Plant pathogenic fungi are a major cause of reduced plant production and post-harvest losses of plant produce. The control of these fungi by some synthetic fungicides is complicated by human and environmental toxicity, the development of resistance by some fungi and high costs, thus prompting the investigation of other means of fungal control. Plant secondary metabolites have a good potential as antifungal agents. The aim of this study is to investigate the potential use of
This article is the translated version, made available to provide access to a larger readership, of which the original English article is available here:
Hierdie artikel is die vertaalde weergawe en is beskikbaar gestel om ‘n breër lesersgroep te bereik. Die oorspronklike Engelse artikel is beskikbaar hier:
Plant fungal pathogens threaten food security worldwide as more than 800 million people have inadequate food supplies and at least 10% of food production is lost to plant diseases (Strange & Scott
Synthetic fungicides are the primary means of controlling plant pathogens. However, safety risks, high costs, side effects and development of resistance towards the use of these fungicides are raising serious concerns (Tripathi & Dubey
Plants are good candidates in the search for fungicidal compounds, since they have to exist under difficult conditions and are attacked by all manner of parasites, especially fungi (Hostettman et al.
In the study we isolated and characterised the main antifungal compound present in the plant extracts and determined the antifungal activity against important plant fungal pathogens and its cytotoxicity.
Leaves of
Powdered dried leaves of
Solvent-solvent extraction is one of the popular techniques used in the preparation of samples for qualitative and quantitative analysis. It is a process of separating one constituent from a mixture by dissolving it into a solvent, in which it is soluble, while the other constituents of the mixture are not, or are at least less soluble (Holden
A bioautographic method, developed in our laboratory (Masoko & Eloff
Rf = distance moved by compound/distance moved by solvent front
The microdilution method developed by Eloff (
Dried residues of the
The samples were tested for cytotoxicity against the Vero monkey kidney cell line (obtained from the Department of Veterinary Tropical Disease, University of Pretoria). The cells were maintained in minimal essential medium (MEM, Highveld Biological, Johannesburg, South Africa) supplemented with 0.1% gentamicin (Virbac) and 5% foetal calf serum (Adcock-Ingram). To prepare the cells for the assay, cell suspensions were prepared from confluent monolayer cultures and plated at a density of 0.5 × 103 cells into each well of a 96-well microtitre plate. After overnight incubation at 37° C in a 5% CO2 incubator, the subconfluent cells in the microtitre plate were used in the cytotoxicity assay. Stock solutions of the plant extract were prepared in growth medium (1 μg/mL–1000 μg/mL). The viable cell growth after 120 h incubation with plant extracts/samples was determined, using the tetrazolium based colorimetric assay (MTT assay) described by Mosmann (
The DCM extract obtained from solvent-solvent extraction was subjected to column chromatography on silica gel 60. The column was eluted with 100% DCM and, subsequently, the polarity of the eluting solvent was increased with methanol (MeOH). A volume of 1000 mL of 100% DCM was initially used, followed by solvent mixture of 10% MeOH, 20% MeOH, 30% MeOH, 40% MeOH, 50% MeOH, 60% MeOH, 80% MeOH, all in DCM, and finally the column was eluted with 100% MeOH. A total of nine fractions, each 1000 ml, were collected. Fractions were pooled according to their similarity in behaviour on TLC. The fractions were concentrated and chromatographed to remove impurities to finally obtain the compound. To confirm that the isolation of the antifungal compound present in the extract is not an artefact of the isolation, bioautography was carried out on the isolated compound and the DCM crude extract.
Nuclear Magnetic Resonance Spectroscopy (1H NMR and 13C NMR) was carried out to determine the structure of the compound, using a Varian Inva 500 MHz spectrometer. The isolated compound from
A quantity of 46.67 g of serially extracted acetone extracts of
The DCM fraction was selected to isolate active compounds by bioassay guided fractionation, based on its activity. Fractions eluted from the DCM extract in open column silica gel chromatography were pooled according to their TLC composition, concentrated and chromatographed to remove impurities which yielded a white precipitate. To examine the activity of the antifungal compounds, bioautography was undertaken of the combined fractions by TLC and the DCM extract that was fractionated and sprayed with
Chromatogram and bioautogram, loaded with dichloromethane fraction, and eluted with benzene/ethanol/ammonium hydroxide 90/9/1 (1) and compound isolated from the extract (2), sprayed with vanillin sulphuric acid spray reagent (a) and
Rf values of active compounds may help in dereplicating isolation of antifungal compounds from other plant extracts. The Rf value of the extract and isolated compound was 0.08 with BEA (benzene/ethanol/ammonium hydroxide 90/9/1) (Kotze & Eloff
The compound was isolated as a white precipitate. NMR spectroscopy identified the compound as oleanolic acid (
Structure of oleanolic acid, isolated from
In isolating bioactive compounds from plant extracts, it is always possible that the major biologically active compounds may be inactivated during the isolation process and that a minor compound may be isolated in the end. To examine this possibility, bioautography was carried out on the crude extract and the isolated oleanolic acid.
The bioautography results (
To evaluate the degree to which the activity has increased by isolating oleanolic acid and the sensitivity towards different fungal pathogens, microdilution assays were carried out against several pathogens.
