**1. Introduction**

Alzheimer's disease (AD) is one type of dementia and a progressive neuro-degenerative disease featured by deposits of extracellular amyloid β (Aβ) peptide and flame-shaped neurofibrillary tangles of hyper-phosphorylated tau protein, inducing neurotoxicity accompanied by cognitive impairment and memory loss [1–4].

The Aβ plaque is composed of Aβ 1–40 and 1–42, major forms of Aβ found in the brains of AD patients. The Aβ 1–42 protein is more neurotoxic and induces more oxidative damage than Aβ 1–40 [5]. An important factor in AD development is considered to be Aβ accumulation, since oxidative stress is followed by Aβ cytotoxicity [1,6]. The overproduction of reactive oxygen species (ROS) induced Aβ accumulation and oxidative stress damages cellular components resulting in structural damage, functional disorder, and cell apoptosis [7].

Cells have an antioxidant defense system protecting them from ROS attacks using various enzymes, such as superoxide dismutase (SOD), glutathione peroxidases (GPx), glutathione reductases (GR) [8]. SOD is known as the first detoxification enzyme and the most powerful endogenous antioxidant in the cell. It acts as a catalyst and converse of the superoxide (O<sup>2</sup>−) radical into ordinary oxygen (O2) and hydrogen peroxide (H2O2), leaving the harmful superoxide anion less dangerous. SOD requires a metal cofactor, such as copper (Cu), zinc (Zn) and manganese (Mn), for activity as a

metalloenzyme [9]. Cu/Zn-SOD (SOD1), which is located in chromosome 21 and encoded in the SOD1 gene located on chromosome 21 and predominantly occurs in the cytosolic compartment. Mn-SOD (SOD2), which is located in chromosome 6 and encoded in the SOD2 gene on chromosome 6, located in the mitochondrial matrix. The reduction of SOD-1 triggers oxidative cellular DNA damage, and SOD-2 protects mitochondrial DNA against damage due to oxidative stress [10]. GPx plays an import role in protecting cells from oxidative damage by converting hydrogen peroxide (H2O2) to H2O with glutathione (GSH) as a substrate, after the oxidation of GSH to Glutathione disulfide (GSSG). GR is known to maintain the reduced GSH level in redox cycle. Furthermore, it resists oxidative stress and maintains a reducing environment inside the cell [11,12].

In addition, Aβ-mediated oxidative stress causes the hyper-phosphorylation of tau protein and affects neurofibrillary tangle formation. The accumulation of neurofibrillary tangles directly correlates with neuro-degeneration and cell death and is closely related to the severity of dementia [13,14].

Apoptosis is mediated by two major pathways, which are divided into extrinsic and intrinsic pathways. Death receptors release several mitochondrial intermembrane proteins by passing death signaling to mitochondria in the intrinsic pathway [15]. A common mitochondria-related apoptotic pathway in neurons is regulated principally by the pro-apoptotic Bcl-2 protein family member, Bax. On receipt of the death signal, Bax is translocated to the mitochondria and interacts with the Bcl-2 family to control the progression of apoptosis [16,17]. Bax activates down-stream e ffector caspases such as caspase-9 and caspase-poly (ADP) ribose polymerase (PARP) in vitro, and in vivo by stimulating cytochrome c release. Notably, Bcl-2 inhibits Bax-induced apoptosis [15,18,19].

*Scrophularia buergeriana* (SB) Miq. (Scrophulariaceae), called Hyun-Sam in Korea, is used to help cure fever, swelling, constipation, pharyngitis, neuritis, and laryngitis as traditional medicine. SB dried root contains various components such as E-harpagoside (CAS: 19210-12-9), 8- *O*-E-p-methoxycinnamoyl harpagide (MCA-Hg), E-p-methoxycinnamic acid (p-MCA, CAS: 830-09-1), cinnamic acid (CAS: 140-10-3), and angoroside C (CAS: 115909-22-3), which displays neuro protective activities [11]. In a previous study, the SB extract (SBE) demonstrated cognition-enhancing activity in a scopolamine-induced short-term memory loss mice model [12]. However, investigation evaluating the effects of SBE in a beta-amyloid induced memory loss model is lacking.

Therefore, we administrated 30 or 100 mg/kg SBE based on the previous study and evaluated the e ffect of SBE on β-amyloid accumulation and tau phosphorylation. Furthermore, we investigated the neuro-protective e ffects against Aβ caused neurotoxicity through antioxidant and anti-apoptotic mechanisms in the Aβ 1–42 injected mice.

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

### *2.1. Sample Preparation*

SBE was obtained from Nutrapharmtec co., Ltd., (Seongnam, Korea). SB dried roots were extracted for 2–4 h at 70–90 ◦C with 70% ethyl alcohol and filter processed, concentrated, and dried. The extraction process was performed with reference to previously described studies [11,12]. For the in vivo study, the standardized SBE was dissolved in 0.5% carboxy-methylcellulose (CMC).

### *2.2. Experimental Animals*

Male C57BL/6N mice (8 weeks old; Orient. Co. Ltd., Gyeonggi, Korea) were used after 7 days adaptation period (20–26 ◦C; 12 h light cycle from 08:00 to 20:00; food, Doo Yeol Biotech, water ad libitum). All studies were conducted according to the animal experiment ethical committee (permission number: 2018-07-008) of ChemOn Inc (Yong in, Korea). The animals were cared based on the guidelines provided by this committee. We monitored changes in body weight, food and water intake once a week. To enhance the well-being of the animals, we provided hygienic and ensured proper breeding and care to prevent disease.

