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. 2024 Jan 23;21(1):26.
doi: 10.1186/s12985-023-02282-x.

A novel film spray containing curcumin inhibits SARS-CoV-2 and influenza virus infection and enhances mucosal immunity

Wipawee Nittayananta  1 , Hatairat Lerdsamran  2 , Nopporn Chutiwitoonchai  3 , Aornrutai Promsong  4 , Teerapol Srichana  5   6 , Kesinee Netsomboon  7 , Jarunee Prasertsopon  2 , Jaruta Kerdto  8
Affiliations

A novel film spray containing curcumin inhibits SARS-CoV-2 and influenza virus infection and enhances mucosal immunity

Wipawee Nittayananta et al. Virol J. .

Abstract

Background: Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza virus is still a major worldwide health concern. Plants are a good source of bioactive compounds to be used as preventive measures for both inhibiting the virus binding and enhancing mucosal innate immunity. Curcumin has been shown to possess antiviral activity and modulate innate immunity. Therefore, the purpose of this study was to develop an oro-nasal film spray containing curcumin and determine its antiviral activity against SARS-CoV-2 and influenza virus infection, as well as its effects on mucosal innate immunity and inflammatory cytokines in vitro.

Methods: The antiviral activity of the film spray against SARS-CoV-2, influenza A/H1N1, A/H3N2, and influenza B was assessed in vitro by plaque reduction assay. Cytotoxicity of the film spray to oral keratinocytes and nasal epithelial cells was assessed by MTT assay, and cytotoxicity to Vero and MDCK cells was assessed by an MTS-based cytotoxicity assay. Oral and nasal innate immune markers in response to the film spray were determined by ELISA and by a commercial Milliplex Map Kit, respectively.

Results: Our data show that the film spray containing curcumin can inhibit both SARS-CoV-2 and influenza virus infections while maintaining cell viability. Results obtained among 4 viruses revealed that curcumin film spray demonstrated the highest inhibitory activity against SARS-CoV-2 with the lowest EC50 of 3.15 µg/ml and the highest SI value of 4.62, followed by influenza B (EC50 = 6.32 µg/ml, SI = 2.04), influenza A/H1N1 (EC50 = 7.24 µg/ml, SI = 1.78), and influenza A/H3N2 (EC50 > 12.5 µg/ml, SI < 1.03), respectively. Antimicrobial peptides LL-37 and HD-5, IL-6 and TNF-α produced by oral keratinocytes were significantly induced by the film spray, while hBD2 was significantly reduced.

Conclusion: Film spray containing curcumin possesses multiple actions against SARS-CoV-2 infection by inhibiting ACE-2 binding in target cells and enhancing mucosal innate immunity. The film spray can also inhibit influenza virus infection. Therefore, the curcumin film spray may be effective in preventing the viral infection of both SARS-CoV-2 and influenza.

Keywords: COVID-19; Curcumin; Influenza; Mucosal immunity; SARS-CoV-2.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Infectivity and cytotoxicity of the curcumin compound at various concentrations. (a) The SARS-CoV-2 spike pseudotyped virus was treated with the compound at 37 °C for 1 h and infected into HEK293-hACE2 cells expressing TMPRSS-2. The virus treated with DMSO served as the control. After 48 h of incubation, viral infectivity was determined by measuring the activity of the luciferase reporter using the Bright-Glo Luciferase Assay System. The cytotoxicity of the compound was determined by the viability of treated cells without infection using the CCK-8 assay. The relative mean values ± SD (%DMSO) from 3–4 independent experiments (each performed in duplicate) are shown. (b) Data from (a) were fitted into dose-response curves using GraphPad Prism software, and the estimated CC50, EC50, and selectivity index are shown
Fig. 2
Fig. 2
Infectivity and cytotoxicity of the curcumin film spray. (a) The SARS-CoV-2 spike pseudotyped virus was treated with the sample at 37 °C for 1 h and infected into HEK293-hACE2 cells expressing TMPRSS-2. The virus treated with the solvent (base) served as the control. After 48 h of incubation, viral infectivity was determined by measuring the activity of the luciferase reporter using the Bright-Glo Luciferase Assay System. The cytotoxicity of the sample was determined by the viability of treated cells without infection using the CCK-8 assay. The relative mean values ± SD (%DMSO) from 2 independent experiments (each performed in duplicate) are shown. (b) Data from (a) were fitted into dose-response curves using GraphPad Prism software, and the estimated CC50, EC50, and selectivity index are shown
Fig. 3
Fig. 3
Cytotoxicity of curcumin film spray to Vero and MDCK cells. Vero cells (a) or MDCK cells (b) were treated with varying concentrations of the curcumin film spray for 72 h. Subsequently, cytotoxicity was determined by measuring viability of the treated cells using the CellTiter 96AQueous One Solution Cell Proliferation Assay kit
Fig. 4
Fig. 4
Antiviral effect of curcumin film spray against SARS-CoV-2 examined by plaque reduction assay. Dose-response curve of antiviral activity of curcumin film spray against; (a) SARS-CoV-2, (b) influenza A/H1N1, (c) influenza A/H3N2, and (d) influenza B viruses, (e) Diagram for antiviral activity screening by plaque reduction assay, (f) Photo of plaque reduction assay experiment. Each concentration of the film spray was performed in triplicate wells
Fig. 5
Fig. 5
Cytotoxicity of the curcumin film spray on oral keratinocytes (a), and nasal epithelial cells (b) by MTT assay. The film spray is safe at concentrations of up to 1.56 µg/ml in oral keratinocytes and 12.5 µg/ml in nasal epithelial cells, respectively
Fig. 6
Fig. 6
Effects of the curcumin film spray formulation on oral and nasal mucosal innate immunity measured by the level of LL-37 (a, b), HD-5 (c, d), and hBD-2 (e, f) proteins produced by oral keratinocytes and nasal epithelial cells. The spray enhanced the production of LL-37 and HD-5 by oral keratinocytes greater than that of the controls, whereas such effects were not found in nasal epithelial cells. P-value was presented as *<0.05 versus untreated control
Fig. 7
Fig. 7
Amount of various cytokines produced by oral keratinocyte and nasal epithelial cells in response to oro-nasal film spray containing curcumin measured by a commercial Milliplex Map Kit; IL-1β (a, b), IL-6 (c, d), IL-10 (e, f), TNF-α (g, h), IFN-γ (i, j). The spray enhanced the production of anti-inflammatory cytokine IL-6 and pro-inflammatory cytokine TNF-α produced by oral keratinocytes greater than that of the controls, whereas such effects were not found in nasal epithelial cells. No significant changes in the production of pro-inflammatory cytokines were observed. P-value was presented as *<0.05 versus untreated control
Fig. 8
Fig. 8
Various actions of the curcumin film spray. Formulation of curcumin film spray shows various actions on prevention of COVID-19; First, it can inhibit SARS-CoV-2 infection by inhibiting ACE-2 binding. Second, the film spray upregulates the production of antimicrobial peptides LL-37 and HD-5 produced by oral epithelial cells, which have been previously reported to inhibit SARS-CoV-2 infection in silico. Third, the film spray induced the production of antiinflammatory cytokines IL-6 and TNF-α produced by oral epithelial cells. In addition, the film spray inhibits influenza virus infection.The infographic was designed by BioRender.com (https://app.biorender.com)
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