Flavonol glycosides from Epimedium pubescens.

Abstract

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

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November 2011 1317Regular Article
Epimedium pubescens MAXIM. (Berberidaceae), together
with other four species E. brevicornum MAXIM., E. sagitta-
tum MAXIM., E. wushanenes T. S. YING, and E. koreanum
NAKAI, is used as the official source of Traditional Chinese
Medicine (TCM) “Yinyanghuo” (Chinese Pharmacopoeia
2010 Edition). It is one of the most well known herbal medi-
cines with tonic, anti-rheumatic and aphrodisiac effects.1,2)
Previous chemical investigations on the geneus Epimedium
have afforded a series of flavonol glycosides,3—7)which pos-
sess multiple biological activities such as androgenic,8)an-
tioxidant,9)antiosteoporosis,10)and antidepressant-like ac-
tions.11)Our further chemical investigation on the aerial parts
of E. pubescens MAXIM. resulted in the isolation of five new
flavonol glycosides (compounds 1, 3, 5—7) and two known
compounds, sagittasine C (2)3)and 4?,5-dihydroxy-8-(3,3-
dimethylallyl)-flavonol 3-O-[b-D-xylopyranosyl(1→3)-4-O-
acetyl-a-L-rhamnopyranoside]-7-O-b-D-glucopyranoside
(4).12)Their structures were elucidated on the basis of spec-
troscopic evidences (Fig. 1).
Compounds 1, 3, 5, 6 and 7 were obtained as yellow amor-
phous powder. The UV absorption bands of all the com-
pounds were at about 205, 270, 320 and 350nm, suggesting
the presence of flavonol skeleton in their structures.
The molecular formula of compound 1 was determined as
C33H40O15based on the high resolution-electrospray ionization-
mass spectrum (HR-ESI-MS) (m/z 677.2448 [C33H41O15]?,
Calcd for 677.2440). The 1H-NMR spectrum of 1 exhibited
two singlet protons at d 12.58 (1H, s, OH-5) and d 6.28 (1H,
s), respectively. Three coupled aromatic protons at d 7.36
(1H, dd, J?8.4, 2.1Hz), 7.34 (1H, d, J?2.1Hz), and 7.08
(1H, d, J?8.4Hz) suggested the presence of a 1,2,4-trisubsti-
tuted benzene ring. A methoxy signal was obversed at d 3.85
(3H, s). A serial of proton signals at d 5.15 (1H, t, J?7.0Hz,
H-12), 3.33 (2H, m, H-11), 1.68 (3H, s, H-15), 1.62 (3H, s,
H-16) correlated with carbon signals at d 122.4, 25.4, 17.7,
21.2 in heteronuclear single quantum coherence (HSQC)
spectrum respectively, suggesting the presence of a prenyl
group. In addition, the proton signals at d 5.34 (1H, d,
J?1.3Hz, H-1?)/0.83 (3H, d, J?6.0Hz, H-6?), and 4.86 (1H,
d, J?1.1Hz, H-1?)/1.08 (3H, d, J?6.2Hz, H-6?) indicated
the existence of two rhamnose moieties. It was further con-
firmed by the acid hydrolysis and HPLC analysis according
to the method of Tanaka et al.13)The absolute configuration
of the sugars was determined to be the L configuration. The
methoxy group at d 3.85 was attached at the C-4? (d 160.5)
due to the characteristic heteronuclear multiple bond connec-
tivity (HMBC) correlation. The aromatic carbon signals at d
158.8, 98.5 and 103.7 were assigned to C-5, C-6 and C-10,
due to their HMBC correlations with the hydroxy group at d
12.58 (OH-5) (Fig. 2). Therefore, the carbon signals at d
162.6 and 105.9 were then assigned to C-7 and C-8 accord-
ing to their HMBC correlations with H-6 (d 6.28). The
prenyl group was located at C-8, which was supported by the
HMBC correlation between H-11 (d 3.33, 2H, m) and C-8 (d
105.9). Moreover, one rhamnose moiety was located at C-3
of the aglycone according to the HMBC correlation of H-
1?/C-3 (d 134.2). And the other rhamnose moiety was lo-
cated at C-2? of the inner one due to the HMBC correlations
of H-1?/C-2? (d 75.6) and H-2? (d 4.10, 1H, m)/C-1? (d
101.6). Thus, the structure of 1 was deduced as 4?-methoxyl-
3?,5,7-trihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-a-L-
rhamnopyranosyl(1→2)-a-L-rhamnopyranoside.
Flavonol Glycosides from Epimedium pubescens
Fengjuan TU,a,#Yi DAI,b,#Zhihong YAO,bXinluan WANG,c,dXinsheng YAO,*,a,band Ling QINc,d
aCollege of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University; Shenyang 110016, P .R. China:
bInstitute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University; Guangzhou
510632, P .R. China: cMusculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, The Chinese
University of Hong Kong; Hong Kong, China: and dTranslational 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 4?,5-dihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-[b b-D-
xylopyranosyl(1→→3)-4-O-acetyl-a a-L-rhamnopyranoside]-7-O-b 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
Chem. Pharm. Bull. 59(11) 1317—1321 (2011)
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed.
#These authors contributed equally to this work.
e-mail: yaoxinsheng@vip.tom.com
Fig. 1.Chemical Structures of 1—7

