Honey and Alzheimer’s Disease—Current Understanding and Future Prospects

Alzheimer’s disease (AD), a leading cause of dementia, has been a global concern. AD is associated with the involvement of the central nervous system that causes the characteristic impaired memory, cognitive deficits, and behavioral abnormalities. These abnormalities caused by AD is known to be attributed by extracellular aggregates of amyloid beta plaques and intracellular neurofibrillary tangles. Additionally, genetic factors such as abnormality in the expression of APOE, APP, BACE1, PSEN-1, and PSEN-2 play a role in the disease. As the current treatment aims to treat the symptoms and to slow the disease progression, there has been a continuous search for new nutraceutical agent or medicine to help prevent and cure AD pathology. In this quest, honey has emerged as a powerful nootropic agent. Numerous studies have demonstrated that the high flavonoids and phenolic acids content in honey exerts its antioxidant, anti-inflammatory, and neuroprotective properties. This review summarizes the effect of main flavonoid compounds found in honey on the physiological functioning of the central nervous system, and the effect of honey intake on memory and cognition in various animal model. This review provides a new insight on the potential of honey to prevent AD pathology, as well as to ameliorate the damage in the developed AD.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder associated with damage to the brain areas such as the cerebral cortex, temporal lobe, hippocampus, amygdala, entorhinal cortex (EC), and parahippocampal region [1,2]. The disease is mainly characterized by impaired memory and cognitive deficits [3,4]. AD is considered the most common cause of dementia, accounting for about 60-70% of the total cases worldwide [5]. In addition to the deficits of memory and cognition, AD is also accompanied by behavioral changes. Since most of the areas affected by the pathology are involved both in cognition and behavior, the predominant behavioral changes, such as agitation, dysphoria, and apathy, are correlated highly with cognitive dysfunction [6].
Previous studies have proposed several effective solutions to reduce the deposition of amyloid fibrils, minimize oxidative stress and neuroinflammation, and/or improve memory and cognition. These can be divided into drugs and antioxidants or neuroprotective agents for ease of discussion. The drugs include N-methyl-d-aspartate (NMDA) receptors antagonists [7,8], agents acting on the acetylcholinergic system (ACh system) [9,10], anti-amyloid [11], and anti-tau [12]. The antioxidants or neuroprotective agents include idebenone (an organic compound from the quinone family) and α-tocopherol [13], estrogen analogues [14,15], and honey [16][17][18]. Although the allopathic medications have shown promising results in attenuating symptoms, they may cause a number of adverse effects Studies suggest that most of honey's antioxidant, anti-inflammatory, and neuroprotective properties are due to its phenolic content [104,105]. Phenolic compounds are comprised of four classes of polyphenols: Phenolic acids, flavonoids, stilbenes, and lignans. Out of these, phenolic acids and flavonoids primarily have the potential to act as antioxidants and reduce oxidative stress [29][30][31] and neuroinflammation [32] that are the mediators of insults to the brain in the neurodegenerative diseases [106,107]. However, this review will focus on the effectiveness of flavonoids and phenolic acids on the CNS and in the prevention/treatment of AD pathology. The main phenolic compounds affecting the physiological functioning and/or the pathophysiology of the CNS are stated in Figure 1. According to the studies on AD models (Refer to Tables 1 and 2), all mentioned flavonoids and phenolic acids exert antioxidant effects and show neuroprotective activity. All agents, except myricetin, were also found to exhibit anti-inflammatory potential. As myricetin possesses an anti-inflammatory ability against post-ischemic neurodegeneration [108], if tested, it may also display similar potential in AD brain. In addition to the antioxidant and anti-inflammatory potential, most polyphenolic components also proved to attenuate AD pathology by decreasing amyloid deposition, with an exception of kaempferol and chlorogenic acid. Additionally, naringenin, naringin, quercetin, caffeic acid, and ellagic acid also reduce levels of p-tau in AD brain. According to the studies on AD models (Refer to Tables 1 and 2), all mentioned flavonoids and phenolic acids exert antioxidant effects and show neuroprotective activity. All agents, except myricetin, were also found to exhibit anti-inflammatory potential. As myricetin possesses an anti-inflammatory ability against post-ischemic neurodegeneration [108], if tested, it may also display similar potential in AD brain. In addition to the antioxidant and anti-inflammatory potential, most polyphenolic components also proved to attenuate AD pathology by decreasing amyloid deposition, with an exception of kaempferol and chlorogenic acid. Additionally, naringenin, naringin, quercetin, caffeic acid, and ellagic acid also reduce levels of p-tau in AD brain.    The results of ellagic acid treatment were: • Better memory in NOR test compared with untreated AD group. • Decreased lipid peroxidation along with increased levels of catalase, GSH, and total antioxidant capacity. • Improved neuronal morphology and lowering of amyloid and tau burden in cerebral cortex. • Co-treatment of ellagic acid with ellagic acid-loaded nanoparticles was more effective in mitigating all the behavioral and brain abnormalities.
