Inhibitory effects and molecular mechanism on mushroom tyrosinase by condensed tannins isolation from the fruit of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow

Article abstract of DOI:10.1016/j.ijbiomac.2020.09.259

The structure of extracted condensed tannin (CT) from the fruit of Sour jujube (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow) and the molecular mechanisms by which CT inhibits the activity of mushroom tyrosinase were investigated. The structure of CT was characterized by high performance liquid chromatography electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The kinetic assays were used to detect inhibition effect, type and mechanism. UV scanning, fluorescence quenching, copper interacting, o-quinone interaction and molecular docking assays were also used to reveal the molecular mechanisms by which CT inhibit tyrosinase. The results showed the structural units of CT containing afzelechin/epiafzelechin, catechin/epicatechin, and gallocatechin/epigallocatechin. Kinetic analysis showed that CT inhibits both the monophenolase and diphenolase activities of tyrosinase and exhibits reversible, mixed type mechanism. The fruit CT interacts primarily with the copper ions and specific amino acid residue (Asn191, Thr203, Ala202, Ser206, Met201, His194, His54, Glu182 and Ile42) in the active site of tyrosinase to disturb oxidation of substrates by tyrosinase. These results suggested the sour jujube fruit is a potential natural source of tyrosinase inhibitors, and has a potential to be used in food preservation, whitening cosmetics.

Full text of DOI:10.1016/j.ijbiomac.2020.09.259

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Inhibitory effects and molecular mechanism on mushroom
tyrosinase by condensed tannins isolation from the fruit of
Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow
Wei Song, Lu-Lu Liu, Yuan-Jing Ren, Shu-Dong Wei, Hai-Bo
Yang
PII:
S0141-8130(20)34622-5
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.09.259
BIOMAC 16889
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
11 July 2020
22 September 2020
30 September 2020
Please cite this article as: W. Song, L.-L. Liu, Y.-J. Ren, et al., Inhibitory effects and
molecular mechanism on mushroom tyrosinase by condensed tannins isolation from the
fruit of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow, International
Journal
of
Biological
Macromolecules
(2018),
https://doi.org/10.1016/
j.ijbiomac.2020.09.259
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Inhibitory effects and molecular mechanism on mushroom tyrosinase
by condensed tannins isolation from the fruit of Ziziphus jujuba Mill.
var. spinosa (Bunge) Hu ex H. F. Chow
Wei Song 1 , Lu-Lu Liu 2, Yuan-Jing Ren 2 , Shu-Dong Wei 2*, Hai-Bo Yang 1,3*
1 School of Life Science and Engineering, Henan University of Urban Construction,
Pingdingshan, Henan 467044, China; songwei@hncj.edu.cn
2 College of Life Science, Yangtze University, Jingzhou, Hubei 434025, China;
2378970509@qq.com, 1060739982@qq.com
3 Forestry College, Henan Agricultural University, Zhengzhou, Henan 450000, China；
30110508@hncj.edu.cn
*Correspondence: 30110508@hncj.edu.cn; weishudong2005@126.com
Abstract: The structure of extracted condensed tannin (CT) from the fruit of Sour
jujube （Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow）and the
molecular mechanisms by which CT inhibits the activity of mushroom tyrosinase were
investigated. The structure of CT was characterized by high performance liquid
chromatography electrospray ionization mass spectrometry, and matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry. The kinetic assays were used
to detect inhibition effect, type and mechanism. UV scanning, fluorescence
quenching, copper interacting, o-quinone interaction and molecular docking assays
were also used to reveal the molecular mechanisms by which CT inhibit tyrosinase. The
results showed the main structural units of CT contain afzelechin/epiafzelechin,
catechin/epicatechin, and atechin/epicatechin. Kinetic analysis showed that CT inhibits
both the monophenolase and diphenolase activities of tyrosinase and exhibits
reversible, mixed type mechanism. The fruit CT interacts primarily with the copper
ions and specific amino acid residue (Asn191, Thr203, Ala202, Ser206, Met201,
His194, His54, Glu182 and Ile42) in the active site of tyrosinase to disturb oxidation of
substrates by tyrosinase. These results suggested the sour jujube fruit is a potential
natural source of tyrosinase inhibitors, and has a potential to be used in food
preservation, whitening cosmetics.

