Synthesis, structural elucidation and antiradical activity of a copper (II) naringenin complex

Article abstract of DOI:10.1007/s10534-019-00187-3

Coupling the extraction and derivatization of flavonoids to the Citrus processing industry is attractive from both the environmental and economic points of view. In the present work, the flavonoid naringin, obtained by “green” extraction with a water:ethanol mixture from waste grapefruit industry, was hydrolyzed to obtain naringenin. This flavonoid was used to synthesize the complex trans-di(aqua) bis(7-hydroxy-2-(4-hydroxyphenyl)-4-oxo-5-chromanolato) copper (II). This compound was characterized by spectroscopic techniques (UV/Vis, IR, Raman, NMR and EPR), and by thermal analysis (TG and DSC). Then, a monocrystal of the complex obtained by dissolution and recrystallization in DMF was analyzed by single crystal X-ray diffraction. This is the first report of the crystal structure of a Citrus flavonoid complex. Additionally, its antiradical activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined and compared with that for naringenin, demonstrating that coordination to copper enhances the antiradicalar activity of naringenin. According to the Mulliken population analysis conducted, by copper favors the delocalization and stabilization of the produced radical, since it acts as an electronic density acceptor.

Full text of DOI:10.1007/s10534-019-00187-3

Biometals
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Synthesis, structural elucidation and antiradical activity
of a copper (II) naringenin complex
.
.
Sebastian A. Suarez Analıa Arias Jose Molina
.
.
´
´
´
Gustavo Celiz
.
Carlos D. Brondino Fabio Doctorovich
Received: 21 August 2018 / Accepted: 9 March 2019
Ó Springer Nature B.V. 2019
Abstract Coupling the extraction and derivatization
of flavonoids to the Citrus processing industry is
attractive from both the environmental and economic
points of view. In the present work, the flavonoid
naringin, obtained by ‘‘green’’ extraction with a
water:ethanol mixture from waste grapefruit industry,
was hydrolyzed to obtain naringenin. This flavonoid
was used to synthesize the complex trans-di(aqua)
bis(7-hydroxy-2-(4-hydroxyphenyl)-4-oxo-5-chro-
manolato) copper (II). This compound was character-
ized by spectroscopic techniques (UV/Vis, IR, Raman,
NMR and EPR), and by thermal analysis (TG and
DSC). Then, a monocrystal of the complex obtained
by dissolution and recrystallization in DMF was
analyzed by single crystal X-ray diffraction. This is
the first report of the crystal structure of a Citrus
flavonoid complex. Additionally, its antiradical activ-
ity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was
determined and compared with that for naringenin,
demonstrating that coordination to copper enhances
the antiradicalar activity of naringenin. According to
the Mulliken population analysis conducted, by cop-
per favors the delocalization and stabilization of the
produced radical, since it acts as an electronic density
acceptor.
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10534-019-00187-3) con-
tains supplementary material, which is available to authorized
users.
G. Celiz (&)
Instituto de Investigaciones para la Industria Quımica
(INIQUI) and Universidad Nacional de Salta, Avenida
Bolivia 5150, A4408FVY Salta, Argentina
e-mail: gceliz@unsa.edu.ar
C. D. Brondino
Departamento de Fısica, Facultad de Bioquımica y
´
CONICET, S3000ZAA Santa Fe, Argentina
´
´
Ciencias Biologicas, Universidad Nacional del Litoral/
´
G. Celiz Á A. Arias Á J. Molina
Facultad de Ciencias Exactas, Universidad Nacional de
Salta, Avenida Bolivia 5150, A4408FVY Salta, Argentina
S. A. Suarez Á F. Doctorovich
´
INQUIMAE, CONICET, Departamento de Quımica
Inorganica, Analıtica y Quımica Fısica, Facultad de
´
´
´
´
Ciencias Exactas y Naturales, Universidad de Buenos
´
Aires, Pabellon II – Ciudad Universitaria - Nun˜ez,
C1428EHA CABA, Argentina
´
123

