UV-Vis, FT-Raman, and 1H NMR Spectroscopies of

cis-trans Carotenoids from Saffron (Crocus sativus L.)

MANOLIS K. ASSIMIADIS, PETROS A. TARANTILIS, and
MOSCHOS G. POLISSIOU*
Laboratory of General Chemistry, Agricultural University of Athens, Iera odos 75, Greece

Stigm ata of Crocus sativus L. were extracted in water, and the extract was analyzed by using a high-pressure liquid chromatography
(HPLC) method. The carotenoids of saffron [cis and trans glycosyl
esters of crocetin or crocins (CRCs)] were isolated, and the two
major isomer compounds (all-trans-digentiobiosyl crocetin and 13cis-digentiobiosyl crocetin) were characterized spectroscopically
with ultraviolet-v isible (UV-vis), Fourier transform (FT)-Raman,
and 1H NMR spectr oscopies. The 13-cis compound showed in a UVvis spectrum an additional peak at 330 nm, in an FT-Raman spectrum the presence of a peak at 1138 cm 2 1, and in the 1H NMR
down eld shifts of protons 12 and 15 and an up eld shift of proton
14, with respect to the all-trans isomer, accord ing to differences in
their skeletal structures. These features are shown also by the pro le of their two-dimensional spectra.
Index Headings: Saffron; Crocus sativus L.; FT-Raman; UV-vis;
NMR; Spectroscopy; cis-trans Carotenoids.

INTRODUCTION
Crocus sativus L. is a ower with a great coloring
power, which is cultivated in Greece, Iran, Morocco,
Spain, India, etc. The dried red stigmata of the stylus of
this ower (identi ed as saffron) are used for avoring
and coloring foods, and they have also been used in perfumery and as a drug in ancient medicine. Because of
their low availability, these stigmata are very expensive.1
The coloring power of Crocus sativus L. comes from
the crocins (CRCs) (glycosyl esters of crocetin), very water soluble carotenoids, mainly composed of digentiobiosyl crocetin. It has been noted2,3 that for every transcrocin there is a corresponding cis-isomer (Fig. 1). However, isolation and characterization of these isomers is not
an easy process, because they are very sensitive to light,
humidity, and high temperatures. The structural study of
these compounds attracts considerable interest because
their biological activity is probably due to a transition
state that is an intermediate between cis- and trans- isomers.2 6
In this work we report the quantitative separation of
cis- and trans-isomers using an isocratic high-pressure
liquid chromatography (HPLC) method and their characterization using ultraviolet-visible (UV-vis), Fourier
transform (FT)-Raman, and 1H NMR spectroscopies.
EXPERIMENTAL
Plant Material. Dried stigmata of Crocus sativus L.
(pure red Greek saffron) were kindly supplied by the Cooperative of Saffron, Krokos Kozanis.
Sample Preparation. We extracted 0.5 g of stigmata
Received 19 June 1997; accepted 8 December 1997.
* Author to whom correspondence should be sent.

