Comp. Biochem. PhysioL Vol. 103B,No. 1, pp. 293-296, 1992 Printed in Great Britain
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
SELECTED BIOLOGICAL ACTIVITIES OF SARAINES V. CAPRIOLI,*G. CIMINO,A. DE GIOLIO,I"A. MADAIO,G. SCOGNAMIGLIOand E. TRIVELLONE Istituto per la Chimica di Molecole di Interesse Biologico del C.N.R., Via Toiano, 6 80072, Arco Felice, Naples, Italy, Fax: 804-1770; and *Agrimont R&D (G. Enichem) Via Fauser, 4 28100, Novara, Italy (Received 19 November 1991) Abstract--l. The Mediterranean sponge Reniera sarai, Pufitzeri-Finali, 1969 (Demospongiae: Haploseleridae: Renieridae) possesses in large amounts a series of unprecedented polycyclic alkaloids, saraines 1-3 and saraines A-C. 2. The structural peculiarities of saraines, their chemical-physical characteristics, along with their relevant abundance in the sponge, prompted a study aimed at investigating their biological properties. 3. Saraines were assayed for their cytotoxic, antibacterial, insecticidal and potential antitumoral activities. These results, along with the growth inhibition of fertilized sea urchin eggs, are reported.
INTRODUCTION Some very unusual nitrogenous metabolites, named saraines, are very abundant (6% of the dry sponge) in the n-butanol soluble fractions from defatted acetone extracts of the Mediterranean sponge Reniera sarai (Cimino et al., 1986, 1989a,b, 1990). The main metabolites exhibit two distinct polycyclic alkaloid skeletons. Saraines 1-3 possess a trans-fused quinolizidone system linked to an unsaturated piperidine ring both directly and by two alkyl chains; saraines A-C, on the contrary, are characterized by a central cage, formally obtained by the coupling of two piperidine rings, surrounded by two alkyl chains. Some minor co-occurring alkaloids, isosaraines 1-3, (Cimino et al., 1989c, 1991) are stereoisomers of saraines 1-3 exhibiting an inverted stereochemistry at the chiral centers 1, 2 and 9. All structures are reported in Table 1. They differ within the same group for the length of one of the alkyl chains (a, b, c), displaying 10, 11 and 12 carbon atoms, respectively. Saraines strongly retain inorganic salts. This property, which complicates terribly their isolation, gives to saraines some catalytic properties comparable to those of the crown-ethers. In fact, they are able to dissolve in organic solvents compounds like potassium acetate enhancing the nucleophilic properties of the anion (Cimino et al., 1986). Until the present the biological role played by saraines in Reniera sarai has not been clear. However, their conspicuous abundance along with the absence of epibionts on the surface tissues of the sponge suggest a possible protective role (Thompson et al. 1985) of saraines against fouling organisms. Trying to support this role we have tested saraines in a series of biological bioassays. Preliminary studies were performed using mixtures (Cimino et al., 1989a) of saraines 1-3 and saraines A-C. Both mixtures displayed a moderate toxicity to the Arthropoda Macrosiphum euphorbiae (Thos.) tAuthor to whom correspondence should be addressed.
(potato aphid) and Tetranychus urticae Koch (twospotted spider mite), whereas the n-butanolic crude extract was highly toxic to Artemia salina (LD50 = 2.2#g/ml). These results prompted further studies on the activity of every single saraine. The six main alkaloids (saraines A - C and saraines 1-3) were evaluated for (1) the toxicity to Artemia salina, (2) the antibacterial activity on Staphylococcus aureus, (3) the insecticidal/acaricidal activity on M. euphorbiae, Aedes aegypti L. (yellow-fever mosquito) and T. uticae, (4) the antitumor effects on the potato disc infected by Agrobacterium tumefaciens and (5) the growth inhibition of the fertilized sea urchin egg development. METHODS AND MATERIALS Extraction and fractionation procedures Reniera sarai was collected by SCUBA divers in the Naples Gulf during January 1990 at the depth of about 40m. Fresh sponge (dry weight 150g) was extracted with acetone according to the previous reports. The n-butanol soluble fraction (9 g) from the defatted acetone extract was fractionated on SiO2 gel (Kieselgel 60, Merck, 0.063-0.200mm, 70-230 mesh) column eluting with an increasing gradient of CH3OH in CHC13. This procedure afforded the mixtures of saraines 1-3 and saraines A-C that were further separated by a series of tedious repetitive chromatographic steps (LiChroprep Si 60 Merck, 0.025-0.040 ram) on Jobin-Yvon MiniPrep LC instruments as previously reported (Cimino et al., 1989a). The fractions obtained were checked by both IH- and 13C-NMR spectra (Bruker WM-500 MHz) and by mass spectrometry (AEI MS-30).
