Abstract

Background:

Chronic stress is known to impair emotional regulation and adaptive behavioral responses through neuroinflammatory activation, oxidative imbalance, and dysregulation of neuroplasticity-related genes. Kiperin Mind Focus, a nootropic nutraceutical containing L-theanine, citicoline, phosphatidylserine, Rhodiola rosea, Ginkgo biloba, caffeine, and Lion’s Mane mushroom extract has been formulated to support stress resilience, mood regulation and neural health. This study aimed to investigate the neuroprotective and neuroregulatory effects of the combined formulation on behavioral, biochemical, histopathological, and molecular parameters in rats exposed to chronic unpredictable mild stress (CUMS).

Methods:

Thirty-two adult male Wistar rats were randomized into four groups (n = 8): Control, Stress, Kiperin Mind Focus (MF), and Stress + Mind Focus (SMF). CUMS was applied for 45 days, and the combined formulation was administered by oral gavage (130 mg/kg/day). Behavioral outcomes were evaluated using the sucrose preference (SPT), open field (OFT), elevated plus maze (EPM), and forced swim (FST) tests. Serum and tissue cytokine levels (IL-1β, IL-6, IL-10, TNF-α) and oxidative stress index (TOS/TAS ratio) were measured. Hippocampal and prefrontal gene expression of FOS, DBH, NMB, BDNF, CREB1, GRIN2A, and GABRB1 was assessed via qPCR, and histopathological changes were semi-quantitatively scored.

Results:

Chronic stress induced anhedonia, anxiety-like behavior, and behavioral despair, accompanied by elevated proinflammatory cytokines, oxidative imbalance, and neuronal degeneration in the hippocampus and prefrontal cortex. The supplementation significantly improved SPT, OFT, EPM, and FST performance, normalized cytokine and oxidative parameters, and reduced neuronal injury scores. At the molecular level, supplementation attenuated stress-induced upregulation of FOS, DBH, and NMB while maintaining neurotrophic (BDNF, CREB1) and GABAergic (GABRB1) expression near control levels.

Conclusion:

Kiperin Mind Focus exerted robust neuroprotective, anti-inflammatory, and antioxidant effects under chronic stress, restoring molecular homeostasis and stabilizing stress-related behavioral outcomes. These findings support its role as a stress-buffering and mood-stabilizing supplement, that promotes emotional regulation and adaptive exploratory behavior under prolonged stress conditions.

Introduction

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.

Materials and methodsEthical approvals and animals

Thirty-two male Wistar albino rats, 3 months old, weighing 300–350 g were procured from Bezmialem Vakif University Experimental Animal Research Center. The animals were housed in groups of three to four per polycarbonate cage under standard laboratory conditions, with a controlled 12-h light/dark cycle, ambient temperature of 22 ± 1°C, and relative humidity maintained at approximately 60%. The rats were provided with ad libitum food and water. All experimental procedures complied with The National and Institutional guidelines for the ethical treatment of laboratory animals. The study protocol was approved by the Laboratory Animals Ethical Committee of Bezmialem Vakif University (No: 2025-8, Date: 26th Feb 2025). All animal experiments were conducted at the Bezmialem Vakif University Experimental Animal Research Center. Biochemical and molecular analyses were performed at Biruni University Research Center.

Chemicals and experimental designChemicals

Mind Focus (Kiperin Pharmaceutical and Food Industry Ltd., İstanbul, Türkiye) contains the following active ingredients per capsule: L-Theanine (150 mg), Lion’s Mane mushroom extract (150 mg), Citicoline (125 mg), Phosphatidylserine (100 mg), Rhodiola extract (75 mg), Caffeine (75 mg), and Ginkgo biloba extract (45 mg). The rat dose was calculated based on the approved human daily dose of 1,440 mg for a 70-kg adult (equivalent to 20.6 mg/kg), as indicated by the Turkish Ministry of Agriculture and Forestry (Janhavi et al., 2019). Dose conversion from human to rat was performed using body surface area (BSA) normalization according to the Reagan–Shaw Km factor method, implemented via the DoseCal virtual dose conversion tool1 (Janhavi et al., 2019). Accordingly, the animal equivalent dose was calculated using the formula: Animal dose (mg/kg) = Human equivalent dose (mg/kg) × (Human Km / Rat Km), where Km values were 37 for humans and 6 for rats. This calculation yielded a rat-equivalent dose of approximately 127 mg/kg, which was rounded to 130 mg/kg. Mind Focus was therefore administered at 130 mg/kg/day, dissolved in 1 mL saline, by oral gavage for 30 days.

