Five new cucurbitane triterpenoids with cytotoxic activity from Hemsleya jinfushanensis

Article abstract of DOI:10.1016/j.phytol.2015.10.019

Five new cucurbitane triterpenoids (hemslepenside B-E (1-3 and 5)), one new cucurbitane (16,25-O-diacetyl-cucurbitane F (4)), and six analogues (6-11) were isolated from the roots of Hemsleya jinfushanensis. The structures of 1-5 were elucidated using infrared absorption spectroscopy (IR), high resolution electrospray ionization mass spectrometry (HR-ESI-MS), and nuclear magnetic resonance spectroscopy (NMR). These five new compounds exhibited cytotoxic effects against lung adenocarcinoma (H460), colon cancer (SW620) and human prostate cancer cells (DU145), and compound 4 showed significant cytotoxic activity, with IC₅₀ values of 0.046, 0.18 and 0.87 μg/mL. This finding suggests that cucurbitane triterpenoids isolated from H. jinfushanensis should be studied further as potential anti-cancer drugs.

Full text of DOI:10.1016/j.phytol.2015.10.019

Phytochemistry Letters 14 (2015) 239–244
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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Five new cucurbitane triterpenoids with cytotoxic activity from
Hemsleya jinfushanensis
Ying Lia,b, Zhongfei Zhenga, Ling Zhoua, Yongjun Liua, Haiyang Wanga, Ling Lia,b
,
Qingqiang Yaoa,
*
a
Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China
b
School of Medicine and Life Sciences, University of Jinan, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 21 May 2015
Received in revised form 22 October 2015
Accepted 23 October 2015
Available online 10 November 2015
Five new cucurbitane triterpenoids (hemslepenside B–E (1–3 and 5)), one new cucurbitane (16,25-O-
diacetyl-cucurbitane F (4)), and six analogues (6–11) were isolated from the roots of Hemsleya
jinfushanensis. The structures of 1–5 were elucidated using infrared absorption spectroscopy (IR), high
resolution electrospray ionization mass spectrometry (HR-ESI-MS), and nuclear magnetic resonance
spectroscopy (NMR). These five new compounds exhibited cytotoxic effects against lung adenocarcinoma
(H460), colon cancer (SW620) and human prostate cancer cells (DU145), and compound 4 showed
significant cytotoxic activity, with IC50 values of 0.046, 0.18 and 0.87 mg/mL. This finding suggests that
cucurbitane triterpenoids isolated from H. jinfushanensis should be studied further as potential anti-
cancer drugs.
Keywords:
Hemsleya jinfushanensis
Cucurbitane triterpenoid
Cytotoxic activity
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
2. Results and discussion
Hemsleya pengxianensis var. jinfushanensis, which is a traditional
Chinese medicinal plant, is widely distributed in southeast China.
This plant was traditionally used for the treatment of bronchitis,
