Journal of Chemical Ecology, Vol. 19, No.

1, 1993

CUCURBITACINS FROM Cucurbita texana: EVIDENCE

FOR THE ROLE OF ISOCUCURBITACINS

F A T H I T. H A L A W E I S H

Delaware Agricultural Experiment Station

Department of Entomology & Applied Ecology
College of Agricultural Sciences
University of Delaware
Newark, Delaware 19717-1303

(Received March 30, 1992; accepted August 19, 1992)

Abstract--In addition to cucurbitacins E and I, cucurbitacins D, 1, 3-epi-

isocucurbitacin D, 2, and B, 3, were isolated from the fruits of Cucurbita
texana and structurally identified by UV, IR, ~H NMR, 13C NMR, and MS,
and 2-O-~-glucopyranosylcucurbitacinI was identified. These compounds have
not been reported previously as constituents of this species. The isolation of
3-epi-isocucurbitacin D 2 together with normal cucurbitacins suggests that
isocucurbitacins occur naturally. Evidence is also discussed that isocucurbi-
tacins are biosynthesized one step ahead of normal cucurbitacin.

Key Words--Cucurbita texana, corn rootworm, phagostimulant, cucurbitacin

D, isocucurbitacin, glycosides.

INTRODUCTION

The cucurbitacins are a group o f highly oxygenated tetracyclic triterpenes having

a unique 19(lO~913)abeo-lO-a-lanostane (cucurbitane) skeleton. Biological
activity o f this group has been recognized for centuries and has been used as a
laxative, an emetic, and in the treatment o f malaria, dysentery, and dysmenor-
rhea (Lavie and Glotter, 1971; Halaweish, 1987). In addition, the cucurbitacins
have received a great deal o f attention because o f their cytotoxic and anticancer
activities (Witkowski et al., 1984; Whithouse and Doskotch, 1969).
Cucurbitacins occur widely in plants o f the family Cucurbitaceae and,
because o f their extreme bitterness, are thought to be involved in plant protection
against herbivores (Metcalf, 1985; Tallamy and Krischik, 1989). Nevertheless,
cucurbitacins are phagostimulants for both adult (Metcalf, 1985) and larval

29

0098-0331/93/01004)029507.00/0 9 1993 Plenum Publishing Corporation

30 HALAWEISH

diabroticite chrysomelid beetles (DeHeer and Tallamy, 1991) and can have
important ecological consequences for plants that possess them. Adult diabro-
ticites can detect cucurbitacins in nanogram quantities and readily devour bitter
plant material. These compounds influence the behavior of a number of impor-
tant crop pests including the banded cucumber beetle (Diabrotica balteata Le
Conte), the northern corn rootworm (D. barberi Smith & Lawrence), the south-
ern corn rootworm (D. undecimpunctata howardii Barber and its western relative
D.u. undecimpunctata Mannerheim) and the western corn rootworrn (D. vir-
gifera virgifera Le Conte).
The pharmacopetic response of diabroticite beetles to cucurbitacins is crit-
ical to the integrated pest management of cucumber beetles and corn rootworms
throughout the world and has led to the development of promising control prod-
ucts containing cucurbitacin baits (Ferguson and Metcalf, 1984; Metcalf and
Rhodes, 1985; Metcalf et al., 1987).
In view of the importance of these compounds to pharmacology and agri-
culture, there is a need to elucidate the chemistry of cucurbitacin-bearing plants.
This is particularly true for species containing high concentrations of cucurbi-
tacins such as the gourd, Cucurbita texana Gray, in which fruits have been
shown to contain concentrations of cucurbitacins E and I of about 0.44 mg/g
fresh weight and 0.75 mg/g cucurbitacin E glycoside (Metcalf et al., 1982).
Since past identification of C. texana cucurbitacins was based mainly on com-
parison with standard materials (Metcalf et al., 1982), we have reexamined the
cucurbitacin chemistry of this gourd to obtain a more complete cucurbitacin
profile.