Biometals
Keywords Naringenin Á Citrus flavonoids Á
Processing waste Á Metal flavonoid complex Á
Crystallography
2014) and cardiovascular illnesses (Ilkay et al. 2015).
These in vivo biological activities are due to its ability
to interact with cell membranes (Selvaraj et al. 2014b),
to quench radicals (Cavia-Saiz et al. 2010), to chelate
transition metals that take part on radical production
(de Souza and De Giovani 2004) and interact specif-
ically in various biological processes.
Introduction
Among the great variety of flavonoid properties, the
most studied has been the antioxidant activity. This
activity is based on the ability of a substance to reduce
the formation of free radicals, and to decompose those
that are formed. The reason why antioxidant activity
deserves interest lies in the fact that free radicals
induce oxidative damage in biomolecules and orga-
nelles, which according to several studies may lead to
diseases such as Parkinson’s disease, heart disease and
even cancer (Klein and Ackerman 2003; Smith et al.
2000; Valko et al. 2006; Waris and Ahsan 2006).
Naringenin is not structurally favored to act as
antiradical, because it does not possess the structural
requirements that favor such activity: (i) ortho-dihy-
droxy substitution in ring B, which allows electronic
delocalization in the radical products; (ii) conjugated
double bond C2=C3 with group C4=O in ring C, which
results in greater electronic delocalization in ring B;
(iii) hydroxyl groups in positions 3 and 5, which can
form hydrogen bonding with the group C4=O (Amic
et al. 2007).
Flavonoids are natural polyphenols that have a struc-
ture of benzo-c-pyrone (C6–C3–C6). More than 8000
compounds with this skeleton have been identified,
which are differentiated by various combinations of
substituents, such as hydroxyls, methoxyls and O-
glycosides. They are exclusive compounds of the plant
kingdom, but species of the animal kingdom can
obtain this polyphenols by eating, for example,
cereals, fruits, and vegetables.
Among the different subclasses of flavonoids,
flavanones occur mainly in Citrus. These compounds
support and enhance the body’s defenses against
oxidative stress and help the organism in the preven-
tion of cardiovascular diseases, atherosclerosis, and
cancer (Barreca et al. 2017). Naringenin (Fig. 1) is a
flavanone that occurs in low concentrations in Citrus
fruits (Erlund 2004), but it can be obtained in large
quantity by hydrolysis of naringin (Pulley 1936), a
flavonoid that can be extracted easily from the solid
waste generated in the industrial grapefruit production
(Poore 1934).
Chelation with metals alters the naringenin elec-
tronic distribution and this will cause changes in its
spectroscopic, physicochemical, and certainly also
biological properties. For that, it is interesting to
Naringenin has biological action on humans. It
could be useful in the treatments of osteoporosis
(Swarnkar et al. 2012), some cancer type (Patel et al.
Fig. 1 Scheme of the synthesis of the naringenin Cu(II) complex starting from naringin
123

Biometals
synthesize metal-naringenin complexes and study
their properties to determine how the metal affects to
the flavonoid. In order to study if the naringenin
antiradical activity can be increased by complexation
to a metal center, the synthesis of a complex of this
flavonoid was proposed. Among the candidate metals,
copper is a good choice, since it is a redox active
metal, biologically relevant, and biocompatible. In
addition, nowadays there are discrepancies among the
different reports with regard to the structural and
spectroscopic characterizations of the product
obtained when naringenin and copper (II) react. This
is probably due to the fact that complexes with
different molar ratios and deprotonation states can be
obtained in the synthesis (Brodowska 2013). More-
over, up to the moment, there is no report of the
crystalline structure for any metal–Citrus flavonoid
complex. For those reasons, the experimental data
presented is focused on the characterization of the
complex obtained, and on the radical scavenging
properties of both naringenin and its Cu(II) complex.
This work presents the synthesis, purification and
characterization of a complex formed between copper
(II) and naringenin (Fig. 1). Spectroscopic properties
(UV–VIS, IR, NMR, RAMAN), thermal analysis and
a single-crystal X-ray diffraction study is presented to
elucidate the three-dimensional structure of this com-
plex. Additionally, an experimental and theoretical
study about the radical scavenging activity of the
complex and naringenin is presented.
acid at 0.8 M concentration, stirring during 2 h at
90 °C. After that, the reaction media was cooled down
to 4 °C, allowing the naringenin precipitation. It was
then washed with cool water and dried at 50 °C. This
process was done for a second time to ensure a
complete conversion.
1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) was
purchased from Sigma (USA). All other reagents were
of analytical grade.
Synthesis and purification of the copper (II)
naringenin complex
A 25 mM solution of naringenin (272.3 g mol-1) in
ethanol:water solution (1:1) and a solution of copper
(II) acetate at the same concentration in water were
prepared. Both solutions were adjusted to pH 7 prior to
the reaction. In order to have a 1:2 (metal:flavonoid)
reactant stoichiometry, 100 mL of the naringenin
solution were stirred and heated at 60 °C, and then
50 mL of copper salt solution was added dropwise.
After the addition of the copper solution, the pH of the
reaction mixture falled down to 6, so it was adjusted to
pH 7–8 again. The reaction media was kept 1 h at
60–65 °C. After that, it was cooled overnight and
filtered through standard filter paper. The solid
obtained was washed first with ethanol:water 1:1
solution and then with acetone, to eliminate reactant
traces (the complex was insoluble in these solvents).
After that, it was filtered again, dried and stored at
room temperature. The synthetized complex shows an
apple-green color, instead of the pale-white color of
naringenin. The obtained mass of purified complex
was 0.7590 g, (yield = 95%).
Materials and methods
Materials
Microanalysis experimental and calculated (in
brackets) for [Cu(NGE)2(H2O)2): C: 56.3% (56.1%)
H: 3.8 (4.0) %. Reinforcing this result, the same molar
ratio metal:ligand, i.e. 1:2, was obtained by the Job’s
method (experimental details and results are provided
in Supplementary Material). ESI Mass Spectrum,
molecular ion: 411.98,568 m/z, expected for
[CuNGE(H2O)3Na]?: 411.898563. In addition, the
product was characterized according to the following
sections.
Naringin was produced from agro-industrial grapefruit
waste using a simple and inexpensive process. It
consists, briefly, on grinding the solid waste to an
average size of 2–4 mm and, after that, performing an
extraction in a fixed bed column with distilled water at
80 °C. Then, the extract obtained is cooled down,
leading to crystallization of the flavanone. The
precipitate was filtered, washed and, finally, dried at
50 °C.
Naringenin (NGE) was obtained from naringin by
acid hydrolysis, using a reported method (Robin et al.
2007). Briefly, the process consists on hydrolyzing a
10% (w/v) aqueous solution of naringin with sulfuric
Spectroscopic characterization
UV–Visible spectra (190–1100 nm) of naringenin and
of the copper (II) naringenin complex were obtained in
123

