Carotenoid Composition of the Fruit of Red Mamey (Pouteria sapota

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Carotenoid Composition of the Fruit of Red Mamey (Pouteria sapota) ENRIQUE MURILLO, Erika Turcsi, Ildiko Szabo, Yesuri Mosquera, Attila Agócs, Veronika Nagy, Gergely Gulyas, and Jozsef Deli J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03146 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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Journal of Agricultural and Food Chemistry

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Carotenoid Composition of the Fruit of Red Mamey (Pouteria sapota)

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Enrique Murillo†,*, Erika Turcsi‡, Ildikó Szabó‡, Yesuri Mosquera†, Attila Agócs‡, Veronika

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Nagy‡, Gergely Gulyás-Fekete§, József Deli‡,§

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†

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of Panamá, 074637 Panamá City, Panamá

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‡

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Szigeti út 12, 7624, Pécs, Hungary

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§

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Department of Biochemistry, Faculty of Exact Natural Sciences and Technology, University

Department of Biochemistry and Medical Chemistry, Medical School, University of Pécs,

Department of Pharmacognosy, Medical School, University of Pécs, Rókus utca 2, 7624,

Pécs, Hungary

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ABSTRACT

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The detailed carotenoid analysis of red mamey (Pouteria sapota) was achieved by HPLC-

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DAD-MS, chemical tests and co-chromatography with authentic samples. Altogether 47

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components were detected and 34 identified from the total extract or after fractionation with

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column chromatography. The main carotenoids were cryptocapsin, sapotexanthin and

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capsanthin 5,6-epoxide. Some further minor components containing the κ-end group with or

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without a hydroxy group, and their 5,6-epoxy precursors were identified. Some comments are

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made about the biosynthesis of κ-carotenoids in red mamey.

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Keywords: mamey, column chromatography, κ-carotenoids, HPLC-DAD-MS, biosynthesis

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INTRODUCTION

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Sapote mamey (Poutiera sapota) is a tropical tree that grows in Central America. Its

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fruit has a high nutrient and vitamin content, orange or red color and a sweet flavor which

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makes it quite popular in this region including South America. There is a high demand for

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mamey in other regions such as Europe, Australia and the Philippines, as well. In Central

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America its juicy flesh is eaten fresh, and used in drinks and desserts.

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Earlier studies showed that carotenoids are responsible for the intensive color of the

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pulp of mamey, but did not achieve the complete separation and identification of the

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individual carotenoids.1,2 Recently, we isolated and used HPLC-MS, UV-vis and 1H- and 13C-

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NMR spectroscopy.3 to characterize a new carotenoid, sapotexanthin (β,κ-carotene-6'-one, 1,

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for formulas see supporting information), as a major carotenoid in mamey, The κ-ring of

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carotenoids, which is rare in Nature, is usually hydroxylated, as in capsanthin, 2, capsorubin,

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3, and cryptocapsin, 4. These carotenoids occur in the ripe fruit of red paprika (Capsicum

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annuum), and in some other natural sources.4

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The first carotenoid containing a κ-ring without a hydroxy group, 3’-deoxycapsanthin,

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5, was isolated in 2004 by Maoka et. al.5 This compound, 5, and its 5,6-epoxy derivative, 3’-

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deoxycapsanthin 5,6-epoxide, 6, together with its regioisomer cryptocapsin 5,6-epoxide, 7,

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were also isolated and characterized from red mamey by our group.6,7 Similarly, two novel

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capsorubin-like carotenoids containing κ-end-group(s) without a hydroxy group, namely 3’-

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deoxycapsorubin, 8, and 3,3’-dideoxycapsorubin, 9, were isolated and characterized from the

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pulp of red mamey.8 These investigations established that red mamey fruit contains several κ

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carotenoids with or without the hydroxy group, and their yellow probable precursor epoxides.

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In 2013, in cooperation with Italian researchers, the carotenoid composition of some

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tropical fruits including red mamey was investigated.9 In red mamey 16 different carotenoids

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were detected by HPLC-DAD-APCI-MS. Sapotexanthin, 1, cryptocapsin, 4, six different

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cryptocapsin monoesters, and a mono ester of cryptocapsin 5,6-epoxide, 7, were identified as

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red components. In addition, β-cryptoxanthin 5,6-epoxide, 10, and β-cryptoxanthin 5,8-

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epoxide, 11, (and their mono esters), β-cryptoxanthin 5,6,5’,6’-diepoxide, 12, and β-carotene


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5,8-epoxide, 13, were detected as yellow carotenoids. In 2016 a two dimensional liquid

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chromatography analysis of red mamey was published.10 By use of a LC x UHPLC-DAD-

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APCI-MS technique, 23 carotenoids with a 2D-HPLC system. Red carotenoids, namely

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cryptocapsin, 4, and its 13Z- and 13’Z-isomers, sapotexanthin, 1, capsanthin 5,6-epoxide, 14,

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capsoneoxanthin, 15, cryptocapsin 5,6-epoxide, 7, 3’-deoxycapsanthin, 5, and a tentatively

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identified new carotenoid iso-3’-deoxycapsanthin, 16 were found.

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However, a complete and extensive identification of the more than 40 carotenoids that

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comprise the carotenoid profile of red mamey was not achieved. This paper now describes the

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identification of all the major and minor carotenoids, quite a number of which have special

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structures and were recently described in some of our previous papers.

