Comparison of the Hepatotoxicities of Raw Chebulae Fructus Immaturus and its Almond Oil-Processed Product

• Integrates traditional almond oil processing with UPLC-Q-TOF-MS to analyze Chebulae Fructus Immaturus (CFI) chemical changes. • Processing alters phytochemical profile: increases pyrogallol/corilagin/gallic acid, decreases granatidine/ellagic acid/quillaic acid. • ACFI significantly reduces hepatotoxicity vs. raw CFI in mice, normalizing ALT/AST and improving liver histopathology. • Mechanism involves CYP2E1 inhibition, lowering oxidative stress, inflammation, and apoptosis via iNOS/Caspase-3 pathways. • Validates traditional detoxification method with modern molecular evidence, bridging Ayurvedic/Uighur medicine with contemporary pharmacology. Almond oil processing is a traditional technique used to reduce the toxicity and enhance the efficacy of Chebulae Fructus Immaturus (CFI, Xiqinguo ), a Chinese herbal medicine for clearing heat, relieving sore throat, and arresting persistent diarrhea with bleeding. However, the chemical and toxicological alterations induced by processing, as well as the mechanisms underlying its detoxification and efficacy enhancement, remain unclear. This study therefore aimed to evaluate the toxicity changes and possible mechanisms of CFI before and after almond oil processing. First, ten batches of CFI were prepared by stir-frying with 0.3 times the amount of almond oil at 160°C for 20 min according to the traditional method. LC-MS analysis in both positive and negative ion modes was performed to profile the chemical changes, and an HPLC method was subsequently established to quantify the most markedly altered components. Subsequently, a repeated-dose oral toxicity test in mice was conducted by administering a dosage of 132 mg/kg, and physiological, hematological, biochemical, and histological analyses in addition to multivariate statistical analyses were performed. A total of 37 components, primarily organic acids, were identified or tentatively characterized in negative ion mode, and quantitative HPLC analysis showed that processed CFI (ACFI) exhibited elevated gallic acid and decreased punicalagins compared to raw CFI. The toxicological studies indicated that CFI induces weight loss, reduces the number of neutrophils, lymphocytes, and monocytes, and causes mild hepatocellular injury with elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Histopathological examination revealed liver injury, manifesting as eosinophilic cytoplasm, pyknosis, and increased expression of Collagen I, CD3, and CD8, suggesting mild hepatocellular necrosis. In contrast, ACFI reduced oxidative stress, inflammation, and apoptosis, as confirmed by western blot and in silico analyses. These findings provide evidence that almond oil processing mitigates CFI hepatotoxicity, likely by modulating the CYP2E1-related pathway.