Total antioxidant capacity , total phenolic content and mineral elements in the fruit peel of Myrciaria cauliflora

The in vitro antioxidant capacity, total phenolic content and mineral elements of the fruit peel of Myrciaria cauliflora were investigated. The antioxidant capacity was analyzed by the diphenylpicrylhydrazyl (DPPH), 2,2’-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP) and β-carotene methods. The assays based on the DPPH (EC50 = 3.18 g sample/g DPPH), ABTS•+ (1017 μmol Trolox/g sample), FRAP (1676 μM Fe2SO4/g sample) and β-carotene/linoleic acid (70% of oxidation inhibition) methods indicated a high antioxidant capacity of the fruit peel extract of the plant. The Folin-Denis method was more efficient in determining the total phenolic compound contents in the different solvents than the Folin-Ciocalteu one. Extractions made with 4:1 methanol-water, 4:1 ethanol-water, 3:2 ethanol-water and 3:2 acetonewater solutions using the Folin-Denis method exhibited high contents of phenolic compounds (18.95, 14.06, 12.93 and 11.99 mg GAE/g, respectively). Potassium was the major element found in the fruit peel, followed by phosphorus, calcium, magnesium and iron, in that order. As a result, the fruit peel of M. cauliflora can be considered as an important source of natural antioxidants and essential elements of easy access for the population and for application in the food industry.

. Ultrasound assisted, agitated bed and soxhlet extractions in ethanol were employed.However, the most trivial extraction process for the general population, i.e. solvent extraction at room temperature, was not considered in any of these studies.Other studies about the fruit peel of M. cauliflora at different ripening stages reported the proximate and mineral compositions, flavonoid content and the inhibition of important enzymes for the carbohydrate metabolism (ALEZANDRO et al., 2013b).Moreover, a recently published study suggested that the fruit peel of M. cauliflora had lipid-lowering effects (ARAÚJO et al., 2013).
Polar extracts obtained from the fruit peel of M. jaboticaba exhibited antiproliferative effects against leukaemia, and the non-polar extracts were active against prostate cancer cells (LEITE- LEGATTI et al., 2012).Beneficial health effects were observed in diabetic rats, improving their lipid profiles and reducing oxidative stress (ALEZANDRO et al., 2013a).
Thus the present work describes a more exhaustive investigation of the total antioxidant capacity, total phenolic content (TPC) and mineral elements of fruit peel extracts of M. cauliflora.The choice of the solvent is an important factor in the extraction process of phenolic compounds from plant materials (ZHAO et al., 2006;IKRAM et al., 2009).Therefore the present work also described the use of different solvent mixtures (including water, methanol, ethanol and acetone) in the extraction process of the phenolic compounds.The antioxidant capacity was analyzed by the diphenylpicrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP) and β-carotene methods.These methods determine different antioxidant powers such as the capacity to scavenge free radicals (DPPH and ABTS methods) and chelate metal ions (FRAP methods).The β-carotene method prevents the formation of radicals and improves the endogenous antioxidant system.The total phenolic content was evaluated using the Folin-Ciocalteu and Folin-Denis methods.The mineral elements were determined by atomic absorption spectroscopy.

