Flavonol glycosides from Epimedium pubescens

Article abstract of DOI:10.1248/cpb.59.1317

Five new flavonol glycosides (1, 3, 5-7) were isolated from the aerial parts of Epimedium pubescens MAXIM., along with two known compounds, sagittasine C (2) and 4′,5-dihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-[β-D-xylopyranosyl(1→3)-4-O-acetyl-α-L-rhamnopyranoside] -7-O-β-D-glucopyranoside (4). The structures were elucidated on the basis of their 1D-, 2D-NMR, MS, UV and IR spectra data.

Full text of DOI:10.1248/cpb.59.1317

November 2011
Regular Article
Chem. Pharm. Bull. 59(11) 1317—1321 (2011)
1317
Flavonol Glycosides from Epimedium pubescens
c, d
Fengjuan TU,a, # Yi DAI,b, # Zhihong YAO,b Xinluan WANG,c, d Xinsheng YAO,*, a, b and Ling QIN
a College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University; Shenyang 110016, P. R. China:
b Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University; Guangzhou
c
510632, P. R. China: Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, The Chinese
University of Hong Kong; Hong Kong, China: and d Translational Medicine Research & Development Center, Shenzhen
Institutes of Advanced Technology, Chinese Academy of Sciences; Shenzhen 518055, China.
Received May 5, 2011; accepted August 1, 2011; published online August 25, 2011
Five new flavonol glycosides (1, 3, 5—7) were isolated from the aerial parts of Epimedium pubescens MAXIM.,
along with two known compounds, sagittasine C (2) and 4ꢀ,5-dihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-[b-D-
xylopyranosyl(1→3)-4-O-acetyl-a-L-rhamnopyranoside]-7-O-b-D-glucopyranoside (4). The structures were eluci-
dated on the basis of their 1D-, 2D-NMR, MS, UV and IR spectra data.
Key words Epimedium pubescens; Berberidaceae; flavonol glycoside
Epimedium pubescens MAXIM. (Berberidaceae), together tuted benzene ring. A methoxy signal was obversed at d 3.85
with other four species E. brevicornum MAXIM., E. sagitta- (3H, s). A serial of proton signals at d 5.15 (1H, t, Jꢂ7.0 Hz,
tum MAXIM., E. wushanenes T. S. YING, and E. koreanum H-12), 3.33 (2H, m, H-11), 1.68 (3H, s, H-15), 1.62 (3H, s,
NAKAI, is used as the official source of Traditional Chinese H-16) correlated with carbon signals at d 122.4, 25.4, 17.7,
Medicine (TCM) “Yinyanghuo” (Chinese Pharmacopoeia 21.2 in heteronuclear single quantum coherence (HSQC)
2010 Edition). It is one of the most well known herbal medi- spectrum respectively, suggesting the presence of a prenyl
cines with tonic, anti-rheumatic and aphrodisiac effects.1,2) group. In addition, the proton signals at d 5.34 (1H, d,
Previous chemical investigations on the geneus Epimedium Jꢂ1.3 Hz, H-1ꢃ)/0.83 (3H, d, Jꢂ6.0 Hz, H-6ꢃ), and 4.86 (1H,
have afforded a series of flavonol glycosides,3—7) which pos- d, Jꢂ1.1 Hz, H-1ꢄ)/1.08 (3H, d, Jꢂ6.2 Hz, H-6ꢄ) indicated
sess multiple biological activities such as androgenic,8) an- the existence of two rhamnose moieties. It was further con-
tioxidant,9) antiosteoporosis,10) and antidepressant-like ac- firmed by the acid hydrolysis and HPLC analysis according
tions.11) Our further chemical investigation on the aerial parts to the method of Tanaka et al.13) The absolute configuration
of E. pubescens MAXIM. resulted in the isolation of five new of the sugars was determined to be the L configuration. The
flavonol glycosides (compounds 1, 3, 5—7) and two known methoxy group at d 3.85 was attached at the C-4ꢀ (d 160.5)
compounds, sagittasine C (2)3) and 4ꢀ,5-dihydroxy-8-(3,3- due to the characteristic heteronuclear multiple bond connec-
dimethylallyl)-flavonol 3-O-[b-D-xylopyranosyl(1→3)-4-O- tivity (HMBC) correlation. The aromatic carbon signals at d
acetyl-a-L-rhamnopyranoside]-7-O-b-D-glucopyranoside 158.8, 98.5 and 103.7 were assigned to C-5, C-6 and C-10,
(4).12) Their structures were elucidated on the basis of spec- due to their HMBC correlations with the hydroxy group at d
troscopic evidences (Fig. 1).
