Three new lignanosides from the aerial parts of Lespedeza cuneata

Article abstract of DOI:10.1080/10286020.2016.1187603

Three new lignanosides (+)-(8S,7′S,8′S)-burselignan-9′-O-β-d-glucopyrano side (1), (+)-(8R,7′S,8′R)-isolariciresinol-9′-O-β-d-fucopyranoside (2), (-)-(8S, 7′R,8′R)-methoxyisoariciresinol-9′-O-α-l-rhamnoside (3), along with four known compounds, were isola

Full text of DOI:10.1080/10286020.2016.1187603

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Three new lignanosides from the aerial parts of
Lespedeza cuneata
Chuang-Feng Zhang, Jian Zhou, Jing-Zhi Yang, Chuang-Jun Li, Jie Ma, Dan
Zhang, Li Li & Dong-Ming Zhang
To cite this article: Chuang-Feng Zhang, Jian Zhou, Jing-Zhi Yang, Chuang-Jun Li, Jie
Ma, Dan Zhang, Li Li & Dong-Ming Zhang (2016): Three new lignanosides from the
aerial parts of Lespedeza cuneata, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2016.1187603
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1187603
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Date: 23 June 2016, At: 17:35

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2016.1187603
Three new lignanosides from the aerial parts of Lespedeza
cuneata
Chuang-Feng Zhanga, Jian Zhoua,b, Jing-Zhi Yanga, Chuang-Jun Li, Jie Maa,
Dan Zhanga, Li Lia and Dong-Ming Zhanga
astate Key laboratory of Bioactive substance and function of natural Medicines, chinese academy of Medical
b
sciences and Peking union Medical college, Beijing 100050, china; department of Pharmacy, the first
affiliated hospital of nanchang university, nanchang 330006, china
ABSTRACT
ARTICLE HISTORY
received 11 March 2016
accepted 4 May 2016
Three new lignanosides (+)-(8S,7′S,8′S)-burselignan-9′-O-β-d-
glucopyrano side (1), (+)-(8R,7′S,8′R)-isolariciresinol-9′-O-β-d-fucopyra-
noside (2), (-)-(8S, 7′R,8′R)-methoxyisoariciresinol-9′-O-α-l-rhamnoside
(3), along with four known compounds, were isolated from the aerial
parts of Lespedeza cuneata (Dum.Cours.) G.Don. The fucopyrano-
side has not been reported in this genus previously. Their structures
and absolute configurations were elucidated on the basis of
comprehensive spectroscopic analyses (UV, IR, HR-ESI-MS, 1D and
2D NMR, CD), as well as by comparison with known analogues in the
literature. Compounds 2 and 6 showed moderate hepatoprotective
activities.
KEYWORDS
Lespedeza cuneata;
lignanoside;
hepatoprotective activities
1. Introduction
Lespedeza cuneata (Dum.Cours.) G.Don is an annual herbaceous plant distributed in many
countries, which named ′ye guan men′ in Chinese, is a very important traditional medicine,
and has been used in the treatments of diabetes [1], hematuria, insomnia and malnutrition [2],
however, the studies on the constituents are very few. Phytochemical studies on the aerial
parts of L. cuneata just led to isolation of isoflavanones, isoflavones, sterols, triterpenoids,
flavonols, flavonol glycosides, and flavone C-glycosides, such as isovitexin, isoorientin,
vicenin-2, and lucenin-2 [3–5]. In our continuing research for constituents from this
important medicinal plant, three new compounds (Figure 1), and four known compounds
were isolated. eir structures were elucidated by various spectroscopic methods (UV, IR,
HR-ESI-MS, 1D and 2D NMR, CD). e isolation and structural elucidation of the new
compounds were described in this paper.
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. e UV spectrum showed absorp-
tion maximum at 204, 284 nm. In the IR spectrum, absorption bands at 3361, 2919, 1601,
CONTACT chuang-feng Zhang
zhangdm@imm.ac.cn
© 2016 informa uK limited, trading as taylor & francis Group

