Abstract: The antimicrobial activities of the acetone, methanol and water extracts from the leaves, stems and roots of this herb were investigated against 10 bacterial and 5 fungal species using the dilution method on solid agar medium. The acetone extracts from the leaf and root were active against Gram-positive bacteria with Minimum Inhibitory Concentration (MIC) ranging between 1.0 and 7.0 mg mL-1, whereas the acetone stem extract was able to inhibit all the bacterial strains at 0.5-7.0 mg mL-1. The methanol extracts of the 3 plant parts showed activity against all the bacterial isolates with MIC values ranging between 0.1 and 10.0 mg mL-1. Again all the extracts exhibited appreciable activity against all the fungi species investigated. The methanol extract of the root showed 100% inhibition against Aspergillus niger, A. flavus and Penicilium notatum at 5 mg mL-1 while it was 88.61% inhibition in Mucor hiemalis. The ability of the extracts of F. muricata to inhibit the growth of several bacteria and fungi is an indication of its broad-spectrum antimicrobial potential which may be employed in the management of bacterial and fungal infections.
INTRODUCTION
Significant number of people in developing countries have limited access to modern drugs for the treatment of common diseases, thus, medicinal plants are their only therapeutic resource (Waller, 1993). The use of traditional herbal remedies as alternative medicine by the indigenous population of South Africa plays a major role in their culture and also features significantly in the primary health care system. With the increasing acceptance of herbal medicine as an alternative form of health care, the screening of medicinal plants for active compounds has become very important as potential sources of novel antibiotic (Meurer-Grimes et al., 1996; Rabe and Van Staden, 1997).
Felicia muricata Thunb. (Asteraceae), locally known as Ubosisi in the Eastern Cape of South Africa, is a small drought resistant perennial herb growing up to 20 cm in height. The leaves are simple and are arranged in alternate fashion.Its name was derived from muricate (rough, with sharp tubercles or protuberances). The species is regarded as an indicator of desertification, becoming increasingly invasive in grassland regions (Jordaan and Kruger, 1993). F. muricata is used by the traditional healers in the management of headaches, stomach catarrh, pains and inflammation (Hutchings, 1989; Hutchings and Van Staden, 1994; McGaw et al., 1997). Information gathered during our preliminary investigation on the local uses of the species also revealed its medicinal importance for the treatment of stomach ache and cancer. Extracts from the plant have been reported to show 80-90% inhibitory activity against cyclooxygenase, an important enzyme in the prostaglandin biosynthesis pathway (McGaw et al., 1997; Okoli and Akah, 2004) and this may be responsible for the anti-inflammatory activity of this plant. Despite the reported medicinal uses of this species, especially for stomach ache and inflammation, the antimicrobial activity of this herb has not been reported in literature. This study investigated the antimicrobial activity of F. muricata by screening its extracts against 10 selected bacterial and 5 fungal strains, with the aim to validate the use of this species in the management of pains and inflammation that could have resulted from microbial infections. According to Mathekga and Meyer (1998), in vitro antimicrobial screening could provide the preliminary observations necessary to select among crude extracts, those with potentially useful properties for further chemical and pharmacological investigations.
MATERIALS AND METHODS
Plant material: Plants used for this study were collected in August 2007 from several populations of F. muricata growing within premises of Alice campus of the University of Fort Hare (33 °11.10`S and 7 ° 10.60`E; altitude 695 m). The mean annual rainfall of this area is about 700 mm and temperature range of 13 to 25 °C. The species was authenticated by Mr. Tony Dold, Selmar Schonland Herbarium, Rhodes University, South Africa. A voucher specimen (AshafaMed.2007/1) was prepared and deposited in the Griffen Herbarium of the University of Fort Hare.
