American Journal of Plant Sciences, 2012, 3, 1573-1580
http://dx.doi.org/10.4236/ajps.2012.311190 Published Online November 2012 (http://www.SciRP.org/journal/ajps)
1573
Biochemical, Antioxidant and Antineoplastic Properties of
Italian Saffron (Crocus sativus L.)
Angelo Gismondi, Mariagiovanna Serio, Lorena Canuti, Antonella Canini*
Department of Biology, University of Rome “Tor Vergata”, Roma, Italy.
Received August 9th, 2012; revised September 18th, 2012; accepted October 19th, 2012
ABSTRACT
Saffron, the most expensive spice in the world, is got by Crocus sativus L. stigmas. The production of this substance has
attracted human interest, since ancient cultures, for its medicinal and culinary properties. Because of saffron high eco-
nomic value, sometimes, since Middle Ages, it is adulterated with other vegetal materials, dyes or synthetic molecules.
Object of this work was the study of one of the best world saffron: Civitaretenga (AQ, Central Italy) C. sativus. Taste,
color and aroma of Civitaretenga spice were determined, according to international standards (ISO/Technical Specifica-
tion 3632), to define its high quality. A biochemical approach was then applied to obtain a secondary metabolite profile
of this product. So, crocins, total phenolic content, flavonoids and phenolic acids were detected by HPLC-DAD and
spectrophotometric analysis. Moreover, in vitro antioxidant properties and in vivo antineoplastic effects, on highly me-
tastatic murine B16-F10 melanoma cell line, were successfully revealed in Civitaretenga C. sativus extract. All these
data confirmed the elevated quality of Civitaretenga saffron and its highly reducing and chemopreventive activity.
Keywords: Crocus sativus L.; Saffron; Plant Secondary Metabolites; Antioxidant; Antineoplastic
1. Introduction
Crocus sativus L. is an herbaceous species of the Irida-
ceae family. It is a monocot plant very diffused in the
Mediterranean Basin and Western Asia [1]. The infertil-
ity of this species has been associated to its triploid ge-
nome (3n), whose origin has not been clarified yet: lit-
erature data suppose that it would be generated from the
evolution, or the hybridization, of other Crocus exem-
plars [2]. Consequently, C. sativus only propagates vege-
tatively [3-5]. Its flower is made up of six violet tepals,
three yellow stamens and a single pistil whose stigma,
defined by three red filaments, is the source of the saf-
fron [6]. This spice is the most expensive in the world
because C. sativus bloom occurs, in a very short period,
only once a year and its harvest should be performed
manually [7]. C. sativus stigmas are characterized by the
presence of sugars, minerals, fats, vitamins and second-
dary metabolites: terpenes, flavonoids, anthocyanins and
carotenoids. Between them, carotenoids are the most
important molecules because they determinate color and
taste of the spice. From this class of compounds, in C.
sativus, we can essentially find lycopene, α- and β-caro-
tene, zeaxanthin and crocetin that are liposoluble and the
hydrosoluble crocins, derived by crocetin esterification
with sugars [8-10]. Principal saffron crocins are trans-cro-
cetin di-(β-D-gentiobiosil) ester (named trans-4-GG), trans-
crocetin (β-D-glucosil)-(β-D-gentiobiosil) ester (named
trans-3-Gg), trans-crocetin (β-D-gentiobiosil) ester (named
trans-2-G), cis-crocetin di-(β-D-gentiobiosil) ester (named
cis-4-GG), trans-crocetin di-(β-D-glucosil) ester (named
trans-2-gg) and cis-crocetin(β-D-glucosil)-(β-D-gentio-
biosil) ester (named cis-3-Gg) [11]. High temperatures
and humidity levels induce crocin oxidation and degra-
dation and consequently the decrease of spice attributes
[12]. Aroma, on the other hand, is determined by the
amount of safranal, a terpenic aldehyde, and picrocrocin,
a glycosidic form of the safranal [9-13]. Saffron has cap-
tured human attention since when past populations em-
ployed it as drug, perfume, dye and aroma [14-17]. Its
adulteration, motivated by the high economic value, has
been performed, since Middle Ages, with natural or syn-
thetic substances [18,19]. For these reasons, international
standards [20] were framed to scientifically determine
saffron quality, to find out spice fraudulent alterations
and for its valorization. According to these conventions,
spectrophotometric and chromatographic investigations
are able to determine spice color, taste and aroma [21-23].
Object of this work has been the identification of Civi-
taretenga (AQ, Central Italy) C. sativus biochemical
components and the quality classification of the saffron
*Corresponding author.

