Bioactive micro-constituents of ackee arilli (Blighia sapida K.D. Koenig)

CARLOS D. GRANDE-TOVAR DELGADO-OSPINA JOHANNES LUISA F. PUERTA GLORIA C. RODRÍGUEZ GIAMPIERO SACCHETTI ANTONELLO PAPARELLA CLEMENCIA CHAVES-LÓPEZ About the authors

Abstract

Abstract: Ackee (Blighia sapida K. D. Koenig) is an exotic fruit widely consumed in the Caribbean countries. While there is extensive research on the presence of hypoglycin A, other bioactive compounds have not been studied. We identified and quantified the changes in bioactive molecules (total phenol, ascorbic acid, hypoglycin A, squalene, D: A-Friedooleanan-7-ol, (7.alpha.), and oleic acid), antioxidant potential, and volatile compounds during two stages of ripe. A clear reduction in hypoglycin A, ascorbic acid, and total polyphenols during the maturation process were observed. On the contrary, oleic acid, squalene, and D: A-Friedooleanan-7-ol, (7.alpha.) contents increased about 12, 12, and 13 times, respectively with advancing maturity. These bioactive molecules were positively correlated with radical scavenging (DDPH and ABTS). Solid phase microextraction (SPME) and gas chromatography coupled mass spectrometry (GC/MS) analysis revealed more than 50 compounds with 3-penten-2-one and hexanal as the major compounds in the fully ripe stage. The results suggested that ripe ackee arilli could serve as an appreciable source of natural bioactive micro-constituents.

Key words
Antioxidants; Friedooleanan; Hypoglycin; Polyphenols; Squalene


INTRODUCTION

Currently, there is a worldwide interest to generate innovative products with high nutritional and functional properties from exotic fruits. Recent studies have shown that frequent consumption of fruits and vegetables is associated with a reduced risk of chronic diseases because of the large amounts of antioxidant compounds they contain including phenolic compounds, carotenoids, anthocyanins and tocopherols (ContrerasCONTRERAS J, CALDERÓN L, GUERRA E and GARCÍA B. 2011. Antioxidant capacity, phenolic content and vitamin C in pulp, peel and seed from 24 exotic fruits from Colombia. Food Res Int 44: 2047-2053. et al. 2011). In particular, phenols are related to the reduction of different deteriorating processes in the human body through their ability to diminish free radical formation after their consumption (Villa-Rodríguez VILLA J, MOLINA F, AYALA J, OLIVAS G and GONZALEZ G. 2011. Effect of maturity stage on the content of fatty acids and antioxidant activity of ´Hass´avocado. Food Res Int 44: 1231-1237.et al. 2011).

The ackee fruit (Blighia sapida K.) was introduced from West Africa to Central America in the eighteenth century, and, in Jamaica, it became an essential part of Jamaican cuisine and eventually gained status as the national fruit of the country (AtolaniATOLANI O, OLATUNJI G and FABIYI O. 2009. Blighia sapida; the plant and its hypoglycins an overview. J Sci Res 39: 15-25. et al. 2009). The fruit has a significant role in the local and even regional and international diets and economies (BenkebliaBENKEBLIA N and LÓPEZ MG. 2015. Saccharides and fructooligosaccharides composition of green and ripe Averrhoa carambola, Blighia sapida and Spondias dulcis fruits. Food Chem 176: 314-318. and López 2015). In fact, its popularity as a culinary delicacy has increased internationally; the fruit in brine is mainly exported to Canada, the UK, and the USA, generating high revenues ($ 13.971 M US dollars in 2015) for the agricultural sector of the country (STATIN 2017STATIN. 2017. Statical Institute of Jamaica. Exports of traditional and Non-Traditional Commodities. Available at http://statinja.gov.jm/Trade-Econ%20Statistics/InternationalMerchandiseTrade/Newtrademore.aspx Accessed April 1, 2017.
http://statinja.gov.jm/Trade-Econ%20Stat...
). In some other countries of the region, especially the north side of Colombia, it is used for ornamental purposes showing good adaptation to the edaphic and climate conditions.

The fruit is pear-shaped and opens spontaneously in three fragments when ripe. The arilli are the cream coloured edible part with a large black seed attached to the end of each piece. Traditional medicine has shown supposed medicinal properties of the ackee arilli which can be used to cure or relieve symptoms like fever, constipation, skin infections, and dysentery (EkuéEKUÉ MRM, SINSIN B, EYOG-MATIG O and FINKELDEY R. 2010. Uses, traditional management, perception of variation and preferences in ackee (Blighia sapida K. D. Koenig) fruit traits in Benin: implications for domestication and conservation. J Ethnobiol Ethnomed 6: 12. et al. 2010, Olusegum and Olutomi 2013OLUSEGUM J and OLUTOMI P. 2013. Chemical, phytochemical and antimicrobial screening of extracts of B. sapida for agricultural and medicinal relevances. Nat Sci 11: 12-17.). Nevertheless, the ackee fruit has some disadvantages since it contains two toxic molecules depending on the maturation stage, hypoglycin A (L-α-amino-β-methylene cyclopropyl propionic acid) and hypoglycin B (γ-L-glutamyl-α-amino-β-methylene cyclopropyl propionic acid). Both are present in the seeds of the fruit, but only hypoglycin A occurs in the arilli (Bowen-ForbesBOWEN-FORBES CS and MINOTT DA. 2011. Tracking hypoglycins A and B over different maturity stages: implications for detoxification of ackee (Blighia sapida KD Koenig) fruits. J Agr Food Chem 59: 3869-3875. and Minott 2011, GaillardGAILLARD Y, CARLIER J, BERSCHT M, MAZOYER C, BEVALOT F, GUITTON J and FANTON L. 2011. Fatal intoxication due to Ackee (Blighia sapida) in Suriname and French Guyana. GC-MS detection and quantification of hypoglycin- A. Forensic Sci Int 206: e103-e107. et al. 2011). The concentration of hypoglycin A is high in the green unripe fruit, but declines as the ripening process advance making it edible. Ingestion of unripe fruit can lead to a toxic condition called “Jamaican vomiting disease” (BlakeBLAKE O, BENNINK M and JACKSON J. 2006. Ackee (Blighia sapida) hypoglycin A toxicity: Dose response assesment in laboratory rats. Food Chem Toxicol 44: 207-213. et al. 2006).

