Bioactive triterpenoid saponins from the tubers of Hemsleya amabilis Diels

Article abstract of DOI:10.1016/j.fitote.2019.104404

Five new cucurbitane-type triterpenoid saponins Xuedanosides A-E (1–5) were isolated from the medicinal plant Hemsleya amabilis Diels by silica gel column, octadecylsilyl (ODS) column, and pre-HPLC techniques. Their structures were determined by spectroscopic analysis and examined alongside existing data from prior studies. Separated compounds were evaluated for their cytotoxic activity in HeLa, HCT-8, MCF-7 and HepG2 human cancer cell lines, and compounds 1 and 2 showed significant effects against HeLa cells with IC₅₀ values of 3.21 and 8.57 μM, respectively.

Full text of DOI:10.1016/j.fitote.2019.104404

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Bioactive triterpenoid saponins from the tubers of Hemsleya
amabilis Diels
Yuexian Jin, Xiaolei Zhou, Junshan Yang, Xudong Xu, Jing
Zhang, Guoxu Ma
PII:
S0367-326X(19)31594-1
DOI:
https://doi.org/10.1016/j.fitote.2019.104404
FITOTE 104404
Reference:
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
8 August 2019
22 October 2019
25 October 2019
Please cite this article as: Y. Jin, X. Zhou, J. Yang, et al., Bioactive triterpenoid saponins
from the tubers of Hemsleya amabilis Diels, Fitoterapia (2019), https://doi.org/10.1016/
j.fitote.2019.104404
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Bioactive triterpenoid saponins from the tubers of Hemsleya amabilis Diels
Yuexian Jin 1,a,b, Xiaolei Zhou 1,c, Junshan Yanga, Xudong Xu a, Jing Zhang b, Guoxu
,a
Ma
Affiliations
a
Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine,
Ministry of Education, Beijing Key Laboratory of Innovative Drug Discovery of Traditional
Chinese Medicine (Natural Medicine) and Translational Medicine, Key Laboratory of Efficacy
Evaluation of Chinese Medicine against Glycolipid Metabolic Disorders, State Administration of
Traditional Chinese Medicine, Institute of Medicinal Plant Development, Peking Union Medical
College and Chinese Academy of Medical Sciences，Beijing 100193, China
b College of Chinese Medicine Materials, Jilin Agricultural University, Changchun 130118, China
c Guangxi Botanical Garden of Medicinal Plants, Nanning, Guangxi Province 530023, China
 Corresponding author. Tel./fax: +86 010 57833296
E-mail address: mgxfl98785@163.com (G.X. Ma)
1These authors contributed equally to this work.

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ABSTRACT
Five new cucurbitane-type triterpenoid saponins Xuedanosides A-E (1-5) were
isolated from the medicinal plant Hemsleya amabilis Diels by silica gel column,
octadecylsilyl (ODS) column, and pre-HPLC techniques. Their structures were
determined by spectroscopic analysis and examined alongside existing data from prior
studies. Separated compounds were evaluated for their cytotoxic activity in HeLa,
HCT-8, MCF-7 and HepG2 human cancer cell lines, and compounds 1 and 2 showed
significant effects against HeLa cells with IC50 values of 3.21 and 8.57 μM,
respectively.
Keywords: Hemsleya amabilis Diels; cucurbitane-type triterpenoid saponins;
cytotoxic activity

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1. Introduction
Hemsleya amabilis Diels, a member of the Cucurbitaceae family, is mainly
distributed in the tropical and subtropical regions of China [1]. The roots of Hemsleya
amabilis, usually called “xue dan” in China, have been an important traditional
Chinese medicine for the treatment of bacillary dysentery, inflammation, ulcers,
jaundice, and tuberculosis [2][3]. Among the isolated structures, the main chemical
constituents of this species are triterpenoid saponins, including oleanane glycosides
and cucurbitane-type glucosides [4], and triterpenoid saponins show in vitro or in vivo
pharmacological effects [5]. In clinical applications, the drugs with the main chemical
component of hemsleyadin possess hepatoprotective, antiinflammatory, purgative,
cardiovascular, antimicrobial, antihelmintic, and antifertility effects and have been
sold on the market,[6][7]. With the purpose of finding new biologically active natural
products from this genus, the 95% EtOH extract of H. amabilis was examined, and 5
new cucurbitane-type triterpenoids, named Xuedanosides A-E (1-5) were obtained
(Figure 1). In this paper, we report the structure elucidation of the new
cucurbitane-type triterpenoids and their cytotoxic activity.
2. Experiments
2.1. General experimental procedures
Optical rotation data were acquired with a Perkin-Elmer 341 digital polarimeter.
UV data were obtained with a Shimadzu UV2550 spectrometer. IR data were
documented with a FTIR-8400S spectrometer. NMR spectra were acquired with a
Bruker AV Ⅲ 600 NMR spectrometer (chemical shift values are shown as δ values
with TMS as the internal standard). HRESIMS was conducted with the LTQ-Obitrap
XL spectrometer. HPLC separation was performed with a Lumiere K-1001 pump, a
Lumiere K-2501 single λ absorbance detector, and a Kromasil (250 × 10 mm)
semipreparative column loaded with C18 (5 μm). C-18 reversed-phase silica gel (40-63
μm, Merck, Darmstadt, Germany) and silica gel (100～200 and 300～400 mesh,
Qingdao Marine Chemical plant, Qingdao, People’s Republic of China) were