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MATERIALS AND METHODS

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Materials. Ripe red mamey fruit (Pouteria sapota) were purchased in the Metropolitan

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public market in Panama City. The varieties were selected based on differences in

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morphology (size and shape of fruit and seed), but they could not be specified, since in

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Panama there are no well-typified varieties of mamey. Analytical grade chemicals were used,

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and authentic carotenoid samples were taken from our collection.

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Pigment Extraction for HPLC Analysis. For each variety of red mamey, 100 g of pulp was

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homogenized in a porcelain mortar with 10 g of sodium bicarbonate and extracted with

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acetone until no more color was released. The extract was diluted with a mixture of diethyl

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ether : n-hexane (1:1), washed with water, and dried over Na2SO4. After filtration, the

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solvents were evaporated. The crude residue was redissolved in ether and saponified with

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30% methanolic KOH overnight. The reaction mixture was washed with water and, after



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drying, was evaporated. The saponified pigments were stored in benzene or hexane at -20 °C

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under nitrogen. The total carotenoid content of mamey reached 0.12 mg/g.

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General Experimental Procedures. The UV/Vis spectra were recorded, in benzene, with a

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Jasco V-530 spectrophotometer. The 1H NMR and

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Bruker DRX Avance II (500/125 MHz for 1H/13C) spectrometer (Bruker Corporation,

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Billerica, MA). Chemical shifts are referenced to internal TMS (1H), or to the residual solvent

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signals (13C). HPLC-DAD separations: gradient pump Dionex P680; detector: Dionex PDA-

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100; detection wavelength: 450 nm; data acquisition was performed by Chromeleon 6.70

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software. The HPLC separation was carried out on an endcapped C30 column (250 x 4.6 mm

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i.d.; YMC C30, 3 µm). Eluents: (A) MeOH : MTBE : H2O (81:15:4); (B) MeOH : MTBE :

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H2O (6:90:4). The chromatography was performed with a linear gradient from 100% eluent A

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to 50% A and B over 45 minutes, with 1 mL/min flow rate. HPLC-MS separations: gradient

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pump Gynkotek 480; detector: Gynkotek UVD 340S; detection wavelength: 450 nm; mass

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spectrometer: Finnigan AQA (Thermo Quest), data acquisition was performed by Chromeleon

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6.40 software. For LC-(APCI)MS positive ion mode was used, with TIC, scanning range 300-

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700 m/z, corona voltage 4 kV, acquisition voltage 20 V, the flow rate of the dried nitrogen as

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nebulizer gas was 600 L/h and the vaporizer temperature was 300 °C. Identification of

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carotenoids: The carotenoids were identified by elution order on the C30 HPLC column, co-

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chromatography with authentic standards, UV-visible spectrum [λmax, spectral fine structure

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(% III/II), and cis peak intensity (% AB/II)], and mass spectrum (molecular ion and

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fragments) compared to standards and data available in the literature.11 Specific chemical tests

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were also used; conversion of the 5,6-epoxide group into the corresponding 5,8-furanoid

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oxide in acidic medium and the reduction of ketones with sodium borohydride.

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Column Chromatography: The extract of ripe fruits was chromatographed on a glass

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column (30 x 6 cm i.d, CaCO3 (Biogal, Hungary) with n-hexane-toluene (6:4) as eluent.

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C NMR spectra were measured on a


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After development seven fractions were visible (in order of decreasing polarity:):

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Fraction 1: 5 mm, ochre band; Fraction 2: 4 mm, red band; Fraction 3: 6 mm pale

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yellow band; Fraction 4: 4 mm pink band.; Fraction 5: 6 mm pink band; Fraction 6: 7

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mm pale yellow band; Fraction 7: 6mm pink band. After processing (extruding the

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column, cutting the column into fractions and extracting with MeOH), the composition of

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the fractions were monitored by HPLC (Figure 2).

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Extraction and Isolation of β-Cryptoxanthin 5,8-epoxides: The extraction and column

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chromatography procedure were performed as described by Gulyás-Fekete et al.7 The

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fraction containing the β-cryptoxanthin 5,8-epoxides was further separated on open

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column chromatography (30 x 6 cm i.d, CaCO3 (Biogal, Hungary) with toluene/n-hexane

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3:7 as eluent. After development three bands were obtained (in order of decreasing polarity):

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Fraction 1: 10 mm pale yellow, β-cryptoxanthin 5,8,5’,8’-diepoxide, 11; Fraction 2: 20 mm

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yellow, β-cryptoxanthin 5,8,5’,6’-diepoxide, 23; Fraction 3: 20 mm yellow β-cryptoxanthin

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5,8-epoxide, 24. After processing (see above) the compounds were crystallized from benzene

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and hexane yielding 0.1-0.2 mg of each compound.



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(3S,5R,8S)-β-Cryptoxanthin 5,8-epoxide [(3S,5R,8S)-5,8-epoxy-5,8-dihydro-β,β-caroten-

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3-ol, 11]: orange crystals, UV-vis (benzene) λmax 416, 438, 465 nm; 1H-NMR (500 MHz,

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CDCl3) δ 6.65 (m, 1H, H-11’), 6.63 (m, 2H, H-15, H-15’), 6.50 (m, 1H, H-11), 6.34 (m, 2H,

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H-12, H-12’), 6.23 (m, 2H, H-14, H-14’), 6.20 (m, 1H, H-10), 6.16 (m, 1H, H-10’), 6.18 (m,

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1H, H-7’), 6.15 (m, 1H, H-8’), 5.31 (m, 1H, H-7), 5.07 (m, 1H, H-8), 4,24 (s, 1H, H-3), 2.11

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(m, 1H, H-4), 2.02 (s, 2H, H-4’), 1.97 (s, 9H, H-20, H-19’, H-20’), 1.90 (s, 1H, H-4), 1.81 (s,

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4H, H-2, H19), 1.72 (s, 3H, H-18’), 1.68 (s, 3H, H-18), 1.63 (s, 2H, H-3’), 1.50 (s, 1H, H-2),

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1.46 (s, 1H, H-2’), 1.45 (s, 1H, H-2’), 1.34 (s, 3H, H-17), 1,20 (s, 3H, H-16), 1.03 (s, 6H, H-

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16’, H-17’).

