Isolation of β-Cryptoxanthin-epoxides, Precursors of Cryptocapsin and

Article pubs.acs.org/JAFC

Isolation of β‑Cryptoxanthin-epoxides, Precursors of Cryptocapsin and 3′-Deoxycapsanthin, from Red Mamey (Pouteria sapota) Erika Turcsi,† Enrique Murillo,‡ Tibor Kurtán,§ Á dám Szappanos,§ Tünde-Zita Illyés,§ Gergely Gulyás-Fekete,# Attila Agócs,† Péter Avar,† and József Deli*,†,# †

Department Department Panamá § Department # Department ‡

of Biochemistry and Medical Chemistry, Medical School, University of Pécs, Szigeti út 12, 7624 Pécs, Hungary of Biochemistry, Faculty of Exact Natural Sciences and Technology, University of Panamá, 074637 Panamá City, of Organic Chemistry, Faculty of Sciences, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary of Pharmacognosy, Medical School, University of Pécs, Rókus utca 2, 7624 Pécs, Hungary

ABSTRACT: From an extract of red mamey (Pouteria sapota) β-cryptoxanthin-5,6-epoxide, β-cryptoxanthin-5′,6′-epoxide, 3′deoxycapsanthin, and cryptocapsin were isolated and characterized by UV−vis spectroscopy, electronic circular dichroism (ECD), nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS). Epoxidation of β-cryptoxanthin delivered the β-(5′R,6′S)- and (5′S,6′R)-cryptoxanthin-5′,6′-epoxides, which were identified by HPLC-ECD analysis. These carotenoids among others are quite common in the fruits of Central America, and as they are natural provitamins A, they should play an important role in the diet of the mostly vitamin A deficient population of this region. KEYWORDS: carotenoids, mamey, tropical fruit, HPLC-ECD analysis

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INTRODUCTION Many foods contain β-cryptoxanthin 5,6-monoepoxide, 1 (Figure 1), and/or the furanoid oxide derivatives (cryptoflavin).1−16 Assignation of the structures was based on the chromatographic properties, the test for furanoid rearrangement, UV−vis spectra, and mass spectrometry data. However, in many cases, the position of the epoxy group [C(5),C(6) or C(5′),C(6′)] remained uncertain. In 1990, isolation of the 5,6monoepoxide derivative, 1, was reported from papaya, and the relative configuration was established by 1H NMR spectroscopy.17 Previously, epoxides of β-cryptoxanthin, 2 (Figure 1), were prepared in our laboratory with monoperoxyphthalic acid.18 After a mild hydrolysis, repeated chromatography, and crystallization, (3S,5R,6S)-, 1, (3S,5S,6R)-5,6-epoxy-, 3 (Figure 1), and (3R,5′R,6′S)-5′,6′-epoxy-β-cryptoxanthin, 4 (Figure 1), as well as (3S,5R,6S,5′R,6′S)-, 5 (Figure 1), and (3S,5S,6R,5′S,6′R)-5,6,5′,6′-diepoxy-β-cryptoxanthin, 6 (Figure 1), were isolated as main products and were fully characterized. Carotenoids with a 5,6-epoxy functionality in the hydroxylated β-ring (antheraxanthin, violaxanthin, etc.) are quite common in nature. The (9Z)-isomers of carotenoid-5,6epoxides are important because they can serve as sources of abscisic acid (ABA), the well-known plant hormone.19,20 On the other hand, carotenoids with 5,6-epoxy groups in a nonhydroxylated β-ring were seldom reported. In certain organs, the 5,6-epoxy-carotenoids transform to 6oxo-κ-carotenoids catalyzed by capsanthin-capsorubin synthase (CCS) enzyme.21 For example, antheraxanthin can transform to capsanthin, violaxanthin can transform to capsanthin 5,6epoxides and to capsorubin in the fruit of red paprika (Capsicum annuum),22−24 in the fruits of Asparagus officinalis25 and Asparagus falcatus,26 and in the flowers of Lilium tigrinum.27 © 2015 American Chemical Society

Recently, we have published the isolation and structure elucidation of some κ-carotenoids without hydroxyl groups, namely, sapotexanthin,28 3′-deoxycapsanthin-5,6-epoxide,29 and 3-deoxy- and 3,3′-dideoxycapsorubin30 from red mamey (Pouteria sapota), which may form from β-carotene-diepoxides and β-cryptoxanthin-diepoxides. Recently, our HPLC analysis of a red mamey extract showed two intense peaks at retention times of 14.5 and 17.5 min with the same UV−vis spectra (445 and 472 nm maxima in the HPLC eluent) and the same molecular mass (568). They were identified tentatively as β-cryptoxanthin-5,6- and -5′6′-epoxides without confirming their planar structures by NMR analysis. These above carotenoids such as sapotexanthin or cryptocapsin can be quite common in central American fruits such as mamey or papaya, and they can act as provitamins A. Vitamin A deficiency is very common in that region, which makes the investigation of these fruits rich in provitamin A even more justified.

