Coumarin glucosides from Cruciata taurica

Article abstract of DOI:10.1016/S0031-9422(01)00471-X

Two new coumarin glycosides (1 and 2) along with two known coumarin glucosides, daphnin (3) and daphnetin glucoside (4) were isolated from the aerial parts of Cruciata taurica. The structures of the new compounds were elucidated by spectral methods and ch

Full text of DOI:10.1016/S0031-9422(01)00471-X

Phytochemistry 59 (2002) 447–450
www.elsevier.com/locate/phytochem
Coumarin glucosides from Cruciata taurica
.
Salvatore De Rosaa,*, Maya Mitovab, Nedjalka Handjievab, Ihsan Calısc
aIstituto per la Chimica di Molecule di Interesse Biologico, CNR, via Campi Flegrei, 34, 80078 Pozzuoli, Napoli, Italy
bInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
cDepartment of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, TR-06100 Ankara, Turkey
Received 31 August 2001; received in revised form 19 November 2001
Abstract
Two new coumarin glycosides (1 and 2) along withtwo known coumarin glucosides, daphnin ( 3) and daphnetin glucoside (4)
were isolated from the aerial parts of Cruciata taurica. The structures of the new compounds were elucidated by spectral methods
and chemical means as 7-O-(60-acetoxy-b-d-glucopyranosyl)-8-hydroxycoumarin (1) and 7-O-[60-O-(300,400-dihydroxycinnamoyl)-b-
d-glucopyranosyl]-8-hydroxycoumarin (2). The phylogenetic significance of coumarins in C. taurica was discussed. # 2002 Elsevier
Science Ltd. All rights reserved.
Keywords: Cruciata taurica; Rubiaceae; Coumarin, Phylogenetic relationships
1. Introduction
2. Results and discussion
Cruciata Mill. (Rubiaceae) is a small genus distributed
in the Europe and Mediterranean region. An extensive
information about morphological, karyological and
ecogeographic differentiation of the genus has been
accumulated (Ehrendorfer and Schonbeck-Temesy,
1982; Ehrendorfer and Krendl, 1976; Ehrendorfer,
1971; Pobedimova, 1958). In continuation of the che-
motaxonomic investigation on the genus Cruciata (De
Rosa et al., 2001; Mitova et al., 1996) herein, we report
the isolation of coumarin glucosides from the extremely
polymorphic polyploid species Cruciata taurica (Pallas
ex Willd.) Ehrend. Coumarin pattern of investigated
Cruciata species showed unexpected relations between
species of this genus.
To our knowledge in C. taurica the coumarins:
umbeliferon, scopoletin (Borisov, 1974) and cruciatin
(Borisov, 1967), flavonoids: hyperoside, quercetin (Bor-
isov, 1967) and rutin (Temizer et al., 1995) and iridoid
glucoside asperuloside (Borisov, 1967) were found.
Recently, we reported the isolation of two new aromatic
monoterpenoid glycosides (De Rosa et al., 2001).
The methanol extract of the aerial parts of C. taurica
was partitioned between chloroform and water. The
water-soluble part, upon repeated droplet counter cur-
rent chromatography (DCCC), column chromato-
graphy (CC) over silica gel and high-pressure liquid
chromatography (HPLC) afforded four pure com-
pounds. By means of spectroscopic data and published
information two of them were identified as the known
coumarin glycosides: daphnin (3) and daphnetin gluco-
side (4) (Jewers and Zirvi, 1978), and two appeared to
be new compounds (1 and 2).
Compound 1, isolated as amorphous solid, was
assigned the molecular formula C17H18O10 as deter-
mined from HRFABMS and NMR data (Table 1). Its
UV spectrum with lmax 266 and 322 nm was character-
istic of a coumarin derivative. The IR spectrum showed
the presence of an ester function and an a–b-unsatu-
rated lactone (ꢀmax 1735 and 1720 cmꢀ1). The 1H NMR
spectrum of 1 showed two proton doublets at ꢁ 7.86 and
6.31 (J=9.5 Hz) characteristic for the H-3 and H-4 of
coumarins (Joseph-Nathan et al., 1984). The presence of
further two proton doublets at ꢁ 7.15 and 7.07 (J=8.7
1
Hz) in the H NMR spectrum, and resonance of car-
bons bearing two oxygen moieties at ꢁ 149.4 (s) and
* Corresponding author. Tel.: +39-081-8675029; fax: +39-081-
8041770.
