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

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.

Microbicide Containing Ellagic Acid Can Inhibit HIV-1 Infection.

OBJECTIVES: Ellagic acid (EA) has a wide range of biological effects. The purpose of this study was to investigate the in vitro effects of EA on HIV-1 replication, viral enzyme activity and cytokine secretion by infected cells. METHODS: The anti-HIV-1 activity of EA in solution was determined in vitro using the infection of TZM-bl cells by the nano luciferase-secreting R5-tropic JRCSF strain of HIV-1, which allows for the quantification of viral growth by measuring nano luciferase in the culture supernatants. The effect of EA on the cytokine secretion of TZM-bl cells was determined by a multiplexed bead array after 48 h of HIV-1 exposure. The antiviral effect of EA in the gel formulation (Ellagel), as would be used for vaginal application, was investigated by the inhibition of infection of UC87.CD4.CCR5 cells with R5-tropic pBaLEnv-recombinant HIV-1. RESULTS: EA in solutions of up to 100 µM was not toxic to TZM-bl cells. EA added either 1 h before or 4 h after HIV-1 exposure suppressed the replication of R5-tropic HIV-1 in TZM-bl cells in a dose-dependent manner, with up to 69% inhibition at 50 µM. EA-containing solutions also exhibited a dose-dependent inhibitory effect on HIV-1 replication in U87 cells. When EA was formulated as a gel, Ellagel containing 25 µM and 50 µM EA inhibited HIV-1 replication in U87 cells by 56% and 84%, respectively. In assays of specific HIV-1 enzyme activity, Ellagel inhibited HIV-1 integrase but not protease. EA did not significantly modulate cytokine secretion. CONCLUSIONS: We conclude that EA either in solution or in a gel form inhibits HIV infection without adverse effects on target cells. Thus, gel containing EA can be tested as a new microbicide against HIV infection.

Vaginal innate immune mediators are modulated by a water extract of Houttuynia cordata Thunb.

BACKGROUND: Vaginal epithelial cells (VECs) produce antimicrobial peptides including human ß-defensin 2 (hBD2) and secretory leukocyte protease inhibitor (SLPI), as well as cytokines and chemokines that play vital roles in mucosal innate immunity of the female reproductive tract. Houttuynia cordata Thunb (H. cordata), a herbal plant found in Asia, possesses various activities including antimicrobial activity and anti-inflammation. As inflammation and infection are commonly found in female reproductive tract, we aimed to investigate the effects of H. cordata water extract in modulating innate immune factors produced by VECs. METHODS: Primary human VECs were cultured and treated with H. cordata at a concentration ranging from 25-200 µg/ml for 6 or 18 h. After treatment, the cells and culture supernatants were harvested. The expression of hBD2 and SLPI mRNA was evaluated by quantitative real-time reverse transcription PCR. Levels of secreted hBD2 and SLPI as well as cytokines and chemokines in the supernatants were measured by ELISA and Luminex assay, respectively. Cytotoxicity of the extract on VECs was assessed by CellTiter-Blue Cell Viability Assay. RESULTS: H. cordata did not cause measurable toxicity on VECs after exposure for 18 h. The expression of hBD2 and SLPI mRNA as well as the secreted hBD2 protein were increased in response to H. cordata exposure for 18 h when compared to the untreated controls. However, treatment with the extract for 6 h had only slight effects on the mRNA expression of hBD2 and SLPI. The secretion of IL-2 and IL-6 proteins by VECs was also increased, while the secretion of CCL5 was decreased after treatment with the extract for 18 h. Treatment with H. cordata extract had some effects on the secretion of IL-4, IL-8, CCL2, and TNF-α, but not statistically significant. CONCLUSIONS: H. cordata water extract modulates the expression of antimicrobial peptides and cytokines produced by VECs, which play an important role in the mucosal innate immunity in the female reproductive tract. Our findings suggest that H. cordata may have immunomodulatory effects on the vaginal mucosa. Further studies should be performed in vivo to determine if it can enhance mucosal immune defenses against microbial pathogens.

Ellagic acid modulates the expression of oral innate immune mediators: potential role in mucosal protection.

BACKGROUND: Ellagic acid (EA) found in various fruits such as pomegranates, blackberries, raspberries, strawberries, and walnuts has different pharmacological functions including antioxidant, antitumor, antiallergic, anti-inflammatory, antibacterial, and antiviral activities. It is not known, however, if EA could enhance mucosal innate immunity. Our goal was to determine the effects of EA on the expression of innate immune mediators produced by oral epithelial cells. METHODS: Culture of primary human gingival epithelial cells (HGEs) was performed in duplicate, and after the primary HGEs had been treated with EA at a concentration ranging from 12.5 to 100 µM for 18 h the cells and supernatants were harvested. The expression of innate immune mediators including human ß-defensin 2 (hBD2), secretory leukocyte protease inhibitor (SLPI), and various cytokines and chemokines was measured at both transcriptional and translational levels by using quantitative real-time PCR, ELISA, and Luminex assay. RESULTS: In the presence of EA, the expression of hBD2-and SLPI mRNA was 3.7-folds and 2.6-folds greater than untreated controls, respectively, and consistent with their secreted protein levels. For cytokines and chemokines, increased expression of RANTES, IL-2, and IL-1ß was found in response to EA. In contrast, EA decreased the expression of IL-6, IL-8, and TNF-α. CONCLUSIONS: This study demonstrated that oral innate immunity is affected by EA found in fruits. Thus, it may play some roles in mucosal innate immunity. The potential of EA for modulating the innate immune mediators may lead to developing a new topical agent to treat and/or prevent immune-mediated oral diseases.