New prenylflavonol glycosides with xanthine oxidase inhibitory activity from the leaves of Cyclocarya paliurus

Article abstract of DOI:10.1016/j.bioorg.2020.104018

Eight new prenylflavonol glycosides (1–8), along with five known analogues (9–13) were isolated from the n-butanol extract of the dried leaves of Cyclocarya paliurus (family Juglandaceae) for the first time. The structures of these compounds were characte

Full text of DOI:10.1016/j.bioorg.2020.104018

Journal Pre-proofs
New prenylflavonol glycosides with xanthine oxidase inhibitory activity from
the leaves of Cyclocarya paliurus
Zi-Jun Ye, Xiao-Ai He, Jian-Ping Wu, Jing Li, Xi-Wen Chang, Jie Tan, Wen-
Yan Lv, Hui Zhu, Hui-Hui Sun, Wen-Xuan Wang, Zu-Hui Chen, Gang-Zhi
Zhu, Kang-Ping Xu
PII:
S0045-2068(20)31315-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.104018
YBIOO 104018
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 February 2020
29 May 2020
12 June 2020
Please cite this article as: Z-J. Ye, X-A. He, J-P. Wu, J. Li, X-W. Chang, J. Tan, W-Y. Lv, H. Zhu, H-H. Sun, W-
X. Wang, Z-H. Chen, G-Z. Zhu, K-P. Xu, New prenylflavonol glycosides with xanthine oxidase inhibitory
activity from the leaves of Cyclocarya paliurus, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
j.bioorg.2020.104018
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New prenylflavonol glycosides with xanthine oxidase inhibitory
activity from the leaves of Cyclocarya paliurus
Zi-Jun Ye a, Xiao-Ai He a, Jian-Ping Wu b, Jing Li b, Xi-Wen Chang b, Jie Tan b,
Wen-Yan Lv b, Hui Zhu b, Hui-Hui Sun b, Wen-Xuan Wang b, Zu-Hui Chen b,c,
Gang-Zhi Zhu a*, Kang-Ping Xu b*
a Department of Pharmacy, Affiliated HaiKou Hospital Xiangya School of Medicine, Central
South University, Haikou, 570100, PR China
b Department of Pharmacochemistry, College of Xiangya Pharmacy, Central South University,
Changsha, 410013, PR China
c Hunan Heran Biotechnology Development company, 410013, Changsha, PR China
* Corresponding author.
E-mail address: csdb2012@126.com (G.Z. Zhu)
* Corresponding author.
E-mail address: xukp395@csu.edu.cn (K.P. Xu)
1

Abstract
Eight new prenylflavonol glycosides (1-8), along with five known analogues
(9-13) were isolated from the n-butanol extract of the dried leaves of Cyclocarya
paliurus (family Juglandaceae) for the first time. The structures of these compounds
were characterized by comprehensive analysis of 1D, 2D NMR, HRESIMS, UV data
and acid hydrolysis. In bioassay, all these thirteen prenylflavonol glycosides exhibited
inhibitory effects on xanthine oxidase (XOD) activity. Especially compounds 2 and 7,
showed outstanding IC50 values of 31.81±2.20 and 29.71±3.69 μM, respectively.
Keywords: Cyclocarya paliurus; prenylflavonol glycosides; xanthine oxidase
inhibitory activities
2

1. Introduction
Xanthine oxidase (XOD) is an important enzyme in the biosynthesis of uric acid,
which catalyzes oxidation of hypoxanthine to yield xanthine and subsequently uric
acid, along with the production of superoxide ion [1]. The over-activity of XOD will
cause overproduction of uric acid, which further lead to hyperuricemia and gout [2-4].
Allopurinol and febuxostat are XOD inhibitors used clinically for the treatment of
gout [5-6]. However, these drugs possess many side effects including systemic
vasculitis, skin rashes, and even renal failure [7]. Therefore, it’s necessary to search
for novel XOD inhibitors with very less adverse effects. Various plant extracts [8-9]
and polyphenolic compounds, especially flavonoids have been reported with
inhibitory effects on XOD [10, 11].
Cyclocarya paliurus (Batal.) Iljinsk is a single species belonging to family
Juglandaceae, widely distributed in south and southeast areas of China [12]. Its leaves
have long been consumed as a traditional tea in China, because of its palatable taste
and many curative effects, such as relieving arthrophlogosis, analgesic,
anti-inflammation, hpyerglycemic, antioxidant and so on [13-15]. Previous chemical
investigations on this plant led to the isolation of various compounds, including
triterpenoids, flavonoids, phenolic acids, polysaccharides and steroids [16-17]. In our
initial investigation, the C. paliurus tea was found to have effect on reducing the
blood uric acid level of people with hyperuricemia and gout. To our best knowledge,
there were no reports regarding that C. paliurus can improve hyperuricemia and
anti-gout by inhibiting xanthine oxidase activity. To explore new bioactive
compounds from C. paliurus, a bioassay-guided method was used to screen XOD
inhibitory compounds from the different extraction portions. Finally, eight new
prenylflavonol glycosides, Icarisids D-K (1−8) (Fig. 1), together with five known
analogues were isolated and identified from the active fraction (n-BuOH extract
fraction) of C. paliurus for the first time. Herein, the isolation, structure elucidation,
and inhibitory effects on XOD of all the prenylflavonol glycosides (1-13) are
discussed.
3

OH
OH
13
OH
O
OH
O
O
O
12
4'
O
HO
HO
O
O
O
OH
OH
1'
8
HO
HO
O
O
HO
HO
O
O
O
1'''
7
O
2
O
O
10
O
O
6
OH
OH
O
OH
O
OH
4
O
OH
O
1''
OH
OH
OH
OH
O
OH
OH
1''''
OH
O
OH
O
OH OH
O
O
OH
OH
OH
OH
HO
OH
OH
1*
3*
2*
14
15
11
OH
O
O
4'
O
O
OH
R
OH
OH
1'
HO
HO
O
O
7
HO
HO
O
O
1'''
HO
HO
O
O
2
10
O
O
O
O
3''''
O
OH
HO
1''''
5
OH
O
OH
COOH
1''
O
O
OH
O
O
OH
OH
OH
O
O
OH
OH
OH
OH
O
OH
O
OH
O
4''''
O
O
OH
OH
OH
OH
6''''
OH
OH
5* R=H
6*
9
R=CH3
4*
OH
OH
O
HO
OH
OH
O
OH
OH
1'''''
O
O
HO
HO
O
O
O
HO
HO
O
O
HO
O
1'''
O
O
O
OH
O
O
O
O
OH
OH
OH
OH
HO
O
1''
OH
OH
O
OH
OH
OH
O
O
OH
O
OH
O
OH
OH
1''''
OH
O
O
O
OH
OH
OH
OH
OH
10
8*
7*
O
O
O
O
OH
OH
OH
HO
HO
O
O
O
HO
HO
O
O
O
HO
HO
O
O
O
O
O
O
O
OH
O
OH
O
OH
OH
O
OH
OH
OH
O
OH
OH
OH
O
OH
OH
OH
O
OH
O
OH
O
O
O
OH
OH
OH
OH
OH
OH
OH
13
11
12
Fig. 1 Chemical structures of compounds 1-13.
4

