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 Journal of Chromatography B, 1002 (2015) 319–328

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Identification of tyrosinase specific inhibitors from Xanthium

strumarium fruit extract using ultrafiltration-high performance liquid
chromatography
Zhiqiang Wang a,b , Seung Hwan Hwang a,b , Bo Huang d , Soon Sung Lim a,b,c,∗
a
Department of Food Science and Nutrition, Hallym University, Chuncheon 200-702, South Korea
b
Institute of Korean Nutrition, Hallym University, Chuncheon 200-702, South Korea
c
Institute of Natural Medicine, Hallym University, Chuncheon 200-702, South Korea
d
College of Food Science and Engineering, Liaoning Medical University, Jinzhou 121-000, China

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a strategy based on ultrafiltration-high performance liquid chromatography coupled with
Received 24 June 2015 diode array detection (UF-HPLC-DAD) was proposed for screening tyrosinase specific inhibitors in Xanthii
Received in revised form 18 August 2015 fructus. The false negatives were distinguished by optimizing the UF-HPLC-DAD parameters to reduce
Accepted 20 August 2015
the background noise; the false positives were distinguished by introducing a blocked tyrosinase in the
Available online 28 August 2015
control group for comparison. To obtain the best blocker, the competitive experiments were performed
using various known ligands. Using this strategy, three competitive inhibitors (protocatechuic acid; 3,5-
Keywords:
di-O-caffeoylquinic acid; and 1,5-di-O-caffeoylquinic acid) and one mixed-type inhibitor (chlorogenic
Ultrafiltration
HPLC acid) were identified. These results were verified using tyrosinase inhibition assay, kinetic analysis, and
Tyrosinase inhibitor structural simulation of the complex. Our experimental results suggest that the proposed strategy could
Binding affinity be useful for high-throughput identification of tyrosinase specific inhibitors in natural products.
Xanthium strumarium © 2015 Elsevier B.V. All rights reserved.

1. Introduction
Tyrosinase (E.C.1.14.18.1) is a multifunctional type-3 copper
containing metalloenzyme ubiquitously present in microorgan-
isms, plants, and animals. It plays a major role in melanin synthesis
and it also has several multi catalytic functions [1]. Products result-
ing from tyrosinase activity can cause deleterious effects, such as
browning reactions in fruits and vegetables, and black spotting in
shrimps and lobsters [2]. The hyperpigmentation of the epidermis
and dermis has also been shown to depend on either an increased
numbers of melanocytes or on the increased activity of the tyrosi-
nase enzyme, and it has also been associated with Parkinson’s
disease [3]. Therefore, tyrosinase inhibitors have potential appli-
cations in medicine, cosmetics, and food industry. To date, a great
number of natural and synthetic tyrosinase inhibitors have been
tested to determine whether they could be effective in tyrosinase
inhibition [4]. Nevertheless, some of their individual activities are
not potent enough to be considered for practical use and do not
comply with the safety regulations for food and cosmetic products
∗ Corresponding author at: Hallym University, 1 Hallymdeahak-gil, Chuncheon,
200-702, South Korea. Fax: +82 33 251 0663.
E-mail address: limss@hallym.ac.kr (S.S. Lim).
[5]. Thus, the process of finding new tyrosinase inhibitors in natural
products is an interesting challenge.
Xanthii fructus, the fruit of Xanthium strumarium L. (Asteraceae),
have been used for the treatment of leukoderma, fever, scrofula,
sinusitis, headache, herpes, and cancer [6]. In a previous study,
we reported that the extracts of these fruits significantly inhibited
aldose reductase activity [7]. As part of our continuing study of this
plant, the inhibitory effects of Xanthii fructus extracts on tyrosinase
activity were investigated in this study.
The conventional bioassay guided fractionation is widely used
to discover new bioactive compounds, but it is a time-consuming,
labor-intensive, and low-efficiency strategy [8]. At present, meth-
ods based on the molecular basis of protein-ligand interactions,
particularly on the binding selectivity and affinity, are being used
with significant success during the early-stage of drug discovery [9].
Consequently, in recent times, many affinity-based methods have
been developed for high-throughput screening, and affinity-based
ultrafiltration method as an indirect detection approach has been
widely applied for identifying enzyme inhibitors from natural prod-
ucts [10–12]. However, these methods have limited resolutions due
to the problems in distinguishing false positives caused by non-
specific ligand binding, and false negatives caused by transient or
covalent interactions. To avoid a large number of false positives,
in a method, competitive experiment was used for distinguishing

