Nutritional and phytochemical composition of Annona cherimola Mill. fruits and by-products: Potential health benefits

Food Chemistry 193 (2016) 187–195

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Analytical Methods

Nutritional and phytochemical composition of Annona cherimola Mill. fruits and by-products: Potential health benefits Tânia Gonçalves Albuquerque a,b,1, Filipa Santos a,1, Ana Sanches-Silva a,c, M. Beatriz Oliveira b, Ana Cristina Bento a, Helena S. Costa a,b,⇑ a b c

Research and Development Unit, Department of Food and Nutrition, National Institute of Health Dr. Ricardo Jorge, I.P., Av. Padre Cruz, 1649-016 Lisbon, Portugal REQUIMTE/Faculdade de Farmácia da Universidade do Porto, R. Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal Centro de Estudos de Ciência Animal (CECA), Universidade do Porto, R.D. Manuel II, Apartado 55142, 4051-401 Porto, Portugal

a r t i c l e

i n f o

Article history: Available online 16 June 2014 Keywords: Annona cherimola Mill. PDO Nutritional composition Bioactive compounds Antioxidant activity By-products

a b s t r a c t Annona cherimola Mill., commonly known as cherimoya, is a tropical fruit well known due to its tasty flavour. In the present study the antioxidant activity of pulp, peel and seeds of four cultivars from A. cherimola Mill. from Madeira Island (Madeira, Funchal, Perry Vidal and Mateus II) was analysed. Moreover, nutritional composition (proximates and vitamins) and bioactive compounds content were determined. The peel of Madeira cultivar showed the highest antioxidant capacity, with an EC50 of 0.97 mg/mL, and total flavonoids (44.7 epicatechin equivalents/100 g). The most abundant carotenoid was lutein, with values ranging from 129 to 232 lg/100 g. The highest L-ascorbic acid content (4.41 mg/100 g) was found in the peel of Perry Vidal cultivar. These results highlight A. cherimola Mill. antioxidant properties, especially in its by-products and encourage their application in cosmetic, pharmaceutical and food processing industries, as added value natural extracts. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Fruits have become increasingly important in human nutrition because of their nutrient composition and potential beneficial health effects. The nutritional value and health related properties of fruits depend not only on the concentration of nutrients and phytochemicals but also on the daily intake and bioavailability (Feliciano et al., 2010). Madeira Island is a Portuguese region with excellent climate conditions for the production of some exotic and tropical fruits, such as, avocado, cherimoya, banana and passion fruit (Valente, Albuquerque, Sanches-Silva, & Costa, 2011). The production of such fruits has increased in the last years due to their attractive sensorial properties and because they are claimed to be good sources of vitamins and other bioactive compounds, like polyphenols or carotenoids. Because of this new and emerging market, there is a growing demand for studies regarding the consumption and potential health benefits of exotic and tropical fruits. However, when compared with other common fruits, there is a large gap to ⇑ Corresponding author at: Research and Development Unit, Department of Food and Nutrition, National Institute of Health Dr. Ricardo Jorge, I.P., Av. Padre Cruz, 1649-016 Lisbon, Portugal. Tel.: +351 217519267; fax: +351 217508153. E-mail address: [email protected] (H.S. Costa). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.foodchem.2014.06.044 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

fill the current lack of knowledge (Pierson et al., 2012). Bioactive compounds are defined as ‘‘inherent non-nutrient constituents of food plants with anticipated health promoting/beneficial and/or toxic effects when ingested’’ (Gry et al., 2007). According to the literature, bioactive compounds concentration varies considerably among the type of plant and cultivar, being influenced by genetic factors, maturity stage, environmental and cultural practices, and postharvest conditions (Odriozola-Serrano, Soliva-Fortuny & MartínBelloso, 2008). The identification of bioactive compounds is of major importance in order to understand the underlying mechanisms of action and interactions of natural products in the human body. Although fruits cultivated in temperate zones have already been and continue to be studied for their nutritional and healthpromoting value, traditional fruits from Madeira Island remain an expansive and potentially novel source of natural products. Nutritional composition data are an essential resource for food researchers and epidemiologists who investigate the relationship between food and disease in populations and require an accurate estimation of nutrient intake, and are also the basis for the development of dietary recommendations (Costa, Vasilopoulou, Trichopoulou, Finglas & Participants of EuroFIR Traditional Foods Work Package 2010). Cherimoya or annona fruit belongs to Annonaceae family and they are native of tropical regions. This is the most exported fruit in Madeira Island, after banana. The cultivars with the highest

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agronomic/commercial interest are Madeira, Funchal, Perry Vidal and Mateus II. Since 2000, the European Union granted cherimoya from Madeira (Annona cherimola Mill.) the Protected Designation of Origin (PDO), becoming the first regional fruit to receive this level of international protection. In 1992, the European Union (EU) created quality product designation systems, known as PDO, Protected Geographical Indication (PGI) and Traditional Speciality Guaranteed (TSG), which protect registered traditional foods and enable producers to market distinctive high quality regional products. These Regulations have been updated and clarified in 2012 (Council of the European Union, 2012). Therefore, when consumers purchase an EU quality labelled product, its quality but also its authenticity is guaranteed. Cherimoya is known to have been cultivated during the times of the Incan Empire, dating back to 1200 BC. It is a subtropical fruit native to the Andes, with a thick green peel, whose pulp is creamy and sweet. Moreover, it is known for its exceptional taste, and it is becoming increasingly important in tropical and subtropical regions, due to its implication in commercial and folk medicine, especially for the treatment of skin disease (Amoo, Emenike, & Akpambang, 2008). In the literature, cherimoya is described as a fruit with high amounts of water, carbohydrates and proteins, and low cholesterol content (Barreca et al., 2011). The focus of this study was to explore the potential health benefits and nutritional components of cherimoya from Madeira Island, as far as we know have never been studied in such detail. The fruit pulp and by-products of four cultivars of A. cherimola Mill. (Funchal, Madeira, Mateus II and Perry Vidal) were selected. The following parameters were evaluated: antioxidant activity (radical 2,2-diphenyl-1-picrylhidrazyl (DPPH) scavenging activity), total phenolics, total flavonoids, proximates (moisture, ash, total protein, total fat and dietary fibre), vitamins A, C and E, and carotenoids. 2. Materials and methods 2.1. Food samples and sample preparation During January 2013, the fruits from four cultivars (Funchal, Madeira, Mateus II and Perry Vidal) of cherimoya were supplied by a company located in Madeira Island (Portugal). Multiple fruits were manually collected and randomly picked from several trees and different parts of each tree, taking into account the ripeness state. To assure that fruits were ripe, the homogenisation was carried out three days after harvest. Moreover, when cherimoyas are ripe, the skin is greener and gives slightly to pressure. To minimise the loss of nutrients, especially vitamins and bioactive compounds, the fruits were placed in appropriate containers and transported in cooler boxes. On the same day, samples were manually divided between edible portion (pulp) and non-edible portions (peel and seeds). Then, the pulp and peel of the fruit were immediately homogenised in a blender (Grindomix GM200, Retsch, Haan, Germany) at 5000 rpm during 1 min. The seeds were grinded using a domestic blender (Taurus, Barcelona, Spain). After homogenisation, samples were stored in the dark at 80 °C, to avoid deterioration until analyses. Moreover, a stabilizing solution was added prior to storage in the case of vitamin C determination (Section 2.5). 2.2. Standards and reagents All chemicals and reagents were purchased from various commercial sources (Sigma–Aldrich, Merck and Prolab) and were, at least, of analytical grade. Trans-retinol, b-carotene, a-tocopherol, lycopene and lutein were acquired from Sigma–Aldrich (Madrid, Spain). Zeaxanthin, b-cryptoxanthin, and a-carotene were obtained from CaroteNature (Lupsingen, Switzerland). L-Ascorbic

