PHYTOTHERAPY RESEARCH

Phytother. Res. 15, 715–717 (2001)

DOI: 10.1002/ptr.865

SHORT COMMUNICATION
Proposed Active Constituents of Dipladenia
martiana

Mário Geraldo de Carvalho,1* Daniela Carvalho Cranchi,1 David G. I. Kingston2 and

Alceni A. Werle3
1
Universidade Federal Rural do Rio de Janeiro, ICE-Departamento de Quı́mica, Seropédica, RJ, 23851-970, Brasil
2
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061-0212, USA
3
UFOP, ICEB Departamento de Quimica, Ouro Preto, MG, Brasil

3b-O-b-D-glucopyranosylsitosterol, pomolic acid, ursolic acid, epicatechin, kaempferol, kaempferol-3-O-

b-D-glucopyranoside (astragalin), quercetin-7-O-b-D-glucopyranoside, quercetin-7-O-b-D-galactopyrano-
side and quebrachitol were isolated by chromatographic fractionation of the methanol extract from the
aerial parts of Dipladenia martiana (Apocynaceae). The hexane extract yielded lupeol and sitostenone.
These compounds are likely to be responsible for the therapeutic effects. Copyright # 2001 John Wiley
& Sons, Ltd.
Keywords: Dipladenia martiana; Apocynaceae; triterpenes; flavanoids.

INTRODUCTION observed by 1H- and 13C-NMR spectral analysis and on a

Dipladenia martiana (Apocynaceae) grows in the
Atlantic forests of Brazil. The genus Dipladenia has not
yet been investigated for its phytochemical constituents
and the Apocynaceae contains species rich in triterpenes,
alkaloids, iridoids and flavonoids (Buckingham, 1994).
The effects of glycosylflavonoids as inhibitors of
histamine release from mast cells and antiinflammatory
effects have been reported (D’Agostino et al., 1998;
Bruneton, 1995). For this reason the present study on the
genus Dipladenia was carried out.
MATERIALS AND METHODS
General. The 1H- and 13C-NMR spectra were recorded
on a Varian Unity 400 spectrometer at 400 and 100 MHz,
respectively. 1H, 1H-COSY, DEPT, 1H,13C-COSY
(HMQC and HMBC) NMR experiments were performed
on the same spectrometer, using standard Varian pulse
sequences; in HMBC determinations a 9 Hz optimization
was employed for the long-range coupling pathways.
Mass spectra were determined with a VG Quattro
instrument. FT-IR spectra were recorded as a film on a
Perkin-Elmer 1500 spectrometer. Chromatography was
performed using silica gel (Merck) for column and
preparative TLC. The purity of the compounds was
* Correspondence to: Dr M. G. de Carvalho, Universidade Federal Rural do
Rio de Janeiro, ICE-Departamento de Quı́mica, Seropédica, RJ 23851-970,
Brazil.
Contract/grant sponsor: Furdação e Auparo a Pesquisa do Estado do Rio de
Janeiro.
Contract/grant sponsor: Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico.
TLC plate, revealed with UV (254 nm) and vanillin–
sulphuric acid as a spray reagent.
Plant material. D. martiana (D. De.) was collected in
Comarinho, Ouro Preto, Brazil and compared with a
herbarium specimen number (No 422) at the Herbarium
José Badini of the Instituto de Ciências Exatas e
Biológicas of the Universidade Federal de Ouro Preto-
M.G., Brazil.
Extraction and isolation of constituents. The aerial
parts were dried, powdered (300 g) and successively
extracted with hexane and methanol at room temperature.
The crude methanol extract (4.0 g) was analysed by TLC
and fractionated on a silica gel column (A) eluted with a
mixture of CHCl3 and methanol (beginning with 10%
methanol and increasing the polarity with methanol to
100%). The fractions A3–6 (138.2 mg) were fractionated
on a silica gel column (B) (CH2Cl2:MeOH, 9:1) and
fractions B3–5 from this column were purified by
preparative TLC (CHCl3:MeOH, 9:1) and crystallization
from AcOEt: MeOH (1:1) to afford a mixture of two
triterpenes (3 ‡ 4, 86.4 mg). 13.0 mg of 3 ‡ 4 was
methylated with diazomethane and fractionated by
preparative TLC (hexane:AcOEt:acetone, 8:1:1) to
obtain pure methyl pomolate (3a, 4.2 mg, mp: 139 °C)
and methyl ursolate (4a, 8.1 mg, mp: 229 °C). Fraction A8
(15.0 mg, from column A) was purified by TLC
(AcOEt : MeOH, 8:2) to obtain the flavonoid kaempferol
(7, 6.2 mg, amorphous solid, mp: 126 °C). Fractions A9–
11 (30.0 mg) were crystallized from hexane : methanol
(1:1) to yield 3-O-b-D-glucopyranosylsitosterol (1,
20.1 mg, mp: 303 °C). Fraction A 18 (80.0 mg, from
column A) was filtered on a Sephadex LH-20 column
using CHCl3 : MeOH (9:1) and fractionated on a silica gel
column (column C) (CHCl3 : MeOH, 8:2) to obtain 15

