4.3. Antibacterial Activity
To the best of our knowledge, the plants utilized in this research have not been previously assessed against
Staphylococcus spp. derived from mastitis. However, various studies have shown the antibacterial activities of extracts from these plants against
Staphylococcus spp. from different sources and other plants against
Staphylococcus spp. isolated from bovine mastitis cases. For instance, the essential oil extracted from
S. lancea has been reported to possess antibacterial activity against
S. aureus, with a MIC value of 0.01 mg/mL [
34], while the 70% methanol extract of the plant’s leaf showed a MIC of 0.06 mg/mL [
35]. Considering the MIC values obtained with the acetone extract of
S. lancea against isolates of
S. aureus in this work, together with these examples, leaf extracts of
S. lancea prepared using different solvents have antibacterial activities against
S. aureus.
The result obtained in this investigation using the acetone extract of
S. leptodictya against the ATCC strain of
S. aureus corresponds with another report of the same MIC value of 0.16 mg/mL against the same strain of
S. aureus as was found in this study [
36]. Meanwhile, another study reported MIC of 0.60 mg/mL with the acetone leaf extract of
T. emetica against
S. aureus [
37], which is higher than the MIC values obtained against six of the
S. aureus strains used in this study with the same extract.
A striking observation from
Table 5 and
Table 6 is that only the acetone extract of
S. lancea showed good activity against all the strains of
S. aureus and NAS. Neither the ethanol extract of
S. lancea nor both (acetone and ethanol) extracts of other plants had good activity against any strain of NAS. The results also suggest that the
S. aureus strains were more susceptible to the extracts than the NAS strains. A similar pattern of susceptibility was reported by other researchers [
38], although they used a disc diffusion method. Significant antibacterial properties against mastitis-causing bacteria were observed with all the extracts obtained from
Liquidambar orientalis leaf using three different solvents (acetone, methanol, and ethanol). However, the antibacterial activity of these extracts was significantly poorer against NAS species than against
S. aureus. The acetone extracts showed the highest antibacterial activity (with a 12 mm inhibition zone diameter) against
S. aureus 17, while the lowest antibacterial activity (with a 7 mm inhibition zone diameter) of acetone extracts was found against NAS-36 [
38]. Another study reported varying susceptibility patterns regarding the essential oils obtained from selected plants [
38]. They reported that some isolates of NAS (
S. epidermidis and
S. xylosus) (MIC = 0.156%) appeared to be less susceptible than some isolates of
S. aureus to the essential oil of
Pelargonium graveolens (MIC = 0.078%). Furthermore, they also reported some isolates of both
S. aureus and NAS that were susceptible at the same concentration to essential oils of other plants, such as
Juniperus virginiana (MIC = 0.010%),
Leptospermum scoparium (MIC = 0.005%),
Pogostemom cablin (MIC = 0.010%), and
Thymus vulgaris (MIC = 0.010%) [
39]. In another study, it was reported that the acetone leaf extract of
Acacia nilotica showed higher antibacterial activity against
S. epidermidis and
S. chromogenes (MIC = 0.156 mg/mL) than
S. aureus (MIC = 0.625 mg/mL) [
40]. This same pattern was also reported [
39], claiming that some isolates of NAS (MIC = 0.020%) appeared to be more susceptible than some isolates of
S. aureus (MIC = 0.156%) to the essential oil of
Cinnamomum cassia. The acetone leaf extract of
Aloe arborescens showed a similar MIC (˃2.5 mg/mL) against
S. aureus and
S. chromogenes, and a lower MIC (2.5 mg/mL) against
S. epidermidis [
40]. Further research needs to be conducted to unravel the reason for these patterns. Knowing that the NAS and STA strains are Gram-positive organisms, this observed pattern may not be due to the cell wall infrastructure of both groups of bacteria. This variation in susceptibility patterns of
S. aureus and NAS isolates may likely be due to the differences in the antibacterial compounds in the different extracts or samples. It might be important to isolate and investigate these compounds and co-formulate them to produce a broad-spectrum product that can be used in the management of bovine mastitis caused by these organisms.
According to
Table 7, NAS strains were more susceptible to the extracts over a shorter MIC range than
S. aureus strains, which are susceptible over a wider MIC range. This is similar also to a previous study which reported geranium oil to have a wider MIC range (0.078–1.25%) for
S. aureus and a narrower MIC range (0.156–1.25%) for NAS [
39]. Knowing that NAS is a group of species of
Staphylococcus, it would be expected that their susceptibility may vary more widely than the strains of
S. aureus which are the same species. To observe the pattern in the opposite direction suggests that the species (or strains) of NAS used in this study have a more similar structural, biochemical, and/or physiological response to the extracts than the strains of
S. aureus used in the study. This may suggest the possibility of extensive genetic variations among the strains of
S. aureus used in this study. This calls for biomolecular investigations to understand the extent of the genetic variations among strains of the same species that are responsible for their varying susceptibility to the same extract.
