A novel dimeric flavonol glycoside from Cynanchum acutum subsp. sibiricum

Article abstract of DOI:10.1080/14786419.2018.1483931

A novel dimeric flavonol glycoside, Cynanflavoside A (1), together with six analogues, kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (2), quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (3), kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside (4), quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside (5), kaempferol-3-O-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside (6), and quercetin-3-O-galactoside (7) were isolated from the n-butyl alcohol extract of Cynanchum acutum subsp. sibiricum. Their structures were determined spectroscopically and compared with previously reported spectral data. All compounds were evaluated for their anti-complementary activity in vitro, and only compound 5 exhibited anti-complement effects with CH₅₀ value of 0.33 mM.

Full text of DOI:10.1080/14786419.2018.1483931

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A novel dimeric flavonol glycoside from
Cynanchum acutum subsp. sibiricum
Si-Wen Yuan, Wei Dai, Xin-Hui Pan, Yan Lu, Dao-Feng Chen & Qi Wang
To cite this article: Si-Wen Yuan, Wei Dai, Xin-Hui Pan, Yan Lu, Dao-Feng Chen & Qi Wang
(2018): A novel dimeric flavonol glycoside from Cynanchum acutum subsp. sibiricum, Natural
Product Research, DOI: 10.1080/14786419.2018.1483931
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Natural Product research, 2018
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2018.1483931
ꢀ
A novel dimeric flavonol glycoside from Cynanchum acutum
subsp. sibiricum
a
a
a
b
b
a
Si-Wen Yuan , Wei Dai , Xin-Hui Pan , Yan Lu , Dao-Feng Chen and Qi Wang
ꢆ
Kꢇy lꢆbꢄꢅꢆꢁꢄꢅy ꢄf Xinjiꢆng Pꢀyꢁꢄmꢇꢃiꢈinꢇ rꢇꢂꢄꢉꢅꢈꢇ ꢆnꢃ uꢁiꢊizꢆꢁiꢄn, sꢈꢀꢄꢄꢊ ꢄf Pꢀꢆꢅmꢆꢈy, Miniꢂꢁꢅy ꢄf
b
eꢃꢉꢈꢆꢁiꢄn, sꢀiꢀꢇzi univꢇꢅꢂiꢁy, sꢀiꢀꢇzi, P.r. cꢀinꢆ; sꢈꢀꢄꢄꢊ ꢄf Pꢀꢆꢅmꢆꢈy, dꢇpꢆꢅꢁmꢇnꢁ ꢄf Pꢀꢆꢅmꢆꢈꢄgnꢄꢂy, Fꢉꢃꢆn
univꢇꢅꢂiꢁy, sꢀꢆngꢀꢆi, P.r. cꢀinꢆ
ABSTRACT
ARTICLE HISTORY
rꢇꢈꢇivꢇꢃ 16 Mꢆꢅꢈꢀ 2018
aꢈꢈꢇpꢁꢇꢃ 29 Mꢆy 2018
A novel dimeric flavonol glycoside, Cynanflavoside A (1), together with
six analogues, kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-
glucopyranoside (2), quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-
glucopyranoside (3), kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-
β-D-xylopyranoside (4), quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-
β-D-xylopyranoside (5), kaempferol-3-O-β-D-glucopyranosyl-7-O-
α-L-rhamnopyranoside (6), and quercetin-3-O-galactoside (7) were
isolated from the n-butyl alcohol extract of Cynanchum acutum subsp.
sibiricum. Their structures were determined spectroscopically and
compared with previously reported spectral data. All compounds
were evaluated for their anti-complementary activity in vitro, and only
compound 5 exhibited anti-complement effects with CH value of
KEYWORDS
aꢂꢈꢊꢇpiꢆꢃꢆꢈꢇꢆꢇ; Cynanchum
acutum ꢂꢉbꢂp. sibiricum;
ꢃimꢇꢅiꢈ flꢆvꢄnꢄꢊ gꢊyꢈꢄꢂiꢃꢇ;
ꢆnꢁi-ꢈꢄmpꢊꢇmꢇnꢁꢆꢅy ꢆꢈꢁiviꢁy
50
0.33 mM.