2
C.-F. ZHaNG ET aL.
6
3
7
9
H3CO
HO
H3CO
HO
H3CO
HO
5
4
OH
O
OH
O
OH
O
1
2
8
8'
7'
1'
OH
OH
OH
OH
9'
2'HO
O
O
O
6'
5'
OH
HO
OH
OH
HO
OCH3
3'
OCH3
H3CO
OCH3
4'
OH
3
OH
1
OH
2
Figure 1. structures of compounds 1–3.
H3CO
HO
H3CO
HO
H3CO
HO
OH
O
OH
O
OH
O
OH
OH
OH
OH
O
O
O
HO
OH
OH
OH
HO
OCH3
HO
H3CO
OCH3
OCH3
OH
3
OH
1
OH
2
Figure 2. Key hMBc(h→c) correlations of compounds 1–3.
1513, and 1450 cm−1 were observed. ese data indicated the presence of hydroxy and
benzene in 1. e molecular formula was determined to be C26H34O11 on the basis of the
pseudo-molecular ion at m/z 545.2005 [M + Na]+. In the 1H NMR spectrum of 1, a set of
AMX-type signals at δH 6.17 (1H, dd, J = 1.2 8.4 Hz, H-6′), 6.57 (1H, d, J = 8.4 Hz, H-5′),
6.82 (1H, d, J = 1.2 Hz, H-2′) were observed, which suggested the existence of 1,3,4-trisub-
stituted benzene ring. Additionally, two methoxy signals at δH 3.73 (3H, s, OMe-5), 3.70 (3H,
s, OMe-3′) and two aromatic proton signals at δH 6.65 (1H, s, H-6), 6.24 (1H, s, H-3) were
demonstrated. e 13C NMR spectrum showed 26 carbon signals. Except for 12 aromatic
carbon signals, six saccharide moiety carbons, two methoxy signals, and a fraction C6H10O2
with one degree of unsaturation were lef. ese NMR spectroscopic data suggested 1 to
be an 2,7′-cyclolign-9,9′-diol system lignan. e anomeric proton signal at δH 4.14 (1H, d,
J = 7.6 Hz, Glc-1) in the 1H NMR spectrum and carbon resonances at δC 102.9, 76.9, 70.9,
76.7, 73.9, 61.1 and the positive optical rotation demonstrated that the sugar was β-d-
glucose. e β-d-glucose was localized at C-9′, which could be confirmed by the HMBC
spectrum (Figure 2). An enhancement of the proton signals H-2′/H-6′ on irradiation of H-8,
the enhancement of H-7′ on irradiation of H-8′ and at the H-9′a, the enhancement of 9′b on
irradiation of H-8 revealed that H-8 and H-7′ are on the different side, and H-8′ /H-7′ are
on the same side (Figure 3). e S-configuration of C-7′ was confirmed as the CD spectrum
showed positive Cotton effect at 275 nm and negative Cotton effect at 293 nm [6]. erefore,
the structure of 1 was elucidated as (+)-(8S,7′S,8′S)-burselignan-9′-O-β-d-glucopyranoside.
Compound 2 was obtained as a white powder, whose molecular formula was determined
to be C26H34O10 on the basis of HR-ESI-MS at m/z 529.2066 [M + Na]+. e UV spectrum
showed absorption maximum at 204, 284 nm. e IR spectrum indicated absorption bands
for hydroxyl group (3353 cm−1), and benzene ring (1600, 1513, and 1448 cm−1). In the 1H
NMR spectrum of 2, a set of AMX-type signals, two methoxy signals, and two aromatic

