Natural Antioxidant Activity and Compounds Content from Wastes of Euterpe edulis Berries

The Euterpe edulis (Juçara) is native to Brazil, which berries and wastes present high antioxidant content. Therefore, in this study, microwave-assisted extraction (MAE) was investigated for antioxidant compounds extraction from E. edulis waste and maximized antioxidant activities using response surface methodology coupled with a central composite design. Three factors were observed: microwave power (400/500/600 W), exposition time (30/60/90 sec) and ethanol concentration solvent (40/60/80%). The extracts were characterized by determination of total phenolic (TPC), flavonoids (TFC), monomeric anthocyanins (TAC), tannins content (TTC), and in vitro antioxidant assay (AA%). The yield of TPC, TFC, TAC, and TTC varied at 595.43-2171.34 mg GAE·100 g DM, 137.36-251.24 mg QE·100 g DM, 179.32-354.38 mg C-3-GE·100 g DM and 0.23-1.00 μg TAE·100 g DM, respectively. The optimal MAE parameters for TPC was microwave power 668.18 W, exposition time 110.45 s and aqueous ethanol concentration 93.64%, for TFC same parameters observed; though for TAC the different parameters were 532.28 W, and for TTC 9.55 s. However, for antioxidant activity, the parameters were 668.18 W, 110.45 s time and 64.41% of aqueous ethanol solvent. Therefore, this methodology was successfully applied for optimal extraction of total phenolics, flavonoids, monomeric anthocyanins and tannins from juçara waste and obtain optimal antioxidant activity.


Introduction
The Juçara palm (Euterpe edulis Martius; Arecaceae), a native plant of Brazilian Atlantic Forest, is widely distributed in Brazil.Mature berries from this plant exhibit a violaceous and globose shape, which are accepted by consumers (Silva et al., 2013).Juçara fruit is commercialized in the form of pulp or juice.During the processing of E. edulis pulp, the seeds with endocarp and epicarp are discarded as waste (Bicudo, Ribani, & Beta, 2014).The violaceous color of E. edulis pulp can be related to the presence of anthocyanins, which belong to the group of flavonoids (Cavalcanti, Santos, & Meireles, 2011).In addition, other bioactive molecules including high antioxidant capacity (Rufino et al., 2011).In addition, Garcia-Mendoza et al (Garcia-Mendoza et al., 2017) demonstrate that industrial residue of juçara presented high concentration of phenolic acids and anthocyanins.In recent years, the use of plant extracts, mainly extract of their waste processing, have gained notable interest in the food industry (Ertas et al., 2015).This fact is related to the search for new therapeutic and preventive agents, as natural antioxidants, for amendments and illness, Alzheimer's disease, and cancer (Zengin, Sarikurkcu, Aktumsek, & Ceylan, 2014).
Natural antioxidants agents present high attention in recent years for their bioactivity and safety (Lu, Qin, Han, Wang, & Li, 2015).The ingestion of this compounds is stimulated by potential neutralization effect on the toxicity of oxidative processes or prevention of prooxidant formation during digestion (Manganaris, Goulas, Vicente, & Terry, 2014;Rahal et al., 2014), which can contribute to reducing or prevent the aforementioned illness.Furthermore, fruits with high antioxidant capacity, such as juçara (Borges et al., 2013) are potential source of bioactive molecules that can be a technological alternative for food industry to prevent the oxidation, providing an increased in food shelf life (Manganaris et al., 2014;Ortega-Ramirez et al., 2014;Tadapaneni, Daryaei, Krishnamurthy, Edirisinghe, & Burton-Freeman, 2014).Therefore, the use of natural antioxidants capable of hindering oxidative processes responsible for losses in the organoleptic characteristics and the nutritional value of food is highly relevant for the food industry (Contini et al., 2014).However, the extraction of natural antioxidants compounds is still critical (Santos, Veggi, & Meireles, 2010), where the evaluation of efficacy and efficiency of each extraction method is extremely important.
Several studies evaluated the influence of the extraction method on different matrices for the isolation of antioxidant compounds (Da Silva Campelo Borges et al., 2011;Dairi et al., 2015;Espinosa-Pardo, Martinez, & Martinez-Correa, 2014;Kukula-Koch et al., 2013;Li, Ngadi, & Ma, 2014).Nonetheless, different approaches and applications do not always provide the same results.Hence, the optimization processing is required (Santos et al., 2010).The optimum combination of power, exposition time, and concentration of extracting solvent to obtain the highest concentration of these compounds is extremely important to ensure efficient utilization of energy, solvents, and food matrix.In this way, microwave-assisted extraction (MAE), an emerging green technology, have demonstrated to be a promising method for the recovery of bioactive compounds such as total phenolics, flavonoids, anthocyanins, and tannins from plants.(Dahmoune et al., 2014;Dahmoune, Nayak, Moussi, Remini, & Madani, 2015;Dairi et al., 2015;Kim et al., 2012;Li et al., 2012Li et al., , 2014;;Zeković, Vladić, Vidović, Adamović, & Pavlić, 2016).Although, to the best of our knowledge the MAE has not been used for extraction of natural antioxidant compounds from E. edulis.
Moreover, response surface methodology (RSM) can be used to optimize the extraction of natural antioxidant.The RSM is the combination of statistical and mathematical techniques which allow the improvement and optimization of processes.In this methodology, the response of interest is influenced by the independent variables or factors (Montgomery, 2004).Previous studies documented that the type of solvent, the temperature of extraction, exposition time, solid to liquid ratio, and microwave power influence the extraction of antioxidant molecules from fruits by MAE (Borges et al., 2011;Dairi et al., 2015).In this context, the aim of the present study was to utilize the antioxidants (total phenolic, flavonoids, anthocyanins, and tannins) present in the waste of pulp juçara processing.Besides, optimize the extraction of these compounds by MAE on the solid waste obtained during juçara berry processing applying RSM.Thereunto, three independent variables, microwave power (W), time exposition (s), and concentration of ethanol (%), were evaluated.

