Diterpene glycosides from the seeds of Pharbitis nil

Article abstract of DOI:10.1021/np900101t

Six new ent-kaurane diterpene glycosides, pharbosides A-F (1-6), and a new ent-gibbane diterpene glycoside, pharboside G (7), together with three known ent-kaurane diterpenoids, 7β,16β,17-trihydroxy-ent-kauran-6α,19- olide (8), 6β,7β,16α,17-tetrahydroxy-ent-kauranoic acid (9), and 6β,7β,16β,17-tetrahydroxy-ent-kauranoic acid (10), were isolated from an ethanolic extract of the seeds of Pharbitis nil. The structures of the new compounds were determined by spectroscopic methods including 1D and 2D NMR analysis. The absolute configurations of the compounds were clarified by CD spectroscopic studies. Full NMR data assignments of the three known compounds (8-10) are reported. The isolated compounds were evaluated for their cytotoxic activities against four human cancer cell lines.

Full text of DOI:10.1021/np900101t

J. Nat. Prod. 2009, 72, 1121–1127
1121
Diterpene Glycosides from the Seeds of Pharbitis nil
Ki Hyun Kim,† Sang Un Choi,‡ and Kang Ro Lee*,†
Natural Products Laboratory, College of Pharmacy, Sungkyunkwan UniVersity, Suwon 440-746, Korea, and Korea Research Institute of
Chemical Technology, Teajeon 305-600, Korea
ReceiVed February 17, 2009
Six new ent-kaurane diterpene glycosides, pharbosides A-F (1-6), and a new ent-gibbane diterpene glycoside, pharboside
G (7), together with three known ent-kaurane diterpenoids, 7ꢀ,16ꢀ,17-trihydroxy-ent-kauran-6R,19-olide (8), 6ꢀ,7ꢀ,16R,17-
tetrahydroxy-ent-kauranoic acid (9), and 6ꢀ,7ꢀ,16ꢀ,17-tetrahydroxy-ent-kauranoic acid (10), were isolated from an
ethanolic extract of the seeds of Pharbitis nil. The structures of the new compounds were determined by spectroscopic
methods including 1D and 2D NMR analysis. The absolute configurations of the compounds were clarified by CD
spectroscopic studies. Full NMR data assignments of the three known compounds (8-10) are reported. The isolated
compounds were evaluated for their cytotoxic activities against four human cancer cell lines.
Pharbitis nil Choisy (Convolvulaceae) is a short-day plant that
is distributed throughout Southeast Asia.1 Pharbitidis Semen, the
seeds of this plant, has been used as a purgative drug in folkloric
medicine.1 Previous phytochemical studies on the seeds and flowers
of this plant have resulted in the isolation of resin glycosides,2-4
gibberellins,5-7 flavonoids, chlorogenic acid derivatives,8 and
anthocyanins.9-11 Seeds of this plant are reported to show antitumor
and antifungal activities.12,13
In a continuing search for bioactive constituents from Korean
medicinal plant sources, we performed a phytochemical investiga-
tion of this herb, and we reported the isolation of three new ent-
kaurane diterpenoids from the CHCl3-soluble fraction of the EtOH
extract.14 In our continuing study on this herb, we have now isolated
a further six new ent-kaurane diterpene glycosides, pharbosides
A-F (1-6), a new ent-gibbane diterpene glycoside, pharboside G
(7), and three known ent-kaurane diterpenoids (8-10) from the
EtOAc-soluble and BuOH-soluble fractions of the ethanolic extract
of P. nil seeds. The structures were determined using spectroscopic
methods including 1D and 2D NMR. All the isolated diterpenoids
were evaluated for their cytotoxic activities against four human
cancer cell lines. Here we report the isolation, structural elucidation,
and cytotoxicity of the isolated compounds.
In particular, NMR data suggested that 1 was the glycoside of ent-
7ꢀ-hydroxykaur-16-en-19-oic acid.16,17 The diterpene skeleton was
reconfirmed by the HMBC spectrum (Figure 1). In the H NMR
1
spectrum, the anomeric proton at δH 5.40 (1H, d, J ) 8.0 Hz)
suggested the presence of a ꢀ-glucopyranosyl moiety. The HMBC
correlation between H-1′ (δH 5.40, 1H, d, J ) 8.0 Hz) and C-19
(δC 178.7) and the NOESY correlation between H-1′ (δH 5.40, 1H,
d, J ) 8.0 Hz) and H3-20 (δH 0.97, 3H, s) indicated that the
ꢀ-glucopyranosyl moiety was located at C-19. Enzymatic hydrolysis
of 1 afforded aglycone 1a and D-glucose. The aglycone 1a was
identified by 1H NMR and MS data.16 The D-glucose was detected
by TLC and identified by the sign of its specific rotation value.
The configuration of 1 was the same as that of ent-7ꢀ-hydroxykaur-
16-en-19-oic acid,16,17 on the basis of its NMR data. The ꢀ-orienta-
tion of the OH group at C-7 was established by the NOESY
experiment (Figure 2), in which correlation of H-7 with H-6a and
H-14b was observed. Thus, the structure of 1 was determined to
be ꢀ-D-glucopyranosyl-ent-7ꢀ-hydroxykaur-16-en-19-oate. The ab-
solute configuration of 1 was determined by measuring the CD
spectrum of 1a. The CD spectrum of 1a showed a negative (λmax
212 nm) Cotton effect, which is similar to the negative (λmax 216
nm) Cotton effect of ent-kaur-16-en-19-oic acid,18 suggesting that
compound 1 possessed the ent-kaurane skeleton.
Pharboside B (2) was obtained as a colorless gum. The molecular
formula was determined to be C26H40O8 from the molecular ion
peak [M + H]+ at m/z 481.2836 (calcd for C26H41O8: 481.2801) in
the positive-ion HRFABMS. The IR, MS, and NMR data of
compound 2 indicated that it was similar in structure to compound
1. The only difference between 1 and 2 was the signal at C-7 in
the NMR data [δH 3.53 (br s)/δC 78.1 in 1; δH 3.43 (m)/δC 72.2 in
2], implying that compounds 1 and 2 are stereoisomers at C-7.17
This was confirmed by the NOESY correlations between H-7 and
H-5, H-9, and H-15. Enzymatic hydrolysis of 2 afforded aglycone
2a and D-glucose. The aglycone 2a was identified by 1H NMR and
MS data.19 Thus, we concluded that compound 2 is ꢀ-D-glucopy-
ranosyl ent-7R-hydroxykaur-16-en-19-oate. The CD spectrum of
2 showed a negative (λmax 202 nm) Cotton effect (λmax 202 nm for
1). Thus, compound 2 is an ent-kaurane diterpenoid.