On the other hand, it has been suggested that a low residual concentration of hypoglycin in ackees may be useful in the development of new therapies in people with specific diseases. The following administration (four weeks) from 100 to 400 mg/kg bodyweight of methanolic extract of Blighia sapida leaves reduced blood glucose level at all doses used and prevented oxidative stress and dyslipidemia in alloxan-induced diabetic rats (Oloyede et al. 2014OLOYEDE OB, AJIBOYE TO, ABDUSSALAM AF and ADELEYE AO. 2014. Blighia sapida leaves halt elevated blood glucose, dyslipidemia and oxidative stress in alloxan-induced diabetic rats. J Ethnopharmacol 157: 309-319.). A great part of the research on the ackee fruit has been focused on the study of hypoglycin A (HGA) and B (HGB), but studies regarding the presence of other phytochemical groups are scarce (Benkeblia and López 2015, Garg and Mitra 1967GARG HS and MITRA CR. 1967. Blighia sapida I. Constituents of the fresh fruit. Planta Med 15: 74-80., AntwiANTWI S, MARTEY ONK, DONKOR K and NII-AYITEY LK. 2009. Anti-diarrhoeal activity of Blighia sapida (Sapindaceae) in rats and mice. J Pharmacol Toxicol 4: 117-125. et al. 2009, Dossou et al. 2014DOSSOU VM, AGBENORHEVI JK, COMBEY S and AFI-KORYOE S. 2014. Ackee (Blighia sapida) fruit arils: Nutritional, phytochemicals and antioxidant properties. Int J Nutr Food Sci 3: 534-537.). Additionally, little is known about the chemical changes occurring during the ripening process of the fruit. Therefore, the present study focused on the identification of some bioactive micro-constituents in ackee arilli from Colombia and their variations during two different ripening stages.

MATERIALS AND METHODS

PLANT MATERIAL

The ackee fruits were randomly collected from trees in Los Farallones National Park (Valle del Cauca, Colombia) and classified into two stages of maturity according to the scale described by Bowen-Forbes and Minott (2011). The fruits were selected according to their appearance, discarding bruised or rotten fruit. Finally, 10 fruits per tree were selected from 20 different trees (Fig.1). The arilli portion was separated from the seeds and lyophilised.

Figure 1
Ackee fruit at different ripening stages. a) Maturation Stage 1 closed fruit, green pod, and green seed and b) Maturation Stage 5, ripe fruit with exposed arilli, open pods, orange-red, and black seeds.

ANTIOXIDANT ACTIVITY

The extraction procedure described by Pérez et al. (2008)PÉREZ J, ARRANZ S, TABERNERO M, DÍAZ E, SERRANO J, GOÑI I and SAURA F. 2008. Updated methology to determine antioxidant capacity in plant food, oils and beverager: Extraction, measurement and expression of results. Food Res Int 41: 274-285. was followed with some modifications: four hundred milligrams (400 mg) of lyophilised ackee arilli were mixed in a vial containing 16 mL of methanol-water (1:1 v/v) with constant stirring for 1 hour. The tubes were centrifuged (2241 × g for 15 min at 25°C), and the supernatant was recovered and filtered (Whatman No. 1 filter). A volume of 16 mL of an acetone-water solution (7:3 v/v) was added to the residue and was then stirred and centrifuged under the same conditions. The supernatant was recovered again.

ABTS assay

The radical scavenging activity was measured by the ABTS radical cation discolouration assay described by Re et al. (1999)RE R, PELLEGRINI N, PROTEGGENETE A, PANNALA A, YANG M and RICE-EVANS C. 1999. Antioxidant activity applying an improved ABTS radical catión decolorization assay. Free Radical Bio Med 26: 1231-1237., as µmol of Trolox equivalents per gram of fresh sample and was calculated by the ratio of the correlation coefficient of the dose-response curve of the sample and the correlation coefficient of the dose-response curve of the Trolox standard.

