In Vitro Antimicrobial Synergism Within Plant Extract Combinations from Three

Journal of Ethnopharmacology 139 (2012) 81–89

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Journal of Ethnopharmacology

journa l homepage: www.elsevier.com/locate/jethpharm

In vitro antimicrobial synergism within plant extract combinations from three

South African medicinal bulbs

∗

B. Ncube, J.F. Finnie, J. Van Staden

Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea are used

Received 27 July 2011

in South African traditional medicine for the treatment of some infectious diseases and other ailments.

Received in revised form

Aim of the study: The study aimed at investigating the antimicrobial efﬁcacies of independent and various

21 September 2011

within-plant extract combinations of three medicinal bulbs to understand the possible pharmacological

Accepted 15 October 2011 interactions.

Available online 30 October 2011

Materials and methods: Bulb and leaf extracts of the three medicinal plants, independently and in com-

binations, were comparatively assessed for antimicrobial activity against two Gram-positive and two

Keywords:

Antimicrobial Gram-negative bacteria and Candida albicans using the microdilution method. The fractional inhibitory

concentration indices (FIC) for two extract combinations were determined.

Extract combination

Interaction Results: At least one extract combination in each plant sample demonstrated good antimicrobial activity

Phytochemical against all the test organisms. The efﬁcacies of the various extract combinations in each plant sample

Synergy varied, with the strongest synergistic effect exhibited by the proportional extract yield combination of

PE and DCM extracts in Merwilla plumbea bulb sample against Staphylococcus aureus (FIC index of 0.1).

Most extract combinations demonstrated either a synergistic, additive or indifferent interaction effect

against the test bacteria with only a few exhibiting antagonistic effects.

Conclusion: The observed antimicrobial efﬁcacy and synergistic interactions indicate the beneﬁcial

aspects of combination chemotherapy of medicinal plant extracts in the treatment of infectious diseases.

1. Introduction with the rapidly changing and potentially damaging external envi-

ronmental factors. Being organisms devoid of mobility, plants have

The continued evolution of infectious diseases and the devel- evolved elaborate alternative defence strategies, which involve an

opment of resistance by pathogens to existing pharmaceuticals, enormous variety of chemical metabolites as tools to overcome

have led to the intensiﬁcation of the search for new novel leads, stress conditions. The ability of plants to carry out combinatorial

against fungal, parasitic, bacterial, and viral infections (Gibbons, chemistry by mixing, matching and evolving the gene products

2004). Despite the recent advances in drug development through required for secondary metabolite biosynthetic pathways, creates

molecular modelling, combinatorial and synthetic chemistry, nat- an unlimited pool of chemical compounds, which humans have

ural plant-derived compounds are still proving to be an invaluable exploited to their beneﬁt. The use of plants by humans in both tra-

source of medicines for humans (Salim et al., 2008). Plant-derived ditional and modern medicinal systems, therefore, largely exploits

antimicrobials have a long history of providing the much needed this principle.

novel therapeutics (Avila et al., 2008). Plants constantly interact A number of traditionally used medicinal plants have to date

been screened for various biological activities in both in vivo and

in vitro models. The chemical investigation and puriﬁcation of

extracts from plants purported to have medicinal properties have

Abbreviations: ATCC, American type culture collection; CFU, colony forming unit; yielded numerous puriﬁed compounds which have proven to be

DCM, dichloromethane; EtOH, ethanol; FIC, fractional inhibitory concentration; INT,

indispensable in the practice of modern medicine (Goldstein et al.,

p-iodonitrotetrazolium chloride; MH, Mueller–Hinton; MIC, minimum inhibitory

1974; Tyler et al., 1988). In traditional medicine, however, these

concentration; MFC, minimum fungicidal concentration; PE, petroleum ether; YM,

compounds are largely utilised as crude extracts in the form of yeast malt.

∗

herbal remedies (Pujol, 1990). In light of the new emerging infec-

Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897.

E-mail address: [email protected] (J. Van Staden). tious diseases and the development of resistance in those with

82 B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89

existing curatives, one of the strategies employed in traditional 2.3. Preparation of saponin-rich extracts

herbal medicine to overcome these mechanisms is the combina-

tion of herbal remedies. Herbal remedies are often prepared from a Saponins were extracted from the plant material as described

combination of several different plant species. The pharmacological by Makkar et al. (2007). The dried and ground plant samples were

effects of such mixtures could be as a result of the total sum of differ- defatted with hexane in a Soxhlet apparatus for 3 h. After air-

ent classes of compounds with diverse mechanisms of action. There drying, saponins were extracted twice from the defatted samples

have been reports of the total contents of a herbal product show- (10 g) in 100 ml of 50% aqueous methanol by incubating at room

ing a signiﬁcantly better effect than an equivalent dose of a single temperature overnight with continuous stirring. The extracts were

isolated active ingredient or a single constituent herb (Williamson, then centrifuged at 3000 rpm for 10 min and the supernatant col-

2001; Nahrstedt and Butterweck, 2010). These ﬁndings suggest that lected. The procedure was repeated with the original residue to

the effects may arise from synergistic mechanisms of herbal ingre- obtain a second supernatant. The ﬁrst and second supernatants

dients. Synergism occurs when two or more compounds interact were combined and ﬁltered under vacuum through Whatman No.

in ways that mutually enhance, amplify or potentiate each other’s 1 ﬁlter paper. Methanol from the ﬁltrate was evaporated from the

◦

effect more signiﬁcantly than the simple sum of these ingredients solution under vacuum at 40 C with the saponin sample in the

(Williamson, 2001). aqueous phase remaining. The aqueous phase was then centrifuged

Although there is signiﬁcant information on the bioactivity at 3000 rpm for 10 min to remove water insoluble materials. The

of the screened medicinal plant extracts, most studies, however, aqueous phase was then transferred into a separating funnel and

report these ﬁndings on the basis of separate classes/groups of extracted three times with an equal volume of chloroform to

compounds extracted using different individual solvents. Many sci- remove pigments. The concentrated saponins in the aqueous solu-

entists perform extraction using solvents with increasing polarity, tion were then extracted twice with an equal volume of n-butanol.

