Chemico-Biological Interactions 239 (2015) 184–191

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Synthesis, characterization and biological evaluation of Rutin–zinc(II)

flavonoid -metal complex
Norma Estefania Andrades Ikeda a,e,1, Estela Maria Novak b,1, Durvanei Augusto Maria c,1,
Adélia Segin Velosa d, Regina Mara Silva Pereira d,⇑
a
Faculty of Medicine, University of São Paulo, São Paulo, Brazil
b
Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
c
Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, Brazil
d
Department of Pharmacy, Anhanguera University of São Paulo, São Paulo, Brazil
e
Department of Biotechnology, Anhanguera University of São Paulo, São Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Synthesis of compounds analogous to natural products from secondary metabolites, such as flavonoids, is
Received 8 December 2014 a promising source of novel drugs. Rutin (quercetin-3-O-rutinoside) is a natural flavone, which has, in its
Received in revised form 19 May 2015 chemical structure, different sites for coordination with transition metals and the complexation with
Accepted 5 June 2015
these metals enhances its biological properties. Rutin–zinc(II), a flavonoid–metal complex, was synthe-
Available online 16 June 2015
sized and characterized by UV–VIS, FT-IR, elemental analysis and 1H NMR. The antioxidant and antitumor
activities, as well as the cytotoxicity and in vivo toxicity of this complex were evaluated and compared
Keywords:
with the free rutin. Rutin–zinc(II) has not shown any cytotoxicity against normal cells (fibroblasts and
Flavonoids
Rutin
HUVECs) or toxicity in BALB/c mice, but has shown antioxidant activity in vitro and cytotoxicity against
Metal complex leukemia (KG1, K562 and Jurkat), multiple myeloma (RPMI8226) and melanoma (B16F10 and SK-Mel-28)
Antitumor cell lines in vitro. In Ehrlich ascites carcinoma model, Rutin–zinc(II) modulated the mitochondrial mem-
brane potential and the expression of genes related to cell cycle progression, angiogenesis and apoptosis.
Ó 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 2. Experimental section

Drug discovery from natural products have brought new clinical
applications of plant secondary metabolites as templates for
synthetic novel drugs [1]. Flavonoids are polyphenolic compounds
found in seeds, fruits, olive oil, tea, and red wine [2–4] that have
demonstrated a wide variety of biological actions [5–7]. Rutin
(quercetin-3-O-rutinoside) is a natural flavone, which has, in its
chemical structure, different sites for coordination with
transition metals and the complexation of rutin with these metals
enhances its biological properties [8]. It has been reported that
rutin has several pharmacological properties including antioxidant,
anti-inflammatory, anticarcinogenic, cytoprotective, antiplatelet,
antithrombotic, vasoprotective, and cardioprotective activities
[9–11].
⇑ Corresponding author. Tel.: +55 11 2967 9147.
E-mail address: rpereira02@hotmail.com (R.M.S. Pereira).
1
These authors contributed equally to this work.
2.1. Synthesis and structural characterization of Rutin–zinc(II)
complex
A solution of [Zn(CH3COO)2]2H2O salt in distilled water was
slowly added dropwise to a solution of dehydrated rutin
(Dimorphandra mollis) in methanol. The mixture was stirred at
90–140 rpm for 24 h at 37–40 °C. The complex was filtered in a
vacuum system, washed with methanol and dried at room temper-
ature. Yield: 25%, M.P. 230 °C, Anal. Cal. for found, exp. (calc.)
C31H48O26Zn2: %C 38.29 (38.62) and %H 4.80 (4.98); IR k (cm-1):
3355(OAH); 1571 (C@O); 1477(C@C); 1270(CAOAC); 804(CAC).
NMR1H (d, ppm): 12.85 (brs, 1H, 5-OH); 12.62 (brs, 1H, 7-OH);
7.50 (brs, 1H, 20 -H); 6.75(brs, 1H, 50 -H); 6.25(d brs, 1H). Melting
points were determined by using a QUIMIS Melting Point
Equipment. The IR spectra were recorded in the solid state in the
range 4000–400 cm1 by using a Nicolet Nexus spectrometer.
The UV–VIS spectra were recorded in aqueous solution (pH 6.5),
in the range 200–500 nm, by using a Femto 800XI spectrometer.
The 1H-NMR spectra were measured at 25 °C in an INOVA –