DPPH radical scavenging activity assay

The radical scavenging activity in the different extracts of ackee arilli was measured using the stable free radical DPPH (Brand-WilliamsBRAND-WILLIAMS W, CUVELIER ME and BERSET C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol 28: 25-30. et al. 1995). The procedure described by Villa et al. (2011)VILLA-RODRÍGUEZ J, MOLINA-CORRAL F, AYALA-ZAVALA F, OLIVAS G and GÓNZALEZ-AGUILAR G. 2011. Effect of maturity stage on the content of fatty acids and antioxidant activity of ´Hass´ avocado. Food Res Int 44: 1231-1237., was followed with some modifications. The radical solution was prepared by mixing 2.5 mg of DPPH radical with 100 mL of pure methanol and was adjusted to 0.70 ± 0.02 absorbance at a wavelength of 515 nm (Genesys 10uv spectrophotometer). Next, 2.0 mL of the radical solution was placed in a test tube and 100 μL of the diluted extract was added. The mixture was shaken in a vortex and kept in the dark for 30 min until the measurement. The results were expressed in EC50 in mg/mL (concentration of antioxidant required to reduce the absorbance of the radical by 50%).

POLYPHENOL EXTRACTION AND QUANTIFICATION

The extraction to determine the polyphenol content (unconjugated + conjugated) in ackee arilli was performed by the method described by VinsonVINSON JA, SU X, ZUBIK L and BOSE P. 2001. Phenol antioxidant quantity and quality in foods: fruits. J Agri Food Chem 49: 5315-5321. et al. (1998) with some modifications. In brief, 4 mL of 70% acetone with 1% hydrochloric acid were added to 0.2 g of the sample and the solution was shaken at 200 rpm for 3 h at room temperature. The mixture was centrifuged at 1089 g for 30 min and the supernatant was filled up to 5 mL with extraction solution. Free polyphenols (unconjugated) were extracted by the same procedure but without hydrochloric acid.

Four millilitres of the supernatants were loaded onto a Strata C18-U cartridge (1g, 6 mL) connected to a vacuum system (Phenomenex Inc., Torrance, CA), previously conditioned with 10 mL of methanol and followed by 25 mL of water. After the complete absorption of the sample, the hydrophilic compounds were eluted twice with 4 mL of 0.1 N sulphuric acid and discharged. The amphiphilic extracts containing polyphenols were eluted twice with 4 mL of 70% acetone and their polyphenol content was quantified after the reaction with Folin-Ciocalteu’s reagent according to Singleton and Rossi (1965)SINGLETON VL and ROSSI JAJR. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticult 16: 144-158.. Extracts (1 mL) were diluted in 50 mL of bi-distilled water in 100 mL flasks and mixed with 5.0 mL of Folin-Ciocalteau’s reagent (Sigma, Steinheim, DE) and, after 5 min, with 20 mL of sodium carbonate (15%). The flasks were mixed and allowed to sit in the dark for 30 min. The absorbance of the solution was measured at 750 nm on a Lambda Bio 20 UV/VIS spectrophotometer (Perkin Elmer, Boston, MA) and the results were expressed as mg of gallic acid equivalents (GAE) per gram of dry weight.

The analysis of individual polyphenols in the extracts was carried out by HPLC using the method described by Schieber et al. (2001)SCHIEBER A, KELLER P and CARLE R. 2001. Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography. J Chromatogr A 910: 265-273.. The separation of phenolic compounds was carried out using a 1200 Agilent Series HPLC (Agilent Technologies, Milano, Italy) equipped with a G1322 degasser, a G1311A quaternary pump, a G136A Column thermostat, a thermo-autosampler injection system, a column oven, and a diode array detector. The system was controlled with Agilent ChemStation for Windows (Agilent Technologies). An Aqua 5 µm C18 (250⋅4.6 mm I.D.) from Phenomenex (Torrance, CA, USA) and a security guard C ODS (433.0 mm I.D.) were used. The column temperature was regulated at 25°C. The mobile phase consisted of 2% (v/v) acetic acid in water (eluent A) and 0.5% acetic acid in water and acetonitrile (1:1, v/v; eluent B). The gradient program was: 10% B to 55% B (50 min), 55% B to 100% B (10 min), 100% B to 10% B (5 min). The injection volume for all samples was 10 mL. Simultaneous monitoring was performed at 280 nm, 320 nm and 370 nm at a flow-rate of 1 mL min-1. Spectra were recorded from 200 to 600 nm.

ORGANIC ACIDS AND SUGAR CONTENT

Lipids were previously removed from the ackee arilli samples in Soxhlet equipment with ether and the samples were homogenised with 25 mL of H2SO4 5 mM in an Ultra-Turrax at 15.000 rpm for 30 seconds. The samples were then centrifuged at 4500 × g for 10 min at 4°C and the supernatants were filtered through a 0.45 µm Millipore filter (Millipore Corporation). Organic acids and sugars were analysed with HPLC equipment (Elite Lachrom, Hitachi) coupled with refractive index detector L-2400. Twenty microlitres of the sample were injected in a column (Agilent Hi-Plex H, 300 mm × 6.5 mm, 8 µm) using H2SO4 5 mM as mobile phase with an operating flow rate of 0.4 mL/min and 65°C.

