Comprehensive evaluation of Pulicaria dysenterica subsp. dysenterica decoction: Phenolic profile by UHPLC-HESI-Orbitrap®-HRMS2, In vitro gastrointestinal stability of biological activities, acute oral toxicity, and antihemorrhoidal potential

Ethnopharmacological relevance: Pulicaria dysenterica Gaertn. subsp. dysenterica has been traditionally used for gastrointestinal and inflammatory disorders; however, its phenolic stability during digestion and its anti-inflammatory efficacy have not been comprehensively investigated. Aim of the study: This study aimed to evaluate the phenolic profile, antioxidant capacity, gastrointestinal stability, acute oral toxicity, and therapeutic efficacy of P. dysenterica subsp. dysenterica decoction extract. Materials and methods: Total phenolic content (TPC) and antioxidant activity (DPPH and CUPRAC assays) were assessed before digestion and after simulated gastric and intestinal digestion. Phenolic compounds were characterized using a validated UHPLC-HESI-Orbitrap®-HRMS2 method. Acute oral toxicity was evaluated according to OECD guideline 423. The therapeutic effects of the extract at two different doses, low dose (PDE-L, 3.2 mg/kg) and high dose (PDE-H, 6.4 mg/kg), were investigated in an experimentally induced hemorrhoid model by assessing rectal-anal coefficient (RAC), inflammatory index, and TNF-α levels in serum and rectal tissues. Results: The undigested extract exhibited the highest TPC (1541.03 ± 17.34 μg GAE/mL) and DPPH activity (616.22 ± 0.39 μg TE/mL), whereas gastric digestion showed superior CUPRAC activity (1255.36 ± 16.14 μg TE/mL). Among 75 screened phenolics, 28 were identified, with chlorogenic acid (8543.88 ± 38.04 μg/g, gastric phase) and caffeic acid (6412.00 ± 96.50 μg/g, intestinal phase) being predominant. No acute oral toxicity was observed up to 2000 mg/kg. PDE-H significantly reduced RAC, inflammatory index, and TNF-α a levels in rats with croton oil-induced hemorrhoids, while PDE-L exhibited limited effects. TNF-α levels correlated positively with inflammation severity. Conclusion: P. dysenterica subsp. dysenterica is a safe, phenolic-rich plant with strong antioxidant and anti-inflammatory properties, supporting its potential as a natural therapeutic candidate for pharmaceutical and nutraceutical applications.