Production of Auroxanthins from Violaxanthin and 9-cis-Violaxanthin

27 Nov 2016 - ... 9-cis-Violaxanthin by Acidic Treatment and the Antioxidant Activities of ... Department of Food and Nutrition, Japan Women's Univers...
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Production of Auroxanthins from Violaxanthin and 9-cis-Violaxanthin by Acidic Treatment and the Antioxidant Activities of Violaxanthin, 9-cis-Violaxanthin, and Auroxanthins Michiko Araki, Naoko Kaku, Momoko Harada, Yuka Ando, Risa Yamaguchi, and Kazutoshi Shindo* Department of Food and Nutrition, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan S Supporting Information *

ABSTRACT: Violaxanthin and 9-cis-violaxanthin (major epoxycarotenoids in fruit) were prepared from mango fruit, purified, and converted to other carotenoids under acidic conditions. The resulting carotenoid structures were then analyzed in detail. Not only violaxanthin but also 9-cis-violaxanthin were found to be converted to (8S,8′S)-, (8S,8′R)-, and (8R,8′R)-auroxanthin at an approximate ratio of 4:6:1. Antioxidant activities of violaxanthin, 9-cis-violaxanthin, (8S,8′S)-auroxanthin, and (8S,8′R)auroxanthin were examined. They possessed potent lipid peroxidation inhibitory and very weak 1O2 quenching activities. KEYWORDS: violaxanthin, 9-cis-violaxanthin, auroxanthin, antioxidant activitity

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INTRODUCTION

In this study, we identifed the acid-catalyzed derivatives of 2 as the auroxanthins 3, 4, and 5. We also examined lipid peroxidation inhibitory (inhibition of rat brain homogenate peroxidation that was initiated by Fe2+−Fe3+-O2 complex14 and advanced by lipid peroxide derived radicals15) and 1O2 quenching (inhibition of methylene blue-sensitized linoleic acid photo-oxidation16) activities of 1−4.

More than 750 carotenoids with various structures have been isolated from natural sources,1 and beneficial medical effects as a result of administration of carotenoids, such as antiarteriosclerosis and anticancer effects, have recently been reported.2,3 Because these effects are mainly derived from the antioxidant activities (singlet oxygen (1O2) quenching activity4,5 and radical quenching activity6) of carotenoids, evaluation of the antioxidant potentials of various carotenoids contained in foods may represent an interesting field in medical research. Violaxanthin, 17 (Figure 1), is a major epoxycarotenoid biosynthesized in higher plants and is present universally in the leaves (photosynthetic organ) as it participates in the xanthophyll cycle. Compound 1 also accumulates in many fruits, mainly in its acylated form.8 9-cis-Violaxanthin, 29 (Figure 1), the geometrical isomer of 1, is also a major epoxycarotenoid produced by higher plants, and acylated compounds of 1 and 2 are contained in several fruits, such as mango, orange, and pineapple, in an approximate ratio of 1:1.10 The enzymatic conversion of 1 to 2 in plants was predicted from the discovery of the isomerization enzyme that converts βcarotene to 9-cis-β-carotene in the strigolactone biosynthetic pathway,11 and 2 was reported to be a precursor of abscisic acid, a plant hormone.12 On the other hand, antioxidant activities of 1 and 2 have been scarcely reported, probably due to their unstable nature during the isolation process. Compound 1 was readily converted to auroxanthins (a mixture of (8S,8′S)-auroxanthin, 3, (8S,8′R)-auroxanthin, 4, and (8R,8′R)-auroxanthin, 5) (Figure 1) via epoxide-furanoid rearrangement by acid catalysis.13 Thus, 1 found in food sources was proposed to be converted to auroxanthins in the stomach after ingestion. Compound 2 was also unstable in acidic conditions, but the structures of acidic catalyzed compounds of 2 have not been reported. Moreover, to our knowledge, the antioxidant activities of auroxanthins have not been examined. © XXXX American Chemical Society