http://dx.doi.org/10.1016/j.jchromb.2015.08.030
1570-0232/© 2015 Elsevier B.V. All rights reserved.
320 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328
specific bindings through blocking the specific site of enzyme by
known ligand, and the results obtained using this method demon-
strated that false positives could be eliminated to a great extent
[13–15]; however, it is not easy to obtain a suitable ligand as
blocker. Moreover, how to effectively decrease the false negative
is still a challenge for many researchers.
In view of the above-mentioned facts, the aim of the present
work was to develop an ultrafiltration-high performance liquid
chromatography-diode array detection (UF-HPLC-DAD) screening
strategy that could distinguish specific tyrosinase inhibitors from
the large number of false positives and false negatives in Xan-
thii fructus extract. Typically, either a mass spectrometer or a UV
detector coupled to HPLC system can be employed. But the latter
combination is widely used for other purposes and many labo-
ratories could perform the ultrafiltration technique without new
instrumentation. In total, four compounds with tyrosinase specific
binding activity were observed and identified through HPLC-DAD
with reference standards. The reliability of this approach was
confirmed using functional assay, kinetic study, and structural sim-
ulation of the complex. Finally, four new tyrosinase inhibitors were
discovered in the present study and two specific ones were distin-
guished by the developed UF-HPLC-DAD strategy.
2. Materials and methods
2.1. Chemicals
Mushroom tyrosinase (120 kDa) was purchased from
Sigma–Aldrich Chemical Co. (St. Louis, MO, US). l-Tyrosine,
arbutin, kojic acid, resveratrol, caffeic acid, 4-hydroxybenzoic
acid, p-anisic acid, cinnamic acid, 4-ethoxybenzoic acid, 4-
propoxybenzoic acid, 4-butoxybenzoic acid, and trolox were
obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, US). The
reference compounds including protocatechuic acid, chlorogenic
acid, 3,5-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid,
1,3-di-O-caffeoylquinic acid, and 1,3,5-tri-O-caffeoylquinic acid
were isolated in our laboratory. Methanol was purchased from
Avantor Performance Materials, Inc. (Center Valley, US). All other
chemicals and solvents, unless otherwise specified, were guaran-
teed reagent grade and purchased from Sigma–Aldrich Chemical
Co. (St. Louis, MO, US).
2.2. Preparation of extract sample and stock solution
Xanthii fructus (fruits of X. strumarium L.) were purchased from
a local market in Chuncheon, Korea. The voucher sample (RIC-
HU1204) has been deposited in the center of efficacy assessment
and development of functional foods and drugs, Hallym University,
Chuncheon, Korea. The specimens were authenticated by Emeritus
Prof. H.J. Chi, Seoul National University, Korea. The Xanthii fruc-
tus were ground, extracted, fractionated, and isolated as previously
described [7].
Individual compounds, including arbutin, kojic acid, resveratrol,
caffeic acid, 4-hydroxybenzoic acid, p-anisic acid, cinnamic acid, 4-
ethoxybenzoic acid, 4-propoxybenzoic acid, 4-butoxybenzoic acid,
and trolox, were dissolved in dimethyl sulfoxide (DMSO) to make
the known ligand solution, and their retention times were obtained
following the HPLC methods described in Section 2.6. For ease of
competitive experiment, above compounds were mixed together
to make the mixed stock solution in a same mole concentration for
each compound.
2.3. Screening procedure of ultrafiltration
