Three new dinormonoterpenoid glucosides from pericarps of Myriopteron extensum

Article abstract of DOI:10.1016/j.phytol.2015.03.018

Three new dinormonoterpenoid glucosides, rel-(3R,4R)-3-(1-hydroxypropan-2-yl)-3,4-epoxypentane-1,5-diol-1-O-β-d-glucopyranoside (1), rel-(3R,4S)-3-(1-hydroxypropan-2-yl)-3,4-epoxypentane-1,5-diol-1-O-β-d-glucopyranoside (2), and rel-(3R,4S)-3-(1-hydroxy-2-propen-2-yl)-3,4-epoxypentane-1,5-diol-1-O-β-d-glucopyranoside (3), were isolated from the edible pericarps of Myriopteron extensum (Wight & Arn.) K. Schum. (Asclepiadaceae). Their structures were elucidated by chemical and spectroscopic methods including HRESIMS, 1D and 2D NMR. Dinormonoterpenoid glucosides were reported from Asclepiadaceae for the first time. Compounds 1-3 were evaluated for their cytotoxicity against five human cancer cell lines HL-60, SMMC-7221, A-549, MCF-7, and SW-480, but they did not exhibit cytotoxicity on the tested cell lines.

Full text of DOI:10.1016/j.phytol.2015.03.018

Phytochemistry Letters 12 (2015) 164–167
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Three new dinormonoterpenoid glucosides from pericarps of
Myriopteron extensum
Qin Dai a,b, Yue-Hu Wang a, Hong-Xia Zhang a, Guo Sun a,b, Zhi-Zhi Du a,
*
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
PR China
b University of Chinese Academy of Sciences, Beijing 100049, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new dinormonoterpenoid glucosides, rel-(3R,4R)-3-(1-hydroxypropan-2-yl)-3,4-epoxypentane-
Received 8 January 2015
Received in revised form 22 March 2015
Accepted 30 March 2015
Available online 13 April 2015
1,5-diol-1-O-
b
-
D
-glucopyranoside (1), rel-(3R,4S)-3-(1-hydroxypropan-2-yl)-3,4-epoxypentane-1,5-di-
-glucopyranoside (2), and rel-(3R,4S)-3-(1-hydroxy-2-propen-2-yl)-3,4-epoxypentane-1,5-
-D-glucopyranoside (3), were isolated from the edible pericarps of Myriopteron extensum
ol-1-O-
b
-
D
diol-1-O-
b
(Wight & Arn.) K. Schum. (Asclepiadaceae). Their structures were elucidated by chemical and
spectroscopic methods including HRESIMS, 1D and 2D NMR. Dinormonoterpenoid glucosides were
reported from Asclepiadaceae for the first time. Compounds 1–3 were evaluated for their cytotoxicity
against five human cancer cell lines HL-60, SMMC-7221, A-549, MCF-7, and SW-480, but they did not
exhibit cytotoxicity on the tested cell lines.
Keywords:
Myriopteron extensum
Asclepiadaceae
Medicinal-edible plant
Dinormonoterpenoid glucosides
ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
that the methanol extract of the whole plant has cytotoxic activity
(Li and Zhang, 2003), and that two new steroidal saponins,
Myriopteron extensum (Wight & Arn.) K. Schum. is the single
species of the genus Myriopteron (Asclepiadaceae), and has its main
distribution in China, India, Indonesia, Myanmar, Thailand and
Vietnam (Li et al., 1995). In the Yunnan Province of China, the aerial
part of the plant in folk medicine is used for cough and
tuberculosis, and the root is used as a remedy for cough, cold,
pulmonary tuberculosis, menorrhagia, uterine prolapse and
prolapse (Editorial Committee of the Administration Bureau of
Traditional Chinese Medicine, 1998). During our ethnobotanical
investigation on medicinal and edible plants used by minority
groups in the Yunnan Province, we found that the Yao people in the
county of Xinping collect fruits of M. extensum in December each
year and eat the pericarps as pickled vegetable. To date, no study
on neither chemistry nor bioactivity of the pericarp of M. extensum
have been published. For those reasons, a chemical study on the
pericarp of M. extensum was conducted, which led to the isolation
of three new dinormonoterpenoid glucosides. The chemical
studies on compounds with this type of structure were rare
(Abe and Yamauchi, 1996; Abe et al., 1996), and no bioactivity
study on them was reported by now. As previous studies revealed
extensumside A with cytotoxic activity and extensumside B, were
obtained in the earlier chemical investigation of the whole plant
(Yang et al., 2004), we conducted the cytotoxic assay of the three
new dinormonoterpenoid glucosides to explore whether this type
of compounds having cytotoxic. In this paper, we report the
structure elucidation and the results of cytotoxicity of the three
new dinormonoterpenoid glucosides.
