.
1979, Vol. 37, pp. 333439, O Hippokrates Verlag GmbH
m?ta
medlca
.Jowmlot tvkdicind plant-
Screening for Antimicrobial Activities-in Marine' Algae from Eastern Sicily1 S. Caccarnese and R. Azzolina lstituto Dipartimentale di Chimica, Universitl, Catania, Italy.
Key Word Index: Rhodophyta; Phaeophyta; Chlorophyta; Antimicrobial Activity; Disc-diffusion Method.
bacterial and antimycotic activity in extracts of marine algae and quantitatiThe distribution of the antimicrobial ve data on the antimicrobial activity of activities against four different classes pure compounds extracted from marine of microorganisms (Gram-positive, algae have appeared [7-91. Specifically, Gram-negative bacteria, yeast and fun- 151 species of British marine algae [lo] gi) in 63 species of marine algae is repor- and twelve seaweeds of Italian waters ted. The bioactivity is not uniformly [I I.] have recently been studied for antidistributed in the various orders, being bacterial activity and data on the anticoncentrated in Dictyotales and Fucales fungal properties of 18 algal extracts of the Phaeophyta and Ceramiales of from Egypt have been reported [12]. the Rhodophyta. A simple extractio- However, t o date no broad-based screening, partitioning and testing procedure ning of Mediterranean algae for antiis described. microbial activity has been reported. Due to this paucity of data for central Mediterranean species and wishing Introduction to relate antimicrobial activity with chemical investigation at an early stage, Marine organisms have recently re- we started a systematic program for ceived attention as potential sources of screening and isolating antimicrobial pharmacologicaIly active agents [I-61. activities in algaewllected along Eastern Some investigators have detected anti- Sicily coasts: to this purpose, we utilized a simple, fast and reliable extraction and testing technique successfully used ' Presented in part at the Second Internatio- by SHAWet al. [13] during the Alpha nal Symposium on Marine Natural Products Helix Baja California Expedition in (IUPAC-SocietA Chimica Italiana), Sorrento, 1974 and experienced by one of us to Italy, September 12-15, 1978.
Abstract
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.
study several new compounds and biological activities from Laurencia species [9, 141. I n this communication, we wish to report the distribution of activities against foui. different classes of microorganisms (Gram-positive, Gram-negative bacteria, yeast and fungi imperfecti) in 63 species belonging to three different phyla (mainly Phaeophyta and Rhodophy ta). Experimental Algae The following collection sites were used, material of many species being taken as duplicate samples: Castelluccio, Avola, Portopalo (limestone sea-shores South of Catania); S. M. La Scala, Capomulini, Acicastello (lava rocks North of Catania). All species were gathered by shore methods including snorkeling at intertidal levels or by scuba diving a t 2-8 meters dcpth, during the summer. Specimens of algae were transported to the laboratory in plastic bags on ice, identified as to species, drained of residual seawater and stored at -20' C. The algal nomenclature and systematic arrangement are those used by CORMACI and FURNARI [15]. Voucher specimens are preserved in 3 O/o formalin in seawater at the authors laboratory. Organisms The following microorganisms were used as test bacteria and fungi: Bacillus subtilis (Difco), Escherichia coli (Department of Plant Pathology), Penicillium digitatum (Department of Plant Pathology), Saccharomyces cerevisiae (baker). These were maintained in opportune agar slants and subcultures were freshly prepa,red before use, except B. subtilis (spore suspensions from Difco were used as received). '
Algal extracts and preparation of samples for testing A previously described [13, 141 extraction procedure was used, with minor changes. An
algal sample (10 g wet weight of plant cleaned from visible epiphytes and calcareous impurities) was homogenized for 10 min in a Waring blendor in 50 ml of toluene-methanol (1:3), then centrifuged to remove insoluble material. An aliquot of this extract (100 pl), corresponding to 20 mg of wet weight of tissue, was transferred to a 13 mm paper disc and applied to an agar plate seeded with one of the four mentioned test organisms. After incubation, the zone of inhibition was measured (see following). To the remaining extract 20 ml of 1 N sodium nitrate solution was added for partition of the lipophylic and hydrophylic components and to desalt the organic layer, which. was separated .by centrifuging, evaporated under nitrogen, and weighed. The lipid weight ranged from 0.1 to 1.5°/o of the weight of the wet algae. A weighed aliquot of this extract was dissolved in methanol and 100 p l of this solution were applied to the paper disc a t concentration levels of 400 ~ ~ 1 mrn 1 3 disc and, for the most active, 80 ,ugh3 mm disc. I t should be mentioned, however, that partitioning and subsequent testing was made only for the toluene-methanol extracts resulted activc against at least one microorganism.
