Understanding the Inhibitory Mechanism of Tea Polyphenols Against Tyrosinase Using Fluorescence Spectroscopy, Cyclic Voltammetry
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RSC Advances View Article Online PAPER View Journal | View Issue Understanding the inhibitory mechanism of tea polyphenols against tyrosinase using fluorescence Cite this: RSC Adv.,2018,8,8310 spectroscopy, cyclic voltammetry, oximetry, and molecular simulations† Haifeng Tang,ace Fengchao Cui, *a Haijuan Li,b Qingrong Huang d and Yunqi Li *a Inhibiting the activity of tyrosinase is a very effective and safe way to prevent enzymatic browning in food and to resist pests in agriculture. Tea polyphenols (TPs), regarded as safe and non-toxic food additives, have been reported due to their potential inhibitory capability against tyrosinase, but their ambiguous inhibitory mechanisms have severely limited their application. In the present work, fluorescence spectroscopy, cyclic voltammetry (CV), oximetry and molecular simulation approaches were employed to shed light on the underlying inhibitory mechanisms of TPs with different structures including (+)-catechin, (À)-epicatechin Creative Commons Attribution 3.0 Unported Licence. gallate (ECG) and (À)-epigallocatechin gallate (EGCG) against tyrosinase. Fluorescence spectra show that the three TPs are capable of binding tyrosinase with a molar proportion of 1 : 1. The analysis of CV curves and oxygen utilization suggests that these three TPs can be oxidized by tyrosinase, revealing that these three TPs are suicide inhibitors of tyrosinase. Furthermore, ECG and catechin make tyrosinase irreversibly inactivated due to their catechol group (ring B) being catalyzed by tyrosinase through Received 24th November 2017 a cresolase-like pathway, while EGCG inhibits the activity of tyrosinase by competing with or delaying Accepted 15th February 2018 the oxidation of substrate. Molecular simulations further confirm that ring B of ECG and catechin makes DOI: 10.1039/c7ra12749a a significant contribution to tyrosinase inhibitory activities, and has a direct interaction with the coupled This article is licensed under a rsc.li/rsc-advances binuclear copper ions in the optimal orientation required by the cresolase-like pathway. 1. Introduction food industry, tyrosinase can expedite the enzymatic browning Open Access Article. Published on 22 February 2018. Downloaded 10/6/2021 11:45:25 AM. of post-harvest produce by catalyzing the oxidation of poly- Tyrosinase (EC 1.14.18.1) containing a catalytic center with bi- phenol compounds,4 leading to the loss of nutritional quality copper ions coordinated by six histidine residues, can catalyze and the shortening of storage life.5 Moreover, the higher tyros- the oxidation of both monophenol (cresolase or mono- inase activity would adversely affect the pest control in agri- phenolase activity) and diphenol (catecholase or diphenolase culture due to promoting the physiological processes of larval activity) to the corresponding ortho-quinones, followed by self- maturity.6,7 Inhibiting tyrosinase bioactivity is therefore favor- polymerizing to dark melanin or another pigment.1–4 In these able to the preservation of agricultural product and pest oxidation reactions, the redox forms of the catalytic center prevention. Although a plenty of natural tyrosinase inhibitors include Cu2–O2 with both monophenolase and diphenolase have been discovered, the understanding of the inhibition activities, Cu2–O with diphenolase activity and Cu2 without any mechanism of tyrosinase inhibitors is still de cient, which have activity, according to the amount of coordinated oxygen.1 In the severely limited their application in food industry and agricul- ture and the development of new inhibitors.8–11 Flavonoids, widely used as dietary supplements to functional aKey Laboratory of Synthetic Rubber, Changchun Institute of Applied Chemistry foods,12 are the largest groups in natural tyrosinase inhibitors (CIAC), Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China. E-mail: which have been discovered,13–17 up to now. Flavonoids were [email protected]; [email protected] usually subdivided into six major groups including avanols, b State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry (CIAC), Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China avones, avonols, avanones, iso avones and anthocyani- 18 cSchool of Life Science, Jilin University, Changchun, Jilin 130012, P. R. China dins. The position and number of hydroxyl group on func- ff dDepartment of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ tional moiety of avonoids have a signi cant e ect on their 08901, USA inhibitory activity and associated mechanism. Some avonols eUniversity of Chinese Academy of Sciences, Beijing 100049, P. R. China with a 3-hydroxyl-4-keto moiety, such as