Biometals
Thermal analysis study
dimethylformamide (at two concentrations) and water.
Spectra in water were performed by adding 20 uL of
flavonoid dissolved in DMF to 3980 uL of water.
Spectra in solid state were carried out using a quartz
cuvette for solids. The equipment used was a UV/Vis
double beam spectrophotometer Lambda 365 with a
50 mm reflectance sphere (Perkin Elmer, USA).
IR spectra were obtained in the 400–4000 cm-1
range using KBr pellet. The Raman spectra were
obtained in the range of 200–3600 cm-1. The spec-
trometer used was a Spectrum GX-FTIR (Perkin
Elmer, USA) with 1 cm-1 resolution and 15 scans per
second.
Differential scanning calorimetry was performed with
a DSC-Q200 apparatus (TA Instruments, USA)
weighing 5–6 mg of sample heated in aluminum pans
with lids at a rate of 10 K/min under a nitrogen gas
flow of 20 mL/min. Thermal gravimetric analysis
(TGA-DTA) was carried out with a Shimadzu DSC-60
instrument (Shimadzu, Japan). Powdered samples
weighing 10–11 mg were heated in open aluminum
pans at a rate of 10 K/min under a nitrogen gas flow of
40 mL/min.
Single-crystal X-ray diffraction study
NMR spectroscopic data were recorded using a
Bruker Avance 250 spectrometer (Bruker, USA)
1
The complex obtained in aqueous ethanol solution was
dissolved in DMF and recrystallized to obtain a
monocrystal. It was analyzed by single crystal X-ray
diffractometry.
operating at 250 MHz for H NMR and 63 MHz for
13C NMR and processed with standard Bruker
software. Shifts are given in ppm and coupling
constants in Hertz.
1
A 170 K temperature data set was collected on a
four circle Oxford Diffraction Gemini CCD S Ultra
diffractometer using a graphite monochromated Mo
Naringenin: H NMR (250 MHz, DMSO) d 12.15
(s, 1H, C5–OH), 10.79 (s, 1H, C7–OH), 9.59 (s, 1H,
C40–OH), 7.31 (d, J = 8.6 Hz, 2H, C20), 6.79 (d,
J = 8.6 Hz, 2H, C30), 5.88 (s, 2H, C6 and C8), 5.44
(dd, J = 12.8, 2.8 Hz, 1H, C2), 3.27 (dd, J = 17.2,
12.8 Hz, 1H, C3a), 2.67 (dd, J = 17.2, 2.8 Hz, 1H,
C3b). 13C NMR (63 MHz, DMSO) d = 196.45 (C4),
166.66, 163.50, 162.96 (C5, C7 and C8a), 157.75
(C40), 128.86 (C10), 128.38 (CH 20and 10), 115.17 (CH
30and 40), 101.78 (C4a), 95.80, 94.98 (CG 6 and 8),
78.45 (CH2), 41.98 (CH2–3).
˚
Ka source (k = 0.71073 A). Measurement yielded
3796 (I) independent reflections of which 2363 were
considered observed [I [ 2r(I)], with an internal
consistency Rint = 0.0519. Data collection strategy
and data reduction followed standard procedures
implemented in ChrysAlis Pro software (CrysAlis
PRO 2015). Structure determination was achieved
routinely by direct methods and difference Fourier.
The structure was refined by least squares on F2, with
anisotropic displacement parameters for non-H atoms.
Hydrogen atoms unambiguously defined by the stere-
ochemistry (C–H’s and O–H’s) were placed at their
calculated positions and allowed to ride on their host
carbon or oxygen atoms with respect to both their
coordinates and their thermal parameters. All calcu-
lations to solve and refine the structures and to obtain
some derived results were carried out with the
computer programs SHELXS97 (Sheldrick 2008),
SHELXL2014 (Sheldrick 2015) and Platon (Spek
2009). MERCURY (Macrae et al. 2006) was used to
draw molecular views and derive further results. Full
use of the CCDC package was also made for searching
at the Cambridge Structural Database, Version 5.37
(Groom et al. 2016).
Electron paramagnetic resonance (EPR) spectra
were performed on a Bruker EMX Plus spectrometer
equipped with a universal high sensitivity cavity
(HSW10819 model) using a Bruker nitrogen contin-
uous-flow cryostat. Spectra were acquired under non-
saturating conditions. Experimental: microwave fre-
quency, 9.45 GHz; modulation field, 100 kHz; mod-
ulation amplitude, 2 G; temperatures are indicated in
the caption to the figures. EPR spectra were simulated
with the EasySpin toolbox based on MATLABÒ (Stoll
and Schweiger 2006). Simulation of the solution
spectrum was performed assuming a Hamiltonian
including Zeeman and Hyperfine interactions, H = lB
S.g.B ? I.A.S, where all the symbols have the usual
meaning, and g- and A-matrices were assumed to be
collinear. EPR spectral intensities were evaluated by
double integration.
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Biometals
