The Beneficial Role of Apigenin against Cognitive and Neurobehavioural Dysfunction: A Systematic Review of Preclinical Investigations

Apigenin is a flavone widely present in different fruits and vegetables and has been suggested to possess neuroprotective effects against some neurological disorders. In this study, we systematically reviewed preclinical studies that investigated the effects of apigenin on learning and memory, locomotion activity, anxiety-like behaviour, depressive-like behaviour and sensorimotor and motor coordination in rats and mice with impaired memory and behaviour. We searched SCOPUS, Web of Science, PubMed and Google Scholar for relevant articles. A total of 34 studies were included in this review. The included studies revealed that apigenin enhanced learning and memory and locomotion activity, exhibited anxiolytic effects, attenuated depressive-like behaviour and improved sensorimotor and motor coordination in animals with cognitive impairment and neurobehavioural deficit. Some of the molecular and biochemical mechanisms of apigenin include activation of the ERK/CREB/BDNF signalling pathway; modulation of neurotransmitter levels and monoaminergic, cholinergic, dopaminergic and serotonergic systems; inhibition of pro-inflammatory cytokine production; and attenuation of oxidative neuronal damage. These results revealed the necessity for further research using established doses and short or long durations to ascertain effective and safe doses of apigenin. These results also point to the need for a clinical experiment to ascertain the therapeutic effect of apigenin.


Introduction
Neurological disorders are diseases that affect the nervous system, ranging from common migraine to severe brain, spine and nerve diseases [1].Some common neurological diseases include dementia, stroke, Parkinson's disease, lateral amyotrophic sclerosis epilepsy and multiple-system atrophy.Neurological diseases have been identified as the leading cause of mortality and disability among adults.These diseases have become a major health burden, especially among aged individuals, as they affect millions of people across the world and the cost of treatment is high [2].The high incidence rate of neurological diseases has been linked to an increase in the age of the population, as this disease is mostly common in adults [3,4].Apart from other pathological characteristics, some neurological disorders, especially neurodegenerative diseases, are accompanied by cognitive and neurobehavioural changes which affect quality of life [5,6].Neuronal degeneration leads to cognitive decline and neurobehavioural impairment and has been identified in patients with AD, PD, epilepsy, stroke and MS [7].Cognitive decline is usually characterized by impaired working memory, learning abilities, executive function and attention memory, while neurobehavioural dysfunction is commonly accompanied by anxiety, depression and loss of motor function and coordination, which are attributed to the loss and death of specific neurons such as cholinergic, dopaminergic and motor neurons in the central nervous system [8].Several therapeutic strategies have been developed to treat the symptoms and mitigate the progression of these impairments to improve quality of life.
In the last few years, the impact of flavonoids on neurological function has been studied extensively.This class of polyphenols has shown potent neuroprotective effects against some neurodegenerative diseases in different experimental models.Apigenin is a flavone-kind of flavonoid present in fruits, teas and vegetables.It is a potent antioxidant and has been shown to exhibit anti-inflammatory, antitumorigenic and antimicrobial activities [9].Its ability to cross the blood-brain barrier is important as it contributes to its pharmacological activity against neurological disorders [10].Kim et al. [10] reported that apigenin exhibited a neuroprotective effect against peripheral nerve degeneration.Some studies have also established that apigenin confers antidepressant activity, which is mediated by its effect on α-adrenergic, dopaminergic and serotonergic receptors [11][12][13][14].Apigenin improved serotonin, dopamine and epinephrine levels, which were altered in depressive animals [15,16].Apigenin further regulates the cAMP-CREB-BDNF signalling pathway and N-methyl-D-aspartate (NMDA) receptors, which play important roles in neuronal survival, synaptic plasticity, cognitive function and mood behaviour [15].Some experimental evidence and expert reviews have shown the pharmacological activities of apigenin, especially against some neurological diseases [17,18].However, no systematic review has assessed the effect of apigenin on cognitive dysfunction and neurobehavioural deficit in preclinical models.This study aims to systematically review preclinical investigations that explored the cognitive-enhancing effects of apigenin and identify research gaps for further studies where needed.In this study, we explored different paradigms, including learning and memory, sensorimotor function and motor coordination, locomotion activity, anxiety-like behaviour and depressive-like behaviour, to assess the effect of apigenin on cognitive impairment and neurobehavioural deficit.We also highlighted some possible biochemical and molecular mechanisms of its therapeutic actions.

