Bioactive compounds and antioxidant activity evolution during the ripening process of 12 Opuntia spp. fruit accessions

doi: 10.9755/ejfa.2016-09-1324


Bioactive compounds and antioxidant activity evolution during the ripening process of 12 Opuntia spp. fruit accessions INTRODUCTION
The genus Opuntia comprises approximately three hundred species.They belong to the family Cactaceae, which are plants that utilize water efficiently, even five times more than conventional crops, requiring low amounts of water.For these reasons, Opuntia spp.are easily adaptable to arid and semiarid regions, and have been considered by the FAO as a valuable alternative for the agricultural development in dry regions where the rains are scarce (Pimienta, 2001).This plant is cultivated in Mexico, Italy, South Africa and other countries, with different species of Opuntia predominating in each country, and it is well known that the weather, region and cultural practices can affect the content of nutrients and mineral composition, as well as quantities of bioactive compounds in plants.However, there are some differences among the cultivars respect to their fruits, such as size, maturation time and number of seeds.Recently, Opuntia spp.has gained considerable attention due to the bioactive components it contains, and many researchers have contributed to the characterization of Opuntia spp.fruits (prickly pear fruits), and they have extracted natural pigments not only from their pulp but also from their skin.Stintzing (2005), published information about antioxidants and phytochemicals present in fruits of different cultivars of Opuntia ficus-indica (L.) Mill.The Opuntia stricta fruit showed an intense purple color due to its high concentration of betacyanins (80 mg/100 g of fresh weight) (Castellar, 2003).Moreover, juice of Opuntia stricta fruit (fresh, dried or concentrated) can be used as a natural food ingredient, and the pigments, antioxidants and others bioactive compounds it contains, help to improve the overall quality of the final food product in which it is added, increasing the acceptability of the consumer (Castellar, 2003;Castellar, 2006;Obón, 2009).These compounds differ depending upon the species and clones studied, and they have not been completely explored.In comparison with beet root, where betalains can also be found, prickly pear fruits offer a wide variety of colors, which could be used in foods due to its natural character (Stintzing et al., 2002).Some species of the genus Opuntia have shown bioactivity as analgesic, anti-inflammatory, antioxidant, hypoglycemic, and neuroprotective effect, and could be useful in cancer chemoprevention (Tesoriere et al., 2005;Bensadón, 2010).The fresh prickly pear fruits are used to elaborate different products as jams, candies, liqueurs, sauces and juices among others.Nevertheless, some species of the genus Opuntia produce red fruits containing big seeds or a large number of seeds, which are not consumed by people but there is a great interest about they in order to obtain natural pigments for food applications, because the opening of new markets for functional food and beverage industry has become an important area of opportunity (Mobhammer et al., 2005 andStintzing, 2007).In order to know what specie and ripening state produces the highest concentration of pigments and bioactive compounds, the record of changes in their concentration along the maturity time is very important.It has been reported that pigmentation of red prickly pear fruits occurs first in the center, long before fruit ripening and skin pigmentation.Pigmentation is full when the fruit is ripe, and it is correlated with the maximum content of total soluble solids (Felker et al., 2008).Therefore, an adequate ripening state at the moment of the harvest is very important for the fruit quality.For these reasons, the aim of this work was to know changes in pigments content and antioxidant compounds occurring along the ripening of twelve accessions of red prickly pear fruits.

Chemicals
Liquid chromatography grade solvents: Acetone, methanol, and acetonitrile were purchased from EMD Millipore Chemicals (Billerica, MA, USA), and formic acid, glacial acetic acid, and phosphoric acid were obtained from Anachemia (Canada).Ultrapure water was obtained from a Millipore Milli-Q water purification system (Billerica, MA, USA).

Fruits harvesting and samples preparation
The twelve prickly pear accessions ꞌCacaloteꞌ (CL), ꞌBondaꞌ (BO), ꞌRojo Tolucaꞌ (RT), ꞌTapón Aguanosoꞌ (TA), ꞌGrifaꞌ (GR), ꞌRojo Insurgentesꞌ (RI), ꞌOrejónꞌ (OR), ꞌSangre de Toroꞌ (ST), ꞌCardonaꞌ (CA), ꞌRojo Pelónꞌ (RP), ꞌSandíaꞌ (SA) and ꞌBurra la Cruzꞌ (BC) were harvested during eight dates after anthesis until their complete ripe.Fifty fruits were randomly collected from five plants (ten of each).Samples were codified as A D , where A means the prickly pear accession, and the subscript D means the days after anthesis, resulting 96 samples.Fruits were ultrafreezed at -70 °C (Ultrafreezer THERMO SCIENTIFIC 303, USA) and lyophilized at 133x10 -3 mBar for quantification of total phenolics, total flavonoids, antioxidant activity, and betalains.Lyophilized samples were ground in a cutter mill (RTSCH GM 200, Germany) at 9000 rpm per 50 s until a fine powder of 150 mm was obtained.

