Structure-activity relationship of xanthones as inhibitors of xanthine oxidase

Article abstract of DOI:10.3390/molecules23020365

Polygala plants contain a large number of xanthones with good physiological activities. In our previous work, 18 xanthones were isolated from Polygala crotalarioides. Extented study of the chemical composition of the other species Polygala sibirica led to the separation of two new xanthones-3-hydroxy-1,2,6,7,8-pentamethoxy xanthone (A) and 6-O--D-glucopyranosyl-1,7- dimethoxy xanthone (C)-together with 14 known xanthones. Among them, some xanthones have a certain xanthine oxidase (XO) inhibitory activity. Furthemore, 14 xanthones as XO inhibitors were selected to develop three-dimensional quantitative structure-activity relationship (3D-QSAR) using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models. The CoMFA model predicted a q2 value of 0.613 and an r2 value of 0.997. The best CoMSIA model predicted a q2 value of 0.608 and an r2 value of 0.997 based on a combination of steric, electrostatic, and hydrophobic effects. The analysis of the contour maps from each model provided insight into the structural requirements for the development of more active XO inhibitors.

Full text of DOI:10.3390/molecules23020365

molecules
Article
Structure–Activity Relationship of Xanthones as
Inhibitors of Xanthine Oxidase
Ling-Yun Zhou 1,2, Jia-Le Peng 3, Jun-Ming Wang 1, Yuan-Yuan Geng 1, Zhi-Li Zuo 3,
*
and Yan Hua 1,
*
1
Key Laboratory for Forest Resources Conservation and Use in the Southwest Mountains of
China (Southwest Forestry University), Ministry of Education, Kunming 650224, China;
zly4321@sina.com (L.-Y.Z.); dawn723@163.com (J.-M.W.); gyy18254369476@163.com (Y.-Y.G.)
Anhui Provincial Engineering Research Center for Polysaccharide Drugs, School of Pharmacy,
Wannan Medical College, Wuhu 241002, China
State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, China; JialePeng2016@163.com
Correspondence: zuozhili@mail.kib.ac.cn (Z.-L.Z.); huayan1216@163.com (Y.H.);
Tel: +86-183-1398-2055 (Z.-L.Z.); +86-138-8866-8877 (Y.H.)
2
3
*
Received: 31 December 2017; Accepted: 7 February 2018; Published: 9 February 2018
Abstract: Polygala plants contain a large number of xanthones with good physiological activities.
In our previous work, 18 xanthones were isolated from Polygala crotalarioides. Extented study
of the chemical composition of the other species Polygala sibirica led to the separation of two
new xanthones—3-hydroxy-1,2,6,7,8-pentamethoxy xanthone (A) and 6-O-β-D-glucopyranosyl-1,7-
dimethoxy xanthone ( )—together with 14 known xanthones. Among them, some xanthones have a
C
certain xanthine oxidase (XO) inhibitory activity. Furthemore, 14 xanthones as XO inhibitors were
selected to develop three-dimensional quantitative structure–activity relationship (3D-QSAR) using
comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis
(CoMSIA) models. The CoMFA model predicted a q2 value of 0.613 and an r2 value of 0.997. The best
CoMSIA model predicted a q2 value of 0.608 and an r2 value of 0.997 based on a combination of steric,
electrostatic, and hydrophobic effects. The analysis of the contour maps from each model provided
insight into the structural requirements for the development of more active XO inhibitors.
Keywords: xanthone; xanthine oxidase inhibitors; 3D-QSAR; gout
1. Introduction
Xanthones are a class of natural products commonly occurring in a few higher plant families [
and have been shown to display a wide range of pharmacological properties [ ]. An important
natural source of these compounds is the Polygalaceae family which includes about 22 genera and
1300 species [ ]. In our previous works [ ], 18 xanthones were isolated from Polygala crotalarioides,
some of which have the inhibitory activitiy against XO. This bioactivity attracted us to investigate
more xanthones from other genus of this family. In the present work, 2 new xanthones (compounds
and , Figure 1) and 14 known xanthones were isolated from Polygala sibirica L. var. megalopha Fr.
1]
2,3
4
5,6
A
C
The bioactivity results showed that all 14 tested xanthones (Figure 2) had a certain inhibition activity
against XO. Today, the ligand-based drug design (LBDD) is an important part in computer-aided
drug design (CADD) [
7,
8]. Comparative molecular field analysis (CoMFA) [
9] and comparative
molecular similarity indices analysis (CoMSIA) [10
,11] represent the successful and widely used
LBDD approaches for guiding the strategies of chemical derivative modifications [12]. Conventional
CoMFA uses electrostatic and steric molecular interaction fields, while CoMSIA includes hydrogen
bonding and hydrophobic fields additionally [13
,
14]. The alignment of molecules was important for
Molecules 2018, 23, 365; doi:10.3390/molecules23020365
www.mdpi.com/journal/molecules

