Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 840719, 8 pages http://dx.doi.org/10.1155/2013/840719 Research Article In Vitro Antimicrobial Activity of Extracts from Plants Used Traditionally in South Africa to Treat Tuberculosis and Related Symptoms Balungile Madikizela, Ashwell Rungano Ndhlala, Jeffrey Franklin Finnie, and Johannes Van Staden Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Correspondence should be addressed to Johannes Van Staden; rcpgd@ukzn.ac.za Received 12 November 2012; Accepted 23 January 2013 Academic Editor: Seong-Gyu Ko Copyright © 2013 Balungile Madikizela et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Respiratory ailments are major human killers, especially in developing countries. Tuberculosis (TB) is an infectious disease causing a threat to human healthcare. Many South African plants are used in the traditional treatment of TB and related symptoms, but there has not been a suicient focus on evaluating their antimicrobial properties. he aim of this study was to evaluate the antimicrobial properties of plants used traditionally to treat TB and related symptoms against microorganisms (Klebsiella pneumoniae, Staphylococcus aureus, and Mycobacterium aurum A+) associated with respiratory infections using the microdilution assay. Ten plants were selected based on a survey of available literature of medicinal plants used in South Africa for the treatment of TB and related symptoms. he petroleum ether, dichloromethane, 80% ethanol, and water extracts of the selected plants were evaluated for antibacterial activity. Out of 68 extracts tested from diferent parts of the 10 plant species, 17 showed good antimicrobial activities against at least one or more of the microbial strains tested, with minimum inhibitory concentration ranging from 0.195 to 12.5 mg/mL. he good antimicrobial properties of Abrus precatorius, Terminalia phanerophlebia, Indigofera arrecta, and Pentanisia prunelloides authenticate their traditional use in the treatment of respiratory diseases. hus, further pharmacological and phytochemical analysis is required. 1. Introduction A few decades ago, there was enormous optimism about the decline of threatening infectious diseases due to advances in technology and science [1]. However, due to a rise in the transitional nature of infectious diseases, such optimism has not yet been met. Worldwide infectious diseases are still the leading cause of death, especially in developing countries, claiming millions of lives yearly despite the enormous improvements made in human healthcare [2]. Globally, infectious diseases are the second leading cause of death of children and adults under the age of 50, placing severe burdens on the developing world [3, 4]. Within the last few decades, there has been the emergence of about 30 threatening infectious diseases with the majority capable of afecting humans [4]. he treatment of infectious diseases is facing a major problem at present with many microbes developing resistance to widely used antibiotics and antiviral therapies [5–7]. A few examples are the pathogens associated with acquired immunodeiciency syndrome (AIDS) and multidrug-resistant TB (MDRTB). In the coming years, emerging diseases will probably increase due to travel, urbanization, overcrowding, and inadequate healthcare leading to new interactions between human beings and animals as well as other diseases [8]. hus, addressing the problem of infectious diseases is now an important and urgent requirement. Respiratory diseases are among the major human killers in the world [9]. TB has been reported to be one of the most serious infectious bacterial diseases, causing a threat to healthcare globally despite the availability of drugs and 2 care centres since the 1940s [7, 9–11]. TB is the most common cause of morbidity and mortality especially when coinfection with HIV-1 occurs and is a pandemic in many parts of the world [12]. TB is the most commonly notiied disease in South Africa and the ith largest cause of death [13, 14]. Approximately 285,000 cases of TB were estimated in South Africa in 2005, and it had the seventh highest population with TB in the world and the second in Africa [14]. Due to the important role medicinal plants play in the process of drug discovery and development, they are widely recognised as sources of active antimicrobial metabolites [11]. Several studies have been carried out in South Africa to record the ethnobotanical uses of plants in the treatment