Received 23 February 1999

Copyright # 2001 John Wiley & Sons, Ltd. Accepted 29 March 2000
716 M. G. DE CARVALHO ET AL.
fractions: fractions C 2–9 yielded the flavan-3-ol 6
(epicatechin, gum, 7.1 mg) and fractions C 10–15 a
mixture of carbohydrates (60.0 mg). Fractions A 19–21
were analysed by 1H- and 13C-NMR (PND and DEPT)
and were shown to contain glycerol (160.0 mg, oil).
Fractions A22–25 were filtered on a Sephadex LH-20
(CHCl3:MeOH, 8:2) followed by HPLC (column D) (C-
18 column, MeOH : H2O, 6:4 v/v, flow 1.0 mL/min)
giving a mixture of quercetin–glycopyranoside (9 ‡ 10,
6.0 mg, gum) and pure glucopyranosyl kaempferol (8,
2.0 mg, gum). A mixture of carbohydrates formed the last
fractions A28–49 (728.0 mg, oil, from column A).
Fractions A33–35 (300.0 mg) were acetylated with
pyridine and Ac2O (1:1). The product (280 mg) was
dissolved in a small amount of MeOH and applied to a
Sephadex LH-20 column that was eluted with MeOH.
The purest fractions (80.0 mg, gum) observed on TLC
were analysed by NMR and GC-EIMS spectra and led us
to identify 11 as its acetyl derivative (11a). The hexane
extract (3.0 g) was subjected to column chromatography
on silica gel using CH2Cl2 (100%) and a mixture with
MeOH increasing the polarity to methanol (100%).
Fractions 2–16 gave a mixture of alkanes and fractions
18–25 (60.0 mg) yielded a mixture of sitostenone (2) and
lupeol (5). The rest of the fractions afforded a mixture of
fatty acids.
Acetylation of 1 and fractions A33/35. Compound 1
(15 mg) and fractions A33–35 (300 mg) were separately
dissolved in a mixture of Ac2O/pyridine (1:1) and the
solutions were allowed to stand for 24 h at room
temperature. The usual work-up yielded 1a (13.0 mg,
mp: 170 °C) and A33–35-Ac (gum, 280.0 mg).
Methylation of triterpene acids. A solution of the
mixture 3 ‡ 4 (pomolic acid ‡ ursolic acid, 13.0 mg) in
MeOH was treated with CH2N2 in Et2O to give methyl
esters which were purified by preparative TLC to yield
methyl ursolate [4a, 8.1 mg, mp: 229 °C, literature
(Buckingham, 1994): 230 °C]. 13C-NMR was similar to
literature values (Pouchert and Behnke, 1993) and methyl
pomolate [3a, 4.2 mg, mp: 130 °C, literature (Bucking-
ham, 1994): 130 °–131 °C].
Copyright # 2001 John Wiley & Sons, Ltd.
RESULTS AND DISCUSSION
The hexane and methanol extracts of D. martiana
afforded glycerol, sitosterol-3b-O-b-D-glucopyranoside
(1), sitostenone (2), pomolic acid (3), ursolic acid (4),
lupeol (5), epicatechin (6), kaempferol (7), kaempferol-
3-O-b-D-glucopyranoside (8), quercetin-7-O-b-D-gluco-
pyranoside (9), quercetin-7-O-b-D-galactopyranoside
(10) and quebrachitol (11).
The known natural glycoside 1 (sitosterol-3-O-b-D-
glucopyranoside), sitostenone (2) and lupeol (5) were
identified mainly by 1H and 13C-NMR data analysis of the
tetra-acetylated derivative 1a, of the natural compounds 2
and 5, and by comparison with literature data (Guevara et
al., 1989; Migliuolo et al., 1990; Mahato and Kundu,
1994).
The triterpenes pomolic acid (3b, 19a-dihydroxy-12-
ursen-28-oic acid, 3) and ursolic acid (3b-hydroxy-12-
ursen-28-oic acid, 4) were identified by 1H and 13C-
NMR. The heteronuclear multiple quantum coherence
(HMQC) and heteronuclear multiple bond correlation
(HMBC) of the methyl-ester derivative 3a showed cross
peaks between H-18 (2.60, s) and 52.3 (C-18, 1JCH),
47.8(C-17, 2JCH), 73.2 (C-19, 2JCH), 138.0 (C-13,2JCH),
129.2 (C-12, 3JCH), 27.4 (C-30, 3JCH), 41.0 (C-20, 3JCH)
and 178.3 (C-28, 3JCH). These data indicated the location
of the alcoholic hydroxyl group at C-19. Comparison of
the 13C chemical shift of 3a with the literature value
(Mahato and Kundu, 1994; Ohtani et al., 1990) allowed
the structure of pomolic acid to be assigned for 3. Thus,