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MATERIALS AND METHODS

General Experimental Procedures. The UV−vis spectra were recorded with a Jasco V-530 spectrophotometer in benzene. ECD and HPLC-ECD spectra were recorded at room temperature with a J-810 spectropolarimeter. UV and ECD data were obtained using a Jasco spectrophotometer and spectropolarimeter (Jasco Analytical Instruments, Easton, MD, USA). The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were measured with a Varian UNITY INOVA 400-WB spectrometer (Varian Medical Systems, Inc., Palo Alto, CA, USA) and on a Bruker Received: Revised: Accepted: Published: 6059

April 17, 2015 June 8, 2015 June 9, 2015 June 9, 2015 DOI: 10.1021/acs.jafc.5b01936 J. Agric. Food Chem. 2015, 63, 6059−6065

Article

Journal of Agricultural and Food Chemistry

voltage of 3500 V. Both sheath and auxiliary gases were nitrogen delivered at 10 and 2 L/min, respectively. The capillary temperature was set to 200 °C, whereas the ESI source heater was set to 40 °C. The RF level of the S-lenses was set to 70. For full MS experiments aimed at exact mass identification, we used the isolation window of m/z 50− 750. By MS/MS experiments performed with the aim of structural elucidation of the β-cryptoxanthin-epoxides, we used the precursor ion isolation window of m/z 567.4−569.4. High-energy collision cell was used for fragmentation at four different collision energy levels, 10, 15, 20, and 25%. In the lists of MS results the first value is always the m/z of the corresponding compound; all other ions are products of fragmentation. The exact mass measurements (HRESITOFMS) were performed using a Waters Q-TOF Premier mass spectrometer (Waters Corp., Milford, MA, USA). The sample was dissolved in MeOH and measured in positive electrospray ionization mode. HPLC-DAD Analysis. A Dionex P680 gradient pump with a Dionex PDA-100 detector and Chromeleon 6.70 software was used. The analytical HPLC separation was carried out on an end-capped C30 column with 250 mm × 4.6 mm i.d., 5 μm, YMC C30 (YMC Co. Ltd.). Eluents were (A) MeOH/methyl tert-butyl ether (MTBE)/H2O (81:15:4) and (B) MeOH/MTBE/H2O (6:90:4). The chromatography was performed in linear gradient from 100% A eluent to 50% B mixture in 45 min, with a 1 mL/min flow. The chiral HPLC-DAD separation was carried out on a 250 × 4.6 mm i.d., 3 μm, Chiralcel OD column (Daicel, Chemical Industries, Ltd.). Eluents were (A) MeOH/EtOH (1:1) and (B) MeCN/EtOH (1:1). The chromatography was performed in linear gradient from 100% A eluent to 100% B mixture in 30 min, with a 1 mL/min flow. HPLC-DAD analysis was performed with a Dionex HPLC system (Thermo Fisher Scientific). Semipreparative HPLC. A model 1050 Hewlett-Packard chromatograph (Hewlett-Packard Development Co., Palo Alto, CA, USA) equipped with a diode array detector and HP ChemStation software was used. The separation was carried out on an end-capped C30 column with a 250 mm × 4.6 mm i.d., 5 μm, YMC C30 with an isocratic system of MeOH/MTBE/H2O (7:16:4), with 2.5 mL/min flow, monitored at 450 nm. HPLC-ECD analysis. Chiral HPLC separations were performed with a Jasco HPLC system using Chiralpak IA column with 250 mm × 4.6 mm i.d., 5 μm (Daicel, Chemical Industries, Ltd.) and n-hexane/ propan-2-ol (9:1) eluent at a flow rate of 1.0 mL/min for 4 and 7 (Figure 1). HPLC-UV and OR chromatograms of the stereoisomeric mixtures were recorded with Jasco MD-910 multiwavelength and OR2090Plus chiral detector, respectively. The online ECD and UV spectra were measured simultaneously by stopping the flow at the UV absorption maximum of each peak. The values of the ECD ellipticity (ϕ) were not corrected for the concentration. Three consecutive scans were recorded and averaged for an HPLC-ECD spectrum with standard sensitivity, 2 nm bandwidth and 1 s response. The background HPLC-ECD spectrum of the eluent was recorded in the same way. Plant Material. Matured fruits were purchased at the Metropolitan public market in Panama City, Panama. Extraction and Isolation. The pulp of red mamey (500 g) was homogenized in a porcelain mortar with 50 g of NaHCO3 and extracted with acetone until no more color was observed. The extract was diluted with a mixture of Et2O/n/hexane (1:1), washed with H2O to remove acetone, dried (Na2SO4), and evaporated. The residue was dissolved in Et2O and saponified with methanolic KOH. After saponification, the ethereal solution was washed to remove alkali and evaporated. The residue was subjected to open column chromatography (Al2O3 Brockman grade III) using an increasing percentage of ether in n-hexane. The mixture of β-cryptoxanthin 5,6- and 5′,6′-epoxide was contained in the fraction that eluted with 15−25% diethyl ether. The specific epoxides were isolated by semiprepative HPLC, with several consecutive injections. Cryptocapsin, 8, was isolated from the fraction that eluted with 50% ether. This fraction was submitted to open column chromatography on