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Keywords: tyrosinase; Sour jujube; inhibitor; molecular mechanism
1. Introduction
Tyrosinase, also known as a copper oxidase, a key enzyme to melanin synthesis,
catalyzes the hydroxylation of monophenol molecules to catechols, which is then
oxidized to DOPA. Excessive accumulation of melanin in the body leads to freckles,
chloasma, senile plaque, and other skin diseases[1]. In fruit and vegetable storage and
transportation, abnormal amounts of melanin can cause fruits and vegetables to brown,
decreasing their taste and nutritional value[2, 3]. Therefore, tyrosinase is closely tied to
our daily lives, and controlling tyrosinase is becoming an increasingly important topic
of study for agriculture, cosmetics, and drug therapy. Due to the ineffectiveness, side
effects from prolonged pharmaceutical use, food, health, and beauty industry usage,
traditionally synthesized tyrosinase inhibitors are being rejected by more and more
people. Meanwhile, due to the growing recognition that plant extracts are easily
degradable, environmentally friendly, have non-toxic side effects, and a relatively
affordable price, plant extracts with functional or active products are increasingly being
favored [4-8]. Therefore, it is of great significance for the food and pharmaceutical
industry to screen the anti- tyrosinase substances from natural products.
Sour jujube (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow) is a
deciduous shrub or small tree with a high nutritional and medicinal value, contains a
variety of bioactive substances, such as Sour jujube polysaccharide, flavonoids,
saponins, triterpenoids, alkaloids, cAMP, CGMP[9-11]. Several substances of jujube
kernel, a mature seed of dried jujube plants have been extracted[12-18]. However, there
are few reports concerning the sour jujube fruit’s bioactivities，in particular from the
fruit’s tannin. To fully understand and thus utilize the Sour jujube it is necessary to
extract CT from the fruit and investigate their bioactivities for anti-tyrosinase.
The CT from natural plant extracts is a kind of polyphenol polymer polymerized by
flavone-3-ol monomers including catechin, epicatechin, gallicatechin and epicatechin
in a certain proportion[19, 20]. CT is widely found in the tube bundle of roots, bark,

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leaves, flowers, fruit, and other organs with a large molecular weight of 1000-20000u
and complex structure. Their chemical properties and biological activities vary greatly
depending on their plant origin, monomer composition, polymerization degree, and
molecular interaction[21-23]. Currently, tannins of different plant species, including
Leucaena[24], pine needles[25], apple[26, 27], longan[28], showed good tyrosinase
inhibition. Unfortunately, some reported natural tyrosinase inhibitors in-vivo use is
restricted due to their insufficient tyrosinase inhibitory activity or the food safety
regulations[29]. Thus, looking for more effective and safer tannins to serve as
tyrosinase inhibitors is indeed feasible.
To the best of our knowledge, the possible effect of CT from Sour jujube fruit on
tyrosinase activity has not been described so far. Therefore, in this study, we chose to
isolate CT and identify its structure from Sour jujube’s fruit. Moreover, CT’s tyrosinase
inhibitory activity and molecular mechanism were investigated. This research would
provide scientific evidence for the utilization of the CT as antityrosinase agents. This
study suggested that CT from Sour jujube fruit is a good tyrosinase inhibitor and has
great potential for usage in fruit and vegetable preservation, medicine, and cosmetics
fields.
2. Materials and Method
2.1 Reagents
Mushroom tyrosinase (3030 U/mg), 3,4-dihydroxyphenylalanine (L-DOPA) and
HPLC standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other
reagents and solvents used were of analytical grade.
2.2 CT sample preparation
The young fruits (about 20 days after flowering) of Sour jujube were collected in
Xiangyun Park, Pingdingshan City, Henan Province, and were subsequently washed
and freeze-dried. The plant samples were ground to a powder and stored at -20℃. The
dried fruit powder (20 g each) was extracted three times with 70% (v/v) acetone at room
temperature, after which the acetone was removed by rotating evaporation at 38℃ The

