Inhibitors of osteoclastogenesis from Lawsonia inermis leaves

Article abstract of DOI:10.1016/j.bmcl.2010.06.118

Ten phenolic compounds (1-10) were isolated from a methanol extract of Lawsonia inermis leaves including two new ones, lawsoniasides A (1) and B (2). Their structures were elucidated by spectroscopic methods (NMR and FTICRMS) in combination with acid hydrolysis and GC analyses. Compounds 4 and 5 showed a significant inhibition on receptor activator for nuclear factor-κB ligand-induced osteoclast formation in murine bone-marrow macrophages.

Full text of DOI:10.1016/j.bmcl.2010.06.118

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4782–4784
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of osteoclastogenesis from Lawsonia inermis leaves
a,
a,b,
a
a
Nguyen Xuan Cuong , Nguyen Xuan Nhiem
, Nguyen Phuong Thao , Nguyen Hoai Nam ,
a
a
a
a,
a
*
Nguyen Tien Dat , Hoang Le Tuan Anh , Le Mai Huong , Phan Van Kiem , Chau Van Minh ,
Ji-Hee Won , Won-Yoon Chung , Young Ho Kim
c
c
b,
*
a
Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Nghiado, Caugiay, Hanoi, Viet Nam
College of Pharmacy, Chungnam National University, Daejeon 305 764, Republic of Korea
Department of Oral Biology, Yonsei University College of Dentistry, Seoul 120 752, Republic of Korea
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Ten phenolic compounds (1–10) were isolated from a methanol extract of Lawsonia inermis leaves includ-
ing two new ones, lawsoniasides A (1) and B (2). Their structures were elucidated by spectroscopic meth-
ods (NMR and FTICRMS) in combination with acid hydrolysis and GC analyses. Compounds 4 and 5
Received 23 March 2010
Revised 12 June 2010
Accepted 22 June 2010
Available online 25 June 2010
showed a significant inhibition on receptor activator for nuclear factor-
mation in murine bone-marrow macrophages.
jB ligand-induced osteoclast for-
Ó 2010 Published by Elsevier Ltd.
Keywords:
Lawsonia inermis
Lythraceae
Osteoporosis
Osteoclast
Bone resorption is necessary in many physiological processes of
the skeleton, and the physiological remodeling of bone in adults is
strictly dependent on it. Normally, a balance exists between bone
formation and bone resorption. When the balance is upset and
bone resorption exceeds bone formation, metabolic bone diseases
will occur, like osteoporosis, which is an important disease com-
monly found among elderly populations, especially postmeno-
pausal women. Osteoporosis is characterized by excessive bone
resorption, which causes changes in the microstructure of the bone
matrix and makes bone prone to fracture. Costs of osteoporosis are
staggering and increasing every year. Actually, antiresorptive ther-
apies and medicines include the estrogen replacement therapies
tion, and evaluation of anti-osteoclastogenic activity of ten pheno-
lic compounds (Fig. 1), from a methanol extract of L. inermis leaves.
Compounds 4 and 5 significantly inhibited receptor activator for
nuclear factor-jB ligand (RANKL)-induced osteoclast formation of
murine bone-marrow macrophages (BMMs).
The leaves of Lawsonia inermis L. was collected at Tam Dao
National Park, Vinh Phuc, Vietnam, during February 2009 and iden-
tified by Dr. Ninh Khac Ban (Institute of Ecology and Biological
Resources, VAST). A voucher specimen (INPC LI-0209) was deposited
at the Herbarium of Institute of Natural Products Chemistry, VAST.
