~ 1 ~ American Journal of Essential Oils and Natural Products 2018; 6(3): 01-09 ISSN: 2321-9114 AJEONP 2018; 6(3): 01-09 © 2018 AkiNik Publications Received: 01-05-2018 Accepted: 05-06-2018 Jiménez Mata Facultad de Ciencias de la Salud, Universidad Internacional de las Américas, 1447-1002 Sede Metropolitana, San José, Costa Rica Villegas-Castro Centro de Investigación en Nutrición Animal (CINA), Universidad de Costa Rica, 11501-2060 Ciudad Universitaria Rodrigo Facio, San José, Costa Rica Granados-Chinchilla Centro de Investigación en Nutrición Animal (CINA), Universidad de Costa Rica, 11501-2060 Ciudad Universitaria Rodrigo Facio, San José, Costa Rica Correspondence Granados-Chinchilla Centro de Investigación en Nutrición Animal (CINA), Universidad de Costa Rica, 11501-2060 Ciudad Universitaria Rodrigo Facio, San José, Costa Rica Costa Rican cashew (Anacardium occidentale L.): Essential oils, carotenoids and bromatological analysis Jiménez Mata, Villegas-Castro, Granados-Chinchilla Abstract Considering cashew tree as economically relevant in Costa Rica, we extracted and characterized the essential oil from leaves and ripe cashew apple. Both oils were tested in vitro for antioxidant potential. The ripe cashew apple was also tested for carotenoid content. We performed nutritional analysis on the nutshell to assess its potential as a supplement for animal feed. Finally, the cashew nut’sfatty acid profile was also determined. Cashew tree leaves exhibited 2, 4-di-tert-butylphenol, 3-hydroxy-1, 3- diphenylpropan-1-one, and 5-methylpyrogallolin 14.14, 6.59, and 6.10 g/100 g, respectively while cashew apple volatile compounds included 2-hydroxy-4-methylvaleric acid, 1, 4-xylene, and 1- nonadeceneat relative concentrations of 13.89, 12.08, and 9.84 g/100 g, respectively. Cashew nut fat profile included oleic and linoleic acids as most prevalent with 56.26 and 14.50 g/100 g, respectively with monounsaturated fatty acids representing an average of (71.97 ± 1.49) g/100 g. Total carotenoids accounted for (2.93 ± 0.07) mg β-carotene/100 g fresh material. It was concluded that the pigment in cashews is imparted, by α-carotene and β-carotene representing 0.24 mg (8.2%) and 1.25 mg (42.5%) per 100 g sample, respectively. We also obtained average values of (2 291.2 ± 35.6) and (6 158.9 ± 136.8) μmol TE/100 g, for leaves and apple essential oils, respectively. Cashew outer shell exhibited a profile rich in fiber (NDF and ADF 38.46 and 27.67 g/100 g, respectively) and crude fat (16.00 g/100 g). Furthermore, we assessed the residue to input a 57.3% of digestible energy. We concluded essential oil to have antioxidant potential and the cashew nut shell to prove potential as a feed supplement. Keywords: Cashew, anacardium occidentale l., leaves, fruit, receptacle, fatty acid profile, carotenoids, essential oils, animal feed 1. Introduction Cashewis a tropical spreading, low-branched, medium-sized (may grow to a height of 5-8 m) evergreen tree [1, 2]. Has alternate oblong-, oval- or sometimes obovate-shaped leathery leaves, borne in terminal clusters that are pale green or reddish when young and become darker when maturing. The fruit is a grey or purple colored kidney-shaped nut consisting of a double-walled shell with a hard exo carp and a thin endocarp, and an edible kernel surrounded by a thin testa [1, 2]. The fruit does not split open at maturity, but once its fruit is fully grown but not ripe, its receptacle swells and becomes a fleshy, juicy, pear or apple-shaped edible hypocarp (or pseudo fruit) which possesses red or yellow coloration when mature [1, 2]. The kernel of the cashew nut, the pseudo fruit (cashew apple) and the leaves are edible [1, 2]. Harvesting areas of 2,742,167 ha have been reported, resulting in1, 600,002 metric ton cashew nut production, worldwide [3]. Cashew is economically relevant as its dry, roasted kernels are sold as a snack [4]. Some studies have been revolving cashew tree. For example, direct solvent extracted phytochemicals and nutritional analysis of cashew tree leaves have already been reported [5], and isolation of several phytochemicals (mostly phenols) have been isolated from cashew tree bark [6]. Several other biological activities have been reported for cashew structures [7] including antimicrobial [8], hypotensive and cardio-inhibitory [9], antioxidant [10]. Additionally, protein isolates and concentrates have been obtained from defatted cashew nut powder [11], and peptides with angiotensin-converting enzyme inhibitory have also been found [12]. Herein we report the extraction and characterization of the essential oil from leaves and ripe cashew apple (receptacle). Both oils were tested for in vitro antioxidant potential. The ripe cashew apple was tested for carotenoid content. As a way to repurpose a byproduct from cashew seed roasting processing, we performed