Arginase inhibition, antibacterial and antioxidant activities of Pitanga seed (Eugenia uniflora L.) extracts from sustainable technologies of high pressure extraction

Food Bioscience 12 (2015) 93–99

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Food Bioscience journal homepage: www.elsevier.com/locate/fbio

Arginase inhibition, antibacterial and antioxidant activities of Pitanga seed (Eugenia uniflora L.) extracts from sustainable technologies of high pressure extraction$ Débora Nascimentoe Santos a, Larissa Lima de Souza a, Carlos Augusto Fernandes de Oliveira a, Edson Roberto Silva b, Alessandra Lopes de Oliveira a,n a Departamento de Engenharia de Alimentos, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo ((FZEA/ USP), Avenida Duque de Caxias Norte, 225, Jardim Elite, CEP 13635-900 Pirassununga, SP, Brazil b Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidad e de São Paulo (FZEA/ USP), Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 19 September 2014 Received in revised form 28 August 2015 Accepted 4 September 2015 Available online 14 September 2015

Pitanga seed extracts from supercritical extraction (SFE), pressurized liquid extraction (PLE) and conventional Soxhlet extraction had their bioactive properties tested in vitro. The essays included antioxidant (reducing power, DPPH
and ABTS
þ ) and antibacterial (S. aureus, E. coli, P. aeruginosa and B. subtilis) activities, as well as the arginase enzyme inhibition test. Purified PLE extracts presented the highest antioxidant capacity (33.37 mM Trolox  g  1 extract) and arginase inhibition (75.96%). All extracts presented the same value of minimum concentration inhibition, 125 mg  g  1, indicating high antibacterial potential. The purified fractions from extracts obtained using SFE and PLE processes had increased arginase from Leishmania inhibition, 48.48% and 75.96% respectively, compared to Soxhlet (37.59%). These findings suggest that using different extraction methods on Pitanga seed can produce compounds with diverse activities. Extracts from Pitanga seed, a residue from the juice processing industry, possess characteristic fruit volatiles and could be used as natural aroma enrichment of Pitanga products or in functional foods formulations since compounds of the extracts present bioactive activities. & 2015 Elsevier Ltd. All rights reserved.

Keywords: Pitanga seed Supercritical extraction Antioxidant Food industry waste Leishmania

1. Introduction Waste from fruit and vegetable processing not only presents a disposal problem for the food industry but also provides promising sources of bioactive compounds that can be utilized for technological or nutritional properties (Ćetković et al., 2012). The Brazilian juice agribusiness disposes tons of Pitanga seed as waste, which is a considerable loss because the seed comprises approximately 30% of the fruit. However, nutritional value and several bioactive compounds in Pitanga fruit have been identified, including pre-vitamin A, carotenoids and flavonoids (Lopes-Filho et al., 2008; Mélo, Lima & Nascimento, 1999). Studies about Pitanga seed showed essential fatty acids (linoleic and α-linolenic acids are present in greater quantities in the seed), volatile and phenolic compounds with biological value (Bagetti, Facco, Rodrigues, Vizzotto & Emanuelli, 2009; Santos, ☆ n

Bioactivity of Pitanga seed (Eugenia uniflora L.) extracts obtained by PLE and SFE. Corresponding author. Fax: þ 55 19 3565 4284. E-mail address: [email protected] (A.L.d. Oliveira).

http://dx.doi.org/10.1016/j.fbio.2015.09.001 2212-4292/& 2015 Elsevier Ltd. All rights reserved.

Souza, Ferreira & Oliveira, 2015). Pitanga leaves have aromatic, anti-fever and anti-dysenteric properties and alcohol extracts from the leaves are used to treat bronchitis, coughs, anxiety and diseases caused by worms (Amorim, Lima, Hovell, Miranda & Rezende, 2009). Brazilian folk medicine uses Pitanga fruit for its therapeutic effects, as for the treatment of diarrhea, hyperglycemia, hyperlipidemia, malaria, and hypertension (Santos, Fortes, Ferri & Santos, 2011). Oliveira, Lopes, Cabral and Eberlin (2006) found similar volatile compounds present in the fruit and leaves. Fruit extract from supercritical fluid extraction (SFE) also revealed the presence of bioactive compounds and volatile sensory analysis identified compounds that confer the characteristic Pitanga aroma (Malaman, Moraes, West, Ferreira & Oliveira, 2011). Generating recovery and sustainable technologies is important to utilize food industry wastes as an alternative source of raw materials with an added benefit, and methods that prevent the loss of compounds present in these residues are essential (Reis et al., 2012). High pressure technologies such as SFE (Supercritical Fluid Extraction) and PLE (Pressurized Liquid Extraction) are available extraction methods for recovery of functional

