Isolation, structure elucidation, and biological evaluation of 16,23-epoxycucurbitacin constituents from eleaocarpus Chinensis

Article abstract of DOI:10.1021/np200879p

Eight new 16,23-epoxycucurbitacin derivatives, designated as elaeocarpucins A-H (1-8), and five known cucurbitacins (9-13) were isolated from the chloroform-soluble partitions of separate methanol extracts of the fruits and stem bark of Elaeocarpus chinensis collected in Vietnam. Isolation work was facilitated using a LC/MS dereplication procedure, and bioassay-guided fractionation was monitored using HT-29 human cancer cells. The structures of compounds 1-8 were determined on the basis of spectroscopic data interpretation, with the absolute configurations of isomers 1 and 2 established by the Mosher ester method. Compounds 1-13 were evaluated in vitro against the HT-29 cell line and using a mitochondrial transmembrane potential assay. Elaeocarpucin C (3), produced by partial synthesis from 16α,23α-epoxy-3β,20β- dihydroxy-10αH,23βH-cucurbit-5,24-dien-11-one (13), was found to be inactive when evaluated in an in vivo hollow fiber assay using three different cancer cell types (dose range 0.5-10 mg/kg/day, ip).

Full text of DOI:10.1021/np200879p

Article
pubs.acs.org/jnp
Isolation, Structure Elucidation, and Biological Evaluation of 16,23-
Epoxycucurbitacin Constituents from Eleaocarpus chinensis
Li Pan,† Yeonjoong Yong,‡ Ye Deng,† Daniel D. Lantvit,§ Tran Ngoc Ninh,⊥ Heebyung Chai,†
Esperanza J. Carcache de Blanco,†,‡ Djaja D. Soejarto,§,∥ Steven M. Swanson,§ and A. Douglas Kinghorn*,†
†Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
‡Division of Pharmacy Practice and Administration, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United
States
§Program for Collaborative Research in the Pharmaceutical Science and Department of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States
⊥Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hoang Quoc Viet, Cau Giay,
Hanoi, Vietnam
∥Department of Botany, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, Illinois 60605, United States
S
* Supporting Information
ABSTRACT: Eight new 16,23-epoxycucurbitacin derivatives,
designated as elaeocarpucins A−H (1−8), and five known
cucurbitacins (9−13) were isolated from the chloroform-
soluble partitions of separate methanol extracts of the fruits
and stem bark of Elaeocarpus chinensis collected in Vietnam.
Isolation work was facilitated using a LC/MS dereplication
procedure, and bioassay-guided fractionation was monitored
using HT-29 human cancer cells. The structures of
compounds 1−8 were determined on the basis of spectro-
scopic data interpretation, with the absolute configurations of isomers 1 and 2 established by the Mosher ester method.
Compounds 1−13 were evaluated in vitro against the HT-29 cell line and using a mitochondrial transmembrane potential assay.
Elaeocarpucin C (3), produced by partial synthesis from 16α,23α-epoxy-3β,20β-dihydroxy-10αH,23βH-cucurbit-5,24-dien-11-
one (13), was found to be inactive when evaluated in an in vivo hollow fiber assay using three different cancer cell types (dose
range 0.5−10 mg/kg/day, ip).
Elaeocarpus chinensis (Gardner & Champ.) Hook.f. ex Benth.
(syn.: Friesia chinensis Gardner & Champ.), an evergreen tree of
the family Elaeocarpaceae, is distributed mainly in subtropical
or tropical areas of Asia, including southern mainland China,
Laos, and Vietnam.1 Besides being grown for ornamental
purposes, E. chinensis is used also as a traditional Chinese herbal
medicine for the treatment of emmeniopathy as well as
extravasated blood and inflammatory edema caused by
traumatic injury.2 Elaeocarpus is a large genus comprising
350−360 species distributed from Madagascar to Oceania, with
the highest concentration occurring in Borneo and Papua New
Guinea.3,4 Previous phytochemical work has resulted in the
isolation of anthocyanins,5 cucurbitacin-type triterpenoids,6−12
flavonoids,13,14 other phenolic derivatives,13 and indolizidine
alkaloids.15−17 Among these compounds, cucurbitacins and
their derivatives are tetracyclic triterpenoids obtained initially
from plants of the family Cucurbitaceae and are reported to
have anticancer, antifertility, anti-inflammatory, cytotoxic, and
purgative activities.18,19 Although the development of cucurbi-
tacins as anticancer drug candidates has been hindered by their
nonspecific cytotoxicity, there is much interest in the
relationship of structure to cytotoxicity within this compound
class.18,19 Some cucurbitacins have been found to affect JAK-
STAP and MAPK signaling pathways in cancer cells and to
show synergistic effects in combination with certain known
anticancer therapeutic agents, such as doxorubicin and
gemcitabine.20,21 Thus far, there have been no studies on the
phytochemical constituents of E. chinensis.
As part of our ongoing program to discover new anticancer
agents from varied natural sources,22,23 a CHCl3 extract of the
fruits of E. chinensis was found to exhibit cytotoxic activity (IC50
0.4 μg/mL) against human colon cancer (HT-29) cells.
Scrutiny of the NAPRALERTSM (Natural Products Alert)
database24 indicated that more than 150 compounds have been
isolated from the genus Elaeocarpus, with most of the cytotoxic
compounds known being cucurbitacin-type triterpenes. In
order to decide whether or not to further pursue this lead,
Special Issue: Special Issue in Honor of Gordon M. Cragg
Received: November 1, 2011
Published: January 12, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Chart 1
the CHCl3-soluble extract of E. chinensis fruits was subjected to
an LC-MS dereplication procedure, which revealed the
probable presence of the known cytotoxic cucurbitacins
cucurbitacin D (9), 3-epi-isocucurbitacin D (10), and 25-O-
acetylcucurbitacin F (11). In addition, certain unknown
cytotoxic compounds corresponding to possible molecular
formulas of C30H44O5 and C30H46O5 were evident. Accordingly,
subsequent bioassay-guided fractionation was conducted using
HT-29 cancer cells to monitor purification and led to the
isolation of six new 16,23-epoxycucurbitacins, elaeocarpucins
A−F (1−6), together with five known cucubitacins inclusive of
cucurbitacin D (9),25,26 3-epi-isocucurbitacin D (10),26 25-O-
acetylcucurbitacin F (11),9,27 cucurbitacin I (12),28 and
16α,23α-epoxy-3β,20β-dihydroxy-10αH,23βH-cucurbit-5,24-
dien-11-one (13).11 Moreover, from the less potently cytotoxic
CHCl3 extract of the stem bark of the same plant, two
additional new cucurbitacins, elaeocarpucins G (7) and H (8),
were purified. Herein, we report the isolation and structure
elucidation of the eight new compounds, 1−8, as well as the
biological assessment of all isolates obtained in this
investigation.
