Sterols from Six Marine Sponges Elena A

Biochemical Systematics and Ecology 32 (2004) 153–167 www.elsevier.com/locate/biochemsyseco

Sterols from six marine sponges Elena A. Santalova, Tatyana N. Makarieva, Irina A. Gorshkova, Andrey S. Dmitrenok, Vladimir B. Krasokhin, Valentin A. Stonik ∗ Laboratory of the Marine Natural Products, Paciﬁc Institute of Bioorganic Chemistry of Far Eastern Branch of the Russian Academy of Sciences, Pr. 100-letya Vladivostoka 159, 690022 Vladivostok, Russia

Received 30 September 2002; accepted 7 April 2003

Abstract

The free sterol fractions from marine sponges Darwinella australiensis, Haliclona sp., Agelas mauritiana, Clathria major, Didiscus aceratus and Teichaxinella labirintica from West- ern Australia were isolated and studied by HPLC, GLC, GLC-MS, and NMR methods. D. australiensis contained ⌬7-, ⌬5-, ⌬5,7-, ⌬5,7,9(11)-sterols, and cholest-7-en-3β-ol was shown to be a main sterol. The free sterols from A. mauritiana proved to be stanols and ⌬7-series compounds, chondrillasterol was identiﬁed as a predominant constituent. Haliclona sp. contained ⌬5-sterols with cholesterol as a main constituent. C. major and D. aceratus contained ⌬5-sterols, and clionasterol was shown to be a main sterol. T. labirintica was shown to contain 3β-hydroxymethyl-A-nor-sterols. Absolute conﬁgurations at C-24 of major sterols from C. major, D. aceratus and A. mauritiana were established by NMR method. Distribution of different sterols in the studied species was discussed to provide additional viewpoint on the probable application of these natural products as chemotaxonomic markers and to understand biological roles of unusual sterols in sponges using an idea of so-called biochemical coordination.  2003 Elsevier Ltd. All rights reserved.

Keywords: Marine sponges; Sterols; Unusual sterols; Didiscus aceratus; Darwinella australiensis; Hal- iclona sp.; Agelas mauritiana; Clathria major; Teichaxinella labirintica

∗ Corresponding author. Tel.: +7-4232-31-1168; fax: +7-4232-31-4050. E-mail address: [email protected] (V.A. Stonik).

1. Introduction

Sponges have proved to be a rich source of sterols with unusual structural features (Djerassi, 1981; Kerr and Baker, 1991; Baker and Kerr, 1993). It is of special interest that, although some sponge species provide the greatest structural diversity of membrane sterols in comparison with any other animals, many of the studied sponges contain the sterol compositions resembling those of animals belonging to other taxa. Why, for example, do some sponge species contain sterol mixtures with a preponderance of cholesterol in them, while another group of sponge species have a very complicated sterol mixture, where cholesterol is minor or absent? Why do some sponge species contain only one or two sterol constituents, with these being unusual structures? Do these unusual sterols play the same role in sponge membranes as cholesterol does in higher animals or carry out additional functions? May data concerning sterol distribution in sponges be utilized to provide information of value for systematic position of one or other sponge species? We hope that the further studies on sponge sterol compositions and sponge ecology may help to answer these ques- tions. Sterols of Darwinella australiensis, Haliclona sp., Clathria major, Didiscus aceratus and Teichaxinella labirintica have not been previously investigated. Agelas mauritiana from another collection, namely from the Great Barrier Reef of Australia, was previously studied by Berquist et al. (1980). Herein we report hemolytic activities of the studied sponge extracts, the isolation of their free sterol fractions and the investigation of those by HPLC, GLC, GLC-MS, and NMR methods to elucidate sterol compositions. Some suggestions concerning a biological role of sponge sterols differing from cholesterol are given.

2. Materials and methods

2.1. Animals

Marine sponges were collected during the 12th scientific cruise of the research vessel “Akademik Oparin” by dredging near the North-Western coast of Australia in November, 1990. Collected sponges were immediately cut, lyophilized and stored at 5 °C. Species identifications were carried out by Mr. V.B. Krasokhin (Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russian Federation). Voucher specimens (012-103, 012-220, 012-263, 012-216, 012-238, 012-74) are on deposit in Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russian Federation.

