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Ethnopharmacological Relevance: Origanum majorana L. is a member of the Lamiaceae family and is commonly used in Egyptian cuisine as a seasoning and flavor enhancer. It is also recognized as a well-known traditional medicine in Egypt and is widely used for treating abdominal colic due to its antispasmodic properties. However, the protective effects of Origanum majorana L. against ulcerative colitis and its underlying mechanisms remain unclear., Aim of the Study: This study aimed to identify the biologically active components present in methanol extracts of Origanum majorana L. using gas chromatography/mass spectrometry (GC/MS). Additionally, it aimed to investigate the therapeutic effects of these extracts on acetic acid-induced ulcerative colitis and elucidate the potential mechanisms involved., Materials and Methods: We conducted a GC-MS analysis of the methanolic extract obtained from Origanum majorana L. Thirty-two male rats were included in the study and divided into four experimental groups, with eight rats in each group: sham, UC, UC + O. majorana, and UC sulfasalazine. After euthanizing the rats, colon tissue samples were collected for gross and microscopic examinations, assessment of oxidative stress, and molecular evaluation. GC-MS analysis identified 15 components in the extracts. Pretreatment with O. majorana L. extract and sulfasalazine significantly improved the disease activity index (DAI) and resulted in notable improvements in macroscopic and microscopic colon findings. Additionally, both treatments demonstrated preventive effects against colonic oxidative damage by reducing the levels of malondialdehyde (MDA) and increasing the levels of the antioxidant systems superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), which operate through the Nrf2/HO-1 signaling pathway. Moreover, these treatments downregulated the colonic inflammatory cascade by inhibiting NFκB, TNFα, IL-1β, IL6, IL23, IL17, COX-2, and iNOS, subsequently leading to downregulation of the JAK2/STAT3 signaling pathway and a decrease in the Th17 cell response. Furthermore, a reduction in the number of apoptotic epithelial cells that expressed caspase-3 was observed., Conclusion: pretreatment with O. majorana L. extract significantly ameliorated acetic acid-induced ulcerative colitis. This effect could be attributed to the protective, antioxidant, anti-inflammatory, and anti-apoptotic properties of the extract., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)