Inhibitory effect of synthetic aromatic heterocycle thiosemicarbazone derivatives on mushroom tyrosinase: Insights from fluorescence, 1H NMR titration and molecular docking studies

Article abstract of DOI:10.1016/j.foodchem.2015.05.124

Three structurally similar aromatic heterocyclic compounds 2-thiophenecarboxaldehyde (a), 2-furaldehyde (b), 2-pyrrolecarboxaldehyde (c) were chosen and a series of their thiosemicarbazone derivatives(1a-3a, 1b-3b and 1c-3c) were synthesized to evaluate t

Full text of DOI:10.1016/j.foodchem.2015.05.124

Food Chemistry 190 (2016) 709–716
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Inhibitory effect of synthetic aromatic heterocycle thiosemicarbazone
derivatives on mushroom tyrosinase: Insights from fluorescence,
1H NMR titration and molecular docking studies
Juan Xie a,b, Huanhuan Dong a, Yanying Yu b, Shuwen Cao a,b,
⇑
a State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, PR China
b Department of Chemistry, Nanchang University, Nanchang 330031, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three structurally similar aromatic heterocyclic compounds 2-thiophenecarboxaldehyde (a),
2-furaldehyde (b), 2-pyrrolecarboxaldehyde (c) were chosen and a series of their thiosemicarbazone
derivatives(1a–3a, 1b–3b and 1c–3c) were synthesized to evaluate their biological activities as mush-
room tyrosinase inhibitors. The inhibitory effects of these compounds on tyrosinase were investigated
by using spectrofluorimetry, 1H NMR titration and molecular docking techniques. From the results of flu-
orescence spectrum and 1H NMR titration, it was found that forming complexes between the sulfur atom
from thiourea and copper ion of enzyme center may play a key role for inhibition activity. Moreover,
investigation of 1H NMR spectra further revealed that formation of hydrogen bond between inhibitor
and enzyme may be helpful to above complexes formation. The results were well coincident with the
suggestion of molecular docking and obviously showed that 2-thiophone N(4)-thiosemicarbazone (1a),
2-furfuran N(4)-thiosemicarbazone (1b) and 2-pyrrole N(4)-thiosemicarbazone (1c) are potential inhibi-
tors which deserves further investigation.
Received 12 January 2015
Received in revised form 24 May 2015
Accepted 29 May 2015
Available online 30 May 2015
Keywords:
Aromatic heterocyclic thiosemicarbazone
derivatives
Tyrosinase inhibitors
Fluorescence spectrum
1H NMR titration
Molecular docking
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Qiu, 2005), thus, preventing this unfavorable browning reaction
has always been a challenge in food science (Arung et al., 2005).
Tyrosinase (EC 1.14.18.1) is a kind of oxidoreductase with din-
uclear copper ions, belong to the type-3 copper protein family,
namely the two copper ions of tyrosinase active center linked
together with three specific histidine residues form a tetrahedron
structure (Li, Wang, Jiang, & Deng, 2009; Yoon, Fujii, & Solomon,
2006). As the rate-limiting enzyme, tyrosinase catalyzes two dis-
tinct reactions of melanin biosynthesis by hydroxylation of
monophenols to o-diphenols and oxidation of o-diphenols to the
corresponding o-quinones (Thanigaimalai et al., 2011), and plays
a key role in processes such as pigmentation in vertebrates and
unfavorable browning in food products (Chen, Huang, & Kubo,
2003). Melanin overproduction may cause hyperpigmentation dis-
eases, such as flecks in mammals (Xue, Luo, Ding, Liu, & Gao, 2008).
In the food industry, browning can cause deleterious changes in
the organoleptic properties of food products, which results in the
loss of quality in fruits and vegetables (Shi, Chen, Wang, Song, &
Accordingly, tyrosinase inhibitors have played important roles in
the fields of medicine, agriculture, food sciences and cosmetics.
Most of the recent research has focused on below three fields,
firstly, finding new naturally occurring inhibitors of tyrosinase
(Lin et al., 2011; Yang, Wang, Wang, & Zhang, 2012), secondly, pro-
ceeding structure modification or synthesis chemical analogs of
target inhibitors for better inhibition activity and lower side effects
(Chen et al., 2012; Wang, Huang, Chen, Chen, & Wang, 2011; Xu,
Yu, Wan, Wan, & Cao, 2014; Zhu, Cao, & Yu, 2013), third, investigat-
ing structure–activity and inhibitory mechanism of tyrosinase
inhibitors (Baek et al., 2012; Ha, Park, Kim et al., 2012; Ha, Park,
Lee et al., 2012).
