jeruk citrus flavonoid
TRANSCRIPT
Accepted Manuscript
C- and O-glycosyl flavonoids in Sanguinello and Tarocco blood orange (Citrussinensis (L.) Osbeck) juice: identification and influence on antioxidant proper-ties and acetylcholinesterase activity
Davide Barreca, Giuseppe Gattuso, Giuseppina Laganà, Ugo Leuzzi, ErsiliaBellocco
PII: S0308-8146(15)01450-8DOI: http://dx.doi.org/10.1016/j.foodchem.2015.09.098Reference: FOCH 18180
To appear in: Food Chemistry
Received Date: 22 June 2015Revised Date: 21 September 2015Accepted Date: 28 September 2015
Please cite this article as: Barreca, D., Gattuso, G., Laganà, G., Leuzzi, U., Bellocco, E., C- and O-glycosyl flavonoidsin Sanguinello and Tarocco blood orange (Citrus sinensis (L.) Osbeck) juice: identification and influence onantioxidant properties and acetylcholinesterase activity, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.09.098
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
C- and O-glycosyl flavonoids in Sanguinello and Tarocco blood
orange (Citrus sinensis (L.) Osbeck) juice: identification and influence
on antioxidant properties and acetylcholinesterase activity
DAVIDE BARRECA, GIUSEPPE GATTUSO*, GIUSEPPINA LAGANÀ, UGO LEUZZI
AND ERSILIA BELLOCCO
Dipartimento di Scienze Chimiche, Università di Messina, Viale F. Stagno d’Alcontres 31,
98166, Messina, Italy.
*Corresponding author.
Telephone +39 0906765241; fax +39 0906765186
E-mail address: [email protected] (G. Gattuso)
__________________________________________________________________
Abstract
2
Sanguinello and Tarocco are the blood orange (Citrus sinensis (L.) Osbeck) cultivars most
diffused worldwide. Reversed phase liquid chromatography coupled with MS-MS analysis
showed that these two varieties have a similar chromatographic pattern, characterized by the
presence of C- and O-glycosyl flavonoids. Of the two, Sanguinello was found to be far richer
in flavonoids than Tarocco. In the juices, twelve individual components were identified for
the first time, namely, four C-glycosyl flavones (lucenin-2, vicenin-2, stellarin-2, lucenin-2
4′-methyl ether and scoparin), three flavonol derivatives (quercetin-3-O-(2-rhamnosyl)-
rutinoside, quercetin-3-O-glycoside, quercetin 3-hydroxy-3-methylglutaryl-glycoside), an O-
triglycosyl flavanone (narirutin 4'-O-glucoside) and a flavone O-glycosides (chrysoeriol 7-O-
neoesperidoside). Moreover, the influence of the identified C- and O-glycosyl flavonoids on
the antioxidant and acetylcholinesterase activity of these juices has been evaluated.
Keywords: Citrus sinensis (L.) Osbeck; Sanguinello and Tarocco blood orange; HPLC-
DAD-ESI-MS-MS; Flavonoids; Antioxidant activity; Acetylcholinesterase activity
3
1. Introduction
Sanguinello and Tarocco (Citrus sinensis (L.) Osbeck) are the most common cultivars of
pigmented (blood) sweet orange. These varieties are widely diffused in the Mediterranean
basin (mainly Southern Italy and Spain) and in the United States of America (California), and
they are consumed worldwide, as fresh or processed products. They are the result of a
spontaneous genetic mutation, occurred many centuries ago in native plants from China and
repeated, perhaps several times, as oranges spread through the Mediterranean basin. In
particular, in Sicily they have been cultivated since the 15th century, becoming a staple of the
local citrus industry, so much so that they have received the Protected Geographical Status
(IGP).
Differently from the typical blond orange, the flesh and the rind of blood oranges are
characterized by the presence of pigmented compounds. The distinctive red flesh colouring is
due to the presence of anthocyanins (cyanidin 3-glucoside, cyanidin 3-6''-malonyl glucoside,
delphinidin 3-glucoside, peonidin 3-glucoside, cyanidin 3-rutinoside and the 6''-malonyl
glucose esters of delphinidin, peonidin and petunidin, Hillebrand, Schwarz, & Winterhalter,
2004; Maccarone, Rapisarda, Fanella, Arena, & Mondello, 1998; Dugo, Mondello, Morabito,
& Dugo, 2003). Recent work described the properties of these compounds, pointing out also
the presence, in significant amount, of vitamin C, along with hydroxycinnamic acids,
hesperidin, narirutin and didymin (Rapisarda & Intelisano, 1996; Rapisarda, Carollo, Fallico,
Tomaselli & Maccarone, 1998; Rapisarda et al., 1999; Kelebek, Canbas, & Selli, 2008;
Berhow, Tisserat, Kanes, & Vandercook, 1998) that confer pigmented orange juice superior
nutraceutical and biological properties with respect to the blond orange varieties (Rapisarda,
Tomaino, Lo Cascio, Bonina, De Pasquale & Saija, 1999; Buscemi et al., 2012; Grosso et al.,
2013). Very little information is available on the minor phenolics components such as C- and
O-glycosyl flavonoids, as we have recently reported for the Moro blood orange variety
(Barreca, Bellocco, Leuzzi, & Gattuso, 2014).
4
In the present paper we report the identification and quantification of twelve compounds in
juice of Sanguinello and Tarocco fruits, carried out simultaneously by means of a single
HPLC-DAD-ESI-MS-MS chromatographic course, as well as an in-depth study on the
antioxidant and acetylcholinesterase activity of the identified C- and O-glycosyl flavonoids
employing selected fractions obtained by preparative RP-HPLC separation.
2. Materials and methods
2.1. Samples
The fruits of Sanguinello and Tarocco blood orange (Citrus sinensis (L.) Osbeck) were
supplied by Vivai Raimondi Spadaro (Messina, Italy). The oblate fruits (average size 5.0-6.2
cm wide and 6.0-7.0 cm high) were harvested in April, about 8 months after the flowering
period. The investigation was carried out on 10 samples from the 2012–2013 fruit season.
2.2. Reagents and standard solutions
HPLC-grade acetonitrile and methanol were supplied by Sigma-Aldrich (St. Louis, MO,
USA), dimethylformamide (DMF) by Carlo Erba (Milano, Italy). Hesperidin and narirutin
were supplied from Extrasynthèse (Genay, France). Vicenin-2, lucenin-2 4'-methyl ether and
scoparin were separated from Citrus limetta (Barreca, Bellocco, Caristi, Leuzzi, & Gattuso,
2011a), lucenin-2, stellarin-2 and chrysoeriol 7-O-neohesperidoside from Citrus bergamia
(Gattuso, Caristi, Gargiulli, Bellocco, Toscano, & Leuzzi, 2006), narirutin 4'-O-glucoside
from Citrus aurantium (Barreca, Bellocco, Caristi, Leuzzi, & Gattuso, 2011d) and they were
used as standards.
