Natural and Synthetic Tyrosinase Inhibitors as ... - Wiley Online Library

Natural and Synthetic Tyrosinase Inhibitors as Antibrowning Agents: An Update M. R. Loizzo, R. Tundis, and F. Menichini

Abstract: Tyrosinase (EC 1.14.18.1), a copper-containing enzyme, can cause enzymatic browning in raw fruits, vegetables, and beverages. Browning is an undesirable reaction that is responsible for less attractive appearance and loss in nutritional quality. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in the food industry. This article reviews recent studies on tyrosinase inhibitors of natural and synthetic origins. The information offered here should help food industry in developing and using potential tyrosinase inhibitors desirable efficacy and safety, and for improving food quality.

Introduction Appearance is one of the attributes that are considered by consumers when they choose a food product. Among them color is a critical determinant for the appearance of fruits, vegetables, and crustaceans. Browning usually impairs the color attribute together with sensory properties such as flavor and texture (softening). However, this process is sometimes desirable, as it can improve the sensory properties of some products such as dark raisins and fermented tea leaves (Martinez and Whitaker 1995). Browning occurs by 2 components: enzymatic and nonenzymatic oxidation. Specifically, reactions of amines amino acids, peptides, and proteins with reducing sugars and vitamin C (nonenzymatic browning, often called Maillard reaction browning), and quinones (enzymatic browning) cause deterioration of food during storage and commercial or domestic processing. The loss of nutritional quality is attributed to the destruction of essential amino acids and a decrease in digestibility and inhibition of proteolytic and glycolytic enzymes. The production of antinutritional and toxic compounds may further reduce the nutritional value and possibly the safety of foods (Mauron 1990). Enzymatic browning results from the action of a group of enzymes namely tyrosinase. This enzyme is widely distributed in nature, including bacteria, fungi, higher plants (with particularly high amounts in mushroom, banana, apple, pear, potato, avocado, and peach), and animals (Mayer 2006). Enzymatic browning of fruits, vegetables, and beverages takes place in the presence of oxygen when tyrosinase and its polyphenolic substrates are mixed after brushing, peeling, and crushing operations, which lead to the rupture of cell structure (Hurrel and Finot 1984). The rate of enzymatic browning depends on the concentration of tyrosinase and phenolic substrates, oxygen availability, pH, temperature, and MS 20111484 Submitted 12/12/2011, Accepted 3/5/2012. Authors are with the Dept. of Pharmaceutical Sciences, Faculty of Pharmacy, Nutrition and Health Sciences, Univ. of Calabria, 87036-I Rende (CS), Italy. Direct inquiries to author Loizzo (E-mail: [email protected]).

so on (Zheng and others 2008a). Enzymatic browning represents one of the food industry’s major problems, especially for fruits, vegetables, and seafood products. In order to prevent browning, use of food additives has been recognized including reducing agents and enzyme inhibitors. Actually, only very few enzyme inhibitors are used in the industry due to off-flavors, food safety, and economic feasibility. The food industry frequently uses ascorbic acid and various forms of sulfite-containing compounds as antibrowning agents. However, sulfite-containing compounds, in addition to causing off-flavors, can cause allergies and, consequently, their application on fresh-cut food products has been banned by the U.S. Food and Drug Administration (US FDA 1986). For this reason the food industry has introduced as antibrowning agents formulations of ascorbic and citric acids that are, however, less effective than sulfiting agents, since ascorbic acid is quickly consumed in the process of reducing quinones formed by tyrosinase (Hsu and others 1988; Santerre and others 1988). Recently, 4-hexylresorcinol was introduced for the prevention of shrimp melanosis and for browning control in fresh and dried fruit slices (McEvily and Iyneger 1992). The importance of safety in the food industry directs researchers in a constant quest for better inhibitors from natural sources as they are largely free of any harmful side effects. The use of alternative methods to avoid browning includes autoclaving and blanching, whereby the food products are immersed in a liquid at 80 to 90 ◦ C for 10 to 12 min or passed through a forced-steam flow. These conventional processes are inherently linked to important weight and nutritional quality losses in the treated product (Konanayakam and Sastry 1988). One of the alternatives that have been proposed are high hydrostatic pressure, irradiation, pulsed electric fields, and microwave energy (Queiroz and others 2008). Some tyrosinase inhibitors have been discovered and commented on. Kim and Uyama (2005) reviewed tyrosinase inhibitors from both synthetic and natural sources for their industrial importance. Likhitwitayawuid (2008) reviewed naturally occurring stilbenes used as tyrosinase inhibitors not only for food

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Tyrosinase inhibitors: an update. . . applications but also as skin whitening agents. That review article also suggests the use of tyrosinase inhibitors as alternative insect control agents, considering that in insects, this enzyme is essential for the sclerotization of the exoskeleton, wound healing, and parasite encapsulation. Recently, Chang (2009) reviewed an update of tyrosinase inhibitors and their inhibitory mechanisms of action. The present review surveys and summarizes tyrosinase inhibitors newly discovered from natural and synthetic sources published from 2009 until today. The mechanisms of action and the structure-activity relationships are also discussed, wherever possible. The knowledge offered in this review should help to provide leads to the ultimate goal of developing new tyrosinase inhibitors of adequate efficacy and safety for the prevention of browning in plant-derived foods and seafood.

Enzymes Several research papers and reviews have already been published on the structural and kinetic aspects of the enzyme tyrosinase (Sánchez-Ferrer and others 1995; Seo and others 2003; Garc´ıaMolina and others 2007; Matoba and others 2006). Therefore, in this section, we will merely introduce the biochemical characteristics of the enzyme and its mechanism of action. Enzymatic browning results from the action of a group of enzymes called tyrosinase (EC 1.14.18.1), catechol oxidase, catacholase, diphenol oxidase, o-diphenolase, phenolase, or polyphenol oxidase. In plants both soluble and membrane-bound tyrosinase have been described. Its gene is encoded in the nucleus and translated in the cytoplasm; the pro-enzyme formed is then transported to the chloroplast where it is cleaved by a protease, producing the active form, while its substrates are contained in the vacuole (Mayer 2006). The term tyrosinase refers to its typical substrate, tyrosine. The enzyme has a higher affinity for the l-isomers of the substrates and its activities appear to have broad substrate-specificities (Rescigno and others 2002). Almost all studies on tyrosinase inhibition conducted so far have used mushroom tyrosinase because it is commercially available. The best-characterized tyrosinases are derived from Streptomyces glausescens, Neurospora crassa, and Agaricus bisporus (Solomon and others 1996). Tyrosinase is a multifunctional copper-containing enzyme, in which copper is bound by 6 or 7 histidine residues and a single cysteine residue. This enzyme possesses both monophenolase activity and diphenolase activity. It is involved in the biosynthesis of melanin and catalyzes the ortho-hydroxylation of tyrosine (monophenol) to 3,4-dihydroxyphenylalanine or DOPA (o-diphenol), and the oxidation of DOPA to dopaquinone (o-quinone) (Cooksey and others 1997). During the browning process this o-quinone can then be converted into brown melanin pigments through a series of enzymatic and nonenzymatic reactions. The obtained o-quinones are powerful electrophiles, which can be nucleophilically attacked by water, other polyphenols, amino acids, peptides, and proteins, leading to Michael-type additions. This enzymatic browning can be prevented by trapping the o-dopaquinone intermediate (Martinez and Whitaker 1995).

Plant Extracts and Isolated Natural Compounds The screening for tyrosinase inhibition of the methanol extracts prepared from the aerial parts of 33 Turkish Scutellaria species was done by Senol and others (2010). Some Scutellaria species are used in nutrition, in particular the young leaves of S. indica and S. baicalensis which are cooked as a vegetable in some Asian countries, whereas the whole plant of S. baicalensis is used as a tea substitute. The methanol extract of S. brevibracteata subsp. subvelutina, S. c 2012 Institute of Food Technologists®

brevibracteata subsp. brevibracteata, S. orientalis subsp. pichleri, S. orientalis subsp. pectinata, S. orientalis subsp. carica, S. brevibracteata subsp. pannosulo, S. albida subsp. Colchica, and S. hastifolia displayed moderate inhibition on tyrosinase with a percentage ranging from 39.57% to 51.58% at 1000 μg/mL for S. albida subsp. colchica and S. brevibracteata subsp. subvelutina, respectively. The stem bark powder of Hesperethusa crenulata syn. Naringi crenulata and Limonia acidissima, common tropical plant species in the Indian subcontinent and Southeast Asia, has been used traditionally as a skin whitening treatment. Dichloromethane extract exhibited the highest IC50 value of 0.546 mg/mL. This IC50 value was approximately 60 times higher than that for kojic acid (IC50 value of 0.0009 mg/mL), a standard tyrosinase inhibitor commonly used in cosmetic and food formulations (Wangthong and others 2010). Sapindus mukorossi, a shrub commonly known to produce soap nuts, generally grows in tropical and sub tropical regions of Asia. Methanol and ethyl acetate extract of these seeds have been evaluated for their mushroom tyrosinase inhibitory activity, which exhibited weak bioactivity with IC50 values of 17.8% and 12.3% at 10 μg/mL (Chen and others 2010). Methanolic extracts of Magnolia denudata and M. denudata var. purpurascens flowers were evaluated for their tyrosinase inhibitory activities, finding IC50 values of 3.34 and 10.55 mg/mL for M. denudata and M. denudata var. purpurascens, respectively, which were about 14- to 46-fold higher than that of ascorbic acid (IC50 value of 0.23 mg/mL) (Jo and others 2011). Polygonatum odoratum, Ampelopsis japonica, and Lindera aggregate, used in traditional Chinese medicine, showed IC50 values of 98.4, 152.1, and 276.3 μg/mL, respectively. As compared to vitamin C, which is widely used as an effective tyrosinase inhibitor, all extracts and Qian-wanghong-bai-san, Qiong-yu-gao, and San-bai-tang formulas showed smaller IC50 values against tyrosinase TOGLIEREI: inhibitory activity. Moreover, as compared with arbutin (IC50 value of 131.4 μg/mL), another known potent tyrosinase inhibitor, extracts from San-bai-tang formula, L. aggregate, and A. japonica had almost similar activity with IC50 values of 80.3, 115.1, and 117.3 μg/mL, respectively (Ye and others 2010). Methanol extract of Citrus grandis fruit tissue inhibited tyrosinase by up to 90.8%, similar to the reference compound kojic acid (95%), at 10 mg/mL (Wu and others 2010). Sixteen tropical vegetables extracted with different solvents were screened for their tyrosinase inhibitory activity. Among them Raphanus sativus (50% propylene glycol) exhibited a percentage of inhibition of 88.50% against tyrosinase, 78.98% was found for Momordica charantia (methanol), 68.73% for Raphanus sativus (ethyl acetate), and 46.57% Cymbopogon citrates (n-hexane) (Kamkaen and others 2007). The effects of pineapple juice (PJ), pineapple shell extract (PSE), and rice bran extract (RBE) on the browning process that occurs in banana slices and puree, compared with citric acid solution at pH 3.8 (pH) and distilled water (DW), were investigated by measuring the color changes in these 2 systems. RBE-treated banana slices had lower browning value than those treated with PJ, PSE, pH, and DW after 3 and 12 h. In fact its browning value after 12 h was 12.05. The luminosity, expressed as L∗ values, of banana slices treated with RBE was higher while a∗ values, which indicate the position on the green (−) to red (+) axis, were lower than those treated with PJ, PSE, pH, and DW after storage 12 h. RBE had an interesting effect also in that it retarded the browning process in banana pure where browning was lower (22.63) than when treated with other extracts after storage for 5 h. The L∗ values of banana puree treated with RBE were higher, whereas its a∗ values were lower than those treated with PJ, PSE, pH, and DW after