[140]   • Improved learning and memory in behavioral tests. • Decreased levels of MDA and increased total thiol levels with restoration of SOD, GPx and catalase activity. [155]

Therapeutic Potential of Flavonoids and Phenolic Acids
Several polyphenols are known to exert protective effects on the nervous system and are suggested to have a role in alleviating symptoms of neurological diseases [159][160][161] including AD [162]. All these phenolic compounds, which are listed in Figure 1, are found to improve cognitive performance in AD pathology, and prevents from cognitive decline when ingested before inception of disease. These compounds are found in different kinds of honey in addition to other sources, and are particularly effective in attenuating oxidative stress along with exerting preventive effects on several other mechanisms in AD pathology. Their potential to reduce AD-induced brain injury is further proven by the microscopic studies and brain assays where they showed neuroprotective effects on the cortex [116,140], hippocampus [124,125], and hypothalamus [119]. Moreover, the consumption of polyphenols resulted in the prevention of hypoperfusion injury [145] with a generalized increment in cell number having normal physiology in the subiculum [122], and hippocampal proper area CA1 [48,136,139,154], CA3, and dentate gyrus [109,120,157], along with preserving normal synapses [112,131,144], the latter is further evident by an increased LTP after polyphenol consumption [154,156,158,163]. Further, the detailed studies of AD-brain animal model showed the potential of the polyphenols to reduce oxidation markers such as MDA [128,153], nitric oxide (NO), and nitrite [112,119,129,130], thereby attenuating free-radical-induced oxidation insults. The observed levels of antioxidants, however, are contrasting, with many studies deducing an elevated level of SOD and catalase, GSH [113,127,140,143,152,155], whereas others concluding decreased expression [115,146,148]. Although the results are contradictory, the studies demonstrating a reduction in the activity of antioxidants claim that this decrease also signifies the attenuation of oxidative injury, which subsequently renders the expression of the anti-inflammatory markers unnecessary. However, despite the discrepancy in the results, all studies conclude that the changes in the expression of these markers lead to reduced oxidative damage and Aβ-plaque accumulation.
Moreover, phenolic compounds can also alter the expression of some critical genes: APP, BACE1, PSEN-1, and Glutathione peroxidase 1 (GPx1). Polyphenols are found to down-regulate the expression of APP [164] and PSEN-1 gene [165], along with either a decrease [142,146,148] or increase in GPx1 expression [116]. As GPx1 is an enzyme that catalyzes the reduction of hydroperoxides and hydrogen peroxide by GSH to attenuate oxidation [166], reduced expression can lead to oxidative injury to cell. Moreover, the expression of BACE1 is also decreased [153] and is thought to be inhibited post-transcription, probably at the protein level [148]. Although not studied yet, a similar down-regulation of PSEN-2 gene expression can be expected by polyphenol consumption [167]. Moreover, polyphenols may also prevent neuritic plaque deposition by increasing α-secretase activity and by reducing cleavage of the APP to amyloidogenic soluble APP-β and β-CTFs [142] and hence, preventing the accumulation of the latter in synapses [148,165]. Taken together, these studies suggest that the polyphenols likely regulate gene expressions to reduce oxidation and formation of Aβ fibrils. Moreover, the polyphenols also increase the expression of the transcription factor Nrf2, which is responsible for regulating the induction of antioxidant genes, thereby improving defense against oxidative injury [139]. To protect the CNS further, the polyphenols reduce the level of pro-inflammatory markers, such as NFκB, TLR4 [139,157], COX-2 [151], MHC class II, TNFα [114,127,146], IL-1α [149], IL-1β [147,151,168], IL-6 [157,169] and increase anti-inflammatory cytokines [115], thereby reducing neuroinflammation. Furthermore, by decreasing neuroinflammation, these substances also attenuate the immunoreactivity of microglia and astroglia in the hippocampus, EC, and amygdala, which is commonly observed in AD neuropathology [115,122,142,146,149,150].