2. Results and discussion
The combined and concentrated extracts of pericarps of M.
extensum (MeOH and 60% MeOH) was partitioned with petroleum
ether and water five times to yield three portions: a petroleum
ether portion, an emulsified portion, and an aqueous portion. The
aqueous portion was concentrated and chromatographed over a
macroporous resin HP-20, silica gel, and Sephadex LH-20 to obtain
compounds 1–3 (Fig. 1).
Compound 1, a colorless gum, had a molecular formula
C
14H26O9 determined on the basis of HRESIMS peak of [M+Na]+
at m/z 361.1482 (calcd. for C14H26O9Na, 361.1475), with two
degrees of unsaturation. In 1H NMR spectrum, an anomeric proton
signal (
CH; 77.9, CH; 71.5, CH; 77.8, CH; 62.7, CH2) in 13C NMR and DEPT
spectra (Table 1) showed the component sugar to be -linked
dH 4.25, d, J = 7.8 Hz) as well as signals (dC 104.3, CH; 75.0,
*
Corresponding author. Tel.: +86 871 65223224; fax: +86 871 65216335.
E-mail address: duzhizhi@mail.kib.ac.cn (Z.-Z. Du).
b
http://dx.doi.org/10.1016/j.phytol.2015.03.018
1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Q. Dai et al. / Phytochemistry Letters 12 (2015) 164–167
165
Fig. 1. The structures of compounds 1–3.
glucopyranose (Abe and Yamauchi, 1996). The aglycone moiety
was considered to be composed of eight carbons, with one degree
of unsaturation. In 13C NMR and DEPT spectra, two carbon signals
(dC-4 62.6 and dC-3 64.7) were observed at slightly higher field than
those of usual carbinols, suggesting the presence of an epoxide
(Abe and Yamauchi, 1996). The correlations signals in 1H–1H COSY
in the HRESIMS spectrum. In the 1D-NMR spectra, the signals of 2
were similar to those of 1 (Table 1). According to the correlations of
1H–1H COSY and HMBC, compound 2 has the same planar structure
as 1. The relative configuration of the 1-hydroxypropan-2-yl group
and hydroxymethyl group of 2 was to be cis determined by
correlations signals of H2-2/H-4 and H2-5/H-6 in ROESY spectrum
of 2 (Fig. 3). Thus, the structure of 2 was characterized as rel-
(3R,4S)-3-(1-hydroxypropan-2-yl)-3,4-epoxypentane-1,5-diol-1-
spectrum showed three fragments: –O–CH2–CH–CH3
(dH-6 1.59–
1.65 and H-7 3.54, 3.72; H-6 1.59–1.65 and H-8 1.02), –CH2–CH2–
d
d
d
O– (dH-1 3.49, 3.89 and dH-2 1.89, 2.14), –O–CH2–CH–O– (dH-4
3.16 and dH-5 3.63, 3.81). The plane structure of aglycone was
suggested to be a dinormonoterpenoid: 3-(1-hydroxypropan-2-
yl)-3,4-epoxypentane-1,5-diol, by the correlations signals (dC-3
64.7 and H-2, 4, 5, 6, 7, 8; dC-2 30.4 and H-4, 6) in the HMBC
spectrum (Fig. 2). The glucosidic linkage was determined by the
O-
b
-
D
-glucopyranoside.
