Antimicrobial activity of southern African medicinal plants with dermatological relevance: From an...

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Antimicrobial activity of southern African medicinal plants withdermatological relevance: From an ethnopharmacological screeningapproach, to combination studies and the isolation of a bioactive compound

8283848586878889

Unathi Mabona a, Alvaro Viljoen b, E. Shikanga c, Andrew Marston d, Sandy Van Vuuren a,n

a Department of Pharmacy and Pharmacology, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africab Department of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africac Department of Chemistry, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africad Department of Chemistry, University of the Free State, Bloemfontein 9300, South Africa

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a r t i c l e i n f o

Article history:Received 20 January 2013Received in revised form19 March 2013Accepted 20 March 2013

Keywords:Antimicrobial screeningAristea eckloniiCombinationsCompound isolationDermatophytesSkin

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41/$ - see front matter & 2013 Published by Ex.doi.org/10.1016/j.jep.2013.03.056

viations: Aq, Aqueous extract; CC, column chroe; HSCCC, high speed counter-current chromary concentration; INT, iodonitrotetrazolium chesponding author. Tel.: +27 11 7172157; fax: +ail address: [email protected] (S. V

e cite this article as: Mabona, U., et aan.... Journal of Ethnopharmacology

a b s t r a c t

Ethnopharmacological relevance: Ethnobotanical reports on more than 100 southern African medicinalplants with dermatological relevance have been highlighted, yet there is still limited scientific data tosupport claims for their antimicrobial effectiveness against skin pathogens. Guided by ethnobotanicaldata, this paper explores the antimicrobial efficacies of southern African medicinal plants used to treatskin ailments.Aim of the study: To investigate the antimicrobial properties of southern African medicinal plants againstdermatologically relevant pathogens. The study also aimed at providing a scientific rationale for thetraditional use of plant combinations to treat skin diseases and the isolation of the bioactive compoundfrom the most active species, Aristea ecklonii (Iridaceae).Materials and methods: Organic and aqueous extracts (132) were prepared from 47 plant species andscreened for antimicrobial properties against dermatologically relevant pathogens using the micro-titre platedilution method. Four different plant combinations were investigated for interactive properties and the sum ofthe fractional inhibitory concentration (ƩFIC) calculated. Isobolograms were used to further investigate theantimicrobial interactive properties of Pentanisia prunelloides combined with Elephantorrhiza elephantina atvaried ratios. A bioactivity-guided fractionation process was adopted to fractionate the organic leaf extract ofAristea ecklonii.Results: Plants demonstrating notable broad-spectrum activities (MIC values ≤1.00 mg/ml) againstthe 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 Warburgiasalutaris. The organic extract of Elephantorrhiza elephantina, a plant reportedly used to treatacne vulgaris, demonstrated noteworthy antimicrobial activity (MIC value of 0.05 mg/ml) againstPropionibacterium acnes. Similarly, Diospyros mespiliformis reported for its traditional use to treatringworm, 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 ofPentanisia prunelloides combined (1:1) with Elephantorrhiza elephantina displayed synergisticinteractions (ƩFIC values 0.31–0.38) against Staphylococcus aureus, gentamycin–methicillin resis-tant Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans. Fractionation of Aristeaecklonii resulted in the isolation of the known bioactive 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 fewexhibiting broad-spectrum activities. Positive antimicrobial effects noted for plants such as Elephantor-rhiza elephantina and Diospyros mespiliformis used for acne vulgaris and ringworm infections, respec-tively, give some validation to their reported traditiona l uses. Synergistic interactions noted forPentanisia prunelloides combined with Elephantorrhiza elephantina validate an enhanced antimicrobial

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lsevier Ireland Ltd.

matography; CFU/ml, colony forming units/ml; D:M, 1:1 mixture of dichloromethane and methanol; DMSO, dimethyltography; MIC, minimum inhibitory concentration; NMR, nuclear magnetic resonance; ΣFIC, the sum of the fractionalloride; TSB, Tryptone Soya broth; UHPLC, ultra-high performance liquid chromatography..27 11 6424355.an Vuuren).

l., Antimicrobial activity of southern African medicinal plants with dermatological relevance:(2013), http://dx.doi.org/10.1016/j.jep.2013.03.056i

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U. Mabona et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎2

Please cite this article as: Mabona, U., et aFrom an.... Journal of Ethnopharmacology

effect when used in combination. Noteworthy antimicrobial activities (MIC range between 2.00 μg/mland 16.00 μg/ml) were observed for plumbagin isolated from Aristea ecklonii.

& 2013 Published by Elsevier Ireland Ltd.

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1. Introduction

The readily-available ethnobotanical literature has reportedover 100 medicinal plants that are used in southern Africa fortreating dermatological disorders (Watt and Breyer-Brandwijk,1962; Hutchings, 1996; Von Koenen, 1996; Felhaber, 1997; Rabeand Van Staden, 1997; Van Wyk et al., 2000, 2009). A review byVan Vuuren (2008), on South African medicinal plants, highlightsnumerous 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 onantimicrobial screening against common pathogens such as, Sta-phylococci species, Pseudomonas aeruginosa and Candida albicans,it has been noted that skin dermatophytes such as Trichophytonmentagrophytes and Microsporum canis have been neglected inmost 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 skindiseases such as ringworm infections, it is necessary to include allthe relevant, including fastidious pathogens.