Additionally, the polyphenols reduce tau hyperphosphorylation and subsequent formation of NFT [170] and decrease the deposition of Aβ-plaques [140,156,171]. They also seem to exert a neuroprotective effect by preventing neuronal injury [136,152] and apoptosis [119,136,151] and by regulating the ACh system, where they increase ACh and choline acetyltransferase (ChAT), and decrease acetylcholinesterase (AChE) [119,123] and butyrylcholinesterase (BChE) [153]. These polyphenols' effects also lead to minimization of deficits of memory and cognitive [172][173][174][175]. As honey contains a number of these polyphenols, its consumption can be expected to have similar potential to prevent and treat CNS pathology in AD. The therapeutic potential of the polyphenols: flavonoids and phenolic acids, are summarized in Tables 1 and 2, respectively.
The effectiveness of honey in minimizing neurodegeneration is attributed to its neuroprotective effects on the brain [30,34], including the prefrontal cortex [176][177][178] and hippocampus [34,35,179,180]. Honey prevents neurodegeneration by attenuating two main phenomena, which are oxidative stress and neuroinflammation [36,132]. The reduction in neuroinflammation [181] is due to the attenuation of oxidative stress [30,31] and the prevention of free radical-mediated injury to the brain tissue [182,183]. This effect is evident by an increase in the antioxidant enzyme such as SOD and a reduction in the oxidative-stress markers, such as plasma MDA and protein carbonyl in aged brains [176,177]. Subsequently, as the hippocampal pyramidal neurons are highly susceptible to oxidative damage, this reduction of oxidative stress probably rescues them from insult and degeneration [34,182].
Further, along with the hippocampus, injury to the medial prefrontal cortex (mPFC) and piriform cortex is commonly observed in AD, both of which are associated with memory and cognition. Unlike other primary sensory cortices, which are minimally affected by the AD pathology, the piriform cortex is possibly affected even before or along with the development of the cognitive symptoms [184][185][186]. Hence, it is also considered a predictive marker of the conversion to AD [184,187]. Similarly, the damage to the mPFC in AD is evident as defective functioning [188,189] and abnormal connectivity with other associated brain areas [190]. Even though no such study has been undertaken to look at the injuries in these areas, the neuroprotective effects of honey may also rescue the mPFC and piriform cortical injury.
Although researchers widely accept the neuroprotective capacity of honey, we still do not have much data on the effects of honey on the physiology and/or anatomy of the human brain, and, therefore, its potential to act on the CNS is not fully understood to date. It is probably due to the late advent of technologies to study the brain in honey-related research and the limitation to researching human CNS. However, to overcome the limitation of experimental access to the human brains and to understand the possible effect of honey on the microscopic level, the research is now predominantly being carried out in rodents.

Effects of Honey on Memory, Cognition, and Behavior
Cortical Aβ deposition exerts effects on temporal lobe atrophy and resultant cognitive impairment in individuals with AD [191]. From psychophysiological perspective, cognition, learning, and memory are believed to be mainly determined by the cortico-hippocampal (C-H) circuit's normal functioning [192,193]. As ACh is the principal neurotransmitter in synapses, the amount of ACh also plays an essential role in learning behavior and cognitive performance [194]. Although relatively constant, the amount of ACh still normally fluctuates, according to the need in the memory processes, such as encoding and retrieval [195]. Essentially, since the integrity of the C-H circuit depends upon the normal physiology of neurons and synapses, the Aβ plaque formation, and therefore AD, may affect the circuit by damaging neurons [196], reducing the number of cholinergic neurons [197], decreasing the ChAT activity [198,199], decreasing ACh release [200], and impairing synapses, which results in defective transmission [72,74]. Surprisingly, ageing and Aβ fibrillogenesis also decrease the AChE activity [201,202]; this finding is unexpected and in contrast to the decreased ACh indicates that the reduction in ACh is likely due to degeneration of the cholinergic neurons along with an increase in another cholinesterase enzyme activity, such as BChE [203] and not due to elevated AChE levels, as the latter is itself hydrolyzed by the former [204,205]. The intake of honey is found to reduce the level of BChE with a further decline in the level of AChE [176,206]. Although the exact mode of action is still not understood, this cholinesterase inhibition, together with neuroprotection, results in improved cognition and memory [207] after honey consumption, as observed in rodents [180,182,208,209] and humans [37,210]. The effects of honey as a nutraceutical agent in improving memory and cognition are further discussed in Table 3 (in rodents) and  Table 4 (in humans). Honey, in a dose-dependent manner, ameliorates the anxiety-like behavior and possibly also acts as an anti-depressant [212] Swiss albino mice Young (3-4 months), aged (12-15 months) The formulation containing honey (400 mL), ghee (800 mL) and gold (288 mg) was given at the dose of 30 mg/kg/daily 15 days The formulation intake improved learning and memory in young and aged mice Decreased activity of AChE in brain [213]