The extract samples were dissolved in methanol to prepare the
extract sample solutions for ultrafiltration screening. Extract sam-
ple solution (at a final concentration of 0.5 mg mL−1 ) and tyrosinase
potassium phosphate buffer (0.1 M, pH 6.6) solution (at a final con-
centration of 25 nM, 100 unit mL−1 ) were mixed in a total volume
of 450 ␮L and incubated at 30 ◦ C for 30 min. The incubated mixture
was filtered through a Microcon YM-10 centrifugal filter unit with a
10,000 MW cut-off membrane (Millipore, Bedford, MA, US) by cen-
trifugation at 5167 × g for 30 min. The filtrate was later subjected
to HPLC analysis. The blank experiment was carried out similarly
but without tyrosinase. The control experiment was carried out
using a resveratrol-blocked tyrosinase. The blocked tyrosinase was
prepared following the methods described in Section 2.5.
2.4. Effect of experimental parameters
The UF screening experimental parameters, including tyrosi-
nase concentrations, incubation times, incubation temperatures,
and centrifugation speeds, were investigated in this study. Specif-
ically, the extract sample solution (at a final concentration of
0.5 mg mL−1 ) was incubated with different concentrations of
tyrosinase (2.2, 10.6, 25, and 50 nM) in a total volume of 450 ␮L at
various time points (10, 20, 30, and 40 min) and temperatures (25,
30, 35, and 40 ◦ C). Next, the incubated mixtures were centrifuged at
different rates (5167, 5741, 6315, and 6887 × g). The filtrates were
analyzed by HPLC to study the effect of the different parameters on
the binding affinities of tyrosinase and samples.
The relative total binding affinities of the ligands from extract
sample towards tyrosinase were defined as ‘total binding degree
(TBD)’, which can be calculated in the following way:
Aa − Ab
TBD% = × 100 (1)
Aa
where Aa and Ab are the peak areas of a compound interacting
without and with active tyrosinase in the HPLC chromatograms,
respectively.
2.5. Competitive experiment
The known ligand solution (22.5 ␮L) and the tyrosinase solution
(405 ␮L, 27.78 nM in phosphate buffer) were mixed and pre-
incubated at 30 ◦ C for 15 min. The blocked tyrosinase solution and
extract sample solution (at a final concentration of 0.5 mg mL−1 )
or mixed stock solution (at a final concentration of 0.08 mM for
each compound) were then mixed in a total volume of 450 ␮L and
incubated at 30 ◦ C for 30 min. The incubated mixture was filtered
through a Microcon YM-10 centrifugal filter unit with a 10,000
MW cut-off membrane (Millipore, Bedford, MA, US) by centrifu-
gation at 5167 × g for 30 min. The filtrate was later subjected to
HPLC analysis.
The tyrosinase competitive binding ability between blocker and
ligands from extract sample was defined as the ‘specific binding
degree (SBD)’, which can be calculated by the following equation:
Ac − Ab
SBD% = × 100 (2)
Aa
where Aa and Ab are the peak areas of a compound interacting
without and with active tyrosinase in the HPLC chromatograms,
respectively; Ac is the peak area of a compound interacting with
known ligand pre-incubated tyrosinase.
2.6. HPLC analysis
The HPLC analysis of each filtrate was performed on an Agilent
1100 series instrument (Agilent Technologies, Seoul, Korea). Sep-
aration was achieved on a C18 column (150 mm length, 4.6 mm
i.d., and 5 ␮m particle size; Phenomenex Gemini), coupled with a
guard column, at 30 ◦ C. Briefly, the samples (10 ␮L) were injected
 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328 321

