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Marine Drugs 2004, 2(1), 8-13;

Article
Antifungal Activity of (+)-Curcuphenol, a Metabolite from the Marine Sponge Didiscus oxeata
Helena Gaspar 1,*, Sonia Savluchinske Feio 1, Ana Isabel Rodrigues 1 and Rob Van Soest 2
1
Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada do Paço do Lumiar, 1649-038, Lisboa, Portugal
2
Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 94766, 1090-GT, Netherlands
*
Author to whom correspondence should be addressed; Tel. (00351)-21-7165141, Fax (00351) 21- 7168100, E-mail: helena.gaspar@ineti.pt
Received: 17 October 2003; in revised form: 26 November 2003 /
Published: 25 February 2004

Abstract

: The antifungal activity of the sesquiterpenoids (+)-curcuphenol and (+)- curcudiol isolated from the Caribbean sponge Didiscus oxeata was evaluated against several filamentous fungi.
Keywords:
Porifera; Didiscus oxeata; antifungal; curcuphenol; curcudiol

Introduction

In the marine environment, sponges (Porifera) are one of the richest sources of biological active secondary metabolites [1]. As part of our general interest in the isolation and characterization of bioactive metabolites from sponges with potential pharmaceutical and agrochemical applications the sesquiterpenoids (+)-curcuphenol (1) and (+)-curcudiol (2) were isolated from the antifungal extract of the Curaçao marine sponge Didiscus oxeata.

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(+)-Curcuphenol and (+)-curcudiol were previously isolated from several marine sponges [28] and were described as ichthyotoxic and antifouling metabolites [3,6]. (+)-Curcuphenol also exhibited antimicrobial activity, antiyeast activity against Candida albicans, cytotoxicity against P- 388 murine leukaemia, A-549 lung, HCT-8 colon, and MDAMB mammary cancer cell lines and strongly inhibited the activity of gastric H, K-ATPase [2,4,8].

Although several biological activities were reported for both (+)-curcuphenol and (+)-curcudiol, there are no studies concerning the antifungal activity against filamentous fungi. So the main aim of this study is to evaluate the antifungal activity of both sesquiterpenes against several filamentous fungi.

Results and Discussion

The antifungal activity of the sesquiterpenoids (+)-curcuphenol and (+)-curcudiol isolated from the Caribbean sponge Didiscus oxeata was evaluated against the filamentous fungi Absidia ramosa, Aspergillus niger, Botrytis cinerea, Cladosporium cucumerinum, Fusarium oxysporum, Penicillium expansum, Rhizopus oryzae, and Trichoderma harzianum at a concentration of 200 μg/disc. (+)- Curcuphenol was also tested, at the same concentration, against the fungi Fusarium solani, Nodulosporium sp., Phytophthora sp., Trichoderma sp., T. koningii, T. lignorum, T. virgatum and Trichophyton mentagrophytes.

The antifungal activity towards several wood surface contaminant fungi was evaluated due the interest of the search for natural compounds for wood protection. Sapstain and mould growth on lumber are serious problems in the forest, pulp and paper industry; although structure damage is minimal to timber, the discolorations are objectionable to buyers. Moreover, the conventional protective solutions are toxic and accumulate in animal tissues [9].

The antidermatophytic effect of (+)-curcuphenol against the human pathogenic fungus Trichophyton mentagrophytes was also evaluated, as dermatophytoses are the most common form of fungal infections found in most countries, affecting skin, hair and nails [10].

While (+)-curcuphenol inhibited the growth of the fungi A. ramosa, A. niger, B. cinerea, C. cucumerinum, F. oxysporum, P. expansum, R. oryzae, T. Harzianum, T. Koningii and T. mentagrophytes, (+)-curcudiol only partially inhibited the growth of A. ramosa (table 1). None of the tested compounds exhibited activity towards Fusarium solani, Nodulosporium sp., Phytophthora sp., Trichoderma sp., T. lignorum and T. virgatum.

