South African Journal of Botany 95 (2014) 97–101

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South African Journal of Botany

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Short communication Antimicrobial and cytotoxic activities of the crude extracts of bicolor leaves, flowers and rhizomes

I.M. Ayoub a, M. El-Shazly a,M.-C.Lub,c, A.N.B. Singab a,⁎ a Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, African Union Organization Street, Cairo 11566, Egypt b Graduate Institute of Marine Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan c National Museum of Marine Biology & Aquarium, Pingtung 944, Taiwan article info abstract

Article history: The crude extracts of leaves, flowers and rhizomes were subjected to comparative antimicrobial Received 11 July 2014 screening against two Gram-positive, two Gram-negative bacteria and four fungal strains using the agar well diffu- Received in revised form 25 August 2014 sion method. The minimum inhibitory concentrations (MIC) of the tested extracts were also determined. Further- Accepted 27 August 2014 more, the cytotoxic activity was evaluated. D. bicolor extracts generally demonstrated notable broad spectrum Available online xxxx antimicrobial activities (MIC values ≤ 500 μg/mL) against all tested pathogens. D. bicolor leaf extract showed potent μ Edited by I Vermaak broad spectrum antimicrobial activity with MIC values ranging between 0.24 and 31.25 g/mL against all tested pathogens. Moreover, the flowers extract exhibited promising antimicrobial activities, displaying MIC values ranging Keywords: between 1.95 and 125 μg/mL against the tested bacteria and fungi. However, the rhizomes extract showed moderate Dietes bicolor antimicrobial activity with MIC values ranging between 31.25 and 500 μg/mL. Despite the potent antimicrobial ac- Yellow wild iris tivity of D. bicolor extracts, they were ineffective as cytotoxic agents against nine tested cancer cell lines, displaying 50% inhibitory concentration (IC50) values above 100 μg/mL. The reported potent antimicrobial activity along with Antimicrobial activity the lack of measurable cytotoxic activity indicated that the antimicrobial activity of D. bicolor crude extracts is me- Cytotoxicity diated through a mechanism other than cytotoxicity. These results suggest that D. bicolor can act as a potential source for natural antibacterial and antifungal agents with a good safety profile at a preliminary level. © 2014 Published by Elsevier B.V. on behalf of SAAB.

1. Introduction pathogens. Among the modern antifungals used nowadays, about 40% are of natural origin (Freiesleben and Jäger, 2014). Infectious diseases are still a leading cause of morbidity and mortal- According to the WHO, 70–95% of the population in developing ity worldwide, despite the great progress in medical technology and sci- countries depend mainly on herbal medicines for their health care entificknowledge(Moellering et al., 2007). Historically, natural (WHO, 2011). Medicinal are freely available, less expensive, products and their derivatives have been an invaluable source of thera- and their use is based on expertise and extensive knowledge among peutic agents. In vitro and in vivo antimicrobial assays have effectively local communities (Mabona and Van Vuuren, 2013; Shai et al., 2008). served as reliable methods to detect several classes of secondary metab- Medicinal plants have been commonly used in traditional medicine olites with potent antimicrobial activity such as penicillins, tetracyclines for the treatment of infectious diseases. Antimicrobials from and glycopeptides (Koehn and Carter, 2005; Silver and Bostian, 1990). sources may exert their activity through mechanisms different from However, resistance to antibiotics is intensifying, thus searching for those of currently used synthetic drugs, thus they can significantly new antimicrobial agents is an urgent unmet goal (Tekwu et al., help in the treatment of resistant microbial strains. Examples of micro- 2012). Besides synthetic small molecules, natural products are still con- organisms that have gained resistance to antimicrobials include Pseudo- sidered as the major sources of innovative new therapeutic entities monas aeruginosa, Escherichia coli, Salmonella typhi, Salmonella targeting a plethora of ailments including infectious diseases (Clardy enteritidis, Shigella dysenteriae, Streptococcus faecalis, Staphylococcus au- and Walsh, 2004). The screening for new antimicrobials often involves reus and Candida albicans (Barbour et al., 2004). plant secondary metabolites exhibiting pharmacological activity against Dietes is a member of family Iridaceae, subfamily Iridoideae (Goldblatt, 1981). It is closely related to both genera Iris and Moraea. The genus Dietes comprises of six species; fivearenativetoSouthAfricaandone(Dietes robinsoniana) is restricted to Lord Howe Island between and Abbreviations:MIC, minimum inhibitory concentration;IC , 50% inhibitory concentra- 50 New Zealand. The five African species include Dietes bicolor, Dietes tion; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. fl ⁎ Corresponding author. Tel.: +20 2 2405 1120; fax: +20 2 2405 1107. iridioides, Dietes flavida, Dietes grandi ora and Dietes butcheriana E-mail address: [email protected] (A.N.B. Singab). (Goldblatt, 1981). D. bicolor (Steud.) Sweet ex Klatt, commonly known

