Inhibition of xanthine oxidase by theaflavin: Possible mechanism for anti-hyperuricaemia effect in mice

Article abstract of DOI:10.1016/j.procbio.2020.06.024

Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Excessive production of uric acid leads to hyperuricaemia. Due to the serious side effects of allopurinol, it is an urgent need to explore new XO inhibitors. Herein, the effects of theaflavin (TF1) on XO and anti-hyperuricaemia effect in hyperuricemic mice were investigated. Kinetic analysis indicate that TF1 is a reversible competitive inhibitor and has a significant inhibitory effect on XO with an IC₅₀ value of 63.17 ± 0.13 μmol/L. Analysis of fluorescence spectra suggests that TF1 causes the obvious fluorescence quenching of XO, which is mainly driven by hydrophobic interactions and hydrogen bonds. Docking studies demonstrate that TF1 interacts with dozens of amino acid residues surrounded in the active cavity of XO, including Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802, Pro-1076, Leu-873, Leu-1014, Asn-768, Leu-648 and Phe-649. The inhibitory mechanism may be the insertion of TF1 into the active site of XO, which hinders the substrate xanthine to enter into the site. Furthermore, the results from animal experiments demonstrate that TF1 is effective in reducing serum uric acid in mice. These findings suggest that TF1 may be a potential drug candidate for the treatment of hyperuricaemia.

Full text of DOI:10.1016/j.procbio.2020.06.024

Journal Pre-proof
Inhibition of xanthine oxidase by theaflavin: possible mechanism for
anti-hyperuricaemia effect in mice
Jianmin Chen (Conceptualization) (Methodology) (Writing - original
draft), Qinglian Li (Software) (Investigation), Yaling Ye (Investigation)
(Data curation), Mengnan Ran (Supervision) (Data curation),
Zhipeng Ruan (Supervision) (Validation), Nan Jin (Writing - review
and editing)
PII:
S1359-5113(20)30145-8
DOI:
https://doi.org/10.1016/j.procbio.2020.06.024
PRBI 12075
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Process Biochemistry
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Revised Date:
Accepted Date:
30 January 2020
13 May 2020
25 June 2020
Please cite this article as: Chen J, Li Q, Ye Y, Ran M, Ruan Z, Jin N, Inhibition of xanthine
oxidase by theaflavin: possible mechanism for anti-hyperuricaemia effect in mice, Process
Biochemistry (2020), doi: https://doi.org/10.1016/j.procbio.2020.06.024
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Inhibition of xanthine oxidase by theaflavin: possible
mechanism for anti-hyperuricaemia effect in mice
Jianmin Chen1,2*, Qinglian Li1, Yaling Ye1, Mengnan Ran1, Zhipeng Ruan1,2, Nan
Jin1,2
1. School of Pharmacy and Medical technology, Putian University, Fujian, China
2. Key Laboratory of Pharmaceutical Analysis and Laboratory Medicine (Putian University),
Fujian Province University, Fujian, China.
*Corresponding author：
School of Pharmacy and Medical technology, Putian University, 351100, Fujian, China
Tel: +86 0594 2787689; Fax: +86 0594 2787689
E-mail address: chenjianmin1985@sina.com ORCID：0000-0003-2934-1493

Graphical abstract
Highlights
 Theaflavin reversibly inhibited xanthine oxidase activity in a
competitive manner.
 Theaflavin quenched the fluorescence of the enzyme by forming
complex with it.
 There was a single class of binding site on xanthine oxidase for
Theaflavin.
 Theaflavin occupied the catalytic site of the enzyme to avoid entry of
xanthine.
 Theaflavin presented uric acid reducing effect in hyperuricemic mice.

