Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive neuronal degeneration in the brain resulting in the impairment of behavior, cognition, memory, language, etc. AD mainly affects individuals in the later years of life and is considered the sixth leading cause of death in elderly patients. In AD, the individual initially shows the loss of short-term episodic and the retention of the long-term memory. However, in the later stages, psychiatric impairment occurs in the patients [1,2,3]. AD can be categorized into two types: familial AD (FAD) presents the minority of AD cases (approx. 1%) and sporadic AD (SAD) or late-onset AD (LOAD) presents the majority of the cases of the AD and aging is considered the main cause of this form of AD [4, 5]. Pathologically, in AD, there is the deposition of senile plaque, accumulation of fibrils, and neurofibrillary tangles (NFTs that ultimately leads to neuronal degeneration in the brain [6, 7]. Amyloid beta (Aβ is the major component of the senile plaques [8], whereas tau protein is the main component of neurofibrillary tangles observed in the brain of AD patients [9]. Aβ is generated as a heterogeneous pool of peptides, and it occurs mainly in two isoforms i.e., Aβ1-40 and Aβ1-42 of which Aβ1-42 peptide is considered more amyloidogenic than Aβ1-40 [10]. Aβ is generated by the sequential cleavage of amyloid precursor protein (APP) by the enzymes, i.e., α and β-secretase [11]. The production of the Aβ is catalyzed by the enzyme γ-secretase [12]. Aβ so produced is mainly non-toxic in its monomeric form but when aggregates to form oligomers followed by the production of senile plaques (fibrils) confers neurotoxicity [11]. Aβ is also responsible for the formation of NFTs by causing the hyperphosphorylation of tau protein [13], inhibition of the binding of the tau with the microtubules [14], leading to their destabilization resulting in the neuronal injury and death [15]. It is suggested that the aggregated tau is more toxic while the soluble and prefibrillar tau is more likely to cause neurodegeneration [16]. It has been reported that oxidative stress may increase the phosphorylation of tau and thus may accelerate the neurodegeneration in the patients of AD [17,18,19]. Further higher levels of phosphorylated tau have been observed in AD patients [20, 21]. In AD, the neurofibrillary degeneration starts in the allocortex and spreads to the associative isocortex [22]. However, the neuronal loss often exceeds NFTs [23]. Therefore, the therapies that inhibit the phosphorylation of tau and its oligomerization may confer therapeutic benefits in patients of AD [12]. However, besides promoting the phosphorylation of tau protein, Aβ also increases the intracellular levels of Ca2+ by opening the calcium channels [24] which further promote the influx of Ca2+ [25] resulting in the Ca2+ overload responsible for the increase production of Aβ (by increasing the expression of β-secretase enzyme), aggregation of Aβ [26, 27], and Aβ-induced membrane damage, fragmentation, and lipid loss [28]. Ca2+ overload also promotes the influx of Ca2+ inside the mitochondria resulting in the mitochondrial outer membrane permeabilization (MOMP), release of pro-apoptotic factors, and subsequent neuronal apoptosis [29]. These facts suggested that reducing the Ca2+ influx may counteract the neurotoxicity confers by the Aβ oligomers in the hippocampus of AD transgenic mice [30]. It has been reported that ROS produced during oxidative stress also utilize the signaling pathways involving Ca2 + to confer mitochondrial and neuronal damage. Further, mitochondrial dysfunction also promotes the production of ROS [31] and the increase production of ROS has been implicated in aging and AD [32]. Antioxidants are the chemical compounds that counteract the oxidative damage conferred by ROS [33, 34]. Preclinical studies suggested that the antioxidant supplementation improves the learning ability [35] and memory functions in rodents [36] by providing protection against extracellular and intracellular ROS [37, 38]. Besides this, the antioxidants inhibit the aggregation of Aβ [39, 40], improve cognition and Mini-Mental State Exam (MMSE) score in AD patients [41, 42], and modulate the therapeutic effect of vitamin B in AD patients [43]. Mitochondria is the cell organelle that is affected mainly by oxidative stress thus the treatment with the mitochondriotropic antioxidants having low cytotoxic effects may improve the outcomes by protecting the mitochondria against the deleterious effects of oxidative stress [44]. However, despite these positive findings, several studies suggested no clear significant effect of antioxidants in AD patients [45,46,47] and convincing evidence regarding their therapeutic efficacy in AD is still lacking [48, 49]. In the present review, the authors describe the production of ROS, ROS-mediated damage in AD, the role of antioxidants, and the therapeutic efficacy of antioxidants in the treatment of AD.

