Slow-Binding Inhibition of Tyrosinase by Ecklonia cava Phlorotannins

Tyrosinase inhibitors improve skin whitening by inhibiting the formation of melanin precursors in the skin. The inhibitory activity of seven phlorotannins (1–7), triphlorethol A (1), eckol (2), 2-phloroeckol (3), phlorofucofuroeckol A (4), 2-O-(2,4,6-trihydroxyphenyl)-6,6′-bieckol (5), 6,8′-bieckol (6), and 8,8′-bieckol (7), from Ecklonia cava was tested against tyrosinase, which converts tyrosine into dihydroxyphenylalanine. Compounds 3 and 5 had IC50 values of 7.0 ± 0.2 and 8.8 ± 0.1 μM, respectively, in competitive mode, with Ki values of 8.2 ± 1.1 and 5.8 ± 0.8 μM. Both compounds showed the characteristics of slow-binding inhibitors over the time course of the enzyme reaction. Compound 3 had a single-step binding mechanism and compound 5 a two-step-binding mechanism. With stable AutoDock scores of −6.59 and −6.68 kcal/mol, respectively, compounds 3 and 5 both interacted with His85 and Asn260 at the active site.


Introduction
Melanin is produced in skin epidermal cells to protect the skin from ultraviolet radiation [1]. The overproduction and accumulation of melanin causes age spots, freckles, melisma, and hyperpigmentation [1,2]. Tyrosinase (EC 1.14.18.1), which belongs to the type 3 copper protein family, is a key enzyme in melanogenesis [1,3]. It is a multifunctional oxide that contains a copper in each of two sets of three histidine residues in the active site. [4]. Furthermore, it catalyzes the hydroxylation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA), and the subsequent oxidation of l-DOPA to DOPA quinone [3,5]. These products are used in melanin biosynthesis [4]. Recently, the tyrosinase inhibitors kojic acid and arbutin were developed to improve skin whitening and as anti-hyperpigmentation agents [3,6]. However, they had undesirable side effects, including dermatitis, skin irritation, and DNA damage [1,4]. Much research has examined a variety of natural plants, with the aim of developing new inhibitors [7]. This study also sought alternative inhibitors without adverse effects.
This study evaluated the tyrosinase inhibitory activity of minor components from E. cava. Among seven isolated compounds (1)(2)(3)(4)(5)(6)(7), two minor compounds (3 and 5) had IC50 values of less than 10 μM, and also exhibited competitive and slow-binding inhibition of tyrosinase. This study also shows how these compounds interact with the catalytic site of tyrosinase via molecular docking.

Inhibition of Phlorotannins on Tyrosinase
Phlorotannins of E. cava and E. stolonifera were reported to have the inhibitory activity on mushroom tyrosinase [12,19]. 7-phloroeckol and dieckol were revealed to be the potential inhibitors within micromole concentration [13,19]. This study evaluated the ability of the isolated phlorotannins 1-7 to suppress the catalytic reaction of tyrosinase over time, in the absence or presence of inhibitor. Their inhibitory activity was calculated using equation (1). A commercial tyrosinase inhibitor was used as a positive control (kojic acid; IC50 = 25.0 ± 0.4 μM). To identify potent inhibitors, the inhibitory activity of all of the isolated compounds at 100 μM was tested against tyrosinase in vitro (Table 1). Of these, compounds 2-5 were confirmed to have inhibitory activity exceeding 50%. Serial dilutions were used to calculate the IC50 values. The tyrosinase inhibitory activity increased in a dosedependent fashion (Figure 2A). Compounds 2-5 showed inhibitory activity, with IC50 values of 7.0 ±

Inhibition of Phlorotannins on Tyrosinase
Phlorotannins of E. cava and E. stolonifera were reported to have the inhibitory activity on mushroom tyrosinase [12,19]. 7-phloroeckol and dieckol were revealed to be the potential inhibitors within micromole concentration [13,19]. This study evaluated the ability of the isolated phlorotannins 1-7 to suppress the catalytic reaction of tyrosinase over time, in the absence or presence of inhibitor. Their inhibitory activity was calculated using equation (1). A commercial tyrosinase inhibitor was used as a positive control (kojic acid; IC 50 = 25.0 ± 0.4 µM). To identify potent inhibitors, the inhibitory activity of all of the isolated compounds at 100 µM was tested against tyrosinase in vitro (Table 1). Of these, compounds 2-5 were confirmed to have inhibitory activity exceeding 50%. Serial dilutions were used to calculate the IC 50 values. The tyrosinase inhibitory activity increased in a dose-dependent fashion ( Figure 2A). Compounds 2-5 showed inhibitory activity, with IC 50 values of 7.0 ± 0.2 to 66.4 ± 0.1 µM (Table 1). Of these, compounds 3 and 5 had inhibitory activity at < 10 µM. Interestingly, the structure of compound 5 contained the moiety of compound 3.
To gain insight into the interaction of the enzyme with phlorotannins, as tyrosinase inhibitors, the products of the catalytic reaction were determined by ultraviolet-visible photometry after the inhibitor was added to the enzyme solution. The IC 50 values of compounds 3 and 5 increased linearly with [S]/Km ( Figure 2B). Furthermore, compounds 3 and 5 were produced with a set of the family liners by various inhibitors at substrate concentrations ranging from 0.156 to 2.50 mM. As shown in Lineweaver-Burk plots ( Figure 2C,D), they had different values of −1/Km and the same 1/Vmax. The interactions with tyrosinase were competitive, with respective K i values of 8.2 ± 1.1 and 5.8 ± 0.8 µM based on Dixon plots ( Figure 2E,F, Table 1). Phlorotannins from seaweeds have been used to develop the non-competitive tyrosinase inhibitors 7-phloroeckol, dieckol, and phlorofucofuroeckol A, and the competitive inhibitors phloroglucinol and eckstolonol [12,13]. We found that compounds 3 and 5 competitively inhibited the catalysis of tyrosinase and might be useful for improving whitening.  (Table 1). Of these, compounds 3 and 5 had inhibitory activity at < 10 μM.
Interestingly, the structure of compound 5 contained the moiety of compound 3.
To gain insight into the interaction of the enzyme with phlorotannins, as tyrosinase inhibitors, the products of the catalytic reaction were determined by ultraviolet-visible photometry after the inhibitor was added to the enzyme solution. The IC50 values of compounds 3 and 5 increased linearly with [S]/Km ( Figure 2B). Furthermore, compounds 3 and 5 were produced with a set of the family liners by various inhibitors at substrate concentrations ranging from 0.156 to 2.50 mM. As shown in Lineweaver-Burk plots ( Figure Table 1). Phlorotannins from seaweeds have been used to develop the non-competitive tyrosinase inhibitors 7-phloroeckol, dieckol, and phlorofucofuroeckol A, and the competitive inhibitors phloroglucinol and eckstolonol [12,13]. We found that compounds 3 and 5 competitively inhibited the catalysis of tyrosinase and might be useful for improving whitening.