Maturity index
The fruits were washed carefully with distilled water in order to eliminate the glochids.The images of each longitudinally cut fruit were recorded in photographs in order to make a comparison between them.These observations together with the external color of the fruits allowed the establishment of a visual scale with the following five values: 1) very immature; 2) immature; 3) moderately mature and 4) mature (Fig. 1).

Total phenolics content
The total phenolics content (TPC) was determined according to the Folin-Ciocalteu method (Waterman and Mole, 1994) with slight modifications.A lyophilized sample of 0.5 g was mixed with 20 mL of 80% methanol and vortexed per 10 min.After which, it was centrifuged at 17500xg per 10 min at 5 °C (centrifuge Thermo Scientific Mod.ST 16R, Germany). 1 mL of the supernatant was mixed with 5 mL of the Folin-Ciocalteu reagent diluted 1:10 (v: v) with distilled water and it was left stand per 7 min.4 mL of a solution of sodium carbonate (7.5 %) was added and left to react in the darkness per 2 h.The absorbance was measured at 760 nm using a spectrophotometer (Jenway 6715 UV/VIS, Jenway Techne Inc. USA).A calibration curve was prepared using gallic acid as standard.Results were expressed as mg Gallic Acid Equivalents (GAE)/g Fresh Weight (FW).Moisture of fresh fruits was used to convert from dry weight to fresh weight.All the experiments were done by triplicate.

Total flavonoids content
The total flavonoids content was determined according to the method described by Dowd and adapted by Arvouet-Grand et al., (1994).Briefly, 0.1 g of lyophilized sample was mixed with 10 mL of 80% methanol; this mixture was vortexed 10 min and filtered through filter paper Whatman grade 1.After which, 2 mL of filtrate was mixed with 2 mL of a methanolic solution of AlCl 3 (2% in methanol).This mixture was left stand for 20 min in the darkness.The absorbance was measured at 415 nm using a spectrophotometer (Jenway 6715 UV/VIS, Jenway Techne Inc. USA).A calibration curve was prepared (0-40 mg/L) using quercetin as standard.Results were expressed as mg Equivalents of Quercetin (EQ)/100 g of Fresh Weight (FW).Moisture of fresh fruits was used to convert from dry weight to fresh weight.
Preparation of samples for radical scavenging capacity assays 0.5 g of lyophilized fruit was mixed with 20 mL of 80% methanol.This mixture was vortexed per 20 min and centrifuged at 17500 xg per 10 min at 5°C in a centrifuge (Thermo Scientific Mod.ST 16R, Germany).The supernatant was used for the determination of radical scavenging assays.

DPPH radical scavenging capacity
The radical scavenging capacity was performed by the DPPH method described by Brand-Williams et al., (1995).Briefly, 0.5 mL of sample was mixed with a methanolic solution of DPPH (6X10 -5 M), which was previously left to stand per 2 h under constant agitation in the darkness.The mixture was vortexed 15 s and left to stand in the darkness per 1 h at 4°C.Absorbance was measured in a spectrophotometer (Jenway 6715 UV/VIS, Jenway Techne Inc. USA) at 515 nm.Trolox was used as standard, and results were expressed as mmol Trolox equivalents (TE)/g fresh weight (FW).Moisture of fresh fruits was used to convert from dry weight to fresh weight.

ABTS radical scavenging capacity
The radical scavenging capacity was determined by the ABTS assay described by Re, et al., (1999) with slight modifications.An aqueous solution of ABTS (7 mM) was mixed with an aqueous solution of potassium persulfate (2.45 mM) at 1:1 (v: v) ratio.This mixture was left in the darkness under constant agitation during 16 h in order to produce the ABTS radical (ABTS + ).The solution containing ABTS + was diluted with acetate buffer until reach an absorbance value of 0.700 ± 0.02 at 754 nm. 100 mL of the extract was added to 3900 mL of the ABTS + solution.The absorbance was measured at 754 nm after 6 min storing the mixture in the darkness.Trolox was used as standard preparing a standard curve (0-15 mM Trolox).Results were expressed as Trolox equivalent antioxidant capacity (TEAC) in µM Trolox equivalent (TE)/100 g fresh weight (FW).Moisture of fresh fruits was used to convert from dry weight to fresh weight.

Analysis of indicaxanthin, betanin and isobetanin Preparation of samples
Lyophilized samples (500mg) were extracted with 80% aqueous methanol (20 mL) in an ultrasonic bath for 20 min at 37°C.Then, samples were vortexed 30 seconds and centrifuged (AllegraTM 25R centrifuge) at 4000 xg for 4 min at room temperature.The supernatant was placed into a 50 mL tube.The residue was re-extracted using the same procedure.The supernatants were combined and completed at 50mL using water.The mixture was subsequently filtered through a 0.2 µm nylon filter and filtrate was assayed for total betalains and specific indicaxanthin, betanin and isobetanin analysis (Kujala et al., 2001).

