TLC Bioautography Guided Detection and Biological Activity of Antifungal Compounds from Medicinal Plant Acorus calamus Linn

摘要

Plant based botanical fungicides are one of the most important alternative for hazardous fungicides. Antifungal activity (AFA), identification and understanding the mechanism of action of predominant antifungal compounds present in medicinal plant (Acorus calamus L) were carried out in this study. The antifungal activity and both n-butanol extract and purified antifungal compound of A. calamus (L) was evaluated against five fungal strains (Fusarium oxysporum, Fusarium solani, Colletotrichum gloesporioides, Botrytis cinerea and Altemaria solani) by using agar overlay techniques and bioautography. Isolation and screen of antifungal compounds (AFC) was carried out by Thin Layer Chromatography (TLC) and bioautography. The AFC were elucidated and identified by using gas chromatography-mass spectrometry. The mode of action of purified AFC from A. calamus was studied by Evans blue staining and generation of Reactive Oxygen Species (ROS). Both n-butanol extracts and purified AFC exhibited different degreesof antifungal activity against all fungal pathogens tested. Both n-butanol extract from leaf and rhizome of A calamus and purified AFC showed prominent one major spot on TLC after detection with p-anisaldehyde. In bioautographic technique, clear inhibition zones at same distance of TLC spot was observed against all fungal strains examined. The major purified antifungal compounds were identified as a p-asarone (94.3%) and a-asarone (3.4%). Fungal cells treated with different concentration of purified AFC showed a rapid production of ROS. The extent of dead cells stained with Evans blue dye seemed to correlate with the level of ROS production. These results suggested both n-butanol extracts and purified compounds from A. calamus show antifungal activityagainst plant pathogenic fungi and main compound attributed for antifungal activity is p-asarone. Furthermore, AFC caused damages to the cell membrane, leading to altering cell morphology and eventually cell death. Thus A. calamus extract has potentialapplication as an alternative or integrated method to chemical control on plant diseases caused by five different plant pathogens examined.