Two new acetylated flavonoid glycosides from Phyllanthus urinaria

Article abstract of DOI:10.1080/10286020.2013.794792

Two new acetylated flavonoid glycosides, quercetin 3-O-α-l-(2,4-di-O- acetyl) rhamnopyranoside-7-O-α-l-rhamnopyranoside (1) and quercetin 3-O-α-l-(3,4-di-O-acetyl) rhamnopyranoside-7-O-α-l-rhamnopyranoside (2), together with two known compounds, quercetin

Full text of DOI:10.1080/10286020.2013.794792

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Two new acetylated flavonoid
glycosides from Phyllanthus urinaria
Chun Wu a , Chun-Shan Wei b , Shao-Fu Yu a , Bai-Lian Liu a , Yao-
Lan Li a , Wen-Cai Ye a , Guang-Dong Tong* a & Guang-Xiong Zhou* b
a Guangdong Province Key Laboratory of Pharmacodynamic
Constituents of TCM and New Drugs Research, Institute of
Traditional Chinese Medicine and Natural Products, Jinan
University , Guangzhou , 510632 , China
b Department of Liver Disease , Shenzhen Hospital Affiliated to
Guangzhou University of Chinese Medicine , Shenzhen , 518033 ,
China
Published online: 16 May 2013.
To cite this article: Chun Wu , Chun-Shan Wei , Shao-Fu Yu , Bai-Lian Liu , Yao-Lan Li , Wen-
Cai Ye , Guang-Dong Tong* & Guang-Xiong Zhou* (2013) Two new acetylated flavonoid glycosides
from Phyllanthus urinaria , Journal of Asian Natural Products Research, 15:7, 703-707, DOI:
10.1080/10286020.2013.794792
To link to this article: http://dx.doi.org/10.1080/10286020.2013.794792
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Journal of Asian Natural Products Research, 2013
Vol. 15, No. 7, 703–707, http://dx.doi.org/10.1080/10286020.2013.794792
Two new acetylated flavonoid glycosides from Phyllanthus urinaria
Chun Wua, Chun-Shan Weib, Shao-Fu Yua, Bai-Lian Liua, Yao-Lan Lia, Wen-Cai Yea,
Guang-Dong Tongb* and Guang-Xiong Zhoua
*
aGuangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs
Research, Institute of Traditional Chinese Medicine and Natural Products, Jinan University,
Guangzhou 510632, China; bDepartment of Liver Disease, Shenzhen Hospital Affiliated to
Guangzhou University of Chinese Medicine, Shenzhen 518033, China
(Received 28 January 2013; final version received 7 April 2013)
Two new acetylated flavonoid glycosides, quercetin 3-O-a-L-(2,4-di-O-acetyl)
rhamnopyranoside-7-O-a-L-rhamnopyranoside (1) and quercetin 3-O-a-L-(3,4-di-O-
acetyl) rhamnopyranoside-7-O-a-L-rhamnopyranoside (2), together with two known
compounds, quercetin (3) and quercetin 3-O-a-L-rhamnopyranoside (4), were isolated
from the ethanol extract of Phyllanthus urinaria. The structures of the new compounds
were determined on the basis of extensive spectroscopic data including IR, HR-ESI-
MS, 1D NMR, and 2D NMR.
Keywords: Euphorbiaceae; Phyllanthus urinaria; acetylated flavonoid glycoside
1. Introduction
nopyranoside (2), together with two
known compounds, quercetin (3) [12]
and quercetin 3-O-a-L-rhamnopyranoside
(4) [13] (Figure 1). In this study, we report
the isolation and structural elucidation of
the new compounds.
Phyllanthus urinaria Linnea (Euphorbia-
ceae) is widely distributed in tropical and
subtropical regions in Asian countries. It is
traditionally believed that this plant can
reduce heat, relieve inflammation, remove
food stagnancy, protect the liver, detoxify
body from poison, and increase the flow of
urine [1]. It has long been used in folk
medicine for liver protection, hepatitis B,
diabetes, nephrolithiasis, jaundice, and
dropsy [2–6]. Earlier chemical studies on
P. urinaria indicated that the major
constituents of the plant are flavonoids,
lignans, tannins, coumarins, and benze-
noids [7–11]. Recently, we carried out a
systematic chemical study on the 95%
ethanol extract of the aerial part of P.
urinaria, which resulted in the isolation of
two new acetylated flavonoid glycosides,
quercetin 3-O-a-L-(2,4-di-O-acetyl) rham-
nopyranoside-7-O-a-L-rhamnopyranoside
(1) and quercetin 3-O-a-L-(3,4-di-O-
acetyl) rhamnopyranoside-7-O-a-L-rham-
2. Results and discussion
Compound 1 was obtained as a yellow
amorphous powder. Its molecular formula
was determined to be C31H34O17 based on
its HR-ESI-MS data. The UV spectrum of
1 exhibited absorption maxima at 265, 346
(sh), and 352 nm, suggesting the presence
of a flavone skeleton. Examination of the
1H and 13C NMR spectral data of 1 (Table
1) indicated that the molecule consisted of
a flavone, two sugars, and two acetyl
moieties. In 1H NMR spectrum, two
doublets were observed at dH 6.39 (d,
J ¼ 1.5 Hz) and 6.63 (d, J ¼ 1.5 Hz),
assignable to H-6 and H-8 of the ring
A of a flavonoid unit. Three ortho- and
meta-coupled aromatic proton signals at
*Corresponding authors. Email: tgd755@163.com; guangxzh@sina.com
q 2013 Taylor & Francis

