Flavone C-glycosides from the flowers of Trollius chinensis and their anti-complementary activity

Article abstract of DOI:10.1080/10286020.2012.760545

Phytochemical investigation of ethanol extract from the flowers of Trollius chinensis Bunge resulted in the isolation of two new flavone C-glycosides (1-2), along with 10 known compounds (3-12). The structures of the new compounds were established as 6?-(

Full text of DOI:10.1080/10286020.2012.760545

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Flavone C-glycosides from the flowers
of Trollius chinensis and their anti-
complementary activity
Jiang-Yun Liu a , Sheng-Yin Li a , Jian-Yong Feng a , Yan Sun a , Jin-
Na Cai a , Xiao-Fei Sun a & Shi-Lin Yang a
a College of Pharmaceutical Sciences, Soochow University, Suzhou,
215123, China
Version of record first published: 22 Apr 2013.
To cite this article: Jiang-Yun Liu , Sheng-Yin Li , Jian-Yong Feng , Yan Sun , Jin-Na Cai ,
Xiao-Fei Sun & Shi-Lin Yang (2013): Flavone C-glycosides from the flowers of Trollius
chinensis and their anti-complementary activity, Journal of Asian Natural Products Research,
DOI:10.1080/10286020.2012.760545
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Journal of Asian Natural Products Research, 2013
http://dx.doi.org/10.1080/10286020.2012.760545
Flavone C-glycosides from the flowers of Trollius chinensis and their
anti-complementary activity
Jiang-Yun Liu*, Sheng-Yin Li, Jian-Yong Feng, Yan Sun, Jin-Na Cai , Xiao-Fei Sun and
*
Shi-Lin Yang
College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China
(Received 6 July 2012; final version received 17 December 2012)
Phytochemical investigation of ethanol extract from the flowers of Trollius chinensis
Bunge resulted in the isolation of two new flavone C-glycosides (1–2), along with 10
known compounds (3–12). The structures of the new compounds were established as
6000-(3-hydroxy-3-methylglutaroyl)-200-O-b-D-galactopyranosyl orientin (1) and 6000-(3-
hydroxy-3-methylglutaroyl)-200-O-b-D-galactopyranosyl vitexin (2) on the basis of
various spectroscopic analysis (including different 1D and 2D NMR spectroscopies,
high-resolution electrospray ionization mass spectrometry) and chemical evidences.
Bioassay showed that eight flavonoids inhibited complement activation on the classic
pathway in vitro, with their IC50 values ranging from 0.88 to 4.02 mM, which may
contribute to the applications of the herb in treatment of acute respiratory distress
syndrome, etc.
Keywords: Trollius chinensis; flavanoid; C-glycoside; anti-complementary activity
1. Introduction
these compounds. Anti-complementary
activities of these compounds on the classic
pathways were also undertaken, which
helps to evaluate the pharmacological
functions of the Trollius flowers.
Trollius chinensis Bunge is a traditional
Chinese medicinal herb widely cultivated
in northern China. Its flowers have been
used as medicinal drugs and tea drinks to
prevent or treat upperrespiratory infections,
pharyngitis, tonsillitis, and bronchitis [1].
Chemical researches have revealed that
orientin, vitexin, and other flavone C-
glycosides are rich in Trollius genus [2–5].
These flavonoids were responsible in major
for diverse bioactivities such as antiviral,
antimicrobial, antioxidant, radioprotection
activities, etc. [2,6]. As part of our ongoing
search of natural ingredients for functional
foods or medicinal drugs, we carried out
phytochemical investigations on this herb.
As a result, two new flavonoids (1–2,
Figure 1) have been obtained from the
ethanol extract, together with 10 known
compounds (3–12). This paper reported
the isolation and identification work of
2. Results and discussion
Compound 1 was obtained as a yellow
powder. The positive HRESIMS exhibited
a pseudomolecular ion peak at m/z
777.1859 [M þ Na]þ, corresponding to
the molecular formula of C33H38O20, and
the degrees of unsaturation were 15. Its UV
spectrum showed a typical absorption of
1
flavonoids at 276 and 338 nm. In the H
NMR spectrum of 1, the occurrence of an
ABX spin system [dH 7.42 (s), 6.84 (1H, d,
J ¼ 8.5 Hz) and 7.52 (1H, d, J ¼ 8.5 Hz)]
characterized an ortho-di-substituted B
ring of the flavone aglycon. Signals at dH
6.58 (1H, s) and 6.24 (1H, s) were attributed
to the aromatic protons at H-3 and H-6
*Corresponding authors. Emails: liujiangyun@suda.edu.cn; caijinna99@yahoo.cn
q 2013 Taylor & Francis