bacillary dysentery and tuberculosis (NPC, 1977). One of the main
types of chemical constituents of this plant, cucurbitane-type
compounds, exhibit a wide range of bioactivities, such as cytotoxic
activity (Xu et al., 2014; Wu et al., 2002) and anti-HIV-1 activity
(Tian et al., 2008). Our studies focus on isolating and investigating
the bioactivity of cucurbitane-type compounds and led to the
isolation of five new cucurbitane triterpenoids (hemslepenside B–
E (1–3, and 5) and 16,25-O-diacetyl-cucurbitane F (4)), together
with six known analogues (scandenogenin A (6) (Kasai et al.,1988),
jinfushanoside G (7) (Chen et al., 2012), jinfushanoside B (8) (Chen
et al., 2005), delavanosides D (9) (Chen et al., 2007), 23,24-dihydro-
cucurbitane F-16,25-diacetate (10) (Qiu and Gao, 2003), and 25-O-
Hemslepenside B (1) (HRESIMS m/z: 636.4474 [M + NH4]+, calcd
for C36H56O8NH4 636.4475) was obtained as a white amorphous
powder and had a molecular formula of C36H58O8. The IR spectrum
(KBr) of 1 exhibited stretching frequencies for OH (3423 cmꢀ1),
CHO (1684 cmꢀ1), and CH CH (1637 cmꢀ1). The 13C NMR (Table 2)
¼
and distortionless enhancement by polarization transfer (DEPT)
spectra showed resonances for seven methyls, nine methylenes,
fourteen methines and six quaternary carbons, including two
olefinic carbons at dC 144.5 and 156.6 and one carbonyl carbon at dC
196.2. The 1H NMR (Table 1) spectrum showed the presence of
seven methyl signals at dH 0.78 (3H, s), 0.82 (3H, s), 0.97 (3H, d,
J = 6.6 Hz), 1.00 (3H, s), 1.10 (3H ꢁ 2, s), and 1.65 (3H, s). Further
features were observed at dH 5.40 (1H, d, J = 5.0 Hz, an olefinic
proton), 5.12 (1H, t, J = 7.1 Hz, an olefinic proton) and dH 9.37 (1H, s,
an aldehyde proton), which suggested that
1 possesses a
acetyl-23,24-dihydro-cucurbitane
F-2-O-
b
-
D
-glucopyranoside
tetracyclic-cucurbitane skeleton. The NMR data obtained for 1
were similar to those obtained for jinfushanoside G (7) (Chen et al.,
2012), except for the presence of an aldehyde proton [dH 9.37 (1H,
s)] at C-26 in 1 instead of a hydroxymethyl in 7. This finding was
confirmed using the heteronuclear multiple bond correlation
(HMBC) (Fig. 2) from dH 9.37 (H-26) to dC 139.2 (C-25) and 9.7 (C-
27). Furthermore, HMBC correlations between dH 4.12 (1H, d,
J = 7.8 Hz, Glc-H-1) and dC 87.36 (C-3), as well as dH 3.27 (1H, H-3,
brs) and dC 106.27 (Glc-C-1), suggested that an additional
(11) (Jiang, 1981)) (Fig. 1). The 11 identified cucurbitane
triterpenoids were evaluated for cytotoxic activity against the
human tumor cell lines H460, SW620 and DU145.
* Corresponding author.
b-orientation of hexose was linked at C-3, and enzymatic
E-mail address: yao_imm@163.com (Q. Yao).
http://dx.doi.org/10.1016/j.phytol.2015.10.019
