Potential anti-gout constituents as xanthine oxidase inhibitor from the fruits of Stauntonia brachyanthera

Article abstract of DOI:10.1016/j.bmc.2017.05.010

With the aim of finding a better xanthine oxidase inhibitor with potential anti-gout properties, the studies on the fruit of Stauntonia brachyanthera were carried out, which led to the isolation of 12 glycosides, including 4 new nor-oleanane triterpenoids. Their structures were determined by comprehensive spectroscopic (NMR and HR MS) analysis. Two compounds (4 and 11) exhibited significant inhibitory activities on xanthine oxidase with IC₅₀ values of 5.22 and 1.60?μM, respectively. Another five compounds (1, 2, 3, 8 and 10) showed qualified activities. The results suggested that the existences of nor-oleanane triterpenoids and flavonoids in the fruits were responsible for the inhibitory activity on xanthine oxidase that could cut off the production of uric acid. Nor-oleanane triterpenoids, a new leading XO inhibitor, is worthy of further studies on molecular biology level for its mechanisms.

Full text of DOI:10.1016/j.bmc.2017.05.010

Accepted Manuscript
Potential anti-gout constituents as xanthine oxidase inhibitor from the fruits of
Stauntonia brachyanthera
Dan Liu, Dandan Wang, Wei Yang, Dali Meng
PII:
S0968-0896(17)30608-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.010
BMC 13728
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
27 March 2017
3 May 2017
5 May 2017
Please cite this article as: Liu, D., Wang, D., Yang, W., Meng, D., Potential anti-gout constituents as xanthine oxidase
inhibitor from the fruits of Stauntonia brachyanthera, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.05.010
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Potential anti-gout constituents as xanthine oxidase inhibitor
from the fruits of Stauntonia brachyanthera
Dan Liua, Dandan Wanga, Wei Yanga, Dali Menga, *
a
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang
Pharmaceutical University), Ministry of Education, School of Traditional Chinese
Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China.
* Corresponding author. E-mail address: mengdl@163.com (D.L. MENG)
Abstract
With the aim of finding a better xanthine oxidase inhibitor with potential
anti-gout properties, the studies on the fruit of Stauntonia brachyanthera were carried
out, which led to the isolation of 12 glycosides, including 4 new nor-oleanane
triterpenoids. Their structures were determined by comprehensive spectroscopic
(NMR and HR MS) analysis. Two compounds (4 and 11) exhibited significant
inhibitory activities on xanthine oxidase with IC50 values of 5.22 and 1.60 µM,
respectively. Another five compounds (1, 2, 3, 8 and 10) showed qualified activities.
The results suggested that the existences of nor-oleanane triterpenoids and flavonoids
in the fruits were responsible for the inhibitory activity on xanthine oxidase that could
cut off the production of uric acid. Nor-oleanane triterpenoids, a new leading XO
inhibitor, is worthy of further studies on molecular biology level for its mechanisms.
Keywords: Stauntonia brachyanthera; fruit; xanthine oxidase inhibitor; nor-oleanane
triterpenoids; gout
1. Introduction
Xanthine oxidase (XO) could catalyze the oxidation of hypoxanthine to xanthine
1
and subsequently to uric acid in the purine nucleotides catabolism 2, which is hard to
excrete and plays a vital role in producing hyperuricemia and gout. Its re-oxidation
involves molecular oxygen which acts as electron acceptor, and during this reaction,
− −
•
superoxide radical (O2 ) and hydrogen peroxide (H2O2) are produced . O2 is
•
3
transformed into H2O2 and O2 either spontaneously or catalyzed by superoxide
1

