Synergistic Effects of Curcumin and Piperine as Potent Acetylcholine and Amyloidogenic Inhibitors With Significant Neuroprotective Activity in SH-SY5Y Cells via Computational Molecular Modeling and in vitro Assay

Abstract

Hallmarks of Alzheimer’s disease (AD) pathology include acetylcholine (ACh) deficiency and plaque deposition. Emerging studies suggest that acetylcholinesterase (AChE) may interact with amyloid β (Aβ) to promote aggregation of insoluble Aβ plaques in brains of patients. Current therapeutic options available for AD patients, such as AChE inhibitors, provide only symptomatic relief. In this study, we screened four natural compounds believed to harbor cognitive benefits—curcumin, piperine, bacoside A, and chebulinic acid. In the first section, preliminary screening through computational molecular docking simulations gauged the suitability of the compounds as novel AChE inhibitors. From here, only compounds that met the in silico selection criteria were selected for the second section through in vitro investigations, including AChE enzyme inhibition assay, 3-(4,5-dimenthylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay, Thioflavin T (ThT) assay, and biochemical analysis via a neuronal cell line model. Of the four compounds screened, only curcumin (−9.6 kcal/mol) and piperine (−10.5 kcal/mol) showed favorable binding affinities and interactions towards AChE and were hence selected. In vitro AChE inhibition demonstrated that combination of curcumin and piperine showed greater AChE inhibition with an IC 50 of 62.81 ± 0.01 μg/ml as compared to individual compounds, i.e., IC 50 of curcumin at 134.5 ± 0.06 μg/ml and IC 50 of piperine at 76.6 ± 0.08 μg/ml. In the SH-SY5Y cell model, this combination preserved cell viability up to 85%, indicating that the compounds protect against Aβ-induced neuronal damage ( p < 0.01). Interestingly, our results also showed that curcumin and piperine achieved a synergistic effect at 35 μM with an synergism quotient (SQ) value of 1.824. Synergistic behavior indicates that the combination of these two compounds at lower concentrations may provide a better outcome than singularly used for Aβ proteins. Combined curcumin and piperine managed to inhibit aggregation (reduced ThT intensity at 0.432 a.u.; p < 0.01) as well as disaggregation (reduced ThT intensity at 0.532 a.u.; p < 0.01) of fibrillar Aβ42. Furthermore, combined curcumin and piperine reversed the Aβ-induced up-regulation of neuronal oxidative stress ( p < 0.01). In conclusion, curcumin and piperine demonstrated promising neuroprotective effects, whereas bacoside A and chebulinic acid may not be suitable lead compounds. These results are hoped to advance the field of natural products research as potentially therapeutic and curative AD agents.

Schisandra Extract and Ascorbic Acid Synergistically Enhance Cognition in Mice through Modulation of Mitochondrial Respiration

“…AA is known to scavenge mitochondria-generated ROS [31], but the effects of SCE or AA on mitochondrial respiration in the brain has not previously been tested. Consistent with previous reports that combinations of plant extracts exhibit augmented biological activity compared with individual plant extracts [32,33], we found that a mixture of SCE and AA at a 4:1 ratio (10 µg/mL) exerted synergistic effects on mitochondrial respiration. However, this effect was absent at lower ratios of SCE in the mixture, implying that not only do SCE and AA in the mixture additively increase mitochondrial activity, the inclusion of AA may contribute to the scavenging of ROS, produced as a result of enhanced mitochondrial respiration.…”

Vicinal difluorination as a C=C surrogate: an analog of piperine with enhanced solubility, photostability, and acetylcholinesterase inhibitory activity

“…One possibility is that analog 2 is induced to adopt the "correct" conformation when binding to both targets (i.e., 2f, Figure 2), and this structure fits well within the AChE active site but poorly within the BACE-1 active site, perhaps due to unfavourable interactions of the fluorine substituents of 2 with BACE-1 active site residues. A second possibility is that 2 adopts an "incorrect" conformation upon binding to both targets (e.g., 2a, Figure 2), and this novel molecular shape is readily accommodated by AChE [35] but not by BACE-1, perhaps due to different levels of flexibility within the enzyme active sites. A third possibility is that the analog 2 adopts different conformations upon binding to AChE vs BACE-1, since the microenvironments within the enzymes' active sites could be different (e.g., more/less polar), the "correct" binding geometry of 2 might be favoured in AChE but not in BACE-1.…”

Explicating anti-amyloidogenic role of curcumin and piperine via amyloid beta (Aβ) explicit pathway: recovery and reversal paradigm effects

“…The preparation of the optimal concentration of pure curcumin, piperine and their mixtures was performed based on an optimized protocol (Manap et al, 2019).…”

“…We had previously reported on the Aβ inhibition and disaggregation assay of selected compounds by using Thioflavin T fluorescence assay (Manap et al, 2019). Thioflavin T (ThT) is a small molecule that emits strong fluorescence upon binding to amyloids (Xue et al, 2017).…”

“…We previously demonstrated neuroprotective effects of combined treatment with curcumin and piperine against Aβ induced degeneration by in silico and in vitro assays (Manap et al, 2019). Curcumin and piperine at 35 µM in combination were able to inhibit neurotoxicities, aggregation and disaggregate Aβ fibrils as well as reversed Aβ-induced neuronal oxidative stress (Manap et al, 2019). In the present study, we continue our investigation at a molecular level by using high-throughput microarray technology in order to elucidate differences in the gene expression profiles between AD and treatment groups.…”