Alpha-Substituted Derivatives of Cinnamaldehyde

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Dec 29, 2014 - Docking results implied that they could not form metal interactions with the copper ions of the enzyme, whereas they could interact with the ...

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Alpha-Substituted Derivatives of Cinnamaldehyde as Tyrosinase Inhibitors: Inhibitory Mechanism and Molecular Analysis Yi Cui, Ge Liang, Yong-Hua Hu, Yan Shi, Yi-Xiang Cai, Huan-Juan Gao, Qing-Xi Chen, and Qin Wang* State Key Laboratory of Cellular Stress Biology and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen 361102, China ABSTRACT: Alpha-substituted derivatives of cinnamaldehyde (alpha-bromocinnamaldehyde, alpha-chlorocinnamaldehyde, and alpha-methylcinnamaldehyde) were used as inhibitors on mushroom tyrosinase. The result showed that three compounds can reduce both monophenolase and diphenolase activity on tyrosinase, and the inhibition was reversible. The IC50 values of alphabromocinnamaldehyde, alpha-chlorocinnamaldehyde, and alpha-methylcinnamaldehyde were 0.075, 0.140, and 0.440 mM on monophenolase and 0.049, 0.110, and 0.450 mM on diphenolase, respectively. The inhibition types and constants on diphenolase for these inhibitors were further studied. The molecular inhibition mechanisms of tyrosinase by the derivatives were investigated by UV-scanning study, fluorescence quenching, and molecular docking. These assays demonstrated that the derivatives could decrease the formation of o-quinones, and all derivatives were static quenchers of mushroom tyrosinase. Docking results implied that they could not form metal interactions with the copper ions of the enzyme, whereas they could interact with the amino acid residues of active site center. This research on alpha-substituted derivatives of cinnamaldehyde as tyrosinase inhibitors would lead to advances in the field of antityrosinase. KEYWORDS: tyrosinase, derivatives of cinnamaldehyde, fluorescence quenching, molecular docking



human colon tumor xenograft in nude mice.12 Yoshio et al.13 reported that alpha-bromocinnamaldehyde has been used widely as an antifungal agent for optical, electric, and musical instruments, and they also proposed the metabolic pathway of alpha-bromocinnamaldehyde in rats. Based on the above, we paid attention to the relationship between derivatives of cinnamaldehyde on alpha-substituted and mushroom tyrosinase. This research aims to understand the mechanism of the alpha-substituted derivatives on tyrosinase and to clarify molecular interaction between derivatives and enzyme structure.

INTRODUCTION Tyrosinase (EC 1.14.18.1) is a copper-containing, mixed function oxidase widely distributed in animals, plants, and microorganisms.1 It catalyzes two distinct reactions of melanin biosynthesis by hydroxylation of monophenols to o-diphenols and oxidation of o-diphenols to the corresponding o-quinones, which are the initial steps whereafter a series of highly reactive quinones are produced to initiate the pigmentation and excessive activation of tyrosinase can cause various dermatological disorders.2 Tyrosinase inhibitors are widely used in dermatological treatments and in cosmetics. Plenty of tyrosinase inhibitors are obtained from natural or synthetic sources, which have been further investigated.3 Cinnamaldehyde, a low molecular weight cinnamic acid analogue with relatively broad distribution in plants, has shown various activities such as antitumor, cytotoxic, and mutagenic.4,5 It has also been proven to have strong activity against a wide variety of wood decay fungi.6,7 Cinnamaldehyde is considered to be a safe fragrant constituent without acute and chronic toxicity, and no case of mutagenecity or genotoxicity and carcinogenicity due to cinnamaldehyde has ever been reported in mammalian.8 In the previous study, Ikuyo et al. 9 reported that cinnamaldehyde inhibited the oxidation of L-DOPA by mushroom tyrosinase with an IC50 of 129 μg/mL (0.98 mM). Several kinds of derivatives of cinnamaldehyde have been reported. 2′-Hydroxycinnamaldehyde derivative isolated from C. cassia has been reported to have an inhibitory effect on farnesyl protein transferase activity and inhibited proliferation of several human cancer cell lines including breast, leukemia, ovarian, lung, and colon tumor.10,11 Dimeric cinnamaldehydes strongly inhibited the growth of human tumor cells through the inducing of apoptosis in tumor cells and in vivo growth of © 2014 American Chemical Society