Introduction
Free radicals are produced during oxidative metabolism in living organisms (RAHMAN and ADCOCK, 2006) or under stress conditions, such as changes in temperature, exposure to UV radiation and attacks by pathogens (DIXON and PAIVA, 1995).The protective mechanism is capable of intercepting or stopping the chain reactions which are typical of free radicals, converting them into less harmful molecules.As a result, antioxidants efficiently repair oxidative damage in cells (DU et al., 2009).Natural antioxidants exhibit protective effects against free radicals.The antioxidant properties of plant foods have been attributed to their terpenoid, carotenoid and phenolic constituents (PÉREZ-JIMÉNEZ et al., 2008).Phenolic compounds are employed in the treatment of cardiovascular diseases (JOSHIPURA et al., 2001;NACZK and SHAHIDI, 2006) and their antioxidant properties have been extensively investigated over the last few years (ABREU et al., 2008;CHOUDHARY et al., 2009;MORAIS et al., 2010;HOSSAIN and RAHMAN, 2011).The high concentration of phenolic compounds in fruit peels and seeds provides an appropriate justification for the use of fruit byproducts as a source of natural antioxidants (FRANCISCO and RESURRECCION, 2009).
The mature fruit is red to blackish purple and exhibits low acidity and a sweet flavour (REYNERTSON et al., 2008), being often eaten fresh or processed as jams, juice and liqueurs.Deterioration of the fruit is observed within two to three days post-harvest, mainly due to pulp fermentation and extreme loss of water.As a consequence, there is a significant amount of natural antioxidants in the peel of the deteriorated fruits of M. cauliflora, that could be better explored by the food and pharmaceutical industries.
The leaves of M. cauliflora have been studied and indicated the presence of phenolic and tannin constituents.The essential oil contains high concentrations of γ-eudesnol, germacrene and elemol (DUARTE et al., 2010).The leaf extract exhibited antimicrobial activity against some bacteria such as Streptococcus mitis, S. mutans, S. sanguinis, S. oralis, S. salivarius and Lactobacillus casei (MACEDO-COSTA et al., 2009).Some sugars, organic acids, minerals, lipids and phenolic compounds were also identified in the fruits of the plant (EINBOND et al., 2004;REYNERTSON et al., 2008).
A few studies about the fruit peel of M. cauliflora can be found in the literature, describing the total phenolic content, total anthocyanin content and the antioxidant activity, as determined by the Folin-Ciocalteu, differential pH and β-carotene methods, respectively http://bjft.ital.sp.gov.brBraz.J. Food Technol mL) were added to 3.90 mL of the 0.06 mM DPPH solution in the absence of light, and the mixture vigorously shaken and then incubated for 30 min at 37 °C.The absorbance data were registered at 515 nm.An antioxidant capacity was evaluated by a 50% concentration (EC 50 ) of free radicals in relation to the initial concentration of free radicals in the DPPH solution.
The ABTS •+ free radical scavenging activity was determined based on the method previously described in the literature (ARNAO et al., 2001).The ABTS •+ radical cation was generated from 5.00 mL stock solution of ABTS containing 88.0 μL of a potassium persulphate solution.The ABTS solution was maintained in the absence of light for 16 h at room temperature, and 1.00 mL then diluted with ethanol to obtain an absorbance of 0.70 at 734 nm.Samples A-D (0.03 mL) were transferred to test tubes containing 3.00 mL of the ABTS •+ radical solution and homogenized.The absorbance was registered after 6 min, and the scavenging activity on the ABTS •+ free radical was compared with a standard Trolox solution (1000 mM), which is a potent antioxidant.
The FRAP determination was based on a method previously described in the literature (FIRUZI et al., 2005).A FRAP solution was prepared by the addition of 25.0 mL of acetate buffer (300 mM) to 2.50 mL of ferric chloride hexahydrate (20 mM) and 2.50 mL of TPTZ (10 mM).Samples A-D (0.09 mL) were added to 0.27 mL of distilled water and 2.70 mL of FRAP reagent.After 30 min of incubation at 37 °C, the absorbance data were registered at 595 nm.The antioxidant capacity of the fruit peel extracts was determined from the calibration curve plotted using FeSO 4 .7H 2 O at concentrations between 500 and 2000 mM as the reference standard.
The test for the inhibition of the oxidation of β-carotene by peroxide radicals was based on the method previously described in the literature (VELIOGLU et al., 1998).An aliquot (0.15 mL) of β-carotene solution in chloroform (20 mg/mL) was added to a mixture of linoleic acid (0.12 mL) and Tween 40 (1.63 mL).The chloroform was removed by evaporation for 2 h at 40 °C.The absorbance data for this mixture were registered at 470 nm and considered as the reference standard.The results were expressed as oxidation inhibition percentages (OI%; as shown in Equation 1), considering the decrease in absorbance of the samples A-D (0.4 mL), where SA 0 and SA 120 are the values for the absorbance of the test sample at zero time and 120 min, respectively, and RA 0 and RA 120 are the values for the absorbance of the reference standard at zero time and 120 min, respectively.Trolox (2.00 mg/mL in methanol) was used in the tests for the inhibition of the oxidation of β-carotene for comparison with the results obtained with the fruit peel of M. cauliflora (RUFINO et al., 2010).
Flame Atomic Absorption Spectrometer.Phosphorus was analyzed using an Analyser 910 Flame Photometer.