2.2. Extraction and isolation of free sterols

Lyophilized sponges were extracted with ethanol at room temperature. Alcoholic extract of each species was evaporated in vacuo to give a dark brown solid. The material was chromatographed using preparative TLC. Obtained sterol fractions were E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 155

acetylated with pyridine-Ac2O (1:1), 16 h. Column chromatography of the sterol acetates over a column with silica gel L 40/100µ (Chemapol, the former Czechoslovakia) eluting with the system hexane–ethylacetate (10:0.1) to give the puriﬁed sterol fractions.

2.3. Separation of free sterol fractions

Free sterols from marine sponges A. mauritiana, D. australiensis and T. labirintica were separated by HPLC analysis using an Altex Ultrasphere-Si column (10 mm × 25 cm) with the system hexane–ethylacetate (5:1) as eluent. All products isolated were monitored as the corresponding acetates by GLC and were analyzed by GLC-MS and NMR. Identiﬁcation of sterols was possible by comparing their relative retention times (RRT) during GLC on a capillary column with those calcu- lated using separation factors for OV-1 phase from Itoh et al. (1982a), and also by using mass and NMR spectra. Cholesterol (Sigma Grade: 99%), β-sitosterol (Sigma Grade: 98%) and clionasterol, isolated from the sponge Baicalospongia bacilifera (Makarieva et al., 1991) were used as standards.

2.4. Analytical methods

GLC analyses were performed on a Sigma 2000 Perkin Elmer chromatograph using a capillary column (50 m × 0.33 mm) with CBP-5 at 290 °C, helium was the carrier gas. GLC-MS analyses were done on Hewlett Packard HP6890 GC System, HP-5MS capillary column (30.0 m × 0.25 mm) at 270 °C, helium was used as the carrier gas, and the ionizing voltage was of 70 eV. 1H NMR spectra were recorded on a Bruker DPX-300 spectrometer in CDCl3 with TMS as an internal standard. HPLC was carried out on a Du Pont Series 8800 Instrument with refractometer index unit RIDK-102 on an ALTEX Ultrasphere-Si column (10 mm × 25 cm) in the system hexane–ethylacetate (5:1). TLC was performed using glass plates (6 × 9 cm) coated with silica gel L 5/40µ (Chemapol, the former Czechoslovakia) in the system chloroform–ethanol (10:0.2) for free sterols. Column chromatography was performed on silica gel L 40/100µ (Chemapol, the former Czechoslovakia) using system hexane–ethylacetate (10:0.1) for sterol acetates.

2.5. Hemolysis

Mouse erythrocytes were washed three times using centrifugation (600×g, 5 min) in cold 150 mM NaCl, 10 mM Tris–HCl (pH 7.4). The pellet was resuspended in the same solution to a ﬁnal concentration of 0.2%. Erythrocytes were incubated with dried residues of ethanol extracts from marine sponges (50–250 µg/ml) for 30 min at room temperature and then centrifuged (600×g, 5 min). Optical densities of super- natants were measured spectrophotometrically at 540 nm. 156 E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167

Table 1 Classiﬁcation and area of collection of the species studied

Species Taxonomic position Locality of collection Depth (m)

Porifera

Demospongiae

Dendroceratida D. australiensis Darwinellidae 14°04,01 S; 121°56,66 E 10 Agelasida A. mauritiana Agelasiidae 14°7,4 S; 121°45,7 E 35 Haplosclerida Haliclona sp. Haliclonidae 14°03,38 S; 121°46,6 E 15 Poecilosclerida C. major Microcionidae 16°44,5 S; 121°16,6 E 48 Halichondrida D. aceratus Desmoxyidae 14°03,38 S; 121°46,6 E 15 Halichondrida T. labirintica Axinellidae 16°41,4 S; 121°09,6 E 54

3. Results

The collected sponges belong to ﬁve different orders of the class Demospongiae. Taxonomic positions and geographical coordinates of areas of collection for the species studied are listed in Table 1. Alcoholic extracts of the sponges collected were tested for hemolytic activities. Results of biotesting showed that extracts of two species (D. australiensis and A. mauritiana) have higher hemolytic properties in comparison with others (Table 2). We isolated free sterol fractions from these sponges and established their sterol compositions by NMR, GLC and GLC-MS methods. Fifty known C26–C29-sterols were identiﬁed (Stonik et al., 1998; Kolesnikova et al., 1992; Itoh et al., 1983; Bohlin