Propionibacterium acnes is an important skin pathogen respon-sible for the chronic inflammatory disease of the sebaceous glandsand hair follicles of the skin. The infection usually results in acnevulgaris, 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 againstacne causing bacteria have been rarely addressed, even thoughattention has been given to this pathogen in other geographicalethnobotanical- relevant studies (Chomnawang et al., 2005, Kimet al., 2007, 2008; Tsai et al., 2010; Balakrishnan et al., 2011).

While many studies have focused on either antimicrobialscreening or the phytochemistry, very little is reported on plant–plant interactions when used in combination, in spite of thetraditional use (Smith, 1895; Hutchings, 1996; Felhaber, 1997).Although some studies have been conducted to evaluate medicinalplant interactions from southern 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 hasbeen sorely neglected.

The identification of bioactive compounds is another importantfactor to be examined to gain insight into the antimicrobialproperties of medicinal plants of dermatological relevance. Pre-viously, a number of antimicrobial related studies have highlightedthe value of identifying the antimicrobial active compound/s (Rabeand 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 plantsaims to present a detailed account of the antimicrobial properties,plant–plant interactive efficacies and isolation of a bioactivenaphthoquinone from one of the most active plant species.

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2. Materials and methods

2.1. Plant collection and identification

Various plant parts (related to traditional use) of 47 differentplant species (representing 38 families) were harvested fromdesignated botanical gardens. Voucher specimens were preparedfor each species and are housed in the Department of Pharmacy

l., Antimicrobial activity of s(2013), http://dx.doi.org/10.1

and Pharmacology, University of the Witwatersrand. Table 1details the plant species collected, reported traditional use, partsof 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 werethen ground to a fine powder using the high speed FritschPulverisette grinder (Labotec). Organic extracts were prepared bysubmerging (720 g) of the dried, crushed plant material in a 1:1mixture of dichloromethane and methanol (D:M) and left on theplatform shaker incubator (Labcon) at 37 1C for 24 h.

Aqueous extracts (Aq) were prepared by submerging themacerated plant material in sterile distilled water, and were thenleft on the platform shaker incubator and kept at ambienttemperature overnight. Thereafter, the liquid extracts were filteredand stored at −80 1C before lyophilisation (Virtis). All extractsamples were stored at room temperature until further use.

2.3. Antimicrobial activity assays

2.3.1. Culture preparationBacterial and fungal test organisms were selected based on

conditions that the plants are reported to treat and on theirprevalence 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 ATCC51663 and anaerobic Propionibacterium acnes ATCC 11827. TheGram-negative bacterium selected for the study was Pseudomonasaeruginosa ATCC 27858. Dermatophytes such as Trichophytonmentagrophytes ATCC 9533, Microsporum canis ATCC 36299 andthe 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, how-ever, was grown in Thioglycolate broth (Oxoid, Ltd) and incubatedunder anaerobic conditions using a candle gas jar for seven days at37 1C.

Dermatophytes, Trichophyton mentagrophytes and Microsporumcanis were grown and maintained on Sabouraud's Dextrose agar(Oxoid, Ltd), incubated at 35 1C for up to seven days in ahumidified environment (Masoko et al., 2007). Candida albicanswas grown in TSB and incubated at 37 1C for 48 h.

2.3.2. Micro-titre plate dilution technique: Minimum inhibitoryconcentration (MIC)

A serial micro-dilution assay was used to quantify the mini-mum inhibitory concentration (MIC) values for plant extractsusing tetrazolium violet reduction as an indicator of growth(Eloff, 1998; NCCLS, 2003). Using aseptic manipulation, 100 μl ofdistilled 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) weretransferred to the first row of the micro-titre plate. The solventDMSO was used when select extracts were insoluble in acetone.Serial dilutions were performed on each plate, and thereafter thecultures (sub-cultured 1:100 in suitable broth) with an approx-imate inoculum size of 1�106 colony forming units/ml (CFU/ml)

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

Plant species Modes of administration and traditionaluses

References Collectedplant part

Collection site andvoucher number

(%) Yield fororganicextractsa

(%) Yield foraqueousextractsa

Acacia erioloba Edgew.,Fabaceae

Wood ash is applied topically for woundhealing

Smith (1996), Von Koenen(1996)

Bark WSBGb UM173 17.7 4.2

Leaf WSBGb UM160 26.8 6.0Acokanthera oppositifolia(Laim.) Codd.,Apocynaceae

Leaf or root pulp is rubbed into thewound, leaf or root is also applied as adressing to swollen parts

Watt and Breyer-Brandwijk (1962),Hutchings (1996),Bethwell (2007)

Leaf WSBGb UM156 20.9 12.9

Aloe arborescens Mill.,Xanthorrhoeaceae

Leaf applied topically to treat wounds,burns and various skin ailments

Bruce (1975), Van Wyket al. (2000)


Athrixia phylicoides DC.,Asteraceae

Plant infusion used to treat sores and boils Hutchings (1996) Leaf Haenertsburg AV999 9.3 4.6

Aristea ecklonii Baker.,Iridaceae

Whole plant preparation applied topicallyfor shingles, also used for the treatment offevers, coughs as well as syphilis

Hutchings (1996),Ngwenya et al. (2003)