we also give here the 13C-NMR data for methyl pomolate
which was not found in the literature.
The flavonoids epicatechin (6), kaempferol (7) and
kaempferol-3-O-b-D-glycopyranoside (8) were identified
by analysis of 1H, 1H, 1H-COSY and 13C-NMR (PND and
DEPT) spectral data and comparison with the 1H-NMR
spectra (Harborne, 1986) and the 13C-NMR data in the
literature (Agrawal, 1989; Breitmaier and Voelter, 1987).
Due to the difficulty of separating the glucosyl
flavonoids from their galactosidic analogues, we ana-
lysed the quercetin glycosides as a mixture (9 ‡ 10). The
compounds contained in the mixture were identified as
quercetin-7-O-b-D-glucopyranoside (9) and quercetin
Phytother. Res. 15, 715–717 (2001)
 ACTIVE CONSTITUENTS OF DIPLADENIA MARTIANA
7-O-b-D-galactopyranoside (10) after analysis of NMR
spectra (1D: 1H, NOEDIFF, 13C and 2D: 1H, 1H-COSY,
HMQC and HMBC) and FAB-mass spectra (M ‡ ‡H:
465). The locations of the glycosides at C-7 were defined
by 1H-NOEDIFF spectral analysis with irradiation at the
chemical shift of both anomeric protons [dH 5.34 (d,
7.6 Hz, H-1@a) and dH 5.40 (d, 7.6 Hz, H-1@b)] to yield
NOE in H-6 (6.29, d, 1.6 Hz) and H-8 (6.49, d, 1.6 Hz).
This information and comparative TLC plate analysis of
the hydrolysis products (butanol:acetone:water, 5:4:1)
were used to confirm the presence of both glycosides in
the mixture.
The carbohydrate quebrachitol (11) was identified by
comparison with literature data (Pouchert and Behnke,
1993). The GC-EIMS of the acetyl derivative showed a
REFERENCES
Agrawal PK. (ed.), 1989. Carbon-13 NMR of Flavonoids.
Central Institute of Medicinal and Aromatic Plants, Else-
vier: New York, 366.
Breitmaier E, Voelter W. 1987. Carbon-13 NMR Spectroscopy:
High-Resolution Methods and Applications in Organic
Chemistry and Biochemistry, 3rdedn, VCH: Weinheim,
294.
Bruneton J. 1995. Pharmacognosy, Phytochemistry Med-
icinal Plants, Lavoisier: Paris, 265.
Buckingham V. 1994. Dictionary of Natural Products. Chap-
man & Hall; London, 373±874.
D'Agostino M, Dini I, Ramundo E, Senatore F. 1998.
Flavonoid glycosides of Alchemilla vulgaris L. Phytother
Res, 12: 162±163.
Guevara AP, Lim-Sylianco CY, Dayrit FM, Finch P. 1989.
Copyright # 2001 John Wiley & Sons, Ltd.
717
peak with a retention time in the GC at 8.27 min (11a)
(relative abundance 100%) and the EI-MS of 11a showed
peaks at m/z (%): 404 (M‡, 4), 362 (M-42, 70), 345 [M-
(42 ‡ 17), 60], 331 [M-(42 ‡ 31), 30], 302 [M-(42 ‡ 60),
55], 301 (100), 285 (30), 182 [404-(3  60 ‡ 420, 65]
that are in agreement with quebrachitol.
Acknowledgements
The authors are grateful to Conselho Nacional de Desenvolvimento
Cientifico e Tecnologico (CNPq) for fellowships to Dr Carvalho’s visit
to Virginia Polytechnic Institute and State University and to Fundação
e Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) for
financial the support. We thank Mr Kim Harich (Virginia Polytechnic
Institute and State University) for mass spectra.
Acylglucosyl sterols from Momordica charantia. Phyto-
chemistry, 28: 1721±1724.
Harborne JB. 1986. The Flavonoids, Advances in Research
Since 1980. Chapman & Hall; London, 455.
Mahato SB, Kundu AP. 1994. 13C NMR spectra of pentacyclic
triterpenoidsÐA compilation and some salient features.
Phytochemistry 37: 1517±1575.
Migliuolo A, Piccialli V, Sica D. 1990. Steroidal ketones from
the sponge Geodia cydonium. J Nat Prod, 53: 1262±1266.
Ohtani K, Miyajima C, Takahasi T et al. 1990. A dimeric
triterpene-glycoside from Rubus coreanus. Phytochem-
istry, 29: 3275±3279.
Pouchert CJ, Behnke J. 1993. The Aldrich Library of 13C- and
1
H-NMR Spectra, (Aldrich Chemical Co., Milwauke, WI)
1st ed, vol. 1: 266.
Phytother. Res. 15, 715–717 (2001)