Materials and Methods
A search was conducted in four different databases (SCOPUS, Google Scholar, PubMed and Web of Science) to identify relevant studies that reported the effect of apigenin on cognitive and neurobehavioural function in preclinical experiments without limitations to regions/location or language using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.The following search terms were used: "Apigenin AND (memor* OR cogniti* OR *behav* OR neuroinflammati* OR neurodegenerat* OR Alzheim* OR Parkinso*)".Interventional studies must list the authority that provided approval and the corresponding ethical approval code.

Study Selection
The selection of studies for inclusion in this review was based on the following criteria: (1) that they are preclinical studies or animal studies that used mice or rats; (2) studies that used any dosage of apigenin administered for any duration versus a control group treated with stress or any chemical capable of inducing memory deficit or neurobehavioural dysfunction; and (3) that they measured outcomes that focused on memory and learning (Morris Water Maze, Barnes maze, Y-maze, T-maze, novel object recognition, passive avoidance test, inhibitory avoidance), anxiety-like behaviour (elevated plus maze, dark-light model of anxiety), depressive-like behaviour (forced swimming test, sucrose preference test, splash test, tail suspension test, sucrose splash test), sensorimotor and motor coordination (rotarod activity, grip strength, catalepsy, SG mount, sensorimotor test) and locomotion activity (open field test, locomotion activity).Articles that reported plant extracts containing apigenin were not included.Also, articles involving the use of other models such as Drosophila melanogaster, Caenorhabditis elegans and cell cultures were excluded from this study.Review articles, theses and conference papers that reported the role of apigenin on cognitive function were also excluded.Studies that reported the use of apigenin in mixtures with other compounds or bioactive constituents were also excluded.Studies that focused on the effect of apigenin on biochemical markers associated with cognitive and neurobehavioural function not indicating relevant behavioural paradigms were also excluded.Two investigators thoroughly reviewed the titles and abstracts of the articles for eligibility for inclusion in the study.Disagreement in the evaluation of eligibility for inclusion was planned to be resolved based on consensus.However, no case of disagreement emerged during the evaluation for eligibility for inclusion.

Data Extraction
One of the authors extracted data from the included studies while the other author checked and confirmed the extracted data.The following information was extracted from the included studies: (1) subjects; (2) dosage and route of administration; (3) duration of experiment; (4) study description; and (5) type of outcome measured.To examine the effect of apigenin using different cognitive and neurobehavioural paradigms, we categorized the measured outcome identified in the included studies into the following: learning and memory; anxiety-like behaviour, depressive-like behaviour; sensorimotor and motor coordination; and locomotion activity.

Study Characteristics
As shown in Figure 1, 461 studies were identified from PubMed, Scopus, Web of Science and Google Scholar.However, 32 studies were included in the systematic review after screening with the eligibility criteria.These studies specifically address the effect of apigenin on cognitive function and behavioural outcomes in preclinical models.The hypothesis was associated with the effect of apigenin on cognitive and behavioural impairment compared to the disease group.All the included studies were performed either in mice or rats, some induced with rotenone, streptozotocin, corticosterone, chronic or mild stress, high-fat diet or fructose, lipopolysaccharide, scopolamine, methotrexate, pentylenetetrazole and acetonitrile (Table 1).The lowest dose of apigenin in the included studies was 2 mg/kg, while 351 mg/kg was the highest.Most studies used 10, 20, 40, 50 and 100 mg/kg.Also, different administration routes were used, including intragastric, intraperitoneal and oral routes, as shown in Table 1.Most of the studies used male subjects, except two studies that reported female animals.All the studies included mice or rats (Sprague Dawley or Wistar strains).The duration of the experiments in the included studies ranged from 4 days to 22 months.Twenty-two (22) 1).

Learning and Memory
In the learning and memory paradigm, eight different cognitive tests were identified in the included studies (Table 2).Of the 32 included studies, only 24 reported the effect of apigenin on learning and memory.Out of these 24 studies, 12 were on the Morris Water Maze, 1 on the Barnes test, 3 studies were on the Y-maze, 2 studies on the T-maze, 2 studies reported novel recognition tests, 3 studies reported passive avoidance tests, while 1 study reported the inhibitory avoidance test.All the studies showed that apigenin improved learning and memory, except for two studies.From the Morris Water Maze tests, eight studies showed that apigenin reduced escape latency [20,30], improved the time spent in the target quadrant [26][27][28]30], reduced first entry to the target quadrant and increased the number of crossings into the platform region [27].Only one study reported the effect of apigenin on spatial memory using the Barnes test.The study showed that apigenin did not affect primary latency or error acquired during acquisition and did not improve spatial memory.Three studies also revealed that apigenin improved spatial working memory in animals using the Y-maze test.In these studies, apigenin restored impaired reference memory and working memory deficit by increasing the percentage of alternating behaviour in the test animals [26,27,31].Two studies also reported that apigenin improved cognitive function using the T-maze test.In these two studies, apigenin increased the exploration of new routes and objects [29] and the percentage of spontaneous alternation [29].Two studies that reported novel recognition tests showed that apigenin improved scopolamine-induced recognition deficit and methotrexate-induced recognition impairment.
The results from the passive avoidance test reported in two studies showed that apigenin improved retention deficit by increasing step-through latency [36] and delayed forgetting of passive avoidance response [10], while one study showed no effect on passive avoidance performance [37].One study also revealed that apigenin did not significantly affect inhibitory avoidance activity.