http://dx.doi.org/10.1016/j.cbi.2015.06.011
0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
 N.E.A. Ikeda et al. / Chemico-Biological Interactions 239 (2015) 184–191
500 Mz Spectrophotometer, in DMSO-d6. The chemical shifts are
referred to DMSO signal at 3.38 ppm.
2.2. Antioxidant activity
The antioxidant activity of Rutin–zinc(II) was tested by two
different methods: superoxide radical scavenger assay and DPPH
assay.
2.2.1. Superoxide radical scavenging assay
The superoxide radicals generated were determined spec-
trophotometrically by monitoring the product of nitroblue
tetrazolium (NBT) [12]. Various concentrations of rutin and
Rutin–zinc(II) solutions (1, 10 e 100 lM) were added to the reaction
mixture containing: 50 lM NBT, 20 lM phenazine methosulfate,
156 lM NADH (adenosine 5-trihydrogen diphosphate), in 0.1 M
phosphate buffer (pH 7.4) making up a final volume of 2.5 mL.
After the incubation of the mixture at 25 °C for 10 min, the
absorbance was read at 560 nm and compared with the control
sample, containing 50 lM NBT. The percentage of superoxide
radical scavenging was calculated from the samples optical density.
All the measurements were replicated 3 times.
2.2.2. DPPH assay
The solution contained 1 mL of DPPH (20 lM) and 1 mL of dif-
ferent concentrations of rutin and Rutin–zinc(II) solution, resulting
in a final concentration of DPPH of 10 lM. The mixtures were vig-
orously mixed and allowed to stand in the dark for 30 min at 25 °C.
The absorbance of the resulting solutions was measured at 517 nm
against a blank sample containing only DPPH, the negative control;
rutin and vitamin C served as the positive control [10,11]. All the
measurements were replicated 3 times.
2.3. Cytotoxicity assay
RPMI 8226, KG1, K562, Jurkat and SK-MEL-28 cell lines were
obtained from ATCC (Rockville, MD, USA). The B16F10,
SK-MEL-28, human fibroblasts and Human Umbilical Vein
Endothelial cells (HUVECs) were provided by Dr. Durvanei
Augusto Maria from the Butantan Institute (São Paulo, Brazil).
The cell lines were routinely cultured in RPMI-1640 medium, con-
taining 10% inactivated fetal bovine serum (supplemented with
2.0 mM glutamine and 50 lg/mL garamycin) at 37 °C in a humidi-
fied incubator with 5% CO2. The cells were treated with Rutin–
zinc(II) and rutin in concentrations ranging from 17.2 lM to
275.6 lM for 24 h and the cell viability was assessed by MTT assay.
The cell viability was determined by MTT [3-(4,5-dimethylthiazo
l-2-yl-2,5-diphenyl tetrazolium bromide] method [13]. At the end
of the treatment, MTT (0.5 mg/mL) diluted in fresh medium was
added. After 4 h of incubation, the blue formazan crystals were dis-
solved with 100 lL acid-isopropanol and the absorbance was mea-
sured at 570 nm using a microplate reader (TiterTek Multiskan
Plus, Flow Laboratories, USA). Experiments were performed in
triplicate.
2.4. In vivo toxicological studies
All animal experiments were performed with the approval of
the Institutional Animal Care and Use Committee of the Butantan
Institute (Permit number: n°611/09). BALB/c female mice,
8-week-old, weighing 20–22 g were maintained in a 12 h
light-and-dark cycle, at a temperature of 22 ± 2 °C and were fed
with standard pellet chow, with free access to water. The Rutin–
zinc(II) complex was solubilized in deionized water and rutin
was solubilized in propylene glycol and diluted in deionized water.
The animals were divided in three groups and treated with
185
Rutin–zinc(II) administered intraperitoneally (i.p.) for 7, 14 and
21 days and observed for 30, 60 and 90 days after treatment.
Toxicology tests were performed with the flavonoid rutin
(5 mg/Kg) in the same conditions. The toxicity was assessed by
body weight measurement, hematological and biochemical analy-
ses and histological examination of liver and kidneys as indicators.
Mice were electively sacrificed by cervical dislocation on day 30, 60
and 90 and blood samples were collected from retro-orbital plexus.
For histopathological studies, tissues were fixed in 10% neutral buf-
fered formalin, dehydrated, and embedded in paraffin by routine
methods and stained with hematoxylin and eosin (H&E).