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implemented for CC, and precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot
Plant of Silica Gel Development, Yantai, China) were used for TLC. All of the
solvents we used were of analytical grade (Beijing Chemical Plant, China).
2.2. Plant material
The entire plants of H. amabilis Diels were collected in Nanning, Guangxi Province,
People’s Republic of China, in October 2017 and were verified by Prof. Xiao-Lei
Zhou. A voucher specimen (CS171024) was submitted to the Institute of Medicinal
Plant Development, Chinese Academy of Medical Sciences.
2.3 Extraction and isolation
The tubers of H. amabilis (5.0 kg) were removed under refluxing three times and
extracted with 95% EtOH three times (each time for 1 h. Removal of the EtOH under
reduced pressure yielded the extract (314 g). The residue was suspended in H2O (1.5
L) and partitioned with petroleum ether (3 × 1 L), EtOAc (3 × 1 L), and n-BuOH (3 ×
1 L), successively. Part of the EtOAc extract (200 g) was subjected to silica gel
chromatography, with a gradient system of CH2Cl2-MeOH (from 1:0 to 0:1) to
produce 10 fractions (Fr. A-J). Fraction C (20.6 g) was eluted after HW-40C column
chromatography with MeOH-H2O (30:70; 60:40; 70:30; 80:20; 90:10; 100:0, v/v)
giving six subfractions (Fr. C1-C6). Fraction C3 (962 mg) was chromatographed by
semipreparative HPLC using MeOH-H2O (60:40, v/v) to yield compounds 1 (8.6 mg,
tR = 16.0 min) and 2 (6.3 mg, tR = 20.9 min). Fraction E (10.9 g) was loaded on an
ODS C18 column eluted with MeOH-H2O (30:60; 60:40; 70:30; 80:20; 90:10; 100:0,
v/v) to give six subfractions (Fr. E1–E6). Fraction E2 (413 mg) was chromatographed
by semipreparative HPLC using MeOH-H2O (65:35, v/v) to yield compound 3 (7.1
mg, tR = 30.2 min). Fraction G (7.0 g) was fractioned by MCI-gel column
chromatography and eluted with MeOH-H2O (30:60; 60:40; 70:30; 80:20; 90:10;
100:0, v/v) to give six subfractions (Fr. G1–G6). Fraction G4 (193 mg) was
chromatographed by preparative HPLC using MeOH-H2O (75:25, v/v) to yield
compounds 4 (5.9 mg, tR = 22.3 min) and 5 (8.1 mg, tR = 35.2 min).
2.3.1. Xuedanoside A (1)
C38H60O14, White amorphous powder; [α]22 + 106.7 (c = 0.1, MeOH); IR (KBr)
D

