Journal of Ethnopharmacology 132 (2010) 506–511 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm The anti-diarrhoeal properties of Breonadia salicina, Syzygium cordatum and Ozoroa sphaerocarpa when used in combination in Swazi traditional medicine Gugu F. Sibandze a,b,∗ , Robyn L. van Zyl a , Sandy F. van Vuuren a a b Department of Pharmacy and Pharmacology, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa Swaziland Institute for Research in Traditional Medicine, Medicinal and Indigenous Food Plants, University of Swaziland, Private Bag 4, Kwaluseni M201, Swaziland a r t i c l e i n f o Article history: Received 7 June 2010 Received in revised form 12 August 2010 Accepted 25 August 2010 Available online 15 September 2010 Keywords: Diarrhea Antimicrobial Anti-diarrheal Medicinal Combination a b s t r a c t Aim of the study: The aim of the study was to determine in vitro activity of the bark of Ozoroa sphaerocarpa R. Fern & A. Fern (Anacardiaceae), Breonadia salicina (Vahl) Hepper & J.I.R. Wood (Rubiaceae) and Syzygium cordatum Hochst ex C Krauss (Myrtaceae) against a diarrhoea-causing pathogen, Escherichia coli; as well as the pharmacological interactions present in their combination. Materials and methods: In consultation with traditional healers, the plants were collected from the wild, dried and extracted with dichloromethane:methanol (1:1). Thereafter, antimicrobial activity of the individual plants and their different combinations was tested using a common diarrhoea pathogen, Escherichia coli by employing the minimum inhibitory concentration assay. Results: Ozoroa sphaerocarpa was the most potent inhibitor of antimicrobial growth (MIC value of 1.2 mg/ml), followed by Syzygium cordatum (MIC value of 1.44 mgl/ml) and lastly Breonadia salicina (MIC value of 10.89 mg/ml). The combination between Syzygium cordatum and Ozoroa sphaerocarpa gave the strongest synergistic interaction (MIC value of 0.33 mg/ml); whilst that between Syzygium cordatum and Breonadia salicina was mildly synergistic (MIC value of 1.00 mg/ml). The triple combination (1:1:1) was also very effective in inhibiting microbial growth (MIC value of 0.44 mg/ml). The combined effect of these plants on toxicity was predominantly synergistic except for the combination of Ozoroa sphaerocarpa and Syzygium cordatum which was predominantly antagonistic (FIC value of 1.48 ± 0.25). The triple combination had a favourable toxicity profile with an IC50 value of 155.76 ± 11.86 ␮g/ml. Conclusion: This study supports the rationale by traditional healers to use the bark of Syzygium cordatum, Breonadia salicina and Ozoroa sphaerocarpa in combination for the treatment of diarrhoea. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Diarrhoeal disease is the third leading cause of death in children in developing countries; accounting for 15.2% of all childhood deaths and enteropathogenic Escherichia coli has been identified as the causative agent for about half of the diarrhoea cases in children. Malnutrition, inadequate water supply and poor sanitation predispose these children to the risk of contracting diarrhoea (Mukherjee et al., 1998; Boutayeb, 2006). In modern medicine, there are four approaches to the treatment of acute diarrhoea; these are maintenance of fluid and electrolyte balance; use of anti-infective agents; anti-diarrhoeal agents; and most recently the use of probiotics or microbial components which have a value in the treatment ∗ Corresponding author at: Swaziland Institute for Research in Traditional Medicine, Medicinal and Indigenous Food, Plants, University of Swaziland, Private Bag 4, Kwaluseni M201, Swaziland. Tel.: +268 518 6816; fax: +268 518 5276. E-mail addresses: sguguster@gmail.com, gsibandze@uniswacc.uniswa.sz (G.F. Sibandze). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.08.050 of rotavirus infections and post antibiotic diarrhoea (Marcos and DuPont, 2007). Traditional healers use a similar approach in the treatment of diarrhoea, by using plants with antimicrobial activity or those that decrease gastric motility. 1.1. Combination therapy in modern medicine The use of combination therapy in clinical practice is very common and is employed for the therapeutic advantages it may provide over single agents. Combination therapy is also employed to increase the spectrum of antimicrobial activity, to prevent treatment failure when antimicrobial resistance is suspected, prevent the development of resistance, to decrease dose-related toxicity by using less of a toxic antimicrobial agent and more of the nontoxic one and to obtain enhanced antimicrobial killing or inhibition. The drugs used in combination may have different mechanisms of action as well as affect different sites of the body; but the overall effect of the combination may either exceed the expected effect (synergism) or nullify each other’s biological effects resulting in a reduced effect (antagonism). An additive interaction is defined as G.F. Sibandze et al. / Journal of Ethnopharmacology 132 (2010) 506–511 the effect where the combined action is equivalent to the sum of the actions of each drug when used alone (Berenbaum, 1989; Boucher and Tam, 2006; Brooks and Carroll, 2007). 