Bol. Soc. Chil. Quím., 47, 235-240 (2002) ISSN 0366-1644

M.PIOVANO¹, J.A.GARBARINO¹, F.A.GIANNINI², E.R.CORRECHE²,
G.FERESIN³, A.TAPIA³, S.ZACCHINO⁴ and R.D.ENRIZ²

¹Departamento de Química, Universidad Técnica Federico Santa María,
Casilla 110-V, Valparaíso, Chile. E-mail: jgarbari@qui.utfsm.cl
²Facultad de Química, Bioquímica y Farmacia, Universidad de San Luis,
Chacabuco y Pedernera 5700, San Luis, Argentina. E-mail: fagian@unsl.edu.ar
³Instituto de Biotecnología, Universidad Nacional de San Juan,
Avenida General San Martín 1009 (O), San Juan (5400), Argentina. E-mail: gferesin@unsj.edu.ar
⁴Cátedra de Farmacognosia, Universidad Nacional de Rosario,
Suipacha 531 (2000), Rosario, Argentina. E-mail: szaabgil@citynet.net.ar

(Received: April 23, 2002 - Accepted: August 7, 2002)

ABSTRACT

RESUMEN

INTRODUCTION

Lichens are a symbiotic association of mostly ascomycetous fungi (mycobiont) and algal or cyanobacterial (photobiont) partners. They occur in a wide variety of habitats and natural environmental conditions such as low temperatures, prolonged darkness, drought, continous light, etc. It has been suggested that in response to these stresses, natural selection has favoured species producing high concentrations of characteristics phenolic compounds¹⁾ (mainly depsides, depsidones).

Since ancient times some lichen extracts have been used medicinally²⁾ and biological investigations have shown that specific secondary lichen metabolites exhibit interesting activities and can be useful as antibiotics, UV absorbents, antivirals, analgesics and antipyretics^3-10).

Nevertheless, in Chile no bibliographic antecedents exist regarding the use of lichen extracts in traditional medicine and existing information relative to metabolites in particular used biologically is extremely poor^11,12).

In this paper we report antifungal and antibacterial activities of several aromatic lichen compounds, such as depsides, depsidones and a diarylether.

Chemical procedures:

Compounds were obtained by quantitative chemical analysis by means of a systematic study, during 20 years, of continental insular and antarctic lichen species.

The method of extracting and isolation of metabolites has been informed in previous papers^13,14).

All compounds were identified by comparison of their spectroscopic data, mainly ¹H NMR, ¹³C NMR, with literature values and by tlc with authentic samples using the Culberson Standardized Solvent System.

Antimicrobial assays

Antifungal assays. In vitro tests were performed using pathogenic and non-pathogenic, filamentous, and dermatophyte fungi. For each compound the minimum inhibitory concentration (MIC) was determinated.

The microorganisms used, Candida albicans ATCC 10231, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 32264, Aspergillus flavus ATCC 9170 and A. niger ATCC 9029, were acquired from the American Collection of Type Cultures (Rockville, MD) and cultivated on Sabouraud chloramphenicol Agar during 48 h at 30ºC. Suspension of the culture in sterile distilled water with a final concentration of 10⁶ viable yeasts/ml was made.

Microsporum cannis C 112, M. gypseum C 115, Trichophyton rubrum C 113, T. mentagrophytes ATCC 9972 and Epidermophyton floccosum C 114, were provided by CEREMIC (Centro de Referencia Micológica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Rosario, Argentina) and cultivated on Sabouraud dextrose Agar (SDA, Oxoid). A suspension of spores was prepared according to a previously described procedure¹⁵⁾ and adjusted to 10⁶ colony forming units/ml. Compounds were diluted in DMSO to concentrations ranging from 0.1 to 250 mg/ml, the final DMSO concentration in the medium not being higher than 2%, which were inoculated with 5 ml of spores suspension. The antifungic agents ketoconazole (Janssen Pharmaceutica) and amphotericin B (Sigma Chemical Co.) were included as positive control . Incubation time of the plates according to their development, was of 24, 48 or 72 h at 30º.

Antibacterial assays: Escherichia coli ATCC 25922, Pseudomona aeruginosa ATCC 27853, Salmonella sp and Staphylococcus aureus (methicillin -sensitive and m. - resistant) strains were used (Laboratorio de Microbiología, Facultad de Ciencias Médicas, U. Nacional de Cuyo, Mendoza, Argentina).

