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Isolation of three new carotenoids and proposed metabolic pathways of carotenoids in hen's egg yolk
Comp. Biochem. Physiol. Vol. 84B, No. 4, pp. 477-481, 1986 Printed in Great Britain
0305-0491/86 $3.00 + 0.00 Pergamon Journals Ltd
ISOLATION OF THREE NEW CAROTENOIDS A N D PROPOSED METABOLIC PATHWAYS OF CAROTENOIDS IN HEN'S EGG YOLK TAKAO MATSUNO, TOSHIHIKO HIRONO, YOSHIHIRO IKUNO, TAKASHI MAOKA, MINORU SHIMIZU and TADAAKI KOMORI Department of Natural Products Research, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan (Received 14 November 1985) Abstract--1. Three new carotenoids, (6R,3'R,6'R)-3'-hydroxy-E,E-caroten-3-one (10), (6S,3'R,6'R)-Yhydroxy-E,E-caroten-3-one (I 1) and (6'RS)-fl,E-caroten-3'-one (12), have been isolated from hen's egg yolk. 2. Based on stereochemical aspects and individual carotenoids, the following metabolic pathways of carotenoids in chicken were proposed. (i) Lutein A (1) to (6R,6'R)-E,E-carotene-3,3'-dione (9) via (6R,3'R,6'R)-3'-hydroxy<,E-caroten-3-one (I0) and/or (3R,6'R)-3-hydroxy-fl,E-caroten-Y-one (5). (ii) Lutein A (I) to (6S,6"R;meso)<,E-carotene-3,3"-dione (8) via (6S,YR,6'R)-Y-hydroxy-E,E-caroten3-one (1 I) and/or (3R,6'R)-3-hydroxy-fl,E-caroten-Y-one (5). (iii) (3R,YR)-Zeaxanthin (2) to (6S,6'R;meso)-E,E-carotene-3,3'-dione (8) via (3R,6'R)-3-hydroxyfl,E-caroten-3'-one (5) and/or (3R,6'S)-3-hydroxy-fl,E-caroten-Y-one (6). (iv) (3R,3'R)-Zeaxanthin (2) to (6S,6'S)<,E-carotene-3,3'-dione (7) via (3R,6'S)-3-hydroxy-fl,~-carotenY-one (6). (v) (3R)-fl-Cryptoxanthin (3) to (6'RS)-fl,E-caroten-Yone (12).
INTRODUCTION
concentrated under reduced pressure in N 2 below 40°C. The concentrated solution was stored at - 20°C overnight. After removal of a sterol by filtration, they were transferred to ether-petroleum ether (p.e.) solution by addition of water. The epiphase was dried over Na 2SO4. The total and individual carotenoid content was determined by the method of McBeth (1972). The extracted solution was concentrated under reduced pressure N 2 below 40°C. The crude carotenoids were separated by column chromatography on silica gel. And then, each fraction was submitted to separation of stereoisomers by high-performance liquid chromatography (HPLC) on a chiral resolution column Sumipax OA-2000 (Maoka et al., 1985; Ikuno et al., 1985).
fl-Carotene, lutein (1), zeaxanthin (2), fl-cryptoxanthin (3), (Grimbleby and Black, 1952) and can° thaxanthin (4) (Steinegger et aL, 1957) have hitherto been known as principal carotenoids in hen's egg yolk. Furthermore, (3R,6'R)-3-hydroxy-fl,E-carotenY-one (5) was obtained from hen's egg yolk by Buchecker and Eugster (1979). Subsequently, Schiedt et al. 0981) isolated further three carotenoids, (3R,6'S)-3-hydroxy-fl,E-caroten-3'-one (6) (from egg yolk), (6S,6'S)-e,E-carotene-3,Y-dione (7) (from egg yolk) and (6S,6"R;meso)-e,E-carotene-3,3'-dione (8) (from liver). In addition to these carotenoids, we have isolated (6R,6'R)-E,E-carotene-3,3'-dione (9) and three new carotenoids (6R,3'R,6'R)-3'-hydroxy-e,E-caroten-3one (10), (6S,YR,6'R)-3'-hydroxy-E,E-caroten-3-one (l l) and (6'RS)-fl,E-caroten-3'-one (12) from hen's egg yolk. The purpose of the present paper is to report the isolation of these carotenoids. Furthermore, taking stereochemical aspects and individual carotenoid into consideration, several new metabolic pathways of lutein A (1), (3 R, 3' R)-zeaxa nt hin (2) and (3R)-fl-cryptoxanthin (3) in hen's egg yolk will be discussed in this paper.
