Two new ent-kaurane-type diterpene glycosides from zucchini (Cucurbita pepo L.) seeds

Article abstract of DOI:10.1016/j.fitote.2015.09.019

Two new ent-kaurane diterpene glycosides; 12α-(β-d-glucopyranosyloxy)-7β-hydroxykaurenolide (1) and 7β-(β-d-glucopyranosyloxy)-12α-hydroxykaurenolide (2), a new steroid; (24S)-stigmasta-7,22E,25-trien-3-one (12), and known compounds (3-11, 13-14) were isolated from zucchini (Cucurbita pepo L.) seeds. The absolute structures of 1 and 2 were determined by acid hydrolysis and application of a modified Moscher's method. Furthermore, isolated compounds (1-14), and a derivative, 1a, were evaluated for their inhibitory effects on macrophage activation by an inhibitory assay of nitric oxide (NO) production.

Full text of DOI:10.1016/j.fitote.2015.09.019

Two new ent-kaurane-type diterpene glycosides from zucchini (Cucurbita
pepo L.) seeds
Takashi Kikuchi, Hiromi Ando, Ken-ichiro Maekawa, Hiroki Arie, Takeshi
Yamada, Reiko Tanaka
PII:
DOI:
Reference:
S0367-326X(15)30093-9
doi: 10.1016/j.fitote.2015.09.019
FITOTE 3272
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
5 August 2015
17 September 2015
18 September 2015
Please cite this article as: Takashi Kikuchi, Hiromi Ando, Ken-ichiro Maekawa,
Hiroki Arie, Takeshi Yamada, Reiko Tanaka, Two new ent-kaurane-type diter-
pene glycosides from zucchini (Cucurbita pepo L.) seeds, Fitoterapia (2015), doi:
10.1016/j.fitote.2015.09.019
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ACCEPTED MANUSCRIPT
Two New ent-Kaurane-type Diterpene Glycosides from Zucchini
(Cucurbita pepo L.) Seeds
Takashi Kikuchi, Hiromi Ando, Ken-ichiro Maekawa, Hiroki Arie, Takeshi Yamada,
Reiko Tanaka*
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka
569-1094, Japan
Key words: Cucurbita pepo L.; ent-Kaurane-type diterpene; Glycoside; Inhibitory
activity on nitric oxide production
Corresponding author. Tel./Fax: +81-72-690-1084; e-mail: tanakar@gly.oups.ac.jp

ACCEPTED MANUSCRIPT
Abstract
Two
new
ent-kaurane
diterpene
glycosides;
and
12-(-D-glucopyranosyloxy)-7-hydroxykaurenolide
7-(-D-glucopyranosyloxy)-12-hydroxykaurenolide
(1)
(2),
a
new
steroid;
(24S)-stigmasta-7,22E,25-trien-3-one (12), and known compounds (3−11, 13−14) were
isolated from zucchini (Cucurbita pepo L.) seeds. The absolute structures of 1 and 2
were determined by acid hydrolysis and application of a modified Moscher’s method.
Furthermore, isolated compounds (1−14), and a derivative, 1a, were evaluated for their
inhibitory effects on macrophage activation by an inhibitory assay of nitric oxide (NO)
production.

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1. Introduction
Pumpkins, including Cucurbita moschata, C. pepo, and C. maxima, are gourd
squashes of the genus Cucurbita and the family Cucurbitaceae. Cucurbita moschata
seeds have been used as an anthelmintic [1], and Cucurbita pepo seeds, as an
anthelmintic and diuretic [2]. Previous studies reported the isolation of
3-p-aminobenzoyl
multiflorane-type
triterpenes:
3-O-p-aminobenzoyl-29-O-benzoylmultifrora-8-ene-3,7,29-triol,
3-O-p-aminobenzoyl-29-O-benzoylmultifrora-7,9(11)-diene-3,29-diol, 7-epi zucchini
factor A, and debenzoyl zucchini factor B, from C. pepo seeds [3, 4]. We recently
isolated five malutiflorane-type triterpenoids, including three new compounds;
3-p-nitrobenzoylmultiflora-7:9(11)-diene-29-benzoate, 3-acetoxymultiflora-7:9(11)-
diene-29-benzoate, and 3-acetoxymultiflora-5(6):7: 9(11)-triene-29-benzoate, from C.
pepo seeds, and evaluated their cytotoxic activities in cancer cell lines as well as their
inhibitory activities on melanogenesis [5]. In a continuing study to explore new
compounds possessing biological activities from C. pepo seeds, we herein isolated two
new
ent-kaurane
diterpene
glycosides;
12-(-D-glucopyranosyloxy)-7-hydroxy-kaurenolide
(1)
and
7-(-D-glucopyranosyloxy)-12-hydroxy-kaurenolide (2), and a new steroid;
(24S)-stigmasta-7,22E,25-trien-3-one (12), along with known compounds (3−11, and
13−14). Furthermore, isolated compounds (1−14), and a derivative, 1a, were evaluated
for their inhibitory effects on macrophage activation by an inhibitory assay of nitric
oxide (NO) production in RAW264.7 mouse macrophages stimulated by
lipopolysaccharide (LPS).

