**3. Discussion**

With respect to the development of anti-AD agents, enzyme inhibition is one of the most promising potential therapeutic strategies. Since BACE1 is the initiating and rate-limiting enzyme in Aβ formation, it is considered as a key target for lowering cerebral Aβ levels [7–9]. Additionally, AChE plays a critical role in cholinergic neurotransmission and participates in non-cholinergic mechanisms such as accelerating Aβ fibril formation through conformational change of Aβ and increasing Aβ toxicity by Aβ–AChE complexes [14–16]. Thus, multi-enzyme target inhibition against BACE1 and AChE may provide a promising therapeutic approach for AD. In search of a candidate for AD prevention and/or treatment, numerous researchers over the past few decades have focused on discovering natural enzyme inhibitors. Several natural inhibitors of BACE1 and AChE such as coumarins, citrus flavanones, triterpenoids, and alkaloids have been reported [36–39]. However, efforts to explore bioactive constituents from marine organisms against BACE1 and AChE have been relatively limited.

In the current study, three effective phlorotannins—eckol, dieckol, and 8,8-bieckol—were studied for their inhibitory properties on BACE1 and AChE. These compounds exhibited powerful inhibitory activities on BACE1 with IC50 values at a range of 1.6–7.7 μM. Several terrestrial plant-derived BACE1 inhibitors, including hespretin, naringenin, and hesperidin, were from citrus fruits with IC50 values ranging from 16.9–30.3 μM. Alkaloids (neferine, liensinine, and vitexin) in *Nelumbo nucifera* (IC50 in the 6.4–28.5 μM range) have been proven to be efficient BACE1 inhibitors. Umbelliferone, isoscopoletin, 7-methoxy coumarin, esculetin, and daphnetin from *Angelica decursiva* with IC50 ranging from 7.7–172.3 μM were identified as BACE1 inhibitors. Compared with those plant-derived BACE1 inhibitors, our compounds demonstrated predominantly inhibitory properties against BACE1 [36,37,39].

Interestingly, the difference in inhibitory properties among phlorotannins is related to the number of hydroxyl groups present. In our new findings, it was shown that 8,8-bieckol containing 11 OH groups had the highest inhibitory efficacy against BACE1 when compared to dieckol (10 OH groups) and eckol (6 OH groups). When phlorotannins from *Eisenia bicyclis*, one of the brown algae, were investigated for their BACE1 inhibitory effects, the result that dieckol was stronger than eckol was similar to that of our present study [40]. Consistent with our result, Ahn and colleagues have reported that the inhibitory effect on HIV-reverse transcriptase of 8,8-bieckol containing a biaryl linkage was tenfold higher than that of 8,4-dieckol with a diphenyl ether linkage [41]. This observation indicated that the steric hindrance of the hydroxyl and aryl groups near the biaryl linkage of 8,8-bieckol noticeably enhanced its inhibitory potency.

Among three phlorotannins, 8,8-bieckol showed the most potent inhibitory activity against AChE. Similar results regarding the correlation between the molecular size of phlorotannins and enzyme inhibitory efficacy was revealed in a previous study. 8,8-bieckol showed more potent activity against hyaluronidase, with an IC50 of 40 μM, than dieckol and eckol (IC50, 120 and >800 μM, respectively) [42]. In addition, the present study first demonstrated the specific molecular docking interaction as well as biological properties of eckol, dieckol, and 8,8-bieckol against AChE.

The BACE1 inhibition kinetics indicated that the tested compounds act as non-competitive inhibitors, which means that these compounds can bind either another regulatory site or to the subsite of BACE1. The inhibition level is dependent on the concentration of the inhibitor but is not reduced by increasing concentrations of substrate. Because of this, Vmax is reduced, but K m is unaffected. In BACE1 inhibitory activity, phlorotannins decreased the Vmax values without affecting the affinity of BACE1 toward the K m, which demonstrated that these compounds exhibited non-competitive inhibition against BACE1. However, AChE kinetics results exhibited that our tested compounds are competitive inhibitors with unchanged Vmax and increased K m. In other words, these compounds interacted directly with the catalytic site of AChE instead of with other allosteric pockets.

In silico docking analysis is a valuable drug discovery tool and can be used to discover prospective, biologically active molecules from natural product databases. The results of the molecular docking score were provided to evaluate the capacity of different protein–ligand complex interactions and to compare the biological activities and the inhibition mode. In the BACE1 docking simulation, multiple hydrogen interactions were observed in the BACE1–phlorotannins complexes. Eckol interacted with both GLY34 and SER36 of BACE1, and dieckol bounded to TRP76, THR232, and LYS321. In addition, 8,8-bieckol formed four hydrogen bonds with BACE1 residues, including LYS107, GLY230, THR231, and SER325. These docking results showed that hydrogen bonds between phlorotannins and allosteric residues of BACE1 play an important role in enzyme inhibition.

AChE docking analysis provides insight into the mechanism underlying active site binding interaction. The hydroxyl group of eckol formed five hydrogen bonds with THR83, TRP86, TYR124, and SER125 of AChE. In particular, the choline-binding site residue (TRP86) of AChE was involved in hydrogen bond interaction with eckol. Dieckol showed four hydrogen-bond interactions with ASN233, THR238, ARG296, and HIS405, whereas 8,8-bieckol made one hydrogen bond with ARG296 located in the active site of AChE. These docking results from the in silico study were in agreemen<sup>t</sup> with our in vitro experimental data.

To date, few studies have investigated the neuroprotective property of phlorotannins. Our previous study demonstrated that phlorotannins ameliorated Aβ25–35 toxicity through the regulation of the apoptotic signal and the NF-kB/MAPKs pathway [34]. Eckol and dieckol suppressed H2O2-induced oxidative stress in murine hippocampus neuronal cells [43]. Moreover, it has been reported in an in vivo study that oral administration of dieckol (10 mg/kg) improved cognitive ability in ethanol-induced memory impairment mice [44].

Nagayama and coworkers demonstrated no significant toxic effects in the oral administration of up to 1,500 mg/kg of phlorotannins for 14 days in male and female Institute of Cancer Research (ICR) mice [45]. In a human study, *E. Cava* extract was shown to be safe for use in food supplements at a maximum daily intake level of 263 mg/day for adults [46]. Collectively, phlorotannins are toxicologically very safe, explaining their traditional and present consumption as foods and medicinal products.

Bioavailability parameters such as biotransformation and conjugation during absorption from the GI tract are principle factors influencing in vivo biological activity. Lipinskis rule of five is a widespread strategy to define bioavailability predictions of drug molecules. According to this predictive model, a compound needs to exhibit optimum GI absorption with a molecular weight of < 500 Da, no more than five hydrogen bond donors, no more than ten hydrogen bond acceptors, and a calculated partition coefficient (LogP) that is no more than five [47]. Fortunately, eckol meets Lipinski s requirements for acceptable oral bioavailability, while dieckol and 8,8-bieckol have limitations on bioavailability [48]. However, the compounds absorbed by specific transporters are an exception to this rule, and a recent study demonstrated that dieckol successfully penetrated into the brain via crossing the blood–brain barrier (BBB), suggesting that the compound may be transported through an unknown mechanism [49]. A study of the permeability of eckol and 8,8-bieckol was limited, but it is likely that similar results might also be predictable as that of dieckol. Overall, our marine compounds

from *E. Cava* are safe, potent, and selective natural dual inhibitors against BACE1 and AChE that can be used for the multi-target, directed agents of AD.

#### **4. Materials and Methods**