acid was purchased from Riedel-de Haën (Seelze, Germany). Ultrapure water from a Milli-Q system (Millipore, Bedford, MA, USA) was used. 2.3. Antioxidant activity 2.3.1. Sample extraction The extracts of cherimoya fruit used for antioxidant activity evaluation were obtained according to the method described by Julián-Loaeza, Santos-Sanchez, Valadez-Blanco, Sanchez-Guzman, and Salas-Coronado (2011), with some modifications. Briefly, 6 g of the fruit parts (peel, pulp or seeds) were mixed with 20 mL of ethanol (90%, v/v) in a Ultra-Turrax (IKAÒ T25, Staufen, Germany) for 10 min at 4500 rpm and then filtered through cotton wool. 2.3.2. Radical DPPH scavenging activity Radical scavenging activity was determined according to the method reported by Julián-Loaeza et al. (2011). Briefly, 2.5 mL of ethanolic sample extract were mixed with 2.5 mL of DPPH ethanolic solution (0.004%, w/v). The peel extract concentrations ranged between 0.025 and 0.5 mg/mL, while for seeds and pulp varied between 0.5 and 5.5 mg/mL, with the exception of Madeira’s cultivar pulp, which was prepared in concentrations between 0.2 and 1.25 mg/mL. Then, the mixture was incubated at room temperature in the dark and stirred (See-saw rocker SSM4, Stuart, Staffordshire, UK) for 30 min. This procedure was also applied to L-ascorbic acid, butylated hydroxytoluene (BHT) and gallic acid, which were employed as controls. The bleaching of DPPH was determined by measuring the absorbance at 517 nm in a spectrophotometer (Thermo Scientific 300 Evolution, Madison, USA). A control was prepared with no extract using 90% aqueous ethanol for baseline correction. For this method a blank solution was used containing 2.5 mL ethanol (90%, v/v) and 2.5 mL of DPPH. The inhibition percentage (IP) was calculated according to the following equation:

IP ¼ ðAbsblank  Abs30min Þ=ðAbsblank Þ  100 where Absblank is the absorbance of the control (blank, without extract) and Abs30min is the absorbance in the presence of the extract after 30 min of reaction. The scavenging activity of radical DPPH was expressed by the EC50 parameter, which means the concentration of the sample that decreases the initial DPPH absorbance by 50%, and these values were calculated by linear regression. All analyses were performed in duplicate. 2.3.3. Total phenolics The total phenolics content of A. cherimola Mill. ethanolic extracts was determined by the Folin-Ciocalteu method (JuliánLoaeza et al. 2011), with some modifications. Briefly, 0.1 mL of sample extract was mixed with 4.6 mL of ultrapure water and 0.1 mL of the Folin-Ciocalteu reagent (2 M). The mixture was left to rest for 3 min in the absence of light and then 0.3 mL of sodium carbonate aqueous solution (2%, w/v) was added. Afterwards, the mixture was stirred and kept incubating in a water bath (40 °C, 60 min). The absorbance was measured at 760 nm against a blank, using a UV/Vis spectrophotometer (Thermo Scientific Evolution 300, Madison, USA). Gallic acid was used as a standard with concentrations ranging from 0.1 to 1.4 mg/mL. The results were expressed in mg of gallic acid equivalents (GAE) per 100 g of sample through the interpolation of the calibration curve of gallic acid. All analyses were performed in duplicate. 2.3.4. Total flavonoids Total flavonoids content was determined according to the method reported by Barreca et al. (2011) with slight modifications. Briefly, 0.1 mL of extract, 2 mL of ultrapure water and 0.15 mL of

