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Acyclic diterpenes and sterols from the genera Bifurcaria and Bifurcariopsis (Cystoseiraceae, Phaeophyceae)
Biochemical Systematics and Ecology 29 (2001) 973–978
Acyclic diterpenes and sterols from the genera Bifurcaria and Bifurcariopsis (Cystoseiraceae, Phaeophyceae) Mohammed Daoudia, Salem Bakkasa, G!erald Culiolib, Annick Ortalo-Magne! b, Louis Piovettib,*, Michael D. Guiryc Laboratoire de Chimie Organique et Bioorganique, Faculte! des Sciences Choua.ıb Doukkali, El Jadida, Morocco b ! Laboratoire de Recherche en Chimie Marine des Organometalliques, Universite! de Toulon et du Var, BP 132, F-83957 La Garde cedex, France c Department of Botany, Martin Ryan Institute, National University of Ireland, Galway, Ireland a
Received 3 October 2000; accepted 4 December 2000
Keywords: Bifurcaria bifurcata; B. galapagensis; B. brassicaeformis; Bifurcariopsis capensis; Acyclic diterpenes; Sterols; Chemotaxonomy
1. Subject and source As a part of our chemotaxonomic studies of the algal family Cystoseiraceae (Culioli et al., 1999a, b; Culioli et al., 2000; Valls and Piovetti, 1995; Valls et al., 1995) we have now investigated lipid extracts from Bifurcaria brassicaeformis . (Kutzing) Barton and Bifurcariopsis capensis (Areschoug) Papenfuss, both of which have not been previously examined. Voucher specimens of these species (no. 10082 for Bifurcaria brassicaeformis from Simons Town [348120 S, 188270 E] in South Africa; no. 10083 for B. brassicaeformis from Clovelly [34870 S, 188260 E] in South Africa; no. 10084 for Bifurcariopsis capensis from Clovelly in South Africa) are deposited in the herbarium of one of us (Phycological Herbarium of M. D. Guiry; GALW). In addition to so that we can complete our studies of geographical variations in the diterpene composition of Bifurcaria bifurcata (Velley) Ross, studies of which were limited to the Moroccan and French Atlantic coasts in our previous
*Corresponding author. Tel.: +33-494-142-346; fax: +33-494-142-168. E-mail address: [email protected] (L. Piovetti). 0305-1978/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 3 0 - 8
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publications (Valls et al., 1993a, b; Valls et al., 1995), we have also reinvestigated the lipid extracts of this species collected off the Irish and Spanish Atlantic coasts. Voucher specimens of this species are deposited in the herbarium of one of us (Pr M. D. Guiry) for the Irish collection: Black Head, Co. Clare [53890 N, 98160 W] (no. 10085), and the herbarium of Dr Pellegrini (Laboratoire de Biologie Marine Fondamentale et Appliqu!ee, Universit!e de la Mer, France) for the Spanish * [438330 N, collections: Vigo [428390 N, 88470 W] (no. P 196.3) and La Coruna 0 8817 W] (no. P 195.5).
2. Previous work The algal family Cystoseiraceae (order Fucales, class Phaeophyceae) is typified by the name-bringing genus Cystoseira, which includes some 66 currently recognised species, 34 of which occur in the Mediterranean and 17 in the NE Atlantic (Guiry, unpubl. data). By contrast, Bifurcaria and Bifurcariopsis are relative small genera of the Cystoseiraceae. Bifurcaria currently includes three species: B. bifurcata (Velley) R. Ross, the generitype (type species) distributed on Atlantic coasts from Morocco (southern limit) to north-western Ireland (northern limit); B. brassicaeformis . (Kutzing) Barton confined to the western (Stegenga et al., 1997) and Indian Ocean . coasts of South Africa (Silva et al., 1996); and B. galapagensis (Piccone & Grunow) Womersley (Womersley, 1964), although the last requires further study as it shows morphological features not typical of the genus as typified by B. bifurcata (Womersley, 1964). Bifurcariopsis is monotypic and the generitype B. capensis (Areschoug) Papenfuss is known only from South Africa (Nizamuddin, 1970; Silva et al., 1996). Of these four species, only two Bifurcaria species have previously been studied chemically, namely, B. bifurcata and B. galapagensis. Previous studies of the secondary metabolites from lipid extracts of B. bifurcata have shown that this species contains a rich array of acyclic diterpenes (Biard et al., 1980; Combaut and Piovetti, 1983; Culioli et al., 1999a, b; Culioli et al., 2000; Hougaard et al., 1991; Semmak et al., 1988; Valls et al., 1986; Valls et al., 1993a, b; Valls et al., 1995). These compounds are geranylgeraniol and geranylgeraniol-derived diterpenes of which the principal ones are (S)-13-hydroxygeranylgeraniol (1), 13-ketogeranylgeraniol (2), the (S)-13-hydroxyfuranoditerpene (3) and (S)-12-hydroxygeranylgeraniol (4). By contrast, B. galapagensis shows the presence of the meroditerpene 5 (bifurcarenone) as the main constituent of the diterpene composition of its lipid extract (Sun et al., 1980; Mori and Uno, 1989).