Antimicrobial testing With regard to B. subtilis, one ml of spbre suspension is added at 45O C to 100 ml of Penassay seed agar. Before congealing, 6 ml portions of the seeded agar medium are added to sterile 10 cm Petri plates, swirling carefully until the agar begins to set. For E. coli, from freshly prepared slants in nutrient agar an inoculum is made in nutrient broth and it is grown for 24 hrs at 37' C. An appropriate amount of inoculum ( 1 x 2 ml) (a too heavy inoculum gives diffuse edges of the inhibition zones) is transferred at 45OC to 100 ml of sterile Penassay seed agar and Petri plates are prepared as described d o v e . For S. cerevisiae, from freshly prepared slants in a opportune yeast maintenance agar [16], an inoculum is prepared in a yeast maintenance broth and it is grown for 24 hrs at 24' C. An appropriate amount of inoculum is transferred a t 45O C to 100 ml of Tryptone soy agar and Petri plates are prepared as described above.
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Caccamese, Azzol ina
Screening for Antimicrobial Activities in Marine Algae
335
Table Algae Tested for Antimicrobial Activity Test microorganismsb
-
Phaeopbyta Du-tyotales Dictyopteris membranacea (STACK.) BAlT. D q o t a dkbotoma (HUDS.) LAMOUR. Dictyota dichotoma var. in&ta (C. AG.) GREV. Dilophus fasciola (ROTH) H O W E Padina pavonitl (L.) THIW Zo~riLl toumefoltii (LAMOUR.) MONT. Fucales Cystoseira compressa (ESPER .) GERL. et NIZAM. Cystoseira rrinita (DESF.) BORY Cystoseira elegans S A W . Cystoseira mediterranea S A W . Cystoseira-sauvageauanaHAMEL Cystoseira s t r h (MONT.)SAUV. Sargassum vulgare C. AG. Ectocatpales Colpomenia sinuosa (MERT.) DERBkS et SOLD. Halogbssum compressurn (GRIFF.) HAMEL Liebmannia leveillei J . AG . Sphacelariules Cladostephus hinutus (L.) PRUDH. V A N REINE Stypocaulon scoparium (L.) KUTZ Desmarestiales Nereid filifonnis (J. AG.) ZANARD. Rhodopbyra Ceramiules Alsidium corallmum C. AG. Ceramium rubrum (HUDS.) C . AG. Gulsonia nodulosa (ERCEG.) J .et G. FELDM. Hafopttys incurvus (HUDS.) B A l T . Laurencia paniculataJ. AG. Lauren& papillosa (FORSK.) GREV. Lawrencia pinnatifda (GMEL.) LAMOUR. '
B. subtilisC
-
E. coli S. cerevisiae . P. digitaturn
-
-
-
+++, + +
++ +
-
+
+++, ++ ++, ++
-
-
+ +
-
-
-
-
+, +
+
f, +
-
+++,+++ +,+++ +, +
-
-
+ +, -
+ -
-
-
+ -
-
+ -
++ -
-
-
+
-
-
-
-
-
-
-
-
-
-
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Phylum Order Speciesa
Caccamese, Azzolina
336
Phylum Order Speciesa
Gigartinales Gigartina acicularir (WULF.) LAMOUR. Gigartina teedii (ROTH) LAMOUR. Gradaria bursa-pastoris (S. G.GMEL.) SILVA Gracilaricr vemrcosa (HUDS.) PAPENFUSS Hypnea musc;fomtis (WULF.) LAMOUR. Phyllophora nervosa (D. C.) GREV. RusoeUa verru~.ulosa(BERT.) J . AG . Schizymenia dubyi (CHAUV.) J . AG. Sphaerococcus coronopifolius (GOOD. et WOODW.) C. AG.
B. srrbtilisC
E. coli S. cerevisiue P. digitatum
+, ++
-
+
+,-
-
-
+++,+++
++
+++, ++
-
+,-
+, '
-
-
++, ++ +, + -
+, +
Nemalionales Lingora viscida (FORSK.) C. AG. Nemalion helminthoides (VELL.) BA'IT. Nemalwn multifuium (WEBER et MOHR) J. AG.
Rhodymeniales Bottyocladia bottyoides (WULF.) J . FELDM. '
Chloropbytu Carrktpales Caulerpa prolifera (FORSK.) LAMOUR.
-
-
+++
-
-
-
-
-
-
-
-
Cyptonemiales Amphiroa rigidd LAMOUR. Corallina elongata ELL. et SOL. Cotallina granfeta ELL. et SOL: Cyptonemia lomution (BERT.) J . AG. Dudresnaya verticilkzta (WITH.) LE JOLIS Grateloupia cosentinii KUTZ. Hdymeniufloresia (CLEM.) C. AG. Janiu rubens (L.) LAMOUR. Peyssonnelia sqrramaria (GMEL.) DEC.
