Carotenoids, carotenoid esters, and anthocyanins of yellow-, orange-, and red-peeled cashew apples (Anacardium occidentale L.)

Food Chemistry 200 (2016) 274–282

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Carotenoids, carotenoid esters, and anthocyanins of yellow-, orange-, and red-peeled cashew apples (Anacardium occidentale L.) Ralf M. Schweiggert a,⇑, Ester Vargas b, Jürgen Conrad c, Judith Hempel a, Claudia C. Gras a, Jochen U. Ziegler a, Angelika Mayer a, Víctor Jiménez b,d, Patricia Esquivel e, Reinhold Carle a,f a

Chair of Plant Foodstuff Technology and Analysis, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany CIGRAS, Universidad de Costa Rica, 2060 San Pedro, Costa Rica Department of Bioorganic Chemistry, Institute of Chemistry, University of Hohenheim, 70599 Stuttgart, Germany d Food Security Center, University of Hohenheim, 70599 Stuttgart, Germany e School of Food Technology, Universidad de Costa Rica, 2060 San Pedro, Costa Rica f King Abdulaziz University, Faculty of Science, Biological Science Department, P. O. Box 80257, Jeddah 21589, Saudi Arabia b c

a r t i c l e

i n f o

Article history: Received 9 July 2015 Received in revised form 7 January 2016 Accepted 10 January 2016

Chemical compounds studied in this article: 7-O-Methylcyanidin 3-O-b-Dgalactopyranoside (PubChem CID: 101765178) a-Carotene (PubChem CID: 4369188) b-Carotene (PubChem CID: 5280489) b-Cryptoxanthin (PubChem CID: 44554791) Lutein (PubChem CID: 16061204) Zeaxanthin (PubChem CID: 5280899)

a b s t r a c t Pigment profiles of yellow-, orange-, and red-peeled cashew (Anacardium occidentale L.) apples were investigated. Among 15 identified carotenoids and carotenoid esters, b-carotene, and b-cryptoxanthin palmitate were the most abundant in peels and pulp of all samples. Total carotenoid concentrations in the pulp of yellow- and red-peeled cashew apples were low (0.69–0.73 mg/100 g FW) compared to that of orange-peeled samples (2.2 mg/100 g FW). The color difference between the equally carotenoid-rich yellow and red colored samples indicated the presence of a further non-carotenoid pigment type in red peels. Among four detected anthocyanins, the major anthocyanin was unambiguously identified as 7-O-methylcyanidin 3-O-b-D-galactopyranoside by NMR spectroscopy. Red and yellow peel color was chiefly determined by the presence and absence of anthocyanins, respectively, while the orange appearance of the peel was mainly caused by increased carotenoid concentrations. Thus, orange-peeled fruits represent a rich source of provitamin A (ca. 124 lg retinol-activity-equivalents/100 g pulp, FW). Ó 2016 Elsevier Ltd. All rights reserved.

Keywords: Cashew Vitamin A b-Carotene b-Cryptoxanthin 7-O-Methylcyanidin Lutein Zeaxanthin

1. Introduction The cashew tree (Anacardium occidentale L., Anacardiaceae) is of considerable economic importance worldwide as a source of two major products – the cashew nut (botanically representing the true fruit) and the cashew apple (the enlarged and fleshy pedicel). According to the Food and Agriculture Organization of the United

⇑ Corresponding author at: University of Hohenheim, Institute of Food Science and Biotechnology, Chair of Plant Foodstuff Technology and Analysis, Garbenstrasse 25, D-70599 Stuttgart, Germany. E-mail address: [email protected] (R.M. Schweiggert). http://dx.doi.org/10.1016/j.foodchem.2016.01.038 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

Nations (FAO), the world production of cashew nuts and apples in 2012 amounted to 4,152,315 and 2,001,301 metric tons, respectively. Most important cashew nut producers were Brazil, Vietnam, Nigeria, India, and Côte d’Ivoire. The highest production of cashew apples was recorded for Brazil, reaching a total of 1,805,000 tons, being equivalent to more than 90% of the total world production in 2012 (FAO, 2015). In Brazil, cashew apples are consumed fresh and approximately 10% of the total produce are commonly processed into juice, pulp, jam, alcoholic beverages, and confectionary according to Assunção and Mercadante (2003). The latter authors also investigated differently colored cashew apples from several geographic regions of Brazil, reporting high levels of vitamin C of

