**3. Results**

### *3.1. Composition of Brainon*

The marker compound in Brainon was confirmed by high-performance liquid chromatography (HPLC) analysis. Quantitative analysis of Brainon revealed that Angoroside C content was determined at approximately 0.5% (Data not shown), and the optimized Brainon was used for the following experiments.

### *3.2. Brainon Recovers Scopolamine-Treated Step-through Latency in the Passive Avoidance Test*

To assess the effectiveness of Brainon on learning and fear-motivated memory, we conducted the passive avoidance task. The SCO-injected group had a considerably shorter step-through latency compared with Normal group (Figure 2). However, Brainon 30 and 100 mg/kg treatment considerably restored (*p* < 0.05) the scopolamine-caused cognitive deficit and GBE 50 mg/kg administration also improved (*p* < 0.05) memory function when comparison with SCO-injected group only.

**Figure 2.** Effects of Brainon scopolamine-treated memory deficit in passive avoidance performance. Values are represented as means ± SEM (*n* = 8). ## *p* < 0.01 vs. Normal group; \* *p* < 0.05 vs. SCO-treated group.

### *3.3. Brainon Improves SCO-Induced Spatial Memory Deficiency in the Morris Water Maze Task*

To examine the effects of Brainon on the spatial memory impairment of mice, escape latency, swim distance, and the platform crossing numbers were assessed in all test groups. Figure 3A revealed that the escape latency of the SCO-treated group was significantly prolonged for 4 days (days 24–27) compared to that in the Normal group (*p* < 0.01). In contrast, 100 mg/kg Brainon and 50 mg/kg GBE shortened (*p* < 0.05) the escape latency on days 26 and 27; 100 mg/kg Brainon also decreased (*p* < 0.05) the escape latency compared to that in the SCO-treated group from day 25. Swimming distance to find the platform in the MWM task showed similar results to those of the escape latency (Figure 3B). The mice in the Brainon 30 (*p* < 0.05), 100 (*p* < 0.01), and GBE 50 (*p* < 0.05) mg/kg groups swam shorter distances to locate the platform in comparison with the SCO-treated groups on day 26. We confirmed that the dose of 30 mg/kg (*p* < 0.05) and 100 mg/kg (*p* < 0.05) Brainon on day 27 reduced the swimming distance in mice when finding the platform. In the probe trials (Figure 3C) on day 28, the platform crossing numbers of the mice in the SCO-treated group was considerably lower comparison with that of the mice in the Normal group (*p* < 0.01). On the other hand, administration with 30 and 100 mg/kg of Brainon (*p* < 0.01) and 50 mg/kg GBE (*p* < 0.01) increased the platform crossing numbers comparison with that observed in the SCO-injected group. Our results demonstrate that Brainon administration enhanced spatial, learning, and memory functions in SCO-treated memory-deficient mice.

**Figure 3.** Effects of Brainon scopolamine treated spatial memory deficiency in the MWM trial. (**A**) The escape latency (s) and (**B**) swim distance of mice was investigated for 4 days (**C**) The platform crossing number was carried out on probe trial. Values are represented as means ± SEM (*n* = 8). ## *p* < 0.01 vs. Normal group; \* *p* < 0.05 and \*\* *p* < 0.01 vs. SCO-treated group.

### *3.4. Brainon Decreases AChE Activity and Increases ACh Levels in SCO-Induced Hippocampal Tissues*

To evaluate the effects of Brainon on AChE and ACh levels, we determined AChE activity and ACh levels in SCO-treated hippocampal tissue. Figure 4A shows that AChE activity in the Normal group was lower than that of the SCO-treated group. Exposure to 100 mg/kg Brainon (*p* < 0.01) and 50 mg/kg GBE (*p* < 0.01) considerably lowered AChE activity comparison with that in the SCO-injected group. However, the ACh levels were found to differ (Figure 4B). SCO treatment significantly downregulated ACh levels in the hippocampus. On the contrary, treatment with 30 (*p* < 0.05) and 100 (*p* < 0.01) mg/kg Brainon increased ACh levels in a dose dependent comparison with that in the SCO-injected group.

### *3.5. Brainon Increases BDNF Protein Expression and CREB Phosphorylation Levels in SCO-Induced Hippocampal Tissue*

To demonstrate the effectiveness of Brainon on the memory-associated proteins in SCO-treated cognitive impairment, we measured the BDNF expression and phosphorylated CREB levels using western blotting. Figure 5 shows that SCO treatment significantly decreased the expression of BDNF and phosphorylated CREB in the hippocampus comparison with that in the Normal group (*p* < 0.01). However, in the hippocampus, 100 mg/kg Brainon (*p* < 0.01) and 50 mg/kg GBE (*p* < 0.01) prevented a decrease in BDNF protein compared to that in the SCO-treated group. In addition, treatment with 30 (*p* < 0.01) and 100 (*p* < 0.01) mg/kg of Brainon increased the levels of phosphorylated CREB in a dose dependent comparison to the hippocampal tissue in the SCO-injected group.

