Article
pubs.acs.org/JAFC
Crocus sativus Petals: Waste or Valuable Resource? The Answer of
High-Resolution and High-Resolution Magic Angle Spinning Nuclear
Magnetic Resonance
Valeria Righi,† Francesca Parenti,‡ Vitaliano Tugnoli,§ Luisa Schenetti,∥ and Adele Mucci*,‡
†
Dipartimento
Dipartimento
Via Giuseppe
§
Dipartimento
‡
di Scienze per la Qualità della Vita, Università di Bologna, Corso D’Augusto 237, 47921 Rimini, Italy
di Scienze Chimiche e Geologiche, and ∥Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia,
Campi 103, 41125 Modena, Italy
di Scienze Biomediche e Neuromotorie, Università di Bologna, Via Belmeloro 8/A, 40123 Bologna, Italy
ABSTRACT: Intact Crocus sativus petals were studied for the first time by high-resolution magic angle spinning nuclear
magnetic resonance (HR-MAS NMR) spectroscopy, revealing the presence of kinsenoside (2) and goodyeroside A (3), together
with 3-hydroxy-γ-butyrolactone (4). These findings were confirmed by HR-NMR analysis of the ethanol extract of fresh petals
and showed that, even though carried out rapidly, partial hydrolysis of glucopyranosyloxybutanolides occurs during extraction.
On the other hand, kaempferol 3-O-sophoroside (1), which is “NMR-silent” in intact petals, is present in extracts. These results
suggest to evaluate the utilization of saffron petals for phytopharmaceutical and nutraceutical purposes to exploit a waste product
of massive production of commercial saffron and point to the application of HR-MAS NMR for monitoring bioactive compounds
directly on intact petals, avoiding the extraction procedure and the consequent hydrolysis reaction.
KEYWORDS: Crocus sativus, saffron, kinsenoside, goodyeroside A, kaempferol-3-O-sophoroside, HR-MAS NMR
■
INTRODUCTION
Crocus sativus (family Iridaceae) is cultivated in some parts of
Italy for the utilization of its stigmas (saffron) in foods and
sweets for both their intense color and strong taste. Moreover,
saffron has also been used in preventive medicine: numerous
pharmacological tests point to specific therapeutic actions, and
many studies are focused on it.1
C. sativus petals represent the main byproduct of saffron
production. Considering that saffron is one of the most
commercialized spices and that 1 kg of saffron is obtained from
more than 160 000 flowers, it is fair to ask whether it is possible
to directly use the petals for phytopharmaceutical or
nutraceutical purposes or as raw material to obtain substances
with pharmacological activity.2 Very recently, a paper proposing
the use of petals of C. sativus as a source of crocin and
kaempferol appeared.3
A number of studies on hepatoprotective, anti-nociceptive,
anti-inflammatory, anti-depressant effects of saffron petal extracts
are reported in the literature.3−5 It is also known that petals of
C. sativus contain kaempferol,6,7 which has been reported to be a
tyrosinase inhibitor,7 and its glycosides, especially kaempferol-3O-sophoroside (1; Figure 1), which exhibit antioxidant and antiinfammatory activities.8,9
Hence, the properties reported for petal extracts could be
simply due to kaempferol derivatives, but they could also be
due to other bioactive species. Sprouts of C. sativus contain
3-(R)-3-β-D-glucopyranosyloxybutanolide [kinsenoside (2);
Figure 1] and 3-(S)-3-β-D-glucopyranosyloxybutanolide [goodyeroside A (3); Figure 1],10 that derive from 3-hydroxy-γbutyrolactone (4; Figure 1) and have been isolated also from
other plants, such as Anoectochilus and Goodyera species
(Orchidaceae).11−14
© 2015 American Chemical Society
Figure 1. Structures of identified compounds in C. sativus petals and
ethanol extract.
