In agar diffusion assays, the inhibitory zone diameter produced is the result of the growth of the test organisms and the diffusion of the test agents through the agar, both events occurring simultaneously [
15] so it then means that any factor which affects the rate of microbial growth or rate of diffusion of the suspected antimicrobial agent under test, will invariably affect the result. The factors which were pertinent to the formation of inhibition zone included the type/nature of the test organisms, the size of inoculum, the culture media (which should be able to support the growth of organisms and not interfere with diffusion or activity of test organisms) and the temperature of incubation [
16]. Secondary metabolites of plants such as saponins, flavonoids, tannins, carbohydrates, cyanogenetic glycosides, reducing sugar and all other active principles of plants have been shown to be responsible for the antimicrobial activities shown by these extracts. [
17,
2]. However from the phytochemical analysis of this plant extract, some of these secondary metabolites were absent (tannins, anthroquinone and flavonoid), some in low and moderate concentrations and a few in high concentration. However, the antimicrobial activities shown by the extract will likely be due to one or more of the several other phytochemical constituents shown to be present in the extract (Table
1). There was no activity against
E. coli for both the typed culture and the clinical isolate, as opposed to information in literature [
8]. This may be due to the absence of some secondary metabolites or the presence of some in low concentration; or it may be due to the type of strains used or a slight change in any of the factors mentioned earlier that are likely to affect rate of microbial growth or rate of diffusion of the test agent. A percentage change in the inhibition zone diameter was used to draw inference on the effects of the interaction on the test organisms. It has been suggested that for a synergistic effect, the diameter of the zone of inhibition in the test plate (plate containing the plant extract and the antibiotics) should be greater than that in the control plate (plate containing extract-free base agar layer) by at least 19%. A percentage increase in the inhibition zone diameter that is lower than 19% indicates additivity. Where the inhibition zone diameters in the test and the control are equal, the combined antibiotics have indifferent effect and if the zone diameter in the test is less than the one in the control, then there is antagonism [
18]. It was also observed that for
S. aureus it was only ampicillin that did not show activity against it. This may be due to the fact that
S. aureus usually develops resistance to β-lactam drugs. There was antagonism in inhibitory zone diameter when streptomycin was combined with the extract. Septrin and ciprofloxacin showed indifferent effects individually when they were combined with the extract. For
P. mirabilis, all the test antibiotics showed activity. However when combined with the plant extract, it was observed that streptomycin showed antagonism. Ciprofloxacin and ampicillin were synergistic when each was combined with the extract. However with septrin, there was additivity. Against
P. aeruginosa all test antibiotics showed activity, but on combining with the plant extract, ciprofloxacin showed synergism while septrin was antagonistic. Ampicillin and streptomycin did not have any individual activity when combined with the extract. The antibiotics were all active against the clinical isolate of
E. coli and the typed culture, when the extract was combined with either ciprofloxacin or septrin, synergistic interaction was observed. Moreover, ampicillin and septrin when combined together with either ciprofloxacin or septrin showed synergistic interaction. Moreover, ampicilin and streptomycin when combined separately with the extract showed antagonism against
E. coli (ATCC 11755). Against the clinical isolate of
E. coli ciprofloxacin was additive, streptomycin was antagonistic while septrin and ampicillin were synergistic. Nakamura
et al [
8] found that the essential oil of
O. gratissimum has antibacterial activity against
Shigella flexineri, E. coli, Klebsiella spp and
Proteus mirabilis. Ketoconazole and nystatin were both active against the clinical isolate of
C. albicans and the type culture. The effect of interaction between the plant extract and the antibiotics was synergistic on
C. albicans. Against
C. albicans (ATCC 90028), ketoconazole showed antagonism while nystatin was additive. In a previous study, using agar dilution technique, Silva
et al [
10] demonstrated that
O. gratissimum exhibited antifungal activities against the dermatophytes:
M. canis, M. gypseum, T. rubrum and T. mentagrophytes. Lemos and co-workers [
11] also showed that the ethanolic crude extract and the ethyl acetate, hexane, chloroform, essential oil and eugenol of
O. gratissimum have antifungal activities against
C. neoformans. Generally, it should be remembered that the study is in vitro, and the likelihood of possibility of change in activity of microflora of a patient cannot be ruled out as it functions in vivo. Also, the possibility of alteration of the presence of other exogenous compound and the significance of this alteration in modifying drug interaction will only be determined by detailed pharmacokinetic studies that relate to the distribution of drugs metabolized by the microflora and their potentially active metabolites to the therapeutic response of the patient to the antibiotic.