Optimal brain health is a multidimensional state encompassing cognitive, emotional, and motor domains supported by lifelong physiological processes that sustain neuronal integrity and mental performance (Chen et al., 2021). Cognitive functions—including attention, memory, executive control, and learning—can decline with age or chronic stress exposure, partly due to cumulative environmental and metabolic influences (American Psychiatric Association, 2013; Aubé, 2018). Age-related neuronal dysfunction and oxidative stress exacerbate this decline, whereas lifestyle and nutritional factors that enhance neuronal resilience may delay or prevent cognitive deterioration (Murman, 2015; Sergiev et al., 2015). As the global aging population increases, nutritional supplements targeting memory and focus have gained attention as adjunct strategies to support mental wellbeing and counteract stress-induced impairments in attention and cognition (Onaolapo et al., 2019).

Plant-derived bioactive compounds have long been recognized for their neuroprotective and adaptogenic properties (Al Akeel et al., 2018; Tamer et al., 2021). Nootropic agents—defined as substances that enhance cognitive performance, learning, and mental clarity—modulate neurotransmitter systems such as dopaminergic, glutamatergic, cholinergic, and serotonergic pathways (Onaolapo et al., 2019). Compared with synthetic psychostimulants, natural nootropics often exhibit fewer adverse effects while promoting neuronal plasticity and stress resilience. Among these, extracts such as Lion’s Mane mushroom, Rhodiola rosea, Ginkgo biloba, and compounds like L-theanine, citicoline, phosphatidylserine, and caffeine have demonstrated synergistic benefits in improving attention, mood, and neurochemical balance through modulation of neurotrophic factors, neurotransmission, and oxidative defense mechanisms (Adibhatla et al., 2002; Dager et al., 1999; Fredholm et al., 1999; Friedman, 2015; Ganzera et al., 2001; Gündoğdu, 2014; Jorissen et al., 2002; Lai et al., 2013; Marchev et al., 2016; Nguyen and Alzahrani, 2023; Wang et al., 2018; Wang et al., 2022; Weinmann et al., 2010).

Mind Focus (Kiperin Pharmaceutical and Food Industry Ltd., İstanbul, Turkiye) is a novel nutraceutical formulation that combines several well-established nootropic and adaptogenic ingredients—including L-theanine, Lion’s Mane mushroom extract, citicoline, phosphatidylserine, Rhodiola extract, caffeine, and Ginkgo biloba—designed to support stress resilience, mood regulation and overall neural health. Although each component has been individually investigated for its neuroprotective and psychotropic properties, their combined action on stress-related behavioral, biochemical, and transcriptional parameters have not been systematically examined. Therefore, the present study aimed to investigate the effects of supplementation on neural activity, neurochemical markers, gene expression profiles, and behavioral alterations in a chronic unpredictable mild stress (CUMS) rat model. Importantly, the behavioral assessment focused on stress-related affective and exploratory domains—including anhedonia, anxiety-like behavior, behavioral despair, and locomotor–exploratory activity—rather than classical attention or learning paradigms. Accordingly, the findings are intended to provide mechanistic insight into the potential behavioral, emotional and stress-buffering and behavioral regulatory effects of the combined formulation as a neuroactive nutritional supplement.

The present study demonstrated that Mind Focus supplementation counteracted CUMS-induced affective disturbances and modulated exploratory activity, as reflected by improvements in anhedonia- (SPT), anxiety-like behavior– (EPM), and behavioral despair–related (FST) measures, together with OFT readouts of locomotion and exploration. Importantly, because no dedicated attention or learning tasks were included (e.g., novel object recognition, set-shifting, 5-CSRTT), these behavioral findings should not be interpreted as direct evidence of enhanced attention or focus. Rather, the observed behavioral changes improved stress-related emotional regulation, behavioral engagement, and exploratory activity under chronic stress conditions. Taken together, these findings suggest that the combined formulation may exert its effects by modulating neural processes associated with affective regulation, arousal, and adaptive behavioral responses, rather than directly enhancing attentional or cognitive performance.