Figure 1. Structures of carotenoids from red mamey and related compounds. DRX Avance II (500/125 MHz for 1H/13C) spectrometer (Bruker Corp., Billerica, MA, USA). Chemical shifts are referenced to internal TMS (1H) or to the residual solvent signals (13C). MS Analysis. Samples were dissolved in MeOH and infused directly using a Fusion 100 T syringe pump (Thermo Fisher, Waltham, MA, USA) to a Q-Exactive quadrupole-orbitrap hybrid mass spectrometer (Thermo Fisher) with HESI source operated in positive ion mode. Data acquisition and analysis were performed by using Xcalibur (version 2.2 SP1.48). The source was operated with a spray 6060

DOI: 10.1021/acs.jafc.5b01936 J. Agric. Food Chem. 2015, 63, 6059−6065

Article

Journal of Agricultural and Food Chemistry

6.15 (1H, m, H-8), 6.16 (1H, d, J = 11.0 Hz, H-10), 6.19 (1H, d, J = 11.0 Hz, H-10′), 6.26 (2H, overlapped, H-14, H-14′), 6.29 (1H, d, J = 15.6 Hz, H-8′), 6.36 (1H, d, J = 15.0 Hz, H-12), 6.37 (1H, d, J = 15.0 Hz, H-12′), 6.62 (4H, overlapped, H-11, H-15, H-11′, H-15′); ECD [hexane, λ (nm) (Δε), c = 1.32 × 10−4 M] 474sh (2.10), 448 (2.49), 412sh (1.68), 333 (3.81), 273 (−13.46), 238 (7.71), 216 (−8.51), 200 (1.33), negative below 198 nm; MS m/z 568.43 [M]+, 553.40 [M − 16 + H]+, 488.36 [M − C6H8]+, 476.36 [M − C7H8]+, 461.34 [M − C7H8O]+, 415.30 [M − 153]+; tR = 14.5 min on a C30 column. Mixture of (3R,5′R,6′S)-β-Cryptoxanthin-5′,6′-epoxide, 4, and (3R,5′S,6′R)-β-Cryptoxanthin-5′,6′-epoxide, 7: orange crystals, UV−vis (benzene) λmax 434 (sh), 457, 487 nm, λmax after acid treatment, 416, 438, 465 nm; 1H NMR (400 MHz, CDCl3) δ 0.94 (3H, s, H-16′), 1.07 (1H, m, H-2′), 1.07 (6H, s, H-16, H-17), 1.10 (3H, s, H-17′), 1.15 (3H, s, H-18′), 1.44 (2H, m, H-3′), 1.48 (2H, m, Hax-2, H-2′), 1.74 (3H, s, H-18), 1.75 (1H, m, H-4′), 1.77 (1H, m, Heq-2), 1.91 (1H, m, H-4′), 1.93 (3H, s, H-19′), 1.96 (6H, s, H-20, H20′), 1.97 (3H, s, H-19), 2.04 (1H, m, Hax-4), 2.41 (1H, m, Heq-4), 4.00 (1H, s, H-3), 5.87 (1H, d, J = 15.6 Hz, H-7′), 6.11 (1H, d, J = 15.4 Hz, H-7), 6.15 (1H, d, J = 15.4 Hz, H-8), 6.16 (1H, d, J = 11.3 Hz, H-10), 6.19 (1H, d, J = 11.7 Hz, H-10′), 6.26 (2H, m, H-14, H14′), 6.29 (1H, d, J = 15.6 Hz, H-8′), 6.36 (1H, d, J = 14.8 Hz, H-12), 6.37 (1H, d, J = 15.1 Hz, H-12′), 6.61 (1H, dd, J = 11.7, 15.1 Hz, H11′), 6.63 (2H, m, H-15, H-15′), 6.64 (1H, dd, J = 11.3, 14.8 Hz, H11); 13C NMR (CDCl3) δ 12.74 (CH3, C-19), 12.79 (CH3, C-20′), 12.81 (CH3, C-20), 13.01 (CH3, C-19′), 17.12 (C-3′), 21.16 (CH3, C18′), 21.60 (CH3, C-18), 25.92 (CH3, C-17′), 26.01 (CH3, C-16′), 28.76 (CH3, C-16), 30.13 (C-4′), 30.29 (CH3, C-17), 33.85 (C-1′), 35.78 (C-2′), 37.13 (C-1), 42.60 (C-4), 48.50 (C-2), 65.10 (C-3), 65.50 (C-5′), 71.38 (C-6′), 124.16 (C-7′), 124.78 (C-11′), 124.96 (C11), 125.59 (C-7), 126.16 (C-5), 130.05 (C-15), 130.19 (C-15′), 131.32 (C-10), 131.94 (C-10′), 132.59 (C-14), 132.76 (C-14′), 134.48 (C-9′), 135.69 (C-9), 136.38 (C-13), 136.54 (C-13′), 137.27 (C-8′), 137.59 (C-12), 137.79 (C-6), 137.98 (C-12′), 138.50 (C-8); MS m/z 568.43 [M]+, 553.40 [M − 16 + H]+, 488.36 [M − C6H8]+, 476.36 [M − C7H8]+, 461.34 [M − C7H8O]+, 415.30 [M − 153]+. 4 tR = 6.55 min and 7 tR = 7.02 min on a Chiralcel OD column. 4 tR = 4.11 min and 7 tR = 3.87 min on a Chiralpak IA column (hexane/propan-2-ol 9:1). 4 HPLC-ECD [hexane/propan-2-ol 9:1, λ (nm) (ϕ)] 332 (4.95), 273 (−19.70), 238 (11.00), 215 (−13.80). 7 HPLC-ECD [hexane/propan2-ol 9:1, λ (nm) (ϕ)] 332 (−4.66), 292sh (−1.14), 266 (7.61), 245sh (2.91), 233 (−2.79), 216 (6.95). 3′-Deoxycapsanthin [(3R,5′R)-3-Hydroxy-β,κ-caroten-6′one, 9]. red crystals, UV−vis (benzene) λmax 484 nm λmax after reduction 434, 457, 487 nm; 1H NMR (500 MHz, CDCl3) δ 0.86 (3H, s, H-16′), 1.08 (6H, s, H-16, H-17), 1.12 (3H, s, H-17′), 1.18 (3H, s, H-18′), 1.48 (1H, m Hax-4′), 1.50 (1H, m, Hax-2), 1.54 (1H, m, Heq2), 1.68 (1H, m, Heq-2′), 1.72 (1H, m H-3′), 1.74 (3H, s, H-18), 1.78 (1H, m Heq-2), 1.97 (3H, s, H-19), 1.98 (6H, s, H-20, H-19′), 1.99 (3H, s, H-20′), 2.06 (1H, m, Hax-4), 2.40 (1H, m, Heq-4), 2.54 (1H, m, Heq-4′), 4.02 (1H, m, H-3), 6.13 (1H, d, H-7), 6.14 (1H, d, H-8), 6.17 (1H, d, H-10, J10/11 = 11.7 Hz), 6.27 (1H, d, H-14, J14/15 = 11.4 Hz), 6.34 (1H, d, H-14′, J14′/15′ = 10.8 Hz), 6.37 (d, 1H, H-12, J11/12 = 14.7 Hz), 6.48 (1H, d, H-7′), 6.52 (1H, d, H-12′, J11′/12′ = 15.4 Hz), 6.56 (1H, d, H-10′, J10′/11′ = 11.3 Hz), 6.61 (1H, m, H-11′), 6.65 (1H, m, H-15′), 6.68 (1H, dd, H-11), 6.69 (1H, m, H-15), 7.34 (1H, d, H-8′, J7′/8′ = 15 Hz); 13C NMR (CDCl3) δ 12.7 (CH3, C-20), 12.8 (CH3, C19, C-20′), 12.9 (CH3, C-19′), 19.5 (C-18′), 19.7 (C-3′), 21.4 (C-18), 24.3 (C-17′), 25.4 (C-16′), 28.5 (C-17), 30.0 (C-16), 34.2 (C-4′), 40.3 (C-2′), 42.3 (C-4), 48.3 (C-2), 65.0 (C-3), 121.1 (CH, C-7′), 123.8 (C-11′), 125.1 (C-11), 125.5 (C-8), 129.6 (C-15′), 131.0 (C10), 131.2 (C-15), 132.1 (C-14), 134.8 (C-14′), 137.1 (C-12), 138.2 (C-7), 140.2 (C-10′), 141.7 (C-12′), 146.2 (C-8′); ECD [hexane, λ (nm) (ϕ)] 347 (3.65), 337sh (2.31), 294 (−15.05), 285sh (−14.00), 253 (9.74), 243sh (7.53), 225 (−8.77), 205 (0.78), negative below 203 nm; MS m/z 568.43 [M]+; HRESITOFMS m/z 568.4262 (calcd for C40H56O2, 568.4280). Cryptocapsin (3′R,5′R)-3′-hydroxy-β,κ-caroten-6′-one, 8): red crystals; UV/vis (benzene) λmax 484 nm, λmax after reduction 434, 457, 487 nm; 1H NMR (400 MHz, CDCl3) δ 0.84 (3H, s, H-17′),

