Offprint Botanica Marina Vol. 43, 2000, pp. 1412145 g 2000 by Walter de Gruyter · Berlin · New York Sterols and Acylglycerols in the Brown Algae Zanardinia prototypus Nardo and Striaria attenuata (Grev.) Grev. from the Black Sea K. Stefanova, St. Dimitrova-Konaklievab, X. Frettea, D. Christovab, Ch. Nikolovac and S. Popova,* a b c Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Faculty of Pharmacy, Medical University, Sofia 1000, Bulgaria Institute of Soil Sciences and Agroecology, “N. Pushkarov”, Shosse Bankya 7, 1080 Sofia, Bulgaria * Corresponding author The sterol composition of these algae appeared to be very complex. While in Zanardinia prototypus the expected fucosterol predominated, Striaria attenuata contained significant amounts of lower sterol homologues (C27- and C28-sterols). Comparisons with the sterol composition of other algae were made and the data obtained was in agreement with some classifications proposed earlier. Significant differences have also been found in the lipid composition of these algae. Zanardinia prototypus contained mainly phospholipids, while the main lipid class in Striaria attenuata appeared to be monogalactosyl diacylglycerols. The concentrations of some of the individual fatty acids in the two algae also showed significant differences. Introduction The more than 265 genera of brown algae (Chromophycota, Phaeophyceae) so far described are grouped into 15 orders (South and Whittick 1987). They are widely spread all over the world and there are many investigations of their chemical composition. In some cases these have resulted in the discovery of new compounds with applications in medicine, the food industry and chemotaxonomy. While some algae have been investigated many times, the research on others has been very limited. For example, the chemical composition of such wide-spread algae as those, belonging to genera Zanardinia and Striaria, have not been investigated until now. Striaria attenuata (Grev.) Grev. belongs to the order Dictyosiphonales, family Striariaceae, members of which are found in all seas, especially these with a temperate or cold climate. Zanardinia prototypus Nardo belongs to the order Cutleriales, family Cutleriaceae. These algae inhabit the sublittoral zone of the south European coasts: Atlantic, Mediterranean and Black Sea. There are only a few complete investigations on the sterol composition of brown algae and fucosterol is accepted in almost all cases as the main sterol (Goad 1978, Elyakov and Stonic 1988). Sterols and lipids are used in taxonomy, because they are constituents of the cell membranes and their composition depends on the taxonomic position of the organism investigated and on the environment. If the algae are collected at locations with similar environmental conditions, as in this case, the sterol and lipid composition could be used for some preliminary taxonomic conclusions. Comparisons with other brown algae could be made in the future, when we obtain more knowledge of sterol and lipid composition of brown algae. Future work will be concentrated on other algae from this group in order to perform a complete investigation of the constituents of the lipid cell membranes of evolutionary lower brown algae. Materials and Methods Plant Material The sample of Zanardinia prototypus Nardo was collected in July 1995 from the southern Black Sea coast of Bulgaria near Varvara village (depth 224 m, water temperature about 22 8C) and voucher number FF 6795. The sample of Striaria attenuata (Grev.) Grev. was collected in May 1994 near the city of Kiten in the same region (depth 122 m, water temperature about 168) and voucher number FF 8594. The samples were washed with tap water, cleaned of epiphytes and dead algal parts, dipped in ethanol and immediately transported to the laboratory. Voucher specimens were deposited in the Faculty of Pharmacy, Medical University at Sofia and identified by Dr St. Dimitrova-Konaklieva. Isolation of lipids The algal material (50260 g wet wt) was homogenised with 180 mL methanol-chloroform (1:1) and refluxed for a few minutes in order to inactivate the enzymes, which can hydrolyse some of the lipids. After filtration the tissue residue was extracted once more with 100 mL methanol-chloroform (1:2) and the combined extracts were used for the analysis of the sterols and of the main lipid classes. 