dismutase. Thus, the over-activity of XO leads to the deposition of uric acid in the
susceptible tissues, which triggers the inflammatory pathways with a concomitant
release of reactive oxygen species. Therefore, in order to reduce the serum uric acid
level below the threshold, the xanthine oxidase inhibitor (XOI) is the most popular
candidate, which can block the biosynthesis of uric acid from purine in vivo.
Triterpenoids have been comprehensively reported for their potential
bioactivities on gout 4-6. And Chinese researchers have found the therapeutic effect of
triterpenoids on rheumatoid arthritis 7. Although the mechanism is still unknown, this
discovery greatly encouraged our further studies on triterpenoids abundant plant.
Fortunately, during investigations, the fruit of Stauntonia brachyanthera Hand-Mazz
attracted our great interests because of its abundant contents of triterpenoids 8, 9
.
S. brachyanthera, an evergreen shrub belonging to the family of Lardizabalaceae,
is well-known for its delicious and nutritious fruits 10-12. The whole plant is
demonstrated to possess definite medicinal values during the treatments of cancer,
inflammation, pain, and also be used as diuretics in China 13. Our previous studies had
revealed the anti-gout effects of the components of its leaves 14. Therefore, it could be
expected that as another part of the same plant, its fruit might also contain the
similar bioactive ingredients.
Based on the above analysis, our group is committed to the studies of natural
biological triterpenoids from this plant which might be helpful to reduce the serum
uric acid level below the threshold by inhibits the activity of xanthine oxidase.
Herewith, the chemical constituents of the fruit of S. brachyanthera, as well as their
potential anti-xanthine oxidase activities and preliminary structure-activity
relationships, will be discussed comprehensively with the aim of finding more
promising natural anti-gout resources.
2. Results and discussion
Compound 1-4 were all white amorphous powder obtained from MeOH solvents.
On the basis of HR-ESI-TOF-MS, their molecular formulas were deduced to be
C40H64O14, C41H66O14, C48H76O19, and C43H68O15 from their quasi-molecular ion peak
at m/z 767.4241 [M-H]- (calcd. 767.4223, C40H63O14), [M-H]- m/z 781.4396 (calcd.
2

