Neuroprotective role of herbal alternatives in circumventing Alzheimer’s disease through multi-targeting approach - a review

ABSTRACT Alzheimer’s disease (AD) is a common form of dementia affecting the elderly worldwide. It is a multifactorial neurodegenerative disorder with no known preventive therapy. Many of the drugs used in the treatment of AD, such as galantamine, rivastigmine, and donepezil, have unpleasant side effects, and hence physicians are keen to find alternatives. Research has shown that plants and their phytochemicals can alleviate AD. These plant products can act through various modes, such as inhibition of amyloid β, acetylcholine, and γ-secretase, modulation of antioxidants, and α-secretase activation, which are known to involve in the improvement of brain functions. A recent approach that has garnered the attention of many researchers in designing a drug against AD is the multi-target-directed ligand (MTDL), wherein the same molecule act on multiple targets. Many studies have reported the potential of herbs to act on multiple targets and display biological properties. The current review summarizes the ongoing evidence on the use of herbs and their derived bioactive molecules in the treatment of AD and in relieving disease-associated pathological events. Currently available plant-derived MTDLs for the treatment or slowing down of the progression of AD are also discussed.


Introduction
Alzheimer's disease (AD) is an irreversible, chronic neurodegenerative disorder characterized by deterioration of cognitive functions and behavioral disturbances [1]. Globally, AD is the most common cause of dementia, affecting approximately 46.8 million people and expected to increase up to 131.5 million by 2050 [2]. The probability of AD aggressively increases with age, more particularly after the age of 65. Thus, age is the primary risk factor Acetylcholine-and glutamate-producing neurons are known to be damaged during AD, thereby affecting the synapses associated with them. This is in agreement with the early cognitive symptoms observed in AD [6]. The main factor for the degeneration of neurons is due to the increased activity of cholinesterases (ChEs), which leads to a decrease in acetylcholine (ACh) levels, which in turn stops the neuronal transmission signals [7]. Studies have also established that acetylcholinesterase (AChE) promote Aβ aggregation and a notable increase in the cortical levels of butyrylcholinesterase (BuChE), which is related to the formation of Aβ plaques and neurofibrillary tangles (NFTs) [8][9][10].
Many natural compounds are known to have neuroprotective effects during AD. A large family of plant isolates has proven to be a modality for treatment by their inhibitory effect on Aβ, cholinesterase, beta, and gamma secretases. Potent activation of alpha secretases by plant products also substantiates the neuroprotective effect.
This review gives a detailed insight into the list of plants and their isolates as neuroprotective agents during AD.

Molecular mechanism of AD
The formation of NFTs and senile plaques are the main histopathological hallmarks of AD [11]. The senile plaques contain amyloid-beta (Aβ) peptide, which consists of 37-49 amino acid residues and are formed by the extracellular and transmembrane domains of amyloid precursor protein (APP) [12]. In plaques, the oligomers might be trapped in fibrillar aggregates. Oligomers may be the hazardous Aβ species that contribute to signaling pathway deregulation (Fyn, FAK, GSK3b, and CDK5), causing changes in cytoskeletal and synaptic proteins, as well as synaptic and neural damage [13] (Figure 1). During sporadic AD, APP is cleaved by gamma and beta secretases to form 4 kDa Aβ peptide. The cleavage product has a strong tendency to form aggregates. Aβ Figure 1. Formation of neurofibrillary tangles (NFTs) and senile plaques. The senile plaques contain amyloid beta (Aβ) peptide, which consists of 37-49 amino acid residues and are formed by the extracellular and transmembrane domains of amyloid precursor protein (APP). Oligomers may be the hazardous Aβ species that contribute to signaling pathway deregulation (Fyn, FAK, GSK3b, and CDK5), causing changes in cytoskeletal and synaptic proteins, as well as synaptic and neural damage. accumulation has been one of the major pathological events resulting from an imbalance between production and clearance [14]. The Aβ aggregation process initiates by selfassembling of Aβ monomers into low molecular weight oligomers, which in turn results in the formation of high molecular weight oligomers known as soluble aggregation intermediates. These further aggregate to form fibrils and accumulate in the brain [15,16]. It is believed that microglia and astrocytes then mount an inflammatory response to clear the amyloid aggregates, and this inflammation likely causes the destruction of adjacent neurons and their neurites.
Other than plaques, the presence of NFTs is considered another characteristic feature in the neuropathological event of AD [17]. These NFTs are insoluble twisted fibers formed by abnormal hyperphosphorylation of a microtubuleassociated protein called 'tau'. NTF in normal form serves as a microtubule-stabilizing protein and plays a role in intracellular (axonal and vesicular) transport [18]. NFT may interfere with the regular axonal transport of components necessary for proper neuronal function and survival, eventually causing neurons to die. In addition, Aβ is thought to trigger neuronal cell death via controlling apoptosis inducers, generating oxidative stress, and increasing free radical-mediated pathways [11].