Results and Discussion
Purification of the EtOH extract of dried seeds of P. nil by
repeated column chromatography led to the isolation of six new
ent-kaurane diterpene glycosides, pharbosides A-F (1-6), a new
ent-gibbane diterpene glycoside, pharboside G (7), and three
known ent-kaurane diterpenoids (8-10).
Pharboside A (1) was obtained as a colorless gum. The molecular
formula was determined to be C26H40O8 from the molecular ion
peak [M]+ at m/z 480.2706 (calcd for C26H40O8: 480.2723) in the
positive-ion HRFABMS. The IR spectrum indicated that 1 possesses
hydroxy (3380 cm-1), ester (1757 cm-1), and CdC double bond
(1664 cm-1) functional groups. In the 13C NMR (including DEPT)
spectrum, a total of 26 carbon signals, composed of two methyls,
eight methylenes, four methines, three quaternary carbons, one
exocyclic double bond, and one carbonyl carbon, including six
signals assignable to the glucose moiety (δC 95.8, 78.8, 78.7, 74.2,
71.2, 62.5),15 were observed. On the basis of the NMR data and
the spectroscopic pattern of diterpenes isolated from this source,14
compound 1 was predicted to be an ent-kaurane diterpene glycoside.
Pharboside C (3) was obtained as a colorless gum. The molecular
formula was determined to be C27H44O11 from the molecular ion
peak [M + H]+ at m/z 545.3001 (calcd for C27H45O11: 545.2962)
in the positive-ion HRFABMS. The IR spectrum indicated that 3
possesses hydroxy (3359 cm-1) and ester (1726 cm-1) functional
1
groups. The H and 13C NMR and DEPT spectra indicated the
* Corresponding author. Tel: +82-31-290-7710. Fax: +82-31-290-7730.
E-mail: krlee@skku.ac.kr.
presence of a glucopyranosyl moiety and three methyls, eight
methylenes, five methines, four quaternary carbons, and one
carbonyl carbon. Thus, on the basis of the NMR data, compound
† Sungkyunkwan University.
‡ Korea Research Institute of Chemical Technology.
10.1021/np900101t CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/12/2009

1122 Journal of Natural Products, 2009, Vol. 72, No. 6
Kim et al.
3 was also thought to be a diterpene glycoside. Comparison of the
1H and 13C NMR data of 3 with those of the known compound
6ꢀ,7ꢀ,16R,17-tetrahydroxy-ent-kauranoic acid (9) indicated that the
only differences between 3 and 9 were the presence of a methoxy
group at C-19 and glucose in 3. The anomeric proton at δH 4.48
(1H, d, J ) 7.5 Hz) in the 1H NMR spectrum suggested the presence
of a ꢀ-glucopyranosyl moiety, located at C-16 by the HMBC
correlation between H-1′ (δH 4.48) and C-16 (δC 87.5). The HMBC
correlation between the methoxy proton (δH 3.70, 3H, s) and C-19
implied that a methoxy group was present at C-19. In addition, the
cross-peaks observed in the HMBC spectrum (Figure 1) from H-6
(δH 4.25, 1H, dd, J ) 11.5, 2.5 Hz) to C-4, C-5, and C-8; from
H-7 (δH 3.36, 1H, d, J ) 2.5 Hz) to C-5, C-8, C-9, and C-15; and
from H-17 (δH 3.39 and 3.60, 1H each, d, J ) 12.0 Hz) to C-13,
C-15, and C-16 reconfirmed the proposed structure of 3. Enzymatic
hydrolysis of 3 afforded D-glucose and aglycone 3a (19-methyl ester
of 9). The aglycone 3a was identified as methyl-ent-6ꢀ,7ꢀ,16R,17-
tetrahydroxykauran-19-oate by NMR data analysis, including data
from 2D NMR (DEPT, HMBC) and HRFABMS. The relative
configuration of 3 was assigned on the basis of the J values in the
1H NMR spectrum and the NOESY experiment (Figure 2). The
coupling constants of H-6 (dd, J5,6 ) 11.5 Hz and J6,7 ) 2.5 Hz)
and H-7 (d, J6,7 ) 2.5 Hz) observed in the 1H NMR spectrum were
the same as those of H-6 (dd, J5,6 ) 11.5 Hz and J6,7 ) 2.5 Hz)
and H-7 (d, J6,7 ) 2.5 Hz) in 6ꢀ,7ꢀ-dihydroxykaurenoic acid.20
Thus, H-6 and H-7 were determined to be in the R-orientation,
which was reconfirmed by NOESY correlations between H-6 and
H-7, H-12b, and H3-20, and between H-7 and H-6, H-12b, and
H-14b. In addition, the NOESY correlation between H-17 and H-15
confirmed that the OH group at C-16 was R-oriented. Thus, 3 was
determined to be methyl-ent-6ꢀ,7ꢀ,16R,17-tetrahydroxykauran-19-
oate-16-O-ꢀ-D-glucopyranoside. The CD spectrum of ent-kaurane-
type diterpenoids shows negative Cotton effects at 206-230 nm.18
The CD spectrum of 3a showed a negative (λmax 225 nm) Cotton
effect, which was similar to the negative (λmax 226 nm) Cotton effect
of 9 corresponding to ent-kauran-19-oic acid. Thus, the skeleton
of 3 is an ent-kaurane diterpenoid.
ion HRFABMS. The IR spectrum indicated that 4 possesses
hydroxy (3360 cm-1) and ester (1725 cm-1) functional groups. The
13C NMR and DEPT spectrum displayed signals indicating the
presence of a hexose moiety and two methyls, eight methylenes,
five methines, four quaternary carbons, and one carbonyl carbon.