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3449-3382, 1666, 1664, 1376 cm-1; and 1H and 13C-NMR (pyridine-d5); see (Tables 1
and 2); HR-ESI-MS m/z 763.8746 [M + Na]+ (calculated for C30H46O8Na+
763.5000).
2.3.2. Xuedanoside B (2)
C38H60O14, White amorphous powder; [α]22 + 112.1 (c = 0.1, MeOH); IR (KBr)
D
3422-3400, 1660, 1648, 1076 cm-1; 1H and 13C-NMR (pyridine-d5); see (Tables 1 and
2); HR-ESI-MS m/z 763.8746 [M + Na]+ (Calculated for C31H50O7Na+ 763.5000).
2.3.3. Xuedanoside C (3)
C38H60O13, White amorphous powder; [α]22 + 60.5 (c = 0.5, MeOH); IR (KBr)
D
3434-3395, 1694, 1643, 1290 cm-1; and 1H and 13C-NMR (pyridine-d5); see (Tables 1
and 2); HR-ESI-MS m/z 747.8752 [M + Na]+ (Calculated for C30H48O8Na+
747.4018).
2.3.4. Xuedanoside D (4)
C36H58O11, White amorphous powder; [α]22 + 83.8 (c = 0.1, MeOH); IR (KBr)
D
1
3410-3300, 1663, 1638, 1363, 1216 cm-1; and H and 13C-NMR (pyridine-d5); see
(Tables 1 and 2); HR-ESI-MS m/z 689.8391 [M + Na]+ (Calculated for C30H46O4Na+
689.3973).
2.3.5. Xuedanoside E (5)
C36H56O11, White amorphous powder; [α]22 + 86.0 (c = 0.1, MeOH); IR (KBr)
D
3464-3366, 1645, 1262, 1110 cm-1; and 1H and 13C-NMR (pyridine-d5); see (Tables 1
and 2); HR-ESI-MS m/z 687.8232 [M + Na]+ (Calculated for C30H50O4Na+
687.3334).
2.4. Cytotoxicity assays
The cytotoxicity of compounds 1-5 was assessed in HeLa, HCT-8, MCF-7 and
HepG2 human cancer cell lines by the MTT method. The cells were incubated in
DMEM supplemented with 10% fetal bovine serum and cultured at a density of
1.2×104 cells/mL in a 96-well microtiter plate. Five different concentrations of each
agent dissolved in dimethyl sulfoxide (DMSO) were then placed in the wells. Each
concentration was evaluated three times. After incubation in a 5% CO2 incubator at 37℃
for 48 h, 10 μL of MTT (4 mg/mL) was placed into each well, and the cells were
incubated for an additional 4 h. The media were removed, and DMSO (200 μL) was

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added into the wells. The absorbance was documented with a microplate reader at a
wavelength of 570 nm. The experiments were conducted a minimum of three times.
2.5. Acid hydrolysis of compounds 1-5
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
evaporated to dryness with ethanol in vacuo at 50°C until the solution is neutral . The
residues were determined in comparison with D-Glucose using TLC (eluted with
CHCl3:MeOH:H2O = 3:2:0.2, and visualized with ethanol-5% H2SO4 spraying).
Furthermore, the absolute configurations of the sugar residues were determined by gas
chromatography. In this method, L-cysteine methyl ester hydrochloride (0.06 mol/L)
and hexamethyldisilazane-trimethylchlorosilane (HMDS-TMCS, 3:1) were added to
the aqueous phase for derivatization. The solution was then centrifuged and the
precipitate was removed. After these processes, n-hexane was used to extract the
derivative and analyzed by GC. D-Glucose (tR = 26.7 min) was detected by
comparison with the authentic monosaccharide.
3. Results and discussion
Compound 1 was acquired as a white amorphous powder with [α]2D2+106.7 (c 0.1,
MeOH), and the Molish and Liebermann-Burchard reactions were positive. The UV
and IR spectra revealed the absorption of hydroxyl groups (3407 cm–1) and carbonyl
groups (1664 cm-1). The HRESIMS spectrum demonstrated a quasi-molecular ion at
+
m/z 763.8746 (Calculated for C38H60NaO14 , 763.5000), which in comparison with the
NMR data, confirmed the molecular formula was C38H60O14, accounting for 9 indices
of hydrogen deficiency. The NMR spectra (Tables 1 and 2) of compound 1 showed
1
the characteristic signals of a triterpenoid saponin. Its H NMR spectrum showed the
presence of 9 angular methyl signals at δH 1.24 (s), 1.37 (s), 1.48 (s), 1.48 (s), 1.50 (s),
1.51 (s), 1.57 (s), 1.71 (s), and 1.91 (s); 9 oxygenated methines at δH 3.62 (m), 3.88
(m), 4.07 (m), 4.18 (m), 4.30 (m), 4.35 (m), 4.48 (m), 4.91 (m), and 5.34 (t, J = 6.0
Hz); and one olefinic proton signal at δH 6.23 (d, J = 6.0 Hz). The 13C NMR spectrum
showed 38 signals isolated by APT investigations into 9 angular methyls δC 19.8, 20.7,
22.2, 22.6, 23.2, 25.5, 25.9, 26.4, and 26.5; 6 methylenes; 13 methines (1 sp2 methines