proton signals attached to four oxygenated methine carbons. In
addition, three olefinic protons were recognized in the H
1
NMR spectrum at δH 4.91 (1H, s, H-27a), 5.04 (1H, s, H-27b),
and 5.67 (1H, d, J = 5.8 Hz, H-6). The 13C NMR spectrum of 1
showed 30 carbon signals, which were classified from DEPT
and HSQC data as seven methyls, six methylenes, three
methines, four quaternary carbons, five oxygen-bearing carbons
(including four secondary and one tertiary), a trisubstituted
double bond, a disubstituted terminal double bond, and a
carbonyl group. The characteristic NMR data of compound 1
were comparable to those of 16α,23α-epoxy-3β,20β-dihydroxy-
10αH,23βH-cucurbit-5,24-dien-11-one (13), a known 16α,23α-
epoxycucurbitane analogue first isolated from Eleaocarpus
hainanensis11 that was also identified in the present
investigation. Comparison of the 1D- and 2D-NMR data of 1
with those of 13 revealed a major change evident in the side
chain located at C-23, with a 2-methylprop-1-ene group in 13
being replaced by a 2-methylprop-2-en-1-ol moiety in 1. The
signals of the latter unit occurred at δH 4.24 (1H, d, J = 2.8 Hz,
H-24), 4.91 (1H, s, Ha-27), 5.04 (1H, s, Hb-27), and 1.72 (3H,
s, H-26) in the 1H NMR spectrum as well as at δC 75.8 (CH, C-
24), 142.7 (C, C-25), 19.5 (CH3, C-26), and 111.7 (CH2, C-
27) in the 13C NMR spectrum. Moreover, key HMBC
correlations from the terminal olefinic methylene protons of
H-27 to C-24, C-25, and C-26, as well as H-24 and H-26 to C-
27, supported the structure assigned for the side-chain moiety.
Thus, the planar structure of 1 could be proposed.
RESULTS AND DISCUSSION
■
Compound 1 was obtained as a white powder. Its molecular
formula was assigned as C30H46O5 on the basis of the [M +
Na]+ ion peak at m/z 509.3225 (calcd 509.3243) in the
HRESIMS. Observed in the 1H NMR spectrum were signals for
seven tertiary methyl groups at δH 0.93 (3H, s, H-18), 1.02
(3H, s, H-28), 1.14 (3H, s, H-19), 1.17 (3H, s, H-29), 1.22
(3H, s, H-30), 1.32 (3H, s, H-21), and 1.72 (3H, s, H-26),
while resonances at δH 3.48 (1H, brs, H-3), 4.40 (1H, ddd, J =
10.4, 10.4, 3.6 Hz, H-16), 4.01 (1H, ddd, J = 12.0, 3.0, 3.0 Hz,
H-23), and 4.24 (1H, d, J = 2.8 Hz, H-24) were attributed to
The Mosher ester procedure was employed to determine the
absolute configuration of the OH groups located at C-3 and C-
24 in compound 1. After treatment with (R)- and (S)-MTPA
chloride, the secondary OH groups at C-3 and C-24 were both
esterified, to afford the (S)- and (R)-MTPA derivatives,
1
respectively. By analyzing the observed H NMR chemical
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shift difference values (ΔδS−R) of certain diagnostic key protons
assigned unambiguously, the absolute configurations of C-3 and
C-24 were both assigned as S (Figure 1). Furthermore, the
group at C-27. The HMBC spectrum of 3 showed key
correlations of H-24 with C-23, C-25, and C-27, as well as H-27
and H-26 with C-25 and C-24, which supported the above
functional group assignment. The trans-configuration of the C-
24, C-25 double bond was deduced on the basis of the key
NOESY correlation between H-24 and H-27. Other observed
NOE effects indicated the relative configuration of the
remaining part of the molecule of 3 to be identical with that
of 1 and other known 16α,23α-epoxycucurbitacins. Thus, the
structure of compound 3 (elaeocarpucin C) was determined as
(3S,8S,9R,10R,13R,14S,16R,17R,21S,23R,24E)-16,23-epoxy-
3,20,27-trihydroxycucurbit-5,24-dien-11-one.
Figure 1. ΔδS−R values of MTPA esters of 1.
Compound 4 was obtained as white, amorphous powder.
The HRESIMS of 4 gave a sodiated molecular ion peak at m/z
507.3073 [M + Na]+, suggesting an elemental formula of
C30H44O5, representing one more degree of unsaturation than
in compounds 1−3. In the 1H NMR spectrum of 4, the proton
signal of an oxygenated CH, assigned as H-24 in compound 1,
was absent, and the corresponding oxygenated carbon was
substituted by a carbonyl group at δC 198.9 in the 13C NMR
spectrum. These differences suggested that the OH group at C-
24 in 1 is oxidized to a carbonyl group in 4. Moreover,
downfield shifts of 0.97 ppm for H-23, 1.09 ppm for Ha-27,
0.82 ppm for Hb-27, and 0.17 ppm for H-26, due to the
deshielding effect caused by the nearby carbonyl group at C-24,
were observed in the 1H NMR spectrum. Data from the
HSQC, HMBC, and NOESY 2D-NMR spectra were consistent
with the above deduction. Thus, the structure of compound 4
( e l a e o c a r p u c i n D ) w a s e l u c i d a t e d a s
(3S,8S,9R,10R,13R,14S,16R,17R,21S,23R)-16,23-epoxy-3,20-di-
hydroxycucurbit-5,25(27)-diene-11,24-dione.
observed NOESY cross-peaks of H-10/H3-28 and H3-30, H-8/
H3-18 and H3-19, H-17/H3-30, H-16/H3-18, H-23/H-15β, and
H3-21/H-17 and H-12α provided evidence that the relative
configurations of the remaining chiral carbons of compound 1
were identical with those of previously reported related
compounds.6−12 Hence, the structure of compound 1 was
d e t e r m i n e d
t o
b e
(3S,8S,9R,10R,13R,14S,16R,17R,21S,23R,24S)-16,23-epoxy-
3,20,24-trihydroxycucurbit-5,25(27)-dien-11-one, and this sub-
stance has been accorded the trivial name elaeocarpucin A.