2
J.-Y. Liu et al.
OH
O
O
HO
O
OH
R1
HO
HO
HO
O
O
OH
OH
HO
O
O
O
HO
OH
R1
OH
H
1
2
Figure 1. Chemical structures of compounds 1–2.
of the A-ring, respectively. Additionally,
the anomeric proton at dH 4.81 (1H, d,
J ¼ 10.0 Hz) and other signals between dH
3.0 and 4.1 can be assumed as the protons of
sugars. Signals at dH 2.42 (2H, s) and 1.20
(3H, s) at high field indicated that there are
protons of some other substitute groups.
The 13C NMR (DEPT) spectral data of 1
revealed the presence of 33 carbons,
including 1 methyl (d 27.3), 4 methylenes
(d 45.2, 45.6, 61.4 £ 2), 10 methines of
sugars (d67.4–81.9, and d 105.5), 15
olefinic carbons of flavones (d 98.1–
181.8), 1 quaternary carbon (d 45.6), as
well as 2 carbonyl carbons at d 169.9 and
172.8. 1H and 13C NMR signals of 1 were
further assigned by extensive analysis of
from Glc-100-H at dH 4.81 to C-7 at dC
162.5/C-8 at dC 103.8/C-9 at dC 156.2, from
Glc-200-H at dH 4.04 to Gal-C-1000 at dC
105.5, from Gal-1000-H at dH 3.91 to Glc-
C-200 at dC 81.3, from both Gal-6000-H at dH
3.27 and 3.48 to HMG-C-10000 at dC 169.9,
from HMG-20000-2H at dH 2.42 to HMG-
C-10000 at dC 169.9, from HMG-60000-3H at dH
1.20 to HMG-C-20000 at dC 45.2/HMG-C-40000
at 45.6, and HMG-40000-2H at dH 2.50 to
HMG-C-50000 at dC 172.8 could be observed,
which confirmed the structural fragments’
connection. On acid hydrolysis, 1 afforded
one sugar moiety that identified as D-
galacose based on the gas chromatography
(GC) analysis of its L-cysteine derivate
[10]. On partial acid hydrolysis, 1 gave
orientin (5) and orientin-200-O-b-D-galac-
topyranoside (3) in HPLC analysis, which
further convinced the absolute configur-
ation of two monosugar moieties. Thus, the
structure of 1 was established as 6000-
(3-hydroxy-3-methylglutaroyl)-200-O-b-D-
galactopyranosyl orientin.
1
HSQC, H-1H COSY and HMBC spectra
(Table 1). The spectral data of 1 showed
rather similarity to those of orientin-200-O-
b-D-galactopyranoside (3) [4], with some
differences at C-5000/6000 of galactosyl (Gal)
group. Moreover, the remaining signals in
the 1H and 13C NMR spectra of 1 indicated
the presence of a 3-hydroxy-3-methylglu-
taroyl (HMG) group (Table 1) [7–9],
which was coincident with its molecular
weight. S-configuration for the C-30000
stereogenic carbon of HMG was deduced
because HMG esters are formed via the
acylation of the hydroxyl group with (S)-
HMG-CoA in natural products. In HMBC
spectrum of 1 (Figure 2), the correlations
Compound 2 was obtained as a yellow
powder. UV spectrum of 2 also showed a
typical absorption of flavonoids at 270 and
330 nm. The molecular formula of 2 was
determined to be C33H38O19 by HR-ESI-
MS at m/z 761.1911 [M þ Na]þ. In the 1H-
NMR spectrum of 2, six aromatic signals
corresponding to H-3 at dH 6.77 (s) and H-6
at dH 6.27 (s), and to an AA0BB0 spin system

