Antimicrobial activity and in vitro cytotoxicity of selected South African Helichrysum species

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<ul><li><p>mytum</p><p>.</p><p>UniUnih Ay ofsonrma</p><p>Received 26 March 2010; received in revised form 14 May 2010; accepted 17 May 2010</p><p>sum species was determined against six microorganisms. Seven of the 36</p><p>Available online at</p><p>ny 71. Introduction</p><p>Helichrysum species are often used to treat conditionsassociated with infections of the skin and the respiratory andgastro-intestinal tracts. For example, root decoctions of H.adenocarpum and H. ecklonis are used to treat diarrhoea inchildren. Different preparations of H. cymosum, H. kraussii andH. odoratissimum are used in the treatment of colds and coughswhile the leaves of H. nudifolium are applied externally towounds and used to treat respiratory infections (Hutchingset al., 1996; Lourens et al., 2008 and references therein; Wattand Breyer-Brandwijk, 1962).</p><p>Although there are several reports on the antimicrobialactivities of Helichrysum extracts, comparative toxicity values</p><p>In a study by Van Vuuren et al. (2006), the antimicrobialactivity of an acetone extract of H. cymosum was determinedagainst ten pathogens and toxicity determined against trans-formed human kidney epithelial cells. The IC50 value of172.01 g/ml reported in the cytotoxicity assay was lower thanthe MIC values (313 g/ml) reported for seven of the screenedmicroorganisms, indicating that cell growth may be inhibited atthe concentrations required to inhibit pathogen growth. Heymanand Meyer (2009) investigated the cytotoxicity of 12 Helichry-sum species and reported IC50 values ranging from b3.125 g/ml to 277 g/ml, while a dichloromethane extract of H.nudifolium roots caused total growth inhibition of three cancercell lines at concentrations below 34 g/ml (Fouche et al.,2008). Furthermore, aqueous methanolic extracts from severalchloroform:methanol (1:1) extracts (leaf and stem extracts for all plants and an additional flower extract for H. rugulosum) exhibited minimuminhibitory concentration (MIC) values lower than 0.1 mg/ml against Bacillus cereus and/or Staphylococcus aureus. The in vitro cytotoxicity[against transformed human kidney epithelial (Graham) cells, MCF-7 breast adenocarcinoma and SF-268 glioblastoma cells] of these extracts wasalso determined at a concentration of 0.1 mg/ml using the sulforhodamine B (SRB) assay. For seven species less than 25% growth was observedfor the Graham and MCF-7 cell lines at the test concentration. 2010 SAAB. Published by Elsevier B.V. All rights reserved.</p><p>Keywords: Antimicrobial activity; Asteraceae; Cytotoxicity; HelichrysumThe antimicrobial activity of 35 indigenous South African HelichryAbstractShort com</p><p>Antimicrobial activity and in vitro cHelichrys</p><p>A.C.U. Lourens a,, S.F. Van Vuurenb, A.Ma Pharmaceutical Chemistry, School of Pharmacy, North-West</p><p>b Department of Pharmacy and Pharmacology, Faculty of Health Sciences,Sout</p><p>c Department of Pharmaceutical Sciences, Tshwane Universitd Department of Biochemistry and Microbiology, PO Box 77000, Nel</p><p>e School of Chemistry, University of KwaZulu-Natal Piete</p><p>South African Journal of Botaare often not available (Drewes and van Vuuren, 2008; Dreweset al., 2006; Lourens et al., 2004; Mathekga and Meyer, 1998).