Page 2

Compound 3 had the molecular formula C33H38O15based
on the HR-ESI-MS (m/z 675.2296 [C33H29O15]?, Calcd for
675.2283). The 1H-NMR spectrum of 3 showed a singlet sig-
nal of OH-5 at d 12.48 (1H, s). A set of ortho-coupled dou-
blet signals of four aromatic protons at d 7.71, 6.93 (each
2H, d, J?7.8Hz) and a one-proton singlet signal at d 6.10
(1H, s) were also observed. The presence of an acetyl group
was supported by a three-proton singlet signal at d 1.97 in
the 1H-NMR spectrum and carbon signals at d 169.7, 20.8 in
the 13C-NMR spectrum. Proton signals at d 5.17 (1H, t,
J?5.9Hz, H-12), 3.34 (2H, m. H-11) as well as two methyl
groups signals at d 1.67 and 1.61 (each 3H, s, H-14, 15) re-
vealed the presence a prenyl group. It was further confirmed
by the carbon signals at d 21.3 (C-11), 123.3 (C-12), 129.9
(C-13), 17.8 (C-14) and 25.4 (C-15) observed in the 13C-
NMR spectrum. Additionally, two sugar moieties were ob-
served with protons d 5.30 (1H, brs)/0.71 (3H, d, J?6.2Hz)
and d 4.22 (1H, d, J?7.6Hz) in the 1H-NMR spectrum.
They were identified as an L-rhamnose and a D-xylose by the
acid hydrolysis experiment and HPLC analysis according to
the method of Tanaka et al.13)The aromatic methylene car-
bon signal at d 99.5 was assigned to C-6, due to its HMBC
correlation with the hydroxy group at d 12.48 (OH-5).
Therefore, the carbon signals at d 158.9 and 106.2 were then
assigned to C-7 and C-8 according to their HMBC correla-
tions with H-6 (d 6.10).The location of the prenyl group at
the C-8 (d 106.2) position was supported by the chemical
shift value of C-6 (d 99.5) in the 13C-NMR spectrum.3,14—16)
It was further confirmed by the HMBC correlation between
H-11 (d 3.34) and C-8. In the HMBC spectrum, a correlation
between the H-1? (d 5.30) and C-3 (d 134.2) were observed,
indicating the rhamnose moiety was attached to C-3 of the
aglycone. The xylose moiety was located at C-3? (d 76.5) of
the inner rhamnose moiety, according to the HMBC correla-
tion of H-1? (d 4.22)/C-3?. The acetyl group was located at
C-4? of the rhamnose moiety due to the correlation between
H-4? (d 4.85) and the carbonyl carbon of the acetyl group at
d 169.7. Therefore, the structure of 3 was elucidated to be
4?,5,7-trihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-b-D-xy-
lopyranosyl(1→3)-4-O-acetyl-a-L-rhamnopyranoside.
The molecular formula of Compound 5 was established as
C37H46O19by HR-ESI-MS (m/z 795.2726 [C37H47O19]?,
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
at d 5.00 (1H, d, J?7.3Hz, H-1??) indicated it to be b-glu-
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-1??. Thus, the structure of 5
was identified as 4?,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
C39H50O20by HR-ESI-MS (m/z 839.2982 [C39H51O20]?,
Calcd for 839.2968). The 1H-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, J?2.1Hz), and
a methoxy group (d 3.85, 3H, s). Proton signals at d 5.37
(1H, m, H-12), 3.73 (2H, brs, H-15), 3.60 (1H, m, H-11a),
3.47 (1H, m, H-11b), 1.66 (3H, s, H-14) in the 1H-NMR
spectrum, correlated with carbon signals at d 120.9 (C-12),
66.1 (C-15), 20.8 (C-11), 13.7 (C-14) in HSQC spectrum re-
spectively, indicating the presence of a hydroxyprenyl group.
Additionally, three anomeric protons were observed at d 5.39
(1H, d, J?1.5Hz, H-1?), 4.88 (1H, d, J?1.1Hz, H-1?), and
5.01 (1H, d, J?7.5Hz, H-1??) respectively, suggesting the
presence of three monosaccharide moieties. They were iden-
tified as two a-L-rhamnose moieties and a b-D-glucose moi-
ety by the acid hydrolysis experiment.13)The methoxy group
was located at C-4? according to the HMBC correlation be-
tween the proton signal at d 3.85 and C-4? (d 161.4). One
rhamnose moiety was located at C-3 of the aglycone due to
the HMBC correlation of H-1?/C-3 (d 134.5). The other
rhamnose moiety was located at C-2? of the inner one due to
the HMBC correlations of H-1?/C-2? (d 75.5) and H-2? (d
4.12, 1H, m)/C-1? (d 101.6). And the glucopyranose moiety
was located at C-7 (d 160.6) according to the ROESY corre-
lation between H-1?? and H-6 (d 6.64) (Fig. 2). Therefore,
compound 6 was elucidated as 4?-methoxyl-5-hydroxyl-8-(3-
methyl-4-hydroxyl-but-2-enyl)-flavonol 3-O-[a-L-rhamnopy-
ranosyl(1→2)-a-L-rhamnopyranoside]-7-O-b-D-glucopyra-
noside.
Compound 7 was obtained with the molecular formula
C34H42O19by HR-ESI-MS (m/z 755.2402 [C34H43O19]?,
Calcd for 755.2393). The NMR spectroscopic data of 7 were
similar with those of 6, except for the loss of the signals of a
hydroxyprenyl group. Thus, the structure of 7 was elucidated
as 4?-methoxyl-5-hydroxyl-flavonol 3-O-[a-L-rhamnopyra-
nosyl(1→2)-a-L-rhamnopyranoside]-7-O-b-D-glucopyra-
noside.
Experimental
General Procedure
UV/Vis spectrometer. IR spectra were obtained using a JASCO FT/IR-480
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 (50mm, YMC,
Japan), HW-40 (Tosoh, Japan) and Sephadex LH-20 (Pharmacia). Thin layer
chromatography (TLC) was performed using percolated silica gel plates
(silica gel GF254, 1mm, 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-
ceutical Co., Ltd., Guiyang, China, and identified by Professor Guang-Xiong
UV spectra were recorded on a JASCO V-550
1318Vol. 59, No. 11
Fig. 2.Key HMBC (→) and ROESY (↔) Correlations of 1 and 6

