Food Research International 44 (2011) 2210–2216

Contents lists available at ScienceDirect

Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Phytochemical and antioxidant characterization of the fruit of black sapote (Diospyros digyna Jacq.) Elhadi M. Yahia ⁎, Fabiola Gutierrez-Orozco, Claudia Arvizu-de Leon Phytochemicals and Nutrition Laboratory, Faculty of Natural Sciences, Autonomous University of Queretaro, Ave de las Ciencias, Juriquilla, 76230, Queretaro, Qro., Mexico

a r t i c l e

i n f o

Article history: Received 9 September 2010 Accepted 11 November 2010 Keywords: Diospyros digyna Black sapote Antioxidant capacity Phytochemicals HPLC Mass spectrometry

a b s t r a c t Consumption of fruits and vegetables has been associated with a healthy state which has been attributed, in part, to their antioxidant capacity. Characterization of the bioactive compounds with antioxidant activity found in fruits cultivated in the tropic is limited. Thus, the objective of the present work was the characterization of phytochemicals and antioxidants of the fruit of black sapote (Diospyros digyna Jacq.). HPLC-DAD-Mass Spectrometry (HPLC-MS) analyses were used to identify and quantify phenolics, carotenoids and tocopherols. Total soluble phenolic content was 247.8 mg GAE/100 g fw (fresh weight). Important phenolics identified were sinapic acid, myricetin, ferulic acid, and catechin. Total carotenoid content was 399.4 μg of β-carotene/100 g fw, and β-carotene and lutein were the main carotenoids identified. δTocopherol concentration was 672.0 μg/100 g dry weight. Antioxidant capacity as measured by the DPPH and FRAP assays was higher in the hydrophilic than in the lipophilic extract, and it is thought to be due mainly to the phenolic content of this fruit. Results suggest that the fruit of black sapote has an antioxidant capacity comparable to other important fruits, and its inclusion in the diet is therefore recommended. © 2010 Published by Elsevier Ltd.

1. Introduction There has been an increase in the development of chronic diseases such as cancer, diabetes, cardiovascular disease, and hypertension in the last few years, which can be explained, at least in part, by factors such as diet and lifestyle (Yahia, 2010). Epidemiological evidence suggests an inverse relationship between a high intake of fruits and vegetables and incidence of these diseases (Lampila, van Lieshout, Gremmen, & Lähteenmäki, 2010; Southon, 2000; WCRF-AICR, 2007; Yahia, 2010). Because of that, the interest for identifying and characterizing phytochemicals in fruits and vegetables that may be related to these effects is of great interest (Barros, Carvalho, & Ferreira, in press). Phytochemicals are a group of compounds present in fruits and vegetables that exert a potential protective role against development of diseases associated with aging like cardiovascular disease, inflammation, and cancer (Temple, 2000; Willett, 1994, 1995). Oxidative stress has been implicated in the development of these disorders (Diaz, Frei, Vita, & Keaney, 1997; Harman, 1995; Smith et al., 1996). Oxidative stress is induced by harmful free radicals damaging lipids, DNA, and proteins (Halliwell & Gutteridge, 1989).

⁎ Corresponding author. Phytochemistry and Nutrition Laboratory, Faculty of Natural Sciences, Autonomous University of Queretaro, Avenida de las Ciencias, Juriquilla, 76230, Queretaro, Qro., Mexico. Tel.: + 52 442 192 1200x5304; fax: + 52 442 234 2958. E-mail address: [email protected] (E.M. Yahia). 0963-9969/$ – see front matter © 2010 Published by Elsevier Ltd. doi:10.1016/j.foodres.2010.11.025

Among phytochemicals, phenolics are secondary plant metabolites which have been shown to be responsible, at least in part, for the observed protective effects of fruit and vegetable consumption (Hertog, 1996). Their antioxidant activity is mediated by inactivation of damaging free radicals (Haslam, 1998). Another group of phytochemicals, carotenoids, have also been shown to have antioxidant activity, besides the well known provitamin A activity of some of them (Yahia & Ornelas-Paz, 2010). Among carotenoids, lycopene has been the object of extensive research which suggests its antioxidant role and antiproliferative activity in cancer cells (Heber, 2004; Obermuller-Jevic et al., 2003). In addition, vitamins C and E have largely been known for their role as antioxidants in the body (Burton & Ingold, 1981; Huang, Ou, & Prior, 2005). Therefore, characterization of phytochemicals in fruits and vegetables is of great importance. Tropical fruits, especially minor fruits such as black sapote, have been little characterized for their phytochemical content. Corral-Aguayo, Yahia, Carrillo-Lopez, and Gonzalez-Aguilar (2008) reported that vitamin E in black sapote fruit was the highest among 8 fruits, and considered black sapote and avocado as relatively good sources for this vitamin. Black sapote (Diospyros digyna Jacq.) belongs to the Ebenaceae family and is native to the south of Mexico and Central America. It is widely consumed in regional markets but considered as an exotic fruit in international markets and thus, potential for commercialization exists. The objective of this study was the identification and quantification of some phytochemicals such as carotenoids, phenolics, and vitamin E in fruits of black sapote and the characterization of their antioxidant activity.

