Oxidative Metabolites of Lycopene and Î³-Carotene in Gac (Momordica

Article pubs.acs.org/JAFC

Oxidative Metabolites of Lycopene and γ‑Carotene in Gac (Momordica cochinchinensis) Takashi Maoka,*,† Yumiko Yamano,‡ Akimori Wada,‡ Tetsuji Etho,§ Yukimasa Terada,∥ Harukuni Tokuda,⊥ and Hoyoku Nishino# †

Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku, Kyoto 606-0805, Japan Kobe Pharmaceutical University, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan § Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan ∥ Center for Computers and Information Technology, Meijo University, Shiogamaguchi, Tempaku, Nagoya 468-8502, Japan ⊥ Department of Complementary and Alternative Medicine, Clinical R&D, Graduate School of Medical Science, Kanazawa University, 13-1, Takara-machi, Kanazawa 920-8640, Japan # Cancer Control Center, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan ‡

ABSTRACT: Three new oxidative metabolites of lycopenes, (erythro)-lycopene-5,6-diol, (threo)-lycopene-5,6-diol, and 1,16dehydro-2,6-cyclolycopene-5-ol B, and four new oxidative metabolites of γ-carotenes, 2′,6′-cyclo-γ-carotene-1′,5′-diol A, 2′,6′cyclo-γ-carotene-1′,5′-diol B, (erythro)-γ-carotene-5,6-diol, and (threo)-γ-carotene-5,6-diol, were isolated as minor components from the aril of gac, Momordica cochinchinensis. These structures were determined on the basis of spectroscopic data, and some of them were compared to the structures of synthetic samples. Furthermore, the oxidative metabolic conversion pathways of lycopene and γ-carotene were discussed. KEYWORDS: carotenoids, gac, Momordica cochinchinensis, lycopene-5,6-diol, 1,16-dehydro-2,6-cyclolycopene-5-ol, γ-carotene-5′,6′-diol, 2′,6′-cyclo-γ-carotene-1′,5′-diol

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INTRODUCTION Lycopene (ψ,ψ-carotene) (1), the major carotenoid in tomato and tomato products, exhibits excellent singlet oxygen quenching activity1,2 and free radical-scavenging activity.3,4 Lycopene also shows growth-inhibiting effects on several human cancer cells and anticarcinogenic effects in mice.5−8 Furthermore, epidemiological studies have shown that the uptake of lycopene reduces the risk of prostate cancer.9−12 Recently, several oxidative metabolites of lycopene, each with a unique five-membered ring end group, including 2,6cyclolycopene-1,5-epoxide (1,5-epoxy-1,2,5,6-tetrahydro-2,6cyclo-ψ,ψ-carotene-1′,5′-diol), 2,6-cyclolycopene-1,5-diol (1,2,5,6-tetrahydro-2,6-cyclo-ψ,ψ-carotene-1′,5′-diol), and 1,16-didehydro-2,6-cyclolycopene-5-ol (1,16-didehydro-1,2,5,6tetrahydro-2,6-cyclo-ψ,ψ-carotene-1′,5′-diol), were isolated from human serum, milk, tomato, and tomato products.13−18 In 1992, Khachik et al.13 first isolated 2,6-cyclolycopene-1,5diol from human serum. Later, 2,6-cyclolycopene-1,5-diol was also isolated from milk and the retina.14,15 Kachik et al.14,15 also demonstrated that the oxidation of lycopene with mchloroperbenzoic acid (MCPBA) yielded 2,6-cyclolycopene1,5-epoxide, which was subsequently converted to 2,6-cyclolycopene-1,5-diol by acidic catalysis. Lu et al.16 reported the partial syntheses of 2,6-cyclolycopene-1,5-diol by oxidation of lycopene with hydrogen peroxide.16 Subsequently, Yokota et al. reported the isolation of 1,5-dihydroxyiridanyl-lycopene (one of the stereoisomers of 2,6-cyclolycopene-1,5-diol)17 and 1,16didehydro-2,6-cyclolycopene-5-ol18 from tomato puree. Gac, Momordica cochinchinensis (Cucurbitaceae), is a deciduous vine that grows in grows Southeast Asia. Tradition© 2015 American Chemical Society

ally, gac has been used as both food and folk medicine in Southeast Asia. Its aril is a bright red color due to the presence of lycopene and β-carotene,19,20 and it is used in Vietnam as a colorant for cooking red glutinous rice. In the present investigation, three new oxidative metabolites of lycopene were isolated from the aril of gac. In addition to lycopene metabolites, four oxidative metabolites of γ-carotene (β,ψ-carotene) (8) were also obtained from M. cochinchinensis. This paper reports the isolation and structural elucidation of these new oxidative metabolites of lycopenes and new oxidative metabolites of γ-carotenes. Furthermore, oxidative metabolic pathways of lycopene in gac are discussed.

