Hepatoprotective glycosides from Leonurus japonicus Houtt.

Article abstract of DOI:10.1016/j.carres.2011.10.034

Two new phenylethanoid glycosides 1 and 2 named leonoside E and leonoside F, and one new sesquiterpene glycoside (3) identified as 7α (H)-eudesmane-4,11 (12)-diene-3-one-2β-hydroxy-13-β-d-glucopyranoside, together with seven known glycosides (4-10), were isolated from the aerial part of Leonurus japonicus Houtt. Their structures were elucidated on the basis of spectroscopic data and chemical evidence. When tested in in vitro assays, compounds 1, 2, 4, and 6 exhibited potent hepatoprotective activity against d-galactosamine-induced toxicity in HL-7702 cells at concentration of 1 × 10^-5 M.

Full text of DOI:10.1016/j.carres.2011.10.034

Carbohydrate Research 348 (2012) 42–46
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Hepatoprotective glycosides from Leonurus japonicus Houtt.
⇑
Yixiu Li, Zhong Chen, Ziming Feng, Yanan Yang, Jianshuang Jiang, Peicheng Zhang
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new phenylethanoid glycosides 1 and 2 named leonoside E and leonoside F, and one new sesquiter-
Received 15 September 2011
Received in revised form 21 October 2011
Accepted 23 October 2011
pene glycoside (3) identified as 7
a (H)-eudesmane-4,11 (12)-diene-3-one-2b-hydroxy-13-b-D-glucopy-
ranoside, together with seven known glycosides (4–10), were isolated from the aerial part of Leonurus
japonicus Houtt. Their structures were elucidated on the basis of spectroscopic data and chemical evi-
dence. When tested in in vitro assays, compounds 1, 2, 4, and 6 exhibited potent hepatoprotective activity
Available online 9 December 2011
against
D
-galactosamine-induced toxicity in HL-7702 cells at concentration of 1 ꢀ 10ꢁ5 M.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Leonurus japonicus Houtt.
Phenylethanoid glycosides
Sesquiterpene glycoside
Hepatoprotective activity
1. Introduction
pounds mentioned above included two new phenylethanoid glyco-
sides, and one new sesquiterpene glycoside. The known compounds
were identified as verbascoside (4),11 2-(3,4-dihydroxyphenethy)-
The genus Leonurus (labiatae) comprises more than 20 species
widespread in Eurasia, from Western Europe to China, and is used
as a traditional Chinese Medicine for regulating menstrual distur-
bance, invigorating blood circulation, diuretics, and dispel edema.1
Previous investigations of this genus revealed the presence of lab-
dane-type diterpenoids,2,3 phenylethanoid glycosides,4,5 iridoids,5
flavonoids,6 and cyclic peptides,7 some of them have shown to ex-
hibit anti-inflammatory,5 anti-platelet aggregation,8 and cytotoxic
activities.9 To systematically search the phytochemistry and bioac-
tivity of the high-polarity fraction of the plants of the genus Leonu-
rus, we investigated Leonurus japonicus Houtt., a species growing
Hebei Province of the People’s Republic of China.10 Two new phe-
nylethanoid glycosides, namely leonoside E and leonoside F, and
O-a-arabinopyranosyl-(1?2)-a-L-rhamnopyranosyl-(1?3)-6-O-b-
D
-glucopyranoside (5),12 cistanoside E (6),13 lavandulifolioside (7),4
staphylionoside E (8),14 citroside A (9),15 9-hydroxy-megastigma-
4,7-dien-3-one-9-O-glucopyranoside (10),16 respectively.