Biochemical, Antioxidant and Antineoplastic Properties of Italian Saffron (Crocus sativus L.) 1574
derived from its stigmas, according to ISO/Technical
Specification 3632 standards. As C. sativus extract is re-
ported in literature showing a large amount of flavonoids,
especially kaempferol, quercetin and naringenin [24,25],
in this study, we also analyzed its antioxidant power and
antiproliferative effects on murine B16-F10 melanoma
cells.
2. Materials and Methods
2.1. Plant Material
Crocus sativus bulbs were obtained from Civitaretenga
cultivars (AQ, Central Italy) and grown in the Botanical
Garden of the University of Rome “Tor Vergata”. For
spice production stigmas of C. sativus were dehydrated at
103˚C for 2, 8 or 16 hours.
2.2. Spice Quality Determination
Spice quality was established, in relation to its taste
(picrocrocin content), aroma (safranal amount) and color
(crocin level),with respect to ISO/TS 3632 standards [20].
Briefly, 5 mg of saffron were resuspended in 20 mL of
ultrapure water at room temperature in the dark for 1
hour and then filtered by a 0.45 µm cellulose filter. Spice
quality was analyzed by spectrophotometric method us-
ing the following formulas:
 spice taste-1%
1 cm
E
 
257 nm Abs20000100% moisture content ,
 safranal content-1%
1 cm
E
 
330 nm Abs20000100% moisture content

,
 color intensity-
1%
1 cm
E

440 nm Abs20000100% moisture content ;
where is 1% molar extinction coefficient of the
solution measured in 1 cm cuvette and 257 nm, 330 nm
and 440 nm are absorption wavelengths respectively of
picrocrocin, safranal and crocins.
1%
1 cm
E
2.3. Secondary Metabolite Extraction
Secondary metabolites extraction was performed on
dried (16 h) stigmas according to [22] method. 50 mg of
dried stigmas were powdered with liquid nitrogen. The
extraction was performed in 10 mL of ddH2O (or metha-
nol) in agitation at 4˚C, in the dark, for 24 hours. After
centrifugation at 30.000 g for 20 minutes, the supernatant
was filtered and analyzed.
2.4. Total Phenolic Content
Total polyphenol determination was carried out by Folin-
Ciocalteau assay [26]. 9 mL of ddH2O (or methanol) and
1 mL of Folin-Ciocalteau reagent (Sigma-Aldrich) were
added to 1 mL of each secondary metabolite extract. Af-
ter 5 minutes of incubation, 10 mL of Na2CO3 7% (w/v)
and 4 mL of ddH2O (or methanol) were added. The solu-
tion was vortexed and incubated at room temperature for
1 hour in the dark. Absorption was determined at 760 nm
by a UV-visible spectrophotometer Cary 50 (Bio Varian).
Total polyphenol concentration was calculated with re-
spect to a caffeic acid calibration curve (20 - 100 mg/L)
and results were expressed as µg of caffeic acid equiva-
lents (µg CAE/mg DW) of dried stigmas.
2.5. HPLD-DAD Analysis
Crocins, flavonoids and phenolic acids, extracted (in wa-
ter) as described before, were identified and quantized by
a high performance liquid chromatography (HPLC, Shi-
madzu) instrument associated with DAD detector
(SPD-M20A), multisolvent delivery system (LC-10AD),
auto-sampler (SIL-10AD), controller module (SCL-10A)
and Class VP 5.02 software. Crocin analysis was per-
formed according to Alonso et al. [27] with some modi-
fications. A Hamilton PRP-10 column (10 µm, 4.6 × 150
mm) was used and the elution gradient was performed
with 10% (v/v) acetonitrile (A) and methanol (B). The
gradient system was 100% A to 90% A in 10 minutes
and then 0% A to 50 minutes, at flow rate of 1 mL/min.
Column re-equilibration between runs was of 10 minutes.
The column was kept at 30˚C. Crocins (trans-4-GG,
trans-3-Gg, cis-4-GG, trans-2-G, cis-3-Gg and trans-2-G)
were measured at 440 nm. Flavonoids and phenolic acids
were analyzed according to Canini et al. [28] with a
Phenomenex Gemini NX C18 column (5 µm, 4.6 × 150
mm). Flavoniods (myricetin, quercetin and kaempferol)
were monitored at 280 nm while phenolic acids (gallic,
chlorogenic and caffeic acids) were monitored at 320 nm.
The eluition gradient was performed with tricloroacetic
acid pH 2.5 (A) and acetonitrile (B). The gradient system
was 85% A to 65% A in 20 minutes and then 20% A in 8
minutes and maintained at this concentration for 5 min-
utes, at flow rate of 1 mL/min. The column was kept at
35˚C. Column re-equilibration between runs was of 10
minutes. For each analysis, 20 μL of each sample was
injected. Peak identification was assured according to
their retention times and by co-elution with authentic
standards (Fluka).
2.6. DPPH Radical Scavenging Test
Aqueous saffron extract antioxidant activity was deter-
mined on the basis of its scavenging activity on the stable
DPPH free radical (Merck). According to Brand-Wil-
liams et al. [29], we monitored absorbance decrease of a
100 µM DPPH methanolic solution, at 517 nm, after 30