Abstract:
Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and then
to uric acid. Excessive production of uric acid leads to hyperuricaemia. Due to the
serious side effects of allopurinol, it is an urgent need to explore new XO inhibitors.
Herein, the effects of theaflavin (TF1) on XO and anti-hyperuricaemia effect in
hyperuricemic mice were investigated. Kinetic analysis indicate that TF1 is a
reversible competitive inhibitor and has a significant inhibitory effect on XO with an
IC50 value of 63.17 ± 0.13 μmol/L. Analysis of fluorescence spectra suggests that TF1
causes the obvious fluorescence quenching of XO, which is mainly driven by
hydrophobic interactions and hydrogen bonds. Docking studies demonstrate that TF1
interacts with dozens of amino acid residues surrounded in the active cavity of XO,
including Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802, Pro-1076,
Leu-873, Leu-1014, Asn-768, Leu-648 and Phe-649. The inhibitory mechanism may
be the insertion of TF1 into the active site of XO, which hinders the substrate xanthine
to enter into the site. Furthermore, the results from animal experiments demonstrate
that TF1 is effective in reducing serum uric acid in mice. These findings suggest that
TF1 may be a potential drug candidate for the treatment of hyperuricaemia.
Key words: xanthine oxidase; theaflavin; inhibitory mechanism; molecular docking;
anti-hyperuricaemia

1 Introduction
Xanthine oxidase (XO, EC 1.1.3.22), a member of xanthine oxidoreductase group, is
found widely in mammalian tissues such as liver and intestine[1, 2]. It is a molecular
mass 290 kD homodimer with each monomer containing two [2Fe-2S] centers, a
flavin adenine dinucleotide cofactor and a molybdenum atom[3]. The molybdopterin
center (Mo-pt) of each monomer acts independently during the process of catalysis,
which catalyze the oxidation of xanthine to uric acid, superoxide anions and hydrogen
peroxide in the presence of molecular oxygen[4]. The overproduction of uric acid in
the serum will lead to hyperuricaemia, which increases the risk of the condition
including gout, cardiovascular disease, chronic kidney disease and other metabolic
disorders[5, 6]. Therefore, XO inhibitors may have the potential for the prevention or
treatment of these diseases. Allopurinol is a commonly used drug for the treatment of
hyperuricaemia and gout, but it may cause some serious side effects, including
hepatitis, kidney disease, allergy and rash[7, 8]. Thus, the screening of new natural
XO inhibitors should be further explored to provide more effective safe drugs to
clinical application.
Flavonoids, various compounds found naturally in many fruits, vegetables, wine,
chocolate and tea, have potential inhibitory effects on XO, and the recent progress in
this field has been summarized [9]. Black tea made from the fresh leaves of Camellia
sinensis via a fermentation process is the most popular tea in the world[10].
Theaflavins (TFs) are formed by the oxidation of flavanols (catechins and
gallocatechins), a process called fermentation during the manufacture of tea, and have

great contribution to the color and flavor of black tea[11]. TFs consist of theaflavin
and its galloyl esters: TF1, theaflavin-3-gallate, theaflavin-3′-gallate, and
theaflavin-3,3′-digallate[11]. TFs have been reported to have various biological
activities, such as antioxidant activity[12], antiviral activity[13], antiobesity
activity[14] , anticancer activity[15], anti-inflammatory effects[16], antidiabetic
effects[17], and the bone loss in models of osteoporosis[18]. Although TFs have been
reported to exhibit inhibitory effects on XO[19], the inhibitory types and mechanisms
of TFs have not been investigated comprehensively so far. In addition, the effect of
black tea aqueous extracts on hyperuricaemia has been reported recently[20], but
whether the single component of the extracts may reduce the level of serum uric acid
in hyperuricaemia animal model has still not been studied.
Therefore, in this work, we investigated the effects of theaflavin (TF1) on the activity
of XO via using spectroscopy and molecular docking, as well as on the level of serum
uric acid in hyperuricemic mice. We choose TF1 as the model drug, because it has the
basic structure of TFs. The purpose of this study is to elucidate the inherent inhibitory
mechanism of TF1 on the activity of XO, which may be helpful to design novel
enzyme inhibitors with higher efficiency and better safety. In addition, the results of
animal experiments will further provide fundamental data for TF1 in the treatment of
hyperuricaemia and other related metabolic diseases.
2. Materials and methods
2.1 Materials
Theaflavin (＞98%) was obtained from Kai-lai Biological Engineering Co., Ltd