Light microscopy observations of the inhibition zone of fungal growth treated with (+)- curcuphenol showed the presence of non-germinated cells and spores presenting morphological alterations, such as reduced germ-tube and multiple germ-tube formation.

Conclusion

(+)-Curcuphenol inhibited the growth of several phytopathogenic and wood surface contaminant fungi as well as the human pathogenic fungus T. mentagrophytes.

Although (+)-curcuphenol showed less efficacy than the commercial products (Amphotericin B, Carbendazim®) it should be worthily studied as these fungicides are toxic and several resistant strains were detected for Carbendazim®.

The results obtained show that (+)-curcuphenol possesses a broad antifungal spectrum when compared with (+)-curcudiol that only partially inhibited the growth of A. ramosa, suggesting that the double bond in the aliphatic chain is required for antifungal activity. Thus, chemical modifications of this group could be a tool in the search for new and safer antifungal agents.

Experimental

Biological material

The marine sponge Didiscus oxeata was collected in June 1998 at a depth of 30 m on the reef of Curaçao (Netherlands Antilles) by M.J. De Kluijver. The specimen was massive-amorphous, with characteristic sinuous grooves at the surface. The surface skeleton is a detachable crust of partly tangentially arranged megascleres. The skeleton of the interior is largely confused, with many loose megascleres and microscleres randomly arranged, but thick ascending spicule tracts may be distinguished. The spicules are oxeas in a large size range, divisible in at least two size categories: 546–1220 × 11–30 μm and 228–366 × 4–7 μm. Microscleres are characteristic didiscorhabds, 60–74 μm long. The specimen conformed in all aspects to previous descriptions. A voucher is kept in the collections of the Zoological Museum Amsterdam under reg. nr. POR.14326.

Isolation of sesquiterpenes

The sponge was immersed in MeOH immediately after collection and kept at −20ºC until extraction. After filtration the sponge was cut into small pieces and successive washed (3× 0.5 L to 1.5 L) with methanol and dichloromethane at room temperature. Evaporation of the organic solvents and the residual sea water yielded the crude extract (18 g). The dicloromethane soluble fraction (4 g) of the crude methanolic extract was subjected to flash column chromatography on Si gel (MN Kieselgel 60 mesh) using a step gradient of n-hexane/EtOAc. The fractions eluting with 10% EtOAc in n-hexane gave (+)-curcuphenol (2 g, 11% methanolic extract) while 30% EtOAc gave (+)-curcudiol (0.2 g, 0,1% methanolic extract). These sesquiterpenes were identified by comparison of their spectroscopic data (NMR, IV, MS, [α]D) with those reported for natural and synthetic compounds [2,11,12] and by HMBC and HMQC.

Biological activity

The wood surface contaminant fungi tested (Fusarium solani, Nodulosporium spp., Phythophtora spp., Trichoderma sp. and Trichoderma virgatum) were isolated from cork. All the other fungi tested were obtained from the Culture Collection of Industrial Microorganisms (CCMI), INETI. The disc-diffusion assay according to Amade et al. [13] was used as a screening test for antifungal activity. The products dissolved in dimethyl sulphoxide (DMSO) were applied on the discs at 200 μg/disc, and set on the plates. Amphotericin B (Sigma) was used as positive control for T. mentagrophytes whereas Carbendazim® was used for all the other fungi. These standards were tested at 100 μg/disc. The fungal inhibition zone around the disc was observed by light microscopy (x 320).

Table Table 1. Antifungal test results.