E-mail address: sderosa@icmib.na.cnr.it (S. De Rosa).
135.7 (s) indicated
(Mikhova and Duddeck, 1996). Thus, the structure of
a
7,8-disubstituted coumarin
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00471-X

448
S. De Rosa et al. / Phytochemistry 59 (2002) 447–450
spectrum, due to the loss of acetate hexose unit, sug-
gested that the acetyl group is located on the glucose
moiety. HMBC correlations observed between the two
protons of glucose methylene group (ꢁ 4.46 and 4.28)
and the acetyl carbonyl group (ꢁ 172.6), and between
the anomeric proton at ꢁ 4.97 and the carbon at ꢁ 149.4,
defined the esterification at C-60 and the glucosidation
at C-7, respectively. Thus, compound 1 is the acetyl
derivative of daphnin (3) and its structure was eluci-
dated as 7-O-(60-acetoxy-b-d-glucopyranosyl)-8-hydroxy
coumarin.
Compound 2, isolated as amorphous solid, was
assigned the molecular formula C24H22O12 as deter-
mined from HRFABMS and NMR data (Table 1). A
preliminary analysis of its spectral data showed that 2 is
related to compound 1. In fact, acid hydrolysis of 2
afforded glucose (TLC; HPAE–FAD), daphnetin and
cinnamic acid. The IR spectrum showed the presence of
an a,b-unsaturated ester function and an a,b-unsatu-
rated lactone (ꢀmax 1725 and 1720 cmꢀ1). Its FABMS
exhibited fragment ion peaks at m/z 341 [MH–162]+
and 179 [MH–162–161]+ indicating the loss of an ester
unit and a hexose. The signal of the anomeric proton at
ꢁ 4.94 (d, J=7.1 Hz) showed that glucose was attached
in the b-configuration. The 13C NMR spectrum of 2
contained 24 carbon signals, six of which were readily
assigned to a glucopyranosyl moiety, nine fitted witha
daphnetin moiety, and the remaining nine carbons to an
aromatic acyl unit. The NMR analysis of the ester unit
of 2 showed a triple-substituted aromatic ring, a double
bond and a carbonyl group. Taking into account the
molecular formula, and the presence of two carbon
singlets at ꢁ 148.6 and 145.7 in the 13C NMR spectrum,
it was assumed that two of the ring substituents were
hydroxyl groups. The signals of the aromatic protons at
ꢁ 7.06 (d, J=1.8 Hz), 7.00 (dd, J=8.5 and 1.8 Hz) and
6.79 (d, J=8.5 Hz) indicated the presence of a 1,3,4-tri-
substituted benzene ring. The coupling constant value
of the olefinic protons (J=15.9 Hz) defined the E-con-
figuration for the double bond. Thus, the aromatic acyl
unit was identified as a 3,4-dihydroxycinnamoyl moiety.
HMBC correlations observed between the two protons
of the glucose methylene group (ꢁ 4.44 and 4.20) and the
ester carbonyl group (ꢁ 166.3), and between the anome-
ric proton at ꢁ 4.94 and the carbon at ꢁ 148.0, defined
the acylation at C-60 and the glucosidation at C-7,
respectively. From the above evidence, compound 2 was
characterised as 7-O-[60-O-(300,400-dihydroxycinnamoyl)-
b-d-glucopyranosyl]-8-hydroxycoumarin.