Both compounds 2 and 3 exhibit hepatoprotective effects;14,15
compound 2 is also antihyperglycemic,12 anti-inflammatory,16
antihyperliposis,17 vascular protective,18 ovariectomy-induced
Received:
Revised:
Accepted:
Published:
8439
July 6, 2015
September 10, 2015
September 14, 2015
September 14, 2015
DOI: 10.1021/acs.jafc.5b03284
J. Agric. Food Chem. 2015, 63, 8439−8444
Article
Journal of Agricultural and Food Chemistry
Figure 2. (a) Water-presaturated 1H HR-MAS NMR spectrum of fresh saffron petals and (b) 1H NMR spectrum of the ethanol (95%) extract of
fresh saffron petals in D2O solution.
bone loss preventive, and osteoclastogenesis suppressing.19 For
these reasons, efforts toward efficient synthesis of compounds 2
and 3 by a chemoenzymatic approach have recently been
reported.20
To gain a deeper insight into this subject and to establish
which are the main metabolites present in C. sativus petals, we
analyzed them by high-resolution magic-angle-spinning nuclear
magnetic resonance (HR-MAS NMR) and their ethanol (95%)
extract by HR-NMR. HR-MAS NMR spectroscopy allows for
the derivation of the biochemical profile of intact (human,
animal, or plant) tissues21 formed by fast-moving small metabolites that give rise to narrow resonances. Through HR-MAS
NMR, we tried to have a look inside a complex row matrix
without disrupting its structure and avoiding hydrolytic
processes that can flank extraction. The purpose of this paper
is to contribute to the utilization of C. sativus petals directly
after the separation from saffron. To the best of our knowledge,
this is the first HR-MAS NMR study of petals.
■
(about 1 h). The extracts were analyzed in HR-NMR and mass
spectrometry (MS).
NMR Measurements. 1H HR-MAS NMR spectra were recorded
with a Bruker Avance 400 (Bruker BioSpin) spectrometer operating at
a frequency of 400.13 MHz. The instrument was equipped with a
1 13
H, C HR-MAS probe for semi-solids and with a broadband inverse
(BBI) probe for liquids. Before HR-MAS examination, intact saffron
petals were introduced in a MAS zirconia rotor (4 mm outer diameter)
and 10 μL of D2O was added to provide deuterium for the lock
system, fitted with a 50 μL cylindrical insert to increase sample homogeneity, and then transferred into the probe cooled to 5 °C. Experiments
were performed at a temperature of 5 °C controlled by a Bruker
cooling unit.
Samples were spun at 4000 Hz, and one-dimensional (1D) and
two-dimensional (2D) spectra were acquired using the sequences
implemented in the Bruker software. The same experiments were
carried out on D2O extract solution at 25 °C, with a lower number of
scans. To record 1H water-presaturated spectrum, a composite pulse
sequence (zgcppr) with 2 s water presaturation during the relaxation
delay, 8 kHz spectral width, 32 000 data points, and 16−8 scans were
used. The 2D correlation spectroscopy (COSY) spectra were acquired
using a standard pulse sequence (cosygpprqf), 1 s water presaturation during relaxation delay, 2−4 kHz spectral width, 2000 data points,
4 scans per increment, and 128 increments. The 2D total correlation
spectroscopy (TOCSY) spectra were acquired using a standard pulse
sequence (mlevgpph19), 1 s water presaturation during relaxation
delay, 100 ms mixing (spin-lock) time, 2−4 kHz spectral width,
4000 data points, 4 scans per increment, and 512 increments. The 2D
heteronuclear single-quantum coherence (HSQC)-edited spectra were
acquired using a standard pulse sequence echo−antiecho phase sensitive
(hsqcedetgp), 0.5 s relaxation delay, 1.725 ms evolution time, 2−4 kHz
spectral width in f2, 2000 data points, 16−8 scans per increment,
15 kHz spectral width in f1, and 256 increments. The 2D heteronuclear
multiple-bond coherence (HMBC) spectra were acquired using a
standard pulse sequence (hmbcgplpndqf), 0.5 s relaxation delay, 50 ms
evolution time, 2−4 kHz spectral width in f2, 2000 data points, 128−32
scans per increment, 22 kHz spectral width in f1, and 256 increments.
Deconvolution of 1H NMR-selected signals was run with
MestReNova 9.1 (2014 Mestrelab Research S.L.).
EXPERIMENTAL SECTION
Plant Materials. C. sativus flowers were collected in Abruzzo
(Italy), in the Aquila Saffron Protected Designation of Origin (PDO)
status, at the Farm “Vigna di More” located in Tione degli Abruzzi, in
autumn 2011. The flowers grew at 700 m above sea level. The bulbs
were planted in August on a ground prepared 1 year before (the soil is
employed every 5 years), and the flowers were harvested in October.