Scavenging activities by the DPPH radical method
Results and discussion
The scavenging activities were carried out by the
method proposed by Brand-Williams et al. (Brand-
Williams et al. 1995). An aliquot of 100 uL of DMF
solution containing different naringenin or its copper
complex at concentrations up to 20 mNNGE (mili
moles of naringenin per liter of solution) was added to
1900 lL of DPPH 65 lM in methanol. The DPPH
absorbance of each tube was read at 514 nm after 8 h
in the dark at 20 °C, when the reaction reaches a semi-
steady state. A control solution of DPPH (100 lL de
methanol and 1900 lL of DPPH solution) was
prepared to estimate the DPPH self-decomposition
during the incubation. For each antioxidant concen-
tration, the percentage of DPPH remaining at the semi-
steady state was calculated as: % DPPH remain-
ing = ([DPPH]S.S/[DPPH]C.S) * 100, where [DPP
H]S.S is the absorbance of the samples after 8 h and
[DPPH]C.S is the absorbance of the control DPPH
solution. These values were plotted versus the antiox-
idant concentrations.
Spectroscopic characterization
The UV–Visible spectrum of the ligand in DMF
(Fig. 2a) presents two bands. The first one is almost
symmetrical, sharp and intense with a maximum at
288 nm (Band II). This band is mainly due to the
electronic p ? p* transition that takes place over the
A and C rings. The second band (Band I) is also due to
a p ? p* transition but on the B ring. This band,
located between 315 and 360 nm, is less intense that
the first one. Both proposed assignments are in
agreement with Shireen et al. who working with
TDDFT assigned to pinocembrin (a flavanone like
naringin but with C40–H, instead C40–OH) the
HOMO-1 ? LUMO transition at 279 nm located in
the p system formed by the A and C rings; and the
HOMO ? LUMO ? 1 transition centered at
324 nm, formed by the p system of the B ring (Ajmala
Shireen et al. 2017). For the complex in DMF, Band II
appears without displacement with respect to narin-
genin, while Band I presents a slight increase in
intensity with respect to the ligand with loss of
symmetry towards the visible zone with absorbance
beyond 360 nm. The spectral modification could be
assigned to charge transfer either ligand–metal or
metal–ligand, transitions mainly produced on the C4–
O–Cu and C5–O–Cu bonds. For the complex in DMF,
but at 7 mM (3900 ppm), a d–d electron transition
band with low intensity is observed centered at
650 nm, while for naringenin in similar conditions
no absorbance over 400 nm is present (Fig. 2c).
In water at neutral pH, the spectrum of naringenin is
the same as in DMF. For the complex, the absorptivity
of Band II appears diminished and its kmax is
displaced to 290 nm, and the intensity of band I
appear increased.
DFT computational studies
Crystalline structures were used as initial configura-
tion to make a structure optimization on naringenin
and its Cu(II) complex. The structures were confirmed
to correspond to a stable ground state by the absence of
imaginary frequency in the force constant calculations
using tools from density functional theory as imple-
mented in the Gaussian 09 package (Bearpark 2009).
The method involving the Becke 3-parameter
exchange functional together with the Lee–Yang–Parr
correlation functional (B3LYP) (Becke 1993; Lee
et al. 1988; Miehlich et al. 1989) was used for all the
calculations. A Mulliken population analysis (charge
and electronic spin density) was conducted with the
aim to correlate these data with the compounds’
antiradicalarial activities of naringenin, the complex
and their relevant radicals using single point. Mulliken
population analysis was made in vacuum and consid-
ering solvent effect (water and methanol) with polar-
izable continuum models of solvation (Scalmania and
Frisch 2010).
In solid state (Fig. 2d), band II is located at
260–300 nm for both compounds, and band I appears
strongly displaced to the visible (from 320–380 nm to
380–490 nm). The complex presents also a band in the
visible region centered at 650 nm due to the d–
d transitions of the Cu(II) (Lever 1984).
The complex was characterized by FT-IR and
Raman spectroscopy. Figures 3 (IR) and 4 (Raman)
present the vibrational spectra of naringenin and of the
copper (II) naringenin complex.
123