DETERMINATION OF THE LIPID FRACTION COMPOSITION

The lipid Fraction was extracted from lyophilised ackee arilli samples with petroleum ether (bp 60−80°C, reflux, two hours) in a Soxhlet apparatus and concentrated in a vacuum. The chemical composition of the lipid fraction was determined by Gas Chromatography coupled to Mass Spectrometry (GC-MS) in a Shimadzu model GCMS QP2010 Ultra system, operated in full scan mode (range 35-500 m/z) with a scan speed of 1000 scans s-1, operated in electron impact (EI) mode at 70 eV. Separation was carried out on a Restek (Bellefont, PA, U.S.A.) chemically bonded Rtx-5MS fused-silica capillary column (30 m×0.25 mm i.d.×0.25 µm film thickness) with temperature program as follows: beginning at 40°C for two minutes, increasing linearly up to 280°C in 20 min, and placed on hold for 20 min. The injector was operated at 280°C in split mode at 1:20 split ratio. Helium was used as the carrier gas at a flow rate of 1.01 mL min−1 and the flow was controlled by the linear velocity at 36.2 cm/s. Samples (1.0 µL) were injected with an AOC-20i+s auto-sampler. The identification of the compounds was established based on their retention index and mass spectra using the NIST11 and WR10 library.

HYPOGLYCIN A

Determination of hypoglycin A in the fruit was performed by the method described by Dundee and Minott, using an internal standard calibration method (Dundee and Minott, 2012DUNDEE S and MINOTT D. 2012. Impact of seed size on residual hypoglycin levels in ackee. Food Res Int 47: 306-309.). The determination was carried out using an HPLC equipment (Elite Lachrom, Hitachi), equipped with a UV detector (254 nm), injected on to a Chromolit RP-18 column (100 × 4.6 mm; Guard cartridge: Chromolit RP-18, 5 × 4.6 mm Merck, Germany). Hypoglycin A in ackee arilli samples was identified by the comparison of the retention times of its phenylisothiocyanate (PITC) derivative with the derivative of the homologue standard (L-Leucine) and was quantified using the respective calibration curve established for the PITC derivative of the homologue standard.

VOLATILE COMPOUNDS

The extraction of volatile compounds was carried out by the procedure indicated by Chaves-LópezCHAVES-LÓPEZ C, MAZZARRINO G, RODRÍGUEZ A, FERNÁNDEZ-LÓPEZ J, PÉREZ-ÁLVAREZ JA and VIUDA-MARTOS M. 2015. Assessment of antioxidant and antibacterial potential of borojo fruit (Borojoa patinoi Cuatrecasas) from the rainforests of South America. Ind Crop Prod 63: 79-86. et al. (2015) with some modifications: 10 g of pulp obtained by hand separation from the peel was added to 30 mL of distilled water and mixed, 10 mL of this sample was mixed with 2.5 g of NaCl and placed in a 50 mL vial containing a micro-stirring bar (Chaves-López et al. 2015). Samples were equilibrated for 40 min at 30°C and a solid-phase microextraction (SPME) manual device equipped with 50/30 divinylbenzene/ carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) fibre (Supelco, Bellefonte, PA) was used to extract free volatile compounds from the ackee arilli juice. The fibre was conditioned in a GC injector port at 270°C for 1 h before use.

The isolation and identification of the volatile compounds were performed on a Varian (Palo Alto, CA) CP3800 gas chromatograph equipped with a 60 m 0.25 mm i.d. DB-1 (df = 0.25 lm; J&W Scientific, Folsom, CA) fused silica capillary column. Analyses were carried out using helium as carrier gas at a column flow of 0.6 mL/min in a split ratio of 1:5 with the following program: (i) 80°C for 0 min, (ii) temperature ramp rate of 3.0°C/min from 80 to 210°C and hold for 1 min followed by, (iii) a temperature ramp rate of 25°C/min from 210 to 300°C and hold for 3 min. The temperatures of the injector and detector were 230 and 300°C, respectively. Several compounds were identified by 3 different analytical methods: (i) retention index (KI), (ii) GC–MS retention times (authentic chemicals), and (iii) mass spectra (authentic chemicals and NIST05 spectral library collection). Identification was considered tentative based only on mass spectrometry data.

STATISTICAL ANALYSIS

Two batches of 10 samples of unripe and ripe fruits were analysed. The analyses were run in triplicate. Data were expressed as means ± SD and statistically analysed by the determination of the least significant difference (LSD at p<0.05) using SAS (2007) program, version 9.2.

RESULTS AND DISCUSSION

HYPOGLYCIN A

Hypoglycin A is an amino acid with the ability to induce hypoglycaemia by inhibiting gluconeogenesis cofactors (CoA and carnitine) that are essential for long-chain fatty acid oxidation. This toxin undergoes dramatic changes in the fruit as described by Gordon and Jackson-Malete (2015)GORDON A and JACKSON-MALETE J. 2015. The life cycle of Ackee (Blighia sapida): Environmental and other influences on toxicity. In: Gordon A (Ed). Food safety and quality systems in developing countries: Volume One: Export challenges and implementation strategies. 1st ed., Jamaica: André Gordon; 2015, p. 33-46.. In fact, the content of hypoglycin A in the ackee arilli decreased from 286.0 ± 31.6 mg/100 g in the unripe fruit to 43.1 ± 4.9 mg/100 g in the ripe fruit. Although some researchers suggested that the geographical location of the trees does not significantly influence the HGA content of the fruits (p>0.05), there is evidence of significant differences in HGA content (Table I shows data regarding the presence of HGA in ackee from different countries) because the physiological conditions of the trees vary according to the place of origin of the fruit and the harvest season (winter or summer) (Bowen-Forbes and Minott 2011, Gaillard et al. 2011, Gordon and Jackson-Malete 2015). However, it is important to highlight that mature ackee arilli on regular-to-medium sized seeds typically contain lower levels of hypoglycin A than mature arilli on very small seeds (Dundee and Minott 2012). It is suggested that lower residual hypoglycin concentrations in ackees may be useful to develop new therapies in people with specific diseases (Oloyede et al. 2014).

