Letter pubs.acs.org/JAFC
Photosensitized E/Z Isomerization of (all‑E)‑Lycopene Aiming at Practical Applications ABSTRACT: Photoisomerization of (all-E)-lycopene to the corresponding Z-isomers was investigated under visible to middleinfrared light irradiation in the presence of several sensitizers, including edible ones. Highly purified (all-E)-lycopene from tomato paste was isomerized to Z-isomers to the extent of 46.4−57.4% after irradiation with the sensitizers for 60 min in acetone, in which a thermodynamically stable isomer of (5Z)-lycopene was predominantly generated, whereas kinetically preferable (9Z)and (13Z)-lycopenes were dominant without sensitizer. Examination of the time course of photoisomerization demonstrated that the highest isomerization efficiency (80.4%) was attained using erythrosine as the sensitizer under 480−600 nm light irradiation in hexane for 60 min, a protocol that successfully suppressed the degradation of lycopene. (5Z)-Lycopene, reported as a more bioavailable isomer, was again predominantly produced with erythrosine and rose bengal in each solvent. KEYWORDS: lycopene, photoisomerization, sensitizer, erythrosine, rose bengal
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chlorophyll a, erythrosine B (erythrosine), and rose bengal (RB) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Preparation of (all-E)-Lycopene. (all-E)-Lycopene was obtained from tomato paste (Kagome Co., Ltd., Tokyo, Japan; lycopene content, 8−12 g/kg) as described in previous papers:11,12 360 mg of fine red crystalline powder from 70.4 g of tomato material; reversedphase HPLC, ≥99.1% purity. Photosensitized Isomerization of (all-E)-Lycopene. To the (all-E)-lycopene solution in acetone (2 × 10−5 mol/L) was added MB, chlorophyll a, erythrosine, or RB at the same molar ratio as lycopene. After nitrogen gas was bubbled through the solutions, samples were irradiated by xenon lamp (XB-50101AA, Ushio Inc., Tokyo) equipped with optical filters corresponding to the absorption wavelength of the individual sensitizers: MB and chlorophyll a, irradiation range ≥600 nm (LV0610 optical filter, Asahi Spectra Co., Ltd., Tokyo, Japan); erythrosine and RB, 480−600 nm (GIF filter, Nikon Corp., Tokyo, Japan). The photosensitization reaction was conducted at 32 °C for 60 min in a 10 × 10 mm glass container with a lid. The reaction mixtures were sampled, and the contents of lycopene isomers were analyzed by reversed-phase HPLC based on the peak areas. The time-dependent photoisomerization behavior in acetone, ethyl acetate, and hexane was also investigated under the same conditions as described above using erythrosine and RB, both of which are legitimate, stable, and sufficiently inexpensive to be applicable for practical use. HPLC Analysis. Z-Isomers of lycopene were separated with a C30 carotenoid column (250 × 4.6 mm i.d., 5 μm particles, YMC, Kyoto, Japan) using two mobile phases consisting of (A) methanol/methyl tert-butyl ether (MTBE)/ H2O (75:15:10, v/v/v) and (B) methanol/ MTBE/H2O (7:90:3, v/v/v). The gradient profile was as follows: 0−3 min, 0−27% B linear; 3−15 min, 27−55% B; 15−25 min, 55−65% B; 25−35 min, 65−85% B; 35−40 min, 85−95% B; 40−48 min, 95− 100% B. Flow rate was 1.0 mL/min, and column temperature was maintained at 25 °C. The quantification of Z-isomers of lycopene was performed by peak area integration at 470 nm using a UV−vis detector (JASCO Co., Tokyo, Japan).5,7,10,12 (13Z)-, (9Z)-, (all-E)-, and (5Z)lycopene were identified by peaks with retention times of 33.8, 37.9, 42.4, and 42.9 min, respectively, with this system.
