Structures of Yellow Xanthophylls and Metabolism of Astaxanthin in the Prawn Penaeus japonicus

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1 Journal of Oleo Science Copyright 2018 by Japan Oil Chemists Society doi : /jos.ess18103 Structures of Yellow Xanthophylls and Metabolism of Astaxanthin in the Prawn Penaeus japonicus Takashi Maoka 1*, Yuki Kawashima 2, and Mikihiro Takaki 2 1 Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku, Kyoto , JAPAN 2 JXTG Nippon Oil & Energy Corporation, High Performance Materials Company, W Building, Konan, Minato-ku, Tokyo , JAPAN Abstract: The dried powder of Paracoccus carotinifaciens (PANAFERD-AX ) contains (3S,3 S)-astaxanthin as a major carotenoid. Administration of PANAFERD-AX for pigmentation of the prawn Penaeus japonicus was examined. Total carotenoid contents in the carapace, muscle, and head of the prawn were dose-dependently increased depending on the amount of PANAFERD-AX administered. Furthermore, not only the amounts of astaxanthins (astaxanthin diester, astaxanthin monoester, and free astaxanthin) but also the amounts of yellow xanthophylls, isoastaxanthin (1), 5,6-dihydropenaeusxanthin (2), penaeusxanthin (3), tetrahydroxypirardixanthin (4), and curstaxanthin (5), were dose-dependently increased with the administration of PANAFERD-AX. 5,6-Dihydropenaeusxanthin (2) and penaeusxanthin (3) are new carotenoids isolated from the prawn P. japonicus. These structures were determined to be (3R,4S,5R,6R,6 S)- 5,6-dihydro-3,4,4 - trihydroxy-β,ε-caroten-3 -one (2) and (3R,4S,6 S)-3,4,4 -trihydroxy-β,ε-caroten-3 -one (3) by UV/vis, ESI TOF MS, 1 H NMR, and CD spectral data. The metabolism of astaxanthin to these yellow xanthophylls in the prawn was discussed. Key words: Penaeus japonicus, astaxanthin, isoastaxanthin, penaeusxanthin, carotenoid metabolism 1 INTRODUCTION The prawn Penaeus japonicus Kurumaebi in Japanese is one of the largest species of prawns and one of the most economically important species of shrimp in Japan. This prawn is a highly regarded food consumed as sashimi, as broiled with salt, as tempura, etc. Cultured specimens account for more than 80 of the prawn market in Japan. They are mainly cultured in Okinawa, Kagoshima, and Kumamoto Prefecture. Not only body weight and flesh quality but also body color is an important commercial quality criterion. It is well-known that the predominant pigment of the prawn is astaxanthin along with some minor yellow colored xanthophylls 1, 2. Therefore, astaxanthin is used for the pigmentation of cultured prawn 3, 4. Paracoccus carotinifaciens 5 is an aerobic gram-negative, halophilic, astaxanthin-producing bacterium and its dry-powder form is referred to PANAFERD-AX, which contains 3S,3 S -astaxanthin as a major carotenoid. It is used for the pigmentation of cultured fish such as salmon and trout, and has on excellent pigmentation-promoting effect on these fish 6. Therefore, we administered PAN- AFERD-AX for pigmentation of the prawn P. japonicus. In the present paper, we describe the accumulation of astaxanthin and yellow xanthophylls, isoastaxanthin 1, 5,6-dihydropenaeusxanthin 2, penaeusxanthin 3, tetrahydroxypirardixanthin 4, and curstaxanthin 5 in the prawn induced by the administration of PANAFERD-AX and structure determination of these yellow xanthophylls 1-5. Furthermore, the metabolism of astaxanthin to these yellow xanthophylls in the prawn is discussed. 2 MATERIALS AND METHODS 2.1 Feeding Experiment The prawn P. japonicus used in this experiment were obtained from HIGASHIMARU Co., Ltd. Prawns were divided four experimental groups. Sixteen specimens of prawns, average body weight 3.6 g, were kept in each 200 L tank. The temperature of sea water was maintained at 24 by heating. The prawns were fed to satiation with experimental food one time a day for 8 weeks. Body weight was monitored at after 4 weeks and 8 weeks of starting experiment. After 8 weeks, prawns were immediately frozen and stored below 80. * Correspondence to: Takashi Maoka, Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku, Kyoto , JAPAN maoka@mbox.kyoto-inet.or.jp Accepted July 19, 2018 (received for review May 28, 2018) Journal of Oleo Science ISSN print / ISSN online