781.4380), [M+Na]+: m/z 979.4872 (calcd. 979.4873), and [M+H]+: m/z 825.4634
(calcd. for 825.4631), respectively. After detail analysis of their 1H and 13C NMR data
(Table 1-2), it could be found that all these compounds had the same aglycon as that
of brachyantheraoside B9 except the sugar units connected to C-24, which were
determined to be one L-arabinose and one D-glucoses in 1, one L-fucose and one
D-glucose in 2, one L-fucose, one D-glucose, and one L-arabinose in 3, and one
D-glucose and one L-fucose in 4 by acid hydrolysis and HPLC analysis.
Table 1 The NMR data of the aglycones of compounds 1–4 (600 MHz for 1H, pyridine-d5; 150 MHz
for 13C, pyridine-d5. J in Hz)
1
2
3
4
Pos.
δ H
δ C
39.4
28.6
79.8
43.6
57.0
19.7
33.9
40.1
48.5
37.6
24.2
122.9
144.7
42.4
28.8
24.3
47.1
44.8
48.4
70.2
36.6
35.5
23.8
74.0
16.1
17.6
26.3
180.4
26.1
δ H
δ C
38.8
27.5
79.5
42.9
56.5
19.1
33.5
39.7
48.0
37.1
24.0
124.3
144.3
42.1
28.3
23.9
46.8
44.5
48.1
69.9
36.2
35.1
23.5
72.8
15.7
17.2
26.0
180.0
25.7
δ H
δ C
39.2
28.5
δ H
δ C
39.2
28.6
1
0.94, 1.48
1.17, 2.10
0.87, dm (12),
1.76, m, 1.94, m
3.42, dd (10.8,
0.88, m, 1.48,
1.74, m, 1.94,
3.44, dd (0.8,
0.93, m, 1.46,
1.18, m, 2.09,
2
3
3.54,dd (10.8,
79.9 3.46, dd (11.2, 79.6
4
43.2
57.0
19.7
34.0
40.1
48.5
37.6
24.3
123.0
144.8
42.5
28.7
24.4
47.2
43.4
56.9
19.9
33.9
40.1
48.4
37.5
24.3
122.9
144.6
42.4
28.6
24.3
47.0
5
0.89
0.85, d (10.8)
1.19, m, 1.56, m
1.25, m, 1.41, m
0.85, m
0.91, m
6
1.35, 1.71
1.30, 1.44
1.37, m, 1.57,
1.24, m, 1.37,
1.43, m,1.71,
1.31, m,1.44,
7
8
9
1.61
1.93, m
1.90, m
1.62, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1.95, 2.20
5.56, brs
1.83, m, 2.14, m
5.52, m
1.84, m, 2.14,
5.68, m
1.89, m,2.25,
5.56, m
1.09, 2.21
1.50, 1.84
1.15, t (12.6),
1.14, dm
1.21, m, 2.17,
1.49, m, 1.84,
2.00, m, 2.24, m
2.00, m, 2.23,
3.37, dd (13.8,
1.94, 2.47
3.35, dd (12.6,
3.31, m
44.8 3.37, dd (14.2, 44.7
1.57, m, 2.45, m
1.57, m; 2.42,
48.5
70.3
36.6
35.6
23.8
73.3
16.2
17.6
26.4
180.5
26.1
1.50, m, 2.46,
48.4
70.2
36.5
35.5
23.7
73.4
16.0
17.6
26.3
180.4
26.0
1.84, 2.08
1.82, 2.03
1.55, s
1.83, m, 2.08, m
1.82, m, 2.06, m
1.51, s
1.81, m, 2.05,
1.84, m, 2.05,
1.44, s
1.79, m, 2.07,
1.82, m,2.03,
1.54, s
3.80, d (12.0),
0.82, s
4.09, d (9.6),
0.73, s
4.20, m, 4.23,
0.80, s
4.12, d (9.7);
0.88, s
0.96, s
0.92, s
0.94, s
0.99, s
1.23, s
1.21, s
1.19, s
1.21, s
1.60, s
1.58, s
1.57, s
1.59, s
The linkages of these sugars were determined respectively through their HMBC
spectra. In the HMBC spectrum of 1, the long range correlations of H-1 (δ 5.29) of
α-arabinose with C-3 (δ 86.9) of β-glucose, H-1 (δ 4.91) of β-glucose with C-24 (δ
74.0) of aglycone, and H-24 (δ 3.80, 4.59) of the aglycone with C-1 (δ 105.3) of
3