Locomotor Activity
Two neurobehavioural paradigms were identified in the included studies, locomotor test and open field, as presented in Table 3.A total of 10 studies reported the effect of apigenin on locomotion using open field tests and locomotion tests.In the open field test, apigenin improved locomotor activity [32,35,37,42,44].However, apigenin did not significantly affect locomotor activity in three studies, as shown by the effect on crossing [22,36], grooming and rearing [33].

Depressive-like Behaviour
Our findings revealed that seven studies reported the effect of apigenin on depressionlike behaviour using the forced swimming test, as shown in Table 4.All the studies showed that apigenin improved immobility time in the forced swimming test.Six studies reported the use of a sucrose preference test to assess the effect of apigenin on depression-like behaviour in the intervention studies, Table 4.The results from these studies revealed that apigenin improved sucrose preference and consumption, and grooming time, and alleviated anhedonic-like behaviour in depressive mice.The tail suspension test was reported in four studies, which revealed that apigenin reduced the duration of immobility time.The splash test was also reported in one study (Table 4).The splash test revealed that apigenin improved grooming activity and locomotion in streptozotocin-induced depressive-like behaviour in a mouse model via an improvement in grooming activity.

Anxiety-like Behaviour Test
Of the five studies that reported the effect of apigenin on anxiety-like behaviour, four studies each reported the impact of the intervention using the elevated plus maze paradigm (Table 5).The four studies showed that apigenin attenuated anxiety-like behaviour by reducing the anxiety index and increasing the time spent in open arms and the number of entries into open arms.One study used the dark-light model of anxiety to examine the effect of apigenin on anxiety-like behaviour (Table 5).The results showed that apigenin did not exhibit an antianxiolytic effect as it did not alter the latency to the first crossing, and not did it show an effect on the time spent in the light compartment.

Sensorimotor Behaviour and Coordination Activity
Four studies reported the impact of apigenin on motor behaviour and performance using the rotarod test (Table 6).All the studies revealed that apigenin improved rotarod performance and enhanced muscle coordination by delaying the fall time.Two studies reported the use of the grip strength test to assess the effect of apigenin on motor coordination and muscular performance.The results showed that apigenin ameliorated the reduction in grip strength performance and improved motor coordination [23,35].Only one study examined the effect of apigenin on motor coordination using the catalepsy test.The result from this study showed that apigenin reversed the change in cataleptic behaviour and postural instability in streptozotocin-induced depression-like behaviour in rats.Other sensorimotor function and behavioural paradigms that revealed the effect of apigenin on muscular coordination include the pole test, spontaneous activity in the cylinder and the challenging beam transversal procedure (Table 6).These tests were reported in a study by Yarim et al. [43], and the results revealed that apigenin reduced total steps but did not show any effect on step errors or error per step.Furthermore, apigenin reduced pole test scores, increased hindlimb and forelimb test scores and improved rearing and grooming in a Parkinson's mouse model.