CaCO3 (Merck) with toluene/hexane (30:70). Five colored zones could be separated: zone 1, 10 mm brick red band (mixture of cryptocapsin 5,8-epoxides29 and 3′-deoxycapsorubin;30 zone 2, 10 mm pink band, cryptocapsin 5,6-epoxide;29 zone 3, 20 mm red band of cryptocapsin, 8; zone 4, 10 mm pink band, 3′-deoxycapsanthin 5,6epoxide;29 zone 5, 3 mm yellow band of cryptoxanthin 5,6,5′,6′diepoxide.29 After processing (cutting the column into pieces and extracting), zones 2−5 were crystallized from benzene by the addition of hexane, yielding 4 mg of cryptocapsin 5,6-epoxide, 8 mg of cryptocapsin, 8, 0.5 mg of 3′-deoxycapsanthin 5,6-epoxide, and 0.5 mg of cryptoxanthin 5,6,5′,6′-diepoxide.29 3′-Deoxycapsanthin, 9, was isolated in pure form by additional column chromatography of a fraction eluted with 30% ether. This fraction was submitted to open column chromatography on CaCO3 with toluene/hexane (20:80). After development, three zones were visible: zone 1, 6 mm pale yellow band, unidentified mixture; zone 2, 50 mm yellow band, mixture of β-cryptoxanthin 5,8- and 5′,8′epoxides; zone 3, 20 mm pink band, 3′-deoxy-capsanthin, 9. After processing, zone 3 was crystallized from benzene by the addition of hexane, yielding 2 mg of 3′-deoxy-capsanthin, 9. Preparation of Semisynthetic β-Cryptoxanthin-5′,6′-epoxides. To a solution of β-cryptoxanthin acetate (108 mg) in Et2O (360 mL) at room temperature was added 9.2 mL of 0.339 M concentration monoperoxyphthalic acid (Et2O solution). The mixture was kept under N2, in the dark. After 12 h, the mixture was washed with 5% aqueous NaHCO3 solution, the organic phase was dried (Na2SO4), and a 30% KOH/MeOH solution (∼100 mL) was added. After 16 h, the solution was washed with H2O until neutral and dried (Na2SO4), and the solvent was evaporated. Column Chromatography of β-Cryptoxanthin-epoxide Mixture. The residue was dissolved in hexane and submitted to open column chromatography on CaCO3 (Biogal, Debrecen, Hungary) with eluent toluene/hexane (20:80). Picture after development: 5 mm bright yellow (zone 1, not identified); 10 mm intermediate zone; 60 mm bright yellow (zone 2, mixture of β-cryptoxanthin-diepoxides and -5,6epoxides); 10 mm pale ochre (zone 3, β-cryptoxanthin 5′,6′-epoxides). After processing, zone 3 was crystallized from benzene by the addition of hexane yielding 6 mg of mixture of β-cryptoxanthin-5′,6′-epoxides, 4 and 7. (3S,5R,6S)-β-Cryptoxanthin-5,6-epoxide [(3S,5R,6S)-3-Hydroxy-5,6-dihydro-5,6-epoxy-β,β-caroten-3-ol, 1]: orange crystals, UV−vis (benzene) λmax 434 (sh), 457, 487 nm, λmax after acid treatment, 418, 438, 465 nm; UV−vis (hexane) λmax (log ε) 475sh (3.93), 447 (4.00), 423sh (3.84), 398sh (3.58), 377sh (3.30), 330 (3.03), 267 (3.33); 1H NMR (400 MHz, CDCl3) δ 0.98 (3H, s, H-16), 1.03 (6H, s, H-16′, H-17′), 1.15 (3H, s, H-17), 1.19 (3H, s, H-18), 1.26 (2H, overlapping, Hax-2, OH), 1.46 (2H, m, H-2′), 1.63 (4H, overlapping, Heq-2, Hax-4, H-3′), 1.72 (3H, s, H-18′), 1.93 (3H, s, H19), 1.96 (3H, s, H-19′), 1.97 (6H, s, H-20, H-20′), 2.02 (2H, t, J = 6.8 Hz, H-4′), 2.39 (1H, m, Heq-4), 3.90 (1H, s, H-3), 5.88 (1H, d, J = 15.4 Hz, H-7), 6.16 (1H, m, H-7′), ∼6.25 (8H, overlapped, H-8, H-10, H-12, H-14, H-8′, H-10′, H-12′, H-14′), ∼6.63 (4H, overlapped, H11, H-15, H-11′, H-15′); ECD [hexane, λ (nm) (Δε), c = 1.98 × 10−4 M] 472sh (1.27), 444 (1.38), 418sh (1.06), 333 (1.82), 270 (−5.41), 238 (1.78), 215 (−4.53), 199 (0.48), negative below 197 nm; MS m/z 568.43 [M]+, 553.40 [M − 16 + H]+, 488.36 [M − C6H8]+, 476.36 [M − C7H8]+, 461.34 [M − C7H8O]+, 415.30 [M − 153]+; tR = 17.5 min on a C30 column. (3R,5′R,6′S)-β-Cryptoxanthin-5′,6′-epoxide [(3R,5′R,6′S)-3Gydroxy-5′,6′-dihydro-5′,6′-epoxy-β,β-caroten-3-ol, 4]: orange crystals, UV−vis (benzene) λmax 434 (sh), 457, 487 nm, λmax after acid treatment, 416, 438, 465 nm; UV−vis (hexane) λmax (log ε) 475sh (3.97), 445 (4.02), 422sh (3.83), 396sh (3.52), 373sh (3.14), 332 (2.77), 267 (3.26), 199 (3.09); 1H NMR (400 MHz, CDCl3) δ 0.94 (3H, s, H-16′), 1.07 (7H, s, H-2′, H-16, H-17), 1.10 (3H, s, H-17′), 1.15 (3H, s, H-18′), 1.26 (1H, s, OH), 1.43 (2H, m, H-3′), 1.48 (2H, m, Hax-2, H-2′), 1.74 (3H, s, H-18), 1.76 (1H, m, H-4′), 1.79 (1H, m, Heq-2), 1.90 (1H, m, H-4′), 1.93 (3H, s, H-19′), 1.96 (6H, s, H-20, H20′), 1.97 (3H, s, H-19), 2.04 (1H, m, Hax-4), 2.38 (1H, m, Heq-4), 4.00 (1H, s, H-3), 5.88 (1H, d, J = 15.6 Hz, H-7′), 6.11 (1H, m, H-7), 6061

