New stigmastane type of steroidal glycosides from the roots of Vernonia cumingiana

Article abstract of DOI:10.1080/07328303.2016.1170137

Two new stigmastane type of steroidal glycosides, vernoniacums A and B (1 and 2), with a Δ^7,9(11) steroidal core were isolated from the roots of Vernonia cumingiana. Their structures were elucidated based on various spectroscopic techniques, including IR, HR-FAB-MS, and ID and 2D NMR. Both compounds were evaluated fortheir cytotoxicity against HeLaand HCT-8 cells, and compound 1 showed mild activity against the tested cell lines with IC50 values of 15.8 and 35.7 pounds were evaluated for their cytotoxicity against HeLa and HCT-8 cells, and compound 1 showed mild activity against the tested cell lines with IC₅₀ values of 15.8 and 35.7 μM, respectively. (Formula Presented).

Full text of DOI:10.1080/07328303.2016.1170137

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
New stigmastane type of steroidal glycosides from
the roots of Vernonia cumingiana
Guo-Xu Ma, Wei Feng, Zhong-Hao Sun, Peng-Fei Li, Nai-Liang Zhu, Jun-Shan
Yang, Xu-Dong Xu & Hai-Feng Wu
To cite this article: Guo-Xu Ma, Wei Feng, Zhong-Hao Sun, Peng-Fei Li, Nai-Liang Zhu, Jun-Shan
Yang, Xu-Dong Xu & Hai-Feng Wu (2016) New stigmastane type of steroidal glycosides from
the roots of Vernonia cumingiana, Journal of Carbohydrate Chemistry, 35:3, 172-179, DOI:
10.1080/07328303.2016.1170137
To link to this article: http://dx.doi.org/10.1080/07328303.2016.1170137
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Date: 11 June 2016, At: 23:09

JOURNAL OF CARBOHYDRATE CHEMISTRY
, VOL. , NO. , –
http://dx.doi.org/./..
New stigmastane type of steroidal glycosides from the roots
of Vernonia cumingiana
Guo-Xu Maa, Wei Fengb, Zhong-Hao Suna, Peng-Fei Lia, Nai-Liang Zhua,
Jun-Shan Yanga, Xu-Dong Xua, and Hai-Feng Wua
aKey Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of
Education, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese
Academy of Medical Sciences, Beijing, P. R. China; bPharmacology Laboratory of Traditional Chinese
Medicine, Hebei University of Chinese Medicine, Shijiazhuang, P. R. China
ABSTRACT
ARTICLE HISTORY
Received  February 
Accepted  March 
Two new stigmastane type of steroidal glycosides, vernoniacums
7,9(11)
A and B (1 and 2), with a ꢀ
steroidal core were isolated from
the roots of Vernonia cumingiana. Their structures were eluci-
dated based on various spectroscopic techniques, including IR,
HR-FAB-MS, and1Dand2DNMR. Bothcompoundswereevaluated
for their cytotoxicity against HeLa and HCT-8 cells, and compound
1 showed mild activity against the tested cell lines with IC50 val-
ues of 15.8 and 35.7 pounds were evaluated for their cytotoxicity
against HeLa and HCT-8 cells, and compound 1 showed mild activ-
ity against the tested cell lines with IC50 values of 15.8 and 35.7 μM,
respectively.
KEYWORDS
Cytotoxic activity; steroidal
glycoside; vernonia
cumingiana
GRAPHICAL ABSTRACT
Introduction
Vernonia cumingiana Benth is a member of the genus Vernonia in the Compositae
family and is widely distributed in the Guangxi, Yunnan, and Guangdong provinces
of China.[1] As a traditional Chinese medicine, this plant has been used histor-
ically for the treatment of rheumatic arthritis, lumbocrural pain, fracture, and
CONTACT Xu-Dong Xu
xdxu@.com; Hai-Feng Wu
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant
hfwu@implad.ac.cn
Key Laboratory of Bioactive
Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing , P. R. China.
Supplemental data for this article can be accessed on the publisher’s website at http://dx.doi.org/./
..
©  Taylor & Francis Group, LLC

