Synthesis of Quercetin-Metal Complexes, In Vitro and In Silico Anticholinesterase and Antioxidant Evaluation, and In Vivo Toxicological and Anxiolitic Activities

Article abstract of DOI:10.1007/s12640-019-00142-7

The level of acetylcholine, a neurotransmitter essential for processing memory and learning, is lower in the brains of patients with Alzheimer’s disease due to the higher concentration of the enzyme acetylcholinesterase. The main compounds used for Alzheimer’s treatment are acetylcholinesterase inhibitors. Quercetin coordination complexes with the metal ions Cu⁺², Zn⁺², Ni⁺², Co⁺², and Fe⁺² were synthesized to investigate their potential use against Alzheimer’s disease, by evaluating the inhibition of acetylcholinesterase in vitro and in silico, as well as the antioxidant activity, toxicity, and anxiolytic action in the zebrafish (Danio rerio) model. The organic complexes were characterized by UV-Vis and FT-IR. The spectral information suggested that coordination of metals occurs with the carbonyl group and OH linked to the C-3 carbon of quercetin. The quercetin-Fe (QFe) complex presented the best antioxidant and antiacetylcholinesterase actions, and these results were confirmed by molecular docking. In the toxicity and locomotor evaluation, the quercetin molecules and the synthesized complexes, mainly QCu and QZn derivatives, showed the highest degree of inhibition of the fish’s locomotor activity, suggesting a possible anxiolytic action. Then, quercetin complexes with metals, mainly with Fe⁺², represent valuable compounds and deserve more investigation as promising agents against Alzheimer’s disease.

Full text of DOI:10.1007/s12640-019-00142-7

Neurotoxicity Research
https://doi.org/10.1007/s12640-019-00142-7
ORIGINAL ARTICLE
Synthesis of Quercetin-Metal Complexes, In Vitro and In Silico
Anticholinesterase and Antioxidant Evaluation, and In Vivo
Toxicological and Anxiolitic Activities
Wildson Max Barbosa da Silva1,2 & Solange de Oliveira Pinheiro3 & Daniela Ribeiro Alves4
&
Jane Eire Silva Alencar de Menezes5 & Francisco Ernani Alves Magalhães6 & Francisca Crislândia Oliveira Silva5
&
Jacilene Silva7 & Emmanuel Silva Marinho7 & Selene Maia de Morais1,4
Received: 29 April 2019 /Revised: 30 October 2019 /Accepted: 21 November 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The level of acetylcholine, a neurotransmitter essential for processing memory and learning, is lower in the brains of patients with
Alzheimer’s disease due to the higher concentration of the enzyme acetylcholinesterase. The main compounds used for
Alzheimer’s treatment are acetylcholinesterase inhibitors. Quercetin coordination complexes with the metal ions Cu+2, Zn+2,
Ni+2, Co+2, and Fe+2 were synthesized to investigate their potential use against Alzheimer’s disease, by evaluating the inhibition
of acetylcholinesterase in vitro and in silico, as well as the antioxidant activity, toxicity, and anxiolytic action in the zebrafish
(Danio rerio) model. The organic complexes were characterized by UV-Vis and FT-IR. The spectral information suggested that
coordination of metals occurs with the carbonyl group and OH linked to the C-3 carbon of quercetin. The quercetin-Fe (QFe)
complex presented the best antioxidant and antiacetylcholinesterase actions, and these results were confirmed by molecular
docking. In the toxicity and locomotor evaluation, the quercetin molecules and the synthesized complexes, mainly QCu and QZn
derivatives, showed the highest degree of inhibition of the fish’s locomotor activity, suggesting a possible anxiolytic action. Then,
quercetin complexes with metals, mainly with Fe+2, represent valuable compounds and deserve more investigation as promising
agents against Alzheimer’s disease.
.
.
Keywords Alzheimer’s disease Coordination compounds Flavonoids
Introduction
* Selene Maia de Morais
Alzheimer’s disease is the most common type of dementia.
Evidence exists for both cholinergic and glutamatergic in-
volvement in the disease’s etiology. Acetylcholine (ACh), a
neurotransmitter essential for processing memory and learn-
ing, is decreased in both concentration and function in patients
with Alzheimer’s disease (Paul and Borah 2017).
Acetylcholinesterase (AChE) inhibitors have proven to be
the most viable therapeutic target for symptomatic improve-
ment of Alzheimer’s disease (AD), because cholinergic deficit
is a consistent and early finding in AD (Mehta et al. 2012).
Individuals with high anxiety have a 53% higher risk of
AD, demonstrating that anxiety is a potential risk for this
disease (Santabarbara et al. 2019). Depression is also related
to AD and is one of the most frequent non-cognitive symp-
toms. The prevalence of depressive symptoms and syndromic
depression in AD depends on the severity of dementia and the
scales used for its detection (Müller-Thomsen et al. 2005).
selenemaiademorais@gmail.com
1
Graduate Program in Biotechnology, Laboratory of Chemistry of
Natural Products (LQPN), Ceará State University, Fortaleza, Ceará,
Brazil
2
Christus University Center (UNICHRISTUS), Fortaleza, Ceará,
Brazil
3
Laboratory of Inorganic Chemistry (LQUIN), Ceará State University,
Fortaleza, Ceará, Brazil
4
Veterinarian Sciences Post Graduation Program, Ceará State
University, Fortaleza, Ceará, Brazil
5
Undergraduate Program in Natural Sciences, Center for Science and
Technology, State University of Ceará, Fortaleza, Brazil
6
Laboratory of Bioprospecting of Natural Products and
Biotechnology (LBPNB), Ceará State University, Tauá, Ceará, Brazil
7
Department of Chemistry, Ceará State University, Limoeiro do
Norte, Ceará, Brazil