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2. Experimental
2.1. General Experimental Procedures
The following chemicals and reagents were purchased: fetal bovine serum (FBS)
from
Invitrogen
Co.
(Carlsbad,
CA,
U.S.A.),
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) from
Sigma-Aldrich Japan Co. (Tokyo, Japan), Dulbecco’s modified Eagle’s medium
(D-MEM), and antibiotics from Nacalai Tesque, Inc. (Kyoto, Japan). All other
chemicals and reagents were of analytical grade. Melting points were determined on a
Yanagimoto micro-melting point apparatus and were uncorrected. Optical rotations
were measured with a JASCO DIP-1000 digital polarimeter. IR spectra were recorded
1
13
on a Perkin-Elmer 1720X FTIR spectrophotometer. H (600 MHz) and C (150 MHz)
NMR spectra were recorded on an Agilent vnmrs600 in CD3OD and CDCl3 with
tetramethylsilane as the internal standard. HR-FAB-MS was recorded on a JEOL
JMS-7000 mass spectrometer. Column chromatography (CC): Diaion HP-20
(Mitsubishi Chemical Co., Tokyo, Japan), Sephadex LH-20 (GE healthcare
Bio-Sciences AB, Uppsala, Sweden), and octadecyl silica gel (ODS; Chromatorex-ODS,
100–200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). HPLC was carried out on an
ODS column [Cosmosil 5C18-PAQ column (Nacalai Tesque, Inc., Kyoto, Japan), 25 cm
 20 mm i.d.] at 35ºC with MeCN / H2O [15 : 85 (HPLC system I), 20 : 80 (HPLC
system II), 25 : 75 (HPLC system III), flow rate 4.0 ml/min], Cosmosil 5C18-MS-II
(Nacalai Tesque, Inc., Kyoto, Japan), 25 cm  20 mm i.d.] at 35ºC {MeCN / H2O [10 :
90 (HPLC system IV), 20 : 80 (HPLC system V)], and (CH3)2CO / H2O [10 : 1 (HPLC
system VI)], flow rate 4.0 ml/min}, and SiO2 column [Cosmosil 5SL-II column (Nacalai

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Tesque, Inc., Kyoto, Japan), 25 cm  20 mm i.d.] at 35ºC with hexane / EtOAc [10 : 1
(flow rate 8.0 ml/min) (HPLC system VII), 5 : 1 (flow rate 4.0 ml/min) (HPLC system
VIII)].
2.2. Plant Material
The seeds of Cucurbita pepo, produced in USA, were purchased from TAKADA
SEEDS Co., Ltd. in 2011. A voucher specimen was deposited in the Herbarium of the
Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.
2.3. Extraction and Isolation
The seeds of cucurbita pepo L. (20 kg) were subjected to extraction with MeOH
under reflux (1 week, 4 times). The MeOH extract (710 g) was then partitioned between
Et2O and H2O, followed by BuOH and H2O.
The Et2O fraction (Fr.) (410 g) was subjected to SiO2 CC [SiO2 (6 kg)] resulting in 8
fractions, Ea—Eh [CH3Cl / EtOAc (1 : 0)→(5 : 1)→(0 : 1) and MeOH], followed by Ed
(59 g), eluted with CH3Cl, was chromatographed by SiO2 (1 kg) to yield 7 fractions,
Ed1—Ed7 [CH3Cl / MeOH (1 : 0)→(10 : 1)→(1 : 1)→(0 : 1)]. Of these, Fr. Ed2 (28 g),
eluted with CHCl3, was subjected to re-chromatography by SiO2 (1 kg) to give 25
fractions, Ed2-1—Ed2-25 [hexane / EtOAc (10 : 1)→(5 : 1)→(2 : 1)→(1 : 1)→(0 : 1)].
SiO2 CC (12 g) of Ed2-21 (324.4 mg), eluted with hexane / EtOAc (2 : 1), gave 13
(50.00 mg) with hexane / EtOAc (5 : 1). Preparative HPLC (HPLC system VII) of
Ed2-7 (10.4 mg), eluted with hexane / EtOAc (10 : 1), gave 12 (1.30 mg; tR 17.0 min).
Ed3 (10 g), eluted with CH3Cl, was subjected to re-chromatography by SiO2 (400 g) to
give 43 fractions, Ed3-1—Ed2-43 [hexane / EtOAc (10 : 1)→(3 : 1)→(1 : 1)→(0 : 1)