Slow-Binding Inhibition
To confirm time-dependent inhibition by the potential inhibitors (compounds 3 and 5), the substrate was added into a mixture of ligand (3.1 μM) that was preincubated with tyrosinase. Over time, their inhibitory activities increased. To calculate the slow-binding parameters (k3, k4, k5, k6, and kapp i), the progress curves were analyzed using equation (2), with increasing concentrations of compounds 3 and 5 ( Figure 3C,D); a replot of kobs was obtained as [I] ( Figure 3E,F). The replot of compound 3 was a straight line that fit equation (3). When forming an encounter complex (EI) of the receptor with the ligand, compound 3 slowly binds to the active site of tyrosinase according to slowbinding mechanism A (a single-step binding mechanism) ( Figure 3E) [20]. In comparison, the replot of kobs for compound 5 fit a hyperbolic equation (5) based on mechanism B (a two-step binding mechanism). This indicates that enzyme isomerization (E * I) results in slow bonding after the ligand rapidly interacts with the receptor [20,21]. Table 2 shows the kinetic parameters for the timedependent inhibition of tyrosinase by the inhibitors. The two strongest inhibitors had different slowbinding mechanisms. Inhibitor 5, which has a higher molecular weight, induced a new conformational state of the enzyme.

Slow-Binding Inhibition
To confirm time-dependent inhibition by the potential inhibitors (compounds 3 and 5), the substrate was added into a mixture of ligand (3.1 µM) that was preincubated with tyrosinase. Over time, their inhibitory activities increased. To calculate the slow-binding parameters (k 3 , k 4 , k 5 , k 6 , and k app i), the progress curves were analyzed using equation (2), with increasing concentrations of compounds 3 and 5 ( Figure 3C,D); a replot of k obs was obtained as [I] ( Figure 3E,F). The replot of compound 3 was a straight line that fit equation (3). When forming an encounter complex (EI) of the receptor with the ligand, compound 3 slowly binds to the active site of tyrosinase according to slow-binding mechanism A (a single-step binding mechanism) ( Figure 3E) [20]. In comparison, the replot of k obs for compound 5 fit a hyperbolic equation (5) based on mechanism B (a two-step binding mechanism). This indicates that enzyme isomerization (E * I) results in slow bonding after the ligand rapidly interacts with the receptor [20,21]. Table 2 shows the kinetic parameters for the time-dependent inhibition of tyrosinase by the inhibitors. The two strongest inhibitors had different slow-binding mechanisms. Inhibitor 5, which has a higher molecular weight, induced a new conformational state of the enzyme. Table 2. Kinetics parameters of tyrosinase by compounds 3 and 5. Compound

Plant Material
E. cava was purchased from a herbal market in Jeju Island, Korea, on May 2015. One of the author (Prof. Y.H. Kim) identified this brown algal species. A voucher specimen (CNU-15005) was deposited at the Herbarium, College of Pharmacy, Chungnam National University (CNU).

Plant Material
E. cava was purchased from a herbal market in Jeju Island, Korea, on May 2015. One of the author (Prof. Y.H. Kim) identified this brown algal species. A voucher specimen (CNU-15005) was deposited at the Herbarium, College of Pharmacy, Chungnam National University (CNU).

Tyrosinase Assay
To evaluate inhibitory activity on tyrosinase with isolated compounds, 130 µL of tyrosinase in 0.05 mM phosphate buffer (pH 6.8) was divided in 96 well plates [19]. 20 µL of compound concentrations ranging from 1-0.015 mM was added. 50 µL of 1.5 mM L-tyrosine in phosphate buffer was diluted into the mixture for calculating the inhibitory activity. 50 µL of 10-0.62 mM L-tyrosine in buffer was added to analyze initial velocity (v 0 ). After starting their reaction for 20 min, an amount of the product was detected at UV-vis 475 nm. The inhibitory activity was analyzed according to Equation (1) Inhibitory activity rate (%) = [(∆C − ∆S)/∆C] × 100 (1)

Slow-Binding Inhibition Analysis
The progress curves were calculated to Equation (2) by Morrison according to time course for 420 s.
[P] = v s t + [(v 0 − v s )/k obs (1 − e −k obst )] where t is time, [P] is product intensity, v 0 and v s are the initial and steady-state reaction velocities, and k obs is the apparent first-order rate concentration.