Methodology
A detailed literature survey was performed using both offline and online resources. Data were mainly collected from various journal publishers, including Elsevier, Springer Nature, Taylor & Francis, Cambridge University Press, Oxford University Press, BioMed Central, and PLOS (Public Library of Science). The online databases such as Google Scholar, Pubget, Medline, PubMed, EMBASE, Mendeley, ScienceDirect, Scopus, and SpringerLink were also used to retrieve literature. The results were then cross-referred to generate the list of references (up to 2018) cited in this review. The Current review methodically summarizes the neuroprotective effects of phytochemicals in various models. Herbal extracts, bioactive constituents, and herbal formulations were included to provide references in the future.

Natural products in AD
Since time immemorial, natural products have been used as medicine for many ailments. Natural products are molecules with diverse functions and have been the source of most active constituents in medicine [19,20]. They are said to be the most successful basis of drug leads with lesser toxicity [21,22]. Natural products may be derived from plants, animals, or microorganisms. Most herbal medicines are complex and constitute many chemical components, which possess diverse biological and pharmacological activities.
Medicinal plants are nature's gift that remains unexplored. The active component present in herbal medicine may serve as the basis for preparing synthetic drugs [22]. Plants can synthesize chemical compounds involved in preventing or curing various diseases, including memory dysfunction and age-related disorders. In modern medicine, plants occupy a very significant place as a source of raw material for synthetic drugs [23]

Cholinesterase inhibitors
The important etiological factor in the pathogenesis of memory deficit in AD is the impairment in cholinergic transmission [24]. The inhibition of AChE increases the levels of acetylcholine in the brain and thus improves the cholinergic functions in AD patients 25. Hence, cholinesterase inhibitors are currently used as standard drugs for treating AD. Tacrine was the first AChE inhibitor drug approved for AD treatment [26]. Later, many other AChE inhibitors such as rivastigmine, galantamine, and donepezil were also developed and approved by the FDA. These drugs alleviate the symptoms but are associated with side effects when used for an extended period [27]. As AD has reached a state of public health burden, the everincreasing reports of side effects from these synthetic and hybrid drugs have driven the research for a novel and safe AchE inhibitors from plant sources.
Plants continue to be the unvaryingly abundant source of therapeutic drugs for AD treatment because of their AChE inhibitory activity. Several plant extracts of various solvents have been reported to show anticholinesterase activity. Aqueous and methanolic root extracts of Acacia nilotica and Withania somnifera possessed moderate anticholinesterase activity (IC50 values of 0.079 and 33.38 µg/ml, respectively) [28,29]. Much lesser inhibitory activity was observed in hydroethanolic seed extracts of Myristica fragrans, which showed 50% enzyme inhibition at a concentration range between 100 and 150 µg/mL [30]. Also, Pinus nigra was used to extracting essential oils possessing 94.4 µg/mL activity [31]. Similarly, different extracts of plants belonging to varied plant families have shown considerable cholinesterase inhibitory activity, which is listed in (Table 1).
Alkaloids derived from various plant extracts show immense potential for AChE inhibitory activity. However, significantly few isolated compounds have been utilized for research and therapeutic purposes. Many isolated compounds from different classes of alkaloids have been considered and tabulated in (Table 2).

