Chlorophyll a and carotenoids of the red alga Erythrotrichia carnea

Biochemical Systematics and Ecology, Vol. 12, No. 3, pp. 279-283, 1984. Printed in Great Britain.

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Chlorophyll a and Carotenoids of the Red Alga Erythrotrichia camea TERJE BJ(Z)RNLAND Department of Marine Biology and Limnology, Section of Madne Botany, University of Oslo, P.O. Box 1069 Blindern, Oelo 3, Norway

Key Word I n d e x - Erythrotrichia carnea; Rhodophycaae; chlorophyll; carotenoids; antheraxenthin; chemowstematics. Abstract - Unialgal, cultured material of the red alga Erythrotn'chia carnea (subclass Bangiophycidae) was found to contain chlorophyll a while chlorophyll d was absent. All carotenoids belonged to the. ~,~-sedes: ~,/~-carotene, ~-cryptoxanthin, zeaxanthin and antheraxanthin. W'rthin the Rhodophyceae this carotenoid composition reflects both pdmitive (/],/J-carotenoids only) and advanced (td-oxygenated carotenoid) features. Within the Rhodophycaae these carotenoid characteristics may not be correlated with chloroplast morphology or with systematic position on the subclass level.

Introduction The carotenoids of red algae are structurally simple in comparison to those of the majority of other algal classes [1-3]. Derivatives of both/3,/Icarotene and/~,£-carotene are present, but in the majority'of species the biochemical elaboration ends at the zeaxanthin and lutein level. Only exceptionally do species possess the ability to synthesize the epoxidized derivatives, antheraxanthin (zeaxanthin-5,6-epoxide) or taraxanthin (lutein-5,6-epoxide). Until now the few reports on such a divergent carotenoid composition refer to species of the subclass Florideophycidae (see [4, 5], systematics according to [6]). The majority of analyses of red algal carotenoids have been done on naturally occurring material. Unfortunately this is often contaminated with epi- or endophytic algae and invertebrates. As the carotenoid content is low in most red algae, even small amounts of biological contaminants may contribute considerably to the total carotenoid amount. The claimed presence of trace carotenoids in such material should therefore be considered with some caution (see [4, 7]). Here is reported the first isolation of an epoxy dihydroxy carotenoid from a member of the red algal subclass Bangiophycidae. The sample examined was obtained from a unialgal laboratorygrown mass culture. (Received for publication 14 October 1983) 279

Results and Discussion The qualitative and quantitative content of chlorophyll and carotenoids in Erythrotrichia carnea is shown in Table 1. As compared with other red algae, E. camea is a relatively rich source of these compounds on a dry weight basis. The chlorophyll pigmentation (chlorophyll a only) and the low proportion of carotenoids in relation to chlorophyll a were in agreement with previous results [4]. The results obtained differ, however, both qualitatively and quantitatively from previously published data on the carotenoid composition of E. camea [8]. Qualitatively, the most apparent TABLE 1. CONTENTOF CHLOROPHYLLa AND CAROTENOIDSIN E.. CARNEA Pigment

Amount mg/g

Chlorophylla t

5.7

/],/I-Carotene /]-CryDtoxanthin Zeaxanthin Antheraxanthin

0.39 0.11 0.05 1.00

Absorption maxima* III/n %

(nm)

(%)

25 7 3 65

(384),(413). 430 574, 615, 663 (428), 449, 478 (425), 446, 473 (427), 448, 478 421, 443, 472

40 33 32 67

Totalcarotenoids1. 1.87 Total carotenoids1" 0.33 Chlorol:)hyll 1" *Solvents-chlorophyll a: Me2CO; individual carotenoids: light petroleum. 1.Amount basedon the UV-visiblespectrum of the total pigment extractin Me2CO.

280

TERJE BJORNLAND

difference was the eadier tentative identification of lutein [8], which is probably a misidentification of antheraxanthin. ~-Cryptoxanthin was not detected by Allen eta/. [8]; this may be explained by the extremely small amounts of pigment obtained in that work (total carotenoids: 40/~g). The most striking quantitative differences were the total content of carotenoids per gram dry weight (0.2 versus 1.87 mg in the present work), and the relative amounts of zeaxanthin and lutein (read: antheraxanthin) (40.5 and 30.5% versus 3 and 65% in the present work). The carotenoids of E. carnea all belong to the p,/]-series. They constituted a reasonable biochemical chain of increasing oxidation level: p,/3carotene, ~J-cryptoxanthin, zeaxanthin and antheraxanthin. The corresponding derivatives within the/3,£-series were absent, as revealed by the combined use of two TLC plates [4] (see Experimental).

o

HO ~x~,.

b

. d

HO .