Comparison of the 1H and 13C NMR data of 4 with those of
6ꢀ,7ꢀ,16ꢀ,17-tetrahydroxy-ent-kauranoic acid (10) showed that the
major difference between these compounds is the presence of a
sugar moiety in 4. The chemical shifts of H-1′ (C-1′) at δH 5.46
(δC 95.9) of glucopyranose are characteristic of an ester-linked
sugar,15 and the HMBC correlation between H-1′ (δH 5.46, 1H, d,
J ) 7.5 Hz) and C-19 (δC 178.9) confirmed that the sugar moiety
is located at C-19. Enzymatic hydrolysis of 4 afforded D-glucose
1
and aglycone 4a, which was identified by comparison of the H
NMR and MS data with those of 10.21 The relative configuration
of 4 was assigned to be the same as those of 3 on the basis of the
NOESY experiment, except for the ꢀ-orientated OH group at C-16,
which was confirmed by the NOESY correlation between H-17 and
H-13. Thus, 4 was determined to be ꢀ-D-glucopyranosyl-ent-
6ꢀ,7ꢀ,16ꢀ,17-tetrahydroxykauran-19-oate.
Pharboside E (5) possessed the same molecular formula as 4,
C26H42O11, based on the molecular ion peak [M]+ at m/z 530.2765
(calcd for C26H42O11: 530.2727) in the HRFABMS. Detailed
analyses of its IR and 1D and 2D NMR data indicated that 5 is an
isomer of 4 that differs with respect to the position of a sugar
moiety. The signal due to C-19 at δC 178.9 in 4 was shifted
downfield to δC 185.8 in 5, and the signal due to C-17 at δC 70.7
in 4 was shifted downfield to δC 78.8 in 5. This information implied
that the glucopyranosyl moiety was at C-17 in 5, which was
confirmed by the HMBC correlation between H-1′ (δH 4.31, 1H,
d, J ) 8.0 Hz) and C-17 (δC 78.8). Enzymatic hydrolysis of 5
yielded D-glucose and aglycone 5a (same as 10).21 Thus, 5 was
determined to be ent-6ꢀ,7ꢀ,16ꢀ,17-tetrahydroxykauran-19-oic acid-
17-O-ꢀ-D-glucopyranoside.
The absolute configurations of 4 and 5 were assigned by
measurement of the CD spectrum of 4a. Its CD spectrum showed
a negative (λmax 226 nm) Cotton effect, similar to the CD spectrum
of the ent-kaurane diterpenoids that shows negative Cotton effects
at 206-230 nm.18
Pharboside D (4) was obtained as a colorless gum with the
molecular formula C26H42O11 based on the molecular ion peak
[M]+ at m/z 530.2712 (calcd for C26H42O11: 530.2727) by positive-

Diterpene Glycosides from Pharbitis nil
Journal of Natural Products, 2009, Vol. 72, No. 6 1123
Figure 1. Key HMBC correlations of compounds 1, 3, and 7.
Pharboside G (7) was obtained as a colorless gum with the
molecular formula C26H38O8, based on the molecular ion peak
[M + Na]+ at m/z 501.2496 (calcd for C26H38NaO8: 501.2464) in
the HRFABMS. The IR spectrum indicated that 7 possesses a
hydroxy group (3359 cm-1), an ester (1725 cm-1), and a CdC
double bond (1635 cm-1). In the 13C NMR (including DEPT)
spectrum, a total of 26 carbon signals, composed of two methyls,
seven methylenes, four methines, three quaternary carbons, one
exocyclic double bond, one carbonyl group, and one aldehyde group,
including six signals assignable to the glucose moiety (δC 96.0, 78.9,
78.8, 74.0, 71.2, 62.5),15 were observed. The NMR data were similar
to those of the related gibberellins.23 Besides the signals for the
glucopyranosyl group, NMR data suggested that the aglycone of 7 is
gibberellin A12-7-aldehyde,24,25 which was reconfirmed through
analysis of the HMBC spectrum (Figure 1). In addition, the HMBC
correlation between H-1′ (δH 5.38, 1H, d, J ) 8.0 Hz) and C-19 (δC
177.4) and the NOESY correlation between H-1′ (δH 5.38, 1H, d, J )
8.0 Hz) and H3-20 (δH 0.80, 3H, s) indicated that the ꢀ-glucopyranosyl
moiety is located at C-19. Enzymatic hydrolysis of 7 afforded D-glucose
24,25
1
and aglycone 7a, as identified by H NMR and MS data.
The
relative configuration of 7 was established by the NOESY experiment
(Figure 2). The NOESY correlations between H-6 and H-14b, H3-20
and between H-7 and H-5, H3-18 suggested that the aldehyde group
(C-7) at C-6 was in the ꢀ-orientation. Thus, 7 was determined to be
ꢀ-D-glucopyranosylgibberellin A12-7-aldehyde-19-oate. The absolute
configuration of 7 was determined by measuring the CD spectrum of
7a. The CD spectrum of 7a showed a negative (λmax 218 nm) Cotton
effect, which was identical to the negative Cotton effect of ent-
gibberellins and their methyl esters at 220-230 nm.26 The CD
spectrum of 7 showed a negative (λmax 208 nm) Cotton effect. This
information indicated that compound 7 possessed the ent-gibberellin
skeleton.
The known ent-kaurane diterpenoids 7ꢀ,16ꢀ,17-trihydroxy-ent-
kauran-6R,19-olide (8),27,28 6ꢀ,7ꢀ,16R,17-tetrahydroxy-ent-kaura-
noic acid (9),22 and 6ꢀ,7ꢀ,16ꢀ,17-tetrahydroxy-ent-kauranoic acid
(10)22 were identified by GC-MS and analysis of MS data. We
performed full NMR data assignments of 8-10 by 2D NMR data
(including DEPT, HMBC, and NOESY) because no published
spectroscopic data of 8-10 were found. The absolute configurations
of these compounds were confirmed by analysis of CD data.18
The cytotoxic activities of the isolated compounds (1-10) against
A549, SK-OV-3, SK-MEL-2, and HCT15 human tumor cell lines
were evaluated using the SRB assay in Vitro. Compounds 4 and 5
exhibited moderate cytotoxic activity against A549, SK-OV-3, SK-
MEL-2, and HCT15 cells (IC50 (4): 25.15, 28.73, 14.29, and 17.23
µM, and IC50 (5): 23.60, 13.77, 15.22, and 22.12 µM, respectively),
while the other compounds showed little cytotoxicity against the
tested cell lines (IC50 > 30 µM).