2.3. Sample preparation
The fruits were preliminarily peeled to avoid contamination by flavedo components
(Gattuso et al., 2006). Peeled fruits were then hand-squeezed, and the juice sample thus
obtained were stored at –20 °C until used for analysis. Prior to analysis, DMF (10.0 mL) was
5
added to the crude juice (10.0 mL) and the mixture was centrifuged for 5 min at 3200 rpm.
The supernatant liquid was then filtered through an Iso-Disc P-34, 3 mm diameter PTFE
membrane, 0.45 µm pore size (Supelco, Bellefonte, PA, USA).
2.4. LC-MS-MS analysis of flavonoids
LC-MS-MS analyses of juice samples were carried out with a ThermoQuest Model LCQ-
Duo equipped with a diode array spectrophotometer and an ion trap mass spectrometer with
an electrospray ionisation source (ESI). Separation of each compound was performed on a
250 mm × 4.6 mm i.d., 5 µm Discovery C18 column, supplied by Supelco (Bellefonte, PA),
equipped with a 20 mm × 4.0 mm guard column. The column was placed in a column oven
set at 30 °C. The injection loop was 20 µL, and the flow-rate was 1.0 mL/min. The mobile
phase consisted of a linear gradient of acetonitrile in H2O as follows: 5–20% (0–15 min), 20–
30% (15–20 min), 30-100% (20–35 min), 100% (35–40 min), 100-5% (40–45 min), and 5%
(45–55 min). UV spectra were recorded between 200 and 450 nm, and simultaneous detection
by diode array was performed at 278 and 325 nm. Operating parameters of the mass
spectrometer were set as follows: capillary temperature 250 °C; spray needle voltage set at
4.50 kV; ES capillary voltage +3 and –47 V for positive and negative polarity, respectively;
tube lens offset 0 and –25 V for positive and negative polarity, respectively. Nitrogen was
used as a sheath gas with a flow of 50 arbitrary units. Mass analysis was carried out in full-
scan mode in the 80-900 amu range, both in positive and negative mode. MS-MS spectra
were obtained using an applied collision energy of 20–30% of instrument maximum. A
source fragmentation of 20 V as a collision energy was used in MS and MS-MS analysis
when required. Errors for m/z ratios for each compound were comprised between 150 and 400
ppm. Each sample was tested three times and gave superimposable chromatograms.
2.5. Identification of compounds
6
Compounds 1–12 were identified by their retention time, UV spectra, MS and MS-MS data
and by comparison with standard samples, where available. Full spectroscopic data are
reported in Table 1.
2.6. Acid hydrolysis
Juice hydrolysis was carried out by following the procedure described by Hertog, Hollman,
& Venema (1992). 6 M HCl (10 mL) in a methanol (25 mL)/water (10 mL) solution was
added to 5 mL of each of the juice samples, to a final concentration of 1.2 M HCl in 50%
aqueous methanol. Ascorbic acid (50 mg) was added as antioxidant. After refluxing at 90 °C
for 20 h under stirring, the solution was allowed to cool to room temperature. The solvents
were then evaporated under reduced pressure, and the residue was suspended in 10 mL
water/DMF (1:1). The mixture was filtered through an Iso-Disc P-34 membrane and analysed
by HPLC.
2.7. Quantitative evaluation of flavonoids content
Quantitative analysis was carried out by mass spectrometry, operating either in positive or
negative ion ESI mode with selected reaction monitoring (SRM). The instrumental
parameters were optimised for maximal generation of the pseudo molecular ions ([M – H]– or
[M + H]+) and of the characteristic fragment ions –indicated as “neutral loss”– employed for
the quantification of each individual compound. Quantification of analytes was obtained by
applying a collision energy of 25% of the instrument maximum for flavone C-glucosides,
20% for flavone-O-glycosides and flavanone-O-glycosides, respectively. For lucenin-2,
vicenin-2, stellarin-2, lucenin-2 4′-methyl ether and scoparin the precursor ions (m/z 609, 593,
623, 623 and 461, respectively) and product ions (m/z 489, 473, 503, 503 and 341,
respectively) were in agreement with a neutral loss of 120 amu, corresponding to the
characteristic fragmentation of the C-linked glucose moiety of the mono- and di-C-glucosyl
flavonoids. The precursor and product ions of chrysoeriol 7-O-neoesperidoside (m/z 609 and
7
301), narirutin (m/z 579 and 271) and hesperidin (m/z 609 and 301) were in agreement with a
neutral loss of 308 amu, corresponding to the characteristic fragment mass of O-diglycoside
(i.e., a neohesperidose or rutinose disaccharide). Narirutin 4'-O-glucoside displayed a neutral
loss of 470 amu, typical of O-triglycosyl flavonoids. The neutral losses of 306, 146 and 162
amu were used for quercetin 3-hydroxy-3-methylglutaryl-O-glycoside, quercetin-3-O-(2-O-
rhamnosyl)-rutinoside and quercetin-3-O-glycoside derivative from the precursor m/z 607,
757 and 463 to product ions at m/z 301, 611 and 301, respectively. The scan width was set to
m/z 1.0. Standard compounds (0.1–600 mg/L) were analysed twice and used for calibration of
the peak areas, by using the Genesis peak detection algorithm integrated in the ThermoQuest
software. To confirm the linearity and reproducibility for the quantification of the analytes,
the standards were analysed twice, and linear calibration curves were constructed from the
averaged peak areas.
This experimental procedure allows to achieve excellent limit of detection (LOD, 0.009–
0.025 µg/mL) and limit of quantification (LOQ, 0.027–0.075 µg/mL). LOD was defined as a
signal to noise ratio of 3. The LOQ was calculated as three times the LOD. Recovery of
analytes was always >98%. Intra- and interday coefficients of variation were below 7% for all
analytes.
2.8. Isolation of flavonoids fraction by preparative HPLC
Preparative LC separation was carried out on a Shimadzu model LC 8 A HPLC instrument
(Shimadzu, Kyoto, Japan) equipped with a Shimadzu SPD-10 A vp UV-vis detector. The
column used was a 250 mm × 21.2 mm i.d., 5 µm, Discovery C18 (Supelco, Bellefonte, PA).