Journal of Asian Natural Products Research
3
d
d
d
d
d
d
d
d
d

4
J.-Y. Liu et al.
OH
8
O
O
HO
HO
OH
OH
O
O
O
1'
OH
HO
1''''
O
HO
O
OH
1''
HO
O
HO
1'''
OH
Figure 2. Key HMBC correlations (H ! C) for compound 1.
of H-20/60 at dH 8.02 (1H £ 2, d,
J ¼ 8.0 Hz) and H-30/50 at dH 6.89
(1H £ 2, d, J ¼ 8.0 Hz) on the B ring of
9 and 12 were isolated from Trollius genus
for the first time.
Seven flavonoids (Table 2) together
with trollioside (11), were evaluated for
their anti-complementary activity on the
classic pathways in vitro [13], while
compounds 4 and 8 were not tested due to
limited amount. Bioassay showed that all
flavonoids inhibited complement acti-
vation on the classic pathway apparently,
with their IC50 data ranging from 0.88 to
4.02 mM, and trollioside (11) showed no
effect in this assay. By comparison with
orientin (5), vitexin (6) with less hydroxyl
group in B ring showed higher activity; 7-
methoxylation in A ring was not favored by
comparison of 7 and 10; esterification of
sugars strengthened anti-complementary
activity apparently, as observed by com-
parison of 7 and 5. Increasing a Gal group
to orientin lowered this activity, as
indicated by 3; however, adding an
additional HMG to 3 increased the activity,
as shown by 1, and 2 showed highest
activity with IC50 value to be 0.88 mM. The
results indicated that esterification of sugar
moiety of these flavone C-glycosides
played important roles for their anti-
complementary activity, while aglycon
substitution also affected their bioactivity.
1
the aglycone were found. The H and 13C
NMR spectral data of 2 resembled those of
1, and the only difference between 2 and 1
(Table 1) was that the hydroxyl group at C-
30 in 1 was replaced by a proton in 2.
Compound 2 afforded D-galacose in GC
analysis [10], and vitexin (6) in HPLC
analysis by partial acid hydrolysis. Accord-
ingly, the structure of 2 was established as
6000-(3-hydroxy-3-methylglutaroyl)-200-O-
b-D- galactopyranosyl vitexin.
In addition, 10 known compounds were
also isolated and identified by comparing
their NMR and MS spectral data with those
reported in the literature. They are assigned
to be orientin-200-O-b-D-galactopyranoside
(3) [4], orientin-200-O-b-D-glucopyrano-
side (4) [11], orientin (5) [12], vitexin
(6) [11], 7-methoxyl-200-O-(2000-methylbu-
tyryl) orientin (7) [5], 7-methoxyl-200-O-
(2000-methylbutyryl) vitexin (8) [5], 7-meth-
oxyl-200-O-(3000,4000-dimethoxybenzoyl)
vitexin (9) [3], 200-O-(2000-methylbutyryl)
isoswertisin (10) [4], trollioside (11) [12],
and 4-(b-D-glucopyranosyloxy)-3-(3-
methyl-2-butenyl) benzoic acid (12) [12],
respectively. Among them, compounds 4,
Table 2. Anticomplementary activity through classical passway of the samples (n ¼ 3, x ^ SD).
ꢀ
Compound
IC50 (mg/ml)
IC50 (mM)
Compound
IC50 (mg/ml)
IC50 (mM)
1
2
3
0.87 ^ 0.059
0.65 ^ 0.018
2.45 ^ 0.125
1.51 ^ 0.081
0.096 ^ 0.025
1.16 ^ 0.08
0.88 ^ 0.02
4.02 ^ 0.20
3.36 ^ 0.18
6
7
9
10
1.35 ^ 0.141
0.56 ^ 0.099
1.28 ^ 0.238
1.43 ^ 0.041
3.12 ^ 0.33
1.01 ^ 0.18
2.04 ^ 0.38
2.70 ^ 0.08
5
Heparin