Extract preparation: The separated leaves, stems and roots of the plant samples were carefully washed under running tap water, air dried at room temperature (30 °C) and pulverized before extraction. Powdered plant material (40 g each) was separately extracted in acetone, methanol and water for 48 h at 30 °C, on an orbital shaker (Stuart Scientific Orbital Shaker, UK). Acetone and methanol were of high analytical grade. The extracts were filtered through Whatman No. 1 filter paper and the filtrate was evaporated to dryness under reduced pressure at 40 °C using a rotary evaporator (Laborota 4000-efficient, Heidolph, Germany). The water extract was freeze-dried using Savant Refrigerated Vapor Trap, (RVT4104, USA). The freeze-dried extract was stored at 4 °C before bioassay. The different extracts were re-dissolved in their respective solvents to give 50 mg mL-1 stock solution (Taylor et al., 1996). This was diluted to the required concentrations of 0.1, 0.5, 1.0, 5.0, 7.0 and 10 mg mL-1 for the bioassay analysis.
Antibacterial assay: Five Gram-positive (Staphylococcus aereus, Staphylococcus epidermidus, Bacillus cereus, Micrococcus kristinae, Streptococcus faecalis) and five Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Shigelia flexneri, Klebsella pneumoniae, Serratia marcescens) bacteria species used in this study were all laboratory isolates. They were obtained from the Department of Biochemistry and Microbiology, University of Fort Hare, South Africa. The organisms were maintained on nutrient agar plates and were revived for bioassay by subculturing in fresh nutrient broth (Biolab, Johannesburg, South Africa) for 24 h before being used.
Nutrient agar (Biolab, Johannesburg, South Africa) was prepared by autoclaving and allowed to cool to 55 °C before the addition of the extracts. The agar medium containing the extracts at final concentrations of 0.1, 0.5, 1.0, 5.0, 7.0 and 10 mg mL-1 were poured into Petri dishes, swirled gently until the agar began to set and left over night for solvent evaporation (Afolayan and Meyer, 1997). Agar plates containing 1% acetone, methanol and water served as controls (Dulger and Ugurlu, 2005). Organisms were streaked in radial pattern on the agar plates (Meyer and Afolayan, 1995). The inoculum size of each test strain was standardized at 5x 105 cfu mL-1 using McFarland Nephelometer standard according to the National Committee for Clinical Laboratory Standards. The plates were incubated under aerobic conditions at 37 °C and examined after 24 and 48 h. Each treatment was performed in triplicate and complete suppression of growth at a specific concentration of an extract was required for it to be declared active (Sindambiwe et al., 1999; Mathekga et al., 2000). Chloramphenicol and streptomycin (standard antibiotics) were used as positive controls in the experiment.
Antifungal assay: Antimycotic activity of F. muricata was investigated using five fungal species (Aspergillus niger, Aspergillus flavus, Penicillium notatum, Mucor hiemalis and Candida albicans). All fungal cultures were maintained on Potato Dextrose Agar (PDA) (Biolab, Johannesburg, South Africa) and were recovered for testing by subculturing on PDA for 3 days at 25 °C prior to bioassay. PDA plates were prepared by autoclaving before the addition of the extracts. Each extract was vortexed with the molten agar at 45 °C to final concentrations of 0.1, 0.5, 1.0, 5.0, 7.0 and 10.0 mg mL-1 and poured into Petri dishes. Blank plates containing only PDA or PDA with the respective solvent served as controls. The prepared plates containing the extracts were inoculated with plugs (5 mm in diameter) obtained from the actively growing portions of the mother fungal plates and incubated at 25 °C for 5 days. The diameter of fungal growth was measured and expressed as percentage growth inhibition of three replicates (Lewu et al., 2006; Koduru et al., 2006). Due to the nature of Candida albicans, the organism was streaked radially like the bacteria.
Statistical analysis: Significant differences within the means of treatments and controls were measured and calculated using the LSD statistical test (Steel and Torrie, 1960). LC50 (the concentration at which 50% of growth was obtained) was calculated by extrapolation.