kaempferol, quercetin † Electronic supplementary information (ESI) available. See DOI: and morin, exhibit the inhibitory activity toward tyrosinase by 10.1039/c7ra12749a 8310 | RSC Adv.,2018,8,8310–8318 This journal is © The Royal Society of Chemistry 2018 View Article Online Paper RSC Advances chelating with the copper ions and occupying the catalytic Chenhua Co., Shanghai, China). Oxygen consumptions of center,15,19 but most of avonols have weaker inhibitory activity tyrosinase interacting with TPs were monitored with YSI 5000 than kojic acid.20 Two isoavone metabolites (8-hydrox- dissolved oxygen meter (YSI Inc., American). ydaidzein and 8-hydroxygenistein), deemed as the suicide substrate of tyrosinase, can make tyrosinase irreversibly inac- 2.2 Inhibitory activity in vitro tivated by substrate-like interaction.14 Tea polyphenols (TPs), such as (+)-catechin (catechin), (À)-epicatechin gallate (ECG) The inhibitory activity of the TPs, i.e., catechin, ECG, and EGCG, and (À)-epigallocatechin-3-O-gallate (EGCG), which belong to and a positive control, i.e., KA, against diphenolase activity of avanols, frequently present in human diet due to their strong tyrosinase was measured with L-DOPA as a substrate using the antioxidant activity. The report has shown that they can reported spectroscopy method.13,24 Assays were conducted in competitively inhibit the monophenolase activity of tyrosinase a 96-well micro-plate and a spectrophotometer reader was used in vitro.21 Particularly, EGCG can penetrate the cell membrane to determine the absorbance.25 Briey, 6 mL of 1000 unit per mL and suppress the activity of tyrosinase in B16 murine melanoma tyrosinase solution was combined with potassium phosphate cell.22 Although the inhibitory activity of these TPs against buffer (pH 6.5) and 1 mM KA or TPs (both dissolved in potas- tyrosinase has been reported, their inhibitory mechanisms are sium phosphate buffer, pH 6.5). Aer pre-incubating at 25 C still elusive. for 5 min, 150 mLof1mML-DOPA aqueous solution was added, In this work, we managed to reveal the inhibitory mecha- followed by incubation at 25 C for 10 min, again, before the nisms of TPs (including catechin, ECG and EGCG as depicting concentration determination of DOPA quinone at 475 nm in Fig. 1) using uorescence spectroscopy, cyclic voltammetry absorbance. The total volume of each reaction system was 300 (CV) and oximetry together with molecular simulations. Fluo- mL. As an exception, the tyrosinase inhibitory activity of catechin rescence spectra determined the nature of interactions between was measured with HPLC rather than spectroscopy, as it TPs and tyrosinase. CV identied the inhibitory mechanism of possessed an overlap adsorption band with DOPA quinone ff which will interfere the analysis of inhibitory activity.26 The Creative Commons Attribution 3.0 Unported Licence. TPs with di erent functional moieties. Oxidation kinetics of tyrosinase discovered the inactivation of TPs against tyrosinase inhibitory activity was expressed by IC50 value, which is equal to by Quintox mechanism. Molecular simulations provided a deep the concentration of inhibitor at 50% inhibitory rate according understanding of their inhibitory mechanism against tyrosi- to the following equation:25 nase at the molecular level. ðA À BÞðC À DÞ ¼ Â % inhibition rate A À B 100% (1) 2. Materials and methods where, A is the optical density (OD475) of potassium phosphate buffer; B, C and D are the OD475 of potassium phosphate buffer 2.1 Chemical reagents and apparatus This article is licensed under a with tyrosinase, with both TPs and tyrosinase, and with TPs, Tyrosinase (TYR, EC1.14.18.1) from mushroom, L-3,4-dihy- respectively. droxyphenylalanine (L-DOPA), kojic acid (KA), catechin, ECG, and EGCG were purchased from Sigma-Aldrich. Nitric acid and Open Access Article. Published on 22 February 2018. Downloaded 10/6/2021 11:45:25 AM. dimethyl formamide (DMF) were purchased from Beijing 2.3 Fluorescence spectroscopy Chemical Works. Multiwalled carbon nanotubes (MWCNTs) The uorescence spectrum of the TP-TYR complex was were purchased from the Nanotech Port Co. Ltd (Shenzhen, measured according to our previous work.27 Briey, 10 mM TP China). Before use, the MWCNTs were acidied by nitric acid.23 was titrated into 3 mL potassium phosphate buffer (pH 6.5) Spectrophotometer reader (BioTek Instruments Inc., USA) containing 10 units tyrosinase under continuous stirring and and High Performance Liquid Chromatograph (HPLC: Shi- nitrogen exists. Aer 5 minutes, tyrosinase was excited at madzu Instruments Inc., Japan) were used to measure the 274 nm and the emission spectrum over the range of 280 nm to inhibitory activity of the tyrosinase inhibitors. CV experiments 400 nm was detected through a 3 nm slit. The emission spec- were performed in a standard three-electrode electrochemical