1
. Introduction
The genus Cynanchum (Asclepiadaceae) includes almost 200 species distributing in east
Africa, Mediterranean region, as well as the tropical, subtropical and temperate areas of
Europe and Asia (Jiang and Li 1977). There are four species in Xinjiang region of China, one
CONTACT dꢆꢄ-Fꢇng cꢀꢇn
ꢃfꢈꢀꢇn@ꢂꢀmꢉ.ꢇꢃꢉ.ꢈn; Qi Wꢆng
qiw16@163.ꢈꢄm
tꢀꢇ ꢂꢉppꢊꢇmꢇnꢁꢆꢊ ꢃꢆꢁꢆ fꢄꢅ ꢁꢀiꢂ ꢆꢅꢁiꢈꢊꢇ ꢈꢆn bꢇ ꢆꢈꢈꢇꢂꢂꢇꢃ ꢆꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2018.1483931.
©
2018 Infꢄꢅmꢆ uK limiꢁꢇꢃ, ꢁꢅꢆꢃing ꢆꢂ tꢆyꢊꢄꢅ & Fꢅꢆnꢈiꢂ Gꢅꢄꢉp

2
S.ꢀW. YUAN ET AL.
of which is Cynanchum acutum subsp. sibiricum (CASS) (Mijit and Pan 2004). Many herbs of
genus Cynanchum have been used as antitussive and expectorants in China for a long time
and CASS is traditionally used in Uygur medicine for the treatment of dysentery and diarrhea
diseases (Mijit and Pan 2004; Pharmacopoeia of the People’s Republic of China’s 2010). In
prior reports, essential oils, flavonoids, alkaloids and C steroidal glycosides have been iso-
2
1
lated from the genus Cynanchum (Zhang and Zhou 1983; Yu et al. 2015; Yildiz et al. 2017).
About the constituents of CASS, only 4 C steroidal glycosides and 6 flavonoid glycosides
2
1
have been reported (Tursunova et al. 1975;Yildiz et al. 2017) and the pharmacological activity
of this plant remains unclear. In our search for the anti-complementary agents from tradi-
tional medicines, an ethanolic extract of the CASS exhibited anti-complementary activity.
Thus, a phytochemical investigation on this plant led to the isolation and characterization
of a novel dimeric flavonol glycoside, Cynanflavoside A (1), together with six analogues (2–7)
from the n-butyl alcohol extract of CASS. Additionally, the anti-complementary activity of
compound 1–7 was evaluated through the classical pathway.
2
. Results and discussion
Compound 1 was obtained as yellow amorphous powder. The HR-ESI-MS gave m/z 1211.3039
+
[
M + Na] , in consistent with the molecular formula C H D O (a couple of H protons were
5
4
56
2
30
irreversibly replaced by D in the Methanol-d after long time placement). Its UV spectrum
4
(
3
λmax 268 and 349 nm) was indicative of its flavonol skeleton. The IR absorption bands at
−1
145, 1658, 1610, 1505 and 1402 cm , indicated the presence of hydroxyl groups, carbonyl
1
13
groups and aromatic rings in the molecule. The H NMR and C NMR spectra (Table S1)
showed two sets of A B -type overlapping doublets, one set at δ 8.07(2H, m)/δ 132.1, δ
2
2
H
C
H
6
.91(2H, m)/δ 116.1, and the other at δ 8.06(2H, m)/δ 131.9, δ 6.89(2H, m)/δ 116.0, which
C H C H C
are attributed to two AA’BB’ systems in two para-substituted aromatic rings. The above
detailed analysis of the 1D and 2D NMR data, it was possible to conclude that one set
belonged to the 2 ’, 6’and 3 ’, 5’protons of the B (unit I)-ring and the other set to the 2 ’, 6’and
3
6
’, 5’ protons of the other B (unit II)-ring. The two singlets at δ 6.38 (1H, s)/δ 94.7 and δ
H C H
.07 (1H, s)/δ 99.7 are characteristic of protons H-8 (unit I), and H-6 (unit II) of the two fla-
C
vones moieties, respectively (The protons of H-8 (unit I) and H-6 (unit II) were irreversibly
replaced by D in the Methanol-d after long time placement, and the signals of H-8 (unit I)
4