nutritional analysis on the nutshell (seed’s outer coating) to assess its potential as a supplement for animal feed. Finally, as the kernel is considered nutrient dense food [4, 11], we also determined its fatty acid profile. To our knowledge, cashew from Costa Rica has not been studied previously. ~ 2 ~ American Journal of Essential Oils and Natural Products 2. Materials and Methods 2.1 Sample collection sites Cashew fruits are seasonal. Ripe cashew apples and nuts and leaves were collected in five different batches from January to February from trees grown along the central Pacific coast of the country (where it occurs more frequently, onaltitudes from 0 to 800 m, 9°54′3.54″N 84°31′46.98″W). In this region, during the recollection time, temperatures ranged from 22.0 to 32.0ºC, relative humidity varied78 to 81%, precipitation averaged from 29.0 to 57.6 mm with4 to 7 days of rain. Only whole leaves were collected. Mature fruits were collected directly from the tree when in season (between January and February).Specimens were identified and selected based on structural characteristics of their leaves and trunks by a biologist with botanical and taxonomical expertise and based on the guidelines previously described [5]. All samples were collected from adult trees and randomly from the tops. Each collection was formed by sampling five different specimens. 2.2 Leaves and cashew apple essential oil extraction The essential oil was extracted by the process of steam distillation using an all-glass still and purified water. Briefly, crushed cashew apples and aerial parts of plant material (ca. 150 g in each case) were placed in a Clevenger type apparatus with 1000 mL flask, oil separator tube, and condenser. A total of 250 mL of purified water was added, and the mixture was vapor distilled (at 96°C at a rate of 20°C/minute and then kept at 96°C for 180 minutes) into a 125-mL Erlenmeyer, which was used to collect the aqueous distillate. The receiving receptacle was kept cold (0°C, using acetone-ice mixture) during the extent of distillation. Finally, liquid-liquid extraction was performed, with diethyl ether (309966, (CH3CH2)2O, for HPLC, ≥ 99.9%, inhibitor-free, Sigma- Aldrich, St. Louis, Mo, USA) as the organic solvent, to recover volatiles. The organic fraction was dried in a rotatory evaporator until an oily substance (invariably, mixtures of volatile organic compounds) was obtained. Only ripened, healthy (without visible scarring), cashew apples were processed. In the samples of cashew apples, paraffin was added to avoid foaming of the non-volatile material in the flask during processing. Type III water with a final conductivity of <10 μScm-1 was obtained using a RiOSTM system (EMD Millipore, Billerica, MA, USA). Oil yields ranged between 0.13 and 0.74 mL oil per 100 grams fresh material; independent components were separated, identified, and quantified using gas chromatography (GC, Agilent 7820A) and mass-spectrometry (MS, Agilent 5977E mass spectrometer). The GC-MS system deployed was equipped with a J&W DBWAX microbore column of 10 m length, 0.1 mm diameter, 0.1 µm film thickness (Agilent Technologies). The carrier gas was helium at a constant flow of 0.3 mL min-1. The column oven temperature was initially held at 50ºC for 0.34 min, then programmed to reach 200ºC at a rate increase of 72.51°C min-1 and held for 0.17 min and, a final temperature increment programmed to reach 230°C at a rate of 8.7°C min-1, held for 7.9 minutes. The total run time was 13.93 min. The split ratio was adjusted at 30:1. The injector temperature was set at 250°C. The mass range was 50-450 m/z. Electron energy was set at 70 eV, 150°C, at positive polarity. Peaks were identified through comparison to spectra contained in the NIST library 14 (Scientific Instrument Services, Inc. ™, Ringoes, NJ, USA). Only hits with a match factor of 80% or above were considered. 2-Hydroxyisocaproic acid (99%, 219819, 3.26 min, M+ 132.1 m/z), 3-hydroxy-1,3- diphenylpropan-1-one(99%, 137731, 3.11 min, M+ 226.1 m/z), 1,4-xylene (≥ 99.5%, 95680, 1.34 min, M+ 107.0 m/z),1-nonadecene(≥ 97%, 284831, 4.19 min, M+ 266.1 m/z). Geraniol (≥99.0%, 48798, 5.85 min, M+ 154.1 m/z) was used as an internal standard. All reagents were acquiredfrom Sigma-Aldrich except 5-methylpyrogallol (Santa Cruz Biotechnology, sc-475292, M+ 140.0 m/z, Dallas, Texas, USA). An example chromatogram is shown (Figure 1A). Additionally, Kovats retention indices (RI) were calculated according to Van den Dool and Kratz [13] using as references a C7 - C30 saturated alkanes (Sigma-Aldrich, 49451-U, 1 000 μg mL-1 each component in hexane, Table 1 and 2). Reference RI values for each compound were also tabulated when available. The pure solvent was injected and used as a blank and subtracted from the oil chromatograms to rule out artifacts. Table 1: Essential oil composition