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water phase was extracted with petroleum ether and ethyl acetate three times. The crude
extracts were dissolved with 50% methanol, and loaded onto the Sephadex LH-20
column (50 cm length × 4 cm i.d). The elution was run with a methanol (A)–water (B)
gradient (50% A, each 500 mL) at a constant flowrate of 1 mL/min. The purified tannin
liquid was freeze-dried and stored at -20℃. The content of CT was determined by the
butanol–HCl method.
2.3 HPLC-ESI-MS and MALDI-TOF MS analysis
Reversed-phase HPLC-ESI-MS analysis was detected by Agilent series 1200
system(Agilent, USA). The determination of MALDI-TOF MS was carried out on the
Burker Reflex III (Germany) with the nitrogen laser, and the laser wavelength were 337
nm. Procedures for HPLC-ESI-MS and MALDI-TOF MS were as described
previously[30].
2.4 Enzyme assay
The monophenolase and diphenolase activities assays were performed as previously
reported[30]. The range concentration of CT was 0-70 μg/mL. All experiments values
were determined at 475 nm using a UV-Vis spectrophotometer (Genesys 10S, USA).
The temperature of enzymatic reaction was controlled at 30 ℃, and water bath was
used to maintain a constant temperature. To describe the mixed-type inhibition
mechanism, the Lineweaver-Burk equation in double reciprocal form can be written
as:
Vm [S]
v 
[I]
[I]
Km (1 ) [S](1
)
KI
KIS
v: Enzymatic reaction rate, Km: Michaelis constant, Ki: inhibition constant of
inhibitor to free enzyme, Kis: inhibition constant of inhibitor on enzyme substrate
complex (ES), [S] : substrate concentration, [I] : inhibitor concentration.
2.5 UV Scanning Study
The experiment was carried out as described by Jimꢀnez-Atiꢀnzar et al. The process
of L-Tyr oxidation by tyrosinase was performed in the absence and presence of CT. The
data were recorded every minute following the addition of L-Tyr and enzyme (16.67

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μg/mL). The scanning wavelength was 200-800 nm.
2.6 Fluorescence Quenching
In this study, fluorescence spectra were measured on a fluorescence
spectrophotometer (F4600, Hitachi, Japan). The measurement conditions were
fluorescence emission wavelengths of 300−450 nm, excitation wavelengths of 290 nm,
and slit widths set to 2.5 nm for both the excitation and emission monochromators. For
the measurement of the enzyme-CT interactions, enzyme (40 μg/mL) in PBS in a final
volume of 0.4 mL was titrated with CT (0, 50, 100, 150 and 200 μg/mL).
2.7 The Chelating Ability between Cu2+ and CT
To detect the interaction between CT and tyrosinase, the chelating ability between
Cu2+ and CT was investigated. Data were acquired after the addition of the same
volume of CuCl2 each time using a UV-Vis spectrophotometer (Genesys 10S, USA).
The absorption spectra were recorded between 240 and 600 nm. The reaction system (3
mL) contained 100 μL of sample (3 mg/mL) solution, 100 μL of copper sulfate
solution with different concentrations (0, 20, 40, 60, 80 and 100 μM), and 2.8 mL of
sodium phosphate buffer (50 mM, pH 6.8).
2.8 Generation of the dopaquinone in absence enzyme
These experiments were carried out as described by Espin and Wichers8. Briefly,
L-DOPA (1 mg/ml) was oxidized by NaIO4 (1 mg/mL), and the yield of dopaquinone
was recorded using a UV-Vis spectrophotometer (Genesys 10S, USA) in the presence
of varying concentrations of CT (50, 100 μg/mL). The scanning wavelength was
240-800 nm.
2.9 Molecular docking
The selection of molecular docking methods was mainly determined by the use of
docking modules for drug design software. The X-ray structure of mushroom
tyrosinase (LWX2, RCSB Protein Data Bank) was used as the initial protein model for
molecular docking. We analyzed Dock with the commonly used software molecular
operation environment 2010 (MOE), for docking screening using drugs. MOE-DOCK
includes the following steps: (1) Import protein crystal structure, hydrogenation and
charge up, and energy minimization. (2) Find the binding site of the protein crystal
structure forms a Gauss contact surface around the binding site to make it a docking