12.58 (OH-5) (Fig. 2). Therefore, the carbon signals at d
Compounds 1, 3, 5, 6 and 7 were obtained as yellow amor- 162.6 and 105.9 were then assigned to C-7 and C-8 accord-
phous powder. The UV absorption bands of all the com- ing to their HMBC correlations with H-6 (d 6.28). The
pounds were at about 205, 270, 320 and 350 nm, suggesting prenyl group was located at C-8, which was supported by the
the presence of flavonol skeleton in their structures.
HMBC correlation between H-11 (d 3.33, 2H, m) and C-8 (d
The molecular formula of compound 1 was determined as 105.9). Moreover, one rhamnose moiety was located at C-3
C33H40O15 based on the high resolution-electrospray ionization- of the aglycone according to the HMBC correlation of H-
mass spectrum (HR-ESI-MS) (m/z 677.2448 [C33H41O15]ꢁ, 1ꢃ/C-3 (d 134.2). And the other rhamnose moiety was lo-
1
Calcd for 677.2440). The H-NMR spectrum of 1 exhibited cated at C-2ꢃ of the inner one due to the HMBC correlations
two singlet protons at d 12.58 (1H, s, OH-5) and d 6.28 (1H, of H-1ꢄ/C-2ꢃ (d 75.6) and H-2ꢃ (d 4.10, 1H, m)/C-1ꢄ (d
s), respectively. Three coupled aromatic protons at d 7.36 101.6). Thus, the structure of 1 was deduced as 4ꢀ-methoxyl-
(1H, dd, Jꢂ8.4, 2.1 Hz), 7.34 (1H, d, Jꢂ2.1 Hz), and 7.08 3ꢀ,5,7-trihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-a-L-
(1H, d, Jꢂ8.4 Hz) suggested the presence of a 1,2,4-trisubsti- rhamnopyranosyl(1→2)-a-L-rhamnopyranoside.
Fig. 1. Chemical Structures of 1—7
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yaoxinsheng@vip.tom.com
These authors contributed equally to this work.
#

1318
Vol. 59, No. 11
cose. The absolute configuration was further elucidated as D
configuration by the acid hydrolysis experiment.13) The
glucose moiety was located at C-7 of the aglycone due to
the rotating frame Overhauser effect spectroscopy (ROESY)
correlation of H-6 (d 6.63)/H-1ꢃꢃ. Thus, the structure of 5
was identified as 4ꢀ,5-dihydroxyl-8-(3,3-dimethylallyl)-flavo-
nol 3-O-[b-D-xylopyranosyl(1→3)-a-L-rhamnopyranozside]-
7-O-b-D-glucopyranoside.
Compound 6 was obtained with the molecular formula
C39H50O20 by HR-ESI-MS (m/z 839.2982 [C39H51O20]ꢁ,
1
Calcd for 839.2968). The H-NMR spectrum of 6 suggested
the presence of a chelated 5-OH group (d 12.61, 1H, s), a
penta-substituted benzene ring (d 6.64, 1H, s), a para-substi-
tuted benzene ring (d 7.12, 7.90, each 2H, d, Jꢂ2.1 Hz), and
a methoxy group (d 3.85, 3H, s). Proton signals at d 5.37
Fig. 2. Key HMBC (→) and ROESY (↔) Correlations of 1 and 6
Compound 3 had the molecular formula C33H38O15 based (1H, m, H-12), 3.73 (2H, br s, H-15), 3.60 (1H, m, H-11a),
1
on the HR-ESI-MS (m/z 675.2296 [C33H29O15]ꢁ, Calcd for 3.47 (1H, m, H-11b), 1.66 (3H, s, H-14) in the H-NMR
675.2283). The 1H-NMR spectrum of 3 showed a singlet sig- spectrum, correlated with carbon signals at d 120.9 (C-12),
nal of OH-5 at d 12.48 (1H, s). A set of ortho-coupled dou- 66.1 (C-15), 20.8 (C-11), 13.7 (C-14) in HSQC spectrum re-
blet signals of four aromatic protons at d 7.71, 6.93 (each spectively, indicating the presence of a hydroxyprenyl group.
