A Novel Keto-Carotenoid from Young Leaves of Two Cycads Abstract ...

Ceratozamiaxanthin: A Novel Keto-Carotenoid from Young Leaves of Two Cycads Antonio Selva,† Franco Cardini,‡,* Mario Chelli,§ and Andrea Mele† †

C.N.R., Centro di Studio sulle Sostanze Organiche Naturali, Dipartimento di

Chimica del Politecnico di Milano, Via Mancinelli, 7, I-20131 Milano, Italy ‡

Dipartimento di Biologia Vegetale, Università di Firenze, Via La Pira 4, I-50121 Firenze, Italy §

C.N.R.-ICCOM, c/o Dipartimento di Chimica Organica "Ugo Schiff", I-50019 Sesto Fiorentino (FI), Italy. Fax: +39-055-282358; E-mail: [email protected]

Abstract The transitory red-brown coloration of the young leaves of two Cycads, (Ceratozamia kuesteriana and forma fuscoviridis of C. mexicana Brongn) is due to a mixture of red ketocarotenoids, present in the chloroplasts within electron-dense plastoglobule-like bodies, diffused in the stroma and absent in the thylakoid membranes. In addition to the four previously characterized very rare or new ketocarotenoids, a novel component C40H58O3, a minor constituent of this mixture, herewith named ceratozamiaxanthin, was isolated, for which we propose the structure of 1,5dihydroxy-1,2,5,6-tetrahydro-β,ψ-caroten-6-one. Moreover two novel minor components, here named kuesteriaxanthin (C40H58O4) and ceratoxanthone (C40H56O2), were also isolated from the same mixture, for which we propose the structure of 3’,4,5-trihydroxy-1,2,5,6-tetrahydro-β,ψcaroten-6-one and 5-hydroxy-3,4-didehydro-1,2,5,6-tetrahydro-β,ψ-caroten-6-one, respectively.

Keywords Cycads; ceratozamiaxanthin; keto-carotenoids; seco-carotenoids.

Introduction We are currently engaged with the characterization of the carotenoids present in the leaves of members of Cycadales, a relict group of ancient gymnosperms.1–5 Ceratozamia is one of the ten still surviving genera of Cycadales; its distribution is today limited to Mexico. Ceratozamia kuesteriana Regel is a definitely attributed species rediscovered in Mexico;6 C. fuscoviridis was renamed C. mexicana Brongn. var. longifolia forma fuscoviridis D.Moore ex Schuster.7 The newly formed leaflets of both C. kuesteriana and of "forma fuscoviridis" of C. mexicana present a transitory red-brown coloration due to

almost the same mixture of several carotenoids.1–5 An investigation8 on the chloroplast ontogenesis has been ultrastructurally carried out just on the red leaflets of "forma fuscoviridis" under study. In very young leaflets the chloroplasts contain, in their stroma, conspicuous electron-dense plasto-globule-like bodies, densely packed around large paracrystalline pro-lamellar bodies; these plastoglobules have been recently isolated and re-investigated; it was concluded that they contain only the mixture of red keto-carotenoids responsible for the typical red-brown colouration of the young leaflets.9 Within this ketomixture we had previously isolated and characterized four seco-carotenoids: the very rare semi-β-carotenone (SBC),1 triphasiaxanthin (TX),3 β-carotenone (BC)5 and the novel 5hydroxy-5,6-seco-β,β-caroten-6-one,2 afterward named ceratoxanthin (CX).4 SBC was the major component, about 67% of total carotenoids, when the newly formed leaflets were markedly red-brown.1 Interestingly, all the three very rare SBC, TX and BC were for the first time found in photosynthetic tissues.1,3,5 In this paper we report the isolation and a study on other three minor components of the above-mentioned keto-mixture, i.e. three new keto-carotenoids, we named ceratozamiaxanthin (CZX), kuesteriaxanthin (KX) and ceratoxanthone (CXO). Isolation and purification of CZX and consequently its structural characterization was particularly difficult, whereas it formed with TX and BC a mixture of at least three minor components. This mixture, preliminarily designated as Y' and reported to as a single compound,10 was only successively resolved, although not completely, by semi-preparative HPLC.2,3 However, the scarcity of the natural source and the difficulty of isolation did not allow to collect a sufficient CZX sample of adequate purity for a complete structural analysis by multidimensional and multinuclear NMR techniques. Therefore we are able to propose a reasonable structure only for CZX, and possible structures for KX and CXO.