2. Materials and methods
2.1 General Experimental Procedures
Optical rotations were measured on a JASCO model 1020 polarimeter (Horiba,
Tokyo, Japan) at room temperature. UV spectra were detected in MeOH using
Probe-2450 (Shimadzu Corporation, Kyoto, Japan). NMR spectra were measured on
Bruker AVANCE 400 and 500 MHz spectrometers (Bruker, Karlsruhe, Germany)
using dimethyl sulfoxide (DMSO-d6) as solvent and tetra methylsilane (TMS) as an
internal standard. High resolution electrospray ionization mass spectroscopy
(HRESIMS) was recorded with an Agilent Technologies liquid chromatograph
connected to Q-TOF mass spectra. Gas Chromatography-Mass Spectrometer
(GC-MS) was measured on GCMS-QP2010 Ultra (Shimadzu Corporation).
Analytical HPLC experiments was carried out on an Agilent 1200 with YMC-Pack
ODS-C18 column (250×4.6 mm, 5 μm, YMC Co. Ltd., Kyoto, Japan) at a flow rate of
1 mL/min. Column chromatographic (CC) separations were carried out on
Macroporous resin HPD-100 (Zhengzhou Qinshi Technology Co., Ltd), polyamide
(80-100 or 30-60 mesh, Taizhou Luqiao Sijia Biochemical Plastics Factory, Taizhou,
China), HW-40C (TOYOPEARL, TOSOH, Tokyo, Japan) and ODS-AA (50 μm,
YMC). Semi-preparative HPLC separations were conducted by using Agilent 1260
system with an ODS-C18 column (250 × 10 mm, 5 μm, YMC).
2.2 Plant Materials
The leaves of Cyclocarya paliurus (Batal.) Iljinsk were collected from Xinning,
Shaoyang, Hunan Province. A voucher specimen (No. 20160820) was identified by
Prof. Kangping Xu from College of Xiangya Pharmacy, Central South University, and
deposited in the Department of Natural Products Chemistry, Xiangya Pharmaceutical
College, Central South University, China.
2.3 Extraction and isolation
The air-dried and powdered leaves of C. paliurus (10.0 kg) were extracted with
5

70% EtOH/H2O under reflux at 100 ℃ (100 L; 2 × 2 h). The solvent was evaporated
under reduced pressure to obtain the crude ethanol extraction, which was then
suspended in water. The aqueous solution (10 L) was partitioned with CH2Cl2, EtOAc
and n-BuOH, respectively. The n-BuOH extract (445 g) was subjected to column
chromatography on Macroporous resin HPD-100 with EtOH-H2O to obtain five
fractions Fr.A1-A5 (0-95%, stepwise). Fr.A3 (107 g) was separated by a polyamide
column using EtOH-H2O afforded to six sub-fractions Fr.B1-B6 (0-95%, stepwise).
Fr.B1 (22.9g) separated to twelve fractions Fr.C1-C12 by HW-40C eluted with a
gradient mixture of MeOH-H2O (from 0:100 to 70:30) on the basis of HPLC analysis.
Combination of fractions Fr.C2 and Fr.C3 (7.5g) was purified by the ODS column
with MeOH-H2O (10-75%, stepwise) system and obtaining 45 sub-fractions
(Fr.E.1-Fr.E45). Fr.E23-25 was separated by Semi-preparative HPLC (ACN-H2O
21%, 0.1% CH3COOH, v/v, 220 nm, 3 mL/min) to give compounds 2 (8.6 mg), 3 (5.9
mg) and 4 (7.4 mg). Fr.E29-30 was separated by Semi-preparative HPLC (ACN-H2O
24%, v/v, 220 nm, 3 mL/min) to give compounds 1 (13.6 mg) and 10 (15.7 mg).
Fr.E35-36 was separated by Semi-preparative HPLC (ACN-H2O 31%, v/v, 0.1%
CH3COOH, 220 nm, 3 mL/min) to give compounds 6 (13.6 mg) and 7 (6.4 mg).
Fr.B2 (20.5 g) was separated to twelve fractions Fr.D1-D12 by HW-40C eluted with a
gradient mixture of MeOH-H2O (0-70%, stepwise) on the basis of HPLC analysis. Fr.
D7 was purified by semi-preparative HPLC (ACN-H2O 31%, v/v, 220 nm, 3 mL/min)
to yield 11 (39.2 mg), 12 (16.4 mg) and 13 (21.7 mg). Combination of sub-fractions
Fr.D2-Fr.D4 (8.7 g) was purified by ODS column with MeOH-H2O (10-75%, step
gradients) system and obtaining 48 sub-fractions (Fr.F1-Fr.F48), Fr.F18-21 were
separated by semi-preparative HPLC (ACN-H2O 19%, 0.1% CH3COOH, v/v, 220
nm, 3 mL/min) and preparative thin-layer chromatography (water saturated n-BuOH)
to afford 8 (7.6 mg). Fr.F25 were separated by semi-preparative HPLC (ACN-H2O
25%, 0.1% CH3COOH, v/v, 220 nm, 3 mL/min) to give 5 (10.6 mg). Fr.F30-33 were
separated by semi-preparative HPLC (ACN-H2O 32%, 0.1% CH3COOH, v/v, 220
nm, 3 mL/min) to give 9 (44.6 mg).
6