Identification The identification of each carotenoid was accomplished by co-HPLC with authentic carotenoids, visible light absorption (VIS), circular dichroism (CD) spectra and mass (MS) spectra, and by chemical treatment such as acetylation (Andrews et al., 1974), allylic-OH test (Karrer and Jirgensons, 1930) and reduction with NaBH 4 (Andrews et al., 1974) in the case of keto carotenoid. Instrument VIS spectra were recorded on a Shimadzu UV-240 spectrophotometer in ether solution. CD spectra were obtained by a JASCO J-500 C spectropolarimeter in ether isopentane-EtOH (5:5:2) (EPA) solution at 20°C. MS spectra were recorded with a Hitachi M-80 mass spectrometer with an ionization energy of 25 eV. HPLC was run on a Waters Model Sl0 instrument with a Waters Lambda Max Model 481 LC spectrophotometer set at 445 nm. The column used was a 300 x 8 mm i.d. stainless steel column packed with Sumipax OA-2000 (particle size 5 #m).
MATERIALS AND METHODS
Biological materials Hen's eggs (500 specimens) were purchased at a market in Kyoto City, Japan. Extraction and isolation According to the method as described previously (Buchecker and Eugster, 1979), the carotenoids were extracted with acetone from egg yolk. The acetone extract was
Authentic samples The authentic carotenoids were prepared from the following materials; lutein A (1) (from gingko), (3R,3'R)-
477
TAKAO MATSUNOet al.
478
zeaxanthin (2) (from cycad), (3R)-fl-cryptoxanthin (3) (from unshiu orange) and canthaxanthin (4) (synthetic sample, F. Hoffmann-La Roche).
30 20
RESULTS
The following nine carotenoids (1), (2), (3), (4), (5), (6), (7), (8) and (9) have been identified.
10
(3R)-fl-Cryptoxanthin (3) This carotenoid was eluted with ether-p.e. (20:80) on silica gel column and showed VIS '~max(425), 449 and 475 rim, MS m/z 552 (M ~ , C40H56O) and CD nm (A~) 224 ( - 6 . 0 ) , 236 (0), 245 ( + 6.0), 260 (0), 280 ( - 10.0), 325 (0) and 350 ( + 3.0) and did not separate from authentic (3) on co-TLC and co-HPLC.
0
Canthaxanthin (4) This carotenoid was eluted with ethe~p.e. (30:70) on silica gel column and showed VIS 2max 468 nm and MS m/z 564 (M +, C40H5202) and did not separate from authentic (4) on co-TLC and co-HPLC,
!
\
/
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/
i I
-30 200
~ 250
_L_ 300
I
350
400
nm
Lutein A (1)
Fig. 1. CD spectra of (6R,3'R,6'R)-3'-hydroxy-~,~-caroten3-one (10) - and (6S,3'R,6'R)-3'-hydroxy-E,~-caroten3-one (ll) in EPA at 20:C.
This carotenoid was eluted with ether on silica gel column and showed VIS 2,,,~ 420, 444 and 472 nm, MS m/z 568 (M +, C40H5602) and CD nm (AE) 220 ( + 2.0), 245 ( + 8.0), 275 (0) and 285 ( - 4.5) and did not separate from authentic (1) on co-TLC and co-HPLC.
respectively. This is the first report of (9) from hen's egg yolk. The following three new carotenoids (10), (11) and (12) have been isolated.
(3R,3'R )-Zeaxanthin (2) This carotenoid was eluted with ether on silica gel column and showed VIS 2max (425), 449 and 475 nm, MS m/z 568 (M +, C40H5602) and CD nm (A~) 224 ( - 18.0), 236 (0), 245 ( + 18.0), 260 (0), 248 ( - 2 4 . 8 ) , 325 (0) and 350 ( + 4 . 0 ) and did not separate from authentic (2) on co-TLC and co-HPLC.
(3R,6'R)-3-Hydroxy-fl,~-caroten-3"-one (5) and (3R,6' S )- 3-hydroxy-fl,E-caroten- 3"-one (6) (5) and (6) were eluted with ether-p.e. (60 : 40) from silica gel column. This fraction showed VIS )~max420, 444 and 472 nm, MS m/z 566 (M +, C40H5402), 548 (M + - 18), 474 (M + - 92) and 460 (M + - 106) and CD nm (AE) 282 (--3.2) (mixture of 6'R and 6'S isomers). However, these stereoisomers could not be separated in any of our HPLC systems, Therefore, the ratio of (5) and (6) was calculated from the CD spectral datum.