Table 1
13C NMR spectral data of compound 1 (CD3OD) and 2 (DMSO-d6),
(ꢁ ppm)a
1
2
2
162.8 s
114.6 d
146.0 d
119.4 d
114.0 d
149.4 s
135.7 s
116.4 s
144.3 s
103.1 d
74.7 d
160.0 s
113.4 d
144.5 d
117.7 d
112.0 d
148.0 s
134.4 s
114.4 s
142.8 s
101.4 d
73.2 d
3
4
5
6
7
8
9
10
10
20
30
77.3 d
71.4 d
75.5 d
70.0 d
40
50
75.6 d
64.6 t
74.0 d
63.2 t
60
COCH3
172.6 s
20.7 q
COCH3
100
200
300
400
500
600
700
800
900
166.3 s
113.6 d
145.3 d
125.3 s
114.8 d
145.7 s
148.6 s
115.8 d
121.4 d
a
Chemical shifts are referred to residual solvents resonance.
Assignments were established by DEPT, HMQC and HMBC spectra.
the aglycone was similar to daphnetin (5) (Mikhova et
al., 1996). Acid hydrolysis of 1 afforded glucose, which
was confirmed by TLC and high performance anion
exchange (HPAE–FAD) and daphnetin. The spectral
data of daphnetin (5) were in excellent agreement with
those reported in the literature (Mikhova et al., 1996).
The signal of the anomeric proton at ꢁ 4.97 (d, J=7.5
Hz), in the 1H NMR spectrum of 1, showed that glucose
was bound in b-configuration. The presence of an acetyl
group [ꢁ 1.95 (3H, s); 20.7 (q) and 172.6 (s)] in the NMR
spectra of 1 and fragment peak (m/z 178), in the EIMS
The coumarin pattern of C. taurica and C. glabra is
very similar. These species contain two common cou-
marins: daphnin (3) and daphnetin glucoside (4) (Mitova
et al., 1996), and additionally C. taurica contain two
daphnin esters (1 and 2). According to the classical tax-
onomical schemes of the genus Cruciata (Ehrendorfer
and Krendl, 1976; Ehrendorfer and Schonbeck-Temesy,

S. De Rosa et al. / Phytochemistry 59 (2002) 447–450
449
1982; Pobedimova, 1958) these species are not con-
sidered closely related. The similar coumarin pattern
could be explained by convergent evolution regarding
the coumarins of C. taurica and C. glabra or by rela-
tionships between these species before the inflorescens
evolution of the genus Cruciata (cymes without brac-
teols, C. glabra; cymes withbracteols, C. taurica). Fur-
thermore, the finding of aromatic monoterpenoid
glycosides in C. taurica (De Rosa et al., 2001), does not
explain this question. The chemical investigation of
more Cruciata species could clarify the relationships in
this genus.
coside (4, 6.2 mg; eluted after 4.8 min) and compound 2
(7.1 mg; eluted after 10.5 min). Compound 1 (58 mg;
with Rf on TLC of 0.92 in CHCl3–MeOH, 7:3) was
obtained after additional purification of frs 46–56 (325
mg) using silica gel column, eluted withCHCl 3–MeOH–
H2O (30:11:2).
3.4. 7-O-(60-Acetoxy-ꢂ-d-glucopyranosyl)-8-
hydroxycoumarin (1)
Amorphous solid, [ꢃ]D=ꢀ76.7ꢂ (MeOH; c 0.004);
UV (MeOH) lmax nm (log "): 322 (4.07), 266 (3.51); IR
ꢀmax (KBr) cmꢀ1 3600–3400, 1735, 1720, 1690, 1620,
1500, 1290, 1200 and 1100; HRFABMS (positive) m/z:
383.1015 [M+H]+ (C17H19O10 requires 383.1004);
EIMS m/z %: 382 [M]+ (0.5), 322 [MꢀAcH]+ (1), 309
[Mꢀ CH2OCOCH3]+ (1), 178 [Mꢀ GlcAc]+ (100), 150
3. Experimental
3.1. General
1
(95); H NMR (CD3OD): ꢁ 7.86 (1H, d, J=9.5 Hz, H–
UV spectra were obtained on a Varian DMS 90 spec-
trophotometer. IR spectra were recorded on a Bio-Rad
FTS-7 FT–IR spectrometer. Optical rotations were
measured on a Jasco DIP 370 polarimeter, using a 10-cm
microcell. FABMS were obtained on a VG-ZAB
4), 7.15 (1H, d, J=8.5 Hz, H-6), 7.07 (1H, d, J=8.5 Hz,
H-5), 6.31 (1H, d, J=9.5 Hz, H-3), 4.97 (1H, d, J=7.5
Hz, H-10), 4.46 (1H, dd, J=11.9, 2.0 Hz, H-6a0), 4.28
(1H, dd, J=11.9, 6.4 Hz, H-6b0), 3.72 (1H, m, H-50),
3.60 (1H, m, H-20), 3.55 (1H, m, H-30), 3.44 (1H, m,
H-40), 1.95 (3H, s, COCH3); 13C NMR: see Table 1.
