A new chiral ligand exchange capillary electrophoresis system based on Zn(ii)-l-leucine complexes coordinating with β-cyclodextrin and its application in screening tyrosinase inhibitors

Article abstract of DOI:10.1039/c4ra09433f

Tyrosinase plays a key role in melanin formation, and it is closely related to hyper pigmentation in animals and enzymatic browning in food. Thus, it is of great significance to screen inhibitors of tyrosinase. In this work, a new chiral ligand exchange-capillary electrophoresis (CLE-CE) system based on the coordination effect of Zn(ii)-l-leucine complexes and β-cyclodextrin (β-CD) was developed for screening the inhibitors of tyrosinase. The effects of the concentration of β-CD, buffer pH, the ratio of l-leucine to Zn(ii), and the complex concentration were investigated with Dns-d,l-tyrosine, Dns-d,l-valine and Dns-d,l-phenylalanine as the tested analytes. The optimum buffer conditions were composed of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(ii), 6.0 mM l-leucine and 4.0 mM β-CD at pH 8.2. It has been found that six pairs of Dns-d,l-AAs could be baseline-separated and five pairs of Dns-d,l-AAs were partly enantioseparated. Then the quantitative analysis of l-tyrosine was conducted and good linearity (r² = 0.992) was obtained with a concentration ranging from 12.95 μM to 413.3 μM. A kinetics study of tyrosinase was realized, and the K_m and V_max were 636 μM and 312 μmol min^-1 mg^-1. Moreover, the proposed method was applied in screening the inhibitors of tyrosinase with four kinds of chalcones as the model inhibitors. The results demonstrated that the developed CLE-CE system was favorable for screening enzyme inhibitors, and showed great potential in related drugs discovery and clinical analysis in the future.

Full text of DOI:10.1039/c4ra09433f

RSC Advances
View Article Online
View Journal | View Issue
PAPER
A new chiral ligand exchange capillary
electrophoresis system based on Zn(II)–L-leucine
complexes coordinating with b-cyclodextrin and its
application in screening tyrosinase inhibitors†
a
Cite this: RSC Adv., 2014, 4, 55280
Yuan Su,ab Xiaoyu Muac and Li Qi
*
Tyrosinase plays a key role in melanin formation, and it is closely related to hyper pigmentation in animals
and enzymatic browning in food. Thus, it is of great significance to screen inhibitors of tyrosinase. In this
work, a new chiral ligand exchange-capillary electrophoresis (CLE-CE) system based on the coordination
effect of Zn(II)–L-leucine complexes and b-cyclodextrin (b-CD) was developed for screening the
inhibitors of tyrosinase. The effects of the concentration of b-CD, buffer pH, the ratio of L-leucine to
Zn(II), and the complex concentration were investigated with Dns-D,L-tyrosine, Dns-D,L-valine and Dns-
D,L-phenylalanine as the tested analytes. The optimum buffer conditions were composed of 100.0 mM
boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(II), 6.0 mM L-leucine and 4.0 mM b-CD at pH 8.2. It
has been found that six pairs of Dns-D,L-AAs could be baseline-separated and five pairs of Dns-D,L-AAs
were partly enantioseparated. Then the quantitative analysis of L-tyrosine was conducted and good
linearity (r2 ¼ 0.992) was obtained with a concentration ranging from 12.95 mM to 413.3 mM. A kinetics
study of tyrosinase was realized, and the Km and Vmax were 636 mM and 312 mmol minꢀ1 mgꢀ1
.
Received 29th August 2014
Accepted 17th October 2014
Moreover, the proposed method was applied in screening the inhibitors of tyrosinase with four kinds of
chalcones as the model inhibitors. The results demonstrated that the developed CLE-CE system was
favorable for screening enzyme inhibitors, and showed great potential in related drugs discovery and
clinical analysis in the future.
DOI: 10.1039/c4ra09433f
www.rsc.org/advances
in clinical treatment, cosmetic industry and food industry.7,8
Both natural and synthetic compounds have been explored as
1. Introduction
the effective inhibitors for tyrosinase, such as kojic acid, ben-
zoic acid, hydroquinone and chalcones.9–12 Kojic acid has been
widely used as the depigmenting agents in cosmetic and food
industry. Meanwhile, chalcones and its derivatives, which are
mainly located in plants, have displayed their great inhibition
effects on tyrosinase.13–15 In the past decades, many protocols
have been developed for screening the inhibitors of tyrosinase,
such as UV spectrophotometry, HPLC, GC-MS and electropho-
retically mediated microanalysis (EMMA).16–18 However, some of
the methods still possessed some disadvantages, such as
complicated sample processing, high sample consumption, and
being susceptible to interference.15 Therefore, exploring new
methods for efficiently screening the inhibitor of tyrosinase is
pressing.
