Evaluation of cytotoxicity and antiviral activity of Rhazya stricta Decne leaves extract against influenza A/PR/8/34 (H1N1)

Abstract

Influenza viruses have developed resistance to the current classes of drugs, which means they could eventually become more virulent and cause more mortality and hospitalization. Our study aims to investigate the antiviral activity of Rhazya stricta Decne leaves extract in vitro and search for new promising drugs from R. stricta identified compounds in silico. The study was performed in vitro by utilizing Madin-Darby Canine Kidney cell line (MDCK) as a substrate for the influenza virus and estimating the inhibition performance of the plant leaves extract. Additionally, in silico screening was conducted to explore the antiviral activity of R. stricta phytochemicals. We investigated the cytotoxicity of R. stricta leaves extract and its antiviral activity against influenza virus (A/Puerto Rico/8/34 (H1N1)) using the MTT assay. The mode of action of the plant leaves extract during the viral life cycle was tested using time-of-addition assay. In silico analyses were performed, including molecular docking, drug-likeness analysis, and toxicity risk assessment, to state the leading compounds to be developed into an anti-influenza virus drug. The 50% cytotoxicity concentration of the leaves extract was CC50: 184.6 µg/mL, and the 50% inhibition concentration was CI50: 19.71 µg\mL. The time of addition assay revealed that R. stricta leaves extract exerted its activity in the late step of the influenza virus replication cycle. In comparison to Oseltamivir, the leading compounds showed better binding affinity and can be developed into oral drugs with low toxicity risk. Isolation and purification of the leading compounds and testing their antiviral activity in vitro and in vivo are required.

Keywords: Cytopathic effect; Molecular docking.; Plant extract; Time of addition assay.

Fig. 1

A and B) Natural photos of R. stricta plant have been captured from the C) collection site at Jeddah-Makkah Road “ 21.544846089411394, 39.642608955967795 ” (color should be used).

Fig. 2

Cell viability of MDCK cells after treated with R. Stricta extract at two-fold dilution concentrations. The experiment was performed as one trial with three replicates.

Fig. 3

Inhibition rate of R. stricta leaves extract against Influenza A virus (A/Puerto Rico/8/34 (H1N1)). The experiment was performed as one trial with four replicates.

Fig. 4

Inhibition rate of R. Stricta leaves extract during different time intervals against Influenza A virus (A/Puerto Rico/8/34 (H1N1)). The horizontal axis represents the times intervals as: Pre-inf 1(-2 – −1h), Pre-inf 2 (-1 – 0 h), Dur-inf (0 – 1 h), Post-inf 1 (2 – 4 h), and Post-inf 2 (4 – 8 h). The experiment was performed as one trial with four replicates.

Fig. 5

3D positions and 2D plotting of R. Stricta top 5 docked compounds into the NA protein active site were compared to the docked position of Oseltamivir. A) Rutin, B) Tetrahydrosecamine, C)16-Hydrorhazisidine, D) Luteolin-7-glucoside, E) kaempferol rhamnoside rutinoside, F) Oseltamivir. (Color should be used).

Fig. 5

3D positions and 2D plotting of R. Stricta top 5 docked compounds into the NA protein active site were compared to the docked position of Oseltamivir. A) Rutin, B) Tetrahydrosecamine, C)16-Hydrorhazisidine, D) Luteolin-7-glucoside, E) kaempferol rhamnoside rutinoside, F) Oseltamivir. (Color should be used).

Fig. 6

Some of the best docked compounds distributed on Log S scale. (Color should be used).