A new isorhamnetin glycoside and other phenolic compounds from Callianthemum taipaicum

Article abstract of DOI:10.3390/molecules17044595

A new flavonol glycoside together with five known phenolic compounds were isolated from the whole herb of Callianthemum taipaicum. The compounds were identified as isorhamnetin-3-O-α-L-arabinoside-7-O-β-D-glucoside (1), isorhamnetin-3-O-β-D-glucoside (2), dibutyl phthalate (3), (+)-1-hydroxylpinoresinol-4'-β-D-glucoside (4), pinoresinol-4'-O-β-D- glucoside (5) and 2-phenylethyl-β-primeveroside (6). Compound 1 was identified as a new flavonol glycoside. The compound 6 was isolated for the first time as natural product. All compounds were isolated for the first time from the Callianthemum genus. Furthermore, the 2D-NMR data of the four known compounds 2-5 are given for the first time in this paper. All the structures were identified on the basis of detailed spectral analysis. The compounds 1 and 4 exhibited certain antifungal activity.

Full text of DOI:10.3390/molecules17044595

Molecules 2012, 17, 4595-4603; doi:10.3390/molecules17044595
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A New Isorhamnetin Glycoside and Other Phenolic Compounds
from Callianthemum taipaicum
Dong-Mei Wang 1,*, Wen-Jun Pu 1, Yong-Hong Wang 2, Yu-Juan Zhang 1 and Shan-Shan Wang 1
1
College of Forestry, Northwest A & F University, Yangling 712100, China;
E-Mails: Yvonne2nadal@163.com (W.-J.P.); hedwig.yujuan@gmail.com (Y.-J.Z.);
121268525@qq.com (S.-S.W.)
2
Shanghai Chempartner Co., LTD, Shanghai 201203, China; E-Mail: Yhwang@chempartner.cn
* Author to whom correspondence should be addressed; E-Mail: dmwli@163.com;
Tel.: +86-29-8703-9015; Fax: +86-29-8708-2256.
Received: 2 December 2011; in revised form: 3 April 2012 / Accepted: 6 April 2012 /
Published: 17 April 2012
Abstract: A new flavonol glycoside together with five known phenolic compounds
were isolated from the whole herb of Callianthemum taipaicum. The compounds were
identified as isorhamnetin-3-O-α-L-arabinoside-7-O-β-D-glucoside (1), isorhamnetin-3-O-
β-D-glucoside (2), dibutyl phthalate (3), (+)-1-hydroxylpinoresinol-4'-β-D-glucoside (4),
pinoresinol-4'-O-β-D-glucoside (5) and 2-phenylethyl-β-primeveroside (6). Compound 1
was identified as a new flavonol glycoside. The compound 6 was isolated for the first time
as natural product. All compounds were isolated for the first time from the Callianthemum
genus. Furthermore, the 2D-NMR data of the four known compounds 2–5 are given for the
first time in this paper. All the structures were identified on the basis of detailed spectral
analysis. The compounds 1 and 4 exhibited certain antifungal activity.
Keywords: Callianthemum taipaicum; isorhamnetin glycoside; phenolic compounds;
antifungal activity
1. Introduction
Plants have been utilized as medicines for thousands of years [1]. About 80% of the World’s
population uses medicinal plants for primary health care [2]. Medicinal plants have attracted more
and more attention for their “safety, cultural acceptability and lesser side effects”, for instance,

Molecules 2012, 17
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Ginkgo biloba for tinnitus, Hypericum perforatum for depression [3]. Natural products isolated from
medicinal plants were used by indigenous populations for physiological and therapeutic effect,
aconitine, atisine, lstrychnine, digoxin, atropine and morphine are some common examples [4].
Isolation and characterization of natural products from medicinal plants has played a significant role in
drug discovery and development.