Biochemical, Antioxidant and Antineoplastic Properties of Italian Saffron (Crocus sativus L.) 1575
minutes of sample addition. Sample antiradical activity
was calculated by the following ratio:

Abs controlAbs sampleAbs control100
;
where Abs control is DPPH solution absorption and Abs
sample is DPPH solution absorption after sample addi-
tion. Antioxidant activity was expressed as IC50 (sample
concentration producing 50% of DPPH activity decrease
with respect to the control).
2.7. FRAP Assay
FRAP assay measures absorbance change at 593 nm ow-
ing to the formation of a blue-colored Fe II—tripyridyl-
triazine compound (Merck), from the colorless oxidized
Fe III form, by the action of electron donating antioxi-
dants [30]. 200 µL of sample was added to 1.8 mL
freshly prepared and pre-warmed (37˚C) FRAP reagent
(10 mM TPTZ in 40 mM HCl, 20 mM FeCl3, 0.3 M ace-
tate buffer pH 3.6; 1:1:10 v/v/v) and incubated at 37˚C
for 10 minutes. Fe II standard solution (50 - 500 µM)
was obtained from ferrous sulphate (FeSO4 in ddH2O).
Results were expressed as mM ferric ions reduced to
ferrous form per gram of sample dried weight, according
to Woidylo et al. [31].
2.8. Cell Cultures and Proliferation Assays
Highly metastatic murine B16-F10 melanoma cell line
was propagated under standard culture conditions [32].
Briefly, cells were cultured in Dulbecco’s modified Ea-
gle’s medium (D-MEM), supplemented with 10% fetal
calf serum (FCS), 200 mM glutamine, 100 U/mL penicil-
lin and 0.1 mg/mL streptomycin, and maintained in hu-
midified atmosphere with 5% CO2 at 37˚C. To test C.
sativus antineoplastic property, B16-F10 cells were
seeded and grown in 35-mm dishes and treated with
aqueous stigma extracts (250, 500 and 1000 µg/ml) for
24, 48, and 72 hours. Cell growth was determinated by
MTT based kit (Sigma). This product was designed for
the spectrophotometric measurement of cell proliferation
rate as a function of mitochondrial activity in living cells.
On the other hand, the proliferation was also analyzed by
counting cells with a Neubauer modified chamber, after
Trypan Blue staining (1%, w/v) for cytotoxicity evalua-
tion. Cell morphology was observed by optical micro-
scope (20×) (Nikon, TE2000-PFS).
2.9. Statistical Analysis
Each experiment was repeated at least three times. Analy-
sis of variance was conducted using one-way ANOVA
test with SPSS (ver.19 ita) for Microsoft and means were
compared by Duncan tests.
3. Results
3.1. Saffron Quality Determination
Stigmas of Civitaretenga C. sativus were dehydrated,
powdered and subjected to aqueous extraction. The dry-
ing process was conducted at different times (2, 8 and 16
hours). To evaluate saffron taste, aroma and color, the
extract was analyzed according to ISO/TS 3632 method.
Saffron values, obtained by spectrophotometric
analysis at different wavelengths, were reported in Table
1: all values remained inside ISO/TS 3632 standard
ranges, except picrocrocin level after 2h of desiccation. 8
and 16 h dehydration processes decreased picrocrocin
amount, respectively of 20% and 37.3%, and increased
safranal content, of 7.4% and 22.2%, with respect to 2 h
procedure. No considerable variation was observed in
crocin amount.
1%
1 cm
E
3.2. Secondary Metabolite Analysis
Aqueous or methanolic extraction was performed on C.
sativus stigmas. Total phenolic content was measured in
both extracts: methanolic solution presented 53.52 ± 1.75
µg CAE/mg DW of total polyphenols with respect to the
aqueous one that was only 31.46 ± 1.05 µg CAE/mg DW.
HPLC-DAD analysis permitted crocin identification in
saffron aqueous extract (Figures 1 and 2): trans-4-GG,
trans-3-Gg, trans-2-gg, cis-4-GG, cis-3-Gg and trans-
2-G isomers were detected, at specific retention times (tR,
min), and quantified (Q, mg/g) with respect to authentic
standards, as reported in Table 2. Trans-4-GG and trans-
3-Gg molecules were the most abundant identified cro-
cins. The chromatographic method was also applied to
reveal the presence, or the absence, of principal saffron
flavonoids (kampferol, quercetin, genistein and myricetin)
and phenolic acids (cumaric, caffeic, chlorogenic and
gallic acid) in the sample (Table 3).
Table 1. Civitaretenga saffron values at 257, 330 and
440 nm for different spice dehydration times. In the lower
part are also reported ISO 3632 standard value
ranges.
E1%
1cm
E1%
1cm
Saffron quality evaluation
Saffron
dehydration time (h)
1%
1 cm
E 257 1%
1 cm
E 330 1%
1 cm
E 440
2 h 75 ± 0.02 27 ± 0.02 94 ± 0.07
8 h 60 ± 0.01 29 ± 0.01 95 ± 0.004
16 h 47 ± 0.02 33 ± 0.01 92 ± 0.03
ISO 3632
standard range 30 < X < 70 20 < X < 50 80 < X < 190