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MATERIALS AND METHODS

Apparatus. The UV/vis spectra were recorded with a Hitachi U2001 spectrophotometer (Hitachi High-Technologies Corporation, Tokyo, Japan) in diethyl ether (Et2O). The positive ion FAB MS spectra were recorded using a JEOL JMS-HX 110A mass spectrometer (JEOL Ltd., Tokyo, Japan) with m-nitrobenzyl alcohol as a matrix. The 1 H NMR (500 MHz) and 13C NMR (125 MHz) spectra were measured with a Varian UNITY INOVA 500 spectrometer (Agilent Technologies, Santa Clara, CA, USA) in CDCl3 with TMS as an internal standard. Preparative HPLC was performed on a Shimadzu LC-6AD with a Shimadzu SPD-6AV spectrophotometer (Shimadzu Corporation, Kyoto, Japan) set at 450 nm. The column used was a 250 × 10 mm i.d., 10 μm Cosnosil 5C18-II (Nacalai Tesque, Kyoto, Japan) Received: Revised: Accepted: Published: 1622

October 15, 2014 January 13, 2015 January 19, 2015 January 29, 2015 DOI: 10.1021/jf505008d J. Agric. Food Chem. 2015, 63, 1622−1630

Journal of Agricultural and Food Chemistry

Article

Table 1. 1H (500 MHz) NMR Data for Compounds 3, 6, and 7 and 13C (125 MHz) NMR Data for Compound 6 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′ 19′ 20′ OH a

6

1

mult (J in Hz)a

2.46 ∼1.84, 1.86 ∼1.84, 1.86

overlapped each m each m

2.46 5.50 6.20

overlapped dd (15, 8) d (15)

6.14 6.60 6.35

d (11) dd (15, 11) d (15)

6.26 6.64 4.69 and 4.71 1.68 1.19 1.92 1.97

d (11) m each br s s s s s

5.12 2.12 2.12

m m m

5.96 6.50 6.25

d (11) dd (15, 11) d (15)

6.19 6.64 6.35

d (11) dd (15, 11) d (15)

6.26 6.64 1.69 1.63 1.82 1.93 1.97

d (11) m s s s s s

position

H

13

1

C

131.8 124.3 22.1 38.8 75.0 78.9 126.2 138.1 134.3 132.8 124.5 138.1 136.3 133.2 129.9 25.7 17.7 21.5 12.6 12.8 131.6 123.9 26.7 40.2 138.6 125.7 124.8 135.4 136.1 131.6 125.2 136.2 136.3 132.5 129.9 25.7 17.7 17.0 12.6 12.8

H

7 mult (J in Hz)

1

H

mult (J in Hz)

5.12 2.12 1.54

m m t (7)

5.12 2.12 1.54

m m t (7)

4.04 5.70 6.38

dd (7.5, 3b) dd (15.5, 7.5) d (15.5)

4.02 5.74 6.38

dd (7.5, 3) dd (15.5, 7.5) d (15.5)

6.20 6.59 6.38

d (11) dd (15, 11) d (15)

6.20 6.59 6.38

d (11) dd (15, 11) d (15)

6.26 6.64 1.69 1.62 1.14 1.93 1.97

d (11) m s s s s s

6.26 6.64 1.69 1.62 1.22 1.93 1.97

d (11) m s s s s s

5.12 2.12 2.12

m m m

5.12 2.12 2.12

m m m

5.96 6.50 6.25

d (11) dd (15, 11) d (15)

5.96 6.50 6.25

d (11) dd (15, 11) d (15)

6.19 6.64 6.35

d (11) dd (15, 11) d (15)

6.19 6.64 6.35

d (11) dd (15, 11) d (15)

6.26 6.64 1.69 1.63 1.82 1.93 1.97 2.07

d (11) m s s s s s br s

6.26 6.64 1.69 1.63 1.82 1.93 1.97 2.07

d (11) m s s s s s br s

s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet; br s, broad singlet. bCoupling with OH.

and a 250 × 10 mm i.d., 10 μm Cosnosil 5SL-II (Nacalai Tesque, Kyoto, Japan). Plant Materials. Gac fruits were purchased at a local vegetable market in Vietnam. Quantitative Analysis of Carotenoids. The total carotenoid content and the amount of carotenoids eluted by column chromatography were calculated using the extinction coefficient of 21 In the HPLC analysis, the relative amounts of E1% cm = 3450 at λmax. individual carotenoids were calculated from the peak area detected at 470 nm. Isolation of Carotenoids. The aril (980 g) of gac was extracted with acetone (Me2CO) at room temperature. The Me2CO extract was evaporated to dryness. The reddish residue was subjected to silica gel 60 (Nacalai Tesque, Kyoto, Japan) column chromatography (300 × 20 mm). The fraction (fr) eluted with 200 mL of ether−hexane (1:9 v/v, fr 1) was subjected to preparative HPLC on ODS (Cosmosil 5C18-II) with a CH3CN flow rate of 2.0 mL/min to yield α-carotene, β-

