The seeds of the plant Psoralea corylifolia were purchased from the local market of Jammu, J&K, India. The starting material used is FSSAI (Food Safety and Standards Authority of India) certified with license number 13319011000193. The dried seeds (2.7 kg) of the plant were coarsely powdered by grinding and extracted with a mixture of DCM (dichloromethane): MeOH (methanol) (1:1, 15 L) at room temperature for 24 h and filtered. The marc was reextracted twice with the same solvent mixture using the same volume ratio of material to solvent. The filtrates were combined and concentrated on a rotary evaporator under reduced pressure at 40°C to yield 322.5 g crude extract with an extractive value (EV) of 11.9%.

DCM: MeOH (1:1) extract was fractionated sequentially with three different solvents, viz hexane, dichloromethane, and methanol by liquid-liquid partition chromatography to yield an enriched fraction of isobavachalcone. 300 g of the dried extract was dissolved in 1.5 L methanol and partitioned with 2 L hexane and this whole procedure was repeated two more times. After separating the upper hexane layer, 1.5 L DCM and 150 mL distilled water were added to the remaining methanolic layer for partitioning and the process was repeated two more times for maximum enrichment. All three fractions; hexane (108.4 g), DCM (119.7 g), and aqueous methanol (69.8 g) thus obtained were monitored by thin layer chromatography (TLC). TLC analysis concluded that the DCM fraction had the highest concentration of isobavachalcone followed by the hexane fraction.

Preferentially, the DCM fraction (100 g) was charged on a silica-gel column (60-120 mesh) and eluted with a gradient of n-hexane-ethyl acetate (95:5–50:50, v/v) which provided several subfractions. These subfractions were further analyzed by TLC using solvent system n-hexane/ethyl acetate (70:30). Based on the identification of isobavachalcone on TLC plates, the subfractions are pooled together and charged on silica gel column (60-120) to furnish the desired compound that is Isobavachalcone (2.1 g).

The purity of the IBC was analyzed using High-Performance Liquid Chromatography (HPLC) (Model: Prominence, Make: Shimadzu). Chromatographic separation was performed in gradient mode on a Purospher STAR RP-18 (250 × 4.6 mm, 5 μm) column. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The flow rate was set to 1 mL/min, with a total run time of 60 min. The sample eluted at 30.3 min, and the detection was carried out at a wavelength of 364 nm. The stock solution was prepared in DMSO and subsequently diluted with methanol for analysis.

Primary astrocytes were cultured from C57BL/6J (Jax:000664) mice pups with the approval of Institutional Animal Ethics Committee (IAEC) of CSIR-Indian Institute of Integrative Medicine (IIIM), Jammu, under IAEC number 287/80/2/2022 and 305/81/8/2022. The astrocytes were isolated from the brain of 3–4 days-old mice pups and were grown in 10% FBS-supplemented DMEM media. Briefly, pups were sacrificed through decapitation and sterilized with 70% ethanol. Brain was exposed and isolated. Olfactory lobes and cerebellum were removed with fine blade. The cortical region separated was collected in incomplete media (media without fetal bovine serum) and cut into small pieces. After that, these cortices were incubated at 37°C in 0.25% trypsin for 30 min, with gentle shaking after every 10 min. Trypsin activity was neutralized with complete media and cells were collected using a 70-micron cell strainer followed by centrifugation at 300 g for 5 min. The pellet was dissolved in fresh media and cells were grown in cell culture flasks and were maintained at 37°C in a CO₂ incubator. Microglia and oligodendrocytes were separated from the underlying astrocyte layer after 6-7 days by shaking the flask for 6 h at 240 rpm. Cultures astrocytes within 2-4 passage were used for all the experiments.