At the molecular and biochemical level, Mind Focus supplementation mitigated the wide-ranging effects of chronic stress on neuroinflammation, oxidative balance, and synaptic signaling. The stress-induced elevations of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in serum, liver, and prefrontal cortex were markedly attenuated by this supplementation, restoring peripheral and central cytokine levels to near-control values. In parallel, the oxidative stress index (TOS/TAS ratio) was significantly reduced in both serum and liver following treatment, indicating improved redox homeostasis. Gene expression analyses revealed that the combined formulation modulated several molecular markers of neural activation (FOS), catecholaminergic signaling (DBH), and neuropeptidergic regulation (NMB), while preserving neuroplasticity-related transcripts such as BDNF, CREB1, GRIN2A, and GABRB1 in both hippocampal and prefrontal regions. Notably, the partial normalization of BDNF and CREB1 expression suggests restoration of neurotrophic support, whereas the stabilization of GABRB1 points to recovery of inhibitory tone, aligning with the observed behavioral improvements.

In the sucrose preference test, Mind Focus supplementation alleviated stress-induced anhedonia, reflecting antidepressant-like effects and improved hedonic motivation under chronic stress. This behavioral improvement is likely mediated by the synergistic activity of its neuroactive constituents that collectively modulate monoaminergic transmission, neurotrophic signaling, and stress hormone regulation. Because we tested only the combined formulation, we cannot attribute these effects to any single ingredient; however, prior studies suggest that L-theanine, a green tea–derived amino acid, has been shown to reverse CUMS-induced depressive-like behaviors by increasing serotonin, dopamine, and norepinephrine levels in limbic–cortical regions (Shen et al., 2019), while citicoline enhances antidepressant efficacy and sucrose preference in stressed rodents through its effects on serotonergic neurotransmission and membrane phospholipid dynamics (Roohi-Azizi et al., 2018). Rhodiola rosea extract, a well-known adaptogen, reduces corticosterone elevation and regulates stress-responsive gene expression in the hippocampus and prefrontal cortex, contributing to enhanced stress resilience (Dinel et al., 2019). Similarly, Ginkgo biloba extract (EGb 761) has been reported to counteract LPS-induced anhedonia and restore dopamine levels in the nucleus accumbens, suggesting a regulatory effect on reward-related pathways (Yeh et al., 2015). In addition, Lion’s Mane mushroom extract contains bioactive compounds such as erinacines and hericenones that promote neurogenesis and synaptic plasticity; while its acute effects on cognition appear domain-specific, improvements in psychomotor performance and attentional engagement observed in healthy adults (Surendran et al., 2025), support its potential to modulate mood and stress resilience. Collectively, prior evidence suggests that several components of the nutraceutical formulation may contribute to the restoration of sucrose preference in treated rats which may reflect a multifaceted enhancement of dopaminergic and serotonergic signaling, neurotrophic activity, and HPA axis regulation, jointly contributing to improved motivation, emotional balance, and exploratory activity control under stress conditions.

In the open field test, Mind Focus supplementation significantly enhanced exploratory activity and reduced anxiety-like behavior in rats subjected to chronic stress, indicating improved emotional regulation and behavioral control. This anxiolytic effect is consistent with the known actions of L-theanine, which has been shown to reverse chronic restraint stress–induced behavioral alterations by normalizing prefrontal neurotransmitter levels, lowering proinflammatory cytokines such as TNF-α and IL-6, and reducing corticosterone elevation (Chandrasekhar et al., 2017). Another key component, Lion’s Mane mushroom extract may have contributed to these behavioral improvements through its stress-reducing and cognition-enhancing properties. In a randomized, placebo-controlled human study (Docherty et al., 2023), reported that both acute and chronic supplementation with H. erinaceus improved task performance speed and tended to reduce perceived stress, suggesting that its bioactive compounds may promote adaptive stress responses and exploratory activity. Additionally, Rhodiola rosea, another major constituent of the supplement, has been shown to attenuate corticosterone elevation and modulate stress-responsive gene expression in the hippocampus and prefrontal cortex, thereby reducing behavioral reactivity to acute stress (Dinel et al., 2019). Likewise, Roohi-Azizi et al. (2018) demonstrated that citicoline administration improved open field performance in chronically stressed rodents, reflecting enhanced exploratory behavior and reduced anxiety when co-administered with antidepressant treatment (Roohi-Azizi et al., 2018). Since the present study tested only the combined formulation, we cannot disentangle the contribution of each component. However, these findings suggest that the supplement alleviates stress-induced anxiety and promotes exploratory motivation by synergistically regulating neuroinflammatory, monoaminergic, and neurotrophic pathways, supporting a more stable and adaptive behavioral profile under chronic stress conditions.

