The solvents used for extraction (in the current study) are of varying polarity (hexane is a non-polar solvent, ethyl acetate is moderately polar while methanol is polar). Since solvents dissolve compounds based on their polarity, this extraction protocol ensures that the compounds in the leaves are separated into non-polar (hexane), medium polar (ethyl acetate), and polar (methanol) compounds. Results obtained from the current study showed that at the same concentrations, the three extracts under investigation showed different activities against the test organisms. Differences in solvent polarities have been reported to account for differences in solubility of active plant principles, and hence, variations in the degree of activity of the extracts [14].
Results from a previous study showed that the methanol extract of the leaves of I. trichantha had zones of inhibition of 19.00 ± 0.58 mm, 15.00 ± 0.58 mm, 25.00 ± 0.58 mm, 21.00 ± 0.58 mm, 4.00 ± 0.58 mm, and 7.00 ± 0.58 mm against S. aureus, B. subtilis, P. aeruginosa, E. coli, A. niger, and C. albicans, respectively, at 20 mg/mL [6]. These results vary widely from those obtained for the same extract at a very similar concentration (25 mg/mL) against the same organisms in the current study (Table 1). The differences obtained could have resulted from the differences in the extraction protocols adopted. In the current study, a preliminary solvent-based fractionation of the compounds present in the leaves of the plant was carried out before antimicrobial testing while in the previous study, a direct extraction using methanol was carried out. The solvent-based fractionation approach (employed in this study) simplifies the process of isolation and characterization of potential bioactive compounds from a sample by narrowing down on the number of compounds likely responsible for an observed activity. While the methanol extract in the current study showed some antimicrobial activity, the results obtained indicated that the most potent compounds responsible for the observed activity mostly reside in the hexane extract.
Generally, the zones of inhibition of the microorganisms increased with the increasing concentration of the extracts. Previous studies have reported similar results. For example, the zones of inhibition of methanol extract of flowers of Nelumbium speciosum against different microorganisms increased with extract concentration [15]. In addition, the results showed that the extracts, at the same concentrations, showed different activities against the microorganisms. The inhibition zone of an extract against an organism depends on the initial population density of the organism, and the nature and diffusion rate of the antimicrobial agent [16, 17].
Of the three extracts, the hexane fraction had the best activity against all the test micro-organisms. It even demonstrated moderate activity against gram-negative bacteria (E.coli, S. typhi, and P. aeruginosa). Gram-negative bacteria are generally more resistant to antibiotics than gram-positive bacteria. Gram-negative bacteria possess an outer membrane which is an asymmetric bilayer of lipopolysaccharides (LPS) and phospholipids, into which are fitted nonspecific porins and specific uptake channels. The LPS-containing bilayers are rigid and slow down passive diffusion of hydrophobic compounds while the narrow pores limit by size the penetration of hydrophilic drugs [18,19,20]. This extract probably contains compounds with the ability to overcome this barrier.
On comparing the MIC results (Table 3) with Tables 1 and 2, some discrepancies were observed. For example, when the MIC values obtained for ICLEE against S. aureus and E. coli was 10 mg/ml (Table 3), it is expected that some microbial growth would occur at 12.5 mg/ml. Similarly, some microbial growth should be observed at 25 mg/ml (Table 1), when the MIC of the extract (ICLEE) was 20 mg/ml for B. subtilis, P. aeruginosa, S. typhi, and K. pneumonia. The MIC values reported in Table 3 are single measurements, whereas those in Tables 1 and 2 are averages of triplicate measurements within limits of experimental error. In reporting the zones of inhibition of the microorganisms (Table 1) at the indicated concentrations (12.5 and 25 mg/ml), it is possible that while some very minimal microbial growth was discernable on a plate, none existed on the two others, and the average of the values is approximately zero. This may be responsible for the apparent discrepancies observed.
The compounds detected in the hexane extract of the leaves of I. trichantha obtained in the current study (Table 4) differ significantly from the results obtained from the GCMS analysis of the same extract previously reported in the literature [11]. The literature report identified the compounds present in the extract as 3,3-dimethyl-2-hexanone, Undecane, Palmitic acid, 5-octadecenoic acid methyl ester, Stearolic acid, Stearic acid, 9,12-octadecadienoic acid, 9,17-octadecadienal, and Eicosanoic acid. This may be due to various factors. Apart from biochemical factors inherent within individual plant species, several external factors such as geographical location, climate, nature of soil, season, and growth conditions have a major influence on plants’ phytoconstituents [21]. For example, phytochemicals in different plants possessing anticancer properties have been known to change quantitatively with seasons [22, 23]. Similarly, new active compounds have been reported from different plant parts collected at different times or from different locations [21].
Plants contain compounds that are responsible for their bioactivities. Ten compounds were identified successfully in the hexane extract of leaves of I. trichantha; however, only two of these compounds (Stigmasterol (7) and β-sitosterol) have been reported (in literature) to possess antimicrobial properties. β-sitosterol (10) has been reported to show activity against S. aureus, B. subtilis, and K. pneumoniae with zones of inhibition of 27 mm, 34 mm and, 26 mm, respectively. It also showed a minimum inhibitory concentration of 25, 12, and 25 µg/mL, respectively, against these organisms. However, it showed no activity against E. coli, P. aeruginosa, and C. albicans [24]. Stigmasterol (7) (at 100 µg/mL) showed 29 mm as the zone of inhibition against S. aureus, 24 mm against E. coli, and 25 mm against C. albicans [25]. Stigmasterol had zones of inhibition of 21 mm against S. aureus, 24 mm against B. subtilis, 21 mm against E. coli, and 21 mm against C. albicans at 50 µg/mL [26]. These literature reports indicate that the compounds, Stigmasterol and β-sitosterol, possess far higher antimicrobial activities (against the indicated organisms) than the crude hexane extract of I. trichantha as shown by the results obtained from the current study. This may be due to the presence of both compounds in small quantities in the crude hexane extract (the peak areas for Stigmasterol and β-sitosterol are 5.86% and 8.70%, respectively). In a previous study, Stigmasterol isolated from the stem bark of N. macrophylla displayed an improved inhibitory effect and lower MIC value when compared to the extract [27]. The antibacterial activity of steroids is attributable to their ability to inhibit ‘sortase’ a participant in pathways involving secretion and anchoring of cell wall proteins [28]. Membrane disruption could be one of the possible mechanisms of the action of sterols on microorganisms [29]. However, the antimicrobial properties of the hexane extract may not be entirely due to these two compounds. As noted earlier, a large percentage of the peaks in ICLHE are unidentifiable (55.29%). The compounds corresponding to these peaks could have made significant contributions to the antimicrobial action of the extract. Furthermore, since GC–MS is unsuitable for analyzing non-volatile plant constituents, these compounds might not have been detected (though they may possess significant antimicrobial activities).