Page 3

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.
Extraction and Isolation
The aerial parts of E. pubescens MAXIM.
(2kg) were extracted twice with 60% ethanol. After removal of the ethanol
in vacuo, the extract (245g) was chromatographied over Diaion HP-20 resin,
eluted with water, 30% and 95% ethanol in successive. The 95% ethanol
eluate (80g) 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 (16mg) 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
to G). The subfraction 11B (eluted with 20% MeOH–H2O) was applied to
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 (21mg) and 7 (10mg).
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
HPLC (UltimateTMXB-C18, 5mm, 21.2?250mm, Welch) with 50%
MeOH–H2O to yield compounds 2 (8mg) and 5 (26mg). Fraction 10G
(eluted with 50% MeOH–H2O) was purified by Sephadex LH-20 with 60%
MeOH–H2O to yield compound 4 (68mg). Fraction 10I (eluted with 50%
MeOH–H2O) was subjected to Sephadex LH-20 eluted with 60%
MeOH–H2O, and then separated by preparative HPLC (UltimateTMXB-C18,
5mm, 21.2?250mm, Welch) with 65% MeOH–H2O to yield compound 1
(9mg).
Acid Hydrolysis and HPLC Analysis
the sugar moieties in the structures were determined by the method of
Tanaka et al.13)Compound 1 (2mg) was hydrolyzed with 2 M HCl for 2h 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
(1ml) containing L-cysteine methyl ester (1mg) (Sigma, U.S.A.) and heated
at 60°C for 1h. Then, o-tolyl isothiocyanate (5ml) (Alfa Aesar, U.K.) was
added to the mixture, which was heated at 60°C for 1h. The reaction mix-
ture was directly analyzed by reversed-phase HPLC. Analytical HPLC was
performed on a Cosmosil 5C18-MS-II column (250?4.6mm i.d., 5mm,
The absolute configuration of
Nacalai Tesque Inc., Japan) at 35°C with isocratic elution of 25% CH3CN
containing 0.1% formic acid for 40min and subsequent washing of the col-
umn with 90% CH3CN at a flow rate 0.8ml/min. Peaks were detected by a
UV detector at 250nm. 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 tR15.9 (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 tR17.2—17.3 (D-Glc) and 29.2—
29.3 (L-Rha) min, respectively. The derivatives of 3 gave two peaks at tR20.1
(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.
4?-Methoxyl-3?,5,7-trihydroxyl-8-(3,3-dimethylallyl)-flavonol
Rhamnopyranosyl(1→2)-a-L-rhamnopyranoside (1): Yellow powder; UV
lmax(MeOH) nm (loge): 205 (4.47), 258 (4.13, sh), 270 (4.17), 346 (3.91);
IR (KBr) cm?1: 3402, 2933, 1653, 1509, 1046; 1H- and 13C-NMR data (see
Tables 1, 2); ESI-MS (positive) m/z: 699 [M?Na]?, 1375 [2M?Na]?, 677
[M?H]?, ESI-MS (negative) m/z: 675 [M?H]?, 1351 [2M?H]?; HR-ESI-
MS m/z: 677.2448 [M?H]?(Calcd for C33H41O15, 677.2440).