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δC 123.2 and 9 oxygenated methines δC 66.5, 70.8, 71.8, 76.3, 79.0, 79.0, 81.0, 83.5,
and 107.0); and 10 quaternary carbons (1 sp2 carbons δC 144.8; 3 carbonyl carbon δC
170.6, 213.8, and 215.5; and 2 oxygenated carbon δC 80.5 and 82.0), of which 32
were assigned to the typical cucurbitacin formations [8] and the remaining 6 were
ascribed to the sugar moiety. The glucose signals at δC 107.0 (d), 76.3 (d), 79.0 (d),
71.8 (d), 79.0 (d), and 62.9 (t), as well as the signal of C-2 being shifted downfield by
12.1 ppm to δC 83.5 (d) in 1, were observed, which indicated the glucopyranosyl
moiety being linked at C-2 in 1. This deduction was confirmed by the HMBC
correlations from the anomeric proton signal at δH 5.34 (d, J = 6.0 Hz, H-1') to δC 83.5
(d, C-2). The coupling value (J = 6.0 Hz) of the anomeric proton suggested the
presence of a β-glucopyranosyl moiety. The NMR data of 1 were similar to those of
2-O-β-D-glucopyranosyl cucurbitacin [9], except for the additional hydroxyl group at
13
C-7 in 1. This observation was confirmed by the C NMR data (δC 66.5) and HMBC
correlations from δH 6.23 (H-6) to δC 66.5 (C-7) and the proton signal at δH 4.48 (m,
H-7) to C-5 (δC 144.8), C-6 (δC 123.2), and C-9 (δC 49.0). In the HMBC spectrum, the
correlations from δH 4.35 (H-2) to δC 81.0 (C-3), δH 1.87 (H-15) to δC 70.8 (C-16), and
δH 2.90 (H-17) to δC 80.5 (C-20) confirmed the presence of other hydroxyl groups at
C-3, C-16, and C-20. Similarly, the correlations from δH 6.23 (H-6) to δC 144.8 (C-5),
δH 3.11 (H-12) to δC 213.8 (C-11), and δH 3.32 (H-23) to δC 35.8 (C-24) and 215.5
(C-22) suggested that the olefinic group was at C-5 and the carbonyl groups were at
C-11, and C-22. Furthermore, the HMBC correlations from δH 2.43 (H-24) and 1.91
(-OAc) to δC 82.0 (C-25) confirmed the acetoxyl group at C-25. Examinations of its
1H-1H COSY and HSQC spectra advanced the establishment of fragments
C-10-C-1-C-2-C-3, C-6-C-7-C-8, C-15-C-16-C-17, and C-23-C-24. The chemical
changes of C-2 and C-3 and the coupling constant between H-2 and H-3 implied a
2,3-cis-diol structure on ring A [10]. The corresponding configuration of the
tetracyclic system of 1 was established with a NOESY experiment, taking into
consideration cucurbitacins’ biogenesis, and by contrasting the coupling constant
pattern with that documented in the literature for comparable compounds [11][12].
The NOE (Figure 3) improvements between H-2 and H-10 and H-3 and H-19