The Qinling Mountains are an important traditional Chinese medicine area in China. They serve as
an important medicinal plant resource. Taibai Mountain, also known as “Herbal Kingdom” and
“Natural Botanical Garden of Asia”, is the highest peak of the Qinling Mountains, with the altitude of
3767.2 m. Due to the special environment, there are many unique medical plants growing in this
area [5]. The Callianthemum genus belongs to the family Ranunculaceae, which include 12 species
worldwide, many of them are found in Asia and Europe. There are five species distributed in west
and northwest China. Callianthemum taipaicum is an endemic species of Taibai Mountain, where it
grows at altitudes between 3,450 to 3,600 m [6]. The whole herb of this plant has detoxifying and
anti-inflammatory activity, and can be used as a medicine to treat various diseases including children’s
pneumonia and drug-fire eyesight [7]. Due to the special environment where Callianthemum taipaicum
grows, it could contain many special phytochemicals. However, a literature survey revealed that no
chemical work has been carried out on this plant or indeed the Callianthemum genus so far. In order to
identify functional compounds from Callianthemum taipaicum, we have isolated a new isorhamnetin
glycoside (1) together with five known phenolic compounds 2–6. The present paper, for the first time,
describes the isolation and structure elucidation of these compounds by detailed analysis of their NMR
spectra. The antifungal activity of compounds 1, 4 is also discussed.
2. Results and Discussion
2.1. Structure Elucidation of the New Compound
Compound 1 obtained as a light yellow amorphous powder. Its molecular formula was established
as C27H30O16 on the basis of HR-ESI-MS: m/z 611.1602 [M+H]+ (calcd. [M+H]+ 611.1607) and ESI-MS
m/z 611.0 [M+H]+. The IR spectrum showed the characteristic absorption bands of hydroxyl (3444.87
and 3429.43 cm−1), carbonyl (1653.00 cm−1) and phenyl group (1600.92 and 1490.97 cm−1) [8].
13C-NMR and DEPT spectrum showed signals for one OCH3, two CH2 (all aliphatic), fourteen CH
(five aromatic, nine aliphatic) and ten quaternary carbons (one carbonyl, five O-bearing and four
1
13
aliphatic) [9]. The H- and C-NMR spectra of compound 1 were compared with the data of known
compounds and showed a typical flavonol pattern with an isorhamnetin aglycon (Table 1) [10–12]. In
addition to the isorhamnetin moiety, mass and NMR spectra showed signals corresponding to two
sugar moieties. Acid hydrolysis of compound 1 gave D-glucose and L-arabinose, identified by
comparision with an authentic sample. The sugar portion was examined by TLC analysis. The
1H-NMR spectrum of compound 1 displayed two doublets at δ 5.57 (1H, d, J = 7.0 Hz) and δ 5.06 (1H, d,
J = 6.6 Hz) for the anomeric protons. Based on the coupling constant J = 7.0 Hz and 6.6Hz greater than
4.0 Hz, the sugar configurations could be identified as α-L-arabinose and β-D-glucose, respectively,
which correlated respectively with signals at 100.71, 100.11 in the HSQC spectrum [13,14]. In the
HMBC (Figure 1), the anomeric proton signal of the arabinose at δ 5.57 (H-1'') correlated with the

Molecules 2012, 17
4597
carbon signal at δ 133.26 (C-3), the proton signal of the glucose at δ 5.06 (H-1''') correlated with the
anomeric carbon signal of the apiose at δ 162.73(C-7). These indicated that the arabinose attached to
C-3 of the aglycone, the glucose attached to C-7 of the aglycone [15]. In addition, in the literature,
there was a remarkable upfield shift (∆δ 2.7) for the C-7 of an isorhamnetin 3,7-diglycoside (δ 161.8)
13
compared to an isorhamnetin 3-glycoside (δ 164.5), the C-NMR spectrum of compound 1 showed
that the chemical shift of δ 162.73 for C-7, was close to the chemical shift of δ 161.8 [11]. Based on
the above evidence and detailed analysis of the NMR spectra, the structure of compound 1 was
determined as isorhamnetin-3-O-α-L-arabinopyranose-7-β-D-glucopyranoside, which had not been
reported previously.
Table 1. 1H-, 13C-NMR and HMBC spectral data of compound 1 (in DMSO, δ in ppm, J in Hz).