Discussion
To the best of our knowledge, this is the first systematic review on the effect of apigenin on cognitive and neurobehavioural outcomes in preclinical studies.Our findings revealed that apigenin exhibited cognitive-enhancing effects and improved neurobehavioural function in stress-induced animals.The studies included in this systematic review showed that apigenin improved cognitive function and neurobehaviour in impaired or stressed animals.In this study, we identified the effects of apigenin on different cognitive and neurobehavioural paradigms: learning and memory, locomotor activity, anxiety-like behaviour, depression-like behaviour and sensorimotor function.
The memory and learning paradigm revealed that apigenin exhibited nootropic effects.A significant improvement in memory and learning abilities was observed in impaired rats.Apigenin improved spatial and long-term memory, short-term and spatial working memory, recognition memory and learning abilities.However, one study reported that apigenin did not improve cognitive function in a chronic neuroinflammatory mouse model induced with glial fibrillary acidic protein-interleukin-6 (GFAP-IL-6), as revealed by the Barnes maze test [21].The authors suggested that though other studies established that apigenin improved memory function in different models, in the context of their study, which involved chronic neuroinflammation, no improvement in cognitive function was observed.
Moreover, the study only employed the Barnes maze and did not explore other learning and memory paradigms.The molecular mechanisms of apigenin's effect on learning and memory have been linked to synaptic transduction of the ERK/CREB/BDNF signalling pathway in the cortical cholinergic system.cAMP response element binding protein (CREB)mediated transcription genes are essential for learning and memory.CREB also mediates the activity of glucagon-like peptide-1 (GLP-1), which in turn activates the CREB-regulated BDNF promoter [48].Impaired ERK/CREB/BDNF signalling pathway and an inhibition of CaMK-II/PKC/PKA-ERK-CREB signalling lead to cognitive deficit and disruption of long-term potentiation in the hippocampus [49].Furthermore, depletion of CREB and withdrawal of BDNF lead to a drastic reduction in synaptic markers in the hippocampus, which may impair long-term potentiation [50,51].Hence, the learning and memory-enhancing effect of apigenin could be linked to its influence on CREB-BDNF signalling.Apigenin improved BDNF levels and enhanced ERK1/2 and CREB expression [21,51].Apigenin also improved GLP-1 expression [51].The memory-enhancing effects of apigenin were also attributed to the attenuation of oxidative stress, inhibition of apoptosis, antiamyloidegonic effects and activation of BDNF.TrkB signalling pathways [29].Treatment with apigenin reduced Bax/Bcl-2 ratio, caspase-3 and PARP expression, hence inhibiting neuronal apoptosis and degeneration.The influence of apigenin on these signalling pathways explains the observed memory-enhancing effects.
Apigenin improved locomotion behaviour in stressed animals, as shown through three major neurobehavioural paradigms.The open field test was commonly used in all the identified studies.Two studies that reported open field tests showed that apigenin had no effect on locomotion in rats with behavioural deficits.Moreover, one of the studies revealed that apigenin did not affect locomotion.In contrast, seven other studies involving open field tests, including one that used the locomotion test, showed that apigenin improved locomotion.
Our findings also showed that apigenin markedly reduced depressive-like behaviour in preclinical studies, as shown by the results of the forced swimming test, sucrose preference test and tail suspension test from 10 included studies.Depressive-like behaviour is associated with low monoamine levels due to high monoamine oxidase activity [17,25,52].The improvement in immobility time exhibited by apigenin in animals with depressivelike behaviour could be due to increased monoamine and serotonin levels.Hence, the mechanism of action of apigenin could be linked to the modulation of monoaminergic systems [25].Apigenin is a potent inhibitor of monoamine oxidase [11,17].Results from the included studies revealed apigenin as an effective natural antidepressant comparable to synthetic compounds.Some findings also attributed the antidepressant effect of apigenin to the modulation of some neurochemicals and proteins in the brain.Yi, Li, Li, Pan, Xu and Kong [16] suggested that the antidepressive effect of apigenin could be associated with multiple biochemical mechanisms, including modulation of brain monoamine, serotonin and dopamine levels; normalization of HPA axis alterations; and downregulation of the cAMP pathway.The antidepressive effect of apigenin has also been linked with its interaction with the serotonergic pathway, increasing serotonin levels and BDNF expression.Other studies also showed that apigenin exhibited antidepressive effects via increased phosphorylation of CREB and elevated levels of BDNF [38,41].After oral treatment with apigenin, changes in the levels of serotonin and high BDNF expression were observed, and these are suggested to activate the PKA-CREB signalling pathways.Experimental investigations have also shown a link between the pathophysiology of depression and neuroinflammation.The production of pro-inflammatory cytokines contributes to the development of depression, and antidepressants have been identified as a potent inhibitor of the production of neuroinflammatory biomarkers such as cytokines.The antidepressant effect of apigenin is partly due to its anti-neuroinflammatory effects [32,33].Apigenin inhibited cytokine production, iNos and COX-2 expression, and NF-kB activation in lipopolysaccharide depressive-like mice.Apigenin also inhibited NLRP3 inflammasome activation through the upregulation of PPAR-γ [33].
The results from the included studies showed that apigenin could be an effective alternative approach for the treatment of anxiety.There are indications that anxiety is linked to alterations in the monoaminergic system [53].The extensive period spent in the open arms in the elevated plus maze test could be due to increased monoamine levels in the brain [54].Amin, Ibrahim, Rizwan-ul-Hasan, Khaliq, Gabr, Muhammad, Khan, Sidhom, Tikmani, Shawky, Ahmad and Abidi [25] reported that apigenin reduced anxiety-like symptoms in rodents, and this was attributed to the modulation of brain monoamine levels and interactions with serotonin receptors.Furthermore, inflammatory processes are triggered by anxiety, which is caused by high levels of pro-inflammatory cytokines such as IL-6, IL-1β and TNF-α.This is due to the impact of these cytokines on neurotransmitters related to the anxiety response.Apigenin also inhibited proinflammatory cytokine production, which could be associated with its anxiolytic effect [11,36].The role of oxidative stress and inflammatory-related transcription factors in the development of anxiety-like disorders has also been established [55,56].Sharma, Sharma and Singh [38] also reported that apigenin exhibited anxiolytic effects via modulation of CREB phosphorylation and elevated BDNF levels.
The results from the sensorimotor tests presented in the included studies revealed that apigenin improved motor coordination and prevented loss of muscle control.In Parkinson's disease and amyotrophic lateral sclerosis models, apigenin improved grip strength and rotarod activity, hence improving sensorimotor function [35,42,43].Impaired motor coordination and balance in Parkinson's disease is linked to alterations in some neurotransmitters such as dopamine, glutamate and γ-amino butyric acid (GABA) [35,57,58].Oxidative stress and neuroinflammatory processes also contribute to motor neuron degeneration, which is important for motor coordination and sensorimotor function [35,42,43].The identified studies showed that apigenin improved sensorimotor function and motor coordination by improving antioxidant defence and the inhibition of neuroinflammation, hence attenuating dopaminergic degeneration and, ultimately, motor neuron degeneration (Figure 2).