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3
suggested OH-2 was β-oriented and OH-3 was α-oriented, respectively. The J
coupling constant (J = 6.0 Hz) also substantiated the antiperiplanar link between H-2
and H-3. The cross-peaks observed between H-7/H3-28 supported a β-orientation of
the OH group at C-7. Similarly, NOE correlations between H-8/H3-19 and H-8/H3-18
and H3-18/H-16 corroborated the β-orientation of these protons. Thus, the structure of
1 was determined to be 3α, 7β, 16α, 20β- tetrahydroxycucurbita- 5 (E)- diene- 11, 22-
dione- 25- O- acetate- 2- O- β- D- glucopyranoside, and named Xuedanoside A.
Compound 2 was obtained as an amorphous white powder with [α]2D2+60.5 (c 0.1,
MeOH), and its molecular formula was established as C38H60O14 from the molecular
1
ion peak at m/z 763.8746 [M + Na]+ in the HRESMS. Its H and 13C NMR data
(Tables 1 and 2) were close to those of 1, which were isomeric, with the exception of
the presence of the sugar group at C-24 (δC 80.3), hydroxyl group at C-25 (δC 77.0)
and acetoxyl group at C-16 (δC 75.2) in 2 instead of the sugar group at C-2 (δC 83.5),
hydroxyl group at C-16 (δC 70.8) and acetoxyl group at C-25 (δC 82.0) in 1, and the
lack of a hydroxyl group at C-7 (δC 24.4). In the HMBC spectrum, the sugar moiety
was located at C-24 on the basis of the correlation between the proton signal at δH
4.44 (H-24) and anomeric carbon at δC 95.6, and the signal for C-24 revealed a
powerful downfield shift to δ 80.3 (+44.5 ppm). Similarly, the correlations from δH
3.02 (H-17) and 2.22 (-OAc) to C-16 (δC 75.2) confirmed the acetoxyl group at C-16.
the correlations of H-24 (δH 4.44), H-26 (δH 1.38) and H-27 (δH 1.36) with the upfield
carbon C-25 (δC 77.0) (compared with C-25 in 1) implied the hydroxyl group at C-25.
Furthermore, in comparison to 1, the signal for C-7 revealed a powerful upfield shift
1
to δ 24.4 (-42.1 ppm), while the hydroxyl group was absent. Taken along with H-1H
COSY, HSQC, HMBC, and NOE spectra, the structure of compound 2 was
determined to be 2β, 3α, 20β, 25- tetrahydroxycucurbita- 5(E)- diene- 11,22- dione-
16- O- acetate- 24- O- β- D- glucopyranoside and named Xuedanoside B.
Compound 3 was separated as a white amorphous powder with [α]2D2+112.1 (c 0.1,
MeOH). Its molecular formula was determined to be C38H60O13 by HRESIMS
(observed m/z 747.8752 [M + Na]+), necessitating nine degrees of unsaturation. Its 1H
and 13C NMR data (Tables 1 and 2) were close to those of 2, with the exception of the

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presence of the sugar group at C-25 (δC 77.0) in 3 instead of the sugar group at C-24
(δC 80.3) in 2. In the HMBC spectrum, the sugar moiety was located at C-25 on the
basis of the correlation between the proton signal at δH 4.95 (H-1’) and oxygenated
carbon at δC 77.0 (C-25), and the signal for C-25 revealed a powerful downfield shift
to δ 83.0 (+5 ppm). Taken along with 1H-1H COSY, HSQC, HMBC, and NOE spectra,
the structure of compound 3 was determined to be 2β, 3α, 20β- trihydroxycucurbita- 5
(E)- diene- 11, 22- dione- 16- O- acetate- 25- O- β- D- glucopyranoside, and named
Xuedanoside C.
Compound 4 was separated as a white amorphous powder with [α]2D2+112.1 (c 0.1,
MeOH). Its molecular formula was determined to be C36H58O11 by HRESIMS
(observed m/z 689.8391 [M + Na]+), necessitating eight degrees of unsaturation. Its
1H and 13C NMR data (Tables 1 and 2) were close to those of 1, with the exception of
the presence of the sugar group at C-3 (δC 87.6) and hydroxyl group at C-25 (δC 77.0)
in 4 instead of the sugar group at C-2 (δC 83.5) and acetoxyl group at C-25 (δC 82.0)
in 1, and the lack of a hydroxyl group at C-7 (δC 24.4). In the HMBC spectrum, the
sugar moiety was located at C-3 on the basis of the correlation between the proton
signal at δH 3.64 (H-3) and the anomeric carbon at δC 107.8, and the signal for C-3
revealed a powerful downfield shift to δ 87.6 (+6.6 ppm). Similarly, the correlations
of H-24 (δH 2.25), H-26 (δH 1.37) and H-27 (δH 1.37) with the upfield carbon C-25 (δC
69.5) (compared with C-25 in 1) implied the hydroxyl group at C-25. Furthermore, in
comparison to 1, the signal for C-7 revealed a powerful upfield shift to δ 24.6 (-41.9
1
ppm), while the hydroxyl group was absent. Taken along with H-1H COSY, HSQC,
HMBC, and NOE spectra, the structure of compound 4 was determined to be 16α, 20β,
25- trihydroxycucurbita- 5 (E)- diene- 11, 22- dione- 3- O- β- D- glucopyranoside and
it was named Xuedanoside D.
Compound 5 was separated as a white amorphous powder with [α]2D2+112.1 (c 0.1,
MeOH). Its molecular formula was determined to be C36H56O11 by HRESIMS
(observed m/z 687.8232 [M + Na]+), necessitating nine degrees of unsaturation. The
IR spectrum revealed strong bands at 3633-3421 cm-1, which is typical of the
1
hydroxyl group. The H NMR (Tables 1 and 2) displayed signals due to 7 tertiary