The total duration of the experiment was 45 days. Prior to the experiment, body weights were measured, and baseline sucrose preference tests were performed to assess hedonic status and ensure balanced group allocation. Rats were randomly assigned to four experimental groups (n = 8 per group):

Control group: The animals were housed under standard conditions with unrestricted access to food and water for 45 days without any stress. Subsequently, 1 ml of saline was administered by oral gavage for 30 days.

Stress group (CUMS): The animals were exposed to the CUMS model for 45 days. During this period, 1 ml of saline was administered by oral gavage for 30 days.

Mind Focus group: During this period, 130 mg/kg combined formulation, dissolved in 1 ml of saline, was administered by oral gavage for 30 days.

Stress + Mind Focus group: The animals were exposed to the CUMS model for 45 days. During this period, 130 mg/kg Mind Focus, dissolved in 1 ml of saline, was administered by oral gavage for 30 days.

Chronic unpredictable mild stress protocol

The rats in stress groups were chronically exposed to various randomly scheduled stressors for 45 days adapted from of the procedure (Table 1; Willner et al., 2019). CUMS was applied daily from days 1 to 45. Mind Focus or saline administration by oral gavage was initiated on day 15 and continued for 30 consecutive days, overlapping with the latter phase of the CUMS protocol. Control rats were housed in a separate room to avoid indirect exposure to stress cues and were handled only for cage cleaning, weighing, and feeding. During stressor application (except for light disruption), animals were monitored every 30 min for signs of distress such as shivering, immobility, or lethargy. Animals showing atypical signs or injuries were removed from the stress protocol and evaluated by the attending veterinarian.

StressorCUMS daysHot plate (45°C, 5 min)16, 28Cold plate (5°C, 5 min)6, 22, 40Wet bedding (24 h)14, 34, 41Restraint stress (4 h)7, 21, 35Social isolation (24 h)8, 19, 36Cage tilting (45°, 24 h)13, 25, 37Tail cramping (1 min)3, 15, 26, 32Crowded housing (24 h)9, 20, 38Different cage (24 h)10, 27, 39Food deprivation (24 h)1, 11, 29Water deprivation (24 h)2, 12, 18, 33Reversal of day (24 h)4, 24, 30Reversal of night (24 h)5, 17, 23, 31

Chronic unpredictable mild stress procedure.

Behavioral assessments (SPT, OFT, EPM, and FST) were conducted during the final week of the experiment. All animals were euthanized on Day 45, and blood and brain tissues were collected at the experimental endpoint for biochemical, molecular, and histopathological analyses. A schematic representation of the experimental timeline is provided in Figure 1.

Experimental timeline of chronic unpredictable mild stress (CUMS) exposure and Mind Focus supplementation. Animals were randomized at baseline (day 0). CUMS was applied from days 1 to 45 in the Stress and Stress + Mind Focus groups. Mind Focus or saline was administered by oral gavage from days 15 to 45. Behavioral tests (SPT, OFT, EPM, and FST) were conducted during the final week, followed by euthanasia and tissue collection at day 45.

Behavioral testsSucrose preference test

The sucrose preference test (SPT) was conducted three times throughout the experiment. Initially, it served to establish the basal anhedonia levels of the animals. Subsequently, tests were performed on the 13th day and at the conclusion of the experiment to verify anhedonia persistence. Before the first test, the animals were individually placed in single cages and accustomed to 1% sucrose solution in a quiet environment. Two bottles of 1% sucrose solution were initially placed in each cage. After 24 h, one sucrose bottle was replaced with drinking water. After the 24-h adaptation period, the animals were subjected to 24-h water and food deprivation. The next day, each cage received two bottles: one with 1% sucrose solution and the other with drinking water. Bottle positions were randomized after 1 h. At the end of 24 h, the weights of consumed sucrose solution and drinking water were recorded. The sucrose preference ratio (%) was calculated using the formula described in previous studies (Fonseca-Rodrigues et al., 2022; Hu et al., 2017).

Open field test

Locomotor activity and exploratory behavior were measured in the open field test (OFT) (Zhang et al., 2020). The test apparatus had a square base (91 × 91 cm), was painted black, and had high walls (40 cm) to prevent the animal from climbing. The test apparatus was divided into a square-shaped middle area (20 × 20 cm) using the EthoVision-XT program. Following 5 min habituation, exploratory behavior was recorded for 5 min using an automated video tracking system (Noldus Information Technology, EthoVision System, Netherlands). The open-field apparatus was rinsed between sessions with 75% alcohol and dried with a towel to prevent any odor clues. During the 5-min session, the total distance traveled, time spent in the center area, transition frequency to the center area, locomotion latency, the frequency of rearing, and the time and frequency of grooming were measured. When evaluating the time spent in the center area, the presence of at least three extremities of rats was determined as a criterion.