JoURNaL oF aSIaN NaTURaL PRoDUCTS RESEaRCH
3
8
8'
6'
7'
2'
1
NOE
Figure 3. important noe (h↔h) enhancement of compound 1.
proton signals were also observed. e 13C NMR spectrum showed 26 carbon signals (includ-
ing 12 aromatic carbon signals, six saccharide moiety carbons and two methoxy signals).
ese NMR spectroscopic data suggested compound 2 has the same skeleton as compound
1. e anomeric proton signal at δH 3.89 (1H, d, 6.6) in the 1H NMR spectrum and carbon
resonances at δC 104.3, 73.5, 71.1, 70.3, 69.8, 16.7 [7] and the positive optical rotation demon-
strated that the sugar was β-d-fucose. e β-d-fucose was localized at C-9′, which could be
confirmed by the HMBC spectrum (Figure 2). An enhancement of the proton signal H-7′ on
irradiation of H-8 and the enhancements of H-9′a, 9′b on irradiation of H-7′ revealed that
H-8 and H-7′ are on the same side, and H-8′/H-7′ are on the different side. In combination
with the positive Cotton effect at 272 nm and negative Cotton effect at 292 nm in the CD
spectrum, the absolute configurations of C-7′ were determined as S. us, the structure of
2 was concluded to be (+)-(8R,7′S,8′R)-isolariciresinol-9′-O-β-d-fucopyranoside.
Compound 3, an white amorphous powder, had the molecular formula C27H36O11 as
established by HR-ESI-MS at m/z 559.2159 [M + Na]+. e UV spectrum showed absorption
maximum at 204, 284 nm. e IR spectrum similarly revealed the presence of hydroxyl
and benzene ring. rough the 1H NMR spectrum, four aromatic proton signals revealed
that two benzene rings were tetrasubstituted. ree methoxy signals were also observed.
e 13C NMR spectrum showed 27 carbon signals (including 12 aromatic carbon signals,
six saccharide moiety carbons and three methoxy signals). ese NMR spectroscopic data
also suggested compound 3 have the same skeleton as compound 1. e anomeric proton
signal at δH 4.47 (1H, br s) in the 1H NMR spectrum and carbon resonances at δC 100.3, 71.9,
70.9, 70.6, 68.6, 17.9 demonstrated that the sugar was α-l-rhamnose. e α-l-rhamnose
was localized at C-9′, which could be confirmed by the HMBC spectrum (Figure 2). An
enhancement of the proton signal H-7′ on irradiation of H-8, the enhancement of H-7′ on
irradiation of H-8′, and the enhancement of H-9′a, 9′b on irradiation of H-9a, 9b revealed
that H-8 and H-7′ are on the same side, and H-8′ and H-7′ are on the same side. Meanwhile,
negative Cotton effect at 271 nm and positive Cotton effect at 291 nm were exhibited in the
CD spectrum, indicating the 7′R configuration for 3. Consequently, the structure of 3 was
determined as (+)-(8S,7′R,8′R)-methoxyisoariciresinol-9′-O-α-l-rhamnoside.
Compounds 1–7 were tested for hepatoprotective activities against APAP (acetami-
nophen)-induced toxicity in HepG2 (human hepatocellular liver carcinoma cell line) cells,
using the hepatoprotective activity drug bicyclol as the positive control [8]. At 10 μM,

4
C.-F. ZHaNG ET aL.
compounds 2 and 6 reduced APAP-induced HepG2 cells damage by increasing the survival
rate, and they are 61.31% (p < 0.01) and 60.76% (p < 0.01), compared with bicyclol’s 57.95%.
As shown in Table 2, compounds 2 and 6 exhibited moderate hepatoprotective activities.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a JASCO P2000 automatic digital polarimeter
(JASCO, Tokyo, Japan). UV spectra were recorded on a JASCO V-650 spectrophoto
meter (JASCO, Tokyo, Japan). CD spectra were measured on a JASCO J-815 spectropola-
rimeter (JASCO, Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 spectrometer
using an FT-IR microscope transmission method (ermo Electron, Madison, WI, USA).
NMR spectra were acquired with Bruker AVIIIHD 600 (Bruker, Billerica, MA, USA), VNS-
600 (Varian, Palo, CA, USA), and Mercury-400 spectrometers (Varian, Palo, CA, USA) in
DMSO-d6 and MeOH-d4. HRESIMS were collected on an Agilent 1100 series LC/MSD
ion trap mass spectrometer (Agilent, Waldbronn, Germany). MPLC system was composed
of two C-605 pumps (Büchi, Flawil, Switzerland), a C-635 UV detector (Büchi, Flawil,
Switzerland), a C-660 fraction collector (Büchi, Flawil, Switzerland), and an ODS column
(450 mm × 60 mm, 50 μm, 400 g; YMC, Kyoto, Japan). Semi-preparative HPLC was con-
ducted using a Shimadzu LC-6AD instrument with an SPD-20A detector (Shimadzu, Kyoto,
Japan) and a Daicel Chiralpak AD-H column (250 × 10 mm, 5 μm). Preparative HPLC was
also performed on a Shimadzu LC-6AD instrument (Shimadzu, Kyoto, Japan) with a YMC-
Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography (CC) was performed
with silica gel (200–300 mesh, Qingdao Haiyang Chemical Inc., Qingdao, China), SF-PRP
512A (100–200 mesh, Beijing Sunflower and Technology Development Co., Beijing, China),
ODS (50 μm, YMC, Kyoto, Japan), and Sephadex LH-20 (Pharmacia, Uppsala, Sweden).
TLC was carried out on glass precoated silica gel GF254 plates. Spots were visualized under
UV light or by spraying with 10% sulfuric acid in EtOH followed by heating.
3.2. Plant material
e aerial parts of Lespedeza cuneata were collected in October 2013 in Xinyang city, Henan
Province, China. e plant material was identified by Professor Ceming Tan. A voucher
specimen is deposited at the Herbarium of the Institute of Material Medical, Chinese
Academy of Medical Sciences and Peking Union Medical College, China (No. ID-22276).
3.3. Extraction and isolation
e aerial parts of Lespedeza cuneata (21 kg) were extracted with 70% EtOH under reflux for
three times. Afer the solvent was evaporated under reduced pressure, the residue (3.68 kg)
was submitted to diatomite column, eluting with ether, CHCl3, EtOAc, CH3COCH3, 95%
EtOH, and 70% EtOH, to afford six corresponding fractions (Fr. 1-6). Fr. 4 (148 g) was
subjected to CC over polyamide resin eluted with H2O and EtOH-H2O (30, 60, and 95%) to
obtain four major fractions (A, B, C, D). Fr. A (77.2 g) was then divided into four subfrac-
tions (A1–A4) via D101 macroporous adsorption resin CC eluted with H2O and EtOH-H2O