Table 2 Hemolytic activities of sponge extracts

µ Species HD50 ( g/ml)

D. australiensis 15 A. mauritiana 50 Haliclona sp. Ͼ100 C. major Ͼ100 T. labirintica Ͼ300 D. aceratus Ͼ500 E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 157 et al., 1981; Bonini et al., 1983; Teshima and Patterson, 1981; Delseth et al., 1979) and their structures are presented in Fig. 1. The percentage sterol composition for each species is given in Table 3.

3.1. Darwinella australiensis

⌬5 ⌬7 ⌬5,7 ⌬5,7,9(11) ⌬7 This sponge contained -, -, - and -sterols. C27 sterol was shown to be the main sterol. The ratio of ⌬5:⌬7:⌬5,7:⌬5,7,9(11) compounds was found to be

3:4:1:1, while the ratio of C27:C28:C29 was 8:5:1.

3.2. Agelas mauritiana

The fraction from A. mauritiana had stanols, ⌬5- and ⌬7-sterols with chondrillasterol (Itoh et al., 1982b) as a major sterol. The ratio of stanols:⌬5:⌬7compounds in this sponge was found to be 19:1:15, while the ratio of C27:C28:C29 was Ϸ2:1:1. Cholesterol was not found and related ⌬5-sterols were very minor in this animal.