142 K. Stefanov et al. Isolation and analysis of sterols A part of the total lipophylic fraction (40 mg) was chromatographed on a preparative silica gel thin layer chromatography (TLC) plate with hexane-acetone (100:8) as the mobile phase. The sterol mixture obtained was investigated by gas liquid chromatography (GLC) with a Hewlett Packard 5890 instrument, on a 12 m SPB-50 glass capillary column, 0.25 mm i. d., 0.25 mm film thickness. The column temperature was increased from 2308 to 270 8C (4 deg · min21). Gas chromatography/mass-spectrometry (GC/MS) investigation was performed on a Hewlett Packard gas chromatograph 5890 series II plus equipped with an Hewlett Packard MS 5972 detector. The column used was an HP5-MS capillary column (30 m 3 0.25 mm i. d., 0.25 mm film thickness). The column temperature was programmed from 2308 to 300 8C (4 deg · min21), followed by 10 min at 300 8C. Helium was used as a carrier gas at 6 psi pressure. The ionisation voltage was 70 eV. Isolation and analysis of the main lipid classes Part of the extract, containing about 40 mg total lipids, was separated by preparative thin layer chromatography (TLC) on silica gel G plates with chloroform-methanol-acetone-acetic acid (35:7:12:0.2) as the mobile phase. The spots of the main lipid classes were identified by their chromatographic behaviour, compared to authentic samples, and by specific spray reagents. The zones of the main lipid classes were scraped off into small glass containers with teflon screw caps. After the addition of an internal standard (heptadecanoic acid) all lipid classes were esterified with 15% acetyl chloride in methanol according to Christie (1973). The analysis of the fatty acid methyl esters (FAME) obtained was carried out by a flameionisation detector 2 gas-liquid chromatography (FID-GLC), on a glass capillary column (30 m, 0.2 d mm i. d., coated with SILAR 10C). The column temperature was increased from 165 8C to 220 8C (2 deg · min21), with nitrogen as a carrier gas at a flow rate 14 mL · s21. To determine the amount of each lipid class the weights of FAME were multiplied by a K-factor (Elenkov et al. 1993) The amounts of the total acylglycerols (TAG) were determined as a sum of the amounts of all acylglycerol classes. In Zanardinia prototypus the sterol composition was characteristic for the brown algae (Elyakov and Stonic 1988), fucosterol (1) being the main sterol component. Other important sterol components appeared to be cholesterol (2) and 24-methylenecholesterol (3). A few minor sterols were identified, such as 22E-dehydrocholesterol (4), cholestanol, desmosterol Table I. Sterol composition of Zanardinia prototypus and Striaria attenuata (% of the total sterol mixture*) Sterol (Fig. 1) Z. prototypus S. attenuata Fucosterol (1) Cholesterol (2) 24-Methylenecholesterol (3) 22E-Dehydrocholesterol (4) Desmosterol (5) Brassicasterol (6) Campesterol (7) Stigmasterol (8) Sitosterol (9) Isofucosterol (10) 11 13 85 6 5 tr.** tr. tr. tr. 2 2 1 tr. 2 16 22 18 2 tr. 2 tr. 11 16 6 2 tr. * Values, obtaines from three parallel measurements ** tr.< 0.5% Results and Discussion Sterols Sterols from the two algae investigated were purified by preparative thin layer chromatography on silica gel. The identification and quantitation of sterols was done by GLC and GC/MS and the results obtained are summarised in Table I. The formulae of the sterols are presented in Figure 1. There were very big differences in the sterol composition of the two algae. Fig. 1. Formulae of the identified sterols Sterols and acylglycerols in brown algae (5), brassicasterol (6), campesterol (7), stigmasterol (8), sitosterol (9) and isofucosterol (10) (stereochemistry at C-24 not determined). Another minor sterol (11) was identified as the sterol found earlier in two gorgonians and one sponge (Carlson et al. 1978). Its lower homologue (12) was found recently in the Black Sea alga Cladophora vagabunda (L.) Hoek (Elenkov et al. 1995). This is the second discovery of a representative of this rare sterol group in algae. The identification of these short-side chain sterols in algae is of importance, because this shows that they can be produced not only by autoxidation in invertebrates, as was proposed earlier (Carlson et al. 1978), but also can come from the diet. Besides desmosterol, its higher homologue, containing two methylene groups more has been detected. From the biogenetic point of view it is probably 24-ethyl-desmosterol. Also, there are traces of two C30-sterols with molecular masses 426 