1.2. Combination therapy in traditional medicine In traditional medicine, different plants are combined for the treatment of a disease. The plants used are often not related in any way or may be used singularly for the treatment of that ailment. However, they may have been found to achieve a better therapeutic effect when in combination (Personal communication with Mr. PP Ndlovu, traditional healer). A number of combination treatments are prepared by traditional healers, such as the use of Terminalia sericea root powder in combination with Vigna unguiculata seeds for the treatment of bilharzia (Neuwinger, 1996). Of the 190 traditional medicine recipes recorded during the Organization of African Unity/Scientific, Technical and Research Commission (OAU/STRC) ethnobotanical survey of Swaziland, 52 (27.4%) were recipes prepared with two or more plant combinations (Adeniji et al., 2000). The bark of Ozoroa sphaerocarpa R. Fern & A. Fern (Anacardiaceae), Breonadia salicina (Vahl) Hepper & J.I.R. Wood (Rubiaceae) and Syzygium cordatum Hochst ex C Krauss (Myrtaceae) are traditionally used in combination for the treatment of diarrhoea (Personal communication with Mr. PP Ndlovu, traditional healer). In the preparation of the traditional remedy, equal amounts of the plants are boiled in water and the concoction drunk for the relief of diarrhoea. 1.3. Implications of combination therapy on toxicity Although it may be beneficial to combine drugs to increase effectiveness or avoid treatment failure in antimicrobial therapy, the implications of the combination on toxicity need to be explored. Two drugs may give the desired synergistic effect when used in combination for the treatment of a microbial infection, but prove to be toxic to the human/host’s cells. As an example, the combined use of two nephrotoxic drugs can result in increased toxicity (synergistic effect) even though the individual doses may not be sufficient to produce such toxicity (Horn, 2007). Such toxicity is not limited only to clinical drugs; herbal preparations can also interact and result in potentiated toxicity. Thus there is a need to investigate the toxic effects or possible interactions that may result when combining plants in the treatment of disease. The aim of this study was to determine the type of pharmacological interaction existing between the bark of Syzygium cordatum, Ozoroa sphaerocarpa and Breonadia salicina when tested against a diarrhoea-causing pathogen; as well as to investigate the implications of the combination treatment on toxicity. 2. Materials and methods 2.1. Plant collection and extraction The bark of Ozoroa sphaerocarpa, Breonadia salicina and Syzygium cordatum were collected in collaboration with a traditional healer from the Manzini region in Swaziland, between March and April 2006. Botanical identification of the plants was done by the Malkerns Research Station, Ministry of Agriculture and Cooperatives in Swaziland and voucher specimen (GM002, GM012 and GM013, respectively) deposited with the Department of Pharmacy and Pharmacology, University of the Witwatersrand, Johannesburg. Details of locality were recorded using a Global Positioning System (GPS). The bark was air-dried separately under shade, crushed into powder using a commercial blender and a known quantity extracted with dichloromethane:methanol (1:1) solvent (both 507 from Rochelle chemicals) for 48 h at room temperature. The solvent was changed twice during this period. The extract was then taken to dryness using a Büchi Rotavapor (R-114). Thereafter, it was air-dried in a fume hood, stored in airtight containers at −20 ◦ C until used. The bark of Breonadia salicina had the highest yield of 12.2% (w/w), followed by Syzygium cordatum (9.1%) and lastly Ozoroa sphaerocarpa (5.0%). 2.2. Antimicrobial combinations To scientifically verify the combined effect of the bark of Ozoroa sphaerocarpa, Breonadia salicina and Syzygium cordatum, against a common diarrhoea-causing pathogen, Escherichia coli (ATCC 25922), a combination study was designed. The antibacterial activity of all three possible plant combinations in various ratios, as well as the triple combination that is traditionally used, was tested to determine the type of pharmacological interaction between these plant combinations. 