INTRODUCTION Lycopene is one of the most widespread and important carotenoids.1,2 In recent years, lycopene has been of interest due to its strong antioxidant properties as well as its potential anticancer and antiarteriosclerosis actions.3 Adequate ingestion of foods rich in lycopene and efficient absorption of the carotenoid through the small intestine are necessary to maintain good health. Lycopene in fresh tomato fruits occurs predominantly as the (all-E)-configuration. On the other hand, Z-isomers of lycopene exist in the human body in considerable quantity; for example, >50% of total lycopene is present as Zisomers in serum and tissues.3−7 These facts suggest that Zisomers of lycopene are more bioavailable than the (all-E)isomer and that the intake of processed foods containing large amounts of Z-isomers would offer health benefits. In fact, both in vitro tests using cultured small intestinal cells and in vivo tests using ferrets strongly support these suggestions.5,8 Moreover, in humans, the ingestion of tomato sauce rich in Z-isomers of lycopene resulted in the practical increase of plasma lycopene concentrations compared to a sample abundant in the (all-E)-isomer.9 In addition, it has been reported that a certain Z-isomer of lycopene has a higher antioxidant capacity than the (all-E)-isomer.3,10 Therefore, the intake of Z-isomers of lycopene rather than the (all-E)-isomer is preferable for health reasons. This highlights the importance of developing appropriate methods for the isomerization of (allE)-lycopene to Z-isomers for practical application to the food and beverage industries, especially for the manufacture of nutritional supplements and edible pigments. Here, we report the first instance of photoisomerization of (all-E)-lycopene using various photosensitizers, including edible ones. This new method is an effective tool for the isomerization of lycopene and will contribute to the understanding of the photoisomerization process on this carotenoid.
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MATERIALS AND METHODS
Chemicals. All solvents were of analytical grade, except for the HPLC grade methanol (Sigma-Aldrich, St. Louis, MO, USA), and used without further purification; hexane was obtained from a solvent dispensing system supplied by Glass Contour (Nikko Hansen & Co., Ltd., Osaka, Japan) under a nitrogen atmosphere. Methylene blue (MB) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and © XXXX American Chemical Society
Received: September 17, 2014 Revised: November 8, 2014 Accepted: November 11, 2014
A
dx.doi.org/10.1021/jf504502t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
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Letter
RESULTS AND DISCUSSION Photoisomerization with Various Sensitizers. To isomerize (all-E)-carotenoids to Z-isomers, heat treatment has been the typical method used so far.3,7,11 Very recently, we reported a detailed study on the thermal isomerization of highpurity (all-E)-lycopene (≥99%), which showed that heating (all-E)-lycopene at 50 °C in hexane produced ca. 30% (5Z)lycopene as a main product, whereas the use of benzene generated ca. 20% (13Z)-lycopene.12 On the other hand, there are some reports on the photosensitized E/Z isomerization of carotenoids, an alternative method to thermal processing, which has been confined to (all-E)-β-carotene.13,14 Under these circumstances, we performed the photoisomerization of (all-E)lycopene to the corresponding Z-forms in acetone in the presence of four kinds of sensitizers using filtered light. After irradiation with 480−600 nm light for 60 min, the concentrations of undecomposed lycopene remaining in solution were determined to be 1.5 × 10−5 mol/L (with MB), 1.7 × 10−5 mol/L (with chlorophyll a), and 1.7 × 10−5 mol/L (without sensitizer) under ≥600 nm light irradiation; 0.7 × 10−5 mol/L (with erythrosine), 1.0 × 10−5 mol/L (with RB), and 1.9 × 10−5 mol/L (without sensitizer) by the HPLC analysis; typical chromatograms are shown in Figure 1. The total amounts of Z-isomers relative to the remaining lycopene after the reaction were 57.4, 51.3, 47.7, and 46.4% with MB, chlorophyll a, erythrosine, and RB, respectively (Table 1). On
the other hand, the blank tests irradiated at the light regions more than 600 nm and between 480 and 600 nm without sensitizer resulted in Z-isomer contents of only 7.4 and 7.7%, respectively. To the best of our knowledge, photoisomerization of (all-E)-lycopene was previously conducted by Stahl et al.15 and Lee and Chen.16 However, Stahl et al.15 performed the iodine-catalyzed isomerization; the iodine is inedible, and the reaction mechanism would contain a radical process principally.17 Then, Lee and Chen16 carried out the isomerization of (all-E)-lycopene by direct light irradiation for 60 min, obtaining only a few percent of Z-isomers. Our results obviously indicated that the use of sensitizer and filtered light resulted in an accelerated isomerization of (all-E)-lycopene to Z-isomers. MB was the most efficient sensitizer for the isomerization among the reagents tested; however, it is not approved for use in food processing. In contrast, chlorophyll a, erythrosine, and RB are usable in such industrial fields, and therefore erythrosine appears to be the most suitable sensitizer for (all-E)-lycopene isomerization. As for the individual Z-isomers generated, several Z-isomers of lycopene were observed in the HPLC chart in addition to (5Z)-, (9Z)-, and (13Z)-lycopene (Figure 1B), which are predominantly contained in processed food.7 In all cases with the above sensitizers after 60 min of irradiation, (5Z)-lycopene showed the greatest increases to 39.9, 31.7, 37.7, and 29.9% relative to the remaining lycopene with MB, chlorophyll a, erythrosine, and RB, respectively (Table 1). On the other hand, (9Z)-, (13Z)-, and other Z-isomers increased to
