Introduction

Microbial resistance, a world health hazard, is dramatically increasing ₁,₂ hence evaluation of natural products to find new, safe and effective active compounds to rotate or substitute with the invalidated ones is one of the scientific strategies to combat drug-resistant pathogens ₃ . One of such plants of medicinal value is Ageratum conyzoides L. Its common name is goat weed ₄ . In Ghana, the following local names are given to the plant: Twi: Guankuro, Adwowa-kuro; Ewe: Nyigbe (cow weed), Mima, (faeces weed: because of its foul smell); Ga: Ntumlumlu; Nzima: Ebuakulo; Krobo: Tokuyowi (he-goat's crown of head; because of its smell) (Dokosi, 1998) ₅ .

The plant is used in Southern Africa for the treatment of fresh wounds and in Central Africa, the leaf is used to aid the healing of burn wounds ₆ . In Ghana, it is used for treating chronic sores, sore eyes and dysentery ₇ . In East Africa, it is used as a styptic ₈ . In Central Africa, it is used to treat pneumonia ₉ . In Cameroon and Congo, the traditional use is to treat fever, rheumatism, headache and colic ₁₀ . In Brazil, aqueous extracts of leaves or whole plants have been used to treat spasms, colds and fevers ₁₁,₁₂,₁₃,₁₄,₁₅ .The purpose of this study is to determine the best solvent for the crude extract extraction and to investigate the antimicrobial properties of the plant against resistant and multiple resistant strains of selected microorganisms (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa).

Materials and methods

Fresh whole plants of Ageratum conyzoides were collected randomly from Cape Coast in August, 2005. The taxonomic identity of this plant was determined by the Department of Botany, University of Cape Coast. The fresh plants were washed under running tap water, air dried for five (5) days and then homogenized to coarse powder. This was stored in air tight bottles.

For aqueous extraction, 100g of air-dried powder was placed in distilled water and boiled for two hours and furthermore left to soak overnight. The resulting suspensions were filtered and evaporated to dryness at 60 ° C in vacuum.

186g of flowers, 98g of leaves, 300g of flowers, 160g of roots and 200g of whole plant were separately and successively soxhlet extracted with 40 to 60% petroleum ether, dichloromethane and methanol. The vacuum dried extracts were reconstituted in the respective solvents used for the extraction. Water extracts were also prepared. These extracts were kept in dark polythene bags and preserved in the refrigerator until needed.

The microbial strains used for the study were made of two reference strains of Staphylococcus aureus and three clinical isolates of Staphylococcus aureus as well as reference strains of Escherichia coli and Pseudomonas aeruginosa. The clinical strains were identified by the analytic profile index (API). The bacterial strains studied were: Oxford Staphylococcus aureus NCTC 6571, methicillin Resistant Staphylococcus aureus NCTC 12493, oxacillin Resistant Staphylococcus aureus, tetracycline and ampicillin Resistant Staphylococcus aureus and cotrimoxazole, penicillin and gentamicin Resistant Staphylococcus aureus, Escherichia coli ATCC 25922, Pseudomonas aeruginosa NCTC 10662.

Well diffusion method using Müeller-Hinton agar plates were used to demonstrate the antimicrobial properties of the crude extracts ₁₆ . A suspension of the bacteria compared to 0.5 Macfarland standard was seeded on the Mueller-Hinton agar plates. Wells of 6mm in diameter and 2cm apart were punctured in the culture media using sterile cork borers. Ten percent concentration of the crude extracts was administered to fullness in each well and the plates were incubated overnight at 37° C. Growth was determined by measuring the diameter of the zone of inhibition. The solvents were used as the negative controls whiles 10?g ampicillin disc (Oxoid) was used as the positive control. The control zones of the solvents were deducted from the zones of inhibition created by the crude extracts.

Results

The methanolic extracts showed considerably more activity on all the strains of Staphylococcus aureus with zone sizes ranging from 26 to 28 mm as shown in Table 1. However, the methanolic extracts were weakly active against Escherichia coli. Pseudomonas aeruginosa was resistant to all the extracts. Water extract and 40-60% petroleum ether extract did not show any antibacterial activity against the test organisms. The dichloromethane extract was weakly active against only Escherichia coli ATCC 25922 with a zone size of 7mm.

Figure 1

Table 1: Activity of the various plant extracts (whole plant)

Figure 2

Figure 1: Inhibition Zone Sizes At 24 and 48 Hours Incubation Are The Same Note: X = 98 % Methanol Solvent, Y = 10µg of Ampicillin

Figure 3

Figure 2: Inhibition Zone Sizes At 24 and 48 Hours Incubation Are The Same Note: X = 98 % Methanol Solvent, Y = 10µg of Ampicillin

Discussion

The preliminary study using 40-60 % petroleum ether extract, dichloromethane extract and methanol extract of the whole plant indicated that the antibacterial properties were found in the polar region of the plant extracts. The results revealed that the non-polar 40-60 % petroleum ether crude extracts and the water extracts did not show any antibacterial effects on the test microorganisms.

The polar methanol extracts of the whole plant were effective against all the strains of Staphylococcus aureus and Escherichia coli. However, they were not effective against Pseudomonas aeruginosa which is an organism causing wound and skin infection. Hence the 98 % methanol had shown to be the best extraction solvent for the antimicrobial constituents in the plant. These findings agreed with the findings of Almagboul et al. (1985). Almagboul et al. (1985) ₁₇ , using methanolic extract of the whole plant demonstrated inhibitory action in the growth of Staphylococcus aureus and Escherichia coli in vitro. Furthermore, the findings of the study using methanolic crude extracts of the flowers, leaves, stems and roots of Ageratum conyzoides against Oxford Staphylococcus aureus NCTC 6571 and Escherichia coli ATCC 25922 revealed that the methanolic crude extract from the stems were weakly active against the test microorganisms as compared to the roots. Comparing antimicrobial potencies of the leaves and the flowers, it was evident that the leaves were more active than the flowers against the test microorganisms. These two microorganisms stated above were selected for this study because among the seven (7) microorganisms used for the preliminary study with the crude methanolic extract of the whole plant, they were susceptible whiles the rest were resistant. The study further showed that the antimicrobial properties of the leaves were strongest followed by the flowers, roots and the stems.

Conclusions

Ageratum conyzoides is known to possess antimicrobial properties. Therapeutically, the leaves of the plant has been found to possess the most active principles against Staphylococcus aureus and Escherichia coli, bacterial associated with skin and wound infections. Methanolic extracts demonstrated considerable antimicrobial properties against the resistant strains of Staphylococcus aureus. However, it was weakly active against E. coli and not active at all against P. aeruginosa. Hence the best solvent to be used for extraction of active compounds of antimicrobial potency is 98% methanol. The plant could be useful in the management of Staphylococcus aureus associated skin and wound infections if further scientific investigations are carried out on them to ensure their safety.