2.5. Antitumor activity evaluation
The antitumor activity of the compounds was evaluated in the
Ehrlich ascites carcinoma (EAC) model. EAC is a spontaneous mur-
ine mammary adenocarcinoma adapted to ascites form and carried
in outbred mice by serial intraperitoneal passages. This model has
been used as a transplantable tumor model to investigate the anti-
neoplastic effects of many substances or compounds [14,15].
2.5.1. Experimental design
Mice were inoculated with 1  104 EAC cells into the peritoneal
cavity. The animals were divided into six groups (n = 10) and trea-
ted with 0.1 mL of the compounds, administered via i.p. route for
14 days. Group A (control group): treated with phosphate-
buffered saline (PBS); Group B: treated with Rutin–zinc(II)
(5 mg/Kg); Group C: treated with rutin (5 mg/Kg); Group D: trea-
ted with Rutin–zinc(II) (5 mg/Kg) in combination with paclitaxel
(15 mg/Kg); Group E: treated with rutin (5 mg/Kg) in combination
with paclitaxel (15 mg/Kg); Group F: treated with paclitaxel
(15 mg/Kg). Rutin–zinc(II) and rutin were solubilized as described
above and paclitaxel was diluted in cremophorÒ. In the groups A, B
and C, mice were treated daily, in the groups D and E received
Rutin–zinc(II) and rutin daily and paclitaxel on day 4, 8, and 12.
Group F received paclitaxel twice a week. The ascitic fluid from
the peritoneal cavity of tumor bearing mice was collected to obtain
the tumor cells at the end of the treatment to perform the DNA
fragmentation, mitochondrial membrane potential and angiogene-
sis, apoptosis and cell cycle-related gene expression analysis of the
EAC cells. Hematological analysis was performed to evaluate the
effect of the compounds on the EAC bearing mice. The survival
rate was determined by Kaplan–Meier method.
2.5.2. DNA fragmentation analysis
The ascitic fluid from the peritoneal cavity of tumor bearing
mice was collected and centrifuged to obtain the tumor cells at
the end of the treatment to perform the flow cytometry analysis
of DNA fragmentation of the EAC cells. The EAC cells were washed
with PBS and centrifuged at 1500 rpm at 4 °C for 5 min. Pellets
were fixed in 70% ice-cold ethanol overnight. The cells were cen-
trifuged, washed with PBS, and incubated in PBS containing
1 mg/mL RNase for 10 min at room temperature. Samples were
stained with 1 mg/mL propidium iodide for 30 min at 4 °C. Data
acquisition was performed by flow cytometry (FACSCalibur) and
analyzed by Cell Quest software.
2.5.3. Mitochondrial membrane potential (Dwm) analysis
The mitochondrial membrane potential (Dwm) was assessed in
EAC cells by flow cytometry using Rhodamine 123 (Invitrogen), a
fluorescent lipophilic cationic dye which preferentially enters into
mitochondria due to the highly negative mitochondrial membrane
potential. Depolarization of DWm results in the loss of
Rhodamine-123 from the mitochondria and a decrease in intracel-
lular fluorescence [16]. The cells were collected from ascitic fluid of
tumor bearing mice, washed with PBS and centrifuged at 1500 rpm
186 N.E.A. Ikeda et al. / Chemico-Biological Interactions 239 (2015) 184–191
at 4 °C for 5 min and incubated in PBS containing 5 mg/mL
Rhodamine 123 for 10 min at 37 °C, in a humidified incubator with
5% CO2 for 30 min. Data acquisition was performed by flow cytom-
etry (FACSCalibur) and analyzed by Cell Quest software.
2.5.4. qRT-PCR analysis of angiogenesis, apoptosis and cell cycle-
related gene expression
Total RNA was isolated from EAC cells using TRIzolÒ reagent
(Invitrogen) according to the protocol supplied by the manufac-
turer. Reverse transcription reaction was carried out using 1 lg
of RNAse-free DNase (Qiagen)-treated total RNA following the pro-
tocol of SuperScript™ III, First-Strand Synthesis SuperMix for
RT-PCR (Invitrogen). The primer sequences of VEGF, Cyclin D1,
Caspase-3 Caspase-8 and GAPDH and cycling conditions used were
previously described [17–20]. Quantitative PCR assays were car-
ried out by using Rotor Gene RG-3000 rotor (Corbett Research,
AUS) with PlatinumÒ SYBRÒ Green qPCR Super Mix-UDG reagent
(Invitrogen). GAPDH was used as a reference for relative quantifi-