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and MeOH]. Preparative HPLC (HPLC system VIII) of Ed3-28 (206.4 mg), eluted with
hexane / EtOAc (10 : 1), gave 14 (3.31 mg; tR 81.5 min).
The BuOH Fr. (40 g) was subjected to Diaion HP-20 CC [Diaion HP-20 (600 g)]
resulting in H2O Fr. (17 g), MeOH Fr. (17 g), and (CH3)2CO Fr. (169 mg). The MeOH
Fr. was chromatographed by ODS (50 g) to yield to 10 fractions, Ma—Mj [MeOH /
H2O (1 : 1)→(7 : 3)→(9 : 1)→(1:0)]. Of these, Fr. Ma (11 g), eluted with MeOH / H2O
(1 : 1), was subjected to re-chromatography with ODS (50 g) to give 16 fractions,
Ma1—Ma16 [MeCN / H2O (1 : 9)→(2 : 8)→(3 : 7)→(4 : 6)→(1 : 0)]. Fr. Ma2 (4643.86
mg), eluted with MeCN / H2O (1 : 9), was separated by sephadex LH-20 CC [sephadex
LH-20 (40 g)] to afford 13 fractions, Ma2-1—Ma2-13 (H2O→MeOH). Preparative
HPLC of Ma2-6 (84.47 mg)(HPLC system I), eluted with H2O, gave 6 (17.59 mg; tR
86.6 min) and 10 (3.27 mg; tR 116.8 min). Fr. Ma3 (1040.96 mg), eluted with MeCN /
H2O (1 : 9), was separated by sephadex LH-20 CC [sephadex LH-20 (40 g)] to afford
21 fractions, Ma3-1—Ma3-21 (H2O→MeOH). Preparative HPLC of Ma3-11 (42.49
mg)(HPLC system I), eluted with MeOH, gave 9 (4.96 mg; tR 110.0 min). Fr. Ma5
(1051.17 mg), eluted with MeCN / H2O (2 : 8), was separated by preparative HPLC
(HPLC system II) to give 5 (12.79 mg; tR 28.2 min), 3 (48.79 mg; tR 32.5 min), 4 (26.30
mg; tR 33.3 min), and 1 (12.83 mg; tR 40.3 min). Fr. Ma6 (192.50 mg), eluted with
MeCN / H2O (2 : 8), was separated by preparative HPLC (HPLC system IV) to give
fifteen fractions, Ma6-1—Ma6-15. Ma6-13 was identified as 7 (8.60 mg; tR 51.4 min).
Fr. Ma6-7 (32.1 mg) was purified with re-preparative HPLC (HPLC system III) to give
1 (24.87 mg) and 8 (4.73 mg; tR 49.4 min). Ma7 (638.86 mg), eluted with MeCN / H2O
(2 : 8), was separated by sephadex LH-20 CC [sephadex LH-20 (30 g)] to afford 13
fractions, Ma7-1—Ma7-12 (H2O→MeOH). Preparative HPLC of Ma7-3 (89.8