Biological bioassays (1) Brine shrimp (Artemia salina) assays were performed in DMSO (1% final volume) using 10 x 3 animals/dose suspended in artificial sea water (5 ml) as previously reported (Meyer et al., 1982; De Giulio et al., 1990). The simple artificial sea water with 1% DMSO was used as control. After 24 hr the data obtained were analyzed by the Finney program (Finney, 1971) which yields L¢50values with 95% confidence intervals. Each dose level and control had three
293
V. CAPRIOLI et al.
294
Table 1.
H (
'
..0 a 6.4/zg/ml) than saraines A - C (4.5 < LDso> 46.7 #g/ml). Of course this could be due to the different alkaloid skeletons exhibited by the two series. However, all the activities were lower than that (LDs0= 2.2/~g/ml) of the crude n-butanol extract of R. sarai. This could be due either to a synergic effect of the saraines or to a dominant activity of some minor co-occurring metabolites. The interesting cytotoxic activities of saraines prompts further studies aimed at ascertaining the presence in R. sarai of more active compounds guiding the isolation work with adequate bioassays. Saraines were also assayed on the sterile potato discs infected by Agrobacterium tumefaciens, used as in-house assay substituting for P388 and 3PS/n vivo antileukemic activity (Ferrigni et al., 1982). Analogously to the brine shrimp assay, the saraines 1 and 3 were the most active compounds. In fact, the
values of inhibition to initiation of the crown gall tumor induced by A. tumefaciens were 51 and 55% for saraines 1 and 3, respectively. Saralnes A, B and C showed comparable values (35, 30 and 40% of inhibition, respectively). The results for saraine B and C are in good agreement with those in brine shrimp assay. The parallelism within the two assays was lacking for saralnes A and 2. In fact, saraine A showed a good inhibition to initiation of the crown gall tumor, whereas saraine 2 was practically inactive, opposite activities were observed in the brine shrimp assay. The data obtained for the inhibition of the fertilized sea urchin egg development revealed interesting activities for almost all saraines. In fact, saraines A-C, saraine 1 and saraine 3 showed a total inhibition of development of the eggs at concentrations between 1.56 and 6.25/ag/ml. On the contrary, saraine 2 assayed at the same concentration was devoid of any activity. On the basis of these data, saraines should be effective in cell duplication (Fusetani, 1987). The antibacterial activity was evaluated recording the MIC against Staphylococcus aureus. Saraines were either inactive (saraine 1), almost inactive (saraine 2) or moderately active (saraines A - C and 3). However, all activities were very modest if compared to those of useful antibiotics, as well as Penicillin-G. Finally the insecticidal/acaricidal evaluation shown in Table 3 summarizes the activities which are insufficient from the point of view of practical application, but are significant and clearly-characterized. For comparisons, only one concentration for test was necessary. Analogously to the brine shrimp assay results, saralnes 1-3 were more active than saraines A-C; that is evident for M. euphorbiae, A. aegypti and juvenile forms of T. urticae. In particular, saraine 1 showed the highest peaks of mortality against two of the three species considered (M. euphorbiae and both motile forms of T. urticae). Although all the investigated saraines were active and showed the same kind
Table 3. Insecticidai/acaricidal potency
Compound Saraine Saraine Saraine Saraine Saraine Saraine
A B C 1 2 3
M. euphorbiae Potato aphid spray at 100 ppm
Aedes aegypti Yellow-fever mosquito dipping in 0.2 ppm
(% mortality)
(% mortality)
20 29 37 68 63 29
24 0 0 20 18 46
Tetranychus urticae Two-spotted spider mite spray at 100 ppm Adults Eggs Juvenile forms (% mortality) (% mortality) (% mortality) 20 19 23 40 32 9