Mitochondrial ROS Formation and Oxidative Stress

Mitochondria are considered the powerhouse of cells and play an important role in the production of energy, thus the mitochondrial impairment results in the impaired energy metabolism, increased production of ROS, and impaired Ca2+ buffering [50]. Mitochondrial formation utilizes the electron transport chain (ETC) and its several complexes for the production of ATP. These complexes are located on the inner mitochondrial membrane together with complex-V [51]. Besides this, the ETC required cytochrome c and co-enzyme Q10. Electrons are transported through a series of complexes and ultimately transferred to the molecular oxygen (O2) resulting in the production of water. The simultaneous translocation of protons from the matrix to intermembrane space creates a proton gradient responsible for the production of ATP, and this reaction is catalyzed by the enzyme ATP synthase [52, 53]. The transfer of the electron across the complexes is a very efficient process,however, when the electron gets leaks from the ETC or its complexes, the electron is captured by the molecular oxygen resulting in the production of ROS. The capturing of the electron by the oxygen molecule is responsible for the production of superoxide radical (O2−), which is then converted into hydrogen peroxides (H2O2) (by the enzyme superoxide dismutase) and the later is converted into hydroxyl radical (OH−) (by the enzyme catalase) [54,55,56,57], and these ROS confer oxidative damage [58, 59] (Fig. 1). Besides increasing the production of ROS, Aβ also depletes endogenous antioxidants of the body [60] resulting in lipoperoxidation and neuronal dysfunctions [61]. Aβ aggregates also promote phosphorylation of tau proteins [62,63,64] which then interferes in the activity of complex-I of ETC, contributing to mitochondrial dysfunction and ROS production [65]. Aβ oligomers mediated ROS formation resulting in the NMDA activation and increased influx of Ca2+ ions through the calcium ion channel responsible for neuronal excitotoxicity and cell death [66,67,68,69]. Besides this, the Aβ itself induces the increase in intracellular Ca2+ concentrations in neuronal cells [70, 71]. The Ca2+ overload causes the impairment in the normal functioning of the mitochondria resulting in the excessive production of ROS responsible for the MOMP and the release of pro-apoptotic factors in the cytoplasm responsible for the neuronal apoptosis [29]. ROS may trigger the opening of mitochondrial permeability transition pore (mPTP) which is involved in the regulation of mitochondrial Ca2+ homeostasis [72] (Fig. 2). ROS induces the opening of mPTP resulting in further release of ROS, and this phenomenon is referred to as “ROS-induced ROS release (RIRR)” [73]. It has been reported that the short and transient opening of mPTP is important for the passage of protons, Ca2+, and other substances [74, 75], but the prolonged opening of mPTP causes mitochondrial disruption and deposition of Ca2+ in the mitochondrial matrix [76]. Besides this, Ca2+ ions also participate in the Aβ-promoted membrane damage and lipid loss [28]. Aging is characterized by the increased production of ROS [77, 78]. ROS further participates in the glycation processes and the formation of advanced glycation end products (AGEs) that has been observed in the plaques of AD patients [79, 80], causing mitochondrial dysfunction [81,82,83,84] and subsequent neuronal degeneration [85,86,87,88,89].

Fig. 1
figure 1

Production of reactiveoxygen species

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Fig. 2
figure 2

ROS-mediated mitochondrial impairment and cell death

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Aβ-Mediated Oxidative Stress and Damage in AD

AD is characterized by the accumulation of Aβ plaques and NFT formation [90] both of which are linked with the production of ROS responsible for the neuronal, synaptic, and mitochondrial dysfunctions in AD patients [50, 91,92,93,94,95]. Oxidative stress facilitates the expression of protein, i.e., amyloid precursor protein (APP) and beta-secretase (BACE1) involved in the increased generation of Aβ [96]. Oxidative stress also induces a pathogenic PS1 conformational change in neurons, resulting in the increased ratio of Aβ42/40 [97]. Oxidative stress may also produce interference in the clearance of Aβ by oxidizing the LRP1. LRP1 is a multifunctional protein that is responsible for the efflux of Aβ across the BBB [98, 99]. Aβ is responsible for the disruption of the normal functioning of the LRP1 protein and thus restricts its clearance and favors its accumulation in the brain [100]. Also, the reduced activity of LRP1 protein has been observed in the brain of AD patients [101]. Aβ activates the JNK/c-jun pathway and induces apoptosis in in vitro models [102,103,104]. JNK has been shown to promote the phosphorylation of tau protein in various cells and animal models [105]. Tau plays a key role in the maintenance of microtubule dynamics and the protection of DNA and RNA from ROS-mediated damage. Thus, the alteration in the tau protein is responsible for the increased oxidation of DNA and RNA [106] and mitochondrial dysfunction in an animal model of AD. Aβ-mediated oxidative stress is responsible for the modification and the alterations in the normal conformation of tau protein resulting in the formation of NFTs [105]. ROS formations facilitate the self-assembly of tau protein into fibrillary polymers [107]. The conformational changes in the tau protein and the formation of the NFTs may further evoke neuronal degeneration [108]. However, it has been reported that the formation of neurofibrillary lesions counteracts the damaging effects of oxidative damage on neuronal cells suggesting neurofibrillary formation is a neuronal adaptation to chronic oxidative stress and functions as the antioxidant to protect the neurons [109]. Besides this, Aβ also induces the rise in the intracellular Ca2+ levels by the activations of calcium channels, glutamate receptors, etc. [110,111,112,113,114]. The increased conductance through the Ca2+ channels resulted in the mitochondrial Ca2+ overload and ROS overproduction resulting in the in mitochondrial outer membrane permeabilization (MOMP) and release of pro-apoptotic responsible for neuronal apoptosis [29].

