Sterols from two marine sedimentary annelids

Comp. Biochem. Physiol. Vol. 70B, pp. 719 to 723, 1981

0305-0491/81/120719-05502.00/0 Copyright © 1981 Pergamon Press Ltd

P~inted in Great Britain. All rights reserved

STEROLS FROM TWO MARINE SEDIMENTARY ANNELIDS D. SICA and G. Dx GIACOMO Istituto di Chimica Organica e Biologica, Universit~t di Napoli, via Mezzocannone 16, Napoli, Italy

(Received 15 April 1981) Abstract--1. The sterol mixtures of two marine sedentary annelids, Chaetopterus variopedatus and Spiro#raphis spallanzani (Phylum Annelida, class Polychaeta) were fractionated by argentation chroma-

tography and were analyzed by combined gas chromatography-mass spectrometry, capfl-lary GLC and coinjection with standards. 2. C. variopedatus and S. spallanzani contain A'-sterols and cholesterol is the major component of the sterol mixtures. In addition to the AS-sterols C. variopedatus also contains five ring saturated sterols, cholestanol being the principal stanol present.

INTRODUCTION In recent years a large number of marine invertebrates have been examined for their sterol content (Goad, 1978; Schmitz, 1978). A number of annelids have been studied and cholesterol has been reported to be the major component of a complex mixture of C26~29AS-mono and diunsaturated sterols (Kobayashi et al., 1973; Kobayashi & Mitsuhashi, 1974; Voogt, 1974; Ballantine et al., 1978) occasionally mixed with lesser amounts of sterols with saturated ring system (Ballantine et al., 1978). F r o m Pseudopotamilla occelata, among other AS-sterols, Kobayashi & Mitsuhashi (1974) isolated 27-nor-(24S)methylcholesta-5,22E-dien-3fl-ol, a sterol with unusual side chain. The present work reports the sterol composition of marine sedentary annelids Spirographis spallanzani Viviani and Chaetopterus variopedatus (Renier) as a part of our work on sterols of marine invertebrates. MATERIALS AND METHODS

Animals Annelids were collected in the Bay of Naples and supplied by the Zoological Station, Naples.

Extraction and separation Fresh tissue from Spirooraphis spallanzani and Chaetopterus variopedatus was homogenized with CHCIa:MeOH (1:1) at room temperature, filtered and the residue extracted a second time with CHCI3. The extracts were saponified by refluxing for 1.5 hr in 10% KOH in 80% aqueous EtOH. The unsaponifiable lipid was separated on a column of silica gel using CH2C12 as eluent. The total sterols were acetylated overnight using acetic anhydridepyridine (1:1) and the acetates were purified over a silica gel column eluted with light petroleum (b.p. 40-70°C)-benzene (7:3). The steryl acetates of S. spallanzani (0.8 g) were separated by column chromatography silica gel (80 g) impregnated with silver nitrate (20 g) which was eluted with light petroleum-benzene as shown in Table 1. The various column fractions (50ml) were monitored by GLC and combined accordingly. The steryl acetates of C. variopedatus (0.3 g) were separated into five fraction (1-5 in order of increasing polarity)by TLC on AgNO3-silica gel (1:9) developed twice with hexane-benzene 1:1. The less polar 719

band 1 was further separated into two fraction (1-a and I-b) by silica gel-AgNO3 plates developed twice with hexane-benzene 1 : 1 (Table 2).