and 428 and fragmentation, similar to those of gorgosterol and its derivatives. In Striaria attenuata the sterol composition differs drastically from that of Zanardinia prototypus. Now the C29-sterols were only about 50% of the total sterol mixture, less than of half of them being fucosterol, the main sterol of most of the brown algae (Goad 1978, Elyakov and Stonic 1988). Striaria. attenuata contains significant amounts of C28-sterols (mainly 24-methylenecholesterol) and C27-sterols (mainly cholesterol). High concentrations of these sterols are characteristic for red algae, but not for brown algae (Goad 1978, Elyakov and Stonic 1988). The C26-D5sterol (13) probably comes from attached phytoplankton or epiphytic algae, which were not completely removed by the washing of the alga after collection. The absence of its more wide-spread analogue with a C-22 double bond is an indication that both C26-sterols could be produced by different organisms. Small amounts of isofucosterol, characteristic for green algae, were also detected. We compared the sterol composition of both algae investigated with those of the recently investigated Colpomenia peregrina (Sauv.) Hamel and Scytosiphon lomentaria (Lyngb.) Link [known recently also as Scytosiphon simplicissimus (Clemente) Cremades] (Stefanov et al. 1996). Both of them belong to the Scytosiphonales. It is evident that they can be separated into two groups. The first one includes Zanardinia prototypus and Scytosiphon lomentaria. They are characterised by high fucosterol concentrations and low concentrations of other sterols. Striaria attenuata and Colpomenia peregrina have a very similar sterol composition close to that of some red algae: very high cholesterol and 24-methylenecholesterol concentrations and unusually low fucosterol concentration. These algae also contain relatively high concentrations of sitosterol and stigmasterol. Some authors (Ribera et al. 1992, Wynne 1981) accept that of the three orders investigated the Dictyosiphonales is the least advanced evolutionary, the 143 Scytosiphonales is more advanced and the Cutleriales is the most advanced order. This is in agreement with our results 2 fucosterol is characteristic for more advanced brown algae(Goad 1978, Elyakov and Stonic 1988, Ribera et al. 1992) and now we find high concentrations of it in Zanardinia prototypus (Cutleriales) and in one representative of the Scytosiphonales (Scytosiphon lomentaria), which according to the same authors is more advanced than Colpomenia peregrina. Further research on other algae from these orders is needed in order to confirm our proposals. Acylglycerols The total lipids were separated into a few main classes 2 triacylglycerols (TAG), monogalactosyl diacylglycerols (MGDG), digalactosyl diacylglycerols (DGDG) and phospholipids (PL). The data regarding the concentrations of the main lipid classes are summarised in Table II. Below we discuss the differences in this main lipid classes in both algae and compare the results obtained from this analysis with those from two other brown algae, Colpomenia peregrina and Scytosiphon lomentaria (Stefanov et al. 1996), which are biologically close to Zanardinia prototypus and Striaria attenuata. Both algae investigated showed differences in the concentrations of the main lipid classes. While the concentrations of TAG appeared to be similar in both species, the concentrations of the polar lipids differed strongly. In Zanardinia prototypus phospholipids predominated, while in Striaria attenuata the main polar lipids appeared to be glycolipids (MGDG and DGDG). In both samples a MGDG/DGDG ratio > 1 was found, which is characteristic for most of Table II. The amounts of the main lipid classes in S. attenuata and Z. prototypus Sample and lipid classes Lipid content mg · g21 dry wt* wt% of total S. attenuata TAG MGDG DGDG PL 10.37 12.8 3.5 6.3 1.0 1.1 0.3 0.6 32.1 38.6 10.4 18.9 Total 33.3 6 3.0 100.0 Z. prototypus TAG MGDG DGDG PL 12.0 5.2 1.5 13.2 1.1 0.5 0.1 1.2 37.8 16.3 4.6 41.3 Total 31.9 6 2.9 100.0 6 6 6 6 6 6 6 6 * Values 6 SD obtained from three parallel TLC separations. 