Air-dried leaves of L. inermis (2.5 kg) were exhaustively extracted
(three times, each 1 h) with hot MeOH (50 °C) under ultrasonic
condition to obtain 100 g MeOH residue. This was suspended in
(
ERT), selective estrogen receptor modulators (SERM), vitamin D,
parathyroid hormone (PTH) analogues, and bisphosphonates,
which show efficacy but reveal serious side effects. Thus, other
nonhormonal and more specific therapies are needed.1
water (1 L) and partitioned in turn with CHCl
3 ꢀ L) giving corresponding extracts: CHCl (C, 26 g), EtOAc (E,
15 g), and a water layer (W). The water layer was passed through a
Dianion HP-20 column using step wise elution with MeOH–H
(0:10, 2:10, 5:10, and 10:0, v/v) to give four corresponding fractions,
3
and EtOAc (each
3
,2
Lawsonia is a single-species genus of the Lythraceae family. In
Vietnam, Lawsonia inermis L. (synonym L. alba Lamk, Vietnamese
name: la mong tay), is used in traditional medicine to treat various
diseases such as menstrual disorder, edema, rheumatism, bronchi-
2
O
8
W1–W4. Lawsoniaside B (2, 15 mg), (+)-pinoresinol di-O-b-D-
9
glucopyranoside (8, 17 mg), and syringaresinol di-O-b-
D
-glucopy-
ranoside (9, 19 mg), were isolated from fraction W2 (7 g) by using
a YMC RP-18 CC with H O–MeOH (5:1, v/v) as eluent, followed by a
silica gel CC eluting with CHCl –MeOH–H O (4:1:0.1, v/v/v).
Fraction W3 (5 g) was further separated on a silica gel CC eluting
with CHCl –acetone–H O (1:6:0.2, v/v/v), followed by YMC RP-18
CC with H O–acetone (4:1, v/v) as eluent to furnish syringinoside
3
10
tis, and hemorrhoids. In continuation of our investigations of Viet-
namese medicinal plant constituents regarding antiosteoporosis
2
4
–7
effects,
this paper deals with the isolation, structural elucida-
3
2
*
Corresponding authors. Tel.: +82 42 821 5933; fax: +82 42 823 6566 (Y.H.K.),
3
2
tel.: +84 4 38363375; fax: +84 4 37564390 (P.V.K.).
2
E-mail addresses: phankiem@vast.ac.vn (P.V. Kiem), yhk@cnu.ac.kr (Y.H. Kim).
These authors contributed equally to this work.
1
1
12
13
(3, 7.0 mg), daphneside (4, 5.0 mg), daphnorin (5, 4.5 mg),
0
960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.06.118

N. X. Cuong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4782–4784
4783
O
OH
OMe
6'
HO
HO
5
8
3
1
1
6
7
4
9
2
10
RO
5'
O
CH3
7
2
3
6
1
H3C
2
1
6
'
1
3
4
5
HO
HO
HO
O
O
O
6
4
3'
1'
5'
O
5
8
OH
MeO
O
OH
6"
7
HO
HO
CH2OH
9
3'
1'
5"
O
O
OH
CH
3
12
2 R = α-D-glucopyranoside
3 R = β-D-glucopyranosyl-(1 6)-β-D-glucopyranoside
HO
HO
1
3"
1"
OH
4
5
'
4'
6
'
4a'
8a'
3'
2'
7
'
7'
8'
6
''
OCH3
H CO
3
O
O
O
HO
HO
3'
4
3
6''
6
7
5
4a
8a
HO
5''
O
9
1'
2
5'
5
''
O
1''
O
4
8
HO
HO
HO
5
8
O
O
3
''
6
4a
8a
3
10
O
OH
H
1
''
7
3
''
O
OH
1
5
6
2''''
OH 4'''
OMe
OH
O
HO
O
6
''' OH
O
3
'
1'''
2
'
4'
5'''
OH
1'
5'
O
O
6'
OH
4
2
R1
3'
5
1
1'
H
H
5'
HO
O
O
6
"
8
7
9
2
3
6
8
R1
HOOC
6
''
6
5
10
5''
1''
2''
5"
O
4
R1 = OMe, R2 = H
7
4''
HO
HO
3''
8 R1 = H, R2 = β-D-glucopyranoside
2
R O
1"
3
"
OH
O
1
= OMe, R2 = β-D-glucopyranoside
OH
9
R
10
OMe
Figure 1. Structures of 1–10.
and (+)-syringaresinol O-b-
ethyl acetate extract (E, 15 g) was separated into five fractions,
E1–E5, by a silica gel CC using gradient elution of CHCl –MeOH
30:1–1:1, v/v). Lawsoniaside A (1, 10 mg), agrimonolide 6-O-b-
D
-glucopyranoside (7, 10 mg).14 The
position of the butanoyl moiety at C-1 and two hydroxyl groups at
C-2 and C-4, confirming the aglycone of 1 as dimethylphlorobutyr-
2
0
3
ophenone. In addition, attachment of the glucose at C-6 was as-
8
0
(
D
-
signed by an HMBC cross peak between H-1 (d 4.52) and C-6
15
16
glucopyranoside (6, 5.0 mg), and isoscutellarin (10, 7.5 mg) were
isolated from fraction E4 (2 g) by using a silica gel CC with CHCl
MeOH–H O (4:1:0.1, v/v/v) as eluent, followed by a YMC RP-18 CC
eluting with H O–MeOH (5:1, v/v).
