1. ANTIFUNGAL ACTIVITY OF FLAVONOIDS ISOLATED FROM ULEX SPECIES P. Máximo, A. Lourenço, S. Feio* and J. Roseiro* Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia Universidade Nova de Lisboa, Quinta da Torre, 2825-114 Caparica, Portugal. *Instituto Nacional de Engenharia e Tecnologia Industrial IBQTA, Laboratório de Microbiologia Industrial Azinhaga dos Lameiros, 1699 Lisboa Codex, Portugal. INTRODUCTION Isoflavonoids are described in Leguminosae as phytoallexins and defence agents against insects. There are different structural factors in isoflavonoid molecules that are important for their antimicrobial activity. The presence of isoprenoid groups proved to increase activity in isoflavons and pterocarpans and the relative acidity and number of hydroxyl groups are important in structure-activity relations [1,2]. References [1] Tahara, S. and Ibrahim, R.K. (1995) Phytochemistry 38, 1073. [2] Tahara, S., Katagiri, Y., Ingham, J.L. and Mizutani, (1994) Phytochemistry 36, 1261. [3] Homans A.L. and Fushs A. (1970) J. Chromatogr. 51, 327-329. Acknowledgments We wish to thank the people from Herbário, Museu, Jardim Botânico, Faculdade de Ciências, Universidade de Lisboa (Portugal) for collecting plant material. One of us (P. M.) wishes to thank FCT (Portugal) for a PRAXIS XXI fellowship. RESULTS AND DISCUSSION The fungicidal activity of 18 isoflavonoids together with 2 chalcones, 1 flavone and 1 isoflavanone, isolated from Ulex species, was tested by the bioautographic method against Cladosporium cucumerinum. Most of the test compounds showed antifungal activity and inhibited the fungal growth at 100 µg concentration. From the experimental results some structure-activity comments can be drawn. Among the 11 isoflavones tested (1-4,13-17,20,21) the structures with an open chain isoprenoid substituent at C-8 were active (1,2,4). The C-6 substituted compounds (13- 15,17) had no activity, except for one dimethylpyrano substituted compound 21 that partially inhibited the fungal growth. All the pterocarpans studied (5-9,12,22) have an 8,9- methylenodioxy group and their structural differences are in ring A substitution. The presence of a C-2 hydroxyl group imparts lack (22) or only partial fungicidal activity (18). The presence of hydroxyl groups seems to play an important role for antifungal behaviour of this kind of compounds. EXPERIMENTAL Cell suspension for thin-layer chromatography (TLC) bioassay Cladosporium cucumerinum CCMI 206 was grown on malt extract agar at 25ºC in pyrex Petri dishes for ten days. The mycelium was harvested from the agar plates in a small volume of fresh Homans and Fuchs nutrient broth [3], filtered through four layers of sterilised gauze and diluted in nutrient broth in order to obtain 10 6 cell ml -1. Bioautographic TLC bioassay Aliquots of the test compounds (100 mg) were spotted, in quadruplicate, on silica gel 60 F 254 TLC plates (Merck 5554), which were eluted with the appropriate eluent for each sample (CHCl 3 -MeOH 0.6% - 1.25% mixts). Developed chromatograms were dried and the spots of each compound were marked under 254 nm UV light. A 20 ml sample of the cell suspension was sprayed evenly over each plate, in a glove box. Plates were incubated in closed pyrex trays lined with moist paper at 25ºC for two-three days, protected from light. Bioautograms were evaluated by clear spots, indicating zones of inhibition. ACTIVE COMPOUNDS _____________________________ NON ACTIVE COMPOUNDS _______________________________________ SLIGHTLY ACTIVE COMPOUNDS ______________________________________________