Journal of Functional Foods 36 (2017) 102–111

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Screening of effective xanthine oxidase inhibitors in dietary

anthocyanins from purple sweet potato (Ipomoea batatas L. Cultivar Eshu
No.8) and deciphering of the underlying mechanisms in vitro
Zi-Cheng Zhang a,b, Hong-Bin Wang c, Qing Zhou d, Ben Hu e, Jia-Hao Wen a, Jiu-Liang Zhang a,b,f,⇑
a
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
b
Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan 430070, China
c
School of Food Engineering, Wuchang Institue of Technology, Wuhan 430065, China
d
Department of Pharmacy, Wuhan City Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, China
e
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
f
Wuhan Engineering Research Center of Bee Products on Quality and Safety Control, Wuhan 430070, China

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

Article history: This study was aimed to screen the effective xanthine oxidase (XO) inhibitors in dietary anthocyanins
Received 3 March 2017 from purple sweet potato and decipher the underlying mechanisms in vitro. Inhibition kinetics demon-
Received in revised form 17 June 2017 strated that anthocyanin-rich purple sweet potato extract (APSPE) has reversible and mixed inhibitory
Accepted 19 June 2017
effect on XO. Among the four fractions purified from APSPE, the fraction with highly-acylated antho-
cyanins showed the most powerful inhibition on XO. Molecular docking results suggested that the inhi-
bition mechanism is the insertion of anthocyanins into the hydrophobic pocket of XO by interacting with
Chemical compounds studied in this article:
some amino acid residues. Fluorescence and CD spectroscopy results showed that the formation of
Xanthine (PubChem CID: 1188)
Allopurinol (PubChem CID: 2094)
anthocyanin-XO complex is the possible quenching mechanism, which changes the secondary structure
Cyanidin-3-sophoroside-5-glucoside of XO. The reaction is entropy-driven and endothermic, with hydrophobic interaction being the major
(PubChem CID: 44256732) driving force. This study indicates that the highly-acylated dietary anthocyanins of purple sweet potato
Cyanidin-3-(60 caffeoyl-600 feruloyl-sophoro would be a promising XO inhibitor in clinical treatment.
side)-5-glucoside (PubChem CID: Ó 2017 Elsevier Ltd. All rights reserved.
44256788)
Peonidin 3-(60 -caffeoyl sophoroside)-5-
glucoside (PubChem CID: 44256858)
Cyanidin 3-(60 -caffeoyl sophoroside)-5-
glucoside (PubChem CID: 44256781)

Keywords:
Dietary anthocyanins
Anthocyanin-rich purple sweet potato
extract (APSPE)
Xanthine oxidase
Inhibition kinetics
Fluorescence quenching
Molecular docking

1. Introduction
Xanthine oxidase (XO), a key enzyme mainly present in the liver
and plasma, catalyzes the oxidation of hypoxanthine to xanthine
⇑ Corresponding author at: College of Food Science and Technology, Huazhong
Agricultural University, Wuhan 430070, China.
E-mail addresses: zzccrystal@163.com (Z.-C. Zhang), whb879694@yeah.net
(H.-B. Wang), qingz_zqing@aliyun.com (Q. Zhou), benhu917@gmail.com (B. Hu),
wjh.auckland@gmail.com (J.-H. Wen), zjl_ljz@mail.hzau.edu.cn (J.-L. Zhang).
and further generates uric acid from xanthine in the purine meta-
bolic pathway (Harrison, 2002; Tung & Chang, 2010). Overproduc-
tion of uric acid in serum will result in hyperuricemia, a common
purine metabolic disorder, and eventually causes uric acid
nephrolithiasis and gouty arthritis (Kramer & Curhan, 2002;
Tomita et al., 2000). Recently, it has been reported that hyper-
uricemia is associated with hypertension, obesity and renal dis-
eases (Lee et al., 2012; Zoccali & Mallamaci, 2013), and XO has
been considered as one of the most promising therapeutic targets.