13 12 10 2 3 14
17 8 12 79 66 11
296
V. CAPRIOLIet al.
of activity (similar action speed, wide spectrum of efficacy, similar external symptomatology of sensitive individuals). N o parallelism is detectable between insecticidal/acaricidal activities against Staphylococcus (representative of bacteria). Probably the prevalent m o d e of action of these so distant target groups is quite different. In conclusion, these selected bioassays revealed a series o f promising activities of saraines. In particular, it seems that the nature of the alkaloid skeletons is determinant for some activities. Generally, the compounds exhibiting the trans-quinolizidone system linked to the piperidine ring seem more active. However, the activity of saraines A - C could be influenced by an optimal p H of the experiment. In fact, they possess in the central cage strongly interacting aldehyde and amine groups that evolve to a pseudobase by acid treatment (Cimino et al., 1990). These first studies stimulate further researches directed both at investigating the bioactivities of saraines in a series of selected controlled conditions and testing also the minor components of R. sarai, isosaraines 1-3. Acknowledgements--This work was partially supported by Progetto flnalizzato Chimica Fine II, CNR Rome. We thank Mr R. Turco for the artwork.
REFERENCES Bettarini F., Massardo P., Piccardi P. and Caprioli V. (1986) Synthesis of some new ether derivatives of hydroquinone and their ovicidal activity against the two-spotted spider nfit¢. Pestic. Sci. 17, 465-472. Caprioli V., Andreoni N , Cappai A. and Daniele E. (1990) Potenzialita in campo antiparassitario di alcuni alcaloidi del lupino. Inform. fitopatol. I, 53-57. Cimino G., De Stefano S., Scognamiglio G., Sodano G. and Trivellone E. (1986) Sarains: a new class of alkaloids from the marine sponge. Reniera sarai. Bull. Soc. Chem. Belg. 95, 783-800. Cimino G., Puliti R., Seognamiglio G., Spinella A.,
Trivellone E., Mattia C. A., and Mazzarella L. (1989a) Amazing new alkaloid skeletons from the marine sponge Reniera sarai. Pure appl. Chem. 61, 535 538. Cimino G., Mattia C. A., Mazzarella L., Puliti R., Scognamiglio G., Spinella A. and Trivellone E. (1989 b) Unprecedented alkaloid skeleton from the Mediterranean sponge Reniera sarai: X-ray structure of an acetate derivative of sarain-A. Tetrahedron 45, 3863-3872. Cimino G., Spinella A. and Trivellone E. (1989c) Isosarain1: a new a I kaloid from the Mediterranean sponge Reniera sarai. Tetrahedron Lett. 30, 133-136. Cimino G., Scognamiglio G., Spinella A. and Trivellone E. (1990) Structural studies on saraine A. J. Nat. Prod. 53, 1519 1525. Cimino G., Fontana A., Madaio A., Scognamiglio and Trivellone E. (1991) Application of two-dimensional shift correlated NMR techniques to the structure determination of an unusual marine alkaloid, isosaraine-2. Magn. Res. Chem. 29, 327-332. De Giulio A., De Rosa S., Di Vincenzo V. and Strazzullo G. (1990). Further bioactive derivative of avarol from Dysidea avara. Tetrahedron 46, 7971 7976. Ferrigni N. R., Putnam J. E., Anderson B., Jacobsen L. B., Nichols D. E., Moore D. S., McLaughlin J. L.. Powell R. G. and Smith C. R. (1982) Modification and evaluation of the potato disc assay and antitumor screening of Euphorbiaeae seeds. J. Nat. Prod. 45, 679~586. Finney D. J. (1971) Probit Analysis. 3rd edn. Cambridge University Press, Cambridge. Fusetani N. (1987) Marine metabolites which inhibit development of echinoderm embryos. Bioorganie Marine Chemistry I (Edited by Scheuer P. J.), p. 61. Springer, Berlin. Meyer B. N., Ferrigni N. R., Putnam J. E., Jacobsen L. B., Nichols D. E. and McLaughlin J. L. (1982) Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 45, 3l 34. Seibert G., Raether W., Dogovic N., Gasic M. J., Zahn R. and Muller W. E. G. (1985) Antibacterial and antifungal activity of avarone and avarol. Zbl. Bakt. Hyg. 260, 379-386. Thompson J. E., Walker R. P. and Faulkner D. J. (1985) Screening and bioassays for biologically-active substances from forty marine sponge species from San Diego, California, USA. Mar. Biol. 88, I I 21.