http://dx.doi.org/10.1016/j.sajb.2014.08.012 0254-6299/© 2014 Published by Elsevier B.V. on behalf of SAAB. 98 I.M. Ayoub et al. / South African Journal of Botany 95 (2014) 97–101 as the Yellow wild iris, Peacock Flower or Butterfly Iris, is a rhizomatous activity, cell culture reagents were obtained from Lonza (Basel, perennial plant with sword-like leaves and Iris-like flowers that are Switzerland). Cancer cell lines including HeLa (cervical cancer cell white to creamy yellow in color and have three dark spots each line), DLD-1 (colorectal adenocarcinoma cell line), HCT 116 (colorectal surrounded by an orange outline (Pooley, 1999; Rudall, 1983). The carcinoma), T47D (human ductal breast epithelial tumor cell line), genus name “Dietes” is derived from the Greek word “dis” which means MCF-7 (breast cancer cell line), MDA-231 (breast cancer cell line), twiceandfromtheLatin“etum” which means a plant association for its K562 (human erythromyeloblastoid leukemia cell line) and Molt4 close relationship to both Iris and Moraea. The species name “bicolor” (human acute lymphoblastic leukemia cell line) were obtained from means two-colored (Cunliff and Teicher, 2005). the Graduate Institute of Natural Products, College of Pharmacy, Family Iridaceae is widely used in traditional medicine to treat flu, Kaohsiung Medical University, Kaohsiung 807, Taiwan. Mucosal mast cold, toothache, malaria, and bruise (Lin et al., 2002). Extracts or active cell-derived rat basophilic leukemia (RBL-2H3) cell line was purchased principles obtained from members of this family have shown a wide from the American Type Culture Collection line (Bioresource Collection range of biological activities including antibacterial, antiprotozoal, anti- and Research Centre, Hsin-Chu, Taiwan). Dulbecco's modified Eagle's viral, antioxidant, antinociceptive, anti-inflammatory cytotoxic, and im- minimal essential medium (DMEM) powder and 3-(4,5-dimethyl- munomodulatory activities (Lucena et al., 2007; Rahman et al., 2003). thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained The major secondary metabolites isolated from members of Iridaceae from Sigma-Aldrich (St. Louis, MO, USA). include isoflavonoids, flavonoids, triterpenoids, naphthoquinones, an- thraquinones, naphthalene derivatives, xanthones and simple phenolics 2.4. Antimicrobial activity (Kukula-Koch et al., 2013; Mahabusarakam et al., 2010). Many plants are used in traditional medicine in the form of crude ex- 2.4.1. Microorganisms used in the antimicrobial assays tracts or infusions to treat various infections without scientific evidence The antimicrobial activity of the tested extracts was assessed against (Noumi and Yomi, 2001). Scientific evidence for medicinal claims repre- standard Gram-positive bacteria including Staphylococcus aureus sents the most important limiting factor for indigenous herbal products (RCMB 000108) and Bacillus subtilis (RCMB 000102) and Gram- development (Van Wyk, 2011). Infusions of the inner part of D. iridioides negative bacteria including Pseudomonas aeruginosa (RCMB 000104) rhizomes are used by traditional healers in South Africa for the treat- and Escherichia coli (RCMB 000107). The tested fungi included Aspergil- ment of diarrhea and dysentery (Pujol, 1990). Despite the traditional lus fumigatus (RCMB 002007), Geotrichum candidum (RCMB 052009), use of Dietes for the treatment of diarrhea and dysentery, there are no Candida albicans (RCMB 005008) and Syncephalastrum racemosum scientific reports studying this effect. We could not even find any data (RCMB 005005). All microorganism cultures were obtained from the in literature regarding the phytochemical or pharmacological properties Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar Uni- of this genus. The scarcity of scientific information on the therapeutic versity, Nasr City, Cairo, Egypt. activity of D. bicolor hinders its full utilization in evidence based comple- mentary medicine and as a source of anti-infective agents. Aiming to 