Journal of Ethnopharmacology 139 (2012) 81–89 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal 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 Article history: Received 27 July 2011 Received in revised form 21 September 2011 Accepted 15 October 2011 Available online 30 October 2011 Keywords: Antimicrobial Extract combination Interaction Phytochemical Synergy a b s t r a c t Ethnopharmacological relevance: Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea are used in South African traditional medicine for the treatment of some infectious diseases and other ailments. Aim of the study: The study aimed at investigating the antimicrobial efficacies of independent and various within-plant extract combinations of three medicinal bulbs to understand the possible pharmacological interactions. Materials and methods: Bulb and leaf extracts of the three medicinal plants, independently and in combinations, were comparatively assessed for antimicrobial activity against two Gram-positive and two Gram-negative bacteria and Candida albicans using the microdilution method. The fractional inhibitory concentration indices (FIC) for two extract combinations were determined. Results: At least one extract combination in each plant sample demonstrated good antimicrobial activity against all the test organisms. The efficacies of the various extract combinations in each plant sample 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 The continued evolution of infectious diseases and the development of resistance by pathogens to existing pharmaceuticals, have led to the intensification of the search for new novel leads, against fungal, parasitic, bacterial, and viral infections (Gibbons, 2004). Despite the recent advances in drug development through molecular modelling, combinatorial and synthetic chemistry, natural plant-derived compounds are still proving to be an invaluable source of medicines for humans (Salim et al., 2008). Plant-derived antimicrobials have a long history of providing the much needed novel therapeutics (Avila et al., 2008). Plants constantly interact Abbreviations: ATCC, American type culture collection; CFU, colony forming unit; DCM, dichloromethane; EtOH, ethanol; FIC, fractional inhibitory concentration; INT, p-iodonitrotetrazolium chloride; MH, Mueller–Hinton; MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration; PE, petroleum ether; YM, yeast malt. ∗ Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897. E-mail address: rcpgd@ukzn.ac.za (J. Van Staden). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.025 with the rapidly changing and potentially damaging external environmental factors. Being organisms devoid of mobility, plants have evolved elaborate alternative defence strategies, which involve an enormous variety of chemical metabolites as tools to overcome stress conditions. The ability of plants to carry out combinatorial chemistry by mixing, matching and evolving the gene products required for secondary metabolite biosynthetic pathways, creates an unlimited pool of chemical compounds, which humans have exploited to their benefit. The use of plants by humans in both traditional and modern medicinal systems, therefore, largely exploits this principle. 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 yielded numerous purified compounds which have proven to be indispensable in the practice of modern medicine (Goldstein et al., 1974; Tyler et al., 1988). In traditional medicine, however, these compounds are largely utilised as crude extracts in the form of herbal remedies (Pujol, 1990). In light of the new emerging infectious diseases and the development of resistance in those with 82 B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 existing curatives, one of the strategies employed in traditional herbal medicine to overcome these mechanisms is the combination of herbal remedies. Herbal remedies are often prepared from a combination of several different plant species. The pharmacological effects of such mixtures could be as a result of the total sum of different classes of compounds with diverse mechanisms of action. There have been reports of the total contents of a herbal product showing a significantly better effect than an equivalent dose of a single isolated active ingredient or a single constituent herb (Williamson, 2001; Nahrstedt and Butterweck, 2010). These findings suggest that the effects may arise from synergistic mechanisms of herbal ingredients. Synergism occurs when two or more compounds interact in ways that mutually enhance, amplify or potentiate each other’s effect more significantly than the simple sum of these ingredients (Williamson, 2001). Although there is significant information on the bioactivity of the screened medicinal plant extracts, most studies, however, report these findings on the basis of separate classes/groups of compounds extracted using different individual solvents. Many scientists perform extraction using solvents with increasing polarity, e.g. petroleum ether, chloroform, ethyl acetate, ethanol and water. The quality of the extracted compounds and their overall quantity in any given plant species would vary largely as a function of the type of solvent used. The question, however, arises: What will the activity be if extracts from one extracting solvent are mixed with those from another of the same plant species? What would be the pharmacodynamic interaction between polar and non-polar extracts of the same plant species? In light of the multiplicity of the phytochemical compounds produced within an individual plant, an investigation into this aspect, could possibly unlock the hidden potentialities of the therapeutic value of the entire set of compounds of a plant extract. We elaborate this knowledge here by assessing the antimicrobial interaction effect of the different extract combinations for three medicinal bulbs. The study extends our previous research on these medicinal bulbs (Ncube et al., 2011a) 2. Materials and methods 2.1. Plant material Bulbs and leaves of Tulbaghia violacea Harv., Hypoxis hemerocallidea Fisch. & C.A. Mey and Merwilla plumbea (Lindl.) Speta were collected in March, from the University of KwaZulu-Natal Botanical Garden, Pietermaritzburg, South Africa and voucher specimens (Table 1) were deposited in the Bews Herbarium (NU) at the University of KwaZulu-Natal, Pietermaritzburg. The samples were separated into bulbs/corms and leaves before being dried in an oven at a constant temperature of 50 ◦ C for five days after which they were ground into fine powders. 