Depending on the age at onset, AD can present either before (early-onset) or after the age of 65 (late-onset). Previous studies have demonstrated that the prevalence of early-onset AD is rather low (< 5%), and commonly associated with autosomal-dominant genetic mutations, which include mutations in the genes encoding for the amyloid-beta precursor protein (APP) (20, 21) as well as in the presenilin 1 (PSEN1) and 2 (PSEN2) genes (22). Additionally, the etiology of early-onset AD has been attributed to the duplication of the APP locus as well as to trisomy of chromosome 21, which causes Down syndrome (23). The etiology of the most common, late-onset disease (sporadic AD) is basically attributed to a variety of genetic and environmental factors. Among them, the ϵ4 allele of the Apolipoprotein E (APOE) gene (24), has been documented to increase the probability of late-onset AD significantly more than various other predisposition genes studied and described in the literature (25, 26). Additional risk genes that contribute to late-onset AD have been recently identified by genome-wide association studies (GWAS), some of them being involved in processes of neuroinflammation (TREM2, TYROB and CD33), memory (CR1, PICALM and BIN1) and lipid metabolism (ABCA7 and CLU) (4). From a molecular point of view, it is important to note that both early- and late-onset AD are essentially characterized by the same traits, i.e. the presence of plaques and tangles (the underlying pathological changes in the brains of AD patients are summarized in Figure 1 ).

Oxidative stress has been shown to be a dominant factor in the progression of AD. Over the years, significant oxidative damage accumulates in the neuronal tissue of AD patient brains, caused by the overproduction of free radicals (49). Free radicals, including ROS, are highly reactive molecules that have unpaired electron(s) in their outer shell, making them highly unstable. They are normally produced by metabolic processes of living organisms such as cellular oxidation, mitochondrial function, regulation and signaling, while they are also produced by specific cells of aerobic organisms such as macrophages, as well as by the electron transport chain in peroxisomes (50). The unpaired electron makes them particularly eager to attack other nearby molecules until a pair is formed. This results in free radical chain reactions and their propagation within the cell, jeopardizing vulnerable biomolecules, such as DNA, lipids and proteins.

The body’s antioxidant activity includes the endogenous and exogenous systems. The endogenous antioxidant system includes both enzymatic and non-enzymatic processes, which are regulated by endogenous molecules such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH), which work by repairing damage resulting from free radicals or by scavenging free radicals. Exogenous antioxidants, such as vitamins C and E, carotenoids and polyphenols, are assimilated into the body through diet and are capable of repairing free radical damage extracellularly, as well as acting as cofactors of endogenous enzymatic antioxidants, enhancing repair or maintaining cellular redox homeostasis (51). Oxidative stress contributes to the pathogenesis and progression of AD in three main ways, as discussed below, which are macromolecule redox modifications, metal ion redox potential, and mitochondrial dysfunction, thus affecting cellular homeostasis, ROS generation, and upregulating the formation of Aβ and tau fibrils (6). Additionally, oxidative stress can further enhance the pathophysiological characteristics of AD by activating JNK and AMPK, which regulate tau phosphorylation and could exacerbate the accumulation of NFTs (52, 53).

NRF2 is abundant in the human brain, primarily in microglia and astrocytes and secondarily in neurons (74). As an antioxidant transcription factor, NRF2 in astrocytes is inactive at basal conditions ( Figure 2A ), but enhances the expression of genes participating in GSH synthesis (75, 76) and downregulates pro-oxidant and pro-inflammatory proteins, including nitric oxide synthase 2 (NOS2), interleukin (IL)-6, cyclooxygenase 2 (COX-2) and tumor necrosis factor-a (TNF-a) (77) with the occurrence of oxidative conditions ( Figure 2B ). It has been shown that the levels and NRF2 activity are affected in the brains of people with neurodegenerative diseases, such as AD. In specific, NRF2 was found in the cytoplasm in the hippocampus of AD patients and its constitutive levels were significantly reduced in the nucleus (78). Similarly, a decrease in the mRNA levels of NRF2 was shown in the cortex of wild-type mice that was age-dependent (79). This finding was corroborated also in other studies, for example in the work of Kanninen et al. who observed that the amounts of Aβ dramatically increased while the levels of NRF2 significantly reduced as the mice aged. This was accompanied by decreased expression of three known targets of the NRF2 pathway, NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase regulatory subunit (GCLM) (80). Bahn et al. demonstrated that, independent of oxidative stress, lowering NRF2 levels enhances BACE1 activity and, consequently, Aβ synthesis (81). This altered regulation of NRF2 in the brains of AD patients leads to the accumulation of misfolded proteins, including the accumulation of Aβ, the hyperphosphorylation of tau and its subsequent aggregation, further exacerbating oxidative stress (80, 82–84) ( Figure 3 ).