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sodium nitrite aqueous solution (5%, w/v) were mixed for 5 min with a vortex (IKAÒ, Staufen, Germany). Then, 0.15 mL of aluminium chloride aqueous solution (10%, w/v) was added and the mixture was vortexed for 1 min. Following this, 1 mL of sodium hydroxide (1 M) was added and stirred for 1 min. The absorbance was immediately read at 490 nm in a UV/Vis spectrophotometer (Thermo Scientific 300 Evolution, Madison, USA). A calibration curve was performed using epicatechin standard with concentrations ranging from 0.1 to 4.0 mg/mL. The results were expressed as mg of epicatechin equivalents (ECE) per 100 g of sample through the interpolation of the calibration curve of epicatechin. All analyses were performed in duplicate. 2.4. Proximates Nutritional analyses were carried out in an accredited laboratory according to ISO/IEC 17025, and this implies accreditation for each nutrient, and taking into account the scope of accreditation and/or successful participation in proficiency-testing schemes. The fruit pulps of four cultivars of A. cherimola Mill. were analysed regarding their content on moisture, ash, total protein, total fat and total dietary fibre. The available carbohydrates (CHO) were calculated as: CHO = 100  (moisture + ash + total protein + total fat + total dietary fibre). Energy values were calculated using the following equations: (1) in kJ, 17  total protein þ 17 available carbohydrates þ 37  total fat þ 8  total dietary fibre and (2) in kcal, 4  total protein þ 4  available carbohydrates þ9  total fat þ 2  total dietary fibre, as indicated in Greenfield and Southgate (2003) for the energy conversion factors and in Regulation (EU) No. 1169/2011 (Regulation, 2011) for the dietary fibre conversion factor. Moisture content was determined by gravimetric method (AOAC 952.08, 2000), using a dry air oven (Memmert, Germany). Total ash analysis was carried out according to AOAC 923.03 (2000). For total nitrogen, the Kjeldahl method in combination with a copper catalyst using a block digestion system Foss Tecator 2006 Digestor and a Foss 2200 Kjeltec Auto Distillation unit (Foss, Hilleroed, Denmark) was used (AOAC 991.20, 2000). The total protein content was calculated by using 6.25 as the nitrogen conversion factor, according to FAO (1973). Total fat determination was performed according to Albuquerque, Sanches-Silva, Santos, and Costa (2012) and AOAC 948.15 (2000), where an acid hydrolysis followed by extraction using a Soxhlet apparatus (Soxtec™ 2050, Foss, Hilleroed, Denmark) with petroleum ether, as the extraction solvent. The content of total dietary fibre was determined by the enzymatic-gravimetric method (AOAC, 2000). 2.5. Total vitamin C Total vitamin C and L-ascorbic acid content of four cultivars of A. cherimola Mill. fruits (pulp, peel and seeds) were determined by a highly sensitive, rapid, precise and accurate high performance liquid chromatography (HPLC) method, previously full validated (Valente, Sanches-Silva, Albuquerque, & Costa, 2014). A standard stock solution of ascorbic acid (1 mg/mL) was freshly prepared on each day of analysis. The working standard solutions were prepared by appropriate dilution to obtain final concentrations of ascorbic acid (1, 20, 40, 60, 80 and 100 lg/mL). The methods described by Valente et al. (2014) and Chebrolu, Jayaprakasha, Yoo, Jifon, and Patil (2012) were applied to determine L-ascorbic acid and total vitamin C content, respectively, in the selected cultivars. Briefly, 4 g of each sample were stabilized with 12 mL of acid solution, 10% (v/v) perchloric acid and 1% (w/v) metaphosphoric acid in ultrapure water. This solution was diluted to 50 mL with mobile phase. For total vitamin C determination, 1 mL of tris(2carboxyethyl)phosphine (TCEP) (5 mM) was added to the previous

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solution. The samples were filtered twice. A volume of 10 lL was injected into the chromatographic system. Separation and quantification was performed on an Alliance 2695 HPLC system (Waters, Milford, MA, USA), equipped with a Waters 2996 photodiode array detector (PDA), using a Synergi™ Hydro-RP (150  4.6 mm i.d., 4.0 lm particle size) analytical column with a SecurityGuard Cartridge AQ C18 (40  2.0 mm i.d., 5.0 lm particle size) also from Phenomenex (Torrance, California, USA). The mobile phase consisted of 20 mM ammonium dihydrogen phosphate, pH 3.5 (adjusted with orthophosphoric acid 85%), and containing 0.015% (w/v) of metaphosphoric acid. The total run time was 6 min at a flow-rate of 0.6 mL/min. Column temperature was kept at 30 °C and the auto-sampler at 4 °C. The detection signal was recorded at 245 nm and the peak areas were quantified and processed with an Empower™ software version 2.0 (Waters, Milford, MA, USA). 2.6. Carotenoids, vitamins A and E The stock standard solutions of the two vitamins and six carotenoids were prepared individually by dissolving each standard in an appropriate solvent with 0.01% (w/v) of BHT and their concentrations were confirmed using a UV/Vis spectrophotometer Evolution 300 (Thermo Scientific, Madison, USA) by reading the absorbance at a given wavelength (k), using the following extinction coefficients (e): zeaxanthin, 2540 (450 nm), lutein, 2550 (445 nm), retinol 1825 (325 nm), a-tocopherol 72 (292 nm) in ethanolic solution; and b-cryptoxanthin, 2460 (451 nm), lycopene, 3450 (472 nm), a-carotene, 2800 (444 nm), b-carotene, 2560 (450 nm) in hexane solution (Hart & Scott, 1995; Heinonen, Ollilainen, Linkola, Varo, & Koivistoinen, 1989; Hulshof, Xu, Bovenkamp, & West, 1997; Olmedilla, Granado, Blanco, & Rojas-Hidalgo, 1992). The standard stock solutions were stored under nitrogen at 80 °C to delay degradation. Six levels of working standard solutions with a mixture of the six carotenoids and vitamins A and E were prepared in mobile phase and were used to obtain the calibration curves. Samples were prepared according to Sanches-Silva et al. (2013). In a 50 mL tube, 5 g of sample (peel, pulp and seeds) were weighed and 120 mg of magnesium carbonate and 30 mL of hexane/ethanol (4:3, v/v) were added. Then the mixture was shaken in a high performance homogeniser, for 5 min at 5600 rpm (Ultra TurraxÒ, IKA, Staufen, Germany). Afterwards, samples were shaken for 30 min at 70 rpm (See-saw rocker,SSM4, Stuart, Staffordshire, UK) in order to allow complete extraction. The residue was separated from the liquid phase by centrifugation at 289  g (5 min and 4 °C) and reextracted under the same conditions. Finally, pooled organic phases were evaporated at 25 °C in a rotary evaporator (Büchi R-210, Labortechnik AG, Switzerland). For the saponification of samples, after evaporation of the organic phase, the extract was dissolved in 20 mL hexane and 20 mL of methanolic potassium hydroxide 10% (w/v), for 4 h at room temperature under light protection. Then, the organic layer was washed with ultrapure water until neutral pH and evaporated as described above. In both cases the residue was dissolved in the organic mobile phase, to a final volume of 5 mL. The samples were filtered using a PTFE syringe filter of 0.22 lm (Millipore, Bedford, MA, USA) and placed in amber vials for analysis. Each sample was injected at least twice. The handling and processing of the samples was always carried out under yellow light and with temperatures below 25 °C, to avoid the compounds’ oxidation and degradation. An Acquity Ultra Performance Liquid Chromatograph (UPLCÒ), (Waters, Milford, MA, USA) equipped with a PDA was used. The detection signal was recorded and the peak areas were quantified and processed with Empower™ software version 2.0.