704
C. Wu et al.
R1
R2
5''
5'''
Me
1 AcO
Me
HO
1''
1''
1'''
1'''
O
O
3''
3'''
HO
HO
HO
OH
AcO
OH
2'
5''
5'''
4'
Me
2 AcO
Me
HO
8
O
O
R2O
O
2
3'''
HO
3''
HO
AcO
6'
HO
3
4
6
OR1
OH
O
3
H
H
5''
Me
HO
1''
O
4
H
3''
HO
HO
Figure 1. Structures of compounds 1–4.
dH 6.92 (H-50, d, J ¼ 8.2 Hz), 7.28 (H-60,
dd, J ¼ 8.2, 1.9 Hz), and 7.34 (H-20, d,
J ¼ 1.9 Hz) indicated the presence of a
30,40-substituted phenyl group in ring B. In
addition, two anomeric protons [dH 5.40
(H-100) and 5.55 (H-1000)] and two methyl
protons [dH 0.75 (H-600, d, J ¼ 6.2 Hz) and
1.13 (H-6000, d, J ¼ 6.0 Hz)] indicated the
presence of two rhamnose residues. Acid
hydrolysis of 1 afforded quercetin (3) and
rhamnose, which were identified by
comparison of the 1H and 13C NMR
spectra and high performance liquid
chromatography (HPLC) Rt value with
those of authentic markers. In 13C NMR
spectrum (Table 1), the carbon signal
assignable to C-2 (dC 158.2) in the
aglycone moiety was shifted to a lower
field by about 11.4 ppm, compared with
that of quercetin (3, dC 146.8), indicating
that one of the two rhamnose residues was
located at C-3 (dC 133.6). The remaining
rhamnose residue was deduced to be
attached at C-7 (dC 161.9), on the basis
of the chemical shifts of H-6 (dH 6.39) and
H-8 (dH 6.63), which were deshielded by
0.20 and 0.22 ppm, respectively, compared
with the corresponding signals of querce-
tin (3, dH 6.19 and 6.41, respectively).
These elucidation were further confirmed
by the HMBC correlations of H-100/C-3
and H-1000/C-7 (Figure 2). Based on 1H–1H
OH
OH
4'
2'
8
O
4
O
2
3
6'
H
5'''
1'''
H3C
HO
O
H
O
3'''
OH
Me
O
5''
HO
HO
O
2''
H3COCO
H
HO
O
H
C
O
CH3
Figure 2. Key HMBC (H ! C) correlations of compound 1.