TABLE I
Hypoglycin A content in ackee arilli (Blighia sapida) fruits.

SUGAR AND ORGANIC ACID CONTENT

Ripening of climacteric fruit usually results in the breakdown of starch to sugars to promote sweetness, a decrease the amount of organic acids and phenols to minimise bitterness and astringency and an increase volatile compounds to produce characteristic flavours and aromas. As evidenced by our results, the sugars (fructose, glucose, and sucrose) and organic acids found in the ackee arilli decreased with the maturation state (Table II). These results are in accordance with researchers which reported that total sugars increased during the first four stages of the ackee ripening process and decreased during the last ripening stage (EmanuelEMANUEL MA, GUTIERREZ-OROZCO F, YAHIA EM and BENKEBLIA N. 2013. Assessment and profiling of the fatty acids in two ackee fruit (Blighia sapida Köenig) varieties during different ripening stages. J Sci Food Agr 93: 722-726. and Benkeblia 2011). In this context, fruits with low sugar content (lower than 1%) are fruits with low carbohydrate content and therefore less sweet flavour. In addition to the changes in sugar concentration during the ackee ripening stages, researchers reported changes in three short-chain fructooligosaccharides identified as 1-kestose (1F-b-D-fructofuranosyl sucrose), nystose (1F(1-b-D-fructofuranosyl)2 sucrose) and DP5 (1F(1-b-D-fructo- furanosyl)3 sucrose) (Benkeblia and López 2015).

TABLE II
Organic acid and sugar content in Ackee arilli (Blighia sapida) fruits.

The organic acid content found in the fruit corresponds to the presence of ascorbic, citric and succinic acid. Ascorbic acid or vitamin C is an important water-soluble vitamin present in foods. In particular, the values of ascorbic acid found in the ackee arilli were 128.1 mg A.A/100g. Although this parameter is difficult to compare because the contents are highly variable, depending on parameters such as the state of maturity, harvesting period, and variety, the ackee arilli presented higher values compared to those found in different exotic fruits like purple passion fruit (36.3 mg A.A/100g), guava (65.8 mg A.A/100g), or the sweet gold pineapple cultivar (61.0 mg A.A/100g), and lower when compared with fruits such as the Cortibel guava fruit cultivar (168.36 mg A.A/100g) (Valente et al. 2011VALENTE A, GONÇALVES-ALBUQUERQUE T, SANCHES-SILVA A and COSTA HS. 2011. Ascorbic acid content in exotic fruits: A contribution to produce quality data for food composition databases. Food Res Int 44: 2237-2242., Soares et al. 2007SOARES FD, PEREIRA T, MARQUES MOM and MONTEIRO AR. 2007. Volatile and non-volatile chemical composition of the white guava fruit (Psidium guajava) at different stages of maturity. Food Chem 100: 15-21.).

LIPIDS

The fatty acid profile of the fruit is important because it has implications in health and in the stability of the oil. The total lipids in the ackee arilli increased during the ripening process. In fact, while unripe fruits showed only 2.20% d.w, the ripe ones showed values of 33.51% d.w. content. These values are higher than those previously reported 28.34% and 20.02% (MachelMACHEL AE, GUTIERREZ-OROZCO F, YAHIA EM and BENKEBLIA N. 2013. Assessment and profiling of the fatty acids in two ackee fruit (Blighia sapida Köenig) varieties during different ripening stages. J Sci Food Agr 93: 722-726. et al. 2013, OladijiOLADIJI AT, SHOREMEKUN KL and YAKUBU MT. 2009. Physicochemical properties of the oil from the fruit of Blighia sapida and toxicological evaluation of the oil-based diet in Wistar rats. J Med Food 12: 1127-1135. et al. 2009), but lower than those reported 55.8% and 46.2% (Goldson et al. 2014GOLDSON A, BREMMER D, NELSON K and MINOTT DA. 2014. Fat profile of jamaican Ackees, oleic acid content and possible health implications. W Indian Med J 63: 9-12., Mitchikpe 2007MITCHIKPE CES. 2007. Towards a food-based approach to improve iron and zinc status of rural Beninese children: en-hancing mineral bioavailability from sorghum-based food. PhD Thesis, Wageningen University, Netherlands, 2007.). These differences could be attributed to the variable ecological conditions including temperature, humidity, soil, etc., as well as the harvest time and ackee varieties.

