Neuroprotective Effects of Epicatechin against Oxidative Stress-Induced Cognitive Impairment: A Systematic Review and Meta-Analysis

Abstract

Oxidative stress is crucial in the pathogenesis of cognitive impairment and neurodegenerative diseases. Epicatechin, a natural flavanol abundant in cocoa, is a promising neuroprotective agent because of its antioxidant and anti-inflammatory properties. This meta-analysis aimed to evaluate the efficacy of epicatechin in mitigating oxidative stress-induced cognitive impairment in animal models. A systematic review of in vivo rodent studies was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, including studies reporting cognitive- and brain-based biochemical outcomes following epicatechin administration. The results indicated that epicatechin significantly improved the cognitive performance of rodents in the Morris water maze test, including improved spatial learning (reduced escape latency), memory retention (increased time spent in the target quadrant), and memory precision (increased island crossings). At the molecular level, epicatechin treatment increased the expression or activity of superoxide dismutase, catalase, and nuclear factor erythroid 2-related factor 2 and reduced the levels of nitric oxide, malondialdehyde, tumor necrosis factor-alpha, and interleukin-1 beta. These findings support the role of epicatechin in enhancing antioxidant defense and modulating neuroinflammation. Collectively, the results highlight that epicatechin has the therapeutic potential for preventing or mitigating oxidative stress-related cognitive dysfunction.

1. Introduction

Oxidative stress arises from an imbalance between reactive oxygen species (ROS) production and the capacity of the antioxidant defense system, ultimately leading to cellular damage. This process is a key pathological mechanism underlying neurodegenerative diseases and cognitive decline. The brain is particularly susceptible to oxidative stress because of its high oxygen consumption, abundant content of polyunsaturated fatty acids, and the limited regenerative capacity of neurons. , While endogenous antioxidant defense mechanisms, such as those mediated by superoxide dismutase (SOD) and glutathione, minimize cellular damage under normal conditions, their efficacy is often diminished under pathological conditions.

Oxidative stress exacerbates disease progression and symptom severity via multiple pathogenic mechanisms in various neurological disorders. In Alzheimer’s disease (AD), the accumulation of amyloid-β (Aβ) contributes to oxidative stress, promoting neuroinflammation and neuronal apoptosis, ultimately exacerbating disease symptoms. In Parkinson’s disease, oxidative stress-mediated damage to dopaminergic neurons is a crucial factor in disease onset and progression. Furthermore, in stroke and ischemic brain injury, large amounts of ROS are generated during the reperfusion phase following ischemia, inducing neuronal necrosis and inflammatory responses, which contribute to cognitive impairment. ,

Due to the detrimental effect of oxidative stress on brain function, modulating oxidative stress is a promising approach for preventing and treating neurological disorders. Among various therapeutic candidates, flavonoids have gained attention for their antioxidant and anti-inflammatory properties, primarily demonstrated in preclinical studies. , In particular, flavanols (flavan-3-ols), distinguished by their C3 hydroxyl (−OH) substitution, exhibit enhanced antioxidant and anti-inflammatory activities in vitro and in vivo. − Additionally, their moderate but favorable bioavailability makes them attractive candidates as therapeutic molecules. While their mechanisms are well described in preclinical models, their physiological relevance and consistency in studies with humans remain under investigation.

Recent studies have highlighted the neuroprotective potential of epicatechin, a major flavanol compound found in cocoa. Preclinical studies have shown that epicatechin scavenges ROS, reduces neuroinflammatory responses, and promotes neuronal survival, thereby supporting its potential as a natural neuroprotective agent. − Furthermore, epicatechin enhances neuroplasticity, potentially improving learning and memory functions. , In addition to these biological effects, epicatechin has attracted increasing interest due to its favorable pharmacokinetic profile, including relatively high oral bioavailability, metabolic stability, and blood–brain barrier permeability, as demonstrated in animal studies. ,

Despite these promising properties, no previous meta-analysis has systematically evaluated the cognitive effects of epicatechin monotherapy in animal models or clinical studies. Although cocoa-derived formulations containing epicatechin have been investigated in clinical trials, no study has isolated and assessed the specific effects of epicatechin on cognitive outcomes in humans. Moreover, inconsistencies remain across studies owing to variations in experimental methodologies, cognitive assessment criteria, and animal models. Therefore, this meta-analysis was conducted to address this gap in the literature and provide mechanistic insights into the therapeutic potential of epicatechin. By integrating findings across diverse experimental designs and model systems, this analysis sought to provide more robust and objective evidence supporting the therapeutic potential of epicatechin for cognitive dysfunction.