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(3S,5R,8S,5’R,6’S)-β-Cryptoxanthin 5,8,5’,6’-diepoxide [(3S,5R,8S,5’R,6’S)-5,8,5’,6’-

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tetrahydro-5,8,5’,6’-diepoxy-β,β-caroten-3-ol, 23]: orange crystals, UV-vis (benzene) λmax

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407, 431, 460 nm; λmax after acidic treatment 388, 410, 436 nm; 1H-NMR (500 MHz, CDCl3)

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δ 6.64 (m, 3H, H-15, H-11’, H-15’), 6.50 (m, 1H, H-11), 6.35 (m, 2H, H-12, H-12’), 6.19-

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6.29 (br m, 5H, H-10, H-14, H-8’, H-10’, H-14’), 5.88 (m, 1H, H-7’), 5.31 (m, 1H, H-7), 5,07

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(m, 1H, H-8), 4.24 (s, 1H, H-3), 2.13 (m, 1H, H-4), 1.96 (m, 6H, H-20, H-20’), 1.92 (m, 4H,

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H-4’, H-19’), 1.89 (m, 1H, H-4), 1.76-1.81 (br m, 5H, H-2, H-19, H-4’), 1.68 (s, 3H, H-18),

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1.49 (m, 2H, H-2, H-2’), 1.43 (m, 2H, H-3’), 1.34 (s, 3H, H-16), 1.20 (s, 3H, H-17), 1.07-1.15

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(br m, 7H, H-2’, H-17’, H-18’), 0.94 (s, 3H, H-16’).

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(3S,5R,8S,5’R,8’RS)-β-Cryptoxanthin

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tetrahydro-5,8,5’,8’-diepoxy-β,β-caroten-3-ol, 24]: orange crystals, UV-vis (benzene) λmax

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388, 410, 436 nm; 1H-NMR (500 MHz, CDCl3) for the 8’RS mixture: δ 5.30 (m, 1H, H-7),

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5.23 (m, 1H, H-7’R), 5.18 (m, 1H, H-7’S), 5.16 (m, 1H, H-8’S), 5.07 (m, 2H, H-8, H-8’R), 4.24

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(m, 1H, H-3), 2.11 (m, 1H, H-4), 1.90 (m, 1H, H-4), 1.81 (s, 3H, H-19’R), 1.80 (m, 1H, H-2),

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1.75 (s, 3H, H-19’S), 1.68 (s, 3H, H-18), 1.48 (m, 1H, H-2), 1.47 (s, 3H, H-18’R), 1.44 (s, 3H,

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H-18’S), 1.34 (s, 3H, H-16), 1.19 (s, 6H, H-17, H-17’R), 1.16 (s, 3H, H-17’S), 1.10 (s, 6H, H-

5,8,5’,8’-epoxide

[(3S,5R,8S,5’R,6’S)-5,8,5’,8’-


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16’S, H-16’R).

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RESULTS AND DISCUSSION

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Identification of main and known carotenoids. A variety (Variety 1., oval form, red pulp,

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weight of 270 g for one piece of fruit) of ripe red mamey was investigated. By use of HPLC-

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DAD and HPLC-MS, 47 compounds were detected in the total extract (Figure 1., Table 1).

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The main component peak 40 was identified as being due to all-trans-cryptocapsin, 4,

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on the basis of its UV-visible spectrum, MS data and co-elution with a standard. Peaks 30 and

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31 were attributed to its 13- and/or 13’-cis isomer of 4, according to the characteristic

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hypsochromic shift, the intensity of the cis peak in the UV-visible spectra, the MS ([M+H]+ at

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m/z 569) and co-chromatography with the iodine-catalysed isomerization mixture of

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cryptocapsin, 4. The carotenoid giving rise to peak 41 was identified as (13/13’Z)-

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sapotexanthin, (13/13’Z)-1, based on the hypsochromic shift and the intensity of its cis peak

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in the UV-visible spectra, and its molecular mass ([M+H]+ at m/z 553), whilst peak 46 was

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tentatively attributed to the (9/9’Z)-isomers of sapotexanthin, 1. The next major peak (45) was

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shown to be due to all-trans-sapotexanthin, 1 because it showed a UV-visible spectrum

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similar to that of cryptocapsin, 4, and a [M+H]+ m/z value of 553, indicating one β and one κ

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end group without any hydroxy group in the molecule.3

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Peak 14 was assigned as all-trans-capsanthin 5,6-epoxide, 14, according to the UV

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characteristics (λmax 464 nm), MS ([M+H]+ at m/z 601) data and co-chromatography with a

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standard isolated from the petals of Lilium tigrinum.12

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All-trans-violaxanthin, 17, (peak 10) and 9-cis-violaxanthin ((9Z)-17) (peak 16)

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showed characteristic UV-visible spectra, for the peak 16 compound a hypsochromic shift of

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4 nm was observed with decreased spectral fine structure and compared to the all-trans



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compound. The identification of these compounds was supported by co-elution with authentic

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standards and by their m/z value ([M+H]+ 601). The peak 13 carotenoid had a similar shaped

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UV-visible spectrum to that of violaxanthin, but with the absorption maxima shifted to lower

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wavelength by about 20 nm. This, the 600 molecular mass and co-chromatography with

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authentic sample, showed that peak 13 is due to the luteoxanthin epimers, 18, 19.