As previously discussed, there is evidence to suggest that NRF2 activity is altered in AD and its dysregulation may contribute to disease progression. NRF2 is a transcription factor that regulates the expression of genes involved in antioxidant cellular defense and inflammatory conditions (85). Several studies have reported that NRF2 activity is reduced in the brains of individuals with AD. The reduction in NRF2 activity is associated with increased levels of oxidative stress and inflammation in the brain. Specifically, it has been found that there is a decrease in the expression of genes that are regulated by NRF2, including genes involved in antioxidant defense and detoxification. Moreover, it has been suggested that the dysregulation of NRF2 activity may contribute to the accumulation of Aβ plaques and NFTs (16). For example, it has been shown that Aβ can inhibit NRF2 activity, which can lead to increased oxidative stress and inflammation (86). Overall, these findings suggest that NRF2 dysregulation may contribute to the pathogenesis of AD, and targeting NRF2 may have therapeutic potential for the treatment of this disease.

Therefore, conducting the present literature review was considered necessary as the treatment of AD is an unmet medical need which affects a large percentage of the aging population, while its pathophysiology is still not fully understood. For this reason, research to find innovative treatments based on newer findings is very important to achieve better outcomes for patients. The role of oxidative stress is considered key in AD progression as oxidative stress contributes significantly to aging and neurodegeneration, which highlights it as a good therapeutic target. The NRF2 transcription factor signaling pathway is an important pathway that activates antioxidant mechanisms in cells, which is impaired in patients with AD and its reactivation is considered to be of critical importance in the treatment of the disease. With this in mind, this literature review focuses on the most recent advances of the last 5 years in antioxidant phytochemicals that are capable of acting as NRF2 activators or potentiators with the aim of preventing or treating AD symptoms.

Polyphenols are a large group of naturally occurring compounds found in plants. They are characterized by the presence of multiple phenol groups in their chemical structure and can be further classified into various subclasses, including flavonoids, phenolic acids, stilbenes, and lignans (87). Polyphenols possess antioxidant and anti-inflammatory properties, and their actions are attributed to their ability to scavenge ROS, modulate enzymatic activity, and regulate gene expression. One of the mechanisms through which polyphenols exert their cellular effects is by activating NRF2 through various pathways, including: 1. Modification of Keap1 (Kelch-like ECH-associated protein 1), leading to the release and nuclear translocation of NRF2, where it binds to the antioxidant response element (ARE) in the DNA, thereby activating the expression of genes involved in antioxidant defenses (88). 2. Kinase signaling pathways: Polyphenols can activate kinases such as protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and PI3K/Akt, which in turn phosphorylate NRF2 or its negative regulator, resulting in the stabilization and nuclear translocation of NRF2 (89). 3. Epigenetic modifications: Some polyphenols can influence the activity of enzymes involved in DNA methylation and histone modification, leading to epigenetic changes that may affect NRF2 gene expression (90). Thus, polyphenols are potential NRF2 activators that could benefit the pathophysiology of AD.

Rosmarinic acid (RosA), a polyphenolic compound found in various culinary herbs, was used against Aβ-induced toxicity in PC12 cells, and the results showed that RosA (10 μM) attenuated Aβ-induced ROS production and reduced lipid hydroperoxides, activating the NRF2-induced cellular antioxidant defense system. More specifically, RosA enhanced the nuclear translocation of NRF2 and its nuclear binding to ARE, activating a set of NRF2 target genes encoding phase II antioxidant enzymes. Mechanistically, the antioxidant effects of RosA were mainly mediated by the Akt/GSK-3β/Fyn pathway through increased NRF2 activity (91).

A polyphenolic extract of Arabidopsis thaliana was tested in vitro on mouse BV-2 microglial cells treated with Aβ_25-35 at a dose of 20 μl/ml. The extract caused a decrease in the production of pro-inflammatory cytokines (IL-6, IL-1β, TNF-a) and an increase in the production of anti-inflammatory cytokines (IL-4, IL-10, IL-13), while it also enhanced the activation of NRF2, leading to upregulation of heme oxygenase-1 (HO-1). The efficacy of the extract was also confirmed in a Drosophila melanogaster model expressing human Aβ_1-42, where it ameliorated Aβ-induced motor dysfunction in flies, indicating neuroprotection in vivo (92).

In vivo studies in an AD experimental model of Caenorhabditis elegans (C. elegans) showed that treatment with a methanolic extract of Romina variety strawberry (100, 500, 1000 μg/mL) (93) and treatment with an olive extract with 20% hydroxytyrosol content (100 μg/mL) (94), both extracts rich in phenolic compounds, led to reduction of oxidative stress and Aβ accumulation, while delaying the Aβ-induced paralysis of C. elegans, without apparent toxicity at these doses. The RNAi technology applied in both studies showed that the efficacy of the methanolic extract of strawberry derives partially from the activation of the DAF-16/FOXO (Forkhead box protein O) and SKN-1 (protein skinhead-1)/NRF2 pathways, while the efficacy of the olive extract derives from the activation of the SKN-1/NRF2 and the overexpression of the C. elegans lifespan biomarker HSP-16.2 (heat shock protein 16.2).