Compound 2 gave the same molecular formula, C30H46O5, as
that of 1 by analysis of the HRESIMS. The NMR spectra of 1
and 2 were closely comparable, with the only differences
1
evident in signals for the side chain at C-23. In the H NMR
spectrum of 2, the H-23 and H-24 resonances appeared at δH
3.84 (ddd, J = 10.7, 8.0, and 2.7 Hz) and 3.87 (d, J = 7.2 Hz),
respectively. Both were shifted upfield and showed a change of
coupling pattern when compared with 1. Correspondingly, in
the 13C NMR spectrum, a downfield shift of approximately 3.0
ppm for the carbon signal of C-24 (δC 78.8) was discernible.
These observed differences suggested that the absolute
configuration of C-24 might be R, which was confirmed
The molecular formula of compound 5 was deduced as
C30H44O5 on the basis of its HRESIMS, the same as that of
1
compound 4. In the H NMR spectrum, a proton singlet
appeared at δH 9.40, and this signal exhibited a correlation in
the HSQC spectrum with a carbonyl group signal at δC 195.2,
which implied the presence of a formyl group. In the HMBC
spectrum, key correlations were observed from the aldehyde
proton to the methyl group carbon at δC 9.9 (C-26) and two
carbon signals of a double bond at δC 151.8 and 139.0 (C-24
and C-25), respectively, which suggested that the formyl group
was at C-25. Comparison of the NMR data of 5 with those of
1
subsequently by calculation of the H NMR chemical shift
differences for the (S)- and (R)-MTPA esters of 2 produced as
a result of the Mosher ester reaction (Figure 2). Thus,
compound 2 (elaeocarpucin B) was determined structurally as
the C-24 epimer of 1.
1
compound 13 showed a downfield H NMR shift of 0.54 ppm
for H-24 and downfield 13C NMR shifts of 26.7 and 3.1 ppm
for C-24 and C-25, respectively. These were consistent with the
substitution of a formyl group at C-27 in 5. The trans-
configuration of the C-24−C-25 double bond was deduced
from the key NOE correlation between the aldehyde proton
(H-27) and H-24. Thus, the structure of compound 5
( e l a e o c a r p u c i n E ) w a s d e d u c e d a s
(3S,8S,9R,10R,13R,14S,16R,17R,21S,23R,24E)-16,23-epoxy-
3,20-dihydroxycucurbit-27-aldehyde-5,24-dien-11-one.
Compound 6 gave a molecular formula of C30H48O5, as
determined by HRESIMS, with one degree of unsaturation less
than compound 1. In comparison of the NMR spectra with
those of compound 1, besides one trisubstituted double bond
ascribed to C-5−C-6, no other double-bond signal was found,
which implied that the side chain of 6 is saturated. This
inference was confirmed by the observed 1H−1H COSY
correlations of H-23 (δH 4.22, 1H, dddd, J = 11.1, 11.1, 2.4, and
2.4 Hz) with two CH2 groups, H-22 (δH 1.31 and 1.45, each
1H) and H-24 (δH 1.42 and 1.87, each 1H). In addition to the
13C NMR signal at δC 72.3 of C-20, another quaternary
Figure 2. ΔδS−R values of MTPA esters of 2.
Compound 3 was obtained as a pale yellow powder. The
HRESIMS gave a sodiated molecular ion peak at m/z 509.3255
[M + Na]+, consistent with a molecular formula of C30H46O5,
the same as those of both compounds 1 and 2. The 1H and 13
C
NMR spectra of 3 were very similar to those of compound 13.
On comparison of the 1H NMR data of these two compounds,
the H3-27 signal at δH 1.68 in compound 13 was absent, while
an oxygenated methylene resonance appeared at δH 4.02 (2H,
s). This suggested that the C-27 methyl group in compound 13
is substituted by a primary alcohol group in compound 3.
Correspondingly, in the 13C NMR spectrum of 3, the observed
downfield shift of 2.7 ppm for C-25 and an upfield shift of 4.0
ppm for C-26 were consistent with the substitution of an OH
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Figure 3. Semisynthesis of compound 3 from compound 13.
oxygenated carbon appeared at δC 70.5 and exhibited HMBC
correlations with the H3-26 (δH 1.20, 3H, s) and H3-27 (δH
1.27, 3H, s) signals, respectively, which indicated a tertiary OH
group at C-25. Analysis of the NOESY spectrum suggested the
relative configuration of the remainder of the molecule of 6 to
be identical with that of 1. Thus, the structure of 6
( e l a e o c a r p u c i n F ) w a s d e t e r m i n e d a s
(3S,8S,9R,10R,13R,14S,16R,17R,21S,23S)-16,23-epoxy-3,20,25-
trihydroxycucurbit-5-en-11-one.
Compound 7 was obtained as a white, amorphous powder,
and its molecular formula was deduced as C30H46O5 from the
HRESIMS. The NMR spectra of 7 were very similar to those of
compound 13, with the only difference being the replacement
of the CH2 group at C-22 by an oxygenated CH. In the COSY
spectrum, a broad singlet of an oxygenated proton at δH 2.95
(H-22) exhibited a weak (due to the small J value of the
coupling constant between H-22 and H-23) but discernible
enhancement with H-23 at δH 4.67 (1H, d, J = 7.8 Hz), and its
corresponding carbon signal at δC 77.6 showed a HMBC
correlation with the methyl proton signal at δH 1.38 (3H, s, H-
21). Thus, it was inferred that an OH group is positioned at C-
22 in compound 7. Correspondingly, a shift to higher field of
around 6 ppm was observed for C-17 in the 13C NMR
spectrum due to the γ-effect caused by this substituent. In the
NOESY spectrum, key NOE cross-peaks of H-22/H-23, H-23/
H-16, H-16/H-18, and H-21/H-17 were observed, which
indicated the α-orientation of the OH group. Accordingly, the
structure of compound 7 (elaeocarpucin G) was assigned as
(3S,8S,9R,10R,13R,14S,16R,17R,21R,22R,23S)-16,23-epoxy-
3,20,22-trihydroxycucurbit-5,24-dien-11-one.
All isolates (1−13) were evaluated their cytotoxic activity
against HT-29 human colon cancer cells. The known
cucurbitacins, cucurbitacin D (9), 3-epi-isocucurbitacin D
(10), 25-O-acetylcucurbitacin F (11), and cucurbitacin I (12),
were found to be the most active in inhibiting the proliferation
of HT-29 cancer cells, with IC50 values ranging from 0.039 to
0.54 μM. Of the eight new 16,23-epoxycucurbitacins,
elaeocarpucin C (3) was found to display potent cytotoxicity
against HT-29 cells with an IC50 value of 0.41 μM, while
elaeocarpucins D (4), G (7), and H (8) were less active against
this same cell line. Thus, a 24(25)-en-27-ol functionality on the
side chain of these new compounds seems to be required for
potent cytotoxicity. In general, when the C-17−C-23 unit is
contained in an epoxide ring, the resultant cytotoxicity is
reduced when compared with known compounds such as 3-epi-
isocucurbitacin D (10) (Table 3).