β-glucose finally lead to the formation of the sugar chains. The β-anomeric
configurations for glucoses and the α-anomeric configuration for arabinose were
determined from their coupling constants [(7.8, glc), (7.2, ara)].
Table 2 The NMR data of the sugar moieties of compounds 1–4 (600 MHz for 1H, pyridine-d5; 150
MHz for 13C, pyridine-d5. J in Hz)
1
2
3
4
Pos.
δ H
inner-glc
4.91, d
δ C
δ H
inner-glc
δ C
δ H
δ C
δ H
δ C
inner-glc
inner-glc
1′
2′
3′
4′
5′
6′
105.9
74.8
86.9
69.6
78.7
62.9
4.87, d
4.18, m
4.26, t
103.7
81.3
78.2
4.77, d
103.8
78.6
86.6
69.8
76.2
64.4
4.81, d
4.02, m
4.12, m
3.89, m
4. 21, m
1.64, d
103.6
82.4
78.2
71.4
78.2
64.1
3.99
4.24
4.06, dd
4.16, m
3.93
4.20, m
3.85, m
4.34, dd
71.5 3.89, t (9.0)
3.96
78.5
62.6
4.12, m
4.66, dd
4.71, dd
terminal-ara
terminal-fuc
middle-fuc
terminal-fuc
1′′
2′′
3′′
4′′
5′′
6′′
5.29, d
4.19
106.0
73.1
74.8
69.8
67.5
5.30, d
4.34, dd
4.06, dd
3.99, d
3.82, q
1.62, d
105.6
73.8
75.4
73.2
71.8
17.3
5.44, d
4.33, dd
3.98, dd
3.93, d (3)
3.75, q
104.2
73.8
75.2
73.9
71.8
17.7
5.25,d
4.45,dd
4.10,dd
4.02, m
3.80,t
106.5
73.4
75.6
73.4
71.4
17.6
4.63
3.87
3.78,4.34
1.53, d
1.54, br.s
terminal-ara
1′′′
2′′′
5.16, d
4.46, t (7.8)
3.84, m
4.22, m
3.72, d
105.9
73.4
3′′′
4′′′
75.0
70.1
5′′′
-COCH3
68.3
1.93, s
171.4, 21.2
1.93, s
171.1, 21.1
For compound 2, the HMBC correlations of the H-1'' (δ 5.44) of the fucose and
the C-2' (δ 81.3) of the glucose, H-1' (δ 4.87) of the glucose and the C-24 (δ 72.8) of
the aglycone, the coupling constants of 7.2 Hz (glc) and 7.5 Hz (fuc), as well as the
13C NMR data of fucose confirmed the composition of the sugar moiety.
The HMBC correlations of compounds 3 between the H-1'' (δ 5.44) of the fucose
and the C-2' (δ 78.6) of the glucose, the H-1''' (δ 5.16) of the arabinose and the C-3' (δ
86.6) of the glucose, and H-1' (δ 4.77) of the glucose and the C-24 (δ 73.3) of the
aglycone finally led to the formation of its sugar moiety. Meanwhile,
the characteristic signals of δ 171.4 for the carbonyl carbon, δ 64.4 for C-6' of the
glucose, as well as the HMBC correlation between the methyl group at δ 1.93 (3H, s)
and the δ 64.4 of C-6' indicated the acetylation of C-6' of the glucose in compound 3.
The β-anomeric configuration of the glucoses and fucose and the α-anomeric
4

configuration of the arabinose were determined from their coupling constants [J = 7.8
1 13
Hz (glc), 7.8 Hz (fuc), 7.2 Hz (ara), respectively] in H NMR and the C NMR data
of fucose.
As for 4, its HMBC correlations between H-1'' (δ 5.25) of the fucose and the C-2'
(δ 82.4) of the glucose, and H-1' (δ 4.81) of the glucose and the C-24 (δ 73.4) of the
aglycone finally confirmed its sugar moiety. At the same time, the diagnostic signals
of the acetyl carbons (δ 171.1, 21.1) and the down-field shifted C-6' of the glucose (δ
64.1), as long as the HMBC correlation between the methyl group at δ 1.93 (3H, s)
and the C-6' indicated the acetylation of C-6' of the glucose in compound 4. The
β-anomeric configuration for the glucoses and fucose were determined from their
1
coupling constants [J = 7.7 Hz (glc), 7.5 Hz (fuc), respectively] in H NMR and the
13C NMR data of fucose.
According to the above analysis, the structures of 1-4 were finally defined as 3β,
→3)
20α, 24-trihydroxy-29-norolean-12-en-28-oic acid 24-O-α-L-arabinopyranosyl-(1
-β-D-glucopyranoside (1), 3β, 20α, 24-trihydroxy-29-norolean-12-en-28-oic acid 24-
O-β-L-fucopyranosyl-(1→2)-β-D-glucopyranoside (2), 3β, 20α, 24-trihydroxy-29-
norolean-12-en-28-oic acid 24-O-β-L-fucopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1
→3)]-6-O-acetyl-β-D-glucopyranoside (3), and 3β, 20α, 24-trihydroxy-29-norolean-
→2)
12-en-28-oic acid 24-O-β-L-fucopyranosyl-(1
-6-O-acetyl-β-D-glucopyranoside
(4) (Fig. 1), which were given the trivial names as brachyantheraoside B1, B2, B3 and
B8, respectively.
By comparing their physical and spectroscopic data with those of the literatures
reported, the rest compounds were identified as YM9 (5) 15, YM10 (6) , brachyanin F
15
17
,
(7) 16, 1′-O-β-D-(3,4-dihydroxyphenyl)-ethyl-6′-O-vanilloylglucopyranoside (8)
brachyanin C (9) 16, saponarin (10) 18, isoquercitrin (11) 19, and vitexin (12) 20 (Fig. 2).
5

