Two new flavone C-glycosides from Trollius ledebourii

Article abstract of DOI:10.1248/cpb.59.1393

Two new flavone C-glycosides, trollisin A (1) and trollisin B (2), along with seven known flavonoids, isoswertisin (3), isoswertiajaponin (4), orientin (5), 2″-O-β-L-galactopyranosylvitexin (6), 2″-O-β-L- galactopyranosylorientin (7), neodiosmin (8) and acacetin-7-O-neohesperidoside (9) were isolated from the flowers of Trollius ledebourii REICHB. The structures of the new compounds were elucidated based on spectral analysis, including MS, 1D- and 2D-NMR experimentation.

Full text of DOI:10.1248/cpb.59.1393

November 2011
Note
Chem. Pharm. Bull. 59(11) 1393—1395 (2011)
1393
Two New Flavone C-Glycosides from Trollius ledebourii
Li-Zhen WU, Hai-Feng WU, Xu-Dong XU,* and Jun-Shan YANG
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College;
Beijing 100193, P. R. China. Received February 24, 2011; accepted July 5, 2011; published online August 15, 2011
Two new flavone C-glycosides, trollisin A (1) and trollisin B (2), along with seven known flavonoids, isoswer-
tisin (3), isoswertiajaponin (4), orientin (5), 2ꢀ-O-b-L-galactopyranosylvitexin (6), 2ꢀ-O-b-L-galactopyranosyl-
orientin (7), neodiosmin (8) and acacetin-7-O-neohesperidoside (9) were isolated from the flowers of Trollius
ledebourii REICHB. The structures of the new compounds were elucidated based on spectral analysis, including
MS, 1D- and 2D-NMR experimentation.
Key words Trollius ledebouri; flavone C-glycoside; trollisin A; trollisin B
Trollius ledebourii REICHB (Ranunculaceae) is a herb ety, which was further confirmed by the correlation of the
(40—100 cm tall) found in damp grassland and widely dis- glucosyl anomeric proton H-1ꢅ (d 4.83, d, Jꢁ10.2 Hz) with
tributed in the north-eastern regions of China. Its flowers, carbon signals at dC 103.6 (C-8), 162.5 (C-7) and 156.2 (C-
called “Jin Lian Hua” in Chinese, are well known for their 9) in the heteronuclear multiple bond connectivity (HMBC)
use as a traditional folk medicine for the treatment of otitis spectrum (Fig. 2). From these data, 1 had a similar pattern to
media acuta, conjunctivitis and upper respiratory tract infec- the known compound 2ꢅ-O-(3ꢆ,4ꢆ-dimethoxybenzoyl) vi-
tion.1) Previous studies on this species have revealed the pres- texin.3) But the signal of C-2ꢅ (dC 81.2) showed a downfield
ence of flavonoids and organic acids, some of which were re- shift of 8.4 ppm compared with the corresponding data (dC
ported to exhibit antiviral, antibacterial and antioxidant activ- 72.8) of 2ꢅ-O-(3ꢆ,4ꢆ-dimethoxybenzoyl) vitexin, which indi-
ities.2) As a follow-up study on the chemical constituents of cated the middle sugar was attached to the C-2ꢅ. This was
T. ledebourii, two new flavone C-glycosides (1, 2) together also confirmed by the long-range correlation between C-1ꢆ
with seven known flavonoids have been isolated.
(dC 105.5) and H-2ꢅ (dH 4.08). Moreover, in the HMBC
spectrum, observation of the cross peaks from H-6ꢅ (dH 3.88,
3.55) to C-7ꢆꢅ (dC 164.8) of the dimethoxybenzoyl moiety
Results and Discussion
Compound 1 was obtained as yellow powder and exhibited suggested that the 1,3,4-trisubstituted aromatic ring was con-
a positive magnesium hydrochloric acid test, indicating a nected to the C-6ꢅ; correlations from H-1ꢅꢅ (dH 4.15) to C-6ꢆ
flavonoid. [a]D20 ꢀ17.1 (cꢁ0.645, MeOH). The molecular (dC 69.1) and from H-6ꢆ (dH 4.21, 3.58) to C-1ꢅꢅ (dC 103.2)
formula was deduced to be C42H48O23 by positive mode high indicated that the terminal sugar was attached to C-6ꢆ. Acid
resolution-electrospray ionization (HR-ESI)-MS data at m/z hydrolysis of 1 yielded D-glucose and L-galactose, which
943.2484 [MꢂNa]ꢂ (Calcd for C42H48O23Na, 943.2484). The
