Journal of Ethnopharmacology 148 (2013) 45–55

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Antimicrobial activity of southern African medicinal plants with

dermatological relevance: From an ethnopharmacological screening
approach, to combination studies and the isolation of a
bioactive compound
Unathi Mabona a, Alvaro Viljoen b, Emmanual Shikanga c, Andrew Marston d,y, Sandy Van
Vuuren a,n
a
Department of Pharmacy and Pharmacology, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa
b
Department of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa
c
Department of Chemistry, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa
d
Department of Chemistry, University of the Free State, Bloemfontein 9300, South Africa

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Ethnobotanical reports on more than 100 southern African medicinal plants
Received 20 January 2013 with dermatological relevance have been highlighted, yet there is still limited scientific data to support claims
Received in revised form for their antimicrobial effectiveness against skin pathogens. Guided by ethnobotanical data, this paper
19 March 2013
explores the antimicrobial efficacies of southern African medicinal plants used to treat skin ailments.
Accepted 20 March 2013
Available online 30 March 2013
Aim of the study: To investigate the antimicrobial properties of southern African medicinal plants against
dermatologically relevant pathogens. The study also aimed at providing a scientific rationale for the traditional
Keywords: use of plant combinations to treat skin diseases and the isolation of the bio-active compound from the most
Antimicrobial screening active species, Aristea ecklonii (Iridaceae).
Aristea ecklonii Materials and methods: Organic and aqueous extracts (132) were prepared from 47 plant species and screened
Combinations
for antimicrobial properties against dermatologically relevant pathogens using the micro-titre plate dilution
Compound isolation
method. Four different plant combinations were investigated for interactive properties and the sum of the
Dermatophytes
Skin
fractional inhibitory concentration (ƩFIC) calculated. Isobolograms were used to further investigate the
antimicrobial interactive properties of Pentanisia prunelloides combined with Elephantorrhiza elephantina at
varied ratios. A bioactivity-guided fractionation process was adopted to fractionate the organic leaf extract of
Aristea ecklonii.
Results: Plants demonstrating notable broad-spectrum activities (MIC values ≤1.00 mg/ml) against
the tested pathogens included extracts from Aristea ecklonii, Chenopodium ambrosioides, Diosp-
yros mespiliformis, Elephantorrhiza elephantina, Eucalyptus camaldulensis, Gunnera perpensa,
Harpephyllum caffrum, Hypericum perforatum, Melianthus comosus, Terminalia sericea and Warburgia
salutaris. The organic extract of Elephantorrhiza elephantina, a plant reportedly used to treat
acne vulgaris, demonstrated noteworthy antimicrobial activity (MIC value of 0.05 mg/ml) against
Propionibacterium acnes. Similarly, Diospyros mespiliformis reported for its traditional use to treat
ringworm, also displayed noteworthy antimicrobial activity against Trichophyton mentagro-
phytes (MIC 0.10 mg/ml) and Microsporum canis (MIC 0.50 mg/ml). The aqueous root extracts of
Pentanisia prunelloides combined (1:1) with Elephantorrhiza elephantina displayed synergistic
interactions (ƩFIC values 0.31–0.38) against Staphylococcus aureus, gentamycin–methicillin resis-
tant Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans. Fractionation of Aristea
ecklonii resulted in the isolation of the known bio-active compound, plumbagin, displa-
ying noteworthy antimicrobial activity (MIC range between 2.00 μg/ml and 16.00 μg/ml).
Conclusion: Most of the plant extracts demonstrated pathogen specific antimicrobial effects with a few
exhibiting broad-spectrum activities. Positive antimicrobial effects noted for plants such as

Abbreviations: Aq., Aqueous extract; CC., column chromatography; CFU/ml., colony forming units/ml; D:M., 1:1 mixture of dichloromethane and methanol; DMSO.,
dimethyl sulfoxide; HSCCC., high speed counter-current chromatography; MIC., minimum inhibitory concentration; NMR., nuclear magnetic resonance; ΣFIC., the sum of the
fractional inhibitory concentration; INT., iodonitrotetrazolium chloride; TSB., Tryptone Soya broth; UHPLC., ultra-high performance liquid chromatography..
n
Corresponding author. Tel.: +27 11 7172157; fax: +27 11 6424355.
E-mail address: Sandy.vanvuuren@wits.ac.za (S. Van Vuuren).
y
Deceased.

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.03.056
46 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55
Elephantorrhiza elephantina and Diospyros mespiliformis used for acne vulgaris and ringworm infections,
respectively, give some validation to their reported traditiona l uses. Synergistic interactions noted for
Pentanisia prunelloides combined with Elephantorrhiza elephantina validate an enhanced antimicrobial
effect when used in combination. Noteworthy antimicrobial activities (MIC range between 2.00 μg/ml
and 16.00 μg/ml) were observed for plumbagin isolated from Aristea ecklonii.
1. Introduction
The readily-available ethnobotanical literature has reported
over 100 medicinal plants that are used in southern Africa for
treating dermatological disorders (Watt and Breyer-Brandwijk,
1962; Hutchings, 1996; Von Koenen, 1996; Felhaber, 1997; Rabe
and Van Staden, 1997; Van Wyk et al., 2000, 2009). A review by
Van Vuuren (2008), on South African medicinal plants, highlights
numerous studies which have focused on evaluating the antimi-
crobial efficacies of plant species used for a variety of ailments,
including skin inflictions. While most studies have focused on
antimicrobial screening against common pathogens such as, Sta-
phylococci species, Pseudomonas aeruginosa and Candida albicans,
it has been noted that skin dermatophytes such as Trichophyton
mentagrophytes and Microsporum canis have been neglected in
most screening assays. This is possibly due to difficulties encoun-
tered with culturing mould dermatophytes, which is a time-
consuming process. However, to validate the antimicrobial effica-
cies of medicinal plants used traditionally to treat common skin
diseases such as ringworm infections, it is necessary to include all
the relevant, including fastidious pathogens.
Propionibacterium acnes is an important skin pathogen respon-
sible for the chronic inflammatory disease of the sebaceous glands
and hair follicles of the skin. The infection usually results in acne
vulgaris, a skin condition common but not exclusive to teenagers,
which has considerable psychological impact (Magin et al., 2006).
The antibacterial effects of South African medicinal plants against
acne causing bacteria have been rarely addressed, even though
attention has been given to this pathogen in other geographical
ethnobotanical-relevant studies (Chomnawang et al., 2005, Kim
et al., 2007, 2008; Tsai et al., 2010; Balakrishnan et al., 2011).
While many studies have focused on either antimicrobial screening
or the phytochemistry, very little is reported on plant–plant interac-
tions when used in combination, in spite of the traditional use (Smith,
1895; Hutchings, 1996; Felhaber, 1997). Although some studies have
been conducted to evaluate medicinal plant interactions from south-
ern African species (Kamatou et al., 2006; Van Vuuren, 2008; Suliman
et al., 2010; Ncube et al., 2012), the use of plant combinations to treat
specific skin ailments has been sorely neglected.
The identification of bio-active compounds is another impor-
tant factor to be examined to gain insight into the antimicrobial
properties of medicinal plants of dermatological relevance. Pre-
viously, a number of antimicrobial related studies have highlighted
the value of identifying the antimicrobial active compound/s (Rabe
and Van Staden, 1997; De Paiva et al., 2003; Van Vuuren et al.,
2006; Shai et al., 2008; Van Vuuren, 2008). Hence, this compre-
hensive study of southern African dermatologically relevant plants
aims to present a detailed account of the antimicrobial properties,
plant–plant interactive efficacies and isolation of a bio-active
naphthoquinone from one of the most active plant species.
2. Materials and methods
2.1. Plant collection and identification
Various plant parts (related to traditional use) of 47 different
plant species (representing 38 families) were harvested from
& 2013 Elsevier Ireland Ltd. All rights reserved.
designated botanical gardens. Voucher specimens were prepared
for each species and are housed in the Department of Pharmacy
and Pharmacology, University of the Witwatersrand. Table 1
details the plant species collected, reported traditional use, parts
of the plants used, voucher numbers and the collection sites.
2.2. Preparation of plant extracts
Plant samples were left to dry at room temperature. They were
then ground to a fine powder using the high speed Fritsch
Pulverisette grinder (Labotec). Organic extracts were prepared by
submerging (720 g) of the dried, crushed plant material in a 1:1
mixture of dichloromethane and methanol (D:M) and left on the
platform shaker incubator (Labcon) at 37 1C for 24 h.
Aqueous extracts (Aq) were prepared by submerging the
macerated plant material in sterile distilled water, and were then
left on the platform shaker incubator and kept at ambient
temperature overnight. Thereafter, the liquid extracts were filtered
and stored at −80 1C before lyophilisation (Virtis). All extract
samples were stored at room temperature until further use.
2.3. Antimicrobial activity assays
2.3.1. Culture preparation
Bacterial and fungal test organisms were selected based on
conditions that the plants are reported to treat and on their
prevalence to cause skin infections. These include aerobic Gram-
positive bacteria; Staphylococcus aureus ATCC 25923, methicillin-
resistant Staphylococcus aureus (MRSA) ATCC 43300, gentamycin–
methicillin-resistant Staphylococcus aureus (GMRSA) ATCC 33592,
Staphylococcus epidermidis ATCC 2223, Brevibacillus agri ATCC
51663 and anaerobic Propionibacterium acnes ATCC 11827. The
Gram-negative bacterium selected for the study was Pseudomonas
aeruginosa ATCC 27858. Dermatophytes such as Trichophyton
mentagrophytes ATCC 9533, Microsporum canis ATCC 36299 and
the yeast Candida albicans ATCC 10231 were also included.
Each bacterial culture was grown in Tryptone Soya broth (TSB)
(Oxoid, Ltd), for 18–24 h at 37 1C. Propionibacterium acnes, however,
was grown in Thioglycolate broth (Oxoid, Ltd) and incubated under
anaerobic conditions using a candle gas jar for seven days at 37 1C.
Dermatophytes, Trichophyton mentagrophytes and Microsporum
canis were grown and maintained on Sabouraud's Dextrose agar
(Oxoid, Ltd), incubated at 35 1C for up to seven days in a
humidified environment (Masoko et al., 2007). Candida albicans
was grown in TSB and incubated at 37 1C for 48 h.
2.3.2. Micro-titre plate dilution technique: Minimum inhibitory
concentration (MIC)
A serial micro-dilution assay was used to quantify the mini-
mum inhibitory concentration (MIC) values for plant extracts
using tetrazolium violet reduction as an indicator of growth
(Eloff, 1998; NCCLS, 2003). Using aseptic manipulation, 100 μl of
distilled sterile water was instilled in each well of a 96 well micro-
titre plate. The plant extracts at starting concentrations of 64 mg/
ml in acetone or dimethyl sulfoxide (DMSO) (Table 2) were
transferred to the first row of the micro-titre plate. The solvent
DMSO was used when select extracts were insoluble in acetone.
 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55 47

