Asiatic acid and madecassic acid cause cardiotoxicity via inflammation and production of excessive reactive oxygen species in zebrafish.

Centella asiatica (L.) Urban is a famous Chinese traditional medicine, which is widely used for treating various chronic inflammatory diseases. Although there are reports that Centella total glycosides exhibit heart-protective properties, our previous experiment showed that it has cardiac toxic effects in zebrafish. The components of Centella total glycosides are complex, so we recommend further research to determine their key components and mechanisms. In this study, sample quantification was done using liquid chromatography-tandem mass spectrometry. The cardiotoxicity of Centella total glycosides, asiaticoside, madecassoside, asiatic acid, and madecassic acid was evaluated using zebrafish and cell models. The zebrafish oxidative stress model and myocarditis model were used to explore further the mechanisms through which cardiotoxicity is achieved. Asiatic acid and madecassic acid caused zebrafish cardiotoxicity and H9C2 cell death. However, no toxicity effects were observed for asiaticoside and madecassoside in zebrafish, until the solution was saturated. The results from the cell model study showed that asiatic acid and madecassic acid changed the expression of apoptosis-related genes in myocardial cells. In the zebrafish model, high concentrations of these components raised the levels of induced systemic inflammation, neutrophils gathered in the heart, and oxidative stress injury. Asiatic acid and madecassic acid are the main components causing cardiotoxicity in zebrafish. This may be due to enhanced inflammation and reactive oxygen species injury, which causes myocardial cell apoptosis, which further leads to cardiac toxicity.

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization.

ETHNOPHARMACOLOGICAL RELEVANCE: Glabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. AIM OF THE STUDY: The study identified the toxic impurities of glabridin and their toxicological mechanism. MATERIALS AND METHODS: In total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. RESULTS: Although glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 µM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. CONCLUSION: Glabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.