Biochemical, Antioxidant and Antineoplastic Properties of Italian Saffron (Crocus sativus L.) 1576
Table 2. Crocin retention times (tR, min) and quantity (Q,
mg/g) detected in Civitaretenga saffron by HPLC-DAD
analysis.
Crocin identification
Crocin tR (min) Q (mg/g)
trans-4-GG 30.51 ± 0.4 45.0 ± 0.2
trans-3-Gg 33.27 ± 0.6 39.2 ± 0.1
trans-2-gg 35.28 ± 0.6 4.5 ± 0.5
cis-4-GG 36.16 ± 0.7 3.9 ± 0.5
cis-3-Gg 37.54 ± 0.4 4.2 ± 0.4
trans-2-G 39.67 ± 0.5 3.2 ± 0.1
Table 3. Specific flavonoid and phenolic acid presence (+)
or absence (−) in Civitaretenga saffron and relative reten-
tion times (tR, min) in HPLC column.
Secondary metabolite survey
Molecules Presence/absence (+/−) tR (min)
Flavonoids
Kampferol + 20.0 ± 0.2
Quercetin + 14.4 ± 0.1
Genistein − ------------
Myricetin + 9.9 ± 0.4
Phenolic acids
Cumaric acid − ------------
Caffeic acid + 11.18 ± 0.6
Chlorogenic acid + 10.59 ± 0.2
Gallic acid + 8.71 ± 0.3
Figure 1. HPLC-DAD chromatogram representing saffron
extract crocins: trans-4-GG (A), trans-3-Gg (B), trans-2-gg
(C), cis-4-GG (D), cis-3-Gg (E) and trans-2-G (F).
3.3. Spice Antioxidant and Antiproliferative
Activity
Civitaretenga C. sativus antioxidant properties were es-
timated by antiradical molecular assays on its stigma
aqueous extract. DPPH test showed a sample IC50 value
of 3.76 mg DW whilst FRAP assay indicated that one
gram of dried sample presented the same antioxidant
power of 2.53 ± 0.15 mM FeSO4. Saffron antiprolifera-
tive effects were tested on highly metastatic murine
B16-F10 melanoma cells for 24, 48 and 72 hours after
treatment with 250, 500 and 1000 µg/ml of spice extract.
As reported in the Figure 3, MTT assay showed cell
growth reduction at all treatments: in particular, with
respect to the control, proliferation decreased of 40.7%,
63.9% and 73.6% respectively after 250, 500 and 1000
µg/ml of treatment at 72 h. Treatment cytotoxic proper-
ties were analyzed by Trypan Blue solution capacity to
stain dead cells: cell mortality was always very reduced
with respect to the control (max < 8%) (Table 4). Cell
morphological observations were also performed by an
optical microscope (Figure 4): 1000 µg/ml treatment for
72 h induced the development of cell dendritic evagina-
tions, with respect to the control cells.
4. Discussion
Saffron derives from C. sativus stigmas. This spice in-
creased its human applications and commercial value
along the time. As Italy, along with Iran, Spain, India,
Greece, Azerbaijan and Morocco, is one of the principal
world saffron producers [33], this work focused on the
determination of Civitaretenga saffron biochemical, or-
ganoleptic and nutraceutical properties. Not all saffron
extracts can be considered equal because various geo-
graphical conditions, genetic factors and temperatures
differently can modulate plant growth and secondary
metabolite synthesis. Food quality, generally, depends on
its chemical composition and preparing techniques. In
saffron production, humidity percentage (about 12%) is
very important to assure spice conservation and excel-
lence: water content, in fact, regulates microbiological
and enzymatic activities reducing spice preciousness [10].
Table 4. Murine B16-F10 melanoma cell toxicity after 24, 48
and 72 hours of treatment with 250, 500 and 1000 µg/ml of
Civitaretenga saffron extract.
Spice cytotoxicity percentages
Treatment
hours Control 250 µg/ml 500 µg/ml 1000 µg/ml
24 h 6.3% 7.1% 11.7% 13.9%
48 h 7.1% 6.5% 13.5% 12.8%
72 h 6.4% 10.4% 12.8% 14.2%