In recent years, capillary electrophoresis (CE) is turned out to
be an available technique for chiral separation in life science,
and this technique has various advantages, including high
efficiency, high speed and low cost.19–22 Among the diverse
separation modes in CE, chiral ligand exchange capillary elec-
trophoresis (CLE-CE) has been widely applied in enantiosepa-
ration of amino acids (AAs) for its obvious advantages, such as
As a polyphenol oxidase, tyrosinase is a copper-containing
enzyme and it is closely related to two reactions in the
synthetic process of melanin: the hydroxylation of mono-
phenols and the oxidation of o-diphenols to o-quinones.1–5 The
excessive accumulation of melanin would lead to both some
skin diseases (such as chloasma, age spots, and sunburn) and a
change in the apparent and sensory properties of food prod-
ucts.6 It is reported that the formation of the melanin could be
controlled by the introduction of tyrosinase inhibitors.6 There-
fore, it is signicant and highly desirable to screen the effective
inhibitors of tyrosinase. Based on the previous studies, the
inhibitors of tyrosinase are becoming more and more important
aBeijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical
Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of
Sciences, No. 2 Zhongguancun Beiyijie, 100190 Beijing, P. R. China. E-mail: qili@
iccas.ac.cn; Tel: +86-10-82627290
bGraduate School, Shandong Agricultural University, 271018 Shandong, P. R. China
cGraduate School, University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing
100049, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra09433f
55280 | RSC Adv., 2014, 4, 55280–55285
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
high convenience, low cost and controllable enantiomer water for 30.0 min in turn. Prior to sample injection, the
migration order.23–26 Nowadays, CLE-CE has been applied in the capillary was rinsed with 0.1 M HCl, 0.1 M NaOH, water and
analysis of drugs,27 foods28 and enzymes.29 However, it still faces running buffer for 2.0 min, respectively. Samples were siphoned
some challenges, including limited available systems and into the capillary at 15.0 cm height for 8.0 s and separated at
narrow applications. Thus, it is essential to construct novel CLE- ꢀ21.0 kV. Sample detection was performed at 254 nm. All
CE systems and broaden their application range. Cyclodextrins operations were conducted at 25 ꢂC. Generally, the running
(CDs) and their derivatives are the most commonly used chiral buffer system was composed of 100.0 mM boric acid, 5.0 mM
selectors in the CE separation. Meanwhile, the previous works ammonium acetate, 3.0 mM zinc sulfate, 6.0 mM L-Leu and 4.0
displayed that CDs could improve the enantioseparation effi- mM b-CD at pH 8.2. All of CLE-CE performance were operated
ciency of CLE-CE system in some extent.29–31 Therefore, con- for multiple measurements (n ¼ 3).
structing the CLE-CE system based on the cooperation effect of
CDs could widen the selection range of chiral ligand and further
widen its application. However, to date, only a few papers have
been published on the enantioseparation by combing CLE-CE
system and CDs system.15,29–34 Furthermore, although it has
been reported that chalcones have inhibition effect on tyrosi-
nase,12,16 to our best knowledge, CLE-CE has not been applied in
screening the inhibitors of tyrosinase with chalcones as the
model inhibitors.
In this work, a new CLE-CE system with L-leucine (L-Leu) as
the chiral ligand, Zn(II) as the central ion and b-CD as the
additive was developed. Various pairs of D,L-AAs, including the
substrate of tyrosinase, D,L-tyrosine (D,L-Tyr), were well enan-
tioseparated under the optimized conditions. In addition, the
inhibitory efficiency of tyrosinase was studied. Moreover, the
proposed CLE-CE method has been applied in screening the
inhibitors of tyrosinase with chalcones as the model
compounds, indicating its great potential in clinical analysis
and drug discovery in the future.
2.3 Sample preparation and AAs derivatization
All aqueous solutions were prepared with triply distilled water
and stored at 4 ꢂC. Standard sample solutions were prepared by
dissolving 2.0 mg of D-, and L-AAs in 40.0 mM lithium carbonate
buffer (adjusted to pH 9.5 with 0.1 M HCl), and they were
diluted to the required concentrations with lithium carbonate
solution in the range of 10–104-fold.
