New acyclic sesquiterpenoid derivatives and a monoterpene disaccharide from Dichondra repens Forst.

Article abstract of DOI:10.1016/j.phytol.2015.08.013

A new highly oxygenated acyclic sesquiterpenoid (2E, 6E)-8,10,11-trihydroxyl-7,11-dimethyl-3-hydroxymethyl-2,6-dodecadienoic acid (1) and its glucoside (2), together with a new pinane monoterpene disaccharide glucoside 6,6-dimethyl-2-methlenebicyclo [3.1.1]hept-3-O-(6-O-apiofuranosyl)-β-d-glucopyranoside (3) were isolated from hydrophilic extract of Dichondra repens. Their structures were elucidated on the basis of spectroscopic analyses and chemical methods. The three compounds did not show any cytotoxic activity (IC50 > 20 μM) against two human lung cancer cell lines (NCI-H661 and A549).

Full text of DOI:10.1016/j.phytol.2015.08.013

Phytochemistry Letters 14 (2015) 23–26
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
New acyclic sesquiterpenoid derivatives and a monoterpene
disaccharide from Dichondra repens Forst.
*
Wen-qiong Wang, Li-jiang Xuan
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park,
Shanghai 201203, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history:
A new highly oxygenated acyclic sesquiterpenoid (2E, 6E)-8,10,11-trihydroxyl-7,11-dimethyl-3-hydrox-
ymethyl-2,6-dodecadienoic acid (1) and its glucoside (2), together with a new pinane monoterpene
disaccharide glucoside 6,6-dimethyl-2-methlenebicyclo [3.1.1]hept-3-O-(6-O-apiofuranosyl)-b-D-glu-
copyranoside (3) were isolated from hydrophilic extract of Dichondra repens. Their structures were
elucidated on the basis of spectroscopic analyses and chemical methods. The three compounds did not
Received 29 May 2015
Received in revised form 20 August 2015
Accepted 24 August 2015
Available online xxx
show any cytotoxic activity (IC50 > 20
A549).
mM) against two human lung cancer cell lines (NCI-H661 and
Keywords:
Dichondra repens
Acyclic sesquiterpenoid
Monoterpene disaccharide glucoside
ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
1. Introduction
HRESIMS pseudomolecular ion at m/z 325.1630 ([M + Na]+,
C
15H26O6Na, calcd 325.1622) and 13C NMR data. The IR spectrum
showed the absorption band of a, b-unsaturated carboxyl group
The genus Dichondra (Convolvulaceae) comprises of eight
species. Dichondra repens Forst., is the only species widely growing
in southern China as common herbaceous cover (Cardin et al.,
2005). In traditional Chinese medicine, D. repens was used for the
treatment of jaundice and dysentery (Cardin et al., 2005).
However, the chemical study on D. repens is rarely reported (Sheu
et al., 2012). We report the isolation and structural elucidation of
the compounds from the hydrophilic extract of D. repens in this
paper. New highly oxygenated acyclic sesquiterpenoid derivatives
(1–2), and a new pinane monoterpene disaccharide (3) (Fig. 1),
along with the known compounds, adicardin (Li et al., 2009),
actinidioionoside (Xu et al., 2005), eleganoside B (Zhang et al.,
2011), tuberonic acid (Nonaka et al., 2010), aegineoside (Ho et al.,
2003), obtusifoside A (Liu et al., 2013), gymnemasaponin IV
(Yoshikawa et al., 1991) were isolated. Their chemical structures
were determined by use of HRESIMS, high resolution NMR
techniques and chemical methods. Moreover, the three new
compounds were evaluated for their cytotoxicity and the results of
which will also be presented.
(1647 cmꢀ1). The 1H NMR spectrum exhibited three tertiary methyl
groups (dH 1.59, 1.13, 1.09) and two olefinic protons (dH 5.92, 5.44).
The 13C NMR and DEPT spectra of 1 displayed three methyls, four
methylenes, four methines, and three quaternary carbons (two
olefinic moieties), and one carbonyl group (dC 170.3). In the 1H–1H
COSY, correlations between H-4/H-5/H-6, H-8/H-9/H-10 were
apparent. In the HMBC (Fig. 2), spectrum revealed that the signal
of the methyl groups at dH 1.13, 1.09 correlated to the quaternary
carbon C-11 (dC 73.5), which further correlated to H-10 (dH 3.24).
The remaining methyl signal (dH 1.59) correlated to the olefinic
carbon C-7 (dC 138.4), which further correlated to H-8 (dH 4.19). The
correlations between H-4 (dH 2.54), H-13 (dH 4.08) and the olefinic
moiety (dC 115.0, 163.0) indicated the linkage of C-4-C3-C-13.
Besides, the correlation between olefinic proton H-2 (dH 5.92) and
the carbonyl group (dC 170.3) showed the linkage between C-1 and
C-2. Thus, the planar structure of 1 was assigned. The absolute
configuration of 1 was determined according to the reported
method (Mondol et al., 2011). Firstly, the relative configuration of 1
was assigned by using Kishi’s Universal NMR Database (Database
2) (Kobayashi et al., 2000). The 13C NMR resonances of C-8/C-
10 were in agreement with a syn arrangement of the 1,3-diol model
system (Fig. 4). Secondly, the absolute configuration of the 8,10-
diol of 1 was determined by modified Mosher’s ester method
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. The
molecular formula was determined as C15H26O6 based on the
(Freire et al., 2005). The chemical shift difference (Dd
=
dS dR)
ꢀ
implied that the configuration is 8R, 10S. Thus, the structure of
compound 1 was elucidated as (2E, 6E)-8,10,11-trihydroxyl-7,11-
dimethyl-3- hydroxymethyl-2,6-dodecadienoic acid.
* Corresponding author. Fax.: +86 21 20231968
E-mail address: ljxuan@mail.shcnc.ac.cn (L.-j. Xuan).
http://dx.doi.org/10.1016/j.phytol.2015.08.013
1874-3900/ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