2. Materials and Methods

2.1. Literature Search Strategy

This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Supplementary Table 1) with the protocol registered in the International Prospective Register of Systematic Reviews (PROSPERO) under registration number CRD420250651154. PubMed, Embase, Cochrane Library, and Web of Science were systematically searched for studies published between February 2024 and January 20, 2025. The search query was as follows: (“Epicatechin” OR “Epicatechol” OR “epi-Catechin”) AND (“Cognitive impairment” OR “Cognitive function” OR “Cognitive enhancement” OR “Cognitive improvement” OR “Cognitive defect” OR “Cognitive dysfunction” OR “Mental performance” OR “Cognition” OR “Memory” OR “Neuroprotective” OR “Neuroinflammation” OR “Alzheimer’s disease” OR “Parkinson’s disease”). A more detailed list of the search terms is provided in Supplementary Table 2.

2.2. Inclusion and Exclusion Criteria

All studies retrieved through the literature search were imported into the Rayyan web tool. Duplicated papers were automatically removed using Rayyan’s built-in function, followed by manual screening of titles and abstracts by two independent reviewers to eliminate the remaining duplicates. The inclusion criteria were as follows: (1) in vivo studies using mice or rats, (2) administration of epicatechin as a single compound, (3) assessment of cognitive impairment along with oxidative stress or neuroinflammation measured in the brain tissue as experimental outcomes, and (4) no restrictions on the age or sex of the animals. Exclusion criteria included (1) unrelated topics; (2) inappropriate study design (e.g., studies on cognitive impairment due to natural aging were excluded, as aging involves multiple physiological factors, making it difficult to isolate the effects of epicatechin); and (3) nonoriginal publication type (e.g., review articles) and studies in which the type of outcome data (e.g., SD or SEM) was not clearly specified.

2.3. Data Extraction

Data were extracted by two independent reviewers. The extracted information included the year and country of publication, baseline conditions of the animals, animal weight, disease model, treatment conditions (including control group intervention), and duration of therapy. Additionally, we collected the outcome measures, mean values, measures of variability (e.g., SD or SEM), and p-values for the meta-analysis. For studies in which the exact numerical values were not reported, data were extracted from published figures using Fiji’s figure calibration plugin. These values were digitized and used for the meta-analysis.

2.4. Quality Assessment

The SYRCLE risk of bias tool was used to assess potential sources of bias and the methodological rigor of each included study. Two independent reviewers performed the assessments, and any disagreements were resolved through discussion with a third reviewer. This tool consists of ten domains designed to identify the risk of bias in animal experiments, including random sequence generation, baseline characteristics, allocation concealment, random housing, blinding of caregivers and outcome assessors, random outcome assessment, incomplete outcome data, selective outcome reporting, and other potential biases. Each domain was rated using predefined signaling questions, and judgments were categorized as “yes” (low risk of bias), “no” (high risk of bias), or “unclear” (insufficient information). These ratings contributed to the overall evaluation of the methodological quality across the studies. The certainty of evidence for each meta-analysis outcome was further assessed using the GRADE approach. As all included studies were randomized in vivo experiments, the initial rating was high. However, the final GRADE scores were downgraded based on risk of bias, inconsistency, indirectness, imprecision, and publication bias domains.

2.5. Meta-Analysis

This meta-analysis evaluated cognitive performance outcomes derived from the Morris water maze (MWM) test along with relevant biochemical markers. Variables reported in fewer than three comparisons were excluded from the analysis because of their limited statistical power. Studies were included if they reported within-group comparisons or provided baseline and postintervention data. Effect sizes were estimated by using a random-effects model based on the inverse variance method. The pooled standardized mean differences (SMDs) and corresponding 95% confidence intervals (CIs) were calculated by synthesizing the differences in the pre- and posttreatment change scores across studies. Review Manager version 5.4 (Nordic Cochrane Center, The Cochrane Collaboration) was used to compute the estimates, including SMDs, CIs, heterogeneity, and overall effect sizes. To assess the potential publication bias, funnel plots were generated and visually examined for asymmetry, which was interpreted as suggestive of a potential bias. Funnel plots were created using custom Python scripts implemented using the matplotlib library (Python 3.12.7) and executed in the Spyder IDE (5.5.1). Graphs were plotted by mapping the standardized effect sizes against their standard errors.

In studies that included multiple epicatechin treatment groups at different doses or time points, each treatment group and its corresponding control group were treated independently. Subgroup analyses were conducted to explore potential confounding factors, specifically stratified by epicatechin dosage. Dosages were categorized as low (<50 mg/kg body weight/day) or high (≥50 mg/kg body weight/day). To maintain an adequate statistical power, subgroup analyses were performed only when at least three comparisons were available for each subgroup.

To examine whether the observed variability in treatment effects could be explained by dose differences, meta-regression analyses were conducted using the Metafor package in R (version 4.5.1.). Standard errors were calculated by using the reported 95% confidence intervals. Following methodological recommendations, a meta-regression was performed for outcomes with at least ten comparisons to minimize the risk of false-positive findings.