Firstly, qualitative antibacterial activity was evaluated by the Agar Well Diffusión Method^16,17) (Müeller _ Hinton Oxoid). To each Agar plate an inocula (100 ml) containing 10⁷ bacteria/ml or a 0.5 optical density of the McFarland Scale was incorporated. The plates were solidified and 6 (six) mm diameter wells were done on each one. Solution of each compound (60 mg) in DMSO, antibacterial agents (Cefotaxime Argentia®., 2 mg/ml) control vehicles (DMSO) were added into the wells. The plates were aerobically incubated at 37 ºC for all organisms during 24 h and four assays under identical conditions were carried out for each one.

Secondly, quantitative analysis was carried out measuring the minimum inhibitory concentration (MIC) using the Agar Dilution Method¹⁸⁾ (Müeller - Hinton Oxoid) for Gram (+) and Gram (-) bacteria. The compounds were dissolved in DMSO and added to the medium in concentrations of 2.5 to 60 mg/ml. The final concentration of DMSO did not exceed 2% and the plates were incubated for 24 h at 37ºC.

RESULTS AND DISCUSSION

In order to achieve leader molecules which could be used as a starting point for discovering new antifungal and antibacterial agents, we have evaluated the activity of 19 (nineteen) aromatic lichen metabolites derived from the acetate-polymalonate route.

The structures of the compounds tested as possible antifungal agents are shown in Fig. 1. Results, showed in Table I indicate, in general, negative activity against yeast and filamentous fungi at concentrations of 250 mg/ml. Nevertheless, divaricatic and difractaic acids (13 and 14) and to a lesser degree lobaric acid (10) presented a moderate but significative activity against M. Gypseum, T. mentagrophytes, T. rubrum and E. foccosum, all of them dermatophyte fungi which cause skin infections.

Antibacterial activity was tested for the compounds presented in Fig. 1 and Fig. 2.

Fig. 1. Secondary lichen metabolites evaluated as antifungal and antibacterial agents.

Table I. MIC of compounds evaluated as possible antifungic agents.

^aCandida albicans, ^bSaccharomyces cerevisae, ^cCryptococcus neoformans, ^dAspergillus flavus, ^eA. niger, ^fMicrosporum canis C 112, ^gMicrosporum gypseum, C 115,^hTrichophyton mentagrophytes ATCC 9972, ⁱT. rubrum C 113,
^jEpidermophyton floccosum C 114. ¹Amp. = amphotericin B.^mKet. = ketoconazole.

Fig. 2. Secondary lichen metabolites evaluated as antibacterial agents.

The qualitative results shown in Table II (expressed in mm, corresponding to the diameter of the halo) indicated that against Gram (-) the 19 (nineteen) compounds are inactive. In contrast against Gram (+) bacterial like S. aureus methicillin - sensitive and S. aureus m. - resistant. A marked action can be observed for compounds 3, 7, 12, 13 14, 16 and 19. According to these results this bioactivity was cuantified by determining the MIC of these metabolites against these germs and their values are shown in Table III, the perlatolic 16 (depside) and epiforelic-1 19 (diarylether) acids being compounds with a very high antibacterial effect, not ignoring the activity of a-collatolic acid 7 (depsidone). It is interesting to observe that these three active compounds are the only ones which possess as substituents in positions 6 and 6' of rings A and B long aliphatic carbon chains which might be indicating that the aliphatic part of the molecule could be making some kind of contribution for these major antibacterial effects.

Table II. Qualitative antibacterial activity.

^aStaphylococcus aureus (methicillin-sensitive), ^bStaphylococcus aureus (methicillin resistant),

^cEscherichia coli, ^dPseudomona aeruginosa, ^eSalmonella sp, ^fAntibacterial Agent (Cefotaxime)

With these results it is possible to demostrate that some lichen metabolites show a moderate antifungal activity against dermatophyte fungi and a significant antibacterial activity against Gram (+) bacteria, which might explain at least in part, the use of extracts in traditional medicine.

Table III. MIC of lichen metabolites with antibacterial bioactivity.

On the other hand the significant antibacterial activity obtained for these molecules, transform these compounds into an interesting starting material for chemical modifications with the purpose of increasing their bioactivity, especially against bacterial strains which by now are becoming resistant against common antibiotics.

ACKNOWLEDGEMENTS

We are grateful to Dirección General de Investigación y Posgrado, DGIYP, Universidad Técnica Federico Santa María for financial support and to Programa de Cooperación CYTED, RED X.A.

We are grateful to the Secretaría de Investigación, Universidad Nacional de San Juan, Argentina for a grant to G.E.F.

U.N.S.L. Dr. R.D.Enriz is a researcher of CONICET.

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