Aqueous Extraction of Hibiscus Flower

Hibiscus flower (Hibiscus rosa-sinensis) were obtained from Premier Herbal Inc. (Toronto, ON, Canada). This aqueous extraction protocol is similar to that previously published with the following modifications [18 (link), 19 (link)]. The flowers were grinded using a coffee grinder into a fine powder. The powder was extracted in boiled double distilled water (ddH₂O) (1 g leaf powder to 10 mL ddH₂O) at 60 °C for 3 h. The extract was then run through a cheese cloth and then filtered via gravity filtration with a P8 coarse filter, followed by vacuum filtration with a 0.45 μm filter (PALL Life Sciences, VWR, Mississauga ON, CA Cat No. 28148–028). The water extract was frozen at − 80 °C, freeze dried using a lyophilizer and then reconstituted in ddH₂O in order to obtain a final stock concentration of 100 mg/mL. Prior to use, the water extract was passed through a 0.22 μm filter (Sarstedt, Montreal, QC, CA Cat No. 83.1826.001) in a biosafety cabinet.