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MATERIALS AND METHODS

Spectroscopic Analysis. HR-ESI-MS spectra were obtained on a JMS-T100LP mass spectrometer (JEOL, Tokyo, Japan), using reserpine as an external standard. NMR spectra were measured by an AVANCE400 (Bruker BioSpin, Karlsruhe, Germany) in CDCl3, using the residual solvent peak as an internal standard (δC 77.0, δH 7.26). Preparation of Violaxanthin, 1, and 9-cis-Violaxanthin, 2. Four hundred grams of mango (Mangifera indica) purchased at a fruit shop in Tokyo was cut into small blocks (3 cm × 3 cm × 3 cm, approximately), suspended in saturated aqueous NaHCO3 water (650 mL), and agitated in a blender for 30 s. One liter of acetone was added to the agitated mango solution, the solution was stirred for 5 min and filtered, and the solid remaining on the filter paper was collected in a 1 L beaker. The solid was added to 600 mL of CH2Cl2/acetone (2:1, v/ v) and stirred for 15 min at room temperature to extract carotenoids (twice). The combined filtrate (1.2 L) was concentrated to a small volume to remove CH2Cl2 and acetone and separated in EtOAc (200 mL) and H2O (200 mL), without pH adjustment. The EtOAc layer was concentrated to dryness to give a crude carotenoid extract (0.80 g) containing diacyl 1 and diacyl 2. To prepare 1 and 2, the carotenoid extract was saponified by resuspension in 20 mL of KOH solution (5 g of KOH/100 mL of 90% EtOH) and 5 mL of CH2Cl2 and stirred for 1 h. The solution was added to a separation funnel containing EtOAc (200 mL) and H2O (200 mL), and the two layers (EtOAc/H2O) were shaken well. The EtOAc layer was collected and dried using anhydrous Na2SO4 and Received: Revised: Accepted: Published: A

