## 3 Results and Discussion
Under an optimal condition, 18.21 (mg/g) of maximum chlorophyll a contents was expected, and actually, from the experiment under the same extraction conditions, 17.98 \(\pm\) 2.02 (mg/g) of chlorophyll a was measured, which indicates that the proposed model was well designed. The predicted concentration of the chlorophyll a from the optimized conditions was higher than all other concentrations obtained from 20 experiments in Table 1, which indicated that the optimization of the ultrasonic extraction seemed to be adequate and efficient in terms of yielding the highest chlorophyll a with a low input frequency, shorter process time, and low temperature. This highest content was also found to be even higher than other reported values from methanol and acetone extraction processes, etc. [12, 30]. The final concentration of chlorophyll a estimated from an optimized process, 18.21 (mg/g), can also be compared with 13.18 (mg/g) from conventional ethanol extraction in terms of its productivity such as 3.72 vs. 0.55 (mg/g/h), respectively, since the extraction time of this process was much shorter than that of conventional extraction (4.91 vs. 24 h). This productivity was also higher than those from other experimental results in Table 1. Moreover, in comparing the final concentrations obtained from a conventional ethanol extraction and experimental extraction conditions in Table 1, the increase of chlorophyll a production through this optimized process must be greatly advantageous, increasing the purification yield and its biological activities as well as reducing the production costs since pure chlorophyll a is one of the most expensive natural substances and is difficult to purify with its intact forms [11, 12]. As also shown in Figure 2, HPLC chromatograms confirmed that the extracts from the optimal process had a higher quality of chlorophyll a and other biologically active substances by comparing Figure 2b,c with the standard chlorophyll a in Figure 2a. We believe that these results confirm that the proposed extraction conditions can be used to extract good amounts of intact chlorophyll a from \begin{table}
\begin{tabular}{c c c c c} \hline  & **Dependent Variables** & \multicolumn{2}{c}{**Response Variables**} \\ \hline
**Time (h)** & **Temperature (\({}^{\circ}\)C)** & **Frequency (kHz)** & **Predicted Value** & **Experimental Value** (mg/g) & **Experimental Value** (mg/g)** \\ \hline
4.91 & 32.59 & 20.52 & 18.21 & 17.98 \(\pm\) 2.02 \\ \hline \end{tabular}
\end{table} 
Table 4: Predicted and experimented values of response variables processed under an optimal extraction condition. Figure 1: 3D response surfaces (**a**) and contour plots (**b**) as a function of ultrasonic frequency, temperature, and process time. _Spirulina_ by a simple ultrasonification process. Therefore, this process can be employed to have more highly purified but less expensive chlorophyll a than from the conventional extraction process, which gives low chlorophyll a extraction yields [11; 12]. In addition to the extraction of high amounts of chlorophyll a from _Spirulina_, the efficacy of the extracts should also be considered to re-confirm the optimal extraction conditions, a result that could be used to expand the utilization of the extracts. First, Figure 3 demonstrates that higher amounts of chlorophyll a reflect better neuroprotective activities of the extract from optimal extraction conditions than from a conventional ethanol extraction at high temperature and with a long process time. In particular, the extract from the optimal conditions enhanced the growth of the glutamate-induced mouse hippocampal neuronal cell line compared to that from a conventional ethanol process: 90.71 \(\pm\) 2.13% vs. 73.01 \(\pm\) 0.03% of the cell growth protection for the control with the addition of 100 (\(\upmu\)g/mL) of the extracts.