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. The pharmacodynamics of drug action in a human body or any living
system is in fact a complex and dynamic process. The results
obtained through in vitro test models therefore, may not always
translate to the same effects when administered into a human
body. Further pharmacological tests using in vivo models are
therefore necessary to help confirm and further ascertain the efficacious properties of such combinations in living systems. A better
understanding of the molecular mechanisms of synergy would
provide a new route to overcome the problem of drug resistant
pathogens.
Acknowledgements
The National Research Foundation (NRF), Pretoria and the University of KwaZulu-Natal are gratefully acknowledged for financial
assistance.
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