2H, d, Jꢂ7.8 Hz) and a one-proton singlet signal at d 6.10 Additionally, three anomeric protons were observed at d 5.39
(1H, s) were also observed. The presence of an acetyl group (1H, d, Jꢂ1.5 Hz, H-1ꢃ), 4.88 (1H, d, Jꢂ1.1 Hz, H-1ꢄ), and
was supported by a three-proton singlet signal at d 1.97 in 5.01 (1H, d, Jꢂ7.5 Hz, H-1ꢃꢃ) respectively, suggesting the
the 1H-NMR spectrum and carbon signals at d 169.7, 20.8 in presence of three monosaccharide moieties. They were iden-
the 13C-NMR spectrum. Proton signals at d 5.17 (1H, t, tified as two a-L-rhamnose moieties and a b-D-glucose moi-
Jꢂ5.9 Hz, H-12), 3.34 (2H, m. H-11) as well as two methyl ety by the acid hydrolysis experiment.13) The methoxy group
groups signals at d 1.67 and 1.61 (each 3H, s, H-14, 15) re- was located at C-4ꢀ according to the HMBC correlation be-
vealed the presence a prenyl group. It was further confirmed tween the proton signal at d 3.85 and C-4ꢀ (d 161.4). One
by the carbon signals at d 21.3 (C-11), 123.3 (C-12), 129.9 rhamnose moiety was located at C-3 of the aglycone due to
(C-13), 17.8 (C-14) and 25.4 (C-15) observed in the 13C- the HMBC correlation of H-1ꢃ/C-3 (d 134.5). The other
NMR spectrum. Additionally, two sugar moieties were ob- rhamnose moiety was located at C-2ꢃ of the inner one due to
served with protons d 5.30 (1H, br s)/0.71 (3H, d, Jꢂ6.2 Hz) the HMBC correlations of H-1ꢄ/C-2ꢃ (d 75.5) and H-2ꢃ (d
1
and d 4.22 (1H, d, Jꢂ7.6 Hz) in the H-NMR spectrum. 4.12, 1H, m)/C-1ꢄ (d 101.6). And the glucopyranose moiety
They were identified as an L-rhamnose and a D-xylose by the was located at C-7 (d 160.6) according to the ROESY corre-
acid hydrolysis experiment and HPLC analysis according to lation between H-1ꢃꢃ and H-6 (d 6.64) (Fig. 2). Therefore,
the method of Tanaka et al.13) The aromatic methylene car- compound 6 was elucidated as 4ꢀ-methoxyl-5-hydroxyl-8-(3-
bon signal at d 99.5 was assigned to C-6, due to its HMBC methyl-4-hydroxyl-but-2-enyl)-flavonol 3-O-[a-L-rhamnopy-
correlation with the hydroxy group at d 12.48 (OH-5). ranosyl(1→2)-a-L-rhamnopyranoside]-7-O-b-D-glucopyra-
Therefore, the carbon signals at d 158.9 and 106.2 were then noside.
assigned to C-7 and C-8 according to their HMBC correla-
Compound 7 was obtained with the molecular formula
tions with H-6 (d 6.10).The location of the prenyl group at C34H42O19 by HR-ESI-MS (m/z 755.2402 [C34H43O19]ꢁ,
the C-8 (d 106.2) position was supported by the chemical Calcd for 755.2393). The NMR spectroscopic data of 7 were
shift value of C-6 (d 99.5) in the 13C-NMR spectrum.3,14—16) similar with those of 6, except for the loss of the signals of a
It was further confirmed by the HMBC correlation between hydroxyprenyl group. Thus, the structure of 7 was elucidated
H-11 (d 3.34) and C-8. In the HMBC spectrum, a correlation as 4ꢀ-methoxyl-5-hydroxyl-flavonol 3-O-[a-L-rhamnopyra-
between the H-1ꢃ (d 5.30) and C-3 (d 134.2) were observed, nosyl(1→2)-a-L-rhamnopyranoside]-7-O-b-D-glucopyra-
indicating the rhamnose moiety was attached to C-3 of the noside.
aglycone. The xylose moiety was located at C-3ꢃ (d 76.5) of
Experimental
the inner rhamnose moiety, according to the HMBC correla-
General Procedure UV spectra were recorded on a JASCO V-550
UV/Vis spectrometer. IR spectra were obtained using a JASCO FT/IR-480
tion of H-1ꢄ (d 4.22)/C-3ꢃ. The acetyl group was located at
C-4ꢃ of the rhamnose moiety due to the correlation between
H-4ꢃ (d 4.85) and the carbonyl carbon of the acetyl group at
d 169.7. Therefore, the structure of 3 was elucidated to be
4ꢀ,5,7-trihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-b-D-xy-
lopyranosyl(1→3)-4-O-acetyl-a-L-rhamnopyranoside.