http://dx.doi.org/10.1016/j.jff.2017.06.048
1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111
As an effective XO inhibitor, allopurinol has become one of the
chemical medicines widely used for the treatment of hyper-
uricemia and gout in clinic (Wu et al., 2010). However, allopurinol
may pose serious threats to the health of the patients, including
allergy, hypersensitivity reactions and nephropathy (Hande,
Noone, & Stone, 1984; Urban et al., 1995). Therefore, it is urgent
to develop safer and more effective bioactive agents or functional
food that can inhibit XO activity with the minimal side effects.
Purple sweet potato (Ipomoea batatas L.), which is widely
planted in China, has been used as a functional food for its high
contents of dietary anthocyanin and phytochemicals beneficial to
human health, and Eshu No.8 is a new species cultivated in Hubei
with good yield (Yang et al., 2011). Several studies have reported
on the structures and biological activities of anthocyanins from
purple sweet potato (Kubow et al., 2016; Wang et al., 2014), but lit-
tle is known about the anti-hyperuricemic activity and XO-
inhibitory activity of dietary anthocyanins. Our previous study
has demonstrated that anthocyanin-rich purple sweet potato
extract (APSPE) has excellent anti-gout and anti-inflammatory
effects through inhibiting XO in vivo (Zhang et al., 2015). However,
the key active components in APSPE for XO inhibition and the inhi-
bitory mechanism still remain largely unknown. Therefore, this
study was aimed to identify and evaluate the key components in
dietary anthocyanins from purple sweet potato that are responsi-
ble for the strong inhibitory activity on XO, and further decipher
the underlying inhibitory mechanisms in vitro. Our results are
expected to facilitate the potential use of dietary anthocyanins
with specific structure as a promising XO inhibitor for clinical pur-
poses and a dietary supplement to relieve the symptom of
hyperuricemia.
2. Materials and methods
2.1. Chemicals and plant materials
Dried purple sweet potato powder (Ipomoea batatas L. cultivar
Eshu No.8) was donated by Puzetian Food Co. Limited (Wuhan,
China). Xanthine oxidase purified from bovine milk was purchased
from Sigma Chemical Co. (St. Louis, MO, USA). Xanthine and allop-
urinol were purchased from Tokyo Chemical Industry Co. (Tokyo,
Japan). Acetonitrile and formic acid (HPLC grade) were obtained
from J.T. Baker Co. (Phillipsburg, New Jersey, USA) and Sinopharm
Chemical Reagent Co. (China), respectively. Water was purified
using a Milli-Q Direct 8 System from Millipore (Billerica, MA,
USA). All other reagents were of analytical grade.
2.2. Extraction and enrichment of anthocyanins
The extraction and enrichment of anthocyanins were performed
using the method described previously with some modifications
(Mi, Park, Dong, & Jung, 2013; Zhang et al., 2015). Dried purple
sweet potato powder was refluxed in 40% anhydrous ethanol at
60 °C for 0.5 h and centrifuged at 2050g for 10 min. Then, the crude
anthocyanin extract was firstly purified on an AB-8 resin column
(weak polarity macro porous resin, 0.3–1.25 mm particle size,
25 mm  100 mm; Nankai Hecheng Science & Technology Co.,
Tianjin, China) to remove sugars, proteins and other impurities.
The crude anthocyanins were loaded and washed with 70% ethanol
including 0.1% TFA and the sample was collected, evaporated,
freeze-dried and named as APSPE. In our previous study, the antho-
cyanin level of APSPE (as Cyanidin-3-Glucoside (C3G) equivalent)
was determined as 3.53  104 mg per 100 g, and the total contents
of polyphenols and flavonoids were 41.0 ± 0.62 mg GAE g1 and
98.2 ± 1.69 mg RE g1, respectively (Zhang et al., 2015).
103
The APSPE received secondary purification on Daisogel SP-120-
30/50-ODS-B column (DAÏSO Co., Osaka, Japan) to generate
different enriched fractions from APSPE. Approximately 5 g of
freeze-dried APSPE was dissolved in 100 mL water and was loaded
on the column. The APSPE sample was then successively washed
with 0.1% TFA added water and 10% methanol (v/v). Fraction 1
was collected with an isocratic elution using 20% methanol (v/v).
Subsequently, 30%, 40% and 50% methanol were used for elution
to generate fraction 2, 3 and 4, respectively. Finally, the different
fractions were evaporated, freeze-dried and stored at 20 °C until
further use.
2.3. HPLC analysis of anthocyanins
APSPE and fraction 1, 2, 3 and 4 were dissolved in water to
1 mg mL1 and filtered through a 0.45 lm cellulose acetate mem-
brane before injection, and HPLC analysis was performed using a
previously described method with some modifications (Wang
et al., in press). The individual anthocyanins were separated by
an Agilent 1100 HPLC (Agilent 145 Technologies Inc., Santa Clara,
CA, USA) with a Diamonsil Plus C18-A column (250 cm  4.6 mm
I.D., 5 lm, Dikma, Shanghai, China). The separation was accom-
plished at 30 °C with the injection volume of 20 lL. Solvent A
was 5% formic acid in water (v/v) and solvent B was purified ace-
tonitrile. The gradients were as followed: 0–10 min, 90–85% A;
10–20 min, 85–80% A; 20–30 min, 80–75% A; 30–40 min, 75–70%
A; 40–45 min, 75–70% A. The eluate was monitored at 525 nm
for all samples, and an additional monitoring at 280 nm was con-
ducted for fraction 1.
2.4. Evaluation of inhibitory activity of anthocyanin on xanthine
oxidase in vitro
APSPE and fraction 1, 2, 3 and 4 were evaluated for their inhibi-
tory activity on commercial xanthine oxidase by spectrophotomet-
rically measuring the formation of uric acid at 295 nm using a
previously reported method with some modification (Jin, Lim, &
Choung, 2013). A series of mixtures consisting of a standard con-
centration of XO (7.5  108 mol L1), phosphate buffer
(0.05 mol L1, pH 7.5) and various amounts of samples (ranging
from 0 to 11.35  105 mol C3G equivalent L1) were incubated
for 0.5 h at 37 °C. Allopurinol was used as control. The activity
was initiated by adding the substrate xanthine (5.0  105 -
mol L1) to the reaction mixture. Then, the enzyme activity was
measured by using micro-plate spectrophotometer (Multiskan
GO, Thermo Scientific, Waltham, USA) to collect the absorbance
value every 30 s. The relative activity of xanthine oxidase was
calculated as follows:
R
Relative Activity ð%Þ ¼  100%; ð1Þ
R0
where R is the reaction rate of inhibitor-containing system and
R0 is the reaction rate of the system without the inhibitor. Then, the
half-maximal inhibitory concentration (IC50) values of the above
two inhibitors were also evaluated separately.
2.5. Inhibition type of anthocyanin on xanthine oxidase by kinetic
analysis
To determine the inhibition type of anthocyanin against XO,
APSPE was tested using the same method in XO activity assay as
described above. Progress curves were obtained at several APSPE
concentrations using various xanthine concentrations. The
Lineweaver-Burk plot equation in double reciprocal form was
written as follows (Lin, Zhang, Pan, & Gong, 2015):
104 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111
 