2 T. Maoka, Y. Kawashima, and M. Takaki 2.2 Test Diets HIGASHIMARU s based feed HIGASHIMARU Co. LTD., Hiki, Kagoshima, Japan was used for control diet indicate as Cont.. This diet was composed of protein 57, lipid 9, ash 18, and other nutrients 7. PANAFERD- AX JXTG Nippon Oil & Energy Corporation, Tokyo, Japan was used as pigmentation source. Carotenoid composition of PANAFERD-AX is as follows; 3S,3 S -astaxanthin 70 of the total carotenoid, 3S -adonirubin 21, canthaxanthin 3, 3S,3 R -adonixanthin 2, and 3S -3-hydroxyechinenone etc 4. The experimental diets containing 25 ppm, 50 ppm, and 100 ppm of 3S,3 S -astaxanthin were prepared by addition of PANAFERD-AX to control diet. They were indicated as Ast 25, Ast 50, and Ast 100, respectively. Each diet was molded 2.7 mm diameter 7 mm length pellet. 2.3 Carotenoid Extraction, Isolation, and Identification The carotenoids were repeatedly extracted from carapace, head, and muscle of the prawn with acetone until the extract became colorless. They were then transferred to n- hexane: Et 2 O 1:1, v/v by addition of water. The n-hexane: Et 2 O phase was washed with water and dehydrated on anhydrous sodium sulphate. The total carotenoid amount were calculated using coefficient of E 1 cm 2100 at λ max 470 nm 7. Then the extract was evaporated to dryness. The residue was dissolved in acetone: n-hexane 2:8, v/v and subjected it to HPLC on silica gel with acetone: n-hexane 2:8, v/v. Six peaks yellow xanthophylls esters retention time 3.8 min, astaxanthin poly unsatulated fatty acid diesters retention time 4.1 min, astaxanthin fatty acid diesters retention time 4.3 min, astaxanthin poly unsatulated fatty acid monoesters retention time 6.2 min, astaxanthin fatty acid monoesters retention time 7.0 min, and astaxanthin retention time 12.6 min were obtained. The identification of individual carotenoids was carried out using our routine method 8. The UV-VIS spectra were recorded with a Hitachi U-2001 spectrophotometer Hitach Field Navigator, Tokyo, Japan in ether. The LC/MS analysis of carotenoids was carried out using a Waters Xevo G2S Q TOF mass spectrometer Waters Corporation, Milford, CT, USA equipped with an Acquity UPLC system. The electrospray ionization ESI time-of-flight TOF MS spectra were acquired by scanning from m/z 100 to 1,500 with a capillary voltage of 3.2 kv, cone voltage of 40 ev, and source temperature of 120. Nitrogen was used as a nebulizing gas at a flow rate of 30 L/h. MS/MS spectra were measured with a quadrupole-tof MS/MS instrument with argon as a collision gas at a collision energy of 30 V. UV-VIS absorption spectra were recorded from 200 to 600 nm using a photodiode-array detector PDA. An Acquity 1.7 μm BEH UPLC C18 column Waters Corporation, Milford, CT, USA was used as a stationary phase and UPLC ODS 80 MeOH- 100 MeOH as a mobile phase, at a flow rate of 0.4 ml/ min for the HPLC system. The 1 H NMR 500 MHz spectrum was measured with a Varian UNITY INOVA 500 spectrometer Varian Corporation, Palo Alto, California USA in CDCl 3 with TMS as an internal standard. The CD spectrum was recorded in Et 2 O at room temperature with a Jasco J-500C spectropolarimeter JASCO Corporation, Hachioji, Tokyo, Japan. Preparative HPLC was performed with a Hitachi L-6000 intelligent pump and an L-4250 UV-VIS detector Hitach Field Navigator, Tokyo, Japan set at 450 nm. The column used was a mm i.d., 5 μm Cosmosil 5SL-II Nacalai Tesque, Kyoto, Japan with acetone:hexane 2:8, v/v as a solvent at a flow rate of 1.0 ml/min. The presence of optical isomers in astaxanthin were determined by the chiral column, Sumichiral OA-2000 Sumika Chemical Analysis Service, Oosaka, Japan with n-hexane:chcl 3 :EtOH 48:16:1, v/v at a flow rate of 1.0 ml/min and