Elevated plus maze

Anxiety-related exploration was assessed using the elevated plus-maze (EPM) by File and co-workers (Pellow et al., 1985). The test apparatus consists of a black wooden platform in the shape of a plus, consisting of two opposite open (50 × 10 × 40 cm) and two closed (50 × 10 × 40 cm) arms. The apparatus was elevated to a height of 50 cm above the floor. At the beginning of the experiment, each rat was placed in the middle compartment (5 × 5 cm) between the open and closed arms with the head facing toward an open arm and allowed to explore for 5 min. The maze was rinsed between sessions with 75% alcohol and dried with a towel to prevent any odor clues. During the experiment, the total time spent in the open arms (%) and closed arms (%) and entries into open arms (%) were recorded with the EthoVision-XT program (Noldus Information Technology, EthoVision System, Netherlands).

Forced swimming test

The forced swimming test (FST), also known as the behavioral despair test, focuses on a rodent’s response to the threat of drowning (Lino-de-Oliveira et al., 2005). Rats were forced to swim in a see-through Plexiglas cylinder that is 45 cm tall and 15 cm in diameter, filled with water (23–27°C, 35 cm deep) for 5 min. The entire experimental procedure was recorded with a camera. During the following 5 min, the duration of immobility time and latency to immobility for every rat were measured by two observers who were blinded to the kind of treatment.

Biochemical analysesSample collections and homogenization

Blood samples were collected at baseline and at the end of the experiment. Serum was separated by centrifugation (3,000 rpm, 10 min) and stored at -80°C until analysis.

Liver, hippocampus and prefrontal cortex tissues were first washed in 0.9% NaCl and each tissue (∼50 mg) samples were placed on ice and transferred into a 2 mL screw-cap tube containing sterile zirconium beads (2.8 mm) and 1.0 mL of cold phosphate buffer (100 mM KH2PO4–K2HPO4, pH 7.4, containing 1.15% KCl) were added (w/v ∼1:10). Homogenization was performed using a bead−mill homogenizer with the following program: 5 m/s speed, 30 s per cycle, 3 cycles, with 30 s cooling on ice between cycles. After homogenization, the tubes were centrifuged at 10.000 × g for 10 min at 4°C and the supernatant collected for downstream analyses (Liang et al., 2011). Total protein concentration in the cleared homogenates was measured using the Bradford assay according to the method described by Bradford (1976).

Measurement of inflammatory cytokines

Frozen serum and tissue homogenates samples were thawed, and IL-6 (Cat No: ER0042), IL-10 (Cat No: ER0033), IL-1β (Cat No: ER1094) and TNF-α (Cat No: ER1393) levels were measured using commercial ELISA kits according to the manufacturers’ instructions. Briefly, samples and standards were added to wells which are pre-coated with related monoclonal antibodies. Biotin was added to all wells and combined with Streptavidin-HRP. The samples were incubated and washed for removing the uncombined enzymes. To stop the reaction, acid was added to the plates and optical density was measured at 450 nm via microplate reader (Thermo Scientific Microplate Reader, United States). The detection range of kits were between 15.625 and 2,000 pg/mL for IL-1β and IL-10, 31.25–4,000 pg/mL for IL-6, and 1.95–250 pg/mL for TNF-α.

Measurement of total antioxidant status and total oxidant status

Serum and tissues total TAS and TOS were determined with commercial kits (Rel Assay Diagnostics kit; Mega Tıp, Gaziantep, Türkiye) (Solakoglu et al., 2017). Additionally oxidative stress index (OSI) values were calculated according to the following formula: OSI = (TOS (μmol ⋅ H2O2 equivalent/g protein) / TAS (mmol.Trolox equivalent/g protein) X 100 (Erel, 2005). Briefly; TAS and TOS were measured in serum samples and tissue homogenates at wavelengths of 240 and 520 nm, respectively, using a plate reader (Thermo Scientific Multiskan FC, 2011-06, United States). For TAS measurements, Trolox, a water-soluble compound of vitamin E was used as a calibrator and the results were expressed as mmol. Trolox equivalent/L. For TOS measurements, H2O2 was used as the standard, and the results were expressed as μmol ⋅ H2O2 equivalent/L. OSI values were expressed as Arbitrary Units.