DOI: 10.1021/acs.jafc.5b01936 J. Agric. Food Chem. 2015, 63, 6059−6065

Article

Journal of Agricultural and Food Chemistry 1.03 (6H, s, H-16, H-17), 1.21 (3H, s, H-16′), 1.37 (3H, s, H-18′), 1.47 (2H, m, H-2), 1.49 (1H, m, Hax-4′), 1.62 (2H, m, H-3), 1.69 (1H, m, Hax-2′), 1.72 (3H, s, H-18), 1.96 (3H, s, H-20), 1.98 (6H, s, H-19′, H-20′), 1.99 (3H, s, H-19), 2.00 (1H, m, Heq-2′), 2.02 (2H, m, H-4), 2.96 (1H, d, J = 14.4 Hz, Heq-4′), 4.51 (1H, m, H-3′), 6.12−6.73 (15H, br overlapping, H-7, H-8, H-10, H-11, H-12, H-14, H-15, H-7′, H-10′, H-11′, H-12′, H-14′, H-15′), 7.33 (1H, d, J = 15.0 Hz, H-8′); MS m/z 568.43 [M]+.

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HCD cell energy were 3 and 6% of the intensity of the precursor ion (568.43) or 0.01 and 0.02% of the total ion chromatogram. In the 1H NMR spectrum of compound 1, methyl group signals were observed at 1.03 ppm (six protons) and 1.72 ppm (three protons) due to the C-16′, C-17′, and C-18′ methyl groups of the unsubstituted β-ring and at 0.98 and 1.15 ppm (C-16, C-17) and 1.19 ppm (C-18) of the 3-hydroxy-5,6-epoxy5,6-dihydro-β-ring.32 The structural elucidation of compounds 1 and 4 was supported by the 1H and 13C NMR measurements, which were in good agreement with the literature data.18,32 In the 3-hydroxy β-end group, the δ(H) of CH2(4) differed significantly for the cis- and trans-epoxide (relative to OH) and, therefore, proved the (3S,5R,6S)-configuration of 1. Because the (5′S,6′R)- or (5′R,6′S)-configuration of the epoxy β-end groups cannot be distinguished in the 1H NMR spectra, the elucidation of the configuration at the nonhydroxylated end group was achieved by electronic circular dichroism (ECD) measurements. Carotenoid-5,6-epoxides exhibit strongly conservative ECD spectra comparable to those of other carotenoids with a substituted β-end group. The influence of additional substituents such as a OH group at C(3) or C(3′) is rather small,33 and thus ECD spectra of the epoxides are governed by the absolute configuration of the epoxy group. The ECD spectra of 1 showed positive Cotton effects (CEs) at 472, 444, 418, 333, 238, and 199 nm and negative ones at 270 and 215 nm. The ECD spectrum of 1 (Figure 3) was compared with those of the related (5R,6S)- and (5S,6R)-5,6-epoxy-β-carotene,34,35 which allowed the assignment of the absolute configuration of 1 as (3S,5R,6S).