2.2.1. Screening for combined activity This constituted the initial screening for antimicrobial activity when the extracts were combined in different ratios. A stock concentration of 85.3 mg/ml in acetone was prepared for each of Breonadia salicina, Ozoroa sphaerocarpa and Syzygium cordatum, and then mixed in separate eppendorf tubes to generate seven different mixtures which were treated separately. The resulting mixture comprising of 28.43 mg/ml of each extract was then plated (100 ␮l) in a 96-well microtitre plate in duplicate and serially diluted in a 1:1 ratio with MilliQTM water to produce a final concentration range of 7.13 mg/ml to 0.06 mg/ml after addition of the microbial culture (100 ␮l). Antimicrobial activity was then determined as previously described by Eloff (1998) and Kamatou et al. (2006) using the MIC assay. Ciprofloxacin (0.01 mg/ml; Sigma) in acetone was used as a positive control. 2.2.2. Two plant combination study The plant extracts were prepared at a concentration of 64 mg/ml in acetone. In separate eppendorf tubes, the different ratios of plant extracts were prepared; 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10. The three possible combinations were used in this study: Syzygium cordatum with Ozoroa sphaerocarpa, Breonadia salicina with Ozoroa sphaerocarpa and Syzygium cordatum with Breonadia salicina. The MIC of each combination was determined as described in Section 2.2.1. 2.2.3. Three plant combination study To determine the combined interaction between the three extracts, a stock of 80 mg/ml extract in acetone was prepared. For each of the eleven ratios, a total of 100 ␮l was prepared by combining various volumes of each extract. In each combination, the volume of extract A and B were altered with extract C being kept constant (20 ␮l). These same ratios as used in Section 2.2.2 were used and the difference was the addition of a constant volume of the third extract. The MIC for each combination was determined using the method described in Section 2.2.1. To determine the effect of varying concentrations of extract C, three concentrations were chosen to observe how the MIC values and isobolograms would be altered. These concentrations were determined from the MIC values of the individual extracts, namely MIC; half the MIC and double the MIC values. By altering the concentration of extract C, three isobolograms were generated for each triple plant combination and each isobologram overlaid for easier comparison. This protocol was repeated for each possible combination such that 9 isobolograms were generated to determine the influence of varying concentrations of each extract in the triple combination regimes used by the traditional healers. 508 G.F. Sibandze et al. / Journal of Ethnopharmacology 132 (2010) 506–511 2.2.4. Data analysis All the extract combinations were plated in duplicate in a single experiment and each experiment was repeated three times and the results averaged. The two combination study isobolograms were constructed to determine the interaction between the plant extracts using the following equation: (X : Y) = MIC of extract B in combination : MIC of extract B alone MIC of extract A in combination MIC of extract A alone (1) The fractional inhibitory concentration (FIC) was also calculated to determine the strength of the interaction using the equation (Bell, 2005): FIC = MIC of extract A in combination with extract B MIC of extract A alone (2) The sum of the FIC values (FIC) was calculated by adding all the FIC values for all the combination ratios and averaged between three individual experiments. The proposed definition by Berenbaum (1978) was used to determine the type and strength of the interaction; where a sum of 1 is an additive interaction, <1 a synergistic interaction, and >1 an antagonistic interaction. The triple plant combination data were analysed in a similar manner to the two plant combination experiments by using the two plants added in different ratios and keeping the third plant as a constant. Three isobolograms were constructed from the data of the three experiments where the initial concentrations were altered (MIC, half or twice the MIC). These were overlaid and plotted together with the isobologram generated from the two plant combination experiments (i.e. without the third extracts). 2.3. Toxicity combination studies 2.3.1. Two plant combination study The different extract combinations were prepared in DMSO and their serial 1:1 dilutions were tested for toxicity against human kidney epithelial cells, using the MTT (3-[4,5-dimethylthiazol2yl]-2,5diphenyltetrazolium bromide) cellular viability assay (Mosmann, 1983; van Zyl and Viljoen, 2002). 