L. belongs to Myrtaceae family and grows naturally in subtropical Latin-American zones [1
]. Its cultivation has extended to other regions outside the American continent with similar climatic conditions. It grows mainly in Brazil, which is why it is known as “Brazilian Cherry”, but it is also grown in Argentina, Paraguay and Uruguay [2
]. Its fruits have a high carbohydrates content (around 38%) where maltose, lactose and fructose are the main identified compounds [3
]. It has a high content of vitamin C, vitamin A, riboflavin (B12) and niacin (B3) [4
]. Immature fruits show a high content of polyphenols that decreases with maturation [5
]. On the other hand, carotenoid content increases with maturation evidenced by an increase in a reddish-orange coloration [6
Pitanga leaves are often used in Brazilian traditional medicine due to its diuretic, antirheumatic, antifebrile, anti-inflammatory and hypocholesterolemic properties [7
]. However, there is little information regarding the medicinal use of the fruit. The fruit has an acidic and sweet flavor and can be consumed fresh, in compotes, jams or juices [6
]. Purple fleshed Pitanga fruit in its latest maturation stage has an edible portion (pulp and skin) of 61.76%, a vitamin C content of 38.35 mg/100 g and total anthocyanin content of 29.60 mg/100 g [11
]. Additionally, a high content of total phenols (799.80 mg of gallic acid/100 g) and total carotenoids (5.86 μg of β-carotene/g) [4
]. Recently, Pitanga juice showed an anti-inflammatory effect on oral gum epithelial cells; these results could be associated to the presence of cyanidin-3-glucoside and oxidoselina-1,3,7(11)-trien-8-one [12
]. Hence, Pitanga’s beneficial properties could be exploited in the nutraceutical industry.
According to DeFelice a nutraceutical can be defined as “a food or part of it, that has some health benefit, including the prevention and/or treatment of a disease” [14
]. Therefore, research has driven evaluation/re-evaluation of foods and their beneficial properties. Antioxidant rich foods have received a lot of interest; promoting their consumption to decrease oxidative stress caused by stress, lack of sleep, poor diet, metabolic problems, etc.
Currently, a large number of different approaches to determine antioxidant activity have been established [15
]. All tests differ in substrates, probes, reaction conditions, instrumentation, and quantification methods. Hence, it is difficult to compare the results obtained by one method or another. Based on the reactions involved, the tests can be further classified into two groups: HAT (Hydrogen Atom Transfer) and SET (Single Electron Transfer) [18
]. In the HAT group, antioxidants must donate a hydrogen atom to stabilize the generated free radicals (a synthetic free radical generator and an oxidizable molecular probe are used to evaluate the kinetics of the reaction). SET-type assays involve a redox reaction where the antioxidant must donate an electron to the generated free radical. In both methods the “competition” with the oxidant radical is measured instead of the antioxidant capacity [18
The ORAC (Oxygen Radical Absorbance Capacity) assay measures the overall antioxidant activity or capacity of a sample’s ability to “quench or neutralize” peroxyl radicals. The peroxyl radicals are reactive species comparable to those ROS (Reactive Oxygen Species) biologically generated in the organism. In the ORAC assay, the peroxyl radicals, generated from the azo-compound AAPH (2,2′-azobis-(2-methylpropionamidine)dihydrochloride) react with fluorescein as a substrate. The fluorescence of the latter compound decreases over time, forming an area under the curve (fluorescence vs. time). In the presence of antioxidant compounds, the area under the curve increases linearly and proportionally to the concentration of antioxidants.
The ORAC assay quantifies via the HAT mechanism and measures the antioxidant activity of polyphenol and non-polyphenolic compounds present in each sample. It is important to note that the antioxidant activity does not have a direct correlation to the polyphenol nature of a sample [19
]. The ORAC test reflects the overall capacity or antioxidant activity of a sample, due to the individual components and their additive, synergistic interactions. The ORAC value is usually expressed as micromoles of Trolox®
equivalents/100 g of sample. Trolox®
is an analogue of vitamin E and is often used as a comparative standard due to its solubility in water [18
The DPPH method name stems from the reactant used (2,2-diphenyl-1-picrylhidracyl) to evaluate the ability of a sample’s antioxidants to “quench or neutralize” a free radical. The DPPH assay utilizes molecules that differ completely from any free radical or reactive species generated by our organism as a source of free radicals. Although the reactant is easy to use, it places this method as a distant analytical approximation of the high reactivity that typically characterizes the ROS generated in biological systems [21
]. This method is classified in the SET group. However, can be classified as a HAT-type assay according to the antioxidant present in the sample [13
]. In this test the sample is incubated for 35 min and the decay of the absorbance is measured at 515 nm.
Based on previous reports of bioactivity [22
] of South-Brazilian Pitanga fruit, the objective of the present work was to extract, identify and quantify the phenolic compounds in mature fruits of Eugenia uniflora
L. The antioxidant capacity was evaluated via ORAC and DPPH; and the phenolic profiles of E. uniflora
from different Uruguayan locations were compared. Pitanga is considered a South American native fruit and its consumption is not among the most common in the region. However, its ethnobotanical characteristics, traditional uses and its antioxidant properties highlights its potential to improve the quality of life of those who consume it [23
]. Hence, this study seeks to explore and further promote the value of the Uruguayan native Pitanga fruit that has not been exploited until now.
2. Materials and Methods
2,2-Diphenyl-1-picrylhidracyl (DPPH), fluorescein, 2,2′-azobis-(2-methylpropionamidine)dihydrochloride (AAPH), monobasic sodium phosphate and dibasic sodium phosphate were obtained from Sigma-Aldrich (Steinheim, Germany). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxilic acid (Trolox®) was purchased from Fluka Chemika (Neu-Ulm, Germany). Cyanidin-3-O-glucoside and rutin were obtained from Polyphenols Laboratories AS (Sandnes, Norway). Ultrapure water was produced using a Millipore Milli-Q® Ultrapure Water Solutions Type 1. All solvents used were HPLC grade from Sigma-Aldrich (Steinheim, Germany).