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mg)(HPLC system III), eluted with H2O, gave 2 (3.76 mg; tR 24.2 min). Preparative
HPLC of Ma7-11 (105.9 mg)(HPLC system V), eluted with MeOH, gave 11 (1.05 mg;
tR 89.7 min).
2.4. Acid Hydrolysis and Determination of Sugar Configurations
The determination of absolute sugar configurations was carried out according to a
method reported previously with slight modifications [6]. A solution of 1 (13.39 mg) in
10% HClaq (1 mL) was heated at 90oC for 5 days. The mixture was extracted with
EtOAc (3 times). The combined organic layers were evapd. in vacuo to furnish a crude
product. The crude product was subjected to CC (SiO2), yielding 1a (3.33 mg). The
H2O layer was concentrated to dryness. The residue was then dissolved in pyridine (0.1
mL) and stirring with L-cysteine methyl ester hydrochloride (0.5 mg) at 60 °C for 1 h.
The reaction mixture was treated with o-tolylisothiocyanate, and heated at 60 °C for 1 h.
The reaction mixture was analyzed by reversed-phase HPLC [column: Cosmosil
5C18-PAQ column (Nacalai Tesque, Inc., Kyoto, Japan), 250 × 4.6 mm i.d. (5 μm);
mobile phase: MeCN−H2O (2:8, v/v) in 1% AcOH; detection: refractive index; flow
rate: 1.0 mL/min; column temperature: 35 °C] to identify the derivatives of D-glucose
by comparison of their retention times with those of authentic samples (tR: D-glucose
28.7 min, L-glucose 26.0 min). The acid hydrolysis of 1 (18.83 mg) was performed
again to give 1a (2.38 mg). The acid hydrolysis of 2 (1.57 mg) was conducted in a
similar manner as described above, and its EtOAc layer gave 1a (0.36 mg). The H2O
layer afforded D-glucose derivative by derivatization with L-cysteine methyl ester
hydrochloride and o-tolylisothiocyanate, followed by HPLC analysis.

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2.5. Preparation of (S)- and (R)- MTPA Esters from 1a
(−)-MTPA-Cl (100 mg) was added to a solution of 1a (2.37 mg) in pyridine. The
mixture was stirred at r.t. for 18 h, poured into H2O, and then extracted with CH2Cl2.
The organic layer was evapd. in vacuo to give a crude product. The crude product was
subjected to CC [SiO2, hexane-EtOAc (5 : 1)] to yield 1b, which was the (S)-MTPA
ester of 1a (2.12 mg). 1c (2.01 mg), which was the (R)-MTPA ester of 1a, was prepared
from 1a (2.48 mg) and (+)-MTPA-Cl (100 mg) in a similar manner as described above.
2.6. Oxidation of 13
13 (10.9 mg) in CH2Cl2 (5 mL) was added to a solution of pyridium chlorochromate
(15.8 mg) and SiO2 (15.8 mg) in CH2Cl2 (5 mL) . The mixture was stirred at r.t. for 4 h,
and then filtrated over SiO2. The filtrate was evapd. in vacuo to furnish a crude product.
Preparative HPLC of the crude extract gave 12 (3.89 mg; tR 43.3 min)(HPLC system
VI).
2.7.12-(-D-glucopyranosyloxy)-7-hydroxykaurenolide (1)
Amorphous solid, []D21 −20.5 (c = 0.26, EtOH); IR maxKBr cm-1: 3434, 2933, 2869,
1754, 1514, 1450, 1370, 1209, 1079; FAB-MS m/z :517 [M+Na]+, 495 [M+H]+;
HR-FAB-MS m/z: 517.2416 (calcd for 517.2414: C26H38O9Na)
2.8. 7-(-D-glucopyranosyloxy)-12-hydroxykaurenolide (2)
19
KBr
Amorphous solid, []D ; −9.4 (c = 0.045, EtOH); IR max
cm-1: 3411, 2923,
2876, 1756, 1649, 1080, 1025; FAB-MS m/z: 517 [M+Na]+, 495 [M+H]+; HR-FAB-MS
m/z: 517.2412 (calcd for 517.2414: C26H38O9Na)