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pocket for proteins. (3) Small molecule structure processing, energy optimization and
conformation generation. (4) Enter docking program．
3 3. Results
3.1 HPLC-ESI-MS Analysis
High-purity CT from Sour jujube fruits were obtained by organic solvent extraction
and column chromatography. Reversed-phase HPLC-ESI-MS further clarified the
chemical composition of the CT. The products of depolymerization were separated and
identified by reversed-phase HPLC-ESI-MS. HPLC showed that CT from the fruit of
Sour jujube was similar in basic structure, contain gallate catechin/epigallate catechin
(GC/EGC) and catechin/epicatechin (C/EC), and have C/EC as the main unit (Fig. 1 a).
3.2 MALDI-TOF MS analysis
MALDI-TOF MS was used to further explain the structural diversity of CT in terms
of the polymer structure. CT is a group of flavane-3 alcohol structure unit connections
by 4-8 (B) or 4-6 C-C key polydisperse polymers and is connected by a C-C and a
C-O-C key of the double bond to a lesser extent (A). As shown in Fig. 1 b, C/EC and
GC/EGC were common in the Plant CT unit, with two hydroxyls in both C3 and C4 are,
with C4 forming one more hydroxyl than C3, indicating that the molecular weight of
C4 is 16 Da more than C3, with 288 Da for the C3, 304 Da for C4, while some CT was
272 Da such as afzelechin/afzelechin (AF/EAF). The mass spectrometry results in Fig.
1b indicate that CT from the trimer (m/z 940.55) to fourteen polymers (m/z 4229.21)
distribution of adjacent poly had a fixed difference, 288 Da, 304 Da or 272 Da, showing
that the main structural units of CT contain C/EC as well as GC/EGC, and some
AF/EAF.

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Figure 1. Structure characterization of the CT. (a) Reversed-phase HPLC–ESI-MS
chromatograms. C/EC terminal unit catechin/epicatechin, C-thio/EC-thio and
GC-thio/EGC-thio represented extender unit catechin/epicatechin and gallate
catechin/epigallate catechin, respectively. BM represented benzyl mercaptan, TB
represented Thiolytic byproduct (b) MALDI-TOF-MS spectra of the CT. DP degree
of polymerization.
3.3 Detection of Inhibitory effect, mechanism and type
The reaction system uses L-Tyr as a substrate to detect the monophenolase activity in
the presence of different concentrations of CT, which is used as the inhibitor. At the
initial stage of the reaction, the product yield increases slowly and then becomes linear.
The reaction system reaches a constant slope, which shows that the reaction is stable.
The steady-state reaction rate of tyrosinase is equal to the slope of the line. The results
show that in the presence of increasing amounts of CT, the steady-state rate was
significantly decreased, indicating that CT can effectively reduce the monophenolase