2.3.1 Icarisid D (1)
Yellow amorphous powder; [α]2D5 -153.0 (c 0.1, MeOH); UV (MeOH) λmax(log ε)
1
13
nm: 204(3.88), 270(1.42), 320(0.77), and 349(0.71) nm; H and C NMR data (500
and 125 MHz in DMSO-d6) see Tables 1 and 3; HRESIMS, m/z 841.3125 [M+H]+
(calcd for C39H53O20, 841.3130).
2.3.2 Icarisid E (2)
Yellow amorphous powder; [α]2D5 -123.3 (c 0.07, MeOH); UV (MeOH) λmax(log
ε) nm: 204(3.87), 270(1.39), 320(0.76) and 349(0.70) nm; 1H and 13C NMR data (400
and 100 MHz in DMSO-d6) see Tables 1 and 3; HRESIMS, m/z 827.2974 [M+H]+
(calcd for C38H51O20, 827.2974).
2.3.3 Icarisid F (3)
Yellow amorphous powder; [α]2D5 -148.4 (c 0.07, MeOH); UV (MeOH) λmax(log
ε) nm: 204(4.00), 271(1.66), 316(0.91), and 350(0.83) nm; 1H and 13C NMR data (500
and 125 MHz in DMSO-d6) see Tables 1 and 3; HRESIMS, m/z 857.3074 [M+H]+
(calcd for C39H53O21, 857.3079).
2.3.4 Icarisid G (4)
Yellow amorphous powder; [α]2D5 -161.8 (c 0.07, MeOH); UV (MeOH) λmax (log
ε) nm: 204(4.15), 271(2.44), 316(1.37), and 350(1.12) nm; 1H and 13C NMR data (500
and 125 MHz in DMSO-d6) see Tables 1 and 3; HRESIMS, m/z 857.3074 [M+H]+
(calcd for C39H53O21, 857.3079).
2.3.5 Icarisid H (5)
Yellow amorphous powder; [α]2D5 -65.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
1
13
nm: 204(3.95), 270(1.52), 320(0.83), and 349(0.76) nm; H and C NMR data (500
and 125 MHz in DMSO-d6) see Tables 2 and 3; HRESIMS, m/z 807.2701 [M+H]+
(calcd for C38H47O19, 807.2712).
2.3.6 Icarisid I (6)
7

Yellow amorphous powder; [α]2D5 -140.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
1
13
nm: 204(4.10), 271(2.30), 316(1.29), and 350(1.05) nm; H and C NMR data (400
and 100 MHz in DMSO-d6) see Tables 2 and 3; HRESIMS, m/z 823.3011 [M+H]+
(calcd for C39H51O19, 823.3025)
2.3.7 Icarisid J (7)
Yellow amorphous powder; [α]2D5 -119.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
1
13
nm: 204(3.95), 270(1.52), 321(0.65), and 349(0.76) nm; H and C NMR data (400
and 100 MHz in DMSO-d6) see Tables 2 and 3; HRESIMS, m/z 955.3442 [M+H]+
(calcd for C44H59O23, 955.3447).
2.3.8 Icarisid K (8)
Yellow amorphous powder; [α]2D5 +71.3 (c 0.09, MeOH); UV (MeOH) λmax (log
ε) nm: 203(3.86), 270(1.20), 321(0.65), and 349(0.60) nm; 1H and 13C NMR data (400
and 100 MHz in DMSO-d6) see Tables 2 and 3; HRESIMS, m/z 825.2827 [M+H]+
(calcd for C38H49O20, 825.2817).
2.4 Acid Hydrolysis and Monosaccharide Determination of Compounds 1−8
The hydrolysis of compounds 1-8 were established according to the published
method with some modifications [18-19]. Compounds 1-8 (each 1.5 mg) were
hydrolyzed in 5M H2SO4 / 80% CH3COOH (1:1.5, V/V) under reflux in the oil bath at
80 ℃ for 12 h. After cooling down, the mixture was neutralized by Na2CO3 and
extracted with CHCl3. The aqueous layer was dried by a rotary evaporator. The dried
powder was dissolved in pyridine (1.5 mL), meanwhile 1.5 mg of L-cysteine methyl
ester hydrochloride was added into it, the mixture was heated in the water bath at 60
℃ with stirring for 1 h. Then trimethylsilylimidazole (1.5 mL) was added and heated
at 60 ℃ for another 30 minutes. The reaction mixture was analyzed by gas
chromatography-mass spectrometry (GC-MS) analysis. Gas chromatography was
conducted on GCMS-QP2010 Ultra equipped with an AOC-20i auto-injector. The
separation conditions are shown below: passed through a SH-Rxi-5Sil MS capillary
8

column (0.25 µm × 30.0 mm, 0.32 mm, Shimadzu Corporation), at a flow rate of 1
mL/min, with a split ratio of 60:1. The carrier gas was Helium and the injection
volume was 1 μL. The front inlet temperature was 300 ℃. The initial oven
temperature was 150 ℃ and raised to 300 ℃ by 15 ℃/min. The sugar configurations
of derivatives were determined by comparing the retention times with authentic
standard sugars under the same conditions.
2.5. In vitro xanthine oxidase inhibitory activity
In the reaction system of Xanthine (X) and Xanthine Oxidase (XOD), the XOD
•-
can catalyze X into uric acid and superoxide ion (O2 ) [20-21]. Accordingly, the
inhibitory effect on XOD can be evaluated by measuring the production of both uric
acid and superoxide ion.
2.5.1 Detection of superoxide ion produced by X / XOD system
•-
The production of O2 was detected spectrophotometrically by the nitrite method
•-
in 96-well plates [22], for the O2 can reduce the Nitrotetrazolium Blue chloride
(NBT) into blue formazan, which had strong absorption at 560nm. Briefly, the assay
mixture was consisted of 10 μL compound solution (final concentration 1.23, 3.70,
11.11, 33.33, 50.00 and 100 µM) and 10μL NBT solution (final concentration 50
μM), 40 μL XOD solution (final concentration 20 U/L) as the substrate, incubated at
37 ℃ for 10 min. The reaction was initiated by adding 40 μL X solution(final
concentration 120 µM), the assay mixture was incubated at 37 ℃ for another 30 min.
Allopurinol was used as the positive control. Both Allopurinol, compounds and XOD
were dissolved in phosphate buffer (PB, pH 7.5), the X was dissolved in 1M NaOH
(100 μL), diluted with PB and neutralized with 2M HCl to pH 7.5. Finally, the
reaction was stopped with the addition of 50 µL of 2 M HCl, the absorbance value at
560 nm was determined by an enzyme marker (CytationTM 5, Bio Tek, USA).
XOD Inhibition rate (%) = [1-(As-Ab)/(Ac-Ab)] × 100%, where As, Ac and Ab
represent the absorbances of the sample, control, and blank groups, respectively.
9

