Inhibitory mechanism of cardanols on tyrosinase

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Inhibitory mechanism of cardanols on tyrosinase Xiang-Ping Yu a,1 , Wei-Chao Su a,1 , Qin Wang a , Jiang-Xing Zhuang b , Rui-Qi Tong a , Qing-Xi Chen a,∗ , Qiong-Hua Chen c,∗ a Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361102, China b College of Medicine, Xiamen University, Xiamen 361102, China c The First Affiliated Hospital of Xiamen University, Xiamen 361003, China

a r t i c l e

i n f o

Article history: Received 22 July 2016 Received in revised form 30 August 2016 Accepted 21 September 2016 Available online xxx Chemical compounds studied in this article: Cardanol triene (PubChem CID:11266523) Cardanol diene (PubChem CID:5356113) Cardanol monoene (PubChem CID:5281854) Keywords: Cardanols Tyrosinase Inhibitory kinetic Fluorescence quenching Molecular docking

a b s t r a c t Cashew nut shell liquid (CNSL), extracted from cashew nut shell, is an abundant natural resource. Cardanols were the major phenolic components isolated from CNSL. In this research, we reported on the inhibitory mechanism of cardanols on tyrosinase for the first time. We studied the functions of cardanols and revealed the underlying mechanism of cardanols as tyrosinase inhibitors. Cardanol triene, cardanol diene and cardanol monoene could decrease the steady-state rate of the tyrosinase diphenolase activity efficiently. The IC50 values of three cardanol compounds were determined to be 40.5 ± 3.7, 52.5 ± 3.2 and 56.0 ± 3.6 ␮M (n = 3), respectively. Meanwhile, the kinetic analysis and the intrinsic/ANS-binding fluoresecence-quenching showed that one cardanol might enter into one tyrosinase. The characteristic values further revealed that cardanols could interact with tyrosinase. Besides, computational study with molecular docking implied that cardanols might affect the amino acid residues of the tyrosinase active site. Collectively, cardanols could moderate inhibitory activities on tyrosinase effectively. © 2016 Published by Elsevier Ltd.

1. Introduction Tyrosinase (EC 1.14.18.1) is widely distributed in nature as o-diphenol-oxygen oxidoreductase, copper-containing polyphenol oxidase [1]. Tyrosinase from various organisms have similar structures and characteristics. Two copper irons with three states (Eoxy Emet Edeoxy ) are the point of the tyrosinase active site [2]. There were two key reactions, the hydroxylation of p-monophenolic amino acid l-tyrosine (l-Tyr) (monophenolase activity of tyrosinase) and the oxidation of o-diphenolic amino acid L-3, 4-dihydroxyphenylalanine (l-DOPA) to the corresponding o-quinone that ultimately transforms to melanin (diphenolase activity of tyrosinase). At present, how to control the melanogenesis becomes a hot topic of life science. Disordered or excessive accumu-

Abbreviations: l-DOPA, l-3,4-dihydroxyphenylalanine; l-Tyr, l-tyrosine; CNSL, cashew nut shell liquid; ANS, 1-anilinonaphthalene-8-sulfonate. ∗ Corresponding authors. E-mail addresses: [email protected] (Q.-X. Chen), [email protected] (Q.-H. Chen). 1 The first two authors contributed equally to this work.

lation of pigmentation leads to various dermatological disorders, such as melasama, age spots and actinic damage [3]. Tyrosinase inhibitors are very important for therapy of clinical phenotypes. A certain amount of inhibitors are not available for clinic treatment for their insolubility, high toxicity or low activity [4]. Therefore, tyrosinase inhibitors extracted from natural products will become a new research direction with broad application prospects [5]. Cashew nut shell liquid (CNSL) is an efficiently available material. CNSL was treated as industrial waste [6]. In recent years, studies show that CNSL has been widely applied in enormous industrial products, polymerization products and combination with other materials. CNSL mainly consists of cardanols (60–65%), cardols (15–20%), polymeric material (10%), and traces of methyl cardol. There are some efficient methods for the separation of different components from technical CNSL [7–9]. Their structures were determined by nuclear magnetic resonance spectroscopic analyses (NMR) [10,11]. In previous studies, ELMER-RICO EMOJICA [12] and ISAO KUBO [13] reported that CNSL showed the significant inhibition of tyrosinase activity. There were many researches on its components, such as anacardic acids and cardols except cardanols [14–16].

http://dx.doi.org/10.1016/j.procbio.2016.09.019 1359-5113/© 2016 Published by Elsevier Ltd.