Table 1
Inhibitory effects of Xanthii fructus extract on tyrosinase.

Fraction Concentration(mg mL−1 ) Inhibition (%) IC50 a (mg mL−1 )

Arbutinb 1 40.08 ± 2.03 c

2.42 ± 0.07
0.5 21.63 ± 1.36
0.1 13.15 ± 1.11
Methanol ext. 1 14.28 ± 0.86 –
Methylene chloride fr. 1 6.85 ± 0.64 –
Ethyl acetate fr. 1 84.25 ± 3.68 0.33 ± 0.02
(EFXF) 0.5 61.52 ± 3.10
0.1 10.79 ± 0.66
n-Butanol fr. 1 70.69 ± 4.21 0.46 ± 0.03
0.5 56.71 ± 2.37
0.1 2.04 ± 0.15
Water fr. 1 15.16 ± 1.17 –
a
IC50 indicates the concentration of extract necessary to decrease the initial concentration of substrate by 50%.
b
Arbutin was used as a positive control.
c
Results are presented as means ± SD.

into the system. The samples were eluted with acidified water To describe the mixed type inhibition mechanism, the
(0.01% trifluoroacetic acid, A) and methanol (B), at a flow rate Lineweaver–Burk equation in double reciprocal form can be
of 0.7 mL min−1 . The optimized gradient chromatographic condi- written as:
tions for extract sample solution were as follows: 10–20% B at 1 1
  Km
  1
[I] [I]
0–5 min; 20–30% B at 5–10 min; 30–50% B at 10–20 min; 50–100% B = 1+ + 1+ (6)
v Vmax ␣Ki Vmax Ki [S]
at 20–25 min. The optimized gradient chromatographic conditions
for mixed stock solutions were as follows: 0–100% B at 0–25 min; Secondary plots can be constructed from:
100% B at 25–30 min. Online UV spectra was obtained under a wave- Km Km [I]
length of 284 nm. Slope = + (7)
Vmax Vmax Ki
And:
2.7. Tyrosinase assay and kinetic study
1 1 1
Y − intercept = app = + [I] (8)
A spectrophotometric tyrosinase assay was performed as Vmax Vmax ˛Ki Vmax
previously described with slight modifications [16]. Briefly, phos- Thus, the Km , Vmax , Ki , and ˛-values can be derived from the above
phate buffer (120 ␮L), sample (20 ␮L, final concentration of equations.
0.1–1 mg mL−1 ), and l-tyrosine buffer (40 ␮L, 1.5 mM) solutions
were mixed in a 96-well plate. Then, 20 ␮L of mushroom tyrosi-
3. Results and discussion
nase solution (500 unit mL−1 in phosphate buffer) was added
to each mixture, which was then incubated for 30 min at 25 ◦ C.
3.1. The tyrosinase inhibitory activity of Xanthii fructus extracts
The absorbance of the mixture was determined at 450 nm using
an EL-800 Universal microplate reader (Bio-Tek Instrument Inc.,
The tyrosinase inhibitory effect of each fraction of Xanthii fructus
Winooski, VT). Arbutin was used as positive control. The inhibitory
methanol extract was tested and the results are shown in Table 1.
activity was calculated as follows:
The results clearly showed that the ethyl acetate fraction was the
 A1 − A2
 most active with an IC50 value of 0.26 mg mL−1 . Although n-butanol
Inhibition% = 1− × 100 (3) fraction also showed a higher activity than arbutin, these results
A3 − A4
suggest that tyrosinase inhibitors are more likely to be present in
where A1 is the absorbance of the test samples with the enzyme, the ethyl acetate fraction of Xanthii fructus methanol extract (EFXF).
A2 is the absorbance of the test samples without the enzyme, A3 is The EFXF was used for further screening and was isolated.
the absorbance of the enzyme without the test sample, and A4 is
the absorbance of the buffer solution without the test samples and 3.2. Screening of tyrosinase inhibitors by UF-HPLC-DAD from
the enzyme. EFXF
For the kinetic study, different concentrations of l-tyrosine
buffer solution (0.3, 0.5, 0.8 mM) were used. The velocities of the As shown in Fig. 1, a strategy based on UF-HPLC-DAD was
enzymatic reaction at different concentrations of the isolated sam- proposed for screening tyrosinase specific inhibitors from natural
ples (0.1, 0.5, and 1 mg mL−1 ) were calculated and compared with products. In our strategy, the experimental conditions including
that of the non-inhibitory enzymatic reaction. The Michaelis con- tyrosinase concentration, incubation time and temperature, and
stant (Km ) and maximal velocity (Vmax ) of the tyrosinase activity centrifugal rate were first optimized to reduce the background sig-
were determined by the Lineweaver–Burk plot with various con- nal noise for distinguishing the false negatives; then, the suitable
centrations of l-tyrosine as a substrate. To describe the competitive blocker was selected through competitive experiment from various
inhibition mechanism, the Lineweaver–Burk equation in double known ligands; last, the tyrosinase specific inhibitors were distin-
reciprocal form can be written as: guished from EFXF by introducing the tyrosinase-blocker complex
1 Km
  1 1
in the control.
[I]
= 1+ + (4)
v Vmax Ki [S] Vmax
3.2.1. Optimizing the UF-HPLC-DAD parameters to reduce
Secondary plots can be constructed from: background noise
We investigated the effect of modifying different UF-HPLC-DAD
app Km [I] parameters, including tyrosinase concentrations, incubation tem-
Km = + Km (5)
Ki peratures, incubation times, and centrifugation speeds, to improve
322 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328
Fig. 1. The strategy for screening tyrosinase inhibitors from natural product extracts based on UF-HPLC-DAD.
the total binding affinity and reduce the background noise. Based on
the liquid chromatograms, the total binding affinity of each ligand
to tyrosinase was represented by TBD, which could be obtained by
dividing the amount of total binding affinity by the original amount
of each compound present in the incubation solution.
First, the EFXF was incubated with 2.2, 10.6, 25, and 50 nM