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Tyrosinase inhibitors: an update. . . 6 h. In conclusion, RBE was effectively reducing the browning process in both systems (Theerakulkait and Sukhonthara 2008). The extracts obtained from leaves and stems of Podocarpus elongatus, P. falcatus, P. henkelii, and P. latifolius, used in traditional medicine in Southern Africa, were investigated for their tyrosinase inhibitory activity. The most active extract was obtained from P. elongatus stem (EC50 value of 0.14 mg/mL), followed by P. falcatus leaves (EC50 value of 0.29 mg/mL) (Abdillahi and others 2011). In Podocarpus genus, amentoflavone (1) and nor- and bisnorditerpenes have been identified. These compounds are able to inhibit tyrosinase (Roy and others 1987; Cheng and others 2007). Moreover, in the Podocarpaceae family an effective inhibitor of tyrosinase, epicatechin, was identified (Kubo and others 2003). Amentoflavone (1) was identified together with naringenin as main constituent of Inulae flos hot water extract. This extract tested against mushroom tyrosinase using dl-DOPA as substrate inhibited the enzyme in a dose-dependent manner with an IC50 value of 4.35 mg/mL. Kinetics analysis by the Lineweaver-Burk plot revealed that I. flos extract act as noncompetitive inhibitor with Ki of 1.48 mM (Wu and others 2010). The tyrosinase inhibitory activity of I. flos could be related to the identified flavonoids since both isolated compounds contain a resorcinol and it is well known that this moiety is crucial for enzyme inhibition. Si and others (2012) studied the inhibitory effects of hesperetin on mushroom tyrosinase using inhibition kinetics and computational simulation. Hesperetin inhibited the enzyme in a dose-dependent manner with an IC50 value of 11.25 mM. The kinetics analysis by the double-reciprocal Lineweaver-Burk plot revealed that this flavanone reversibly inhibited tyrosinase in a competitive-manner with a Ki value of 4.03 mM. Hesperetin probably interacts with the enzyme in the same way as other flavonoids through a copper chelator activity. Through computational docking simulation studies authors hypothesized that hesperetin could bind the enzyme through residues localized in the active site (Met280, His61, His85, and His259). The flavonoid chrysontemin, isolated from the leaves of Diospyros kaki, showed a weak inhibitory activity with an IC50 of 211 μM. For the same plant hyperoside, isoquercitrin, quercetin3-O-(2 -O-galloyl-β-d-glucopyranoside), trifolin, astragalin, and kaempferol-3-O-(2 -O-galloyl-β-d-glucopyranoside) were isolated but did not exhibit any inhibitory activity against the enzyme. Structure Activity Relationship (SAR) analysis evidenced that chrysontemin, isoquercitrin, and astragalin present a glucopyranoside moiety at the C-3 position of the flavonoid moiety but this function is not indispensable for the bioactivity. On the contrary, the structure of isoquercitrin is different from chrysontemin for the 4-keto group of the C ring, which seems to be responsible for the antityrosinase activity. Moreover, it seems that the 3 ,4 dihydroxy groups of isoquercitrin cannot be inserted into the active site of the enzyme. Similarly in hyperoside and quercetin-3-O(2 -O-galloyl-β-d-glucopyranoside) the steric hindrance effects could hinder the entry into the active pocket. Lineweaver-Burk plot revealed that chrysontemin acted as competitive inhibitor, acting probably through chelation of binuclear copper localized in the catalytic center of the enzyme (Xue and others 2011). Salvia is a large genus distributed throughout the world. S. cryptantha and S. cyanescens were tested for their tyrosinase inhibitory activity, finding that it had low inhibitory activity as compared to the reference kojic acid (Süntar and others 2011). This might presumably be related to the phytochemical content, although these species are known to contain phenolics in remarkable amounts. Nerya and others (2004) stated that the most important factor in efficacy of the chalcones against tyrosinase is

the location of the hydroxyl groups on aromatic rings, while Lee and others (2004) reported that prenylation with isoprenyl group or vinylation of the flavonoid molecules did not enhance tyrosinase inhibitory activity. In fact, among prenylated compounds tested kuwanon C (2) and sanggenon D (3) were found to possess considerable inhibitory activity with IC50 values of 49.2 and 7.3 μM, respectively. Conversely, the addition of a vinyl moiety did not result in obtain bioactivity. Previously, Kang and others (2004) reported the ability of rosmarinic acid and its methyl ester, isolated from S. miltiorrhiza to inhibit mushroom tyrosinase with IC50 values of 16.8 and 21.5 μM, respectively. They found bioactivity to be comparable to kojic acid (IC50 value of 22.4 μM). Both compounds acted as competitive inhibitors with Ki values of 2.4 × 10−5 and 1.5 × 10−5 M for rosmarinic acid and its methyl ester, respectively. Successively, Lin and others (2011) reported comparative inhibitory activity against tyrosinase of rosmarinic acid, methyl rosmarinate, and pedalitin (4) isolated from Rabdosia serra. Compound 4 exhibited the most promising activity with an IC50 value of 0.28 mM followed by methyl rosmarinate. The inhibitory effect of methyl rosmarinate was higher to that of rosmarinic acid. SAR analysis revealed that methoxy substitution could increase the bioactivity. Both rosmarinic acid and methyl rosmarinate were considered as competitive inhibitors of tyrosinase, while pedalitin was suggested to be a mixed-type inhibitor of tyrosinase. Biofractionation of Anastatica hierochuntica, an Egyptian herbal medicine led to the isolation of silybin A (5) and B (6), isosilybin A (7) and B (8), kaempferol, and quercetin, which inhibited mushroom tyrosinase with percentages of inhibition of 24.2%, 200 μM), which has only one isoprenyl group in the same position. Kuwanon U, an isogeranyl flavanone, showed much lower inhibitory activity than kuwanon E (30) with IC50 values > 200 and 77.99 μM, respectively. This observation suggested that the substitution of a methyl group at the 4 -OH group on the B-ring, as in kuwanon U, compromises the bioactivity. Among the tested compounds were also 3 stylbene glycosides that inihibited the enzyme: oxyresveratrol-3 -O-β-d-glucopyranoside (IC50 value of 1.64 μM), oxyresveratrol-2-O-β-d-glucopyranoside (IC50 value of 29.75 μM), and mulberroside A (IC50 value > 200 μM). SAR analysis revealed that glycosidation at the 2 or 4-position substantially weakened activity to a much larger extent

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than at the 3 or 5 position. Among the isoprenyl-substituted 2-arylbenzofuran derivatives, moracin N exhibited the highest inhibitory activity with an IC50 value of 30.52 μM. In this compound the isoprenyl group remains intact, which might contribute to its higher tyrosinase inhibitory activity relative to moracin O (IC50 value of 93.58 μM) in which the isoprenyl group forms a 5-membered ring with the -OH group at the 6-position. The ethanolic extract of Greyia flanaganii leaves showed an interesting tyrosinase inhibitory activity (IC50 of 32.62 μg/mL). Following this observation, bioassay-guided fractionation was done which led to the isolation of only one bioactive constituent, 2 ,4 ,6 -trihydroxydihydrochalcone; it showed the highest activity with an IC50 value of 69.15 μM. (Mapunya and others 2011). From Anacardium occidentale nuts cardol triene was isolated. This compound reveal strong irreversible competitive tyrosinase inhibitory activity (IC50 value of 22.5 μM) (Zhuang and others 2010). The SAR analysis revealed that the presence of a resorcinol-moiety could drastically enhance its enzyme inhibitory activity. The inhibitory kinetics studies showed that cardol triene is a mixed-type inhibitor. The tyrosinase inhibitory activity of ethanolic extract of Hibiscus cannabinus leaf was evaluated before and after subjecting it to far-infrared (FIR) irradiation. Several reports have shown the potential of FIR treatments for enhancing the activity levels and the content of functional components in food products (Lee and others 2005, 2006; Eom and others 2009). Purification of extract led to the isolation of kaempferitrin as the main component. Prior to FIR irradiation, no tyrosinase inhibitory activity was detected. On the contrary, after FIR treatement (for 1 h at 60 ◦ C) significant tyrosinase inhibitory activity was observed (IC50 value of 3500 ppm). HPLC analysis of this extract identified kaempferol, afzelin, and α-rhamnoisorobin as derhamnosylation products. Among them kaempferol exhibited an IC50 value of 171.4 μM (Rho and others 2010a). Mango (Mangifera indica) is the most cultivated fruit in Thailand. Several studies have shown that mango seed kernels contain various phenolic compounds including gallotannins, epicatechin, and condensed tannin-related polyphenols (Arogba 2000; Puravankara and others 2000; Abdalla and others 2007). Refluxing in acidified


