2.1. Optimization of Antibacterial Activity by EVOP-fs
The experimental conditions used in the first set of experiments, the corresponding antibacterial activities of cycle I and II, their differences and average values are presented in
Table 1. The extraction temperature, extraction time and ethanol concentration of central point in first set (E
10, E
20) were 45 °C, 12 h and 45%, respectively [
16]. The error limits, effects and the change in the mean effect were calculated and the results are shown in
Table 2.
In the first set, the error limits for average, effects and changes in mean were 0.1739, 0.1235 and 0.1096, respectively. The change in mean effect was −0.2115. According to the decision-making procedure, after calculating the change in the mean effect and error limit, an examination was necessary to determine whether any change in the control (search level) experimental conditions would help to improve the objective function [
10]. The optimum condition was achieved when the effect was smaller than the error limit, while the change in the mean effect was large. Moreover, because the dependent variables are the population of
S. aureus ATCC6538, in which growth was suppressed by addition of
P. frutescens var. acuta leaf extract, the optimum point was reached when the code of mean effect was negative.
The determination of the magnitude of the change in mean effect, which is negative and large, compared to the error limit, is a requirement in order to confirm the achievement of the optimum condition. Such a situation, where some of the effects are larger in comparison to the error limit, does not ensure that the condition at the search region (E10, E20) of first set is the actual optimum and a second set of experiments is called for.
In the second set (set II), the search level (E
10, E
20) was fixed at the best condition of Set I, at a level of E
14 in which the number of
S. aureus ATCC6538 was 6.050 log CFU/mL. The extraction temperature, extraction time and ethanol concentration of central point in second set (E
10, E
20) were 60 °C, 18 h and 60%, respectively. The experimental conditions and the results of Set II experiments are presented in
Table 3, and the effects and error limits are shown in
Table 4. In the second set, the error limits for average, effects and changes in mean was 0.1739, 0.1235 and 0.1096, respectively. The change in mean effect was 0.0870. Most effective antibacterial activity (4.890 log CFU/mL) was obtained at E
14. The extraction temperature, extraction time and ethanol concentration of E
14 point in second set were 75 °C, 24 h and 45%, respectively. In this case, not all of the effects were smaller than error limit, and the change in mean effect was smaller compared to the error limit even though it is positive. It has been reported that if all or any of the effects are larger than the error limits, the change in the experimental conditions may yield better results [
9].
Under the above conditions, a third set of experiments was designed in which the best condition of Set II (E
14) was selected as the search level (E
10, E
20) for Set III. The experimental conditions and the results of Set III are shown in
Table 5, and the calculated effects and error limits are presented in
Table 6. In the EVOP-factorial design, the effects remain smaller than the error limits while the changes in the mean effect remain larger and positive so as to reach the optimum level. Thus, in the experiments of third set (set III), we were able to arrive at the proper optimum condition, in which all effects were smaller than error limit and the changes in mean effect were large and positive. As shown in
Figure 1, the population of
S. aureus ATCC6538 decreased from 7.535 log CFU/mL in the initial set to 4.865 log CFU/ml in the third set.
In this study, it was shown that higher antibacterial activity was achieved in a higher extraction temperature of 75 °C (R = −0.7904**) and in a longer extraction time of 24 h (R = −0.7273**). However, antibacterial activity of
P. frutescens var. acuta leaf extract against
S. aureus ATCC6538 was not affected by differentiation of ethanol concentration in the extraction solvent (R = 0.0635) as shown in
Figure 2. Therefore, the maximum antibacterial activity of
P. frutescens var. acuta leaf against
S. aureus ATCC6538 determined by the EVOP-factorial technique was obtained at 75 °C extraction temperature, 24 h extraction time and 45% ethanol concentration.
P. frutescens var. acuta leaf extracted at optimum extraction condition (75 °C, 24 h, 45% ethanol concentration) was then extracted with 70% ethanol (MeOH), hexane, chloroform (CHCl
3) and ethyl acetate (EtOAc) to identify
in vitro antibacterial activities of
P. frutescens var. acuta leaf against
S. aureus ATCC6538. The effects of the
P. frutescens var. acuta leaf extract on the growth of
S. aureus ATCC6538 demonstrated the reduced viability. The ethyl acetate extracts exerted potential effect of antibacterial activity against
S. aureus ATCC6538. The ethyl acetate extract exerted potential effect of antibacterial activity against
S. aureus ATCC6538 followed by chloroform extract. More than 99% inhibition of tested pathogen was observed by the ethyl acetate extract (
Figure 3A). Hexane and methanol extracts did not reveal significant effect of antibacterial activity against
S. aureus ATCC6538.
Hexane extract and methanol extract did not reveal significant effect of antibacterial activity against S. aureus ATCC6538.
2.2. Total Phenolic Contents
The amount of total phenolic contents of the leaf extracts (n-hexane, chloroform, ethyl acetate and methanol) of
P. frutescens var. acuta leaf was tested, and occurred in the range of 5.9–111.3 mg% dry sample (
Figure 3B). The total phenolic contents of the leaf extracts of methanol, hexane, chloroform and ethyl acetate were noted to be 38.0 ± 6.7, 5.9 ± 3.2, 49.1 ± 4.3 and 111.3 ± 8.6 mg% of dry sample, respectively. These results showed that the total phenolic contents in ethyl acetate extract (111.3 ± 8.6 mg% of dry sample) were the highest as compared to the other extracts.