Characterization of Volatile Oil Constituents from Aurantii Fructus Immaturus (Zhishi) Based on Geographical Origin, Extraction Method, and Processing Differences and Its Mechanism of Ameliorating Slow Transit Constipation via Regulation of the PERK/CHOP-Autophagy Axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Aurantii Fructus Immaturus (Zhishi) is traditionally used to treat constipation, and bran-frying moderates its drastic effects. Processing-related changes in volatile oil and their relationship with ER stress and autophagy in slow-transit constipation (STC) remain unclear. AIM OF THE STUDY: To characterize variations in Zhishi volatile oil by origin, extraction method, and bran-processing, and to investigate its effects in STC in relation to the PERK/CHOP-autophagy axis. MATERIALS AND METHODS: Volatile oils from raw and bran-processed Zhishi of different origins were analyzed by HS-GC-MS and GC-MS. PCA, OPLS-DA, VIP, and GLM assessed influencing factors. Antioxidant activity was evaluated by DPPH· and ABTS•+ assays. Loperamide-induced STC rats received raw or processed oil, D-limonene, or mosapride. Defecation, intestinal propulsion, colonic histology, and biochemical and molecular markers were evaluated. RESULTS: Seventy-seven constituents were identified, with Jiangxi Zhishi showing the greatest diversity. Bran-frying decreased several monoterpene hydrocarbons, relatively enriched selected oxygenated constituents, and increased the number of detected constituents. Hydrodistillation produced the highest yield and diversity (P < 0.05). Processed oil showed stronger DPPH· scavenging activity than raw oil and D-limonene (P < 0.01); raw and processed oils had comparable ABTS•+ activity, and both outperformed D-limonene (P < 0.01). In STC rats, both oils improved defecation and intestinal propulsion, protected colonic tissue, increased c-kit, ATP, LC3B, 5-HT4R, and SOD, and decreased GRP78, CHOP, p-PERK/PERK, and MDA. Processed oil was more effective than raw oil, whereas D-limonene was weakest. Exploratory Spearman analysis linked selected VIP markers with pharmacodynamic endpoints, while D-limonene showed weak correlations. CONCLUSION: Origin, processing, and extraction method influence Zhishi volatile oil composition. Bran-frying enhanced antioxidant activity and therapeutic effects against STC, potentially through attenuation of PERK/CHOP-mediated ER stress, modulation of LC3B-associated autophagy-related processes and oxidative stress, protection of interstitial cells of Cajal, and modulation of 5-HT4R signaling.