JOURNAL OF CARBOHYDRATE CHEMISTRY
173
Figure . Structures of compounds –.
malaria in southern China.[2] Previous phytochemical investigations on this genus
have disclosed the presence of sesquiterpene lactones and steroidal glycosides that
have extensive biological activities, such as cytotoxic, antibacterial, and antimalar-
ial effects, etc.[3–10] Among these components, steroidal glycosides possessing the
ꢀ
7,9(11) steroidal core with typical UV characteristics and representative 1H and 13C
NMR data for the respective conjugated diene system were not common in reported
natural products. For the purpose of finding new bioactive agents from medicinal
plants, our team examined the methanol extract of V. cumingiana and isolated two
new stigmastane type of steroidal glycosides, vernoniacums A and B (1 and 2, Fig. 1)
with the ꢀ7,9(11) steroidal core. In this paper, we reported the isolation and structure
elucidation of the new compounds and their cytotoxic activities.
Results and discussions
Compound 1 was obtained as an amorphous solid that had [α]2D0 + 58.4 (c = 0.07,
MeOH). Its positive reaction with the Liebermann–Burchard and Molisch reagents
indicated that it might be a saponin.[11–12] Its HR-FAB-MS in the positive-ion mode
gave a quasi-molecular ion peak at m/z 671.3471 [M + Na]+ (calcd. 671.3407 for
C35H52O11Na). In conjunction with the analysis of 1H and 13C NMR (Table 1) spec-
tra, the formula of compound 1 was deduced as C35H52O11. Fragment ion at m/z
509.27 indicated the loss of a hexose moiety. UV spectrum displayed absorptions at
234, 243, and 250 nm, as reported for the ꢀ7,9(11) stigmastane type of glycoside.[13]
IR spectrum indicated the presence of a carbonyl group (1,764 cm−1) and a dou-
ble bond (1,641 cm−1). The 1H NMR spectrum (Table 1) of 1 showed the angular
signals at δ 0.89 (s, H3-18) and 1.22 (3H, s, H-19), two olefinic protons signals at
H
δ 5.43 (1H, t, J = 1.8 Hz, H-7) and 5.59 (1H, t, J = 2.4 Hz, H-11), and the dis-
H
tinctive H-3 proton signal at δ 3.95 (m), as well as a set of proton signals for the
H
sugar moiety with its anomeric proton at δ 5.01 (1H, d, J = 8.4 Hz, H-1ꢁ). The
H
13C NMR spectrum (Table 1) displayed 35 carbon signals including six for a sugar
moiety and 29 for the aglycone unit, which exhibited a conjugated diene consist-
ing of two trisubstituted carbon signals at δ 120.4 (C-7) and 119.8 (C-11), two
C

174
G.-X. MA ET AL.
Table . NMR data ( MHz, in pyridine-d) for compounds 1 and 2.
1
2
Position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)

., CH
., CH
., CH
., CH
., CH
., CH
., CH
., C
., C
., m., m
., m., m
., m
., m., m
., m
., m., m
., t (.)
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., C
., m., m
., m., m
., m
., m., m
., m
., m., m
., t (.)








., C




., C
., C
., CH
., CH
., C
., t (.)
., dd (., .)., d (.)
., CH
., CH
., C
., t (.)
., dd (., .)., d (.)








., CH
., CH
., CH
., CH
., CH
., CH
., CH
., C
., m
., m., m
., m
., dd (., .)
., s
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., C
., m
., m., m
., m
., dd (., .)
., s
., s
., m
., d (.)
., dd (., .)
., d (.)
., s
., m



., CH
., CH
., C
., dd (., .)
., d (.)




., CH
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., m
., d (.)
., d (.)
., q (.)
., d (.)
., d (.)
., m
., m
., m
., m
., CH
., CH
., CH
., C
., CH
., CH
., CH
., CH
., CH
., CH
., CH
., C
., m
., d (.)
., d (.)