Quantification of total betalains
Betalains content was calculated according to Tsai et al. (2011) with slight modifications (Equation 3).All determinations were performed on a UV/Vis spectrometer (Helios, Thermo).The UV/vis absorption spectra were recorded at 536 nm.
Where Abs is the absorbance value at a wavelength of 536 nm for betacyanins, DF is the dilution factor, V is the volume of solution (mL), W is the dry weight (g), and L is the path-length (1 cm) of the cuvette.The molecular weight (MW= 550 g/mol) and the molar extinction coefficient (ε= 61,600 L/mol cm) were applied in order to quantify betalains.

Statistical analysis
A completely randomized design was used.Means and standard deviations were calculated.The statistical software SAS (software JMP 5.0, V. 9.0) (2002) was used for the analysis of variance (ANOVA).The Tukey test was used for the comparison of means (P≤ 0.05) with a 95% of confidence.Correlation analysis was carried out using Pearson's correlation coefficient.All the experiments except maturity, size and color were done by triplicate.

Changes in pigments concentration during the ripening
Pigments (isobetanins, betanins, and indicaxanthins) were detected in immature fruits BO 63 , RT 63 , CL 77 , RI 77 , OR 77 and CA 77 (Table 1).Nevertheless, in the other accessions, the pigments appeared until the fruits were under-ripe, as in TA 100 , GR 100 , RP 100 , SA 100 and BC 100 or even until they were moderately ripe as ST 116 .Betacyanin was the main pigment found in these fruits, meanwhile lower levels of isobetanins, betanins and indicaxanthins were found.The highest concentration of isobetanins was found in ripe fruits, being of 3.58 ± 0.15 mg/100 g dry basis (DB) and 4.58 ± 0.38 mg/100 g DB for BO 143 and RT 143 respectively.The accessions BO 143 and RT 143 showed also the highest concentrations of betanins (45.71 and 38.09 ±1.81 mg/100 g DB respectively) and indicaxanthins (29.67 ± 0.45 and 27.20 ± 0.10 mg/100 g DB respectively).Therefore, the highest concentration of total betalains was found in BO 143 and RT 143 , being of 78.96 ± 0.54 and 69.87 ± 0.29 mg 100 -1 g DB respectively.Several studies highlight the antioxidant properties of betalains and the importance of Opuntia spp.fruits containing high levels of these bioactive compounds (Fernández-López et al., 2010;Sumaya-Martínez et al., 2011).Moreover, significantly positive correlations were found between the DPPH or ABTS radical scavenging activity and pigments concentration for these two accessions (Tables 4 and 5).These results indicate that the free radical scavenging activity was increased directly proportional to the increment of pigments concentration in all accessions.So it can be inferred that these pigments play an important role on the antioxidant activity showed by BO and RT accessions, nevertheless the Opuntia spp.fruits contain other compounds with antioxidant activity such as phenolics.These pigments are appreciated as nutraceutical ingredients since they promote health and prevent illnesses due to their effect against oxidative stress (Livrea and Tesoriere, 2006).Moreover, the highest content of total betalains can be an indicator of ripeness in prickly pear fruits.

Changes in total phenolics content during the ripening
TPC increased over the ripening process, ranging between 24.93 ± 0.26 mg GAE/100 g FW for RT 15 , to 257.77 ± 0.17 mg GAE/100 g FW for GR 143 (Table 2).Nevertheless, the most significantly increment in TPC was found between 77 and 100 DAA, when the fruit was from immature to under-ripe for almost all the accessions except for CL, RT, and TA, whose maximum change in TPC was found between 116 and 143 DAA, when the fruit changed from moderately ripe to ripe, and for GR and ST, whose maximum changes in TPC were between 49 and 63 DAA for the former and between 63 and 77 DAA for the latter, when both accessions were immature.The TPC for ripe fruits varied widely among the accessions, showing as low contents as 65.04 ± 0.17 mg GAE/100 g FW in BO 143 .Nevertheless, they were found also high levels of TPC in six out of the twelve accessions studied (GR Rosa) and nectarine (Prunus persica var.Nectarin) (Gil, et al., 2002;Cantín, et al., 2009;Belhadj, et al., 2016).These high concentrations of phenolics were significantly correlated (positively) with DPPH and/or ABTS radical scavenging activity, except for OR accession, which was not significant neither for DPPH nor for ABTS radical scavenging activity.This result can be due to the antioxidant activity in OR may be influenced mainly by other compounds such as flavonoids or betalains.Variations in TPC could be due to chemical changes of phenolic compounds as the maturation process occurs, and due to genetic variability among the different accessions, since the cultivation conditions were controlled and the climate conditions were the same for all the accessions.