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Peaks 8 and 15 gave a UV-visible spectrum similar to that of peak 10. The molecular

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masses ([M+H]+), detected at m/z 601 and 585, respectively, seemed to correspond to all-

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trans-neoxanthin,

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assumptions were confirmed by co-elution with the authentic standards. Peak 9, the shoulder

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of peak 10, was tentatively attributed to neochrome, 21, by UV-visible and mass spectra

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([M+H]+ at m/z 601). Co-chromatography with the product from acid treatment of β-

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cryptoxanthin 5,6,5’,6’-diepoxide, 12, (containing 5,6,5’,8’-, 22, 5,8,5’,6’-, 23, and 5,8,5’,8’-

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diepoxides, 24, of β-cryptoxanthin), allowed the identification of the carotenoid of peak 24 as

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β-cryptoxanthin 5,8,5’,8’-diepoxide, 24, and the carotenoids of peaks 19 and 20 as β-

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cryptoxanthin 5,6,5’,8’-diepoxide, 22, and/or β-cryptoxanthin 5,8,5’,6’-diepoxide, 23. At the

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time we could not distinguish between the 5,6,5’,8’- and 5,8,5’,6’- epimers. The UV-visible

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and mass spectra ([M+H]+ at m/z 585) confirmed the identifications.

20,

and

all-trans-β-cryptoxanthin

5,6,5’,6’-diepoxide,

12.

These

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Peaks 42 and 44 were identified as being due to β-carotene 5,6-epoxide, 25, and 5,8-

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epoxide, 13, respectively, on the basis of the UV-visible and MS data ([M+H]+ at m/z 553).

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Although β-carotene, 26 (peak 47) was found in traces, we could not detect β-cryptoxanthin,

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zeaxanthin, α-carotene, α-cryptoxanthin or lutein.

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Column chromatography on calcium carbonate. To obtain the full evidence for the

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unidentified and mixed peaks, the whole extract of mamey was separated by column

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chromatography (CaCO3) with a mixture of hexane-toluene (6:4) to give seven fractions (see


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method above) containing carotenoids with different polarities (in order of decreasing

190

polarity):

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Fraction 1. In Fraction 1 (Figure 2.) seven peaks were detected by HPLC. Six of

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these could be identified. The main carotenoids were neoxanthin, 20, (peak 8) and (9Z)-

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violaxanthin ((9Z)-17, peak 16). The third identified compound was neochrome, 21, (peak 9).

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Peak 13a, which appeared under peak 13 in the total chromatogram, was due to a mixture of

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mainly furanoid isomers. UV-vis spectra of peak 24a and the apex and downslope of the Peak

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12 showed a single broad band at 462 and 467 nm, respectively. In addition, Peak 24a showed

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the weak cis peak at 352 nm, indicating a 9Z configuration. The molecular ions ([M+H]+)

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were at 601 m/z for both compounds. After repeated column chromatography, the carotenoid

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corresponding to peak 12 was obtained in pure state. Its UV-vis spectrum revealed maxima at

200

508 and 478 nm (in benzene, no cis peak), identical with the spectrum of capsanthin 5,6-

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epoxide, 14. However, there was no reaction with HCl:AcOH, indicating that a 5,6-epoxy

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group was not present. In an earlier work, we described the isolation of capsoneoxanthin, 15,

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a carotenoid with κ- and allenic end group from the buds of Asparagus falcatus.13 The

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chromatographic and characteristic spectral properties of capsoneoxanthin, 15, and those of

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the peak 12 carotenoid were identical, thus we tentatively identified this component as

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capsoneoxanthin, 15. Unfortunately we had no authentic sample, and could not separate

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enough amount of the compound for NMR analysis. Peak 24a which is covered by peak 24 in

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the chromatogram of the total sample was tentatively attributed to the (9Z)-isomer of

209

capsanthin 5,6-epoxide, ((9Z)-14) because reaction with HCl:AcOH indicated that this

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compound contained a 5,6-epoxy group. This fraction also contained capsorubin, 3, (Tr = 14.8

211

min, [M+H]+ at m/z = 601, λmax = 481 nm) in traces, which appeared as a component of peak

212

21 in the total extract.



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Some minor components (peaks 4-6) with very short retention times had a UV-visible

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spectrum similar to that of violaxanthin. We assumed that these compounds might be

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latoxanthin epimers, 27 and 28. Co-chromatography with 5,6-diepilatoxanthin, 27, isolated

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from red paprika,14 confirmed that peak 4 was due to this epimer. Because of lack of the other

217

epimer standards, we assumed that one of peaks 5 and 6 might be due to 6-epilatoxanthin, 28,

218

while the identity of the other remained unknown. Peak 7 showed a characteristic UV-visible

219

spectrum, with a hypsochromic shift of 4 nm and less fine structure compared to peak 4, so it

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was tentatively identified as (9/9’Z) isomer of 5,6-diepilatoxanthin, 27.