carotene, 9Z-β-carotene, γ-carotene, lycopene (1), 9Z-lycopene, and 13Z-lycopene. The fraction eluted with ether-hexane (5:5 v/v, fr 2) was subjected to preparative HPLC on silica gel (Cosmosil 5Si) with acetone− hexane (15:85 v/v) to yield new carotenoid 3 (retention time 18.4 min) and 1,16-dehydro-2,6-cyclolycopene-5-ol A (rt 20.5 min). The fraction eluted with ether−hexane (8:2 v/v, fr 3) was subjected to preparative HPLC on silica gel (Cosmosil 5Si) with acetone− hexane (25:75 v/v) to yield isocryptoxanthin (rt 9.4 min), βcryptoxanthin (rt 10.4 min), and new carotenoids 11 (rt 12.0 min), 12 (rt 14.0 min), 6 (rt 16.0 min), and 7 (rt 17.0 min). The fraction eluted with ether (fr 4) was subjected to preparative HPLC on silica gel (Cosmosil 5 Si) with acetone−hexane (25:75 v/v) to yield new carotenoids 10 (rt 12.0 min) and 9 (rt 14.0 min), 2,6-cyclolycopene1,5-diol B (rt 17.0 min) and 2,6-cyclolycopene-1,5-diol A (rt 20.0 min), lutein (25.0 min), and zeaxanthin (27.0 min). Identification and Characterization of Carotenoids. αCarotene, β-carotene, 9Z-β-carotene, γ-carotene (8), lycopene (1), 1623

DOI: 10.1021/jf505008d J. Agric. Food Chem. 2015, 63, 1622−1630


Article

Table 2. 1H (500 MHz) NMR Data for Compounds 9−12 in CDCl3 9 position 2 3 4 7 8 10 11 12 14 15 16 17 18 19 20 2′ 3′ 4′ 6′ 7′ 8′ 10′ 11′ 12′ 14′ 15′ 16′ 17′ 18′ 19′ 20′ a

10

H

mult (J in Hz)a

∼1.46 ∼1.62 2.02 6.16 6.15 6.15 6.65 6.35 6.26 6.64 1.03 1.03 1.71 1.96 1.97 2.31 1.53 and 2.21 1.67 and 1.80 2.24 5.73 6.25 6.18 6.60 6.36 6.24 6.64 1.24 1.17 1.18 1.94 1.98

m m t (7) d (15) d (15) d (11) dd (15, 11) d (15) d (11) m s s s s s m m m d (10, 6) dd (16, 10) d (16) d (11) dd (15, 11) d (15) d (11) m s s s s s

1

1

H

11 mult (J in Hz)

∼1.46 ∼1.62 2.02 6.16 6.15 6.15 6.65 6.35 6.26 6.64 1.03 1.03 1.71 1.96 1.97 ∼1.96 1.78 and 1.80 1.68 and 1.71 2.52 5.48 6.15 6.13 6.60 6.36 6.26 6.64 1.21 1.25 1.17 1.91 1.97

m m t (7) d (15) d (15) d (11) dd (15, d (15) d (11) dd (15, s s s s s m m m dd (10, dd (15, d (15) d (11) dd (15, d (15) d (11) m s s s s s

11)

11)

6) 11)

11)

1

H

mult (J in Hz)

∼1.46 ∼1.62 2.02 6.16 6.15 6.15 6.65 6.35 6.26 6.64 1.03 1.03 1.71 1.97 1.97 5.12 2.12 1.54 4.04 5.70 6.38 6.20 6.59 6.38 6.26 6.64 1.69 1.62 1.14 1.93 1.97

m m t (7) d (15) d (15) d (11) dd (15, 11) d (15) d (11) dd (15, 11) s s s s s m m t (7) dd (7.5, 3) dd (15.5, 7.5) d (15.5) d (11) dd (15, 11) d (15) d (11) m s s s s s

12 1

H

mult (J in Hz)

∼1.46 ∼1.62 2.02 6.16 6.15 6.15 6.65 6.35 6.26 6.64 1.03 1.03 1.71 1.97 1.97 5.12 2.12 1.54 4.02 5.74 6.38 6.20 6.59 6.38 6.26 6.64 1.69 1.62 1.22 1.93 1.97

m m t (7) d (15) d (15) d (11) dd (15, 11) d (15) d (11) dd (15, 11) s s s s s m m t (7) dd (7.5, 3) dd (15.5, 7.5) d (15.5) d (11) dd (15, 11) d (15) d (11) m s s s s s

s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. (erythro)-γ-Carotene-5′,6′-diol (12). Yield: 0.2 mg. UV/vis (Et2O): 420, 445, 472 nm. HR FAB MS m/z: [M+] calcd for C40H58O2, 570.4437; found 570.4439. 1H NMR (Table 2). Ab Initio Molecular Orbital Calculation. Ab initio molecular orbital calculations were performed using the Gaussian 03 program (Gaussian, Inc., Wallingford, CT). The starting coordinates were generated by inspection of the molecular model and were fully optimized using a Fujitsu HX600 supercomputer at the Information Technology Center of Nagoya University. The position of methyl group and hydroxyl group were represented as follows. The position of methyl group was represented by the average of coordinates of three hydrogens that were obtained by the ab initio molecular orbital calculations. Similarly, the position of the hydroxyl group was represented by the coordinates of the base of a perpendicular line drawn from the hydrogen atom to the extension of the C-6 and O bond. In Vitro Epstein−Barr Virus (EBV) Early Antigen Activation Induction Effect. EBV genome-carrying lymphoblastoid cells (Raji cells) derived from Burkitt’s lymphoma were cultivated in RPMI-1640 medium with 10% fetal bovine serum (FBS). The Raji cells were incubated for 48 h at 37 °C in a medium containing n-butyric acid (4 nmol), TPA (32 pmol), and various amounts of test compounds. Smears were made from the cell suspension, and we employed an indirect immunofluorescence technique. Details of the in vitro assay on EBV-EA induction have been reported previously.23,24