1
2
3
4
Fig. 1 The structures and the key HMBC correlations of compounds 1-4
5
6
R1=β-D-glc-(1→3)-α-L-rha-(1→2)-α-L-ara-; R2=α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-
→2)
R1=α-L-rha-(1
→4)
→6)
-β-D-glc-(1
-α-L-ara-; R2=α-L-rha-(1
-β-D-glc
7
8
9
10 R1=-H;
11 R1=-H;
R2=β-D-glc-; R3=β-D-glc-; R4=-H;
R5=-H
R2=-H;
R3=-H;
R3=-H;
R4=-O-β-D-glc-; R5=-OH
R4=-H; R5=-H
12 R1=β-D-glc-; R2=-H;
Fig. 2 The structures of compounds 5-12
The xanthine oxidase inhibitory activities of all the glycosides were evaluated in
vitro and the results were listed in Table 3. Allopurinol was used as the positive
control. Among them, 4 and 11 exhibited significant inhibitory activities with the IC50
6

values of 5.22 and 1.60 µM, respectively, which were all stronger than that of positive
control (IC50 50.15 µM). In addition, compounds 1, 2, 3, 8 and 10 also showed the
inhibitory activities against XO in a certain degree with IC50 values of 57.42, 56.68,
33.70, 61.10 and 50.24 µM, respectively, which were close to that of the positive
control.
Table 3 The inhibitory activities of the isolated compounds on XO in vitro
Compoounds.
IC50 (µM)
57.42
56.68
33.70
5.22
-
1
2
3
4
5
6
97.30
86.22
61.10
-
7
8
9
10
50.24
1.60
-
11
12
allopurinol
50.15
* IC50 > 100 µM
These compounds could be divided into two groups, nor-oleanane glycosides and
phenol glycosides. From Table 3, it could be found that the nor-oleanane glycosides
had better inhibitory behaviors than those of the others. By comparing the structures
of 1, 2, 3 and 4 to those of the others (5 and 6), it could be confirmed that the
inhibitory activities will be significantly weakened when the C-28 was substituted by
the sugar moieties and the free carboxyl group at C-28 was very necessary to the
14
anti-gout activity . The acetyl substitution on sugar moieties could lower down the
polarity of the molecule and subsequently reduce the abilities, such as 3 and 4. And
the more glycosyls it had in the structure, the less inhibition it produced (3 and 4).
Flavonoids, phenols, and their glycosides have been reported to have inhibitory
effects on XO 21-23. Among the six phenolic glycosides obtained in our present studies,
the XO inhibitory effect of 11 (IC50=1.60 µM) was significantly higher than the others,
and 10 (IC50=50.24 µM) also expressed a considerable capability. The results
indicated that flavonoids were much powerful than the simple phenolics. Preliminary
7

