Acylated flavone 8-C-glucosides from the flowers of Trollius chinensis

Article abstract of DOI:10.1016/j.phytol.2018.04.019

Ten new flavone 8-C-glucosides, trollichinensides A–J, with the substitution of various acyls at C-2″ C-3″ or C-6″ of the glucose moiety, together with thirteen known ones were isolated from the flowers of Trollius chinensis. Spectroscopic analyses including NMR and HRESIMS resulted in the establishment of the structures of these constituents and electronic circular dichroism (ECD) experiment was employed for the determination of the absolute configuration of the glucose residue in these molecules.

Full text of DOI:10.1016/j.phytol.2018.04.019

PhytochemistryLetters25(2018)156–162
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Acylated flavone 8-C-glucosides from the flowers of Trollius chinensis
⁎
Jin-Xia Weia,b, Dan-Yi Lib, Zhan-Lin Lia,b,
a
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, 110016, PR
China
b
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, 110016, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ten new flavone 8-C-glucosides, trollichinensides A–J, with the substitution of various acyls at C-2″, C-3″, or C-
6″ of the glucose moiety, together with thirteen known ones were isolated from the flowers of Trollius chinensis.
Spectroscopic analyses including NMR and HRESIMS resulted in the establishment of the structures of these
constituents and electronic circular dichroism (ECD) experiment was employed for the determination of the
absolute configuration of the glucose residue in these molecules.
Trollius chinensis
Ranunculaceae
Flavone-8-C-glucoside
Acyl
Electronic circular dichroism
1. Introduction
vanilloylvitexin (Zou et al., 2005), 2″-O-veratroylvitexin (Zou et al.,
2004), 3″-O-(2‴-methylbutyryl)vitexin (Li et al., 2009), 2″-O-acetylor-
The plants of Trollius genus (Ranunculaceae), which are mainly
distributed in north China and widely used as folk medicines for the
treatment of upper respiratory infections, are rich in organic acids,
flavone C-glycosides, and the assembly of these two structural moieties,
acylated flavone 8-C-glycosides, which have been isolated from T. le-
debouri (Wu et al., 2006, 2009, 2011a; Zou et al., 2004, 2005) and T.
chinensis (Cai et al., 2006; Li et al., 2009), as well as some pheny-
lethanols (Liu and Luo, 2010; Wu et al., 2011a, 2011b), alkaloids
(Wang et al., 2004a), and diterpenoids (Zou et al., 2006). A preliminary
phytochemical investigation by us into the flavone 8-C-glycosides from
T. chinensis led to the isolation of six ones (Li et al., 2009). While,
considering of the existence of four flavone 8-C-glucosides including
vitexin, orientin, isoswertisin, and isoswertiajaponin in T. chinensis
(Wang et al., 2004b), together with various organic acids like veratric
acid, benzoic acid, vanillic acid, and 4-hydroxybenzoic acid, the
structural diversity of the acylated flavone 8-C-glucosides in T. chinensis
was subjected to reexamination, resulting in ten new acylated flavone
8-C-glucosides and thirteen known ones whose isolation and structural
elucidation are reported as follows.
ientin (Kato and Morita, 1990), 2″-O-(2‴-methylbutyryl)orientin (Zou
et al., 2004), 2″-O-veratroylorientin (Zou et al., 2004), 6″-O-acetylor-
ientin (Hori et al., 1987), 2″-O-(2‴-methylbutyryl)isoswertisin (Zou
et al., 2004), 3″-O-(2‴-methylbutyryl)isoswertisin (Zou et al., 2004),
2″-O-(2‴-methylbutyryl)isoswertiajaponin (trollisin I) (Cai et al., 2006;
Wu et al., 2006), and 2″-O-veratroylisoswertiajaponin (trollisin II) (Cai
et al., 2006) which were identified by comparison of their NMR data
with those reported.
The issue needs to be addressed in the structural elucidation of
acylated flavone 8-C-glucosides from Trollius genus by NMR technique
is the substituting position of acyl moieties. Till now, acylation has
occurred at C-2″, 3″, and 6″ of glucose. Although HMBC is the most
accurate spectroscopic method to assign the acyl, proton signal on the
acylated carbon of glucose measured in deuterated dimethyl sulfoxide
can be employed as another indicator. The chemical shifts of H-2″ of C-
2″-acylated flavone 8-C-glucosides were observed at the range from δH
5.30–5.50 as a triplet with the coupling constant of 9–10 Hz, and the
resonances for H-3″ of C-3″-acylated ones were at δH 4.85–5.10 as a
triplet. The 6″-acylated flavone 8-C-glucosides could be readily identi-
fied from the resonances for H-6″ at δH 4.00–4.50 or for C-6″ at about δC
64.5. What’s more interesting, the veratroyl (Li et al., 2009; Zou et al.,
2004), vanilloyl (Zou et al., 2005), or 4-hydroxylbenzoyl connected to
C-2″, or feruloyl linked to C-3″of the glucose moiety of flavone-8-C-
glucoside exhibited long-range shielding effect leading to the up-field
shift of H-6 by about 0.1–0.2 chemical shift. For instance, H-6 of vitexin
at δH 6.26 (Wang et al., 2004b) up-field shifted to δH 6.09 (Zou et al.,
2004) and H-6 for isoswertisin at δH 6.52 (Wang et al., 2004b) shifted to
2. Results and discussion
Detailed separation and purification by chromatographic methods
on the ethyl acetate extract of the flowers of T. chinensis yielded ten new
flavone 8-C-glucosides (1–10, Fig. 1) and thirteen known ones in-
cluding 2″-O-(2‴-methylbutyryl)vitexin (Zou et al., 2004), 2″-O-(4‴-
hyroxybenzoyl)vitexin (Brum-Bousquet et al., 1977), 2″-O-
⁎
Corresponding author at: Shenyang Pharmaceutical University, Box 81, Shenyang, 110016, PR China.