Compounds 1−3, 6, 9, 10, and 13 were also evaluated in a
HT-29 cell-based mitochondrial transmembrane potential
assay, but none of these substances were found to be active
(IC50 >10 μM).
The initial cytotoxic activity of compound 3 encouraged
further biological evaluation of this compound. A sufficient
amount of 3 (>25 mg) was generated from the known inactive
compound 13 by selectively oxidizing the allylic methyl group
(C-27) into a primary alcohol (Figure 3), for evaluation in the
in vivo hollow fiber assay. This method may be used as a
secondary discriminator to prioritize compounds possessing
promising in vitro activity for potential further testing in a
relevant in vivo xenograft model.29−32 The human cancer cell
lines evaluated using ip administration comprised MDA-MB-
435 (melanoma), MCF-7 (breast), and HT-29 (colon) for the
in vivo hollow fiber assay. However, no inhibition of
proliferation by 3 was observed over the course of the study
for any of the cancer cell types, which were administrated at a
dose range of 0.5 to 10 mg/kg/day.
The molecular formula of compound 8 was determined as
C30H44O5 from the protonated molecular ion peak at m/z
485.3261 [M + H]+ in the HRESIMS. The NMR data of 8 were
again similar to those of 13. When comparing the 13C NMR
spectra of these two compounds, the signal of a CH2 group at
δC 24.0 ascribed to C-7 in compound 13 was absent, with a
carbonyl group signal appearing at δC 199.6 instead, thus
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured using a Fisher Scientific melting point apparatus and are
uncorrected. Optical rotations were recorded on a Perkin-Elmer 343
automatic polarimeter. UV spectra were measured with a Perkin-Elmer
Lambda 10 UV/vis spectrometer. IR spectra were obtained on a
Thermo Scientific Nicolet 6700 FT-IR spectrometer. NMR spectro-
scopic data were run at room temperature on Bruker Avance DRX-400
or 600 MHz spectrometers, and the data were processed using
MestReNova 6.0 software (Mestrelab Research SL, Santiago de
Compostela, Spain). Accurate mass values were performed on a
Micromass LCT ESI spectrometer. Sodium iodide was used for mass
calibration for a calibration range of m/z 100−2000. LC-MS
experiments were performed on a liquid chromatographic/autosam-
pler system that consisted of a Waters Alliance 2690 Separations
Module (Waters, Milford, MA, USA) and a Micromass LC-TOF II
mass spectrometer (Micromass, Wythenshawe, UK) equipped with an
orthogonal electrospray source (Z-spray). Column chromatography
was carried out with silica gel (230−400 Mesh; Sorbent Technologies,
1
suggesting a carbonyl group at C-7 in 8. In the H NMR
spectrum, downfield shifts of approximately 0.5 ppm for H-6
and 0.6 ppm for H-8 were observed due to deshielding effects
caused by the carbonyl group at C-7. These were consistent
with the downfield shifts of around 27 and 5 ppm for C-5 (δC
167.0) and C-6 (δC 125.6), respectively, as well as ca. 15 ppm
for C-8 (δC 58.1) in the 13C NMR spectrum. Key HMBC
correlations of H-3, H-10, H-28, and H-29 to C-5, and H-8 to
C-7, were observed to support the structure proposed. Further
analysis of the NOESY experiment revealed the consistent
relative configuration of 8 with other cucurbitacin analogues
isolated in this investigation. Thus, the structure of compound
8
( e l a e o c a r p u c i n H ) w a s d e d u c e d a s
(3S,8S,9S,10R,13R,14S,16R,17R,21S,23R)-16,23-epoxy-3,20-di-
hydroxycucurbit-5,24-diene-7,11-dione.
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a
1
Table 1. H NMR Chemical Shifts of Compounds 1−8
position
1
1
2
3
4
5
6
7
8
b
b
1.47, m
1.47, m
1.42, m
1.43, m
1.49, m
1.49, m
1.48, m
1.66
1.78
2α
2β
3
1.73, m
1.67, m
1.74, m
1.66, m
1.73, m
1.68, m
1.73, m
1.68, m
1.74, m
1.68, m
1.74, m
1.65, m
3.48, brs
1.75, m
1.68, m
1.78, m
3.48, brs
5.67, d (5.8)
1.88, m
3.48, brs
5.67, d (5.8)
1.89, m
3.48, brs
5.67, d (5.8)
1.91, m
3.48, brs
3.48, brs
3.48, brs
5.67, d (5.6)
1.93, m
3.65, brs
6
5.67, d (5.6)
1.90, m
5.68, d (6.0)
1.91, m
5.67, d (5.8)
1.90, m
6.17, d (brs)
7α
7β
8
2.46, m
2.44, m
2.44, m
2.44, m
2.44, m
2.44, m
2.44, m
b
b
b
b
b
1.96
1.94
1.96
1.92, d (8.3)
2.25, m
1.96, d (8.0)
2.25, m
1.91
1.94
2.54, brs
2.67, m
10
12α
12β
15α
15β
16
2.25, m
2.25, m
2.25, m
2.24, m
2.24, m
3.03, d (14.8)
2.43, d (14.8)
1.48, m
3.03, d (14.8)
2.42, d (14.8)
1.48, m
3.03, d (14.8)
2.43, d (14.8)
1.47, m
3.03, d (14.8)
2.42, d (14.8)
1.50, m