E.M. Yahia et al. / Food Research International 44 (2011) 2210–2216

2. Materials and methods 2.1. Reagents Reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise stated and standard purity was ≥97% for β-carotene, ≥79% for lutein, ≥90% lycopene, ≥95 and ≥90% for α- and δtocopherol, respectively. Phenolic acids standards were HPLC (highperformance liquid chromatography) grade. HPLC-grade methanol, acetone, n-hexane, toluene, methyl tert-butyl ether (MTBE), methylene chloride, acetonitrile, and ethanol were purchased from J.T.Baker (Baker Mallinckrodt, Mexico). All other solvents were ACS grade. HPLC-grade water was prepared by a Milli-Qplus purification system (Millipore Corp., Bedford, MA).

2211

plates were incubated for 30 min in the dark, and read in a MRX microplate reader (Dynex Technology, Chantilly, VA) set at 490 nm. A calibration curve was prepared using ascorbic acid as a standard and results (AOC) were expressed as ascorbic acid equivalents (AAE) in mg/100 g fw (Rice-Evans & Miller, 1994). For FRAP (ferric ion reducing antioxidant power) assay (Benzie & Strain, 1996), aliquots of 280 μL of FRAP reagent were placed in 96well plates, and 20 μL of extracts, diluted to different concentrations, were added. The plates were incubated for 30 min protected from light and read at 630 nm in a MRX microplate reader (Dynex Technology, Chantilly, VA). Calibration curves were prepared using ascorbic acid as a standard, and results were expressed as AAE in mg/ 100 g fw. FRAP reagent was prepared by mixing 50 mL of 300 mM acetate buffer (pH 3.6), 5 mL of 10 mM 2,4,6-tripyridyl-2-triazine (TPTZ) in 40 mM of HCl, and 5 mL of 20 mM FeCl3.

2.2. Samples 2.4. Analysis of antioxidant compounds About 5 kg of fruits of black sapote were obtained from the local markets in Queretaro, Mexico and selected on the basis of ripeness, freedom of defects, and uniformity of size and color, and were taken to the Laboratory of Phytochemicals and Nutrition of the Faculty of Natural Sciences of the Autonomous University of Queretaro. Fruit were used at its overripe stage, the maturity preferred by consumers for their fresh consumption. Fruit were washed and physically and chemically characterized by measuring weight, internal and external color, and total soluble solids content (TSS, °Brix), and representative samples were freeze-dried until reaching a constant weight, for moisture content analysis. °Brix was measured in the juice obtained from a representative portion of each fruit, using a temperature adjusted hand refractometer (ATAGO, Co. Ltd., Osaka, Japan). Color was measured with a Minolta spectrophotometer (Minolta, Co. Ltd., Osaka, Japan), which was calibrated with the white pattern during each sampling time. External color was longitudinally determined on three points of each flat side of the fruit (six points for each fruit). For flesh (internal) color, a big slice from a flat side of each fruit was obtained and color was determined longitudinally on three equidistant points. L*, a*, b*, C*, and h° values were recorded. Samples were then frozen with liquid nitrogen and kept at −80 °C until lyophilization before analysis. 2.3. Extracts preparation and antioxidant capacity (AOC) analysis Lipophilic (LPE) and hydrophilic extracts (HPE) were obtained as reported by Wu et al. (2004) with some modifications. Samples of 1 g of freeze-dried tissue were homogenized in 10 mL of hexane/ dichloromethane (1:1, v/v) using an Ultra Turrax model T25 Basic homogenizer (IKA Works, Willmington, NC). The homogenate was sonicated for 5 min in a Bransonic 2510 (Bransonic Ultrasonic Co., Danbury, CT) and then centrifuged at 15,000 g for 10 min at 4 °C. The supernatant was collected, and the sediment was subjected to an additional extraction using the same procedure. Both supernatants were mixed and rotoevaporated at 40 °C. The residue was reconstituted in 10 mL of HPLC acetone. This constituted the LPE. Aliquots were filtered through a 0.45 μm nylon membrane for analysis. The pellet, after the second extraction process, was homogenized in 20 mL of acetone/water/acetic acid (70:29.5:0.5, v/v/v), sonicated, and centrifuged using the same conditions mentioned previously. The supernatant was collected, and the sediment was subjected to extraction again. Both supernatants were mixed which constituted the hydrophilic extract (HPE). LPE and HPE were used for AOC analysis. DPPH (2,2′-diphenyl-1-picrylhydrazyl) assay was performed as previously reported (Kim, Lee, Lee, & Lee, 2002), with some modifications, using a microplate reader. Aliquots of 280 μL of 100 μM DPPH/methanol solution per well were placed in a 96-well plate, and 20 μL of extracts, diluted to different concentrations, were added to each well. Aliquots of 300 μL of methanol were used as a blank. The