In the forced swim test, Mind Focus supplementation significantly reduced immobility time compared with the stress group, suggesting an antidepressant-like effect under chronic stress conditions. This behavioral improvement is consistent with the known neurochemical and neurotrophic actions of several key components within the formulation, although we cannot disentangle the contribution of each component. L-theanine has previously been shown to ameliorate depressive-like behavior in CUMS models by enhancing serotonergic, dopaminergic, and noradrenergic transmission in the prefrontal cortex and hippocampus, accompanied by reduced plasma corticosterone levels (Shen et al., 2019). Similarly, Rhodiola rosea extract has been demonstrated to reduce corticosterone elevation and modulate stress-responsive gene expression in limbic regions, thereby normalizing HPA axis reactivity and improving mood stability (Dinel et al., 2019). Citicoline has also been reported to potentiate antidepressant efficacy by improving sucrose preference, locomotor activity, and behavioral resilience in stressed rodents (Roohi-Azizi et al., 2018), likely through enhanced monoaminergic and membrane phospholipid metabolism. In addition, Ginkgo biloba extract exerts protective effects against chronic stress-induced depressive phenotypes by regulating hippocampal BDNF expression and preventing its stress-related methylation-dependent downregulation (Ayatollahi et al., 2020). Together, these findings suggest that the supplementation may counteract the stress-induced depressive behavior by integrating serotonergic, dopaminergic, and neurotrophic mechanisms, maintaining hippocampal plasticity and reducing behavioral despair in response to chronic stress.

Biochemical results indicate that chronic stress induced marked peripheral and central inflammatory activation, particularly reflected by elevated IL-1β, IL-6, IL-10, and TNF-α levels. Mind Focus supplementation, especially in the SMF group, effectively attenuated these changes, maintaining cytokine profiles near control values and suggesting notable immunomodulatory and anti-inflammatory properties. In parallel, the oxidative stress index (TOS/TAS ratio) was markedly increased in both liver and serum of stressed animals, indicating a pronounced systemic oxidative imbalance. The administration of supplement, particularly in the MF and SMF groups, significantly reduced the TOS/TAS ratio toward control levels, demonstrating its antioxidant efficacy and capacity to restore redox homeostasis under chronic stress conditions. These findings are in agreement with previous evidence highlighting the immunomodulatory potential of L-theanine, a key component of the combined formulation, although we cannot disentangle the contribution of each component L-theanine has been shown to regulate immune and oxidative pathways by suppressing the overproduction of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, while enhancing antioxidant defense through glutathione upregulation and free-radical scavenging activity (Chen et al., 2023). Moreover, its ability to attenuate stress-related activation of the hypothalamic–pituitary–adrenal (HPA) axis and inhibit NF-κB signaling contributes to reduced neuroinflammation and improved neuronal resilience under chronic stress. In addition to these mechanisms, Lion’s Mane mushroom extract—also included in the supplement—has been shown in a recent randomized controlled human trial to increase neurotrophic and antioxidant biomarkers, improve psychomotor speed, and reduce subjective stress when taken daily (Contato and Conte-Junior, 2025). Furthermore, Rhodiola rosea extract has been reported to exert adaptogenic, anti-inflammatory and antioxidant effects by normalizing stress hormone release, down-regulating stress-responsive cytokines and enhancing resilience to oxidative and inflammatory insult (Pu et al., 2020). Ginkgo biloba extract, another constituent of the supplement, has demonstrated robust neuroprotective and anti-inflammatory effects by improving cerebral microvascular perfusion, attenuating microglial activation and lowering oxidative stress markers in the brain; it has also been shown to modulate both pro-inflammatory cytokines and neurotrophic factors in stress and neurodegenerative contexts (Mousavi et al., 2022). Collectively, these results suggest that the anti-inflammatory and antioxidant effects of the supplement may reflect the combined actions of L-theanine, Lion’s Mane, Rhodiola rosea and Ginkgo biloba, together with other bioactive compounds, thereby restoring systemic and neural homeostasis disrupted by prolonged stress exposure.