MATERIALS AND METHODS

Materials. Mushroom tyrosinase (EC 1.14.18.1) from mushroom (Agaricus bisporus) was the product of Sigma-Aldrich (St. Louis, MO). The specific activity of the enzyme was 6680 U/mg. L-3,4Dihydroxyphenylalanine (L-DOPA), L-tyrosine (L-Tyr), alpha-bromocinnamaldehyde (aBCA), alpha-chlorocinnamaldehyde (aCCA), alpha-methylcinnamaldehyde (aMCA), dimethylsulfoxide (DMSO), and NaIO4 were also obtained from Sigma-Aldrich. Other reagents with standard analytical purity were acquired from Sinopharm (Shanghai, China). The water used was redistilled and ion-free. Enzyme Activity Assay. Monophenolase and diphenolase activity assay were performed as previously reported.14 In this reaction, L-Tyr and L-DOPA were used as the substrate for monophenolase and diphenolase activity assay, respectively. The reaction media (3 mL) for enzyme activity assay contained 1 mM L-Tyr or 0.5 mM L-DOPA in 50 mM Na2HPO4−NaH2PO4 buffer (PH 6.8) and 100 mL of different concentrations of inhibitor. The final concentrations of mushroom tyrosinase were 33.33 and 3.33 μg/mL for monophenolase and Received: Revised: Accepted: Published: 716

November 12, 2014 December 29, 2014 December 29, 2014 December 29, 2014 DOI: 10.1021/jf505469k J. Agric. Food Chem. 2015, 63, 716−722

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of the derivatives of cinnamaldehyde. (A), (B), and (C) represent aBCA, aCCA, and aMCA, respectively. diphenolase activity, respectively.15,16 Enzyme assay was performed by following the increase of optical density at 475 nm (ε = 3700 M−1 cm−1) accompanying the oxidation of substrates. The reaction was carried out at 30 °C. A Beckman DU800 spectophotometer was used for absorbance and kinetic measure.17 The inhibitor was dissolved in 3.3% DMSO and diluted to appropriate concentration. The controls without inhibitor but containing 3.3% DMSO in the reaction media were routinely carried out. The inhibitory effects of inhibitors on the enzyme were expressed as the concentrations that inhibited 50% of the enzyme activity (IC50). The inhibition types were obtained by the Line-weaver Burk plot, and the inhibition constants were determined by the secondary plots of the apparent Km/Vm or 1/Vm versus the concentrations of the inhibitors.18 UV Scanning Study. The experiment was carried out according to the method of Jiménez-Atiénzar et al.19 with slight modifications. The oxidation of L-DOPA was carried out with and without the presence of inhibitor. The reaction media (3 mL) contained 0.5 mM L-DOPA in 50 mM Na2HPO4−NaH2PO4 buffer (pH 6.8) and 0.1 mL of inhibitor (dissolved in DMSO). The final concentration of the mushroom tyrosinase was 16.67 μg/mL. The UV spectra were recorded using a Beckman DU-800 spectrophotometer. Nonenzymatic Generation of the Quinones. The experiment was carried out by methods of Espin and Wichers.20 L-DOPA (100 μM) was oxidized by NaIO4 (100 μM). The spectra in the absence and presence of the inhibitor (9, 18, 27, and 36 μM) were recorded with the DU800 spectrophotometer. Fluorescence Spectrum Quenching. The experiment was performed with reference to Kim et al.21 Fluorescence spectra were recorded using an Cary Eclipse fluorescence spectrophotometer. Fluorescence emission spectra were recorded at wavelengths of 300− 420 nm upon excitation at 280 nm. The excitation and emission bandwidths were both 5 nm.22,23 For each data point, 10 μL of inhibitor solution was added to 2 mL of tyrosinase solution to make sure each inhibitor was at the appropriate range of concentration. Each measurement was done thrice, and the means were calculated. The fluorescence quenching data were plotted as the fluorescence intensity against the inhibitor concentration. Fluorescence quenching was described by the Stern−Volmer equation24