Figure 2. Key NOESY correlations of compounds 1, 3, and 7.
Pharboside F (6) had the molecular formula C26H42O11
(HRFABMS) from the molecular ion peak [M + H]+ at m/z
1
531.2845 (calcd for C26H43O11: 531.2805). The H and 13C NMR
data showed that 6 was related to 3, with the only major difference
being the disappearance of the 19-methyl group in 6. Enzymatic
hydrolysis of 6 afforded D-glucose and aglycone 6a. The structure
1
of 6a was identified by comparison of its H NMR and MS data
Experimental Section
with those of 9.22 Moreover, the cross-peaks in the NOESY
spectrum of 6 indicated that the corresponding substituents in 6
have the same orientations as those in 3. Thus, 6 was determined
to be ent-6ꢀ,7ꢀ,16R,17-tetrahydroxykauran-19-oic acid-16-O-ꢀ-D-
glucopyranoside. Finally, we determined that compound 6 possesses
the same absolute configuration as 3 on the basis of the CD
spectrum of 6, which showed a negative Cotton effect at 204 nm.
General Experimental Procedures. Optical rotations were measured
on a JASCO P-1020 polarimeter. CD spectra were measured on a
JASCO J-715 spectropolarimeter. IR spectra were recorded on a Bruker
IFS-66/S FT-IR spectrometer. NMR spectra were recorded on a Varian
UNITY INOVA 500 NMR spectrometer operating at 500 MHz (1H)
and 125 MHz (13C), respectively, with chemical shifts given in ppm
(δ) using TMS as an internal standard. FAB and HR-FAB mass spectra

1124 Journal of Natural Products, 2009, Vol. 72, No. 6
Kim et al.
Table 1. 1H NMR Data of Compounds 1-5 (methanol-d4, 500 MHz, δ in ppm, J in Hz)a
H
1
2
3
4
5
1a
1b
2a
2b
3a
3b
5
6a
6b
7
9
11a
11b
12a
12b
13
14a
14b
0.91, m
1.40, m
1.57, m
1.94, m
1.14, m
1.28, m
1.78, d (11.5)
1.91, m
2.20, m
3.53, br s
1.45, m
1.16, m
1.57, m
1.43, m
0.92, m
1.43, m
1.54, m
1.95, m
1.14, m
1.31, m
1.82, d (11.5)
1.94, m
2.12, m
3.43, m
1.52, m
1.15, m
1.58, m
1.43, m
1.57, m
2.64, br s
1.85, m
2.15, m
2.28, br s
0.92, m
1.78, m
1.48, m
2.16, m
1.82, m
1.99, m
0.92, m
1.77, m
1.46, m
2.13, m
1.84, m
1.97, m
0.88, m
1.82, m
1.47, m
2.13, m
1.82, m
1.99, m
1.81, d (11.5)
4.25, dd (2.5, 11.5)
1.83, d (11.5)
4.42, dd (2.0, 11.5)
1.84, d (11.5)
4.26, dd (2.0, 11.5)
3.36, d (2.5)
1.44, m
1.51, m
1.81, m
1.48, m
1.85, m
2.32, br s
1.49, m
3.42, d (2.0)
1.53, m
1.50, m
1.84, m
1.48, m
1.87, m
2.08, br s
1.48, m
3.39, d (2.0)
1.47, m
1.50, m
1.84, m
1.49, m
1.88, m
2.11, br s
1.50, m
1.58, m
2.60, br s
1.83, m
2.18, m
2.21, br s
1.84, m
1.80, m
1.84, m
15a
15b
17a
17b
18
20
2.02, d (12.5)
2.13, d (12.5)
3.39, d (12.0)
3.60, d (12.0)
1.43, s
1.89, d (12.5)
2.16, d (12.5)
3.40, d (11.0)
3.47, d (11.0)
1.48, s
1.90, d (12.5)
2.13, d (12.5)
3.50, d (11.0)
3.78, d (11.0)
1.37, s
4.75, s
4.78, s
1.20, s
0.97, s
4.78, s
4.86, s
1.50, s
1.01, s
0.89, s
1.01, s
1.07, s
OMe
Glc-1′
Glc-2′
Glc-3′
Glc-4′
Glc-5′
Glc-6′a
Glc-6′b
3.70, s
4.48, d (7.5)
3.35, m
3.37, m
3.39, m
3.26, m
3.70, dd (3.5, 11.5)
3.87, dd (1.5, 11.5)
5.40, d (8.0)
3.35, m
3.37, m
3.39, m
3.30, m
5.46, d (8.0)
3.35, m
3.40, m
3.42, m
3.30, m
5.46, d (7.5)
3.35, m
3.40, m
3.42, m
3.30, m
4.31, d (8.0)
3.35, m
3.37, m
3.39, m
3.25, m
3.70, dd (3.5, 11.5)
3.83, br d (11.5)
3.72, dd (3.5, 11.5)
3.83, dd (1.5, 11.5)
3.72, dd (3.5, 11.5)
3.84, dd (1.5, 11.5)
3.69, dd (3.5, 11.5)
3.89, dd (1.5, 11.5)
a
The assignments were based on DEPT, H,1H-COSY, and HMBC experiments.
1
were obtained on a JEOL JMS700 mass spectrometer. ESI mass spectra
were obtained on a VG BIOTECH platform LC-mass spectrometer.
Preparative HPLC was performed using a Gilson 306 pump with a
Shodex refractive index detector and an Apollo Silica 5 µm column
(250 × 22 mm) or Econosil RP-18 10 µm column (250 × 22 mm).
Silica gel 60 (Merck Co., Germany, 70-230 mesh and 230-400 mesh)
was used for column chromatography. TLC was performed using Merck
precoated silica gel F254 plates and RP-18 F254s plates. The packing
material for molecular sieve column chromatography was Sephadex
LH-20 (Pharmacia Co., Sweden). Low-pressure liquid chromatography
was performed over a LiChroprep Lobar-A RP-18 (240 × 10 mm)
column with a FMI QSY-0 pump (ISCO).