RESULTS AND DISCUSSION

The extraction of red mamey was performed as described previously.28 Column chromatography of the extract on aluminum oxide and calcium carbonate resulted in 1 mg of β-cryptoxanthin-5,6-epoxide, 1, 1.5 mg of β-cryptoxanthin5′,6′-epoxide, 4, 2 mg of 3′-deoxycapsanthin, 9, and 8 mg of cryptocapsin, 8. Structure Elucidation of Natural β-Cryptoxanthin 5,6and 5′,6′-Epoxides. The β-cryptoxanthin monoepoxides, 1 and 4 exhibited UV−vis maxima at (434), 457, and 487 nm in benzene, which was in accordance with the 5,6- or 5′,6′-epoxy group. Upon acidification with dilute HCl solution, a 20 nm hypsochromic shift of the above transition was observed, which was characteristic of the conversion to the corresponding furanoid oxide. Mass spectrometry helped to identify the location of the epoxide groups unequivocally. The compound eluted at the retention time of 14.5 min had the molecular ion at (m/z) 568.43. Fragmentation of this mass even with relatively low fragmentation energies produced a large number of ions. The fragmentation profile was very similar to that reported earlier for β-cryptoxanthin.31 Characteristic ions were observed at values of m/z 553.40 ([M − 16 + H]+ or protonated βcryptoxanthin), 488.36 ([M − C6H8]+), 476.36 ([M − C7H8]+), 461.34 ([M − C7H8O]+), and 415.30 ([M − 153]+), loss of the hydroxylated ring with cleavage at the 7,8 carbon−carbon bond from the protonated molecule, and several fragment ions corresponding to cleavages of the polyene chain. The other compound eluting at the retention time of 17.5 min had the molecular ion at (m/z) 568.43, as well. The fragmentation of this compound seemed to be almost identical to the fragmentation of the other compound with the retention time of 14.5 min. It contained the same high abundant ions, but the most intensive differed. To clarify the position of the epoxidation we calculated the theoretical masses of two ions (169.12340 and 171.13769), which contain the six-membered ring, four methyl groups, the epoxide, and the hydroxyl groups as they all together could occur in the tandem spectra only if the position of the epoxidation is 5,6. All of the tandem spectra were analyzed using mass isolation ranges of 169.11−169.13 and 171.13−171.15, and these two ions (Figure 2) were observed only in the spectra of the compound with the retention time of 17.5 min. Their intensities applying 25%

Figure 3. ECD and UV−vis spectra of (3S,5R,6S)-β-cryptoxanthin-5,6epoxide, 1 (blue curves), and (3R,5′R,6′S)-β-cryptoxanthin-5′,6′epoxide, 4 (red curves), in acetonitrile.

Although the β-cryptoxanthin-5′,6′-epoxide, 4, contained the epoxide moiety in the other end group, its ECD spectrum showed the same CEs for the corresponding transitions as the β-cyryptoxanthin 5,6-epoxide, 1, which determined the absolute configuration as (3R,5′R,6′S). In this way, the compound eluted at 14.5 min was identified as (3R,5′R,6′S)-β-cryptoxanthin 5′,6′-epoxide, 3, whereas the compound eluted at 17.4 min was (3S,5R,6S)-β-cryptoxanthin 5,6-epoxide, 1. Investigation of Semisynthetic β-Cryptoxanthin-5′,6′epoxides. Earlier, from a mixture of synthetic epoxidation products of β-cryptoxanthin, we could isolate and crystallize a

Figure 2. Two ions, m/z 169.12 and 171.14, used for the differentiation of 5,6- and 5′,6′-epoxides. 6062

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Figure 4. (A) HPLC-UV and HPLC-OR chromatograms of a mixture of (3R,5′R,6′S)- and (3R,5′S,6′R)-cryptoxanthin-5′,6′-epoxide diastereomers, 4 and 7, on a Chiralpak IA column (hexane/isopropanol 9:1). (B) HPLC-ECD spectra of cryptoxanthin-5′,6′-epoxide diastereomers (3R,5′R,6′S)-, 4 (blue curve), and (3R,5′S,6′R)-, 7 (red curve), in hexane/isopropanol 9:1.

Structure Elucidation of Cryptocapsin 8 and 3′Deoxycapsanthin 9. The isolation of cryptocapsin was described earlier from red mamey.29 All of the spectroscopic data of the isolated compound were identical with the data reported in the literature.31,32 The UV−vis spectrum of 3'-deoxycapsanthin, 9 (λmax 484 nm in benzene, 498 and 469 nm in hexane) was in agreement with the chromophore conjugated with a carbonyl group. Reduction of 9 with NaBH4 gave an approximately 1:1 mixture of the corresponding diastereomeric alcohols. The UV−vis spectrum of this mixture showed the expected fine structure and a hypsochromic shift (λmax 434, 457, 487 nm in benzene). The molecular formula of 9 was determined to be C40H56O2 by HPLC-MS. The 13C and 1H NMR assignments for 9 were made on the basis of 1D (1H) and 2D (COSY, HSQC) experiments and by comparison with capsanthin and sapotexanthin. In compound 9, methyl group signals could be attributed to an unsubstituted κ-ring. The 13C and 1H NMR data of 9 were similar to those of sapotexanthin, except for the β-end group signals (δ H-3, δ C-3).28 The 1H signal at δ 4.02 and the 13C signal at δ 65.0 indicated that the hydroxy group is attached to the cyclohexane and not the κ-ring.32,36 The connection of H-2, H-3, and H-4 protons from the β-end group and the connection of H-2′, H-3′, and H-4′ protons from the κend group were confirmed on the basis of COSY measurements. All other 1H and 13C data and all-E polyene chain were in good accordance with literature data.32,36 The ECD spectrum of 9 showed positive CEs at 347, 253, and 205 nm and negative ones at 294 and 225 nm with pronounced shoulders (Figure 5). These ECD transitions are governed by the C-5′ chirality center, which is adjacent to the carbonyl group conjugating with the decaene moiety. On the basis of the ECD spectrum, the absolute configuration of 9 could be determined as (3R,5′R).36 If β-cryptoxanthin-5,6,5′,6′-diepoxide is present in high amounts in a plant, it usually triggers the production of cryptocapsin-5,6-epoxide and 3′-deoxycapsanthin-5,6-epoxide. The 3-hydroxy κ-end group forms from a 3-hydroxy-5,6-epoxyβ end group by pinacol rearrangement, which is a common process in carotenoid biosynthesis. In this way the cryptocapsin, 8, could be formed from β-cryptoxanthin 5,6-epoxide, 1, in red