tyrosinase (8.8, 42.4, 100, and 200 units mL−1 , respectively). After
separation through the UF unit, the ultrafiltrates were analyzed
by HPLC. Fig. 2a displays the differences in TBD of each compo-
nent incubated with different concentrations of tyrosinase. Most
of the ligands showed increased TBD as tyrosinase concentrations
increased. In the case of insufficient enzymes during incubation,
competition between ligands may occur which could lower the
total binding affinities or even lead to candidates being missed
[17]. Therefore, adequate enzyme concentrations are required to
ensure the detection of all potential ligands. Even though most of
the compounds showed their highest TBD when incubated with
50 nM tyrosinase, the TBD only decreased slightly when incubated
with 25 nM tyrosinase. To improve cost efficiency, further incuba-
tion experiments were performed using a tyrosinase concentration
of 25 nM.
The EFXF was incubated with tyrosinase at 25, 30, 35, and
40 ◦ C to determine the appropriate incubation temperature for the
assays, and the ultrafiltrates were subjected to HPLC analysis. The
TBDs of each component after incubation at different tempera-
tures are shown in Fig. 2b. In general, the effect of temperature
on enzymatic reactions is complex; enzyme activity increases with
an increase in temperature, and the rate of enzyme thermal denat-
uration also increases [18]. The same also applies to the binding
assay. In our results, all compounds showed high TBD when incu-
bated at different temperatures except compound 1, 3 and 4, and
30 ◦ C was selected for further incubation experiments.
Fig. 2c shows the effect of incubation time on TBD. The TBDs of
most ligands increased in a time-dependent manner. The need for
sufficient time to achieve ligand-enzyme equilibrium could explain
this phenomenon. Taking into account our results and the screen-
ing efficiency, 30 min of the incubation time was used in further
experiments.
Fig. 2d shows the effect of centrifugation speed on TBD. The
results clearly show that the lowest speed is the best. During the
binding assay, many ligand-enzyme complexes would be formed
through a weak connection and these complexes could be bro-
ken down easily by external forces. However, the ligand will not
be separated from the ligand-enzyme complexes if the speed is
low enough. Rotation speeds of over 5000 × g are sufficient for UF
separation, and therefore, a speed of 5167 × g was used in further
experiments.
In the present method, critical to the successful detection of
ligands is attaining spectra with adequate signal-to-noise ratios.
The transient or covalent interactions of free enzymes and chemi-
cals in the mixture might result in synergistic and/or antagonistic
effects, which will affect the total binding affinities of the enzyme
and ligands in the mixture, thus, creating background noise. These
interactions will change when the mixture components change and
cannot be eliminated from the mixtures. In principle, the synergis-
tic effects will not influence the screening results and the hits will
be distinguished by comparing them with the control group; how-
ever, if antagonistic effects exist, the hits will be missed as false
negatives due to a reduction in the total binding affinity. How-
ever, the total binding affinities were significantly increased and
the background noise was dramatically reduced after optimization.
As results, the hits were distinguished much clearer and the false
negative hit (compound 3) was distinguished (Fig. S1).
3.2.2. Competitive experiment for selecting blocker
False positives could be distinguished by using a known ligand
as blocker to provide an additional control by occupying the specific
binding site of the enzyme to prevent the binding of other chemi-
cals. However, it is not easy to obtain a suitable blocker. Therefore,
four known tyrosinase competitive inhibitors (arbutin [19], kojic
acid [19], 4-hydroxybenzoic acid [20], and resveratrol [19]), two
non-competitive inhibitors (caffeic acid [21] and p-anisic acid [21]),
two un-competitive inhibitors (4-propoxybenzoic acid [20] and 4-
butoxybenzoic acid [20]), two-mixed type inhibitors (cinnamic acid
[21] and 4-ethoxybenzoic acid [20]), and one non-inhibitor (trolox
[22]) were selected to perform the competitive experiments for
investigating their effects on UF-HPLC-DAD analysis. As shown in
Fig. 3a and b, arbutin, kojic, resveratrol, caffeic acid, and trolox
showed high binding affinity to active tyrosinase and were blocker
candidates. However, caffeic acid-blocked tyrosinase showed no
selectivity to distinguish the specific ligands from the mixed
stock solution (Fig. 3d); in contrast, resveratrol-blocked tyrosinase
showed excellent selectivity to distinguish specific ligands from
the mixed stock solution, and trolox also was distinguished as a
false positive hit by resveratrol-blocked tyrosinase (Fig. 3c). These
results suggested that the high affinity competitive inhibitors are
blocker candidates.
Three high affinity competitive inhibitors (arbutin, kojic acid,
and resveratrol) were tested as blockers to investigate their effect
on UF-HPLC-DAD analysis resolution of EFXF. Based on the liquid
chromatograms, the competitive binding affinities of each ligand
bind toward tyrosinase against to the blockers were presented by
SBD. As shown in Fig. 4, the SBD of ligands differed depending on
the blocker used and the blockers could be ordered from best to
 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328 323

