B/ac/m,m~ Syslem~:s and Eco/ogy, Vol. 13, No. 2, p~. 167-168, 1985. 0305-1978/85 $3.00+0.00 Printed in Great Britain. Pergamo~ Press Ltd.

Sterols of Four Mediterranean Hydroids

E. FA'I'I'ORUSSO, V. LANZOI"rl, S. MAGNO and E. NOVELLINO Dipart imento di Chimica delle Sostanze Naturali-Universit8 di Napoli, Via L Rodinb 22. 1-80138 Napoli, Italy

K e y W o r d I n d e x ~ l e n t e r a t a ; hydroids; tecata; atecata; Aa-sterols.

A b ~ t r a c t m T h e distribution of sterols in four Mediterranean hydroids has been examined. Cholesterol is the principal sterol in these organisms which uniquely contain Ae-sterols, including a rather uncommon C== compound.

Int roduct ion Although during the last decade many species of the marine invertebrates have been examined for their sterol contents and a host of novel com- pounds have been characterized, only a few data have so far been reported in the literature on the sterol composition of hydroids, which is the simplest class belonging to the phylum Coelenterata [1,2]. This is largely due to the difficulties of collecting and identifying the biological material.

Very recently, as a part of a program to investigate the natural products from marine sources of the Mediterranean Sea, we had the

opportunity of examining four species of hydroid colonies [3], Aglaophenia p/uma and Sertularella crassicaulis, beloning to the subor- der Tecata, and Halocordyle disticha and Eudendrium glomeratumto Atecata, collected in the Bay of Naples and we report here their sterol composition.

Results and Discussion Our results, listed in Table 1, revealed that the four hydroids uniquely contain Ae-sterols, in- cluding a rather uncommon C=e compound. Like data obtained with the majority of other

"[able 1. STEROL COMPOSITION OF HYDROIDS (mg/kg dry material)

Eudendn'um Ha/ocordyle Aglaophenia Sertularella Sterol (R,) g/omeraturn disticha pluma cra$$icaulis

1 24-Norcholesta-5,22-dien-3,8ool (0.69) 4 4 31 7 2 27-Nor-24-methylcholesta-5,22-dien-3.8-ol (0.87) 4 4 11 8 ,3 Cholesta-5,22-dien-3~'-ol (0.91) 19 27 40 35 4 Cholesterol (1,00) 107 90 133 93 6 Cholesta-5,24-dien-3~'-ol (1.08) - - 11 - - - - 6 24-Methylcholesta-5,22-dien-3#-ot (1.09) 36 7 33 23 7 24.Methylenecholest.5.en.3/?-ol (1.24) 13 4 10 11 S 24-Methylcholest-5-en-3#-ol (1.26) 3 3 5 5 9 24-Ethylcholesta-5,22-dien-3,8-ol (1.31) 6 1 3 3

10 24-Ethylcholest-5-en-3,8-ol (1.48) 9 9 9 16 11 (24Z)-24-Ethylcholesta-5,24(28)-dien-31g-ol (1.50) 7 5 4 18 12 Cholesta - 5,23- diene- 3#,25-diol - - t - - - - 13 Choleeta - 5,25- diene-3~',24-diol - - t - - - -

Retention time (R,) of acetate derivatives relative to cholesteryl acetate (1.00) on OV-101 capillary column, t.

(Received 14 March 1 984)

167

Trace amounts.

168 E. FA'I'I'ORUSSO, V. LANZOTTI, S. MAGNO AND E. NOVELLINO

Coelenterata, the predominant sterol is choles- terol; also present in substantial quantities are 24-methylcholesta - 5,22-dien- 3/~'-ol, cholesta- 5,22-dien-3#-ol and 24-methylenecholest- 5-en-3~'-ol, which are also found in other marine invertebrates.

Examination of the sterol profiles indicates that all the species of hydroids under investi- gation, which are taxonomically very different, have a similar composition, the only exception being the presence in H. disticha of desmosterol (5) and trace amounts of the sterols 12 and 13 (identified by spectra) which could be artifacts caused by autoxidation of § during the air- drying of the fresh material [6].

This was though likely since the biological material was unprotected from aerial degrad- ation during extraction and purification. As a confirmation, fresh material was harvested again and extracted immediately following air-drying in the dark. Only trace amounts of liagosterol (12) and its isomer (13) could then be found.

This hypothesis was substantiated when additional quantities of 12 and 13 were obtained from the sterol 'pool', which had been exposed to air for several weeks.

Experimental Co//action and extraction. Colonies of the four hydroids, listed =n Table1, were collected in the Bay of Naples (November 1982-February 1983) and freed by hand from macroscopic epibionts. A freshly collected sample of each spec'~s (ca. 500 g) was freeze dried and extracted with CHCI., (3 x 600 m[) at room temp. After removal of the solvent to dryness, the combined extracts were saponified with 10% KOH in 80% aqueous EtOH (50ml). The non- saponifiable fraction was chromatographed, under pressure,

on a silica gel column using C6H~-Et20 (4:1) as the eluent and the appropriate fractions were bulked and taken to dryness.

Identification of the sterol$. The crude sterol fraction of each organism, obtained as above, was aoatylated (Ac=O-CsHeN, 1:1 for 12 h at room temp.) and further purified by a SiO= column using as eluent 40-70 ° light petroleum-Cell 8, 7:3 and analysed by GC/MS using a Hewlett-Packard 5995 A instrument equipped with a glass capillary column (15 m x 0.2 mm i.d.) coated with OV-101, f low of N=:1.5 ml/min, oven temp. 250 °.

The identification of the steryl acetates was based upon their GCR, and comparison of the GC/MS spectra, with those of authentic specimens.

The quantitation of the sterols was performed by a programmable integrator using 5o-cholsetane as an internal standard.

TLC (SiO=, eluent CeHe-Et=O, 1:1) of an aliquot of the crude sterol fraction from H. disticha and elution of the bands Rt 0.65 and 0.55 gave 12 and 13, respectively, which were identified by comparison of their spectral properties with those of authentic samples [4, 51.

Acknowledgements - -Th is work was supported by "Ministero della Pubblica Istruzione, Italia'. We thank Dr. G. Valentini (Istituto Chimica Organice Industriate UniversitY, Pisa) for GC/MS analyses and Dr. M. Pansini for identifying the hydroids.

References 1. Goad, L. J. (1978) in MarinelVaturalProduct$ (Scheuer,

P. J., ed.). Vol. 2, p. 76. Academic Press, New York. 2. Crist, B. V., Li, X., Bergquist, P. R. and Djeressi, C. (1983)

J. Org. Chem. 48, 44-72 (and preceding papers of the series).

3. De Napoli, L., Fattorusso, E., Magno, S. and Mayol, L. (1984) Biochem. Syst. EcoL 12, 321-322.

4. Morisaki, M., Kidooka, S. and Ikekava, N. (1976) Chem. Pharm. Bu/I. 24, 3214.

5. Fattorusso, E., Magno, S., Santacroce, C., Sica, D., Impellizzeri, G., Mangiafico, S., Oriente, G., Piattelli, M. and Sciuto, S. (1975) Phytochemistry 14, 1579.

6. Francisco, C., Combaut, G., Taste, J., Tarchini, C and Djerassi, C. (1979) Steroids 34, 2462.