cation and data were analyzed according to the Livak method [21].
2.6. Statistical analyses
Statistical analyses were performed by using One-way Analysis
of Variance (ANOVA) and Tukey–Kramer post hoc test for multiple
comparisons. Statistical significance was set at p < 0.05.
3. Results
3.1. Structural characterization
The complex Rutin–zinc(II) was obtained when a solution of
[Zn(CH3COO)2].2H2O salt in distilled water was slowly added drop-
wise to a solution of dehydrated rutin in methanol. A new com-
pound Rutin–zinc(II) was characterized by UV–VIS and FT-IR
spectroscopy. Comparing the electronic spectra (200–500 nm) of
rutin and Rutin–zinc(II), a band shift at 353 nm to 393 nm (band
I of rutin) and another at 259 nm to 269 nm (band II of rutin) were
observed (Fig. 1). These shifts suggest that two metal ions may be
occupying the coordination sites 4, 5 and 40 , 50 (Fig. 1). The IR spec-
trum of Rutin–zinc(II) showed a band shift in 1655 Rutin–zinc(II)
to 1627 cm1 indicating an interaction of the Zn(II) ion via the car-
bonyl group, in position 4 (Fig. 1). In comparison with the rutin fre-
quencies of mOH, mC@C, mCAOAC and mCAC bands (3351, 1597, 1295 e
808 cm1, respectively), the complex Rutin–zinc(II) shows bands
in 3322, 1571, 1272 e 805 cm1, respectively, shifting to lower fre-
quencies, indicating also the Zn(II) ion coordination at position 30
and 40 as well. The complexation of Zn(II) to rutin was also verified
Fig. 1. Spectrum of UV–VIS in methanol solution of 50 lM of rutin and Rutin–zinc(II) (A) and schematic structure of Rutin–zinc(II) (B).
by 1H NMR. The proton signals of Rutin–zinc(II) shifted to lower
frequencies relative to the free flavonoid. The coordination with
the Zn(II) ion decreases the electron density of the flavonoid
around 5-OH, probably due to the interaction with Zn(II) (a Lewis
acid). The 1H NMR signal assigned to 3’ and 4’ hydroxyl in Rutin–
zinc (II) becomes broader when compared with free rutin. These
data are in agreement with the IR and the elemental analyses data
demonstrating metallic ion coordination in positions 4, 5 and 30 , 40 ,
and four water molecule are coordinated in each ion (Fig. 1).
3.2. Antioxidant activity
Superoxide radicals generated from dissolved oxygen by
PMS-NADH coupling can be measured by their ability to reduce
NBT. As shown in Fig. 3, in a concentration of 10 lM Rutin–
zinc(II) showed better effect than rutin. However, in a concentra-
tion of 100 lM Rutin–zinc(II) superoxide radical scavenging was
25% higher than the rutin scavenging ability. The percentage inhi-
bition of Rutin–zinc(II) and rutin at 100 lM was 70%, and 55%,
respectively. DPPH assay showed that Rutin–zinc(II) was more
effective than the free flavonoid rutin, which inhibited 80% at
30.4 lM in comparison with free rutin, which inhibited 45% in
the same concentration. The Rutin–zinc(II) effect is comparable
with vitamin C (Fig. 2).
3.3. Cytotoxicity
The cytotoxic effect of Rutin–zinc(II) in comparison with free
rutin was determined by MTT assay. Rutin–zinc(II) showed cyto-
toxicity against all tumor cell lines tested after 24 h of treatment,
but, in fibroblasts and HUVECs normal cell lines, no cytotoxic
effects were observed. The human acute myeloid leukemia cell line
(KG1) showed higher sensitivity to the complex with an IC50 of
91.4 lM than RPMI8226 multiple myeloma cell line (IC50 of
196.6 lM) as shown in Table 1. The flavonoid rutin showed no
cytotoxicity against tumor and normal cell lines tested.
3.4. Toxicity of Rutin–zinc(II) in mice
Subchronic toxicity tests were performed to determine Rutin–
zinc(II) toxicity. No behavioral changes or physical signs of pain,
discomfort or neurological impairment such as pilo-erection,
vocalization, motility alterations, dyspnea, cyanosis and convul-
sions were observed in the mice, and all groups of treatment
showed progressive weight gain throughout the experiment in
comparison with the control group (Table 2). The effect of
5 mg/Kg Rutin–zinc(II) and rutin on red blood cells, white blood
 N.E.A. Ikeda et al. / Chemico-Biological Interactions 239 (2015) 184–191 187