2.2. Preparation of plant extracts The ground samples (20 g) were sequentially extracted with 20 ml/g of petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water in a sonication bath containing ice for 1 h. The crude extracts were then filtered under vacuum through Whatman No. 1 filter paper and the organic extracts concentrated in vacuo at 35 ◦ C using a rotary evaporator. The concentrated extracts were subsequently dried at room temperature under a stream of cold air. Water extracts were freeze-dried and kept in airtight containers. The percentage yield of extracts from each extracting solvent was calculated as the ratio of the mass of the dried extract to the mass of the ground plant sample. 2.3. Preparation of saponin-rich extracts Saponins were extracted from the plant material as described by Makkar et al. (2007). The dried and ground plant samples were defatted with hexane in a Soxhlet apparatus for 3 h. After airdrying, saponins were extracted twice from the defatted samples (10 g) in 100 ml of 50% aqueous methanol by incubating at room temperature overnight with continuous stirring. The extracts were then centrifuged at 3000 rpm for 10 min and the supernatant collected. The procedure was repeated with the original residue to obtain a second supernatant. The first and second supernatants were combined and filtered under vacuum through Whatman No. 1 filter paper. Methanol from the filtrate was evaporated from the solution under vacuum at 40 ◦ C with the saponin sample in the aqueous phase remaining. The aqueous phase was then centrifuged at 3000 rpm for 10 min to remove water insoluble materials. The aqueous phase was then transferred into a separating funnel and extracted three times with an equal volume of chloroform to remove pigments. The concentrated saponins in the aqueous solution were then extracted twice with an equal volume of n-butanol. The n-butanol was evaporated under vacuum at 45 ◦ C. The dried fractions containing saponins were dissolved in 10 ml of distilled water and freeze-dried. 2.4. Preparation of phenolic-rich extracts Phenolic compounds were extracted from plant material as described by Makkar (1999) with modifications. Dried plant samples (2 g) were extracted with 10 ml of 50% aqueous methanol by sonication on ice for 20 min. The extracts were then filtered under vacuum through Whatman No. 1 filter paper. The concentrated phenolic-rich extracts were subsequently dried at room temperature under a stream of cold air. 2.5. Antibacterial activity Minimum inhibitory concentrations (MIC) of extracts for antibacterial activity were determined using the microdilution bioassay as described by Eloff (1998). Overnight cultures (incubated at 37 ◦ C in a water bath with an orbital shaker) of two Gram-positive (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600) and two Gram-negative (Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883) bacterial strains were diluted with sterile Mueller–Hinton (MH) broth to give final inocula of approximately 106 CFU/ml (colony forming units). The dried crude organic plant extracts (PE, DCM, and ethanol) were resuspended in 70% ethanol to known concentrations while saponin and phenolic extracts were redisolved in 50% methanol and water extracts in distilled water to the same concentrations. One hundred microlitres of each extract were serially diluted two-fold with sterile distilled water in a 96well microtitre plate for each of the four bacterial strains. A similar two-fold serial dilution of neomycin (Sigma Aldrich, Germany) (0.1 mg/ml) was used as a positive control against each bacterium. One hundred microlitres of each bacterial culture were added to each well. Water, 50% methanol and 70% ethanol were included as negative and solvent controls respectively. The plates were covered with parafilm and incubated at 37 ◦ C for 24 h. Bacterial growth was indicated by adding 50 ␮l of 0.2 mg/ml p-iodonitrotetrazolium chloride (INT) (Sigma–Aldrich, Germany) with further incubation at 37 ◦ C for 2 h. Since the colourless tetrazolium salt is biologically reduced to a red product due to the presence of active organisms, the MIC values were recorded as the concentrations in the last wells in which no colour change was observed after adding the INT indicator. Bacterial growth in the wells was indicated by a reddish-pink colour. The assay was repeated twice with two replicates per assay. B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 83 Table 1 Medicinal bulbs used in this study and their traditional medicinal application. Family Species Voucher number Medicinal uses Alliaceae Tulbaghia violacea Harv. NCUBE 04 NU Hypoxidaceae Hypoxis hemerocallidea Fisch. & C.A. Mey NCUBE 01 NU Hyacinthaceae Merwilla plumbea (Lindl.) Speta NCUBE 02 NU Bulbs and leaves are used for the treatment of gastrointestinal ailments, asthma, constipation, oesophageal cancer, tuberculosis, colds and fever, HIV/AIDS (Hutchings et