The D,L-AAs were derivatized as described in the previous
study.30 Briey, 20.0 mL D,L-AAs (2.0 mg mLꢀ1), 20.0 mL 40.0 mM
lithium carbonate and 20.0 mL Dns-Cl (1.5 mg mLꢀ1 in acetone)
were mixed in a 200.0 mL vial and kept at room temperature for
30 min. In order to terminate the reaction, 5.0 mL 2% ethꢂyl-
amine was added. All Dns-D,L-AAs samples were stored at 4 C
before use.
2.4 Enzyme incubation
All enzymatic reactions were carried out in 100 mM NaH2PO4
buffer (pH 6.8) at 25 ꢂC with a nal tyrosinase concentration of
565 U mgꢀ1. The enzyme incubation experiments were con-
ducted as following: 40.0 mL of different concentrations of l-Tyr,
2. Materials and methods
2.1 Reagents and chemicals
40.0 mL NaH2PO4 buffer and 40.0 mL tyrosinase (565 U mgꢀ1
)
ꢂ
were mixed at 25 C and incubated for 15 min, then the reac-
tions were ended by heating in boiling water for 10 min, fol-
lowed by centrifugation for 10 min at 10 000 rpm. Aer that, the
supernatant were gathered and derivatized with Dns-Cl by the
same processes as that of the standard AAs.
Chalcone, 4-hydroxychalcone (4-HC), 40-hydroxychalcone (40-
HC), 2-hydroxychalcone (2-HC), 20-hydroxychalcone (20-HC)
were purchased from J&K Chemistry Company (Beijing, China).
Boric acid, ammonium acetate, zinc sulfate, sodium hydroxide,
lithium carbonate, tris(hydroxymethyl) aminomethane (Tris),
acetone, b-cyclodextrin (b-CD), g-cyclodextrin (g-CD), a-cyclo-
dextrin (a-CD) and other reagents were obtained from Beijing
Chemical Factory (Beijing, China). Tyrosinase (source: mush-
rooms) was bought from Express Technology Co., Ltd. (565 U
mgꢀ1, Beijing, China). Dansyl chloride (Dns-Cl), D,L-Tyr, D,L-Leu,
D,L-valine (D,L-Val), D,L-phenylalanine (D,L-Phe) and other D,L-AAs
were from Sigma-Aldrich Chemical Company (St. Louis, USA).
In order to screening the inhibitors of tyrosinase, IC50
(concentration of a substance with half of the maximal inhibi-
tory) of different inhibitors was investigated. The experiments
were conducted as following: different concentrations of
inhibitors (4-HC, 40-HC, 2-HC, 20-HC, chalcone) were mixed with
equivoluminal L-Tyr solution (333 mM), then the mixture was
incubated with tyrosinase solution for 15 min at 25 ꢂC. Aer
that, the reactions were treated with the same steps as described
above.
2.2 Instruments and separation conditions
The CE apparatus used in this work consisted of a 1229-High 2.5 Calculation
Performance Capillary Electrophoresis (HPCE) analyzer (Beijing
The inhibition efficiency was calculated according to the
following equation:
Institute of New Technology and Application, Beijing, China), a
UV detector (Rilips Photoelectricity Factory, Beijing, China), an
uncoated fused-silica capillary (Yongnian Optical Fiber Factory,
Hebei, China) and a HW-2000 chromatography workstation
(Qianpu Soware, Nanjing, China).
I ¼ (C2 ꢀ C1)/(C0 ꢀ C1)
where I is the inhibition efficiency, C0 represents the concen-
The bare fused-silica capillary (60.0 cm ꢁ 75.0 mm id, effec- tration of L-Tyr in the absence of tyrosinase and inhibitor, C1
tive length 45.0 cm to detector) was washed by 0.1 M NaOH and and C2 are the remaining concentration of L-Tyr in the absence
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55280–55285 | 55281

View Article Online
RSC Advances
Paper
of the inhibitor and in the presence of the inhibitor,
respectively.
