Feddes Repertorium 116 (2005) 3 – 4 , 195 – 200 DOI: 10.1002/fedr.200411068 Weinheim, August 2005 Medical University, Department of General Biology, Białystok Universidad Nacional del Comahue, Bariloche B.CZECZUGA; E.CZECZUGA-SEMENIUK; S. CALVELO & S. LIBERATORE Carotenoids in representatives of the Protousnea (Parmeliaceae), endemic genus from South America With one Figure and 2 Tables Summary Zusammenfassung Column, thin-layer and reverse-phase high-performance liquid chromatography revealed the presence of the following carotenoids in the thalli of 7 lichen species representatives of the Protousnea (Parmeliaceae) endemic genus from South America: α-carotene, β-carotene, retrodehydro-β-carotene, α-cryptoxanthin, β-cryptoxanthin, lycopene-5,6-epoxide, lutein, 3′-epilutein, zeaxanthin, antheraxanthin, cryptoflavin, loroxanthin, capsochrome, neoxanthin, violaxanthin, 3-hydroxyechinenone, adonixanthin, canthaxanthin, astaxanthin, rhodoxanthin, citranaxanthin and torularhodin methyl ester. The total content of carotenoids ranged from 32.45 (Protousnea dusenii) to 48.21 µg g–1 dry weight (Protousnea teretiuscula). Carotinoide in Vertretern der Protousnea (Parmeliaceae), einer in Südamerika endemischen Gattung Introduction In the study on carotenoids in the thalli of various lichen species from southern part of South America (CZECZUGA & FERRARO DE CORONA 1987; CZECZUGA & CALVELO 1994, 1995, 1996; CZECZUGA et al. 1991, 1999a, 2003) we established the presence of a number of rare carotenoids. Particularly interesting were the results concerning the thalli of some Pseudocyphellaria species from Argentina (CZECZUGA et al. 1999a). The genus Protousnea (MOTYKA) KROG has a fruticose and generally pendulous thallus. It is Untersuchungen mittels Säulen-, Dünnschicht- und Hochleistungs-Flüssig-Chromatographie erbrachte den Nachweis folgender Carotinoide in den Thalli von sieben repräsentativen Arten von Protousnea (Parmeliaceae), einer in Südamerika endemischen Gattung: α-Caroten, β-Caroten, Retrodehydro-β-Caroten, α-Cryptoxanthin, β-Cryptoxanthin, Lycopen5,6-epoxid, Lutein, 3′-Epilutein, Zeaxanthin, Antheraxanthin, Cryptoflavin, Loroxanthin, Capsochrom, Neoxanthin, Violaxanthin, 3-Hydroxyechinenon, Adonixanthin, Canthaxanthin, Astaxanthin, Rhodoxanthin, Citranaxanthin und Torularhodin Methyl Ester. Der Gesamt-Carotin-Gehalt reicht von 32,45 µg–1 (Protousnea dusenii) bis 48,21 µg–1 Trockengewicht (Protouesnea teretiuscula). an endemic genus from south-western South America and the Islas Malvinas (KROG 1976). Seven species are known up to now, all of which have been reported from Argentina (CALVELO & LIBERATORE 2002). The present report is a supplement to study on carotenoids in lichens from South America. Materials and methods The seven lichen species namely Protousnea alectoroides (MOTYKA) KROG, P. dusenii (DU RIETZ) KROG, P. magellanica (MONT.) KROG, P. malacea © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0014-8962/05/3-408-0195 196 (STIRT.) KROG, P. poeppigii (NEES & FLOT.) KROG, P. scrobiculata (SAMBO) KROG and P. teretiuscula KROG were collected from the south-western Argentina. They grow as epiphytic on branches of Nothofagus spp. or Austrocedrus chilensis trees, and occasionally on shrubs of the understory, in southern South America forests. The species of investigated lichens are deposited in the Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, 8400 Bariloche RN Argentina. The carotenoid pigments were isolated using column chromatography (CC), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Prior to chromatography, the material was homogenized with acetone