In today's society, the prevalence of various degenerative illnesses is soaring as the average human lifetime rises (World Population Ageing 2013, 2013). These degenerative diseases are characterized by oxidative stress and low-level chronic inflammation (Jenny, 2012). The average human lifespan is increasing, which is one of the major risk factors for dementia, including Alzheimer's disease (AD) and other notable neurodegenerative disorders (Yankner et al., 2008); (Behl et al., 2021). Dementia and other neurodegenerative disorders are complex and poorly understood. Oxidative stress is one of the factors that contribute to the aging process and is linked to neurodegenerative illnesses (Khansari et al., 2009).

Oxidative stress is generated by an imbalance between the generation of ROS and the capacity of biological systems to detoxify them. Brain is one of the most susceptible organs to ROS due to its high oxygen demand and availability of peroxide-sensitive lipids. H2O2, one of the numerous ROS molecules, contributes greatly to the production of oxidative stress. The presence of excessive levels of ROS produces an increase in cellular oxidative stress, which leads to inactivation of cellular enzymes, oxidation and denaturation of proteins, DNA damage and lipid peroxidation thus threatening the cellular integrity and function. When brain cells are exposed to H2O2, it may lead to cellular death in a manner comparable to apoptosis or necrosis (Chen et al., 2009; Tian et al., 2020; Toman et al., 2002). Previous research has revealed that oxidative stress has a key role in a number of neurodegenerative illnesses, including depressive-like behaviour, memory impairment, anxiety, Parkinson's disease, AD and several others (Hoogendam et al., 2017; Yang et al., 2014). Furthermore, there is evidence that antioxidants may minimize the amount of damage caused by oxidative stress to neuronal cells (Kelsey et al., 2010).

Some of the first mentions of dementia and herbal therapies have been reported in the ancient Indian medicinal system known as Ayurveda (Manyam, 1999). The effects of Bacopa monnieri on the central nervous system are explicitly mentioned in both the Charaka Samhita (2500 BCE) and the Susruta Samhita (2300 BCE) (Dash and Kashyap, 1980; Singh and Dhawan, 1997). Additionally, Bacopa has widely been used as a dietary supplement in India mainly because of the low toxicity concerns it poses and the apparent therapeutic effects it has as a nootropic. Bacopa can increase cognition functions by enhancing mental focus, memory performance, and learning behaviour.

Bacosides are the major constituents of Bacopa monnieri. They are a kind of triterpenoid saponin composed of dammarane and glycone units with jujubogenin or pseudojujubogenin moieties. The major alkaloids include brahmine, nicotine, herpestine, apigenin, hersaponin, cucurbitacins, monnierasides I-III, D-mannitol, and plantainoside B. Bacosides are a family of twelve known analogs. Bacopasides I-XII are novel saponins identified recently. Bacoside A, a mixture of bacoside A3, bacopaside II, bacopasaponin C, and jujubogenin, has been the topic of research since some time (Aguiar and Borowski, 2013). A wide range of advantages are associated with Bacopa monnieri, which has been studied extensively for its pharmacological properties. Some of these benefits include protecting neurons, improving memory and learning, reducing inflammation, promoting adaptability, protecting the heart, and even alleviating epilepsy and depression (Fatima et al., 2022). Bacopa monnieri has been reported to be used in an Ayurvedic formulation known as Brāhmī Ghṛita, containing Brahmi (Bacopa monnieri), Vacā (Acorus calamus), Kuṣṭha (Saussurea lappa), Shankhpushpi (Convolvulus pluricaulis), and Purāṇa Ghṛita (old clarified butter/old ghee). The old clarified butter or old ghee has been described in Ayurveda as a memory enhancer, anticonvulsant, healer of mind and anti-inflammatory agent, and for the cure and management of several neurological disorders such as anxiety, dementia and depression (Yadav and Reddy, 2012; Yadav et al., 2014; Dhawan and Singh, 1996; Chandre et al., 2004; Farooqui et al., 2018). The molecular mechanisms associated with the beneficial effects of Brāhmī Ghṛita are not fully understood. However, it is reported that this may act not only by reversing cholinergic abnormalities in the frontal cortex and hippocampus (Russo and Borrelli, 2005), but also via alleviating cholinergic neurodegeneration (Sara, 1989), lowering norepinephrine, and increasing 5-hydroxytryptamine levels in the hippocampus, hypothalamus, and cerebral cortex (Saraf et al., 2010) and delaying aging of human brain and improving cognitive functions (Sharma et al., 2022). However, its mechanism of action and pharmacokinetics is not yet studied well (Farooqui et al., 2018).