2.4.2. Antibacterial activity tackle this problem, we evaluated the in vitro antimicrobial activity of The antibacterial activity was evaluated in vitro using the agar well D. bicolor crude extracts obtained from various parts including leaves, diffusion method. Centrifuged pellets of bacteria from a 24 h old culture flowers and rhizomes. Moreover, the cytotoxic activity of these extracts containing approximately 104–106 CFU (colony forming unit) per mL on nine different cell lines was also evaluated. were spread on the surface of the nutrient agar (NaCl 0.5%, tryptone 1%, yeast extract 0.5%, agar 1%, 1000 mL of distilled water, pH 7.0) 2. Materials and methods which was autoclaved at 121 °C for at least 20 min then cooled down to 45 °C, poured into Petri dishes and allowed to settle. After solidifica- 2.1. Plant material tion, wells of 6 mm diameter were cut in the medium with the help of a sterile metallic borer. Extracts were prepared at a concentration of D. bicolor leaves, flowers and rhizomes were collected in April, 2011 10 mg/mL in dimethyl sulfoxide (DMSO). The tested samples (100 μL) from a botanical garden on Cairo–Alexandria road and were kindly were loaded into the wells. DMSO was loaded as the negative control. identified and authenticated by Mrs Therease Labib, Consultant of All plates were observed for a zone of inhibition at 37 °C for 24 h. The Plant Taxonomy at the Ministry of Agriculture and Director of Orman activity was determined by measuring the diameter of the inhibition Botanical Garden, Giza, Egypt. Voucher specimens were deposited in zone (in mm). Each inhibition zone was measured three times by cali- the herbarium of the Department of Pharmacognosy, Faculty of Phar- per to get an average value. The test was carried out three times for macy, Ain Shams University (voucher specimen number IA-10411). each bacterium culture (Rahman et al., 2001). Gentamicin was used as the positive control at a concentration of 30 μg/mL. 2.2. Preparation of D. bicolor extracts 2.4.3. Antifungal activity Plant material (leaves, flowers and rhizomes) were air-dried and The tested samples were separately screened for their antifungal ac- ground into coarse powder. Fifty grams from each powder were extract- tivity in vitro against A. fumigatus, G. candidum, C. albicans and ed with 80% methanol (Kuete et al., 2012) for 3 days at room tempera- S. racemosum using agar well diffusion method on Sabouraud dextrose ture. Extracts were filtered, evaporated under vacuum at 45 °C, then agar plates (Rathore et al., 2000). The fungi culture was purified by a sin- lyophilized and kept in the refrigerator for further biological studies. gle spore isolation technique (Choi et al., 1999). Sabouraud dextrose agar plates were prepared using a homogeneous mixture of glucose–pep- 2.3. Chemicals and reagents tone–agar (40:10:15) sterilized by autoclaving at 121 °C for 20 min. The sterilized solution (25 mL) was poured in each sterilized Petri dish Solvents used in the study were of analytical grade. All microorgan- in the laminar flow and left for 20 min to form the solidified Sabouraud isms and culture media for the antibacterial and antifungal screening as- dextrose agar plate. These plates were inverted and kept at 30 °C in the says were supplied by the Regional Center for Mycology and incubator to remove moisture and to check for any contamination. Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo, Egypt. Clo- trimazole, the antifungal standard drug, was obtained from Bayer 2.4.4. Antifungal assay Health Care Pharmaceuticals, Germany. Gentamicin, the antibacterial Fungal strain was grown in 5 mL Sabouraud dextrose broth (glucose: standard drug, was obtained from Memphis Co. for Pharmaceutical peptone; 40:10) for 3 to 4 days to achieve 105 CFU/mL cells. The fungal and Chemical Industries, Cairo, Egypt. For the assessment of cytotoxic culture (0.1 mL) was spread out uniformly on the Sabouraud dextrose I.M. Ayoub et al. / South African Journal of Botany 95 (2014) 97–101 99