Biometals
Fig. 2 UV/Vis spectra of the copper (II) naringenin complex
(full line), and naringenin (dashed line). a in DMF: naringenin
0.05 mM and copper (II) naringenin complex 0.025 mM; b in
water at neutral pH: naringenin 0.05 mM and copper (II)
naringenin complex 0.025 mM; c in DMF: naringenin 15 mM
and copper (II) naringenin complex 8 mM; d in solid state by
diffuse reflectance spectroscopy
Fig. 3 IR spectra for naringenin (black—bottom line) and for the copper (II) naringenin complex (gray—upper line)
The band assignments were made according to
Unsalan et al. (Unsalan et al. 2009), who studied
experimentally and theoretically the molecular struc-
tures of naringenin. The most intense absorption for
the phenolic groups stretching appears at 3290 cm-1
for naringenin and at 3201 cm-1 for the complex. The
complex shows an additional band at 3412 cm-1
,
which would correspond to the O–H stretching
123

Biometals
Fig. 4 Raman spectra for
naringenin (black—bottom
line) and for the copper (II)
naringenin complex (gray—
upper line)
assigned to coordinated water (Uivarosi et al. 2016).
The IR absorption between 3200 and 2800 cm-1
correspond, for both molecules, to C–H stretches. By
Raman resonance, these bands were more defined.
The most intense absorption, due to the C=O
stretching, occurred in the region of 1635 cm-1 (IR)
for naringenin, whereas for the complex it appeared at
1615 cm-1 (IR). The displacement to lower frequen-
cies indicates weakening of the double bond by
coordination to the copper ion. Tan et al. synthesized
and characterized naringenin, hesperetin and apigenin
copper (II) complexes. They presented similar IR
assignments and reported the same displacement of
the carbonyl signal at lower frequencies when the
flavonoids were coordinated to copper (Tan et al.
2009). Centered at 1602 (IR) (1618–1555 cm-1 in
Raman) for naringenin, and at 1557 cm-1 (IR)
(1591–1555 cm-1 in Raman) for the complex, stretch-
ing bands corresponding to C=C are observed.
A particular band centered at 1316 cm-1 (IR and
Raman) for naringenin, is not present in the complex.
We suggest that it can be assigned to a C5–OH
deformation and it could be a main vibrational
evidence of the complex formation at the C4–O and
C5–O position. This assumption is in accordance with
Unsalan et al. who studied by DFT the FT-IR of
naringenin and assigned this absorption at 1314 cm-1
to dC–OH ? mCO (Unsalan et al. 2009).
assignment is supported by Nakamoto, who studying
the FT-IR of complexes of b-diketones and Cu(II),
assigned the IR region close to 610 cm-1 to dM-
? mM–O (Nakamoto 2009).
The H NMR spectra obtained for naringenin and
ring
1
for the complex are shown in Fig. 5. The naringenin
13C NMR spectra are in agreement with other
previously reported (Agrawal and Schneider 1983).
The naringenin 1H NMR spectra was identical to other
previously reported (Pouchert and Behnke 1993). For
the complex, it was obtained a drastically different
spectral profile with wide signals due to the presence
of the paramagnetic copper atom. The signals appear
enlarged; this fact prevents integration and determi-
nation of the multiplicities of the hydrogen atoms.
Despite of this, we could see the absence of the proton
located on the 5-OH hydroxyl group (d 12.15 in
naringenin), indicating that it is in the form of
alkoxide. In addition, we suggest that the band
centered at d 9.6 corresponds to the hydroxyl group
40-OH (which does not undergo displacement with
respect to naringenin), and to the signal of the 7-OH
that was displaced due to the presence of the alkoxide
formed in the same ring. These results are compatible
with chelation and charge neutralization by the copper
(II) metal center in the 5-alkoxi 4-keto position. In
addition, no ethanol signals (typical at 1.1 and around
4), or acetate ion (near to 2 ppm) are observed, so they
are not part of the complex.
Further evidence for complex formation was a low-
intensity band at 596 cm-1 (IR), present in the
complex but not in the naringenin spectrum. This
band was attributed to a M–O stretching. This
Wang et al. (2006) synthetized a complex with
Zn(II) and naringenin, They report
a similar
123

Biometals
Fig. 5 H1 NMR spectrum for naringenin (defined signal) and its Cu(II) complex (broad signal). The 2D structure of naringenin is
shown as inset
assignment that us with broadening of the signals and
loss of the C5–OH signal in the complex (Wang et al.
2006).
X-band EPR spectra of a frozen solution in DMSO
and a polycrystalline sample of the copper (II)
naringenin complex are shown in Fig. 6.
The EPR spectrum of the frozen DMSO solution of
the Cu2? complex (S = 1/2) (spectrum a) shows nearly
axial symmetry with well solved hyperfine structure
with the Cu nucleus (I = 3/2) at g//. Simulation of this
spectrum (dashed line) yielded g1,2,3 = 2.290, 2.060,
2.053 and A1,2,3 = 15.8, 2.4, n.d. mT (n.d., non-
detectable). These values, which are within the typical
ones reported for Cu (II) complexes in nearly square
planar coordination with O-ligands, indicate a dx2–y2
Fig. 6 X-band EPR spectra of the copper (II) naringenin
complex: a- in DMSO frozen solution (140 K) and b- solid state
at room temperature; dashed line: theoretical simulation
magnetic ground state for the Cu(II) ions. In line with
the solution complex, the EPR spectrum of the
polycrystalline sample at room temperature (spectrum
No significant differences were observed in the
EPR spectra of the solid taken in the range of 120 K–
room temperature (Fig. 7), apart from the typical
b in Fig. 6) shows also axial symmetry with
detectable hyperfine structure at g//but broadened
due to inter-copper dipole–dipole interactions.
123