Sample preparation
Mature fruits were harvested in the City of Diamantina (State of Minas Gerais, Brazil) at a latitude of S 18° 14' 58" and longitude of W 43° 36' 01" in November 2009.The identity of a voucher specimen was checked by a morphological analysis and compared with authentic material deposited in the herbarium of the Instituto de Ciências Biológicas of the Federal University of Minas Gerais, under the code B1987.The newly harvested fresh fruits were washed and sanitized by immersion in a sodium hypochlorite solution (200 mg/L) for 10 min.The fruit peel and pulps were manually separated.The fruit peel was dried in an oven with forced air circulation for seven days at 40 ± 5 °C.The dried fruit peel was crushed and sieved (80 mesh).

Evaluation of the antioxidant activity
The dried and powered fruit peel (1.00 g) was homogenized in 40.0 mL of (1:1) methanol-water solution for 1 h at room temperature.This mixture was centrifuged at 3000 rpm (approximately 500 g-force) for 15 min.The supernatant was collected and denominated Extract 1.The solid residue was submitted to the same homogenization and centrifugation process using 40.0 mL of (7:3) acetone-water solution, obtaining Extract 2. The two extracts were combined and the solvents evaporated off.The solid extract was diluted in methanol at different concentrations, providing samples A (0.50 mg/mL), B (1.00 mg/mL), C (1.50 mg/mL) and D (2.00 mg/mL).All experiments were carried out in triplicate.
The DPPH free radical scavenging activity was determined based on the method previously described in the literature (BRAND-WILLIAMS et al., 1995;SÁNCHEZ-MORENO et al., 1998) (RUFINO et al., 2010), considering that lower EC 50 values correspond to higher antioxidant activities (YEH et al., 2011).Moreover, the antioxidant capacity of the fruit peel of M. cauliflora as measured by the DPPH radical capture method was significantly higher than that of other plant species, such as the peels of Citrus paradise, C. sinensis, C. limon and C. aurantiifolia (EC 50 = 5.15, 4.99, 3.77 and 1.72 mg dried sample/mL DPPH, respectively) (GUIMARÃES et al., 2010) and the fruit extracts of Ficus glomerata (9.04 g of crude extract/g DPPH) (VERMA et al., 2010).
The antioxidant activity of the fruit peel of M. cauliflora as measured by the FRAP method (1676.80μmol Fe 2 SO 4 /g), see Table 1, was higher than the corresponding activity observed for the fruit (pulp and peel) extract of the plant (635 μmol Fe 2 SO 4 /g)

Determination of the total phenolic content
The total phenolic content (TPC) was determined by spectrophotometry as previously described in the literature (ZIELINSKI and KOZLOWSKA, 2000).Acetone, acetone-water (4:1), acetone-water (3:2), ethanol, ethanolwater (4:1), ethanol-water (3:2), methanol, methanol-water (4:1), methanol-water (3:2) and water were used as the solvents to extract the TPC.Samples of dried and powered fruit peel (1.00 g) were submitted to extraction with each of the eluents described above (25.0mL), shaking for 4 h and then centrifuging for 5 min at 1400 rpm (approximately 110 g-force).Each extract was filtered, and aliquots of the resultant solution (1.00 mL each) added to 9.00 mL of water and 1.00 mL of Folin-Ciocalteu or Folin-Denis reagent.After 5 min, 10.0 mL of a 7% sodium carbonate solution were added.The mixture was diluted to 25.0 mL with water and homogenized.After 60 min, the absorbance data were registered at 725 nm.The TPC was measured using a gallic acid standard and expressed as mg of gallic acid/g (GAE/g).All the experiments were carried out in triplicate.