According to various studies, caffeic acid (CA), along with its ester derivatives, has shown significant therapeutic effects against AD. In an in vivo and in vitro study by Colonnello et al. (95), rats and C. elegans were exposed to various toxins, and then administered CA at doses of 100 μM and 25 mM, respectively. The results showed that CA prevented cell dysfunction, oxidative damage and transcriptional regulation in the rat cortex, and restored the activity of NRF2/ARE, while in C. elegans it reduced the loss of survival and motor alterations caused by the toxins. The compounds CA phenethyl ester (CAPE) and CA phenethyl ester 4-O-glucoside (FA-97) were also examined for their potential activity against AD. CAPE showed a protective effect against the memory impairment induced by intracerebral injection of Aβ_O (amyloid-β oligomers) in mice, preventing the increase of ROS levels, neurodegeneration and neuroinflammation. Its administration was carried out at a dose of 10 mg/kg intraperitoneally (i.p.) after intracerebroventricular (i.c.v.) injection of Aβ_O and led to the neutralization of oxidative stress through the induction of NRF2/HO-1 pathway in the hippocampus of mice (96). FA-97 protected against hydrogen peroxide (H₂O₂)-induced apoptosis, decreased ROS and MDA (malondialdehyde) levels, and increased GSH and SOD levels in SH-SY5Y and PC12 cells through activation of NRF2/HO-1 signaling. In vivo, FA-97 prevented scopolamine-induced cognitive impairment by reducing neuronal apoptosis, protecting against cholinergic system dysfunction, and reversing elevated MDA level and low total antioxidant capacity (T-AOC) in mouse brains (97) ( Table 1 ).

Flavonoids are a diverse group of antioxidant polyphenolic compounds that are widely distributed in plants. They are characterized by a common structure consisting of two aromatic rings (A and B rings) connected by a three-carbon bridge (C ring). The C ring often contains oxygen atoms, forming various subgroups of flavonoids, including flavones, flavonols, flavanones, flavanols (catechins), and anthocyanins. Biologically, flavonoids exhibit anti-inflammatory, antioxidant, anti-cancer, cardioprotective, and neuroprotective activities (98). Flavonoids can modulate various cellular signaling pathways, gene expression, enzyme activities, and cellular functions, contributing to their biological effects, while they activate NRF2 signaling through similar mechanisms as polyphenols.

Quercetin is one of the main flavonoid compounds with potential therapeutic activity against various diseases, including neurodegenerative diseases (99). When administered i.p. at a dose of 50 mg/kg to a rat model of streptozotocin (STZ)-induced AD, it provided neuroprotection by activating the α7 nAChR (nicotinic acetylcholine receptor)/NRF2/HO-1 pathway, improving cognitive deficits, and reducing acetylcholinesterase (AChE) activity, Aβ aggregation and mitochondrial toxicity (100). A different study in a PC12 cell model that had undergone Aβ_25-35-induced cytotoxicity, showed that the use of quercetin at different concentrations (0, 10, 20, 40 and 80 μmol/L) increased cell survival and proliferation, T-AOC, as well as the levels of the antioxidant enzymes SOD, GPx, CAT, sirtuin 1 and NRF2. It also reduced LDH (lactate dehydrogenase), AChE, MDA and HO-1 levels (101). In addition, the combination of quercetin with sitagliptin (Sita), using quercetin as pretreatment dissolved in olive oil at a dose of 100 mg/kg, showed good results in rats with Aβ-induced AD. The combination significantly increased escape latency in the Morris water maze (MWM) test of cognitive function, decreased Aβ_1-42 levels, and increased NRF2/HO-1 expression and activity in rat brain (102).

Cyanidin-3-glucoside (C3G), a major anthocyanin found in berries, reduced L-glutamic acid (L-Glu)-induced oxidative damage and apoptosis, in mouse hippocampal HT22 neuronal cells, according to a study by Sukprasansap et al. (103). C3G exerted neuroprotective and antioxidant activities by inhibiting the expression of oxidative proteins, such as calpain and caspase-12, regulating the expression of survival proteins, such as NRF2 and ERK (extracellular signal-related kinase), and enhancing the activation of the endogenous antioxidant system, including proteins SOD, CAT, and GPx.