A 80
B 100

superoxide scavenger (%)

80

DPPH inhibition (%)

60

60
40

40

20
20

0 0
1 μM 10 μM 100 μM 1 μM 10 μM 100 μM Vitamin C Rutin Rutin-zinc(II)
Rutin Rutin-zinc(II)

Fig. 2. Superoxide radical scavenging activity of rutin and Rutin–zinc(II) at 1, 10 and 100 lM (A). DPPH inhibition from vitamin C, rutin and Rutin–zinc(II) at 30.4 lM (B).

Fig. 3. Histological analysis of liver (a), kidney (b) and lung (c) of BALB-c mice treated with Rutin–zinc(II) for 21 days and sacrificed 90 days after treatment. H&E stain (x400).

Table 1 3.5. The combination of rutin and Rutin–zinc(II) with paclitaxel

Determination of Rutin–zinc(II) IC50 by MTT assay.
Cell lines
B16F10
SK-MEL 28
KG1
RPMI 8226
JURKAT
K562
Fibroblasts
HUVECs
cells, platelets and liver enzymes was assessed after 7, 14 and
21 days of treatment and 30,60 and 90 days after treatment. An
increase in the number of platelets, red blood cells and white blood
cells was observed after 21 days of treatment in the groups treated
with rutin and Rutin–zinc(II) (Table 2). However, 90 days after
treatment all parameters returned to normal levels. No significant
changes in ALT and AST levels were observed (Table 2).
Histopathological examinations of the tissues indicated that there
were no detectable abnormalities and alterations in the micro-
scopic examination of the liver, kidney and lungs in comparison
to the control (Fig. 3).
enhanced DNA fragmentation
Rutin–zinc(II)
IC50 (lM) The effects of Rutin–zinc(II), rutin, paclitaxel and combinations
160.7 on DNA fragmentation in EAC cells, in vivo, were investigated. The
194.0 percentages of DNA fragmentation of the untreated control group
91.4 (16.6 ± 3.7%), Rutin–zinc(II) (31.5 ± 5.7%), rutin (16.9 ± 8.8%), pacli-
196.6 taxel (36.0 ± 1.8%), rutin in combination with paclitaxel
150.2
173.2
(63.4 ± 3.5%) and Rutin–zinc(II) in combination with paclitaxel
>275.6 (60.7 ± 8.1%) demonstrates that the combination with Rutin–
>275.6 zinc(II) and rutin significantly enhanced paclitaxel antitumor activ-
ity (Fig. 4).
3.6. Loss of mitochondrial membrane potential (Dwm) in EAC cells
The changes in the mitochondrial membrane potential (Dwm)
was assessed by flow cytometry using Rhodamine 123 fluorescent
probe. The Fig. 5 shows the percentages of Dwm in the EAC cells in
all groups of treatment: rutin (30.0 ± 8.0%), Rutin–zinc(II)
(8.7 ± 6.3%), paclitaxel (2.4 ± 1.2%), rutin in combiation with pacli-
taxel (2.7 ± 0.3%) and Rutin–zinc(II) in combination with paclitaxel
(2.8 ± 0.8%) and untreated control group (86.7 ± 8.3%). The results
demonstrate a significant loss of mitochondrial membrane

Table 2
Effect of intraperitoneal administration of rutin and Rutin–zinc(II) at the dose of 5 mg/Kg on hematological parameters, liver enzymes and body weight in BALB/c mice.