Isorhamnetin 3-O-β-D-
Compound 1
glucoside-7-O-α-L-rhamnoside
Structure Position
δ H
δ C
HMBC
δ H
δ C
2
3
4
5
6
7
8
156.79
133.26
177.56
160.85
99.27
156.9
133.3
177.6
161.0
99.4
12.6 (1H, s)
C-5,6,10
6.43 (1H, d, 1.5 Hz)
C-5,7,8,10 6.44 (1H, d, 2.0 Hz)
162.73
94.49
161.6
94.7
6.97 (1H, d, 1.5 Hz)
C-6,7,9,10 6.83(1H,d, 2.0 Hz)
Flavonol
group
9
10
1'
2'
3'
4'
5'
6'
155.97
105.59
120.92
113.54
149.58
146.90
156.0
105.7
121.0
113.5
149.7
147.0
115.3
122.4
55.8
7.97 (1H, s)
9.87 (1H, s)
C-2,3',6'
7.94 (1H, s)
C-3',5'
C-1',3'
C-2,2',3'
C-3'
6.93 (1H, d, 8.5 Hz) 115.20
7.54 (1H, d, 8.5 Hz) 122.19
6.93 (1H, d, 8.5 Hz)
7.55 (1H, d, 8.5 Hz)
3.83 (3H, s)
3'-OCH3 3.84 (3H, s)
55.69
1''
2''
5.57 (1H, d, 7.0 Hz) 100.71
C-3
5.57 (1H, d, 6.7 Hz)
3.2 (1H, m)
100.8
74.4
3.0–3.8 (1H, m)
3.0–3.8 (1H, m)
3.0–3.8 (1H, m)
3.0–3.8 (2H, m)
-------
74.32
67.32
69.81
60.60
-----
Sugar
at C-3
3''
3.2 (1H, m)
76.5
4''
3.1 (1H, m)
70.1
5''
3.1 (1H, m)
77.6
6''
-----
C-7
3.57, 3.45
60.6
1'''
2'''
3'''
4'''
5'''
6'''
5.06 (1H, d, 6.6 Hz) 100.11
5.55 (1H, s)
98.4
3.0–3.8 (1H, m)
3.0–3.8 (1H, m)
3.0–3.8 (1H, m)
3.0–3.8 (1H, m)
3.0–3.8 (2H, m)
70.00
76.40
72.33
77.45
65.72
3.83 (1H, s)
70.1
Sugar
at C-7
3.63 (1H, d, 3.2 Hz)
3.3 (1H, m)
70.3
71.7
3.4 (1H, m)
69.9
1.10 (1H, d, 6.1 Hz)
18.0

Molecules 2012, 17
Figure 1. The structure and the key HMBC correlations of compounds 1–6.
4598
OCH3
OCH3
OH
3'
OH
OH
3'
4'
2'
1'
4'
2'
1'
6'''
8
4'''
5'''
2'''
O
HO
O
O
8
9
7
5'
O
O
HO
HO
1'''
9
7
5'
6'
6'
3'''
OH
HO
6
3'
1'
2'
5'
3
6
10
O
HO
OH
OH
3
5
10
3''
O
2''
5
OH
4''
O
1''
OH
4'
OH
O
5''
O
6'
2
1
OH
HO
OH
6''
5'
4''
5''
2''
O
O
4'
3'
6'
1'
HO
HO
1''
O
3''
OH
O
7'
8'
9'
8
10
6
3
9
H3CO
2'
1
2
7
5
O
O
11
9
8
H
H
2
4
7
OCH3
OH
3
4
1
HO
O
6
4
5
3
OCH3
3
OH
2
4
4''
O
5''
2''
O
5
7
9
8'
HO
HO
1
6
O
3
6
8
6'
5'
2
1
H
H
3''
OH
1''
4
2'
5'
H3CO
OH
4'
1'
3'
6''
9'
O
HO
HO
7'
4''
5''
2''
O
O
O
5
HO
1''
2'
HO
3'
7
6'
1'
4'
3''
OH
OH
O
6
5
2.2. Antifungal Activity of Compounds
Due to the insufficient quantities of compounds 2, 3, 5, 6, we only tested compounds 1 and 4 for
their antifungal activities using amphotericin as positive control (Table 2). The results showed that the
tested compounds exhibited certain antifungal activity. Compound 1 showed inhibitory activity against
the growth of Rhizoctonia cerealis, Botrytis cinerrea and Thanatephorus cucumeris with EC50 was
1.92 ± 0.30, 1.38 ± 0.26, 1.39 ± 0.19 mg/mL, respectively. Compound 4 showed obviously inhibitory
activity against the growth of Rhizoctonia cerealis, Botrytis cinerrea and Valsa mali with EC50 was
0.75 ± 0.37, 0.82 ± 0.05, 1.28 ± 0.34 mg/mL, respectively.
Table 2. Antifungal results of compounds 1 and 4.