◦

e.g. petroleum ether, chloroform, ethyl acetate, ethanol and water. The n-butanol was evaporated under vacuum at 45 C. The dried

The quality of the extracted compounds and their overall quan- fractions containing saponins were dissolved in 10 ml of distilled

tity in any given plant species would vary largely as a function of water and freeze-dried.

the type of solvent used. The question, however, arises: What will

the activity be if extracts from one extracting solvent are mixed

2.4. Preparation of phenolic-rich extracts

with those from another of the same plant species? What would

be the pharmacodynamic interaction between polar and non-polar

Phenolic compounds were extracted from plant material as

extracts of the same plant species? In light of the multiplicity

described by Makkar (1999) with modiﬁcations. Dried plant sam-

of the phytochemical compounds produced within an individual

ples (2 g) were extracted with 10 ml of 50% aqueous methanol by

plant, an investigation into this aspect, could possibly unlock the

sonication on ice for 20 min. The extracts were then ﬁltered under

hidden potentialities of the therapeutic value of the entire set of

vacuum through Whatman No. 1 ﬁlter paper. The concentrated

compounds of a plant extract. We elaborate this knowledge here

phenolic-rich extracts were subsequently dried at room temper-

by assessing the antimicrobial interaction effect of the different

ature under a stream of cold air.

extract combinations for three medicinal bulbs. The study extends

our previous research on these medicinal bulbs (Ncube et al.,

2011a) 2.5. Antibacterial activity

Minimum inhibitory concentrations (MIC) of extracts for

2. Materials and methods antibacterial activity were determined using the microdilution

bioassay as described by Eloff (1998). Overnight cultures (incubated

◦

2.1. Plant material at 37 C in a water bath with an orbital shaker) of two Gram-positive

(Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600)

Bulbs and leaves of Tulbaghia violacea Harv., Hypoxis heme- and two Gram-negative (Escherichia coli ATCC 11775 and Klebsiella

rocallidea Fisch. & C.A. Mey and Merwilla plumbea (Lindl.) Speta pneumoniae ATCC 13883) bacterial strains were diluted with sterile

were collected in March, from the University of KwaZulu-Natal Mueller–Hinton (MH) broth to give ﬁnal inocula of approximately

Botanical Garden, Pietermaritzburg, South Africa and voucher spec- 10 CFU/ml (colony forming units). The dried crude organic plant

imens (Table 1) were deposited in the Bews Herbarium (NU) at the extracts (PE, DCM, and ethanol) were resuspended in 70% ethanol

University of KwaZulu-Natal, Pietermaritzburg. The samples were to known concentrations while saponin and phenolic extracts were

separated into bulbs/corms and leaves before being dried in an oven redisolved in 50% methanol and water extracts in distilled water to

◦

at a constant temperature of 50 C for ﬁve days after which they the same concentrations. One hundred microlitres of each extract

were ground into ﬁne powders. were serially diluted two-fold with sterile distilled water in a 96-

well microtitre plate for each of the four bacterial strains. A similar

two-fold serial dilution of neomycin (Sigma Aldrich, Germany)

2.2. Preparation of plant extracts (0.1 mg/ml) was used as a positive control against each bacterium.

One hundred microlitres of each bacterial culture were added to

The ground samples (20 g) were sequentially extracted with each well. Water, 50% methanol and 70% ethanol were included

20 ml/g of petroleum ether (PE), dichloromethane (DCM), 80% as negative and solvent controls respectively. The plates were cov-

◦

ethanol (EtOH) and water in a sonication bath containing ice for ered with paraﬁlm and incubated at 37 C for 24 h. Bacterial growth

␮

1 h. The crude extracts were then ﬁltered under vacuum through was indicated by adding 50 l of 0.2 mg/ml p-iodonitrotetrazolium

Whatman No. 1 ﬁlter paper and the organic extracts concen- chloride (INT) (Sigma–Aldrich, Germany) with further incubation

◦ ◦

trated in vacuo at 35 C using a rotary evaporator. The concentrated at 37 C for 2 h. Since the colourless tetrazolium salt is biologically

extracts were subsequently dried at room temperature under a reduced to a red product due to the presence of active organisms,

stream of cold air. Water extracts were freeze-dried and kept in the MIC values were recorded as the concentrations in the last wells

airtight containers. The percentage yield of extracts from each in which no colour change was observed after adding the INT indi-

extracting solvent was calculated as the ratio of the mass of the cator. Bacterial growth in the wells was indicated by a reddish-pink

dried extract to the mass of the ground plant sample. 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 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)

Hypoxidaceae Hypoxis hemerocallidea NCUBE 01 NU Plant decoctions have purging effects and boost the immune system. Corms

Fisch. & C.A. Mey used for the treatment of inﬂammation, testicular tumours, urinary

complaints, cancer and HIV/AIDS (Watt and Breyer-Brandwijk, 1962; Crouch

et al., 2006)

Hyacinthaceae Merwilla plumbea (Lindl.) NCUBE 02 NU Decoctions are used for wound healing, boils, sores, sprains, to remove scar

Speta tissue, cleaning and rejuvenating the body. Enhances, male potency and libido

(Crouch et al., 2006; Van Wyk et al., 2009)

2.6. Anticandidal activity above. The fractional inhibitory concentration indices (FIC) calcu-

lated was limited to combinations with two extracts only. The FIC

A microdilution method as described by Eloff (1998) and mod- index expresses the interaction of two or more agents in which the

iﬁed for fungi (Masoko et al., 2007) was used to determine the concentration of each test agent in combination is expressed as a

antifungal activity of the extracts against Candida albicans (ATCC fraction of the concentration that would produce the same effect

10231). An overnight fungal culture was prepared in Yeast Malt when used independently (Berenbaum, 1978). The FIC was calcu-

(YM) broth. Four hundred microlitres of the overnight culture were lated as the MIC of the combination divided by the MIC of each

added to 4 ml of sterile saline and absorbance was read at 530 nm. individual component extract. The FIC index was then calculated as

The absorbance was adjusted with sterile saline to match that of the sum of each component FIC in a combination and interpreted

a 0.5 M McFarland standard solution. From this standardised fun- as either synergistic (≤0.5), additive (0.5–1.0), indifferent (1–4.0)

≥ gal stock, a 1:1000 dilution with sterile YM broth was prepared or antagonistic ( 4.0) (Schelz et al., 2006).

giving a ﬁnal inoculum of approximately 10 CFU/ml. Dried crude

organic plant extracts (PE, DCM, and ethanol) were resuspended in

70% ethanol to known concentrations while saponin and phenolic 3. Results and discussion

extracts were redisolved in 50% methanol and water extracts in dis-

tilled water to the same concentrations. One hundred microlitres The results in this study are discussed in the context of both the

of each extract were serially diluted two-fold with sterile water in interaction effects and the antimicrobial efﬁcacy of the different

a 96-well microtitre plate. A similar two-fold dilution of Ampho- extract combinations. Ríos and Recio (2005) suggested that MIC

tericin B (Sigma–Aldrich, Germany) (2.5 mg/ml) was used as the greater than 1 mg/ml for crude extracts or 0.1 mg/ml for isolated

positive control while water and 70% ethanol were used as nega- compounds should be avoided and proposed that activity would be

tive and solvent controls respectively. One hundred microlitres of very interesting in MICs of 0.1 mg/ml and 0.01 mg/ml for extracts

the dilute fungal culture were added to each well. The plates were and isolated compounds respectively. On the other hand, Fabry