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up to 121 mg/100 g FW, irrespective of their origin and peel color. Furthermore, the fruit was reported to contain provitamin A carotenoids such as b-carotene and b-cryptoxanthin, although occurring at rather low levels (17–68 and 8–64 lg/100 g FW, respectively) as compared to other fruits like papaya (200– 554 lg b-carotene and 160–494 lg b-cryptoxanthin per 100 g FW; Schweiggert, Steingass, Esquivel, & Carle, 2012). Other carotenoids found in cashew apples were lutein and zeaxanthin (Assunção & Mercadante, 2003), similarly being present at very low levels (5–14 and 1–3 lg/100 g FW) when compared to other fruit and vegetables like green leafy vegetables or green and yellow bell pepper, all commonly reaching far more than 2000 lg/100 g FW (Schweiggert & Carle, 2015). Lutein and zeaxanthin most recently received considerable attention due to their accumulation in neural human tissues such as the brain and the macula (Krinsky & Johnson, 2005; Vishwanathan, Kuchan, Sen, & Johnson, 2014). Consequently, a protective role of these antioxidant and photoprotective compounds in visual and cognitive health has been proposed and a sufficient dietary supply of these carotenoids may help to delay the onset or even prevent chronic diseases, e.g. age-related macular degeneration and age-related cognitive impairment (Johnson, 2012). Although yellow-, orange-, and red-colored fruits are available, only a few studies dealt with their natural pigments (Assunção & Mercadante, 2003; de Abreu et al., 2013; de Brito, Pessanha de Araújo, Lin, & Harnly, 2007) and several biochemical aspects remained unclear to date. For instance, only the profiles of saponified carotenoid extracts have been reported and, thus, the presence and eventual identity of carotenoid esters remained unknown. Carotenoid esters were recently reported to aggregate in a particular presumably liquid-crystalline form in vitro (Hempel, Schädle, Leptihn, Carle, & Schweiggert, 2016). Plant foods having carotenoid esters were consistently observed to contain highly similar liquidcrystalline carotenoid aggregates, which have been proposed to be more bioavailable than solid-crystalline forms as present, e.g., in carrots and tomatoes (Schweiggert & Carle, 2015). Thus, the presence and yet unknown identity of carotenoid esters in cashew apples might be of interest for other researchers and nutritionists. In addition, a rare methylated anthocyanin was previously proposed to be the main anthocyanin in the peels of cashew apples (de Brito et al., 2007), although only being identified by LC–PDA– ESI/MS yet, not allowing the unambiguous determination of the position of the methyl group. The correct localization is of considerable interest, since 7-O-methylated anthocyanins have been proposed to be a potential chemotaxonomic trait of members of the Anacardiaceae (Feuereisen et al., 2014). Besides elucidating the origin of the different colors of cashew apples, quantitative data on the provitamin A-active b-carotene and, in particular, the esters of the nutritionally relevant b-cryptoxanthin, lutein and zeaxanthin might help to assess the nutritional potential of cashew apples, which are consumed daily in Brazil and other South and Central American countries. Therefore, the present study aimed at comparing the genuine profiles of carotenoids, carotenoid esters, and anthocyanins of differently colored cashew apples, including the elucidation of the main cashew anthocyanin by 2D NMR experiments.

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different origins of red- and yellow-peeled pseudo-fruits were analyzed, while two samples of one single origin of the orange-peeled pseudo-fruit were available. A photograph of the differently colored cashew apples is shown in Fig. 1. The peel of the receptacles was removed manually, and the pulp was cut into small cubes (ca. 0.5
0.5
0.5 cm3). Peels and pulp were separately frozen using liquid nitrogen, freeze-dried, packed into aluminum-coated plastic pouches, and sealed under vacuum. Samples were stored at 80 °C until carotenoid and anthocyanin analyses. Deionized water was used throughout unless stated otherwise. All reagents or solvents were of analytical or HPLC grade. Ammonium acetate, hydrochloric acid (37%), light petroleum (b.p. 40–60 °C), methanol, methyl tert-butyl ether (MTBE), potassium hydroxide (KOH), and sodium chloride (NaCl) were purchased from VWR International (Darmstadt, Germany), L-ascorbic acid, D-galactose, L-cysteine methyl ester hydrochloride, and the silylation mixture (10% (v/v) hexamethyldisilazane/-trimethylchlorosilane (HMDS/TMCS), 2/1, v/v, in pyridine) from Sigma–Aldrich (Steinheim, Germany), L-galactose

from Carbosynth (Compton, UK), D2O from Deutero (Kastellaun, Germany), and acetone, calcium carbonate, citric acid monohydrate, disodium hydrogen phosphate, ethyl acetate, and trifluoroacetic acid (TFA) from Merck (Darmstadt, Germany). Standards of (all-E)-a-carotene, (all-E)-antheraxanthin, (all-E)b-cryptoxanthin, (all-E)-b-carotene, (all-E)-lutein, (all-E)mutatoxanthin (mixture of two isomers), (all-E)-neoxanthin, phytoene, phytofluene (mixture of two isomers), (all-E)violaxanthin, (all-E)-zeaxanthin, and (all-E)-zeaxanthin dipalmitate were obtained from CaroteNature (Ostermundigen, Switzerland). Lutein esters were isolated from a Tagetes erecta L. extract and added to cashew carotenoid extracts to confirm the identification of lutein dipalmitate, the major ester in T. erecta L. flowers (Ziegler et al., 2015). 2.2. Carotenoid extraction and saponification All procedures described below were carried out under dim light as follows. Prior to carotenoid extraction, the defrosted samples were homogenized to obtain a fine powder. An aliquot of 100 ± 10 mg of the ground peel and 10 mg of calcium carbonate (CaCO3) or 200 ± 10 mg of the ground pulp and 20 mg of CaCO3 were extracted with 3 mL of extraction solvent (methanol/ethyl acetate/light petroleum, 1:1:1, v/v/v) using an Ultra-Turrax laboratory homogenizer (IKA Labortechnik, Staufen, Germany). After centrifugation, the extraction solvent was collected and the extraction

2. Materials and methods 2.1. Plant material and reagents Yellow-, red-, and orange-peeled pseudo-fruits (receptacles) of cashew (A. occidentale L.) were acquired at different local markets in Guacalillo (Puntarenas, Costa Rica). All pseudo-fruits were obtained in a ready-to-eat ripeness stage. Each two fruits of three

Fig. 1. Photograph of yellow-, orange-, and red-peeled cashew fruits. Brightness and contrast were adjusted with Adobe Photoshop (CS4).

