**3. Results**

### *3.1. Effects of the Scrophularia Buergeriana Extract (SBE) on the Passive Avoidance Test in A*β *1–42 Treated Mice*

To determine the effect of SBE on memory deficits, we conducted the passive avoidance test in the Aβ-induced mouse model. Aβ-injected mice demonstrated a remarkable reduction in the time to move from the illuminated compartment to the shaded area compared to normal mice (*p* < 0.01), implying that the learning capacity was lowered. However, the step-through latency was dose-dependently increased in mice administered with SBE 30 mg/kg (*p* < 0.05) and 100 mg/kg (*p* < 0.01) (Figure 2) compared to Aβ-injected mice.

**Figure 2.** Effects of *Scrophularia buergeriana* extract (SBE) on the step-through latency of passive avoidance performance in Aβ 1–42 treated mice. The data are expressed as means ± standard error of the mean (SEM) of independent experiments (*n* = 8). ## *p* < 0.01 vs. Control group; \* *p* < 0.05 and \*\* *p* < 0.01 vs. Aβ (1–42) group.

### *3.2. E*ff*ects of the SBE on the MWM Test in A*β *1–42 Induced Memory Deficit Mice*

To investigate the memory-enhancing effect of SBE, we next performed the MWM test in C57BL/6 mice. Normal mice quickly located the platform during four consecutive behavioral tests. The Aβ-injected group exhibited a significantly delayed escape latency compared to the normal group from days 25–27. However, mice treated with SBE 30 mg/kg and 100 mg/kg demonstrated a shortened escape latency from days 25–27 (Figure 3A). Furthermore, we observed that Aβ-injected mice demonstrated significantly increased swim distances to find the platform compared to the normal control mice (Figure 3B). We confirmed that SBE 30 mg/kg (*p* < 0.05) and 100 mg/kg (*p* < 0.01) on day 27 decreased the distance traveled to locate the platform. In the probe trials (Figure 3C), the Aβ-treated mice reported a decreased number of crossings over the previous platform position against as the control mice. The number of crossings was recovered by treatment with SBE 30 mg/kg (*p* < 0.05) and 100 mg/kg (*p* < 0.01) compared to the Aβ-injected mice and the recovery was significant. Therefore, we confirmed that SBE enhanced spatial recognition in the MWM test.

### *3.3. E*ff*ects of the SBE on Glutathione Reductase (GR) Activity in the Hippocampus*

To evaluate the effects of SBE on GR activity of Aβ treatment mice, GR activity was measured in the hippocampal tissue. GR activity was significantly reduced in mice treated with Aβ compared with the normal group. However, mice treated with SBE 30 mg/kg (*p* < 0.05) and 100 mg/kg (*p* < 0.05) significantly increased hippocampal GR activity compared to the Aβ-treated group. Furthermore, the efficacy of SBE was comparable to the normal group mice (Figure 4).

**Figure 3.** Effects of SBE on Aβ 1–42 induced memory impairment in the Morris water maze (MWM). (**A**) The escape latency and (**B**) Swim distance was recorded across 4 days (Day 24–Day 27). (**C**) The number of crossings in a probe trial performed on Day 28. The data are expressed as means ± SEM of independent experiments (*n* = 8). # *p* < 0.05 and ## *p* < 0.01 vs. Control group; \$ < 0.05 and \$\$ < 0.01 vs. Control group; \* *p* < 0.05 and \*\* *p* < 0.01 vs. Aβ (1–42) group.

**Figure 4.** Effects of SBE on glutathione reductase (GR) activity in the mouse hippocampus. The hippocampus was lysed, and the supernatant was used to measurement GR activity. The results were calculated as a unit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidized per protein and expressed as means ± SEM of independent experiments (*n* = 3). ## *p* < 0.01 vs. Control group; \$\$ < 0.01 vs. Control group; \* *p* < 0.05 vs. Aβ (1–42) group.

### *3.4. E*ff*ects of the SBE on the Antioxidant Enzymes in Hippocampus of A*β *1–42 Treated Mice*

To investigate the effects of SBE on antioxidant enzymes in the hippocampus of Aβ treated mice, the protein levels of SOD1, SOD2, and GPx-1 were evaluated. The protein levels of SOD1, SOD2, and GPx-1 were markedly decreased in mice treated with Aβ against the control group. Furthermore, treatment with SBE 30 mg/kg increased GPx-1 protein expression by 1.3-fold. In addition, SOD1, SOD2, and GPx-1 protein levels were further increased in mice administered SBE 100 mg/kg by 4.2-, 1.7- and 2.9-fold, respectively, compared to the Aβ-treated group (Figure 5).

