Introduction

Hesperidin is a natural flavanone glycoside and one of the most biologically active flavonoids in citrus fruit [1]. Apart from being identified in Citrus sp., hesperidin is also present in banana peel, bergamot fruit, lemon fruit and peels. It is lipophilic and can easily cross the blood-brain barrier. Structurally, hesperidin has an aglycone known as hesperetin, which binds to rutinose [6-O-(α-l-Rhamnopyranosyl)-D-glucopyranose] and/or [6-O-(α-l-Rhamnosyl)-D-glucose]. It is also referred to as β-7-rutinoside of hesperetin [2].

Previous investigations revealed that hesperidin possesses several biological activities, including anti-inflammatory, anti-cancer and antioxidant activities [3]. It also exhibits beneficial effects on hypercholesterolemia, infertility and hypertension [4,5,6]. Several studies have shown the impact of hesperidin on central nervous system disorders. Diet supplemented with hesperidin improved cerebral blood flow, cognitive function and memory performance. Hesperidin reduced depressive symptoms in Parkinson’s model via modulation of serotonergic and kappa-opioid receptors and enhancement of dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid levels [7]. Also, hesperidin improved learning and memory and neurobehavioural functions in Alzheimer’s disease mouse model [8]. Hesperidin suppressed amyloid precursor protein expression and inhibited accumulation of β-amyloid protein and expression of γ-secretase. There are previous reviews on the neuroprotective mechanisms of hesperidin and its beneficial effects on central nervous system disorders [9]. However, currently, there has been no attempt to systematically synthesize available evidence on the effect of hesperidin on cognitive and neurobehavioural function. This systematic review and meta analysis evaluates current pre-clinical evidence of the effect of hesperidin on cognitive performance and neurobehavioural function. Studies that investigated the impact of hesperidin on learning and memory, anxiety, motor coordination and locomotion were included, and meta-analysis was performed to evaluate its effect quantitatively.

Materials and methods

Study Design

Primary animal-based studies that assayed hesperidin as therapeutic against cognitive and neurobehavioural disorders were rigorously searched in Scopus, EBSCOhost (including MEDLINE, CINAHL, APA PsycInfo etc.), Web of Science (WoS), Proquest, and PubMed via topic-based query-algorithm without regional, temporal or language confinements using the “Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines” [10] (Fig. 1). The topical algorithm was defined with the key elements as “hesperidin AND (memor* OR cogniti* OR *behav* OR neuroinflammati* OR neurodegenerat* OR Alzheim* OR Parkinso*)” and where necessary, adapted to different databases requirements for optimum retrieval. New study(ies) was(were) thereafter sought via email-alert recording until final analysis. The data querying and acquisition coupled with preprocessing involving merging and duplicate removal of multiple occurrences of a document in Endnote version 20 and Excel 2016 was done by E.T.C. Then, the initial screening of the studies for inclusion and exclusion decision-making was title and abstract oriented as per the screening protocol and criteria pre-set for this study. Full-text of the studies that passed the abstract/title screening was downloaded for _targeted variables extraction in predesigned data collection forms created as per _targeted data criteria. Finally, external records were further sought from manual review of citations in the eligible studies to ensure the non-missing of relevant studies. The workflow is presented in Figure S1.

Fig. 1
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Schema for selecting studies on effect of hesperidin and cognitive performance and neurobehavioural outcomes

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Inclusion Criteria

The chief criterion for any study included in the current investigation was to report in vivo models that assayed hesperidin effects on cognitive performance and neurobehaviour of pathologically induced neurodegenerative disease in rats. Only studies that evaluated metrics of learning and memory, anxiety, sensorimotor and locomotion in rats were selected. The study design must include a placebo group (untreated groups) and experimental groups involving untreated pathologic (induced neurogenerative disease) control group and intervention group (hesperidin-treated induced neurodegenerative disease group). All studies were included irrespective of hesperidin doses used. Reasonable outcomes (provided in measurable metrics) related to cognitive performance and neurobehavioural function and an adequate description of the corresponding assay must be presented.