(6S,6'S)-E,E-carotene-3,Y-dione (7), (6S,6'R;meso)~,E-carotene-3,3'-dione (8) and (6R,6'R)-E,E-carotene3,3'-dione (9) (7), (8) and (9) were eluted with ether-p.e. (50: 50) from a silica gel column. The separation of (7), (8) and (9) was achieved by HPLC (Ikuno et al., 1985). These carotenoids showed the same VIS Lm~x418, 440 and 469nm and MS m/z 564 (M +, C40H520~), 472 (M + - 92) and 458 (M ~ - 106) spectral data. CD (A~ at 268 rim) of (7), (8) and (9) showed ( - 4 0 ) , (no optical activity) and ( + 40), respectively. Therefore, (7), (8) and (9) have been identified with (6S,6'S)-, (6S,6"R;meso)- and (6R,6'R)-E,E-carotene-3,3'-dione,
(6R,3'R,6'R)-3'-Hydroxy-~,~.-caroten-3-one (10) and (6S,3"R,6"R )-3'-hydroxv-e,E -caroten-3-one (11) (10) and (11) were eluted with ether-p.e. (65:35) from a silica gel column. The separation of two stereo-isomers, (10) and (11), was achieved by HPLC with a chiral resolution column, Sumipax OA-2000, eluting with n-hexane-CH 2C12-EtOH (54: 10 : 0.3). Both (10) and (11) showed the same VIS )~ma~417, 439 and 468 nm and MS m/z 566 (M +, C40H5402), 548 (M + - 18), 474 (M ~ - 92) and 460 (M + - 106), CD spectra of(9) and (10) are shown in Fig. 1. Both (10) and (11) gave a monoacetate by acetylation and were positive for allylic OH test. Sodium borohydride reduction of (10) gave tunaxanthin F, (3R,6R,YR,6'R)-E,e-carotene-3,3'-diol and tunaxanthin I, (3S,6R,YR,6'R)-~,E-carotene-3,Y-diol (Fig. 2). In a same manner, (11) yielded tunaxanthin E (3S,6S,YR,6'R)-E,E-carotene-3,Y-diol and tunaxanthin H (3R,6S,YR,6'R)<,~.-carotene-3,3'-diol (Fig. 2). The identification of tunaxanthin was carried out by co-HPLC (Ikuno et al., 1985) and comparison of CD spectra with those of authentic samples. Therefore, based on the results described above the structures of (10) and (11) have been established to be (6R,3'R,6'R)-3'-hydroxy-E,e-caroten-3-one and (6,S,3'R,6'R)-3'-hydroxy-~,~-caroten-3-one, respectively.
(6'RS)-fl,~ -caroten-3"-one (12) (12) was eluted with ether-p.e. (15: 85) from a silica gel column. (12) had VIS 2,1,x 422, 445 and 472 nm and MS rn/z 550 (M*, C40H540), 458 (M + - 92) and 444 (M ÷ - 1 0 6 ) . (12) gave neither acetate nor trimethylsilanate. The sodium borohydride reduction product of (12) showed VIS 2 .... 422, 455 and 472 nm
Carotenoids in hen's egg yolk
479
Table 1. Content and composition of carotenoids in hen's egg yolk
ua A
? =.
I I
Total carotenoids (mg/100 g)
2.5
(3R)-fl-Cryptoxanthin (6'RS)-fl,E-Caroten-Y-one Canthaxanthin (6S,6'S)-E,E-Carotene-3,Y-dione (6S,6"R;meso)-~,~ -Carotene-3,Y-dione (6R,6'R)-~,~ -Carotene-3,Y-dione (6R,Y R,6'R)-3'-Hydroxy<,~ -caroten-3-one (6S,3'R,6'R)-3'-Hydroxy<,~-caroten-3-one (3 R,6'R)-3-Hydroxy-/~,~ -caroten-3'-one (3 R,6'S)- 3-Hydroxy-fl,~ -caroten-3"-one Lutein A (3R,3'R)-Zeaxanthin
17.3% 0.5 17.9 0.5 0.3 0.2 0.4 0.6 1.3 1.2 40.0 19.8
[-
and MS m/z 552 (M ÷, C 4 0 H 5 6 0 ). This reduction product gave mono acetate and was positive for allylic OH test. Thus the structure of (12) has been assumed to be (6'RS)-/?,E-caroten-3'-one. This is probably the same product once mentioned by Buchecker and Eugster (1979). Table 1 shows the content and percentage composition of carotenoids in hen's egg yolk.