Table 1
Southern African medicinal plants commonly used for dermatological purposes.

Plant species Modes of administration and traditional References Collected Collection site and (%) Yield for (%) Yield for
uses plant part voucher number organic aqueous
extractsa extractsa

Acacia erioloba Edgew., Wood ash is applied topically for wound Smith (1996), Von Koenen Bark WSBGb UM173 17.7 4.2
Fabaceae healing (1996)
b
Leaf WSBG UM160 26.8 6.0
Acokanthera oppositifolia Leaf or root pulp is rubbed into the Watt and Breyer- Leaf WSBGb UM156 20.9 12.9
(Laim.) Codd., wound, leaf or root is also applied as a Brandwijk (1962),
Apocynaceae dressing to swollen parts Hutchings (1996),
Bethwell (2007)
Aloe arborescens Mill., Leaf applied topically to treat wounds, Bruce (1975), Van Wyk Leaf WSBGb UM152 19.7 16.5
Xanthorrhoeaceae burns and various skin ailments et al. (2000)
Athrixia phylicoides DC., Plant infusion used to treat sores and boils Hutchings (1996) Leaf Haenertsburg AV999 9.3 4.6
Asteraceae
Aristea ecklonii Baker., Whole plant preparation applied topically Hutchings (1996), Leaf Random Harvest 12.3 5.9
Iridaceae for shingles, also used for the treatment of Ngwenya et al. (2003) Indigenous nursery
fevers, coughs as well as syphilis UM163
Roots Random Harvest 64.0 11.4
Indigenous nursery
UM164
Bauhinia macranthera Leaf extract used to treat wounds Von Koenen (1996) Leaf WSBGb UM155 25.4 20.5
Benth. ex Hemsl.,
Fabaceae
Boophane disticha L.f., Bulb preparation applied topically to treat Watt and Breyer- Leaf WSBGb UM165 55.9 6.5
Amaryllidaceae septic wounds, boils, external sores and Brandwijk (1962), Van
rheumatism Wyk et al. (2000)
Bridelia micrantha Baill., Bark decoction used to treat burns and Mabogo (1990), Hutchings Bark WSBGb UM149 0.3 1.0
Euphorbiaceae wounds (1996), Van Wyk et al.
(2011)
Leaf WSBGb UM150 33.4 3.6
Chenopodium Whole plant decoction use to treat Hutchings (1996), Pesewu Leaf Near Rayton 3.7 7.1
ambrosioides Bert. ex eczema, wounds and skin infections et al. (2008) (Gauteng) B & F 14
Steud., Chenopodiaceae
Cissampelos capensis Rhizomes, roots and/or leaf paste used for Van Wyk et al. (2000) Leaf Sun valley (Western 10.6 4.1
Thunb., boils, snakebite wounds, ulcers and cape) UM128
Menispermaceae syphilis sores
Cotyledon orbiculata Externally apply juice for wart removal, or Watt and Breyer- Leaf WSBGb UM135 14.4 18.2
Forssk., Crassulaceae place the hot leaf directly to the swollen Brandwijk (1962), Bhat
part of the body also for corns, warts, boils and Jacobs (1995),
Felhaber (1997), Van Wyk
et al. (2000)
Dicoma anomala Sond., Charred roots, stems and/or leaf paste Hutchings (1996), Tuber WSBGb UM167 13.0 17.6
Asteraceae used for wounds, ulcers, ringworm and Felhaber (1997)
head sores. The tuber is used in
combination with Elephantorrhiza
elephantina to treat acne.
Dioscorea dregeana T. Small piece of root boiled in water and Watt and Breyer- Tuber WSBGb UM174 6.1 8.8
Durand & Schinz., applied externally used for cuts and sores Brandwijk (1962), Pujol
Dioscoreaceae (1990), Van Wyk et al.
(2000)
Diospyros mespiliformis Root or leaf decoction used for scars, skin Von Koenen (1996), Van Leaf WSBGb UM151 25.6 9.4
Hochst. ex A.DC., rash, bruises, wounds and ringworm Wyk et al. (2011)
Ebenaceae
Dodonaea angustifolia L. Leaves and tips of twigs boiled in water, Watt and Breyer- Leaf Pretoria–Villieria 22.6 7.1
f., Sapindaceae filtered and applied externally to treat Brandwijk (1962), Smith UM125
boils; used as a dressing. (1996), Van Wyk et al.
(2000)
Ekebergia capensis Bark infusion used for abscesses, boils and Pujol (1990), Van Wyk Bark WSBGb UM139 12.6 0.6
Sparrm., Meliaceae acne et al. (2000, 2011)
Leaf WSBGb UM138 9.3 11.1
Elephantorrhiza Roots and rhizomes boiled in water for Pujol (1990), Felhaber Leaf WSBGb UM171 50.1 11.3
elephantina (Burch.) external use to treat acne and other skin (1997), Van Wyk et al.
Skeels, Fabaceae diseases. Also used in combination with (2000)
Pentanisia prunelloides to treat eczema
Roots and WSBGb UM172 5.0 8.9
rhizomes
Embelia ruminate (E. Leaf paste used to treat open wounds and Kumara Swamy et al. Leaf WSBGb UM175 31.7 5.3
Mey. ex A.Dc.) Mez, leprosy (2007)
Myrsinaceae
Erythrina lysistemon Bark applied as poultice used for sores, Coates Palgrave (1977), Leaf Pretoria–Villieria 18.7 4.4
Hutch., Fabaceae abscesses and open wounds Pujol (1990), Hutchings UM132
(1996), Van Wyk et al.
(2000), Van Wyk et al.
(2011)
Eucalyptus camaldulensis Bark infusion used as a wash, to treat acne Hutchings (1996) Bark Pretoria–Villieria 14.0 7.2
Dehnh., Myrtaceae UM122
48 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55