</p><p> Corresponding author. Tel.: +27 18 299 2266; fax: +27 18 299 4243.E-mail address: (A.C.U. Lourens).</p><p>0254-6299/$ - see front matter 2010 SAAB. Published by Elsevier B.V. All righdoi:10.1016/j.sajb.2010.05.006unication</p><p>otoxicity of selected South Africanspecies</p><p>Viljoenc, H. Davidsd, F.R. Van Heerdene</p><p>versity, Private Bag X6001, Potchefstroom 2520, South Africaversity of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg,fricaTechnology, Private Bag X680, Pretoria 0001, South AfricaMandela Metropolitan University, Port Elizabeth 6031, South Africaritzburg, Private Bag X01, Scottsville 3209, South Africa</p><p>7 (2011) species exhibited mutagenicity (Elgorashi et al.,2008; Reid et al., 2006; Verschaeve and Van Staden, 2008). Onthe other hand, an aqueous extract of H. aureonitens did notexhibit any cytotoxic effects at a concentration as high as8.44 mg/ml against HF cells (Meyer et al., 1996).</p><p>ts reserved.</p></li><li><p>Table 1Antimicrobial activity (MIC, mg/ml) and cytotoxicity (average at 0.1 mg/mlstandard deviation) of Helichrysum extracts.</p><p>Plant species Antimicrobial activity a</p><p>Test organismsCytotoxicitya,b</p><p>C. neoformansATCC 90112</p><p>P. aeruginosaATCC 9027</p><p>S. aureusATCC 12600</p><p>B. cereusATCC 11778</p><p>S. epidermidisATCC 2223</p><p>K. pneumoniaeNCTC 9633</p><p>% T/C of Graham cells % T/C of SF-268 cells % T/C of MCF-7 cells</p><p>H. acutatum DC.A. Lourens and A. Viljoen 1Graskop</p><p>NS c NS 0.5 1 NS NS 0.10.1 15.81.0 6.60.1</p><p>H. adenocarpum DC. subsp adenocarpumS.N.Unknown locality</p><p>NS NS NS NS NS NS 98.30.6 88.13.6 37.23.2</p><p>H. appendiculatum (L.f.) LessA. Lourens and A. Viljoen 28Graskop</p><p>NS NS NS 4 NS NS 91.72.1 81.02.8 37.52.4</p><p>H. aureonitens Sch. Bip.J.E. Victor 2439Amatolas</p><p>NS NS 4 4 4 NS 75.60.9 76.04.7 37.53.7</p><p>H. aureum (Houtt.) Merril var. aureumJ.E. Victor 2428Amatolas</p><p>NS NS 0.02 0.01 NS NS 5.00.2 35.91.9 7.0 d</p><p>H. callicomum Harv.DBH 6565Transkei</p><p>NS NS 1 0.25 4 NS 27.32.9 56.12.8 11.2 d</p><p>H. cephaloideum DC.A. Lourens and A. Viljoen 20Underberg</p><p>NS NS NS 1 4 NS 93.74.6 83.82.6 46.1 d</p><p>H. dasyanthum (Willd.) SweetA. Lourens (ex J. Manning) 33Cape Agulhas</p><p>NS NS NS 4 NS NS 91.30.7 81.73.6 52.8 d</p><p>H. excisum (Thunb.) LessJ. Vlok 2830Oudtshoorn</p><p>NS NS 0.2 0.03 0.1 NS 63.61.1 71.93.1 31.40.8</p><p>H. felinum Less.J. Vlok 2828Oudtshoorn</p><p>NS NS 2 0.16 2 NS 48.01.4 54.12.3 23.20.2</p><p>H. cf. foetidum (L.) MoenchJ. Vlok 2833George</p><p>NS NS 0.5 0.01 NS NS 32.72.1 57.82.1 24.90.4</p><p>H. herbaceum (Andr.) SweetA. Lourens and A. Viljoen 17Northern Drakensberg</p><p>0.5 NS 0.5 1 4 NS 50.83.0 46.41.6 22.90.3</p><p>H. indicum (L.) GriersonA. Lourens (ex. J. Manning) 34Cape Agulhas</p><p>NS NS 1 0.5 4 NS 81.12.3 70.64.2 35.80.8</p><p>H. kraussii Sch. Bip.A. Lourens and A. Viljoen 4Lydenburg</p><p>NS NS 0.5 0.004 4 2 28.61.4 45.24.4 9.0 d</p><p>H. melanacme DC.A. Lourens and A. Viljoen 12Clarens</p><p>NS NS 0.5 0.25 4 2 18.10.4 53.32.7 12.2 d</p><p>230A.C.U.Lourens</p><p>etal.