4.1 Chemistry4.1.1 Isolation and identification of 5α-EAL from Inula macrophylla

Whole plant collections are from Uzbekistan, in 2021 and were recognized as Inula macrophylla by Dr. K. A. Eshbakova. The specimen of the voucher was archived at the Academy of Sciences of the Republic of Uzbekistan.

10.0 kg of the Inula macrophylla were air-dried, powdered, and extracted with 90% ethanol. After evaporation, the residue was suspended in H2O and successively partitioned with EtOAc. The EtOAc layer (308 g) was fractionated into five fractions (A‒E) via silica gel column chromatography eluting with CHCl3–MeOH (from 60:1 to 3:1, v/v). Fr. B (18 g) was fractionated using medium-pressure liquid chromatography with a MeOH–H2O mobile phase (from 10:90 to 90:10, v/v) over a period of 50 min, resulting in the isolation of five fractions. Fr. B2 was purified by preparative HPLC (MeCN–H2O from 40:60 to 60:40, v/v, 60 min) to obtain 5α-EAL. The 1D NMR spectra were detected via a Bruker Avance III 400 instrument (Figs. S1 and S2).

4.2 In vitro activity4.2.1 Cell viability assay in BV-2 cells

Murine microglial BV-2 cells were plated at a concentration of 8 × 103 cells/well in 96-well plates and cultured overnight. After treatment with different levels of 5α-EAL (1.25, 2.5, 5, 10 and 20 µM) for 24 h, 100 µL of MTT solution (5 mg/mL) from Solarbio in China was pipetted into each well. The plates were then incubated at 37 °C for 2 h. The blue formazan products in the cells were diluted in DMSO (200 µL/well) and mixed thoroughly. The optical density of the reaction medium was read at a wavelength of 570 nm using a microplate reader.

4.2.2 Inhibition of nitric oxide production in BV-2 cells

NO production inhibitory was measured according to the previous method [8]. Briefly, 96-well plates were plated with murine microglial BV-2 cells (2 × 105/well) and treated for 24 h with or without 1 μg/mL LPS (Escherichia coli 0111: B4, Sigma, U.S.) by adding 5α-EAL. The NO concentration in the supernatant was determined with an NO content assay kit (Solarbio, China).

4.2.3 ELISA assay

After treatment of LPS-stimulated BV-2 cells with 5α-EAL for 24 h, pro-inflammatory (TNF-α, PGE2) and anti-inflammatory (IL-10) mediator levels in culture supernatants were measured using ELISA kits according to the manufacturer’s method. (TNF-α: SU-BN20852, IL-10: SU-BN20162, R&D, USA; PGE2: KA0326, Abnova, China, Arg-1: ab269541, Abcam, USA).

4.2.4 Western blotting assay

The western blotting assays were measured according to the previous method [26]. In short, BV-2 cells were either exposed to 1 μg/mL LPS or left untreated, followed by treatment with DMSO or 5α-EAL at various concentrations. After 24 h, the cells were rinsed with chilled PBS (Solarbio). The chilled lysis buffer containing protease inhibitor and phosphatase inhibitor mixture (AbMole BioScience, China) was added to the cells and then gently scratched off. After centrifuging at 12,000 rpm for 10 min at 4 °C, Protein concentrations were assessed utilizing a bicinchoninic acid (BCA) assay kit from TIANGEN, China. Equivalent quantities of protein were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels from EpiZyme, China, and subsequently transferred to NC membranes manufactured by GVS, USA. Following this, the membranes underwent blocking with 5% nonfat milk from BD for 1 h before being incubated overnight at 4 °C with primary antibody diluted appropriately. Subsequent to washing, the membranes were exposed to a specific secondary antibody for a duration of 2 h. The chemiluminescence western blotting detection system (Thermo Fisher Scientific, USA) was used to visualize the immunoreactive bands. ImageJ software was then used for band analysis. The main primary antibodies used in this study were rabbit monoclonal anti-iNOS (1:1000, Abcam, USA), rabbit monoclonal anti-COX-1 (1:1000, CST, USA), rabbit monoclonal anti-COX-2 (1:1000, CST, USA) and rabbit monoclonal anti-GAPDH (1:1000, CST). The secondary HRP-conjugated antibody was goat anti-rabbit IgG (1:5000, SAB).

4.2.5 Immunofluorescence for NF-κB translocation assay

Immunofluorescence experiment was conducted according to the previous method to evaluate the nuclear translocation of NF-κB p65 [26]. In brief, the BV-2 cells were plated in 8-well plates and incubated for 24 h. The cells were pretreated with 5α-EAL (10 μM) for 1 h and then activated by LPS (1 μg/mL). Then, the fixed cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min in 4 °C and washed three times with PBS containing 5% BSA and blocked with 5% bovine serum albumin (BSA in PBS) for 1 h at room temperature. The permeabilized cells were incubated with primary antibody (anti-p-NF-κB p65) overnight at 4 °C. After removing the primary antibody, the cells were incubated with Anti-rabbit IgG (H+L), F (ab′)2 Fragment (Alexa Fluor® 647 Conjugate) for 1 h at room temperature. After removing the secondary antibody, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL in PBS) for 10 min. All images were obtained using a fluorescence microscope (Leica German).