., s
., d (.)
., m
., m
., m
., m
., m., m
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
., m., m
-OCOCH
-OCOCH
-OCH
., CH
., CH
., s
., s
tetrasubstituted ones at δ 136.4 (C-8) and 144.1 (C-19), and two angular carbons at
C
δ 19.6 (C-18) and 13.6 (C-19). All of the proton signals were assigned to the corre-
C
sponding carbons through direct 1H and 13C correlations in the HMQC spectrum.
The above-mentioned data proved that compound 1 was a ꢀ7,9(11) stigmastane type
7,9(11)
of glycoside.[14–15] Excluding the seven degrees of unsaturation due to the ꢀ
stigmastane type of steroidal skeleton and the sugar unit, three degrees of unsat-
uration remained for the C10H15O4 side chain which included three methyl, five
methine, and two quaternary carbons including an ester carbonyl group (δ 178.2).
C
For the side chain, the Heteronuclear Multiple Bond Correlation (HMBC) correla-
tions (Fig. 2) between δ 2.55 (H-20) and C-21 (δ 178.2) or C-22 (δ 81.1), between
H
C
C
δ 5.78 (H-22) and C-20 (δ 52.8) or C-23 (δ 86.8), between δ 4.88 (H-23) and
H
C
C
H
C-21 (δ 178.2) or C-24 (δ 85.1), and between δ 4.52 (H-28) and C-22 (δ 81.1),
C
C
H
C
C-23 (δ 86.8) or C-24 (δ 85.1) indicated the presence of butyrolactone and furan
C
C
rings, which were linked by C-22 and C-23. This partial structure was also deduced

JOURNAL OF CARBOHYDRATE CHEMISTRY
175
Figure . Key HMBC correlations of compound .
1
by H-1H COSY spectrum. Meanwhile, the proton signals at δ 1.07 (3H, d, J =
H
7.2 Hz, H3-26), 1.28 (3H, d, J = 7.2 Hz, H3-27), 1.35 (3H, d, J = 7.8 Hz, H3-29), and
2.32 (1H, m, H-25), together with three methyl resonances at δ 17.0 (C-26), 18.0
C
(C-27), and 15.9 (C-29), and a methenyl carbon signal at δ 28.7 (C-25) revealed the
C
presence of an isopropyl moiety and a methyl group. These two groups were deter-
mined to be attached to C-24 and C-28, respectively, on the basis of HMBC corre-
lations between δ 2.32 (H-25) to C-24 (δ 85.1) and δ 1.35 (H-29) to C-28 (δ
H
C
H
C
85.0). The sugar moiety was located at C-3 on the basis of the correlation between
the proton signal at δ 3.95 (1H, m, H-3) and the anomeric carbon at δ 102.3. The
H
C
type and absolute configuration of the sugars was identified as D-glucose on the
basis of comparison with authentic monosaccharide (CHCl3:MeOH:H2O = 3:2:0.2,
visualized with ethanol-5% H2SO4 spraying) by Thin Layer Chromatography (TLC)
method, followed by gas chromatography. The stereochemistry of the different ring
junctions and substituents was determined via a 2D Nuclear Overhauser Effect Spec-
troscopy (NOESY) experiment. The cross-peaks observed between H-5/H-1α, H3-
19/H-1β, H-14/H-15α, H3-18/H-12β, H-20/H-22, H-22/H-23 established the trans
fusion of the A/B and C/D rings junction, and the cis fusion of the E/F ring junc-
tion. Additionally, the NOE correlations between H-3 and H-5 and between H-16
and H3-18 indicated the β-orientation of sugar moiety at C-3 and the α-orientation
of the hydroxyl group at C-16. The large coupling constant (J = 7.8 Hz) between H-
1ꢁ and H-2ꢁ also supported the beta-linkage of the sugar moiety. Furthermore, the
association of H-22 with H3-29 and H-23 with H-25 suggested that the methyl group
at C-28 was β-oriented and the hydroxyl group at C-24 was α-oriented. Therefore,
the structure of compound 1 was established as shown in Figure 1 and named as
vernoniacum A.
Compound 2 was isolated as a white amorphous powder which had [α]2D0 + 37.9
(c = 0.08, MeOH). Its molecular formula was calculated as C38H58O13 based on
the analysis of its HR-FAB-MS data (m/z: 745.3813 [M + Na]+, calcd. 745.3775 for
C38H58O13Na). Its 1H NMR spectrum (Table 1) displayed two methyl signals at δ
H
0.63 (3H, s, H3-18) and 0.81 (3H, s, H3-19), two olefinic protons signals at δ 5.34
H