detection at 470 nm. Esterified astaxanthins underwent hydrolysis with lipase to form free astaxanthin. They were then submitted to chiral HPLC Identification of Individual Carotenoids Astaxanthin diester UV-VIS: 470 nm Et 2 O. ESI TOF MS: m/z M H calcd for C 82 H 113 O 6, astaxanthin-c22:6 and C20:4 fatty acid diester, m/z M H calcd for C 82 H 111 O 6, astaxanthin-c22:6 and C20:5 fatty acid diester, m/z M H calcd for C 80 H 111 O 6, astaxanthin-c20:5 and C20:4 fatty acid diester, m/z M H calcd for C 82 H 109 O 6, astaxanthin-c20:5 and C20:5 fatty acid diester, m/z M H calcd for C 78 H 115 O 6, astaxanthin-c22:6 and C16:0 or C20:5 and C18:1 fatty acid diesters, m/z M H calcd for C 78 H 113 O 6, astaxanthin-c22:6 and C16:1 or C20:5 and C18:2 fatty acid diesters, m/z M H calcd for C 78 H 109 O 6, astaxanthin-c20:5 and C18:0 fatty acid diester, m/z M H calcd for C 76 H 113 O 6, astaxanthin C20:5 and C16:0 fatty acid diesters, 1 H NMR Chemical shift and spin coupling values were in agreement with previously published value 9. Chiral resolution revealed that astaxanthin was consisted with 3R,3 R, meso, and 3S,3 S optical isomer at the ratio of 12:40:48 in the case of carapace 100 ppm astaxanthin administrate group Astaxanthin monoester UV-VIS: 470 nm Et 2 O. ESI TOF MS m/z M H calcd for C 62 H 87 O 5, astaxanthin-c22:4 fatty acid ester, m/z M H calcd for C 62 H 83 O 5, astaxanthin-c22:6 fatty acid ester, m/z M H calcd for C 60 H 89 O 5, astaxanthin-c20:1 fatty acid ester, m/z M H calcd for C 60 H 85 O 5, astaxanthin-c20:3 fatty acid ester, m/z M H calcd for C 60 H 83 O 5, astaxanthin-c20:4 1426

3 Yellow Xanthophylls in the Prawn fatty acid ester, m/z M H calcd for C 60 H 81 O 5, astaxanthin-c20:5 fatty acid ester, m/z M H calcd for C 58 H 87 O 5, astaxanthin-c18:0 fatty acid ester, m/z M H calcd for C 58 H 85 O 5, astaxanthin-c18:1 fatty acid ester, m/z M H calcd for C 58 H 83 O 5, astaxanthin-c18:2 fatty acid ester, m/z M H calcd for C 57 H 85 O 5, astaxanthin-c17:0 fatty acid ester, m/z M H calcd for C 56 H 83 O 5, astaxanthin-c16:0 fatty acid ester, m/z M H calcd for C 56 H 81 O 5, astaxanthin-c16:1 fatty acid ester, m/z M H calcd for C 54 H 79 O 5, astaxanthin-c14:0 fatty acid ester, m/z M H calcd for C 52 H 75 O 5, astaxanthin-c12:0 fatty acid ester. 1 H NMR Chemical shift and spin coupling values were in agreement with previously published value 9. Chiral resolution revealed that astaxanthin was consisted with 3R,3 R, meso, and 3S,3S optical isomer at the ratio of 12:40:48 in the case of carapace 100 ppm astaxanthin administrate group Astaxanthin UV-VIS: 470 Et 2 O. ESI TOF MS: M H, calcd. for C 40 H 53 O 4, , m/z M Na, calcd. for C 40 H 52 O 4 Na, H NMR Chemical shift and spin coupling values were in agreement with previously published value 9. Chiral resolution revealed that astaxanthin was consisted with 3R,3 R, meso, and 3S,3S optical isomer at the ratio of 12:40:48 in the case of carapace 100 ppm astaxanthin administrate group. 2.5 Characterization of yellow xanthophylls Yellow xanthophylls fraction obtained from preparative HPLC was saponified with 5 KOH/MeOH at room temperature for 3 hrs, as usual manner 8. They were re-chromotographed by silica gel HPLC Cosmosil 5SL-II with acetone: n-hexane 4:6, v/v as a solvent at a flow rate of 1.0 ml/min afforded five yellow xanthophylls, isoastaxanthin retention time 8.3 min, 5,5-dihydropenaeusxanthin retention time 13.0 min, penaeusanthin retention time 16.8 min, tetrahydropirardixanthin retention time 20.3 min, and crustaxanthin retention time 22.3 min Isoastaxanthin 1 UV-VIS: 419, 439, 468 nm. ESI TOF MS: m/z M Na, calcd. for C 40 H 52 O 4 Na, , m/z M H, calcd. for C 40 H 53 O 4, Product ions of ESI TOF MS/MS from M H : m/z 579 M H-H 2 O, 561 M H-2H 2 O, 523, 445, 377, 225, 211, 197, 153, 139, H NMR δ CDCl 3 : H. s, CH 3-17, 17, H, s, CH 3-16, 16, H, s, CH 3-18, 18, H, s, CH 3-19, 19, H, s, CH 3-20, 20, H, d, J 17 Hz, H-2, 2 ax, H, d, J 17 Hz, H-2, 2 eq, H, d, J 9 