Gelidhales Gelidiu& spathulatum (KUIZ.) BORNET Pterocladia pinnuta (HUDS.) PAPENFUSS
+
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,
Lautenk obtusa (HUDS.) LAMOUR. Polysiphoniu subulata (DUCL.) J . AG. Rytiphloea tinctok (CLEM.) C. AG. Sphondylothamnion multifrdum (HUDS.) NAEG. Spyridid fihmentosa (WULF.) HARV. Vidaliu volub& (L.) J. AG. Wrangelia penin'hta C . AG.
Test microorganismsb
-
-
+,++
+++
+
-
++, ++
Screening
for Antimicrobial Activities in Marine A l g a e
Phylum
337
Test microorganismsb
Order Speciesa
B. subtilisc
Codiales Codium buna (L.) C. AG. Codium decorticatum (WOODW.) HOWE Codium tomentosum (HUDS.) STACKH.
-
-
E. coli S. cerevisiae P digitatum
-
-
-
-
-
Dasycladales Dasycladus vemicuhric (SCOP.) KRASSER C. AG .
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Ulvales Ulva rig&
'Species in the same order are arranged alphabetically. blnhibition using disc-diffusion method (see Experimental); - No inhibition zones; + Zones less than 16mm diameter; ++ Zones greater than 16mm diameter; +++ Zones greater than 19mm diameter. 'First entry, before panitioning; second entry. after partitioning.
Finally, for P. digitatum, 10-20 mg of welldried spores are homogenized in 2 in1 of 0.1010 Triton X-100 solution. This suspension is transferred to Tryptone soy agar and Petri plates are prepared as .described above. After preparing the Petri plates seeded with each of the four test microorganisms, the discs impregnated with the extract are gently placed on the Petri plates. Each species is screened in duplicate and all assay plates are incubated for 24 hrs at 24i.(26O C (E. coli-seeded plates are incubated a t 37O C). Those algae which contain antimicrobial substances show a distinct zone of inhibition around the disc (see Table). Control discs containing standard concentrations of antibiotics are used to check the sensitivity and the size of inoculum. Streptomycin sulfate is used as standard for B. subtilis-seeded plates (6 pglml) and E. coli-seeded plates (60 pg/ml). Filipin is used as standard antifungal for S, cerevisiae and P. digitatum-seeded plates (240 pg/ ml). Methanol, used as solvent to test the extracts after partitioning, shows no inhibition of the cultures. The media used in this work are dehydrated culture media from Difco and are sterilized in the usual fashion by autoclaving.
Results and Discussion
The procedure for extracting and testing algal extracts described above is fast and reliable and the activity data obtained are quite significant in view of two observations. First, partitioning extracts of the algae between sodium nitrate solution and toluene-methanol gives an enrichment of antimicrobial activity in one of the phases, according to lipophylic o r hydrophylic character of the active compound(s); second, each among the four microorganisms used for testing utilizes different mode of action. The table includes the algae tested and the appropriate antimicrobial activities found. A t first, from the data in the table, it appears evident that the response against Bacillus subtilis (Grampositive bacterium) and Penicillium digitatum (imperfect fungus) is much
'
higher than that against Escheridn'a coli (Gramnegative bacterium) and Saccharomyces cerevisiae (yeast). Attention should be given to the few algae active against the last two mentioned organisms. These are Dictyota dichotoma, Zonaria tournefovtii, Cystoseira elegans, Laurencia obtusa, Rytiphloea tinctoria, Pterocladia pinnata, Caulerpa prolifera, Sphaerococcus coronopifolius. In fact, especially needed for clinical use, as well as in plant pathology, are antibiotics effective against these two classes of microorganisms. Moreover, the table shows that the partition procedure gives, in general, toluene extracts with retained or enhanced activity against B. subtilis. This fact agrees with the known presence, in some cases, of unusual lipophylic compounds. As an example, from the active alga Dictyota dichotoma pachydictyol A, J , has been isolated [.17]. I t was reported to be active against Staphylococcus aureus, and other structurally related diterpene alcohols, probably also responsible for the activity, have also been isolated [IS, 191. The largest number of active species, was found in the Phaeophyta and, remarkably, within the phyla the antimicrobial activity is not uniformly distributed in the various orders. For example, in the Phaeophyta (table) most of the species with antibacterial