The Bhavprakasa Varg-Prakara medication classification from the 16th century classified Bacopa monnieri as a brain tonic, which means it has therapeutic importance for neurological health and cognitive well-being (Dash and Kashyap, 1980). Several new animal and clinical studies support these long-held ideas. Long-term Bacopa treatment resulted in improved learning and memory retention in animals (Balaji et al., 2015; Charles et al., 2011; Das et al., 2002; Preethi et al., 2012; Prisila Dulcy et al., 2012; Reddy et al., 2014; Singh and Dhawan, 1982; Vollala et al., 2011a, 2011b). Prolonged oral administration of Bacopa (over 12 weeks) to healthy volunteers increased information processing speed, free recall, verbal memory, and learning, according to clinical studies and a meta-analysis of randomised controlled trials. Several studies revealed that the drugs made from Bacopa lowered anxiety while increasing learning (Kongkeaw et al., 2014; Lloyd et al., 2001; Peth-Nui et al., 2012; Review et al., 2012; Roodenrys et al., 2002; Stough et al., 2012, 2015). Furthermore, other publications have shown Bacopa's effect in reducing oxidative stress which is one of the major causes of neurodegenerative disorders (Hosamani, 2009; Limpeanchob et al., 2008; Shinomol, 2011; Shinomol and Bharath, 2012; Singh et al., 2013, 2017; Sumathi et al., 2012). According to various research, Bacopa encounters and reduces oxidative damage caused by H2O2, one of the many ROS components, by directly quenching the free radicals or activating survival signalling and anti-apoptotic pathways (Anand and Bhat, 2016; Bhatia et al., 2017; Castelli et al., 2020; Ramachandran et al., 2014; Singh et al., 2010; Yamchuen et al., 2017). However, to the best of our knowledge, to date the effect of Bacopa on the anti-oxidant machinery of the neuronal cells has been sparsely explored without much detailed molecular mechanisms. Therefore, a thorough assessment of mechanisms of action of Bacopa monnieri at the molecular level on the anti-oxidant machinery when exposed to oxidative stress is much needed.

In view of the current scenario, the neuroprotective capacity of Bacopa monnieri (L.) Wettst. and its metabolite cocktail Bacoside-A against H2O2-driven oxidative stress was investigated in this study employing a variety of in vitro, in siloco and in vivo experiments. This study attempted to provide information on the precise molecular mechanism of action of Bacopa monnieri on the antioxidant system of neuronal cells when subjected to acute oxidative stress using these experimental approaches. Molecular docking revealed that Bacoside-A can prevent the proteasomal degradation of Nrf2, a regulator of cellular resistance to oxidants, by inhibiting its interaction with Keap1, an adaptor subunit of Cullin 3-based E3 ubiquitin ligase. This allows Nrf2 to enter the nucleus and bind to the antioxidant response element (ARE), present at the promoter region of antioxidant enzyme genes. This activates antioxidant-responsive genes, which repair oxidatively stressed neural cells and protect it from stress-induced damage. The result of this in silico study was validated through in vitro experiments employing immunoblot and immunofluorescence studies in an artificially induced oxidatively stressed cellular model. The robustness of our findings was further confirmed through in vivo studies involving rodent models, making this study unique from other earlier studies. This comprehensive and multi-level analysis of mechanistic pathways underscores the novelty of our study, providing valuable insights for potential target-based therapeutic analysis of potential phytochemicals from Bacopa monnieri.