structure-activity relationships analysis showed that the unsubstituted C-8 was
necessary for the flavonoids to practice as a XOI, and the sugar substitution at C-3
could enhance the inhibition capability. Among the simple phenolic glycosides, 7 and
8 exhibited different activities, while 9 had no effect at all. So, the oxidation on
phenolic structure would increase the XO inhibitory effect, which was consistent with
the previous report 24. From an overall perspective, compared with nor-oleanane
glycosides, the activities of phenolic glycosides didn’t perform a good XOI activity.
But, as a main constituent in the fruits of S. brachyanthera, this type of chemical
constituent would also play an active role for the XO inhibitory effects of the fruit in
vivo due to their multiple-factors and multiple-targets properties.
As mentioned above, the leaves of the plant had been reported in our previous
study for its anti-gout bioactivity due to the existence of nor-oleanane triterpenoids 14.
However, besides nor-oleanane triterpenoids, the flavonoids in the fruits also
contribute a lot to the effect. Therefore, combined the studies on its leaves and fruits,
it could be believed that both nor-oleanane triterpenoids and flavonoids are naturally
occurring bioactive compounds that exerts prominent anti-gout effects, which enrich
the structures and resources of gout suppressants.
3. Conclusion
The current study reported four new and eight known compounds from the
delicious fruit of S. brachyanthera. Seven of them showed obvious inhibitory
activities on xanthine oxidase and the effects of two ones were even stronger than that
of positive control. Therefore, this fruit could exert its XO inhibitory functions with
the existences of nor-oleanane triterpenoids and flavonoids. Especially nor-oleanane
triterpenoids, a new leading XO inhibitor, is worthy of further studies from
molecular biology level for its mechanism.
4. Experimental section
4.1 Instruments and reagents
The nuclear magnetic resonance (NMR) spectra were acquired using Bruker
ARX-600 spectrometer (Bruker Biospin, Rheinstetten, Germany). Chemical shifts (δ
ppm) are relative to TMS (Tetramethylsilane) as an internal standard. HR-TOF-MS
8

was measured on a micro TOFQ Bruker mass spectrometer. Analytical HPLC was
performed on a kromasil-ODS column (4.6×200 mm, AkzoNobel Global, Bohus,
Sweden) consisting with a refractive index detector (Shimadzu RID-10A, Kyoto,
Japan). Signs of optical rotation were observed by an optical rotation detector (Jasco
LC2000P, Tokyo, Japan). Preparative HPLC was equipped with an YMC-ODS
column (YMC-Pack ODS-A, 250×20 mm, 5 µm) and a refractive index detector
(Shimadzu RID-10A). Column chromatographic (CC) purifications were performed
on silica gel (200-300 mesh, Qingdao Haiyang Chemical Group Corporation, Qingdao,
China), as well as Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), and
Macroporous resin HPD100 (Cangzhou Bon adsorber Technology Co., Ltd.,
Cangzhou, China). Xanthine Oxidase from milk (X4875-10UN, Grade IV, ammonium
sulfate suspension, 0.1-0.4 units/mg protein), D-glucose, L-fucose, L-arabinose, and
L-rhamnose were all purchased from Sigma-Aldrich (Sigma-Aldrich China, Shanghai,
China). Silica gel GF254 (Qingdao Haiyang Chemical Group Corporation, Qingdao,
China) were used for analytical and preparative TLC. And xanthine was
purchased from Beijing Coupling Technology Co., Ltd. All solvents and chemicals
used here were analytical grade, while other reagents were of HPLC grade or of the
highest grade commercially available.
4.2 Plant material
The plants of S. brachyanthera were collected on October, 2009 in Hunan
Province, and were identified by Prof. Jincai Lu, School of Traditional Chinese
Materia Medica, Shenyang Pharmaceutical University, China. The specimen
(NO.HLG-0910) was deposited in the School of Traditional Chinese Material Medica,
Shenyang pharmaceutical University, China.
4.3 Extraction and isolation
The air-dried fruits of S. brachyanthera (7.0 kg), whose seeds were separated,
were crushed into good pieces and were decocted with EtOH-H2O (7:3, v/v, 35 L)
under refluxing for 4 hours for 4 times, and the total extraction was retrieved after
filtered. After a recuperation of the solvent from the mixture in vacuo, the final
aqueous fractions (2.7 kg) were dissolved in water and passed through macroporous
9