RESULTS AND DISCUSSION
Antibacterial activity: The Minimum Inhibitory Concentration (MIC)
values of the reference drugs, acetone, methanol and water extracts from
the leaves, stems and roots of F. muricata against the tested organisms
are shown in Table 1. The acetone extract of the leaves
was active only against Gram-positive bacteria with MIC ranging between
1.0-5.0 mg mL-1, whereas the methanol extract of the leaves
suppressed the growth of all tested organisms with inhibition range of
5.0-10.0 mg mL-1. Both the acetone and methanol extracts of
the stem showed activity against all the bacteria strains with MIC ranging
between 0.1 - 7.0 mg mL-1. The acetone extract of the roots
was active only against Gram-positive bacteria apart from P. eruginosa
that was inhibited at 10.0 mg mL-1 which was the highest concentration
tested, whereas the methanol extract of the roots inhibited all the bacteria
tested at inhibition range of 1.0 to 10.0 mg mL-1. Water extracts
from all the plant parts were not active against any of the organisms
except M. kristinae that was inhibited at 5 mg mL-1
by extract from the stem. Generally, the methanol extracts were more active
than other extracts. This may be associated with the presence of soluble
phenolic and polyphenolic compounds (Kowalski and Kedzia, 2007).
| Table 1: | Antibacterial activity of the extracts from the leaves, stems and roots of Felicia muricata |
| +: Positive, -: Negative, MIC: Minimum Inhibitory Concentration, Chl: Chloranphenicol, Strep.: Streptomycin, *Not active at 10 mg mL-1, which was the highest concentration tested | |
The acetone and methanol extracts were active against Bacillus cereus, a respiratory pathogen commonly associated with colds and flu (Viljoen et al., 2004) at MIC values of 0.5-5.0 mg mL-1. The inhibitory property of extracts of F. muricata against pathogenic bacterial strains can introduce the plant as a candidate for drug development for the treatment of ailments caused by these pathogens. The non-activity of the water extract against most bacterial strains investigated is in accordance with the reported cases of earlier workers who showed that aqueous extracts of plants generally showed little or no antibacterial activities (Madamonbe and Afolayan, 2003; Koduru et al., 2006; Aliero et al., 2006). Traditionally, however, plant extracts are prepared with water as infusions, decoctions and poultices; therefore it seems unlikely that the traditional healer is able to extract those compounds which are responsible for activity in the acetone and methanol extracts. Gram-negative bacteria have been reported to be more resistant to plant extracts than the Gram-positive strains (Rabe and Van Staden, 1997; Grierson and Afolayan, 1999; Afolayan, 2003). The ability of the stem extracts to inhibit all Gram-negative bacteria strains tested was an indication of the plants broad spectrum of the antimicrobial property.
Antifungal activity: The majority of the extracts showed broad
antimycotic activity against the tested organisms at 0.5 mg mL-1
or lower (Table 2). Only the acetone extract of the
leaves and the methanol extract of the roots showed inhibition (100%)
against C. albicans at 10 mg mL-1, which was the highest
concentration tested in this study. Table 2 does not
include the column for C. albicans. Extracts from the roots were
inhibitor to the fungi species than the leaf and stem extracts. Apart
from the water extract of the leaves which did not show any activity against
any of the fungi, all the extracts were fungicidal (100% inhibition) on
P. notatum and M. hiemalis. The susceptibility of
A. flavus to the extracts of F. muricata is noteworthy, as
the fungus has recently been implicated in cases of immuno-compromised
patients that frequently develop opportunistic and superficial mycosis
(Portillo et al., 2001). In general, the methanol extract had the
highest activity against both bacteria and fungi strains. This was followed
by the acetone extract and the least activity was observed in the water
extract. The ability of the extracts of this plant to inhibit the growth
of several bacteria and fungi species is an indication of the broad spectrum
antimicrobial potential of F. muricata, which makes the species
a candidate for bioprospecting for antibiotic drugs. Work is therefore
in progress on the isolation, purification and structural elucidation
of the bioactive compounds in this plant.
| Table 2: | Antifungal activity of extracts from the leaves, stems and roots of Felicia muricata |
| Values are means of percentage growth inhibition of three replicates. Values within a column followed by the same superscript are not significantly different at p<0.05. LC50 values in mg mL-1 were calculated by extrapolation | |
ACKNOWLEDGMENTS
This research was supported by the National Research Foundation of South Africa and Govan Mbeki Research and Development Centre of the University of Fort Hare.