As is known to all, research on structure–activity relationship
has a key role and important scientific significance in discovery
of drug and bioactive compounds. Recent researches of Sanghun
Jung research group (Lee et al., 2010; Thanigaimalai et al., 2012)
suggested that aromatic and naphthaldehyde thiosemicarbazones
may be a new kind of potential inhibitors of tyrosinase due to their
special structure containing sulfur atom in thiourea and p-planar
connection to thiourea unit without steric hindrance which was
identified as a main structural requirement. It is an obstacle for
⇑
Corresponding author at: State Key Laboratory of Food Science and Technology,
Nanchang University, Nanchang 330047, PR China.
E-mail addresses: 15870622557@163.com (J. Xie), donghuanhuan@outlook.com
(H. Dong), yuyanying@ncu.edu.cn (Y. Yu), caosw@ncu.edu.cn (S. Cao).
http://dx.doi.org/10.1016/j.foodchem.2015.05.124
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

710
J. Xie et al. / Food Chemistry 190 (2016) 709–716
its further application due to its lack of a firm assistance from
visual and direct experimental data in spite of these research
results do have certain theoretical guiding significance in new inhi-
bitor discovery of tyrosinase. Obviously, there is a developing need
for understanding how to affect each other between inhibitor and
tyrosinase by means of reliable and direct experiments.
1H NMR (600 MHz, DMSO) d 11.46 (s, 1H, NH), 8.24 (s, 1H, CH),
8.15 (d, J = 4.3 Hz, 1H, C-NH), 7.65 (d, J = 5.0 Hz, 1H,
thiophene-H), 7.43 (d, J = 3.5 Hz, 1H, thiophene-H), 7.11 (dd,
J = 4.8, 3.8 Hz, 1H, thiophene-H), 3.00 (d, J = 4.6 Hz, 3H, CH3); MS
(ESI): m/z 200 [MꢀH]+.
2-Thiophone N(4)-phenylthiosemicarbazone (3a): Yellow crys-
tals, yield 89.2%; mp: 168–171 °C; IR (KBr) 3141, 1547,
The fluorescence quenching titration is so far a valuable and
reliable method to explore interaction of inhibitor and tyrosinase
(Wang, Zhang, Yan, & Gong, 2014; Zhu et al., 2013). Similarly, the
interaction between inhibitor and tyrosinase may be received
more directly by investigating the variation of 1H NMR spectrum
of inhibitor hydrogen under gradual titration of tyrosinase. By
combinating results of 1H NMR titration and molecular docking,
it was understood clearly how inhibitor and tyrosinase affect each
other by forming complex and hydrogen bond between inhibitor
and enzyme, and many information of three dimension space
was received in the same time. Aromatic heterocyclic compounds
possess structural similarity to aromatic and naphthaldehyde,
but until present, we have very few knowledge on the inhibition
activity and mechanism of aromatic heterocyclic thiosemicar-
bazones on tyrosinase. Based on this, three structurally similar aro-
matic heterocyclic compounds, 2-thiophenecarboxaldehyde (a),
2-furaldehyde (b), 2-pyrrolecarboxaldehyde (c) were chosen and
their thiosemicarbazone derivatives (1a–3a, 1b–3b, 1c–3c) were
synthesized and their inhibitory effect and mechanism were inves-
tigated by fluorescence spectrum, 1H NMR titration and molecular
docking.
706 cmꢀ1 1H NMR (600 MHz, DMSO-d6) d 11.81 (s, 1H, NH), 9.79
;
(s, 1H, C-NH), 8.35 (s, 1H, CH), 7.70 (d, J = 5.0 Hz, 1H,
thiophene-H), 7.57 (d, J = 7.5 Hz, 2H, Ph-H), 7.55–7.52 (m, 1H,
thiophene-H), 7.40–7.33 (m, 2H, Ph-H), 7.20 (d, J = 7.4 Hz, 1H,
Ph-H), 7.14 (dd, J = 5.0, 3.6 Hz, 1H, thiophene-H); MS (ESI): m/z
262 [MꢀH]+.
2-Furfuran N(4)-thiosemicarbazone (1b): yellow crystals, yield
96.3%; mp: 147–149 °C; IR (KBr) 3140, 1525, 762 cmꢀ1 1H NMR
;
(600 MHz, DMSO-d6) d 11.41 (s, 1H, NH), 7.97 (s, 1H, CH), 8.19,
7.61 (s, 1H, NH2), 7.81 (d, J = 1.1 Hz, 1H, furfuran-H), 6.97 (d,
J = 3.4 Hz, 1H, furfuran-H), 6.62 (dd, J = 3.4, 1.7 Hz, 1H,
furfuran-H); MS (ESI): m/z 170 [MꢀH]+.