In several cases the
MIC values in μg/ml of
Microorganisms | Oleanolic acid | Amphotericin B | |
---|---|---|---|
160 | 250 | 0.78 | |
120 | 250 | 0.31 | |
120 | 190 | 7.5 | |
470 | 250 | 40 | |
20 | 8 | 7.5 | |
20 | 2 | 25 | |
20 | 50 | 20 | |
20 | 20 | 40 | |
60 | 16 | 40 | |
Average | 130 | 130 | 18.1 |
Because the activity of the extract was comparable with the activity of the compound, it means that despite removing approximately 99% of ‘supposedly inactives’ from the crude extract, based on bioautography on a mass basis, the activity did not increase 100 fold. The most probable explanation for this is that there must be a large degree of synergism taking place. The other compounds acting synergistically were not active after they were separated from other compounds during bioautography. An alternative explanation is that other volatile compounds may have been present in the crude extract and that these evaporated during the long period of removing the solvent from the chromatograms before spraying with the fungi. Alternatively, some antifungal compounds may have been destroyed during the isolation process.
Amphotericin B was active with an average MIC value of 18 μg/mL, but in some cases against certain fungi the crude extract had a higher activity than amphotericin B. This points to the possible use of crude extracts in protecting plants against fungi.
If the conclusion reached in the previous paragraph is correct, that there must be substantial activity residing in compounds outside oleanolic acid, an analysis of the total activity of the crude extract and the isolated compound can be made (Eloff
Total activity of the
Samples | Mass in mg | Average MIC in mg/mL | Total activity in mL |
---|---|---|---|
DCM extract | 7820 | 0.13 | 60 154 |
Oleanolic acid | 1204 | 0.13 | 9 262 |
DCM, dichloromethane.
The motivation for determining the cytotoxicity of the extract and oleanolic acid is that if the cytotoxicity is low, there is a possibility that it could be used to treat fungal pathogens on edible plants. If it has a high toxicity, it could still be useful in the horticultural industry (Eloff, Angeh & McGaw
Cellular cytotoxicity of the
Cellular toxicity can also be expressed by the LC50 values, calculated from the regression curve.
Many antifungal compounds are toxic, because several more similar metabolic pathways exist between fungi and mammals than between bacteria and mammals. Therefore there are fewer specific targets that might be addressed. The important question is how the toxicity to the target organism relates to the cellular toxicity. The selectivity index can be defined here as the LC50 in (μg/ml)/MIC in (μg/ml). The higher this value is, the safer the product is to use under controlled conditions (
Selectivity index of crude and oleanolic acid against 10 fungi.
Microorganisms | Crude extract LC50 (μg/mL) | Crude extract MIC (μg/mL) | Selectivity index (crude extract) | Oleanolic acid LC50 (μg/mL) | Oleanolic acid MIC (μg/mL) | Selectivity index (oleanolic acid) |
---|---|---|---|---|---|---|
413 | 310 | 1.0 | 129 | 250 | 0.5 | |
413 | 160 | 2.5 | 129 | 250 | 0.5 | |
413 | 120 | 3.0 | 129 | 250 | 0.5 | |
413 | 120 | 3.0 | 129 | 190 | 0.7 | |
413 | 470 | 0.9 | 129 | 250 | 0.5 | |
413 | 20 | 21.0 | 129 | 8 | 16.0 | |
413 | 20 | 21.0 | 129 | 2 | 64.0 | |
413 | 20 | 21.0 | 129 | 50 | 2.6 | |
413 | 20 | 21.0 | 129 | 20 | 6.5 | |
413 | 60 | 7.0 | 129 | 16 | 8.0 |
The crude extract had a high selectivity index value of 21 against microorganisms
The high selectivity index of 16 and 64 of oleanolic acid against
It appears that there may be greater potential in using plant extracts than the isolated oleanolic acid. The crude plant extract would cost much less to produce. There were major differences in the sensitivity of the different pathogens to the crude extract. This may be valuable because it indicates that the activity is on a basic metabolic target present in all the fungi.
Because many antifungal agents are also highly toxic, it was important to determine the cellular toxicity and the selectivity index. Under controlled conditions the crude extract and oleanolic acid may be used against infections caused by some pathogens with a reasonably good selectivity index value; therefore posing relatively low toxicity threats. In the case of other pathogens, neither the crude extract nor oleanolic acid would be of any potential use, due to its toxicity. One should, however, keep in mind that cellular toxicity does not necessarily correspond to toxicity via other routes. If the toxic compound is ingested and it is destroyed at the low gut pH, or not taken up from the gut or rapidly detoxified, the toxicity may be much lower. It is also possible that a more toxic compound could be produced after uptake of the extract, stressing the importance of
We are grateful to the curator of the Lowveld National Botanical Garden for allowing us to collect plant material. Prof. Lise Korsten (Department of Plant Pathology, University of Pretoria), who provided cultures of the phytopathogens used. This research was supported by the National Research Foundation (NRF) and the University of Pretoria.
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.
M.M. did the work as part of a M.Sc. study. L.M. helped in isolating the active compound and elucidated the structure. J.N.E. identified the project, supervised the study and revised the manuscript and submitted it.
Nuclear Magnetic Resonance spectrum of isolated compound.