*4.1. General*

Fluorescence and optical density were measured by Bio-TEK ELISA fluorescence reader FLx 800 and Bio-TEK ELx 808, respectively (Winooski, VT, USA). Eckol (>95%), dieckol (>95%), and 8,8-bieckol (>95%), were bought from National Development Institute of Korean Medicine (Gyeongsangbuk-do, Korea). The BACE1 assay kit was purchased from Invitrogen (Pan Vera, Madison, WI, USA). TACE and substrate were bought from R&D Systems (Minneapolis, MN, USA). AChE from *Electrophorus electricus* (electric eel), 5,5-dithiobis-(2 nitrobenzoic acid) (DTNB), resveratrol, galantamine, trypsin, chymotrypsin, elastase, and their substrates, including N-benzoyl-L-Arg-pNA, N-benzoyl-L-Tyr-pNA, and N-succinyl-Ala-Ala-Ala-p NA, were from Sigma-Aldrich (St. Louis, MO, USA).

#### *4.2. Enzyme inhibition Studies*

Fluorometric assays with a recombinant human BACE1 or TACE were conducted according to manufacturer instructions. Briefly, reaction mixtures containing human recombinant BACE1 (1.0 U/mL), the substrate (75 μM in 50 mM ammonium bicarbonate), and phlorotannins dissolved in an assay buffer (50 mM sodium acetate, pH 4.5) were incubated in darkness for 60 min at 25 ◦C in well plates. The increase in fluorescence intensity produced by substrate hydrolysis was observed on a fluorescence microplate reader with excitation and emission wavelengths of 545 and 590 nm, respectively. The inhibition ratio was obtained using the following equation:

$$\text{Inhibtition (\%)}=\{1-(S-S\_0)/(C-C\_0)\}\times 100$$

where *C* was the fluorescence of control (enzyme, assay buffer, and substrate) after 60 min of incubation, *C*0 was the fluorescence of control at time 0, *S* was the fluorescence of tested samples (enzyme, sample solution, and substrate) after 60 min of incubation, and *S*0 was the fluorescence of the tested samples at time 0.

A human recombinant TACE (0.1 ppm in 25 mM Tris buffer), the substrate (APP peptide YEVHHQKLV using EDANS/DABCYL), and phlorotannins were dissolved in an assay buffer, which were then combined and incubated for 60 min in the dark at 25 ◦C. The increase in fluorescence intensity produced by substrate hydrolysis was observed on a fluorescence microplate reader with excitation and emission wavelengths of 320 and 405 nm, respectively.

The colorimetric assays, including AChE, trypsin, chymotrypsin, and elastase were assayed according to previously described methods [36]. The hydrolysis of AChE was monitored according to the formation of yellow 5-thio-2-nitrobenzoate anions at 405 nm for 15 min, which were produced by the reaction of DTNB with thiocholine released from ACh. All reactions were performed in 96-well plates in triplicate and recorded using a microplate spectrophotometer.

*<sup>N</sup>*-benzoyl-L-Arg-*p*NA, *<sup>N</sup>*-benzoyl-L-Tyr-*p*NA, and *<sup>N</sup>*-succinyl-Ala-Ala-Ala-*p*NA were used as substrates to assay the inhibition of trypsin, chymotrypsin, and elastase, respectively. Enzyme, Tris-HCl buffer (0.05 M, in 0.02 M CaCl2, pH 8.2), and phlorotannins were incubated for 10 min at 25 ◦C; then, substrate was added for 30 min at 37 ◦C. The absorbance was recorded at 410 nm. The inhibition ratio was obtained using the following equation:

> Inhibition (%) = {[1 − (A − B)]/control} × 100

where A was the absorbance of the control (enzyme, assay buffer, and substrate) after 60 min of incubation, and B was the absorbance of tested sample (assay buffer and sample solution) after 60 min of incubation.

#### *4.3. Enzyme Kinetic Study*

To evaluate the kinetic mechanisms of phlorotannins towards BACE1 and AChE, Dixon and Lineweaver–Burk plots were conducted by various concentrations of substrate and inhibitors. Kinetic parameters such as *K*i, Vmax, and K m values were calculated by Sigma Plot 12.3 (Systat Software, Inc., San Jose, CA, USA)

#### *4.4. Molecular Docking Study*

X-ray crystal structures of human BACE1 (PDB code: 2WJO) and AChE (PDB code: 4PQE) were retrieved from the Protein Data Bank (PDB, http://www.rcsb.org/). Three-dimensional (3D) structures of eckol, dieckol, and 8,8-bieckol were obtained from PubChem with compound identification number (CID) of 145937, 3008868, and 3008867, respectively. The Autodock Vina software version 1.1.2 (The Scripps Research Institute, La Jolla, CA, USA,) was used to conduct molecular docking analysis. The dimensions of the grid were 30 × 30 × 30 Å, the cluster radius was 1 Å, and the C α coordinates in each selected backbone binding residue of the protein receptor was used for the center of docking space. Other options for docking simulations were used as defaults. The atomic coordinates of the ligands were drawn and displayed using Marvin sketch (5.11.4, 2012, ChemAxon, One Broadway Cambridge, MA, USA).

## *4.5. Statistical Analysis*

All results were presented as the mean ± SD of three independent experiments. Statistical significance was assessed by Duncans multiple range tests using Statistical Analysis System (SAS) version 9.3 (SAS Institute, Cary, NC, USA).
