Search for new antifungal compounds from higher plants

The increasing occumnce of opportunistic systemic mycoses associated with the use of immunosuppressive drugs and AIDS has led to new efforts in the search for novel antifungal compounds. At the same time, there is continuing interest in the discovery of antifungal agents which are effective against plant pathogenic fungi. Since the plant kingdom provides a useful source of lead compounds of novel structure, a wide-scale investigation of species from the tropics has been undertaken. TLC bioautography allows the screening of plant material and subsequent bioassay-guided fractionation and isolation. Treatments with immunosuppressive drugs and the spread of AIDS have meant that diseases caused by weaknesses in the immunitary systems of humans are becoming more and more prevalent. Associated with these problems is an increasing predisposition to fungal attack. The infections commonly observed in the immuno-compromised host include candidiasis (Candida albicans and other species) of the oesophagus and mouth, cryptococcosis (Candida neoformans) and aspergillosis (AspergillusfZavus, A. fimigatus, A. niger). As there are few really effective antifungal preparations currently available for the treatment of systemic mycoses and as the efficacy of existing drugs is rather limited, it is important to find new sources of antifungal agents. In the past, efforts have been mainly directed towards compounds active against plant pathogenic fungi (and research along these lines in agrochemistry is still of high interest) but it has been demonstrated that plant-derived constituents may offer potential leads for novel agents against systemic mycoses (1). Although other techniques exist, bioautography is the method of choice when searching for antifungal compounds from plants. This is because it allows the combination of a bioassay in situ and, at the same time, localization of active constituents on the TLC plate employed for the assay. Spore-producing fungi, such as Aspergillus, Penicillium and Cladosporium spp. can all be employed as target organisms in direct bioautographic procedures (2, 3). After migration of an extract or sample on a TLC plate, the plate is sprayed with the microorganism and incubated in a humid atmosphere. Zones of inhibition appear where spore growth is prevented by the active constituents of the plant extract. Bioautography with the plant pathogenic fungus Cladosporium cucumerinum has been employed to guide the fractionation and isolation of numerous new natural products with fungicidal activity from African and other tropical plants (4). These compounds have many different chemical structures, varying from naphthoquinones to saponins. Since direct bioautography is not possible with yeasts such as Candida albicans, a simple and rapid agar overlay assay has been developed (5). This technique, which is a hybrid of direct and contact bioautography, relies on the transfer of active compounds by a diffusion process from the stationary phase into an agar layer containing the microorganism. When the plate is sprayed with methylthiazoyltetrazolium chloride (MTT), an MTT formazan is produced and inhibition zones are observed against a purple background.