Histopathology

From each animal, the hippocampus and prefrontal cortex were sampled, snap-frozen at –80°C and, at the time of histopathology, fixed in 10% neutral-buffered formalin. Tissues underwent routine processing (graded ethanol dehydration, xylene clearing, paraffin embedding) (Bancroft and Gamble, 2008); 5-μm paraffin sections were cut, mounted, deparaffinized/rehydrated, stained with hematoxylin–eosin (H&E), dehydrated, cleared, and coverslipped. Bright-field images were acquired at 20 × under constant exposure/white balance. Evaluation was blinded. A fixed, centrally placed square region of interest (ROI) of identical pixel size across images (calibrated at 20 × ) was analyzed per section. Exactly 20 pyramidal neurons within the ROI were inspected; k denotes the number that met “injured” criteria (red-neuron morphology: shrunken/triangular soma with hypereosinophilic cytoplasm and pyknotic nucleus; or ghost/shadow neuron: pale soma with no discernible nucleus). Perineuronal halo/vacuolization was visually estimated as percentage of the ROI. A 0–3 ordinal score was assigned as follows: 0 if k ≤ 1 and halo < 15%; 1 if k ≥ 2 and/or halo 15–30%; 2 if k = 5–9 and/or halo 30–60%; 3 if k ≥ 10 and/or halo > 60%.

RNA isolation and relative expression levels evaluated by qPCR

Brain tissue samples were washed with PBS and stored at –80°C for RNA isolation. The samples were physically broken down by using a mortar and pestle and crushed in liquid nitrogen. Subsequently, samples were resuspended with TRIzol Reagent (Invitrogen, United States), and the total RNA isolation was performed according to the manufacturer’s protocol. Concentration and purity of the isolated RNA were assessed using a NanoDrop ND-2000c spectrophotometer (Thermo Fisher Scientific Inc., United States).

The relative expression levels of FOS, DBH, NMB, and GRIN2A were evaluated using quantitative real-time polymerase chain reaction (qPCR). cDNA synthesis was performed using 1,000 ng of total RNA with the OneScript® Plus cDNA Synthesis Kit [Applied Biological Materials Inc. (Abm), Canada] according to the manufacturer’s instructions, in a T100 thermal cycler PCR machine (Bio-Rad, Singapore). qPCR experiments were conducted in duplicate using the BlasTaq™ 2X qPCR Master Mix (Abm, Canada) and the LightCycler 480 instrument (Roche, Germany). GAPDH was used as the endogenous control for normalization of gene expression. The primers used were as follows: for FOS, forward 5’- TACTACCATTCCCCAGCCGA-3’and reverse 5’-GCTGTCACCGTGGGGATAAA-3’; for DBH, forward 5’-CCTTCCCCATGTTCAACGGA-3’ and reverse 5’- ACCGGCTTCTTCTGGGTAG-3’; for NMB, forward 5’- CCCAGAGGGAGCAGAGACTA-3’ and reverse 5’-TGGACCACTGAGGTTCATGC-3’; for GRIN2A, forward 5’- CCCAGGCTTGTGGTGATCGT-3’ and reverse 5’-CGAAG GGGGCTTCCTCCAAG-3’ for GAPDH (housekeeping gene), forward 5’-TTCACCACCATGGAGAAGGC-3’ and reverse 5’-CTCGTGGTTCACACCCATCA-3’. Relative gene expression levels were calculated using the 2–ΔΔCt method. All reactions included no-template controls to assess contamination and nonspecific amplification.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, United States) and R version 4.5.1 with the ggplot2 (v4.0.0), FSA (v0.10.0), ggsignif (v0.6.4), and effsize (v0.8.1) packages. Data distribution was assessed for normality using the Kolmogorov–Smirnov test. For normally distributed data, comparisons among the four independent experimental groups were conducted using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison post hoc test. For non-normally distributed data, the Kruskal–Wallis test with Dunn’s post hoc analysis and Holm correction was applied. Descriptive statistics are presented as mean ± standard deviation (SD).