Table 1 (continued )

Plant species Modes of administration and traditional References Collected Collection site and (%) Yield for (%) Yield for
uses plant part voucher number organic aqueous
extractsa extractsa

Ficus natalensis Hochst., Leaves used as hot compress for wounds, Hutchings (1996), Van Leaf Pretoria–Villieria 3.6 8.2
Moraceae boils and warts Wyk et al. (2011) UM131
Ficus sur Forssk., Bark used as a compress for boils Palmer and Pitman (1972) Bark WSBGb UM140 1.3 1.3
Moraceae Hutchings (1996)
b
Leaf WSBG UM141 8.9 6.3
Gunnera perpensa L., Root or rhizome and leaf are used as Hutchings (1996), Leaf WSBGb UM168 7.0 13.1
Gunneraceae infusion or decoction for dressing wounds Felhaber (1997), Drewes
and to treat psoriasis et al. (2005), Van Wyk
et al. (2009)
Rhizomes WSBGb UM176 18.5 10.5
Halleria lucida L., Unspecified parts used topically for Pooley (1993), Hutchings Leaf WSBGb UM177 11.9 7.3
Scrophulariaceae various skin complaints (1996)
Stem WSBGb UM178 4.6 3.2
Harpephyllum caffrum Bark applied externally to treat acne and Pujol (1990), Van Wyk Bark Pretoria–Villieria 10.4 9.7
Bernh. ex Krauss, eczema et al. (2000, 2011) UM128
Anacardiaceae
Hypericum perforatum L., Above ground parts applied externally to Van Wyk et al. (2000) Leaf UWc UM162 40.6 6.2
Hypericaceae treat wounds and first degree burns

Ilex mitis Radlk., Ground bark applied as paste or decoction Hutchings (1996), Van Bark WSBGb UM144 0.1 6.3
Aquifoliaceae for skin rash and sores on the face Wyk et al. (2011)
Leaf WSBGb UM145 28.7 6.1
Kigelia africana (Lam.) Externally applied to treat ulcers, sores, Watt and Breyer- Fruit Zululand UM161 5.4 11.0
Benth., Bignoniaceae abscesses and rheumatism Brandwijk (1962), Coates
Palgrave (1977),
Hutchings (1996), Van
Wyk et al. (2000, 2011)
Lannea discolor Engl., Bark applied externally to treat boils and Watt and Breyer- Leaf Pretoria–Villieria 17.0 9.7
Anacardiaceae abscesses Brandwijk (1962), UM121
Hutchings (1996), Van
Wyk et al. (2000)
Lantana rugosa Thunb., Leaf, stem or ripe fruits used as a paste for Smith (1895), Roberts Leaf Rayton (Gauteng)B & 7.7 4.1
Verbenaceae festering sores and cuts (1990), Hutchings (1996) F10

Malva parviflora L., Leaf paste combined with Pelargonium Smith (1895) Leaf WSBGb UM166 17.2 18.7
Malvaceae alchemilloides to treat wounds and
abscesses
Melianthus comosus Leaf poultice and leaf decoction used to Gerstner (1938), Watt and Leaf WSBGb UM147 21.7 9.8
Vahl., Melianthaceae treat bed sores, sceptic wounds, reduce Breyer-Brandwijk (1962),
swellings Hutchings (1996)
Melianthus major L., Leaf poultice and leaf decoction used for Van Wyk et al. (2009) Leaf WSBGb UM142 15.7 17.8
Melianthaceae septic wounds, sores and bruises
Mentha longifolia Huds., Leaves applied topically to treat wounds Van Wyk et al. (2000) Leaf WSBGb UM148 8.4 10.4
Lamiaceae
Opuntia ficus-indica Leaves applied topically for skin rash, Smith (1996), Von Koenen Leaf Pretoria–Villieria 5.9 12.0
Mill., Cactaceae ulcers, furuncles, fresh wounds and warts (1996) UM120
Pellaea calomelanos Decoction or infusions of leaves and/or Watt and Breyer- Leaf WSBGb UM146 16.1 9.4
Link., Adiantaceae rhizomes applied externally for boils and Brandwijk (1962), Pujol
abscesses (1990), Hutchings (1996),
Van Wyk et al. (2000)
Rhizomes WSBGb UM179 30.6 3.8
Pentanisia prunelloides Roots applied externally for burns and Felhaber (1997), Van Wyk Root bark PNBGd UM182 10.6 10.7
Walp., Rubiaceae swellings. Also used in combination with et al. (2000)
Dicoma anomala to treat insect stings and
bites
Roots PNBGd UM183 5.8 4.8
stripped
Pittosporum viridiflorum Roots or leaves combined with Momordica Hutchings (1996) Leaf WSBGb UM159 16.0 7.7
Sims., Pittosporaceae foetida and Vernonia natalensis in a
decoction used to treat boils
Rauvolfia caffra Sond., Bark applied topically to treat measles, Gerstner (1938), Leaf WSBGb UM137 11.6 6.6
Apocynaceae urticaria and other skin rashes Hutchings (1996)
Rothmannia capensis Sap from fruit applied topically for burns
Arnold and Gulumian Leaf WSBGb UM157 13.3 2.5
Thunb., Rubiaceae and wounds (1984), Hutchings (1996)
Scadoxus puniceus (L.) Bulbs and root decoction applied topically
Watt and Breyer- Roots and WSBGb UM143 7.4 10.3
Friis & Nordal, to treat wounds, ulcers, sores and allergies
Brandwijk (1962), rhizomes
Amaryllidaceae Hutchings (1996), Van
Wyk et al. (2000)
Solanum incanum L., Leaves or roots applied topically to treat Gerstner (1938), Leaf WSBGb UM158 4.3 6.6
Solanaceae wounds, furuncles and ringworm Hutchings (1996), Von
Koenen (1996)
Terminalia sericea Burch. Root sap or bark applied externally as an Watt and Breyer- Roots Swaziland AdCAV21 33.1 15.2
ex DC., Combretaceae antiseptic for wounds and to treat leprosy Brandwijk (1962), Pujol
and snakebites
 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55 49

Table 1 (continued )

Plant species Modes of administration and traditional References Collected Collection site and (%) Yield for (%) Yield for
uses plant part voucher number organic aqueous
extractsa extractsa

(1990), Hutchings (1996),

Van Wyk et al. (2000)
Trichilia emetica Vahl., Leaves or fruits used as poultice for Van Wyk et al. (2011) Leaf WSBGb UM169 13.6 14.2
Meliaceae bruises and eczema
b
Vernonia natalensis Sch. Roots or leaves combined with Momordica Hutchings (1996) Leaf WSBG UM170 10.5 4.0
Bip. ex Walp., foetida and Pittosporum viridiflorum in a
Asteraceae decoction to treat boils
Roots WSBGb UM180 10.2 6.6
Viscum capense L.f., Whole plant applied externally to treat Hutchings (1996) Leaf Magaliesburg UM119 15.4 9.1
Santalaceae warts and other skin complaints
Warburgia salutaris (G. Leaf and stalk lotion in combination with Hutchings (1996) Bark WSBGb UM181 7.9 5.1
Bertol.) Chiov., Hibiscus surattensis, used as an anti-
Canellaceae inflammatory and to treat sores and skin
irritation
Leaf WSBGb UM154 15.8 7.6
Zantedeschia aethiopica Leaf applied directly to the skin to treat Watt and Breyer- Leaf WSBGb UM136 20.4 7.6
Spreng., Araceae wounds, boils and sores Brandwijk (1962), Van
Wyk et al. (2000)
Ziziphus mucronata Leaf, root or bark decoction applied Watt and Breyer- Bark Pretoria–Villieria 10.0 8.6
Willd., Rhamnaceae topically to treat boils, sores and swellings Brandwijk (1962), UM126
Hutchings (1996), Van
Wyk et al. (2000, 2011)
Leaf Pretoria–Villieria 10.6 21.9
UM127

a
Percentage yield expressed for organic (dichloromethane: methanol, 1:1 v/v) and aqueous extracts per dry weight of grounded plant material weighed.
b
Walter Sisulu Botanical Garden, Johannesburg, South Africa.
c
University of the Witwatersrand Medicinal Garden, Johannesburg, South Africa.
d
Pretoria National Botanical Garden, Pretoria, South Africa.