Sagittasine C (2): Yellow powder; UV lmax(MeOH) nm (loge): 205
(4.50), 258 (4.14, sh), 269 (4.15), 346 (3.91); IR (KBr) cm?1: 3388, 2925,
1651, 1599, 1076; 1H-NMR (DMSO-d6, 400MHz) d: 12.59 (1H, s, 5-OH),
7.40 (1H, dd, J?8.5, 2.1Hz, H-6?), 7.38 (1H, d, J?2.1Hz, H-2?), 7.09 (1H,
d, J?8.5Hz, H-5?), 6.62 (1H, s, H-6), 5.25 (1H, d, J?1.3Hz, H-1?), 5.17
(1H, t, J?7.0Hz, H-12), 5.00 (1H, d, J?7.5Hz, H-1?), 4.00 (1H, brs, H-2?),
3.86 (3H, s, 4?-OCH3), 3.72 (1H, m, H-6??a), 3.54 (1H, m, H-11a), 3.53 (1H,
m, H-3?), 3.48 (1H, m, H-6??b), 3.43 (2H, m, H-11b, H-5?), 3.35—3.28
(overlapped in HDO, H-2?, 3?), 3.26—3.12 (3H, m, H-4?, H-4?, H-5?), 1.70
(3H, s, H-14), 1.61 (3H, s, H-15), 0.81 (3H, d, J?6.0Hz, H-6?); 13C-NMR
data (DMSO-d6, 100MHz) d: 178.3 (C-4), 160.5 (C-7), 159.0 (C-5), 157.4
(C-2), 152.9 (C-9), 150.2 (C-4?), 146.4 (C-3?), 134.6 (C-3), 131.1 (C-13),
122.4 (C-1?), 122.1 (C-12), 120.8 (C-6?), 115.5 (C-2?), 111.7 (C-5?), 108.3
(C-8), 105.5 (C-10), 102.0 (C-1?), 100.6 (C-1?), 98.1 (C-6), 77.2 (C-5?),
76.6 (C-3?), 73.3 (C-2?), 71.2 (C-4?), 70.6 (C-5?), 70.3 (C-3?), 70.0 (C-2?),
69.7 (C-4?), 60.6 (C-6?), 55.7 (4?-OCH3), 25.4 (C-15), 21.4 (C-11), 17.7
(C-14), 17.4 (C-6?); ESI-MS (positive) m/z: 715 [M?Na]?, 1407
[2M?Na]?, 693 [M?H]?, ESI-MS (negative) m/z: 691 [M?H]?, 1383
[2M?H]?; HR-ESI-MS m/z: 693.2404 [M?H]?(Calcd for C33H41O16,
693.2389).
4?,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 (loge): 204 (4.43, sh), 270 (4.25), 316 (3.97, sh), 350 (3.91);
3-O-a-L-
November 20111319
Table 1. NMR Data of Aglycone Moiety for Compounds 1, 3, 5—7 (in DMSO-d6)
13567
No.
dC
dH(J in Hz)
dC
dH(J in Hz)
dC
dH(J in Hz)
dC
dH(J in Hz)
dC
dH(J in Hz)
2
3
4
5
6
7
8
9
156.6
134.2
177.7
158.8
98.5
162.6
105.9
153.7
103.7
21.2
155.9
133.1
176.8
157.9
99.5
158.9
106.2
153.9
102.0
21.3
157.7
134.0
178.1
159.0
98.1
160.4
108.3
152.9
105.5
21.4
157.3
134.5
178.2
159.1
98.1
160.6
108.1
153.0
105.1
20.8
157.4
134.8
177.9
160.9
99.4
163.1
94.7
156.1
105.8
6.28 s 6.10 s6.63 s 6.64 s6.48 d (2.1)
6.78 d (2.1)
10
113.38 m 3.34 m3.34 m
3.55 m
5.16 t (6.9)
3.47 m
3.60 m
5.37 m 12
13
14
15
122.4
130.9
17.7
25.4
122.5
115.4
146.4
150.0
111.7
120.6
55.7
5.15 t (7.0)123.3
129.9
17.8
25.4
120.6
130.2
115.3
160.3
115.3
130.2
5.17 t (5.9) 122.2
131.0
17.8
25.4
120.3
130.7
115.4
160.4
115.4
130.7
120.9
135.6
13.7
66.1
122.1
130.6
114.0
161.4
114.0
130.6
55.5
1.68 s
1.62 s
1.67 s
1.61 s
1.69 s
1.60 s
1.66 s
3.73 brs
1?
2?
3?
4?
5?
6?
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)
7.36 dd (8.4, 2.1)
3.85 s
12.58 s
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
12.61 s
7.12 d (8.9)
7.88 d (8.9)
3.85 s
12.65 s
4?-OCH3
5-OH12.48 s12.57 s