(Xi'an, China). XO (35.7 units/mL, EC 1.17.3.2) derived from bovine milk was
purchased from Sigma-Aldrich (St. Louis, MO, USA) without further purification.
Xanthine, uric acid, phosphotungstic acid, lithium sulfate and potassium oxonate were
bought from Aladdin Bio-Chem Technology Co.,LTD (Shanghai, China). All other
chemicals used in this study were all analytical grade. During the whole research
process, a Milli-Q-Plus ultra-pure water system of Millipore (Sartorius 611, Germany)
was used to obtain the water needed in the experiments.
2.2 Inhibitory effects of TF1 on the activity of XO
The activity of XO was tested according to the method previously described with
some modifications[21]. Briefly, 100 μL of the test compound in DMSO and 200 μL
of XO (0.04 U/mL) in 0.2 mol/L PBS were mixed, and the mixture was stand for 5
min at room temperature. The control group was carried out by adding the equal
volume solvent instead of compound itself. The reaction was started by pouring 2.7
mL of 0.1 mmol/L xanthine in PBS to the mixture. The OD (optical density) values of
the mixtures were recorded at the wavelength of 290 nm per minute at room
temperature, where allopurinol was used as a positive control. Each test compound
was repeated three times. Enzyme activity determined in the absence of inhibitor was
defined as 100%. The inhibition was calculated according to the following formula:
inhibition (%) = [(reaction rate of control - reaction rate of sample)/ reaction rate of
control]×100%.
2.3 Kinetic analysis for the inhibition of competitive type
To describe and study the inhibition mechanism of competitive type, the

Lineweaver–Burk (LB) equation can be expressed as:
1
퐾
1
1
푚
=
(1 + [퐾I]) [S]
+
(1)
(2)
ν
V
max
V
푖
max
Secondary plots can be constructed from
퐾
[I]
퐾
푚
퐾mapp
=
+
,
max
m
V
max
퐾
푖
V
Where ν, Km and Ki represent the rate of the enzymatic reaction, the
Michaelis–Menten constant and inhibition constant, respectively. The concentrations
of inhibitors and substrates are expressed in terms of [I] and [S], respectively. The
linear regression plot of 1/ν vs. 1/[S] can be drawn to obtain the value of apparent
Michaelis–Menten constant (Kampp ). If the secondary plot of Kampp vs. [I] is linearly fitted,
suggesting that a single inhibition site or a single class of inhibition site[22].
2.4 Intrinsic fluorescence quenching
Spectrofluorometer FS5 (Edinburgh Instruments, England) equipped with a
thermostat bath were employed to record the fluorescence emission spectra. Add a 2.0
mL solution containing 0.02 U/mL XO to a quartz cuvette, and then use a
micropipette to continuously add 0.2 mmol/L TF1 solution for giving a concentration
ranged from 0 to 57.14 μmol/L. The mixture was then put in the thermostatic bath for
5 minutes to reach equilibrium at various temperatures (288, 298 or 308 K). After
excitation at 280 nm, fluorescence emission spectra were obtained at 290–500 nm. To
correct the background fluorescence, the fluorescence of PBS was subtracted. In
consideration of absorption of excitation light and re-absorption of emitted light, all
data of the fluorescence obtained from these experiments were corrected by the
following equation: [23]

퐹 = 퐹푚푒(퐴 +퐴 )/2
(3)
1
2
푐
The corrected and measured fluorescence are expressed in the terms of Fc and Fm,
respectively. A1 and A2 are the absorbance of TF1 at excitation and emission
wavelengths, respectively.
2.5 Molecular simulation
The probable interaction between TF1 and XO was further studied by using molecular
docking technology (AutoDock, version 4.2.6). The 3D conformer of TF1 was
downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/compound), and the
structure of XO (PDB ID: 1FIQ) was obtained from the RCSB PDB
(http://www.rcsb.org/pdb)[3]. In order to perform the docking simulation, a grid box
with dimensions of 90 Å×90 Å×120 Å was defined to make sure the whole active
center enclosed. The grid maps with a grid spacing of 0.375 Å were calculated via
using AutoGrid. Lamarckian genetic algorithm (LGA) was used for docking
calculation, and the number of runs was set to 100 times. The docking conformation
with the lowest binding energy among all clusters and/or in the largest cluster was
selected for further analysis, which adopted the PyMOL molecular graphics system.
LigPlot analysis is usually performed to understand the hydrophobic interaction
between the ligand and enzyme[24], so the interaction between TF1 and XO was
further analyzed via using software LigPlot+ (version 1.4.5).
2.6 Effects of TF1 on hyperuricaemia in mice
All animal experiments were performed according to the agreement of the Animal
Protection and Use Committee of Putian University [NO. 2019(14)] and the ARRIVE

