Protocatechuic acid (3, 4-Dihydroxybenzoic acid) (3, 4-dihydroxybenzoic acid) is a natural phenolic compound found in many edible and medicinal plants. Recent studies indicate that it could be used as a protective agent against cardiovascular diseases and neoplasms. The mechanism of its action is mostly associated with antioxidant activity, including inhibition of generation as well as scavenging of free radicals and up-regulating enzymes which participate in their neutralization.

Protocatechuic acid 抑制Aβ和αS的聚集，破坏其已形成的纤维结构，预防由Aβ和αS诱导的毒性引起的PC12细胞死亡[3]。

Protocatechuic acid 能在不改变小鼠运动活动的情况下，防止应激诱导的强迫游泳试验中的静止时间。此外，Protocatechuic acid 还能减轻 ARS 小鼠血清皮质酮的升高和脂质过氧化反应，并恢复大脑皮层和海马中的酶抗氧化剂[1]。给大鼠注射镉并用前列腺素和Protocatechuic acid （10-20 mg/kg）治疗后，其 BChE 活性明显降低。镉和前列腺素或Protocatechuic acid （10-20 mg/kg）处理大鼠后，MDA 水平明显下降[2]。

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°C, 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°C and using a UV/Visible spectrophotometer.

Protocatechuic acid is dissolved in DMSO. 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°C, 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%.