The injection loop was 2.0 mL, and the flow rate was 10.0 mL/min at room temperature (20
°C). UV-vis detection was carried out at 278 nm. The mobile phase consisted of a linear
gradient of acetonitrile in H2O as follows: 25% (0–25 min), 25–100% (25–35 min), 100%
(35–40 min), 100-5% (40–45 min). 2.0 mL of crude juice was diluted 1:1 (v/v) with DMF,
8
centrifuged for 5 min at 3200 rpm, the supernatant was filtered through an Iso-Disc P-34, 3
mm diameter PTFE membrane, 0.45 µm pore size (Supelco, Bellefonte, PA, USA) and then
injected and flavonoids collected in the HPLC course time range of 11–28 min. Collected
fractions were joined, the solvents were removed under reduced pressure, and the resulting
residue was redissolved in 2.0 mL H2O/DMF 1:1 v/v. Prior to analysis, the volumes were
adjusted to match the concentrations they had in the original crude juices, as confirmed by
their qualitative and quantitative HPLC profiles before and after the separation.
2.8. DPPH• assay
The antioxidant ability of the crude juice and of selected fractions was assessed using the
stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•). The assay was carried out
according to a literature procedure (Rapisarda et al., 1999) with minor modifications. The
juices were centrifuged at 4000 rpm for 10 min and the supernatant was collected for analysis,
whereas fraction collected from preparative HPLC were redissolved in 2.0 mL H2O/DMF 1:1
v/v to restore the concentration the analytes had in the original crude juice. 20 µL of sample
solution was mixed with 63 µM of DPPH• in methanol, to a final volume of 4.0 mL.
The stability of the DPPH radical over the analysis time lapse (0–50 min) was determined
with a control containing all the components except the samples. After the addition of the
samples to the reaction mixtures, the absorbance of each sample at 517 nm was measured and
used as the starting point, and then the changes in absorbance at the same wavelenght were
monitored over 50 min. The inhibition percentage, I(%), of radical scavenging activity was
calculated as I(%) = 100 × (A0 – AS) / A0, where A0 is the absorbance of the control
immediately after the addition of the sample to be tested and AS is absorbance of the sample
after 50 minute of incubation. All tests were run in triplicate and the results expressed as
means ± standard deviation (SD). Trolox (1–100 µM) was chosen as a standard antioxidant,
and juice activity expressed in µM of Trolox equivalents (µM TE).
9
2.9. Trolox equivalent antioxidant capacity (TEAC) assay
The assessment is based on the comparison of the ability of a sample solution to quench
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cations (ABTS•+), with respect
to efficiency of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). A stable
stock solution of ABTS•+ was produced by reacting a 7 mM aqueous solution of ABTS (final
concentration) with 2.45 mM ammonium persulfate (final concentration), and allowing the
mixture to stand in the dark at room temperature for 12–16 h before use (Pellegrini, Re, Yang,
Rice-Evans, & Lester, 1999). At the beginning of the analysis day, an ABTS•+ working
solution was obtained by diluting the stock solution, to an absorbance of 0.70 ± 0.02 Abs at
734 nm, verified with a Varian Cary 50 Scan UV-Visible spectrophotometer (Varian,
Waldbronn, Germany). Sanguinello and Tarocco juices were centrifuged at 4000 rpm for 10
min and the supernatant was collected for analysis, whereas fraction collected from
preparative HPLC were redissolved in 2.0 mL H2O/DMF 1:1 v/v to restore the concentration
the analytes had in the original crude juice. 5 µL of each sample was added to the cuvette and
the absorbance was measured after 6 min at 734 nm. Due to the high activity, the samples
obtained from the juices were first diluted 1:5 with distilled water prior of the analysis. All
tests were run in triplicate and the results expressed as means ± standard deviation (SD). The
inhibition percentage I(%) of radical scavenging activity was calculated as I(%) = 100 × (A0 –
AS) / A0, where A0 is the absorbance of the control immediately after the addition of the
sample to be tested and AS is absorbance of the sample after 4 minute of incubation. Trolox
(in the range from 1.0 to 50 µM) was used as a reference standard. TEAC was expressed as
µM of Trolox equivalents (µM TE).
2.10. Ferric reducing antioxidant power (FRAP).
The FRAP assay was performed according to a literature procedure with minor
modification (Benzie & Strain, 1996). Fresh working FRAP reagent was prepared daily by
10
mixing 25 mL acetate buffer (300 mM, pH 3.6), 2.5 mL TPTZ solution (10 mM in 40 mM
HCl) and 2.5 mL of FeCl3⋅6H2O solution (20 mM). The reagent was warmed to 37 °C, then
1500 µL were placed in a cuvette and the initial absorbance was measured. 5 µL of sample
was added to the cuvette and the absorbance was measured immediately and after 4 min at
593 nm. All tests were run in triplicate and the results expressed as means ± standard
deviation (SD). Trolox was chosen as a standard antioxidant, and extracts activity was
expressed in µM Trolox equivalents (µM TE).
2.11. Hydroxyl radical scavenging assay.
Hydroxyl radical was assayed as described by Halliwell, Gutteridge, & Aruoma (1987)
with a slight modification. This assay is based on quantification of the degradation product of
2-deoxyribose by condensation with thiobarbituric acid (TBA). The reaction mixture
contained (in a final volume of 1.0 mL) 2.8 mM deoxyribose, 10 mM phosphate buffer (pH
7.4), 25 mM FeCl3, 100 mM EDTA, 2.8 mM H2O2, 100 mM ascorbic acid and 25 µL of the
tested sample. Each mixture was incubated for 1 h at 37 °C in a water bath. At the end of the
incubation period, 1 mL of 1% (w/v) TBA was added to each mixture, followed by the
addition of 1 mL of 2.8% (w/v) trichloroacetic acid. The solutions were heated in a water bath
at 100 °C for 15 min to develop the pink-coloured malondialdehyde-thiobarbituric acid
adduct. After cooling, the absorbance was measured at 532 nm against an appropriate blank
solution. All tests were run in triplicate and the results expressed as means ± standard
deviation (SD). Trolox was chosen as a standard antioxidant, and extracts activity was
expressed in µM Trolox equivalents (µM TE).
2.12. AChE inhibition assay.
AChE inhibition by the flavonoids present in juices obtained from the two varieties was
evaluated as described by Ellman, Courtney, Andres, & Featherstone (1961). The assay is
based on the spectrophotometric measurement of the increase in the yellow colour produced
11
by thiocholine upon reaction with dithiobisnitrobenzoate. The increase in the absorbance
value due to the spontaneous hydrolysis of the substrate was corrected by subtracting the
absorbance of blank reaction without added enzyme.