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Chromatographic separation was performed with an Acquity UPLCÒ BEH C18 analytical column (50  2.1 mm i.d., 1.7 lm particle size) and an Acquity UPLCÒ BEH C18 guard-column (5  2.1 mm i.d., 1.7 lm particle size) from Waters (Milford, MA, USA). The column temperature was kept at 20 °C and the auto-sampler at 5 °C. Mobile phases were (A) acetonitrile/methanol (containing 0.05 M ammonium acetate)/dichloromethane (75:20:5, v/v/v) and (B) ultrapure water. The gradient program started with A-B (75:25), after 6 min, A-B (78:22), then changed to 100% A for 7 min, and finally returned to A-B (75:25) until 18 min. The flow rate was isocratic at 0.5 mL/ min and an injection volume of 10 lL was applied. The concentration of each carotenoid was expressed as lg per 100 g of sample, and vitamin E was expressed as mg per 100 g of sample. Vitamin A activity was expressed as lg of retinol activity equivalents (RAE) per 100 g of sample. 2.7. Statistical analysis The statistical analyses of data were performed using Microsoft Office ExcelÒ 2010. Results are expressed as mean ± standard deviation (SD) or as percentage. Values presented in the tables are the average values of three individual samples (n = 3). Moreover the results for pulps of cherimoya are presented per 100 g of edible portion in fresh weight basis. For multiple comparisons of normally distributed data, parametric one-way analysis of variance (ANOVA) followed by Tukey test was used. A value of P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Antioxidant activity 3.1.1. Radical DPPH scavenging activity The DPPH method is widely used to evaluate antioxidant activity of a food matrix. It is based in the reducing ability of the sample antioxidants on the DPPH through an electron transfer reaction, which is measured by the decrease in absorbance at 517 nm. Lower EC50 values indicate higher antioxidant activity. The EC50 values for DPPH radical scavenging activity of ethanolic extracts of four cultivars of A. cherimola Mill. fruit (pulp, peel and seeds) are presented in Table 1. The results of our study have shown that for pulp, the lowest EC50 was observed for Madeira cultivar (0.97 ± 0.01 mg/mL), revealing a much higher antioxidant activity than for the other three cultivars. Julián-Loaeza et al. (2011) reported EC50 values

for Annona diversifolia pulp varying between 1.70 and 1.99 mg/ mL. The EC50 found in the present study for the pulp of A. cherimola Mill. fruit (Madeira cultivar) is lower than the values reported in the literature, which indicates that this species of cherimoya has a higher antioxidant activity than the fruit pulp of A. diversifolia. With respect to the peel, Madeira’s cultivar showed the best result with an EC50 of 0.18 mg/mL, whereas Perry Vidal, Funchal and Mateus II cultivars showed an EC50 2.1, 1.8 and 1.3 times higher than Madeira’s cultivar, respectively. Our results are consistent with the results reported in the literature, as many of the bioactive compounds present in fruits are located in the peel (Balasundram, Sundram, & Sammar, 2006). Loizzo et al. (2012) found an EC50 of 0.06 mg/mL for the fruit peel of A. cherimola Mill., but the cultivar is not identified. Nonetheless, the reported results are in agreement with our study, since the peel of cherimoya showed higher antioxidant activity than the pulp. For the analysed seeds, the cultivar with the highest EC50 was Perry Vidal (4.24 mg/mL), followed by Mateus II (3.22 mg/mL) and Madeira (3.19 mg/mL). When comparing the EC50 values reported by Lima, Pimenta, and Amelia (2010) for the ethanolic extracts of fruit seeds from Annona cornifolia, with the results found in the present study the antioxidant activity was lower. For comparative purposes, the standard controls, L-ascorbic acid, gallic acid and BHT, often used as antioxidants for food and cosmetics were also tested. The highest EC50 was found for BHT (0.854 mg/mL) followed by L-ascorbic acid (0.004 mg/mL) and gallic acid (0.002 mg/mL). The EC50 value of the ethanolic extracts of the fruit peel of A. cherimola Mill. Madeira cultivar was 5 times higher than the EC50 of BHT, indicating a higher antioxidant activity than BHT. 3.1.2. Total phenolics The concentration of total phenolics in fruit pulp, peel and seeds of A. cherimola Mill. are presented in Table 1. For the analysed pulps, the total phenolics content ranged from 3.06 to 12.0 mg GAE/100 g of edible portion, for Funchal and Madeira cultivars, respectively. The highest total phenolics value for the peel was found for Mateus II cultivar (19.6 mg GAE/100 g of sample). The seeds of the analysed cultivars showed similar values, varying between 3.35 mg GAE/100 g (Perry Vidal cultivar) and 4.16 mg GAE/100 g (Mateus II cultivar). The pulp of Madeira cultivar has total phenolics content about 3–4 times higher than those found for the other cultivars analysed in the present study. These concentrations are lower than those reported in the literature for the fruit pulp of Annona muricata, 42 mg GAE/100 g (Lako et al., 2007) and 120 mg GAE/100 g (Hassimoto, Genovese, & Lajolo, 2005).

Table 1 DPPH radical scavenging activity (EC50), total phenolics and total flavonoids content of different parts of Annona cherimola Mill. fruit. Part of the fruit Pulp

Peel

Seeds

Cultivar Funchal Madeira Mateus II Perry Vidal Funchal Madeira Mateus II Perry Vidal Madeira Mateus II Perry Vidal

EC50A (mg/mL) a

4.52 ± 0.01 0.97b ± 0.01 3.45c ± 0.01 4.59a ± 0.06 0.33d ± 0.00 0.18d ± 0.00 0.23d ± 0.00 0.37d ± 0.01 3.19e ± 0.08 3.22c,e ± 0.08 4.24f ± 0.31

Total phenolics (mg GAEB/100 g) a

3.06 ± 0.03 12.0b ± 0.04 4.31c ± 0.02 3.43d ± 0.03 18.5e ± 0.10 19.5f ± 0.13 19.6f ± 0.15 17.0g ± 0.12 3.61d,h ± 0.02 4.16c,i ± 0.02 3.35d,j ± 0.01

Total flavonoids (mg ECEC/100 g) 1.31a ± 0.02 15.0b ± 0.01 3.19c ± 0.01 1.33a ± 0.01 33.0d ± 0.04 44.7e ± 0.10 41.9f ± 0.05 36.7g ± 0.04 4.62h ± 0.02 3.06i ± 0.02 6.75j ± 0.05

Values are average of three individual samples (n = 3), expressed as mean ± standard deviation. Different letters (a, b, c) within columns denote statistically significant differences (P < 0.05) by Tukey test. For pulp data are expressed as mg/100 g of edible portion in fresh weight basis. For peel and seeds data are expressed as mg/100 g of sample in fresh weight basis. A EC50, concentration of the sample that decreases the initial DPPH absorbance by 50%. B GAE, gallic acid equivalents. C ECE, epicatechin equivalents.