al., 1996; Crouch et al., 2006; Van Wyk et al., 2009, Klos et al., 2009) Plant decoctions have purging effects and boost the immune system. Corms used for the treatment of inflammation, testicular tumours, urinary complaints, cancer and HIV/AIDS (Watt and Breyer-Brandwijk, 1962; Crouch et al., 2006) Decoctions are used for wound healing, boils, sores, sprains, to remove scar tissue, cleaning and rejuvenating the body. Enhances, male potency and libido (Crouch et al., 2006; Van Wyk et al., 2009) 2.6. Anticandidal activity A microdilution method as described by Eloff (1998) and modified for fungi (Masoko et al., 2007) was used to determine the antifungal activity of the extracts against Candida albicans (ATCC 10231). An overnight fungal culture was prepared in Yeast Malt (YM) broth. Four hundred microlitres of the overnight culture were added to 4 ml of sterile saline and absorbance was read at 530 nm. The absorbance was adjusted with sterile saline to match that of a 0.5 M McFarland standard solution. From this standardised fungal stock, a 1:1000 dilution with sterile YM broth was prepared giving a final inoculum of approximately 106 CFU/ml. Dried crude organic plant extracts (PE, DCM, and ethanol) were resuspended in 70% ethanol to known concentrations while saponin and phenolic extracts were redisolved in 50% methanol and water extracts in distilled water to the same concentrations. One hundred microlitres of each extract were serially diluted two-fold with sterile water in a 96-well microtitre plate. A similar two-fold dilution of Amphotericin B (Sigma–Aldrich, Germany) (2.5 mg/ml) was used as the positive control while water and 70% ethanol were used as negative and solvent controls respectively. One hundred microlitres of the dilute fungal culture were added to each well. The plates were covered with parafilm and incubated at 37 ◦ C for 24 h, after which 50 ␮l (0.2 mg/ml) INT were added and incubated for a further 24 h at 37 ◦ C. The wells remained clear where there was inhibition of fungal growth. MIC values were recorded as the lowest concentrations that inhibited fungal growth after 48 h. To determine the fungicidal activity, 50 ␮l of sterile YM broth were added to all the clear wells and further incubated at 37 ◦ C for 24 h after which the minimum fungicidal concentrations (MFC) were recorded as the last clear wells. The assay was repeated twice with two replicates per assay. 2.7. Checkerboard method for the interaction effect Stock solutions (50 mg/ml) of each individual extract from each plant part were prepared by redisolving water extracts in distilled water, saponin and phenolic extracts in 50% methanol and PE, DCM and ethanol extracts in 70% ethanol. In the case of 1:1 test combinations, equal aliquots of 50 ␮l each of the two extracts were mixed to make up to a volume of 100 ␮l in the first wells of a 96-well microtitre plate while 1:1:1 and 1:1:1:1 combinations had each extract contributing 33.3 ␮l and 25 ␮l respectively to make up 100 ␮l in the first wells of a 96-well microtitre plate. The percentage yields of PE, DCM, ethanol and water extracts in each plant part sample were noted and combinations of extracts in the proportion of their extract yields from each sample were prepared by mixing stock solutions in these respective ratios to make up a volume of 100 ␮l in the microtitre plate wells. MIC values were determined for each of these combinations to establish any interaction effect following the antibacterial and antifungal assays described above. The fractional inhibitory concentration indices (FIC) calculated was limited to combinations with two extracts only. The FIC index expresses the interaction of two or more agents in which the concentration of each test agent in combination is expressed as a fraction of the concentration that would produce the same effect when used independently (Berenbaum, 1978). The FIC was calculated as the MIC of the combination divided by the MIC of each individual component extract. The FIC index was then calculated as the sum of each component FIC in a combination and interpreted as either synergistic (≤0.5), additive (0.5–1.0), indifferent (1–4.0) or antagonistic (≥4.0) (Schelz et al., 2006). 3. Results and discussion The results in this study are discussed in the context of both the interaction effects and the antimicrobial efficacy of the different extract combinations. Ríos and Recio (2005) suggested that MIC greater than 1 mg/ml for crude extracts or 0.1 mg/ml for isolated compounds should be avoided and proposed that activity would be very interesting in MICs of 0.1 mg/ml and 0.01 mg/ml for extracts and isolated compounds respectively. On the other hand, Fabry et al. (1998) defined active crude extracts as those having MIC values <8 mg/ml. In this study, however, MIC and MFC values of less than 1 mg/ml were considered to be of good activity. The in vitro phytochemical synergy is evident in most of the extract combinations from the three different plant species studied (Tables 2–6). Although very few of the independent extracts exhibited good antimicrobial activity (MIC < 1 mg/ml) in both anticandidal and antibacterial bioassays, a number of extract combinations demonstrated good activity, even in cases where none of the independent component extracts were active (Tables 2–6). Of the independent sample extracts, only DCM extracts showed good antibacterial activity against at least one bacterial strain in all samples except Hypoxis hemerocallidea corms (Table 2). On the other hand, good antibacterial activity was only recorded in Tulbaghia violacea bulb extracts against Bacillus subtilis and Staphylococcus aureus, and Hypoxis hemerocallidea leaf extracts against Bacillus subtilis among all the PE sample extracts. All