Sample
Species
Rhizoctonia cerealis
Botrytis cinerrea
Thanatephorus cucumeris
Rhizoctonia cerealis
Botrytis cinerrea
Valsa mali
Rhizoctonia cerealis
Botrytis cinerrea
Thanatephorus cucumeris
Valsa mali
Toxicity egressions
y = 16.004x + 19.444
y = 31.509x + 6.5604
y = 31.145x + 6.793
y = 62.96x + 10.36
y = 48.363x + 10.255
y = 21.612x + 23.711
y = 637.44x − 6.3644
y = 575.85x − 17.957
y = 268.36x + 29.736
y = 115.12x + 31.014
r
EC50 mg/mL
1.92 ± 0.30
1.38 ± 0.26
1.39 ± 0.19
0.75 ± 0.37
0.82 ± 0.05
1.28 ± 0.34
0.13 ± 0.003
0.17 ± 0.041
0.09 ± 0.008
0.14 ± 0.008
0.9945
0.9322
0.9366
0.9958
0.9768
0.9819
0.8097
0.9008
0.9767
0.8673
Compound 1
Compound 4
Amphotericin

Molecules 2012, 17
4599
3. Experimental
3.1. General
ESI-MS was performed on an Agilent 1200 HPLC with the 6130 SQD system, with a Waters
XBridge C18 column (50 × 4.6 mm, 3.5 μm, Waters). HR- ESI-MS was performed on Accurate-Mass-
Q-TOF-LC/MS 6520 (Agilent, USA). HPLC separation was performed on a Gilson GX-281 HPLC
with a PRC-ODS column (20 × 250 mm, 15 μm, Shimadzu). The mobile phase for HPLC was 10 mM
NH4CO3 water solution (A) and MeCN (B) for base method, 0.05% TFA water solution(A) and MeCN
1
13
(B) for acid method, respectively. H- and C- spectra were measured with a Bruker AVANCE III
spectrometer (500 MHz) and a Bruker AVANCE III spectrometer (400 MHz). The FTIR spectra was
measured with a Shimadzu Prestige-21 spectrometer. Thin layer silica gel was purchased from
Qingdao Ocean Company. All solvents were analytical grade.
3.2. Plant Material
A 5-year-old whole Callianthemum taipaicum plant was collected in Taibai Mountain, Shaanxi
Province, China, in September 2010. The voucher specimen (NO. 148) has been deposited at the
Herbarium of the Northwest Sci-Tech University of Agriculture and Forestry.
3.3. Extraction and Isolation
The air-dried and powdered plant (988 g) of Callianthemum taipaicum was extracted five times
with 95% ethanol (1 L, 12 h) at room temperature. The filtrates were combined and evaporated to
dryness under vacuum. The residue (188 g) was suspended in water (1.8 L) and partitioned with
petroleum ether, ethyl acetate and n-BuOH, successively.
The n-BuOH extract (14.0 g) was fractionated by column chromatography over silica gel eluting
with CH2Cl2-MeOH (20:1 to 0:100) to yield five fractions (Fr. 1–Fr. 5). Fr. 2 (4.3 g) was submitted to
RP-HPLC on PRC-ODS column (20 × 250 mm, 15 μm) with MeCN-H2O base method for 0–5 min
(5:95) and 5–20 min (50:50) to yield seven subfractions. The subfractions 4–6 were submitted to
RP-HPLC on PRC-ODS column (20 × 250 mm, 15 μm) with MeCN-H2O (80:20) to yield compound 1
(100 mg), compound 4 (34 mg) and compound 5 (7 mg), respectively. Fr. 3 (2.7 g) was submitted to
RP-HPLC on PRC-ODS column (20 × 250 mm, 15 μm) with MeCN-H2O base method for 0–5 min
(5:95) and 5–20 min (17:83) to yield nine subfractions. Subfraction 5 was submitted to RP-HPLC on
PRC-ODS column (20 × 250 mm, 15 μm) with MeCN-H2O base method (80:20) to yield compound 6
(14 mg).
The ethyl acetate extract (1.2 g) was submitted to RP-HPLC on PRC-ODS column (20 × 250 mm,
15 μm) with MeCN-H2O base method for 0–7.5 min (30:70 to 90:10) to yield five fractions (Fr. 1–Fr. 5).