◦

covered with paraﬁlm and incubated at 37 C for 24 h, after which et al. (1998) deﬁned active crude extracts as those having MIC val-

50 ␮l (0.2 mg/ml) INT were added and incubated for a further 24 h ues <8 mg/ml. In this study, however, MIC and MFC values of less

◦

at 37 C. The wells remained clear where there was inhibition of than 1 mg/ml were considered to be of good activity. The in vitro

fungal growth. MIC values were recorded as the lowest concen- phytochemical synergy is evident in most of the extract combina-

trations that inhibited fungal growth after 48 h. To determine the tions from the three different plant species studied (Tables 2–6).

fungicidal activity, 50 ␮l of sterile YM broth were added to all the Although very few of the independent extracts exhibited good

◦

clear wells and further incubated at 37 C for 24 h after which the antimicrobial activity (MIC < 1 mg/ml) in both anticandidal and

minimum fungicidal concentrations (MFC) were recorded as the antibacterial bioassays, a number of extract combinations demon-

last clear wells. The assay was repeated twice with two replicates strated good activity, even in cases where none of the independent

per assay. 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

2.7. Checkerboard method for the interaction effect all samples except Hypoxis hemerocallidea corms (Table 2). On

the other hand, good antibacterial activity was only recorded

Stock solutions (50 mg/ml) of each individual extract from each in Tulbaghia violacea bulb extracts against Bacillus subtilis and

plant part were prepared by redisolving water extracts in distilled Staphylococcus aureus, and Hypoxis hemerocallidea leaf extracts

water, saponin and phenolic extracts in 50% methanol and PE, DCM against Bacillus subtilis among all the PE sample extracts. All

and ethanol extracts in 70% ethanol. In the case of 1:1 test com- water and ethanol extracts exhibited poor antibacterial activ-

binations, equal aliquots of 50 ␮l each of the two extracts were ity (MIC > 1 mg/ml). Hypoxis hemerocallidea leaf extracts against

mixed to make up to a volume of 100 ␮l in the ﬁrst wells of a Staphylococcus aureus was the only exception. Despite the lower

96-well microtitre plate while 1:1:1 and 1:1:1:1 combinations had number of active extracts recorded independently for the PE and

each extract contributing 33.3 ␮l and 25 ␮l respectively to make up DCM extracts in this study, a combination of these two extracts in

100 ␮l in the ﬁrst wells of a 96-well microtitre plate. The percent- equal proportions (1:1) (Table 2) yielded very good antibacterial

age yields of PE, DCM, ethanol and water extracts in each plant part activity (MIC < 1 mg/ml) against most strains in all samples. This

sample were noted and combinations of extracts in the proportion tremendous increase in bioactivity was mostly recorded in sam-

of their extract yields from each sample were prepared by mix- ples where none of the two independent extracts exhibited good

ing stock solutions in these respective ratios to make up a volume antibacterial activity, with a signiﬁcant number of them show-

of 100 ␮l in the microtitre plate wells. MIC values were deter- ing a synergistic effect (FIC ≤ 0.5). In all 1:1 extract combinations,

mined for each of these combinations to establish any interaction except those with water extracts, a combination that included

effect following the antibacterial and antifungal assays described either PE or DCM extracts showed good activity against at least two

84 B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 written

0.8 0.4 0.4 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.8 0.4 0.4 0.8 0.8 0.4 0.4 P/D/E/W 0.2 0.2 0.1 0.2 0.2 0.2 0.2 boldly

) )

) ) ) ) ) )

0.4 0.5 0.3 0.3 0.3 0.4 0.3 0.3 0.4 ( ( values

0.5(0.8) 0.8( 0.8( 0.8( 0.8(0.6) 0.8( 0.8( 0.8( E/W 3.1(0.6) 6.3(2.3) 3.1( 3.1(0.7) 6.3(2.5) 6.3(2.0) 6.3(1.0) 1.6(0.6) 1.6(1.5) 3.1(1.2) 3.1(1.2) 3.1(1.2) 1.6(1.1) 1.6(0.6) 0.4 0.4 FIC

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 D/E/W 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.3 brackets.

) ) ) ) ) ) ) ) ) extracts.

0.3 0.3 0.3 0.3 0.5 0.2 0.5 0.6 0.3 shown (

0.8(1.1) 0.8( 0.8(1.3) 0.8(1.1) 0.8( 0.8(0.6) 0.8(0.6) 0.8( 0.8(2.1) 0.8( 0.8( 0.8( 0.8(1.1) 0.8(0.6) 0.8( 0.8(1.1) D/W 1.6(2.0) 1.6(2.1) 1.6(0.6) 1.6(0.6) 1.6(0.6) 1.6( 1.6(0.6) 0.4 are

sample

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) case).

values

0.3 0.3 0.2 0.4 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.3 0.5 0.5 0.5

(0.6) ( (0.8) (0.6) ( ( (0.6) ( ( (1.3) ( ( (0.8) ( ( ( ( ( plumbea FIC

each

0.8(1.1) 0.8( 0.8( 0.8(0.6) 0.8( 0.8(1.3) D/E 0.4 0.4 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.4 0.4 0.2 0.4 0.2 0.4 0.4 0.2 0.4 in

water.

= Merwilla equal

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 P/E/W 1.0 1.0 1.0 1.0 1.0 0.3 0.3 and

values

ethanol,

(MIC

0.5 0.5 0.5 0.5 0.5 0.5 P/D/W 1.0 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 80%

E hemerocallidea

0.5 0.5 0.5 0.5 0.5 P/D/E 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 combination

Hypoxis

in ,

) ) ) ) ) ) ) ) ) ) ) ) )

0.4 0.4 0.4 0.4 0.5 0.4 0.3 0.3 0.5 0.3 0.5 0.3 0.2 extract

violacea

dichloromethane, 0.8(1.0) 0.8( 0.8(0.8) 0.8(1.0) 0.8( 0.8( 0.8(1.0) 0.8( 0.8( 0.8( 0.8( P/W 1.6(1.1) 1.6(0.6 1.6(1.0) 1.6( 1.6( 1.6(0.6) 1.6( 3.1(0.7) 1.6( 3.1( 1.6( 1.6(0.6)

) 0.8(0.6) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

Tulbaghia

0.3 0.3 0.3 0.3 0.3 0.2 0.4 0.4 0.3 0.2 0.2 0.2 0.3 0.4 0.5 0.4 0.4 0.4 component ether;

( (0.6 ( ( ( ( ( ( ( ( (0.8) (0.6) ( ( ( (

0.8(1.0) 0.8( 0.8( 0.8(0.8) 0.8( 0.8(0.6) 0.8( 0.8( P/E 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.4 0.8 0.4 0.4 0.4 0.4 0.4 0.4 0.4 each

mg/ml).