3. Results and discussion
3.1. Identification of flavonoids
As a part of our on-going studies on the Citrus genus we have turned our attention to the
separation and identification of the flavonoids present in the two most widespread cultivars of
blood orange, namely, Sanguinello and Tarocco. As in the case of the Moro cultivar (Barreca
et al., 2014), the chromatograms recorded at 278 and 325 nm (Fig. 1) on the juices obtained
from Sanguinello and Tarocco fruits –by following the procedure described above– showed
the presence of several well defined peaks belonging to flavonoid derivatives. The most
common flavonoid derivatives present in Citrus juices are flavanone and flavone compounds
(Gattuso, Barreca, Gargiulli, Leuzzi, & Caristi, 2007; Barreca et al., 2011a; Barreca,
Bellocco, Caristi, Leuzzi, & Gattuso, 2011c; Barreca et al., 2013). A direct comparison of
peak intensity in the chromatograms recorded at 280 and 325 nm provided a preliminary
screening of the flavonoid aglycone subclasses. In fact, both flavanones and flavones have
intense absorptions in the 270–280 nm region (the so-called band II), whereas in the 320–330
nm region (band I) only flavones and flavonols show significant absorption leading to the
assignment of chromatographic peaks either to the flavanone or flavone/flavonol subclass. In
addition, the chromatogram recorded for crude juice samples after acidic hydrolysis (not
shown), revealed that compounds 1–4 and 8 were resistant to HCl treatment, whereas the rest
were not, providing evidence that the former flavonoids possess C-linked saccharide moieties,
whereas the latter bear O-linked glycosyl substituents.
12
The UV spectra recorded for each of the peaks present in the chromatograms revealed the
presence of a flavone skeleton for compounds 1–4, 8 and 11, a flavonol skeleton for 6,7 and
9 and, finally, a flavanone core for compounds 5, 10 and 12. Moreover, reversed phase
chromatographic inspection of the two juices after acidic hydrolysis (data not shown) showed
the presence of C-glycosidic linkages in compounds 1–4 and 8 (Gattuso et al., 2006).
In addition, compounds 1–4 showed the unequivocal fragmentation pattern –characterized
by the losses of 18, 90, 120 amu ions along with the appearance of the highly diagnostic
aglycone A + 113 and A + 83 ions (Gattuso et al., 2006)– of the di-C-glycosyl flavones. Their
tR and UV-vis data, as well as their pseudo molecular ion in positive ion mode ([M+H]+, m/z
611, 595, 625 and 625) and in negative ion mode ([M–H]–, m/z 609, 593, 623 and 623) for
compounds 1, 2, 3 and 4, respectively, led us to identify compounds 1 as lucenin-2, 2 as
vicenin-2, 3 as stellarin-2 and 4 as lucenin-2 4′-methyl ether. Comparison to standard
compounds confirmed these assignments.
Compound 8 shows the typical UV spectrum of a flavone structure. According to its tR,
UV-vis and MS data (characterized by the presence in negative ionization mode of [M–H–
90]– and [M–H–120]– ion peaks) it was identified as scoparin (chrysoeriol-8-C-glucoside).
This was further supported by the absence of the fragment peak at m/z [M–H–18]– in MS-MS
spectrum, that suggested that the sugar moiety is attached to the 8-position. Remarkably,
given their ubiquitous presence in citrus juices, this is the first time that mono- and di-C-
glycosyl flavones have been identified in Sanguinello and Tarocco juices.
The UV spectrum of compound 11 is compatible with a chrysoeriol derivative. The MS-
MS spectrum in positive ionization mode showed the presence of a disaccharide composed of
rhamnose and glucose, due to the consecutive loss of 146 and 162 mass units. The higher
abundance of the [M+H–308]+ fragment than the [M+H–146]+ fragment and the presence of
the fragment [M–H–120]– in negative ionization mode revealed the presence of a (1→2)
13
interglycosidic linkage between the two monosaccharides. The aglycone showed a well-
defined peak fragment with m/z 299 and according to literature data (Gattuso et al., 2006), the
MS-MS negative mode fragmentation focused on the m/z 299 ion allowed us to confirm the
UV-vis assignment, and to ultimately identify the aglycone as chrysoeriol. Therefore,
compound 11 was identified as chrysoeriol 7-O-neohesperidoside, which had not been
previously reported as a component of the juice obtained from these two blood orange
varieties.
The UV spectrum of compound 5 showed an hypsochromic shifts on band I compatible
with the presence of a substituent in the 4' position on a flavanone skeleton. The MS–MS
spectrum, focused on the [M+H]+ pseudomolecular ion at m/z 743, is characterized by the
fragmentation pattern typical of O-triglycosyl flavonoids with m/z 581, 435 and 273
(aglycone). The glucose–rhamnose–glucose fragmentation indicates the probable presence of
a rutinose group. On the basis of the excellent agreement of these data with those present in
the literature (Ma, Cuyckens, Van den Heuvel, & Cleys, 2001), compound 5 was identified as
the flavanone narirutin 4'-O-glucoside, found for the first time in these two juices.
Compounds 6, 7 and 9 showed the presence of two well-defined bands at ~260 and 350 nm
along with two shoulders at ~270 and 300 nm, suggesting the probable presence of a flavonol
skeleton. Moreover, the hypsochromic shift of band I absorbance maximum suggests the
presence of a glycosyl moiety attached to the 3-, 5- or 4'-hydroxyl group. The MS spectra of
these compounds are characterized by a pseudo molecular ion in positive ion mode ([M+H]+)
at m/z 757, 463 and 609 and in negative ion mode ([M–H]–) of 755, 461 and 607,
respectively. MS-MS analysis revealed the presence of the same aglycone structure for all the
three derivatives (m/z 303 in positive and 301 in negative ionization mode). Compound 6,
according to its UV spectrum and MS-MS fragmentation in the positive ionization mode,
indicative of subsequent losses of a rhamnose (m/z 611, [M–H–146]+) and a rhamnose-
14
glucose disaccharide unit (m/z 303, [M–H–308]+), led us to identify the compound as
quercetin-3-O-(2-rhamnosyl)-rutinoside. The presence of the rutinoside structure was deduced
by the absence of the [M–H–120]– fragment in the negative ionization mode, that indicates
the presence of a neohesperidoside disaccharide. This fragmentation pattern is also in
agreement with the one reported by Farias, & Mendez (2014) for the same compound.
Compound 7, on the other hand, is characterized by the losses, in the MS-MS spectrum, of a
single hexosyl unit (m/z 303, [M–H–162]+ and (m/z 301, [M–H–162]–) and according to its
UV spectrum and fragmentation pattern was tentatively identified as quercetin-3-O-glycoside.
The negative ionization mode MS-MS spectrum of compound 9, focused on the pseudo
molecular ion at m/z 607, showed the appearance of peaks corresponding to the loss of
C4H6O3 (m/z 505, [M–H–102]–) and C6H8O4 (m/z 463, [M–H–144]–), both compatible with
the presence of a 3-hydroxy-3-methylglutaryl substituent on the saccharide residue.
