J Hattori Bot. Lab. No. 86: 235- 241(July 1999) CAROTENOIDS IN LICHENS FROM BENNET ISLAND LYING IN THE EAST-SIBERIAN SEA 1 2 1 BAZYLI CZECZUGA , MIKHAIL ZHURBENK0 , EWA CZECZUGA-SEMENIUK AND MARILYN D. WALKER3 ABSTRACT . Column and thin-layer chromatography revealed the presence of the following carotenoids in the thalli of 14 lichen species (18 specimens) from Bennet Island (East-Siberian Sea): a-, /3-carotene, /3-cryptoxanthin, lutein, zeaxanthin, lutein epoxide, antheraxanthin, violaxanthin, mu­ tatoxanthin, neoxanthin, 3 '-hydroxyechinenone, canthaxanthin, astaxanthin, rhodoxanthin, cap­ sochrome and /3-citraurin. The total content of carotenoids ranged from 46.8 in Sphaerophorus globosus to 87.5 µg g- 1 dry wt in Cetraria nigricans. The amount of the carotenoid content in the form of the provitamin of vita­ min A ranged from 12.5 to 68.3% of the total carotenoid content. INTRODUCTION Lichens in the far north play an important role in the life both of the animals living in these areas, particularly reindeer, and the people (Turner 1977, Donkin 1981 , Richardson and Young 1977, Richardson 1988). During the long winters, they provide practically the only food available to the reindeer and from the lichens these animals assimilate microele­ ments and vitamins (Pulliainen 1971 ). Certain carotenoids produced by plants, including lichens, are known to be a source of vitamin A. Study of the carotenoid content of common species of lichen in this region is therefore not only of theoretical but also of practical sig­ nificance. MATERIALS AND METHODS The lichen species studied were collected in July- August 1989 from Bennet Island (76°S, 149°E; habitat soil and stone; altitude 240-260 m) belonging to the Novosibirsk Is­ lands which are classified as being in the "high" Arctic Circle, lying in the East-Siberian Sea. The carotenoid pigments were isolated using column and thin-layer chromatography. 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 l 0% KOH in ethanol at 20°C for 24 h in the dark under nitrogen. Column and thin-layer chromatography (Czeczuga 1980) were used to separate the carotenoids, 1 Department of General Biology, Medical University, Kilinskiego I, PL-15-230 Biafystok 8, Poland. 2 Botanical Institute, Academy of Science of the Russia, Professor Popov 2, 197-022 Sankt Peters­ burg, Russia. 3 Institute of Arctic and Alpine Researche, University of Colorado at Boulder, Campus Box 450, Boulder, Colorado 80309-0450, U. S.A. 236 J. Hattori Bot. Lab. No. 86 I 9 9 9 which were identified by comparison with standard compounds (Hoffman-La Roche and Sigma Company) by a) the behaviour on column chromatography; b) their UV-VIS spectra (Beckman 2400); c) their partition between n-hexane and 95% ethanol; d) their R1 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 spectrum (cf. Vetter et al. 1971 ). Quantita­ tive determinations were determined by UV, VIS spectroscopy (Davies 1976). For the structures of carotenoids see Straub (1987) and Czeczuga (1988). Quantitative ratios of the carotenoids being vitamin A precursors were estimated ac­ cording to the method of Bauernfeind (1972). RESULTS AND DISCUSSION Sixteen carotenoids were isolated from the thalli of 14 species, including rhodoxan­ thin, capsochrome and f3-citraurin (Table 1). The predominant carotenoids were f3-crypto­ xanthin ( 1), lutein epoxide ( 5), violaxanthin (2), mutatoxanthin ( 6), canthaxanthin (2) and astaxanthin (2) (Table 2). The total carotenoid content in the material studied ranged from 46.8 (Sphaerophorus g/obosus) to 87 .5 µg g- 1 of dry mass ( Cetraria nigricans collected in July). In the thalli of all the lichen species studied the presence of /3-carotene and lutein epoxide was determined, and in I 4 of the 18 specimens investigated, the presence of asta­ xanthin. It was interesting to note the presence of rhodoxanthin ( Cetraria fastigiata and C. ni­ gricans), capsochrome (Cetraria andrejevii, C. fastigiata, C. nigricans and Neuropogon sulphureus) and /3-citraurin (Cetraria nigricascens) in the thalli, which are carotenoids most frequently been found to date in lichens growing under extreme conditions. As re­ gards rhodoxanthin, this carotenoid was found previously in the thalli of Cetraria delisei and C. nivalis from the Kamchatka (Far East) (Czeczuga et al. 1989) and of Umbilicaria proboscidea, Cetraria laevigata and Hypogymnia pseudophysodes from the mountains of Chichibu in Central Japan (Czeczuga and Yoshida 1991); it has also been found in a few species from the Antarctic (Czeczuga and Xavier-Filho 1987), the region of Taimyr (north