2.5.2 Detection of uric acid produced by X / XOD system
The concentration of uric acid was measured at 295 nm in 96-well plates
according to the published method with some modifications [23-25]. The method was
similar to the method above, excepted the mixture contained 20 μL compound
solution (final concentration 1.23, 3.70, 11.11, 33.33, 50.00 and 100 µM), without
NBT and final absorbance value was detected at 295 nm.
XOD Inhibition rate (%) = [1-(As-Asb)/(Ac-Ab)] × 100%, where As, Asb, Ac and
Ab represent the absorbances of the sample, sample blank, control, and blank groups,
respectively.
2.6 Statistical analysis
All the experiments were carried out three times and all data were expressed as
means ± SD. Besides, the statistical analyses were performed using one-way ANOVA
with Student’s t-test in Prism 5 software (GraphPad software, San Diego, CA, USA),
and p < 0.05 was considered to be statistically significant.
10

Table 1
1H NMR data of 1–4 (δ in ppm, J in Hz).
Position
6
1b
2a
3b
4b
6.63 (s)
6.63 (s)
6.61 (s)
6.62 (s)
2', 6'
3', 5'
11
7.95 (d, 8.2)
7.11 (d, 8.2)
2.81 (o)
7.98 (d, 8.7)
7.14 (d, 8.6)
2.82 (o)
7.98 (d, 8.2)
7.13 (d, 8.2)
2.82 (o)
7.98 (d, 8.5)
7.13 (d, 8.5)
2.82 (o)
12
1.55, 1.61 (m)
1.17 (s)
1.53, 1.61 (m)
1.17 (s)
1.53, 1.62 (m)
1.17 (s)
1.54, 1.62 (m)
1.17 (s)
14
15
1.17 (s)
1.17 (s)
1.17 (s)
1.17 (s)
-OCH3
1''
3.85 (s)
3.86 (s)
3.85 (s)
3.85 (s)
5.39 (br s)
4.14(br s)
3.64 (o)
5.37 (br s)
4.05 (br s)
3.57 (o)
5.44 (br s)
4.16 (br s)
3.54 (o)
5.55 (br s)
4.13 (br s)
3.40 (o)
2''
3''
4''
3.16 (o)
3.14 (o)
3.59 (o)
3.14 (o)
5''
3.34 (o)
3.49 (o)
3.37 (o)
3.59 (o)
6''
0.82 (d, 4.6)
4.98 (d, 4.9)
3.31(o)
0.89 (d, 6.1)
4.99 (d, 6.4)
3.31 (o)
0.87 (d, 6.1)
4.98 (d, 6.0)
3.32 (o)
0.86 (d, 6.1)
4.98 (d, 6.5)
3.32 (o)
1'''
2'''
3'''
3.32(o)
3.31 (o)
3.40 (o)
3.32 (o)
4'''
3.18(o)
3.17 (o)
3.14 (o)
3.16 (o)
5'''
3.43(o)
3.43 (o)
3.59 (o)
3.43 (o)
6'''
3.47, 3.71(o)
4.89 (br s)
3.68 (o)
3.49, 3.72(o)
4.19 (d, 7.6)
2.97 (o)
3.49, 3.70 (o)
4.29 (d, 7.0)
3.01 (o)
3.46, 3.71 (o)
4.25 (d, 7.5)
2.98 (o)
1''''
2''''
3''''
4''''
3.34 (o)
3.08 (o)
3.15 (o)
3.15 (o)
3.16 (o)
3.19 (o)
3.39 (o)
3.16 (o)
11

5''''
6''''
3.37 (o)
2.89, 3.47 (o)
3.15 (o)
2.98 (o)
1.09 (d, 5.6)
3.49, 3.70 (o)
3.46, 3.71(o)
a Measured in DMSO-d6 at 400 MHz.
b Measured in DMSO-d6 at 500 MHz.
Overlapped signals indicated by (o).
12

Table 2
1H NMR data of 5-8 (δ in ppm, J in Hz).
Position
6
5b
6a
7a
8a
6.62 (s)
6.63 (s)
6.65 (s)
6.59 (s)
2', 6'
3', 5'
11
7.81 (d, 8.5)
6.94 (d, 8.4)
3.43, 3.57 (o)
5.16 (t, 7.0)
1.60 (s)
7.93 (d, 8.8)
7.16 (d, 8.9)
3.42, 3.60 (o)
5.18 (t, 7.0)
1.61 (s)
7.90 (d, 8.8)
7.16 (d, 8.9)
3.41, 3.58 (o)
5.16 (t, 6.8)
1.62 (s)
7.82 (d, 8.7)
6.94 (d, 8.6)
3.42, 3.54 (o)
5.15 (t, 6.8)
1.60 (s)
12
14
15
1.69 (s)
1.70 (s)
1.70 (s)
1.69 (s)
-OCH3
1''
3.86 (s)
3.86 (s)
5.51 (br s)
3.96 (br s)
3.40 (o)
5.46 (br s)
4.06 (br s)
3.55 (o)
5.36 (br s)
4.03 (br s)
3.58 (o)
5.38 (br s)
4.22 (br s)
3.58 (o)
2''
3''
4''
3.05 (o)
3.13 (o)
3.15 (o)
3.16 (o)
5''
3.18 (o)
3.43 (o)
3.47 (o)
3.25 (o)
6''
0.80 (d, 4.5)
5.00 (d, 5.5)
3.31 (o)
0.87 (d, 6)
5.00 (d, 6.4)
3.12 (o)
0.89 (d, 6.1)
5.32 (d, 7.2)
3.52 (o)
0.84 (d, 6.0)
4.99(d, 5.6)
3.29 (o)
1'''
2'''
3'''
3.31 (o)
3.31 (o)
3.45 (o)
3.19 (o)
4'''
3.17 (o)
3.18 (o)
3.40 (o)
3.15 (o)
5'''
3.43 (o)
3.42 (o)
3.52 (o)
3.20 (o)
6'''
3.47, 3.71 (o)
4.74 (br s)
3.48, 3.71 (o)
4.21 (d, 7.7)
2.99 (o)
3.45, 3.67 (o)
4.18 (d, 7.7)
2.96 (o)
3.49, 3.69 (o)
4.33 (d, 7.7)
3.07 (o)
1''''
2''''
3''''
4''''
2.75 (o)
3.07 (o)
3.29 (o)
1.82, 1.96 (m)
3.09 (o)
3.20 (o)
3.36 (o)
13