Histopathological evaluation further supports the neuroprotective potential of Mind Focus under chronic stress conditions. Sections from stressed rats displayed characteristic features of stress-related neuronal injury, including triangular perineuronal halos, cytoplasmic eosinophilia, and shrunken “dark” neurons in both the hippocampus and prefrontal cortex. In contrast, tissues from the Stress + Mind Focus group exhibited substantially preserved neuronal morphology with fewer degenerative changes, as reflected by significantly lower semi-quantitative injury scores in both regions. These findings suggest that the supplementation may attenuate the stress-induced neuronal damage, possibly through its combined antioxidant, anti-inflammatory, and neurotrophic actions. Although the contribution of each component cannot be disentangled, a previous study has reported that L-theanine counteracted the oxidative and enzymatic disturbances in the brain by restoring glutathione levels, normalizing membrane-bound ATPase activity, and reversing cortical and hippocampal histopathological alterations in toxin-induced injury models (Sumathi et al., 2015). In the literature, the components such as L-theanine and Ginkgo biloba have been shown to reduce neuronal apoptosis, suppress NF-κB–mediated microglial activation, and preserve synaptic integrity under oxidative or glucocorticoid stress (Chandrasekhar et al., 2017; Chen et al., 2023; Nguyen and Alzahrani, 2023; Shen et al., 2019; Sumathi et al., 2015; Wang et al., 2022; Weinmann et al., 2010; Yeh et al., 2015), while Rhodiola rosea and Lion’s Mane extracts enhance neurogenesis and neuronal resilience through BDNF-linked signaling pathways (Dinel et al., 2019; Ganzera et al., 2001; Marchev et al., 2016). Recent evidence indicates that Lion’s Mane mushroom extract also attenuates neuroinflammatory responses and oxidative stress by downregulating proinflammatory cytokines, upregulating antioxidant enzymes such as SOD and catalase, and promoting synaptic plasticity and memory-related signaling in the hippocampus (Zhang et al., 2020). Moreover, Rhodiola extract has been shown to exert strong neuroprotective and anti-apoptotic effects by preserving mitochondrial integrity, restoring ATP and cytochrome c oxidase levels, and preventing hippocampal neuronal apoptosis in experimental models of neurodegeneration and chronic stress (Wang et al., 2017). Similarly, phosphatidylserine supplementation has been demonstrated to reduce hippocampal TNF-α and IL-10 expression, enhance neuronal survival, and alleviate depression-like behaviors through its anti-inflammatory and neuroprotective properties in post-stroke models (Partoazar et al., 2021). Together, these effects likely contribute to the observed morphological protection in cortical and hippocampal circuits.

In the present study, hippocampal and prefrontal expression analyses revealed that CUMS induced significant upregulation of FOS, DBH, and NMB, while Mind Focus supplementation was associated with non-significant trend toward control-like values, and with a significant reduction in NMB expression compared to stressed animals. These findings are consistent with the hypothesis that chronic stress activates hippocampal stress-responsive transcriptional programs and noradrenergic signaling, which can be modulated by neuroactive nutritional interventions.