Conclusions
Apigenin is a less toxic flavone widely present in many fruits and vegetables.This systematic review of preclinical trials adds to existing individual studies suggesting that apigenin can improve cognitive and neurobehavioural function.Apigenin improved learning and memory, locomotion activity, sensorimotor and motor coordination and depressive-like and anxiety-like behaviour.The learning and memory-enhancing effects of apigenin could be attributed to the modulation of BDNF levels and expression of ERL1/1 and CREB.Apigenin improved ERK/CREB/BDNF signalling which, in turn, contributed to long-term potentiation and memory function.Apigenin also attenuated hippocampal cholinergic deficit and improved acetylcholine levels, which are important for cholinergic neurotransmission and required for memory function.In addition, the antidepressive and

Conclusions
Apigenin is a less toxic flavone widely present in many fruits and vegetables.This systematic review of preclinical trials adds to existing individual studies suggesting that apigenin can improve cognitive and neurobehavioural function.Apigenin improved learning and memory, locomotion activity, sensorimotor and motor coordination and depressivelike and anxiety-like behaviour.The learning and memory-enhancing effects of apigenin could be attributed to the modulation of BDNF levels and expression of ERL1/1 and CREB.Apigenin improved ERK/CREB/BDNF signalling which, in turn, contributed to long-term potentiation and memory function.Apigenin also attenuated hippocampal cholinergic deficit and improved acetylcholine levels, which are important for cholinergic neurotransmission and required for memory function.In addition, the antidepressive and antianxiolytic effects of apigenin are related to the inhibition of inflammatory markers (TNFα, IL-6, IL-1β, iNOS and COX-2) and NF-kB activation; the modulation of monoamine, dopamine and serotonin levels; the normalization of HPA axis alterations; and downregulation of the cAMP pathway.Apigenin also improved sensorimotor function and motor coordination via attenuation of motor and dopaminergic degeneration; modulation of dopamine, glutamate and γ-amino butyric acid (GABA); and inhibition of ROS production and neuroinflammation.
These results show very good potential for exploring apigenin in clinical studies.Further studies can employ clinical experiments in different populations using short-and long-term trials to examine the effect of apigenin on cognitive and neurobehavioural disorders.Furthermore, different delivery systems can also be examined to establish therapeutic efficacy and minimize challenges associated with absorption, distribution and metabolism.Also, different doses of apigenin can be further explored to ascertain the safety levels around its use in treatment and its consumption through food substances.

Figure 2 .
Figure 2. Biochemical and molecular mechanisms of apigenin against cognitive and neurobehavioural deficit.

Figure 2 .
Figure 2. Biochemical and molecular mechanisms of apigenin against cognitive and neurobehavioural deficit.

Table 1 .
Description of included studies.

Table 2 .
Effect of apigenin on learning and memory.

Table 3 .
Effect of apigenin on locomotor behaviour.

Table 4 .
Effect of apigenin on depressive-like behaviour.

Table 5 .
Effect of apigenin on anxiety-like behaviours.

Table 6 .
Effect of apigenin on sensorimotor function and motor coordination.