Tyrosinase inhibitors: an update. . . ethanol extract of sun-dried mango seed kernels was characterized by the highest total phenolics content and tyrosinase inhibitory activity with an ID50 value of 4.13 mg/mL. This extract acts as a competitive inhibitor of the enzyme. Moreover, it was also shown to be a good chelator of copper, which is the metal at the center of the active site of tyrosinase (Maisuthisakul and Gordon 2009). Recently, Ko and others (2011) investigated the potential tyrosinase inhibitory activity of 70% ethanol extract obtained from the branches of Distylium racemosum. EtOAc and n-butanol fraction showed the stronger mushroom tyrosinase inhibitory activity, using l-tyrosine as the substrate with IC50 values of 27.1 and 29.6 μg/mL. From the EtOAc fraction 20 compounds were isolated. Among them gallocatechin showed a strong tyrosinase inhibitory activity with an IC50 value of 4.8 μg/mL. Other constituents such as epi-gallocatechin gallate, methyl gallate, and quercitrin exhibited interesting IC50 values of 30.2, 40.5, and 37.3 μg/mL, respectively. The extracts of dog rose (Rosa canina) hips and pomegranate (Punica granatum) arils were assayed for inhibition of tyrosinase derived from mushrooms and vegetables and polyphenol oxidase activity. In addition to the in vitro studies, melanosis in foods such as artichokes, mushrooms, and pear juice were evaluated. The results revealed that dog rose hip extract was more effective than pomegranate aril extract and this bioactivity could be related to the total phenolics content since in the pomegranate extract it was 1.45 mg gallic acid equivalent/mL, while in dog rose extracts it was 9.16 mg gallic acid equivalent/mL. Dog rose extract analyzed by HPLC showed a high content of epigallocatechin (8 mM), which has been reported as a competitive inhibitor of tyrosinase (Zocca and others 2011). Another flavanol that possesses similar properties is epigallocatechin gallate found in pomegranate extract, it contained an average of 335 mg/L (0.7 mM) of epigallocatechin gallate, a value higher than the IC50 value (0.034 mM) reported by Kim and Uyama (2005). The same research group reported also an IC50 value of 0.017 mM for epicatechin gallate when inhibiting tyrosinase; in the dog rose extract, epicatechin gallate was found to have a concentration of 230 mg/L (0.52 mM). Another phenolic compound found in dog rose extract that possesses antipolyphenol oxidase/tyrosinase activity is p-hydroxybenzoic acid (485 mg/L, 3.5 mM). The hydroxybenzoic acids also include ellagic acid, which has antityrosinase activity that was detected only in the dog rose extract (177 mg/L) (Yoshimura and others 2005; Zocca and others 2011). Previously, Lin and others (2010) had reported on the inhibitory activities of p-propylbenzoic acid, p-butylbenzoic acid, p-pentylbenzoic acid, p-hexylbenzoic acid, p-heptylbenzoic acid, and p-octylbenzoic acid on potato polyphenol oxidase, finding IC50 values from 0.047 to 0.213 mM for p-octylbenzoic acid, and p-propylbenzoic acid, respectively. All benzoic acids are reversible inhibitors, but the inhibitory types were determined to be noncompetitive. The result of the inhibitory type lead the authors to hypothesize that the inhibitors attach to the enzyme at a site different from the active site and hinder the binding of substrate to the enzyme through steric hindrance or by changing the protein conformation. On the basis of their observations the authors speculated that when substrate, it can bind the enzyme and will induce a new hydrophobic pocket in the enzyme-substrate complex, and the para-position hydrocarbon chain can then be inserted into the pocket. Among these tested compounds, p-octylbenzoic acid was the most potent inhibitor, suggesting that the hydrophobic pocket accepts the 8 hydrocarbon chain well. The results pointed out that the inhibitor could be embraced by the enzyme hydrophobic pocket. This mechanism, in addition,

is probably involved in the tyrosinase inhibitory activity of phydroxybenzoic acid and methyl p-hydroxybenzoate isolated from the branches of Ficus erecta var. sieboldii. Both compounds showed promising bioactivity with IC50 values of 0.98 and 0.66 mM for p-hydroxybenzoic acid and methyl p-hydroxybenzoate, respectively. Kinetics analysis by the Lineweaver-Burk plot indicated that both compounds were competitive inhibitors of diphenolase of mushroom tyrosinase (Park and others 2011). Zocca and others (2010) reported the ability of processing water prepared by cooking Brassica oleracea leaves to inhibit tyrosinase. In particular, for mushroom tyrosinase the IC50 value was 200 μL of processing water. This water contained high total sulfur content (0.218 g/L) together with phytochemicals such as glucosinolates, phenols, antocyanins, and organic acids and is known to inhibit the browning process Rojas-Graü and others 2008; Volden and others 2009). Similar results were previously obtained with another member of the Brassicaceae family, Barbarea orthocerus, which contains the tyrosinase inhibitor barbarin showed an IC50 value of 4.2 × 10−5 M against mushroom tyrosinase (Seo and others 1999). Processing water was also freeze-dried in order to increase its utility and effectiveness. However, this process resulted in a loss of bioactivity, probably due to the stresses of the process, which can result in a degradation of biomolecules as reported by Kamath (2006). The addition of ascorbic acid to processing water resulted in complete inhibition of grape polyphenoloxidases, an effect which was 34% stronger than the acid alone. One possible mechanism for this synergistic effect is that processing water may specifically interact with polyphenoloxidases rendering it unable to catalyze the enzymatic reaction, while ascorbic acid reduces the quinones generated by polyphenoloxidases. From Ecklonia cava, a brown alga largely used as a food ingredient, dieckol was isolated. This phlorotannin is able to inhibit mushroom tyrosinase in a dose-dependent manner (IC50 value of 20 μM). The inhibition kinetics revealed that dieckol behaved as a noncompetitive inhibitor. Molecular modeling studies found that this compound could interact with residues His208, Met215, and Gly46 in the active site of the enzyme that were more important than those of compound arbutin (His208, Gly216, and Asn205), and were the main contributors to the receptor-ligand interaction (Kang and others 2012). Crustaceans are widely consumed all over the world because of their delicious and nutritional value. Shrimp and shrimp products of Thailand are well known for their long-standing excellent reputation worldwide, owing to outstanding quality, freshness, variety, and taste (Rattanasatheirn and others 2008). Pacific white shrimp (Litopenaeus vannamei) is an important commercial species characterized by a very short shelf-life span, due to melanosis. To maintain the quality and to avoid melanosis of shrimp or other crustaceans, sulfite and 4-hexylresorcinol are widely used (Martinez-Alverez and others 2005). Shrimp treated with 5 g/L of green tea ethanolic extract, with prior chlorophyll removal, brought about lower melanosis, compared with the control, and showed similar scores to those treated with sodium metabisulfite. The main flavonoids present in green tea include catechins (flavan3-ols) (Cabrera and others 2006). Catechin probably acted as a competitive inhibitor for polyphenoloxidase because of its structural similarity with the substrate (Nirmal and Benjakul 2009). Furthermore, the ethanolic green tea extract, with prior chlorophyll removal, had no adverse impact on the sensory attributes of the treated shrimp (Nirmal and Benjakul, 2011a). On the cephalothorax of Pacific white shrimp mimosine (β-(3-hydroxy4-pyridon-1-yl)-l-alanine) obtained from Leucaena leucocephala



Tyrosinase inhibitors: an update. . . OH O

HN OH N H

30

OMe HO

OH O

MeO O

O

HO HO HO

R O

31 R= H 32 R= OMe OH O

O

O

OH

O

MeO

33

OMe

34

OH

Figure 4–Structures of compounds 30–34.

was investigated by Nirmal and Benjakul (2011b). This nonprotein amino acid showed a dose-dependent manner inhibitory activity towardpolyphenoloxidase with an apparent molecular weight of 210 kDa. Kinetics analysis by Lineweaver-Burk plot indicated that mimosine could bind both the enzyme and the enzyme-substrate complex, but with different affinities. Moreover, it showed copper reduction and chelating capacity in a dose-dependent manner. Mimosine could react with the intermediate browning product, thereby rendering lower red-brown coloring formation. From Saccharum officinarum processing desugared sugar cane extract (DSE) was obtained and fractionated (Chung and others 2011). Among other isolated constituents lariciresinol 4-O-β-dglucoside and threo-guaiacylglycerol-7-O-β-d-glucopyranoside exhibited a promising activity with IC50 values of 42.59 and 57.72 μM. SAR analysis revealed that active compounds are characterized by the presence of a free hydroxyl group at 4-position in the aromatic ring. At the same time the presence of free carboxylic groups that could be forming internal hydrogen binding to the free hydroxyl groups reduced drastically the bioactivity. A perusal analysis of literature revealed the presence of several oxindole alkaloids isolated from Isatis costata as tyrosinase inhibitors. Costinones A, B, isatinones A, isatinones B, indirubin, and trisindoline exhibited a significant activity with IC50 values ranging from 7.21 to 17.34 μM (Ahmad and others 2010). Thymol and carvacrol are members of monoterpene phenol class and are the main constituents of thyme oil. Both compounds are known as antioxidants and were used as additives in protecting food qualities. The potential use of thyme oil was analyzed using mushroom tyrosinase and l-tyrosine as substrate. As well as the oil thymol and carvacrol both exhibited a concentration-dependent inhibitory activity against dopachrome formation and oxygen consumption. SAR analysis revealed that replacement of the hydroxyl group with the methoxy group, drastically reduced the effect on the enzyme suggesting the crucial role of hydroxyl group in the enzyme interaction. Interestingly, another study suggested that thymol inhibits the redox reaction between dopaquinone and leukodopachrome instead of enzymatic reaction. In particular, thymol successfully inhibited oxidation of l-DOPA to dopaquinone, coupled with reduction of p-benzoquinone. Hence, the suppres-


sion of dopachrome formation by thymol is due to the inhibition of conversion of leukodopachrome to dopachrome. On the basis of results the authors conclude that the antibrowning effect of thymol is due to the inhibition of the redox reaction without any interaction with the enzyme (Satooka and Kubo 2011). Far Eastern sea cucumber (Stichopus japonicas) extracts were screened for their tyrosinase inhibition using different analytical methods (Husni and others 2011). The 70% ethanolic extract (IC50 value of 0.49 to 0.61 mg/mL) was more capable in inhibiting tyrosinase activity than the water extract (IC50 value 1.80 to 1.99 mg/mL). Lower IC50 values were seen with the spectrophotometric method. Kinetic analysis revealed that extracts are reversible and mixed-type inhibitors. Through bioactivity-guided fractionation ethyl-α-d-glucopyranoside (IC50 value of 0.19 mg/mL) and adenosine (IC50 value of 0.13 mg/mL) were identified as bioactive constituents. Sasa quelpaertensis dried leaves have been used to make a leaf tea. Bioactivity-guided fractionation of S. quelpaertensis ethyl acetate fraction led to the isolation of N-p-coumaroylserotonin (31) and N-feruloylserotonin (32) that showed strong tyrosinase inhibitory activity with IC50 values of 0.027 mM and 0.026 mM, respectively. Interesting activity was also obtained with 3-O-pcoumaroyl-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone (33, IC50 value of 0.055 mM) and 3-O-p-coumaroyl-1-(4hydroxy-3,5-dimethoxyphenyl)-1-O-β-glucopyranosylpropanol (34, IC50 value of 0.053 mM) considering that arbutin used as the control showed an IC50 value of 0.048 mM (Sultana and Lee 2009) (Figure 4). Arabinose is an aldopentose having both sugar and aldehyde groups in its structure. This compound significantly inhibited tyrosinase with IC50 0.1 mM, and this was accompanied by conformational changes in enzyme structure. Arabinose induced mixed-type inhibition with Ki value of 0.22 mM. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence showed that arabinose induced tyrosinase to unfold and expose inner hydrophobic regions. Computational docking simulation analysis revealed that this inhibitor could interact with the catalytic site through residues His61, Asn260, and Met280 (Hu and others 2012) (Table 1).