Leaf Random HarvestIndigenous nurseryUM163

12.3 5.9

Roots Random HarvestIndigenous nurseryUM164

64.0 11.4

Bauhinia macrantheraBenth. ex Hemsl.,Fabaceae

Leaf extract used to treat wounds Von Koenen (1996) Leaf WSBGb UM155 25.4 20.5

Boophane disticha L.f.,Amaryllidaceae

Bulb preparation applied topically to treatseptic wounds, boils, external sores andrheumatism

Watt and Breyer-Brandwijk (1962), VanWyk et al. (2000)


Bridelia micrantha Baill.,Euphorbiaceae

Bark decoction used to treat burns andwounds

Mabogo (1990), Hutchings(1996), Van Wyk et al.(2011)


Leaf WSBGb UM150 33.4 3.6Chenopodiumambrosioides Bert. exSteud., Chenopodiaceae

Whole plant decoction use to treateczema, wounds and skin infections

Hutchings (1996), Pesewuet al. (2008)

Leaf Near Rayton(Gauteng) B & F 14

3.7 7.1

Cissampelos capensisThunb.,Menispermaceae

Rhizomes, roots and/or leaf paste used forboils, snakebite wound, ulcers and syphilissores

Van Wyk et al. (2000) Leaf Sun valley (Westerncape) UM128

10.6 4.1

Cotyledon orbiculataForssk., Crassulaceae

Externally apply juice for wart removal, orplace the hot leaf directly to the swollenpart of the body also for corns, warts, boils

Watt and Breyer-Brandwijk (1962), Bhatand Jacobs (1995),Felhaber (1997), Van Wyket al. (2000)


Dicoma anomala Sond.,Asteraceae

Charred roots, stems and/or leaf pasteused for wounds, ulcers, ringworm andhead sores. The tuber is used incombination with Elephantorrhizaelephantina to treat acne.

Hutchings (1996),Felhaber (1997)

Tuber WSBGb UM167 13.0 17.6

Dioscorea dregeana T.Durand & Schinz.,Dioscoreaceae

Small piece of root boiled in water andapplied externally used for cuts and sores

Watt and Breyer-Brandwijk (1962), Pujol(1990), Van Wyk et al.(2000)

Tuber WSBGb UM174 6.1 8.8

Diospyros mespiliformisHochst. ex A.DC.,Ebenaceae

Root or leaf decoction used for scars, skinrash, bruises, wounds and ringworm

Von Koenen (1996), VanWyk et al. (2011)


Dodonaea angustifolia L.f., Sapindaceae

Leaves and tips of twigs boiled in water,filtered and applied externally to treatboils; used as a dressing.

Watt and Breyer-Brandwijk (1962), Smith(1996), Van Wyk et al.(2000)

Leaf Pretoria–VillieriaUM125

22.6 7.1

Ekebergia capensisSparrm., Meliaceae

Bark infusion used for abscesses, boils andacne

Pujol (1990), Van Wyket al. (2000, 2011)


Leaf WSBGb UM138 9.3 11.1Elephantorrhizaelephantina (Burch.)Skeels, Fabaceae

Roots and rhizomes boiled in water forexternal use to treat acne and other skindiseases. Also used in combination withPentanisia prunelloides to treat eczema

Pujol (1990), Felhaber(1997), Van Wyk et al.(2000)


Roots andrhizomes

WSBGb UM172 5.0 8.9

Embelia ruminate (E.Mey. ex A.Dc.) Mez,Myrsinaceae

Leaf paste used to treat open wounds andleprosy

Kumara Swamy et al.(2007)


Erythrina lysistemonHutch., Fabaceae

Bark applied as poultice used for sores,abscesses and open wounds

Coates Palgrave (1977),Pujol (1990), Hutchings(1996), Van Wyk et al.(2000), Van Wyk et al.(2011)


18.7 4.4

Eucalyptus camaldulensisDehnh., Myrtaceae

Bark infusion used as a wash, to treatpimples

Hutchings (1996) Bark Pretoria–VillieriaUM122

14.0 7.2

U. Mabona et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

Please cite this article as: Mabona, U., et al., Antimicrobial activity of southern African medicinal plants with dermatological relevance:From an.... Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.03.056i




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Table 1 (continued )






Ficus natalensis Hochst.,Moraceae

Leaves used as hot compress for wounds,boils and warts

Hutchings (1996), VanWyk et al. (2011)


3.6 8.2

Ficus sur Forssk.,Moraceae

Bark used as a compress for boils Palmer and Pitman (1972)Hutchings (1996)


Leaf WSBGb UM141 8.9 6.3Gunnera perpensa L.,Gunneraceae

Root or rhizome and leaf are used asinfusion or decoction for dressing woundsand to treat psoriasis

Hutchings (1996),Felhaber (1997), Dreweset al. (2005), Van Wyket al. (2009)


Rhizomes WSBGb UM176 18.5 10.5Halleria lucida L.,Scrophulariaceae

Unspecified parts used topically forvarious skin complaints

Pooley (1993), Hutchings(1996)


Stem WSBGb UM178 4.6 3.2Harpephyllum caffrumBernh. ex Krauss,Anacardiaceae

Bark applied externally to treat acne andeczema

Pujol (1990), Van Wyket al. (2000, 2011)