Honey on Dopaminergic Neurons-Important Players in Memory Deficits in AD
In addition to ACh, dopamine plays a significant role in learning and memory functions. Besides being secreted from dopaminergic neurons (DN) of the Ventral tegmental area (VTA) and substantia nigra pars compacta (SNpC) in the midbrain [216,217], dopamine is also released by locus coeruleus (LC), located in the brainstem, which co-releases dopamine along with noradrenaline [218,219]; this released dopamine from LC innervates CA3 [220], and is thought to be the primary source of supply to the dorsal hippocampus [218,221]; however, a recent study suggests that the midbrain modulate dopaminergic innervation to the dorsal hippocampus and this stimulation is sufficient to arouse aversive memory even in the absence of input from LC [216]. To aid with the understanding of the contrasting source of dopamine, Takeuchi et al. proposed that although the projection of dopaminergic fibers from LC is denser than the midbrain [221], the midbrain and LC both modulate dorsal hippocampus in different kinds of memory consolidation processes [222]. Since the DN in the VTA are the primary site for dopamine synthesis, the DN in the midbrain-hippocampal (M-H) loop are vital in learning, memory formation, and consolidation [223,224]. The DN, and the secreted dopamine, modulate synaptic plasticity and contribute to the LTP in the hippocampus [225], thereby playing an essential part in the genesis and fortification of the episodic [226], aversive [216], and spatial memories [224]. Moreover, dopamine, together with norepinephrine, is crucial for the recognition memory [227,228]. In non-diseased brains, the number of DN and, therefore, the functional connectivity of the midbrain tends to decline with age [229,230], which may appear as deficits in learning and memory [231]. Similarly, as AD is a disease of old age, there is degeneration of DN [232,233]; However, due to the Aβ pathology, probably more damage occurs to the dopaminergic synapses in the M-H loop. Due to the mentioned insult, there is a decrease in dopamine, leading to impaired synaptic plasticity [234,235] and deficits of memory [233,236]. Polyphenols are found to prevent the degeneration of dopaminergic neurons and increase dopamine levels [137,[237][238][239][240][241]. Although all these studies discuss the attenuation of neuroinflammation with/without Parkinson's disease, similar results are expected in the AD model. As the insults to DN and reduced dopamine in AD are recently being studied in detail, more research is encouraged to be conducted on the AD M-H loop to understand the effect of honey and its constituent polyphenols on memory improvement in AD.

Honey as a Nootropic Agent-Prevention, Treatment, or Both?
In light of the previous research, it is evident that by acting on the CNS and working through various mechanisms, honey acts as a nootropic agent (refer Figures 2 and 3). Now the question arises of the right time to utilize these nootropic properties to alleviate AD symptoms. Another similar issue is understanding whether honey consumption is effective in preventing the development/conversion of mild cognitive impairment into AD, mitigating the damage during ongoing AD disease pathology or reversing the injury done to the brain by AD. Although, to our knowledge, this aspect is not assessed to date, the studies on polyphenols (discussed in Tables 1 and 2) may suggest some possible effects. The intake of phenolic compounds before initiation of the AD neuropathology is found to halt the progression of the CNS disease, protect neurons, reduce neuroinflammation and oxidative damage, and minimize memory and cognitive deficits, as seen in the studies on the AD-rodent models [48,109,111,112,117]. Likewise, honey ingestion in subjects with developed AD may also cause effects similar to those observed in the polyphenols-treated AD model [121,122,125,126]. Since these polyphenols are abundant in honey, we can expect the same benefits with honey consumption in human subjects.
Although honey is loaded with various kinds of polyphenols [100,102,103], the protective or curative effect of honey can be enhanced further by consuming it in combination with some nutraceutical agent [182,210,242]. Moreover, the synthesis of dimer by combining caffeic acid and ferulic acid [243], and the use of an amino acid (glutamine) conjugated with phenolic acid [244], both of which proved to be more efficient than the polyphenol alone, have paved a path for the likelihood of the advent of similar new combinations with honey that might emerge as the novel therapies for the prevention of AD. Moreover, a mixture of honey with other nutraceutical substances has proven to be effective in AD (for review: [245][246][247]) and can be appraised in prevention and/or management of AD.