1
instrument, using glycerol as matrix. H and 13C NMR
spectra were recorded at 500 and 125 MHz, respectively,
on a Bruker AM 500 instrument, under Aspect X32
control. The 2D NMR spectra were obtained using
Bruker’s microprograms. DCCC was performed on a
Buchi 670 apparatus by ascending mode. Aluminum
sheets silica-gel 60 F254 were used for TLC. Merck
silica-gel 60 (0.063–0.2) were used for CC. Preparative
HPLC purifications were carried out on a Waters
apparatus equipped witha Kromasil C-18 column (7.8
mm i.d.ꢁ30 cm) and withUV detector.
3.5. 7-O-[60-O-(300,400-Dihydroxycinnamoyl)-ꢂ-d-
glucopyranosyl]-8-hydroxycoumarin (2)
Amorphous solid, [ꢃ]D=ꢀ25.9ꢂ (MeOH; c 0.009);
UV (MeOH) lmax nm (log "): 320 (4.52), 264 (3.48), 227
(4.03); IR ꢀmax (KBr) cmꢀ1 3600–3400, 1725, 1720,
1690, 1620, 1500, 1490, 1290, 1200 and 1060; HRF
ABMS (positive) m/z: 503.1210 [M+H]+ (C24H23O12
requires 503.1221), 341 [MHꢀ162]+, 179 [MHꢀ162–
1
161]+; H NMR (CD3OD): ꢁ 7.81 (1H, d, J=9.7 Hz,
3.2. Plant material
H-4), 7.47 (1H, d, J=15.9 Hz, H-300), 7.09 (1H, d, J=8.5
Hz, H-6), 7.06 (1H, d, J=1.8 Hz, H-500), 7.00 (1H, dd,
J=8.5, 1.8 Hz, H-900), 6.98 (1H, d, J=8.5 Hz, H-5), 6.79
(1H, d, J=8.5 Hz, H-800), 6.29 (1H, d, J=9.7 Hz, H-3),
6.27 (1H, d, J=15.9 Hz, H-200), 4.94 (1H, d, J=7.1 Hz,
H-10), 4.44 (1H, bd, J=11.4 Hz, H-6a0), 4.20 (1H, dd,
J=11.4, 6.4 Hz, H-6b0) 3.73 (1H, m, H-50) 3.42–3.30
(3H, m, H-20, H-30 and H-40); 13C NMR: see Table 1.
C. taurica was collected at florescence, in May 1999,
at Hatay, Turkey and dried in shade at room tempera-
ture. The voucher specimens CO434 was deposited in
the herbarium of the Institute of Botany, Bulgarian
Academy of Sciences (SOM).
3.3. Extraction and isolation
3.6. Acid hydrolysis
Dry aerial parts (27 g) were extracted twice with
MeOH, and the concentrated extract (4.0 g) partitioned
between CH3Cl and H2O. Ascending DCCC with
CHCl3–MeOH–H2O–n-PrOH separated the aqueous
phase (3.0 g) (9:12:8:2). The flow-rate was 25 ml/h.
Fractions of 12.5 ml were collected. The MeOH soluble
part, of fractions 15–19, contained daphnin (3, 386 mg).