### *3.6. Brainon Upregulates the Expression of SOD-1 and SOD-2 in SCO-Induced Hippocampal Tissue*

To examine the effects of Brainon on antioxidant enzymes, we determined SOD-1 and SOD-2 levels in SCO-treated hippocampal tissue of mice. SCO treatment significantly decreased SOD-1 and SOD-2 levels in comparison with that in the Normal group. However, administration with 30 (*p* < 0.01) and 100 (*p* < 0.01) mg/kg Brainon increased SOD-1 levels by 1.6- and 3.6-fold, respectively, whereas administration with 50 mg/kg (*p* < 0.01) of GBE recovered the decreased SOD-1 levels 3.9-fold with SCO treatment. Furthermore, treatment with 100 mg/kg (*p* < 0.01) Brainon and 50 mg/kg (*p* < 0.01) GBE also enhanced SOD-2 levels by 1.9- and 2.1-fold, respectively, in comparison with the mice in the SCO-injected group (Figure 6).

**Figure 5.** Effects of Brainon on CREB-BDNF pathway in the memory deficit mice hippocampal tissues. (**A**) Brain-derived neurotrophic factor (BDNF) and phosphorylation of cAMP-response element-binding protein (CREB) was measured via western blotting. (**B**) The protein band density was determined using the software. Phosphorylation levels of the CREB protein were normalized using the CREB, and BDNF protein was normalized using the GAPDH. Values are expressed as means ± SEM (*n* = 3). ## *p* < 0.01 vs. Normal group; \*\* *p* < 0.01 vs. SCO-treated group.

**Figure 6.** Effects of Brainon on anti-oxidant related protein levels in the hippocampal tissues of memory deficit mice. (**A**) Superoxide dismutase (SOD)-1, and SOD-2 protein expression were evaluated via western blotting. (**B**) The protein band density was measured utilizing by the software. Protein was normalized using the GAPDH. Values are represented as means ± SEM (*n* = 3). ## *p* < 0.01 vs. Normal group; \*\* *p* < 0.01 vs. SCO-treated group.

### *3.7. Brainon Decreases Interleukin (IL)-1β, IL-6, and Tumor Necrosis Factor (TNF)-α mRNA Expression in SCO-Induced Hippocampal Tissue*

The effects of Brainon on the expression of IL-1β, IL-6, and TNF-α mRNA in the hippocampi of SCO-treated mice were confirmed using real-time qPCR (Figure 7). SCO treatment significantly increased expression of IL-1β, IL-6, and TNF-α mRNA compared to that in the Normal group (*p* < 0.01). Figure 7 shows that the expression of IL-1β mRNA significantly decreased after treatment with 100 mg/kg Brainon (*p* < 0.01) and 50 mg/kg GBE (*p* < 0.01) by 30% and 18%, respectively, compared to the SCO-injected group. In comparison with the SCO-injected group, expression of IL-6 mRNA was reduced dose dependently after treatment with 30 (*p* < 0.05) and 100 (*p* < 0.01) mg/kg Brainon by 25% and 29%, respectively, and even further reduced by 35% after treatment with 50 (*p* < 0.01) mg/kg GBE. Treatment with 100 mg/kg Brainon (*p* < 0.01) and 50 mg/kg GBE (*p* < 0.01) considerably alleviated TNF-α mRNA expression by 22% and 28%, respectively, compared to that in the SCO-injected group.

**Figure 7.** Effects of Brainon on anti-inflammation related mRNA expression in the memory deficit mice hippocampal tissues. Interleukin (IL)-1β, IL-6, and tumor factor necrosis (TNF)-α was evaluated by real-time qPCR. The levels of IL-1β, IL-6, and TNF-α mRNA were normalized utilizing the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control. Values are represented as means ± SEM (*n* = 3). ## *p* < 0.01 vs. Normal group; \* *p* < 0.05 and \*\* *p* < 0.01 vs. SCO-treated group.

### *3.8. Brainon Suppresses Apoptosis-Related Protein Levels in SCO-Induced Hippocampal Tissue*

To demonstrate the effects of Brainon on SCO-induced apoptosis, we examined Bax, Bcl-2, cleaved caspase-9, and cleaved-PARP protein expression in the mouse hippocampus. As shown in Figure 8, SCO injection markedly upregulated protein levels of Bax, cleaved caspase-9, and cleaved-PARP comparison with that in the Normal control group. On the contrary, Bcl-2 protein levels downregulated in the SCO-treated group. Meanwhile, treatment with 30 (*p* < 0.01) and 100 mg/kg Brainon (*p* < 0.01), and 50 mg/kg GBE (*p* < 0.01) significantly prevented the SCO-caused increase in the expression of Bax and cleaved-PARP proteins. Treatment with 100 mg/kg (*p* < 0.01) Brainon also decreased the expression of cleaved-caspase 9 proteins but 50 mg/kg GBE did not reduce cleavedcaspase 9. Furthermore, treatment with 100 mg/kg Brainon (*p* < 0.01) restored the SCOcaused reduction in Bcl-2 protein expression in the hippocampi.

**Figure 8.** Effects of Brainon on cell death-related protein expression in the memory deficit mice hippocampal tissues. (**A**) Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9 and cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) protein was analyzed using western blotting. (**B**) The density of the protein bands was determined utilizing the software. Protein was normalized using the GAPDH. Values are represented as means ± SEM (*n* = 3). ## *p* < 0.01 vs. Normal group; \*\* *p* < 0.01 vs. SCO-treated group.