guidelines. Before the experiments, ICR mice (25-28g) were acclimated for at least 1
week. The method used to evaluate the effects of TF1 on animal hyperuricemic model
was followed by the previous reported method with some adjustments[25]. There
were five groups with six mice per group (n = 6) totally. Four groups, including the
control, TF1 (100 mg/kg), TF1 (200 mg/kg) and allopurinol group, were injected
intraperitoneally with potassium oxonate (PO) at a dosage of 250 mg/kg to raise the
level of serum uric acid. Besides, a normal group was injected intraperitoneally with 1
ml 0.9% NaCl. After one hour, the mice in the TF1 (100 mg/kg) and TF1 (200 mg/kg)
group were administrated TF1 intragastrically; in allopurinol group were
administrated intragastrically in a dose of 20 mg/kg; in control group were
administrated saline intragastrically. Two hours after PO induction, the whole blood
samples of mice were collected. Blood was coagulated at room temperature for 1 hour,
and then centrifuged at 3000 rpm for 5 mins to obtain serum. The level of serum uric
acid was tested by the phosphotungstic acid method reported previously with some
modifications[26].
2.7 Statistical analysis
The in vitro effects of TF1 on the activity of XO were evaluated three times (n = 3),
while the animal studies included six mice in each group (n = 6). The results obtained
were expressed as means ± standard deviation (SD). To determine the significant
difference at p < 0.05 or p < 0.01, one-way analysis of variance was performed by
using EXCEL software (version 2007, Microsoft, USA) followed by multiple tests.
3 Results and discussion

3.1 Reversible inhibition effect of TF1 on XO
Fig. 1A shows the inhibitory effects of allopurinol (positive control) and TF1 on the
activity of XO, indicating that both of them significantly inhibited the activity of XO
with a positive correlation between the effects and dosages. Specifically, TF1
decreased the activity of XO to approximately 20% when its concentration reached to
166.67 μmol/L. Loss of 50% enzyme activity (IC50) was occurred when the
concentration of TF1 was 63.17 ± 0.13 μmol/L (that of allopurinol was 4.43 ± 0.06
μmol/L), suggesting that TF1 was also an effective XO inhibitor. TF1 and its galloyl
esters account for about 2% of the dried water extract of black tea, and they are
proved to be safe by tea consumption over a long history worldwide[27]. Therefore,
due to the serious side effects of allopurinol[7], TF1 may be an alternative in the
treatment of hyperuricaemia and gout.
As shown in Fig.1B, the curves of ν vs. [XO] were obtained at different
concentrations of TF1, and proved that it mediated inhibition was reversible. The
characteristic of good linearity is observed in the curves, all of which pass through the
origin. In addition, with an increase of the concentration of TF1, the slope of the plots
declines and it suggest that TF1 does not reduce the quantity of effective enzymes, but
decrease the activity of all enzymes available. These results indicate that the
inhibitory effect of TF1 on the activity of XO through non-covalent intermolecular
interactions is reversible.
3.2 Inhibition kinetic of TF1 on the activity of XO
LB double reciprocal plots were employed to analyze the inhibitory type of TF1