JoURNaL oF aSIaN NaTURaL PRoDUCTS RESEaRCH
5
(30, 60, and 95%). Fr. A2 (19.8 g) was fractionated by Sephadex LH-20 CC eluted with
CH3OH to furnish sixteen fractions (A2-1–A2-16), of which fraction A2–2 (7.4 g) was
further separated by silica gel CC eluted with CHCl3-MeOH (4:1) to yield four fractions (A2-
2-1–A2-2-4). Separation of Fr. A2-2-2 (2.1 g) over reversed-phase C18 silica gel CC eluted
with MeOH-H2O (20–60%) to yield three fractions (a1~a3). Fr. a2 (91 mg) was purified by
semi-preparative HPLC (3.0 ml/min, 25% MeOH-H2O isocratic elution, detected at 210,
tR = 31.6 min and 47.2 min) to yield compounds 1 (7 mg) and 2 (15 mg). Fr. a3 (64 mg) was
purified by semi-preparative HPLC (3.0 ml/min, 30% MeOH-H2O isocratic elution, detected
at 210 nm, tR = 22.4 min) to yield compound 3 (6 mg). We also got five known compounds
from Fr. a2 and Fr. a3, namely, (-)-lyoniresinol-3α-O-β-d-glucopyranoside [9](4), (+)-
isolariciresinol-9′-O-β-d-glucopyranoside[10](5), (-)-5’-methoxyisolariciresinol-3-O-β-d-glu-
copyranoside [11](6), ( )-5,5′-dimethoxylariciresi nol-4′-O-β-d-glucopyranoside (7) [12].
3.3.1. (+)-(8S,7′S,8′S)-Burselignan-9′-O-β-d-glucopyranoside (1)
White amorphous powder,[ꢀ]2D0 + 85.5(c 0.12, MeOH); UV (MeOH) λmax (logε): 204 (3.16),
284 (2.19) nm; IR (KBr) νmax: 3361, 2919, 1601, 1513, 1450, 1372, 1248, 1162, 1121, 1078,
1031 cm−1; CD (MeOH): Δε218nm + 7.52, Δε238nm + 8.40, Δε275nm + 3.49, Δε297nm − 2.0; For
1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 545.2005 [M + Na]+
(calcd for C26H34O11Na, 545.1993).
3.3.2. (+)-(8R,7′S,8′R)-Isolariciresinol-9′-O-β-d-fucopyranoside (2)
White amorphous, [ꢀ]2D0 + 18.9 (c 0.1, MeOH); UV (MeOH) λ (logε): 204 (3.73), 284
max
(2.22) nm; IR (KBr) νmax: 3353, 2927, 1668, 1600, 1513, 1448, 1372, 1272, 1174, 1127, 1069,
1031 cm−1; CD (MeOH): Δε216nm + 5.25, Δε238nm + 2.44, Δε272nm + 2.49, Δε292nm − 2.72; For
1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 529.2066 [M + Na]+
(calcd for C26H34O10Na, 529.2044).
3.3.3. (-)-(8S,7′R,8′R)-Methoxyisoariciresinol-9′-O-α-l-rhamnoside (3)
White amorphous powder,[ꢀ]2D0 − 72.8(c 0.17, MeOH); UV (MeOH) λmax (logε): 204 (3.41),
284 (3.12) nm; IR (KBr) νmax: 3377, 2925, 1672, 1604, 1513, 1459, 1426, 1377, 1325, 1243,
1116, 1050 cm−1; CD (MeOH): Δε222 nm − 4.18, Δε241 nm − 5.29, Δε271 nm − 2.17, Δε291 nm + 0.57;
For 1H and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS: m/z 559.2159 [M + Na]+
(calcd for C27H36O11Na, 559.2150).
3.4. Determination of absolute configurations of the sugar moieties in 1–3
Compound 1 (5.0 mg) was dissolved in H2O (2 ml) with snailase at 37 °C for 4 h. Afer
extraction with EtOAc (3 × 2 ml) to remove the aglycone, the aqueous layer was evaporated
to furnish a monosaccharide residue. e residue was purified on preparative silica gel
thin layer chromatography [CHCl3-MeOH-H2O (2:1:0.5)] to afford glucose (1.3 mg). e
glucose was subjected to the test of optical rotations to confirm the D-form in compound
1. Hydrolysis of 2–3 and sugar identification were performed according to the procedure
described for 1, which lead to d-fucose (1.1 mg) and l-rhamnose (1.5 mg). e[ꢀ]2D0 values
of d-glucose, d-fucose, and l-rhamnose were + 52.1 (c 0.13, H2O),+73.4 (c 0.11, H2O),
and + 7.6 (c 0.15, H2O), respectively.

