Significance of Astragaloside IV from the Roots of Astragalus mongholicus as an Acetylcholinesterase Inhibitor-From the Computational and Biomimetic Analyses to the In Vitro and In Vivo Studies of Safety

Abstract

The main aim of the study was to assess the acetylcholinesterase-inhibitory potential of triterpenoid saponins (astragalosides) found in the roots of Astragalus mongholicus. For this purpose, the TLC bioautography method was applied and then the IC₅₀ values were calculated for astragalosides II, III and IV (5.9 μM; 4.2 μM, and 4.0 μM, respectively). Moreover, molecular dynamics simulations were carried outto assess the affinity of the tested compounds for POPC and POPG-containing lipid bilayers, which in this case are the models of the blood-brain barrier (BBB). All determined free energy profiles confirmed that astragalosides exhibit great affinity for the lipid bilayer. A good correlation was obtained when comparing the logarithm of n-octanol/water partition coefficient (logPow) lipophilicity descriptor values with the smallest values of free energy of the determined 1D profiles. The affinity for the lipid bilayers changes in the same order as the corresponding logPow values, i.e.,: I > II > III~IV. All compounds exhibit a high and also relatively similar magnitude of binding energies, varying from ca. -55 to -51 kJ/mol. Apositive correlation between the experimentally-determined IC₅₀ values and the theoretically-predicted binding energies expressed by the correlation coefficient value equal 0.956 was observed.

Keywords: IC50; SH-SY5Y; acetylcholinesterase; free energy; lipophilicity; molecular docking; safety; zebrafish.

Figure 1

Total ion chromatogram of 50% methanolic extract from the roots of Astragalus mongholicus in the positive ionisation mode (A), extracted ion chromatogram of astragaloside III and IV (m/z of 785) (B), mass spectrum of astragaloside IV (C), mass spectrum of astragaloside III (D).

Figure 2

1D free energy profiles associated with the permeability of astragalosides I–IV through the lipid bilayer. Two types of homogeneous bilayers were considered: composed either of POPC (red lines, l-h-s panels) or POPG (green lines, r-h-s panels). The error values (vertical bars) were estimated by the bootstrapping method.

Figure 3

Results of the TLC-bioautography assay for the inhibition of acetylcholinesterase enzyme in daylight on the silica-gel-covered TLC normal phase. The TLC plate shows different concentrations of the tested astragalosides: II (A II), III (A III), and IV (A IV).

Figure 4

(A) Correlation between the logP_ow values (taken from [32]) and the smallest value of the free energy profiles associated with the permeability of the studied compounds through the lipid bilayer. The lipid bilayer was composed of either POPC molecules (red points) or POPG molecules (green points). The error bars correspond to the combined bootstrapping errors determined for the smallest value of the energy and the value for the molecule located outside the bilayer. (B) The same as in (A) but the correlation calculated for the logPS_Fubrain values. (C) Correlation between experimentally-determined IC₅₀ values (recalculated as log(IC₅₀)) and the theoretical ligand-AChE binding energies.

Figure 5

(A) Superposition of the most favourable poses of all ligands interacting with AChE. (B) The most favourable location of the astragaloside IV molecule bound to AChE. The ligand molecule is shown in ball-and-stick representation whereas all the closest amino-acid residues (of the distance not larger than 0.38 nm) are represented by thin sticks. The description of the interaction types is given in the text. The residue numbering is compatible with the PDB:3EVE record.

Figure 6

Effect of astragaloside IV (6, 12.5, and 25 μg/mL) on viability of SY-SY5Y human neuroblastoma cells; the histogram shows the mean cellular viability ± SD in comparison with the control saline-treated cells.

Figure 7

Representative photo of control and astragaloside IV-treated (25 µg/mL) larvae after the 95 h long incubation. The scale bar 1 mm.