Bark Pretoria–VillieriaUM128

10.4 9.7

Hypericum perforatum L.,Hypericaceae

Above ground parts applied externally totreat wounds

and first degree burns Van Wyk et al. (2000) Leaf UWc UM162 40.6 6.2Ilex mitis Radlk.,Aquifoliaceae

Ground bark applied as paste or decoctionfor skin rash and sores on the face

Hutchings (1996), VanWyk et al. (2011)


Leaf WSBGb UM145 28.7 6.1Kigelia africana (Lam.)Benth., Bignoniaceae

Externally applied to treat ulcers, sores,abscesses and rheumatism

Watt and Breyer-Brandwijk (1962), CoatesPalgrave (1977),Hutchings (1996), VanWyk et al. (2000, 2011)

Fruit Zululand UM161 5.4 11.0

Lannea discolor Engl.,Anacardiaceae

Bark applied externally to treat boils andabscesses

Watt and Breyer-Brandwijk (1962),Hutchings (1996), VanWyk et al. (2000)


17.0 9.7

Lantana rugosa Thunb.,Verbenaceae

Leaf, stem or ripe fruits used as a paste forfestering sores and cuts

Smith (1895), Roberts(1990), Hutchings (1996)

Leaf Rayton (Gauteng)

B & F10 7.7 4.1Malva parviflora L.,Malvaceae

Leaf paste combined with Pelargoniumalchemilloides to treat wounds andabscesses

Smith (1895) Leaf WSBGb UM166 17.2 18.7

Melianthus comosusVahl., Melianthaceae

Leaf poultice and leaf decoction used totreat bad sores, sceptic wounds, reduceswellings

Gerstner (1938), Watt andBreyer-Brandwijk (1962),Hutchings (1996)


Melianthus major L.,Melianthaceae

Leaf poultice and leaf decoction used forseptic wounds, sores and bruises

Van Wyk et al. (2009) Leaf WSBGb UM142 15.7 17.8

Mentha longifolia Huds.,Lamiaceae

Leaves applied topically to treat wounds Van Wyk et al. (2000) Leaf WSBGb UM148 8.4 10.4

Opuntia ficus-indicaMill., Cactaceae

Leaves applied topically for skin rash,ulcers, furuncles, fresh wounds and warts

Smith (1996), Von Koenen(1996)


5.9 12.0

Pellaea calomelanosLink., Adiantaceae

Decoction or infusions of leaves and/orrhizomes applied externally for boils andabscesses

Watt and Breyer-Brandwijk (1962), Pujol(1990), Hutchings (1996),Van Wyk et al. (2000)


Rhizomes WSBGb UM179 30.6 3.8Pentanisia prunelloidesWalp., Rubiaceae

Roots applied externally for burns andswellings. Also used in combination withDicoma anomala to treat insect stings andbites

Felhaber (1997), Van Wyket al. (2000)

Root bark PNBGd UM182 10.6 10.7

Rootsstripped

PNBGd UM183 5.8 4.8

Pittosporum viridiflorumSims., Pittosporaceae

Roots or leaves combined with Momordicafoetida and Vernonia natalensis in adecoction used to treat boils

Hutchings (1996) Leaf WSBGb UM159 16.0 7.7

Rauvolfia caffra Sond.,Apocynaceae

Bark applied topically to treat measles,urticaria and other skin rashes

Gerstner (1938),Hutchings (1996)


Rothmannia capensisThunb., Rubiaceae

Sap from fruit applied topically for burnsand wounds

Arnold and Gulumian(1984), Hutchings (1996)


Scadoxus puniceus (L.)Friis & Nordal,Amaryllidaceae

Bulbs and root decoction applied topicallyto treat wounds, ulcers, sores and allergies

Watt and Breyer-Brandwijk (1962),Hutchings (1996), VanWyk et al. (2000)

Roots andrhizomes

WSBGb UM143 7.4 10.3

Solanum incanum L.,Solanaceae

Leaves or roots applied topically to treatwounds, furuncles and ringworm

Gerstner (1938),Hutchings (1996), VonKoenen (1996)


Terminalia sericea Burch.ex DC., Combretaceae

Root sap or bark applied externally as anantiseptic for wounds and to treat leprosyand snakebites

Watt and Breyer-Brandwijk (1962), Pujol

Roots Swaziland AdCAV21 33.1 15.2


Please cite this article as: Mabona, U., et al., Antimicrobial activity of southern African medicinal plants with dermatological relevance:From an.... Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.03.056i




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100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132

Table 1 (continued )






(1990), Hutchings (1996),Van Wyk et al. (2000)

Trichilia emetica Vahl.,Meliaceae

Leaves or fruits used as poultice forbruises and eczema

Van Wyk et al. (2011) Leaf WSBGb UM169 13.6 14.2

Vernonia natalensis Sch.Bip. ex Walp.,Asteraceae

Roots or leaves combined with Momordicafoetida and Pittosporum viridiflorum in adecoction to treat boils

Hutchings (1996) Leaf WSBGb UM170 10.5 4.0

Roots WSBGb UM180 10.2 6.6Viscum capense L.f.,Santalaceae

Whole plant applied externally to treatwarts and other skin complaints

Hutchings (1996) Leaf Magaliesburg UM119 15.4 9.1

Warburgia salutaris (G.Bertol.) Chiov.,Canellaceae

Leaf and stalk lotion in combination withHibiscus surattensis, used as an anti-inflammatory and to treat sores and skinirritation

