Kinase experiment:

AChE activity investigation is carried out in a reaction mixture containing 50 μL of tissue homogenate, 50 μL of 5, 5'-dithiobis-(2-nitrobenzoic) acid (DTNB), 1175 μL of 0.1 M phosphate-buffered solution, pH 8.0. After incubation for 20 min at 25℃, 25 μL of acetylthiocholine iodide solution is added as the substrate. The AChE activity is determined as changes in absorbance reading at 412 nm for 3 min at 25℃ and using a UV/Visible spectrophotometer.

Cell experiment:

Dilutions of Protocatechuic acid (2, 5, 10, 20, 50, and 100 μM) are prepared from stock solutions, with serum-free culture medium. Equal volumes of each solution are mixed with Aβ1-42 (10 μM), then incubated for 24 h on a thermoblock, with continuous agitation, and then exposed to PC12 cells for 24 h to test whether Protocatechuic acid can prevent cell death triggered by Aβ. Cell viability is determined by MTT reduction assay. Cells are treated with 200 μL per well of MTT solution (final concentration, 0.5 mg/mL in DMEM-Glutamax medium) for 3 h, at 37℃, with 5% CO2. The dark blue formazan crystals that formed are solubilized with 100 μL per well of DMSO, for 30 min. Absorbance is measured at 540 nm, with a microplate reader. Results are expressed as the percentage of MTT reduction in relation to the absorbance of control cells at 100%.

Protocatechuic acid is a phenolic compound which exhibits neuroprotective effect.

Protocatechuic acid inhibits the aggregation of Aβ and αS and destabilizes their preformed fibrils. Protocatechuic acid prevents the death of PC12 cells triggered by Aβ- and αS-induced toxicity[3].

Protocatechuic acid is able to prevent stress induced immobility time in forced swim test without altering locomotor activity in mice. Further, Protocatechuic acid treatment attenuates the elevation of serum corticosterone, lipid peroxidation and restores enzymatic antioxidants in cerebral cortex and hippocampus in ARS mice[1]. Rat administered cadmium and treated with prostigmine and doses of Protocatechuic acid (10–20 mg/kg) has significantly reduced BChE activity. Cadmium and either prostigmine or Protocatechuic acid (10–20 mg/kg) treated rats shows significant reduction in MDA level[2].

References:
[1]. Thakare VN, et al. Attenuation of acute restraint stress-induced depressive like behavior and hippocampal alterations with protocatechuic acid treatment in mice. Metab Brain Dis. 2016 Oct 26.
[2]. Adefegha SA, et al. Alterations of Na+/K+-ATPase, cholinergic and antioxidant enzymes activity by protocatechuic acid in cadmium-induced neurotoxicity and oxidative stress in Wistar rats. Biomed Pharmacother. 2016 Oct;83:559-568.
[3]. Hornedo-Ortega R, et al. Protocatechuic Acid: Inhibition of Fibril Formation, Destabilization of Preformed Fibrils of Amyloid-β and α-Synuclein, and Neuroprotection. J Agric Food Chem. 2016 Oct 10.