plus spectrometer. ESI-MS spectra were recorded on a Finnigan LCQ Ad-
vantage MAX mass spectrometer. HR-ESI-MS spectra were acquired using
Thermo-fishier LTQ Orbitrap XL mass spectrometer. 1D- and 2D-NMR
spectra were measured with a Bruker AV-400 spectrometer. Open column
chromatography was performed using silica gel (200—300 mesh, Qingdao
Haiyang Chemical Group Corp., Qingdao, China), ODS (50 mm, YMC,
Japan), HW-40 (Tosoh, Japan) and Sephadex LH-20 (Pharmacia). Thin layer
The molecular formula of Compound 5 was established as
C37H46O19 by HR-ESI-MS (m/z 795.2726 [C37H47O19]ꢁ, chromatography (TLC) was performed using percolated silica gel plates
Calcd for 795.2706). The NMR data of 5 were similar with
those of 3, except for the loss of the acetyl group signals and
additional signals of a glucose moiety. The anomeric proton
(silica gel GF254, 1 mm, Yantai). An agilent series 1200 HPLC instrument
equipped with a quaternary pump, a multiple wavelength detector, an auto
sampler and a column compartment was used.
Plant Material The plant was supplied by Guizhou Tongjitang Pharma-
at d 5.00 (1H, d, Jꢂ7.3 Hz, H-1ꢃꢃ) indicated it to be b-glu-
ceutical Co., Ltd., Guiyang, China, and identified by Professor Guang-Xiong

November 2011
1319
Table 1. NMR Data of Aglycone Moiety for Compounds 1, 3, 5—7 (in DMSO-d6)
1
3
5
6
7
No.
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
d
H (J in Hz)
2
3
4
5
6
156.6
134.2
177.7
158.8
98.5
155.9
133.1
176.8
157.9
99.5
157.7
134.0
178.1
159.0
98.1
157.3
134.5
178.2
159.1
98.1
157.4
134.8
177.9
160.9
99.4
6.28 s
6.10 s
6.63 s
6.64 s
6.48 d (2.1)
6.78 d (2.1)
7
8
9
10
11
162.6
105.9
153.7
103.7
21.2
158.9
106.2
153.9
102.0
21.3
160.4
108.3
152.9
105.5
21.4
160.6
108.1
153.0
105.1
20.8
163.1
94.7
156.1
105.8
3.38 m
3.34 m
3.34 m
3.55 m
5.16 t (6.9)
3.47 m
3.60 m
5.37 m
12
13
14
122.4
130.9
17.7
5.15 t (7.0)
123.3
129.9
17.8
5.17 t (5.9)
122.2
131.0
17.8
120.9
135.6
13.7
1.68 s
1.62 s
1.67 s
1.61 s
1.69 s
1.60 s
1.66 s
3.73 br s
15
25.4
25.4
25.4
66.1
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
122.5
115.4
146.4
150.0
111.7
120.6
130.2
115.3
160.3
115.3
120.3
130.7
115.4
160.4
115.4
130.7
122.1
130.6
114.0
161.4
114.0
130.6
55.5
121.9
130.6
114.1
161.4
114.1
130.6
55.5
7.34 d (2.1)
7.71 d (8.7)
6.93 d (8.7)
7.80 d (8.7)
6.94 d (8.7)
7.90 d (9.0)
7.12 d (9.0)
7.88 d (8.9)
7.12 d (8.9)
7.08 d (8.4)
6.93 d (8.7)
7.71 d (8.7)
6.94 d (8.7)
7.80 d (8.7)
7.12 d (9.0)
7.90 d (9.0)
3.85 s
7.12 d (8.9)
7.88 d (8.9)
3.85 s
120.6 7.36 dd (8.4, 2.1) 130.2
55.7
4ꢀ-OCH3
5-OH
3.85 s
12.58 s
12.48 s
12.57 s
12.61 s
12.65 s
Zhou, College of Pharmacy, Jinan University. A voucher specimen was de-
posited in the Institute of Traditional Chinese Medicine and Natural Prod-
ucts, Jinan University, Guangzhou, China.
Nacalai Tesque Inc., Japan) at 35 °C with isocratic elution of 25% CH3CN
containing 0.1% formic acid for 40 min and subsequent washing of the col-
umn with 90% CH3CN at a flow rate 0.8 ml/min. Peaks were detected by a
UV detector at 250 nm. One peak of the derivatives of 1 was obversed at tR
29.1 (L-Rha) min. The mixture of standard monosaccharides, such as L-
rhamnose, D-glucose, L-glucose, D-xylose, and L-xylose (Sigma, U.S.A.),
were subjected to the same method. The peaks of the standard monosaccha-
ride derivatives were recorded at tR 15.9 (L-Glc), 17.2 (D-Glc), 18.7 (L-Xyl),
20.0 (D-Xyl), and 29.2 (L-Rha) min. Following the above procedure, the de-
rivatives of 2, 6 and 7 gave two peaks at tR 17.2—17.3 (D-Glc) and 29.2—
29.3 (L-Rha) min, respectively. The derivatives of 3 gave two peaks at tR 20.1
(D-Xyl) and 29.3 (L-Rha) min. Those of 4 and 5 both gave three peaks at tR
17.2—17.3 (D-Glc), 20.0—20.1 (D-Xyl), and 29.1—29.2 (L-Rha) min.