Page 4

IR (KBr) cm?1: 3419, 2926, 1651, 1612, 1044; 1H- and 13C-NMR data (see
Tables 1, 2); ESI-MS (positive) m/z: 697 [M?Na]?, 1371 [2M?Na]?, ESI-
MS (negative) m/z: 673 [M?H]?; HR-ESI-MS m/z: 675.2296 [M?H]?
(Calcd for C33H39O15, 675.2283).
4?,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 (loge): 204 (4.59, sh), 270 (4.34), 318
(4.08, sh), 348 (4.03); IR (KBr) cm?1: 3420, 2926, 1651, 1601, 1074; 1H-
NMR (DMSO-d6, 400MHz) d: 12.53 (1H, s, 5-OH), 7.78 (2H, d, J?8.8Hz,
H-2?, 6?), 6.96 (2H, d, J?8.8Hz, H-3?, 5?), 6.63 (1H, s, H-6), 5.32 (1H, brs,
H-1?), 5.17 (1H, t, J?7.0Hz, H-12), 5.00 (1H, d, J?7.4Hz, H-1??), 4.85
(1H, t, J?10.0Hz, H-4?), 4.22 (1H, d, J?7.6Hz, H-1?), 4.14 (1H, brs, H-
2?), 3.80 (1H, dd, J?10.1, 2.3Hz, H-3?), 3.77 (1H, m, H-5?a), 3.73 (1H, m,
H-6??a), 3.57 (1H, m, H-11a), 3.48 (1H, m, H-6??b), 3.44 (1H, m, H-11b),
3.43 (1H, m, H-5??), 3.40—3.25 (overlapped in HDO, H-4?, H-5?, H-2??, H-
3??), 3.17 (1H, m, H-4??), 3.14 (1H, m, H-5?b), 3.08 (1H, m, H-3?), 2.98
(1H, m, H-2?), 1.97 (3H, s, 4?-OAc), 1.68 (3H, s, H-14), 1.60 (3H, s, H-15),
0.72 (3H, d, J?6.2Hz, H-6?); 13C-NMR data (DMSO-d6, 100MHz) d: 178.1
(C-4), 169.7 (4?-O-COCH3), 160.5 (C-7), 160.5 (C-4?), 159.0 (C-5), 157.7
(C-2), 153.0 (C-9), 133.8 (C-3), 131.0 (C-13), 130.6 (C-2?, 6?), 122.1 (C-
12), 120.3 (C-1?), 115.4 (C-3?, 5?), 108.3 (C-8), 105.6 (C-1?), 105.5 (C-10),
101.3 (C-1?), 100.6 (C-1??), 98.1 (C-6), 77.2 (C-5??), 76.7 (C-3?), 76.6 (C-
3??), 76.5 (C-3?), 73.3 (C-2??), 72.9 (C-2?), 71.3 (C-4?), 69.6 (C-4??), 69.6
(C-2?), 69.5 (C-4?), 68.4 (C-5?), 65.8 (C-5?), 60.6 (C-6??), 25.4 (C-15), 21.4
(C-11), 20.8 (4?-O-COCH3), 17.8 (C-14), 17.0 (C-6?); ESI-MS (positive)
m/z: 859 [M?Na]?, 1695 [2M?Na]?, 837 [M?H]?, ESI-MS (negative)
m/z: 835 [M?H]?; HR-ESI-MS m/z: 837.2825 [M?H]?(Calcd for
C39H49O20, 837.2812).
4?,5-Dihydroxyl-8-(3,3-dimethylallyl)-flavonol 3-O-[b-D-Xylopyranosyl-
(1→3)-a-L-rhamnopyranoside]-7-O-b-D-glucopyranoside (5): Yellow pow-
der; UV lmax(MeOH) nm (loge): 204 (4.38, sh), 270 (4.13), 321 (3.87, sh),
350 (3.85); IR (KBr) cm?1: 3409, 2924, 1651, 1600, 1075; 1H- and 13C-
NMR data (see Tables 1, 2); ESI-MS (positive) m/z: 817 [M?Na]?, 1611