Tyrosinase inhibitors: an update. . . Table 1–Plant extract with tyrosinase inhibitory activity. Extracts S. albida subsp. colchica S. brevibracteata subsp. subvelutina Hesperethusa crenulata Sapindus mukorossi Sapindus mukorossi Magnolia denudate Magnolia denudata var. purpurascens Mangifera indica Podocarpus elongatus Podocarpus falcatus Polygonatum odoratum Ampelopsis japonica Lindera aggregata Citrus grandis Stichopus japonicas Stichopus japonicas Raphanus sativus Raphanus sativus Momordica charantia Cymbopogon citrates Brassica oleracea Dalea elegans Lithrea molleoides Greyia flanaganii Cudrania cochinchinensis Cudrania cochinchinensis Soygerm koji fermented with Aspergillus oryzae BCRC 32288 Distylium racemosum Distylium racemosum Inulae Flos

Part of plant

Extraction solvent

IC50 -ID50-EC50

Aerial parts Aerial parts Stem bark Seeds Seeds Flowers Flowers Seed kernels Stem Leaves Rhizomes Roots Leaves Fruit tissue Body without visceral organ and fluids Body without visceral organ and fluids Whole plant Whole plant Whole plant Whole plant Leaves Aerial parts Aerial parts Leaves Stem Roots Seeds Branches Branches Aerial parts (Tea)

MeOH MeOH CH2 Cl2 MeOH EtOAc MeOH MeOH EtOH MeOH MeOH 30% EtOH 30% EtOH 30% EtOH MeOH 70% EtOH H2 O 50% Propylene glycol EtOAc MeOH n-Hexane Processing H2 O EtOH EtOH EtOH MeOH MeOH EtOAc EtOAc n-BuOH Hot H2 O

39.57% (at 1000 μg/mL) 51.58% (at 1000 μg/mL) 0.546 mg/mL 17.8% (at 10 μg/mL) 12.3% (at 10 μg/mL) 3.34 mg/mL 10.55 mg/mL 4.13 mg/mL 0.14 mg/mL 0.29 mg/mL 98.4 μg/mL 152.1 μg/mL 276.3 μg/mL 90.8 (at 10 mg/mL) 0.49–0.61 mg/mL 1.80–1.99 mg/mL 88.50% (at 5 mg/well) 68.73% (at 5 mg/well) 78.98% (at 5 mg/well) 46.57% (at 5 mg/well) 200 μL 0.48 μg/mL 3.77 μg/mL 32.62 μg/mL 36.3 μg/mL 56.2 μg/mL 0.19 mg/mL 27.1 μg/mL 29.6 μg/mL 4.35 mg/mL

Synthetic Compounds It is well known that tyrosinase can be inhibited by aromatic acids (Robit and others 1997). The effects of cinnamic acid and its derivatives on the activity of mushroom tyrosinase have been studied by Shi and others (2005). All these compounds strongly inhibited the diphenolase activity of mushroom tyrosinase with IC50 values of 2.10, 0.50, and 0.42 mM, for cinnamic acid, 4hydroxycinnamic acid, and 4-methoxycinnamic acid, respectively. In particular, cinnamic acid and 4-methoxycinnamic acid were noncompetitive inhibitors (Ki 1.994 and 0.458 mM, respectively), while the 4-hydroxycinnamic acid acted as competitive inhibitor with Ki 0.244 mM. Qiu and others (2009) analyzed the effects of the cinnamic acid analog α-cyano-4-hydroxycinnamic acid on both the monophenolase and diphenolase activity of mushroom tyrosinase. For the monophenolase activity, when the concentration of this analog reached 80 μM, the lag time was lengthened from 20 to 150 s and the steady-state activity was lost by about 75%. The IC50 value was estimated to be 48 μM. For the diphenolase activity, the inhibitory effect of this analog was lower (IC50 value of 2.17 mM). The kinetic analysis revealed that this analog acted on the diphenolase activity as reversible and competitive inhibitors. Because the π-conjugated framework of hydroxycinnamic acid is much smaller than that of this analog, this compound could easily bind to the enzyme active site. Moreover, the presence of the cyano-group on this analog makes the binding with the active site of tyrosinase even more difficult. The IC50 values of 4-hydroxycinnamic acid and α-cyano-4-hydroxycinnamic acid for diphenolase activity were 0.50 and 2.17 mM, respectively. The binding affinity of α-cyano-4-hydroxycinnamic acid to the diphenolase was not as tight as that of 4-hydroxycinnamic acid probably due to the presence of the cyano-group and the ethylenic linkage in the α-cyano-4-hydroxycinnamic acid structure. So the inhibition of α-cyano-4-hydroxycinnamic acid on the diphenolase is weaker than 4-hydroxycinnamic acid. Both 4-

hydroxycinnamic acid and α-cyano-4-hydroxycinnamic acid acted on the diphenolase activity of tyrosinase as competitive inhibitors. Kojic acid is largely used as a food additive to prevent enzymatic browning. On the basis of this evidence a series of kojic acid derivatives was synthetized. Among them, 2(cyclohexylthiomethyl)-5-hydroxy-4H-pyran-4-one (35) showed the most potent activity (IC50 value of 0.087 μM) followed by 2-(pentylthiomethyl)-5-hydroxy-4H-pyran-4-one with an IC50 value of 0.097 μM (Rho and others 2010b). A series of oxadiazole and triazolothiadiazole derivatives were synthesized and tested as mushroom tyrosinase inhibitors (Lam and others 2010). Among them, 5 derivatives showed high tyrosinase inhibition with IC50 values ranging from 0.87 to 1.49 μM. SAR analysis attributed an important role to the cycloamine moiety attached at N-3 of the oxadiazole ring (piperazine > piperidine > pyrrolidine > morpholine > methylpiperazines). Ghani and Ullah (2010) projected a series of 1,3,4-thiadiazole-2(3H)thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole5(4H)-thiones, and substituted hydrazides. The Ki values of thiadiazoles, triazoles, oxadiazoles, and substituted hydrazides were in the range of 0.19 to 5.2 μM, 1.01 to2.4 μM, 1.35 to 69.4 μM, and 49 to 177.2 μM, respectively. 1,3,4-Thiadiazole-2(3H)-thiones were found to be the most potent inhibitors. Among them the most potent was the compound containing a 5-(4-hydroxyphenyl) substitution. The tyrosinase inhibitory activity was drastically reduced when the hydroxyl group on the 4-position of the phenyl ring was removed. Two more substitutions at the same position of the phenyl ring with hydrophobic groups yielded better inhibitors. The inhibitory activities of triazoles were comparable with those of some inhibitors of the thiadiazole and oxadiazole series. In the 1,3,4-oxadiazole-2(3H)-thiones series, the compound characterized by a 4-benzyloxyphenyl group on the oxadiazole ring was found to be the most potent inhibitor (K i value of 1.35 μM). Comparison of this compound with its positional isomer showed



Tyrosinase inhibitors: an update. . . a 3-fold lower activity. Substitution of a 4-hydroxyphenyl group at 5-position of the oxadiazole ring was the second most potent inhibitor in this series. SAR analysis revealed that both the absence of a hydroxyl group from the phenyl ring and the presence of a cyclohexyl ring as a substituent at the 5-position drastically reduced the enzyme inhibitory activity. This behavior was similar to what was observed earlier in the thiadiazole and triazole series in which the presence of a 5-(4-hydroxy) phenyl group increased the tyrosinase inhibitory activity. However, a comparison of structures indicated that the potency varied depending on the class of compounds and the type of substitutions. Hydroxy- and/or alkoxy-substituted phenyl-benzo[d]thiazole derivatives were recently synthesized and evaluated for their ability to inhibit mushroom tyrosinase by Ha and others (2011). Among them, compounds 36 and 37 exhibited much higher tyrosinase inhibition activity (45.36% to 73.07% and 49.94% to 94.17% tested at 0.01 to 20 μM, respectively) than the reference compound kojic acid (9.29% to 50.80% tested at 1.25 to 20 μM). The kinetic behaviors of the enzyme in the presence of both compounds were investigated. Using compound 36 at a concentration of 1.25 and 20 μM the enzyme showed Km values of 1.02 and 5.65 mM, respectively, and the same Vmax value (1.54 × 10−2 ). While in the presence of compound 37, at the same concentration the enzyme exhibited Km values of 1.16 and 1.46 mM, respectively, and the same Vmax value (1.37 × 10−2 ). This enzyme behavior suggested that both compounds are competitive inhibitors of mushroom tyrosinase. Recently, Ha and others (2012) reported the synthesis and the biological evaluation of a series of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives. Compounds 38 and 39 exhibited around 40% of inhibition at 20 mM similar to that of kojic acid. SAR analysis revealed that compound 38 (an hydroxyl group at 3-position and a methoxyl group at 4-position on the phenyl ring) has a similar structure to DOPA while compound 39 (an hydroxyl group at 4-position on the phenyl ring) showed a structure similar to tyrosine. The crucial role of the hydroxyl group at 4-position could be deduced observing the tyrosinase inhibitory activity of compound 40, characterized by two methoxy substituents at 3-position and 4-position on the phenyl ring that showed a lower activity (around 10% of inhibition at 20 mM). At the same time analog 41 (2 methoxy substituents at positions 2 and 4) exhibited the greatest inhibition of the l-DOPA oxidase activity of mushroom tyrosinase with an IC50 of 5.05 μM which was 5.31-time fold lower than resveratrol used as reference compound. Docking simulation investigation predicted that compound 41 interacts with residue Ser254, Glu258, and Asp262 in the active pocket through formation of the hydrogen and/or ionic bonding. Moreover, kinetic analysis indicates that this compound acted as a competitive inhibitor of mushroom tyrosinase. p-Hydroxybenzaldehyde thiosemicarbazone (HBT) and p-methoxybenzaldehyde thiosemicarbazone (MBT) were synthesized and evaluated for their inhibition activities on mushroom tyrosinase. Results evidenced that both compounds exhibited strong inhibitory monophenolase activity of the enzyme with IC50 values of 0.76 and 7.0 μM, respectively. Moreover, HBT and MBT exhibited potent inhibitory effect against diphenolase activity in a dose-dependent manner with IC50 values of 3.80 and 2.62 μM, respectively. Kinetic analyses by Lineweaver-Burk double reciprocal plots evidenced that both compounds acted as mixed-type inhibitors and reversible (Chen and others 2012). The thiosemicarbazone (Z)-2-(naphthalen-1-ylmethylene) hydrazinecarbothioamide showed the strongest inhibitory activity (IC50 value of 1.1 μM) (Lee and others 2010). SAR analysis evidenced that the position of hydrophobic substituents on the c 2012 Institute of Food Technologists®