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D.N. Santos et al. / Food Bioscience 12 (2015) 93–99

Table 1 Pitanga seed extract reducing power using TPC assay and antioxidant activity using DPPH
and ABTS
þ . Essay

Variables in SFE P (kgf  cm  2) T (°C)

TPCa in the extract (mg  g  1 GAE)

TPCa in the seeds (mg  100 g  1 seeds GAE)

DPPH
-IC50 (mg  g  1 extracts)

ABTS
þ a (mM Trolox  g  1 extract)

SFE 01 SFE 02 SFE 03 SFE 04 SFE 05 SFE 06 SFE 07 SFE 08 SFE 09 SFE 10 SFE 11 Soxhlet SFE-EtOH Purified SFEEtOH Purified PLE

110 240 110 240 83 267 175 175 175 175 175

35 35 55 55 45 45 31 59 45 45 45

175 175

45 45

49.74 73.48 44.41 39.79 18.08 66.75 61.59 61.42 48.76 42.65 23.35 – 51.08 27.34

0.014 0.034 0.010 0.019 0.003 0.028 0.024 0.028 0.021 0.017 0.011 – 0.018 –

41,881.9 31,915.2 50,246.3 23,461.0 30,139.3 23,403.7 33,028.6 38,120.5 33,205.9 35,864.5 38,343.1 12,955.2 nd nd

12.56 5.84 4.52 5.95 6.41 7.45 5.32 6.07 5.72 6.07 5.32 18.66 19.29 11.82

102

60

–

–

4601.17

33.37

a

Mean of three replicates; total phenolic content (TPC); supercritical extract (SFE); supercritical extract with ethanol as co-solvent (SFE-EtOH), purified fraction from SFE with co-solvent (Purified SFE-EtOH), purified fraction from PLE extraction (Purified PLE). nd – not determined.

compounds from plant and animal matrices. In these systems, the solvent actively modifies the matrix, deforming cellular structures and denaturing proteins. Additionally, the solubility increases with pressure, subsequently extractions carried out at high pressure have high mass transfer rates (Jun, 2009; Prasad, Yang, Yi, Zhao & Jiang, 2009). SFE using carbon dioxide as a solvent has been routinely used for the recovery of compounds beneficial to health; therefore, its application in the food and pharmaceutical industries is appropriate. Carbon dioxide is an ideal solvent for the extraction of natural products because it is non-explosive, readily available, easy to remove from extracted products and has low toxicity. The use of CO2 is environmentally friendly because it allows for extraction without the use of organic solvents and has a critical temperature of 31 °C, which enables the recovery of thermo sensitive natural compounds with minimal damage, preserving their bioactive properties (Reverchon & De Marco, 2006). PLE method has also been used for several materials. According to Li, Dong, Shim, and Kannan (2007), the main advantages include ease of automation, speed and efficiency, with little consumption of solvent. The increased extraction temperature improves the solubility, diffusion rate and mass transfer (Polovka, Št’avíková, Hohnová, Karásek & Roth, 2010). The objective of this study was to obtain Pitanga seed extracts using high pressure technology and generally recognized as safe (GRAS) solvents and show compounds with some active properties in these extracts. Successful production of extracts from SFE and PLE that are free of organic solvents residues and could enable use as raw material for functional foods and pharmaceuticals. The result would be enhancement of these seeds, so far regarded as a food industry processing waste.

