J Food Sci Technol DOI 10.1007/s13197-015-1821-4
ORIGINAL ARTICLE
Physicochemical, bioactive and functional evaluation of the exotic fruits Opuntia ficus-indica AND Pilosocereus pachycladus Ritter from the Brazilian caatinga Rosane Liége Alves de Souza 1 & Maristela F. S. Santana 2 & Edangelo M. S. de Macedo 3 & Edy Sousa de Brito 3 & Roberta T. P. Correia 1
Revised: 17 March 2015 / Accepted: 23 March 2015 # Association of Food Scientists & Technologists (India) 2015
Abstract The Brazilian caatinga is characterized as one of the most populated and biologically diverse semi-arid regions in the world. Two exotic fruits collected in this unique bioma, the prickly pear (Opuntia ficus-indica) and facheiro (Philosocereus pachycladus Ritter), were investigated in regard to their physicochemical, bioactive and functional characteristics. Four different extracts were prepared and investigated: water (W), ethanol/ water 70:100 (E70), ethanol/water 80:100 (E80), and ethanol only (E100). The betalain compounds were investigated using the LC-DAD-ESI-MS technique. The prickly pear fruits are sweeter and less acidic when compared to facheiro fruits, but they also have lower total solids, ash and protein. The total phenolic content (TPC) of water and ethanolic extracts ranged from 8.89 to 12.34 mg GAE/100 g and 82.23 to 107.67 mg GAE/100 g for Opuntia and facheiro fruits, respectively. The identification of betanin and isobetanin derivatives, as well as Research highlights > Betanin and isobetanin derivatives of facheiro fruits were shown for the first time; > Prickly pear and facheiro extracts presented in vitro antienzymatic effects; > The total phenolic content of exotic facheiro fruit was shown for the first time; > Facheiro fruit has high bioactive content and expressive colorant potential. * Roberta T. P. Correia
[email protected] 1
Laboratory of Food Bioactive Compounds, Chemical Engineering Department, Federal University of Rio Grande do Norte, Campus Lagoa Nova, 59075-180 Natal, RN, Brazil
2
Centro de Tecnologia da Informação Renato Archer, Ministério da Ciência, Tecnologia e Inovação, 60511-110 Fortaleza, CE, Brazil
3
EMBRAPA – Postharvest Physiology and Technology Laboratory, Embrapa Tropical Agroindustry, R. Dra. Sara Mesquita, 2270, Pici, 60511-110 Fortaleza, CE, Brazil
the TPC, antioxidant and antienzymatic activities of facheiro fruits were reported for the first time. All extracts presented some degree of alpha-amylase and alpha-glucosidase inhibition, with the exception of the facheiro water extracts. Keywords Caatinga . Cactaceae fruits . Phenolics . Betalains
Introduction The caatinga ecosystem, also referred as the Bdry forest^, stretches for more than 800,000 Km2 and is considered to be one of the 37 Wilderness Areas of the World (Leite and Machado 2010). Many flora and fauna species are found in no other part of the world, except for the caatinga territory (Schober 2002). Regardless of the reported use of some medicinal plants for folk medicine or for the manufacture of phytochemical products (Albuquerque et al. 2007), several edible vegetables and fruits from caatinga remain completely unexploited in regard to their technological, bioactive and functional characteristics, despite their potential economic value. Several Cactaceae grow in this semi-arid region, either native or adapted species. Opuntia fícus indica fruits also known as Indian fig Opuntia and prickly pear, have edible cladodes, but its sweet and colored fruits are the most desirable and commercially valuable parts of this plant and present economic relevance throughout arid and semi-arid areas of the world (Stintzing et al. 2001; Yahia and Mondragon-Jacobo 2011). The fruits of Opuntia species are generally low in acid and sweet (Medina et al. 2007), but compositional and functional differences between fruits collected in different regions are expected due to the variability of ecotypes, plant physiology and growth conditions (Khatabi et al. 2013; Yahia and Mondragon-Jacobo 2011). Due to the attractive color and sweetness, these semi-arid fruits can be consumed fresh (Cefola et al. 2014) or as ingredients for the manufacturing of several other products (Atef et al. 2013;
J Food Sci Technol