1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

240
Y. Li et al. / Phytochemistry Letters 14 (2015) 239–244
Fig. 1. Structures of compounds 1–5.
Table 1
1H (400 MHz) data for compounds 1–5 in d6-DMSO.
Position
1
1
2
3
4
5
1.01(1H, m)
2.21(1H, m)
1.68(1H, m)
1.85(1H, m)
3.27(1H, brs)
1.27(1H, m)
2.22(1H,m)
1.71(1H, m)
1.85(1H, m)
3.26(1H, brs)
1.31(1H, m)
1.84(1H, m)
1.46(1H, m)
1.85(1H, m)
3.27(1H, brs)
0.83(1H, m)
1.57(1H, m)
4.45(3H, s)
0.83(1H, m)
1.56(1H, m)
3.27(1H, m)
2
3
4
5
6
7
4.58(3H, s)
2.83(1H, d, 9.2)
5.34(1H, brd, 5.4)
1.67(1H, m)
2.35(1H, m)
5.34(1H, brd, 5.6)
1.72(1H, m)
2.32(1H, m)
5.47(1H, d, 5.0)
1.87(1H, m)
2.09(1H, m)
1.84(1H, m)
5.59(1H, m)
1.87(1H, m)
2.30(1H, m)
1.85(1H, m)
5.64(1H, m)
1.84(1H, m)
2.27(1H, m)
1.82(1H, m)
8
1.53(1H, m)
1.47(1H, m)
9
10
11
12
2.35(1H, m)
3.65(1H, m)
1.67(1H, m)
2.5(1H, m)
2.34(1H, m)
3.09(1H, m)
1.67(1H, m)
2.51(1H, m)
1.85(1H, m)
2.35(1H, m)
2.36(1H, m)
2.19(1H, d, 14.1)
3.09(1H, d, 14.4)
2.38(1H, m)
3.34(1H, m)
2.32(1H, m)
3.36(1H, m)
13
14
15
1.00(1H, m)
1.21(1H, m)
0.99(1H, m)
1.27(1H, m)
1.55(1H, s)
1.02(1H, m)
1.10(1H, m)
0.96(1H, m)
1.23(1H, m)
1.48(1H, m)
0.81(3H, s)
1.23(1H, m)
1.34(1H, m)
1.81(1H, m)
2.00(1H, m)
1.61(1H, m)
0.64(3H, s)
1.21(1H, m)
1.79(1H, m)
5.27(1H, t, 12.0)
1.22(1H, m)
1.84(1H, m)
5.25(1H, t, 12.0)
16
17
18
19
20
21
22
2.61(1H, m)
0.82(3H, s)
0.94(3H, s)
2.62(1H, d, 7.6)
0.83(3H, s)
0.94(3H, s)
0.82(3H, s)
1.00(3H, s)
1.01(3H, s)
0.95(3H, s)
1.56(1H, m)
0.94(3H, d, 8.0)
1.54(1H, m)
2.37(1H, m)
1.58(1H, m)
2.24(1H, m)
6.65(1H, t, 7.2)
1.38(1H, m)
0.89(3H, d, 6.2)
1.00(1H, m)
1.35(1H, m)
1.85(1H, m)
2.02(1H, m)
5.11(1H, t, 14)
2.32(1H, m)
0.84(3H, d, 6.2)
0.99(1H, m)
1.30(1H, m)
1.81(1H, m)
2.32(1H, m)
5.12(1H, t, 7.1)
1.27(3H, s)
1.27(3H, s)
23
6.79(1H, d, 16.0)
6.95(1H, d, 16.0)
6.74(1H, d, 16.0)
6.94(1H, d, 16.0)
24
25
26
27
28
29
9.37(1H, s)
1.65(3H, s)
0.78(3H, s)
0.97(3H, s)
1.10(3H, s)
1.66(3H, s)
3.89(2H, d, 5.1)
0.77(3H, s)
1.11(3H, s)
1.66(3H, s)
3.89(2H, d, 5.2)
0.97(3H, s)
0.99(3H, s)
1.14(3H, s)
1.48(3H, s)
1.49(3H, s)
1.13(3H, s)
1.07(3H, s)
0.85(3H, s)
1.48(3H, s)
1.49(3H, s)
1.13(3H, s)
1.18(3H, s)
0.88(3H, s)
30
0.97(3H, s)
16-OOCCH3
1.78(3H, s)
1.95(3H, s)
1.78(3H, s)
1.86(3H, s)
25-OOCCH3
3-glc
10
4.12(1H, d, 7.8)
2.92(1H, m)
3.14(1H, m)
2.97(1H, m)
3.02(1H, m)
3.61(1H, m)
3.4(1H, m)
4.14(1H, d, 7.8)
2.91(1H, m)
3.07(1H, m)
3.02(1H, m)
3.04(1H, m)
3.61(1H, m)
3.41(1H, m)
4.12(1H, d, 7.8)
3.01(1H, m)
3.15(1H, m)
3.11(1H, m)
3.06(1H, m)
3.66(1H, m)
3.48(1H, m)
4.32(1H, d, 7.8)
3.07(1H, m)
3.16(1H, m)
3.01(1H, m)
3.13(1H, m)
3.63(1H, m)
3.36(1H, m)
20
30
40
50
60

Y. Li et al. / Phytochemistry Letters 14 (2015) 239–244
241
Table 3
IC50 values (
lines.
Table 2
13C (150 MHz) data for compounds 1–5 in d6-DMSO.