Fr. 2 was submitted to RP-HPLC on PRC-ODS column (20 × 250 mm, 15 μm) with MeCN-H2O base
method (40:60) to yield compound 2 (18 mg). Fr. 5 was submitted to RP-HPLC on PRC-ODS column
(20 × 250 mm, 15 μm) with MeCN-H2O acid method (5:95) to yield compound 3 (20 mg).

Molecules 2012, 17
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Compound 1 obtained as a light yellow amorphous powder. Its molecular formula was established
as C27H30O16 on the basis of HR-ESI-MS: m/z 611.1602 [M+H]+ (calcd. [M+H]+ 611.1607) and
ESI-MS m/z 611.0 [M+H]+. The IR spectrum showed the characteristic absorption bands of hydroxyl
(3444.87 and 3429.43 cm−1), carbonyl (1653.00 cm−1) and phenyl groups (1600.92 and 1490.97 cm−1).
1H-NMR (DMSO) and 13C-NMR (DMSO): Table 1.
Compound 2 was obtained as a light yellow amorphous powder. Compound 2 gave D-glucose on
acid hydrolysis, identified by comparison with an authentic sample by TLC analysis. ESI-MS: m/z
479.0 [M+H]+. 1H-NMR (DMSO): δ = 12.61 (s, 1H), 10.83 (s, 1H), 9.80 (s, 1H), 7.94 (d, J = 2.0 Hz,
1H), 7.49 (dd, J = 8.0, 2.0, 1H), 6.91 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 6.21 (d, J = 2.0 Hz,
1H), 5.57 (d, J = 7.2 Hz, 1H), 3.83 (s, 3H), 3.11–3.58 (d, J = 11.5 Hz, 2H), 3.11–3.58 ppm (m, 1H);
13C-NMR (DMSO): δ = 177.38 (C-4), 164.12 (C-7), 161.20 (C-5), 156.35 (C-9), 156.25 (C-2), 149.37
(C-4'), 146.85 (C-5'), 132.93 (C-3), 122.00 (C-2'), 121.04 (C-1'), 115.18 (C-3'), 113.46 (C-6'), 104.01
(C-10), 100.75 (C-1 of Glc), 98.68 (C-6), 93.67 (C-8), 77.43 (C-5 of Glc), 76.38 (C-2 of Glc), 74.31
(C-4 of Glc), 69.79 (C-3 of Glc), 60.56 (C-6 of Glc), 55.64 ppm (C-21 of OCH3). The HMBC
spectrum revealed significant correlations of H-6 with C-10; H-8 with C-9 and C-10; H-2' with C-4';
H-3' with C-1' and C-5'; H-5' with C-2' and C-4'; H-1'' with C-3; 5-OH with C-5, C-6 and C-10; 7-OH
with C-6, C-7 and C-8; 20-OH with C-3' and C-4'. Based on the above evidence and detailed analysis
of the NMR spectra, the structure of compound 2 was determined as isorhamnetin-3-O-β-D-
glucopyranoside, which had been isolated from Eupatorium tinifolium [16].
Compound 3 was obtained as a colorless oily solid. Its molecular formula was established as
C16H22O4 by means of ESI-MS m/z 279.1 [M+H]+. 1H-NMR (CDCl3): δ = 7.71 (m, 1H), 7.71 (m, 1H),
7.52 (m, 1H), 7.52 (m, 1H), 4.30 (t, J = 6.7 Hz, 2H), 1.72 (m, 2H), 1.43 (m, 2H), 0.96 ppm (t,
13
J = 7.5 Hz, 3H); C-NMR (CDCl3): δ = 167.71 (C-7), 132.32 (C-1, C-2), 130.91 (C-4, C-5), 128.84
(C-6), 65.56 (C-8), 30.58 (C-9), 19.18 (C-10), 13.72 ppm (C-11). The HMBC spectrum revealed
significant correlations of H-4 with C-2 and C-3; H-5 with C-1 and C-6; H-6 with C-5 and C-7; H-8
with C-7, C-9 and C-10; H-9 with C-8, C-10 and C-11; H-10 with C-8, C-9 and C-11; H-11 with C-9
and C-10, which supported the proposed structure of compound 3. Therefore, the structure of
compound 3 was identified as dibutylphthalate, which had been synthesized [17].
