Journal of Ethnopharmacology 139 (2012) 81–89 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journa l homepage: www.elsevier.com/locate/jethpharm In vitro antimicrobial synergism within plant extract combinations from three South African medicinal bulbs ∗ B. Ncube, J.F. Finnie, J. Van Staden Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa a r t i c l e i n f o a b s t r a c t Article history: Ethnopharmacological relevance: Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea are used Received 27 July 2011 in South African traditional medicine for the treatment of some infectious diseases and other ailments. Received in revised form Aim of the study: The study aimed at investigating the antimicrobial efficacies of independent and various 21 September 2011 within-plant extract combinations of three medicinal bulbs to understand the possible pharmacological Accepted 15 October 2011 interactions. Available online 30 October 2011 Materials and methods: Bulb and leaf extracts of the three medicinal plants, independently and in com- binations, were comparatively assessed for antimicrobial activity against two Gram-positive and two Keywords: Antimicrobial Gram-negative bacteria and Candida albicans using the microdilution method. The fractional inhibitory concentration indices (FIC) for two extract combinations were determined. Extract combination Interaction Results: At least one extract combination in each plant sample demonstrated good antimicrobial activity Phytochemical against all the test organisms. The efficacies of the various extract combinations in each plant sample Synergy varied, with the strongest synergistic effect exhibited by the proportional extract yield combination of PE and DCM extracts in Merwilla plumbea bulb sample against Staphylococcus aureus (FIC index of 0.1). Most extract combinations demonstrated either a synergistic, additive or indifferent interaction effect against the test bacteria with only a few exhibiting antagonistic effects. Conclusion: The observed antimicrobial efficacy and synergistic interactions indicate the beneficial aspects of combination chemotherapy of medicinal plant extracts in the treatment of infectious diseases. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction with the rapidly changing and potentially damaging external envi- ronmental factors. Being organisms devoid of mobility, plants have The continued evolution of infectious diseases and the devel- evolved elaborate alternative defence strategies, which involve an opment of resistance by pathogens to existing pharmaceuticals, enormous variety of chemical metabolites as tools to overcome have led to the intensification of the search for new novel leads, stress conditions. The ability of plants to carry out combinatorial against fungal, parasitic, bacterial, and viral infections (Gibbons, chemistry by mixing, matching and evolving the gene products 2004). Despite the recent advances in drug development through required for secondary metabolite biosynthetic pathways, creates molecular modelling, combinatorial and synthetic chemistry, nat- an unlimited pool of chemical compounds, which humans have ural plant-derived compounds are still proving to be an invaluable exploited to their benefit. The use of plants by humans in both tra- source of medicines for humans (Salim et al., 2008). Plant-derived ditional and modern medicinal systems, therefore, largely exploits antimicrobials have a long history of providing the much needed this principle. novel therapeutics (Avila et al., 2008). Plants constantly interact A number of traditionally used medicinal plants have to date been screened for various biological activities in both in vivo and in vitro models. The chemical investigation and purification of extracts from plants purported to have medicinal properties have Abbreviations: ATCC, American type culture collection; CFU, colony forming unit; yielded numerous purified compounds which have proven to be DCM, dichloromethane; EtOH, ethanol; FIC, fractional inhibitory concentration; INT, indispensable in the practice of modern medicine (Goldstein et al., p-iodonitrotetrazolium chloride; MH, Mueller–Hinton; MIC, minimum inhibitory 1974; Tyler et al., 1988). In traditional medicine, however, these concentration; MFC, minimum fungicidal concentration; PE, petroleum ether; YM, compounds are largely utilised as crude extracts in the form of yeast malt. ∗ herbal remedies (Pujol, 1990). In light of the new emerging infec- Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897. E-mail address: [email protected] (J. Van Staden). tious diseases and the development of resistance in those with 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.025 82 B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 existing curatives, one of the strategies employed in traditional 2.3. Preparation of saponin-rich extracts herbal medicine to overcome these mechanisms is the combina- tion of herbal remedies. Herbal remedies are often prepared from a Saponins were extracted from the plant material as described combination of several different plant species. The pharmacological by Makkar et al. (2007). The dried and ground plant samples were effects of such mixtures could be as a result of the total sum of differ- defatted with hexane in a Soxhlet apparatus for 3 h. After air- ent classes of compounds with diverse mechanisms of action. There drying, saponins were extracted twice from the defatted samples have been reports of the total contents of a herbal product show- (10 g) in 100 ml of 50% aqueous methanol by incubating at room ing a significantly better effect than an equivalent dose of a single temperature overnight with continuous stirring. The extracts were isolated active ingredient or a single constituent herb (Williamson, then centrifuged at 3000 rpm for 10 min and the supernatant col- 2001; Nahrstedt and Butterweck, 2010). These findings suggest that lected. The procedure was repeated with the original residue to the effects may arise from synergistic mechanisms of herbal ingre- obtain a second supernatant. The first and second supernatants dients. Synergism occurs when two or more compounds interact were combined and filtered under vacuum through Whatman No. in ways that mutually enhance, amplify or potentiate each other’s 1 filter paper. Methanol from the filtrate was evaporated from the ◦ effect more significantly than the simple sum of these ingredients solution under vacuum at 40 C with the saponin sample in the (Williamson, 2001). aqueous phase remaining. The aqueous phase was then centrifuged Although there is significant information on the bioactivity at 3000 rpm for 10 min to remove water insoluble materials. The of the screened medicinal plant extracts, most studies, however, aqueous phase was then transferred into a separating funnel and report these findings on the basis of separate classes/groups of extracted three times with an equal volume of chloroform to compounds extracted using different individual solvents. Many sci- remove pigments. The concentrated saponins in the aqueous solu- entists perform extraction using solvents with increasing polarity, tion were then extracted twice with an equal volume of n-butanol. ◦ e.g. petroleum ether, chloroform, ethyl acetate, ethanol and water. The n-butanol was evaporated under vacuum at 45 C. The dried The quality of the extracted compounds and their overall quan- fractions containing saponins were dissolved in 10 ml of distilled tity in any given plant species would vary largely as a function of water and freeze-dried. the type of solvent used. The question, however, arises: What will the activity be if extracts from one extracting solvent are mixed 2.4. Preparation of phenolic-rich extracts with those from another of the same plant species? What would be the pharmacodynamic interaction between polar and non-polar Phenolic compounds were extracted from plant material as extracts of the same plant species? In light of the multiplicity described by Makkar (1999) with modifications. Dried plant sam- of the phytochemical compounds produced within an individual ples (2 g) were extracted with 10 ml of 50% aqueous methanol by plant, an investigation into this aspect, could possibly unlock the sonication on ice for 20 min. The extracts were then filtered under hidden potentialities of the therapeutic value of the entire set of vacuum through Whatman No. 1 filter paper. The concentrated compounds of a plant extract. We elaborate this knowledge here phenolic-rich extracts were subsequently dried at room temper- by assessing the antimicrobial interaction effect of the different ature under a stream of cold air. extract combinations for three medicinal bulbs. The study extends our previous research on these medicinal bulbs (Ncube et al., 2011a) 2.5. Antibacterial activity Minimum inhibitory concentrations (MIC) of extracts for 2. Materials and methods antibacterial activity were determined using the microdilution bioassay as described by Eloff (1998). Overnight cultures (incubated ◦ 2.1. Plant material at 37 C in a water bath with an orbital shaker) of two Gram-positive (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600) Bulbs and leaves of Tulbaghia violacea Harv., Hypoxis heme- and two Gram-negative (Escherichia coli ATCC 11775 and Klebsiella rocallidea