of tuberculosis and related symptoms such as coughing, respiratory ailments, and fever [11]. Examples of such detailed studies are recorded in useful reviews of medicinal plants by Watt and Breyer-Brandwijk [36]; Hutchings et al. [15], as well as Van Wyk et al. [17, 29]. hus, there is a great potential in inding medicinal plants with activity against microorganisms related to respiratory infections. Most studies done in South Africa on plants used traditionally to treat respiratory ailments have focused on evaluating their antimycobacterial activity against TB causing bacterial strains [13, 14, 37] but with little emphasis on the related symptoms [38, 39]. herefore, this current study was aimed at evaluating the antimicrobial activity of selected plants against bacteria related to respiratory infection. 2. Materials and Methods 2.1. Selection and Preparation of Plants for Bioassays. Ten plants were selected based on the available literature of medicinal plants used by various South African tribes in the treatment of TB and related symptoms [11, 15, 17, 20, 29]. he plant materials were collected from the University of KwaZulu-Natal Botanical Garden and Ukulinga Research Farm. Voucher specimens were deposited in the University of KwaZulu-Natal (Pietermaritzburg) Herbarium (NU) for botanical authentication. he species name, family, traditional uses, voucher numbers, parts used, and previously screened activity of the plants used in the study are given in Table 1. he plant materials were oven-dried at 50∘ C, ground into powders, and stored in airtight containers. Dried plant material (10 g) was extracted sequentially with 200 mL of each of the four solvents, petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH), and water (from polar to nonpolar) by sonication for 1 h at 15∘ C with temperature being maintained by the addition of ice. he samples were then iltered through Whatman No. 1 ilter paper and concentrated to dryness under reduced pressure using a rotary evaporator to obtain crude extracts. he aqueous extracts were dried under reduced pressure by means of a freeze-drier. Crude extracts were then stored in the dark at 10∘ C until use. he percentage yield for each extract was determined. 2.2. Bacterial Strains and Culture Conditions. hree strains of bacteria were used for antibacterial evaluation against the extracts. he strains were Klebsiella pneumoniae (ATCC Evidence-Based Complementary and Alternative Medicine 13883), Staphylococcus aureus (ATCC 12600), which were obtained from the American type culture collection, and Mycobacterium aurum A+ from the Microbiology Laboratory, Division of Pharmacology, University of Cape Town. he test organisms were selected because of their respiratory pathogenesis and fast growth rates [14, 40]. he strains were maintained on Mueller-Hinton (MH) agar except for M. aurum A+ which was maintained on Middlebrook 7H10 agar supplemented with 0.5% glycerol and 10% OADC (oleic acid, albumin, dextrose, and catalase). 2.3. Antimicrobial Activity Assays. he minimum inhibitory concentrations (MIC) of plant extracts were determined using the broth microdilution method in 96-well microtitre plates for S. aureus and K. pneumoniae as described by Elof [41] and Jadaun et al. [42] for M. aurum A+. he extracts were dissolved in 10% DMSO except for aqueous extracts which were dissolved in sterile distilled water. he test organisms (S. aureus and K. pneumoniae) were cultured in MH broth for 24 h, while Middlebrook broth supplemented with 10% OADC and 0.5% glycerol was used to culture M. aurum A+ for 72 h at 37∘ C. Neomycin and streptomycin were used as the positive controls whereas broth, 10% DMSO, and water were used as negative controls. For antibacterial testing against S. aureus and K. pneumonia, 100 �L of water were added in each well and Middlebrook broth supplemented with 0.5% glycerol, and 10% OADC was used for M. aurum A+. One hundred microliters of solvent controls and test samples were added to the irst wells of the microplate starting with a concentration of 100 mg/mL of extracts and 5 mg/mL for the positive control and then two-fold serially diluted down the wells. he optical density of S. aureus and K. pneumoniae was adjusted with MH broth to match that of McFarland standard, equivalent to 108 colony forming units at 600 nm, and that of M. aurum A+ was adjusted to 0.125 at 550 nm using Middlebrook broth enriched with 0.5% glycerol and 10% OADC growth supplement. One hundred microliters of the diluted culture were added to all the wells. he microtitre plates were then incubated for 24 h at 37∘ C (S. aureus and K. pneumoniae) and for 72 h at 37∘ C (M. aurum A+). Ater incubation, 40 �L of p-iodonitrotetrazolium violet (INT) indicator (S. aureus and K. pneumoniae) and resazurin (M. aurum A+) were added to evaluate growth inhibition. he results were observed ater 30 min for S. aureus and K. pneumoniae and 24 h for M. aurum A+. he lowest concentration containing blue (M. aurum A+) and clear wells (S. aureus and K. pneumoniae) were considered as the MIC values. he assay was repeated twice with two replicates per assay. 