24
W.- Wang, L.- Xuan / Phytochemistry Letters 14 (2015) 23–26
Fig. 1. Structure of compounds 1–3.
Fig. 2. Key HMBC correlations of 1–3.
Compound 2 was obtained as a white amorphous powder. The
molecular formula was determined as C21H36O11 based on the
HRESIMS pseudomolecular ion at m/z 487.2144 ([M + Na]+,
C
21H36O11Na, calcd 487.2150) and 13C NMR data. Its IR spectrum
revealed similar absorption bands to 1. Comparison of the 1H and
13C data with that of 1 showed that 2 contained an additional sugar
unit. After acid hydrolysis, 2 afforded 1 and D-glucose by HPLC and
rotation analyses. Therefore, 2 was the glycoside of 1. The long-
range correlation between the anomeric proton dH 4.28 (d,
J = 7.7 Hz) and C-13 (dC 72.2) suggested
D-glucose located at C-13
Fig. 4. Assignment of the relative configuration of 1 based on Kishi’s Universal NMR
Database.
(Fig. 2). The configuration of -glucose was deduced from the
b
D
coupling constant. Thus, the structure of 2 was determined as (2E,
6E)-8,10,11-trihydroxyl-7,11-dimethyl-3-hydroxymethyl-2,6-
dodecadienoic acid 13-O-b-D-glucopyranoside.
which further correlated to H-1. Therefore, the aglycone of 3 was 3-
hydroxy-camphene. The ROESY spectrum (Fig. 3) revealed
correlations of H-1/H-9/H-5 indicated H-1, H-9, H-5 to be
Compound 3 was obtained as a white amorphous powder. The
molecular formula was determined as C21H34O10 based on the
HRESIMS pseudomolecular ion at m/z 469.2082 ([M + Na]+, calcd.
469.2044) and 13C NMR data. The 1H NMR spectrum exhibited two
tertiary methyl groups (dH 1.28, 0.66), two olefinic protons (dH
4.97) and two anomeric protons (dH 4.50, 5.01). The 13C NMR and
DEPT spectra of 3 displayed two sugar moieties, two methyls, three
methylenes, three methines, and two quaternary carbon atoms.
The HMBC experiments showed correlation between the methyl
groups (dH 1.28 and dH 0.66) and C-6 (dC 42.7), which further
correlated to H-1 and H-5 (dH 2.43 and dH 1.93). H-3 (dH 4.53)
correlated to C-4 (dC 33.9), which further correlated to H-5 (dH
1.93). H-3 also correlated to C-10 (dC 115.3) and C-2 (dC 149.0),
b
-oriented. In addition, correlations of H-8/H-3/H-7 indicated
H-3, H-8 to be -oriented. After acid hydrolysis, 3 afforded
glucose and -apiose, which were identified by HPLC analysis and
rotation analyses. configuration of Glc and configuration of Api
a
D-
D
b
a
were suggested from the J values of H-1 of Glc (dH 4.50, J = 7.8 Hz),
H-1 of Api (dH 5.01, J = 2.5 Hz). The correlation between H-1 of Api
(dH 5.01) and C-6 of Glc (dC 68.8) suggested that the sugar moiety
was Apiosyl-(1 !6)-glucosyl. Furthermore, the HMBC correlation
between H-1 of Glc (dH 4.50) and C-3 (dC 72.8) showed Glc linked to
C-3. Therefore, compound 3 was characterized as 6,6-dimethyl-2-
methlenebicyclo[3.1.1]hept-3-O-(6-O-apiofuranosyl)-b-D-gluco-
pyranoside.
We evaluated the cytotoxic activity of these three new
compounds against two human lung cancer cell lines (NCI-
H661 and A549) by a SRB method. All of the compounds showed no
significant cytotoxic activity (IC50 > 20 mM).
3. Conclusion
The phytochemical study of the aerial part of D. repens led to the
isolation of an unusual new highly oxygenated acyclic sesquiter-
penoid (1) and its glucoside (2), along with a new pinane
monterpene disaccharide glucoside (3). Their cytotoxic activity
on NCI-H661 and A549 cell lines were evaluated and they all
showed no toxic (IC50 > 20 mM) activity.
Fig. 3. Selected ROESY correlations of 3.