176
G.-X. MA ET AL.
(1H, t, J = 1.8 Hz, H-7) and 5.47 (1H, t, J = 2.4 Hz, H-11), and one anomeric proton
at δ 5.02 (1H, d, J = 7.8 Hz, Glu-1), corresponding to the ꢀ7,9(11) stigmastane type
H
of glycoside. Its 13C NMR (Table 1) spectrum displayed 38 resonance signals due to
one sugar moiety, five methyl groups (δ 14.7, 19.8, 17.7, 18.9, and 17.9), one acetyl
C
group (δ 22.2, 170.9), one methoxy group (δ 51.6), two double bonds (δ 122.1,
C
C
C
135.4, 144.3, 119.1), and other 20 carbons. The NMR data for this compound were
similar to that of 1, except for the downfield-shifted C-16 (δ 78.8) and C-28 (δ
C
C
113.1) and the upfield-shifted C-21 (δ 99.4) for 2, in contrast to the C-16 (δ 72.1),
C
C
C-28 (δ 85.0), and C-21 (δ 178.2) for 1. In the HMBC spectrum, the correlations
C
C
between methine H-16 (δ 5.57) and δ 170.9 and between methoxyl group δ 3.31
H
C
H
and C-28 (δ 113.1) or C-29 (δ 17.9) suggested the presence of acetyl group at C-16
C
C
and methoxy and methyl groups at C-28 for 2. Additionally, the HMBC correlations
between C-21 (δ 99.4) and H-20 or H-22 displayed that the ester carbonyl at C-21
C
in compound 1 was reduced to hydroxyl group in compound 2. The remaining cor-
relations observed in the NOESY spectrum of 2 were in same as that of 1, indicating
that the trans fusion of the A/B and C/D rings and cis fusion of the E/F ring junctions.
The NOE enhancements from H3-29 to H-17 indicated that the methyl group at C-
28 was α-oriented and the methoxyl group at C-28 was β-oriented. Accordingly, the
structure of 2 was assigned as shown in Figure 1 and named as vernoniacum B.
The isolated compounds 1 and 2 were tested for cytotoxic activity against HeLa
(cervical cancer) and HCT-8 (colon cancer) human cancer cell lines using the 3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method with
adriamycin as a positive control. The result showed that compound 2 was inac-
tive against the two cell lines, while compound 1 showed mild cytotoxic activities
towards Hela and HCT-8 cells with IC50 values of 15.8 and 35.7 μM, respectively, as
compared to the IC50 value of adriamycin (0.86, 1.24 μM).
Conclusions
Two new stigmastane type of steroidal glycosides, vernoniacums A and B (1 and 2)
with the ꢀ7,9(11) steroidal core were isolated from the roots of V. cumingiana. Their
structures were elucidated by extensive spectroscopic analysis. Compound 1 showed
mild cytotoxic activities towards Hela and HCT-8 cells with IC50 values of 15.8 and
35.7 μM, respectively.
Experimental section
General experimental procedures
Optical rotation data were obtained using a Perkin-Elmer 341 digital polarimeter.
UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrom-
eter, respectively. NMR spectra were obtained with a Bruker AV III 600 NMR spec-
trometer (chemical shift values are presented as δ values with TMS as the internal
standard and the temperature is conducted at 300.0 K). HR-ESI-MS spectra were