1 Km ½I 1 1 the fluorescence spectra of these solutions were measured at three
¼ 1þ þ ;
m V max K i ½S V max
where m is the enzyme reaction rate in the absence and presence of
APSPE, Ki and Km are the inhibition constant and Michaelis-Menten
constant, respectively, [I] and [S] are the concentrations of inhibitor
APSPE and substrate xanthine, respectively.
The secondary plot of the slopes of the straight lines or vertical
intercept versus [I] as well as the constants for the binding of
APSPE with free XO or enzyme-substrate complex (Ki and Kis) were
obtained from the following equations (Kim et al., 2014):
K m ½I Km
Slop ¼ þ ð3Þ
K i V max V max
½I 1 Each spectrum was collected by the average of three consecutive
Intercept ¼ þ ð4Þ
K is V max V max
2.6. Molecular docking
The molecular docking was performed using Surflex-Dock mod-
ule of Sybyl-x 2.0 (Tripos, Inc., St. Louis, MO, USA). In the docking
process, semiflexible docking mode was adopted to treat the pro-
tein and small molecules, which meant that the conformation of
protein was rigid and that of small molecules was flexible. The
crystal structure of bovine milk xanthine oxidase with quercetin
inhibitor binding (PDB code 3NVY) was retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb) (Cao, Pauff, & Hille,
2014). The structure of XO protein was prepared by the following
steps: removing the quercetin inhibitor in the binding pocket;
removing all water molecules except for the two close to the
Arg880 in the active site (Tung et al., 2010); modifying the ends
of the main chain and side chain; adding hydrogen and changing
the protonation state of amino acid residues at pH = 7. AMBER7
FF99 force field was used for the structural optimization of XO.
In order to compare the XO inhibition activity of anthocyanins
in fraction 2 and 3 and to explore the possible binding modes
between XO and anthocyanins with different structures, three rep-
resentative small molecules were selected for molecular docking,
including Cyanidin 3-sophoroside-5-glucoside (No.8 individual
anthocyanin in Supplementary Table 1) from fraction 2, Cyanidin
3-(60 caffeoyl-600 feruloyl sophoroside)-5-glucoside and Peonidin 3-
(60 caffeoyl-600 feruloyl sophoroside)-5-glucoside (No.12 and No.15
individual anthocyanins in Supplementary Table 1) from fraction
3. The structures of these three molecules were firstly standardized
in Sybyl-x 2.0, and their 3D conformations were generated by the
Concord program. Then, the atom charges were calculated by the
Gasteiger Hückel method. Finally, energy minimization was per-
formed using the Tripos force field and the Powell method with
the RMS force difference of 0.005 kcal mol1 Å1 and the maxi-
mum iterations of 10,000.
In the Surflex-Dock docking process, the prototype molecules
were set as ligand mode and the initial conformation of the ligand
was set as 5. The total scores of docking results output by Surflex-
Dock were used to compare the binding abilities of anthocyanins
with different structures to XO.
2.7. Fluorescence measurements
The fluorescence spectra were detected by F-4600 FL Spec-
trophotometer (Hitachi, Tokyo, Japan) equipped with a thermostat
water bath, a Xe lamp and a 1.0 cm path-length quartz cell to char-
acterize the binding activity of fraction 3 with XO. 2.0 mL solution
containing 5.0  107 mol L1 XO was titrated by various concen-
trations of fraction 3, ranging from 0 to 7.5  103 mg mL1. The
resulting solutions were allowed to equilibrate for 5 min and then
ð2Þ
different temperatures (298, 304 and 310 K) in the wavelength
range of 300–500 nm with an excitation wavelength at 280 nm.
The widths of both the excitation and emission slits were set at
2.5 nm and the individual spectrum of fraction 3 in sodium phos-
phate buffer (0.05 mol L1, pH 7.5) was measured and subtracted
to correct fluorescence background.
2.8. Circular dichroic (CD) spectroscopy
The far-UV CD spectra measurements were carried out by a cir-
cular dichroism spectrometer (J 1500, Jasco Corp, Tokyo, Japan) in a
wavelength range of 200–240 nm with a 1-mm path length quartz
cuvette at a speed of 50 nm min1 under constant nitrogen flush.
scans, and the pH 7.5 sodium phosphate buffer running under
the same conditions was taken as a blank. The concentration of
XO was 1.0  106 mol L1 and the fraction 3/XO molar ratios were
0:1, 2:1, 4:1 and 6:1, respectively. All the results were expressed as
ellipticity in mill degrees, and Yang’s reference software (Jasco
Corp, Tokyo, Japan) was used to calculate the percentages of differ-
ent secondary structures (a-helix, b-sheet, b-turn and random coil)
of XO.
2.9. Statistical analysis
The results were presented as the average of three replications
and expressed as mean ± SD values. One-way analysis of variance
(ANOVA) was performed by using Origin version 9.0 for Windows
followed by multiple tests in order to determine the significant dif-
ference at p < 0.05.
3. Results and discussion
3.1. Inhibitory effects of APSPE on xanthine oxidase activity in vitro
The inhibitory effects of APSPE and allopurinol on XO are
shown in Fig. 1. Fig. 1A illustrates that the relative activity of
XO showed a decreasing trend with the increasing concentration
of APSPE, indicating that the concentration of APSPE is negatively
correlated with the relative activity of XO, and APSPE inhibits XO
activity in a dose-dependent manner. The half-maximal inhibitory
concentration (IC50) values of APSPE and allopurinol were esti-
mated to be 7.194 ± 0.858  105 mol C3G equivalents L1 and
5.391 ± 0.425  106 mol L1, respectively. Although the inhibi-
tory effect of APSPE on XO was not as strong as that of allopuri-
nol, our results indicate the high potential of APSPE to be used as
an XO inhibitor. Besides, our previous study has demonstrated the
strong side effect of allopurinol on hyperuricemic mice, which
could cause inflammatory cells infiltrating in renal interstitium,
the loss of brush border and dilation of renal tubes (Zhang
et al., 2015). Taken all evidences into consideration, APSPE is
believed to be a promising XO inhibitor with strong inhibitory
effect and little side effect.
3.2. Inhibition type of APSPE on xanthine oxidase by kinetic analysis
To determine whether the inhibitory activity of APSPE on XO is
reversible, the plots of m vs. [XO] at different concentrations of
APSPE were constructed and displayed in Fig. 1B. The slope of
the straight line in the plot was decreased with the increasing
amount of APSPE, suggesting that the inhibition of APSPE on XO
is reversible.
Reversible inhibition could be classified into competitive inhibi-
tion, non-competitive inhibition, uncompetitive inhibition and
 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111 105