In another study using the APP/PS1 AD mouse model and HT22 cells, anthocyanin from Korean black beans supplementation was observed to control the PI3K/Akt/GSK-3 pathway which subsequently reduced Aβ_O-induced oxidative stress and neurotoxicity by activating the NRF2 antioxidant system. The supplement improved memory capabilities and pre- and postsynaptic protein markers in vivo, highlighting it as a potent neuroprotective and antioxidant agent in AD (18).

Another flavonoid, folecitin, which is extracted from Hypericum oblongifolium Wall, was investigated for its therapeutic potential against lipopolysaccharide (LPS)-induced memory impairment caused by Aβ production in a mouse model. LPS was administered i.p. at a dose of 250 μg/kg/day for 3 consecutive weeks, followed by co-administration of folecitin (30 mg/kg/day) with LPS for the last two weeks. The results showed that folecitin significantly reduced LPS-induced apoptotic proteins, inhibited BACE1, and restored both presynaptic and postsynaptic connections, ameliorating memory impairment. Folecitin also activated the endogenous antioxidant proteins NRF2 and HO-1, by inducing phosphorylation of Akt pathway proteins (104).

Treatment with 10 µM and 20 µM isoliquiritigenin (ISL), a natural flavonoid found in licorice root, counteracted oxidative stress and inflammation in BV-2 cells stimulated with Aβ_O, as it significantly reduced the production of inflammatory cytokines and nitric oxide (NO) and restored the morphological changes induced by Aβ_O. In addition, it activated NRF2 signaling and suppressed nuclear factor kappa beta (NF-κB) signaling (105).

Nobiletin, a bioflavonoid derived from citrus peels, and hawthorn leaf flavonoids (HLF) were explored in different studies for their effect on Aβ- and Aβ_25-35-induced toxicity, respectively, in rat models, where they showed equally significant therapeutic effects. Nobiletin was administered at a dose of 10 mg/kg/day from day 0 to day 7 after Aβ injection, and was shown to improve cognitive function and neuronal loss in the hippocampus, while reducing oxidative damage and neuroinflammation. These effects, according to the researchers, were partially attributed to the regulation of the toll-like receptor 4 (TLR4)/NF-κB/NRF2 pathways (106). HLF was administered at doses of 50, 100, and 200 mg/kg for a period of 30 days and showed a significant improvement in neuronal damage and memory deficits, an increase in the activity and levels of the antioxidant enzymes SOD, CAT, and GSH, and a decrease in MDA. HLF reduced oxidative stress specifically through activation of the NRF2/ARE signaling pathway (107).

Farrerol, an important bioactive component of rhododendron belonging to the class of flavones, was also found to be able to reduce Aβ-induced oxidative stress and inflammation in mouse BV-2 microglial cells at various concentrations (1.5 and 10 µM), through enhancing the activation of the NRF2/Keap1 pathway (108). Similarly, genistein, an isoflavone, was able to exert in vitro neuroprotective activity via the NRF2/HO-1/PI3K signaling in the SH-SY5Y cell line, which had undergone Aβ_25-35-induced toxicity at doses of 10, 30, and 50 µM (109).

The methoxy-flavonoid, umuhengerin, was administered to an AD mouse model induced by intracerebral injection of STZ. Mice were orally administered umuhengerin at a dose of 30 mg/kg or donepezil as a positive control at a dose of 2.5 mg/kg for 21 days, and the results showed that umuhengerin attenuated STZ-induced neuroinflammation and oxidative stress by upregulating NRF2 and downregulating Keap1 expression, resulting in improved cognitive function. In addition, umuhengerin reduced the expression of β-secretase and NF-κB, as well as AChE activity, leading to reduced Aβ formation (110).

Hesperetin, a bioactive flavonoid produced from hesperidin, significantly reduced oxidative stress, apoptosis, and neurodegeneration in vivo at a dose of 50 mg/kg in the brain of mice that had received an i.c.v. injection of Aβ 24 h earlier. Similar results were also found in vitro at concentrations of 10, 20 or 50 µM hesperetin in HT22 and BV-2 cells challenged with Aβ, through reduction of ROS levels and lipid peroxidation and increase of NRF2/HO-1 expression (111).

Isoastilbin (IAB) was used against AD in a mouse model induced by AlCl₃ and D-lactose, as well as in PC12 cells induced by L-Glu, and the results showed that it reduced oxidative damage in both models, while also reducing the deposition of Aβ and phosphorylated tau in the brain of mice. IAB further enhanced the nuclear levels of NRF2 and increased the expression of antioxidant enzymes regulated by it (112).

Finally, the flavonoid compound complanatoside A (CA), in an in vivo study, had the ability to extend the lifespan of C. elegans in a dose-dependent manner, with the 50 μM concentration exerting the maximum increase in lifespan by 16.87%. The mechanism found after further investigation was based on the involvement of DAF-16/FOXO, SKN-1 and heat shock factor 1 (HSF-1) signaling pathways. CA also caused a reduction in the aggregation of toxic proteins Aβ and α-synuclein, and improved the motility of the organisms (113) ( Table 2 ).