Siberia) (Czeczuga and Shchelkunova 1986) and Greenland (Czeczuga and Jacobsen I 993). In 4 species of Parmeliaceae collected from the Chichibu Mountains, the presence of f3-citraurin was determined. Capsochrome was identified in the thalli of Cladina rangiferina from Yakut, ASR (Czeczuga et al. 1991 ). It also occurred in a few species of Peltigera collected from the mountains of North India (Czeczuga and Upreti 1991). As can be seen from Table 2, individuals of such species as Cetraria andrejevii, C. fastigiata, C. nigricans and C. nigricascens collected for analysis at two different times (July and August) differed from each other to a greater or lesser degree in their carotenoid composition. Carotenoids common to the thalli of two species of the Sphaerophorus genus were found to be f3-carotene, f3-cryptoxanthin, lutein epoxide, violaxanthin and astaxanthin. On the other hand, f3-carotene, lutein epoxide and antheraxanthin occurred in the thalli of all the individuals of the 4 species of Cetraria. Furthermore, the carotenoids common to two B. Cz ECZUGA et al.: Carotenoids in lichens from Bennet Island 237 Table I. Carotenoids found in lichens from Bennet Islands. Summary Structure No. Carotenoid Semisystematic name formula (see Fig. 1) I. a-Carotene C40H56 A- r- B /3. £-Carotene 2. /3-Carotene C40H 56 A- r- A {3,/3-Carotene 3. /3-Cryptoxanthin C40H560 A- r- C /3,/3-Carotene-3-o I 4. Lutein C40H s60 2 C- r- 0 /3,e-Carotene-3,3 '-diol 5. Zeaxanthin C40Hs602 C- r- C /3,/3-Carotene-3,3 '-diol 6. Lutein epoxide C40H560 3 D- r- E 5, 6-Epoxy-5 ,6-dihydro-{3,s-carotene- 3,3 '-diol 7. Antheraxanthin C40H5603 C- r- E 5,6-Epoxy-5, 6-dihydro-/3,{3-carotene- 3,3'-diol 8. Violaxanthin C40H5604 E- r- E 5,6,5' ,6' -Diepoxy-5,6,5' ,6' -tetrahydro- {3,/3-carotene-3,3 '-diol 9. Mutatoxanthin C40H560 3 C- r1- F 5,8-Epoxy-5 ,8-dihydro-/3,/3-carotene- 3,3'-diol 10. Neoxanthin C40Hs604 E- r1- G 5,5-Epoxy-6,7-didehydro-5,6,5' ,6' - tetrahydro-/3,{3-carotene-3,5,3 '-trio I 11. 3 '-Hydroxyechinenone C40H 5402 C- r- H 3 '-Hydroxy-/3,/3-carotene-4-one 12. Canthaxanthin C40Hs202 H- r- H {3,{3-Carotene-4,4 '-di one 13 . Astaxanthin C40H5204 I- r- 1 3,3 '-Dihydroxy-{3,{3-carotene-4,4' - di one 14. Rhodoxanthin C40H5002 K- r2- K 4' ,5 '-Dihydro-4,5 '-retro-/3,/3-carotene- 3,3'-dione 15. Capsochrome C40H5604 F- r1- L 5,8-Epoxy-3,3 '-dihydroxy-5,8-dihydro- {3,x-caroten-6' -one 16. /3-Citraurin C30H400 2 C- r1- M 3-Hydroxy-8' -apo-/3-caroten-8' -al species of Hypogymnia were {3-carotene, lutein epoxide and mytatoxanthin whi le /3- carotene, {3-cryptoxanthin, lutein epoxide, violaxanthin and astaxanthin occurred in the thall i of three species of Dactylina. In order to compare the carotenoid content of Dactylina madreporiformis thalli, a sample of this lichen was taken from the Colorado Mountains (altitude 3,660 m). The re­ sults were compared with the data obtained from the thalli of this species from Bennet Is­ land. It was found that the thalli from both the Colorado Mountains and Bennet Island contained /3-carotene, {3-cryptoxanthin, lutein epoxide and astaxanthin. Such carotenoids as lutein, 3 ' - epilutein and mutatoxanthin, on the other hand, occurred only in the D. madreporiformis thalli from the Colorado Mountains. The thalli of this lichen from the Colorado Mountains did not contain zeaxanthin and violaxanthin, which were present in the thall i from Bennet Island. As is known, the carotenoids determined in these studies, a-, {3-carotene, and {3-cryp­ toxanthin, are vitamin A provitamins. This depends on the number of structurally non­ modified /3-rings, {3-carotene having two rings, while a-carotene and {3-cryptoxanthin have N (;.) 00 Table 2. Carotenoid distribution in lichens from Bennet Island. Major Total content o/c f .t . Carotenoid detected Constant _ 1 d o o v1 amm Family and species carotenoids (see Table I) carotenoid (µg g ry A precursors (%) weight) Sphaerophoraceae Sphaerophorus fragilis (L.) Pers. 2,3, 4,6, 8, 9, 10, II , 13 9 (39.8) 40.4 20.4 Sphaerophorus globosus (Huds.) Vain. 2, 3, 6, 8, 12, 13 2, 3, 6, 8, 13 13 (33.6) 46.8 31.8 Pertusariaceae Pertusaria ocu/ata (Dicks.) Th. Fr. 2, 6, 8, 9, 12 , 13 8 (34.0) 84.9 18.2 Parmeliaceae ~ Cetraria andrejevii Oxn. (July, 1989) 2,4, 6, 7,9, II, 13 6(23.4) 80.3 14.6 :i:: "';:i Cetraria andrejevii Oxn. (August, 1989) I, 2, 3, 4, 6, 7, 8, 9, 15 6(21.3) 44.6 45.0 8. Cetrariafastigiata (Del.) Karnef. (July, 1989) 2, 3, 4, 5, 6, 7, 9, 12, 13, 15 6(19.9) 48.9 34.8 o:i Cetrariafastigiata (Del.) Karnef. (August, 1989) 2,3,5,6, 7,8, 11 , 13 , 14 2, 6, 7 3(21.2) 51.3 68.3 ~ Cetraria nigricans (Retz.) Ny!.