Toxicity of Tart Cherry Extract of C2C12 and U937 Cell Lines

Abstract

Oxidative stress and inflammation are implicated in secondary damage from eccentrically-biased muscle contractions. Research suggests that the antioxidant/anti-inflammatory compounds in tart cherry juice may accelerate strength recovery following damage; however, the underlying mechanisms remain unclear. PURPOSE: To investigate the effects of tart cherry extract (TCE) on skeletal muscle (C2C12) and immune cells (U937) and to determine maximum effective dose for subsequent muscle damage experiments.\ METHODS: TCE was purified using a water-based extraction method and evaluated for total phenolic content using the Folin-Ciocalteau’s assay. C2C12 myotube cultures were differentiated for 5d, and U937 monocytes were grown to 1x106 cells/mL and then preconditioned with TCE for 24-48h. Cultures were then incubated with 50% ethanol (EtOH) to induce cell death. Reactive oxygen species (ROS) scavenging activity of TCE was assessed using a DPPH assay. Toxicity of TCE and EtOH was assessed using an XTT assay. RESULTS: ROS inhibition was observed at all [TCE] (7-100μg/mL) in both C2C12 and U937 cell culture media (p<0.05), and ROS inhibition was highest in C2C12 media vs. U937 media (p<0.05). Treatment with EtOH decreased cell viability in both C2C12 (-81±5%) and U937 (-69±11%) cells, relative to untreated control cultures (p<0.05). Pretreatment of C2C12 cells with TCE (7-35μg/mL) for 48h attenuated EtOH-induced toxicity by 18±6%-26±5% (p<0.02), indicating a protective effect of TCE on myotube cultures. Higher [TCE] (50-100μg/mL) decreased viability by 14±3%-20±4%, compared with untreated controls (p<0.02), indicating a toxic threshold for TCE. Pretreatment with TCE for 24h (50-100μg/mL) and 48h (35-100μg/mL) rescued U937 cell viability after the EtOH challenge (p<0.02). Interestingly, U937 cell counts increased when preconditioned with TCE at [21.5-100μg/mL] for 24h vs. untreated controls (p<0.02), suggesting a stimulatory effect on cell proliferation. CONCLUSION: These data help establish an experimental range of TCE for future in vitro studies, and demonstrate that TCE has protective antioxidant effects on cultured skeletal muscle cells and monocytes. Funding Source: Combat Feeding Research & Engineering Program / Framingham State University Department of Chemistry and Food Science.