METHODS AND MATERIALS

Plant Material. C. texana (Cucurbitaceae) was grown to maturity in

25-cm pots in a greenhouse (Agriculture Experiment Station, University of Del-
aware, Newark, Delaware) and plants were hand pollinated. Fruits were col-
lected when ripe and held in a freezer until extraction.
Extraction. The fruits (1.3 kg) were cut into small pieces, homogenized in
chloroform (4 • 300 ml) for 10 min and filtered. The filtrate was concentrated
in a rotary evaporator to about 100 ml (aqueous), then partitioned with hexane
(4 x 150 ml) and CHC13 (4 X 150 ml). The CHC13 extract was concentrated
and evaporated to dryness (1.6 g). The residue was adsorbed onto the top of a
silica gel column 60 A (80 g, 70-230 mesh ASTM) and chromatographed using
hexane-EtOAc [100, 9: 1, 4:2, 3:2, 2:3, 1:8, 400 ml each, EtOAc-MeOH
(1:1) 600 ml]. Fractions of 100 ml were collected, screened with TLC, and
grouped by similarity. Silica gel GFz54 plates (20 x 10 0.2 mm thickness) were
used and developed with toluene-EtOAc (25:75) and examined under UV.
CUCURBITACINS 31

Fractions 11-18 showed cucurbitacin E (Rf0.8), but in very low concentration

and highly contaminated with chlorophyll; fractions 19-22 provided a yellowish
residue, which was further purified with preparative HPLC (described below)
to give compound 3 (Rf 0.7). Fractions 23-25, 26-30, and 31-36 all provided
compounds with Rfvalues of 0.56 (cucurbitacin I), 0.47, 2, and 0.37, 1, respec-
tively. All compounds were purified by preparative HPLC and crystallized from
MeOH. HPLC separation was performed with a Perkin-Elmer S-100 with UV
detector at 229 nm, preparative column Spherisorb-ODS-2 5/zm (20 cm • 10
mm ID) with gradient separation using solvent A (ACN-H20, 30:70) and sol-
vent B (ACN-H20, 45:50) with B 0% to 100% in 30 rain at a flow rate of 2
ml/min. When developed further with EtOAc-MeOH (1 : 1), fractions 37-44
showed the presence of two glycosides, which, after preparative TLC (solvent,
CHCL3-MeOH 9 : 1), were shown to be the glycosides of cucurbitacin E and I
(2-O-/3-o-glucopyranosyl-cucurbitacin E, and 2-0-/3- o-gtucopyranosyl-cucur-
bitacin I, respectively) by comparison with standard samples (Rf 0.34 and 0.24,
respectively).
Visualization. Vanillin-phosphoric acid reagent was used (Duncan et al.,
1968). TLC plates were heated at 120~ for 10 rain and examined under UV
light at 365 nm. Cucurbitacins appeared as red or yellow spots, c~-Ketol cucur-
bitacins were visualized by spraying with triphenyltetrazolium chloride reagent
(TTC) (Duncan et al., 1968) and appeared as red spots after heating to 100~
for 5 min, which intensified over steam. Diosphenol cucurbitacins were visu-
alized with FeCL3 reagent (Polman, 1975) and appeared as blue spots.
UV analysis was performed with a Hewlett Packard 8452A Diode Array
Spectrophotometer. IR spectra were analyzed by Perkin-Elmer FT-IR Spectrom-
eter 1720X. NMR spectra were examined on a Bmcker AM 250 MHz, and MS
spectra on a VG 7070F, E1 70 eV. Melting points were measured using a Kofler
hot-stage microscope. Optical rotation was measured on Perkin-Elmer model
241 polarimeter.
Compound I (cucurbitacin D) (Fig. 1). White crystals (84 mg from MeOH),
mp 150-152~ [cr 5 + 55 (c = 1, MeOH), positive reaction with TTC reagent
(i.e., c~-ketol system in ring A), Xmax (MeOH) 230 nm; IR (KBr) 3450, 1725,
1705, 1685, 1450, 1375, 1225 and 1085 cml; MS m/z (rel. int.) 516.31 (0.5),
499.30 (M + - H 2 0 + H) (3.9), 481.29 (M § + H) (1.8), 464.26 (1.6),
403.23 (3.9), 369.20 (6.1), 203.12 (7.2), 189.10 (10.4), 166.07 (11.3), 113.05
(87.7), 111.05 (75), and 96.04 (100%).
Compound 2 (3-epi-isocucurbitacin D) (Fig. 1). White crystals (15 mg,
from MeOH), mp 178-180~ [c~]~5 - 78 ~ (c = 1, CHC13), positive reaction
with TTC reagent (i.e., a-ketol system in ring A), kma~ (MeOH) 230 nm, IR
(KBr), 3450, 1725, 1705, 1685, 1450, 1375, 1235, 1085 cm -I. MS m/z (rel.
int.): 516.31 (0.4), 499.30 ( M + - H 2 0 + H) (4.8), 480.29 (2.1), 464.26 (1.6),
403.23 (2.7), 369.20 (8), 203.12 (8.8), 189.10 (13.6), 166.07 (8.3), 115 (22.6),
113.05 (95.7), and 111.05 (100%).
32 HALAWEISH