activity were found in the Dictyotales and Fucales. In the Rhodophyza (table) the order Ceramiales was the richest in active species, while the Cryptonemiales was nearly devoid of active species. Conceivably, this pattern of activity could be of taxonomic significance. A subdivision in families was not possible, because of too few species for each one, but as we intend to encompass the screening to deeply-growing and uncommon species further data will give a statistically significant pattern of bioactivity at the level of orders and families. Gratifying enough, theageneralizations established from the table'agree completely with those drawn for algae from Baja California [I31 thus dernonstrating the validity of the screening scheme when a correct and reliable experimental procedure is applied to investigations in different regions of the world. In fact, we believe that our extractioning, partitioning and testing procedure is more selective and reproducible than a procedure used for testing samples of thallus [lo] [(in Desmarestia ligulata activity can be due t o free sulphuric acid (20)], or a procedure in which one ml of algal extract is tested against cultures of leather moulds [12]. Finally, tests on algal extracts after partitioning were carried out at 400~cg and 80 p g per disc: this concentration range was already used for testing pure compounds with the disc-diffusion method [9]. Thus, reproducible activity'of extracts a t these levels suggests either good potency or high concentration of a given agent. The authors are actively investigating some of 'the most potent algae, using bioassay against B. subtilis as a ,,traceru
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Caccamese, Azzolina
Screening for Antimicrobial Activities in Marine Algae
339
Drugs and Foods from the Sea, p. 187, Norman, OK, 1978, The University of Oklahoma Press. 10. Hornsey, I. S, and D. Hide, Br. Phycol. J., . 9, 353 (1974). 11. Porzi, G. and E. Minelli Bertazzoni, Boll. Acknowledgements Soc. It. Biol. Sper., 51, 1591 (1975). 12. Khaleafa, A. F., M. A. M. Kharboush, A. This work was supported in part by a reMetwally, A. F. Mohsen and A. Serwi, search grant (SRG 16) from NATO. Bot. Mar., 18, 163 (1975). We thank Drs G. FURNARI and M. CORMACI 13. Shaw, .P. D., W. 0.Mc Clure, G . van (Institute of Botany, University of Catania) Blaricom, J. Sims, W. Fenical and J. Rude, for helpful advice in identifying the material, in H. H. Webber and G . D. Ruggieri (eds.), and Professor K. L. RINEHART of the UniverFood-Drugs from the Sea Proceedings 1974, sity of Illinois for stimulating discussion. p. 429, Washington, D. C., Marine Technology Society. 14. Caccamese, S., L. P. Hager, K. L. Rinehart, jr. and R. B. Setzer, Bot. Mar., 22, References 41 (1979). 15. Cormaci, M. and G. Furnari: Inform. Bot. 1. Baslow, M. H., Marine Pharmacology, Ital., 11, 000 (1979). Huntington, N. Y., 1977, Krieger Publ. Co. 16. Collins, C. H. and P. M. Lyne, Microbio2. Burkholder, P. R., in 0.A. Jones and R. logical Methods, p. 151, London, 1970, ButEndean (eds.), Biology and Geology of Coterworths. ral Reefs, Vol. 11, p. 117, New York, 1973, 17. Hirschfeld, D. R., W. Fenical, G . H. Y. Academic Press. Lyn, R. M. Wing, P. Radlick and J. J. 3. Rinehart, K. L., jr. and L. Shield, in M. J. Sims, J. Am. Chem. Soc., 91, 4050 (1973). Weinstein and G. H. Wagman (eds.), Anti18. Fattorusso, E., S. Magno, L. Mayol, C. Sanbiotics: Isolation, Sepaiation and Purificatacroce, D. Sica, V. Amico, G . Oriente, M. tion, Amsterdam, 1978, Elsevier. Piattelli and C. Tringali, J. C. S. Chem. 4. Bhakuni, D. S, and M. Silva, Bot. Mar., Commun., 575 (1976). 17, 40 (1974). 5. Grant, P. T. and A.M. Mackie, Nature, 267, 19. Danise, B., L. Minale, R. Riccio, V. Amico, G. Oriente, M. Piattelli, C. Tringali, E. 786 (1977). Fattorusso, S. Magno and L. Mayol, Ex6. Nisizawa, K., Jap. J. Phycol., 26, 73 (1978). perientia, 33, 413 (1977). 7. Sims, J. J., M. S. Donnell, J. V. Leary and G. H. Lacy, Antimicrob. Agents Chemo- 20. Carlberg, G . E., E. Percival and M. A. Rhaman, Phytochemistry, 17, 1289 (1978). ther., 7, 320 (1975). 8. Glombitza, K.-W., H . Stoffelen, U. Murawsky, J. Bielaczek and H. Egge, Planta Med., Address: S . Caccamese, 21, 105 (1974). Istituto Dipartimentale di Chimica, 9. Caccamese, S. and K. L. Rinehart, jr., in UniversitLi, Catania, Italy . P. N. Kaul and C. J. Sinderman (eds.),
Downloaded by: Universite Laval. Copyrighted material.
during separation procedures, and the results o f these studies will be reported in due course.