3.04, d (14.4)
2.44, d (14.4)
1.48, m
3.01, d (14.4)
2.42, d (14.4)
1.42, m
3.04, d (14.8)
2.42 (14.8)
1.48, m
3.04, d (14.8)
2.55 (14.8)
1.36, m
1.85, m
1.84, m
1.85, m
1.84, m
1.85, m
1.82, m
1.85, m
2.40, m
4.40, ddd (10.4, 4.33, ddd (10.4, 4.38, ddd (10.1, 4.42, ddd (10.1, 4.43, ddd (10.4, 4.37, ddd (10.4,
4.37, ddd (10.0, 4.37, ddd (10.4,
10.4, 3.6)
10.4, 3.7)
10.1, 3.9)
1.99, d (10.5)
0.94, s
10.1, 3.9)
2.00, d (10.5)
0.94, s
10.4, 3.8)
1.95, d (10.1)
0.96, s
10.4, 3.6)
1.95, d (10.4)
0.93, s
10.0, 3.6)
2.34, d (10.1)
0.95, s
10.4, 3.6)
1.90, d (10.6)
0.98, s
b
b
17
1.95
1.95
18
0.93, s
1.14, s
1.32, s
0.93, s
1.14, s
1.31, s
1.36, m
19
1.13, s
1.14, s
1.15, s
1.13, s
1.14, s
1.17, s
21
1.32, s
1.36, s
1.36, s
1.31, s
1.38, s
1.32, s
22α
1.57, dd (12.0,
14.0)
1.52, m
1.67, m
1.52, m
1.45, m
2.95, brs
1.49, m
22β
1.25, m
1.26, m
1.38, m
1.52, m
1.41, m
1.31, m
1.43, m
23
4.01, ddd (12.0, 3.84, ddd (10.7, 4.61, ddd (11.1, 4.98, dd (11.8,
4.86, ddd (11.1, 4.22, dddd (11.1,
4.67, d (7.8)
4.22, ddd (11.2,
8.0, 2.4)
3.0, 3.0)
8.0, 2.7)
8.5, 2.8)
2.8)
7.2, 2.7)
11.1, 2.4, 2.4)
24
4.24, d (2.8)
3.87, d (7.2)
5.44, d (8.3)
5.66, dd (7.6,
1.4)
1.87, m
5.34, brd (8.0)
5.12, d (8.0)
1.42, m
26
1.72, s
4.91, s
5.04, s
1.02, s
1.17, s
1.22, s
1.72, s
4.92, s
5.04, s
1.02, s
1.17, s
1.22, s
1.72, s
4.02, s
1.89, s
6.10, s
5.86, s
1.01, s
1.17, s
1.24, s
1.77, d (1.2)
9.40, s
1.20, s
1.72 (brs)
1.77 (s)
1.69, s
1.71, s
27a
27b
28
1.27, s
1.02, s
1.17, s
1.21, s
1.02, s
1.18, s
1.25, s
1.01, s
1.17, s
1.20, s
1.01, s
1.17, s
1.24, s
1.14, s
1.28, s
1.24, s
29
30
a
Measured at 400 MHz; obtained in CDCl3 with TMS as internal standard; J values (Hz) are given in parentheses. Assignments are based on 1H−1H
b
COSY, HSQC, and HMBC spectroscopic data. Overlapping signals.
Atlanta, GA, USA). Analytical TLC was conducted on precoated 250
μm thick silica gel UV254 aluminum-backed plates (Sorbent
Technologies). Waters Atlantis (4.6 × 150 mm) and semipreparative
(10 × 150 mm) C18 (5 μm) columns were used for analytical and
semipreparative HPLC, respectively, as conducted on a Waters system
comprised of a 600 controller, a 717 Plus autosampler, and a 2487
dual-wavelength absorbance detector.
Plant Material. The fruits and stems of E. chinensis were collected
in Honba Forest Reserve (12°06.953′ N; 109°00.072′E; Alt. 275 m),
Khanh Hoa Province, Vietnam, by T.N.N., Vuong Tan Tu, and D.D.S.
in November 2008, who also identified this plant. A voucher specimen
(original collection DDS et al. 13583; re-collection 114330) has been
deposited in the John G. Searle Herbarium of the Field Museum of
Natural History (under accession number FM 2287877), Chicago,
Illinois.
LC-MS Dereplication Procedure. LC-UV Conditions. Sample
concentration, 10 mg/mL MeOH solution; mobile phase, gradient
elution of MeOH−H2O (0−10 min, from 50:50 to 70:30; 11−30 min,
100% MeOH); UV detection wavelength, 220 nm; flow rate, 0.75 mL/
min; injection volume, 45 μL for the 96-well plate with sample
concentration of ca. 20 μg/mL, and 11.3 μL for the 96-well plate with
sample concentration of ca. 5 μg/mL, respectively.
Cytotoxicity Assay Screening. Fractions were collected into two
96-well plates (250 μL/well × 90 and negative control/well × 6) with
sample concentrations of 20 and 5 μg/mL, respectively, and were
tested for HT-29 cell growth inhibition activity, according to an
established protocol.33
LC-MS Conditions. HPLC conditions: mobile phase, a gradient
elution of MeOH−H2O (0−10 min, from 50:50 to 70:30; 11−30 min,
100% MeOH); injection volume, 45 μL (10 mg/mL). The mobile
phase flow rate was maintained at 0.75 mL/min and was split post-
column using a microsplitter valve (Upchurch Scientific, Oak Harbor,
WA, USA) to ca. 20 μL/min for introduction to the ESI source.
Optimal ESI conditions: capillary voltage, 3000 V; source temperature,
110 °C; cone voltage, 55 V. Q1 was set to optimally pass ions from m/
z 100−2000, and all ions transmitted into the pusher region of the
TOF analyzer were scanned over m/z (100−1000 range) with a 1 s
integration time. Data were acquired in a continuum mode during the
LC run.
Data Analysis. Using a combination search of proposed molecular
formulas corresponding to the major active peaks and the key word
“Eleaocarpus” in the SciFinder database (Chemical Abstracts Service,
Columbus, OH, USA), the peaks with unknown molecular formulas
were designated for further fractionation.