mg/mL) of compounds 1–11 against three different human cancer cell
Position
1
2
3
4
5
a
Compound
IC50
(
mg/mL)
1
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
26.53
26.55
22.02
34.46
70.25
80.74
42.64
142.6
118.39
24.21
42.8
33.82
70.88
93.16
42.54
H460
SW620
DU145
29.09
87.36
42.32
144.5
118.07
24.73
43.54
39.89
36.41
77.48
41.01
47.51
49.83
34.75
28.35
50.58
17.35
26.43
35.17
19.2
36.18
26.3
156.63
139.22
196.21
9.75
19.57
28.06
26.43
29.1
87.38
42.33
144.51
118.08
24.74
43.56
39.9
37.19
77.48
40.93
47.46
49.82
34.76
28.4
50.63
17.36
26.43
36.08
19.34
36.41
24.64
127.14
136.04
22.13
62.11
19.58
26.43
28.07
28.14
86.76
42.14
141.68
118.31
24.42
44.13
49.02
35.92
214.5
49.32
49.32
49.66
34.88
28.13
49.95
17.48
20.62
35.5
18.87
37.06
24.54
127.14
136.17
22.19
60.37
19.16
28.88
26.11
1
2
3
4
5
6
7
8
2.12 ꢂ 0.26
6.9 ꢂ 0.13
2.41 ꢂ 0.08
0.046 ꢂ 0.28
9.18 ꢂ 0.14
1.38 ꢂ 0.68
2.22 ꢂ 0.16
1.51 ꢂ 0.11
5.53 ꢂ 0.35
3.98 ꢂ 0.46
3.96 ꢂ 0.28
0.12 ꢂ 0.10
0
0.82 ꢂ 0.24
2.22 ꢂ 0.23
1.21 ꢂ 0.38
0.18 ꢂ 0.10
100.69 ꢂ 0.34
3.23 ꢂ 0.27
347.53 ꢂ 0.59
2.44 ꢂ 0.39
2.2 ꢂ 0.10
2.14 ꢂ 0.35
6.3 ꢂ 0.36
3.83 ꢂ 0.53
0.87 ꢂ 0.12
79.25 ꢂ 0.24
2.24 ꢂ 0.94
179.5 ꢂ 0.63
4.06 ꢂ 0.45
5.48 ꢂ 0.35
15.8 ꢂ 0.10
81.48 ꢂ 0.45
0.34 ꢂ 0.43
0
142.09
119.25
24.31
42.68
48.17
33.49
213.39
49.49
48.52
50.73
43.84
74.19
55.49
20.63
20.63
79.21
25.22
204.07
121.55
151.18
80.28
27.25
27.33
19.17
48.13
33.79
213.31
49.39
48.51
50.69
43.82
74.14
55.42
20.56
20.67
79.16
25.17
203.98
121.53
151.09
80.22
27.21
27.29
19.19
25.78
22.64
170.63
22.57
170.24
21.7
9
10
11
4.36 ꢂ 0.19
5.68 ꢂ 0.20
0.28 ꢂ 0.71
0
Cisplatinb
DMSO (0.8%)
a
Means ꢂ S.D. from three independent experiments (n = 3).
b
Positive control.
(3H, CH3-27) in 7. The results of the NOESY experiments confirmed
that 2 possesses the cis configuration and 7 possesses the trans
configuration. Enzymatic hydrolysis of 2 afforded
the composition sugar. The signal at dH 4.14 (1H, d, J = 7.8 Hz)
suggested the -orientation for the anomeric proton. Thus,
compound 2 was determined to be 3 -hydroxycucurbita-5,24
-glucopyranoside,
D-(+)-glucose as
b
b
(E)-diene-11,27-hydroxy-26-methyl-3-O-b-D
25.61
23.7
170.71
22.61
170.32
21.75
which is also named hemslepenside C.
Hemslepenside D (3) was determined to be a white amorphous
powder with the molecular formula of C36H58O8 based on positive
ion HRESIMS [m/z: 636.4476 [M + NH4]+, calcd for 636.4475. The
molecular weight of 3 was equal to that of delavanosides D (9)
(Chen et al., 2007), and the main difference in the NMR data
(Tables 1 and 2) between 3 and 9 exists in the end of the side
chains, with dC-26 shifted upfield from 14 in 9 to 59.87 in 3 and dC-27
shifted downfield from 68.0 in 9 to 21.51 in 3. Based on these
findings, we deduced that 3 and 9 were a pair of cis and trans
16-OOCCH3
25-OOCCH3
3-glc
10
106.27
74.75
78.11
71.07
77.48
62.12
106.27
74.75
78.11
71.06
77.51
60.31
106.19
74.81
78.14
71.12
77.57
62.16
106.4
75.6
77.91
70.57
77.83
62.01
20
30
40
50
60
isomers of
D
24(25). The NOESY experiments for 3 and 9 further
confirmed this deduction. dH 5.12 (1H, H-24) and dH 1.66 (3H, CH3-
26) in 3 showed a significant correlation, while no correlation was
observed between dH 5.12 (1H, H-24) and dH 3.89 (2H, CH2OH-27)
in 3. In contrast, the NOESY (Fig. 2) correlation was observed
between dH 5.71 (1H, H-24) and dH 4.31 (2H, CH2OH-26) in 9, and
there was no correlation between dH 5.71 (1H, H-24) and dH 1.82
(3H, CH3-27) in 9. These results demonstrated that 3 possesses the
cis configuration and 9 possesses the trans configuration. HMBC
correlations between dH 4.12 (Glc-H-1) and dC 86.76 (C-3)
suggested that an additional hexose was linked at C-3, with the
hydrolysis of 1 afforded
compound 1 was determined to be 3
diene-11-hydroxy-26-al-3-O-b-D
named hemslepenside B.