Plant Material. The seeds of P. nil were purchased at Kyungdong
herbal market, Seoul, Korea, in July 2006, and were identified by one
of the authors (K.R.L.). A voucher specimen (SKKU 2006-7) was
deposited in the herbarium of the College of Pharmacy, Sungkyunkwan
University, Suwon, Korea.
Extraction and Isolation. The dried seeds (10 kg) were extracted
with 50% EtOH (3 × 4 L, each 3 days) at room temperature and filtered.
The filtrate was evaporated in Vacuo to obtain EtOH extracts (1.4 kg),
which were suspended in distilled H2O (7.2 L) and then successively
partitioned with n-hexane, CHCl3, EtOAc, and n-BuOH, yielding 10,
7, 10, and 550 g, respectively. The EtOAc-soluble fraction (10 g) was
chromatographed on a silica gel (230-400 mesh, 300 g) column and
eluted with CHCl3-MeOH (10:1 f 1:1, gradient system) to yield five
fractions (A-E). Fraction C (2.7 g) was chromatographed further on
an RP-C18 silica gel (230-400 mesh, 150 g) column and eluted with
MeOH-H2O (3:2 f 1:0, gradient system) to give eight subfractions
(C1-C8). Fraction C7 (1.5 g) was subjected to repeated column
chromatography (including silica gel column, Sephadex LH-20) and
purified by preparative reversed-phase HPLC using a solvent system
of 60% MeCN to yield 7 (6 mg). Compounds 8 (6 mg) and 9 (5 mg)
were obtained from fraction D (2.5 g) by separation of preparative
reversed-phase HPLC, using a solvent system of 30% MeCN. Fraction
E (3.0 g) was chromatographed further on an RP-C18 silica gel
(230-400 mesh, 150 g) column and eluted with MeOH-H2O (1:1 f
1:0, gradient system) to give 11 subfractions (E1-E11). Fractions E5
(80 mg), E10 (100 mg), and E11 (850 mg) were subjected to repeated
column chromatography (including silica gel column, Sephadex LH-
20, Lobar-A RP-18 column) and further purified by preparative
reversed-phase HPLC (MeCN-H2O, 2:3 or 1:4) to yield compounds
1 (7 mg) and 2 (4 mg) from fraction E10, 3 (50 mg) from fraction
E11, and 10 (30 mg) from fraction E5. The BuOH-soluble fraction
(50 g) was chromatographed on a DIAION HP-20 resin column using
a gradient solvent system of 100% distilled H2O and 100% MeOH to
yield four fractions (F-I). Fraction G (7.0 g) was subjected to a
silica gel (230-400 mesh, 150 g) column and eluted with
CHCl3-MeOH-H2O (7:3:0.5) to give three subfractions (G1-G3).
Fraction G2 (1.5 g) was chromatographed further on an RP-C18 silica
gel (230-400 mesh, 50 g) column, eluted with MeOH-H2O (1:1),
and further purified by preparative reversed-phase HPLC, using a
solvent system of 30% MeOH to obtain 4 (30 mg), 5 (4 mg), and 6 (7
mg).
Pharboside A (1): colorless gum; [R]25 -22.6 (c 0.18, MeOH);
D
CD (MeOH) λmax (∆ꢀ) 202 (-11.8) nm; IR (KBr) νmax 3380, 2947,
1
2835, 1757, 1664, 1451, 1030, 699 cm-1; H NMR, see Table 1; 13C
NMR, see Table 3; positive ESIMS m/z 983 [2 M + Na]+; positive
HRFABMS m/z 480.2706 [M]+ (calcd for C26H40O8, 480.2723).
Pharboside B (2): colorless gum; [R]25D +5.1 (c 0.10, MeOH); CD
(MeOH) λmax (∆ꢀ) 202 (-24.9) nm; IR (KBr) νmax 3382, 2946, 2835,
1
1725, 1665, 1454, 1031, 699 cm-1; H NMR, see Table 1; 13C NMR,
see Table 3; positive FABMS m/z 481 [M + H]+; positive HRFABMS
m/z 481.2836 [M + H]+ (calcd for C26H41O8, 481.2801).
Pharboside C (3): colorless gum; [R]25 -36.3 (c 0.25, MeOH);
D
CD (MeOH) λmax (∆ꢀ) 202 (-19.4) nm; IR (KBr) νmax 3359, 2946,
1
2834, 1726, 1451, 1030, 699 cm-1; H NMR, see Table 1; 13C NMR,
see Table 3; positive ESIMS m/z 1111 [2 M + Na]+; positive
HRFABMS m/z 545.3001 [M + H]+ (calcd for C27H45O11, 545.2962).
Pharboside D (4): colorless gum; [R]25 -104.5 (c 0.84, MeOH);
D
CD (MeOH) λmax (∆ꢀ) 223 (-40.6) nm; IR (KBr) νmax 3360, 2946,
1
2835, 1725, 1451, 1030, 699 cm-1; H NMR, see Table 1; 13C NMR,
see Table 3; positive FABMS m/z 530 [M]+; positive HRFABMS m/z
530.2712 [M]+ (calcd for C26H42O11, 530.2727).
Pharboside E (5): colorless gum; [R]25 -10.5 (c 0.05, MeOH);
D
CD (MeOH) λmax (∆ꢀ) 222 (-7.95) nm; IR (KBr) νmax 3358, 2946,
1
2834, 1451, 1030, 699 cm-1; H NMR, see Table 1; 13C NMR, see
Table 3; positive FABMS m/z 530 [M]+; positive HRFABMS m/z
530.2765 [M]+ (calcd for C26H42O11, 530.2727).