1979, Vol. 37, pp. 333439, O Hippokrates Verlag GmbH
m?ta
medlca
.Jowmlot tvkdicind plant-
Screening for Antimicrobial Activities-in Marine' Algae from Eastern Sicily1 S. Caccarnese and R. Azzolina lstituto Dipartimentale di Chimica, Universitl, Catania, Italy.
Key Word Index: Rhodophyta; Phaeophyta; Chlorophyta; Antimicrobial Activity; Disc-diffusion Method.
bacterial and antimycotic activity in extracts of marine algae and quantitatiThe distribution of the antimicrobial ve data on the antimicrobial activity of activities against four different classes pure compounds extracted from marine of microorganisms (Gram-positive, algae have appeared [7-91. Specifically, Gram-negative bacteria, yeast and fun- 151 species of British marine algae [lo] gi) in 63 species of marine algae is repor- and twelve seaweeds of Italian waters ted. The bioactivity is not uniformly [I I.] have recently been studied for antidistributed in the various orders, being bacterial activity and data on the anticoncentrated in Dictyotales and Fucales fungal properties of 18 algal extracts of the Phaeophyta and Ceramiales of from Egypt have been reported [12]. the Rhodophyta. A simple extractio- However, t o date no broad-based screening, partitioning and testing procedure ning of Mediterranean algae for antiis described. microbial activity has been reported. Due to this paucity of data for central Mediterranean species and wishing Introduction to relate antimicrobial activity with chemical investigation at an early stage, Marine organisms have recently re- we started a systematic program for ceived attention as potential sources of screening and isolating antimicrobial pharmacologicaIly active agents [I-61. activities in algaewllected along Eastern Some investigators have detected anti- Sicily coasts: to this purpose, we utilized a simple, fast and reliable extraction and testing technique successfully used ' Presented in part at the Second Internatio- by SHAWet al. [13] during the Alpha nal Symposium on Marine Natural Products Helix Baja California Expedition in (IUPAC-SocietA Chimica Italiana), Sorrento, 1974 and experienced by one of us to Italy, September 12-15, 1978.
Abstract
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.
study several new compounds and biological activities from Laurencia species [9, 141. I n this communication, we wish to report the distribution of activities against foui. different classes of microorganisms (Gram-positive, Gram-negative bacteria, yeast and fungi imperfecti) in 63 species belonging to three different phyla (mainly Phaeophyta and Rhodophy ta). Experimental Algae The following collection sites were used, material of many species being taken as duplicate samples: Castelluccio, Avola, Portopalo (limestone sea-shores South of Catania); S. M. La Scala, Capomulini, Acicastello (lava rocks North of Catania). All species were gathered by shore methods including snorkeling at intertidal levels or by scuba diving a t 2-8 meters dcpth, during the summer. Specimens of algae were transported to the laboratory in plastic bags on ice, identified as to species, drained of residual seawater and stored at -20' C. The algal nomenclature and systematic arrangement are those used by CORMACI and FURNARI [15]. Voucher specimens are preserved in 3 O/o formalin in seawater at the authors laboratory. Organisms The following microorganisms were used as test bacteria and fungi: Bacillus subtilis (Difco), Escherichia coli (Department of Plant Pathology), Penicillium digitatum (Department of Plant Pathology), Saccharomyces cerevisiae (baker). These were maintained in opportune agar slants and subcultures were freshly prepa,red before use, except B. subtilis (spore suspensions from Difco were used as received). '
Algal extracts and preparation of samples for testing A previously described [13, 141 extraction procedure was used, with minor changes. An
algal sample (10 g wet weight of plant cleaned from visible epiphytes and calcareous impurities) was homogenized for 10 min in a Waring blendor in 50 ml of toluene-methanol (1:3), then centrifuged to remove insoluble material. An aliquot of this extract (100 pl), corresponding to 20 mg of wet weight of tissue, was transferred to a 13 mm paper disc and applied to an agar plate seeded with one of the four mentioned test organisms. After incubation, the zone of inhibition was measured (see following). To the remaining extract 20 ml of 1 N sodium nitrate solution was added for partition of the lipophylic and hydrophylic components and to desalt the organic layer, which. was separated .by centrifuging, evaporated under nitrogen, and weighed. The lipid weight ranged from 0.1 to 1.5°/o of the weight of the wet algae. A weighed aliquot of this extract was dissolved in methanol and 100 p l of this solution were applied to the paper disc a t concentration levels of 400 ~ ~ 1 mrn 1 3 disc and, for the most active, 80 ,ugh3 mm disc. I t should be mentioned, however, that partitioning and subsequent testing was made only for the toluene-methanol extracts resulted activc against at least one microorganism.