Vitamin E was determined as described previously (Ornelas-Paz, Yahia, & Gardea-Bejar, 2007). Samples of 0.5 g of lyophilized powder were homogenized in 10 mL of HPLC methanol, stirred at 55 rpm in a water bath at 30 °C for 15 min, and centrifuged at 5000 g for 5 min. The supernatant was filtered through a 0.45 μm nylon membrane, and aliquots of 20 μL were injected into the HPLC (HP 1100 Series, Hewlett-Packard/Agilent Technologies Co., Palo Alto, CA). A 150 × 4.6 mm i.d., 3.5 μm, Symmetry C18 column (Waters Co., Milford, CT) was used. HPLC methanol (100%) was employed as the mobile phase at a flow rate of 0.8 mL/min. For α and δ-tocopherols detection, a model FLD G1321A fluorescence detector (Agilent Technologies Co., Palo Alto, CA) at an excitation wavelength of 294 nm and emission wavelength of 325 nm was used. Calibration curves for quantification were prepared using standards of α- and δ-tocopherols, respectively. The concentration range and correlation coefficients (r2) for the calibration curves were 0–0.1 mg/mL and 0.9989 and 0–0.1 mg/mL and 0.9987 for α- and δ-tocopherols, respectively. Total soluble phenols (TSP) were extracted as described previously by Wolfe, Xianzhong, and Liu (2003), with some modifications. This method measures a sample's reducing capacity and can be considered as another (electron transfer) antioxidant capacity assay (Huang et al., 2005; Prior, Wu, & Schaichet, 2005). Briefly, 1 g of lyophilized sample was homogenized in 20 mL of 80% acetone using an Ultra Turrax model T25 basic homogenizer (IKA Works, Willmington, NC) at ambient temperature. The homogenate was sonicated for 5 min in a Bransonic 2510 (Bransonic Ultrasonic Co., Danbury, CT) and centrifuged at 19,000 g for 15 min at 2 °C in a Hermle Z323K centrifuge (Labortechnik, Wehingen, Germany). The supernatant was collected, and an additional extraction was done in the sediment following the same procedure. Both supernatants were mixed and evaporated at 40 °C using a rotary evaporator (Buchi R-205, Labortechnik, Switzerland). The residue was reconstituted in 25 mL of methanol and taken to 50 mL with HPLC water. An aliquot was filtered through a 0.45 μm membrane for analysis. Extractions were performed in triplicate. For TSP quantification, 30 μL-aliquots were diluted with HPLC water (1:10) and was placed in 96-well plates. Then, 150 μL of Folin–Ciocalteu reagent (dilution 1:10) and 120 μL of 7.5% Na2CO3 were added. The plates were incubated for 2 h protected from light, and absorbance was measured at 630 nm using a Dynex MRX microplate reader (Dynex Technol. Chantilly, VA). Results were expressed as milligrams of gallic acid equivalents (GAE)/100 g fw. Identification and quantification of individual phenolics were carried out using HPLC. Phenolic extracts were prepared as mentioned previously and aliquots of 20 μL were injected in the HPLC system (HP 1100 Series, Hewlett-Packard/Agilent Technologies Co., Palo Alto, CA). equipped with a diode array detector (DAD) set at 280 and 320 nm. A 250 × 4.6 mm i.d., 5 μm, X-terra RP18 column (Waters Co., Milford, CT) was used. Mobile phase consisted of 1% formic acid (98%) (A) and

2212

E.M. Yahia et al. / Food Research International 44 (2011) 2210–2216

acetonitrile (2%) (B), at a flow rate of 0.5 mL/min. Elution gradient was 2 to 100% (B) from 0 to 70 min. The following individual phenolic standards were purchased from Sigma Aldrich: cinnamic, gallic, protocatechuic, catechin, chlorogenic, sinapic, quercetin, p-hydroxybenzoic, p-coumaric, caffeic, kaempferol, ferulic, myricetin, vanillic, epicatechin, isoramnethin, and o-coumaric. Calibration curves for each standard were prepared for quantification. Characterization of the phenolic extracts was carried out by coupling the HPLC system to an HP 6210 time of flight mass spectrometer (MS-TOF, Agilent, Palo Alto, CA), equipped with an electrospray ionization interface (ESI) operating at the negative ionization mode with the following settings: drying gas temperature (nitrogen): 350 °C, drying gas flow rate: 9 L/min, nebulizer pressure: 40 psig, fragmentor voltage: 220 V, skimmer: 60 V, capillary voltage: 3500 V, scan range of m/z: 50–1000. Carotene and TC extractions were performed according to the 970.64-AOAC method (AOAC, 2000), with some modifications (SotoZamora, Yahia, Brecht, & Gardea, 2005). Briefly, 0.5 g of lyophilized sample was mixed with 10 mL of hexane/acetone/toluene/ethanol (10:7:7:6, v/v/v/v) solution and 1 mL of 40% KOH in methanol, stirred at 56 °C for 20 min, and cooled with tap water and 10 mL of hexane were added. After that, 10 mL of 10% Na2SO4 were added, stirred, and the mixture was incubated with protection from light until phase separation. The top phase was taken for analysis. For TC quantification, a Beckman DU-65 spectrophotometer set at 450 nm was used. A calibration curve was prepared using β-carotene in hexane as the standard and hexane as the blank. For β-carotene and lutein quantification, aliquots were filtered through a 0.45 μm nylon membrane (Pall Co., Ann Arbor, MI) and injected (20 μL) into the HPLC, equipped with a DAD set at 470 nm. A 150 × 4.6 mm i.d., YMC C30 column (YMC Inc., Wilmington, NC) was used. The mobile phase consisted of methanol (100%) and methyl tert-butyl ether (MTBE, 0%). The elution gradient was 0–100% MTBE in 35 min at a flow rate of 1 mL/min. Analysis were monitored using HP ChemStation software version A.06.03 (Hewlett-Packard/Agilent Technologies Co., Palo Alto, CA). A calibration curve was prepared with β-carotene and lutein, and measurements were performed in triplicate. For characterization of the carotenoid extracts by MS, an atmospheric pressure chemical ionization (ApCI+) source operating at the positive ionization mode was used. Ionization parameters were as follows: drying gas temperature (nitrogen): 350 °C, drying gas flow rate: 5 L/min, corona voltage: 4.0 μA, nebulizer pressure: 20 psig, fragmentor voltage: 200 V, skimmer: 60 V, capillary voltage: 4000 V, scan range of m/z: 100–1000. 2.5. Data analysis