Compound 3 was isolated as a colorless gum. The molecular
formula is C14H24O9, determined by the [M+Na]+ peak at m/z
359.1318 (calcd. for C14H24O9Na, 359.1318) in the HRESIMS
spectrum, indicating that compound 3 has two protons less and
one degree of unsaturation more than compounds 1 and 2. On
comparing the 1D-NMR data (Table 1) of compound 3 with those of
1 and 2, compound 3 showed two typical signals for a terminal
double bond (dC 145.9, qC; 114.2, CH2, dH 5.13, s, 5.28, s), rather
than the signals for a –CHCH3 group in 1 and 2. Further confirmed
by 1H–1H COSY and HMBC spectra, the planar structure of 3 was
predicted to be 3-(1-hydroxy-2-propen-2-yl)-3,4-epoxypentane-
HMBC correlations between the anomeric proton at
at 66.1 (Fig. 2).
The relative configuration of the 1-hydroxypropan-2-yl group
d 4.25 and C-1
d
and hydroxymethyl group was determined to be trans by the
correlations signals of H2-2/H2-5 and H-4/H-6 in the ROESY
spectrum of 1 (Fig. 3). The absolute configuration of the sugar was
determined to be
D
-glucose by GC analysis to compare of the
1,5-diol-1-O-b-D-glucopyranoside. The relative configuration of
retention time of the derivative obtained from the acylation
reaction of hydrolyzed 1 with its authentic sugar derivatized in a
similar way, which showed retention time at 21.985 min. Thus, the
structure of 1 was characterized as rel-(3R,4R)-3-(1-hydroxypro-
the 1-hydroxy-2-propen-2-yl group and hydroxymethyl group of
compound 3 was to be cis by the correlation signals of H2-2/H-4
and H2-5/H2-8 in the ROESY spectrum of 3 (Fig. 3). Thus, the
structure of 3 was characterized as rel-(3R,4S)-3-(1-hydroxy-2-
pan-2-yl)-3,4-epoxypentane-1,5-diol-1-O-
b-
D-glucopyranoside.
propen-2-yl)-3,4-epoxypentane-1,5-diol-1-O-b-D-glucopyrano-
Compound 2 was isolated as a colorless gum. It has the same
molecular formula C14H26O9 as that of compound 1, determined by
peak of [M+Na]+ at m/z 361.1482 (calcd. for C14H26O9Na, 361.1475)
side.
Compounds 1–3 were evaluated for their cytotoxicity against
HL-60, SMMC-7221, A-549, MCF-7, and SW-480 human cancer cell
Table 1
1H (600 MHz, CD3OD) and 13C (100 MHz) NMR data of compounds 1–3,
d
in ppm.