2. Materials and methods 2.1. Pitanga seed, SFE, PLE and Soxhlet conventional methods The seeds from ripe native Pitanga fruit were recovered, washed to remove traces of pulp and dried in an oven with air circulation at 38 °C for 54 h. The dried seeds were peeled, crushed and frozen at  20 °C (Santos et al., 2015). Ether extract was obtained via the hot lipid extraction method using a Soxhlet extractor and petroleum ether (Synth, Diadema, Brazil) as the organic solvent (Instituto Adolfo Lutz, 2008). In the SFE procedure, contact between the crushed seeds,

which were packed in a fixed-bed extractor with a volume of 300 cm3, and supercritical CO2 to pre-set pressure (P) and temperature (T) was promoted using a central composite design (CCD). The seed extract was separated from the gas and collected in a flask collector. At the end of the process, the extracts were weighed and compared to the mass of dry seeds to calculate the percent yield. For all of the tests, a standardized time of 16 h was used to establish static equilibrium and the dynamic extraction period was 6 h. The condition that presented highest yield was made again, using ethanol (EtOH, Synth, Diadema, Brazil) as cosolvent. The extract obtained was purified and, crude and purified extracts were evaluated (Santos et al., 2015). PLE was performed using an ASE 100 accelerated solvent extraction system (Dionex, Voisins le Bretonneaux, France) with 34 ml stainless steel ASE vessels. Anhydrous ethanol (EtOH) was used as the solvent. In the optimization process, four parameters were studied: T, static time (ST), the number of cycles of solvent washes per sample (C) and the necessary solvent volume (VF) in a 24 CCD, as reported by Oliveira, Destandau, Fougère, and Lafosse (2014). Dried and crushed seeds (5 g) were packed in the vessel extractor with 5 g of sodium sulfate as the adsorbent material used to disperse the vegetal matrix in the extraction cell, allowing improved contact with the solvent and extract clarity. The ethanolic extract obtained by PLE (crude extract) was evaporated and purified. 2.2. Bioactivity tests Fifteen different extracts were obtained: 11 from SFE in the CCD, one using conventional extraction by Soxhlet, one from SFEEtOH, one from the purified SFE-EtOH fraction, and one from the purified PLE fraction (Table 1). Antioxidant capacity was analyzed using DPPH radical and ABTS
þ methods, and the reducing power of these extracts was determined as total amount of phenolic compounds (TPC) using the Folin–Ciocalteu method. Antimicrobial capacity and arginase inhibition assays were also performed. 2.2.1. Reducing power determination using Folin–Ciocalteu reagent The reducing power of the compounds in the extracts was determined using the method described by Singleton and RossiJúnior (1965). One ml of extract diluted in methanol (MeOH; 1,000 mg extract/l methanol, JT. Baker, Center Valley, United States), and 5 ml of Folin–Ciocalteu (Haloquímica, Tatuapé, Brazil) was diluted in distilled water (1:10, v–v). After 10 min, 4 ml of aqueous