3.1.2 Effect of pH. According to the mechanism of CLE-CE,
the ionization equilibrium between the analytes and chiral
ligands is closely related to pH of the running buffer. In addi-
tion, pH of the running buffer can obviously affect the silicon
hydroxyl dissociation on the capillary wall and the electroos-
motic ow (EOF) in CLE-CE system. Therefore, it is greatly
necessary for studying the inuence of pH on Rs in this work. As
shown in Fig. 2, it could be observed that the Rs increased
obviously with the increasing of pH from 7.9 to 8.3. Meanwhile,
the migration times of the test analytes were greatly prolonged
(Fig. S1B†). Moreover, the current of the CLE-CE system was
increased to a higher pH value, which would affect the Rs and
peak shapes. Thus, taking all of the factors into consideration,
the buffer pH at 8.2 was nally selected for further analysis.
3.1.3 Effect of Zn(II)–L-Leu. The complexation reaction in
CLE-CE is between the chiral ligand and the central ion, thus
the ratio of the central ion to the ligand should have a signi-
cant effect on the Rs of the test analytes. Therefore, it is essential
to investigate the effect of the ratio of Zn(II) to L-Leu on Rs. In
this work, the concentration of Zn(II) was xed at 3.0 mM and
the concentration of L-Leu was changed from 1.5 mM to 9.0 mM.
As exhibited in Fig. 3, it could be found that the Rs increased
when the ratio of Zn(II) to L-Leu was changed from 2 : 1 to 1 : 2.5.
Moreover, the migration times of the test analytes were pro-
longed with the ratio increasing (Fig. S1C†). In order to get
higher Rs in short migration time, the concentration ratio of
Zn(II) to L-Leu at 1 : 2 was chosen for further investigation.
3.1.4 Effect of complex concentration. The complex
concentration was also an important parameter in the forma-
tion of ternary mixed metal complexes. Thus, when the ratio of
Zn(II) to L-Leu was kept at 1 : 2, the inuence of the complex
concentration on Rs was investigated with the concentration of
Zn(II) ranging from 2.0 mM to 6.0 mM. Fig. 4 demonstrated that
the separation efficiency decreased with the concentration of
Zn(II) increasing. Meanwhile, the migration times of the test
analytes were also shortened (Fig. S1D†). In view of the higher
resolution and the shorter migration time, 3.0 mM Zn(II) and 6.0
mM L-Leu were selected as the nally complex concentration for
further work.
3. Results and discussion
3.1 Optimization of separation condition
A new CLE-CE system with L-Leu as the chiral ligand and Zn(II)
as the central ion was developed in this work. In this CLE-CE
system, b-CD was used as the additive. Several key parameters
including pH, the ratio of central ion to ligand, the concentra-
tions of the complex and b-CD were investigated in detail with
Dns-D,L-Val, Dns-D,L-Tyr and Dns-D,L-Phe (which represented the
aromatic D,L-AAs and aliphatic D,L-AAs) as the test analytes to get
the optimal separation conditions.
3.1.1 Effect of CDs. CDs have been widely applied in chiral
separation owing to its intrinsic chirality.30 The separation
performance of different kinds of CDs was investigated by the
chiral separation efficiency (Rs) and the plate numbers. As
shown in Table S1,† it could be observed that a-CD displayed no
enantioseparation ability for the test analytes. Meanwhile, other
CDs were all suitable for enantioseparation. Although the Rs
and plate numbers of g-CD were higher than that of b-CD, the
migration times were prolonged (Table S1†). Thus, taking the
key factors (Rs, migration times, plate numbers) into consider-
ation, b-CD was the best choice for further study.
It has been reported that the enantioseparation of D,L-AAs in
CLE-CE system could be improved in cooperation with b-CD.28,29
Therefore, the inuence of the concentration of b-CD (0–5.0 mM)
on the chiral recognition was studied. As displayed in Fig. 1, the
separation efficiency obviously became better with the addition
of b-CD in the buffer solution. We found that when the
concentration of b-CD was 4.0 mM, the resolution (Rs) of the
three test pairs of Dns-D,L-AAs was higher than 1.5. However, the
migration times of the test analytes were greatly prolonged with
increasing the concentration of b-CD (Fig. S1A†). Based on the
CLE-CE requirement of high Rs and shorter migration times, 4.0
mM b-CD was nally chosen for further study.