3. Results

3.1. Study Selection

A total of 1290 articles were identified through a systematic search of four databases. After removing duplicates, 1046 studies remained. Screening of title and abstracts led to the exclusion of 1012 studies based on the prespecified exclusion criteria, leaving 34 articles for full-text assessment. Of these, 22 additional studies were excluded, resulting in 12 studies included in the final systematic review and meta-analysis. The detailed study selection process is shown in Figure .

1.

PRISMA flowchart of the study selection process (Created in BioRender. Cho, I. (2025) https://BioRender.com/t7w7k4j).

3.2. Characteristics of Included Studies

This systematic review and meta-analysis analyzed 12 in vivo animal studies published between 2009 and 2024. These studies were conducted across diverse geographical regions, including China, Mexico, the United States, Egypt, and Iran. Various rodent models have been employed, such as C57BL/6, , C57BL/6J, ICR, SPF Kunming, NMRI (Naval Medical Research Institute), and transgenic APP695/PS1-dE9 (APP/PS1) mice, as well as Sprague–Dawley and Wistar rats. −

The duration of epicatechin administration varied considerably, ranging from 1 day to 9 months. Most studies (n = 11) administered epicatechin orally, − whereas one study used intraperitoneal injections. The dosage regimens also differed widely from 1 to 200 mg/kg body weight/day.

The included studies modeled cognitive impairment resulting from diverse etiologies, including AD, ,, sepsis-associated encephalopathy, exposure to extremely low-frequency electromagnetic fields (ELF-EMF), lead-induced neurotoxicity, Gulf War Illness (GWI), traumatic brain injury (TBI), , high-fat diet (HFD)-induced obesity-related cognitive decline, global ischemia/reperfusion injury (I/R), and arsenic-induced neurotoxicity.

Various behavioral tests have been employed to assess cognitive function, including the MWM test, novel object recognition (NOR), eight-arm radial maze, water maze spatial task, wire hanging, forelimb placement, rotarod, forced swim, tail suspension, open field, and passive avoidance. However, owing to the limited number of studies utilizing assessments other than the MWM test, only data from the MWM were included in the quantitative analysis.

To investigate the potential mechanisms by which epicatechin alleviates cognitive deficits, we measured several biochemical markers that are broadly categorized into antioxidant defense systems, oxidative stress, and neuroinflammation domains. A detailed summary of the study characteristics and extracted data is given in Table .

1. Characteristics of the Studies Included in the Meta-Analysis .

year (reference)	country	animal	model	number (F/M)	duration	administration	dose (mg/kg body weight/day)	collected outcome
2024	China	Sprague–Dawley rats	traumatic brain injury	0/NA	7 days	oral	1.5	escape latency, Time in the spent target quadrant, Island crossings, swimming speed, IL-1β, TNF-α
2009	Mexico	Wistar rats	Alzheimer’s disease	0/98	single dose	oral	30	MDA
2019	Mexico	Wistar rats	Alzheimer’s disease	0/60	4 days	oral	200	escape latency, SOD, MDA, IL-1β, TNF-α
2024	USA	Wistar rats	Gulf War Illness	0/28	14 days	oral	1	MDA, IL-1β, TNF-α
2019	Egypt	Wistar rats	global ischemia/reperfusion injury	0/75	6 days	intraperitoneal	30	escape latency, Time spent in the target quadrant, NF-κB
2016	China	C57BL/6 mice	traumatic brain injury	0/16	28 days	oral	5, 15, 45	Nrf2
2022	China	C57BL/6 mice	sepsis-associated encephalopathy	0/45	3 doses (0.5, 24, 48 h)	oral	50	escape latency, Time spent in the target quadrant, Island crossings, swimming speed, SOD, MDA, IL-1β, TNF-α
2022	USA	C57BL/6J mice	high-fat diet-induced obesity	0/20	24 weeks	oral	2, 20	escape latency, time spent in the target quadrant
2017	China	ICR mice	brain injury induced by extremely low-frequency electromagnetic fields	0/90	30 days	oral	30, 60, 90	escape latency, time spent in the target quadrant, SOD, CAT, NO, MDA
2024	China	SPF Kunming mice	lead-induced cognitive impairment	0/32	10 weeks	oral	50	escape latency, Island crossings, swimming speed, SOD, CAT, Nrf2, MDA
2024	Iran	NMRI mice	arsenic-induced neurotoxicity	0/60	2 weeks	oral	25, 50, 100	SOD, CAT, Nrf2, NO, MDA, NF-κB, TNF-α
2016	China	APP/PS1 mice	Alzheimer’s disease	0/16	4 months	oral	50	Escape latency, SOD, CAT

^aAbbreviations: CAT, catalase; IL-1β, interleukin-1 beta; MDA, malondialdehyde; NF- κB, nuclear factor kappa B; Nrf2, nuclear factor erythroid 2-related factor 2; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-alpha.

^bNot available (NA): The number of animals used in each result is provided; however, the total number used across all experiments could not be determined due to incomplete reporting in the original study.