100% 0.020
Allopurinol (B)
APSPE
80% a
0.015
Relative Activity (%)

60% d

v (ΔOD / min)
0.010
40%

0.005
20%

(A)
0% 0.000
0 2 4 6 8 10 12 2.5 5.0 7.5 10.0 12.5
[I] / (10 mol L )
-5 -1
[XO] / (10 mol L )
-8 -1

(C) 500

400
500
slop

300

200
400
100

0
c
0.00 0.35 0.70

1/v (ΔOD / min)

[I] / (10 mol L )
-2 -1

200 300
a
Intercept

100
200

0
0.00 0.35 0.70
-2
[I] / ( 10 mol L )
-1
100

0
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4
-5 -1
1/ [S] (10 mol L )

Fig. 1. (A) Inhibitory effects of APSPE and allopurinol on XO activity. T = 310 K, pH 7.5, c (XO) = 7.5  108 mol L1, and c (xanthine) = 5.0  105 mol L1; c (APSPE) was
presented as mol C3G equivalent L1. (B) Plots of m vs. [XO]. c (xanthine) = 5.0  105 mol L1, and c (APSPE) = 1.1, 2.2, 2.75, 3.3  102 mol C3G equivalent L1 for curve a-d,
respectively. (C) Lineweaver-Burk plots. c (XO) = 7.5  108 mol L1, and c (APSPE) = 0, 0.35, 0.70  102 mol C3G equivalent L1 for curve a-c, respectively; Insert (I) and (II)
are the secondary plots of slope and intercept vs. [APSPE], respectively. Values are expressed as mean ± SD (n = 3).

mixed inhibition based on the relationships among the enzyme, APSPE can be assigned as a reversible and mixed inhibitor of XO
substrate and inhibitor (Marangoni, 2003, chap. 4). To investigate (Chu, Chen, Wu, & Hsieh, 2014).
the kinetics of the enzyme in the presence of APSPE, the
Lineweaver-Burk plot equation in double reciprocal form was used 3.3. HPLC analysis of different fractions separated from APSPE
and the results were displayed in Fig. 1C. From curves a to c, with
the increasing concentration of APSPE, the slopes of the straight In previous studies, dietary individual anthocyanins had been
lines and the intercepts on the left of the vertical axis were all sig- separated by HPLC under the same conditions. The results showed
nificantly increased, indicating the significant decrease of Vmax and that there were 19 individual anthocyanins in the compound
increase of Km. Also, the intersection of the Lineweaver-Burk plot APSPE, and the structures of the 19 anthocyanins were also identi-
was located at the second quadrant and was close to the y-axis. fied by HPLC-ESI-MS/MS (Wang et al., in press; Zhang et al., 2015).
All these phenomena indicate that APSPE exhibits mixed inhibition As our HPLC assay produced the same results as in these studies,
on XO, with competitive inhibition being dominant (Dong et al., we did not display these results in the paper. Based on our previous
2016). In addition, the secondary plots are linearly fitted (insert HPLC-ESI-MS/MS database (Wang et al., in press; Zhang et al.,
in Fig. 1C), indicating that only one inhibition site is observed 2015) and the HPLC assay results of the four fractions (Fig. 2),
(Yan, Zhang, Hu, & Ma, 2013). The parameters for the binding of the composition of dietary individual anthocyanins of fraction 1,
APSPE with free XO (Ki) and with enzyme-substrate complex (Kis) 2, 3 and 4 was clearly identified through comparing the retention
could be fitted and obtained from Eqs. (2) and (3), respectively. times (tR) of anthocyanins in the HPLC-ESI-MS/MS database and
The calculation results show that the Ki and Kis values are those from the HPLC assay results (Fig. 2). Fraction 1 was found
4.19  103 mol L1 and 7.50  103 mol L1, respectively. The to contain very little anthocyanins under the monitoring at
lower Ki value represents a tighter binding mode of APSPE with 525 nm (Fig. 2A-(I)), but the detection at 280 nm showed that
XO, indicating that the affinity of APSPE for free enzyme is stronger there was much copigment such as flavonoid in fraction 1
than that for the enzyme-substrate complex. Taken together, (Fig. 2A-(II)), which could be easily eluted and enriched by 20%
106 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111