Fig. 1 Influence of b-CD concentration on Rs. Buffer conditions:
100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(II),
6.0 mM L-Leu, adjusted pH to 8.2, and different concentrations of
b-CD ranging from 0–5.0 mM. Capillary: 60.0 cm, 45.0 cm effective ꢁ
75.0 mm id; injection: siphoned for 8.0 s at 15.0 cm; voltage: ꢀ21.0 kV; Fig. 2 Influence of pH on Rs. Buffer conditions: 100.0 mM boric acid,
UV detection: 254 nm; temperature: 25 ꢂC. Rs1: resolution of Dns-D,L- 5.0 mM ammonium acetate, 3.0 mM Zn(II), 6.0 mM L-Leu and 4.0 mM
Val; Rs2: resolution of Dns-D,L-Tyr; Rs3: resolution of Dns-D,L-Phe.
55282 | RSC Adv., 2014, 4, 55280–55285
b-CD at different pH values. Other conditions are the same as Fig. 1.
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Table
1
Enantioseparation of Dns-D,L-AAs under the optimum
conditiona
b
Dns-D,L-AAs
Rs
tLc/min
tDc/min
Dns-D,L-Thr
Dns-D,L-Phe
Dns-D,L-Val
Dns-D,L-Tyr
Dns-D,L-Met
Dns-D,L-Asn
Dns-D,L-Trp
Dns-D,L-Ala
Dns-D,L-Pro
Dns-D,L-Ser
Dns-D,L-Asp
Dns-D,L-Glu
Dns-D,L-Ile
Dns-D,L-Cys
Dns-D,L-His
Dns-D,L-Leu
Dns-D,L-Arg
2.31
2.05
1.96
1.84
1.65
1.54
1.05
1.03
0.87
0.45
0.37
0
48.67
53.96
38.46
47.60
62.41
65.01
86.12
56.23
37.23
62.70
27.99
26.97
42.57
24.97
21.71
24.99
19.75
51.40
55.95
40.08
49.44
65.08
67.34
89.50
57.90
38.35
63.29
28.22
26.97
42.57
24.97
21.71
24.99
19.75
Fig. 3 Influence of concentration ratio of Zn(II) to L-Leu on Rs. Buffer
conditions: 100.0 mM boric acid, 5.0 mM ammonium acetate, 4.0 mM
b-CD, the concentration ratio of Zn(II) to L-Leu was from 2 : 1 to 1 : 2.5
with Zn(II) kept at 3.0 mM, adjusted pH to 8.2. Other conditions are the
same as Fig. 1.
0
0
0
0
0
a
Running buffer: 100.0 mM boric acid, 5.0 mM NH4 AC, 3.0 mM Zn(II),
6.0 mM L-Leu and 4 mM b-CD at pH 8.2. b Rs ¼ 2(tD ꢀ tL)/(WD + WL). c t:
migration time.
EMO of Dns-D,L-Tyrs was also much dependent on b-CD in the
proposed CLE-CE system. In other words, the EMO of Dns-D,L-
AAs should be Dns-L-AAs > Dns-D-AAs, which could not be
tuned if b-CD was used as the coordinators in CLE-CE system
(Fig. 2, Table 1 and Fig. S2†).
Fig. 4 Influence of complex concentration on Rs. Buffer conditions:
100.0 mM boric acid, 5.0 mM ammonium acetate, 4.0 mM b-CD and
different concentrations of Zn(II) complex with L-Leu in the range of
2.0–6.0 mM, the ratio of Zn(II) to L-Leu was kept at 1 : 2, adjusted pH to
8.2. Other conditions are the same as Fig. 1.
3.2 Discussion on chiral recognition mechanism
In this study, the results exhibited in Fig. 1 and Table S2† clearly
displayed that the three test analytes could not be enantiose-
parated without b-CD. On the other hand, only Dns-D,L-Tyr
could be partly enantioseparated among the three test analy-
tes (Table S2†) while only b-CD was added in the buffer solution.
Therefore, we assumed that the coordination reaction between
Zn(II)–L-Leu complex and b-CD could achieve the chiral
resolution.
The separation mechanism of ligand exchange is based on
the formation of diastereomeric ternary complex between the
analytes and the mental–ligand complexes. The principle of
CLE-CE is shown as follows:
Based on the above experimental results, the optimum
buffer was composed of 100.0 mM boric acid, 5.0 mM ammo-
nium acetate, 3.0 mM Zn(II), 6.0 mM L-Leu and 4.0 mM b-CD at
pH 8.2. As shown in Table 1 and Fig. 5, we observed that six
pairs of Dns-D,L-AAs (containing Dns-D,L-Val, Dns-D,L-Tyr, Dns-
D,L-Phe) were baseline-separated, and ve pairs of Dns-D,L-AAs
were partly separated. It should be noted that under the
optimum conditions, although eleven pairs of Dns-D,L-AAs
could be well enantioseparated, six pairs of the analytes still
could not be enantioseparated as exhibited in Table 1. The
reason is unclear, and further exploration should be conducted
in the future.