against XO. As shown in Fig.1C, with the increase of TF1 concentration, the vertical
axis intercept (1/Vmax) of the LB plots remains unchanged, whereas the horizontal
axis intercept (-1/Km) increases; these results indicate that TF1 locates directly in the
active center of XO as a typical competitive inhibitor to compete with the substrate
(xanthine) in the catalytic process[28]. The secondary plot (Fig.1D) is linearly fitted,
indicating only a single class of inhibition sites for TF1 against XO. The value of
inhibition constant (Ki) was calculated to be (9.96 ± 0.94) × 10-5 mol/L.
3.3 Effects of TF1 on the fluorescence of XO
Fluorescence quenching was used to further study the interaction between TF1 and
XO. The fluorescence emission spectra of XO in the absence and presence of TF1 are
shown in Fig.2A and 2B. Obviously, due to the tryptophan and tyrosine residues, XO
exhibits a strong intrinsic fluorescence emission peak at 334 nm[29]. When
the concentration of TF1 was increased, the fluorescence intensity of XO decreased
dramatically with the emission peak unchanged, suggesting that TF1 interacted with
XO and quenched its fluorescence.
In order to confirm the quenching mechanism of the combination of TF1 and XO, the
data of fluorescence quenching had been further processed by the following equation
(Stern-Volmer):
⁄
[ ]
퐹0 퐹 = 1 + 퐾qτ0 Q = 1 + 퐾sv[Q]
(4)
where F0 and F are the fluorescence intensities before and after the addition of TF1,
respectively. Kq, τ0, [Q] and KSV are the quenching rate constant of the biomolecule,
the average lifetime of the fluorophore (τ0=10-8 s), the concentration of TF1 and the

dynamic quenching constant of Stern–Volmer, respectively. As shown in Fig.2C, the
plots of F0/ F vs. [Q] all have excellent linearity within the studied concentrations,
indicating that the formation of TF1–XO complex is a single quenching process by a
manner of static or dynamic quenching. Then KSV at 288, 298 and 308 K were
obtained from the linear regress plots, the values of which were 7.69 ×104, 5.73 ×104
and 4.25 ×104 L/mol, respectively, inversely correlating with temperature. Thus, it is
easy to calculate the values of Kq, which were 7.69 ×1012, 5.73 ×1012 and 4.25 ×1012
L/(mol·s) at 288, 298 and 308 K, respectively. It is obvious that they are much greater
than the maximum scatter collision quenching constant [2.0×1010 L/(mol·s)][30].
Thus, it is safely to be inferred that static quenching plays a more significant role in
the TF1–XO interaction[31], indicating the formation of a stable TF1–XO complex.
3.4 Number of Binding Sites of TF1 on XO
Given that it is a static quenching process, the apparent binding constant (Ka) and the
number of binding sites (n) were obtained from a regression curve plotted according
to the equation:
log 퐹 −퐹 = log퐾a + nlog[Q]
(5)
0
퐹
The regression curve is presented in Fig.2D. The intercept and slope values of the
curve were used to calculate Ka and n, which were 2.11×104 L/mol and 1.29 (R =
0.996) at 288 K，2.00×104 L/mol and 1.23 (R = 0.994) at 298 K, and 1.64×104 L/mol
and 1.22 (R = 0.998) at 308 K. The values of n for the TF1–XO complex are all equal
to 1 independent with the temperatures, suggesting that there is a single class of TF1
binding sites on XO[32].

3.5 Thermodynamic analysis
The thermodynamic parameters (enthalpy change △Ho and entropy change △So) are
very important, because they can help to elucidate the major forces in the formation of
TF1–XO complex. If there is no significant change in temperature, the value of △Ho
and △So can be considered as a constant and determined by the van’t Hoff equation
below:
표
표
∆퐻
∆푆
logKa = − 2.303푅푇
+
(6)
2.303푅
Where Ka is the binding constant at the corresponding temperature (288, 298 and 308
K); T and R represent the experimental temperature (K) and the universal gas constant
[8.314 J/(mol·K)], respectively. Then the value of △Go (free energy change) can be
calculated via using the equation below:
∆퐺표 = ∆퐻표 − 푇∆푆표
(7)
The calculated thermodynamic parameters are listed in Table 1. According to the
negative values of △Go and △Ho, a spontaneous and exothermic process between TF1
and XO is proposed. Moreover, the positive value of △So and the negative value of
△Ho indicated that there are mainly hydrophobic effects and hydrogen bonds between
TF1 and XO[33, 34].
3.6 Computational docking analysis
A re-docked method with some modifications was employed to validate the docking
procedure[6]. In brief, the original ligand salicylic acid was re-docked into XO using
the procedure described in the section 2.5. The results showed that the docked
conformation of salicylic acid was similar with the original X-ray structure[3],