Exclusion Criteria

Articles other than primary studies reporting the use of hesperidin in combination with other substances or intervention, were excluded in this study.

Data Extraction

The _targeted data extracted for the present study from the final database of the eligible studies include the study characteristics (authors’ name, publication year, sample size from each group), outcome variables (mean and standard deviation) in the hesperidin-treated group, untreated pathologic group, and placebo group, dose, and route of administration. Data were executed and validated by OTA and ETC. Disagreement was resolved by discussion in any case.

Statistical Analysis

Outcome variables (effect sizes) datasets related to the intervention (hesperidin treated) group versus untreated (pathologic) group and intervention group versus placebo group were fitted in a mixed-effects model to compute the SMD (bias-corrected standardised mean difference) using the inverse variance technique based on the exact formulae [11]. The SMD confidence intervals were adjusted using the Knapp-Hartung protocol [12]. The between-studies variance (τ2) estimate was achieved via the restricted maximum likelihood estimator [13]. Assessment of heterogeneity between studies was based on I2-statistic acceptable limit set as I² ≯75% [14]. Outcome variables with ≥ 3 studies were meta-analysed. Finally, the sensitivity of the model was tested via leave-one-study-out mixed-effects meta-analytic cross-validation (Viechtbauer and Cheung, 2010). The final outcomes were summarized in forest plots. In all cases, results from the random-effects model were interpreted in lieu of common-effects model. Small study effects or publication bias were assessed using limit meta-analysis (Rücker et al. 2011). Models were fitted in R version 4.3.0 (2023-04-21 ucrt) based on metasens v.1.5-2, metafor v.4.0–0, meta v.6.2-1, dmetar v.0.0.9000, and performance analytics v.2.0.4 packages [13, 15,16,17].

Results

Selection of Articles and Study Characteristics

The search results revealed that 1069 documents were retrieved from 5 databases: PubMed (n = 150), Web of Science (n = 257), Scopus (n – 339), ProQuest (n = 90) and EBSCOhost (n = 233), as shown in Fig. 1. Out of the 1069 articles retrieved, 46 studies were included in the systematic and meta-analysis review based on the inclusion and exclusion criteria. The characteristics of the included studies are shown in Table 1. A total of 35 studies presented in Table 1 used Sprague Dawley rats, while 11 studies used mice. Six studies used the aluminium chloride and diabetes–induced cognitive impairment model while 5 studies employed stress-induced model. Two studies used valproic acid model, 6-hydroxydopamine model, lipopolysaccharide model, APPswedish transgenic model, ketamine and 3-nitropropionic acid model. One study each used scopolamine, sodium fluoride, D-galactose, methothrexate, traumatic brain, nitric oxide, formaldehyde, thioacetamide, febrile seizure, ischemia, post-partum, neuropathic pain, methionine, quinolinic acid, emamectin benzoate, homocysteine, olfactory bulbectomy, paclitaxel, pentylenetetrazole, rotenone, 5xFAD models. The study duration varied in the included studies from 5 to 112 days as shown in Table 1.

Table 1 Study characteristics of articles included in the systematic review and meta-analysis
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Hesperidin (Sample Control) vs. Placebo (Normal Control)

Figure 2 revealed the analysis of 16 studies that reported elevated plus maze in normal rats treated with hesperidin and placebo. The result shows no significant difference after treatment with hesperidin when compared to the placebo group with SMD = 0.04 and 95% CI -2.66;2.75 and I2 = 84%. The pooled mean difference from the Y-Maze test between hesperidin-treated groups (Sample control group) and placebo (normal control) also revealed no significant difference (SMD = 0.27, 95% CI [-0.79;1.32], P < 0.01) in the fixed and random effect models with I2 = 76% as shown in Fig. 3. Similar results were obtained for Morris Water Maze (SMD = 0.50, 95% CI = -1.69;2.69, I2 = 83) and object recognition tests (SMD = -1.32), 95% CI = -4.72–2.01), I2 = 91%) measured in hesperidin (sample control) and placebo groups as shown in Figs. 4 and 5. Figure 6 shows the forest plot of pooled SMD data from the passive avoidance test between the treated group and placebo. No significant difference was observed between the groups tested, as revealed by the SMD (5.07) and 95% CI [-17.51; 27.65] with I2 = 89%.