obtained from cashew tree leaves Analyte Mean ± SD (relative abundance, g/100 g)a RI literatureb RI calculatedc Major components 1 2,4-di-tert-butylphenol 14.14 ± 2.47 2280 2277 2 3-Hydroxy-1,3-diphenylpropan-1-one 6.59 ± 1.84 - 1647 3 5-Methylpyrogallol 6.10 ± 0.78 - 1726 4 1,3-Xylene 5.91 ± 1.25 1132 1132 5 1-Nonadecene 5.71 ± 2.87 1938 1938 6 Methyl (E)-octadec-9-enoate 4.07 ± 0.68 2426 2420 7 1-Tridecene 3.75 ± 1.30 1337 1339 8 Methyl (E)-octadec-15-enoate 3.55 ± 0.18 - 2536 9 2-Phenylacetaldehyde 3.52 ± 0.47 1648 1649 10 1-Heptadecanol 3.13 ± 0.16 2451 2457 11 1-Docosene 2.96 ± 1.54 - 2219 12 1,4-Xylene 2.86 ± 1.95 1130 1133 13 (E)-5-Octadecene 2.46 ± 1.97 1811 1814 14 1-Undecanol 2.26 ± 0.80 1840 1836 15 6-Methylhept-5-en-2-one 1.80 ± 0.28 1341 1341 16 1-Octanol 1.78 ± 0.10 1565 1567 17 1-heptyl-2-methylcyclopropane 1.76 ± 0.13 - 1626 18 9-methyl-1-Decene 1.75 ± 0.28 - 1303 19 1-O-octadecyl 2-O-prop-2-enyl oxalate 1.54 ± 1.29 - 2992 20 2,6-bis(1,1-dimethylethyl)-4-hydroxy-4-methyl-2,5- cyclohexadien-1-one 1.44 ± 0.29 2117 2113 21 (Z)-Hex-3-en-1-ol 1.38 ± 0.46 1386 1387 ~ 3 ~ American Journal of Essential Oils and Natural Products 22 2-(4-methoxyphenyl)propan-2-ol 1.34 ± 0.47 - 1626 23 Methyl Octadecanoate 1.25 ± 0.53 2419 2417 24 5,5-Dimethylhex-1-ene 1.21 ± 0.56 1204 1202 25 Cyclotetradecane 1.15 ± 0.06 2073 2074 26 Methyl hexadecanoate 1.14 ± 0.59 2202 2203 27 β-Caryophyllene 1.07 ± 0.12 1591 1590 28 (E)-pent-3-en-2-one 1.04 ± 0.30 1133 1130 29 1-Hexadecanol 0.99 ± 0.22 2363 2359 30 2,3 Pyridinedicarboxylic anhydride 0.95 ± 0.32 - 815 31 8-methyl-1-undecene 0.94 ± 0.33 1124 1123 32 Cyclopentyl 4-ethylbenzoate 0.85 ± 0.19 1835 1821 33 1-Decanol 0.85 ± 0.09 1760 1751 34 Nonylcyclopropane 0.84 ± 0.05 - 1075 35 Octyl propanoate 0.80 ± 0.25 1542 1540 36 Phenylmethanol 0.64 ± 0.05 - 1679 37 (E)-5-methylundec-4-ene 0.60 ± 0.08 - 1880 38 2-(4-methylcyclohexa-2,4-dien-1-yl)propan-2-ol 0.59 ± 0.13 1814 1810 39 1-Tridecyn-4-ol 0.57 ± 0.27 - 2494 40 3-Methoxypropane-1,2-diol 0.54 ± 0.23 - 1562 41 Bis(2-ethylhexyl) hexanedioate 0.46 ± 0.32 1892 1879 42 N-hydroxyacetamide 0.40 ± 0.49 - 1239 43 (Z)-3-Hexenyl benzoate 0.40 ± 0.39 2093 2081 44 3-Methylhex-1-ene 0.34 ± 0.08 - 1562 45 Oxalic acid, allyl hexadecyl ester 0.32 ± 0.09 - 1782 46 2-Methylbutan-1-ol 0.31 ± 0.04 1208 1203 47 5-methylidenetridecane 0.30 ± 0.11 - 940 48 1,1,3-Trimethylcyclopentane 0.28 ± 0.04 1219 1221 49 1-Methyl-2-(3-methylpentyl)cyclopropane 0.25 ± 0.08 - 1448 50 N-nitro-1-pentanamine 0.23 ± 0.06 1390 1384 51 1,3-dimethoxypropan-2-ol 0.22 ± 0.09 1403 1416 52 Octylcyclopropane 0.19 ± 0.11 - 794 53 Ethenyl-3-methyloxirane 0.15 ± 0.04 - 1001 54 Diphenylmethanone 0.15 ± 0.03 - 1313 55 1,1-dimethyl-cyclopentane 0.12 ± 0.05 869 866 56 1-methyl-cyclopentene 0.10 ± 0.12 782 770 Total area sum, g/100 g Alkanes/Cycloalkanes 34.84 ± 1.88 Carboxylic acids 34.57 ± 1.18 Alcohols/Phenols 15.33 ± 1.56 Ketones 11.02 ± 0.86 Aldehydes 3.52 ± 0.20 aStandard deviation (SD) reported as the result of the variation among five independent batches of leaves. bKovats standard polar retention indices obtained from the NIST Standard Reference Database 1A v17. https://www.nist.gov/srd/nist-standard-reference-database-1a-v17. cNonisothermal Kovats retention indices calculated as RIx = 100n + 100(tx-tn)/(tn+1 − tn). Table 2: Essential oil profile and composition obtained from cashew apples Analyte Mean ± SD (relative abundance, g/100 g)a RI literatureb RI calculatedc Major compounds 1 2-Hydroxy-4-methylvaleric acid 13.89 ± 0.63 - 2151 2 1,4-xylene 12.08 ± 1.30 1130 1137 3 1-Nonadecene 9.84 ± 4.89 1938 1942 4 1-Docosene 7.83 ± 4.24 - 2049 5 (E)-3-Octadecene 6.47 ± 2.03 1896 1904 6 2,4-Ditert-butyl-phenol 6.35 ± 0.39 2280 2291 7 (E)-5-Eicosene 6.20 ± 2.37 2047 2055 8 1-Tridecene 6.10 ± 2.75 1337 1338 9 Cyclotetradecane 1.89 ± 0.30 - 2249 10 Methyl 14-methylpentadecanoate 1.64 ± 0.47 2166 2166 11 1-Hexadecanol 1.63 ± 0.60 2363 2363 12 1-Decanol 1.54 ± 0.22 1760 1762 13 1-O-octadecyl 2-O-prop-2-enyl oxalate 1.54 ± 0.14 - 2393 14 1,1-Dimethylcyclopentane 1.53 ± 0.77 - 1506 15 1,1,3-Trimethylcyclopentane 1.44 ± 1.14 - 1481 16 Nonylcyclopropane 1.39 ± 0.85 - 861 17 Methyl (Z)-N-hydroxybenzenecarboximidate 1.34 ± 0.46 - 1184 18 2,3 Pyridinedicarboxylic anhydride 1.26 ± 0.57 - 653 19 3,4-Dimethylpentan-1-ol 1.26 ± 0.40 1412 1414 20 Methyl hexadecanoate 1.04 ± 0.05 2202 2204 ~ 4 ~ American Journal of Essential Oils and Natural Products 21 3-Methoxypropane-1,2-diol 0.95 ± 0.20 - 1273 22 3,7-Dimethyloct-1-ene 0.92 ± 0.34 - 1044 23 2-methyloct-1-ene 0.92 ± 0.30 - 636 24 2-(1,2,4-triazol-1-yl) ethanol 0.90 ± 0.36 - 1481 25 Hexanedioic acid bis (2-ethylhexyl) ester 0.86 ± 0.17 1892 1893 26 5-methyl-(triazolylethanol)-1-hexanol 0.86 ± 0.06 1442 1438 27 Methyl octadecanoate 0.83 ± 0.28 2419 2419 28 2-Methylbutan-1-ol 0.80 ± 0.36 1208 1205 29 1-(3,4-dihydro-2H-pyrrol-5-yl) ethenone 