Cognitive impairment and mood disturbances are increasingly recognized as major contributors to reduced quality of life and productivity across populations. These conditions arise from complex and interacting biological pathways, including oxidative stress, neurotransmitter imbalance, mitochondrial dysfunction, and neuro-inflammation, each of which can disrupt normal neural signaling and cognitive processing. Although a range of pharmacological treatments, such as antidepressants, anxiolytics, and cognition-enhancing agents, are available, their effectiveness is often constrained by several limitations. Many therapies yield only modest symptomatic improvement, require prolonged treatment periods before benefits emerge, and may produce undesirable side effects that reduce adherence. In addition, access to specialized psychiatric or neurological care can be limited in many regions, further diminishing the reach of conventional interventions. These challenges have contributed to rising interest in complementary, nutraceutical, and plant-derived strategies aimed at supporting neurocognitive function and emotional well-being (1–4).

Botanical ingredients have been widely investigated for their potential benefits in cognitive function, mood regulation, and overall emotional well-being. Among these, Ginkgo biloba (GB) has attracted substantial scientific attention for health benefits due to its worldwide long-standing use in traditional medicine and a steadily expanding body of modern recent pharmacological evidence supporting its potential role in cognitive performance and emotional well-being (5–19). GB has a particularly long history of traditional use in East Asia, especially in China, Japan, and Korea, where its leaves and seeds have been incorporated into medicinal practices for centuries. Today, the plant is cultivated across Europe, the Americas, and other regions, and its standardized extracts are used in botanical preparations for neurocognitive and emotional support.

A growing body of clinical and preclinical evidence supports the potential efficacy of standardized G. biloba extract (GBE) in enhancing memory, executive function, and mood in both healthy individuals and those with mild cognitive or mood disturbances. Extracts derived from the leaves of GB are among the most extensively studied botanical preparations and have been evaluated for their potential to support cognitive performance and to contribute to the management of age-related neurodegenerative conditions, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and dementia (7, 12–15).

The potential health benefits of GBE are largely attributed to its diverse bioactive compounds, including terpenoids, flavonoids, polyphenols, and organic acids (6, 20). GBE is particularly rich in bilobalide, ginkgolides (A, B, and C), and flavone glycosides such as quercetin, isorhamnetin, and kaempferol. These constituents have been associated with antioxidant, anti-inflammatory, neuroprotective, and anti-apoptotic properties, which may act additively or synergistically to mitigate oxidative stress, modulate neurotransmitter systems, and improve cerebral blood flow, thereby supporting cognitive function and emotional well-being (6–9, 12, 17, 19, 20).

Alongside GBE, a considerable body of clinical and mechanistic evidence also suggests the potential of phosphatidylserine (PS) in supporting brain health (21–31). PS is an essential phospholipid and a key structural component of neuronal membranes, where it contributes to membrane fluidity, receptor dynamics, intracellular signaling, and neurotransmitter release. Supplementation with PS has been associated with improvements in memory, attention, and overall cognitive performance, and its role in maintaining synaptic integrity, modulating stress responses and supporting cholinergic and dopaminergic pathways further underscores its relevance to both cognitive function and emotional well-being.

Given the potential for additive or synergistic effects, there is increasing scientific interest in the association of GBE and PS for cognitive health and emotional well-being. In addition to its intrinsic neuroprotective and cognition-supporting properties, PS may exert a complementary role by optimizing the interaction of bioactive compounds into neuronal membranes (28). This dual action provides a clear rationale for formulating GBE and PS together to optimize outcomes related to cognitive performance and emotional well-being. Previous clinical findings have reported promising preliminary outcomes for this innovative association (27, 32).

In the present study, we investigated the mechanistic basis of the association of a standardized GBE and PS (hereafter referred to as GBP). We assessed its potential antioxidant and neuroprotective properties in both neuronal cellular and cell-free systems, along with its effects on key neurotransmitter-related pathways. These analyses aim to provide supportive evidence for the potential of this GBP association as an effective natural approach for promoting cognitive function and emotional well-being.