E-mail address: lzl1030@hotmail.com (Z.-L. Li).
https://doi.org/10.1016/j.phytol.2018.04.019
Received 27 January 2018; Received in revised form 3 March 2018; Accepted 3 April 2018
1874-3900/©2018PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

J.-X. Wei et al.
PhytochemistryLetters25(2018)156–162
Fig. 1. Structures of flavone-8-C-glucosides from Trollius chinensis.
δH 6.33 (Li et al., 2009) when a veratroyl was connected to C-2″.
Compound 1, an amorphous yellow powder, exhibited the mole-
cular formula of C30H28O13 determined from HRESIMS (m/z 597.1601
[M+H]+, 619.1423 [M+Na]+). 1H NMR of 1 displayed the chelated
phenolic hydroxyl at C-5 at δH 13.16 (1H, brs) and H-3 proton signal at
The following three compounds including 5, 6, and 7 had the mo-
lecular formulae of C23H22O12 (HRESIMS m/z 491.1188 [M+H]+),
C
26H28O12 (m/z 533.1653 (M+H)+), and C30H28O14 (m/z 613.1545 [M
+H)+), respectively. All of these three compounds were assigned to be
3″-O-acylated orientin derivatives for their 1H NMR data, especially the
H-3″ for compounds 5 at δH 4.84 (1H, t, J = 9.2 Hz), 6 at δH 4.88 (1H, t,
δ
H 6.79 (1H, s), which were characteristic markers for flavone skeleton
instead of flavonol, and an AA′BB′ coupling system at ring-B composed
of two sets of doublet signals at δH 8.06 (2H, d, J = 8.5 Hz, H-2′, 6′) and
6.94 (2H, d, J = 8.5 Hz, H-3′, 5′). Additionally, the chemical shifts for
H-6 at δH 6.25 and C-6 at δ 98.4, as well as the anomeric proton at δH
4.86 (1H, d, J = 9.8 Hz) and the six oxygenated aliphatic carbons at δC
73.6, 68.8, 80.4, 68.6, 81.6, and 60.8 assigned 1 a vitexin scaffold. The
acyl moiety in 1 was determined to be veratroyl from the proton signals
of an ABX system at δH 7.50 (1H, brd, J = 8.3 Hz), 7.37 (1H, brs), and
7.03 (1H, d, J = 8.3 Hz) and two methoxyl group signals at δH 3.79 and
3.71. Furthermore, the characteristic proton at δH 5.10 (1H, t,
J = 9.6 Hz) was assigned to H-3″ and its long-range correlation with the
carbonyl of veratroyl moiety at δC 165.1 established the structure of 1
as 3″-O-veratroylvitexin, given the trivial name of trollichinenside A.
Compound 2, with the molecular formula of C31H28O13 determined
from the HRESIMS (m/z 609.1601 [M+H]+), was obtained as an
amorphous yellow powder. 1H and 13C NMR spectra of 2 displayed the
3″-O-acylated vitexin structure, as well as the proton and carbon signals
for E-feruloyl moiety including the trans-coupling olefinic protons at δH
7.48 and 6.46 (J = 15.9 Hz). NOESY experiment was used to assign the
methoxy group on C-3‴ of the feruloyl group. Finally, 2 was determined
to be 3″-O-feruloylvitexin, named trollichinenside B.
Based on the quasi-molecular ion peak in HRESIMS at m/z 597.1600
[M+H]+, compound 3, a yellow amorphous powder, was given a
molecular formula of C30H28O13 and assigned a acylated vitexin deri-
vative from the 1H and 13C NMR data. The acyl moiety of 3 could be
unambiguously deduced to be veratroyl from the ABX coupling aro-
matic protons at δH 7.40 (1H, dd, J = 8.4, 1.9 Hz), 7.32 (1H, d,
J = 1.9 Hz), and 6.82 (1H, d, J = 8.4 Hz), as well as two methoxyl
groups at δH 3.77 and 3.69. Additionally, the resonances for H-6″ at δH
4.52 (1H, d, J = 11.5 Hz) and 4.33 (1H, m), and C-6″ at δC 64.7 showed
the substitution of veratroyl at C-6″ of glucose. Consequently, the
J = 9.2 Hz), and 7 at δH 5.09 (1H, t, J = 8.6 Hz), respectively.
Furthermore, the acyls in these three compounds were identified as
acetyl for compound 5 (δH 1.97 in 1H and δC 169.9 and 21.2 in 13C
NMR), 2-methylbutyryl for 6 (δC 175.4, 40.4, 26.4, 16.6, and 11.3 in
13C NMR), and veratroyl for 7 (δC 165.1, 152.6, 148.2, 123.2, 122.7,
111.8, 111.0, 55.7, and 55.4 in 13C NMR). Finally, HMBC experiments
unambiguously determined the structures for compounds 5 as 3″-O-
acetylorientin (trollichinenside E), 6 as 3″-O-(2‴-methylbutyryl)or-
ientin (trollichinenside F), and 7 as 3″-O-veratroylorientin (trollichi-
nenside G).