suggesting that docking procedure is corroborated. And then, molecular docking study
was performed to further understand the binding mode of the complexes formed
between TF1 and XO. According to the cluster analysis, the conformation with the
lowest binding energy (-7.81 kcal/mol) in all the clusters was considered to be the
optimal conformation, while the conformation with the lowest binding energy (-7.18
kcal/mol) in the biggest cluster was considered as the suboptimal conformation. The
binding energy of TF1 conformations are almost equal to that of the substrate
xanthine (-7.1 kcal/mol)[35], suggesting that TF1 may have roughly the same binding
affinity as xanthine with XO, which may be the reason why the inhibitory effects of
TF1 on XO is relatively low. Moreover, the binding energy of either the optimal or
suboptimal conformation is slightly lower than △Go (−5.83 kcal/mol, equal to −24.41
kJ/mol listed in table 1). Although the calculated value is slightly different from the
experimental value, it shows that the interaction between TF1 and XO is spontaneous.
As presented in Fig. 3A and 3C, both the optimal and suboptimal conformations of
TF1 appear in the active cavity (marked in color) of XO. The active center, mainly
composed of various residues such as Leu-648，Asn-768，Glu-802，Ser-876，Glu-879,
Arg-880, Phe-914，Phe-1009，Thr-1010，Val-1011，Leu-1014 and Pro-1076, is a long
narrow path, through which the substrate may reach the catalytic site easily (Mo-pt
cofactor)[36, 37]. When an inhibitor TF1 located in the active cavity, the path was
mostly blocked. Thus it may prevent the substrate (xanthine) from entering the
catalytic site and ultimately hindering its oxidation[36]. As for the optimal
conformation (Fig.3B), TF1 is located in the active cavity by forming 5 hydrogen

bonds with residues Leu-648 (d=2.0 Å), Asn-768 (d=2.0 Å), Glu-802 (d=2.0 Å),
Glu-879 (d=1.8 Å) and Thr-1010 (d=1.9 Å), which are believed to form the active
cavity[36]. The suboptimal conformation (Fig.3D) also binds with XO via forming 6
hydrogen bonds with amino acid residues Asn-768 (d=1.7 Å and 2.5 Å), Lys-771
(d=2.1 Å and 2.6 Å) and Glu-802 (d=1.7 Å and 2.2 Å), some of which also take part
in forming the active cavity[36]. These findings indicate that hydrogen bonding plays
a key role in the binding of TF1 to XO. In addition, it has been proved that Glu-802
plays an important role in the hydroxylation of xanthine[38], suggesting that both the
optimal and suboptimal conformations have the similar binding region as the substrate.
The second interaction force between TF1 and XO was from hydrophobic
interactions[39], which were analyzed by LigPlot+. The results (Fig.3E) show the
hydrophobic interactions between the optimal conformation of TF1 and the 12
residues of XO, including Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802,
Pro-1076, Leu-873, Leu-1014, Asn-768, Leu-648 and Phe-649. As shown in Fig.3F,
the suboptimal conformation of TF1 is surrounded by 14 residues of XO, including
Asn-768, Leu-1014, Ser-1075, Pro-1076, Met-770, Lys-771, Phe-649, Phe-1013,
Val-1011, His-875, Ser-876, Leu-648, Leu-873 and Glu-802. Most of these residues,
such as Glu-802, Thr-1010, Leu-873, Val-1011 and Leu-648, were described as
catalytic residues[40], and the others were all distributed around the Mo-pt center. All
these hydrophobic interactions help TF1 to hold in the binding site of the XO. The
docking results suggest that TF1 binds to XO through hydrogen bonds and
hydrophobic interactions, producing a steric impediment to the entry of the substrate

