Lignans from the stems of Clerodendrum inerme Gaertn
Cite this paper: Vietnam J. Chem., 2021, 59(2), 187-191
DOI: 10.1002/vjch.202000164
Article
Lignans from the stems of Clerodendrum inerme Gaertn.
Tran Thi Minh*, Nguyen Thi Minh Thuong
School of Chemical Engineering, Hanoi University of Science and Technology
1 Dai Co Viet, Hai Ba Trung, Hanoi 10000, Viet Nam
Submitted September 19, 2020; Accepted November 11, 2020
Abstract
The polar constituents of Clerodendrum inerme collected from coastal area of Thai Binh province were
investigated. From the water soluble fraction of the C. inerme stems, four lignan and lignan glucosides (1-4) along with
a phenolic glucoside (5) were isolated. Their chemical structures were established as icariol A2 (1), syringaresinol-4-O-
-glucopyranoside
(2),
lariciresinol-4-O--D-glucopyranoside
(3),
dehydrodiconiferyl
alcohol-4-O--D-
glucopyranoside (4) and leonuriside A (5) by NMR spectroscopic and mass spectrometric analysis as well as
comparison with those reported in the literature. Compounds 1, 2, 3 and 5 were found for the first time from this plant.
Keywords. Clerodendrum inerme, stems, lignan, lignan glucosides.
1. INTRODUCTION
Silica gel 60 (0.04-0.063 mm, Merck), RP-18 resins
(150 m, YMC), Diaion HP 20 (Mitsubishi
chemical Co.), and Sephadex LH-20 (25-100 m,
Clerodendrum inerme Gaertn. (Verbenaceae) is
widely distributed near the seashore from the north
to the south of Vietnam.[1] This plant has been used
as a folk medicine in Thailan, China, and Vietnam
for treatment of various diseases such as skin
diseases, topical burns, malaria, rheumatism and
hepatitis.[2] This species has been reported to contain
Sigma-Aldrich)
chromatography
were
used
The
for
thin
column
layer
(CC).
chromatography (TLC) was performed on Merck
precoated TLC DC-Alufolien silica gel 60F254 and
RP-18F254S. The plates were visualized under UV
fluorescence or by dipping in 1 % vanillin-H2SO4
and heating at 100 oC for 1-2 min.
flavonoids,[3]
glycosides,[6,7]
diterpenes,[4,5]
megastigmane
phenylethanoid
and iridoid
glycosides.[8] Previously, we have reported the
2.2. Plant materials
isolation
and
structural
determination
of
andrographolide and lupeol hexacosanoate ester
from the ethyl acetate fraction of methanolic extract
obtained from the leaves of C. inerme.[9] As a
continuation of our investigation on this plant, we
wish to describe the isolation and structural
determination of five polar compounds comprising
four lignan and lignan glucosides (1-4) along with a
phenolic glucoside (5) from the water layer of
methanolic extract of C. inerme stems collected in
the beach of Thai Binh province.
The stems of C. inerme (Verbenaceae) were
collected from the coastal area of Thai Binh
province, Vietnam, in 2018. The plant was identified
by Prof. Tran Huy Thai, Institute of Ecology and
Biological Resources, Vietnam Academy of Science
and Technology (VAST). A voucher specimen
(HUST.N02) was deposited in the laboratory of the
Organic Department, Hanoi University of Science
and Technology (HUST), Vietnam.
2.3. Extraction and isolation
2. MATERIALS AND METHODS
The dry and powdered stems of C. inerme (5.0 kg)
were extracted three times with 80 % aqueous
methanol at 50 oC using a sonicator. After
evaporation of the solvent under reduced pressure,
the methanol extract (185.0 g) was suspended in
water (3 L) and partitioned with n-hexane followed
by ethyl acetate (EtOAc) to give n-hexane (27.1 g),
2.1. General experimental procedures
ESI-MS were recorded on an ESI-LC/MS/MS-Xevo
TQMS spectrometer. NMR spectra were recorded on
a Bruker Avance 500 MHz spectrometer with
tetramethylsilane (TMS) as an internal standard.