D
-(+)-glucose as the sugar moiety. Thus,
-hydroxycucurbita-5,24(E)-
-glucopyranoside, which is also
b
Hemslepenside C (2) was obtained as a white amorphous
powder. The molecular formula of this compound was determined
to be C36H60O8 based on positive ion HRESIMS m/z: [M + NH4]+
638.4630, calcd for 638.4632. IR absorptions at 3383, 2930 and
1629 cmꢀ1 indicated the presence of hydroxyl, methyl and ethenyl
groups. The NMR data (Tables 1 and 2) obtained for 2 were very
similar to those obtained for jinfushanoside G (7) (Chen et al.,
2012), except for the obvious variations at the end of the side
chains; dC-26 was shifted upfield from 68.1 in 7 to 22.1 in 2, while dC-
27 was shifted downfield from 14.0 in 7 to 62.1 in 2, which led to the
deduction of the interchanging of groups of CH2OH and CH3 at C-
26 and C-27 in compounds 2 and 7, respectively. Combined with
the fact that these two compounds have the same molecular
formula, we deduced that these compounds were a pair of cis and
signal at dH 4.12 (1H, d, J = 7.8 Hz), suggesting the
the anomeric proton, and enzymatic hydrolysis of 3 afforded
(+)-glucose as the composition sugar. Thus, compound 3 was
determined to be 3 -hydroxycucurbita-5,24(E)-diene-11-one-26-
methy-27-hydroxyl-3-O- -glucopyranoside, which is also
named hemslepenside D.
b-orientation for
D-
b
b-D
16,25-O-Diacetyl-cucurbitane F (4) was isolated as a white
amorphous powder and had a molecular formula of C34H50O9
(HRESIMS m/z: 620.3796 [M + NH4]+, calcd for C34H50O9 NH4
620.3799). The IR spectrum exhibited resonances for OH
trans isomers of
D
24(25). To confirm this deduction, 2 and 7 were
(3447 cmꢀ1), C O (1738 and 1692 cmꢀ1). The 1H NMR (Table 1)
¼
and 13C NMR (Table 2) spectra and DEPT experiments exhibited ten
methyl groups (dC: 18.97, 20.34, 20.45, 21.48, 22.35, 22.42, 23.99,
25.56, and 26.99), four methylene groups, nine methine groups,
subjected to nuclear overhauser enhancement spectroscopy
(NOESY) experiments. The NOESY (Fig. 2) correlation was observed
between dH 5.11 (1H, H-24) and dH 1.66 (3H, CH3-26) in 2, and there
was no correlation between dH 5.11 (1H, H-24) and dH 3.89 (2H,
CH2OH-27) in 2. In contrast, the NOESY correlation was observed
between dH 5.71 (1H, H-24) and dH 4.30 (2H, CH2OH-26) in 7, and
there was no correlation between dH 5.71 (1H, H-24) and dH 1.83
and eleven quaternary carbons, which were assigned to
a
triterpene skeleton. A comparison of the 13C and 1H NMR spectra
of this compound with those of 16,25-O-diacetyl-cucurbitane F-2-
O-b-D-glucopyranoside (Xu et al., 2014) revealed that they were

242
Y. Li et al. / Phytochemistry Letters 14 (2015) 239–244
similar, except for the presence of a
D
-glucopyranoside. The signal
SW620 and DU145. As shown in Table 3, most of the compounds
displayed cytotoxic activity against the cancer cell lines, and
compound 4 exhibited the strongest cytotoxicity (IC50 = 0.046, 0.18,
of C-2 was shifted upfield from 83.3 to dC 70.04 (t), which further
confirmed that no glucopyranosyl moiety was linked at C-2 in 4.