phenyl ring of benzaldehydethiosemicarbazones enhances the inhibitory activity. Furthermore, the aromatic group of benzaldehyde thiosemicarbazones can be replaced with the sterically bulky cyclohexyl group. Thus, hydrophobicity of the aryl or alkyl group on hydrazine of thiosemicarbazones is the determinant factor for their inhibitory activity rather than planarity. Following this observation, Yi and others (2011) modified thiosemicarbazone pharmacophore with the aim to optimize the structure and to increase its bioactivity. All obtained compounds exhibited dose-dependent inhibitory activity on the diphenolase of mushroom tyrosinase. The compound 1-benzylidenethiosemicarbazone exhibited a stronger inhibitory activity than kojic acid which was used as reference (9 μM instead of 102 μM). SAR analysis pointed out that the addition of a methylene group did slightly enhance the inhibitory activity, whereas the introduction of an ethylene group led to drastical bioactivity reduction. A critical aspect for the bioactivity is the distance that seperates the thiosemicarbazone group from the aromatic ring. In fact the introduction of a 2-pyridyl substituent, instead of the phenyl group, produces a much less active analog. On the contrary, the introduction of a 2-pyrolidinyl or 2-furanyl substituent slightly increased the bioactivity. Using the phenylmethylenethiosemicarbazones skeleton as a starting point, Yi and others (2009) synthetized and tested a series of hydroxyor methoxy-substituted phenylmethylenethiosemicarbazones. All tested compounds exhibited an interesting inhibitory activity against mushroom tyrosinase using l-DOPA as substrate. Among them compounds 42 (IC50 value of 0.38 μM), 43 (IC50 value of 0.28 μM), 44 (IC50 value of 0.33 μM), and 45 (IC50 value of 0.18 μM) were the most active. In phenylmethylenethiosemicarbazones series, compound 46, characterized by a phenyl ring without any type of substituents, exhibited a strong inhibitory activity (IC50 value of 1.93 μM). In contrast, compounds characterized by the presence of hydroxyl groups on 2-position and 4-position of the phenyl ring showed a lower bioactivity. This bioactivity was drastically reduced if the substituent is in 3-position. A decrease in inhibitor potency was observed also when the hydroxyl group in 4-position was replaced with methoxy group. On the contrary the introduction of a bromine substituent increase the bioactivity. Compound 45 exhibited the strongest bioactivity probably due to the 2,4-resorcinol and thiosemicarbazide moieties in its structure. Authors hypothesized that the presence of an hydrophobic subunit connected to the resorcinol aromatic ring could help 45 to bind residues His190 and Ala202 located in the hydrophobic pocket of the enzyme. When the hydroxyl groups were replaced by methoxy substituents a drastical reduction in the inhibitory activity was observed. Moreover, disubstituted compounds showed stronger inhibitory activity than trisubstituted compounds indicating that the third substituent might hinder the correct docking of the inhibitor to the active site of the enzyme. SAR analysis revealed also that the replacement of benzene moiety of 46 with furan group as in 2-(2-furanylmethylene)-thiosemicarbazone (47) (IC50 value of 0.45 μM) determined a high potency suggesting that the electron-rich aromatic ring might be more favorable for the interaction between the active site of the enzyme and the inhibitor. In continuing searching of novel tyrosinase inhibitors Yi and others (2010a) synthesized and tested a series of 4-O-substituted phenylmethylenethiosemicarbazones. The (1-(1-(4-(2-(2methoxyethoxyl)ethoxyl))benzyliene)thiosemicarbazide) (48) exhibited a strong inhibitory activity with an IC50 value of 0.34 μM. SAR analysis revealed that changement in the number of oxygen atom and the length of alkyl group could be reduced the bioactivity of 48 probably due the steric hindrance that


Tyrosinase inhibitors: an update. . . R O HO O

S

cyclohexyl

35

R1 42 43 44 45 46

R1= OH, R2= H R1= H, R2=Br R1= OH, R2= Br R1=R2= OH R1=R2= H

N

R2

36 R= H, R1= OH 37 R= R1= OH

S C N NH C NH2 H

R2

S

R1

O

S N NH C NH2 C H 47

R3

38 39 40 41

R1 H

S N H

OH

R4

R1= R4= H, R2= OH, R3= OMe R1= H, R2=R4= OMe, R3= OH R1= R4= H, R2= OMe, R3= OH R1= OMe, R2=R4= H, R3= OMe

S C N NH C NH2 H

CH3O(CH2)2O(CH2)2O 48 Figure 5–Structures of compounds 35–48.

impede the interaction between inhibitor and the active site of the enzyme. The elongation of alkyl chain determined the loss of the inhibition further supporting the evidence that the chain attached on 4-position of phenylmethylenethiosemicarbazone might play a crucial role in the inhibitory activity (Figure 5). Moreover, the introduction of secondary alkyl group in the side chain could be favorable for the enzyme inhibition and the presence of the –CH2 CH=CH2 group in the side chain was better than those the introduction of –CH(CH3 )2 group, this is probably due the possible interaction between the –CH2 CH=CH2 group with the hydrophobic site of the eznyme. The introduction of a benzoxyl group had no influence in terms of reduction the activity while the introduction of a pyridine methyleneoxyl group to 4position of 2-(phenylmethylene)-thiosemicarbazone determined an increase in the IC50 value. Previous investigation demonstrated that 1-(1-arylethylidene)thiosemicarbazide derivatives showed strong inhibitory activities against mushroom tyrosinase (Liu and others 2008). A new series of alkylidenethiosemicarbazide compounds was synthesized and tested against the diphenolase activity of mushroom tyrosinase. In particular, 1-(propan-2ylidene)thiosemicarbazide (49) (IC50 value of 0.086 μM), 1-propylidenethiosemicarbazide (IC50 value of 0.20 μM), 1-ethylidenethiosemicarbazide (IC50 value of 0.23 μM), and 1-(butan-2-ylidene)thiosemicarbazide (IC50 value of 0.28 μM) showed strongest inhibitory activity. SAR analysis demonstrated that the increase of the length of alkyl chain determined a decrease in the activity probably due the stereo hindrance that prevents the binding with the catalytic site of the enzyme. Another important structure requirement is the saturated substituents since compounds with unsaturated substituents are weaker inhibitors. A decrease in inhibitory activity was observed also in derivatives with a carbonyl group corresponding to thiosemicarbazides without a carbonyl group (Liu and others 2009). Yi and others (2010b) tested different 4-hydroxybenzaldehyde derivatives as tyrosinase inhibitors and found the IC50 values

ranged from 0.059 to 3.26 mM. Among the synthetized compounds, the analog with a dimethoxyl phosphate substituent was the most potent inhibitor. Replacement of a methoxy group with an ethoxy group drastically reduced the tyrosinase inhibitory activity, probably due to the dimension of this substituent that hinders the binding of the compound with the enzyme. The substitution of the methoxy and methoxyethoxy groups at 4-position of the phenyl ring led to 2 new tyrosinase inhibitors with IC50 values of 0.66 and 0.9 mM. Moreover, the terminal methoxy group contributed to tyrosinase inhibitory activity, but the elongation of alkyl chain makes the binding with active site of the enzyme more difficult. The compound characterized by ethylene oxide moiety in the side chain showed potent tyrosinase inhibitory activities, whereas its opened-ring congener exhibited a weaker inhibitory effect. Summarizing such evidence applied to all synthetized structures, SAR analysis pointed out that: (1) the numbers of oxygen atoms contained in the chain substituted at 4-position of benzaldehyde modified the enzyme bioactivity; (2) the elongation of the alkyl chain might retard the binding of inhibitors with the active site of tyrosinase; (3) the molecular symmetry significantly influenced the enzyme activities; and (4) the introduction of l-glycine, l-alanine, or l-tryptophan enhanced the enzyme inhibitory activities. Another research group synthesized, and evaluated for its tyrosinase inhibitory activity, a series of bis-salicylaldehydes (Delogu and others 2010). 5,5 -Methylene-bis-salicylaldehyde (50) exerted the highest activity (IC50 value of 0.074 mM) which was 10-fold higher than for benzaldehyde. SAR analysis pointed out that the introduction of a methoxy group in the para position of compound 50 reduced its bioactivity (IC50 value of 0.117 mM) while the introduction of an hydroxy group in the same position led to good inhibitory activity (IC50 value of 0.076 mM) similar to 50. Pan and others (2011) synthesized and identified as tyrosinase inhibitors three 3,4-dihydroxybenzoates. The 3,4dihydroxybenzoate exhibited the strongest inhibitory activity with



Tyrosinase inhibitors: an update. . . an IC50 value of 81 μM followed by octyl 3,4-dihydroxybenzoate and heptyl 3,4-dihydroxybenzoate (IC50 values of 129 and 316 μM, respectively). The bioactivity was potentiated increasing length of hydrocarbon chains. Determination of the inhibitory mechanism of heptyl 3,4-dihydroxybenzoate and 3,4-dihydroxybenzoate revealed that the 1st was reversible inhibitor while the 2nd was an irreversible inhibitor. In particular, the mechanish of inhibition of octyl 3,4-dihydroxybenzoate depended on the concentration since it was observed that when the concentration was lower than 50 μM, it showed a reversible inhibitory mechanism that became irreversible around 75 μM, so octyl 3,4-dihydroxybenzoate is a mixed-type inhibitor. Analysis of the double-reciprocal plots indicated that compounds are uncompetitive inhibitors on the enzyme that means that 3,4dihydroxybenzoates could only bind the tyrosinase in the form of enzyme-substrate complex and not in the free form. Phlorizin is a flavonoid distributed in fruitss such as apples and pears. Its analogs were synthesized and tested against mushroom tyrosinase (Fang and others 2011). 4-Hydroxybenzyl 3,5-dihydroxybenzoate, 4-hydroxybenzyl 2,4-dihydroxybenzoate, 4-hydroxybenzyl 2,4,6dihydroxybenzoate, 3-hydroxybenzyl 3,5-dihydroxybenzoate, and 3-hydroxybenzyl 2,4-dihydroxybenzoate exhibited inhibitory activity with IC50 less than 10 μM. In particular, 4-hydroxybenzyl 2,4-dihydroxybenzoate exhibited the strongest avctivity with an IC50 value of 4.95 μM. These 5 compounds were competitive inhibitors of tyrosinase and the SAR clearly showed that the position of hydroxyl substituted on ring B remarkably affected the inhibition. The effect of a series of 4-(phenylurenyl)chalcone derivatives against banana tyrosinase using cathecol as substrate were investigated by Sonmez and others (2011). The result showed that all tested compounds acted in a competitive-manner as revealed by Lineweaver-Burk double reciprocal plots, with IC50 values ranging from 0.133 to 0.289 μM. Matos and others (2011) modified the 3-phenylcoumarin scaffold introducing methoxyl, ethoxyl, hydroxyl, and/or bromo at the 6, 8, and 4 positions as substituents with the aim to find out any structural features for the tyrosinase inhibitory activity. The compound characterized by a bromo atom and 2 hydroxyl groups in the 3-phenylcoumarin moiety showed the highest potency (IC50 value of 215 mM). The inhibitory mechanism of this compound was determined using a Lineweaver-Burk double reciprocal plot. With an increase in compound concentration, the Vmax value decreased, whereas the K m value was unchanged, suggesting that this compound is a noncompetitive tyrosinase inhibitor. SAR analysis revealed that in the phenylcoumarin series the methoxyl and ethoxyl substitutions reduced the activity against the enzyme. Seo and others (2010) reported a series of chalcone derivatives as tyrosinase inhitors. Among them, 4 ,4-dihydroxychalcone (51) and 4 -amino-4hydroxychalcone (52) showed IC50 values of 4.8 and 8.3 mM, respectively. Kinetics analysis by Lineweare-Burk plot indicated that all compounds acted as competitive inhibitors. Critical for the srong bioactivity of all synthtized chalcones apparently is the presence of sterically unencumbering groups on the A ring. Curcumin is a major ingredient of turmeric, a spice powder obtained from the rhizome of Curcuma longa. Du and others (2011) isolated and tested curcumin, demethylcurcumin, and bis-demethylcurcumin against mushroom tyrosinase finding IC50 values of 94.73, 53.03, and 33.50 μM. Using these natural curcuminoids, the authors designed several analogs possessing m-diphenols and o-diphenols. All tested compounds resulted in more activity than kojic acid used as reference compound. In particular, 1,5-bis-(2,4-dihydroxybenzylidene)penta-1,4–3-one c 2012 Institute of Food Technologists®