Primary astrocytes were first treated with microglia conditional media to induce astrogliosis (Liddelow et al., 2017). For this, the microglia (N9) cell line, provided by Dr. Anirban Basu (National Brain Research Centre, Gurgaon, India) was cultured in RPMI-1640. N9 cells (Passage 28-35) were seeded in 24 well plate at density 0.05 × 10⁶ per well. The cells were treated with 1 μg/ml LPS, and supernatant was collected after 24 h. Seeded astrocytes were then treated with the LPS stimulated N9 cells supernatant for 24 h. Thereafter, the treatment for NLRP3 inflammasome activation was given, including priming with fresh 1 μg/ml LPS for 4 h, followed by washing and IBC or MCC950 (100 nM) treatment for 1 h in incomplete media (media without fetal bovine serum). ATP at 5 mM concentration was used as secondary signal for 30 min.

The experiment was terminated by collecting the cell supernatant.

After the termination of experiments, the supernatant was collected and analyzed for levels of cytokines. For measuring IL-1β and IL-18, supernatant collected after NLRP3 inflammasome activation as mentioned above, was subjected to ELISA. For detection of IL-1β in mice cortex, mice cortices were isolated and homogenized in RIPA buffer to measure IL-1β in homogenates. The manufacturer’s protocol was followed for measuring the cytokines. The levels of measured cytokines were normalized with the total protein content of the cells. For total protein content of cell pellet lysed with lysis buffer (0.2 N NaCl and 1% Triton-X 100) was measured.

IC50 of Isobavachalcone for IL-1β and IL-18 was calculated by using GraphPad Prism 8 software using non-linear regression analysis model. Log (inhibitor) vs. response curve was plotted.

Following treatment with 5 and 10 μM IBC (1 h) and NLRP3 inflammasome activation in primary astrocytes, cells were lysed with ice-cold buffer (KCl [150 mM], HEPES-KOH [20 mM, pH 7.5], NP-40 [1%], sodium orthovanadate [1 mM], PMSF [0.1 mM] and protease inhibitor cocktail [1%]). Cells were centrifuged at 300 g for 10 min. The supernatant was collected for western blot analysis of total ASC protein. The pellet was washed with cold PBS and resuspended in 500 µL cold PBS. For the crosslinking of ASC, 2 mM of suberic acid was added to the resuspended pellet and incubated at 37°C for 30 min in water bath. After centrifugation, the supernatant was discarded, and a 2X Laemmli buffer was added to the pellet. The samples were heated at 95°C for 5 min and subjected to western blotting for analyzing ASC oligomers.

For cell viability assay, primary astrocytes were seeded in 96 well plate and IBC treatment at different concentration was given for 24 h. For MTT assay, cells were treated with 1.25, 2.5, 5, 10, 20, 40, 80 and 100 μM of IBC, while for the SRB assay, 0.625, 1.25, 2.5, 5, 100, 20, 40, 80 and 100 μM IBC treatment was given. MTT dye at a concentration of 2.5 mg/ml was added 4 h before experiment termination. Formazan crystals thus formed were dissolved in DMSO and absorbance was measured at 570 nm.

For the SRB assay, the experiment was terminated by adding 50% chilled TCA into the wells for 1 h. Pellets were washed five times with distilled water and kept drying. Cells were then incubated with SRB dye for 30 min at RT, followed by washing with 1% acetic acid thrice. After drying, pellets were dissolved in 10 mM Tris-HCl (pH-10.5) and absorbance was measured at 540 nm.

Levels of cleaved IL-1β and caspase-1 were analyzed in cellular supernatant. The supernatant collected after the end of the experiment was incubated with StartaClean resin for 1 h at 4°C. Samples were kept on rotation so that the resin could bind properly with proteins. The concentrated resin pellet was obtained by centrifuging samples at 300 g for 10 min 2X Laemmli buffer was added to the resin pellet, heated at 95°C for 10 min, and subjected to western blotting.