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2.9. 16,17-dihydro-7-hydroxy-12-oxokaurenolide (1a)
23
KBr
Amorphous solid; []D −79.8 (c = 0.11, EtOH); IR max
cm-1: 3463, 2954,
2882, 1759, 1699, 1452, 1196, 1064; EI-MS m/z : 332 [M]+ (100), 314 (9), 288 (10),
273 (21), 255 (30), 165 (25), 137 (45), 109 (74); HR-EI-MS m/z: 332.1984 (calcd for
332.1987: C20H28O4)
2.10. (24S)-stigmasta-7,22E,25-trien-3-one (12).
22
KBr
[]D +17.9 (c = 0.57, CHCl3); IR max
cm-1: 2963, 2868, 1718, 1645, 1445,
1382, 967, 885; EIMS m/z (rel. int. ): 408 (21) [M]+, 379 (21), 325 (3), 298 (19), 269
(100), 229 (8); HREIMS m/z: 408.3392 [M]+ (calcd for 408.3392: C29H44O)
2.11. Cell culture
The RAW264.7 cell line was grown in D-MEM medium, which was supplemented
with 10% FBS and antibiotics (100 units/mL penicillin and 100 g/mL streptomycin).
Cells were incubated at 37°C in a 5% CO2 humidified incubator.
2.12. Cytotoxicity assay
Cytotoxicity assays were performed according to a method reported previously [7]
Briefly, RAW264.7 cells (5 × 104 cells in 100 L), which were pre-incubated for 24h on
a 96-well microplate, were treated for 24 h with test samples dissolved in dimethyl
sulfoxide (DMSO) at a final concentration of 30, 10, 3 M, and 1 M, and MTT
solution was added. After a 3 h incubation, 20% SDS containing 0.1 M HCl was added

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to dissolve the formazan produced by the cells. The absorbance of each well was read at
570 nm using a microplate reader.
2.13. Inhibitory assay of NO production
An inhibitory assay of NO production was performed according to a method reported
previously [8] with slight modifications. Briefly, RAW264.7 cells (5 × 104 cells in 100
L) were seeded on 96-well microplates and incubated for 24h. D-MEM (100 L)
containing the test samples (final concentration of 30, 10, 3, or 1 M) dissolved in
DMSO (final concentration 0.2 %) and LPS (final concentration of 5 g / mL) was
added. After cells had been treated for 24 h, the supernatant of the cultured medium
(150 L) was transferred to a 96-well microplate, and 75 L of 0.1 %
N-(1-naphtyl)ethylenediamine in H2O and 75 L of 1 % sulfanilamide in 5 %
phosphoric acid were then added. After being incubated for 30 min, the absorbance of
each well was read at 570 nm using a microplate reader. The optical density of vehicle
control cells was assumed to be 100%.
3. Results and discussion
Compounds 1−13 were isolated from the MeOH extract of C. pepo seeds (Fig. 1).
Compound 1 had an [M+Na]+ ion in the HR-FAB-MS at m/z 517.2416, which was
consistent with the molecular formula C26H38O9Na (calcd 517.2414). Its IR spectrum
showed absorption that indicated a hydroxy group (3434 cm-1) and a -lactone (1754
1
13
cm-1). H and C NMR spectra indicated two tertiary methyls [H 0.88 (s), 1.28 (s)],
three oxymethines [H 3.82 (brt), 4.30 (d), 4.63 (t); C 72.3 (d), 84.5 (d), 85.3 (d)], an

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Fig 1. Structures of compounds 1−14 from C. pepo seeds.

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Table 1
1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of Compounds 1—3a.
1b
2b
H
1ac
No.
1
H
(J in Hz)
C
(J in Hz)
C
H
(J in Hz)
C
1.58 m
1.04 m
1.56 m
38.4
t
t
t
1.58 dt
1.01 m
1.53 m
(5.0, 12.6)
38.2
t
t
t
1.54 m
1.01 m
1.54 m
1.63 m
2.09 m
1.45 m
37.0
t
t
t






2
3
(2H)
18.4
29.3
(2H)
18.7
29.8
16.9
27.7
2.04 m
1.42 m
2.09 dt
(5.3, 14.3)
1.41 ddd (6.7, 9.9, 14.3)
4
43.1
53.0
85.3
72.3
46.7
52.2
35.2
27.1
s
d
d
d
s
42.9
53.2
84.2
83.1
46.3
52.1
34.9
28.4
s
d
d
d
s
41.6
52.6
83.3
71.5
46.3
51.4
34.2
35.8
s
5
1.89 d
4.63 t
4.30 d
(6.5)
(6.5)
(6.5)
1.91 d
4.96 t
4.36 d
(6.2)
(6.2)
(6.2)
1.79 d
4.67 t
4.43 d
(6.7)
d
d
d
s
6
(6.7)
(6.7)

7

8
9
1.01 dd
(4.1, 14.7)
d
s
1.03 m
d
s
1.36 dd (5.3, 13.8)
d
s
10
11
1.40 m
1.85 m
3.82 brt
t
1.23 m
1.79 m
3.66 m
2.49 d
t
2.24 dd (5.3, 16.5)
2.33 dd (13.8, 16.5)
t