and H-6 (unit II) were still not appeared when the Methanol-d was replaced by DMSO). The
4
fact that the molecule is non-symmetrical and that the A (unit I) and A (unit II)-ring protons
did not show any coupling, meant that the interflavonoid linkage must be of the
Spectaflavoside A type (6 (unit I)-8(unit II)) (Sivasothy et al. 2012). These spectral features
imply that compound 1 was a dimer of kaempferol, in which the interflavonol linkage was
the type 6 (unit I)-8 (unit II) (Ofman et al. 1995; Sivasothy et al. 2012), and both units were
very similar to kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (2) (Wu
1
13
et al. 2009). With the help of the H NMR, C NMR and HSQC spectra of compound 1, we
confirmed four anomeric signals of sugars at δ 5.82 (H-1’’(unit I), 1H, d, J = 7.6 Hz)/δ 100.2
H
C
(
C-1’’(unit I)), δ 5.24 (H-1’’’(unit I), 1H, s)/δ 102.9 (C-1’’’(unit I)), δ 5.76(H-1’’ (unit II), 1H, d,
H C H
J = 7.6 Hz)/δ 99.7 (C-1’’(unit II)) and δ 5.20 (H-1’’’(unit II), 1H, s)/δ 102.6 (C-1’’’(unit II)). The
C
H
C
configurations of two β-conformation glucose units were deduced by analysis the coupling
constants of the anomeric H-atoms at δ 5.82 (1H, d, J = 7.6 Hz), δ 5.76 (1H, d, J = 7.6 Hz)
H
H

NATURAL PRODUCT RESEARCH
3
1
13
and the H and C NMR spectra data of compound 1. Two anomeric proton signals (δ
H
1
13
5
.24/5.20, each s), two methyl signals (δ 0.97/0.95, overlapping), and the H and C NMR
H
data of compound 1 revealed two α-conformation rhamnose units. The absolute configu-
ration of sugars was confirmed to be D-glucose and L-rhamnose by acid hydrolysis and TLC
analysis. The location of two sugar residues were arranged by the HMBC spectrum, in which
the anomeric protons δ 5.82 (1H, d, J = 7.6 Hz, H-1’’) and δ 5.24 (1H, s, H-1’’’) exhibited
H
H
connectivities with δ 134.4 (C-3) and δ 80.7 (C-2’’), respectively, so the glucose was linked
C
C
to the flavonoid core at C-3 position, and the rhamnose was linked to the glucose core at
C-2’’position in both unit I and II. Consequently, the structure of compound 1 was concluded
to be kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside-(I-6,II-8)-kaemp-
ferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (named Cynanflavoside A).
Dimeric flavonol glycosides constitute a new class of bisflavonol glycosides, and there are
only two types, Kunzeagin A and Spectaflavoside A, to be reported till present. Unlike
Kunzeagin A in which a methylene bridge connects its two flavonol glycoside monomers,
similar to Spectaflavoside A, the two flavonol glycoside monomers in Cynanflavoside A were
linked together by a C-C bond (Ito et al. 2004; Sivasothy et al. 2012).
kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (2) (Wu et al. 2009),
quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (3) (da Silva et al. 2000),
kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside (4) (Ogunlana and
Ogunlana 2015), quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside (5) (Kazuma
et al. 2003), kaempferol-3-O-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside (6) (Lee and
Der Marderosian 2010) and quercetin-3-O-galactoside (7) (Ma et al. 2007) were also isolated
from the n-butyl alcohol extract of CASS. This paper is the first report of compounds 1, 3,
5
–7 from CASS.