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DISCUSSION
Schiedt et al. (1981) confirmed that (3R,YR)zeaxanthin (2) could be transformed to (6S,6'S)E,E-caroten-3,Y-dione (7) via (3R,6'S)-3-hydroxyE,E-caroten-Y-one (6) in hen's egg by an isotope experiment. When lutein A (1) diester was also fed to broilers, the two corresponding metabolites could be isolated from the liver and determined as (3R,6'R)3-hydroxy-fl,E-caroten-Y-one (5) and (6S,6'R;meso)E,E-carotene-3,Y-dione (8). According to these results, they proposed the following carotenoids metabolic pathways in chicken. (i) (3R,3'R)-Zeaxanthin (2)~(3R,6'S)-3-hydroxyfl,E-caroten-3'-one (6) ~ (6S,6'S)-~,E -carotene- 3,3'dione (7). (ii) Lutein A (1) diester~(3R,6'R)-3-hydroxy-fl,Ecaroten-3'-one (5)~(6S,6'R;meso )-E,E-carotene-3,3'dione (8). In addition to (1), (2), (5), (6), (7) and (8) reported by Schiedt et al. (1981), we have isolated (9) and three new carotenoids (10), (11) and (12). Taking the metabolic pathways of carotenoids in chicken proposed by Schiedt et al. (1981), stereochemical aspects and individual carotenoids into consideration, we have assumed the following metabolic pathways for carotenoids (Fig. 3). (i) Lutein A (1) to (6R,6'R)-e,E-carotene-3,Y-dione (9) via (6R,3'R,6'R)-3'-hydroxy<,E-caroten-3-one (10) and/or (3R,6'R)-3-hydroxy-fl,E-caroten-Y-one (5). (ii) Lutein A (1) to (6S,6"R;meso)-E,E-carotene3,Y-dione (8) via (6S,3'R,6'R)-Y-hydroxy-E,~caroten-3-one (11) and/or (3R,6'R)-3-hydroxy-l~,Ecaroten-Y-one (5). (iii) (3R,YR)-Zeaxanthin (2) to (6S,6"R;meso)E,E-carotene-3,Y-dione (8) via (3R,6'R)-3-hydroxyB,E-caroten-Y-one (5) and/or (3R,6'S)-3-hydroxy/~,E-caroten-Y-one (6). (iv) (3R,YR)-Zeaxanthin (2) to (6S,6'S)-E,Ecarotene-3,Y-dione (7) via (3R,6'S)-3-hydroxy-fl,Ecaroten-Y-one (6).
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Carotenoids in hen's egg yolk (v) (3R)-fl-Cryptoxanthin (3) to (6'RS)-fl,E -carotenY-one (12). In conclusion, the secondary hydroxy moieties located on C-3 and C-3' in lutein A (1), (3R,3'R)zeaxanthin (2) and (3R)-fl-cryptoxanthin (3) (lacked of hydroxyl at C-3') undergo stepwise alternative oxidation along with E-type to E-type end group transformation until (6S,6'S)-E,E-carotene-3,3'dione (7), (6S,6'R;meso)-E,E-carotene-3,3'-dione (8), (6R,6'R)-E,E-carotene-3,3'-dione (9) and (6'RS)-fl,Ecaroten-3'-one (12). Acknowledgements--We wish to thank Mr Takeshi Ueda and Miss Sakiko Ooe for their assistance. REFERENCES
Andrews A., Borch G., Liaaen-Jensen S. and Snatzke G. (1974) Animal carotenoids. 9. On the configuration of astaxanthin and actinioerythrin. Acta Chem. Scand. B2g, 730-736. Buchecker R. and Eugster C. H. (1979) Eine Suche nach 3'-Epilutein (= (3R,3'S,6'R)-fl,E-carotin-3,3'-diol) und 3'-
481
O-Didehydrolutein (= (3R,6'R)-3-Hydroxy-/3,E-carotin3'-on) in Eigelb, in Bluten von Caltha palustris und in Herbstblattern. Heir. Chim. Acta 62, 2817-2824. Grimbleby F. H. and Black D. J. G. (1952) Variations in the composition of egg-yolk pigment. Br. J. Nutrition 6, 393-397. Ikuno Y., Maoka T., Shimizu M., Komori T. and Matsuno T. (1985) Direct diastereomeric resolution of carotenoids II. All ten stereoisomers of tunaxanthin (E,E-carotene3,3'-diol). J. Chromatogr. 328, 387-391. Karrer P. and Jirgensons B. (1930) Pflanzenfarbstoffe XXVII. t]ber die Methylierung des Xanthophylls. Heir. Chim. Acta 13, 1102 1103. McBeth J. W. (1972) Carotenoids from nudibranchs. Comp. Biochem. Physiol. 41B, 55-68. Maoka T., Komori T. and Matsuno T. (1985) Direct diastereomeric resolution of carotenoids I. 3-Hydroxy4-oxo-fl-end group. J. Chromatogr. 318, 122-124. Schiedt K., Englert G., Noack K., Vecchi M. and Leuenberger F. J. (1981) Xanthophyll conversion in the chicken. Abst. Sixth lnternat. Symp. Carotenoids. Steinegger P., Streiff K. and Zeller P. (1957) Pigmentation and color of egg yolk in poultry after administration of synthetic carotenoids. Mitt. Gebiete Lebensm. u. Hyg. 48, 445-450.