3. Present study Ether extracts of dried Bifurcaria brassicaeformis and Bifurcariopsis capensis collected at Simons Town and Clovelly (South Africa) were investigated in order to study the lipophilic secondary metabolites and to compare the results with those of previous studies on Bifurcaria bifurcata and B. galapagensis.
M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
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M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
Table 1 Determination of sterols and diterpenes (mg g1 algal dry wt) Species
Sterolsa
Diterpenesb
Bifurcaria brassicaeformis Simons town Clovelly
5.2 5.7
} }
Bifurcariopsis capensis Clovelly
1.9
}
Bifurcaria bifurcata Quiberon Roscoff El Jadida Oualidia
1.5 1.5 1.7 1.6
6.1 5.9 6.6 4.5
a b
Totally analysed as fucosterol. Total amount of diterpenes.
Crude extracts (1.2% and 0.4% of algal dry weight for Bifurcaria brassicaeformis and Bifurcariopsis capensis, respectively) were fractionated by column chromatography using silica gel as adsorbent and Et2O as eluant. After evaporation, each residue was analysed by normal-phase HPLC (EtOAc–isooctane, 1 : 1), using a method previously described for the determination of sterols and diterpenoids from Cystoseiraceae (Piovetti et al., 1991). This study (Table 1) showed the total absence of diterpenes in both species, and the only sterol detected was fucosterol (main sterol of the brown algae). For comparison, ether extracts of Bifurcaria bifurcata collected at different locations (French collections: Quiberon [478290 N, 3870 W] and Roscoff [488440 N, 38590 W]; Moroccan collections: El Jadida [338150 N, 88310 W] and Oualidia [328490 N, 88570 W]) were analysed in the same way (Table 1). Ether extracts of dried Bifurcaria bifurcata collected at three separate locations} * (Spain)}were investigated to Black Head (Ireland), and Vigo and La Coruna complete the study of the geographical variations in the diterpene composition of this species. The different collections were treated and extracted in an identical manner (Valls et al., 1993a). Each extract was analysed by HPLC (EtOAc–isooctane, 1 : 1) with the method of Piovetti et al. (1991), leading to the results listed in Table 2. For the identification of compounds analysed, extracts were fractionated by liquid chromatography using silica gel. These fractions were subsequently subjected to semi-preparative normal-phase HPLC (EtOAc–isooctane, 2 : 3 and then 1 : 1), leading to pure geranylgeraniol (about 10 mg for each extract), 6 (about 20 mg for each extract), 2 (about 500 mg for each extract) and 1 (about 200 mg for each extract). Their structures have been established by means of spectral methods (IR, MS, NMR) and compared with data of authentic samples isolated from other extracts of this species previously studied. It must be specified that 6 is a minor constituent of the diterpene composition of B. bifurcata which was previously
M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
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Table 2 Geographical variations in the diterpene composition of Bifurcaria bifurcata Compounds (mg g1 algal dry wt) Locations Black Head (Ireland) Vigo (Spain) * (Spain) La Coruna Quiberona (France) a b
1 1.1 0.9 0.7 1.4
2
6
Geranylgeraniol
5.0 5.3 5.5 4.7
b
b
b
b
b
b
b
b
Added for comparison. Present as trace (amount not determined).
Table 3 Chemical groups of species of the genera Bifurcaria and Bifurcariopsis Chemical groups
Secondary metabolites
Species
I
No diterpenoids (sterolic metabolism privileged) Linear diterpenes (geranylgeraniol metabolism) Meroditerpenes (shikimic acid and geranylgeraniol pathways) Linear meroditerpenes Cyclic meroditerpenes Rearranged meroditerpenes
Bifurcaria brassicaeformis Bifurcariopsis capensis Bifurcaria bifurcata
II III IIIA IIIB IIIC
} Bifurcaria galapagensis }
described in the lipid extract of a specimen collected at Quiberon (Culioli et al., 1999b).
