Proﬁling of lipophilic and phenolic phytochemicals of four cultivars from cherimoya ( Annona cherimola

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Introduction
Annona, a plant genus from the family Annonaceae, comprises 119 species of which Annona cherimola Mill., Annona muricata L., Annona squamosa L., Annona reticulata L. and the interspecific hybrid A. squamosa L. Â A. cherimola Mill. are of significant commercial importance (Pareek, Yahia, Pareek, & Kaushik, 2011;Pinto et al., 2005). Among these, A. cherimola Mill., commonly known as cherimoya, is the one with the strongest consumer demand (Pinto et al., 2005). Although native from South America and Antilles, A. cherimola is now cultivated in several tropical and subtropical areas around the world. Spain, Peru and Chile are the main producers of cherimoya while small production areas exist in California, Israel and Madeira Island, Portugal (Pinto et al., 2005).
The cherimoya shrub or small-tree is well adapted to the edaphoclimatic conditions of Madeira Island, Portugal, where the estimated production in 2014 was 1104 ton in an area of 115 ha (DRE, 2015), supporting the Portuguese demand for this fruit. After cherimoya introduction in the island in 1897 (Arun Jyothi, Venkatesh, Chakrapani, & Roja Rani, 2011), propagation by seeds prevailed and originated diverse vigorous plants from which cultivars were developed and improved (Nunes, 1997). Nowadays, there are several cherimoya cultivars, being 'Madeira', 'Perry Vidal', 'Mateus I' and 'Funchal' the most important, with a high potential to be commercialized in national and international markets. Fruits are heart-shaped, the skin is thin and delicate, differing in coloration at maturity being yellowish-green in ''Funchal" and brownish-green in the others cultivars (Agripérola Cooperativa Agrícola CRL, 1998;Caldeira, Araujo, & Nunes, 1995).
However, there is limited and/or absent data regarding the characterization of cherimoya fruits pulp from 'Perry Vidal', 'Mateus I', 'Mateus III' and 'Funchal' cultivars. As far as we could ascertain, there is only one study reporting the volatile composition of cherimoya fruits from these cultivars (Ferreira et al., 2009).
The increasing recognition of cherimoya nutritional value highlights the importance of this fruit as a valuable supplement for diets, as well as for industrial applications. In addition to its nutritional value, the lipophilic and phenolic profiles of this fruit can be useful to determine its economic and health potential. Hence, and given that the phytochemicals or phytonutrients are dependent on the cultivar, geographic origin or climacteric conditions, the present study aims to evaluate the lipophilic and phenolic fractions composition of the ripe mesocarp of four cherimoya cultivars from Madeira Island, namely 'Perry Vidal', 'Mateus I', 'Mateus III' and 'Funchal', by gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis, respectively.

Samples preparation and physicochemical parameters
Cherimoya (Annona cherimola Mill.) without evidence of physical or pathological injuries were selected from Centro de Fruticultura Subtropical do Funchal, Madeira Island, Portugal. Mature green fruits from 'Perry Vidal', 'Mateus I', 'Mateus III' and 'Funchal' cultivars were hand harvested in January 2015 (winter season) and then left to reach full ripeness at room temperature (20-23°C). Ripe fruits (n = 6) were then peeled (peel was fully discarded), sliced and quick-frozen in liquid nitrogen. Frozen samples were lyophilized, milled to pass through a 40-60 mesh sieve and stored (humidity ca. 5%) in a freezer at À18°C for further analyses.
Fruit firmness was determined after removing the skin on two opposite sides (n = 12) in the middle of each fruit using a pressure-testing instrument (Model FT 327) fitted with an 11.3 mm cylindrical plunger. The force required to penetrate into the flesh was expressed in Newtons (N). Fresh slices of each sample (n = 6) were used to measure fruit water content through a Gibertini-Eurotherm balance at 105°C, as well as to determine the total soluble solids (TSS) or Brix percentage in a digital brix refractometer from ATAGO.
Data are reported as mean ± SD and analysed by one way analysis of variance (ANOVA) to determine differences between means at 5% confidence level. Statistical analyses were performed using SPSS v 23 for Windows.