Serial dilutions were performed on each plate, and thereafter the
cultures (sub-cultured 1:100 in suitable broth) with an approx-
imate inoculum size of 1  106 colony forming units/ml (CFU/ml)
were introduced. A volume of 100 μl of the culture was added to all
the wells. Tests were performed at least in duplicate. Each plate was
subsequently sealed with a sterile adhesive sealing film. All micro-titre
plates were incubated in suitable conditions as detailed in Section
2.3.1. When testing for antimicrobial properties of plant extracts
against the more fastidious pathogens such as Propionibacterium
acnes, Trichophyton mentagrophytes and Microsporum canis, modifica-
tions to the standard MIC method were undertaken. Micro-titre plates
for Propionibacterium acnes were incubated without the sterile adhe-
sive seal film on the micro-titre plates. When testing the dermato-
phytes, the growth indicator was added to the micro-titre plate prior
to incubation (Masoko et al., 2007).
When testing for bacteria and the yeast, after incubation, 40 ml
(0.04% w/v) of p-iodonitrotetrazolium chloride (INT) (Sigma
Aldrich) was added as an indicator for microbial growth to each
well of the micro-titre plates. The minimum inhibitory concentra-
tion was defined as the lowest concentration of the test sample
where there is no visible microbial growth.
Positive and negative controls were included in each essay.
Positive controls included ciprofloxacin and amphotericin B dis-
playing antimicrobial efficacy towards bacteria and fungi, respec-
tively. Negative controls included acetone or DMSO (final starting
concentration of 12.5% v/v) to ascertain if any growth inhibition
was attributed to the solvent and culture control (pathogen
growing independently). The culture growing independently was
used to monitor viability and as a comparative standard when
reading MIC values after INT was added.
2.4. Interactive combination studies
The synergistic, additive or antagonistic interaction between
plants reported to be used in combination were investigated using
two approaches. First, plant extracts with a starting concentration
of 64 mg/ml were mixed in 1:1 ratios. The MIC values were
determined for each combination to establish the interaction and
the sum of the fractional inhibitory concentration (ƩFIC) was
calculated for each combination using the following equation;
MIC ðaÞ in combination with ðbÞ
FIC ðiÞ ¼
MIC ðaÞ independently
MIC ðbÞ in combination with ðaÞ
FIC ðiiÞ ¼
MIC ðbÞ independently
(i) and (ii) in this study represents the different plants in
combination. The sum of the FIC, known as the FIC index was
thus calculated as ƩFIC¼FIC(i)+FIC(ii). This may be classified as
either synergistic (≤0.50), additive (0.50–1.00), indifferent
(41.00–4.00) or antagonistic (44.00) (Van Vuuren and Viljoen,
2008).
Combinations with notable interactions were further investi-
gated at various ratios against a selection of the pathogens. The
MIC assay was conducted on nine ratios i.e. 9:1; 8:2; 7:3; 6:4; 5:5;
4:6; 3:7; 2:8; 1:9 of the plants in combination. The results were
then plotted on an isobologram using GraphPad Prisms software
(Version 5), allowing for a figurative representation of the inter-
actions. The isobolograms were interpreted by examining the data
points of the ratios where the MIC for each concentration is
determined in relation to the independent MICs. Data points
falling below or on the 0.50 line on the isbolologram were
interpreted as synergistic. Points between 0.50 and/or on the
1.00 line were interpreted as additive and points 41.00–≤4.00
line were defined as either non-interactive or antagonistic ( 44.0)
(Van Vuuren and Viljoen, 2011). For all assays, positive and
negative controls were included in all repetitions (Section 2.3.2).
Assays were undertaken at least in duplicate and the mean
values noted.
 50
Table 2
Screening of plant extracts from South African medicinal plants for antimicrobial activity against common skin pathogens (MIC recorded in mg/ml).

c d
Plant samples Staphylococcus MRSA GMRSA Staphylococcus Pseudomonas Candida Brevibacillus Propionibacterium Trichophyton Microsporum
aureus ATCC 43300 ATCC 33592 epidermidis aeruginosa albicans agri acnes mentagrophytes canis
ATCC 25923 ATCC 2223 ATCC 27858 ATCC 10231 ATCC 51663 ATCC 11827 ATCC 9533 ATCC 36299

a a a a a a
D:M Aq D:M Aq D: Aq D:M Aq D: Aq D:M Aq D:M Aq D:M Aq D:M Aq D:M Aq
M M

Acacia erioloba barkb 0.50 4.00 1.00 2.00 1.00 4.00 1.00 4.00 2.00 8.00 1.00 2.00 0.50 2.00 0.20 0.25 1.00 8.00 1.00 1.00
Acacia erioloba leaf 1.00 4.00 2.00 4.00 2.00 4.00 2.00 8.00 2.00 16.00 2.00 2.00 0.50 8.00 0.20 0.50 0.50 8.00 2.00 8.00
Acokanthera oppositifolia leaf 0.75 16.00 4.00 8.00 4.00 4.00 4.00 416.00 1.50 416.00 2.00 16.00 0.50 16.00 4.00 2.00 2.00 416.00 2.00 1.00
Aloe arborescens leaf 2.00 4.00 2.00 4.00 1.00 4.00 1.00 4.00 1.00 416.00 1.00 416.00 2.00 16.00 0.50 4.00 0.25 8.00 16.00 8.00
Athrixia phylicoides leafb 1.00 4.00 2.00 4.00 2.00 4.00 2.00 8.00 2.00 4.00 2.00 4.00 3.00 8.00 2.00 2.00 1.00 416.00 1.00 416.00
Aristea ecklonii leaf 0.20 2.00 0.20 4.00 0.20 4.00 0.10 2.00 0.20 4.00 0.30 8.00 8.00 0.75 0.05 1.50 0.05 1.00 16.00 2.00
Aristea ecklonii roots 0.01 2.00 0.05 1.00 0.05 1.00 0.05 1.00 0.20 1.00 0.05 4.00 2.00 2.00 0.03 4.00 0.03 0.50 4.00 1.00
Bauhinia macranthera leaf 2.00 16.00 2.00 416.00 2.00 416.00 2.00 416.00 0.50 416.00 2.00 416.00 4.00 4.00 0.50 1.00 1.00 4.00 2.00 2.00
Boophane disticha leaf 4.00 416.00 2.00 416.00 2.00 416.00 0.50 416.00 1.00 416.00 0.50 16.00 8.00 416.00 0.50 1.00 2.00 16.00 2.00 416.00