UV spectrum showed lmax (MeOH) (log e) at 217 (4.45), 267
(4.14), 296 (3.97), and 332 (3.96) nm characteristic of a
flavone. The IR spectrum revealed the presence of hydroxy
(3355 cmꢀ1), carbonyl (1653 cmꢀ1) and aromatic groups
1
(1605, 1577, 1512, 1438 cmꢀ1). The H-NMR spectrum of 1
(Table 1) showed resonances at dH 7.92 (2H, d, Jꢁ8.4 Hz)
and 6.94 (2H, d, Jꢁ8.4 Hz), suggesting the presence of an
AAꢃBBꢃ system on the B-ring, corresponding to the protons
H-2ꢃ, 6ꢃ and H-3ꢃ, 5ꢃ, respectively. Two singlets at dH 6.68
R1
R2
R3
R4
and 6.24 were due to the protons at C-3 and C-6 in rings C
and A of a flavone, respectively. In addition, signals due to
two aromatic methoxy groups at dH 3.84 (3H, s) and 3.80
(3H, s) and an ABX system [dH 7.50 (1H, dd, Jꢁ8.4,
1.8 Hz), dH 7.38 (1H, d, Jꢁ1.8 Hz), dH 7.03 (1H, d,
Jꢁ8.4 Hz)] corresponding to a 1, 3,4-trisubstituted aromatic
ring were also revealed, indicating the presence of a
dimethoxybenzoyl group. The 13C-NMR spectrum (Table 1)
revealed 38 carbon signals, indicating the presence of a
flavonoid moiety, three saccharide moieties and a dimethoxy-
1
2
3
4
5
6
7
H
CH3
CH3
CH3
H
H
H
H
OH
OH
H
Glc (1→6) Gal
veratroyl
Glc
H
H
H
H
H
Gal
Gal
H
H
H
H
H
H
OH
1
benzoyl moiety in 1. From analysis of the H- and 13C-NMR
data of the sugar moieties, 1 was deduced to be a flavone C-
glycoside and three sugar moieties were determined to be b
(JH of anomeric carbons ꢄ7.0 Hz). Signals at dC 98.3 (C-6),
103.6 (C-8) and 80.1 (C-5ꢅ) revealed that the site of the sugar
linkage in 1 should be at the C-8 position of the aglycon moi-
Fig. 1. Structures of Compounds 1—9
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: xdxu@implad.ac.cn

1394
Vol. 59, No. 11
Table 1. 1H- (600 MHz) and 13C-NMR (150 MHz) Data of Compounds 1
and 2a,b)
1.
1 (DMSO-d6)
2 (DMSO-d6)
2.
No.
dH
dC
dH
dC
2
3
4
5
6
7
8
9
10
1ꢃ
2ꢃ
3ꢃ
4ꢃ
163.5
102.7
181.8
160.6
98.3
162.5
103.6
156.2
103.6
121.7
128.6
115.8
161.1
115.8
128.6
164.3
102.5
182.3
161.4
95.0
163.3
105.5
155.1
104.4
121.4
128.8
115.9
161.3
115.9
128.8
56.5
6.68 s
6.24 s
6.83 s
6.52 s
Fig. 2. Key HMBC Correlations of Compounds 1 and 2
a positive magnesium hydrochloric acid test, indicating a
flavonoid. [a]D20 ꢂ2.18 (cꢁ0.046, MeOH). The molecular
formula was established to be C28H32O15 by HR-ESI-MS at
m/z 631.1639 [MꢂNa]ꢂ (Calcd for C28H32O15Na, 631.1624).
The UV spectrum showed lmax (MeOH) (log e) at 214 (4.36),
269 (4.08) and 333 (4.08) nm characteristic of a flavone. The
IR spectrum showed the presence of hydroxy (3326 cmꢀ1),
carbonyl (1653 cmꢀ1) and aromatic (1602, 1575, 1447 cmꢀ1)
7.92 (d, 8.4)
6.94 (d, 8.4)
7.99 (d, 8.4)
6.95 (d, 8.4)
5ꢃ
6ꢃ
7-OCH3
6.94 (d, 8.4)
7.92 (d, 8.4)
6.95 (d, 8.4)
7.99 (d, 8.4)
3.88 s
Glc
Gal
Glc
1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
6ꢅ
4.83 (d, 10.2)
4.08 (t, 9.0)
3.56 m
3.24 m
3.46 m
71.5
81.2
78.4
71.2
80.1
61.5
4.72 (d, 10.2)
3.86 (t, 9.0)
3.27 (t, 8.4)
3.40 m
73.2
70.7
78.4
70.6
80.2
1
groups. The H-NMR spectrum of 2 (Table 1) indicated the
presence of an AAꢃBBꢃ system [dH 7.99 (2H, d, Jꢁ8.4 Hz),
6.95 (2H, d, Jꢁ8.4 Hz)] on the B-ring, the same as 1. The
13C-NMR spectrum of 2 (Table 1) revealed 28 carbon sig-
nals, of which there were two sugar moieties and a methoxyl
group besides the flavone skeleton. According to above men-
tioned data, Compound 2 was determined to have an analo-
gous pattern with the known compound isoswertisin.3) By an-
alyzing the 13C-NMR data with isoswertisin, the signal of C-
6ꢅ (dC 69.3) of 2 was shifted downfield by 8.1 ppm, suggest-
ing that the middle sugar was attached to C-6ꢅ, which was
further confirmed by the correlation between H-1ꢆ (dH 4.14)
and C-6ꢅ (dC 69.3) in the HMBC spectrum (Fig. 2). There-
fore, the structure of 2 was evaluated as shown in Fig. 1,
named trollisin B.