Ant.heroxonthin : e-R~-c B , / ~ - c a r o t e n e :o-R,-o B, = - corotane : o-R~-b e-cryptoxonthin : c-R~-b - crytoxonthin : c-Rl-o L u t e i n : c-Ri-d Mutot.oxon~.hin : f-Rz- c T o r a x o n t h i n : e-R,-d V i o L o x o n + , h i n : e-R~-e Z a o x 0 n t h i n : c - R I- c

c

. e

/3,p-Carotene,/~-cryptoxanthin and zeaxanthin possess UV-visible properties consistent with nine in-chain and two endo-cyclic double bonds. Their identities were further supported by the m / z of the molecular ion and the mass spectral degradation products [9]. p-Cryptoxanthin and zeaxanthin possess one and two primary/secondary hydroxyl groups respectively, as revealed by acetylation and subsequent MS of their acetates. The identity of fJ,/~-carotenewas further confirmed by ~H NMR. The 1H NMR data for p-cryptoxanthin and zeaxanthin will be presented in a separate paper on the chirality of red algal carotenoids [10]. Antheraxanthin possessed UV-visible spectral properties typical for carotenoids with nine inchain and one endo-cyclic double bonds. The m / z of 584 indicated a three-oxygenated xanthophyll. Two of the oxygen functions were primary/secondary hydroxyl groups as

H O ~ ' , , I I

0

f

RI

Ra RG. 1. CAROTENOID STRUCTURES. The chirality of red alga carotenoids has been as.sumed to be the same as in other biological material [27]. This assumption is valid for the red alga carotenoids examined for chiroptic properties until now (I0].

CAROTENOIDS OF ERYTHROTRICHIACARNEA

demonslxated by acetylation (kinetics followed by PC) and subsequent MS of the final acetylation product. The oxidation test with p-CI-anil and Nalight [11] was negative and excluded allylic positions for the hydroxyl groups. The third oxygen atom was present as an epoxide group, as demonstrated by the yellow-green colour when the xanthophyll was treated with HCI in Et20 [12]. Further support for its epoxidic nature was obtained from the MS of antheraxanthin itself and its diacetate, notably the prominent fragments at m / z 504, 221 and 181 (588, 263 and 223 for the diacetate) [9]. In addition, treatment of antheraxanthin ex E. carnea with 1% c'rtric acid in Et20-MeOH [12] gave a more polar furanoid rearrangement product (mutatoxanthin) Which behaved chromatographically homogeneously on two supports, but split into two components on the third (TLP-II) (compare [15, 16]). Similar treatment of antheraxanthin from anthers of Lilium tigrinum gave an identical mixture of mutatoxanthins (UV-visible and MS). Acetylation of each of the two mixtures was followed by PC, and was found identical in respect of kinetics as wall as polarity of intermediates and final product. Antheraxanthin ex E. carnea was, however, slightly less polar than antheraxanthin from anthers of L. tigrinum When co-chromatographed on TLP-I, but co-chromatographed with antheraxanthin ex Lactuca sativa and green grass in the same system. This discrepancy may arise from cis-trans isomerism as epoxidic xanthophylls from flower parts are often present as cis-isomers [13-15]. IR of a crystalline sample ex E. carnea showed no bends Which suggested anything other than an antheraxanthin structure. Recent studies (including CD and 1H NMR) have established its structure as all-/Tans antheraxanthin [10]. As a member of the subclass Bangiophycidae, E. carnea possesses several features considered to be primitive within the Rhodophyceae (see [6]). Most relevant for the following discussion is the occurrence of a single, centrally located chloroplast with a stellate appearance. This archaic cell organization is common within the Bangiophycidae but is rare within the Florideophycidae. In the latter subclass it is restricted to the Nemaliales, the most primitive order of the Florideophycidae. The chloroplasts of E. carnea possess caro-

281

tenoid pigmentation charectadzed by the following two features of potential chemosystematic and phylogenetic importance: Rmtly, all carotenoids belong to the/3,/3-series while representatives of the p,E-series are cornpletely lacking. Both biochemically and when compared with the majority of other red algae inveslJ~ted for carotenoids (see [3]), this represents a simple carotenoid pattam. An attempt to correlate this feature with systematic position (Bangiophycidae versus Florideophycidea) and chloroplast morphology turns out to be negative: within the Bangiophycidae a carotenoid complement restricted to the /],/3-series has been found in the genera Asterocy~ [19] and Porphyridium [19, 20]. However, this simple carotenoid feature is also possessed by soma members of the Florideophycidae, v/z. species of Gracilaria (Gigartinales) [5, 21], Acanthophora spic/fera (Ceremialea) [21] and Polysiphonia brod/aei and P. urceolata (Ceramiales) [4]. The trace amounts of lutein reported in A. spicifera [21] may probably be ascribed to biological contaminants in the naturally occurring material (see Introduction). As to chloroplast morphology, this carotenoid feature is not restricted to stellate chloroplasts. While both Asterocytis and Porphyridium have a chloroplast which is stellate, all the genera of the Rorideophycidae mentioned above should, according to their systematic position within this subclass, have non-stellate chloroplasts (compare [6]). Furthermore, species with a stellata chloroplast may also contain carotenoids of the /~],~-series as shown for both naturally occurring and unialgally cultured Bangia fuscopurpurea (subcJaas Bangiophycidae) [4]. Secondly, the carotenoids of E. carnea have been biochemically evolved beyond the dihydroxy level, as the main xanthophyll also possesses a 5,6-epoxide moiety. In this respect, the carotenoid pigmentation may be considered as advanced, and E. carnea is the first representative of the subclass Bangiophycidae reported to contain an epoxidized dihydroxy xanthophyll. Previously, antheraxanthin has been detected within the Acanthophora [21] and Gracilaria [5, 21] of the Florideophycidae, in several cases also together with the di-epoxidized xanthophyll violaxanthin. Tarexanthin, the f],£-analogue of antheraxanthin, has been reported from Gelidium corneum (Gelidiales), Grateloupia filicinus and G.

