Anti-Influenza Virus (H5N1) Activity Screening on the Phloroglucinols from Rhizomes of Dryopteris crassirhizoma

Abstract

For screening the active phloroglucinols on influenza virus (H5N1) from Dryopteris crassirhizoma NaKai, a database was established including twenty-three phloroglucinols that had been isolated from Dryopteris crassirhizoma. Their inhibitory effect on the neuraminidase (NA) of influenza virus H5N1 was screened by molecular docking. As a result, three candidates were selected. The rhizomes of D. crassirhizoma were subjected to isolation and purification processes to obtain the inhibitor candidates. Thirteen phloroglucinols were obtained, including three selected candidates and two new phloroglucinols. The five phloroglucinols were investigated for their inhibitory activity on NA in vitro. The results showed that dryocrassin ABBA and filixic acid ABA exhibited inhibitory effects on NA with IC₅₀ as 18.59 ± 4.53 and 29.57 ± 2.48 μM, respectively, and the other three phloroglucinols showed moderate inhibitory activity. Moreover, the anti-influenza virus (H5N1) activity and cytotoxicity of dryocrassin ABBA and filixic acid ABA were tested on Madin-Darby canine kidney (MDCK) cells with the cell counting kit-8 (CCK8) method. The results confirmed that dryocrassin ABBA exhibited an inhibitory activity with low cytotoxicity (TC₅₀ > 400 μM) against influenza virus (H5N1) which will have to be investigated in further detail. In conclusion, phloroglucinols from D. crassirhizoma were shown to have anti-influenza virus activity, and especially dryocrassin ABBA, one of the phloroglucinols, may have the potential to control influenza virus (H5N1) infection.

Keywords: Dryopteris crassirhizoma; dryocrassin ABBA; influenza virus H5N1; neuraminidase; phloroglucinols.

Figure 1

Co-crystallized (green) and redocked (pink) oseltamivir. The reliability of molecular docking was validated using the known X-ray structure of neuraminidase (NA) complexed with oseltamivir. The result demonstrated that the low root mean-square deviation (RMSD) was 1.12 Å between the docked and the crystal conformation of oseltamivir, which indicated the high reliability of AutoDock Vina.

Figure 2

Binding modes of trimeric, tetrameric phloroglucinols (A) and monomeric, dimer phloroglucinols (B) in the active site and ‘150-cavity’ of NA.

Figure 3

Binding modes of dryocrassin ABBA (A); filixic acid ABA (B) and filixic acid ABB (C) with key residues of NA.

Figure 3

Binding modes of dryocrassin ABBA (A); filixic acid ABA (B) and filixic acid ABB (C) with key residues of NA.

Figure 4

Structure of compound 7 and its HMBC correlations.

Scheme 1

Electrospray Ionization Mass Spectroscopy (ESIMS) fragments of compound 7.

Figure 5

Structure of compound 13 and its HMBC correlations.

Scheme 2

ESIMS fragments of compound 13.

Figure 6

Structures of the 11 known phloroglucinols isolated from Dryopteris crassirhizoma (DC).

Figure 6

Structures of the 11 known phloroglucinols isolated from Dryopteris crassirhizoma (DC).

Figure 7

Inhibitory effect of dryocrassin ABBA on H5N1 virus in Madin-Darby canine kidney (MDCK) cells. Oseltamivir was used as a positive control. Culture medium in 96-well plates was removed and cells were washed with PBS. Then, 100 μL sample solution at different concentrations and 100 μL of virus at 100 TCID₅₀ were added. After incubation at 37 °C in 5% CO₂ for 72 h, the antiviral activity was investigated by cell counting kit-8 (CCK8) and cytopathic effect (CPE) reduction assay.

Figure 8

The toxicity of dryocrassin ABBA on MDCK cells. Culture medium in 96-well plates were removed and cells were washed with PBS. Then, 100 μL dryocrassin ABBA solution at different concentrations were added. After incubation at 37 °C in 5% CO₂ for 72 h, the relative cell viability was investigated by CCK8 assay.