Fig. 1. Structures of the sterols identified in sponges. 158 E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 ) tr. – 0.52 0.29 1.91 6.84 2.91 12.26 – 0.14 – – – aceratus labirintica 0.85 2.91 continued on next page ( 0.16 sp. tr. 0.93 0.06 –––– – –––– 1.27 2.22 1.28 tr. – ––––– ––––– ––––– ––––– 1.00) = D.australiensis mauritiana A. Haliclona C. major D. T. –– ––––– ––––– ––––– – ––––– ––––– ––––– –––– ––––– –––– 0.695 0.69 0.87 0.895 tr. 0.89 0.92 0.92 0.925 5.700.93 0.950.985 tr. 5.10 tr.1.001.00 tr. 1.00 13.50 4.48 6.851.02 2.511.03 tr.1.04 8.691.06 7.65 26.431.07 0.611.07 28.72 20.481.09 tr. tr. tr. 1.10 6.80 0.87 5.31 7.65 tr. 0.69 tr. 0.24 tr. 15.83 21.12 11.55 tr. 22 22 22 15 0 ⌬ ⌬ ⌬ ⌬ ⌬ 26 27 27 27 27 5,22 2F 5,22 22 5,22 22 5,7,9(11) 7,22 5 0 5,22 7,22 22 5 5,7 5,7,9(11),22 5,22 ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ 26 27 27 27 27 27 27 27 27 27 27 27 28 27 27 28 28 designation ) A-nor-C 6a )C )C 3b 5g )C ) A-nor-C )C -cholest- A-nor-C )C ) A-nor-C α -ol ( 1b 6c 2b 1e β 7d -ol ( β )C -ol ( -ol ( )C -ol ( β 1f β β 1g )C -ol ( )C 1a β -ol ( )C 1d β 3c -ol ( )C β -cholest-22E-en ( -cholest-15-en ( -cholestan (A-nor- A-nor-C )C 1c )C α α α -ol ( 2c β 4d )C -ol ( -methyl-27-nor-A-nor-5 -cholest-22E-en-3 -cholesta-7,22E-dien-3 -ol ( β ξ α α 2d β -ol ( β -ol ( ) -ol (cholesterol) ( β β 6d -methyl-5 -methylcholesta-5,22E-dien-3 -methyl-5 ) ξ ξ ξ ed sterol C ed sterol C ed sterol C 6b fi fi fi -24,26-Cyclocholest-5-en-3 -24,26-Cyclocholest-5,22-dien-3 ξ ξ ,25 ,25 -Methylcholesta-5,7,9(11),22E-tetraen-3 -Methylcholesta-5,22E-dien-3 -Cholest-22E-en-3 -Cholesta-7,22E-dien-3 -Cholestan-3 -Hydroxymethyl-A-nor-5 -Hydroxymethyl-24-nor-A-nor-cholesta-5,22E-dien ( -Hydroxymethyl-A-nor-5 -Hydroxymethyl-A-nor-5 -Hydroxymethyl-24 ξ ξ ξ ξ β β α α α β β β 22E-en ( cholestanol) ( 6 27-Nor-24 73 18 Unidenti 11 Unidenti N Sterol1 24-Nor-cholesta-5,22E-dien-3 Short RRT Fraction (%) 23 34 Unidenti 27-Nor-24 8 Cholesta-5,22E-dien-3 10 5 17 5 19 24 20 Cholesta-5,7-dien-3 15 5 93 14 3 16 24 53 12 27-Nor-24 13 Cholest-5-en-3 21 24 22 24 Table 3 Sterol compositions of sponges studied (RRT are given for acetates, RRT of cholesterol acetate E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 159 ) – 29.77 tr. 0.49 – 19.35 tr. tr. – 8.36 tr. tr. tr. –– 0.34 aceratus labirintica – tr. tr. –– continued on next page ( tr. – –– 0.32 sp. – –––– ––– –– –––– 0.42 tr. tr. tr. 9.03 tr. 2.04 1.46 ––––– –––– – ––––– ––––– ––––– D.australiensis mauritiana A. Haliclona C. major D. T. ––––– – ––––– ––––– – ––––– ––––– – – ––– ––––– 1.10 1.121.13 20.961.185 10.47 1.21 9.45 1.23 tr.1.241.24 tr.1.25 tr. 7.651.271.27 tr. 1.29 tr. 5.22 13.921.31 2.091.33 tr. 13.911.33 tr. 1.33 tr. 3.40 2.871.35 1.37 tr. 9.43 tr.1.391.40 tr. 1.45 2.551.48 3.82 tr. 6.01 4.74 4.3 22 0 22 22 ⌬ ⌬ ⌬ ⌬ 28 28 29 29 7 22 5,7,22 F,22 5,24(28) 24(28) 5 7,22 24(28) 0 5,22 5,7,9(11),22 5,22 22 22 7,24(28) 7 5,F 7,22 ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ 27 28 28 28 28 28 28 28 28 28 29 29 29 29 29 28 28 29 29 designation ) A-nor-C 6i )C )C )C )C 2j 1j 3j )C 5k -cholest- A-nor-C 3h )C α -ol ( -ol ( -ol ( )C 3g β β β -ol ( )C )C β -ol ( -cholestan ( -cholest-22E-en A-nor-C 4g )C 1h β -cholest-24(28)- A-nor- 1.23 2h )C α α )C -cholest-22E-en A-nor-C -ol ( 2g α α )C β 1k 2k -ol ( -ol ( 3i -ol ( β )C β β -ol ( )C 2i -ol ( β -ol ( β -ol ( 1i β β -ol ( β -ol ( )C -cholest-22E-en-3 β α 3d -methyl-A-nor-5 -methyl-A-nor-5 -ethyl-A-nor-5 ξ ξ ξ -cholesta-5,22E-dien-3 -cholesta-7,22E-dien-3 -ol ( α α β -cholest-22E-en-3 -cholesta-7,22E-dien-3 -cholesta-7,24(28)-dien-3 -cholestan-3 -cholest-7-en-3 -cholest-24(28)-en-3 α α α α α ) -cholest-22E-en-3 α α ) ed sterol C ed sterol C 6j fi fi )C 6h continued -Methyl-5 -Ethylcholesta-5,7,9(11),22E-tetraen-3 -Methylcholest-5-en-3 -Methylcholesta-5,7,22E-trien-3 -Methyl-5 -Ethyl-5 -Methyl-5 -Methyl-5 -Ethylcholesta-5,22E-dien-3 -Methyl-5 ) ) -Cholest-7-en-3 -Hydroxymethyl-24 -Hydroxymethyl-24 -Hydroxymethyl-23,24R-dimethyl-A-nor-5 -Hydroxymethyl-24 -Hydroxymethyl-24-methyl-A-nor-5 ξ ξ ξ ξ ξ ξ ξ ξ ξ ξ α β β β β β 6g 6k ( 22E-en ( ( en ( 24 5 40 (24R)-23,24-Dimethyl-5 25 24 32 3 44 Unidenti 37 24 N Sterol23 3 30 24 Short RRT Fraction (%) 26 24 28 24-Methylcholesta-5,24(28)-dien-3 31 24 33 24-Methyl-5 35 3 39 3 41 24 36 23,24R-Dimethyl-5 29 3 42 24 45 23,24R-Dimethyl-5 27 Unidenti 34 24 38 24 43 24 Table 3 ( 160 E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 – – – 14.70 0.42 0.33 0.45 – tr. – tr. tr. tr. aceratus labirintica tr. tr. – tr. tr.– tr. – sp. –––– 1.87 – –– 0.35 1.20 tr. – ––––– D.australiensis mauritiana A. Haliclona C. major D. T. ––––– –– – ––––– ––––– ––––– –– ––––– –––– 1.49 1.495 2.551.501.50 0.851.51 2.231.55 tr. 1.55 2.23 1.63 9.981.58 tr. 1.68 1.69 34.881.69 tr.1.71 60.441.79 tr. 20.57 0 ⌬ 29 5 7,22 5,24(28) 5,24(28) 0 24(28) 2F F 24(28) 7 5,24(28) 24(28) 5,24(28) ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ 29 29 29 29 29 30 29 29 30 29 30 29 30 designation ) A-nor-C 6l )C )C )C )C )C )C 3k 1o 1p 1m 1n 2n -cholestan ( α -ol ( -ol ( -ol ( )C β -ol ( -ol ( β β -ol ( β β 3l β )C )C 2l 1l -ol ( β -ol ( β -ol ( β -ethyl-A-nor-5 ξ ) -cholestan-3 -cholest-7-en-3 -cholesta-7,22E-dien-3 -cholest-24(28)Z-en-3 α α α α ed sterol C ed steroled sterol C C ed sterol C fi fi fi fi continued -Ethyl-5 -Ethylcholest-5-en-3 -Ethyl-5 -Ethyl-5 -Hydroxymethyl-24 ξ ξ ξ ξ β 49 24-Ethylcholesta-5,24(28)E-dien-3 52 Unidenti 50 24-Ethylcholesta-5,24(28)Z-dien-3 51 24 58 24-Ethyl-5 N Sterol46 3 Short RRT Fraction (%) 47 24 53 Unidenti 55 Unidenti 56 24 57 24-Propylcholesta-5,24(28)E-dien-3 59 24-Propylcholesta-5,24(28)Z-dien-3 48 24 54 Unidenti Table 3 ( E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 161