Fig. 2. Effect of tyrosinase concentration a, incubation temperature b, incubation time c, and centrifugation speed d on TBD%. Different letters in one group indicate significant
differences, p < 0.05.
324 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328

Fig. 3. UF-HPLC-DAD analysis of the mixed stock solution incubated with buffer a, active tyrosinase b, resveratrol-blocked tyrosinase c, and caffeic-blocked tyrosinase d.
Structures of compounds in the mixed stock solution e.

Fig. 4. Effect of arbutin, kojic acid, and resveratrol on SBD %. Different letters in one group indicate significant differences, p < 0.05.
 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328 325

Fig. 5. UF-HPLC-DAD analysis of EFXF for identifying the ligands binding to tyrosinase a and the structures of identified ligands b. Stars indicate candidate ligands, including
protocatechuic acid (3), chlorogenic acid (5), 3,5-di-O-caffeoylquinic acid (10), and 1,5-di-O-caffeoylquinic acid (11).

Fig. 6. Docking model of resveratrol a, protocatechuic acid b, chlorogenic acid c, 3,5-di-O-caffeoylquinic acid d, and 1,5-di-O-caffeoylquinic acid e. The structure of tyrosinase
is colored in green; the structures of ligands are colored in magentas; the yellow balls are Cu2+ ; the residues interacted with ligands are colored by orange. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 326
Table 2
Results of UF-HPLC-DAD analysis, tyrosinase assay, and kinetic analysis.

Peak no. Compounds UF affinity assay Tyrosinase assay Kinetic analysis

TBD (%) SBD (%) Screening results Conc. (mg mL−1 ) Inhibition (%) IC50 e (mM) Conc. (mg mL−1 ) Vmaxf (mOD min−1 ) Km f (mM) Ki f (mM) ˛-Valuef Inhibition type

3 Protocatechuic acid 41.85 ± 0.85 a

28.62 ± 1.30 + c
1 83.08 ± 8.03 2.53 ± 0.06 None 39.06 0.27 9.28 g
/ Competitive
0.5 72.54 ± 6.94 0.5 38.46 0.32
0.1 14.99 ± 2.09 0.1 39.84 0.47

5 Chlorogenic acid 61.77 ± 0.11 18.98 ± 1.09 + 1 80.24 ± 4.99 1.05 ± 0.06 None 42.37 0.31 0.90 2.78 Mixed-
0.5 62.82 ± 3.84 0.1 33.90 0.56 type
0.1 4.86 ± 0.21 0.5 25.84 0.65

10 3,5-di-O- 98.19 ± 0.01 10.83 ± 0.70 + 1 89.56 ± 8.56 1.07 ± 0.08 None 24.13 0.05 0.34 / Competitive
caffeoylquinic

Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328

acid
0.5 66.46 ± 5.94 0.1 24.08 0.29
0.1 13.37 ± 2.54 0.5 24.15 0.61

11 1,5-di-O- 96.89 ± 0.01 15.44 ± 1.05 + 1 84.70 ± 2.12 1.19 ± 0.03 None 28.41 0.09 1.10 / Competitive
Caffeoylquinic
acid
0.5 59.57 ± 1.54 0.1 28.99 0.34
0.1 14.18 ± 0.79 1 27.40 0.64

12 1,3-di-O- 79.87 ± 1.02 12.61 ± 2.76 −d 1 66.87 ± 3.57 1.67 ± 0.08 None 25.06 0.04 0.82 8.66 Mixed-
Caffeoylquinic type
acid
0.5 44.58 ± 2.64 0.1 20.00 0.29
0.1 10.54 ± 3.84 0.5 19.46 0.32