under nitrogen in dark glass bottles and the extracts kept in a refrigerator until analysed. Saponification was carried out with 10% KOH in ethanol at 20 °C for 24 h in the dark under nitrogen. Column and thin-layer chromatography (CZECZUGA 1985; KRAUS & KOCH 1996) were used to separate the carotenoids, which were identified by comparison with standard compounds by a) the behaviour on column chromatography; b) their UV-VIS spectra (Beckman 2400); c) their partition between n-heksane and 95% ethanol; d) their Rf-values on thin-layer chromatography; e) the presence of allylic OH-group determined by the acid CHCL3 test; f) the epoxide test and g) the mass Feddes Repert., Weinheim 116 (2005) 3 – 4 spectrum (cf. VETTER et al. 1971). Carotenoid pigments were determined also by ion-pairing, reversephase HPLC. To 1000 µl of the clear extract, 300 µl of ion-pairing reagent was added according to MANTOURA & LLEWELLYN (1983). The HPLC equipment consisted of Shimadzu LC-6A double-system pump, driven by a gradient programmer Shimadzu SCL-6B and Rheodyne 7125 injector equipped with a 20 µl loop. Detection was by a Shimadzu SPD-6AV UVVIS spectrophotometric detector set on 440 nm and Shimadzu RF-535 fluorescence detector. Carotenoids as pigment standards were obtained from Hoffman La Roche Company, Switzerland, International Agency for 14C Determinations, Denmark and Sigma Chemical Company, USA. Quantitative determinations were determined by UV, VIS spectroscopy. For the structures of carotenoids see STRAUB (1987) and CZECZUGA (1988). Results In the thalli of seven species of lichens from the south-western Argentina, 22 carotenoids were identified (Fig. 1; Table 1), which had previously been noted in the thalli of many other lichen species from South America ex- Fig. 1 Structural features of carotenoids from investigated materials (see Table 1) © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 197 B. CZECZUGA et al.: Catotenoids in Protousnea from South America Table 1 List of the carotenoids from the investigated materials Carotenoid Summary formula Structure (see Fig. 1) Semisystematic name C40H56 C40H56 C40H56 C40H56O C40H56O C40H56O C40H56O2 C40H56O2 C40H56O2 C40H56O3 C40H56O3 C40H56O3 C40H56O4 A-r-B B-r-B A-r1-A A-r-C B-r-C D-r-E C-r-F C-r-F C-r-C C-r-G B-r2-H F-r3-I H-r2-K 14. neoxanthin C40H56O4 G-r2-L 15. violaxanthin C40H56O4 G-r-G 16. 17. 18. 19. 20. C40H54O2 C40H54O3 C40H52O2 C40H52O4 C40H50O2 B-r-M C-r-M N-r-N M-r-M O-r1-O C33H44O C41H54O2 B-r2-P B-r-R ε,ε-Carotene β,β-Carotene 4′,5′-Didehydro-4,5′-retro-β,β-carotene β,ε-Caroten-3-ol β,β-Caroten-3-ol 5,6-Epoxy-5,6-dihydro-ϕ,ϕ-carotene β,ε-Carotene-3,3′-diol β,ε-Carotene-3,3′-diol (stereisomeric) β,β-Carotene-3,3′-diol 5,6-Epoxy-5,6-dihydro-β,β-carotene-3,3′-diol 5,8-Epoxy-5,8-dihydro-β,β-caroten-3-ol β,ε-Carotene-3,19,3′-triol 5,8-Epoxy-3,3′-dihydroxy-5,8-dihydroβ,x-caroten-6′-one 5,6′-Epoxy-6,7-didehydro-5,6,5′,6′-tetrahydroβ,β-carotene-3,5,3′-triol 5,6,5′,6′-Diepoxy-5,6,5′,6′-tetrahydroβ,β-carotene-3,3′-diol 3-Hydroxy-β,β-caroten-4-one 3,3′-Dihydroxy-β,β-caroten-4-one β,β-Carotene-4,4′-dione 3,3′-Dihydroxy-β,β-carotene-4,4′-dione 4′,5′-Didehydro-4,5′-retro-β,β-carotene3,3′-dione 5′,6′-Dihydro-5′-apo-18′-nor-β-caroten-6′-one Methyl-3′,4′-didehydro-β,ϕ-caroten-16-oate 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. α-carotene β-carotene retrodehydro-β-carotene α-cryptoxanthin β-cryptoxanthin lycopene-5,6-epoxide lutein 3′-epilutein zeaxanthin antheraxanthin cryptoflavin loroxanthin capsochrome 3′-hydroxyechinenone adonixanthin canthaxanthin astaxanthin rhodoxanthin 21. citranaxanthin 22. torularhodin methyl ester