Pharmaceutical Chemistry Journal, Vol. 45, No. 10, January, 2012 (Russian Original Vol. 45, No.

10, September, 2011)

ISOLATION AND CHARACTERIZATION OF A NEW ANTICANCER

DITERPENOID FROM JATROPHA GOSSYPIFOLIA
A. Falodun,

1,*

Qiu Sheng-Xiang, G. Parkinson, and S. Gibbons

Original article submitted March 31, 2011.

Jatropha gossypifolia root bark is used in ethnomedicine for the treatment of various diseases and
infections. We have studied the phytochemistry and evaluated the anticancer activity of J. gossypifolia root
extract. Phytochemical investigation of the root of J. gossypifolia resulted in the isolation and
characterization of one new diterpenoid along with four well-known compounds. The new compound was
1
13
established by 1D and 2D NMR spectra and x-ray analysis, while spectral ( H, C NMR, and ESI-MS)
characteristics of the known compounds were compared with reported data. The new compound showed
potent proliferation inhibitory ac-tivity against A-549 human cancer cell line.
Key words: Jatropha gossypifolia, root extract, anticancer activity, lathyrane diterpenes, cytotoxicity

In this paper, we report

on
the
isolation,
characterization, structure
Bellyache bush or Jatropha gossypifolia L. is a medici-nalelucidation, and in vitro
plant used in ethnomedicine for the treatment of several anticancer activity of one
diseases and infections. The ethnomedicinal use ranges from new and several known
diterpenes
anti-inflammatory, analgesic, and anticancer to the treatment oflathyrane-type
isolated
from
J.
gossypifolia
gastric disorders. Bellyache bush is a small shrub belong-ing to
the Euphorbiaceae family that is widely distributed in manyroot.
tropical countries, where it has been introduced as or-namental
EXPERIMENTAL PART
and medicinal plants [1]. It forms dense thickets, competing
with useful species, hindering mustering, and re-ducing pasture
Physicochemical
productivity in rangelands, particularly in ri-parian zones [2].
methods. Melting points
All parts of J. gossypifolia plant are highly toxic, causing thewere mea-sured using a
death of animals that consumed it, espe-cially during drought Buchi Model 510 instrument
[1]. It is an erect shrub up to 3 m high, evergreen, perennial, and remained un-corrected.
reproducing by seeds and vegetative re-generation. It was alsoOptical
rotations
were
used in ancient medicine for the treat-ment of cancerous determined with a Jasco
DIP-360 digital polarimeter.
growth [3, 4] and pesticidal purposes [5 7].
1
The H NMR (400 MHz)
The reported phytochemical constituents include lignans
13
and several diterpenoids [8, 9]. Some new diterpenes and theirand C NMR (400 MHz)
analogs have been isolated and characterized, includingspectra of the purified
jatropholones A and B, as well as lignans, jatrophane and compound were recorded on
a Bruker DRX-400 NMR
venkatasin [10 12].
spectrometer (Bruker Co.,
1
Germany),
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Univer-sity of Benin, Rheinstetten,
Benin City, Nigeria.
using
deuterated
2
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, dimethylsul-foxide (DMSOPeoples Republic of China.
d6) as the solvent and the
solvent residual peak as a
3 The School of Pharmacy, University of London, 29 39 Brunswick Square, London reference. Chemical shifts
( ) are expressed in parts per
WC1N 1AX, United Kingdom.
million (ppm) relative to
TMS.
INTRODUCTION

* e-mail: faloabi@uniben.edu, faloabi25@yahoo.com.

mass spectroscopy (ESIMS) analysis was performed

on an Applied Biosystems
API2000
LC/MS/MS
System (ABI, Foster City,
CA). The mass spec-tra were
measured using electrospray
ionization in both posi-tive
and negative ion modes. The
capillary 4500 V (negative)
and 5500 V (positive) were
used in this study. The
electrospray probe flow was
adjusted to 20 ml/min.
Continu-ous mass spectra
were recorded by scanning
from 100 to 800 m/z.
Plant Material. Fresh
plant
samples
of
J.
gossypifolia was collected at
Otukpo in Benue State in
March 2009, iden-tified by
Mr. Simon Peters, and
authenticated by Prof. M. Idu
(Department
of
Botany,
Faculty of Life Science,
University