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Fraction 2. The main carotenoid of this fraction was capsanthin 5,6-epoxide (14, peak

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14). After repeated column chromatography the pure capsanthin 5,6-epoxide, 14, showed

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absorption maxima at 506, and 478 nm (461 nm after acidic treatment) in benzene, which is

224

identical with data published earlier.13 This fraction contained a luteoxanthin epimer, 18 or

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19, (peak 13), and probably the (13Z) and/or (13’Z)-isomer of capsanthin 5,6-epoxide, 14,

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(λmax 349, 451 nm), which could not be detected in the total extract.

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Fraction 3. The main carotenoids of Fraction 3 were violaxanthin, 17, and the other

228

luteoxanthin epimer, 19 or 18. The luteoxanthin epimers (8’R and 8’S, 18 and 19) cannot be

229

separated on a C30 stationary phase, but they can be separated by column chromatography on

230

CaCO3.15 This fraction contained capsanthin, 2, (HPLC Tr = 15,5 min, [M+H]+ at m/z = 585,

231

λmax = 475 nm, peak 23a) in trace amounts. The other minor peak (peak 34) had a capsorubin-

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like UV-VIS spectrum with absorption maxima at long wavelength (λmax = 481 nm) and with

233

[M+H]+ at m/z 585, and it was identified as 3’-deoxycapsorubin, 8. This new carotenoid has

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been isolated in pure form and its structure elucidated.8

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The carotenoid corresponding to peak 25 was also found in this fraction; it had one

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absorption maximum at 448 nm in the UV-vis spectrum indicating a 5,8-epoxy and a κ-end

237

group. Based on the spectral properties and chromatographic behaviour15 this peak was


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identified as cryptocapsin 5,8-epoxide, 29. This compound together with cryptocapsin 5,6-

239

epoxide, 7, have been isolated and characterized in our laboratory by spectroscopic methods.7

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It is known that the carotenoid diastereomers with non-hydroxylated (5R,8S)- and (5S,8R)-

241

5,8-epoxy-β end-group cannot be separated by open column chromatography or RP-HPLC.

242

Thus we separated them on chiral stationary phase, and identified the diastereomers by

243

HPLC-CD technique.7

244

Fraction 4. The main component of Fraction 4 (peak 22) had a UV/Vis spectrum (λmax

245

= 463 nm) identical to that of capsanthin 5,6-epoxide, 14, but its molecular mass ([M+H]+)

246

was only m/z 585. Reaction with HCl:AcOH indicated that a 5,6-epoxy group was present in

247

the molecule. This compound has also been isolated on a milligram scale and proven to be

248

cryptocapsin 5,6-epoxide, 7.7

249

Fraction 5. The Fraction 5 contained the main carotenoid of mamey, cryptocapsin, 4,

250

(peak 40) and its 13Z- and 13’Z-isomers ((13Z)-4 and (13’Z)-4), which showed characteristic

251

cis peaks at 355 nm. The fourth peak showed absorption maxima at 398, 421, 448 nm, which

252

are characteristic for the semi-furanoid (5,6,5’,8’/5,8,5’,6’-diepoxy) derivatives. The

253

molecular mass ([M+H]+) m/z 585 confirmed that these furanoids are β-cryptoxanthin

254

5,6,5’,8’-diepoxide, 22 and/or 5,8,5’,6’-diepoxide, 23, (peak 19).

255

Fraction 6. The Fraction 6 containing apolar carotenoids showed a quite complex

256

picture. The main components were β-cryptoxanthin 5,6,5’,6’-diepoxide, 12, (peak 15), and

257

the other β-cryptoxanthin 5,8,5’,6’- or 5,6,5’,8’-diepoxides (peaks 19, 20), which were

258

identified in the total extract. The third peak (peak 23) showed a similar UV-Vis spectrum

259

(λmax 463) and molecular mass ([M+H]+ at m/z 585) to peak 22. Reaction with HCl:AcOH

260

indicated that this compound also contained a 5,6-epoxy group in the molecule. This

261

compound has also isolated on a milligram scale and proven to be 3’-deoxycapsanthin-5,6-

262

epoxide, 6.7 The next two peaks showed a similar UV-vis spectrum, with λmax at 427 and 451



Page 12 of 25

263

nm. This, and the molecular mass ([M+H]+ at m/z 569) indicated one 5,8-epoxy and one

264

hydroxy group in the molecules. Thus these compounds were identified as β-cryptoxanthin-

265

5’,8’-epoxide, 30, (peak 37), and β-cryptoxanthin-5,8-epoxide, 11, (peak 38). Our earlier

266

study on the behaviour of carotenoids on different stationary phases15 showed that β-

267

cryptoxanthin 5’,8’-epoxide, 30, is eluted earlier than β-cryptoxanthin 5,8-epoxide, 11 on a

268

C30 stationary phase. The last minor peak (peak 43) had a capsorubin-like UV-vis spectrum

269

with long-wavelength absorption maximum (λmax = 481 nm) and with [M+H]+ at m/z 569; it

270

was thus identified as 3,3’-dideoxycapsorubin, 9. This new carotenoid has been isolated in

271

pure form and its structure was elucidated.8

272

Fraction 7. The main component here was sapotexanthin, 1, (peak 45), which was

273

isolated earlier in crystalline state and characterized.3 Otherwise β-carotene-5,6-epoxide, 25,

274

(peak 42) and 5,8-epoxide, 13, (peak 44) could be detected.