Compound 9, according to these observations was therefore tentatively identified as quercetin
3-hydroxy-3-methylglutaryl-glycoside.
Compounds 10 and 12 were identified as flavonoid derivatives whose presence in red
blood juice had been already reported in previous studies (Rapisarda et al., 1999; Kelebek et
al., 2008). The comparison of their UV, MS, MS-MS data and retention times to those already
reported in literature led to the unambiguous identification of the rutinosyl-flavanones
narirutin (10) and hesperidin (12). In Fig. 2 the structures of the flavonoids 1–5, 8 and 10–12
are depicted.
3.2. Quantitative Evaluation
In both varieties, hesperidin (12) is by far the most abundant derivative (426.07 ± 10.10
mg/L in Sanguinello and 106.4 ± 5.74 mg/L in Tarocco), followed by vicenin-2 (2) and
narirutin (10), albeit they are present in a much lower amount (Table 2). As far as the
flavonoid classes are concerned, O-glycosyl flavanones are the most abundant derivatives,
15
followed by C-glycoside flavones and finally by O-glycosyl flavonols. Overall, the total
content of flavonoids identified was found to be 568.58 ± 21.79 and 165.75 ± 8.74 mg/L for
Sanguinello and Tarocco, respectively. These findings led us to classify the Sanguinello
variety among the richest citrus juices in terms of flavonoid content, along with Moro sweet
orange, lemon (C. limon), clementine (C. clementina) and bergamot (C. bergamia), all
containing, on average, more than 500 mg/L total flavonoids (Gattuso et al., 2007).
3.3. Antioxidant assays
Nutraceutical research has gained considerable momentum over the past few years,
supported by several epidemiological studies that correlate food composition and properties
of the relevant bioactive compounds to the prevention od several pathological conditions and
ultimately to general good health (Benavente-García & Castillo, 2008; Barreca, Laganà,
Bruno, Magazù, & Bellocco, 2013; Grosso et al., 2013; Patil, Jayaprakasha, Murthy, &
Vikram, 2009; Barreca et al., 2009; Bellocco et al., 2009). For instance, consumption of red
orange juice has been associated with an increase of nutraceuticals in plasma (such as vitamin
C, carotenoids and flavonoids) and reduced cardiovascular risk, as evidenced by studies
carried out on healthy subjects (Riso et al., 2005; Buscemi et al., 2012).
The evaluation of the nutraceutical efficacy of a given single compound or of a complex
matrix (such as a juice or its solvent extracts) is often based on several different methods that,
taken together, can supply a clear picture of the role and of the function exerted by the
samples under investigation. In the literature, it is possible to find many in vitro studies to
evaluate, in various oxidation and solvent conditions, the scavenging efficiency of the
analysed samples (Pérez-Jimenéz et al., 2008). In this work we performed different in vitro
assays such as DPPH•, FRAP assay, ABTS•+, and OH•- scavenging activity on crude juice and
on a selected fraction obtained by reversed phase HPLC separation, along with the evaluation
of the influence of the flavonoids herein identified on acetylcholinesterase activity.
16
The chromatographic separation and identification provides a picture in which the
Sanguinello variety is much richer in flavonoid derivatives than the Tarocco one, both from
the qualitative and quantitative points of view. To evaluate the impact of the flavonoid
components on the assays performed, we separated, by preparative HPLC, a broad fraction
containing all the flavonoids detected (henceforth referred to as flavonoid pool, FP, Fig. 3).
The first evidence of a direct involvement of the compounds identified was obtained by the
DPPH• assay. As depicted in Fig. 4, both juices showed significant antioxidant power against
DPPH• radicals. The Sanguinello juice had the highest activity, reaching up to ~65% of
inhibition (corresponding to 14.7±0.24 µM TE) followed by the Tarocco one with ~49% of
inhibition (11.4±0.63 µM TE). The FP fraction (3.49±0.60 µM TE) obtained from
Sanguinello accounts for ~15.7% of the activity of crude juice, whereas in Tarocco the
corresponding FP fraction (0.90±0.25 µM TE) accounts for only ~3.75% of the activity.
In line with DPPH• results, Sanguinello juice and its FP fraction were found to outperform
Tarocco and its flavonoid pool in quenching all the radicals tested. In fact, the former juice
quenched ABTS•+ with an activity corresponding to 32.55±1.18 and 19.08±0.75 µM TE (juice
and FP, respectively), and 16.40±0.85 and 4.05±0.38 µM TE (juice and FP, respectively)
against the hydroxyl radical (Fig. 4). Tarocco juice and its FP fraction, on the other hand,
reached only 24.19±2.27 and 7.87±0.35 µM TE (juice and FP, respectively) in the ABTS•+
assay and 13.00±1.27 and 1.55±0.47 µM TE (juice and FP, respectively) in the hydroxyl
radical scavenging assay.
This ability to scavenge different kind of reactive species is fundamental to their role in the
detoxification mechanisms of living organism, where primary and relative weak oxidants
(such as O2•-) can combine with other species, e.g. nitric oxide, to give very dangerous
reactive compounds (hydrogen peroxide, hydroxyl radical, singlet oxygen, peroxynitric
radical). Therefore, the effects of compounds present in the two extracts were further analysed
17
by FRAP to measure their capacity to reduce iron. Crude Sanguinello and Tarocco juice had a
ferric reducing antioxidant power corresponding to 11.45±0.66 and 7.54±0.51 µM TE,
respectively, using Trolox as the reference antioxidant. FP fractions accounts for ~27% and
19.6% of this activity as far as Sanguinello and Tarocco varieties are concerned.
The results obtained reveal the potential of these juices to be powerful scavengers of
neutral, negative and positively charged radicals, with a primary antioxidant activity that may
be ascribed to their reducing ability. Based on previous studies, it is most probable that the
flavonoid derivatives contributing the most are those bearing a catecholic substitution at the
B-ring (i.e., two vicinal OH groups or, to a lesser extent, vicinal OH and OMe substituents),
as well as those bearing a double bond at the 2,3 position of the C ring, (conjugated with the
4-oxo group, Rice-Evans, Miller, & Paganga, 1996; Barreca et al. 2009; Barreca et al., 2011a,
2011c, 2011d; Bellocco et al., 2009). The difference between the antioxidant activity of the
juices and the relative FP fractions can be most probably ascribed to other substances such as
anthocyans, vitamins and phenolic acids present in the crude juice (Barreca et al., 2013) that
are removed during sample preparation and chromatographic separation of the DMF-diluted
juice.