Compound 4 was obtained as a white amorphous powder. Compound 4 gave D-glucose on acid
hydrolysis, identified by comparison with an authentic sample by TLC analysis. ESI-MS: m/z 540.2
[M+NH4]+. 1H-NMR (DMSO): δ = 7.05 (d, J = 8.5 Hz, 1H), 6.95 (d, J = 1.8 Hz, 1H), 6.86 (dd, J = 8.6,
1.8 Hz, 1H), 6.74 (d, J = 1.2 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 6.55 (dd, J = 8.0, 1.2 Hz, 1H), 4.86 (d,
J = 7.6 Hz, 1H), 4.69 (m, 1H), 3.87 (t, J = 7.7 Hz, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.64 (m, 1H), 3.55 (t,
J = 7.7 Hz, 1H), 3.47 (m, 1H), 3.10–3.30 (m, 4H), 2.78 (dd, J = 13.2, 4.4 Hz, 1H), 2.55 (m, 1H), 2.40
(dd, J = 13.2, 11.2 Hz, 1H), 2.18 (m, 1H); 13C-NMR (DMSO): δ = 155.06 (C-5'), 148.74 (C-3'), 147.43
(C-3), 145.51 (C-4'), 144.55 (C-4), 137.67 (C-1'), 131.66 (C-1), 120.55 (C-6), 117.73 (C-6'), 115.36
(C-5), 112.70 (C-2), 110.20 (C-2'), 100.16 (C-1 of Glu), 81.60 (C-7'), 76.99 (C-2 of Glu), 76.84 (C-3
of Glu), 73.21 (C-4 of Glu), 71.83 (C-9), 69.68 (C-5 of Glu), 60.66 (C-6 of Glu), 58.58 (C-9'), 55.66
(C-OMe), 55.54 (C-OMe), 52.47 (C-8'), 41.93 (C-8), 32.08 ppm (C-7). The HMBC spectrum revealed
significant correlations of H-2 with C-4 and C-6; H-5 with C-1 and C-3; H-6 with C-2 and C-4; H-2'
with C-1' and C-3'; H-5' with C-1', C-3' and C-4'; H-6' with C-4'; H-7' with C-9, C-2', C-8' and C-9';
H-1''with C-4'. Based on the above evidence and detailed analysis of the NMR spectra, the structure of

Molecules 2012, 17
4601
compound 4 was identified as (+)-1-hydroxyl pinoresinol-4'-β-D-glucopyranoside, which had been
isolated from another plant, Saussurea japonica [18].
Compound 5 was obtained as a light yellow amorphous powder. Compound 5 gave D-glucose on
acid hydrolysis, identified by comparison with an authentic sample by TLC analysis. ESI-MS: m/z
538.0 [M+NH4]+. 1H-NMR (DMSO): δ = 8.76 (dd, J = 8.2, 1.2Hz, 1H), 8.73 (d, J = 8.3 Hz, 1H), 7.05
(d, J = 8.5 Hz, 1H), 6.95 (d, J = 1.8 Hz), 6.89 (d, J = 1.1 Hz, 1H), 4.88 (d, J = 3.3 Hz, 1H), 4.67 (d,
J = 3.6 Hz, 1H), 4.61 (d, J = 4.1 Hz, 1H), 4.14 (m, 1H), 3.77 (s, 6H), 3.75 (m, 1H), 3.66 (d, J = 11.9 Hz,
13
1H), 3.45 (dd, J = 11.9, 4.5 Hz, 1H), 3.28 (m, 1H), 3.18 (m, 1H), 3.05 ppm (m, 1H); C-NMR
(DMSO): δ= 148.88 (C-3'), 147.48 (C-3), 145.88 (C-4), 145.78 (C-4'), 135.14 (C-1'), 132.11 (C-1),
118.60 (C-6), 118.08 (C-6'), 115.09 (C-5'), 110.45 (C-2'), 110.32 (C-2), 100.06 (C-1 of Glu), 85.13
(C-7), 84.82 ( C-7'), 76.98 (C-5 of Glu), 76.81 (C-3 of Glu), 73.15 (C-2 of Glu), 70.97 (C-9), 69.61
(C-4 of Glu), 60.61 (C-6 of Glu), 55.62 (C-OMe), 55.53 (C-OMe), 53.67 (C-8'), 53.51 ppm (C-8). The
HMBC spectrum revealed significant correlations of H-2 with C-1, C-3, C-4, C-6 and C-7; H-7 with
C-1, C-2, C-6, C-8 and C-9; H-9 with C-7 and C-8; H-2' with C-1', C-3', C-4', C-6' and C-7'; H-5' with
C-1', C-3', C-4' and C-6'; H-6' with C-2', C-4', C-5' and C-7'; H-7' with C-1', C-2', C-6', C-8' and C-9';
H-9' with C-7' and C-8'; H-1'' with C-4'. Thus, the structure of compound 5 was identified as
pinoresinol-4'-O-β-D-glucopyranoside, which had been isolated from the plant of Rhus javanica var.
roxburghiana [19].