Compounds 1–7 were evaluated for in vitro anti-complementary activity, but only com-
pound 5 showed anti-complementary activity with CH values of 0.33 mM.
5
0
3
. Experimental
3
.1. General
1
13
H NMR (600 MHz, 400 MHz), C NMR (150 MHz, 100 MHz) and 2D NMR (HSQC, HMBC) spec
-
tra were recorded at room temperature on DRX-600 spectrometer and DRX-400 spectrometer
Bruker, Germany) in CD OD and DMSO. UV spectra were measured on a Shimadzu UV-2600
(
3
spectrophotometer in absolute MeOH. IR spectra were recorded using an Avatar 360 FT-IR
ESP spectrometer (Nicolet, USA) at room temperature. Optical rotations were measured on
a JASCO P-1020 polarimeter. HR-ESI-MS spectra were acquired using an Agilent 6210 ESI/
TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). L-rhamnose and
D-glucose (Alfa Aesar, Great Britain). Analytical TLC were run on silica gel plates (GF254, Yantai
Institute of Chemical Technology, Yantai, China). Spots on the plates were observed under
UV light and visualized by spraying with 10% H SO , followed by heating. Column chroma-
2
4
tography was performed on silica gel (200~300 mesh and 300~400 mesh; Qingdao Marine
Chemical Factory, Qingdao, China), Lichroprep RP18 gel (40~60 μm, Merck, Darmstadt,
Germany) and reversed-phase MCI gel (CHP20P, Mitsubishi Chemical Inc., Fukuoka, Japan).
Distilled solvents were used for chromatographic separations.

4
S.ꢀW. YUAN ET AL.
3
.2. Plant material
The drugs were collected in August 2015 in Shihezi region of Xinjiang Province, P. R. China
and identified as Cynanchum acutum subsp. sibiricum (willd.) Rech.f. by associate professor
Peng Li, School of Pharmacy of Shihezi University. The voucher specimens (WQ-CASS-15–1)
were deposited in the Herbarium, School of Pharmacy, Shihezi University.
3
.3. Extraction and isolation
CASS (aerial parts) was shade dried and powdered. Powdered plant material (14.8 kg) was
extracted with 95% EtOH (3 × 20 L, each for 7 d) at room temperature and filtered. The EtOH
extract was evaporated in vacuo to give a residue (900 g), a portion of which (850 g) was
suspended in H O (1.0 L) and partitioned successively with petroleum ether, ethyl acetate,
2
and n-butyl alcohol (each 3 × 2 L). The n-butyl alcohol extract (178 g) was chromatographyed
on a Silica gel (200~300 mesh, 2.5 kg, 14 × 120 cm) column, eluting successively with chlo-
roform-methanol (from 100:0 to 0:100) to yield ten fractions based onTLC profiles. Compound
7
(100 mg) was obtained as a yellow powder from Fraction 2 (10.3 g), which was purified by
column chromatography using MCI gel with MeOH-H O (from 10:90 to 90:10). Fraction 5
2
(
12.0 g) was subjected to MCI column using MeOH-H O (from 10:90 to 60:40) as eluent,
2
followed by repeated column chromatography on RP gel with MeOH-H O (35:65), to give
1
8
2
2
(5 mg), 4 (35 mg) and 6 (6 mg). Fraction 8 (6 g) was subjected to MCI column using MeOH-
H O (from 10:90 to 50:50) as eluent, followed by repeated column chromatography on RP
2
18
gel with MeOH-H O (20:80), to yield 1 (2.5 mg), 3 (18 mg) and 5 (20 mg).