adsorptive resin (HPD100), eluted seriatim with H2O, 30% EtOH, 60% EtOH and
90% EtOH, respectively. The 60% EtOH eluates (210 g) were dealt with silica gel
column chromatography (CC) (1000 mm×100 mm i.d.) with a successive
CH2Cl2-MeOH system (100:1-0:100, v/v) to give eight fractions (1-8). Comp. 12 (3.4
mg) was obtained directly from Fr. 5 by the recrystallization method; Comp. 1 (54.1
mg) and 11 (10.6 mg) were separated from Fr. 6 employing ODS CC (300 mm×50
mm i.d.) with MeOH-H2O (33:67, v/v) as eluant; Comp. 5 (20.0 mg), 6 (14.3 mg),
and 10 (155.5 mg) were all obtained from Fr. 7 by the same method as 11. Comp. 2-4
(5.2, 14.9, 15.4, 6.6 mg, respectively) and 7-9 (10.9, 11.1, 8.7 mg, respectively) were
purified from Fr. 4 and Fr. 8, respectively, employing ODS CC (500 mm×60 mm i.d.)
with MeOH-H2O (30:70-45:55, v/v) as eluant and followed by preparative HPLC with
MeOH-H2O (80:20-55:45, v/v) as mobile phase.
4.4 Acid hydrolysis
The operation was carried out with a modified method reported 8, 16. In brief, the
solution of sample (each about 4.0 mg) is homogenized in methanol (2.5 mL),
associated with 1 M H2SO4 (2.5 mL). Then the solution was heated at 90 ℃ for 7 h
(kept sealed) and was neutralized with 1 M NaOH to give cloudy solution. An
extraction with EtOAc (saturated with H2O, 5 mL×2) was done when the mixture was
cool down, of which the aqueous solution was retrieved under reduced pressure. The
H2O fraction was diluted and passed through a Sep-Pak C18 cartridge, which then
analyzed by HPLC under the following method: column, Capcell Pak NH2 UG80;
solvent, MeCN-H2O (3:1); flow rate, 0.5 mL/min; detection, RI and OR. The
identifications of D-glucose, L-arabinose, L-rhamnose, and L-fucose presenting in the
polysaccharide parts were recognized by the polarities with those of authentic samples.
They were detected at 589 nm on a polarimeter that the optical rotation of 0.2% (w/v)
of D-glucose, L-arabinose, L-rhamnose, and L-fucose consisting with a homologous
standard distilled water solution.
4.5 In vitro xanthine oxidase inhibitory activity
The XO inhibition assay was carried out as the modified method reported 14, 25, 26
.
The uric acid concentration was measured at 290 nm by spectrophotometric on 96
10

well plates (Fisher Science, US). The compounds were previously prepared into 1.5,
0.15, 0.015, 0.0015 mg/mL solutions, respectively. The tubes were incubated at 37 °C
for 15 min, then, 60 µL of xanthine solution was added into control (10 µL PBS (0.2
M, pH 7.4) solution and 30 µL xanthine oxidase solution (0.48 M)) and test groups
(10 µL compound solution and 30 µL xanthine oxidase solution), respectively, while
60 µL of PBS was added into sample controls (10 µL compound solution and 30 µL
PBS). The absorption was then measured immediately. The XO inhibition rate was
determined by measuring the difference in absorbance that correlates with uric acid
formation. Allopurinol was used as positive control. All experiments were performed
in triplicate and averaged.
XO inhibition rate = (1-(At-As)/Ac) × 100%, where Ac, As, and At are the
absorbances of the control, sample control, and test groups, respectively.
4.6 Statistical analysis
All computations were made with the statistical software of Statistical Product
and Service Solutions (SPSS, version 17.0), and the level of significance was set at
P<0.05.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(Grant No.81073154, 81374061, 81573694), the Program for Innovative Research
Team of the Ministry of Education and Program for Liaoning Innovative Research
Team in University, and the Program for Innovation team of Liaoning province (Grant
No. LT2015027).
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13

Potential anti-gout constituents as xanthine oxidase inhibitor
from the fruits of Stauntonia brachyanthera
Dan Liua, Dandan Wanga, Wei Yanga, Dali Menga, *
a Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang
Pharmaceutical University), Ministry of Education, School of Traditional Chinese
Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China.
* Corresponding author. E-mail address: mengdl@163.com (D.L. MENG)
14