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activity in a dose-dependent manner (Fig. 2 a). The inhibitory concentration for the
monophenolase activity decreased to 50% was 136.22 μg/mL.
The relationship between mushroom tyrosinase and CT is shown as Fig. 2 b. The
results showed that L-DOPA was oxidized without lag time and that diphenolase
activities were remarkably decreased in the presence of an increasing CT concentration.
The concentration of inhibitors leading to half activity of enzyme was 57.67 μg/mL.
There was a set of straight lines that all passed through the origin (Fig. 2 c). With the
concentration of CT increasing, the slope of line was decreased, indicating that the
effect of CT on tyrosinase was reversible. As shown in Fig. 2 d, a set of straight lines
intersects with the second quadrant. The Km values were extended, and the Vm values
were reduced in the presence of increasing amounts of CT. We calculated the values of
the inhibition constants (KI, 24.44 μg/mL) and of the enzyme−substrate complexes
(KIS, 70.97 µg/mL) independently. KI< KIS and KIS is 2.9 times as large as KI.

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Figure 2. Determination of the inhibitory effects of CT on the monophenolase and
diphenolase activity of mushroom tyrosinase. (a) Inhibitory activity of the CT on
monophenolase activity of mushroom tyrosinase. (I) Progress curve for the oxidation of
L-Tyr by the enzyme. The concentrations of the inhibitor for curves 0-4 are 0, 40, 66.67,
106.67, and 133.33 µg/mL. (II) Effects of condensed tannins on the lag time of mushroom
tyrosinase. (III) Effects of condensed tannins or the steady-state rate of monophenolase;
Vss represented the steady-state rate of monophenolase. (b) Inhibitory activity of the
condensed tannins on diphenolase activity of mushroom tyrosinase. (c-d) Determination
of the inhibitory mechanism (c) and type (d) of condensed tannins extracted from the fruit
of Sour jujube. L-dopa was used as the substrate to detect the inhibition type and
mechanism of of tyrosinase diphenolase. (I) Lineweaver-Burk plots for diphenolase
activity. (II) The plot of slope versus the concentration of the condensed tannins for
determining the inhibition constants KI. (III) The plot of intercept versus the
concentration of the condensed tannins for determining the inhibition constants KIS. 0-4
represents the concentrations of the condensed tannins, which were 0, 13.33, 26.67, and
53.33, 66.67 µg/mL.
3.4 Hydroxylation of L-Lyr and Oxidation of L-DOPA influenced by the Presence of
CT
To further confirm that CT from the fruit of Sour jujube interfered with the
hydroxylation of L-Lyr by tyrosinase, spectra were obtained during the oxidation of
L-Tyr in the absence and presence of CT (100 μg/mL). We found that a series of
absorption peaks appear at 475 nm when CT is not added. However, in the presence of
CT, the intensity of the peak gradually decreased (Fig.3 b).
The oxidization of L-DOPA by NaIO4 in the absence and presence of the CT was
also investigated. As shown in Fig. 3 c, there were no characteristic peaks without
NaIO4; Conversely, a characteristic peak appeared at 475 nm while NaIO4 was
involved. However, the intensity of the peak shows an obvious decrease with the
addition of varying concentrations of CT (50 and 100 μg/mL).

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Figure 3. The change in the spectra obtained during the oxidation of L-Tyr and
L-DOPA with the CT by mushroom tyrosinase. (a-b) The oxidation of L-Tyr absence
and presence of CT (100 μg/mL) respectively (c) UV-Vis spectra for the oxidation
of L-DOPA. The assay was performed in 3 ml of 50 mM sodium phosphate buffer
(pH 6.8) at 30℃.
3.5 The Chelating Ability between Cu2+ and CT
In this study, the direct interaction between CT and copper ions was detected by full
wavelength scanning (240-600 nm). The results showed that with the increase of
copper ion concentration，the interaction of CT with copper ions appeared as a slight
redshift phenomenon (Fig. 4), suggesting that CT had good copper ion reduction ability
and metal chelation ability on the enzyme.