It has been reported that the enantiomer migration order
(EMO) of D,L-AAs was related to the different chiral ligand.30,35 As
shown in Fig. S2† in this study, Dns-L-Tyr always migrated
ahead of Dns-D-Tyr, no matter the ligand was D-Leu or L-Leu,
which is different from the reported experiments.30,35 Thus it
was speculated that although the stabilization of the formed
ternary complexes with L-Leu or D-Leu as the chiral ligand was
different, the enantiorecognition ability of the ternary complexes
was much improved by coordinating with b-CD. Meanwhile, the
Zn(L-Sel)2 + L-A / (L-Sel)Zn(L-A) + L-Sel
Zn(L-Sel)2 + D-A / (L-Sel)Zn(D-A) + L-Sel
Sel: selector; A: analyte
Resolution is due to the difference in complex stability
constants of the mixed complexes with analyte enantiomers. In
this work, the three test analytes could not be baseline sepa-
rated when the CLE-CE system with Zn(II)-L-Leu as the chiral
selector worked alone. The results indicated the differences in
the stability of the formed complexes were limited and the
satisfactory enantioseparation result could not be obtained.26,36
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55280–55285 | 55283

View Article Online
RSC Advances
Paper
AAs could not be enantioseparated (Table 1). The reason was
remained unknown and further study on the chiral recognition
mechanism should be carried out in the future.
3.3 Application in screening the inhibitors of tyrosinase
3.3.1 Quantitative analysis of L-Tyr. Since L-Tyr was selected
as the substrate of tyrosinase, it is essential to conduct the
quantitative analysis of L-Tyr. A series of standard solutions of
Dns-D,L-Tyr ranging from 12.95 mM to 413.3 mM were examined.
The linearity was investigated by plotting the peak area versus
analyte concentration. The obtained equation of linearity
regression of L-Tyr was shown as follows: y ¼ 5648x + 5816 (R2 ¼
0.992). The limit of detection (LOD, S/N ¼ 3) of the substrate was
6.5 mM.
The good linearity of L-Tyr demonstrated that the proposed
CLE-CE method is available to determine the enzymatic activity
of tyrosinase and further screen its inhibitors.
3.3.2 Kinetics study of tyrosinase. Then the enzyme
kinetics was estimated by the Michaelis–Menten equation as
shown following:
V0 ¼ Vmax [S]/(Km + [S])
where, V0 is the initial rate of enzyme reaction, [S] is the
substrate concentration, Vmax is the maximum rate, and Km is
the Michaelis constant.43 As shown in Fig. S3,† the Line-weaver
Burk plot was obtained with good linearity (R2 ¼ 0.994). It was
calculated that the Km and Vmax were 636 mM and 312 mmol
minꢀ1 mgꢀ1, respectively.
3.3.3 Inhibitors screening. Based on the experimental
Fig.
5 Entioseparation of the three test Dns-D,L-AAs under the results, the proposed CLE-CE method could be applied in
optimum conditions. (A) Dns-D,L-Val (5.69 mM); (B) Dns-D,L-Tyr
(3.68 mM); (C) Dns-D,L-Phe (4.04 mM). Buffer conditions: 100.0 mM
boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(II), 6.0 mM L-Leu
and 4.0 mM b-CD at pH 8.2.
screening the inhibitors of tyrosinase. Meanwhile, it has been
reported that the chalcones and its derivatives, which are
mainly located in plants, have displayed their great inhibition
effects on tyrosinase.13–15 Therefore, the obtained IC50 of the ve
inhibitors were conducted to screening the inhibitors of tyros-
inase. As displayed in Fig. S4† and Table 2, it could be found
that the IC50 of 4-HC was the lowest one among the ve inhib-
itors, indicating the best inhibition efficiency. In addition, the
inhibition efficiency order of the selected inhibitors was shown
as follows: 4-HC [ 40-HC > 20-HC > chalcone > 2-HC, which was
coincident with the previous work.12 Moreover, the results
demonstrated that the proposed CLE-CE method based on
cooperation effect of b-CD is indeed suitable for screening the
inhibitors of tyrosinase.