TABLE 1. tH NMR SmFTS OF RELEVANTPROTONS OF COMPOUNDS1, 2 AND 3 a

Compound

Proton No. 1 2 3

2 4.45 dd(14, 7) 4.44dd(14, 7)

3 4.10
6 5.75 m 5.75m 5.75m
12~ 2.74 br d(15) 2.72 br d(15) 2.74 br d(15)
12c~ 3.28 br d(15) 3.20 br d(15) 3.20 br d(15)
16 4.34 br t(7) 4.33 br t(7) 4.34 br t(7)
17 2.52 d(7) 2.40 d(7) 2.60 d(7)
23 6.62 br d(15) 6.62 br d(15) 6.62 br d(15)
24 7.12 br d(15) 7.12 br d(15) 7.12 br d(15)
Methyls:
18 0.95 0.95 0.98
19 1.09 1.10 1.08
21 1.34 1.34 1.35
26 1.34 1.36 1.40
27 1.38 1.38 1.40
28 1.32 1.36 1.25
29 1.34 1.34 1.35
30 1.30 1.34 1.35
25-OAc 2.02

"Coupling constant (J) in Hz is quoted in parentheses. Signals are singlets unless otherwise indi-
cated.

Compound 3 (cucurbitacin B) (Fig. 1). White crystals (30 mg, from MeOH),
mp 186-188~ [lit. (Jacobs et al., 1990) mp 184~ [ot]~5 + 83 (c ; eq 1,
MeOH), positive reaction with TTC reagent (i.e., c~-ketol in ring A); Xmax
(MeOH) 230 nm; IR (KBr) 3450, 1725, 1707, 1690, 1450, 1375, 1225, and
1085 c m - 1 ; MS m/z (rel. int.), 499.30 ( M § acid + H) (0.7), 498.30
(4), 480.30 (2.7), 465.25 (4.6), 403.23 (1.5), 369.20 (18.5), 203.12 (10.9),
189.10 (23.9), 166.07 (16.3), 113.05 (21.2), 111.05 (89.5), 96.04 (99.4), 60.03
(32.5), and 43.02 (CH3CO) (100%).
The 1H N M R and 13C N M R data o f compounds 1, 2, and 3 are presented
in Tables 1 and 2.

RESULTS AND DISCUSSION

The mass spectrum o f compound 1 showed the (M +) peak at m/z 516.3098

(C3oH4407) with the two fragments at m/z 499.2988 and 480.2879, which is in
accordance with the molecular formulas o f C3oH4206 + H and C3oH4oO5,
CUCURBITACINS 33

TABLE 2. 13C N M R SHIFTS OF COMPOUNDS 1, 2 AND 3 a

Compound

Carbon No. 1 2 3

1 36.02 36.45 35.98

2 71.16 210.98 71.63
3 212.38 79.45 212.51
4 50.67 47.86 50.66
5 140.35 140.13 140.41
6 120.10 121.3 120.45
7 24.28 24.36 23.85
8 41.32 42.4 42.37
9 48.30 48.73 48.42
10 34.63 32.36 33.72
11 213.12 212.39 213.07
12 48.27 48.83 48.09
!3 50.24 50.63 50.21
14 48.27 48.35 48.63
15 45.42 45.63 45.21
16 70.40 71.14 71.25
17 58.10 57.43 58.19
18 20.17 19.98 19.81
19 20.20 20.40 20.08
20 77.98 78.09 77.20
21 23.76 23.94 23.94
22 202.44 202.68 202.51
23 119.01 119.01 120.33
24 154.80 155.73 152.00
25 72.68 72.41 79.30
26 30.31 29.51 26.38
27 28.30 29.01 26.00
28 28.26 28.69 29.33
29 21.01 19.98 21.22
30 19.04 18.68 18.98
25-OAc 170.25

aShifts in ppm, solvent CDC13 and TMS as standard.

r e s p e c t i v e l y , w h i c h arise d u e to t h e c o n s e c u t i v e loss o f t w o m o l e c u l e s o f water.