Extraction and Isolation. The air-dried and milled fruits (480 g)
of E. chinensis were extracted by maceration with MeOH (3 × 2 L) at
room temperature for two days each. After removing the solvent under
reduced pressure, the combined and concentrated MeOH extract was
suspended in a mixture of 80% MeOH−H2O (1 L), then partitioned
with hexane and CHCl3 in turn, to afford hexane- (20 g) and CHCl3
(7 g)-soluble extracts. The CHCl3-soluble extract, with an IC50 value
of 0.4 μg/mL against HT-29 cells, was subjected to a LC-MS
dereplication procedure, in which the effluent from the HPLC
chromatography was split, with part passed into a mass spectrometer
and part collected in a 96-well plate. The latter was subjected to
448
dx.doi.org/10.1021/np200879p | J. Nat. Prod. 2012, 75, 444−452

Journal of Natural Products
Article
a b
,
Table 2. 13C NMR Chemical Shifts of Compounds 1−8
position
1
2
3
4
5
6
7
8
1
20.5
28.4
76.0
42.0
139.6
120.3
23.9
42.7
49.3
35.1
213.4
48.2
48.5
47.9
40.6
76.7
55.2
19.8
20.3
72.0
29.3
41.1
76.5
75.8
142.7
19.5
111.7
27.2
25.3
21.2
20.5
28.5
76.1
41.7
139.7
120.4
23.9
42.7
49.4
35.2
213.4
48.3
48.5
48.0
40.6
76.6
55.3
19.8
20.3
72.1
29.3
45.0
76.7
78.8
143.7
17.7
114.7
27.2
25.4
21.1
20.5
28.5
76.0
41.7
139.6
120.4
23.9
42.7
49.3
35.1
213.4
48.3
48.3
48.1
40.7
77.3
54.9
19.9
20.3
72.4
29.3
48.7
72.5
124.8
138.6
14.2
67.9
27.2
25.3
21.1
20.5
28.6
76.1
41.7
139.6
120.3
24.0
42.7
49.3
35.2
213.2
48.2
48.4
47.9
40.3
77.0
54.9
19.9
20.4
72.3
29.2
45.3
77.2
198.9
143.0
18.1
126.4
27.2
25.4
21.2
20.7
28.7
76.2
41.8
139.8
120.5
24.3
42.8
49.5
35.3
213.5
48.4
48.6
48.2
40.7
76.6
55.1
20.1
20.5
72.5
29.4
47.2
73.2
151.8
139.0
9.9
21.0
28.6
76.0
42.0
139.6
120.4
24.9
42.7
49.3
35.1
213.4
48.2
48.4
47.9
40.7
76.5
55.0
19.8
20.3
72.3
29.2
49.5
74.2
46.9
70.5
28.0
31.0
27.1
25.3
21.1
20.5
28.5
76.1
41.7
139.6
120.3
23.9
42.7
49.3
35.2
213.5
48.2
48.5
47.5
40.5
76.6
48.7
20.3
20.4
74.8
25.7
77.6
75.4
120.8
138.2
18.8
26.0
27.1
25.3
21.3
20.5
28.3
76.1
43.0
167.0
125.6
199.6
58.1
49.5
37.4
211.0
48.3
47.8
47.5
40.5
76.5
54.7
19.9
21.1
72.2
29.7
49.1
72.8
124.8
136.6
18.5
25.8
27.8
24.8
21.1
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
195.2
27.3
25.6
21.3
a
Measured at 100 MHz; obtained in CDCl3 with TMS as internal standard. Assignments are based on HSQC and HMBC NMR spectra.
Multiplicity obtained from the DEPT spectrum.
b
cytotoxicity screening using HT-29 cancer cells. ESIMS analysis
indicated that the active peaks were at m/z 560, 516, 484, and 486, of
which those at 516 and 560 amu corresponded to the presence of
three known cytotoxic cucurbitacins, cucurbitacin D (9), 3-epi-
isocucurbitacin D (10), and 25-O-acetylcucurbitacin F (11). However,
peaks at m/z 484 and 486, with possible molecular formulas of
C30H44O5 and C30H46O5, respectively, did not seem to match those of
any known cucurbitacin triterpenes. Accordingly, bioassay-guided
fractionation was used to facilitate the isolation process.
The CHCl3-soluble extract was subjected to chromatography over a
silica gel column and eluted with a CH2Cl2−acetone gradient to afford
10 fractions (F1−F10). Fractions F3, F4, and F5 were active against
HT-29 cells with IC50 values of 0.3, <0.16, and 0.2 μg/mL,
respectively. Fraction F3 (220 mg) was chromatographed over an
open C18 column (2.2 × 20 cm) using MeOH−H2O mixtures (70:30
to 100% MeOH) for elution, to give three subfractions (F301−F303).
F302 was purified by HPLC on a semipreparative RP-18 column, using
MeOH−H2O (60:40) as solvent, to afford 1 (10 mg), 2 (9.0 mg), 6
(7.0 mg), and a mixture of two compounds, which was subjected to
further separation by HPLC, using CH3CN−H2O (33:67) for elution,
to give 4 (4.0 mg) and 5 (1.0 mg), respectively. Fraction F4 (250 mg)
was fractionated over an open C18 column (2.2 × 20 cm), eluted with
MeOH−H2O (70:30 to 100% MeOH), to afford five subfractions
(F401−F405). Cucurbitacin D (9, 35 mg) was obtained as crystals
from a MeOH−H2O (ca. 70:30) solution of F401. Further purification
of combined fractions F402−4 was conducted on a semipreparative
RP-18 HPLC column, using MeOH−H2O (60:40) as solvent, to yield
3-epi-isocucurbitacin D (10, 2.5 mg), cucurbitacin I (12, 1.0 mg), and
25-O-acetylcucurbitacin F (11, 3.5 mg). Elaeocarpucin C (3, 4.0 mg)
was purified from fraction F5 (270 mg) by repeated separation on a
semipreparative RP-18 HPLC column, using MeOH−H2O (60:40)
and CH3CN−H2O (35:65) sequentially for elution. In addition,
compound 13 (10 mg) was recrystallized from the inactive fraction F2
using acetone as solvent. In order to obtain a sufficient amount of 13
as starting material to support the semisynthesis of elaeocarpucin C
(3), the residue of F2 was chromatographed over a silica gel column
and eluted with CH2Cl2−acetone mixtures (20:1 to 5:1) to afford an
additional 200 mg quantity of this compound.
The stems of E. chinensis were also investigated in the present study.
A CHCl3-soluble extract (11 g) was prepared from the air-dried and
then powdered stems (900 g) by following the extraction and partition
procedures described above for the fruits. However, this was less
cytotoxic (IC50 10.1 μg/mL, HT-29 cells) than the CHCl3-soluble
extract of the fruits. This extract was fractionated over a silica gel
column using CH2Cl2−acetone mixtures of increasing polarity to yield
eight fractions (F1′−F8′). All fractions were analyzed using HPLC and
TLC, and F4′ was found to be rich in cucurbitacins and was
determined to contain cucurbitacin D (9), 3-epi-isocucurbitacin D
(10), 25-O-acetylcucurbitacin F (11), and unknown cucurbitacin
analogues. Separation of F4′ over a semipreparative RP-18 HPLC
column using MeOH−H2O (60:40) led to the purification of
compounds 7 (2.0 mg) and 8 (1.8 mg).
Elaeocarpucin A (1): white powder; mp 258−260 °C; [α]20
D
+144.0 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 207 (3.73) nm;
IR (film) νmax 3445, 1687, 1456, 1375, 1215, 1097, 1075, 1028, 755
cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz,
CDCl3) data, see Tables 1 and 2; HRESIMS m/z 509.3225 [M + Na]+
(calcd for C30H46O5Na, 509.3243).