Lipophilic and phenolic compounds extraction
Three grinded fruits (20 g) of each cultivar were Soxhlet extracted with dichloromethane during 6 h. The solvent was evaporated to dryness by low-pressure evaporation, the lipophilic extracts were weighted and the results were expressed in percent of dry material (% DW).
Subsequently, the solid residues from the dichloromethane extraction were suspended (m/v 1:100) in a methanol/water (MeOH:H 2 O, 50:50 v/v) mixture, at room temperature for 24 h, under constant stirring. Then, the suspension was filtered, MeOH removed by low-pressure evaporation, and the phenolic extracts freeze-dried. The extraction yields were expressed in percent of dry material (% DW) .

GC-MS analysis
Before GC-MS analysis, two aliquots of each dried lipophilic extract (20 mg each) and an accurate amount of internal standard (tetracosane, 0.50 mg) were dissolved in 250 lL of pyridine. The compounds containing hydroxyl and carboxyl groups were converted into their trimethylsilyl (TMS) ethers and esters derivatives, respectively, by adding 250 lL of N,O-bis(trimethylsilyl)trifluoroa cetamide and 50 lL of trimethylchlorosilane, standing the mixture at 70°C for 30 min (Freire, Silvestre, Neto, & Cavaleiro, 2002). The derivatized extracts were analyzed by GC-MS on a Trace Gas Chromatograph 2000 Series, equipped with a Thermo Scientific DSQ II single-quadrupole mass spectrometer. The following conditions were used: electron impact energy 70 eV; collection rate: 1 scan s À1 ; ion source temperature: 250°C; m/z range: 33-700. The column used was a DB-1 J&W capillary column (30 m Â 0.32 mm inner diameter, 0.25 lm film thickness) and helium was used as carrier gas (35 cm s À1 ). The chromatographic conditions were as follows: initial temperature: 80°C for 5 min; temperature gradient: 4°C min À1 ; final temperature: 260°C; temperature gradient: 2°C min À1 ; final temperature: 285°C for 8 min; injector temperature: 250°C; transfer-line temperature: 290°C; split ratio: 1:33.
Compounds were identified as TMS derivatives by comparing their mass spectra with the GC-MS spectral library (Wiley-NIST Mass Spectral Library 1999), with data from literature (AOCS Lipid Library, 2013;Oliveira et al., 2005), and, in some cases, by the injection of standards.
For semi-quantitative analysis, GC-MS was calibrated with pure reference compounds, representative of the major lipophilic extractive families (stigmasterol, octadecanoic acid and nonadecan-1-ol) relative to tetracosane. The respective response factors were calculated as an average of six GC-MS runs. For tocopherol and kaurene diterpenes the response factor of stigmasterol was used. Each aliquot was injected in triplicate. The presented results are the average of the concordant values obtained for the six aliquots (less than 5% variation between injections of the same aliquot and between aliquots of the same cherimoya variety extracts).

ESI-MS n analysis
The HPLC was coupled to a LCQ Fleet ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA), equipped with an electrospray ionization source and operating in negative mode. The nitrogen sheath and auxiliary gas were 50 and 10 (arbitrary units), respectively. The spray voltage was 5 kV and the capillary temperature was 360°C. The capillary and tune lens voltages were set at À28 V and À115 V, respectively. CID-MS n experiments were performed on mass-selected precursor ions in the range of m/z 100-1500. The isolation width of precursor ions was 1.0 mass units and the scan time was equal to 100 ms and the collision energy was optimized between 15 and 40 (arbitrary units), using helium as collision gas. The data acquisition was carried out by using Xcalibur Ò data system (ThermoFinnigan, San Jose, CA, USA).

Phenolic compounds quantification by UHPLC-UV
Catechin was used as reference compound for quantitative analysis, with the UV detector monitored at 280 nm. Calibration curve was obtained by UHPLC injection of standard solutions in MeOH:H 2 O, with concentrations between 5 and 80 lg mL À1 . Besides the linearity, the limits of detection (LOD = 8.01 lg mL À1 ) and quantification (LOQ = 26.72 lg mL À1 ) were also estimated using the S/N approach (n = 5). The calibration curve obtained was y = 45783x À 68,057 (y = area, x = C (lg mL À1 ); R 2 = 0.9943). The concentrations were calculated based on triplicate injections and the mean value determined in each case (less than 5% variation between injections).