water and ethanol extracts exhibited poor antibacterial activity (MIC > 1 mg/ml). Hypoxis hemerocallidea leaf extracts against Staphylococcus aureus was the only exception. Despite the lower number of active extracts recorded independently for the PE and DCM extracts in this study, a combination of these two extracts in equal proportions (1:1) (Table 2) yielded very good antibacterial activity (MIC < 1 mg/ml) against most strains in all samples. This tremendous increase in bioactivity was mostly recorded in samples where none of the two independent extracts exhibited good antibacterial activity, with a significant number of them showing a synergistic effect (FIC ≤ 0.5). In all 1:1 extract combinations, except those with water extracts, a combination that included either PE or DCM extracts showed good activity against at least two 84 Table 2 Antibacterial activity (MIC mg/ml) and FIC values for the independent and equal ratio extract combinations of Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea sample extracts. Plant part Tulbaghia violacea Bulb Bacterial strain MIC (mg/ml) and FIC indices P Hypoxis hemerocallidea Merwilla plumbea Neomycin (␮g/ml) D Bs 0.8 0.8 3.1 Ec 3.1 0.8 Kp 1.6 Sa 0.8 0.8 0.8 Leaf Bs 1.6 Ec 3.1 3.1 1.6 1.6 Kp 0.8 Sa 3.1 Corm Bs 1.6 1.6 Ec 6.3 3.1 3.1 Kp 3.1 Sa 6.3 3.1 Bs 0.8 0.4 Leaf 3.1 6.3 Ec 3.1 Kp 3.1 Sa 3.1 1.6 Bulb Bs 6.3 0.8 3.1 Ec 6.3 Kp 6.3 3.1 12.5 12.5 Sa Bs 6.3 1.6 Leaf 3.1 Ec 6.3 Kp 3.1 3.1 6.3 0.8 Sa Bs: 0.098; Ec: 3.13; Kp: 1.56; Sa: 1.56 E W P/D P/E P/W P/D/E P/D/W P/E/W D/E D/W D/E/W E/W P/D/E/W 6.3 3.1 1.6 12.5 6.3 3.1 12.5 12.5 3.1 3.1 1.6 3.1 1.6 3.1 1.6 0.8 3.1 3.1 3.1 1.6 3.1 3.1 3.1 3.1 18.8 18.8 3.1 18.8 18.8 12.5 12.5 12.5 18.8 12.5 3.1 12.5 18.8 18.8 3.1 18.8 18.8 18.8 18.8 12.5 6.3 12.5 18.8 12.5 0.4(1.0)a 0.4(0.3) 0.8(1.5) 0.4(1.0) 0.4(0.8) 0.4(0.3) 0.2(0.3) 0.4(0.6) 0.4(0.5) 0.4(0.2) 0.4(0.3) 0.8(0.4) 0.8(3.0) 0.4(0.2) 0.8(0.5) 0.4(0.4) 0.8(1.1) 0.4(0.2) 0.8(0.4) 0.8(0.1) 0.4(0.3) 0.4(0.2) 0.4(0.3) 0.4(0.6) 0.4(0.6 0.4(0.3) 0.8(1.0) 0.2(0.3) 0.4(0.3) 0.4(0.3) 0.4(0.3) 0.4(0.2) 0.4(0.4) 0.8(0.4) 0.4(0.3) 0.8(0.4) 0.4(0.8) 0.8(0.5) 0.8(0.8) 0.4(0.6) 0.4(0.2) 0.8(0.4) 0.4(0.2) 0.8(0.6) 0.4(0.2) 0.8(0.4) 0.4(0.3) 0.8(0.4) 0.8(1.0) 0.8(0.3) 0.8(0.8) 0.8(1.0) 0.8(0.6) 0.8(0.3) 1.6(1.1) 1.6(0.6 1.6(1.0) 1.6(0.4) 0.8(0.5) 1.6(0.4) 0.8(1.0) 0.8(0.3) 0.8(0.5) 1.6(0.6) 1.6(0.4) 3.1(0.7) 1.6(0.4) 3.1(0.5) 0.8(0.3) 1.6(0.4) 1.6(0.6) 0.8(0.2) 0.1 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.5 0.5 0.5 0.5 0.5 0.3 0.3 0.3 0.3 0.3 0.5 0.3 0.3 0.5 0.3 0.5 0.3 0.3 0.3 0.5 0.3 0.3 0.5 0.5 1.0 0.3 0.3 0.3 0.3 0.5 0.5 0.5 0.5 0.5 1.0 0.5 0.5 0.5 0.5 1.0 0.5 0.5 0.5 0.5 0.5 0.5 1.0 1.0 1.0 0.3 0.5 0.3 0.5 0.4(0.6) 0.4(0.3) 0.4(0.8) 0.8(1.1) 0.4(0.6) 0.4(0.3) 0.2(0.2) 0.4(0.6) 0.4(0.4) 0.8(0.5) 0.4(0.4) 0.8(0.5) 0.4(1.3) 0.4(0.2) 0.2(0.2) 0.4(0.8) 0.2(0.3) 0.4(0.3) 0.4(0.3) 0.8(0.6) 0.2(0.2) 0.8(0.5) 0.4(0.3) 0.8(1.3) 0.8(1.1) 0.8(0.3) 0.8(1.3) 1.6(2.0) 0.8(1.1) 0.8(0.3) 0.8(0.6) 1.6(2.1) 0.8(0.6) 1.6(0.6) 0.8(0.5) 1.6(0.6) 0.8(2.1) 0.8(0.2) 0.8(0.5) 0.8(0.6) 0.8(1.1) 1.6(0.6) 0.8(0.6) 1.6(0.3) 0.4(0.3) 1.6(0.6) 0.8(0.3) 0.8(1.1) 0.5 0.5 0.5 1.0 0.5 1.0 1.0 0.5 0.5 1.0 1.0 1.0 0.3 0.5 0.5 0.5 1.0 1.0 1.0 1.0 0.5 0.5 1.0 0.5 3.1(0.6) 6.3(2.3) 0.5(0.8) 3.1(0.4) 3.1(0.7) 6.3(2.5) 6.3(2.0) 6.3(1.0) 0.8(0.3 0.8(0.3) 0.4(0.4) 0.8(0.3) 0.8(0.6) 1.6(0.6) 1.6(1.5) 0.4(0.5) 3.1(1.2) 3.1(1.2) 3.1(1.2) 1.6(1.1) 0.8(0.4) 1.6(0.6) 0.8(0.3) 0.8(0.3) 0.8 0.4 0.4 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.2 0.8 0.4 0.2 0.4 0.1 0.8 0.8 0.4 0.4 0.2 0.2 0.2 0.2 Bs = Bacillus subtilis, Ec = Escherichia coli, Kp = Klebsiella pneumonia, Sa = Staphylococcus aureus, P = petroleum ether; D = dichloromethane, E = 80% ethanol, W = water. FIC values are shown in brackets. FIC values boldly written indicate a synergistic interaction effect while boldly written MIC values are considered very active (<1 mg/ml). a The MIC value recorded for 1:1, 1:1:1 and 1:1:1:1 extract combinations represent the MIC value for each component extract in combination (MIC values equal in each case). B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 Plant species B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 85 Table 3 Extract yields (%) and antibacterial activity (MIC, mg/ml) and FIC values and for the proportional extract yield combinations of Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea sample extracts. Plant species Tulbaghia violacea Hypoxis hemerocallidea Merwilla plumbea Neomycin (␮g/ml) Plant part Extract yields (%) E Bacterial strain P D W Bulb 0.6 0.3 7.3 36.5 Leaf 1.5 1.0 24.0 16.9 Corm 0.3 0.2 21.5 9.1 Leaf 0.8 0.6 11.4 9.4 Bulb 0.5 0.6 17.3 13.6 Leaf 1.3 1.1 16.4 15.5 MIC (mg/ml) and FIC indices P/D P/E P/W D/E D/W E/W Bs Ec Kp Sa Bs Ec Kp Sa Bs 0.5/0.3(1.0) 0.5/0.3(0.3) 0.5/0.3(0.8) 0.5/0.3(1.0) 0.2/0.2(0.7) 0.5/0.3(0.3) 0.5/0.3(0.5) 0.5/0.3(0.7) 0.5/0.3(0.5) 0.1/1.5(1.2) 0.1/1.5(0.5) 0.1/1.5(1.0) 0.1/1.5(1.1) 0.1/1.5(0.3) 0.1/1.5(0.5) 0.1/1.5(0.2) 0.1/1.5(0.2) 0.01/1.6(1.0) 0.1/3.0(0.3) 0.1/6.2(0.4) 0.03/1.6(0.5) 0.03/1.6(0.1) 0.1/1.5(0.8) 0.3/2.8(0.3) 0.1/1.5(0.8) 0.1/1.5(0.2) 0.1/3.0(0.2) 0.1/1.5(0.4) 0.1/1.5(0.5) 0.03/0.8(0.5) 0.03/.08(0.1) 0.1/1.5(0.4) 0.1/1.5(0.5) 0.03/0.8(0.1) 0.1/1.5(0.2) 0.01/1.6(0.5) 0.03/3.1(0.2) 0.03/3.1(0.2) 0.03/3.1(1.0) 0.03/3.1(0.2) 0.4/5.9(0.8) 0.2/2.9(0.3) 0.4/5.9(0.7) 0.2/2.9(0.5) 0.02/1.6(0.1) 1.1/5.2(0.5) 1.1/5.2(0.6) 0.5/2.6(1.2) 1.1/5.2(0.4) 1.8/1.3(0.4) 1.8/1.3(0.7) 1.8/1.3(0.2) 1.8/1.3(0.2) 1.1/0.5(0.4) Ec Kp Sa Bs Ec Kp Sa Bs Ec Kp Sa Bs Ec Kp Sa 0.5/0.3(0.2) 0.5/0.3(0.3) 1.0/0.6(0.4) 0.5/0.3(1.5) 0.5/0.3(0.2) 0.5/0.3(0.3) 0.5/0.3(0.4 0.4/0.4(0.6) 0.4/0.4(0.2) 0.4/0.4(0.2) 0.4/0.4(0.1) 0.4/0.4(0.3) 0.4/0.4(0.2) 0.4/0.4(0.3) 0.4/0.4(0.6) 0.01/1.6(0.5) 0.01/1.6(1.0) 0.01/1.6(0.5) 