Diterpene Glycosides from Pharbitis nil
Journal of Natural Products, 2009, Vol. 72, No. 6 1125
Table 2. 1H NMR Data of Compounds 6-10 (methanol-d4, 500 MHz, δ in ppm, J in Hz)a
H
6
7
8
9
10
1a
1b
2a
2b
3a
3b
5
6
7
9
0.89, m
1.83, m
1.48, m
2.14, m
1.82, m
1.99, m
1.84, d (11.5)
4.23, dd (2.0, 11.5)
3.39, d (2.0)
1.48, m
0.90, m
1.42, m
1.40, m
1.74, m
1.36, m
2.07, m
2.05, d (12.0)
3.38, dd (5.5, 12.0)
9.78, d (5.5)
1.52, m
1.08, m
1.60, m
1.52, m
1.57, m
1.41, m
2.02, m
1.87, d (6.5)
4.57, t (6.5)
4.17, d (6.5)
1.55, m
0.90, m
1.83, m
1.42, m
2.09, m
1.82, m
1.99, m
1.78, d (12.5)
4.26, dd (2.0, 11.5)
3.36, d (2.0)
1.47, m
0.86, m
1.69, m
1.41, m
2.12, m
1.81, m
1.97, m
1.84, d (11.5)
4.32, dd (2.0, 11.5)
3.47, d (2.0)
1.48, m
11a
1.51, m
1.40, m
1.53, m
1.51, m
1.53, m
11b
1.82, m
1.72, m
1.70, m
1.84, m
1.82, m
12a
1.48, m
1.41, m
1.41, m
1.49, m
1.45, m
12b
1.88, m
1.53, m
1.59, m
1.88, m
1.92, m
13
14a
2.30, br s
1.49, m
2.50, t (1.5)
1.87, m
2.00, m
1.02, m
2.05, m
1.46, m
2.10, m
1.49, m
14b
1.81, m
2.16, m
1.54, m
1.78, m
1.85, m
15a
15b
17a
17b
18
20
1.95, d (12.5)
2.14, d (12.5)
3.39, d (12.0)
3.59, d (12.0)
1.35, s
1.52, d (12.5)
2.13, d (12.5)
4.85, s
4.93, s
1.16, s
1.60, d (12.5)
2.11, d (12.5)
3.39, d (11.0)
3.44, d (11.0)
1.27, s
1.88, d (12.5)
2.09, d (12.5)
3.39, d (11.0)
3.43, d (11.0)
1.45, s
1.92, d (12.5)
2.12, d (12.5)
3.64, d (11.0)
1.41, s
1.00, s
1.08, s
0.80, s
0.84, s
1.02, s
Glc-1′
Glc-2′
Glc-3′
Glc-4′
Glc-5′
Glc-6′a
Glc-6′b
4.50, d (8.0)
3.35, m
3.37, m
3.39, m
3.25, m
5.38, d (8.0)
3.35, m
3.37, m
3.39, m
3.25, m
3.70, dd (3.5, 11.5)
3.87, dd (1.5, 11.5)
3.69, dd (3.5, 11.5)
3.80, dd (1.5, 11.5)
a
The assignments were based on DEPT, H,1H-COSY, and HMBC experiments.
1
Table 3. 13C NMR Data of Compounds 1-10 (methanol-d4, 125 MHz, δ in ppm)
carbon
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
OMe
Glc-1′
Glc-2′
Glc-3′
Glc-4′
Glc-5′
Glc-6′
41.8 t
20.3 t
39.2 t
44.8 s
50.5 d
30.3 t
78.1 d
49.5 s
48.8 d
40.6 s
19.2 t
34.7 t
45.3 d
39.9 t
46.7 t
157.0 s
103.8 t
28.9 q
178.7 s
16.4 q
41.9 t
20.1 t
40.9 t
44.9 s
53.3 d
34.6 t
72.2 d
49.9 s
54.7 d
41.5 s
19.3 t
34.0 t
45.1 d
30.0 t
46.8 t
156.3 s
104.0 t
32.6 q
179.0 s
17.6 q
41.8 t
19.0 t
41.0 t
45.6 s
52.0 d
72.2 d
83.3 d
49.7 s
50.7 d
42.0 s
20.2 t
27.7 t
41.5 d
37.5 t
46.0 t
87.5 s
66.9 t
32.7 q
180.4 s
17.2 q
52.8 q
100.0 d
75.7 d
78.5 d
71.6 d
78.1 d
62.9 t
42.0 t
19.7 t
40.6 t
45.7 s
50.6 d
72.0 d
83.4 d
49.6 s
53.2 d
41.8 s
20.1 t
28.3 t
42.0 d
37.7 t
49.8 t
80.6 s
70.7 t
32.5 q
178.9 s
17.4 q
42.0 t
19.2 t
41.2 t
45.5 s
52.4 d
73.4 d
82.6 d
49.7 s
51.7 d
42.3 s
19.9 t
28.0 t
41.8 d
38.7 t
47.2 t
80.2 s
78.8 t
34.4 q
185.8 s
17.2 q
42.0 t
18.8 t
41.5 t
45.8 s
52.9 d
73.5 d
82.6 d
49.6 s
51.8 d
42.4 s
20.6 t
27.8 t
41.6 d
38.5 t
46.1 t
87.7 s
66.9 t
35.0 q
185.5 s
17.1 q
41.1 t
20.9 t
38.1 t
45.6 s
58.4 d
59.2 d
208.9 d
51.5 s
59.5 d
46.2 s
18.3 t
33.3 t
39.6 d
38.6 t
44.4 t
158.7 s
106.9 t
30.2 q
177.4 s
16.0 q
38.6 t
17.9 t
29.4 t
43.2 s
52.9 d
85.4 d
72.5 d
46.9 s
57.4 d
35.4 s
18.5 t
22.0 t
38.8 d
34.7 t
45.9 t
83.0 s
71.2 t
26.0 q
185.2 s
21.1 q
41.9 t
19.9 t
40.7 t
45.3 s
51.2 d
72.9 d
83.2 d
49.6 s
52.6 d
42.0 s
20.3 t
27.5 t
41.9 d
38.3 t
49.6 t
79.3 s
64.1 t
33.3 q
182.6 s
17.3 q
41.9 t
19.5 t
40.9 t
45.1 s
50.9 d
72.4 d
83.0 d
49.6 s
52.5 d
41.9 s
20.1 t
28.2 t
41.9 d
38.1 t
49.6 t
80.5 s
70.6 t
33.1 q
182.3 s
17.1 q
95.8 d
74.2 d
78.8 d
71.2 d
78.8 d
62.5 t
95.9 d
74.0 d
78.7 d
71.1 d
78.8 d
62.4 t
95.9 d
74.0 d
78.7 d
71.1 d
78.8 d
62.3 t
105.1 d
75.3 d
78.0 d
71.7 d
78.1 d
62.8 t
100.0 d
75.8 d
78.5 d
71.6 d
78.1 d
63.0 t
96.0 d
74.0 d
78.8 d
71.2 d
78.9 d
62.5 t
Pharboside F (6): colorless gum; [R]25 -59.3 (c 0.21, MeOH);
7ꢀ,16ꢀ,17-Trihydroxy-ent-kauran-6r,19-olide (8): colorless gum;
[R]25D +1.9 (c 0.27, MeOH); CD (MeOH) λmax (∆ꢀ) 201 (-16.4), 208
(+24.1), 219 (+12.9) nm; IR (KBr) νmax 3381, 2949, 1757, 1664, 1451,
1029, 699 cm-1; 1H NMR, see Table 2; 13C NMR, see Table 3; positive
FABMS m/z 351 [M + H]+; positive HRFABMS m/z 351.2198
[M + H]+ (calcd for C20H31O5, 351.2172).