for

(<1 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

petroleum

0.3 0.3 0.3 0.5 0.2 0.3 0.2 0.4 0.2 0.3 0.2 0.3 0.4 0.5 0.4 0.1

( (1.0) (0.8) ( ( (0.6) ( ( ( ( ( ( ( ( ( (0.6) P value

combinations ,

active

0.8(1.5) 0.8( 0.8(3.0) 0.8( 0.8(1.1) 0.8( 0.8( P/D 0.4(1.0) 0.4 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 MIC

very

aureus

the extract

3.1 3.1 3.1 6.3 W 18.8 18.8 18.8 18.8 12.5 12.5 12.5 18.8 12.5 12.5 18.8 18.8 18.8 18.8 18.8 18.8 12.5 12.5 18.8 12.5 ratio

indices

represent considered 6.3 3.1 1.6 6.3 3.1 3.1 3.1 1.6 3.1 1.6 3.1 1.6 3.1 3.1 3.1 1.6 3.1 3.1 3.1 3.1 0.8

equal FIC E 12.5 12.5 12.5

are

Staphylococcus

and and

3.1 3.1 1.6 1.6 3.1 3.1 3.1 6.3 3.1 1.6 3.1 3.1 1.6 3.1 3.1 0.8 0.8 0.8 0.8 0.8 0.4 0.8 0.8

, 12.5 values

1.56 combinations (mg/ml)

MIC

Sa: 1.6 1.6 6.3 3.1 3.1 6.3 6.3 6.3 3.1 6.3 0.8 0.8 0.8

independent MIC PD 12.5

pneumonia

extract

the 1.56;

written

for

Kp:

1:1:1:1

boldly

Klebsiella

3.13; Bacterial strain Bs Bs Bs Bs EcKpSa Ec 3.1 Kp 1.6 SaEc 3.1 Kp 3.1 Ec 6.3 3.1 Sa EcKp Sa 6.3 Sa Kp 3.1 EcKp Sa 6.3 values

and

, Ec: while FIC

coli 1:1:1

and part

effect

0.098;

1:1,

Bulb Bs Plant Bulb Bs: Corm Leaf BsLeaf 1.6 Leaf for

mg/ml)

Escherichia

interaction (MIC

, recorded

g/ml)

␮ activity

( value subtilis

violacea

plumbea

synergistic hemerocallidea

a MIC

species

Bacillus The

a Plant Tulbaghia Hypoxis Merwilla Neomycin Table Bs Antibacterial indicate

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 Plant Extract yields (%) Bacterial MIC (mg/ml) and FIC indices

part strain

P D E W P/D P/E P/W D/E D/W E/W

Tulbaghia violacea Bulb 0.6 0.3 7.3 36.5 Bs 0.5/0.3(1.0) 0.1/1.5(1.2) 0.1/3.0(0.3) 0.1/1.5(0.4) 0.03/3.1(0.2) 1.1/5.2(0.5)

Ec 0.5/0.3(0.3) 0.1/1.5(0.5) 0.1/6.2(0.4) 0.1/1.5(0.5) 0.03/3.1(0.2) 1.1/5.2(0.6)

Kp 0.5/0.3(0.8) 0.1/1.5(1.0) 0.03/1.6(0.5) 0.03/0.8(0.5) 0.03/3.1(1.0) 0.5/2.6(1.2)

Sa 0.5/0.3(1.0) 0.1/1.5(1.1) 0.03/1.6(0.1) 0.03/.08(0.1) 0.03/3.1(0.2) 1.1/5.2(0.4)

Leaf 1.5 1.0 24.0 16.9 Bs 0.2/0.2(0.7) 0.1/1.5(0.3) 0.1/1.5(0.8) 0.1/1.5(0.4) 0.4/5.9(0.8) 1.8/1.3(0.4)

Ec 0.5/0.3(0.3) 0.1/1.5(0.5) 0.3/2.8(0.3) 0.1/1.5(0.5) 0.2/2.9(0.3) 1.8/1.3(0.7)

Kp 0.5/0.3(0.5) 0.1/1.5(0.2) 0.1/1.5(0.8) 0.03/0.8(0.1) 0.4/5.9(0.7) 1.8/1.3(0.2)

Sa 0.5/0.3(0.7) 0.1/1.5(0.2) 0.1/1.5(0.2) 0.1/1.5(0.2) 0.2/2.9(0.5) 1.8/1.3(0.2)

Hypoxis Corm 0.3 0.2 21.5 9.1 Bs 0.5/0.3(0.5) 0.01/1.6(1.0) 0.1/3.0(0.2) 0.01/1.6(0.5) 0.02/1.6(0.1) 1.1/0.5(0.4)

hemerocallidea

Ec 0.5/0.3(0.2) 0.01/1.6(0.5) 0.2/6.1(0.5) 0.01/0.8(0.3) 0.02/1.6(0.1) 1.1/0.5(0.4)

Kp 0.5/0.3(0.3) 0.01/1.6(1.0) 0.1/3.0(1.0) 0.01/0.8(0.5) 0.1/3.0(1.0) 0.6/0.2(0.4)

Sa 1.0/0.6(0.4) 0.01/1.6(0.5) 0.1/3.0(0.3) 0.01/1.6(0.5) 0.02/1.6(0.1) 1.1/0.5(0.4)

Leaf 0.8 0.6 11.4 9.4 Bs 0.5/0.3(1.5) 0.1/1.5(1.1) 0.1/1.5(0.2) 0.04/0.8(0.6) 0.1/1.5(0.3) 0.9/0.5(0.3)

Ec 0.5/0.3(0.2) 0.1/1.5(0.5) 0.2/2.9(0.2) 0.04/0.8(0.3) 0.1/1.5(0.1) 1.1/2.0(0.5)

Kp 0.5/0.3(0.3) 0.1/1.5(1.0) 0.1/1.5(0.5) 0.04/0.8(0.5) 0.1/1.5(0.5) 0.9/0.5(0.7)

Sa 0.5/0.3(0.4 0.1/1.5(2.0) 0.1/1.5(0.2) 0.1/1.5(2.0) 0.1/1.5(0.1) 0.4/0.4(0.2)

Merwilla plumbea Bulb 0.5 0.6 17.3 13.6 Bs 0.4/0.4(0.6) 0.04/1.6(0.5) 0.1/1.5(0.1) 0.03/0.8(0.3) 0.3/6.0(0.7) 1.7/1.4(0.6)

Ec 0.4/0.4(0.2) 0.04/1.6(0.5) 0.1/3.0(0.2) 0.1/1.5(0.5) 0.3/6.0(0.4) 1.7/1.4(0.6)