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3.1.3. Total flavonoids Comparing with the other analysed cultivars, Madeira’s cultivar showed the highest total flavonoids content in pulp and peel (Table 1). For the analysed seeds the values for total flavonoids varied between 3.06 to 6.75 mg ECE/100 g for Mateus II and Perry Vidal cultivars, respectively. Similar results were reported by Onyechi, Ibeanu, Eme, and Kelechi (2012), for the fruit pulp of A. muricata (9.32 mg ECE/100 g). Julián-Loaeza et al. (2011) presented higher contents for total flavonoids of A. diversifolia pulp, ranging between 107.4 and 152.0 mg of catechin/100 g. 3.2. Proximates Table 2, presents the limits of detection and quantification for each of the components analysed within our study. In general, the pulp of cherimoya is rich in water and has lower contents of total protein and total fat when compared with other fruits (Amoo et al., 2008). Therefore, this type of fruit has low energy content, but it is considered a good source of bioactive compounds, such as, phenolics and carotenoids. The energy content of the fruit pulps varied between 81 kcal (345 kJ) and 102 kcal (429 kJ) per 100 g of edible portion, for Perry Vidal and Madeira cultivars, respectively. Water is essential to maintain homeostasis in the body and to allow nutrient transport to cells, as well as removing waste products of metabolism (Thomas, 2001). The moisture content of the analysed cultivars from A. cherimola Mill. fruits was in the range 71.5–78.3 g/100 g of edible portion (Table 3). The lowest ash content was found for Funchal cultivar (0.372 ± 0.0 g/100 g of edible portion) and the highest was for Madeira cultivar (0.556 ± 0.0 g/100 g of edible portion). Amoo et al. (2008) presented lower values (0.25 g/100 g of sample) for the ash content in the fruit juice of A. cherimola Mill. collected in Nigeria. Julián-Loaeza et al. (2011) reported similar values for the ash content in the pulp of A. diversifolia collected in Mexico (0.89; 0.92 and 0.94 g/100 g of edible portion, for deep pink, pink and white varieties, respectively). For the pulp of A. muricata from Brazil, Souza, Vieira, Silva, and Lima (2011) reported an ash content of 0.48 g/100 g. Despite the different species of cherimoya and the different geographic origins, values reported in the literature are similar to the values presented in our study. Protein is the structural component of all cells in the body, and a moderate protein intake could easily prevent weight gain more than reducing fat or carbohydrates, because it can increase shortterm satiety and suppress food intake (Thomas, 2001). The highest

Table 2 List of components with limits of detection (LOD) and quantification (LOQ) used for the analyses of Annona Cherimola Mill. fruit and its by-products. Components

LOD

LOQ

Moisture Ash Total N (protein) Total fat Total dietary fibre Ascorbic acid Vitamin E (a-tocopherol) Retinol Lutein Zeaxanthin b-cryptoxanthin Lycopene a-carotene b-carotene

0.03 g/100 g 0.03 g/100 g 0.03 g/100 g 0.03 g/100 g 0.1 g/100 g 0.044 mg/100 g 0.03 mg/100 g 2 lg/100 g 6.2 lg/100 g 3.2 lg/100 g 3.2 lg/100 g 2.2 lg/100 g 2.8 lg/100 g 2.0 lg/100 g

0.1 g/100 g 0.1 g/100 g 0.1 g/100 g 0.1 g/100 g 0.4 g/100 g 0.115 mg/100 g 0.12 mg/100 g 6.2 lg/100 g 22 lg/100 g 12 lg/100 g 12 lg/100 g 8.2 lg/100 g 9.2 lg/100 g 6.2 lg/100 g

Values are average of three individual samples (n = 3), expressed as mean ± standard deviation. LOD, Limit of detection. LOQ, Limit of quantification.

Table 3 Nutritional composition and energy value (per 100 g of edible portion in fresh weight basis) of the four analysed cultivars of Annona cherimola Mill. fruits pulp. Components

Energy (kJ (kcal)) Moisture (g) Ash (g) Total protein (g) (NCF = 6.25) Total fat (g) Available carbohydrates (g) Total dietary fibre (g)

Cultivars Funchal

Madeira

Mateus II

Perry Vidal

376 (89) 76.1 ± 0.9 0.372 ± 0.0 1.96 ± 0.0

429 (102) 71.5 ± 0.0 0.556 ± 0.0 1.48 ± 0.1

378 (89) 75.9 ± 0.3 0.547 ± 0.0 1.78 ± 0.0

345 (81) 78.3 ± 0.1 0.510 ± 0.0 1.36 ± 0.0

0.176 ± 0.0 18.2 ± 0.9

0.103 ± 0.0 21.1 ± 0.2

0.133 ± 0.0 18.8 ± 0.4

0.156 ± 0.0 17.6 ± 0.1

3.21 ± 0.1

5.32 ± 0.2

2.84 ± 0.1

2.09 ± 0.0

Values are average of three individual samples (n = 3), expressed as mean ± standard deviation. NCF, Nitrogen conversion factor.

total protein content per 100 g of edible portion found in the analysed cultivars was for Funchal cultivar (1.96 g/100 g of edible portion), followed by Mateus II cultivar (1.78 g/100 g of edible portion), Madeira cultivar (1.48 g/100 g of edible portion) and Perry Vidal cultivar (1.36 g/100 g of edible portion). In the literature, Julián-Loaeza et al. (2011) reported lower protein values for the fruit pulp of A. diversifolia (0.89–1.14 g/100 g) and Souza et al. (2011) presented values for the pulp of A. muricata (1.09 g/ 100 g). Moreover, USDA National Nutrient Database (U.S., 2010) and Saxholt et al. (2008) presented values of 1.57 g/100 g and 1.7 g/100 g of edible portion, respectively for the pulp of A. cherimola Mill. Other food composition databases have reported values for cherimoya nutritional composition but a detailed comparison was not possible since the species are not identified. The total fat content was the lowest of all the analysed components in our study, varying between 0.103 ± 0.0 g/100 g of edible portion for Madeira cultivar and 0.176 g/100 g of edible portion for Funchal cultivar (Table 3). However, our findings are similar with the values reported in USDA Nutrient Database (U.S., 2010) (0.68 g/100 g) and Saxholt et al. (2008) (0.6 g/100 g). Available carbohydrates in the analysed samples varied between 18.2 ± 0.9 and 21.1 ± 0.2 g/100 g of edible portion. Julián-Loaeza et al. (2011) have reported values but for the total carbohydrates content of A. diversifolia Safford fruit pulp, pink and deep pink varieties (18.4 ± 0.1 and 20.3 ± 0.1 g/100 g of edible portion, respectively), whereas the pulp from white variety of A. diversifolia has a lower content (13.6 ± 0.2 g/100 g of edible portion) when compared with the results obtained within our study. In the work developed by Souza et al. (2011), the total carbohydrates content was lower (12.99 g/100 g of edible portion) for the pulp of A. muricata L. than for A. cherimola Mill. fruit under study. Fruits and vegetables are important contributors to the intake of dietary fibre and provide about one-third of the total intake (Thomas, 2001). According to our results (Table 3), the pulp of A. cherimola Mill. fruit (Madeira cultivar) had the highest total dietary fibre content (5.32 g/100 g of edible portion) and Perry Vidal cultivar had the lowest content (2.09 g/100 g of edible portion). The total dietary fibre content is in agreement with the values reported in the literature for the same species. Unfortunately, it was not possible to compare the present data with other cherimoya species due to a lack of values in the literature and because the existent values are presented as crude fibre. 3.3. Total vitamin C The content of vitamin C in foods is extremely important to understand the relationship of dietary intake and human health