Hz, H-6, 6, H, dd, J 15.5, 9.5, H-7, 7, H, br.s, OH-4,4, H, d, J 11 Hz, H-10, 10, H, d, J 15 Hz, H-8, 8, H, m, H-14, 14, H, d, J 15Hz, H-12, 12, H, dd, J 15, 11 Hz, H-11, 11, H, m, H-15, H-15. CD λ Δε : 235 0, , 268 0, , ,6-Dihydropenaeusxanthin 2 UV-VIS: 419, 439, 468 nm. ESI TOF MS: m/z M Na, calcd. for C 40 H 56 O 4 Na, , m/z M H, calcd. for C 40 H 57 O 4, H NMR δ CDCl 3 : H. s, CH 3-16, H. d, J 6.5 Hz, CH 3-18, H. s, CH 3-17, H. s, CH 3-17, H, s, CH 3-16, H, dd, J 15, 3 Hz, H-2ax, H, dd, J 9.5, 9.5 Hz, H-6, H, s, CH 3-18, H, overlapped, H-2 eq and H-5, H, s, CH 3-19, H, s, CH 3-19, H, s, CH 3-20, 20, H, d, J 17 Hz, H-2 ax, H, d, J 17 Hz, H-2 eq, H, d, J 9 Hz, H-6, H, dd, J 10, 3.5 Hz, H-4, H, m, H-3, H, dd, J 15.5, 9.5, H-7, H, dd, J 15.5, 9.5, H-7, H, d, J 15.5 Hz, H-8, H, br.s, OH-4, H, d, J 11 Hz, H-11, H, d, J 11 Hz, H-10, H, d, J 15 Hz, H-8, H, d, J 11 Hz, H-14, H, d, J 11 Hz, H-14, H, d, J 15Hz, H-12, H, d, J 15Hz, H-12, H, dd, J 15, 11 Hz, H-11 or 11, H, dd, J 15, 11 Hz, H-11 or 11, H, m, H-15, H-15. CD λ Δε : 225 0, , 266 0, , , Penaeusxanthin 3 UV-VIS: 425, 445, 473 nm. ESI TOF MS: m/z M, calcd. for C 40 H 54 O 4, H NMR δ CDCl 3 : H. s, CH 3-17, H, s, CH 3-16, H, s, CH 3-17, H, s, CH 3-16, H, dd, J 12.5, 7.5 Hz, H-2ax, H, dd, J 12.5, 12.5 Hz, H-2eq, H, s, CH 3-18, H, s, CH 3-18, H, s, CH 3-19, H, s, CH 3-20, 20, H, s, CH 3-19, H, d, J 17 Hz, H-2 ax, H, d, J 17 Hz, H-2 eq, H, d, J 9 Hz, H-6, H, m, H-3, H, d, J 3.5 Hz, H-4, H, dd, J 15.5, 9.5 Hz, H-7, H, br.s, OH-4, H, d, J 15 Hz, H-7, H, d, J 11 Hz, H-10, H, d, J 15 Hz, H-8, H, d, J 15Hz, H-8, H, m, H-14, 14, H, d, J 15 Hz, H-12, 12, H, dd, J 15, 11 Hz, H-11 or 11, H, dd, J 15, 11 Hz, H-11 or 11, H, m, H-15, H-15. CD λ Δε : 245 0, , 275 0, , , R,4S,5R,6R,3 R,4 S,5 R,6 R -Tetrahydroxypirardixanthin 4 UV-VIS: 419, 439, 468 nm. ESI TOF MS: m/z M, calcd. for C 40 H 60 O H NMR δ CDCl 3 : H. s, CH 3-16, 16, H. d, J 6.5 Hz, CH 3-18, 18, H. s, CH 3-17, 17, H, dd, J 15, 3 Hz, H-2, 2 ax, H, dd, J 9.5, 9.5 Hz, H-6, 6, H, overlapped, H-2, 2 eq and H-5, 5, H, s, H-19, 19, H, s, CH 3-20, 20, H, dd, J 10, 3.5 Hz, H-4, 4, H, m, H-3, 3, H, dd, J 15.5, 9.5, H-7, 7, H, d, J 15.5 Hz, H-8, 8, H, d, J 11 Hz, H-10, 10, H, m, H-14, 14, H, d, J 15Hz, H-12, 12, H, dd, J 15, 11 Hz, H-11, 11, H, m, H-15,

4 T. Maoka, Y. Kawashima, and M. Takaki CD λ Δε : 238 0, , R,4S,3 R,4 S -Crustaxanthin 5 UV-VIS: 425, 449, 475 nm. ESI TOF MS: m/z M, calcd. for C 40 H 56 O H NMR δ CDCl 3 : H, s, CH 3-16, 16, H, s, CH 3-17, 17, H, dd, J 12.5, 7.5 Hz, H-2, 2 ax, H, dd, J 12.5, 12.5 Hz, H-2, 2 eq, H, s, CH 3-18, 18, H, s, CH 3-20, 20, H, s, CH 3-19, 19, H, m, H-3, 3, H, d, J 3.5 Hz, H-4, 4, H, d, J 15 Hz, H-7, 7, H, d, J 15 Hz, H-8, 8, H, m, H-14, 14, H, d, J 15 Hz, H-12, 12, H, dd, J 15, 11 Hz, H-11 or 11, H, m, H-15, H-15. CD λ Δε : , 236 0, , 260 0, , 320 0, RESULTS AND DISCUSSION 3.1 Accumulation of administered carotenoids in the prawn The prawns in all experimental groups grew normally. The average body weight of the prawns increased from 3.5 to 13.0 g during the 8 weeks of the feeding experiment. There were no differences in the growth rate between the experimental groups. The total amounts of carotenoid, astaxanthin disester, astaxanthin monoester, astaxanthin, and yellow xanthophylls in the carapace of prawns receiving PANAFERD- AX were increased dose-dependently as shown in Fig. 1. PANAFERD-AX contained not only 3S,3 S -astaxanthin 70 of the total carotenoid but also 3S -adonirubin 21, canthaxanthin 3, 3S,3 R -adonixanthin 2, and 3S -3-hydroxyechinenone etc. 4 as minor carotenoids. In the present investigation, adonirubin, canthaxanthin, adonixanthin, and 3-hydroxyechinenone were not found in the prawn. Adonirubin, canthaxanthin, and 3-hydroxyechinenone are known biosynthetic and metabolic intermediates of β-carotene to astaxanthin 1, 2, 5, 6. Similarly, adonixanthin is an intermediate during the metabolism of zeaxanthin to astaxanthin 1, 2, 5, 6. Therefore, these intermediate carotenoids were