0.72 ± 0.32 - 1273 30 Pentadecanal 0.69 ± 0.06 2016 2019 31 [(Z)-dodec-9-enyl] acetate 0.64 ± 0.40 1986 1988 32 2,2,4-Trimethylpentane 0.58 ± 0.24 698 698 33 4-Methylhexan-1-ol 0.47 ± 0.06 1414 1410 34 6-Methylhept-1-ene 0.44 ± 0.07 - 1044 35 (3S)-3,4-dimethylpentan-1-ol 0.43 ± 0.07 1412 1414 36 (Z)-dodec-2-en-1-ol 0.36 ± 0.17 - 1642 37 Octylcyclopropane 0.33 ± 0.18 - 636 38 4-methylhexan-2-ol 0.32 ± 0.22 - 819 39 2,2,3-trimethylcyclobutan-1-one 0.26 ± 0.08 - 1252 40 (E)-2,2-dimethyldec-3-ene 0.22 ± 0.05 - 1731 41 1-Hexanol 0.21 ± 0.12 1360 1360 42 N-hydroxyacetamide 0.21 ± 0.12 - 1413 43 (Z)-3-Hexen-1-ol 0.20 ± 0.05 1386 1376 44 3-hydroxybutan-2-one 0.18 ± 0.08 1289 1296 45 4,4-Dimethylpent-1-ene 0.18 ± 0.08 - 1672 46 (Z)-2-Butene-1,4-diol 0.15 ± 0.07 - 1667 47 1,3-Dimethoxypropan-2-ol 0.14 ± 0.12 - 942 Total area sum, g/100 g Alkanes/Cycloalkanes 59.90 ± 3.35 Carboxylic acids 22.66 ± 1.47 Alcohols/Phenols 16.67 ± 1.27 Ketones 1.16 ± 0.05 aStandard deviation (SD) reported as the result of the variation among five independent batches of cashew apples.bKovats standard polar retention indices obtained from the NIST Standard Reference Database 1A v17. https://www.nist.gov/srd/nist-standard-reference-database-1a- v17.cNon-isothermal Kovats retention indices calculated as RIx = 100n + 100(tx-tn)/(tn+1 − tn). 2.3 Cashew nut fatty acid profiling Twenty-five nuts per batch (five different batches in total) were mechanically pressed using an oil expeller (PITEBA, Scheemb derzwaag, Scheemda, The Netherlands). Twenty µL of the resulting oil was diluted 100-fold using diethyl ether, after mild methanolysis, using an organic catalyst (tetramethyl ammonium hydroxide, 334901, 25 wt. % solution in methanol, Sigma-Aldrich, St. Louis, MO, USA), the resulting methyl-ester fatty acids were separated, identified, and quantified using gas chromatography using the conditions mentioned at 2.2.Tetradecanoic (6.16min; M+ 227.6 m/z), pentadecanoic (6.72 min; M+ 243.4 m/z), hexadecenoic (7.58 min; M+ 256.3 m/z), octadecanoic (9.70 min; M+ 285.5 m/z), 9Z-octadecenoic (7.78 min; M+ 284.1 m/z), and (Z,Z)-9,12- octadecadienoic (10.86 min; M+ 280.0 m/z) acids from Nu- Chek Prep (Elysian, MN, USA) were used as standards. An example chromatogram is shown (Figure 1B). Fig 1: Example chromatograms of A. Fatty acid profile from cashew nut B. Essential oil (volatile) profile of cashew tree leaves. MM2 minimized 3D structures of the most abundant compounds found are shown for each panel ~ 5 ~ American Journal of Essential Oils and Natural Products 2.4 Total carotenoid content and HPLC carotenoid identification assay. Carotene assays were performed according to Biehler and coworkers [14] with some modifications. Briefly, 5g of a thawed sample weighed into a 50-mL high-density polyethylene centrifuge tube (BD Biosciences, CA, and USA). Then, fivemL ofmethanol (chromatographic grade, J.T. Baker, Avantor Materials, PA, and USA) and 1 g magnesium carbonate (USP, M7179, Sigma-Aldrich, and St. Louis, MO, USA) were added. The mixture was forced into contact and homogenized using a digital Ultra-turrax® at 18 000 rpm (T25, IKA®WerkeGmbH& Co. KG, Staufen im Breisgau, Germany) during 1-3 min. Later, mixtures were incubated for 15min on ice; samples were centrifuged(Thermo Scientific TM Sorvall TM ST 16R Thermo Fisher Scientific, Inc. Waltham, MA, USA) at 10ºC for 5 min at 2500 × g. The supernatant was decanted into another 50-mL centrifuge tube, extraction was repeated twice with eight mL of a mixture of hexane: acetone (1:1, both chromatographic grade, J.T. Baker, Avantor Materials, PA, and USA) and organic fractions were combined. To the combined extracts, 25 mL of saturated aqueous sodium chloride (ACS grade, 1064045000, Sigma- Aldrich, St. Louis, MO, USA) solution was added, and the mixture was shaken. The supernatant hexane phase was transferred into a 50-mL centrifuge tube, and the lower aqueous phase was re extracted with eight mL of hexane and combined with the 1st extract. Hexane extracts were weighed exactly for volume determination. A 5-mL aliquot was then pipette from the combined extracts into a 12-mL glass vial, evaporated to dryness under vacuum at 10ºC (Centrivap, LABCONCO, Kansas City, MO, USA), and purged with argon (ultra-high purity, 99.999%, Praxair, Danbury, CT, USA), and sealed. 2.4.1 Spectrophotometric analyses Dried extracts were reconstituted in 1 to 10 mL of hexane and sonicated for 2 min. Absorbance values are measured at 450 nm using a 1-mL quartz cuvette in a UV/Visible spectrophotometer (Shimadzu Pharmaspec UV-1700); concentrations achieved by comparing against a 0.5 to 10 mg β-carotene L-1 7-point standard calibration curve. 