4. Chemotaxonomic significance As shown in Table 1, the chemical study of two previously unstudied species belonging to the genera Bifurcaria (B. brassicaeformis) and Bifurcariopsis (B. capensis) shows the total absence of diterpenes in their lipid extracts. In these entities, the secondary metabolism of the seaweeds seems to prioritise the synthesis of sterols instead of diterpenoids. The only significant chemical difference between them is the large difference existing in the yield of their lipid extract (1.2% for Bifurcaria brassicaeformis and 0.4% for Bifurcariopsis capensis). They can be precisely distinguished from the closely related species Bifurcaria bifurcata and B. galapagensis since, by contrast, the former contains a rich array of acyclic diterpenes and the latter a cyclic meroditerpene (5), perhaps indicating a degree of phylogenetic unrelatedness that requires further investigation. Accordingly, we therefore include these two previously uninvestigated species in the chemical group (I) of our classification of Cystoseiraceae (Valls and Piovetti, 1995) (Table 3).
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M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
By contrast, the results listed in Table 2 show that the diterpene composition of Bifurcaria bifurcata collected at three new locations off the Irish and Spanish coasts is closely related to that of Quiberon in France. These specimens can be clearly included in the chemical type 1 previously described in the study of the geographical variations in the diterpenoid composition of B. bifurcata (Valls et al., 1995). This type was characterized by the presence of 2 and 1 as the main diterpenoids of the lipid extract.
Acknowledgements The authors wish to thank Ms Lorna Kelly and Dr John Bolton (Martin Ryan Institute, National University of Ireland, Galway, Ireland; Department of Botany, University of Cape Town, South Africa) for identification and collection of Bifurcaria brassicaeformis and Bifurcariopsis capensis in South Africa, and Pr Robert Valls (Laboratoire des Organo-Phosphor!es, Universit!e d’Aix-Marseille, France) for identification and collection of Bifurcaria bifurcata in Spain.
References Biard, J.F., Verbist, J.F., Floch, R., Letourneux, Y., 1980. Tetrahedron Lett. 21, 1849. Combaut, G., Piovetti, L., 1983. Phytochemistry 22, 1787. Culioli, G., Mesguiche, V., Piovetti, L., Valls, R., 1999a. Biochem. Syst. Ecol. 27, 665. Culioli, G., Daoudi, M., Mesguiche, V., Valls, R., Piovetti, L., 1999b. Phytochemistry 52, 1447. Culioli, G., Di Guardia, S., Valls, R., Piovetti, L., 2000. Biochem. Syst. Ecol. 28, 185. Hougaard, L., Anthoni, U., Christophersen, C., Nielsen, P.H., 1991. Phytochemistry 30, 3049. Mori, K., Uno, T., 1989. Tetrahedron 45, 1945. Nizamuddin, M., 1970. Bot. Mar. 13, 131. Piovetti, L., Deffo, P., Valls, R., Peiffer, G., 1991. J. Chromatogr. 588, 99. Semmak, L., Zerzouf, A., Valls, R., Banaigs, B., Jeanty, G., Francisco, C., 1988. Phytochemistry 27, 2347. Silva, P.C., Basson, P.W., Moe, R.L., 1996. Univ. Calif. Berkeley Publ. Bot. 79, 1. Stegenga, H., Bolton, J.J., Anderson, R.J., 1997. Bolus Herbarium. University of Cape Town, Cape Town. Sun, H.H., Ferrara, N.M., McConnell, O.J., Fenical, W., 1980. Tetrahedron Lett. 21, 3123. Valls, R., Banaigs, B., Francisco, C., Codomier, L., Cave, A., 1986. Phytochemistry 25, 751. Valls, R., Banaigs, B., Piovetti, L., Archavlis, A., Artaud, J., 1993a. Phytochemistry 34, 1585. Valls, R., Banaigs, B., Piovetti, L., Zerzouf, A., 1993b. Ann. Inst. Oc!ean. 69, 215. Valls, R., Piovetti, L., 1995. Biochem. Syst. Ecol. 23, 723. Valls, R., Piovetti, L., Banaigs, B., Archavlis, A., Pellegrini, M., 1995. Phytochemistry 39, 145. Womersley, H.B.S., 1964. Aust. J. Bot. 12, 53.