144 K. Stefanov et al. Table III. Fatty acid composition of S.attenuata and Z.prototypus (wt% of total*) Fatty acids S. attenuata TAG Z. prototypus MGDG DGDG PL TAG MGDG DGDG PL 14:0 8.9 6.8 7.9 8.7 14.1 12.7 14.0 7.6 16:0 16:1 31.5 5.2 51.7 1.8 51.9 4.0 55.0 3.2 26.6 11.2 29.0 7.8 26.7 14.0 29.3 7.5 18:0 18:1** 18:2 18:3 18:4 2 18.9 16.5 4.9 3.2 4.6 19.1 2.1 0.5 0.5 4.1 15.1 6.6 2.2 3.0 2.8 17.6 5.8 1.5 0.7 7.8 22.6 6.2 1.4 1.2 3.7 18.9 8.5 2.3 5.5 10.0 14.0 4.8 4.8 2 11.6 23.2 6.6 1.5 2.8 20:4** 20:5 22:1 1.0 6.1 3.7 8.0 3.0 0.9 2.2 1.5 1.5 3.0 2 1.5 1.5 7.3 2 2.6 9.0 2 11.3 2 2 4.2 1.8 3.8 * Values, obtained from three parallel TLC separations ** More than one isomer presented the plants. The significant differences obtained in the composition of polar lipids of both algae are an indication that their cell membranes could be stabilised by different ways (the polar lipids are very important constituents of the lipid cell membranes and their composition determines the cell membrane functions). The fatty acid composition of the different lipid classes also differed in the two algae investigated (Table III). The differences were relatively small in TAG, which could be connected with the similar concentrations of TAG in these algae. The most significant difference obtained was the higher concentrations of linoleic and linolenic acids in Striaria attenuata, which is an indication for the higher activity of the fatty acid desaturases in this alga. In Colpomenia peregrina and Scytosiphon lomentaria also the fatty acid composition of TAG shows negligible differences (Stefanov et al. 1996). Analogously to other plants the fatty acid differences in the polar lipids of the two algae investigated were more significant. While the saturated acid concentrations were similar, in the unsaturated acids we found extremely big differences. Striaria attenuata contained in its MGDG about ten times more 20:1 acid than Zanardinia prototypus. The remaining unsaturated acids in MGDG were in higher concentrations in Zanardinia prototypus. In the DGDG of Striaria attenuata the 16:1 acid was four times more than in Zanardinia prototypus, while the last alga contained more 20:1 acid. There was some similarity between the fatty acid composition of MGDG of Zanardinia prototypus and Colpomenia. peregrina (Stefanov et al. 1996). The fatty acid composition of the phospholipids in the both algae investigated also showed significant differences. Contrary to the other lipid classes these differences were mainly in the palmitic acid concentrations, the last one being twice as much as in Striaria attenuata. Only in the PL we found 22:1 acid in Zanardinia prototypus and now it is twenty times more than in Striaria attenuata phospholipids. Acknowledgements This investigation has been completed with the financial support of the National Foundation for Scientific Research of Bulgaria (Contract X-710) Accepted 19 October 1999. References Christie, W. 1973. Lipid Analysis. Pergamon Press, Oxford, pp 38239. Carlson R., S. Popov, I. Massey, C. Delseth, E. Ayanoglu, T. Varkony and C. Djerassi. 1978. Minor and trace sterols in marine invertebrates VI. Occurence and possible origins of sterols possessing unusually short hydrocarbon side chains. Bioorganic Chem. 7: 4532479. Elenkov, I., A. Ivanova, K. Stefanov, K. Seizova and S. Popov. 1993. A quantitative determination of lipid classes in higher plants and algae by a gas-chroma- tographic procedure, Bulg. Chem.Commun., 26: 982 103 Elenkov I., T. Georgieva, P. Hadjieva, S. Dimitrova, and S. Popov. 1995. Terpenoids and sterols in Cladophora vagabunda., Phytochemitry 38: 4572459. Elyakov, G. B. and V. A. Stonic. 1988. In: (A. V. Kamernitskii, ed.) Steroids from Marine Organisms. Moscow, Nauka [in Russian] pp 52256. Goad L. 1978. The sterols of marine invertebrates: Composition, biosynthesis and metabolites. In: (P. J. Scheuer, Sterols and acylglycerols in brown algae ed.) Marine Natural Products, Chemical and Biological Perspectives. Academic Press, New York, San Francisco, London. pp 80281. Ribera M. A., A. G. Garreta, T. Gallardo, M. Cormaci, G. Furnari and G. Giacone. 1992. Check-list of Mediterranean seaweeds. I. Fucophyceae (Warming, 1884). Bot. Mar. 35: 1092130. South, G. R. and A. Whittick. 1987. Introduction to Phycology. Blackwell Scientific Publications, Oxford, London. pp 529. 145 Stefanov, K., V. Bankova, S. Dimitrova-Konaklieva, R. Aldinova and S. Popov. 1996. Sterols and acylglycerols in the brown algae Colpomenia peregrina (Sauv.) Hamel and Scytosiphon lomentaria (Lyngb.) Link. Bot. Mar. 39: 4752478. Wynne, M. J. 1981. Phaeophyta: Morphology and classification. In: (C. S. Loban and M. J. Wynne, eds). The Biology of Seaweeds. Univ. California Press, Berkeley and Los Angeles, pp 70280.