Cellular lysates were prepared using radio immuno precipitation assay (RIPA) buffer for protein analysis. Briefly, the cell pellet was dissolved in RIPA buffer containing sodium fluoride, sodium orthovanadate, phenyl methyl sulfonyl fluoride (PMSF) and 1% protease inhibitor cocktail for 1 h with vertexing sample every 15 min. The supernatant was collected after high-speed centrifugation of samples for 20 min at 4°C. For analysis of proteins in mice hippocampi, the hippocampi isolated from mice brains was immediately washed with saline and stored in liquid nitrogen. For lysates preparation, tissue was homogenized in RIPA buffer substituted with sodium fluoride, sodium orthovanadate, phenyl methyl sulfonyl fluoride and 1% protease inhibitor cocktail. Total protein was measured using Bradford assay. Proteins were separated using SDS-PAGE and then transferred from gel to PVDF membrane. The protein of interest was labeled with a specific primary antibody overnight at 4°C. Further, the blot was incubated with corresponding HRP-conjugated secondary antibody for 1 h at room temperature. The band were visualized in the Genaxy Chemi Doc imaging system (Make: Syngene, Maryland, USA; Model: G: BOX, XT-4). Densitometry analysis of all the immunoblots was performed by using ImageJ software. Band density of proteins was normalized with β-actin. The phosphorylated proteins were normalized with their total forms.

Cells were seeded on coverslips in 6 well plates. After treatments, cells were fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.1% Triton-X 100 for 7 min. Blocking was done for 30 min with 2% BSA, followed by overnight incubation with primary antibody. Alexa fluor conjugated secondary antibody incubation was given to cells for 1 h for labeling. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI) (1 μg/mL) for 10 min. Following staining, the coverslips were mounted on slides using mounting media (PBS and glycerol at a ratio of 1:9). Cells were washed thrice with PBS after each incubation. The images were acquired in a CQ1 high-throughput imaging system.

Aβ₄₂-HiLyte fluor488 peptide was prepared following the manufacturer’s protocol (Anaspec Inc.). Primary astrocytes seeded on coverslips were treated with IBC (10 μM) or rapamycin (24 h). After 12 h, 2 μg/ml fluorochrome tagged (Hilyte fluor488) Aβ₄₂ protein was added to cells for another 12 h. Bafilomycin A₁ was given 3 h prior to experiment termination. After the end of the assay, cells were washed with PBS and cells were fixed with 4% PFA, permeabilized with Triton-X 100, and the nucleus was stained with DAPI, as mentioned above. Similarly, slides were prepared, and images were taken and analyzed in the CQ1 high throughput imaging system.

AMPK expression was knocked down from the cells by using siRNA against AMPK using FuGENE HD for 24 h. Primary astrocytes seeded in 6 well plates were incubated in OPTI-MEM media for 30 min prior to transfection. The transfection mixture was prepared and kept in sterile condition for 20 min before being added to the cells. Following this, cells were treated for NLRP3 inflammasome activation as described above.

All animal experiments were conducted with the approval of the Institutional Animal Ethics Committee of CSIR-IIIM, Jammu (IAEC No. 305/81/8/2022), adhering to the ethical guidelines of CCSEA (https://ccsea.gov.in). The mice were housed in IVCs (Individually ventilated cages) under controlled environmental conditions with a temperature 25°C ± 2°C, Relative Humidity 50%-60%, and a light cycle of 12 h light/dark. Gamma-irradiated feed and autoclaved RO water were provided as ad libitum to all experimental animals. At the end of experiments, animals were humanely euthanized by CO₂ asphyxiation.

C57BL/6J (Jax:000664) male mice were used for pharmacokinetic analysis of IBC. Mice were randomly sorted into 9 groups (n = 5). 30 mg/kg oral dose of IBC (1% DMSO +70% PEG-200 + 29% water) was given to mice. Blood or brain samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h. The plasma (100 µL) sample was processed with ethyl acetate (200 µL) containing IS (150 ng/mL) and centrifuged at 300 g for 10 min. The organic layer was separated and dried in a vacuum, reconstituted with acetonitrile, and analyzed by LC-MS/MS [SRM transition of IBC and IS (Phenacetin) is 325.2 & gt; 149.0 and 180.2 & gt; 110.2 in Positive mode]. Brain homogenate was prepared using the weighted amount of brain tissue in water at the concentration level of 350 mg/mL and processed similarly to plasma samples.

