Antiviral activity of Sambucus FormosanaNakai ethanol extract and related phenolic acid constituents against human coronavirus NL63

Abstract

Human coronavirus NL63 (HCoV-NL63), one of the main circulating HCoVs worldwide, causes respiratory tract illnesses like runny nose, cough, bronchiolitis and pneumonia. Recently, a severe respiratory illness outbreak of HCoV-NL63 has been reported in a long-term care facility. Sambucus FormosanaNakai, a species of elderberry, is a traditional medicinal herb with anti-inflammatory and antiviral potential. The study investigated the antiviral activity of Sambucus FormosanaNakai stem ethanol extract and some phenolic acid constituents against HCoV-NL63. The extract was less cytotoxic and concentration-dependently increased anti-HCoV-NL63 activities, including cytopathicity, sub-G1 fraction, virus yield (IC50 = 1.17 μg/ml), plaque formation (IC50 = 4.67 μg/ml) and virus attachment (IC50 = 15.75 μg/ml). Among the phenolic acid constituents in Sambucus FormosanaNakai extract, caffeic acid, chlorogenic acid and gallic acid sustained the anti-HCoV-NL63 activity that was ranked in the following order of virus yield reduction: caffeic acid (IC₅₀ = 3.54 μM) > chlorogenic acid (IC₅₀ = 43.45 μM) > coumaric acid (IC₅₀ = 71.48 μM). Caffeic acid significantly inhibited the replication of HCoV-NL63 in a cell-type independent manner, and specifically blocked virus attachment (IC₅₀ = 8.1 μM). Therefore, the results revealed that Sambucus Formosana Nakai stem ethanol extract displayed the strong anti-HCoV-NL63 potential; caffeic acid could be the vital component with anti-HCoV-NL63 activity. The finding could be helpful for developing antivirals against HCoV-NL63.

Keywords: Antiviral; Attachment inhibition; Caffeic acid; Human coronavirus NL63; Sambucus FormosanaNakai; Virus yield.

Fig. 1

Inhibitory effects of Sambucus Formosana Nakai extract on viral cytopathicity and Sub-G1 fraction in HCoV-NL63 infected cells. HCoV-NL63 at MOI of 0.1 was mixed with the extract, and immediately added to LLC-MK-2 cell culture. Virus-induced cytopathic effect was photographed 36 h post-infection by microscopy (A). Infected cells in the presence or absence of the extract were harvested 36 hpi, stained using propidium iodide, and then examined using flow cytometry. The flow cytometry histograms (top) and the percentage of sub-G1 phase (bottom) in HCoV-NL63 infected cells was displayed (B). **, p value < 0.01 compared with mock-treated infected cells.

Fig. 2

Reduction of HCoV-NL63 yield in MK-2 cells by Sambucus Formosana Nakai extract. Supernatant of HCoV-NL63-infected cells in the presence or absence of the extract was harvested 36 hpi and HCoV-NL63 yield in the supernatant was determined by plaque assay (A). The rate of virus yield reduction was calculated based on the ratio of loss particle number to mock-treated group (B). **, p value < 0.01. compared with mock-treated infected cells.

Fig. 3

Effects of Sambucus Formosana Nakai extract on plaque formation, virucidal activity and virus attachment. MK-2 cell monolayer was infected with HCoV-NL63, simultaneously treated with the extract for 1 h, and then covered with the agarose overlay medium. After 3-day incubation at 37 °C in a CO₂ incubator, plaques were determined after crystal violet staining. The inhibitory activity of the extract on the plaque formation was according to on the ratio of loss plaque number to mock-treated group (A). In the virucidal assay, the extract was mixed with HCoV-NL63 (10⁶ pfu), then incubated at 37 °C for 1 h. The extract/virus mixture was diluted by 1000-fold dilution and examined for the residual infectivity by plaque assay (B). In the attachment assay, HCoV-NL63 was mixed with the extract, then immediately added onto MK2 cell monolayer for 1 h at 4 °C. After washing, the cell monolayer was overlaid with 0.75% agarose medium for 3 days at 37 °C in CO₂ incubator. Attachment inhibition was determined based on the residual plaques (C). *, p value < 0.05; **, p value < 0.01 compared with mock-treated cells.

Fig. 4

Inhibitory effects of the phenolic acid constituents on viral cytopathicity and virus yield in HCoV-NL63 infected cells. HCoV-NL63 at MOI of 0.1 was added to LLC-MK-2 cell culture and then immediately treated with the phenolic acid constituents. Virus-induced cytopathic effect was photographed 36 h post-infection by microscopy (A). Supernatant of HCoV-NL63-infected/treated cells was harvested 36 hpi; virus yield in supernatant was determined plaque assay. The rate of virus yield reduction was calculated based on the ratio of loss particle number to mock-treated group (B). CA, caffeic acid; CH, chlorogenic acid; CO, coumaric acid; FE, ferulic acid; GA, gallic acid. **, p value < 0.01, ***, p value < 0.001 compared with mock-treated infected cells.

Fig. 5

Effects of caffeic acid and chlorogenic acid on plaque formation, virucidal activity and virus attachment. MK-2 cell monolayer was infected with HCoV-NL63, simultaneously treated with the caffeic acid or chlorogenic acid for 1 h, and then covered with the agarose overlay medium for 3-day at 37 °C in a CO₂ incubator. After crystal violet staining, the inhibitory activity of caffeic acid and chlorogenic acid on the plaque formation was according to on the ratio of loss plaque number to mock-treated group (A). In the virucidal assay, the caffeic acid or chlorogenic acid was mixed with HCoV-NL63 (10⁶ pfu), then incubated at 37 °C for 1 h. The 1000-fold dilution of the compound/virus mixture was examined for the residual infectivity by plaque assay (B). In the attachment assay, MK2 cell monolayer was infected with HCoV-NL63 (100 pfu), immediately treated with the caffeic acid or chlorogenic acid for 1 h at 4 °C, washed, and overlaid with 0.75% agarose medium for 3 days at 37 °C in CO₂ incubator. Attachment inhibition was determined based on the residual plaques (C). CA. caffeic acid; CH, chlorogenic acid. *, p value < 0.05;**, p value < 0.01; ***, p value < 0.001 compared with un-treated infected cells.

Fig. 6

Inhibition of HCoV-NL63 infectivity in human airway epithelial cells by caffeic acid. Calu-3 cells were infected with HCoV-NL63 at a MOI of 0.05 and immediately treated with caffeic acid for 36 h at 32 °C. Images of virus-induced CPE effect were photographed by a light microscope (A, top). In addition, mock, infected, and infected/treated cells were performed using immunofluorescence staining anti-HCoV-NL63 immunized sera and secondary antibody Alexa Fluor anti-mouse IgG (A, middle); total cells were stained with DAPI (A, bottom). Infectivity was determined according to the ratio of HCoV-NL63-positive cells to total cells (B). **, p value < 0.01; ***, p value < 0.001 compared with un-treated infected cells.