Table 1 Total soluble solids (°Brix), weight, firmness, and internal color of fruits of black sapote.a Total soluble solids (°Brix) Weight (g) Color L a b Hue (h) Croma (C) a

17.87 ± 0.98 179.97 ± 17.34 22.59 ± 4.82 16.50 ± 3.59 2.99 ± 1.91 10.25 ± 4.75 16.83 ± 3.77

Data are means of three repetitions ± standard deviation (SD).

cladodes, and strawberry (Corral-Aguayo et al., 2008). In this study, differences were observed between the AOC values of the HPE obtained by FRAP and DPPH. DPPH is a widely accepted method because it can give stable and reproducible results, in addition to its simplicity (Katsube et al., 2004). In addition, since fruits in general are characterized by higher hydrophilic than lipophilic AOC activity, FRAP and DPPH have been used, instead of the well-known ORAC assay commonly used to measure lipophilic AOC activity (Corral-Aguayo et al., 2008; Huang et al., 2005; Schlesier, Harwat, Böhm, & Bitsch, 2002). The low AOC found in the lipophilic extract when measured by DPPH and the fact that no AOC was observed in the LPE when FRAP was used may be explained by the limitations that DPPH and FRAP present when used to analyze LPE, since these methods may not be able to detect compounds with antioxidant activity such as carotenoids (Benzie & Strain, 1999). It might be necessary then to use another assay for measuring AOC in LPE of black sapote. Furthermore, the great variety of assays, with different substrates and reaction kinetics, used to evaluate the antioxidant capacity of fruits and vegetables (Benzie & Strain, 1999; Cao & Prior, 1999; Evelson, Travacio, & Repetto, 2001; Gil, Tomas-Barberan, Hess-Pierce, Holeroft, & Kader, 2000; Van den Berg, Haenen, Van den Berg, & Bast, 1999), makes comparison and interpretation of results difficult among different reports. No α-tocopherol was detected in black sapote, while the content of δ-tocopherol was higher than what has been found for some other fruits such as mango, strawberries, papaya and prickly pear fruit, but lower than avocado and guava (Corral-Aguayo et al., 2008) (Table 2). The recommended dietary intake for vitamin E for the Mexican population is 10 mg equiv α-tocopherol (Instituto Nacional de la Nutricion, 2001) which means that 100 g of black sapote would contribute with about 5% of this value per day. The importance of vitamin E as antioxidant relies on their synergistic activity with other compounds such as lycopene, β-carotene and vitamin C (Liu, Shi, Ibarra, Kakuda, & Xue, 2008).

Results are presented as means of three replications and the standard deviation from the mean. All data analyses were performed using Sigma Plot 10.0 (Systat Software Inc, San Jose, CA). 3. Results and discussion Physical characteristics of the fruit of black sapote used in this study such as color, weight, and total soluble solids (°Brix) are presented in Table 1. The importance of using more than one assay to evaluate AOC has been shown before (Ikram et al., 2009). Thus, we determined AOC in black sapote using DPPH and FRAP. AOC of the HPE was higher than that of the LPE using the two assays (HPE: 302.7 ± 15.9, LPE: 2.2 ± 0.7 by DPPH; and HPE: 501.5 ± 26.4 mg AAE/100 g fw, LPE: not detected by FRAP) (Fig. 1). Higher values of AOC in the HPE have been reported by others, as measured by different assays such as DPPH, FRAP, trolox equivalent antioxidant capacity (TEAC), oxygen radical absorbance capacity (ORAC), total oxidant scavenging capacity (TOSC), and N,N-dimethyl-p-phenylendiamine (DMPD) for fruits like guava, avocado, black sapote, mango, papaya, prickly pear fruit,

Fig. 1. Antioxidant capacity of hydrophilic and lipophilic extracts of black sapote measured by DPPH and FRAP assays.

E.M. Yahia et al. / Food Research International 44 (2011) 2210–2216 Table 2 Content of antioxidant compounds in black sapote.a

2213

Table 3 Content of phenolic compounds identified in black sapote by HPLC analyses.a

Compound

Content

Peak

Compound

Content (mg/100 g dw)

TSP (mg GAE/100 g fw) TC (μg β-carotene/100 g fw) δ-Tocopherol (mg/100 g dw)

247.816 ± 16.500 399.409 ± 7.192 0.716 ± 0.063

1 2 3 4 5 6 7 8 9 10

Cinnamic acid Catechin Epicatechin p-hydroxybenzoic acid Caffeic acid Sinapic acid Ferulic acid o-Coumaric acid Myricetin Protocatechuic acid

9.84 ± 3.33 79.941 ± 9.89 18.53 ± 1.11 24.10 ± 5.77 25.23 ± 3.27 110.71 ± 26.90 82.00 ± 6.59 8.64 ± 1.03 85.01 ± 8.78 4.75 ± 1.10

TSP: Total soluble phenols. TC: Total carotenoids. a Data are means of three repetitions ± standard deviation (SD).

