Pleiotropic Potential of Evernia prunastri Extracts and Their Main Compounds Evernic Acid and Atranorin: In Vitro and In Silico Studies

Abstract

Evernia prunastri is a lichen widely distributed in the Northern Hemisphere. Its biological properties still need to be discovered. Therefore, our paper focuses on studies of E. prunastri extracts, including its main metabolites evernic acid (EA) or atranorin (ATR). Phytochemical profiles using chromatographic analysis were confirmed. The antioxidant activity was evaluated using in vitro chemical tests and in vitro enzymatic cells-free tests, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT). The anti-inflammatory potential using cyclooxygenase-2 (COX-2) and hyaluronidase were determined. The neuroprotective potential using acetylcholinesterase, (AChE), butyrylcholinesterase (BChE), and tyrosinase (Tyr) was estimated. The hypoglycemic activity was also confirmed (α-glucosidase). Principal component analysis was performed to determine the relationship between the biological activity of extracts. The inhibitory effect of EA and ATR on COX-2 AChE, BChE, Tyr, and α-glucosidase was evaluated using molecular docking techniques and confirmed for EA and ATR (besides α-glucosidase). The penetration of EA and ATR from extracts through the blood-brain barrier was confirmed using the parallel artificial membrane permeability assay blood-brain barrier test. In conclusion, depending on chemical surroundings and the concentration, the E. prunastri extracts, EA or ATR, showed attractive pleiotropic properties, which should be further investigated.

Keywords: enzyme inhibition; lichen; molecular docking; neurodegenerative diseases; oak moss.

Figure 1

The chemical structures of the main secondary metabolites of E. prunastri.

Figure 2

The chromatogram of extracts from Evernia prunastri shows the identified compounds, i.e., evernic acid, atranorin, and (+)-usnic acid, respectively. The compounds were characterized by Rt = 6.99 min, 8.44 min, and 8.28 min, respectively (Rt—retention time): dichloromethane (0.5 mg/mL) (a), acetone (0.5 mg/mL) (b), and methanol (1 mg/mL) (c).

Figure 2

The chromatogram of extracts from Evernia prunastri shows the identified compounds, i.e., evernic acid, atranorin, and (+)-usnic acid, respectively. The compounds were characterized by Rt = 6.99 min, 8.44 min, and 8.28 min, respectively (Rt—retention time): dichloromethane (0.5 mg/mL) (a), acetone (0.5 mg/mL) (b), and methanol (1 mg/mL) (c).

Figure 3

The polyphenolic content in Evernia prunastri extracts expressed as mg gallic acid equivalent/g of extract (GAE/g of extract). The mean values ± SD from n = 6 measurements are presented. Hex—hexane extract, DCM—dichloromethane extract, Ace—acetone extract, MeOH—methanol extract, MeOH-H₂O—methanol-water extract, H₂O—water extract.

Figure 4

Inhibition of hyaluronidase by the extracts of Evernia prunastri. The mean values expressed as a % of inhibition ± SD from n = 3 are presented; “na”—not active hexane extract at concentration 3.125 mg/mL; Hex—hexane extract, DCM—dichloromethane extract, Ace—acetone extract, MeOH—methanol extract, MeOH-H₂O—methanol-water extract 1:1 v/v, H₂O—water extract, EA—evernic acid.

Figure 5

Inhibition of acetylcholinesterase (AChE) and butylcholinesterase (BChE) by the extracts of Evernia prunastri. The mean values expressed as a % of inhibition ± SD from n = 4 measurements for extracts and n = 3 measurements for lichen metabolites are presented; “*”—samples measured at concentration 10 mg/mL; Hex—hexane extract, DCM—dichloromethane extract, Ace—acetone extract, MeOH—methanol extract, MeOH-H₂O—methanol-water, H₂O—water extract, EA—evernic acid, ATR—atranorin.

Figure 6

Inhibition of tyrosinase by the extracts of Evernia prunastri. The mean values expressed as a % of inhibition ± SD from n = 6 measurements for extracts and azelaic acid and n = 3 for evernic acid and atranorin are presented; “na”—not active hexane extract at concentration 0.8 mg/mL; Hex—hexane extract, DCM—dichloromethane extract, Ace—acetone extract, MeOH—methanol extract, MeOH-H₂O—methanol-water extract, H₂O—water extract, EA—evernic acid, ATR—atranorin, AA—azelaic acid.