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
SELECTED BIOLOGICAL ACTIVITIES OF SARAINES V. CAPRIOLI,*G. CIMINO,A. DE GIOLIO,I"A. MADAIO,G. SCOGNAMIGLIOand E. TRIVELLONE Istituto per la Chimica di Molecole di Interesse Biologico del C.N.R., Via Toiano, 6 80072, Arco Felice, Naples, Italy, Fax: 804-1770; and *Agrimont R&D (G. Enichem) Via Fauser, 4 28100, Novara, Italy (Received 19 November 1991) Abstract--l. The Mediterranean sponge Reniera sarai, Pufitzeri-Finali, 1969 (Demospongiae: Haploseleridae: Renieridae) possesses in large amounts a series of unprecedented polycyclic alkaloids, saraines 1-3 and saraines A-C. 2. The structural peculiarities of saraines, their chemical-physical characteristics, along with their relevant abundance in the sponge, prompted a study aimed at investigating their biological properties. 3. Saraines were assayed for their cytotoxic, antibacterial, insecticidal and potential antitumoral activities. These results, along with the growth inhibition of fertilized sea urchin eggs, are reported.
INTRODUCTION Some very unusual nitrogenous metabolites, named saraines, are very abundant (6% of the dry sponge) in the n-butanol soluble fractions from defatted acetone extracts of the Mediterranean sponge Reniera sarai (Cimino et al., 1986, 1989a,b, 1990). The main metabolites exhibit two distinct polycyclic alkaloid skeletons. Saraines 1-3 possess a trans-fused quinolizidone system linked to an unsaturated piperidine ring both directly and by two alkyl chains; saraines A-C, on the contrary, are characterized by a central cage, formally obtained by the coupling of two piperidine rings, surrounded by two alkyl chains. Some minor co-occurring alkaloids, isosaraines 1-3, (Cimino et al., 1989c, 1991) are stereoisomers of saraines 1-3 exhibiting an inverted stereochemistry at the chiral centers 1, 2 and 9. All structures are reported in Table 1. They differ within the same group for the length of one of the alkyl chains (a, b, c), displaying 10, 11 and 12 carbon atoms, respectively. Saraines strongly retain inorganic salts. This property, which complicates terribly their isolation, gives to saraines some catalytic properties comparable to those of the crown-ethers. In fact, they are able to dissolve in organic solvents compounds like potassium acetate enhancing the nucleophilic properties of the anion (Cimino et al., 1986). Until the present the biological role played by saraines in Reniera sarai has not been clear. However, their conspicuous abundance along with the absence of epibionts on the surface tissues of the sponge suggest a possible protective role (Thompson et al. 1985) of saraines against fouling organisms. Trying to support this role we have tested saraines in a series of biological bioassays. Preliminary studies were performed using mixtures (Cimino et al., 1989a) of saraines 1-3 and saraines A-C. Both mixtures displayed a moderate toxicity to the Arthropoda Macrosiphum euphorbiae (Thos.) tAuthor to whom correspondence should be addressed.
(potato aphid) and Tetranychus urticae Koch (twospotted spider mite), whereas the n-butanolic crude extract was highly toxic to Artemia salina (LD50 = 2.2#g/ml). These results prompted further studies on the activity of every single saraine. The six main alkaloids (saraines A - C and saraines 1-3) were evaluated for (1) the toxicity to Artemia salina, (2) the antibacterial activity on Staphylococcus aureus, (3) the insecticidal/acaricidal activity on M. euphorbiae, Aedes aegypti L. (yellow-fever mosquito) and T. uticae, (4) the antitumor effects on the potato disc infected by Agrobacterium tumefaciens and (5) the growth inhibition of the fertilized sea urchin egg development. METHODS AND MATERIALS Extraction and fractionation procedures Reniera sarai was collected by SCUBA divers in the Naples Gulf during January 1990 at the depth of about 40m. Fresh sponge (dry weight 150g) was extracted with acetone according to the previous reports. The n-butanol soluble fraction (9 g) from the defatted acetone extract was fractionated on SiO2 gel (Kieselgel 60, Merck, 0.063-0.200mm, 70-230 mesh) column eluting with an increasing gradient of CH3OH in CHC13. This procedure afforded the mixtures of saraines 1-3 and saraines A-C that were further separated by a series of tedious repetitive chromatographic steps (LiChroprep Si 60 Merck, 0.025-0.040 ram) on Jobin-Yvon MiniPrep LC instruments as previously reported (Cimino et al., 1989a). The fractions obtained were checked by both IH- and 13C-NMR spectra (Bruker WM-500 MHz) and by mass spectrometry (AEI MS-30).