Molecules 2018, 23, 365
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QSAR modeling. Especially, the alignment based on docking was normally more reasonable than on
common structures as it takes the binding pose in the receptor into consideration. It also provides
us convenience for docking as many crystal structures of XO have been released [15–18]. Herein, we
studied the structure–activity relationship (SAR) of xanthones as inhibitors of XO through 3D-QSAR
techniques based on molecular docking. We expect that our work could provide complementary and
useful information to discover novel potent XO inhibitors.
O
OMe
OMe
O
OMe
MeO
HO
MeO
MeO
OMe
OH
O
O
A
B
O
OMe
MeO
O
OH
COSY
HMBC
O
HO
O
HO
OH
C
Figure 1. The stucture of compounds A–C from Polygala sibirica.
OH
O
OMe
O
O
OH
OMe O
OH
8
9a
4a
8a
2
1
OMe
OMe
MeO
HO
OMe
OH
MeO
HO
9
O
O
7
3
6
HO
4b O
4
O
5
1
2
3
OMe O
OH
O
OH
O
OMe
MeO
HO
OMe
OH
HO
OMe
OMe
MeO
OMe
OH
O
O
O
4
5
6
O
O
OMe
OH
O
OH
O
OH
MeO
OMe
OMe
O
O
HO
MeO
HO
HO
OH
O
O
OMe
7
8
9
O
OH
O
OMe
OH
O
OMe
MeO
MeO
MeO
HO
OMe
OH
O
O
O
OH
OMe
10
11
12
O
OH
OH
O
OH
HO
O
O
O
HO
O
13
14
Figure 2. The stucture of the 14 xanthones studied.
2. Results and Discussion
2.1. Identification of New Compounds
Compound was obtained as a yellow amorphous powder and its molecular formula (C18H18O8)
was determined based on the HR-ESI-MS data (m/z 575.1667 [M + Na]+ calcd. for 575.1741). The UV
spectrum revealed maxima at 208, 245, 277, and 308 nm. The IR exhibited absorption bands at
A
ν
max

Molecules 2018, 23, 365
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3425 (O–H), 1611 (chelated C=O), 1510 (aromatic ring) cm−1. Its 13C-NMR spectral data (Table 1)
showed the presence of five methoxyl signals, 1 carbonylcarbon signal, as well as 12 aromatic carbons.
These spectral data indicated the presence of a hexasubstituted xanthone. The carbon signal at δC 172.8
is characteristic for a doubly unchelated carbonyl [19] and four of five methoxyl signals (56.6, 60.9,
61.0, 61.7, and 61.8) were typical of di-ortho-substituted methoxyl groups (δC > 60) [20]. The 1H-NMR
spectrum of
respectively. The structure of
of the above evidence, the structure of
A
showed two aromatic proton singlets at δH 6.63 and 6.87, assignable to H-4 and H-5,
was further confirmed by the HMBC spectrum (Figure 1). On the basis
was elucidated as 3-hydroxy-1,2,6,7,8-pentamethoxyxanthone.
A
A
Table 1. The 1H (600 MHz) and 13C-NMR (125 MHz) data of A in DMSO-d6.
Position
δ
δC
H
1
2
3
4
5
6
7
8
9
153.2 s
138.6 s
156.6 s
98.8 d
96.2 d
157.7 s
138.9 s
152.8 s
172.8 s
152.8 s
110.4 s
109.6 s
152.9 s
61.7 q
61.0 q
56.5 q
60.9 q
61.8 q
6.63 (s)
6.87 (s)
4a
8a
9a
10a
OMe-1
OMe-2
OMe-6
OMe-7
OMe-8
3.80 (s)
3.74 (s)
3.89 (s)
3.72 (s)
3.81 (s)
Compound
B was isolated as an amorphous yellow powder. Based on the ESI-MS of B indicating
[M + Na]+ at m/z 311, its molecular formula was deduced to be C15H12O5. The UV spectrum showed
absorption maxima at 205, 245, 285, and 358 nm, suggesting the presence of a xanthone skeleton.
1
The H-NMR spectrum of
B showed the presence of three methoxyl signals at 3.84 and 3.86, two
singlets of aromatic protons at 6.84 (s, H-5) and 7.39 (s, H-8) and three mutual coupling signals of
aromatic protons. Finally, compound
comparison of the literature [21].
B was determined as 6-hydroxy-l,7-dimethoxyxanthone by the
Compound
457.1110 [M + Na]+ (calcd. for 457.1105). The 1H-NMR and 13C-NMR spectra of
similar to those of , except that signals corresponding to the sugar moiety included an anomeric
proton signal ( = 5.88 (1H, d, J = 7.5 Hz)) and anomeric carbon signal ( = 101.6). On acid hydrolysis,
gave a D-glucose unit by comparison with the authentic sample on GC. The relative configuration
of the glucose residue was deduced to be by the coupling constants (J) of 7.5 Hz. In the HMBC
spectrum, the glucosyl group was propamorphous yellow powderosed at C-6 by the correlations from
C
, amorphous yellow powder, was analyzed for C21H22O10 by HR-ESI-MS at m/z
C
(Table 2) were
B
δ
δ
C
β
Glc-H-1 (δH 5.88, J = 7.5 Hz) to C-6 (
δ
c 153.2). Furthermore, the structure of compound
C
was further
confirmed by the HMBC and 1H-1H COSY spectrum. Therefore, the structure of
C was formulated as
6-O-β-D-glucopyranosyl-1,7-dimethoxyxanthone.