4.2.6 Blood–brain barrier permeability assay

The BBB permeability of 5α-EAL was assessed using the parallel artificial membrane permeation assay (PAMPA) method, as previously described. The pre-coated PAMPA plate system (Cat. No. 353015) from Coning, U.S., was used to perform the permeability assays. Carbamazepine and hydrocortisone were supplied by Sigma. 5α-EAL was diluted in DMSO at a concentration of 8 mg/mL to form a stock solution, which was then diluted in PBS to a final concentration of 400 μg/mL. The solution was filtered through 0.22 μM syringe filters and pipetted into the donor wells (300 μL/well), while PBS was added to the wells of the recipient plate (200 μL/well). The filter plate was then assembled with the acceptor plate, and the entire assembly was incubated at room temperature for 16 h without agitation. At the end of the incubation, 150 μL of supernatant was removed from the donor and acceptor well and quantified by HPLC with a UV screening detector at 210 nm.

4.3 In vivo activity4.3.1 Animals and ethical statement

Male C57BL/6J mice were obtained from the Fourth Military Medical University, Shaanxi, China (n = 10/group) at 6–8 weeks of age and 25–30 g in weight. Animals were placed in a dedicated germ-free animal room under conditions of 12/12 light/dark cyclicity. They were provided with standardized nutrition, which consisted of pure water and AIN-93M. All animal procedures were performed under the principles and guidelines outlined in the Care and Use of Laboratory Animals.

In this study, all mice were randomized into six groups (n = 10/group) as shown below: group I, control (WT + Vehicle); group II, control + high dose 5α-EAL (30 mg/kg per day, WT + 5α-EAL-high); group III, SCOP (SCOP + Vehicle); group IV, SCOP + low-dose 5α-EAL (10 mg/kg per day, SCOP + 5α-EAL-low); group V, SCOP + high-dose 5α-EAL (30 mg/kg per day, SCOP + 5α-EAL-high); group VI, SCOP + DNP (3 mg/kg per day, SCOP + DNP). 5α-EAL (10 and 30 mg/kg per day) was dissolved in a solution of water containing 10% Tween 80, 10% ethanol, and 80% distilled water (0.1% w/vol). Donepezil Hydrochloride was dissolved in saline. The aforementioned groups were administered to 8-month-old WT mice for a period of 3 weeks. The WT + Vehicle, WT + 5α-EAL-high, SCOP + 5α-EAL-low, and SCOP + 5α-EAL-high groups received treatment via intraperitoneal (i.p.) injection of 5α-EAL, while the DNP group was treated with an oral gavage (i.g.) of DNP.

4.3.2 Barnes maze task

The Barnes Maze is the most widely used test in behavioral neuroscience to study spatial learning and memory in rodents. Briefly, mice were acclimated to the experimental room for 30 min before each daily experiment. Subsequently, they were placed in the center of the Barnes Maze in a starting box for 20 s. The start box was taken away and the mice were permitted to explore the Barnes maze for 3 min. If the mice did not locate the destination box on the first day, they were guided to find the hole and then remain in the box for 1 min. There was no guidance provided on days 2–4. Mice were under study for 4 consecutive days. On the fifth day, the dark box of the target hole was withdrawn, and the latency time, residence time in the destination quadrant, number of times of exploring the target hole, and the movement trajectory of the mice were recorded in the 90 s period. The table was wiped with 75% disinfectant alcohol before and after the test to remove odor interference.

4.3.3 Y-maze task

The Y-maze experiment is a common experimental tool for assessing working memory. The Y-maze comprises three equal arms (40 cm × 15 cm × 9 cm). Mice were placed in the center of the Y-maze and given 5 min to explore the three arms. Their motion was automatically recorded with a video tracker.

4.3.4 Measurement of AChE activity

After the behavior tests on day 28, Mice were executed by cervical dislocation. The mouse cortex and hippocampus were collected, washed with cold sterile saline, and stored at − 80 °C prior to use. The tissues of cortices and hippocampi were weighed, homogenized, and centrifuged at 4 °C 4000 rpm for 5 min. After 10 min collect the supernatant. The measurement of AChE activity was conducted following the instruction of acetylcholinesterase assay kit. This assay was conducted in a 2 mL centrifuge tube, adding 30 µL of 10% tissue solution, 500 µL of substrate buffer, and 500 µL of coloring liquid for each group. Incubate at 37 °C for 6 min, then add 30 µL of inhibitor and 100 µL of transparent agent, respectively. After incubation for 15 min, the OD value was measured at 412 nm using a microplate reader, and then activity was calculated.

4.3.5 Statistical analysis

GraphPad 8.0 software was used for statistical analysis. The experimental data were presented as mean ± SEM for each group. Student’s t-test or two-way analysis of variance (ANOVA) followed by Tukey post hoc test was used for all statistical analyses. *p < 0.05, **p < 0.01, and ***p < 0.001 were regarded as statistically significant for between-group differences.