Elaeocarpucin B (2): white powder; mp 270−272 °C; [α]20D +94.0
(c 0.08, MeOH); UV (MeOH) λmax (log ε) 207 (3.63) nm; IR (film)
449
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Journal of Natural Products
Article
(S)-MTPA ester of 1 were recorded directly after each reaction and
were assigned on the basis of COSY and NOESY experiments, with
ambiguous and overlapping signals not used for the ΔδS−R
calculation.34,35 1H NMR data of (R)-MTPA ester of 1 (400 MHz,
pyridine-d5): δ 5.923 (1H, d, J = 4.4 Hz, H-24), 5.547 (1H, d, J = 5.6
Hz, H-6), 5.255 (1H, s, H-27a), 5.139 (1H, brs, H-3), 5.069 (1H, s, H-
27b), 4.752 (1H, ddd, J = 9.4, 9.4, 2.3 Hz, H-16), 4.512 (1H, m, H-
23), 1.830 (3H, s, H-26), 2.072 (1H, d, J = 9.4 Hz, H-17), 1.442 (3H,
s, H-21), 1.282 (3H, s, H-30), 1.258 (3H, s, H-18), 1.216 (3H, s, H-
Table 3. Cytotoxicity of Compounds Isolated from E.
chinensis
a
b
compound
HT-29
3
0.41
5.6
4
7
2.9
8
2.7
9
0.12
0.039
0.54
0.19
0.006
1
29), 1.132 (3H, s, H-19), 1.097 (3H, s, H-28). H NMR data of (S)-
10
11
12
MTPA ester of 1 (400 MHz, pyridine-d5): δ 5.889 (1H, d, J = 2.7 Hz,
H-24), 5.636 (1H, d, J = 5.6 Hz, H-6), 5.077 (1H, brs, H-3), 5.001
(1H, s, H-27a), 4.963 (1H, s, H-27b), 4.810 (1H, ddd, J = 9.4, 9.4, 2.3
Hz, H-16), 4.547 (1H, m, H-23), 2.107 (1H, d, J = 9.4 Hz, H-17),
1.759 (3H, s, H-26), 1.463 (3H, s, H-21), 1.327 (3H, s, H-30), 1.253
(3H, s, H-29), 1.221 (3H, s, H-18), 1.131 (3H, s, H-28), 1.123 (3H, s,
H-19).
c
paclitaxel
a
Compounds 1, 2, 5, 6, and 13 were not cytotoxic against HT-29 cells
(IC50 > 10 μM), using a standard protocol.31 Results are expressed as
IC50 values (μM). Used as a positive control substance.
b
c
Preparation of the (R)- and (S)-MTPA Ester Derivatives of
Compound 2. The (R)-MTPA ester and the (S)-MTPA ester of 2
were produced by following the same Mosher reaction procedure
νmax 3442, 1687, 1457, 1375, 1216, 1097, 1075, 1022, 754 cm−1; H
1
NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see
Tables 1 and 2; HRESIMS m/z 509.3264 [M + Na]+ (calcd for
C30H46O5Na, 509.3243).
1
applied to compound 1. H NMR data of (R)-MTPA ester of 1 (400
MHz, pyridine-d5): δ 5.855 (1H, d, J = 8.2 Hz, H-24), 5.546 (1H, d, J
= 5.2 Hz, H-6), 5.216 (1H, s, H-27a), 5.140 (1H, brs, H-3), 5.014 (1H,
s, H-27b), 4.745 (1H, m, H-16), 4.467 (1H, m, H-23), 2.119 (1H, d, J
= 9.6 Hz, H-17), 1.615 (3H, s, H-26), 1.483 (3H, s, H-21), 1.271 (3H,
s, H-29), 1.265 (3H, s, H-30), 1.229 (3H, s, H-18), 1.138 (3H, s, H-
19), 1.109 (3H, s, H-28). 1H NMR data of (S)-MTPA ester of 1 (400
MHz, pyridine-d5): δ 5.924 (1H, d, J = 8.0 Hz, H-24), 5.649 (1H, d, J
= 4.8 Hz, H-6), 5.244 (1H, s, H-27a), 5.082 (1H, brs, H-3), 5.034 (1H,
s, H-27b), 4.647 (1H, m, H-16), 4.381 (1H, m, H-23), 2.071 (1H, d, J
= 9.7 Hz, H-17), 1.739 (3H, s, H-26), 1.458 (3H, s, H-21), 1.283 (3H,
s, H-29), 1.264 (3H, s, H-30), 1.176 (3H, s, H-28), 1.161 (3H, s, H-
18), 1.198 (3H, s, H-19).
Elaeocarpucin C (3): pale yellow, amorphous powder; [α]20D +91.0
(c 0.04, MeOH); UV (MeOH) λmax (log ε) 206 (3.87), 219 (3.48)
nm; IR (film) νmax 3435, 1685, 1465, 1375, 1215, 1067, 755 cm−1; 1H
NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see
Tables 1 and 2; HRESIMS m/z 509.3255 [M + Na]+ (calcd for
C30H46O5Na, 509.3243).
Elaeocarpucin D (4): white, amorphous powder; [α]20 +136.0 (c
D
0.09, MeOH); UV (MeOH) λmax (log ε) 213 (3.84) nm; IR (film)
1
νmax 3471, 1685, 1462, 1375, 1096, 754 cm−1; H NMR (400 MHz,
CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2;
HRESIMS m/z 507.3073 [M + Na]+ (calcd for C30H44O5Na,
507.3086).
Generation of Compound 3 from Compound 13. Selenium
dioxide (SeO2, 500 mg) was dissolved in 2.5 mL of distilled water, and
then 12.5 mL of MeOH was added to give a clear solution. Next, 10 g
of silica gel was added to this solution to form a slurry, with the solvent
evaporated under reduced pressure to afford a silica gel powder
containing 5% selenium dioxide.36 A portion of this pretreated silica
gel (1 g) was suspended in 7 mL of CH2Cl2 with 0.5 mL of t-BuOOH
(5.0−6.0 M in decane) and stirred for 15 min at room temperature.
Compound 13 (200 mg) was dissolved in a mixture of CH2Cl2−
MeOH (4:1, 20 mL), and the solution obtained was added dropwise
to the above-mentioned oxidizing reagent. The mixture was sealed and
stirred overnight at room temperature, with the product analyzed by
TLC (CH2Cl2−acetone, 5:1; Rf 0.2). After the reaction, the mixture
was filtered and the residue was washed with CHCl3. The filtrate was
partitioned with water, and the organic phase was evaporated under
reduced pressure after washing with saturated NaCl water, to give a
mixture of compound 3 and unchanged compound 13. This mixture
was subjected to chromatography on an open reversed-phase C18
column, using a gradient of MeOH−H2O (70:30 to 100% MeOH) for
elution, to afford 32 mg of 3 and 150 mg of 13. The yield of this
reaction was around 15−20%, and the unchanged 13 could be recycled
(Figure 3). When performing this selective oxidation procedure,
modification of the reaction by raising the temperature used,
prolonging the reaction time, or increasing the amount of oxidant
did not increase the yield of the desired primary alcohol (3), but led to
the generation of an α,β-unsaturated aldehyde derivative, as an
undesired side product, which was identified as compound 5.