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Table 2. The 1H (600 MHz) and 13C-NMR (125 MHz) data of C in DMSO-d5.
Position
δ
δC
H
1
2
3
4
5
6
7
8
160.8 s
110.0 d
134.4 d
105.9 d
103.8 d
153.2 s
147.3 s
106.6 d
174.7 s
158.3 s
117.2 s
112.5 s
150.8 s
56.1 q
6.97 (d, 8.2)
7.51 (t, 8.2)
6.76 (d, 8.2)
7.59 (s)
7.89 (s)
9
4a
8a
9a
10a
OMe-1
OMe-7
3.83 (s)
3.67 (s)
55.7 q
Glc-1
5.88 (d, 7.5)
4.42 (m)
4.44 (m)
4.40 (m)
4.35 (m)
101.6 d
74.5 d
78.3 d
70.9 d
79.2 d
62.2 t
2
3
4
5
6
4.57 (d, 11.7) 4.40 (m)
2.2. CoMFA and CoMSIA Analysis
Table 3 showed the predicted pIC50 values for 14 xanthones (Figure 2), and the differences between
the predicted and experimental pIC50 values are given as well. The CoMFA model was obtained using
the training set of 14 xanthones, and all corresponding statistical parameters were listed in Table 4.
Figure 3 showed the relationship between the CoMFA
Table 3. Xanthones and their observed and predicted (pre.) XO inhibition activities.
CoMFA
CoMSIA
pIC50
Number
pre.pIC50
Residue
pre.pIC50
Residue
1
2
3
4
5
6
7
8
9
10
11
12
13
14
3.66
3.64
3.79
3.71
3.55
3.60
3.62
3.72
3.57
3.57
3.55
3.61
3.53
3.70
3.657
3.648
3.789
3.705
3.551
3.599
3.615
3.722
3.569
3.572
3.555
3.613
3.524
3.702
−0.003
3.654
3.636
3.785
3.721
3.547
3.601
3.619
3.719
3.57
3.575
3.553
3.61
3.528
3.701
−0.006
−0.004
−0.005
0.011
−0.003
0.001
−0.001
−0.001
0
0.008
−0.001
−0.005
0.001
−0.001
−0.005
0.002
−0.001
0.002
0.005
0.003
0
0.005
0.003
−0.006
−0.002
0.002
0.001
Predicted and experimental pIC50 values, from which it can be seen that all points are located
near the diagonal line. The high q2, r2 and F-values, along with the low SEE value, indicated a good
statistical correlation and reasonable predictability of the CoMFA model. The best CoMSIA model was
developed using three descriptor fields: steric, electrostatic and hydrophobic fields. According to the
COMFA analysis method, a good statistical correlation, and reasonable predictability of the CoMSIA
model were also indicated from Table 4 and Figure 3.