4ꢀ-Methoxyl-3ꢀ,5,7-trihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-a-L-
Rhamnopyranosyl(1→2)-a-L-rhamnopyranoside (1): Yellow powder; UV
Extraction and Isolation The aerial parts of E. pubescens MAXIM.
(2 kg) were extracted twice with 60% ethanol. After removal of the ethanol
in vacuo, the extract (245 g) was chromatographied over Diaion HP-20 resin,
eluted with water, 30% and 95% ethanol in successive. The 95% ethanol
eluate (80 g) was then subjected to a silica-gel column chromatography (CC)
eluted with chloroform–methanol in gradient to give fourteen fractions.
Fraction 7 (CHCl3–MeOH 9 : 1 eluent) was then subjected to ODS CC
eluted with MeOH–H2O in gradient. And nine subfractions were obtained
(7A—I). The subfraction 7H (eluted with 60% MeOH–H2O) was further
separated by Sephadex LH-20 eluted with CHCl3–MeOH (1 : 1). Compound
3 (16 mg) was obtained after the purification by HW-40 CC eluted with 50%
MeOH–H2O. Fraction 11 (CHCl3–MeOH 8 : 2 eluent) was chromatogra-
phied on ODS CC with MeOH–H2O in gradient to yield 7 subfractions (11A
l
max (MeOH) nm (log e): 205 (4.47), 258 (4.13, sh), 270 (4.17), 346 (3.91);
to G). The subfraction 11B (eluted with 20% MeOH–H2O) was applied to IR (KBr) cmꢆ1: 3402, 2933, 1653, 1509, 1046; H- and 13C-NMR data (see
1
repeated Sephadex LH-20 CC eluted with CHCl3–MeOH (1 : 1) and 35%
MeOH–H2O, respectively. Then the eluate was separated by preparative
HPLC with 50% MeOH–H2O to yield compounds 6 (21 mg) and 7 (10 mg).
Fraction 10 (CHCl3–MeOH 8 : 2 eluent) was also subjected to ODS CC
eluted with MeOH–H2O in gradient to yield 9 subfractions (10A—I). Frac-
tion 10F (eluted with 50% MeOH–H2O) was further subjected to Sephadex
LH-20 eluted with 55% MeOH–H2O and then separated by preparative
Tables 1, 2); ESI-MS (positive) m/z: 699 [MꢁNa]ꢁ, 1375 [2MꢁNa]ꢁ, 677
[MꢁH]ꢁ, ESI-MS (negative) m/z: 675 [MꢆH]ꢆ, 1351 [2MꢆH]ꢆ; HR-ESI-
MS m/z: 677.2448 [MꢁH]ꢁ (Calcd for C33H41O15, 677.2440).
Sagittasine C (2): Yellow powder; UV lmax (MeOH) nm (log e): 205
(4.50), 258 (4.14, sh), 269 (4.15), 346 (3.91); IR (KBr) cmꢆ1: 3388, 2925,
1
1651, 1599, 1076; H-NMR (DMSO-d6, 400 MHz) d: 12.59 (1H, s, 5-OH),
7.40 (1H, dd, Jꢂ8.5, 2.1 Hz, H-6ꢀ), 7.38 (1H, d, Jꢂ2.1 Hz, H-2ꢀ), 7.09 (1H,
HPLC (UltimateTM XB-C18, 5 mm, 21.2ꢅ250 mm, Welch) with 50% d, Jꢂ8.5 Hz, H-5ꢀ), 6.62 (1H, s, H-6), 5.25 (1H, d, Jꢂ1.3 Hz, H-1ꢃ), 5.17
MeOH–H2O to yield compounds 2 (8 mg) and 5 (26 mg). Fraction 10G (1H, t, Jꢂ7.0 Hz, H-12), 5.00 (1H, d, Jꢂ7.5 Hz, H-1ꢄ), 4.00 (1H, br s, H-2ꢃ),
(eluted with 50% MeOH–H2O) was purified by Sephadex LH-20 with 60%
MeOH–H2O to yield compound 4 (68 mg). Fraction 10I (eluted with 50%
MeOH–H2O) was subjected to Sephadex LH-20 eluted with 60%
MeOH–H2O, and then separated by preparative HPLC (UltimateTM XB-C18,
5 mm, 21.2ꢅ250 mm, Welch) with 65% MeOH–H2O to yield compound 1
(9 mg).