282

proteus (Cryptonemiales) and Schizymeniadubyi (Gigartinales) [22] (see also [4]), but its identity has still not been established with modem physical methods. Accordingly, the 5,6-epoxide slzucture element has a scattered occurrence within the Rhodophyceae and may be correlated neither with systematic position at the subclass level nor with chloroplast morphology. In conclusion, care should be taken not to construct far reaching phylogenetic lines within the Rhodophyceae based on the two carotenoid features discussed above (compare [5, 21]). Experimental Erythrottichia carnea (Dillw.) J. Ag. (class: Rhodophyceae; subclass: Bangiophycidso) from the Osiofjord, Norway, was brought into uniaigal culture by Jan Rueness. Mass cultures for pigment analyses were grown in 100 polystyrene dishes (Heger Plastics A/S, type 10100) at 18°.-Fhe light sources were equal numbers of Philips fluorescent tubes TL/33 and TL/68. The light intensity was 11 ~E m - 2 s- 1 as measured with an L1-188integrating quantum photometer fitted with an LI-190s cosinus sensor (Lambda Instr. Corp.), and the LD photoperiod was 14:10. Each culture dish contained about 140 ml of enriched 20%0 seawater medium [23]. The medium was renewed weekly. 245 g fr. wt (34.8 g dry wt) of alga were harvested after 10 months. The fresh algal material was extracted in a Warring blendor with Me2CO and MeOH-Me2CO (3:7). The extracted pigments were separated by TLC on silica gel G-CaCO3 (1:1 ) (TLP-I) with petroI-Me2CO-C6Hc-iso-PrOH (78:20:4:1) as developing solvent. Each of the carotenoid fractions obtained was rechromatogrephed on silica gel G-Ca(OH)2MgO-CaSO4 (10:4:3:1) (TLP-II) with an appropriate mixture of light petroleum, Me2CO and iso-PrOH as a developing solvent. Authentic carotanoids for co-chromatography were isolated from the following sources: /],/]-carotene and /],~carotene: Deucua carota; o-cryptoxanthin: Capsicum annuum flavum; /]-cryptoxanthin and zeaxanthin: calyx of Physalls alkekengi; luteln: Medicago satire; antheraxanthin: Lactuca saliva and green grass (total extracts) and anthers of L#ium tigdnum. Chlorophyll a was isolated from Hordeum vulgare. Co-chromatography was carried out on TLP-I (chlorophyll a and carotenoids), TLP-II (carotenoids) and on paper Schleicher Et SchOll No. 287 (SEtS 287) [24] (chlorophyll a and acetylation products of antheraxanthin and mutatoxanthin). UV-visible spectra were recorded in Me,CO (total extract and chlorophyll a) and n-CsH14, light petroleum or Me2CO (carotenoids). E]%cm-valuesapplied for quantitative calculations were: 890 (chlorophyll a) [26], 2020 (total carotanoids, absorption maximum at 478 nm for the total extract) and 2500 (TLC-pure carotenoids). The IR spectrum was recorded in a KBr disc, mess spectra were obtained at 70 eV and 190-210 ° and 1H NMR at 200 MHz (CDCIs, TMS). The epoxide test and the acid catalysed epoxide-furenoid rearrangement were carried out with HCI in Et20 and with 1% citric acid in Et20-MeOH, respectively [12], acetylation