Acyclic diterpenes and sterols from the genera Bifurcaria and Bifurcariopsis (Cystoseiraceae, Phaeophyceae) Mohammed Daoudia, Salem Bakkasa, G!erald Culiolib, Annick Ortalo-Magne! b, Louis Piovettib,*, Michael D. Guiryc Laboratoire de Chimie Organique et Bioorganique, Faculte! des Sciences Choua.ıb Doukkali, El Jadida, Morocco b ! Laboratoire de Recherche en Chimie Marine des Organometalliques, Universite! de Toulon et du Var, BP 132, F-83957 La Garde cedex, France c Department of Botany, Martin Ryan Institute, National University of Ireland, Galway, Ireland a
Received 3 October 2000; accepted 4 December 2000
Keywords: Bifurcaria bifurcata; B. galapagensis; B. brassicaeformis; Bifurcariopsis capensis; Acyclic diterpenes; Sterols; Chemotaxonomy
1. Subject and source As a part of our chemotaxonomic studies of the algal family Cystoseiraceae (Culioli et al., 1999a, b; Culioli et al., 2000; Valls and Piovetti, 1995; Valls et al., 1995) we have now investigated lipid extracts from Bifurcaria brassicaeformis . (Kutzing) Barton and Bifurcariopsis capensis (Areschoug) Papenfuss, both of which have not been previously examined. Voucher specimens of these species (no. 10082 for Bifurcaria brassicaeformis from Simons Town [348120 S, 188270 E] in South Africa; no. 10083 for B. brassicaeformis from Clovelly [34870 S, 188260 E] in South Africa; no. 10084 for Bifurcariopsis capensis from Clovelly in South Africa) are deposited in the herbarium of one of us (Phycological Herbarium of M. D. Guiry; GALW). In addition to so that we can complete our studies of geographical variations in the diterpene composition of Bifurcaria bifurcata (Velley) Ross, studies of which were limited to the Moroccan and French Atlantic coasts in our previous
*Corresponding author. Tel.: +33-494-142-346; fax: +33-494-142-168. E-mail address: [email protected] (L. Piovetti). 0305-1978/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 3 0 - 8
974
M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
publications (Valls et al., 1993a, b; Valls et al., 1995), we have also reinvestigated the lipid extracts of this species collected off the Irish and Spanish Atlantic coasts. Voucher specimens of this species are deposited in the herbarium of one of us (Pr M. D. Guiry) for the Irish collection: Black Head, Co. Clare [53890 N, 98160 W] (no. 10085), and the herbarium of Dr Pellegrini (Laboratoire de Biologie Marine Fondamentale et Appliqu!ee, Universit!e de la Mer, France) for the Spanish * [438330 N, collections: Vigo [428390 N, 88470 W] (no. P 196.3) and La Coruna 0 8817 W] (no. P 195.5).
2. Previous work The algal family Cystoseiraceae (order Fucales, class Phaeophyceae) is typified by the name-bringing genus Cystoseira, which includes some 66 currently recognised species, 34 of which occur in the Mediterranean and 17 in the NE Atlantic (Guiry, unpubl. data). By contrast, Bifurcaria and Bifurcariopsis are relative small genera of the Cystoseiraceae. Bifurcaria currently includes three species: B. bifurcata (Velley) R. Ross, the generitype (type species) distributed on Atlantic coasts from Morocco (southern limit) to north-western Ireland (northern limit); B. brassicaeformis . (Kutzing) Barton confined to the western (Stegenga et al., 1997) and Indian Ocean . coasts of South Africa (Silva et al., 1996); and B. galapagensis (Piccone & Grunow) Womersley (Womersley, 1964), although the last requires further study as it shows morphological features not typical of the genus as typified by B. bifurcata (Womersley, 1964). Bifurcariopsis is monotypic and the generitype B. capensis (Areschoug) Papenfuss is known only from South Africa (Nizamuddin, 1970; Silva et al., 1996). Of these four species, only two Bifurcaria species have previously been studied chemically, namely, B. bifurcata and B. galapagensis. Previous studies of the secondary metabolites from lipid extracts of B. bifurcata have shown that this species contains a rich array of acyclic diterpenes (Biard et al., 1980; Combaut and Piovetti, 1983; Culioli et al., 1999a, b; Culioli et al., 2000; Hougaard et al., 1991; Semmak et al., 1988; Valls et al., 1986; Valls et al., 1993a, b; Valls et al., 1995). These compounds are geranylgeraniol and geranylgeraniol-derived diterpenes of which the principal ones are (S)-13-hydroxygeranylgeraniol (1), 13-ketogeranylgeraniol (2), the (S)-13-hydroxyfuranoditerpene (3) and (S)-12-hydroxygeranylgeraniol (4). By contrast, B. galapagensis shows the presence of the meroditerpene 5 (bifurcarenone) as the main constituent of the diterpene composition of its lipid extract (Sun et al., 1980; Mori and Uno, 1989).