A c u c u r b i t a n e n u c l e u s w a s s h o w n b y 1H N M R d a t a ( T a b l e 1) s u c h as 6 5.75
(m), ~ 3 . 2 8 (d, J = 15), ~5 2 . 7 4 (d, J = 15) a n d / i 4 . 3 4 (t, J = 7) a s s i g n e d for
H - 6 , 12c~-H, 1 2 ~ - H a n d H - 1 6 , r e s p e c t i v e l y ( V e l d e a n d L a v i e , 1983). I n addi-
t i o n , t h e o l e f i n i c c e n t e r in t h e side c h a i n at C - 2 3 , 2 4 w a s identified at 15 6.61
(d, J = 15) a n d ~ 7 . 1 2 (d, J = 15) a s s i g n e d for H - 2 3 a n d H - 2 4 , r e s p e c t i v e l y ,
a n d t h e d o u b l e d o u b l e t at ~ 4 . 4 5 [dd, J = 14, 7] w a s a s s i g n e d f o r H - 2 in t h e
34 HALAWEISH

Ho /~ ',/~-. I ~

1 R= H
3 R= COCH3

o ~H

HO / ~ ~--"

HO" / / ~ "

FIG. 1. Structures of cucurbitacins D, 1, 3-epi-isocucurbitacin D, 2, and B, 3, and the

cucurbitacin precursor 10c~-cucurbitan-5,24-dien-3/3-ol, 4.

ot-ketol system in ring A (Velde and Lavie, 1983; Jacobs et al., 1990). The
latter statement indicated that compound 1 was a normal cucurbitacin (i.e.,
2-hydroxy-3-oxo) in ring A. The rest of the proton shifts were assigned by
comparison, which all support the structure of compound 1 as cucurbitacin D
(Velde and Lavie, 1983; Jacobs et al., 1990). 13C NMR data (Table 2) showed
CUCURBITACINS 35