Figure 7

The relationship of Evernia prunastri extracts on the factorial plane formed by the first two principal components, where individual varieties were denoted as extracts—hexane, dichloromethane, acetone, methanol, methanol-water, and water; AChE—acetylcholinesterase inhibition, Atranorin—atranorin content, BChE—butyrylcholinesterase inhibition, CAT—catalase inhibition, COX-2—cyclooxygenase-2 inhibition, CUPRAC—CUPRAC analysis, Cu²⁺—chelating activity of Cu²⁺, DPPH—DPPH analysis, Evernic acid—evernic acid content, Fe²⁺—chelating activity of Fe²⁺, Glu—α-glucosidase inhibition, GPX—glutathione peroxidase inhibition, GR—glutathione reductase inhibition, Hyal—hyaluronidase inhibition, SOD—superoxide dismutase inhibition, TPC—total phenolic content, Tyr—tyrosinase inhibition, Usnic acid—usnic acid content.

Figure 8

Principal component analysis (PCA) showing the factor loading plot, where individual varieties were denoted as extracts—hexane (1), dichloromethane (2), acetone (3), methanol (4), methanol-water (5), and water (6); AChE—acetylcholinesterase inhibition, Atranorin—atranorin content, BChE—butyrylcholinesterase inhibition, CAT—catalase inhibition, COX-2—cyclooxygenase-2 inhibition, CUPRAC—CUPRAC analysis, Cu²⁺—chelating activity of Cu²⁺, DPPH—DPPH analysis, Evernic acid—evernic acid content, Fe²⁺—chelating activity of Fe²⁺, Glu—α-glucosidase inhibition, GPX—glutathione peroxidase inhibition, GR—glutathione reductase inhibition, Hyal—hyaluronidase inhibition, SOD—superoxide dismutase inhibition, TPC—total phenolic content, Tyr—tyrosinase inhibition, Usnic acid—usnic acid content.

Figure 9

Proposed binding mode of evernic acid with COX-2 (PDB id: 5F1A). The key interactions of evernic acid with residues in the active sites of COX-2: hydrogen bonds (blue solid lines), hydrophobic interactions (grey dashed lines), π-stacking (green dashed line), and a salt bridge (yellow dashed line) (a); Proposed binding mode of atranorin with COX-2 (PDB id: 5F1A). The key interactions of atranorin with residues in the active sites of COX-2: hydrogen bonds (blue solid lines), hydrophobic interactions (grey dashed lines), π-stacking (green dashed line), and salt bridges (yellow dashed line) (b).

Figure 10

Proposed binding mode of evernic acid with AChE (PDB id: 4BDT). The key interactions of evernic acid with residues in the active sites of AChE: hydrophobic interactions (grey dashed lines), hydrogen bonds (blue solid lines), π-stacking (green dashed line), and salt bridges (yellow dashed line) (a); Proposed binding mode of atranorin with AChE (PDB id: 4BDT). The key interactions of atranorin with residues in the active sites of AChE: hydrophobic interactions (grey dashed lines), hydrogen bonds (blue solid lines), and π-stacking (green dashed line) (b).

Figure 11

Proposed binding mode of evernic acid with BChE (PDB id: 4BDS). The key interactions of evernic acid with residues in the active sites of BChE: hydrophobic interactions (grey dashed lines), hydrogen bonds (blue solid lines), π-stacking (green dashed line), and salt bridges (yellow dashed line) (a). Proposed binding mode of atranorin with BChE (PDB id: 4BDS). The key interactions of atranorin with residues in the active sites of BChE: hydrogen bonds (blue solid lines), π-stacking (green dashed line), and salt bridges (yellow dashed line) (b).

Figure 12

Proposed binding mode of evernic acid with tyrosinase (PDB id: 2Y9X). The key interactions of evernic acid with residues in the active sites of tyrosinase: hydrophobic interactions (grey dashed lines), hydrogen bonds (blue solid lines), and salt bridges (yellow dashed line) (a). Proposed binding mode of atranorin with tyrosinase (PDB id: 2Y9X). The key interactions of atranorin with residues in the active sites of tyrosinase: hydrophobic interactions (grey dashed lines), hydrogen bonds (blue solid lines) π-stacking (green dashed line), and salt bridges (yellow dashed line) (b).

Figure 13

Proposed binding mode of evernic acid with α-glucosidase (PDB id: 2QMJ). The key interactions of evernic acid with residues in the active sites of α-glucosidase: hydrophobic interactions (grey dashed lines), hydrogen bonds (blue solid lines), π-stacking (green dashed line), and salt bridges (yellow dashed line).