The mitochondrial electron transport chain plays a key role in the cell’s energy production via a series of electron transfer reactions (Rich and Maréchal, 2010). Under physiological conditions, up to 4% of electrons may escape and react directly with dissolved oxygen in the cytoplasm, giving rise to reactive oxygen species (ROS). The latter are either free radicals such as superoxide anion (O₂^.–) or hydroxyl radical (OH^.–), molecules such as hydrogen peroxide (H₂O₂), or singlet oxygen (¹O₂) (Haleng et al., 2007). Although they are involved in several complex physiological processes such as growth regulation, apoptosis, blood pressure regulation, cognitive function, and immune defense (Brieger et al., 2012), their elevated levels generate a state of oxidative stress, which plays a major role in the occurrence of chronic and degenerative diseases, among them neurotoxicity (Pham-Huy et al., 2008).

The body has several defense mechanisms to control the cellular ROS levels to avoid getting into a status of oxidative stress. These include endogenous antioxidants, as a first defense line, such as catalase, superoxide dismutase, and glutathione peroxidase. Antioxidants are the second defense line comprising ascorbic acid, uric acid, glutathione, α-tocopherol, and ubiquinol and the third defense line includes DNA repair enzyme systems and proteolytic enzymes (Ighodaro and Akinloye, 2018). However, in case of elevated ROS levels, a supply of exogenous antioxidants, such as natural plant extracts, polyphenols, vitamins, is crucial to reduce ROS dependent damage.

The genus Mentha, a member of the mint family, Lamiaceae, contains around 42 species with a sub-cosmopolitan distribution across all agroclimatic conditions. This genus is widely known for its essential oils that are valued at over US$ 400 million (Bounatirou et al., 2007). Different preparations (Tinctures, decoction, and tablets) of the genus are traditionally used to treat several ailments, such as bad breath, gingivitis and ondotalgies, menstrual cramps, respiratory and gastrointestinal infections. These preparations are also used as sedative, diuretic, carminative and antispasmodic agents (El Hassani, 2020).

The Cuban mint, Mentha rotundifolia (L.) Huds. is a hybrid perennial herb derived from a cross between M. suaveolens and M. longifolia (Denslow and Poindexter, 2009). This plant is traditionally used as a remedy for pain with antiseptic and anti-inflammatory agents to treat wounds and infections (Riahi et al., 2019). Furthermore, it owns antioxidant, anti-tyrosinase, anti-acetylcholinesterase and insecticidal activities (Fatiha et al., 2015; Ben Haj Yahia et al., 2019). These properties are mainly attributed to the presence of several phenolic compounds with rosmarinic acid as a dominant metabolite (Siham et al., 2019).

Several antioxidant therapeutic approaches are currently being investigated for the treatment of oxidative stress-related pathologies, among them neurological diseases. Some of which are currently undergoing clinical trials (Forman and Zhang, 2021). In fact, treatment with free radical scavengers or antioxidants to enhance the antioxidant defenses or decrease the pro-oxidant production may be effective in preventing, treating, or even stopping many neurological diseases (Teleanu et al., 2019). In this context, we aim to study, in vivo, the antioxidant effect of M. rotundifolia leaves aqueous extract. We here explored the antioxidant properties of M. rotundifolia leaves aqueous extract in vitro using ABTS and DPPH assays and in vivo against H₂O₂-induced neurotoxicity and oxidative stress. Behavioral changes and hepatic biomarkers (AST, ALT, and bilirubin) were also assayed. Finally, we characterized the phytoconstituents of the aqueous extract of M. rotundifolia using liquid chromatography coupled with mass spectroscopy technique (LC-MS/MS).

H₂O₂ is generated by several sources of oxidative stress. It’s considered as a signaling biological messenger due to its ability to diffuse freely into many types of tissues and membranes (Gammella et al., 2016). It damages important classes of biological macromolecules in cells directly by oxidizing lipids, proteins, and nucleic acids, which result in many diseases (Guesmi et al., 2018). The cells’ antioxidant defenses counteract H₂O₂ and ROS that have crossed the cell membrane. However, once they reach a threshold, cells eventually succumb to severe oxidative damage, leading to cell death (Law et al., 2014). Oxidative stress has been also linked to cognitive decline in several medical conditions, including aging, traumatic brain injury, and Alzheimer’s disease. In fact, most studies on cognitive impairment in Alzheimer’s disease link it to oxidative stress through decreased levels of antioxidant enzymes (Alzoubi et al., 2012).