The molecular formula of C32H30O13 was give to compound 8, on
the basis of its HRESIMS data (m/z 623.1762 [M+H]+). 8 was de-
termined to be an isoswertisin derivative (Wang et al., 2004b) by
analyses on 1H and 13C NMR data. Furthermore, the proton resonance
for H-2″ at δH 5.43 (1H, t, J = 9.6 Hz) and the NMR data for a feruloyl
moiety, as well as detailed NOESY and HMBC analyses, established the
structure of compound 8 as 2″-O-feruloylisoswertisin, given the trivial
name of trollichinenside H.
The molecular formulae of compounds 9 and 10 were established as
C
29H26O12 (HRESIMS m/z 567.1485 [M+H]+) and C32H30O14 (m/z
639.1709 [M+H]+), respectively. These two compounds could be de-
termined to be 2″-O-acylated isoswertiajaponin derivatives since the
protons of an ABX coupling system on ring-B of flavone skeleton were
observed in the 1H NMR spectra and one methoxyl group signal was
observed to display NOESY correlation with the proton singlet of H-6.
Furthermore, 9 was determined to be 2″-O-benzoylisoswertiajaponin
(trollichinenside I) for the proton signals of a benzoyl moiety at δH 7.68
(2H, d, J = 7.3 Hz), 7.57 (1H, m), and 7.43 (2H, t, J = 7.7 Hz), and
compound 10 to be 2″-O-feruloylisoswertiajaponin (trollichinenside J)
by NMR analyses.
Although the sugar moiety of all these flavone-8-C-glycosides could
be readily determined as glucose with a β-configuration for the glyco-
sidic bond judged by the 13C NMR data and coupling constant of the
anomeric proton, the issue of the absolute configuration of the glucose
moiety in these molecules remained unsolved.
There has been one report on the establishment of the glucose in C-
glucosides as d-form by oxidative hydrolysis with ferric chloride fol-
lowed by GC detection (Shimamura et al., 2006). However, a non-
consumable approach would be preferred by the chemists focusing on
natural products. Around 1980s, Dr. Gaffield reported the application
structure of compound
3 could be elucidated to be 6″-O-vera-
troylvitexin, with the trivial name of trollichinenside C.
The 1H and 13C NMR spectra of compound 4 with the molecular
formula of C29H26O14 determined from HRESIMS (m/z 599.1390 [M
+H]+), displayed signals for orientin and vanilloyl. The characteristic
proton triplet for H-2″ at δH 5.49 (1H, t, J = 9.7 Hz), together with its
HMBC correlation with the carbonyl carbon of vanilloyl group at δC
164.7, established 4 as 2″-O-vanilloylorientin, named trollichinenside
D.
157

J.-X. Wei et al.
PhytochemistryLetters25(2018)156–162
Fig. 2. Rotational conformers of flavone 8-C-β-D-glucoside and 6-C-β-D-glucoside and their projections in quadrant sector diagram.
of electronic circular dichroism (ECD) experiment to determine the
glycosidation position of flavone 6 or 8-C-glycosides according to their
Cotton effect at 250–275 nm (Gaffield et al., 1978), and further the
absolute configuration of the anomeric carbon of arabinose residues
(Gaffield et al., 1984), providing an alternative way to determine the
absolute configuration of the glucose moiety in flavone-8-C-glucosides.
First of all, the sign of the ECD band (250–275 nm) related to the
charge-transfer transition of ring-A and 4-carbonyl (Gaffield et al.,
1978) was determined by whichever sector was predominantly occu-
pied by the asymmetric carbon nearest to the glycosidic bond (C-2″) in
the glucose moiety attached to C-6 or C-8 of the flavone, in the pos-
tulated quadrant viewing from the 4-carbonyl to C-10 (Fig. 2). The
second issue to be addressed was the existence of two rotational con-
formers for flavone 6-C-glucosides (conformers I and II in Fig. 2) or 8-C-
glucosides (III and IV in Fig. 2) with the plane of the sugar moiety
nearly perpendicular to ring-A, which should be differentiated by
NOESY experiment before the analysis on the ECD data. Finally, the
conformer established was put into the quadrant to analyze the sector
contribution and correlated with the ECD data.
no NOE correlation observed between the protons in the sugar moiety
and those on ring-A in the NOESY spectrum, isoorientin was judged to
adopt predominantly conformation I (Fig. 2) with reference to the
crystal structure reported for swertisin (Ohba et al., 2004). Therefore,
D-configuration was assigned to the glucose in isoorientin by the po-
sitive Cotton effect at 260–275 nm (Fig. S63).
The results above enriched the structural diversity of flavone-8-C-
glucosides from Trollius genus by reporting firstly the substitution of
phenolic acyls at C-3″ of the glucose moiety and that of benzoyl on the
sugar residue. Furthermore, it intrigues us to explore that if all of the
organic acids in the plant share the possibility to be attached to C-2″, C-
3″, and C-6″ of glucose and there is the existence of the substitution of
acyls at C-4″. Additionally, ECD experiment was applied for the de-
termination of the absolute configuration of sugar moiety in flavone-8-
C-glucosides.
