**2. Results**

#### *2.1. In Vitro Inhibitory Study of Phlorotannins on BACE1 and AChE*

The chemical structures of eckol [4-(3,5-dihydroxyphenoxy)dibenzo-p-dioxin-1,3,6,8-tetrol], dieckol [4-[4-[6-(3,5-dihydroxyphenoxy)-4,7,9-trihydroxydibenzo-p-dioxin-2-yl]oxy-3,5-dihydroxyp henoxy]dibenzo-p-dioxin-1,3,6,8-tetrol], and 8,8-bieckol [9-(3,5-dihydroxyphenoxy)-2-[9-(3,5-dihy droxyphenoxy)- 1,3,6,8-tetrahydroxydibenzo-p-dioxin-2-yl]dibenzo-p-dioxin-1,3,6,8-tetrol] were shown in Figure 1. Eckol is a trimer of phloroglucinol (1,3,5-trihydroxybenzene) units, while dieckol and 8,8-bieckol are hexamer. As presented in Table 1 and Figure 2, 8,8-bieckol exhibited the strongest BACE1 inhibition (IC50, 1.62 ± 0.14 μM), followed by dieckol (IC50, 2.34 ± 0.10 μM) and eckol (IC50, 7.67 ± 0.71 μM). Interestingly, the IC50 value of all of the tested compounds were lower than that of resveratrol (IC50, 14.89 ± 0.54 μM), which is a well-known BACE1 inhibitor that was used as a positive control.

**Figure 1.** The chemical structures of (**A**) eckol, (**B**) dieckol, and (**C**) 8,8-bieckol.


a The IC50 values (μM) were calculated from a log dose inhibition curve and expressed as the mean ± standard deviation (SD). All assays were performed in three independent experiments. DMSO was used as a negative control in the BACE1 and AChE assays. b Resveratrol and c galantamine were used as positive controls in the BACE1 and AChE assays, respectively. d Inhibition constant (*K*i) and e inhibition mode were determined using Dixon plot and Lineweaver–Burk plot, respectively.

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Ʒ **Figure 2.** (**A**) β-Secretase (BACE1) and (**B**) acetylcholinesterase (AChE) inhibitory activities of eckol (-), dieckol (), and 8,8-bieckol (). All assays were performed in three independent experiments. Dimethyl sulfoxide (DMSO) was used as negative controls in the BACE1 and AChE assays.

Three phlorotannins displayed high potencies as AChE inhibitors (Table 1 and Figure 2). Both dieckol and 8,8-bieckol exhibited potent AChE inhibitory activity (IC50 values of 5.69 ± 0.42 and 4.59 ± 0.32 μM, respectively) and about twofold greater than the eckol (IC50, 10.03 ± 0.94 μM).

To demonstrate the specificity of the targeted enzymes, all compounds were tested against tumor necrosis-converting enzyme (TACE), which is a candidate for α-secretase, and other serine proteases, including trypsin, chymotrypsin, and elastase (Table 2). With serine proteases being found in nearly all body tissues and involved in various physiological functions, including digestion, reproduction and immune response, off-target activity causing severe side effects is possible and likely if an inhibitor is not specific to BACE1 and AChE [35]. Therefore, to determine whether phlorotannins inhibited only targeted enzymes without affecting the normal pathway, all compounds were tested against TACE and serine proteases. Up to 100 μM, our tested compounds did not show significant inhibition against the above enzymes, indicating that three phlorotannins appeared to be relatively specific inhibitors of BACE1 and AChE.


**Table 2.** Inhibitory activities of phlorotannins against tumor necrosis-converting enzyme (TACE), trypsin, chymotrypsin, and elastase a,b.

a The inhibitory activity (%) was expressed as the mean ± SD of three independent experiments. DMSO was used as a negative control in TACE and serine proteases assays. b Comparison of concentration level in each sample is not significantly different.

#### *2.2. Kinetic Study of BACE1 and AChE Inhibition*

As shown in Table 1 and Figure 3, Lineweaver–Burk plots for the inhibition of BACE1 by eckol, dieckol, and 8,8-bieckol were fitted well to the noncompetitive inhibition mode in visual inspection, and the *K*i values of eckol, dieckol, and 8,8-bieckol were 31.2, 20.1, and 13.9 μM, respectively. On the other hand, our tested compounds were competitive inhibitors of AChE, where the Lineweaver– Burk plots intersected a common point on the *y*-axis (Table 1 and Figure 4). The *K*i values of eckol, dieckol, and 8,8-bieckol were 37.3, 12.3, and 11.4 μM, respectively, and were obtained from the Dixon plot.