Hemizygous 5x-FAD transgenic mice (B6.Cg-Tg(APPSwFlLon, PSEN1*M146L*L286V) 6799Vas/Mmjax; MMRRC Strain: 034848-JAX) on congenic C57BL/6J genetic background were used for experiments. The 5x-FAD mice were genotyped using APP-specific qPCR probes to detect the transgene as per the protocol of Jackson Laboratory. Six months old 5x-FAD mice were randomly divided into three groups (n = 5), based on their body weights, with three males and two females in each group. We used mixed-cohort of males and females for our study to exclude any gender biased effect of IBC. Also, AD symptoms are more pronounced in females than in males (Aggarwal and Mielke, 2023), making it possible that males and females respond differently to the drug and treatment. Considering this, it is important to show effect of IBC on both sexes. At this age, Aβ deposition and neuroinflammation has already progressed in these mice. We have used IBC as a curative drug rather than a preventive drug. Prior to the initiation of the study, all the animals were acclimatized for 1 week under standard laboratory conditions; animals were drug naive with no prior procedures performed. All testing were performed from 1 to 4 p.m. IBC was orally (OD) given to two groups at different doses, 25 mg/kg (1% DMSO, 2% Tween 80, 19% PEG200, and 78% distilled water) and 50 mg/kg (1% DMSO, 5% Tween 80, 19% PEG200% and 75% distilled water) using oral gavage. 5x-FAD control mice were administered with vehicle (1% DMSO, 5% Tween 80, 19% PEG200% and 75% distilled water). For behavioral and Immunohistochemistry assays, experimenters were blinded to animal identity while carrying out the procedures.

To check the effect of IBC on the behavior of 5x-FAD mice, open field test (OFT), rotarod (Make; Ugo Basile, rota-rod for mice 7650) and radial arm maze (Make; Ugo Basile) tests were performed. All behavioral assays and testing were performed by an experimenter blinded to the treatment groups. To check the effect of IBC on motor coordination and balancing rotarod test was performed. After acclimatizing mice with rotarod for 5 days, their latency to fall was calculated on the day of analysis. The given final values are the mean of three independent attempts of each mouse. OFT was performed to assess exploratory and locomotor activity in a white box (45 × 60 cm). Distance traveled, speed, time spent in corner and center zones and mobile episodes were recorded through an automated video tracking system using AnyMaze software. To check the effect of IBC on the spatial memory of mice, the eight-armed radial arm maze test was done. The mice were acclimatized to the maze for 5 days before the experiment. Each mouse was trained to navigate the maze for five consecutive days. The mouse was placed on the end of one arm (entry arm). For spatial memory assessment, a food reward (100 mg of butter cookies) was placed in the selected arm, which was considered as entry arm. The movement of mice was tracked with the help of automated AnyMaze software connected with a tracking camera and various memory retention parameters were assessed.

Mice’s hippocampus tissue was fixed in 10% formalin. Dehydrated tissue slices were made and placed on slides. For analysis, tissue was rehydrated following subsequent incubation in xylene, xylene: ethanol, 100% ethanol, 90% ethanol, 70% and 50% ethanol. After tissue rehydration, cells were permeabilized using 0.4% Triton-X100 for 10 min. Blocking was done in 2% BSA, followed by incubation with primary and secondary antibodies. Images were taken in a CQ1 high throughput imaging system and analyzed using cell pathfinder software.

After the end of the experiment, blood was collected from the retinal orbital plexus of mice in EDTA-coated vials. Samples were centrifuged, and plasma was separated. The levels of Aβ in the plasma were measured by using the protein-specific ELISA kit.

Statistical analyses were performed using GraphPad Prism 8 software. All in-vitro experiments were performed thrice. The response of independent mice from the group was noted for animal experiments and the mean was calculated. The data presented here represents the mean ± SD. Statistical significance of the experiment was calculated using one-way ANOVA analysis and Bonferroni method was used as a post-hoc test. p value less than 0.05 was taken as significant.