5''''
6''''
4.17 (m)
3.04 (o)
2.89, 3.47 (o)
3.40 (o)
1.11 (d, 5.6)
0.99 (d, 6.0)
3.50, 3.69 (o)
1'''''
2'''''
3'''''
4'''''
5'''''
6'''''
5.23 (br s)
3.74 (o)
3.40 (o)
3.15 (o)
3.56 (o)
1.06 (d, 6.1)
a Measured in DMSO-d6 at 400 MHz.
b Measured in DMSO-d6 at 500 MHz.
Overlapped signals indicated by (o)
14

Table 3
13C NMR data of 1–8 (δ in ppm).
Position
1b
2a
3b
4b
5b
6a
7a
8a
2
3
157.12
134.63
178.39
159.03
98.20
156.88
134.61
178.42
158.99
98.13
156.89
134.21
178.30
159.14
98.13
156.90
135.01
178.46
158.99
98.17
157.60
134.14
178.17
159.08
98.06
157.04
134.95
178.33
159.19
98.23
157.09
134.69
178.37
159.04
98.09
157.72
133.91
178.22
160.61
98.30
4
5
6
7
160.80
109.84
152.95
105.61
122.27
130.68
114.19
161.61
17.52
160.68
109.74
152.83
105.49
122.11
130.53
114.18
161.58
17.42
160.68
109.67
152.86
105.53
122.21
130.64
114.17
161.50
17.45
160.73
109.78
152.90
105.60
122.24
130.65
114.19
161.60
17.49
160.27
108.28
152.96
105.47
120.45
130.58
115.51
160.47
21.43
160.61
108.35
153.03
105.56
122.14
130.52
114.23
161.57
21.39
159.92
108.59
153.17
105.60
122.05
130.40
114.18
161.51
21.50
160.61
108.37
153.17
105.00
120.36
130.84
115.74
160.77
21.57
8
9
10
1'
2', 6'
3', 5'
4'
11
12
13
14
15
-OCH3
1''
42.95
42.91
42.88
42.98
122.23
131.05
25.46
122.22
131.18
25.50
122.19
131.12
25.39
122.42
131.18
25.62
69.21
69.05
69.03
69.17
29.33
29.37
29.28
29.36
28.71
28.60
28.62
28.67
17.86
17.91
17.90
18.00
55.61
55.50
55.49
55.58
55.54
55.46
100.90
75.74
101.06
80.60
101.76
70.45
101.26
81.36
100.11
77.75
70.35
71.89
69.67
101.23
81.38
100.96
80.51
101.57
69.35
81.27
69.78
70.57
2''
3''
70.23
70.31
69.85
70.58
70.22
68.67
4''
72.05
71.63
81.11
71.73
71.63
71.61
5''
70.85
70.46
69.85
70.24
70.54
70.39
15

6''
1'''
17.58
100.65
73.48
76.55
69.78
77.24
60.76
101.72
70.33
70.59
71.49
68.90
17.69
17.42
100.58
73.40
76.48
69.67
77.18
60.67
106.42
73.74
76.24
69.31
65.81
17.45
100.58
73.92
76.47
69.17
77.15
60.66
104.85
73.35
76.30
69.66
76.88
61.00
17.43
100.64
73.46
76.53
69.32
77.22
60.48
106.17
73.89
76.31
69.74
76.70
60.73
17.45
100.50
73.39
76.60
70.35
77.19
60.65
109.39
83.89
172.05
41.12
74.85
22.98
17.43
100.64
73.41
76.63
69.67
77.25
60.66
106.00
74.04
75.14
75.95
71.71
17.77
17.38
97.97
77.53
76.94
70.26
77.38
60.51
106.35
73.73
76.22
69.29
65.78
17.64
100.63
73.50
76.38
69.90
76.87
60.76
105.78
74.07
76.72
69.98
77.30
61.04
2'''
3'''
4'''
5'''
6'''
1''''
2''''
3''''
4''''
5''''
6''''
1'''''
2'''''
3'''''
4'''''
5'''''
6'''''
100.50
70.50
70.35
71.99
69.79
18.00
a Measured in DMSO-d6 at 400 MHz.
b Measured in DMSO-d6 at 500 MHz.
16

Table 4
The GC retention time of compounds 1-8 and reference substances.
Compounds
L-Rha
Retention time (min)
Peak 1: 6.520 ~ 6.590
Peak 2: 7.115 ~ 7.170
Peak 1: 8.495 ~ 8.540
Peak 2: 8.960 ~ 9.005
Peak 1: 7.275 ~ 7.325
Peak 2: 7.645 ~ 7.695
Peak 1: 7.530 ~ 7.580
Peak 2: 7.875 ~ 7.930
L-Rha: 6.560, 7.147
D-Glc: 8.515, 8.982
L-Rha: 6.558, 7.145
D-Glc: 8.512, 8.980
D-Xyl: 7.285, 7.664
L-Rha: 6.558, 7.145
D-Glc: 8.513, 8.981
L-Rha: 6.562, 7.148
D-Glc: 8.516, 8.983
L-Rha: 6.559, 7.145
D-Glc: 8.513, 8.980
L-Rha: 6.559, 7.148
D-Glc: 8.513, 8.981
D-Qui: 7.552, 7.899
L-Rha: 6.558, 7.145
D-Glc: 8.512, 8.980
Type of the monosaccharide
—
D-Glc
D-Xyl
D-Qui
1*
—
—
—
L-Rha, D-Glc
2*
L-Rha, D-Glc, D-Xyl
3*
4*
5*
L-Rha, D-Glc
L-Rha, D-Glc
L-Rha, D-Glc
6*
7*
L-Rha, D-Glc, D-Qui
L-Rha, D-Glc, D-Xyl
17