5′,6′-epoxide derivative, which had been reported as the (3R,5′R,6′S)-epoxide, 4.18 To have a further identification aid of the β-cryptoxanthin 5′,6′-epoxide, the compound was prepared again semisynthetically from β-cryptoxanthin by oxidation with monoperoxy phthalic acid. This furnished the necessary amount for the NMR investigations. After isolation of the mixture of the synand anti-mono-5′,6′-epoxides, which are nondistinguishable by nuclear magnetic resonance, the assignment of the 1H and 13C signals furnished by 2D techniques (1H−13C HSQC, 1H−13C HMBC measurements, 1H−1H COSY) was possible. The endgroup carrying the epoxide moiety has shown the known ambivalent behavior considering the assignment of the proton signals, whereas the signal of the 3-OH-β end-group was assigned unequivocally. However, the resulting chemical shifts were in good accordance with the published data.18 The identification of β-cryptoxanthin 5′,6′-epoxide of natural origin was confirmed by semisynthetic counterpart in the further analytical investigations. HPLC reinvestigation of this semisynthetic compound with a chiral stationary phase showed that this sample contains two components with identical UV−vis spectra and sign of optical rotation. The epoxidation of β-cryptoxanthin could produce two diastereomeric 5′,6′-epoxy derivatives, which could not be separated by column chromatography using calcium carbonate or HPLC using C18 or C30 columns. However, separation of (5′R,6′S)- and (5′S,6′R)-cryptoxanthin-5′,6′-epoxide diastereomers, 4 and 7, respectively, was achieved on Chiralcel OD and Chiralpak IA HPLC columns, which showed approximately equal amounts of the two diastereomers (Figure 4). Comparison of the retention times of the semisynthetic compounds with that of natural β-cryptoxanthin-5′,6′-epoxide, 4, on Chiralcel OD column showed that the first-eluting diastereomer was the natural one. On the Chiralpak IA column, this elution order was apparently reversed. The HPLC-ECD spectra of the diastereomeric (3R,5′R,6′S)-, 4, and (3R,5′S,6′R)-, 7, showed opposite CEs for the corresponding ECD transitions, but they were not mirror image curves (Figure 4B). On the basis of the ECD spectra of natural (3R,5′R,6′S)-, 4, the (3R,5′R,6′S) absolute configuration was assigned to the second-eluting diastereomer. 6063

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nonhydroxylated rings and the enzyme that catalyzes the rearrangement of the epoxides should be highly active to produce the detected carotenoids in such a high concentration.21,28,29 It has been also confirmed that natural 5,6-epoxy carotenoids bear the 5R,6S configuration independent of the presence or absence of the hydroxyl group on the sixmembered ring. During the synthesis of semisynthetic epoxides the compound with 5S,6R configuration is obtained, as well. Our study has also shown that the two epoxide isomers can be separated only on a chiral column if the ring is not hydroxylated. In the light of these new findings old data about similar compounds should be rechecked.

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AUTHOR INFORMATION

Corresponding Author

*(J.D.) Phone: +36-72-503-650, ext. 28833. Fax: +36-72-503650, ext. 28826. E-mail: [email protected].

Figure 5. ECD spectrum of 9 in hexane.

Funding

mamey. Similarly, the nonhydroxylated 5,6-epoxy-β-ring could result in the nonhydroxylated κ-ring, for example, 3′deoxycapsanthin 9 forms from β-cryptoxanthin 5′,6′-epoxide 4 (Figure 6). Our results confirm recently published observations: In red mamey, both the enzyme responsible for the epoxidation of the

Financial support was achieved by OTKA Grants K 83898, 105871, 105459, and 115931 (Hungarian National Research Foundation). Notes

The authors declare no competing financial interest.

Figure 6. Proposed biosynthetic pathway. 6064

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ACKNOWLEDGMENTS We thank Dr. L. Drahos for performing the HR-ESI-TOF-MS measurement. We also thank J. Rigó, R. Lukács, and Zs. Götz for their assistance. The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary.

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