3. Present study Ether extracts of dried Bifurcaria brassicaeformis and Bifurcariopsis capensis collected at Simons Town and Clovelly (South Africa) were investigated in order to study the lipophilic secondary metabolites and to compare the results with those of previous studies on Bifurcaria bifurcata and B. galapagensis.
M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
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Table 1 Determination of sterols and diterpenes (mg g1 algal dry wt) Species
Sterolsa
Diterpenesb
Bifurcaria brassicaeformis Simons town Clovelly
5.2 5.7
} }
Bifurcariopsis capensis Clovelly
1.9
}
Bifurcaria bifurcata Quiberon Roscoff El Jadida Oualidia
1.5 1.5 1.7 1.6
6.1 5.9 6.6 4.5
a b
Totally analysed as fucosterol. Total amount of diterpenes.
Crude extracts (1.2% and 0.4% of algal dry weight for Bifurcaria brassicaeformis and Bifurcariopsis capensis, respectively) were fractionated by column chromatography using silica gel as adsorbent and Et2O as eluant. After evaporation, each residue was analysed by normal-phase HPLC (EtOAc–isooctane, 1 : 1), using a method previously described for the determination of sterols and diterpenoids from Cystoseiraceae (Piovetti et al., 1991). This study (Table 1) showed the total absence of diterpenes in both species, and the only sterol detected was fucosterol (main sterol of the brown algae). For comparison, ether extracts of Bifurcaria bifurcata collected at different locations (French collections: Quiberon [478290 N, 3870 W] and Roscoff [488440 N, 38590 W]; Moroccan collections: El Jadida [338150 N, 88310 W] and Oualidia [328490 N, 88570 W]) were analysed in the same way (Table 1). Ether extracts of dried Bifurcaria bifurcata collected at three separate locations} * (Spain)}were investigated to Black Head (Ireland), and Vigo and La Coruna complete the study of the geographical variations in the diterpene composition of this species. The different collections were treated and extracted in an identical manner (Valls et al., 1993a). Each extract was analysed by HPLC (EtOAc–isooctane, 1 : 1) with the method of Piovetti et al. (1991), leading to the results listed in Table 2. For the identification of compounds analysed, extracts were fractionated by liquid chromatography using silica gel. These fractions were subsequently subjected to semi-preparative normal-phase HPLC (EtOAc–isooctane, 2 : 3 and then 1 : 1), leading to pure geranylgeraniol (about 10 mg for each extract), 6 (about 20 mg for each extract), 2 (about 500 mg for each extract) and 1 (about 200 mg for each extract). Their structures have been established by means of spectral methods (IR, MS, NMR) and compared with data of authentic samples isolated from other extracts of this species previously studied. It must be specified that 6 is a minor constituent of the diterpene composition of B. bifurcata which was previously
M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
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Table 2 Geographical variations in the diterpene composition of Bifurcaria bifurcata Compounds (mg g1 algal dry wt) Locations Black Head (Ireland) Vigo (Spain) * (Spain) La Coruna Quiberona (France) a b
1 1.1 0.9 0.7 1.4
2
6
Geranylgeraniol
5.0 5.3 5.5 4.7
b
b
b
b
b
b
b
b
Added for comparison. Present as trace (amount not determined).
Table 3 Chemical groups of species of the genera Bifurcaria and Bifurcariopsis Chemical groups
Secondary metabolites
Species
I
No diterpenoids (sterolic metabolism privileged) Linear diterpenes (geranylgeraniol metabolism) Meroditerpenes (shikimic acid and geranylgeraniol pathways) Linear meroditerpenes Cyclic meroditerpenes Rearranged meroditerpenes
Bifurcaria brassicaeformis Bifurcariopsis capensis Bifurcaria bifurcata
II III IIIA IIIB IIIC
} Bifurcaria galapagensis }
described in the lipid extract of a specimen collected at Quiberon (Culioli et al., 1999b).
