Hydroethanolic extract of Schinus terebinthifolia as a promising source of anti-influenza agents: Phytochemical profiling, cheminformatics, molecular docking and dynamics simulations

Abstract

Although Schinus terebinthifolia (commonly known as Brazilian peppertree) has been documented to possess various biological activities, such as anticancer, antibacterial, and antioxidant properties, its anti-influenza activity has not yet been documented. Here, an aqueous-ethanolic extract (30% v/v ethanol solution), prepared from its aerial parts (leaves and stalks), was established to determine whether it is a rich source of antiviral agents. The hydroethanolic plant extract, with a TPC value of 264.11 mg (GAE)/g DE, exhibits a promising IC50 value of 16.33 μg/mL, similar to that of authentic quercetin (IC50 = 12.72 μg/mL), and approximately 5.34 times higher than that of gallic acid (IC50 = 3.06 μg/mL) as determined by the DPPH assay. This extract contains 1.71 mg of gallic acid (representative marker) per gram of dried plant material, according to HPLC analysis. Using untargeted metabolomics analysis coupled with a series of cheminformatics tools (MetFrag, SIRIUS, CSI:FingerID, and CANOPUS), we ultimately proved that the S. terebinthifolia hydroethanolic extract contains simple phenolics (e.g., methyl gallate, ethyl gallate, and chlorogenic acid), flavonoids (afzelin and myricitrin), dicarboxylic acids, and germacrone. As anticipated, the plant extract exhibited anti-influenza activity with an IC50 of 2.21 μg/mL (CC50 > 50 μg/mL) and did not exert hemolytic activity at the concentration of 2000 μg/mL, underscoring its efficacy as a safe antiviral solution. In silico molecular docking and dynamic simulations suggest that neuraminidase and the cap-binding domain of influenza RNA polymerase (PB2) are preferentially targeted for inhibition by the detected metabolites. Owing to the diverse therapeutic effects of secondary metabolites, the anti-H5N1 activity of the newly developed plant extract is currently under investigation.

Fig 1. HPLC analysis of gallic acid as representative marker in S. terebinthifolia hydroethanolic extract.

(A) 30% hydroethanolic extract. (B) standard gallic (250 µg/mL) acid and gallic acid standard curve.

Fig 2. Structural annotation of putative gallic acid, methyl gallate and ethyl gallate detected from the S. terebinthifolia hydroethanolic extract.

Fig 3. Structural annotation of putative shikimic acid and 3-Dehydroshikimic acid detected from the S. terebinthifolia hydroethanolic extract.

Fig 4. Structural annotation of putative catechol, pyrogallol and quinic acid detected from the S. terebinthifolia hydroethanolic extract.

Fig 5. Structural annotation of putative chlorogenic acid (m/z 353.0910 [M-H]^-) detected from the S. terebinthifolia hydroethanolic extract.

Fig 6. Structural annotation of putative myricitrin and afzelin detected from the S. terebinthifolia hydroethanolic extract.

Fig 7. Structural annotation of putative hexadecanedioic acid, germacrone and myoinositol detected from the S. terebinthifolia hydroethanolic extract.

Fig 8. Antiviral activity of S. terebinthifolia hydroethanolic extract, where IC₅₀ defines the concentration of a substance that required to inhibit viral replication by 50%.

The lower IC₅₀ values signify a greater antiviral potency; (C) CC₅₀ is used in the framework of cytotoxicity assays, meaning the concentration of a plant extract and/or the authentic GA (1) that cause a 50% reduction in the viability of the MDCK cells. In this case, the IC₅₀ value of S. terebinthifolia aqueous ethanol extract is significantly less than its CC₅₀ value. This indicated its effective antiviral without inducing any harmful effects on the host cells.

Fig 9. A) The three-dimensional structure depicting the structural alignment of polyphenols found in the S. terebinthifolius hydroethanolic extract (gallic acid, methyl gallate, ethyl gallate, chlorogenic acid, afzelin, and myricitrin) with the reference ligand drug (oseltamivir carboxylic acid) the active site of NA structure.

B) The potential inhibitory effects of various phenolics towards the catalytic amino acid residues of NA structure.

Fig 10. A) Structural alignment of selected dicarboxylic acids (undecanedioic acid, hexadecanedioic acid and azelaic acid) exhibiting comparable binding energies with two reference ligands, oseltamivir carboxylic acid and Isorhamnetin.

B) Their 2D structures hypothesize potential inhibitory effects against the catalytic residues of the NA structure.

Fig 11. A) Structural orientation of terpenoid derived natural products in drug-recognition site of neuraminidase.

Oseltamivir carboxylic acid and germacrone. B) Their 2D structures theorize potential inhibitory effects against the catalytic residues of the NA structure.

Fig 12. A) Structural orientation of phenolic substances detected in the S. terebinthifolia hydroethanolic extract (gallic acid, methyl gallate, ethyl gallate, chlorogenic acid, afzelin, myricitrin) and three positive ligands (quercetin, mGTP, and favipiravir-RTP) in the cap-binding domain of PB2 subunit of influenza RNA polymerase.

B) 2D ligand-receptor interactions of chosen phenolic compounds with the PB2 subunit.

Fig 13. A) Structural orientation of non-phenolic substances detected in the S. terebinthifolia aqueous ethanol extract with positive ligands lining in the cap-binding domain of PB2 subunit of influenza RNA polymerase.

Germacrone, referent ligand – Gallic acid, hexadecanedioic acid, undecanedioic acid, azelaic acid, referent ligands – quercetin, favipiravir-RTP, and mGTP. B) 2D ligand-receptor interactions of selected metabolites with the PB2 subunit.

Fig 14. MD simulation analysis of PB2 subunit with selected metabolites from hydroethanolic S. terebinthifolia extract (quercetin (control), myricitrin, afzelin, hexadecanedioic acid).

A) RMSD. B) RMSF. C) Radius of Gyration. D) Solvent accessible surface area.

Fig 15. MD simulation analysis of NA protein with selected metabolites from hydroethanolic S. terebinthifolia extract (quercetin (control), afzelin, hexadecanedioic acid, undecanedioic acid).

A) RMSD. B) RMSF. C) Radius of Gyration. D) Solvent accessible surface area.