October 9, 2016 November 26, 2016 November 27, 2016 November 27, 2016 DOI: 10.1021/acs.jafc.6b04506 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Physicochemical Data for Compounds 3−5. (8S,8′S)-Auroxanthin, 3: yellow solid; [α]D24 +23 (c 0.10, CHCl3); UV−vis (MeOH) λmax 379, 410, 424 nm; %III/II (%OD424/OD410) 95; HR-ESI-MS (negative) m/z 599.40932 [M − H]− (calcd for C40H55O4) 599.41003; 1H NMR (CDCl3) δ 1.17 (s, 6H, H-16 and H-16′), 1.33 (s, 6H, H-17 and H-17′), 1.51 (dd, J = 3.4, 14.5 Hz, 2H, H-2b and H-2′b), 1.61 (s, 6H, H-18 and H-18′), 1.71 (s, 6H, H-19 and H-19′), 1.76 (dd, J = 3.8, 14.5 Hz, 2H, H-2a and H-2′a), 1.94 (s, 6H, H-20 and H-20′), 1.99 (dd, J = 4.2, 13.8 Hz, 2H, H-4b and H-4′b), 2.11 (dd, J = 4.2, 13.8 Hz, 2H, H-4a and H-4′a), 4.25 (m, 2H, H-3 and H-3′), 5.16 (s, 2H, H-8 and H-8′), 5.25 (s, 2H, H-7 and H-7′), 6.18 (d, J = 11.8 Hz, 2H, H-10 and H-10′), 6.21 (d, J = 11.2 Hz, 2H, H-14 and H-14′), 6.31 (d, J = 15.1 Hz, 2H, H-12 and H-12′), 6.48 (dd, J = 11.8, 15.1 Hz, 2H, H-11 and H-11′), 6.60 (m, 2H, H-15 and H-15′); 13C NMR (CDCl3) δ 12.6 (C-19 and C-19′), 12.8 (C-20 and C-20′), 28.9 (C-17 and C-17′), 29.0 (C-18 and C-18′), 31.4 (C-16 and C-16′), 33.7 (C-1 and C-1′), 46.7 (C-4 and C-4′), 47.4 (C-2 and C-2′), 67.7 (C-3 and C3′), 86.8 (C-5 and C-5′), 87.7 (C-8 and C-8′), 119.9 (C-7 and C-7′), 124.3 (C-11 and C-11′), 127.2 (C-10 and C-10′), 129.9 (C-15 and C15′), 132.3 (C-14 and C-14′), 136.2 (C-13 and C-13′), 137.6 (C-12 and C-12′), 137.8 (C-9 and C-9′), 154.0 (C-6 and C-6′). (8S,8′R)-Auroxanthin, 4: yellow solid; [α]D24 −22 (c 0.13, CHCl3); UV−vis (MeOH) λmax 378, 400, 425 nm; %III/II (%OD425/OD400) 92; HR-ESI-MS (negative) m/z 599.41268 [M − H]− (calcd for C40H55O4) 599.41003; 1H NMR (CDCl3) δ 1.17 (s, 3H, H-16) 1.19 (s, 3H, H-16′), 1.33 (s, 3H, H-17), 1.34 (s, 3H, H-17′), 1.46−1.53 (2H, H-2b and H-2′b), 1.61 (s, 3H, H-18), 1.68 (s, 3H, H-18′), 1.71 (s, 3H, H-19), 1.74−1.82 (2H, H-2a and H-2′a), 1.80 (s, 3H, H-19′), 1.94 (s, 6H, H-20 and H-20′), 1.90 (m, 1H, H-4′b), 1.99 (m, 1H, H4b), 2.09−2.14 (2H, H-4a and H-4′a), 4.23−4.27 (2H, H-3 and H-3′), 5.07 (s, 1H, H-8′), 5.16 (s, 1H, H-8), 5.25 (s, 1H, H-7), 5.30 (s, 1H, H-7′), 6.18 (d, J = 11.8 Hz, 2H, H-10 and H-10′), 6.21 (d, J = 11.2 Hz, 2H, H-14 and H-14′), 6.31 (d, J = 15.1 Hz, 2H, H-12 and H-12′), 6.48 (dd, J = 11.8, 15.1 Hz, 2H, H-11 and H-11′), 6.60 (m, 2H, H-15 and H-15′); 13C NMR (CDCl3) δ 12.6 (C-19), 12.8 (C-20 and C-20′), 13.4 (C-19′), 28.1 (C-17′), 28.9 (C-17), 29.0 (C-18), 30.6 (C-18′), 31.3 (C-16′), 31.4 (C-16), 33.7 (C-1), 34.2 (C-1′), 46.7 (C-4), 47.4 (C-2′, C-2, and C-4′), 67.7 (C-3), 67.9 (C-3′), 86.8 (C-5), 87.2 (C5′), 87.7 (C-8), 88.4 (C-8′), 118.8 (C-7′), 119.9 (C-7), 124.3 (C-11), 124.4 (C-11′), 126.2 (C-10′), 127.2 (C-10), 129.8 (C-15′), 129.9 (C15), 132.1 (C-14′), 132.3 (C-14), 136.1 (C-13′), 136.2 (C-13), 137.4 (C-12′), 137.6 (C-12), 137.8 (C-9), 138.6 (C-9′), 153.2 (C-6′), 154.0 (C-6). (8R,8′R)-Auroxanthin, 5: yellow solid; UV−vis (MeOH) λmax 376, 398, 423 nm; %III/II (%OD398/OD423) 74; HR-ESI-MS (negative) m/ z 599.41320 [M − H]− (calcd for C40H55O4) 599.41003; 1H NMR (CDCl3) δ 1.19 (s, 6H, H-16 and H-16′), 1.34 (s, 6H, H-17 and H17′), 1.48 (m, 2H, H-2b and H-2′b), 1.68 (s, 6H, H-18 and H-18′), 1.80 (s, 6H, H-19 and H-19′), 1.80 (m, 2H, H-2a and H-2′a), 1.90 (m, 2H, H-4b and H-4′b), 1.94 (s, 6H, H-20 and H-20′), 2.11 (m, 2H, H4a and H-4′a), 4.24 (m, 2H, H-3 and H-3′), 5.07 (s, 2H, H-8 and H8′), 5.31 (s, 2H, H-7 and H-7′), 6.18 (d, J = 11.8 Hz, 2H, H-10 and H10′), 6.21 (d, J = 11.2 Hz, 2H, H-14 and H-14′), 6.31 (d, J = 15.1 Hz, 2H, H-12 and H-12′), 6.48 (dd, J = 11.8, 15.1 Hz, 2H, H-11 and H11′), 6.60 (m, 2H, H-15 and H-15′); 13C NMR (CDCl3) δ 12.8 (C-20 and C-20′), 13.4 (C-19 and C-19′), 28.1 (C-17 and C-17′), 30.6 (C-18 and C-18′), 31.3 (C-16 and C-16′), 34.2 (C-1 and C-1′), 47.4 (C-2, C2′, C-4, and C-4′), 67.9 (C-3 and C-3′), 87.2 (C-5 and C-5′), 88.4 (C8 and C-8′), 118.8 (C-7 and C-7′), 124.4 (C-11 and C-11′), 126.2 (C10 and C-10′), 129.8 (C-15 and C-15′), 132.1 (C-14 and C-14′), 136.1 (C-13 and C-13′), 137.4 (C-12 and C-12′), 138.6 (C-9 and C9′), 153.2 (C-6 and C-6′). 1 O2 Quenching Experiment. Eighty microliters of 25 μM methylene blue and 100 μL of 0.24 M linoleic acid with or without 40 μL of carotenoid (final concentration = 1−100 μM) (each dissolved in ethanol) were added to small glass test tubes (5 mL). Tubes were mixed well and were illuminated at 7000 lx at 22 °C for 3 h in a styrofoam box, then 50 μL of the reaction mixture was removed and diluted to 1.5 mL with ethanol, and OD235 was measured to estimate the formation of conjugated dienes.17 The OD235 in the