Sawaya et al. 1983). On the other hand, the fruits of Pilosocereus pachycladus Ritter, locally known as facheiro, are virtually ignored worldwide. This exotic example of Cactaceae found in the Brazilian caatinga produces purple colored fruits approximately 3 cm in size, with fleshy and succulent pulp and plenty of black obovate seeds (Abud et al. 2010). The scientific information about this species of cactus is extremely scarce, but its use for animal feeding during drought times and local consumption by rural people has been reported (Nascimento et al. 2011). Tropical and exotic fruits contain a wide range of bioactive compounds with health-relevant activities (Dembitsky et al. 2011). With regard to phenolic compounds, besides the widely recognized health effects, they are also able to modulate the breakdown of carbohydrates by inhibiting the enzymes alphaamylase (E.C.3.2.1.1) e alpha-glucosidase (E.C.3.2.1.20) and help in managing early stages of diabetes type 2 (Ankolekar et al. 2011; Correia et al. 2012; Gonçalves et al. 2010). Betalains are also important bioactive substances widely found in Cactaceae species which bring attractive orange to purple colors to cactus fruits (Khatabi et al. 2013; Stintzing et al. 2002). Although the Pilosocereus fruits also have similar intense purplish colors, to the best of our knowledge, no studies focusing on its betalain content or other bioactive compounds were found in the literature. Obtaining scientific data about these little studied species would bring light into promising plants that would contribute to the diversification of local pharmacopeia and appreciation of local resources (Alencar et al. 2010). Several bioactive compounds have their biosynthesis and bioactivity affected by abiotic factors such as UV radiation, water availability, temperature and soil composition. Therefore, harsh environmental conditions such as the ones found in the Brazilian caatinga may induce metabolic changes that would result in higher phytochemical production and accumulation in stressed vegetables (Pavarini et al. 2012). This finding brings a fresh perspective to the research of edible plants grown in challenging climatic environments. Therefore, this paper has the objective of investigating the Opuntia ficus-indica and Pilosocereus pachycladus Ritter fruit pulps in regard to their physicochemical, bioactive and functional characteristics. This study presents new data concerning selected functional substances found in facheiro pulp and explores the bioactive value and technological potential of these exotic caatinga fruits.
Brazil). Several lots were harvested and mixed in order to form a single batch. All fruits were harvested at physiological maturity, which was established according to the color of their surface, using an arbitrary scale: fruits with 50 % or more of their surface with yellow-orange color for prickly pear and orange-red color for facheiro were selected for this study. The fruits were washed, screened for uniformity, the absence of physical defects and manually peeled. The prickly pear fruits were processed into fruit pulp by using a fruit processor (Itametal, Brazil). The pulp of facheiro fruits was obtained manually. All fruit pulps were kept frozen (−10 °C) until use. Extracts preparation The extracts were prepared using 30 g of prickly pear fruit pulp or 5 g of facheiro fruit pulp and 100 ml of solvent (water (W), ethanol/water 70:100 (E70), ethanol/water 80:100 (E80), and ethanol only (E100)). The material (fruit pulp + solvent) was homogenized in a stainless steel blender (Waring, USA) at the highest speed for 1 min. The material was vacuum filtered through a Whatman no. 1 paper and centrifuged (SH 701, Solab, Brazil) at 1000×g for 5 min. Physicochemical characterization Total solids, ash, protein, pH and titratable acidity were determined according to methods 920.151, 940.26, 920.152, 981.12 and 942.15A, respectively (AOAC 1998). Total sugars were analyzed as described by Miller (1959), with modifications. Total polyphenol content
Materials and methods
The analysis was performed according to Correia et al. (2012) with some modifications. Briefly, aliquots of 1 ml of the extract were transferred to assay tubes to which were added in this sequence: 1 ml of 95 % ethanol, 5 ml of distilled water and 0.5 ml of Folin-Ciocalteau 1N reagent. The samples were homogenized and 5 min later, 1 ml of sodium carbonate 5 % (w/v) was added, followed by a new homogenization. The assay tubes were kept in a dark chamber for 1 h, and the samples had the absorbance measured at 725 nm against a blank consisting of a solution of 95 % ethanol. A calibration curve built with different concentrations of gallic acid was used in order to convert the absorbance as milligrams of gallic acid equivalent (GAE) per 100 g of sample (mg GAE/100 g).