0305-0491/86 $3.00 + 0.00 Pergamon Journals Ltd
ISOLATION OF THREE NEW CAROTENOIDS A N D PROPOSED METABOLIC PATHWAYS OF CAROTENOIDS IN HEN'S EGG YOLK TAKAO MATSUNO, TOSHIHIKO HIRONO, YOSHIHIRO IKUNO, TAKASHI MAOKA, MINORU SHIMIZU and TADAAKI KOMORI Department of Natural Products Research, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan (Received 14 November 1985) Abstract--1. Three new carotenoids, (6R,3'R,6'R)-3'-hydroxy-E,E-caroten-3-one (10), (6S,3'R,6'R)-Yhydroxy-E,E-caroten-3-one (I 1) and (6'RS)-fl,E-caroten-3'-one (12), have been isolated from hen's egg yolk. 2. Based on stereochemical aspects and individual carotenoids, the following metabolic pathways of carotenoids in chicken were proposed. (i) Lutein A (1) to (6R,6'R)-E,E-carotene-3,3'-dione (9) via (6R,3'R,6'R)-3'-hydroxy<,E-caroten-3-one (I0) and/or (3R,6'R)-3-hydroxy-fl,E-caroten-Y-one (5). (ii) Lutein A (I) to (6S,6"R;meso)<,E-carotene-3,3"-dione (8) via (6S,YR,6'R)-Y-hydroxy-E,E-caroten3-one (1 I) and/or (3R,6'R)-3-hydroxy-fl,E-caroten-Y-one (5). (iii) (3R,YR)-Zeaxanthin (2) to (6S,6'R;meso)-E,E-carotene-3,3'-dione (8) via (3R,6'R)-3-hydroxyfl,E-caroten-3'-one (5) and/or (3R,6'S)-3-hydroxy-fl,E-caroten-Y-one (6). (iv) (3R,3'R)-Zeaxanthin (2) to (6S,6'S)<,E-carotene-3,3'-dione (7) via (3R,6'S)-3-hydroxy-fl,~-carotenY-one (6). (v) (3R)-fl-Cryptoxanthin (3) to (6'RS)-fl,E-caroten-Yone (12).
INTRODUCTION
concentrated under reduced pressure in N 2 below 40°C. The concentrated solution was stored at - 20°C overnight. After removal of a sterol by filtration, they were transferred to ether-petroleum ether (p.e.) solution by addition of water. The epiphase was dried over Na 2SO4. The total and individual carotenoid content was determined by the method of McBeth (1972). The extracted solution was concentrated under reduced pressure N 2 below 40°C. The crude carotenoids were separated by column chromatography on silica gel. And then, each fraction was submitted to separation of stereoisomers by high-performance liquid chromatography (HPLC) on a chiral resolution column Sumipax OA-2000 (Maoka et al., 1985; Ikuno et al., 1985).
fl-Carotene, lutein (1), zeaxanthin (2), fl-cryptoxanthin (3), (Grimbleby and Black, 1952) and can° thaxanthin (4) (Steinegger et aL, 1957) have hitherto been known as principal carotenoids in hen's egg yolk. Furthermore, (3R,6'R)-3-hydroxy-fl,E-carotenY-one (5) was obtained from hen's egg yolk by Buchecker and Eugster (1979). Subsequently, Schiedt et al. 0981) isolated further three carotenoids, (3R,6'S)-3-hydroxy-fl,E-caroten-3'-one (6) (from egg yolk), (6S,6'S)-e,E-carotene-3,Y-dione (7) (from egg yolk) and (6S,6"R;meso)-e,E-carotene-3,3'-dione (8) (from liver). In addition to these carotenoids, we have isolated (6R,6'R)-E,E-carotene-3,3'-dione (9) and three new carotenoids (6R,3'R,6'R)-3'-hydroxy-e,E-caroten-3one (10), (6S,YR,6'R)-3'-hydroxy-E,E-caroten-3-one (l l) and (6'RS)-fl,E-caroten-3'-one (12) from hen's egg yolk. The purpose of the present paper is to report the isolation of these carotenoids. Furthermore, taking stereochemical aspects and individual carotenoid into consideration, several new metabolic pathways of lutein A (1), (3 R, 3' R)-zeaxa nt hin (2) and (3R)-fl-cryptoxanthin (3) in hen's egg yolk will be discussed in this paper.
