Evaluation of green tea extract as a safe personal hygiene against viral infections

Abstract

Background: Viral infections often pose tremendous public health concerns as well as economic burdens. Despite the availability of vaccines or antiviral drugs, personal hygiene is considered as effective means as the first-hand measure against viral infections. The green tea catechins, in particular, epigallocatechin-3-gallate (EGCG), are known to exert potent antiviral activity. In this study, we evaluated the green tea extract as a safe personal hygiene against viral infections.

Results: Using the influenza virus A/Puerto Rico/8/34 (H1N1) as a model, we examined the duration of the viral inactivating activity of green tea extract (GTE) under prolonged storage at various temperature conditions. Even after the storage for 56 days at different temperatures, 0.1% GTE completely inactivated 10⁶ PFU of the virus (6 log₁₀ reduction), and 0.01% and 0.05% GTE resulted in 2 log₁₀ reduction of the viral titers. When supplemented with 2% citric acid, 0.1% sodium benzoate, and 0.2% ascorbic acid as anti-oxidant, the inactivating activity of GTE was temporarily compromised during earlier times of storage. However, the antiviral activity of the GTE was steadily recovered up to similar levels with those of the same concentrations of GTE without the supplements, effectively prolonging the duration of the virucidal function over extended period. Cryo-EM and DLS analyses showed a slight increase in the overall size of virus particles by GTE treatment. The results suggest that the virucidal activity of GTE is mediated by oxidative crosslinking of catechins to the viral proteins and the change of physical properties of viral membranes.

Conclusions: The durability of antiviral effects of GTE was examined as solution type and powder types over extended periods at various temperature conditions using human influenza A/H1N1 virus. GTE with supplements demonstrated potent viral inactivating activity, resulting in greater than 4 log₁₀ reduction of viral titers even after storage for up to two months at a wide range of temperatures. These data suggest that GTE-based antiviral agents could be formulated as a safe and environmentally friendly personal hygiene against viral infections.

Keywords: Antioxidant; Antiviral; Catechins; Green tea extract; Influenza virus.

Fig. 1

Chemical structures of green tea catechins. Chemical structures of epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG) are shown

Fig. 2

Maintenance of viral inactivation activity of GTE. a-c Maintenance of viral inactivation activity of GTE solution. 0.01, 0.05, and 0.1% GTE solutions were stored at 4 °C (a), 25 °C (b), and 37 °C (c), and 0.001% GTE was incubated at 25 °C. The GTE solutions incubated for different time points were mixed with 10⁶ PFU of PR8 virus and further incubated for six h at 25 °C for viral inactivation. Residual viral titers were determined by plaque assay on MDCK cells. d Maintenance of viral inactivation activity of GTE powder. GTE powder was stored for 1 − 16 weeks at 25 °C, and then dissolved in distilled water to make 0.1% GTE solutions. The GTE solutions were incubated with 10⁶ PFU of PR8 virus for six h at 25 °C for viral inactivation. Residual viral titers were determined by plaque assay on MDCK cells. As a control, the virus was incubated with PBS under the same conditions, and the viral titer was indicated by upper dashed lines. Data are the mean of two independent experiments. Detection limit of plaque assay is 5, and error bars indicate the standard deviation (SD)

Fig. 3

Maintenance of viral inactivation activity of GTE solution with supplements. 0.01, 0.05, and 0.1% GTE solutions were incubated at 25 °C (a) or 37 °C (b) in the presence of 2% citric acid, 0.1% sodium benzoate, and 0.2% ascorbic acid (GTE-mix). The GTE-mix solutions incubated for different times were mixed with 10⁶ PF of PR8 virus for six h at 25 °C for viral inactivation. Residual viral titers were determined by plaque assay on MDCK cells. As a control, the virus was incubated with PBS, and the viral titer was indicated by upper dashed lines. c As another controls, the virus was treated with the individuals of the supplement or with the mixture of three agents. Data are the mean of two independent experiments. Detection limit of plaque assay is 5, and error bars indicate the SD

Fig. 4

Effects of supplements on the kinetics of viral inactivation by GTE. a Chemical stability of green tea catechins. 0.1% GTE and GTE-mix solutions were stored at 25 °C for up to 56 days. The samples were obtained at various time-points and catechins concentration was measure by GC. b Kinetics of viral inactivation by GTE and GTE-mix. 0.1% GTE and GTE-mix solutions were incubated at for 24 h at 25 °C. After the incubation, the GTE and GTE-mix solutions were mixed with 10⁶ PFU of PR8 virus and further incubated at 25 °C for 0, 1, 5, 10, 30, 120, and 360 mins for viral inactivation. Residual viral titers at each time-point were determined by plaque assay on MDCK cells. Data are the mean of two independent experiments. Detection limit of plaque assay is 5, and error bars indicate the SD

Fig. 5

Morphology of influenza virus treated with GTE. a Cryo-EM images of PR8 virus treated with PBS or GTE. 5 × 10⁷ PFU of PR8 virus was incubated with PBS or 0.1% GTE 0.1% for 24 h at 25 °C before observation under cryo-EM. Two images of the virus treated either PBS or GTE 0.1% are shown. b Analysis of the size distribution of PR8 virus treated with GTE or PBS by dynamic light scattering (DLS). 5 × 10⁷ PFU of PR8 virus was incubated with PBS or GTE (0.01, 0.05, and 0.1%) for 24 h at 25 °C. After the incubation, the size distribution of the virus was analyzed by DLS. The results of intensity distribution are shown. The red box indicates the particle size at the highest intensity