Results The mixture Y' was continuously extracted and isolated from red-brown leaflets of "forma fuscoviridis" and also of C. kuesteriana Regel by the usual TLC system11 and accumulated. The two Y' mixture samples from the two Cycads showed on TLC the same Rf and a single co-chromatographic spot. The Y' sample was the most abundant of the minor keto-components in the keto-mixture from both the two Cycads. In "forma fuscoviridis" Y' mixture represented about 8% of the total carotenoids. By reversed-phase HPLC both Y' samples provided almost the same three main components, even though not in the same percentages. In the "forma fuscoviridis" Y' 2

composition was: TX (30-35%),3 BC (3-5%)5 and CZX (55-65%). CZX represented ca. 4.8% of the total carotenoids. However, the incomplete separation of the Y' components, achieved by reversed-phase HPLC, did not provide an absolutely pure sample of CZX, even after reiterated attempts. On TLC,11 CZX showed a middle polarity (Rf ≈ 0.48), being a little more epiphasic than lutein (Rf ≈ 0.42) and more polar than both CX (Rf = 0.51–0.54)2 and SBC (Rf = 0.690.73).1 Also on reversed phase HPLC, CZX performed as a middle polarity compound, with a retention time (Rt = 13.8 min) longer than that of the less polar TX (Rt = 10.3 min) and shorter than that of the more polar CX (Rt = 14.7 min). On silica gel layer CZX showed a marked red cyclamen coloration, a little different from the intense red coloration of SBC and the red coloration with some light tone of brown of both CX1 and TX.3 The more polar KX represented only 1.6% of the total carotenoids and showed a Rf = 0.17 (Rt = 9.2 min). On the other hand, the less polar CXO (present only as 1.2%) showed a Rf = 0.72 (Rt = 19.2 min). Chemistry. The acid isomerization test for the presence of a 5,6-epoxy-β-ring was negative both for KX and CXO. Acetylation by standard procedures were unsuccessful for CZX, while for KX gave the monoacetyl derivative. Samples of CZX, KX and CXO reacted with NaBH4 in EtOH and with LiAlH4 in anhydrous ether giving products with a TLC polarity increase consistent with the reduction of a carbonyl group. Visible spectra. The visible spectral data of CZX recorded in five organic solvents are shown in Table 1; remarkably, the spectral fine structure was completely lost with polar solvents while it was just partially present in n-hexane [λmax 478 and 499 (sh) nm]. The reduction of CZX with NaBH4 or LiAlH4, gave a product which absorbed at 418 sh, 440 and 467 nm (n-hexane), with an hypsochromic shift of 38 nm respect the parent compound. The spectra of KX and CXO showed the same complete loss of fine structure in polar solvents, while their fine structure was partially retained in n-hexane (Table 1).

3

Table 1. Absorbance (λmax) of CZX and reduced CZX compared with five ketocarotenoids quoted in the text. Abs. EtOH

n-hexane

C6H6

CS2

CHCl3

Ceratozamiaxanthin (CZX) Reduced CZX

478 418 sh, 440, 467

491 –

512 –

495 –

476 –

Semi-β-carotenone (SBC) Reduced SBC

466 420 sh, 443, 470

481 –

499 –

481 –

470 424 sh, 445, 473

464 –

477 –

494 –

478 –

465 443

Triphasiaxanthin (TX) Reduced TX

465, 485 sh 424 sh, 443, 472

482 –

499 –

482 –

470 –

Kuesteriaxanthin (KX) Reduced KX

464 sh, 475, 502 sh 422, 444, 473

488 –

508 –

491 –

473 –

Ceratoxanthone (CXO) Reduced CXO

456-7 sh, 475 416 sh, 440, 468

487

507

487

471

COMPOUND

Ceratoxanthin (CX) Reduced CX

H3C OH

HO

O 1,5-Dihydroxy-1,2,5,6-tetrahydro-β,ψ-caroten-6-one (ceratozamiaxanthin, CZX) C40H58O3 Exact Mass: 586.4386 Mol. Wt.: 586.8867 17

16

18

H3C OH

1

5

OH

19

18'

20

OH 3'

6

1'

O

20'

19'

16'

17'

3',4,5-Trihydroxy-1,2,5,6-tetrahydro-β,ψ-caroten-6-one (kuesteriaxanthin, KX) C40H58O4 Exact Mass: 602.4335 Mol. Wt.: 602.8861 H3C OH