Hutchings (1996) Bark WSBGb UM181 7.9 5.1

Leaf WSBGb UM154 15.8 7.6Zantedeschia aethiopicaSpreng., Araceae

Leaf applied directly to the skin to treatwounds, boils and sores

Watt and Breyer-Brandwijk (1962), VanWyk et al. (2000)


Ziziphus mucronataWilld., Rhamnaceae

Leaf, root or bark decoction appliedtopically to treat boils, sores and swellings

Watt and Breyer-Brandwijk (1962),Hutchings (1996), VanWyk et al. (2000, 2011)

Bark Pretoria–VillieriaUM126

10.0 8.6


10.6 21.9

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.


were introduced. A volume of 100 μl of the culture was added to allthe wells. Tests were performed at least in duplicate. Each platewas subsequently sealed with a sterile adhesive sealing film. Allmicro-titre plates were incubated in suitable conditions asdetailed in Section 2.3.1. When testing for antimicrobial propertiesof plant extracts against the more fastidious pathogens such asPropionibacterium acnes, Trichophyton mentagrophytes and Micro-sporum canis, modifications to the standard MIC method wereundertaken. Micro-titre plates for Propionibacterium acnes wereincubated without the sterile adhesive seal film on the micro-titreplates. When testing the dermatophytes, the growth indicator wasadded 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) (SigmaAldrich) was added as an indicator for microbial growth to eachwell of the micro-titre plates. The minimum inhibitory concentra-tion was defined as the lowest concentration of the test samplewhere 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 startingconcentration of 12.5% v/v) to ascertain if any growth inhibitionwas attributed to the solvent and culture control (pathogengrowing independently). The culture growing independently wasused to monitor viability and as a comparative standard whenreading MIC values after INT was added.

2.4. Interactive combination studies

The synergistic, additive or antagonistic interaction betweenplants reported to be used in combination were investigated usingtwo approaches. First, plant extracts with a starting concentration

Please cite this article as: Mabona, U., et al., Antimicrobial activity of sFrom an.... Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1

of 64 mg/ml were mixed in 1:1 ratios. The MIC values weredetermined for each combination to establish the interaction andthe sum of the fractional inhibitory concentration (ƩFIC) wascalculated for each combination using the following equation;

FIC ðiÞ ¼ MIC ðaÞ in combination with ðbÞMIC ðaÞ independently

FIC ðiiÞ ¼ MIC ðbÞ in combination with ðaÞMIC ðbÞ independently

(i) and (ii) in this study represents the different plants incombination. The sum of the FIC, known as the FIC index wasthus calculated as ƩFIC¼FIC(i)+FIC(ii). This may be classified aseither 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. TheMIC 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 werethen 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 datapoints of the ratios where the MIC for each concentration isdetermined in relation to the independent MIC's. Data pointsfalling below or on the 0.50 line on the isbolologram wereinterpreted as synergistic. Points between 0.50 and/or on the1.00 line were interpreted as additive and points 41.00–≤4.00line were defined as either non-interactive or antagonistic (44.0)(Van Vuuren and Viljoen, 2011). For all assays, positive andnegative controls were included in all repetitions (Section 2.3.2).Assays were undertaken at least in duplicate and the meanvalues noted.





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676869707172737475767778798081828384858687888990919293949596979899

100

101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132

Table 2Screening of plant extracts from South African medicinal plants for antimicrobial activity against common skin pathogens (MIC recorded in mg/ml).

Plant samples Staphylococcusaureus ATCC25923

cMRSA ATCC43300

dGMRSAATCC 33592

StaphylococcusepidermidisATCC 2223

PseudomonasaeruginosaATCC 27858

Candidaalbicans ATCC10231

Brevibacillusagri ATCC51663

Propionibacteriumacnes ATCC 11827

TrichophytonmentagrophytesATCC 9533

Microsporumcanis ATCC 36299

aD:M Aq aD:M Aq aD:M

Aq aD:M Aq aD:M

Aq aD:M Aq D:M Aq D:M Aq D:M Aq D:M Aq

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 1.00Acacia 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 8.00Acokanthera 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 1.00Aloe 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 8.00Athrixia 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 1.00 416.00Aristea 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 2.00Aristea 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 1.00Bauhinia 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 2.00Boophane 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 2.00 416.00Bridelia 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 2.00Bridelia 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 4.00Chenopodium 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 4.00Cissampelos 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 8.00Cotyledon 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 8.00Dicoma 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 8.00Dioscorea 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 4416.00 4.00 416.00Diospyros 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 4.00Dodonaea 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 4.00Ekebergia 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 8.00Ekebergia 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 4.00Elephantorrhiza 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 8.00Elephantorrhiza elephantine roots+rhizomes b 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 4.00Embelia 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 8.00Erythrina 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 16.00Eucalyptus 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 2.00Ficus 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 4.00Ficus 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 8.00 416.00 416.00Ficus 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 4.00Gunnera 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 1.00Gunnera 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 16.00Halleria 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 2.00Halleria 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 8.00Harpephyllum 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 4.00Hypericum 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 4.00Ilex 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 16.00Ilex 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 1.00 416.00Kigelia 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 8.00 416.00Lantana 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 4.00Lannea 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 2.00Malva 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 4.00 416.00 416.00Melianthus 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 1.00Melianthus 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 4.00Mentha 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 2.00Opuntia 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 16.00Pellaea 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 4.00Pellaea 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 16.00Pentanisia 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 16.00Pentanisia 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 8.00Pittosporum 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 1.00Rauvolfia 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