D
CD (MeOH) λmax (∆ꢀ) 204 (-18.0) nm; IR (KBr) νmax 3382, 2947,
1
2834, 1453, 1030, 699 cm-1; H NMR, see Table 2; 13C NMR, see
Table 3; positive FABMS m/z 531 [M + H]+; positive HRFABMS
m/z 531.2845 [M + H]+ (calcd for C26H43O11, 531.2805).
Pharboside G (7): colorless gum; [R]25 -33.5 (c 0.14, MeOH);
D
CD (MeOH) λmax (∆ꢀ) 208 (-26.7) nm; IR (KBr) νmax 3359, 2946,
6ꢀ,7ꢀ,16r,17-Tetrahydroxy-ent-kauranoic acid (9): colorless gum;
1
2834, 1725, 1635, 1451, 1020, 699 cm-1; H NMR, see Table 2; 13C
[R]25 -167.6 (c 0.80, MeOH); CD (MeOH) λmax (∆ꢀ) 226 (-30.4)
D
NMR, see Table 3; positive FABMS m/z 501 [M + Na]+; positive
HRFABMS m/z 501.2496 [M + Na]+ (calcd for C26H38NaO8, 501.2464).
nm; IR (KBr) νmax 3379, 2947, 2834, 1664, 1452, 1029, 669 cm-1; 1H
NMR, see Table 2; 13C NMR, see Table 3; positive FABMS m/z 391

1126 Journal of Natural Products, 2009, Vol. 72, No. 6
Kim et al.
[M + Na]+; positive HRFABMS m/z 391.2122 [M + Na]+ (calcd for
C20H32NaO6, 391.2097).
) 12.5 Hz, H-15b), 1.27 (3H, s, H-18), 0.80 (3H, s, H-20); positive
FABMS m/z 339 [M + Na]+.
The aqueous phase of the hydrolysates of 1-7 were subjected
separately to column chromatography over silica gel eluted with
MeCN-H2O (8:1) to yield glucose with positive specific rotation. The
specific rotations of the sugar obtained from 1-7 ranged from +42.5
to +49.7; [R]25D +42.5 (c 0.05, H2O) of 1, [R]25D +44.6 (c 0.02, H2O)
of 2, [R]25 +48.6 (c 0.20, H2O) of 3, [R]25 +47.8 (c 0.20, H2O) of
6ꢀ,7ꢀ,16ꢀ,17-Tetrahydroxy-ent-kauranoic acid (10): colorless
gum; [R]25D -70.8 (c 0.25, MeOH); CD (MeOH) λmax (∆ꢀ) 226 (-23.9)
nm; IR (KBr) νmax 3382, 2945, 2335, 1662, 1026, 699 cm-1; 1H NMR,
see Table 2; 13C NMR, see Table 3; positive FABMS m/z 369
[M + H]+; positive HRFABMS m/z 369.2278 [M + H]+ (calcd for
C20H33O6, 369.2277).
D
D
4, [R]25 +49.7 (c 0.02, H2O) of 5, [R]25 +46.1 (c 0.05, H2O) of 6,
Enzymatic Hydrolysis of 1-7. A solution of each sample in H2O
(3 mL) was individually hydrolyzed with crude hesperidinase (20 mg,
from Aspergillus niger, Sigma-Aldrich) at 37 °C for 72 h. Each reaction
mixture was extracted with EtOAc (3 × 5 mL) to yield the individual
EtOAc extract and H2O phase after removing the solvents. The
combined EtOAc layers from 1 (3 mg), 2 (1 mg), and 7 (2 mg) were
chromatographed separately over silica gel Waters Sep-Pak Vac 6 cc
(CHCl3-MeOH, 30:1) to give aglycones 1a (1 mg), 2a (0.5 mg), and
7a (1 mg), respectively. The combined EtOAc layers from 3 (10 mg),
4 (10 mg), 5 (1 mg), and 6 (2 mg) were chromatographed separately
over silica gel Waters Sep-Pak Vac 6 CC (CHCl3-MeOH, 5:1) to give
aglycones 3a (4 mg), 4a (5 mg), 5a (0.4 mg), and 6a (1 mg),
respectively. The aglycone of each compound was identified by 1H
NMR and MS data. In particular, aglycone 3a was identified by NMR
data analysis, including 2D NMR data (DEPT, HMBC), because no
spectroscopic data of 3a had been reported in the literature.
1a: colorless gum; [R]25D -15.0 (c 0.05, MeOH); CD (MeOH) λmax
D
D
25
and [R]D +45.6 (c 0.05, H2O) of 7. MeCN-H2O (6:1) was used as
the solvent system for TLC identification of glucose (Rf, 0.33).29,30
In Vitro Cytotoxicity Test. A sulforhodamin B bioassay (SRB) was
used to determine the cytotoxicity of each compound against four
cultured human cancer cell lines.31 The assays were performed at the
Korea Research Institute of Chemical Technology. The cell lines used
were A549 (non-small cell lung carcinoma), SK-OV-3 (ovary malignant
ascites), SK-MEL-2 (skin melanoma), and HCT (colon adenocarci-
noma). Doxorubicin was used as a positive control. The cytotoxicities
of doxorubicin against A549, SK-OV-3, SK-MEL-2, and HCT cell lines
were IC50 0.007, 0.056, 0.117, and 0.164 µM, respectively.