Physicochemical characterization of cherimoya fruit
Cherimoya fruits used in this study were comparable in weight, water content and firmness (Table 1). In general, fruit weight ranged from 522.1 g to 587.1 g in 'Funchal' and 'Perry Vidal', respectively. In what concerns water content, cultivars 'Mateus III' (66.7%) and 'Perry Vidal' (62.0%) were quite similar, showing a slightly higher content than 'Mateus I' (57.9%) and 'Funchal' (56.3%). The pulp firmness was evaluated in the mature stage, and 'Perry Vidal' displayed the highest pulp firmness with 1.09 N. Regarding the total soluble solids (TSS), which are predominantly made of sugars, the lowest value was observed for 'Funchal' (17.0°B rix) and the highest one for 'Perry Vidal' (26.5°Brix). All the values obtained are comparable to data reported elsewhere for other species and cultivars (Andrade, Zoghbi, Maia, Fabricius, & Marx, 2001;Arun Jyothi et al., 2011;Pareek et al., 2011), except the TSS of 'Perry Vidal' which indicates that this cultivar is sweeter than the others evaluated in this study, and in other previously studied varieties (Caldeira et al., 1995).
The ripe mesocarp of the four cherimoya cultivars studied here presented similar lipophilic extractives yields (Table 1) with values ranging from 0.57 to 0.94% of dry material for 'Mateus III' and 'Mateus I, respectively. These contents are of the same order of those found on the pulp of Annona muricata (Melot, Fall, Gleye, & Champy, 2009) and, also, on other tropical fruits, e.g. in the ripe pulp of mango fruits (Vilela et al., 2013) and in the unripe pulp of banana (Lúcia Oliveira, Freire, Silvestre, & Cordeiro, 2008).
The yields of the MeOH:H 2 O ripe mesocarp extracts of the studied cherimoya cultivars ranged between 58.53% of dry weight for 'Perry Vidal' and 72.21% for 'Mateus III' ( Table 1). The high yield of polar extractives obtained from fruit pulp was expectable, due to the presence of sugars or other water-soluble components, as suggested by the TSS values. In fact, an extraction yield of about 45% was already described for loquat fruit pulp (Delfanian, Esmaeilzadeh Kenari, & Sahari, 2015).