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Table 4. Statistical parameters of CoMFA and CoMSIA model.
Method
CoMFA
CoMSIA
PLS statistics
q2
SEP
0.613
0.065
6
0.997
0.005
426
0.608
0.066
6
0.997
0.006
0.997
Optimum components
r2
SEE
F-ratio
Field distribution
Steric
Electrostatic
Hydrophobic
0.632
0.368
0.152
0.316
0.532
Figure 3. Plot of experimental against predicted pIC50 by CoMFA and CoMSIA.
2.3. Verification of Model Reliability
Using the established model, we predicted the relative values of all 14 xanthones and sorted their
values. From the order of the original activity, the prediction of the model and the order of docking
scoring (Table 5), it was found that the order of activity of Compounds 4, 8, and 14 with relatively
high activity was relatively in front, and compound 13 with the ranking of the lowest active was still
backward, and the order of the predicted value was basically the same as that of the original activity.
The results showed that the activity value predicted by the model is reliable.
Table 5. The relative experimental and predicted (pre.) values order of 14 xanthones.
Original
Activity
Order
CoMFA pre.
Activity
Order
CoMSIA
pre. Activity
Order
Docking
Score
Order
coMFA-pre. coMSIA-pre.
Docking
Score
Number pIC50
pIC50
pIC50
1
2
3
4
5
6
7
8
9
10
11
12
13
14
3.66
3.64
3.79
3.71
3.55
3.6
3.62
3.72
3.57
3.57
3.55
3.61
3.53
3.7
3.657
3.648
3.789
3.705
3.551
3.599
3.615
3.722
3.569
3.572
3.555
3.613
3.524
3.702
3.654
3.636
3.785
3.721
3.547
3.601
3.619
3.719
3.570
3.575
3.553
3.610
3.528
3.701
3
8
4
14
1
2
7
12
6
9
10
5
8
4
14
2
3
7
12
6
10
9
11
5
1
4
8
14
2
3
7
12
6
1
10
9
11
5
72.23
71.41
60.48
60.59
53.7
54.48
45.98
64.35
62.93
54.32
50.12
49.86
38.95
61.05
2
8
9
14
4
3
6
10
5
1
11
12
7
11
13
13
13
13

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2.4. CoMFA and CoMSIA Contour Maps
Contour maps of CoMFA and CoMSIA (Figures 4 and 5) gave a semi-quantitative information
for the structure of all 14 xanthones. Compound was used as the reference molecule. The green
3
contours represent regions where bulky substituents would increase the activity, while the yellow
contours represent regions where the steric bulky group would be unfavorable. Furthermore, the blue
and red contours depict the position where positively charged groups and negatively charged groups
would be favorable, respectively. In addition, the purple and orange regions represent the effect of
the electrostatic field on the activity of the compounds. The introduction of hydrophobic groups in
the purple region can improve the activity of the compound, and the introduction of the hydrophilic
group in the orange region is beneficial to the activity.
Figure 4. Contour maps of CoMFA: steric (a) and electrostatic (b) based on Compound 3.
(a)
(b)
(c)
Figure 5. Contour maps of CoMSIA: steric (a); electrostatic (b); and hydrophobic (c) based on
Compound 3.
Compound 3 has the highest activity value, and its various maps are representative. However,
because the molecular docking is not as regular as the skeleton overlap, the situation of each molecule
placed into the active cavity docking is different. In order to analyze comprehensively, each molecule
would be placed in the three-dimensional field, electrostatic field, and hydrophobic field.
As shown in Figure 6, each molecule was placed in a steric field and analyzed in detail. It was
found that the yellow region tended to be close to the 1, 8 position of the compound, indicating that
the two positions should be not suitable for introduction of groups. This may be the reason why
Compound
3 with no substitutions at 8-position was more potent than Compound 4 with a methoxy
substituent. The green region tended to approach the 2-, 3-, 6-, and 7-position of the compound,
indicating that these positions may be suitably introduced into the group and that the larger groups
may favor the activity increase, which explain why Compound 13 without the glycosyl group at
7-position had weak activity compared to Compound
information of the compound was not obvious.
8 with a glycosyl substitution. The 4,5-position
As shown in Figure 7, each molecule was placed in an electrostatic field and analyzed in detail.
It was found that the blue region tended to be close to the 1, 8 position of the compound, indicating that
the introduction of positively charged groups at this site should increase the activity. Compound 3 with