FOS is an immediate-early gene that serves as a marker of neuronal activation and stress responsiveness. Its upregulation in the hippocampus following chronic stress exposure aligns with previous reports demonstrating increased neuronal activity in stress-related circuits and impaired inhibitory regulation under prolonged stress conditions. Lower FOS expression observed in the Mind Focus-treated groups relative to the stress group suggests a potential dampening of stress-induced hyperactivation, possibly through anxiolytic and neuroregulatory effects of L-theanine, phosphatidylserine, and Rhodiola extracts, which have been shown to modulate HPA axis reactivity and glutamatergic signaling. Although the contribution of each component cannot be disentangled, a previous study by Kawada et al. has reported that L-theanine suppresses NMDA-induced c-Fos expression and inhibits mTOR-p70S6K pathway activation in the hippocampus, thereby reducing excitotoxic neuronal activity and contributing to its sedative and neuroprotective effects (Kawada et al., 2025). Moreover, Wong et al. demonstrated that daily administration of Lion’s Mane mushroom extract enhances functional and structural nerve recovery after peripheral nerve injury by upregulating Akt and MAPK signaling as well as c-Fos expression, suggesting that Lion’s Mane may support neuronal survival and regeneration through similar activity-dependent transcriptional pathways (Wong et al., 2012). Consistently, Xia et al. (2016) found that Rhodiola rosea reduced stress-induced c-Fos expression in the hypothalamus and normalized corticosterone and CRH levels, indicating that its adaptogenic and anti-stress effects may partly operate via downregulation of stress-responsive immediate-early genes (Xia et al., 2016). In addition, Bennett and Semba (1998) reported that caffeine induces region-specific and dose-dependent changes in c-Fos expression, with low stimulant doses enhancing locomotion without widespread neuronal activation, and higher doses triggering marked c-Fos induction in striatal, limbic, and hypothalamic regions, suggesting a nuanced, dose-regulated modulation of neuronal excitability (Bennett and Semba, 1998). Collectively, these findings suggest that the combined action of Mind Focus constituents, from literature-based hypotheses about individual components, may contribute to the neuronal excitability and may limit maladaptive stress-induced transcriptional activation, leading to a more balanced neural network response that supports behavioral stability, emotional regulation, and stress resilience under chronic stress conditions.

DBH, the rate-limiting enzyme in the conversion of dopamine to norepinephrine, serves as a key marker of catecholaminergic turnover and stress responsiveness. In our study, hippocampal and prefrontal DBH expression was significantly elevated in the stress group compared to controls, consistent with hyperactivation of noradrenergic circuits under chronic stress. Mind Focus supplementation was associated with DBH expression levels closer to control group in both regions, suggesting a stabilizing effect on catecholaminergic neurotransmission. This modulation could plausibly involve combined effects of its bioactive components; however, attribution to a specific component is not possible. Previous studies reported that the caffeine, L-theanine, and citicoline independently influenced the dopaminergic–adrenergic balance. Specifically, Kim (2002) demonstrated that caffeine, through antagonism of adenosine A1 and A2A receptors co-localized with dopamine D1 and D2 receptors in striatal neurons, enhances dopaminergic signaling and restores locomotor and feeding behavior even in dopamine-depleted (Dbh-/-) mice (Kim, 2002). Within this framework, our findings are consistent with the notion that adenosine receptor blockade by caffeine facilitates downstream dopaminergic and noradrenergic activation, potentially attenuating stress-induced catecholaminergic dysregulation, although no statistically significant normalization was demonstrated. Additionally, Lion’s Mane mushroom extract has been shown to enhance dopaminergic function and neuronal plasticity by promoting neurotrophin-mediated survival pathways, particularly via activation of Akt and MAPK cascades (Wong et al., 2012). These mechanisms not only support catecholaminergic neuron integrity but also facilitate adaptive neuronal signaling under stress. Rhodiola rosea, a well-characterized adaptogen, has similarly been reported to regulate catecholamine metabolism by reducing corticosterone-induced overactivation of the HPA axis and restoring monoamine turnover in stress-exposed rodents (Dinel et al., 2019). Although the contribution of each component cannot be disentangled, these findings suggest that the combined effects of caffeine, H. erinaceus, and R. rosea may act to stabilize dopamine–norepinephrine balance through both receptor-mediated and neurotrophic pathways, thereby mitigating stress-related catecholaminergic overdrive and contributing to improved behavioral stability and emotional regulation. Factorial designs (single-ingredient arms and their combinations) or head-to-head comparisons would be required to determine whether any specific component drives this transcriptional pattern or whether synergistic interactions are involved.

