**1. Introduction**

The characterization of Alzheimer's diseases (AD) represents amyloid β accumulation, hyperphosphorylation of the tau protein, increased inflammation and oxidative stress leading to neuronal death, which is accompanied by an impairment in memory and cognitive abilities [1,2]. Moreover, a decrease in the neurotransmitter, acetylcholine (Ach), in the AD brain is a crucial factor responsible for dementia [1].

Scopolamine (SCO) is a muscarinic receptor blocker and tropane alkaloid drug that interrupts cholinergic nerve transmission, causing memory disability and cognitive deficits in the central nervous system (CNS) [1,3–6]. Normal cholinergic activity in the CNS influences hippocampal nerve regeneration and memory impairment via the cAMP response elementbinding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling [7]. SCO administration causes cholinergic neuronal pathway dysregulation, including increased acetylcholinesterase (AChE) activity, suppression of ACh release and impairment of the memory circuit, and decreased CREB-BDNF levels. Moreover, the neuronal inflammatory

cascade, oxidative stress generation, and neuron cell death, including cholinergic malfunction, are causative factors in patients with neurodegenerative disorders. The hippocampus is susceptible to oxidative stress, and excessive oxidative stress induces memory deficiency by damaging synaptic plasticity and causing inflammation and neuronal cell death [7,8]. SCO-treated mice showed oxidative stress-induced neuronal inflammation and apoptosis in the brain [9]. Therefore, SCO injection of animals is utilized as a useful pharmacological experimental model for cognitive degeneration and memory disorders in AD [2–5].

*Scrophularia buergeriana* Miquel (SB), belonging to the Scrophulariaceae family, is indigenous to Korea and also found abundantly in China and Japan. In traditional medicine, dried SB roots have long been used to alleviate high fever, swollen skin, obstipation, pharyngitis, neuro-inflammation, and throat infection [10]. SB roots have been found to contains various components such as E-harpagoside, 8- *O*-E-p-methoxycinnamoyl harpagide (MCA-Hg), E-p-methoxy-cinnamic acid (p-MCA), cinnamic acid, and angoroside C. Moreover, we confirmed that Brainon exhibited significant neuroprotective effects against neurotoxicity caused SH-SY5Y cells by glutamate [11]. We previously demonstrated that Brainon exerts anti-amnesic effects via inhibition of amyloid beta accumulation and hyperphosphorylation of tau protein in memory deficit mice by β-amyloid [12].

Therefore, we administrated 30 or 100 mg/kg Brainon based on the previous study [12] and confirmed the cognitive-improving activities of Brainon using passive avoidance performance and Morris water maze (MWM) task based on this research. To demonstrate the potential mechanism of Brainon, we confirmed the CREB-BDNF signaling pathway, AChE activity, and ACh levels in mice hippocampal tissue. Moreover, the anti-inflammatory and antioxidant effects of Brainon, as well as its effects on neuronal death, were evaluated in SCO-caused memory-impaired mice.

### **2. Materials and Methods**

### *2.1. Preparation of Sample*

Brainon was supplied by Nutrapharm Tech Co., Ltd., (Seongnam, Korea). SB roots were dried and extracted using 70% EtOH. The resulting solution was filtered, concentrated, and dried to yield Brainon extract (SB extract; SBE). The detailed process has been described previously [11,12]. To dissolve the Brainon, we used 0.5% carboxy-methylcellulose (CMC) and then applied for the in vivo study.