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2.3. UV scanning study The experiment contained 3 mL reaction media and was measured described by Jiménez-Atiénzar et al. [27]. The mixture included 0.5 mM l-DOPA and 100 ␮L of inhibitors (dissolved in DMSO) in 50 mM sodium phosphate buffer (pH 6.8). The oxidation process of l-DOPA was recorded at the absence and presence of inhibitors. These assays were monitored for the formation of dopachrome at 475 nm using a Beckman DU-800 spectrophotometer [28]. The final concentration of tyrosinase was 16.67 ␮g/mL. 2.4. Inhibitory kinetic course of tyrosinase

Fig. 1. Chemical structures of cardanols from cashew nut shell liquid (CNSL).

Though cardanols and various their derivatives have been associated with various of biological effects [15,17,18], the inhibitory mechanism on regulating tyrosinase activity is still unknown. Cardanols are rich sources of long-chain alkyl substituted salicylic acid and resorcinol [19,20]. Their chemical structures were showed in Fig. 1. We suggested that they might suppress the generation of melanin by specifically inhibiting tyrosinase. Zhuang et al. [21] have demonstrated that the inhibitory mechanism of cardol triene on tyrosinase. In this work, we continued to investigate the inhibitory kinetic course of tyrosinase by cardanols and explored their interaction mechanism. This study will provide a comprehensive understanding of the inhibitory regulation by cardanols in vivo.

2. Materials and methods 2.1. Materials Cardanols were separated and purified from CNSL [21], which was provided by Xiamen Welso Co., Ltd. Mushroom tyrosinase (Agaricus bisporus) was the product of Sigma-Aldrich (St. Louis, MO, USA). The specific activity of the enzyme was 6680 U/mg. lDOPA, l-Tyr, dimethylsulfoxide (DMSO) were also obtained from Sigma-Aldrich. Other reagents were to be analytical grade. The water used was redistilled and ion-free.

2.2. Enzyme activity assay The reaction media (3 mL) for enzyme activity assay was previously reported [21,22]. It contained 0.5 mM l-DOPA and 100 ␮L different concentrations of inhibitors dissolved in 3.3% DMSO. The reaction was added the substrate (l-DOPA). Along with the oxidation of l-DOPA, the density at 475 nm (␧ = 3700 M−1 cm −1 ) was checked at each concentration of inhibitors. Tyrosinase was firstly incubated with inhibitors using a Beckman DU800 spectophotometer to make the absorbance and kinetic measurements [23]. Temperature was controlled at 37 ◦ C to keep the stability of the system [2]. All were performed in 50 mM sodium phosphate buffer (pH 6.8). In this method, the effects of inhibitors can be evaluated [24]. The reaction at each concentration was carried out along with controls of 3.3% DMSO without inhibitors. The values of IC50 (the inhibited 50% of the enzyme activity) were calculated from triplicate measurements to express the inhibitory effects of inhibitors on the enzyme. The inhibition types (irreversible & reversible) and the inhibition constants were obtained using the method described by Chen et al. [25,26].

The experiment was performed based on the method in a previous study [21]. l-DOPA was used as the substrate. The substrate-enzyme reaction was tested to detect the inhibitory kinetic, and kinetic course of the enzyme was carried out on the following conditions. The 3.0 mL reaction contained 50 ␮L of enzyme and 100 ␮L of different concentrations of inhibitors. All tests were conducted at a constant temperature of 30 ◦ C, and reacted in 0.1 M sodium phosphate buffer (pH 6.8). During the oxidation of l-DOPA, the absorbance at 475 nm (␧ = 3700 M−1 cm−1 ) was monitored to determine the enzyme activity. The final concentration of tyrosinase was 16.67 ␮g/mL. The kinetic constants were obtained with reference to Chen et al. [29]. 2.5. Intrinsic and ANS-binding fluorescence measurement Fluorescence spectra were measured by the methods of Kim [30] and Ionit¸a˘ [31] with some modification. To investigate the interaction of inhibitors and tyrosinase, the fluorescence intensities were recorded using a Varian Cary Eclipse fluorophotometer with an excitation wavelength (␭ex ) of 280 nm and a range of emission wavelength (␭em ) from 300 to 450 nm. The excitation and emission slit widths were both 5 nm, and the scan speed was 600 nm/min. Assays of system contained 2.0 mL mixture with tyrosinase and different concentrations of inhibitors [32]. The change of the fluorescence emission intensity was measured with 1 min blending. Each measurement was recorded in triplicate. The final concentrations of tyrosinase were 33.33 ␮g/mL. In addition, the ANS-binding fluorescence intensity of tyrosinase was studied by the emission wavelength ranged from 400 to 600 nm with an excitation wavelength of 350 nm. Tyrosinase was labled with 100 ␮M ANS for 5 min first, and the other measurements were the same with the intrinsic fluorescence experiments [33,34]. 2.6. Molecular docking with ligand The molecular operation environment software (MOE) is an effective tool to study the interaction of tyrosinase and ligands. In the docking, the parameters should be set accurately consulted with the previous research [26]. After the parameter setting, the tertiary structures of inhibitors and tyrosinase were energy minimized. The receptor and site denote the receptor atoms and dummy atoms, respectively. The docked conformations are improved with the highest score. 3. Results and discussion 3.1. Effects of cardanols on the diphenolase activity of tyrosinase Effects of cardanols on the oxidation of l-DOPA by tyrosinase were studied. Cardanols inhibited tyrosinase activities dramatically with a dose-dependent manner. The relative activities of enzyme were all reduced to around 40% by cardanol triene, cardanol diene

