Detection of antimicrobial compounds by bioautography of different extracts of leaves of selected South African tree species

Abstract

The hexane, acetone, dichloromethane and methanol extracts of Combretum vendae A.E. van Wyk (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae) and Protorhus longifolia (Bernh. Ex C. Krauss) Engl. (Anacardiaceae) were screened for their antimicrobial activity. The test organisms included bacteria (Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus), and fungi (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Microsporum canis and Sporothrix schenckii). A simple bioautographic procedure, involving spraying suspensions of the bacteria or fungi on thin layer chromatography (TLC) plates developed in solvents of varying polarities was used to detect the number of antibacterial and antifungal compounds present in the extracts. All the extracts had antimicrobial activity against at least one of the test microorganisms. This activity was denoted by white spots against a red-purple background on the TLC plates after spraying with tetrazolium violet. Twenty seven TLC plates; 9 for each solvent system and 3 different solvent systems per organism were tested in the bioautographic procedure. Of the bacteria tested, S. aureus was inhibited by the most compounds separated on the TLC plates from all the tested plants. Similarly, growth of the fungus C. neoformans was also inhibited by many compounds present in the extracts. Loxostylis alata appeared to be the plant extract with the highest number of inhibition bands when compared with other plants tested against both bacteria and fungi. This species was selected for in depth further study.

Keywords: Antibacterial; Antifungal; Bioautography; Medicinal plants; Synergism.

Figure 1

Chromatograms of extracts of hexane (H), acetone (A), dichloromethane (D) and methanol (M) developed in Benzene/Ethanol/Ammonia hydroxide: 90:10:1 [BEA] (non-polar/basic), Chloroform/Ethyl acetate/Formic acid: 5:4:1 [CEF] (intermediate polar/acidic) and Ethyl acetate/Methanol/Water: 40:5.4:4 [EMW] (polar/neutral) and sprayed with vanillin in concentrated sulphuric acid. PL = P. longifolia, KW = Kirkia wilmsii, ON = Ochna natalitia, KA= Khaya anthotheca, LA = Loxostylis alata, CH = Commiphora harveyi, CV = Combretum vendae.

Figure 2

Hexane (H), acetone (A), dichloromethane (D), and methanol (M) extracts of Ochna natalitia (ON), Khaya anthotheca (KA) Loxostylis alata (LA) and Commiphora harveyi (CH) separated on TLC plates using EMW, BEA and CEF, sprayed with bacterial organisms and 24 hrs later by INT. White areas indicate inhibition of bacterial growth by compounds of the plant extract after 60 mins of incubation at 37°C. ON and KA were sprayed with E. coli while LA and CH were sprayed with S. aureus.

Figure 3

Hexane (H), acetone (A), dichloromethane (D), and methanol (M) extracts of Loxostylis alata (LA), Kirkia wilmsii (KW) and Combretum vendae (CV) separated on TLC plates using EMW, BEA and CEF, sprayed with fungal organisms and 24 hours later by INT. White areas indicate inhibition of fungal growth by compounds of the plant extract after 24 hrs of incubation at 37 °C. LA, KW and CV were sprayed with A. fumigatus, C. albicans and S.schenkii, respectively.