The bulbs were put at 2−3 cm distance from each other, at least 8 cm
depth in rows of two or three, according to the space and land
available. Flowers were hand-picked early in the morning, before
sunrise before perianth opening, and were placed in traditional wicker
baskets. After collection, they were stored at 5 °C and send to the lab,
where intact petals were used for HR-MAS NMR measurements
within 3 days after collection.
Extraction. Fresh petals (10 g) were extracted with 95% ethanol
(Sigma-Aldrich), and the extract was vacuum-evaporated (150 mg).
The whole process was carried out quickly at room temperature
8440
DOI: 10.1021/acs.jafc.5b03284
J. Agric. Food Chem. 2015, 63, 8439−8444
Article
Journal of Agricultural and Food Chemistry
Figure 3. Enlarged (a) carbohydrate and (b) aliphatic regions of the HSQC HR-MAS NMR spectrum of fresh saffron petals. ChoCC = cholinecontaining compounds.
Electrospray Ionization (ESI) Ion-Trap (IT) MS Measurements.
The negative-ion ESI mass spectra and ESI−MS/MS data (collision
energies of 10, 15, 20, or 35 eV) were acquired with a LC−MS(n) IT
6310A (Agilent Technologies) coupled with a HPLC Agilent Series
1200 equipped with a Zorbax SB-C18 column, 30 × 2.1 mm inner
diameter, 3.5 μm particle size (Agilent). Eluents were acetonitrile, H2O,
and 1% formic acid. Chromatographic runs were performed using a
gradient of 1% formic acid in acetonitrile (98 → 10%) and 1% formic
acid in water (2 → 90%). The solvent flow rate was 0.2 mL/min; the
temperature kept at 25 °C; and the injector volume selected was 2 μL.
Total ion current (TIC) chromatograms were acquired in both positive
and negative mode in the mass range between m/z 100 and 1400.
He was used as the collision gas in MS2 experiments.
broad and weak signals are found in the aromatic region, at
about 6.9 and 7.7 ppm, where multiplets from kaempferol
derivatives are expected. These two broad resonances are sometimes assigned to polyphenols, but they have also been attributed
to NH protons.22 On the other hand, some multiplets in the
range of 3.1−2.5 ppm, outside the common carbohydrate
region, are detected, and other low signals mainly attributable to
free amino acids and lipids are present at lower parts per million
(ppm).
To disentangle the complex spectral pattern, it was necessary
to use 2D NMR homo- and heterocorrelated experiments, such
as COSY, TOCSY, HSQC, and HMBC. We were thus able to
reconstruct molecular skeletons and compare the chemical shifts
and the correlation patterns to those reported in the literature
and in NMR databanks, such as the Human Metabolome
Database (HMDB; http://www.hmdb.ca/). Using this approach,
we found that the majority of signals in the 1H spectrum are due
■
RESULTS AND DISCUSSION
The water-presaturated 1H HR-MAS NMR spectrum of saffron
petals (Figure 2a) shows the overlapped signals from a number
of metabolites, mainly in the carbohydrate region, whereas only
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DOI: 10.1021/acs.jafc.5b03284
J. Agric. Food Chem. 2015, 63, 8439−8444
4.55
4
8442
103.9
75.6
78.4
f
f
f
77.4
77.9
182.5
38.2
C
13
C
4.40e (d, 7.8 Hz)
3.19
3.36
3.29
3.29