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was repeated 2–3 times until the solid residue was colorless. The combined organic extracts were evaporated to dryness under a gentle nitrogen stream and stored at 80 °C until HPLC analysis. Prior to HPLC analyses, the dried extracts were re-dissolved in 1 mL of a mixture (1:1, v/v) of methanol and MTBE and membrane-filtered (PTFE, 0.45 lm, Macherey-Nagel, Düren, Germany) into amber vials. For saponification, the method of Aschoff et al. (2015) was followed with slight modifications. Briefly, the above mentioned dried extracts were re-dissolved in 3 mL of light petroleum, and 3 mL of methanolic KOH (30% w/v) were added. After stirring for 23 h, 2 mL of water were added and the mixture was centrifuged to enhance phase separation. The organic layer was separated and washed twice with water. After adding 1 mL of aqueous sodium chloride (30% w/v), the remaining methanolic phase was re-extracted with 2 mL of MTBE, which was washed with water once. All separated organic phases were combined, evaporated to dryness under a gentle stream of nitrogen, and stored at 80 °C until HPLC analyses.

2.3. Carotenoid analyses by HPLC–PDA–MSn For carotenoid identification, a series 1100 HPLC (Agilent, Waldbronn, Germany) with a G1315B photodiode array detector (PDA) was interfaced with a Bruker Esquire 3000+ ion trap mass spectrometer (Bruker, Bremen, Germany) using an atmospheric pressure chemical ionization (APCI) source operated in both positive and negative mode. Mass spectra were collected from m/z 100 to 1200 at a scan rate of 13,000 Th/s. Carotenoid quantification was carried out on a Waters 2695 separations module (Waters, Eschborn, Germany) and a 2996 photodiode array detector. The used column, solvents, elution gradients, and all further settings of both used HPLC systems were identical as described below. Chromatographic separation was achieved with a YMC C30 reversed phase column (150
3.0 mm i.d., 3 lm particle size, YMC Europe, Dinslaken, Germany) protected by a YMC C30 guard column of the same material. Carotenoids in non-saponified samples were separated using a method of Hempel et al. (2014). Briefly, HPLC solvents consisted of methanol/MTBE/water (80:18:2, v/v/v, eluent A) and methanol/MTBE/water (8:90:2, v/v/v, eluent B), both containing 0.4 g/L of ammonium acetate. The elution gradient was as follows: isocratic at 100% A for 5 min, from 100% to 90% A in 2 min, from 90% to 50% A in 20 min, from 50% to 0% A in 0.5 min, isocratic at 0% A for 1 min, from 0% to 100% A in 1 min, and isocratic at 100% A for 2.5 min. Total run time was 32 min at a flow rate of 0.95 mL/min and a column temperature of 40 °C. Injection volume was 10 lL. Carotenoids were monitored at 450 nm, recording additional UV/Vis spectra in the range of 200–700 nm. When using this method for HPLC–MSn measurements by coupling to the above mentioned mass spectrometer, the settings were as follows. APCI (+/): drying temperature, 350 °C; vaporizer temperature, 450 °C; drying and nebulizing gas, nitrogen at 5.0 L/min and 65 psi, resp.; capillary potential, +4000 V/2800 V; collision gas, helium; fragmentation amplitude, 0.8–1.2 V. Regarding saponified samples, HPLC solvents consisted of methanol/water (90:10, v/v, eluent A) and methanol/MTBE/water (78:20:2, v/v/v, eluent B), both containing 0.4 g/L ammonium acetate. The elution gradient was as follows: from 100% to 93% A in 2 min, from 93% to 77% A in 14 min, from 77% to 55% A in 4 min, from 55% to 22% A in 8 min, from 22% to 0% in 1 min, isocratic at 0% A for 2 min, from 0% to 100% A in 3 min, and isocratic at 100% A for 2 min. Total run time was 36 min at a flow rate of 0.75 mL/ min and a column temperature of 40 °C. Injection volume was 10 lL. Carotenoids were monitored at 450 nm, recording additional UV/Vis spectra in the range of 200–700 nm. For HPLC–MSn

analyses, the settings of the mass spectrometer were identical to those described above. Identification of individual carotenoids was carried out by comparing retention times, UV/Vis absorption and mass spectra to those of authentic standards. When reference compounds were unavailable, obtained UV/Vis absorption and mass spectra were compared to those reported in literature (Britton, 1995; de Rosso & Mercadante, 2007; Meléndez-Martínez, Stinco, Liu, & Wang, 2013; Ornelas-Paz, Yahia, & Gardea-Bejar, 2007; Schweiggert, Kammerer, Carle, & Schieber, 2005; Schweiggert et al., 2012). For the identification of b-carotene (Z)-isomers, DB/DII ratios were determined according to Britton (1995) and compared to literature data (Meléndez-Martínez et al., 2013; Molnár & Szabolcs, 1993). Prior to quantitation by HPLC–PDA, the concentrations of stock solutions of b-carotene, b-cryptoxanthin, lutein, and violaxanthin were determined spectrophotometrically using their specific absorption coefficients according to Britton (1995) in order to elaborate linear calibration curves. The violaxanthin calibration was used for the quantitation of violaxanthin, (9Z)-violaxanthin, and antheraxanthin. Lutein, lutein esters, and a-carotene were quantitated by the lutein calibration. The b-carotene calibration was used for the quantitation of b-carotene, b-carotene (Z)-isomers, and zeaxanthin, while b-cryptoxanthin and b-cryptoxanthin esters were quantitated by the b-cryptoxanthin calibration. When the mass concentration of the above mentioned carotenoids was derived from related calibration curves, molecular weight correction factors were used to account for differing molecular weights where appropriate. Retinol activity equivalents (RAE) were calculated according to the guidelines of the US Institute of Medicine (2001). 2.4. Extraction and HPLC–PDA–MSn analyses of anthocyanins An aliquot of 300 ± 10 mg lyophilized peels was extracted with 3 mL of aqueous HCl (pH 2), using an Ultra-Turrax laboratory homogenizer (30 s, 25 °C). After centrifugation, the aqueous phase was collected and the solid residues were re-extracted four times as described above. The extracts were concentrated in vacuo and made up to 2 mL using McIlvaine’s buffer (pH 3.5) prior to HPLC analyses. A series 1100 HPLC (Agilent, Waldbronn, Germany) was equipped with a G1315B PDA and an analytical Kinetex C18 column (250
4.6 mm i.d., 5 lm particle size, 100 Å pore size, Torrance, CA, USA) protected by a C18 guard column (4.0
2.0 mm, Torrance, CA, USA), both operated at 25 °C. HPLC solvents, gradient program, and flow rate were set as described by Gras, Carle, and Schweiggert (2015). Injection volume was 50 lL. Anthocyanins were monitored at 520 nm and additional UV/Vis spectra were recorded in the range of 200–700 nm. For HPLC–ESI(+)-MSn measurements, the above described HPLC system was interfaced with a Bruker Esquire 3000+ ion trap mass spectrometer (Bruker, Bremen, Germany) fitted with an ESI source. The mass spectrometer was operated in positive ion mode, using nitrogen as drying gas at a flow rate of 10 L/min. Nebulizing gas was nitrogen at a pressure of 60 psi. Nebulizer temperature was set at 365 °C. Helium was used for collision induced dissociation (CID) experiments at a fragmentation amplitude of 0.8 V. 2.5. Preparative isolation of anthocyanins Anthocyanins were isolated from freeze-dried cashew peels by extraction with aqueous acetone (80% v/v, 1% w/v ascorbic acid) and subsequent purification by solid phase extraction (SPE) according to a previously reported procedure (Berardini et al., 2005). However, in contrast to the reported procedure, extraction by stirring for 3 h was replaced by an extraction using a probesonicator with a MS72 microtip (HD 3100, Bandelin electronic,