Lawsoniaside A (1) was obtained as a white powder. Its molec-
ular formula, C18 , was determined by the FTICRMS peak at m/
z 409.14679 [M+Na] (calcd for C18
(d 154.88). Thus, 1 was elucidated as 1-butanoyl-3,5-dimethyl-
3
–
phloroglucinyl-6-O-b-
D-glucopyranoside, named lawsoniaside A.
2
The molecular formula of lawsoniaside B (2) was determined to
+
2
be C17
for C17
H
H
24
O
9
by FTICRMS result at m/z 395.13183 [M+Na] (calcd
1
3
24
9
O Na, 395.13180). The C NMR data of 2 (see Table 1)
were identical to those of syringin, except for signals of the glu-
cosyl moiety. The small coupling constant of the anomeric proton
at d 4.91 (1H, d, J = 5.0 Hz, H-1 ) and sugar carbon signals at d
102.58 (C-1 ), 74.15 (CH, C-2 ), 76.51 (CH, C-3 ), 69.93 (CH, C-4 ),
2
1
H
26
O
9
+
1
26 9
H O Na, 409.14745). The H
0
NMR spectrum of 1 (see Table 1) showed typical signals of two aryl
methyls and one terminal methyl at d 2.05 (s, H-11), 2.21 (s, H-12),
0
0
0
0
0
0
and 0.94 (t, J = 7.5 Hz, H-10), respectively. An anomeric proton at d
77.14 (CH, C-5 ), and 60.89 (CH
anosyl unit. Moreover, acid hydrolysis of 2 afforded
2
, C-6 ), indicated an
a
-D
-glucopyr-
-glucose
0
22
4
.52 (1H, d, J = 8.0 Hz, H-1 ) confirmed a b-glycosidic linkage. The
C NMR spectrum of 1 revealed 18 carbon signals, including seven
D
1
3
0
(see Supplementary data). The anomeric proton H-1 (d 4.91)
showed HMBC correlation with C-4 (d 133.92), confirming attach-
ment of the glucose at C-4 (Fig. 2). Consequently, the new structure
quaternary, five methine, three methylene, and three methyl car-
bons, detected by DEPT experiments. Of these, a fully substituted
aromatic ring was suggested by six quaternary carbon signals at
d 112.72 (C-1), 159.96 (C-2), 109.18 (C-3), 161.82 (C-4), 111.61
3-(4-O-a-D-glucopyranosyl-3,5-dimethoxy) phenyl-2E-propenol
was elucidated for 2, named lawsoniaside B.
(
C-5), and 154.88 (C-6). The presence of a butanoyl moiety was
identified by carbon signals at d 210.09 (C, C-7), 46.95 (CH , C-8),
9.62 (CH , C-9), and 14.33 (CH , C-10), and two aryl methyls were
Since potentiated bone resorption by osteoclasts is one of the
major causes of osteoporosis, inhibitors of osteoclasts may there-
fore present useful agents to prevent the excessive bone resorption
associated with osteoporosis. Thus, an inhibitory activity of all
compounds on TRAP-positive osteoclast formation was assayed
(see Supplementary data). Among isolated compounds, 4 and 5 sig-
nificantly suppressed osteoclast formation by 20% and 13% at a
2
1
2
3
17,18
at d 8.27 (C-11) and 9.81 (C-12).
nals at d 105.72 (CH, C-1 ), 75.70 (CH, C-2 ), 77.88 (CH, C-3 ),
Moreover, sugar carbon sig-
0
0
0
0
0
0
7
1.91 (CH, C-4 ), 78.02 (CH, C-5 ), and 62.93 (CH
with the spin-coupling pattern of the sugar proton signals (J
= 8.0 Hz, J = 9.0 Hz, and J = 9.0 Hz) indicated a b- -gluco-
-glucose was confirmed by acid hydrolysis
see Supplementary data).