Chalcones are a class of flavonoid compounds consisting of two aromatic rings connected by a three-carbon α,β-unsaturated carbonyl system. They often contain hydroxyl (-OH) groups and other functional groups on the rings. The presence of the α,β-unsaturated carbonyl system imparts reactivity to chalcones, allowing them to undergo various chemical transformations (114). Chalcones have demonstrated a range of pharmacological properties, including antioxidant, anti-inflammatory, anticancer, antimicrobial, and neuroprotective activities. They interact with multiple cellular targets and signaling pathways, contributing to their diverse biological effects, while they activate NRF2 similarly to the previous categories.

2,3-dihydroxy-4,6-dimethoxychalcone (DDC) was shown to be protective against cellular oxidative damage and to reduce Aβ accumulation in primary mouse cortical neurons when administered at a dose of 30 µM in a 3-hour treatment. In a 24-hour treatment it appeared to increase HO-1 expression through activation of the NRF2/ARE pathway. However, neuronal death was not affected by DCC in either condition, suggesting that this mechanism is not the main neuroprotective mechanism of DCC (115).

Another compound of the chalcone class, hesperidin methylchalcone (HMC), was evaluated for its effect on the Neuro-2a cell model and in a Wistar rat model, where it showed a significant effect in reducing Aβ accumulation, protecting the cells from Aβ-induced toxicity. Also, HMC improved cognitive deficits in rats through enhancing cholinergic effects, inhibiting AChE, β-secretase, and caspase-3. Finally, HCM reduced Aβ-derived oxidative stress and reduced neuroinflammation by suppressing the NF-κB pathway and activating the NRF2/HO-1 pathway (116) ( Table 3 ).

Terpenoids are produced by plants, fungi, and some animals. Chemically, terpenoids are derived from a basic building block called isoprene. Isoprene units are combined to form different-sized carbon chains, which determine the classification of terpenoids. They are involved in plant defense mechanisms, serving as components of essential oils and contributing to the plant’s resistance against pathogens and herbivores. Terpenoids also have medicinal properties and are used in traditional medicine for their antimicrobial, anti-inflammatory, antioxidant, and anticancer effects (117). Similar to previous categories, terpenoids upregulate NRF2 leading to induction of phase II detoxifying enzymes and modulation of redox signaling.

Cycloastragenol, a triterpenoid saponin isolated from various legume species of the Astragalus genus, was administered orally at a dose of 20 mg/kg/day for 6 weeks to an AD mouse model induced by intracerebral injection of Aβ (5 μg/mouse). Its administration resulted in the enhancement of the expression of NRF2, HO-1, brain-derived neurotrophic factor (BDNF), phosphorylated tropomyosin-related kinase B receptor (p-TrkB) and neuronal nuclear protein (NeuN), while it reduced the expression of activated microglial cells and inflammatory cytokines. In addition, cycloastragenol improved apoptotic cell death and memory impairment (118).

Another terpenoid compound that showed therapeutic effect in AD is andrographolide (Andro), a diterpenoid isolated from the stem and leaves of Andrographis paniculata. Andro was tested in PC12 cells subjected to toxicity induced by aluminum maltolate [Al(mal)₃] (700 µM) at doses of 1.25, 2.5, 5, 10, 20 and 40 µM and the results showed that the groups treated with 5 and 10 µM Andro had significantly increased cell viability from 67.4% to 91.9% and 91.2%, respectively, decreased expression of proteins related to neurotoxicity, such as APP and BACE1, while they had increased expression of NRF2, p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). Finally, an increase in the nuclear translocation of NRF2 and p62 and a decrease in the expression of Keap1 in the cytoplasm were observed (119).

Quinovic acid (QA), also a member of the terpene family, was studied for its activity against intracerebral Aβ_1-42 injection-induced toxicity, cognitive deficits, and cholesterol dyshomeostasis in male C57BL/6J mice. The results showed that mice treated with Aβ_1-42 had increased formation of Aβ oligomers, cholesterol accumulation in the brain and oxidative stress. However, QA administration significantly improved these outcomes by preventing oxidative stress through upregulating the NRF2/HO-1 pathway, reducing gliosis, neuroinflammatory mediators and apoptotic markers, and improving synaptic indices and spatial learning behaviors and memory (120).