D.N. Santos et al. / Food Bioscience 12 (2015) 93–99

solution containing 7.5 g  100 ml  1 anhydrous sodium carbonate (Synth, Diadema, Brazil) was added. After 2 h at room temperature in the dark, absorbance was measured at 756 nm using a Biospectro SP 22 spectrophotometer (Curitiba, Brazil). The blank was prepared with 1 ml of MeOH rather than extract. Gallic acid (Fluka, Bellefonte, United States) was used to determine the standard curve (y¼ 0.0104xþ 0.00169, R2 ¼0.9998). Reducing power was calculated as TPC and expressed in mg of gallic acid equivalents (GAE) per 100 g of dry Pitanga seed. 2.2.2. Antioxidant capacity determination by the inhibition of DPPH radical (DPPH
) In a test-tube, 3.9 ml of DPPH
(2,2-diphenyl-1-picrylhydrazyl, Sigma Aldrich, Munich, Germany) diluted in MeOH (6  10  5 M) and 0.1 ml of extract were combined. Readings at 515 nm were performed in triplicate using a Biospectro SP 22 spectrophotometer (Curitiba, Brazil) and included a control without extract. The decrease in the optical density of DPPH
due to the extracts was compared with the control, and the percentage of DPPH
discoloration for the different extracts was calculated. The half-maximal effective concentration (EC50) was determined using the midpoint between the extracts percentage inhibition and baseline concentration (Brand-Williams, Cuvelier & Berset, 1995). 2.2.3. Trolox equivalent antioxidant capacity (TEAC) The TEAC was determined using the methodology described by Re et al. (1999) with adaptations. ABTS
þ (2,2′-Azino-bis(3ethylbenzothiazoline-6-sulfonic acid, Sigma Aldrich, St. Louis, United States) was prepared using 7 mM ABTS
þ and 140 mM of potassium persulfate (Synth, Diadema, Brazil) incubated at room temperature without light for 16 h. Then, the solution was diluted with ethanol until it reached an absorbance of 0.700 ( 70.02) at 734 nm. For antioxidant capacity measurements, 30 ml of extract was diluted in MeOH from 1000 to 10,000 mg  ml  1. Three mL of ABTS
þ solution was added to each dilution and the absorbance at 734 nm was determined using a Biospectro SP 22 spectrophotometer (Curitiba, Brazil). The synthetic antioxidant Trolox (Sigma Aldrich, St. Louis, United States) was used as a standard solution for the calibration curve (y¼  0.00029x þ0.6838, R2 ¼0.9804). The results were expressed as mM of Trolox per g of extract. 2.2.4. Antimicrobial capacity-minimum inhibitory concentration (MIC) The antimicrobial effects against Pseudomonas aeruginosa ATCC 15442, Bacillus subtilis ATCC 6623, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 (CEFAR, São Paulo, Brazil) were tested according to the methodology described by Andrade et al. (2010) with some modifications. The pure bacterial strains were subcultured in 10 ml of nutrient broth (Oxoid, Cambridge, United Kingdom) and incubated at 35 °C for 24 h. Then, the absorbance of the broth with the microorganisms was read at 625 nm using an UV densitometer (Suspension Turbidity Detector, DEN-1, Riga, Latvia) and compared with a 0.5 McFarland turbidity standard, equivalent to 1.5  108 UFC  ml  1. To test the antimicrobial capacity, the extracts were aseptically inoculated in Mueller–Hinton broth (Oxoid, Cambridge, United Kingdom) previously containing the incubated bacteria (1.5  108 UFC  ml  1). Positive (growth medium þstrains) and negative (growth medium alone) controls were also made. After 24 h of incubation at 37 °C, the minimum inhibitory concentration (MIC), defined as the lowest concentration where no microbial growth occurs, was determined by comparison with the negative control. Assays were performed in triplicate.

95

2.2.5. Arginase inhibition Recombinant arginase from Leishmania amazonensis (ARG-L) was prepared and purified as described (Silva, Maquiaveli & Magalhães, 2012). Rat liver arginase (ARG-1) was prepared by disruption of the hepatic tissue in 50 mM Tris–HCl buffer, 1 mM EDTA and PMSF at pH 7.2 using a blender. ARG-1 was fully activated by the addition of 10 mM MnSO4 to the homogenate and heating of the solution at 60 °C for 10 min. The suspension obtained was clarified by centrifugation at 20,000g, and the supernatant was used to perform the arginase inhibition tests. The inhibition tests for ARG-L and ARG-1 were performed at pH 9.5 in 50 mM CHES buffer and 50 mM L-arginine (pH 9.5), L. amazonensis recombinant arginase, and the Pitanga extract (SFE, SFE-EtOH, PLE and Soxhlet). The reaction mixtures were incubated in a 37 °C water bath for 15 min. The quantity of enzyme used was adjusted to 10% of the maximum consumption of L-arginine substrate. Quantification of urea production was performed by the method described by Berthelot (Fawcett & Scott, 1960). Briefly, arginase catalytic capacity was stopped by transferring 10 ml of the reaction mixture into 750 ml of reagent A (20 mM phosphate buffer pH 7, containing 60 mM salicylate, 1 mM sodium nitroprusside and 500 IU of urease). This mixture was incubated at 37 °C for 5 min. Next, 750 mL of reagent B (10 mM sodium hypochlorite and 150 mM NaOH) was added, and the samples were incubated at 37 °C for 10 min. Absorbance measurements were carried out at 600 nm using a Hitachi 2810U spectrophotometer (Tokyo, Japan). The positive and negative controls were performed under the same conditions in the absence of inhibitor. The experiments were performed in triplicate in at least two independent experiments. 2.3. Statistical analysis Operational conditions (pressure and temperature) for SFE were studied according to a central composite design (CCD) and the results of antioxidant capacity (TPC, DPPH
, ABTS
þ ) were analyzed by response surface analysis (RSA). This analysis includes verification of independent factors (P and T) in relation to the response by means of predictive mathematical models when they are significant (Barros, Scarminio, Bruns & 1996). The response surface analysis was performed using Statistica v.7.0 program (Tulsa, United States). For testing the antimicrobial capacity and inhibition of enzyme arginase, results were obtained in triplicate and expressed as mean 7 standard deviation (Santos et al., 2015). For PLE, operational conditions (temperature, cycle numbers, volume of solvent and, the solvent contact time with Pitanga seed) were also studied according to a CCD and analyzed by RSA (Oliveira et al., 2014).