3. Experimental
3.1. General experimental procedures
Supposing that the glucose moiety in compounds 1–10 had the D-
configuration resulting in a 4C1 conformation, the observation of the
major nuclear Overhauser effects (NOEs) between H-2″, H-4″, and H-6″
of the glucose moiety and H-2′ and H-6′ on ring-B, and minor NOE cross
peaks between H-2′ and H-6′ and H-1″, H-3″, and H-5″ in the NOESY
spectra of compounds 2, 3, 4, 8, 9, and 10, disclosed that these flavone
8-C-β-glucosides were mixtures of two rotational conformers with one
(III) in predominance. This established conformer (III) with C-2″ in the
upper right sector would lead to a negative ECD absorption at
250–275 nm, resembling the ECD data of compounds 8 (Fig. S47), 7
(Fig. S40), and 3 (Fig. S18) with the substitution of acyls at C-2″, C-3″,
or C-6″ of the glucose moiety, as well as that of isoswertisin without the
substation of acyl (Fig. S61). Therefore, the absolute configuration of
the glucose moiety in 1–10 was confirmed to be D-form, for that those
with the substitution of L-glucose (1C4 conformation) and the same
NOESY properties would set C-2″ to the lower right sector, resulting in
the positive Cotton effect.
Optical rotations were measured with a Perkin-Elmer 241 MC po-
larimeter. IR spectra were obtained on a Bruker IFS-55 spectrometer
with KBr pellets. ECD spectra were obtained on a Bio-Logic MOS-450
spectrometer. Mass spectra were performed on an Agilent 1100 mass
spectrometer (ESIMS) and
a Bruker micrOTOF-Q spectrometer
(HRESIMS). The 1D and 2D NMR spectra were recorded in DMSO-d6 on
a Bruker ARX-300 and AV-600 NMR spectrometers with TMS as an
internal standard. Semi-preparative HPLC was carried out using a RP-
C18 column (YMC ODS-A, 20 × 250 mm, 5 μm), a Shimadzu LC-6AD
pump, and a Shimadzu SPD-20A UV detector. The detector wavelength
was set at 210 nm. The mobile phase was MeOH-H2O system with or
without 0.1% acetic acid. Silica gel (Qingdao Haiyang Chemical Co.,
Ltd.), Sephadex LH-20 (GE Healthcare), and RP-C18 (50 μm, YMC Co.,
Ltd, Kyoto, Japan) were used for column chromatography.
3.2. Plant material
In order to verify this proposal, the flavone-6-C-glucoside, iso-
orientin was prepared from orientin isolated from T. chinensis, by the
Wesley-Moser rearrangement reaction followed by HPLC separation.
The glycosidic bond was unambiguously determined to be β-form by
the coupling constant value of the anomeric proton. Although there was
The air-dried flowers of Trollius Chinensis Bunge were purchased
from Tongrentang drugstore, Shenyang, Liaoning Province, China, in
November 2005, and were authenticated by Professor Qi-Shi Sun
(Shenyang Pharmaceutical University). A voucher specimen (JLH-
158

J.-X. Wei et al.
PhytochemistryLetters25(2018)156–162
200530) has been deposited in the Department of Natural Products
Chemistry, Shenyang Pharmaceutical University, Shenyang, China.
subjected to passage over a silica gel column eluted with CH2Cl2-MeOH
in a gradient (100:0–100:10) to give ten fractions (Fr. E2-A to Fr. E2-J).
Subfraction Fr. E2-C was chromatographed on a column of reversed-
phase C18 silica gel, eluted with MeOH-H2O (0:1–9:1) to give three
subfractions (Fr. E2-C1 to Fr. E2-C3). Subfraction Fr. E2-C2 was sepa-
rated over Sephadex LH-20 eluted with MeOH, to give 2″-O-(2‴-me-
thylbutyryl)vitexin (468.2 mg). Subfraction Fr. E2-C3 was separated by
semi-preparative HPLC, using MeOH-H2O (59:41, containing 0.1%
acetic acid, 3.0 mL/min) as the mobile phase, to give 7 (7.4 mg,
27.72 min), 1 (17.0 mg, 33.47 min), 6 (13.7 mg, 37.25 min), trollisin II
(53.5 mg, 45.55 min), 3″-O-(2‴-methylbutyryl)vitexin (10.8 mg,
49.96 min), 8 (46.8 mg, 53.18 min), 9 (22.4 mg, 65.33 min), 3 (8.2 mg,
88.57 min). Subfraction Fr. E2-D was separated over ODS, using MeOH-
H2O (0:1–9:1) to yield three subfractions (Fr. E2-D1 to Fr. E2-D3).
Subfraction Fr. E1-D1 was separated by semi-preparative HPLC on an
ODS column eluted with MeOH-H2O (52:48, containing 0.1% acetic
acid, 3.0 mL/min) as the mobile phase, and then every fraction was put
on Sephadex LH-20 eluted with MeOH for further purification, to give 5
(5 mg, 27.96 min), 2″-O-acetylorientin (20.9 mg, 31.44 min), 4 (9.3 mg,
39.82 min), 6″-O-acetylorientin (29.8 mg, 43.72 min), 2″-O-(4‴-hyrox-
ybenzoyl)vitexin (9.6 mg, 46.85 min), 2″-O-vanilloylvitexin (27.2 mg,
49.13 min), 2″-O-(2‴-methylbutyryl)orientin (64.9 mg, 67.25 min).
Subfraction Fr. E2-D2 was put on a Sephadex LH-20 and then separated
by a preparative TLC with a solvent system of CH2Cl2-MeOH-HAc
(5:1:0.1) to provide compounds 2″-O-veratroylorientin (61.9 mg) and
2″-O-veratroylvitexin (30.7 mg). Subfraction Fr. E2-D3 was separated
by semi-preparative HPLC using MeOH-H2O (57:43, containing 0.1%
acetic acid, 3.0 mL/min) as the mobile phase to give 2 (6.5 mg,
3.3. Extraction and isolation
The air-dried flowers (5.0 kg) were decocted with water for three
times. The extract was concentrated and added to EtOH until the con-
centration of EtOH was 80% (v/v). After being laid aside overnight at
room temperature, the supernatant was evaporated in vacuo to give a
brown gum (920 g). Half of the gum was suspended in water (1.5 L),
and then partitioned successively with CH2Cl2 (3 × 1.5 L), EtOAc
(3 × 1.5 L) and n-BuOH (3 × 1.5 L). The EtOAc extract (30 g) was
subjected to silica gel column chromatography eluted with CH2Cl2-
MeOH in a gradient (100:5–0:100) to afford four fractions (Fr. E1-Fr.