(53) member of m-diphenol group exhibited the strongest inhibitory activity with an IC50 value of 0.65 μM. When a methoxy group replaced an hydroxyl group in 4-position the bioactivity decreased. The crucial role of 4-position is clearly visible. Moreover, the order of bioactivity of compounds depend on the linkers so compounds with acetone linkers > compounds with cyclopentanone > compounds with tetrahydropyran-4one > compounds with cyclohexanone > compounds with tetrahydrothiopyran-4-one > piperidin-4-one. The inhibition kinetics, analyzed by Lineweaver–Burk plot, revealed that compounds with o-diphenol acted as noncompetitive inhibitors, while compounds m-diphenols could be bound only by the free form of the enzyme. Data regard the kinetics are controversial since previously described compounds characterized by m-diphenols showed competitive inhibitory activity (Song and others 2006; Baek and others 2009). Molecular docking study revealed that both 2,5-bis-(3,4-dihydroxybenzylidene)cyclopentanone (54) and 2,5-bis-(2,4-dihydroxybenzylidene)cyclopentanone (55) formed a π-π link between one aromatic group and His194 localized in the active pocket of the enzyme together with multiple hydrogen bonds that involved the hydroxylic groups. In particular, o-diphenol compounds such as compound 54 formed four hydrogen bond with Gly183, Trp184, Ser206 while m-diphenol compounds such as compound 55 formed seven hydrogen bond with Ser206, Ala202, Asn191, Asn188, Thr203, and Ser146 residues. The number of hydrogen bonds with the active site of the enzyme could explain the stronger inhibitory activity of m-diphenol compounds respect the o-diphenol compounds. Successively, Hosoya and others (2012) synthetized 47 curcumin-like diarylpentanoid analogues as potential tyrosinase inhibitors. Among them (1E,4E)-1,5-bis(4-hydroxyphenyl)penta1,4-dien-3-one exhibited an IC50 value of 6.4 μM. Compound (1E,4E)-1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3one, that possesses as curcumin a methoxy group at 3-position and an hydroxyl group at 4-position, showed an higher inhibitory activity (IC50 value of 75.5 μM) than its parent compound curcumin. In agreement with Du and others (2011), SAR analysis revealed that the substitution of an hydroxyl group at the 4-position is crucial for a strong inhibitory activity (Figure 6). Choi and others (2010) synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (56), which showed potent tyrosinase inhibitory activity (IC50 value of 2.95 μM). Kinetics analysis by the Lineweare–Burk plot indicated that compound 56 acts as noncompetitive inhibitor when l-tyrosine was used as substrate. From pinapple, the 2 sulfur-containing compounds N-lγ -glutamyl-S-sinapyl-l-cysteine and S-sinapyl-l-cysteine were isolated and tested for tyrosinase inhibitory activity, finding IC50 values of 237.33 and 199.04 μM, respectively (Zheng and others 2010b). A series of bibenzyl glycosides was synthesized and evaluated for their tyrosinase inhibitory activity. All of the synthesized bibenzyl glycosides exhibited strong inhibitory activities (Tajima and others 2011). In particular, 2,2 ,4 -trihydroxy-4-β-dxylopyranosylbibenzyl (57), characterized by bulky bibenzyl glycosides, exhibited an IC50 value of 0.43 μM that was 17 times higher than that of kojic acid used as reference. Chemical characteristics of compound 57 suggest that the enzyme might possess a hydrophilic cavity at its catalytic site. Takahashi and Miyazawa (2011) synthesized a series of phenylpropanoid amides of serotonin and analyzed their ability to inhibit the tyrosinase. Among the tested derivatives, N-Isoferuloyl serotonin (58) showed the


Tyrosinase inhibitors: an update. . . O C H

S N NH C NH2

OHC

49

CHO

HO

R

OH

50

51 R= NH2 52 R= OH

O

R3

R1 R2

OH

O R1

R3 R2

HO

OH

HO

OH

55

53 R2= H, R1= R3= OH 54 R1= H, R2= R3= OH Figure 6–Structures of compounds 49–55.

most promising inhibitory activity (IC50 of 5.4 μM). LineweaverBurk plot revealed that compound 58 acted as a noncompetitive inhibitor of mushroom tyrosinase. Previously, Bao and others (2010) synthetized a series of natural product biphenyl glycoside fortuneanoside E analogs. Among them, 3,4 -dihydroxy-3 ,5 dimethoxybiphenyl-4-carboxylic acid (59) exhibited the highest bioactivity with an IC50 value of 0.02 mM acting as competitive inhibitor (Ki = 0.015 mM). This activity was 7 times higher than that of fortuneanoside E (IC50 value of 0.14 mM) and 10 times higher than that of arbutin a known as potent tyrosinase inhibitors. SAR analysis showed that the presence of a 4-hydroxy-3,5dimethoxyphenyl moiety was critical for the inhibitory activity. Several 5-(substituted benzylidene)hydantoin derivatives were synthesized and tested against mushroom tyrosinase enzyme (Ha and others 2011). In terms of the SAR analysis, compound 60, characterized by 2 hydroxy groups on the phenyl ring despite having a structure similar to DOPA, did not inhibit mushroom tyrosinase. Furthermore, compound 61, with a hydroxyl group on the phenyl ring, showed low activity despite having a structure similar to tyrosine. Compound 62, characterized by a phenyl ring substituted with a hydroxyl group and a methoxy group showed the highest inhibitory activity. Compound 63, with these groups inverted and compound 64 with 2 methoxy groups, demonstrated poor inhibitory activity. A docking study brought evidence that compound 62 interacts strongly with the enzyme as a competitive tyrosinase inhibitor. Several SAR studies have identified hydroxyl as the functional group required for tyrosinase inhibitory activity (Yokota and others 1998; Shiino and others 2001, 2003; Kim and others 2006). The hydroxyl groups in compounds carry out the nucleophilic attack on the copper atoms in the active site of the enzyme and are directly involved in transferring protons during catalysis, which then result in inactivation of the tyrosinase. In this context, using inhibition kinetics and computational simulation the reversible inhibition of tyrosinase by isophthalic acid was studied (Si and others 2011). Isophthalic acid inhibited tyrosinase in a complex manner with K i of 17.8 mM, without modification of the tertiary protein structure. In this study, the authors predicted that isophthalic acid could bind to the Pro175 or Val190. Some of the synthesized compounds of a series of dihydropyrimidin-(2H)-one analogs and rhodanine deriva-

tives exhibited significant inhibitory tyrosinase activity (Liu and others 2011). Rhodanine derivatives inhibited the enzyme more than the dihydropyrimidin-(2H)-one analogues, which indicated that the smaller size of the ring could help the molecules to interact with the active site of the enzyme. Among this series compound 65 characterized by a hydroxyethoxyl group at position-4 of the phenyl ring, exhibited the most potent inhibitory activity (IC50 value of 0.56 mM). The kinetics analysis demonstrated that the inhibitory effect of compound 65 was irreversible. Comparing it to the enzyme inhibitory activities of 3,4-dihydropyrimidin-2-(1H)-thione analogs, as previously hypothesized compounds with a hydroxyl group in the benzene ring showed stronger inhibitory activities probably due the ability of hydroxyl groups to create a metal-ligand binding interaction with the dicopper nucleus (Ghani and Ullah 2010). Moreover, the tyrosynase inhibitory activity was higher when the hydroxyl group was introduced in the p-position with respect to the o-position. If the substituent is introduced in the m-position on the phenyl rings a decrease in inhibitory activities is observed (Figure 7). Eight vic-substituted o-aminophenols belonging to 2 isomeric series were investigated for their tyrosinase inhibitory activity by Rescigno and others (2011). Four o-aminophenolic compounds derived from the 3-hydroxyorthanilic acid scaffold and their 4 counterparts derived from the isomeric 2-hydroxymethanilic acid were investigated as mushroom tyrosinase inhibitors. Among the synthetized compounds, hydroxymethanilic compounds acted as substrates for the enzyme, which oxidized them to the corresponding phenoxazinone derivatives. Analysis of the results revealed that aromatic amines, o-aminophenols, and even o-diamines could represent tyrosinase substrates. In fact, amines are hydroxylated to the corresponding o-aminophenols, and o-aminophenols are oxidized to the corresponding o-quinoneimines. A similar behavior was observed also with o-diamines, o-aminophenols, and o-diphenols. On the contrary, the substitution of nitrogen for oxygen caused a drastic reduction in the reaction speed. Arbutin (4-hydroxylphenyl-O-β-d-glucopyranoside) is a wellknown tyrosinase inhibitor. Yi and others (2008) designed, synthesized, and tested a series of helicid (4-formylphenyl-O-βd-allopyranoside) derivatives. These compounds are characterized by analogy with arbutin’s chemical structure. 4-Formylphenyl (4,6-O-benzylidene)-β-d-allopyranoside (66) demonstrated the



Tyrosinase inhibitors: an update. . .

