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Cucurbitane and hexanorcucurbitane glycosides from
the fruits of Cucurbita pepo cv dayangua
DA-CHENG WANG†, HONG-YU PAN‡, XU-MING DENG‡, HUA XIANG‡,
HUI-YUAN GAO†, HUI CAI‡ and LI-JUN WU†*
†School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shengyang
110016, China
‡Jilin University, Changchun 130062, China
(Received 3 June 2005; revised 7 September 2005; in final form 4 April 2006)
Phytochemical investigation of the fruits of Cucurbita pepo cv dayangua has led to the isolation of two
cucurbitane glycosides: cucurbitacin L 2-O-b-
D-glucopyranoside (1), cucurbitacin K 2-O-b-D-glucopyrano-
side (2) and two hexanorcucurbitane glycosides: 2,16-dihydroxy-22,23,24,25,26,27-hexanorcucurbit-5-en-
11,20-dione 2-O-b-
D-glucopyranoside (3) and 16-hydroxy-22,23,24,25,26,27-hexanorcucurbit-5-en-
11,20-dione 3-O-a-
L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside (4). Compounds 1, 2 and 3 were
isolated from Cucurbita genus for the first time, while compound 4 is a new one. Their structures were
determined on the basis of chemical and spectroscopic evidence.
Keywords: Cucurbita pepo cv dayangua; Cucurbitane glycoside; Hexanorcucurbitane glycoside; Cucurbita
1. Introduction
Cucurbita pepo cv dayangua, which is distributed in the Duo lun county of the autonomous
region of Mongolia, has been employed in folk medicine to treat colds and alleviate aches
[1,2]. Previous pharmacological tests showed that it possessed antibacterial, antiviral, anti-
inflammatory and analgesic effects [3,4]. During our search for new anti-tumour agents, the
ethanolic extract of fruits of the plant exhibited a significant dose-dependent inhibitory effect
against HeLa and HepG cell growth. Phytochemical investigation of the ethanolic extract of
the fruits has led to the isolation of two cucurbitane glycosides and two hexanorcucurbitane
glycosides. The present paper deals with the isolation and structure elucidation of the four
cucurbitacin saponins.
2. Results and discussio n
Compounds 1, 2 and 3 were identified as the previously known, cucurbitacin L 2-O-b-
D-
glucopyranosid e, cucurbitacin K 2-O-b-
D-glucopyranosid e and 2,16-dihyd roxy-
Journal of Asian Natural Products Research
ISSN 1028-6020 print/ISSN 1477-2213 online q 2007 Taylor & Francis
http://www.tandf.co.uk/journals
DOI: 10.1080/10286020600782538
*Corresponding author. Email: wdc9928@yahoo.com.cn
Journal of Asian Natural Products Research, Vol. 9, No. 6, September 2007, 525–529
22,23,24,25,26,27-hexanorcucurbit-5-en-11,20-dione 2-O-b-D-glucopyranoside, respectively
(figure 1) by comparison of their spectroscopic data with those reported in the literature [5].
Compound 4 was obtained as white amorphous powder (MeOH). It showed positivity
to Molish and Liebermann–Burchard tests and the molecular formula was determined as
C
36
H
56
O
13
by HRESI-MS (quasi-molecular ion peak at m/z 719.3643 [M þ Na]
þ
). The
sugar moieties were identified as glucose and rhamnose by TLC with authentic samples after
acid hydrolysis. The
1
H NMR spectrum of 4 exhibited signals characteristic for six methyl
protons at
d
0.76, 1.05, 1.38, 1.51, 1.15, 1.52, one trisubstituted olefinic proton at
d
5.94 and
two anomeric protons at
d
4.89, 6.74. The
13
C NMR and DEPT spectra of 4 showed 36
carbons (table 2), including a pair of olefinic carbons at
d
139.6 and 119.7, two carbonyl
carbons at
d
212.3 and 208.7, two anomeric carbons at
d
105.2 and 101.1. The
1
H NMR and
13
C NMR spectral data of 4 suggested that it was a hexanorcucurbitane glycoside [6,7].
Comparison of the
13
C NMR data with those of compound 3 (table 1) revealed the absence of
two olefinic and one carbonyl carbon signals at
d
120.7, 146.9 and 196.9, due to C-1, C-2 and
C-3 of compound 3 respectively, indicating the differences in ring A between the two
compounds. In the HMBC spectrum, the two methyl protons at
d
1.05 and 1.51 showed
Figure 1. The structures of compounds 1, 2 and 3.