No formal statistical outlier detection tests (e.g., Grubbs’ test or ROUT method) were applied to behavioral, biochemical, or gene expression datasets. All data points obtained from individual animals were retained and included in the final analyses. To account for potential variability and small group sizes, data distribution and variance assumptions were evaluated using Brown–Forsythe and Bartlett’s tests, and Welch’s correction was applied when appropriate. Non-parametric tests were used where distributional assumptions were not met. Analyses were therefore conducted without exclusion of extreme values, ensuring that the reported results reflect the full biological variability of the experimental groups. A p < 0.05 was considered statistically significant for all analyses. Given the number of molecular endpoints assessed across multiple tissues, the gene expression analyses were considered exploratory in nature, and results—particularly those with marginal p-values—should be interpreted with caution due to the increased risk of type I error associated with multiple comparisons.

ResultsBody weight

Throughout the experimental period, body weight was regularly monitored (Figure 2). Body weights of all groups significantly increased during the 7-week experimental period (p < 0.001). In contrast, treatment with MF either alone or in combination with stress, maintained body weights comparable to controls, indicating that supplementation may mitigate stress-induced weight loss.

Changes in body weight over the experimental period. Weekly body weight measurements of rats in the control (C), stress (S), Mind Focus (MF), and stress + Mind Focus (SMF) groups throughout the 9-week experimental period. Data is presented as mean ± SD.

Sucrose preference test

The sucrose preference test was performed to assess anhedonic behavior. Rats exposed to the CUMS protocol displayed a significant reduction in sucrose preference compared to controls (p < 0.01) at the 13th day of the CUMS period, indicating the development of anhedonia (Figure 3A). Sucrose preference significantly decreased in the S (p < 0.001) and SMF (p < 0.05) groups than in the controls and significantly increased in the MF group compared to the S group (p < 0.001) (Figure 3B).

Sucrose preference test results. (A) Sucrose preference percentage on day 15 and (B) on the last day of the experiment in control (C), stress (S), Mind Focus (MF), and stress + Mind Focus (SMF) groups. Data are presented as mean ± SD. *p < 0.05, ***p < 0.001 compared to control group; +p < 0.05, +++p < 0.001 compared to S group.

Open field test

Open field test was performed to measure animals’ anxiety levels and locomotor activity. Total distance was significantly increased in the S (p < 0.05), MF (p < 0.001) and SMF (p < 0.05) groups compared to the controls (Figure 4A). Similarly, time spent in the center area was significantly increased in the SMF (p < 0.05), MF (p < 0.001) and SMF (p < 0.001) groups than in the controls (Figure 4B). The lowest values of velocity (p < 0.01) (Figure 4C) and transition frequency to the center area (p < 0.05) (Figure 4D) were detected in the MF group compared to the controls.

Open field test results. (A) Total distance traveled (cm), (B) time spent in the center zone (s), (C) velocity (cm/s), (D) transition frequency to the center zone (E) rearing time (s), (F) grooming time (s), and (G) movement latency (s) in the control (C), stress (S), Mind Focus (MF), and stress + Mind Focus (SMF) groups. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control group; +p < 0.1 compared to S group.

Rearing time significantly decreased in the S group (p < 0.001) than in the control group (Figure 4E). Additionally, grooming time of animals significantly decreased in the MF group (p < 0.05) than in the S group (Figure 4F). Lastly, movement latency of animals significantly increased in the S group (p < 0.05) compared to the controls (Figure 4G).

Elevated plus maze

In the EPM, time spent in open arms of animals significantly decreased (p < 0.05) in the S group compared to the controls and significantly increased (p < 0.01) in the MF group than in the S group (Figure 5A). To support this finding, time spent in closed arms of animals significantly increased in the S group (p < 0.01) than in the control group and significantly decreased (p’s < 0.05) in MF and SMF groups compared to the S group (Figure 5B). Time spent in the center areas significantly increased (p’s < 0.05) in both MF and SMF groups than in the S group (Figure 5C). Lastly, entries into open arms significantly decreased (p < 0.05) in the S group compared to the controls and significantly increased (p < 0.01) in the SMF group than in the S group (Figure 5D).

Elevated Plus Maze test results. (A) Percentage of time spent in the open arms, (B) percentage of time spent in the closed arms, (C) percentage of time spent in the center area, and (D) percentage of entries into the open arms in the control (C), stress (S), Mind Focus (MF), and stress + Mind Focus (SMF) groups. Data are presented as mean ± SD. *p < 0.05, **p < 0.01 compared to control group; +p < 0.05, ++p < 0.01 compared to S group.

Forced swim test

In the forced swim test, rats subjected to chronic stress exhibited a significant increase in immobility duration compared to controls (p < 0.01), indicating a depression-like phenotype. Rats in the MF group showed significantly shorter immobility times compared to S group (p < 0.05), suggesting an antidepressant-like effect of the supplement under chronic stress conditions (Figure 6).