Volume 52, Number 4, 1998

of saffron in the dark, with 5 mL of water, in an ultrasonic bath for 10 min at 25 8 C. For the extraction of the
stigmata of Crocus sativus L., we used only water (even
if the best solvent for these carotenoids is a 1:1 mixture
of water/methanol) in order to avoid the presence of lipids in the fractions. The extract was ltered with a 0.45
mm lter (Schleicher & Schuell disposable lter holders).
The HPLC instrument was a Jasco HPLC pump (PU980) connected with a UV-vis detector (Jasco UV-vis detector UV-970/975). A loop 0.2 mL in volume was used
for the injections.
The mobile phase was comprised of two isocratic solvents: from 0 to 7 min, 20% acetonitrile in water and,
from 7 to 25 min, 40% acetonitrile in water. For each
chromatographic separation, 0.2 mL of extract was injected onto a Kromasil C18 10 m m reversed-phase column
(250 3 4 mm). The wavelength detection was at 440 nm.
Fractions corresponding to each peak were collected,
concentrated, and re-injected for further puri cation. After the solvent evaporation with nitrogen gas ow, the
fractions were used for spectral characterization.
UV-Vis Spectroscopy. A UV-vis spectrophotometer
was used (Jasco UV-vis spectrophotometer V-550). The
scanning range was from 200 to 600 nm at a scanning
speed of 400 nm/min.
FT-Raman Spectroscopy. A Nicolet 750 magna IR
with Raman accessory was used. The beamsplitter was
CaF2, the source power was 0.5 W, and the wavelength
was 1064 nm (near-IR).
NMR Spectroscopy. A Varian-Unity 600 was used for
NMR experiments: (1) 1H 1D in CD3OD, proton frequency 600.042 MHz, and (2) 1H 1H 2D COSY.
RESULTS AND DISCUSSION
Four fractions of the crocins can be isolated quantitatively from the HPLC analysis: T1 (all-trans-digentiobiosyl crocin), T2 (all-trans-gentiobiosyl glycosyl crocin), C1
(13-cis-digentiobiosyl crocin), and C2 (13-cis-gentiobiosyl glycosyl crocin).6
The T1, T2 fractions showed characteristic UV-vis
spectra of trans-carotenoids with peaks at 260 and 440
nm. C1 and C2 showed peaks at 260, 330, and 440 nm.
The peak at 330 nm is characteristic of the cis carotenoids.6
FT-Raman spectra were recorded in the solid state,
from the two major fractions of isomers, the T1 (all-trans)
and C1 (cis) fractions, (Fig. 2). At the FT-Raman spectrum of the T1 compound, a strong single peak at 1535
cm2 1 is present, corresponding to stretching vibrations of
the C5 C bonds. For the C1 compound this peak shifts to
1547 cm2 1, and one more peak is observed at 1581 cm2 1.

0003-7028 / 98 / 5204-0519$2.00 / 0
q 1998 Society for Applied Spectroscopy

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519

FIG. 2. FT-Raman spectra of all-trans and 13-cis digentiobiosyl ester

of crocetin.

FIG. 1. Molecular structure of carotenoids from saffron (all-trans glycosylester of crocetin, 13-cis glycosylester of crocetin).

Important differences are also observed in the region of

the stretching vibrations of the C C bonds (1100 1300
cm2 1). For the all-trans compound there is a medium
peak at 1165 cm2 1 , characteristic of the all-trans carotenoids, and a weak one at 1209 cm2 1 . In the FT-Raman
spectrum of the cis isomer there is one more peak at 1138
cm2 1, and the intensity of the peak at 1166 cm2 1 is decreased; these observations are characteristic of the 13cis isomers.7
The simplicity of the FT-Raman spectrum for the alltrans crocin is due to the high symmetry of this isomer.
In fact, the crystal structure information of its dimethyl
derivative has shown that these molecules are linear, symmetrical, and planar with most of their single (C C) and
double (C5 C) bonds equivalent. The bond lengths for
carbons C(9) C(10), C(11) C(12), C(13) C(14), and
, and the bond lengths
C(15) C(15 9 ) are 1.344 1.353 A
between carbons C(10) C(11), C(12) C(13), and C(14)
.8 In terms of the 13-cis isomer,
C(15) are 1.431 1.444 A
changing the symmetry in the central part of the molecule
causes differentiation of the single and double bonds;
subsequently their spectrum shows more peaks, decreased in intensity and shifted to higher wavelengths,
with respect to the spectrum of the all-trans isomer.9
All the above observations have been con rmed by the
600 MHz 1H NMR data. The most remarkable differences
between the 1H NMR spectra of the all-trans and the 13-

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Volume 52, Number 4, 1998

cis crocin appear at the signals of the ole nic protons,

which is a consequence of the loss of symmetry in the
central part of the molecule that contains the conjugated
double bonds.
The values of the chemical shifts and coupling constants for the protons of the unsaturated chain and glucose units, derived by rst-order analysis, are summarized in Table I, and 1H NMR and 2D (COSY) data for
the ole nic region of the two isomers are illustrated in
Figs. 3 and 4.
In the 1H NMR spectrum of the all-trans isomer there
TABLE I. 1H NMR data of all-trans and 13-cis digentiobiosyl ester
of crocetin.
T1
Proton

d (ppm)
7.43

10
10 9
11
6.66
11 9
12
6.75
12 9
14
6.49
14 9
15
6.83
15 9
19
1.98
19 9
20
2.01
20 9
1
5.53
19
4.32
2,3,4,5
3.25 3.40
2 9 ,3 9 ,4 9 ,5 9
6x
4.16
6x 9
3.83
6a
3.77
6a 9
3.65