AMPK is a primary regulator of autophagy and NLRP3 inflammasome-mediated inflammation, and both mechanisms are dysregulated in AD. With this information, we identified IBC (purity-98.8%; Supplementary Figure S1) as an inducer of AMPK-mediated autophagy and an inhibitor of NLRP3 inflammasome. To prove the effect of IBC on these processes, we used mouse primary astrocytes for all the in vitro experiments. The purity of the culture was tested by using markers of astrocytes (glial fibrillary acidic protein; GFAP), neurons (neuronal nuclear protein; Neu N) and microglial cells (ionized calcium-binding adapter molecule 1; IBA1) (Supplementary Figures S2A, B). To analyze the effect of IBC on AMPK phosphorylation at Thr 172, the cells were treated for 24 h with IBC. The western blot analysis revealed a concentration-dependent increase in the expression of AMPK (Thr 172) (p < 0.0001 at 5, 10 and 20 μM; Figure 1B). We further found that the cells treated with IBC showed a significant increase in the levels of autophagy marker LC3B-II (Light chain 3) (p < 0.0001 at 5, 10 and 20 μM; Figure 1C). The induction of autophagy by IBC was also supported by reducing levels of another autophagy marker, SQSTM1 (sequestosome 1), with the increase of IBC concentration (p < 0.0001 at 10 and 20 μM; Figure 1C).

After confirming the induction of autophagy by IBC, we proceeded to know whether the ability of IBC to induce AMPK-mediated autophagy can lead to the inhibition of NLRP3 inflammasome. We found through ELISA that the primary astrocytes primed with media from LPS-stimulated microglial N9 cells, when activated with LPS (lipopolysaccharide) and ATP (5′ adenosine triphosphate), led to the release of IL-1β and IL-18. The release of both IL-1β and IL-18 was significantly inhibited with IC50 values 2.95 µM and 2.75 µM, respectively, when the cells were treated with IBC for 1.5 h, which clearly indicated the inhibition of NLRP3 inflammasome (Figures 1D, E). MCC950 (100 nM) was used as a standard inhibitor of NLRP3 inflammasome in the experiments. We further confirmed the inhibition of NLRP3 inflammasome by measuring the cleaved and active forms of IL-1β and CASP1 from the supernatant of cells treated with IBC by using western blotting. The data clearly indicated the reduced release of IL-1β and CASP1 (p < 0.0001 at 5 and 10 μM) from the cells and, thus, the inhibition of NLRP3 inflammasome, though there was no change in the expression of NLRP3 protein (Figure 1F). Further, to check the effect of IBC on NLRP3 inflammasome complex formation, we analyzed the levels of ASC protein oligomers. Results showed that IBC-treated cells inhibited ASC monomer, dimer, and oligomer formation (Supplementary Figures S2C, D). Further, the IBC treatment at 5 μM and 10 µM concentrations showed a highly significant (p < 0.0001) reduction of speck formation in cells, in comparison to the cells induced with ATP, indicating reduced oligomerization (Supplementary Figures S2E, F).