Compound 1 was obtained as a white powder, its molecular for-
mula, C26H40O16, was established on the basis of HRESIMS at m/z
631.2209 [M+Na]+ (calcd for 631.2214). Its IR spectra showed
absorption bands for hydroxyl groups (3372 cmꢁ1), –CH2–
(2932 cmꢁ1), aromatic rings (1592, 1513, and 1440 cmꢁ1). The 1H
NMR spectrum (Table 1) of 1 exhibited characteristic signals
belonging to 3-methoxyl-4-hydroxyphenylethanol moieties: one
set of ABX system [d 6.68 (1H, d, J = 2.0 Hz, H-2), 6.81 (1H, d,
J = 8.0 Hz, H-5), and 6.63 (1H, dd, J = 8.0, 2.0 Hz, H-6)], two diaste-
reotopic protons at d 3.56 (1H, dd, J = 11.5, 3.5 Hz) and d 3.89 (1H,
dd, J = 11.5, 3.0 Hz) of the side-chain of the aglycon moiety, and
one methoxy group at d 3.72 (3H, s). In addition, three signals of
anomeric protons appearing as d at d 4.21 (1H, J = 8.0 Hz), 5.25
(1H, J = 1.0 Hz), 4.26 (1H, J = 7.0 Hz), which were attributed to
Glu-10, Rha-100 and Ara-1000, respectively. This monosaccharide anal-
ysis was confirmed by acid hydrolysis, which afforded glucose,
rhamnose, and arabinose, identified by TLC. The 13C NMR spectrum
(Table 1) of 1 showed twenty-six carbon signals including nine car-
bons that could be assigned to the aglycon moiety, and seventeen
for three monosaccharide groups. An unambiguous determination
of the sequence and linkage sites was obtained from the HMBC cor-
relations (Fig. 2), in which the correlations of H-7 (d 2.70) with C-2
(d 116.2), C-6 (d 119.3), of H-10 (d 4.21) with C-8 (d 69.8), of H-100 (d
5.25) with C-30 (d 81.6), and of H-1000 (d 4.26) with C-200 (d 80.8)
one new sesquiterpene glycoside identified as 7
mane-4,11 (12)-diene-3-one-2b-hydroxy-13-b- -glucopyranoside,
were isolated, along with seven known compounds. The hepato-
protective activities evaluation of the isolated compounds has been
investigated.
a (H)-eudes-
D
2. Results and discussion
The plant extract was subjected to HP-20 macroporous resin
using a H2O–EtOH step gradient to give six fractions. The 15% and
30% fractions were further subjected to column chromatography
and preparative HPLC to yield ten compounds (Fig. 1). The new com-
⇑
Corresponding author. Tel: +86 10 63165231; fax: +86 10 63017757.
E-mail address: pczhang@imm.ac.cn (P. Zhang).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.10.034

Y. Li et al. / Carbohydrate Research 348 (2012) 42–46
43
14
1
9
2
HO
O
8
7
10
3
HO
HO
12
5
4
6
11
7
β
HO
HO
O
2
α
HO
13
O
O
3
1'''
O
O
1'
OCH3
OH
1
15
O
8
OH
HO
O
OH
1''
2
α
6
7
β
HO
HO
O
1
O
1'
3
4
OCH3
OH
4
O
1''
HO
OH
5
O
O
8
OH
O
OH
1'''
6
O
HO
HO
HO
5
HO
OH
HO
OH
1
3
2
HO
HO
O
O
HO
HO
HO
O
HO
O
O
OH
OH
O
O
O
O
OH
OH
O
OH
O
HO
HO
O
OH
HO
O
HO
OH
O
HO
OH
OH
4
5
HO
HO
HO
O
O
O
O
O
HO
OH
OH
HO
O
OH
O
O
OCH3
OH
O
HO
HO
HO
OH
O
OH
O
HO
O
HO
OH
OH
6
7
OH
C
HO
OH
OH
O
O
O
OH
O
O
OH
O
HO
HO
HO
O
HO
HO
OH
OH
O
OH
10
9
8
Figure 1. The structures of 1–10.
were observed. On the basis of above evidence and related litera-
experiment, a correlation between H-100 (d 5.02) and C-30 (d 81.7)
was observed (Fig. 2). On the basis of above evidence and related
literature,18 the structure of compound 2 was determined as 2-
ture,17 the structure of 1 was deduced to 2-(3-methoxyl-4-
hydroxyphenyl)-ethanol-O-
a-
L
-arabinopyranosyl-(1?2)-
a-L-
rhamnopyranosyl-(1?3)-b-
leonoside E.
Compound 2 was obtained as a white powder. The molecular
formula was C27H42O17, as indicated by HRESIMS. The IR spectra
similarly indicated hydroxyl, -CH2-, and aromatic groups. The 13C
NMR and 1H NMR data (Table 1) were closely similar to those of
D
-glucopyranoside (Fig. 1), and named
(3-methoxyl-4-hydroxyphenyl) ethanol-O-
(1?3) [b- -glucopyranosyl-(1?6)]-b- -glucopyranoside,
named leonoside F (Fig. 1).