agar plates by a sterilized triangular folded glass rod. Plates were left for (10 cm cell culture dishes) in a humidified chamber with 5% (v/v) CO2 5–10 min so that the culture was properly adsorbed on the surface of in air at 37 °C. The cells were maintained as monolayer culture by serial Sabouraud dextrose agar plates. Small wells (4 mm × 2 mm) were cut subculturing. All experiments were carried out using cells in the loga- into the plates with the help of a well cutter and the bottom of the rithmic growth phase. wells was sealed with 0.8% soft agar to prevent the flow of the tested sample at the bottom of the well. The tested samples were prepared 2.5.2. Cytotoxicity assay in DMSO at a concentration of 10 mg/mL. Tested samples (100 μL) The sensitivity of human cancer cells to the tested D. bicolor aqueous were loaded into the wells. DMSO was loaded as the negative control. methanolic extracts was determined using the MTT assay (Marks et al., The plates were incubated at 30 °C for 3 to 4 days and then examined 1992; Van de Loosdrecht et al., 1991). The assay depends on the fact for the formation of the inhibition zone. Each inhibition zone was mea- that MTT, a yellow colored compound, is converted by viable cells into sured three times by a caliper to get an average value. The test was per- formazan, a purple derivative. Freshly trypsinized cell suspensions were formed three times for each fungus. Clotrimazole (30 μg/mL) was used seeded at densities of 7000 cells per well (for HeLa, DLD-1, HCT 116, as the positive control (Rathore et al., 2000). T47D, MCF-7, MDA-231), 2 × 104 cells per well (for K562, RBL-2H3) and 4 × 104 cells per well (for Molt4) in a 96-well culture plate and incu- 2.4.5. Determination of the minimum inhibitory concentration (MIC) bated overnight. The tested extracts were dissolved in DMSO to form The agar plate method was used to determine the MIC of the tested stock solutions of 1 mg/mL. Cells were treated with various concentra- μ extracts. Nutrient agar (for bacteria) and Sabouraud dextrose agar (for tions of the tested samples (1, 10 and 100 g/mL).Theappropriatedilu- fungi) were heated in the autoclave for 25 min at 121 °C and allowed tions of each extract were prepared in the culture medium and added fi to cool to 45 °C. Two-fold serial dilutions of each sample were added to the cells (the nal concentration of DMSO was less than 0.2% in the cul- to the medium immediately before it was poured into the Petri dishes. ture well) and were incubated for 72 h at 37 °C under 5% (v/v) CO2.The DMSO was used as the negative control. The culture of each organism medium was removed from each well. A stock MTT solution (5 mg/mL) μ in the nutrient broth (beef extract 5 g/L and peptone 10 g/L, pH = was diluted 1:10 in culture medium and was added to the wells (100 L 7.0) for bacteria and Sabouraud dextrose broth for fungi was diluted per well). Cells were then incubated for 4 h. The medium was removed with sterile distilled water to 105–106 CFU/mL. A loop of each suspen- and the formed formazan crystals were dissolved in DMSO and the absor- sion was inoculated in the appropriate medium with the sample or bance was recorded at 550 nm. The assay was performed in triplicate for the control added. After inoculation, the plates were incubated at 37 °C each sample concentration. The % cell viability of three independent ex- for 24 h for bacteria, and at 30 °C for three to four days for fungi. The periments was calculated by the following formula: MIC was considered to be the lowest concentration that completely inhibited the visible growth of a microorganism compared with the con- O:Doftreatedcells−O:D of culture medium % cell viability ¼ 100 trol (Rahman et al., 2001). Each test was performed in duplicate. The an- O:Dofuntreatedcells−O:D of culture medium tibiotics gentamicin and clotrimazole were used as the positive controls. where O.D = optical density.