Fig. 2
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Forest plot of preclinical investigations on the effect of hesperidin on anxiogenic behaviour in elevated plus maze of Sample control vs. placebo

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Fig. 3
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Forest plot of preclinical investigations on the effect of hesperidin on cognitive performance in Y-maze tests of Sample control vs. placebo

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Fig. 4
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Forest plot of preclinical investigations on the effect of hesperidin on cognitive performance in Morris Water Maze Test of Sample control vs. placebo

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Fig. 5
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Forest plot of preclinical investigations on the effect of hesperidin on cognitive performance in novel recognition test of Sample control vs. placebo

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The result of the open field test of 16 eligible studies is shown in Fig. 7. The forest plot revealed no significant difference between the hesperidin (Sample control) and placebo groups with SMD = 0.49 and 95% CI [-0.17;1.16] and I2 = 59%. The forest plot for pooled standard mean difference (SMD) from the rotarod test measured between hesperidin-treated rats (sample control) and placebo (normal control) is shown in Fig. 8. The result showed a significant difference between hesperidin (Sample control) and placebo groups with SMD = -4.10 and 95% CI [-7.92;-0.29] and I2 = 89%. In the forced swimming test, no significant difference was observed between the hesperidin-treated group and placebo as shown in the forest plot (SMD = -1.33; 95% CI [-3.41; 0.42]; I2 = 86%) in Fig. 9. The result of the meta-analysis of locomotor activity between hesperidin-treated rats and placebo showed no significant difference (SMD = -2.31, 95% CI = -7.64;3.02, I2 = 78%) as shown in Fig. 10.

Fig. 6
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Forest plot of preclinical investigations on the effect of hesperidin on cognitive performance in passive avoidance test of Sample control vs. placebo

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Fig. 7
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Forest plot of preclinical investigations on the effect of hesperidin on cognitive performance in open field test of Sample control vs. placebo

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Fig. 8
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Forest plot of preclinical investigations on the effect of hesperidin on rotarod performance test of Sample control vs. placebo

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Fig. 9
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Forest plot of preclinical investigations on the effect of hesperidin on forced swimming test of Sample control vs. placebo

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Fig. 10
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Forest plot of preclinical investigations on the effect of hesperidin on locomotor activity of Sample control vs. placebo

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Hesperidin (Intervention Group) vs. Cognitive Impaired Group

Our findings from the forest plot pooled data from 16 included studies that reported elevated plus maze test showed no significant difference (SMD = -0.21, 95%CI = -3.12; 2.69, I2 = 93%) after intervention with hesperidin as shown in Supplementary Fig. 1. However, results from the Y-Maze test showed a significant difference between the hesperidin-treated cognitively impaired rats and the untreated group (SMD = 8.31; 95%CI = 4.55–12.6; I2 = 93%), as revealed by the forest plot in Supplementary Fig. 2. Similarly, a significant difference was also observed between the hesperidin-treated group and untreated cognitive impaired rats (SMD = -5.90, 95% CI = − 11.18; -0.61, I2 = 83%) from the Morris Water Maze test of 13 included studies as shown in the forest plot in Supplementary Fig. 3. The result of the novel recognition test showed a significant difference (SMD = -3.59, 95% CI = -6.82; -0.35, I2 = 92%) between the hesperidin-treated and untreated cognitively impaired rats, respectively, as shown in Supplemented Fig. 4. For the passive avoidance test, there was no significant difference (SMD = 8.66, 95% CI = -0.20; 17.53) between hesperidin-treated and untreated cognitively impaired rats, as shown in Supplementary Fig. 5.