Table 4 GC-MS data of sterols and structure assignments

Notation Sterol M+ MS data (typical fragments) Relative structure retention data

C26 ⌬5,22 -C26 1a – 352, 337, 282, 267, 255, 213 0.695

C27 ⌬5,22 -C27 1b – 366, 351, 282, 267, 255, 213 0.895 ⌬22 -C27 2b 428 344, 315, 257 0.92 ⌬5,22 -C27 1c – 366, 351, 282, 267, 255, 213 0.925 22 ⌬ -C27 2c 428 368, 344, 315, 269, 257, 215 0.95 7,22 ⌬ -C27 3b 426 411, 342, 313, 255, 229, 213 1.00 5 ⌬ -C27 1d – 368, 353, 260, 255, 247, 213 1.00 0 ⌬ -C27 2d 430 370, 355, 315, 275, 257, 215 1.02 5,22 ⌬ -C27 1e – 364, 349, 282, 267, 253, 213,109 1.03 7,22 ⌬ -C27 3c 426 411, 342, 313, 255, 229, 213 1.04 5 ⌬ -C27 1f – 366, 351, 281, 255, 213 1.07 5,7 ⌬ -C27 4d 426 366, 351, 325, 313, 253, 211 1.07 7 ⌬ -C27 3d 428 413, 315, 273, 255, 229, 213 1.12