Acknowledgment. This work was supported by grant no. PF 2-1 of
the Plant Diversity Research Center from the Ministry of Science &
Technology in Korea. We thank Drs. E. J. Bang, S. G. Kim, and J. J.
Seo at the Korea Basic Science Institute for their aid in the NMR and
MS spectra measurements.
1
(∆ꢀ) 212 (-19.8) nm; H NMR (CDCl3, 500 MHz) δ 4.85 (1H, br s,
H-17a), 4.82 (1H, br s, H-17b), 3.65 (1H, t, J ) 3.0 Hz, H-7), 2.70
(1H, br s, H-13), 2.26 (2H, br s, H-15), 1.80 (1H, d, J ) 11.5 Hz,
H-5), 1.26 (3H, s, H-18), 0.98 (3H, s, H-20); positive FABMS m/z
Supporting Information Available: 1D and 2D NMR data of 1
and 3, 1D NMR data of 2 and 4-6, and 1D, 2D NMR and MS data of
7. This material is available free of charge via the Internet at http://
pubs.acs.org.
319 [M + H]+.
1
2a: colorless gum; [R]25 +2.2 (c 0.02, MeOH); H NMR (CDCl3,
D
500 MHz) δ 4.90 (1H, br s, H-17a), 4.85 (1H, br s, H-17b), 3.63 (1H,
m, H-7), 2.75 (1H, br s, H-13), 2.21 (2H, br s, H-15), 1.79 (1H, d,
J ) 11.5 Hz, H-5), 1.27 (3H, s, H-18), 0.86 (3H, s, H-20); positive
FABMS m/z 319 [M + H]+.
References and Notes
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D
1
(-43.4) nm; H NMR (CDCl3, 500 MHz) δ 4.25 (1H, dd, J ) 2.0,
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3.47 (1H, d, J ) 1.5 Hz, H-7), 3.46 (1H, d, J ) 11.5 Hz, H-17b), 2.15
(1H, d, J ) 12.5 Hz, H-15a), 2.14 (1H, br s, H-13), 2.10 (1H, m, H-2a),
1.92 (1H, m, H-15b), 1.90 (1H, m, H-3a), 1.84 (1H, m, H-3b), 1.83
(1H, d, J ) 11.5 Hz, H-5), 1.82 (1H, m, H-12a), 1.81 (1H, m, H-11a),
1.78 (1H, m, H-1a), 1.77 (1H, m, H-14a), 1.54 (1H, m, H-2b), 1.50
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13C NMR (CD3OD, 125 MHz) δ 180.4 (C-19), 82.3 (C-7), 79.2 (C-
16), 70.9 (C-6), 65.0 (C-17), 53.1 (OCH3-19), 52.1 (C-9), 50.6 (C-5),
49.1 (C-15), 48.4 (C-8), 45.8 (C-4), 41.9 (C-10), 41.8 (C-1), 40.6 (C-
13), 39.7 (C-3), 36.7 (C-14), 31.4 (C-18), 29.5 (C-12), 20.1 (C-11),
18.9 (C-2), 18.3 (C-20); HMBC correlations for H-5 of C-4, C-6, C-7,
and C-19, for H-7 of C-5, C-6, C-9, and C-15, for H-17 of C-13 and
C-15, for H-18 of C-3 and C-19, for H-20 of C-1, C-5, and C-9, for
19-OCH3 of C-19; positive FABMS m/z 383 [M + H]+; positive
HRFABMS m/z 383.2412 [M + H]+ (calcd for C21H35O6, 383.2434).
4a, 5a: colorless gum; [R]25D -134.2 (c 0.25, MeOH); CD (MeOH)
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λ
max (∆ꢀ) 226 (-12.7) nm; 1H NMR (CD3OD, 500 MHz) δ 4.28 (1H,
dd, J ) 1.5, 11.5 Hz, H-6), 3.44 (1H, d, J ) 11.5 Hz, H-17a), 3.38
(1H, d, J ) 11.5 Hz, H-17b), 3.32 (1H, d, J ) 1.5 Hz, H-7), 2.12 (1H,
d, J ) 12.5 Hz, H-15a), 2.09 (1H, br s, H-13), 1.93 (1H, d, J ) 12.5
Hz, H-15b), 1.78 (1H, d, J ) 11.5 Hz, H-5), 1.45 (3H, s, H-18), 1.02
(3H, s, H-20); positive FABMS m/z 391 [M + Na]+.
6a: colorless gum; [R]25D -34.2 (c 0.05, MeOH); 1H NMR (CDCl3,
500 MHz) δ 4.26 (1H, dd, J ) 1.5, 11.5 Hz, H-6), 3.44 (1H, d, J )
11.5 Hz, H-17a), 3.38 (1H, d, J ) 11.5 Hz, H-17b), 3.33 (1H, d, J )
1.5 Hz, H-7), 2.18 (1H, d, J ) 12.5 Hz, H-15a), 2.13 (1H, br s, H-13),
1.98 (1H, d, J ) 12.5 Hz, H-15b), 1.79 (1H, d, J ) 11.5 Hz, H-5),
1.35 (3H, s, H-18), 0.91 (3H, s, H-20); positive FABMS m/z 369
[M + H]+.
7a: colorless gum; [R]25D -45.0 (c 0.05, MeOH); CD (MeOH) λmax
(∆ꢀ) 218 (-47.3) nm; 1H NMR (CDCl3, 500 MHz) δ 9.79 (1H, d, J )
5.5 Hz, H-7), 4.96 (1H, s, H-17a), 4.85 (1H, s, H-17b), 3.25 (1H, dd,
J ) 5.5, 12.0 Hz, H-6), 2.56 (1H, t, J ) 1.5 Hz, H-13), 2.18 (1H, d,
J ) 12.5 Hz, H-15a), 1.98 (1H, d, J ) 11.5 Hz, H-5), 1.45 (1H, d, J
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783.
(23) Yamaguchi, I.; Takahashi, N. J. Chem. Soc., Perkin Trans. 1 1975,
992–996.

Diterpene Glycosides from Pharbitis nil
Journal of Natural Products, 2009, Vol. 72, No. 6 1127
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