Experimental Design
The optimal extractions conditions can be obtained by the ratio of responses based on variables in the process through the Response surface methodology (RSM) (Karacabey & Mazza, 2010).A central composite design was utilized to determine the optimized condition in MAE extraction of total phenolic content (TPC), total flavonoids content (TFC), total monomeric anthocyanin content (TAC), total tannins content (TTC) and antioxidant activity from the juçara waste; non-coded and coded factors (microwave power, exposition time, and solvent concentration) are exhibited in Table 1.Although several factors such as microwave power, microwave temperature, exposure time, composition of solvent, solids to solid ratio and extraction cycles, can affect the extraction efficiency in MAE, studies show as the main independent variables microwave power, exposure time and solvent concentration (Li et al., 2012(Li et al., , 2013;;Zeković et al., 2016).To predict the optimal conditions of the extraction process experimental design software (Minitab ® 17.1.0,USA) package was used for the regression analysis of the data to fit a second-order polynomial equation (Equation 1) for the regression analysis of the data. (1) Where, TPC, TFC, TAC, and TTC values denote Y 1 , Y 2 , Y 3, and Y 4 , respectively; whereas the three independent variables (or factors) were microwave power (X 1 ), exposition time (X 2 ), and ethanol concentration (X 3 ).β 0 is the model constant, β i is the linear coefficient, β ii is the quadratic coefficient, β ij is the two factors interaction coefficient, and X i and X j are independent variables (factors) level.According to the analysis of variance (ANOVA), the regression coefficients of individual linear, quadratic and interaction terms were determined.

Microwave-Assisted Extraction (MAE)
MAE present advantages compared with conventional extraction, which is considered a green technology (Zeković et al., 2016).Powder of Juçara waste was subjected to MAE utilizing a DGT 100 Plus system (Provecto Analytics Ltd., Jundiaí, SP, Brazil) for antioxidant extraction.Briefly, 500 mg of waste were added to 25 mL of aqueous ethanol solution, sealed into the extraction vessels, and subjected to extraction protocol following the experimental design (Table 1).After each extraction, the vessels were centrifuged at 1,400 × g for 10 min at 4 ºC and cooled to 25 ºC.The precipitate was re-extracted with an additional 25 mL of the same ethanol solution and at the same MAE conditions; the supernatants were pooled and stored in amber vials at 4 ºC.

Determination of Total Phenolic Content (TPC)
TPC of the extracts of E. edulis waste was estimated based on Folin-Ciocalteu method described by Ainsworth & Gillespie (2007).The absorbance value at 765 nm was recorded using a Spectrophotometer UV-1800 (Shimadzu Corporation, Kyoto, Japan) and the results were calculated based on a calibration curve of gallic acid (0.00-1.25 mg L -1 ).The results were expressed as mg gallic acid equivalent (GAE) per 100 g of dry matter (DM).

Determination of Total Flavonoids Content (TFC)
The TFC extracts of E. edulis waste were estimated by a colorimetric method developed by Chang, Yang, Wen, and Chern (2002) utilizing aluminum chloride.The absorbance was read at 415 and 700 nm using Spectrophotometer UV-1800 (Shimadzu Corporation, Kyoto, Japan); the latter wavelength was utilized to correct the influence of haze.A calibration curve utilizing quercetin as standard (0-50 mg L -1 ) was used and the data was expressed as mg of quercetin equivalents (QE) per g of dry matter (DM).

