Orientadora: Profa. Dra. Rosane Marina Peralta

Data da Defesa: 02/08/2019

INTRODUCTION: Euterpe edulis Martius, popularly known as “Juçara”, is a native palm tree of the Atlantic Rainforest found predominantly in the states of the southern and southeastern regions of Brazil. The Juçara palm produces fruits, globular berries weighing about 1g, purple in color. The pulp of the fruit presents a composition very similar in terms of texture, color, flavor and nutritional composition to that of açaí (Euterpe oleracea Mart). Therefore, the fruits of Juçara are processed and exclusively marketed as frozen pulp. During the industrial production of pulp, where the epicarp and the mesocarp are separated from the seeds, the fruits are macerated and mixed with different amounts of water. Thus, the industrial production of Juçara pulp generates solid waste that is often discarded. A proper use of these residues could become important because they are rich in bioactive compounds, such as phenolics. Pereskia aculeata Miller, popularly known as 'ora-pro-nobis', is an unconventional vegetable belonging to the family Cactaceae, native to South America. Its leaves are rich in proteins with high tryptophan content, for this reason they are called meat of the poor. They are widely used in traditional cooking, serving as an ingredient of several sweet and savory dishes. The leaves are used in salads, soups, omelets or pies, as well as to enrich breads, cakes and pastas. The fruits are used for juices, jellies, mousses and liqueurs. Besides its use as a food, P. aculeata has application in folk medicine. Its leaves are used as emollients in the treatment of cutaneous wounds and inflammatory processes. In spite of the studies that evidence the nutritional and ethnopharmacological relevance of ora-pro-nobis leaves, information on their phytochemical constitution remains limited.

AIMS: The objectives of the present work were to evaluate the phytochemical profiles and biological activities of the fruit peel of Euterpe edulis and the leaves of Pereskia aculeata. Within this context, the hydroethanolic extracts were characterized in terms of their phenolic compounds, and antioxidant, antibacterial and hepatotoxicity activities.

MATERIAL AND METHODS: E. edulis berries were harvested in the city of Marialva (Paraná, Brazil) and immediately taken to the Laboratory, where they were sorted to exclude those unripe or with cracks. Next, the berries were washed in running potable water and sanitized with sodium hypochlorite (200.0 mg/L). Then the berries were manually de-pulped and the peels separated from pulp and seeds. The obtained peels were dried (40 ± 5 °C) for 24 h in a forced air circulation oven, subsequently mashed in a blender and sieved (80 mesh) to obtain a homogeneous flour. Finally, the material was stored away from light and at room temperature (28 °C). The proximate composition of the E. edulis peel flour (moisture, protein, fat, ash and carbohydrates) was determined using standard procedures (AOAC, 2016). Leaves of Pereskia aculeata (ora-pro-nobis), in the form of a flour, were obtained from a reliable commercial source (Ora Pro Nobis Brasil, Joinville, Santa Catarina, Brazil). The extracts were prepared using a flour/solvent ratio of 1:20, with an extractor solution composed of 70% ethanol in deionized water. The vials were sealed and shaken for 2 h at 130 rpm, at room temperature and protected from light. This procedure was repeated three times. Thereupon the samples were centrifuged at 10,000 rpm during 15 min, and the obtained supernatants were evaporated at 45 °C to remove ethanol. In the end, the aqueous phase was lyophilized and stored in a freezer at −20 °C. Phenolic compounds were identified and quantified by liquid chromatography coupled to mass spectrometry. The identification of the phenolic components was accomplished by employing standard compounds (when available) and by comparison with available literature data. Quantification was achieved utilizing the most fitting compound, with results expressed as mg per g of extract. To evaluate the antioxidant activity of the extracts, cell-based methods and traditional methods were used in vitro. Cell-based method is the oxidative hemolysis inhibition assay (OxHLIA). Traditional in vitro methods are the DPPH and ABTS, assays. The sulforhodamine B assay was carried out to evaluate the extract’s hepatotoxic potential in a porcine liver cell culture (PLP2), using ellipticine as positive control. Results were given in GI50 values, which corresponded to the concentration that constrained 50% of the net cell growth. Five Gramnegative bacteria and three Gram-positive bacteria were used to evaluate the antibacterial activity. The minimum inhibitory concentration (MIC) for all bacterial strains was

determined using a colorimetric assay. The MIC value was defined as the lowest concentration that inhibits visible bacterial growth. The minimum bactericidal concentration (MBC) was also determined by measuring the lowest concentration required to kill a microorganism. The results were expressed as mean values and standard deviations (SD) as an outcome of three repetitions for each sample.