12
13
14
(8.2)
84.5
46.0
33.0
d
d
t
77.3
49.6
32.5
d
d
t
214.7
55.2
33.7
s
d
t
2.75 brd (5.0)
(5.3)
2.27 dd (1.2, 4.7)
1.65 dd (1.2, 12.6)
2.09 dd (4.7, 12.6)
1.74 dd (7.6, 11.7)
1.37 m
1.72 m
(2H)
1.76 m
2.02 dd
2.76 brd
1.78 m




(4.7, 11.8)
(14.6)
15
2.60 dt
1.71 m
(2.3, 14.7)
43.4
t
44.5
t
42.6
t
16
17
155.6
110.0
s
t
155.9
109.4
s
t
2.11 m
36.0
22.6
d
q
A
B
5.02 brs
5.09 brs
4.98 brs
5.09 brs
1.16 d
(7.0)
18
19
20
1'
184.9
25.9
20.9
102.5
75.1
78.2
71.8
78.1
62.8
s
q
q
d
d
d
d
d
t
184.2
26.0
20.2
106.0
75.6
78.5
71.8
77.5
63.0
s
q
q
d
d
d
d
d
t
181.8
25.6
20.0
s
1.28 s
1.26 s
1.32 s
0.92 s
q
q
0.88 s
0.86 s
4.44 d
3.16 dd
3.37 m
3.28 m
3.28 m
3.66 m
3.86 dd
(8.0)
4.43 d
3.23 dd
3.35 t
(8.0)
2'
(8.0, 9.1)
(8.0, 9.1)
(9.1)
3'
4'
3.30 m
3.30 m
3.66 m
3.83 dd
5'
6'
A
B
(1.5, 12.0)
(2.1, 11.4)
aAssignments were based on 1H-1H COSY, HMQC, HMBC, and NOESY spectroscopic data.
bMeasured in CD3OD.
cMeasured in CDCl3.

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exo-methylene [H 5.02 (brs), 5.09 (brs); C 110.0 (t), 155.6 (s)], and a terminal glucose
[H 4.44 (d); C 62.8 (t), 71.8 (d), 75.1 (d), 78.1 (d), 78.2 (d), 102.5 (d)]. The
configuration of glucose at C-1′ was determined to be  based on the the coupling
constants of H-1′ [H 4.44 (d), J1’, 2’=8.0 Hz]. The following correlations were observed
in the HMBC spectrum; Me-19 [H 1.28 (s)]/C-3, C-4, C-5, and C-18 [C 184.9 (s)];
Me-20 [H 0.88 (s)]/C-1, C-5, C-9, and C-10; H-6 [H 4.63 (t)]/C-5 and C-7 [C 72.3
(d)] ; H-7 [H 4.30 (d)]/C-8; H-13 / C-12 [C 84.5 (d)] and C-16 [C 155.6 (s)]; H2-17
[H 5.02 (brs), 5.09 (brs)]/C-13 and C-15; and H-1’ [H 4.44 (d)]/C-12 (Fig. 2). In the
1H-1H COSY spectrum, the correlations of H2-1−H2-2−H2-3; H-5−H-6−H-7; and
H2-11−H-12; H-13−H2-14 were observed (Fig. 2).
1
Fig. 2. Key HMBC (
1–2, 1a, and 12.
) and H-1H COSY (
) correlations of compounds

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These results indicated that 1 was a kaurane-type diterpene or its enantiomer, an
ent-kaurane-type, as shown in Figure 2. The relative structures of the 6, 7, and 12
positions were characterized by NOESY experiments, which observed the following
correlations; H-6/Me-19; H-7/Me-20; and H-12/H-17B (Fig. 3). The acid hydrolysis
of 1 was performed to determine the configurations of glucose and aglycon (Fig. 4).
Fig. 3. Selected NOE correlations of compound 1.