OH

OH

OH

HO

H N

O N H

HO

XylO

56

OH

OH

HOOC

60 61 62 63 64

59

OH 58

O

O

NH

NH

HN

R1

OMe

MeO OH

R

OMe HO

OH

57

S

S

O R= R1= OH R= H, R1= OH R= OH, R1= OMe R= OMe, R1= OH R= OMe, R1= OMe

OCH2CH2OH 65

Figure 7. Structures of compounds 56–65.

OAc Ph

O

O

HO

O OH O

CHO

H2N

HN

N

HO

O

O

O OAc AcO

OAc O OAc

OH

OH O

OH

OAc

AcO AcO

68 R=

OH

O OAc

OR

66

O

AcO AcO

67 R=

S

O O

HO

69

O

OH

OH O OH

OH O

70

Figure 8–Structures of compounds 66–70.

strongest enzyme inhibitory activity (IC50 value of 0.052 mM). SAR analysis indicated that the presence of 4,6-O-benzylidene, 2,3-O-isopropylidene, and 6-O-dimethyl phosphate into sugar moiety could help the interaction with the enzyme probably due the lipophilic character of these substituents. Moreover, a β-d-glucopyranoside moiety was better than β-d-allopyranoside moiety in the enzyme inhibitory activity. In regards to the type of sugar it is clear that the ribose moiety was more preferable than glucose moiety. Kinetics analysis showed that helicid and analogues were competitive inhibitors of the mushroom tryrosinase while the circular dichroism spectra indicated that those compounds induced conformational changes of mushroom tyrosinase upon binding. Based on the evidence that the modification of glycosyl moiety of arbutin might facilitate the inhibitory activity against tyrosinase, the same research group designed, synthesized and evaluated a series of novel 4-functionalized phenyl-


O-β-d-glycosides using thiosemicarbazide, oxime, and methyloxime skeleton (Yi and others 2009). SAR analysis revealed that compounds characterized by thiosemicarbazide moiety demonstrated significant antityrosinase activity. In particular, p-phenyltetra-O-acetyl-β-d-glucopyranoside benzaldehyde (67) showed an IC50 value of 0.31 μM. The presence of β-d-allopyranoside or β-d-glucopyranoside moiety did not influenced the activity. On the contrary the presence of tetra-O-acetyl-β-d-glucopyranoside moiety was favorable for the enzyme interaction. Interestingly, the replacement of this sugar moiety with tetra-O-acetyl-β-dgalactopyranoside moiety did not affect the bioactivity. At the same time the replacement of acetyl substituents with benzoyl groups determinated a drastically reduction in the inhibitory potency. In order to investigate if the volume of the sugar moiety could be influenced, the enzyme inhibitory activity of hepta-O-acetyl-βd-lactoside analog was designed and tested, finding an IC50 value


Tyrosinase inhibitors: an update. . . Table 2–Isolated natural compounds with tyrosinase inhibitory activity. Compounds Kuwanon C Sanggenon D Rosmarinic acid Rosmarinic acid methyl ester Pedalitin Silybin A Isosilybin A Isosilybin B Luteolin Kaempferol Quercetin Hesperetin Hierochin A (+)-Dehydrodiconiferyl alcohol (+)-Balanophonin Hierochin B 3,4-Dihydroxybenzaldehyde Arbutin N-p-Coumaroylserotonin N-Feruloylserotonin 3-O-p-Coumaroyl-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone 3-O-p-Coumaroyl-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-O-β-glucopyranosylpropanol Ethyl-α-d-glucopyranoside Adenosine Barbarin Epigallocatechin Epigallocatechin gallate Gallocatechin epi-Gallocatechin gallate Methyl gallate Quercitrin p-Octyl benzoic acid p-Propyl benzoic acid p-Hydroxybenzoic acid Methyl p-hydroxybenzoate 3,5,7,4 -Tetrahydroxy-30-(2-hydroxy-3-methylbut-3-enyl)flavones Uralenol Broussoflavonol F Artocarpfuranol Dihydromorin Steppogenin Norartocarpetin Artocarpanone Artocarpesin Isoartocarpesin (Z,Z)-5-(Trideca-4,7-dienyl)-resorcinol Dalenin Sophoraflavanone G Kurarinone Kurarinol 5 -Geranyl-5,7,2 ,4 -tetrahydroxyflavone 2,4,2 ,4 -Tetrahydroxychalcone Morachalcone A Kuwanon E Moracin C Moracin N 2 ,4 ,6 -trihydroxydihydrochalcone Cardol triene 2,3-cis-Dihydromorin 2,3-trans-Dihydromorin Oxyresveratrol Quercetin-7-O-β-d-glucoside Kaempferol-7-O-β glucopyranoside Morin-7-O-β-d-glucoside Quercetin-3,7-di-O-β-d-glucoside 6,7,4-Trihydroxyisoflavone Genistin Daidzein Glycitein Daidzin Vitexin Isovitexin Lariciresinol 4-O-β-d-glucoside Threo-guaiacylglycerol-7-O-β-d-glucopyranoside Erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol-4 -O-β-d-glucopyranoside Threo-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol 4 -O-β-d-glucopyranoside 2-(3-methoxyl-4-hydroxyphenyl)-3-Hydroxylmethyl-5-trans-carboxylethylene-7-methoxyl-2,3dihydrobenzofuran


IC50 49.2 μM 7.3 μM 16.8 μM 21.5 μM 0.28 mM 21 μM 10 μM 6.1 μM 14 μM 25 μM 15 μM 11.25 mM 25 μM 16 μM 15 μM 30 μM 17 μM 174 μM 0.027 mM 0.027 mM 0.055 mM 0.053 mM 0.19 mg/mL 0.13 mg/mL 4.2 × 10−5 M 8 mM 0.7 mM 4.8 μg/mL 30.2 μg/mL 40.5 μg/mL 37.3 μg/mL 0.047 mM 0.213 mM 0.98 mM 0.66 mM 96.6 μM 49.5 μM 82.3 μM 47.93 μM 10.3 μM 0.57 μM 0.46 μM 1.54 μM 0.52 μM 0.66 μM 0.449 μg/mL 0.26 μM 4.7 μM 2.2 μM 0.1 μM 37.09 μM 0.062 μM 0.14 μM 77.9 μM 111.47 μM 30.52 μM 69.15 μM 22.5 μM 31.1 μM 21.1 μM 2.33 μM 143.07 μM 161.54 μM 196.33 μM > 1000 μM 0.009 mM 0.343 mM 0.203 mM 0.218 mM 0.267 mM 6.3 mg/mL 5.6 mg/mL 42.59 μM 57.72 μM 85.87 μM 95.81 μM 798.02 μM

Type of inhibition Competitive Competitive Competitive Competitive Mixed-type NR NR NR NR NR NR Competitive NR NR NR NR NR NR NR NR NR NR NR NR NR Competitive Competitive NR NR NR NR Noncompetitive Noncompetitive Competitive Competitive NR NR NR NR NR NR NR NR NR NR NR Noncompetitive Noncompetitive Noncompetitive Noncompetitive NR NR NR NR NR NR NR Competitive NR NR NR NR NR NR NR Competitive Competitive Competitive Competitive Competitive NR NR NR NR NR NR NR


Tyrosinase inhibitors: an update. . . Table 2–Continued. Compounds Arabinose Dieckol Curcumin Demethylcurcumin bis-Demethylcurcumin Chrysontemin Mimosine Helicid Costinones A Costinones B Isatinones A Isatinones B Indirubin Trisindoline

IC50 0.1 mM 20 μM 94.73 μM 53.03 μM 33.50 μM 211 μM 3.70 μM 2.54 mM 7.21 μM 9.40 μM 11.51 μM 12.53 μM 14.29 μM 17.34 μM

Type of inhibition Mixed-type Noncompetitive NR NR NR Competitive Mixed-type Competitive NR NR NR NR NR NR

NR: not reported.

of 0.65 μM p-phenyl-hepta-O-acetyl-β-d-lactoside benzaldehyde (68). Collectively all these observations revealed that the configuration and bond type of sugar moiety played a very important role in the enzyme inhibitory activity and that the lipophilic character of acetylated sugar moiety could increase the potency of the inhibitors. Both 67 and 68 demonstrated to act as reversible inhibitors. This finding is in disagreement with the mode of action of 1-phenylthiourea and 1-(1-phenylethylidene) thiosemicarbazide analogs that acted as irreversible inhibitors. Particularly, the Lineweaver-Burk double reciprocal plots showed that 67 was a competitive inhibitor of mushroom tyrosinase whereas 68 could be considered as mixed-II-type inhibitor. The kinetics behavior of 67 lead the authors to hypothesize that compound 67 could interact with the enzyme through two different portions of its structure: the sulfur atom of the thiosemicarbazide moiety chelate, the binuclear copper of the enzyme, and such interaction acted as a bridge to link the acetylated glucose moiety and the hydrophobic protein pocket, which facilitated the acetylated glucose moiety to approach the hydrophobic protein pocket. Whereas the large size of lactoside moiety that characterized compound 68 prevented by binding to the active site of the complex enzyme-lDOPA. 5-Benzylidene barbiturate and thiobarbiturate derivatives were synthetized and tested by Yan and others (2009). These compounds displayed more potent tyrosinase inhibitory activities than that parent compounds barbituric acid, thiobarbituric acid, and 4-hydroxybenzylaldehyde. In the most active series compounds 5(4-(2-methoxyethoxy)benzylidene)-2-thioxodihydropyrimidine4,6(1H,5H)-dione, and 5-(4-(2-(2-methoxyethoxy)ethoxy) benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione displayed interesting IC50 values of 28.43 and 34.20 μM, respectively. SAR analysis clearly revealed that the thiobarbiturate moiety was more favorable than barbiturate moiety in inhibiting the diphenolase activity of mushroom tyrosinase. As previously evidenced in different types of compounds an hydroxyl group at 4-position of phenyl ring determined a strong inhibitory activity as compounds 5-(4-hydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione and 5-(4-hydroxybenzylidene)-2-thioxo-dihydropyrimidine-4,6 (1H,5H)-dione with IC50 values of 13.98 and 14.49 μM, respectively. According to previous investigation the replacement of the hydroxyl group with alkyl chain drastically reduced the bioactivity and this reduction is consequent also to the length of alkyl chain since it causes a steric hindrance that prevents the binding with the catalytic site of the enzyme. Kinetics studies revealed that the most potent inhibitor 5-(4-hydroxybenzylidene)pyrimidine-