1
Compound 6 was obtained as a white amorphous powder. ESI-MS: m/z 425.142 [M+Na]+. H-
NMR (acetone d6): δ = 7.27–7.4 (m, 1H), 4.46 (d, J = 7.6 Hz, 1H), 4.46 (d, J = 5.7 Hz, 1H), 4.10 (d,
J = 11.1, 2.3 Hz, 1H), 3.85 (m, 1H), 3.74 (d, J = 11.1, 5.9 Hz, 1H), 3.68 (m, 1H), 3.61 (m, 1H), 3.54
13
(m, 1H), 3.48–3.51 (m, 1H), 3.37–3.43 (m, 1H), 3.27 ppm (m, 1H); C-NMR (DMSO): δ = 138.4
(C-1), 130.4 (C-2, C-3, C-5, C-6), 130.2 (C-4), 105.3 (C-1''), 103.0 (C-1'), 77.3 (C-3'), 76.6 (C-5'), 74.8
(C-2''), 74.7 (C-2'), 73.2 (C-7), 70.9 (C-4'), 70.3 (C-6'), 66.8 ppm (C-5''). Compound 6 was identified
as 2-phenylethyl-β-primeveroside, which had been synthesized [20]. It is reported here as an isolated
natural product for the first time.
3.4. Acid Hydrolysis of Compound 1
Compound 1 (5.0 mg) was dissolved in 5.0 mL of 2 N HCl, heated at 90 °C for 2 h, and then
partitioned between ethyl acetate and water. Aglycon isorhametin was recovered from the ethyl acetate
layer and idertified by direct comparison with an authentic sample. The water layers identified by
comparison with an authentic sample by TLC analysis. Sugars liberated from compound 1 were
identified as arabinose and glucose [21].
3.5. Antisepsis Activity
Antifungal activities were assayed using amphotericin as positive control. Four fungi (Rhizoctonia
cerealis, Botrytis cinerrea, Thanatephorus cucumeris, Valsa mali) were obtained from the Microbe
Laboratory of Northwest Sci-Tech University of Agriculture and Forestry, and were identified by
Li Xiao-Ming, senior laboratorian. The growth of the hyphae method was adopted to study the
compound’s antifungal activity [22]. Every treatment was repeated three times, after cultivation for 72 h
at 28.8 °C in constant temperature culture box.

Molecules 2012, 17
4602
4. Conclusions
In this paper, one new flavonol glycoside, one natural product isolated for the first time and other
phenolic compounds were isolated from the Callianthemum genus. The 2-D NMR data of four known
compounds 2–4 were first given. The compounds 1 and 4 exhibited certain antifungal activity. This
research provides strong support for further study of Callianthemum taipaicum.
Acknowledgements
This work was supported by the program from the Forestry Research Foundation for the Public
Service Industry of China (200904004) and the Program for Youth Backbone Talents of Northwest
A&F University (Z111020902).
References
1. Samuelsson, G. Drugs of Natural Origin: A Textbook of Pharmacognosy, 5th ed.; Swedish
Pharmaceutical Press: Stockholm, Sweden, 2004.
2. Cordell, G.A. Changing strategies in natural products chemistry. Phytochemistry 1995, 40,
1585–1612.
3. Delang, C.O. The role of medicinal plants in the provision of health care in Lao PDR. J. Med.
Plants Res. 2007, 1, 50–59.
4. Balunas, M.J.; Kinghorn, A.D. Drug discovery from medicinal plants. Life Sci. 2005, 78, 431–441.
5. Ma, N.X. Taibai Mountain the Peak of Qinling Mountains; Shaanxi Science and Technology
Press: Xi'an, China,1982.
6. Ren, Y. Taibai Mountain Nature Reserve Biodiversity Research and Management; China Forestry
Publishing House: Beijing, China, 2006.