Mineral analyses
The concentrations of the mineral elements Ca, Cu, Fe, K, Mg, Mn and Zn were determined by flame atomic absorption spectroscopic analysis as previously described in the literature (NANDA et al., 2003).The phosphorus concentration was determined by spectrophotometry as described by Rowell (1994), and the absorbance of the samples was measured at 725 nm.Samples of dried and powered fruit peel (0.20 g) were incinerated in a muffle furnace at 550 °C to complete oxidation of all carbonaceous material.The ash was cooled to room temperature, dissolved in 10.0 mL of HCl (0.1 N), transferred to a 50.0 mL volumetric flask and the volume completed with water.All experiments were carried out in triplicate.

Statistical analyses
All data were expressed as the mean ± standard deviation of triplicate experiments.The statistical analysis was carried out using Statistica 7.0 software (STATSOFT, 2001).Differences were tested for significance using the ANOVA procedure, with a significance level of p < 0.05.
The FRAP antioxidant test indicates that the fruit peel of M. cauliflora acts as a reducing agent by donating a hydrogen atom to the ferric complex, inhibiting the radical chain reactions.
Table 2 shows the antioxidant capacity of the fruit peel of M. cauliflora based on the oxidation inhibition percentage with different β-carotene concentrations.The increase in the β-carotene concentration (to 0.5, 1.0, 1.5 and 2.0 mg.mL -1 ) was related to the oxidation inhibition percentage (OI%) of the samples (OI% = 72.1,75.9, 82.5 and 87.5, respectively).The antioxidant activity of the fruit peel extract at 2.0 mg.mL -1 (OI% = 87.5%) was similar to value obtained with Trolox at the same concentration (OI% = 89.5%),confirming the powerful antioxidant capacity of this plant material.In fact, the antioxidant capacity of the fruit peel of M. cauliflora was higher than the corresponding capacity of fruits of Punica granatum at 0.2 mg/mL (OI% = 76.6)(ÇAM and HISIL, 2010) and the fruit extract of Ficus glomerata (OI% = 49.75)(VERMA et al., 2010).
The mechanism of bleaching β-carotene is mediated by hydroperoxide free radicals, which are formed from the abstraction of hydrogen atoms from the diallylic methylene groups of linoleic acid.The linoleic acid free radicals attack the highly unsaturated system of the β-carotene.As a result, the β-carotene molecules undergo rapid discoloration in the absence of an antioxidant (JAYAPRAKASHA et al., 2007).In the present study, the fruit peel extracts of M. cauliflora prevented the β-carotene bleaching by neutralizing the linoleate-free radical and other free radicals formed in the test mixture.

Determination of the total phenolic content
Table 3 shows the total phenolic content found in the extracts of the fruit peel of M. cauliflora using the Folin-Denis and Folin-Ciocalteau methods with different solvent mixtures.The extraction of the phenolic compounds was more efficient using the Folin-Denis method than using the Folin-Ciocalteau one.In fact, the total phenolic content as measured by the Folin-Ciocalteau method gave low values (1.35 mg GAE/g for acetone and 4.43 mg GAE/g for 4:1 ethanol-water solution), and the use of different solvent mixtures did not promote significant statistical variations in the total phenolic contents of the fruit peel extracts of M. cauliflora.
On the other hand, the total phenolic content as measured by the Folin-Denis method promoted significant Table 2. Antioxidant activity as determined by β-carotene bleaching for the fruit peel of M. cauliflora.
The mineral elements are essential regulators of physiological processes.Calcium, zinc and magnesium are important as cofactors in enzymatic processes, mainly in the structure of the DNA repair system (KONCZAK and ROULLE, 2011).Magnesium is also important in the change of energy in the mitochondria.Iron is essential to the transport of oxygen in the bloodstream.Some minerals are components of important enzymes such as Mn, Cu and Zn for superoxide dismutase and Fe for catalase (EVANS and HALLIWELL, 2001;BARROS et al., 2012).Both these enzymes protect the cell membranes from oxidative damage.The high concentration of these mineral elements in the fruit peel of M. cauliflora must promote the formation of these enzymes, and as a consequence provide its antioxidant capacity.

