2.1. Optimization of Antibacterial Activity by EVOP-Factorial System
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 error limits, effects and the change in the mean effect were calculated and the results are shown in Table 2
. The extraction temperature, extraction time and ethanol concentration of the central point in first set (E10
) were 50 °C, 14 h and 50%, respectively.
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 −0.2105. 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 P. aeruginosa
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 conditions in the search region (E10, E20) of the first set is the actual optimum and a second set of experiments is called for.
In the second set, the search level (E10
) was fixed at the best condition of Set I, at a level of E21
, in which the number of P. aeruginosa
was 4.955 log CFU/mL. The extraction temperature, extraction time and ethanol concentration of the central point in the second set (E10
) were 65 °C, 20 h and 75%, respectively. The experimental conditions and the results of the 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 0.1612, 0.1145 and 0.1016, respectively. The change in mean effect was −0.0685. The most effective antibacterial activity (4.095 log CFU/mL) was obtained at E14
. The extraction temperature, extraction time and ethanol concentration of the E14
point in the second set were 80 °C, 26 h and 50%, respectively. In this case, not all of the effects were smaller than the 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 [10
From the above situation, the third set of experiments was designed in which the best condition of Set II (E14
) was selected as the search level (E10
) 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, we were able to arrive at the proper optimum condition, in which all effects were smaller than the error limit and the change in mean effect is large and positive. As shown in Figure 1
, the population of P. aeruginosa
decreased from 6.660 log CFU/mL in the initial set to 4.060 log CFU/mL in the third set.
In this study, it was shown that higher antibacterial activity was achieved at a higher extraction temperature of 80 °C (R = −0.800**
) and in a longer extraction time of 26 h (R = −0.731**
). However, antibacterial activity of P. frutescens var. acuta
leaf extract against P. aeruginosa
was not affected by the presence of different ethanol concentrations in the extraction solvent (R = −0.076), as shown in Figure 2
. Therefore, the maximum antibacterial activity of P. frutescens var. acuta
leaf against P. aeruginosa
, determined by the EVOP-factorial technique, was obtained at 80 °C extraction temperature, 26 h extraction time and 50% ethanol concentration. These conditions were also found in our previous study conducted on Coptidis rhizoma
against Streptococcus mutans
Although the optimal condition of P. frutescens var. acuta on antibacterial activity is well described in this study, the extraction yield and bioactive compounds in P. frutescens var. acuta are highly variable depending on environmental conditions. Therefore, further studies are needed to compare the effects of various seasonal samples of P. frutescens var. acuta leaf on antibacterial activity using EVOP-factorial design technique.