Biological bioassays (1) Brine shrimp (Artemia salina) assays were performed in DMSO (1% final volume) using 10 x 3 animals/dose suspended in artificial sea water (5 ml) as previously reported (Meyer et al., 1982; De Giulio et al., 1990). The simple artificial sea water with 1% DMSO was used as control. After 24 hr the data obtained were analyzed by the Finney program (Finney, 1971) which yields L¢50values with 95% confidence intervals. Each dose level and control had three
293
V. CAPRIOLI et al.
294
Table 1.
H (
'
..0 a 6.4/zg/ml) than saraines A - C (4.5 < LDso> 46.7 #g/ml). Of course this could be due to the different alkaloid skeletons exhibited by the two series. However, all the activities were lower than that (LDs0= 2.2/~g/ml) of the crude n-butanol extract of R. sarai. This could be due either to a synergic effect of the saraines or to a dominant activity of some minor co-occurring metabolites. The interesting cytotoxic activities of saraines prompts further studies aimed at ascertaining the presence in R. sarai of more active compounds guiding the isolation work with adequate bioassays. Saraines were also assayed on the sterile potato discs infected by Agrobacterium tumefaciens, used as in-house assay substituting for P388 and 3PS/n vivo antileukemic activity (Ferrigni et al., 1982). Analogously to the brine shrimp assay, the saraines 1 and 3 were the most active compounds. In fact, the
values of inhibition to initiation of the crown gall tumor induced by A. tumefaciens were 51 and 55% for saraines 1 and 3, respectively. Saralnes A, B and C showed comparable values (35, 30 and 40% of inhibition, respectively). The results for saraine B and C are in good agreement with those in brine shrimp assay. The parallelism within the two assays was lacking for saralnes A and 2. In fact, saraine A showed a good inhibition to initiation of the crown gall tumor, whereas saraine 2 was practically inactive, opposite activities were observed in the brine shrimp assay. The data obtained for the inhibition of the fertilized sea urchin egg development revealed interesting activities for almost all saraines. In fact, saraines A-C, saraine 1 and saraine 3 showed a total inhibition of development of the eggs at concentrations between 1.56 and 6.25/ag/ml. On the contrary, saraine 2 assayed at the same concentration was devoid of any activity. On the basis of these data, saraines should be effective in cell duplication (Fusetani, 1987). The antibacterial activity was evaluated recording the MIC against Staphylococcus aureus. Saraines were either inactive (saraine 1), almost inactive (saraine 2) or moderately active (saraines A - C and 3). However, all activities were very modest if compared to those of useful antibiotics, as well as Penicillin-G. Finally the insecticidal/acaricidal evaluation shown in Table 3 summarizes the activities which are insufficient from the point of view of practical application, but are significant and clearly-characterized. For comparisons, only one concentration for test was necessary. Analogously to the brine shrimp assay results, saralnes 1-3 were more active than saraines A-C; that is evident for M. euphorbiae, A. aegypti and juvenile forms of T. urticae. In particular, saraine 1 showed the highest peaks of mortality against two of the three species considered (M. euphorbiae and both motile forms of T. urticae). Although all the investigated saraines were active and showed the same kind
Table 3. Insecticidai/acaricidal potency
Compound Saraine Saraine Saraine Saraine Saraine Saraine
A B C 1 2 3
M. euphorbiae Potato aphid spray at 100 ppm
Aedes aegypti Yellow-fever mosquito dipping in 0.2 ppm
(% mortality)
(% mortality)
20 29 37 68 63 29
24 0 0 20 18 46
Tetranychus urticae Two-spotted spider mite spray at 100 ppm Adults Eggs Juvenile forms (% mortality) (% mortality) (% mortality) 20 19 23 40 32 9