**Figure 5.** Effects of SBE on antioxidant protein expression levels in the hippocampus of memory-impaired mice. (**A**) The expression of superoxide dismutase 1 (SOD1), SOD2, and glutathione peroxidase-1 (GPx-1) were detected by Western blotting analysis. (**B**) The protein bands were quantified and calculated using the relevant software. Protein expression levels were normalized on β-actin level. The data are expressed as means ± SEM of independent experiments (*n* = 3). ## *p* < 0.01 vs. Control group; \$ < 0.05 and \$\$ < 0.01 vs. Control group; \*\* *p* < 0.01 vs. Aβ (1–42) group.

### *3.5. E*ff*ects of the SBE on Apoptosis in the Hippocampus of A*β *1–42 Treated Mice*

To demonstrate the protective effect of SBE on Aβ-induced apoptosis, the protein expression levels of Bax, Bcl-2, caspase-9, and cleaved PARP were analyzed in the hippocampal tissue. Aβ treatment significantly increased the protein levels of Bax, cleaved Caspase-9, and cleaved PARP. Conversely, the Bcl-2 protein levels decreased in the Aβ-treated group. Bcl-2/Bax are known as important proteins related to apoptosis in the mitochondria. The Bax protein level was dose-dependently reduced with SBE treatment by 31% and 51%, and SBE 100 mg/kg (*p* < 0.01) significantly increased the levels of Bcl-2 and Bcl-2/Bax by 1.41- and 2.87-fold, respectively, compared to the Aβ-treated group in Figure 6. SBE 30 mg/kg and 100 mg/kg significantly reduced the levels of cleaved caspase-9 by 61%, and 76%, respectively, and reduced cleaved PARP by 33% and, 74%, respectively, compared to the Aβ treatment group. Therefore, we confirmed that SBE exhibited a prominent neuro-protective effect.

**Figure 6.** Effects of SBE on apoptotic protein expression levels in the hippocampus of memory-impaired mice. (**A**) The expression of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9 and cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) was detected by Western blotting analysis. (**B**) The protein bands were quantified and calculated using the software. Protein expression levels were normalized on β-actin level. The data are expressed as means ± SEM of independent experiments (*n* = 3). ## *p* < 0.01 vs. Control group; \$ < 0.05 and \$\$ < 0.01 vs. Control group; \*\* *p* < 0.01 vs. Aβ (1–42) group.

### *3.6. E*ff*ects of the SBE on A*β *Accumulation and Hyper-Phosphorylation of Tau Proteins in Hippocampus of A*β *1–42 Treated Mice*

The pathological hallmarks of AD include Aβ accumulation and tau hyper-phosphorylation. The hyper-phosphorylation of the tau protein, can contribute to neuronal degeneration. Using western blot analysis, we confirmed that the Aβ injection promoted Aβ accumulation in the mouse hippocampus (Figure 7). The administration of SBE 30 mg/kg (*p* < 0.01) and 100 mg/kg (*p* < 0.01) significantly reduced Aβ accumulation in the hippocampus against as the Aβ injection group by 22% and 53%, respectively (Figure 7B). Based on the protective effect of SBE on Aβ accumulation, we confirmed the effect of SBE on tau phosphorylation in Aβ treated mice. Aβ treatment increased the tau protein phosphorylation compared to the normal group, and treatment with SBE 30 mg/kg and 100 mg/kg significantly attenuated Aβ induced hyper-phosphorylation of tau by 69% and 71%, respectively, compared to the Aβ treatment group.

**Figure 7.** Effects of SBE on amyloid-beta protein expression levels and phosphorylation of tau protein levels in the hippocampus of memory-impaired mice. (**A**) The expression of amyloid-beta (Aβ) and phospho (p)-tau was detected by Western blotting analysis. (**B**) The protein bands were quantified and calculated using the software. Phosphorylation levels of the tau protein were normalized to those of tau, and Aβ protein expression level was normalized on β-actin level. The data are expressed as means ± SEM of independent experiments (*n* = 3). ## *p* < 0.01 vs. Control group; \$\$ < 0.01 vs. Control group; \*\* *p* < 0.01 vs. Aβ (1–42) group.
