**Background:** Oxidative stress, resulting from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, plays a causal role in neurodegenerative diseases. The genus Mentha is traditionally used for various ailments, and Mentha rotundifolia (L.) Huds. (Cuban mint) is known for its analgesic, antiseptic, and anti-inflammatory properties. This study aimed to evaluate the antioxidant and neuroprotective effects of M. rotundifolia aqueous extract both in vitro and in vivo.
**Methods:** The aqueous extract of M. rotundifolia leaves was prepared by infusion (100 g dried leaves in 1000 mL water at 90°C for 15 min), yielding 13.43 ± 1.04% crude extract. Chemical composition was analyzed using LC-MS/MS with a Shimadzu system coupled to an MS 8050 triple quadrupole mass spectrometer. In vitro antioxidant activity was assessed using DPPH and ABTS assays. For the in vivo study, 48 Wistar rats (180–200 g) were divided into 8 groups (n=6): Group 1 (saline + tap water), Group 2 (saline + 2% H2O2), Groups 3–4 (62.5 mg/kg extract ± H2O2), Groups 5–6 (125 mg/kg extract ± H2O2), and Groups 7–8 (250 mg/kg extract ± H2O2). Extract was administered intraperitoneally for 21 days; 2% H2O2 was added to drinking water from day 8. Behavioral tests included open field (locomotion and anxiety), rotarod (motor coordination), Y-maze (spatial working and reference memory), and tail immersion test (hyperalgesia). Hepatic biomarkers (ALT, AST, total bilirubin) were measured in serum. Data were analyzed using two-way ANOVA with Bonferroni's post-hoc test.
**Key Results:** LC-MS/MS identified 40 phytoconstituents, with kaempferol glucuronide dominating at 85% relative abundance. The extract showed IC50 values of 26.47 ± 1.68 μg/mL (DPPH) and 41.21 ± 2.54 μg/mL (ABTS). H2O2 exposure significantly decreased body weight from day 15, which was attenuated by the extract at 125 and 250 mg/kg. In the open field test, H2O2 significantly reduced locomotion (crossed squares and sit-ups, p<0.0001), which was reversed by the extract at 62.5 and 125 mg/kg for crossed squares and at all three doses for sit-ups. H2O2 also decreased entries into and time spent in the inner zone (p<0.0001), indicating anxiety-like behavior; these effects were reversed at 125 mg/kg (entries) and 125 and 250 mg/kg (time). In the rotarod test, H2O2 significantly impaired motor coordination (p≤0.05), which was improved by the extract at 62.5 and 125 mg/kg (p≤0.05). In the Y-maze, H2O2 significantly decreased spontaneous alternation (working memory, p≤0.05) and novel arm entries (reference memory, p≤0.05); these were reversed at 125 mg/kg. The tail immersion test showed H2O2 significantly decreased tail withdrawal latency (p<0.0001), which was attenuated at 125 and 250 mg/kg. Extract-treated groups also showed significantly higher latency independent of H2O2 exposure, suggesting an analgesic effect. Hepatic biomarkers: H2O2 elevated ALT (48.56 ± 2.90 vs. 33.23 ± 2.35 UI/L in controls), AST (71.15 ± 2.82 vs. 48.85 ± 1.92 UI/L), and total bilirubin (35.42 ± 7.00 vs. 0.77 ± 0.09 mg/dL). The extract at 125 and 250 mg/kg significantly reduced ALT and AST; bilirubin was reduced at all three doses. At 250 mg/kg, AST and ALT levels in H2O2-exposed animals were comparable to tap water controls.
**Clinical Implications:** This study provides the first in vivo evidence of the neuroprotective potential of M. rotundifolia aqueous extract against oxidative stress-induced behavioral and biochemical changes. The extract's high kaempferol glucuronide content (85%) and strong antioxidant activity suggest it may be a promising candidate for developing nutraceutical interventions for oxidative stress-related disorders, including neurodegenerative diseases. The observed mild elevation in bilirubin with extract treatment may have therapeutic potential, as mild hyperbilirubinemia has been associated with reduced oxidative stress. However, further studies are needed to isolate individual components, determine therapeutic doses, and elucidate the underlying mechanisms.