Molecular Docking with Ligand. In this research, molecular operation environment 2010 software (MOE) was used for protein− ligand docking. The method was consulted with reference to Chen.27 Before docking, the 3D structures of the inhibitors and tyrosinase were energy minimized. Hydrogens were added using the energy minimization module of the MOE. The parameters for molecular docking were as follows: the receptor and site were set to receptor atoms and dummy atoms, respectively; the refinement was set to force field; the retain of both the first and second scoring were set to 10; both the first and second rescoring were set to London dG; the MM/ GBVI binding free energy scoring was used to rank the docking poses. A more negative value reflects a stronger interaction. Other parameters used were the default settings of the software. The docked conformation, which had the highest score, was selected to analyze the mode of binding.



RESULTS Effects of Derivatives of Cinnamaldehyde on the Monophenolase Activity of Mushroom Tyrosinase. Taking aBCA, aCCA, and aMCA (see Figure 1 for structures) as the effectors, we probed the effects of these compounds on the activity of mushroom tyrosinase for the oxidation of L-Tyr. The lag time and characteristic of monophenolase activity were observed. The reaction system reached a steady state after the lag time. After the steady state, the curve of product increased linearly with increasing reaction time. The results are shown in Figure 2. Increasing concentrations of these effectors not only changed the lag time of the enzyme (Figure 2A-II, Figure 2B-II, and Figure 2C-II), but also the steady-state rates decreased obviously (Figure 2A-III, Figure 2B-III, and Figure 2C-III). When the concentrations of effectors were increased to 0.2 mM, the lag time was extended by aBCA, aCCA, and aMCA up to 115%, 75%, and 9%, respectively. As shown in Table 1, the concentrations for aBCA, aCCA, and aMCA leading to 50% enzyme activity (IC50) were 0.075, 0.140, and 0.440 mM, respectively. Effects of Derivatives of Cinnamaldehyde on the Diphenolase Activity. Effects of the derivatives on the oxidation of L-DOPA catalyzed by mushroom tyrosinase were investigated. The results showed that the inhibition was dosedependent, while the relative activity of the enzyme reaction was reduced with increasing concentration of the inhibitors (Figure 3). The concentrations of aBCA, aCCA, and aMCA for leading to 50% enzyme activity loss (IC50) were 0.049, 0.110, and 0.450 mM, respectively. The values are given in Table 1 as well. Inhibition Mechanism of Derivatives of Cinnamaldehyde on the Diphenolase Activity. To ascertain the inhibition mechanism of derivatives of cinnamaldehyde, the oxidation of L-DOPA by derivatives was studied. Figure 4 shows that the plots of the remaining enzyme activity versus the concentrations of enzyme at different concentrations of aBCA gave a family of straight lines, which all passed through the origin. The slopes of the lines descended with an increase of the

F0/F = 1 + Kqτ0[Q] = 1 + KSV[Q] where F0 and F are the fluorescence intensities before and after the addition of the quencher, respectively, Kq is the bimolecular quenching constant, τ0 (10−8 s) is the lifetime of the fluorophore in the absence of the quencher, [Q] is the concentration of the quencher, and KSV is the Stern−Volmer quenching constant. Hence, the above equation was applied to determine the KSV using linear regression of a plot of F0/F against [Q]. For the calculation of bimolecular quenching constants, the data were plotted as F0/F against [Q], with the bimolecular quenching constants calculated by linear regression. There were two types of fluorescence-quenching mechanisms: static (by complex formation) and dynamic (by collisional processes).25 When the type was static, the apparent binding constant (KA) and the binding affinity (n) were estimated by plotting log[(F0 − F)/F] against log[Q] with the following equation26 log[(F0 − F )/F ] = log KA + n log[Q] where [Q] is the quencher concentration 717