Cytotoxicity Assay. Compounds 1−13 were evaluated against
human colon cancer cells (HT-29), according to a previously
described protocol.33
Elaeocarpucin E (5): white, amorphous powder; [α]20 +82.0 (c
D
0.05, MeOH); UV (MeOH) λmax (log ε) 218 (4.12) nm; IR (film)
νmax 3458, 1703, 1688, 1460, 1213, 1376, 1072, 1021, 755 cm−1; H
1
NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see
Tables 1 and 2; HRESIMS m/z 507.3112 [M + Na]+ (calcd for
C30H44O5Na, 507.3086).
Elaeocarpucin F (6): white, amorphous powder; [α]20 +79.0 (c
D
0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.57) nm; IR (film) νmax
1
3432, 1688, 1462, 1213, 1391, 1376, 1162, 1072, 755 cm−1; H NMR
(400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see
Tables 1 and 2; HRESIMS m/z 511.3399 [M + Na]+ (calcd for
C30H44O5Na, 511.3388).
Elaeocarpucin G (7): white, amorphous powder; [α]20 +55.0 (c
D
0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.50) nm; IR (film) νmax
1
3476, 2948, 2917, 2845, 1687, 1462, 1380, 1059, 755 cm−1; H NMR
(400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see
Tables 1 and 2; HRESIMS m/z 487.3423 [M + H]+ (calcd for
C30H47O5, 487.3423).
Elaeocarpucin H (8): white powder; mp 244−246 °C; [α]20D +68.0
(c 0.17, MeOH); UV (MeOH) λmax (log ε) 228 (3.95) nm; IR (film)
νmax 3429, 2968, 2925, 2855, 1695, 1647, 1458, 1377, 1026, 756 cm−1;
1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data,
see Tables 1 and 2; HRESIMS m/z 485.3261 [M + H]+ (calcd for
C30H45O5, 485.3267).
Preparation of the (R)- and (S)-MTPA Ester Derivatives of
Compound 1. Portions of compound 1 (1.0 mg of each) were added
into two NMR tubes and dried under a vacuum overnight at room
temperature. Deuterated pyridine (1 mL) was transferred to each tube
to give a clear solution. (S)-(+)-α-Methoxy-α-(trifluoromethyl)-
phenylacetyl (MTPA) chloride (10 μL) or (R)-MTPA chloride (10
μL) was injected into the NMR tubes separately under a N2 gas steam
and mixed quickly with the dissolved sample. The NMR tubes with
reagents were sealed and stored overnight in a dryer until the reaction
was completed, with 1H NMR spectroscopy used to monitor the
reaction. The 1H NMR chemical shifts of the (R)-MTPA ester and the
Mitochondrial Transmembrane Potential Assay. A JC-1
mitochondrial membrane potential assay kit obtained from Cayman
Chemicals was used to detect the ΔΨ. Experiments were conducted
according to the protocol established previously.37,38
In Vivo Hollow Fiber Assay. The hollow fiber assay was
conducted as described previously,30−32,39 and is summarized here.
Human cancer cell lines designated HT29, MCF-7, and MDA-MB-435
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were propagated in RPMI-1640 medium supplemented with fetal
bovine serum (5% v/v) and 2 mM glutamine at 37 °C in a 5% CO2
atmosphere. Monolayer cultures in late log-phase growth were
released by digestion with trypsin, and suspended in medium. Sterile
conditioned30 polyvinylidene fluoride hollow fibers perforated with
500 kDa molecular weight exclusion pores were filled with the cells
(HT29: 1 × 106; MCF-7: 5 × 106 and MDA-MB- 435: 1 × 106 per
fiber). The fibers were then heat sealed at two-cm intervals and cut to
generate the fibers used for the study. Prior to implantation, the fibers
were cultured overnight at 37 °C in a 5% CO2 atmosphere. On the
following day (day zero) a set of fibers representative of each cell line
was evaluated for viable cell mass by a modified MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.40 An-
other set of fibers remained in culture to confirm sterility. The
remaining fibers were transplanted into immunodeficient female NCr
nu/nu mice. For intraperitoneal (ip) implants, a small incision was
made through the skin and musculature of the dorsal abdominal wall,
the fiber samples were inserted into the peritoneal cavity in a
craniocaudal direction, and the incision was closed with skin staples.
On day three, the mice were treated with elaeocarpucin C (3) at 0.5, 1,
5, and 10 mg/kg in four daily ip injections on days 3, 4, 5 and 6
followed by fiber retrieval on day 7. Elaeocarpucin C (3) was initially
dissolved in EtOH and subsequently diluted (1:1) with Tween 80.
This mixture was then diluted with saline to its final injection state
which consisted of only 5% EtOH. Paclitaxel was administered at 2
mg/kg in a 10% EtOH-Tween 80 (1:1) solution. The vehicle group
received the 10% EtOH-Tween 80 (1:1) vehicle. On day 7 of the
study, all mice were sacrificed and the fibers were retrieved and viable
cell mass was evaluated by MTT assay. The percent net growth for
each cell line in each treatment group was calculated by subtracting the
day-zero absorbance from the day 7 absorbance and dividing this
difference by the net growth in the day 7 vehicle-treated controls
minus the day-zero values.
DEDICATION
■
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, Frederick, Mary-
land, for his pioneering work on the development of natural
product anticancer agents.
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* Supporting Information
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1-614-247-8094. Fax: +1-614-247-8119. E-mail:
kinghorn@pharmacy.ohio-state.edu.
ACKNOWLEDGMENTS
■
This study was supported by grant P01 CA125066 (awarded to
A.D.K.) from NCI, NIH. E. chinensis samples were collected
under the terms of agreement between the University of Illinois
at Chicago and the Institute of Ecology and Biological
Resources of the Vietnam Academy of Science and Technology,
Hanoi, Vietnam. We acknowledge Mr. J. Fowble, College of
Pharmacy, The Ohio State University (OSU), and Dr. C.-H.
Yuan, Campus Chemical Instrument Center, OSU, for
facilitating the acquisition of the 400 and 600 MHz NMR
spectra. We thank Ms. N. Kleinholz, Mr. M. Apsega and Dr. K.
Green-Church, Campus Chemical Instrument Center, OSU, for
the mass spectrometric measurements. We are very grateful to
Dr. G. M. Cragg, formerly of NCI-Frederick, for serving for
several years as NCI Program Director of a forerunner of the
above-cited program project, namely, grant U19 CA52956 from
NCI, NIH.
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