Before moving further, we wanted to know if IBC has any toxicity in primary astrocytes. Therefore, we assessed the toxicity using two different assays, MTT and SRB. In both assays, IBC showed no toxicity in astrocytes even at 100 μM concentration (Supplementary Figure S3A). The activation of AMPK is regulated by two main kinases, LKB1 (liver kinase B1) and CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2). To ascertain the cause of AMPK activation, we checked the phosphorylation and activation of these kinases after treating primary astrocytes with IBC for 24 h. Interestingly, there was no change in the levels of pLKB1 (Ser 428), whereas pCAMKK2 (Ser 511) was found to be significantly upregulated after concentration-dependent treatment with IBC (p < 0.05 at 5 μM, 0.01 at 10 μM and 0.0001 at 20 μM; Figures 2A, B). However, we found no change in MTOR (mechanistic target of rapamycin) levels. ULK1 (unc-51-like kinase 1), which is a direct target of AMPK, was found to be phosphorylated at Ser 317, marking its activation; which was also confirmed indirectly through increased phosphorylation of FIP200, another protein present in the ULK1 complex (Figures 2A, B). Further, the downstream protein BECN1 (beclin 1, autophagy related) did not show any change in the expression. However, BCL2 (B-cell lymphoma), which is known to interact with BECN1 and downregulate autophagy, showed a marked decline, thereby tilting the ratio in favor of BECN1 activation (p < 0.05 at 5 μM, and 0.0001 at 10 and 20 μM; Figures 2A, B). The level of proteins ATG5 (autophagy related 5) and ATG7 (autophagy related 7), involved in the maturation of the autophagosome, was also found to be upregulated, thus confirming the induction of autophagy by IBC in primary astrocytes (Figures 2A, B). To confirm the fusion of autophagosome with the lysosome, we did a confocal microscopy analysis for the co-localization of LC3B and LAMP1 (lysosomal-associated membrane protein 1), membrane marker proteins for autophagosome and lysosome, respectively. The data showed the proximity of LC3B and LAMP1 after the treatment of primary astrocytes with IBC (p < 0.0001 at 10 μM; Figures 2C, D; Supplementary Figure S3B). In all these experiments, rapamycin (Rap) at a concentration of 250 nM was used as a standard drug for the induction of autophagy.

After confirming the induction of autophagy by IBC, we wanted to know if it plays any role in inhibiting NLRP3 inflammasome in primary astrocytes. In our earlier assays, autophagy was checked after 24 h of treatment, and the inhibition of NLRP3 inflammasome was analyzed after 90 min of treatment with IBC. Therefore, we first checked whether IBC could induce autophagy under the conditions used for the NLRP3 inhibition assay. Interestingly, cells treated with IBC in the presence of LPS and ATP showed significantly enhanced phosphorylation of AMPK at Thr 172, along with that a marked increase in the level of autophagy marker LC3B-II was observed (p < 0.0001 at 5 and10 μM; Figure 3A). Further, the additional autophagy marker SQSTM1 levels also showed a significant decline after treatment with IBC (p < 0.0001 at 5 and10 μM; Figure 3A). To confirm the involvement of autophagy in NLRP3 inhibition by IBC, we inhibited the autophagy by end-stage autophagy inhibitor bafilomycin A₁ and checked its effect on NLRP3 inflammasome mediated release of IL-1β. We first checked the inhibition of autophagy by analyzing the expression of LC3B-II, which showed an accumulation of the protein in the presence of bafilomycin A₁, thus confirming the inhibition of autophagy. The inhibition of autophagy completely reversed the inhibitory effect of IBC on the release of IL-1β, which was almost equal to the levels in the cells treated with LPS + ATP as confirmed by both ELISA and western blotting (Figures 3B, C). These results clearly indicated the autophagic regulation of IBC-mediated inhibition of NLRP3 inflammasome.

Our earlier data showed that AMPK plays an essential role in autophagy induced by IBC. Therefore, we wanted to know if the knockdown of AMPK by using siRNA can affect the inhibition of NLRP3 inflammasome by IBC. We found that under the conditions of NLRP3 inhibition assay, the siRNA-mediated inhibition of AMPK completely reversed the inhibitory effect of IBC on two important proteins (IL-1b and CASP1) of the NLRP3 inflammasome complex. The release of both these proteins after treatment with IBC in the presence of siAMPK was almost similar to that of astrocytes treated with LPS + ATP (Figures 3D, E). Interestingly, there was no change in the expression of NLRP3 protein in the presence or absence of siAMPK, whereas its negative effect on autophagy marker LC3B-II was clearly visible (Figures 3D, E). The effect of siAMPK on the assembly proteins involved in NLRP3 complex formation also showed a similar trend. Treating astrocytes with IBC reduced the co-localization of CASP1 and NLRP3 compared to those treated with LPS + ATP (p < 0.0001 at 10 μM; Figures 3F, G; Supplementary Figure S4A). However, in the presence of siAMPK, the cells treated with IBC did not affect the co-localization of CASP1 and NLRP3 as observed through confocal microscopy (Figures 3F, G; Supplementary Figure S4A). Western blotting and confocal microscopy confirmed the knockdown effect of siAMPK on the AMPK (Thr 172) in all the experiments (Supplementary Figures S4B, C, D).