Groups RBC (103/mm3) WBC (103/mm3) PLT (103/mm3) ALT (U/mL) AST (U/mL) Weight (g)
Control 9.05 ± 0.66 4.91 ± 0.79 687.5 ± 97.08 19.4 ± 1.7 17.6 ± 1.7 24.34 ± 0.70
Rutin 13.13 ± 1.31⁄⁄ 9.95 ± 1.63⁄⁄⁄ 2595 ± 432.60⁄⁄⁄ 19.2 ± 1.5 16.1 ± 0.4 22.48 ± 1.15
Rutin–zinc(II) 12.48 ± 1.74⁄ 8.32 ± 0.58⁄⁄ 2036 ± 94.54⁄ 19.4 ± 1.2 17.2 ± 1.0 23.39 ± 1.32

Data showed as mean ± SD, n = 10. ***p < 0.0001, **p < 0.001, *p < 0.05, compared with untreated control (ANOVA and Tukey–Kramer post-hoc test) after 21 days of treatment.
RBC: red blood cell, WBC: white blood cell, PLT: platelets, AST: aspartate aminotransferase, ALT: alanine amionotransferase.
188 N.E.A. Ikeda et al. / Chemico-Biological Interactions 239 (2015) 184–191

A Control
B C Paclitaxel
Control
Rutin Rutin -zinc(II) Rutin-zinc(II)+Paclitaxel
Rutin +Paclitaxel

80
D
***
***

60
EAC cells (%)

40 ***
***

20

0
1

1
S

.
N /M

N /M

S
N /M

N /M

N /M

N /M

.
ag

ag

ag

ag

ag

ag
G

G
2

2
0/

0/

0/

0/

0/

0/
fr

fr

fr

fr

fr

fr
G

G
G

G
A

A
D

D
Control Rutin Rutin-Zn(II) Paclitaxel Rutin+paclitaxel Ru-Zn(II)+paclitaxel
(untreated)

Fig. 4. DNA fragmentation analysis (A, B, C and D) of EAC cells collected from tumor bearing mice treated with rutin, Rutin–zinc(II), paclitaxel, paclitaxel associated with rutin
and Rutin–zinc(II) in comparison with untreated control group. ANOVA (p < 0.0001), ***p < 0.0001 (Tukey–Kramer post-hoc test).

Control
B Rutin
A 100 Rutin-Zn(II)
mitochondrial membrane potential-ΔΨm (%)

80

60 Control
Paclitaxel
C Rutin+paclitaxel
Rutin-Zn(II)+paclitacel
40 ***

20
***
*** *** ***
0
Control Rutin Rutin-Zn(II) Paclitaxel Rutin+paclitaxel Rutin-Zn(II)+paclitaxel
(untreated)

Fig. 5. Mitochondrial membrane potential analysis (A, B and C) of EAC cells collected from tumor bearing mice treated with rutin, Rutin–zinc(II), paclitaxel, paclitaxel
associated with rutin and Rutin–zinc(II) in comparison with untreated control group. ANOVA (p < 0.0001), ***p < 0.0001 (Tukey–Kramer post-hoc test).

potential in the ECA cells of the groups treated with paclitaxel and the tested compounds significantly down-regulated VEGF and
combinations in comparison with the single flavonoid rutin and Cyclin D1 and up-regulated Caspase-3 and Caspase-8 gene expres-
Rutin–zinc(II) (see Table 3). sion in comparison with untreated control group.