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Table 4 Total vitamin C, L-ascorbic acid and dehydroascorbic acid content of four analysed cultivars of Annona cherimola Mill. fruit. Part of the fruit Pulp

Peel

Seeds

Cultivar Funchal Madeira Mateus II Perry Vidal Funchal Madeira Mateus II Perry Vidal Funchal Madeira Mateus II Perry Vidal

L-Ascorbic

acid (mg/100 g)

a

0.795 ± 0.0 0.485b ± 0.0 1.33c ± 0.0 1.25c ± 0.1 0.544b ± 0.0 0.227d ± 0.0 3.22e ± 0.1 4.41f ± 0.1 1.58g ± 0.0 1.17c,h ± 0.1 0.665i ± 0.1 0.782ª,j ± 0.1

Dehydroascorbic acid (mg/100 g) a

1.33 ± 0.1 6.24b ± 0.1 3.16c ± 0.3 1.38d ± 0.1 3.65e ± 0.0 4.22c ± 0.1 1.88f ± 0.1 0.755g ± 0.1 0.292h ± 0.0 0.862g ± 0.1 0.894g ± 0.1 0.761g ± 0.1

Total vitamin C (mg/100 g) 2.13a ± 0.1 6.73b ± 0.1 4.49c ± 0.3 2.63d ± 0.1 4.19e ± 0.0 4.45c ± 0.2 5.10f ± 0.0 5.17f ± 0.1 1.87g ± 0.0 2.03a,g ± 0.1 1.56h ± 0.0 1.54h ± 0.1

Values are average of three individual samples (n = 3), expressed as mean ± standard deviation. Different letters (a, b, c) within columns denote statistically significant differences (P < 0.05) by Tukey test. For pulp data are expressed as mg/100 g of edible portion in fresh weight basis. For peel and seeds data are expressed as mg/100 g of sample in fresh weight basis.

(Valente et al., 2011). Simultaneous detection of L-ascorbic acid and dehydroascorbic acid is very difficult, due to the low absorption of dehydroascorbic acid in the ultraviolet range of the spectrum (Spínola, Mendes, Câmara, & Castilho, 2012). Therefore, usually it is determined indirectly, by difference of total vitamin C (obtained after dehydroascorbic acid reduction) and the L-ascorbic acid content of the sample. In the present study, the concentration of the TCEP reducing agent was optimised (1.25; 2.5; 5.0 and 10.0 mM), and the best results were obtained with the concentration of 5 mM. The dietary reference intake (DRI) for vitamin C is 90 mg/day for males and 75 for females (Institute of Medicine & Food & Nutrition Board, 2013). Considering a serving size of 160 g/day, cherimoya pulp from Annona Cherimola Mill. Madeira cultivar can contribute 12% and 14% of DRI for vitamin C for adult males and females, respectively. The total vitamin C, L-ascorbic acid and dehydroascorbic acid results are shown in Table 4, and the limits of detection and quantification for ascorbic acid are presented in Table 2. Concentrations of total vitamin C in the analysed pulps of the four cultivars of A. cherimola Mill. fruits varied between 2.13 and 6.73 mg/100 g of edible portion, for Funchal and Madeira cultivars, respectively. However, in comparison with the other parts of the fruit, the pulp showed lower values of L-ascorbic acid, ranging from 0.485 to 1.33 mg/100 g of edible portion, for Madeira and Mateus II cultivars, respectively. The only exception was found in the peel from Madeira cultivar that presented the lowest L-ascorbic acid content (0.227 mg/100 g of edible portion). In the literature, there are some values reported for vitamin C content of pulps from A. cherimola Mill. species. USDA National Nutrient Database (U.S., 2010) reported a value of 12.6 mg/100 g for total ascorbic acid content of pulps from A. cherimola Mill., Saxholt et al. (2008) presented a L-ascorbic acid content of 11.5 mg/100 g for the same species. Julián-Loaeza et al. (2011) reported a range of values that varied between 1.51 and 2.38 mg/100 g of pulp from A. diversifolia fruits, but a spectrophotometric method was used for the determination of these values. In the literature, spectrophotometric methods are recognised as giving underestimate values, thereafter liquid chromatographic methods have gained popularity due to its high throughput and accuracy over spectrophotometric methods (Chebrolu et al., 2012). For the pulp of A. muricata, Souza et al. (2011) reported a vitamin C content of 64.4 mg/100 g, but it was determined using a titration methodology. The peel of Perry Vidal cultivar presented the highest content of L-ascorbic acid (4.41 mg/100 g) from all the analysed samples. The mean content of total vitamin C quantified in seeds, was approximately 2 times lower than for the other parts (peel and pulp).