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Fig. 4. Acid hydrolysis of compounds 1 and 2, and application of a modified Moscher’s
method for 1a.
Acid hydrolysis by 10% HCl yielded an unexpected 12-oxo product, 1a, and D-glucose.
The formation of 1a from 1 was inferred as follows: (I) was produced from 1 by acid
hydrolysis.  bond at 16 and 17 positions in (I) attacked H+ ion. And then H− ion from
C-12 attacked carbocation at C-16, followed by carbonyl group generation at C-12 gave
1a. D-glucose was identified by derivatization with L-cysteine methyl ester
hydrochloride and o-tolylisothiocyanate, followed by a HPLC analysis [6]. The relative
structures of the 6 and 7 positions in 1a were the same as those in 1 because the
following NOE correlations were observed; H-6/Me-19 and Me-20/H-7 and H-14pro-R

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(Fig. 5). The absolute configuration of 1a was characterized by the application of a
modified Moscher’s method (Fig. 4). The  (S−R) values for H-6 (−0.09), Me-19
(−0.11), and Me-20 (−0.01) were negative, while those of H-13 (+0.08), H-16 (+0.10),
and Me-17 (+0.19) were positive (Fig. 4). Therefore 1a, and its substance, 1 possessed a
7-hydroxy group, and 1 was established as the 6,7,12-trihydroxy-ent-
kaur-16-en-18-oic acid -lactone 12-O--D-glucopyranoside (Table 1).
Fig. 5. Selected NOE correlations of compound 1.
Compound 2 had an [M+Na]+ ion in the HR-FAB-MS at m/z 517.2412, which was
consistent with the molecular formula C26H38O9Na (calcd 517.2415), similar to 1. Its IR
spectrum showed absorption, which indicated a hydroxy group (3411 cm-1) and a
1
13
-lactone (1756 cm-1). H and C NMR spectra indicated that the structure of 2 was
similar to that of 1, except for a 7-hyhdroxy group [H 4.30 (d); C 72.3 (d) in 1] and
12-(-glucopyranosyloxy) group [H 3.82 (brt); C 84.5 (d) in 1], as well as the
presence of a 7-(-glucopyranosyloxy) group [H 4.36 (d); C 83.1 (d) in 2] and

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12-hydroxy group [H 3.66 (m); C 77.3 (d) in 2]. These results were confirmed by
1
HMBC and H-1H COSY experiments (Fig. 2). The acid hydrolysis of 2 gave the same
products as those of 1, namely 1a (Fig. 4), and D-glucose. D-glucose was identified by
derivatization with L-cysteine methyl ester hydrochloride and o-tolylisothiocyanate,
followed by a HPLC analysis [6]. Therefore the absolute configuration of 2 was the
same
as
that
of
1,
and
2
was
established
as
the
6,7,12-trihydroxy-ent-kaur-16-en-18-oic acid -lactone 7-O--D-glucopyranoside
(Fig. 1).
Compound 12 had an [M]+ ion in the HR-EI-MS at m/z 408.3392, which was
consistent with the molecular formula C29H44O (calcd 408.3392). Its IR spectrum
1
showed the presence of a carbonyl group (1718 cm-1). H and 13C NMR spectra
indicated that the presence of three tertiary methyls [H 0.58 (s), 1.02 (s), 1.65 (s)], a
secondary methyl [H 1.03 (d)], a primary methyl [H 0.84 (t)], a trisubstituted olefin [H
5.18 (m); C 117.0 (d), 139.5 (s)], a disubstituted olefin [H 5.19 (dd), 5.25 (dd); C
130.3 (d), 136.9 (d)], an exo-methylene [H 4.71 (2H, m); C 109.6 (t), 148.6 (s)], and a
ketone [C 212.0 (s)] (Table 2). The 1H and 13C NMR signals of 12 were similar to those
of the known compound 13, except for the absence of a hydroxy group and existence of
a carbonyl group at C-3 [C 212.0 (s)]. This was confirmed by HMBC and 1H-1H COSY
experiments (Fig. 2). Furthermore, the oxidation of 13 by PCC gave 12 (Fig. 6).
Therefore, compound 12 was established as (24S)-stigmasta-7,22E,25-trien-3-one.