</p><p>/South</p><p>African</p><p>Journalof</p><p>Botany</p><p>77(2011)</p><p>229235</p></li><li><p>Table 1 (continued).</p><p>Plant species Antimicrobial activity a</p><p>Test organismsCytotoxicitya,b</p><p>C. neoformansATCC 90112</p><p>P. aeruginosaATCC 9027</p><p>S. aureusATCC 12600</p><p>B. cereusATCC 11778</p><p>S. epidermidisATCC 2223</p><p>K. pneumoniaeNCTC 9633</p><p>% T/C of Graham ce % T/C of SF-268 cells % T/C of MCF-7 cells</p><p>H. miconiifolium DC.A. Lourens and A.Viljoen 24Himeville</p><p>NS NS 2 1 4 NS 37.96.6 52.41.8 20.9 d</p><p>H. montanum DC. NS NS 1 4 NS NS 29.40.7 46.14.0 19.2 d</p><p>F. Van Heerden 1Murraysburg</p><p>H. nudifolium (L.) LessA. Lourens and A. Viljoen 13Clarens</p><p>NS NS NS 4 NS NS 73.12.5 83.91.8 35.30.4</p><p>H. odoratissimum (L.) SweetA. Lourens and A. Viljoen 6Pilgrims Rest</p><p>NS NS 0.02 0.03 4 2 17.50.4 48.21.4 7.40.7</p><p>H. oreophilum KlattA. Lourens and A. Viljoen 10Draaikraal</p><p>NS NS 4 4 NS NS 63.42.2 82.93.0 34.80.1</p><p>H. pallidum DC.A. Lourens and A. Viljoen 7Graskop</p><p>NS NS NS NS NS NS 88.60.9 83.99.7 49.01.7</p><p>H. pandurifolium SchrankA. Lourens (ex J. Manning) 35Cape Agulhas</p><p>NS NS 2 4 NS NS 57.11.2 71.80.8 34.20.1</p><p>H. patulum (L.) D. DonA. Lourens (ex J. Manning) 36Cape Agulhas</p><p>NS NS 4 4 NS NS 63.81.3 75.22.0 37.8 d</p><p>H. petiolare Hilliard and BurttJ. Vlok 2827Oudtshoorn</p><p>NS NS 4 2 NS NS 59.33.4 76.63.0 33.4 d</p><p>H. platypterum DC.A. Lourens and A. Viljoen 30Dullstroom</p><p>NS NS 0.05 0.5 NS NS 0.80.3 35.11.5 4.6 d</p><p>H. psilolepis Harv.A. Lourens and A. Viljoen 11Clarens</p><p>NS NS 1 1 NS NS 25.91.9 58.47.4 23.1 d</p><p>H. retortum (L.) Willd.A. Lourens (ex J. Manning) 37Cape Agulhas</p><p>ND NS 4 NS NS NS 79.63.4 87.34.8 NDe</p><p>H. rosum (Berg.) Less. cf. var. rosumJ.E. Victor 2436Amatolas</p><p>NS NS 1 1 4 NS 47.51.8 54.85.0 22.50.7</p><p>H. ruderale Hilliard &amp; Burtt.A. Lourens and A. Viljoen 32Hilton</p><p>ND NS 2 1 NS NS 45.11.2 75.83.9 ND</p><p>(continued on next page) 231A.C.U.Lourens</p><p>etal.</p><p>/South</p><p>African</p><p>Journalof</p><p>Botany</p><p>77(2011)</p><p>229235lls</p></li><li><p>Table 1 (continued).</p><p>Plant species Antimicrobial activity a</p><p>Test organismsCytotoxicitya,b</p><p>C. neoformansATCC 90112</p><p>P. aeruginosaATCC 9027</p><p>S. aureusATCC 12600</p><p>B. cereusATCC 11778</p><p>S. epidermidisATCC 2223</p><p>K. pneumoniaeNCTC 9633</p><p>% T/C of Graham cells % T/C of SF-268 cells % T/C of MCF-7 cells</p><p>H. rugulosum Less.Stems and leavesA. Lourens and A. Viljoen 8Ventersdorp</p><p>NS NS 0.5 0.25 4 NS 3.01.2 44.73.6 12.72.7</p><p>H. rugulosum Less.FlowersA. Lourens and A. Viljoen 8Ventersdorp</p><p>1.0 NS 0.01 0.33 0.01 0.22 ND ND ND</p><p>H. scitilum Hilliard &amp; Burtt.F. Van Heerden 2Murraysburg</p><p>NS NS NS NS 4 NS 58.61.2 85.36.8 46.8 d</p><p>H. simillimum DC.A. Lourens and A. Viljoen 23Himeville</p><p>NS NS 1 4 4 NS 54.62.6 66.83.9 26.8 d</p><p>H. splendidum (Thunb.) LessA.Lourens and A. Viljoen 2Graskop</p><p>NS NS 4 NS NS NS 82.33.2 72.83.3 49.22.5</p><p>H. wilmsii MoeserA. Lourens and A.Viljoen 5Pilgrims Rest</p><p>NS NS 1 1 4 NS 22.52.1 64.16.1 15.30.5</p><p>H. zeyheri Less.F. Van Heerden 3Koueveld Mountains</p><p>NS NS 2 4 NS NS 54.49.5 58.23.1 37.4 