D.-C. Wang et al.526
correlations to the methine carbon at
d
86.0, quarter nary carbon at
d
42.1 and olefinic carbon
at
d
139.6; the latter was characteristically due to C-5 of cucurbitane skeleton [5]. Thus the
first two carbons were assigned to C-3 and C-4 respectively. The methylenic proton at
d
1.70
(
d
28.8) showed long-range correlation (HMBC) to carbons at
d
86.0 (C-3) and 42.1 (C-4),
indicating that the methylene (
d
28.8) was connected to C-3. The long-range correlation
between the methylenic proton at
d
1.56 (
d
22.5) and carbon at
d
86.0 (C-3) elucidated that
the carbon at
d
22.5 was connec ted to the carbon at
d
28.8. Thus the two methylenic carbons
at
d
22.5 and 28.8 were assigned to C-1 and C-2, respectively. Finall y the carbons of the ring
Aof4 were assigned as shown in table 2. From the comparison of
13
C NMR data of 4 with
Table 1.
13
C NMR spectral data of compounds 1, 2 and 3 (150 MHz, C
5
D
5
N).
C 123C 123
1 120.9 120.9 120.7 19 18.3 18.3 18.3
2 146.8 146.8 146.9 20 80.1 80.3 208.4
3 197.0 197.0 196.9 21 25.5 25.4 31.6
4 49.5 49.5 49.5 22 216.1 215.6
5 137.0 137.0 137.0 23 32.8 40.9
6 120.5 120.5 120.4 24 38.5 74.8
7 23.9 23.9 24.0 25 69.0 72.2
8 41.8 41.8 42.1 26 29.9 25.1
9 50.9 51.1 50.2 27 30.1 27.6
10 35.6 35.6 35.6 28 20.2 20.2 20.1
11 214.0 214.0 212.8 29 27.6 27.2 27.4
12 49.7 49.7 49.7 30 20.8 20.8 20.8
13 49.3 49.3 49.0 Glc-1
0
100.6 100.6 100.6
14 48.7 48.6 47.8 2
0
74.4 74.4 74.4
15 46.5 46.4 46.2 3
0
78.5 78.5 78.5
16 70.6 70.6 71.4 4
0
70.3 70.4 70.7
17 59.0 58.9 67.9 5
0
78.8 78.8 78.7
18 20.4 20.5 19.9 6
0
61.9 61.9 62.0
Table 2.
1
H NMR and
13
C NMR spectral data and key HMBC correlations of compound 4 (600 Hz for
1
H and
150 MHz for
13
C, C
5
D
5
N).
C
d
C
d
H
HMBC C
d
C
d
H
HMBC
1 22.5 1.56 m, 1.83 m C-3 19 20.5 1.38 s C-10, C-11
C-8, C-9
2 28.8 1.85 m, 1.70 m C-3, C-10, C-4 20 208.7
3 86.0 3.62 brs C-1, C-5, C-1
0
21 31.7 1.15 s C-20, C-17
4 42.1 22 19.4 1.52 s C-4, C-3, C-5
5 139.6 23 28.1 1.05 s C-3, C-4, C-5
6 119.7 5.94 d 5.6 C-7, C-10, C-4, C-8 24 25.5 1.51 s C-8, C-15, C-14, C-13
7 24.5 2.76 dd 18.8,8.1 C-6, C-5, C-9 1
0
105.2 4.89 d 7.6 C-3
1.93 m
8 43.7 1.90 m 2
0
76.2
9 49.6 3
0
80.5 C-2
0
, C-4
0
10 35.8 2.51 m C-9, C-11 4
0
71.9 4.17 t 9.2 C-3
0
, C-5
0
, C-6
0
11 212.3 5
0
78.2 3.85 m
12 47.5 2.54 d 14.4 C-11, C-13, C-14 6
0
62.7 C-4
0
3.30 d 14.4
13 50.5 1
00
101.1 6.74 brs C-5
00
, C-2
0
14 49.2 2
00
72.6 4.75 d 2.4 C-3
00
, C-4
00
15 46.2 1.76 m, 1.93 m C-16 3
00
72.4
16 71.4 3.50 m C-20, C-14 4
00
74.1
17 68.1 3.48 d 6.5 C-20, C-13, C-16 5
00
69.6
18 20.0 0.76 s C-12, C-14 6
00
19.4 1.70 d 6.1 C-5
00
, C-4
00
C-17, C-13
Cucurbitane and hexanorcucurbitane glycosides 527
those of 3 (table 1) and the analysis of HMQC and HMBC spectra of 4, carbons of the ring B,
C and D of the aglycone were also assigned (table 2).