2.3.2. Three plant combination study To determine the type of interaction between the three extracts in combination (1:1:1), an extract concentration of 66.67 ␮g/ml of each of the extracts was prepared in DMSO, before being mixed in a 1:1:1 ratio. This was tested using the MTT assay as described in Section 2.3.1. 2.3.3. Data analysis The IC50 values of the different extract combinations were determined by plotting log sigmoid dose–response curves using the Enzfitter® program. Thereafter isobolograms were constructed and interpreted. 3. Results and discussion Ozoroa sphaerocarpa and Syzygium cordatum had some antimicrobial activity against the bacterial strain Escherichia coli (MIC values of 1.20 and 1.44 mg/ml, respectively); whilst Breonadia salicina had no appreciable activity (MIC value of 10.89 mg/ml). When combining drugs in therapy, synergism is the most desired effect because this mechanism of interaction results in more effectiveness of the combination than the individual drugs (Berenbaum, 1978). The combined effect of the plants was predominantly synergistic, regardless of the ratio and plant combination. The combination between Breonadia salicina and Ozoroa sphaerocarpa gave an overall synergistic interaction (FIC value of 0.89 ± 0.13) whereas when viewing the isobologram, most of the points lie in the additive/antagonism region (Fig. 1). The 1:1 combination of Breonadia salicina and Ozoroa sphaerocarpa gave a lower MIC value (1.67 mg/ml) compared to Breonadia salicina alone (Table 1). This combination was the least effective in inhibiting the growth of Escherichia coli. The addition of different concentrations of Syzygium cordatum to this combination resulted in a more antagonistic interaction (Fig. 1), especially at the MIC concentration of Syzygium cordatum (FIC = 1.81). When dealing with toxicity studies for combination treatments, the desired effect is antagonism because this interaction results in reduced toxicity of either of the extracts. With regards to toxicity, the combination between Breonadia salicina and Ozoroa sphaerocarpa gave an overall additive interaction (FIC = 1.02 ± 0.27) with some ratios interacting synergistically and antagonistically when tested against transformed human kidney epithelial cells (Fig. 2). The ratios that resulted in antagonism were the higher concentrations of Ozoroa sphaerocarpa, compared to Breonadia salicina, such as those concentrations between 20 and 50 ␮g/ml. Between 1 and 10 ␮g/ml of Ozoroa sphaerocarpa, the predominant interaction was synergism (Fig. 2). Breonadia salicina and Syzygium cordatum displayed a strong synergistic interaction in inhibiting the growth of Escherichia coli, with FIC value of 0.73 ± 0.04 (Fig. 1). This is also evident in the 1:1 combination of these plants (Table 1), where the MIC value of both extracts was significantly reduced when added together (1.00 mg/ml). The antimicrobial activity of Breonadia salicina and Syzygium cordatum was improved upon the addition of Ozoroa sphaerocarpa (Fig. 1). However, the relationship between Breonadia salicina and Syzygium cordatum with respect to toxicity was strongly synergistic, with FIC value of 0.46 ± 0.01 (Fig. 2), indicating increased toxicity of the combination. The combination between Syzygium cordatum and Ozoroa sphaerocarpa was synergistic (FIC = 0.56 ± 0.17) with regards to antimicrobial activity (Fig. 1). The 1:1 combination of these plants greatly lowered their individual MIC values (Table 1). This did not change even with the addition of Breonadia salicina which resulted in even stronger synergism, with FIC values of 0.49, 0.63 and 0.24 for half the MIC, MIC and double the MIC of Breonadia salicina (Fig. 1). The toxicity results of this combination were additive/antagonistic, with a FIC value of 1.48 ± 0.25 (Fig. 2). Therefore, the more favourable two plant combination was found to be that between Ozoroa sphaerocarpa and Syzygium cordatum, which gave the best antimicrobial activity (FIC = 0.56 ± 0.16) and also displayed a safer toxicity profile. The triple combination, as used in the traditional preparation (1:1:1) was found to have potent antimicrobial activity (MIC value of 0.44 mg/ml). It was also interesting to note that this combination had a safer toxicity profile when tested against human kidney epithelial cells (IC50 value of 155.76 ± 11.86 ␮g/ml). It is noteworthy that combinations involving Breonadia salicina were not favourable, both with reference to toxicity and antimicrobial activity, with the exception of the antimicrobial activity of Breonadia salicina in combination with Syzygium cordatum which displayed the desired synergistic interaction (Fig. 1). The inclusion