O 5-Hydroxy-3,4-didehydro-1,2,5,6-tetrahydro-β,ψ-caroten-6-one (ceratoxanthone, CXO) C40H56O2 Exact Mass: 568.4280 Mol. Wt.: 568.8714

Figure 1. Possible structures of ceratozamiaxanthin (CZX), kuesteriaxanthin (KX) and ceratoxanthone (CXO). 4

Mass spectrometry. A C40H58O3 molecular formula of 586.4386 a.m.u. was in agreement with the accurate mass of CZX molecular ion (M+, m/z 586.4380) measured by high resolution positive-ion (+) electron-impact ionization (EI) mass spectrometry (MS). Analogous results were also obtained using fast atom bombardment ionization. The 70 eV EI(+) mass spectrum of CZX showed the most significant fragments at m/z 571, (M+ – CH3), 568 (M+ – H2O), 550 (M+ – 2H2O); also both the typical (M+ – 92) and (M+ – 106) peaks at m/z 494 and 480, respectively, were present with an abundance ratio R (= IM+ – 92/IM+ – 106)

of ~0.2, in good agreement with the characteristic low values of R reported for

many monocyclic or acyclic carotenoidic structures.12 The EIMS analysis, however, revealed the poor purity of the CZX sample that could be reached even with reiterate purification by TLC and HPLC, whereas M+ (m/z 586) with a reliable MS spectrum could only be attained within a narrow window of temperature (ca. 200 °C), after that the more volatile contaminants could have been evaporated at lower, progressively increasing probe temperature. At higher probe temperature other less volatile contaminants or degradation products were observed. Therefore a further purification of the CZX M+ was needed, which was performed by tandem-MS,4 for instance by the mass analyzed ion kinetic energy (MIKE) method: metastable and/or collisionally activated (CA) M+, dissociating in the second field-free region of a reversed geometry double-focusing mass spectrometer, showed a very strong water loss (m/z 568), the characteristic peaks at m/z 494 (M+ – 92) and 480 (M+ – 106) with the typical reversed abundance ratio R with respect to the normal mass spectrum,4 and both the structurally significant α-cleavages to the keto group at m/z 413 and 441, respectively. The MIKE spectrum of CZX M+ did not show a relevant second water loss; however a loss of water (m/z 550) could be observed in the MIKE spectrum of the (M+ – H2O) fragment (m/z 568), in agreement with the presence of two OH groups on the M+ structure of CZX.4 The CA-MIKE spectrum of (M+ – H2O) fragment (m/z 568) of CZX and of isobaric M+ of SBC showed different dissociation patterns.4 The 70 eV EI mass spectrum of reduced CZX with NaBH4 or LiAlH4 did not show any detectable M+ peak. However, operating in negative ion mode (see experimental), CZX by electron capture process gave an abundant molecular anion (M–, m/z 586) and the fragments: M– – H2O (m/z 568); M– – 2H2O (m/z 550) and the typical M– – 92 and M– – 106 (m/z 494 and 480, respectively) with an abundance ratio R of ~0.1, i.e. a low value as that observed in the 70 eV EI mass spectrum (see above). The reduced CZX sample gave a weak M– at m/z 588, showing an increase of two mass units with respect to CZX molecular