2,4,6(1H,3H,5H)-trione and 5-(4-hydroxybenzylidene)-2thioxo-dihydropyrimidine-4,6(1H,5H)-dione acted as irreversible inhibitors. Moreover, the circular dichroism spectra showed that both compounds induced conformational changes of the enzyme (Yan and others 2009). A series of caffeoyl-amino acidyl-hydroxamic acid derivatives showed an interesting enzyme inhibitory activity compared to their parent compound In particular, CA-L/d-Phe-NHOH, CA-Phg-NHOH, CA-Leu-NHOH, CA-Ala-NHOH, and CAPro-NHOH exhibited higher inhibitory activity than derivatives CA-β-Ala-NHOH and CA-Gly-NHOH. This different behavior could be explained considering that different character of amino acids inserted in derivatives structure. In particular, hydrophobic alkyl chain of Leu and or the aromatic ring of Phe could make easier the chelation of dinuclear copper located on the active site of the enzyme by hydroxamic acid. Interestingly, CA-Pro-NHOH has hydrophobic character that allows to permit the binding to the active site of the enzyme (Kwak and others 2011). The mushroom tyrosinase inhibitory activity of 2phenylethanol, 2-phenylacetaldehyde, and 2-phenylacetic acid has recently been investigated (Zhu and others 2011). All compounds acted as reversible inhibitors; furthermore, 2-phenylacetaldehyde and 2-phenylacetic acid were shown to be uncompetitive inhibitors and 2-phenylethanol a mixed-type inhibitor. The inhibiting ability was influenced by the position of the functional group on the benzene ring, in particular 2-phenylacetaldehyde which inhibited the enzyme more than 2-phenylacetic acid or 2-phenylethanol. Recently, Nesterov and others (2008) synthetized and tested the natural product 1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy3-methylphenyl) propane isolated for the 1st time in Dianella ensifolia. This compound was 22 times more potent than kojic acid for inhibiting mushroom tyrosinase activity in a competitive and reversible fashion (Ki = 0.3 μM). It is well known that vitamin C has potent tyrosinase inhibitory activity and is largely used in the food industry for this purpose. Based on this consideration Yi and others (2009) reported the strong inhibitory activity of vitamin C esters namely d-ascorbic acid-6-p-hydroxybenzoic acid ester (69, IC50 value of 0.58 mM) and d-ascorbic acid-6-(3,4,5-hydroxybenzoic acid) ester (70, IC50 value of 0.16 mM) on the diphenolase activity of mushroom tyrosinase. The kinetic analysis revealed that the 2 esters behaved differently since compound 70 was a reversible inhibitor and its mechanism was mixed type and ester 69 was an irreversible inhibitor (Figure 8 and Tables 2 and 3).


Tyrosinase inhibitors: an update. . . Table 3–Synthetic compounds with tyrosinase inhibitory activity. Compounds Cinnamic acid 4-Hydroxycinnamic acid 4-Methoxycinnamic acid α-Cyano-4-hydroxycinnamic acid Rosmarinic acid methyl ester 2-(Cyclohexylthiomethyl)-5-hydroxy-4H-pyran-4-one 2-(Pentylthiomethyl)-5-hydroxy-4H-pyran-4-one p-Hydroxybenzaldehyde thiosemicarbazone p-Methoxybenzaldehyde thiosemicarbazone (Z)-2-(Naphthalen-1-ylmethylene)hydrazinecarbothioamide 1-Benzylidenethiosemicarbazone 2-[(2-Hydroxyphenyl)methylene]-thiosemicarbazone 2-[(4-Bromophenyl)methylene]-thiosemicarbazone 2-[(2-Hydroxy-4-bromophenyl)methylene]thiosemicarbazone 2-[(2,4-Dihydroxyphenyl)methylene]-thiosemicarbazone 2-(Phenylmethylene)-thiosemicarbazone 2-(2-Furanylmethylene)-thiosemicarbazone (1-(1-(4-(2-(2-Methoxyethoxyl)ethoxyl))benzyliene)thiosemicarbazide) 1-(Propan-2-ylidene)thiosemicarbazide 1-Propylidenethiosemicarbazide 1-Ethylidenethiosemicarbazide 1-(Butan-2-ylidene)thiosemicarbazide 5,5 -Methylene-bis-salicylaldehyde 4,4 -Dimethoxy-5,5 -methylene-bis-salicylaldehyde 4,4 -Dihydroxy-5,5 -methylene-bis-salicylaldehyde 3,4-Dihydroxybenzoate octyl 3,4-Dihydroxybenzoate heptyl 3,4-Dihydroxybenzoate 4-Hydroxybenzyl 2,4-dihydroxybenzoate 4 ,4-Dihydroxychalcone 4 -Amino-4-hydroxychalcone Curcumin Demethylcurcumin bis-Demethylcurcumin 1,5-bis-(2,4-Dihydroxybenzylidene)penta-1,4–3-one 2,5-bis-(3,4-Dihydroxybenzylidene)cyclopentanone 2,5-bis-(2,4-Dihydroxybenzylidene)cyclopentanone (1E,4E)-1,5-bis(4-Hydroxyphenyl)penta-1,4-dien-3-one 5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol N-L-γ -Glutamyl-S-sinapyl-l-cysteine S-Sinapyl-l-cysteine 2,2 ,4 -Trihydroxy-4-β-d-xylopyranosylbibenzyl N-Isoferuloyl serotonin 3,4 -Dihydroxy-3 ,5 -dimethoxybiphenyl-4-carboxylic acid Fortuneanoside E 4-Hydroxyethoxyldihydropyrimidin-(2H)-one 4-Formylphenyl (4,6-O-benzylidene)-β-d-allopyranoside p-Phenyl-tetra-O-acetyl-β-d-glucopyranoside benzaldeide p- Phenyl-hepta-O-acetyl-b-d-lactoside benzaldeide 5-(4-(2-Methoxyethoxy)benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione 5-(4-(2-(2-Methoxyethoxy)ethoxy)benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione 5-(4-Hydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione 5-(4-Hydroxybenzylidene)-2-thioxo-dihydropyrimidine-4,6(1H,5H)-dione d-Ascorbic acid-6-p-hydroxybenzoic acid ester d-Ascorbic acid-6-(3,4,5-hydroxybenzoic acid) ester

IC50 2.10 mM 0.5 mM 0.42 mM 0.28 mM 2.17 mM 0.087 μM 0.097 μM 0.76 μM (monophenolase); 3.8 μM (diphenolase) 7 μM (monophenolase); 2.62 μM (diphenolase) 1.1 μM 1 μM 0.38 μM 0.28 μM 0.33μM 0.18 μM 1.93 μM 0.45 μM 0.34 μM 0.086 μM 0.20 μM 0.23 μM 0.28 μM 0.074 mM 0.117 mM 0.076 mM 81 μM 129 μM 316 μM 4.95 μM 4.8 mM 8.3 mM 94.73 μM 53.03 μM 33.50 μM 0.65 μM 1.19 μM 0.78 μM 6.4 μM 2.95 μM 237.33 μM 199.04 μM 0.43 μM 5.4 μM 0.02 mM 0.14 mM 0.56 mM 0.052 mM 0.31 μM 0.65 μM 28.43 μM 34.20 μM 13.98 μM 14.49 μM 0.58 mM 0.16 mM

Type of inhibition Noncompetitive Competitive Noncompetitive Competitive Competitive NR NR Reversible/ Mixed-type Reversible/ Mixed-type NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR Irreversible Mixed type/Uncompetitive Reversible/Uncompetitive NR Competitive Competitive NR NR NR Competitive Noncompetitive Competitive NR Noncompetitive NR NR NR Noncompetitive Competitive NR Irreversible Competitive Reversible Reversible NR NR Irreversible Irreversible Irreversible Mixed-type

NR: not reported.

Concluding Remarks The object of this review article was to gather and present an up-to-date display of natural and synthetic compounds able to inhibit tyrosinase. This enzyme is regarded to play a critical role during food handling, storage, and commercial or domestic processing. In particular, in plant foods it causes undesirable enzymatic browning, especially in bruised or cut fruits and vegetables, which subsequently leads to a significant decrease in nutritional and market values. The information offered in this review should help to provide leads to the ultimate goal of developing new antityrosinase inhibitors (plant extracts, natural, and synthetic compounds) satisfactory efficacy, safety, and useful for the food industry.

The extracts from Dalea elegans, Lithrea molleoides, Greyia flanaganii, Cudrania cochinchinensis, and Distylium racemosum could be indicated among the most active tyrosinase inhibitors from natural sources. All these extracts showed flavonoids as main constituents. This class of natural occurring compounds is the largest group in tyrosinase inhibitors. Several studies have been demonstrated that the number and position of hydroxyl group of the B ring as well as the substituents with steric hindrance jock a key role in the tyrosinase inhibitory activity. The hydroxyl groups in compounds carry out the nucleophilic attack on the copper atoms in the active site of the enzyme and are directly involved in transferring protons during catalysis, which then result in inactivation of the tyrosinase. Regard



Tyrosinase inhibitors: an update. . . the steric hindrance of prenyl group, sugars, or alkyl chain, all these substituent could be hindering the entry of the inhibitor into the active pocket of the enzyme. Our data collection clearly evidenced that steppogenin, artocarpesin, norartocarpetin, artocarpanone, isoartocarpesin, dalenin, and kurarinol were the most active natural compounds with IC50 values ranging from 0.1 to 1.54 μM. Many efforts have been spent in the search for effective and safe tyrosinase inhibitors, and a large number of synthetic tyrosinase inhibitors have already been reported. Among them 4-formylphenyl (4,6-O-benzylidene)-β-d-allopyranoside (IC50 value of 0.052 μM), 1-(propan-2-ylidene)thiosemicarbazide (IC50 value of 0.086 μM), 2-(cyclohexylthiomethyl)-5-hydroxy-4Hpyran-4-one (IC50 value of 0.087 μM), and 2-(pentylthiomethyl)5-hydroxy-4H-pyran-4-one (IC50 value of 0.097 μM) exhibited the highest inhibitory activity. The 4-formylphenyl (4,6-Obenzylidene)-β-d-allopyranoside is an helicid analog which presents analogy with a well-known tyrosinase inhibitor arbutin’s chemical structure (4-hydroxylphenyl-O-β-d-glucopyranoside). Results evidenced that the modification of glycosyl moiety of arbutin might facilitate the inhibitory activity against tyrosinase. The 1-(propan-2-ylidene)thiosemicarbazide is a member of alkylidenethiosemicarbazide series. In thiosemicarbazide compounds the position of hydrophobic substituents on the phenyl ring enhances the inhibitory activity. Another critical aspect is the distance that separates the thiosemicarbazone group from the aromatic ring. Moreover, important structure requirements are the presence of saturated substituents. 2-(Cyclohexylthiomethyl)-5-hydroxy-4H-pyran-4-one and 2-(pentylthiomethyl)-5-hydroxy-4H-pyran-4-one are kojic acid derivatives. The presence of thioether linkage and lipophilic acid moiety was critical for the tyrosinase inhibitory activity. However, more research is needed in investigating if the potent inhibitors reported in this review article could be introduced for pratical use and are compatible with the safety regulations in order to be commercialized as food additives.

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