Fig. 2. HPLC-DAD chromatograms of (A)-(I) fraction 1, (B)-(I) fraction 2, (C) fraction 3 and (D) fraction 4 separated from APSPE detected at 525 nm; (A)-(II) is HPLC assay result
of fraction 1 detected at 280 nm; (B)-(II) is the HPLC result of fraction 2 detected at 525 nm with adjusted HPLC elution program. Insert in (B)-(I) and (C) are structures of
Cyanidin 3-sophoroside-5-glucoside (No.8), Cyanidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-glucoside (No.12) and Peonidin 3-(60 caffeoyl-600 feruloyl sophoroside)-5-
glucoside (No.15), respectively.

methanol. Fig. 2B-(I) (and Supplementary Table 1) exhibits the
composition of dietary individual anthocyanins in fraction 2.
No.1 to No.8 individual anthocyanins could be detected but with
a low degree of separation. In order to achieve satisfying separation
of fraction 2, we adjusted the HPLC elution program as follows:
0–20 min, 90–85% A; 20–40 min, 85–80% A; 40–45 min, 80–70%
A. The HPLC fingerprint of fraction 2 was finally obtained with
favorable separation (Fig. 2B-(II)). These 8 individual anthocyanins
were all with a low degree of acylation and a low molecular weight
of 916.38 g mol1 in average. The structure of No.8 individual
anthocyanin in fraction 2 was Cyanidin 3-sophoroside-5-
glucoside (insert in Fig. 2B-(I)), whose chemical structure has been
reported previously (Wang et al., in press). Fig. 2C (and Supple-
mentary Table 1) exhibits the composition of individual antho-
cyanins of fraction 3, including No.9 to No.19 individual
anthocyanins. The average molecular weight of these anthocyanins
was 1081.20 g mol1 and they were all with a high degree of acy-
lation, as could be reflected from the structure of No.12 and No.15
individual anthocyanins (insert in Fig. 2C). In addition, the results
in Fig. 2D show that fraction 4 contained very little anthocyanins,
indicating that most of the anthocyanins were eluted and enriched
by 30% and 40% methanol.
 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111
Fig. 2 (continued)
3.4. Inhibitory effects of different fractions on xanthine oxidase activity
in vitro
The inhibitory effects of different fractions separated from
APSPE on XO activity were compared. As shown in Fig. 3, with the
same concentration at 5  103 mg mL1, the inhibitory effects of
different fractions were totally different. The relative activity of
XO with different fractions and APSPE followed the order of fraction
3 (55.02 ± 1.54%) < fraction 2 (68.81 ± 1.61%) < APSPE (79.56 ±
1.49%) < fraction 1 (82.21 ± 1.78%)  fraction 4 (82.39 ± 1.02%). This
phenomenon suggests that faction 3 has the most powerful inhibi-
tory effect on XO activity, followed by fraction 2. In combination
with the results of HPLC analysis, it could be inferred that the main
inhibitory effect may be attributed to dietary anthocyanins while
Fig. 3. Inhibitory effects of different fractions on XO activity. T = 310 K, pH 7.5, c
(XO) = 7.5  108 mol L1, c (xanthine) = 5.0  105 mol L1, and c (samples)
= 5.0  103 mg mL1. Values are expressed as mean ± SD (n = 3) and different
letters marked above the bar are significantly different by ANOVA multiple test
(p < 0.05).
107
the copigment in fraction 1 may have little contribution to the inhi-
bition. Moreover, the anthocyanins with a higher degree of acyla-
tion and higher molecular weight were more effective than those
with a lower degree of acylation, which could be deduced according
to the result that the inhibitory effect of fraction 3 was stronger
than that of fraction 2.
3.5. Molecular docking results
To provide the theoretical evidence to prove that fraction 3 has a
stronger inhibitory effect on XO than fraction 2 and to explore the
possible binding modes among XO, its substrate and dietary antho-
cyanins to form complexes, Cyanidin 3-sophoroside-5-glucoside
(No.8 individual anthocyanin in Supplementary Table 1) and
Cyanidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-glucoside
(No.12 individual anthocyanin in Supplementary Table 1) were used
as the representative materials for fraction 2 and 3 to perform
molecular docking experiment. Peonidin 3-(60 caffeoyl-600 feruloyl
sophoroside)-5-glucoside (No.15 individual anthocyanin in Supple-
mentary Table 1) was used to clarify how the changes in the parent
nucleus structure of anthocyanins influence their binding with XO.
The results show that all the three small molecules could bind with
the enzyme. However, the total scores of Cyanidin 3-(60 caffeoyl-
600 -feruloyl sophoroside)-5-glucoside and Cyanidin 3-sophoroside-
5-glucoside were 9.7435 and 3.3544, respectively, demonstrating
that the former has stronger binding ability with XO. Moreover, both
the number of hydrogen bonds and the amino acid residues that
formed the hydrogen bonds were also different between these two
small molecules. Cyanidin 3-sophoroside-5-glucoside formed 7
hydrogen bonds with ASN768, GLU802, SER876 and GLN1122, while
Cyanidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-glucoside
formed 10 hydrogen bonds with LEU648, ARG871, GLU879,
ARG880, THR1010 and GLN1122. These results theoretically explain
why fraction 3 has stronger inhibitory effect on XO than fraction 2.
Through comparing the structures of these two molecules, we found
that Cyanidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-glucoside
has two acylated groups (insert in Fig. 2C), the 60 -caffeoyl group
and the 600 -feruloyl group on the sophoroside, which were not
108 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111
observed in Cyanidin 3-sophoroside-5-glucoside (insert in Fig. 2B-
(I)). Therefore, we infer that the acylated groups on the sophoroside
might play a critical role in the interaction between anthocyanins
and XO. Meanwhile, there was no significant difference in binding
with XO between Cyanidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-