2.4.2 Chromatographic analyses All samples were stored at −70ºC until HPLC analysis; dried extracts were re dissolved using one mL MTBE and transferred to an HPLC 2 mL capacity vial. Chromatographic separation was achieved using a 150 mm x 4.6 mm, 5 µm analytical column (YMC Co. Ltd., Carotenoid C30, Kyoto Prefecture, Japan) and a solvent system that included MeOH (solvent A) and MTBE.A modular HPLC system (Shimadzu Prominence, Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) equipped with a degasser (DGU-20A5), quaternary pump (LC-20AT), an autosampler (SIL-20A HT), a system controller (CBM-20A), a column oven (CTO-20A), and photodiode array detector (SPD-M20AV) was used for analysis. Chromatographic data management was performed using LC Solutions (Version. 5.2). Gradient elution was set as follow: 0-5 min 80% A (5 min), 5-7 min 73% A, 7-15 min 62.5% A, 15-20 62,5 A, 20-30 min 45% A, 30-35 10% A, 35- 40 10% A, 40-45 min 80% A. Solvent flow and column compartment temperature, detector wavelength and sample injection volume were kept constant during elution at 0.6 mL min-1, 30°C, 450 nm (using 472 nm as reference wavelength), and 3 µL, respectively. 2.5 Lipophilic ORACFL assay The determinationwas performed according to prior and co- workers [15]. Briefly, ten µL leaves and cashew apple essential oil was dissolved in 25 µL of acetone and then diluted with 65 µL of a seven g/100 g water and acetone (1:1) solution of randomly methylated β-cyclodextrin. All aliquots are mixed in a black flat-bottomed polystyrene 96-well micro liter plates (Thermo Scientific™ Nunc™ FluoroNunc™, Roskilde, Denmark). Forty µL of fluorescein solution was added by injectors in the micro plate reader, followed by 150 µL of 2, 2′-azobis (2-amidino-propane) dihydrochloride (17.2 mg mL- 1, 9.4 µmol well-1); readings were initiated immediately. Fluorescence was measured with a SynergyTMBiotek HT microplate reader at λex= 485 nm and λem= 520 nm andthe Gen 5TMsoftware (BioTek Instruments Inc., Winooski, VT, USA).Analyses were performed in triplicate. 2.6 Proximate analysis of cashew nutshell Dry matter (DM, loss of drying/moisture), crude protein (CP), fat (EE), fiber (CF), and ash, as well as calcium, phosphorus, neutral detergent fiber (NDF), Acid detergent fiber (ADF), lignin, and gross energy assayswere performed to assess the nutritional quality of each of the animal by-products meals collected. All tests were performed using ISO 17025 accredited methods based on AOAC 930.15, 990.03, 920.39, 962.09, 942.05, 968.08/975.03/985.35, 965.17/986.24, 935.13, 2002.04, 973.18 and ISO 9831:1998 respectively. Neutral and aciddetergent insoluble nitrogen (NDIN/ADIN) were determined as previously described [16]. 3. Results and Discussion 3.1 Characterization of cashew tree leaves and apples essential oil Few articles have described volatile components of cashew leaves or apple reports. Experiments have been conducted and reported from Brazil [17, 18], and Venezuela [19] with compounds such as palmitic/oleic acid, ethyl 2-hydroxy-4- methylpentanoate, and car-3-ene recounted for pseudo fruits, respectively. In our case, cashew tree leaves exhibited 2, 4-di- tert-butylphenol, 3-hydroxy-1, 3-diphenylpropan-1-one, and 5-methylpyrogallolin 14.14, 6.59, and 6.10 g/100 g, respectively. On a previous species, we already discussed the presence of 2, 4-di-tert-butylphenol in tree leaves [20]. However, in this case, cashew as an evergreen tree, the compound may be related to stress and UV injury as it grows near the coasts where temperatures are relatively high. Additionally, 3-hydroxy-1, 3-diphenylpropan-1-one may be responsible for some of the leaves yellow-tones coloration. As this is naturally a highly resinous tree, some of these aromatic compounds may be used as building blocks for gum-resins, then, the pyrogallol related compound may be a precursor for more complex plant structures such as hydrolyzabletannins [21]. Further, chalcone analogs have been described previously in plants as a response to different stimuli and have several described physiological functions [22]. On another hand, cashew apple volatile compounds included 2-hydroxy-4- methylvaleric acid,1,4-xylene, and 1-nonadeceneat relative concentrations of 13.89, 12.08, and 9.84 g/100 g, respectively.2-Hydroxy-4-methylvaleric acid has been isolated previously from wine [23], and a similar compound was also isolated from cashew apple [17]. Also, recently, 1, 4- xylene has been isolated from Chrysophyllum albidum G. Don [24]. Interestingly, 1-nonadecene has been described recently as a component of the solvent extracts from Zygophyllum coccineum L. leaves [25] and Terminalia travancorensis Wight ~ 6 ~ American Journal of Essential Oils and Natural Products &Arn bark [25]. The sum of the four most abundant components represented 32.74% in tree leaves and 43.64% for cashew apple essential oil (Figure 2 A, B), and 96.3% of the fatty acid profile from mechanically extracted cashew nut oil (Figure 2 C). Fig 2: Distribution of the four most