3.3. Effect of Epicatechin on Cognitive Impairment

The impact of epicatechin on cognitive function was evaluated across eight studies employing the MWM test, a widely used behavioral paradigm for assessing spatial learning and memory in rodent models. Four primary MWM test outcome measures were identified: escape latency, time spent in the target quadrant, island crossings (also termed platform crossings), and swimming speed. Escape latency reflects the time required for the subject to locate the hidden platform during the acquisition phase and serves as an indicator of spatial learning and search efficiency, with a shorter escape latency indicating better learning performance. Time spent in the target quadrant represents the proportion of time the participant spent in the quadrant where the platform was previously located during the probe trial; longer durations suggest enhanced retention of spatial memory. Island crossings assess how frequently a subject crosses the former platform location, thereby reflecting the precision of spatial memory retrieval, with higher frequencies interpreted as improved recall accuracy. In addition, the swimming speed was measured to account for potential differences in locomotor ability that could confound the interpretation of cognitive outcomes. The lack of significant differences in swimming speed between the groups helps to ensure that changes in escape latency or quadrant time reflect genuine cognitive effects rather than motor impairments.

Eleven comparisons were included in the meta-analysis of escape latency. − ,,,, Accordingly, both the control and epicatechin-treated groups consisted of 109 animals. The pooled SMD was −0.59 (95% CI: −0.97 to −0.21; Z = 3.03, p = 0.002, I ² = 44%) (Figure A). Subgroup analyses stratified by epicatechin dosage showed that high-dose epicatechin (≥50 mg/kg body weight/day) produced a larger effect size (SMD = −0.70, 95% CI: −1.23 to −0.16; Z = 2.56, p = 0.01, I ² = 53%) than low-dose (<50 mg/kg body weight/day) effect size (SMD = −0.44, 95% CI: −1.02 to 0.13; Z = 1.51, p = 0.13, I ² = 37%) (Supplementary Figure 1A). Nevertheless, subgroup differences were not statistically significant (Chi² = 0.40, p = 0.53, I ² = 0%).

2.

Meta-analysis results of Morris water maze test: (A) escape latency, (B) time spent in the target quadrant, (C) island crossings, and (D) swimming speed.

For the time spent in the target quadrant, 85 and 86 animals in the control and epicatechin-treated groups, respectively, were included in the analysis, based on a total of nine comparisons. − ,, The meta-analysis yielded a significant effect favoring epicatechin, with an SMD of 0.77 (95% CI: 0.11 to 1.42; Z = 2.28, p = 0.02, I ² = 73%) (Figure B). Subgroup analysis based on epicatechin dosage indicated that high-dose treatment (≥50 mg/kg body weight/day) resulted in a greater increase in time spent in target quadrant (SMD = 1.29, 95% CI: −0.07 to 2.51; Z = 2.07, p = 0.04, I ² = 82%) than low-dose treatment (<50 mg/kg body weight/day) (SMD = 0.43, 95% CI: −0.35 to 1.21; Z = 1.08, p = 0.28, I ² = 63%) (Supplementary Figure 1B). However, no statistically significant difference was observed between the low- and high-dose subgroups (Chi² = 1.35, p = 0.25, I ² = 25.9%).

For island crossings, 29 animals in the control group and 30 in the treatment group were analyzed, derived from three independent studies. ,, The pooled result also demonstrated a statistically significant increase in island crossings following epicatechin administration (SMD = 0.76, 95% CI: 0.03 to 1.49; Z = 2.05, p = 0.04, I ² = 38%) (Figure C).

Swimming speed was analyzed based on three studies, including a total of 60 animals included in the analysis, consisting of 30 in each of the control and epicatechin-treated groups. ,, The meta-analysis showed no statistically significant difference between the groups (SMD = 0.63, 95% CI: −0.66 to 1.93; Z = 0.96, p = 0.34, I ² = 79%) (Figure D). Due to the limited number of comparisons available, subgroup analyses were not performed for island crossings and swimming speeds.

3.4. Effect of Epicatechin on the Antioxidant Defense System

To evaluate the effects of epicatechin on the antioxidant mechanisms in the brain, this meta-analysis focused on three key molecular markers: SOD, catalase (CAT), and nuclear factor erythroid 2-related factor 2 (Nrf2). These markers were selected because of their well-established roles in neutralizing ROS and maintaining redox balance in neural tissue. Their activity and expression were measured by using brain tissue samples from the included studies, providing insights into the molecular-level antioxidant responses induced by epicatechin treatment.

SOD activity in brain tissue was evaluated in six studies, ,− with one study further distinguishing between the Zn-SOD and Mn-SOD subtypes. For meta-analysis, data from both subtypes were combined. A total of 83 animals were included in the control and epicatechin-treated groups. SOD catalyzes the dismutation of superoxide radicals into hydrogen peroxide, serving as a key component of the antioxidant defense system. The meta-analysis revealed a significantly higher SOD activity in epicatechin-treated groups than in the controls (SMD = 3.57, 95% CI: 2.12 to 5.02; Z = 4.83, p < 0.00001, I ² = 87%) (Figure A).