Antimicrobial testing With regard to B. subtilis, one ml of spbre suspension is added at 45O C to 100 ml of Penassay seed agar. Before congealing, 6 ml portions of the seeded agar medium are added to sterile 10 cm Petri plates, swirling carefully until the agar begins to set. For E. coli, from freshly prepared slants in nutrient agar an inoculum is made in nutrient broth and it is grown for 24 hrs at 37' C. An appropriate amount of inoculum ( 1 x 2 ml) (a too heavy inoculum gives diffuse edges of the inhibition zones) is transferred at 45OC to 100 ml of sterile Penassay seed agar and Petri plates are prepared as described d o v e . For S. cerevisiae, from freshly prepared slants in a opportune yeast maintenance agar [16], an inoculum is prepared in a yeast maintenance broth and it is grown for 24 hrs at 24' C. An appropriate amount of inoculum is transferred a t 45O C to 100 ml of Tryptone soy agar and Petri plates are prepared as described above.
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Caccamese, Azzol ina
Screening for Antimicrobial Activities in Marine Algae
335
Table Algae Tested for Antimicrobial Activity Test microorganismsb
-
Phaeopbyta Du-tyotales Dictyopteris membranacea (STACK.) BAlT. D q o t a dkbotoma (HUDS.) LAMOUR. Dictyota dichotoma var. in&ta (C. AG.) GREV. Dilophus fasciola (ROTH) H O W E Padina pavonitl (L.) THIW Zo~riLl toumefoltii (LAMOUR.) MONT. Fucales Cystoseira compressa (ESPER .) GERL. et NIZAM. Cystoseira rrinita (DESF.) BORY Cystoseira elegans S A W . Cystoseira mediterranea S A W . Cystoseira-sauvageauanaHAMEL Cystoseira s t r h (MONT.)SAUV. Sargassum vulgare C. AG. Ectocatpales Colpomenia sinuosa (MERT.) DERBkS et SOLD. Halogbssum compressurn (GRIFF.) HAMEL Liebmannia leveillei J . AG . Sphacelariules Cladostephus hinutus (L.) PRUDH. V A N REINE Stypocaulon scoparium (L.) KUTZ Desmarestiales Nereid filifonnis (J. AG.) ZANARD. Rhodopbyra Ceramiules Alsidium corallmum C. AG. Ceramium rubrum (HUDS.) C . AG. Gulsonia nodulosa (ERCEG.) J .et G. FELDM. Hafopttys incurvus (HUDS.) B A l T . Laurencia paniculataJ. AG. Lauren& papillosa (FORSK.) GREV. Lawrencia pinnatifda (GMEL.) LAMOUR. '
B. subtilisC
-
E. coli S. cerevisiae . P. digitaturn
-
-
-
+++, + +
++ +
-
+
+++, ++ ++, ++
-
-
+ +
-
-
-
-
+, +
+
f, +
-
+++,+++ +,+++ +, +
-
-
+ +, -
+ -
-
-
+ -
-
+ -
++ -
-
-
+
-
-
-
-
-
-
-
-
-
-
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Phylum Order Speciesa
Caccamese, Azzolina
336
Phylum Order Speciesa
Gigartinales Gigartina acicularir (WULF.) LAMOUR. Gigartina teedii (ROTH) LAMOUR. Gradaria bursa-pastoris (S. G.GMEL.) SILVA Gracilaricr vemrcosa (HUDS.) PAPENFUSS Hypnea musc;fomtis (WULF.) LAMOUR. Phyllophora nervosa (D. C.) GREV. RusoeUa verru~.ulosa(BERT.) J . AG . Schizymenia dubyi (CHAUV.) J . AG. Sphaerococcus coronopifolius (GOOD. et WOODW.) C. AG.
B. srrbtilisC
E. coli S. cerevisiue P. digitatum
+, ++
-
+
+,-
-
-
+++,+++
++
+++, ++
-
+,-
+, '
-
-
++, ++ +, + -
+, +
Nemalionales Lingora viscida (FORSK.) C. AG. Nemalion helminthoides (VELL.) BA'IT. Nemalwn multifuium (WEBER et MOHR) J. AG.
Rhodymeniales Bottyocladia bottyoides (WULF.) J . FELDM. '
Chloropbytu Carrktpales Caulerpa prolifera (FORSK.) LAMOUR.
-
-
+++
-
-
-
-
-
-
-
-
Cyptonemiales Amphiroa rigidd LAMOUR. Corallina elongata ELL. et SOL. Cotallina granfeta ELL. et SOL: Cyptonemia lomution (BERT.) J . AG. Dudresnaya verticilkzta (WITH.) LE JOLIS Grateloupia cosentinii KUTZ. Hdymeniufloresia (CLEM.) C. AG. Janiu rubens (L.) LAMOUR. Peyssonnelia sqrramaria (GMEL.) DEC.