Flavonoids identified in Sanguinello and Tarocco juices exhibit a remarkable diversity of
both chemical structures and biological activities, making them a promising source of lead
compounds for new drugs. Acetylcholinesterase (AChE) is the target of cholinesterase
inhibitors employed for the treatment of senile dementia, myasthenia gravis, Parkinson’s
disease and ataxia. Considering that the inhibition of AChE has been among the most
common approaches to neurodegenerative disorders treatment, and that existing drugs are
effective only against mild to moderate types of disease, the search for new efficient and
selective antiacetylcholinesterase agents attracts considerable attention. Our results show the
potential of the flavonoids present in the FP fraction to inhibit AChE activity. 25 µL of FP
18
fraction obtained by Sanguinello variety is able to induce ~33.5% of AChE inhibition, while
the fraction obtained from Tarocco reached ~21.0% of inhibition (Fig. 4).
4. Conclusions
After our recent report on Moro blood orange, with the identification of the flavonoids
present in Sanguinello and Tarocco juices we completed our investigations on the flavonoid
composition of the juices from blood orange varieties. HPLC-DAD-ESI-MS-MS separation
allowed for the identification in these juices, for the first time, of lucenin-2, vicenin-2,
stellarin-2, lucenin-2 4′-methyl ether, narirutin 4′-O-glucoside, quercetin-3-O-(2-rhamnosyl)-
rutinoside, quercetin-3-O-hexoside, scoparin, quercetin 3-hydroxy-3-methylglutaryl-
glycoside, chrysoeriol and 7-O-neohesperidoside. In addition, our study supplied new
evidence for their health promoting properties, showing also a remarkable difference between
Sanguinello and Tarocco. The analysis of the activity of the FP fractions provided an
experimental evidence of the significant role played by the identified compounds on the total
antioxidant activity. In particular, their high reactivity towards one of the most dangerous in
vivo radical (i.e., OH•), and the potential to inhibit AChE activity was revealed.
Our studies have added solid evidence, to that already present in the literature, that blood
orange juice is an outstanding source of bioactive compounds. These juices are extremely rich
in secondary metabolites that exhibit a remarkable diversity of both chemical structures and
biological activities. They may be recognized as a valid dietary component –confirming the
ancient common knowledge that drinking orange juice helps to leads a healthy life– and as a
source of compounds with the potential to help and support endogenous antioxidant and
defence systems.
Abbreviation Used
19
ABTS – 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); AChE –
Acetylcholinesterase; DPPH – 2,2-diphenyl-1-picrylhydrazyl; Trolox – 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid.
References
Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2011a). Flavonoid profile
and radical-scavenging activity of Mediterranean sweet lemon (Citrus limetta Risso) juice.
Food Chemistry, 129(2), 417–422.
Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2011b). Elucidation of the
flavonoid and furocoumarin composition and radical-scavenging activity of green and ripe
chinotto (Citrus myrtifolia Raf.) fruit tissues, leaves and seeds. Food Chemistry, 129(4),
1504–1512.
Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2011c). Kumquat
(Fortunella japonica Swingle) juice: flavonoid distribution and antioxidant properties. Food
Research International, 44(7), 2190–2197.
Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2011d). Distribution of C-
and O-glycosyl flavonoids, (3-hydroxy-3-methylglutaryl)glycosyl flavanones and
furocoumarins in Citrus aurantium L. juice. Food Chemistry, 124(2), 576–582.
Barreca, D., Bisignano, C., Ginestra, G., Bisignano, G., Bellocco, E., Leuzzi, U., & Gattuso,
G. (2013). Polymethoxylated, C- and O-glycosyl flavonoids in Tangelo (C. reticulata × C.
paradisi) juice and their influence on antioxidant properties. Food Chemistry, 141(2), 1481–
1488.
20
Barreca, D., Laganà, G., Bruno, G., Magazù, S., & Bellocco, E. (2013). Diosmin binding to
human serum albumin and its preventive action against degradation due to oxidative injuries.
Biochimie, 95(11), 2042–2049.
Barreca, D., Bellocco, E., Leuzzi, U., & Gattuso, G. (2014). First evidence of C- and O-
glycosyl flavone in blood orange (Citrus sinensis (L.) Osbeck) juice and their influence on
antioxidant properties. Food Chemistry, 149, 244–252.
Barreca, D., Laganà, G., Tellone, E., Ficarra, S., Leuzzi, U., Galtieri, A., & Bellocco, E.
(2009). Influences of flavonoids on erythrocyte membrane and metabolic implication through
anionic exchange modulation. Journal of Membrane Biology, 230(3), 163–171.
Bellocco, E., Barreca, D., Laganà, G., Leuzzi, U., Tellone, E., Ficarra, S. Kotyk, A., &
Galtieri, A. (2009). Influence of L-rhamnosyl-D-glucosyl derivatives on properties and
biological interaction of flavonoids. Molecular and Cellular Biochemistry, 321(1-2), 165–
171.
Benavente-García, O., & Castillo, J. (2008). Update on uses and properties of Citrus
flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity.
Journal of Agricultural and Food Chemistry, 56(15), 6185–6205.
Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a
measure of “antioxidant power”: the FRAP assay. Analytical Biochemistry, 239(1), 70–76.
Berhow, M., Tisserat, B., Kanes, K., & Vandercook, C. (1998). Survey of phenolic
compounds produced in Citrus. USDA ARS Technical Bulletin, 1856, 1–154, available also
at http://www.ars.usda.gov/is/np/phenolics/title.htm.
Buscemi, S., Rosafio, G., Arcoleo G., Mattina, A., Canino, B., Montana, M., Verga, S., &
Rini, G. (2012). Effects of red orange juice intake on endothelial function and inflammatory
21
markers in adult subjects with increased cardiovascular risk. American Journal of Clinical
Nutrition, 95(5), 1089–1095.
Dugo, P., Mondello, L., Morabito, D., & Dugo, G. (2003). Characterization of anthocyanin
fraction of Sicilian blood orange juice by Micro-HPLC-ESI/MS. Journal of Agricultural and
Food Chemistry, 51, 1373–1376.
Ellman, G.L., Courtney, K.D., Andres, J.V., Featherstone, R.M., 1961. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7,
88–95.
Farias, L. D. S., & Mendez, A. S. L. (2014). LC/ESI-MS method applied to characterization
of flavonoids glycosides in B. forficata subsp. pruinosa. Quimica Nova, 37(3), 483–486.
Gattuso, G., Caristi, C., Gargiulli, C., Bellocco, E., Toscano, G., & Leuzzi, U. (2006).
Flavonoid glycosides in bergamot juice (Citrus bergamia Risso). Journal of Agricultural and
Food Chemistry, 54(11), 3929–3935.