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Table 1 Diphenolase inhibition constants of cardanols on tyrosinase. Inhibitors

cardanol triene cardanol diene cardanol monoene

IC50 (␮M)

40.5 ± 3.7 52.5 ± 3.2 56.0 ± 3.6

Inhibition (␮M)

Inhibition constants (␮M)

mechanism

type

KI

KIS

reversible reversible reversible

mixed mixed mixed

77.1 ± 1.2 78.4 ± 1.4 74.8 ± 1.4

46.4 ± 0.9 64.6 ± 0.7 53.5 ± 0.4

Fig. 2. Effects on the diphenolase activities of tyrosinase by cardanols: (I) The indicated cardanols concentration were added to the assay system when tyrosinase was preincubated with cardanols for 2 h at 25 ◦ C (b) or not (a); (II) Effects on the oxidation of l-DOPA by concentrations of tyrosinase at different concentrations of cardanols. The concentrations of cardanols for curves 1–5 were 0, 14.0, 28.0, 42.0, and 56.0 ␮M. A, B and C represented cardanol triene, cardanol diene, and cardanol monoene, respectively.

and cardanol monoene, respectively (Fig. 2a). Cardanols inhibited tyrosinase activities dramatically with a dose-dependent manner. The IC50 of cardanol triene, cardanol diene and cardanol monoene were 40.5 ± 3.7, 52.5 ± 3.2 and 56.0 ± 3.6 ␮M (n = 3), respectively. Their inhibition on tyrosinase were about 40 times more potent than that of arbutin (IC50 = 2700 ␮M)[35] and 10 times of kojic acid (IC50 = 400 ␮M)[36] which was often uesd as a positive standard. In the meanwhile, we found that when tyrosinase was preincubated with cardanols for 2 h at 25 ◦ C, the tyrosianse activity remained around 10% at the equilibrium state by cardanols (Fig. 2b). We indicated that cardanols reversibly inhibited the tyrosinase. To ascertain the inhibitory mechanism of cardanols behaved, the oxidation of l-DOPA by tyrosinase was studied. Increasing concentrations of cardanols generated a family of straight lines, which

Fig. 3. Determination of the inhibitory types and constants of cardanols on tyrosinase. The concentrations of cardanols for curves 1–5 were 0, 14.0, 28.0, 42.0, and 56.0 ␮M. A, B and C represented cardanol triene, cardanol diene, and cardanol monoene, respectively.

all passed through the origin (Fig. 2A-II, B-II and C-II). The results showed that the presence of cardanols did not reduce the amount of active enzyme, but depressed the enzyme activities. Lineweaver-Burk plot analysis (the plots of 1/v versus 1/[lDOPA]) gave a family of straight lines to evaluate the type of inhibition. As shown in Fig. 3, the results demonstrated that they were all mixed-type inhibitors. From the plot of the slopes versus the concentrations of cardanols, inhibition constant (KI ) was obtained. The enzyme-substrate complex (KIS ) was also obtained from the plot of the vertical intercepts versus the concentrations of cardanols. The values of cardanols tested were summarized in Table 1 for comparison. 3.2. Oxidation process of l-DOPA in the presence and absence of cardanols The influences of cardanols on the oxidation of l-DOPA were shown in Fig. 4. The spectra of wavelength scan were obtained at the absence and presence of cardanols. In the absence of cardanols,

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Fig. 4. Consecutive wavelength scan of the oxidation of l-DOPA at the absence (A), and the presence of cardanols (0.2 mM) by tyrosinase. (B, C and D represented cardanol triene, cardanol diene and cardanol monoene, respectively.) Curves 1–8 represented 0–7 min after the addition of tyrosinase.