a
-
L
-rhamnopyranosyl-
D
D
and
Compound 3 was isolated as colorless oil, the molecular formula
21H32O8 of 3 was determined on the basis of the HRESIMS at m/z
C
435.2002 [M+Na]+ (calcd for 435.1989). The IR showed signals for
1, except for a
a-L
-arabinopyranosyl unit in 1 was replaced by a
hydroxyl groups (3380 cmꢁ1
) and a terminal double bond
b- -glucopyranoside group, which was further confirmed based
D
(1665 cmꢁ1). In the 1H NMR spectrum of 3 (Table 1), two methyl
groups including a vinyl methyl signal at d 1.26, five methylene
signals including a O-bearing methylene group at d 4.29/4.08,
two methine signals, one pair of olefinic proton signals at d 5.13/
5.02, and an anomeric proton at d 4.15 (1H, d, J = 7.5) indicating
a b-linkage, were observed. The 13C NMR spectrum of 3 (Table 1)
on the observation that acid hydrolysis of 2 gave only glucose
and rhamnose, identified by TLC. Furthermore, the location of lat-
ter one was confirmed at C-60 of inner glycosyl unit by +7.6 ppm
shift of C-60 observed from 13C NMR spectrum and the HMBC cor-
relation from H-1000 (d 4.23) to C-60 (d 68.5). In addition, in an HMBC

44
Y. Li et al. / Carbohydrate Research 348 (2012) 42–46
Table 1
1H NMR (500 Hz) and 13C NMR (125 Hz) data of compounds 1–3 (in DMSO-d6)a
No.
Compound 1
Compound 2
No.
1
Compound 3
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
46.5
1
2
3
4
5
6
7
8
131.1
116.2
146.2
146.2
112.2
119.3
35.4
131.6
116.7
146.6
146.4
112.8
119.8
35.4
1.94dd (13.0, 5.5)
1.59t (13.0)
4.19dt (13.0, 5.0)
6.68d (2.0)
6.68d (2.0)
2
3
4
5
6
68.5
200.4
125.8
162.1
33.1
6.81d (8.0)
6.81d (8.0)
6.63dd (8.0, 2.0)
2.70 br s
3.56m
6.64dd (8.0, 2.0)
2.71td (6.5, 3.0)
3.61m
2.68d (11.5)
2.12overlap
2.14 br s
1.66m
69.8
71.0
3.89m
3.72s
Glc
3.87m
3.73s
Glc (inner)
4.21d (7.0)
3.09m
7
8
9
41.7
27.3
42.7
–OMe
55.8
56.1
1.66m
1.38m
10
20
30
40
50
60
4.21d (8.0)
3.09dd (8.0, 2.5)
3.36t (5.0)
3.87 br s
3.15
103.3
73.9
81.6
68.4
76.7
60.9
102.9
74.5
81.7
68.8
76.0
68.5
10
11
12
37.4
149.7
111.5
3.38m
3.19 br s
3.19 br s
3.60m
5.13d (1.5)
5.02d (1.5)
4.29d (13.0)
4.08d (13.0)
1.71s
3.46dd (11.5, 5.0)
3.65m
13
70.4
3.97d (11.0)
Rha
Rha
14
15
23.0
11.3
100
200
300
400
500
600
5.25d (1.0)
3.77dd (2.0, 3.0)
3.57m
99.2
80.8
70.5
72.5
67.8
17.7
5.02d (1.0)
3.50m
3.17m
3.16m
3.91m
101.1
71.0
70.5
72.5
68.8
18.3
1.26s
Glc
4.15d (7.5)
2.99m
3.13m
10
20
30
40
50
60
102.6
74.2
77.3
70.7
77.6
61.8
3.16m
3.60m
1.09d (6.0)
Ara
4.26d (7.0)
3.17m
3.18m
3.66m
3.60m
1.10d (6.0)
Glc (terminal)
4.23d (8.0)
2.97dd (6.5, 5.5)
3.15m
3.06m
3.06m
1000
2000
3000
4000
5000
105.7
71.1
72.6
67.6
65.5
103.8
74.0
77.1
70.3
77.3
3.44dd (12.0, 5.0)
3.68m
3.09m
3.10m
3.39m
6000
3.44dd (11.5, 5.0)
3.66m
61.5
a
NMR data (d) were measured in DMSO-d6 at 500 MHz for 1H and 125 MHz for 13C. Coupling constants (J) in Hz are given in parentheses. The assignments are based on
HMBC experiments.