In this context, the present study was conducted to evaluate the protective effect of the aqueous extract of M. rotundifolia leaves against H₂O₂ induced toxicity. H₂O₂ exposure significantly decreased the locomotion activity evaluated by the OF and Rotarod tests as it was shown with other substances inducing oxidative stress such as aspartame, clomipramine, and paraquat (Niveditha et al., 2017; Onaolapo et al., 2017; Othman et al., 2019). It also induced Anxiety-like symptoms defined by the number of entries and the time spent in the inner zone of the OF. In the Y-maze test, the spatial working memory and the spatial reference memory were represented by the spontaneous alternance and the number of entries in the novel arm, respectively, which was significantly decreased due to H₂O₂ exposure. As awaited, the plant treated groups showed less oxidative stress related behavior impairments especially at the doses of 125 mg/kg.

These findings may be attributed to the high content of neuroprotective compounds in M. rotundifolia extract. It was shown that kaempferol, identified in the extract, act as neuroprotective agent against rotenone-induced Parkinson’s disease model of rats by preventing the loss of tyrosine hydroxylase expression (Pan et al., 2020). In addition, its administration protected against Chlorpyrifos-induced oxidative stress and memory deficits in rats via GSK3β-Nrf2 signaling pathway and increased the activities of antioxidant enzymes and AChE (Hussein et al., 2018). Furthermore, the intracerebroventricular Micro-injection of Kaempferol reduced anxiety through the GABAergic mechanism by binding to specific sites of benzodiazepines (Zarei et al., 2021). However, in H₂O₂ conditions, it was observed that the group received a 250 mg/kg dose of M. rotundifolia extract had significantly less locomotor activity. It was thought that this was related to the toxicity or the sedative effect of the extract, but according to the literature, the plant is safe even at high doses and has no psychotropic effect (Bounihi, 2016). Further investigations should be carried out to explain these results.

Hyperalgesia was assessed using the tail immersion test. H₂O₂ exposure significantly decreased the latency of the tail reflex. Several studies have related hyperalgesia to oxidative stress and showed that it can be attenuated by antioxidant treatments (Mohajjel Nayebi et al., 2021). The extract treated groups, whether in “Tap water” or “H₂O₂” conditions showed dose-dependent responses. Tail withdrawal latency was maximal at 250 mg/kg suggesting an analgesic effect of M. rotundifolia extract. It was previously shown that at a dose of 600 mg/kg, M. rotundifolia extract had a greater effect than aspirin on acetic acid induced writhing in mice (Boussouf et al., 2017).

The liver is involved in the detoxification and elimination of xenobiotics, some of which are potentially toxic. This leads to oxidative damage of hepatocytes, reflected by the increase of hepatic enzymes. Additionally, liver plays a key role in the metabolism of orally and intra-peritoneally administered drugs (Turner et al., 2011), the two routes we chose for the administration of H₂O₂ and M. rotundifolia extract, respectively. It was even shown that impaired liver function was predisposing factor for encephalopathy and neurodegenerative diseases (Butterworth, 2013). Furthermore, in a cohort study of 1,581 older adults, elevated AST and ALT levels were associated with the diagnosis of Alzheimer’s disease, poor cognition, and reduced glucose metabolism in the brain (Nho et al., 2019). Thus, substances able to regulate liver biomarkers may be effective in preventing neurodegenerative diseases. The major antioxidant enzymes that can eliminate H₂O₂ are catalase, glutathione peroxidase and peroxiredoxins (Phaniendra et al., 2015). It was previously demonstrated that M. rotundifolia extract significantly improved the activities of these enzymes in the liver homogenate after carrageenan injection (Mohajjel Nayebi et al., 2021). Hepatic biomarkers (ALT, AST, and Bilirubin) were assayed as well in this work. The end products of some metabolic pathways such as uric acid, taurine, and bilirubin can have cytoprotective effects, but at the same time, they are harmful at high levels (Glazer, 1989). In the present study, we focused on the antioxidant effect of bilirubin. H₂O₂ exposure induced a significant hyperbilirubinemia accompanied by a significant increase in AST and ALT levels. M. rotundifolia extract significantly decreased the elevated levels in a dose-dependent manner. At a dose of 250 mg/kg, AST and ALT levels in H₂O₂ groups were comparable to those in “Tap water” groups.