This evidence suggested that oxidative and nitrosative stress play a key role in the pathogenesis and damage in AD [115, 116]. Oxidative stress is imposed by the overproduction and the increased levels of the Aβ which is responsible for the oxidation of proteins, lipids, and nucleic acids in the hippocampus and cortex region of the brain [117]. This is further confirmed by the finding suggesting the increased oxidation of protein and lipid in the brain regions rich in Aβ [118, 119]. The increased levels of the carbonylated proteins in the hippocampus and parietal cortex suggested protein oxidation in the AD brain [120]. Further, the membrane proteins are damaged more as compared to the cytoplasmic proteins [121]. Protein modification resulting from the reaction of the proteins with 4-hydroxynonenal (4-HNE) and peroxynitrite (ONOO–) increases the proteosomal degradation of brain proteins [122]. Further, the evidence suggesting the lipid oxidation is suggested by the increased concentrations of 4-HNE in the brain hippocampus of AD patients [123]. 4-HNE is also responsible for the oxidative modification of lipoic acid [124] “apparently” evident in hippocampal regions of the brain [125]. Oxidative stress also confers the oxidative damage to the nuclear and mitochondrial DNA in AD in frontal, parietal, and temporal cortex [126, 127]. It is further confirmed by the studies suggesting the increased levels of 8-hydroxyguanine in the hippocampus of AD patients in the preclinical stage of disease [128]. The oxidative DNA damage has also been observed in the peripheral blood cells of AD patients [129]. RNA oxidation especially mRNA oxidation has been reported in the frontal cortex region of the brain [130]. Oxidative stress is also responsible for the reduced production of ATP and brain energy metabolism both of which have been considered the key event in AD pathogenesis [131]. The reduction of the ATP levels causes the leakage of the electron from the ETC resulting in the overproduction of ROS [132]. Oxidative stress reduces the expression or inactivates the enzyme ATP synthase in the AD brain [133,134,135]. It has been reported that the accumulated Aβ in the AD brain directly interact with the ATP synthase, inhibit it [136], and thus impaired the ATP production in AD [137] further suggesting the link between AD and diabetes [118, 119].

Oxidative stress also influences the expression of ApoE4 [138] which is involved in the regulation of aggregation and clearance of Aβ [139, 140], mitochondrial dysfunction, calcium homeostasis [141, 142], synaptic architecture, and functioning [143,144,145]. It has been reported that the impaired ApoE expression is linked to oxidative stress implicated in AD [146,147,148,149]. The role of ROS in the emergence of AD and the consequences of Aβ deposition and its role in AD-related neuronal degeneration are presented in Fig. 3a, b.

Fig. 3
figure 3

a Factors contributingto the ROS Production and Alzheimer’s disease. b Amyloid beta-mediated neuronal dysfunction and neuronal cell death in AD

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Oxidative Stress Induced by AD Inducing Agents in the Preclinical Models of AD

AlCl3 is mainly used to induce Alzheimer-like disease in laboratory animals. Al also binds with the Aβ promoting its precipitation in a β-sheet conformation responsible for its retention and accumulation in brain tissue [150]. Al also promotes the oligomerization of Aβ and promotes the Ca2+ overload, inducing ER stress and apoptotic pathway governed by specific caspase-12 [151]. It has been reported that the administration of AlCl3 enhances ROS production and oxidative stress, LPO, and NO levels but reduces the GSH levels as compared to control [152]. Further, the administration of AlCl3 led to the deposition of aluminum in the hippocampus and cortex. This is responsible for the induction of memory impairment novel object recognition test and passive avoidance test. It has been reported that the transferrin favors the transport of aluminum across the BBB responsible for its entry into the brain. The deposition of aluminum in the brain is responsible for neuronal structural damage, neuronal cell death, and memory deterioration [153]. This is further in line with the evidence that the AlCl3 significantly reduces contextual memory in the passive avoidance test and spatial memory in the Morris water maze test. AlCl3 is known to upregulate the expression of proapoptotic protein and downregulate the expression of an anti-apoptotic protein. AlCl3 also promotes the translocation of cyt c and increases the expression of enzymes capsase-9 and capsase-3 to cause neuronal cell death [154].

STZ is a glucosamine-nitrosourea derivative, used frequently for the induction of AD-like pathology in experimental animals. STZ administration resulted in the impaired metabolism of glucose in the brain responsible for neuronal degeneration in AD. Administration of STZ (icv route) resulted in acute memory impairment, partial recovery of memory, and then chronic slow memory impairment. However, the administration of higher doses of STZ resulted in the severe and faster degeneration of neurons resulting in more cognitive deficits. It is in line with the evidence that the administration of STZ (≤ 1 mg/kg, icv) promotes slower neuronal degeneration, STZ (2 mg/kg, icv) administration induces cognitive impairment and neurodegeneration while STZ (≥ 3 mg/kg, icv) produces interference in locomotor activity, neurotoxicity, and faster severe neurodegeneration [155]. The findings from the behavioral studies suggested that the administration of STZ resulted in the impairment in learning and memory in the passive avoidance test and Morris water maze (MWM) test [156]. The further administration of STZ resulted in the emergence of oxidative stress evident by the increased LPO, nitrite levels and decreased GSH and SOD levels [156]. The pathological investigation suggested that the administration of STZ increases the hippocampal Aβ level by increasing the expression of APP [156] and increases the total cerebral Aβ fragments, tau protein, and Aβ deposits [157]. Insulin is known to negatively regulate the metabolism of Aβ and tau proteins [157]. STZ (3 mg/kg, icv) administration does not interfere with the peripheral blood glucose level, induces significant cognitive impairment and resulted in the insulin-resistance state which is analogous to AD [158]. STZ (3 mg/kg, icv) further decreases the mitochondrial transmembrane potential, ATP content, and calcium-induced mitochondrial permeability transition responsible for neuronal cell death [159].