D-Xyl: 7.293, 7.665
L-Rha: 6.559, 7.146
D-Glc: 8.513, 8.981
8*
L-Rha, D-Glc
18

3. Result and discussion
3.1 Structure elucidation of compounds 1-8
Icarisid D (1) was obtained as yellow, amorphous powder. Its chemical formula:
C39H52O20 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
841.3125 (calcd for C39H53O20, 841.3130). The UV spectrum showed absorptions at
204, 270, 320, and 349 nm. The 1H and 13C NMR spectra displayed a set of signals at
[δH 7.95 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H); δC 122.27 (C-1′), 130.68
(CH-2′, 6′), 114.19 (CH-3′, 5′), 161.61 (C-4′)] suggested the existing of a
p-substituted benzene ring system; an aromatic methine [δH 6.63 (s); δC 98.20 (CH-6)]
indicated the existence of a pentasubstituted ring A; a methoxy group signal at [δH
3.85(s); δC 55.61 (4′-OMe)]; a series of signals at [δH 2.81 (overlapped, 2H),
1.54-1.60 (m, 2H), 1.17 (s, 6H); δC 17.5 (CH2-11), 42.9 (CH2-12), 69.2 (C-13), 29.33
(CH3-14), 28.71 (CH3-15)] ascribed to a (γ-hydroxyl-γ, γ-dimethyl)-propyl moiety;
the characteristic peaks at [δH/δC 5.39 (br s)/100.90 (CH-1′′) and 0.82 (d, J = 4.6
Hz)/17.58 (CH3-6′′)], [δH/δC 4.89 (br s)/101.72 (CH-1′′′′) and 0.87 (d, J = 5.7
Hz)/17.36 (CH3-6′′′′)] and [δH/δC 4.98 (d, J = 4.9 Hz)/100.65 (CH-1′′′) and 3.47-3.71
(m, 2H)/60.76 (CH2-6′′′)] indicated the existence of two α-rhamnopyranosyl and a
1
13
β-glucopyranosyl units. The assignment of H and C NMR spectroscopic data for 1
1
(Tables 1 and 3) was based on H-1H COSY, HSQC and HMBC correlations (Fig.
2). The HMBC correlations between 4′-OCH3 (δH 3.85) and C-4′ (δc 161.98)
suggested that C-4′ was substituted by a methoxy group. The location of
(γ-hydroxyl-γ, γ-dimethyl)-propyl moiety to C-8 was proved by the cross-peaks of
H-11 (δH 2.81) with C-7 (δC 160.80), C-8 (δC 109.84), and C-9 (δC 152.92). Moreover,
the correlations between H-6 (δH 6.63) and C-5 (δC 159.03), C-7 (δC 160.80), C-8 (δC
109.84), C-10 (δC 105.61) confirmed that the proton singlet at δH 6.63 was assignable
as H-6. The correlations of δH 5.39 (δH 1′′)/C-3 (δC 134.63), 4.89 (δH 1′′′′)/C-2′′ (δC
75.74) and δH 4.98 (δH 1′′′)/δC 160.80 (C-7) in the HMBC indicated the linkage of
sugar
moieties
is
3-O-α-rhamnopyranosyl-(2→1)-α-rhamnopyranosyl-flavonol-7-O-β-glucopyranoside
19

1
13
and the assignments of their H and C NMR data (Tables 1 and 2) were based on
1
the HSQC and H-1H COSY (Fig. 2) data analysis. The absolute configuration of
α-rhamnopyranosyl and β-glucopyranose in 1 was determined to be D and L, which
was based on the GC-MS analysis of sugar derivatives (see Table 4 and supporting
information). Therefore, the structure of 1 was determined as 4′-methoxy-5-hydroxyl
-8-(γ-hydroxyl-γ,γ-dimethyl)-propyl-3-O-α-rhamnopyranosyl-(1→2)-α-rhamnopyran
osyl-flavonol-7-O-β-D-glucopyranoside.
Icarisid E (2) was obtained as yellow, amorphous powder. Its chemical formula:
C38H50O20 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
827.2974 (calcd for C38H51O20, 827.2974). The 1D, 2D NMR data were very similar
to 1, excepted the β-xylopyranose replaced the α-rhamnopyranosyl′. The HMBC (Fig.
2) correlations between δH 4.19 (d, J = 7.6 Hz, H-1′′′′) and δC 80.60 (CH-2′′)
suggested that the β-xylopyranose was located at C-2′′. Besides, the assignments of
1
sugar units were also based on HSQC and H-1H COSY (Fig. 2) data analysis. The
absolute configuration of rhamnopyranosyl, xylopyranose and glucopyranoside was
detected to be L, D, D by the acid hydrolysis and GC-MS analysis of sugar
derivatives (see Table 4 and supporting information). Therefore, compound 2 was
determined
as
4′-methoxy-5-hydroxyl-8-(γ-hydroxyl-γ,γ-dimethyl)-propyl-3-O-β-D-xylopyranose-(1
→2)-α-L-rhamnopyranosyl-flavonol-7-O-β-D-glucopyranoside.
Icarisid F (3) was obtained as yellow, amorphous powder. Its chemical formula:
C39H52O21 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
1
13
857.3074 (calcd for C39H53O21, 857.3079). The H and C NMR data (Tables 1 and
3) were very similar to compound 2, excepted a β-glucopyranoside′ replaced the
β-xylopyranose. The HMBC (Fig. 2) correlations between δC 4.29 (d, J = 7.0 Hz,
H-1′′′′) and δC 81.11 (CH-4′′) suggested that the β-glucopyranoside′ was located at
C-4′′. The assignments of sugar units were based on HSQC and 1H-1H COSY (Fig. 2)
data analysis. The absolute configuration of glucopyranoside and rhamnopyranosyl
was conducted as D, L by using acid hydrolysis and GC-MS analysis of sugar
20