5-glucoside and Peonidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-
glucoside, since the total scores of them were 9.7435 and 9.4835,
respectively. This result indicates that whether it is a hydroxyl or a
methoxy group on the C-30 site of the B ring of anthocyanins, the
docking results would not be affected.
As shown in Fig. 4, the simulated molecular structure of Cyani-
din 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-glucoside displayed
an excellent match with the active cavity of the enzyme, and 5
hydrogen bonds were formed between the small molecule and
the residues of Leu 648, Glu 879, Arg 871 and Gln1122 in the active
cavity site, suggesting that anthocyanins could occupy the catalytic
center of the enzyme by competing with the substrate and hydro-
gen bonds may play a dominant role in the binding with XO (Wang,
Zhang, Pan, & Gong, 2015). More specifically, the 60 -caffeoyl group
was inserted into the hydrophobic region, a long and narrow chan-
nel (marked in orange and yellow in Fig. 4) that connects the sub-
strate with the Mo center of XO as reported by Lin, Zhang, Pan et al.
(2015), and another 5 hydrogen bonds were formed there to pro-
mote the interaction of the 60 -caffeoyl group with residues Arg
880 and Thr 1010. On the other hand, a previous study has
reported that some amino acid residues such as His 875, Ser 876,
Glu 879, Arg 880 and Phe 1013 are related to the formations of
a-helix (Pauff, Cao, & Hille, 2009), and the hydrogen bonds
between the Cyanidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-
glucoside and the enzyme may increase the content of a-helix in
XO and change its conformation. This speculation was then testi-
fied by CD spectroscopy.
Thus, it could be concluded that dietary anthocyanins of purple
sweet potato could be an efficient inhibitor of XO and fraction 3
has a stronger inhibitory effect than fraction 2. The principal inhi-
bitory mechanism of dietary anthocyanins on XO activity could be
the insertion of acylated group of anthocyanins into the hydropho-
bic pocket of XO as well as the occupation of anthocyanins on the
catalytic center of the enzyme by competing with the substrate.
Moreover, the formation of the hydrogen bonds with some special
amino acid residues could cause the conformational changes in XO.
The docking results could be important evidence on the structure-
effect relationship between dietary anthocyanin of purple sweet
potato and XO, and fraction 3 would be used in further studies to
illustrate the interaction of anthocyanins with XO by fluorescence
and CD spectroscopy experiments.
3.6. Intrinsic fluorescence quenching of XO by fraction 3
As shown in Fig. 5, the binding mechanism of fraction 3 on XO
was further investigated by fluorescence spectroscopy experiment.
Fig. 4. Computational visualization of the optimal docking conformation of Cyanidin 3-(60 caffeoyl-600 -feruloyl sophoroside)-5-glucoside with the active site of XO. Carbon is
in white, nitrogen in blue, oxygen in red for anthocyanins. Yellow dotted lines represent hydrogen bonds. Different residues are in different colors and the residue numbers
are also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
When the excitation wavelength was set at 280 nm, the character-
istic fluorescence emission peak of XO was observed at about
340 nm, which is mainly attributed to its intrinsic fluorescent
amino acid residues (tryptophan, tyrosine and phenylalanine)
(Peng, Ding, Jiang, Sun, & Peng, 2014). Also, the fluorescence inten-
sity was significantly reduced without any emission peak shifts
with increasing amount of fraction 3 added to XO solution (curve
a–g), which is a powerful evidence for the interaction between
fraction 3 and XO (Lin, Zhang, Liao, & Pan, 2015). Then, the data
of the fluorescence quenching were analyzed by the Stern-
Volmer equation (Mohammadi & Moeeni, 2015; Shahabadi,
Maghsudi, Kiani, & Pourfoulad, 2011):
F0
¼ 1 þ K sv ½Q  ¼ 1 þ K q s0 ½Q ; ð5Þ
F
where F0 and F symbolize the fluorescence intensity of XO in the
absence and presence of fraction 3, respectively, [Q] is the concen-
tration of fraction 3 and Ksv is the Stern-Volmer quenching constant,
which was detected by the biomolecule quenching rate constant
Kq and the average lifetime of biomolecule with no quencher s0
(Ksv = Kqs0 , for biomolecules, s0 = 108 s).
There are several pathways for the fluorescence quenching,
which are generally classified into two ways, dynamic quenching
and static quenching. The dependence of reaction on temperature
and excited-state lifetime is used to differentiate dynamic and sta-
tic quenching (Yan et al., 2013). The curves of the Stern–Volmer
were linear within the studied concentrations, suggesting that
there is a single type of quenching existed in the interaction
between fraction 3 and XO.
As shown in Table 1, the Ksv values decreased with increasing
temperature in the solution system. Besides, the Kq values were
calculated to be much higher than the maximum scatter collision
quenching constant of various quenchers with biopolymers
(2.0  1010 L mol1 s1) (Zhang, Ma, Wang, Zhang, & Zhou, 2012).
These results indicate that the possible quenching mechanism of
interaction between fraction 3 and XO may be static quenching
accompanied by the formation of fraction 3-XO complex.
3.7. Binding constant and number of binding sites of fraction 3-XO
complex
The data of fluorescence measurements were further evaluated
by the modified Stern-Volmer equation (Li, Zhao, Su, Lin, & Wang,
2016) to calculated the binding constant (Ka) for the quencher-
acceptor system and the number of binding sites (n):
F0 1 1 1
¼ þ ; ð6Þ
F 0  F f a K a ½Q  f a
where Ka represents the modified Stern-Volmer quenching constant
for the accessible fluorophores and fa is the fraction of accessible
fluorescence. The dependence of F0/(F0  F) on the reciprocal value
 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111 109