Electrospray ionization
636
0091-150X/12/4510-0636

2012 Springer Science+Business Media, Inc.

Isolation and Characterization of a New Anticancer Diterpenoid

637

Crude methanol extract

of Benin, Benin City, Nigeria). A voucher specimen of her- was partitioned with pet
ether
(60

90C),
barium number IFE 2700 was deposited.
chloroform, ethylacetate and
butanol with 6.8, 2.5, 7 and
Biological Screening
3
g
fraction
yield,
respectively.
Cell line and culture. A-549 (human lung adenocarciThe pet ether fraction was
noma epithelial cell line) cell culture was obtained from
subjected to chromatography
Biomedicine Research and Development Centre of Jinan
University (Guangzhou, China). The cell lines were cultured inover repacked column solid
growth medium (RPMI-1640 medium, pH 7.4), supple-mented silica gel (200 300 mesh)
with 10 % fetal bovine serum (FBS) and antibiotics [penicillin and eluted with solvents of
(100 units/ml) and streptomycin sulfate (100 g/ml)]. The cell increasing polarity using a
2
lines were grown in 50 cm tissue culture flasks (Corning, NY,stepwise gradient of EtOAc (0
100%, v/v) in pet ether. A
USA) and used for cytotoxicity assay.
total of 25 fractions were

Cytotoxicity assay. The cytotoxicity of the samples wascollected. Fractions of similar

studied using the method [13] based on MTT (3-(4,5-dime- compositions, as determined
thyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. In by thin-layer chromatography
4
brief, human cancer cells were plated at 2 10 cells per well in(TLC) and reac-tion with
96-well microtiter plates (Costar 3599, Corning, NY, USA) H2SO4 spray reagent, were
with 100 l of RPMI-1640 growth medium and incu-bated for pooled together to give ten
24 h at 37C with 5% CO2 in a humidified atmo-sphere (Incu-combined fractions. Then,
column
Safe, Sanyo, Japan), during which period a par-tial monolayer repeated
chromatogra-phy
gave
pure
was formed. Then, the medium was removed and fresh growth
compounds,
which
were
remedium containing various concentrations (100, 50, 25, 12.5,
6.5, and 3.125 g/ml) of the test com-pound was added. After a crystallized from an acetone
ether (1 : 4) mixture. One new
2-day incubation at 37C with 5% CO2, the growth medium
compound (1) was subjected
was removed and MTT reagent (0.1 mg/ml) was added. Uponto spectroscopic analysis by
the incubation at 37C for 4 h, the MTT reagent was removed 1D and 2D NMR, ESI, and IR
and DMSO (100 l) was added to each well and then shaken for techniques. Four known
15 min. Then, the absorbance was determined by an ELISA compounds obtained from
reader (Bio-Rad, USA) at a wavelength of 492 nm. Controldifferent solvent systems
wells contained only the incubation medium without test included jatropholone-B (1.02
compound. The conventional anticancer drug, cisplatin, was g,
2),
(4E)used as a posi-tive control in this study. The inhibition of jatrogrossidentadione acetate
cellular growth by the test compound was calculated as the (10 mg, 3), jatrophone (9 mg,
percentage inhibi-tory activity and expressed as the IC 50 value4), and citlalitrione (15 mg,
(concentration of the test compound necessary to inhibit cells 5). The com-pounds were
growth by 50%).
analyzed using ID- and 2DNMR

spectroscopy

Statistical analysis. Data were expressed as meantechniques in comparison to

standard deviation (SD) for three replicate determinations and
data reported in the literature.
then analyzed using SPSS V.13 software (SPSS Inc., Chicago,
Compound 2. White
USA). One-way analysis of variance (ANOVA) and the
solid;
m.p. 152 154C;
22
Duncans new multiple-range test were used to deter-mine the D , 261 (CHCl ; c,
3
differences among these means; confidence values P < 0.050.001); UV (MeOH; max,
nm): 256; EIMS (probe, 70
were regarded as significant.
eV),+ m/z (rel. int.): 332+
{M } (5), 314 (M-H2O)
EXTRACTION AND ISOLATION
(2), 304 (3), 223 (7), 176
(14), 163 (16); HREIMS
+
The air-dry powdered sample of J. gossypifolia root wasobsd. (m/z), 332.1981 (M ),
calcd.
for
C
H
O
20 28 4,
reduced to fine powder using a mechanical mill. The dried
1
13
332.1987); H and C NMR
ground root (500 g) was macerated with a methanol (2.5 L) at data [14].
room temperature for 48 h. The extract was filtered and
Compound 3. White
concentrated using a rotary evaporator at reduced pressure tosolid; m.p., 54 56C; D22,
give a thick brown mass (25 g). The crude extract was stored in 175 (CHCl3; c, 0.026);
UV (MeOH; max, nm): 262;
a refrigerator at 4C until use.
EIMS (probe, 70 eV), m/z
638

