Raw264.7 is a murine macrophage-like cell line commonly used to study the anti-inflammatory efficacy of natural compounds. However, the impacts of long-time incubation on the tested compounds are often inappropriately ignored. Among 77 natural phenolic compounds (mainly flavonoids), only 36 remain more than 70% after a 15-h incubation in cell culture medium at 37 °C. Interestingly, for those compounds with a relatively good chemical stability, the presence of Raw264.7 cells could accelerate their disappearance in the medium, indicating that cellular metabolism occurred. As a representative phenolic, hesperetin was found to be efficiently metabolized by Raw264.7 cells and the metabolite was identified as a glucuronide in the further investigation. The glucuronidation activity is constitutive in this cell line. At certain concentration levels of hesperetin, the ability of hesperetin to inhibit PGD2 production in LPS-induced Raw264.7 cells was significantly enhanced by introducing β-glucuronidase, which can hydrolyze hesperetin glucuronide, into the incubation medium. The results indicate that glucuronidation and excretion of hesperetin can significantly impact its bioactivity in Raw264.7 cells.
Keywords: Raw264.7; degradation; hesperetin; in vitro; metabolism; natural phenolic compounds.