U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55

Bridelia micrantha bark 2.00 8.00 2.00 6.00 2.00 4.00 2.00 8.00 2.00 416.00 4.00 4.00 4.00 2.00 1.00 0.25 2.00 416.00 2.00 2.00
Bridelia micrantha leaf 2.00 8.00 1.00 4.00 1.00 16.00 2.00 16.00 2.00 416.00 2.00 16.00 2.00 0.50 1.00 1.00 1.00 8.00 1.00 4.00
Chenopodium ambrosioides leaf 0.80 4.00 0.25 8.00 0.50 8.00 0.50 16.00 0.25 416.00 2.00 8.00 0.50 8.00 0.40 2.00 0.25 2.00 4.00 4.00
Cissampelos capensis leaf 2.00 416.00 4.00 416.00 2.00 416.00 2.00 416.00 2.00 416.00 2.00 8.00 1.00 416.00 0.25 0.50 1.00 2.00 1.00 8.00
Cotyledon orbiculata leaf 1.50 416.00 4.00 416.00 1.00 416.00 0.38 416.00 0.50 416.00 0.25 416.00 4.00 12.00 0.25 16.00 2.00 416.00 1.00 8.00
Dicoma anomala tuberb 0.50 4.00 0.50 8.00 0.50 8.00 0.50 8.00 8.00 8.00 2.00 8.00 4.00 8.00 4.00 16.00 0.03 4.00 4.00 8.00
Dioscorea dregeana tuberb 2.00 416.00 416.00 416.00 1.00 416.00 1.25 416.00 2.00 416.00 2.00 416.00 0.25 416.00 2.00 2.00 2.00 4 416.00 4.00 416.00
Diospyros mespiliformis leaf 1.00 1.75 1.00 4.00 1.00 2.00 1.00 4.00 1.00 2.00 1.00 8.00 0.50 0.50 0.05 2.00 0.10 4.00 0.50 4.00
Dodonaea angustifolia leafb 1.60 0.50 0.50 1.00 1.60 1.00 4.00 4.00 2.00 416.00 4.00 4.00 1.00 3.00 2.00 4.00 0.50 2.00 2.00 4.00
Ekebergia capensis bark 1.00 4.00 1.00 2.00 2.00 4.00 0.38 2.00 0.75 16.00 1.00 2.00 2.00 16.00 1.00 4.00 1.00 8.00 1.00 8.00
Ekebergia capensis leaf 0.50 416.00 8.00 6.00 0.50 8.00 0.50 8.00 1.00 16.00 1.00 16.00 4.00 2.00 1.00 4.00 2.00 4.00 2.00 4.00
Elephantorrhiza elephantina leaf 0.50 16.00 1.00 8.00 0.50 8.00 0.38 16.00 1.00 12.00 1.00 16.00 2.00 416.00 1.00 0.25 2.00 416.00 16.00 8.00
b
Elephantorrhiza elephantine roots+rhizomes 0.50 2.00 0.50 1.00 0.50 2.00 1.00 4.00 2.00 4.00 1.00 4.00 0.50 0.50 0.05 2.00 1.00 4.00 0.50 4.00
Embelia ruminate leaf 2.00 3.00 1.50 4.00 1.00 0.25 0.38 416.00 0.75 8.00 1.00 0.40 4.00 416.00 1.00 416.00 4.00 416.00 2.00 8.00
Erythrina lysistemon leaf 0.20 8.00 0.20 8.00 0.20 8.00 0.20 8.00 0.20 416.00 2.00 16.00 8.00 16.00 0.08 0.25 1.00 8.00 2.00 16.00
Eucalyptus camaldulensis barkb 0.50 0.63 0.50 0.50 0.25 1.00 0.50 2.00 2.00 4.00 0.50 2.00 0.25 0.20 0.10 2.00 1.00 1.00 4.00 2.00
Ficus natalensis leaf 0.25 4.00 0.25 2.00 0.50 4.00 4.00 4.00 4.00 416.00 2.00 8.00 2.00 4.00 8.00 1.00 0.50 4.00 4.00 4.00
Ficus sur bark 0.75 416.00 1.00 416.00 1.25 416.00 2.00 416.00 2.00 416.00 8.00 416.00 8.00 416.00 2.00 0.25 1.00 8.00 416.00 416.00
Ficus sur leaf 4.00 416.00 2.00 416.00 4.00 416.00 4.00 416.00 1.00 416.00 2.00 4.00 2.00 416.00 4.00 0.25 0.25 16.00 1.00 4.00
Gunnera perpensa leaf 0.40 0.50 0.25 1.00 0.20 1.00 0.25 2.00 1.00 8.00 0.50 1.60 0.38 0.10 0.03 2.00 0.03 0.25 1.00 1.00
Gunnera perpensa rhizomesb 0.50 4.00 8.00 4.00 2.00 4.00 2.00 8.00 2.00 8.00 2.00 0.50 4.00 4.00 0.25 1.00 1.00 8.00 4.00 16.00
Halleria lucida leaf 0.40 0.50 0.25 1.00 0.40 2.00 1.00 4.00 0.50 8.00 4.00 4.00 1.00 1.00 0.38 1.00 1.00 16.00 2.00 2.00
Halleria lucida stem 0.25 2.00 1.00 8.00 0.50 4.00 2.00 16.00 2.00 8.00 2.00 8.00 1.00 8.00 2.00 0.25 2.00 416.00 2.00 8.00
Harpephyllum caffrum bark 0.40 1.00 0.50 0.25 0.50 0.25 0.50 1.00 0.25 2.00 1.00 0.25 0.50 0.50 0.18 0.50 0.50 2.00 1.00 4.00
Hypericum perforatum leaf 0.50 1.00 6.00 0.50 4.00 1.00 0.13 1.00 0.50 4.00 1.00 0.40 1.00 1.00 1.00 0.50 1.00 8.00 1.00 4.00
Ilex mitis bark 4.00 416.00 4.00 6.00 2.00 6.00 3.00 8.00 1.50 8.00 6.00 8.00 4.00 4.00 4.00 2.00 4.00 2.00 4.00 16.00
Ilex mitis leaf 4.00 8.00 8.00 8.00 4.00 8.00 2.00 8.00 2.00 16.00 4.00 8.00 3.00 4.00 3.00 1.00 2.00 8.00 1.00 416.00
Kigelia africana fruit 4.00 16.00 4.00 416.00 4.00 416.00 1.50 416.00 2.00 16.00 1.00 416.00 1.00 416.00 1.00 2.00 4.00 16.00 8.00 416.00
Lantana rugosa leaf 2.00 4.00 2.00 4.00 2.00 8.00 1.50 8.00 2.00 416.00 3.00 8.00 0.50 4.00 0.50 1.00 0.05 4.00 2.00 4.00
Lannea discolor leaf 2.00 16.00 1.00 16.00 2.00 4.00 2.00 16.00 1.00 12.00 2.00 8.00 1.00 4.00 1.00 1.00 0.05 16.00 4.00 2.00
Malva parviflora leaf 0.50 8.00 2.00 4.00 0.50 4.00 2.00 416.00 1.00 416.00 2.00 16.00 4.00 416.00 8.00 0.25 0.05 4.00 416.00 416.00
Melianthus comosus leaf 0.40 1.60 0.50 0.25 0.25 0.25 0.25 0.25 0.10 2.00 0.50 0.25 0.25 2.00 0.10 0.25 0.05 0.50 0.50 1.00
Melianthus major leaf 1.00 0.50 2.00 0.50 1.00 0.50 2.00 1.00 1.25 2.00 0.50 4.00 0.25 2.00 0.10 1.00 0.05 1.00 0.50 4.00
Mentha longifolia leaf 1.00 2.00 1.00 4.00 2.00 4.00 1.00 8.00 2.00 4.00 2.00 8.00 2.00 4.00 0.50 1.00 0.80 2.00 1.00 2.00
Opuntia ficus-indica leaf 16.00 416.00 16.00 416.00 8.00 416.00 4.00 416.00 4.00 416.00 4.00 416.00 8.00 416.00 4.00 2.00 2.00 16.00 8.00 16.00
Pellaea calomelanos leaf 0.75 4.00 2.00 8.00 0.50 4.00 0.02 4.00 0.75 8.00 0.50 12.00 4.00 2.00 0.50 4.00 1.00 8.00 2.00 4.00
Pellaea calomelanos rhizomes 3.00 416.00 0.25 416.00 2.00 416.00 2.50 416.00 1.00 416.00 4.00 416.00 4.00 416.00 4.00 2.00 2.00 8.00 8.00 16.00
Pentanisia prunelloides root barkb 4.00 8.00 4.00 16.00 8.00 416.00 4.00 416.00 8.00 416.00 8.00 8.00 4.00 8.00 1.00 4.00 2.00 416.00 2.00 16.00
Pentanisia prunelloides roots strippedb 4.00 4.00 4.00 8.00 8.00 4.00 8.00 16.00 8.00 416.00 8.00 16.00 2.00 4.00 0.50 4.00 4.00 4.00 1.00 8.00
Pittosporum viridiflorum leafb 4.00 4.00 8.00 8.00 8.00 4.00 8.00 4.00 8.00 416.00 2.00 2.00 2.00 8.00 8.00 0.25 0.50 1.00 1.00 1.00
Rauvolfia caffra leaf 2.00 8.00 4.00 8.00 4.00 4.00 4.00 16.00 2.00 416.00 4.00 416.00 0.50 4.00 4.00 2.00 2.00 8.00 1.00 1.00
 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55 51