E4). The fraction Fr. E1 (15 g) was chromatographed over silica gel
using a gradient system of CH2Cl2-MeOH (100:0–100:5). The collected
fractions were combined on the basis of their TLC characteristics to
yield ten fractions (Fr. E1-A to Fr. E1-J). The fraction Fr. E1-E was put
on a Sephadex LH-20 column chromatography eluted with MeOH and
combined into three fractions (Fr. E1-E1 to Fr. E1–E3), monitored by
TLC. Subfraction Fr. E1–E2 was separated by semi-preparative HPLC
using MeOH-H2O (61:39, 6.0 mL/min) as the mobile phase to afford
compounds of 3″-O-(2‴-methylbutyryl)isoswertisin (41.1 mg, 37 min)
and 2″-O-(2‴-methylbutyryl)isoswertisin (176.9 mg, 45 min). The
fraction Fr. E1-F was isolated by semi-preparative HPLC using MeOH-
H2O (47:53, 6.0 mL/min) to give Fr. E1-F1 fraction, which was further
purified by a preparative TLC with solvent system of CH2Cl2-MeOH
(8:1) to provide trollisin I (379.7 mg). The fraction E2 (11 g) was
Table 1
1H NMR data of compounds 1–5 in DMSO-d6.
1a
2b
3a
4a
5a
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
2
3
4
5
6
6.79 s
6.25 s
6.63 s
6.09 s
6.74 s
6.17 s
6.65 s
6.07 s
6.64 s
6.26 s
7
8
9
10
1′
2′
3′
8.06 d (8.5)
6.94 d (8.5)
8.00 d (8.7)
6.91 d (8.7)
7.94 d (8.7)
6.89 d (8.7)
7.55 d (2.2)
7.47 brs
4′
5′
6′
6.94 d (8.5)
8.06 d (8.5)
13.16 brs
6.91 d (8.7)
8.00 d (8.7)
13.12 brs
6.89 d (8.7)
7.94 d (8.7)
13.17 brs
6.89 d (8.4)
7.62 dd (8.4, 2.2)
13.10 brs
6.87 d (8.0)
7.53 brd (8.0)
13.16 brs
5-OH
7-OCH3
1″
2″
3″
4″
5″
6″
4.86 d (9.8)
4.24 m
5.10 t (9.6)
3.74 m
3.40 m
3.76 m
4.85 d (9.4)
4.10 m
5.01 t (9.2)
3.62 m
3.38 m
3.76 m
4.81 d (9.8)
4.04 m
3.63 m
3.45 m
3.34 m
5.00 d (10.1)
5.49 t (9.7)
3.61 m
3.51 t (9.4)
3.40 m
4.77 d (9.8)
3.98 t (9.5)
4.84 t (9.2)
3.55 t (9.3)
3.36 m
4.52 d (11.5)
4.33 m
3.85 brd (10.7)
3.64 m
3.78 brd (11.0)
3.61 dd (11.0, 5.7)
3.59 m
3.59 m
1‴
2‴
7.37 brs
7.28 brs
7.32 d (1.9)
7.19 d (1.9)
1.97 s
3‴
4‴
5‴
6‴
7‴
8‴
7.03 d (8.3)
7.50 brd (8.3)
6.75 d (8.1)
7.06 brd (8.1)
7.48 d (15.9)
6.46 d (15.9)
6.82 d (8.4)
7.40 dd (8.4, 1.9)
6.74 d (8.3)
7.23 dd (8.3, 1.9)
9‴
3‴-OCH3
4‴-OCH3
3.71 s
3.79 s
3.77 s
3.69 s
3.77 s
3.74 s
a
Data collected at 600 MHz.
Data collected at 300 MHz.
b
159

J.-X. Wei et al.
PhytochemistryLetters25(2018)156–162
35.00 min) and 10 (21.8 mg, 48.17 min).
[M−H − vanillic acid]−; HRESIMS m/z 599.1390 [M+H]+ (calcd. for
C29H27O14, 599.1395).
3.3.1. Trollichinenside A (1)
Yellow powder; [α]D20 −98 (c 0.02, MeOH); IR (KBr) νmax 3414,
3.3.5. Trollichinenside E (5)
2978, 2841, 1651, 1452, 1407 cm−1
;
1H NMR (DMSO-d6, 600 MHz)
Yellow powder; [α]D20 −22 (c 0.13, MeOH); IR (KBr) νmax 3353,
and 13C NMR (DMSO-d6, 150 MHz) spectroscopic data, see Tables 1 and
3; HRESIMS m/z 597.1601 [M+H]+ (calcd. for C30H29O13, 597.1603),
619.1423 [M+Na]+ (calcd. for C30H28O13Na, 619.1422).
1725, 1662, 1611, 1579, 1516, 1434, 1366, 1253 cm−1 1H NMR
.
(DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectroscopic
data, see Tables 1 and 3; (−)-ESIMS m/z 489.0 [M−H]−, 428.9 [M−H
−
acetic acid]− HRESIMS m/z 491.1188 [M+H]+ (calcd. for
;
3.3.2. Trollichinenside B (2)
C23H23O12, 491.1184).
Yellow powder; [α]2D0 −101 (c 0.03, MeOH); 1H NMR (DMSO-d6,
300 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectroscopic data, see
3.3.6. Trollichinenside F (6)
Tables
1
and 3; HRESIMS m/z 609.1601 [M+H]+ (calcd. for
Yellow powder; [α]D20 −48 (c 0.03, MeOH); IR (KBr) νmax 3438,
C
31H29O13, 609.1603).
2975, 1653, 1514, 1364, 1262, 1191 cm−1 1H NMR (DMSO-d6,
.
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectroscopic data, see
Tables
and 3; HRESIMS m/z 533.1653 [M+H]+ (calcd. for
26H29O12, 533.1654).
3.3.3. Trollichinenside C (3)
2
Yellow powder; [α]2D0 −47 (c 0.02, MeOH); ECD (MeOH): 265
(−2.5); IR (KBr) νmax 3438, 2974, 1755, 1654, 1604, 1513, 1452, 1383,
1271, 1180 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-
d6, 150 MHz) spectroscopic data, see Tables 1 and 3; HRESIMS m/z
597.1600 [M+H]+ (calcd. for C30H29O13, 597.1603).
C
3.3.7. Trollichinenside G (7)
Yellow powder; [α]2D0 −173 (c 0.01, MeOH); ECD (MeOH): 271
(−3.0). IR (KBr) νmax 3438, 2978, 2841, 1750, 1644, 1516, 1451, 1382,
1273, 1180 cm−1. 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-
d6, 150 MHz) spectroscopic data, see Tables 2 and 3; HRESIMS m/z
613.1545 [M+H]+ (calcd. for C30H29O14, 613.1552).
3.3.4. Trollichinenside D (4)
Yellow powder; [α]D20 −81 (c 0.16, MeOH); IR (KBr) νmax 3378,
2970, 1656, 1606, 1515, 1372, 1282 cm−1 1H NMR (DMSO-d6,
.
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectroscopic data, see
3.3.8. Trollichinenside H (8)
Tables 1 and 3; (+)-ESIMS m/z 599.2 [M+H]+, 621.1 [M+Na]+
,
Yellow powder; [α]2D0 −212 (c 0.10, MeOH); ECD (MeOH): 261
(−5.2), 279 (−4.8). IR (KBr) νmax 3414, 2977, 1753, 1654, 1602,
431.1 [M+H − vanillic acid]+; (–)-ESIMS m/z 597.0 [M−H]−, 428.9
Table 2
1H NMR data of compounds 6–10 in DMSO-d6.
6a
7a
8a
9a
10b
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
2
3
4
5
6
6.51 s
6.12 s
6.66 s
6.26 s
6.86 s
6.42 s
6.73 s
6.30 s
6.72 s
6.40 s
7
8
9
10
1′
2′
3′
7.41 brs
7.49 brs
8.13 d (8.8)
6.94 d (8.8)
7.58 brs
7.57 brs
4′
5′
6′
6.81 d (7.9)
7.48 brd (7.9)
13.19 brs
6.92 d (8.4)
7.58 brd (8.4)
13.19 brs
6.94 d (8.8)
8.13 d (8.8)
13.32 brs
3.83 s
4.96 d (10.1)
5.43 t (9.6)
3.59 m
3.54 t (9.2)
3.38 m
3.82 m
6.92 d (8.4)
7.66 dd (8.4, 2.0)
13.28 brs
6.90 d (8.4)
7.63 brd (8.4)
13.32 brs
3.83 s
4.95 d (10.1)
5.42 t (9.4)
3.57 m
3.51 m
3.37 m
3.85 m
3.64 m
5-OH
7-OCH3
1″
2″
3″
4″
5″
6″
3.76 s
4.81 d (9.3)
4.01 m
4.88 t (9.2)
3.52 t (9.4)
3.36 m
4.85 d (9.8)
4.27 t (9.3)
5.09 t (8.6)
3.73 m
3.40 m
3.76 m
5.05 d (10.1)
5.51 t (9.5)
3.65 m
3.55 t (9.4)
3.42 m
3.76 m
3.58 dd (11.8, 5.9)
3.87 dd (13.0, 10.9)
3.66 m
3.57 m
3.61 m
1‴
2‴
3‴
2.32 m
1.55 m
7.39 brs
7.21 d (1.7)
7.68 d (7.3)
7.43 t (7.7)
7.20 brs
1.35 m
4‴
5‴
6‴
7‴
0.80 t (6.7)
1.05 d, (6.7)
7.57 m
7.43 t (7.7)
7.68 d (7.3)
7.03 d (8.4)
7.53 brd (8.4)
6.75 d (8.2)
6.75 d (8.2)
7.01 brd (8.2)
7.26 d (15.9)
6.16 d (15.9)
7.02 dd (8.2, 1.7)
7.27 d (15.9)
6.17 d (15.9)
8‴
9‴
3‴-OCH3
4‴-OCH3
3.71 s
3.79 s
3.77 s
3.77 s
a
Data collected at 600 MHz.
Data collected at 300 MHz.
b
160

J.-X. Wei et al.
PhytochemistryLetters25(2018)156–162
Table 3
13C NMR data of compounds 1–10 in DMSO-d6.