The present in vitro studies demonstrate that the association of GBE and PS can potentially modulate neurotransmitter-related pathways and safely exert complementary antioxidant and neuroprotective effects, consistent with mechanisms implicated in cognitive function and emotional well-being. Importantly, GBP showed no intrinsic cytotoxicity up to the highest concentrations tested, supporting its safety in neuronal models. To our knowledge, this represents the first investigation of the supporting mechanistic evidence for this association, providing novel insights that complement existing clinical findings (27, 32).

Neurotransmitter modulation was a prominent finding in this study. GBP inhibited AChE and MAO-A enzymes in a concentration-dependent manner, suggesting potential to enhance cholinergic signaling and preserve monoamine tone—mechanisms directly relevant to memory, attention, and mood regulation. Importantly, the magnitude of AChE and MAO-A inhibition observed in vitro was modest when compared with potent pharmaceutical reference inhibitors, and is therefore more consistent with a supportive neuro-modulatory role rather than a drug-like pharmacological effect. Such moderate enzyme modulation may nonetheless contribute to cumulative functional benefits when combined with complementary mechanisms such as antioxidant protection and neurotrophic support. Previous studies have shown that GBEs inhibit both MAO-A and MAO-B activity (36), while PS specifically inhibits Monoamine oxidase B (MAO-B) (37). These independent findings provide biological plausibility for the monoaminergic effects observed here and highlight the potential complementarity of the GBE and PS within the formulation. By contrast, GABA-T activity was unaffected, and GABA-A receptor binding was weak and only evident at high concentrations. This observation is consistent with evidence that Ginkgo-derived terpene trilactones, such as ginkgolides and bilobalide, function as antagonists or negative modulators at GABA-A receptors (38, 39). Such interactions appear to be modest relative to their actions on cholinergic and monoaminergic systems and are more likely to contribute to the regulation of excitatory-inhibitory balance and neuroprotection under stress conditions rather than to direct GABAergic modulation. Taken together, these findings suggest that the potential neuro-modulatory effects of GBP are primarily mediated through cholinergic and monoaminergic pathways, with only minor contributions from GABAergic activity. These mechanistic findings provide a biological context for previous clinical evidence linking both GBE and PS to improvements in memory performance, attentional control, and emotional regulation (6, 8, 9, 13, 15, 17, 24, 27–29, 32, 40), as modulation of cholinergic and monoaminergic pathways may support cognitive processing and mood regulation, while complementary antioxidant and neurotrophic mechanisms may contribute to stress resilience. The selection of cholinergic, monoaminergic, and GABAergic targets in the present study was guided by their well-established roles in cognitive performance, mood regulation, and stress resilience, as well as by prior experimental and clinical evidence indicating that both standardized GBEs and PS interact with these neurotransmitter systems. While the present work focused on these pathways, other mechanisms, including glutamatergic signaling and neuro-inflammatory processes, may also contribute to the overall neurocognitive effects of GBP and warrant investigation in future studies.

Oxidative stress and free radical damage are central mechanisms in neurodegenerative processes, cognitive decline, and mood disorders (40–42). In the present study, GBP demonstrated clear neuroprotective activity in cellular models, where it protected SH-SY5Y neuroblastoma cells from H₂O₂-induced oxidative stress. GBP treatment restored cell viability and reduced membrane damage, indicating preservation of neuronal integrity under oxidative challenge. These effects confirm its capacity to counteract oxidative stress–induced cytotoxicity, a mechanism directly relevant to the prevention of neuronal injury and functional decline.

Complementing these cellular findings, GBP also exhibited robust antioxidant activity across multiple cell-free assays, including DPPH, ABTS, FRAP, ORAC, HORAC, TAS and TPC. Together, these results indicate broad-spectrum radical-scavenging and reducing potential, reflecting the contribution of polyphenolic constituents to the extract. By neutralizing reactive oxygen species (ROS) and supporting redox homeostasis, GBP may mitigate one of the key pathophysiological drivers of cognitive decline and emotional disturbances such as low mood and impaired stress resilience. Although the antioxidant capacity of GB-based preparations has been previously reported, the use of multiple complementary cell-free antioxidant assays in the present study was used to mechanistically contextualize the observed neuroprotective effects under oxidative stress conditions in neuronal cells.