considered to be oxidatively metabolized to astaxanthin in the prawn 1, 2. Furthermore, contents of yellow xanthophylls were also dose-dependently increased by the administration of PANAFERD-AX, as shown in Fig. 1 and Table 1. These results indicated that these yellow xanthophylls were metabolized from astaxanthin in the prawn. Similar results as carotenoid content of carapace were obtained in the cases of muscle and head. Namely, total carotenoid content in head of control group, Ast 25, Ast 50, and Ast 100 were μg/g, μg/g, μg/g, and μg/g, respectively. Total yellow xanthophylls content in head of control group, Ast 25, Ast 50, and Ast 100 were μg/g, μg/g, μg/g, and μg/g, respectively. Similarly, total carotenoid content in muscle of each experimental group were as follows, control group μg/g, Ast μg/ g, Ast μg/g, and Ast μg/g. Total yellow xanthophylls content in muscle of each experimental group were as follows, control group μg/ g, Ast μg/g, Ast μg/g, and Ast μg/g. Fig Carotenoids contents μg/g in the control and PANAFERD-AX administrated groups of the Prawn. Cont: control group, Ast 25: contained 25 ppm astaxanthin to control diets, Ast 50: contained 50 ppm astaxanthin to control diets, Ast 100: contained 100 ppm astaxanthin to control diets. Others: β-carotene other minor carotenoids, Yellow-Xan-est: Yellow xanthophylls 1-5 esters, Asta-diest: Astaxanthin diester, Ast-monoest; Astaxanthin monoester, Ast; Astaxanthin. Each experimental group consisting 16 specimens of prawns. Error bars indicate standard division of total carotenoid content in each experimental group n 16. Different small letters indicate significant differences of total carotenoid content in each experimental group p The data were analyzed by one-way ANOVA, followed by the Tukey-Kramer test and the paired Student s t- test.

5 Yellow Xanthophylls in the Prawn Table 1 Contents μg/g of total yellow xanthophylls, isoastaxanthin 1, 5,6-dihydropenaeusxanthin 2, penaeusxanthin 3, tetrahydropirardixanthin 4, and crustaxanthin 5 in the carapace of the each experimental group of the prawn. Exp. Group Total Y. X Cont Ast * Ast ** Ast ** Total Y. X.: Total yellow xanthophylls, Cont: control group, Ast 25: contained 25 ppm astaxanthin to control diets, Ast 50: contained 50 ppm astaxanthin to control diets, Ast 100: contained 100 ppm astaxanthin to control diets. Each experimental group consisting 16 specimens of prawn. Significance: *p < 0.05, **p < 0.01 vs Control group by Student s t-test. 3.2 Racemization of administered astaxanthin in the prawn Schiedt et al. 10, 11 demonstrated the racemization of 3S,3 S -astaxanthin in the prawn in a radioisotope-labeling experiment using 3 H -labeled astaxanthin. Namely, 3 H -labeled 3S,3 S -astaxanthin was converted to 3R,3 R -, meso -, and 3S,3 S -isomers at an approximate ratio of 1:2:1 in this prawn. The amounts of 3R,3R -, meso -, and 3S,3 S -astaxanthin in the control group and 100 ppm astaxanthin-administered group in the carapace of the prawn are shown in Table 2. As the results, not only the amount of 3S,3 S astaxanthin but also those of meso - and 3R,3 R -astaxanthin were increased by the administration of 100 ppm 3S,3 S -astaxanthin. This indicated that a part of 3S,3 S astaxanthin ingested by the prawn was converted to meso - and 3R,3 R -astaxanthin, as reported by Schiedt 10, 11 et al. 3.3 Structures of yellow xanthophylls Yellow xanthophylls obtained from the prawn were esterified with fatty acids. Therefore, the yellow xanthophyll fraction was saponified with 5 KOH/MeOH and submitted for preparative HPLC. Five yellow xanthophylls, including two new compounds, were obtained. Carotenoid 1, with a molecular formula of C 40 H 52 O 4, Table 2 Content μg/g of optical isomers of astaxanthin in the carapace of control group and 100 ppm astaxanthin administration group. Exp. Group (3R,3 R) (meso) (3S,3 S) Cont Ast Cont: control group, Ast 100: contained 100 ppm astaxanthin to control diets. Each experimental group consisting 16 specimens of prawn. showed UV-VIS absorption maxima at 419, 439, and 468 nm indicating the presence of the ε,ε-carotene-type conjugated double-bond system 7. Characteristic product ions of the MS/MS spectrum at m/z 579 M H-H 2 O and 561 M H-2H 2 O indicated the presence of two hydroxy groups in the molecule. 