Tyrosinase Inhibition Ability Provided by Hop Tannins: A Mechanistic Investigation ^†

Abstract

The hop is rich in tannins used as a conventional additive in the beer industry, but other applications are limited. This study investigated the tyrosinase inhibition activity of extracted hop tannins and the associated structure–function activity. The tannins were extracted and subjected to a gel permeation chromatography (GPC), a nuclear magnetic resonance (NMR) and an acid-cleavage coupled HPLC-ESI-MS/MS analysis to obtain the structural information of the tannins. Then, tyrosinase inhibition kinetic assays, inductively coupled plasma optical emission spectrometer and antioxidant (ICP-OES), circular dichroism (CD) as well as molecular docking analysis were applied to investigate the inhibition mechanism. Furthermore, the intracellular inhibition ability of hop tannins was assessed with B16-F10 cells. The results indicated that hop tannins were composed of (epi)catechin as extensional units and (epi)gallocatechin as terminal units and can be classified as prodelphenidins. The tyrosinase inhibition assays showed the hop tannin had a IC₅₀ = 76.52 ± 6.56 µM, meanwhile it inhibited the tyrosinase through a competitive–noncompetitive mixed way. The tannins were found to bind on the surface of tyrosinase via forming hydrogen bonding and consequently changed the secondary structure of tyrosinase. The fluorescence and antioxidant assay indicated the tannin had both copper ion chelating and antioxidant ability, which may also contribute to the inhibition. The intracellular inhibition analysis showed that activity of tyrosinase was reduced by 66.67% and melanin production was found to be reduced by 34.50% while 10 µM hop tannins were applied. These results indicated that the hops are not only important in the beer industry, but that hop tannins can be also applied as whitening agents in the cosmetic industry.

1. Introduction

Tyrosinase is a copper-containing enzyme that is involved in melanin hyperpigmentation in humans, molting in insects, and undesirable browning in fruits and vegetables [1]. At present, a variety of tyrosinase inhibitors have been developed and widely used in the market. However, due to safety and stability factors, only a few inhibitors were used in commercial production [2].

Condensed tannins are classified to polyphenols, which are widely distributed in plant tissues [3]. The hop is rich in tannins used as a conventional additive in the beer industry, but other applications are limited. In order to expand the industrial utilization of the hops, and also provide a new tyrosinase inhibitor for the cosmetic industry, the structure-related tyrosinase inhibition mechanisms of the hop tannins were studied. The tannins were extracted and purified with a Sephadex LH-20 column, then the structures of the hop tannins were characterized through a gel permeation chromatography (GPC), a nuclear magnetic resonance (NMR) and an acid-cleavage coupled HPLC-ESI-MS/MS analysis. Then, the inhibition mechanism of the hop tannins was evaluated through tyrosinase inhibition kinetic assay, Circular Dichroism (CD) Spectroscopy, molecular docking (MD), antioxidant assay as well as an inductively coupled plasma-optical emission spectroscopy (ICP-OES). The tyrosinase inhibition ability was also observed on the B16 melanoma cell model.

2. Materials and Methods

2.1. Preparation of Hop Tannins Extract

The method for extraction and purification of the hop tannins was the same as our previous report [4].

2.2. GPC

GPC was used to obtain the average molecular mass of the purified tannin according to our previous report and Kennedy’s report [5,6].

2.3. ¹³C-NMR Analysis

Purified hops powder (30 mg) was dissolved with 750 µL CD₄O:D₂O (1:1, v/v), and loaded into a nuclear magnetic tube for ¹³C-NMR analysis at a frequency of 100.60 MHz, acquisition time of 1.36 s.

2.4. Acid-Cleavage Coupled with HPLC-ESI-MS/MS Analysis

The acid-cleavage and reversed-phase HPLC-ESI-MS/MS analysis was applied, MS detection was done using a positive ionization in multiple-reaction monitoring (MRM) mode.

2.5. Tyrosinase Inhibition Kinetic Assay

The effects of the hops tannins on mushroom tyrosinase activity was assayed by a spectrophotometric method according to our previous report [4].

2.6. CD

The CD spectra were obtained on a MOS-450 AF-CD CD spectropolarimeter to investigate the influence of hop tannins on the secondary structure in mushroom tyrosinase [7,8].

2.7. MD

Enzyme-substrate molecule docking experiments were performed using AutoDock Vina software (DeLano Scientific LLC, Palo Alto, CA, USA). AutoDock Vina was selected as docking method to perform the blind docking simulations within a grid box that was centered on the two copper ions of the active site. All visualizations were analyzed with PyMOL 2.2 (DeLano Scientific LLC, Palo Alto, CA, USA).

2.8. Antioxidant Ability

The antioxidant activity of the hops tannin was assessed through 3-ethylbenzthiazolin-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The hop tannins against DPPH radical was analyzed by the method described in Brand-Williams’s report [9,10], while the ABTS radical was assessed as described by Re et al. [11].

2.9. ICP-OES

The chelating ability between Cu²⁺ and hops tannin was measured using ICP-OES as previously described [12].

2.10. Cell Culture and Cytotoxicity Assay

B16-F10 cells were grown in Dulbecco’s modified eagle medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin and cultured in a cell incubator with humidified atmosphere of 37 °C and 5% CO₂. The medium was changed every 2 days. Cells were digested with 0.25% trypsin and subcultured when the cells were about 80% confluent. The cytotoxicity levels of hop tannins on melanoma B16F10 cells were determined according to the standard method of CCK-8 assay.