TSP content (247.8 mg GAE/100 g fw) was lower than what has been reported for other tropical fruits such as guava, carambola, mamey sapote, sapodilla and mango (Mahattanatawee et al., 2006) (Table 2). However, Corral-Aguayo, Yahia, Carrillo-Lopez, and Gonzalez-Aguilar (2008) reported a higher TSP value in black sapote as compared to papaya, mango, and avocado. HPLC chromatogram of black sapote phenolics extract is shown in Fig. 2. For peak identification and corresponding content please refer to Table 3. Ten phenolic compounds were identified by comparison with retention times of the standards. From these, five were hydroxycinnamic acids (sinapic, ferulic, caffeic, cinnamic and ocoumaric); two were flavan-3-ols (catechin and epicatechin); two were hydroxybenzoic acids (p-hydroxybenzoic and protocatequic); and one flavonol (myricetin). The four major phenolics found in black sapote were sinapic with a concentration of 110.7 mg/100 g dw (peak 6), followed by myricetin (peak 9, 85.0 mg/100 g dw), ferulic acid (peak 7, 82.0 mg/100 g dw), and catechin (peak 2, 79.9 mg/100 g dw). The highest concentrations of sinapic acid have been reported in cranberries (0–21.2 mg/100 g fw) and the skin and seeds of citrus (3.0–95.4 mg/100 g fw) (Bocco, Cuvelier, Richard, & Berset, 1998; Zuo, Wang, & Zhan, 2002). Based on this, black sapote could be considered a good source of this phenolic compound. Sinapic acid has been suggested to have anti-inflammatory properties by inhibiting the activation and expression of genes involved in inflammation (Yun et al., 2008). The flavonol myricetin levels found in black sapote are higher than what has been reported for fruits like berries and grapes (5.7 and 0.4 mg/100 g fw, respectively) (US Department of Agriculture, 2007). Myricetin has been shown to be beneficial against inflammation, atherosclerosis and thrombosis (Ong & Khoo, 1997). Although low levels of ferulic acid have been reported in fruits, here we found that it was almost 75% of the main phenolic sinapic acid, and higher than the concentrations reported for blackberries and cranberries (Sellappan, Akoh, & Krewer, 2002; Zuo et al., 2002). The

a Data are means of three repetitions ± standard deviation (SD). Peak numbers correspond to Fig. 2.

flavan-3-ols, catechin and epicatechin, found in black sapote have also been reported in other fruits. Higher levels of catechin exist in black sapote as compared to peaches and red grapes (12.2 and 10.1 mg/ 100 g fw, respectively), while epicatechin values reported for apricot and red grapes (5.5 and 8.7 mg/100 g fw, respectively) are below what has been found here (US Department of Agriculture, 2007). Caffeic acid is the most common hydroxycinnamic acid in fruit and vegetables. In cranberries, for instance, its concentration has been reported in the range of 2.2 to 25.4 mg/100 g fw (Zuo et al., 2002). Although caffeic acid in black sapote was not the main phenolic, its level is higher when compared to other fruits. White wine constitutes one of the best sources of protocatechuic acid (Li, Wang, Wang, & Wu, 1993) and in raspberry a concentration of 6–10 mg/100 g fw has been reported (Macheix, Fleuriet, & Billot, 1990). Here we found this phenolic acid in the lowest concentration in relation to the other phenolics identified in black sapote. Phenolic compounds in black sapote were identified by LC-ESI-MS and are labeled as peaks 1–13 following the elution order in the LC-MS total ion chromatogram (TIC) (Fig. 3) and their identification is presented in Table 4. Peak 1 was tentatively identified as dicoumaroylhexose-deoxyhexoside with an ion at m/z 472 and fragment at m/z 164 corresponding to coumaric acid. Peak 2 was identified as a cinnamic acid derivative with fragment ions at m/z 214, 196, and 148, according to what has been previously reported (Rubilar, Pinelo, Shene, Sineiro, & Nuñez, 2007) and retention time of the standard compound. Peak 3 was identified as catechin by comparison with retention time and fragmentation pattern of the standard with fragment ions at m/z 578 and 289 which may suggest that catechin

Fig. 2. HPLC chromatogram at 280 nm of black sapote phenolic extract: 1: cinnamic acid; 2: catechin; 3: epicatechin; 4: p-hydroxybenzoic acid; 5: caffeic acid; 6: sinapic acid; 7: ferulic acid; 8: o-coumaric acid; 9: myricetin; 10: protocatechuic acid.

2214

E.M. Yahia et al. / Food Research International 44 (2011) 2210–2216

Fig. 3. Total Ion Chromatogram (TIC) of phenolic extracts of black sapote. For peak identification refer to Table 4.

Table 4 Phenolic compounds identified in black sapote by LC-ESI-MS analysesa Peak

TR (min)

m/z

1 2 3 4 5 6 7 8 9 10 11 12 13

16.86 17.50 19.16 20.30 21.32 22.96 24.61 25.80 27.53 29.19 30.08 34.79 42.35

492, 214, 578, 289, 194, 275, 353, 385, 681, 154, 701, 518, 616,

a

Compound name 472, 196, 289 245 176, 138 258 224 556, 136 270 315 565

164 148

148

232

Dicoumaroylhexose-deoxyhexose Cinnamic acid derivative Catechin Epicatechin Ferulic acid Dimer of p-Hydroxybenzoic acid Derivative of 3-caffeoylquinic acid Sinapic acid hexoside Galloylated caffeic acid hexoside Protocatechuic acid Diapigenin hexoside Isoramnethin hexose-malonate Dimyricetin hexose-malonate

Peak numbers and retention times correspond to Fig. 3.

