Cytotoxicity Evaluation of Rhubarb Extracts

Cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well. After 16–24 h, cells were treated with the extracts from petioles and roots of R. rhaponticum and R. rhabarbarum and stilbenes (RHPG and RHPT), at concentrations of 1–100 µg/mL, for 24 h. After incubation, the cell culture medium was removed, and wells were rinsed twice with 0.02 M phosphate-buffered saline (PBS) containing Ca²⁺/Mg²⁺ (0.8 mM/0.4 mM), incubated in PBS containing Ca²⁺/Mg²⁺, 5.5 mM glucose, and 0.0125 mg/mL resazurin. HUVECs viability was estimated by measurements of the ability of live cells to reduce non-fluorescent resazurin to resorufin, a fluorescent product. After a 3 h incubation, resorufin fluorescence was measured (λ_ex = 530 nm, λ_em = 590 nm), using the Fluoroscan Ascent microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) [20 (link)]. The metabolic activity of control HUVECs (untreated with the examined extracts and stilbenes) was assumed as 100% of cell viability. Samples treated with 1% Triton-X100 were reference samples, with no viable cells (0% of viability). In cell samples treated with the examined extracts or stilbenes, a decrease in cell viability ≥ 20% (compared to control/untreated HUVECs) was assumed as a cytotoxic effect.