Analytical methods Analytical GLC of steryl acetates was performed with a Carlo Erba 2920 gas chromatograph using a SE-30 fused silica capillary column at 248°C. The retention time relative to that of cholesteryl acetate (1.00) on GLC for the standard samples of fucosteryl acetate and 28-isofucosteryl acetate were 1.60 and 1.65, respectively. NMR spectra were recorded with a Bruker WH-270 in CDCI3 with TMS as internal standard. Combined GC-MS analysis was performed on an LKB 2091 S GC-MS instrument using a glass column packed with OV-61 at 250°C. Diagnostic ions present in the GC-mass spectra of the steryl acetates from annelids were as follows. Steroi lb m/e (rel. int.): 352 (100) M÷-60, 337 (10), 255 (54), 253 (~, 228 (7), 213 (12), 282 (6), 267 (3), 244 (5), 231 (7), 97 (82); sterol 3b: 366 (100) M+-60, 351 (12), 255 (55), 253 (8), 228 (7), 213 (12), 282 (8), 267 (4), 245 (5), 111 (40); sterol 4b: 428 (30) M ÷, 413 (5), 368 (2), 353 (5), 315 (37), 25"/ ( 1 ~ , 253 (14), 229 (7), 274 (6), 215 (16), 344 (63), 329 (22), 284 (4), 269 (11); sterol __5b:368 (100) M+-60, 353 (25), 255 (22), 228 (5), 213 (19), 260 (22), 247 (26); sterol 6b: 430 (30) M +, 415 (11), 370 (26), 355 (23), 257 (5), 290 (6), 230 (27), 275 (32), 276 (32), 215 (100), 316 (3), 247 (4); sterol 7b: 366 (100) M÷-60, 351 (24), 255 (12), 253 (38), 228 (12), 213 (18), 282 (7), 281 (8), 245 (14), 69 (100); sterol __8b:380 (100) M+-60, 365 (8), 255 (72), 253 (14), 228 (14), 213 (16), 337 (12), 282 (12), 267 (5), 272 (4), 259 (4); sterol __9b:380 (100) M+-60, 365 (19), 255 (14), 253 (26), 228 (13), 213 (25), 296 (54), 281 (19), 338 (4), 337 (2), 272 (8), 259 (13); sterol lOb: 442 (6) M +, 427 (10), 382 (2), 367 (9), 315 (43), 257 (6), 255 (23), 229 (22), 275 (15), 215 (52), 358 (100), 343 (21), 298 (8), 283 (13); sterol ll._bb: 382 (100) M+-60, 367 (38), 255 (29), 228 (8), 213 (25), 274 (28), 261 (28); sterol 12b: 444 (27) M +, 429 (18), 384 (21), 369 (9), 257 (21), 290 (14), 230 (29), 275 (35), 276 (40), 215 (100), 247 (6); sterol 13b: 394 (1120)M+-60, 379 (9), 255 (73), 253 (19), 228 (18), 213 (18), 351 (26), 282 (14), 267 (6), 286 (4), 273 (4); sterol 14b: 396 (100) M÷-60, 381 (19), 255 (18), 228 (5), 213 (17), 288 (14), 275 (15); sterol 1510:458 (63) M +, 443 (14), 398 (45), 383 (43), 257 (5), 290 (20), 230 (25), 275 (34), 276 (35), 215 (100), 247 (9); sterol 1610:394 (20) M +-60, 379 (3), 255 (4), 253 (9), 228 (12), 213 (15), 296 (100), 281 (16), 273 (4), 55 (54); sterol 17b: 408 (13) M+-60, 393 (4), 255 (4), 253 (10), 228 (6), 213 (6), 296 (100), 281 (16). The mass spectral fragmentations of the above-mentioned steryl acetates were in agreement with published

720

D. SIcx and G. Dl GIACOMO

9 s u O d S O J

J0pJ039J

E> ,,.-~ Q m

, o

~

~ __...._-.--~

o~ ::a c:

E

O'g

aso0dsaJ

J0pJ090J --o

%

O

F

]

I

E

2

Sterols from C. variopedatus and Sp. spallanzani

721

Table 1. Column chromatography on silica gel-AgNO3 of the steryl acetates from Spirooraphis spallanzani Fractions

Eluent p:b* v/v

wt, mg

61-73

85-15

355

Steryl acetatest 5b (88~), 11....bb(3%) and 14__._bb(99/0).

74-76

10

5b(40~), 11.._b.b(10~), 13__._bb(20~) and 14__._bb(309/0).

77-86

11

5b (9~o) and 13_.._bb(91%).

9

8b (89~o) and 13__.bb(11%).