Iron plays a key role in the pathogenesis of AD [160] by increasing the production of ROS by participating in the Fenton reaction [161, 162]. It has been reported that Fe3+ reacts with water to form hydroxyl radicals in the presence of H2O2. The hydroxyl radicals so formed causes the conversion of soluble fibrinogen into insoluble fibrin which is resistant to hydrolytic actions of enzymes and is thus responsible for the neurological consequences [163, 164]. The iron itself is also responsible for the oxidation of the cellular molecules [165, 166] and causes neuronal cell damage in the AD and related conditions [167]. The iron-mediated neuronal cell death is known as ferroptosis, a recent mechanism that is targeted by the researchers in the context of AD [168, 169]. Besides this, iron itself increases the production of and aggregation of Aβ [160, 170,171,172,173], the later causes the conversion of Fe3+ form to Fe2+ further responsible for the excessive generation of ROS and subsequent neuronal cell death [174, 175]. Further studies have suggested that the excess of iron induces cognitive deficits, and interacting with Aβ increases ROS production and contributes to neuronal cell death [176, 177].

Endogenous Antioxidant Defense and the Emergence of AD

Brain is the organ that is most susceptible to the damage confer by the ROS, and it is due to the increased production of ROS, low levels of antioxidants, low repair capacity, and non-replicating natures of neurons [178]. Antioxidants are the chemical compounds that have the ability to counteract the oxidative damage confer by ROS [33, 34]. Thus, the antioxidant either endogenous or exogenous mitigates the oxidative damage by maintaining the balance between pro-oxidant and antioxidant defense [179]. The antioxidants may be enzymatic and non-enzymatic, but they perform the same function that is the mitigation of oxidative damage [180, 181]. The antioxidants confer the antioxidant effect either by trapping the oxygen, by chelation with the prooxidative metals [182,183,184], or by inhibiting the production of ROS or by converting the ROS into stable compounds [183, 185, 186]. Thus, the treatment of neurodegenerative disorders by antioxidants may provide benefits and halt the progression of the disease to advance stages by counteracting the process of neuronal degeneration [187]. However, despite of the beneficial effects imposed by the antioxidants, the findings from the clinical studies suggested inconsistent results which might be due to poor study design, selection of the wrong antioxidant, inability of the molecule to cross the BBB, treatment in the advanced state of disease, etc.[188].

Endogenous Antioxidants

Glutathione

Glutathione is the most prevalent antioxidantof the cell existing in two isoforms, thiol-reduced (GSH) and disulfide-oxidized form (GSSG). In the reduced form, the glutathione reacts with ROS, and in this process, it is converted into the oxidized form. The conversion of the reduced form to the oxidized form is catalyzed by the enzyme glutathione peroxidase (GPx) whereas the conversion of oxidized form into reduced form is catalyzed by the enzyme glutathione reductase (GR). Thus, GSH/GSSG ratio is frequently used as a frequent indicator of oxidative stress in the cells [189, 190]. The results from the biochemical analysis of the AD brain suggested the reduced level of GSH and increased levels of GSSG in the frontal cortex region and the hippocampus region of the brain as compared to control [191,192,193]. Further, the extent of the changes in the levels of the oxidized and reduced form is closely related to the severity of the disease [191, 193].

Superoxide Dismutase

Superoxide dismutase (SOD) is an enzyme that catalyzes the conversion of the superoxide radicals into hydrogen peroxide [194,195,196]. Superoxide dismutase exists in three forms (includes copper/zinc-superoxide dismutase, manganese-superoxide dismutase, and extracellular superoxide dismutase) in the body (mainly in the cytoplasm and mitochondria) [196,197,198]. It has been reported that AD is characterized by the reduced expression and the activity of enzyme SOD in the hippocampus region of the brain [199, 200].

Catalase

Catalase is an enzyme present in the peroxisomes [201] responsible for the conversion of approx. 6 million molecules of H2O2 into H2O and O2 every minute [202]. The findings from the preclinical studies suggested the reduced activity of catalase in the STZ-induced AD in rats [203]. Further, the findings from the clinical studies suggested the reductions in CAT activity in several regions of the brain of AD patients [204]. It has been reported that the Aβ in AD promotes the accumulation of H2O2 in the hippocampal neurons [79, 205] by inhibiting the enzyme catalase [206]. It may happen that Aβ directly inhibits the enzyme catalase or it may inhibit the catalase by promoting oxidative stress [207].

Methionine Sulfoxide Reductase

Methionine sulfoxide reductase (MsrA) is responsible for the conversion of methionine sulfoxide into methionine, and the reduced expression of the MsrA activity has been reported in several regions of the brain AD [208].

Nuclear Factor E2-Related Factor 2

E2-related factor 2 (Nrf2) regulates the endogenous antioxidant response in response to oxidative stress. The interaction of Nrf2 with the kelch-like ECH-associated protein (Keap1) sequesters it into the cytoplasm, leading to ubiquitination and subsequent degradation. Nrf2 in its free form stabilizes and translocates inside the nucleus to carry out the gene transcription for the antioxidant enzymes and to confer protection against oxidative damage. However, when the antioxidant response is not required, Nrf2 is removed from the nucleus by a nuclear export sequence near its nuclear localization signal [209]. It has been reported that the Nrf2 increases the expression of the various endogenous enzyme (heme oxygenase-1 (HMOX1), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), NAD(P)H dehydrogenase (quinone) 1 (NQO1)) to confer the protection against the oxidative stress [210,211,212,213,214]. NRF2 also regulates the expression of anti-inflammatory mediators such as interleukin (IL)-17D, CD36, etc. [215,216,217] and reduces the expression of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, etc. [218, 219]. Nrf2 signaling impairment has been implicated in the pathogenesis of AD [209]. It has been reported that in AD, Nrf2 is predominantly localized to the cytoplasm in hippocampal neurons and failed to get translocated inside the nucleus. It is further accompanied by the reduced levels of nuclear Nrf2 in AD. Further, in aged individuals, Nrf2 often fails to perform its normal functions responsible for the emergence of oxidative damage and further neuronal degeneration in later stages [209].