3. Results and discussion 3.1. Reducing power of Pitanga seed extracts using TPC The reducing power demonstrated by the total phenolic content (TPC) of the supercritical extracts from Pitanga seed ranged from 18.08 to 73.48 mg  g  1 GAE or from 0.003 to 0.034 mg GAE  100 g  1 Pitanga seed (Table 1). These extracts have a considerable amount of phenolic compounds even when using supercritical CO2 as a solvent at low polarity. However, according to Oliveira et al. (2014) PLE ethanol extracts had a higher concentration of total phenolic compounds, ranging from 0.42 to 1.68 mg GAE  100 g  1 Pitanga seed. In Pitanga seed extracts from SFE, the process variables (P and T) did not influence the total amount of phenolic compound. Analysis of variance (ANOVA) of P and T influence on the TPC was not significant at 95% confidence for both linear and quadratic

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Table 2 Analysis of variance (ANOVA) for first and second order factorial experimental design for the total phenolic content (TPC) and antioxidant activity of Pitanga seed extracts using ABTS
þ a (mM Trolox  g  1 extract) Source of variation

Model (R) Residue (r) Total Model (R) Residue (r) Total

Sum square (SS) TPC 673.11 668.98 1342.09 TPC 2,130.73 849.83 2,980.56

ABTS 39.37 4.21 43.57 ABTS 28.42 17.66 46.08

Ftab

Degree of freedom (DF)

Mean square (MS)

Fcalc

TPC 3 3 6 TPC 5 5 10

TPC 224.37 222.99

ABTS 39.37 0.84

TPC 1.00

ABTS 46.80

TPC 3.18

ABTS 6.61

TPC 426.15 169.97

ABTS 14.21 2.21

TPC 2.50

ABTS 6.43

TPC 5.05

ABTS 4.46

ABTS 1 5 6 ABTS 2 8 10

Fcalc ¼MSR/MSr. Order to be significant, Fcal 4Ftab at 95% confidence interval. a

Significant parameters of the model presented.

models (Table 2). The coefficients of determination (R2) were 0.50 for the linear and 0.71 for the quadratic model. The R2 values also indicated that the generated models did not have the predictive capacity to describe the behavior of the TPC in the extracts when different T and P conditions are employed. 3.2. Antioxidant capacity The antioxidant capacity of the extracts from SFE and Soxhlet when analyzed using DPPH
were very low (IC50 mean greater than 30,000 mg  g  1 extract, Table 1). Similar values were found for rambutan extracts obtained by SFE (Palanisamy et al., 2008) and for supercritical extracts from Pitanga leaves (Martinez-Correa et al., 2011). High reduction potential values were found for SFEEtOH extracts and purified extract from PLE. The DPPH
method is not the most suitable for SFE extracts, as this radical interacts specifically with hydrophilic compounds, and supercritical CO2 extracts without polarity modifiers are generally hydrophobic. The use of EtOH as a polarity modifier and cosolvent in SFE resulted in an increased extract antioxidant capacity, with a radical inhibition capacity of 94.20% for 5000 mg  g  1 extract. However, when the extract concentration was increased to 7500 mg  g  1, the antioxidant capacity decreased to 24.94% (Fig. 1). This behavior is typical of pro-oxidant capacity and results from the mechanism of antioxidant action, which according to Dai and Mumper (2010) occurs when the maximum antioxidant concentration is surpassed and the excess antioxidant assumes an opposite characteristic and induces oxidation mechanisms. Phenolic antioxidants behave like pro-oxidants under conditions that favor their autoxidation; for example, at high pH, high concentrations of metal ions and in the presence of oxygen (Dai and Mumper, 2010). Rahman (2007) affirm that carotenoids, in particular β-carotene, exhibit pro-oxidant properties at high concentrations and, the flavonoids could also presents pro-oxidant activity. Although pro-oxidant activities could be related to unsafe effects on health, the resveratrol in pro-oxidant conditions provided a significant anticancer activity in the study of apoptosis induction (Lastra & Villegas, 2007). Because the antioxidant capacity was lower for higher extract concentrations, the concentrations of the SFE-EtOH extract were likely in the transition range from antioxidant to pro-oxidant. The purified SFE-EtOH extract also retained this characteristic, but with a lower antioxidant capacity of 34.52% (Fig. 1). The extract purification eliminated tannins with a high degree of polymerization; therefore, SFE-EtOH and PLE purified extracts did not have high amounts of tannins (Oliveira et al., 2014), and smaller antioxidant and pro-oxidant activities were expected. Pitanga seed extracts from PLE using EtOH as solvent at high temperatures presented high TPC and antioxidant capacity. This PLE extract and its purified fraction (Purified PLE) were analyzed