Figure 1. Structures of violaxanthin, 1, 9-cis-violaxanthin, 2, and auroxanthins, 3, 4, and 5. concentrated to dryness to give a red oil containing 1 and 2 (310 mg). The red oil was subjected to chromatography on a 10 × 2 cm column of Chromatorex FL60D silica gel (Fuji Silysia Chemical Ltd., Aichi, Japan) and eluted with hexane/acetone (3:1, v/v) (300 mL). The fractions containing 1 and 2 were collected and concentrated to dryness to give a red powder (9.9 mg). Finally, the red powder was separated by preparative HPLC (HPLC equipment: Hitachi L-7000 HPLC system (Hitachi High-Technologies Co., Tokyo, Japan); column, Cosmosil 5SL (Nakarai Tesque, Inc., Kyoto, Japan), 250 mm × 10 mm i.d.; solvent, hexane/acetone (7:3, v/v); flow rate, 3.0 mL/min; detection, photodiode array (PDA), monitored at 250−700 nm). Under these conditions, pure 1 and 2 were eluted at 15.0 and 18.5 min, respectively. The fluorescent lamps above the laboratory bench were switched off when the above experiments were performed to prevent geometrical isomerization of 1 and 2. Acid Conversion of Violaxanthin, 1, and 9-cis-Violaxanthin, 2, and Purification of the Products. Compounds 1 and 2 (each 2.0 mg) were dissolved in 2 mL of 0.1 M HCl in 50% EtOH and stirred for 10 min at room temperature. The solution was transferred to a test tube containing 5 mL of EtOAc and 5 mL of H2O and mixed well using a vortex mixer. The EtOAc layer was dried over anhydrous Na2SO4 and concentrated to dryness to give a mixture of auroxanthins (a mixture of (8S,8′S)-auroxanthin, 3, (8S,8′R)-auroxanthin, 4, and (8R,8′R)-auroxanthin, 5) in each experiment. The auroxanthin mixtures obtained from 1 and 2 were combined and separated by HPLC (column, Cosmosil 5SL, 250 mm × 100 mm i.d.; solvent, hexane/EtOAc (1:1, v/v); flow rate, 3.0 mL/min; detection, PDA, monitored at 250−700 nm), and the three peaks eluting at 17.0 min (3), 20.5 min (4), and 23.5 min (5) were collected. B