4. Chemotaxonomic significance As shown in Table 1, the chemical study of two previously unstudied species belonging to the genera Bifurcaria (B. brassicaeformis) and Bifurcariopsis (B. capensis) shows the total absence of diterpenes in their lipid extracts. In these entities, the secondary metabolism of the seaweeds seems to prioritise the synthesis of sterols instead of diterpenoids. The only significant chemical difference between them is the large difference existing in the yield of their lipid extract (1.2% for Bifurcaria brassicaeformis and 0.4% for Bifurcariopsis capensis). They can be precisely distinguished from the closely related species Bifurcaria bifurcata and B. galapagensis since, by contrast, the former contains a rich array of acyclic diterpenes and the latter a cyclic meroditerpene (5), perhaps indicating a degree of phylogenetic unrelatedness that requires further investigation. Accordingly, we therefore include these two previously uninvestigated species in the chemical group (I) of our classification of Cystoseiraceae (Valls and Piovetti, 1995) (Table 3).
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M. Daoudi et al. / Biochemical Systematics and Ecology 29 (2001) 973–978
By contrast, the results listed in Table 2 show that the diterpene composition of Bifurcaria bifurcata collected at three new locations off the Irish and Spanish coasts is closely related to that of Quiberon in France. These specimens can be clearly included in the chemical type 1 previously described in the study of the geographical variations in the diterpenoid composition of B. bifurcata (Valls et al., 1995). This type was characterized by the presence of 2 and 1 as the main diterpenoids of the lipid extract.
Acknowledgements The authors wish to thank Ms Lorna Kelly and Dr John Bolton (Martin Ryan Institute, National University of Ireland, Galway, Ireland; Department of Botany, University of Cape Town, South Africa) for identification and collection of Bifurcaria brassicaeformis and Bifurcariopsis capensis in South Africa, and Pr Robert Valls (Laboratoire des Organo-Phosphor!es, Universit!e d’Aix-Marseille, France) for identification and collection of Bifurcaria bifurcata in Spain.
References Biard, J.F., Verbist, J.F., Floch, R., Letourneux, Y., 1980. Tetrahedron Lett. 21, 1849. Combaut, G., Piovetti, L., 1983. Phytochemistry 22, 1787. Culioli, G., Mesguiche, V., Piovetti, L., Valls, R., 1999a. Biochem. Syst. Ecol. 27, 665. Culioli, G., Daoudi, M., Mesguiche, V., Valls, R., Piovetti, L., 1999b. Phytochemistry 52, 1447. Culioli, G., Di Guardia, S., Valls, R., Piovetti, L., 2000. Biochem. Syst. Ecol. 28, 185. Hougaard, L., Anthoni, U., Christophersen, C., Nielsen, P.H., 1991. Phytochemistry 30, 3049. Mori, K., Uno, T., 1989. Tetrahedron 45, 1945. Nizamuddin, M., 1970. Bot. Mar. 13, 131. Piovetti, L., Deffo, P., Valls, R., Peiffer, G., 1991. J. Chromatogr. 588, 99. Semmak, L., Zerzouf, A., Valls, R., Banaigs, B., Jeanty, G., Francisco, C., 1988. Phytochemistry 27, 2347. Silva, P.C., Basson, P.W., Moe, R.L., 1996. Univ. Calif. Berkeley Publ. Bot. 79, 1. Stegenga, H., Bolton, J.J., Anderson, R.J., 1997. Bolus Herbarium. University of Cape Town, Cape Town. Sun, H.H., Ferrara, N.M., McConnell, O.J., Fenical, W., 1980. Tetrahedron Lett. 21, 3123. Valls, R., Banaigs, B., Francisco, C., Codomier, L., Cave, A., 1986. Phytochemistry 25, 751. Valls, R., Banaigs, B., Piovetti, L., Archavlis, A., Artaud, J., 1993a. Phytochemistry 34, 1585. Valls, R., Banaigs, B., Piovetti, L., Zerzouf, A., 1993b. Ann. Inst. Oc!ean. 69, 215. Valls, R., Piovetti, L., 1995. Biochem. Syst. Ecol. 23, 723. Valls, R., Piovetti, L., Banaigs, B., Archavlis, A., Pellegrini, M., 1995. Phytochemistry 39, 145. Womersley, H.B.S., 1964. Aust. J. Bot. 12, 53.