Neurotox Res
Oxidative stress caused by free radicals has also been
shown to be a major contributor to the development of AD
(Yar et al. 2015). Oxidative stress, an increased process in the
aging brain, is induced by an imbalance in the redox state,
involving the generation of excess reactive oxygen species
(ROS) or dysfunction of the antioxidant system.
The zebrafish (Danio rerio) has long been used as an ani-
mal model for biomedical research, particularly in genetic and
developmental studies. It is physiologically homologous to
humans, allowing researchers to probe the pathways and
mechanisms relevant to human pathogenesis and clinical
treatments (Egan et al. 2009). Zebrafish is fast becoming a
promising model organism for research into anxiety and stress
(Collier et al. 2017). Indeed, this species is demonstrating the
potential to be an “exceptional” animal to investigate experi-
mental, genetic, and pharmacological models of neurobehav-
ioral disorders, such as depression (Pittman and Piato 2017).
Inhibition of AChE has proven to be the most viable ther-
apeutic treatment for the symptomatic improvement of
Alzheimer’s disease (Geldmacher 2007), and flavonoids are
promising natural compounds for the development of
multipotent drugs against AD. These compounds have advan-
tages, such as their high AChE inhibitory and antioxidant
activities and low toxicity (Uriarte-Pueyo and Calvo 2011).
In the brains of patients with Alzheimer’s disease, the
levels of metals like iron and copper decrease in the affected
areas (Magaki et al. 2007; Cassetta et al. 2012), leading to the
hypothesis that deficiency of these elements contributes to the
pathogenesis. Brocardo et al. (2005) showed zinc’s inhibitory
effect on AChE activity of the cerebral cortex and hippocam-
pus of rats using a quercetin-Zn complex, with increased in-
hibition of AChE.
Fig. 1 Representation of the quercetin structure, showing the chelating
sites
enhance mitochondrial biogenesis in dopaminergic cells (Ay
et al. 2017), while prophylactically protecting neuronal cells
of neurotoxic action induced by chlorpyrifos, a powerful crys-
talline insecticide of the organophosphate class (Kaur et al.
2019). Our results suggest that quercetin is a promising neu-
roprotective agent, especially when combined with metal co-
ordination, as shown below.
This article describes efforts to obtain a useful product for
the treatment of Alzheimer’s disease through the synthesis of
quercetin complexes with five metals (Fe2+, Ni2+, Co2+, Zn2+,
and Cu2+) and compares their antioxidant, anxiolytic, and ace-
tylcholinesterase inhibition actions and toxicity in zebrafish.
Materials and Methods
Typically, flavonoid-metal complexes are more active com-
pared with free binders, since flavonoids play an important
role in the bioavailability of metal ions, which are present at
low levels in the human body (Park et al. 2018).
All reagents and solvents employed in the syntheses and anal-
yses were purchased from commercial sources and used with-
out prior purification.
Quercetin is known as a versatile molecule because of its
wide clinical effects, such as carcinogenic inhibition, decrease
of cardiovascular disease, antiobesity effect, reduced risk of
cataracts, and protective effects against neurodegenerative dis-
eases (Kazemipour et al. 2018). Quercetin is one of the most
abundant flavonoids and is found in fruits, leaves, and other
plant parts. Due to the presence of chelating sites in its struc-
ture, quercetin is able to form complexes with a series of
cations (de Castilho et al. 2018).
The chelating properties of quercetin (Fig. 1) are deter-
mined by their chemical structure, with two aromatic rings
(benzoyl ring A and cinnamoyl ring B) having various hy-
droxyl groups, joined by O-heterocycle. The possible chelat-
ing sites are the 3-hydroxychromone, 5-hydroxychromone,
and 3′,4′-dihydroxyl groups (Kalinowska et al. 2016).
Studies suggest that quercetin can effectively activate sur-
vival neurotransmitters (PKD1/CREB/BDNF) as well as
Quercetin Complex Synthesis
Syntheses of the complexes were performed with the metal
salts FeSO4·7H2O, Cu(CH3COO)2, Zn(CH2COO)2, NiCl2,
and CoCl2 with quercetin (Q) as ligand in a stoichiometric
ratio of 1:2. The quercetin binder was dissolved in 10 mL of
methanol and the metal salt in 10 mL of distilled water. The
solutions were added to a 50-mL flask under stirring at room
temperature. After 20 min of reaction, 3 drops of triethylamine
were added to the solution, which immediately changed the
color of the solution. The reacting solution was stirred for 3 h.
After this period, the solution was left at low temperature
(refrigerator) for 2 days for the total precipitation of the syn-
thesized compound, which was then filtered through a porous
plate funnel and stored in a desiccator under vacuum.