(rel. int.): 314 (M-H2O) (2),

299 (2), 271 (11), 161 (34),
109 (26),1367 (50), 55 (59),
43(100); C NMR data[15].
Compound 22
4. Light
yellow oil; D , 15
(CHCl3; c, 0.002); UV
(MeOH; max, nm): 252;
EIMS (probe, 70 eV), m/z
(rel. int.): 322 M+ (1), 314
(M-H2O) (1), 299 (2), 271
(9), 161 (6), 123 (7), 109
(11), 67
(20),1355 (29), 43
1
(100); H and C NMR data
[15].
Compound
5. Light
22
yellow oil; D ; 7 (CHCl3;
c, 0.001); UV (MeOH; max,
nm): 253; EIMS (probe, 70+
eV), m/z (rel. int.): 332 M
(1), 314 (M-H2O) (1), 299
(2), 271 (9), 161 (6), 95
(16), 81
(16),13 55 (32), 43
1
(100); H and C NMR data
[15].
X-ray crystallographic
analysis of compound 1
crystal-lized from pet ether
acetone showed that the
crystal
system
was
orthorhombic.
RESULTS AND
DISCUSSION
The pet ether fraction
obtained from J. gossypifolia
ex-tract was first purified on a
silica gel column, from which
ten fractions were collected
and pooled into 4 major
fractions as monitored by
TLC and reaction to H2SO4
spray reagent (fractions 1
4). Among these, fraction 4
was found to show three spots
on TLC plate and then
additionally purified by silica
gel column. Four fractions
were obtained and then
grouped into two subfractions
(2A and 2B). The major subfraction (2A, 40 mg) was
finally obtained using the re-

A. Falodun et al.
17

12
10

O
14
1
16

19

18

O
5

H
H

OH

Fig. 1. Structure of compound 1.

H
H

peated column chromatography with a burette-like glass column to give compound 1, crystalline solid.
The new compound isolated from J. gossypifolia was
identified as lathyrane diterpene, called falodone (Fig. 1), on
1
13
the basis of ESI-MS, H and C NMR, and x-ray analyses.
The compound was obtained as white solid with m.p. 247C.
The ESI-MS in the positive and negative ion modes exhib+
ited signals of quasi-molecular ions at m/z = 285 [M + H] ,
which corresponded to a molecular formula of C 19H24O2 in
13
combination of its nineteen carbon signals in the C NMR
spectrum (CHCl3-d6, 400 MHz). The molecular formula
C19H24O2 assigned to compound 1 was determined by
13
analy-sis of the C NMR data associated with HR ESI (+)
MS. The IR spectrum indicated the presence of hydroxyl
1
1
group (3407 cm ), ketone carbonyl group (1718 cm ),
1
and aro-matic ring (1612 and 775 cm ).
1
The H NMR spectrum (Table 1) exhibited signals for
one aromatic proton at = 5.24 ppm (s) and suggested a
1,2,3,4,5-substituted aromatic ring in the molecule. The
spectrum also displayed signals for four vinyl methyl groups

TABLE 1. H and
for Compound 1
Position

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

13

C NMR (CDCl3 at 400 MHz) Chemical Shifts

H NMR

Multiplicity (J, Hz)

1.83
2.55

5.24

1.60
0.91
0.93
1.62
1.58
3.22

1.26
1.28
0.81
1.24

d(13.6)
m

m
m
d (8.0)
m

s
s
s
s

13

C NMR

208.2
42.6
30.1
134.3
150.1
114.8
137.1
25.9
16.1
19.5
15.8
33.5
42.4
131.8
145.6
13.3
28.2
17.1
21.4

HO

Fig. 2. HMBC correlations of structure 1.