3.67, 3.88
104.8
75.9
79.2
72.8
79.2
63.9
77.3
4.54,d 4.49
179.7
37.2
13
77.2
2c
2.87, 2.65 (dd,
18.0, 6.4 Hz;
ddd, 18.0,
1.9, 0.8 Hz)
4.72
H
1
3b
4.58
3.27
3.49
f
f
f
4.59
4.86
3.03, 2.78 dd,
18.6, 6.1 Hz;
d, 18.6 Hz)
H
1
103.7
75.6
78.4
f
f
f
77.9
77.9
182.5
38.5
C
13
H
3c
4.38e (d, 7.8 Hz)
3.19
3.36
3.29
3.29
3.67, 3.88
4.47d
2.89, 2.73 (dd,
18.2, 1.4 Hz;
dd, 18.2,
6.4 Hz)
4.73
1
C
105.1
75.9
79.2
72.8
79.2
63.9
76.4
77.4
180.1
38.1
13
4b
4.54, 4.36
(d, 10.3 Hz)
4.71 (t, 4.6 Hz)
2.97, 2.51 (dd,
18.1, 6.1 Hz;
d, 18.1 Hz)
H
1
79.8
70.0
183.0
40.1
C
13
H
4c
2.83, 2.37 (dd,
17.7, 5.8 Hz;
dt, 17.7,
1.3 Hz)
4.57 (ddt, 4.3,
5.8, 1.3 Hz)
4.42, 4.22d
(dd, 10.0, 4.3
Hz; dt, 10.0,
1.2 Hz)
1
C
78.9
69.5
180.3
39.7
13
1c
6.92
8.05
5.43g
3.74h
3.61
3.36
3.20
3.49, 3.69
4.78
3.37
3.41−3.30
3.69, 3.80
8.05
6.92
6.42
6.22
H
1
C
63.5
79.5
63.7
106.0
76.8
106.9
164.2
101.0
167.2
95.9
159.6
123.9
133.5
117.5
162.8
117.5
133.5
102.2
84.3
79.0
nd
136.1
160.3
13
Chemical shifts refer to sucrose 1-H at 5.40 and 94.8 ppm, respectively. bIntact petals. cPetals extracted with ethanol (95%). dLong-range correlation with CO. eLong-range correlations at 4.40/77.2
and 4.38/77.4 ppm in the HMBC spectrum. fOverlapped to β-Glc. gLong-range correlations with 136.0 ppm of carbon in the HMBC spectrum. hLong-range correlations with 102.2 and 106.0 ppm of
carbon in the HMBC spectrum.
a
4.55
3.27
3.49
f
f
f
4.86
4a
5
6
7
8
8a
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴, 4‴, 5″’
6‴
2b
2.99, 2.71 dd,
18.1, 6.1 Hz;
d, 18.1 Hz)
3
1
2
H
1
Table 1. 1H and 13C NMR Data of Compounds 2−4 (ppma) in C. sativus Petals As Obtained by HR-MAS NMR and of Compounds 1−4 Extracted with Ethanol (95%) (D2O)
Journal of Agricultural and Food Chemistry
Article
DOI: 10.1021/acs.jafc.5b03284
J. Agric. Food Chem. 2015, 63, 8439−8444
Article
Journal of Agricultural and Food Chemistry
Figure 4. ESI−IT MS/MS spectra of (left) compound 1 and (right) minor kaempferol derivative.
time, the free glucose signals enhance with respect to those
of sucrose (1-Hα‑Glc/1-Hsuc passes from 0.4 to 1.2). Hence, the
extraction process, even though carried out rapidly and without
acidic conditions, promoted hydrolysis of compounds 2 and 3
to compound 4 and free Glc.
ESI−IT MS was finally employed to confirm the presence of
compound 1 and to clarify the structure of the minor flavonol
detected in the ethanol extract (Figure 4). The two major
peaks detected in negative mode correspond to m/z 609 and
771 [M − H]− pseudo-molecular ions. The collision-induced
dissociation (CID) with He gas of m/z 609 (highest) peak gave
m/z 429 [M − C6H12O6 − H]− and m/z 285 [kaempferol − H]−
as expected for two subsequent hexose losses starting from
compound 1. On the other hand, CID of m/z 771 gave m/z
609, corresponding to a loss of C6H10O5, demonstrating that
this derivative contains a further hexose unit. Minor peaks,
corresponding to pseudo-molecular species of m/z 625 (which
decomposes to m/z 463 through hexose loss) and m/z 651
(which gives m/z 471 and 285 under CID), were also detected.
The former could be due to quercetin sophoroside, and the
latter is probably an acetylated derivative.