From frs 26–34 (112 mg), after silica-gel column, eluted
Compound 1 (5 mg) or compound 2 (2 mg) dissolved
in 0.5 ml of 2 N HCl was refluxed for 1 h. The reaction
mixture was neutralised and extracted withEtOAc to
obtain aglycone. The water-soluble residue was ana-
lysed by TLC [mobile phase—iPrOH–toluen–EtOAc–
H2O (50:10:25:12.5)], and HPAE–PAD (Dionex) equip-
ped witha Carbopac PA1 column eluted with15 mM
NaOH (1 ml minꢀ1) and witha Pulsed Amperometric
detector, giving d-glucose, identified by comparison of
its retention time with that of the authentic sample.
withCHCl –MeOH–H2O (30:11:2) and purification on
3
HPLC Kromasil C-18 column, eluted withCH 3CN–
H2O (1:3; flow 2 ml/min) were obtained daphnetin glu-

450
S. De Rosa et al. / Phytochemistry 59 (2002) 447–450
De Rosa, S., Mitova, M., Handjieva, N., Ersaz, T., Calis, I., 2001.
Compound 1 gave daphnetin (5) as aglycone. Com-
Monoterpenoid glucosides from Cruciata taurica. J. Nat. Prod.
Lett., submitted.
pound 2 gave daphnetin (5) and 3,4-dihydroxycinnamic
acid. The aglycones were identified by TLC [mobile
phase—CHCl3–MeOH–H2O (60:15:4, lower layer)] in
comparison withauthentic samples.
Ehrendorfer, F., 1971. Evolution and eco-geographical differentiation
in some South-West Asiatic Rubiaceae. In: Davis, P., Harper, P.,
Hedge, I. (Eds.), Plant Life of South-West Asia. University Press,
Edinburgh, pp. 195–215.
Ehrendorfer, F., Krendl, F., 1976. Cruciata. In: Tutin, T.G. (Ed.),
Flora Europea, Vol. 4. University Press, Cambridge, pp. 36–37.
Ehrendorfer, F., Schonbeck-Temesy, E., 1982. Cruciata. In: Davis, P.
(Ed.), Flora of Turkey, Vol. 7. University Press, Edinburgh, pp.
850–855.
Acknowledgements
This research is a part of the joint project Bulgaria–
Turkey (1999–2001): ‘‘Terpenoids, flavanoids and phe-
nylethanoids in Scrophulariacea, Lamiacea and Rubia-
ceae plants from Bulgaria and Turkey’’. The authors are
grateful to the Bulgarian National Foundation for Sci-
entific Research(Project B-1001) and CNR Rome for
the partly financial support of this work. Mass spectra
and NMR spectra were provided by ‘‘Servizio di Spet-
trometria di Massa del CNR-Napoli’’ and ‘‘Servizio
NMR, ICMIB–CNR’’, respectively. The assistance of
Mr. V. Mirra is gratefully acknowledged.
Jewers, K., Zirvi, K., 1978. The coumarin glycosides of Daphne acu-
minata: use of 13C-NMR spectroscopy for their identification.
Planta Med. 33, 403–406.
Joseph-Nathan, P., Dominguez, M., Ortega, D., 1984. Shift reagent
1H NMR study of methoxycoumarins. Heterocyclic Chem. 21,
1141–1144.
Mikhova, B., Duddeck, H., 1996. 13C-NMR spectroscopy of coumar-
ines and their derivatives: a comprehensive review. In: Atta-ur-
Rahman (Ed.), Studies in Natural Products Chemistry, Vol. 18.
Elsevier Science, Amsterdam, pp. 971–1080.
Mitova, M., Anchev, M., Panev, St., Handjieva, N., Popov, S.,
1996. Coumarins and iridoids from Crucianella graeca, Cruciata
glabra, Cruciata laevipes and Cruciata pedemontana (Rubiaceae). Z.
Naturforsch. 51c, 631–634.
Pobedimova, E., 1958. Sect. Cruciatae. In: Shishkin, B. (Ed.), Flora
USSR, Vol. 23. Moskva, Leningrad, pp. 314–326.
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