Kp 0.4/0.4(0.2) 0.04/1.6(0.5) 0.1/1.5(0.1) 0.1/1.5(0.5) 0.3/6.0(0.4) 1.7/1.4(0.6)

Sa 0.4/0.4(0.1) 0.1/3.0(2.0) 0.1/3.0(0.2) 0.1/1.5(1.0) 0.3/6.0(0.5) 0.9/0.7(0.6)

Leaf 1.3 1.1 16.4 15.5 Bs 0.4/0.4(0.3) 0.1/1.5(0.5) 0.2/2.9(0.5) 0.1/1.5(0.5) 0.1/1.5(0.3) 0.8/0.8(0.4)

Ec 0.4/0.4(0.2) 0.1/1.5(0.5) 0.2/2.9(0.3) 0.1/1.5(0.5) 0.1/1.5(0.2) 1.6/1.5(0.6)

Kp 0.4/0.4(0.3) 0.1/0.7(0.3) 0.1/1.5(0.1) 0.1/1.5(0.5) 0.1/1.5(0.1) 1.6/1.5(0.6)

Sa 0.4/0.4(0.6) 0.1/1.5(0.5) 0.2/2.9(0.3) 0.1/1.5(0.6) 0.1/1.5(0.2) 1.6/1.5(0.6)

␮

Neomycin ( g/ml) 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 syner- in these crude extracts are among the lipophilic (non-polar) group

gistic or additive interaction effect. When extracts were combined of compounds. Although PE extracts more highly non-polar com-

in a 1:1:1 combination, antibacterial efﬁcacy was only enhanced in pounds than DCM, the two solvents generally extract non-polar

combinations that included both PE and DCM extracts in all samples classes of compounds compared to water and ethanol which extract

except for Merwilla plumbea leaf extract. The trend in antibacterial mostly polar compounds. However, the weak potency recorded

activity observed in 1:1, 1:1:1 and 1:1:1:1 extract combinations when the two extracts were tested independently, and the cor-

in this study leads to the conclusion that the active constituents respondingly 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 MIC and MFC (mg/ml) and FIC indices

part

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

Tulbaghia violacea Bulb MIC 6.3 3.1 6.3 18.8 0.4 0.8 1.6 0.3 1.0 0.5 0.4 0.8 0.3 1.6 0.8

MFC 6.3 6.3 12.5 18.8 0.8 0.8 1.6 0.5 1.0 1.0 0.4 0.8 0.3 1.6 0.8

FIC 0.3 0.2 0.3 0.1 0.2 0.2

Leaf MIC 0.8 1.6 3.1 12.5 0.4 0.4 3.1 0.3 0.3 0.5 0.4 1.6 0.3 1.6 0.8

MFC 1.6 1.6 6.3 12.5 0.4 0.4 3.1 0.3 0.5 0.5 0.4 1.6 0.3 1.6 0.8

FIC 0.5 0.4 2.2 0.3 1.1 0.4

Hypoxis Corm MIC 6.3 6.3 3.1 3.1 0.8 0.8 3.1 0.5 0.5 0.5 0.4 3.1 0.3 0.4 0.8

hemerocallidea

MFC 6.3 6.3 3.1 3.1 1.6 0.8 3.1 1.0 0.5 0.5 0.4 3.1 0.3 0.4 0.8

FIC 0.5 0.4 1.6 0.2 1.5 0.3

Leaf MIC 6.3 0.8 0.8 0.8 0.8 0.8 3.1 0.5 0.5 0.3 0.4 1.6 0.3 0.4 0.8

MFC 6.3 3.1 1.6 0.8 0.8 0.8 3.1 0.5 0.5 0.3 0.4 1.6 0.3 0.4 0.8

FIC 0.4 0.7 4.4 0.4 2.5 0.8

Merwilla plumbea Bulb MIC 6.3 6.3 6.3 18.8 0.4 0.4 1.6 0.5 0.5 1.0 0.4 3.1 0.5 1.6 0.8

MFC 6.3 6.3 6.3 18.8 0.4 0.8 3.1 0.5 1.0 1.0 0.4 3.1 0.5 3.1 0.8

FIC 0.2 0.3 0.7 0.2 0.7 0.7

Leaf MIC 3.1 0.4 3.1 6.3 0.8 0.4 3.1 1.0 1.0 1.0 0.8 1.6 0.3 1.6 0.8

MFC 6.3 0.8 6.3 6.3 0.8 0.8 3.1 1.0 1.0 1.0 0.8 3.1 0.3 1.6 0.8

FIC 1.2 0.3 1.0 1.2 4.4 0.5

Amphotericin B MIC: 9.77; MFC: 78.1

(␮g/ml)

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 Plant part Extract yields (%) MIC/MFC (mg/ml) and FIC indices

P D E W P/D P/E P/W D/E D/W E/W

Tulbaghia violacea Bulb MIC 0.5/0.3 0.1/1.5 0.1/6.2 0.03/0.8 0.1/6.2 2.1/10.4

0.6 0.3 7.3 36.5 MFC 0.5/0.3 0.1/1.5 0.1/6.2 0.03/.08 0.1/12.4 2.1/10.4

FIC 0.1 0.3 0.3 0.1 1.0 0.7

Leaf MIC 0.5/0.3 0.1/1.5 0.2/2.9 0.0.3/0.8 0.2/2.9 3.7/2.6

1.5 1.0 24.0 16.9 MFC 0.5/0.3 0.1/1.5 0.2/2.9 0.03/0.8 0.4/5.9 7.3/5.2

FIC 0.5 0.3 0.4 0.1 0.7 1.6

Hypoxis Corm MIC 1.0/0.6 0.02/1.6 0.1/1.5 0.01/0.8 0.01/1.6 1.1/0.5

hemerocallidea

0.3 0.2 21.5 9.1 MFC 1.0/0.6 0.02/1.6 0.1/1.5 0.01/0.8 0.01/1.6 1.1/0.5

FIC 0.1 0.5 0.5 0.3 0.5 0.5

Leaf MIC 0.9/0.7 0.1/1.5 0.1/0.7 0.04/0.7 0.05/0.7 0.4/0.4

0.8 0.6 11.4 9.4 MFC 0.9/0.7 0.1/1.5 0.1/0.7 0.04/0.7 0.05/0.7 0.4/0.4

FIC 0.4 1.0 0.9 0.5 0.9 0.8

Merwilla plumbea Bulb MIC 0.4/0.4 0.2/6.1 0.2/6.1 0.2/6.1 0.1/3.0 1.7/1.4

0.5 0.6 17.3 13.6 MFC 0.4/0.4 0.2/6.1 0.2/6.1 0.2/6.1 0.3/6.0 1.7/1.4

FIC 0.2 1.0 0.4 1.0 0.4 0.3

Leaf MIC 0.4/0.4 0.1/1.5 0.2/2.9 0.1/1.5 0.1/1.5 1.2/1.9

1.3 1.1 16.4 15.5 MFC 0.9/0.7 0.1/1.5 0.5/5.8 0.1/1.5 0.1/1.5 1.2/1.9

FIC 1.0 0.3 1.0 0.4 0.4 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 from those of the equal combinations, with most combinations that

action is potentiated by the accompanying compounds in each of included both PE and DCM extracts showing enhanced activity. A

the two extracts and those in ethanol extracts. The compounds closer comparison of the proportional extract yield combinations

from all water extracts displayed a diluting effect characteristic in of PE and DCM, with that of a 1:1 ratio combination (Tables 2 and 3)

all combinations as evidenced by the low efﬁcacy in most extract reveals that the 1:1 combination is a less active combination than

combinations that included a water extract. the proportional yield combination. The proportional extract yield

