Pimenta dioica (L.) Merr. Bioactive Constituents Exert Anti-SARS-CoV-2 and Anti-Inflammatory Activities: Molecular Docking and Dynamics, In Vitro, and In Vivo Studies

Abstract

In response to the urgent need to control Coronavirus disease 19 (COVID-19), this study aims to explore potential anti-SARS-CoV-2 agents from natural sources. Moreover, cytokine immunological responses to the viral infection could lead to acute respiratory distress which is considered a critical and life-threatening complication associated with the infection. Therefore, the anti-viral and anti-inflammatory agents can be key to the management of patients with COVID-19. Four bioactive compounds, namely ferulic acid 1, rutin 2, gallic acid 3, and chlorogenic acid 4 were isolated from the leaves of Pimenta dioica (L.) Merr (ethyl acetate extract) and identified using spectroscopic evidence. Furthermore, molecular docking and dynamics simulations were performed for the isolated and identified compounds (1-4) against SARS-CoV-2 main protease (Mpro) as a proposed mechanism of action. Furthermore, all compounds were tested for their half-maximal cytotoxicity (CC₅₀) and SARS-CoV-2 inhibitory concentrations (IC₅₀). Additionally, lung toxicity was induced in rats by mercuric chloride and the effects of treatment with P. dioca aqueous extract, ferulic acid 1, rutin 2, gallic acid 3, and chlorogenic acid 4 were recorded through measuring TNF-α, IL-1β, IL-2, IL-10, G-CSF, and genetic expression of miRNA 21-3P and miRNA-155 levels to assess their anti-inflammatory effects essential for COVID-19 patients. Interestingly, rutin 2, gallic acid 3, and chlorogenic acid 4 showed remarkable anti-SARS-CoV-2 activities with IC₅₀ values of 31 µg/mL, 108 μg/mL, and 360 µg/mL, respectively. Moreover, the anti-inflammatory effects were found to be better in ferulic acid 1 and rutin 2 treatments. Our results could be promising for more advanced preclinical and clinical studies especially on rutin 2 either alone or in combination with other isolates for COVID-19 management.

Keywords: Pimenta; anti-SARS-CoV-2; docking and dynamics simulations; in vitro and in vivo studies; rutin.

Figure 1

Chemical structures of the isolated bioactive phenolic compounds (ferulic acid 1, rutin 2, gallic acid 3, and chlorogenic acid 4) from the leaves of Pimenta dioica (L.) Merr.

Figure 2

Top: R_g of the protein molecules in the six MD runs. Bottom: RMSD of the protein backbone atoms in the four MD runs. (Green: complex 1 (ferulic acid), blue: complex 2 (rutin), yellow: complex 3 (gallic acid), red: complex 4 (chlorogenic acid), black: complex 5 (co-crystallized N3), gray: complex 6 (docked N3)).

Figure 3

Per residue, RMSF for the protein residues in the six MD runs. (Green: complex 1 (ferulic acid), blue: complex 2 (rutin), yellow: complex 3 (gallic acid), red: complex 4 (chlorogenic acid), black: complex 5 (co-crystallized N3), gray: complex 6 (docked N3)). The binding site amino acids are highlighted with a grey background.

Figure 4

RMSD of each compound in the six MD runs. (Green: complex 1 (ferulic acid), blue: complex 2 (rutin), yellow: complex 3 (gallic acid), red: complex 4 (chlorogenic acid), black: complex 5 (co-crystallized N3), gray: complex 6 (docked N3)).

Figure 5

The number of H-bonds formed between each compound and the corresponding protein complex. (Green: complex 1 (ferulic acid), blue: complex 2 (rutin), yellow: complex 3 (gallic acid), red: complex 4 (chlorogenic acid), black: complex 5 (co-crystallized N3), gray: complex 6 (docked N3)).

Figure 6

CC₅₀ and IC₅₀. The cytotoxicity values for studied compounds were assessed on Vero E6 cells while their antiviral activities were evaluated against SARS-CoV-2 (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820).

Figure 7

Effect of P. dioica aqueous extract and its isolated bioactive compounds on lung miRNA 21-3p expression in rats. Data (n = 10 per group) are presented as fold of control considering the normal control one.

Figure 8

Effect of P. dioica aqueous extract and its isolated bioactive compounds on lung miRNA 155 expression in rats. Data (n = 10 per group) are presented as fold of control considering the normal control one.

Figure 9

Sections stained with hematoxylin and eosin (H&E; 200 X) histological examination of rats’ lungs of different groups compared to control group (a) Negative control group (I); (b) Group II: positive control: (was received HgCl₂ (1 mg/kg) for a 15-day period. (c) Group III: Was treated with HgCl₂ (1 mg/kg.) + ferulic acid (50 mg/kg) for a 15-day period. (d) Group IV: Was treated with HgCl₂ (1 mg/kg.) + rutin (75 mg/kg) for a 15-day period. (e) Group V: Was treated with HgCl₂ (1 mg/kg.) + gallic acid (50 mg/kg) for a 15-day period; (f) Group VI: Was treated with HgCl₂ (1 mg/kg.) + chlorogenic acid (50 mg/kg) for a 15-day period. (g): Group VII: Was treated with HgCl₂ (1 mg/kg.) + P. dioica extract (50 mg/kg) for a 15-day period.

Figure 10

Superimposed poses of the docked N3 inhibitor (represented in green color) over the native co-crystallized one (represented in red color) produced from the redocking process inside the Mpro binding pocket. Left (2D) and right (3D) graphical representations.