In vitro evaluation of the neuroprotective potential of Olea dioica against Aβ peptide-induced toxicity in human neuroblastoma SH-SY5Y cells

Abstract

Alzheimer's disease (AD) is a type of neurodegenerative disease, associated with the hastening of ROS, acetylcholinesterase (AChE) activity, and amyloid β peptides plaques in the brain. The limitations and side effects of existing synthetic drugs incline toward natural sources. In the present communication active principles of methanolic extract of Olea dioica Roxb, leaves are explored as an antioxidant, AChE inhibitor, and anti-amyloidogenic. Furthermore, neuroprotection against the amyloid beta-peptide has been studied. The bioactive principles were identified by GC-MS and LC-MS and further subjected to antioxidant (DPPH and FRAP) and neuroprotection (AChE inhibition, ThT binding, and MTT assay, DCFH-DA and lipid peroxidation (LPO) assay using neuroblastoma (SHSY-5Y) cell lines) assays. Methanolic extract of O. dioica Roxb, leaves was found to contain polyphenols and flavonoids. In vitro assays exhibited potential antioxidant and anti-AChE (˃50%) activities. ThT binding assay indicated protection against amyloid-beta aggregation. MTT assay, Aβ1-40 (10 µM) with extract increase the cell viability (˃50%) and showed significant cytotoxicity to SHSY-5Y cells. ROS level (˃25%) significantly decreased in the Aβ1-40 (10 µM) + extract (15 and 20 μM/mL) and LPO assay (˃50%) suggesting prevention of cell damage. Results advocate that O. dioica leaves are a good source of antioxidants, anti-AChE, and anti-amyloidogenic compounds which may be further evaluated as a natural medicine for the treatment of AD.

Keywords: GC-LCMS; SHSY-5Y; acetylcholinesterase; alzeheimer’s disease; amyloid; beta; olea dioica roxb.

FIGURE 1

GC-MS (A) and LC-MS (B) analyses of methanol extract of O. dioica leaves.

FIGURE 2

FTIR analysis (A) and UV- Absorbance (B) of methanol extract of O. dioica leaves.

FIGURE 3

DPPH radical scavenging activity (A) and FRAP assay (B).

FIGURE 4

Bio-autographic assay of AChE inhibition activity in the TLC plates. (A) TLC plates at UV-light. (B) and (C) White spots over a yellow background develops as an indication of acetylcholinesterase inhibition zones at different time interval.

FIGURE 5

AChE inhibition by plant extracts positive control (A) and Thioflavin-T assay of plant extract and positive control (B).

FIGURE 6

Neuroprotective effect of plant extract against Aβ_1-40 at different concentrations of inducing cell toxicity in SHSY-5Y cells. Gal (Galantamine) was used as a positive control.

FIGURE 7

Cell viability in SHSY-5Y cells which were exposed to different concentrations of plant extract for 24 h. Cell viability was assessed by MTT assay.

FIGURE 8

ROS measurement by fluorescence microscope (A) and ROS levels in control (Aβ_1-40) and treated groups of extract + amyloid beta (B). The treated groups decrease the level of ROS in extract treated groups compared to control. The results were expressed in fluorescence intensity. Significance was checked by one-way ANOVA. The error bars represent the Mean ± SD. The p-value *p < 0.01 and **p < 0.001, were considered as significant while comparing Aβ_1-40 treated group and Aβ_1-40+ extract.

FIGURE 9

LPO in SHSY-5Y cells were treated with Aβ_1-40 at different concentrations (5 and 10 µM) (A). The effect of extract on treated groups showed a decreasing LPO activity in Aβ_1-40+ extract treated group. Quantitative evaluation of the extent of inhibition of acetylcholinesterase activity (AChE) by plant extract (5, 15, and 20 μM/mL) and the standard AChE inhibitor galantamine (5 μM/mL) (B). The data shown are with Mean ± SD. The p-value **p < 0.001, and ***p < 0.001 were considered as significant while comparing Aβ_1-40 treated group and Aβ_1-40+ extract.