Identification The identification of each carotenoid was accomplished by co-HPLC with authentic carotenoids, visible light absorption (VIS), circular dichroism (CD) spectra and mass (MS) spectra, and by chemical treatment such as acetylation (Andrews et al., 1974), allylic-OH test (Karrer and Jirgensons, 1930) and reduction with NaBH 4 (Andrews et al., 1974) in the case of keto carotenoid. Instrument VIS spectra were recorded on a Shimadzu UV-240 spectrophotometer in ether solution. CD spectra were obtained by a JASCO J-500 C spectropolarimeter in ether isopentane-EtOH (5:5:2) (EPA) solution at 20°C. MS spectra were recorded with a Hitachi M-80 mass spectrometer with an ionization energy of 25 eV. HPLC was run on a Waters Model Sl0 instrument with a Waters Lambda Max Model 481 LC spectrophotometer set at 445 nm. The column used was a 300 x 8 mm i.d. stainless steel column packed with Sumipax OA-2000 (particle size 5 #m).
MATERIALS AND METHODS
Biological materials Hen's eggs (500 specimens) were purchased at a market in Kyoto City, Japan. Extraction and isolation According to the method as described previously (Buchecker and Eugster, 1979), the carotenoids were extracted with acetone from egg yolk. The acetone extract was
Authentic samples The authentic carotenoids were prepared from the following materials; lutein A (1) (from gingko), (3R,3'R)-
477
TAKAO MATSUNOet al.
478
zeaxanthin (2) (from cycad), (3R)-fl-cryptoxanthin (3) (from unshiu orange) and canthaxanthin (4) (synthetic sample, F. Hoffmann-La Roche).
30 20
RESULTS
The following nine carotenoids (1), (2), (3), (4), (5), (6), (7), (8) and (9) have been identified.
10
(3R)-fl-Cryptoxanthin (3) This carotenoid was eluted with ether-p.e. (20:80) on silica gel column and showed VIS '~max(425), 449 and 475 rim, MS m/z 552 (M ~ , C40H56O) and CD nm (A~) 224 ( - 6 . 0 ) , 236 (0), 245 ( + 6.0), 260 (0), 280 ( - 10.0), 325 (0) and 350 ( + 3.0) and did not separate from authentic (3) on co-TLC and co-HPLC.
0
Canthaxanthin (4) This carotenoid was eluted with ethe~p.e. (30:70) on silica gel column and showed VIS 2max 468 nm and MS m/z 564 (M +, C40H5202) and did not separate from authentic (4) on co-TLC and co-HPLC,
!
\
/
\
/
i I
-30 200
~ 250
_L_ 300
I
350
400
nm
Lutein A (1)
Fig. 1. CD spectra of (6R,3'R,6'R)-3'-hydroxy-~,~-caroten3-one (10) - and (6S,3'R,6'R)-3'-hydroxy-E,~-caroten3-one (ll) in EPA at 20:C.
This carotenoid was eluted with ether on silica gel column and showed VIS 2,,,~ 420, 444 and 472 nm, MS m/z 568 (M +, C40H5602) and CD nm (AE) 220 ( + 2.0), 245 ( + 8.0), 275 (0) and 285 ( - 4.5) and did not separate from authentic (1) on co-TLC and co-HPLC.
respectively. This is the first report of (9) from hen's egg yolk. The following three new carotenoids (10), (11) and (12) have been isolated.
(3R,3'R )-Zeaxanthin (2) This carotenoid was eluted with ether on silica gel column and showed VIS 2max (425), 449 and 475 nm, MS m/z 568 (M +, C40H5602) and CD nm (A~) 224 ( - 18.0), 236 (0), 245 ( + 18.0), 260 (0), 248 ( - 2 4 . 8 ) , 325 (0) and 350 ( + 4 . 0 ) and did not separate from authentic (2) on co-TLC and co-HPLC.
(3R,6'R)-3-Hydroxy-fl,~-caroten-3"-one (5) and (3R,6' S )- 3-hydroxy-fl,E-caroten- 3"-one (6) (5) and (6) were eluted with ether-p.e. (60 : 40) from silica gel column. This fraction showed VIS )~max420, 444 and 472 nm, MS m/z 566 (M +, C40H5402), 548 (M + - 18), 474 (M + - 92) and 460 (M + - 106) and CD nm (AE) 282 (--3.2) (mixture of 6'R and 6'S isomers). However, these stereoisomers could not be separated in any of our HPLC systems, Therefore, the ratio of (5) and (6) was calculated from the CD spectral datum.