The forest plot from the rotarod test also revealed a significant difference between hesperidin-treated (intervention) and untreated cognitive impaired rats (pathological) (SMD = -9.72, 95% CI = -13.71;-5.73 and I2 = 83%) (Supplementary Fig. 6). The result of the open field test in Supplementary Fig. 7 showed a significant difference (SMD = 3.47 95% CI = 0.51; 6.43 I2 = 94%) between the hesperidin-treated and untreated cognitively impaired rats. The forest plot of the forced swimming test presented in Supplementary Fig. 8 also revealed a significant difference between hesperidin-treated and untreated cognitive impaired rats (SMD = -3.86, 95% CI = − 6.24; -1.47, I2 = 94%). Also, a significant difference was observed in the locomotion activity of hesperidin-treated groups compared to the untreated cognitive impaired group (SMD = 5.85, 95% CI = 0.64; 11.07, I2 = 94%) as shown in Supplementary Fig. 9.

Discussion

This is the first systematic review and meta-analysis on the effect of hesperidin on cognitive function and neurobehavioural outcomes in animal studies. The efficacy of hesperidin was assessed in two different studies. First, we assessed the effect of hesperidin on normal animals (sample control vs. placebo). In the second study, a meta-analysis was conducted on the effect of hesperidin on cognitively impaired animals (hesperidin-treated group (intervention) vs. disease (pathological) group).

Results from the first meta-analytic study revealed that treatment with hesperidin did not significantly affect cognitive and neurobehavioural outcomes in normal rats except in the studies on rotarod tests. This result suggests that hesperidin may improve muscular coordination and motor function in normal rats.

The overall outcome of the second study revealed that hesperidin showed a significant effect on cognitive performance and neurohavioural function in cognitively impaired rats. Using the eligible studies, we investigated the effect of hesperidin on neurobehavioural impairments. Some of the parameters used for the investigation include Y-Maze, Morris Water Maze Passive avoidance test and novel object recognition tests (Learning and memory tests), elevated plus maze (anxiety-related behavioural test), open field and rotarod tests (sensorimotor tests) as well as forced swimming test and locomotor activity (locomotor test).

Effect of Hesperidin on Anxiety-Related Outcomes

The meta-analytic results showed that hesperidin did not improve anxiety-related behaviour in rats. It was observed from the forest plot data pooled from 16 included studies that hesperidin did not exhibit an effect on anxiety, which is contrary to individual studies. Furthermore, data pooled from 4 studies showed that hesperidin did not improve cognitive performance associated with aversive stimuli, as revealed by results from the passive avoidance test.

Hesperidin Improved Learning and Memory Function

Our findings revealed that hesperidin showed significant learning and memory-enhancing effects as revealed by the pooled data from the Y-Maze, Morris water maze and novel object recognition test. The meta-regression data confirmed a significant association between treatment with hesperidin and memory function in cognitively impaired rats. These results suggest that hesperidin specifically improved spatial learning and memory, spatial memory and long-term memory, as well as recognition memory in cognitive impaired rats, which correlates with the results of the individual studies included in this study. The ability of hesperidin to improve cognitive performance in memory-impaired rats may be due to different biochemical mechanisms, especially its antioxidant and anti-neuroinflammatory effects. Memory and learning-enhancing effects of hesperidin have also been linked to the improvement of cholinergic function in cognitive impaired rats. The degeneration of cholinergic neurons and depletion of acetylcholine levels in the brain is associated with cognitive impairment. Hesperidin inhibited acetylcholinesterase activity and improved cholinergic function in cognitive-impaired rats [20, 38]. Oxidative stress also contributes to poor neurobehavioural outcomes. The brain is susceptible to oxidative damage and may cause radical-induced neuronal damage, leading to loss of synaptic connections, accumulation of protein aggregates, neurodegeneration and ultimately memory impairment [61, 62]. Impaired neurogenesis may contribute to memory impairment because it involves the development of neuronal cells from neural stem cells that are progenitor cells.