Composition of the lipophilic extracts
The chemical composition of the lipophilic extracts of the four cherimoya cultivars was studied by GC-MS analysis; the detailed identification and quantification of the main lipophilic compounds present in the ripe mesocarps are summarized in Table 2. The predominant compounds present in these extracts were two kaurene diterpenes, followed by a series of free fatty acids (C12 to C22), sterols and minor amounts of long-chain aliphatic alcohols (C16 to C30), among others. The relative abundance of the identified compounds and their families differ somewhat between cultivars, as depicted in Fig. 1.
Among the fatty acids family, polyunsaturated fatty acids, like octadeca-9,12-dienoic (x-6) and octadeca-9,12,15-trienoic (x-3) acids, are essential nutrients that must be obtained from the diet since they are not produced in the human body (Sánchez-Moreno, De Pascual-Teresa, De Ancos, & Cano, 2012). The role of fatty acids, particularly x-3 and x-6 fatty acids, in the human health is essentially related with the prevention, delay, or treatment of chronic and acute diseases, including cancer, cardiovascular diseases, osteoporosis, and immune disorders (Chen, McClements, & Decker, 2013;Simopoulos, 1999;Simopoulos, 2008). The studied cherimoya fruits can contribute to the intake of ca. 3.8 mg ('Perry Vidal') and 4.7 mg ('Funchal') per 100 g of fresh   fruit of x-3 and x-6 fatty acids, respectively. When compared with other fruits, the ripe cherimoya mesocarp has a higher content than e.g. mangoes (ca. 3.4 mg of x-3 and 0.9 mg of x-6 fatty acids per 100 g of fresh fruit) (Vilela et al., 2013), but a lower content than e.g. bananas (ca. 12.1 mg of x-3 and 5.9 mg of x-6 fatty acids per 100 g of fresh fruit) (Vilela et al., 2014). Notwithstanding, as expected, the content of x-3 and x-6 fatty acids in ripe cherimoya mesocarp is far from those found in well-known edible sources of these components, such as for example purslane and spinach (Simopoulos, 2004). Free sterols, a very important group of lipophilic compounds given their broad range of biological functions and activities (Piironen, Lindsay, Miettinen, Toivo, & Lampi, 2000), account for 9.6-23.7% of the identified lipophilic components of ripe cherimoya mesocarps. b-Sitosterol is the major component of this family (124-277 mg kg À1 DW) present in all mesocarp samples, representing between 48.8% ('Perry Vidal') and 58.7% ('Mateus III') of total sterols contents and between 5.2% ('Perry Vidal' and 'Funchal') and 13.9% ('Mateus III') of the total lipophilic extractives ( Table 2). Other sterols include stigmasterol (54-183 mg kg À1 DW) and campesterol (28-70 mg kg À1 DW). These three sterols have been detected in the fruits of the cultivar 'Madeira' , and b-sitosterol along with stigmasterol were isolated from the fresh fruits of A. glabra (Hsieh et al., 2004). Smaller amounts of 24-methylenecholesterol (0-3 mg kg À1 DW), fucosterol (4-18 mg kg À1 DW) and 24-methylenecycloartenol (6-13 mg kg À1 DW) were also identified in all cultivars investigated. As far as our literature survey could ascertain, these three sterols were identified for the first time in Annona species. Still, they were already reported in mangoes (Vilela et al., 2013). Noteworthy is the fact that these cherimoya fruits can contribute to the daily intake of free phytosterols with ca. 8.7-24.6 mg per 100 g of fresh fruit, which seem to be a practical and safe option for reducing cholesterol levels in the population as discussed elsewhere (Piironen et al., 2000;Quílez, García-Lorda, & Salas-Salvadó, 2003).
Additionally, other compounds like long-chain aliphatic alcohols (LCAA), 1-monopalmitin, squalene and a-tocopherol were also detected in the extracts, although in minor amounts (Table 2). Only four LCAA were identified, namely hexadecan-1-ol, octadecan-1-ol, octacosan-1-ol and triacontan-1-ol, representing between 15 and 48 mg kg À1 of dry material. The presence of LCAA in the cherimoya mesocarp has been previously identified in the fruits of the cultivar 'Madeira' . This class of compounds was reported to lower the plasma cholesterol in humans with their regular consumption (Hargrove, Greenspan, & Hartle, 2004). Squalene (Fig. 2), a natural polyunsaturated triterpene with the ability to inhibit the development of various tumours (Reddy & Couvreur, 2009), was only detected in significant amounts (136 mg kg À1 DW) in the cultivar 'Mateus I'. a-Tocopherol, the most bioactive form of vitamin E (Bramley et al., 2000), was the only tocopherol detected in the all the studied cherimoya mesocarps, accounting for 2-12 mg kg À1 DW, with the highest values observed for 'Mateus III' and 'Mateus I', respectively (Table 2). Lastly, the lipophilic extracts of all cherimoya mesocarps were also analyzed by GC-MS with a short length (15 m) column, using chromatographic conditions that enable the elution and detection of esterified fatty acids (diglycerides, triglycerides and steryl esters) and steryl glucosides (Freire et al., 2002). As an illustrative example, Fig. S1 (Supplementary material) shows the typical GC-MS chromatogram of the derivatized lipophilic extract of the ripe mesocarp of 'Mateus I' cultivar, obtained by a short length column. Only small amounts of steryl glucosides, namely campesteryl 3b-Dglucopyranose, stigmasteryl 3b-D-glucopyranoside and sitosteryl 3b-D-glucopyranoside (41.97, 42.22 and 42.67 min, respectively), and steryl esters were detected in the four cultivars. Worth noting is the fact that these compounds are also present in small amounts in fruits like bananas (Vilela et al., 2014) and mangoes (Vilela et al., 2013). In addition, stigmasteryl 3b-D-glucopyranoside along with sitosteryl 3b-D-glucopyranoside were isolated from the fresh fruits of A. glabra (Hsieh et al., 2004). The presence of free and esterified sterols is important to improve lipid metabolism and immune function (Moreau, Whitaker, & Hicks, 2002).