After confirming the dual pharmacological activity of IBC, we further wanted to determine whether the autophagic activity of IBC can help glial cells to clear Aβ. Therefore, we treated the astrocytes with IBC for 24 h and added Aβ₄₂-HiLyte fluor488 to the culture 12 h before the termination of the experiment. The confocal image analysis revealed that the cells with IBC showed significantly low fluorescence compared to the cells with Aβ alone, indicating reduced intracellular levels of Aβ in the IBC-treated cells (p < 0.0001). However, when we inhibited the autophagy with bafilomycin A₁, the levels of Aβ were found to be increased in the treated cells, which confirmed the involvement of autophagy in the clearance of Aβ by IBC (Figures 4A, C). These results were further consolidated when we inhibited autophagy at an early stage by using siRNA against AMPK. The cells treated with IBC displayed a marked decline in intracellular levels of Aβ compared to Aβ control. The cells that were treated with siAMPK showed a complete reversal of the IBC effect on the clearance of Aβ (Figures 4B, D). The treatment plan for these experiments is given in Figure 4E.

Based on in vitro data, we decided to investigate the effect of IBC on the pathology of AD in the transgenic mouse model (5x-FAD). Before going for a long-term study to analyze the effect of IBC on AD pathology, we first confirmed the penetration of IBC into the brain after a single dose (p.o.). Therefore, we compared the plasma and brain pharmacokinetic (PK) profile of IBC after a single dose of 30 mg/kg (p.o). The blood samples collected at different time points through 24 h showed plasma C_max value of 208 ± 46 ng/ml (Figure 5A; Table 1). The brain PK studies revealed that IBC crossed the blood-brain barrier with low penetration and showed a C_max value of 34 ± 04 ng/g of brain tissue (Figure 5B; Table 2). We determined the brain-to-plasma ratio of 0.12–0.16. To observe the effect of IBC on AD pathology, we decided to treat the 6 months old 5x-FAD mice at two different doses that are 25 mg/kg and 50 mg/kg, for 2 months (Experiment timeline is given in Figure 5C and detailed plan is illustrated in Supplementary Figure S5).

After 6 weeks of dosing, the animals were trained for 5 days each for the radial arm maze and rotarod tests. We generated the baseline behavior data by comparing the age-matched wild-type C57BL/6J mice group with the 5x-FAD control group and found a significant deviation in all the parameters tested during behavior analysis. In a radial arm maze test, the animals treated with IBC at both the doses 25 and 50 mg/kg took significantly less time (16.58 and 14.36 s, respectively) to enter the baited arm in comparison to the control group, which took an average of 32.3 s (p < 0.01; Figure 5D). Furthermore, the animals treated with IBC spent a significantly longer time in the baited arm than the control group (p < 0.01 at 25 mg/kg and <0.0001 at 50 mg/kg; Figure 5E). The treated animals also traveled significantly less distance to find the baited arm and showed a smaller number of entries into the non-baited arm (Figures 5F, G). These data clearly indicated improvement in spatial memory of 5x-FAD mice treated with IBC. We also tested the exploratory behavior of mice. The 5x-FAD mice displayed a significant reduction in their desire to explore the surrounding environment, which represents the behavior associated with AD. However, the animals treated with IBC showed improved exploratory behavior, which was evident from less time spent in the center, more time spent in the corners, and being in the mobile state for a longer time at higher speed along with greater distance traveled as compared to the 5x-FAD control group (p < 0.05; Figures 5I–M). The track plots of the mice in the radial arm maze test and open field test, which were drawn using AnyMaze software, are shown in Figures 5H, N, respectively. The treatment of 5x-FAD mice with IBC also improved their neuro-muscular coordination and stamina as evaluated by the rotarod test. The animals treated with IBC were able to stay on moving rods for a significantly longer time (92 s at 25 mg/kg and 118 s at 50 mg/kg) in comparison to the 5x-FAD control group, which could stay on rods for about 63 s only (p < 0.001 at 25 mg/kg and <0.0001 at 50 mg/kg; Figure 5O).