(6S,6'S)-E,E-carotene-3,Y-dione (7), (6S,6'R;meso)~,E-carotene-3,3'-dione (8) and (6R,6'R)-E,E-carotene3,3'-dione (9) (7), (8) and (9) were eluted with ether-p.e. (50: 50) from a silica gel column. The separation of (7), (8) and (9) was achieved by HPLC (Ikuno et al., 1985). These carotenoids showed the same VIS Lm~x418, 440 and 469nm and MS m/z 564 (M +, C40H520~), 472 (M + - 92) and 458 (M ~ - 106) spectral data. CD (A~ at 268 rim) of (7), (8) and (9) showed ( - 4 0 ) , (no optical activity) and ( + 40), respectively. Therefore, (7), (8) and (9) have been identified with (6S,6'S)-, (6S,6"R;meso)- and (6R,6'R)-E,E-carotene-3,3'-dione,
(6R,3'R,6'R)-3'-Hydroxy-~,~.-caroten-3-one (10) and (6S,3"R,6"R )-3'-hydroxv-e,E -caroten-3-one (11) (10) and (11) were eluted with ether-p.e. (65:35) from a silica gel column. The separation of two stereo-isomers, (10) and (11), was achieved by HPLC with a chiral resolution column, Sumipax OA-2000, eluting with n-hexane-CH 2C12-EtOH (54: 10 : 0.3). Both (10) and (11) showed the same VIS )~ma~417, 439 and 468 nm and MS m/z 566 (M +, C40H5402), 548 (M + - 18), 474 (M ~ - 92) and 460 (M + - 106), CD spectra of(9) and (10) are shown in Fig. 1. Both (10) and (11) gave a monoacetate by acetylation and were positive for allylic OH test. Sodium borohydride reduction of (10) gave tunaxanthin F, (3R,6R,YR,6'R)-E,e-carotene-3,3'-diol and tunaxanthin I, (3S,6R,YR,6'R)-~,E-carotene-3,Y-diol (Fig. 2). In a same manner, (11) yielded tunaxanthin E (3S,6S,YR,6'R)-E,E-carotene-3,Y-diol and tunaxanthin H (3R,6S,YR,6'R)<,~.-carotene-3,3'-diol (Fig. 2). The identification of tunaxanthin was carried out by co-HPLC (Ikuno et al., 1985) and comparison of CD spectra with those of authentic samples. Therefore, based on the results described above the structures of (10) and (11) have been established to be (6R,3'R,6'R)-3'-hydroxy-E,e-caroten-3-one and (6,S,3'R,6'R)-3'-hydroxy-~,~-caroten-3-one, respectively.
(6'RS)-fl,~ -caroten-3"-one (12) (12) was eluted with ether-p.e. (15: 85) from a silica gel column. (12) had VIS 2,1,x 422, 445 and 472 nm and MS rn/z 550 (M*, C40H540), 458 (M + - 92) and 444 (M ÷ - 1 0 6 ) . (12) gave neither acetate nor trimethylsilanate. The sodium borohydride reduction product of (12) showed VIS 2 .... 422, 455 and 472 nm
Carotenoids in hen's egg yolk
479
Table 1. Content and composition of carotenoids in hen's egg yolk
ua A
? =.
I I
Total carotenoids (mg/100 g)
2.5
(3R)-fl-Cryptoxanthin (6'RS)-fl,E-Caroten-Y-one Canthaxanthin (6S,6'S)-E,E-Carotene-3,Y-dione (6S,6"R;meso)-~,~ -Carotene-3,Y-dione (6R,6'R)-~,~ -Carotene-3,Y-dione (6R,Y R,6'R)-3'-Hydroxy<,~ -caroten-3-one (6S,3'R,6'R)-3'-Hydroxy<,~-caroten-3-one (3 R,6'R)-3-Hydroxy-/~,~ -caroten-3'-one (3 R,6'S)- 3-Hydroxy-fl,~ -caroten-3"-one Lutein A (3R,3'R)-Zeaxanthin
17.3% 0.5 17.9 0.5 0.3 0.2 0.4 0.6 1.3 1.2 40.0 19.8
[-
and MS m/z 552 (M ÷, C 4 0 H 5 6 0 ). This reduction product gave mono acetate and was positive for allylic OH test. Thus the structure of (12) has been assumed to be (6'RS)-/?,E-caroten-3'-one. This is probably the same product once mentioned by Buchecker and Eugster (1979). Table 1 shows the content and percentage composition of carotenoids in hen's egg yolk.
