Antifungal activity and cytotoxicity of isolated compounds from leaves of Breonadia salicina

Abstract

Ethnopharmacological relevance: Breonadia salicina is used traditionally to treat wounds, ulcers, fevers, headaches, and fungal infections. The aim of this study was to investigate the antifungal activity of the plant extract and compounds isolated there from.

Materials and methods: Leaf extracts of Breonadia salicina were screened for antifungal activity against seven plant pathogens: Aspergillus niger, Aspergillus parasiticus, Colletotrichum gloeosporioides, Trichoderma harzianum, Penicillium expansum, Penicillium janthinellum and Fusarium oxysporum. Bioautography assay was used to determine the presence and number of antifungal compounds of the plant extracts. Bioassay-guided fractionation using column chromatography of the chloroform extract led to the isolation of four antifungal compounds. Nuclear Magnetic Resonance (NMR) spectroscopy, Mass Spectrometry (MS) and Electron Impact Mass Spectrometry (EIMS) were used for the identification of antifungal compounds. Cytotoxicity of the chloroform crude extract and isolated compounds was determined using the MTT (3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide) assay against Vero monkey kidney cells.

Results: Compound 1 was identified as ursolic acid, while compounds 2, 3 and 4 were not identified conclusively owing to the presence of mixtures of long chain fatty acids. Compounds 3 and 4 had good antifungal activity against Aspergillus parasiticus and Penicillium janthinellum with MIC values of 10 and 16 µg/ml respectively. Compound 2 and ursolic acid had some activity with MIC values ranging between 20 and 250 µg/ml. The crude extract was less toxic to the Vero cells (LC50=82 µg/ml) than ursolic acid (LC50=25 µg/ml). Compounds 2 and 3 were not toxic at the highest concentration tested (LC50=200 µg/ml). Compound 4 was the most toxic to the cells with an LC50 of 35 µg/ml.

Conclusions: The results support the traditional use of Breonadia salicina for antifungal applications, and demonstrate the potential value of developing antifungal compounds from plant natural products. Indications of toxicity should be evaluated at an early stage as the selectivity of the product in affecting fungi preferentially to plant or mammalian cells should be identified when assessing the potential usefulness of the product.

Keywords: Antifungal activity; Breonadia salicina; Cytotoxicity; Minimum inhibitory concentration; Ursolic acid.