abundant compounds found in A. cashew tree leaves essential oil B. cashew apple essential oil and C. fatty acid profile from mechanically extracted cashew nut oil.Key:2,4-di-tert-butylphenol (2,4-DTBP), 3-hydroxy-1,3-diphenylpropan-1- one (3-H-1,3-DP), 2-hydroxy-4-methylvaleric acid (2-H-4-MVA) 3.2 Fat fatty profile of the cashew nut We obtained a profile that included oleic and linoleic acids as most prevalent with 56.26 and 14.50 g/100 g, respectively. Monounsaturated fatty acids represent an average of 71.97 ± 1.49 (Table 3). These values are in line with those obtained previously [27]. The fatty acid composition containinga combination of palmitic (C16:0) and oleic acids (C18:1), hint toward a potential use of cashew nut oil as long chain fatty acid supplement for dairy cows [28]. Table 3: Fatty acid profile analysis of fresh cashew seeds Fatty acida (shorthand nomenclature) Mean ± SD (total area sum, g/100 g) C18:1 56.26 ± 3.14 C18:2 14.50 ± 1.48 C18:0 14.28 ± 1.05 C16:0 11.26 ± 0.41 Sum of saturated fatty acids 26.69 ± 1.13 Sum of monounsaturated fatty acids (MUFA) 71.97 ± 1.49 Sum of polyunsaturated fatty acids (PUFA) 1.34 ± 0.29 aIdentified compounds with a total area sum ≤ 1 g/100 g also included C7:0, C8:0, C9:0, 9:0-diacid, C10:0, 9c-C12:1,C14:0, 11c- C16:1, C17:0, C20:0, C22:0 and C24:0. 3.3 Carotenes Total carotenoids were measured on three independent batches of colored cashew apples, (2.93 ± 0.07) mg β- carotene/100 g fresh material. Additionally, HPLC carotenoid analysis show six major pigment signals evidencing that, for the most part, the pigment in cashews is imparted, in fact, by α-carotene (retention time of 14.351, figure 3) and β-carotene (retention time of 17.111, figure 3) representing 0.24 mg (8.2%) and 1.25 mg (42.5%)per 100 g sample, respectively. This data is in line with carotenoid contents for another Anacardiaceae genus [29, 30]. β-Carotene esters can be observed24 to 28 min region (Figure 3). However, no conclusive identification was achieved for these signals. Other spectrometric approaches (e.g., NMR, MS) may be applied in the future, to characterize said components. ~ 7 ~ American Journal of Essential Oils and Natural Products Fig 3: Chromatographs of A.Chloroform diluted standards of β-cryptoxanthin (Rt = 11.344 min), β-carotene (Rt = 17.047 min) and lycopene (Rt = 36.891 min). B. Cashew apple chloroform extract exhibiting α-carotene (Rt = 14.351 min), β-carotene (Rt = 17.111 min, area sum 8.20%), unknown signals at 24.201 (area sum 7.46%), 25.825 (area sum 11.94%), 26.183 (area sum 8.95%) and 27.925 min (area sum 20.89%) 3.4 Cashew apple and leaves essential oil antioxidant potential Based on the compounds found during the profiling, we used a lipophilic ORAC using Trolox equivalents as a suggestive assay to assess both cashew apple and leaves essential oil potential for radical scavenging capabilities; we obtained average values of (2 291.2 ± 35.6) and (6 158.9 ± 136.8) μmol TE/100 g, respectively. Cashew apple has already been reported as a cellular mediated and direct scavenging potential [10]. Aromatic and phenol-based structures found in the leaves’ oil account for the high in vitro potential for radical scavenging. 3.5 Cashew outer shell, potential as animal feed As expected nutshell exhibits a profile rich in fiber (NDF and ADF 38.46 and 27.67 g/100 g, respectively). Also, crude fat content is considerable, i.e., 16.00 g/100 g (Table 4). The average energy of the cashew residue that is digestible corresponds to 57.3%, being moderately useful. Cashew nut shell is a remnant available in the country which operation units needed for processing are few, which means that meal production from this residue is cost-effective, thus making its use asanimal feed, especially ruminants, a feasible option. Cashewnutshell meal could be combined with other residues or forages to supplement the diet of the animal. However, an additional the elimination of the irritant anacardic acid/cashew ~ 8 ~ American Journal of Essential Oils and Natural Products nut shell liquid may be necessary before hand. In this regard, other studies have included cashew nut shell liquid [31] and cashew apple essential oil into feed as an additive [32, 33] successfully. Cashew bagasse and pulp has already been considered as a feed ingredient for ruminants [34], and cashew nut testa has been considered as pig feed [35]. Using an evaluation of ration balancing system (National Research Council (NRC) Nutrient Requirements [36]), we concluded that a maximum of 4 kg (ration inclusion of ca. 8.4%) of cashew nut outer shell can be incorporated to a dairy cattle’s diet if an animal of 450 kg with daily milk production of 16 kg per day and a ration that included 20 kg silage, 1.5 kg citrus pulp, 6 kg compound feed, 1 kg molasses and 10 kg star grass [Cynodon dactylon (L.) Pers.] are considered. Table 4: Proximate analysis and bromatological data for cashew nutshell Assay Concentration, g/100 ga Proximate analysis Crude fat 16.00 ± 0.24 Dry matter/Loss on drying 93.73 ± 0.30 Crude protein 5.31 ± 0.26 Crude ash 1.19 ± 0.08 Fiber and protein fractionation Neutral detergent fiber (NDF) 38.46 ± 2.07 Acid detergent fiber (ADF) 27.67 ± 1.55 Lignin 3.91 ± 0.61 Neutral detergent nitrogen (NDIN) 0.42 ± 0.06 Acid detergent nitrogen (ADIN) 0.29 ± 0.05 Mineral Calcium (mg kg-1) 533.01 ± 101.22 Phosphorus (mg kg-1) 415.23 ± 39.14 Energy input Gross energy (kJ kg-1) 4 760.51 ± 355.74 aStandard deviation (SD) reported as the result of the variation among five independent batches of cashew nut shells. 