1a
2a
3a
4a
5a
6a
7a
8a
9a
10b
2
3
4
5
164.0
102.6
182.1
160.7
98.4
164.0
102.3
182.0
160.6
98.8
163.3
102.4
181.7
160.6
98.5
164.2
102.4
181.9
160.6
97.8
164.1
102.6
182.0
160.6
98.2
163.8
102.1
181.6
160.7
98.8
164.6
102.5
182.0
160.7
98.3
164.6
102.5
182.3
161.7
94.9
164.7
102.5
182.2
161.7
94.7
164.7
102.4
182.2
161.7
94.8
6
7
8
9
10
1′
2′
3′
4′
163.2
103.8
156.2
104.0
121.7
129.1
115.9
161.4
115.9
129.1
163.2
104.0
156.4
104.0
121.8
128.8
116.0
161.3
116.0
128.8
163.3
104.2
156.2
104.2
121.5
128.4
116.0
161.3
116.0
128.4
162.5
102.4
156.3
103.7
122.0
114.1
145.9
149.8
115.7
119.5
162.8
103.7
156.0
104.0
122.0
114.0
145.9
149.7
115.8
119.4
163.8
103.8
156.3
103.8
121.4
113.6
146.2
150.7
115.8
119.3
163.1
103.7
156.2
103.9
121.9
114.0
146.0
149.9
115.6
119.4
162.9
103.6
155.7
104.4
121.5
129.3
116.0
161.5
116.0
129.3
56.8
162.7
103.2
155.6
104.3
121.8
114.2
146.0
150.1
115.8
119.7
56.6
162.8
103.5
155.7
104.4
121.8
114.2
146.0
150.2
115.9
119.7
56.8
5′
6′
7-OCH3
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
6‴
7‴
8‴
9‴
3‴-OCH3
4‴-OCH3
73.6
68.8
80.4
68.6
81.6
60.8
122.7
111.8
148.2
152.7
111.0
123.2
165.1
74.0
68.9
79.7
68.5
81.6
60.8
125.7
110.9
148.0
149.3
115.4
123.2
144.6
115.4
166.5
55.7
73.8
70.9
78.6
70.7
78.6
64.7
121.8
111.7
148.3
152.8
110.8
123.2
165.6
71.1
72.5
76.1
70.8
82.3
61.4
120.8
112.5
147.1
151.2
114.9
123.3
164.7
73.5
68.5
79.9
68.5
81.8
61.1
169.9
21.2
73.8
68.6
79.4
68.6
81.9
61.3
175.4
40.4
26.4
11.3
16.6
73.5
68.7
80.4
68.8
81.8
61.2
122.7
111.8
148.2
152.6
111.0
123.2
165.1
71.0
72.3
75.8
70.6
82.2
61.0
125.5
111.1
148.0
149.4
115.6
123.1
144.8
114.2
165.6
55.7
70.9
73.2
75.9
70.7
82.5
61.3
129.7
128.9
128.6
133.2
128.6
128.9
164.8
71.0
72.2
75.9
70.8
82.4
61.4
125.5
111.2
148.0
149.4
115.6
123.0
144.7
114.2
165.5
55.7
55.4
55.7
55.4
55.6
55.6
55.4
55.7
a
Data collected at 150 MHz.
Data collected at 75 MHz.
b
1514, 1448, 1368, 1336, 1275, 1248 cm−1
.
1H NMR (DMSO-d6,
n-butanol to give a yellow powder, which was separated by semi-pre-
parative HPLC on a RP-C18 column (YMC ODS-A, 20 × 250 mm, 5 μm)
eluted with MeOH-H2O (44:56, containing 0.1% acetic acid, 3.0 mL/
min) as the mobile phase to give orientin (19.0 mg, 36.62 min) and
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectroscopic data, see
Tables
32H31O13, 623.1759).
2
and 3; HRESIMS m/z 623.1762 [M + H]+ (calcd. for
C
isoorientin (14.3 mg, 39.23 min). (–)-ESIMS: 447.0 [M−H]− 1H NMR
;
(DMSO-d6, 300 MHz): δH 13.60 (1H, brs, 5-OH), 7.33 (1H, brd,
J = 8.4 Hz, H-6′), 7.29 (1H, brs, H-2′), 6.75 (1H, d, J = 8.4 Hz, H-5′),
6.47 (1H, s, H-3), 6.25 (1H, s, H-8), 4.55 (1H, d, J = 9.9 Hz, H-1″), 4.06
(1H, t, J = 8.9 Hz, H-2″), 3.66 (1H, d, J = 11.2 Hz, H-6″a), 3.41 (1H,
brd, J = 11.2 Hz, H-6″b), 3.10–3.20 (3H, H-3″, 4″, 5″). ECD (MeOH):
270 (+1.2).
3.3.9. Trollichinenside I (9)
Yellow powder; [α]D20 −125 (c 0.02, MeOH); IR (KBr) νmax 3438,
2981, 1752, 1645, 1450, 1366, 1268, 1206, 1126 cm−1 1H NMR
.
(DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectroscopic
data, see Tables 2 and 3; HRESIMS m/z 567.1485 [M+H]+ (calcd. for
C
29H27O12, 567.1497).
Conflict of interest
3.3.10. Trollichinenside J (10)
Yellow powder; [α]D20 −99 (c 0.53, MeOH); IR (KBr) νmax 3406,
2923, 1737, 1703, 1653, 1638, 1598, 1455, 1352, 1253 cm−1. 1H NMR
(DMSO-d6, 300 MHz) and 13C NMR (DMSO-d6, 75 MHz) spectroscopic
data, see Tables 2 and 3; (+)-ESIMS m/z 639.2 [M+H]+, 661.1 [M
+Na]+; (–)-ESIMS m/z 637.0 [M−H]−; HRESIMS m/z 639.1709 [M
+H]+ (calcd. for C32H31O14, 639.1708).