Thus, other kinds of chiral selectors should be added into the
CLE-CE system as the chiral additives to improve the chiral
separation efficiency.
It's well known that b-CD is the traditional chiral additive as
its intrinsic chirality and unique structure.37,38 The analytes
could be contained to this special structure of b-CD, and then
react with the hydroxyl of b-CD, forming the inclusion
complexes. The different formation constants in the formation
of the inclusion complexes for enantiomers make the analytes
separate.39–41 However, as displayed in Table S2,† none of the
three test analytes could be baseline separated when b-CD
worked alone.
Table 2 IC50 of the five inhibitors of tyrosinasea
It has been reported that CDs could act as an additional
ligand toward the central metal ion of the complexes in CLE-
CE.42 Therefore, we assumed that the coordinating ability of
CDs could be utilized for the CLE-CE system. As shown in Table 1,
the coordinating ability of b-CD in the CLE-CE system was
clearly proved. It should be mentioned that in the proposed
CLE-CE system, although b-CD depicted its well effect in
assisting enantioseparation, there were still six pairs of Dns-D,L-
Inhibitions
IC50/mM
4-HC
0.82
2.65
16.99
23.91
43.14
40-HC
20-HC
Chalcone
2-HC
a
Incubation conditions: 333 mM L-Tyr as the substrate and different
inhibitors were incubated with tyrosinase for 15.0 min at 25 ꢂC.
55284 | RSC Adv., 2014, 4, 55280–55285
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
18 W. H. Zhang, Z. H. Lv, T. F. Jiang, Y. H. Wang and L. H. Guo,
J. Food Drug Anal., 2012, 20, 159–163.
4. Conclusion
19 Z. L. Chen, K. Uchiyama and T. Hobo, Anal. Chim. Acta, 2004,
523, 1–7.
In this work, a new CLE-CE system based on Zn(II)–L-Leu
complex with the cooperation of b-CD was developed. The
system has been successfully explored for enantioseparation of
Dns-D,L-AAs and further applied in screening the inhibitiors of
tyrosinase with chalcones as the model compounds. The
obtained results demonstrated the method is feasible for ena-
tioseparation of Dns-D,L-AAs and screening the inhibitors of
tyrosinase. Moreover, this proposed CLE-CE system can provide
a new strategy for screening the inhibitors of tyrosinase, and it
has good potential for relevant drug discovery and clinical
analysis in future.
20 Z. Q. Li, C. C. Liu, X. M. Dou, Y. Ni, J. X. Wang and
Y. Yamaguchi, J. Chromatogr. A, 2014, 1331, 100–107.
21 Z. Q. Li, S. X. Chen, C. C. Liu, D. W. Zhang, X. M. Dou and
Y. Yamaguchi, J. Chromatogr. A, 2014, 1361, 286–290.
22 Z. Q. Li, X. M. Dou, Y. Ni, Q. M. Chen, S. Y. Cheng and
Y. Yamaguchi, J. Chromatogr. A, 2012, 1229, 274–279.
23 Z. X. Zheng, J. M. Lin and F. Qu, J. Chromatogr. A, 2003, 1007,
189–196.
24 T. D. Wang and J. W. Kang, Electrophoresis, 2009, 30, 1349–
1354.
25 P. T. T. Ha, J. Hoogmartens and A. V. Schepdael, J. Pharm.
Biomed. Anal., 2006, 41, 1–11.
Acknowledgements
We are grateful for the nancial support from National Natural
Science Foundation of China (no. 21175138, no. 21375132 and
no. 21321003).
¨
26 M. G. Schmid, N. Grobuschek, O. Lecnik and G. Gubitz, J.
Biochem. Biophys. Methods, 2001, 48, 143–154.
¨
¨
27 H. Hodl, A. Krainer, K. Holzmuller, J. Koidl, M. G. Schmid
¨
and G. Gubitz, Electrophoresis, 2007, 28, 2675–2682.
28 S. Kodama, A. Yamamoto, A. Matsunaga and K. Hayakawa, J.
Chromatogr. A, 2001, 932, 139–143.
29 L. Qi, J. Qiao, G. L. Yang and Y. Chen, Electrophoresis, 2009,
30, 2266–2272.
Notes and references
1 I. Kubo, Q. X. Chen and K. Nihei, Food Chem., 2003, 81, 241–
247.
2 Q. K. Chen and I. Kubo, J. Agric. Food Chem., 2002, 50, 4108–
4112.