275

Isolation of β-cryptoxanthin-5,8-epoxides. As an alternative to calcium carbonate,

276

alumina (Al2O3) can be used as adsorbent in open column chromatography to give fractions

277

with different pigment composition.7 In this case, by increasing the percentage of Et2O in n-

278

hexane, eleven fractions were obtained from the total extract. By repeated column

279

chromatography

280

deoxycapsanthin6, 5, cryptocapsin 5,6-epoxide7, 7, cryptocapsin 5,8-epoxide7, 29, 3’-

281

deoxycapsanthin 5,6-epoxide7, 6, 3’-deoxycapsorubin8, 8, 3,3’-dideoxycapsorubin8, 9, and

282

their 5,6-epoxy precursors (β-cryptoxanthin 5,6-epoxide6, 10, β-cryptoxanthin 5’,6’-epoxide6,

283

31, β-cryptoxanthin 5,6,5’,6’-epoxide7, 12, have been isolated and characterized in other

284

studies. One of the fractions eluted with 40% Et2O in n-hexane contained mainly β-

285

cryptoxanthin 5,8- and 5’,8’-epoxides. Column chromatography on CaCO3 of this latter

286

fraction gave ca. 0.1-0.2 mg-s of β-cryptoxanthin 5,8-epoxide, 11, β-cryptoxanthin 5,8,5’,6’-

287

diepoxide, 23, and β-cryptoxanthin 5,8,5’,8’-epoxide, 24, respectively. The HPLC analysis on

some

κ-carotenoids

(sapotexanthin3,

1,


cryptocapsin6,

4,

3’-

Page 13 of 25


288

a chiral column showed that all the three isolated compounds were pure diastereomers. Due to

289

the very small amounts no CD data are available for compounds 11, 23 and 24. However, the

290

1

291

epoxides as (5R,8S)-β-cryptoxanthin 5,8-epoxide, 11, (5R,8S,5’R,6’S)-β-cryptoxanthin

292

5,8,5’,6’-diepoxide, 23. Due to the minute amount of 24 available for the 1H-NMR-

293

measurement and difficulties regarding the purification of the substance (no separation of

294

diastereomers) only some well-resolved signals could be used to identify the isomers in the

295

mixture.16 Thus the third compound was identified as a mixture of the 8’-diastereomers,

296

(5R,8S,5’R,8’RS) of β-cryptoxanthin 5,8,5’,8’-epoxide.

H NMR investigations allowed the assignments of the configuration for the first two

297

During carotenoid biosynthesis in higher plants lycopene is cyclized the to give α- and

298

β-carotene. Xanthophylls are formed enzymatically as oxidation products from carotenes. The

299

most common oxygen containing groups, originate from molecular oxygen, found in plant

300

xanthophylls are hydroxyl at C(3), epoxy at 5,6 position of the ring, and keto-group. In the

301

ripe mamey pulp carotenoids with ε-end group (e.g. α-carotene, α-cryptoxanthin, lutein) were

302

not observed, only carotenoids with β- and κ-end groups. Previously, red paprika extracts

303

contained mainly hydroxylated carotenoids, which indicated the following biosynthetic route:

304

β-carotene → β-cryptoxanthin → zeaxanthin → antheraxanthin → capsanthin → violaxanthin

305

→ capsanthin 5,6-epoxide → capsorubin. This suggests that the rate of the hydroxylation

306

reaction is greater than that of the epoxidation reaction, thus first the hydroxy and then the

307

epoxy derivative forms, which transforms to κ-carotenoid by pinacolic rearrangement. In red

308

mamey, we detected some non-hydroxylated κ-carotenoids in larger amounts, and the non-

309

hydroxylated 5,6-epoxy-β precursors of them. In this case, the first reaction should be the

310

epoxidation, which is followed by pinacolic rearrangement and hydroxylation. Based on these

311

findings we assume that the enzymes responsible for the epoxidation of the non-hydroxylated



312

β-rings and the pinacol rearrangement should be highly active in this fruit. The proposed

313

plausible pathway of biosynthetic routes in red mamey is shown on Figure 3.

314

Giuffrida et al. suggested in their paper the existence of a new carotenoid iso-3’-

315

deoxycapsanthin, 16, which we could not find during our extensive chromatographic study.

316

We think that the existence of such kind of isomer is highly improbable, the proposed

317

biosynthetic route also rules it out.

318

The Central American fruit mamey sapote (Poutiera sapota) is especially rich in

319

carotenoids, both in amount and diversity. With consecutive chromatographic separations and

320

HPLC analysis we managed to identify more than 40 major and minor carotenoids including

321

new type of pigments with deoxy kappa end-groups. Some of these new compounds could be

322

isolated in high purity and full structure elucidation has been achieved. Because of the

323

complexity of the extract there are still minor components that could not be identified. Mamey

324

fruit is a good source of provitamin A carotenoids as well, so its consumption is advisable in

325

the regions where it grows. Although the carotenoid composition of mamey fruit from

326

different locations and in different stages of ripening is similar, the proportion of the pigments

327

can vary drastically.17 These differences will be elaborated in another paper.

328 329

SUPPORTING INFORMATION. Additional figures and data are available free of charge

330

via the Internet at http://pubs.acs.org.

331 332

ACKNOWLEDGEMENTS

333

This study was financed by the Hungarian Scientific Research Fund (Grant: OTKA 115931)

334

and the PTE ÁOK (No: KA-2015-18). Veronika Nagy thanks the János Bolyai Research

335

Scholarship of the Hungarian Academy of Sciences for support. The authors are grateful to

336

Mrs. Judit Rigó, Ms. Zsuzsanna Götz and Mr. Roland Lukács for their skilful assistance. The


Page 14 of 25

Page 15 of 25


337

authors thank prof. George Britton for his useful comments. The present scientific

338

contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs,

339

Hungary.

