Honeysuckle extracts as a potential inhibitor of SARS-CoV-2 infection

Abstract

Background: In the current era of coronavirus disease 2019 (COVID-19), we were interested in searching for medications other than the currently available antiviral drugs Paxlovid and Molnupiravir that cause minimal side effects and do not harm the human body. Honeysuckle extract (HSE) is a traditional Chinese medicine (TCM) that has been shown to exert antiviral effects in other studies. However, no studies have indicated whether HSE has an inhibitory effect on SARS-CoV-2.

Methods: We prepared HSEs from dried honeysuckle flowers. We performed a cell viability assay, median tissue culture infection dose (TCID₅₀) assay, and qRT‒PCR, and calculated the virus titers using the Reed-Muench method to evaluate the inhibitory effects of aqueous and alcohol HSEs on SARS‒CoV‒2 and explore the possible underlying mechanisms.

Results: In this study, post-treatment with HSE resulted in dose-dependent decreases in both the RNA levels and TCID₅₀ of SARS-CoV-2 in Vero E6 cells; treatment with 50 μg/ml and 100 μg/ml alcohol HSEs achieved up to 95.323% and 92.587% inhibition, respectively. Moreover, pre-treatment with aqueous HSEs effectively reduced the RNA levels, and TCID₅₀ of SARS-CoV-2 by up to 99.684%, and alcohol HSEs achieved up to 99.921% inhibition; both of these effects occurred in a dose-dependent manner.

Conclusion: The results suggest that HSEs may have the potential to prevent SARS-CoV-2 infection.

Keywords: COVID-19; Lonicera japonica Thunb.; SARS-CoV-2; extracts; honeysuckle.

FIGURE 1

Steps to prepare and determine the highest non-lethal dose of HSEs in Vero E6 cells, as well as to assess the impact of different extraction methods on SARS-CoV-2 replication inhibition and reduction of infection potency, as outlined in a flowchart. The results indicate that pre-treatment yields better outcomes for both SARS-CoV-2 replication inhibition and reduction of infection potency.

FIGURE 2

The highest established nonlethal dose of HSE in Vero E6 cells, for which SARS-CoV-2 was isolated. Vero E6 cells were exposed to various concentrations of HSE for 24 h and 48 h, aiming to determine the nonlethal dose. The cytotoxicity of these substances on Vero E6 cells was assessed using CCK-8 assays. The Y-axis of the graphs shows the mean % cell viability normalized to that in the control group (0 μg/ml; 100%). Each experiment was conducted in quadruplicate. Treatment with aqueous HSE for 24 h (A) and 48 h (B), treatment with honeysuckle alcohol extracts for 24 h (C) and 48 h (D).

FIGURE 3

Relative SARS-CoV-2 RNA levels were decreased in a dose-dependent manner after a 24-h pre-treatment with aqueous HSE. Vero E6 cells were pretreated with HSE at concentrations of 0, 50, or 100 μg/ml for 24 h. Subsequently, the cells were washed with 1x PBS and infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 for an additional 24 h. The viral yield in the cell supernatant was then quantified using qRT‒PCR and normalized to that in the control group (0 μg/ml; 100%). The Y-axis of the graphs represents the mean % relative viral RNA levels, which were normalized to that in the control group (0 μg/ml; 100%).