Exogenous Antioxidants in the Treatment of AD

Antioxidants provide protection against ROS and counteract the deleterious effects of oxidative stress [37]. Antioxidant, e.g., luteolin has been shown to significantly inhibit the production of ROS and increases the neuronal cell survival by improving the antioxidant status [220]. Further, luteolin and epigallocatechin-3-gallate (EGCG) have the mitochondrial restorative capability and suppress the burden of Aβ in the brain to confer the therapeutic benefits in the AD [221]. Numerous antioxidants have been screened for their anti-AD potential, but the antioxidants which are used mainly in the clinical studies are given below:

Curcumin

Curcumin is a polyphenolic natural compound that has shown its beneficial therapeutic effect in AD and related conditions in various preclinical and clinical studies. A metanalysis of six clinical trials with a total of 289 subjects suggested the beneficial effect of curcumin on cognitive function [222]. The findings from the preclinical studies suggested that the curcumin treatment counteracts the deleterious effects of oxidative stress and reverses the memory impairment [38]. The results from a study in which the participants were administered with curcumin (180 mg/day for 12 weeks) suggested that the curcumin treatment reduces GSK-3β activity, IAPP, and insulin resistance significantly as compared to the placebo group [223]. In a study of 34 AD patients receiving curcumin (1 g/day or 4 g/day for 6 months), curcumin treatment did not show any significant effect as compared to placebo [41]. Another study suggested that the daily administration of curcumin 2–4 g/day for 24 weeks did not produce any significant alterations in plasma and CFS levels of Aβ and tau levels [42]. Further studies have reported that the curcumin inhibits the aggregation of Aβ [39, 40], reduces the plaque burden [224, 225] and improve cognition and MMSE score [41, 42].

Omega-3 Fatty Acids

Omega-3 fatty acids (OFA) is one of the key molecules with diverse antioxidant properties and has been shown to reduce the risk of several neurological disorders. Docosahexaenoic acid (DHA) is the most important component of OFA. In the brain, the enzyme phospholipase A2 and lipoxygenase convert DHA into neuroprotection 1 (NPD1) which exerts neuroprotective effects [226]. DHA also regulate neurogenesis, neuronal survival, and normal cognitive functions [227]. DHA supplementation thus improves learning ability [35] and memory functions in aged rats [36]. Besides this, DHA also confers protection against the deleterious effects of ROS [228, 229], enhances the endogenous antioxidant system [230], provide neuroprotection against Aβ-induced neurotoxicity [231, 232], limit the production and accumulation of Aβ [233, 234], and increases the cerebral acetylcholine levels [235, 236]. Several studies have reported the therapeutic benefits of OFA and its component DHA in AD patients. In a study on 44 individuals, OFA supplementation has been shown to improve the recall of objects as compared with placebo [237]. A study on 204 AD patients on acetylcholine esterase inhibitor treatment having a MMSE score of 15 points were supplemented with OFA for 6 months. It was observed that OFA supplementation did not delay the rate of cognitive decline but improve MMSE score in a smaller group of patients further suggesting the beneficial effect of OFA [238].A study on 174 AD patients receiving OFA for 6 months suggested that the OFA supplementation lowers the rate of deterioration and preserves the cognition in the AD patients [239]. In a study on 39 AD patients with OFA supplementation for 12 months, it was observed that the supplementation slowed down the cognitive and functional decline in the AD patients [240]. A double study on 204 AD patients suggested that the OFA supplementation for 6 months positively improve weight and appetite AD patients [241]. Oral supplementation with OFA results in the reduction of Ab burden, total and phosphorylated tau levels in CSF, inflammatory biomarkers and neuronal loss in AD patients [242,243,244]. Phillips et al. [245] reported no benefit of OFA supplementation for 4 months in AD patients. OFA is known to modulate the therapeutic effect of vitamin B such that when the levels of OFA are low, then vitamin B produces no effect on cognition,however, when the levels of OFA are higher then the vitamin B, treatment may slow down the cognitive dysfunctioning in the AD patients [43]. Further, it has been reported that vitamin B in the presence of OFA confers beneficial effects on brain atrophy [246].