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methyl groups at δH 1.21 (s), 1.24 (s), 1.26 (s), 1.31 (s), 1.41 (s), 1.53 (s), and 1.99 (s);
9 oxygenated methines at δH 3.37 (d, J = 9.0 Hz), 3.94 (m), 4.12 (m), 4.21 (m), 4.24
(m), 4.29 (m), 4.98 (m), 5.12 (m), and 5.19 (m); and 2 olefinic proton signals at δH
5.68 (d, J = 6.0 Hz) and δH 6.68 (d, J = 6.0 Hz). The 13C NMR spectrum of 5 revealed
36 carbon signals corresponding to 7 angular methyls, 7 methylenes (2 oxygenated
methylenes), 14 methines (2 sp2 methines and 9 oxygenated methines), and 8
quaternary carbons (2 sp2 carbons, 1 carbonyl carbon, and 1 oxygenated carbon). The
NMR data of 1 were similar to those of Xuedanencins B, except for the additional
glucopyranosyl moiety at C-3 in 5 which was confirmed by the HMBC correlations
from the anomeric proton signal at δH 5.19 (d, J = 8.4 Hz, H-1') to δC 94.3 (d, C-3).
1
The H and 13C NMR spectra of 5 substantiated the substructure for another ring
created by the cyclization of the side chain by an ether linkage [13][14]. Additionally,
13
a C-1H long-rang correlation signal between H-16 and C-23 showed that C-16 and
C-23 were connected via an ether bond to create a pyranoid structural element. In the
HMBC, 13C-1H long-range correlation signals at H-23/C-25 and H3-26 and
H2-27/C-24 put the olefinic bond between C-24 and C-25, and an OH on one of the
terminal methyl groups. The NOE cross-peaks at H-24/H-16 and H3-28/H-17
suggested that H-23/H-17 was α-oriented. The NOE cross-peaks at H-2/H-10,
H-3/H-1α, H3-18/H-16, and H3-21/H-23 showed that OH-2, Oglc-3, H-16, and OH-20
were β-oriented and OH-3 was α-oriented. The NOE correlation between H2-27 and
H-24 showed the E configuration of the olefinic bond; therefore, 5 was typified as 2β,
20β, 26- trihydroxycucurbita- 16α- 23α- epoxy- 5, 24 (E)- diene- 11- one- 3- O- α- D-
glucopyranoside, and was named Xuedanoside E.
Compounds 1-5 were evaluated to establish their cytotoxic activity according to the
MTT procedure with HeLa, HCT-8, MCF-7 and HepG2 human cancer cell lines. The
outcomes of their cytotoxic activity are shown in Table 3 (see Tables). As it can be
seen, compounds 1 and 2 showed significant cytotoxic effects against HeLa cells with
IC50 values of 3.21 and 8.57 μM, while compounds 3-5 had moderate effects.
Compound 1 showed moderate effects against HepG2 cells with IC50 values of 18.62
μM. Compound 5 exhibited moderate cytotoxicity against HCT-8 cells with IC50

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values of 19.36 μM. All compounds demonstrated mild or no effects against MCF-7
cells. These above results indicated that the isolates could selectively enhance the
cytotoxicity against the tested HeLa cells line to a certain degree. Furthermore,
cucurbitacins were prepared to establish the structure–activity relationships for
cytotoxicity against the HeLa cells.
Conflict of interest
We declare that we have no financial and personal relationships with individuals or
organizations that can inappropriately influence our work, and there is no professional
or personal interest of any kind in any product, service and/or company that could be
construed as influencing the results presented in, or the review of, the present
manuscript.
Acknowledgements
The work was financially supported by the CAMS Innovation Fund for Medical
Sciences (CIFMS) (No. 2017-I2M-1-013) and the National Natural Sciences
Foundation of China (No. 81502945).
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Chen J , Niu X , Li Z , et al. Four New Cucurbitane Glycosides fromHemsleya jinfushanensis[J].
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Li P , Zhu N , Hu M , et al. New cucurbitane triterpenoids with cytotoxic activities from,
Hemsleya penxianensis[J]. Fitoterapia, 2017, 120:158-163.
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Rui HK, Yuan MY, Yu QM, Ye XQ, Qian GH, Wang KL. Chemical structure of eueurbitaein
glyeosides. Acta Pharmacol Sin 1981;16:445–7.
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Che, C. T., Fang X. D., PhonbeJr, C. H., Kinghorn, A. D., Farnsworth, N. R., 1985. Hecht SM:
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FROM HEMSLEYA AMABILIS[J]. Chinese Journal of Magnetic Resonance, 2002, 19(3):247-251.

