Abstract: Extracts of seventeen traditional Australian medicinal plants used to treat infections and respiratory conditions were tested for anti-mycobacterial activity against the fast-growing strains, Mycobacterium fortuitum and M. smegmatis. Four extracts, the aerial parts of Pterocaulon sphacelatum (Asteraceae), the bark and leaves of Acacia ligulata (Mimosaceae), the leaves and stems of Eremophila alternifolia (Myoporaceae) and the leaves of Eremophila longifolia, showed activity against M. smegmatis only, while the two Eremophila extracts were also active against M. fortuitum. The minimum inhibitory concentrations ranged from 20-66 mg mL-1. The identification of the anti-mycobacterial compounds from these extracts may yield new and effective agents to combat diseases caused by Mycobacterium species.
INTRODUCTION
Tuberculosis (TB) is a serious respiratory infection and one of the most important diseases worldwide. Epidemiological evidence indicates that one third of the world`s population is infected with the causative bacterial agent, Mycobacterium tuberculosis, eight million cases emerge annually and about 2 million people die from the disease (Gautam et al., 2007). Recent data from the World Health Organization support these estimates with 8.8 million new TB cases and 1.6 million people deaths, including 195,000 patients infected with HIV, in 2005 (WHO, 2007). By 2020, it has been estimated that nearly 1 billion people will become infected, 200 million will develop disease and 35 million will die from TB, although recent figures suggest that the global epidemic is on the threshold of decline (WHO, 2007). However, the emergence and spread of drug-resistant M. tuberculosis, especially multidrug-resistant (MDR) strains resistant to standard TB drugs such as isoniazid and rifampin, reduces the efficacy of current treatment regimes and threatens programs aimed at controlling TB (Sacchettini et al., 2008). Drug-resistant TB is caused by a number of factors including slow-acting drugs and inadequate health-care systems, leading to poor compliance, incomplete treatment and relapse (Sacchettini et al., 2008). Treatment of MDR-TB requires the use of at least three drugs not prescribed previously to the patient and in some cases four or more anti-TB agents are necessary. Nevertheless, not all cases are cured. As a result, there is an urgent need for potent new drugs to deal properly with cases of MDR-TB. Moreover, the increasing occurrence of infections caused by other species of Mycobacterium is of additional concern (Bodle et al., 2008).
The increased incidence of new and reemerging infectious diseases, such as TB, has led to the need for new antimicrobial compounds that have diverse chemical structures and different mechanisms of action. Natural products, such as those found in plants, or their semi-synthetic derivatives, may provide novel compounds that can be developed into antimicrobial drugs (Bisht et al., 2006; McRae et al., 2007). Numerous plant species have been used as traditional medicines by the Aboriginal peoples of Australia (Lassak and McCarthy, 2001). However, few have been systematically investigated for their therapeutic, pharmacological and chemical properties. Previously, we found that compounds isolated from the leaves of the Australian tropical medicinal plant, Planchonia careya, were active against fast-growing strains of mycobacteria (McRae et al., 2008). In the present study, we have broadened our search in the hope of discovering new potential anti-mycobacterial phytochemicals. Given the unique nature of Australia`s native flora, it is likely that new or alternative anti-TB agents will be discovered among plants known to have medicinal properties. To increase the probability of finding appropriate drugs, the plants chosen for investigation have been used as traditional medicines to treat respiratory infections.
MATERIALS AND METHODS
The ethanol extracts used in this study are part of a larger collection that has been described previously (Palombo and Semple, 2001). The extracts studied here were derived from 17 plants belonging to 10 families and were selected because of their traditional use in the treatment of infections, particularly respiratory illness (Table 1). Extracts were prepared as follows. Plant material was freeze-dried and milled to a coarse powder.
| Table 1: | Botanical names of plant species screened, parts extracted and traditional medicinal uses |
| aFamily names follow Cronquist (1981). bGenus and species names follow Jessop (1993). cPlant parts extracted: AP: Aerial Parts; B: Bark; L: Leaves; RB: Root Bark; S: Stem; SB: Stem Base; WP: Whole Plant | |
Powdered material was placed in a glass column plugged with cotton wool and extracted with ethanol at room temperature for 24 h. A second extraction with ethanol was carried out and the two portions combined and concentrated in vacuo. The concentrated extract was centrifuged at 10,000 x g for 20 min at 20°C. The supernatant was recovered, filter sterilised and stored at -70°C.