In general, the characterized phytoconstituents have been already reported in prior works for their pharmacological properties. Quinic and chlorogenic acids have a wide range of biological activities such as antidiabetic, DNA protective, neuroprotective, hepatoprotective, and antifungal effects (Ma and Ma, 2015; Zuo et al., 2015). Caffeoyl shikimic acid showed an anti-hyperuricemia effect and ameliorated kidney injury in mice (Zhang et al., 2021). Kaempferol and its derivatives are flavonols with promising bioactivities including anti-cancer, antiviral, antimicrobial, and neuroprotective properties (Bangar et al., 2022). Additionally, all these compounds have been shown to possess an important antioxidant activity (Sato et al., 2011; Uzun et al., 2017; Riahi et al., 2019), which may explain the strong antioxidant potential of M. rotundifolia aqueous extract in DPPH and ABTS assays, as it was previously confirmed (Fatiha et al., 2015; Siham et al., 2019).

Noteworthy, when bilirubin acts as an antioxidant, it can protect the cells against up to 10,000 times higher concentrations of H₂O₂ (Doré et al., 1999). In the current work, administration of M. rotundifolia extract resulted in a mild elevation in bilirubin level. This may be due to the richness of M. rotundifolia in flavonoids as it was previously demonstrated with isolated flavonoids from of the Milk thistle, Silybum marianum L., induced elevated bilirubin concentrations (Šuk et al., 2019). Patients with Gilbert’s syndrome, which is characterized by mild elevated bilirubin, have been shown to have a significant lower level of oxidative stress without experiencing symptoms of toxicity related to elevated bilirubin levels (Maruhashi et al., 2012). Furthermore, it was found that the induction of mild hyperbilirubinemia may have an important therapeutic potential specially to prevent diseases related to oxidative stress such as cardiovascular disease, type 2 diabetes, metabolic syndrome, and neurodegenerative disorders (Vitek et al., 2019). These findings suggest that the M. rotundifolia leaves have an important antioxidant potential, and their phytoconstituents may be good candidates to develop new drugs for oxidative stress comorbidities.

Substantial differences were found at the chemical composition of M. rotundifolia from the Moroccan flora compared to those from other countries. In the current work, kaempferol glucuronide (accounted for 85%) dominated the extract along with other minor compounds, among them quinic acid, chlorogenic acid, caffeoyl shikimic acid, and kaempferol rutinoside. Noteworthy, rosmarinic acid was detected at the extract as a minor compound; however, it was among the major compounds from the Algerian and Tunisian species (Fatiha et al., 2015; Siham et al., 2019). While eriocitrin was predominant in Saudi Arabian flora (Alharbi et al., 2021). These variations may be explained by several factors such as geographical distribution, temperature, water availability and soil composition (Kabtni et al., 2020).

Mentha rotundifolia L. is traditionally used for its analgesic, antispasmodic, and anti-inflammatory properties. The current study is the first of its kind carried out for this plant in terms of its neuroprotective potential in an in vivo model. M. rotundifolia extract furnished in vitro antioxidant activities, reduced the effects of H₂O₂-induced oxidative stress, and attenuated the associated behavioral changes. These activities may explain its traditional uses in health and nutrition supplements and promotes its valorization as a nutraceutical. However, further analysis should be performed for better understanding of its neuroprotective effect pathway, isolate its individual components, and determine its therapeutic dose, onset and duration of action.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

All animal procedures were conducted in accordance with international ethical standards (European Union Directive 2010/63/EU) legislation and ARRIVE (Animal Research Reporting of In Vivo Experiments) guidelines.

KB, NB, and MS designed the study, analyzed the data, and contributed to the writing of the manuscript. MT and KT supervised and provided critical feedback that shaped the final version of the manuscript. All authors contributed to the article and approved the submitted version.