Gattuso, G., Barreca, D., Gargiulli, C., Leuzzi, U., & Caristi, C. (2007). Flavonoid
composition of Citrus juices. Molecules, 12(8), 1641–1673.
Grosso, G., Galvano, F., Mistretta, A., Marventano, S., Nolfo, F., Calabrese, G., Buscemi, S.,
Drago, F., Veronesi, U., & Scuderi, A. (2013). Red Orange: Experimental Models and
Epidemiological Evidence of Its Benefits on Human Health. Oxidative Medicine and Cellular
Longevity, 157240
Halliwell, B., Gutteridge, J. M. C., & Aruoma, O. L. (1987). The deoxyribose method: a
simple test-tube assay for determination of rate constants for reactions of hydroxyl radicals.
Analytical Biochemistry, 165(1), 215–219.
22
Hertog, M. G. L., Hollman, P. C. H., & Venema, D. P. (1992). Optimization of a quantitative
HPLC determination of potentially anticarcinogenic flavonoids in vegetables and fruits.
Journal of Agricultural and Food Chemistry, 40(9), 1591–1598.
Hillebrand, S., Schwarz, M., & Winterhalter, P. (2004). Characterization of anthocyanins and
pyranoanthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juice. Journal of
Agricultural and Food Chemistry, 52, 7331–7338.
Kelebek, H., Canbas, A., & Selli, S. (2008). Determination of phenolic composition and
antioxidant capacity of blood orange juices obtained from cvs. Moro and Sanguinello (Citrus
sinensis (L.) Osbeck) grown in Turkey. Food Chemistry, 107(4), 1710–1716.
Ma, Y.-L., Cuyckens, F., Van den Heuvel, H., Claeys, M. (2001). Mass spectrometric
methods for the characterization and differentiation of isomeric O-diglycosyl flavonoids.
Phytochemical Analysis, 12(3), 159–165.
Maccarone, E., Rapisarda, P., Fanella, F., Arena, E., & Mondello, L. (1998). Cyanidin-3-(6''-
malonyl)-glucoside. An important anthocyanin of blood orange juice. Italian Journal of Food
Science, 10(4), 367–372.
Riso, P., Visioli, F., Gardana, C., Grande, S., Brusamolino, A., Galvano F., Galvano, G., &
Porrini, M. (2005). Effects of blood orange juice intake on antioxidant bioavailability and on
different markers related to oxidative stress. Journal of Agricultural and Food Chemistry,
53(4), 941–947.
Patil, B. S., Jayaprakasha, G. K., Murthy, K. N. C., & Vikram, A. (2009). Bioactive
compounds: Historical perspectives, opportunities, and challenges. Journal of Agricultural
and Food Chemistry, 57(18), 8142–8160.
Pellegrini, N., Re, R., Yang, M., Rice-Evans, C. A., & Lester, P. (1999). Screening of dietary
carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying the 2,2'-
23
azobis(3-ethylenebenzothiazoline-6-sulfonic acid) radical cation decolorization assay.
Methods in Enzymology, 299, 379–389.
Pérez-Jimenéz, J., Arranz, S., Tabernero, M., Díaz-Rubio, M. E., Serrano, J., Goñi, I., &
Saura-Calixto, F. (2008). Updated methodology to determine antioxidant capacity in plant
foods, oils and beverages: Extraction, measurement and expression of results. Food Research
International, 41(3), 274−285.
Rapisarda, P., & Intelisano, S. (1996). Sample preparation for vitamin C analysis of
pigmented orange juices. Italian Journal of Food Science, 8(3), 251-256.
Rapisarda, P., Carollo, G., Fallico, B., Tomaselli, F., & Maccarone, E. (1998).
Hydroxycinnamic acids as markers of Italian blood orange juices. Journal of Agricultural and
Food Chemistry, 46(2), 464-470.
Rapisarda, P., Tomaino, A., Cascio, R., Bonina, F., Pasquale, A., & Saija, A. (1999).
Antioxidant effectiveness as influenced by phenolic content of fresh orange juices. Journal of
Agricultural and Food Chemistry, 47(11), 4718–4723.
Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure–antioxidant activity
relationships of flavonoids and phenolic acids. Free Radical Biology & Medicine, 20(7),
933−966.
24
Table 1. Retention time, UV spectra and MS-MS data for compounds 1–12.
tR (min) UV-vis (nm) MS2 focused on [M + H]+ (m/z) a MS2 focused on [M – H]– (m/z) a 1 13.12 270, 349 611 [M + H]+ (2), 593 [M + H – 18]+ (100), 575 [M + H – 36]+ (9),
545 [M + H – 66]+ (8), 539 [M + H – 72]+ (3), 527 [M + H – 84]+ (2), 491 [M + H – 120]+ (6), 473 [M + H – 138]+ (14)
609 [M – H] – (2), 591 [M – H – 18] – (8), 519 [M – H – 90] – (28), 489 [M – H – 120] – (100), 399 [M – H – 210] – (A +
113) (2), 369 [M – H – 240] – (A + 83) (3) 2 14.62 271, 335 595 [M+H]+ (1), 577 [M+H–18]+ (100), 559 [M+H–36]+ (14),
529 [M+H–66]+ (9), 523 [M+H–72]+ (2), 511 [M+H–84]+ (4), 475 [M+H–120]+ (7), 457 [M+H–138]+ (19)
593 [M–H]– (6), 575 [M–H–18]– (11), 503 [M– H–90]– (30), 473 [M–H–120]– (100), 383 [M–H–210]– (A+113) (5),
353 [M–H–240]– (A+83) (11)
3 15.12 271, 347 625 [M + H]+ (2), 607 [M + H – 18]+ (100), 589 [M + H – 36]+ (8), 559 [M + H – 66]+ (2), 505 [M + H – 120]+ (4), 487 [M + H – 138]+ (6)
623 [M – H]– (25), 605 [M – H – 18]– (2), 533 [M – H – 90]– (15), 503 [M – H – 120]– (100), 413 [M – H – 210] ]–
(A+113) (4), 383[M – H – 240] ]– (A+83) (14) 4 15.57 257 (sh), 271, 348 625 [M+H]+ (2), 607 [M+H–18]+ (100), 589 [M+H–36]+ (11),