1 H NMR signals of H-2 2, OH-4 4, H-6 6, H-16 16, H-17 17, H indicated the presence of a 3-keto-4-hydroxy-ε-end group and these of H-7 7 to H and H and H indicated the presence of an all-trans-polyene chain 12. The CD spectrum of this compound showed a positive maximum at 256 nm Δε 20.3 and a negative maximum at 285 nm Δε These spectral data were in agreement with 6S,6 S isoastaxanthin 4,4 -dihydroxy-ε,ε-carotene-3.3 -dione reported by Schiedt et al. 10, 11. Therefore, this carotenoid was identified as 6S, 6 S -isoastaxanthin 1. The yellow carotenoid 4, with a molecular formula of C 40 H 60 O 4, showed absorption maxima at 419, 439, and 468 nm. UV-VIS, ESI TOF MS, and 1 H NMR data on this carotenoid were identical to those of 3S,4R,5S,6S,3 S,4 R,5 S,6 S - tetrahydroxypirardixanthin 5,6,5,6 -tetrahydro-β,βcarotene-3,4,3,4 -tetraol isolated from the spindle shell Fushinus perplexus 13. On the other hand, this compound showed a mirror image of the CD spectrum in comparison with 3S,4R,5S,6S,3 S,4 R,5 S,6 S - tetrahydroxypirardixanthin 13. Namely, 3S,4R,5S,6S,3 S,4 R,5 S,6 S -tetrahydroxy- pirardixanthin showed a positive maximum Δε 4.0 at 263 nm 13 While, this compound showed a negative maximum Δε 4.0 at 263 nm. This clearly indicated that this compound was the enantiomer of 3S,4R,5S,6S,3 S,4 R,5 S,6 S -tetrahydroxypirardixanthin. Therefore, 3R,4S,5R,6R,3 R,4 S,5 R,6 R stereochemistry was assigned for this compound. These results are in agreement with those reported by Schiedt et al. 10, 11 However, detailed spectral data on 3R,4S,5R,6R,3 R,4 S,5 R,6 R -tetrahydroxypirardixanthin have not been reported 14. Therefore, UV-VIS, ESI TOF MS, 1 H NMR, and CD spectral data of this compound are described in this manuscript for the 1429

6 T. Maoka, Y. Kawashima, and M. Takaki first time. Tetrahydroxypirardixanthin with other configurations was not found in the prawn. The spectral data of yellow carotenoid 5 were in agreement with those of crustaxanthin β,β-carotene-3,4,3,4 tetrol with 3R,4S,3 R,4 S configurations 12, 15. Crustaxanthin, with other configurations, was not found in the prawn. Yellow carotenoid 3, with a molecular formula of C 40 H 54 O 4, is a new carotenoid. This carotenoid showed UV-VIS absorption maxima at 425, 445, 473 nm indicating the presence of the β,ε-carotene-type conjugated doublebond system 7. The molecular formula of this compound was determined as C 40 H 54 O 4 by HR ESI TOF MS. 1 H NMR signals of this compound showed the presence of a 3,4-cis-3,4-dihydroxy-β-end group H-2 to H-4, H-16, H-17, and H-18, a 3-keto-4-hydroxy-ε-end group H-2, OH-4, H-6, H-16, H-17, and H-18 and an all-trans-polyene chain H-7 to H15, H-7 to H-15, H-19, 20, 19, and This was also confirmed by COSY and NOESY experiments. The relative stereochemistry of this compound was confirmed by NOESY correlation data, as shown in Fig. 2. From these spectral data, the structures of this carotenoid Fig. 2 Structure of yellow xanthophylls, 6S,6 S -isoastaxanthin 1, 3R,4S,5R,6R,6 S -5,6-dihydropenaeusxanthin 2, 3R,4S,6 S -penaeusxanthin 3 3R,4S,5R,6R,3 R,4 S,5 R,6 R -tetrahydroxypirardixanthin 4, and 3R,4S,3 R,4 S crustaxanthin 5 isolated from the prawn and key NOESY correlations of 2 and