Vitamin E

Vitamin E is a well-known antioxidant that is known to confer neuroprotection in AD [247, 248]. The antioxidant potential of vitamin E is due to the presence of the hydroxyl group in the phenolic ring structure [249]. Further, the studies have suggested the reduced level of vitamin E in the patients of AD [46, 47, 250]. Various clinical studies suggested the beneficial effect of vitamin E in AD. However, some of the studies present either no effect of vitamin E or the worsening of the condition upon vitamin E treatment. A recent meta-analysis suggested that vitamin E exhibit the most pronounced protective effects [251]. A study on 613 AD patients receiving vitamin E 2000 IU/day (n = 152) for 2 years suggested that the vitamin E delays clinical progression, functional decline, and caregiver burden [252]. Another study on 341 patients also suggested that vitamin E at 2000 IU a day for 2 years slows the progression of AD [253]. In another study on 57 patients receiving vitamin E 800 at IU per day for 6 months suggested the beneficial effect of vitamin E treatment on the cognitive functions of AD patients [254]. However, some of the studies suggested that the vitamin E treatment did not affect the progression of AD. For example, a study on 516 AD patients receiving 1000 IU of alpha-tocopherol twice daily for 3 years suggested no effect on the progression from MCI to Alzheimer’s dementia over 3 years [255]. Similar results have been reported by another study on 756 AD patients receiving donepezil and vitamin E. The results suggested that the treatment with vitamin E did not delay progression to AD whereas treatment with donepezil delayed the progression [256]. Similarly, in another double-blind study on 769 patients receiving 2000 IU of vitamin E daily shows that 10 mg of donepezil daily for 3 years suggested no significant effect of vitamin E treatment on the progression to AD whereas the donepezil therapy reduces the rate of progression to AD [257]. One of the studies on 40 AD patients receiving vitamin E at 2000 IU/day suggested the worsening of neuropsychologic test scores upon vitamin E treatment [258]. Mechanistically, vitamin E treatment counteracts the lipid peroxidation by neutralizing the peroxyl radicals [259]; suppresses the Aβ-mediated production of ROS [260], Aβ-induced neurotoxicity, and cognitive impairment [261,262,263],manages acetylcholinesterase activity [262, 264,265,266],improves cognitive functions [267],and delays the progression of AD [236, 252, 261, 262, 268,269,270]. Thus,vitamin E supplementation may provide therapeutic benefits in AD patients [46, 47, 252], reduce the emergence of AD [267], and slow down the functional decline in AD patients [252].

Vitamin C

Vitamin C is a well-known antioxidant. It is suggested that the level of vitamin C decreases in the brain of the cognitively impaired and AD individuals [271]. It has been reported that the AD patients are characterized by increased level and activity of ROS [272] due to the deposition of Aβ [273], activation of microglia cells [274], neuronal mitochondrial dysfunctioning [275], disturbance in the redox metal metabolic pathway [63, 64, 276], and the deficiencies of neurotransmitters such as noradrenaline and serotonin [277]. Further, the observation from the studies suggested that the CSF:plasma ratio of vitamin C rises significantly in the AD patients compared to the controls, suggesting the increased consumption of antioxidants by the brain in AD patients [278]. Vitamin C acts as a first-line barrier to ROS [279] and thus might be an important therapeutic option in the pharmacotherapy of AD. Vitamin C has been shown to improve cognition, and various studies suggested the relationship between plasma concentration of vitamin C and cognitive performance [280,281,282]. Further, some of the studies suggested no relationship or failed to show the relationship between the plasma concentration of vitamin C and cognitive performance [283,284,285]. The findings from the clinical studies and trials suggested that the higher level of vitamin C improves cognition and MMSE score in AD patients [281, 286,287,288,289]. However, a study suggested that there is no correlation between vitamin C concentrations and MMSE cognitive function in cognitively impaired individuals [290].

Vitamin C and Vitamin E Combination Therapy

The effect of the combined treatment with vitamins C and E has been evaluated in various studies. A study on two groups of AD patients supplemented with vitamin E 400 IU and vitamin C 1000 daily for 1 month was conducted. It was observed that the supplementation increased the concentrations of both vitamins in plasma and CSF. The study suggested that the combination is superior over the alone treatment in AD on the basis of the biochemical findings of the study [291]. One of the observational studies on 4740 individuals receiving vitamin E 400 IU daily and vitamin C 500 mg daily for 3 years suggested that the combination reduces the prevalence and the incidence of AD [292].

Others

Selenium (Se)

It plays a main role in the maintenance of health and treatment of neurologic disorders including AD [293, 294]. The level of the Se decreases with the increase in age [295] and contributes to the emergence of AD [296]. The reduced levels of Se have been described in AD patients [297]. Further, the treatment with Se slows down the cognitive decline [298,299,300] and reduces the Aβ burden [301], tau pathology, and neurodegeneration in AD patients [302].

Melatonin or N-Acetyl-5-methoxytryptamine

Melatoninis are neurohormones secreted by the pineal gland and confer antioxidant effect [303,304,305,306]. It has been reported that aging is accompanied by the reduction of CSF melatonin level, and this may favor the emergence of AD and related conditions [307]. Melatonin inhibits the aggregation and deposition of Aβ [308] and Aβ-induced oxidative damage, mitochondria dysfunction, and caspase-3 activity [309, 310]. Melatonin also activates GABAA receptors to counteract excitotoxicity and corrects the circadian rhythm in AD patients [311]. Melatonin (6 mg/day, daily) treatment has been demonstrated to enhance sleep quality and mood in MCI patients [312].

Estrogens

Estrogens also confer neuroprotection against oxidative stress and excitotoxicity [313]. Estrogen may confer the beneficial effect in AD by enhancing the trafficking of APP in the trans-Golgi network [314], by reducing the expression of BACE1 [313], by enhancing the uptake of Aβ in cortical microglia [315], and by preventing the aggregation of Aβ [316]. Further, selective estrogen receptor modulators have been shown to confer neuroprotective effects in the neuronal cultures by preventing Aβ-induced neurotoxicity [317]. Estrogen treatment also improves memory function in AD women and sustains neuronal viability [318].

Coenzyme Q10

Coenzyme Q10 (CoQ10) is a potent antioxidant in mitochondrial membranes [319] and exerts a neuroprotective effect by increasing the stability of membranes, decreasing the DNA damage, and potentiating the endogenous antioxidants [320]. It has been reported that the levels of CoQ10 decrease with the increase in age, making the mitochondria more susceptible to damage [321]. CoQ10 further exerts anti-inflammatory effects by inhibiting NF-κβ [322], by reducing the cortical levels [323] and deposition of Aβ [324], and by improving the brain energetics [325].

