& Bioch ial em b ic ro a c l i T M e f c Magwenzi et al., J Microbial Biochem Technol 2014, S3 h o Journal of n l o a n l o r DOI: 10.4172/1948-5948.S3-003 g u y o J ISSN: 1948-5948 Microbial & Biochemical Technology Research Article Article OpenOpen Access Access The Effect of Selected Combretum Species from Zimbabwe on the Growth and Drug Efflux Systems of Mycobacterium aurum and Mycobacterium smegmatis Ruvimbo Magwenzi, Colet Nyakunu and Stanley Mukanganyama* Department of Biochemistry, University of Zimbabwe, Mt. Pleasant, Harare, Zimbabwe Abstract Treatment of tuberculosis has become a challenge due to the rapid increase of multidrug and extensive drug resistant Mycobacterium tuberculosis. Medicinal plants might represent a possible source for new potent antibacterials to which pathogen strains are not resistant. In this study, five Combretum plant species-Combretum imberbe, Combretum zeyheri, Combretum hereroense, Combretum elaeagnoides and Combretum platypetalum used in traditional medicine were exposed to susceptibility tests to determine the effects of drug accumulation in avirulent M. smegmatis and M. aurum. The MIC values of the potent plant species was then determined. Using the agar disc diffusion assay, it was found out that the only the ethanolic extract from Combretum imberbe was active on M. smegmatis and it had an MIC of 125 µg/ml in the broth microdilution assay. However, using Sabouraud dextrose broth, Combretum platypetalum was found to have antimycobacterial effects which were not detected when using the agar disc diffusion assay. MIC of 63 and 125 µg/ml and MBCs of 250 and 500 µg/ml were obtained for C. platypetalum for M. smegmatis and M. aurum respectively. C. imberbe extract gave an MIC of 125 µg/ml but did not produce MBCs suggesting that it is bacteriostatic and not bactericidal. Drug accumulation transport assays were performed on C. imberbe, C. hereroense and C. platypetalum and results show that extracts from two plants are efflux pump inhibitors. Determination of the IC50 for the transport process was as conducted onCombretum imberbe using CCCP as the standard inhibitor. Both plants extract in addition to C. hereroense maybe potential sources for leads for efflux pump inhibitor in mycobacteria. Keywords: Tuberculosis; Mycobacteria; Combretum species; contributed to the resurgence of tuberculosis [6]. Resistance decreases Antituberculosis; Drug efflux the effectiveness of most available antitubercular agents, and therefore, there is an urgent need to develop new drugs to help reduce the global Introduction burden of tuberculosis [6]. Tuberculosis (TB) has threatened humanity for thousands of Plants comprise the largest component of the diverse therapeutic years and remains one of the deadliest diseases on planet earth [1]. elements of traditional health care practices in both humans and It is the leading cause of death worldwide from a single pathogen, animals. Plants are rich in a wide variety of secondary metabolites, claiming more lives than AIDS, malaria, diarrhea, leprosy, and all other such as tannins, terpenoids, alkaloids, and flavonoids, which have tropical diseases combined [2]. TB is believed to be largely a disease been found in vitro to have antimicrobial properties [7]. A number of poverty, with the highest incidences occurring in Asia and Africa of interesting outcomes have been found with the use of a mixture of [3]. The increase in TB incidence in Africa is strongly associated with natural products to treat diseases, most notably the synergistic effects the prevalence of HIV infection among TB patients, exceeding 60% and polypharmacological application of plant extracts [8]. Since in South Africa, Botswana, Zambia, and Zimbabwe [4]. Zimbabwe is the advent of antibiotics, in the 1950s, the use of plant derivatives as ranked 16th on the list of 22 high burden TB countries in the world. antimicrobials has been virtually nonexistent. The interest in using plant It has indicated that Zimbabwe had 71,961 tuberculosis cases in 2007 extracts for treatment of microbial infections has increased in the late and an estimated incidence rate of 540 cases per 100,000 populations 1990s, as conventional antibiotics become ineffective [7]. Antimicrobial [5]. Human tuberculosis (TB) is mainly caused by Mycobacterium compounds from plants may inhibit bacteria/fungi through different tuberculosis. The mycobacterium is aerobic, pathogenic and usually mechanisms than conventional antibiotics, and could therefore be establishes its infection in the lungs [2]. of clinical value in the treatment of resistant microbes [9]. Natural Treatment of TB requires multi-drug therapy comprising; an initial intensive phase of rifampicin, isoniazid, pyrazinamide, and ethambutol daily for two months and a continuation phase of rifampicin and *Corresponding author: Dr. Stanley Mukanganyama, Biomolecular Interactions isoniazid for a further 4 months, either daily or 3 times a week. Analyses Group, Department of Biochemistry, University of Zimbabwe, P.O. Isoniazid and