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RESULTS AND DISCUSSION (the table 1 and 2 are showned in the section of the pictures)
Fresh leaves of O. gratissimum yielded 0.21% of EO. The analysis of the EO by CG/MS revealed a major component (67%) with a retention time of 14.22 min
Table I reports the inhibition zones of OE determined for six strains of Gram-positive or Gram-negative bacteria using the diffusion technique on solid media. Proteus, Klebsiella, Escherichia, Salmonella, Staphylococcus and Shigella showed inhibition zones ranging from 13 to 25 mm. P. aeruginosa was considered resistant since no inhibition zone was observed.
MICs for all bacterial test are reported in Table II. The EO inhibited S. aureus at a concentration of 0.75 mg/ml. In contrast to the relatively low MIC of EO for Gram-positive bacteria, Gram-negative bacteria belonging to the genera Shigella, Salmonella, Escherichia, Klebsiella, and Proteus were inhibited by EO with MICs ranging from 3 to 12 mg/ml. Pseudomonas was not inhibited by EO at concentrations as high as 24 mg/ml. The MICs of the reference drugs used in this study were similar to those presented in other reports (data not shown). The minimum concentration of antimicrobial necessary to kill an organism, MBC, should be equal to or greater than the MIC for that microbe. In this study six bacterial strains presented MBCs which were within one twofold dilution of the MIC obtained for these organisms.
The activity of EO from O. gratissimum against S. aureus was higher than that of the other bacteria tested. EO was also active against members of the family Enterobacteriaceae. The MICs for Shigella, Salmonella, Escherichia, Klebsiella, and Proteus ranged from 3 to 12 mg/ml.
The EO was subjected to successive cc on silica gel and the resulting fractions were analyzed by TLC on silica gel. Chromatograms were run in duplicate and one set was used as the reference chromatogram (Fig.1A). The other set was assayed for bioautography and showed inhibition zone against S. aureus indicating the presence of active compounds (Fig.1B). The compound that showed antibacterial activity, was identified as eugenol (Moffat et al. 1986) by 1H and 13C NMR, supported by GC/MS, by retention time, and by comparison with an authentic sample.
Jedlickova et al. (1992) studied the antibacterial properties of Vietnamese cajeput oil and ocimum oil in combination with antibacterial agents. According to these authors, the plant products were found to be effective medicines for local application in modern medicine practice. They also suggested on the basis of in vitro tests the synergistic action of these two kinds of medicines.
Recently, Lima et al. (1993) tested in vitro antifungal activity of thirteen EO obtained from plants against dermatophytes. Of the tested oils, O. gratissimum was found to be the most active, inhibiting 80% of the dermatophyte strains tested and producing zones greater than 10 mm in diameter. More recently, Nwosu and Okafor (1995) reported the antifungal activities of extracts of ten medicinal plants collected from southeastern Nigeria against seven pathogenic fungi. According to these authors, O. gratissimum inhibited the growth of Trichophyton rubrum and T. mentagrophytes. They also suggested the possible use of certain plant extracts in the treatment of subcutaneous phycomycosis in humans and animals. Other reports have shown smooth muscle contracting lipid-soluble principles (Onajobi 1986) and antimutagenic activity (Obaseiki-Ebor et al. 1993) in organic solvent extracts of leaves of O. gratissimum.
Ilori et al. (1996) have reported the antidiar-rhoeal activities of leaf extracts of O. gratissimum investigated by disc diffusion and tube dilution methods. These authors have shown that the extracts were active against Aeromonas sobria, E. coli, P. shigelloides, S. typhi and S. dysenteriae. They have also shown that the MIC for those organisms ranged from 8 to 50 mg/ml, while the MBC were from 8 to 62 mg/ml. Our present study show that gram-negative bacteria belonging to the genera Proteus, Klebsiella, Salmonella, Escherichia, and Shigella were inhibited by EO with MICs ranging from 3 to 12 mg/ml. Other reports have shown MIC results similar to or higher than ours (Ramonoelina et al. 1987, Janssen et al. 1989). These differences may be explained by susceptibility testing conditions, physicochemical characteristics of the oil, and even strain-to-strain differences. In vivo data may be helpful in determining the potential usefulness of the essential oil from O. gratissimum
Thin-layer chromatography plates were run in duplicate and one set was visualized by Vanillin/Sulfuric acid spray reagent (A). The other set was used for bioautography with Staphylococcus aureus (B) as described in Material and Methods. EO: essential oil; F6, F15, F17, and F19 fractions.