C28 ⌬5,7,9(11),22 -C28 5g 436 376, 361, 350, 277, 251, 235, 209 1.09 ⌬5,22 -C28 1g – 380, 365, 282, 267, 255, 213 1.10 ⌬22 -C28 2g 442 382, 344, 315, 269, 257, 215 1.13 ⌬5,7,22 -C28 4g 438 378, 363, 335, 313, 253, 211 1.185 ⌬5,24(28) -C28 1h – 380, 365, 296, 281, 253, 213 1.23 ⌬5 -C28 1i – 382, 367, 261, 255, 213 1.24 ⌬7,22 -C28 3g 440 425, 342, 313, 255, 229, 213 1.24 ⌬24(28) -C28 2h 442 427, 358, 315, 298, 255, 215 1.27 ⌬0 -C28 2i 444 384, 369, 315, 276, 257, 215 1.27 ⌬7,24(28) -C28 3h 440 425, 356, 313, 255, 229, 213 1.39 ⌬7 -C28 3i 442 427, 315, 273, 255, 229, 213 1.40

C29 ⌬5,22 -C29 1j – 394, 282, 267, 255, 213, 69 1.31 ⌬5,7,9(11),22 -C29 5k – 390, 375, 363, 251, 235, 209 1.33 5,22 ⌬ -C29 1k – 394, 379, 282, 267, 255, 213 1.33 22 ⌬ -C29 2j 456 344, 315, 269, 257, 217, 69 1.35 22 ⌬ -C29 2k 456 396, 344, 315, 269, 257, 215 1.37 7,22 ⌬ -C29 3k 454 342, 313, 255, 229, 213, 69 1.48 5 ⌬ -C29 1l – 399, 381, 275, 255, 213 1.495 7,22 ⌬ -C29 3k 454 439, 342, 313, 255, 229, 213 1.50 5,24(28)E ⌬ -C29 1m – 394, 379, 296, 281, 253, 213 1.50 5,24(28)Z ⌬ -C29 1n – 394, 379, 296, 281, 253, 213 1.51 0 ⌬ -C29 2l 458 398, 383, 315, 275, 257, 215 1.55 ⌬7 -C29 3l 456 441, 315, 273, 255, 229, 213 1.69 ⌬24(28)Z -C29 2n 456 358, 315, 298, 255, 215 1.71 (continued on next page) 162 E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167

Table 4 (continued)

Notation Sterol M+ MS data (typical fragments) Relative structure retention data

C30 ⌬5,24(28)E -C30 1o – 408, 393, 296, 281, 253, 213 1.69 ⌬5,24(28)Z -C30 1p – 408, 393, 296, 281, 253, 213 1.79 A-nor-sterols

C26 ⌬22 -C26 5a 414 354, 344, 315, 269, 257, 215, 55 0.69

C27 22 ⌬ -C27 5b 428 368, 344, 315, 269, 257, 215, 55 0.89 22 ⌬ -C27 5c 428 368, 344, 315, 269, 257, 215, 95 0.92 15 ⌬ -C27 6d 428 368, 315, 255, 215, 206, 202, 93 0.93 0 ⌬ -C27 5d 430 370, 355, 315, 275, 257, 215 1.00

C28 22 ⌬ -C28 5g 442 382, 344, 315, 269, 257, 215, 69 1.10 24(28) ⌬ -C28 5h 442 427, 358, 315, 255, 215, 69 1.23 0 ⌬ -C28 5i 444 384, 369, 275, 257, 215 1.25

C29 ⌬22 -C29 5j 456 344, 315, 270, 257, 213, 69 1.29 ⌬22 -C29 5k 456 396, 344, 315, 270, 257, 215, 55 1.33 ⌬0 -C29 5l 458 398, 383, 275, 257, 215 1.49

3.3. Haliclona sp.

The free sterol fraction from Haliclona sp. contained ⌬5-sterols and stanols, cholesterol was shown to be a main sterol. The ratio of stanols:⌬5 compounds in this Ϸ sponge was found to be 1:100, while the ratio of C27:C28:C29:C30 was 100:125:50:1.

3.4. Clathria major

The free sterols from C. major included ⌬5-sterols and stanols. The ratio of stanols:⌬5 compounds in this sponge was found to be 1:100, while the ratio of

C26:C27:C28:C29 was 1:5:4:6. A main sterol was identiﬁed as clionasterol by analysis of its NMR spectra and determination of its 24S conﬁguration as described earlier by Rubinstein et al. (1976).

