**1. Introduction**

Alzheimers disease (AD) is a progressive and irreversible neurodegenerative disorder with characteristic features of cognitive dysfunction, memory impairment, and behavior disturbances. The neuropathological hallmarks of AD patients are the presence of extracellular deposits of amyloid plaques and intracellular filamentous neurofibrillary tangles in the brain [1]. Amyloid plaques and neurofibrillary tangles are aggregates of amyloid-β peptide (Aβ) and hyperphosphorylated tau protein, respectively. In recent years, the "amyloid hypothesis" has arisen as the major pathological mechanism in AD, and the evidence from transgenic mice models revealed that Aβ triggered tau phosphorylation and neurofibrillary tangles formation [2].

Aβ is generated by the sequential proteolytic cleavage of two aspartic proteases, β- and γ–secretase, in the amyloidogenic pathway. β-Secretase (BACE1) initially cleaves amyloid precursor protein (APP) at the N-terminus of the Aβ peptide domain, which is followed by the cleavage of γ–secretase in the transmembrane region of APP, leading to the production of Aβ peptide [3]. Therefore, these secretases have been suggested as potential targets to hinder Aβ formation and thereby delay or stop the progression of AD. γ-Secretase inhibitors have shown severe toxicity problems because of on-target interference with Notch signaling, and genetic deletion of catalytically active subunit presinilin-1 (PS-1) was found to be lethal in embryonic mice [4–6]. In contrast, BACE1 inhibition powerfully lowers the level of Aβ in the central nervous system (CNS) of both transgenic mice models and AD patients [7–9]. Recent studies have exhibited few mechanism-based side effects of BACE1 inhibition with chronic administration in animal models, but these are relatively weak and mild compared to γ-secretase-inhibitor-induced deficits [10–12]. Unlike BACE1, which leads to the formation of Aβ, α-secretase acts within the Aβ domain to preclude the Aβ generation in the non-amyloidogenic pathway. The α-secretase and BACE1 compete for the same APP substrate, with an increase in one cleavage event leading to a decrease in the other. As BACE1 initiates the amyloidogenic pathway and is putatively rate-limiting, it is a critical target for lowering cerebral Aβ levels in the treatment and/or prevention of AD.

In the past, it has been found that acetylcholinesterase (AChE) is involved in the degradation of the neurotransmitter. The observation of a significant loss of cholinergic neurons in AD patients is the major correlate of cognitive impairment. Cholinesterase inhibitors can increase acetylcholine (ACh) levels in the synaptic cleft and partially ameliorate cognitive symptoms for patients with mild to severe AD [13]. Recent findings supported that AChE is associated predominantly with pre-amyloid diffuse deposits, amyloid cores of mature amyloid plaques, and cerebral blood vessels in an AD patient brain. In addition, it triggers the Aβ fibrillogenesis via the formation of stable Aβ– AChE complexes [14,15]. Neurons treated with these complexes exhibited a disrupted neurite network compared to neurons treated with Aβ alone [16]. Based on these findings, the suppression of both enzymes is a very desirable feature of AD therapy.

Current AD therapies are mainly palliative and temporarily slow cognitive decline, and treatments based on the underlying pathologic mechanisms of AD are totally limited [17]. Several therapeutic approaches have recently revealed promising results in clinical trials, such as BACE1 and γ-secretase inhibitors, inhibition of Aβ plaque formation, passive Aβ immunotherapy, etc. However, the clinical use of these agents needs further careful assessment of their effectiveness on cognitive decline and their adverse effects [18]. Another strategy for AD therapy is the use of natural products, which are more effective, safer and have fewer adverse effects than synthesized drugs [19]. Neuroprotective natural compounds such as (-)-epigallocatechin-3-gallate (EGCG) from green tea, resveratrol from grape, curcumin from tumeric, and quercetin from apples revealed significant therapeutic potential toward the amelioration and prevention of AD [20].

Marine organisms are a rich source of several natural molecules, including polyphenol, polysaccharide, sterol, and peptide, which have many biological properties such as antioxidant, anti-inflammatory, anti-hypertensive, anti-obesity, anti-diabetes, and anti-cancer effects [21–25]. *Ecklonia cava* (*E. cava*) is an edible brown seaweed which is distributed in Japan and the southern coast of Korea, and it is recognized as a rich source of bioactive derivatives, containing 3.1% crude phlorotannins [26,27]. Phlorotannins are unique polyphenolic compounds containing a dibenzo[1,4]dioxin element as the core structure not found in terrestrial plants. The compounds consist of phloroglucinol units linked to each other in several ways. Based on the type of linkage, phlorotannins are classified into four subgroups: eckols (phlorotannins with a dibenzodioxin linkage), fuhalols and phlorethols (with an ether linkage), fucols (with a phenyl linkage), and fucophloroethols (with an ether and phenyl linkage) [27].

Recently, it has been reported that phlorotannins possess various bioactivities such as antioxidant, antidiabetic, anti-hypertensive, anti-human-immunodeficiency-virus type-1 (HIV-1), and radioprotective activities [28–32]. Regarding the study of neuroprotective effects, phlorotannins-rich *E. cava* extract ameliorated the Aβ formation by modulating α- and γ-secretase expression and inhibiting Aβ-induced neurotoxicity [19,33]. In our previous study, three major phlorotannins of *E. cava*—eckol, dieckol, and 8,8-bieckol—exhibited anti-apoptotic and anti-neuroinflammatory properties against Aβ-induced cellular damage, which led to our interest in the study of phlorotannins-mediated suppression of related enzymes in Aβ production and aggregation [34]. Therefore, the purpose of the present study is to evaluate the inhibitory effects of these compounds against both BACE1 and AChE through in vitro and in silico approaches.