9Z-lycopene, 13Z-lycopene, 1,16-didehydro-2,6-cyclolycopene-5-ol A (2), 2,6-cyclolycopene-1,5-diol A (4), and 2,6-cyclolycopene-1,5-diol B (5) were identified based on UV/vis, 1H NMR, and FAB MS data.14−18,21,22 1,16-Didehydro-2,6-cyclolycopene-5-ol B (3). Yield: 1 mg. UV/vis (Et2O): 431, 455, 486 nm. HR FAB MS m/z: 552.4344 [M+] (calcd for C40H56O, 552.4331). 1H NMR (Table 1). (threo)-Lycopene-5,6-diol (6). Yield: 3 mg. UV/vis (Et2O): 431, 455, 486 nm. HR FAB MS m/z: 570.4436 [M+] (calcd for C40H58O2, 570.4437). FAB MS/MS m/z 552 [M-18] +, 501 [M-69] +, 478 [M92] +, 443 [M-127] +, 374 [M-96−127] +. 1H and 13C NMR (Table 1). Acetylation of 6 with acetic anhydride in pyridine at room temperature for 1 h gave a monoacetate of 6. FAB MS m/z 612 [M+]. (erythro)-Lycopene-5,6-diol (7). Yield: 1 mg. UV/vis (Et2O): 431, 455, 486 nm. HR FAB MS m/z: 570.4437 [M+] (calcd for C40H58O2, 570.4437). 1H NMR (Table 1). Acetylation of 7 with acetic anhydride in pyridine at room temperature for 1 h gave a monoacetate of 7. FAB MS m/z 612 [M+]. 2′,6′-cyclo-γ-Carotene-1′,5′-diol A (9). Yield: 0.2 mg. UV/vis (Et2O): 420, 445, 472 nm. HR FAB MS m/z: 570.4430 [M+] (calcd for C40H58O2, 570.4437). 1H NMR (Table 2). 2′,6′-cyclo-γ-Carotene-1′,5′-diol B (10). Yield: 0.2 mg. UV/vis (Et2O): 420, 445, 472 nm. HR FAB MS m/z: [M+] calcd for C40H58O2, 570.4437; found 570.4429. 1H NMR (Table 2). (threo)-γ-Carotene-5′,6′-diol (11). Yield: 0.1 mg. UV/vis (Et2O): 420, 445, 472 nm. HR FAB MS m/z: [M+] calcd for C40H58O2, 570.4437; found 570.4440. 1H NMR (Table 2). 1624



Article

Figure 1. Lycopene (1) and its oxidative metabolites (2−7) in gac. The relative stereochemistry was indicated as R* and S*.

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RESULTS AND DISCUSSION Three new carotenoids, 3, 6, and 7, were isolated as minor components along with lycopene (1), 1,16-didehydro-2,6cyclolycopene-5-ol A (2), 2,6-cyclolycopene-1,5-diol A (4), and 2,6-cyclolycopene-1,5-diol B (5) from the aril of gac, as shown in Figure 1. Relative stereochemistry of these compounds is indicated as R* and S*. The new carotenoid 3 showed absorption maxima at 431, 455, and 486 nm, indicating the presence of a decene conjugated double bond system.22 The molecular formula of 3 was determined as C40H56O by HR FAB MS. The 1H NMR spectral data for 3, assigned by COSY and NOESY analyses, are shown in Table 1. The 1H NMR spectroscopic data were almost the same as those of 1,16-didehydro-2,6-cyclolycopene5-ol A (2),18 except for the H-5, H-6, H-7, and H-18 positions.

This suggested that 3 might be an epimer of 1,16-didehydro2,6-cyclolycopene-5-ol A (2), having a (2S*,5S*,6R*) configuration. The relative configuration of 3 was elucidated by performing a NOESY experiment. In the case of 2, a NOESY correlation for CH3-18/H-7 was not noted. However, a NOESY correlation for CH3-18/H-7 was observed in the case of 3 (Figure 2). This clearly indicated that 3 is a 6-epimer of 2. Therefore, a (2S*,5S*,6S*) configuration was postulated for 3. Thus, compound 3 was named 1,16-didehydro-2,6-cyclolycopene-5-ol B ((2S*,5S*,6S*)-1,16-didehydro-1,2,5,6-tetrahydro-2,6-cyclo-ψ,ψ-carotene-1′,5′-diol). This stereostructure was also confirmed using the ab initio molecular orbital method employing the Gaussian 03 program. Both new carotenoids 6 and 7 showed the same absorption maxima at 431, 455, and 486 nm, indicating the presence of a 1625



Article

Figure 2. NOESY correlations of 3, 6, and 7.