With the exception of Hypoxis hemerocallidea corm extracts combination closely mimic the natural interaction effect of these

against Staphylococcus aureus, a combination of PE and DCM compounds in plants, and as such may best serve to explain the

extracts in the proportion of their extract yields (Table 3) showed successful defence mechanism developed by plants. On the other

good antibacterial activity in all plant samples. The FIC indices in hand, a 1:1:1 combination of PE, DCM and ethanol extracts gave

most of these extract combinations showed either a synergistic or good antibacterial activity in all the tested plant samples except

additive effect, with only a few exhibiting an indifferent interaction for Merwilla plumbea leaf samples against all bacterial strains

effect. A look at the yields of these two extracts (Table 3) indicates and Merwilla plumbea bulbs against Staphylococcus aureus, com-

that the percentage extract yields in all samples except Merwilla pared to their corresponding proportion extract yield combinations

plumbea bulbs were slightly higher for PE than DCM extracts. This (Tables 2 and 6). The results indicate that the efﬁcacious inter-

therefore, translates to this proportional extract yield combination action effect may be dependent on the precise concentrations of

having a slightly higher concentration of the PE extract constituents certain compounds in an extract combination. In spite of the fact

than those of DCM. For example, in the case of Tulbaghia violacea that most independent plant-derived extracts have shown weak

bulb extracts with 0.6% and 0.3% extract yields for PE and DCM potency against pathogenic bacteria compared to antibiotics, plants

respectively, would have a proportional yield combination of 67 ␮l almost always ﬁght infections successfully in their natural environ-

PE extract stock solution with 33 ␮l of DCM extract stock solution. ment. The aspect of synergistic mechanisms becomes the apparent

The PE and DCM proportional extract yield combination of Merwilla strategy employed by plants, hence the improved efﬁcacy demon-

plumbea bulbs demonstrated the strongest synergistic inhibitory strated by combining the within-plants extracts in this study. The

effect against Staphylococcus aureus, with an FIC index value as low generally good antibacterial activity shown by both proportional

as 0.1. This is despite the fact that each of the two individual compo- extract yields and equal extract combinations of PE and DCM in

nent extracts had an independent MIC of 12.5 mg/ml each against almost all samples in this study, suggests that non-polar com-

the same bacterial strain. This, being the only PE:DCM extract compounds interact more synergistically than the polar compounds.

bination with more of the DCM extract constituents, indicates that The trend is, however, consistent with most of the ﬁndings in other

this phenomenon signiﬁcantly potentiates the two extract efﬁca- non-interaction studies, in which non-polar extracts demonstrated

cies. Such synergistic combinations may often, translate to superior better antimicrobial activity than polar ones (Rabe and Van Staden,

therapeutic effects and even lower dosage requirement for each 1997; McGaw et al., 2001; Ncube et al., 2011b).

extract, thereby reducing the likelihood of dose-dependent toxic- Considering the scarcity of plant extracts with good antibacte-

ity experienced with most herbal remedies (Klippel, 1990; Boucher rial activity against Gram-negative bacteria in most of the previous

and Tam, 2006). In a study with Hypericum perforatum, Nahrstedt studies with different plant materials (Rabe and Van Staden, 1997;

and Butterweck (2010) demonstrated that the bioavailability of Shale et al., 1999), these extract combinations offer good and

certain active constituents, such as hypericin, can be improved promising prospects for the treatment of diseases caused by these

by the accompanying compounds present in the same botanical bacteria in traditional medicine. Escherichia coli and Klebsiella pneu-

product, a principle which most herbal therapeutics lends credence moniae, for example, are known causative agents for diseases

to. such as, urinary tract, gastrointestinal tract and wound infections,

The overall trend in the antibacterial efﬁcacy and interaction bacteriaemia, pneumonia septicaemia and meningitis in humans

effect of the proportional extract yield combination differed slightly (Sleigh and Timbury, 1998; Einstein, 2000). Tulbaghia violacea,

B. Ncube et al. / Journal of Ethnopharmacology 139 (2012) 81–89 87 active

very

0.2/0.1/2.0/10.2 0.2/0.1/2.0/10.2 0.2/1.4/3.5/2.5 0.2/1.4/3.5/2.5 0.03/0.02/2.1/0.9 0.1/0.04/4.4/1.8 0.1/0.04/0.8/0.7 0.1/0.04/0.8/0.7 0.2/0.2/6.8/5.3 0.2/0.2/6.8/5.3 0.1/0.1/1.5/1.4 0.1/0.1/1.5/1.4 P/D/E/W

considered

are

0.04/1.0/5.2 0.04/1.0/5.2 0.1/1.8/1.3 0.1/1.8/1.3 0.01/1.1/0.5 0.01/1.1/0.5 0.04/0.9/0.7 0.1/3.5/2.7 0.1/3.5/2.7 0.1/0.8/0.8 0.1/1.5/1.5 D/E/W 0.02/0.4/0.4 written

extracts. boldly

0.2/2.1/10.3 0.2/2.1/10.3 0.1/1.8/1.2 0.2/3.6/2.5 0.03/2.2/0.9 0.03/2.2/0.9 0.05/1.7/1.3 0.1/3.5/2.7 0.2/3.1/2.9 0.2/3.1/2.9 P/E/W 0.03/0.4/0.3 0.03/0.4/0.3 values 78.1

sample

MFC

MFC:

albicans

and plumbea

9.77;

MIC

Candida

0.05/0.02/2.9 0.05/0.02/2.9 0.1/0.1/1.4 0.1/0.03/1.5 0.1/0.03/1.5 0.2/0.3/5.8 0.2/0.3/5.8 0.1/0.1/1.4 0.2/0.2/2.7 P/D/W 0.0.1/0.04/0.7 0.01/0.04/0.7 0.01/0.04/0.7 MIC:

Merwilla

water.