Table 2 Carotenoid distribution in lichens Species Carotenoid (see Table 1 and Fig. 1) Major carotenoid (%) Total (µg g–1) dry weight Protousnea alectoroides (MOTYKA) KROG Protousnea dusenii (DU RIETZ) KROG Protousnea magellanica (MONT.) KROG Protousnea malacea (STIRT.) KROG Protousnea poeppigii (NEES & FLOT.) KROG Protousnea scrobiculata (SAMBO) KROG Protousnea teretiuscula KROG 1,2,4,5,7,10,13,15,16,18,19,21 15(24.12) 41.18 2,3,5,7,9,10,12,15,17,19,21 2,4,5,7,10,11,14,15,17,19,22 15(20.08) 15(18.46) 32.45 38.25 1,2,5,7,8,10,13,15,16,18,19 2,3,5,7,9,10,12,15,17,19,21 15(29.17) 15(17.95) 45.08 40.34 2,4,5,7,10,11,14,15,18,19,22 15(30.14) 39.72 2,5,6,7,8,10,12,15,19,20,21 15(21.02) 48.21 Constant carotenoids 2,5,7,10,15,19 ception retrodehydro-β-carotene, α-cryptoxanthin, loroxanthin, citranaxanthin, and torularhodin methyl ester. Β-Carotene, β-cryptoxanthin, lutein, antheraxanthin, violaxanthin and astaxanthin were found in all investigated species of Protousnea (Table 2). The total carotenoid content in the investigated materials ranged from 32.45 (Protousnea dusenii) to 48.21 µg g–1 dry weight (Protousnea teretiuscula). © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 198 Discussion In the thalli of seven species of Protousnea endemic genus from South America, the presence of 22 carotenoids was established. This all carotenoids were found already in other species of Parmeliaceae family (CZECZUGA 1980; CZECZUGA et al. 1999b, 2000). As regards of the groups of rare carotenoids, they have been found occasionally in investigated lichens. The most noteworthy are retrodehydro-β-carotene, α-cryptoxanthin, loroxanthin, cytranaxanthin and torularhodin methyl ester. Retrodehydro-β-carotene is carotenoid characteristic of fungi (GOODWIN 1980). This carotenoids, also termed isocarotene belongs to the group of hydrocarbons (STRAUB 1987) and react with acids. The proton is eliminated from the vinylogous position at the opposite end of the polyene chain so that, for example, isocryptoxanthin is converted into the retro-derivative, retrodehydro-β-carotene (GOODWIN 1980). Dehydro-β-carotene is present in the fungus Epicoccum nigrum (FOPPEN & GRIBANOVSKI-SASSU 1969). Isocarotene was detected in the thalli of Parmelia glabra from Polizzi Generosa in Sicily, collected from Corylus avellana at an altitude of 1010 m (CZECZUGA et al. 1999b). As regards α-cryptoxanthin, it is a derivative of α-carotene and like this carotene, α-cryptoxanthin has rarely been found in lichens to date. We found α-cryptoxanthin in the thalli of Parmelia sulcata in the Białowieska Forest (CZECZUGA 1994). Loroxanthin belongs to the group of polyhydroxy compounds and was first described from unicellular green algae Chlorella vulgaris and Scenedesmus obliquus later from Cladophora trichotoma (STRAIN 1958; AITZETMÜLLER et al. 1969), and then found in other algae (NITSCHE 1974). This carotenoid has been identified as a derivative of lutein in which the C-19 methyl group has been converted into hydroxymethyl (WEEDON 1971). In the lichens from Sicily, loraxanthin was also encountered in the thalli of Parmelia conspersa collected from basaltic rock in Buccheri at an altitude of 400 m (CZECZUGA et al. 1999b). Citranaxanthin belongs to group of apocarotenales. This carotenoid was first isolated from the trigeneric hybrid Sinton cirangequat (of Fortunella margarita with Poncirus trifo- © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Feddes Repert., Weinheim 116 (2005) 3 – 4 liata × Citrus sinensis) (YOKOYAMA & WHITE 1965, 1966). We detected citranaxanthin in the thalli of Cladonia fimbriata, Diploschistes scruposus