## 3 Results and Discussion
### Western Blot  
Western blot was performed using the same amount of protein. BDNF levels of hippocampus tissues in E100 and E300 were significantly higher than those in the control and in the positive control (\(p<0.05\); Figure 5). SZS has various biological activities, and many studies have reported its neuroprotective [6], sedative and hypnotic [8], and anxiety suppression [25] effects, as well as anti-Alzheimer's disease activities [12; 13; 34; 35; 36]. However, little information is available about the antidepressant activity of SZS. Very recently, _Ziziplus muconata_ showed an antidepressant effect in chronically stressed rats due to its capacities for antioxidant activity, and probably the modulation of monoamines transmission [37]. Spinosin, a major constituent of SZS, has been reported to have the activities of neurogenesis for cognitive improvement [38; 39; 40], sleep reduction for the treatment of insomnia [41], neuroprotection [42], and cardioprotection [43]. However, the antidepressant activity of spinosin was not previously reported. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Group**} & \multicolumn{4}{c}{**Normalized Value (\(u=7\))**} \\ \cline{2-5}  & **MAO-A** & **MAO-B** & **AChE** & **BChE** \\ \hline C & 1.00 \(\pm\) 0.70 & 1.00 \(\pm\) 0.55 & 1.00 \(\pm\) 0.72 & 1.00 \(\pm\) 1.11 \\ N & 1.65 \(\pm\) 0.97 & 0.99 \(\pm\) 0.48 & 1.65 \(\pm\) 0.78 & 0.65 \(\pm\) 0.26 \\ P & 1.06 \(\pm\) 0.31 & 1.00 \(\pm\) 0.37 & 2.33 \(\pm\) 1.37 & 0.98 \(\pm\) 0.86 \\ E100 & 1.08 \(\pm\) 0.37 & 0.75 \(\pm\) 0.43 & 1.52 \(\pm\) 0.76 & 0.42 \(\pm\) 0.23 \\ E300 & 1.22 \(\pm\) 0.26 & 0.56 \(\pm\) 0.47 & 0.99 \(\pm\) 0.20 & 0.42 \(\pm\) 0.22 \\ \hline \hline \end{tabular} C: normal control; N: negative control (CUMS); P: positive control (CUMS + 15 mg/kg fluoxetine); E100 and E300: doses of the extract (CUMS + 100 and 300 mg/kg, respectively).  
\end{table} 
Table 2: MAO-A, MAO-B, AChE, and BChE activities of the hippocampus in normalized values. Figure 4: LC-MS/MS analysis of neurotransmitters in the hippocampal of mouse brains after behavioral tests by intraperitoneal injection of fluoxetine and oral administration of the extract into mice (\(n=7\)). C: normal control; N: negative control (CUMS); P: positive control (CUMS + 15 mg/kg fluoxetine); E100 and E300: doses of the extract (CUMS + 100 and 300 mg/kg, respectively). GABA: \(\gamma\)-amino butyric acid. #: Kruskal-Wallis test; *: Mann-Whitney test. #: \(p<0.05\); ##: \(p<0.01\); *: \(p<0.05\); **: \(p<0.01\). **SZS showed MAO-B and AChE inhibitory activities in vitro; however, MAO-B activity in the hippocampus was low and AChE activity was detected, but differences between the control and dosed groups were not significant. These results suggest that the antidepressant activity of SZS is mediated via pathways other than MAO-B or AChE inhibitions, similar to the results of novel herbal treatment [44]. The CUMS method is widely used to investigate antidepressant activity in animal models [45]. CUMS can induce behavioral changes resembling clinical depression, and these parameters can be reversed or prevented by chronic antidepressant treatment [46]. In addition, the TST, FST, and OFT were performed as behavioral tests to investigate antidepressant effects, as many other studies have reported [47; 48; 49]. FST and TST are the two most validated behavioral assays for assessing antidepressant potential [50; 51]. To avoid false-positive results, OFT is used to evaluate locomotor activity [30]. In this study, the tests were carried out using CUMS-treated mice. The TST experiments showed significant decreases in immobility times, and the FST experiments also exhibited decreases in immobility times in the SZS-extract-treated groups. In the OFT, total and zone distances were significantly increased with a dose dependency. In addition, neurotransmitter monoamines levels in the hippocampus tissues were significantly increased in the extract-treated groups.