Kinetic Characterization of Tyrosinase-Catalyzed Oxidation of Four Polyphenols*

Current Medical Science 40(2):239-248,2020 DOICurrent https://doi.org/10.1007/s11596-020-2186-0 Medical Science 40(2):2020 239 Kinetic Characterization of Tyrosinase-catalyzed Oxidation of Four Polyphenols* Wan-yu LIU1†, Cong-ming ZOU2†, Jian-hua HU1, Zi-jun XU1, Lu-qin SI1, Jun-jun LIU1#, Jian-geng HUANG1# 1School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650021, China The Author(s) 2020, corrected publication July 2020 Summary: Phenolic compounds such as chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and caffeic acid are widely distributed in fruits, vegetables and traditional Chinese medicines with a wide range of biological activities. Tyrosinase plays a critical role in the food industry, but recent studies have proposed unexplored aspects of clinical application. Tyrosinase-catalyzed oxidation of four polyphenols as well as its underlying mechanism remains unclear. In the current work, we investigated the kinetic properties of tyrosinase-catalyzed oxidation of the four polyphenols of interest. To measure the unstable o-quinone products, an analytical method using 3-methyl-2-benzothiazolinone hydrazone (MBTH) was established. The optimal incubation time, buffer pH, temperature and enzyme concentration for the enzyme activity in the presence of each polyphenol of interest were investigated. Under the final optimized conditions, the kinetics and substrate specificity of four polyphenols were examined. Kinetic data showed that tyrosinase had the greatest substrate affinity to chlorogenic acid compared with its isomers and caffeic acid. The catalytic efficiency with chlorogenic acid was 8- to 15-fold higher than that with the other 3 polyphenols. Molecular docking study demonstrated that the tight binding of chlorogenic acid at the peripheral site should be the major reason for the specificity to chlorogenic acid. In light of this, the rational design of high-affinity inhibitors against tyrosinase may focus on the binding of both the Cu site and peripheral site. This study will supply a basis for the selection of phenolic acids in food industry and health care. Key words: polyphenols; tyrosinase; kinetic characterization; molecular docking Daily diet plays an important role in disease determined by a variety of genetic and environmental prevention. Evidence shows that people can benefit factors[3]. Phenolics not only contribute to the color from plant phenolic compounds which are often and sensory characteristics of vegetables and fruits, absorbed from the diet[1]. Phenolic compounds are but also provide protection against pathogens and ubiquitous in plants and consist of a large number of predators. More importantly, phenolic chemicals are a secondary metabolites derived from pentose phosphate, major source of the intake of natural antioxidants in the shikimate and phenylpropanoid pathways[2]. The human diet[4]. composition and amount of phenolic substances are Typical plant-derived phenolic compounds occurring in the human diet include flavonoids, The original version of this article was revised due to a phenolic acids, tannins, stilbens and lignans, etc. It is retrospective Open Access order. worthy to note that major classes of phenolic acids are Wan-yu LIU, E-mail: [email protected]; Cong- hydroxycinnamic acids and hydroxybenzoic acids. The ming ZOU, E-mail: [email protected] main polyphenolic representatives of hydroxycinnamic † The authors contributed equally to this study. acids are chlorogenic acid and caffeic acid[5]. The # Corresponding authors, Jun-jun LIU, E-mail: junjun.liu@ latter is found in foods largely as an ester form with hust.edu.cn; Jian-geng HUANG, E-mail: jiangenghuang@ quinic acid called chlorogenic acid[6]. Chlorogenic hust.edu.cn *The study was supported by grants from the National acid, also known as 3-O-caffeoyl quinine, is widely Natural Science Foundation of China (No. 81773811), distributed in fruits (apple), vegetables (potato), drinks [7] [8] Yunnan Applied Basic Research Project (No. 2017FB074), such as tea, coffee , wine , traditional Chinese the Yunnan Provincial Tobacco Monopoly Bureau Grants medicines such as honeysuckle[9], gardenia[10] and even (No. 2017YN09) and the Fundamental Research Funds for tobacco[11]. According to different esterification sites the Central Universities (No. 2020kfyXGYJ061). of quinine, 4-O-caffeoyl quinine (cryptochlorogenic 240 Current Medical Science 40(2):2020 acid) and 5-O-caffeoyl quinine (neochlorogenic food industry and health care. acid) are two important isomers of chlorogenic acid. Recently, an increasing number of research papers 1 MATERIALS AND METHODS have been published to ascertain the nutritional benefits and physiological effect[8]. Scientific 