Immunomodulatory effect of Cannabis root extract on inflammatory cascades via endocannabinoid system regulation

Cannabis roots have been widely used in traditional medicine, with documented references in classical texts describing their use for the treatment of various inflammatory diseases and pain. Despite their longstanding ethnopharmacological significance, the bioactive compounds responsible for these effects and their underlying mechanisms remain unexplored. The present study was conducted to evaluate the unique anti-inflammatory mechanisms of Cannabis sativa root fractions, and moreover, to investigate its mechanism related with the endocannabinoid system (ECS). Antioxidant activities and phenol contents of various Cannabis root fractions were determined by chemical assays. The effects of cannabis root fractions on inflammatory markers and endocannabinoid receptor (CB1, CB2) levels were evaluated in LPS-stimulated RAW 264.7 cells. Intracellular 2-arachidonoylglycerol (2-AG) levels were measured using LC-MS/MS. The fraction with the highest potential was further investigated to elucidate its mechanism using endocannabinoid receptor antagonists. Among the fractions, ethyl acetate fraction (CSREA) demonstrated the highest potential in both antioxidant and anti-inflammatory effects. However, its effect was not attributed to the inhibition of NF-κB signaling pathways. LC-MS/MS analysis showed that CSREA affected intracellular 2-AG levels, supporting its potential via the ECS. CSREA also effectively suppressed ERK phosphorylation, a critical inflammatory signaling pathway modulated by ECS. However, CSREA activity was reduced by co-treatment with a CB1 antagonist. This study demonstrates that CSREA suppresses inflammatory responses and restores cellular homeostasis primarily by regulating the endocannabinoid system. However, its exclusive use of an acute in vitro inflammation model represents a limitation, and the effects of CSREA in chronic and in vivo settings require further investigation.