3. Results and Discussion he plants selected in this study are all used in South African traditional medicine for the treatment of TB and related symptoms. he percentage yield and MIC values of the plant extract against K. pneumoniae, S. aureus and M. aurum A+ are presented in Table 2. he highest quantity of extract yield was observed from the EtOH extract of Evidence-Based Complementary and Alternative Medicine 3 Table 1: Medicinal plants used traditionally in South Africa to treat TB and related symptoms. Family, species Amaryllidaceae Brunsvigia grandilora Lindl. Asparagaceae Asparagus africanus Lam. Asparagaceae Asparagus falcatus (L.) Oberm. Fabaceae Abrus precatorius subsp. africanus Verdc. Combretaceae Terminalia phanerophlebia Engl. & Diels Leguminosae Indigofera arrecta Benth. ex Harv. & Sond. Moraceae Ficus sur Forssk. Polygalaceae Polygala fruticosa P. J. Bergius Rubiaceae Pentanisia prunelloides Schinz Lamiaceae Leonotis intermedia Lindl. Voucher specimen number BALUNGI 31 BALUNGI 36 BALUNGI 44 Traditional use Coughs and colds Tuberculosis Tuberculosis Parts used Previously screened activity References Bulbs Mutagenic and antimutagenic activities of bulbs [15, 16] Analgesic and anti-inlammatory activities of roots. Two antiprotozoal compounds were isolated [15, 17–19] Shoots Leaves, roots Caryophyllene-type sesquiterpene lactone isolated from the leaves was found to have remarkable efect on the proliferation (IC50 1.82 uM). Antibacterial activity of the roots [20–22] Sedative efects of roots. Antibacterial and cytotoxicity activity of seeds. Five compounds were isolated and identiied as isolavan, quinones, and hydroquinones [15, 23–26] BALUNGI 43 Tuberculosis, bronchitis, whooping cough, chest complaints, and asthma BALUNGI 37 Tuberculosis Roots Antibacterial and antifungal activities of the leaves [20, 27] BALUNGI 41 Tuberculosis Root Antibacterial activity of the leaves [20, 28] BALUNGI 39 Tuberculosis/ulceration of the lung BALUNGI 35 Tuberculosis, blood puriication, intestinal sores,sinusitis, and gonorrhoea BALUNGI 40 and 42 Tuberculosis, chest pain, fever, and toothache BALUNGI 38 Colds, coughs, bronchitis, asthma, tuberculosis, high blood pressure, and jaundice Leaves, roots Bark, root Whole plant Root Leaves, stem Antimalarial and antibacterial activities of the leaves. Antibacterial activity against Gardnerella vaginalis and toxicity [15, 29–31] [11, 29, 32, 33] Antibacterial activity against B. subtilis, S. aureus, E. coli,and K. pneumonia. Antiviral activity of leaves and roots [15, 29, 34] Anti-inlammatory activity [11, 35] 4 Evidence-Based Complementary and Alternative Medicine Table 2: he antibacterial (MIC values) efects of plants used traditionally as remedies for the treatment of tuberculosis and related symptoms in South Africa. Plant species Plant part Extract Extract yield % (DW) L PE DCM 80% EtOH 1.03 1.24 3.54 Water 12.50 0.78 0.195 12.50 12.50 12.50 1.56 2.66 12.50 12.50 3.13 6.25 S PE DCM 80% EtOH Water 2.28 2.87 3.42 2.43 12.50 12.50 6.25 1.56 12.50 6.25 0.78 1.56 6.25 3.13 1.56 3.13 L PE DCM 80% EtOH Water 0.40 0.50 1.50 6.80 12.50 12.50 6.25 1.56 6.25 6.25 0.39 6.25 12.50 12.50 6.25 12.50 L PE DCM 80% EtOH Water 0.80 0.62 2.76 1.00 12.50 12.50 6.25 3.13 3.13 1.56 0.39 2.56 12.50 12.50 3.13 1.56 Bb PE DCM 80% EtOH Water 2.83 0.98 4.47 3.42 12.50 12.50 3.13 3.13 12.50 3.13 3.13 3.13 12.50 6.25 6.25 6.25 B PE DCM 80% EtOH Water 0.50 0.50 0.60 0.40 12.50 12.50 6.25 6.25 6.25 6.35 3.13 6.25 12.50 12.50 6.25 12.50 R PE DCM 80% EtOH Water 3.20 0.70 7.70 2.10 6.25 6.25 0.78 12.50 12.5 6.25 3.13 3.13 12.50 12.50 0.195 3.13 L PE DCM 80% EtOH 2.66 1.79 11.15 9.69 12.50 6.25 0.39 0.78 12.50 12.50 0.39 Water 6.25 12.50 0.78 12.50 R PE DCM 80% EtOH Water 0.60 0.70 11.40 0.80 12.50 12.50 3.13 6.35 6.25 1.56 1.56 12.50 12.50 12.50 1.56 12.50 L PE DCM 80% EtOH Water 1.02 