87-90

75-25

91-95

66

96-99

70-30

54

100-104

60-40

134

106-113

57

8b.

I

2b (60~), 3b (31~o) and 8b (9~o). l b (13~), 2b (5%), 3b (59%), 1619(18~o) and 17__.bb(5~). 9b.

I

* p = 40-70 ° Light petroleum; b = benzene. t See formulae.

data (Knights, 1967; Wyllie & Djerassi, 1968; Knights & Brooks, 1969; Sheikh & Djerassi, 1974; Zaretskii, 1976; Djerassi, 1978). RESULTS

AND

DISCUSSION

The unsaponifiable fraction from the chloroformmethanol extract of each annelid was chromatographed on a silica gel column and the fraction containing sterols was subsequently acetylated. Gas chromatograms of the mixed steryl acetates obtained from Spirographis spallanzani and Chaetopterus variopedatus are reported in Figs 1 and 2 respectively. The steryl acetates of S. spallanzani were separated by column chromatography on silica gel impregnated with AgNO3 (Table 1). Individual steryl acetates of C. variopedatus were identified after separation of the mixture into six

zones by argentation TLC (Table 2). The identification of sterols was based on relative G L C retention times, coinjection and comparison of the G C - M S spectra with those of authentic samples obtained from other marine invertebrates (Sica et al., 1978, 1980, 1981; Sica, 1980). The percent composition of sterol mixtures and the retention times of the acetate derivatives are shown in Table 3. The annelids S. spallanzani and C. variopedatus contain a complex mixture of sterols widely distributed in the marine environment, cholesterol being the principal sterol present. The steroi composition of S. spallanzani obtained from the Bay of Naples is similar to that found by Voogt (1974) in a specimen collected at Banyuls-sur-mer, France. In addition to the common AS-sterols C. variopedatus also contains five 5~-stanols among which cholestanol is the major

Table 2. Argentic TLC of the steryl acetates from Chaetopterus

variopedatus TLC Band

wt, mg

Stanyl and steryl acetates

1-a

37

6b (909/0), 12b (6%) and 15b (4%).

1-b

103

.5b (81~o), l l b (7%) and 14b (12%).

2

75

lb (2%), 2b (9%), 3b (24%), 4b (4%), 5b (21%), 8b (33~o) and 13b (7%).

3

16

l b (19%), 3b (25%), 7b (21%), 16b (29~) and 17b (~/o).

4

5

5

17

9b (20%), lOb (61%) and 16b (19%). 9b.

722

D. SIcx and G. D] GIACOMO

R

R

R

R ' O ' R'O/~ aR = ' Hb, R'=COCH3 H

9

10

11

12

13

14

16

17

15

~

723

Sterols from C. variopedatus and Sp. spallanzani Table 3. Sterol composition of annelids (%) GC peak A B C D E F G H 1 I J J K L L M N

RRT* 0.66 0.89 0.92 0.93 1.00 1.02 1.08 1.11 1.26 1.28 1.30 1.31 1.40 1.60 1.61 1.65 1.92

Sterol 24-Norcholesta-5,22-dien-3fl-ol (la) 27-Nor-24-methylcholesta-5,22-dien-3fl-ol (2a)1" Choiesta-5,22-dien-3fl-ol (3a) 5ct-Cholest-22-en-3fl-ol (4a) Cholesterol (5a) 5a-Cholestan-3fl-ol (6a) Cholesta-5,24-dien-3fl-ol (7a) 24-Methylcholesta-5,22-dien-3fl-ol (8a) 24- Methylenecholesterol (9a) 24-Methyl-5ct-cholest-24(28)-en-3fl-ol (10a) 24-Methylcholest-5-en-3fl-ol (lla) 24-Methyl-5~t-cholestan-3fl-ol (12a) 24-Ethylcholesta-5,22-dien-3fl-ol (13a) 24-Et hylcholest-5-en-3fl-ol (14a) 24-Et hyl-5~t-cholestan-3fl-ol (15a) (24Z)-24-Ethylcholesta-5,24(28)-dien-3fl-ol (16a) 24-Propylidenecholest-5-en-3fl-ol (17a)