3.5. Didiscus aceratus

The sponge D. aceratus contained ⌬5-sterols and stanols. The ratio of stanols:⌬5 Ϸ compounds in this sponge was found to be 1:400, while the ratio of C26:C27:C28:C29 was 1:200:350:1000. Therefore, C29-sterols were predominant constituents. A main sterol was identiﬁed as clionasterol in the same manner as in C. major. E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 163

3.6. Teichaxinella labirintica

The free sterol fraction of this sponge contained 3β-hydroxy-A-nor-sterols along with minor common 3β-hydroxy-sterols (3.5% in total fraction). The ratio of A-nor- stanols:⌬5 compounds in this sponge was found to be Ϸ45:1, while the ratio of

C26:C27:C28:C29:C30 was 1:47:96:45:2 (Table 4).

4. Discussion

The sponges represent one of the most ancient groups of animals and some of them contain perhaps the greatest diversity of sterols. We now report a variety of sterols from six Australian sponges. Fig. 2 demonstrates that the studied species contain six different series of sterols, namely ⌬5-sterols, stanols, ⌬7-, ⌬5,7-, ⌬5,7,9(11)- sterols and 3β-hydroxy-A-nor-sterols. T. labirintica belongs to the order Halichondrida. Bergquist et al. (1980) charac- terized this order from the sterol data as an extremely diverse group. Nevertheless, the sterol content from the related species, namely Teichaxinella morchella from the Gulf of Mexico, previously investigated by Djerassi’s group (Bohlin et al., 1981), proved to be very similar to that of T. labirintica. Both sponges have very rare A- nor-sterols with modiﬁed steroidal nuclei. But, in contrast to T. morchella, T. labirintica was found to contain trace amount of usual sterols. Haliclona sp. from the order Haplosclerida has a similar sterol proﬁle with those of other species belonging to the same genus (Bergquist et al., 1980; Sheikh and Djerassi, 1974; Ballantine et al., 1977). All these sponges contain ⌬5-sterols and cholesterol as a dominant constituent. D. aceratus belonging to the order Halichondrida contains ⌬5-sterols with a pre-

