Extraction, Separation and Partial Identification of ‘Ataulfo’ Mango Fruit Carotenoids

Abstract

Carotenoids in fruits and vegetables are excellent sources for antioxidants and vitamin A. A reversed-phase HPLC method has been developed in our laboratory for separation and extraction of the carotenoids in crude and saponified extracts from ‘Ataulfo’ mango fruit grown in México. Fruit were obtained from local markets at adequate stage of ripening and evaluated for objective color, soluble solids and carotenoids extraction, separation and identification. The qualitative chromatographic analysis of crude extracts highlighted the existence of three main peaks which had UV-Vis spectra similar of those reported for all-trans-violaxanthin, 9-cis-violaxanthin and all-trans-β-carotene. A saponification step of crude extracts indicated that all-trans-violaxanthin and 9-cis-violaxanthin existed in esterified form while all-trans-β-carotene was found in a free state. Further LC/TOF-Mass spectroscopy analysis from the main esters of all-trans-violaxanthin and 9-cisviolaxanthin revealed that both were dibutyrates. The identity of all-trans-β-carotene was also confirmed by mass spectroscopy. The all-trans-violaxanthin levels were higher than 9-cis-violaxanthin levels, both as dibutyrates. ‘Ataulfo’ mangoes had high content of all-trans-β-carotene and low levels of all-trans-violaxanthin and 9cis-violaxanthin. We conclude that the main xanthophylls from ‘Ataulfo’ mango are all-trans-violaxanthin dibutyrate and 9-cis-violaxanthin dibutyrate while all-trans-βcarotene was the more abundant carotene detected. These data are important to assess the value of mango carotenoids for human nutrition and health. INTRODUCTION Mango (Mangifera indica L.) is a popular fruit in several countries. Mexico is the fourth biggest producer and the biggest exporter of this fruit (Yahia et al., 2006). The per cápita consumption of mango in Mexico is high (Yahia et al., 2006), and ‘Ataulfo’ is becoming one of the most important cultivar produced and consumed in the country and also exported to some other countries. Therefore, mango fruit consumption in Mexico contribute several beneficial nutritional and health compounds such as dietary fiber, poliphenols, ascorbic acid, tocopherols, and especially carotenoids. The yellow-orange color of mango is due to its high content of carotenoids, which are correlated with several health benefits such as prevention against some types of cancer and heart diseases. Some carotenoids such as all-trans-β-carotene are also precursors for vitamin A. The most abundant carotene in most mango cultivars is all-trans-β-carotene, while the most important xantophylls seem to be violaxanthin and its isomers (Chen et al., 2004). Several monoesters and diesters of violaxanthin and neolutein were tentatively identified in crude extracts of ‘Alphonso’ mango (Cano and de Ancos, 1994). Burns et al. (2003) found three violaxanthin esters in mango from Costa Rica, but no identification was provided. The predominant xantophyll in crude extracts of ‘Kent’ mangoes was identified as violaxanthin ester (Pott et al., 2003a), and two predominant xantophyll esters Proc. IVth IC on MQUIC Eds. A.C. Purvis et al. Acta Hort. 712, ISHS 2006 334 were identified as dibutyrates of all-trans-violaxanthin and 9-cis-violaxanthin by LC(APcI) MS analyses (Pott et al., 2003b). The objective of this study was to develop a chromatographic method to extract, separate, identify and quantify the most important carotenoids in mango fruit. MATERIALS AND METHODS High-performance liquid chromatography (HPLC) grade methanol, acetone, nhexanes, 2-propanol, reactive grade benzene, anhydrous granular sodium sulphate, calcium carbonate and Na2S2O3 were purchased from J.T. Baker (Baker Mallinckrodt, Mexico). BHT was obtained from Merck KGaA (Darmstadt, Germany). Diethyl ether, tert-butyl-methyl ether (MTBE) HPLC grade, mixture of all-trans-α/β-carotene from carrots, αand β-tocopherol were supplied by Sigma-Aldrich (St. Louis, MO) while alltrans-violaxanthin was obtained from CaroteNature GmbH (Lupsingen, Switzerland). HPLC grade water was prepared by a Milli-Qplus purification system (Millipore Corp., Bedford, MA). Fresh ripe ‘Ataulfo’ mango fruit were obtained from a local market in Queretaro, Mexico, and selected for homogenized size and color, and freedom from defects. Fruit maturity was objectively determined by °Brix using a hand refractometer, and color using a Minolta spectrophotometer, and variables L*, a*, b*, C* and h° were recorded. The extraction procedure of carotenoids was carried out as described by Pott et al., (2003a) with slight modifications. Fresh mango pulp from each fruit (6 g) was homogenized in presence of calcium carbonate (0.2 g) and methanol (15 ml). The homogenate was filtered adding methanol until retained solid became colorless. The methanolic extract was mixed with 50 ml of a mixture of hexane-acetone (1:1, v/v) containing 0.1% of BHT, and after a vigorous stirring 40 ml of 10% sodium sulphate were added for phase separation. The upper layer was separated, washed several times with water and evaporated in rotavapor at 35°C. When saponification step was necessary the residue was dissolved in diethyl ether (30 ml), added 0.2 ml of 40% methanolic KOH, and the mixture was left for 16 h in the dark at room temperature. After completion of saponification the extract was washed with water and evaporated as described above. Saponified and unsaponified residues were dissolved in 2 ml of 2-propanol, filtered through a polyethylene membrane of 0.45 μm of pore and 25 μL were injected to the HPLC system. Cis isomers of violaxanthin were generated for identification purposes according to Molnár et al., (2004). A quantity of 0.11 mg of all-trans-violaxanthin was dissolved in 1 ml of benzene containing 0.002 mg of I2 and the obtained solution was exposed at daylight until equilibrium was reached within 40 min. The mixture was then washed with 5% Na2SO3 (50 ml), evaporated at reduced pressure (35°C) and re-dissolved in 2-propanol (2 ml) previous to HPLC analysis. Samples were injected automatically to an HP 1100 series HPLC system equipped with an inline degasser, 100 μL loop, a diode array detector (DAD) and a fluorescence detector (FLD). Spectras for all separated peaks were recorded between 200 and 500 nm (each 2 nm) as well as individual signals for 9cis-violaxanthin, all-trans-violaxanthin and all-trans-β-carotene at 436, 440 and 452 nm by DAD, respectively. HPLC system was fitted to C30 reversed-phase high resolution column (4.6 x 150 mm) with a spherical particle size of 3 μm, which was kept at 15°C. After some pilot studies, the most appropriate mobile phase was found to be composed of water (A), methanol (B) and MTBE (C) with the following gradient elution: 4% A, 95.2% B and 0.8% C in the beginning and decreased at 4% A, 55.3% B and 40.7% C within 78 min at a flow rate of 0.75 ml/min. The most important carotenoids were identified by comparing retention time and absorption spectra of unknown peaks with reference standards as well as co-chromatography with added standards, while minor peaks were tentatively identified based on the spectrum characteristics as reported in the literature. Quantitative data for all-trans carotenoids were obtained by calibration curves constructed with pure compounds. Quantification of cis isomers of carotenoids was based on calibration curves of their parent trans carotenoids. Tukey-Kramer Honestly Significant Difference test was used as a comparison of

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@inproceedings{Yahia2006ExtractionSA, title={Extraction, Separation and Partial Identification of ‘Ataulfo’ Mango Fruit Carotenoids}, author={Elhadi M. Yahia}, year={2006} }