Thus, compound 4 was determined to be 16,25-O-diacetyl-
cucurbitane F.
and 0.87
m
g/ml). Compound 4 was even more active than cisplatin
g/ml) against the H460 and SW620 cell
(IC50 = 0.12, 0.28, and 0.84
m
Hemslepenside E (5) was obtained as a white amorphous
powder with the molecular formula of C40H60O14, in agreement
with the positive ion HRESIMS (positive) m/z: 782.4329 [M + NH4]+,
calcd for 782.4327. A comparison of the NMR data (Tables 1 and 2)
obtained for 5 and those obtained for 4 indicated that the two
compounds were very similar, except for the presence of one
additional sugar unit in 5. It was apparent that the signal of C-3 in 5
was shifted downfield by 12.6 in comparison to 4, which indicated
lines, while 5, which contains the same aglycone as 4 except for one
inactive glucopyranoside, was inactive. This finding suggested that
glycosidation may reduce the cytotoxic activity. Based on the
cytotoxicity results, the relationship between elementary struc-
ture and activity could be elucidated as follows. By comparing the
structure and cytotoxic activities of 5 with those of 4, we confirmed
that the glycosidation at C-3 may reduce cytotoxic activity. A
comparison of the structure and cytotoxic activities of two pairs of
isomer compounds (2 and 7; 3 and 9) confirmed that the cis
configuration at 24 exhibited stronger activity than its isomer with
the trans configuration. By comparing the structure and cytotoxic
activities of 1 with those of 7, we deduced that the aldehyde
function at C-26 would enhance cytotoxicity. The IC50 values of
that
D-glucose was linked to C-3. The above deduction was
confirmed by the HMBC (Fig. 2) correlation between dH 4.32 (1H,
Glc-H-1) and dC 93.14 (C-3). Partial enzymatic hydrolysis of 5 also
yielded 4, together with
determined based on a comparison with a trade sample. Thus,
the structure of 5 was determined to be 2 ,3 ,20-trihydroxycu-
D-(+)-glucose, and the sugar was
b
a
compound 1 were 2.12, 0.82, and 2.14 mg/ml, and the cytotoxicity
curbita-5,23(E)-diene-11,22-dione-16,25-diacetyl-3-O-
pyranoside, which is also named hemslepenside E.
The in vitro experiment revealed that the isolated compounds
(1–11) exhibited obvious cytotoxic activity against H460,
b
-
D
-gluco-
of 1 may be related to the aldehyde function at C-26, which is a
unique structural feature of this compound in comparison to its
analogues. Compound 2, which possesses the cis configuration,
exhibited stronger cytotoxicity against the SW620 and DU145 cell
Fig. 2. Key HMBC and NOESY correlations of compounds 1–5,7, and 9.

Y. Li et al. / Phytochemistry Letters 14 (2015) 239–244
243
lines than 7, which has the trans configuration. In addition, in
another pair of isomers, compound 3 (cis) showed stronger activity
than 9 (trans) against all of the tested cell lines, which suggested
that the cis configuration may increase activity. Compounds 6, 8
and 10 showed moderate cytotoxic potency, and 5, 7, and 11 were
tR = 36.3 min). Fr.C-4 was further subjected to pre-HPLC to give
compound 11 (48.0 mg, 65:35, tR = 24.4 min). Fr.D (45 g) was eluted
with CHCl2, CHCl2–MeOH (99:1, 98:2, 95:5, 93:7, 90:10, 85:15,
80:20, and 100:0, each 1.5 L), resulting in 11 subfractions (Fr.D1–Fr.
D11). Compounds 2 (15.0 mg, tR = 10.9 min), 7 (46.0 mg, tR = 21.6
min) and 8 (62.0 mg, tR = 28.9 min) were obtained from Fr.D-
2 using ODS CC and pre-HPLC (MeOH:H2O = 75:25, 7 mL/min,
210 nm).
inactive in some cell lines (IC50 > 20
mg/ml).
3. Experimental
3.1. General
3.4. Structural elucidation of compounds 1–5
3.4.1. Hemslepenside B (1)
IR spectra were measured using a Thermo Nicolet NEXUS-
670 FTIR spectrometer. Optical rotations were run on a JASCO P-
2000 polarimeter. HR-ESI-MS was determined using Thermo
Scientific Accela PDA and LTQ-Orbitrap XL mass spectrometers
(Thermo Fisher Scientific, Germany). NMR spectra were performed
on a Bruker Avance III HD NMR spectrometer (Bruker BioSpin).
White amorphous powder; [a D
(KBr) nmax 3423, 2947, 2705, 1684, 1637, 1458, 1382, 1251, 1167,
]
25: +28.15 (c = 0.1, MeOH); IR
1074 and 1033 cmꢀ1 1H NMR data see Table 1; 13C NMR data see
;
Table 2; and ESI-HRMS m/z 636.4474 ([M + NH4]+) (calcd for
C36H58O8NH4, 636.4475).