5

anion, and the fragments M– – H2O, M– – 2H2O and M– – 3H2O at m/z 570, 552 and 534, respectively (see Experimental). Mass spectra of samples of KX and CXO. The EI mass spectra (70 eV, 2 mA) of KX and CXO were recorded by direct introduction of the samples into the ion source kept between 220 and 240 °C without heating the probe. Compound KX gave a molecular peak at m/z 602.4328 (6%), consistent with the molecular formula C40H58O4 (calcd. 602.4335), and the fragments 584 (2%, M+ – H2O), 566 (1%, M+ – 2H2O), 550 (4%, M+ – 52), 510 (0.5%, M+ – 92) and 496 (3%, M+ – 106) with an abundance ratio R of ca. 0.17 (typical for monocyclic or acyclic carotenoids), 478 (1%, M+ – 106 – H2O), 443 (1%, M+ – 159), 429 (4%, M+ – C9H17O3), and 412 (1%, M+ – 190). Monoacetylated KX gave m/z peaks at 644 (M+), 626 (M+ – H2O), 552 (M+ – 92), 538 (M+ – 106), 523 (M+ – 121), and 471 (M+ – C9H17O3). The MIKE spectrum, performed as for CZX (see above), showed the following peaks: m/z 602 (94%, M+), 587 (8%, M+ – CH3), 585 (19%, M+ – 17), 559 (37%, M+ – C3H7), 545 (18%, M+ – C4H9), 531 (21%, M+ – C5H11), 510 (6%, M+ – 92), 496 (3%, M+ – 106), 463 (16%, M+ – C9H15O), 435 (22%, M+ – 167), 393 (100%, M+ – 209), and 337 (38%, M+ – C16H25O3). Compound CXO gave a molecular peak at m/z 568.4277 (28%), consistent with the molecular formula C40H56O2 (calcd. 568.4280) and fragments: 550 (13%, M+ – H2O), 531 (5%, M+ – 37), 523 (7%, M+ – 45), 509 (5%, M+ – 59), 476 (1%, M+ – 92) and 462 (10%, M+ – 106) with a low abundance ratio R of 0.10 (monocyclic or acyclic carotenoid), 441 (5%, C32H41O2, α-cheto cleavage), 430 (10%, M+ – 138), 413 (4%, M+ – C9H15O2). Spectrum by tandem MS showed significant peaks at m/z 568 (95%), 550 (50%, M+ – H2O), 525 (48%, M+ – C3H7), 476 (90%, M+ – 92), 462 (22%, M+ – 106), 441 (20%, M+ – 127), 413 (30%, M+ – C9H15O2). 1

H-NMR Spectroscopy. The powerful structural analysis capabability of multidimensional

and multinuclear NMR techniques was severely hampered by the obtainment of only very small amounts of non completely purified CZX sample. However, reasonable 1H-NMR spectra were recorded and a selection of the most significant bands (Table 2) were recognized tentatively by comparison of the observed chemical shifts with those of specific structural frames reported in Englert’s monograph13 as discussed below.

6

Table 2. Selection of 1H-NMR data of CZX. 1.39 (or 1.43) 1.17

1.98

1.72

1.98

2.03 1.60

OH

HO

1.46 1.18

O 1.98

7.60 (or 7.55)

1.03 1.04

1.98

Chemical shift (δ, ppm, int. ref. TMS)

Assignments and comments

1.03, 1.04

Two diasterotopic methyl groups at C-1’

1.17, 1.18

Two diasterotopic methyl groups at C-1

1.25

Broad singlet due to lipidic impurities

1.39 or 1.43

Methyl group at C-5 (uncertain assignment, see line below)

1.46

Broad multiplet, methylene group at C-2’. Chemical shift of CH2 groups at C-2 and C-3 is also expected in the range 1.41.5 (see ref. 13, structure e111)

1.60

Broad multiplet, methylene group at C-3’

1.72

Methyl group at C-5’

1.98

Methyl groups at C-9, C-9’, C-13, C-13’

2.03

Broad multiplet, methylene group at C-4’

7.60 or 7.55

Vinylic proton at C-8, namely β to carbonyl group and γ to COH (see ref. 13, structure e119)

Structure and discussion The ensemble of the chemical, chromatographic, MS, UV-Visible and 1H-NMR spectral data above let us to propose the reasonable structure of 1,5-dihydroxy-6-oxo1,2,5,6-tetrahydro-β,ψ-carotene (Fig. 1) for CZX. 17

HO 16

1

18

H 3C

OH *

19

18'

20

6

1'

5

O

20'

19'

16'

17'

Figure 2. Proposed structure for ceratozamiaxanthin (CZX), Y’b. Such a structure appears indeed consistent with all of the experimental data above. In detail, the relevant points of fitting are: (i) the C40H58O3 molecular formula determined by accurate mass measurements of M+; (ii) no acetylation reaction for the absence of primary or secondary hydroxylic groups; 7

