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

Lee, Yun Ha; Jang, Yo Han; Kim, Young-Seok; Kim, Jinku; Seong, Baik Lin

GTE powder was dissolved in distilled water to make 1% (10 mg/ml) solution and serially diluted to make 0.1%, 0.05%, 0.01%, and 0.001% solutions. The solutions were stored at various temperatures (4°C, 25°C, and 37°C), and aliquots were taken at predetermined time-points (6 hours (h), 10 h, 1 day (d), 2 d, 4 d, 7 d, 14 d, 28 d, and 56 d) and immediately kept frozen at −20°C until use. Equal volume of PR8 virus (10 6 PFU dissolved in PBS) was added to the GTE solutions and incubated for six h at 25°C before determining the viral titers. Residual viral titers were determined by plaque assay on MDCK cells at 37°C, as described previously [33, 34] . MDCK cells in 12-well culture plates were infected with 200 μl of 10-fold serially diluted virus samples and incubated for 1 h on a shaker at room temperature (RT). The inoculums were removed and the cells were overlaid with medium containing 1% low-melting agarose, DMEM, and 10 μg/ml trypsin. The 12-well plates were incubated at 37°C in 5% CO 2 incubator. After three days of the incubation, the virus plaques were counted. The GTE power was stored at 25°C and was taken at 1, 4, 8, and 16 weeks and kept at −20°C until use. The GTE powders were dissolved in distilled water to make 1% (10 mg/ml) solution and diluted to 0.1% concentration. Then, equal volume of PR8 virus solution (10 6 PFU/ml) was added, and the mixture was further incubated for six h at 25°C. Residual viral titers were determined by plaque assay on MDCK cells at 37°C. Antiviral effect and stability of GTE with supplements GTE solutions (0.01, 0.05, and 0.1%) were supplemented with 2% citric acid, 0.1% sodium benzoate, and 0.2% ascorbic acid to make GTE-mix. The GTE-mix solutions were stored at 25°C and 37°C for up to 56 days. The GTE-mix taken at various time-points were incubated with 10 6 PFU of PR8 virus at 25°C for six h for viral inactivation. Residual viral titers were measured by plaque assay on MDCK cells. To determine the chemical stability of green tea catechins, 0.1% GTE and GTE-mix were stored at 25°C for up to 56 days. Aliquots were taken at pre-determined time-points and were kept frozen at −20°C until use. The concentrations of catechins at various time-points were analyzed by gas chromatography (GC) with a Hewlett-Packard (HP) column equipped with a FID detector, using helium as a carrier gas. Identification and quantification of the GC peaks was accomplished by GC/MS analysis with an Agilent HP 5973 GC/Mass Spectrometer (Agilent Technologies, USA).