2-Furfuran N(4)-methylthiosemicarbazone (2b): yellow crys-
tals, yield 71.5%; mp: 152–154 °C; IR (KBr) 3158, 1529,
752 cmꢀ1 1H NMR (600 MHz, DMSO-d6) d 11.43 (s, 1H, NH), 8.20
;
(s, 1H, C-NH), 7.96 (d, J = 4.4 Hz, 1H, CH), 7.81 (d, J = 1.2 Hz, 1H,
furfuran-H), 6.93 (d, J = 3.4 Hz, 1H, furfuran-H), 6.63 (dd, J = 3.4,
1.8 Hz, 1H, furfuran-H), 2.99 (d, J = 4.6 Hz, 3H, CH3); MS (ESI):
m/z 184[MꢀH]+.
2-Furfuran N(4)-phenylthiosemicarbazone (3b): yellow crys-
tals, yield 87.0%; mp: 175–177 °C; IR (KBr) 3134, 1539,
739 cmꢀ1 1H NMR (600 MHz, DMSO-d6) d 11.83 (s, 1H, NH), 9.86
;
2. Materials and methods
(s, 1H, NH), 8.08 (s, 1H, CH), 7.86 (d, J = 0.8 Hz, 1H, furfuran-H),
7.59 (d, J = 7.9 Hz, 2H, Ph-H), 7.36 (t, J = 7.8 Hz, 2H, Ph-H), 7.19 (t,
J = 7.4 Hz, 1H, Ph-H), 7.09 (d, J = 3.4 Hz, 1H, furfuran-H), 6.66 (dd,
J = 3.3, 1.7 Hz, 1H, furfuran-H); MS (ESI): m/z 246 [MꢀH]+.
2-Pyrrole N(4)-thiosemicarbazone (1c): purple crystals, yield
2.1. Chemicals and reagents
Mushroom tyrosinase (EC 1.14.18.1) and L-3,4-dihydroxy
phenyl-alanine (L-DOPA) were purchased from Sigma (St. Louis,
MO, USA). 2-thiophenecarboxaldehyde (a), 2-furaldehyde (b),
2-pyrrolecarboxaldehyde (c) were obtained from J&K Chemical
Co (Shanghai, China). All other reagents were local and of analytical
grade. The water used was re-distilled and ion-free.
71.2%; mp: 183–186 °C; IR (KBr) 3156, 1589, 740 cmꢀ1 1H NMR
;
(600 MHz, DMSO-d6) d 11.35 (s, 1H, pyrrole-N-H), 11.26 (s, 1H,
NH), 8.07, 7.95 (s, 1H, NH2), 7.83 (d, J = 2.4 Hz, 1H, CH), 6.98 (d,
J = 1.2 Hz, 1H, pyrrole-H), 6.42–6.37 (m, 1H, pyrrole-H), 6.10 (dd,
J = 5.8, 2.5 Hz, 1H, pyrrole-H); MS (ESI): m/z 169[MꢀH]+.
2-Pyrrole N(4)-methylthiosemicarbazone (2c): purple crystals,
2.2. Synthesis
yield 67.8%; mp: 186–188 °C; IR (KBr) 3146, 1556, 741 cmꢀ1 1H
;
NMR (600 MHz, DMSO-d6) d 11.31 (s, 1H, NH), 11.30 (s, 1H,
pyrrole-N-H), 8.44 (d, J = 4.5 Hz, 1H, NH), 7.83 (s, 1H, CH), 7.01 (s,
1H, pyrrole-H), 6.40 (s, 1H, pyrrole-H), 6.11 (dd, J = 5.7, 2.6 Hz,
1H, pyrrole-H), 3.03 (d, J = 4.6 Hz, 3H, CH3); MS (ESI): m/z 183
[MꢀH]+.
The synthesis pathways are described in Fig. 1, as reference (Li,
Zhang, Zhang, & Niu, 2010) described. The products were purified
by recrystallization from ethanol detected by HPLC as one peak
and identified by ESI–MS and 1H NMR analyses. ESI–MS data were
obtained on a Bruker ESQUIRE-LC (Germany), and NMR data were
acquired on a 600 MHz NMR spectrometer (AV400) from Bruker
(Germany).
2-Pyrrole N(4)-phenylthiosemicarbazone (3c): purple crystals,
yield 63.9%; mp: 170–172 °C; IR (KBr) 3141, 1535, 729 cmꢀ1;1H
NMR (600 MHz, DMSO-d6) d 11.67 (s, 1H, NH), 11.54 (s, 1H,
pyrrole-N-H), 10.03 (s, 1H, NH), 7.95 (s, 1H, CH), 7.60 (d,
J = 7.8 Hz, 2H, Ph-H), 7.39 (t, J = 7.7 Hz, 2H, Ph-H), 7.21 (t,
J = 7.3 Hz, 1H, Ph-H), 7.05 (s, 1H, pyrrole-H), 6.48 (s, 1H,
pyrrole-H), 6.14 (dd, J = 5.4, 2.7 Hz, 1H, pyrrole-H); MS (ESI): m/z
245 [MꢀH]+.