Click here to display table

Table 1. Antifungal test results.
Organism(+)-CurcuphenolCarbendazim®Amphotericin B
Filamentous Fungi
Absidia ramosa4.5 cm6.0 cm-
Aspergillus niger1.0 cm3.5 cm-
Botrytis cinera±2.0 cm-
Cladosporium cucumerinum2.0 cm3.7 cm-
Fusarium oxysporum±2.0 cm-
Penicillium expansum±4.0 cm-
Rhizopus oryzae1.6 cm3.5 cm-
Trichoderma harzianum±4.0 cm-
Trichoderma koningii1.0 cm4.2 cm-
Trichophyton mentagrophytes1.0 cm-5.0 cm

± partial inhibition of fungal growth, - not tested

Acknowledgements

This work was funded in part by the European Community under the contract MAS-3-CT97- 0144 Symbiosponge.

  • Sample Availability: Samples are available from the authors.

References

  1. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Marine Natural Products. Nat. Prod. Rep 2003, 20, 1–48 a.nd previous reviews by Faulkner.. [Google Scholar]
  2. Wright, A. M.; Pomponi, S. A.; McConnell, O. J.; Kohmoto, S.; McCarthy. (+)-Curcuphenol and (+)-Curcudiol, Sesquiterpene Phenols from Shallow and Deep Water Collections of the Marine Sponge Didiscus oxeata. J. Nat. Prod 1987, 50, 976–978. [Google Scholar]
  3. Braekman, J. C.; Daloze, D.; Moussiaux, B.; Stoller, C.; Deneubourg, F. Sponge Secondary Metabolites: New Results. Pure & Appl. Chem 1989, 61, 509–512. [Google Scholar]
  4. Duque, C.; Zea, S.; De Silvestri; Calderon, A.; Medina, A. Biological Activity vs. Chemical Composition of the Chloroform Extract of the Marine Sponge Didiscus oxeata. Rev. Colomb. Quim 1988, 17, 39–46.: 113, 208508y.. [Google Scholar]
  5. Butler, M. S.; Capon, R. J. Aromatic Bisabolenes from an Australian Marine Sponge, Arenochalina sp. J Nat Prod 1991, 54, 619–623. [Google Scholar]
  6. Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N. Antifouling Terpenes and Steroids Against Barnacle Larvae from Marine Sponges. Biofouling 1997, 11, 283–291. [Google Scholar]
  7. Rodríguez, D. A.; Vera, B. New Diterpenes from the Caribbean Sponge Epipolasis reiswigi. J. Org. Chem 2001, 66, 6364–6368. [Google Scholar]
  8. Fusetani, N.; Sugano, M.; Matsunaga, S.; Hashimoto, K. (+)-Curcuphenol and Dehydrocurcuphenol, Novel Sesquiterpenes wich Inhibit H, K-ATPase, from Sponge Epipolasis sp. Experentia 1987, 43, 1234–1235. [Google Scholar]
  9. Podile, A.; Prakash, A. Lysis and Biological Control of Aspergillus niger by Bacillus subtilis AF 1. Can. J. Microbiol 1996, 42, 533–538. [Google Scholar]
  10. Gadhi, C.; Benharref, A.; Jana, M.; Basile, A.; Contet-Audonneau, N.; Fortier, B. Antidermatophytic Properties of Extracts from the Leaves of Aristolochia paucinervis Pomel. Phytother. Res 2001, 15, 79–81. [Google Scholar]
  11. Ono, M.; Yamamoto, Y.; Akita, H. Reactions of Methyl 4,5-Epoxy-(2E)-pentenoate with Arenes. II. Application to the Synthesis of (±)-Curcudiol, (±)-Curcuphenol, (±)-Curcuhydroquinone, and (±)-Curcuquinone. Chem. Pharm. Bull 1995, 43, 553–558. [Google Scholar]
  12. Ono, M.; Ogura, Y.; Hatogai, K.; Akita, H. Total Synthesis of (S)-(+)-Curcudiol, and (S)-(+) and (R)-(−)-Curcuphenol. Chem. Pharm. Bull 2001, 49, 1581–1585. [Google Scholar]
  13. Amade, P.; Pesando, D.; Chevolot, L. Antimicrobial Activities of Marine Sponges from French Polynesia and Brittany. Marine Biology 1982, 70, 223–228. [Google Scholar]
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