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Berlin, Germany) at 75% amplitude for 30 s, which was repeated three times. In further contrast to the reported procedure, the purified methanolic anthocyanin-rich SPE eluate was only concentrated instead of evaporating to dryness. The concentrated extract was diluted with acidified water (0.1% TFA) to a final solvent ratio of ca. 50:50 (v/v, water:methanol). Further purification was carried out by preparative HPLC using a 250
21.1 mm i.d., 5 lm particle size, 125 Å pore size, Aqua C18 reversed-phase column (Phenomenex Aschaffenburg, Germany). The mobile phase consisted of water (0.1% v/v TFA) and methanol (0.1% v/v TFA) as solvents A and B, respectively. Separation was achieved by gradient elution from 40% B to 50% B in 20 min, followed by a returning gradient from 50% to 40% B in 5 min. The flow rate was 9.5 mL/min. Compounds were monitored at 280 nm. The combined collected fraction containing compound 25 was evaporated in vacuo to dryness, lyophilized, and stored at 80 °C under nitrogen atmosphere until NMR and GC–MS analyses. 2.6. NMR spectroscopy NMR spectroscopy was carried out as described previously (Berardini et al., 2005). A Varian Unity Inova 500 MHz spectrometer (Darmstadt, Germany) was used to record 1H and 13C NMR spectra of the isolated anthocyanin, which was dissolved in 1% (v/v) aqueous TFA. The 1H chemical shifts were referenced to residual solvent signal at dH 4.7 (D2O) relative to TMS. Adiabatic 2D broadband and band-selective gHSQC and gHMBC, gDQFCOSY, as well as ROESY were recorded using CHEMPACK 4.0 pulse sequences (implemented in Varian VnmrJ 2.1B software). The isolated compound 25 was identified as 7-O-methylcyanidin 3-O-b-Dgalactopyranoside, since the obtained 1D and 2D NMR data were in agreement with our previously published data on 7-Omethylcyanidin 3-O-b-D-galactopyranoside isolated from mango peels (Berardini et al., 2005). 2.7. Determination of the absolute configuration by GC–MS Sugar configuration was determined by GC–MS after hydrolysis with aqueous TFA (2 M), derivatization with L-cysteine methyl ester hydrochloride, and trimethylsilylation as described previously in detail (Maier, Conrad, Carle, Weiss, & Schweiggert, 2015). According to these analyses, the sugar derivative was identified as D-galactose with the retention time of 20.75 min, being identical to the respective retention time of derivatized authentic D-galactose.

Under the same conditions, the L-galactose derivative revealed a retention time of 21.40 min. 2.8. Statistical analyses Analysis of variance (ANOVA) and Tukey’s HSD (Honestly Significant Difference) test were used for determination of significantly different means (P < 0.05) using SAS 9.1 (SAS Institute, Cary, NC, USA). All values are reported as means ± standard deviation. 3. Results and discussion 3.1. Identification of carotenoids and carotenoid esters The qualitative carotenoid composition was highly similar in the peels and pulps of all differently colored cashew apples. As illustrated by the exemplary chromatograms of peel from an orange-peeled fruit (Fig. 2A and B), violaxanthin (1), neoxanthin (2), antheraxanthin (5), and lutein (6) were only detected in the saponified samples, comparing retention times, UV/Vis and mass spectra (Table 1) to those of authentic standards. Similarly, (9Z)-

