Potentising and application of an extract of Melianthus comosus against plant fungal pathogens

Abstract

Due to consumer resistance to the use of synthetic chemicals in agriculture, the aim of this research was to develop an antifungal extract from the leaves of Melianthus comosus that could be marketed as an organic fungicide by Healthtech Laboratories (Pty) Ltd. Ten solvents of varying polarities (hexane, carbon tetrachloride, diethyl ether, dichloromethane, chloroform, acetone, ethanol, ethyl acetate, methanol and water) were used to extract the dried and powdered leaves of Melianthus comosus. The antifungal activity of each extract was tested against 10 plant pathogenic fungi (Rhizoctonia solani, Fusarium oxysporum, Penicillium janthinelum, Penicillium expansum, Colletotrichum gloeosporioides, Trichoderma harzianum, Pythium ultimum, Phytophthora nicotiana, Aspergillus niger, and Aspergillus parasiticus). The acetone extract had the highest antifungal activity (average MIC of 0.10 mg/ml against 8 pathogens), followed by the ethanol extract with an average MIC of 0.22 mg/ml against 8 pathogens. Bioautography was used to determine the number of antifungal compounds in the extracts and to locate the active compounds on bioautograms, to facilitate bioassay guided isolation. The main active compound against all organisms was present in the acetone extract and was of intermediate polarity with an Rf value of 0.33 in BEA. There was another compound that was non-polar with an Rf value of 0.86 that had moderate antifungal activity against Fusarium oxysporum. The inactive compounds were generally of high polarity (4) and some were also non-polar (5). Enrichment procedures removing inactive compounds were developed to increase the concentration of the active compounds. Acetone was the best primary extractant for enrichment based on antifungal activity. Two pathways were used to enrich the acetone extract. Pathway 1 included the use of water to extract the highly polar inactive compounds from the plant material. The water-“washed” marc was dried and “washed” (extracted once) separately with hexane or dichloromethane or ethyl acetate. The respective marcs were again dried and extracted with acetone. All extracts were tested for antifungal activity. The acetone extract following the water and dichloromethane “washes” was the most active (average MIC of 0.088 mg/ml against all 10 fungal organisms) and was termed “HT01”. In pathway 2, a solvent-solvent extraction method was used. The plant material was first extracted with acetone and the acetone extract dried. The dried extract was dissolved in a 1:1 mixture of ethyl acetate and water in a separating funnel. The ethyl acetate fraction was extracted with water several times to remove all the very polar inactive compounds. The water and the ethyl acetate fractions were both dried and used for phytochemical analysis and bioassays. The water fraction was relatively inactive against all organisms. The ethyl acetate fraction was termed “HT02” and had an average MIC of 0.066 mg/ml against all 10 fungal organisms. Work was continued on HT02 including a field trial and cytotoxicity assays as it was the most active extract. The HT02 extract was used for a field trial (done in collaboration with the Healthtech Laboratories) on the plant Symphytum officinale (Comfrey) infected by rust. When the in vitro activity of HT02 was compared with six commonly used fungicides, it had the most activity against Penicillium expansum and the second highest activity against Fusarium oxysporum. After nearly two months plants treated with HT02 (0.2 mg/ml) had c. 50 infected leaves. Plants treated with a commercial fungicide “Bravo 500” (containing 1.5 mg/ml chlorothalonil) had c. 250 infected leaves and untreated plants had extensive infection. Moreover, plants treated with HT02 had flourished when compared to the other treatments. The HT02 extract was more effective against the particular fungus at a six times lower concentration than “Bravo 500”. The cytotoxicity of HT02 was determined using the brine shrimp and cell line toxicity assays. The LC50 of HT02 was 4.5 mg/ml against brine shrimp larvae and 0.0445 mg/ml against Vero cell lines. The toxic principles present in the extract were tentatively identified on TLC using a 20% solution of antimony-III-chloride in ethanol and Kedde’s reagent as cardiac glycosides of Rf values: 0, 0.08, 0.23, 0.34 and 0.58 in EMW solvent system. Lead precipitation and vacuum column chromatography were used to remove the cardiac glycosides from the extract, hence reducing the cytotoxicity of the extract. Lead precipitation removed all five cardiac glycosides but the antifungal activity of the extract was reduced four-fold. Fractionation of HT02 by means of column chromatography gave nine fractions. The antifungal fractions (F3 with MIC values of 0.04 and 0.08 mg/ml against Fusarium oxysporum and Penicillium janthinellum respectively, and F2 with MIC values of 0.08 and 0.16 mg/ml against Fusarium oxysporum and Penicillium janthinellum) were combined and showed no apparent cytotoxicity against the Vero cell lines. The inactive fractions were combined and had an LC50 of 0.0861 mg/ml against the Vero cell lines. To isolate the main antifungal compound, gradient vacuum liquid chromatography and gravity assisted column chromatography was used in the bioassay-guided fractionation of the acetone extract. The major active compound of Rf 0.33 in BEA, 0.71 in CEF and 0.44 in hexane: ethyl acetate (7:3, v/v) was isolated and identified as the triterpene 3-hydroxy-12-oleanen-28-oic acid (oleanolic acid). The compound had high antifungal activity with MIC values ranging from 7.8 to 15.6 ìg/ml against fungal pathogens used. This compound has been isolated from the root bark of Melianthus comosus and other plants but this is the first description of its antifungal activity against plant pathogenic fungi. The compound had no apparent cytotoxicity on Vero cell lines Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Angeh, IE 2006, Potentising and application of an extract of Melanthus Comosus against plant fungal pathogens, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02232012-172854 / > E506/gm