C1
J (Hz)

d(11.4)
dd(15; 11.4)
d(15)

d (ppm)
7.50
7.44
6.65

J (Hz)
d(11.7)
d(11.7)
dd(11.7; 15)

7.33
6.75
6.36
6.48
7.02
6.73
2.01

d(15)
d(15)
d(11.7)
d(11.7)
dd(11.7; 14.3)
dd(11.7; 14.3)
s

2.02

dd(7.7; 2.6)
dd(7.7; 2.6)

d(7.7)
d(7.7)
dd(11.4;
dd(11.7;
dd(11.4;
dd(12.1;

5.54
4.33
3.26 3.40
1.8)
2.2)
5.1)
5.5)

4.16
3.84
3.78
3.65

d(7.7)
d(7.7)
dd(11.4; 1.8)
(11.7; 2.2)
dd(11.4; 5.1)
dd(12.1; 5.5)

FIG. 3. 600 MHz 1H 1D NMR and 1H 1H 2D COSY NMR spectra of

the ole nic region of all-trans digentiobiosyl ester of crocetin.

is a down eld doublet at 7.43 ppm coming from proton

10 coupling with proton 11 (J 5 11.4 Hz), a doublet of
doublets at 6.66 ppm due to proton 11, which is coupled
with proton 12 (J 5 15 Hz), and also a doublet of doublets at 6.49 ppm for protons 14 and 14 9 as a result of
coupling with protons 15 and 15 9 (J 5 7.7 Hz; 2.6 Hz).
There is also a similar coupling for protons 15 and 15 9
at 6.83 ppm.
In the case of the 13-cis isomer spectrum, there are
remarkable differences for protons 10, 12, and 15, which
are shifted down eld by 0.07, 0.58, and 0.19 ppm respectively, and an up eld shift for proton 14 by 0.13
ppm, compared to the trans isomer spectrum. The coupling constants for the 13-cis isomer are J10 11 5 J10 9 11 9
5 11.7 Hz; J11 12 5 J11 9 12 9 5 15 Hz; and J14 15 5 J14 9 15 9
5 11.7 Hz. These differences are due to the change of
the spin system between protons 19 10 11 12 and 19 9
10 9 11 9 12 9 . In the all-trans con guration, proton 12 is
very close to and interacts with proton 14, while proton
15 is very close to and interacts with methyl group 20.
In the 13-cis con guration the location of the above protons is different; these interactions do not exist, and a
new interaction can be observed between proton 14 and
methyl group 20. According to the above observations,
the cis double bond is con rmed to be located between
carbons 13 and 14.
The 2D NMR (COSY) spectrum of the unsaturated
chain clearly records these structural differentiations. In
fact, the 13-cis isomer shows a completely different pro le, with the appearance of a second set of signals of
ole nic protons in addition to the pattern shown by the
trans isomer. In addition, the attribution of peaks con rmed by the 2D NMR for the unsaturated protons revealed some discrepancies with respect to attributions
given by Speranza et al.,2 who attributed proton 12 to

FIG. 4. 600 MHz 1H 1D NMR and 1H 1H 2D COSY NMR spectra of

the ole nic region of 13-cis digentiobiosyl ester of crocetin. The asterisk
(*) stands for byproducts.

7.50 ppm instead of 7.33 ppm and proton 10 to 7.33 ppm

instead of 7.50 ppm.
The chemical shifts for the sugar (gentibiose) protons
1 and 1 9 , which are 5.53 ppm and 4.32 ppm, respectively,
and their large coupling constants (7.7 Hz) place them in
axial positions. This observation proves that the glucose
units are in the b -anomeric form. Moreover, the chemical
shift of proton 1 at a lower eld than proton 1 9 proves
that the rst anomeric proton can be attributed to the
glucose unit that has the ester linkage.
It should also be noted that the chemical shifts of exocyclic protons 6a and 6x are found at lower eld than
protons 6a 9 and 6x 9 , because they belong to the rst glucose unit (that with the ester linkage). The overlap of
protons 2, 3, 4, and 5 and 2 9 , 3 9 , 4 9 , and 5 9 prevents their
total rst-order analysis by 1H NMR.
Generally, in glucopyranozide derivatives, the exocyclic part of the sugar molecule adopts three con rmations
around the C(5) and C(6) bond, the gg (gauche gauche),
gt (gauche trans), and tg (trans gauche) (Fig. 5). In the
solid state only the gt conformation is observed, because
the steric hindrance of this form is less than the other two
rotamers (gg and tg). In solution, an equilibrium is es-

FIG. 5. Three rotamers about the C1 9 C6 linkage of gentiobiose.

