3.6. Enzyme Inhibition Assay of Isolated Flavonoids

The compounds 5281780, 5280443, and 188316 with higher binding affinity and stable interactions were selected for further in vitro enzyme inhibition assay. These compounds were reported from A. paniculata; hence, they were isolated [67] from the corresponding plant source and further subjected to inhibition studies.

The assay was performed according to the manufacturer protocol supplied in BACE-1 activity (Sigma, BACE-1FRET assay kit). The enzyme activity was observed with Varioskan Flash (version 4.00.53) using SkanIt Software (2.4.5 RE for Varioskan Flash) of NCL, Pune. The BACE-1 substrate solution (50 μM), a BACE-1 enzyme solution (2 μL), and inhibitor solutions were prepared as per the manufacturer’s protocol [6,136,137,138].

BACE-1 substrate solution was prepared with 1 mg/mL (500 μM) by adding 0.5 ml of DMSO to BACE-1 substrate. 7-Methoxycumarin-4-acetyl-Asn670, Lue 671-Amyloid β/A4 Precursor Protein 770 Fragment 667-(2,4 dinitrophenyl), and Lys-Arg-Arg amide trifluorophenyl salt was used as a substrate for BACE-1. A BACE-1 enzyme solution of ~0.3 unit/μL was prepared. The molecule quercetin was purchased from the local vendor of Sigma and was used as a known inhibitor: a control for BACE-1 inhibitors. Then, 20 μM of quercetin inhibitor solution was prepared with 1% DMSO solution. The isolated compounds were used for the isolated inhibitor solution preparation. The 5281780 solution (20 μM) with 1% DMSO was prepared after heating the solution at 55 °C for 5 min. The 188316 solution (20 μM) was prepared with 1% DMSO. The 5280443 solution was also prepared with concentration of 20 μM in 1% DMSO. These inhibitor solutions with variable concentrations (1 μM, 2 μM, 3 μM, 4 μM) were used for testing BACE-1 activities. The fluorescence assay buffer (78 μL, 79 μL, 80 μL, 81 μL), a BACE-1 enzyme solution (2 μL), the substrate solution (20 μL), and inhibitor solutions (0 μL, 1 μL, 2 μL, 3 μL, 4 μL) were added to a 96-well plate. Each concentration was considered in triplicate. The fluorescence readings were noted at 0 min as the “time zero” reading at an excitation of 490 nm and emission of 520 nm. The plate was incubated for 120 min at 37 °C, and the readings were taken. The readings were noted as S120. The percent inhibition was calculated using the formula, percent inhibition = [1 − (S₁₂₀ − S₀)/(C₁₂₀ − C₀)] × 100 [139].

The inhibitory activities of both AChE and BChE were measured using the spectrophotometric method developed by Ellman [140]. Acetylcholinesterase (Human), butyrylcholinesterase (Human) with acetylcholine iodide (ACh), and butyrylchlorine chloride (BCh) were used as their substrates, respectively. The enzyme activity was observed using Varioskan Flash (version 4.00.53), and the reading was observed using SkanIt Software (2.4.5 RE for Varioskan Flash). AChE and BChE solutions of ~0.15 unit/μL were prepared. The stock solution of AChE substrate (2.5 μM) and BChE substrate (2.5 μM) solution was prepared with DMSO, respectively. The compound eserine was purchased from Sigma vendor and was used as a known inhibitor as a control for both AChE and BChE assay. A 20-μM concentration of eserine inhibitor solution was prepared with 1% DMSO solution.

The isolated compound inhibitor solutions were prepared using the same method as discussed in the BACE-1 assay. The sodium phosphate buffer (pH 8) (78 μL, 79 μL, 80 μL, 81 μL), 20 μL of the 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB), AChE, and BChE enzyme solution (2 μL), substrate solution (20 μL), and inhibitor solutions (0 μL, 1 μL, 2 μL, 3 μL, 4 μL) were added to a 96-well plate. Each concentration was considered in triplicate. The plate was incubated for 15 min at 25 °C, and the readings were taken at 412 nm. The percentage inhibition was calculated using the formula percentage inhibition = (1 − C/I) × 100, where C and I were the enzyme activities without and with the test sample, respectively.