2-Thiophone N(4)-thiosemicarbazone (1a): yellow crystals,
yield 72.0%; mp: 178–180 °C; IR(KBr) 3148, 1537, 711 cmꢀ1 1H
;
NMR (600 MHz, DMSO-d6) d 11.44 (s, 1H, NH), 8.22 (s, 1H, CH),
8.17, 7.52 (d, J = 14.4 Hz, 2H, NH2), 7.65 (d, J = 5.0 Hz, 1H,
thiophene-H), 7.46–7.42 (m, 1H, thiophene-H), 7.10 (dd, J = 5.0,
3.6 Hz, 1H, thiophene-H); MS (ESI): m/z 186 [MꢀH]+.
2-Thiophone N(4)-methylthiosemicarbazone (2a): yellow crys-
tals, yield 89.3%; mp: 156–159 °C; IR (KBr) 3171, 1547, 711 cmꢀ1
;
2.3. Enzyme activity assay
The assay of the enzyme activity was performed as described
previously with minor modification (Huang et al., 2006).
was used as substrate for the enzyme activity assay. The reaction
media (3 mL) for activity assay contained 2.8 mL 0.5 mM -DOPA
L-DOPA
L
in 50 mM Na2HPO4-NaH2PO4 buffer (pH 6.8) and 0.1 mL of differ-
ent concentrations of inhibitor (dissolved in DMSO previously).
0.1 mL of the aqueous solution of mushroom tyrosinase was added
to the mixture. The solution was immediately monitored by mea-
suring the linear increase in optical density at 475 nm of formation
Fig. 1. Synthesis of aromatic heterocyclic thiosemicarbazone derivatives 1a–1c, 2a–
2c and 3a–3c. Reagents and condition: (i) methanol, reflux, 3–5 h.

J. Xie et al. / Food Chemistry 190 (2016) 709–716
711
of the DOPA chrome for 150 s using a Shimadzu UV-2450 spec-
trophotometer (Japan). The extent of inhibition by the addition of
the sample was expressed as the percentage necessary for 50%
inhibition (IC50), calculated by SPSS19. The value of relative enzy-
matic activity can be calculated by the following equation:
binding constant (Ka) and the number of binding sites (n) can be
calculated by the following equation (Shahabadi, Maghsudi, &
Rouhani, 2012):
F0 ꢀ F
log
¼ log Ka þ n log½Qꢂ
F
Relative enzymatic activity ð%Þ ¼ OD1=OD2 ꢁ 100%
2.6. Molecular docking study
where OD1 is the slope of reaction kinetics equation obtained from
reaction with inhibitor; OD2 is the slope of reaction kinetics equa-
tion obtained from reaction with reagent blank. Kojic acid was used
as a reference standard inhibitor for comparison.
Molecular docking is an application wherein molecular model-
ing techniques are used to predict how a protein (enzyme) inter-
acts with small molecules (ligands) (Fradera, Knegtel, & Mestres,
2000; Gopalakrishnan, Aparna, Jeevan, Ravi, & Desiraju, 2005;
Perola, 2006). The active site was docked with inhibitors using
the dock suite of Accelrys Discovery Studio 2.0 software
(Accelrys, Inc.). The 3D structure of compound 1a was generated
in Chem3D Ultra 8.0, and the X-ray crystal structure of Agaricus
bisporus tyrosinase (PDB ID: 2Y9X) was retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb) (Hu et al., 2012).
All crystallographic water molecules, solvent molecules and ions
were removed from the protein structure. The forcefield
CHARMm function was used to roughly search the conformations
when compounds docked into tyrosinase, and then the modified
tyrosinase was selected as the binding site for the study and
expanded to make the cavity. Subsequently, the conformations of
compounds were optimized using the same forcefield function, fol-
lowed by flexibly being docked in a stepwise manner with the pro-
tocol of Dock ligands. The docked conformation, which had the
highest score, was selected to analyze the mode of binding.
2.4. Determination of the inhibition mechanism and the inhibition type
Inhibitory mechanism assay was assayed by maintaining the
concentration of L-DOPA and changing the concentration of the
enzyme in reaction medium. The enzyme activity was measured
for different concentrations of inhibitor.