187 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH
Vietnam Journal of Chemistry
Tran Thi Minh et al.
EtOAc (17.0 g), and water (95.5 g) residues.
4.65 (1H, d, J = 7.5 Hz, H-1’), 3.69 (6H, s, 2,6-
OCH3), 3.61 (1H, m, H-6’a), 3.43 (1H, m, H-6’b),
3.19 (2H, m, H-2’, H-5’), 3.14 (1H, m, H-4’), 3.02
(1H, m, H-3’); 13C-NMR (500MHz, DMSO)
(ppm): 153.9 (C-4), 153.1 (C-2, C-6), 127.5 (C-1),
103.5 (C-1’), 93.8 (C-3, C-5), 77.0 (C-3’), 76.4 (C-
5’), 74.2 (C-2’), 70.0 (C-4’), 61.0 (C-6’), 56.1 (2,6-
OCH3).
The water soluble fraction (95.5 g) was
subjected to column chromatography on a Diaion
HP 20 eluting with the solvent systems of
methanol/water, increasing concentration of
methanol in water (0, 20, 40, 60, 100 %). The
fraction eluted with 40-60 % methanol (14.6 g) was
chromatographed on a silica gel column eluting with
a
gradient
solvent
system
of
dichloromethane/methanol (10/1, 5/1, 2.5/1, 1/1,
v/v) to give eight sub-fractions (CI.1-CI.8). Sub-
fractions CI.2, CI.3, CI.6, and CI.7 were further
separated on sephadex LH 20 column eluting with
methanol, then followed by RP-18 column eluting
with methanol/water (1/2, v/v) to afford compounds
1 (55.9 mg), 2 (17.8 mg), 4 (12.5 mg), and 5 (62.1
mg), respectively. CI.5 (1.27 g) was similarly
separated on a Sephadex LH 20 column eluting with
methanol, then purified on a RP-18 column eluting
with methanol/water (1/2.5, v/v) to give compound 3
(16.7 mg).
3. RESULTS AND DISCUSSION
The separation of water residue of C. inerme stems
using chromatography methods yielded five
compounds 1-5 (figure 1). The chemical structures
of all five compounds were identified based on
comparison of spectral data with those published.
Compound 1 was obtained as a white amorphous
powder. The molecular formula C22H28O9 was
determined by ESI-MS and NMR spectroscopic
data. The 13C-NMR spectrum of 1 showed eight
carbon signals indicating the presence of two
symmetrical halves to the molecule, which was
further confirmed by the symmetrical proton signals
Icariol A2 (1): White amorphous powder, ESI-
MS (positive): m/z 419 [M+H-H2O]+, 401 [M+H-
1
1
on H-NMR. The H-NMR spectrum of 1 indicated
proton signals of two 1,3,4,5-tetrasubstituted
benzene rings [H 6.64 (4H, s, H-2, H-6, H-2’, H-
6’)], two oxymethine groups [H 4.83 (2H, d, J = 8.0
Hz, H-7, H-7’)], four methoxy groups [H 3.76 (12H,
s, OCH3)], two oxymethylene groups [H 3.49 (4H,
m, H2-9, H2-9’)], and two methine groups [H 2.14
1
2H2O]+; H-NMR (500 MHz, DMSO) H: 6.64 (4H,
s, H-2, H-6, H-2’, H-6’), 4.83 (2H, d, J = 8.0 Hz, H-
7, H-7’), 3.76 (12H, s, OCH3), 3.49 (4H, m, H2-9,
H2-9’), 2.14 (2H, m, H-8, H-8’); 13C-NMR (125
MHz, DMSO) C: 147.8 (C-3, C-5, C-3’, C-5’),
134.6 (C-1, C-1’), 133.2 (C-4, C-4’), 103.7 (C-2, C-
6, C-2’, C-6’), 82.0 (C-7, C-7’), 60.4 (C-9, C-9’),
55.9 (3, 5, 3’, 5’-OCH3), 53.4 (C-8, C-8’).