Journal of Asian Natural Products Research
705
Table 1. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectral data for compounds 1 and 2
(DMSO-d6, d in ppm, J in Hz).
1
2
No.
dH
dC
dH
dC
2
3
4
5
6
7
8
–
–
–
158.2
133.6
177.6
160.9
99.7
161.9
99.8
156.2
105.8
120.2
115.8
145.5
149.0
115.6
120.3
98.2
71.8
66.0
73.0
68.0
17.1
98.5
69.8
70.3
71.2
69.9
18.2
170.0
20.8
–
–
–
–
158.1
134.5
177.8
161.0
98.5
162.1
99.6
156.2
105.8
120.4
115.7
145.5
148.9
115.5
121.2
101.5
67.4
70.3
69.9
68.0
16.9
98.5
69.8
70.1
71.6
69.7
17.9
–
–
6.39 (d, 1.5)
–
6.63 (d, 1.5)
–
6.45 (d. 1.9)
–
6.76 (d. 1.9)
9
10
–
–
–
–
10
–
7.34 (d, 1.9)
20
7.31 (d, 1.8)
–
–
6.91 (d, 8.2)
30
–
40
–
6.92 (d, 8.2)
500
600
7.28 (dd, 8.2, 1.9)
5.40 (d, 1.8)
5. 34 (dd, 3.4, 1.8)
3.92 (dd, 9.7, 3.4)
4.63 (t, 9.7)
3.37 (m)
0.75 (d, 6.2)
5.55 (br s)
3.85 (dd, 3.0, 1.9)
3.64 (dd, 9.2, 3.0)
3.32 (t, 9.2)
3.43 (m)
7.27 (dd, 8.2, 1.8)
5.68 (d, 1.7)
4.26 (dd, 2.8, 1.7)
5.15 (dd, 9.9, 2.8)
4.63 (t, 9.9)
3.29 (m)
0.78 (d, 6.2)
5.56 (br s)
3.85 (dd, 3.0, 1.9)
3.65 (dd, 9.7, 3.0)
3.31 (t, 9.7)
3.42 (m)
100
200
300
400
500
6
1000
2000
3000
4000
5000
6000
1.13 (d, 6.0)
2.01 (s)
1.13 (d, 6.1)
–
–
1.99 (s)
–
2.02 (s)
200-COCH3
200-COCH3
300-COCH3
300COCH3
400COCH3
400-COCH3
–
–
–
169.9
20.5
169.7
20.8
–
2.05 (s)
–
169.7
20.9
–
–
correlation spectroscopy (COSY) spec-
trum, and compared with the correspond-
ing 1H NMR signals of quercetin 3-O-a-L-
rhamnopyranoside (4) which was unac-
etylated (Table 1), the connectivities of the
acetyl groups of 1 were established. Two
protons H-200 (dH 5.34) and H-400 (dH 4.63)
of the one rhamnose residue were shifted
to a lower field by 1.11 and 1.22 ppm from
4 [H-200 (dH 4.23) and H-400 (dH 3.41)],
convinced that two hydroxyl groups at C-
200 and C-400 of rhamnose residue were
acetylated. This was further supported by
HMBC experiment (Figure 2), in which
long-range correlations of H-200/acetoxyl
carbon at dC 170.0 and H-400/acetoxyl
carbon at dC 169.7 were observed. Thus,
the structure of 1 was elucidated as
quercetin 3-O-a-L-(2,4-di-O-acetyl) rham-
nopyranoside-7-O-a-L-rhamnopyrano-
side.
Compound 2, a yellow amorphous
powder, had a molecular formula of
C31H34O17 on the basis of the HR-ESI-
MS data. The UV spectrum of 2 exhibited
absorption maxima at 268, 339 (sh), and
1
356 nm. The H and 13C NMR data of 2
(Table 1) were similar to those of 1,