The inhibition type was assayed by the Lineweaver–Burk plot,
the equation can be written as:
ꢀ
ꢁ
ꢀ
ꢁ
1
v
Km
Vm
½Iꢂ
1
Vm
½Iꢂ
¼
1 þ
þ
1 þ
KI
KIS
where
v is the reaction velocity; Km is the Michaelis constant; Vm is
the maximal velocity; [I] is the concentration of inhibitor; [S] is the
concentration of substrate; KI is the constants for the inhibitor bind-
ing with the free enzyme and KIS is the constants for the inhibitor
binding with enzyme substrate complex, They were obtained from
the slope or the vertical intercept versus the inhibitor concentra-
tion, respectively.
2.7. 1H NMR titration
2.5. Fluorescence quenching titration
1H NMR titration studies were performed to investigate the
interaction between the compounds 1a–3a and tyrosinase. A
0.6 mL 15 mM solution of compounds 1a–3a in DMSO-d6 was pre-
pared and titrated with tyrosinase solution by using a micropipette
The experiment was performed with reference (Wang et al.,
2014). A 2.0 mL solution containing 5.0 lg/mL tyrosinase was
added to the quartz cuvette, then titrated by successive addition
of compound 1a, compound 1b, compound 1c and 1a-Cu2+ com-
plex (the constituent ratio of ligant and Cu2+ was 1.5:1, the chela-
tion ratio has been determined by continuous variation method)
(Karikari, Mather, & Long, 2007) solution using a pipette (to final
(to final concentrations ranging from 0.17 to 0.67 mg/lL). After
each addition of tyrosinase, the 1H NMR spectrum was recorded
and the changes in the chemical shift of the protons were noted.
3. Results and discussion
concentrations ranging from 0 to 60 lM). Fluorescence intensities
were recorded using a Hitachi F-4600 spectrofluorophotometer
(Japan) at three different temperatures (25, 30 and 35 °C) with
an excitation wavelength of 280 nm and excitation and emission
slit widths of 5 nm.
In this study, all of the fluorescence intensities were corrected
for the absorption of exciting light and reabsorption of emitted
light. The following formula was used to correct the inner-filter
effect (Bi, Yan, Wang, Pang, & Wang, 2012):
3.1. Effect and Inhibition mechanism of thiosemicarbazone derivatives
on the diphenolase activity of mushroom tyrosinase
The IC50 values of the 12 compounds a–3a, b–3b and c–3c for
comparison were listed in Table 1, kojic acid as comparison control.
As shown in Table 1, when thiosemicarbazide group was intro-
duced at the aldehyde side of index compounds, noticeable activity
enhancement was presented on mushroom tyrosinase. However, a
hydrogen atom of N-terminal on thiourea groups replaced by
methyl or phenyl, the inhibitory activity of compounds 2a, 2b,
2c, 3a, 3b and 3c on tyrosinase was significantly reduced with
1þ A2Þ=2
Fcorr ¼ FobseðA
Fcorr and Fobs are the corrected and observed fluorescence inten-
sities, respectively. A1 and A2 are the absorbances of inhibitor at the
excitation and emission wavelengths, respectively.
IC50 > 50 lM. More interestingly, although compound 1a, com-
pound 1b and compound 1c have the same thiosemicarbazide resi-
dues, but their inhibitory activity was much different, which may
explained by different vicinal effects induced by the hetero atoms
from hetrocyclic ring. The inhibitory activity of compound 1a
Fluorescence quenching was described by the Stern–Volmer
equation (Eftink & Ghiron, 1981):
F0
F
¼ 1 þ Kqs0½Qꢂ ¼ 1 þ KSV½Qꢂ
(IC50 = 0.43
was strongest, followed by compound 1b (IC50 = 2.35
oxygen hetero atom in the heterocyclic ring, compound 1c
(IC50 = 3.99 M) with nitrogen hetero atom in the heterocyclic ring.
lM) with sulfur hetero atom in the heterocyclic ring
where F0 and F are the fluorescence intensities before and after the
addition of the quencher, respectively, Kq is the bimolecular
quenching constant, s0 (10ꢀ8 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.
For the static quenching interaction, if it is assumed that there
are similar and independent sites in the biomolecule, the apparent
l
M) with
l
These might have been due to three hetero atoms have different
vicinal effects on the forming of complexes between sulfur atoms
from thiourea of inhibitor and the dicopper nucleus in the active
site of tyrosinase. From knowlege of basic organic chemistry, we

712
J. Xie et al. / Food Chemistry 190 (2016) 709–716
Table 1
Structure and inhibitory activity of compounds a–3a, b–3b and c–3c on mushroom tyrosinase.
