Benth . ) Benth . ( Leguminosae-Papilionoideae ) Inhibitory activity of acetylcholinesterase by Pterodon pubescens ( Benth . ) Benth . ( Leguminosae-Papilionoideae ) leaf extracts

Pterodon pubescens (Benth.) Benth is native species from the Brazilian Cerrado, popularly known as “sucupira-branca,” and rich in bioactive metabolites. This study aimed to assess the inhibitory activity of the enzyme acetylcholinesterase by leaf extracts of P. pubescens Benth. Phenolic constituents and flavonoids were analyzed by LC-DAD and LCMS. The qualitative and quantitative enzymatic inhibitory profile was performed by 96-well microplate in ELISA reader. The statistical analysis was reached with a significance limit of p < 0.05 applied by the Tukey’s test. Bioactive components detected were terpenoids, phenolic acids (gallic acid, ferulic acid, and rosmarinic acid), and flavonoids (kaempferol, luteolin, apigenin, rutin, and quercetin). There were no significant differences of inhibition between the crude methanolic and ethyl acetate extracts and between the hydroalcoholic and ethyl acetate phases at the concentration of 80 μg mL. The phytochemical profile in LCMS are related to the acetylcholinesterase inhibitory effect. This species has an importance for future in vivo studies, purification, and isolation of molecules with a possible biopesticidal effect.

Hexane phase was rotavaporated and transferred to amber flasks. This procedure was repeated three times. The flask containing the hexane phase was maintained open in a gas exhaust hood to allow evaporating the solvent. After evaporation, these flasks were capped and stored.
In the previously used hydroalcoholic phase, 50 mL of ethyl acetate solution was added. This procedure was repeated two more times in the separatory funnel. The ethyl acetate and hydroalcoholic phases were obtained, being subsequently rotavaporated and transferred to amber flasks. These flasks were maintained in a gas exhaust hood and, after solvent evaporation, they were capped and stored. For the test of acetylcholinesterase inhibition in TLC described by Martson (2002), plates were eluted in the mobile phase 8:2 (v/v) chloroform/methanol and subsequently submitted to the 96 well microplate inhibition test.

Acetylcholinesterase inhibition test in 96-well microplate
This test was carried out by using an adaptation of the Fosberg (1984) method, differing in concentration and number of samples. The enzyme acetylcholinesterase (E.C. 3.1.1.7 electric ell code C3389) was purchased from Sigma Aldrich in 2 KU. The enzyme AChE was diluted in 30 mL buffer A 50 mM Tris-HCl pH 7.8, resulting in a concentration of 66.6 U mL -1 , being added 1% albumin for stabilization, aliquoted in 2 mL Eppendorf, and stored in a freezer.
Buffer B was prepared by using 0.067 M sodium phosphate at pH 6.85. Acetonitrile was the solvent used for preparing 1 mM para-nitrophenyl acetate substrate, which is insoluble above 3 mM. Concentrations of crude ethyl acetate and methanolic extracts and their respective hydroalcoholic and ethyl acetate phases were first prepared in DMSO at 40 mg mL -1 (stock solution) for further dilution and have a solution of 8 mg mL -1 to prepare serial dilutions. To optimize the inhibition test, the used concentrations were added in buffer B. In the first dilution, 979 µL of the buffer and 21 µL of the inhibitor were added to 8 mg mL -1 in DMSO, followed by serial dilutions in the factor of 2, totaling six concentrations.
The final highest concentration in the well was 160 µg mL -1 in a decreasing manner.
In the first reaction step, 94 µL buffer B and 6 µL enzyme with 1% albumin were added for enzyme control. Then, 94 µL buffer B and 6 µL Tris-HCl buffer with 1% albumin were added to the blank of enzyme control to maintain enzyme conditions. After, 94 µL buffer B with inhibitor and 6 µL Tris-HCl buffer with 1% albumin were added to the blank of inhibitor. Research, Society and Development, v. 9, n. 9, e689997739, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7739 7 Finally, 94 µL buffer B with inhibitor and 6 µL enzyme with 1% albumin at 2 U mL -1 were added to the inhibitor test.
Subsequently, it was conditioned in a BOD at 25 °C for 10 minutes to interact the inhibitor with the enzyme. In the second step, the reaction volume was completed with 98 µL of buffer B and 2 µL of 1 mM PNPA substrate, which was added every 20 seconds. After the reaction was completed with a volume of 200 µL, readings started after 2 minutes and 30 seconds on the Elisa plate reader every 20 seconds in a period of 5 minutes and at wavelengths of 405 nm. Yellow staining is the formed product para-nitrophenol, derived from the hydrolysis of nitrophenyl acetate by acetylcholinesterase.

Statistical analysis
The results presented here correspond to the average of three replications (n = 3) ± standard deviation of the mean. The significance limit for all statistical analyses was (p <