Molecules 2018, 23, 365
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no substituent at the 8-position had better activity compared to Compound
4
being oxygen-substituted.
This also confirmed the previous inference that its 1- and 8-position should be no substituent. The red
region tended to be close to the 7-position of the compound, indicating that the introduction of a
negatively charged group can increase the activity of the compound.
Figure 6. Steric field views of 14 xanthones.
Figure 7. Electrostatic field views of 14 xanthones.
The incorporation of a hydrophilic group at the 2, 3, 6, 7 position of the compound (as shown
in the orange region of Figure 8) should enhance the activity while the purple region tended to be
close to 1, 4, indicating the introduction of hydrophobicity at both positions groups were conducive to
increasing the activity.
Figure 8. Hydrophobic field views of 14 xanthones.

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3. Experimental Section
3.1. General Information
Optical rotations were recorded using a P-1020 digital polarimeter (Jasco, Tokyo, Japan). The UV
spectra were measured on a UV-2401PC spectrophotometer (Shimadzu, Suzhou, China). The IR spectra
were recorded as KBr pellets on a Tensor-27 spectrometer (Bruker, Bremen, Germany). The NMR
spectra were recorded on an AM-400 spectrometer (Bruker) with TMS as an internal standard. ESI-MS
and HR-EI-MS-TOF-MS were measured on a Bruker HTC/Esquire spectrometer and a Bruker Daltonics
Flex spectrometer, respectively. GC analysis was carried out on an HP-5890 II system (Gentech Scientific,
Alto, CA, USA) equipped with a FID detector and a HP-20M capillary column (25 m
Column chromatography was performed with silica gel (Qingdao Marine Chemical Industry Factory,
Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Fairfield, CT, USA) and
×
0.32 mm
×
0.3 µm).
reversed-phase C18 silica gel (40–60 µm, Merck, Darmstadt, Germany). TLC was performed with silica
gel GF254 (Qingdao Marine Chemical Industry Factory). Fractions were monitored by TLC and spots
were visualized by heating after spraying with 5% H2SO4 in ethanol.
3.2. Plant Material
The whole plant of P. Sibirica L. Var megalopha Fr. were collected in Yongshan, Yunnan Province,
People’s Republic of China. A voucher specimen (HUA002) was identified by Prof. Fan Du (Southwest
Forestry University) and was deposited at the College of Forestry, Southwest Forestry University.
3.3. Extraction, Isolation, and Characterization
The 75% ethanol extract of the whole dry plant of Polygala sibirica (5.0 kg) was purified using
chromatography with D101 macroporous resin and the column contents were eluted with H2O,
35% EtOH, 65% EtOH, and 95% EtOH, using a gradient. The 65% EtOH elute was concentrated
in vacuo to afford a residue (480 g), which was subjected to silica gel column chromatography,
using a CHCl3–MeOH gradient (50:1
Fr2 (8 g) was applied to MCI-gel column with MeOH–H2O gradient (1:1
fractions (Fr2A-Fr2D). Fr2B (1.9 g) was chromatographed on a silica gel column and eluted in a
step gradient manner with a CHCl3:MeOH (15:1 0:1) gradient system to afford five fractions 2B1-2B5.
Subfraction 2B2 (0.9 g) was further purified by Sephadex LH-20 (CHCl3:MeOH = 1:1) chromatography
and semi-prepared HPLC (CH3CN:H2O = 40:60, = 254 nm) to produce compound (retention
time = 11.5 min, 5.6 mg) and compound (retention time = 8.2 min, 10.4 mg). Subfraction 2B5 (0.5 g)
was further purified by Sephadex LH-20 (CHCl3:MeOH = 1:1) chromatography and semi-prepared
HPLC (CH3CN:H2O = 30:70, = 254 nm, retention time = 14.1 min) to produce compound (8.6 mg).
3-hydroxy-1,2,6,7,8-pentamethoxyxanthone ( ): yellow amorphous powder; HR-ESI-MS m/z: 385.0899
[M + Na]+ (calcd. for 385.0894, C18H18O8Na+); [α]2D0 22.59◦ (c 0.9, MeOH); UV (CH3OH)
(log ):
→
0:1) as the eluent to separate it into 13 fractions (Fr1–Fr13).
→
1:0) to afford four
→
λ
A
B
λ
C
A
−
λ
ε
max
308 (4.29), 277 (4.07), 245 (4.62), 208 (4.46); IR (KBr) νmax: 3425, 2936, 2853, 1611, 1510, 1468, 1424, 1384,
1273, 1123, 1080, 1061, 993 cm-1; 1H-NMR and 13C-NMR data see Table 1.
6-Hydroxy-l,7-Dimethoxyxanthone (
B
): yellow amorphous powder; ESI-MS m/z: 317 [M + K]+, 311
(log ): 358 (4.022), 285 (3.987), 245 (4.186), 205 (4.517);
: 7.62 (1H, t, J = 8.5 Hz, H-3), 7.39 (1H, s, H-8), 7.04 (1H, d, J = 7.8 Hz,
H-4), 6.90 (1H, d, J = 8.2 Hz, H-2), 6.84 (1H, s, H-5), 3.86 (3H, s, OMe-1), 3.84 (3H, s, OMe-7); 13C-NMR
(100 MHz, DMSO-d6) : 160.0 (s, C-l), 106.1 (d, C-2), 134.4 (d, C-3), 109.5 (d, C-4), 102.3 (d, C-5), 153.8 (s,
[M + Na]+ and 289 [M + H]+; UV (CH3OH)
λ
max
ε
1H-NMR (400 MHz, DMSO-d6)
δ
δ
C-6), 146.0 (s, C-7), 105.6 (d, C-8), 173.6 (s, C-9), 157.4 (s, C-4a), 114.4 (s, C-8a), 111.3 (s, C-9a), 150.5 (s,
C-l0a), 56.1 (q, OMe-1), 56.8 (q, OMe-7).
6-O-
β
-D-Glucopyranosyl-1,7-Dimethoxyxanthone (
C
): yellow amorphous powder; HR-ESI-MS m/z:
112.78◦ (c 0.8, MeOH); UV (CH3OH)
457.1110 [M + Na]+ (calcd. for 457.1105, C21H22O10Na+); [α]2D0
−