1.1 Materials evidence shows that chlorogenic acid has a wide Mushroom tyrosinase (2687 units/mg) was pur- range of biological activities, such as antioxidant[12] chased from Sigma-Aldrich (USA). Chlorogenic acid, and anti-mutagenesis[13], cardiovascular protection[14], cryptochlorogenic acid, neochlorogenic acid, caffeic antibacteria and antivirus[15], immune regulation, acid and 3-methyl-2-benzothiazolinone hydrazone lowering blood glucose and blood lipids[16]. Moreover, (MBTH) hydrochloride monohydrate were all purchased pharmacological studies have also demonstrated that from Macklin (China). N,N-dimethylformamide (DMF) both cryptochlorogenic acid and neochlorogenic acid was obtained from Sinopharm Chemical Reagent Co., exhibit excellent antioxidative, antibacterial, antiviral Ltd (China). All the chemicals were of analytical grade. and antipyretic activities[17]. In addition, caffeic acid 1.2 Methods has also demonstrated antioxidant activity in the 1.2.1 MBTH Assay of the Reaction Products The prevention of premature aging and antimicrobial reaction products were determined by MBTH assay activity in the treatment of dermal diseases[18]. described by Estelle Zeyer with minor modifications[32]. Tyrosinase is a copper-containing monooxygenase, The reaction mixture consisting of 750 μL of substrate widely distributed in plants, micro-organisms and solution, 750 μL of 24 mmol/L aqueous MBTH mammals[19]. Tyrosinase catalyzes two types of reaction, solution, and 750 μL of 100 mmol/L phosphate buffer by which monophenols and o-diphenols are oxidized (pH 7.2) containing 8% of DMF (analysis buffer) was to o-diphenols and o-quinones, respectively[20]. The pre-incubated at 37°C for 10 min. Then, 750 μL of 100 role of tyrosinase for triggering enzymatic browning U/mL enzyme solution was added to start the reaction. reactions has been well established in the food industry. Absorbance was recorded over 20 min using a UV- Searching potent tyrosinase inhibitors is of great 2100 spectrophotometer in 10-mm quartz cuvettes. importance for postponing the discoloration process. The maximum absorbance wavelength of the reactions To gain this goal, a large number of chemicals from for all the substrates was applied to all experiments both natural and synthetic sources have been tried[21–24]. below. Since the standards of the unstable reaction However, more efforts are still required to identify products were not commercially available, calibration better inhibitors without obvious adverse side effect. curves were constructed after sufficient incubation of Recently, a variety of studies have proposed several each substrate of specific concentration with excessive previously unexplored aspects of tyrosinase in clinical enzyme. studies. Tyrosinase also plays a key role in the pigments 1.2.2 Optimization of Reaction Conditions A synthesis such as melanin, which regulates the color of reaction mixture consisting of 50 μL of substrate hair and skin[25]. Tyrosinase is considered as a target in the solution, 50 μL of 24 mmol/L MBTH and 50 μL of treatment of specific dermatological diseases associated analysis buffer was pre-incubated for 10 min. The with melanin hyperpigmentation[26, 27]. Tyrosinase is analysis buffer consisted of 100 mmol/L phosphate also known as an autoantigen in various autoimmune buffer containing 8% of DMF. The reaction was disease and serves as a marker for vitiligo[28]. Despite initiated by the addition of enzyme and terminated by contradictory opinion concerning the role of tyrosinase adding 200 μL ice-cold acetonitrile. After vortex and in mutagenicity and tumor suppression, melanocyte- centrifugation, 200 µL of the supernatant was applied directed tyrosinase prodrug therapy might offer a for absorbance determination of the mixture system by highly selective drug delivery approach for malignant microplate reader. Prior to kinetic study of tyrosinase- melanoma[29, 30]. catalyzed oxidation of four polyphenols, the effect of Chlorogenic acid has been reported as a substrate incubation time, incubation buffer pH, temperature and of polyphenol oxidase derived from apple[7] and dill enzyme concentration was investigated to optimize (Anethum graveolens)[31]. The dill-derived polyphenol the reaction conditions. The incubation time was oxidase exhibited better affinity to chlorogenic acid optimized over a time range of 0 to 120 min. The than catechol and dopamine[31]. However, very little reaction was started by the addition of enzyme and attention has been devoted to polyphenol oxidase the absorbance was immediately measured during mediated oxidation of the isomers of chlorogenic acid the predefined time range by microplate reader. The such as cryptochlorogenic

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