No. of C/H
1
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
2
66.1
3.49 (ddd, 9.8, 7.5, 6.4)
3.89 (dt, 10.1, 6.1)
1.89 (dt, 15.1, 5.9)
2.14 (dt, 14.6, 7.0)
66.3
3.49 (m)
66.8
3.62 (dt, 9.9, 7.2)
3.92 (dt, 9.9, 6.0)
1.87 (dt, 15.1, 5.8)
2.17 (ddd, 14.6, 7.8, 6.5)
3.97 (ddd, 9.9, 7.7, 5.9)
1.83 (dt, 14.5, 7.2)
30.4
29.7
36.2
2.30 (ddd, 13.7, 7.5, 5.9)
3
4
5
64.7
62.6
61.0
63.9
63.9
61.6
64.0
64.0
61.5
3.16 (dd, 6.3, 4.7)
3.63 (dd, 12.0, 6.4)
3.81 (dd, 12.0, 4.5)
1.61 (m)
3.29 (m)
3.14 (m)
3.64 (dd, 12.0, 6.6)
3.81 (dd, 12.1, 5.1)
1.75 (m)
3.44 (dd, 12.0, 6.3)
3.55 (dd, 12.0, 5.3)
6
7
40.4
65.0
39.9
64.9
145.9
63.8
3.54 (dd, 10.8, 7.4)
3.72 (dd, 10.7, 6.1)
1.02 (d,7.1)
3.49 (m)
4.11 (d, 14.1)
4.19 (d, 14.1)
5.13 (s)
3.56 (m)
8
14.1
12.5
1.00 (d, 6.9)
114.2
5.28 (s)
10
20
30
40
50
60
104.3
75.0
77.9
71.5
77.8
62.7
4.25 (d, 7.8)
3.17 (m)
104.4
75.0
77.9
71.6
77.9
62.7
4.23 (d, 7.9)
3.17 (dd, 9.0, 7.9)
3.35 (m)
104.5
75.0
77.9
71.6
77.9
62.7
4.23 (d, 7.8)
3.16 (m)
3.36 (m)
3.34 (m)
3.28 (m)
3.27 (m)
3.31 (m)
3.28 (m)
3.27 (m)
3.27 (m)
3.67 (dd, 11.9, 4.8)
3.85 (dd, 11.6, 4.4)
3.67 (m)
3.67 (dd, 11.9, 5.3)
3.86 (m)
3.86 (d, 12.0)

166
Q. Dai et al. / Phytochemistry Letters 12 (2015) 164–167
CH3
H
3.3. Extraction and isolation
O
HOH2C
O
The air-dried and powered pericarps (without seeds and
tomenta inside) of M. extensum (1.4 kg) were extracted with
MeOH at room temperature (15 L ꢀ 3 days ꢀ 3 times), then with
60% MeOH under ultrasonic waves (8 L ꢀ 30 min ꢀ 3 times). The
combined extract was concentrated under reduced pressure. The
residue was partitioned with petroleum ether and water five times
to yield petroleum ether portion (24.45 g), emulsified portion
(45.24 g), and aqueous portion (about 300 g). The aqueous portion
was chromatographed over a macroporous resin HP-20 column
(2 kg), and eluted with H2O, 30% MeOH, 50% MeOH, 70% MeOH,
and MeOH, successively. The 30% MeOH–H2O fraction (10.26 g)
was chromatographed on silica gel column (200–300 mush, 500 g),
eluting with CHCl3–MeOH (9:1 ! 8:2 ! 7:3) to get fractions 1–10.
Fr. 7 (1.811 g) was subjected to silica gel column chromatography
(200–300 mush, 150 g) again, eluted with CHCl3–MeOH–H2O
(9:1:0.05) to Fr. 7-1 to 7-8. Fr. 7-3 (97 mg), Fr. 7-5 (60 mg), and Fr.
7-8 (723 mg) were further purified by Sephadex LH-20 column
chromatography, eluted with methanol to obtain compounds 2
(20 mg), 3 (18 mg), and 1 (126 mg), respectively.
CH2OH
HOH2C
HO
HO
O
OH
Fig. 2. Selected 1H–1H COSY (bold lines) and HMBC (H ! C) correlations of
compound 1.
lines. However, they were all inactive under the tested concentra-
tions (IC50 > 40
cancer cell lines are 2.22
7.25 M (A-549), 12.16
respectively. The IC50 values of paclitaxel against the five cancer
m
M). The IC50 values of cisplatin against the five
M (HL-60), 10.21 M (SMMC-7221),
M (MCF-7), and 10.07 M (SW-480),
m
m
m
m
m
cell lines are less than 0.008
positive controls).