3.

Meta-analysis results of antioxidant defense system markers: (A) SOD (superoxide dismutase), (B) CAT (catalase), and (C) Nrf2 (nuclear factor erythroid 2-related factor 2).

CAT activity was assessed in four studies using brain tissue. − CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen, functioning as another key antioxidant enzyme implicated in neuroprotection. A total of 64 animals were included in the control and treatment groups. The meta-analysis showed a significantly increased CAT activity following epicatechin administration (SMD = 3.22, 95% CI: 1.52 to 4.92; Z = 3.71, p = 0.0002, I ² = 89%) (Figure B).

Nrf2 expression in brain tissue was examined in three studies. ,, Nrf2 regulates the expression of antioxidant and detoxifying genes, and its activation indicates an upregulated antioxidant system. In the study by Cheng et al. (2016), Nrf2 levels were reported separately in the cytosol and nucleus; for the meta-analysis, combined values were used. A total of 42 animals were assigned to the control and treatment groups. The meta-analysis showed a significant increase in Nrf2 expression in the epicatechin-treated group (SMD = 3.38, 95% CI: 1.00 to 5.75; Z = 2.79, p = 0.005, I ² = 89%) (Figure C). Due to the limited number of available comparisons, subgroup analyses were not performed for SOD, CAT, and Nrf2.

3.5. Effect of Epicatechin on Oxidative Stress

To evaluate the effects of epicatechin on oxidative stress in the brain, we focused on two commonly used molecular indicators, NO and MDA (malondialdehyde). , Both are widely recognized biomarkers that reflect oxidative damage at the cellular level. When overproduced, NO can be converted into reactive nitrogen species (RNS), contributing to neuronal toxicity. MDA, a byproduct of lipid peroxidation, serves as a key indicator of membrane damage caused by oxidative stress.

NO levels in the brain tissue were assessed in two studies, yielding six comparisons for the meta-analysis as a result of varying doses. , A total of 48 animals were included in the control and treatment groups. The meta-analysis revealed that NO concentrations were significantly reduced in the epicatechin-treated groups compared to those in the controls (SMD = −3.59, 95% CI: −5.33 to −1.85; Z = 4.04, p < 0.0001, I ² = 83%) (Figure A). A subgroup analysis was not performed for NO because of the limited number of available comparisons.

4.

Meta-analysis results of oxidative stress markers: (A) NO (nitric oxide) and (B) MDA (malondialdehyde).

MDA levels were assessed in seven studies, reflecting differences in both dosing and timing of measurement after epicatechin administration. ,− ,− ,− A total of 86 animals were included in the control and epicatechin-treated groups. MDA, a terminal product of lipid peroxidation, was measured using a thiobarbituric acid reactive substance (TBARS) assay, with increased levels indicating greater oxidative damage. The meta-analysis revealed a significant reduction in MDA levels in the epicatechin-treated groups compared to those in the controls (SMD = −2.70, 95% CI: −3.71 to −1.69; Z = 5.22, p < 0.0001, I ² = 78%) (Figure B). Subgroup analysis based on epicatechin dosage showed that treatment with a high dose (≥50 mg/kg body weight/day) was associated with a greater effect (SMD = −3.66, 95% CI: −5.23 to −2.09; Z = 4.57, p < 0.00001, I ² = 78%) than that with a low dose (<50 mg/kg body weight/day) (SMD = −1.73, 95% CI: −2.96 to −0.51; Z = 2.77, p = 0.006, I ² = 74%) (Supplementary Figure 2). However, the comparison between the low- and high-dose subgroups did not show a statistically significant difference (Chi² = 3.59, p = 0.06, I ² = 72.1%).

3.6. Effect of Epicatechin on Neuroinflammation

Neuroinflammation is essential in the progression of cognitive decline and neuronal damage. Among the key regulators of this process is NF-κB (nuclear factor kappa B), a transcription factor that promotes the expression of pro-inflammatory cytokines such as IL-1β (interleukin-1 beta) and TNF-α (tumor necrosis factor-alpha). Sustained activation of this pathway exacerbates neuroinflammatory responses that impair neuronal function and contribute to the pathogenesis of neurodegenerative disorders. In this meta-analysis, we investigated the effects of epicatechin on NF-κB signaling and its downstream inflammatory mediators, particularly IL-1β and TNF-α in brain tissue.

NF-κB levels in brain tissue were evaluated in two independent studies, comprising a total of four comparisons. , A total of 18 animals were included in each of the control and epicatechin-treated groups. Although NF-κB is a central transcription factor in neuroinflammatory signaling, the meta-analysis did not show a statistically significant effect of epicatechin on its expression (SMD = – 1.50, 95% CI: – 5.21 to 2.21; Z = 0.79, p = 0.43, I ² = 88%) (Figure A). This inconsistency may reflect variability under the experimental conditions or the limited number of available comparisons.