Preparative HPLC was run on
a LC-6AD Shimadzu Liquid
chromatograph equipped with an SPD-20A UV/VIS Detector and
3.4.2. Hemslepenside C (2)
an ODS column (YMC-Pack ODS-A column, 250 mm ꢁ 20 mm,
White amorphous powder; [a]D
25: +44.37 (c = 0.1, MeOH); IR
5
m
m). Column chromatography (CC) was performed using silica
(KBr) nmax 3383, 2930, 1629, 1466, 1381, 1168, 1075, 1010 and
943 cmꢀ1; 1H data see Table 1; 13C NMR data see Table 2; and ESI-
HRMS m/z 638.4630 ([M + NH4]+) (calcd for C36H60O8 NH4,
638.4632).
gel (300–400 mesh, Qingdao Marine Chemistry Ltd., China), an
ODS C18 (50 m, YMC Co., Ltd., Kyoto, Japan).
m
3.2. Plant material
3.4.3. Hemslepenside D (3)
Rhizomes of H. jinfushanensis were collected from Chongqing in
the People’s Republic of China in September 2012 and identified by
Dr. Si-rong Yi from the Chongqing Institute of Pharmaceutical
Plants. A voucher specimen (voucher number: HA201209B) was
deposited in the Institute of Materia Medica at the Shandong
Academy of Medical Science in China.
White amorphous powder; [a]
25: +44.53 (c = 0.1, MeOH); IR
D
(KBr) nmax 3406, 2963, 2882, 1691, 1462, 1384, 1167, 1074 and
1015 cmꢀ1; 1H data see Table 1; 13C NMR data see Table 2; and ESI-
HRMS m/z 636.4476 ([M + NH4]+) (calcd for C36H58O8 NH4,
636.4475).
3.4.4. 16,25-O-Diacetyl-cucurbitane F (4)
3.3. Extraction and isolation
White amorphous powder; [
(KBr) nmax 3450, 2976, 2930, 1738, 1692, 1631, 1431, 1371, 1248,
1058 and 1024 cmꢀ1 1H data see Table 1; 13C NMR data see
Table 2; and ESI-HRMS m/z 620.3796 ([M + NH4]+) (calcd for
34H49O9 NH4, 620.3799).
a]
25: +11.38 (c = 0.1, MeOH); IR
D
Air-dried rhizomes (3.0 kg) of H. jinfushanensis were cut into
slices, minced, extracted exhaustively with 95% EtOH under reflux
(3 ꢁ10 L) and filtered. The extract was concentrated to dryness in a
vacuum using a rotatory evaporator, concentrated to an aqueous
suspension and further extracted successively with EtOAc, CHCl3
and n-BuOH. The EtOAc fraction (200 g) was absorbed on 300 g of
silica gel (200–300 mesh) and chromatographed over a silica gel
(10 ꢁ120 cm, 2 kg) column using a gradient system of CHCl2 (8 L),
CHCl2–MeOH (99:1/8 L, 98:2/10 L, 95:5/12 L, 93:7/10 L, 90:10/12 L,
85:15/8 L, 80:20/6 L, and 100:0/5 L) to give five fractions (Fr.A–Fr.E).
Fr.A (25 g) was separated by a silica gel (300–400 mesh) and eluted
with a gradient system of CHCl2–MeOH (99:1, 98:2, 95:5, 93:7,
90:10, 85:15, 80:20, and 100:0, each 800 mL), resulting in six
fractions (Fr.A1–Fr.A6). Fr.A-2 (6.8 g) was absorbed on silica gel
(10 g) and eluted with CHCl2–MeOH (99:1, 98:2, 95:5, 93:7, 90:10,
85:15, 80:20, and 100:0, each 500 mL) to afford five subfractions
(Fr.A-2-1 to Fr.A-2-5). Subfraction A-2-4 (900 mg) was purified via
pre-HPLC (MeOH:H2O = 65:35, 7 mL/min, 210 nm) to give com-
pounds 4 (30 mg, tR = 35.7 min), 6 (21 mg, tR = 40.2 min) and 10
(15 mg, tR = 44.9 min). Fr.B (15.3 g) was absorbed on 20 g of silica gel
and chromatographed with CHCl2–MeOH (99:1, 98:2, 95:5, 93:7,
90:10, 85:15, 80:20, and 100:0, each 300 mL) to give Fr. B1–B4. Fr.