Forced Swim Test results. Immobility time (s) during the Forced Swim Test in the control (C), stress (S), Mind Focus (MF), and stress + Mind Focus (SMF) groups. Data are presented as mean ± SD. ***p < 0.001 compared to control group; +p < 0.05 compared to S group.

Quantitative levels of inflammation biomarkers

Quantitative assessment of proinflammatory and anti-inflammatory cytokines revealed distinct systemic and regional patterns across the experimental groups (Figure 7). Chronic stress markedly elevated IL-1β levels in the liver, serum, and prefrontal cortex compared with the control group (p < 0.0001), indicating pronounced systemic and neuroinflammatory activation. In contrast, hippocampal IL-1β levels showed a mild but non-significant increase. Mind Focus treatment effectively mitigated these stress-induced elevations. Both MF and SMF significantly reduced hepatic and serum IL-1β levels compared with the stress group (p < 0.05 for all), while maintaining values comparable to controls. In the prefrontal cortex, MF treatment normalized IL-1β expression to near-control levels, and SMF administration also significantly attenuated the stress-induced increase (p < 0.001).

Quantitative levels of inflammation markers IL-1β, IL-6, IL-10 and TNF-α in serum, liver, prefrontal cortex and hippocampus. C, control group; S, stress group; MF, Mind Focus group; SMF, Stress+ Mind Focus group; *p < 0.05, **p < 0.005, ***p < 0.0005, and **** p < 0.0001 vs. control group; + p < 0.05, ++ p < 0.005, +++p < 0.0005 and ++++ p < 0.0001 vs. stress group.

Similar to IL-1β, IL-6 concentrations were significantly increased in the liver and serum of stressed animals (p < 0.0001), reflecting systemic inflammatory activation (Figure 7). However, IL-6 levels in the prefrontal cortex and hippocampus remained unchanged across groups (p > 0.05). Both MF and SMF treatments significantly reduced hepatic and serum IL-6 levels compared to the stress condition (p < 0.01), with the MF group showing slightly stronger normalization. These findings indicate that the supplement primarily mitigated peripheral IL-6 upregulation associated with chronic stress.

In peripheral tissues, IL-10 levels were also significantly elevated in the stress group (p < 0.0001), suggesting compensatory anti-inflammatory activation (Figure 7). MF administration maintained IL-10 concentrations comparable to controls, whereas SMF treatment resulted in a moderate but significant increase. Nevertheless, both treatment groups displayed markedly lower IL-10 levels than the stress group (p < 0.0001), indicating effective modulation of peripheral immune balance. In the prefrontal cortex and hippocampus, IL-10 levels remained stable without significant changes among groups (p > 0.05).

Chronic stress exposure significantly increased TNF-α levels in the liver, serum, and prefrontal cortex (p < 0.0001), while hippocampal concentrations were less affected (Figure 7). Mind Focus administration effectively attenuated this proinflammatory response. MF treatment maintained TNF-α levels comparable to controls across all tissues, whereas SMF showed mild but significant elevations relative to control values. However, both MF and SMF treatments produced significantly lower TNF-α levels than the stress group (p < 0.001), confirming a substantial anti-inflammatory effect.

Quantitative levels of oxidative stress indexes

OSI values in liver and serum revealed distinct alterations among the experimental groups (Figure 8). In both tissues, chronic stress produced a marked elevation in OSI compared with the control group (p < 0.0001), indicating a clear shift toward oxidative imbalance under stress conditions. MF treatment alone did not significantly differ from control values (p > 0.05) in either liver or serum, while SMF exposure yielded a notable decrease in OSI relative to the stress group (p < 0.0001). Both treatment groups exhibited substantially lower OSI values compared with the stress condition (p < 0.0001 for all comparisons). A significant difference between the MF and SMF groups was also observed in serum samples (p < 0.0001), whereas their hepatic OSI values remained comparable (p > 0.05).

Quantitative levels of oxidative stress indexes (TAS/TOS) in liver tissue (A) and serum (B) of the control (C), stress (S), Mind Focus (MF), and stress + Mind Focus (SMF) groups. ****p < 0.0001 vs. control group; ++++p < 0.0001 vs. stress group.

Histopathological findings

Figure 9 represents the micrographs of hippocampus and prefrontal cortex from control and stress induced groups. Control sections showed preserved neuronal morphology while the sections from stressed animals indicated