Fruit pulp preparation
DPPH radical scavenging activity
The prickly pear and Pilosocereus fruits were collected in January–March 2012 in Northeastern Brazil and were generously donated by Instituto Nacional do Semiárido (INSA,
The antioxidant activity was determined according to Thaipong et al. (2006). Briefly, a stock solution was prepared by dissolving 24 mg DPPH (Sigma-Aldrich, USA) and
J Food Sci Technol
100 ml methanol (VETEC, Brazil) and stored at −20 °C until needed. For the analyses, a fresh solution was prepared using 10 ml stock solution and 45 ml methanol to obtain an absorbance of 1.1 ± 0.1 at 515 nm using a spectrophotometer (Genesys 10S UV–VIS series, Thermo Scientific, USA). Pulp extracts (150 μL) were allowed to react with 2850 μL of the fresh DPPH solution for 24 h in the dark and the absorbance was read at 515 nm. The standard curve was built with different Trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2carboxylic acid, Sigma-Aldrich, USA) concentrations (25 to 800 μM Trolox). Results were expressed in μM Trolox eq. (TE)/g sample. Betalain determination Standards and reagents Formic acid (Sigma Aldrich, USA), HPLC grade acetonitrile and methanol (Tedia, Btazil) were used in this study. HPLC grade water was prepared from distilled water using a Milli-Q system (Millipore Lab, Bedford, MA). Sample preparation The fruits of Opuntia fícus indica and Pilosocereus pachycladus Ritter were milled, centrifuged and filtered using nylon 0.45 μm membranes (Millipore, Trasadingen, Switzerland). LC-DAD-ESI-MS conditions The instrument used was Varian 250 HPLC (Varian Inc., CA, USA) coupled to a 335 diode array detector (DAD) and a mass spectrometer 500-IT MS (Varian, CA, USA). A Symetr C18 column (Waters, USA - 3 μm, 2×250 mm) was used with a 400 μL/min flow at 30 °C. The mobile phase consisted of the combination of two different solutions: A (0.1 % formic acid in water) and B (0.1 % formic acid in acetonitrile). The gradient varied linearly from 10 % B to 26 % (v/v) in 40 min, 26 % B to 100 % (v/v) in 50 min and held up to 100 % B 55 min. The selected wavelengths were 350, 270 and 520 nm, the spectrum of UV / VIS was collected continuously in the range of wavelengths 190–650 nm. The mass spectrum was obtained using electrospray ionization in positive modes (PI) in a fragmentation voltage of 80 V for a mass range from 100 to 2000a. The drying gas pressure was 35 psi, the pressure of the nebulizer 40 psi, the temperature of the drying gas 370 °C, voltage 4000 V for PI, and the shield voltage of 600 V. The LC system was directly coupled to the MSD. The identification of compounds was primarily based on the mass spectra of ions and molecular ions derived from MS-MS data and compared with the literature (Vicent and Scholz 1978). The method described by Shwartz and Elbe (1980) was used for the betalain
quantification and the extinction coefficient of 1120 cm−1 was used for betalains (Vicent and Scholz 1978).
Alpha-amylase (EC 3.2.1.1) inhibition The alpha-amylase inhibitory activity was determined using the chromogenic method described by Gonçalves et al. (2010). Briefly, 40 μl of sample, 160 μl of distilled water and 400 μL of starch solution (0.5 % w/v in 20 mM phosphate buffer pH 6.9 containing 6.7 mM sodium chloride) were mixed in a screw-top plastic tube. The reaction was started by the addition of 200 μL of the enzyme solution of porcine pancreatic alpha-amylase (Sigma-Aldrich, USA) 4 unit/ml. The tubes were incubated at 25 °C for 3 min. After this, 200 μl aliquots were transferred to a separate tube containing 100 μL DNS reagent solution (96 mM DNS, 5.3 M sodium potassium tartrate in 2 M NaOH) and placed into an 85 °C water bath. After 15 min, this mixture was diluted with 900 μL distilled water and the alpha-amylase activity was determined by measuring the absorbance at 540 nm. A control sample consisting of 40 μL of methanol instead of the fruit sample, and a blank consisting of 200 μL of distilled water instead of the enzyme solution was prepared. The standard curve was built with different maltose concentrations (0–0.1 %). The absorbance due to maltose was calculated using the standard curve and the % inhibition was calculated by using the equation: % alpha-amylase inhibition = 100 − (% reaction after 3 min). The % reaction was obtained by % reaction = (% maltose sample/ % maltose control) × 100.
Alpha-glucosidase (EC 3.2.1.20) inhibition The alpha-glucosidase inhibitory activity was determined according to Ankolekar et al. (2011). Alpha-glucosidase was assayed by using 50 μL of fruit extracts and 100 μL of 0.1 M phosphate buffer (pH 6.9) containing an alphaglucosidase solution (1 U/ml) incubated in 96-well plates at 25 °C for 10 min. After preincubation, 50 μL of 5 mM pnitrophenyl-alpha-D-glucopyranoside (Sigma-Aldrich, USA) solution in 0.1 M phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated at 25 °C for 5 min. Before and after the incubation, the sample absorbance was recorded at 405 nm (Sto and St5, respectively) using a microplate reader (BioChrom ASYS UVM340, Cambridge, UK) and were compared to the control absorbance (Cto and Ct5), which consisted of 50 μL of buffer solution in place of the extract. Results were expressed as % inhibition according to the equation: % alphaglucosidase inhibition = [((ACt5 − ACto) − (ASt5 − ASto)) / (ACt5 − ACto)] × 100.
J Food Sci Technol
Statistical analyses In this study, all analyses were performed in triplicate (n=9) and results are presented as mean ± standard deviation. Analysis of variance (one way-ANOVA) and Tukey’s test were performed using the software Statistica® 7.0. The significance level for the difference between means was of 5 % (p