559 [M+H–66]+ (6), 541 [M+H–84]+ (1), 505 [M+H–120]+ (6), 487 [(M+H–138]+ (11)
623 [M–H]– (44), 605 [M–H–18]– (4), 533 [M–H–90]– (12), 503 [M–H–120]– (100), 413 [M–H–210]– (A+113) (3),
383 [M–H–240]– (A+83) (10) 5 15.71 283, 323 743 [M+H]+ (35), 581 [(M+H)–162]+ (42), 435 [M+H–308]+ (8),
273 [M+H–308–162]+ (A) (10) 741 [M–H]− (43), 579 [M–H–162]− (18), 433 [M–H–308]−
(5), 271 [M–H–308–162]− (A) (25) 6 16.38 255, 270, 348 757 [M+H]+ (100), 611 [M+H–146]+ (70),
465 [M+H–146-146]+ (70), 303 [M+H–146–146–162]+ (75) 755 [M–H]– (100), 301[M–H–454]– (63)
7 16.57 255, 270, 348 465 [M+H]+ (100), 303 [M+H–162]+ (35) 463 [M+H] – (60) 8 19.75 256, 268, 347 463 [M+H]+ (1), 445 [M+H–18]+ (100), 427 [M+H–36]+ (18),
409 [M+H–54]+ (2), 397 [M+H–66]+ (17), 373 [M+H–90]+ (1), 367 [M+H–96]+ (3), 343 [M+H–120]+ (9), 325 [M+H–138]+ (1), 313
[M+H–150]+ (1)
461 [M–H]– (4), 371 [M–H–90]– (A+71) (8),
341 [M–H–120]– (A+41) (100)
9 20.40 259, 268(sh), 301(sh), 343
609 [M+H]+ (100), 303 [M+H–306]+ (A) (60) 607 [M–H]– (56), 505 [M–H–102]– (12), 463 [M–H–144]– (23), 301 [M–H–306]– (100)
10 21.50 283, 323 581 [M+H]+ b
579 [M–H]– b
11 22.42 252, 268,347 609 [M+H]+ (12), 463 [M+H–146]+ (21), 301 [M + H – 308]+ (A) (100)
607 [M–H]– (75), 487 [M–H–120]– (2), 461 [M–H–146]– (1), 341 [M–H–266]– (1), 299 [M–H–308]– (A)(100), 284 [M–
H–308-15]– (A-15)(12) 12 22.97 283, 324 611 [M+H]+ (40), 465 [M+H–146]+ (24), 449 [M+H–162]+ (68), 303
[M + H – 308]+ (A) (100) 609 [M – H]– (42), 463 [M – H – 146]– (27), 301 [M–H–
308]– (A)(100) a Values in brackets represent relative abundance. b MS2 data could not be obtained.
25
Table 2. Flavones C-glycosides (1-4, 8), flavonols O-glycoside (6-7, 9), flavones O-glycosides (11)
and flavanones O-glycosides (5, 10 and 12) content (mg/L) in Citrus sinensis (L.) Osbeck juice.
Sanguinello Tarocco
Mean SD Mean SD
1 Lucenin-2 4.50 0.42 7.23 0.34
2 Vicenin-2 36.20 2.40 32.22 1.31
3 Stellarin-2 6.46 1.64 0.78 0.02
4 Lucenin-2 4′-methyl ether 0.70 0.03 0.26 0.01
5 Narirutin 4′-O-glucoside 10.93 0.85 0.52 0.02
6 Quercetin-3-O-(2-rhamnosyl)-rutinoside 2.36 0.15 0.97 0.21
7 Quercetin-3-O-glycoside 4.13 0.21 0.66 0.15
8 Scoparin 7.43 0.90 n.d. n.d.
9 Quercetin 3-hydroxy-3-methylglutaryl-glycoside 7.97 0.64 2.08 0.11
10 Narirutin 61.33 1.68 14.40 0.81
11 Chrysoeriol 7-O-neoesperidoside 0.50 0.07 0.23 0.01
12 Hesperidin 426.07 10.64 106.40 5.74
26
Captions to figures
Fig. 1. Typical DAD chromatograms of Citrus sinensis (L.) Osbeck juice detected at 278 (A) and
325 (B). Components 1–12 were identified as follows: lucenin-2 (1), vicenin-2 (2), stellarin-2 (3),
lucenin-2 4′-methyl ether (4), narirutin 4′-O-glucoside (5), quercetin-3-O-(2-rhamnosyl)-rutinoside
(6), quercetin-3-O-glycoside (7), scoparin (8), quercetin 3-hydroxy-3-methylglutaryl-glycoside (9),
narirutin (10), chrysoeriol 7-O-neoesperidoside (11) and hesperidin (12).
Fig. 2. Structures for the flavonoids 1–6, 8 and 10–12.
Fig. 3. FP fractions collected from the juices by preparative HPLC (λ = 278 nm).
Fig. 4. DPPH• (A), ABTS•+ (B), hydroxyl radical (C) scavenging activity and AChE (D) inhibitory
activity of crude juices and FP fraction expressed in µM Trolox equivalents. (a) Sanguinello crude
juice; (b) Tarocco crude juice; (c) Sanguinello FP fraction; (d) Tarocco FP fraction. Each value
represents means ± SD (n = 3).
27
FIGURE 1
RT: 10.00 - 30.00
10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
50000000
55000000
60000000
65000000
70000000
uA
U
NL:7.00E7
Channel A UV aranciarossa_020219142526
RT: 10.00 - 30.00
10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
50000000
55000000
60000000
65000000
70000000
uA
U
NL:7.00E7
Channel A UV SuccoTarocco17-3-04
RT: 10.00 - 30.00
10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
11000000
12000000
13000000
14000000
15000000
16000000
uA
U
NL:1.60E7
Channel C UV aranciarossa_020219142526
RT: 10.00 - 30.00
10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
11000000
12000000
13000000
14000000
15000000
16000000
uA
U
NL:1.60E7
Channel C UV SuccoTarocco17-3-04
Sanguinello Tarocco278 nm 278 nm
325 nm 325 nm
1
2
3
5
89
10
11
12
6 71
2
3
5
9
10
11
12
6 7
44
28
FIGURE 2
29
FIGURE 3
RT: 10.00 - 30.00
10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
50000000
55000000
60000000
65000000
70000000
uA
U
NL:7.00E7
Channel A UV aranciarossa_020219142526
Broad fraction containing all the detected flavonoids
(FP)
30
FIGURE 4
c d a b0
102030405060708090
100
Inh
ibit
ion
(%)
A B
C D
a b c d0123456789
101112131415
TE
(µµ µµ
M)
a b c d0.02.55.07.5
10.012.515.017.520.022.525.027.530.032.535.0
TE
( µµ µµM
)
a b c d0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
TE
(µµ µµ
M)
Highlights
Twelve flavonoids were identified/quantified in Sanguinello and Tarocco blood orange Flavonoids strongly influence the antioxidant activity of Sanguinello and Tarocco Sanguinello and Tarocco juices display remarkable acetylcholinesterase inhibition