Biometals
dependence of the EPR signal intensity with temper-
ature, the larger the temperature, the lower the
intensity. The fact that the hyperfine structure is
partially solved in these spectra indicates that the
Cu(II) ions are very weakly coupled by exchange. This
is in line with the crystal lattice of Cu(II) ions observed
in Fig. 10, which is an 1-D arrangement linked by non-
covalent interactions, that form a weakly exchange
coupled systems (Neuman et al. 2012; Rizzi et al.
2016). This conclusion is also reinforced by the
temperature variation of the intensity (I) of the solid
state EPR spectra (Fig. 7).
The EPR spectrum of the solid shows also a broad
weak resonance line at * 160 mT (see inset Supple-
mentary material, Figure EPR). This transition, which
must not be observed in an isolated mononuclear
copper (II) compound, has been detected in a few
cases in 1D—systems containing Cu(II) ions coupled
by dipole–dipole interactions, as could be the case of
Cu2? complex with naringenin according to Fig. 8
(Bencini and Gatteschi 1990). The analysis of this
phenomenon is beyond of the aims of this paper.
Thermal study
Note that the intensity of S = 1/2 EPR spectra under
non-saturating conditions are proportional the mag-
netic susceptibility v. These data, analyzed assuming a
Curie–Weiss behavior (I = constant/(T-H), where the
constant is proportional to the Curie constant C, and H
is the Weiss temperature, a parameter proportional to
the magnitude to the exchange interaction between
copper ions, yielded a null H-value within the
experimental error. The fact that the positions of the
of the g//- and A//-features in both solid and solution
complexes are rather similar (Fig. 6) constitutes a
strong evidence that the structure of the complex in
solid state determined by X-ray crystallography (pre-
sented below) is kept upon dissolution in DMSO
solvent.
The thermal behavior of the complex was determined
by TGA and DSC. First, the DSC thermogram for
naringenin (Fig. 8) presents 3 peaks: the first one at
202 °C is related to the glass transition; the second
endothermic peak at 250 °C without loss of mass is
related to the melting point of naringenin, which is
consistent with the value reported in the literature
(CRC Handbook of Chemistry and Physics 93th
edition); the third endothermic event (centered at
283 °C) was attributed to thermal decomposition of
naringenin, which was previously reported for similar
flavanones (Ferreira et al. 2017). For the complex, an
endothermic peak at 161 °C can be ascribed to the loss
of 2 coordinated water molecules, which agree with a
weight loss of 5.1% by TGA. The thermal event at
202 °C is related to the melting point of the complex,
showing a decrease of the reticular energy comparing
with naringenin. The last endothermic event with
Fig. 7 Plot of 1/I versus T, where I is the intensity of the EPR
spectra evaluated by double integration. A least square linear fit
with a Curie–Weiss model to the data yielded slope = 0.00158
(3), intercept = - 0.08(8), square R = 0.995, where the slope
and the intercept are proportional to C-1 (C = Curie constant)
and H, respectively (see main text for more details)
Fig. 8 DSC of naringenin (black line) and copper (II)
naringenin complex (grey line). under nitrogen atmosphere,
heating rate 10 °C min-1. Sample mass 5.83 mg of complex and
5.05 mg of naringenin
123

Biometals
matter loss centered at 291 °C is related to thermal
decomposition of the ligand.
Cu(II) hexacoordinated center (Fig. 9). The coordina-
tion sphere is formed by two oxygen atoms from two
bidentate naringenin ligands and one oxygen atom
from two dimethylformamide molecules, with metal-
Single-crystal X-ray diffraction study
˚
O distances in the range 1.900(2)–2.560(5) A
The crystals obtained by recrystallization in DMF
were analyzed by single crystal X-ray. These crystals
(Table 2). According to this result the nomenclature
of
the
complex
is
trans-di(N,N-dimethyl-
˚
are triclinic, space group P-1, with a = 7.3581(7) A,
methanamide)bis(7-hydroxy-2-(4-hydroxyphenyl)-4-
oxo-5-chromanolato) copper (II).
˚
˚
b = 9.6904(7) A, c = 12.2937(8) A, a = 86.098(6)°,
3
˚
b = 73.088(7)°, c = 85.224(7)°, V = 834.87(12) A
The crystal structure is supported by H-bonds
between the uncoordinated naringenin OH and the
oxygen atom of the DMF (O(7)–H(7)–O(6)), showing
columns along an axis (Table 3, Fig. 10a). In spite of
and Z = 4. Crystallographic results are given in
Table 1. The molecular structure of the complex,
[Cu(C15H7O5)2(C3H7O1N1)2], is monomeric with a
Table 1 Crystal and structure refinement data
Crystal data
C
36H36CuN2O12
Z = 1
Mr = 752.22
F(000) = 391
Dx = 1.496 Mg m-3
¯
Triclinic, P1
˚
Hall symbol: -P 1
Mo Ka radiation, k = 0.71073 A
˚
a = 7.3581 (7) A
Cell parameters from 1162 reflections
˚
b = 9.6904 (7) A
h = 4.3–26.2°
l = 0.72 mm-1
˚
c = 12.2937 (8) A
a = 86.098 (6)°
b = 73.088 (7)°
c = 85.224 (7)°
T = 173 K
Prism, red
0.3 9 0.3 9 0.2 mm3
3
˚
V = 834.87 (12) A
Data collection
Oxford diffraction Gemini CCD S ultra diffractometer
Radiation source: fine-focus sealed tube
Graphite monochromator
3796 Independent reflections
2363 Reflections with I [ 2r(I)
Rint = 0.052
x scans, thick slices
hmax = 29.1°, hmin = 3.7°
h = - 5 ? 9
Absorption correction: multi-scan CrysAlis PRO, Oxford
Diffraction (2009)
Tmin = 0.928, Tmax = 1.000
6620 measured reflections
Refinement
k = - 12 ? 12
l = - 15 ? 15
Refinement on F2
Zero restraints
Least-squares matrix: full
R[F2 [ 2r(F2)] = 0.071
Hydrogen site location: mixed
H atoms treated by a mixture of independent and constrained
refinement
wR(F2) = 0.153
w = 1/[r2(F2o) ? (0.0441P)2 ? 0.2509P] where
P = (F2o ? 2F2c)/3
S = 1.06
(D/r)max \ 0.001
-3
˚
3796 Reflections
246 Parameters
Dimax = 0.63 e A
-3
˚
Dimin = - 0.57 e A
123