340 341

REFERENCES

342 343

(1) Alia-Tejacal, I.; Villanueva-Arce, R.; Pelayo-Zaldivar, C.; Colinas-Leon, M.T.; López-

344

Martínez, V.; Bautista-Baños, S. Postharvest physiology and technology of sapote mamey

345

fruit (Pouteria sapota (Jacq.) H.E. More & Stearn). Postharvest Biology and Technology,

346

2007, 45, 285-297.

347

(2) Alia-Tejacal, I.; Soto-Hernandez, R.M.; Colinas-Leon, M.T.; Martínez-Damian, M.T.

348

Análisis preliminary de carotenoides y compuestos fenólicos en frutos de zapote mamey

349

(Pouteria sapota (Jacq.) H.E. More & Stearn) Rev. Chapingo Serie Hort. 2005, 11, 225-231.

350

(3) Murillo, E.; McLean, L.; Britton, G.; Agócs, A.; Nagy, V.; Deli, J. Sapotexanthin, an A-

351

provitamin carotenoid isolated from mamey (Pouteria sapota). J. Nat. Prod. 2011, 74, (2)

352

283-285.

353

(4) Murillo, E.; Nagy, V.; Agócs, A.; Deli, J. Carotenoids with κ-end group. In: Carotenoids:

354

Food Sources, Production and Health Benefits ed.: Yamaguchi, M. Chapter 3. pp. 49-78.

355

Nova Science Publishers, Inc. (2013)

356

(5) Maoka, T.; Akimoto, N.; Fujiwara, Y.; Hashimoto, K. Sructure of new carotenoids with

357

the 6-oxo-κ end group from the fruits of paprika, Capsicum annuum. J. Nat. Prod. 2004, 67,

358

115-117.

359

(6) Turcsi, E.; Murillo, E.; Kurtán, T.; Szappanos, Á.; Illyés, T.Z.; Gulyás-Fekete, G.; Agócs,

360

A., Avar, P.; Deli, J. Isolation of β-cryptoxanthin-epoxides, precursors of cryptocapsin and 3’-



361

deoxycapsanthin, from red mamey (Pouteria sapota). J. Agric. Food Chem. 2015, 63 (26),

362

6059-6065.

363

(7) Gulyás-Fekete, G.; Murillo, E.; Kurtán, T.; Papp, T.; Illyés, T.Z.; Drahos, L.; Visy, J.;

364

Agócs, A.; Turcsi, E.; Deli, J. Cryptocapsinepoxide-type carotenoids from red mamey,

365

Pouteria sapota. J. Nat. Prod. 2013, 76 (4) 607-614.

366

(8) Murillo, E.; Mosquera, Y., Kurtán, T.; Gulyás-Fekete, G.; Nagy, V.; Deli, J. Isolation and

367

characterization of two novel capsorubin like carotenoids from the red mamey (Pouteria

368

sapota). Helv. Chim. Acta 2012, 95 (6), 983-988.

369

(9) Murillo, E.; Giuffrida, D.; Menchaca, D.; Dugo, P.; Torre, G.; Melendez-Martinez, A.J.;

370

Mondello, L. Native carotenoids compositions of some tropical fruits. Food Chem. 2013, 140,

371

825-836.

372

(10) Cacciola, F.; Giuffrida, D.; Utezas, M.; Mangraviti, D.; Dugo, P.; Menchaca, D.; Murillo,

373

E.; Mondello, L. Application of comprehensive two-dimensional liquid chromatography for

374

carotenoid analysis in red mamey (Pouteria sapote) fruit. Food Anal. Methods 2016, 9, 2335-

375

2341, DOI 10.1007/s12161-016-0416-7

376

(11) Eugster, C.H. Chemical Derivatization. In Carotenoids; Britton, G., Liaaen-Jensen, S.,

377

Pfander, H., Eds.; Birkhauser: Basel, Switzerland, 1995; Vol. 1A., 71-80.

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(12) Deli, J.; Molnár, P.; Pfander, H.; Tóth, G.; Isolation of capsanthin 5,6-epoxide from

379

Lilium tigrinum. Acta Bot. Hung. 1999/2000, 42, 105-110.

380

(13) Deli, J.; Molnár P.; Ősz E., Tóth, G. Capsoneoxanthin, a new carotenoid, isolated from

381

the fruits of Asparagus falcatus. Tetrahedron Lett. 2000, 41, 8153-8155.

382

(14) Deli, J.; Molnár, P.; Matus, Z.; Tóth G.; Steck, A.; Pfander, H. Isolation of carotenoids

383

with 3,5,6-trihydroxy-5,6-dihydro-β-end groups from red paprika (Capsicum annuum). Helv.

384

Chim. Acta 1998, 81, 1233-1241.


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(15) Turcsi, E.; Nagy V.; Deli J. Study on the elution order of carotenoids on endcapped C18

386

and C30 reverse silica stationary phase. A review of the database. J. Food Comp. Anal. 2016,

387

47, 101-112.

388

(16) Acemoglu, M.; Eugster, C.H. Luteochrome; spektroskopische, chiroptische und

389

chromatographische Eigenschaften. Helv. Chim. Acta 1984, 67, 2226-2230.

390

(17) S. Arias 1, R; Martínez-Castillo, J.; Sobolev, V.S.; Blancarte-Jasso, N.H.; Simpson,

391

S.A.; Ballard, L.L.; Duke, M.V.; Liu, X.F.; Irish, B.M.; Scheffler, B.E. Development of a

392

Large Set of Microsatellite Markers in Zapote Mamey (Pouteria sapota (Jacq.) H.E. Moore &

393

Stearn) and Their Potential Use in the Study of the Species. Molecules 2015, 20, 11400-

394

11417.