Neurotox Res
Spectroscopic Measurements
Locomotor Activity of Zebrafish (Open Field Test)
and In Vivo Measuring of Acetylcholinesterase
Inhibition
Spectrophotometric analyses were performed with a
Shimadzu 1800 UV-Vis spectrophotometer. Infrared spectra
were obtained using a Nicolet iS5 spectrophotometer from
Thermo Scientific. The samples were prepared as KBr pellets
in the ratio 1:20 (m/m) (sample: KBr), and the spectra were
recorded in the range of 4000 to 400 cm −1, using 32 scans and
4 cm−1.
The open field test was performed to evaluate the possible
alteration of the motor coordination of the animals, through
sedation and/or muscle relaxation. Initially, the fish (n = 6/
group) were treated (v.o.) with a test sample (4.0, 12, or
40 mg/kg) of the vehicle (DMSO 3%, 20 μL) or diazepam
(DZP, sedative control; 2.5 mg/kg.). A group of animals with-
out treatment was included (naive). After 1 h of treatment, the
animals were placed in glass Petri dishes (10 × 15 cm) con-
taining the same water as the aquarium, marked with four
quadrants, and the locomotor activity was analyzed by
counting the number of crossing lines. Using the CL value
of the naive group as the baseline (100%), the percentage of
locomotor activity (%LA) was calculated individually for 0–
5 min.
After the zebrafish locomotor activity test, open field test,
the fish was anesthetized in cold water and euthanized by
decapitation. Shortly afterwards, the brains were dissected
and homogenized. The hydrolysis rate of 0.8 mM
acetylthiocholine was determined in a final volume of 2 mL
with 100 mM phosphate buffer pH 7.5 and 1.0 mM DTNB.
Prior to substrate addition, 10 μg of protein sample was
preincubated with the reaction medium described above for
10 min at − 18 °C. Acetylthiocholine hydrolysis was moni-
tored by formation of DTNB dianion thiolate at 412 nm for
3 min at intervals of 30 s (de Abreu et al. 2019).
Antioxidant Activity
The antioxidant activity was measured in 96-well flat bottom
plates using a BioTek ELISA reader, model ELX 800, using
the Gen5 V2.04.11 software, based on the method described
by Wang (2017). In the 96-well plates, the following solutions
were used per well: 180 μL of DPPH (2,2-diphenyl-1-
picrylhydrazyl) methanolic solution and 20 μL of the extract
sample dissolved in methanol and diluted 10 times to obtain
the final concentration of 0.2 mg.mL−1.
The percent inhibition (PI%) was calculated by comparing
the reaction rates of the samples relative to the controls. The
standard used as the positive control was the quercetin mole-
cule. The results were expressed as percent inhibition, calcu-
lated with the following formula:
AC−AS
PI% ¼
 100
AC
Sampling and Determination of Oxidative Status
AC absorbance of DPPH control at time 0
AS absorbance of sample containing DPPH after 60 min
The whole body of the animals was homogenized in pH 7.4
phosphate buffer. Then, the samples were centrifuged (3000g
at 4 °C for 10 min). The precipitate was discarded and the
supernatant was used for biochemical analysis. The oxidative
mediator profile of the animals was expressed in relation to the
protein concentration obtained in each homogenate. This con-
centration was determined by the Bradford method.
Antiacetylcholinesterase Activity
The inhibitory activity of the enzyme acetylcholinesterase
(AChE) was measured in 96-well flat bottom plates using
the same BioTek ELISA reader and software identified above,
based on the method described by Ellman et al. (2002).
The dilutions of the samples and of the positive standards
used in the quantitative microplate assays were based on
20 mg/mL standard solution to prepare the other solutions:
200, 100, 50, 25, 12.5, 6.25, 3.12, 1,56, and 0.78 μg mL−1.
The parameters referring to the natural color of the extracts
were dropped from the analysis. The percent inhibition of
acetylcholinesterase was calculated by comparing the reaction
rates (substrate hydrolysis) of the samples against the blank
(considered total AChE activity, 100%). The standard used as
a positive control was galantamine.
Catalase
Catalase activity was evaluated as previously described (Li
and Schellhorn 2007), where H2O2 consumption is measured
by reducing absorbance at 240 nm. Fifty microliters of sample
was mixed with 2.9 mL phosphate buffer solution (pH 7.4)
and 50 μL substrate (0.3 M H2O2). Absorbances at time zero
and 60 s after substrate addition were measured, and absor-
bance reduction was used to calculate the concentration of
H2O2 produced per minute. For conversion, it was considered
that 1 mmol of H2O2 produced every minute is equivalent to
1 IU/mL of enzymatic activity.