Gelidhales Gelidiu& spathulatum (KUIZ.) BORNET Pterocladia pinnuta (HUDS.) PAPENFUSS
+
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,
Lautenk obtusa (HUDS.) LAMOUR. Polysiphoniu subulata (DUCL.) J . AG. Rytiphloea tinctok (CLEM.) C. AG. Sphondylothamnion multifrdum (HUDS.) NAEG. Spyridid fihmentosa (WULF.) HARV. Vidaliu volub& (L.) J. AG. Wrangelia penin'hta C . AG.
Test microorganismsb
-
-
+,++
+++
+
-
++, ++
Screening
for Antimicrobial Activities in Marine A l g a e
Phylum
337
Test microorganismsb
Order Speciesa
B. subtilisc
Codiales Codium buna (L.) C. AG. Codium decorticatum (WOODW.) HOWE Codium tomentosum (HUDS.) STACKH.
-
-
E. coli S. cerevisiae P digitatum
-
-
-
-
-
Dasycladales Dasycladus vemicuhric (SCOP.) KRASSER C. AG .
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Ulvales Ulva rig&
'Species in the same order are arranged alphabetically. blnhibition using disc-diffusion method (see Experimental); - No inhibition zones; + Zones less than 16mm diameter; ++ Zones greater than 16mm diameter; +++ Zones greater than 19mm diameter. 'First entry, before panitioning; second entry. after partitioning.
Finally, for P. digitatum, 10-20 mg of welldried spores are homogenized in 2 in1 of 0.1010 Triton X-100 solution. This suspension is transferred to Tryptone soy agar and Petri plates are prepared as .described above. After preparing the Petri plates seeded with each of the four test microorganisms, the discs impregnated with the extract are gently placed on the Petri plates. Each species is screened in duplicate and all assay plates are incubated for 24 hrs at 24i.(26O C (E. coli-seeded plates are incubated a t 37O C). Those algae which contain antimicrobial substances show a distinct zone of inhibition around the disc (see Table). Control discs containing standard concentrations of antibiotics are used to check the sensitivity and the size of inoculum. Streptomycin sulfate is used as standard for B. subtilis-seeded plates (6 pglml) and E. coli-seeded plates (60 pg/ml). Filipin is used as standard antifungal for S, cerevisiae and P. digitatum-seeded plates (240 pg/ ml). Methanol, used as solvent to test the extracts after partitioning, shows no inhibition of the cultures. The media used in this work are dehydrated culture media from Difco and are sterilized in the usual fashion by autoclaving.
Results and Discussion
The procedure for extracting and testing algal extracts described above is fast and reliable and the activity data obtained are quite significant in view of two observations. First, partitioning extracts of the algae between sodium nitrate solution and toluene-methanol gives an enrichment of antimicrobial activity in one of the phases, according to lipophylic o r hydrophylic character of the active compound(s); second, each among the four microorganisms used for testing utilizes different mode of action. The table includes the algae tested and the appropriate antimicrobial activities found. A t first, from the data in the table, it appears evident that the response against Bacillus subtilis (Grampositive bacterium) and Penicillium digitatum (imperfect fungus) is much
'
higher than that against Escheridn'a coli (Gramnegative bacterium) and Saccharomyces cerevisiae (yeast). Attention should be given to the few algae active against the last two mentioned organisms. These are Dictyota dichotoma, Zonaria tournefovtii, Cystoseira elegans, Laurencia obtusa, Rytiphloea tinctoria, Pterocladia pinnata, Caulerpa prolifera, Sphaerococcus coronopifolius. In fact, especially needed for clinical use, as well as in plant pathology, are antibiotics effective against these two classes of microorganisms. Moreover, the table shows that the partition procedure gives, in general, toluene extracts with retained or enhanced activity against B. subtilis. This fact agrees with the known presence, in some cases, of unusual lipophylic compounds. As an example, from the active alga Dictyota dichotoma pachydictyol A, J , has been isolated [.17]. I t was reported to be active against Staphylococcus aureus, and other structurally related diterpene alcohols, probably also responsible for the activity, have also been isolated [IS, 191. The largest number of active species, was found in the Phaeophyta and, remarkably, within the phyla the antimicrobial activity is not uniformly distributed in the various orders. For example, in the Phaeophyta (table) most of the species with antibacterial
