Glabrol—an impurity in licorice extract—causes toxicity in muscle, bone, and immune tissues through activation of the AP-1 signaling pathway

Licorice ( Glycyrrhiza genus ) is a traditional medicinal herb that has also been widely used in the food and cosmetic industries, leading to widespread human exposure. Currently, many components have been identified as active ingredients in licorice; however, the toxic impurities and quality markers still require further investigation. Glabrol has been identified as a potentially toxic component in glabridin (an extract from licorice). In this study, we sought to evaluate the toxicity of glabrol in commercial licorice extracts and investigated the toxicological mechanism. The content of glabrol and the acute toxicity in ten commercial licorice extracts from different vendors were quantified using HPLC. The toxicity was further verified in zebrafish, cells in vitro and mammals in vivo mouse models . For the in vivo experiments, C57BL/6 mice received daily oral gavage of licorice extracts with (Sample C: 319.23 μg/g) o low (Sample B: 1.54 μg/g) glabrol content for 7 days. Locomotion was assessed via Open Field Test and Elevated Plus Maze, followed by blood and organ collection for pathological and biochemical analyses. To investigate the toxicological mechanism of glabrol, RNA - seq was performed on zebrafish embryos exposed to glabrol. Morphological and histopathological evaluations in zebrafish treated with the glabrol standard were carried out using phalloidin staining, transmission electron microscopy, and alizarin red staining. Our results indicated that glabrol was detected in all tested commercial licorice extracts, and its content showed a significant positive correlation with toxicity in cells and zebrafish. In mice, licorice extracts with higher glabrol levels led to low survival rates, hypoactivity, acute liver and kidney injury, and significantly elevated plasma inflammatory cytokines. Transcriptomic and mechanistic studies revealed that glabrol disrupted AP-1 signaling pathways and may impair myo-fiber organization, osteoclast differentiation, and inflammatory responses. This study establishes glabrol as a prevalent risk-associated impurity in licorice extracts and reveals that its toxicity is mediated via the AP-1 signaling pathway.