Determination of Total Monomeric Anthocyanin Content (TAC)
TAC of extracts of the juçara waste was estimated by the pH differential method.In solution, at pH 1.0 anthocyanins exhibit predominantly the colored oxonium form whereas, at pH 4.5 there is a shift towards the colorless hemiketal form; therefore, it is possible to estimate TAC by the difference between absorbance values at 520 nm (Lee, Durst, & Wrolstad, 2005).Absorbance values at 520 and 700 nm were evaluated using Spectrophotometer UV-1800 (Shimadzu Corporation, Kyoto, Japan).The wavelength of 700 nm was utilized to correct the influence of haze on sample absorbance.The TAC value was calculated as follows (Equation 2): Where, A equals the difference between (Abs 520 nm (pH 1.0)-Abs 700 nm (pH 1.0)) and (Abs 520 nm (pH 4.5)-Abs 700 nm pH (4.5)); MW is the molecular weight 449.2 g mol -1 of cyanidin-3-glucoside (cyd-3-glu); DF is dilution factor of each sample; 10 3 is the unit conversion from g to mg; ε is 26,900 molar extinction coefficient, in L mol -1 cm -1 , for cyd-3-glu; and l is light path length in cm.The results were expressed as mg cyanidin-3-glucoside equivalents per 100 g of dry matter (DM).Note.All results are the means ± SD (n = 3).a-b Same letters prescribe that there was no difference between the experimental and predicted results within the analysis; different letters determine the difference.

Determination of Total Tannin Content (TTC)
TAC of the E. edulis waste extract was estimated according to the methodology of Makkar (2003).This method is based on the precipitation of condensed tannins using PVPP.In the first step was measured the content of total phenolics at the absorbance value at 725 nm using Spectrophotometer UV-1800 (Shimadzu Corporation, Kyoto, Japan).Whereas for the second step, were precipitated tannins and the absorbance value of the decantate was evaluated at 725 nm using Spectrophotometer UV-1800 (Shimadzu Corporation, Kyoto, Japan).The difference between the absorbance values between the first and the second steps was utilized to estimate the TTC value through a calibration curve of tannic acid as standard (0-14 µg mL -1 ) and expressed per 100 g of dry matter (DM).

Antioxidant Activity
β-carotene bleaching assay was used to determine the antioxidant activity of extracts by the β-carotene-linoleic acid model system (Siraichi et al., 2013) with modifications.Briefly, 1 mL of β-carotene (0.2 mg/mL) was pipetted into a glass tube with 20 mL of linoleic acid, 200 mg of Tween 40.The chloroform was completely evaporated by using a rotary evaporator (QUIMIS, Brazil).After, 50 mL of distilled water were added to the flask with vigorous stirring.Another emulsion was made without β-carotene.Aliquots (4.8 mL) of the prepared emulsions were transferred to a series of tubes containing 0.2 mL of extracts.The tubes were placed in a water bath at 50 ºC for 2 h.
The absorbance of each sample was measured using a Spectrophotometer UV-1800 (Shimadzu Corporation, Kyoto, Japan) set at 470 and 700 nm immediately after sample preparation (t = 0 min) and at 30-min intervals until the end (t = 120 min) of the experiment; the latter wavelength was utilized to correct the influence of haze.Emulsion without β-carotene using like blank.Water and BHT (3 mg/mL) were used as negative and positive controls, respectively.The antioxidant activity of extracts was expressed as (Equation 3): (3)