HO
O
HO
HO
HO
HO
O
HO
O
O
OCH3
OH
O
O
O
H3C
HO
OH
O
OH
HO
O
O
OCH3
OH
O
HO
OH
O
O
OH
H3C
HO
O
OH
HO
O
HO
HO
HO
OH
HO
OH
1
3
2
Figure 2. Key HMBC correlations in compounds 1–3.
exhibited twenty-one carbon resonances, six of which were as-
signed to a glucopyranosyl moiety, fifteen for the sesquiterpene
aglycon moiety. A ketone carbonyl (d 200.4), two pairs of olefinic
signals with one cyclic and one exo-cyclic double bond at d 125.8
(162.1) and 149.7 (111.5), which were assigned C-4 (5), and C-11
(12) respectively, and a quaternary carbon signal at d 37.4, were
observed in the 13C NMR spectrum of 3. Thus, these data above al-
lowed the skeleton of 3 to be deduced as an eudesmanolide-type
sesquiterpene19 derivative. Furthermore, an HMBC experiment
showed 1H/13C correlations between C-3 and H-2, H-1, and be-
tween Glc-10 and H-13. The relative stereochemistry of 3 was
determined by ROESY experiment, in which the correlation of
type sesquiterpene is defined in the b-configuration, H-C (7) was
deduced to be -oriented, which was further supported by the fact
a
that correlation between H-C (7) and H -C (6), the latter has no
a
correlation with 14-Me. Additionally, a strong correlation between
H-C (2) and H -C (1) suggested the H-C (2) has the
a
-configuration.
a
As a result, the structure of compound 3 can be concluded to be 7
a
(H)-eudesmane-4,11 (12)-diene-3-one-2b-hydroxy-13-b-D-gluco-
pyranoside (Fig. 2).
The hepatoprotective activities for compounds 1–10 were eval-
uated against -galactosamine-induced toxicity in HL-7702 cells.
D
Compounds 1, 2, 4, and 6 displayed a promising hepatoprotective
activity at 1 ꢀ 10ꢁ5 M in vitro by comparison of the cell survival
rate and inhibitory rate with those of positive control substance
bicyclol as shown in Table 2.
H -C (9) with H -C (1), and H-C (7), of 14-Me with Hb-C (9) and
a
a
Hb-C (6), were observed. Because the 14-Me of eudesmanolide-

Y. Li et al. / Carbohydrate Research 348 (2012) 42–46
45
Table 2
bile phase, respectively, to yield compound 1 (10 mg), 6 (15 mg),
8 (15 mg), 9 (12 mg). The 30% EtOH fraction (99 g) was subjected
to chromatography over Sephadex LH-20 column using a H2O–
MeOH step gradient to give 10 fractions (C1–C10), fraction C2
(40 g) was repeatedly applied to chromatography over Sephadex
LH-20 column and further preparative HPLC using MeOH–H2O
(40:60) to afford compound 3 (10 mg) and 10 (30 mg). Fraction
C4 (0.8 g) was further purified by reversed-phase preparative HPLC
with MeOH–H2O (30:70) to give compound 2 (12 mg). Fraction C7
was further subjected to chromatography over Sephadex LH-20
column to give 20 subfractions (C7-1–C7-20). Subfraction C7-7
(10 g) was purified by reversed-phase preparative HPLC with
MeOH–H2O (30:70, 40:60, 40:60) to give compound 4 (25 mg), 5
(15 mg), and 7 (30 mg).
Hepatoprotective effects of compounds 1, 2, 4 and 6 against D-galactosamine-induced
toxicity in HL-7702 cellsa
Compounds
Cell survival (%)
Inhibition (%)
Normal
100
55***
66**
68**
69**
66*
Control
Bicyclolb
25.3
30.0
30.9
24.4
31.7
Compound 1*
Compound 2*
Compound 4
Compound 6
69**
a
b
Results are expressed as mean SD (n = 3; for normal and control, n = 6).
Positive control substance.
p <0.05.
p <0.01.
p <0.001.
*
**
***
3.4. Characterization data
3.4.1. Leonoside E (1)
3. Experimental
White powder; ½a D20
ꢁ32.67 (c 0.056, MeOH); UV (MeOH) kmax
ꢂ
3.1. General experimental procedures
218, 330 nm; IR mmax 3372, 2932, 1647, 1592, 1513, 1440, 1129,
1074, 1039 cmꢁ1 1H NMR (DMSO-d6, 500 MHz), and 13C NMR
;
(DMSO-d6, 125 MHz) data, see Table 1; ESIMS: m/z 631[M+Na]+,
607[MꢁH]; (+)-HRESIMS: calcd for C26H40O16Na [M+Na]+, m/z
631.2214; found m/z 631.2209.
Optical rotations were measured on a Jasco P-2000 polarimeter.
The UV spectra were scanned by a Jasco V650 spectrophotometer.