6
C.-F. ZHaNG ET aL.

JoURNaL oF aSIaN NaTURaL PRoDUCTS RESEaRCH
7
Table 2. hepatoprotective effects of compounds 1–7 (1 × 10−5 mol/l) against aPaP-induced toxicity in
hepG2 cells.
Compound
oD(Mean SD)
Cell survival rate (percentage of normal)
control
aPaP 8 mM
2.228 0.067
1.257 0.024***
1.207 0.019
1.366 0.049##
1.157 0.023
1.158 0.009
1.223 0.008
1.354 0.032##
1.147 0.004
1.343 0.045##
100.00
56.42
54.19
61.31
51.94
51.99
54.89
60.76
51.50
57.95
1
2
3
4
5
6
7
Bicyclola
***p＜0.001, compared with control; #p＜0.05, ##p＜0.01, compared with model (aPaP). aPositive control substance.
Compounds 1–3 (2.0 mg) were separately dissolved in 2 M HCl-H2O (2 ml) and heated at
70 °C for 12 h. Afer extraction with EtOAc (3 × 2 ml) to remove the aglycone, the aqueous
layer was evaporated to afford a neutral residue. e dried sugar residue was diluted in anhy-
drous pyridine (1 ml), to which l-cysteine methyl ester hydrochloride (2 mg) was added.
e mixture was stirred at 60 °C for 2 h, and then treated with N-trimethylsilylimidazole
(0.2 ml). e mixture was then heated to dryness at 60 °C for another 2 h. e dried
reactant was partitioned between n-hexane (2 ml) and H2O (2 ml) for three times. e
n-hexane layer was concentrated (1 ml) and subjected to GC analysis (column: HP-5,
60 m × 0.25 mm × 0.25 μm, Dikma; detector: FID; detector temperature: 280 °C; injector
temperature: 250 °C; carrier: N2; Temperature-programmed: From 200 °C to 280 °C in 2 min
and maintained the final temperature 30 min). e saccharides of compounds 1–3 were
determined by GC analysis of the derivatives to be d-glucose, d-fucose, and l-rhamnose,
of which the retention times were 18.508, 15.107, and 14.696 min, respectively.
3.5. Hepatoprotective activity assay
Hepatoprotective activities against APAP induced toxicity of the seven compounds (1–7)
were examined in HepG2 (human hepatocellular liver carcinoma cell line) cells. Compounds
2 and 6 exhibited moderate hepatoprotective activities at 10 μM compared with bicyclol.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is project was financially supported by the National Mega-project for Innovative Drugs [grant
number 2012ZX09301002-002] and National Natural Science Foundation of China [grant number
81560632], [grant number 81202546].
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