In conclusion, this work shows that HR-MAS NMR can be
employed to monitor bioactive compounds freely tumbling
in the cell environment, directly on intact petals. HR-MAS
NMR allows for detection of the presence of compounds 2, 3,
and 4 directly on intact C. sativus petals, without the need of
extraction processes. Their content was estimated by HR-MAS
NMR to be roughly 0.6%. HR-MAS NMR does not detect
instead flavonols, especially compound 1, that are still contained
in petals in a not negligible amount, as demonstrated by 1H
NMR analysis of the ethanol extract. This implies that flavonol
derivatives in saffron petals have low tumbling rates, which
could be due to their close association to macromolecular
species in cell walls.24
The 3-hydroxybutyrolactone derivatives 2−4 contained in
C. sativus petals are biologically active species that, together with
compound 1, probably contribute to confer hepatoprotective,
antihyperglycemic, anti-inflammatory, and other effects to petal
to sucrose (Suc), glucose (Glc), and fructose (Fru), as can be
better seen observing HSQC correlations (Figure 3). Apart from
those sugars, other resonances as a result of compounds 2−4 are
detected and assigned, through 2D experiments, as reported in
Table 1. The chemical shifts determined by us on intact petals
were parallel to those reported in pyridine-d5 by Zhang et al.20
Remarkably, even 2D NMR spectra did not highlight signals
from kaempferol or its derivatives in the aromatic region,
meaning that, in the cell environment of fresh petals, these
molecules are not freely tumbling but are probably associated
with rigid structures within the cell. Other minor resonances
were confirmed to derive from free amino acids (mainly alanine,
Ala, glutamine, Gln, and valine, Val).
The molar ratio of total 2−4 derivatives relative to the
water signal, estimated through deconvolution of 2-H signals
of compounds 2−4 at 2.8−2.5 ppm and H2O at 4.97 ppm
in the not presaturated 1H NMR spectrum, resulted in around
0.3 mol %. It can be roughly converted to 0.8% in weight
with respect to water and then, considering 75% water in petals,
to 0.6% of compounds 2−4 in intact petals.
Fresh petals were then extracted rapidly with 95% ethanol;
the vacuum-evaporated residue was solubilized in D2O; and 1D
and 2D NMR and ESI MS/MS spectra were acquired.
The 1H NMR spectrum of the extract is reported in Figure 2b,
and it clearly displays signals in the aromatic region that compare
well to those reported by Wolfram et al.23 and were assigned,
together with other resonances in the carbohydrate region,
to kaempferol 3-O-sophoroside (1; Table 1) and to a minor
derivative that differs mainly for the 2′,6′-H signals that are found
at 8.09 ppm, 8-H at 6.78 ppm (d, 2.2 Hz), 6-H at 6.50 ppm
(d, 2.2 Hz), and 1-HGlc at 5.48 ppm (d, 7.6 Hz) and could be
a 7-substituted derivative of compound 1.
Apart from differences in the chemical shifts, as a result of a
change from the cell environment to D2O solution, resonances
from compounds 2−4 were also detected in the ethanol extract,
even though in a ratio different with respect to that observed
in intact petals: the (2 + 3)/4 molar ratio passes from 2.5:1 in
intact petals to less that 1:1 in the extracts, and at the same
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extracts. The extraction process leads to partial hydrolysis of
petal constituents; hence, it could be useful to evaluate the
direct employment of saffron petals for phytopharmaceutical
and nutraceutical purposes, to better exploit a waste product of
massive production of commercial saffron.
The data presented in this study strongly support the idea
that petals of C. sativus are not a waste but can be used for
their biological activity in specific foods as well as in herbalist
products. This can have a major impact for the community of
saffron, the cultivation of which represents an opportunity to
improve and develop the economy of the agricultural sector in
poor European regions. The exploitation of not only stigmas
but also petals as raw material for health-enhancing products
would further enhance the economical value of this activity and
reduce waste production.
■
AUTHOR INFORMATION
Corresponding Author
*Telephone: 00390592058636. E-mail: [email protected].
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors are very grateful to Prof. Dario Iarossi and the “Vigna
di More” farm, located in Tione degli Abruzzi, for the supply of
C. sativus petals (http://www.valleaterno.it/vignadimore/).
■
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DOI: 10.1021/acs.jafc.5b03284
J. Agric. Food Chem. 2015, 63, 8439−8444