Photosensitized E/Z Isomerization of (all‑E)‑Lycopene Aiming at Practical Applications ABSTRACT: Photoisomerization of (all-E)-lycopene to the corresponding Z-isomers was investigated under visible to middleinfrared light irradiation in the presence of several sensitizers, including edible ones. Highly purified (all-E)-lycopene from tomato paste was isomerized to Z-isomers to the extent of 46.4−57.4% after irradiation with the sensitizers for 60 min in acetone, in which a thermodynamically stable isomer of (5Z)-lycopene was predominantly generated, whereas kinetically preferable (9Z)and (13Z)-lycopenes were dominant without sensitizer. Examination of the time course of photoisomerization demonstrated that the highest isomerization efficiency (80.4%) was attained using erythrosine as the sensitizer under 480−600 nm light irradiation in hexane for 60 min, a protocol that successfully suppressed the degradation of lycopene. (5Z)-Lycopene, reported as a more bioavailable isomer, was again predominantly produced with erythrosine and rose bengal in each solvent. KEYWORDS: lycopene, photoisomerization, sensitizer, erythrosine, rose bengal
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chlorophyll a, erythrosine B (erythrosine), and rose bengal (RB) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Preparation of (all-E)-Lycopene. (all-E)-Lycopene was obtained from tomato paste (Kagome Co., Ltd., Tokyo, Japan; lycopene content, 8−12 g/kg) as described in previous papers:11,12 360 mg of fine red crystalline powder from 70.4 g of tomato material; reversedphase HPLC, ≥99.1% purity. Photosensitized Isomerization of (all-E)-Lycopene. To the (all-E)-lycopene solution in acetone (2 × 10−5 mol/L) was added MB, chlorophyll a, erythrosine, or RB at the same molar ratio as lycopene. After nitrogen gas was bubbled through the solutions, samples were irradiated by xenon lamp (XB-50101AA, Ushio Inc., Tokyo) equipped with optical filters corresponding to the absorption wavelength of the individual sensitizers: MB and chlorophyll a, irradiation range ≥600 nm (LV0610 optical filter, Asahi Spectra Co., Ltd., Tokyo, Japan); erythrosine and RB, 480−600 nm (GIF filter, Nikon Corp., Tokyo, Japan). The photosensitization reaction was conducted at 32 °C for 60 min in a 10 × 10 mm glass container with a lid. The reaction mixtures were sampled, and the contents of lycopene isomers were analyzed by reversed-phase HPLC based on the peak areas. The time-dependent photoisomerization behavior in acetone, ethyl acetate, and hexane was also investigated under the same conditions as described above using erythrosine and RB, both of which are legitimate, stable, and sufficiently inexpensive to be applicable for practical use. HPLC Analysis. Z-Isomers of lycopene were separated with a C30 carotenoid column (250 × 4.6 mm i.d., 5 μm particles, YMC, Kyoto, Japan) using two mobile phases consisting of (A) methanol/methyl tert-butyl ether (MTBE)/ H2O (75:15:10, v/v/v) and (B) methanol/ MTBE/H2O (7:90:3, v/v/v). The gradient profile was as follows: 0−3 min, 0−27% B linear; 3−15 min, 27−55% B; 15−25 min, 55−65% B; 25−35 min, 65−85% B; 35−40 min, 85−95% B; 40−48 min, 95− 100% B. Flow rate was 1.0 mL/min, and column temperature was maintained at 25 °C. The quantification of Z-isomers of lycopene was performed by peak area integration at 470 nm using a UV−vis detector (JASCO Co., Tokyo, Japan).5,7,10,12 (13Z)-, (9Z)-, (all-E)-, and (5Z)lycopene were identified by peaks with retention times of 33.8, 37.9, 42.4, and 42.9 min, respectively, with this system.
INTRODUCTION Lycopene is one of the most widespread and important carotenoids.1,2 In recent years, lycopene has been of interest due to its strong antioxidant properties as well as its potential anticancer and antiarteriosclerosis actions.3 Adequate ingestion of foods rich in lycopene and efficient absorption of the carotenoid through the small intestine are necessary to maintain good health. Lycopene in fresh tomato fruits occurs predominantly as the (all-E)-configuration. On the other hand, Z-isomers of lycopene exist in the human body in considerable quantity; for example, >50% of total lycopene is present as Zisomers in serum and tissues.3−7 These facts suggest that Zisomers of lycopene are more bioavailable than the (all-E)isomer and that the intake of processed foods containing large amounts of Z-isomers would offer health benefits. In fact, both in vitro tests using cultured small intestinal cells and in vivo tests using ferrets strongly support these suggestions.5,8 Moreover, in humans, the ingestion of tomato sauce rich in Z-isomers of lycopene resulted in the practical increase of plasma lycopene concentrations compared to a sample abundant in the (all-E)-isomer.9 In addition, it has been reported that a certain Z-isomer of lycopene has a higher antioxidant capacity than the (all-E)-isomer.3,10 Therefore, the intake of Z-isomers of lycopene rather than the (all-E)-isomer is preferable for health reasons. This highlights the importance of developing appropriate methods for the isomerization of (allE)-lycopene to Z-isomers for practical application to the food and beverage industries, especially for the manufacture of nutritional supplements and edible pigments. Here, we report the first instance of photoisomerization of (all-E)-lycopene using various photosensitizers, including edible ones. This new method is an effective tool for the isomerization of lycopene and will contribute to the understanding of the photoisomerization process on this carotenoid.