DOI: 10.1021/acs.jafc.6b04506 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Plausible reaction mechanism for the conversion of violaxanthin, 1, and 9-cis-violaxanthin, 2, to auroxanthins, 3, 4, and 5. absence of carotenoid was measured as negative control (no 1O2 quenching activity), and the 1O2 quenching activity of carotenoid was calculated from OD235 in the presence of carotenoid relative to this reference value. The activity was indicated as the IC50 value, which represents the concentration at which 50% inhibition was observed. Inhibitory Experiment of Lipid Peroxidation in Rat Brain Homogenate. Rat brain was homogenized according to the method of Kubo et al.18 with some modifications. Frozen rat brain (Wistar, 8 weeks, male), purchased from Funakoshi (Tokyo, Japan), was defrosted in ice-cold 0.1 M phosphate buffer at pH 7.2, and then 0.8 g of the brain was mixed with 30 mL of ice-cold phosphate buffer for 2 min in a Teflon homogenizer. Two hundred microliters of the homogenate, 0.6 mL of 0.1 M phosphate buffer at pH 7.2, 0.1 mL of 1 mM sodium ascorbate, and 50 μL of carotenoid solutions dissolved in methanol (final concentration = 0.1−100 μM) were added to small glass test tubes (5 mL) and mixed well. Tubes were incubated at 37 °C for 1 h under reciprocal agitation. Malondialdehyde was produced in the reaction mixture according to the concentrations of the lipid peroxides and was then reacted with thiobarbituric acid to quantify the amount by OD532. Percent inhibition was calculated as follows: (1 − (T − B)/(C − B)) × 100 (%), where T, C, and B are the OD532 readings of the treated carotenoid, the control (peroxidation with no carotenoid), and the zero-time control (no peroxidation without homogenate), respectively.

absorbances at 17.0, 20.5, and 23.5 min, and the peak area ratio was approximately 4:6:1. The three peaks derived from 1 and 2 were shown to be identical by cochromatography of the acid conversion products of 1 and 2 (the identities of the three peaks were further substantiated by 1H NMR spectra (Figure S7−S-9)). We combined the EtOAc layers of 1 and 2, isolated each peak by preparative silica gel HPLC using the conditions described above, and analyzed them by HR-ESI-MS and 1D (1H and 13C) and 2D (1H−1H DQF COSY, HMQC, HMBC, and NOESY) NMR spectroscopy. The peaks at 17.0, 20.5, and 23.5 min were identified to be (8S,8′S)-auroxanthin, 3 (0.5 mg), (8R,8′S)-auroxanthin, 4 (0.8 mg), and (8R,8′R)-auroxanthin, 5 (0.1 mg), respectively, by detailed analyses of the 1D and 2D NMR spectra based on the previously reported partially assigned 1H and 13C NMR data.1 This is the first paper showing that 2 is converted to auroxanthins, 3, 4, and 5 by acid catalysis. A plausible reaction mechanism for the conversion of 2 to auroxanthins is shown in Figure 2. The cation generated at epoxide oxygen by protonation conjugates to the olefin structure in the intermediate. Then, the 9-cis olefin structure isomerizes to the more stable 9-trans olefin. This finding clearly showed that 9-cis-violaxanthin was converted to auroxanthins in the stomach and also implied that cis isomers of violaxanthin are converted to auroxanthins by acid treatment. We measured the lipid peroxidation inhibitory and 1O2 quenching activities of 1 − 4 (we could not test the antioxidant activities of 5 because the purity of 5 was not sufficient due to its low yield) and compared them with the activities of βcarotene and astaxanthin. The results (IC50) are shown in Table 1. As shown in Table 1, 1−4 showed potent lipid peroxidation inhibitory activities (IC50 = 0.43−2.1 μM) superior to those of β-carotene (IC50 = 72 μM) and astaxanthin (IC50 > 100 μM), indicating that they may be good candidates as lipid-related radical quenching agents from food sources. In contrast, 1 and 2 possessed weaker 1O2 quenching activities (IC50 = 9.8 and 18