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676869707172737475767778798081828384858687888990919293949596979899

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2.5. Bioactivity-guided isolation of Aristea ecklonii leaf material

A combination of column chromatography (CC) and high speedcounter-current chromatography (HPCCC) (Dynamic ExtractionsLtd.) was used to fractionate the extract and purify the bio-activecompound. Column chromatography fractionation was achievedby gradient elution, which involved eluting the column withvarying polarities of a DCM and methanol mixture (100:0; 95:5;90:10). A two phase solvent system for HPCCC fractionation wasprepared containing n-hexane, ethyl acetate, methanol and dis-tilled water (8:8:5:5, v/v/v/v). The sample solution for HPCCCseparation was prepared by dissolving approximately 120 mg ofthe dry extract of Fraction, F3 from CC separation into 2 ml of thesolvent mixture consisting equal volumes of the two phases. Thinlayer chromatography (TLC) was used to monitor the chemicalprofile of the fractions obtained from the CC and HPCCC. Auto-biography assays (Van Vuuren et al., 2006) were used as a guide toidentify 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 of1 μl was applied and the column temperature was adjusted to40 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 wasemployed, starting with 90% A and 10% B, changing to 50% B in15 min, then changing to 100% B in 1 min, with a post-run time of1 min.

Structure elucidation was undertaken using nuclear magneticresonance (NMR). The NMR spectra were recorded on a Bruker600 Avance II NMR at 600 MHz for 1H NMR and 150 MHz for 13Cand distortionless enhancement through polarisation transfer(DEPT) NMR.

Prior to fractionation of Aristea ecklonii, a bulk sample waspurchased. Leaf material was dried, ground (50 g) and seriallyextracted using 500 ml (�10) of CH2Cl2: MeOH (1:1; V/V).

The micro-titre plate dilution assay as detailed in Section 2.3.2,was used to confirm the antimicrobial effects of Aristea eckloniicrude extract, fractions from CC and the isolated compoundagainst Staphylococcal species, Pseudomonas aeruginosa and Can-dida albicans.

107108109110111112113114115116117118119120121122123124125126127128129130131132

3. Results and discussion

3.1. Antimicrobial screening

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 afew plant species (as detailed hereafter) showed interestingresults specifically as the positive antimicrobial efficacy had adirect correlation to the traditional use. Aristea ecklonii is usedtraditionally to treat shingles (Ngwenya et al., 2003) (Table 1).Other antimicrobial-related uses not specific to skin diseases havebeen reported i.e. the treatment of fevers, coughs as well as theuse for syphilis (Hutchings, 1996). This prompted the investigationagainst other related skin pathogens with the hypothesis thatAristea ecklonii may offer other additional antimicrobial efficaciesnot previously documented. The current findings demonstratenoteworthy antimicrobial properties for Aristea ecklonii, whereorganic and aqueous extracts of leaf and root displayed thegreatest antimicrobial effect (lowest MIC value of 0.01 mg/ml forD:M against Staphylococcus aureus). The antifungal properties ofAristea ecklonii against plant pathogens have been reported byPretorius et al. (2002), where 100% antifungal inhibition was





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676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132


observed against all strains tested. This is the first report forefficacy against skin pathogens.

Chenopodium ambrosioides has been reported for its traditionaluse 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 boththe tested bacteria and Trichophyton mentagrophytes (MIC valuesbetween 0.25 mg/ml and 0.80 mg/ml). The broad-spectrum anti-microbial efficacy of Chenopodium ambrosioides was corroboratedby earlier findings noted by Suliman et al. (2010).

Dicoma anomala has been used traditionally as a paste to treatwounds, ulcers, ringworm and head sores (Hutchings, 1996;Felhaber, 1997). The current findings show noteworthy antimicro-bial activities of the organic extract against Staphylococcal speciesand Trichophyton mentagrophytes with a MIC value of 0.50 mg/mland 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 similarantimicrobial effects against Staphylococcus aureus and Pseudomo-nas aeruginosa of Dicoma anomala, however, no previous reportscould be found where efficacy was tested on Trichophyton menta-grophytes and Microsporum canis.

Diospyros mespiliformis has been reported for its use to treat avariety of skin ailments such as scars, skin rashes, bruises, woundsand ringworm (Von Koenen, 1996; Van Wyk et al., 2011) (Table 1).The organic extract demonstrated MIC values between 0.05 mg/mland 1.00 mg/ml. The antimicrobial effects of this plant against thedermatophytes give some validation to the traditional use espe-cially for treating ringworm as it demonstrated noteworthy anti-microbial effects against Trichophyton mentagrophytes (MIC0.10 mg/ml) and Microsporum canis (MIC 0.50 mg/ml). Thebroad-spectrum antimicrobial effects of Diospyros mespiliformishave also been confirmed by Adeniyi et al. (1996).