into the active site.
3.7 The anti-hyperuricaemia effect of TF1 in mice
Given that the inhibitory effects of TF1 on XO, the anti-hyperuricaemia effect of TF1
in mice was further investigated. Mouse model of hyperuricaemia was induced by
intraperitoneal injection of potassium oxonate (PO, an uricase inhibitor). The results
presented in Fig.4 show that the normal serum uric acid level in mice is 2.39 mg/dL.
After injection of PO for 2 hours, the level of uric acid in control group reaches to
5.59 mg/dL, which is significantly higher than the normal group. After intragastric
administration of TF1 (100 mg/kg) in hyperuricemic mice, the value of serum uric
acid drops to 5.17 mg/dL, but there is no significant difference when it compared with
the control. When the dosage reaches to 200 mg/kg in hyperuricemic mice, the value
of serum uric acid drops to 3.61 mg/dL, which shows a significant decrease compared
to the control group. After intragastric administration of allopurinol (20 mg/kg), the
serum uric acid of mice drops significantly to 2.59 mg/dl. The results suggest that TF1
indeed has anti-hyperuricaemia effect, but its effect is lower than that of allopurinol.
This may be due to the different inhibitory effects of TF1 and allopurinol on XO,
which has been confirmed in section 2.2 of this study. However, due to the confirmed
long-term safety of TF1, it may have potential clinical applications in the treatment of
gout and other metabolic disorders.
4. Conclusions
The inhibitory mechanism of TF1 on XO and its anti-hyperuricaemia effect in mice
have been investigated in this study. In summary, the key founding of our study are as

follows: (i) TF1 inhibited the activity of XO in a reversible and competitive manner
with an IC50 value of 63.17 ± 0.13 μmol/L and Ki value of (9.96 ± 0.94) × 10-5 mol/L;
(ii) a static and strong fluorescence quenching of XO occurred because of the
formation of TF1-XO complexes; (iii) thermodynamic analysis showed that the
formation of TF1-XO complexes was a spontaneous process mainly driven by
hydrogen bonds and hydrophobic interactions; (iv) molecular simulation suggested
that TF1 located in the catalytic center via interacting with some residues, such as
Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802, Pro-1076, Leu-873,
Leu-1014, Asn-768, Leu-648 and Phe-649; and (v) the results of animal experiments
suggested that TF1 exhibited anti-hyperuricaemia effect in hyperuricemic mice. As a
consequence, the possible reason for the inhibition of XO activity is the insertion of
TF1 into the active center of XO, thus hindering the entry of xanthine. We propose a
novel inhibition mechanism that provides new insights into the interaction between
TF1 and XO and confirm the anti-hyperuricaemia effect in mice, indicating that TF1
can be a promising XO inhibitor for the treatment of hyperuricaemia and other related
metabolic diseases.
CRediT author statement
Jianmin Chen: Conceptualization, Methodology, Writing- Original draft preparation；
Qinglian Li: Software，Investigation； Yaling Ye: Investigation，Data curation；
Mengnan Ran: Supervision，Data curation: Zhipeng Ruan: Supervision, Validation；
Nan Jin: Reviewing and Editing。

Conflict of interest
We declare that we do not have any commercial or associative interest that represents
a conflict of interest in connection with the work submitted.
Acknowledgements
This work was supported by the Natural Science Foundation of Fujian Province
(Grant No.2019J01806), the Putian Science and Technology Bureau (Grant No.
2018SP3004) and Training Program of Innovation and Entrepreneurship for
Undergraduates (Grant No. 201911498055, 201811498003 and 201811498027).
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Figure Legends
Fig.1. (A) Inhibitory effects of allopurinol and theaflavin (TF1) on the activity of
xanthine oxidase (XO) (pH 7.4, T = 298 K). The structures of allopurinol and TF1 are
presented in the inset. The concentrations of xanthine and XO used in these
experiments were 1.0×10-4 mol/L and 0.04 U/mL, respectively. (B) Plots of υ vs. [XO].
c(xanthine) = 1.0×10-4 mol/L, and c(TF1) = 0, 20.83, 41.67, 83.33 and 166.67 μmol/L
for curves a→e, respectively. (C) LB plots. c(XO) = 0.04 U/mL, and c(TF1) = 0,
20.83, 41.67, 83.33 and 166.67 μmol/L for curve a→e, respectively. (D) The
secondary plot of Kampp (slope) vs. [TF1].
Fig.2. (A) Intrinsic fluorescence emission spectra of xanthine oxidase (XO)
under the existence of theaflavin (TF1) with different concentrations, which were 0,
9.52, 18.18, 26.08, 33.33, 40.00, 46.15, 51.85 and 57.14 μmol/L for curves (a–i),
respectively. (B) Relative fluorescence intensity (%) of XO in the presence of TF1
with different concentrations (298K). (C) The plots based on Stern–Volmer equation
for studying the quenching effects of TF1 on the intrinsic fluorescence of XO at
different temperatures (288, 298 and 308 K). (D) Secondary plots of log [(F0−F)/F] vs.
log [Q] at different temperatures (288, 298 and 308 K).
Fig.3. Molecular docking of the interactions between theaflavin (TF1) and xanthine
oxidase (XO): (A) the magenta sphere represent Mo ions, which is surrounded by
various residues (marked in color) to develop the active cavity; the coarser stick in