3.86 (3H, s, 4ꢀ-OCH3), 3.72 (1H, m, H-6ꢃꢃa), 3.54 (1H, m, H-11a), 3.53 (1H,
m, H-3ꢃ), 3.48 (1H, m, H-6ꢃꢃb), 3.43 (2H, m, H-11b, H-5ꢄ), 3.35—3.28
(overlapped in HDO, H-2ꢄ, 3ꢄ), 3.26—3.12 (3H, m, H-4ꢄ, H-4ꢃ, H-5ꢃ), 1.70
(3H, s, H-14), 1.61 (3H, s, H-15), 0.81 (3H, d, Jꢂ6.0 Hz, H-6ꢃ); 13C-NMR
data (DMSO-d6, 100 MHz) d: 178.3 (C-4), 160.5 (C-7), 159.0 (C-5), 157.4
(C-2), 152.9 (C-9), 150.2 (C-4ꢀ), 146.4 (C-3ꢀ), 134.6 (C-3), 131.1 (C-13),
Acid Hydrolysis and HPLC Analysis The absolute configuration of 122.4 (C-1ꢀ), 122.1 (C-12), 120.8 (C-6ꢀ), 115.5 (C-2ꢀ), 111.7 (C-5ꢀ), 108.3
the sugar moieties in the structures were determined by the method of
Tanaka et al.13) Compound 1 (2 mg) was hydrolyzed with 2 M HCl for 2 h at
90 °C. The mixture was evaporated to dryness under a vacuum, and then the
residue was dissolved in H2O and extracted with CHCl3. The aqueous layer
was collected. After drying in vacuo, the residue was dissolved in pyridine
(1 ml) containing L-cysteine methyl ester (1 mg) (Sigma, U.S.A.) and heated
at 60 °C for 1 h. Then, o-tolyl isothiocyanate (5 ml) (Alfa Aesar, U.K.) was
added to the mixture, which was heated at 60 °C for 1 h. The reaction mix-
ture was directly analyzed by reversed-phase HPLC. Analytical HPLC was
performed on a Cosmosil 5C18-MS-II column (250ꢅ4.6 mm i.d., 5 mm,
(C-8), 105.5 (C-10), 102.0 (C-1ꢃ), 100.6 (C-1ꢄ), 98.1 (C-6), 77.2 (C-5ꢄ),
76.6 (C-3ꢄ), 73.3 (C-2ꢄ), 71.2 (C-4ꢃ), 70.6 (C-5ꢃ), 70.3 (C-3ꢃ), 70.0 (C-2ꢃ),
69.7 (C-4ꢄ), 60.6 (C-6ꢄ), 55.7 (4ꢀ-OCH3), 25.4 (C-15), 21.4 (C-11), 17.7
(C-14), 17.4 (C-6ꢃ); ESI-MS (positive) m/z: 715 [MꢁNa]ꢁ, 1407
[2MꢁNa]ꢁ, 693 [MꢁH]ꢁ, ESI-MS (negative) m/z: 691 [MꢆH]ꢆ, 1383
[2MꢆH]ꢆ; HR-ESI-MS m/z: 693.2404 [MꢁH]ꢁ (Calcd for C33H41O16,
693.2389).
4ꢀ,5,7-Trihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-b-D-Xylopyranosyl-
(1→3)-4-O-acetyl-a-L-rhamnopyranoside (3): Yellow powder; UV lmax
(MeOH) nm (log e): 204 (4.43, sh), 270 (4.25), 316 (3.97, sh), 350 (3.91);

1320
Vol. 59, No. 11
Table 2. NMR Data of Sugar Moieties for Compounds 1, 3, 5—7 (in DMSO-d6)
1
3
5
6
7
No.