7. Institute of Botany. Qinling Flora; Chinese Science Press: Beijing, China, 1974.
8. Wang, X.L.; Liu, B.R.; Chen, C.K.; Wang, J.R. Four new fluorenone alkaloids and one new
dihydroazafluoranthene alkaloid from Caulophyllum robustum Maxim. Fitoterapia 2011, 82,
793–797.
9. Rodriguez, E.; Shen, M.C.; Mabry, T.J.; Domfnguez, X.A. Isorhamnetin-3-O-glucoside-7-O-
arabinoside from Eschscholzia mexicana. Phytochemistry 1973, 8, 2069–2071.
1
10. Markham, K.; Geiger, H. H nuclear magnetic resonance spectroscopy of flavonoids and their
glycosides in hexadeuterodimethylsulfoxide. In The Flavonoids; Chapman & Hall: London, UK,
1994; pp. 441–498.
11. Rosch, D.; Krumbein, A.; Mugge, C.; Kroh, L.W. Structural investigations of flavonol glycosides
from sea buckthorn (Hippopha rhamnoides) pomace by NMR spectroscopy and HPLC-ESI-MS.
J. Agric. Food Chem. 2004, 13, 4039–4046.
12. Zou, Y.P.; Tan, C.H.; Wang, B.D.; Jiang, S.H.; Zhu, D.Y. Flavonoid Glycosides from Ranunculus
chinensis Bge. Helv. Chim. Acta 2007, 10, 1940–1945.
13. Vitalini, S.; Braca, A.; Passarella, D.; Fico, G. New flavonol glycosides from Aconitum burnatii
Gáyer and Aconitum variegatum L. Fitoterapia 2010, 7, 940–947.

Molecules 2012, 17
4603
14. Xu, C.L.; Zhang, Y.N.; Wang, J.Y.; Li, X.; Sun, G.Z. The structure of two flavonol glycosides
from leaves of Salix Raddeana Laksch. Chin. J. Anal. Chem. 2004, 12, 1663–1666.
15. Kim, H.Y.; Moon, B.H.; Lee, H.J.; Choi, D.H. Flavonol glycosides from the leaves of Eucommia
ulmoides O. with glycation inhibitory activity. J. Ethnopharm. 2004, 93, 227–230.
16. D’Agostino, M.; Simone, F.D.; Dine, A.; Ramundo, E.; Zollo, F. Flavonol glycosides from
Eupatorium tinifolium. Phytochemistry 1990, 1, 353–354.
17. McNulty, J.; Nair, J.J.; Cheekoori, S.; Larichev, V.; Capretta, A.; Robertson, A.J. Scope and
mechanistic insights into the use of tetradecyl (trihexyl) phosphonium bistriflimide: A remarkably
selective ionic liquid solvent for substitution reactions. Chem. A Eur. J. 2006, 36, 9314–9322.
18. Shi, Y.P.; Ma, J. Identification the structure of lignan glycosides of Saussurea and research the
law of replacement of the carbon chemical shift displacement. Zhong Cao Yao 2002, 9, 772–775.
19. Ouyang, M.A.; Wein, Y.S.; Zhang, Z.K.; Kuo, Y.H. Inhibitory activity against tobacco mosaic
virus (TMV) replication of pinoresinol and syringaresinol lignans and their glycosides from the
root of Rhus javanica var. roxburghiana. J. Agric. Food Chem. 2007, 16, 6460–6465.
20. Tsuruhami, K.; Mori, S.; Sakata, K.; Amarume, S.; saruwatari, S.; Murata, T.; Usui, T. Efficient
Synthesis of Primeverosides as Aroma Precursors by Transglycosylation of Diglycosidase from
Penicillium multicolor. J. Carbohydr. Chem. 2005, 24, 849–863.
21. Oshima, R.; Yamauchi, Y.; Kumanotani, J. Resolution of the enantiomers of aldoses by liquid
chromatography of diastereoisomeric 1-(N-acetyl-[α]-methylbenzylamino)-1-deoxyalditol acetates.
Carbohydr. Res. 1982, 2, 169–176.
22. Hsu, F.L.; Chen, P.S.; Chang, H.T.; Chang, S.T. Effects of alkyl chain length of gallates on their
antifungal property and potency as an environmentally benign preservative against wood-decay
fungi. Int. Biodeterior. Biodegrad. 2009, 5, 543–547.
Sample Availability: Samples of the compounds are available from the authors.
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