of Breonadia salicina in the combination might serve another purpose other than its inhibitory effect on diarrhoeal pathogens. However, on its own, it displayed the best toxicity profile of the three plants, with the highest IC50 value against human kidney epithelial cells (Table 1). It is interesting to note that Breonadia salicina is traditionally used to treat diarrhoea as well as wounds or injuries (Neuwinger, 1996; Venter and Venter, 2002), however, in this study; it did not inhibit the test pathogen. It is possible that it mediates its activity in these areas through another mechanism 1.5 1.0 0.5 0.0 0.0 0.5 1.0 1.5 1.0 0.5 0.0 0.0 1.5 O. sphaerocarpa bark (MIC in combination /MIC alone) 0.5 1.0 1.5 O. sphaerocarpa bark (MIC in combination/ MIC alone) S. cordatum & B. salicina, with O. sphaerocarpa 1.5 S. cordatum bark (MIC in combination/ MIC alone) 509 B. salicina & O. sphaerocarpa, with S. cordatum S. cordatum & O. sphaerocarpa, with B. salicina B. salicina bark (MIC in combination/ MIC alone) S. cordatum bark (MIC in combination /MIC alone) G.F. Sibandze et al. / Journal of Ethnopharmacology 132 (2010) 506–511 Figure legend 1.0 Two plant combination At MIC At ½ MIC 0.5 At 2x MIC 0.0 0.0 0.5 1.0 1.5 B. salicina bark (MIC in combination/ MIC alone) Fig. 1. The interaction between the three plants in combination, with the third plant at constant concentration, either MIC, 1/2 MIC or 2× MIC. The isobologram for the two plant combination has been overlayed for comparison. of action such as acting as an adsorbent, like isphaghula husk to produce its anti-diarrhoeal effect. Syzygium species have, for a long time, been reported to possess anti-diarrhoeal properties and are used in many countries for the management of diarrhoea. As an example, Syzygium samarangense is used by the Philippines in the treatment of diarrhoea (Ghayur et al., 2006) and in Southern Africa, Syzygium cordatum is used for the treatment of stomach troubles, cold and fever and diarrhoea (Amusan et al., 2002; Samie et al., 2005; Mathabe et al., Table 1 In vitro antimicrobial activity (MIC, mg/ml) and cytotoxicity (with percent cell death in parenthesis) of the traditionally used medicinal plant extracts and their combinations. Plant name or combinations MIC (mg/ml) Cytotoxicity IC50 (␮g/ml) ± s.d. Ozoroa sphaerocarpa Syzygium cordatum Breonadia salicina Ozoroa sphaerocarpa: Breonadia salicina (1:1) Ozoroa sphaerocarpa: Syzygium cordatum (1:1) Breonadia salicina: Syzygium cordatum (1:1) Ozoroa sphaerocarpa: Breonadia salicina: Syzygium cordatum (1:1:1) Ciprofloxacin 1.20 1.44 10.89 1.67 8.11 ± 2.80 26.80 ± 2.54 >200 (70.84 ± 2.72%) n.d.a a n.d., not determined. 0.33 n.d. 1.00 n.d. 0.44 155.76 ± 11.86 4.4 × 10−4 >100 (67.59 ± 1.12%) 2006). In the present investigation, the anti-Escherichia coli activity of Syzygium cordatum in combination with either Breonadia salicina or Ozoroa sphaerocarpa is reported. Alone, Syzygium cordatum was able to inhibit microbial growth of Escherichia coli (Table 1), and in combination, acted synergistically to inhibit Escherichia coli (Fig. 1). The activity of this plant is supported by reports on the antimicrobial/anti-Escherichia coli/anti-diarrhoeal effects of Syzygium species (Hammer et al., 1999; Dorman and Deans, 2000; Djoukeng et al., 2005; Samie et al., 2005; Mathabe et al., 2006). The anti-diarrhoeal activity of Syzygium samarangense has been attributed to the presence of flavonoids which have been found to possess relaxant activity on isolated rabbit jejunum, mediated through the blockade of calcium influx (Ghayur et al., 2006), as well as possess antibacterial properties (Palombo, 2006). Ozoroa sphaerocarpa displayed good antimicrobial activity against Escherichia coli when used alone, with an MIC value of 1.20 mg/ml (Table 1). Mathabe et al. (2006) demonstrated anti-Escherichia coli activity of Ozoroa insignis with an MIC of 0.078 mg/ml. Apart from being used to treat diarrhoea, Ozoroa insignis is used to treat tapeworm and hookworm (Mølgaard et al., 2001; Rea et al., 2003). When in combination, Ozoroa sphaerocarpa was able to suppress Escherichia coli growth effectively, when combined with Syzygium cordatum. The antimicrobial activity of the combination between Ozoroa sphaerocarpa and Syzygium cordatum was also more favourable, displaying synergism, which was also improved by the addition of increasing concentrations of Breonadia salicina, resulting in a more synergistic interaction (Fig. 1). Though the traditional remedy involves using all three plants in equal ratios, this investigation G.F. Sibandze et al. / Journal of Ethnopharmacology 132 (2010) 506–511 S. cordatum and O.sphaerocarpa 3 B. salicina (IC50 in combination/IC50 