5.

Meta-analysis results of neuroinflammation markers: (A) NF-κB (nuclear factor kappa B), (B) IL-1β (interleukin-1 beta), and (C) TNF-α (tumor necrosis factor-alpha).

IL-1β levels in the brain tissue were assessed in four studies, including 23 animals in each of the control and epicatechin treatment groups. ,,, The meta-analysis showed a statistically significant reduction in IL-1β levels following epicatechin administration compared to those in controls (SMD = – 1.35, 95% CI: – 2.05 to −0.65; Z = 3.79, p = 0.0001, I ² = 0%) (Figure B). Subgroup analyses for NF-κB and IL-1β were not performed because of the small number of comparisons available.

TNF-α levels in the brain tissue were investigated across five studies comprising seven group comparisons, with 41 animals included in each of the control and treatment groups. ,,,, The pooled analysis revealed a significant reduction in TNF-α levels in the epicatechin-treated group compared with those in the controls (SMD = – 2.63, 95% CI: – 4.02 to −1.24; Z = 3.70, p = 0.0002, I ² = 75%) (Figure C). Following stratification based on epicatechin dosage, high-dose treatment (≥50 mg/kg body weight/day) demonstrated a greater reduction (SMD = −3.61, 95% CI: −6.47 to −0.76; Z = 2.48, p = 0.01, I ² = 84%) than low-dose treatment (<50 mg/kg body weight/day) (SMD = −2.17, 95% CI: −3.60 to −0.73; Z = 2.96, p = 0.003, I ² = 55%) (Supplementary Figure 3). However, the difference between subgroups was not statistically significant (Chi² = 0.79, p = 0.37, I ² = 0%).

3.7. Quality Assessment

The risk of bias was assessed using the SYRCLE tool, which evaluates internal validity across ten domains specific to in vivo studies. Table provides an overview of the risk of bias ratings across the ten domains assessed using the SYRCLE tool for each included study. Domains related to baseline characteristics, incomplete outcome data, and other sources of bias were judged to be at low risk. By contrast, the remaining domains, including sequence generation, allocation concealment, random housing, and blinding procedures, were rated as unclear because of insufficient reporting. These assessments revealed the risk of bias in the overall evaluation of the evidence quality.

2. SYRCLE’s Risk of Bias Tool for Animal Studies .

year (reference)	1	2	3	4	5	6	7	8	9	10
2024	U	Y	U	U	U	U	U	Y	U	Y
2009	U	Y	U	U	U	U	U	Y	U	Y
2019	U	Y	U	U	U	U	U	Y	U	Y
2024	U	Y	U	U	U	U	U	Y	U	Y
2019	U	Y	U	U	U	U	U	Y	U	Y
2016	U	Y	U	U	U	U	U	Y	U	Y
2022	U	Y	U	U	U	U	U	Y	U	Y
2022	U	Y	U	U	U	U	U	Y	U	Y
2017	U	Y	U	U	U	U	U	Y	U	Y
2024	U	Y	U	U	U	U	U	Y	U	Y
2024	U	Y	U	U	U	U	U	Y	U	Y
2016	U	Y	U	U	U	U	U	Y	U	Y

^aThe used domains include (1) sequence generation, (2) baseline characteristics, (3) allocation concealment, (4) random housing, (5) blinding of investigators, (6) random outcome assessment, (7) blinding of outcome assessors, (8) incomplete outcome data, (9) selective outcome reporting, and (10) other sources of bias. The risk of bias for each domain was rated as yes (Y, low risk) or unclear (U, insufficient information).

The quality of evidence for each outcome was assessed by using the GRADE approach. All outcomes were downgraded for indirectness owing to the exclusive reliance on animal models, which may limit the generalizability of the findings to the human population. Inconsistency was also a concern for most outcomes, as indicated by the substantial heterogeneity (I ²) observed across studies. Some outcomes were further downgraded for imprecision owing to the wide CI and small sample sizes. Finally, although formal statistical tests for publication bias could not be performed (no outcome included more than ten independent studies), visual inspection of the funnel plots revealed asymmetrical distributions for most outcomes, suggesting a potential risk of publication bias (Supplementary Figure 4).

4. Discussion

This meta-analysis provides comprehensive evidence that epicatechin exerts neuroprotective effects in various animal models of oxidative stress-induced cognitive impairment. Behavioral outcomes derived from the MWM test consistently indicated improved cognitive performance following epicatechin administration, including reduced escape latency, increased time spent in the target quadrant, and more frequent island crossings. Although the swimming speed has been assessed in only a few studies, it has shown no statistically significant changes, suggesting that improvements in spatial learning and memory are unlikely to be confounded by changes in locomotor function. The concurrent improvement in escape latency and target quadrant time, two outcomes of distinct motor demands, provides further support for the cognition-specific effects of epicatechin.