Figure 3. Outline of the total synthesis of (5S,6R)- and (5S,6S)-lycopene-5,6-diol.

decene conjugated double bond system.22 The molecular formulas of both 6 and 7 were determined as C40H58O2 by HR FAB MS. Acetylation of both compounds yielded a monoacetate. The 1H and 13C NMR data for 6, which were assigned by COSY, NOESY, HSQC, and HMBC analyses, are shown in Table 1. These 1H and 13C NMR spectroscopic data were almost the same as those of lycopene (1), except for the

C-5, C-6, and C-18 positions. The presence of one secondary hydroxy group (δC 78.9 and δH 4.04) and tertiary hydroxy group (δC 75.0) in 6 was revealed by NMR data. The presence of a tertiary hydroxy group at C-5 and secondary hydroxy group at C-6 was determined by HMBC data. HMBC correlations for CH3-18/C-5, CH3-18/C-6, H-3/C-5, and H-7/C-6 were in agreement with this structure. Thus, the planar structure of 6 1626



Article

Figure 4. γ-Carotene (8) and its oxidative metabolites (9−12) in gac.

was determined as 5,6-dihydro-ψ,ψ-carotene-5,6-diol. The 1H NMR data for 7 were almost the same as those for 6, except for the H-6 and H-18 positions. The COSY experiment also revealed the planar structure 5,6-dihydro-ψ,ψ-carotene-5,6-diol for 6. We decided on lycopene-5,6-diol to refer to the carotenoid of 5,6-dihydro-ψ,ψ-carotene-5,6-diol. Concerning the relative stereochemistry of lycopene-5,6-diol, the threo form (indicated as 5S*,6S* configuration) and erythro form (indicated as 5S*,6R* configuration) can be considered (Figure 1). The relative stereochemistry of 6 and 7 was elucidated by NOESY and NOE difference experiments. Strong NOE (>5%) was observed between protons of CH3-18 and OH-6 in compound 6, and NOE was not noted between protons of CH3-18 and OH-6 in compound 7. Furthermore, strong NOE between protons of CH3-18 and H-6 was not observed in compound 6 but it was observed in compound 7. Molecular calculation using the ab initio molecular orbital method employing the Gaussian 03 program indicated that, in the case of the threo isomer, distances of CH3-18 to H-6 and CH318 to OH-6 are 3.81 and 3.28 Å, respectively. Additionally, in the case of the erythro isomer the distances of CH3-18 and H-6 and CH3-18 and OH-6 are 3.16 and 3.91 Å, respectively. It has been reported that NOE is observed at both protons pairs, located within 3.5 Å.25 Therefore, threo stereochemistry for 6 and erythro stereochemistry for 7 could be postulated. These

stereochemistries were also confirmed with a synthetic approach. Namely, (5S,6R)-(erythro)-lycopene-5,6-diol was synthesized by a stepwise C15 + C10 + C15 double Wittig reaction. The key compound, (5S,6R)-C15-dihydroxy aldehyde, was prepared via Sharpless asymmetric epoxidation of geraniol and subsequent acidic hydrolysis of the epoxide as shown in Figure 3. The enantiomeric purity of the epoxy geraniol was improved by recrystallization of its dinitrobenzoate. In the same procedure, (5S,6S)-(threo)-lycopene-5,6-diol was synthesized starting from nerol. The enantiomerically enriched compound was obtained by recrystallization of the dihdroxy dinitrobenzoate instead of the epoxy dinitrobenzoate. The outline of the total synthesis of (5S,6R)- and (5S,6S)- lycopene-5,6-diol is shown in Figure 3. The detailed synthetic procedures of these compounds will be described elsewhere. The 1H NMR and 13C NMR data of 6 were identical to those of synthetic (threo)-lycopene-5,6-diol, and the 1H NMR data of 7 were identical to those of (erythro)-lycopene-5,6-diol. Therefore, structures of 6 and 7 were determined to be (threo-) and (erythro)-lycopene-5,6-diol, respectively. Four oxidative metabolites of γ-carotenes, 9, 10, 11, and 12, were isolated as very minor components. The new carotenoid 9 showed absorption maxima at 420, 445, and 472 nm. The molecular formula of 9 was determined as C40H58O2 by HR FAB MS. The 1H NMR data for 9 are shown in Table 2. The 1627



Article

Figure 5. Possible metabolic conversion pathways of lycopene in gac.