0.1/1.5(1.1) 0.1/1.5(0.5) 0.1/1.5(1.0) 0.1/1.5(2.0) 0.04/1.6(0.5) 0.04/1.6(0.5) 0.04/1.6(0.5) 0.1/3.0(2.0) 0.1/1.5(0.5) 0.1/1.5(0.5) 0.1/0.7(0.3) 0.1/1.5(0.5) 0.2/6.1(0.5) 0.1/3.0(1.0) 0.1/3.0(0.3) 0.1/1.5(0.2) 0.2/2.9(0.2) 0.1/1.5(0.5) 0.1/1.5(0.2) 0.1/1.5(0.1) 0.1/3.0(0.2) 0.1/1.5(0.1) 0.1/3.0(0.2) 0.2/2.9(0.5) 0.2/2.9(0.3) 0.1/1.5(0.1) 0.2/2.9(0.3) 0.01/0.8(0.3) 0.01/0.8(0.5) 0.01/1.6(0.5) 0.04/0.8(0.6) 0.04/0.8(0.3) 0.04/0.8(0.5) 0.1/1.5(2.0) 0.03/0.8(0.3) 0.1/1.5(0.5) 0.1/1.5(0.5) 0.1/1.5(1.0) 0.1/1.5(0.5) 0.1/1.5(0.5) 0.1/1.5(0.5) 0.1/1.5(0.6) 0.02/1.6(0.1) 0.1/3.0(1.0) 0.02/1.6(0.1) 0.1/1.5(0.3) 0.1/1.5(0.1) 0.1/1.5(0.5) 0.1/1.5(0.1) 0.3/6.0(0.7) 0.3/6.0(0.4) 0.3/6.0(0.4) 0.3/6.0(0.5) 0.1/1.5(0.3) 0.1/1.5(0.2) 0.1/1.5(0.1) 0.1/1.5(0.2) 1.1/0.5(0.4) 0.6/0.2(0.4) 1.1/0.5(0.4) 0.9/0.5(0.3) 1.1/2.0(0.5) 0.9/0.5(0.7) 0.4/0.4(0.2) 1.7/1.4(0.6) 1.7/1.4(0.6) 1.7/1.4(0.6) 0.9/0.7(0.6) 0.8/0.8(0.4) 1.6/1.5(0.6) 1.6/1.5(0.6) 1.6/1.5(0.6) Bs: 0.098; Ec: 3.13; Kp: 1.56; Sa: 1.56 Bs = Bacillus subtilis, Ec = Escherichia coli, Kp = Klebsiella pneumonia, Sa = Staphylococcus aureus, P = petroleum ether; D = dichloromethane, E = 80% ethanol, W = water. FIC values are shown in brackets. FIC values boldly written indicate a synergistic interaction effect while boldly written MIC values are considered very active (<1 mg/ml). bacterial strains in each sample, with all exhibiting either a synergistic or additive interaction effect. When extracts were combined in a 1:1:1 combination, antibacterial efficacy was only enhanced in combinations that included both PE and DCM extracts in all samples except for Merwilla plumbea leaf extract. The trend in antibacterial activity observed in 1:1, 1:1:1 and 1:1:1:1 extract combinations in this study leads to the conclusion that the active constituents in these crude extracts are among the lipophilic (non-polar) group of compounds. Although PE extracts more highly non-polar compounds than DCM, the two solvents generally extract non-polar classes of compounds compared to water and ethanol which extract mostly polar compounds. However, the weak potency recorded when the two extracts were tested independently, and the correspondingly good activity when combined with each other and Table 4 Anticandidal activity (MIC and MFC, mg/ml) and FIC values for the independent and equal ratio extract combinations of Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea sample extracts. Plant species Plant part Tulbaghia violacea Bulb P D E W P/D P/E P/W P/D/E P/D/W P/E/W D/E D/W D/E/W E/W P/D/E/W 6.3 6.3 3.1 6.3 6.3 12.5 18.8 18.8 0.5 1.0 0.3 0.3 0.3 0.5 0.5 0.5 6.3 6.3 3.1 3.1 0.5 0.5 0.5 0.8 0.8 0.2 1.6 1.6 1.1 3.1 1.6 1.6 0.2 1.6 1.6 0.4 0.4 0.8 0.8 12.5 12.5 0.4 0.4 0.1 0.4 0.4 0.3 0.4 0.3 0.3 3.1 6.3 1.6 1.6 0.3 3.1 3.1 2.2 3.1 1.0 1.0 1.6 1.6 0.8 0.8 0.2 0.4 0.4 0.4 0.8 0.3 0.5 0.8 1.6 0.4 0.8 0.3 0.4 0.4 0.5 0.8 MFC 6.3 FIC MIC 6.3 Leaf MFC 6.3 FIC Bulb MIC 6.3 6.3 MFC FIC Leaf MIC 3.1 MFC 6.3 FIC MIC: 9.77; MFC: 78.1 6.3 3.1 3.1 0.5 0.5 0.5 0.3 0.3 6.3 6.3 6.3 6.3 18.8 18.8 0.5 0.5 0.5 1.0 1.0 1.0 0.4 0.8 3.1 6.3 6.3 6.3 1.0 1.0 1.0 1.0 1.0 1.0 3.1 1.5 1.6 1.6 2.5 3.1 3.1 0.7 1.6 3.1 4.4 0.4 0.3 0.4 0.4 0.8 1.6 3.1 0.7 1.6 1.6 0.5 0.8 0.5 0.5 0.4 0.2 0.4 0.4 0.4 0.4 0.4 0.2 0.8 0.8 1.2 0.3 0.8 0.8 3.1 1.6 3.1 3.1 4.4 1.6 3.1 0.7 3.1 3.1 1.0 0.5 0.8 1.6 0.8 0.4 0.8 0.8 0.7 0.4 0.8 0.3 0.4 0.8 0.3 1.0 0.8 3.1 1.6 0.5 0.8 0.8 0.4 0.4 0.4 0.2 0.8 0.8 1.2 Leaf Hypoxis hemerocallidea Merwilla plumbea Amphotericin B (␮g/ml) MIC and MFC (mg/ml) and FIC indices Corm MIC MFC FIC MIC MFC FIC MIC 0.3 0.3 0.3 0.3 0.3 0.5 0.5 0.3 0.3 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 P = petroleum ether; D = dichloromethane, E = 80% ethanol, W = water. FIC values boldly written indicate a synergistic interaction effect while boldly written MIC and MFC values are considered very active (<1 mg/ml). FIC values are calculated for MFC only. MIC and MFC values recorded for 1:1, 1:1:1 and 1:1:1:1 extract combinations represent the MIC and MFC values for each component extract in combination. 86 B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 Table 5 Extract yields (%) and anticandidal activity (MIC and MFC, mg/ml) and FIC values for the proportional extract yield combinations of Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea sample extracts. Plant species Tulbaghia violacea Plant part Extract yields (%) MIC/MFC (mg/ml) and FIC indices P D E W 0.6 0.3 7.3 36.5 1.5 1.0 24.0 16.9 0.3 0.2 21.5 9.1 0.8 0.6 11.4 9.4 0.5 0.6 17.3 13.6 1.3 1.1 16.4 15.5 Bulb Leaf Hypoxis hemerocallidea Corm Leaf Merwilla plumbea Bulb Leaf P/D P/E P/W D/E D/W E/W MIC MFC FIC MIC MFC FIC MIC 0.5/0.3 0.5/0.3 0.1 0.5/0.3 0.5/0.3 0.5 1.0/0.6 0.1/1.5 0.1/1.5 0.3 0.1/1.5 0.1/1.5 0.3 0.02/1.6 0.1/6.2 0.1/6.2 0.3 0.2/2.9 0.2/2.9 0.4 0.1/1.5 0.03/0.8 0.03/.08 0.1 0.0.3/0.8 0.03/0.8 0.1 0.01/0.8 0.1/6.2 0.1/12.4 1.0 0.2/2.9 0.4/5.9 0.7 0.01/1.6 2.1/10.4 2.1/10.4 0.7 3.7/2.6 7.3/5.2 1.6 1.1/0.5 MFC FIC MIC MFC FIC MIC MFC FIC MIC MFC FIC 1.0/0.6 0.1 0.9/0.7 0.9/0.7 0.4 0.4/0.4 0.4/0.4 0.2 0.4/0.4 0.9/0.7 1.0 0.02/1.6 0.5 0.1/1.5 0.1/1.5 1.0 0.2/6.1 0.2/6.1 1.0 0.1/1.5 0.1/1.5 0.3 0.1/1.5 0.5 0.1/0.7 0.1/0.7 0.9 0.2/6.1 0.2/6.1 0.4 0.2/2.9 0.5/5.8 1.0 0.01/0.8 0.3 0.04/0.7 0.04/0.7 0.5 0.2/6.1 0.2/6.1 1.0 0.1/1.5 0.1/1.5 0.4 0.01/1.6 0.5 0.05/0.7 0.05/0.7 0.9 0.1/3.0 0.3/6.0 0.4 0.1/1.5 0.1/1.5 0.4 1.1/0.5 0.5 0.4/0.4 0.4/0.4 0.8 1.7/1.4 1.7/1.4 0.3 1.2/1.9 1.2/1.9 0.5 P = petroleum ether; D = dichloromethane, E = 80% ethanol, W = water. FIC values boldly written indicate a synergistic interaction effect while boldly written MIC and MFC values are considered very active (<1 mg/ml). FIC values are calculated for MFC only. with ethanol extracts, strongly suggest that their mechanism of action is