4. Conclusion Essential oils fromleavesand cashew apple demonstrated the presence of phenolic and aromatic compounds and hence demonstrate a potential for radical scavenging, other applications or biological activities may further be investigated. As a colored fleshy product, cashew has a good source of provitamin a, which may improve its antioxidant potential drastically. Cashew nutshell is the residue left after the kernel has been removed for toasting and future consumption or oil extraction, we demonstrated that the residue has the potential of being repurposed. Fatty acid from nut oil has an excellent profile and the potential both as an animal feed supplement and oil for human consumption especially as it is highly monounsaturated. We contribute new comparative data on a tree that is common in the coastline of Costa Rica. 5. Acknowledgments The Office of the Vice Provost for Research of the University of Costa Rica supported this initiative, financially, using the grant B6257.Adrian Martinez and Astrid Leiva are acknowledged for their help with the calculation of digestible energy and energy balance, respectively. Special thanks to Carolina Cortés and Graciela Artavia for their contributions measuring β-carotene, total carotenoids, and ORAC. 6. References 1. Morton JF. Cashew apple, Anacardium occidentale L. Fruits of warm climates. Lafayette, Indiana. Center for New Crops and Plant Products, Department of Horticulture and Landscape Architecture, Purdue University, 1987, 239-240. 2. Zamora N, Jiménez Q, Poveda L. Árboles de Costa Rica Volumen 2. Heredia. Instituto Nacional de Biodiversidad (IN Bio), 2000, 190. 3. Clay, JW. World agriculture and the environment: a commodity-by-commodity guide to impacts and practices. Washington, DC. Island Press, 2004, 263-282. 4. Ros E. Health Benefits of Nut Consumption. Nutrients. 2010; 2:652-682. 5. Jaiswal Y, Naik V, Tatke P, Gabhe S, Vaidya A. Pharmacognostic and preliminary phytochemical investigations of Anacardium occidentale (Linn.) leaves. International Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4:625-631. 6. Fadeyi OE, Olatunji GA, Ogundele VA. Isolation and characterization of the chemical constituents of Anacardium occidentale cracked bark. Natural Products Chemistry and Research. 2015; 3:192. 7. Baptista A, Gonçalves RV, Bressan J, Peluzio MCG. Antioxidant and antimicrobial activities of crude extracts and fractions of cashew (Anacardium occidentale L.), cajui (Anacardium microcarpum) and pequi (Caryocar brasilienseC). A systematic review. Oxidative Medicine and Cellular Longevity. 2018; 2018:1-10. 8. Agedah CE, Ba Wo DDS, Nyananyo BL. Identification of antimicrobial properties of cashew, Anacardium occidentale L. (Family Anacardiaceae). Journal of Applied Sciences and Environmental Management. 2010; 14:25-27. 9. Tchikaya FO, Bantsielé GB, Kouakou-Siransy G, Datté JY, Ypo PA, Zirihi NG et al. Anacardium occidentale Linn. (Anacardiaceae) stem bark extract induces hypotensive and cardio-inhibitory effects in experimental animal model. 2011; 8:452-461. 10. González E, Vaillant F, Pérez A, Rojas G. In vitro cell- mediated antioxidant protection of human erythrocytes by some common tropical fruits. Nutrition and Food Sciences. 2012; 2:139. 11. Ogunwolu SO, Henshaw FO, Mock H-P, Santros A, Awonorin SO. Functional properties of protein concentrate and isolates produced from cashew (Anacardium occidentale L.) nut. Food Chemistry. 2009; 115:852-858. 12. Yao G-I, Chai Y, Chen J, Wu Y-G. Separation and identification of ACE inhibitory peptides from cashew nut (Anacardium occidentale Linnaeus) protein. International Journal of Food Properties. 2017; 20:S981- S991. 13. van Den Dool H, Kratz PD. A generalization of the retention index system including linear temperature programmed gas—liquid partition chromatography. Journal of Chromatography A. 1963; 11:463-471. 14. Biehler E, Mayer F, Hoffmann L, Krause E, Bohn T. Comparison of 3 spectrophotometric methods for carotenoid determination in frequently consumed fruits and vegetables. Journal of Food Science. 2010; 75:C55- C61. 