By comparison with the NMR data reported in litera-
ture,3—6) the known compounds 3—9 were identified as
isoswertisin (3), isoswertiajaponin (4), orientin (5), 2ꢅ-O-b-L-
galactopyranosylvitexin (6), 2ꢅ-O-b-L-galactopyranosylori-
entin (7), neodiosmin (8) and acacetin-7-O-neohesperidoside
(9).
3.44 m
3.88 (t, 9.6)
3.55 m
4.20 (br d, 10.2) 69.3
3.56 m
1ꢆ
2ꢆ
3ꢆ
4ꢆ
5ꢆ
6ꢆ
4.03 (d, 7.2)
3.42 m
3.16 m
3.47 m
3.15 m
105.5
70.2
72.6
67.3
71.5
69.1
4.14 (d, 7.8)
2.95 (t, 8.4)
3.08 (t, 8.4)
3.02 m
103.2
73.3
76.8
69.8
76.8
3.03 m
4.21(br d, 10.8)
3.58 m
3.62 (br d, 10.8) 60.9
3.40 m
1ꢅꢅ
2ꢅꢅ
3ꢅꢅ
4ꢅꢅ
5ꢅꢅ
6ꢅꢅ
4.15 (d, 7.8)
2.95 (t, 8.4)
3.09 (t, 8.4)
3.04 m
3.02 m
3.63 (br d, 10.2)
3.40 m
103.2
73.2
76.8
69.9
76.8
60.9
Veratroyl 1ꢆꢅ
121.6
111.8
152.9
148.3
111.0
2ꢆꢅ
3ꢆꢅ
4ꢆꢅ
5ꢆꢅ
6ꢆꢅ
7ꢆꢅ
7.38 (d, 1.8)
7.03 (d, 8.4)
Experimental
General NMR spectra were obtained with a Bruker AV III 600 NMR
spectrometer (chemical shift values are presented as d values with tetra-
methylsilane (TMS) as the internal standard). UV and IR spectra were
recorded on Shimadzu UV2550 and FTIR-8400S spectrometer, respectively.
The optical rotations were obtained in MeOH at 20 °C on a Perkin-Elmer
341 digital polarimeter. HR-ESI-MS spectra were performed on an LTQ-
Obitrap XL spectrometer. GC analysis was carried out on a GC-7890: col-
umn, DB-5 (30 mꢇ0.32 mmꢇ0.25 mm); detector, FID-6850 (Agilent, Cali-
fornia, U.S.A.). C18 reversed-phase silica gel (40—63 mm, Merck, Darm-
stadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel
(CHP 20P, 75—150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan)
and silica gel (100—200 mesh, Qingdao Marine Chemical Plant, Qingdao,
People’s Republic of China) were used for column chromatography. Pre-
coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel
Development, Yantai, People’s Republic of China) were used for TLC. All
solvents used were of analytical grade (Beijing Chemical Plant).
7.50 (dd, 8.4, 1.8) 123.2
164.8
3ꢆꢅ-OCH3 3.84 s
4ꢆꢅ-OCH3 3.80 s
55.7
55.6
a) Chemical shift values were in ppm and J values (in Hz) were presented in paren-
theses. b) The assignments were based on HSQC, HMBC and 1H–1H TOCSY experi-
ments.
were confirmed by PC and GC analysis. 1D- and 2D-nuclear
Overhauser effect (NOE) analysis were also carried out. The
correlation between the proton at dH 4.03 and the proton at
dH 4.08 indicated that the anomeric proton of the middle
sugar was on the same side as the proton of C-2ꢅ. Together
with the correlations of 1ꢆ-H/3ꢆ-H, 5ꢆ-H, the middle sugar
moiety was confirmed to be L-galactose. Thus, the structure
of 1 was established as shown in Fig. 1, named trollisin A.
Compound 2 was obtained as yellow powder and exhibited
Plant Material The flowers of T. ledebourii were collected from
Chengde, Heibei (P. R. China) and identified by Prof. Wen-Yan Lian. A
voucher specimen (HB-02-0128) has been deposited at the Institute of Medi-
cinal Plant Development.