g/ml) and

(mg/ml) ␮

W (

ethanol, 0.02/0.01/1.5 MIC/MFC P/D/E 0.1/0.03/0.7 0.1/0.03/0.7 0.05/0.03/0.7 0.05/0.03/0.7 0.01/0.01/0.7 0.01/0.04/0.7 0.01/0.04/0.7 0.02/0.03/0.8 0.02/0.03/0.8 0.1/0.05/0.7 0.1/0.05/0.7

hemerocallidea 80%

E MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC Amphotericin

Hypoxis

violacea

dichloromethane,

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 0.1/0.04/0.8/0.7 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 P/D/E/W 0.01/0.01/0.6 0.002/0.001/0.1/0.1 0.01/0.01/0.6 0.002/0.001/0.1/0.1 0.03/0.02/0.4/0.3 0.03/0.02/0.4/0.3 0.01/0.01/0.2/0.2 0.03/0.03/0.4/0.4 0.02/0.01/0.2/0.2 0.03/0.03/0.4/0.4 0.03/0.03/0.4/0.4 Tulbaghia

ether;

0.01/0.3/1.3 0.01/0.3/1.3 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 0.01/1.1/0.5 0.01/1.1/0.5 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 D/E/W 0.01/0.1/0.7 0.01/0.6/0.2 0.02/0.4/0.4 0.03/0.4/0.4 petroleum

combinations

yield

aureus

extract

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 0.02/1.1/0.5 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 0.05/1.7/1.3 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 P/E/W 0.03/0.4/0.3

Staphylococcus

proportional

the ,

0.03/0.01/1.5 0.1/0.1/1.4 0.1/0.1/1.4 0.1/0.1/1.5 0.1/0.1/1.5 0.1/0.1/1.5 0.1/0.1/1.4 0.1/0.1/1.4 P/D/W 0.01/0.1/0.8 0.01/0.1/0.8 0.01/0.1/0.8 0.0.1/0.04/0.7 0.0.1/0.04/0.7 0.0.1/0.04/0.7 0.03/0.02/0.8 0.03/0.02/0.8 0.03/0.02/0.8 0.03/0.02/0.8 0.01/0.04/0.7 0.01/0.04/0.7 0.01/0.04/0.7 0.03/0.03/0.7 0.1/0.05/0.7 0.1/0.05/0.7 for

1.56

pneumonia mg/ml)

Sa:

(mg/ml)

MFC,

1.56;

0.1/0.1/1.4 0.1/0.1/1.4 0.1/0.1/1.4 0.02/0.01/1.5 0.1/0.1/1.4 0.1/0.1/1.4 0.1/0.1/1.4 0.04/0.1/1.5 0.04/0.1/1.5 0.1/0.1/1.4 0.1/0.1/1.4 MIC P/D/E 0.1/0.03/0.7 0.1/0.03/0.7 0.05/0.03/0.7 0.05/0.03/0.7 0.01/0.01/0.7 0.01/0.01/0.7 0.01/0.01/0.7 0.01/0.04/0.7 0.02/0.03/0.8 0.1/0.05/0.7 0.1/0.05/0.7 Klebsiella

and

Kp: =

, (MIC

3.13;

coli

Ec:

Bacterial strain Bs Bs Bs Bs Bs Bs Sa Sa Sa Sa Sa Sa 0.04/0.1/1.5 Ec Kp Ec Kp 0.1/0.1/1.4 Ec Kp Ec Kp Ec Kp Ec Kp activity

0.098;

Escherichia

Bulb Plant part Bulb Bs: Leaf Leaf Leaf Corm =

, anticandidal

g/ml) and

␮

subtilis violacea

plumbea

species

hemerocallidea Bacillus

mg/ml).

Plant Tulbaghia Merwilla Neomycin Hypoxis Table Bs Antibacterial (<1

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 Ec Kp Sa MIC MFC

Phe 3.1 3.1 3.1 6.3 6.3 12.5

Bulb Sap 3.1 6.3 3.1 6.3 12.5 12.5

Tulbaghia violacea Phe/Sap 6.3/6.3 (4.1) 6.3/6.3 (3.0) 3.1/3.1(2.0) 3.1/3.1 (1.0) 6.3/6.3 6.3/6.3 (1.0)

Phe 6.3 3.1 3.1 6.3 6.3 6.3

Leaf Sap 3.1 3.1 1.6 6.3 12.5 12.5

Phe/Sap 9.4/9.4 (4.5) 9.4/9.4 (6.1) 6.3/6.3(5.9) 6.3/6.3 (2.0) 3.1/3.1 3.1/3.1 (0.8)

Phe 0.8 0.8 0.8 0.8 1.6 1.6

Corm Sap 3.1 1.6 1.6 3.1 3.1 3.1

Hypoxis Phe/Sap 0.8/0.8 (1.3) 0.2/0.2 (0.4) 0.4/0.4(0.8) 0.2/0.2 (0.3) 0.4/0.4 0.4/0.4 (0.4)

hemerocallidea Phe 0.8 0.8 1.6 0.8 1.6 1.6

Leaf Sap 3.1 1.6 1.6 1.6 3.1 3.1

Phe/Sap 1.6/1.6 (2.5) 0.4/0.4 (0.8) 0.8/0.8 (1.0) 0.2/0.2 (0.4) 0.4/0.4 0.4/0.4 (0.4)

Phe 3.1 6.3 6.3 6.3 12.5 12.5

Bulb Sap 6.3 6.3 12.5 6.3 12.5 12.5

Merwilla Phe/Sap 3.1/3.1 (1.5) 6.3/6.3 (2.0) 1.6/1.6(0.4) 1.6/1.6(0.5) 3.1/3.1 3.1/3.1 (0.5)

plumbea Phe 3.1 6.3 6.3 6.3 6.3 12.5

Leaf Sap 6.3 6.3 3.1 6.3 6.3 6.3

Phe/Sap 3.1/3.1 (0.8) 3.1/3.1 (1.0) 1.6/1.6(0.8) 1.6/1.6(0.5) 6.3/6.3 6.3/6.3 (1.5)

␮ ␮

Neomycin ( g/ml) Bs: 0.098; Ec: 3.13; Kp: 1.56; Sa: 1.56 Amphotericin B ( g/ml) MIC: 9.77; MFC: 78.1

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 fungicidal activity (Tables 4 and 5) in all samples except for Mer-

African traditional medicine against some of these ailments. In light willa plumbea leaves. A 1:1:1 combination of DCM, ethanol and

of the global threat by the emerging antibiotic resistant bacterial water extracts showed good fungicidal activity in all plant sam-

strains which cause infectious diseases, the results of this study ples except for Merwilla plumbea bulb extracts, while a proportional

provides an insight into the potential sources of such remedies and extract yield combination of PE, DCM and ethanol gave good

further conﬁrm the efﬁcacy of mixing medicinal plant extracts. fungicidal activity in all samples except for Hypoxis hemerocallidea