Compound
R1
R2
IC50
(lM)
Inhibition mechanism
Inhibition type
Inhibition constants
KI (lM)
KIS (lM)
a
1a
2a
3a
b
1b
2b
3b
c
1c
2c
3c
S
S
S
S
O
O
O
O
NH
NH
NH
NH
–
–
H
Me
Ph
–
628.26 219.74
0.43 0.11
174.07 20.97
55.48 18.62
1163.03 266.90
2.35 0.80
162.56 52.51
311.44 34.09
3202.13 703.31
3.99 1.27
344.83 42.40
94.23 20.74
17.94 3.75
–
–
Mix
–
–
–
Mix
–
–
–
Mix
–
–
0.08
–
–
–
0.60
–
–
–
1.67
–
–
0.29
–
–
–
1.18
–
–
–
Reversible
–
–
–
Reversible
–
–
–
H
Me
Ph
–
H
Reversible
2.59
–
–
Me
Ph
–
–
–
–
–
–
–
–
Kojic acid
–
Results are presented as the mean (n = 3) standard deviation (SD).
Fig. 2. Fluorescence spectra of tyrosinase in the presence of compound 1a, 1b, 1c and 1a–Cu2+ complex at 25 °C. Concentrations for curves were 0, 6, 12, 18, 24, 30, 36, 42, 48,
54 and 60
M, respectively. The inset depicts Stern–Volmer plots for the fluorescence quenching of tyrosinase by compound 1a, 1b, 1c at 25, 30 and 35 °C and 1a–Cu2+
l
complex at 25 °C.
know that oxygen, nitrogen and sulfur possess both inductive
effect, O (3.5) > N (3.0) > S (2.6), and conjugative effect, N > O > S,
and the final results should be the balance of these different effects
in their vicinal effects on the forming of complex, where S pos-
sesses 3p occupied orbital easy overlapping with and contributing
electrons to the 3d vacant orbital of Cu2+ from tyrosinase active
center, and weak inductive effect compared to other two atoms
of N and O, so it should have better vicinal effect. As to N and O
heterocycles, if just the inductive effects were considered, the vic-
inal effect of N heterocycle should be better than that of O hetero-
cycle due to the former possesses weak inductive effect, but the
truth is opposite. This could be explained by that the final vicinal
effects should be the balance of inductive and conjugative effects
of these two compounds where O atom has better conjugative

J. Xie et al. / Food Chemistry 190 (2016) 709–716
713
Table 2
The Stern–Volmer quenching constants KSV, the quenching rate constants Kq and the binding constant Ka at different temperatures.
Compound
Temperature (°C)
KSV (103 L molꢀ1
)
Kq (1011 L molꢀ1 sꢀ1
)
Ka (103 L molꢀ1 sꢀ1
)
1a
25
30
35
25
30
35
25
30
35
25
9.06
8.86
8.79
8.69
8.51
7.31
8.40
8.05
6.88
7.03
9.06
8.86
8.79
8.69
8.51
7.31
8.40
8.05
6.88
7.03
1.63
1.35
1.03
0.73
0.71
0.45
0.58
0.47
0.31
0.34
1b
1c
1a–Cu2+
Fig. 3. (A) 2D structure for the interaction of compound 1a with tyrosinase. Green dotted lines denoted metal interactions between the ligands and the copper irons at the
catalytic center of the tyrosinase. (B) Tertiary structure for the interaction of compound 1a with tyrosinase residues. (C) Tertiary structure for the interaction of compound 2a
with tyrosinase residues. (D) Tertiary structure for the interaction of compound 3a with tyrosinase residues. The compound was stick in the center with green color. The
orange spheres indicated the copper atoms (CU400, CU401). The dashed green line depicts the corresponding hydrogenbonding. The brow color represents hydrophobicity
and blue color represents hydrophily. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
effect compared to N atom which surpasses the inductive effect of
them (Xing, Pei, Xu, & Pei, 2005). Therefore, the compound 1a has
the strongest inhibiting ability, follow by compound 1b and com-
pound 1c.
KIS were obtained from the Lineweaver–Burk plot and their values
were listed in Table 1.
3.2. Spectrofluorimetry studies
Moreover, the inhibition mechanism by compounds 1a–1c
against the tyrosinase for the oxidation of L-DOPA was studied.
The relationship between the enzyme activity and the concentra-
tion of inhibitors gave a family of straight lines passed through
the origin (Fig. S1), indicating that the inhibition was reversible.
The inhibitory kinetics of mushroom tyrosinase by compounds
1a–1c had been studied. The double-reciprocal plots yielded a
family of straight lines intersected at the second quadrant
(Fig. S2), indicating that they were mixed-type inhibitors. KI and
The technique of fluorescence quenching has generally been
applied to investigate the interactions of ligands and proteins
(Mu, Li, & Hu, 2013). To assess the interaction of compounds
1a–1c with tyrosinase, the fluorescence emission spectra of the
tyrosinase in the absence and presence of compounds 1a–1c
excited at 280 nm are shown in Fig. 2. The concentration increase
of compounds 1a–1c led to a dramatic decrease in the intensity
of fluorescence maximum emission bond at 369 nm, indicating

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J. Xie et al. / Food Chemistry 190 (2016) 709–716
hydrogen bonding dominated, then a significant decrease in com-
plex formation and KSV would be expected due to weakening of
hydrogen bridges at higher temperatures (Joye, Davidov-Pardo,
Ludescher, & McClements, 2015).