at H = 0.81, 0.93, 1.24 and 1.26 ppm; four allylic methylene

groups manifested by multiplets at H = 0.81 0.82 and 0.93
0.94, a doublet at = 1.26 ppm (2H, J = 8.0 Hz), and a
singlet at 1.24 ppm. Five methane protons were also
13
observed. The C NMR (400 MHz) spectra of compound 1
revealed the presence of 19 non-equivalent carbon atoms.
The protonated carbon were assigned from the HSQC experiment and characterized by DEPT experiment, while the
non-protonated carbons were detected by the HMBC experi13
ment. The C NMR spectrum in combination with the
above data conspicuously showed the signals for one
carbonyl, aro-matic double bonds, four methyl groups and
two oxygenated quaternary carbons (Table 1).
1

13

Thus, the H and C NMR spectra (Table 1) of compound 1 suggested that it is a lathyrane-like diterpenoid [15].
1
13
All the signals for the protons and carbons in the H and C
NMR spectral respectively were assigned from 2D NMR
1
1
( H H COSY, HSQC, HMBC, and NOSEY) and DEPT
experiments. The compound contains one hydroxyl group at
position 5. The methyl groups were present in positions 2,
13 and 9 (two methyl groups). The HMBC experiment
showed that the H-3 (2.55) was correlated to C-2, C-4
(134.3). H-17 (1.26) to C-12 (33.5) and C-13 (42.4), H11(15.8) to C-10 (19.5) and C-12 (33.5), H-16 (1.24) to C-2
(42.6), C-1) 208.4) and C-3 (C-3). These correlations
suggested the pres-ence of a cyclopentanone ring involving
C-1, C-2, C-3, C-4 and C-15. The HMBC spectrum also
showed a correlation of M-18 ( , 0.93 ppm) and Me-19 ( ,
0.81 ppm) with C-8 ( , 25.9 ppm) and C-10 ( , 19.5 ppm),
suggesting the presence of a cyclopropane moiety at C-8 and
C-10 gem-dimethyl groups at C-9.
All these data allowed full assignment for all hydrogen and
carbon atoms, and established the structure of compound 1 as
TABLE 2. Anticancer activity of compound 1
Compound

IC50 (g/mL)
A-549 cell line

120.0 0.9

Cisplatin*

101.3 1.2

Notes: IC50 value is the concentration of the test sample necessary

to kill 50% of cancer cells (A-549 human lung adenocarcinoma
epithe-lial cell line); * positive control.

Isolation and Characterization of a New Anticancer Diterpenoid

639

REFERENCES

20

O
HO

14

O
3

1
0

O
1
8

HO

O
13

Fig. 3. x-Ray crystallographic structure of compound 1

4
O

O
6

HO

cyclopropa[3, 4]cyclohepta[1,2-e]inde-6-one, 1,1a,4,5,7,8,9

17
3
octahydro-3-hydroxy-1,1,5,7-tetramethyl. Thus, the structure
and stereochemistry of falodone were clearly established.
Finally, the proposed structure of falodone (Fig. 3) was confirmed by the data of x-ray crystallographic analysis:
C19H24O2; M = 284.38; monoclinic system.
5
The structures of known compounds (Fig. 4) were estab- Fig. 4. Structures of compounds
lished by comparison of their physical and spectral proper-ties 2 5.
with data reported in the literature. The occurrence of these
compounds in J. gossypifolia is reported here for the first time.
The anticancer activity of lathyrane
The authors
was reported by Schmeda [15]. In this
are
grateful
to the
study, the anticancer activity of comChinese
Academy
pound 1 with reference to cisplatin was
Sci-ences
investigated using the MTT assay on A-of
549 human lung cancer cell line. A mi-(CAS) and the
World
tochondrial enzyme in living cells,Third
Academy
of
succinate-dehydrogenase, cleaves the
Sciences
(TWAS)
tetrazolium ring and converts the MTT to
an in-soluble purple formazan, and the for the Fellowship
amount of formazan pro-duced is directlygrant for Dr.
proportional to the number of viable cells. Abiodun Falodun.
Support from the
Table 2 shows that 1 (at IC50 of 120University
of
g/mL), caused inhibi-tion of A-549 cell Benin is also
growth, thus indicating that it could be a appreciated.
potential anticancer agent.
Special thanks to
Rev.
Father
Adodo of the Ewu
Monastery
for
In the present study, one newdirective to the
compound was isolated from J. plant.
gossypifolia, purified, and identified as
falodone. It was established that the
isolated compound exhibited anticancer
activity.
CONCLUSION

ACKNOWLEDGEMENT

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M.
Csurh
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ache
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(Jatro
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gossy
pifoli
a) in
Quee
nslan
d,
Pest
Revie
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Series

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