activity were found in the Dictyotales and Fucales. In the Rhodophyza (table) the order Ceramiales was the richest in active species, while the Cryptonemiales was nearly devoid of active species. Conceivably, this pattern of activity could be of taxonomic significance. A subdivision in families was not possible, because of too few species for each one, but as we intend to encompass the screening to deeply-growing and uncommon species further data will give a statistically significant pattern of bioactivity at the level of orders and families. Gratifying enough, theageneralizations established from the table'agree completely with those drawn for algae from Baja California [I31 thus dernonstrating the validity of the screening scheme when a correct and reliable experimental procedure is applied to investigations in different regions of the world. In fact, we believe that our extractioning, partitioning and testing procedure is more selective and reproducible than a procedure used for testing samples of thallus [lo] [(in Desmarestia ligulata activity can be due t o free sulphuric acid (20)], or a procedure in which one ml of algal extract is tested against cultures of leather moulds [12]. Finally, tests on algal extracts after partitioning were carried out at 400~cg and 80 p g per disc: this concentration range was already used for testing pure compounds with the disc-diffusion method [9]. Thus, reproducible activity'of extracts a t these levels suggests either good potency or high concentration of a given agent. The authors are actively investigating some of 'the most potent algae, using bioassay against B. subtilis as a ,,traceru
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Caccamese, Azzolina
Screening for Antimicrobial Activities in Marine Algae
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Drugs and Foods from the Sea, p. 187, Norman, OK, 1978, The University of Oklahoma Press. 10. Hornsey, I. S, and D. Hide, Br. Phycol. J., . 9, 353 (1974). 11. Porzi, G. and E. Minelli Bertazzoni, Boll. Acknowledgements Soc. It. Biol. Sper., 51, 1591 (1975). 12. Khaleafa, A. F., M. A. M. Kharboush, A. This work was supported in part by a reMetwally, A. F. Mohsen and A. Serwi, search grant (SRG 16) from NATO. Bot. Mar., 18, 163 (1975). We thank Drs G. FURNARI and M. CORMACI 13. Shaw, .P. D., W. 0.Mc Clure, G . van (Institute of Botany, University of Catania) Blaricom, J. Sims, W. Fenical and J. Rude, for helpful advice in identifying the material, in H. H. Webber and G . D. Ruggieri (eds.), and Professor K. L. RINEHART of the UniverFood-Drugs from the Sea Proceedings 1974, sity of Illinois for stimulating discussion. p. 429, Washington, D. C., Marine Technology Society. 14. Caccamese, S., L. P. Hager, K. L. Rinehart, jr. and R. B. Setzer, Bot. Mar., 22, References 41 (1979). 15. Cormaci, M. and G. Furnari: Inform. Bot. 1. Baslow, M. H., Marine Pharmacology, Ital., 11, 000 (1979). Huntington, N. Y., 1977, Krieger Publ. Co. 16. Collins, C. H. and P. M. Lyne, Microbio2. Burkholder, P. R., in 0.A. Jones and R. logical Methods, p. 151, London, 1970, ButEndean (eds.), Biology and Geology of Coterworths. ral Reefs, Vol. 11, p. 117, New York, 1973, 17. Hirschfeld, D. R., W. Fenical, G . H. Y. Academic Press. Lyn, R. M. Wing, P. Radlick and J. J. 3. Rinehart, K. L., jr. and L. Shield, in M. J. Sims, J. Am. Chem. Soc., 91, 4050 (1973). Weinstein and G. H. Wagman (eds.), Anti18. Fattorusso, E., S. Magno, L. Mayol, C. Sanbiotics: Isolation, Sepaiation and Purificatacroce, D. Sica, V. Amico, G . Oriente, M. tion, Amsterdam, 1978, Elsevier. Piattelli and C. Tringali, J. C. S. Chem. 4. Bhakuni, D. S, and M. Silva, Bot. Mar., Commun., 575 (1976). 17, 40 (1974). 5. Grant, P. T. and A.M. Mackie, Nature, 267, 19. Danise, B., L. Minale, R. Riccio, V. Amico, G. Oriente, M. Piattelli, C. Tringali, E. 786 (1977). Fattorusso, S. Magno and L. Mayol, Ex6. Nisizawa, K., Jap. J. Phycol., 26, 73 (1978). perientia, 33, 413 (1977). 7. Sims, J. J., M. S. Donnell, J. V. Leary and G. H. Lacy, Antimicrob. Agents Chemo- 20. Carlberg, G . E., E. Percival and M. A. Rhaman, Phytochemistry, 17, 1289 (1978). ther., 7, 320 (1975). 8. Glombitza, K.-W., H . Stoffelen, U. Murawsky, J. Bielaczek and H. Egge, Planta Med., Address: S . Caccamese, 21, 105 (1974). Istituto Dipartimentale di Chimica, 9. Caccamese, S. and K. L. Rinehart, jr., in UniversitLi, Catania, Italy . P. N. Kaul and C. J. Sinderman (eds.),
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during separation procedures, and the results o f these studies will be reported in due course.