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
dH (J in Hz)
Rhamnose-1
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
100.7
75.6
70.1
71.4
70.4
17.5
5.34 d (1.3)
4.10 m
3.63 m
3.26—3.10 o
3.26—3.10 o
0.83 d (6.0)
101.2
69.6
76.5
71.4
68.2
17.0
169.7
20.8
5.30 br s
4.12 br s
3.80 m
4.84 t (10.0)
3.32 m
101.8
69.5
80.9
69.9
70.5
17.4
5.30 br s
4.13 br s
3.56 m
3.40—3.27 o
3.27—3.05 o
0.84 d (6.1)
100.6
75.5
70.1
71.3
70.4
17.5
5.41 d (1.3)
4.11 m
3.59 m
3.23—3.07 o
3.23—3.07 o
0.81 d (5.6)
100.7
75.5
70.1
71.3
70.4
17.4
5.39 d (1.5)
4.12 m
3.61 m
3.22—3.08 o
3.22—3.08 o
0.81 d (5.6)
0.71 d (6.2)
4ꢃ-O-COCH3
1.97 s
Rhamnose-2
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
101.6
70.2
70.6
71.9
68.7
17.5
4.86 d (1.1)
3.68 m
3.50—3.31 o
3.26—3.10 o
3.50—3.31 o
1.08 d (6.2)
101.6
70.2
70.7
71.9
68.8
17.6
4.88 br s
3.68 m
3.36 m
3.23—3.07 o
3.39 m
1.11 d (6.2)
101.6
70.2
70.7
71.9
68.8
17.6
4.88 d (1.1)
3.68 m
3.36 m
3.22—3.08 o
3.39 m
1.10 d (6.1)
6ꢄ
Xylose
1ꢄ
2ꢄ
3ꢄ
4ꢄ
105.5
72.9
76.7
69.5
65.7
4.22 d (7.6)
2.98 m
3.11 m
3.30 m
3.77 m
105.8
73.8
76.2
69.5
65.7
4.32 d (7.3)
3.08 m
3.14 m
3.40—3.27 o
3.75 m
5ꢄ
3.14 m
3.12 m
Glucose
1ꢃꢃ
2ꢃꢃ
3ꢃꢃ
4ꢃꢃ
100.5
73.3
76.6
69.6
77.2
60.6
5.07 d (7.3)
3.30 m
3.30 m
3.27—3.05 o
3.43 m
3.71 m
99.9
73.1
76.4
69.5
77.2
60.6
5.01 d (7.5)
3.27 m
3.30 m
3.23—3.07 o
3.44 m
3.72 m
100.6
73.3
76.6
69.6
77.1
60.6
5.07 d (7.3)
3.30 m
3.31 m
3.22—3.08 o
3.43 m
3.72 m
5ꢃꢃ
6ꢃꢃ
3.48 m
3.48 m
3.48 m
“o” refers to peaks overlapped with other signals.
1
IR (KBr) cmꢆ1: 3419, 2926, 1651, 1612, 1044; H- and 13C-NMR data (see
Tables 1, 2); ESI-MS (positive) m/z: 697 [MꢁNa]ꢁ, 1371 [2MꢁNa]ꢁ, ESI-
MS (negative) m/z: 673 [MꢆH]ꢆ; HR-ESI-MS m/z: 675.2296 [MꢁH]ꢁ
(Calcd for C33H39O15, 675.2283).
[2MꢆH]ꢆ; HR-ESI-MS m/z: 795.2726 [MꢁH]ꢁ (Calcd for C37H47O19,
795.2706).
4ꢀ-Methoxyl-5-hydroxyl-8-(3-methyl-4-hydroxyl-but-2-enyl)-flavonol 3-
O-[a-L-Rhamnopyranosyl(1→2)-a-L-rhamnopyranoside]-7-O-b-D-glucopy-
ranoside (6): Yellow powder; UV lmax (MeOH) nm (log e): 204 (4.62, sh),
270 (4.38), 316 (4.14, sh), 348 (4.07); IR (KBr) cmꢆ1: 3408, 2930, 1651,
1597, 1062; 1H- and 13C-NMR data (see Tables 1, 2); ESI-MS (positive) m/z:
861 [MꢁNa]ꢁ, 839 [MꢁH]ꢁ, ESI-MS (negative) m/z: 873 [MꢁCl]ꢆ; HR-
ESI-MS m/z: 839.2982 [MꢁH]ꢁ (Calcd for C39H51O20, 839.2968).