Fig. 2. Distribution of free sterols in the sponges studied. 164 E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 ponderance of clionasterol. We have not found in the literature any data concerning sterols from the sponges of this genus. Other species of this order are known to have a variety of sterols, including stanols, ⌬5- and ⌬7-compounds with usual and sometimes unusual side chain structures (Bergquist et al., 1980; Makarieva et al., 1995; Shubina et al., 1984, 1985a, b). D. australiensis belongs to the order Dendroceratida, which lacks a mineral skel- eton, and the sterol profile of this animal shows remarkable variations when com- pared with those of D. gardineri and D. oxeata from New Zealand, studied by Bergquist et al. (1991). C. major from the order Poecilosclerida has a sterol fraction resemble that of D. aceratus with a preponderance of clionasterol. Another species belonging to the same genus, namely Clathria sp. from Western Australia, was earlier investigated (Bergquist et al., 1986). However, sterols of these animals were different: stanols and ⌬7-sterols were identified in Clathria sp. with cholestanol as a dominant sterol. A. mauritiana of our collection has the almost identical sterol mixture as the same animal collected from the Great Barrier Reef by Bergquist et al. (1980). Both specimens contain stanols and ⌬7-sterols. Our data confirm the opinion of Bergquist et al. (1980, 1986, 1991) that particular combinations of sterols of certain structural types appeared to characterize some orders and families of the Porifera. This conclusion seems to be even more sound for some genera belonging to different families. For example, we obtained some additional evidence for the genera Haliclona, Teichaxinella, Agelas. Cholesterol is a predominant sterol of cellular membranes in the animal kingdom, especially in vertebrates. However, such marine invertebrates as holothurians (sea cucumbers), starfish and a series of sponges contain, instead of cholesterol, sterols with modified skeleta or with additional alkyl groups in side chain. The predomi- nance of unusual marine sterols in free sterol fractions may, in many cases, be observed in organisms producing membranolytic agents. Cholesterol is frequently a minor sterol or absent in these species. For example, it is well known that holothurians and starfish, containing toxic triterpene or steroidal glycosides, have a high percentage of stanols, ⌬7-sterols or 14α-methylsterols instead of cholesterol (Stonik and Elyakov, 1988; Makarieva et al., 1993; Stonik et al., 1992, 1998). In a series of toxic sponges we have previously found unusual sterols with highly alkylated side chains or cyclopropane ring in the side chain (Shubina et al., 1984, 1985a, b; Makari- eva et al., 1995, 1996). We have suggested that the peculiarities of free sterol fractions in toxic marine animals would be connected with adaptation to the membranolytic activities of their own toxins. This connection appears to exist in some sponges as in echinoderms. Introduction of unusual sterols including some stanols, ⌬7-sterols and highly alkylated in side chains ⌬5-sterols into cellular membranes makes membranes less sensitive to the actions of the species own membranolytic toxins. For example, the preincubation of cell suspensions (mouse erythrocytes and mouse Ehrlich carcinoma cells) with cyclopropane-containing sterol from the marine sponge Rhizochalina incrustata decreased the cytotoxic and hemolytic effects of rhizochalin, a toxic constituent from the same sponge (Makarieva et al., 1998). We have called the phenomenon, which involves the interdependent presence of two different series E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167 165 of secondary metabolites in living organisms, “biochemical coordination”. Several cases of “biochemical coordination” of the type “membranolytic toxins—unusual sterols” are evidenced on analysis of literature data concerning marine sponges. For example, Theonella swinhoei, containing the toxic macrolide swinholide (Carmely and Kashman, 1985) has unusual 4-methylene-sterols as predominant sterol constituents (Kho et al., 1981); R. incrustata, containing the cytotoxic rhizochalin (Makarieva et al., 1989a, b) has (24R)-24,25-methylene-5α-cholestane-3β-ol as a main sterol (Makarieva et al., 1996); Sarcotragus spinulosus, producing toxic sarcohydroquin- ines and sarcochromenols (Stonik et al., 1992) contains terpenoids instead of sterols (Ponamarenko et al., 1998); Trachyopsis halichondroides and Cymbastela coralliophila, producers of membranolytic trisulphated steroids (Makarieva et al., 1995), contain unusual sterols with additional alkylations in side chains and have no cholesterol in free sterol fractions. Two sponge species examined in this study had ethanol extracts that had stronger membranolytic (hemolytic) activities than the other four sponge extracts (Table 2). Apparently both D. australiensis and A. mauritiana may be considered as additional cases of biochemical coordination “toxins—unusual sterols”. The sterol fractions from A. mauritiana and D. australiensis include significant amounts of ⌬7-sterols, which are similar to the sterol compositions of toxic starfish and holothurians (Stonik and Elyakov, 1988; Stonik et al., 1999). This means that the presence of membranolytic toxins may influence sterol compositions of sponges in some cases. We suggest that unusual sterols may protect cellular membranes of toxic marine animals against action of their toxins. Earlier Djerassi’s group distinguished some sponges containing unusual addition- ally alkylated side chains in sterols as also having the branched demospongic fatty acids in their membrane phospholipids. It was suggested that such “an unusual fea- ture of the membrane may represent a response to stress from sponge’s biotic environment, or it may reflect a specialized requirement of certain membrane-bound enzymes for internal fluidity” (Walkap et al., 1981). We consider that this is not only a response to stress from external factors, but to stress from the species own toxins. But in any case it may be supposed that alterations in membrane compositions accompany toxicity of many sponges.

Acknowledgements

Authors are very grateful to Dr. Olga P. Moiseenko, Dr. Ekaterina G. Lyakhova, Dr. Ludmila P. Ponomarenko for LREIMS measurements and GC analysis. The research described in this publication was possible in part by award No. REC-003 of the US Civilion Research & Development Foundation for the Independent States of the Former Soviet Union (CRDF) and by grants No. 00-15-97806 and No. 01- 04-96907 of the Russian Foundation for Basic Research. 166 E.A. Santalova et al. / Biochemical Systematics and Ecology 32 (2004) 153Ð167

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