Table 2 Stern-Volmer equation for the interaction between cardanols and tyrosinase. Inhibitors cardanol triene cardanol diene cardanol monoene

Type of quenching

KSV (M−1 )

KA (M−1 )

n

static static static

6.6 × 10 ± 126.3 7.0 × 103 ± 113.7 6.7 × 103 ± 158.3

1584.9 ± 23.4 1584.9 ± 22.8 1158.9 ± 23.3

0.93 ± 0.12 0.84 ± 0.08 0.81 ± 0.11

the maximum value of absorbance at 475 nm was 0.64 (Fig. 4A). At the first 7 min, the peak intensities were reduced by 38.3%, 28.2% and 11.6%, respectively, with the addition of cardanol triene, cardanol diene and cardanol monoene (Fig. 4B–D). It was surprisingly demonstrated that the lower saturation of C15 -alkyl side-chain did lead to enhancing inhibitory activities: cardanol triene was the noticeable inhibitor. The results reflected the same inhibitory pattern with the enzyme activity assay: cardanol triene > cardanol diene > cardanol monoene. Muralidhara et al. [37] and Kim et al. [30] reported that the location of the hydroxyl group of inhibitors disturbed inhibitory activities by the combinational steric hindrance [38]. We predicted that the contribution of C15 -alkyl side chains to cardanols at similar positions interfered with inhibitory activities to varying degrees.

3.3. Inhibitory kinetic analysis of tyrosinase by cardanols The substrate-enzyme kinetic course has built a model to analysis quantitatively. Kinetic analysis was applied to investigate the interaction of tyrosinase and cardanols. According to the following Schemes 1 and 2, the inhibitory kinetic of tyrosinase by cardanols can be studied. E and I represent the native enzyme and inactivator, and n denotes the number of effective inactivator that combines with each enzyme and makes the enzyme inactivated. The values

3

E

k1

E'

Scheme 1. The first-order inactivation of the enzyme.

E + nI

k2

EIn

Scheme 2. The second-order inactivation of the enzyme.

of n are given from the straight lines in the plot of log(k1 ) versus log[cardanol]. Shown in Fig. 5, the semilogarithmic plots for the inhibitory reaction were represented straight lines. The kinetic rate constants were given from the slopes of the straight lines, and got the same trend of the concentrations of cardanols. The n of cardanol triene, cardanol diene and cardanol monoene were 0.98, 0.91 and 0.91, respectively (Fig. 5A-II, B-II and C-II). These results declared that the kinetic course acted under a first-order reaction[39]. We predicted that one cardanol could enter into one tyrosinase fully, and

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Fig. 5. Inhibitory kinetics course of tyrosinase by cardanols (A, B and C represented cardanol triene, cardanol diene and cardanol monoene, respectively.): (I) Effects of kinetics course of tyrosinase by cardanols (42 ␮M); (II) The plot of inhibitory kinetic rate constants (k) of tyrosinase versus log[cardanol]. Tyrosinase was preincubated with the indicated cardanols concentration for 2 h at 25 ◦ C.

Fig. 6. Changes in the intrinsic tyrosinase fluorescence with increasing the concentrations of cardanol triene at ␭ex = 280 nm: (A) The quenching effect of tyrosinase; (B) The Stern-Volmer plot of the tyrosinase quenching; (C) The plot of lg[(F0 -F)/F] against lg[cardanol triene].

was preceded by a relatively reversible binding into the tyrosinase active site. 3.4. Effects of cardanols on the conformation of tyrosinase 3.4.1. Intrinsic fluorescence-quenching analysis It was observed that the tryptophan fluorescence could show whether the conformation of tyrosinase changes [30,40]. The tryptophan residues of tyrosinase can be examined under 280 nm of ␭ex , and has a strong fluorescence in 337 nm of ␭em . Shown in Fig. 6, the fluorescence intensities of tyrosinase were collected, with the increasing concentrations of cardanols. There was a decreasing change of fluorescence intensitiy with the accumulation of cardanol triene, but not dramatic changable in the emission spectra (Fig. 6A). The results indicated that cardanol triene combined tyrosinase depending on the accumulation of concentrations merely. According to the previous reports[33], the fact implied that cardanol triene loosened the structure of tyrosinase. The Stern–Volmer plot in Fig. 6B measured the quenching constants of cardanol triene, and Fig. 6C showed the plot of lg[(F0 -F)/F] against lg[cardanol triene] for tyrosinase with concentrations of cardanol triene [41]. (F0 and F are the fluorescence intensities before and after the addition of the