potentiated by the accompanying compounds in each of the two extracts and those in ethanol extracts. The compounds from all water extracts displayed a diluting effect characteristic in all combinations as evidenced by the low efficacy in most extract combinations that included a water extract. With the exception of Hypoxis hemerocallidea corm extracts against Staphylococcus aureus, a combination of PE and DCM extracts in the proportion of their extract yields (Table 3) showed good antibacterial activity in all plant samples. The FIC indices in most of these extract combinations showed either a synergistic or additive effect, with only a few exhibiting an indifferent interaction effect. A look at the yields of these two extracts (Table 3) indicates that the percentage extract yields in all samples except Merwilla plumbea bulbs were slightly higher for PE than DCM extracts. This therefore, translates to this proportional extract yield combination having a slightly higher concentration of the PE extract constituents than those of DCM. For example, in the case of Tulbaghia violacea bulb extracts with 0.6% and 0.3% extract yields for PE and DCM respectively, would have a proportional yield combination of 67 ␮l PE extract stock solution with 33 ␮l of DCM extract stock solution. The PE and DCM proportional extract yield combination of Merwilla plumbea bulbs demonstrated the strongest synergistic inhibitory effect against Staphylococcus aureus, with an FIC index value as low as 0.1. This is despite the fact that each of the two individual component extracts had an independent MIC of 12.5 mg/ml each against the same bacterial strain. This, being the only PE:DCM extract combination with more of the DCM extract constituents, indicates that this phenomenon significantly potentiates the two extract efficacies. Such synergistic combinations may often, translate to superior therapeutic effects and even lower dosage requirement for each extract, thereby reducing the likelihood of dose-dependent toxicity experienced with most herbal remedies (Klippel, 1990; Boucher and Tam, 2006). In a study with Hypericum perforatum, Nahrstedt and Butterweck (2010) demonstrated that the bioavailability of certain active constituents, such as hypericin, can be improved by the accompanying compounds present in the same botanical product, a principle which most herbal therapeutics lends credence to. The overall trend in the antibacterial efficacy and interaction effect of the proportional extract yield combination differed slightly from those of the equal combinations, with most combinations that included both PE and DCM extracts showing enhanced activity. A closer comparison of the proportional extract yield combinations of PE and DCM, with that of a 1:1 ratio combination (Tables 2 and 3) reveals that the 1:1 combination is a less active combination than the proportional yield combination. The proportional extract yield combination closely mimic the natural interaction effect of these compounds in plants, and as such may best serve to explain the successful defence mechanism developed by plants. On the other hand, a 1:1:1 combination of PE, DCM and ethanol extracts gave good antibacterial activity in all the tested plant samples except for Merwilla plumbea leaf samples against all bacterial strains and Merwilla plumbea bulbs against Staphylococcus aureus, compared to their corresponding proportion extract yield combinations (Tables 2 and 6). The results indicate that the efficacious interaction effect may be dependent on the precise concentrations of certain compounds in an extract combination. In spite of the fact that most independent plant-derived extracts have shown weak potency against pathogenic bacteria compared to antibiotics, plants almost always fight infections successfully in their natural environment. The aspect of synergistic mechanisms becomes the apparent strategy employed by plants, hence the improved efficacy demonstrated by combining the within-plants extracts in this study. The generally good antibacterial activity shown by both proportional extract yields and equal extract combinations of PE and DCM in almost all samples in this study, suggests that non-polar compounds interact more synergistically than the polar compounds. The trend is, however, consistent with most of the findings in other non-interaction studies, in which non-polar extracts demonstrated better antimicrobial activity than polar ones (Rabe and Van Staden, 1997; McGaw et al., 2001; Ncube et al., 2011b). Considering the scarcity of plant extracts with good antibacterial activity against Gram-negative bacteria in most of the previous studies with different plant materials (Rabe and Van Staden, 1997; Shale et al., 1999), these extract combinations offer good and promising prospects for the treatment of diseases caused by these bacteria in traditional medicine. Escherichia coli and Klebsiella pneumoniae, for example, are known causative agents for diseases such as, urinary tract, gastrointestinal tract and wound infections, bacteriaemia, pneumonia septicaemia and meningitis in humans (Sleigh and Timbury, 1998; Einstein, 2000). Tulbaghia violacea, Table 6 Antibacterial and anticandidal activity (MIC and MFC, mg/ml) for the proportional extract yield combinations of Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea sample extracts. Plant part Bacterial strain Tulbaghia violacea Bulb Bs Ec Kp Sa Bs Ec Kp Sa Bs Leaf Hypoxis hemerocallidea Merwilla plumbea Neomycin ␮g/ml) Corm MIC (mg/ml) MIC/MFC (mg/ml) Candida albicans P/D/E P/D/W P/E/W D/E/W P/D/E/W P/D/E P/D/W P/E/W D/E/W P/D/E/W 0.1/0.1/1.4 0.1/0.03/0.7 0.1/0.03/0.7 0.1/0.1/1.4 0.05/0.03/0.7 0.05/0.03/0.7 0.1/0.1/1.4 0.1/0.1/1.4 0.01/0.01/0.7 0.01/0.1/0.8 0.01/0.1/0.8 