3.7. Angiogenesis, apoptosis and cell cycle-related gene expression 4. Discussion

analysis
Natural products like flavonoids represent an attractive source
The expression of VEGF, Cyclin D1, Caspase-3 Caspase-8 genes of varied structures that exhibit potent biological activities and
in EAC cells was assessed by qRT-PCR. Data presented in Fig. 6 desirable pharmacological profiles [22,23]. Furthermore, flavo-
shows the effect of the different treatments in EAC in vivo. All of noids can form complexes with transition metal ions and these
 N.E.A. Ikeda et al. / Chemico-Biological Interactions 239 (2015) 184–191 189

Table 3 concentrations and experimental conditions. The flavonoid

Hematological parameters of EAC bearing mice. metal-complex Rutin–zinc(II) was synthesized and characterized
Groups RBC (103/ WBC (103/ PLT (103/mm3) by UV–VIS, FT-IR spectroscopy and 1H NMR indicating that the
mm3) mm3) Zn(II) ion is coordinated to the benzopyrane ring of rutin (positions
Control (normal) 8.87 ± 0.85 3.91 ± 0.45 687.5 ± 97.0 4 and 5), and to the aromatic ring (30 and 40 positions). Rutin–
Control (untreated) 4.00 ± 0.55 5.37 ± 0.35 967.0 ± 90.5 zinc(II) has shown cytotoxicity against leukemia, multiple mye-
Rutin 6.87 ± 0.30⁄⁄⁄a 8.42 ± 0.43⁄⁄⁄a 984.0 ± 25.97 loma and melanoma cell lines, but did not show any cytotoxicity
Rutin–Zn(II) 6.26 ± 0.5⁄⁄⁄a 7.10 ± 0.18⁄⁄⁄a 924.0 ± 63.69
Paclitaxel 2.99 ± 0.24 2.97 ± 0.12 594.0 ± 30,51
against normal cells in vitro. However, at the same concentrations,
Rutin + paclitaxel 2.12 ± 0.15 2.10 ± 0.17⁄⁄b 766.0 ± 34.0⁄b free rutin did not show cytotoxicity against normal and cancer
Rutin– 5.97 ± 0.38⁄⁄⁄b 3.57 ± 0.33 1286 ± 73.15⁄⁄⁄b cells. Regarding the cytotoxicity of rutin against the cancer cells
Zn(II)+paclitaxel evaluated in this work, there are no reports of cytotoxicity against
The data were expressed as mean ± SD, n = 10. ***p < 0.0001, **p < 0.001, *p < 0.05, RPMI 8226 and KG1 cell line and the flavonoid showed no cytotox-
compared with (a) untreated control group and (b) paclitaxel treated group icity against SK-ML-28 cell line [25]. Rutin from Pupalia lappacea
(ANOVA and Tukey–Kramer post-hoc test). extract showed cytotoxicity against a K562 cell line [26] and the
flavonoid isolated from Leonurus heterophyllus showed no cytotox-
icity against a K562 cell line [27], wolfberry fractions containing
complexes possess higher scavenging and anti-inflammatory activ- rutin showed cytotoxicity against Jurkat cell line [28]. Rutin
ities and cytoprotective effects than the parent flavonoid [24]. The demonstrated antimetastatic activity in vivo in B16F10 melanoma
synthesis of a new Zn(II) complex bearing a rutin ligand started [29]. Matsuo et al. [30] evaluated the cytotoxicity of various flavo-
from the premise that this polyphenol demonstrated anticancer noids, including rutin, against normal cells. The results demon-
activity in several works carried out with different rutin sources, strated that rutin did not show cytotoxicity against HUVEC