Journal of Asian Natural Products Research
5
However, further anti-complementary
assays should be undertaken to test this
result. Accumulating data have suggested
that excessive activation of complement is
involved in the pathogenesis of many auto-
immune disorders, inflammatory diseases,
and inflammation responses, including
acute respiratory distress syndrome
[13,14]. The results suggested that these
flavone C-glycosides should be main
representative constituents for the appli-
cations of the herb in treatments of acute
respiratory distress syndrome, etc.
applied by Qingdao Haiyang Chemical,
Ltd., Qingdao, China. Cosmosil C18
reversed-phase silica gel (75 mm, Nacalai
tesque, Inc., Kyoto, Japan) was used for
column chromatography.
3.2 Plant material
The flowers of T. chinensis Bunge were
collected from Chengde, Hebei Province,
China, in August 2006, and the botanical
origin of material was identified by Doctor
Jinna Cai (Shanghai Mediclon Inc., Shang-
hai, China). The voucher specimens (No.
TC060901) are deposited at the herbarium
of College of Pharmaceutical Sciences,
Soochow University, Suzhou, China.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on
Perkin-Elmer PL341 polarimeter. UV
spectra were measured on Perkin-Elmer
Lambda 25 uv/vis spectrometer instrument
(Perkin-Elmer, Inc., Waltham, MA, USA).
NMR spectra were recorded on a Varian
400M NMR System (Varian, Inc., Palo
Alto, CA, USA). HRESI mass spectra
were performed with a Finnigan LCQ-
Deca high-resolution mass spectrometer
(Thermo-Finnigan, Inc., San Jose, CA,
USA). GC analysis was carried out on a
Shimadzu GC-14C gas chromatograph
with FID detection, using a Rtx-1 capillary
column (30 m £ 0.25 mm, i.d.; Shimadzu
Corporation, Tokyo, Japan). HPLC separ-
ations were performed on Shimadzu LC-
20A series apparatus with a SPD-20A UV
detector (Shimadzu Corporation, Tokyo,
Japan), equipped with a 250 £ 20 mm i.d.
preparative Cosmosil AR-II C18 column
(Nacalai Tesque, Inc., Kyoto, Japan);
Medium pressure liquid chromatography
(MPLC) was performed on a Lisure HP
Purifier apparatus with a UV detector
(Lisure science Corporation, Suzhou,
China). Polyamide chromatography
(100–200 mesh) was manufactured by
Taizhou Luqiao Biochemical Co., Taiz-
hou, China. Silica gel for column chroma-
tography and precoated silica gel plates 60
F254 for thin layer chromatography were
3.3 Extraction and isolation
The flowers (5 kg) were extracted with
70% aqueous ethanol (v/v) for two times
(50 liters, 1 h each) under reflux. After
evaporation, the residue was suspended in
H2O (10 liters) and filtered. The liquor
solution was subjected to AB-8 macro-
porous resin column, eluting with H2O,
10%, 70%, and 95% ethanol (each
30 liters) successively. The 70 and 95%
eluants were combined and concentrated
under reduced pressure to give a residue,
which was suspended in H2O and parti-
tioned using EtOAc (5 £ 4 l) to give
EtOAc fraction (TE, 126 g) and water
fraction (TW, 165 g). TW was applied to
vacuum silica gel column eluting with
EtOAc–EtOH (7:3, 5:5, 1:9) to afford
three fractions (JW-1 , 3). JW-1 (8.2 g)
was subjected to silica MPLC using a
stepwise gradient elution of CHCl3 –
MeOH (0.1% CH3COOH) (7:3, 5:5, 2:8),
purified by Sephadex LH-20 eluting with
90% MeOH to give 5 (70 mg) and 6
(50 mg). JW-2 (21.0 g) was subjected to
polyamide MPLC eluting with EtOAc–
EtOH (7:3, 5:5, 2:8) to afford three
fractions (JW-2-1 , 3). JW-2-2 (4.8 g)
was subjected to ODS column eluting
with CH3CN–H2O (0.1% CH3COOH)