■
MATERIALS AND METHODS
Chemicals. All solvents were of analytical grade, except for the HPLC grade methanol (Sigma-Aldrich, St. Louis, MO, USA), and used without further purification; hexane was obtained from a solvent dispensing system supplied by Glass Contour (Nikko Hansen & Co., Ltd., Osaka, Japan) under a nitrogen atmosphere. Methylene blue (MB) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and © XXXX American Chemical Society
Received: September 17, 2014 Revised: November 8, 2014 Accepted: November 11, 2014
A
dx.doi.org/10.1021/jf504502t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
■
Letter
RESULTS AND DISCUSSION Photoisomerization with Various Sensitizers. To isomerize (all-E)-carotenoids to Z-isomers, heat treatment has been the typical method used so far.3,7,11 Very recently, we reported a detailed study on the thermal isomerization of highpurity (all-E)-lycopene (≥99%), which showed that heating (all-E)-lycopene at 50 °C in hexane produced ca. 30% (5Z)lycopene as a main product, whereas the use of benzene generated ca. 20% (13Z)-lycopene.12 On the other hand, there are some reports on the photosensitized E/Z isomerization of carotenoids, an alternative method to thermal processing, which has been confined to (all-E)-β-carotene.13,14 Under these circumstances, we performed the photoisomerization of (all-E)lycopene to the corresponding Z-forms in acetone in the presence of four kinds of sensitizers using filtered light. After irradiation with 480−600 nm light for 60 min, the concentrations of undecomposed lycopene remaining in solution were determined to be 1.5 × 10−5 mol/L (with MB), 1.7 × 10−5 mol/L (with chlorophyll a), and 1.7 × 10−5 mol/L (without sensitizer) under ≥600 nm light irradiation; 0.7 × 10−5 mol/L (with erythrosine), 1.0 × 10−5 mol/L (with RB), and 1.9 × 10−5 mol/L (without sensitizer) by the HPLC analysis; typical chromatograms are shown in Figure 1. The total amounts of Z-isomers relative to the remaining lycopene after the reaction were 57.4, 51.3, 47.7, and 46.4% with MB, chlorophyll a, erythrosine, and RB, respectively (Table 1). On
the other hand, the blank tests irradiated at the light regions more than 600 nm and between 480 and 600 nm without sensitizer resulted in Z-isomer contents of only 7.4 and 7.7%, respectively. To the best of our knowledge, photoisomerization of (all-E)-lycopene was previously conducted by Stahl et al.15 and Lee and Chen.16 However, Stahl et al.15 performed the iodine-catalyzed isomerization; the iodine is inedible, and the reaction mechanism would contain a radical process principally.17 Then, Lee and Chen16 carried out the isomerization of (all-E)-lycopene by direct light irradiation for 60 min, obtaining only a few percent of Z-isomers. Our results obviously indicated that the use of sensitizer and filtered light resulted in an accelerated isomerization of (all-E)-lycopene to Z-isomers. MB was the most efficient sensitizer for the isomerization among the reagents tested; however, it is not approved for use in food processing. In contrast, chlorophyll a, erythrosine, and RB are usable in such industrial fields, and therefore erythrosine appears to be the most suitable sensitizer for (all-E)-lycopene isomerization. As for the individual Z-isomers generated, several Z-isomers of lycopene were observed in the HPLC chart in addition to (5Z)-, (9Z)-, and (13Z)-lycopene (Figure 1B), which are predominantly contained in processed food.7 In all cases with the above sensitizers after 60 min of irradiation, (5Z)-lycopene showed the greatest increases to 39.9, 31.7, 37.7, and 29.9% relative to the remaining lycopene with MB, chlorophyll a, erythrosine, and RB, respectively (Table 1). On the other hand, (9Z)-, (13Z)-, and other Z-isomers increased to