γ-and β-secretase inhibitors
Many plant extracts and their derived compounds are found to influence the Aβ production pathways, mainly by interacting with brain enzymes like β-and γ-secretases [112,113]. As explained earlier, both β-and γ-secretases are involved in the synthesis of Aβ. β-secretase cleaves the APP to form a transmembrane C-99 fragment with the N-terminus of the Aβ peptide ( Figure 2) followed by the action of γsecretase, which cleaves C-99 fragment in the transmembrane domain to make the C-terminus of Aβ [112].
In addition to β-APP processing, γ-secretase also plays a vital role in the cleavage of the Notch family of cell-surface receptors, a protein mainly required for transcriptional regulation during neuron development [114]. As a result, the use of γ-secretase inhibitors has provided insights into proteolytic activity and suggests that such inhibition might be a useful strategy for AD therapeutics [115]. A triterpene isolated from Actaea racemosa reduced the formation of Aβ toxicity through the modulation of γ-secretase activity. Thus, it suggests that the isolated compound may bind to γ-secretase APP complex, modulating the cleavage of APP and hence lowering the formation of Aβ peptides. 116,demonstrated that the use of green tea polyphenol epigallocatechin-3-gallate (EGCG) inhibited LPS-induced Aβ elevation levels through the suppression of LPS-induced β-and γ-secretase activities [116]. However, the inhibition of Notch protein by γ-Secretase inhibitors affects neuronal development, as Notch has multiple substrates that are involved in neuronal development [117]. Hence, β-secretase, also referred to as β-site APP cleaving enzyme 1 (BACE-1), a transmembrane aspartic protease secreted in almost all tissues but present in higher amounts in neurons of the brain [115], is considered as the most promising target for pharmaceutical research on AD, compared to γ-secretase.

α-secretase activators
α-secretase enzyme proteolytically cleaves the APP via the non-amyloidogenic pathway at L688 residue located within the Aβ sequence and thereby preventing the formation of Aβ ( Figure 2). The first enzyme for α-secretase was proposed in 1998, when ADAM17, also known as tumor necrosis factor-converting enzyme (TACE), was reported to possess α-secretase activity [118]. Later, ADAM9 and ADAM10    were also shown to have α secretase activity [119]. These three proteins belong to the ADAM (a disintegrin and metalloprotease) family. It is reported that mutations in ADAM10 alter the processing of APP and lead to AD by increasing Aβ levels [120]. Thus, a promising yet underestimated approach to overcome AD would be, activating α-secretase processing of βAPP. Moderate overexpression of ADAM10 in an APP mouse model showed a decreased level of Aβ, and prevented its accumulation. Such Catechin, quercetin Flavonols Eugenia dysenterica decreased levels of Aβ are found to alleviate cognitive deficits [121,122]. Various studies have corroborated that several drugs currently used in the treatment of AD promote α-secretase activity by activating associated signaling cascades. Thus, it has been considered as one of the best therapeutic approaches in AD [123][124][125].

Aβ inhibitors
Bioactivity-guided isolation has led to the discovery of novel bioactive compounds from plants, which are useful in preventing Aβ-induced neuronal cells [126]. In vitro assays were widely used to assess the activity of isolated compounds. It is observed that phenolic compounds, alkaloids, and glycosides comprise the major part of the isolated compounds with Aβ inhibitory activity. Their antioxidant activity and lipophilicity make it easy for them to cross the blood-brain barrier [126]. A list of compounds with Aβ inhibitory activity is provided in (Table 3 and 4).

Antioxidants in AD
Oxidative stress is a process of ROS generation, which plays a central role in cellular injuries and various clinical disorders, including neurodegen . β-and γ-secretases are involved in the synthesis of Aβ. β-secretase cleaves the APP to form transmembrane C-99 fragment with the N-terminus of the Aβ peptide. This is followed by the action of γsecretase, which cleaves C-99 fragment in the transmembrane domain to make the C-terminus of Aβ.  [142] (Continued)  [160] Zingiber officinale

Zingiberaceae Seed
In vitro ThT and primary hippocampal neuron Prevented the formation of oligomers and dis-aggregated the pre formed fibrils.