The fatty acid percentages present in ackee arilli are shown in Table III. The major oil components in the ackee arilli sampled in this study corresponded to oleic acid (C18:1) (63.45%) followed by palmitic acid (16:0) (21.35%) and stearic acid (18:0) (3.51%) in oil. Studies conducted in ripe ackee arilli from Jamaica reported that palmitic, stearic, and linoleic (18:1) acids were the predominant fatty acids (OdutugaODUTUGA AA, ASEMOTA HN, MUSAC I, GOLDEN KD and KEAN EA. 1992. Fatty acid composition of arilli from ackee fruit (Blighia sapida L.). Jamaican J Sci Technol 3: 30-32. et al. 1992). The same authors suggested that the presence of sunlight during the opening of the fruits increased the lipid content. Also, other researchers noted that ackee oil of Nigeria is rich in behenic, palmitoleic, oleic, gadoleic, erucic, and 9, 12-eicosanoic acids (Oladiji et al. 2009). On the other hand, it was reported that the major acid in the arilli from Jamaica was oleic acid (Δ9-cis-oleic acid, an omega n-9) (EmanuelEMANUEL MA and BENKEBLIA N. 2011. Ackee fruit (Blighia sapida Konig). In: Yahia EM (Ed), Postharvest biology and technology of tropical and subtropical fruits. Cambrige, UK: Woodhead Publishing; 2011, p. 54-64. et al. 2013). A diet with high contents of oleic acid (polyunsaturated fatty acid) has been estimated to reduce the risk of suffering coronary heart disease, and to reduce the risk factors for cardiovascular disease such as those related to thrombogenesis, in vitro LDL oxidative susceptibility, and insulin sensitivity (Lopez-Huertas 2010LOPEZ-HUERTAS E. 2010. Health effects of oleic acid and long chain omega-3 fatty acids (EPA and DHA) enriched milks. A review of intervention studies. Pharmacol Res 61: 200-207.).

TABLE III
Contents lipids in Ackee arilli (Blighia sapida) fruits.

It is important to acknowledge the presence of two important terpenes in the lipid fraction of Colombian ackee arilli: D:A-Friedooleanan-7-ol, (7.alpha.), and squalene (317 ± 29 and 448 ± 28 mg/100 g Ackee, respectively). To our knowledge, this is the first time that these two compounds are reported in ackee arilli. The squalene, a functional lipid, is highly unsaturated hydrocarbon with antioxidant activity that contributes to the reduction of cholesterol, triglyceride levels in serum, and protects against a variety of cancers (Xiao et al. 2016XIAO H, YAO Z, PENG Q, NI F, SUN Y, ZHANG CX and ZHONG ZX. 2016. Extraction of squalene from camellia oil by silver ion complexation. Sep Purif Technol 169: 196-201.). In addition, squalene has many physiological functions, such as the promotion of superoxide dismutase activity in vivo, enhancement of immune responses and membrane stabilising properties (BhattacharjeeBHATTACHARJEE P and SINGHAL RS. 2003. Extraction of squalene from yeast by supercritical carbon dioxide. World J Microb Biot 19: 605-608. and Singhal 2003, Ko et al. 2002KO TF, WENG YM and CHIOU RYY. 2002. Squalene content and antioxidant activity of Terminalia catappa leaves and seeds. J Agri Food Chem 50: 5343-5348.). However, since squalene is an intermediate in endogenous cholesterol synthesis, it has been suggested that it can lead to an increase in cholesterol, which translates to a greater risk for the development of atherosclerosis (Salvo et al. 2017SALVO A, LA TORRE GL, DI STEFANO V, CAPOCCHIANO V, MANGANO V, SAIJA E, PELLIZZERI V, CASALE KE and DUGO G. 2017. Fast UPLC/PDA determination of squalene in Sicilian P.D.O. pistachio from Bronte: Optimization of oil extraction method and analytical characterization. Food Chem 221: 1631-1636.).

Squalene is produced by both animals and plants as a biochemical intermediate. In particular, it is present in high quantities in Amaranthus cruentus (6000-8000 mg/100 g) (Popa et al. 2015POPA O, BĂBEANU NE, POPA I, NIȚĂ S and DINU-PÂRVU CE. 2015. Methods for obtaining and determination of squalene from natural sources. BioMed Res Int 2015: 1-16.), in the brazil nut (13.8 mg/100 g oil) (Ryan et al. 2009RYAN E, GALVIN K, O’CONNOR TP, MAGUIRE AR and O’BRIEN NM. 2009. Fatty acid profile, tocopherol, squalene and phytosterol content of brazil, pecan, pine, pistachio and cashew nuts. Int J Food Sci Nut 57: 219-228.), in Pistacia vera L. (up to 21.8 mg/100g) and in Prunus armeniaca L. kernel oils (12.6–43.9 mg/100 g of oil) (Salvo et al. 2017, Rudzińska et al. 2017RUDZIŃSKA M, GÓRNAŚ P, RACZYK M and SOLIVEN A. 2017. Sterols and squalene in apricot (Prunus armeniaca L.) kernel oils: the variety as a key factor. Nat Prod Res 31: 84-88.). Several studies suggested that the content of these compounds is significantly affected by the variety among the species and different climate conditions (Salvo et al. 2017, Rudzińska et al. 2017). On the other hand, researchers found that squalene can be used to treat S. aureus infections, but we found no evidence of inhibitory activity against S. aureus of ackee extracts in vitro (Sri-Charan-Bindu et al. 2015SRI-CHARAN-BINDU B, MISHRA DP and NARAYAN B. 2015. Inhibition of virulence of Staphylococcus aureus – a food borne pathogen – by squalene, a functional lipid. J Funct Food 18: 224-234.).