Decoctions or infusions of root or rhizome and leaf of Gunneraperpensa are traditionally used as a dressing for wounds and forpsoriasis (Table 1). The organic extract of Gunnera perpensa leafexhibited antimicrobial activity (MIC values between 0.03 mg/mland 1.00 mg/ml) against the tested pathogens, with similarities inantimicrobial efficacy noted for the aqueous extract againstStaphylococcus aureus and respective resistant strains (MIC valuesbetween 0.50 mg/ml and 1.00 mg/ml). Therefore, the findingscorroborate with the traditional use as an antiseptic and dressingfor wounds. Findings were similar to those reported in literature(Steenkamp et al., 2004; Drewes et al., 2005; Buwa and VanStaden, 2006; Nkomo and Kambizi, 2009), however, this reportprovides new evidence for efficacy against Propionibacteriumacnes, Brevibacillus agri, Trichophyton mentagrophytes and Micro-sporum canis.

Terminalia sericea is used for a variety of ailments (Table 1) andthe antimicrobial activity has been extensively studied especiallyfor conditions associated with diarrhoea and respiratory ailments(Eloff, 1999; Fyhrquist et al., 2002; Steenkamp et al., 2004; Eldeenet al., 2005; Masoko et al., 2005; Moshi and Mbwambo, 2005;Tshikalange et al., 2005; Eldeen and Van Staden, 2007; Sulimanet al., 2010). In the current study, Terminalia sericea exhibitedmostly noteworthy broad-spectrum antimicrobial effects againstskin 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 withHibiscus surattensis to treat sores and skin irritations (Table 1).The organic bark extract displayed noteworthy antimicrobialactivity presenting with MIC values between 0.03 mg/ml and1.00 mg/ml against tested pathogens, with exception of Breviba-cillus agri and Microsporum canis. It is worth noting that the barkonly had a better overall effect for the organic extracts when


compared to the leaf samples. As the traditional use is usuallyaqueous by nature, the possibility of substitution of leaf materialfor bark may be warranted. This could possibly protect this plantspecies, which is rapidly dwindling in numbers in the wild. Similarnoteworthy activity for Warburgia salutaris methanol extractagainst Staphylococcus aureus has been reported by Rabe andVan 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 activityagainst the relevant pathogen Propionibacterium acnes with MICvalues between 0.05 mg/ml and 1.00 mg/ml.

3.2. Combination studies

Four different plant combinations were analysed for interactiveproperties. The mean MIC's and ƩFIC's of these combinations againstthe six pathogens are presented in Table 3. Plant species incorporatedin the combinations were Dicoma anomala, Elephantorrhiza elephantinaand Pentanisia prunelloides. These plants have also been reported to beused independently and in combination for a variety of skin ailments(Table 1). Mostly, non-interactive effects were noted. However, someinteractions worth highlighting are the D:M combination of Dicomaanomala with Elephantorrhiza elephantina (ƩFIC value of 4.0 borderingon an antagonistic effect) and the combinations of Pentanisia prunel-loides (root) with either Elephantorrhiza elephantina or Dicoma anomalawhere selective synergistic interactions were observed. One interest-ing combination was that of Pentanisia prunelloides with Elephantor-rhiza elephantina (roots). Even though Pentanisia prunelloides (root) didnot exert any noteworthy antimicrobial effects when screened inde-pendently, synergistic interactions were noted when the aqueousextract 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 notan exact science (where formulations are accurately measured to theexact mg or mg quantity), this combination was combined in variousratios to determine if variations of the concentration of the two plantsin the mixture may result in different interactions (Fig. 1). Noantagonistic interactions were observed and several synergistic inter-actions were predominant for the aqueous extracts at varying ratiosagainst Staphylococcus aureus, GMRSA and Staphylococcus epidermidis.Furthermore, more favourable interactions were observed for theaqueous extracts, irrespective of the ratio at which these two plantsare combined. It was also worth noting that the 1:1 combinations ofthe aqueous extracts demonstrated the most synergistic interactions,lending some credibility to the traditional use of water preparationsfor medicinal purposes. While examining the aqueous and organicextract ratio combinations of Pentanisia prunelloides and Elephantor-rhiza elephantina in more depth, the enhanced efficacy of thecombination was mainly attributed to higher Elephantorrhiza elephan-tina concentrations.

3.3. Antimicrobial activity and bioactivity-guided isolation of Aristeaecklonii leaf

A bio-autographic assay of the crude extract indicated thecompound with RF 0.72 to be the most active against the testedbacterial strains, thus it was targeted for isolation. This compoundhad low polarity and was extracted from the dichloromethanephase 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) containingmainly plumbagin was then purified using HPCCC to obtain 35 mgof the compound. The isolated compound appeared as orange





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S. aureusATCC 25923

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.000.00

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GMRSAATCC 33592

0.00 0.25 0.50 0.75 1.00 1.25 1.500.00

0.25

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S. epidermidisATCC 2223

0.00 0.25 0.50 0.75 1.00 1.25 1.500.00

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MIC P. prunelloides in combination with E. elephantina/MIC