derivatives (see Table 4 and supporting information). Therefore, compound 3 was
determined as 4′-methoxy-5-hydroxyl-8-(γ-hydroxyl-γ,γ-dimethyl)-propyl-3-O-β-D-
glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-flavonol-7-O-β-D-glucopyranoside.
Icarisid G (4) was obtained as yellow, amorphous powder. Its chemical formula:
C39H52O21 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
857.3074 (calcd for C39H53O21, 857.3079). The 1D, 2D NMR data were almost same
as compound 3. Both two compounds have two β-glucopyranosides and a
13
rhamnopyranosyl, but the C NMR data of 4 appeared a signal δC 71.73 (CH-4′′),
1
which was characteristic signal of C-4′′ in rhamnopyranosyl. Furthermore, the H-1H
COSY (Fig. 2) between δC 5.55 (br s, H-1′′) and δC 4.13 (br s, H-2′′), the HMBC (Fig.
2) correlations between δC 4.25(d, J = 7.5 Hz, H-1′′′′) and δC 81.36 (CH-2′′) suggested
that the β-glucopyranoside′ was located at C-2′′. The assignments of sugar units were
1
based on HSQC and H-1H COSY (Fig. 2) data analysis. The absolute configuration
of sugars was detected by acid hydrolysis and GC-MS analysis of sugar derivatives
(see Table 4 and supporting information). Therefore, compound 4 was determined as
4′-methoxy-5-hydroxyl-8-(γ-hydroxyl-γ,γ-dimethyl)-propyl-3-O-β-D-glucopyranosyl-
(1→2)-α-L-rhamnopyranosyl-flavonol-7-O-β-D-glucopyranoside.
Icarisid H (5) was obtained as yellow, amorphous powder. Its chemical formula:
C38H46O19 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
807.2701 (calcd for C38H47O19, 807.2712). The 1D, 2D NMR spectra were almost
identical
with
compound
9,
which
was
determined
as
8-(3,3-dimethylallyl)-5,7-dihydroxy-4′-methoxyflavonol-3-[O-2-C-carboxy-3,5-dideo
xy-β-D-erythro-pentofuranosyl-(1→2)-α-L-rhamnopyranoside]-7-(β-D-glucopyranosi
de) [26], excepted for the methoxy group at C-4′ was replaced by a hydroxide radical.
1
13
The H and C NMR data (Tables 2 and 3) displayed a set of signals at [δH 3.43,
3.57 (overlapped, each 1H), δH 5.16 (t, J = 7.0 Hz, 1H), 1.69 (s, 3H), 1.60 (s, 3H); δC
21.43 (CH2-11), 122.23 (CH-12), 131.05 (C-13), 25.46 (CH3-14), 17.86 (CH3-15)]
indicated the existence of a prenyl unit. The HMBC cross-peaks from H-11 [δH 3.43,
3.57 (overlapped, each 1H)] to C-7 (δC 160.27), C-8 (δC 108.28), C-9 (δC 152.96) and
21

C-12 (δC 122.23) proved the prenyl unit was linked at C-8. The sugar subunits were
also proved by the HRESIMS spectrum at m/z 663.2280 [(M-144)+H]+, 517.1701
[(M-144-146)+H]+, 355.1173 [(M-144-146-162)+H]+ and the assignments of the
1
sugar units were also based on HSQC and H-1H COSY (Fig. 2) data analysis. The
dideoxyfuranose subunit is common in cardenolides, and its configuration was
already confirmed by the single-crystal X-ray diffraction analysis [27-28]. Moreover,
the absolute configuration of glucopyranoside and rhamnopyranosyl was detected by
acid hydrolysis and GC-MS analysis of sugar derivatives (see Table 4 and supporting
information).
Therefore,
compound
5
was
determined
as
4′,
5-dihydroxyl-8-prenyl-3-[O-2-C-carboxy-3,5-dideoxy-β-D-erythro-pentofuranosyl-(1
→2)-α-L-rhamnopyranosyl]-flavonol-7-O-β-D-glucopyranoside.
Icarisid I (6) was obtained as yellow, amorphous powder. Its chemical formula:
C39H50O19 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
1
13
823.3011 (calcd for C39H51O19, 823.3025). The H and C NMR data (Tables 2 and
3) were very similar to those of 5 excepted the dideoxyfuranose was replaced by a
β-quinovopyranosyl unit and existence of a methoxy group. The HMBC (Fig. 2)
correlations between δH 4.21 (d, J = 7.7 Hz, H-1′′′′) and δC 81.38 (CH-2′′) indicated
that the quinovopyranosyl was located at C-2′′, the correlations between δH 3.85(s)
with C-4′ (δC 161.57) showed the methoxy group was located at C-4′ and the
1
assignments of the sugar units were also based on HSQC and H-1H COSY (Fig. 2)
data analysis. Further acid hydrolysis and GC-MS analysis of sugar derivatives
proved the absolute configuration of rhamnopyranose, glucopyranose and
quinovopyranosyl was L, D, D (see Table 4 and supporting information). Therefore,
compound
6
was
proved
to
be
4′-methoxy-5-hydroxyl-8-prenyl-3-O-β-D-quinovopyranosyl-(1→2)-α-L-rhamnopyra
nosyl-flavonol-7-O-β-D-glucopyranoside.
Icarisid J (7) was obtained as yellow, amorphous powder. Its chemical formula:
C44H58O23 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
955.3442 (calcd for C44H59O23, 955.3447). The 1D, 2D NMR spectra were very
22

similar to those of 6 excepted the signals of β-xylopyranose and α-rhamnopyranosy
units replaced the quinovopyranosyl. According to the HMBC (Fig. 2) correlations
between 4.18 (d, J = 7.7 Hz, H-1′′′′) and δC 80.51 (C-2′′) suggested that the
β-xylopyranose was located at C-2′′, and the correlations between δH 5.23 (br s,
H-1′′′′′) and δC 77.53 (C-2′′′) indicated that the α-rhamnopyranosy was located at
1
C-2′′′ and the assignments of the sugar units were based on HSQC and H-1H COSY
(Fig. 2) data analysis. Furthermore, the absolute configuration of sugars was detected
by acid hydrolysis and GC-MS analysis of sugar derivatives (see Table 4 and
supporting information). Therefore, compound
7
was determined as
4′-methoxy-5-hydroxyl-8-prenyl-3-O-β-D-xylopyranose-(1→2)-α-L-rhamnopyranosy
l-flavonol-7-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside.
Icarisid K (8) was obtained as yellow, amorphous powder. Its chemical formula:
C38H48O20 was determined by the positive-ion HRESIMS data as [M+H]+ at m/z
825.2827 (calcd for C38H49O20, 825.2817). The 1D, 2D NMR spectra were very
similar to 5 excepted the signals of dideoxyfuranose was replaced by
β-glucopyranoside′. The HMBC (Fig. 2) correlations between δH 4.33 (d, J = 7.7 Hz,
H-1′′′′) and δC 81.27 (CH-3′′) suggested that the β-glucopyranoside′ was located at
C-3′′. Moreover, the assignments of sugar units were also based on HSQC and 1H-1H
COSY (Fig. 2) data analysis. The absolute configuration of sugars was detected by
acid hydrolysis and GC-MS analysis of sugar derivatives (see Table 4 and supporting
information).
Therefore,
compound
8
was
determined
as
4′
，
5-dihydroxyl-8-prenyl-3-O-β-D-glucopyranoside-(1→3)-α-L-rhamnopyranosyl-flavo
nol-7-O-β-D-glucopyranoside.
The five reported compounds (9-13) were confirmed by comparing their
spectroscopic data with their literature data, and was identified as
8-(3,3-dimethylallyl)-5,7-dihydroxy-4′-methoxyflavonol-3-[O-2-C-carboxy-3,5-dideo
xy-β-D-erythro-pentofuranosyl-(1-2)-α-L-rhamnopyranoside]-7-(β-D-glucopyranosid
e) (9) [26], Icarisid B (10) [29], epimedin C (11) [30], epimedin B (12) [31],
Epimedin A (13) [32].
23