D:A-Friedooleanan-7-ol, (7.alpha.) is poorly known, however, it is reported that friedelane-type triterpenes might have potent anti-diabetic activity as suppressors of hepatic glucose production in insulin-resistant states (ArdilesARDILES AE, GONZÁLEZ-RODRÍGUEZ A, NÚÑEZ MJ, PERESTELO NR, PARDO V, JIMÉNEZ IA, VALVERDE AM and BAZZOCCHI IL. 2012. Studies of naturally occurring friedelane triterpenoids as insulin sensitizers in the treatment type 2 diabetes mellitus. Phytochemistry 84: 116-124. et al. 2012). To our knowledge, this is the first report of the presence of D:A-Friedooleanan-7-ol, (7.alpha.) and squalene in ackee fruit.

PHENOLIC CONTENT

The phenolic content of fully ripe fruits was about 6-fold lower than the unripe with 1.5 mg GAE g-1d.w. and 2.1 mg GAE g-1d.w. of free and total polyphenols, respectively (Table IV). These values are slightly lower than those reported for apples using the same extraction solvent (Sacchetti et al. 2008SACCHETTI G, COCCI E, PINNAVAIA GG, MASTROCOLA D and DALLA-ROSA M. 2008. Influence of processing and storage on the antioxidant activity of apple derivatives. Int J Food Sci Technol 43: 797-804.). Since ripe ackee shows lower moisture content than that of apples (68.1 g 100g-1f.w.) when expressed as fresh weight, the total polyphenol content is higher than that of apples (0.67 mg GAE g-1f.w.), but this data only allows ackee to be classified as a fruit with low polyphenol content (VinsonVINSON JA, HAO Y, SU X and ZUBIK L. 1998. Phenol antioxidant quantity and quality in foods: vegetables. J Agr Food Chem 46: 3630-3634. et al. 2001).

TABLE IV
Total polyphenol and single polyphenol content of unripe and fully ripe ackee fruits.

When calculated based on total polyphenols data, the percentage of conjugation of polyphenols is about 30% and is lower than that of apples, which is about 50% (Vinson et al. 2001). Previous research reported that the level of total phenolic compounds is somewhat higher in the arilli of unripe ackee fruits, but decreases by 30% during the ripening process. The level of total phenolic compounds ranged from 10.59 mg g−1 fresh weight at the unripe stage 1 to 7.38 mg g−1 fresh weight at the ripe stage 5 (Emanuel and Benkeblia 2011).

Polyphenols in Ackee were also determined by HPLC analysis, and the most abundant ones (in terms of peak intensity) were catechin, epicatechin, and phloridzin which was found in trace amounts. Upon ripening, the abundance of catechin and epicatechin decreased, whilst the content of phloridzin increased. The percentage of conjugation of catechins in ripe ackee arilli is about 30%, similar to that of apples, whilst phloridzin shows a percentage of conjugation of 46%.

Catechin and epicatechin are polyphenols commonly found in tropical fruits (such as cacao) and other common fruits such as apple, plum, and grapevine. On the contrary, phloridzin is not a common polyphenol since it is found primarily in apples; closely related species from the Rosaceae family do not contain phloridzin and only trace amounts were reported in strawberry. The abundance of catechin and epicatechin in ackee fruit is interesting from the nutritional point of view since these polyphenols are bioavailable and abundant antioxidants in foods with widely recognised functional properties, such as cacao, chocolate, and tea (Crozier et al. 2012CROZIER A, ASHIHARA H and TOMÁS-BARBÉRAN F (Eds). 2012. Teas, cocoa and coffee: Plant secondary metabolites and health. Chichester, UK: Wiley-Blackwell; 2012.).

ANTIOXIDANT ACTIVITY

The antioxidant capacity of the ackee arilli measured by ABTS and DPPH methods increased during the ripe process, as reported in Fig. 2, and the values were higher than those reported for other exotic fruits from South America (Contreras et al. 2011, Vasco et al. 2008VASCO C, RUALES J and KAMAL-ELDIN A. 2008. Total phenolic compounds and antioxidant capacities of major fruits from Ecuador. Food Chem 111: 816-823.). This corroborates the findings of HamzahHAMZAH RU, EGWIN EC, KABIRU AY and MUAZU MB. 2013. Phytochemical and in vitro antioxidant properties of the methanolic extract of fruit of Blighia sapida, Vitellaria paradoxa and vitex doniana. Oxid Antioxid Med Sci 2: 215-221. et al. (2013) who stated that this fruit could be a potential source of natural antioxidants. Previous research has reported that the increase of the antioxidant activity is associated to physiological factors such as maturity, as well as technological factors like storage conditions and processing (Lindley 1998LINDLEY MG. 1998. The impact of food processing on antioxidants in vegetable oils, fruits and vegetables. Trends Food Sci Tech 9: 336-340., Heyles and LugasiHEYLES L and LUGASI A. 2006. Formation of certain compounds having technological and nutriotional importance in tomato fruits during maturation. Acta Aliment 35: 183-193. 2006). Thus, we can hypothesize that ripe Ackee arilli could serve as a considerable source of natural antioxidants.