Astaxanthin (ATX), mainly found in algae (Haematococcus pluvialis), yeasts (in yeast fungus Xanthophyllomyces dendrorhous) and animal species including trout and salmon, is a dark red xanthophylls carotenoid. It is also the most abundant flavonoid in propolis. In this study by Hafez et al. (121), it was investigated in AD-like rats induced by hydrated aluminum chloride (AlCl₃ ^·6H₂O) solution in doses ranging from 5 to 15 mg/kg p.o. for 6 weeks. ATX was found to improve the MWM performance of rats, accompanied by suppressed accumulation of Aβ_1-42 and MDA. It also inhibited AChE and monoamine oxidase, as well as BACE1. Finally, ATX also upregulated acetylcholine, serotonin, and NRF2, along with miRNA-124, proposing a good anti-AD activity (121). These neuroprotective effects were also corroborated in other studies which highlighted the implication of PI3K/Akt/GSK-3β/NRF2 signaling pathway in the mechanism of action of this phytochemical (122).

Furthermore, in vivo research in an experimental model of C. elegans showed that the compounds 2-butoxytetrahydrofuran (2-BTHF) and palmitic acid, diterpene glycosides derived from the sea cucumber Holothuria scabra, were able to extend the lifespan of C. elegans and enhance oxidative resistance, mainly through activation of DAF-16/FOXO/insulin/IGF (insulin-growth factor) and SKN-1/NRF2 signaling pathways (123) ( Table 4 ). It is important to note that palmitic acid was administered at the rather low dose of 1 μg/mL, which might explain its beneficial effect, since this saturated fatty acid has been typically associated with negative effects on cell viability.

Alkaloids are another group of NRF2 activators. They are characterized by their nitrogen-containing heterocyclic structures and often exhibit potent physiological and pharmacological effects, as they can act as analgesics, stimulants, sedatives, anti-inflammatory agents, antimicrobials, anticancer agents, etc. (124).

A study investigating the beneficial effects of fangchinoline (FAN), a natural alkaloid, on HT22 cells with L-Glu-induced oxidative damage showed that FAN (1, 3, 5 μM) inhibited cell apoptosis in a dose-dependent manner and significantly decreased oxidative damage, reducing intracellular ROS levels and enhancing the activity of SOD and the endogenous antioxidant system. The underlying mechanism involved in these actions was the upregulation of NRF2/HO-1 levels through the downregulation of Keap1 protein expression (125).

Berberine, another natural alkaloid, was shown to decrease the activity of NF-κB and enhance the activity of the NRF2/HO-1 antioxidant system in vitro in HT22 cells exposed to Aβ_25-35, providing neuroprotective effects. This mechanism was found in combination with other mechanisms, such as inhibition of cell apoptosis and reduction of oxidative stress, as well as improvement of mitochondrial membrane potential (126).

Finally, according to a study by Jiang et al. (127), i.p. administration of Rhynchophylline (Rhy) (10 or 20 mg/kg), an alkaloid found in the Uncaria species, to Aβ_1-42-induced AD mice resulted in amelioration of cognitive impairment, limiting oxidative damage by reducing ROS, MDA and GSH levels and restoring the expression of NRF2 and the antioxidant enzymes it regulates in the frontal cortex and hippocampus of mice. Rhy was shown to exert its protective effects through activation of NRF2-ARE, as, when cells were transfected with NRF2 siRNA, Rhy activity was abolished ( Table 5 ).

Polysaccharides are complex carbohydrates composed of long chains of the same or different monosaccharide units joined together by glycosidic bonds. They are abundant in nature and play crucial roles as structural components and energy storage molecules in organisms. In plants, they function as structural components in cell walls (e.g., cellulose) or as storage molecules (e.g., starch). In animals, polysaccharides such as glycogen serve as a storage form of glucose. Polysaccharides also play roles in cell recognition, immune response modulation, and as energy sources (128). The activation of NRF2 by polysaccharides has been reported in several studies, some of them described below.

Polysaccharides have shown therapeutic potential in reducing the pathogenesis of AD by demonstrating significant neuroprotective effects. According to a study by Murphy et al. (129), in which intranasal application of the polysaccharide Mini-GAGR, a 0.7-kDa cleavage product of low-acyl gellan gum, was administered, the levels and nuclear translocation of NRF2 were increased, through enhanced dissociation from its inhibitor, Keap1. An upregulation in NRF2-dependent antioxidant enzymes and growth associated protein 43 (GAP43) was also found. Administration was performed at a dose of 100 nmol/40μl Mini-GAGR (20μl/nostril) in 3xTg-AD mouse models (with an average weight of 25.5g) once daily for a period of 20 days. After 20 days of treatment, a reduction in p-tau and Aβ levels was observed in neurons in vivo, and memory improvement was also observed.

In a different study investigating the neuroprotective properties of Amanita caesarea polysaccharides (ACPS), it was found that pre-treatment with ACPS (2.5 or 5 μg/ml) in HT22 cells 3 hours before their exposure to L-Glu (25 mM), improved cell viability and oxidative stress by enhancing the nuclear levels of NRF2 and reducing the cytoplasmic levels of NRF2 and cytochrome C. In addition, it suppressed the expression of Keap1 protein and enhanced the expression of antioxidant enzymes such as HO-1, SOD and GCLC (130) ( Table 6 ).