14 1,3,5-tri-O- 100.00 ± 0.00 4.72 ± 0.13 − 1 80.24 ± 4.24 1.16 ± 0.06 None 24.51 0.05 0.85 5.93 Mixed-
Caffeoylquinic type
acid
0.5 52.68 ± 2.12 0.1 17.45 0.31
0.1 4.46 ± 0.22 0.5 17.06 0.33

Arbutinb 1 35.98 ± 1.95 27.99 ± 1.14

0.5 23.43 ± 1.01
0.1 15.31 ± 0.94
a
Results are presented as means ± SD.
b
Arbutin was used as a positive control in tyrosinase assay.
c
‘+’ indicates that this compound was screened out by the UF-HPLC-DAD assay in this study.
d
‘−’ indicates that this compound was not screened out by the UF-HPLC-DAD assay in this study.
e
‘IC50 ’ indicates the concentration of extract necessary to decrease the initial concentration of substrate by 50%.
f
The kinetic parameters Vmax , Km , Ki , and ˛ values were calculated from the Lineweaver–Burk plot which are shown in Fig. S3.
g
There was no ˛ value in the competitive-type inhibitor.
 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328 327

worst as follows: resveratrol >kojic acid> arbutin. Similar patterns

were obtained after introducing arbutin, kojic acid, and resveratrol
in the control group, and the ligands were easily distinguished in
the resveratrol group (Fig. S2).
In theory, high-affinity competitive ligands will tightly bind to
the active site of the enzyme and prevent the binding of other com-
petitive ligands. However, noncompetitive and mixed-type ligands
generally cause an allosteric effect by binding to another inhibitory
site and, therefore, they have very limited use as enzyme blockers
even though they are also able to bind to the active site. Uncom-
petitive ligands were not considered because they bind only to
substrate-enzyme complexes and do not work in affinity assays.
Resveratrol, a high affinity competitive tyrosinase inhibitor, was
used as blocker in further experiments because it provided the
highest resolution.

3.2.3. Identification of tyrosinase specific inhibitors

The UF-HPLC-DAD screening for tyrosinase ligands from EFXF
was performed under above optimized conditions. The ultrafiltrate
was then subjected to HPLC analysis. The control experiment was
carried out with tyrosinase pre-incubated with resveratrol. The
results are shown in Fig. 5. Finally, compound 3 (protocatechuic
acid), 5 (chlorogenic acid), 10 (3,5-di-O-caffeoylquinic acid), and
11 (1,5-di-O-caffeoylquinic acid) were distinguished as candidate
ligands from a total of 14 peaks and identified by reference stan-
dards.