Molecules 2018, 23, 365
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λ
(logε): 360 (4.137), 283 (4.215), 247 (4.586), 203 (4.717); IR (KBr) νmax: 3417, 2925, 1620, 1503, 1476,
max
1433, 1385, 1270, 1177, 1077, 806, 583 cm-1; 1H-NMR and 13C-NMR data, see Table 2.
3.4. Acid Hydrolysis of Compound C
◦
A solution of
C
(3 mg) in 1 M HCl (3 mL) was heated in a water bath at 70 C for 6 h. After cooling,
the reaction mixture was neutralized with NaHCO3 and extracted with CHCl3. The aqueous solution
was further concentrated to dryness and dissolved in 1 mL pyridine. Then hydroxyl-ammonium
hydrochloride (1 mg) was added. The solution were kept in a 80 ◦C water bath for 30 min, followed
◦
by treatment with 1 mL Ac2O and kept in a 90 C water bath for another 40 min. The authentic
monosaccharide samples were treated similarly as the hydrolysis products. Finally, 1
µL of these
acetylated sugars were injected into a HP-20M capillary column using N2 as carrier (oven temp.
◦
◦
210 C) to be analyzed by GC using a FID detector (detector temp. 280 C). Ultimately, the sugar
was determined to be D-glucose by comparing the retention time with that of standard sample: tR:
D-glucose 8.686 min.
3.5. Bioassay of Xanthine Oxidase Inhibitory Activity
The enzyme xanthine oxidase catalyzes the oxidation of xanthine to uric acid. Inhibition of
xanthine oxidase results in a decreased production of uric acid. The uric acid production was measured
according to the increasing absorbance at 290 nm. Test solutions were prepared by adding xanthine
(final concentrations 50
µM), hydroxylamine (final concen-tration 0.2 mM), EDTA (final concentration
0.1 mM), and flavonol glycosides in four concentrations (2.5, 5.0, 10.0, 20.0
µM). The reaction was
started by adding 0.2 mL of xanthine oxidase (6.25 mU/mL) in a phosphate buffer solution (pH = 7.50,
◦
200 mM). The mixture (total 1 mL) was incubated for 30 min at 37 C. Prior to the measurement of uric
acid prodution, the reaction was stopped by adding 0.1 mL of HCl (0.58 M) [22]. The IC50 values were
calculated by the mean data values from three determinations.
3.6. Dataset Preparation
All 14 xanthones and their biological activities were performed by our own. Two-dimensional
structures of xanthones were sketched by Chem-Draw 12.0 software and then converted into
three-dimensional structures by GOLD software (Version 3.1, Genetic Optimization of Ligand Docking,
London, UK). Correctly re-docking in the protein crystal structure is a key requirement to predict
the binding poses. Since the XO protein (PDB entry: 1N5X) was downloaded from the protein data
bank [1N5X], it was prepared by removing water and solvent molecules, adding hydrogen atoms,
loading charges, and amending amino acid residues. The original molecule TEI-6720 in the protein
was extracted as a reference ligand in the docking process to explore the root mean square deviation
(RMSD). As a result, the scoring function of CHEMPLP and the docking template of chemscore
kinase were selected as this combination afforded a RMSD of 0.506. Therefore, these 14 compounds
were docked by the selected docking template and scoring function to obtain the poses which were
employed in the QSAR modeling later. Then the activities of all xanthones along with its pIC50 values
were transformed and were given in Table 3. The values of pIC50 of the dataset set ranging from 3.53
to 3.79 are supposed to contribute to the appropriateness of the training set xanthones.
3.7. CoMFA and CoMSIA Studies
The 3D-QSAR modelling was carried out in Sybyl-X 2.0 (Tripos, Inc., St. Louis, MO, USA).
The CoMFA models were developed on the training set of 14 molecules and validated by the test set of
the same molecules, in which Lennard-Jones and Coulomb potentials were employed to calculate the
CoMFA steric and electrostatic fields respectively, using the sp3 carbon as the probe atom having a + 1
charge to determine the magnitude of the field values. The steric and electrostatic energy cutoff was
set to 33 kcal/mol with the column-filtering value set to 2.0 kcal/mol. The CoMSIA was similar to