Composition of the phenolic extracts
The identification of the main components of the MeOH:H 2 O extracts of ripe mesocarp from the cherimoya cultivars 'Perry Vidal', 'Mateus', 'Mateus III' and 'Funchal' was carried out by UHPLC-DAD and UHPLC-MS n analysis. Table 3 summarizes the phenolic compounds identified in each extract, their retention time, the molecular ion [MÀH] À and the main product ions obtained by MS n . Compounds were identified by comparing their fragmentation profiles with reference compounds run under the same experimental conditions, or, when standard were not available, their identifications were corroborated with the literature, as indicated in Table 3 and discussed below.
The quantification of the phenolic compounds detected in the ripe mesocarp of the four cherimoya cultivars is presented in Table 4. 'Mateus I' and 'Mateus III' cultivars show the higher phenolic compounds content, accounting 9603 and 6299 mg kg À1 of dry weight, respectively. There is no information regarding the content of flavan-3-ols on cherimoya fruits, for comparative purposes. However, when compared with those described for the pulp of other tropical fruits, such as avocado (which also presented catechin derivatives and procyanidins as the major constituents), these contents are considerably higher (Rodríguez-Carpena, Morcuende, Andrade, Kylli, & Estévez, 2011). The phenolic fractions of the mesocarp of 'Mateus I' and 'Mateus III' cultivars are very similar, with procyanidin dimer isomer 7 as the major component, followed by a procyanidin trimer isomer 11 and epicatechin 6 ( Fig. 2). Besides the procyanidin isomers, it was also identified (epi)catechin-(epi) gallocatechin 3 and (epi)gallocatechin 5 in 'Mateus I' cultivar and (epi)afzelechin-(epi)catechin 10 in 'Mateus III' cultivar. Procyanidin dimer isomer 7 was also identified as the major component of the phenolic fraction of 'Funchal' cultivar mesocarp (706 mg kg À1 DW), followed by a procyanidin trimer isomer 11 (404 mg kg À1 DW) and procyanidin tetramer 12 and epicatechin 6 (both with 147 mg kg À1 DW). 'Perry Vidal' is the cultivar presenting the lowest  phenolic compounds content, accounting for 485 mg kg À1 DW. Epicatechin 6 (189 mg kg À1 DW), two procyanidin dimer isomers 2 and 4 (87 and 117 mg kg À1 DW, respectively) and a procyanidin trimer isomer 11 (92 mg kg À1 DW) were the only detected compounds in the phenolic fraction of this mesocarp. Reports on the role of these bioactive phenolic compounds on human health suggest a relevant contribution to prevent cardiovascular health problems (De Pascual-Teresa, Moreno, & García-Viguera, 2010). Higher intakes of flavan-3-ols and their polymers were also associated with a significant reduction in the concentration of oxidative stress biomarkers (Cassidy et al., 2015).

Conclusions
The lipophilic fractions of the four cherimoya cultivars used in this study are mainly composed of kaurane diterpenes, namely kaurenoic acid and kaurenol, followed by fatty acids and sterols.
In addition, the mesocarp of cherimoya fruits from the investigated cultivars also contains a high variety of flavan-3-ols, including both galloylated and non-galloylated components. Five phenolic compounds are identified for the first time as constituents of cherimoya fruits, viz. catechin, (epi)catechin-(epi)gallocatechin, (epi)gallocatechin, (epi)afzelechin-(epi)catechin and procyanidin tetramer. In general, the mesocarp of 'Mateus I' contains a higher quantity of lipophilic and phenolic compounds. Our results support the use of this exotic fruit as a rich source of health-promoting components, with the capacity to prevent or slow down the progress of various oxidative-stress related disorders.