is present in black sapote as a dimer. Peak 4 was definitely identified as epicatechin based on its fragmentation profile (m/z 289 and 245) and retention time of the standard compound. Peak 5 corresponded to ferulic acid as seen by the presence of an ion at m/z 194 and a fragment at m/z 176 as a result of the loss of a water molecule. p-Hydroxybenzoic acid was identified as a dimer in peak 6 by the presence of ions at m/z 275 and the free molecule at m/z 138. Peak 7 presented a fragment ion at m/z 353 and was tentatively identified as a derivative of 3-caffeoylquinic acid based on previous reports (Lin & Harnly, 2008). Based on retention time of the standard compound and

fragment ions at m/z 385 and 224, peak 8 was identified as sinapic acid hexoside. Peak 9 was only tentatively identified as galloylated caffeic acid hexoside based on its fragment ions at m/z 681, 556 (loss of gallic acid) and 214 (loss of caffeic acid hexoside). Protocatechuic acid was identified in peak 10 based on the presence of the fragment ions at m/z 154 and 136, corresponding to the free mass of the acid and the acid minus a water molecule. Peak 11 was tentatively identified as a dimer of apigenin linked to a hexose based on the presence of the base ion at m/z 701 and a fragment ion at m/z 270. Although isoramnethin was not identified by HPLC analysis, peak 12 corresponded to isoramnethin hexose-malonate based on the ion at m/z 518 and a fragment at m/z 315. Peak 13 was identified as dimyricetin hexose-malonate based on the ion at m/z 616 which corresponds to myricetin dimer and the fragment at m/z 565 (myricetin linked to hexose-malonate). TC content was 399.4 ± 7. 2 μg β-carotene/100 g fw. HPLC chromatogram of carotenoid extracts of black sapote is shown in Fig. 4. By comparison with standard compounds, peak 1 and peak 2 were identified as lutein and β-carotene, respectively, and their contents are shown in Table 5. Mass spectrometric analysis was performed in the carotenoid extracts and their fragmentation patterns compared with those of the standards. Lutein monoepoxide was identified by means of its fragmentation profile and retention time (7.3 min) of the standard compound (Fig. 5). The UV spectra of lutein epoxides have been reported before with three λmax: 416, 440, ad 470 nm which corresponds to what has been found here (Fig. 5) (Breithaupt & Bamedi, 2002). In addition, fragmentation pattern was the same as the standard compound with the protonated molecule at m/z 571 and base ion at m/z 553 as a result of the loss of water which is typical for

Fig. 4. HPLC chromatogram at 470 nm of black sapote carotenoids extracts: 1: lutein; 2: β-carotene.

E.M. Yahia et al. / Food Research International 44 (2011) 2210–2216 Table 5 Content of carotenoid compounds identified in black sapote by HPLC analyses.a Peak

Compound name

Content (mg/100 g dw)

1 2

Lutein β-carotene

0.29 ± 0.10 5.13 ± 1.90

a Data are means of three repetitions ± standard deviation (SD). Peak numbers correspond to Fig. 4.

lutein (Breithaupt, Wirt, & Bamedi, 2002). Identification of β-carotene was carried out by comparison of its retention time (16.0 min). However, based on its fragmentation profile, it might correspond to a β-carotene isomer (Ornelas-Paz et al., 2007). Carotenoids are believed to be responsible, at least in part, for the beneficial effects of consuming fruits and vegetables on preventing diseases associated with aging. In addition, carotenoids are important precursors of vitamin A and their antioxidant activity has been recognized (Astrog, Gradelet, Berges, & Suschetet, 1997; Paiva & Russell, 1999). Although TC content of black sapote found in this study is lower than that of other fruits such as mango and papaya, it is higher than the levels found in strawberry, guava and prickly pear (Corral-Aguayo, Yahia, Carrillo-Lopez, & Gonzalez-Aguilar, 2008). The lutein concentration found in black sapote is comparable to that of blueberry and black currant, and higher than what has been reported for apricot, watermelon, and strawberry (Marinova & Ribarova, 2007; Setiawan, Sulaeman, Giraud, & Driskell, 2001). The concentration of β-carotene found in black sapote is below the values reported for other tropical fruits like mango, papaya, and peach, but higher than guava (CorralAguayo, Yahia, Carrillo-Lopez, & Gonzalez-Aguilar, 2008; Pott, Breithaupt, & Carl, 2003; Setiawan et al., 2001; Yahia, Ornelas-Paz, & Gardea, 2006). Carotenoids have been recognized to possess antioxidant activity by quenching singlet oxygen (Stahl & Sies, 2003) and from here their importance in human health. Although black sapote cannot be considered a rich source of carotenoids, these might add to the major contribution of phenolic compounds to the antioxidant capacity of this fruit. In addition, although in low concentration, this study demonstrates that carotenoids such as lutein and β-carotene, exist as part of the biologically active compounds in black sapote.

4. Conclusion In the present study, HPLC-MS analyses were successfully applied for the identification and quantification of antioxidant compounds in black sapote. To the best of our knowledge no similar reports on the identification of phytochemicals exist for this fruit. The antioxidant capacity observed in black sapote might be attributed to the presence of phytochemicals that can contribute to a healthy diet.