1
(2H, m, H-8, H-8’)]. Analysis of ESI-MS, H and
13C-NMR data indicated the structure of 1 was
identical to icariol A2.[10] The HMBC spectrum was
examined to confirm this structure. The HMBC
correlations from H-7/H-7’ to C-8/C-8’, C-9/C-9’,
C-2,6/C-2’,6’, and from H-8/H-8’ to C-1/C-1’
confirmed the presence of 7,7’-monoepoxy type
lignan skeleton. The positions of methoxy groups at
C-3,5 and C-3’,5’ were determined by the HMBC
correlation between proton signal of methoxy groups
(H 3.76) to C-3,5/C-3’5’. The large coupling
constant of H-7/H-7’ (J = 8.0 Hz) suggested trans
orientation between H-7/H-8 and H-7’/H-8’. Thus,
the chemical structure of compound 1 was
determined as icariol A2.[10] This compound was
found in the Neoalsomitra integrifoliola genus and
showed weak anti-inflammatory activity.[ 11]
Syringaresinol-4-O--glucopyranoside
(2):
White amorphous powder, ESI-MS (positive): m/z
1
419 [M+H-Glucose]+; H-NMR (500 MHz, CD3OD)
and 13C-NMR (125 MHz, CD3OD) (ppm): given in
table 1.
Lariciresinol-4-O--D-glucopyranoside
(3):
White amorphous powder, ESI-MS (positive): m/z
1
343 [M+H-Glucose]+, 523 [M+H]+; H-NMR (500
MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD)
(ppm): given in table 1.
Dehydrodiconiferylalcohol-4-O--D-glucopyranoside
(4): White amorphous powder, ESI-MS (positive):
1
m/z 341 [M+H-Glucose]+, 521 [M+H]+; H-NMR
(500 MHz, CD3OD) and 13C-NMR (125 MHz,
CD3OD) (ppm): given in table 1.
Compound 2 was obtained as a white amorphous
powder. The molecular formula of 2 was clarified as
C28H36O13 based on the ion peak at m/z 419 [M+H-
Glucose]+ in ESI-MS and NMR spectral data. The
Leonuriside A (5): White amorphous powder,
1
ESI-MS (positive): m/z 355 [M+Na]+; H-NMR
(500MHz, DMSO) (ppm): 6.07 (2H, s, H-3, H-5),
Vietnam Journal of Chemistry
Lignans from the stems of…
1H NMR spectrum of 2 showed the proton signals of was confirmed by the HMBC correlation between
two symmetric 1,3,4,5-tetrasubstituted benzene rings
[δH 6.73 (2H, s, H-2, H-6) and 6.67 (2H, s, H-2’, H-
6’)], two oxymethylene groups [δH 4.30 (2H, dd, J =
8.5, 5.0 Hz, H-9a, H-9a’) and 3.93 (2H, dd, J = 9.0,
3.0 Hz, H-9b, H-9b’)], two oxymethine groups [δH
4.87 (1H, d, J = 3.6 Hz, H-7) and 4.73 (1H, d, J =
4.3 Hz, H-7’)], two methine groups [δH 3.14 (2H, m,
H-8, H-8’)], four methoxy groups [δH 3.87 and 3.86
(each, 6H, s)], an anomeric proton signal [δH 4.88
(1H, d, J = 7.5 Hz, H-1”)], and other proton signals
the anomeric proton H-1” (H 4.90) and C-4 (C
147.3). The relative configuration of 3 was indicated
based on NOESY spectroscopic analysis. The
NOESY correlation of H-8 (H 2.37)/H-8’(H 2.73)
and the absence of the NOESY correlation of H-
7/H-8 indicated cis orientation between H-8/H-8’
and trans orientation between H-7/H-8, respectively.