cyan represents for TF1 lies in the active cavity. (B) Details between TF1 (the optimal
conformation) and XO, the dashed line in yellow represents hydrogen-bond. (C) The
suboptimal conformation lies in the active cavity. (D) Details between TF1 (the
suboptimal conformation) and XO. (E, F) Hydrophobic interactions between TF1 [the
optimal (E) and suboptimal conformation (F)] and XO.
Fig.4. Dose-dependent anti-hyperuricaemia effects of theaflavin (TF1) and allopurinol
in mice. Normal: mice without treatment; Control: mice treated with potassium
oxonate (PO); Allopurinol: mice treated with PO followed by administering 20 mg/kg
allopurinol intragastrically; TF1 (100 mg/kg): mice treated with PO followed by
administering 100 mg/kg TF1 intragastrically; TF1 (200 mg/kg): mice treated with PO
followed by administering 200 mg/kg TF1 intragastrically. The data were presented as
mean ± SD. *P0.05, **P0.01 when the drug treated group was compared with the
hyperuricemic control group.

Table 1 Kinetic and thermodynamic parameters of the interaction between theaflavin
(TF1) and xanthine oxidase (XO) under different temperatures.
T(K)
288
298
308
Ksv(×104L/mol)
7.688±0.04
5.730±0.03
4.246±0.03
Ra
Ka(×104L/mol)
Rb
n
△Ho(kJ/mol)
△Go(kJ/mol)
-23.91±0.24
-24.41±0.22
-24.92±0.31
△So[J/(mol·K)]
0.9905
0.9857
0.9931
2.11±0.02
0.9959
0.9941
0.9986
1.291±0.03
1.231±0.03
1.215±0.02
-9.28±0.16
50.79±0.12
2.00±0.02
1.64±0.01
Ra and Rb represent the correlation coefficient for the KSV and Ka values, respectively.

Figures
Fig.1
100
0
0.08
A
B
20.83 mol/L
41.67 mol/L
83.33 mol/L
166.67 mol/L
Theaflavin
Allopurinol
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
80
60
40
20
0
a
e
0
20 40 60 80 100 120 140 160 180
0
1
2
3
4
5
6
7
[XO] /(10-3U/mL)
Concentration（ mol/L）
120
C
2500
D
e
a
100
80
60
40
20
0
2000
1500
1000
500
0
y = a + b*x
Equation
Weight
Instrumental
Residual 6.15443
Sum of
Squares
0.99497
0.98661
Pearson's
Adj. R-Sq
Value Standard
Intercept 795.52 28.65249
Slope 10.802 0.62808
B
-0.03 -0.02 -0.01 0.00 0.01 0.02 0.03 0.04
0
20 40 60 80 100 120 140 160 180
1/[Xantine(10-6mol/L)]
[Theaflavin] (mol/L)

Fig.2
7000
100
90
80
70
60
50
40
30
20
10
0
a
i
B
A
6000
5000
4000
3000
2000
1000
0
300
350
400
450
500
0
10
20
30
40
50
60
Wavelength /nm
[Theaflavin] (mol/L)
6
5
4
3
2
1
288K
298K
308K
D
0.6
0.4
C
288K
298K
308K
0.2
0.0
-0.2
-0.4
-0.6
1.0
1.2
1.4
Log[Q]
1.6
1.8
10
20
30
40
50
60
[Q](mol/L)

Fig.3
E
F

Fig.4
n=6
6
5
4
3
2
1
0


ol
tr
Con
rinol
Normal
mg/kg) mg/kg)
TF
00
00
(2
Allopu
(1
TF