Extraction and Isolation Dried flowers (15 kg) were extracted twice

November 2011
1395
with 95% ethanol. The concentrated extract obtained under reduced pressure
was suspended in water and extracted with petroleum ether, ethyl acetate
the sugar residue and then reacted in anhydrous pyridine (0.5 ml) on a mag-
netic stirrer at 90 °C for 0.5 h in an oil bath. After cooling, acetic anhydride
and 1-butanol, successively. The water fraction (500 g) was subjected to (0.5 ml) was added to the reaction mixture and heated at 90 °C for 0.5 h, then
polyamide (100—200 mesh) column chromatography, and eluted with
MeOH–H2O (0 : 1—1 : 0, v/v) to give 7 fractions. Fraction 2 (50 g) was chro-
matographed on a polyamide column and eluted with MeOH–CHCl3 (0 : 1—
3 : 4, v/v), then purified with CC over silica gel (MeOH–CHCl3) and
Sephadex LH-20 (MeOH) to afford 6 (33 mg) and 7 (24 mg); Fraction 4
(4 g) was isolated by reversed-phase MCI gel and MPLC elution with
MeOH–H2O (50 : 50, v/v), then purified by Sephadex LH-20 (MeOH) to
give 3 (4 mg), 4 (5 mg), 5 (5 mg), 8 (3 mg) and 9 (4 mg) and by preparative
the solution was dried by N2 at 70 °C. The acetylated derivative was dis-
solved in CHCl3 and analyzed by GC. The authentic sugars were processed
using the same method as above. Column temperature 210 °C; injection tem-
perature 240 °C; detection temperature 250 °C; carrier gas N2 at a flow of
1.5 ml/min; D-glucose, L-galactose, 16.0 and 16.4 min, respectively.
Acknowledgment This project was financially supported by the General
Program of Natural Science Foundation of Beijing, China (No. 7082059);
HPLC elution with MeOH–H2O (45 : 55, v/v) to afford 1 (13 mg) and 2 and by the technological large platform for comprehensive research and de-
(2 mg).
Trollisin A (1): Yellow powder; [a]D20 ꢀ17.1 (cꢁ0.645, MeOH); UV lmax
(MeOH) (log e): 217 (4.45), 267 (4.14), 296 (3.97), 332 (3.96) nm; IR (KBr)
velopment of new drugs in the Eleventh Five-Year “Significant New Drugs
Created” Science and Technology Major Projects (No. 2009ZX09301-003).
cmꢀ1: 3355, 2923, 1653, 1605, 1577, 1512, 1438, 1357, 1272, 1227, 1175, References
1076, 1022, 840, 764; HR-ESI-MS m/z: 943.2484 [MꢂNa]ꢂ (Calcd for
C42H48O23Na, 943.2484). 1H- and 13C-NMR data, see Table 1.
1) Jiangsu New Medical College, “The Dictionary of Chinese Herbal
Medicine,” Vol. 1, Shanghai Science and Technology Press, Shanghai,
1997, pp. 1398—1399.
Trollisin B (2): Yellow powder; [a]D20 ꢂ2.18 (cꢁ0.046, MeOH); UV lmax
(MeOH) (log e): 214 (4.36), 269 (4.08), 333 (4.08) nm; IR (KBr) cmꢀ1
:
2) Lin Q. F., Feng S. Q., Li Y. L., Cen Y. Z., Yang Y. T., Wang L. Y., J.
Zhejiang University (Sci. Ed.), 31, 412—415 (2004).
3) Wang R. F., Yang X. W., Ma C. M., Liu H. Y., Shang M. Y., Zhang Q.
Y., Cai S. Q., Park J. H., J. Asian Nat. Prod. Res., 6, 139—144 (2004).
4) Zou J. H., Yang J. S., Dong Y. S., Zhou L., Lin G., Phytochemistry, 66,
1121—1125 (2005).
5) Rio J. A. D., Benavente O., Castillo J., Borrego F., Phytochemistry, 31,
723—724 (1992).
6) Wang Z. Y., Li N., Wang K. J., Dai H. F., J. Plant Res. Environ., 19,
92—94 (2010).
3326, 2923, 1653, 1602, 1575, 1447, 1362, 1244, 1178, 1076, 1019, 833;
HR-ESI-MS m/z: 631.1639 [MꢂNa]ꢂ (Calcd for C28H32O15Na, 631.1624).
1H- and 13C-NMR data, see Table 1.
Determination of Sugar Components Compound 1 (2 mg) in 2%
H2SO4 was heated at 100 °C for 4 h in a water bath. The reaction mixture
was neutralized with Ba2CO3, filtered and then extracted with ethyl acetate.
After concentration, the H2O layer was examined by PC with 1-
butanol–acetic acid–H2O (4 : 1 : 5, choose the upper layer) and compared
with authentic samples. Hydroxylamine hydrochloride (2 mg) was added to