The anti-periplanar orientation of H-7 and H-8 was
further confirmed by the large coupling constants of
H-7 (J = 6.5 Hz). Thus, compound 3 was identified
13
of sugar from 3.22 to 3.55 ppm. The C-NMR and
as
lariciresinol-4-O--D-glucopyranoside
by
HSQC spectra of 2 showed twenty eight carbon
signals comprising six carbon signals of a hexose
unit and 22 carbon signals of aglycone moiety. The
sugar unit was identified as -D-glucose based on
the chemical shift of carbons (δC 105.4, 78.3, 77.8,
75.7, 71.3, 62.6) and the coupling constant of
anomeric proton (J = 7.5 Hz).[12] The above
mentioned NMR data indicated 2 to be a furofuran
lignan glucoside. Moreover, the presence of
furofuran lignan skeleton was further confirmed by
comparison of its NMR spectral data with those
published.[13]
Compound 4 had a molecular formula of
C26H32O11, which was suggested by its ESI-MS,
NMR and DEPT data. The 13C NMR and DEPT
spectra of 4 indicated the presence of a hexose unit
and 20 carbon signals for the aglycone moiety. The
1H NMR spectrum of 4 showed signals of five
aromatic protons [δH 7.17 (d, J = 8.0 Hz), 7.05 (d, J
= 2.0 Hz), 6.95 (dd, J = 8.0, 2.0 Hz), 6.96 and 6.97
(each, 1H, s)], two methoxy groups [δH 3.85 and
3.90 (each, 3H, s)], two trans-olefinic protons which
appeared as AB part of an ABX2 spin system [δH
6.57 (d, J = 16.0 Hz) and 6.24 (dt, J = 15.5, 6.0 Hz)],
a dihydrobenzofuran ring, and an anomeric proton
[δH 4.91 (d, J = 7.5 Hz)]. These data suggested that
1
the significant H-1H COSY cross peaks of H-7/H-
8/H-9 and H-7’/H-8’/H-9’ along with the key
HMBC correlations of H-7/C-2,6, C-9; H-7’/C-2’,6’,
C-9’; and H-8,8’/C-1, C-1’. The positions of
methoxy groups at C-3,5 and C-3’,5’ were
determined by HMBC correlations from the proton
signals of methoxy groups (δH 3.87, 3.86) to C-3,5
and C-3’,5’. The linkage of glucose at C-4 was
determined by the HMBC correlation from H-1” (H
the aglycone of compound
4
was
a
dehydrodiconiferyl alcohol type lignan.[14] The 13C
NMR data of the sugar moiety were consistent with
4.88) to C-4 (C 135.6). The small coupling those of β-D-glucose.[12-14] The long-range
constants of H-7 (J = 3.6 Hz) and H-7’(J = 4.3 Hz)
indicated cis orientation between H-7/H-8 and
H-7’/H-8’. Thus, the structure of compound 2 was
correlation from H-1” (H 4.91) to C-4 (C 147.7)
confirmed that the -D-glucopyranosyl was attached
to C-4 of the aglycone. The trans orientation of H-7
and H-8 was indicated by the large coupling
constants of H-7 (J = 6.5 Hz). Thus, compound 4
was identified as dehydrodiconiferyl alcohol-4-O--
D-glucoside by comparison of its NMR spectral data
with those published.[14]
Compound 5 was identified as leonuriside A, via
the comparison of its NMR spectral data with those
published.[15] To our best knowledge, this is the first
isolation of compounds 1, 2, 3 and 5 from C. inerme,
whereas the compound 4 was previously isolated
from this genus growing in Thailand.[8]
identified
as
syringaresinol-4-O--D-
glucopyranoside by comparison of its NMR spectral
data with those published.[12]
Compound 3 was obtained as a white amorphous
powder. The molecular formula of C26H34O11 was
derived from its pseudomolecular ion peak at m/z
523 [M+H]+ in ESI-MS and NMR spectral data. The
13
1H and C NMR spectra (table 1) of 3 showed the
presence of two 1,3,4-trisubstituted benzene rings,
two methoxy groups, two oxymethylene groups, one
oxymethine group, three methine groups, and a
hexose moiety. The sugar unit was identified as β-D-
1
glucopyranose by comparing the H and 13C NMR Acknowledgements. This research is funded by
1
data with those published.[12,13] The analysis of H Vietnam National Foundation for Science and
13
and C NMR data indicated 3 to be a lariciresinol
Technology Development (NAFOSTED) under grant
lignan glucoside.[13] The linkage of glucose at C-4 number 104.01-2018.36.