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CoMFA about the fields around the aligned molecules, and the other descriptor named hydrophobic
effect was calculated together.
3.8. Validation of 3D-QSAR Models
Internal validation was performed using partial least square (PLS) analysis on the training set,
in which leave-one-out (LOO) method was used to determine the cross-validated q2 and the optimal
number of components (ONC), and non-cross-validation was performed to calculate conventional
r2 using the same number of components. The external validation (r2pred) was recommended for the
assessment of the predictive abilities of CoMFA and CoMSIA models on the test set. Predictive rp2red
were calculated as
r2pred = 1 − PRESS/SD
(1)
in which SD is the sum of squared deviations between the experimental inhibitory activity of the test
set and the mean experimental inhibitory activity of the training set molecules, PRESS is the sum of
squared deviations between the predicted and experimental activity values for every molecule in the
test set.
4. Conclusions
The chemical constituents of Polygala sibirica led to two new xanthones. Combined with the
previously isolated xanthones from the Polygala crotalarioides, we investigated the activity of xanthones
against XO. On the basis of these data, three-dimensional quantitative structure–activity relationships
(3D-QSAR) models using CoMFA and CoMSIA were utilized to investigate structural requirements
for improving potency of xathone derivatives as XO inhibitors. Authentic CoMFA model (q2 = 0.613,
r2 = 0.997) and CoMSIA model (q2 = 0.608, r2 = 0.997) were developed and validated. Analysis of
model parameters and contour maps provided details of the structure–activity relationship. We expect
this is quite helpful to develop new xathone XO inhibitory lead compounds, which can provide a
promising approach to treat gout.
Acknowledgments: The authors are grateful for supports from Yunnan Forest Resources Cultivation and
Utilization Collaborative Innovation Center, National Natural Science Foundation of China (no. 31260083),
and Wannan Medical College Cultivating Foundation (no. WK2016Z06).
Author Contributions: L.-Y.Z., Z.-L.Z., and Y.H. designed research; L.-Y.Z. and Y.-Y.G. performed the isolation
and purification of plants, then analyzed the data; L.-Y.Z., J.-L.P., and J.-M.W. conducted the bioassay of xanthine
oxidase inhibitory activity and establish structure–activity relationship; L.-Y.Z. and J.-L.P. wrote the paper.
All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds
limited amount.
A and B are not available from the authors because of the
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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