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Fig. 4 The metal chelating ability of CT . The concentration of CT is 100 μg/mL. a, b,
and c represented CT, catechin (positive control), and PBS (negative control).
3.6 Analysis of fluorescence intensity change
As shown in Fig. 5 a, an obvious peak appeared at 337 nm with an excitation of 290
nm. As the CT concentration increased, the fluorescence intensity of tyrosinase
weakened significantly (Fig. 5 c). However, there was no significant red shift appeared
(Fig. 5 b). A good linear is obtained by Stern-Volmer plot in Fig. 5 d, which could
calculate the quenching constant（Kq）and the Stern–Volmer quenching constan（t Ksv）
values of 3.35×1013 LM-1s-1 and 3.35×103 M-1, respectively. The Kq value is much
-1
higher than 2.0 × 1010 L Mol s -1, so the quenching process is a static quenching
process.

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Figure 5. Changes in tyrosinase intrinsic fluorescence. (a) Fluorescence emission
spectra of mushroom tyrosinase in the presence of CT with different concentrations. (b)
Maximum florescence intensity changes (%); (c) Relative florescence intensity
changes; (d) the Stern–Volmer plot for the fluorescence quenching of tyrosinase. F0 and
F are the fluorescence intensities before and after the addition of the CT.
3.7 Molecular docking
The protein crystal structure was optimized and the ligand was connected to the
target active cavity. The binding force was analyzed according to the docking results, as
shown in Fig. 6. L-Tyr, L-Dopa, catechin (monomers of the CT) and target (tyrosinase)
were selected for the docking simulation to detect the binding of small molecular
ligands to the target active sites. The energy scores of the most stable docking models
for L-Tyr, L-dopa, catechin and catechin trimer were - 11.43, - 14.39, - 18.48, - 22.59
kcal / mol, respectively.

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Figure 6. Molecular dock between condensed tannins and mushroom tyrosinase. L-Tyr
(a), L-Dopa (b), catechin (c), catechin trimer (d).
The molecular docking results showed that the ligands could also chelate the
copper ions in the enzyme active site(Fig. 6). Furthermore, the hydroxyl radical and the
benzene ring structure in the structure of the catechin ligand were the key factors for the
interaction between ligands and protein. Among them, the 3 position hydroxyl attached
to hydroxylphenyl formed H-bonds with Thr203, Ala202 and Met201; the 4 position
hydroxyl formed hydrogen bonds with Met201; the 3 position hydroxyl in benzopyran
formed H-bonds with Glu182, as well as the 5 position hydroxyl with Ile42; oxygen and
hydroxyl oxygen connected to the benzopyran skeleton formed weak electrostatic

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interactions with Asn191 and His54; formed a π-π stacking interaction between His194
and benzene in ligands. In addition, the catechin ligands can be well placed within the
hydrophobic pocket consisting of Asn191, Thr203, Ala202, Ser206, Met201, His194,
His54, Glu182 and Ile42, to realize the strong interaction between protein and ligand.
4. Discussion
Tyrosinase is a copper oxidase that has three states of tyrosinase, including oxidation,
reduction and deoxidization. It is a key enzyme in the synthesis of melanin, which
catalyzes the hydroxylation of monophenol to catechol, which is then oxidized to
DOPA[1, 31].This study reported the inhibitory effect of CT from the fruit of Sour
jujubeon both monophenolic dehydrogenase and bisphenol oxygenase activity of
tyrosinase. Regarding monophenolase activity, CT can effectively reduce the
monophenolase steady-state activity remarkable in a dose-dependent manner,with
changing the lag time (Fig. 2). The presence of o-diphenol in the monophenolase
reaction system shortens the lag time, because the lag time is the time required to reach
the steady o-diphenol concentration. For diphenolase activity, types of tyrosinase
inhibition mechanisms under the presence of CT from the fruit of Sour jujube was
reversible suggesting that CT just reduced the speed of the enzymatic reaction, but did
not affect the occurrence of the reaction. The concentration of inhibitors leading to the
half activity of the enzyme was 57.67 μg/mL suggesting that CT from the fruit of Sour
jujube had a strong tyrosinase-inhibiting activity, the several studies showed methanol
extracts from sorghum distillery residue [32], ergothioneine [33] and arbutin[34] all
can inhibit tyrosinase activity, with IC 50 were 580 μg/mL, 1025 μg/mL, 462.4 μg/mL
respectively. Obviously, CT from the fruit of Sour jujube had better tyrosinase
inhibition ability than some inhibitors in the previous studies.
Further investigated the kinetic mechanism showed that the CT changed the
maximum reaction rate of the enzymatic reaction velocity (Vm) and Michaelis constant
(Km) and was a mix type inhibitor. As shown in Fig. 4b, a group of slope intercept
changes but intersects in the two quadrants, which indicates that Km increases with the
increase of concentration, and Vm decreases with the increase of concentration, which
is a mixed competitive inhibition. The results show that the CT not only competes with
the substrate for the active site of enzyme, but also binds to the enzyme substrate