a
600

Fluorescence intensity
500

400

300 g

200

100

0
300 350 400 450 500
Wavelength / nm

Fig. 5. Fluorescence spectra of XO in the presence of fraction 3 at different concentrations. T = 298 K, pH 7.5, c (XO) = 5  107 mol L1 and c (fraction 3) is varied in the range
of 0, 1.25, 2.5, 3.75, 5.0, 6.25, 7.5  103 mg mL1 from curves a-g, respectively.

Table 1
Binding interaction and relative thermodynamic parameters of fraction 3 with XO at different temperatures.

T (K) Ksv/(104 L mol1) Kq/(1012 L mol1 s1) Ra Ka/(104 L mol1) Rb DH/(kJ mol1) DG/(kJ mol1) DS/(J mol1 K1) Rc
298 7.38 ± 0.01 7.38 ± 0.01 0.9956 4.13 ± 0.02 0.9973 131.818 ± 0.03 60.68 ± 0.01 645.97642 ± 0.22 0.9848
304 6.92 ± 0.03 6.92 ± 0.03 0.9982 6.64 ± 0.05 0.9997 64.56 ± 0.01
310 5.94 ± 0.02 5.94 ± 0.02 0.9961 10.09 ± 0.03 0.9958 68.44 ± 0.01

Ra is the correlation coefficient for the Ksv and Kq values; Rb is the correlation coefficient for the Ka values; Rc is the correlation coefficient for the van’t Hoff plot.

of quencher concentration 1/[Q] is linear, and the constant Ka for the
fraction 3-XO complex at different temperatures was obtained
using the intercept and slope (Table 1). The results show that with
the increase of temperature, the Ka showed an increasing trend,
indicating that there is a strong binding force in the formation of
fraction 3-XO complex, and the binding of fraction 3 to XO is an
entropy-driven and endothermic reaction (Yan et al., 2013).
When there is a static quenching interaction of a small molecule
binding independently to the equivalent sites on a macromolecule,
the number of binding sites (n) for fraction 3-XO complex could be
obtained according to the following equation (Xiao et al., 2011):
F0  F
log ¼ log K b þ n log½Q 
F
Based on this equation, the values of n at 298 K, 304 K and 310 K
were calculated to be 0.97 ± 0.01, 1.26 ± 0.03 and 1.32 ± 0.02,
which were all approximately equal to 1, suggesting that there is
only one binding site in XO for fraction 3.
action of fraction 3 with XO is spontaneous. The positive values of
both DH and DS are usually considered as the evidence for a
hydrophobic interaction (Ross & Subramanian, 1981) and the bind-
ing process is predominately enthalpy-driven with hydrophobic
interaction being the major driving force.
3.9. Circular dichroism (CD) spectroscopy
CD spectroscopy is a useful technique to investigate the changes
in the secondary structure of proteins in the absence/presence of
small molecules, and far-UV CD can provide the information about
the conformation of the proteins (Akram, Bhat, & Kabir-ud-Din,
ð7Þ 2016). As shown in Fig. 6, at the wavelength of 216 nm, the spectra
of XO displayed a negative band in the UV region which has been
proved to be the characteristic of b-sheet of XO in previous studies
10