We have quantified higher amounts of dehydroascorbic acid than L-ascorbic acid in some parts of the fruit. This is in accordance with Spínola et al. (2012) who concluded that cherimoya had one of the highest percentages of dehydroascorbic acid in relation to total vitamin C among several fruits and vegetables. In the literature, several studies verified that L-ascorbic acid significantly decreases with time, beginning to be degraded immediately after harvesting and continuing during storage. In the present study, in order to minimise the losses, a stabilization solution was added to the samples. Therefore, the differences found among our results and those achieved by other authors may be related with other factors, such as maturity degree and cultivar. Another aspect to take into account when determining vitamin C is the reducing agent used. In the present study, TCEP has been used as reducing agent, however, in the literature other agents such as dithiothreitol or bmercaptoethanol have been used. According to Chebrolu et al. (2012) these two reducing agents are not as efficient in the reduction of ascorbic acid as TCEP. 3.4. Carotenoids, retinol equivalents activity and vitamin E Cherimoya cultivars from Madeira Island were also analysed by UHPLC, in order to determine their content in carotenoids, vitamin E and their provitamin A activity. The applied method allows chromatograms with high resolution in the separation of six carotenoids and two vitamins (Fig. 1A and B). Despite the difficulty in the separation of zeaxanthin and lutein peaks, due to the fact that these carotenoids differ only in the position of the double bond in one of the end rings (Rivera, Villaró & Canela, 2011), in the present study a good separation was obtained (Fig. 1A). Typical chromatograms (without and with saponification) of the carotenoids of the peel of A. cherimola Mill. Madeira’s cultivar are shown in Fig. 1(C and D). In Table 2, the limits of detection and quantification for each of the analysed carotenoids are shown. As far as we know, up to now this is the first study that presents the content of carotenoids of A. cherimola Mill. for four cultivars (Funchal, Madeira, Mateus II and Perry Vidal) and their by-products, therefore, it was not possible to make comparisons with data from other studies. Carotenoids content in fruits and vegetables depends on several factors such as cultivar, maturity, postharvest storage, processing and preparation (Quirós & Costa, 2006). The results obtained with respect to carotenoids content (Table 5), indicate that neither pulp nor seeds of cherimoya have carotenoids that could be quantified under the present study. Nonetheless, in the peel of the analysed samples it was possible to quantify three carotenoids (lutein, b-cryptoxanthin and b-carotene). Saponification allowed the hydrolysis of carotenoid esters

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Fig. 1. Chromatograms of carotenoids and vitamin E stock standard solution (A and B). (1) lutein, (2) zeaxanthin, (3) b-cryptoxanthin, (4) lycopene, (5) a-carotene, (6) bcarotene, (7) a-tocopherol. (A) k = 450 nm. (B) k = 295 nm. AU – absorbance. Chromatogram of the analysed fruit peel from A. cherimola Mill. Madeira cultivar. (C) Without saponification. (D) With saponification. k = 450 nm. AU – absorbance. Table 5 Carotenoids and retinol activity equivalents (lg/100 g) of the fruit peel from four cultivars of Annona cherimola Mill. Cultivars

Saponification

Lutein Mean ± SD (lg/100 g)

b-cryptoxanthin Mean ± SD (lg/100 g)

b-carotene Mean ± SD (lg/100 g)

RAE Mean (lg/100 g)

Funchal Madeira Mateus II Perry Vidal

Without

137ABC ± 15 172AB ± 3 232A ± 26 129C ± 4

n.d. n.d. n.d. n.d.

87.5B ± 5 139AB ± 14 174A ± 11 87.3B ± 9

7.29B 11.6AB 14.5A 7.28B

Funchal Madeira Mateus II Perry Vidal

With

33.3a ± 1 40.7a ± 4 61.6a ± 12 39.0a ± 2

14.5a ± 1 12.2a ± 1 12.3a ± 1 n.d.

84.8ab ± 9 98.3a ± 3 117a ± 21 64.7b ± 3

7.67ab 8.66a 10.3a 5.39b

n.d., not detected (<3.2 lg/100 g). Values are average of three individual samples (n = 3), expressed as mean ± standard deviation. RAE, retinol activity equivalents. Different letters (A, B, C) within columns from samples without saponification denote statistically significant differences (P < 0.05) by Tukey test. Different letters (a, b, c) within columns from samples with saponification denote statistically significant differences (P < 0.05) by Tukey test.

and the removal of chlorophyll and other substances that could interfere with the analysis (Inbaraj et al., 2008). However, as previously mentioned by Hart and Scott (1995), this procedure can sometimes result in the loss of some carotenoids. Therefore, both extraction procedures (with and without saponification) were carried out for all the analysed samples. Lutein and b-carotene showed considerable losses after saponification, like it was reported in other matrices (Oliver, Palou, & Pons, 1998). These losses occur mainly during the washing with potassium hydroxide, due to the formation of an emulsion. However, in our study the saponification process allowed quantification of b-cryptoxanthin in the analysed samples. The lutein content in the analysed samples ranged from 129 ± 4 to 232 ± 26 lg/100 g, for samples without saponification, and from 33.3 ± 1 to 61.6 ± 12 lg/100 g for samples with saponification. The highest content of lutein was observed in Mateus II cultivar and it was the most abundant carotenoid in all the analysed samples. Isabelle et al. (2010) have reported a lutein content of 6.0 lg/100 g in the pulp of A. muricata, without saponification and Murillo, Meléndez-Martínez, and Portugal (2010) presented 230 lg/100 g for the pulp of Annona purpurea, with

saponification. In the USDA National Nutrient Database (U.S., 2010) a content of 6.0 lg/100 g for the pulp of A. cherimola Mill. is reported, but it is the sum of lutein and zeaxanthin carotenoids. From the three identified carotenoids, b-cryptoxanthin was the less abundant carotenoid in the peel of A. cherimola Mill. fruit being only possible to quantify after saponification. The values for b-cryptoxanthin were very similar among the different analysed cultivars, varying between 12.2 and 14.5 lg/100 g for Madeira and Funchal cultivars. In our study, for the peel of A. cherimola Mill., the content was ten times higher than the value described in the USDA National Nutrient Database (U.S., 2010) for the pulp of the same species. In the literature, b-cryptoxanthin content of the pulp of A. muricata fruit was 5 lg/100 g (without saponification) (Isabelle et al., 2010). According to our results, saponification proved to be essential for the quantification of b-cryptoxanthin, since as we can observe in Fig. 1C two great peaks appear in the chromatogram and one of these has the same retention time of b-cryptoxanthin, but the spectrum does not match with the standard. We have carefully checked this situation and it seems that those peaks correspond to chlorophylls.