2.5. Cytotoxicity studies Cytotoxicity was expressed as IC50 which is the concentration that re- duces the absorbance value of the treated cells by 50% compared to the 2.5.1. Cell culture control (untreated cell). Doxorubicin was used as the positive control. The cytotoxicity of D. bicolor aqueous methanolic extracts was ex- amined on nine cancer cell lines including HeLa, DLD-1, HCT 116, 2.6. Data analysis T47D, MCF-7, MDA-231, K562, Molt4 and RBL-2H3. Cells were main- tained in DMEM supplemented with 10% heat-inactivated fetal bovine Data were measured in triplicates (n = 3) and expressed as serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL pen- means ± S.D. The 50% inhibitory concentration (IC50) was calculated icillin, and 100 μg/mL streptomycin. Cells were cultured in Cellstar by calcuSyn software (Biosoft, Ferguson, MO, USA).

Table 1 Mean inhibition zones and minimum inhibitory concentrations (MICs) of various extracts against different microorganisms.

Microorganism DBLa DBRb DBFc Standard

Inhibition zone MIC Inhibition zone MIC Inhibition zone MIC Inhibition zone MIC (mm)d (μg/mL) (mm) (μg/mL) (mm) (μg/mL) (mm) (μg/mL)

Gram positive bacteria Gent.e Bacillus subtilis (RCMBf 000102) 25.3 ± 0.7 0.24 16.2 ± 0.6 31.25 20.3 ± 0.7 1.95 29.1 ± 0.04 0.03 Staphylococcus aureus (RCMB 20.4 ± 0.9 1.95 14.1 ± 0.2 62.5 18.8 ± 0.5 7.8 25.1 ± 0.08 0.24 000108) Gram negative bacteria Gent. Escherichia coli (RCMB 000107) 19.7 ± 0.6 3.9 15.7 ± 0.3 31.25 17.4 ± 0.5 15.62 25.6 ± 0.04 0.12 Pseudomonas aeruginosa (RCMB 17.2 ± 0.3 15.62 12.2 ± 0.3 125 14.3 ± 0.1 62.5 24.3 ± 0.08 0.24 000104) Fungi Clot.g Aspergillus fumigatus (RCMB 002007) 22.1 ± 0.5 0.48 16.1 ± 0.2 31.25 18.2 ± 0.9 7.8 26.1 ± 0.01 0.12 Candida albicans (RCMB 005008) 18.8 ± 0.1 7.8 10.2 ± 0.8 250 14.4 ± 0.1 62.5 18.3 ± 0.01 3.9 Geotrichum candidum (RCMB 052009) 19.2 ± 0.4 1.95 12.4 ± 0.6 125 15.4 ± 0.5 62.5 23.1 ± 0.03 0.48 Syncephalastrum racemosum (RCMB 16.3 ± 0.1 31.25 9.7 ± 0.3 500 13.1 ± 0.3 125 20.5 ± 0.02 0.97 005005)

a D. bicolor leaf extract. b D. bicolor rhizomes extract. c D. bicolor flowers extract. d Data are measured in triplicate (n = 3) and presented as means ± S.D., well diameter: 6.0 mm (100 μL was tested). e Gentamicin. f Regional Centre of Mycology and Biotechnology, Al-Azhar University. g Clotrimazole. 100 I.M. Ayoub et al. / South African Journal of Botany 95 (2014) 97–101

3. Results and discussion reported antimicrobial activity with the lack of measurable cytotoxicity indicates that D. bicolor extracts act as antimicrobial agents through a 3.1. Antibacterial activity mechanism other than cytotoxicity.