The fruits (purple fleshed Pitanga breeding lines) were collected in different Uruguayan locations (described below), they were identified and kept at the Jose Arechavaleta Herbarium in the Faculty of Chemistry, UdelaR, Uruguay (Voucher number MVFQ 4427). Samples 1, 5, 6 & 7 were collected in the north of Montevideo Department (−34.804951, −56.230206) in December 2014, November 2015, December 2015 and April 2016, respectively. Sample 2 was collected in the south of Montevideo Department (−34.884536, −56.073039), sample 3 in Paysandú Department (−32.322604, −58.088243) and sample 4 in Ciudad de la Costa, Canelones Department (−34.799105, −55.908381); samples 2, 3 & 4 were collected in December 2014. All samples were freeze-dried, and the water content calculated by weight before and after lyophilization.
Each lyophilized grinded sample (100 mg) was mixed with 1 mL of methanol/water/formic acid (70:29:1, v/v/v ) in a 2 mL conical polypropylene tube (Eppendorf, Madrid, Spain). Then, the samples were vortexed and subjected to indirect sonication in an ultrasound cleaning bath for 60 min (BRANSONIC® Ultrasonic cleaner mod. 5510E-MTH, Ultrasonic frequency: 135 W or 42 KHz ± 6%). The samples were kept overnight at 4 °C and sonicated again for 60 min. The supernatant was separated from the solid residue after centrifugation (9500× g, 15 min), filtered using a 0.45 µm PVDF filter (Millex HV13, Millipore, Bedford, MA, USA) and stored at 4 °C until they were analyzed.
2.4. Identification of Phenolic Compounds Via HPLC-DAD-ESI/MSn and Quantification Via RP-HPLC-DAD
The identification analyses were carried out using an Agilent HPLC 1100 series model equipped with a photodiode array detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany). It consisted of a binary pump (model G1312A), a degasser (model G1322A), an autosampler (model G1313A), and a photodiode array detector (model G1315B). The HPLC system was controlled by ChemStation for LC 3D Systems software Rev. B.01.03-SR2 (204) (Agilent Technologies Spain S.L., Madrid, Spain). The mass detector was an ion trap spectrometer (model G2445A) equipped with an electrospray ionization interface and was controlled by LC/MS software (Esquire Control Ver. 6.1. Build No. 534.1., Bruker Daltoniks GmbH, Bremen, Germany). The ionization conditions were 350 °C capillary temperature and 4 kV voltage, the nebulizer pressure was 65.0 psi and the nitrogen flow rate was 11 L/min. Full-Scan mass covered the range of m/z from 100 to 1200. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 to 2 V. The mass spectrometry data were acquired in the positive ionization mode for anthocyanins and in the negative ionization mode for other flavonoids. The MSn was carried out in the automatic mode on the more abundant fragment ion in MS(n-1). A Luna C18 column (250 × 4.6 mm, 5 µm particle diameter; Phenomenex, Macclesfield, UK) was used. Mobile phase A: water/formic acid (99:1, v/v), mobile phase B: acetonitrile, initial conditions: 8% solvent B, reaching 15% solvent B at 25 min, 22% at 55 min, and 40% at 60 min, which was maintained isocratic until 70 min. The flow rate was 0.8 mL/min during the whole run, all gradients were linear, and the injection volume was 7 µL. Chromatograms were recorded at 280, 320, 360 and 520 nm.
For quantification experiments the same conditions were applied, except for the injection volume was set to 20 µL and the flow rate was 0.9 mL/min. Anthocyanins were quantified as cyanidin 3-O-glucoside at 520 nm and flavonols as rutin at 360 nm.
2.5. DPPH Antioxidant Activity Measurements
Experiments were carried out using 96-well micro plates (Nunc, Roskilde, Denmark) and an Infinite® M200 micro plate reader (Tecan, Grödig, Austria). The antioxidant activity was evaluated measuring the change of the absorbance at 515 nm after 35 min of reaction with the radical DPPH˙ (2 µL of the sample + 250 µL of DPPH˙ solution). The results were expressed as µmol of Trolox®/100 g dry weight.
2.6. ORAC Antioxidant Activity Measurements
The antioxidant assay was performed using black-walled 96-well plates (Nunc, Roskilde, Denmark) and an Infinite® M200 micro plate reader (Tecan, Grödig, Austria). Each well with a final volume of 200 μL. 10 mM phosphate buffer (pH 7.4) was used to prepare 1 μM fluorescein and 250 mM AAPH solutions. Each well received 150 μL of fluorescein solution and 25 μL of phosphate buffer, Trolox® solutions or sample solution to measure the blank, the curve or the samples respectively. The plate was placed into the microplate reader and after 30 min of incubation at 37 °C, 25 μL AAPH solution were added to each well and fluorescence was recorded every 5 min for 120 min using an excitation wavelength of 485 nm and an emission wavelength of 520 nm. ORAC values were calculated using the difference in Areas Under the Fluorescein Decay Curve (AUC) between the blank and a sample. The results were expressed as µmol of Trolox®/100 g dry weight.
2.7. Statistical Analysis
Data shown are mean values (n
= 3), subjected to Analysis of Variance (ANOVA) and multiple range test (Tukey’s test), using RStudio software (Version 1.1.383, RStudio, Boston, MA, USA) and InfoStat (Version 2017, Universidad Nacional de Córdoba, Córdoba, Argentina) [24