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violaxanthin (4) was tentatively identified by its characteristic UV/Vis and mass spectral behavior, including a comparison of its DB/DII ratio to data reported previously (Molnár & Szabolcs, 1993). Since these compounds (1–6) were absent in genuine samples but present in saponified samples, their occurrence as xanthophyll esters appears likely. However, we were unable to find the respective xanthophyll esters, possibly due to co-elutions of the predominant b-cryptoxanthin, lutein, and zeaxanthin esters (Table 1, see results below). In both saponified and genuine extracts, zeaxanthin (7), b-cryptoxanthin (8), phytoene (9) and two phytofluene isomers (10, 11) were assigned by comparing the retention times, UV/Vis and mass spectra of the respective compounds to those of authentic standards. Noteworthy, phytoene (9) and phytofluene isomer 1 (10) eluted after b-cryptoxanthin (8) when using the longer method for non-saponified samples, while they eluted before b-cryptoxanthin using the shorter method for saponified samples (Table 1 and Fig. 2). The two major compounds 13 and 14 were unambiguously identified as a-carotene and b-carotene by the aid of authentic standards, respectively. In addition, (13Z)- and (9Z)-b-carotene (compounds 12 and 15, respectively) were tentatively identified according to their elution order (Meléndez-Martínez et al., 2013), their UV/Vis spectra including characteristic DB/DII values (Meléndez-Martínez et al., 2013), and their b-carotene-like mass spectral behavior, i.e. a molecular ion [M] at m/z 536 and pseudo-molecular ion [M+H]+ at m/z 537 (Table 1). Regarding the above mentioned carotenoid esters, b-cryptoxanthin myristate (compound 16) was identified due to its characteristic UV/Vis absorption maxima being identical to those of unesterified b-cryptoxanthin, its molecular ion [M] at m/z 762, and its pseudo-molecular ion [M+H]+ at m/z 763. In agreement, the latter ion produced a characteristic CID fragment [M +H228 (myristic acid)]+ at m/z 535 and further carotenoid specific ions (Table 1). By analogy, compound 18 was identified to be b-cryptoxanthin palmitate, representing a major carotenoid ester in most cashew apples, while compound 17 remained unknown. Several compounds (19–21) revealed characteristic UV/Vis spectra with absorption maxima at 422, 446, and 472 nm, confirming the presence of lutein esters as indicated by the appearance of lutein after saponification. In agreement, these compounds (19–21) produced CID fragments at m/z 533, possibly representing the lutein ester after loss of two fatty acid moieties. Compound 20 showed a molecular ion [M] at m/z 1044 and a pseudo-molecular ion [M +H]+ at m/z 1045. The latter produced a predominant CID fragment [M+H256 (palmitic acid)]+ at m/z 789. Fragments indicating the loss of other fatty acid species were not detected. In addition, compound 20 co-eluted with the major ester (lutein dipalmitate) present in a T. erecta L. extract (Ziegler et al., 2015) and, consequently, was assigned to lutein dipalmitate. Compound 21 was identified as lutein 30 -O-palmitate 3-O-oleate due to its characteristic mass spectral behavior, which was in agreement with Ziegler et al. (2015). Compound 22 represented a further major ester of orangepeeled cashew apples. Its UV/Vis absorption spectrum identical to free zeaxanthin, its molecular ion [M] at m/z 1044, its pseudo-molecular ion [M+H]+ at m/z 1045, and its predominant fragment ion [M+H256 (palmitic acid)]+ indicated the presence of a zeaxanthin palmitate ester. Due to the absence of other fatty acid-specific fragment ions and its co-elution with an authentic zeaxanthin dipalmitate standard, compound 22 was assigned to zeaxanthin dipalmitate. 3.2. Concentration of carotenoids and carotenoid esters Predominant carotenoids in all types of cashew apples were the provitamin A carotenoids b-carotene (44–69% of total carotenoids),

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Fig. 2. HPLC separation of saponified (A) and non-saponified (B) carotenoid extracts of orange-peeled cashew apple, also depicting the structures of a-carotene (13), bcarotene (14), b-cryptoxanthin (8), and b-cryptoxanthin palmitate (18).

a-carotene (0–14%), and the carotenoid ester b-cryptoxanthin palmitate (7–37%, see Online Supplementary material for quantitative carotenoid ester data, Table S1). The presence of relevant amounts of the latter was confirmed by the appearance of high amounts of free b-cryptoxanthin after saponification (Table 2 and Fig. 2B), being almost absent in genuine samples (Table 2). Since the molar concentration of b-cryptoxanthin in most saponified samples (4.0–30.4 lmol/100 g FW) exceeded those of b-cryptoxanthin and its esters in the corresponding non-

saponified samples (4.1–24.1 lmol/100 g FW), we assume the presence of further b-cryptoxanthin esters which remained unidentified in our study. Therefore, retinol activity equivalents (RAE) were estimated using data of saponified samples (Table 2). Highest RAE values were generally found in the peels, although 135 lg/100 g FW were reached in the pulp of a single orangepeeled fruit. The mean range determined in the pulp of all types was 37–124 lg RAE/100 g FW (Table 2), being comparable to those of papaya (39–127 lg/100 g, Schweiggert et al., 2012) and mango

Table 1 HPLC retention times, UV/Vis spectra, and MS spectral data of carotenoids from peels and pulp of differently colored cashew apples. Rt (min) (method for saponified extracts)

Rt (min) (method for non-saponified extracts)

Compound identity

HPLC–PDA UV/Vis absorption maxima (nm)

[M] m/z

[M+H]+ m/z

HPLC/APCI(+)MSn experiment m/z

STDe

1 2

8.7 9.2

n.d. n.d.

Violaxanthin Neoxanthin

416/438/468 416/438/466

n.a. n.a.

[601]: 391 (100), 316 (93), 220 (86), 583 (80), 584 (62), 475 (55) n.a.