2.5. Bioactivity-guided isolation of Aristea ecklonii leaf material

1.00
8.00
8.00
4.00

8.00
8.00
16.00
2.00
4.00
8.00
2.00
4.00
4.00 416.00
12.50
A combination of column chromatography (CC) and high speed
≥16. 00

counter-current chromatography (HSCCC) (Dynamic Extractions

1.00

1.00
4.00

2.00

2.00
4.00
4.00

2.00
4.00

2.00
0.50

12.50
Ltd.) was used to fractionate the extract and purify the bio-active
compound. Column chromatography fractionation was achieved
8.00
4.00
2.00

16.00
16.00
16.00
8.00
8.00
16.00
4.00
4.00
416.00
25.00
0.03

by gradient elution, which involved eluting the column with

varying polarities of a DCM and methanol mixture (100:0; 95:5;
90:10). A two phase solvent system for HSCCC fractionation was
prepared containing n-hexane, ethyl acetate, methanol and dis-
1.00

1.00
1.00
1.00
1.00
2.00
4.00

2.00
2.00
25.00
0.03

0.03
0.50
1.50

tilled water (8:8:5:5, v/v/v/v). The sample solution for HSCCC

separation was prepared by dissolving approximately 120 mg of

GMRSA gentamycin methicillin resistant Staphylococcus aureus; written in bold are noteworthy activities; MIC values for negative controls (acetone or DMSO) were 8:00-16:00.
the dry extract of Fraction, F3 from CC separation into 2 ml of the
1.00

1.00

1.00
4.00
4.00
2.00

2.00

4.00
2.00
0.25

0.50

0.50

0.50
1.25

solvent mixture consisting equal volumes of the two phases. Thin

layer chromatography (TLC) was used to monitor the chemical
profile of the fractions obtained from the CC and HSCCC. Auto-
8.00 1.00

8.00 1.00

8.00 1.00
2.00 8.00
3.00 16.00 416.00 8.00
16.00 4.00

416.00 4.00 416.00 2.00 416.00 4.00

8.00 2.00

16.00 4.00

416.00 4.00 416.00 2.00 416.00 8.00

8.00 0.03
416.00 2.00 416.00 4.00 416.00 0.04

2.00 0.20

0.16 1.25

biography assays (Van Vuuren et al., 2006) were used as a guide to

identify the active antimicrobial compound.
The purity of the isolated compound was monitored with ultra-
high performance liquid chromatography (UHPLC) (Waters™),
coupled to a photo diode array detector. An injection volume of
4.00 1.00

416.00 2.00 416.00 1.00

8.00 1.00

4.00 1.00
8.00 8.00

416.00 0.50 416.00 2.00

16.00 2.00
2.00 8.00
0.80 0.50

1.25 0.16

1 μl was applied and the column temperature was adjusted to

40 1C. The mobile phase consisted of (A) 0.5% acetic acid and
(B) acetonitrile at a flow rate of 0.3 ml/min. Gradient elution was
employed, starting with 90% A and 10% B, changing to 50% B in
4.00 1.00
416.00 2.00
416.00 8.00

416.00 16.00
416.00 4.00

416.00 2.00

16.00 4.00
4.00 0.50

1.25 1.25

15 min, then changing to 100% B in 1 min, with a post-run time of

1 min.
Structure elucidation was undertaken using nuclear magnetic
resonance (NMR). The NMR spectra were recorded on a Bruker
600 Avance II NMR at 600 MHz for 1H NMR and 150 MHz for 13C
1.00

1.00
4.00
2.00

4.00
8.00
4.00
0.50
0.25
0.03

0.50

0.50
0.31
0.10

and distortionless enhancement through polarisation transfer

(DEPT) NMR.
8.00
416.00
416.00

416.00
416.00
8.00
416.00
8.00
16.00
4.00
2.00
416.00
0.25

1.25

Prior to fractionation of Aristea ecklonii, a bulk sample was

purchased. Leaf material was dried, ground (50 g) and serially
extracted using 500 ml (  10) of D:M (1:1; V/V).
1.00
4.00

8.00
6.00
4.00

1.00
4.00
2.00
0.25
0.20

0.50
1.50
0.50

0.47

The micro-titre plate dilution assay as detailed in Section 2.3.2,

was used to confirm the antimicrobial effects of Aristea ecklonii
4.00
416.00
416.00

416.00
416.00
6.00
8.00
4.00
8.00
416.00

8.00
0.25

0.25

0.63

crude extract, fractions from CC and the isolated compound

against Staphylococcal species, Pseudomonas aeruginosa and Can-
dida albicans.
Plant sample dissolved in DMSO where the crude extracts were insoluble in acetone.
1.00

1.00
2.00
4.00

4.00
8.00
4.00

4.00
2.00
0.80
0.40

0.50
0.50

0.55
4.00
12.00
416.00

416.00
416.00
8.00
416.00
4.00
4.00
4.00

8.00
0.25

0.25

1.25

3. Results and discussion

1.00
4.00
8.00

8.00
6.00
4.00

2.00
4.00
2.00
2.00
0.50
0.40

0.50

0.83

3.1. Antimicrobial screening

4.00
416.00
416.00

416.00
416.00
4.00
4.00
4.00
4.00
8.00

4.00
0.25
1.60

1.25

The antimicrobial activities of the plant extracts against der-

matologically relevant pathogens are shown in Table 2. Generally,
activity varied greatly depending on the pathogen studied, but a
1.00
8.00

4.00
4.00
2.00

4.00
0.50

0.50

0.40

0.40
0.80

0.50
1.60

0.69

MRSA methicillin resistant Staphylococcus aureus.

few plant species (as detailed hereafter) showed interesting

results specifically as the positive antimicrobial efficacy had a
Ciprofloxacin/amphotericin B positive control

direct correlation to the traditional use. Aristea ecklonii is used

traditionally to treat shingles (Ngwenya et al., 2003) (Table 1).
Other antimicrobial-related uses not specific to skin diseases have
D:M dichloromethane: methanol.
Scadoxus puniceus rhizomes+rootsb

been reported i.e. the treatment of fevers, coughs as well as the

use for syphilis (Hutchings, 1996). This prompted the investigation
against other related skin pathogens with the hypothesis that
Zantedeschia aethiopica leaf
Ziziphus mucronata barkb
Rothmannia capensis leaf

Vernonia natalensis roots

Warburgia salutaris bark

Aristea ecklonii may offer other additional antimicrobial efficacies

Terminalia sericea rootsb

Vernonia natalensis leafb

Warburgia salutaris leaf

Ziziphus mucronata leaf

Solanum incanum leaf

not previously documented. The current findings demonstrate

Trichilia emetica leaf

Viscum capense leaf

noteworthy antimicrobial properties for Aristea ecklonii, where

organic and aqueous extracts of leaf and root displayed the
greatest antimicrobial effect (lowest MIC value of 0.01 mg/ml for
(μg/ml)