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Table 1. 1H NMR Spectroscopic Data (600 MHz) for Compounds 1–5 (δH in ppm, J in Hz).
Position
1
2
3
4
5
2.67 (1H, m)
1.75 (1H, m)
2.40(1H, m)
1.52(1H, m)
1.63(1H, m)
1.49(1H, m)
2.43(1H, d, 12.0)
1.86(1H, m)
2.61(1H, m)
1.43(1H, m)
2.42(1H, m)
1.52(1H, m)
1
2
3
4.35 (1H, m)
4.03(1H, m)
4.21(1H, m)
4.10(1H, m)
3.42(1H, m)
3.42 (1H, d,
9.0)
3.37(1H, d,
9.0)
3.62 (1H, d, 6.0)
3.64(1H, s)
4
5
6
6.23 (1H, d, 6.0)
4.48 (1H, m)
2.88 (1H, m)
5.72(1H, d, 6.0)
2.28(1H, m)
5.50(1H, d, 12.0)
2.16(1H, m)
5.68(1H, d, 6.0)
2.26(1H, m)
1.82(1H, m)
1.93(1H, m)
5.73(1H, d, 6.0)
2.29(1H, m)
1.79(1H, m)
1.88(1H, m)
7
1.78(1H, m)
1.79(1H, m)
8
9
1.86(1H, d, 7.8)
1.81(1H, m)
10
11
2.52 (1H, s)
2.63(1H, d, 14.4)
2.52(1H, d, 14.4)
2.42(1H, d, 14.4)
2.64(1H, m)
3.11 (1H, d, 18.0)
2.73 (1H, d, 12.0)
3.21(1H, d, 14.4)
2.79(1H, d, 14.4)
3.20(1H, d, 14.4)
2.75(1H, d, 14.4)
3.08(1H, d, 14.4)
2.61(1H, d, 14.4)
3.18(1H, d, 12.0)
2.75(1H, d, 14.4)
12
13
14
2.17 (1H, m)
1.87 (1H, m)
4.91 (1H, m)
2.90 (1H, m)
1.24 (3H, s)
1.71 (3H, s)
2.03(1H, m)
1.51(1H, m)
5.96(1H, t, 6.0)
3.02(1H, d, 7.2)
1.17(3H, s)
1.84(1H, m)
1.68(1H, m)
4.87(1H, m)
2.97(1H, d, 6.0)
1.20(3H, s)
1.88(1H, m)
1.57(1H, m)
5.12(1H, m)
2.12(1H, d, 14.4)
1.26(3H, s)
2.02(1H, m)
1.44(1H, m)
5.90(1H, t, 12.0)
3.05(1H, m)
1.15(3H, s)
15
16
17
18
19
20
21
1.16(3H, s)
1.15(3H, s)
1.24(3H, s)
1.20(3H, s)
1.57 (3H, s)
1.69(3H, s)
1.59(3H, s)
1.41(3H, s)
1.95(1H, m)
1.57(3H, s)
22
23
24
1.74(1H, d, 13.8)
3.32 (1H, m)
3.06 (1H, m)
2.43 (1H, m)
2.32 (1H, m)
3.17(1H, m)
3.09(1H, m)
3.50(1H, m)
3.27(1H, m)
2.25(1H, m)
2.19(1H, m)
3.40(1H, m)
3.27(1H, m)
4.98(1H, m)
2.21(1H, m)
2.11(1H, m)
4.44(1H, d, 7.8 )
6.68(1H, d, 6)
25
26
1.48(3H, s)
1.48(3H, s)
1.38(3H, s)
1.36(3H, s)
1.37(3H, s)
1.37(3H, s)
1.99(3H, s)
4.60(1H, m)
4.50(1H, m)
1.31(3H, s)
1.53(3H, s)
1.21(3H, s)
1.44(3H, s)
1.44(3H, s)
27
28
29
30
1.50(3H, s)
1.37(3H, s)
1.51(3H, s)
1.91(3H, s)
1.26(3H, s)
1.31(3H, s)
1.50(3H, s)
2.22(3H, s)
1.48(3H, s)
1.09(3H, s)
1.54(3H, s)
1.27(3H, s)
1.33(3H, s)
1.50(3H, s)
2.14(3H, s)
OAc-25