2215

References Association of Official Analytical Chemists (AOAC) (2000). Vitamin and other nutrient. In W. Horwitz (Ed.), Official methods of analysis of the Association of Analytical Chemists International (pp. 4−5). (17th ed). Gaithersburg, MD: AOAC. Astrog, P., Gradelet, S., Berges, R., & Suschetet, M. (1997). Dietary lycopene decreases initiation of liver preneoplastic foci by diethylnitrosamine in rat. Nutrition and Cancer, 29, 60−68. Barros, L., Carvalho, A.M., & Ferreira, I.C.F.R. in press. Exotic fruit as a source of important phytochemicals: Improving the traditional use of Rosa canina fruits in Portugal. Food Research International. Benzie, F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239, 70−76. Benzie, F. F., & Strain, J. J. (1999). Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods in Enzymology, 299, 15−27. Bocco, A., Cuvelier, M. E., Richard, H., & Berset, C. (1998). Antioxidant activity and phenolic composition of citrus peel and seed extracts. Journal of Agricultural and Food Chemistry, 46(6), 2123−2129. Breithaupt, D. E., & Bamedi, A. (2002). Carotenoids and carotenoid esters in potatoes (Solanum tuberosum L.): New insights into an ancient vegetable. Journal of Agricultural and Food Chemistry, 50, 7175−7181. Breithaupt, D. E., Wirt, U., & Bamedi, A. (2002). Differentiation between lutein monoester regioisomers and detection of lutein diesters from marigold flowers (Tagetes erecta L.) and several fruits by liquid chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry, 50, 66−70. Burton, G. W., & Ingold, K. U. (1981). Autoxidation of biological molecules. The autoxidation of vitamin E and related chainbreaking antioxidants in vitro. Journal of the American Chemical Society, 103, 6472−6477. Cao, G., & Prior, R. L. (1999). Measurement of oxygen radical absorbance capacity in biological samples. Methods in Enzymology, 299, 50−62. Corral-Aguayo, R., Yahia, E. M., Carrillo-Lopez, A., & Gonzalez-Aguilar, G. (2008). Correlation between some nutritional components and the total antioxidant capacity measured with six different assays in eight horticultural crops. Journal of Agricultural and Food Chemistry, 56, 10498−10504. Diaz, M. N., Frei, B., Vita, J. A., & Keaney, J. F. (1997). Antioxidants and atherosclerotic heart disease. The New England Journal of Medicine, 337, 408−416. Evelson, P., Travacio, M., & Repetto, M. (2001). Evaluation of total reactive antioxidant potential (TRAP) of tissue homogenates and their cytosols. Archives of Biochemistry and Biophysics, 388, 261−266. Gil, M. I., Tomas-Barberan, F. A., Hess-Pierce, B., Holeroft, D. M., & Kader, A. A. (2000). Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agricultural and Food Chemistry, 48, 4581−4589. Halliwell, B., & Gutteridge, J. M. (1989). Free radicals in biology and medicine. Oxford, UK: Oxford University Press. Harman, D. (1995). Role of antioxidant nutrients in aging: Overview. Age, 18, 51−62. Haslam, E. (1998). Practical polyphenolics—From structure to molecular recognition and physiological action. Cambridge, UK: University Press, Cambridge. Heber, D. (2004). Vegetables, fruits and phytoestrogens in the prevention of diseases. Journal of Postgraduate Medicine, 50(2), 145−149. Hertog, M. G. L. (1996). Epidemiological evidence on potential health properties of flavonoids. Proceeding of the Nutrition Society, 55, 385−397. Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53, 1841−1856. Ikram, E. H. K., Eng, K. H., Jalil, A. M. M., Ismail, A., Idris, S., Azlan, A., et al. (2009). Antioxidant capacity and total phenolic content of Malaysian underutilized fruits. Journal of Food Composition and Analysis, 22, 388−393. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (2001). Ingestión diaria recomendada de proteínas, vitaminas y nutrimentos inorgánicos para la población mexicana. http://quetzal1.innsz.mx/docs/idrinn.pdf. Retrieved March 18, 2010 from.

Fig. 5. Mass and UV–visible absorption spectra of peak 1 in Fig. 4.

2216

E.M. Yahia et al. / Food Research International 44 (2011) 2210–2216

Katsube, T., Tabata, H., Ohta, Y., Yamasaki, Y., Anuurad, E., Shiwaku, K., et al. (2004). Screening for antioxidant activity inedible plant products: Comparison of lowdensity lipoprotein oxidation assay, DPPH radical scavenging assay, and Folin– Ciocalteu assay. Journal of Agricultural and Food Chemistry, 52, 2391−2396. Kim, D. O., Lee, K. W., Lee, H. J., & Lee, C. Y. (2002). Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. Journal of Agricultural and Food Chemistry, 50, 3713−3717. Li, P., Wang, X. Q., Wang, H. Z., & Wu, Y. N. (1993). High performance liquid chromatography determination of phenolic acids in fruits and vegetables. Biomedical and Environmental Sciences, 6(4), 389−398. Lin, L. Z., & Harnly, J. M. (2008). Phenolic compounds and chromatographic profiles of pear skins (Pyrus spp.). Journal of Agricultural and Food Chemistry, 56(19), 9094−9101. Liu, D., Shi, J., Ibarra, A. C., Kakuda, Y., & Xue, S. J. (2008). The scavenging capacity and synergistic effects of lycopene, Vitamin E, Vitamin C, and β-carotene mixtures on the DPPH free radical. Food Science and Technology, 41, 1344−1349. Lampila, P., van Lieshout, M., Gremmen, P., & Lähteenmäki, L. (2010). Consumer attitudes towards enhanced flavonoid content in fruit. Food Research International, 42, 122−129. Macheix, J. J., Fleuriet, A., & Billot, J. (1990). Fruit phenolics. Boca Raton, FL: CRC Press. Mahattanatawee, K., Manthey, J. A., Luzio, G., Talcott, S. T., Goodner, K., & Baldwin, E. A. (2006). Total antioxidant activity and fiber content of select Florida-grown tropical fruits. Journal of Agricultural and Food Chemistry, 54(19), 7355−7363. Marinova, D., & Ribarova, F. (2007). HPLC determination of carotenoids in Bulgarian berries. Journal of Food Composition and Analysis, 20, 370−374. Obermuller-Jevic, U. C., Olano-Martin, E., Corbacho, A. M., Eiserich, J. P., van der Vliet, A., Valacchi, G., et al. (2003). Lycopene inhibits the growth of normal human prostate epithelial cells in vitro. Journal of Nutrition, 133, 3356−3360. Ong, K. C., & Khoo, H. E. (1997). Biological effects of myricetin. General Pharmacology, 29(2), 121−126. Ornelas-Paz, J. J., Yahia, E. M., & Gardea-Bejar, A. (2007). Identification and quantification of xanthophylls esters, carotenes, and tocopherols in the fruit of seven Mexican mango cultivars by liquid chromatography-atmospheric pressure chemical ionization-time of flight mass spectrometry (LC-APCI+) MS. Journal of Agricultural and Food Chemistry, 55, 6628−6635. Paiva, S., & Russell, R. (1999). Beta carotene and other carotenoids as antioxidants. Journal of the American College of Nutrition, 18, 426−433. Pott, I., Breithaupt, D. E., & Carl, R. (2003). Detection of unusual carotenoids in fresh mango (Mangifera indica L. Cv. Kent). Phytochemistry, 64, 825−829. Prior, R. L., Wu, X., & Schaichet, K. (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 53, 4290−4302. Rice-Evans, C., & Miller, N. J. (1994). Total antioxidant status in plasma and body fluids. Methods in Enzymology, 234, 279−293. Rubilar, M., Pinelo, M., Shene, C., Sineiro, J., & Nuñez, M. J. (2007). Separation and HPLCMS identification of phenolic antioxidants from agricultural residues: Almond hulls and grape pomace. Journal of Agricultural and Food Chemistry, 55, 10101−10109. Schlesier, K., Harwat, M., Böhm, V., & Bitsch, R. (2002). Assessment of antioxidant activity by using different in vitro methods. Free Radical Research, 36(2), 177−187.