2
3
.4. Cynanflavoside A
2
2
−1
Yellow amorphous powder; [α] : −105 (c = 0.02, MeOH); IR (KBr) λ cm : 3145, 1658, 1610,
D
max
1
1
505, 1402, 1178, 1066; H NMR (CD OD, 600 MHz): Unit I: δ 8.07 (2H, m, H-2′, 6′), 6.91 (2H,
3
H
m, H-3′, 5′), 6.38 (1H, s, H-8), 5.82 (1H, d, J = 7.6 Hz, H-1’’), 5.24 (1H, s, H-1’’’), 4.09 (1H, m, H-5’’’),
4.01 (1H, s, H-3’’’), 3.76 (1H, m, H-2’’’), 3.68 (1H, m, H -6a’’), 3.62 (1H, m, H-3’’), 3.58 (2H, m, H-2’’,
4’’), 3.52 (1H, m, H-6b’’), 3.37 (1H, m, H-4’’’), 3.25 (1H, m, H-5’’), 0.97 (3H, m, H-6’’’); Unit II: δH
8.06 (2H, m, H-2′, 6′), 6.89 (2H, m, H-3′,5′), 6.07 (1H, s, H-6), 5.76 (1H, d, J = 7.6 Hz, H-1’’), 5.20
(
1H, s, H-1’’’), 4.07 (1H, m, H-5’’’), 3.96 (1H, m, H-3’’’), 3.74 (1H, m, H-2’’’), 3.63 (1H, m, H-6a’’),
3
.62 (1H, m, H-3’’), 3.57 (2H, m, H-2’ ’, 4’’), 3.52 (1H, m, H-6b’’), 3.35 (1H, m, H-4’’’), 3.25 (1H, m,
1
H-5’’), 0.95 (3H, m, H-6’’’). H NMR (DMSO-d , 600 MHz): Unit I: δ 7.97 (2H, m, H-2′, 6′), 6.84
6
H
(
3
2H, m, H-3′,5′), 5.70 (1H, m, H-1’’), 5.08 (1H, m, H-1’’’), 3.73 (1H, m, H-3’’’), 3.72 (1H, m, H-5’’’),
.56 (1H, m, H -6a’’), 3.47 (2H, m, H-5’ ’, 4’’’), 3.41 (1H, m, H-2’’), 3.35 (1H, m, H-3’’), 3.30 (1H, m,
H-6b’’), 3.13 (1H, m, H-2’’’), 3.09 (1H, m, H-4’’), 0.74 (3H, m, H-6’’’); Unit II: δ 7.97 (2H, m, H-2′,
H
6′), 6.84 (2H, m, H-3′,5′), 5.67 (1H, d, J = 7.6 Hz, H-1’’), 5.08 (1H, m, H-1’’’), 3.73 (1H, m, H-3’’’),
3
.72 (1H, m, H-5’’’), 3.54 (1H, m, H -6a’’), 3.45 (2H, m, H-5’ ’, 4’’’), 3.40 (1H, m, H-2’’), 3.35 (1H, m,
1
3
H-3’’), 3.30 (1H, m, H-6b’’), 3.12 (1H, m, H-2’’’), 3.09 (1H, m, H-4’’), 0.74 (3H, m, H-6’’’) C NMR
(
CD OD, 151 MHz): Unit I: δ 179.3 (C, C-4), 166.0 (C, C-7), 163.8 (C, C-5), 161.3 (C, C-4’), 158.5
3
C
(C, C-9), 158.4 (C, C-2), 134.4 (C, C-3), 132.1 (CH, C-2 ’, 6’), 123.4 (C, C-1’), 116.1 (CH, C-3 ’, 5’),
1
7
6
09.8 (C, C-6), 105.8 (C, C-10), 102.9 (CH, C-1’’’), 100.2 (CH, C-1’’), 94.7 (CH, C-8), 80.7 (CH, C-2’’),
9.0 (CH, C-3’’), 78.6 (CH, C-5’’), 74.1 (CH, C-4’’’), 72.4 (CH, C-4’’), 72.3 (CH, C-3’’’), 71.9 (CH, C-2’’’),
9.9 (CH, C-5’’’), 62.7 (CH , C-6’’), 17.5 (CH , C-6’’’); Unit II: δ 178.0 (C, C-4), 165.9 (C, C-7), 163.0
2
3
C

NATURAL PRODUCT RESEARCH
5
(
(
C, C-5), 161.0 (C, C-4’), 158.3 (C, C-9), 157.2 (C, C-2), 134.1 (C, C-3), 131.9 (CH, C-2 ’, 6’), 123.1
C, C-1’), 116.0 (CH, C-3 ’, 5’), 105.9 (C, C-10), 102.6 (CH, C-1’’’), 99.7 (CH, C-6), 99.7 (CH, C-1’’),
9
7
9.6(C, C-8), 80.1 (CH, C-2’’), 78.9 (CH, C-3’’), 78.4 (CH, C-5’’), 74.0 (CH, C-4’’’), 72.3 (CH, C-4’’),
2.2 (CH, C-3’’’), 71.8 (CH, C-2’’’), 69.7 (CH, C-5’’’), 62.6 (CH , C-6’’), 17.5 (CH , C-6’’’). HR-ESI-MS:
2
3
+
m/z 1211.3039 [M + Na] (Calcd for C H D O Na 1211.3031).