### *2.3. A*β *1–42 Injection and Drug Administration*

The experimental animals were grouped as follows: normal control group, Aβ 1–42 treated group, and co-treated groups with Aβ 1–42 and the SBE (30 or 100 mg/kg/day). SBE was dissolved in 0.5% CMC and orally administered for 4 weeks (Day 28). The passive avoidance test and Morris water maze (MWM) were performed for 3 (Days 15–17) and 7 (Days 22–28) days. The Aβ peptide was dissolved in sterile 0.1 M phosphate-buffered saline (pH 7.4) and pre-incubated at 37 ◦C for 7 days. Zoletil ® (Virbac, Carros, France) and xylazine (Bayer, Leverkusen, Germany) (4:1, *v*/*v*) were used for mice to anesthetize and Aβ 1–42 was injected (5 μL/2.5 min, i.c.v.) using stereotax ic apparatus coordinates (Anterior/Posterior (AP), −1.0 mm; Mediolateral/Lateral (ML), +1.0 mm; Dorsal/Ventral (DV), −2.5 mm).

### *2.4. Passive Avoidance Test*

The passive avoidance test equipment (Twin County Med Associates, Hudson, NY, USA) was used to perform passive avoidance experiments. This apparatus is divided into light and dark compartments with a guillotine door in the middle. The bottom is gridded to administer an electric shock. The tests were carried out for 3 days at the same time every day at 24 h intervals. For adaptation training, the animals were placed in the shaded area for 2 min, and then placed back in the illuminated area. When the animal moved into the shaded area, it was immediately placed in the illuminated compartment (day 15). Twenty-four hours later (day 16), two training sessions were performed every 2 min. After 60 s of adaptation, the animals were allowed to move between the two compartments freely for 120 s. However, on moving to the shaded area, the guillotine door was closed and a 0.20 mA scrambled shock was applied for 2 s. The animals that failed to move were excluded from and 8 mice per group participated in the experiments. On the last day of testing, (day 17), the animal was located on the illuminated area and the guillotine door was opened. The time taken to move to the shaded area was measured.

### *2.5. Morris Water Maze (MWM) Test*

The MWM test was performed 22 days after the Aβ 1–42 peptide injection. The platform was placed in one of the four designated release points in the water pool, allowing the animal a search time of 60 s. The mice that found the platform were left on it for 30 s, but the mice that failed to find the platform within 60 s were placed on the platform and allowed to rest for about 30 s. All mice tested twice a day, and the position of the platform was randomly changed in the water pool. Using this method, we measured the time taken to locate the platform by repeating the test for 7 consecutive days (training: 2 days, behavioral test: 4 days, probe trial: 1 day). On the last 7 days (Day 28), a probe trial was conducted. Here, 1 h after vehicle or drug administration, the platform was taken out of the pool, and the number of times the mice passed the platform was measured for 60 s on Day 28. The entire experiment plan is exhibited in Figure 1.

**Figure 1.** Experimental plan of this study.

### *2.6. Preparation of Tissue Samples*

The mice were anesthetized and sacrificed after behavioral tasks for biochemical studies. The hippocampus was separated from the brain tissue and then immediately stored at −80 ◦C until further assessment.

### *2.7. Glutathione Reductase (GR) Activity*

GR enzyme activity was determined using the Glutathione Reductase Assay Kit (Abcam, Cambridge, UK). Hippocampal tissues were homogenized in cold assay buffer, and centrifuged at 10,000× *g* for 15 min at 4 ◦C. The separated supernatant from hippocampus was taken and kept at −80 ◦C until further analysis. This assay is based on the reduction of glutathione by nicotinamide adenine dinucleotide phosphate (NADP)H in the presence of GR. GR activity can be detected by measuring the change in absorbance at 405 nm.

### *2.8. Western Blotting*

Hippocampal tissues were homogenized with RIPA buffer and 1% protease inhibitor cocktail (Roche, Mannheim, Germany) and the lysate was centrifuged at 10,000× *g* for 15 min at 4 ◦C. The separated supernatant from hippocampus was taken, and protein concentrations were measured using a BCA protein assay kit (Thermo, Waltham, MA, USA). The proteins were separated using 8% or 12% SDS-PAGE and were then moved to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were initially incubated to block with 5% non-fat skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 30 min. Next, they were incubated with specific primary antibodies against SOD-1, SOD-2, GPx-1, Aβ (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax, Bcl-2, cleaved Caspase-9, cleaved PARP, phospho-tau, tau, and β-actin (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at 23 ◦C. The membranes were then incubated in the corresponding horseradish peroxidase-conjugated anti-rabbit, anti-mouse immunoglobulin G (1:10000; GenDEPOT, Barker, TX, USA) for 1 h at 23 ◦C. The membrane was detected using the ECL system (Atto, Tokyo, Japan). The intensity of the bands on the membrane was detected by Image-Pro Plus software (6.0 Version; Media Cybernetics, Silver Spring, MD, USA)

### *2.9. Statistical Analysis*

The experimental results are expressed as standard error of the mean (SEM) and were assessed using the SPSS program (version 22.0, SPSS Inc., Chicago, IL, USA). Difference value of between treatment groups were analyzed by Student's *t*-test and one-way analysis of variance (ANOVA), and performed following multiple comparisons correction using Dunnett's post-hoc test using Origin 7.0 software (OriginLab, Northampton, MA, USA). *p* < 0.05 indicates that there is a statistical difference, and *p* < 0.01 was considered statistically highly significant between mean values.