The improved memory behavior of 5x-FAD mice after 2 months of treatment with IBC led us to explore the molecular changes in the brains of these mice. Therefore, we analyzed the effect of IBC on the deposition of Aβ, which is a key pathological feature of AD responsible for cognitive impairment. We evaluated the Aβ levels in the hippocampi of the 5x-FAD mice by immunohistochemical (IHC) analysis. We observed a huge load of Aβ plaques (Average number of plaques = 55) in the 5x-FAD control mice group. The counting of Aβ plaques, using the cell pathfinder software, revealed that the average number of plaques was reduced to 21 and 15 in the mice that were treated with IBC at 25 and 50 mg/kg, respectively (p < 0.0001; Figures 6A, B; Supplementary Figures S6A, B). This was further validated by the fluorescence analysis of samples, which displayed a highly significant decline in the treated groups (Figures 6A, C). Further, the analysis through ELISA also exhibited reduced levels of Aβ₄₂ in the blood plasma of mice treated with IBC compared to the untreated control group, indicating the decline in the Aβ load in these mice (p < 0.0001; Figure 6D).

Deposition of Aβ₄₂ is strongly linked with increased neuroinflammation, which is reflected in the form of reactive astrogliosis and activation of NLRP3 inflammasome. Thus, we intended to know if the IBC meditated decline in Aβ₄₂ has reduced brain inflammation. The IHC analysis of the hippocampi for GFAP showed a large number of astrocytes expressing elevated levels of GFAP in the control group. However, in the mice treated with 25 and 50 mg/kg of IBC, the expression of GFAP was significantly reduced (p < 0.0001; Figures 6E, F; Supplementary Figure S6C). Also, Aβ, being one of the damage-associated molecular patterns (DAMP) known to activate NLRP3 inflammasome, we checked if the dwindling load of Aβ after treatment with IBC could reduce the activation of NLRP3 inflammasome. For that, we analyzed the levels of IL-1β in the cortices in the brains of these mice using ELISA. The data indicated a highly significant decline in the levels of IL-1β in the 5x-FAD mice treated with IBC at both doses 25 and 50 mg/kg, compared to the control group (p < 0.001 at 25 mg/kg and <0.0001 at 50 mg/kg; Figure 6G).

After confirming the clearance of Aβ deposits and alleviation of inflammation from the brain of 5x-FAD mice, we analyzed the expression of some of the proteins that are directly involved in the autophagic pathway. The western blot analysis clearly showed increased expression of pAMPK and ATG7, whereas the expression of SQSTM1 was significantly decreased in the mice treated with IBC at 25 and 50 mg/kg (p < 0.0001; Figure 7A). Further, the animals treated with IBC also expressed reduced levels of NLRP3 inflammasome proteins. The western blot analysis showed significant abrogation of CASP1 (p < 0.0001) and ASC (p < 0.001 at 25 mg/kg and <0.0001 at 50 mg/kg) in mice treated with IBC. This was further reflected in the reduced cleavage of IL-1β in these mice (p < 0.0001; Figure 7B). The treatment of mice with IBC also inhibited the level of the reactive phenotype of astrocytes and microglial cells, evidenced by decreased expression of GFAP (p < 0.01 at 25 mg/kg and <0.0001 at 50 mg/kg) and IBA1 (p < 0.0001) (Figure 7B).