ethambutol, eradicate most of the rapidly replicating Box MP 167, Mt. Pleasant Harare, Zimbabwe, Tel: 263-4-2917638/09; E-mail: [email protected], [email protected] bacilli in the first 2 weeks of treatment. Thereafter, rifampicin and pyrazinamide have an important role in the sterilization of lesions by Received April 15, 2014; Accepted May 07, 2014; Published May 14, 2014 eradicating organisms; these two drugs are crucial for the successful Citation: Magwenzi R, Nyakunu C, Mukanganyama S (2014) The Effect of 6-month treatment regimens. Rifampicin and isoniazid are the main Selected Combretum Species from Zimbabwe on the Growth and Drug Efflux drugs used today, rifampicin being the more important agent in Systems of Mycobacterium aurum and Mycobacterium smegmatis. J Microbial Biochem Technol S3: 003. doi:10.4172/1948-5948.S3-003 terms of reducing the duration of treatment and assuring favorable outcomes. However, these drugs are being rendered useless because of Copyright: © 2014 Magwenzi R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits the emergence of drug-resistant TB, such as multi-drug resistant TB unrestricted use, distribution, and reproduction in any medium, provided the (MDR-TB) and extensively drug-resistant TB (XDR-TB) and this has original author and source are credited J Microbial Biochem Technol Bacteria: Biochemical Physiology ISSN:1948-5948 JMBT, an open access journal Citation: Magwenzi R, Nyakunu C, Mukanganyama S (2014) The Effect of Selected Combretum Species from Zimbabwe on the Growth and Drug Efflux Systems of Mycobacterium aurum and Mycobacterium smegmatis. J Microbial Biochem Technol S3: 003. doi:10.4172/1948-5948. S3-003 Page 2 of 7 products continue to play a most significant role in the drug discovery E7 for C. platypetalum were kept in the Department of Biochemistry, and development process, and plants are recognized as a useful source University of Zimbabwe. The plant leaves were oven dried at 50°C and of highly active antimycobacterial metabolites [10]. Antimicrobial ground to a fine powder in a two-speed electric blender. A volume of 8 resistance in medicine is now recognized as a major emerging problem ml of ethanol was added to two grams of the powder and the mixture of public health importance [11]. The search and development of new, was shaken in a vortex mixer for 5 minutes. The mixture was left to more efficient and safe antimycobacterial drugs is warranted. At least sit for an hour and was then filtered using a syringe that had a piece twenty four species of Combretum are well known in African traditional of cloth inserted in it and sterilization of the suspension was done by medicine and are used for the treatment of a variety of ailments and using a millipore 45 µm sterile filter and 2 ml syringe into a labeled diseases, ranging from scorpion and snake bites, mental problems, small glass vial. The solvent was evaporated under a stream of air in heart and worm remedies to fever and microbial infections. The wide a fume cupboard at room temperature to quantify the extraction. The spread use of Combretum species in treatment of TB related symptoms extract was then dissolved in absolute ethanol for use. and other ailments makes them potential sources of lead compounds that can be developed as antitubercular drugs. Many species of Growth of bacteria Combretum have been found to possess powerful antibacterial and Middlebrook 7H9 media was made up with 5.2 g/L Middlebrook antifungal effects. The large number of antimicrobial compounds 7H9 base supplemented with 1 g/L casein hydrosylate (CAH) was used found in species of Combretum might explain why they are so widely for the growth of mycobacteria. The media components were dissolved used in African traditional medicine. Among antimicrobial active in 1 liter of boiled distilled water and autoclaved to sterilize them. compounds isolated from Combretum species are combretastatins Mycobacterium aurum and Mycobacterium smegmatis were grown in (bibenzyle compounds), acidic tetracyclic and pentacyclic triterpenes/ Middlebrook 7H9 media supplemented with casein acid hydrosylate triterpenoids, ellagitannins, phenanthrenes, flavonoids, saponins and and in Middlebrook 7H9 solid media at 37°C under aerobic conditions. cycloartane glycosides [12]. The leaves, roots and stem bark of C. zeyheri are used medicinally. The leaves are used frequently and have Determination of antimycobacterial activity a variety of uses in African traditional medicine. The smoke of burnt To determine the activity of the plant extracts as antimycobacterials, leaves is inhaled for treatment of coughs. The leaves of C. imberbe are initial screening was carried out using the agar disc diffusion method used in the treatment of diarrhea and cough, symptoms that can be [13]. Once identified as being potent, growth inhibition parameters related to bacterial and fungal infection. were determined using the broth microdilution method. The method The study was undertaken to test the antimycobacterial activity of used was modified from that of [13].