Figure 2
Antioxidant capacity of the ackee arilli (Blighia sapida) fruit in two maturation stages. ABTS expressed in TEAC (Trolox equivalent antioxidant capacity) as µmol of Trolox equivalents per gram of fresh sample. DPPH expressed in EC50 (concentration of ackee extract required to reduce the absorbance of the radical by 50%) in mg/mL.

During ripe, fruit suffers physiological and biochemical changes, including the biosynthesis and accumulation of pigments, lipids, vitamins, and antioxidants, among others (Villa-Rodríguez et al. 2011, Goulao and Oliveira 2008GOULAO LF and OLIVEIRA CM. 2008. Cell wall modifications during fruit ripening: when a fruit is not the fruit. Trends Food Sci Tech 19: 4-25.). There are also other factors that are present in the physiology such as exposure to light and air, and the presence of pathogens, stress and environmental changes. At these stages, the plant raises physical and chemical barriers, such as the development of secondary metabolites or other types of molecules, where the fruit is protected by a defensive reaction that increases the concentration of antioxidant compounds. In this study, we observed that ackee arilli showed considerable amounts of squalene that probably contributed to the antioxidant activity.

VOLATILE COMPOUNDS

It is well known that the aroma of fruits comes from some components formed during the fruit ripening process with a low aroma threshold value and high relative content. Many factors affect the volatile composition of fruit, including the degree of maturity and ripening stages, environmental conditions, postharvest handling and storage conditions, as well as the genetic composition of the plant. To the best of our knowledge, information related to the volatile compounds content that contributes to the aroma of the ackee fruit has not been reported yet. The volatile fraction present in the two maturation stages of ackee fruit is reported in Table V. For all the compounds, identification was based on chromatographic peak RI and similarity index (SI) higher than 90%.

TABLE V
Volatile fraction composition in two maturation stages of ackee fruits (expressed as peak area %).

The successful identification of 54 compounds during the two stages of maturation yielded different chemical classes of volatile compounds like aldehydes (15), esters (15), carboxylic acids (7), alcohols (6), ketones (4), monoterpenes (3) and alkanes (4). The composition of the volatile fraction changed throughout the ripening stages as evidenced during the present research, where several volatile compounds showed an increase in the relative peak area during ripeness. In particular, there was an increase in ketones, esters, and alkanes. On the contrary, aldehyde and alcohol content decreased during the ripening process.

Among the volatile compounds detected in the unripe ackee arilli fruit, aldehydes correspond to 40% of the volatile content, followed by alcohols (19%), acids (16%), and esters (10%). In this stage of maturation, hexanal, acetic acid, and benzaldehyde were the most abundant compounds, representing nearly 19%, 14% and 13% of the volatile fraction, respectively. The contribution of 1-octen 3-ol, dodecane, spiropentanoic acid methyl ester, and 1-hexanol, was also pronounced and ranged between 5% and 9.6% of the relative area. Aldehydes, alcohols, and esters may result from enzymatic reactions during fruit ripening. In particular, the presence of C6 aldehydes, alcohols, and esters suggests that the lipoxygenase pathway may be activated during the ripening process (Sansone-Land et al. 2014SANSONE-LAND A, TAKEOKA GR and SHOEMAKER CF. 2014. Volatile constituents of commercial imported and domestic black-ripe table olives (Olea europaea). Food Chem 149: 285-295.). Hexanal was found in both maturation stages with a high area percentage. Other C6 compounds were found at different concentrations such as 1-hexanol, 3-hexen-ol, 2-hexenal (E), hexanoic acid-2-penthylethyl ester and hexanoic acid methyl ester. In addition, other products derived from the enzymatic degradation of fatty acids like heptanal, octanal, 3-penten-2-one and 2-heptanone were present.

In the ripe stage, aldehydes were the most abundant compounds with 25%, followed by ketones, acids, and esters with 20%, 17%, and 15%, respectively. The volatile fraction was dominated by 3-penten-2-one (14.95%), which is usually described to have a sharp, acetone-like and fruity odor, hexanal (13.51%), that could be correlated with the characteristic odour of unripe fruits, dodecane (7.1%) and n-hexadecanoic acid (6%), that confer waxy notes, acetophenone (5.6%) which provides aromatic notes described as sweet flowers, and nonaldehyde (5%).

The compounds 3-penten-2-one, n-propyl acetate, 2-butanone 4hydroxy-3-methyl, 2-butenal-2-methyl (E), 2-heptanal and gamma terpinene were present only in the fully ripe stage of fruits, contributing to the overall aroma of ripe ackee fruit. However, it is important to underline that no single compound or a simple combination of these compounds has the typical smell of ripe fruit.

CONCLUSION

The present study represents a contribution to the chemical and functional characterisation of ackee fruit. Although polyphenols are reduced during the ripe stage, ackee arilli could serve as an appreciable source of natural antioxidants like citric acid, squalene, and oleic acid. Thus, ripened ackee fruit can be categorised as a functional food that could compete with other tropical fruits how banana, mango, papaya, passion fruit, or pineapple. In this work, for the first time, we identified and quantified the squalene and D: A-Friedooleanan-7-ol, (7.alpha.). In addition, the volatile profile of unripe and ripe arilli was identified.

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Publication Dates

  • Publication in this collection
    09 Sept 2019
  • Date of issue
    2019

History

  • Received
    9 Feb 2018
  • Accepted
    30 Apr 2018
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