1.00 5.62 3.20 12.50 12.50 6.25 3.13 6.25 12.50 0.195 3.13 12.50 12.50 0.78 1.56 St PE DCM 80% EtOH Water 0.50 0.60 4.80 3.00 6.25 6.25 3.13 3.13 3.13 12.50 1.56 1.56 12.50 12.50 1.56 6.25 A. precatorius subsp. africanus A. africanus A. falcatus B. grandilora Antibacterial activity MIC (mg/mL) Klebsiella Mycobacterium Staphylococcus pneumoniae aurum A+ aureus F. sur I. arrecta L. intermedia 3.13 6.25 Evidence-Based Complementary and Alternative Medicine 5 Table 2: Continued. Plant species Extract Extract yield % (DW) L PE DCM 80% EtOH Water 1.30 1.40 4.40 1.50 6.25 12.50 3.13 3.13 6.25 12.50 0.39 1.56 12.50 6.25 0.195 0.39 R PE DCM 80% EtOH Water 0.20 0.20 11.40 6.76 6.25 6.25 0.39 1.56 3.13 12.50 0.78 1.56 12.50 12.50 0.78 1.56 Wp PE DCM 80% EtOH Water 0.73 1.04 16.67 4.84 6.25 6.25 3.13 3.13 1.56 1.56 3.13 3.13 6.25 6.25 1.56 6.25 L PE DCM 80% EtOH 0.50 1.20 16.00 9.80 6.25 6.25 1.56 0.39 6.25 6.25 0.195 Water 6.25 6.25 0.195 0.39 PE DCM 80% EtOH 0.10 0.30 12.90 1.70 6.25 3.13 3.13 6.25 12.50 6.25 0.195 Water 12.50 6.25 1.56 3.13 PE DCM 80% EtOH Water 1.00 0.40 11.00 3.70 6.25 6.25 0.195 1.56 3.13 1.56 0.195 1.56 6.25 3.13 0.39 1.56 P. prunelloides P. fruticosa T. phanerophlebia Antibacterial activity MIC (mg/mL) Klebsiella Mycobacterium Staphylococcus pneumoniae aurum A+ aureus Plant part R T Neomycin Streptomycin 0.0975 0.098 0.39 0.0975 0.195 L: leaves, R: roots, B: bark, Bb: bulb, S: seeds, St: stem, Wp: whole plant, T: twigs, MIC: minimum inhibitory concentration, DCM: dichloromethane, PE: petroleum ether, EtOH: ethanol, % DW: percentage dry weight Values boldly written are considered very active. P. fruticosa, whilst the lowest one was from PE extracts of the roots of T. phanerophlebia. Generally, EtOH was the best extractant giving the highest mass of extracts while PE yielded the lowest mass. he masses of the water extracts were the second highest to EtOH for most of the species extracted. In this study, only MIC values equal to or less than 1.0 mg/mL for crude extracts were considered as exhibiting good activity [43]. Out of 68 extracts tested from diferent parts of 10 species, 17 were found to have good antimicrobial activities at least against one or more of the strains tested. Twelve extracts showed good activity against M. aurum A+ and 11 against S. aureus. It was observed that M. aurum A+ was the most susceptible bacterium while K. pneumoniae was the most resistant with only 6 extracts showing good activity. Due to lipopolysaccharides present on their outer membrane, Gram-negative bacteria are usually impermeable to most antibacterial compounds [44, 45]. his can explain the low number of active extracts against K. pneumoniae. he water extract of T. phanerophlebia (leaf) showed the best activity with the lowest MIC value of 0.098 mg/mL among the extracts tested. For water extracts only T. phanerophlebia leaves exhibited good activity against all bacterial species tested with MIC values ranging from 0.098 to 0.39 mg/mL. he water extracts of I. arrecta (leaf), P. prunelloides (leaf), and T. phanerophlebia (root) also showed good activity against at least one bacterial strain with MIC values ranging from 0.39 to 0.78 mg/mL. his was encouraging as many reports oten state that water extracts lack bioactivity [46]. he evaluation of aqueous extracts aims to mimic the traditional use; therefore, the observed antibacterial activities exhibited by water extracts could be of interest. Such plants are candidates for further studies such as antimycobacterial evaluation against TB causing bacterial strains. Comparing the root and leaf extracts of I. arrecta, the EtOH extracts of the leaf showed good antibacterial activity against all bacterial species tested with MIC values ranging from 0.39 to 0.78 mg/mL. For P. prunelloides leaves and 6 roots, the EtOH extracts showed good activity with MIC values ranging from 0.195 to 0.78 mg/mL against almost all bacterial species tested. his is noteworthy as it indicates the presence of bioactive compounds in these plants that might help to isolate drugs that cure ailments related to respiratory infection. he palmitic acid previously isolated from roots of P. prunelloides by Yf et al. [34] could be responsible for the antimicrobial activity of this plant observed in this study. Yf et al. [34] also did an antiviral test on the root decoction of P. prunelloides and reported the inhibition of Inluenza A virus. hese results make this plant a potentially efective remedy against respiratory