The relative stereochemistry of 4 was determined by NOESY analysis. In the NOESY
spectrum H
3
-23 showed correlations to H-3 and H-10, while H
3
-24 was correlated with H-10
and H-17. The H-10 was a orientated according to the study about cucurbitacin biogenesis [8].
So the H-3, H
3
-23, H
3
-24, H-17 should be a orientated. Because of undisplayed NOE
correlations between H-16 and H-24, 17 in the NOESY spectrum, H-16 should be b orientated.
The direct and long-range connections between protons and carbons were assigned
(table 2), and carbons at
d
105.2, 76.2, 80.5, 71.9, 78.2, 62.7 were assigned to the glucose
unit, while carbons at
d
101.1, 72.6, 72.4, 74.1, 69.6, 19.4 were assigned to the rhamnose
unit. The anomeric configuration of the sugar moieties were determined to be b for glucose
on basis of the coupling constants (J
1
0
,2
0
¼ 7.6 Hz), while a for rhamnos e due to the chemical
shift value of C-5
00
(
d
69.6) [9]. The HMBC correlation was obser ved between an anomeric
proton at
d
4.89 (H-1
0
) and carbon at
d
86.0 due to C-3 of the aglycone. Another anomeric
proton at
d
5.38 (H-1
00
) was correlated with carbon at
d
76.2 (C-2
0
), together with the
downfield shift of C-3
0
(þ 2.0 ppm) and the upfield shift of C-4
0
(2 1.5 ppm), suggesting
the rhamnopyranosyl unit was attached to C-2
0
of the glucopyranosyl moiety. Therefore, the
structure of 4 was determined to be 16-hydroxy-22,23,24,25,26,27-hexanorcucurbit-5-en-
11,20-dione 3-O-a-
L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside (figur e 2).
3. Experimental
3.1 General experimental procedures
1
H NMR (600 MHz, C
5
D
5
N) and
13
C NMR (150 MHz, C
5
D
5
N) spectra were recorded on a
Bruker ARX-600 spectrometer with TMS as an internal standard. HRESI-MS data were
measured with a Bruker AOEXIII 7.0 TESLA FT-MS. Column chromatography was carried
out on silica gel (Qingdao Haiyang Chemical Co. Ltd., 200–300 mesh), Sephadex LH-20
(Amersham Pharmacia Biotech AB). Spots (on TLC) were visualised by spraying with
vanillin (1%) and H
2
SO
4
(10%) in EtOH and heating (1108C, 5 min).
Figure 2. The structure and key HMBC correlations of compound 4.R¼ -a-L-rhamnopyranosyl-(1 ! 2)-b-D-
glucopyranoside.
D.-C. Wang et al.528
3.2 Plant material
The fruits of Cucurbita pepo cv dayangua were collected from the planting base of Jilin
University, Jilin Province of China in August 2002. The specimen was botanically identified
by An-min Lu (Institute of Medicinal Plant Development of Chinese Academy of Medi cal
Science, China). A voucher specimen has been deposited in the Herbarium of the Institute of
Medicinal Plant Development of Chinese Academy of Medical Science, China.
3.3 Extraction and isolation
The air-dried fruits (15 kg) were extracted with 95% ethanol for three times under reflux. The
combined solution was concentrated under vacuum and subjected to a column of
macroporous absorption resin (AB-8) eluted with 70% EtOH, then the solution was
evaporated to dryness under vacuum to give a residue (200 g). The residue was
chromatographed on silica gel with CHCl
3
/MeOH (in gradient) to give 15 fractions.
Fractions 6 and 7 (13 g together) were repeatedly chromatographed on silica with a solvent
system of CHCl
3
/MeOH/H
2
O (85:15:10) to give 1 (32 mg), 2 (40 mg) and 3 (25 mg). Fraction
13 (8 g) was further separated by Sephadex LH-20 eluting with 80% MeOH to give 4
(12 mg).
3.3.1 Compound 4. White amorphous powder (MeOH), HRESI-MS m/z 719.3643
[M þ Na]
þ
(calculated for C
36
H
56
O
13
Na, 719.3627).
1
H NMR,
13
C NMR, and HMBC data:
see table 2.
Acknowledgements
The authors are grateful to A. Zeper for mass spectra measurement, and Wen Li and Yi Sha
for NMR measurement. Financial support is from the National Natural Science Foundation
of China (No. 30371083).
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Cucurbitane and hexanorcucurbitane glycosides 529