7 Yellow Xanthophylls in the Prawn was determined as 3,4,4 -trihydroxy-β,ε-caroten-3 -one with 3,4-cis-configuration, and this carotenoid was named penaeusxanthin. The CD spectrum of this compound was almost similar to the additive CD spectra of 3R,4S,3 R,4 S crustaxanthin 5 and 6S, 6 S -isoastaxanthin 1 with half intensity, as shown in Fig. 3. According to the additive rule of CD spectra of carotenoid 8, 15, the 3R,4S,6 S configuration was postulated for this compound. The yellow carotenoid 2, with a molecular formula of C 40 H 56 O 4, is a new carotenoid. This carotenoid showed UV-VIS absorption maxima at 419, 439, and 468 nm The molecular formula of this compound was determined as C 40 H 56 O 4 by HR ESI TOF MS. 1 H-NMR signals of this compound showed the presence of a 3,4-cis-3,4-dihydroxy-5,6- dihydro-β-end group H-2 to H-6, H-16, H-17, H-18, a 3-keto-4-hydroxy-ε-end group H-2, OH-4, H-6, H-16, H-17, and H-18 and an all-trans-polyene chain H-7 to H15, H-7 to H-15, H-19, 20, 19, and This was also confirmed by COSY and NOESY experiments. The relative stereochemistry of this compound was confirmed by NOESY correlation data as shown in Fig. 2. From these spectral data, the structure of this carotenoid was determined to be 5,6-dihydro-3,4,4 -trihydroxy-β,ε-caroten-3 -one with 3,4-cis-configuration. This structure corresponded to the 5,6-dihydro derivative of penaeusxanthin 3 described above. Therefore, this compound was named 5,6-dihydropenaeusxanthin 2. The CD spectrum of this compound was almost similar to the additive CD spectra of 3R,4S,5R,6R,3 R,4 S,5 R,6 R -tetrahydroxypirardixanthin 4 and 6S, 6 S -isoastaxanthin 1 with half intensity, as shown in Fig. 3. According to the additive rule of CD spectra of carotenoid 8, 15, the 3R,4S,5R,6R,6 S configuration was postulated for this compound. 3.4 Metabolism of astaxanthin to yellow xanthophylls in the prawn A carotenoid feeding experiment using a radioisotope-labeled compound in the prawn revealed that β-carotene was oxidatively metabolized to astaxanthin through echinenone, canthaxanthin, and adonirubin 1, 2. Similarly, zeaxanthin was oxidatively converted to astaxanthin via adonixanthin 1, 2. On the other hand, Katagiri et al isolated tetrahydroxypirardixanthin in the prawn and proposed a reductive metabolic pathway from astaxanthin to tetrahydroxypirardixanthin 16. Subsequently, Schiedt et al., isolated isoastaxanthin 1, tetrahydroxypirardixanthin 4, and curstaxanthin 5, as metabolites of astaxanthin from the prawns P. japonicus and P. vannamei in an astaxanthin administration experiment 10, 11. In the present investigation, we isolated a series of yellow xanthophylls, isoastaxanthin 1, 5,6-dihydropenaeusxanthin 4, penaeusxanthin 3, tetrahydroxypirardixanthin 2, and crustaxanthin 5, from the prawn. The amounts of these yellow xanthophylls were increased by the administration amount of astaxanthin dose-dependently, as shown in Table 1. Therefore, these yellow xanthophylls were considered to be metabolites of astaxanthin. As reported by Schiedt et al. 10, 11, the chiral conversion Fig. 3 A CD spectra of penaeusxanthin 3, 6S,6 S -isoastaxanthin 1, 3R,4S,3 R,4 S -crustaxanthin 5 in ether at room temperature, and additive spectrum of 6S,6 S -isoastaxanthin and 2R,4S,3 R,4 S crustaxanthin half intensity. B CD spectra of 5,6-dihydropenaeusxanthin 4, 6S,6 S isoastaxanthin 1, 3R,4S,5R,6R,3 R,4 S,5 R,6 R -tetrahydropirardixanthin 4 in ether at room temperature, and additive spectrum of 6S,6 S -isoastaxanthin and 3R,4S,5R,6R,3 R,4 S,5 R,6 R tetrahydropirardixanthin half intensity. 1431

8 T. Maoka, Y. Kawashima, and M. Takaki of administered 3S,3 S -astaxanthin was observed in the present investigation, as described above. Chiral conversion at the 3 3 -hydroxy group in astaxanthin could be explained by the presence of isoastaxanthin 1, having a 3-keto-4-hydroxy-ε-end group, as an intermediate. Namely, the 3 3 -hydroxy group in astaxanthin was oxidized once to a carbonyl group with double-bond translation from C5-C6 C5 -C6 to C4-C5 C4 -C5 to form isoastaxanthin 1. Then, the 3-keto-4-hydroxy-ε-end group in isoastaxanthin 1 was reversibly converted to the 3-hydroxy-4-keto-β-end group to form astaxanthin. Another possible mechanism of astaxanthin racemization is keto-enol tautomerization of the 3 3 -hydroxy group, as shown in Fig. 4. Through these conversions, the chirality of the 3 3 -hydroxy group of astaxanthin