Ginsenosides are a group of bioactive compounds found in the roots of Panax ginseng and related plants. They are classified as triterpene saponins, consisting of a dammarane triterpene backbone with sugar moieties attached, and are known for their diverse pharmacological properties and health benefits. They have been studied for their anti-diabetic, neuroprotective, anti-inflammatory, antioxidant, anticancer, and immune-modulating properties, among others (131).

According to studies in AD mouse models, two steroidal glycosides of the Ginsenoside family, Ginsenoside K (CK) and Ginsenoside Rk3 (GRk3), showed promising neuroprotective and antioxidant effects. CK (20 and 40 mg/kg) was found to significantly improve memory and reduce cell apoptosis and oxidative stress in a mouse model of scopolamine hydrobromide-induced memory impairment. CK also showed the ability to inhibit the expression of Aβ, as well as the ability to enhance the activation of the NRF2/Keap1 pathway and the activity of the endogenous antioxidant system (132). GRk3, according to a study in a model of APP/PS1 mice, restored mitochondrial membrane potential, reduced intracellular ROS production and reduced neuronal apoptosis, through activation of the AMPK/NRF2 signaling pathway. It also improved learning and memory deficits in vivo (133). Additionally, another compound of the same family, Ginsenoside Re, was evaluated for its effect against Aβ-induced cytotoxicity and apoptosis in SH-SY5Y cells, and the results showed that at doses of 20, 25, and 30 μM, it was able to inhibit Aβ-mediated mitochondrial apoptosis, reduce cytochrome C release, and inactivate caspase-3/9, while it was also found to enhance NRF2 activation (134). Finally, gintonin, a glycolipoprotein also derived from ginseng root, was administered as a treatment to Aβ-induced mice at a dose of 100 mg/kg/day (p.o.) for four weeks. It resulted in the reduction of oxidative stress by enhancing the expression of NRF2 and HO-1, which led to a reduction in ROS levels and lipid peroxidation. Furthermore, it suppressed activated microglial cells and inflammatory mediators, thereby improving synaptic and memory functions in the brain of Aβ-compromised mice (135) ( Table 7 ).

Bacopa monnieri extract, which contains various compounds such as the alkaloids brahmin, nicotine, herpestine, bacosides A and B, saponins A, B and C, triterpenoid saponins, stigmastanol, β-stigasterol, α-alanine, aspartic acid, glutamic acid, and serine and pseudojumbogenin glycosides (136, 137), was tested at doses from 10 to 100 µg/ml and was shown to inhibit tau protein aggregation in vitro and acted as an antioxidant by restoring NRF2 levels in Neuro-2a cells. It also reduced p-tau load and GSK-3β phosphorylation in formaldehyde-stressed cells, suggesting that Bacopa monnieri could be considered a potent herb against tau protein phosphorylation and aggregation (138).

Another compound tested for its therapeutic activity against tau pathology in mouse fibroblasts is dimethyl fumarate (DMF), which is an inducer of the transcription factor NRF2. The results showed that DMF, at a dose of 100 mg/kg intragastrically (i.g.) daily for 3 weeks, induces NRF2 transcription through a mechanism involving Keap1 and PI3K/Akt/GSK-3β pathways. DMF modulates hippocampal GSK-3β activity, tau phosphorylation, neuronal injury, and inflammatory processes involved in astrogliosis, microgliosis, and proinflammatory cytokine production. These findings suggest that DMF may have neuroprotective effects beyond the Keap1/NRF2 axis, by inhibiting GSK-3β in a mouse model of tauopathy (75).

Melatonin, which, in addition to bacteria and eukaryotic cells, is also found in plant organisms (139), was able to inhibit p-tau aggregation-induced toxicity in Neuro-2a and N9 cells, as well as to reduce the levels of p-tau. It also enhanced the cellular levels of NRF2 and its nuclear translocation in response to oxidative stress. Melatonin also upregulated anti-inflammatory cytokines and activated MAP3K (mitogen activated protein kinase kinase kinase) in tau-compromised microglial cells and neurons, while downregulating the pro-inflammatory proteins IL-1β and COX-2 (140).

Oxyphylla A, a compound extracted from Alpinia oxyphylla, was tested in in vivo and in vitro AD models at various concentrations (12.5-100 µM) and the results showed that it could reduce the expression of APP and Aβ and alleviate cognitive deficits in SAMP8 mice. In addition, oxyphylla A exerted antioxidant activity through the Akt/GSK-3β and NRF2/Keap1/HO-1 pathways (141).