(iii) the middle TLC polarity of native CZX and the polarity increase of the reduced product both supporting the presence of one conjugated C=O and two tertiary OH groups; (iv) EI (+) and (–) MS data showing: for native CZX relevant losses of H2O and 2H2O from both M+ and M-; the presence of both the typical (M-92) and (M – 106) peaks with low abundance ratios (R) of ~0.2, or 0.1, respectively, as for many monocyclic or acyclic carotenoidic structures;12 for reduced CZX the detection of a weak but significant M- with an increase of two mass units with respect to CZX M-, according to the reduction of one C=O group, and the fragments M– – H2O, M- – 2H2O and M– – 3H2O. (v) Tandem-MS (MIKE) data4 showing: from CZX M+ a very strong water loss (m/z 568), the characteristic peaks M+ – 92 and M+ – 106 with the typical reversed abundance ratio (R) with respect to the normal EI MS spectrum,4 and both the structurally significant αcleavages to the keto group at m/z 413 and 441, respectively. As expected these MIKE spectra did not show a relevant second water loss, whereas this could only be a consecutive process; in fact the MIKE spectra of the (M+ – H2O) fragment (m/z 568) of CZX did show a further loss of water (m/z 550), in agreement with the presence of two OH groups on the M+ structure of CZX.4 The (M+ – H2O) fragment (m/z 568) of CZX appeared to be isomeric of M+ of SBC, whereas their collision-activated dissociation patterns (CA-MIKE spectra) were different.4 Besides, we have not been able to obtain metastable or CA MIKE spectra of CZX M- probably because no metastable M- species could be generated by electron attachment while collisional activation of M- would easily induce an electrondetachment process leading to the neutral (and so undetectable) molecule. (vi) Visible spectral data of CZX recorded in five organic solvents (Table 1) showing: the spectral fine structure completely lost with polar solvents, just partially present with nhexane, as for a keto-conjugated structure. In spite of the same keto-conjugated chromophore of the proposed CZX structure as of those of SBC, CX and TX, the λmax values of CZX are always shifted to longer wavelenghts for a few nm with respect to those of other three compounds,1–3 while all the reduced products of CZX, SBC, CX and TX show very close λmax values; consequently the reduction of C=O induces a greater hypsochromic shift for CZX (37–38 nm, in n-hexane) than for the other three ketocarotenoids (20–22 nm, in n-hexane or ethanol).1–3 Reasonably, we would attribute both the longer wavelenghts of native CZX and the larger hypsochromic shift by C=O reduction, to specific effects (H-bonding or hyperconjugation) of the end-group on C-6, linked to the same keto-conjugated chromophore of the other three compounds. In the CZX proposed

structure

such

specific

effects 8

(in

particular

H-bonding)

could

be

straightforwardly induced by the tertiary OH on C-5, in α position to the keto group, not present in the end-groups of the other three compounds. For instance, a comparably large hypsochromic shift by a conjugated C=O reduction (~34 nm, in ethanol) had already been reported14 for ‘desmethylspheroidenone’ (1-hydroxy-3,4-didehydro-1,2,7’,8’-tetrahydroψ,ψ-caroten-2-one), which presents indeed a tertiary OH in α position to the keto group, conjugated to the chromophore, like for CZX. (vii) 1H-NMR spectroscopy data showing: (a) two pairs of diastereotopic methyl groups were clearly recognisable at 1.03, 1.04 and 1.17, 1.18 ppm. The former pair, unambiguously assigned to the non-equivalent geminal methyl groups on C-1’, does confidently support an unsubstituted β ring end-group, like for SBC and CX; the latter pair was assigned to the geminal methyl groups on C-1 of the proposed CZX structure (Fig. 1). In the latter case the assignment was supported by literature data on similar end-group (see ref. 13, compound e138). The doubling of signals due to geminal methyl group indicates the presence of a stereocenter in C-6, consistent with the proposed structure. (b) the neat singlet at 1.39 ppm can be assigned to a methyl group attached to C(5)-OH. A similar chemical shift (1.43 ppm) was reported for a structurally related compound (see ref. 13, structure e119) (c) the low field signals (7.55 or 7.60 ppm) suggest the existence of a H-C=C-(CO)-C(OH) partial structure, i.e. one vinylic proton in a 1,3 syn-coplanar relationship with a carbonyl group and in γ position with respect to an hydroxylated carbon atom. Similar values of chemical shifts are reported for structurally related reference compounds (see ref. 13, structure e119)