d</p><p>Antimicrobial control f 2.5103 0.3103 0.3103 1.0103 2.5103 0.7103</p><p>DMSO control g 2 4 8 8 16 4a Experiments at least done in duplicate.b At a concentration of 0.1 mg/ml of 5-flourouracil, more than 80% growth was observed for the SF-268 cells, while an IC50 of 1.1 g/ml was determined for the same drug against the MCF-7 cell line (Kamatou et al.,</p><p>2008).c Not susceptible, MIC observed equal to that of solvent control.d Experiments only done once.e Not determined.f Ciprofloxacin used as a positive control against bacteria and Amphotericin B used as a positive control against the yeast.g Solvent control.</p><p>232A.C.U.Lourens</p><p>etal.</p><p>/South</p><p>African</p><p>Journalof</p><p>Botany</p><p>77(2011)</p><p>229235</p></li><li><p>n JoThe above-mentioned results prompted us to investigate theantimicrobial activity and in vitro cytotoxicity of chloroform:methanol (1:1) extracts of Helichrysum species against threedifferent cell lines at a single concentration of 0.1 mg/ml. Theaim was to obtain a general preliminary idea of the cytotoxicityof extracts screened for antimicrobial activity.</p><p>2. Materials and methods</p><p>2.1. Plant material</p><p>The Helichrysum species (Asteraceae) were collected inseveral provinces of South Africa. Voucher specimens weredeposited at the University of KwaZulu-Natal herbarium (NU)in Pietermaritzburg, South Africa (voucher numbers, Table 1).Plants were identified at the National Herbarium at the NationalBiodiversity Institute in Pretoria, South Africa.</p><p>2.2. Preparation of extracts</p><p>Leaves and stems were air-dried (protected from sunlight),ground and extracted twice with chloroform:methanol (1:1) for24 h at room temperature. Thereafter the solvent was removedunder vacuum.</p><p>2.3. Antimicrobial activity</p><p>Six microorganisms, including three Gram-positive bacteria(Bacillus cereus, ATCC 11778, Staphylococcus aureus, ATCC12600, and Staphylococcus epidermidis, ATCC 2223), twoGram-negative bacteria (Klebsiella pneumoniae, NCTC 9633and Pseudomonas aeruginosa, ATCC 9027), and a yeast(Cryptococcus neoformans, ATCC 90112) were used in thescreening of the crude extracts. Selection of test organisms wereundertaken according to the traditional use of species of thisgenus i.e. treatment of respiratory disorders and wounds. Thereference stock cultures were obtained from the National HealthLaboratory Service (NHLS) (Johannesburg) and were main-tained in the microbiology laboratory of the Department ofPharmacy and Pharmacology, University of the Witwatersrand,Johannesburg, South Africa.</p><p>MIC values were determined by the 96-well microplatemethod as described by Eloff (1998) and adapted by Mageeet al. (2006). Stock solutions were prepared by dissolvingsamples in dimethyl sulfoxide (DMSO, 64 mg/ml) sincedissolution problems occurred with solvents such as acetone.These stock solutions were serially diluted to obtain a finalconcentration range of 16 to 0.125 mg/ml of extract afteraddition of the bacterial culture. A fixed bacterial culture of anapproximate inoculum size of 1106 colony forming units(CFU)/ml in Tryptone Soya broth was added to all wells.Further dilutions were prepared when activity below theseranges were observed.