Galantamine

Galantamine is an effective scavenger of ROS and confers neuroprotective effects by inhibiting the activation of P2X7 receptors, protects the mitochondria from the damaging effect of ROS, increases the levels of acetylcholine by inhibiting the expression of acetylcholinesterase, and also inhibits the enzymatic activity of γ-secretase responsible for the production of Aβ [326].

Berberine

Berberine is known to confer antioxidant effects, reduces the levels of ROS, potentiates the level and the signaling of BDNF, reduces the levels of apoptotic proteins Bax/Bcl-2, down-regulates the activities of caspases, and suppresses neuronal apoptosis [327].

Edaravone

Edaravone is a potent free radical scavenger, and it has been reported that the edaravone administration (5 mg/kg) prevented the deleterious effect of Ab on memory and the acetylcholine levels of rats [328]. Administration of edaravone also reduces the production of Aβ by inhibiting the expression of enzyme BACE1, reduces the deposition of Aβ and inhibits the Aβ-mediated hyperphosphorylation of Tau, inhibits neuronal loss and neuronal degeneration, and restores the cognition in aged APPswe/PS1dE9 (APP/PS1) mice [329]. Edaravone at 25 μM counteracted the ZnO NP-induced reduction of ATP level and the activity of complexes I and V of the mitochondria and lipid peroxidation and blocked the expression of NF-κβ to confer protective effects in SH-SY5Y cells [330].

Hyperoside

Hyperoside has been shown to inhibit the Aβ-induced ROS production, mitochondrial dysfunction cytotoxicity, apoptosis, etc. Hyperoside also increases the expression of PI3K/Akt pathway and inhibits the expression of Bad and Bcl(XL) and caspase to suppress the neuronal cell death, suggesting its potential in the treatment of AD in the preclinical and clinical settings [331].

Miscellaneous: Molecular Hydrogen Therapy

Molecular hydrogen is made up of two protons and two electrons, and its antioxidant potential was first studied in 2007 [332]. It has been reported that molecular hydrogen therapy may confer therapeutic benefits in neurodegenerative disorders [333]. It is suggested that less than 4% of hydrogen shows greater efficacy and a better safety profile [334]. The greatest advantage of molecular hydrogen therapy is that the hydrogen even at higher concentrations is still non-toxic [335] and the inhalation of the hydrogen gas has no prominent adverse effects [332, 336]. Further, hydrogen-rich water has been considered safe for administration by many researchers [337,338,339]. In comparison with the conventional antioxidants, the molecular hydrogen only reduces ·OH and therefore showed lesser side effects [332]. Treatment with the molecular hydrogen suppresses the proinflammatory cytokines such as IL-1, IL-6, IL-10, and TNF-a [143, 144, 340], neutralizes Aβ-induced ROS generation and mitochondrial dysfunction [341], modulates Ca2+ signal transduction [342], inhibits the activation of NLRP3 [343], stimulates energy metabolism by stimulating AMPK-Sirt1-FoxO3a pathway [341], maintains cell homeostasis [344], inhibits the translocation of BAX, and suppresses the phosphorylation of JNK responsible for neuronal cell apoptosis [345, 346]. Molecular hydrogen therapy further improves learning and memory ability [347], improves cognition [343], and confers anti-inflammatory and anti-oxidant effects [348, 349]. Besides this, the previous study has shown that hydrogen-rich water improves cognition in the mice model [350]. Further, the administration of hydrogen-rich saline for 14 days has been shown to counteract the cognitive decline induced by i.c.v. administration of Aβ and improve performance in MWM and preserved LTP by inhibiting the IL-1β, JNK, and NF-κβ [351, 352]. Molecular hydrogen suppresses the inflammatory responses and oxidative stress, which are more pronounced in female than in male AD mice [343]. Hippocampus plays a key role in the regulation of learning and memory-related activities [353, 354]. Hydrogen water has been shown to restore the proliferating cells in the dentate gyrus following restraint stress [350], suggesting the beneficial effect. Further, another study suggested that the administration of hydrogen-rich water for 30 days improves the performance in MWM and counteracts the hippocampal neuronal degeneration upon 18 weeks of treatment [355].

A study on 73 MCI subjects receiving 300 mL of H2-water (water infused with H2) for 1 year suggested that the improvement of ADAS-cog score after 1 year in the APOE4 carriers [356]. Another study on 20 adults receiving hydrogen-rich water for 4 weeks suggested that the administration of hydrogen-rich water increases the BAP in the individuals aged ≥ 30 years, suggesting the antioxidant effect [357]. These lines of evidence suggested that molecular hydrogen therapy offers a new direction for the treatment of AD. However, the therapy is in the initial phase and very few clinical studies have been conducted till now. Further, the molecular hydrogen modulates all the pathways which are modulated by the antioxidants, but the antioxidants are in clinical use for a very long period of time. So, the exact superiority between the molecular hydrogen therapy and the conventional antioxidants can be elucidated only after the suitable clinical studies on them.