5
4
56
2
30
3
.5. Anti-complementary activity
Assays were based on the degree of hemolysis of erythrocytes after complement activation
Xu et al. 2007). Each tested sample was dissolved in dimethyl sulfoxide (DMSO) and then
(
2
+
diluted with Barbital buffer saline (BBS) (pH 7.0, containing 0.1% gelatin, 0.5 mM Mg and
2
+
0
1
.15 mM Ca ) to various concentrations. The final concentration of DMSO did not exceed
% and showed no interference with the anti-complementary activity. Sensitized erythro-
cytes (EA) were prepared by incubating sheep erythrocytes (4.0 × 108 cells/mL) with rabbit
anti-sheep erythrocyte antibody. Various dilutions of tested samples (200 μL) were mixed
with 200 μL of diluted guinea pig serum (1:80) and 200 μL EA. The mixture was incubated
at 37 °C for 30 min and centrifuged at 1800 g for 5 min. The optical density of the supernatant
(
200 μL) was measured at 405 nm with a spectrophotometer (Wellscan MK3, Labsystems
Dragon). Anti-complementary activity was determined as the mean of triplicate measure-
ments and was expressed as CH , which corresponded to the concentration that inhibited
5
0
50% of hemolysis of the complement-dependent lysis control. Heparin sodium salt was used
as the positive control. And the sample controls, the blank control, the complement-de-
pendent lysis control were all prepared under the same conditions.
3
.6. Determination of absolute configuration of sugars
The absolute configuration of sugars was carried out by acidic hydrolysis and TLC analysis.
Compounds 1 (2.5 mg) was hydrolyzed with concentrated AcOH (3.5 mL), concentrated HCl
(
1 mL), and H O (5.5 mL) at 100 °C for 2 h. The mixture was extracted with EtOAc, and the
2
remaining aqueous phase evaporated to dryness. TLC analysis was performed over silica gel
with n-butyl alcohol-methanol-chloroform-acetic acid-water (12.5:5:4.5:1.5:1.5, v/v/v/v/v) as
mobile phase and the result consistent with authentic samples of D-glucose and L-rhamnose
(
Figure S10). Compound 1 revealed D-Glc and L-Rha.
4
. Conclusion
A novel dimeric flavonol glycoside, Cynanflavoside A (1), together with six known flavonol
glycosides were isolated from the n-butyl alcohol extract of CASS. Compounds 1, 3, 5–7
were isolated from this plant for the first time. The anti-complementary activity of com-
pounds 1–7 was evaluated through the classical pathway and compound 5 showed mod-
erate anti-complementary activity.
Disclosure statement
No potential conflict of interest was reported by the authors.

6
S.ꢀW. YUAN ET AL.
Funding
This work was supported by the National Natural Science Foundation of China.
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