JOURNAL OF CARBOHYDRATE CHEMISTRY
177
performed on a LTQ-Obitrap XL spectrometer. C18 reversed-phase silica gel (40–
63 μm, Merk, Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Swe-
den), MCI gel (CHP 20P, 75–150 μm, Mitsubishi Chemical Corporation, Tokyo,
Japan) and silica gel (100–200 and 300–400 mesh, Qingdao Marine Chemical plant,
Qingdao, People’s Republic of China) were used for column chromatography. And
precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Devel-
opment, Yantai, People’s Republic of China) were used for TLC. All solvents used
were of analytical grade (Beijing Chemical Works).
Plant material
The roots of V. cumingiana were collected in September 2014 from Nanning city,
Guangxi province, China, and identified by Prof. Jing-Quan Yuan, Department
of Pharmaceutical Chemistry, Guangxi Botanical Garden of Medicinal Plant. A
voucher specimen (NO. 140905) was deposited at the Guangxi Botanical Garden
of Medicinal Plant.
Extraction and isolation
The roots of V. cumingiana (4.2 kg) were powdered and extracted three times with
methanol. Removal of the methanol under reduced pressure yielded a methanol
extract (523 g). The residue was dissolved in water and extracted with petroleum
ether (3×1,000 mL), chloroform (3×1,000 mL), ethyl acetate (3×1,000 mL) and n-
butanol (3×1,000 mL), respectively. The n-butanol-soluble fraction (68 g) was sub-
jected to silica gel column chromatography using a CHCl3-MeOH gradient (from
1:0 to 0:1) as eluent, to yield eleven fractions (Fr.VC1–VC5). The Fr. VC2 (8.6 g)
was separated to ODS MPLC eluting with MeOH-H2O (30:70; 50:50; 70:30; 80:20;
90:10; 100:0, v/v) to obtain six sub-fractions, VI1–VI6. Sub-fraction VI4 was pre-
pared by HPLC using MeOH-H2O (60:40, v/v) on YMC-Pack ODS-A column to
give compound 1 (6.7 mg, tR = 24.6 min). Sub-fraction VI5 was prepared by HPLC
using MeOH-H2O (58:42, v/v) on YMC-Pack ODS-A column to give compound 2
(7.9 mg, tR = 29.7 min).
Vernoniacum A (1)
Amorphous solid; [α]2D0 + 58.4 (c = 0.07, MeOH); UV (MeOH) λ
(log ε) 234
max
(4.16), 243 (4.20), 250 (4.04) nm; IR (KBr) cm−1 3,428, 1,764, 1,641; H and 13C
NMR (Pyridine-d5): see (Table 1); HR-FAB-MS m/z 671.3471 [M + Na]+ (calcd.
C35H52O11Na for 671.3407).
1
Vernoniacum B (2)
Amorphous solid; [α]2D0 + 37.9 (c = 0.08, MeOH); UV (MeOH) λ
(log ε) 234
max
(3.87), 243 (3.91), 250 (3.73); IR (KBr) cm−1 3,457, 2,945, 2,874, 1,698, 1,656; 1H

178
G.-X. MA ET AL.
and 13C NMR (Pyridine-d5): see (Table 1); HR-FAB-MS m/z 745.3813 [M + Na]+
(calcd. C38H58O13Na for 745.3775).
Acid hydrolysis of –
Each compound (3.0 mg) was heated in 3 mol/L CF3COOH (4 mL) for 3 h in a water
bath. Each mixture was then extracted with EtOAc. The aqueous layer was evapo-
rated to dryness with ethanol in vacuo at 50°C until neutral. The residues were deter-
mined in comparison with D-Glucose using TLC (CHCl3: MeOH: H2O = 3:2:0.2,
visualization with ethanol-5% H2SO4 spraying). Furthermore, the absolute configu-
rations of the sugars were determined by gas chromatography according to a method
previously described.[13–14] By this method, L-cysteine methyl ester hydrochloride
(0.06 mol/L) and hexamethyldisilazane-trimethylchlorosilane (HMDS-TMCS, 3:1)
were added to the aqueous residue for derivatization. The solution was then cen-
trifuged and the precipitate was removed. After these processes, n-hexane was used
to extract derivate and analyzed by GC. D-Glucose (tR = 25.3 min) was detected by
comparing with authentic monosaccharide.
Cytotoxicity bioassays
Antiproliferative activity of compounds 1–2 were assessed by MTT method using
HeLa and HCT-8 human cancer cell lines. Each cell was sent on to 96-well microtiter
plates at a density of 6 × 104 cells/mL per well. Cells were preincubated for 24 h at
37°C in 5% CO2 for 24 h. Then five different concentrations of each compound dis-
solved in dimethyl sulfoxide (DMSO) were added to each well. Each concentration
was tested in triplicate. After 48 h, 10 μL of MTT (4 mg/mL) was added to each
well and incubated for another 4 h. Then removed the liquid in the well and added
DMSO (200 μL) to each well. The absorbance was recorded on a microplate reader
at a wavelength of 570 nm.
Funding
The work was financially supported by the Technological Large Platform for Comprehensive
Research and Development of New Drugs in the Twelfth Five-Year “Significant New Drugs Cre-
ated” Science and Technology Major Projects (No. 2012ZX09301-002-001-026), the National
Natural Sciences Foundation of China (No. 81502945), and the PUMC Youth Fund (No.
3332015141).
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