6
J.-Y. Liu et al.
(16:84) to afford three subfractions (JW-2-
2-1 , 3). JW-2-2-3 (0.6 g) was subjected
to preparative HPLC, using a mobile phase
of CH3CN–H2O (0.1% CH3COOH)
(16:84) to give 1 (21 mg) and 2 (19 mg).
By similar separation procedures, com-
pound 3 (20 mg) from JW-2-2-1 (0.9 g),
and 4 (15 mg) from JW-2-3 (1.2 g) were
also obtained. Part of the EtOAc fraction
(20.0 g) was applied to polyamide MPLC
using a stepwise gradient elution of
CHCl3–EtOAc (8:2, 5:5, 4:6, 2:8) to
afford five fractions (JE-1 , 5). JE-3
(3.8 g) was subjected to reverse MPLC
eluting with MeOH–H2O (0.1% HAc)
(35:65, 40:60) to give 10 (70 mg), 11
(45 mg), and 9 (34 mg). By similar
separation procedures, compounds 12
(25 mg), 8 (28 mg), and 7 (24 mg) were
also separated from JE-4 (4.5 g).
derivative with that of D-galacose
(13.4 min) standard sample. As for partial
acid hydrolysis, compounds 1 and 2 (3 mg
each) were solved separately in 2 M HCl
(3 ml) and heated at 608C on a water bath
for 2 h, neutralized with 2 M NaOH to get
sample solutions. The two samples were
analyzed on HPLC by comparison of the
retention times with those of reference
compounds, with acetonitrile–water (con-
tained 1% acetic acid) as eluant using a
gradient elution of 13–15% acetonitrile in
25 min, and the detection wavelength was
set at 340 nm. The mobile phase flow rate
was 1.0 ml/min. Orientin-200-O-b-D-galac-
topyranoside (tR 16.9 min) and orientin (tR
20.9 min) were detected from hydrolyte of
1, and vitexin (tR 28.5 min) was detected
from hydrolyte of 2, respectively.
3.5 Anti-complementary activity assay
through the classical pathway
3.3.1 Compound 1
20
The bioassay was carried out according to
the method described [13]. Each com-
pound (3 mg, dissolved with DMSO) was
applied for this assay, with heparin
(sodium salt, 160 IU/mg) used as positive
control. The 50% percent inhibition was
calculated as shown in Table 2. Trollioside
showed no effect in this assay.
Yellow amorphous powder, ½aꢀD 212 (c
0.042,MeOH). UV lmax (MeOH) nm: 276,
C
338. For 1H (400 MHz, DMSO-d6) and 13
NMR (100 MHz, DMSO-d6) spectral data,
see Table 1. HRESIMS: m/z 777.1859
[M þ Na]þ (calcd for C33H38O20 Na,
777.1854).
3.3.2 Compound 2
20
Yellow amorphous powder, ½aꢀD 225 (c
330. For 1H (400 MHz, DMSO-d6) and 13
Acknowledgements
0.056,MeOH). UV lmax (MeOH) nm: 270,
C
The authors are grateful to Prof. Guoqiang Song
of Shanghai Institute of Materia Medica,
Chinese Academy of Sciences for the NMR
measurements, and Prof. Daofeng Chen of
School of Pharmacy, Fudan University for the
anti-complementary activity assay. This work
was financially supported by a project funded
by the Program of Improving the Standard of
Pharmacopoeia of the People’s Republic of
China (2015), and by the Priority Academic
Program Development of Jiangsu Higher
Education Institutions (PAPD).
NMR (100 MHz, DMSO-d6) spectral data,
see Table 1. HRESIMS: m/z 761.1911
[M þ Na]þ (calcd for C33H38O19Na,
761.1905).
3.4 Acid hydrolysis and GC and HPLC
analysis of 1–2
The GC analysis was carried out according
to the method described [10]. The absolute
configuration of the monosaccharide was
confirmed to be D-galacose by comparison
of the retention time of its L-cysteine
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