In vivo
Transgenic 2576 mice Inhibition of oligomer and fibril formation from monomer and destabilization of preformed fibrils Inhibition of Aβ production in the rat brain [150] Phenolics Pomiferin  [196] Salvianolic acid B

Rosmarinus officinalis
(Lamiaceae) Aβ induced toxicity rats Increased the learning and behavior and increased healthy neurons in CA region in brain [213] Cryptotanshinone Salvia miltiorrhiza (Lamiaceae) In vivo APP/PS1 transgenic mice Swe/APP cortical neurons increased release of sAPP and reduction in levels of Aβ [214] Eugenol Acorus calamus (Acoraceae) In vitro PC12 cells Inhibition of Aβ-induced Ca 2+ intake [154] erative diseases [215]. The brain cells are continuously exposed to a surplus of free radicals, which leads to oxidative stress. Thus, ROSinduced oxidative stress in the brain is one of the most common etiologies of neurodegenerative disorders, including AD [216,217]. The oxidative stress not only mediates neurotoxicity induced by Aβ, but also enhances the production of Aβ [218]. Thus, oxidative stress is a prime contributing factor for AD development, and antioxidants can be considered therapeutic approaches.

MTDL: A new therapeutic approach for AD
For 15 years, AD had been treated symptomatically, and the therapeutic approaches are of modest efficacy [219]. The approved drugs fall into two categories: AChE inhibitors and N-methyl D-aspartate (NMDA) receptor antagonists, with four and one drug in each group, respectively [220,221]. These cholinergic drugs increased cholinergic system deficiency by inhibiting the AChE enzyme, which degrades acetylcholine. One of the important drugs belonging to this class is donepezil. Many evidence infers that AChE inhibition reinstates the cholinergic system and mediates the disease progression [222].
On the other hand, the excessive NMDA glutamate receptor activity observed in AD was inhibited by a low-affinity, non-competitive and open channel blocker, memantine, which is frequently used with AChE inhibitors [219]. These drugs are insufficient for AD therapy, and this warrants more research towards finding drugs against AD. 223, suggested that identification of Aβ or tau proteins as a target in AD created two groups of researchers, referred to as 'baptists' and 'tauists' [223]. However, both these groups failed to develop the potential drugs which can cure the disease. Moreover, along with Aβ, antagonistic AChE also targets other aspects of AD.
Over the past nine decades, researchers have been targeting one factor at a time, which did not result in any drug to cure AD. Efficient pharmacotherapy may require simultaneous action on several targets involved in its pathogenesis due to the complexity of AD. Such effects may be achieved by administering a drug cocktail or a multicomponent drug. Besides AD, other neurological disorders such as depression, allergies, hypertension, schizophrenia, inflammation, and metabolic diseases can also be treated by this combination of drugs [224]. But, this approach carries the risk of potentially hazardous drugdrug interactions caused by specific pharmacokinetic and pharmacodynamic properties of individual components. It would be ideal if a single molecule could simultaneously act on multiple targets with greater efficacy and safety profile. In 2005, Morphy and Rankovic proposed this innovative strategy to develop MTDLs as potential drug candidates. This approach can be more relevant and practical since AD is a complex neurological disorder with multiple causative factors.
Further, to reduce the side effects, many reports suggest using herbal alternatives to enhance the efficacy of the therapy in the future [225]. Thus, identifying novel pharmacological neuroprotective MTDLs from plants is the new hope for treating AD. These natural products can simultaneously act on multiple targets associated with AD (enhance α-secretase activity; decrease βand γ-secretase activity; inhibit Aβ; prevent oxidative stress and inflammation). Some plant products possessing multiple targets against AD are presented in tabulated in Table 5. The summary of the role of plant extracts and their phytochemicals in circumventing AD is represented in Figure 3.

Conclusion and prospects
Natural products have tremendous potential to act against AD and have given hope to the scientific fraternity as sources of drugs. Though the cause of AD is not clearly understood, natural products with multiple activities like AChE inhibition, NMDAR antagonist, antioxidant ability, amyloid inhibition, and anti-inflammation have the potential to be used as drugs. The healing power of culinary herbs and medicinal plants Table 5. Plant products with multiple targets against AD.

Disclosure statement
No potential conflict of interest was reported by the author(s).