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RESULTS AND DISCUSSION Diacyl violaxanthin and diacyl 9-cis-violaxanthin in mango were extracted in CH2Cl2/acetone (2:1) under alkaline conditions, and the extract was saponified to afford crude violaxanthin, 1, and 9-cis-violaxanthin, 2. Compounds 1 and 2 were purified by partitioning between EtOAc and H2O, silica gel column chromatography, and preparative silica gel HPLC. From 400 g of mango, pure 1 (3.2 mg) and 2 (1.7 mg) were isolated. To obtain the products of 1 and 2 after acid conversion, 1 and 2 (each 2.0 mg) were dissolved in 0.1 M HCl in 50% EtOH and stirred for 10 min at room temperature. The solution was partitioned between EtOAc and H2O, and EtOAc layers containing the converted products were analyzed by silica gel HPLC using hexane/EtOAc (1:1) as the solvent. For both 1 and 2, HPLC analysis showed three peaks with similar UV−vis C

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of carotenoids and their antioxidant and anti-inflammatory effects in cardiovascular care. Mediators Inflammation 2013, 2013, 1. (3) Fiedor, J.; Burda, K. Potential role of carotenoids as antioxidants in human health and disease. Nutrients 2014, 6, 466−488. (4) Ramel, F.; Birtic, S.; Cuiné, S.; Triantaphylidés, C.; Ravanat, J.; Havaux, M. Chemical quenching of singlet oxygen by carotenoids in plants. Plant Physiol. 2012, 158, 1267−1278. (5) Hirayama, O.; Nakamura, K.; Hamada, S.; Kobayashi, Y. Singlet oxygen quenching ability of naturally occurring carotenoids. Lipids 1994, 29, 149−150. (6) El-Agamey, A.; Lowe, M. G.; McGarvey, J. D.; Mortensen, A.; Phillip, M. D.; Truscott, T. G.; Young, J. A. Carotenoid radical chemistry and antioxidant/pro-oxidant properties. Arch. Biochem. Biophys. 2004, 430, 37−48. (7) Kuhn, R.; Winterstein, A. Viola-xanthin, das xanthophyll des gelben stiefmütterchens (Viola tricolor) (Ü ber konjugierte Doppelbindungen, XVI.). Ber. Dtsch. Chem. Ges. B 1931, 64, 326−332. (8) Müller, H. Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection. Z. Lebensm. Unters. For. 1997, 204, 88−94. (9) Molár, P.; Szabolcs, J.; Radics, L. Isolation and configuration determination of mono- and di-cis-violaxanthins. Magy. Kem. Foly. 1987, 93, 122−128. (10) Yano, M.; Kato, M.; Ikoma, Y.; Kawasaki, A.; Fukazawa, Y.; Sugiura, M.; Matsumoto, H.; Oohara, Y.; Nagao, A.; Ogawa, K. Quantitation of carotenoids in raw and processed fruits in Japan. Food Sci. Technol. Res. 2005, 11, 13−19. (11) Alder, A.; Jamil, M.; Marzorati, M.; Bruno, M.; Vermathen, M.; Bigler, P.; Ghisia, S.; Bouwmeester, H.; Beyer, P.; Al-Babilli, S. The path from β-carotene to a carlactone, a strigolactone-like plant hormone. Science 2002, 335, 1348−1351. (12) Qin, X.; Zeevaart, D. J. A. The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water stressed bean. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 15354−15361. (13) Strain, H. H. Leaf xanthophylls: The action of acids on violaxanthin, violeoxanthin, taraxanthin, and tareoxanthin. Arch. Biochem. Biophys. 1954, 48, 458−468. (14) Aruoma, O. I.; Halliwell, B.; Laughton, J. M.; Quinlan, J. G.; Gutteridge, M. C. J. The mechanism of initiation of lipid peroxidation. Evidence against a requirement for an iron(II)-iron(III) complex. Biochem. J. 1989, 258, 617−620. (15) Buettner, R. G. The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate. Arch. Biochem. Biophys. 1993, 300, 535−543. (16) Kobayashi, M.; Sakamoto, Y. Singlet oxygen quenching ability of astaxanthin esters from the green alga Haematococcus pluvialis. Biotechnol. Lett. 1999, 21, 265−269. (17) Hirayama, O.; Nakamura, K.; Hamada, S.; Kobayashi, Y. Singlet oxygen quenching ability of naturally occurring carotenoids. Lipids 1994, 29, 149−150. (18) Kubo, K.; Yoshitake, I.; Kumada, Y.; Shuto, J.; Nakamizo, N. Radical scavenging action of flunarizine in rat brain in vitro. Arch. Int. Pharmacodyn. Ther. 1984, 272, 283−295. (19) Shimidzu, N.; Goto, M.; Miki, W. Carotenoids as singlet oxygen quenchers in marine organisms. Fish. Sci. 1996, 62, 134−137. (20) Müller, L.; Fröhlich, K.; Böhm, V. Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (αTEAC), DPPH assay and peroxy radical scavenging assay. Food Chem. 2011, 129, 139−148. (21) Frankel, N. E. Antioxidants in lipid foods and their impact on food quality. Food Chem. 1996, 57, 51−55.