3.3. Validation of the tyrosinase inhibiting activities of the Fig. 7. a Calculation of the TBD for a compound with Ki = 1 mM by the equilibrium
identified compounds (13); b correlation between TBD and Ki , red circles are the mixed-type inhibitors did
not identify from the EFXF by UF-HPLC-DAD assay, purple circle is the mixed-type
inhibitor identified from EFXF by UF-HPLC-DAD assay, black circles are the com-
In order to validate the screened ligands, the components of
petitive inhibitors identified from EFXF by UF-HPLC-DAD assay. (For interpretation
EFXF were isolated and assessed by tyrosinase assay. As shown in of the references to colour in this figure legend, the reader is referred to the web
Table 2, compound 3, 5, 10, 11, 12 (1,3-di-O-caffeoylquinic acid), version of this article.)
and 14 (1,3,5-tri-O-caffeoylquinic acid) showed greater inhibitory
false positives. Our strategy for screening tyrosinase inhibitor from
activities than arbutin. And the tyrosinase inhibitory activities of
natural products is reliable.
the compound 10, 11, 12, and 14 were first time reported in the
present study. However, our results from UF-HPLC-DAD screening
3.4. Binding affinity of the identified compounds
only identified compounds 3, 5, 10, and 11 as candidate ligands. The
competitive inhibitor, resveratrol, was used as tyrosinase blocker
In the present study, a term, TBD, is defined which represents
and it tightly bound to the specific site of the enzyme to prevent
the binding affinity of the ligand towards tyrosinase. TBD can be
the binding of other specific ligands. Thus, in theory, only spe-
calculated by the equilibrium (1) showed in Section 2.4, in which
cific inhibitors should be distinguished using our UF-HPLC-DAD
Aa and Ab , the peak areas of free ligand in filtrate detected by HPLC,
method.
can represent the concentration of free ligand in the filtrate in the
A kinetic analysis was performed to confirm our hypothesis,
absence and presence of tyrosinase. Thus, Aa and Ab can be replaced
and the kinetic parameters and the Lineweaver-Burk plots are pre-
by ligand initial concentration [L]0 and ligand concentration [L],
sented in Table 2 and Fig. S3, respectively. These results show that
respectively. A new equilibrium was obtained as follows:
compounds 3, 10, and 11 were indeed competitive inhibitors while
compounds 5, 12, and 14 were mixed-type inhibitors. All competi- [L]0 − [L]
TBD (%) = × 100% (9)
tive inhibitors were identified by our UF-HPLC-DAD method as well [L]0
as one mixed-type inhibitor. We suggest that the identification of
Ki is the measurement of ligand binding affinity to enzyme and was
compound 5 was possible because mixed-type inhibitors can also
calculated by the Michaelis–Menten equation in present study. Ki
bind to the active site. Nevertheless, other mixed-type inhibitors
also can be represented by the equation showed below:
were not distinguished perhaps because they bind preferably to
another allosteric site rather than to the active site. [E] [L]
Ki = (10)
To obtain a direct three-dimensional view of the interaction [E × L]
between tyrosinase and the UF-HPLC-DAD assay identified ligands,
where [E] is the enzyme concentration, [E × L] is the enzyme-ligand
we calculated a simulated model of the complexes via the molec-
complex concentration. Moreover,
ular docking method (Fig. 6). According to the docking model,
coppers, which in tyrosinase play important roles for remaining [E] = [E]0 − [E × L] (11)
tyrosinase catalysis activity, showed interactions with all ligands;
[L] = [L]0 − [E × L] (12)
and all ligands identified from EFXF bind to the active site which
is same with resveratrol, even though their interacted residues are where [E]0 is the enzyme initial concentration. So that
different. These implications from the simulated complex struc- 
tures are in good accordance with our previous experimental data, [E]0 + [L]0 + Ki − ([E]0 − [L]0 + K i )2 + 4[L]0 K i
TBD(%) = × 100%
which resveratrol can prevent other ligands bind to the active site 2[L]0
and therefore can be used to distinguish the specific inhibitors from (13)
328 Z. Wang et al. / J. Chromatogr. B 1002 (2015) 319–328
The relationship between TBD, [E]0 , [L]0 , and Ki was established.
But we still cannot build the effective correlations between TBD and
Ki for comparison in the natural product affinity system, because
the [L]0 of each ligand is unknown, and the relative [E]0 to each lig-
and is also unknown. However, when we consolidate the Ki value,
we found that the higher [E]0 , the higher TBD; the lower [L]0 , the
higher TBD. As shown in Fig. 7a, the TBD will close to a constant once
[E]0 high than 20 nM and [L]0 lower than 0.7 mM. In our experiment,
the 25 nM of tyrosinase and 0.5 mg mL−1 of EFXF were used, which
can be considered that Ki will be the primary parameter affect the
TBD only. The correlations between Ki and TBD were calculated by
the experimental data and the computational data. As shown in
Fig. 7b, even though the experimental correlation is lower than the
computational one, the trends of them are same. And good cor-
relation between Ki and TBD was obtained in our strategy, which
higher affinity compound will have a higher TBD. The differences
between computational and experimental results may be caused
by the effect of substrate in Ki calculation.
4. Conclusion
In this study, a UF-HPLC-DAD screening strategy was proposed
to identify tyrosinase specific inhibitors from EFXF. Using this strat-
egy, protocatechuic acid, chlorogenic acid, 3,5-di-O-caffeoylquinic
acid, and 1,5-di-O-caffeoylquinic acid were successfully distin-
guished as tyrosinase inhibitors from the false negatives and the
false positives in a high resolution. Moreover, the data obtained
from experiment have high correlations to the inhibitory activi-
ties. The proposed strategy is simple, fast, cheap, high resolution,
and it could be used in high-throughput screening. In conclusion,
this strategy could greatly accelerate the discovery of new specific
active compounds from natural products.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgments
Thanks for Park Yoon Ha in the Institute of Natural Medicine,
Hallym University supporting us in this research. This research was
supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of
Education (NRF-2012R1A1A2008842) and Priority Research Cen-
ters Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Tech-
nology (NRF-2009-0094071), Hallym University Research Fund
(HRF-201504-008).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jchromb.2015.
08.030.
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