DOI: 10.1021/jf505469k J. Agric. Food Chem. 2015, 63, 716−722

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Journal of Agricultural and Food Chemistry

led to a decrease in the enzyme activity. Therefore, the inhibition of tyrosinase by aBCA was reversible. We also studied the inhibition mechanisms of aCCA and aMCA, as above. Results indicated that both aCCA and aMCA were reversible inhibitors of mushroom tyrosinase. Inhibition Types and Constants on the Diphenolase Activity by Derivatives of Cinnamaldehyde. The plots of 1/v versus 1/[S] gave a family of straight lines indicating that aBCA was mixed type inhibitor (Figure 5A-I). Figure 5B-I indicated that aCCA was a noncompetitive inhibitor of the enzyme. As shown in Figure 5C-I, the result domenstrated that aMCA was an uncompetitive inhibitor of mushroom tyrosinase. The inhibition constants (KI) were obtained from the plots of the slopes versus the concentrations of the inhibitors (Figure 5A-II and Figure 5B-II), and the enzyme−substrate complexes (KIS) were obtained from the vertical intercepts versus the concentrations of these compounds (Figure 5A-III and Figure 5C-II). The values obtained are summarized in Table 1 for comparison. Oxidation of L-DOPA in the Presence and Absence of Derivatives of Cinnamaldehyde. The spectra obtained during the oxidation of L-DOPA in the absence (Figure 6A) and presence (Figure 6B, Figure 6C, and Figure 6D) of aBCA, aCCA, and aMCA by mushroom tyrosinase are given. In the absence of these compounds, the absorbance of the typical peak at 475 nm was 0.75. With the existence of aBCA, aCCA, and aMCA, at 10 min after the addition of the enzyme the peak intensity was reduced by 67%, 48%, and 24%, respectively. The results were consistent with the enzyme activity assay. Interaction of Derivatives of Cinnamaldehyde with oQuinones. Ultraviolet−visible scan was used to research the inhibitory mechanism of the compounds on mushroom tyrosinase. The o-diphenol (L-DOPA) was oxidized by sodiumperiodate (NaIO4) in the absence and presence of the inhibitors. Figure 7A, curve 1 shows spectrum of L-DOPA without any characteristic peaks. Curves 2−5 show the oxidation of L-DOPA by NaIO4 for 1−4 min with a characteristic peak at 475 nm. Because the absorption did not increase as time goes by, L-DOPA (100 μM) was considered to be totally oxidized by NaIO4 (100 μM). Curves 1 in Figure 7B, 7C, and 7D are spectra of aBCA, aCCA, and aMCA, respectively. None of them showed characteristic peak at 475 nm. However, when the compounds were added to the mixture of L-DOPA and NaIO4, a decrease of absorbance at the peak occurred (Figure 7B, Figure 7C, and Figure 7D curves 2−5). Oxidation of L-DOPA by tyrosinase and NaIO4 had the same typical peak at 475 nm indicating that they produced the same products. After addition of effectors, both the enzymatic and nonenzymatic reactions, the product peak became lower. The results indicated that effectors could bring down the oxidative product of L-DOPA, meaning that they prevented the formation of o-quinones, so the generation of melanin had

Figure 2. Inhibitory activity of the derivatives of cinnamaldehyde on monophenolase activity of mushroom tyrosinase. (I) Progress curves for the oxidation of L-Tyr by the enzyme. (II) Effects on the lag time of monophenolase. (III) Effects on the stable activity of L-Tyr by the enzyme. (A), (B), and (C) represent aBCA, aCCA, and aMCA, respectively. The concentrations of aBCA for curves 1−6 were 0, 0.04, 0.08, 0.12, 0.16, and 0.20 mM. The concentrations of aCCA for curves 1−7 were 0, 0.05, 0.1, 0.15, 0.2, 0.3, and 0.4 mM. The concentrations of aMCA for curves 1−6 were 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mM.

