidation and increased hepatic glutathione-S-transferase activity. It also induced a pro-inflammatory effect and promoted a pro-apoptotic environment in these animals (Campos et al. 2018). Such results demonstrate the in vivo antioxidant protection of 'tucum' fruit while demonstrating that it can also affect the levels of antioxidants.
Although specific classes of phenolic compounds present in 'tucum' are known (Rosa et al. 2016), there are no studies assigning individual compounds to the antioxidant activity of the extract or describing antioxidant mechanisms of 'tucum' extract compounds. Therefore, the aim of the present study was to investigate the antioxidant capacity of 'tucum' extracts' by the ferric reducing antioxidant power (FRAP) method and scavenging of hydroxyl radicals by the 2-deoxyribose (2-DR) degradation assay.
The antioxidant activity of an extract can be determined by two main mechanisms: chelation of metals or scavenging of free radicals. The 2-DR assay is commonly used to investigate the pro-oxidant or antioxidant activities of pure compounds from plant extracts. It can also be used to study antioxidant mechanisms (Genaro-Mattos et al. 2009;Lopes et al. 1999). Figure S1 shows that EAqL has a protective effect in vitro at different reaction times, except at the concentration of 0.5 mg/ml, at which the extract did not provide protection to 2-DR at any of the times tested ( Figure S1a-b). Furthermore, the oxidative damage to 2-DR was inversely proportional to the concentration of EAqL, which has a dose-dependent antioxidant effect (Figure S1c-d). The results obtained by varying the concentration of 2-DR suggest that the mechanism of action of EAqL is of the scavenger type, as its performance was similar to that of mannitol, a classic free radical scavenger ( Figure S1e-f).
The efficiency of EAqL against 2-DR degradation decreased with increasing concentrations of 2-DR, which works as the hydroxyl radical ( OH) detector molecule. This finding suggests that the extract has an OH capture activity. 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' peel extract has high antioxidant activity when compared to other 'Cerrado' fruits and apple (Rosa et al. 2016, Siqueira et al. 2013. In order to relate the antioxidant activity with specific bioactive compounds, we performed the fractionation of the EAqL extract by HPLC ( Figure S2), followed by in vitro antioxidant tests of the fractions. A total of 19 samples were collected from the HPLC and were numbered from 1 to 19. All 19 extract fractions showed antioxidant activity in the 2-DR degradation assay (Table S1). Five fractions were chosen for further analysis: 06, 09, 15, 17, and 18. In the 2-DR assay, fractions 09 and 18 showed the highest antioxidant activity when the percentage of protection was divided by ng/mL (Table S1). Fractions 15 and 17 showed the highest peaks in the chromatographic profile ( Figure S2). Fraction 06 showed intermediate antioxidant activity among the 19 fractions tested with the 2-DR assay (Table S1). Fractions 06 and 09 were identified by HPLC and by comparison of retention time and UV-visible spectral data from the standards, while fractions 15, 17 and 18 were identified by HPLC and mass spectrometry (MS) analysis in ion mode positive (Table S2). The UV spectral data of fractions 06 and 09 were similar to the UV spectral data of the catechin standard ( Figures S3-4). MS analysis of fraction 15 showed two peaks at m/z 449 and a fragment ion at m/z 287 ( Figure S5), corresponding to cyanidin-3-glycoside and cyanidin, respectively (Liu et al. 2012). The mass ion (m/z ¼ 301) of fraction 17 corresponded to a peonidin fragment (Liu et al. 2012) and fraction 18, with mass ion (m/z ¼ 303), was identified as a fragment of quercetin (Petrovic et al. 2010) (Figures S6-7). A study to characterize phenolic compounds in different extracts of 'tucum' peel detected catechin and cyanidin compounds in all extracts, quercetin was found only in the aqueous extract with methanol/water partition (1:3), and peonidin was not identified (Rosa et al. 2016). These results suggest that lyophilization of the peel before extract preparation optimizes the extraction of phenolic compounds.

Conclusions
The 'tucum' fruit is a relevant source of antioxidants and there are few studies about it. Studies on plant-derived antioxidants require an understanding of their antioxidant mechanism, i.e. they may act as free radical scavengers or metal chelating agents. The present work shows that the aqueous extract of 'tucum' presents a predominant free radical scavenger activity in vitro. Isolation and identification of 'tucum' antioxidants open opportunities to investigate them in depth and to study their potential application in the nutraceutical and phytopharmaceutical industry.