Combinative effects of akarkara root-derived metabolites on anti-inflammatory and anti-alzheimer key enzymes: integrating bioassay-guided fractionation, GC-MS analysis, and in silico studies

Abstract

Background: Anacyclus pyrethrum L. (Akarkara root), a valuable Ayurvedic remedy, is reported to exhibit various pharmacological activities. Akarkara root was subjected to bioassay-guided fractionation, to isolate its active constituents and discover their potential bioactivities, followed by computational analysis.

Methods: The methanol extract and its fractions, methylene chloride, and butanol, were assessed for their antioxidant, anti-inflammatory, and anticholinergic potentials. The antioxidant activity was determined using DPPH, ABTS, FRAP, and ORAC assays. The in vitro anticholinergic effect was evaluated via acetyl- and butyryl-cholinesterase inhibition, while anti-inflammatory effect weas determined using COX-2 and 5-LOX inhibitory assays. The methylene chloride fraction was subjected to GC/MS analysis and chromatographic fractionation to isolate its major compounds. The inhibitory effect on iNOS and various inflammatory mediators in LPS-activated RAW 264.7 macrophages was investigated. In silico computational analyses (molecular docking, ADME, BBB permeability prediction, and molecular dynamics) were performed.

Results: Forty-one compounds were identified and quantified and the major compounds, namely, oleamide (A1), stigmasterol (A2), 2E,4E-deca-2,4-dienoic acid 2-phenylethyl amide (A3), and pellitorine (A4) were isolated from the methylene chloride fraction, the most active in all assays. All compounds showed significant in vitro antioxidant, anticholinergic and anti-inflammatory effects. They inhibited the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in activated RAW macrophages. The isolated compounds showed good fitting in the active sites of acetylcholinesterase and COX-2 with high docking scores. The ADME study revealed proper pharmacokinetics and drug likeness properties for the isolated compounds. The isolated compounds demonstrated high ability to cross the BBB and penetrate the CNS with values ranging from 1.596 to -1.651 in comparison with Donepezil (-1.464). Molecular dynamics simulation revealed stable conformations and binding patterns of the isolated compounds with the active sites of COX-2 and acetyl cholinesterase.

Conclusions: Ultimately, our results specify Akarkara compounds as promising candidates for the treatment of inflammatory and neurodegenerative diseases.

Keywords: ADME; Akarkara root; Anti-inflammatory; Anticholinergic; GC-MS; Molecular docking.

Fig. 1

Antioxidant activity of A. pyrethrum roots methanolic extract (ME), methylene chloride (MCF), and butanol (BF) fractions using (A) DPPH represented by a line graph, (B) ABTS, FRAP, and ORAC, (C) metal chelating assay, and (D) anticholinergic activity (AChE and BChE). µM: micromolar; TE: Trolox; Data are expressed as mean ± standard deviation of three replicates; equivalent. Different letters on the bar indicate significant differences at P < 0.0001 with Tukey’s test

Fig. 2

Anti-inflammatory activities of A. pyrethrum roots methanolic extract (ME), methylene chloride (MCF), and butanol (BF) fractions in vitro against COX-2 and 5-LOX, as well as in LPS-induced RAW264.7 macrophages (iNOS). Different letters on the bar indicate significant differences at P < 0.0001 with Tukey’s test. Standards: Celecoxib (COX 2), Zileuton (5-LOX), and parthenolide (iNOS) are serving as positive controls

Fig. 3

Antioxidant activity using (A) DPPH represented by a line graph, (B) ABTS, FRAP, and ORAC assays, (C) metal chelating assay, and (D) anticholinergic activity (AChE and BChE) of the isolated compounds (A1-A4). µM: micromolar; TE: Trolox equivalent. Data are expressed as mean ± standard deviation of three replicates; Different letters on the bar indicate significant differences at P < 0.05 with Tukey’s test

Fig. 4

(A) In vitro COX-2 and 5-LOX, as well as in LPS-induced RAW264.7 macrophages (iNOs) anti-inflammatory activities, and (B) the inhibitory effect of pro-inflammatory cytokines (TNF- α, IL-1β, and IL-6) secretion in LPS-stimulated RAW264.7 macrophage cells of the isolated compounds (A1-A4). Different letters on the bar indicate significant differences at P < 0.0001. * Significant from negative control at P < 0.0001. ^# Significant from positive control at P < 0.0001 with Tukey’s test

Fig. 5

3D interaction diagrams of (A) oleamide (A1), (B) stigmasterol (A2), (C) deca-2E,4E-dienoic acid 2-phenylethylamide (A3), and (D) pellitorine (A4) in AChE binding site

Fig. 6

3D interaction diagrams of (A) oleamide (A1), (B) stigmasterol (A2), (C) deca-2E,4E-dienoic acid 2-phenylethylamide (A3), and (D) pellitorine (A4) in COX-2 binding site

Fig. 7

Bioavailability radar chart of the isolated compounds; oleamide (A), stigmasterol (A2), deca-2E,4E-dienoic acid 2-phenylethylamide (A3), and pellitorine (A4), respectively. The pink zone signifies the range of the optimal property values for oral bioavailability and the red line is the compounds’ predicted properties. Saturation (INSATU), size (SIZE), polarity (POLAR), solubility (INSOLU), lipophilicity (LIPO), and flexibility (FLEX)

Fig. 8

(A) RMSD of Cα atoms of the protein backbone atoms. (B) RMSF of each residue of the protein backbone Cα atoms. (C) RoG of Cα atoms of protein residues of the backbone atoms relative (black) to the starting minimized over 60 ns for the acetylcholinesterase receptor (AChE) receptor enzymes protein with oleamide (red)

Fig. 9

(A) RMSD of Cα atoms of the protein backbone atoms. (B) RMSF of each residue of the protein backbone Cα atoms (C) RoG of Cα atoms of protein residues of the backbone atoms relative (black) to the starting minimized over 60 ns for the cyclooxygenase-2 receptor enzymes protein with stigmasterol (green)