Y Y

3 4 5 6 7 8 9 10 11 12 13 14 15 16

10.3 12.3 12.7 15.8 17.6 22.9 23.0 23.6 24.4 25.0 25.4 26.0 26.5 n.d.

n.d. n.d. n.d. n.d. 2.9 5.8 4.6 5.4 6.5 8.0 8.8 10.4 11.3 17.0

400/422/448 326/412/436/464 420/444/472 422/446/472 426/452/476 426/452/478 276/286/296 332/348/366 332/348/366 338/422/444/472 422/446/472 426/452/476 424/448/472 426/452/478

n.a. n.a. n.a. 568 568 552 n.a. n.a. n.a. 536 536 536 536 762

[601]: [601]: [585]: [551]: [569]: [553]: n.a. [543]: n.a. [537]: [537]: [537]: [537]: [763]:

n.a. n.a. Y Y Y Y Y Y Y n.a.c Y Y n.a.c n.a.

17

n.a.

17.5

426/448/474

n.a.

n.a.

n.a.

n.a.

18

n.d.

19.0

426/452/478

790

791

[791]: 535 (100), 443 (8), 699 (7), 413 (6)

n.a.

19

n.d.

22.2

422/446/474

1016

1017

n.d. n.d.

23.8 24.0

422/446/472 422/446/472

1044 1070

1045 1071

[1017]: 759 (100), 997(97), 731(82), 697(61), 761(61), 760(60), 789(50), 669(46), 925 (45), 533(38), 473(20), 459(13) [1045]: 789(100), 697(93), 533(20), 953(12) [1071]: 815(100), 789(63), 533(37), 723(17), 979(13), 697(8), 487(8)

n.a.d

20 21 22

n.d.

25.2

Unidentified (9Z)-Violaxanthina Antheraxanthin Lutein Zeaxanthin b-Cryptoxanthin Phytoene Phytofluene isomer 1 Phytofluene isomer 2 (13Z)-b-Carotenec a-Carotene b-Carotene (9Z)-b-Carotenec b-Cryptoxanthin myristate Unknown carotenoid ester b-Cryptoxanthin palmitate Lutein ester (identity unknown)d Lutein dipalmitate Lutein 30 -O-palmitate 3O-oleate Zeaxanthin dipalmitate

601 601, 583b 601 601 585 551b 569 553 545 543 543 537 537 537 537 763

426/452/476

1044

1045

[1045]: 789(100), 697(93), 441(25), 953(24), 533 (20)

Y

583 422 567 533 413 535

(100), (100), (100), (100), (100), (100),

484 583 375 457 411 239

(53), (63), (27), (46), (87), (41),

393 565 444 209 476 319

(49), (62), (17) (26), (82), (35),

414 (46), 519 (30) 491 (46), 221 (46), 260 (46) 208 (22), 509 (22), 495 (19), 451 (19), 499 (19) 551 (62) 460 (30), 348 (27), 415 (26)

461 (100), 433 (40), 283 (40), 363 (38), 487 (34), 525 (34) 445 413 399 401 535

(100), (100), (100), (100), (100),

415 445 445 265 760

(48), 359 (39), 413 (25), 399 (16) (99), 399 (52) (96), 413 (82), 441 (79), 455 (67), 444 (65) (67), 157 (63), 253 (57), 295 (52), 444 (13), 413 (20) (8), 533 (8), 413 (4), 443 (4), 479 (3)

n.d.: not detected. n.a.: not available. a Tentatively identified by comparing the DB/DII value of the putative (9Z)-violaxanthin (0.06) to that reported by Molnár and Szabolcs (1993), i.e. 0.06 (Q = 17.50). b In source fragment [MH2O+H]+ in agreement with reference compound. c Tentatively identified by comparing the DB/DII values of the putative (9Z)-b-carotene (0.06) and (13Z)-b-carotene (0.42) to those reported by Meléndez-Martínez et al. (2013), i.e. 0.13 and 0.43, respectively. d Tentative identification according to UV/Vis spectra. The obtained mass spectra were highly ambiguous, hindering its reliable identification. e ‘‘Y” indicates that the presented analytical data were in agreement with an authentic reference compound.

Y n.a.

R.M. Schweiggert et al. / Food Chemistry 200 (2016) 274–282

No.

279

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R.M. Schweiggert et al. / Food Chemistry 200 (2016) 274–282

Table 2 Content of carotenoids and retinol activity equivalents as determined after saponification of extracts from differently colored cashew apples (in lg/100 g FW). Compound

Violaxanthin (9Z)-Violaxanthin Antheraxanthin Lutein Zeaxanthin b-Cryptoxanthin (13Z)-b-Carotene a-Carotene b-Carotene Total carotenoidsa Retinol activity equivalentsb

Red-peeled cashew apple

Orange-peeled cashew apple

Yellow-peeled cashew apple

Peel

Pulp

Peel

Pulp

Peel

Pulp

132 ± 54 A 134 ± 21 A 143 ± 8 A 556 ± 75 A 116 ± 22 B 220 ± 39 DC 125 ± 40 A 367 ± 38 AB 1387 ± 130 B 3250 ± 385 B 148 ± 15 B

30 ± 26 A 45 ± 15 B 28 ± 5 B 29 ± 12 C 4±2C 60 ± 22 D 51 ± 11 B 50 ± 5 C 430 ± 41 C 734 ± 21 C 43 ± 4 C

tr. 157 ± 27 A 195 ± 39 A 323 ± 48 B 269 ± 32 A 1676 ± 113 A 132 ± 10 A 283 ± 8 B 2238 ± 148 A 5278 ± 406 A 274 ± 18 A

90 ± 29 A 49 ± 3 B 142 ± 18 A 2±1 C 58 ± 5 BC 742 ± 48 B 39 ± 4 B 4±1 C 1101 ± 87 B 2228 ± 346 B 124 ± 9 B

142 ± 97 A 109 ± 27 A 105 ± 25 A 312 ± 134 B 51 ± 35 C 333 ± 11 C 154 ± 51 A 383 ± 53 A 1196 ± 365 B 2815 ± 795 B 137 ± 37 B