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DISCUSSION
Schiedt et al. (1981) confirmed that (3R,YR)zeaxanthin (2) could be transformed to (6S,6'S)E,E-caroten-3,Y-dione (7) via (3R,6'S)-3-hydroxyE,E-caroten-Y-one (6) in hen's egg by an isotope experiment. When lutein A (1) diester was also fed to broilers, the two corresponding metabolites could be isolated from the liver and determined as (3R,6'R)3-hydroxy-fl,E-caroten-Y-one (5) and (6S,6'R;meso)E,E-carotene-3,Y-dione (8). According to these results, they proposed the following carotenoids metabolic pathways in chicken. (i) (3R,3'R)-Zeaxanthin (2)~(3R,6'S)-3-hydroxyfl,E-caroten-3'-one (6) ~ (6S,6'S)-~,E -carotene- 3,3'dione (7). (ii) Lutein A (1) diester~(3R,6'R)-3-hydroxy-fl,Ecaroten-3'-one (5)~(6S,6'R;meso )-E,E-carotene-3,3'dione (8). In addition to (1), (2), (5), (6), (7) and (8) reported by Schiedt et al. (1981), we have isolated (9) and three new carotenoids (10), (11) and (12). Taking the metabolic pathways of carotenoids in chicken proposed by Schiedt et al. (1981), stereochemical aspects and individual carotenoids into consideration, we have assumed the following metabolic pathways for carotenoids (Fig. 3). (i) Lutein A (1) to (6R,6'R)-e,E-carotene-3,Y-dione (9) via (6R,3'R,6'R)-3'-hydroxy<,E-caroten-3-one (10) and/or (3R,6'R)-3-hydroxy-fl,E-caroten-Y-one (5). (ii) Lutein A (1) to (6S,6"R;meso)-E,E-carotene3,Y-dione (8) via (6S,3'R,6'R)-Y-hydroxy-E,~caroten-3-one (11) and/or (3R,6'R)-3-hydroxy-l~,Ecaroten-Y-one (5). (iii) (3R,YR)-Zeaxanthin (2) to (6S,6"R;meso)E,E-carotene-3,Y-dione (8) via (3R,6'R)-3-hydroxyB,E-caroten-Y-one (5) and/or (3R,6'S)-3-hydroxy/~,E-caroten-Y-one (6). (iv) (3R,YR)-Zeaxanthin (2) to (6S,6'S)-E,Ecarotene-3,Y-dione (7) via (3R,6'S)-3-hydroxy-fl,Ecaroten-Y-one (6).
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3. P o s s i b l e
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Carotenoids in hen's egg yolk (v) (3R)-fl-Cryptoxanthin (3) to (6'RS)-fl,E -carotenY-one (12). In conclusion, the secondary hydroxy moieties located on C-3 and C-3' in lutein A (1), (3R,3'R)zeaxanthin (2) and (3R)-fl-cryptoxanthin (3) (lacked of hydroxyl at C-3') undergo stepwise alternative oxidation along with E-type to E-type end group transformation until (6S,6'S)-E,E-carotene-3,3'dione (7), (6S,6'R;meso)-E,E-carotene-3,3'-dione (8), (6R,6'R)-E,E-carotene-3,3'-dione (9) and (6'RS)-fl,Ecaroten-3'-one (12). Acknowledgements--We wish to thank Mr Takeshi Ueda and Miss Sakiko Ooe for their assistance. REFERENCES
Andrews A., Borch G., Liaaen-Jensen S. and Snatzke G. (1974) Animal carotenoids. 9. On the configuration of astaxanthin and actinioerythrin. Acta Chem. Scand. B2g, 730-736. Buchecker R. and Eugster C. H. (1979) Eine Suche nach 3'-Epilutein (= (3R,3'S,6'R)-fl,E-carotin-3,3'-diol) und 3'-
481
O-Didehydrolutein (= (3R,6'R)-3-Hydroxy-/3,E-carotin3'-on) in Eigelb, in Bluten von Caltha palustris und in Herbstblattern. Heir. Chim. Acta 62, 2817-2824. Grimbleby F. H. and Black D. J. G. (1952) Variations in the composition of egg-yolk pigment. Br. J. Nutrition 6, 393-397. Ikuno Y., Maoka T., Shimizu M., Komori T. and Matsuno T. (1985) Direct diastereomeric resolution of carotenoids II. All ten stereoisomers of tunaxanthin (E,E-carotene3,3'-diol). J. Chromatogr. 328, 387-391. Karrer P. and Jirgensons B. (1930) Pflanzenfarbstoffe XXVII. t]ber die Methylierung des Xanthophylls. Heir. Chim. Acta 13, 1102 1103. McBeth J. W. (1972) Carotenoids from nudibranchs. Comp. Biochem. Physiol. 41B, 55-68. Maoka T., Komori T. and Matsuno T. (1985) Direct diastereomeric resolution of carotenoids I. 3-Hydroxy4-oxo-fl-end group. J. Chromatogr. 318, 122-124. Schiedt K., Englert G., Noack K., Vecchi M. and Leuenberger F. J. (1981) Xanthophyll conversion in the chicken. Abst. Sixth lnternat. Symp. Carotenoids. Steinegger P., Streiff K. and Zeller P. (1957) Pigmentation and color of egg yolk in poultry after administration of synthetic carotenoids. Mitt. Gebiete Lebensm. u. Hyg. 48, 445-450.