Fig. 1. Inhibition capacity of DPPH radical of Pitanga seed extracts in different concentrations.

by ESI/MS and HPLC/ESI/HRMS. Phenolic glycosides as pentoside ellagic acid, quercetin hexoside, ellagic acid deoxyhexoside and kaempferol pentoside, and phenolic compounds like ellagic acid and quercetrin were identified in these samples (Oliveira et al., 2014). These compounds have known biological activities (Ferreres, Grosso, Gil-Izquierdo, Valentão & Andrade, 2013; Sójka, Guyot, Kołodziejczyk, Król & Baron, 2009). Therefore, it can be expected that antioxidant capacity displayed in SFE-EtOH, Purified SFE and Purified PLE is related to the presence of these compounds in the extracts. Antioxidant capacity measured using the ABTS radical method expressed the antioxidant capacity as mM Trolox equivalent in 1 g extract. Extracts from SFE ranged from 4.52 to 19.29 mM Trolox equivalents (Table 1). Despite applying ethanol as a cosolvent, the reducing power (expressed in phenolic compounds) of the SFEEtOH extract is less than some supercritical extracts. However, the antioxidant capacity observed for the radical inhibition was superior compared to other extracts. This result is due to the differences in extract components when a polarity modifier is used. In the case of SFE-EtOH extracts, the compounds present have greater antioxidant capacity compared to compounds in supercritical extracts without polarity modifiers. The influence of extraction variables P and T was especially apparent with respect to antioxidant capacity. ANOVA indicated that the first order effect of these variables showed a significant T effect and a significant interaction between T and P (Table 2). The temperature effect was negative, indicating that the extracts obtained at low temperatures retained components with antioxidant properties. The negative effect of T and the positive effect of P  T are shown in the Pareto Diagram (Fig. 2). ANOVA of the second order model describing the influence of the variables on antioxidant capacity was not significant for P, T, or the interaction between them. Thus, the significant first order model (Eq. 1) was used to generate the response surface (Fig. 3), illustrating the

D.N. Santos et al. / Food Bioscience 12 (2015) 93–99

Fig. 2. Pareto chart shows the effect of independent variables P (pressure) and T (temperature) on the antioxidant activity (ABTS
þ ) of Pitanga seed extracts from SFE (supercritical fluid extraction).

Fig. 3. Response surface analyses of temperature and pressure (T and P) effects on the antioxidant activity (ABTS
þ ) of Pitanga seed extracts from SFE (supercritical fluid extraction).

influence of these variables on the range of SFE extract antioxidant capacity values studied in this work.