Building on these observations, we further examined whether GBP could influence BDNF expression under oxidative stress. BDNF is a critical mediator of synaptic plasticity and memory consolidation, and reduced levels have been implicated in both cognitive impairment and depressive symptoms (43). GBP preserved BDNF expression in SH-SY5Y cells exposed to H₂O₂, indicating a role in maintaining neurotrophic support under stress conditions. These findings are consistent with in vivo studies showing that standardized GBEs upregulate BDNF expression in the hippocampus, thereby promoting long-term memory persistence and enhancing learning outcomes (44, 45). By preserving both redox balance and BDNF expression, GBP may contribute to enhanced cognitive resilience and emotional well-being, complementing its neuro-modulatory effects.

The present mechanistic data provides a framework for interpreting clinical findings with the formulation of GBE and PS in Virtiva™ Plus. Controlled clinical trials have demonstrated improvements in memory, attention, and mental flexibility in healthy adults, as well as beneficial effects on emotional distress and physiological responses to prolonged exertion (27, 32). The current results offer mechanistic support for these outcomes, linking the observed cognitive and emotional benefits to cholinergic and monoaminergic modulation, antioxidant defense, and neurotrophic preservation. It is important to note that the concentrations employed in the present in vitro assays do not directly reflect achievable plasma or brain levels following oral supplementation. As is common in mechanistic in vitro studies of botanical preparations, higher concentrations are often required to overcome limitations related to cellular uptake, metabolic degradation, protein binding, and the absence of physiological accumulation processes (46–50). Accordingly, the concentrations used here were intended to explore biological pathways and mechanistic potential rather than to model in vivo exposure. These findings should therefore be interpreted as providing mechanistic plausibility supporting potential in vivo effects, which require confirmation in appropriately designed pharmacokinetic and clinical studies.

The inclusion of two active ingredients in GBP provides a rational explanation for the multifaceted biological effects observed in the present study. GB extract supplies flavonoids and terpenoids with antioxidants, anti-inflammatory, and cerebrovascular activity, while PS contributes to membrane fluidity, receptor function, and neurotransmitter release. PS may also optimize the incorporation of bioactive compounds into neuronal membranes. The complementary biological activities of these two agents likely account for the additive or synergistic benefits observed in vitro.

GBP potentially exerts mild but multi-targeted actions with the potential for fewer adverse effects, in respect to other conventional supports. By combining moderate enzyme inhibition with antioxidants and neurotrophic properties, it may provide a broader profile suited for preventive or adjuvant use. These characteristics are relevant not only to memory and executive function but also to emotional well-being and low mood. While nutraceuticals cannot replace established therapies in conditions such as AD or major depression, they may serve as useful adjuncts or preventive strategies to support cognition, stress resilience, and emotional health in otherwise health populations, not using pharmacological therapies. Safety is an essential consideration for nutraceutical strategies intended for long-term use. In this study, GBP showed no intrinsic cytotoxicity in SH-SY5Y cells across concentrations up to 1,500 μg/mL, consistent with the well-documented safety of both components in preclinical and clinical studies.

Taken together, these findings provide mechanistic evidence that GBP potentially supports neurotransmitter function, redox balance, and neurotrophic signaling processes that collectively underpin memory performance, executive function, and emotional well-being. These results add to the growing body of mechanistic evidence supporting the potential role of the supplementation of GB and PS as a natural strategy for maintaining cognitive health and emotional resilience.

This study has several limitations. The in vitro assays were designed to explore mechanistic interactions rather than to model physiological exposure levels. The complex composition of botanical extracts also precludes attribution of observed effects to individual constituents. These limitations are inherent to mechanistic food chemistry studies and highlight the need for future integrative approaches combining compositional analysis with in vivo validation.

This study provides mechanistic evidence supporting the biological plausibility of the association of GBE and PS in modulating neurotransmitter-related pathways, counteracting oxidative stress, and preserving neurotrophic support under adverse conditions. These complementary actions, possibly, acting additively and/or synergistically, underpin processes central to memory, executive function, and emotional well-being. To our knowledge, this is the first investigation to characterize the mechanistic basis of GBP, offering a biological rationale that supports its potential application as a nutraceutical strategy for cognitive and emotional health.