Evidence from the Studies on Nutraceutical Formulations or Dietary Supplements in AD Patients

Oxidative stress performs major function in the pathogenesis of AD and thus the supplementation of the antioxidant may confer therapeutic benefits in AD patients. Further, the findings from the AD patients suggested that they often have some nutritional deficiency due to the impaired availability of those nutrients [358]. It has been found that the MCI is a risk factor that makes the patients prone to AD and the supplementation of antioxidant in the MCI lowers the risk of further development of AD and related dementia [286]. It has been reported that the correction of oxidative stress and the consumption of tocopherol, ascorbic acid, and β-carotene lowers the risk of AD [251]. Further, AD patients are characterized by the reduced plasma levels of tocopherol, ascorbic acid, and β-carotene, suggesting the importance of these interventions in the pharmacotherapy of AD patients [359]. A study in which several vitamins, antioxidants, amino acids, nutrients, etc. are administered in the form of a pill to the AD patients suggested the reduction in the oxidative stress parameters. The study also suggested that the above pill when used in combination with the donepezil improves the condition of AD patients significantly [360]. Another study on the nutraceutical formulation also claimed that the use of the formulation in AD patients improves cognitive functions [361]. In another study, the authors suggested that the use of the nutraceuticals in AD patients for 3–6 months improves cognition and mood, further suggesting therapeutic benefits of these formulations [362]. Another study by the same author on 24 patients of AD suggested that the daily supplementation with the nutraceutical for 1 year improves the cognitive performance [363]. However, a previous study in which the participants were supplemented with various vitamins and antioxidants for 5 years reported no statistically significant differences in the scores [364]. Another double-blind randomized clinical study on 3786 AD patients receiving vitamin E 400 IU, selenium 200 IU per day, or placebo suggested that neither supplement prevented dementia [365].

Limitations and Failure of Antioxidant Therapy in AD

Oxidative stress has been concerned in the pathogenesis of AD, but despite of this, the role of antioxidants in the pharmacotherapeutics of AD is still controversial. AD patients are characterized by enhanced lipid peroxidation, cognitive dysfunction, abnormal heavy metal metabolism, and a decrease in the total antioxidant capacity. However, the studies have revealed that there is no significant decrease in the levels of the major internal enzymatic and non-enzymatic antioxidants in the body of AD patients. Therefore, the antioxidant therapy should be initiated in the patients followed by the determination of the cognitive outcomes and oxidative parameters in the patients [366]. Theoretically, it is suggested that the antioxidants provide therapeutic benefit in AD patients by providing protection against the ROS and by preventing the accumulation of Aβ [367]. Various studies have described the beneficial effects of antioxidants, suggesting that the treatment with the antioxidants may improve the cognitive performance in AD patients [179, 368, 369]. However, some of the studies suggested no clear significant effect of antioxidants in AD patients [45,46,47]. Thus, despite of the beneficial effects of these compounds in in vitro and in vivo studies, convincing evidence regarding their therapeutic efficacy in AD is still lacking [48, 49]. The result is that only a few studies supported the therapeutic benefits of antioxidants in AD patients [48, 370, 371]. The lack of evidence regarding the therapeutic effects of antioxidant treatment might be due to several reasons, as follows:

First Is the Lack of a Suitable and Relevant Animal Model

The preclinical studies on some antioxidants show their excellent therapeutic potential, but the same compound did not show any significant or promising effect in the clinical studies. These types of incidence suggesting that relevant animal models of AD should be developed to screen the therapeutic potential of the compounds targeted for AD treatment. Further, the difference in the methodology used in the preclinical and clinical studies on the same compound further affects their clinical value or therapeutic efficacy [372]. Further, the dose of antioxidants used in the preclinical studies is very high, and it is impractical to extrapolate that dose to human studies [368]. It has been found that Aβ deposition is the characteristic of AD. However, the findings from the animal models suggested that oxidative stress is responsible for the overproduction and accumulation of Aβ in the brain and vice versa [373,374,375].

Second Is the Clinical Study Design, Protocol, and Their Conflicting Results

The therapeutic potential of the antioxidants remains of question because sometimes they are started at the stages where the disease already progressed to the last stages or sometimes they are started too early. Further, some of the antioxidants were studied at very high doses or in very low doses. Sometimes, antioxidants were used in the combinations where their true potential always remains hidden. These constraints may greatly affect the therapeutic effectiveness of antioxidants for the treatment of AD [368, 376].Various studies suggested that the antioxidants including vitamin C, vitamin E, melatonin, CoQ, etc. failed to inhibit the process of neuronal degeneration, deposition of Aβ, any significant improvement in cognitive functions, etc. [26, 27, 377]. A population-based investigation suggested a relationship between the GPx activity and cognitive decline [378], but the results were inconsistent when observed in AD patients [293]. Further, the confusing and conflicting results have been reported by the studies on SOD and catalase activities in AD patients [199, 200, 379].

Third Is the Nature of Antioxidants and Their Antioxidant Potential

Some of the antioxidants can also act as pro-oxidants under deleterious conditions, and thus, they may potentiate oxidative stress and thus their antioxidant activity remains questionable in these conditions [380]. Consistent with these findings, some of the agents (e.g., CoQ) which has been suggested and speculated to confer therapeutic benefits in AD patients cannot cross the BBB and failed to produce any change in the cognitive functions in AD patients [381, 382]. For CoQ, it has been reported that to exert its therapeutic effect, CoQ must be retained in the mitochondrial ETC. Currently available antioxidants also suffer from similar problems. For example, vitamin E has very slow and lesser bioavailability in the human brain to inhibit AD [383].

Future Directions

Antioxidant therapies have several limitations (as discussed above) for the treatment of AD. Therefore, it further demands in-depth studies to explore and delineate the efficacy of antioxidants for the treatment of AD. This particular review paper will certainly help the researchers/scientists to substantiate/explore the efficacy of antioxidants in the treatment of AD.