ABTS•+ = 6.57* − 1.32P − 1.98T * + 2.04P × T *

(1)

SFE extract antioxidant capacity values are higher than values obtained from other fruit pulp (2.0 mM Trolox  g  1 cupuaçu pulp, and 8.2 mM  g  1 Trolox guava pulp) (Kuskoski, Asuero, Troncoso, Mancini-Filho & Fett, 2005). The SFE-EtOH extract (19.29 mM  g  1 extract) and PLE extract (33.37 mM  g  1 extract) presented the highest concentrations of Trolox equivalents. According to Wootton-Beard, Moran, and Ryan (2011) there are differences between the results obtained by DPPH
and ABTS
þ which result from the reaction mechanisms. In ABTS
þ analyses, a transfer of electrons occurs and different antioxidant compounds provide electrons to reduce the radical cation. Despite the potential of the antioxidant compound, these compounds have time to react completely, allowing an accurate measurement of total antioxidant capacity. The inhibition of DPPH
is based on the reaction of a hydrogen atom transfer that occurs between antioxidants and peroxyl radicals. In this method, nitrogen radicals are created instead of peroxyl radicals, which are more stable and less transient. Some antioxidant compounds react more slowly than peroxyl radicals, which results in lower levels of antioxidant capacity. 3.3. Antimicrobial capacity The antimicrobial capacity for Pitanga seed extracts using SFE,

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SFE-EtOH, purified SFE-EtOH and Soxhlet was tested using E. coli, S. aureus, P. aeruginosa, and B. subtilis to determine the minimum inhibitory concentration (MIC) for the different extraction methods. Extracts rich in terpene compounds were obtained using Soxhlet or supercritical CO2, and compounds with higher polarity were found in SFE-EtOH and purified SFE-EtOH extracts (Santos et al., 2015). The MIC (lowest concentration where no microbial growth occurs) was 125 mg  mL  1 for all extracts. Although Gram (  ) bacteria are more resistant to antibiotic substances from plants than Gram ( þ) bacteria due to the greater complexity of the cell wall, no differences in the antimicrobial capacity of the extracts were observed between strains. Similarly, Bouzada et al. (2009) did not observe higher sensitivity of Gram (þ) to Pitanga leaf extracts. Physical and chemical characterization of essential oils from the same Pitanga seed obtained by SFE, showed as the major volatile compounds the sesquiterpenes germacrone, у-elemene and furanodiene (Santos et al., 2015). These compounds are recognized for their antibacterial and antifungal properties (Adio, 2009). Consequently, the presence of these compounds in the extracts contributes to the inhibition of bacterial growth. The SFE Pitanga seed extracts contained properties similar to essential oils (Santos et al., 2015). Some studies suggest that essential oils have a higher antibacterial capacity than a mixture of the main components from Pitanga seed extracts. The minor components present in essential oils produce a synergistic effect similar to effects contributed by major components and are important for the inhibitory capacity. For example, camphor and eucalyptol, general components in essential oils from many plants, have oxygenated groups in their structure, which increase the antimicrobial capacity of terpenoids. Consequently, combinations of different biochemical components in essential oils may increase antimicrobial efficacy (Lv, Liang, Yuan & Li, 2011). The MIC of SFE extracts from shiitake mushrooms was tested by Kitzberger, Smânia, Pedrosa, and Ferreira (2007) using Micrococcus luteus and Bacillus cereus, and the lowest MIC was 250 mg  ml  1. By comparison, the results from Pitanga seed extracts are promising, indicating high inhibition of microorganism growth. Bouzada et al. (2009) used methanol for the extraction of Pitanga leaves and achieved antimicrobial capacity by inhibiting 1.9 and 2.2 cm zones of Salmonella typhimurium and Shigella sonnei, respectively. However, the ethanol extract from Pitanga leaves did not demonstrate antimicrobial capacity against E. coli, P. aeruginosa and S. aureus (Coutinho, Costa, Lima & Siqueira-Júnior, 2010). This difference in results derived from the same matrix reinforces the importance of the extraction method to the biological activities. 3.4. Arginase inhibition In protozoa, arginase is responsible for the production of ornithine, an amino acid precursor of polyamines required in parasite proliferation. Therefore, inhibiting the action of arginase inhibits parasite proliferation and disease. Table 3 displays the percent inhibition of arginase isolated from rat liver and produced from Leishmania for different Pitanga seed extracts determined by spectrophotometric analyses. The highest inhibition (76%) was obtained for arginase from Leishmania (ARG-L) when it was tested Purified PLE extract followed by SFE-EtOH extract that showed high arginase inhibition (48%) (Table 3). This behavior indicates that compounds with higher polarity, extracted when ethanol is used, are more effective enzyme inhibitors. Certain substances can interfere with the enzyme–substrate complex formation between arginase and its substrate, L-arginine, displacing the hydroxide ion linked to manganese (Mn2 þ ) in the active site of the enzyme and causing loss of