30 L. Qi, Y. L. Han, M. Zuo and Y. Chen, Electrophoresis, 2007,
28, 2629–2634.
3 H. S. Lee, J. Agric. Food Chem., 2002, 50, 1400–1403.
4 J. Z. Zhang, B. Li, G. B. Xu, G. J. Cheng and S. J. Dong, Analyst,
1999, 124, 699–703.
5 X. Chen, G. J. Cheng and S. J. Dong, Analyst, 2001, 126, 1728–
1732.
31 L. Qi, G. L. Yang, H. Z. Zhang and J. Qiao, Talanta, 2010, 81,
1554–1559.
32 Z. X. Zheng, F. Qu and J. M. Lin, Chin. J. Chem., 2003, 21,
1478–1484.
33 A. Giuffrida, V. Cucinotta, G. Maccarrone, M. Messina,
E. Rizzarelli and G. Vecchio, Eur. J. Inorg. Chem., 2014,
2014, 377–383.
´
6 M. Jimenez, S. Chazarra, J. Escribano, J. Cabanes and
´
F. Garcıa-Carmona, J. Agric. Food Chem., 2001, 49, 4060–4063.
7 I. Kubo, I. Kinst-Hori and Y. Yokokawa, J. Nat. Prod., 1994,
57, 545–551.
34 T. Horimai, M. Ohara and M. Ichinose, J. Chromatogr. A,
1997, 760, 235–244.
8 I. Kubo, Y. Yokokawa and I. Kinst-Hori, J. Nat. Prod., 1995,
58, 739–743.
35 H. Z. Zhang, L. Qi, J. Qiao and L. Q. Mao, Anal. Chim. Acta,
2011, 691, 103–109.
9 Z. P. Zheng, K. W. Cheng, Q. Zhu, X. C. Wang, Z. X. Lin and
M. F. Wang, J. Agric. Food Chem., 2010, 58, 5368–5373.
10 S. Parvez, M. Kang, H. S. Chung and H. Bae, Phytother. Res.,
2007, 21, 805–816.
¨
36 M. G. Schmid and G. Gubitz, Anal. Bioanal. Chem., 2011, 400,
2305–2316.
37 W. X. Zhu, Q. Y. Wang, K. F. Du, S. Yao and H. Song, Chin.
Sci. Bull., 2013, 58, 3390–3397.
11 O. Nerya, R. Musa, S. Khatib, S. Tamir and J. Vaya,
Phytochemistry, 2004, 65, 1389–1395.
38 J. Wang and I. M. Warner, J. Chromatogr. A, 1995, 711, 297–
304.
12 F. Sonmez, S. Sevmezler, A. Atahan, M. Ceylan, D. Demir,
N. Gencer, O. Arslan and M. Kucukislamoglu, Bioorg. Med.
Chem., 2011, 21, 7479–7482.
13 J. B. Liu, C. H. Chen, F. Y. Wu and L. Z. Zhao, Chem. Biol.
Drug Des., 2013, 82, 39–47.
¨
39 G. Gubitz and M. G. Schmid, Electrophoresis, 2000, 21, 4112–
4135.
40 M. Blanco and I. Valverde, Trends Anal. Chem., 2003, 22, 428–
439.
41 C. M. Boone, J. C. M. Waterval, H. Lingeman, K. Ensing and
W. J. M. Underberg, J. Pharm. Biomed. Anal., 1999, 20, 831–
863.
14 X. D. Zhang, X. Hu, A. J. Hou and H. Y. Wang, Biol. Pharm.
Bull., 2009, 32, 86–90.
15 B. B. Sun, L. Qi, X. Y. Mu, J. Qiao and M. L. Wang, Talanta,
2013, 116, 1121–1125.
16 S. Khatib, O. Nerya, R. Musa, M. Shmuel, S. Tamir and
J. Vaya, Bioorg. Med. Chem., 2005, 13, 433–441.
17 N. Jun, G. Hong and K. Jun, Bioorg. Med. Chem., 2007, 15,
2396–2402.
42 B. D. Wu, Q. Q. Wang, L. Guo, R. Shen, J. W. Xie, L. H. Yun
and B. H. Zhong, Anal. Chim. Acta, 2006, 558, 80–85.
43 T. S. Chang, H. Y. Ding and H. C. Lin, Biosci., Biotechnol.,
Biochem., 2005, 69, 1999–2001.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55280–55285 | 55285