A complex of 5-hydroxypyrrolidin-2-one and pyrimidine-2,4-dione isolated from Jatropha curcas

Article abstract of DOI:10.1016/S0031-9422(98)00531-7

A complex of 5-hydroxypyrrolidin-2-one and pyrimidine-2,4-dione was isolated from the leaves of Jatropha curcas L. by extraction with ethyl acetate and subsequent fractionation of the extract by column chromatography on Sephadex LH20, silica gel and fractogel TSK HW 40. Final purification was carried out by HPLC on a preparative RP-18 column and the structure was proposed mainly by mass spectrometry, ¹H- and ¹³C-NMR.

Full text of DOI:10.1016/S0031-9422(98)00531-7

PERGAMON
Phytochemistry 50 (1999) 337±338
A complex of 5-hydroxypyrrolidin-2-one and pyrimidine-2,4-dione
isolated from Jatropha curcas
Ruth Staubmanna, *, Manfred Schubert-Zsilaveczc, Alois Hiermannb,
Theodor Kartnigb
aInstitut fur Biotechnologie der Technischen Universitat Graz, Petersgasse 12/1, A-8010 Graz, Austria
È
È
bInstitut fur Pharmakognosie der Universitat Graz, Universitatsplatz 4, A-8010 Graz, Austria
È
È
È
cInstitut fur Pharmazeutische Chemie der Johann Wolfgang Goethe-Universitat Frankfurt am Main, Marie-Curie-Straûe 9, D-60439 Frankfurt am
Main, Germany
È
È
Revised 8 July 1998
Abstract
A complex of 5-hydroxypyrrolidin-2-one and pyrimidine-2,4-dione was isolated from the leaves of Jatropha curcas L. by
extraction with ethyl acetate and subsequent fractionation of the extract by column chromatography on Sephadex LH20, silica
gel and fractogel TSK HW 40. Final puri®cation was carried out by HPLC on a preparative RP-18 column and the structure
1
was proposed mainly by mass spectrometry, H- and 13C-NMR. # 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Jatropha curcas L; Euphorbiaceae; Leaves; Isolation; Pyrrolidinones; 5-Hydroxypyrrolidin-2-one
1. Introduction
2. Results and discussion
Jatropha curcas L. is cultivated as a medicinal plant
in many tropical and subtropical countries. The leaves
and also the other parts of the plant are used for the
treatment of various diseases. Compounds that have
been isolated from J. curcas leaves include the ¯avo-
noids apigenin and its glycosides vitexin and isovitexin,
the sterols stigmasterol, b-D-sitosterol and its b-D-glu-
Fresh leaves were extracted with ethyl acetate and
the extract subsequently fractionated on Sephadex LH
20, silica gel and fractogel TSK HW40. Bio-assay-
guided isolation was done by testing the anti-in¯am-
matory using a carrageenan-induced rat paw edema-
test (Winter, Risley, & Nuss, 1962). Final puri®cation
of the substance was achieved by HPLC on a prepara-
tive RP-18 column. The pure compound was white,
crystalline and soluble in water and methanol.
coside (Chhabra, Mahunnah,
&
Mshiu, 1990).
Furthermore J. curcas leaves were reported to contain
steroid sapogenins, alkaloids, the triterpenae alcohol,
1-triacontanol and a dimer of a triterpene alcohol
(Neuwinger, 1994).
Although Kuhnt, Probstle, Rimpler, Bauer, and
Heinrich, 1995 isolated (R)-5-hydroxypyrrolidin-2-one
from the leaves of Hyptis verticillata Jacq. (Labiatae)
(Kuhnt et al., 1995), there is no report in the literature
concerning the isolation of this compound from plants
belonging to the Euphorbiaceae.
The use of 2-D NMR techniques allowed the assign-
ment of all the 1H- and 13C-NMR resonances and a
nonambiguous determination of the structure of 5-
hydroxypyrrolidine-2-one (1) and pyrimidin-2,4-dione
(uracil) (2). By means of an H,H-COSY (Aue,
Bartholdi, & Ernst, 1976) spectrum the resonances
were assigned to two spin systems (3-H2/4-H2/5-H, 1;
1
45-H/6-H, 2). On the basis of the known H chemical
shifts and proton±proton couplings, the unambiguous
assignment of the protonated carbon atoms was car-
ried out using a HSQC (Bodenhausen & Ruben, 1980)
spectrum, which reveals the one-bond C±H connectiv-
ities. The HMBC (Bax & Summers, 1986) spectrum
enabled us to assign the nonprotonated carbon atoms.
* Corresponding author.
0031-9422/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00531-7