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Table 6 Vitamin E (a-tocopherol) content (mg/100 g) of the four cultivars of Annona cherimola Mill. Cultivars

Saponification

Vitamin E (a-tocopherol) Peel Mean ± SD (mg/100 g)

Pulp Mean ± SD (mg/100 g)

Seeds Mean ± SD (mg/100 g)

Funchal Madeira Mateus II Perry Vidal

Without

0.48A ± 0.06 0.50A ± 0.06 0.51A ± 0.12 0.62A ± 0.04

0.22A ± 0.01 0.23A ± 0.01 0.15B ± 0.01 0.071C ± 0.00

n.a. 0.74A ± 0.11 0.80A ± 0.11 0.34B ± 0.05

Funchal Madeira Mateus II Perry Vidal

With

0.24a ± 0.02 0.28a ± 0.02 0.30a ± 0.05 0.36a ± 0.09

0.13a ± 0.03 0.11a ± 0.01 0.069b ± 0.01 0.056b ± 0.00

n.a. 1.0a ± 0.04 1.1a ± 0.07 0.76a ± 0.13

n.a., not analysed. Values are average of three individual samples (n = 3), expressed as mean ± standard deviation. For pulp data are expressed as mg/100 g of edible portion in fresh weight basis. For peel and seeds data are expressed as mg/100 g of sample in fresh weight basis. Different letters (A, B, C) within columns from samples without saponification denote statistically significant differences (P < 0.05) by Tukey test. Different letters (a, b, c) within columns from samples with saponification denote statistically significant differences (P < 0.05) by Tukey test.

With respect to b-carotene, the values ranged from 87.3 ± 9 to 174 ± 11 lg/100 g, without saponification, and from 64.7 ± 3 to 117 ± 21 lg/100 g with saponification. Mateus II cultivar presented the highest content and Perry Vidal the lowest for both procedures. Isabelle et al. (2010) analysed the pulp of A. muricata fruits without saponification and reported a value of 5 lg/100 g, while in the USDA National Nutrient Database (U.S., 2010) a value of 2 lg/ 100 g is reported for the fruit pulp of A. cherimola Mill. Vitamin A (retinol) plays a role in a variety of functions throughout the body, such as, visual system, immune, growth and reproduction function and its dietary requirements can be supplied by provitamin A carotenoids in fruit. Vitamin A activity is expressed as RAE, where 1 RAE is equivalent to 1 lg retinol, 2 lg b-carotene dissolved in oil, 12 lg b-carotene or 24 lg of other provitamin A carotenoids (Institute of Medicine, Food & Nutrition Board, 2013). The highest amount of RAE (Table 5) was found for Mateus II cultivar (14.5 lg/100 g), followed by Madeira cultivar (11.6 lg/100 g), Funchal cultivar (7.29 lg/100 g) and Perry Vidal cultivar (7.28 lg/100 g). Institute of Medicine, Food and Nutrition Board (2013) established a DRI for vitamin E of 15 mg/day for adult males and females. For cherimoya pulp, consumption of 160 g/day can supply 2.5% of the vitamin E daily requirement. Vitamin E is a fat-soluble vitamin that includes eight forms: four tocopherols and four tocotrienols. Of the many different forms of vitamin E, a-tocopherol is the most abundant form in nature and has the highest biological activity (Herrera & Barbas, 2001). According to the obtained results (Table 6), in most samples, saponification resulted in the loss of a-tocopherol, except for the fruit seeds of A. cherimola Mill. The limits of detection and quantification for a-tocopherol are presented in Table 2. For the analysed pulps, a-tocopherol content ranged between 0.071 and 0.23 mg/ 100 g of edible portion (without saponification), while in the peel values varied from 0.48 to 0.62 mg/100 g of sample (without saponification). The highest a-tocopherol content (1.1 ± 0.07 mg/ 100 g) was found for the seeds from Mateus II cultivar (with saponification). Isabelle et al. (2010) have reported a a-tocopherol content of 0.012 mg/100 g in the pulp from A. muricata (without saponification) and USDA National Nutrient Database (U.S., 2010) presented a content of 0.27 mg/100 g of edible portion for the pulp of A. cherimola Mill.

4. Conclusions In the present study a detailed analysis of the antioxidant activity, nutritional composition and bioactive compounds of the

fruits of four A. cherimola Mill. cultivars including its by-products has been performed. As far as we know, up to now, this is the first study that reports data on these components for the studied cultivars (Funchal, Madeira, Mateus II and Perry Vidal). According to the obtained results, this species of cherimoya, which is registered as PDO, has great antioxidant potential, especially its by-products. Moreover, considerable amounts of bioactive compounds, namely, carotenoids and vitamins were present in this fruit. Further studies on isolation and characterisation of individual phenols and flavonoids would be a valuable contribution for the characterisation of functional properties of this species. These results highlight A. cherimola Mill. antioxidant properties, especially from its byproducts and support their employment as added value natural extracts in cosmetic, pharmaceutical and food processing industries. Furthermore, this work will contribute to maintain the biodiversity and to promote the sustainable development and exploitation of traditional fruits of Madeira’s Island. Acknowledgements Authors would like to thank ‘‘Terra Cidade’’ company and ‘‘Direcção Regional de Agricultura e Desenvolvimento Rural’’ from Madeira Island, which kindly provided cherimoya cultivars. This work was funded by National Institute of Health Dr. Ricardo Jorge (INSA), I.P. under the project ‘‘Bioactive compounds and its potential health benefits’’ (2012DAN730). Tânia Gonçalves Albuquerque is grateful for the research grant (BRJ/DAN-2012) funded by INSA. References Albuquerque, T. G., Sanches-Silva, A., Santos, L., & Costa, H. S. (2012). An update on potato crisps contents of moisture, fat, salt and fatty acids (including trans-fatty acids) with special emphasis on new oils/fats used for frying. International Journal of Food Sciences and Nutrition, 63, 713–717. Amoo, I. A., Emenike, A. E., & Akpambang, V. O. E. (2008). Compositional evaluation of Annona cherimoya (Custard Apple) fruit. Trends in Applied Sciences Research, 3(2), 216–220. AOAC. (2000). Association of official analytical chemists. In Official methods of analysis chemists (17th ed.). Washington, DC. Balasundram, N., Sundram, K., & Sammar, S. (2006). Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chemistry, 68, 191–203. Barreca, D., Laganà, G., Ficarra, S., Tellone, E., Leuzzi, U., Galtieri, A., & Bellocco, E. (2011). Evaluation of the antioxidant and cytoprotective proprieties of the exotic fruit Annona cherimola Mill. (Annonaceae). Food Research International, 44, 2302–2310. Chebrolu, K. K., Jayaprakasha, G. K., Yoo, K. S., Jifon, J. L., & Patil, B. S. (2012). An improved sample preparation method for quantification of ascorbic acid and dehydroascorbic acid by HPLC. Food Science and Technology, 47, 443–449. Costa, H. S., Vasilopoulou, E., Trichopoulou, A., Finglas, P., & Participants of EuroFIR Traditional Foods Work Package (2010). New nutritional data on traditional foods for European food composition databases. European Journal of Clinical Nutrition, 64, 73–81.

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