At the molecular level, these behavioral improvements were accompanied by modulation of biomarkers related to oxidative stress and neuroinflammation. Epicatechin significantly increased the activity of endogenous antioxidant enzymes, such as SOD and CAT, and upregulated Nrf2 expression, particularly in the nucleus, indicating activation of the antioxidant defense system. Concurrently, the levels of NO and MDA, indicators of oxidative damage, were significantly reduced, supporting the antioxidative role of epicatechin in preserving neuronal integrity. Inflammatory markers, including TNF-α and IL-1β, were also consistently reduced following epicatechin treatment, reinforcing its role in modulating neuroinflammatory pathways. However, the effect on NF-κB expression was inconsistent across studies, possibly due to differences in experimental designs, time points of measurement, or the possibility that epicatechin mediates anti-inflammatory effects through NF-κB-independent mechanisms.

Although these molecular and behavioral outcomes were assessed separately, their concurrent improvement suggests a potential mechanistic link, whereby the antioxidant effect of epicatechin contributes to the attenuation of neuroinflammation, ultimately resulting in enhanced cognitive function. Although direct causal links between these biological domains could not be confirmed from the available data, the pattern of results was consistent with this sequence of action and warrants further mechanistic investigation.

Subgroup analyses were performed to explore the potential dose–response effects of epicatechin. High-dose treatments (≥50 mg/kg body weight/day) were generally associated with stronger effects than those of low-dose treatments (<50 mg/kg body weight/day) in multiple outcomes, including escape latency, time spent in the target quadrant, and MDA and TNF-α levels. However, none of the subgroup comparisons reached statistical significance (all p > 0.05). This pattern suggests a potential trend toward increased efficacy at higher doses; however, the lack of statistical confirmation highlights this uncertainty. This may stem from the moderate-to-high heterogeneity observed across studies or the limited number of available comparisons, thus potentially reducing the statistical power and obscuring subgroup-level distinctions. For outcomes such as island crossings, swimming speed, and levels of specific biochemical markers (e.g., SOD, CAT, Nrf2, IL-1β, and NF-κB), the number of comparisons was insufficient to permit subgroup analysis.

To further explore potential dose–response relationships that may not have been captured through subgroup analysis, meta-regression analyses were conducted using dose as a continuous moderator. In line with standard recommendations, meta-regression was applied only to outcomes with at least ten comparisons, including escape latency, SOD, and MDA, to ensure sufficient statistical power and minimize the risk of false-positive findings. Among these, only the model for escape latency showed a marginal trend, with higher doses associated with greater effects (β = −0.0043, p = 0.0766), and residual heterogeneity reduced to zero (I ² = 0%, R ² = 100%) (Supplementary Figure 5A). This suggests that the dose may explain most of the variations between studies in this outcome. However, the lack of statistical significance indicates that this trend remains unclear. In contrast, the meta-regression models for SOD and MDA did not show any meaningful slope or improvement in heterogeneity, with wide confidence intervals and nonsignificant p-values (Supplementary Figure 5B,C). These results suggest no consistent evidence for dose responsiveness across outcomes, underscoring the need for a cautious interpretation of the dose–response relationship.

This meta-analysis had limitations. First, human clinical trials could not be included because of an insufficient number of eligible studies identified during the systematic search. Therefore, this analysis was limited to in vivo animal models, which may reduce the generalizability of the findings to human populations. Risk-of-bias assessments using the SYRCLE tool revealed that many studies failed to report key methodological details, including randomization, allocation concealment, and blinding. These reporting deficiencies resulted in many domains being rated as “unclear,” potentially compromising the internal validity of the findings. Moreover, high heterogeneity was observed in many of the pooled analyses, suggesting variability in the study design, animal models, or measurement techniques. Funnel plot asymmetry suggested potential publication bias, although formal tests (Egger’s regression and Begg’s rank tests) could not be conducted because of the small number of studies per outcome.

Although this meta-analysis focused exclusively on preclinical studies involving epicatechin monotherapy, related findings from the broader literature help contextualize our results. Currently, no clinical studies have directly investigated the cognitive effects of epicatechin administered as a single compound. However, several human trials using cocoa flavanol extracts containing high levels of epicatechin have reported improvements in cognitive performance (Table ). ,− These effects are generally attributed to vascular mechanisms such as enhanced cerebral blood flow and are confounded by the presence of other bioactive compounds. Similar patterns have been observed in preclinical studies using cocoa flavanol mixtures, which have demonstrated neuroprotective and cognitive benefits through antioxidant and anti-inflammatory pathways. For example, Nehlig et al. reviewed several animal studies showing that cocoa flavanol-rich extracts can improve spatial memory, increase antioxidant enzyme activity, and upregulate signaling pathways, such as Nrf2/HO-1 and CREB; these mechanisms are closely aligned with those highlighted in our analysis. However, as with human studies, the use of multicompound interventions in animal studies limits the ability to attribute the specific effects to epicatechin. Collectively, these findings reinforce the need for mechanism-driven studies to isolate the effects of epicatechin and highlight the importance of conducting well-controlled clinical trials to evaluate its translational potential in humans.