Sellappan, S., Akoh, C. C., & Krewer, G. (2002). Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. Journal of Agricultural and Food Chemistry, 50(8), 2432−2438. Setiawan, B., Sulaeman, A., Giraud, D. W., & Driskell, J. A. (2001). Carotenoid content of selected Indonesian fruits. Journal of Food Composition and Analysis, 14, 169−176. Smith, M. A., Perry, G., Richey, P. L., Sayre, L. M., Anderson, V. E., Beal, M. F., et al. (1996). Oxidative damage in Alzheimer's. Nature, 382, 120−121. Soto-Zamora, G., Yahia, E. M., Brecht, J. K., & Gardea, A. (2005). Effects of postharvest hot air treatments on the quality and antioxidant levels in tomato fruit. LebensmittelWissenschaft & Technologie, 38, 657−663. Southon, S. (2000). Increased fruit and vegetable consumption within the EU: Potential health benefits. Food Research International, 33, 211−217. Stahl, W., & Sies, H. (2003). Antioxidant activity of carotenoids. Molecular Aspects of Medicine, 24, 345−351. Temple, N. J. (2000). Antioxidants and disease: More questions than answers. Nutrition Research, 20, 449−559. US Department of Agriculture (2007). USDA database for the flavonoids content of selected foods. Beltsville, MD: USDA. Van den Berg, R., Haenen, G. R. M. M., Van den Berg, H., & Bast, A. (1999). Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chemistry, 66, 511−517. WCRF-AICR (2007). Food, nutrition, physical activity, and the prevention of cancer: A global perspective. Washington (DC): World Cancer Research Fund-American Institute for Cancer Research. Willett, W. C. (1994). Diet and health: What should we eat? Science, 254, 532−537. Willett, W. C. (1995). Diet, nutrition and avoidable cancer. Environmental Health Perspectives, 103, 165−710. Wolfe, K., Xianzhong, W., & Liu, R. H. (2003). Antioxidant activity of apple peels. Journal of Agricultural and Food Chemistry, 51, 609−614. Wu, X., Gu, L., Holden, J., Haytowitz, D. B., Gebhardt, S. E., Beecher, G., et al. (2004). Development of a database for total antioxidant capacity in foods: A preliminary study. Journal of Food Composition and Analysis, 17, 407−422. Yahia, E. M. (2010). The contribution of fruit and vegetable consumption to human health. Phytochemicals: Chemistry, Nutricional and Stability (pp. 3−51). : WileyBlackwell Chapter 1. Yahia, E. M., & Ornelas-Paz, J. J. (2010). Chemistry, stability and biological actions of carotenoids. In L. A., E., & G. A. (Eds.), Fruit and vegetable phytochemicals: Chemistry, nutritional value and stability. Publication, USA: Wiley-Blackwell, A John Wiley & Sons, Inc. Yahia, E. M., Ornelas-Paz, J. J., & Gardea, A. (2006). Extraction, separation and partial identification of ‘Ataulfo’ mango fruit carotenoids. Acta Horticulturae, 712, 333−338. Yun, K. J., Koh, D. J., Kim, S. H., Park, S. J., Ryu, J. H., Kim, D. G., et al. (2008). Antiinflammatory effects of sinapic acid through the suppression of inducible nitric oxide synthase cyclooxygenase-2, and proinflammatory cytokines expressions via nuclear factor-kB inactivation. Journal of Agricultural and Food Chemistry, 56, 10265−10272. Zuo, Y., Wang, C., & Zhan, J. (2002). Separation, characterization, and quantitation of benzoic and phenolic antioxidants in American cranberry fruit by GC-MS. Journal of Agricultural and Food Chemistry, 50(13), 3789−3794.