alone) S. cordatum (IC50 in combination/IC50 alone) 510 2 1 0 0 1 2 3 B. salicina and O. sphaerocarpa 3 2 1 0 0 2 3 O. sphaerocarpa (IC50 in combination/IC50 alone) O. sphaerocarpa (IC50 in combination/IC50 alone) S. cordatum (IC50 in combination/IC50 alone) 1 S. cordatum and B. salicina 3 2 1 0 0 1 2 3 B. salicina (IC50 in combination/IC50 alone) Fig. 2. The interactions between the different extracts tested against human kidney epithelial cells. has shown that changing the concentrations of the extracts may result in improved activity, as illustrated by the increase in synergy upon addition of a higher concentration of the third extract (Fig. 1). Work carried by other researchers has shown the presence of synergism between different plant combinations (Boik, 2001; Kamatou et al., 2006; Okusa et al., 2007). In addition, synergistic/additive effects were exhibited by extracts of Kola nitida seed in combination with some fluoroquinolines when tested against Escherichia coli. This is beneficial in delaying possible resistance to fluoroquinolines and is essential information to avoid therapeutic failure when treating a patient who has initially received Kola nitida before fluoroquinolines are administered (Ibezim et al., 2006). Another study, using Cordia gilletii De Wild with tetracycline and streptomycin, gave additive and synergistic interactions when tested against Escherichia coli and Staphylococcus aureus (Okusa et al., 2007). The presence of synergy between plant extracts or between plants and standard microbial drugs emphasizes the need for further research into combination work due to the increased risk of patients developing resistance to the agents when used as monotherapy. In the present study, all possible interactions have been displayed at various concentration ratios; however, interaction with standard antimicrobial agents has not been investigated. There is need to investigate the interaction of these plants with standard antimicrobials and this was outside the scope of this study as our aims were to validate the effectiveness of the three plants in combination against a diarrhoea-causing pathogen. Plants have been effectively used to combat diarrhoea for centuries, especially in the African setting. Studies on anti-diarrhoeal efficacy of some of these plants have shown variable mechanisms of action. Some of the anti-diarrhoeal activity is mediated through the action on diarrhoea pathogens like Escherichia coli, Salmonella spp and Shigella spp (Alanís et al., 2005), whilst others play an important role as smooth muscle relaxants as well as by inhibiting prostaglandin synthesis (Agunu et al., 2005; Gutiérrez et al., 2007). Others do not appear to interfere with any of the above, but are still excellent anti-diarrhoeal agents which stimulate water re-absorption or reduction of intraluminal fluid accumulation (Gutiérrez et al., 2007). Though not all possible modes of action have been investigated in the present study, it can be concluded that Syzygium cordatum and Ozoroa sphaerocarpa possibly possess anti-diarrhoeal activity mediated by inhibiting the growth of Escherichia coli. The combination of the two is also active at all concentrations tested. Breonadia salicina, on the other hand, though possibly contributing some activity, lacked anti-Escherichia coli activity, but its anti-diarrhoeal activity may be due to some other mechanisms, as previously discussed. The results of the combination experiments support the traditional use of these three plants in combination for the treatment of diarrhoea and also prove that traditional medicine is a reliable source of knowledge for the development of new drugs. Acknowledgements The authors would like to thank the National Research Foundation (Thuthuka Fund) South Africa, United Nations University Fellowship, Belgian Technical Corporation Fellowship, Postgraduate Merit Award and the Faculty Research Committee Grant, University of the Witwatersrand, South Africa for the financial support. Our gratitude also goes to the traditional healer who assisted us with the recipe and plant collection, Mr PP Ndlovu (deceased). G.F. Sibandze et al. / Journal of Ethnopharmacology 132 (2010) 506–511 References Adeniji, K.O., Amusan, O.O.G., Dlamini, P.S., Enow-Orock, E.G., Gamedze, S.T., Gbile, Z.O., Langa, A.D., Makubu, L.P., Mahunnah, R.L.A., Mshana, R.N., Sofowa, A., Vilane, M.J., 2000. Traditional medicine and pharmacopoeia contribution to ethnobotanical and floristic studies in Swaziland. Scientific Technical and Research Commission of the Organization of African Unity (OAU/STRC). Agunu, A., Yusuf, S., Andrew, G.O., Zezi, A.U., Abduranhman, E.M., 2005. 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