8
3.8. Binding forces between fraction 3 and XO
6
There are essentially four kinds of interaction forces between
4
small molecules and biomolecules, including hydrogen bonds,
CD(mdeg)

van der Waals forces, electrostatic and hydrophobic interactions 2

(Shahabadi, Fili, & Kheirdoosh, 2013). The determination of the
thermodynamic parameters such as enthalpy change DH and 0
entropy change DS of the binding reaction can indicate the interac- -2
d
tion force of the fraction 3-XO complex. The values of the thermo-
dynamic parameters were calculated based on the van’t Hoff -4
a
equation (Krause-Heuer, Price, & Aldrich-Wright, 2012):
-6
DH DS
log K a ¼  þ ð8Þ -8
2:303RT 2:303R 200 210 220 230 240
Wavelength / nm
DG ¼ DH  TDS; ð9Þ
Fig. 6. Far-UV CD spectra of XO in the presence of increasing amount of fraction 3. c
where Ka is the binding constant at different temperatures (298 K, (XO) = 1  106 mol L1 and the fraction 3/XO molar ratios are 0:1, 2:1, 4:1 and 6:1
304 K and 310 K) and R is the gas constant (8.314 J mol1 K1). from curves a-d, respectively. The contents of different secondary structures of free
The negative values of DG shown in Table 1 suggest that the inter- XO and fraction 3-XO complex (CD spectra) at pH 7.5 and 298 K are in the insert.
110 Z.-C. Zhang et al. / Journal of Functional Foods 36 (2017) 102–111
(Mir, Khan, Khan, Dar, & Rather, 2012). Besides, the CD intensity of
XO was significantly decreased and shifted to zero level with
increasing molar ratio of fraction 3 to XO (from 0:1 to 6:1, curve
a to d), indicating the changes in the protein secondary structure
with the increase of a-helical content (Hadizadeh, Keyhani,
Keyhani, & Khodadadi, 2009). Moreover, the effects of fraction 3
on the XO secondary structure calculated by Yang’s reference soft-
ware are shown in the insert of Fig. 6. It can be observed that when
the molar ratio of fraction 3/XO was increased to 6:1, a-helix was
increased from 40.3% to 51.9% with b-sheet and b-turn decreasing
from 9.9% to 2.6% and from 34.7% to 31.5%, respectively. All the
above results confirm that anthocyanins could increase the content
of a-helix in XO through the hydrogen bonds between antho-
cyanins and the amino acid residues of XO such as Glu 879 and
Arg 880, which has been proved by the results of molecular dock-
ing in Fig. 4. The combination of fraction 3 and polypeptide chains
of XO may alter the hydrogen bond networks and the secondary
structures of XO molecules. Besides, with the addition of fraction
3, XO might form a more compact structure and make it harder
for xanthine to bind to the active site, which finally leads to a
reduction in enzymatic activity (Yan, Zhang, Pan, & Wang, 2014).
4. Conclusion
The present study demonstrates that APSPE can effectively inhi-
bit the activity of XO with an IC50 value of 7.194 ± 0.858  105
mol C3G equivalent L1. The inhibition type of APSPE on XO is
reversible and mixed inhibition, with competitive inhibition being
dominant. HPLC analysis of the 4 fractions separated from APSPE
show that dietary anthocyanins are enriched in fraction 2 (antho-
cyanins with low acylation) and 3 (anthocyanins with high acyla-
tion). Besides, the inhibitory activities of different fractions
against XO follow the order of fraction 3 > fraction
2 > APSPE > fraction 1 and 4, indicating that the inhibitory effect
may be attributed to anthocyanins. Molecular docking results fur-
ther confirm that fraction 3 has a stronger inhibition on XO than
fraction 2. The principal inhibitory mechanism of dietary antho-
cyanins on XO activity could be the insertion of acylated group of
anthocyanins into the hydrophobic pocket of XO and the occupa-
tion of anthocyanins on the catalytic center of the enzyme by com-
peting with the substrate. The acylated groups on the sophoroside
may play an important role in the binding process of anthocyanin
with XO, and the category of anthocyanins (Cyanidin or Peonidin)
does not affect the inhibition. Both the fluorescence and CD spec-
troscopy results show that the formation of anthocyanin-XO com-
plex may be the possible inhibition mechanism. After the complex
is formed, it is harder for xanthine to bind to the active site, which
finally leads to a reduction in enzymatic activity.
This study provides fresh insight into the key effective xanthine
oxidase (XO) inhibitors in the dietary anthocyanins from purple
sweet potato and the underlying mechanisms in vitro. In combina-
tion with the results in our previous study, high-acylated dietary
anthocyanins from purple sweet potato can be a promising XO
inhibitor both in vitro and in vivo for clinical purposes and as a diet-
ary supplement in hyperuricemia treatment. Moreover, this study
may provide implications for assessing the health benefits of
anthocyanin-rich foods.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This research is supported by the Fundamental Research Funds
for the Central Universities of China (No. 2013PY095), the Clinical
Research Project of Health and Family Planning Commission of
Wuhan Municipality (No. WX13A05), the Research Project of
Wuhan City Central Hospital (No. YQ15A04) and National Under-
graduate Innovative Test Program (No. 201510504070). The
authors would like to thank Mr. Zuo-xiong Liu for giving advice
on language for this paper.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jff.2017.06.048.
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