</p><p>The plates were incubated at 37 C for 24 h for bacteria and</p><p>A.C.U. Lourens et al. / South Africa48 h for the yeast. MIC values were determined at least induplicate. Positive controls included ciprofloxacin for bacteriaand amphotericin B for the yeast. A solvent control wasincluded with the assay since DMSO has antimicrobial activityand samples with MIC values equal to that found for DMSOwere considered not susceptible (NS, Table 1). After theincubation period, 50 l of a 0.4 mg/ml p-iodonitrotetrazoliumviolet (INT) solution was added to all wells and left for 6 h(except C. neoformans which was left for 12 h) at roomtemperature before reading the MIC's.</p><p>2.4. Cytotoxicity assay</p><p>Cytotoxicity was determined with the sulforhodamine B(SRB) assay (Kamatou et al., 2008; Monks et al., 1991).Transformed human kidney epithelial cells (Graham cells,obtained from Dr. R Van Zyl, University of the Witwatersrand),MCF-7 breast adenocarcinoma and SF-268 glioblastoma cells(obtained from the National Cancer Institute (NCI), USA) wereused to determine cytotoxicity. The Graham cells were includedas a representative of a non-cancerous cell line.</p><p>The Graham cells were cultured in Ham's F10 mediacontaining 5% (v/v) heat inactivated fetal calf serum (FCS) and0.1% gentamicin. The SF-268 and MCF-7 cells weremaintained in RPMI-1640 media containing 5% FCS and0.01% L-glutamine, while media used in the assay included0.1% gentamicin.</p><p>The Graham cells were seeded at 25 000 cells/well, while theMCF-7 and SF-268 cells were seeded at 15 000 cells/well. Cellswere incubated for 24 h at 37 C in 5% CO2 at 100% relativehumidity to allow for attachment to the 96 well plates.</p><p>Crude extracts were weighed and dissolved in DMSO toprepare stock solutions of ca. 10 mg/ml. These were furtherdiluted with appropriate culture media, before addition to themicrotitre plate (after the 24 h incubation period) to obtainconcentrations of 0.1 mg/ml extract per well. Samples wereplated out in triplicate or quadruplicate.</p><p>After a 48 h incubation period, cells were fixed with 50%trichloroacetic acid (TCA) and incubated for 1 h at 4 C. Fixedcells were stained with a 0.4% (w/v) SRB solution for 10 min atroom temperature after drying. The bound dye was solubilisedthrough the addition of 200 l Tris base. The absorbance wasread at 492 nm (Labsystems iEMS reader MF), connected toAscent (version 2.4) software. Cytotoxicity results wereexpressed as average standard deviation of duplicateexperiments.</p><p>The percentage treated cell growth in reference to controlgrowth was calculated as follows:</p><p>%T = C =meanabstestsamplemeanabsbackground 100</p><p>meanabscontrolmeanabsblank </p><p>where mean abs test sample refers to the mean absorbance at492 nm obtained for the three/four similar sample wells.</p><p>mean abs background refers to the mean absorbance at492 nm of the wells containing only sample and media.</p><p>233urnal of Botany 77 (2011) 229235mean abs control refers to absorbance of wells containingonly cell suspension and no sample.</p><p>mean abs blank refers to wells containing only media.</p></li><li><p>Jo3. Results and discussion</p><p>The an...</p></li></ul>


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