1.0
A 1.0 B Cyclin D1 relative gene expression
VEGF relative gene expression

0.8
0.8

0.6
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0.4 ***

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Fig. 6. VEGF (A), Cyclin D1 (B), Caspase-3 (C) and Caspase-8 (D) gene expression analysis of EAC cells collected from tumor bearing mice treated with with rutin, Rutin–
zinc(II), paclitaxel, paclitaxel in combination with rutin and Rutin–zinc(II). ANOVA (p < 0.0001), ***p < 0.0001 (Tukey–Kramer post-hoc test).
190 N.E.A. Ikeda et al. / Chemico-Biological Interactions 239 (2015) 184–191
(200 lM) and fibroblasts (500 lM), which corroborates our find-
ings [30]. The cytotoxic activity showed by Rutin–zinc(II) was also
observed in a Quercetin–zinc(II) complex that has the property to
intercalate into C–G DNA sequences suggesting metal ion coordi-
nation with oxygen atom in the phosphodiester backbone resulting
in cleavage of DNA in vitro [31]. Transition metal complexes like
platinum complexes have been utilized in cancer treatment based
on their successful antitumor activity derived from their ability to
form covalent adducts with DNA by ligand exchange [32]. DNA is
generally the major biological target of platinum compounds and
their cytotoxicity is correlated with the formation of
1,2-intrastrand adducts between the N7 atoms of two adjacent
purine residues [33]. The in vivo studies in EAC bearing mice
demonstrated that in comparison with rutin, Rutin–zinc(II) signif-
icantly induced DNA fragmentation in tumor cells. However, in
combination with paclitaxel, both rutin and Rutin–zinc(II) signifi-
cantly enhanced DNA fragmentation. Paclitaxel is a diterpenoid
isolated from Taxus brevifolia, used clinically for the treatment of
ovarian and breast cancer and primarily kills cancer cells via
microtubule stabilization [34,35]. The loss of mitochondrial mem-
brane potential can activate caspase signaling pathways triggering
the release of cytochrome c and other apoptotic factors to the cyto-
plasm [36]. Although the treatment with rutin and Rutin–zinc(II)
significantly decreased the mitochondrial membrane potential
the effects of the combination with paclitaxel were the strongest.
The treatments demonstrated antiproliferative and antiangiogenic
activity modulating genes related to cell cycle progression, angio-
genesis and apoptosis with decreased cyclin D1 and VEGF expres-
sion and increased expression of initiator and executor caspases-8
and -3. However, the combination of Rutin–zinc(II) with paclitaxel
showed synergistic antitumor activity, preventing anemia and
myelossuppression, undesirable side effects of cancer and
chemotherapy, and in addition increased lifespan of EAC bearing
mice as observed in Fig. 7. Metal–flavonoid chelates, like com-
plexes of rutin with Fe(II), Fe(III), Cu(II) and Zn(II), play an impor-
tant role in protecting biological macromolecules, such as proteins,
lipids and DNA, from oxidative stress that contribute to cancer,
atherosclerosis, cardiovascular diseases, aging and inflammatory
diseases [37,38]. Within the same in situ environment, malignancy
develops under conditions in which the malignant cells are suscep-
tible to the potential cytotoxic effects of the cellular zinc levels that
exist in the normal cells. Sliwinsky and colleagues proposed that
the mechanism of such action of zinc involves enhanced expres-
sion of anti-apoptotic Bcl-2 and Bcl-XL proteins and increased
expression of proteins of mitogenic signaling pathways, including
p53, AKT and MAP kinases [39]. The results demonstrated that
the flavonoid metal-complex Rutin–zinc(II) could be a potential
chemopreventive and therapeutic anticancer agent.
100
80
Percent survival
60
40
20
0 [14] A.M. Boldi, Libraries from natural product-like scaffolds, Curr. Opin. Chem.
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (days)
Fig. 7. Kaplan–Meier survival curve for EAC bearing mice treated with rutin, Rutin–
zinc(II), paclitaxel, paclitaxel associated with rutin and Rutin–zinc(II).
p = 0.0043(Log-Rank test).
5. Conclusion
In the present study, the results demonstrated that the newly
synthesized molecule Rutin–zinc(II) is more efficient as an antiox-
idant agent than free rutin. Moreover, Rutin–zinc (II) has not
shown any cytotoxicity against normal cells or toxicity in BALB/c
mice, but has shown antioxidant activity and cytotoxicity against
cancer cell lines in vitro and synergistic antitumor activity prevent-
ing side effects of chemotherapy. The data obtained from the UV–
VIS, FT-IR, mass spectrometry and NMR studies indicate that Zn(II)
is coordinated to rutin in the benzopyrane ring (positions 4 and 5)
and in the aromatic ring (30 and 40 positions). The increase in bio-
logical activity of Rutin–zinc(II) complex could be associated with
the coordination of Zn(II) in those positions.
Conflict of interest
The authors declare that there is no conflict of interests regard-
ing the publication of this paper.
Transparency Document
The Transparency document associated with this article can be
found in the online version.
Acknowledgements
Financial support from FAPESP is gratefully acknowledged.
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