13 12 10 2 3 14
17 8 12 79 66 11
296
V. CAPRIOLIet al.
of activity (similar action speed, wide spectrum of efficacy, similar external symptomatology of sensitive individuals). N o parallelism is detectable between insecticidal/acaricidal activities against Staphylococcus (representative of bacteria). Probably the prevalent m o d e of action of these so distant target groups is quite different. In conclusion, these selected bioassays revealed a series o f promising activities of saraines. In particular, it seems that the nature of the alkaloid skeletons is determinant for some activities. Generally, the compounds exhibiting the trans-quinolizidone system linked to the piperidine ring seem more active. However, the activity of saraines A - C could be influenced by an optimal p H of the experiment. In fact, they possess in the central cage strongly interacting aldehyde and amine groups that evolve to a pseudobase by acid treatment (Cimino et al., 1990). These first studies stimulate further researches directed both at investigating the bioactivities of saraines in a series of selected controlled conditions and testing also the minor components of R. sarai, isosaraines 1-3. Acknowledgements--This work was partially supported by Progetto flnalizzato Chimica Fine II, CNR Rome. We thank Mr R. Turco for the artwork.
REFERENCES Bettarini F., Massardo P., Piccardi P. and Caprioli V. (1986) Synthesis of some new ether derivatives of hydroquinone and their ovicidal activity against the two-spotted spider nfit¢. Pestic. Sci. 17, 465-472. Caprioli V., Andreoni N , Cappai A. and Daniele E. (1990) Potenzialita in campo antiparassitario di alcuni alcaloidi del lupino. Inform. fitopatol. I, 53-57. Cimino G., De Stefano S., Scognamiglio G., Sodano G. and Trivellone E. (1986) Sarains: a new class of alkaloids from the marine sponge. Reniera sarai. Bull. Soc. Chem. Belg. 95, 783-800. Cimino G., Puliti R., Seognamiglio G., Spinella A.,
Trivellone E., Mattia C. A., and Mazzarella L. (1989a) Amazing new alkaloid skeletons from the marine sponge Reniera sarai. Pure appl. Chem. 61, 535 538. Cimino G., Mattia C. A., Mazzarella L., Puliti R., Scognamiglio G., Spinella A. and Trivellone E. (1989 b) Unprecedented alkaloid skeleton from the Mediterranean sponge Reniera sarai: X-ray structure of an acetate derivative of sarain-A. Tetrahedron 45, 3863-3872. Cimino G., Spinella A. and Trivellone E. (1989c) Isosarain1: a new a I kaloid from the Mediterranean sponge Reniera sarai. Tetrahedron Lett. 30, 133-136. Cimino G., Scognamiglio G., Spinella A. and Trivellone E. (1990) Structural studies on saraine A. J. Nat. Prod. 53, 1519 1525. Cimino G., Fontana A., Madaio A., Scognamiglio and Trivellone E. (1991) Application of two-dimensional shift correlated NMR techniques to the structure determination of an unusual marine alkaloid, isosaraine-2. Magn. Res. Chem. 29, 327-332. De Giulio A., De Rosa S., Di Vincenzo V. and Strazzullo G. (1990). Further bioactive derivative of avarol from Dysidea avara. Tetrahedron 46, 7971 7976. Ferrigni N. R., Putnam J. E., Anderson B., Jacobsen L. B., Nichols D. E., Moore D. S., McLaughlin J. L.. Powell R. G. and Smith C. R. (1982) Modification and evaluation of the potato disc assay and antitumor screening of Euphorbiaeae seeds. J. Nat. Prod. 45, 679~586. Finney D. J. (1971) Probit Analysis. 3rd edn. Cambridge University Press, Cambridge. Fusetani N. (1987) Marine metabolites which inhibit development of echinoderm embryos. Bioorganie Marine Chemistry I (Edited by Scheuer P. J.), p. 61. Springer, Berlin. Meyer B. N., Ferrigni N. R., Putnam J. E., Jacobsen L. B., Nichols D. E. and McLaughlin J. L. (1982) Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 45, 3l 34. Seibert G., Raether W., Dogovic N., Gasic M. J., Zahn R. and Muller W. E. G. (1985) Antibacterial and antifungal activity of avarone and avarol. Zbl. Bakt. Hyg. 260, 379-386. Thompson J. E., Walker R. P. and Faulkner D. J. (1985) Screening and bioassays for biologically-active substances from forty marine sponge species from San Diego, California, USA. Mar. Biol. 88, I I 21.