Table 3. Percent Relative Rate of EBV-EA Activationa with Respect to the Positive Control in the Presence of Carotenoids 1, 4, 6, 8, 9, and 11b concentration (mol ratio/TPA)c compounds lycopene (1) 2,6-cyclolycopene-1,5-diol A (4) lycopene-5,6-diol (6) γ-carotene (8) 2,6-cyclo-γ-carotene-1′,5′-diol A (9) γ-carotene-5′,6′-diol (11)

1000 d

15.5 (60) 9.6 (60) 11.3 (60) 12.0 (70) 9.2 (70) 6.2 (70)

500

100

10

IC50e

43 31.8 34.4 34.9 31.9 32.0

86.7 79.8 80.8 81.2 78.6 79.8

100 100 100 100 100 100

406 355 374 379 349 352

a Values represent the percentage relative to the positive control values (100%). bThere are significant differences (P < 0.01) in inhibitory capacity in treatment groups of carotenoids 1, 4, 6, 8, 9, and 11 with the control group. cTPA concentration was 20 ng (32 pmol/mL). dValues in parentheses are percentage viability of Raji cells. eUnits are nmol.

H NMR spectroscopic data showed the presence of a βcarotene moiety (H-2 to H-20). The signals of the remaining part (H-2′ to H-20′) were the same as those of H-2 to H-20 positions of 2,6-cyclolycopene-1,5-diol A (4), having a (2S*,5S*,6R*) configuration.14 This structure was confirmed by COSY and NOESY experiments. Therefore, the structure of 9 was determined as (2′S*,5′S*,6′R*)-1′,2′,5′,6′-tetrahydro2′,6′-cyclo-β,ψ-carotene-1′,5′-diol. Like 2,6-cyclolycopene-1,5diol, this structure corresponds to a cyclic oxidative metabolite of γ-carotene. Therefore, we referred to 9 as 2′,6′-cyclo-γcarotene-1′,5′-diol A (Figure 4). The new carotenoid 10 showed the same UV/vis and MS spectra as those of 9. The 1H NMR spectroscopic data were almost the same as those of 9 except for the H-2′ to H-6′ and H-16′ to H-18′ positions. This suggested that 10 might be an epimer of 2′,6′-cyclo-β,ψ-carotene-1′,5′-diol A (9). The 1H NMR spectroscopic data of H-2′ to H-6′ and H-16 to H-18 were the same as those of H-2 to H-6 and H-16′ to H-18′ of 2,6-cyclolycopene-1,5-diol B (5), having a (2′S*,5′S*,6′S*) configuration.14 Therefore, the structure of 10 was determined as (2′S*,5′S*,6′S*)-1′,2′,5′,6′- tetrahydro-2′,6′-cyclo-β,ψ-carotene-1′,5′-diol and was named 2′,6′-cyclo-β,ψ-carotene-1′,5′1

diol B (Figure 4). Very recently, Crawford et al. synthesized 1,2,5,6-tetrahydro-2,6-cyclo-γ-carotene-1,5-diol, an oxidation product of γ-carotene.26 This compound has the same relative stereochemistry as that of 9 and the spectral data of 1,2,5,6tetrahydro-2,6-cyclo-γ-carotene-1,5-diol synthesized by Crawford et al. were identical to those of 9. Both compounds 11 and 12 showed the same absorption maxima at 420, 445, and 472 nm and the same molecular formula of C40H58O2. 1H NMR data of both compounds were similar to each other except for H-5′ and H-18′. 1H NMR data of 11 indicated the presence of a β-carotene moiety (H-2 to H20) and (threo)-lycopene-5,6-diol moiety (H-2′ to H-20′). Therefore, the structure of 11 was determined as (threo)-5′,6′dihydro-β,ψ-carotene-5′,6′-diol, which was confirmed by COSY and NOESY experiments, and was named (threo)-γ-carotene5′,6′-diol (Figure 4). Similarly, the structure of 12 was determined as (erythro)5′,6′-dihydro-β,ψ- carotene-5′,6′-diol by comparison of the 1H NMR data with β-carotene and (erythro)-lycopene-5,6-diol (7), and was named (erythro)-γ-carotene-5′,6′-diol (Figure 4). Based on the results of the present investigation and the previous literature,13−18 possible metabolic conversion path1628



Article

and compositions in gac are shown in Table 4. Gac is a good dietary source of lycopene, as is tomato.19,20

Table 4. Carotenoid Compositions in Aril of Gac carotenoid

% composition

α-carotene β-carotene 9Z-β-carotene γ-carotene (2) lycopene (1) 9Z-lycopene 13Z-lycopene 1,16-didehydro-2,6-cyclolycopene-5-ol A (2) 1,16-didehydro-2,6-cyclolycopene-5-ol B (3) 2,6-cyclolycopene-1′,5′-diol A (4) 2,6-cyclolycopene-1,5-diol B (5) (threo)-lycopene-5,6-diol A (6) (erythro)-lycopene-5,6-diol B (7) 2′,6′-cyclo-γ-carotene-1′,5′-diol A (9) 2′,6′-cyclo-γ-carotene-1′,5′-diol B (10) (threo)-γ-carotene-5′,6′-diol (11) (erythro)-γ-carotene-5′,6′-diol (12) other carotenoidsa