mM (cisplatin and paclitaxel as
3. Experimental
3.3.1. rel-(3R,4R)-3-(1-Hydroxypropan-2-yl)-3,4-epoxypentane-1,5-
diol-1-O-
Colorless gum; [
max (nm) (log e): 209 (2.23), 194 (1.92); IR (KBr) n
max cmꢁ1: 3418,
b-D-glucopyranoside (1)
3.1. General experimental procedures
a
]
D
21 = ꢁ31.0 (c = 0.20, MeOH); UV (MeOH)
l
Optical rotations were measured with a Horiba Sepa-300
polarimeter. UV spectra were obtained using a Shimadzu UV-
2401A spectrophotometer. A tensor 27 spectrophotometer was
used for scanning IR spectroscopy using KBr pellets. 1D and 2D
NMR spectra were measured on Brucker AM-400 and DRX-500,
Avance III 600 with tetramethylsilane (TMS) as internal standard.
2929, 1636, 1384, 1036, 630; 1H and 13C NMR data see Table 1;
HRESI-MS m/z 361.1482 [M+Na]+ (calc. 361.1475 for C14H26O9Na).
3.3.2. rel-(3R,4S)-3-(1-Hydroxypropan-2-yl)-3,4-epoxypentane-1,5-
diol-1-O-
Colorless gum; [
max (nm) (log e): 204 (2.67); IR (KBr) n
max cmꢁ1: 3427, 2928, 1635,
b-D-glucopyranoside (2)
a]
21 = ꢁ34.0 (c = 0.19, MeOH); UV (MeOH)
Unless otherwise specified, chemical shifts (d) were expressed in
D
l
ppm with reference to the solvent signals. ESIMS was obtained on a
Bruker HTC/Esquire spectrometer. HRESIMS was recorded on an
Agilent G6230 TOF MS spectrometer.
1384, 1035, 582; 1H and 13C NMR data see Table 1; HRESI-MS m/z
361.1471 [M+Na]+ (calc. 361.1475 for C14H26O9Na).
Column chromatography (CC) was done using silica gel (200–
300 mesh, Qingdao Marine Chemical Ltd. Co., China) and Sephadex
LH-20 (GE Healthcare bio-sciences AB, Sweden), TLC was
performed on silica gel GF254 (Qingdao Marine Chemical Led.
Co., China), and spots were visualized by heating silica gel plates
sprayed with 10% H2SO4 in ethanol. GC analysis was performed on
an Agilent 7890A gas chromatography equipped with an H2 flame
ionization detector (FID).
3.3.3. rel-(3R,4S)-3-(1-Hydroxy-2-propen-2-yl)-3,4-epoxypentane-
1,5-diol-1-O-
Colorless gum; [
max (nm) (log e): 223 (2.47), 201 (3.14); IR (KBr) n
max cmꢁ1: 3427,
b-D-glucopyranoside (3)
a
]
D
21 = ꢁ61.4 (c = 0.14, MeOH); UV (MeOH)
l
2929, 1634, 1034, 576; 1H and 13C NMR data see Table 1; HRESI-MS
m/z 359.1318 [M+Na]+ (calc. 359.1318 for C14H24O9Na).
3.4. Acid hydrolysis of compounds 1–3
3.2. Plant material
Compounds 1–3 (2 mg) were dissolved in 1 M HCl (dioxane–
H2O, 1:1, 2 mL) and heated at 95 8C for 2 h, respectively. After
drying under a stream of nitrogen, the reaction mixtures were
extracted with EtOAc (ꢀ3). The aqueous layer was neutralized with
NaHCO3 and evaporated under vacuum to furnish a neutral residue
(Wang et al., 2006). The residue was dissolved in anhydrous
The fresh fruits of M. extensum were collected from Xinping
County of Yunnan Province, China in December 2011 and were
identifiedby Prof.Li-SongWang.Avoucherspecimen(KUN0309000)
has been deposited with the Herbarium of the Kunming Institute of
Botany, Chinese Academy of Sciences.