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complex. It is indicating that CT affected not only the affinity of the enzyme for the
substrate, but also the ability of enzyme catalysis. This mode of the inhibitory
mechanism also appeared in many other tyrosinase inhibitors from plants[35-38].
Furthermore, KI< KIS indicating that the binding capacity of inhibitors to free enzymes
was much stronger than binding to the enzyme-substrate complex[39].
After we knew inhibition type and kinetic mechanism of CT from the fruit of Sour
jujube on tyrosinase activity, however, what is its specific molecular mechanism? So
the UV scanning, o-quinone interaction, copper chelating, fluorescence quenching, and
molecular docking assays were to further unravel and confirm the molecular
mechanism of anti-tyrosinase by CT.
Tyrosinase has a unique dual catalytic function. Firstly, it catalyzes the
hydroxylation of L-tyrosine to form L-dopa, then oxidizes L-dopa to form DOPA
quinone, and finally forms melanin[40]. UV scanning and o-quinone interaction assay
showed that hydroxylation of L-Lyr and oxidation of L-DOPA influenced by the
Presence of CT. Particularly, L-DOPA alone did not show any characteristic peaks,
while the oxidation of L-DOPA by NaIO4 gave rises to the corresponding o-quinones
with a characteristic peak at 475 nm, the peak value was negatively correlated with the
concentration of CT. It revealed that CT inhibits tyrosinase activity by not only
disturbing the formation of L-DOPA, but also reducing o-quinone generation. It
suggests that the inhibition of CT on the double catalysis of tyrosinase is
omnidirectional.
Most of the tyrosinase inhibitors have the same characteristics of binding copper ions
in the active center of the enzyme, for instance, kojic acid and chalcones. Moreover, it
is reported that CT has chelated metal ions[41-43], and the two copper ions are within
the active site of tyrosinase. Thus, we asked if the inhibition mechanism of CT involves
interacting with copper ions of the enzyme. The copper chelating assay performed to
confirm the direct interaction between CT copper ions. The results indicated CT alters
the conformation of tyrosinase by form a CT-copper complex, which may be one of the
main reasons for CT inhibition of tyrosinase activity.
The hydroxyl groups of CT have a critical effect on tyrosinase activity[44, 45].
Through HPLC and MALDI analysis, we found that CT from Sour jujube fruit is
mainly composed of catechin and epicatechin containing two hydroxyl groups, which