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Table 3 Percent inhibition (%) of arginase activity isolated from rat liver and Leishmania (L.) amazonensis by Pitanga seed extracts. Arginase from rat liver Extract

SFE

14.99 79.20 Arginase from Leishmania Extract SFE 21.01 7 6.78

SFE-EtOH

Purified SFE-EtOH

Soxhlet

Purified PLE

42.377 3.93

23.66 75.0

33.657 7.05

25.88 7 13.00

SFE-EtOH 48.487 10.55

Purified SFE-EtOH 17.93 7 4.64

Soxhlet 37.59 7 8.62

Purified PLE 75.96 7 5.46

All results presented as the means of three replicates values7 SEM (Standard error of the mean). supercritical extract (SFE); supercritical extract with ethanol as co-solvent (SFE-EtOH), purified fraction from SFE with co-solvent (Purified SFE-EtOH), Purified fraction from PLE extraction (Purified PLE).

arginase function (Tormanen, 2003). Rat arginase inhibition, which simulates the mammalian liver enzyme, was lower for all extracts, less than 50% enzyme inhibition (Table 3). Recent study showed that pure phenolic compounds tested for arginase inhibition capacity in rat and Leishmania presented low inhibition for rat arginase and high for ARG-L due to differences in the molecular structures, which reflects in the activation energy that is lower for ARG-L (Manjolin, Reis, Maquiaveli, Santos-Filho & Silva, 2013). Purified PLE Pitanga seed extracts are rich in phenolic compounds and contain the flavonoids quercetin and quercitrin (Oliveira et al., 2014). In a recent study, Silva et al. (2012) demonstrated that the phenolic compounds quercetin, quercitrin and isoquercitrin inhibited arginase. The quercitrin presented an EC50 of 10 mM or 4.48 mg  ml  1, while the quercetin presented an EC50 of 4.3 mM or 1.76 mg  ml  1. The inhibitory effect of PLE Pitanga seed extracts that contain these flavonoids, show an EC50 of 42.46 mg  ml  1. The importance of arginase inhibition is twofold. First, inactivation of arginase affects the vital metabolism of the protozoan Leishmania, contributing to the control of Leishmaniose disease (Silva, Castilho, Pioker, Silva & Floeter-Winter, 2002). Second, the inhibition of arginase from host increases endothelial function and lowers blood pressure, which may contribute to the development of new drugs that would combat hypertension (Bagnost et al., 2009). Supercritical extracts from Pitanga seed, especially those obtained with ethanol as a cosolvent, can be investigated in future studies that may identify new drugs that inhibit arginase and are useful for the treatment of leishmaniasis or hypertension.

4. Conclusions All extracts demonstrated high capacity against pathogenic microorganisms and moderate antioxidant capacity. High antimicrobial activity was expected because these seeds extracts presented similar characteristics of leaves and fruit essential oils, whose activity is proven. The compounds of the Pitanga seed essential oils presents activity and therefore can be used as a natural preservative in food formulation or even as a medicament. Particularly, the SFE and SFE-EtOH extracts showed high MIC, exceeding values obtained for many other plant extracts usually employed for this purpose. Pitanga seed, so far treated as waste of pulp and juice processing industry, could be exploited for this purpose and be destined to a new and important application. Additionally, these extracts contain phenolic compounds and showed inhibitory activity of arginase, especially PLE extract that confirmed higher antioxidant capacity than the other. The further study of extract capacity can lead to the development of new drugs to control pathogenic bacteria, treat leishmaniasis or elucidate the role of antioxidant compounds in the human diet and cosmetics.

Acknowledgments The authors would like to thank FAPESP (State of São Paulo Research Foundation, Brazil) for financial support under the research projects 2008/00148-0 and 2010/16665-3 and CAPES for the MSc scholarship.

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