B-2 was further fractionated on an RP-18 column and eluted with
MeOH-H2O (MeOH:H2O = 75:25, 7 mL/min, 210 nm) to afford
compounds 1 (28.0 mg, tR = 51.1 min) and 5 (18.0 mg, tR = 32.7 min).
Fraction C (31.7 g) was fractionated using silica gel column
chromatography (CC) with a gradient elution of CHCl2, CHCl2–
MeOH (99:1, 98:2, 95:5, 93:7, 90:10, 85:15, 80:20, and 100:0, each
1.2 L) to yield 6 fractions (Fr.C1–Fr.C6). Fr.C-3 was further purified
via ODS CC and pre-HPLC (MeOH:H2O = 70:30, 7 mL/min, 210 nm)
to give compounds 3 (32.5 mg, tR = 29.2 min) and 9 (43.0 mg,
;
C
3.4.5. Hemslepenside E (5)
White amorphous powder; [a D
]
25: +47.15 (c = 0.1, MeOH); IR
(KBr) nmax 3447, 2975, 2879, 1738, 1692, 1629, 1432, 1371, 1252,
1024 cmꢀ1; 1H data see Table 1; 13C NMR data see Table 2; and ESI-
HRMS m/z 782.4329 ([M + NH4]+) (calcd for C40H60O14 NH4,
782.4327).
3.5. Determination of the glucose configuration in compounds 1–3
and 5
3.5.1. Enzymatic hydrolysis
Compounds 1–3 and 5 (4 mg) were hydrolyzed with cellulase
(Worthington, USA, each 20 mg) in H2O (each 4 mL), and the
mixtures were incubated for 120 h at 40 ꢃC. The reaction mixture
was extracted with EtOAc, and the aqueous layers were evaporated
to dryness to obtain the sugar fractions. The sugar residue and
authentic samples of
dissolved in H2O (each 1 mL) and mixed with EtOH (each 1 mL)
-methylbenzylamine (7 l) and NaBH3CN
D-(+)-glucose and
L-(ꢀ)-glucose were
that contained (S)-(ꢀ)-
a
m
(8 mg), respectively. The mixture was incubated for 4 h at 40 ꢃC.
Glacial acetic acid (0.2 mL) was then added and concentrated to
dryness. Acetic anhydride (0.3 mL) and pyridine (0.3 mL) were
added to the residue and acetylated at room temperature for 24 h.
After the pyridine was removed, the aqueous solution of the
reaction mixture was subjected to a Cleanert C18-N column
(Agela), and H2O, 20% CH3CN and 50% CH3CN (15, 15 and 10 mL)
were used as the eluents.

244
Y. Li et al. / Phytochemistry Letters 14 (2015) 239–244
3.5.2. HPLC analysis of compounds 1–3 and 5
after the incubation period. All 11 compounds were evaluated, and
the obtained data are presented as the mean of three independent
experiments. In addition, each compound was tested in three
parallel wells.
The 50% CH3CN fraction was analyzed via HPLC under the
following conditions: Agilent SB-C18 column (4.6 ꢁ 250 mm,
5 mm); mobile phase, 40% CH3CN; flow rate, 0.8 mL/min; and
DAD detection, 230 nm. The absolute configuration of the
monosaccharide was determined by comparing the retention time
tR (min) of the derivatives (1-[(S)-N-acetyl-a-methylbenzylamino]-
1-deoxyglucitol acetate derivatives) with those of authentic
Acknowledgment
This project was supported by the Key Laboratory for Rare and
Uncommon Diseases of Shandong Province, PR China.
samples: 20.31 min (derivative of
(derivative of -glucose). All of the sugar moieties were determined
to be -(+)-glucose based on the finding that the retention time of
the sugars in compounds 1, 2, 3 and 5 were 20.63, 20.67, 20.62,
D-glucose) and 19.35 min
L
D
Appendix A. Supplementary data
20.65 min, which was determined to correspond to
D-(+)-glucose.
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2015.10.019.
3.6. Cytotoxicity assay
The H460, SW620 and DU145 cell lines were obtained from the
Institute of Materia Medica in the Chinese Academy of Medical
Sciences. The cells were cultured in RPMI-1640 medium (Gibco,
USA) containing 15% heat-inactivated fetal bovine serum (Tian-
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