E. elephantina independently

MICP. prunelloides in combination with E. elephantina/MIC






C. albicansATCC 10231

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

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

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

Combinations Staphylococcusaureus ATCC25923

aMRSAATCC 43300

bGMRSAATCC 33592

Staphylococcusepidermidis ATCC2223

Pseudomonasaeruginosa ATCC27858

Candidaalbicans ATCC10231

MIC and ƩFICmean values

cD:M dAq cD:M dAq cD:M dAq cD:M dAq cD:M dAq cD:M dAq cD:M dAq

Elephantorrhiza elephantina+Dicomaanomala tuber

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

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 1.42Elephantorrhiza elephantina+Pentanisiaprunelloides root

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

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 0.39Pentanisia prunelloides root bark+Dicomaanomala tuber

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

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 1.00Pentanisia prunelloides root+Dicomaanomala tuber

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

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.


needle-like crystals at room temperature (25 1C). Ultra highperformance liquid chromatography analysis of the compoundindicated a purity of 99% and a maximum absorption wavelength(ʎmax) of 267 nm.


After structure elucidation of the compound using comprehen-sive 1 and 2D 1H and 13C NMR, this compound was identifiedas plumbagin, which is a naphthoquinone. Isolated plumbaginshowed the following NMR signals; 1H NMR (CDCl3) δ: 6.85 (1H, s,





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

Plant fractions StaphylococcusaureusATCC 25923

MRSAATCC43300

GMRSAATCC33592

StaphylococcusepidermidisATCC 2223

PseudomonasaeruginosaATCC 27858

CandidaalbicansATCC 10231

Crude extract 156.00 156.00 156.00 78.00 156.0 313.00DCM fraction 39.00 78.00 39.00 20.00 78.00 78.00Fraction F3 20.00 39.00 20.00 80.00 20.00 39.00Plumbagin (compound) 8.00 16.00 16.00 4.00 8.00 2.00Ciprofloxacin/amphotericin B positivecontrol

0.31 0.63 0.31 0.31 0.31 2.00

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, J¼7.5, 8.4 Hz,H-7), 7.57 (1H, d, J¼7.5 Hz, H-8), 2.15 (3H, s, Me-2), 11.97 (–OH);13CNMR(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 ofpathogens for Aristea ecklonii (bulk) crude extract, fraction andthe isolated compound is shown in Table 4. Isolated compoundswhich demonstrate MIC values below 100 μg/ml are considered tohave some clinical relevance and an isolated compound is con-sidered to be of interest when MIC values are below 10 μg/ml (Riosand Recio, 2005). Thus, the bio-active compound, plumbagin,displayed noteworthy antimicrobial activity (MIC range between2.00 μg/ml and 16.00 μg/ml) against the tested pathogens. Further-more, the antimicrobial effect of plumbagin against Candida albicanswas comparable, having the same MIC value of 2.00 μg/ml tothe commercial antifungal, amphotericin B. The antimicrobialproperties of Aristea ecklonii and chemical constituents againstskin relevant pathogens have not been investigated previously,however, the presence of plumbagin in both the roots and leavesof the plants was first reported by Kumar et al. (1985). Resultsobserved from previous studies on plumbagin demonstrated verylow MIC values (1.56 μg/ml and 0.78 μg/ml against Staphylococcusaureus and Candida albicans, respectively) (De Paiva et al., 2003).A diffusion assay by Jeyachandran et al. (2009) on plumbaginisolated from the root extract of Plumbago zeylanica also showednoteworthy activity against a variety of pathogens includingStaphylococcus aureus (MIC o1.00 μg/disc) and Pseudomonas aer-uginosa (MIC 43.00 μg/disc). Plumbagin isolated from Plumbagospecies such as Plumbago zeylanica and Plumbago scandens hasbeen reported to possess anti-carcinogenic and concentrationdependant toxicity in human peripheral blood cells (De Paivaet al., 2003; Wang et al., 2008; Seshadri et al., 2011; Bothirajaet al., 2011), which may warrant caution when used in higherconcentrations.

116117118119120121122123124125126127128129130131132

4. Conclusion

When investigating the antimicrobial activities of plantsused for skin infections it was found that most of the plantextracts demonstrated pathogen specific antimicrobial effects,with about 8.3% exhibiting broad-spectrum activities against thetested pathogens. Notable antimicrobial effects for plants such asElephantorrhiza elephantina and Diospyros mespiliformis used foracne vulgaris and ringworm infections, respectively, give somevalidation for their reported traditional uses. Synergistic interac-tions noted for Pentanisia prunelloides combined with Elephantor-rhiza elephantina validate their antimicrobial effects of aqueouspreparations used in combination. As this plant combination istraditionally also used as a treatment for eczema, further investi-gation into the possible additive anti-inflammatory effects is


warranted. Enhanced antimicrobial activities were observed forthe naphthoquinone plumbagin isolated from Aristea ecklonii.

This comprehensive study gives some validation towards thetraditional use of some medicinal plants for the treatment of skininflictions. Additionally, selective plants could be targeted forfuture study on wound healing proliferation which plays animportant 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 ofHealth Sciences (University of the Witwatersrand) for assistedSPARC funding and NRF free standing bursary funding. We wouldalso like to convey our sincere gratitude to the personnel of WalterSisulu Botanical Garden for their assistance in plant collecting.Also, we are grateful for assistance from Dr Wei Chen (Departmentof Pharmaceutical Sciences, Tshwane University of Technology),for assisting with UPLC and HPTLC analysis.

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