24

OH
OH
12
OH
O
OH
O
O
O
4'
HO
HO
O
O
O
11
OH
OH
1'
HO
HO
O
O
HO
HO
O
O
O
1'''
O
O
O
9
2
O
O
OH
OH
6
4
O
OH
O
OH
OH
O
1''
OH
O
O 1''''
OH
OH
OH
O
OH
OH
OH
O
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
2*
3*
HO
1*
OH
OH
O
OH
OH
O
OH
OH
HO
HO
O
O
O
HO
O
O
HO
O
O
O
O
OH
O
HO
O
HO
HO
O
OH
COOH
OH
O
O
OH
OH
O
OH
O
OH
O
O
OH
OH
OH
OH
O
O
OH
O
O
O
OH
O
OH
OH
OH
OH
OH
OH
4*
6*
5*
OH
HO
OH
O
OH
O
OH
O
O
O
HO
HO
O
O
O
HO
O
O
OH
O
O
HO
O
OH
OH
OH
OH
OH
OH
O
O
O
OH
OH
OH
OH
O
O
O
OH
OH
OH
OH
8*
7*
1H-1H COSY
HMBC
Fig. 2 Key 1H-1H COSY and HMBC correlations of compounds 1-8.
25

3.2. XOD inhibitory effects of the prenylflavonol glycosides
The xanthine oxidase inhibitory effects of all the prenylflavonol glycosides
(1-13) and Allopurinol (positive control) have been evaluated by measuring the
production of superoxide ion and uric acid, and the results were listed in Table 5.
Both in the assays of superoxide ion and uric acid production, compounds 1, 2, 6 and
7 exhibited significant inhibitory activities on XOD with IC50 values ranging from 29
to 55 µM, compounds 3, 4 and 10 exhibited moderate inhibition on XOD with IC50
values ranging from 57 to 88 µM. Moreover, other tested compounds showed weak
effects on uric acid production with IC50 > 100 µM.
Regarding the basic chemical structure of flavonols, the hydroxyl groups at C5
and C7 along with the double bond between C2 and C3 were found to be essential for
high inhibitory activity on XOD [33-34]. It is noteworthy that all the compounds were
flavonols with the unsubstituted hydroxyl group at C5, double bond between C2 and
C3, whereas the hydroxyl groups at C7 and C3 are substituted with the glycosides.
The substitution of hydroxyl groups at C7 is responsible for the lower inhibition of
1-13 than positive control allopurinol [35]. Besides, among the prenylflavonol
glycosides showing inhibitory activities on XOD, compound 7 displayed higher
inhibitory effect, indicating that glycosylation of flavonols affected the inhibitory
effect on XOD depending on the conjugation site, number and the class of sugar
moiety [35-36]. In addition, when the glycosyl moiety was same, the occurrence of
(γ-hydroxyl-γ,γ-dimethyl)-propyl unit showed a better inhibitory activity than those of
prenyl unit (1 Vs 11, 2 Vs 12, 4 Vs 13).
26

Table 5
Inhibitory effects of 1–13 on XOD activity in vitro.
IC50 (μM)
Compound
superoxide ion
45.32±2.43
31.81±2.20
68.18±4.56
56.22±2.64
77.34±4.40
43.65±2.27
29.71±3.69
>100
uric acid
53.94±3.68
42.03±2.03
74.96±1.02
88.04±3.56
>100
1
2
3
4
5
6
55.64±1.71
39.26±3.67
>100
7
8
9
85.22±5.38
75.21±4.65
>100
>100
10
68.62±2.72
>100
11
12
93.47±4.26
82.59±2.88
8.54±1.58
>100
13
>100
Allopurinol
10.76±1.06
IC50 values in μM are means ± SD from three independent experiments.
27

4. Conclusion
In the present study, eight new prenylflavonol glycosides named Icarisids D-K
(1-8), together with five known analogues (9-13) were discovered and isolated from
the active site (n-BuOH extraction) of C. paliurus. The potential anti-gout activity of
C. paliurus was evaluated by the XOD inhibition assays, and compounds 1, 2, 6 and 7
exhibited significant XOD inhibitory activities, with IC50 values of 45.32±2.43,
31.81±2.20, 43.65±2.27 and 29.71±3.69 μM. It is worthy noted that compound 1-13
are the first discovery of prenylflavonol glycosides from Cyclocarya paliurus. Here,
we also firstly found inhibitory effect of C. paliurus on XOD. The present research
enriches the structural diversity of C. paliurus and indicates that prenylflavonols
could be developed as new chemical agents for the treatment of hyperuricemia and
gout in the future.
Conflict of interest
No potential conflict of interest was reported by the authors.
Acknowledgements
This research was supported by the Hunan Province Ordinary Higher Education
Teaching Reform Research Project, Central South University Deepening Innovation
and Entrepreneurship Education Reform Research Project (No. 2019CG006), Central
South University College Students Innovation and Entrepreneurship Project (No.
GS201910533126X; No. GS201910533022X; No. XCX20190632), Central South
University postgraduate independent exploration and innovation project (No.
2019zzts751; No. 2018zzts239), Hunan Graduate Research and Innovation Project
(No. CX20190247), Changsha Science and Technology Plan (No. kq1801072), Open
Sharing Fund for the Large-scale Instruments and Equipments of Central South
University. We are very grateful to Prof. Guogen Liu (Modern Analysis and Testing
Centre of Central South University) and Prof. Hongping Long (High Resolution Mass
28

Spectrometry Laboratory of Advanced Research Center in Hunan University of
Traditional Chinese Medicine) for the NMR and HRESIMS tests.
Appendix A. Supporting Information
The HRESIMS, 1D and 2D NMR, UV spectra and GC-MS analysis of sugar residue
derivatives of compounds 1-8 are available in the supplementary data.
29