Table 1. Antioxidant Activities of Violaxanthin, 1, 9-cisViolaxanthin, 2, (8S,8′S)-Auroxanthin, 3, and (8S,8′R)Auroxanthin, 4 compound

lipid peroxidation inhibitory activity (IC50, μM)

1 O2 quenching activity (IC50, μM)

violaxanthin, 1 9-cis-violaxanthin, 2 (8S,8′S)-auroxanthin, 3 (8S,8′R)-auroxanthin, 4 β-carotene astaxanthin

0.46 0.43 2.1 0.79 72 >100

9.8 18 >100 >100 12 1.2

μM) than β-carotene (IC50 = 12 μM) and astaxanthin (IC50 = 1.2 μM), and 3 and 4 did not show any 1O2 quenching activity. The weaker 1O2 quenching activities of 1 and 2 relative to that of astaxanthin can be explained by the decrease in conjugated double-bond number,19 and the loss of 1O2 quenching activities in 3 and 4 also can be explained in the same way. On the other hand, lipid peroxidation inhibitory activities of 3 and 4 were almost identical to those of 1 and 2, and the activities of 1−4 were superior to those of β-carotene and astaxanthin. Compound 1, β-carotene, and astaxanthin were reported to possess almost equivalent peroxy radical quenching activity in a previous study.20 Therefore, reactions other than peroxy radicals may mainly contribute to rat brain homogenate peroxidation. Furthermore, the rat brain peroxidation inhibitory activities of 1−4 may be derived from 5,6- or 5,8-epoxide structures due to the structural differences among 1−4, β-carotene, and astaxanthin. Because 1−5 are ingested in daily meals, their antioxidant activities may have some beneficial effects on maintaining food quality21 and also health. Further biological evaluations of 1−5 are in progress.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04506. 1 H and 13C NMR spectra of (8S,8′S)-auroxanthin (3), (8S,8′R)-auroxanthin (4), and (8R,8′R)-auroxanthin (5) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(K.S.) Phone: +81-3-5981-3433. Fax: +81-3-5981-3433. Email: [email protected]. ORCID

Kazutoshi Shindo: 0000-0002-9281-0822 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Norihiko Misawa, Ishikawa Prefectural University, for a critical reading of the manuscript. We also thank Prof. Hiroyuki Takemura, Japan Women’s University, for helpful suggestions on the chemical reactions.

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REFERENCES

(1) Britton, G.; Liaaen-Jensen, S.; Pfander, H. Carotenoids Handbook; Birhăuser Verlag: Basel, Switzerland, 2004. (2) Ciccone, M. M.; Cortese, F.; Gesualdo, M.; Carbonara, S.; Zito, A.; Ricci, G.; Pascalis, D. F.; Scichitano, P.; Riccioni, G. Dietary intake D

DOI: 10.1021/acs.jafc.6b04506 J. Agric. Food Chem. XXXX, XXX, XXX−XXX