RESUMO
The majority of antifungal compounds reported so far target the cell wall or cell membrane of fungi, suggesting that other types of antibiotics cannot exert their activity because they cannot penetrate into the cells. Therefore, if the permeability of the cell membrane could be enhanced, many antibiotics might be found to have antifungal activity. We here used the polyene antibiotic nystatin, which binds to ergosterol and forms pores at the cell membrane, to enhance the cellular permeability. In the presence of nystatin, many culture extracts from entomopathogenic fungi displayed antifungal activity. Among all the active extracts, two active components were purified and identified as helvolic acid and terramide A. Because the minimum inhibitory concentration of either compound was reduced four-fold in the presence of nystatin, it can be concluded that this screening method is useful for detecting novel antifungal activity.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Nistatina/farmacologia , Polienos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antifúngicos/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dicetopiperazinas/isolamento & purificação , Dicetopiperazinas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Ergosterol/química , Fungos/química , Fungos/citologia , Fungos/efeitos dos fármacos , Ácido Fusídico/análogos & derivados , Ácido Fusídico/isolamento & purificação , Ácido Fusídico/farmacologia , Lactamas/isolamento & purificação , Lactamas/farmacologia , Testes de Sensibilidade Microbiana , Nistatina/química , Polienos/químicaRESUMO
High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane-ethyl acetate-methanol-water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min(-1) and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, (1)H-NMR and (13)C-NMR spectra.