Conclusions
Several tests can be used to evaluate the antioxidant activity of plant materials, exhibiting different action mechanisms.The fruit peel of M. cauliflora showed high antioxidant capacity for the different methods.Moreover, the fruit peel of M. cauliflora was a good source of natural antioxidants such as the phenolic compounds, which were better estimated using the Folin-Denis method and 4:1 methanol-water, 4:1 ethanol-water, 3:2 ethanol-water or 3:2 acetone-water solutions as the solvent.The fruit peel of M. cauliflora was also a good source of minerals, potential catalysts of oxidative reactions.As a result, their consumption could contribute with substantial amounts of the dietary reference intakes of these compounds, suggesting their incorporation into the diet for food enrichment.
The TPC values found in the present work were low (553.6 mg GAE/ g) when compared to a previous study with Myrciaria jaboticaba peels (LEITE-LEGATTI et al., 2012).In that study, fresh fruits were employed, providing higher TPC values than the corresponding values obtained for the TPC of the dried fruit.The high TPC values registered in these studies may be an indication of the antioxidant activity of the plants (SILVA et al., 2012).

Analyses of the mineral elements
The concentration of mineral elements in the fruit peel of M. cauliflora is shown in Table 4. Potassium was the most abundant mineral in the fruit peel (1.060 mg/g of sample).This mineral is an essential dietary nutrient and fundamental to the regulation of the acid-base and water balances (SULAIMAN et al., 2011).The minerals phosphorus and calcium were also abundant (0.377 and 0.113 mg/g of sample, respectively).Other minerals were detected at low concentrations, such as Mg, Fe, Zn, Cu and Mn (0.065, 0.032, 0.026, 0.014 and 0.009 mg/g of sample, respectively).The concentrations of P, Mn, Zn, Fe and Cu in the fruit peel of M. cauliflora were higher as compared to the fruit pulps from this same plant (0.0200, 0.0028, 0.0019, 0.0033 and 0.0006 mg/g of sample, respectively) (LETERME et al., 2006).
The minerals K, Mg, and Ca were found in higher concentrations in the fruit peel of M. cauliflora than in the fruit pulps of Seel lime (1.012, 0.048, and trace, respectively).Higher values for Fe, Mn, Zn and Cu were also registered for the fruit peel of M. cauliflora when compared to the fruits of other plants.The concentration of Fe in the fruit peels of Lima orante, Sweet lime, Tahiti lime, Pera orange and Ponkan mandarin was 1008.6, 943.4, 768.7, 731.0, and 321.5 μg/100 g of sample, respectively (BARROS et al., 2012).The concentration of Mn in the fruits of Quandong, Finger lime (green) and Finger lime (pink) were 0.0029, 0.0045 and 0.0004 mg/g of sample, respectively.The concentration of Zn in the . Aliquots of the samples A-D (0.10 303 , Campinas, v. 16, n. 4, p. 301-309, out./dez.2013 Total antioxidant capacity, total phenolic content and mineral elements in the fruit peel of Myrciaria cauliflora Araújo, C. R. R. et al. http://bjft.ital.sp.gov.brBraz.J. Food Technol the corresponding value for the fruit extracts (pulp and peel, without seeds), EC 50 = 138.0g dried sample/g DPPH

Table 1 .
Antioxidant capacity of the fruit peel of M. cauliflora using different analytical methods.

Table 4 .
Concentration of mineral elements in the fruit peel of M. cauliflora.