The obtained KSV values seemed to be weakly temperature
-dependent and of little decrease instead of significant decrease
or increase, which indicated that the change of complex formation
between tyrosinase and inhibitors is weakly dependent on temper-
ature under tested temperature. The fact may be explained by
speculating that there is a kind of dominated and much stronger
complex interaction existing stably under tested temperatures
besides hydrophobic interactions and hydrogen bridges between
tyrosinase and inhibitor, which was corroborated in subsequent
experiment.
The static quenching binding constants (Ka) of compounds
1a–1c were calculated to be 1.63 ꢁ 103, 0.73 ꢁ 103 and
0.58 ꢁ 103 L molꢀ1 at 25 °C, respectively, which showed that the
interaction of compound 1a with tyrosinase was the strongest, fol-
lowed by the compound 1b, compound 1c. These results were con-
sistent with inhibitory activity of tyrosinase. In order to further
verify inhibitor and enzyme acting site associated with copper,
the fluorescence titration of tyrosinase with 1a–Cu2+ complex
was processed. As depicted Fig. 2D, the concentration increase of
1a–Cu2+ complex led to a weaker decrease in the intensity of emis-
sion band at 369 nm compared with compound 1a. The static
quenching binding constant (Ka) of 1a–Cu2+ complex was
0.34 ꢁ 103 L molꢀ1 at 25 °C. The result showed that copper ion
might occupy inhibitor activity site after inhibitor and copper coor-
dination, so that inhibition ability of inhibitor to Cu2+ of tyrosinase
was attenuated.
3.3. Molecular docking study
In order to understand the right site and interaction forces of
inhibitor binding to tyrosinase, the molecular docking of com-
pound 1a–3a to tyrosinase was carried out using AAccelrys
Discovery Studio 2.0 software. The dominating configuration of
the binding complexes of tyrosinase with compound 1a–3a, which
were the lowest binding free energy (ꢀ19.46, ꢀ19.29,
ꢀ19.47 kcal molꢀ1, respectively), was shown in Fig. 3.
Fig. 4. (A) 1H NMR titration of compound 1a, 2a and 3a (15 mM) with tyrosinase in
From Fig. 3A, it was revealed that as a ligand, the sulfur atom of
thiourea group could coordinate to the dicopper ions of the
enzyme active center. Moreover, it was found that a hydrophobic-
ity zone predicted by the software (Fig. 3A) was formed by the fol-
lowing residues: Ala 286, Asn 260 and His 244. All of these were
well coincident with the results of flurescence quenching
experiment(Chen et al., 2013). From Fig. 3B–D, a hydrogen bonding
interaction between the terminal amino proton of compound 1a
and tyrosinase active-site residue His 244 was found, whereas,
no direct hydrogen bonding interactions between the compounds
2a and compound 3a and tyrosinase, which was coincident with
the latter experiment result of 1H NMR titration.
DMSO-d6 solvent. Concentrations of tyrosinase for curves 0–4 were 0, 0.17, 0.33, 0.5
and 0.67 mg/lL, respectively. (B) Plausible binding mode of compound 1a with
tyrosinase.
compounds 1a–1c react with tyrosinase and quench its intrinsic
fluorescence.
The values of KSV and Kq based on the Stern–Volmer plots and Ka
based on equation used by Shahabadi et al. were got, listed in
Table 2. It was found that the every value of Kq at different temper-
atures was much greater than the maximum quenching constant of
scatter collision of various quenchers with biopolymers,
2.0 ꢁ 1010 L molꢀ1 sꢀ1 (Lakowicz, 2009). Thus, it was definitely
believed that static quenching was dominant in the inhibitor–
tyrosinase interaction.