W.- Wang, L.- Xuan / Phytochemistry Letters 14 (2015) 23–26
25
Table 1
4. Experimental
1H NMR (500 MHz) and 13C NMR (125 MHz) Data of 1-2 (CD3OD)
4.1. General
Position
1
2
dH (J in Hz)
dc
dH (J in Hz)
dc
Optical rotation was measured by PerkinElmer 341 polarimeter.
IR was obtained on Hitachi 275-50 spectrometer.1H NMR,13C NMR,
and 2D NMR spectrum were recorded on Bruker AM-500 spec-
trometer (1H: 500 MHz and 13C: 125 MHz). HRESIMS was recorded
on Finnigan LCQ-DECA spectrometer. Column chromatography
1
2
3
4
5
6
7
8
170.3
115.0
163.0
30.0
28.0
127.6
138.4
77.8
36.7
77.9
73.5
24.8
65.9
170.5
117.3
158.1
30.0
27.7
127.7
138.3
77.8
36.7
77.9
73.5
24.9
72.2
5.92 s
6.00 s
2.54 dd (14.0, 7.6)
2.23 dd (14.0, 7.6)
5.44 t (7.6)
2.53 m
2.20 m
5.40 t (7.0)
was done on MCI CHP-20P gel (75–150
Industries Co., Ltd.), and Cosmosil 75C18-OPN (20–45
Tesque Inc.). Prep-HPLC was performed on Waters 4000 Prep-HPLC
m
m; Mitsubishi Chemical
mm; Nacalai
4.19 t (7.0)
1.74 m, 1.51 m
3.24 d (10.4)
4.14 m
1.71 m, 1.56 m
3.20 m
9
10
11
12
13
using a YMC C18 column (10 ꢁ 250 mm, 5
mm).
1.09 s
4.08 s
1.06 s
4.2. Plant material
4.39 d (15.9)
4.11 d (15.9)
1.56 s
1.09 s
4.25 d (7.5)
3.17 m
3.23 m
3.20 m
3.30 m
3.81 dd (11.6, 3.3),
3.60 dd (11.6,3.3)
14
15
Glc-1
2
3
4
5
6
1.59 s
1.13 s
10.8
25.7
10.9
25.5
103.6
75.0
77.9
71.5
77.8
62.7
The aerial parts of D. repens Forst. were collected from Guangxi,
China, in September 2011, and identified by Prof. Heming Yang. A
voucher specimen (No. SIMM811) was deposited at the Herbarium
of the Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, and People’s Republic of China.
4.3. Extraction and isolation
Air-dried, powdered aerial parts of D. repens (5.0 kg) were
extracted with H2O/acetone 2:8 (v/v) at room temperature for
7 ꢁ24 h. After removal of organic solvent, the sample was
suspended in H2O and sequentially extracted with petroleum
ether, CHCl3. The water-soluble fraction was subjected to a column
of MCI and eluted with H2O, 25%, 50%, 75%, and 100% MeOH
successively. Six major fractions (A–F) were obtained. Fraction C
was further subjected to a C18 column eluted with MeOH/H2O
(10–50%) and yielded 2 (40.6 mg). Fraction D was separated by a C18
column eluted with MeOH/H2O (10–80%) to give fractions
(D1–D3). Fraction D1 was further chromatographed on a Sephadex
LH-20 with MeOH and yield 1 (12.3 mg). Fraction F was subjected
to a C18 column eluted with MeOH/H2O (50–100%) to give 3
(27.9 mg).
concentrated, and dissolved in H2O. The elutes were identified as
D-
glucose [a 23D = +52 (c = 0.1, H2O), D-apiose [a 23
]
]
D = +6 (c = 0.1, H2O)
in comparisons with their specific rotations with those of the
corresponding authentic samples.
4.6. Physical and spectroscopic data of new compound
(2E, 6E)-8,10,11-trihydroxyl-7,11-dimethyl-3-hydroxymethyl-
2,6-dodecadienoic acid (1)
White amorphous powder. [a 24
] D = +5 (c = 0.1, MeOH). HRE-
SIMS: m/z 325.1630 ([M + Na]+, C15H26O6Na+, calcd. 325.1622). UV