Statistical Analysis
All analyses are carried out in triplicate and results reported as mean values with standard deviation.ANOVA with Turkey test was performed using XLSTAT version 2013.2.03 (Addinsoft, Paris, France).RSM was performed using the Minitab ® software (version 17.1.0,USA).The regression coefficients of linear, square and two-way interaction terms were evaluated by analysis of variance, and the relevant (p < 0.05) terms were utilized to generate the surface and contour plots.The fitted polynomial equation indicated the optimal conditions for the TPC, TFC, TAC and TTC response variables.Differences in phenolic, flavonoids, monomeric anthocyanin and tannins compounds were considered significant at p < 0.05., you may refer the reader to that source and simply give a brief synopsis of the method in this section.
The RSM indicated the empirical relationship between TPC (Equation 4), TFC (Equation 5), TAC (Equation 6), TTC (Equation 7) and AA% (Equation 8) value with the extraction conditions were generated as follows: Moreover, ANOVA for experimental results show the quadratic polynomial model for TPC, TFC, TAC, TTC and AA% was significant highly (F = 30.78,F = 46.03,F = 50.81,F = 9.01, F = 8.89, respectively), with p < 0.001 for TPC, TFC and TAC; p = 0.001 for TTC; p = 0.002 for AA%, (Table 2).There is only a 0.01 to 0.002% chance that a ''Model F-Value'' this large could occur due to noise, recommended the significant of the model.R 2 and R 2 adjusted (Table 2) values for the model did not differ considerably, this confirms an adequate statistical model.However, a large value of R 2 not necessarily designates that the regression model is a sound one.Therefore, it is better to use the R 2 adjusted to evaluate the model adequacy, as that the addition of a variable to the model R 2 increase with the significant or non-significant variable (Karazhiyan, Razavi, & Phillips, 2011).The absence of lack of fit and the value of pure error indicated good reproducibility of the experimental data (Table 2).The model could work well for the prediction of TPC, TFC, TAC, TTC and AA% extract from E. edulis waste powder.Note. a Degree of freedom; b (mg GAE•100 g DM -1 ); c (mg QE•g DM -1 ); d (mg C3QE•100 g DM -1 ); e (mg TAE•100 g DM -1 ); R 2 A = R 2 Adjusted; R 2 P = R 2 Predicted; X 1 = Power (W); X 2 = Time (s); X 3 = Ethanol Concentration (%).

Interactions of the Studied Factors
The interaction between microwave power, exposition time and ethanol aqueous solution between TPC are shown in three-dimensional surface and contour plots (Figure 1A).The contour plots indicate the nature and extent of interactions of different components (Prakash, Talat, Hasan, & Pandey, 2008).The maximum point of each three-dimension plot subjected is the optimum point for the two factors presented in the chart.The effect between the exposition time (X 2 ) and ethanol concentration (X 3 ) are presented in Figure 1-A1, this can be observed the time and ethanol concentration is directly proportional to TPC yield, same behavior was observed in juçara pulp MAE (Cavalcanti et al., 2011), in coriander seed extracts (Zekovi et al., 2016), in Cammelia oleifera fruit (Zhang et al., 2011) and in Vitis coignetiae (Kim et al., 2012).Thus, the highest ethanol concentration increased extraction of compounds (Kim et al., 2012), this may be attributed the difference in dielectric properties of solvent towards microwave heating (Dahmoune et al., 2014).
The effect between the ratio of microwave power (X 1 ) and ethanol concentration (X 3 ) are represented in Figure 1-A2.First, the extraction combination decreased efficiency by raise power but after 550 W increased.Probably because increased diffusion rate and solubility of the target compounds in the solvent were affected by temperature (Fernández-Ponce, Casas, Mantell, & Martínez de la Ossa, 2015) and higher power must be excited phenolic molecules (Zeković et al., 2016).Also, an increase of ethanol concentration increase TPC yield, probably for the fact of solvent polarity declined and solubility of molecules increased.The same behavior has observed in Pistacia lentiscus and Vitis coignetiae (Dahmoune et al., 2014;Kim et al., 2012).This behavior was observed for phenolic compounds from Euterpe edulis peels and pulp near the seeds by Garcia-Mendonza et al. (Garcia-Mendoza et al., 2017).According to Fernández-Ponce et al. (2015) and Santos et al. (2012), others fruits extracts presented the same behaviors for temperature action.The interaction of ratio of microwave power (X 1 ) and exposition time (X 2 ) are presented in Figure 1-A3.The yield of TPC decreases with the increase of power, probably due to thermal degradation of phenolic compounds (Dahmoune et al., 2015;Dairi et al., 2015).

Table 1 .
Extraction conditions of the experimental design and results of total phenolic content (TPC), total flavonoids content (TFC), total monomeric anthocyanin content (TAC), total tannins content (TTC) and antioxidant activity (AA%)

Table 2 .
Analysis of variance (ANOVA) and regression coefficients for the quadratic polynomial model for the experimental results of total phenolic (TPC), total flavonoids (TFC), total monomeric anthocyanin (TAC), total tannins content (TTC) and antioxidant activity (AA%) from Euterpe edulis waste extract The experimentally optimized conditions described by the model to selected for maximum TPC and TFC extraction is microwave power 668.18W, exposition time of 110.45 seconds and aqueous ethanol concentration 93.64%.For maximum TAC extraction is microwave power 532.28 W, exposition time of 110.45 seconds and aqueous ethanol concentration 93.64%.For TTC extraction is microwave power 668.18W, exposition time of 9.55 seconds and aqueous ethanol concentration 93.64%.To obtain the optimum maximum extraction of antioxidants molecules studied, the parameter was determined: 668.18 W, 110.45 seconds and 93.64% by the model.And for antioxidant activity is microwave power 668.18W, exposition time of 110.45 seconds and aqueous ethanol 64.41%.