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may be one of the important reasons for the inhibition of tyrosinase by CT. Several
types of researches showed fluorescence quenching assay often used in the interaction
between protein and small molecules. With the CT concentration increased, the
fluorescence intensity of tyrosinase weakened significantly indicating that the quencher
interacted with the fluorophore. However, there was no apparent blueshift phenomenon
appeared in the maximum emission spectra of tyrosinase in the presence of CT.
Therefore, the combination of tyrosinase and CT only affected the fluorophore
microenvironment of the tyrosinase, but not changed the direction and structure of the
tyrosinase.
To further explain inhibitory mechanism between CT with mushroom tyrosinase,
MOE2010 was adopted to stimulated the tyrosinase with L-Tyr, L-DOPA, catechin,
and trimer of catechin. The docking simulation revealed that the L-DOPA, catechin
and trimer of catechin could chelate the copper ions in the enzyme active site, whereas
L-Tyr could not chelate the copper ions. These results were supported the detection
result of copper chelating capacity. Additionally, there are six relatively conserved
histidine residues within the active site of tyrosinase, each of the three subunits is
complexed with one copper ion to form a coordination structure of binuclear copper as
the auxiliary group. In contrast, the two copper ions are connected by oxygen
bridges[46-48]. Because of the formation of the van der Waals force, the hydrophobic
and hydrogen bonding interactions between ligand compounds, and the amino acid
residues around the target protein receptor pockets, the ligand compounds can be
closely integrated with the target protein. It is also possible that the presence of these
forces enables compounds to exert their biological activity. The amino acids in
tyrosinase forming interaction with catechin ligands were Asn191, Thr203, Ala202,
Ser206, Met201, His194, His54, Glu182 and Ile42. The molecular docking results well
support the mixed inhibition type we observed, because this inhibition type generally
appeared when the inhibitor has multiple possible binding sites. The model of
molecular docking and corresponding inhibition mechanism was also found in those
reported in hot spots[42, 43, 49]. Therefore, the molecular docking model of tyrosinase
with CT was credible and could be valid and feasible for screening and identification of
more lead complexes of tyrosinase inhibitors.
5. Conclusion

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In conclusion, the CT from the Sour jujube fruit displayed efficient both
monophenolase and diphenolase inhibitory activities of tyrosinase. The inhibitory
mechanism by which the CT inhibits tyrosinase may be through binding to the specific
amino acid residues or to the copper ion of the active site of tyrosinase, which are two
ways to disperse the acylation of L-Try and the oxidation of L-Dopa, ultimately affect
the synthesis of melanin. Therefore, The sour jujube fruit is a potential natural source of
tyrosinase inhibitors, and has the potential to be used in food preservation, whitening
cosmetics, and so on. This research contributed to better understand the inhibition
mechanism of tyrosinase inhibitors by the interaction between tyrosinase and the
inhibitors.
Funding: This work was supported by grants from the the Science and Technology Research Project
from Hubei Provincial Education Department (D20191301), National Nature Science Foundation of
China Grant (No. 81402309), the Key scientific research projects of colleges and universities in Henan
Province (No. 21A180003) and Henan University of Urban Construction Academic technology leader
program（xs2019006-HY）.
Author Contributions: Data curation, Wei Song; Formal analysis, Yuan-Jing Ren; Funding
acquisition, Wei Song and Hai-Bo Yang; Investigation, Wei Song and Yuan-Jing Ren; Project
administration, Lu-Lu Liu, Shu-Dong Wei and Hai-Bo Yang; Resources, Lu-Lu Liu; Supervision,
Shu-Dong Wei and Hai-Bo Yang; Writing – original draft, Wei Song; Writing – review & editing,
Hai-Bo Yang.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations:
CT
soluble condensed tannin
Sour jujube
KI
Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow
inhibition constant
KIS
inhibition constant
NaIO4
Sodium periodate
HPLC-ESI-MS high performance liquid chromatography electrospray ionization mass spectrometry
GC/EGC
C/EC
Kq
gallate catechin/epigallate catechin
catechin/epicatechin
the quenching constant
Ksv
the Stern–Volmer quenching constant
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Author statement:
Song Wei：Data curation，Funding acquisition，Investigation，Writing – original draft. Ren Yuan-Jing:
Formal analysis, Investigation. Liu Lu-Lu: Resources, Project administration. Wei Shu-Dong: Project
administration , Writing – review & editing; Yang Hai-Bo: Project administration, Supervision, Funding
acquisition , Writing – review & editing

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Graphical abstract
CT from the fruit of Sour jujube inhibit the activity of tyrosinase through simultaneous
interaction with its copper ions and specific amino acid residues.