**4. Discussion**

Previously, we reported that SBE extracts demonstrate potent anti-amnesic activity and anti-apoptotic effects in a scopolamine-induced memory impairment mouse model and in the SH-SY5Y cell line [11,12]. In vitro studies have shown that SBE treatment inhibited cell death induced by glutamate. Furthermore, the in vivo study indicates that acute and prolonged treatment with SBE demonstrates the highest anti-oxidant activity in the scopolamine-induced memory impairment model. However, research on amyloid-beta, which affects in the development of AD, is insufficient. The first report aimed to identify the neuro-protective activities, including amyloid-beta accumulation prevention effects and related mechanisms of chronic SBE administration in the Aβ 1–42-induced cognitive deficit mice model.

AD is a progressive, neurodegenerative disorder that causes damage to the brain and is characterized by cognitive decline, and irreversible memory loss. Amyloid-beta peptides, and hyper-phosphorylated tau protein comprising neurofibrillary tangles have been considered critical factors of pathological hallmarks of AD [10]. Indeed, Aβ levels were higher in the brain of AD patients than normal aged brain samples, indicating clinical signs such as spatial memory loss [20,21].

To assess cognitive deficits, passive avoidance and MWM tasks were performed to investigate learning and memory skills. The passive avoidance task is used to confirm the memory function by measuring the escape time from the space. The MWM task is the most widely used laboratory behavioral test to assess hippocampal-dependent cognitive deficits and learning functions in mice and rats [20,22,23]. Injection of Aβ 1–42 resulted in severe cognitive impairments and memory loss in the passive avoidance test and MWM, as well as neurodegeneration. However, SBE treatment dose-dependently increased the step-through latency in the passive avoidance task and decreased escape latency and swim distance significantly for 4 days of MWM trials. This implied that SBE

administration improves cognitive performance and ameliorates memory deficits, although not as completely as the normal model that did not cause memory loss.

In the previous study, we have demonstrated that neuronal cell death and cognitive deficiency due to oxidative stress are related to Aβ accumulation in the brain. [24]. Aβ is a known neuro-toxic peptide that promotes oxidative stress and lipid peroxidation in the intermembrane and also causes ROS generation by serving as a source of ROS [5,6,25]. The accumulation of Aβ may result in an increased production of ROS, subsequently leading to neuronal death through apoptotic pathways [25,26]. To prevent ROS generation and regulate the steady-state O<sup>2</sup> concentration, cells have anti-oxidant defense systems such as SODs, GR and GPx-1 enzymes [11,12,27]. Aβ 1–42 injection attenuated hippocampal antioxidant enzyme activities and protein expression levels, the same as in previous studies [28,29]. Nevertheless, we observed that the administration of SBE (30, 100 mg/kg) could significantly enhance GR activity and SODs, GPx-1 protein levels in the Aβ 1–42-treated mouse hippocampus.

The mitochondria are much more sensitive to oxidative stress and Aβ-mediated oxidative stress increased mitochondrial dysfunction leading to apoptotic cell death. The accumulated Aβ triggers neuronal death in the hippocampus by releasing caspase activators [24,29,30]. Aβ-induced apoptosis is regulated by mediators such as caspases, Bcl-2, and Bax [10]. The proportion of Bax and Bcl-2 affects whether a cell undergoes or escapes apoptosis. The caspase pathway promotes apoptosis and releases apoptosis-promoting factors when the Bax/Bcl-2 ratio is increased [20]. In our study, SBE treatment significantly lowered Bax, cleaved caspase-9, and cleaved PARP protein expression levels and upregulated Bcl-2 protein expression levels at the same time in mice hippocampal tissue.

Aβ injections increased the accumulation of Aβ and induced hyper-phosphorylation of the tau protein in the brain. The brains of AD patients demonstrate abnormally phosphorylated tau, 4–8-fold higher than normal brains [31]. In this study, the Aβ-injection increased the phosphorylated tau protein expression levels about 4–5 times as against the normal control. We observed that the Aβ and phosphorylated tau protein expression levels dose-dependently decreased in response to treatment with SBE compared to the normal group.

However, this pre-clinical study has some limitations and we will confirm the anti-inflammatory effects of SBE in a mouse model induced neuroinflammation and memory loss in a further study. Further studies are also needed for immunohistochemical analysis and the effect of SBE in clinical trials.

Collectively, our results indicate that SBE treatment improved memory impairment through reduction of Aβ accumulation and the regulation of oxidative stress, anti-apoptotic pathways, and tau protein hyper-phosphorylation in the Aβ 1–42 memory impairment-induced hippocampal tissue.