4ꢀ-Methoxyl-5-hydroxyl-flavonol 3-O-[a-L-Rhamnopyranosyl(1→2)-a-L-
rhamnopyranoside]-7-O-b-D-glucopyranoside (7): Yellow powder; UV lmax
(MeOH) nm (log e): 204 (4.41, sh), 266 (4.17), 317 (3.97, sh), 342 (3.98);
4ꢀ,5-Dihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-[b-D-Xylopyranosyl-
(1→3)-4-O-acetyl-a-L-rhamnopyranoside]-7-O-b-D-glucopyranoside (4):
Yellow powder; UV lmax (MeOH) nm (log e): 204 (4.59, sh), 270 (4.34), 318
(4.08, sh), 348 (4.03); IR (KBr) cmꢆ1: 3420, 2926, 1651, 1601, 1074; H-
1
NMR (DMSO-d6, 400 MHz) d: 12.53 (1H, s, 5-OH), 7.78 (2H, d, Jꢂ8.8 Hz,
H-2ꢀ, 6ꢀ), 6.96 (2H, d, Jꢂ8.8 Hz, H-3ꢀ, 5ꢀ), 6.63 (1H, s, H-6), 5.32 (1H, br s,
H-1ꢃ), 5.17 (1H, t, Jꢂ7.0 Hz, H-12), 5.00 (1H, d, Jꢂ7.4 Hz, H-1ꢃꢃ), 4.85
(1H, t, Jꢂ10.0 Hz, H-4ꢃ), 4.22 (1H, d, Jꢂ7.6 Hz, H-1ꢄ), 4.14 (1H, br s, H-
2ꢃ), 3.80 (1H, dd, Jꢂ10.1, 2.3 Hz, H-3ꢃ), 3.77 (1H, m, H-5ꢄa), 3.73 (1H, m,
H-6ꢃꢃa), 3.57 (1H, m, H-11a), 3.48 (1H, m, H-6ꢃꢃb), 3.44 (1H, m, H-11b),
3.43 (1H, m, H-5ꢃꢃ), 3.40—3.25 (overlapped in HDO, H-4ꢄ, H-5ꢃ, H-2ꢃꢃ, H-
3ꢃꢃ), 3.17 (1H, m, H-4ꢃꢃ), 3.14 (1H, m, H-5ꢄb), 3.08 (1H, m, H-3ꢄ), 2.98
(1H, m, H-2ꢄ), 1.97 (3H, s, 4ꢃ-OAc), 1.68 (3H, s, H-14), 1.60 (3H, s, H-15),
0.72 (3H, d, Jꢂ6.2 Hz, H-6ꢃ); 13C-NMR data (DMSO-d6, 100 MHz) d: 178.1
(C-4), 169.7 (4ꢃ-O-COCH3), 160.5 (C-7), 160.5 (C-4ꢀ), 159.0 (C-5), 157.7
(C-2), 153.0 (C-9), 133.8 (C-3), 131.0 (C-13), 130.6 (C-2ꢀ, 6ꢀ), 122.1 (C-
12), 120.3 (C-1ꢀ), 115.4 (C-3ꢀ, 5ꢀ), 108.3 (C-8), 105.6 (C-1ꢄ), 105.5 (C-10),
101.3 (C-1ꢃ), 100.6 (C-1ꢃꢃ), 98.1 (C-6), 77.2 (C-5ꢃꢃ), 76.7 (C-3ꢄ), 76.6 (C-
3ꢃꢃ), 76.5 (C-3ꢃ), 73.3 (C-2ꢃꢃ), 72.9 (C-2ꢄ), 71.3 (C-4ꢃ), 69.6 (C-4ꢃꢃ), 69.6
(C-2ꢃ), 69.5 (C-4ꢄ), 68.4 (C-5ꢃ), 65.8 (C-5ꢄ), 60.6 (C-6ꢃꢃ), 25.4 (C-15), 21.4
(C-11), 20.8 (4ꢃ-O-COCH3), 17.8 (C-14), 17.0 (C-6ꢃ); ESI-MS (positive)
m/z: 859 [MꢁNa]ꢁ, 1695 [2MꢁNa]ꢁ, 837 [MꢁH]ꢁ, ESI-MS (negative)
m/z: 835 [MꢆH]ꢆ; HR-ESI-MS m/z: 837.2825 [MꢁH]ꢁ (Calcd for
C39H49O20, 837.2812).
1
IR (KBr) cmꢆ: 3420, 2931, 1654, 1603, 1066; H- and 13C-NMR data (see
Tables 1, 2); ESI-MS (positive) m/z: 777 [MꢁNa]ꢁ, 755 [MꢁH]ꢁ, ESI-MS
(negative) m/z: 789 [MꢁCl]ꢆ, 1507 [2MꢆH]ꢆ; HR-ESI-MS m/z: 755.2402
[MꢁH]ꢁ (Calcd for C34H43O19, 755.2393).
Acknowledgements We thank Wen Xu (Laboratory of Chinese Drugs
Pharmaceutics, Guangdong Hospital of Traditional Chinese Medicine,
Guangzhou, China) for measuring the HR-ESI-MS data. We also thank
Zhen-qiang Mu and Ming-yan Liu for measuring the NMR and ESI-MS
data. And we are grateful to Guizhou Tongjitang Pharmaceutical Co., Ltd.
for providing the plant material. This work was supported by Grants from
the National Natural Science Foundation of China (NSFC-RGC-
30831160510).
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