RESULTS AND DISCUSSION: The centesimal composition of the fruit peel of Juçara was 7.41% fat, 7.12% protein, 40.10% fiber and 2.39% ash, very similar to that found in the literature for the whole fruit. The E. edulis peel presented considerable energy density (2.67 kcal/g d.w) due to its lipid content. Chemical analysis of the hydroalcoholic extract of Juçara allowed identifying nineteen phenolic compounds. Seventeen are non-anthocyanin phenolic compounds, including two phenolic acids, four flavanonols, six flavones and five flavonols. The two anthocyanin molecules are cyanidin glycoside derivatives. Among the nonanthocyanin compounds, apigenin-6-C-pentoside-8-C-hexoside isomers were the dominant components, representing 23% of the total phenolic non-anthocyanin compounds, followed by a apigenin-C-glucoside derivative (11.84%), kaempferol-O-hexosyl-deoxyhexoside (7.54%), and taxifolin-O-hexoside isomer 2 (7.18%). The major components of the peel extract were the anthocyanins cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside (5.32 and 6.23 mg/g), which together accounted for more than 87% of the extract’s total phenolic content. In the hydroethanolic extract of P. aculeata leaves a total of ten phenolic compounds were identified, among which two were phenolic acids (caffeic acid derivatives) and eight were flavonoids (quercetin, kaempferol and isorhamnetin glycoside derivatives). Caftaric acid was the major extracts’ constituent accounting for more than 49% of the phenolic content (11.72 mg/g), followed by quercetin-3-O-rutinoside (3.56 mg/g) and isorhamnetin- O-pentoside-O-rutinoside (2.27 mg/g). The total phenolic content found in our extract was 23.75 mg/g, with the amounts of phenolic acids and flavonoids being almost equivalent. The results of the cell-based antioxidant activity assays are expressed as the concentration of extract required to inhibit (delay) 50% of hemolysis. For comparison purposes, Trolox, a synthetic antioxidant, was also assessed. We found an IC50 value (60 min) of 42 μg/mL and IC50 (120 min) of 107 μg/mL, for the hydroalcoholic extract of Juçara. Trolox exhibited superior protection ability with an IC50 (60 min) of 5.5 μg/mL and IC50 (120 min) of 20.4 μg/mL. For the hydroalcoholic extract of P. aculeata leaves we found a value of IC50 (30 min) was 57.0 μg/mL, twice as low as the IC50 value (60 min) which was 131.0 μg/ mL. Trolox, used as a positive control, had a protective capacity superior, with an IC50 value (60 min) of 20,4 μg/mL. The extracts were also evaluated by DPPH and ABTS assays. The hydroalcoholic extract of Juçara presented a DPPH radical scavenging capacity of 13,107μmol TE/g, higher than that found in the literature for the Juçara pulp (745 μmol TE/ g). However, our sample showed significantly lower ABTS radical elimination activity (23 μmol TE/mg) than the pulp (677 μmol TE/mg). The P. aculeata hydroethanolic extract had an antioxidant capacity superior to Trolox, with IC50 values of 72.9 μg/mL for the DPPH assay and IC50 of 40.5 μg/mL for the assay of ABTS. Both extracts evaluated showed no toxicity against the primary liver culture PLP2. In analyzing the results of the inhibitory activity exhibited by the extracts against bacteria, we found that both were more active against gram-positive bacteria than against gram-negative bacteria.

CONCLUSIONS: The results presented suggest the possibility of the exploitation of residues of the Juçara fruit to obtain bioactive compounds. More specifically, the fruit peels present the potential of being used to produce food additives with high added value. The information reported here not only corroborates the importance of the production and consumption of P. aculeata leaves but also reinforces their potential as a sustainable source of nutraceuticals and promising food ingredients to be used for food enrichment.

Key words: Euterpe edulis; Pereskia aculeata; phenolic compounds; antioxidant potential.

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