NMB, a bombesin-like neuropeptide implicated in stress, thermoregulation, and emotional processing, exhibited robust upregulation in the hippocampus under chronic stress. The significant reduction of NMB expression in the Stress + Mind Focus group offers a possible mechanistic hypothesis that the supplementation may partially modulate the stress-related peptidergic signaling. Although the contribution of each component cannot be disentangled, this effect may be mediated through indirect modulation of limbic circuits and neuroendocrine feedback mechanisms, consistent with previous evidence linking nutritional bioactives such as Ginkgo biloba and Rhodiola rosea to the regulation of neuropeptide expression and neuronal plasticity under stress. In line with these findings, Williams and Naidoo (2020) highlighted that endoplasmic reticulum (ER) lipid homeostasis is crucial for neuropeptidergic vesicle production and circadian regulation (Williams and Naidoo, 2020). They demonstrated that ER lipid defects in neuropeptidergic neurons disrupt phosphatidylserine metabolism, impairing vesicular neuropeptide transport and contributing to stress- and sleep-related neuronal dysfunction. Remarkably, phosphatidylserine supplementation restored neuropeptide vesicle formation and normalized behavioral rhythms in Drosophila models. Our findings offer a possible mechanistic hypothesis suggesting that components of the supplement may restore neuropeptidergic balance under chronic stress by improving ER homeostasis and maintaining vesicular signaling integrity, ultimately supporting adaptive emotional and physiological regulation.

Chronic stress is well known to suppress neuroplasticity-related signaling in corticolimbic circuits, leading to structural and functional impairments associated with anxiety, anhedonia, and cognitive decline. Ginkgo biloba, Rhodiola rosea, and Lion’s Mane mushroom extract were previously reported to enhance BDNF expression and CREB phosphorylation through modulation of MAPK and PI3K/Akt pathways, thereby promoting neuronal survival and synaptic remodeling under stress conditions (Ayatollahi et al., 2020; Wang et al., 2017; Wong et al., 2012). In the present study, hippocampal and prefrontal BDNF and CREB1 expression exhibited a downward trend under chronic stress, consistent with impaired neurotrophin signaling and transcriptional regulation of synaptic genes. Mind Focus supplementation suggesting a restorative effect on BDNF and CREB1 expressions at control-like levels, suggesting a non-significant trend toward control-like values on neurotrophic and transcriptional homeostasis. However, these differences did not reach statistical significance and should therefore be interpreted as trends rather than demonstrated effects.

It should also be acknowledged that the qPCR panel comprised multiple stress- and neuroplasticity-related genes assessed across two brain regions, increasing the potential for false-positive findings despite post-hoc corrections. Accordingly, the molecular analyses were conducted in an exploratory framework. Genes showing consistent stress-related changes with clear statistical support—such as FOS and NMB in the hippocampus and BDNF in the prefrontal cortex—may be considered more robust findings, whereas non-significant or trend-level changes should be interpreted as hypothesis-generating and warrant validation in independent cohorts or targeted follow-up studies.

