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Tuberculosis is a disease of worldwide occurrence that affects millions of people annually. One of the top 10 causes of death and the leading cause from a single infectious agent. Resistance is developing against current an- tituberculosis drugs, which also have toxicity concerns. Some plants from the Menispermaceae family have been used traditionally to treat tuberculo- sis and cough related symptoms. In this study, the antimycobacterial effect of Cissampelos owariensis, Cissampelos mucronata and Tinospora fragosa from the Menispermaceae family were investigated. Acetone, methanol:water (4:1), hot water and dichloromethane:methanol (1:1) extracts of Cissampelos owar- iensis whole plant, Cissampelos mucronata leaves, and stem, Tinospora fra- gosa aerial part were tested against non-pathogenic and pathogenic myco- bacterial strains (M. smegmatis, M. fortuitum, M. aurum, and M. bovis) using a two-fold serial microdilution assay and thiazolyl blue tetrazolium bromide (MTT) as the growth indicator. Cytotoxicity of the active extracts was deter- mined using a tetrazolium based colorimetric assay against Vero monkey kidney cells. The selectivity index of the active extracts was also evaluated. The acetone extracts were most active with minimum inhibitory concentration (MIC) values between 30 - 940 μg/ml. Cissampelos mucronata (leaves and stem) and Cissampelos owariensis were toxic to Vero cells with LC50 values of 20, 30, 40 μg/ml respectively while Tinospora fragosa was safer with LC50 of 3180 μg/ml. Tinospora fragosa also had the highest selectivity index of 39.75. Further studies are on-going on the isolation of the bioactive compounds of the extracts.
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Faculty of
Veterinary Science
Fakulteit Veeartsenykunde
Lefapha la Diseanse tša Bongakadiruiwa
Introduction
Tuberculosis (TB) is a disease of worldwide occurrence that affects millions of
people globally. Prevalence of resistant strains and comorbidity with other
disease conditions makes tuberculosis a disease of public health concern.
Plants offer potential novel sources of potent antimicrobial agents in the form
of bioactive secondary metabolites (Cowan, 1999). Anti-tubercular activity has
been reported in some plants as well as in lichen species (Jimenez-Arellanes, et
al., 2003; Gupta et al., 2007). Some plants from the Menispermaceae family
are used traditionally to treat cough and TB-related symptoms hence the need
for scientific enquiry into their potential anti-tuberculosis activity. The aim of
this study was to investigate and isolate potential antimycobacterial
compounds from selected species of the Menispermaceae family.
Methodology
§The antimycobacterial and cytotoxic effect of Cissampelos mucronata, and
two other species from the Menispermaceae family was evaluated;
§Antimycobacterial activity was determined against non-pathogenic and
pathogenic Mycobacterium strains using a two-fold serial dilution assay and
thiazolyl blue tetrazolium bromide (MTT) as the growth indicator (Eloff,
1998).
§Cytotoxicity was determined against Vero African monkey kidney cells using
a tetrazolium based colorimetric assay (Mosmann, 1983).
§The acetone extracts of C. mucronata leaves and stem were the most active
against the strains used i.e. Mycobacterium smegmatis, M. fortuitum, M.
aurum and M. bovis.
§Fractionation of the acetone extract C. mucronata (aerial parts), was carried
out using vacuum liquid chromatography with hexane, ethyl acetate,
acetone and methanol:chloroform (3:7)
§Bioassay-guided isolation of the active compounds was performed on the
ethyl acetate fraction using column chromatography.
Results and Discussion
§The ethyl acetate fraction of C. mucronata was most active with minimum
inhibitory concentration (MIC) of 0.015-0.08 mg/ml against all the
mycobacterial strains. The MIC was equal or close to the MIC of the positive
controls (rifampicin and streptomycin) with most of the strains (Table 1).
§The hexane fraction was not toxic to Vero cells (Table 2) compared to
doxorubicin (positive control).
§One compound was isolated and is in the process of being identified
(Figure 1).
1,2 1 1 3 4 1
R. T. Akande , I. Famuyide , S. Nkadimeng , A. Aro , T. Hlokwe , L. J. McGaw
1 2 3
Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Sciences, University of Pretoria, Pretoria, South Africa. Nigeria Atomic Energy Commission, Abuja, Nigeria. Department of Agriculture and Animal
4
Health, College of Agriculture and Environmental Sciences, University of South Africa, Florida, South Africa. Bacteriology Section, Agricultural Research Council—Onderstepoort Veterinary Institute, Onderstepoort, Pretoria, South Africa.
References
Cowan, M.M. 1999. Clinical Microbiology Reviews 12, 564-582.
Eloff, J.N. 1998. Planta Medica 64, 711-713.
Gupta, V.K. et al. 2007. Pharmaceutical Biology 45, 200-204.
Jimenez-Arellanes, A. et al. 2003. Phytotherapy Research 17,
903-908.
Mosmann, T. 1983. Journal of Immunological Methods 65, 55-63.
Table 1: Minimum inhibitory concentration of fractions against pathogenic and non-
pathogenic strains (mg/ml)
Table 2: Cytotoxicity and selectivity index of fractions against Vero cells
Conclusion
The extract and fractions were active against the non-pathogenic and
pathogenic mycobacteria strains in line with the traditional use of the
plants against TB-related symptoms. The fractions were relatively
cytotoxic except for the hexane fraction which had a good selectivity
index against the mycobacteria. Structural elucidation and biological
evaluation of the isolated compound is ongoing.
Figure 1: Thin layer chromatography of the Cissampelos mucronata crude extract (CM),
ethyl acetate fraction (ET), and isolated compound (CPD)
The antimycobacterial and cytotoxic effect of Menispermaceae species
#2303
CM ET cpd
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The increase of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) demands the search for alternative antimycobacterial drugs. The aim of this study was to evaluate plants used in Mexican traditional medicine to treat respiratory diseases for activity against MDR-TB. A group of 22 plants was screened for activity against Mycobacterium tuberculosis H37Rv and Mycobacterium avium at concentrations from 50 to 200 microg/mL. The antimycobacterial effect was determined by a microcolorimetric assay with Alamar blue dye. None of the aqueous extracts had antimycobacterial activity. Hexane extracts from Artemisia ludoviciana, Chamaedora tepejilote, Lantana hispida, Juniperus communis and Malva parviflora, and methanol extracts from Artemisia ludoviciana and Juniperus communis inhibited the growth of Mycobacterium tuberculosis. Mycobacterium avium was inhibited by Juniperus communis hexane extract and by Malva parviflora methanol extract. The active extracts were tested against monoresistant variants of Mycobacterium tuberculosis H37Rv (isoniazid, rifampin, streptomycin and ethambutol resistant) and the hexane extract of Lantana hispida showed the best activity. Lantana hispida hexane extract was also active against a group of MDR-TB clinical isolates. In contrast, it did not inhibit the growth of non-tuberculous mycobacteria. The hexane extract of Lantana hispida was fractionated by column chromatography and one of its fractions (FVI) inhibited the growth of all the MDR-TB clinical isolates at concentrations up to 25 microg/mL. This study supports the fact that selecting plants by ethnobotanical criteria enhances the probability of finding species with activity against mycobacteria, and our results point to Lantana hispida as an important source of potential compounds against MDR-TB.
  • J N Eloff
Eloff, J.N. 1998. Planta Medica 64, 711-713.
  • V K Gupta
Gupta, V.K. et al. 2007. Pharmaceutical Biology 45, 200-204.
  • T Mosmann
Mosmann, T. 1983. Journal of Immunological Methods 65, 55-63.