0.01/0.1/0.8 0.03/0.01/1.5 0.0.1/0.04/0.7 0.0.1/0.04/0.7 0.0.1/0.04/0.7 0.1/0.1/1.4 0.03/0.02/0.8 0.04/0.5/2.6 0.04/0.5/2.6 0.02/0.3/1.3 0.04/0.5/2.6 0.1/0.9/0.6 0.1/0.9/0.6 0.1/1.8/1.2 0.1/0.9/0.6 0.02/1.1/0.5 0.01/0.3/1.3 0.01/0.3/1.3 0.01/0.1/0.7 0.01/0.3/1.3 0.04/0.9/0.6 0.04/0.9/0.6 0.1/1.8/1.3 0.04/0.9/0.6 0.02/2.1/0.9 0.2/0.1/2.0/10.2 0.2/0.1/2.0/10.2 0.04/0.02/0.5/2.5 0.2/0.1/2.0/10.2 0.2/1.4/3.5/2.5 0.4/0.3/6.9/4.9 0.1/0.1/1.7/1.2 0.1/0.1/1.7/1.2 0.01/0.01/0.6 MIC MFC 0.1/0.03/0.7 0.1/0.03/0.7 0.05/0.02/2.9 0.05/0.02/2.9 0.2/2.1/10.3 0.2/2.1/10.3 0.04/1.0/5.2 0.04/1.0/5.2 0.2/0.1/2.0/10.2 0.2/0.1/2.0/10.2 MIC MFC 0.05/0.03/0.7 0.05/0.03/0.7 0.0.1/0.04/0.7 0.1/0.1/1.4 0.1/1.8/1.2 0.2/3.6/2.5 0.1/1.8/1.3 0.1/1.8/1.3 0.2/1.4/3.5/2.5 0.2/1.4/3.5/2.5 MIC 0.01/0.01/0.7 0.1/0.03/1.5 0.03/2.2/0.9 0.01/1.1/0.5 0.03/0.02/2.1/0.9 0.03/0.02/0.8 0.03/0.02/0.8 0.03/0.02/0.8 0.01/0.04/0.7 0.1/0.1/1.4 0.01/0.04/0.7 0.01/0.04/0.7 0.1/0.1/1.5 0.03/0.03/0.7 0.1/0.1/1.5 0.1/0.1/1.5 0.1/0.05/0.7 0.1/0.1/1.4 0.1/0.1/1.4 0.1/0.05/0.7 0.02/1.1/0.5 0.02/1.1/0.5 0.02/1.1/0.5 0.1/0.8/0.7 0.1/0.8/0.7 0.1/0.8/0.7 0.03/0.4/0.3 0.05/1.7/1.3 0.03/0.9/0.7 0.03/0.9/0.7 0.03/0.9/0.7 0.1/1.5/0.5 0.1/1.5/0.5 0.1/0.8/0.8 0.1/1.5/0.5 0.01/1.1/0.5 0.01/0.6/0.2 0.01/1.1/0.5 0.02/0.4/0.4 0.1/1.7/1.4 0.04/0.9/0.7 0.04/0.9/0.7 0.03/0.9/0.7 0.03/0.9/0.7 0.03/0.9/0.7 0.1/1.7/0.4 0.1/0.8/0.8 0.1/0.8/0.8 0.1/0.8/0.8 0.03/0.4/0.4 0.002/0.001/0.1/0.1 0.01/0.01/0.6 0.002/0.001/0.1/0.1 0.1/0.04/0.8/0.7 0.03/0.02/0.4/0.3 0.03/0.02/0.4/0.3 0.01/0.01/0.2/0.2 0.03/0.03/0.9/0.7 0.05/0.1/1.7/1.3 0.03/0.03/0.9/0.7 0.1/0.1/3.4/2.7 0.03/0.03/0.4/0.4 0.02/0.01/0.2/0.2 0.03/0.03/0.4/0.4 0.03/0.03/0.4/0.4 MFC 0.02/0.01/1.5 0.1/0.03/1.5 0.03/2.2/0.9 0.01/1.1/0.5 0.1/0.04/4.4/1.8 MIC MFC 0.01/0.04/0.7 0.01/0.04/0.7 0.01/0.04/0.7 0.01/0.04/0.7 0.03/0.4/0.3 0.03/0.4/0.3 0.02/0.4/0.4 0.04/0.9/0.7 0.1/0.04/0.8/0.7 0.1/0.04/0.8/0.7 MIC MFC 0.02/0.03/0.8 0.02/0.03/0.8 0.2/0.3/5.8 0.2/0.3/5.8 0.05/1.7/1.3 0.1/3.5/2.7 0.1/3.5/2.7 0.1/3.5/2.7 0.2/0.2/6.8/5.3 0.2/0.2/6.8/5.3 MIC MFC 0.1/0.05/0.7 0.1/0.05/0.7 0.1/0.1/1.4 0.2/0.2/2.7 0.2/3.1/2.9 0.2/3.1/2.9 0.1/0.8/0.8 0.1/1.5/1.5 0.1/0.1/1.5/1.4 0.1/0.1/1.5/1.4 Ec 0.01/0.01/0.7 Kp 0.01/0.01/0.7 0.02/0.01/1.5 Sa Bs 0.1/0.1/1.4 Leaf Ec 0.01/0.04/0.7 0.1/0.1/1.4 Kp 0.1/0.1/1.4 Sa Bulb Bs 0.04/0.1/1.5 Ec 0.02/0.03/0.8 0.04/0.1/1.5 Kp Sa 0.04/0.1/1.5 Leaf Bs 0.1/0.05/0.7 0.1/0.05/0.7 Ec 0.1/0.1/1.4 Kp 0.1/0.1/1.4 Sa Bs: 0.098; Ec: 3.13; Kp: 1.56; Sa: 1.56 B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 Plant species Amphotericin B (␮g/ml) MIC: 9.77; MFC: 78.1 Bs = Bacillus subtilis, Ec = Escherichia coli, Kp = Klebsiella pneumonia, Sa = Staphylococcus aureus, P = petroleum ether; D = dichloromethane, E = 80% ethanol, W = water. MIC and MFC values boldly written are considered very active (<1 mg/ml). 87 88 B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 Table 7 Antibacterial and anticandidal activity (MIC/MFC, mg/ml) and FIC values for the independent and 1:1 extract combinations of phenolic- and saponin-rich extracts of Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea sample extracts. Plant species Plant part Extract MIC (mg/ml) and FIC indices MIC/MFC (mg/ml) and FIC indices Bacteria Candida albicans Bs Tulbaghia violacea Hypoxis hemerocallidea Merwilla plumbea Neomycin (␮g/ml) Phe 3.1 Bulb Sap 3.1 Phe/Sap 6.3/6.3 (4.1) Phe 6.3 Leaf Sap 3.1 9.4/9.4 (4.5) Phe/Sap 0.8 Phe Corm Sap 3.1 Phe/Sap 0.8/0.8 (1.3) 0.8 Phe Leaf Sap 3.1 1.6/1.6 (2.5) Phe/Sap Phe 3.1 Bulb Sap 6.3 Phe/Sap 3.1/3.1 (1.5) Phe 3.1 Sap 6.3 Leaf 3.1/3.1 (0.8) Phe/Sap Bs: 0.098; Ec: 3.13; Kp: 1.56; Sa: 1.56 Ec Kp 3.1 6.3 6.3/6.3 (3.0) 3.1 3.1 9.4/9.4 (6.1) 0.8 1.6 0.2/0.2 (0.4) 0.8 1.6 0.4/0.4 (0.8) 6.3 6.3 6.3/6.3 (2.0) 6.3 6.3 3.1/3.1 (1.0) Sa MIC 3.1 6.3 6.3 3.1 6.3 12.5 3.1/3.1(2.0) 3.1/3.1 (1.0) 6.3/6.3 3.1 6.3 6.3 1.6 6.3 12.5 6.3/6.3(5.9) 6.3/6.3 (2.0) 3.1/3.1 0.8 0.8 1.6 1.6 3.1 3.1 0.4/0.4(0.8) 0.2/0.2 (0.3) 0.4/0.4 1.6 0.8 1.6 1.6 1.6 3.1 0.8/0.8 (1.0) 0.2/0.2 (0.4) 0.4/0.4 6.3 6.3 12.5 12.5 6.3 12.5 1.6/1.6(0.4) 1.6/1.6(0.5) 3.1/3.1 6.3 6.3 6.3 3.1 6.3 6.3 1.6/1.6(0.8) 1.6/1.6(0.5) 6.3/6.3 Amphotericin B (␮g/ml) MIC: 9.77; MFC: 78.1 MFC 12.5 12.5 6.3/6.3 (1.0) 6.3 12.5 3.1/3.1 (0.8) 1.6 3.1 0.4/0.4 (0.4) 1.6 3.1 0.4/0.4 (0.4) 12.5 12.5 3.1/3.1 (0.5) 12.5 6.3 6.3/6.3 (1.5) FIC values are shown in brackets. Boldly written MIC values are considered very active (<1 mg/ml). Hypoxis hemerocallidea and Merwilla plumbea are used in the South African traditional medicine against some of these ailments. In light of the global threat by the emerging antibiotic resistant bacterial strains which cause infectious diseases, the results of this study provides an insight into the potential sources of such remedies and further confirm the efficacy of mixing medicinal plant extracts. Although most of the extract combinations demonstrated either synergistic or additive, interaction effects in the antibacterial test in this study, there were a few cases where extract combinations exhibited indifferent effects, for example, the 1:1 combination of PE and DCM, extracts of Hypoxis hemerocallidea leaf samples against Bacillus subtilis (FIC index 3.0). The PE, DCM, ethanol and water proportional extract yield combination of Hypoxis hemerocallidea corm extracts, on the other hand, demonstrated the best antibacterial activity, with the highest component MIC (water) of 0.1 against both Escherichia coli and Staphylococcus aureus. Such a scenario, however, highlights the complexity of the interaction effects of a suite of chemical compounds found within a plant, and the potential dangers associated with the widely held perception by herbal medicinal users that herbal mixtures, almost always have an enhanced efficacy. Combination