According to a study by Wang et al. (142), who investigated the therapeutic effects of the extract of Ginkgo biloba 761 (EGb761) against Aβ-induced cell damage in an in vitro model of AD, it was found that EGb761 (25, 50, 100 and 250 μg/ml) resulted in ameliorating cell damage by inhibiting apoptosis, reducing ROS production and blood-brain barrier permeability, while enhancing the expression of Akt, NRF2 and HO-1.

Forsythoside A (FA) was also studied in vitro in an array of cell lines (Aβ_1-42-treated N2a cells, erastin-treated HT22 cells and LPS-induced BV-2 cells), where it showed similar results. Specifically, it provoked decreased lipid peroxidation and iron deposition, and enhanced mitochondrial activity and dopaminergic signaling. Pro-inflammatory cytokines were found downregulated, including IL-6, NO and IL-1, contrasting the levels of anti-inflammatory cytokines, while NF-kB signaling was also diminished. The in vivo results in male APP/PS1 double transgenic AD mice proposed an amelioration of memory and cognitive deficits, accompanied by reduced Aβ deposition and p-tau levels in the brain. All these results significantly implicated the NRF2/GPx4 (glutathione peroxidase 4) axis (143).

In vitro studies in PC12 cells that had undergone oxidative damage and apoptosis induced by Aβ_25-35 showed that the compounds (1E,4E)-1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one (CB) and (1E,4E)-1-(3,4-dimethoxyphenyl)-5-(4-hydroxy-3,5-dimethoxyphenyl)penta-1,4-dien-3-one (FE), two curcumin analogues, as well as sargahydroquinoic acid (SHQA), a marine natural compound found in various species of the brown macroalga genus Sargassum, showed significant neuroprotective effects by enhancing the expression and translocation of NRF2, as well as the expression of NRF2-regulated antioxidant enzymes (19, 144).

A study in AD rats administered with Benincasa hispida fruit extract, an extract rich in alkaloids, flavonoids, vitamins, volatile polysaccharides, hemicellulose polysaccharides, terpenes, sterols, amino acids and fatty acids (145), in doses of 250 and 500 mg/kg/day for a period of 16 days, showed, among other findings, that the antioxidant genes Keap1/NRF2/HO-1 were upregulated (146).

3H-1,2-dithiole-3-thione (D3T) is a compound found naturally in cruciferous plants such as broccoli and cauliflower. When i.p. administered (10 and 20 mg/kg) in a Tg2576 mouse model of AD, it significantly improved cognitive deficit, reduced Aβ accumulation and oxidative damage, and upregulated NRF2 and HO-1 levels, promoting neurogenesis in the mouse hippocampus (147).

According to a study in N2a/APPswe cells (mouse Neuro 2a cells expressing human APP harboring the Swedish mutation), it was found that sulforaphane at concentrations of 1.25 and 2.5 µM increased the expression and promoted the nuclear translocation of NRF2 by reducing the DNA demethylation levels of the NRF2 promoter, while a significant reduction in the intracellular levels of Aβ_1-40 and Aβ_1-42 and an increase in SOD were also observed (148).

In vivo and in vitro research on the potential actions of osthole (OST), a coumarin derivative, against AD showed that OST had good neuroprotective effects, particularly through the NRF2 pathway. In L-Glu-induced HT22 cells, preincubation with 20 and 40 μM OST helped to enhance cell viability, reduce apoptosis, and inhibit caspase-3, caspase-8, and caspase-9. OST also reduced intracellular ROS levels, restored mitochondrial membrane potential, and upregulated the expression of NRF2 and its regulated proteins. In APP/PS1 transgenic mice, administration of 15 and 30 mg/kg OST for 8 weeks resulted in improved memory and cognitive behaviors, decreased Aβ and p-tau accumulation, and enhanced NRF2 and downstream proteins expression levels, including SOD-1 and HO-1 (149).

According to the results of the study by Nakhate et al. (150), i.p. administration of 0.5 and 1 mg/kg of the compound 5-hydroxy-2-methyl-1,4-naphthoquinone or plumbagin one hour before the first dose of STZ in a mouse model of STZ-induced AD, resulted in improving cognitive deficits, as assessed by MWM, through inhibition of the β-secretase enzyme and activation of the NRF2/ARE pathway.

Finally, treatment with Hibiscus sabdariffa L. extract (HSE), hydroxycitric acid (HCA) and isocitric acid (ICA) in a C. elegans model showed that HSE and HCA prolonged the lifespan of C. elegans by 24% and 6%, respectively, while ICA did not show similar efficacy. HSE and HCA also enhanced the nuclear localization of the transcription factors DAF-16/FOXO and SKN-1/NRF2 (151) ( Table 8 ).

The main mechanisms of action of the previously discussed phytochemicals are summarized in Figure 4 .