The neural progenitor cells resident in the dentate gyrus of the brain’s hippocampal region are important for neurogenesis during the embryonic stage and adulthood. It has been shown that they also contribute to maintaining memory and learning functions. The work of Aranarochana [8] and Naewla et al. [47] revealed that hesperidin attenuated memory impairment caused by valproic acid and methotrexate–induced impaired neurogenesis.

Hesperidin Improved Sensorimotor Function

The effect of hesperidin on sensorimotor function was examined as shown by the pooled data from 23 eligible studies that reported open field (15) and rotarod (7) tests. Our findings suggest that hesperidin improved sensorimotor coordination and motor learning in cognitive-impaired rats. In the open field tests, cognitive-impaired animals were characterized by decreased movement and activities [20, 63]. Hesperidin improved motor function, locomotive and exploratory activities of cognitively impaired rats [22, 34, 64]. Hesperidin exhibited good behavioural outcomes via attenuation of sensorimotor deficit, motor dysfunction, impaired grip strength, and muscular coordination skills in the rotarod test [38, 49, 57].

Effect of Hesperidin on Locomotor Activity

Our findings also revealed that hesperidin improved locomotive activity in cognitively impaired rats, as shown by the results from the forced swimming and locomotion activity tests. This result correlates with previous findings [29, 25, 27, 50]. Kumar et al. [25] reported that impairment in motor function was triggered by degeneration of dopaminergic neurons in the striatum, impaired antioxidant defense mechanism in the neurons and microglia activation. However, hesperidin showed significant inhibition of neuroinflammation and oxidative damage to neurons, contributing to improved locomotive behaviour. Hesperidin also reduced immobility and improved cognition in memory-impaired rats [27].

Evidence Gaps and Future Directions

One of the limitations of this study is the variation in the doses of hesperidin observed in the included studies. Different doses were observed in the included studies, though some authors reported the same hesperidin dose in the investigation. Furthermore, different study designs were combined from the identified studies in the meta-analysis. There were variations in the study design, which involved the use of different chemical compounds to induce cognitive and behavioural impairments in the animals. Furthermore, species effects may also affect the study as we combined results from investigations that used rats and mice in the meta-analysis. Hence, this study did not consider variation in doses, specificity in the study design, route of administration of hesperidin and chemical compounds used for the study design. A strength of the study was that we examined the effects of hesperidin on cognitive performance and behavioural function in preclinical models using different learning and memory, sensorimotor and locomotive paradigms. This study provided the opportunity to quantitatively examine the consistency of the memory-enhancing effects of hesperidin in mice and rats. The role of hesperidin in cognitive performance and neurobehaviour outcomes requires further investigation, especially in human studies. Studies on the nootropic effects of hesperidin in childhood and adulthood and sensitivity to sexes should also be explored. More studies are required to determine the dose range at which hesperidin can exert positive cognitive and neurobehaviour outcomes in neurologically impaired subjects.

Conclusion

This study highlighted that hesperidin improved learning and memory, sensorimotor and locomotive functions in cognitive-impaired rats. These results correlate with previous findings from individual studies. However, our findings did not reveal a positive effect on the anxiety-related behavioural outcome, which contradicts reports from individual studies. These reported actions of hesperidin have been linked to its neuroprotective effects via inhibition of acetylcholinesterase activity and cholinergic impairment, attenuation of neuroinflammation, inhibition of oxidative stress-induced neuronal damage and mitochondrial dysfunction. These findings suggest that hesperidin treatment could be a potentially suitable neuroprotective therapy capable of improving cognitive performance and neurobehavioural outcomes. Further studies are suggested especially n the effect of hesperidin on anxiety-like behaviour. These results will provide scientific information with potential translational value from animal studies to clinical trials.