The dried leaves and branches of F. erecta were kindly provided by the Korean Seed Association (Seongnam, Korea) and identified by Professor Joo-Hwan Kim (Gachon University, Seongnam, Korea). Voucher specimens (SCD-A-114-1 and SCD-A-114-2) were deposited at the Clinical Medicine Division, Korea Institute of Oriental Medicine (Daejeon, Korea). The dried leaves (3.4 kg) and branches (4 kg) of F. erecta were extracted twice with aqueous ethanol using an electric extractor (COSMOS-660, Kyungseo Machine Co., Incheon, Korea) for 3 h at 80 ± 2℃. The filtered extracted solution was concentrated using a rotary evaporator under vacuum and freeze-dried to obtain powdered leaf and branch extracts (636.13 and 293.31 g, respectively). The yield of EEFE was 18.71% (leaf extracts) and 7.33% (branch extracts).
In vitro Aβ1–42 aggregation assay
The Aβ1–42 aggregation assay was performed as described in our previous report  using the Thioflavin T β-Amyloid Aggregation kit (Cat. AS-72214, Anaspec, Inc., Fermont, CA, USA). The mixture of Aβ1–42 peptide solution and EEFE was combined with thioflavin T. Then, fluorescence signals were read at an excitation/emission wavelength = 440/484 nm at 37℃ using a Multi-Mode microplate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA, USA). Morin (Cat. M4008-2G, Sigma-Aldrich, St. Louis, MO, USA) was used as an Aβ aggregation inhibitor, positive control. Experiments were performed in triplicate and repeated three times. The inhibition (%) of Aβ1–42 aggregation was calculated by following equation:
Animals and intracerebroventricular injection of Aβ aggregates
Male C57BL6 mice weighing 24 ± 2 g were obtained from Orient Bio (Seoul, South Korea), and acclimated for 1 week prior to the study. Animals were given standard rodent diet (2918C; ENVIGO, Blackthorn, UK) and water ad libitum. They were maintained in individual acrylic cages at a temperature of 23 ± 3℃, relative humidity of 55 ± 15%, ventilation frequency of 10–20 times/h, illumination intensity of 150–300 lux, and lights on from 8 AM until 8 PM (12:12 h light:dark cycle). The experiments were approved by the Institutional Animal Care and Use Committee of Nonclinical Research Institute, Chemon Inc. (IACUC Approval No. 18-M111) and was performed according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
The Aβ1–42 peptide was obtained from Tocris Bioscience (Cat. 1428, Pittsburgh, PA, USA), dissolved to a concentration of 1 µg/µL in sterilized 0.1 M phosphate-buffered saline (PBS; pH 7.4), and incubated at -20℃ for 4 weeks to encourage the formation of Aβ aggregates. Mice were anesthetized with mixed zoletil (tiletamine–zolazepam; Verbac, Carros, France) and rompun (Bayer, Leverkusen, Germany) (4:1 v/v) at dose of 1 mL/kg before Aβ injection. The incubated Aβ aggregates were injected in the intracerebroventricular (ICV) area using a stereotaxic apparatus (Harvard Apparatus, Panlab, Sandiego, CA, USA) fixed at the following coordinates: anterior/posterior (AP) -1.0 mm, dorsal/ventral − 2.5 mm, and mediolateral/lateral + 1.0 mm. Aggregated Aβ1–42 (5 µL) was infused at a rate of 2 µL/min speeds . Saline was used as a control. At 24 h after the injection, experimental mice were assorted into five groups (n = 8 per group): normal control mice (NOR), Aβ1–42-injected mice (Aβ), Aβ + EEFE-injected mice at 50 or 150 mg/kg/day (EEFE-50 or EEFE-150), and Aβ + morin-injected mice at 10 mg/kg/day (M-10). Animals received a nontoxic concentration of EEFE for 20 days. The experimental schedule, including Aβ injection, EEFE administration, and behavioral testing, is shown in Fig. 1.
Preparation of brain tissue sections for immunohistochemistry and Nissl staining
At the end of experimental period, mouse from each group were sacrificed under deep anesthesia. Three mouse brain from each group were perfused transcardially with saline and then fixed in 4% paraformaldehyde. Each of hippocampal and cortical tissues isolated from five mouse of each group were immediately stored at -80 °C until further analysis. Brain paraffin blocks were sliced into 4-µm-thick sections. The slides were deparaffinized and hydrated with xylene and sequentially of ethanol solutions, respectively. Slides were treated with 0.3% hydrogen peroxide in methanol for 25 min and briefly rinsed in PBS for quenching endogenous peroxidase activity. For antigen retrieval, slides were boiled in citrate buffer (pH 6.0) in a microwave for 15 min. Slides were incubated with horse serum for blocking for 1 h at 37℃ and then probed with primary anti-Aβ (AB_10988723, 1:250 dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-neuronal nuclei (NeuN) (AB_10711153, 1:250 dilution; Abcam, Cambridge, UK), and anti-ionized calcium-binding adaptor molecule 1 (Iba-1) (AB_839504, 1:100 dilution; Wako Pure Chemicals, Osaka, Japan) antibodies for overnight at 4 °C. To detect Aβ, NeuN, and Iba-1, the slides were incubated with labeled streptavidin–biotin for 30 min and visualized using diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories, Burlingame, CA, USA). Slides were immersed in 1% cresyl violet acetate solution for Nissl staining, washed with water, and dehydrated with 90% and 100% ethanol for 5 min before mounting in xylene. Mounted slides were then captured at 400 × magnification using a microscope (Olympus DP71, Tokyo, Japan). Image analysis was performed by blinded investigators using Image J software program (Java-based image processing program, NIH, Bethesda, MD).