Experimental Extraction and isolation. Fresh red-brown leaflets of C. kuesteriana and of "forma fuscoviridis" were obtained from the collections of two Botanical Gardens of Naples and Florence Universities respectively. Limbs of both the Cycads were completely extracted and treated with the same procedure.1–5 The Y'-mixture, obtained as a TLC fraction of Rf ~0.48, was isolated from the extracts on semi-preparative silica gel layers with the classic developing mixture11 and continuously accumulated.1–5 For the successive steps of purification the Y'-mixture was subjected to silica gel column and the major fraction, eluted by Et2O/40-60° petroleum ether (4 : 1), was collected. A reversed phase HPLC system was eventually employed for 9

the separation of CZX from the Y'-mixture, for the preliminary identification and for the final purification of the samples for MS and 1H NMR analyses, as previously reported.1–3 A Spherisorb column S5 ODS2 (250 × 4.8 mm) was used with a linear gradient of 0 to 80% EtOAc in MeCN/H2O (9 : 1) at 1 ml/min flow. Positive (+) and negative (-) ion EI MS, and tandem-MS (MIKE)4,5 spectra were registered with a reversed geometry double-focousing VG-Micromass ZAB-2F instrument, fitted with a dual EI/chemical ionization (CI) ion source, heated progressively from room temperature to 220 °C; the samples were introduced directly into the ion source without heating the insertion probe. EI(+) mass spectrum of CZX (70eV, 2mA) showed the following significant peaks: m/z (% Rel. Int.): 586 (M+) (17), 571 (2), 568 (M+ – H2O) (7), 550 (M+ – 2H2O) (5), 494 (3), 480 (15), 412 (7), 69 (100). The tandem-MS (MIKE) spectra of both (M+) and (M+ – H2O) of CZX and the experimental details thereof had already been reported as for an unknown component4 of Y'-mixture; also the MIKE spectra of (M+) of SBC were reported4 for a comparison with the latter. EI(–) spectra of both CZX and its reduction product were obtained in CI mode using CO2 in the source at a pressure of ~10–4 torr as moderating gas for the electron capture process efficiency. Significant peaks, [m/z (% Rel. Int.)], of EI(–) spectra of: (i) CZX: 586 (M–) (70), 568 (M– – H2O) (100), 550 (M– – 2H2O) (57), 494 (4), 480 (65), 462 (95), 444 (28); and of (ii) CZX reduction product: 588 (M–) (3.5), 570 (M– – H2O) (15), 552 (M– – 2H2O) (32), 534 (M– – 3H2O) (14), 442 (100), 414 (53). Accurate mass measurements of CZX M+ were performed with a VG 70 SQ instrument set at 7,000 resolving power, equipped with an EI source, and with a Kratos MS-50 at 12,000 resolving power, with a fast atom bombardment source, using atomic Xe at 8KV and glycerol added with acetic acid as matrix. 1

H-NMR spectrum was recorded on a Bruker Avance 500 spectrometer at room

temperature and using CDCl3 as solvent. Chemical shifts (δ, ppm) were referenced to internal tetramethylsilane (TMS).

Aknowledgements We thank Prof. F. Fabbri and P.V.Arrigoni and Prof.G. Cellai Ciuffi (Botanical Garden of Florence University) for the supply of C. fuscoviridis, Prof. A. Moretti and, particularly, Prof. G. De Luca (Botanical Garden of Naples University) for the supply of C. kuesteriana,

10

Mr. Walter Panzeri (CNR) for technical assistance. This work was financially supported by Ministero della Pubblica Istruzione, 1994.

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2

F. Cardini, G. Britton, A. Selva (1989) Phytochemistry, 28, 2793.

3

F. Cardini, G. Britton, A. Selva, M. Chelli (1990) Biochem. System. Ecol., 18, 317.

4

A. Selva, F. Cardini (1993) Org. Mass Spectrom., 28, 570.

5

A. Selva, F. Cardini, M. Chelli (1994) Org. Mass Spectrom., 29, 695.

6

A. Moretti, S. Sabato, M. Vazquez Torres (1982), Brittonia, 34, 185.

7

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8

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9

L. Morassi Bonzi, P. Medeghini Bonatti, C. Paoletti, L. Pazzagli, G. Cappugi, M.A. Pancani, F. Cardini (1996) J. Plant Physiol., 149, 663.

10

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11

Eichenberger, E.C. Grob (1962) Helv. Chim. Acta, 45, 974.

12

C.R. Enzell, I. Wahlberg (1980) in Biochemical Applications of Mass Spectrometry (G.R. Waller, O.C. Dermer, eds.), p. 407, Wiley, New York.

13

G. Englert (1995) in Carotenoids, Vol. 1B, Spectroscopy (G. Britton, S. Liaaen-Jensen, H. Pfander, eds.), Birkauser Verlag, Basel.

14

J. Manwaring, E.H. Evans, G. Britton, D.R. Schneider (1980) FEBS Letters, 110, 47.

11