Satureja macrostema is a plant that is located in various regions of Mexico and is used in a traditional way against illness. Essential oils (EOs) were obtained from leaves Satureja macrostema and the chemical composition was evaluated by gas chromatography–mass spectrometry (GC-MS). The antioxidant effect of the oil was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and by Trolox Equivalent Antioxidant Capacity (TEAC). In vitro antibacterial activity against Escherichia coli and Staphylococcus aureus was determined using a broth microdilution assay and thin layer chromatography–direct bioautography (TLC-DB) to identify active antibacterial compounds. The EOs analysis showed 21 compounds, 99% terpenes, and 96% oxygenated monoterpenes, with trans-piperitone epoxide (46%), cis-piperitone epoxide (22%), and piperitenone oxide (11%) as more abundant compounds. Likewise, S. macrostema EOs showed an antioxidant activity of DPPH = 82%, with 50% free radical scavenging (IC50) = 7 mg/mL and TEAC = 0.005, an antibacterial effect against E. coli of 73% inhibition, and 81% over S. aureus at dose of 100 µL of undiluted crude oil. The TLC-DB assay showed that the most active compounds were derived from piperitone. The comparison with other studies on S. macrostema shows variability in the compounds and their abundances, which can be attributed to climatic factors and the maturity of plants with similar antioxidant and antibacterial activities.
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