MIC values are commonly used as the selectivity criterion in screen- 4. Conclusion ing for new antimicrobials. A crude extract is considered to be promis- ing with MIC below 100 μg/mL, meanwhile, a pure compound The worldwide increase in the antimicrobial drug resistance and the showing MIC of 4 to 16 μg/mL is considered as a promising antimicrobial promising antimicrobial activity of D. bicolor extracts are encouraging agent with optimal value less than or equal to 1 μg/mL (Cos et al., 2006; factors to investigate the possibility of their utilization as a future source Gibbons, 2008). In general, the crude extracts of D. bicolor leaves, for antimicrobial drug candidates. It is obvious that the crude extracts of flowers and rhizomes inhibited the growth of all tested standard D. bicolor leaves, flowers and rhizomes possess promising broad spec- Gram-positive and Gram-negative bacteria in the diffusion assay. The trum antimicrobial activity against certain Gram-positive and Gram- leaf extract showed the most potent activity against S. aureus and negative bacteria as well as all fungi, especially the leaf extract which B. subtilis showing inhibition zones of 20.4 and 25.3 mm, respectively. exerted the most potent activity. These findings provide scientificevi- The MIC values ranged between 0.24 and 1.95 μg/mL against the tested dence to support the ethno-medicinal uses of Dietes sp. for the treat- Gram-positive bacteria (Table 1). The leaf extract was also effective ment of diarrhea and dysentery. Thus, these findings merits further against the tested Gram-negative bacteria showing inhibition zones of studies to isolate and identify the bioactive compounds responsible for 17.2 and 19.7 mm against P. aeruginosa and E. coli, respectively, with the observed antimicrobial activities. The lack of measurable cytotoxic- MIC values of 15.62 and 3.9 μg/mL. The aqueous methanolic extract of ity against all the nine tested cancer cell lines, suggests the use of the flowers also showed promising antimicrobial activity with MIC D. bicolor as a potential natural antibacterial and antifungal drug with values ranging between 7.8 and 62.5 μg/mL against all tested bacteria. a good safety profile. Meanwhile, the rhizomes extract exhibited good activity against S. aureus, B. subtilis and E. coli with MIC values ranging between 31.25 and 62.5 μg/mL whereas a moderate antimicrobial activity was ob- Conflict of interest served against P. aeruginosa with MIC value of 125 μg/mL. The authors declare that there are no conflicts of interest. 3.2. Antifungal activity Acknowledgment The availability of safe antifungal agents is limited, thus the search for new candidates from natural sources is mandatory. The promising This work was partially funded by the grant offered to Prof Abdel antibacterial activities exhibited by D. bicolor crude extracts, encouraged Nasser B. Singab from the Science and Technology Development Fund us to assess their antifungal activities. In general, all extracts were effec- in Egypt (STDF), grant no. 5251. tive against all tested fungi (Table 1). The aqueous methanolic extract of the leaves exhibited the most potent antifungal activity with zones of inhibition approaching that of clotrimazole (at the used concentrations) References μ and MIC values ranging between 0.48 and 31.25 g/mL. The inhibition Al-Khateeb, E., Finjan, S., Maraqa, A., 2013. Antioxidant and antimicrobial activities of Iris zone of the leaf extract (10 mg/mL) against C. albicans was 18.8 mm, nigricans methanolic extracts containing phenolic compounds. European Scientific while the inhibition zone of the well-known antifungal agent, clotrima- Journal 9, 83–91. μ Barbour, E.K., Al Sharif, M., Sagherian, V.K., Habre, A.N., Talhouk, R.S., Talhouk, S.N., 2004. zole (30 g/mL), was 18.3 mm. Furthermore, the MIC value of the leaves Screening of selected indigenous plants of Lebanon for antimicrobial activity. Journal extract against C. albicans was 7.8 μg/mL which was found to be twice of Ethnopharmacology 93, 1–7. that of clotrimazole (3.9 μg/mL). These data suggest that the Choi, Y.