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 IC₅₀ of 62.81 ± 0.01 μg/ml as compared to individual compounds, i.e., IC₅₀ of curcumin at 134.5 ± 0.06 μg/ml and IC₅₀ 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.

Keywords: Alzheimer’s disease; acetylcholinesterase; amyloid beta; curcumin; piperine.

Figure 1

Summary of computational and experimental approach conducted in this study.

Figure 2

Relative position of AutoGrid box in acetylcholinesterase (AChE) encompassing both catalytic action site (CAS) and peripheral anionic site (PAS) docking sites.

Figure 3

Binding interactions of ligands to AChE. Amino acids in orange box represent CAS residues, blue box for PAS, while the remaining amino acids are residues that were involved in stabilizing the binding within the AChE gorge.

Figure 4

Color coding of non-covalent interactions linking ligand–receptor pairs. The interaction can be categorized into five groups, namely, hydrogen bonds, electrostatic, hydrophobic, and unfavorable linkage.

Figure 5

Hydrogen bond molecular surface mapped the locations of hydrogen bonds in ligands when bound to AChE. Curcumin and piperine demonstrated a high number of hydrogen bond donors and hydrogen bond acceptors. Meanwhile, bacoside A showed a higher number of hydrogen bond donors while chebulinic acid presented with a higher number of hydrogen bond acceptors.

Figure 6

Degree of hydrophobicity on the molecular surface of ligands when bound to AChE. Curcumin and piperine both have similar sizes of areas with low hydrophobicity and areas of low hydrophilicity. Meanwhile, bacoside A has a larger area of hydrophobicity while chebulinic acid has a smaller area of hydrophobicity.

Figure 7

Dose–response relationships for curcumin, piperine, and donepezil used singularly in various concentration gradients from 2.5 μM to 100 μM. Each curve represents the increase in cell viability as a function of the dose. Effective concentration (EC) was calculated using GraphPad Prism.

Figure 8

Effect of varying concentrations of curcumin and piperine used in combination in SH-SY5Y cells proliferation after 24 h. Synergism quotient (SQ) values were calculated to determine if there was any synergistic activity in the treatment. The results presented are averages of three independent experiments each done in triplicate and expressed as the mean ± SEM. *p < 0.05, **p < 0.01, one-way ANOVA with Dunnett’s test compared to combined curcumin and piperine group (CuR: Pip).

Figure 9

Effects of amyloid β (Aβ), Donepezil + Aβ, curcumin + Aβ (CuR + Aβ), piperine + Aβ (Pip + Aβ), and combined curcumin and piperine + Aβ [(CuR + Pip) + Aβ] in SH-SY5Y cell proliferation after 24 h treatment. The cell viability percentage was calculated relative to the untreated group and expressed as the mean ± SEM. ^ap < 0.05 compared to untreated cells, ^bp < 0.01 compared to the Aβ group, ^cp < 0.01 compared to the (CuR: Pip) + Aβ group (one-way ANOVA followed by Tukey’s test).

Figure 10

Thioflavin T (ThT) fluorescence assay of SH-SY5Y cells treated with Aβ, Donepezil + Aβ, curcumin + Aβ (CuR + Aβ), piperine + AB (Pip + Aβ), and combined curcumin and piperine + AB [(CuR + Pip) + Aβ] on (A) inhibition and (B) disaggregation of preformed Aβ fibrils (n = 3, *p < 0.05, **p < 0.01 compared to the Aβ group, one-way ANOVA followed by Tukey’s test).

Figure 11

Biochemical assay of SH-SY5Y cells treated with Aβ, Donepezil + Aβ, curcumin + Aβ (CuR + Aβ), piperine + Aβ (Pip + Aβ), and combined curcumin and piperine + Aβ [(CuR + Pip) + Aβ]. (A) Lipid peroxidation assay, (B) glutathione assay, (C) catalase assay. Values were presented as mean ± SD, from three independent experiments. *p < 0.01, compared to untreated group. ^#p < 0.05, ^##p < 0.01, compared to the Aβ group (one-way ANOVA followed by Tukey’s test). MDA, malondialdehyde; GSH, glutathione; Cat, catalase.