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Glc
1`
5.34(1H, d, 6.0)
4.07(1H, t, 6)
4.18 (1H, t, 6)
4.30 (1H, m)
4.98(1H, d, 6.0)
4.95(1H, d, 7.8)
3.93(1H, m)
4.20(1H, m)
4.19(1H, m)
3.88(1H, m)
4.46(1H, m)
4.32(1H, m)
4.89(1H, d, 6)
3.98(1H, m)
4.22(1H, d, 6)
4.21(1H, m)
3.96(1H, m)
4.55(1H, d, 12)
4.39 (1H, m)
5.19(1H, d, 8.4)
4.12(1H, m)
4.29(1H, m)
4.24(1H, m)
3.94 (1H, m)
4.56(1H, d, 12)
4.35(1H, m)
2`
3.55(1H, t, 1.8)
4.17(1H, m)
4.02(1H, m)
4.16(1H, m)
4.52(1H, d, 12)
4.33(1H, m)
3`
4`
5`
3.88 (1H, m)
4.50 (1H, m)
6`
4.38 (1H, d, 12.0)
Table 2. 13C NMR (150MHz, in pyridine-d5) spectral data of compounds 1-5.
Position
1
2
3
4
5
1
33.6
83.5
81.0
43.2
144.8
123.2
66.5
53.5
49.0
35.5
213.8
49.8
48.4
50.7
46.9
70.8
59.3
20.7
22.2
80.5
25.5
215.5
32.6
35.8
82.0
26.5
26.4
19.8
23.2
25.9
170.6
22.6
35.0
71.4
81.8
43.3
142.8
119.0
24.4
43.0
48.5
34.8
212.9
49.1
49.4
50.9
44.2
75.2
55.8
20.7
20.8
80.4
25.4
213.1
40.1
80.3
77.0
19.4
19.3
22.9
26.7
26.0
171.1
21.8
22.5
29.0
87.6
42.5
141.7
118.9
24.6
43.6
49.2
36.3
213.7
49.6
49.7
51.6
46.9
70.8
59.2
20.7
20.8
80.6
25.9
216.6
33.2
38.9
69.5
30.5
30.4
19.5
28.8
26.3
34.3
71.6
94.3
42.9
142.3
119.8
24.6
43.4
49.3
34.3
213.4
49.1
49.1
49.6
42.1
71.3
56.5
20.5
21.0
72.8
30.6
47.1
70.9
129.3
139.0
22.2
61.2
21.5
25.7
23.7
35.0
71.4
81.8
44.3
142.9
119.0
24.4
43.0
48.5
34.8
213.1
49.1
49.4
51.0
43.3
75.1
55.7
20.5
20.8
80.3
25.5
216.4
33.0
36.6
77.0
27.6
27.4
19.4
19.3
26.0
171.0
21.8
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OAc-25
Glc

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1`
2`
3`
4`
5`
6`
107.0
76.3
79.0
71.8
79.0
62.9
95.6
82.5
75.3
80.7
72.8
63.1
99.3
75.7
79.2
72.2
78.6
63.3
107.8
75.9
79.2
72.2
78.7
63.4
107.7
76.7
79.2
72.2
79.1
63.2
Table 3. The IC50 values of compounds 1–5 against cancer cell lines.
IC50
Compounds
HeLa
HCT-8
24.72 ± 0.52
＞50
MCF-7
＞50
HepG2
18.62 ± 167
＞50
3.21 ± 0.13
8.57 ± 0.34
12.86 ± 0.25
18.37 ± 0.35
10.29 ± 0.44
1.08 ± 0.06
1
＞50
2
36.24± 1.27
＞50
＞50
＞50
3
＞50
＞50
4
19.36± 0.92
3.27 ± 0.64
＞50
37.44 ± 2.16
4.28 ± 0.40
5
Doxorubicinb
7.55 ± 0.17
a Value present mean ± SD of triplicate experiments. b Positive control substance.

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Fig. 1 Structure of Compounds 1-5
Fig. 2 Key HMBC and 1H-1H COSY correlations for 1
Fig. 3 Key NOESY correlations for Compound 1

Figure 1

Figure 2

Figure 3