S. spallanzani

C. variopedatus

2.5 5.6 14.0 t~ 45.6 t -11.3 8.2 t 1.6 -1.8 5.0 -3.5 0.9

1.6 2.7 8.7 1.2 39.5 13.0 1.2 9.9 7.1 1.1 2.8 0.9 2.0 4.8 0.5 2.3 0.4

* RRT, retention time of acetate derivatives relative to cholesteryl acetate (1.00) on SE-30 capillary column. l" Deduced from the RRT. Indicates trace amounts.

component, in agreement with previous reports for other annelids (Ballantine et al., 1978). In S. spallanzani AS-sterols are present a n d traces of stanols could be detected in c h r o m a t o g r a m s a n d mass spectra.

Acknowledgements--This work was supported by the "Progetto Finalizzato per l'Oceanografia ed i Fondi Marini", C.N.R. We thank Professor L. Boniforti (Istituto Superiore di SanitY, Roma) for GC-MS determinations and Zoological Station (Napoli) for supplying with annelids. REFERENCES BALLANTINE J. A., LAVIS A., ROBERTS J. C., MORRIS R. J., ELSWORTH J. F. & CRAGG G. M. L. (1978) Marine sterols---VII. The sterol compositions of oceanic and coastal marine Annelida species. Comp. Biochem. Physiol. 61B, 43-47. DJERASSI C. (1978) Recent advances in the mass spectrometry of steroids. Pure Appl. Chem. 50, 171-184. GOAD L. J. (1978) The sterols of marine invertebrates: composition, biosynthesis and metabolites. In Marine Natural Products (Edited by SCHEUER P. J.), Vol. II, pp. 75-172, Academic Press, New York. K~IGnTS B. A. (1967) Identification of plant sterols using combined GLC/mass spectrometry. J. gas. chromat. 5, 273-282. KNIGHTS B. A. & BROOKS C. J. W. (1969) Isomers of 24-ethylidenecholesterol: gas chromatografic and mass spectrometric characterization. Phytochemistry 8, 463-467.

KOBAYASHI M. & MITSUHASHI H. (1974) Marine sterols V. Isolation and structure of occelasterol, a new 27-norergostane-type sterol, from an Annelida, Pseudopotamilla occelata. Steroids 24, 399-410. KOBAYASHIM., NISmZAWA M., Totx) K. & MITSUHASHI H. (1973) Marine sterois I. Sterols of Annelida, Pseudopotamilla occelata Moore. Chem. Pharm. Bull. 21, 323-328. SCHMITZ F. J. (1978) Uncommon marine steroids. In Marine Natural Products (Edited by SCHEUER P. J.), Vol. 1, pp. 241-297, Academic Press, New York. SHEIKH Y. M. & DJERASSl C. (1974) Steroids from sponges. Tetrahedron 30, 4095-4103. SICA D. (1980) Sterols from some molluscs. Comp. Biochem. Physiol. 65B, 407-410. SICA D., BONWORTI L. & DI GIACOMO G. (1981) Sterol composition of two Actiniaria. Comp. Biochem. Physiol. 70, 153-156. SICA D., DE SIMONE F. & ZOLLO F. (1978) Sterols from the sponge Chondrilla nucula. Gaz. Chim. Ital. 108, 575-578. SICA D., DE SIMONE F., SENATORE F. & ZOLLO F. (1980) Synthesis of two stanols identified in the sponge Suberites carnosus. Gazz. Chim. Ital. 110, 147-150. VOOGT P. A. (1974) Biosynthesis and composition of sterols in Annelida I. Investigations on some polychaetes. Netherl. J. Zool. 24, 22-31. WILLIE S. G. & DJERASSI C. (1968) Mass spectrometry in structural and stereochemical problems CXLVL Mass spectrometric fragmentations typical of sterols with unsaturated side chains. J. org Chem. 33, 305-313. ZARETSKn Z. V. (1976) Mass Spectrometry of Steroids. Israel Universities Press, Jerusalem.