D:M against Staphylococcus aureus). The antifungal properties of

Aristea ecklonii against plant pathogens have been reported by
d
b
a

Pretorius et al. (2002), where 100% antifungal inhibition was

52 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55
observed against all strains tested. This is the first report for
efficacy against skin pathogens.
Chenopodium ambrosioides has been reported for its traditional
use to treat eczema, wounds and skin infections (Hutchings, 1996;
Pesewu et al., 2008). The organic extract of Chenopodium ambro-
sioides exhibited noteworthy antimicrobial activity against both
the tested bacteria and Trichophyton mentagrophytes (MIC values
between 0.25 mg/ml and 0.80 mg/ml). The broad-spectrum anti-
microbial efficacy of Chenopodium ambrosioides was corroborated
by earlier findings noted by Suliman et al. (2010).
Dicoma anomala has been used traditionally as a paste to treat
wounds, ulcers, ringworm and head sores (Hutchings, 1996;
Felhaber, 1997). The current findings show noteworthy antimicro-
bial activities of the organic extract against Staphylococcal species
and Trichophyton mentagrophytes with a MIC value of 0.50 mg/ml
and 0.03 mg/ml, respectively. The activity towards the dermato-
phyte gives some validation for its traditional use to treat ring-
worm infections. Steenkamp et al. (2004) demonstrated similar
antimicrobial effects against Staphylococcus aureus and Pseudomo-
nas aeruginosa of Dicoma anomala, however, no previous reports
could be found where efficacy was tested on Trichophyton menta-
grophytes and Microsporum canis.
Diospyros mespiliformis has been reported for its use to treat a
variety of skin ailments such as scars, skin rashes, bruises, wounds
and ringworm (Von Koenen, 1996; Van Wyk et al., 2011) (Table 1).
The organic extract demonstrated MIC values between 0.05 mg/ml
and 1.00 mg/ml. The antimicrobial effects of this plant against the
dermatophytes give some validation to the traditional use espe-
cially for treating ringworm as it demonstrated noteworthy anti-
microbial effects against Trichophyton mentagrophytes (MIC
0.10 mg/ml) and Microsporum canis (MIC 0.50 mg/ml). The
broad-spectrum antimicrobial effects of Diospyros mespiliformis
have also been confirmed by Adeniyi et al. (1996).
Decoctions or infusions of root or rhizome and leaf of Gunnera
perpensa are traditionally used as a dressing for wounds and for
psoriasis (Table 1). The organic extract of Gunnera perpensa leaf
exhibited antimicrobial activity (MIC values between 0.03 mg/ml
and 1.00 mg/ml) against the tested pathogens, with similarities in
antimicrobial efficacy noted for the aqueous extract against
Staphylococcus aureus and respective resistant strains (MIC values
between 0.50 mg/ml and 1.00 mg/ml). Therefore, the findings
corroborate with the traditional use as an antiseptic and dressing
for wounds. Findings were similar to those reported in literature
(Steenkamp et al., 2004; Drewes et al., 2005; Buwa and Van
Staden, 2006; Nkomo and Kambizi, 2009), however, this report
provides new evidence for efficacy against Propionibacterium
acnes, Brevibacillus agri, Trichophyton mentagrophytes and Micro-
sporum canis.
Terminalia sericea is used for a variety of ailments (Table 1) and
the antimicrobial activity has been extensively studied especially
for conditions associated with diarrhoea and respiratory ailments
(Eloff, 1999; Fyhrquist et al., 2002; Steenkamp et al., 2004; Eldeen
et al., 2005; Masoko et al., 2005; Moshi and Mbwambo, 2005;
Tshikalange et al., 2005; Eldeen and Van Staden, 2007; Suliman
et al., 2010). In the current study, Terminalia sericea exhibited
mostly noteworthy broad-spectrum antimicrobial effects against
skin relevant pathogens, hence supporting its use for dermatolo-
gically related ailments.
Warburgia salutaris is traditionally used for a variety of ailments
(Van Wyk, 2008) and has been known to be combined with
Hibiscus surattensis to treat sores and skin irritations (Table 1).
The organic bark extract displayed noteworthy antimicrobial
activity presenting with MIC values between 0.03 mg/ml and
1.00 mg/ml against tested pathogens, with exception of Breviba-
cillus agri and Microsporum canis. It is worth noting that the bark
only had a better overall effect for the organic extracts when
compared to the leaf samples. As the traditional use is usually
aqueous by nature, the possibility of substitution of leaf material
for bark may be warranted. This could possibly protect this plant
species, which is rapidly dwindling in numbers in the wild. Similar
noteworthy activity for Warburgia salutaris methanol extract
against Staphylococcus aureus has been reported by Rabe and
Van Staden (1997).
The organic extracts of medicinal plants reported to be tradi-
tionally used (Table 1) to treat acne vulgaris and pimples
(Elephantorrhiza elephantina, Ekebergia capensis, Eucalyptus camal-
dulensis and Harpephyllum caffrum) displayed noteworthy activity
against the relevant pathogen Propionibacterium acnes with MIC
values between 0.05 mg/ml and 1.00 mg/ml.
3.2. Combination studies
Four different plant combinations were analysed for interactive
properties. The mean MICs and ƩFICs of these combinations against
the six pathogens are presented in Table 3. Plant species incorporated
in the combinations were Dicoma anomala, Elephantorrhiza elephantina
and Pentanisia prunelloides. These plants have also been reported to be
used independently and in combination for a variety of skin ailments
(Table 1). Mostly, non-interactive effects were noted. However, some
interactions worth highlighting are the D:M combination of Dicoma
anomala with Elephantorrhiza elephantina (ƩFIC value of 4.0 bordering
on an antagonistic effect) and the combinations of Pentanisia prunel-
loides (root) with either Elephantorrhiza elephantina or Dicoma anomala
where selective synergistic interactions were observed. One interest-
ing combination was that of Pentanisia prunelloides with Elephantor-
rhiza elephantina (roots). Even though Pentanisia prunelloides (root) did
not exert any noteworthy antimicrobial effects when screened inde-
pendently, synergistic interactions were noted when the aqueous
extract of the plant was combined with Elephantorrhiza elephantina
(root and rhizome), presenting with a mean ƩFIC value of 0.39.
Considering that the traditional use of plants in combination is not
an exact science (where formulations are accurately measured to the
exact mg or mg quantity), this combination was combined in various
ratios to determine if variations of the concentration of the two plants
in the mixture may result in different interactions (Fig. 1). No
antagonistic interactions were observed and several synergistic inter-
actions were predominant for the aqueous extracts at varying ratios
against Staphylococcus aureus, GMRSA and Staphylococcus epidermidis.
Furthermore, more favourable interactions were observed for the
aqueous extracts, irrespective of the ratio at which these two plants
are combined. It was also worth noting that the 1:1 combinations of
the aqueous extracts demonstrated the most synergistic interactions,
lending some credibility to the traditional use of water preparations
for medicinal purposes. While examining the aqueous and organic
extract ratio combinations of Pentanisia prunelloides and Elephantor-
rhiza elephantina in more depth, the enhanced efficacy of the
combination was mainly attributed to higher Elephantorrhiza elephan-
tina concentrations.
3.3. Antimicrobial activity and bioactivity-guided isolation of Aristea
ecklonii leaf
A bio-autographic assay of the crude extract indicated the
compound with RF 0.72 to be the most active against the tested
bacterial strains, thus it was targeted for isolation. This compound
had low polarity and was extracted from the dichloromethane
phase during the liquid–liquid partitioning of the crude extract.
This dichloromethane phase (2.31 g) also had lower MIC values
(20–78 μg/ml) than that of the aqueous phase.
From column chromatography, Fraction F3 (120 mg) containing
mainly plumbagin was then purified using HSCCC to obtain 35 mg
of the compound. The isolated compound appeared as orange
 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55 53

Table 3
Average MIC (expressed in mg/ml) and ∑FIC values for 1:1 plant combinations.

a b
Combinations Staphylococcus MRSA GMRSA Staphylococcus Pseudomonas Candida MIC and ƩFIC
aureus ATCC ATCC 43300 ATCC 33592 epidermidis ATCC aeruginosa ATCC albicans ATCC mean values
25923 2223 27858 10231

c d c d c d c d c d c d c d
D:M Aq D:M Aq D:M Aq D:M Aq D:M Aq D:M Aq D:M Aq

Elephantorrhiza elephantina+Dicoma MIC 1.00 4.00 2.00 4.00 1.00 4.00 1.00 8.00 2.00 8.00 2.00 8.00 1.50 6.00
anomala tuber
ƩFIC 2.00 2.50 4.00 1.25 2.00 0.75 1.50 1.50 0.63 1.50 2.13 1.00 2.04 1.42
Elephantorrhiza elephantina+Pentanisia MIC 1.00 1.00 2.00 1.00 0.50 1.00 1.00 2.00 2.00 416.00 2.00 2.00 1.42 1.40
prunelloides root
ƩFIC 1.13 0.38 2.25 0.56 0.53 0.38 0.56 0.31 0.63 ND 1.13 0.31 1.04 0.39
Pentanisia prunelloides root bark+Dicoma MIC 1.00 4.00 1.00 8.00 1.50 416.00 1.00 16.00 8.00 416.00 4.00 8.00 2.75 9.00
anomala tuber
ƩFIC 1.13 0.75 1.13 0.75 1.69 ND 1.13 1.50 1.00 ND 2.13 1.00 1.37 1.00
Pentanisia prunelloides root+Dicoma MIC 1.00 8.00 1.00 2.00 1.00 8.00 1.00 4.00 4.00 8.00 4.00 16.00 2.00 7.67
anomala tuber
ƩFIC 1.13 2.00 1.13 0.38 1.06 1.50 1.06 0.38 0.50 0.75 1.56 3.00 1.07 1.34

a
MRSA methicillin-resistant Staphylococcus aureus.
b
GMRSA gentamycin–methicillin-resistant Staphylococcus aureus.
c
D:M dichloromethane: methanol (1:1).
d
Aq aqueous extracts; ND ƩFIC index not determined as MIC values 416.00 mg/ml and excluded in mean values.