706
C. Wu et al.
suggesting that the molecule of 2 also
consisted of quercetin as an aglycone, two
rhamnosyl and two acetyl groups. Com-
UT, USA) and a reversed-phase (RP)
C18 column (5 mm, 20 mm £ 250 mm;
Cosmosil). Thin-layer chromatography
(TLC) was carried out using precoated
silica gel plates (GF254, Yantai Chemical
Industry Research Institute, Yantai,
China). All the reagents were purchased
from Tianjin Damao Chemical Company
(Tianjin, China).
1
parison of the H NMR spectral (Table 1)
data of 2 with those of quercetin 3-O-a-L-
rhamnopyranoside (4, H-300 at dH 3.75 and
H-400 at dH 3.41, respectively) revealed that
two protons at H-300 (dH 5.15) and H-400 (dH
4.63) of the one rhamnose residue were
shifted to a lower field by 1.40 and
1.22 ppm (more than 1 ppm), respectively.
This indicated that two hydroxyl groups at
C-300 and C-400 of rhamnose residue were
acetylated. This conclusion was further
confirmed by HMBC correlations of H-
300/acetoxyl carbon at dC 169.9 and H-
400/acetoxyl carbon at dC 169.7. Finally, the
structure of 2 was determined to be
quercetin 3-O-a-L-(3,4-di-O-acetyl) rham-
nopyranoside-7-O-a-L-rhamnopyrano-
side.
3.2 Plant material
The plant material was collected from
Shenzhen, Guangdong, China, and was
authenticated by G.X. Zhou at the Depart-
ment of Pharmacognosy, College of
Pharmacy, Jinan University. A voucher
specimen (No. 2012YXZ0213) was also
deposited in the Department.
3.3 Extraction and isolation
The dried aerial parts of P. urinaria
(10 kg) were soaked in 95% ethanol and
extracted by percolation. The extract
(900 g) was obtained from the soaked
solution after evaporation of the solvent
under reduced pressure. This extract was
suspended in distilled water, and then
partitioned with petroleum ether, chloro-
form, and ethyl acetate (EtOAc),
respectively.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured using a
JASCO P-1030 automatic digital polari-
meter (JASCO, Tokyo, Japan). UV spectra
were measured using a JASCO V-550 UV-
Vis spectrophotometer. IR spectra were
measured using a JASCO FT/IR-480 plus
infrared spectrometer with KBr pellets. 1D
and 2D NMR spectra were recorded by
Bruker AV-400 spectrometer with tetra-
methylsilane as the internal standard, and
chemical shifts were expressed in d values
(ppm). HR-ESI-MS data were obtained
from an Agilent 6210 liquid chromatog-
raphy mass spectrometer detector time of
flight (LC/MSD TOF) (Agilent, Pala Alto,
CA, USA). Silica gel (200–300 mesh,
Qingdao Marine Chemical, Inc., Qingdao,
China), octadecylsilyl (ODS; 50 mm,
YMC, Tokyo, Japan), and Sephadex LH-
20 (25–100 mm, Fluka, Buchs, Switzer-
land) were used for column chromatog-
raphy (CC). Preparative HPLC was carried
out by a Varian instrument equipped with
UV detectors (Varian, Salt Lake City,
The EtOAc extract (130 g) was sub-
jected to silica gel CC (200–300 mesh,
1.3 kg) eluting with CHCl3 –CH3OH
(100:0 to 0:100) in gradient to yield eight
fractions (fractions A–H) based on TLC
patterns. Fraction C (3.8 g) was chromato-
graphed on ODS column eluting with
CH3OH–H2O (1:9 to 1:0) to afford three
subfractions (C1–C3). Subfraction C2
(900 mg) was subjected to Sephadex LH-
20 CC with the eluent of 70% CH3OH–
H2O to yield compounds 3 (20.1 mg) and 4
(19.8 mg). Fraction D (4.5 g) was sub-
jected to an ODS column eluting with
CH3OH–H2O (1:9 to 1:0) in gradient
to afford four subfractions (D1–D4).
Subfraction D3 (1.0 g) was applied to

Journal of Asian Natural Products Research
707
S&T Special Project on Major New Drug
Innovation (2011ZX09307-002-01). The
authors are grateful to Dr Ying-Hui Duan, Mr
Lai-Ming Li, and Mr Xiao-Jie Zen, Institute of
Traditional Chinese Medicine and Natural
Products, Jinan University, for measuring the
HR-ESI-MS and NMR spectral data.
Sephadex LH-20 CC by eluting with
CH3OH to afford three fractions (D3A–
D3C). Fraction D3A (200 mg) was purified
by semi-preparative RP-C18 HPLC with
the eluent 25% CH3CN–H2O (254 nm,
4 ml/min) to give compound 1 (14.9 mg,
tR ¼ 25.6 min) and compound 2 (15.5 mg,
tR ¼ 33.4 min)
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3.3.1 Quercetin 3-O-a-L-(2,4-di-O-
acetyl) rhamnopyranoside-7-O-a-L-
rhamnopyranoside (1)
Yellow amorphous powder; C31H34O17;
20
½aꢀD 2127.2 (c ¼ 0.11, CH3OH); UV
(MeOH) lmax (log 1): 265 (4.4), 346 (sh),
and 352 (4.5) nm; IR (KBr) nmax: 3440,
1
1745, 1650, and 1610 cm–1; H and 13C
NMR spectral data (DMSO-d6): see Table
1. HR-ESI-MS: m/z 679.1863 [M þ H]þ
(calcd for C31H35O17, 679.1874).
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3.3.2 Quercetin 3-O-a-L-(3,4-di-O-
acetyl) rhamnopyranoside-7-O-a-L-
rhamnopyranoside (2)
Yellow amorphous powder; C31H34O17;
20
½aꢀD 2129.5 (c ¼ 0.12, CH3OH); UV
(MeOH) lmax (log 1): 266 (4.3), 339 (sh),
and 356 (4.6) nm; IR (KBr) nmax: 3415,
1
1720, 1650, and 1602 cm–1; H and 13C
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and F. Miao, Acta Pharmacol. Sin. 39,
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42, 428 (2011).
NMR spectral data (DMSO-d6): see Table
1. HR-ESI-MS: m/z 679.1865 [M þ H]þ
(calcd for C31H35O17, 679.1874).
Acknowledgments
This research was financially supported by
the National Natural Science Foundation of
China (No. 30873245), the Science and
Technology Project of Shenzhen city of China
(No. 201202154), and the Chinese National