Whole structure of 1 was elucidated by HSQC and HMBC
2
, C-6 ), together
0
1 –
0
0
0
0 0
3 –4
D
10 lM concentration, respectively, as revealed by the decreased
2
2 –3
pyranosyl unit. The
D
number of TRAP-positive multinuclear osteoclasts (Fig. 3). These
compounds may reduce osteoclastogenesis by blocking the fusion
of RANKL-treated BMMs rather than cell viability. In addition, the
(
experiments. The aryl methyl protons H-11 (d 2.05) showed HMBC
correlations with C-2 (d 159.96)/C-3 (d 109.18)/C-4 (d 161.82), and
H-12 (d 2.21) correlated with C-4 (d 161.82)/C-5 (d 111.61)/C-6 (d
compounds did not show cytotoxic activity on BMMs at 10 lM.
These results suggest that 4 and 5 have anti-osteoclastogenic
activities. This is the first report on anti-osteoclastogenic constitu-
ents of Lawsonia inmermis, suggesting potential usage of this plant
and its secondary metabolites for prevention and treatment osteo-
porosis and related bone diseases.
1
54.88), confirming carbon positions of the aromatic ring (Fig. 2).
1
3
Comparison of the C NMR chemical shifts for the aromatic ring
of 1 with those of dryopteroside18 and eucalmainoside E, indicated
19

4
784
N. X. Cuong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4782–4784
HO
O
OH
O
HO
HO
CH3
H C
OMe
3
OH
HO
HO
O
O
O
OH
HO
OH
CH3
MeO
CH OH
2
2
1
Figure 2. Key HMBC correlations of 1 and 2.
Acknowledgments
This work was supported by VAST and partly by Priority
Research Centers Program through the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education, Science
and Technology (2009-0093815), Republic of Korea. The authors
are grateful to Institute of Chemistry, VAST and KBSI for provision
of spectroscopic instrument.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.118.
References and notes
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7
The NMR spectroscopic data of 1 and 2
2
5
1a
2b
(J in Hz)
8.
Lawsoniaside A (1): amorphous white powder, ½
a
ꢁ
+1.5 (c 0.50, MeOH); IR(KBr)
Position
D
m
max 3428 (OH), 2925 (CH), 1730 (C@O), 1645 (aromatic ring), 1075 (C–O–
ꢂ1
1
1
3
d
C
d
H
(J in Hz)
d
C
d
H
C) cm ; H NMR (CD OD, 500 MHz) and C NMR (CD OD, 125 MHz) are given
3
3
+
+
in Table 1; ESIMS m/z 409 [M+Na] ; FTICRMS m/z 409.14679 [M+Na] (calcd for
Aglycone
25
D
C
18
H
26
O
9
Na, 409.14745). Lawsoniaside B (2): amorphous white powder, ½
a
ꢁ
1
2
3
4
5
6
7
8
9
112.72
159.96
109.18
161.82
111.61
154.88
210.09
46.95
19.62
14.33
8.27
—
—
—
—
—
—
—
132.57
104.51
152.67
133.92
152.67
104.51
128.41
130.12
61.41
—
6.72 s
—
—
—
6.72 s
6.47 d (16.0)
6.33 dt (16.0, 5.0)
4.11 t (5.0)
ꢂ15.5 (c 0.50, MeOH); IR(KBr)
m
max 3435 (OH), 2917 (CH), 1652 (aromatic ring),
ꢂ1
1
13
1
1
3
084 (C–O–C) cm
;
6 6
H NMR (DMSO-d , 500 MHz) and C NMR (DMSO-d ,
25 MHz) are given in Table 1; ESIMS m/z 395 [M+Na]+; FTICRMS m/z
+
95.13183 [M+Na] (calcd for C17
H
24
O
9
Na, 395.13180).
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1.66 m
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56.34
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0
1
105.72
75.70
77.88
71.91
78.02
62.93
4.52 d (8.0)
3.56 dd (8.0, 9.0)
3.42 t (9.0)
3.35 t (9.0)
3.12 m
102.58
74.15
76.51
69.93
77.14
60.89
4.91 d (5.0)
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3.21
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3.20c
3
0
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4
3.15 m
3.05 m
3.42 dd (5.0, 12.0)
3.59 dd (2.0, 12.0)
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0
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3.60 dd (5.5, 12.0)
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2
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Measured in CD
Measured in DMSO-d
Overlapped signals, assignments were done by HSQC and HMBC experiments.
3
OD.
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6
.