-W., Hyde, K.D., Ho, W., 1999. Single spore isolation of fungi. Fungal Diversity 3, – anticandidal activity of the leaf extract is very promising and compara- 29 38. Clardy, J., Walsh, C., 2004. Lessons from natural molecules. Nature 432, 829–837. ble to that of clotrimazole. The aqueous methanolic extract of the Cos, P., Vlietinck, A.J., Berghe, D.V., Maes, L., 2006. Anti-infective potential of natural prod- flowers showed the most potent activity against A. fumigatus (MIC ucts: how to develop a stronger in vitro ‘proof-of-concept’. Journal of – 7.8 μg/mL). However, a moderate activity was observed against Ethnopharmacology 106, 290 302. μ Cunliff, J., Teicher, U., 2005. Botanical Names and Their MeaningsAvailable from www. S. racemosum (MIC 125 g/mL). On the other hand, the rhizomes exhib- biosciencecommunications.dk/botanical/A-K.doc. ited a moderate to weak antifungal activity with MIC values ranging be- Freiesleben, S., Jäger, A., 2014. Correlation between plant secondary metabolites and their tween 31.25 and 500 μg/mL against the tested fungi. The results antifungal mechanisms — a review. Medicinal & Aromatic Plants 3, 154. Gibbons, S., 2008. Phytochemicals for bacterial resistance—strengths, weaknesses and op- obtained in this study were consistent with the reports on the antimi- portunities. Planta Medica 74, 594–602. crobial activity of the crude alcoholic extracts of different species be- Goldblatt, P., 1981. Systematics, phylogeny and evolution of Dietes (Iridaceae). Annals of longing to family Iridaceae (Al-Khateeb et al., 2013; Ifesan et al., 2010; the Missouri Botanical Garden 68, 132–153. Ifesan, B.O., Ibrahim, D., Voravuthikunchai, S.P., 2010. Antimicrobial activity of crude Ramtin et al., 2013; Rani and Nair, 2011). ethanolic extract from Eleutherine americana. Journal of Food, Agriculture & Environ- ment 8, 1233–1236. 3.3. Cytotoxicity Koehn, F.E., Carter, G.T., 2005. The evolving role of natural products in drug discovery. Na- ture Reviews Drug Discovery 4, 206–220. Kuete, V., Wiench, B., Hegazy, M.E., Mohamed, T.A., Fankam, A.G., Shahat, A.A., Efferth, T., MTT assay was used to evaluate the cytotoxic activity of the crude 2012. Antibacterial activity and cytotoxicity of selected Egyptian medicinal plants. extracts of D. bicolor leaves, flowers and rhizomes on nine different can- Planta Medica 78, 193–199. ł ź cer cell lines, namely HeLa, DLD-1, HCT 116, T47D, MCF-7, MDA-231, Kukula-Koch, W., Sieniawska, E., Widelski, J., Urjin, O., G owniak, P., Skalicka-Wo niak, K., 2013. Major secondary metabolites of Iris spp. Phytochemistry Reviews 1–30. K562, Molt4 and RBL-2H3. None of the D. bicolor extracts exhibited cy- Lin, J., Puckree, T., Mvelase, T.P., 2002. Anti-diarrhoeal evaluation of some medicinal plants totoxicity against all cancer cell lines after 72 h treatment, compared used by Zulu traditional healers. Journal of Ethnopharmacology 79, 53–56. Lucena, G.M.R.S., Gadotti, V.M., Maffi, L.C., Silva, G.S., Azevedo, M.S., Santos, A.R.S., 2007. to the control. The IC50 values were found to be greater than fl μ Antinociceptive and anti-in ammatory properties from the bulbs of Cipura paludosa 100 g/mL for all extracts against the tested cancer cell lines. Mean- Aubl. Journal of Ethnopharmacology 112, 19–. 25 while, doxorubicin the positive control, exhibited IC50 values ranging Mabona, U., Van Vuuren, S.F., 2013. Southern African medicinal plants used to treat skin between 0.001 and 0.20 μg/ml. Despite the antimicrobial activity exhib- diseases. South African Journal of Botany 87, 175–193. fl Mahabusarakam, W., Hemtasin, C., Chakthong, S., Voravuthikunchai, S.P., Olawumi, I.B., ited by the crude extracts of D. bicolor leaves, owers and rhizomes, they 2010. Naphthoquinones, anthraquinones and naphthalene derivatives from the were ineffective as cytotoxic agents at doses up to 100 μg/mL. The bulbs of Eleutherine americana. Planta Medica 76, 345–349. I.M. Ayoub et al. / South African Journal of Botany 95 (2014) 97–101 101