15. Prior RL, Hoang H, Gu L, Wu X, Bacchiocca M, Howard L et al. Assays for hydrophilic and lipophilic antioxidant capacity(oxygen radical absorbance capacity (ORACFL)) of plasma and other biological and food samples. Journal of Agricultural and Food Chemistry. 2003; 51:3273- 3279. ~ 9 ~ American Journal of Essential Oils and Natural Products 16. Licitra G, Hernández TM, Van Soest PJ. Standardization of procedures for nitrogen fractionation of ruminant feeds. Animal Feed Science and Technology. 1996; 57:347-358. 17. Maia JGS, Andrade EHA, Zoghbi MGB. Volatile constituents of the leaves, fruits, and flowers of cashew (Anacardium occidentale L.) Journal of Food Composition and Analysis. 2000; 13:227-232. 18. Bicalho B, Rezende CM. Volatile Compounds of Cashew Apple (Anacardium occidentale L.) Zeitschrift für Naturforschung. 2001; 56c:35-39. 19. MacLeod AJ, Troconis NG. Volatile flavor components of cashew ‘apple’ (Anacardium occidentale). Phyto chemistry. 1982; 10:2527-2530. 20. Granados-Chinchilla F, Villegas E, Molina A, Arias C. Composition, Chemical Fingerprinting and Antimicrobial Assessment of Costa Rican Cultivated Guavas (Psidium friedrichsthalianum (O. Berg) Nied. and Psidium guajavaL.) Essential Oils from Leaves and Fruits. Natural Products Chemistry & Research, 2016. 21. Gross GG. Enzymes in the Biosynthesis of Hydrolyzable Tannins In Plant Polyphenols Synthesis, Properties, Significance. Eds. Hemmingway RW, Laks PE. New York. Springer Science, 1992, 43-60. 22. Cazarolli LH, Kappel VD, Zanatta AP, Suzuki DOH, Yunes RA, Nunes RJ. Natural and Synthetic Chalcones: Tools for the Studyof Targets of Action—Insulin Secretagogue or Insulin Mimetic? In the Studies in Natural Products Chemistry Volume 39. Ed. Atta-ur- Rahman, FRS. The Netherlands. Elsevier, 2013, 47-89. 23. Gracia-Moreno E, Lopez R, Ferreira V. Quantitative determination of five hydroxy acids, precursors of relevant wine aroma compounds in wine and other alcoholic beverages. Analytical and Bio analytical Chemistry. 2015; 407:7925-34. 24. Ishola FT, Aboaba SA, Choudhary MI, Ekundayo O. Chemical and Biological Assessments of the Essential Oils of Chrysophyllum albidum G. Don. Journal of Agricultural Science and Technology A. 2017; 7:234- 245. 25. El-Shora HM, El-Amier YA, Awad MH. Antioxidant Activity of Leaf Extracts from Zygophyllum coccineum L.Collected from Desert and Coastal Habitats of Egypt. International Journal of Current Microbiology and Applied Sciences. 2016; 5:635-641. 26. GC-MS analysis of the chloroform extract of bark of Terminalia travancorensis Wight &Arn. (Combretaceae). International Journal of Pharmaceutical Sciences and Research. 2017; 8:794-798. 27. Ryan E, Galvin K, O’Connor TP, Maguire AR, O’Brien NM. Fatty acid profile, tocopherol, squalene and phytosterol content of Brazil, pecan, pine, pistachio and cashewnuts. International Journal of Food Sciences and Nutrition. 2006; 3/4:219-228. 28. Loften JR, Linn JG, Drackley JK, Jenkins TC, Soderholm CG, Kertz AF. Invited review: Palmitic and stearic acid metabolism in lactating dairy cows. Journal of Dairy Science. 2014; 97:4661-4674. 29. Assunção RB, Mercadante AZ. Carotenoids and ascorbic acid from cashew apple (Anacardium occidentale L.): variety and geographic effects. Food Chemistry. 2003; 81:495-502. 30. Khoo H-E, Prasad KN, Kong K-W, Jiang Y, Ismail A. Carotenoids and their isomers: color pigments in fruits and vegetables. Molecules. 2011; 16:1710-1738. 31. López CAA, Lima KRS, Manno MC, Tavares FB, Fernandes Neto DL, Jesús MLC et al. Effects of cashew nut shell liquid (CNSL) on the performanceof broiler chickens.Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 2012; 4:1027-1035. 32. Barreto Cruz OT, Valero MV, Zawadzki F, Rivaroli DC, do Prado RM, Lima BS et al. Effect of glycerine and essential oils (Anacardium occidentale and Ricinus communis) on animal performance, feed efficiency and carcass characteristics of crossbred bulls finished in a feedlot system. Italian Journal of Animal Science. 2014; 13:3492. 33. Valero MV, do Prado RM, Zawadski F, Eiras CE, Madrona GS, do Pardo IN. Propolis and essential oils additives in the diets improved animal performance and feed efficiency of bulls finished in feedlot. Acta Scientiarum. 2014; 36:419-426. 34. Geron LJV, Trautmann-Machado RJ, Moura DC, Marques FM, Souza OM, Paula Junior EH. Cashews, canola, barley, cupuacu and their waste used in ruminant nutrition. PUBVET2013; 7:1549. 35. Donkoh A, Attoh-Kotoku V, Kwame RO, Gascar R. Evaluation of nutritional quality of dried cashew nut testa using laboratory rat as a model for pigs. The Scientific Journal. 2012; 2012:1-5. 36. White RR, Roman-Garcia Y, Firkins JL, VandeHaar MJ, Armentano LE, Weiss WP et al. Evaluation of the National Research Council (2001) dairy model and derivation of new prediction equations. 1. Digestibility of fiber, fat, protein, and non fiber carbohydrate. Journal of Dairy Science. 2015; 100:3591-3610.