THE ANTIMICROBIAL AND ANTIMYCOBACTERIAL ACTIVITY OF PLANTS USED FOR THE TREATMENT OF RESPIRATORY AILMENTS IN SOUTHERN AFRICA AND THE ISOLATION OF ANACARDIC ACID FROM OZOROA PANICULOSA

Abstract

Tuberculosis (TB) is one of the leading causes of mortality in the developing world. Mycobacterium tuberculosis, the causative organism, infects approximately a third of the world’s population. With high rates of HIV infection, particularly in sub-Saharan Africa, TB rates are inevitably soaring. Treatment regimens are based on multi-drug therapy taken over a prolo nged period, leading to poor patient compliance, in turn resulting in the development of multi-drug resistant TB (MDR-TB) which is difficult to treat. The need for new anti-TB drugs that can decrease the period of treatment or the number of doses and that will be effective against latent and MDR-TB is desperate. An added advantage would be the ability of a new class of anti-TB drugs to have a novel target to avoid potential crossresistance to existing drugs. Various approaches have been taken to antituberculosis drug development, including the high-throughput screening of plants, which represent an enormous, largely untapped resource of novel compounds. A further parameter to increase the chances of finding promising lead compounds is to focus research on plants that have traditionally been used to treat symptoms associated with TB. Traditional herbalists prescribing plant-based treatments have long played an important role in the provision of primary healthcare, especially in rural areas where most of the population is poor and unable to afford modern drugs. The aim of this study is to research literature resources pertaining to medicinal plants in southern Africa used to treat symptoms associated with TB, collect these plants, prepare methanol and acetone extracts for the antimicrobial and antimycobacterial testing, identify a plant with promising activity, and to isolate the active principle/s. These compounds in turn would be identified structurally and tested for activity against a range of micro-organisms, including mycobacteria, as well as for cytotoxicity. Twenty-three plant parts from nineteen different species were collected and 46 extracts prepared. These extracts were tested against a range of Gram-positive and –negative bacteria and fungi using the disc diffusion and broth micro-dilution methods. The effects of these extracts were also tested against non-pathogenic mycobacteria using the broth microdilution method, and against M. tuberculosis using the radiometric BACTEC 460 ABSTRACT V method. Te n of the nineteen species exhibited activity against two or more of the eleven organisms tested, namely Xerophyta retinervis bark, Helichrysum odoratissimum leaves, Ozoroa paniculosa bark, Eriocephalus africanus leaves, Siphonochilus aethiopicus roots, Conyza scabrida leaves, Syzigium cordatum bark, Tetradenia riparia leaves, Datura stramonium leaves and Dioscorea sylvatica tubers. The acetone extract of O. paniculosa was further pursued for the isolation of its active principles as it exhibited potent activity against Enterococcus faecalis, Pseudomonas aeruginosa, M. tuberculosis and Mycobacterium aurum with MIC’s of less than 1mg/ml. Previous work on related species has indicated anti-cancer, anti-helminthic, anti- malarial and anti-schistosomiasis activities, but no antimicrobial or antimycobacterial properties have been researched. Moronic, anacardic, and ginkgolic acids have previously been isolated from related species. Bio-assay guided fractionation led to the isolation of a known C15:1 anacardic acid (compound 1) and two HPLC fractions (HPLC2 and HPLC3) of which the predominant components of HPLC3 is the saturated analogue of anacardic acid. NMR data of HPLC2 suggest it is also a C15:1 anacardic acid, although the location of its double bond is as yet unkown. These compounds were cytotoxic to CHO cells at 44.9 – 64μg/ml. Compound 1 and HPLC2 were moderately active against M. tuberculosis with MIC’s of 125μg/ml, while HPLC3 had increased activity with an MIC of 31.3μg/ml, work not previously reported. Similarly, HPLC3 had the greatest activity a gainst M. smegmatis with an IC50 value of 22.1μg/ml. M. aurum had higher IC 50 values ranging from 98.4 to 112.5μg/ml for all three compounds. Compound 1 had potent activity against a range of Grampositive bacteria with IC50 values of 1.3, 2.1 and 6.5μg/ml against Bacillus cereus, E. faecalis and Staphylococcus aureus respectively. Furthermore, this compound had good activity against one of two drug-resistant strains of S. aureus tested with IC50’s of 6.9 and 43.2μg/ml. The saturated anacardic acid (HPLC3) in general had poorer activity against Gram-positives than its unsaturated analogues, consistent with reported literature. The anacardic acids had decreased activity against yeast and Gram-negative organisms tested, with IC50 values of around 80μg/ml against Candida albicans, 19 ->250μg/ml for Serratia odorifera, 26 - >250μg/ml for Klebsiella pneumoniae and 17 – 68μg/ml for ABSTRACT VI Pseudomonas aeruginosa, results comparable to those previously reported. The saturated anacardic acid appeared to have better activity than its unsaturated analogues against Gram- negatives and yeast. The current literature suggests that lipophilic compounds have greater activity against M. tuberculosis. Results obtained in this project are in support of these findings, as the saturated anacardic acid, believed to be the major constituent of HPLC3 based on NMR data available, had increased activity against this organism compared to the unsaturated and less hydrophobic compound 1 and HPLC2. It is possible that these findings implicate anacardic acid in the disruption of the mycobacterial cell wall.