ANTIBIOFILM EFFECTS OF PARTIALLY PURIFIED FRACTION OF ETHYLACETATE EXTRACT OF ACALYPHA WILKESIANA LEAVES ON CLINICAL ISOLATES OF CANDIDA ALBICANS I, II AND CANDIDA PARAPSILOSIS

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Department of Medicine

ABSTRACT
Candida infection is the most common hospital-acquired infection caused by fungal pathogen which is frequently involved in biofilm growth, exhibiting to increased resistance to antifungal drugs. This research is aimed at evaluating the biofilm eradication activity of purified fraction of ethylacetate extract of Acalypha wilkesiana leaves on biofilms of C. albicans I, II and C. parapsilosis. Column Chromatography-TLC was used to fractionate and partially purify the extract using a bioassay guided fractionation. The most potent fraction was partially purified using preparative TLC. The minimum inhibitory concentration (MIC) , and median inhibitory concentration (IC50) were carried out using micro dilution method. Fungicidal activity (MFC) of the purified fraction was determined by agar well diffusion method. Adhered cells penetrability of the extracts was carried out by forming adhered cells on membrane polycarbonate filters, and determining the concentration of the antifungals that penetrated the adhered cells with time. Median adhered cells (AdEC50) and median biofilm eradication concentration (BEC50) were determined by microdilution method. Antibiofilm activity of the purified fraction on the morphology of the sessile cells of Candida spp was evaluated by scanning electron microscopy. The mode of action was predicted by the use of sorbitol and ergosterol to evaluate their effect on the IC50 of the antifungal and comparing it with the IC50 of caspofungin and Amphotericin B which are standard antifungals with known mode of actions. The result revealed that ethylacetate extract was the most potent with antifungal activities compared to n-hexane, methanol and aqueous extracts of A. wilkesiana leaves. The purified fraction of the ethylacetate extract had MIC of 1.25mg/ml on C. albicans I, II and C. parapsilosis, respectively. MFC of 2.5mg/ml on C. albicans I and II but had no fungicidal activity on C. parapsilosis. The partially purified fraction had minimum values of IC50 and BEC50 compared to other fractions. There was reduction in clusters formed by proliferation of the sessile cells on the morphology of C. albicans I, II and C. parapsilosis in all treated plates compared to the control. There was increase in the IC50 of the purified fraction and caspofungin in the presence of sorbitol. In conclusion, the partially purified fraction of ethylacetate extract had antibiofilm activity against the three strains of Candida used. Increased in IC50 of the purified fraction and caspofungin implicated the fungal cell wall as the possible target of the partially purified fraction of ethylacetate extract of A. wilkesiana leaves.