IR spectra were recorded on an IMPACT 400 (KBr) spectrometer. 1H
NMR (500 MHz), 13C NMR (125 MHz), and 2D NMR spectra were
run on an INOVA-500 spectrometer and values were given in
ppm. HRESIMS spectra were performed on a Finnigan LTQ FT mass
spectrometer. The ESI mass spectra were recorded by an Agilent
1100 series LC/MSD TOF from Agilent Technologies. Column chro-
matography was performed with Macroporous resin (Diaion HP-
3.4.2. Leonoside F (2)
White powder; ½a D20
ꢁ73.2 (c 0.05, MeOH); UV (MeOH) kmax
ꢂ
220, 330 nm; IR mmax 3367, 2929, 1624, 1512, 1440, 1273, 1073,
1041 cmꢁ1 1H NMR (DMSO-d6, 500 MHz), and 13C NMR (DMSO-
;
d6, 125 MHz) data, see Table 1; (+)-HRESIMS: calcd for
27H42O17Na [M+Na]+, m/z 661.2314; found m/z 661.2322.
20, Mitsubishi Chemical Corp. Tokyo, Japan), Rp-18 (50 lm; YMC,
C
Kyoto, Japan), and Sephadex LH-20 (Pharmacia Fine Chemicals,
Uppsala, Sweden). Preparative HPLC was carried out on a Shimadzu
LC-6AD instrument with a SPD-20A detector, using a YMC-Pack
3.4.3. 7a (H)-Eudesmane-4,11 (12)-diene-3-one-2b-hydroxy-13-
b-
D
-glucopyranoside (3)
Colorless oil; ½a D20
ꢂ
ꢁ6.65 (c 0.124, MeOH); UV (MeOH) kmax
ODS-A column (250 ꢀ 20 mm, 5
lm). TLC was carried out with
glass precoated silica gel GF254 plates. Spots were visualized under
UV light or by spraying with 10% sulfuric acid in EtOH followed by
heating.
253 nm; IR mmax 3380, 2928, 2877, 1665, 1611, 1411, 1294, 1075,
1043 cmꢁ1 1H NMR (DMSO-d6, 500 MHz), and 13C NMR (DMSO-
;
d6, 125 MHz) data, see Table 1; (+)-HRESIMS: calcd for C21H32O8Na
[M+Na]+, m/z 435.1989; found m/z 435.2002.
3.2. Plant material
3.5. Acid hydrolysis of leonurus D–E (1–2)
The dried aerial part of Leonurus japonicus Houtt. was purchased
from Tongrentong Pharmacy, and identified by Professor Lin Ma of
the Chinese Academy of Medical Sciences. A voucher specimen
(NO. ID-S-2387) was deposited at the Institute of Materia Medica,
Chinese Academy of Medical Sciences.
Based on the reported procedure,20 each (5 mg) of the com-
pounds 1 and 2 were dissolved 0.2 N H2SO4 (5 mL) and heated at
100 °C for 2 h. After cooling, the reaction mixture was extracted
with EtOAc. The aqueous layer was concentrated to dryness, then
analyzed on silica gel TLC (EtOAc–MeOH–H2O–AcOH 13:3:3:4)
by comparison with authentic samples.
3.3. Extraction and isolation
The dried aerial parts of Leonurus japonicus Houtt. (23 kg) were
extracted with 30% EtOH under reflux for 2 ꢀ 2 h. After evapora-
tion of EtOH in vacuo, the concentrated extract (3020 g) was sus-
pended in H2O (1000 mL), then 20 L of 95% EtOH were added.
The resulting precipitate was removed and the supernatant solu-
tion was concentrated to give residue (2000 g). The residue was
subjected to column chromatography over macroporous resin
two times, eluting successively with H2O, 15% EtOH, 30% EtOH,
50% EtOH, 70% EtOH, and 95% EtOH (20 L each). After removing
the solvents, 15% EtOH fraction (62 g) was subjected to chromatog-
raphy over ODS eluting with H2O–MeOH mixtures of increasing ra-
tio of MeOH gave five fractions (B1-5) on the basis of HPLC-DAD
analysis. Fraction B2 (28 g) was further separated by Sephadex
LH-20 column chromatography with 15%MeOH/H2O as the mobile
phase to yield four fractions (B2-1–B2-4). Subfractions B2-3 (10 g)
were further purified by reversed-phase preparative HPLC, using
different MeOH–H2O (32:68, 32:68, 34:66, and 33:67) as the mo-
3.6. Bioassays for hepatoprotective activity
The hepatoprotective activities for compounds 1–10 were
determined by
a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) colorimetric assay in HL-7702 cells.
Acknowledgments
This research was supported by the Nation Science and Tech-
nology Project of China (No. 2009ZX09301-003-10-1). We thank
Mrs. Yinghong Wang and Mr. Hongyue Liu for NMR measurements.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.10.034.

46
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