The average MIC of the extracts against all bacteria (
Table 7; column “STA & NAS strains”) showed that only the acetone extract of
S. lancea showed excellent antibacterial activity (mean MIC = 0.03 mg/mL). The acetone extracts of the other plants showed only moderate activity, while the ethanol extracts of all the plants showed moderate to poor activity. The average MIC values of all the acetone extracts of each of the plants were lower than those of their ethanol counterparts, except for
E. caffra. The average MIC value of the ethanol extract of
E. caffra (mean MIC = 0.18 mg/mL) for each of the bacteria appears to be less than half of the average MIC value of its acetone extract (mean MIC = 0.45 mg/mL). This is an interesting observation since, though acetone is mostly the preferred extractant due to the observed better antibacterial activity, ethanol remains the suitable solvent due to the potential to commercialize the findings of this study. This is due to the fact that ethanol is not as flammable and less dangerous to work with in large quantities compared to acetone. Moreover, ethanol is able to effectively permeate cell membranes, which facilitates the extraction of a wide range of polar and non-polar components from inside the cell, permitting the extraction of higher levels of polar and non-polar endo-cellular components [
33]. Therefore, the ethanol extract of
E. caffra should be further investigated. The observation of better activities with the acetone extract of the other plants is consistent with various studies, which have suggested acetone to be the preferred solvent of extraction for antimicrobial investigation of plants since acetone can extract compounds of a wider range of polarity [
18,
19].
Against the ATCC strain of
S. aureus, only the acetone extracts of
S. lancea had good activity (MIC = 0.07 mg/mL). Both ethanol and acetone extracts of the other plants showed moderate activities, except for the ethanol extracts of
P. capensis (MIC = 0.94 mg/mL),
Z. mucronata (MIC = 0.83 mg/mL), and
T. emetica (MIC = 0.83 mg/mL) which showed poor activities. It generally appears that the isolates were more susceptible to the extracts than the ATCC strain, which is an interesting result, while the ATCC strain was more susceptible to gentamicin (positive control) than the isolates (
Table 5 and
Table 6).
The antibacterial investigation suggests the potential usefulness of the extract of S. lancea as an antibacterial agent with a broad spectrum of activity against staphylococci implicated in causing bovine mastitis.
4.4. Cytotoxicity
When the LC
50 is 0.02 mg/mL or lower, a plant extract is classified as cytotoxic [
32]. Based on the above definition, all the tested plant extracts in this study were relatively non-cytotoxic as the lowest value obtained was 0.08 mg/mL (
Table 9), which is above the cut-off value.
However, another study reported the LC
50 of acetone and ethanol extracts of
S. lancea extracts to be below 0.05 mg/mL, which is lower compared to the values obtained in this work [
41]. They observed that the acetone and ethanol extracts of
S. lancea exhibited moderate toxicity toward Vero cells at concentrations of 0.25 and 0.05 mg/mL, respectively. In contrast, the ethyl acetate extract of
S. lancea showed no toxicity at the same concentrations. Therefore, they hypothesized that the presence of alkaloids and saponins in
S. lancea could be responsible for its observed toxicity [
41]. LC
50 values of 0.025, 0.022, and 0.051 mg/mL against Vero cells by other species of the
Searsia genus, such as
S. leptodictya,
S. pendulina, and
S. pentheri, respectively have been reported [
42]. Moreover, the cytotoxic activity of water extracts of
S. lancea leaf material has been reported against brine shrimp with LC
50 value of 0.6 mg/mL [
43].
According to reports, the n-hexane and ethyl acetate extracts derived from the root bark of
E. caffra demonstrated cytotoxic effects on human cervical carcinoma cells at concentrations of 0.11 mg/mL and 0.06 mg/mL, respectively [
44]. Another study found that the ethanol extract of the stem bark of
A. venosum exhibited greater toxicity (LC
50 = 0.026–0.041 mg/mL) compared to the root extract (LC
50 = 0.063–0.080 mg/mL) against brine shrimp [
45]. However, it should be noted that in vitro cellular toxicity may not necessarily reflect in vivo toxicity due to various factors, such as gut interactions and bioavailability. Therefore, further studies involving acute and chronic animal toxicity testing are necessary to confirm the safety of these plant extracts [
42]. The LC
50 of ethanol extracts of the plants in this study were higher than those of their acetone counterparts, which suggests that the ethanol extracts are relatively less toxic compared to their acetone counterparts. The results obtained by [
45] also favor ethanol extracts over acetone extracts. This is significant for potential commercialization as ethanol is preferred as a solvent for industrial use since it is less flammable and dangerous to work with in large quantities.
To determine the safety margin of a plant extract, the selectivity index (SI) is calculated using two variables: Cytotoxicity (mg/mL) and minimum inhibitory concentration (MIC) values [
46]. If the selectivity index (SI) value is greater than 1, it indicates that the plant extract is more harmful to the pathogen than to the mammalian cells tested for cytotoxicity. A higher SI value is more encouraging since it suggests that the plant extract’s activity is not due to general toxicity. Consequently, the higher the SI, the greater the potential for the plant extract to be developed into a safe herbal product. The acetone extracts of
S. lancea, which showed the most promising antibacterial activity against all the organisms tested, also had a very promising LC
50 value of 0.15 ± 0.02, SI range of 2.14 to 10.23, and mean SI of 4.69 ± 0.89. Based on the results obtained, it can be concluded that although the extracts of all three plants tested have the potential to be developed as safe and effective herbal remedies for treating microbial infections,
S. lancea seems to be the most promising candidate.