3.6 7.7 1.2 4.1 40.7 11.8 4.5 2.5 1.4 4.5 2.5 6.0 2.0 0.8 0.8 0.5 0.5 4.9

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AUTHOR INFORMATION

Corresponding Author

*T.M. telephone: +81-75-781-1107. Fax: +81-75-791-7659. Email: [email protected]. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Mitani Sangyo Co., Ltd. for providing gac fruits. REFERENCES

(1) Di Mascio, P.; Kaiser, S.; Sies, H. Lycopene is the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 2000, 72, 990−997. (2) Oshima, S.; Ojima, F.; Sakamato, H.; Ishiguro, Y.; Terao, J. Supplementation with carotenoids inhibits singlet oxygen-mediated oxidation of human plasma low-density lipoprotein. J. Agric. Food Chem. 1999, 44, 2306−2309. (3) Böhm, F.; Tinkler, J. H.; Truscott, T. G. Carotenoids protect against cell membrane damage by the nitrogen dioxide radical. Nat. Med. (N. Y., NY, U. S. A.) 1995, 1, 98−99. (4) Böhm, F.; Edge, R.; Burk, M.; Truscott, T. G. Dietary uptake of lycopene protects human cells from singlet oxygen and nitrogen dioxide - ROS components from cigarette smoke. J. Photochem. Photobiol., B 2001, 64, 176−179. (5) Levy, J.; Danilenko, M.; Shatoi, Y. The Tomato Carotenoid Lycopene and Cancer. In Food Factors for Cancer Prevention; Ohigashi, H., Osawa, T., Terao, J., Watanabe, S., Yoshikawa, T., Eds.; Springer: Tokyo, 1997; pp 209−212. (6) Nishino, H.; Tokuda, H.; Satomi, Y.; Masuda, M.; Bu, P.; Onozuka, M.; Yamaguchi, S.; Okuda, Y.; Takayama, Y.; Tsuruta, J.; Okuda, M.; Ichiishi, E.; Murakoshi, M.; Yano, M. Cancer prevention by carotenoids. Pure. Appl. Chem. 1999, 71, 2273−2278. (7) Nishino, H.; Murakoshi, M.; Ii, T.; Takemura, M.; Kuchide, M.; Kanazawa, M.; Mou, Y. M.; Wada, S.; Masuda, M.; Ohsaka, Y.; Yogosawa, S.; Satomi, Y.; Jinno, K. Carotenoids in cancer chemoprevention. Cancer Metastasis Rev. 2002, 21, 257−264. (8) Nishino, H.; Murakoshi, M.; Tokuda, H.; Satomi, Y. Cancer prevention by carotenoids. Arch. Biochem. Biophys. 2009, 483, 165− 168. (9) Gann, P. H.; Ma, J.; Giovannucci, E.; Willett, W.; Sacks, F. M.; Hennekens, C. H.; Stampfer, M. J. Lower prostate cancer risk in men with elevated plasma lycopene levels: results of a prospective analysis. Cancer Res. 1999, 59, 1225−1230. (10) Giovannucci, E. Tomatoes, tomato-based products, lycopene, and cancer: Review of the epidemiologic literature. J. Natl. Cancer Inst. 1999, 91, 317−331. (11) Giovannucci, E.; Ascherio, A.; Rimm, E. B.; Stamofer, M. J.; Colditz, G. A.; Willett, W. C. A prospective study on tomato products, lycopene, and prostate cancer risk. J. Natl. Cancer Inst. 1995, 94, 391− 398. (12) Gerster, H. The potential role of lycopene for human health. J. Am. Coll. Nutr. 1997, 176, 109−126. (13) Khachik, F.; Beecher, G. R.; Goli, M. B.; Lusby, W. R.; Smith, J. C., Jr. Separation and identification of carotenoids and their oxidation products in the extracts of human plasma. Anal. Chem. 1992, 64, 2111−2122. (14) Khachik, F.; Steck, A.; Niggli, U. A.; Pfander, H. Partial synthesis and structural elucidation of the oxidative metabolites of lycopene identified in tomato paste, tomato juice and human serum. J. Agric. Food Chem. 1998, 46, 4885−4874−4884. (15) Khachik, F.; Pfander, H.; Traber, B. Proposed mechanism for the formation of synthetic and naturally occurring metabolites of

a β-Cryptoxanthin, isocryptoxanthin, zeaxanthin, lutein. Carotenoid content 0.61 mg/g.