chemotherapy is often employed in clinical practice for the treatment of infectious diseases. Substantial research trials have been done to investigate the interaction effects of medicinal plant extracts with known clinical drugs in the treatment of various ailments, with several of them yielding positive interaction effects (Sato et al., 2004; Filoche et al., 2005; Prabhakar and Doble, 2009). Combinations of different medicinal plant components in the herbal formulation remedies are increasingly becoming a common phenomenon in most traditional medicinal systems. In an attempt to understand the antimicrobial efficacy of such herbal combinations, Van Vuuren and Viljoen (2008) and Ndhlala et al. (2009) investigated this aspect with different plant parts and herbal preparations respectively, and reported both positive and negative interaction effects. While extract combinations may, in some cases, result in an enhanced efficacy, it must, however, be pointed out that synergism should not always be assumed, as this and other previous studies have shown. In the anticandidal test, a 1:1 and proportional extract yields combinations of DCM and ethanol extracts resulted in good fungicidal activity (Tables 4 and 5) in all samples except for Merwilla plumbea leaves. A 1:1:1 combination of DCM, ethanol and water extracts showed good fungicidal activity in all plant samples except for Merwilla plumbea bulb extracts, while a proportional extract yield combination of PE, DCM and ethanol gave good fungicidal activity in all samples except for Hypoxis hemerocallidea corms (Tables 4 and 6). In general, a combination that included an ethanol extract, except in the 1:1:1:1 combination of all the extracts, resulted in better antifungal efficacy than any other combination (Table 4). This may be an indication that the candidate compounds for the observed anticandidal activity are enhanced from the ethanol extracts. The efficacy and interaction effect in the proportional extract yield combination followed an almost similar trend to that of equal combinations. Candida albicans has proved to be more resistant to most plant extracts (Heisey and Gorham, 1992; Buwa and Van Staden, 2006; Ncube et al., 2011a), and the good fungicidal activity demonstrated by these extract combinations offer promising prospects for the treatment of candidiasis particularly for HIV/AIDS patients. Candidiasis is a common opportunistic infection among HIV/AIDS patients and is one of the major causes of death in developing countries (Reichart, 2003). Tulbaghia violacea is commonly used in South African traditional medicine among HIV/AIDS patients (Klos et al., 2009) and Hypoxis hemerocallidea is used as an immune system booster. The good fungicidal activity demonstrated by some extract combinations of these two plant species, provide a basis for their use in the treatment of such opportunistic infections in traditional medicine. The good antibacterial activity demonstrated by the phenolicrich extracts of Hypoxis hemerocallidea samples (Table 7), corresponds with the high phenolic content previously identified in these samples (Ncube et al., 2011a). Chances are, however, high that the good activity shown by these extracts in this study may be as a result of these phenolic compounds. A 1:1 combination of phenolic-rich and saponin-rich extracts resulted in a synergistic interaction effect against Escherichia coli and Staphylococcus aureus in Hypoxis hemerocallidea corm, and against Staphylococcus aureus in leaf extract samples respectively. A positive fungicidal interaction effect was also exhibited by a combination of saponin-rich and phenolic-rich extracts in both corm and leaf samples of Hypoxis hemerocallidea, each with an MFC value of 0.4 and an FIC value of B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 0.4 in each sample respectively. This is despite the fact that none of the two independent extracts showed good fungicidal activity. A similar extract combination in Tulbaghia violacea leaf samples resulted in antagonistic interaction effects (FIC index > 4.0) against Bacillus subtilis, Escherichia coli and K. pneumonia, with an indifferent effect against Staphylococcus aureus (FIC index of 2.0). In Merwilla plumbea, the antibacterial interaction effect of the two extracts ranged from a synergistic to indifferent interaction effect, although none of the combinations showed good activity against any of the tested bacterial strains. 4. Conclusions In all the plant samples tested in this study, at least one of the extract combinations exhibited good antibacterial and fungicidal activity. These results demonstrate the potential phytotherapeutic value of a total set of phytochemical compounds within a single medicinal plant. Although a lot of research has been done on the bioactivity of a number of medicinal plants, the results of this study reveals that potential for novel leads might still remain locked in these plants if analyses and conclusions remain drawn from only individual extracts. While the screening of independent plant extracts is in itself a research area of major significance in traditional medicine, the results of this study provides clear evidence that the full potential therapeutic value of a medicinal plant lies in the manipulation of the entire extract sets together in various combinations. Be that as it may, it must, however, be emphasised that due to the diversity and multitude of chemical compounds in each plant extract, their interaction effects are equally diverse and may lead to some toxic effects within the human body. For this reason, toxicological tests for such extract combinations are necessary to avoid detrimental effects. 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