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.phytol.2018.04.019.
3.3.11. Isoswertisin
1H NMR (DMSO-d6, 300 MHz): δH 13.36 (1H, brs, 5-OH), 10.32 (1H,
brs, 4′-OH), 8.04 (2H, d, J = 8.7 Hz, H-2′, 6′), 6.90 (2H, d, J = 8.7 Hz,
H-3′, 5′), 6.83 (1H, s, H-3), 6.52 (1H, s, H-6), 4.72 (1H, d, J = 9.6 Hz, H-
1″), 3.88 (3H, s, 7-OCH3). ECD (MeOH): 266 (– 4.6).
References
Brum-Bousquet, M., Tillequin, F., Paris, R.R., 1977. The C-flavonosides of Sarothamnus
scoparius. Isolation of a new compound, the 6″-O-acetylscoparoside. Lloydia 40,
591–592.
Cai, S.Q., Wang, R.F., Yang, X.W., Shang, M.Y., Ma, C.M., Shoyama, Y., 2006. Antiviral
flavonoid-type C-glycosides from the flowers of Trollius chinensis. Chem. Biodivers. 3,
343–348.
3.4. Preparation and identification of isoorientin
Gaffield, W., Horowitz, R.M., Gentili, B., Chopin, J., Bouillant, M.L., 1978. Circular di-
chroism of C-glycosylflavones, a chiroptical method for differentiating 6-C-, 8-C- and
6, 8-di-C-β-glycosyl isomers. Tetrahedron 34, 3089–3096.
Orientin of 50 mg was suspended in 50 mL 6% aqueous hydro-
chloride solution which was refluxed for 7 hs. The reactant solution was
neutralized by sodium carbonate to pH = 7 and then partitioned with
Gaffield, W., Besson, E., Chopin, J., 1984. Effect of conformation and configuration of
161

J.-X. Wei et al.
PhytochemistryLetters25(2018)156–162
J.H., 2004b. Trollioside, a new compound from the flowers of Trollius chinensis. J.
Asian Nat. Prod. Res. 6, 139–144.
arabinose residues on the circular dichroism of C-glycosylflavones. Phytochemistry
23, 1317–1322.
Hori, K., Satake, T., Yamaguchi, H., Saiki, Y., Murakami, T., Chen, C.M., 1987. Chemical
and chemotaxonomical studies of filices. LXXII. Chemical studies on the constituents
of Odontosoria gymnogrammoides Christ. Yakugaku Zasshi 107, 774–779.
Kato, T., Morita, Y., 1990. C-glycosylflavones with acetyl substitution from Rumex acetosa
L. Chem. Pharm. Bull. 38, 2277–2280.
Li, Z.L., Li, D.Y., Hua, H.M., Chen, X.H., Kim, C.S., 2009. Three new acylated flavone C-
glycosides from the flowers of Trollius chinensis. J. Asian Nat. Prod. Res. 11, 426–432.
Liu, Z.Y., Luo, D.Q., 2010. Chemical constituents of Trollius chinensis. Chin. Tradit. Herb.
Drugs 41, 370–373.
Wu, X.A., Zhao, Y.M., Yu, N.J., 2006. Flavone c-glycosides from Trollius ledebouri reichb.
J. Asian Nat. Prod. Res. 8, 541–544.
Wu, X.A., Zhao, Y.M., Zhu, J., Wu, J., Zhu, S.Q., Xu, G.S., 2009. Acylated flavone C-
glycosides from Trollius ledebouri Reichb. Nat. Prod. Res. Dev. 21, 1–5.
Wu, L.Z., Wu, H.F., Xu, X.D., Yang, J.S., 2011a. Two new flavone C-glycosides from
Trollius ledebourii. Chem. Pharm. Bull. 59, 1393–1395.
Wu, L.Z., Xu, X.D., Yang, J.S., 2011b. Chemical constituents of water soluble fraction of
Trollius ledebouri Reichb. Chin. Pharm. J. 46, 745–747.
Zou, J.H., Yang, J.S., Zhou, L., 2004. Acylated flavone C-glycosides from Trollius ledebouri.
J. Nat. Prod. 67, 664–667.
Zou, J.H., Yang, J.S., Dong, Y.S., Zhou, L., Lin, G., 2005. Flavone C-glycosides from
flowers of Trollius ledebouri. Phytochemistry 66, 1121–1125.
Ohba, S., Yoshida, K., Kondo, T., 2004. Swertisin dihydrate. Acta Cryst. C60, 893–896.
Shimamura, N., Miyase, T., Umehara, K., Warashina, T., Fujii, S., 2006. Phytoestrogens
from Aspalathus linearis. Biol. Pharm. Bull. 29, 1271–1274.
Wang, R.F., Yang, X.W., Ma, C.M., Cai, S.Q., Li, J.N., Shoyama, Y., 2004a. A bioactive
alkaloid from the flowers of Trollius chinensis. Heterocycles 63, 1443–1448.
Wang, R.F., Yang, X.W., Ma, C.M., Liu, H.Y., Shang, M.Y., Zhang, Q.Y., Cai, S.Q., Park,
Zou, J.H., Yang, J.S., Zhou, L., Lin, G., 2006. A new labdane type diterpenoid from Trollius
ledebouri. Nat. Prod. Res. 20, 1031–1035.
162