TERJE BJQRNLAND with Ac20 in dry pyridine [26] and allylic oxidation with p-CI-anil under Na-light [11]. Chlorophyll a. UV-visible x~M~jxCOnm: (384), (413), 430, 574, 815 and 663. p,/]-Carotene. UV-visible X~P~x nm: (426), 449 and 478; III/11 (%)=40. MS m/z (rel. int.): 536 [M] + (100%), 444 (9) and 430 (1). 1H NMR (200 MHz, CDCI3, TMS): dl.03s (12H, Me-l, 1, 1', 1'), 1.72s (6H, Me-5, 5') and 1.97s (12H, Me-9, 13, 9', 13'). /]-Cryptoxanthin. UV-visible ~P~x nm: (425), 446 and 473; III/11 (%)=33. MS m/z (rel. int.): 552 [M] + (100%), 534 (0.8), 460 (21) and 446 (1). /]-Cryptoxanthin-3-acetate. Partially synthetic from pcryptoxanthin. UV-visible iMe2Co nm: (429), 451 and 476; III/11 (%)= 17. MS m/z (rel. int.): 594 [M] + (100%), 534 (4), 502 (11), 488 (1), 442 (10) and 426 (0.9). Zeaxanthin. UV-visible X~P~°= x nm: (427), 448 and 475; III/11 (%)=32. MS m/z (ref. int.): 568 [M] + (100%), 550 (0.8), 476 (17) and 452 (1). Zeaxanthin-3,3'-diacetate. Partially synthetic from zeaxanthin. MS m/z (rel. int.): 652 [M] + (100%), 592 (8), 560 (5), 546 (1), 532 (1), 500 (9), 486 (0.8), 440 (5) and 426 (0.2). Antheraxanthin. UV-visible X~t~ nm: 421,443 and 472; III/11 (%)=57. IR ]Ker -r~x cm-1: 3400s (OH), 3040w, 2660s, 2930s and 2630m (CH), 1680w, 1570w (C=C), 1450m (ring-CH2), 1368s (gem.Me), 1185w (C-O), 1150w, 1125w, 1050s (C-O), 1010w, 968s (C=C), 840w (trisubstituted C=C), 810w and 710w. MS m/z (tel. int.): 584 [M] + (100%), 582 (2), 568 (4), 566 (4), 504 (29), 492 (9), 478 (1), 438 (6), 352 (17), 221 (40) and 181 (17). Antheraxanthin-3,3"-diacetate. Partially synthetic from entheraxenthin. UV-visible ~mM~ax c° nm: (428), 448 and 477; III/ll (%) = 59. MS m/z (rel. int.): 668 [M] + (44%), 666 (0.7), 652 (0.6), 608 (6), 588 (29), 576 (10), 562 (0.8), 548 (0.5), 528 (2), 516 (2), 263 (26), 223 (10) and 43 (100). Mutatoxanthin. Partially synthetic from antheraxanthin (2.0 mg) ex E. carnea. The reaction product was homogeneous on TLP-I and paper SEtS 287. UV-visible: ~'maxr~hexane nm: (406), 426 and 453; III/11 (%)=61. By chromatography on TLP-II with petroI-Me2CO-MeOH (55:42:3) as a developing solvent the reaction product split into two components: Mutetoxanthin I: yield, 17%; Rf 0.17; UV-visible xM~xCOrim: (402), 426 and 451; III/11 (%) =45; MS m/z (rel. int.): 584 [M] + (14%), 566 (9), 550 (4), 504 (14), 492 (4), 486 (3), 221 (59), 181 (36) and 43 (100). Mutatoxanthin I1: yield, 14%; Rf0.11; UV-visible xMx~::Orim: (402), 428 and 453; III/11 (%) =43; MS m/z (rel. int.): 584 [M]+ (12%), 566 (9), 550 (4), 504 (13), 492 (4), 486 (2), 221 (62), 181 (30) and 43 (100). Mutatoxanthin. Partially synthetic from entheraxanthin (0.68 mg) ex anthers of L. tigrinum. Mutatoxanthin I: yield, 15%; UV-visible Z~P~°xi nm: 403, 425 and 452; III/11 (%)=65; MS (contaminated) m/z (rel. int.): 584 [M] + , 504 and 492. Mutatoxanthin II: yield, 12%; UV-visible ,petrol nm: 403, 425 ",msx and 451; III/II (%)=62; MS (contaminated) m/z (rel. int.): 594 [M] + , 504 and 492.

Acknowledgements - A specimen culture of E. camea was provided by Jan Rueness, Section of Madne Botany, Universiw of Oslo, who also gave valuable suggestions conceming the culture conditions and commented on the

CAROTENOIDSOF ERYTHROTRICHIACARNEA botanical part of the manuscript. SynnCve Liaaen-Jensen and Arna Jensen, Norwegian Institute of Technology, University of Trondheim, are acknowledged for valuable discussions during part of the study. 1H NMR was recorded at NAVF's National NMR Laboratory, University of Oslo, and MS partly at the Department of Chemistry, University of Oslo and partly at the Norwegian Institute of Technology, University of Trondheim. The project was financially supported by Niders Forskningsfond and the Norwegian Council for Science and Humanities (NAVF) (grant D.50. 42-13).

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