GRIN2A, a key NMDA receptor subunit critical for excitatory neurotransmission and synaptic plasticity, showed mild downregulation under stress with no significant differences between stress-induced and Mind Focus treated rats. This finding counteracts the stress-induced transcriptional dysregulationon glutamatergic activity mediated by L-theanine and citicoline, which have been shown to regulate glutamate–GABA balance and protect against excitotoxicity via NMDA receptor modulation (Chandrasekhar et al., 2017; Roohi-Azizi et al., 2018). On the other hand, the significant reduction in hippocampal GABRB1 expression observed in the stress group reflects weakened inhibitory GABAergic tone, a hallmark of chronic stress–induced hyperexcitability. The supplementation did not significantly restore but sustained hippocampal GABRB1 expression compared to the stress-induced rats, indicating that possible modulation of inhibitory signaling that may underlie its anxiolytic and mood-stabilizing behavioral effects observed in the open field, elevated plus maze, and forced swim tests. Collectively, these findings suggest that the combined formulation exerts neuroprotective and neuroregulatory actions by sustaining the expression of key genes governing neurotrophic support, excitatory–inhibitory balance, and synaptic adaptability. Through the combined effects of its bioactive constituents—including L-theanine’s glutamatergic modulation, phosphatidylserine’s membrane-stabilizing role, citicoline’s enhancement of phospholipid turnover, and Lion’s Mane mushroom extract and Rhodiola rosea’s neurotrophic actions—the supplement appears to mitigate stress-induced transcriptional dysregulation within hippocampal and prefrontal networks, thereby promoting neural resilience, exploratory and emotional stability.

Despite the comprehensive multimodal design integrating behavioral, biochemical, histopathological, and molecular endpoints, several methodological limitations should be acknowledged. First, the use of a single dose and treatment duration (130 mg/kg/day for 30 days) restricts the ability to establish dose–response relationships and optimal treatment windows for Mind Focus. In addition, because only the combined formulation was evaluated, the study cannot determine the relative contribution of individual ingredients or potential synergistic interactions. Another limitation is that only male Wistar rats were included. Sex differences in stress responsivity, neuroinflammatory signaling, and behavioral responses to psychotropic and nutraceutical interventions are well documented. Therefore, the present findings may not fully generalize to females, and future studies should explicitly examine potential sex-dependent effects of Mind Focus supplementation using factorial designs (single-ingredient arms and their combinations) or head-to-head comparisons which employ multiple dosage regimens and post-stress intervention models to dissect component-specific versus synergistic effects. Second, brain tissues were snap-frozen at –80°C prior to fixation for histological analysis. While this approach preserved molecular integrity for gene expression assays, it may have introduced freezing artifacts such as vacuolation, perineuronal halo formation, or dark neuron appearance, potentially confounding morphological interpretation. Although freezing-related artifacts such as vacuolization, perineuronal halos, and dark neuron appearance may affect absolute morphological interpretation, all experimental groups were processed using identical tissue handling and blinded scoring procedures; therefore, relative differences in histopathological injury scores between groups remain informative. For more reliable neuropathological evaluation, future studies should prioritize immediate perfusion or immersion fixation and complement hematoxylin–eosin staining with neurodegeneration and synaptic integrity markers (e.g., NeuN, Fluoro-Jade C, cleaved caspase-3, synaptophysin, MAP2). Third, behavioral assessments were limited to validated paradigms (SPT, OFT, EPM, and FST), which primarily reflect affective and exploratory dimensions. Although these tests collectively capture stress-induced behavioral alterations, they do not provide direct measures of attention, learning, or executive function. Accordingly, any interpretation of potential effects on focus or attentional performance should be considered hypothesis-generating rather than demonstrated outcomes. Future studies incorporating dedicated cognitive paradigms such as novel object recognition, attentional set-shifting, or the five-choice serial reaction time task would be required to directly assess these domains. However, stress and focus are linked in a “sweet spot” way: a little or moderate stress can sharpen attention, but too much or long-lasting stress tends to scatter it and harm concentration. The type (acute vs. chronic), intensity, and duration of stress all matter (Liu et al., 2020). Acute high stress can disrupt top-down control from the prefrontal cortex, making attention more driven by sudden, emotionally salient stimuli instead of task goals, which increases distractibility (Shields et al., 2016). Experiments show that people under acute stress make more errors on attention tasks that require ignoring distractions and are more likely to mind wander and understand material less well (Sänger et al., 2014). Excessive or prolonged stress, especially with strong anxiety and high cortisol, usually impairs focus, learning, and complex thinking (Liu et al., 2020). In light of these findings, it is thought that Kiperin Mind Focus reduces stress or increases stress tolerance, thereby limiting the negative effects of excessive stress on attention and executive functions. Thanks to this indirect effect, it is anticipated that concentration, focus and cognitive performance can be supported. Fourth, although transcriptional data indicated modulation of key neuroplasticity and stress-related genes, protein-level validation (e.g., Western blot or immunohistochemistry for BDNF, CREB, or GABRB1) was not performed. Consequently, post-transcriptional or translational regulation cannot be excluded. Similarly, neurotransmitter quantification (dopamine, serotonin, glutamate, GABA) and hormonal profiling (corticosterone) were not included, limiting mechanistic linkage between gene expression and functional outcomes. Finally, the study duration captured only short-term neurobehavioral and molecular outcomes following supplementation. Longitudinal investigations incorporating post-treatment withdrawal periods, electrophysiological recordings, and advanced neuroimaging (e.g., MRI volumetry, PET-based neuroinflammation markers) would further clarify the persistence and clinical relevance of Mind Focus-induced neuroprotection.