In the same notion, the accurate dose of honey to prevent and/or treat AD has not been deduced to date. One of the important reasons of inability to draw conclusions is the fact that most studies are conducted on rodents, with very few studies on human subjects. Moreover, many confounding factors need to be addressed, such as the type of honey to be ingested, the therapeutic dose of honey, the minimum duration of honey intake, stage of AD (if given for treatment). Since few studies mentioned in Table 4 use a formulation of honey and other nootropic agents, another question arises whether combinations with such agents are more effective in terms of dosage and duration in improving human cognition. To our knowledge, currently there are no known studies on primates that observe the benefits of honey on cognition, or sporadic AD which is best modeled by the rhesus monkeys [248]. Comparative studies are encouraged in these areas, with possible usage of primates such as chimpanzees etc., to observe and deduce invaluable conclusions. Antioxidants 2023, 12, x FOR PEER REVIEW 20 of 34   Although honey is loaded with various kinds of polyphenols [100,102,103], the protective or curative effect of honey can be enhanced further by consuming it in combination with some nutraceutical agent [182,210,242]. Moreover, the synthesis of dimer by combining caffeic acid and ferulic acid [243], and the use of an amino acid (glutamine) conjugated with phenolic acid [244], both of which proved to be more efficient than the polyphenol alone, have paved a path for the likelihood of the advent of similar new combinations with honey that might emerge as the novel therapies for the prevention of AD. Moreover, a mixture of honey with other nutraceutical substances has proven to be effective in AD (for review: [245][246][247]) and can be appraised in prevention and/or management of AD.
In the same notion, the accurate dose of honey to prevent and/or treat AD has not been deduced to date. One of the important reasons of inability to draw conclusions is the

Conclusions and Future Directions
The phenolic compounds prevent damage to the neurons while promoting apoptosis in dysfunctional or cancer cells, which points toward different mechanisms of action in the brain cells than the rest of the body. On the same notion, the polyphenols are believed to exhibit oxidation-promoting properties for review: [249], rendering it necessary to explore the reasons for the switch between pro-oxidant and antioxidant characteristics to utilize these novel qualities appropriately. However, as various polyphenols have anti-amyloid and anti-tau potential, there is a possibility that they function as antioxidants in the cells that contain (or may contain) the Aβ aggregates and NFTs, whereas they promote oxidation in other abnormal cells; this aspect of these substances, as well as that of honey, needs further elaboration. Some recent studies have demonstrated the potential of flavonoids in prevention of memory decline in elderly individuals [250][251][252][253]. Similarly, the research on animal models of AD (mentioned in Tables 1 and 2) have shown an effectiveness of polyphenols in prevent and/or treat AD symptoms. However, the source of phenolic compounds in all these studies are variable. Considering the fact that the phenolic compounds can be obtained from various sources, notably fruits, vegetables, beverages, and nuts [254,255], the same polyphenols from distinct sources may have different pharmacological activities, and their polyphenolic potential, especially the capacity to act as an anti-oxidant and anti-inflammatory agent, may also vary accordingly. In the light of these considerations, new studies focusing primarily on the flavonoids and phenolic acids derived from honey are highly encouraged to understand the polyphenolic potential of honey.
Although an increased level of AChE is observed in cerebrospinal fluid (CSF) of AD patients [256], an overall reduction of AChE concentration is found in the AD brain [201,202]. The reduced amount of AChE suggests that AD pathology can be due to the reduction of some neuroprotective variant of AChE (e.g., AChE-R) in the absence of an increase in AChE activity [202]. Taking this into consideration, although the most reliable drug to treat mild to moderate AD is donepezil, an AChE inhibitor, it paradoxically increases the amount of AChE in the CSF [257,258]. These findings suggest that the AChE could have different properties inside the brain and within the CSF, pointing toward the possibility that the effect of honey on memory and cognition is due to neuroprotection with/without some mechanism other than AChE inhibition.
The role of dopaminergic system has been studied for a long time; however, its importance in memory deficits in AD was not clear. With the new studies on the role of dopaminergic system in learning and memory, a decline in dopamine levels with the damage in synapses is observed in AD. Furthermore, it is found that restoration of the dopaminergic levels is associated with improvement of memory deficits in AD [259]. For this purpose, dopamine agonists are being tested and proven to reverse the memory-related symptoms [260,261]. Moreover, the dopamine and its derivatives are found effective to reverse oxidative stress, inflammation, and Aβ load [262]. However, the dopamine replacement methods have given promising results, the effects of polyphenols and honey are still not being elucidated. Considering the polyphenol composition of honey, it can be more effective in treating various aspects of AD neuropathology compared to dopamine alone. On the same note, despite the fact that honey consumption is found to have a miraculous role in treating various diseases [263][264][265], its effectiveness in neurodegenerative diseases is still under evaluation. Having being proven to positively affect cognition and memory, the antioxidant capacity of honey also suggests its potential to manage neurological disorders and neurodegenerative diseases. More studies are needed to be conducted using animal models of neurodegenerative diseases, such as AD, to study the benefits of honey in its treatment and management.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.