**Figure 3.** Dixon plot of BACE1 inhibition by (**A**) eckol, (**C**) dieckol, and (**E**) 8,8-bieckol in the presence of different substrate concentrations: 250 nM (•), 500 nM (-), and 750 nM (). Lineweaver–Burk plot of BACE1 inhibition by (**B**) eckol, (**D**) dieckol, and (**F**) 8,8-bieckol in the presence of different inhibitor concentrations: 0.3 μM (•), 3 μM (-), 30 μM (), and 50 μM () for eckol; 0.3 μM (•), 1.5 μM (-), 3 μM (), and 15 μM () for dieckol; 0.3 μM (•), 3 μM (-), 7.5 μM (), and 15 μM () for 8,8-bieckol. All assays were performed in three independent experiments.

**Figure 4.** Dixon plot of AChE inhibition by (**A**) eckol, (**C**) dieckol, and (**E**) 8,8-bieckol in the presence of different substrate concentrations: 250 μM (•), 500 μM (-), and 750 μM (). Lineweaver–Burk plot of AChE inhibition by (**B**) eckol, (**D**) dieckol, and (**F**) 8,8-bieckol in the presence of different inhibitor concentrations: 1 μM (•), 10 μM (-), 25 μM (), and 100 μM () for eckol; 0.1 μM (•), 10 μM (-), 25 μM (), and 50 μM () for dieckol and 8,8-bieckol. All assays were performed in three independent experiments.

#### *2.3. In Silico Docking Study of the Inhibition of BACE1 and AChE by Phlorotannins*

According to the in silico docking simulation results, BACE1 and phlorotannins complexes had an allosteric inhibition mode (Table 3 and Figure 5). GLY34 and SER36 of BACE1 formed two hydrogen bonds with the hydroxyl group of eckol with bonding distances of 3.277 and 3.239 Å, respectively. In the dieckol–BACE1 complex, TRP76, THR232, and LYS321 participated in three hydrogen bonds

(bonding distance: 2.960, 3.149, and 3.488 Å, respectively). 8,8-Bieckol–BACE1 complex had four hydrogen bonding interactions with residues LYS107, GLY230, THR231, and SER325 (bonding distance: 3.120, 2.773, 3.098, and 2.887 Å, respectively). In addition, the lowest binding energy of the three tested compounds were negative values: -8.8 kcal/mol for eckol, -10.1 kcal/mol for dieckol, and -9.0 kcal/mol for 8,8-bieckol.

**Figure 5.** In silico docking simulation of BACE1 inhibition by (**A**) eckol, (**B**) dieckol, and (**C**) 8,8-bieckol. View of the binding site magnified from (**D**) eckol, (**E**) dieckol, and (**F**) 8,8-bieckol. Hydrogen interaction diagram of (**G**) eckol, (**H**) dieckol, and (**I**) 8,8-bieckol.


**Table 3.** Molecular interactions of BACE with eckol, dieckol, and 8,8-bieckol.

As indicated in Table 4 and Figure 6, the docking results for eckol, dieckol, and 8,8-bieckol indicated negative binding energies of −8.8, −9.5, and −9.2 kcal/mol, respectively. Hydrogen bonding interactions between eckol and THR83, TRP86, TYR124, and SER125 of AChE were observed by five hydrogen bonds (bonding distance of 2.855, 2.712, 3.134, 2.883, and 3.313 Å, respectively). Dieckol was bound at the ASN233, THR238, ARG296, and HIS405 of AChE, linked by four hydrogen bonds with bonding distances of 3.399, 2.837, 3.344, and 3.181 Å, respectively, while 8,8-bieckol had one hydrogen bond with the ARG296 residue of AChE (bonding distance: 3.151 Å).

**Figure 6.** In silico docking simulation of AChE inhibition by (**A**) eckol, (**B**) dieckol, and (**C**) 8,8-bieckol. View of the binding site magnified from (**D**) eckol, (**E**) dieckol, and (**F**) 8,8-bieckol. Hydrogen interaction diagram of (**G**) eckol, (**H**) dieckol, and (**I**) 8,8-bieckol.


**Table 4.** Molecular interactions of AChE with eckol, dieckol, and 8,8-bieckol.