3. Summary of Human Clinical Trials Investigating the Cognitive Effects of Cocoa Flavanol Intake .

year (reference)	design	subject and number (F/M)	intervention	duration	cognitive measures	proposed cognitive mechanism
2011	RCT crossover	healthy adults 30 (22/8)	–35 g dark chocolate (773 mg CF)	single dose	visual spatial working memory, choice reaction time	↑ cerebral perfusion and possibly retinal blood flow
			–35 g white chocolate
2010	RCT crossover	healthy adults 30 (17/13)	–994 mg CF drink	single dose	cognitive demand battery (serial threes, serial sevens, rapid visual information processing)	↑ cerebral perfusion and substrate supply
			–520 mg CF drink
			–low CF drink
2012	RCT	MCI 90 (NA/NA)	–990 mg/day CF	8 weeks	mini-mental state examination, trail making test A & B, verbal fluency	↑ endothelial function and cerebral blood flow
			–520 mg/day CF
			–45 mg/day CF
2014	RCT	healthy adults 37 (27/10)	–900 mg/day CF	3 months	modified Benton task	↑ dentate gyrus capillary density → ↑ cerebral blood volume → ↑ dentate gyrus function
			–45 mg/day CF
2015	RCT	healthy adults 30 (18/12)	–993 mg/day CF	8 weeks	mini-mental state examination, trail making test A and B, verbal fluency	↓ insulin resistance; ↑ cerebral perfusion
			– 520 mg/day CF
			–48 mg/day CF
2016	RCT crossover	healthy adults 32 (16/16)	–flavanol-rich chocolate (520 mg EC)	single dose	psychomotor vigilance task, 2-back working memory task	↑ endothelial function → ↑ cerebral perfusion
			–flavanol-poor chocolate (66 mg EC)
2016	RCT crossover	healthy adults 40 (18/22)	–494 mg/day CF	4 weeks	executive function (Go-NoGo, Stroop, Trails, Plus-Minus, Letter memory), memory (episodic, working, spatial, implicit), digit symbol substitution test, rapid visual information processing	↑ BDNF expression in brain and periphery via MAPK and PI3K signaling
			–23 mg/day CF
2017	RCT crossover	healthy adults 23 (6/17)	–brewed cocoa (499 mg CF, 21 mg caffeine)	single dose	Bakan dual task, serial subtraction, continuous performance task	↑ cerebral blood flow
			–caffeinated cocoa (455 mg CF, 70 mg caffeine)
			–caffeine-only (66 mg caffeine)
			– placebo
2018	RCT crossover	healthy adults 10 (0/10)	–563 mg CF	single dose	color-word stroop task, face-name matching task	↑ cerebral perfusion and neural activation
			+exercise
			–38 mg CF
			+exercise

^aAbbreviations: CF, cocoa flavanol; EC, epicatechin; MCI, mild cognitive impairment; RCT, randomized controlled trial.

^bNot available (NA): The total number of participants could not be determined due to incomplete reporting in the original study.

^cArrows in the “Proposed cognitive mechanism” column show the direction of effect: ↑ for increase, ↓ for decrease, and → for a mechanistic sequence or association.

In conclusion, the findings of this meta-analysis support the neuroprotective potential of epicatechin in the mitigation of oxidative stress-induced cognitive impairment in animal models. The observed improvements in cognitive performance appear to be partially mediated by enhanced antioxidant capacity and suppressed neuroinflammatory responses. Although higher doses of epicatechin are generally associated with stronger effects across multiple outcomes, the evidence for dose dependency remains inconclusive, owing to limitations in statistical power and heterogeneity. These results highlighted the therapeutic potential of epicatechin. However, its applicability to human health remains to be established. As all data were derived from preclinical models, well-controlled clinical trials are needed to validate the translational relevance of these findings and assess the efficacy and safety of epicatechin in human populations.

All data generated or analyzed during this study are included in this article and its Supporting Information. Additional information is available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.5c05536.

• PRISMA checklist 2020 (Table S1); search terms and filters used in literature search (Table S2); subgroup analyses based on epicatechin dose: Morris Water Maze outcomes (Figure S1), MDA (Figure S2), TNF-α (Figure S3); publication bias assessments using funnel plots (Figure S4); and meta-regression analyses for epicatechin dose and cognitive/biochemical outcomes (Figure S5) (PDF)

Young Cheol Yoon and J. Min contributed equally to this work.

Y.C.Y.: Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. Y.J.M.: Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. H.M.P.: Writing – review and editing. I.H.C.: Writing – review and editing. C.W.J.: Writing – review and editing, Investigation. S.J.K.: Writing – review and editing. I.G.H.: Writing – review and editing. H.-H.J.: Writing – review and editing. S.W.K.: Writing – review and editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

The authors declare no competing financial interest.