Western blot analysis
Homogenized brain lysates (30 µg) were resolved on polyacrylamide gels and then transferred on 0.2-µm PVDF membranes using the Trans-Blot transfer system (Bio-Rad, Hercules, CA, USA). Blocked membranes with 5% nonfat milk washed with Tris-buffered saline with 0.1% tween 20 (TBST) and then incubated with primary anti-BDNF (AB_10862052), anti-phospho-CREB (AB_731734), anti-total CREB (AB_ 2827810, Abcam), and anti-Aβ (1:3000 dilution; Santa Cruz Biotechnology Inc.) antibodies for overnight at 4℃. Washed membranes with TBST were incubated with secondary antibodies anti-rabbit- horseradish peroxidase (HRP). Immuno-reactive membrane was developed using the chemiluminescent substrate reagents (Cat. 34577, Amersham Bioscience, Piscataway, NJ, USA). Membranes were also incubated with anti-β-actin (AB_476743, Sigma-Aldrich, Saint Louis, MO, USA) for loading control. Protein bands were detected by using imaging analyzer (Las-4000 MINI; Fuji Photo, Tokyo, Japan) and quantified with Image J software (NIH).
As we have described previously , memory and learning function of experimental mice was conducted using passive avoidance task (PAT) and Morris water maze (MWM) tests. The PAT was performed using an electronic shock generator with lightened and darkened compartments (Jeungdo Bio & Plant Co. Ltd., Seoul, Korea) on days 8–10 after Aβ injection. Transfer latency time was recorded as the amount of time (within 5 min) the mice remained in the lightened compartment. The MWM was conducted on days 15–21 after Aβ injection. Mice were put in a water pool containing four designated release points and allowed to find the escape platform for 60 s. After finding the platform, the animals were allowed to rest for 30 s on the platform. If mice failed to find the platform within 60 s, they were guided to rest on the platform for 30 s. After all animals finished the first trial, the next trial was started. The release points were randomly chosen every time without overlap. Time taken to find the platform (escape latency) was monitored (training: 2 days, behavioral tests: 4 days, and probe trial: 1 day). In the last day (day 7), the platform was removed 1 h after EEFE administration and the probe trial was performed for 60 sec to measure the number of times the mice crossed the platform. Experimental mice were given EEFE or morin prior to behavioral test.
Quantitative analysis of inflammatory cytokines in brain tissues
Mouse pro-inflammatory cytokines, TNF-α and IL-1β were measured in brain lysates using commercial competitive ELISA (Cat. MBS2500421, MyBioSource, San Diego, CA, USA) and (Cat. AB100705, Abcam), respectively. Supernatants of brain lysates were collected by according to manufacture’s protocol. The concentration of TNF-α and IL-1β was calculated from the standard curves. The optical density (OD) was measured using a microplate reader (BioTek Instrument, Winooski, VT, USA) at 450 nm.
Chemicals, Reagents, And Sample Preparation For Quantitative Analysis
Three standard compounds (rutin, chlorogenic acid, and kaempferol-3-O-rutinoside) and Biopurify Phytochemicals were purchased from ChemFaces Biochemical (Wuhan, China) and (Chengdu, China) (purity > 98% by high-performance liquid chromatography [HPLC]), respectively. Acetonitrile and water of HPLC grade (J. T. Baker Chemical, Phillipsburg, NJ, USA), and trifluoroacetic acid (TFA, Sigma-Aldrich) were used for quantitative analysis, respectively. For quantitative analysis, powdered EEFE leaves were dissolved in 80% aqueous methanol to a final concentration of 10 mg/mL and filtered through a syringe filter (0.45-µm pore size). To make a standard mixture, the stock solutions of the three standard compounds (1.0 mg/mL) were mixed with methanol to final concentration of 0.1 mg/mL and diluted to obtain various concentrations of standard solution before chromatographic analysis.
For HPLC analysis, we used the HPLC system equipped with a photodiode array (PDA) detector (Waters Alliance e2695, PDA #2998, Waters Corp., Milford, MA, USA). The data were acquired and processed using Empower software (version 3; Waters Corp.). For chromatographic separation of the three compounds, we used a Sunfire C18 analytical column (250 × 4.6 mm, 5 µm, Waters Corp.), which we maintained at 35 °C. The gradient conditions were 10–23% B for 30 min, 23–100% B for 10 min, and 100% B for 10 min. The mobile phases consisted of two solvents: 0.1% (v/v) TFA in water (A) and acetonitrile (B). For scanning chromatograms, PDA detection was performed at 210–400 nm. Column were used at flow rate of 1.0 mL/min and injected volume of 10 µL, respectively.
All data were expressed as the mean ± SEM. A value of p < 0.05 was considered to indicate statistical significance. Prism software (Graph Pad, version 8.4.1, San Diego, CA, USA) was used for all analyses. Statistically significant differences were evaluated with one-way analysis of variance (ANOVA) for comparison three or more groups, and with an unpaired or paired Student’s t-test for comparison between two groups. All experiments were performed individually at least three times.