Isolates of the fast-growing mycobacterial strains, Mycobacterium fortuitum and M. smegmatis, were obtained from the Department of Microbiology, Monash University, Victoria, Australia. M. fortuitum and M. smegmatis are commonly used as a lternatives to the virulent species, M. tuberculosis, to indicate the anti-mycobacterial potential of plant extracts and other natural products (Gautam et al., 2007). Bacteria were cultivated in Brain Heart Infusion (BHI) broth and on BHI agar (Oxoid, Ltd.) at 37°C for 5 days. To determine anti-mycobacterial activity of extracts, plate-hole diffusion assays were used. Briefly, molten BHI agar inoculated with bacterial culture was poured into a petri dish and allowed to solidify. A sterile cork-borer (5 mm diamete) was used to make holes in the agar. Plant extracts (10 μL) were added to the holes and the plates left for 30 min to allow the extracts to absorb into the agar, after which the plates were incubated for 5 days at 37°C. Ethanol was used as a negative control. To aid in the visualization of zones of growth inhibition around the holes, a 2 mg mL-1 solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was sprayed over the agar. The plates were further incubated at 37°C for 30 min, after which the zones of inhibition (clear areas amongst a background of purple) were measured. For the extracts which showed activity, the Minimum Inhibitory Concentrations (MIC) were determined using two-fold serial dilutions of the extracts in plate-hole diffusion assays (McRae et al., 2008).
RESULTS AND DISCUSSION
Screening of all extracts using plate-hole diffusion assays revealed that four displayed anti-mycobacterial activity (zones of inhibition ranged from 10-18 mm; data not shown). The results of MIC assays are presented in Table 2. As indicated from the plate-hole diffusion assays, four of the extracts showed activity against M. smegmatis and two were also active against M. fortuitum. The greater level of activity towards M. smegmatis was also reflected in the MIC results which showed that, for the extracts which were active against both species, the MIC levels were greater for M. fortuitum. Of particular interest is that the Eremophila extracts exhibited broader activity compared to the other plants. These and other members of the Eremophila genus have previously been shown to have considerable broad-spectrum activity against Gram positive bacteria, including antibiotic-resistant strains (Palombo and Semple, 2001, 2002; Ndi et al., 2007a). However, this is the first study to demonstrate activity against species of Mycobacterium. The antibacterial activity of an extract of Eremophila duttonii against Staphylococcus aureus was shown to be the result of increased cell permeability due to cell membrane damage (Tomlinson and Palombo, 2005) while the antimicrobial constituents of some Eremophila species have recently been identified as o-naphthoquinones and serrulatane diterpenoids (Ndi et al., 2007b, c; Smith et al., 2007). Since terpenoids exert their antimicrobial activity by destabilization and permeabilization of bacterial membranes (Smith et al., 2007), it would be of interest to determine if the active extracts identified in this study exert similar damage to the mycobacterial cell membrane. There are limited reports of traditional uses of Australian plants in the treatment of TB (Lassak and McCarthy, 2001), although specific activity against bacteria has not been determined.
| Table 2: | Anti-mycobacterial activity of Australian medicinal plant extracts |
| -: No activity | |
The present study is the first extensive screening of Australian medicinal plants for anti-mycobacterial activity and it provides scientific evidence of specific activity that supports the traditional use of plants to treat respiratory illness. However, additional testing against the respiratory pathogen, M. tuberculosis, is needed to confirm that the observed activity extends to this species. Future research will also aim to determine the active compounds in these extracts.
ACKNOWLEDGMENTS
We are grateful to Dr. Susan Semple, University of South Australia, for providing plant extracts and Dr. Tim Stinear, Monash University, for providing strains of Mycobacterium.