This study provides comprehensive evidence that Mind Focus supplementation exerts multifaceted neuroprotective, anti-inflammatory, and stress-buffering effects in rats exposed to CUMS. Across behavioral paradigms, supplementation significantly ameliorated stress-induced anhedonia, anxiety, and behavioral despair, as demonstrated by improved performance in the sucrose preference, open field, elevated plus maze, and forced swim tests. These behavioral outcomes indicate that the combined formulation enhances emotional stability, exploratory motivation, and stress resilience under chronic stress conditions.

Biochemically, chronic stress induced robust systemic and neuroinflammatory activation, reflected by elevated IL-1β, IL-6, IL-10, and TNF-α levels, along with increased oxidative stress index (TOS/TAS ratio). Mind Focus—particularly in its SMF—effectively normalized cytokine and oxidative parameters, supporting robust anti-inflammatory and antioxidant capacities. Histopathological analyses further confirmed structural neuroprotection, with treated groups exhibiting preserved neuronal morphology and significantly lower injury scores in both the hippocampus and prefrontal cortex compared to stressed animals.

At the molecular level, Mind Focus was associated with attenuation of CUMS-induced upregulation of FOS and NMB, while other neuroplasticity-related genes (DBH, BDNF, CREB1, GRIN2A, and GABRB1) showed no statistically significant differences across groups, despite mean expression values in treated animals being numerically closer to control levels. Collectively, these transcriptional patterns are consistent with the possibility that the combined formulation may contribute to limiting stress-related neuronal hyperactivation and peptidergic dysregulation, while exerting modulatory effects on neurotrophic and inhibitory signaling pathways within corticolimbic circuits under chronic stress conditions.

Together, these effects converge to sustain synaptic integrity, maintain redox and neurotransmitter balance, and protect neural circuits from chronic stress–induced degeneration. In summary, Mind Focus acts a neuroprotective, mood-stabilizing, and stress-resilience–promoting supplement that preserves neuronal structure and function under chronic stress conditions. These findings highlight its potential relevance as a nutraceutical strategy for mitigating the neurobehavioral consequences of sustained psychological stress and supporting stress adaptation and emotional regulation, and mental wellbeing. It is believed that Kiperin Mind Focus may indirectly support attention, concentration and focus by reducing stress or increasing stress tolerance. Future research should extend these findings through longitudinal and translational studies exploring dose–response relationships, sex-dependent differences, and the long-term neurobehavioral effects of the supplementation. Integrating advanced imaging, electrophysiological, and proteomic approaches will be crucial to delineate the cellular and circuit-level mechanisms underlying its neuroprotective actions. Moreover, clinical trials in human populations experiencing chronic psychological stress, burnout, or stress-related disorders could clarify its therapeutic potential as a nutraceutical intervention for enhancing stress resilience, emotional resilience, and behavioral stability under stress.