Biometals
Fig. 9 Thermal ellipsoids of trans-di(N,N-dimethylmethanamide)bis(7-Hydroxy-2-(4-hydroxyphenyl)-4-oxo-5-chromanolato) cop-
per (II) drawn at the 50% probability level. H atoms were removed for clarification
˚
Table 2 Selected bond distances (A)
flavonoid alone. It is because in the complex these
groups are interacting with the copper but not are
participating in intermolecular attractions, what yes
occure in the naringenin crystaline structure (Table 4).
It is well known that the acid strength of the
ionizable hydrogens of naringenin follows the order:
7-OH [ 40-OH [ 5-OH (Agrawal and Schneider
1983). However, in the crystal structure the only
ionized hydrogen is the corresponding to C5-OH. This
is because copper induces the loss of the proton
located at the C5-hydroxy C4-keto position, which is
the unique position where naringenin can act as a bi-
dentate chelate.
Interaction
Cu(II)–naringenin
Naringenina
Cu–O4
1.933(5)
1.900(2)
2.560(3)
1.260(5)
1.303(5)
–
Cu–O5
–
Cu–O6
–
C4–O4 (C=O)
C5–O5 (C-OH)
1.265(5)
1.353(5)
aPreviously reported, CSD refcode: DOLRIF
the presence of aromatic rings there are no p–p
interactions in the structure, while the packing is
assisted by weak C–HÁÁÁO interactions (Table 3)
between naringenin’s phenyl group and one of the
DMF’s methyl group (Fig. 10b); and by CH–p
interactions in c direction (Fig. 10c). The overall
effect of these weak interactions, uniformly dis-
tributed in space, is the formation of a three-dimen-
sional structure where each molecule is linked to ten
different neighbors. Other interesting result is that the
bond lenghs in the crystal of C4=O4 and C5-OH5
groups are shorter in the complex with respect to the
Scavenging activities of naringenin and its copper
(II) complex
Flavanones are not among the flavonoids that have
higher antioxidant capability (Rice-Evans et al. 1996).
This is because the phenolic rings A and B are not
conjugated, since C2 and C3 have sp3 hybridization.
Therefore, ring B is almost perpendicular to rings A
and C. Figure 11 shows that naringenin had higher
Table 3 Selected
interactions distances (A)
Donor–H
Acceptor
D–H
HÁÁÁA
DÁÁÁA
D–HÁÁÁA
˚
O(7)–H(7)
O(6)a
O(5)b
O(40)c
Cg(1)d
Cg(2)d
0.77(5)
0.91(7)
0.98
1.98(5)
1.81(7)
2.86(2)
2.90(3)
2.86(3)
2.729(5)
2.708(4)
3.189(6)
3.573(6)
3.618(5)
167(5)
169(7)
92(6)
O(40)–H(40)
C(16)–H(16A)
C(17)–H(17C)
C(3’)–H(3’)
a1 - x, - y, - z
b- 1 ? x, y, 1 ? z
c- 1 ? x, - 1 ? y, 1 ? z
d- x, - y, 1 - z
0.98
127(5)
137(6)
0.98
123

Biometals
Fig. 10 View of three-
dimensional structure
a Packing view projected
down a showing H-bond
between O(7)–H(7) and
O(6)1, b C(16)–H(16)–
O(40)2 weak interaction
c C(17)–H(17C)–p (Cg1)3
and C(30)-H(30)–p (Cg2)3
along c axis. 11 - x, - y,
- z, 2- 1 ? x, - 1 ? y,
1 ? z, 3- x, - y, 1- z
Table 4 Selected interactions distances in both compounds
˚
(A)
Atoms
Cu(II)Naringenin
1.260(5)
Naringenina
1.265(5)
C4–O4
(C=O)
C5–O5
(C–OH)
1.303(5)
1.353(5)
aPreviously reported, CSD refcode: DOLRIF
antiradical activity when is chelating Cu(II) than when
it is free. De Sousa et al. has previously screened the
antiradicalarial activity of quercetin, rutin and 3-hy-
droxyflavone and some parent complexes of Cu(II)
and Fe(II) by using DPPH. They found that the
complexed flavonoids were more effective radical
scavengers than the free flavonoids (de Souza and De
Giovani 2004). Pekal et al. also reported that the
quercetin copper (II) complex is much more effective
as free radical scavenger than the flavonoid alone
(Pe˛kal et al. 2011). They did not make additional
experiments to understand the reason.
Fig. 11 Remaining DPPH (%) at the end of the reaction as a
function of the concentration of naringenin free (full line) or
chelating Cu(II) (dashed line). Mean and SD of two independent
values are presented
affect the reactivity of the C40–OH group, which is the
main reactive group of naringenin against radicals
(Jabbari et al. 2014) and therefore, naringenin free or
complexed must have the same antiradical activity.
The second phenolic group that can react with
DPPH is the C7–OH. According to the Mulliken
population analysis (Table 5), in the complex the C5–
O- charge is delocalized reducing the net charge of
copper and increasing the negative charge on C4–O
Due to the lack of resonance between the B ring and
the rest of the molecule, complex formation should not
123