Marks, D.C., Belov, L., Davey, M.W., Davey, R.A., Kidman, A.D., 1992. The MTT cell viability Rathore, H., Mittal, S., Kumar, S., 2000. Synthesis, characterization and antifungal activities assay for cytotoxicity testing in multidrug-resistant human leukemic cells. Leukemia of 3d-transition metal complexes of 1-acetylpiperazinyldithiocarbamate, M (acpdtc) Research 16, 1165–1173. 2. Pesticide Research Journal 12, 103–107. Moellering Jr., R.C., Graybill, J.R., McGowan Jr., J.E., Corey, L., 2007. Antimicrobial resistance Rudall, P., 1983. Leaf anatomy and relationships of Dietes (Iridaceae). Nordic Journal of prevention initiative—an update: proceedings of an expert panel on resistance. The Botany 3, 471–477. American Journal of Medicine 120, S4–S25. Shai, L.J., McGaw, L.J., Masoko, P., Eloff, J.N., 2008. Antifungal and antibacterial activity of Noumi, E., Yomi, A., 2001. Medicinal plants used for intestinal diseases in Mbalmayo Re- seven traditionally used South African plant species active against Candida albicans. gion, Central Province, Cameroon. Fitoterapia 72, 246–254. South African Journal of Botany 74, 677–684. Pooley, E., 1999. A Field Guide to Wild Flowers KwaZulu-Natal and the Eastern Region. Silver, L., Bostian, K., 1990. Screening of natural products for antimicrobial agents. Natal Flora Publications Trust, Durban and Scottsville. European Journal of Clinical Microbiology & Infectious Diseases 9, 455–461. Pujol, J., 1990. NaturAfrica: the herbalist handbook: African Flora, Medicinal Plants. J. Pujol Tekwu, E.M., Pieme, A.C., Beng, V.P., 2012. Investigations of antimicrobial activity of some Natural Healers Foundation. Cameroonian medicinal plant extracts against bacteria and yeast with gastrointesti- Rahman, A.-u, Choudhary, M.I., Thomsen, W.J., 2001. Bioassay Techniques for Drug Devel- nal relevance. Journal of Ethnopharmacology 142, 265–273. opment. Harwood Academic Publishers, The Netherlands. Van de Loosdrecht, A.A., Nennie, E., Ossenkoppele, G.J., Beelen, R.H., Langenhuijsen, M.M., Rahman, A.-u, Nasim, S., Baig, I., Jalil, S., Orhan, I., Sener, B., Choudhary, M.I., 2003. Anti- 1991. Cell mediated cytotoxicity against U 937 cells by human monocytes and mac- inflammatory isoflavonoids from the rhizomes of Iris germanica. Journal of rophages in a modified colorimetric MTT assay: a methodological study. Journal of Ethnopharmacology 86, 177–180. Immunological Methods 141, 15–22. Ramtin, M., Massiha, A., Khoshkholgh-Pahlaviani, M.R.M., Issazadeh, K., Assmar, M., Van Wyk, B.E., 2011. The potential of South African plants in the development of new me- Zarrabi, S., 2013. In vitro antimicrobial activity of Iris Pseudacorus and Urtica Dioica. dicinal products. South African Journal of Botany 77, 812–829. Zahedan Journal of Research in Medical Sciences 35–39. WHO, 2011. The World Medicines Situation, Traditional Medicines: Global Situation, Is- Rani, V.S., Nair, B.R., 2011. Antimicrobial effects of crude extracts of Eleutherine bulbosa. sues and Challenges. World Health Organization, Geneva. Journal of Medicinal and Aromatic Plant Sciences 33, 46–52.