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Table 2
NMR spectral data for 12 in CDCl3.a
No.
1
(J in Hz)
No.
15
(J in Hz)
H
C
38.8
H
1.51 m
1.41 m
C
23.0
1.46 m
t
t
t






2.13 ddd (2.6, 5.8, 13.2)
2
2.28 m
2.43 m
38.1
16
17
18
19
20
21
22
23
24
25
26
27
28
1.75 m (2H)
1.29 m
28.3
55.8
t
d
q
q
d
d
d
d
d
s
3
4
5
6
212.0
44.2
42.9
30.0
s
t
0.58 s
12.1
2.23 m (2H)
1.81 m
1.02 s
12.4
d
t
2.04 m
40.5
1.26 m
1.03 d (6.8)
5.25 dd (8.2, 15.3)
5.19 dd (8.4, 15.3)
2.43 m
20.9


1.83 m
136.9
130.3
52.0
7
8
5.18 m
117.0
139.5
48.8
34.4
21.7
39.3
d
s
d
s
t
9
1.75 m
148.6
109.6
20.2
10
11
12
4.71 m (2H)
1.65 s
t
1.63 m (2H)
1.27 m
q
t
t
A
B
1.41 m
25.7


2.04 m
1.48 m
13
14
43.3
55.0
s
29
0.84 t (7.4)
12.1
q
1.83 m
d
aAssignments were based on 1H-1H COSY, HMQC, HMBC, and NOESY spectroscopic data.
Fig. 6. Preparation of 12 by the oxidation of 13.

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Known compounds were identified as cucurbitoside F (3) [9], cucurbitoside A (4)
[10], cucurbitoside C (5) [10], cucurbitoside D (6) [10], (+)-lariciresinol (7) [11],
(−)-secoisolariciresinol (8) [11], lariciresinol-4′-O--D-glucoside (9) [12],
lariciresinol-4-O--D-glucoside
(10)
[12],
isolariciresinol
(11)
[13],
(24S)-stigmasta-7,22E,25-trien-3-ol (13) [14], and 3-hydroxycholest-7-en-24-one
(14) [15] by comparisons with spectroscopic data of those previously reported.
Macrophages may be a potential therapeutic target for inflammatory diseases [16].
Activated macrophages release pro-inflammatory mediators, such as NO, reactive
oxygen, interleukin-1 beta, tumor necrosis factor-alpha, and other inflammatory
mediators, which play important roles in biological defense. However, the
overexpression of these mediators had been implicated in diseases such as osteoarthritis,
rheumatoid arthritis, and diabetes because the increased production of pro-inflammatory
mediators has been shown to induce severe or chronic inflammation [16]. Isolated
compounds (1−14), the derivative, 1a, and L-NMMA, which was a NO synthase
inhibitor and used as a positive control, were evaluated for macrophage activation by
the inhibitory assay of NO production in RAW264.7 mouse macrophages stimulated by
LPS. Among the compounds tested, 3-hydroxycholest-7-en-24-one (14) exhibited
inhibitory effects on macrophage activation at 3−30 M (produced NO: 81.3% at 3 M,
61.2% at 10 M, and 33.9% at 30 M). The IC50 value of 14 (IC50 15.5 M) was
superior to those of L-NMMA (IC50 23.9 M). Compound 14 exhibited low cytotoxicity
at 30 M (cell viability: 85.9% at 30 M), but not at effective concentrations, namely 3
and 10 M. Compounds 1−13 and 1a did not exhibit inhibitory effects on macrophage
activation (IC50 >30 M). These results suggested that compound 14 has potential as an
anti-inflammatory disease agent.

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Table 3
Inhibitory Effects of Macrophage Activation by Compound 14 and L-NMMA from
Cucurbita pepo L. Seeds.a
NO produced % (Cell viability %)
IC50
(M)
15.5
1 M
 M
10 M
 M
104.3±4.5
(96.1±0.8)
81.3±4.5
(97.8±0.4)
91.4±0.8
(101.9±0.4)
61.2±4.0**
(94.6±1.3)
68.9±4.5
(98.5±0.9)
33.9±2.6**
(84.9±0.8)
43.1±1.1
14
L-NMMA 93.3±2.2
(101.5±0.9)
23.9
(109.4±0.5)
a NO Produced (%) and cell viability (%) were determined based on the absorbance at
570 nm by comparisons with values for DMSO (100%). Each value represents the
mean ± standard error (S.E.) of four determinations. The concentration of DMSO in
the sample solution was 2 l/ml. Significant differences from the vehicle control group
were shown as **: p<0.01, and *p<0.05.
b Positive control.
Acknowledgments
We thank Dr. Katsuhiko Minoura and Dr. Mihoyo Fujitake (Osaka University of
Pharmaceutical Sciences) for the NMR and MS measurements.
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Graphical abstract

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Conflict of interest
The authors have no conflict of interest directly relevant to the content of this article.