lmax(MeOH) nm: 206. IR(KBr): 3419, 2918, 1647, 1431, 1373, 1165,
1078 cmꢀ1. 1H and 13C NMR (CD3OD): see Table 1.
(2E, 6E)-8,10,11-trihydroxyl-7,11-dimethyl-3-hydroxymethyl-
2,6-dodecadienoic acid 13-O-b-D-glucopyranoside (2)
4.4. Preparation of Mosher’s esters
Compound 1 (2 mg) was treated with (S)-MPTA chloride and
(R)-MPTA chloride in Pyridine-d5, separately, and stood for 8 h at
room temperature. After reaction, the chemical shift difference
Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) Data of 3 (CD3OD)
(Dd
=
dS
ꢀ
dR) observed, which made it possible to conclude that the
Position
3
absolute configuration of compound 1 was 8R, 10S configuration
(
Dd15 = +0.090, Dd14 = ꢀ0.215, Dd12 = +0.119, Dd10 = +0.002, Dd8
=
dH (J in Hz)
2.43 t (5.4)
dc
ꢀ0.003).
1
2
3
4
5
6
7
8
51.8
S-MPTA ester of 1: 1H NMR (in Pyridine-d5, 400 MHz):
d
1.439
149.0
72.8
33.9
40.9
42.7
27.1
22.3
26.5
115.3
99.4
74.9
78.2
71.9
76.6
65.6
111.0
78.1
80.5
68.8
74.
4.53 d (7.3)
2.01 m, 2.23 m
1.93 m
(3H, s, Me-15),1.602 (3H, s, Me-14), 1.429 (3H, s, Me-12), 3.377 (1H,
m, CH-10), 4.873 (1H, m, CH-8).
R-MPTA ester of 1: 1H NMR (in Pyridine-d5, 400 MHz):
d 1.329
(3H, s, Me-15), 1.817 (3H, s, Me-14), 1.348 (3H, s, Me-12), 3.375 (1H,
m, CH-10), 4.876 (1H, m, CH-8).
2.27m, 1.91 m
0.66 s
1.28 s
4.97 dd (17.8, 1.7)
4.50 d (7.8)
3.22 m
3.31 m
3.25 m
3.31 m
9
10
Glc-1
2
3
4
5
6
4.5. Sugar analysis
Compounds 2 (10 mg) and 3 (10 mg) were hydrolyzed with 1 N
H2SO4 in H2O and heated for 4 h in an 85 ꢂC water-bath, separately.
After cooling, the reaction mixture was neutralized with 10%
Na2CO3 and extracted with CHCl3 twice. The aqueous layer was
desalted by Sephadex LH-20 with MeOH to afford a sugar residue.
The residue was dissolved in H2O and directly analyzed by HPLC
with authentic samples (MeCN–H2O, 95/5):
21.1 min from Compounds 2 and 3, and -apiose at 24.0 min from
Compound 3. Each of these eluates was individually collected,
3.51 m, 3.86 m
5.01 d (2.5)
3.90 d (2.5)
Api-1
2
3
4
5
3.59 m, 4.00 m
3.76 d (9.7), 3.97 d (9.7)
D-glucose eluted at
D

26
W.- Wang, L.- Xuan / Phytochemistry Letters 14 (2015) 23–26
White amorphous powder. [a 24
]
D = ꢀ10 (c = 0.1, MeOH). HRE-
References
SIMS: m/z 487.2144 ([M + Na]+, C21H36O11Na+, calcd. 487.2150). UV
lmax(MeOH) nm: 205. IR(KBr): 3430, 2918, 1649, 1431, 1373, 1165,
1081 cmꢀ1. 1H and 13C NMR (CD3OD): see Table 1.
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]
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assay as described in the literature (Skehan et al., 1990).
Doxorubicin (Sigma) was used as positive control.
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The authors gratefully acknowledge grants from the National
Science & Technology Major Project “Key New Drug Creation and
Manufacturing Program”, China (Number: 2009ZX09301-001), the
National Natural Sciences Foundation of China (No. 81473111).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2015.08.013.