concentrations of inhibitor, indicating that the presence of aBCA did not bring down the amount of active enzyme but just Table 1. Inhibition Constants of Cinnamaldehyde Derivatives IC50 (mM)

inhibition (mM)

inhibition constants (mM)

compd

monophenolase

diphenolase

mechanism

type

KI

KIS

aBCA aCCA aMCA

0.075 0.140 0.440

0.049 0.110 0.450

reversible reversible reversible

mixed noncompetitive uncompetitive

0.044 0.116

0.063 0.116 0.194

718

DOI: 10.1021/jf505469k J. Agric. Food Chem. 2015, 63, 716−722

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Journal of Agricultural and Food Chemistry

Figure 3. Inhibitory activity of the derivatives of cinnamaldehyde on diphenolase activity of tyrosinase. (A), (B), and (C) represent aBCA, aCCA and aMCA, respectively.

Figure 4. Effect of concentrations of mushroom tyrosinase on its activity for the oxidation of L-DOPA at different concentrations of aBCA. The concentrations of aBCA for curves 1−5 were 0, 0.0125, 0.025, 0.0375, and 0.05 mM.

been reduced. This was the other process to restrain the activity of tyrosinase. Analysis of Fluorescence Quenching of Tyrosinase with Derivatives of Cinnamaldehyde. Fluorescence quenching was applied to investigate the interactions of ligands and proteins.28 The fluorescence emission spectra of tyrosinase in the absence and presence of aBCA are shown in Figure 8A. The fluorescence intensities of the emission peaks at 338 nm were inversely decreased with increasing concentration of aBCA (Figure 8B). Figure 8C is the Stern−Volmer plot which measured the Stern−Volmer quenching constants of aBCA. The results, shown in Table 2, suggested a good linear Stern− Volmer plot with Kq and KSV values of 3.77 × 1012 M−1 s−1 and 3.77 × 104 M−1, respectively. Due to the fact that the KSV for dynamic cannot be larger than 100 L/M,26 the KSV we got demonstrated that the type of fluorescence-quenching mechanism between tyrosinase and aBCA was static. Figure 8D shows the plot of log[(F0 − F)/F] against log[Q] for tyrosinase with various concentrations of aBCA. From this graph, the values of KA and n were obtained from the intercept and the slope. We obtain an equation of linear regression: log[(F0 − F )/F ] = 5.89 + 1.29 log[Q],

R2 = 0.9965

We have studied the fluorescence quenching of tyrosinase with aCCA and aMCA as well. Results were similar to aBCA, which indicated that both aCCA and aMCA were static quenchers of mushroom tyrosinase. All the values of KSV, Kq, KA, and n are shown in Table 2. Molecular Docking Analysis. Docking simulations were further performed to understand the mechanism underlying the

Figure 5. Determination of the inhibition type and constants of the compounds of tyrosinase. (A), (B), and (C) represent aBCA, aCCA and aMCA, respectively. The concentrations of aBCA for curves 1−5 were 0, 0.0125, 0.0250, 0.0375, and 0.0500 mM. The concentrations of aCCA for curves 1−5 were 0, 0.5, 1.0, 1.5, and 2.0 mM. The concentrations of aMCA for curves 1−5 were 0, 0.15, 0.30, 0.45, and 0.60 mM. 719

DOI: 10.1021/jf505469k J. Agric. Food Chem. 2015, 63, 716−722

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Journal of Agricultural and Food Chemistry

Figure 8. Changes in intrinsic tyrosinase fluorescence at different concentrations of aBCA. (A) Emission spectra 1−6 of tyrosinase in the presence of aBCA for 0, 0.005, 0.010, 0.015, 0.025, and 0.035 μM, respectively; (B) maximum florescence intensity changes; (C) Stern− Volmer plot describing the tyrosinase quenching caused by association with quencher; (D) plot of log[(F0 − F)/F] against log[Q] for tyrosinase and various concentrations of quencher. F0 and F are the fluorescence intensities before and after the addition of the quencher.

