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. Evaluation of five medicinal plants used in diarrhoea treatment in Nigeria. Journal
of Ethnopharmacology 101, 27–30.
Alanís, A.D., Calzada, F., Cervantes, J.A., Torres, J., Ceballos, G.M., 2005. Antibacterial
properties of some plants used in Mexican traditional medicine for the treatment
of gastrointestinal disorders. Journal of Ethnopharmacology 100, 153–157.
Amusan, O.O.G., Dlamini, P.S., Msonthi, J.D., Makhubu, L.P., 2002. Some herbal
remedies from Manzini region of Swaziland. Journal of Ethnopharmacology 79,
109–112.
Bell, A., 2005. Antimalarial drug synergism and antagonism: mechanistic and clinical
significance. FEMS Microbiology Letters 253, 171–184.
Berenbaum, M.C., 1978. A method for testing for synergy with any number of agents.
The Journal of Infectious Diseases 137, 122–130.
Berenbaum, M.C., 1989. What is synergy? American Society for Pharmacology and
Experimental Therapeutics 41, 93–141.
Boik, J., 2001. Natural Compounds in Cancer Therapy. Oregon Medical Press, LCC,
USA.
Boucher, A.N., Tam, V.H., 2006. Mathematical formulation of additivity for antimicrobial agents. Diagnostic Microbiology and Infectious Disease 55, 319–325.
Boutayeb, A., 2006. The double burden of communicable and non-communicable
diseases in developing countries. Transactions of the Royal Society of Tropical
Medicine and Hygiene 100, 191–199.
Brooks, G.F., Carroll, K.C., 2007. Bacteriology. In: Brooks, G.F., Carroll, K.C., Butel, J.S.,
Morse, S.A. (Eds.), Jawetz, Melnick & Adelberg’s Medical Microbiology, 24th ed.
McGraw-Hill Companies Inc..
Djoukeng, J.D., Abou-Mansour, E., Tabacchi, R., Tapondjou, A.L., Bouda, H., Lonsti, D.,
2005. Antibacterial triterpenes from Syzygium guineense (Myrtaceae). Journal of
Ethnopharmacology 101, 283–286.
Dorman, H.J.D., Deans, S.G., 2000. Antimicrobial agents from plants: antibacterial
activity of plant volatile oils. Journal of Applied Microbiology 88, 308–316.
Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64,
711–713.
Ghayur, M.N., Gilani, A.H., Khan, A., Amor, E.C., Villaseňor, I.M., Choudhary, M.I.,
2006. Presence of calcium antagonist activity explains the use of Syzygium samarangense in diarrhoea. Phytotherapy Research 20, 49–52.
Gutiérrez, S.P., Sánchez, M.A.Z., González, C.P., García, L.A., 2007. Antidiarrhoeal
activity of different plants used in traditional medicine. African Journal of
Biotechnology 6, 2988–2994.
511
Hammer, K.A., Carson, C.F., Riley, T.V., 1999. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology 86, 985–
990.
Horn, J.R., 2007. Appendix II: important drug interactions & their mechanisms. In:
Katzung, B.G. (Ed.), Basic & Clinical Pharmacology, 10th ed. McGraw-Hill Companies Inc., United States of America.
Ibezim, E.C., Esimone, C.O., Nnamani, P.O., Onyishi, I.V., Brown, S.A., Obodo, C.E., 2006.
In vitro study of the interaction between some fluoroquinolones and extracts of
Kola nitida seed. African Journal of Biotechnology 5, 1781–1784.
Kamatou, G.P.P., Viljoen, A.M., van Vuuren, S.F., van Zyl, R.L., 2006. In vitro evidence
of antimicrobial synergy between Salvia chamelaeagnea and Leonotis leonurus.
South African Journal of Botany 72, 634–636.
Marcos, L.A., DuPont, H.L., 2007. Advances in defining etiology and new therapeutic
approaches in acute diarrhea. Journal of Infection 55, 385–393.
Mathabe, M.C., Nikolova, R.V., Lall, N., Nyazema, N.Z., 2006. Antibacterial activities
of medicinal plants used for the treatment of diarrhoea in Limpompo Province,
South Africa. Journal of Ethnopharmacology 105, 286–293.
Mølgaard, P., Nielsen, S.B., Rasmussen, D.E., Drummond, R.B., Makaza, N., Andreasse,
J., 2001. Antihelmintic screening of Zimbabwean plants traditionally used
against schistosomiasis. Journal of Ethnopharmacology 74, 257–264.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. Journal of Immunological
Methods 65, 55–63.
Mukherjee, P.K., Saha, K., Murugesan, T., Mandal, S.C., Pal, M., Saha, B.P., 1998. Screening of anti-diarrhoeal profile of some plant extracts of a specific region of West
Bengal, India. Journal of Ethnopharmacology 60, 85–89.
Neuwinger, H.D., 1996. African Ethnobotany Poisons and Drugs. Chapman and Hall,
Weinheim.
Okusa, P.N., Penge, O., Debleeschouwer, M., Duez, P., 2007. Direct and indirect
antimicrobial effects and antioxidant activity of Cordia gilletii De Wild (Boraginaceae). Journal of Ethnopharmacology 112, 476–481.
Palombo, E.A., 2006. Traditional plant and herbal remedies used in the treatment
of diarrhoeal disease: mode of action, quality, efficacy, and safety considerations. In: Ahmed, I., Aqil, F., Owais, M. (Eds.), Modern Phytomedicine.
Turning Medicinal Plants into Drugs. Wiley-VCH Verlag GmbH & Co. KGaA,
Weiheim.
Rea, A.I., Schmidt, J.M., Setzer, W.N., Sibanda, S., Taylor, C., Gwebu, E.T., 2003. Cytotoxicity of Ozoroa insignis from Zimbabwe. Fitoterapia 74, 732–735.
Samie, A., Obi, C.L., Bessong, P.O., Namrita, L., 2005. Activity profiles of fourteen selected medicinal plants from Rural Venda communities in South Africa
against fifteen clinical bacterial species. African Journal of Biotechnology 4,
1443–1451.
van Zyl, R.L., Viljoen, A.M., 2002. In vitro activity of Aloe extracts against Plasmodium
falciparum. South African Journal of Botany 68, 106–110.
Venter, F., Venter, J.A., 2002. Making The Most of Indigenous Trees, 2nd ed. Briza
Publications, Pretoria, South Africa.