ways of 1,16-didehydro-2,6-cyclolycopene-5-ols (2 and 3), 2,6cyclolycopene-1,5-diols (4 and 5), and lycopene-5,6-diols (6 and 7) from lycopene (1) are proposed, as shown in Figure 5. Nevertheless, lycopene-5,6-epoxide was not detected in gac, and it was assumed that compounds 2−7 might be formed through lycopene-5,6-epoxide. Epoxidation of lycopene provided lycopene-5,6-epoxide. Lycopene-5,6-epoxide was reported as a reaction product of lycopene with MCPB,14 but it has not been isolated and characterized by spectroscopy including NMR.14−18 Lycopene-5,6-epoxide might be a very unstable compound and soon converted to other metabolites. Immediately, lycopene-5,6-epoxide was converted to 2,6cyclolycopene-1,5-diols (4 and 5) through epoxy ring cleavage and cyclization of the end group by acid catalysis. Subsequently, dihydroxylation of the hydroxy group at C-1 in 4 and 5 yielded 1,16-didehydro-2,6-cyclolycopene-5-ols (2 and 3). However, hydrolytic cleavage of the epoxide ring in lycopene-5,6-epoxide led to two diastereomeric pairs of lycopene-5,6-diols, 6 and 7. Similarly, 2′,6′-cyclo-γ-carotene-1′,5′-diols (9 and 10) and γcarotene-5′,6′-diols (11 and 12) were assumed to be formed from γ-carotene through γ-carotene-5′,6′-epoxide, which was not detected in gac, like lycopene-5,6-diol. The antitumor-promoting activity of lycopene (1), 2,6cyclolycopene-1,5-diol A (4), lycopene-5,6-diol (6), γ-carotene (8), 2′,6′-cyclo-γ-carotene-1′,5′-diol (9), and γ-carotene-5′,6′diol (11) was examined using the Epstein−Barr virus (EBV) activation assay in Raji cells. The results are shown in Table 3. These compounds showed inhibitory effects on the EBV-EA induction of Raji cells without significant cytotoxicity (more than 60% viability of Raji cells) in this assay. The lycopene oxidative metabolites 2,6-cyclolycopene-1,5-diol A (4) and lycopene-5,6-diol (6) showed slightly higher activity than lycopene (1), which is a well-known and excellent antitumorpromoter.5−8 Similarly, 2′,6′-cyclo-γ-carotene-1′,5′-diol (9) and γ-carotene-5′,6′-diol (11) also showed slightly higher activity than γ-carotene (8). These results revealed that oxidative metabolites of lycopene and γ-carotene exhibit anticarcinogenic activity as well as lycopene and γ-carotene. Carotenoid contents 1629



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lycopene in tomato products and human serum. J. Agric. Food Chem. 1998, 46, 4885−4890. (16) Lu, Y.; Etoh, H.; Watanabe, N.; Ina, K.; Ukai, N.; Oshima, S.; Ojima, F.; Sakamato, H.; Ishiguro, Y. A new carotenoid, hydrogen peroxide oxidation products from lycopene. Biosci., Biotechnol., Biochem. 1995, 59, 2153−2155. (17) Yokota, T.; Etoh, H.; Ukai, N.; Oshima, S.; Sakamoto, H.; Ishiguro, Y. 1,5-Dihydroxyiridanyl-lycopene in tomato puree. Biosci., Biotechnol., Biochem. 1997, 61, 549−550. (18) Yokota, T.; Etoh, H.; Oshima, S.; Hayakawa, K.; Ishiguro, Y. Oxygenated lycopene and dehydrated lutein in tomato puree. Biosci., Biotechnol., Biochem. 2003, 67, 2644−2647. (19) Aoki, H.; Kieu, T. M.; Kuze, N.; Tomisaka, K.; Chuene, N. V. Carotenoid pigments in gac fruit (Momordica cochinchinensis Spreng). Biosci., Biotechnol., Biochem. 2002, 66, 2479−2482. (20) Ishida, B. K.; Turner, C.; Chapman, M. H.; McKeron, T. A. Fatty acid and carotenoid composition of gac (Momordica cochinchinensis Spreng) fruit. J. Agric. Food Chem. 2004, 52, 274−279. (21) Schiedt, K.; Liaaen-Jensen, S. Isolation and Analysis. In Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser: Basel, 1995; Vol. 1A, pp 81−108. (22) Britton, G. UV/Visible Spectroscopy. In Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser: Basel, 1995; Vol. 1B, pp 13−62. (23) Tsushima, M.; Maoka, T.; Katsuyama, M.; Kozuka, M.; Matsuno, T.; Tokuda, H.; Nishino, H.; Iwashima, A. Inhibitory effect of natural carotenoids on Epstein-Barr virus activation activity of a tumor promoter in Raji cells. A screening study for anti-tumor promoters. Biol. Pharm. Bull. 1995, 18, 227−233. (24) Maoka, T.; Mochida, K.; Kozuka, M.; Ito, Y.; Fujiwara, Y.; Hashimoto, K.; Enjo, F.; Ogata, M.; Nobukuni, Y.; Tokuda, H.; Nishino, H. Cancer chemopreventive activity of carotenoids in the fruits of red paprika Capsicum annuum L. Cancer Lett. 2001, 172, 103− 109. (25) Englert, G. NMR Spectroscopy. In Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser: Basel, 1995; Vol. 1B, pp147−160. (26) Crawford, K.; Mazzola, E.; Khachik, F. Total synthesis of 1,2,5,6tetrahydro-2,6-cyclo-γ-carotene-1,5-diol, an oxidation product of γcarotene. Synthesis 2014, 46, 635−645.

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Oxidative Metabolites of Lycopene and Î³-Carotene in Gac (Momordica

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