Figure 6. Consecutive spectra obtained during the oxidation of LDOPA in the absence (A) and presence (B), (C), and (D) of inhibitors (0.2 mM) by mushroom tyrosinase. (B), (C), and (D) represent aBCA, aCCA, and aMCA, respectively. Curves 1−11 represent 0−10 min after the addition of the enzyme.

Table 2. Stern−Volmer Equation for the Interaction between Quenchers and Tyrosinase compd aBCA aCCA aMCA

type of quenching

KSV (M−1)

Kq (M−1 s−1)

KA (M−1)

n

static static static

3.77 × 10 3.84 × 104 4.86 × 104

3.77 × 10 3.84 × 1012 4.86 × 1012

7.76 × 105 5.75 × 105 3.47 × 10

1.29 1.05 0.97

4

12

enzyme. whereas all of them could interact the amino acid residues in the center of active site. The oxygen for aBCA on the aldehyde group could directly act on His85 and His61. Besides, aBCA could form intercations with tyrosinase residues, such asAla 286 , His 244 , Glu 256 , Phe 264 , Asn 260 , Phe 292 , His263,Val283, His295, and Ser282. (Figure 9A). The same analyses were carried out on aCCA and aMCA, which indicated the oxygen on the aldehyde group for aCCA and aMCA could directly act on His94 (Figure 9B) and His85 (Figure 9C), respectively. In addition, aCCA and aMCA could also interact with another 10 (Figure 9B) or 9 (Figure 9C) amino acid residues in tyrosinase. These interactions might enhance the inhibitory potency of derivatives on the enzyme.

Figure 7. UV−vis spectra for the oxidation of L-DOPA. Line 1 is 100 μM L-DOPA or compounds alone. Curves 2−5 in (A) represent 1−4 min after adding NaIO4; curves 2−5 in (B), (C), and (D) are different concentrations (9, 18, 27, and 36 μM) of aBCA, aCCA, and aMCA, respectively.



DISCUSSION This paper explained the relationship between the cinnamaldehyde derivatives and mushroom tyrosinase. For monophenolase activity, three derivatives could extend the lag time and inhibit the steady-state activity. Comparing the inhibitory effects of three derivatives, we found that aBCA was better than aCCA, and aMCA was weakest. With respect to diphenolase activity, the IC50 showed the same results with the inhibitory effects of monophenolase.

antityrosinase activities of the derivatives of cinnamaldehyde. The docking modes of aBCA, aCCA, and aMCA were examined in the enzyme catalytic site. The docked conformations revealed that all compounds have similar mechnism with mushroom tyrosinase (Figure 9). They could not form metal interactions with the copper irons of the 720

DOI: 10.1021/jf505469k J. Agric. Food Chem. 2015, 63, 716−722

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Journal of Agricultural and Food Chemistry