Vietnam Journal of Chemistry
Tran Thi Minh et al.
Table 1: 1H and 13C-NMR spectral data of compounds 2-4 (in CD3OD)
2
3
4
C
position
C
139.5
104.9 6.73 (s)
154.4
135.6
154.4
H (mult., J in Hz)
-
C
139.5
111.4 7.01 (s)
150.9
147.3
118.0 7.16 (d, 8.0)
119.6 6.90 (d, 8.0)
83.8
54.1
60.5
H (mult., J in Hz)
-
C
138.1
111.2 7.05 (d, 2.0)
151.0
147.7
118.1 7.17 (d, 8.0)
H (mult., J in Hz)
-
1
2
3
4
5
6
7
8
9
-
-
-
-
-
-
-
104.9 6.73 (s)
119.4 6.95 (dd, 8.0, 2.0)
87.1
55.5
72.8
4.78 (d, 3.6)
3.14 (m)
3.93 (dd, 9.0, 3.0)
4.30 (dd, 8.5, 5.0)
4.86 (d, 6.5)
2.37 (m)
3.89 (m), 3.67 (m)
88.8
55.3
64.9
5.60 (d, 6.5)
3.48 (m)
3.79 (dd, 11.0, 7.5)
3.86 (m)
3-OCH3 56.9
5-OCH3 56.9
3.87 (s)
3.87 (s)
-
56.7
-
133.5
113.4 6.81 (s)
149.0
145.8
116.2 6.74 (d, 8.0)
122.1 6.66 (d, 8.0)
33.6
43.8
73.7
3.88 (s)
-
-
56.7
-
132.7
3.85 (s)
-
-
1’
2’
3’
4’
5’
6’
7’
8’
9’
133.1
104.6 6.67 (s)
149.3
136.3
149.3
112.2 6.97 (s)
-
-
-
-
-
145.3
149.2
130.1
-
-
-
104.6 6.67 (s)
116.5 6.96 (s)
131.9 6.57 (d, 16.0)
127.6 6.24 (dt, 15.5, 6.0)
87.5
55.7
72.9
4.73 (d, 4.3)
3.14 (m)
3.93 (dd, 9.0, 3.0)
4.30 (dd, 8.5, 5.0)
3.86 (s)
3.86 (s)
2.94 (m), 2.52 (m)
2.73 (m)
4.02 (m)
3.75 (m)
3.85 (s)
-
63.8
4.22 (dd, 5.5, 1.0)
3’-OCH3 57.1
5’-OCH3 57.1
56.4
-
56.8
-
3.90 (s)
-
1”
2”
3”
4”
5”
6”
105.4 4.88 (d, 7.5)
102.9 4.90 (d, 7.0)
102.8 4.91 (d, 7.5)
75.7
77.8
71.3
78.3
62.6
3.50 (m)
3.44 (m)
3.44 (m)
3.22 (m)
3.69 (dd, 12.0, 5.0) 62.5
3.80 (dd, 12.0, 2.5)
74.9
77.9
71.4
78.2
3.49 (m)
3.49 (m)
3.41 (m)
3.41 (m)
74.9
77.8
71.3
78.2
62.5
3.51 (m)
3.49 (m)
3.41 (m)
3.41 (m)
3.69 (m), 3.89 (m)
3.70 (m), 3.89 (m)
Figure 1: Structure of compounds 1-5 from C. inerme stems
Vietnam Journal of Chemistry
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Corresponding author: Tran Thi Minh
School of Chemical Engineering
Hanoi University of Science and Technology
1 Dai Co Viet, Hai Ba Trung, Hanoi 10000, Viet Nam
E-mail: minh.tranthi@hust.edu.vn
Tel.: +84- 988557877.
Figure 2: The key HMBC correlations of compounds 1-3
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