82 ± 12 A 37 ± 16 B 20 ± 1 B 19 ± 14 C 6±2 C 97 ± 61 D 33 ± 3 B 39 ± 30 C 360 ± 60 C 693 ± 102 C 37 ± 6 C

tr., trace amount. Different letters within a row indicate significant differences of means (P < 0.05). Six fruits of red- and yellow-peeled (n = 6) and two fruits of orange-peeled (n = 2) cashew apples were analyzed in duplicate. a Excluding eventually unidentified carotenoids (see chromatogram in Fig. 2). b As calculated according to the guidelines of the US Institute of Medicine (2001).

(54 lg/100 g, USDA nutrient data base (2015)), but lower than those found in carrot (835 lg/100 g, USDA nutrient data base (2015)). Besides vitamin A precursor carotenoids, cashew apples were found to contain the human macular pigments lutein and zeaxanthin. Quantitative determination of lutein and zeaxanthin esters was conducted when PDA signals indicated the absence or only low levels of co-eluting compounds (compounds 20–22), but several unidentified carotenoid esters (e.g., compound 17) may have masked the presence of further lutein and zeaxanthin esters. In agreement, we found higher molar concentrations of lutein and zeaxanthin after saponification (5.5–9.8 and 0.9–2.0 lmol/100 g FW, resp.) as expected according to the molar concentrations of the respective esters (4.0–5.9 and 0.0–0.4 lmol/100 g FW, resp.). Noteworthy, both lutein and zeaxanthin levels were rather low in both peels and pulp (2–556 lg lutein and 4–269 lg zeaxanthin per 100 g FW, Table 2) as compared to other foods. For instance, green leafy vegetables like curly kale contain more than 2000 lg lutein per 100 g FW. Zeaxanthin is found at levels >2000 lg/100 g in Chinese wolfberries (Britton & Khachik, 2009) and, as a more common part of the human diet, occurs in egg yolk at variable concentrations, e.g., at ca. 565–640 lg/100 g (Nimalaratne, Lopes-Lutz, Schieber, & Wu, 2012). Higher levels of total carotenoids were consistently found in the peels (2.8–5.3 mg/100 g FW) when compared to the pulp (0.7– 2.2 mg/100 g FW) of all cashew apple types studied (Table 2). While the peels of orange fruits exhibited the highest content in carotenoids (5.3 mg/100 g FW), the absolute contents in individual and total carotenoids were highly similar in yellow and red peels (P > 0.05) as shown in Table 2. These findings indicated that the presence of a further non-carotenoid pigment type might be responsible for the obvious color differences, such as the below described anthocyanins. In agreement with our results, Assunção and Mercadante (2003) analyzed saponified extracts of homogenized cashew peel and pulp, similarly reporting b-cryptoxanthin (8–64 lg/100 g FW) as well as a- and b-carotene (6–52 and 17–68 lg/100 g FW, resp.) to be the major carotenoids in cashew fruits. By analogy, main carotenoids in processed cashew apple juices were previously found to be b-cryptoxanthin and b-carotene, including some (Z)isomers of b-cryptoxanthin and b-carotene (Zepka & Mercadante, 2009). In the study of Assunção and Mercadante (2003), elongated red varieties contained only slightly higher total carotenoid levels (155–204 lg/100 g FW) when compared to the elongated yellow varieties (99–174 lg/100 g FW), while the investigated roundshaped red variety even contained significantly lower carotenoid

levels (119 lg/100 g FW, Assunção and Mercadante (2003)). These findings confirm that the red and yellow color of cashew fruits is not necessarily an indicator for high and low carotenoid and provitamin A contents, respectively, indicating the contribution of an additional pigment type – the anthocyanins mentioned below. In brief conclusion, orange-peeled cashew apples represent rich sources of provitamin A carotenoids like b-carotene and several b-cryptoxanthin esters, while yellow- and red-peeled types contained significantly lower amounts of provitamin A. Although containing rather low levels as compared to other lutein- and zeaxanthin-rich foods, cashew apples also supply nutritionally relevant amounts of lutein and zeaxanthin. To date, cashew apples are mostly seen as a waste or by-product of cashew nut production. Due to their content in nutritionally valuable compounds, their use and consumption might be encouraged in the future. 3.3. Cashew anthocyanins and chemotaxonomic considerations Fig. 3 shows HPLC chromatograms of anthocyanin extracts obtained from yellow-, orange-, and red-peeled cashew apples,

Fig. 3. HPLC separation of anthocyanins from the peels of differently colored cashew apples (monitored at 520 nm). In addition, the structure of 7-O-methylcyanidin 3-O-b-D-galactopyranoside (25) is shown.

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R.M. Schweiggert et al. / Food Chemistry 200 (2016) 274–282 Table 3 Identification parameters of anthocyanins and their abundance in differently colored cashew apples. No.

23 24 25 26

Compound identity

Cyanidin 3-O-hexoside# Unknown methylated delphinidin 3-O-hexoside# 7-O-Methylcyanidin 3-O-b-Dgalactopyranoside* Unknown

HPLC–PDA Vis absorption maxima (nm)

[M]+ m/z

15.6 16.8

519 526

18.5 21.1

Rt (min)

HPLC/ESI(+)-MSn experiment m/z

Present in Red-peeled cashew apple

Orange-peeled cashew apple

Yellow-peeled cashew apple

449 479

[449]: 287 (100) [479]: 317(100)

+ +

tr. tr.

n.d. n.d.