complete similarity with published data (Velde and Lavie, 1983; Jacobs et al.,
1990) and also confirmed that compound 1 is cucurbitacin D.
The mass spectrum of compound 2 showed the (M +) peak at m/z 516.3083
(C3oH4407) with the mass fragments at 499.2872 [ M + - H 2 0 + H] and m/z
480.2981 (M + - 2 H 2 0 ) , which is in accordance with the molecular formulas of
{ C 30H4206 and C30H4005 and confirms the (M +) peak. The main features of
its 1H NMR (Table 1) spectrum were of a cucurbitane nucleus, such as 6 5.75
for H-6, ~ 3.28 (d, J = 15) for 12a-H, b 2.70 (D, J = 15) for 12fl-H, b 4.3
(t, J = 7) for H-16, and A23'24 protons at 6 6.60 (d, J = 15) and 6 7.12 (d, J
= 15) for H-23 and 24, respectively (Velde and Lavie, 1983; Jacobs et al.,
1990). The latter unsaturated bond was confirmed by the presence of the major
peak in the MS spectra at m/z 111.043 (100%) (Kupchan et al., 1970). A sharp
singlet at 6 4.1 was assigned for the C-3 methine hydrogen in the
c~-ketol and indicated that compound 2 is a member of the isocucurbitacin series
(Kupchan et al., 1978; Hylands and Magd, 1986). The 2-hydroxyl-3-oxo-cucur-
bitacins, such as 1, show the signal for H-2 at 3 4.45 as a double doublet (Velde
and Lavie, 1983). The fact that H-3 in Compound 2 resonated at 6 4.1 is evidence
for an axial hydrogen (i.e., equatorial hydroxy group) and indicative of an
epimeric isocucurbitacin known as 3-epi-isocucurbitacin D 2, which was reported
previously from Phormium tenax Forest (Arisawa et al., 1984). The structure
of the rest of the molecule was established primarily by comparison of its 1H
NMR data with those known for cucurbitacins and further confirmed with 13C
NMR (Table 2), which showed complete similarity to cucurbitacin data (Velde
and Lavie, 1983; Jacobs et al., 1990). Furthermore, the c~-ketol system in ring
A was positively confirmed by reaction with TTC reagent (Duncan et al., 1968).
All of these data proved compound 2 to be 3-epi-isocucurbitacin D.
Compound 3 showed chemical shifts (Tables 1 and 2) identical with those
of cucurbitacin D (compound 1), except for a sharp singlet at 6 2.02 assigned
to an acetate group and accommodated at C-25 hydroxyl, which was also con-
firmed by 13C NMR shifts at 170.25 and 20.25 ppm. (Velde and Lavie, 1983;
Jacobs et al., 1990). This indicated that compound 3 is cucurbitacin B, which
was also proven through comparison of the physical and MS published data
(Arisawa et al., 1984).
This represents the first isolation and identification of cucurbitacins B, D,
and 3-epi-isocucurbitacin D, and the identification of cucurbitacin I glucosides
from C. texana. The latter glucoside was identified by comparison with known
standard. It is also the first isolation of 3-epi-isocucurbitacin D 3 from any
member of the genus Cucurbita. Of further significance is the presence and
isolation of 3-epi-isocucurbitacin D 3 under the mildest conditions of extraction
and preparation, confirming previous suggestions (Halaweish, 1987; Hylands
and Magd, 1986) that isocucurbitacins are naturally occurring compounds rather
than artifacts (Hylands and Mansour, 1983; Kupchan et al., 1978; Arisawa et
36 HALAWEISH

al., 1984). The presence of 3-epi-isocucurbitacin D 3 in C. texana tissues was

also confirmed in a fresh fruit sample extract (screened with HPTLC and HPLC),
which excludes the possibility of formation during the purification processes.
The results of this study suggest an important step in the biosynthetic
pathway of cucurbitacins which, to date, has not been considered in as much
detail as have the pathways of other related groups of triterpenes and steroids.
The order of oxidation in ring A primarily determines the configuration of
cucurbitacins as normal or iso. From our results and some earlier work (Hal-
aweish, 1987), we offer the following support for the hypothesis that isocucur-
bitacins are formed one step prior to normal cucurbitacins. Cucurbitacin
formation begins with squalene-2, 3-epoxide and proceeds through other inter-
mediates to the main cucurbitacin precursor, 10o~-cucurbita-5,24-dien-3/~-ol 4
(Fig. 1) (Balliano et al., 1983). The latter compound 4 and isocucurbitacins are
hydroxylated at C-3 rather than C-2. Other related aglycones and glycosides
such as bryogenin and bryosides (isolated from Bryonia dioica) are also hydrox-
ylated only at C-3 in ring A (Hylands and Kosugi, 1982). Further evidence is
provided by the transformation of the intermediate 4 to cucurbitacin C after
incubation with seedlings of Cucumis sativus L; cucurbitacin C has only C-3
OH in ring A (Balliano et al., 1983). Moreover, cucurbitacins such as F, O, P
and Q (2,3 diol in ring A) (Lavie and Glotter, 1971) provide a possible inter-
mediate in the process of transformation of iso to normal cucurbitacin. Perhaps
the best evidence comes from the fact that labeled isocucurbitacin B has been
retrieved from seedlings and tissue cultures of B. dioica previously fed with
labeled precursors (Halaweish, 1987). All of these data suggest that hydrox-
ylation takes place at C-3 first rather than at C-2 and indicate that isocucurbitacin
formation occurs one step ahead of normal cucurbitacin biosynthesis.

Acknowledgments--I am very grateful for the kind support and expertise of Dr. Douglas
Tallamy in producing this work. Designated Miscellaneous Paper 1048 of the Delaware Agricultural
Experiment Station, Contribution No. 627 of the Department of Entomology and Applied Ecology,
University of Delaware, Newark, Delaware, USA.

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