338
R. Staubmann et al. / Phytochemistry 50 (1999) 337±338
The NMR data of 1 are in good agreement with those
of the synthetic racemate (Farina, Martin, Paredes,
Ortega, & Tito, 1984) and the natural (R)-5-hydroxy-
pyrroldin-2-one (Kuhnt et al., 1995). Additionally, the
structures of 1 and 2 were corroborated by GC±MS
analysis after trimethylsilylation.
From the foregoing, it was assumed that the two
substances which occur in the ratio of 1:1 form a com-
plex, which could not be separated by HPLC and
TLC. Further investigations on the pharmacological
action of this novel complex are in progress.
bined and the compound puri®ed by CC (fractogel
TSK HW(40), isocratic elution with 40% EtOH).
Fractions of 10 ml were collected and monitored by
TLC. Fraction 11 showed a pure compound on TLC.
Final puri®cation was carried out by HPLC on a pre-
parative RP-18 column (isocratic elution with 10%
CH3CN) to give 3.0 mg pure white compound.
1
5-Hydroxypyrrolidin-2-one (1): C4H7NO2; H-NMR:
3
3
d =5.24 (dd, J5,4peq =1.7 Hz, J5,4pax =6.3 Hz, H-5),
2.48 (m, J3pax,3peq =16.7 Hz, J3pax,4pax = 9.9 Hz,
2
3
2
3J3pax,4peq =7.9 Hz, H-3pax), 2.37 (m, J4pax,4peq
=
13.5 Hz, J4pax,3pax = 9.9 Hz, J4pax,3peq =9.7 Hz,
3
3
2
3J4pax,5 =6.3 Hz, H-4pax), 2.19 (m, J3peq,3ax =16.7
3
3
3. Experimental
Hz, J3peq,4pax =9.7 Hz, J3peq,4peq =3.5 Hz, H-3peq),
3
2
1.90 (m, J4peq,4pax =13.5 Hz, J4peq,3pax = 7.9 Hz,
3
3.1. General
3J4peq,3pax =3.5 Hz, J4peq,5 =1.7 Hz); 13C-NMR:
d =182.3 (C-2), 80.9 (C-5), 31.3 (C-4), 29.5 (C-3).
Pyrimidine-2,4-dione (uracil) (2): C4H4N2O2; 1H-
NMR: NMR spectra were recorded with a Varian
UNITYplus-400, 5 mm inverse probe head; solvent:
[D4]MeOH; T 303 K. The MeOH signal was used as
an internal standard (1H: d= 3.3, 13C, d 49.0). The
parameters were as follows: COSY: 458 mixing pulse.
HMBC: phase-sensitive using TPPI, delay to achieve
long-range couplings 71 ms.
GC±MS: Finigan MAT 312 system with Icis data
system, EI mode, 70 eV, and Hewlett Packard 5890
Series II, using a 30 m  0.32 mm DB-05 fused silica
capillary column; helium was used as carrier gas;
temp. program: 3 min isotherm. at 50, 50±1008C (48C
min 1, 100±3008C (38C min 1).
3
NMR: d =7.38 (d, J6,5 = 7.6 Hz, H-6), 5.59 (d,
3J5,6 =7.6 Hz, H-5); 13C-NMR: d= 167.8 (C-4), 153.9
(C-2), 143.5 (C-6), 101.7 (C-5).
GC±MS analysis: 0.2 mg of the complex were
transformed (3 h, room temperature) with 10 ml N-
methyl-N-(trimethylsilyl)-tri¯uoroacetamide (MSTFA).
2,4-bis[(trimethylsilyl)oxy]-pyrimidine: C10H20N2O2Si2,
m/z (rel. int.): 256 (M + , 58), 241 (100), 207 (10), 147
(30). N-trimethylsilyl-5-trimethylsilyloxy-pyrrolidin-2-on:
C10H23NO2Si2, m/z (rel. int.): 245 (M + , 30), 230 (27),
156 (20), 147 (100).
3.2. Plant material
Acknowledgements
Fresh leaves of cultivated J. curcas were obtained
from Proyecto Biomasa, Universidad Nacional de
Ingenierõa, Managua, Nicaragua. A voucher specimen
is deposited at the Institute of Pharmacognosy,
University of Graz, (Herbar No. J95/01).
This project was supported by the company Sucher
& Holzer and the Austrian government.
References
3.3. Isolation of the compound
Aue, W. P., Bartholdi, E., & Ernst, R. R. (1976). Journal of Chemistry
and Physics, 64, 2229.
Fresh leaves (200 g) were defatted with petrol in a
Soxhlet apparatus, extracted with EtOAc at room tem-
perature and the marc ®nally Soxhlet extracted with
EtOAc. The extract was concentrated in vacuo and
subjected to CC (Sephadex LH 20, isocratic elution
with EtOAc±MeOH 3:2). Fractions of 20 ml were col-
lected and monitored by TLC. Fraction 1±20 were
combined, and the compounds separated by CC (silica
gel, elution with EtOAc±MeCOEt 5:3, EtOAc±
MeCOEt±MeOH 5:3:2, EtOAc±MeCOEt±MeOH±
HCOOH 5:3:2:0.5). Fractions of 20 ml were collected
and monitored by TLC. Fraction 26±28 were com-
Bax, A., & Summers, M. F. (1986). Journal of the American Chemical
Society, 108, 2093.
Bodenhausen, G., & Ruben, D. J. (1980). Chemical and Physical
Letters, 69, 185.
Chhabra, S. C., Mahunnah, R. L. A., & Mshiu, E. N. (1990). Journal
of Ethnopharmacology, 28, 255.
Farina, F., Martin, M. V., Paredes, M. C., Ortega, M. C., & Tito, A.
(1984). Heterocycles, 22, 1733.
Kuhnt, M., Probstle, A., Rimpler, H., Bauer, R., & Heinrich, M.
(1995). Planta Medica, 61, 227.
Neuwinger, H.D. (1994). Afrikanische Arzneip¯anzen und Jagdgifte
(p. 450). WV GesmbH, Germany.
Winter, C. A., Risley, E. A., & Nuss, G. W. (1962). Proceedings of the
Society of Experimental Biology and Medicine, 111, 544.