Changes in total flavonoids content during the ripening
Significantly statistical differences were found in TFC during the ripening process of fruits, ranging from 0.56 ± 0.02 mg EQ/100g FW for SA 15 to 19.45 ± 0.07 mg EQ/100g FW for OR 143 (Table 3).Six out of the twelve accessions studied (GR, RI, OR, CA, RP, and SA) exhibited the biggest change in TFC between 100 and 116 DAA, when the fruits changed from under-ripe to moderately ripe.The accessions CA, ST, and BC showed the major change in TFC between 36 and 49 DAA, when the fruit changed from very immature to immature.The accessions RT and TA showed the most marked change between 116 and 143 DAA, when the fruits changed from moderately ripe to ripe.The concentration of flavonoids was significantly correlated (positively) with DPPH and/ or ABTS radical scavenging activity for almost all the accessions, except for BO, which showed a significant correlation between pigments or TPC and DPPH or ABTS radical scavenging activity (as discussed above).The BO accession displayed a marked change in TFC between 77 and 100 DAA, when the fruits change from immature to under-ripe.Times at which the major changes occurred in TPC did not coincide with those occurred in TFC, this behavior can be displayed because flavonoids are not the unique type of phenolics present in prickly pear fruits.Opuntia spp.fruits contain flavonoids commonly found in other fruits and vegetables, the type of flavonoid and its content depends on the species and accession (Miean and Mohamed, 2001).

Radical scavenging activity
Independently of the assay (DPPH or ABTS), it was found an increment of the antioxidant activity along the ripening process.This increment was due to the presence of different kind of chemical compounds with antioxidant activity in prickly pear fruits.The study of correlation between the bioactive compounds presenting antioxidant activity and the DPPH or ABTS assay reveals that the free radical scavenging activity was dependent on the accession studied, since each one synthetizes different compounds at different stages of the ripening process, leading a higher antioxidant activity as the maturity occurs.
Results about DPPH assay showed an increment of the radical scavenging activity over the ripening process in the twelve accessions of prickly pear fruits studied (Table 4), for example, the radical scavenging activity for RT ranged from1.25 ± 0.02 µmol TE/g FW at 15 DAA to 6.30 ± 0.02 µmol TE/g FW at 143 DAA.In fact, all Results about ABTS assay (Table 5) were expressed in the same units used for DPPH in order to make a better comparison between them.It was found the same trend in ABTS than DPPH assay over the ripening process.Nevertheless, the ABTS assay displayed higher values of radical scavenging activity when compared with DPPH.
For the same example given above, RT 15 displayed 2.69 ± 0.03 µmol TE/g FW increasing until 9.19 ± 0.02 µmol TE/g FW for RT 143 .For ABTS assay, as in DPPH, all the accessions reached a similar value of free radical scavenging capacity at 143 DAA, ranging from 8.06 ± 0.02 µmol TE/g FW for TA 143 to that shown in RT 143 .These greater values found when the ABTS radical was used can be explained by the higher selectivity of the DPPH radical.The ABTS radical reacts with any hydroxylated aromatic molecule, independently of its real antioxidant potential (Kopjar et al., 2015).

CONCLUSIONS
The antioxidant activity of prickly pear fruits increases along the ripening process, this increment is positively correlated with the increase in concentration of the bioactive compounds present in fruits such as phenolics, flavonoids and pigments.The pigments appeared in different stages of the ripening process, depending on the accession studied, nevertheless, once the fruit is ripe, their concentration remains constant.This fact makes possible to propose the measurement of betalains concentration as a parameter of ripeness, additionally to traditional parameters as total soluble solids concentration, titratable acidity and

Table 3 : Evolution of total flavonoids content of twelve red prickly pear fruit accessions from very immature to ripe
Means in each column with different letters are statistically different (Tukey, P≤0.05).*: Significant at P≤0.05.Values are means±standard deviations

Table 4 : Evolution of DPPH radical scavenging capacity of twelve red prickly pear fruits from very immature to ripe
Means in each column with different letters are statistically different (Tukey, P≤0.05).Values are means±standard deviations

Table 5 : Evolution of ABTS radical scavenging capacity of twelve red prickly pear fruit accessions from very immature to ripe
color.It is hoped that this work contributes to select the accessions with the better potential to be established in arid zones of Mexico in order to take in advantage these fruits as an important source of pigments.Moreover, this work can contribute to the knowledge of the stage of the ripening process at which would be possible to obtain a major concentration of bioactive compounds taking as reference the anthesis.Means in each column with different letters are statistically different (Tukey, P≤0.05).Values are means±standard deviations (Contd...)