and Parmelia pulla from Sicily (CZECZUGA et al. 1999b) and in the thalli of Umbilicaria esculenta from Yunnan in China collected from rocks at an altitude of 3800 m (CZECZUGA et al. 2000). Citranaxanthin was also found in the thalli of Pseudocyphellaria corrifolia from the Nahuel Huapi National Park in Argentina, altitude 1350 m (CZECZUGA et al. 1999a). The former originated as a result of βcarotene degradation in natural conditions, the latter due to β-cryptoxanthin degradation. Torularhodin methyl ester is derivative of torularhodin, the pigment of the red yeast Torula rubra (LEDERER 1934), and this last rare carotenoid was detected in the thalli of Parmelia conspersa collected on Etna at an altitude of 1100 m from epilithic mosses (CZECZUGA et al. 1999b). Torularhodin originates as a result of multiple transformations of torulene (GOODWIN 1980). Torularhodin itself occurs both in lower (ARPIN & LIAAEN-JENSEN 1967) and higher fungi (CZECZUGA 1978, 1979). The carotenoids found to be common to seven species of Protousnea from southern South America forests were found β-carotene, β-cryptoxanthin, lutein, antheraxanthin, violaxanthin and astaxanthin. In all investigated specimens of Protousnea species violaxanthin was found to occur in large amounts. This carotenoid variety is likely to be associated with such environmental factor as insolation. The contents of violaxanthin and zeaxanthin are known to change under varying intensites of light. Under strong light the zeaxanthin content increases and the violaxanthin decreases, whereas in shady conditions the relative concentrations are reversed. So this is termed violaxanthin or xanthophylls cycle which is observed in higher plants but is also characteristic to lichens from green algae as phycobionts (CZECZUGA et al. 2004a). Violaxanthin is formed from zeaxanthin via antheraxanthin. Insolation environmental factor also increases to other carotenoids and on total content. Our previous studies demonstrated that the presence of β-carotene or lutein in large amounts is associated with poor lighting, that is, in shaded areas. In the thalli from the shadier sites the total carotenoid content was also higher. A B. CZECZUGA et al.: Catotenoids in Protousnea from South America similar observation was made in lichen species from other families and applied not only to carotenoids but also to chlorophylls in phycobionts (CZECZUGA 1988, 1993; CZECZUGA et al. 2004 c). As regards of the groups of ketocarotenoids, they have been found in investigated lichens. There are such as 3′-hydroxyechinenone, adonixanthin, canthaxanthin and astaxanthin. Adonixanthin is a carotenoid which is rare in lichens. All other ketocarotenoids from this group are present more often in lichens (CZECZUGA 1988; 1993; CZECZUGA et al. 2004b). References AITZETMÜLLER, K.; STRAIN, H. H.; SVEC, W. A.; GRANDOLFO, M. & KATZ, J. J. 1969: Loroxanthin, a unique xanthophyll from Scenedesmus obliquus and Chlorella vulgaris. – Phytochemistry 8: 1761 – 1770. ARPIN, N. & LIAAEN-JENSEN, S. 1967: Recherches chimiotaxinomiques sur les champignons. III. Fungal carotenoids. – Phytochemistry 6: 995 – 1005. 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Addresses of the authors: Professor Dr. Bazyli C z e c z u g a , Dr. Ewa C z e c z u g a - S e m e n i u k , Medical University, Department of General Biology, Kilińskiego 1, PL – 15-089 Białystok, Poland; Dr. Susana C a l v e l o , Dr. Sandra L i b e r a t o r e , Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, 8400 Bariloche, Rio Negro, Argentyna. e-mail: Bazzylio@poczta.onet.pl Manuscript received: December 21st 2004.