S. aureus GMRSA
2.00 ATCC 25923 combination with E. elephantina /MIC 1.50 ATCC 33592
combination with E. elephantina /MIC

1.75
1.25
P. prunelloides independently
P. prunelloides independently

MIC P. prunelloides in
MIC P. prunelloides in

1.50
1.00
1.25

1.00 0.75

0.75
0.50
0.50
0.25
0.25

0.00 0.00
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 0.00 0.25 0.50 0.75 1.00 1.25 1.50
MICP. prunelloides in MICP. prunelloides in
combination with E. elephantina/MIC combination with E. elephantina/MIC
E. elephantina independently E. elephantina independently

S. epidermidis C. albicans
1.50 ATCC 2223 3.5 ATCC 10231
combination with E. elephantina /MIC

combination with E. elephantina /MIC

1.25 3.0
P. prunelloides independently

P. prunelloides independently
MIC P. prunelloides in

MIC P. prunelloides in

2.5
1.00
2.0
0.75
1.5
0.50
1.0

0.25 0.5

0.00 0.0
0.00 0.250.50 0.75 1.00 1.25 1.50 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
MIC P. prunelloides in MICP. prunelloides in
combination with E. elephantina/MIC combination with E. elephantina/MIC
E. elephantina independently E. elephantina independently

Fig. 1. Isobologram representation of Pentanisia prunelloides and Elephantorrhiza elephantina combination against Staphylococcus aureus, GMRSA, Staphylococcus epidermidis
and Candida albicans. Ratio combinations for aqueous extracts ; ratio combinations for D:M extracts ; 1:1 combination for aqueous and organic extracts .

needle-like crystals at room temperature (25 1C). Ultra high After structure elucidation of the compound using comprehen-
performance liquid chromatography analysis of the compound sive 1 and 2D 1H and 13C NMR, this compound was identified
indicated a purity of 99% and a maximum absorption wavelength as plumbagin, which is a naphthoquinone. Isolated plumbagin
(ʎmax) of 267 nm. showed the following NMR signals; 1H NMR (CDCl3) δ: 6.85 (1H, s,
54 U. Mabona et al. / Journal of Ethnopharmacology 148 (2013) 45–55

Table 4
Minimum inhibitory concentrations of Aristea ecklonii crude (bulk) extract, fractions and plumbagin compound (MIC recorded in μg/ml).

Plant fractions Staphylococcus MRSA GMRSA Staphylococcus Pseudomonas Candida

aureus ATCC ATCC epidermidis aeruginosa albicans
ATCC 25923 43300 33592 ATCC 2223 ATCC 27858 ATCC 10231

Crude extract 156.00 156.00 156.00 78.00 156.0 313.00

DCM fraction 39.00 78.00 39.00 20.00 78.00 78.00
Fraction F3 20.00 39.00 20.00 80.00 20.00 39.00
Plumbagin (compound) 8.00 16.00 16.00 4.00 8.00 2.00
Ciprofloxacin/amphotericin B positive 0.31 0.63 0.31 0.31 0.31 2.00
control
Acetone negative control 16  103 416  103 416  103 416  103 16  103 416  103

H-3), 7.24 (1H, d, j¼7.5, 8.4 Hz, H-6), 7.66 (1H, dd, J ¼7.5, 8.4 Hz,
H-7), 7.57 (1H, d, J ¼7.5 Hz, H-8), 2.15 (3H, s, Me-2), 11.97 (–OH);
13
CNMR(CDCl3), δ: 184.6 (C-1), 149.6 (C-2), 135.1 (C-3), 90.3 (C-4);
160.3 (C-5), 123.5 (C-6), 136.0 (C-7), 118.6 (C-8), 132.1 (C-8a), 114.9
(C-4a), 14.9 (2-CH3).
Minimum inhibitory concentrations against a selection of
pathogens for Aristea ecklonii (bulk) crude extract, fraction and
the isolated compound is shown in Table 4. Isolated compounds
which demonstrate MIC values below 100 μg/ml are considered to
have some clinical relevance and an isolated compound is con-
sidered to be of interest when MIC values are below 10 μg/ml (Rios
and Recio, 2005). Thus, the bio-active compound, plumbagin,
displayed noteworthy antimicrobial activity (MIC range between
2.00 μg/ml and 16.00 μg/ml) against the tested pathogens. Further-
more, the antimicrobial effect of plumbagin against Candida albicans
was comparable, having the same MIC value of 2.00 μg/ml to
the commercial antifungal, amphotericin B. The antimicrobial
properties of Aristea ecklonii and chemical constituents against
skin relevant pathogens have not been investigated previously,
however, the presence of plumbagin in both the roots and leaves
of the plants was first reported by Kumar et al. (1985). Results
observed from previous studies on plumbagin demonstrated very
low MIC values (1.56 μg/ml and 0.78 μg/ml against Staphylococcus
aureus and Candida albicans, respectively) (De Paiva et al., 2003).
A diffusion assay by Jeyachandran et al. (2009) on plumbagin
isolated from the root extract of Plumbago zeylanica also showed
noteworthy activity against a variety of pathogens including
Staphylococcus aureus (MIC o1.00 μg/disc) and Pseudomonas aer-
uginosa (MIC 43.00 μg/disc). Plumbagin isolated from Plumbago
species such as Plumbago zeylanica and Plumbago scandens has
been reported to possess anti-carcinogenic and concentration
dependant toxicity in human peripheral blood cells (De Paiva
et al., 2003; Wang et al., 2008; Seshadri et al., 2011; Bothiraja
et al., 2011), which may warrant caution when used in higher
concentrations.
4. Conclusion
When investigating the antimicrobial activities of plants
used for skin infections it was found that most of the plant
extracts demonstrated pathogen specific antimicrobial effects,
with about 8.3% exhibiting broad-spectrum activities against the
tested pathogens. Notable antimicrobial effects for plants such as
Elephantorrhiza elephantina and Diospyros mespiliformis used for
acne vulgaris and ringworm infections, respectively, give some
validation for their reported traditional uses. Synergistic interac-
tions noted for Pentanisia prunelloides combined with Elephantor-
rhiza elephantina validate their antimicrobial effects of aqueous
preparations used in combination. As this plant combination is
traditionally also used as a treatment for eczema, further investi-
gation into the possible additive anti-inflammatory effects is
warranted. Enhanced antimicrobial activities were observed for
the naphthoquinone plumbagin isolated from Aristea ecklonii.
This comprehensive study gives some validation towards the
traditional use of some medicinal plants for the treatment of skin
inflictions. Additionally, selective plants could be targeted for
future study on wound healing proliferation which plays an
important curative role in the overall health of the skin.
Acknowledgements
Financial assistance from Carnegie and Faculty Research Com-
mittee (University of the Witwatersrand) research grants is grate-
fully acknowledged. The authors would like to thank the Faculty of
Health Sciences (University of the Witwatersrand) for assisted
SPARC funding and NRF free standing bursary funding. We would
also like to convey our sincere gratitude to the personnel of Walter
Sisulu Botanical Garden for their assistance in plant collecting.
Also, we are grateful for assistance from Dr Wei Chen (Department
of Pharmaceutical Sciences, Tshwane University of Technology),
for assisting with UPLC and HPTLC analysis.
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