Antiviral and anti-inflammatory efficacy of nanoencapsulated brazilian green propolis against SARS-CoV-2

Abstract

The global COVID-19 pandemic, caused by SARS-CoV-2, continues to pose a significant threat to public health and the economy. SARS-CoV-2 is highly contagious, transmitted primarily through direct contact or inhalation of droplets, and can cause severe respiratory illnesses and other health complications, including post-acute COVID-19 syndrome. This study explored the antiviral potential of Brazilian green propolis, a natural product rich in flavonoids and phenolic compounds encapsulated in a microemulsion, to enhance its stability and antiviral effects. Brazilian green propolis extract was encapsulated in a microemulsion (ME-GP) and characterized using various physicochemical techniques. Furthermore, the antiviral and anti-inflammatory activities of ME-GP was evaluated in vitro and ex-vivo against SARS-CoV-2. For this, cells or tonsils were treated with ME-GP followed by infection with SARS-CoV-2. The microemulsion showed a size of approximately 217 nm, negative zeta potential, high encapsulation efficiency for artepillin C and baccharin (< 99%), and a spherical morphology. The ME-GP formulation was evaluated for antiviral activity against multiple SARS-CoV-2 variants (Wuhan, Gamma, Delta, and Omicron) in Caco-2 cells. The results demonstrated a significant reduction in viral load, particularly for the Wuhan and Delta variants, with up to a 99% reduction in viral load under prophylactic treatment conditions. Time-of-addition assays revealed that ME-GP acts at an early stage in the viral life cycle, likely by interfering with viral entry or immediate post-entry events. Additionally, ME-GP was evaluated in human tonsils, demonstrating an 80% reduction in viral load, suggesting its potential to reduce the transmission and progression of infection. Furthermore, ME-GP exhibited anti-inflammatory activity in human tonsils, significantly decreasing IL-1β, IL-6, TNF-α, and TNF-β levels. Thus, this study highlights the promising prophylactic and therapeutic potential of nanoencapsulated green propolis for combating SARS-CoV-2 and its variants, providing a natural adjunct in COVID-19 therapy.

Keywords: Green propolis; Microemulsion; SARS-CoV-2; Variants.

Fig. 1

Chemical structures of artepillin C and baccharin, the main compounds in Brazilian green propolis.

Fig. 2

Stability of ME-GP over time.

Fig. 3

TEM image of ME-GP. Scale bar: 500 nm.

Fig. 4

Virucidal activity of free and nanoencapsulated Brazilian green propolis against SARS-CoV-2, measured by viral titers using the TCID₅₀ after 2 h of treatment with the compounds. The positive control represents the experimental group with viral infection without treatment; GP represents the group of cells infected with SARS-CoV-2 and treated with green propolis extract, and ME-GP indicates the group of cells infected with SARS-CoV-2 and treated with the microemulsion.

Fig. 5

Virucidal activity of free and nanoencapsulated green propolis against SARS-CoV-2, measured by viral titers using the TCID50 after 2 h of treatment with the compounds. * p < 0.05, compared to positive controls.

Fig. 6

Time-of-addition assay of ME-GP during SARS-CoV-2 infection. Vero cells were infected at an MOI of 1 for 1 h, and ME-GP (15 µg/mL) was added at 0, 3, 6, 9, 12, or 24 h post-infection. Viral RNA was quantified 24 h post-infection and expressed relative to vehicle-treated infected controls. A significant reduction in viral RNA was observed only when ME-GP was added at time 0. Data are presented as the mean ± SD (n = 3). ****p < 0.0001.

Fig. 7

Antiviral activity of artepillin C against SARS-CoV-2 in Caco-2 cells. * p < 0.05, compared to vehicle.

Fig. 8

(A) Antiviral activity in human tonsils treated with Brazilian green propolis encapsulated in microemulsion (ME-GP, 125 µg/mL) for 5 days, (B) Levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in human tonsils treated with ME-GP. Data represent the mean ± SD of two independent experiments performed in triplicates. Where: C+ (positive control) is viral inoculation in the tonsil without any treatment, followed by incubation under the same conditions as the experimental group. *p < 0.05, compared to the positive control (C+).