3.4. 1H NMR titration studies
1H NMR titration is a useful method to identify interaction
between different molecules by determining chemical shift of
hydrogen atoms(Gromov et al., 2014; Liu et al., 2015). The interac-
tions between compounds 1a–3a and tyrosinase were further
observed from 1H NMR titrations in DMSO-d6. The titrations were
carried out by addition of tyrosinase to inhibitor in different con-
centrations and their NMR plots (Fig. 4A) were collected. Upon
Another strategy to investigate the nature of the quenching of
inhibitors on tyrosinase fluorescence is studying the temperature
dependence of the quenching phenomenon. If static quenching
was prevalent by complex formation between the tyrosinase and
inhibitor, a less straightforward relation between KSV and temper-
ature would be found as, depending on the forces involved in the
interaction, an increase in temperature would increase or decrease
complex formation between tyrosinase and inhibitor. For example,
if hydrophobic interactions dominated, then an increase in com-
plex formation and KSV would be expected due to strengthening
of hydrophobic forces at elevated temperatures. Conversely, if
the addition of 0.5 mg/lL of tyrosinase to compound 1a or 2a or
3a, the peaks at 11.4 ppm (of –CSNH–, Hb) almost completely dis-
appeared. The disappearance of –CSNH– proton may be given rise
by the generation of tautomer from proton transfer between amino

J. Xie et al. / Food Chemistry 190 (2016) 709–716
715
Chen, Q. X., Huang, H.,
&
Kubo, I. (2003). Inactivation kinetics of mushroom
and thiol, and then sulfur atom from the thiosemicarbazide group
donated hydrogen and formed complex with enzymic Cu2+. The
peaks at 8.17 ppm (amino proton, –NH2, Hc’) gradually shifted
upfield for compound 1a, which indicated that the increase of
the electron density on amino proton due to formation of hydrogen
bond between –NH2 group proton and tyrosinase. However, the
peaks at 8.15 ppm for compound 2a and 9.79 ppm for compound
3a (amino proton, –NH–, Hc’) almost be no shift, which suggested
no formation of hydrogen bond between –NH– group proton and
tyrosinase due to stereo-hindrance of methyl or phenyl. Similar
changes were identified in case of molecular docking study. It is
believed that formation of hydrogen between inhibitor and
enzyme will make the sulfur and copper coordination more stable,
which enhances the inhibitory ability of compound 1a. Based on
the above investigation results, a plausible binding mode of com-
pound 1a with the tyrosinase has been proposed as given in Fig. 4B.
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Various methods were used to investigate the interaction of
inhibitor (the aromatic heterocyclic thiosemicarbazone) and
tyrosinase in this research. Fluorescence quenching studies
revealed that inhibitor binds to tyrosinase with a static process
and forms complexes with the copper ions at the active site of
tyrosinase with which inhibitor exerts its inhibiting activity.
Furthermore, the binding site of inhibitor-tyrosinase complexes
between sulfur atom of thiourea group in inhibitor and dicopper
ions of the enzyme active center, and formation of hydrogen bind
between hydrogen atom of –NH2 group in thiourea moiety of inhi-
bitor and tyrosinase amino acid residue His 244, were successfully
predicted by molecular docking and further confirmed by 1H NMR
titration, by which the inhibitory ability of inhibitor was effectively
enhanced. The results suggest that fluorescence quenching and 1H
NMR titration are firm and relatively direct experiment methods to
investigate and understand the interaction between inhibitor and
enzyme, and the aromatic heterocyclic thiosemicarbazone may
be a potential novel class of tyrosinase inhibitor which deserves
further research.
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Acknowledgments
This work was supported by National Natural Sciences
Foundation of China (No. 20962014) and the free searching foun-
dation of State Key Laboratory of Food Science and Technology
(Nanchang University) in China (SKLF-TS-200914).
Perola, E. (2006). Minimizing false positives in kinase virtual screens. Proteins:
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Appendix A. Supplementary data
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colourant quinoline yellow with bovine serum albumin by spectroscopic
techniques. Food Chemistry, 135, 1836–1841.
Shi, Y., Chen, Q. X., Wang, Q., Song, K. K., & Qiu, L. (2005). Inhibitory effects of
cinnamic acid and its derivatives on the diphenolase activity of mushroom
(Agaricus bisporus) tyrosinase. Food Chemistry, 92, 707–712.
Thanigaimalai, P., Lee, K. C., Sharma, V. K., Joo, C., Cho, W. J., Roh, E., et al. (2011).
Structural requirement of phenylthiourea analogs for their inhibitory activity of
melanogenesis and tyrosinase. Bioorganic and Medicinal Chemistry Letters, 21,
6824–6828.
Thanigaimalai, P., Rao, E. V., Lee, K. C., Sharma, V. K., Roh, E., Kim, Y., et al. (2012).
Structure-activity relationship of naphthaldehydethiosemicarbazones in
melanogenesis inhibition. Bioorganic and Medicinal Chemistry Letters, 22,
886–889.
Wang, Y. J., Zhang, G. W., Yan, J. K., & Gong, D. M. (2014). Inhibitory effect of morin
on tyrosinase: insights from spectroscopic and molecular docking studies. Food
Chemistry, 163, 226–233.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
05.124.
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