Biometals
Table 5 Charge and
electronic spin density (in
parentheses) from Mulliken
population analysis in
vacuum (white), methanol
(bold) and water (italics) for
naringenin, naringenin
Cu(II) complex and some of
their relevant phenolic
radicals
Relevant atoms
C5–O
C4–O
C40–O
C7–O
Cu
Naringenin
- 0.347
–
- 0.389
–
- 0.358
- 0.345
–
–
–
–
2 0.366
–
2 0.419
–
2 0.385
2 0.366
–
–
–
–
- 0.367
–
- 0.42
–
- 0.386
- 0.367
–
–
–
–
Naringenin C7–O*
(without 7-H*)
- 0.338
(- 0.016)
2 0.357
(2 0.018)
- 0.358
(- 0.018)
- 0.264
(0.617)
2 0.343
(0.536)
- 0.346
(0.534)
- 0.650
(0.017)
2 0.645
(0.070)
- 0.645
(0.070)
- 0.621
(0.073)
2 0.634
(0.065)
- 0.635
(0.064)
- 0.363
(0.081)
2 0.385
(0.084)
- 0.385
(0.085)
- 0.271
(0.091)
2 0.339
(0.044)
- 0.341
(0.043)
- 0.528
(0.021)
2 0.532
(0.062)
- 0.532
(0.062)
- 0.495
(0.115)
2 0.506
(0.121)
- 0.506
(0.121)
- 0.356
- 0.307
(0.648)
2 0.364
(0.606)
- 0.366
(0.604)
- 0.346
(- 0.012)
–0.362
(2 0.017)
- 0.363
(- 0.017)
- 0.351
(0.000)
2 0.370
(0.000)
- 0.370
(0.000)
- 0.313
(0.650)
2 0.368
(0.611)
- 0.369
(0.609)
–
–
–
2 0.385
–
–
–
–0.385
–
–
–
Naringenin C5–O*
(without 5-H*)
- 0.357
–
–
–
2 0.385
–
–
–
- 0.385
–
–
–
Complex
(without 5-H?)
- 0.359
1.342
(0.840)
1.254
(0.711)
1.253
(0.711)
1.261
(0.711)
1.257
(0.712)
1.257
(0.712)
–
2 0.385
–
- 0.386
–
Complex C7–O*
(without 5-H?, neither 7-H*)
- 0.357
–
2 0.385
–
- 0.386
–
and C7–O, what favors the homolytic cleavage of the
C7O–H bond. For another hand, the charge density of
Cu in the complex is 1342 (in vacuum). When the
homolytic rupture occurs and C7–O* radical is
formed, the charge density on copper decreases further
(to 1.253 in vacuum). This indicates that part of the
unpaired electron charge is being transferred to
copper. So, it acts as an electronic density acceptor.
These two effects justify theoretically the experimen-
tal fact that naringenin has greater antiradical activity
when it is forming the copper complex. In simulated
media with solvents (water and ethanol), this behavior
is also evident, but with slight differences in the charge
and spin densities.
In addition to the reported in this paper, it must be
taken into account that when Cu(II) is present it could
exist coupled reaction that stabilize either, reaction
intermediates or products. Furthermore, the antiradi-
calarial enhancement could even be associated to
reactions where the Cu(II)–Cu(I)–Cu-(II) cycle is
present (Arif et al. 2017).
Chelation of metals with flavonoids reduces the
redox potential of both. This, in turn, retard the metal
propensity to promote free radical formation (Selvaraj
et al. 2014a), and enhance the interaction of the
123

Biometals
flavonoid with biological membranes (Selvaraj et al.
2014b). Moreover, the antioxidant enhancement of
naringenin and hesperetin when they are forming
Cu(II) complexes with 1,10-phenanthroline as co-
ligand would seem to be the reason of the improve-
ment of the apoptotic action against malignant A549
cells by a mitochondria-independent pathway (Ta-
mayo et al. 2016).
copper acts as an electronic density acceptor stabiliz-
ing the produced radical. So, it is interesting to suspect
that the delivery of naringenin in the form of Cu(II)
complex could help to increase the efficacy of this
ligand in the treatment of this and other similar
neurodegenerative diseases. The next step is to assay
properties and toxicity of the complex to test its scope.
Acknowledgements We thank Universidad Nacional de Salta
It has been demonstrated that naringenin amelio-
rates Alzheimer’s disease type neurodegeneration
with cognitive impairment in rat models (Khan et al.
2012) This disease is oxidative stress mediated. So, it
is interesting to suspect that the delivery of naringenin
in the form of Cu(II) complex, with better antiradi-
calarial activity, could help to increase its cytotoxicity
against malignant cells and more efficacies in the
treatment of neurodegenerative diseases oxidative
stress mediated.
´
(Proyecto CIUNSa No 2227), Agencia Nacional de Promocion
Cientıfica y Tecnologica of Argentina (PICT 2012 No 0696) and
´
´
´
´
Secretarıa de Polıticas Universitarias (PNo. 33-63-029-2014)
for financial support. The authors acknowledge ANPCyT
(Project No. PME 2006-01113) for the purchase of the Oxford
Gemini CCD diffractometer. We also acknowledge
´
Departamento de Fısica de la Materia Condensada, CNEA for
the use of TGA facilities and Dra. Gabriela Levy for her helpful
assistance. Universidad Nacional de Catamarca (UNCa) is also
´
appreciated for their computation supports. G. Celiz, S. Suarez,
C. D. Brondino, and F. Doctorovich are members of the
Research Career of CONICET.
Conclusions
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