Fig. 7. Dose-response plot for maximum ANS-fluorescence intensity of tyrosinase in the presence of increasing concentrations of cardanols at ␭ex = 350 nm (The inset showed the ANS-fluorescence of tyrosinase changes at different cardanol concentrations.).

various concentrations of cardanol triene.) A good linear agreement with the plots suggested that cardanol triene was a good quencher of the fluorophore in tyrosinase. Meanwhile, we studied the effects of cardanol diene and cardanol monoene, as well. The results of

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Fig. 8. Tertiary structure and secondary structure for the interaction of cardanols with the tyrosinase. A, B and C represented cardanol triene, cardanol diene, and cardanol monoene, respectively. In the graph, the size and intensity of the turquoise discs surrounding the tyrosinase residues showed the different exposures, which were changed by cardanols.

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fluorescence-quenching experiments did agree with the kinetic analysis.The average binding constants (KA ) and the binding sites (n) were obtained from the equation of linear regression. As listed in Table 2, the types of quenching mechanism between tyrosinase and cardanols were all static, due to the average dynamic quenching constants (KSV ) were greater than 100 L/M. On the basis of the static type of quenching mechanism, we predicted that cardanols went into the catalytic domain of tyrosinase, and formed relative state complexes with no fluorescence [32]. The binding sites of cardanol triene, cardanol diene and cardanol monoene were got to be 0.93 ± 0.12, 0.84 ± 0.08 and 0.81 ± 0.11 (n = 3), respectively. It could be concluded that cardanols might alter the conformation of tyrosinase, and one cardanol interacted with one tyrosinase. 3.4.2. ANS-binding fluorescence-quenching analysis ANS dye is sensitive to the hydrophobic surfaces within tyrosinase and binds to the amino acid residues of tyrosinase. We monitored the ANS-fluorescence intensities to detect the hydrophobicity domain changes of tyrosinase [42]. The results revealed that the ANS-fluorescence intensities were increasing with the continuing addition of cardanols, and all cardanols generated similar effects (Fig. 7). We predicted that cardanols might change the conformation of tyrosinase to a certain extent. 3.5. Molecular docking analysis To clarify the inhibitory mechanism of cardanols on tyrosinase, docking simulation was further performed on the basis of anti-tyrosinase activities by cardanols. Shown in Fig. 8, the results revealed that cardanols interacted with residues in proximity to the tyrosinase active [43]. These residues are thought to be involved in the stage of cardanol binding [44]. In addition, the simulation results were also consistent with the fluorescence-quenching measurements. Cardanols were not simply binding to the copper irons, but affected the conformation of tyrosinase in a mixed-type. We found that the hydroxy group for cardanol triene on the phenol group could directly act on Asn81 (Fig. 8A); the hydroxy group of cardanol diene could interact with His85 (Fig. 8B); however, cardanol monoene had no direct effect on tyrosinase (Fig. 8C). The docking analysis demonstrated that cardanols focused on making interactions with a dozen of tyrosinase residues, such as: His(61,85,94,259,263 ), Glu256 , Phe264 , Val(248,283 ), Asn(81,260 ). These simulation binding interactions might make a step forward to know the inhibitory mechanism of cardanols on tyrosinase. 4. Conclusions In this work, we made a comprehensive study on the inhibitory mechanism of cardanols on tyrosinase. Their inhibitory effects were investigated by enzyme activity assay and UV scanning study. These results revealed that cardanols inhibited tyrosinase activities effectively, and the inhibitory abilities of cardanols might affected by the combinational steric hindrance of C15 -alkyl side chains. Moreover, the inhibitory kinetic and fluorescence-quenching analysis showed that about one cardanol can interact with one tyrosinase consistently. The molecular docking simulation revealed that cardanols might interact with the amino acid residues in the tyrosinase active site not the copper irons. Base on the above, these in-vitro experiments demonstrated that cardanols were all efficient tyrosinase inhibitors. However, the potent inhibitory activity of enzyme is not always consistent with the capacity of reducing pigmentation in cells. In the development of new agents for treating hyperpigmentation, we should make more studies on cardanols, and confirm their safety and efficiency simultaneously.

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