effectors and tyrosinase only affected the microenvironment in close proximity to the fluorophore of tyrosinase, as no other changes in the immediate surroundings of the quenched residues were observed.31 This mode was also observed by Mu et al.32 in fluorescence quenching between 4-methoxycinnamic acid and tyrosinase. Moreover, tricin30 and thiobarbituric acid33 showed similar quenching mechanisms. Compared with the values in Table 2, for aBCA, aCCA, and aMCA, the KSV increased in turn; meanwhile, the binding constant (KA) and the binding affinity (n) decreased in turn. All the trends of the changes declared that tyrosinase and aBCA had the strongest interaction while the interaction between aMCA and enzyme was the weakest. The results were consistent with enzyme assay. The results of molecular docking by MOE suggested that the inhibition on tyrosinase by three derivatives represented a different strategy for inactivating tyrosinase compared to simple copper chelators. They did not bind directly to the copper ions of the active site. An example of such inhibitor was 4methoxycinnamic acid, which can occupy the position of CuA to suppress the monophenolase activity.32 The scores for aBCA, aCCA, and aMCA docking with tyrosinase were −9.59, −9.35,l and −8.84 kcal/mol, respectively. The score meant that the docking between tyrosinase and all compounds was successful. The molecular docking showed that aMCA could bind residues within free enzyme whereas the inhibition type demonstrated that aMCA could not interact with enzyme directly. L-DOPA may reduce the affinity of aMCA and enzyme, and aMCA prefers to combine with the complex rather than free enzyme. Taken together, three derivatives of cinnamaldehyde could inhibit the acitvity of tyrosinase in two ways. First, the effectors combined with enzyme to affect the microenvironment in close proximity to the active site of tyrosinase. At the same time, alpha-substituent group enhanced the reciprocity between effectors and the residues within active site, which caused activity of tyrosinase to be restricted. The second way was enzyme, inhibitor, and the substrate to form ternary compound, and effectors could prevent the oxidative process of substrate, which brings down the formation of melanin. This was the other approach was to restrict the activity of tyrosinase. Among three derivatives, aBCA had the best inhibitory effect on tyrosinase. In addition, Lu et al.34 reported that aBCA was a low toxical compound, with slight nonspecific inflammatory irritation effects on the skin and eye. The minimun effective dose of subacute toxicity experiment (oral) was 369 mg/kg body weight. In conclusion, aBCA was a good tyrosinase inhibitor and could be widely used as insecticide, bactericide, and so on.

Figure 9. Binding mode of the ligands with tyrosinase residues. The receptor exposure differences were shown by the size and intensity of the turquoise discs surrounding the residues. (A), (B), and (C) represent aBCA, aCCA, and aMCA, respectively.

aBCA was the strongest inhibitor, and aMCA was the weakest. The inhibitions of three derivatives were reversible demonstrating that the effectors did not reduce the amount of the efficient enzyme, but just decreased the activity of tyrosinase.29 It was interesting that each inhibition type of three compounds was different and this phenomenon may result in the different inhibitory capacities for derivatives. Compared with cinnamaldehyde, whose IC50 of diphenolase was 0.98 mM,9 alphasubstituted derivatives showed better inhibitory effects. Due to the existence of alpha substituents, the terpenes on derivatives became unstable and easier to interfere with tyrosinase. Moreover, Xiao et al.30 reported that alpha halogeno group can enhance cinnamaldehyde antibacterial ability greatly. Perhaps the substituents hindered the activity of enzyme and thus restrained bacterial growth. Fluorescence quenching was used to investigate the interactions of ligands and proteins. The presented results showed that the tyrosinase fluorescence intensity decreased in the presence of derivatives; however, there was no apparent λem shift in the fluorescence emission spectra of tyrosinase with the addition of derivatives. Therefore, the interaction between



AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86-592-2184648. E-mail: [email protected] Funding

The present investigation was supported by the Natural Science Foundation of China (Grant No. 31271952 and 31371857), the Fundamental Research Funds for the Central Universities (Grant No. 20720140541), and the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant No. J1310027). 721

DOI: 10.1021/jf505469k J. Agric. Food Chem. 2015, 63, 716−722

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Journal of Agricultural and Food Chemistry Notes

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The authors declare no competing financial interest.



ABBREVIATIONS USED aBCA, alpha-bromocinnamaldehyde; aCCA, alpha-chlorocinnamaldehyde; aMCA, alpha-methylcinnamaldehyde; L-DOPA, L3, 4-dihydroxyphenylalanine; L-Tyr, L-tyrosine; DMSO, dimethylsulfoxide; Km, Michaelis−Menten constant; KI, equilibrium constant of the inhibitor combining with the free enzyme; KIS, equilibrium constant of the inhibitor combining with the enzyme−substrate complex



REFERENCES

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DOI: 10.1021/jf505469k J. Agric. Food Chem. 2015, 63, 716−722

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