[2M?Na]?, 795 [M?H]?, ESI-MS (negative) m/z: 793 [M?H]?, 1587
[2M?H]?; HR-ESI-MS m/z: 795.2726 [M?H]?(Calcd for C37H47O19,
795.2706).
4?-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 (loge): 204 (4.62, sh),
270 (4.38), 316 (4.14, sh), 348 (4.07); IR (KBr) cm?1: 3408, 2930, 1651,
1597, 1062; 1H- and 13C-NMR data (see Tables 1, 2); ESI-MS (positive) m/z:
861 [M?Na]?, 839 [M?H]?, ESI-MS (negative) m/z: 873 [M?Cl]?; HR-
ESI-MS m/z: 839.2982 [M?H]?(Calcd for C39H51O20, 839.2968).
4?-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 (loge): 204 (4.41, sh), 266 (4.17), 317 (3.97, sh), 342 (3.98);
IR (KBr) cm?: 3420, 2931, 1654, 1603, 1066; 1H- and 13C-NMR data (see
Tables 1, 2); ESI-MS (positive) m/z: 777 [M?Na]?, 755 [M?H]?, ESI-MS
(negative) m/z: 789 [M?Cl]?, 1507 [2M?H]?; HR-ESI-MS m/z: 755.2402
[M?H]?(Calcd for C34H43O19, 755.2393).
Acknowledgements
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).
We thank Wen Xu (Laboratory of Chinese Drugs
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Table 2.NMR Data of Sugar Moieties for Compounds 1, 3, 5—7 (in DMSO-d6)
13567
No.
dC
dH(J in Hz)
dC
dH(J in Hz)
dC
dH(J in Hz)
dC
dH(J in Hz)
dC
dH(J in Hz)
Rhamnose-1
1?
2?
3?
4?
5?
6?
4?-O-COCH3
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 brs
4.12 brs
3.80 m
4.84 t (10.0)
3.32 m
0.71 d (6.2)
101.8
69.5
80.9
69.9
70.5
17.4
5.30 brs
4.13 brs
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
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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)
1.97 s
Rhamnose-2
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2?
3?
4?
5?
6?
Xylose
1?
2?
3?
4?
5?
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 brs
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)
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
3.14 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
3.12 m
Glucose
1??
2??
3??
4??
5??
6??
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
3.48 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
3.48 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
3.48 m
“o” refers to peaks overlapped with other signals.

Page 5

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November 20111321