Curcumin Inhibits In Vitro SARS-CoV-2 Infection In Vero E6 Cells through Multiple Antiviral Mechanisms

Abstract

Due to the scarcity of therapeutic approaches for COVID-19, we investigated the antiviral and anti-inflammatory properties of curcumin against SARS-CoV-2 using in vitro models. The cytotoxicity of curcumin was evaluated using MTT assay in Vero E6 cells. The antiviral activity of this compound against SARS-CoV-2 was evaluated using four treatment strategies (i. pre-post infection treatment, ii. co-treatment, iii. pre-infection, and iv. post-infection). The D614G strain and Delta variant of SARS-CoV-2 were used, and the viral titer was quantified by plaque assay. The anti-inflammatory effect was evaluated in peripheral blood mononuclear cells (PBMCs) using qPCR and ELISA. By pre-post infection treatment, Curcumin (10 µg/mL) exhibited antiviral effect of 99% and 99.8% against DG614 strain and Delta variant, respectively. Curcumin also inhibited D614G strain by pre-infection and post-infection treatment. In addition, curcumin showed a virucidal effect against D614G strain and Delta variant. Finally, the pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) released by PBMCs triggered by SARS-CoV-2 were decreased after treatment with curcumin. Our results suggest that curcumin affects the SARS-CoV-2 replicative cycle and exhibits virucidal effect with a variant/strain independent antiviral effect and immune-modulatory properties. This is the first study that showed a combined (antiviral/anti-inflammatory) effect of curcumin during SARS-CoV-2 infection. However, additional studies are required to define its use as a treatment for the COVID-19.

Keywords: COVID-19; D614G strain; Delta variant; SARS-CoV-2; antiviral; curcumin; immune response; inflammation.

Figure 1

Cytotoxicity of curcumin on Vero E6. Viability of Vero E6 after 48 h of curcumin treatment (from 1.25 to 40 µg/mL). Data were presented as Mean ± SEM. The viability percentages of the treated cell were calculated based on untreated control (n = 8).

Figure 2

Positive controls of viral inhibition showed low cytotoxicity on Vero E6. The figure represents the viability percentage of Vero E6 cells after 48 h of treatment with (A) CQ (6.3–100 µM) and (B) heparin (6.3–100 µg/mL). Bars represent mean values ± SEM. Two independent experiments with four replicates each experiment was performed (n = 8).

Figure 3

Antiviral effect of curcumin against SARS-CoV-2 by pre–post infection treatment. (A) Representative scheme of pre–post infection treatment. (B)The figure shows the reduction of D614G strain titer (PFU/mL) on Vero E6 supernatants after pre–post infection treatment with curcumin (n = 4). Chloroquine (CQ) was included as a positive control of viral inhibition. Data were presented as median ± IQR (interquartile range). Mann–Whitney test ** p  ≤  0.01. Inhibition percentages of 99%, 51.3%, 22.2%, and 27.8% were obtained at 10, 5, 2.5, and 1.25 µg/mL of curcumin, respectively. (C) Representative plaques on Vero E6 cells of pre–post infection treatment of curcumin against D614G strain.

Figure 4

Curcumin inhibited SARS-CoV-2 by pre-infection treatment strategy. (A) Representative scheme of pre-infection treatment. (B) The figure represents the reduction of D614G strain titer (PFU/mL) on Vero E6 supernatants after pre-treatment with curcumin (from 1.25 to 10 µg/mL). Heparin was used as a positive control of viral inhibition. Data were presented as median ± IQR (n = 4). Mann–Whitney test ** p  ≤  0.01. Inhibition percentages of 99.2%, 39.3%, −12.8%, and −0.4% were obtained at 10, 5, 2.5, and 1.25 µg/mL of curcumin, respectively. (C) Representative plaques on Vero E6 cells of pre-treatment of curcumin against D614G strain.

Figure 5

Curcumin inhibited the SARS-CoV-2 by post-infection treatment strategy. (A) Representative scheme of post-infection treatment. (B) The figure represents the reduction of D614G strain titer (PFU/mL) on Vero E6 supernatants after post-infection treatment with curcumin (from 1.25 to 10 µg/mL). Chloroquine (CQ) was included as a positive control of viral inhibition. Data were presented as median ± IQR (n = 4). Mann–Whitney test ** p  ≤  0.01. Inhibition percentages of 84.4%, 31.7%, 21.9%, and 14.8% were obtained at 10, 5, 2.5, and 1.25 µg/mL of curcumin, respectively. (C) Representative plaques on Vero E6 cells of post-infection treatment of curcumin against D614G strain.

Figure 6

Curcumin inhibited SARS-CoV-2 infectivity under the co-treatment condition. (A) Representative scheme of co-treatment strategy. (B) The figure represents the reduction of D614G strain titer (PFU/mL) on Vero E6 supernatants after co-treatment treatment with curcumin (from 1.25 to 10 µg/mL). Chloroquine (CQ) was used as a positive control of viral inhibition. Data were presented as median ± IQR (n = 4). Mann–Whitney test ** p  ≤  0.01. Inhibition percentages of 92%, 60.4%, 39.3%, and 2.3% were obtained at 10, 5, 2.5, and 1.25 µg/mL of curcumin, respectively. (C) Representative plaques on Vero E6 cells of the co-treatment strategy of curcumin against SARS-CoV-2 D614G strain.

Figure 7

Treatment with curcumin inhibited the infection by SARS-CoV-2 Delta variant. (A) The figure represents the reduction of Delta variant titer (PFU/mL) on Vero E6 supernatants after pre–post infection treatment with curcumin (from 1.25 to 10 µg/mL). Inhibition percentages of 99.8%, 98.4%, 98.9%, and 62.9% were obtained at 10, 5, 2.5, and 1.25 µg/mL of curcumin, respectively. (B) Representative plaques on Vero E6 cells of the pre–post infection strategy of curcumin against SARS-CoV-2 Delta variant. (C) The figure shows the reduction of Delta variant titer (PFU/mL) on Vero E6 supernatants after co-treatment with curcumin (from 1.25 to 10 µg/mL). Inhibition percentages of 99.9%, 99.1%, 31.9%, and 56.5% were obtained at 10, 5, 2.5, and 1.25 µg/mL of curcumin, respectively. (D) Representative plaques on Vero E6 cells of the co-treatment of curcumin against SARS-CoV-2 Delta variant. Chloroquine (CQ) was used as a positive control of viral inhibition. Data were presented as median ± IQR (n = 4). Mann–Whitney test ** p  ≤  0.01, * p  ≤  0.05. *** p ≤  0.001.

Figure 8

Anti-inflammatory effect of curcumin in PBMCs stimulated with SARS-CoV-2. Gene expression of Inflammatory cytokines was quantified in PBMCs by real-time PCR. The figure represents the fold change of (A) IL-1β, (B) IL-6, (C) IL-8, (D) MCP-1, and (E) TNF-α. Cells untreated were used as a negative control. Data were represented as median ± IQR (n = 6). Mann–Whitney test ** p  ≤  0.01, *** p  ≤  0.001.

Figure 9

Anti-inflammatory effect of curcumin in PBMCs stimulated with SARS-CoV-2. Inflammatory cytokines were quantified in PBMCs supernatants by ELISA. The figure represents the concentrations of (A) IL-1β, (B) IL-6, and (C) IL-8. Cells untreated were used as a negative control. Data were represented as median ± IQR (n = 6). Mann–Whitney test ** p  ≤  0.01, *** p ≤  0.001.