could be converted. Furthermore, a series of yellow xanthophylls with a 3R,4S -3,4-dihydroxy-β-end group penaeusxanthin 2 and crustaxanthin 5 and 3R,4S,5R,6R -5,6-dihydro-3,4- dihydroxy-β-end group 5,6-dihydropenaeusxanthin 2 and tetrahydroxypirardixanthin 4 could be considered as metabolites of isoastaxanthin 1. Namely, the 3-keto-4-hydroxy-ε-end group in isoastaxanthin 1 was stereoselectively converted to the 3R,4S -3,4-dihydroxy-β-end group with reduction of the carbonyl group at C-3 and doublebond translation from C4-C5 to C5-C6 to form penaeusxanthin 3 and crustaxanthin 5. Then, reduction of the doubl-bond at C5-C6 to a single bond in penaeusxanthin 3 and crustaxanthin 5 formed 5,6-dihydropenaeusxanthin 2 and tetrahydroxypirardixanthin 4, as shown in Fig. 3. Therefore, isoastaxanthin 1, with a 3-keto-4-hydroxy-ε-end group, was considered as a key intermediate for the racemization of astaxanthin and reductive metabolism of astaxanthin in the prawn. 4 CONCLUSION Not only the amounts of astaxanthins astaxanthin diester, astaxanthin monoester, and free astaxanthin but also the amounts of yellow xanthophylls, isoastaxanthin 1, 5,6-dihydropenaeusxanthin 2, penaeusxanthin 3, tetrahydroxypirardixanthin 4, and curstaxanthin 5, were dose-dependently increased with the administration of PANAFERD-AX as source of 3S,3 S -astaxanthin. Based on the results of the present investigation, the reductive metabolic pathways of astaxanthin to these yellow xanthophylls in the prawn can be proposed, as shown in Fig. 4. References 1 Katayama T.; Hirata, K.; Chichester, C.O. The biosynthesis of astaxanthin-iv. The carotenoids in the prawn, Penaeus japonicus Bate Part I. Bull. Jpn. Soc. Sci. Fish. 37, Katayama T.; Kitama, T.; Chichester, C.O. The biosynthesis of astaxanthin in the prawn, Penaeus japonicus Bate Part II. Int. J. Biochem. 3, Latscha, T. The role of astaxanthin in shrimp pigmentation. Advances in tropical aquaculture Tahiti Aquacop Ifremer Actes de Colloque 9, Fig. 4 Possible metabolic pathway of astaxanthin to yellow xanthophylls in the prawn. 1432

9 Yellow Xanthophylls in the Prawn Yamada, S.; Tanaka, Y.; Sameshima, M.; Ito, Y. Pigmentation of prawn Penaeus japonicus with carotenoids: I. Effect of dietary astaxanthin, β-carotene and canthaxanthin on pigmentation. Aquaculture 87, Tsubokura, A.; Yoneda, H.; Mizuta, H. Paracoccus carotinifaciens sp. nov., a new aerobic gram-negative astaxanthin-producing bacterium. J. Syst. Bacteriol. 49, Ishibashi, T. Manufacture production of carotenoid from Paracoccus bacterium in Japanese. Seibutsukogaku kaishi 93, Britton, G. UV/Visible Spectrometry. in Carotenoid Britton, G.; Liaaen-Jensen, S.; Pfander, H. eds.. Brikhauser Verlag, Basel, Vol. 1B, pp Maoka, T. Structural studies of carotenoids in plants, animals, and food products. in Carotenoids Nutrition. Analysis and Technology Kaczor, A.; Baranska, M. eds.. Wiley Blackwell, UK, pp Maoka, T.; Akimoto, N. Carotenoids and their fatty acid esters of spiny lobster Panulirus japonicus. J. Oleo Sci. 57, Schiedt, K.; Bischof, S.; Glinz, E. Recent progress on carotenoid metabolism in animals. Pure Appl. Chem. 63, Schiedt, K.; Bischof, S.; Glinz, E. Metabolism of carotenoids and in vivo racemization of 3S,3 S -astaxanthin in the crustacean Penaeus. Methods Enzymol. 214, Englert, G. NMR Spectrometry. in Carotenoids Britton, G.; Liaaen-Jensen, S.; Pfander, H. eds.. Brikhauser Verlag, Basel, Vol. 1B, pp Tsushima, M.; Maoka, T.; Matsuno, T. Structure of carotenoids with 5,6-dihydro-β-end groups from the spindle shell Fushinus perplexus. J. Nat. Prod. 64, Carotenoids Handbook Britton, G.; Liaaen-Jensen, S.; Pfander, H. eds.. Birkhäuser, Basel Bucheker, R.; Noack, K. Circular dichroism. in Carotenoids Britton, G.; Liaaen-Jensen, S.; Pfander, H. eds.. Brikhauser Verlag, Basel, Vol. 1B, pp Katagiri, K.; Koshino, Y.; Takashi Maoka, T.; Matsuno, T. Occurrence of pirardixanthin derivatives in the prawn, Penaeus japonicas. Comp. Biochem. Physiol. 87B,