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521

tablished essentialy between gt and gg. The ``attractive

gauche effect between C4 O4 and C6 O6 seems to
slightly favor the gg rotamer.10 This situation seems to be
the same in the case of disaccharides.11
With the use of the coupling constant values JH5 H6x 5
1.8 Hz, JH5 H6a 5 5.1 Hz, JH5 9 H6x 9 5 2.2 Hz, and JH5 9 H6a 9
5 5.5 Hz, which are also given in Table I, the population
of the rotamers Pgg, Pgt, and Ptg can be estimated for
the rst and the second glucose unit of gentiobiose as
follows:
First glucose unit: Pgg (%) 5 61, Pgt (%) 5 36, Ptg
(%) 5 3.
Second glucose unit: Pgg (%) 5 53, Pgt (%) 5 40, Ptg
(%) 5 7.
These percentages of rotamer populations have been
calculated according to the appropriate Karplus-type
equations:10
Pgg 5 100 2 Pgt 2 Ptg, Pgt 5 (JH5,H6a 2 1.5) 3 100/
9.9,
Ptg 5 (JH5,H6x 2 1.5) 3 100/9.9.
The above results of gentibiose con rm the ndings with
respect to the other disaccharides;11 i.e., in solution the
equilibrium between gg and gt rotamers is adopted, as in
the case of the monosaccharides.10
CONCLUSION
The aim of the present work was to obtain and characterize the cis-trans carotenoids from saffron (Crocus
sativus L.) by the most appropriate spectroscopic methods. The cis and trans glycosyl esters of crocetin have
been isolated and puri ed by HPLC. Their UV-vis, 1H
NMR, and FT-Raman spectra were recorded in solution
and in the solid state.
The 13-cis isomer is easily differentiated from the alltrans isomer with the following spectral features: (1) additional absorption at 330 nm (so-called cis peak) in its
UV-vis spectrum, which is absent in the UV-vis spectrum
of the trans isomer; (2) the presence of a peak at 1138
cm2 1 in the FT-Raman spectrum with a simultaneous reduced intensity of the 1166 cm2 1 peak; (3) in its 1H NMR

522

Volume 52, Number 4, 1998

spectrum differentiation of almost all the protons. The

most remarkable are the down eld-shifted protons 12 and
15 and the up eld-shifted proton 14, in comparison to
the spectrum of the trans compound.
Finally FT-Raman spectroscopy is revealed to be a
suitable method for characterizing 13-cis crocetin because of the differences it shows in the region of the
stretching vibrations of the C C bonds (1100 1300 cm2 1)
and the suitability of using it in both solutions and in the
solid form. Moreover, new Raman techniques such as micro-FT-SERS (surface-enhanced Raman spectroscopy)
exhibit spectra with very high mass sensitivity, allowing
us to record Raman spectra of crocetin at concentrations
of less than 102 6 M.12
AKNOWLEDGMENTS
We thank Claude Maerschal for the 1 H NMR experiments at the
University of Brussels (U.L.B.), and ``Cooperative of Saffron, Krokos
Kozanis for providing the stigmata of saffron.
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3. P. A. Tarantilis, M. Polissiou, and M. Manfait, J. Chromatogr. 664,
55 (1994).
4. H. Pfander and M. Rychener, J. Chromatogr. 234, 443 (1982).
5. V. Sujata, G. A. Ravishankar, and V. L. Venkataraman, J. Chromatogr. 648, 187 (1993).
6. P. A. Tarantilis, G. Tsoupras, and M. Polissiou, J. Chromatogr. 699,
107 (1995).
7. Y. Kouama, ``Proton Nuclear Magnetic Resonance and Raman
Spectroscopies of Cis-Trans Carotenoids from Pigment Protein
Complexes , in Methods in Enzymology, Vol. 213, Carotenoids, L.
Packer, Ed. (Academic Press, New York, 1992), Part A, Section II,
Chap. 27, pp. 298 305.
8. P. A. Tarantilis, M. Polissiou, D. Mentzafos, and M. Manfait, J.
Chem. Crystallogr. 24, 739 (1994).
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