Although most of the extract combinations demonstrated either corms (Tables 4 and 6). In general, a combination that included

synergistic or additive, interaction effects in the antibacterial test an ethanol extract, except in the 1:1:1:1 combination of all the

in this study, there were a few cases where extract combinations extracts, resulted in better antifungal efﬁcacy than any other com-

exhibited indifferent effects, for example, the 1:1 combination bination (Table 4). This may be an indication that the candidate

of PE and DCM, extracts of Hypoxis hemerocallidea leaf samples compounds for the observed anticandidal activity are enhanced

against Bacillus subtilis (FIC index 3.0). The PE, DCM, ethanol and from the ethanol extracts. The efﬁcacy and interaction effect in the

water proportional extract yield combination of Hypoxis hemero- proportional extract yield combination followed an almost similar

callidea corm extracts, on the other hand, demonstrated the best trend to that of equal combinations. Candida albicans has proved

antibacterial activity, with the highest component MIC (water) of to be more resistant to most plant extracts (Heisey and Gorham,

0.1 against both Escherichia coli and Staphylococcus aureus. Such 1992; Buwa and Van Staden, 2006; Ncube et al., 2011a), and the

a scenario, however, highlights the complexity of the interaction good fungicidal activity demonstrated by these extract combina-

effects of a suite of chemical compounds found within a plant, tions offer promising prospects for the treatment of candidiasis

and the potential dangers associated with the widely held per- particularly for HIV/AIDS patients. Candidiasis is a common oppor-

ception by herbal medicinal users that herbal mixtures, almost tunistic infection among HIV/AIDS patients and is one of the major

always have an enhanced efﬁcacy. Combination chemotherapy is causes of death in developing countries (Reichart, 2003). Tulbaghia

often employed in clinical practice for the treatment of infectious violacea is commonly used in South African traditional medicine

diseases. Substantial research trials have been done to investi- among HIV/AIDS patients (Klos et al., 2009) and Hypoxis hemero-

gate the interaction effects of medicinal plant extracts with known callidea is used as an immune system booster. The good fungicidal

clinical drugs in the treatment of various ailments, with several activity demonstrated by some extract combinations of these two

of them yielding positive interaction effects (Sato et al., 2004; plant species, provide a basis for their use in the treatment of such

Filoche et al., 2005; Prabhakar and Doble, 2009). Combinations of opportunistic infections in traditional medicine.

different medicinal plant components in the herbal formulation The good antibacterial activity demonstrated by the phenolic-

remedies are increasingly becoming a common phenomenon in rich extracts of Hypoxis hemerocallidea samples (Table 7),

most traditional medicinal systems. In an attempt to understand corresponds with the high phenolic content previously identiﬁed

the antimicrobial efﬁcacy of such herbal combinations, Van Vuuren in these samples (Ncube et al., 2011a). Chances are, however, high

and Viljoen (2008) and Ndhlala et al. (2009) investigated this aspect that the good activity shown by these extracts in this study may

with different plant parts and herbal preparations respectively, be as a result of these phenolic compounds. A 1:1 combination of

and reported both positive and negative interaction effects. While phenolic-rich and saponin-rich extracts resulted in a synergistic

extract combinations may, in some cases, result in an enhanced interaction effect against Escherichia coli and Staphylococcus aureus

efﬁcacy, it must, however, be pointed out that synergism should in Hypoxis hemerocallidea corm, and against Staphylococcus aureus

not always be assumed, as this and other previous studies have in leaf extract samples respectively. A positive fungicidal interac-

shown. tion effect was also exhibited by a combination of saponin-rich and

In the anticandidal test, a 1:1 and proportional extract yields phenolic-rich extracts in both corm and leaf samples of Hypoxis

combinations of DCM and ethanol extracts resulted in good 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 89

0.4 in each sample respectively. This is despite the fact that none Eloff, J.N., 1998. A sensitive and quick microplate method to determine the min-

imal inhibitory concentration of plant extracts for bacteria. Planta Medica 64,

of the two independent extracts showed good fungicidal activity.

711–713.

A similar extract combination in Tulbaghia violacea leaf samples

Fabry, W., Okemo, P.O., Ansorg, R., 1998. Antibacterial activity of East African medic-

resulted in antagonistic interaction effects (FIC index > 4.0) against inal plants. Journal of Ethnopharmacology 60, 79–84.

Filoche, S.K., Soma, K., Sissons, C.H., 2005. Antimicrobial effects of essential oils in

Bacillus subtilis, Escherichia coli and K. pneumonia, with an indif-

combination with chlorhexidine digluconate. Oral Microbiology and Immunol-

ferent effect against Staphylococcus aureus (FIC index of 2.0). In

ogy 20, 221–225.

Merwilla plumbea, the antibacterial interaction effect of the two Gibbons, S., 2004. Anti-staphylococcal plant natural products. Natural Product

Reports 21, 263–277.

extracts ranged from a synergistic to indifferent interaction effect,

Goldstein, A., Aronow, L., Kalman, S.M., 1974. Principles of Drug Action: the Basis of

although none of the combinations showed good activity against

Pharmacology, 2nd ed. John Wiley and Sons, New York.

any of the tested bacterial strains. Heisey, R.M., Gorham, B.K., 1992. Antimicrobial elects of plant extracts on Streptococ-

cus mutans, Candida albicans, Trichophyton rubrum and other microorganisms.

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4. Conclusions

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Klos, M., Van de Venter, M., Milne, P.J., Traore, H.N., Meyer, D., Oosthuizen, V., 2009.

activity. These results demonstrate the potential phytotherapeutic

In vitro anti-HIV activity of ﬁve selected South African medicinal plant extracts.

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Makkar, H.P.S., 1999. Quantiﬁcation of Tannins in Tree Foliage: a Laboratory Manual

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Techniques to Develop Simple Tannin Assay for Predicting and Improving the

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evidence that the full potential therapeutic value of a medicinal

An investigation on the biological activity of Combretum species. Journal of

plant lies in the manipulation of the entire extract sets together Ethnopharmacology 75, 45–50.

Nahrstedt, A., Butterweck, V., 2010. Lessons learned from herbal medicinal products:

in various combinations. Be that as it may, it must, however, be

the example of St. John’s wort. Journal of Natural Products 73, 1015–1021.

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Ncube, B., Finnie, J.F., Van Staden, J., 2011a. Seasonal variation in antimicrobial and

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equally diverse and may lead to some toxic effects within the South Africa. South African Journal of Botany 77, 387–396.

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pathogens. rial infections Kaposi’s sarcoma. Medical Microbiology and Immunology 192,

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