diseases, and the evaluation of palmitic acid against respiratory microbes is required. For T. phanerophlebia leaf, root, and twig extracts, the EtOH extracts showed good activity against almost all bacterial strains tested with MIC values ranging from 0.098 to 0.39 mg/mL. his, however, was excluding the leaf extract that did not exhibit good activity against one strain and root extract that also did not show good activity against two strains. In a study on antimicrobial properties done by Shai et al. [27] against S. aureus, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa, the leaf extracts of T. phanerophlebia exhibited good antimicrobial activity which was also the case in our study against the tested bacteria. In addition, in a literature investigation conducted by Nair et al. [47], several alkaloids were reported to have been isolated from the stem of T. phanerophlebia which exhibited cyclooxygenase-2 enzyme activity. he antimicrobial activity observed from diferent parts of this plant might be due to those alkaloids (cholestane triterpenoids �-sitostenone, stigmast-4-ene-3,6-dione, and �-sitosterol). It is, therefore, important to evaluate these compounds in bioassays against these microorganisms. Among the bark and root extracts of F. sur, only the EtOH extract (root) showed good activity with an MIC value of 0.78 mg/mL against K. pneumoniae and 0.195 mg/mL against S. aureus. he activity of this plant could be due to the triterpene compounds found in the latex isolated in a previous study by Feleke and Brehane [48], which also warrants testing in similar bioassays. Among the leaf and stem of L. intermedia, the EtOH extract (leaf) showed good activity with MIC value of 0.195 against M. aurum A+ and 0.78 against S. aureus. hese indings are very interesting as they indicate the antimicrobial activity of this plant. Of all the extracts of A. africanus and A. falcatus, good activity was observed only in the EtOH extracts with both having an MIC value of 0.39 mg/mL against M. aurum A+. Asparagus species are known to have steroidal saponins as their major bioactive constituents; therefore, the antimycobacterial activity of these two species of Asparagus could be due to saponins [49]. However, among all extracts of the leaves and seeds of A. precatorius subsp. africanus, only the EtOH (leaves and seeds) and DCM extracts (leaves) showed good activity with MIC values ranging from 0.195 to 0.78 mg/mL against M. aurum A+ only. Phytochemical research done in a previous study by Taur and Patil [50] from the root and aerial part of this plant showed the presence of triterpenoids and saponins. Saponins are known to have broad spectrum of pharmacological activities, including antimicrobial properties [51]. In view of the fact that these Evidence-Based Complementary and Alternative Medicine plants were selected based on their traditional uses against TB and related symptoms, these indings are noteworthy. However, all the extracts of B. grandilora and P. fruticosa did not show good activity against all bacterial species tested despite being reported to be used in the treatment of TB and related symptoms. However, bioactivity cannot be completely ruled out from such plant species as they could be active against other bacterial strains that cause respiratory ailments. he other possible explanation could be that antimicrobial efects of these plants are not mediated through direct inhibition on microbial growth but rather through immunostimulation or the compounds potentially active require metabolic activation by certain enzyme in vivo [52]. 4. Conclusion Two or more extracts of A. precatorius subsp. africanus, T. phanerophlebia, I. arrecta, and P. prunelloides showed good antimicrobial activity against at least one or more of the bacterial strains tested. herefore, the good antimicrobial properties of A. precatorius subsp. africanus, T. phanerophlebia, I. arrecta, and P. prunelloides found in this study form a good basis for further pharmacological (such as antimycobacterial evaluation against infectious TB microorganisms) and phytochemical investigation, and they validate the traditional use of these plants in the treatment of respiratory diseases in South Africa. he fact that some species of plants did not display good antimicrobial activity does not mean that they may have the same efect in vivo; thus, it should be noted that they only demonstrated weak activity in vitro. 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