516

463

[419]: 301 (100)

+++

+

n.d.

n.a.

n.a.

n.a.

+

tr.

n.d.

n.d., not detected. n.a., not available due to interfering signals of co-eluting compounds. +++, present in high amounts; +, present in low amounts; tr., trace amount, i.e., identification based on mass spectra only due to low and ambiguous UV/Vis signals. See corresponding chromatogram in Fig. 3. * Identity confirmed by NMR experiments. # Tentatively identified.

clearly indicating the lack of anthocyanins in yellow peels. Anthocyanins were detected in orange-peeled cashew apples, although at comparably low signal intensity. Red-peeled samples contained a total of four anthocyanins (compounds 23–26). According to our HPLC–PDA–MSn data (Table 3), compound 23 might represent a cyanidin 3-O-hexoside due to its UV/Vis absorption maximum at 519 nm, its molecular ion [M]+ at m/z 449, and a CID loss of a hexose fragment, i.e. [M162]+ at m/z 287. Compound 24 revealed a UV/Vis absorption maximum at 526 nm and a molecular ion [M]+ at m/z 479. By analogy to the hexose loss described for compound 23, the parent ion produced a predominant CID fragment [M162]+ at m/z 317. The latter fragment may represent a methylated delphinidin, but we were unable to determine the position of methylation. The major compound 25 exhibited an absorption maximum at 516 nm and a molecular ion [M]+ at m/z 463, which produced a CID daughter ion [M162]+ at m/z 301. Due to its importance for the red color of red-peeled cashew fruits, we isolated the pigment by solid phase extraction and preparative HPLC for structure elucidation by 1D and 2D NMR spectroscopy and GC–MS as described above. In contrast to a previous LC–DA D–ESI/MS-based assumption of a 5-methyl cyanidin hexoside (de Brito et al., 2007), the compound was unambiguously identified as 7-O-methylcyanidin 3-O-b-D-galactopyranoside by 1D and 2D NMR. Such rarely encountered 7-O-methylated anthocyanins were previously proposed to be typical pigments of the Anacardiaceae family, possibly being even a chemotaxonomic trait (Feuereisen et al., 2014). The 7-O-methylcyanidin 3-O-b-D-galactopyranoside found in cashew was previously described to be the predominant pigment in red-colored peels of mango (Mangifera indica L., Anacardiaceae, Berardini et al., 2005). The same pigment was also present in the exocarp of Brazilian pepper (Schinus terebinthifolius Raddi, Anacardiaceae), although 7-O-methylpelargonidin 3-O-b-Dgalactopyranoside was prevailing (Feuereisen et al., 2014). Furthermore, Staghorn sumac (Rhus typhina L., Anacardiaceae) was also reported to contain a number of 7-O-methylated derivatives of cyanidin and delphinidin (Kirby, Wu, Tsao, & McCallum, 2013). The present report about the main anthocyanin in cashew apples, the 7-O-methylcyanidin 3-O-b-D-galactopyranoside, adds further evidence to the above mentioned hypothesis of Feuereisen et al. (2014). This hypothesis merits further investigation, since it might be helpful to clarify the taxonomic position of further economically important genera of the Anacardiaceae. For instance, the taxonomic position of the genus Pistacia has been under debate (Pell, 2004). While it may be seen to represent a separate family (Pistaciaceae, Adans) due to several morphological features aberrant for most members of the Anacardiaceae, many authors classified them

into the Anacardiaceae tribe Rhoeae based on molecular markers (Pell, 2004; Yi, Wen, Golan-Goldhirsh, & Parfitt, 2008). The proposed Anacardiaceae-specific 7-O-methyl anthocyanins have not been mentioned in reports on anthocyanins from Pistacia species yet. For instance, Longo, Scardino, and Vasapollo (2007) reported 3-O-glucosides of delphinidin and cyanidin to be the major pigments in the berries of Pistacia lentiscus L., and cyanidin 3-Ogalactoside was found in kernels of P. vera (Bellomo & Fallico, 2007; Wu & Prior, 2005). Furthermore, fruits of Pleiogynium timorense DC Leenh. (Burdekin plum) were previously reported to contain cyanidin 3-glucoside as major anthocyanin (Netzel, Netzel, Tian, Schwartz, & Konczak, 2006). Noteworthy, the complete absence of 7-O-methylated anthocyanins in the mentioned Anacardiaceae members should not be deduced from these reports and targeted analyses of 7-O-methyl anthocyanins in colored plant tissues of other Anacardiaceae members are of highest relevance to elucidate their qualification as taxonomic markers within this complex and disputable family. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We thank the Alexander von Humboldt Foundation (Bonn, Germany) for partially funding this study in the framework of the Research Group Linkage Program. E.V. acknowledges support from the Baden-Württemberg Stiftung (Stuttgart, Germany), the Sistema de Estudios de Posgrado of the University of Costa Rica and the National Scientific and Technological Research Council of Costa Rica (CONICIT) in form of a short-time scholarship. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.foodchem.2016. 01.038. References Aschoff, J. K., Kaufmann, S., Kalkan, O., Neidhart, S., Carle, R., & Schweiggert, R. M. (2015). In vitro-bioaccessibility of carotenoids, flavonoids and vitamin c from differently processed oranges and orange juices (Citrus sinensis (L.) Osbeck). Journal of Agricultural and Food Chemistry, 63(2), 578–587. Assunção, R. B., & Mercadante, A. Z. (2003). Carotenoids and ascorbic acid from cashew apple (Anacardium occidentale L.): Variety and geographic effects. Food Chemistry, 81(4), 495–502.

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