CH2
HOH2C
H
CH3
HOH2C
H
CH3
H
O
O
O
HOH2C
O
CH2OH
CH2OH
CH2OH
HOH2C
HO
HO
HOH2C
HO
HO
HOH2C
O
O
O
HO
O
O
HO
OH
OH
OH
3
2
1
Fig. 3. Selected ROESY correlations of compounds 1–3.

Q. Dai et al. / Phytochemistry Letters 12 (2015) 164–167
167
pyridine (1 mL), to which 2 mg of
L
-cysteine methyl ester
Key Laboratory of Phytochemistry and Plant Resources in West
China, Kunming Institute of Botany, for measuring all the spectra
data, and Professor Yan Li’s group for cytotoxicity bioassay.
hydrochloride was added. The mixture was stirred at 60 8C for
2 h, and after evaporation in vacuo to dryness, 0.2 mL of N-
trimethylsilylimidazole was added; the mixture was kept at 60 8C
for another 2 h (Dong et al., 2011). The reaction mixture
was partitioned between n-hexane and H2O (2 mL each), and
the n-hexane extract analyzed by GC under the following
conditions: capillary column, HP-5 (5% phenyl methyl siloxan,
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.
03.018.
50 m ꢀ 0.32 mm, with a 0.52
mm film, Agilent); detection, FID;
detector temperature, 250 8C; injection temperature, 250 8C;
initial temperature 160 8C, then raised to 280 at 5 8C/min; carrier,
He gas; flow rate: 2.219 mL/min.
References
3.5. Cytotoxicity assay
Abe, F., et al., 1996. Dinormonoterpenoids and their apiosylglucosides from
Thevetia peruviana. Phytochemistry 43, 161–163.
Abe, F., Yamauchi, T., 1996. 10-Carboxyloganin, normonoterpenoid glucosides and
dinormonoterpenoid glucosides from the leaves of Cerbera manghas. Chem.
Pharm. Bull. 44, 1797–1800.
Alley, M.C., et al., 1988. Feasibility of drug screening with panels of human tumor
cell lines using a microculture tetrazolium assay. Cancer Res. 48, 589–601.
Dong, L., et al., 2011. Cytotoxic triterpenoid saponins from Lysimachia clethroides. J.
Nat. Prod. 74, 2128–2136.
The cytotoxicity assay was performed using the MTT method,
and the following human tumor cell lines were used: HL-60,
SMMC-7221, A-549, MCF-7, and SW-480. Each tumor cell line was
exposed to each test compound at various concentrations in
triplicate for 48 h, with cisplatin and paclitaxel (Sigma) used as
positive controls (Alley et al., 1988).
Editorial Committee of the Administration Bureau of Traditional Chinese Medicine,
1998. Chinese Materia Medica (Zhonghua Bencao), vol. 17. Shanghai Press of
Science and Technology, Shanghai, pp. 5711–5713.
Acknowledgements
Li, P.T., Gilbert, M.G., Stevens, W.D., 1995. Asclepiadaceae. In: Flora of China 16, Flora
of China Editorial Committee of Chinese Academy of SciencesScience Press,
Beijing, pp. 195.
Li, Y.Y., Zhang, G.L., 2003. Chemical constitutes and the cytotoxic activity of
Myriopteron extensum. Nat. Prod. Res. Dev. 15, 113–115.
Wang, J.S., et al., 2006. Isoflavone diglycosides from Glycosmis pentaphylla. J. Nat.
Prod. 69, 778–782.
Yang, M.F., et al., 2004. Steroidal saponins from Myriopteron extensum and their
cytotoxic activity. Planta Med. 70, 556–560.
This work was sponsored by grants from the Natural Science
Foundation of the Yunnan Province (2013FB065), the 45th
Scientific Research Foundation for the Returned Overseas Chinese
Scholars from State Education Ministry and the National Science
and Technology Support Program of China (2013BAI11B02). The
authors are grateful to members of the analytical group in State