Biochemical, Antioxidant and Antineoplastic Properties of Italian Saffron (Crocus sativus L.) 1577
Figure 2. DAD crocin absorbance spectra: trans-4-GG (A); trans-3-Gg (B); trans-2-gg (C); cis-4-GG (D); cis-3-Gg (E) and
trans-2-G (F).
Figure 3. Murine B16-F10 melanoma cell growth determi-
nation by MTT assay at 24, 48 and 72 h after treatment
with 250, 500 and 1000 µg/ml of saffron extract.
Civitaretenga saffron quality was established according
to international standardization [20] (Table 1). This
study observed that desiccation procedure affected spice
quality: long dehydration times (8 and 16 h) highly re-
duced saffron taste (picrocrocin level) and weakly in-
creased spice aroma (safranal content), with respect to
short desiccation processes (2 h), but, interestingly, they
didn’t significantly modify its color (crocin amount).
These results could be explained because safranal mole-
cule comes from picrocrocin degradation, following ex-
tensive drying periods [34]. On the other hand, we con-
cluded that spice color modulation and crocin stability,
correlated to high temperature variations [35], would not
be associated to different desiccation times. Secondary
metabolite production in plants can vary from 6.8 to 32.1
µg CAE/mg DW [36]. Total polyphenolic content found
in Civitaretenga stigmas was very high: aqueous extract
phenol quantity resulted within literature reported ranges
whilst methanolic solution showed a 1.7-fold increased
concentration, surely, due to the organic solvent capacity
to capture less polar molecules, as flavonoids and pheno-
lic acids. Oxidative reactions are essential processes in
cell metabolism; however, they induce cell structural
damages, apoptosis and several pathologies (cancer,
atherosclerosis, diabetes, etc.), stimulating reactive oxy-
gen species (ROS) synthesis. A lot of plant food com-
pounds were recognized as antiradical substance sable to
prevent and rescue cell oxidative stress and several pa-
thologies [37-39]. To give more information about saf-

Biochemical, Antioxidant and Antineoplastic Properties of Italian Saffron (Crocus sativus L.) 1578
Figure 4. Morphological evidences of differentiation were
identified in treated (1000 µg/ml) B16-F10 melanoma cells
(B), with respect to the control ones (A) (20× images).
fron antioxidant elements with respect to human diet,
even if saffron methanolic extract had shown a great
phenolic content, we decided to only analyze the exact
secondary metabolite composition of aqueous solution,
because of its major cell bioavailability and absorption
capacity. Crocin levels (Figure 1 and Table 2) were
quite elevated with respect to the average values studied
in other scientific reports [27]. Saffron secondary me-
tabolite profile was characterized by the presence of dif-
ferent flavonoids (kampferol, quercetin and myricetin)
and phenolic acids (caffeic, chlorogenic and gallic acid);
nevertheless, HPLC-DAD analysis didn’t detect any
genistein or cumaric acid traces (Table 3). These results
suggested us to investigate spice antioxidant properties.
DPPH and FRAP assays revealed a good saffron anti-
radical effect, though not superior to literature data [40].
Different research groups published saffron antineoplas-
tic effects [41]. Obtained results (Figure 3) demonstrated
that Civitaretenga stigma solution showed a great anti-
proliferative effect, on murine B16-F10 melanoma cell
line, but also a very low citotoxicity (Table 4). Cell pro-
liferation reduction could be associated with C. sativus
extract differentiating properties. This hypothesis was,
preliminary, supported by cell shape morphological evi-
dences (Figure 4): with respect to the control, typical
dendrite-like structures [42] were observed in cells, in
particular, after 72 h of 1000 µg/ml treatment. All these
data support the idea that Civitaretenga C. sativus saffron
is a spice of elevated quality and that it contains highly
reducing and chemopreventive agents.
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