395 396 397

Legends

398

Table 1. Carotenoid composition of red mamey extract by HPLC. UV-vis and MS data of

399

carotenoids obtained from HPLC-DAD-MS. Total carotenoid content was 120 mg.

400 401 402 403

Figure 1. HPLC chromatogram of red mamey extract (YMC C30, 3 µm, detection at 450 nm, other conditions as in text, for peak numbers see Table 1.) Figure 2. Chromatograms of fractions of red mamey extract after open column

404

chromatography (YMC C30, 3 µm, detection at 450 nm, other conditions as in text, for

405

peak numbers see Table 1.)

406

Figure 3. Proposed biosynthetic pathway

407 408 409



410 411 412 413 414


Page 18 of 25

Page 19 of 25

415


Table. 1. UV-Vis

Peak Carotenoid

MS (m/z)

%

No

( λ nm)

1

Unidentified

0.8

419, 444

2

Unidentified

0.2

416, 431

3

Unidentified

0.4

440, 466

4

5,6-Diepilatoxanthin

0.3

416, 441, 468

5

6-Epilatoxanthin

0.1

414, 438, 467

6

Unidentified

0.3

439, 467

7

(9/9'Z)-5,6-Diepilatoxanthin

0.3

412, 436, 463

8

Neoxanthin

3.8

416, 440, 468

9

Neochrome

+

601.3 [M+H] ; 583.3 [M+H-H2O]

402, 423, 448

+

601.3 [M+H]+

1.8 416, 440, 468

601.2 [M+H] ; 583.2 [M+H-H2O]

+

+

601.3 [M+H]

+

10

Violaxanthin

11

Unidentified furanoid

1.1

398, 421, 448

12

Capsoneoxanthin

1.7

463

13

Luteoxanthin

3.5

398, 421, 448

601.3 [M+H]+, 531.3, 425.2

14

Capsanthin 5,6-epoxide

9.2

464

601.3 [M+H] ; 583.2 [M+H-H2O] , 425.2

15

β-Cryptoxanthin 5,6,5’,6’-diepoxide

4.0

415, 438, 468

585.3 [M+H] ; 567.4 [M+H-H2O]

16

(9Z)-Violaxanthin

411, 434, 463

601.3 [M+H]+; 583.3 [M+H-H2O]+

17

Unidentified (9/9'Z) isomer

18

Unidentified furanoid

+

+

+

+

3.0 433, 462 396, 419, 446 4.3 19


20


21

Capsorubin + (13Z/13’Z)-unidentified

0.3

+

+

398, 421, 448

585.2 [M+H] , 567.4 [M+H-H2O] , 425.1

398, 421, 448

585.2 [M+H]+

481 and 454 6.8

22

585.3 [M+H]+; 567.3 [M+H-H2O]+

Cryptocapsin 5,6-epoxide

467

3’-Deoxycapsanthin 5,6-epoxide +

467

585.3 [M+H]

+

475

585.3 [M+H]

+

6.2 23

Capsanthin



β-Cryptoxanthin 5,8,5’,8’-diepoxide +

388, 402, 426

Page 20 of 25

+

555.3 [M+H] ;

460

(9Z)-Capsanthin 5,6-epoxide

25

Cryptocapsin 5,8-epoxide

0.1

448

26

Unidentified

0.1

421, 447, 475

27

Red (13Z)-unidentified

28

Red (13Z)-unidentified

29

β-Carotene 5,6,5’,8’-diepoxide

30

(13Z/13’Z)-Cryptocapsin

463

569.3 [M+H]

31

(13Z/13’Z)-Cryptocapsin

463

569.3 [M+H]+

32

β-Cryptoxanthin 5,6-epoxide

444, 472

569.3 [M+H]+

33

Unidentified mixture

1.0

450, 475

34

3’-Deoxycapsorubin

0.3

482

585.2 [M+H]+

35

3’-Deoxy-capsanthin

0.6

473

569.3 [M+H]

+

36

Unidentified

427, 451

569.3 [M+H]

+

427, 451

569.3 [M+H]+

468

601.3 [M+H]

+

24

569.4 [M+H]+, 551.3 [M+H-H2O]+, 425.1

1.1 468 569.4 [M+H]+

398, 421, 447 3.0

+

2.9

445, 470 2.6

37


38


39

Unidentified

1.7

469 21.4

40

Cryptocapsin

41

(13Z/13'Z)-Sapotexanthin

42

β-Carotene 5,6-epoxide

472 2.3

569.3 [M+H]+, 551.3

354, 461

553.3 [M+H]

+

420, 444, 472 0.3 482

3,3’-Dideoxycapsorubin

44

β-Carotene 5,8-epoxide

2.8

427, 452

553.4 [M+H] , 516.3, 425.4

45

Sapotexanthin

11.2

473

553.3 [M+H]+, 531.2

46

(9/9'Z)-Sapotexanthin

0.1

468

47

β-Carotene

0.3

450, 476

416 417


569.3 [M+H]

+

43

+

Page 21 of 25

418


Figure 1.

419 420 421 422 423 424 425 426 427 428 429 430 431 432



433

Figure 2.

434 435

436 437

438 439


Page 22 of 25

Page 23 of 25


440 441 442



Figure 3.

24

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TOC Graphic:


Carotenoid Composition of the Fruit of Red Mamey (Pouteria sapota

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