2.11. The Effect of Hop Tannins on Intracellular Melanin Production and Tyrosinase Activity of B16F10 Melanoma Cells

Melanin content assay was performed to evaluate the inhibitory effect of the hop tannins on melanogenesis according to method of Si [13]. The biochemical enzyme method used was as described by Si et al. [13] with some modifications. First, 2 mL of B16F10 mouse melanoma cells (1 × 10⁶ cells) were seeded in a 6-well plate. After cell adherence, the hops tannins were added for 48 h. After that, the cells were washed thrice with PBS, and then broken with 500 μL of PBS buffer (pH 6.8) containing 1% (w/v) Triton X-100.

2.12. Statistical Analysis

One-way analysis of variance (ANOVA) was performed to determine differences between the experimental samples (triplicated) using Minitab 18 (Minitab Inc., York, PA, USA). Tukey’s post hoc test was used to infer a specific difference between the individual samples, and different letters indicate a significant difference where p < 0.05.

3. Results and Discussion

3.1. Structural Analysis

Hop tannin structure was elucidated using ¹³C NMR to obtain the composition of the structural moieties. Characteristic signals of (epi)catechin and (epi)gallocatechin were detected. Acid-cleavage analysis coupled HPLC-ESI-MS/MS results indicated that hop tannins were composed of (epi)catechin as extensional units and (epi)gallocatechin as terminal units. GPC result showed the average molecular weight of hop tannins was 3581 g/mol.

3.2. Inhibitory Effect, Mechanism, and Type of Hop Tannins on the Tyrosinase

Figure 1A showed that catalytic activity of the tyrosinase was reduced while increasing hop tannin concentrations, implying the inhibition ability from hop tannins. The IC₅₀ of hop tannins was determined as 76.52 ± 6.56 µM (

Table 1. Tyrosinase inhibition activity of the hop tannins ¹.

	IC50 (mg/mL)	Inhibition Mechanism	Inhibition Type	Inhibition Constants (mM)
Hop tannin	76.52 ± 6.56 µM a	reversible	mixed	KIS = 2.61 ± 0.16	KI = 1.79 ± 0.12
Kojic acid	49.54 ± 2.08 µM b	--	--	--	--

¹ Data are expressed as the mean of three replicates ± standard deviation; the data were compared by one-way ANOVA and Tukey’s post hoc test; different letters within a column indicate a significant difference, p < 0.05.

), which was comparable to kojic acid (49.54 ± 2.08 µM). The inhibition kinetic analysis showed a set of straight lines that all passed through the origin (Figure 1B), indicating that the hop tannins inhibit the tyrosinase through a reversible inhibition. The inhibitory type was further evaluated through Lineweaver–Burk plot (Figure 1C), second quadrant intersected straight lines were observed. It implied hop tannins inhibit tyrosinase through a competitive–noncompetitive mixed way. K_I (1.79 ± 0.12) showed smaller value than K_IS (2.61 ± 0.16), indicating that the affinity of the inhibitor for free enzyme was stronger than enzyme–substrate complex.

3.3. CD

Structural conformations are crucial for function and activity of tyrosinase. However, the secondary structure of tyrosinase were changed after interact with hop tannins (Figure 2). This conformation changes may lead by the non-selective binding of tannin and result in a decrease of tyrosinase catalytic activity [14].

3.4. Copper Ion Chelating and Antioxidant Abilities

The copper ions are responsible for: (1) remaining the conformation of active cite of tyrosinase and; (2) participating in the catalyze reaction (redox reaction). In the current study, the hop tannins not only showed antioxidant abilities, but were also found able to chelate with copper ions (

Table 2. Copper ion chelating and antioxidant abilities of hop tannins ¹.

	Cu2+ Chelating (%)	DPPH Free Radical Scavenging Activity (IC50 μM)	ABTS Free Radical Scavenging Activity (IC50 μM)
Hop tannins	44.77 ± 0.45	1.17 ± 0.08 b	1.52 ± 0.02 b
Ascorbic acid	-	22.04 ± 0.28 a	17.83 ± 0.30 a

¹ Data are expressed as the mean of three replicates ± standard deviation; the data were compared by one-way ANOVA and Tukey’s post hoc test; different letters within a column indicate a significant difference, p < 0.05.

). These results indicated hop tannins could either alter the conformation of tyrosinase by forming tannin–copper complexes, or hinder the redox reaction.

3.5. Molecular Docking Analysis

Molecular docking analysis suggested that the structural units of hop tannins were observed embedding into the target active cavity of the tyrosinase and interacted with the surrounding amino acid residues (Figure 3). The results also found that the hydrogen bonds were mainly responsible for the tannin-tyrosinase binding.

3.6. Effect of Hop Tannin on Cell Viability, Melanin Synthesis, and Tyrosinase Activity in B16F10 Mouse Melanoma Cells

Within non-toxic dosages (0–10 μM, Figure 4A), melanin content and intracellular tyrosinase activity were observed reducing with increasing hop tannin concentrations. Especially when 10 μM hop tannins were applied, tyrosinase activity was reduced by 66.67% and melanin production was reduced by 34.50%. These findings demonstrated that hop tannins regulated tyrosinase and subsequently suppressed melanin synthesis in B16F10 mouse melanoma cells at nontoxic doses.

4. Conclusions

Condensed tannins were extracted from hops and characterized as prodelphenidins ((epi)catechin as extensional units and (epi)gallocatechin as terminal units). The hop tannins showed outstanding tyrosinase inhibition ability through a competitive–noncompetitive mixed way. This inhibition ability can be attributed to: (1) changing the conformation of tyrosinase after binding process; (2) chelating with copper ions; (3) hinder the redox reaction of the copper ions; and (4) blocking the active sites through forming hydrogen bonds with amino acid residues. On the aspect of melanoma cell, the hop tannins were also found able to regulate tyrosinase and subsequently suppressed melanin synthesis.