Neurotox Res
Determination of TBARS Levels
with cycles of 500 interactions, and the steepest descent algo-
rithm, having the convergence limit of 10e−7.
Thiobarbituric acid reactive substances (TBARS), represented
mainly by malondialdehyde (MDA), are formed as a
byproduct of lipid peroxidation that can be detected by the
TBARS assay using thiobarbituric acid as a reagent (Kil
et al. 2014). MDA concentration was determined as an indi-
cator of lipid peroxidation as described above using the
TBARS method.
To perform the assay, 100 μL of the homogenate was
mixed with 100 μL of 40% trichloroacetic acid and 400 μL
of 60% thiobarbituric acid. The mixture was incubated at
96 °C for 30 min and then kept in an ice bath. To stop the
reaction, 200 μL of glacial acetic acid was added to the cen-
trifuged suspension (1700g, 30 min). The obtained superna-
tant was read on a spectrophotometer (ASYS UV 340,
Biochrom, Cambridge, UK) in a 530-nm reader. TBARS con-
centrations were then calculated using a calibration curve
using MDA as standard and expressed in μg MDA/mg
protein.
The structure of acetylcholinesterase was obtained from the
Protein Data Bank (http://www.rcsb.org/pdb/home/home.do),
deposited with the code PDB 4EY6 generated from X-ray
diffraction with resolution of 2.3 Å, classified as a hydrolase
inhibitor, Homo sapiens expression system and organism,
complexed with the ligand (galantamine) GNT (Cheung
et al. 2012). The proteins for the molecular docking were
prepared by removing the residues (H2O, FUC, EDO, PE8,
NO3, NAG, and GNT), then adding the polar (H) hydrogens,
after which the structure was saved in PDBQT format. After
the preparation of the ligands and the receptor, the molecular
docking was performed using the code AutoDock Vina (Trott
and Olson 2010) considering the rigid protein and the flexible
ligands. Molecular and redocking graphs were plotted using
the UCSF Chimera package (Pettersen et al. 2004).
Statistical Analysis
The results were expressed as mean standard error of the
mean for each group of 6 animals. After confirming the nor-
mality of distribution and homogeneity of variance of the data,
the differences between the groups were submitted to analysis
of variance (one-way ANOVA), followed by the Tukey test.
All analyses were performed using GraphPad Prism v. 5.0,
with 5% statistical significance (p < 0.05).
Determination of GSH Levels
For the determination of reduced glutathione (γ-glutamil-L-
cisteinilglicin, GSH), 400 μL of cell lysate was added to
800 μL of Tris-HCl buffer (0.4 M, pH 8.9) and 20 μL of the
DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)) chromogen, also
called Ellman’s reagent (Sigma-Aldrich, St. Louis, MO,
USA). The absorbance was measured by spectrophotometry
at 412 nm (UVASYS 340, Biochrom, Cambridge, UK), using
a GSH standard as a calibration curve. The results were
expressed as nmol of GSH/mg of protein (Sharma et al. 1997).
Results and Discussion
Electronic Spectroscopy in the Ultraviolet and Visible
Region
Acute Toxicity
The electronic spectrum of quercetin in methanol has two
absorption bands related to transitions π → π*, one at
378 nm, called band I, corresponding to the conjugated system
between ring B and carbonyl of ring C (cinnamoyl system),
and another at 260 nm, band II, referring to the conjugated
system between ring A and carbonyl of ring C (benzoyl sys-
tem). It is still possible to identify another band in the region of
215 nm in the electronic spectrum in the ultraviolet region of
the molecule quercetin, attributed to the electronic transitions
of the aromatic ring (Birjees Bukhari et al. 2008). The electron
spectra of the complexes QCo, QCu, QFe, QNi, and QZn in
dimethyl sulfoxide (DMSO) presented a bathochromic shift
occurring in band I (Table 1), which can be explained by the
interaction of the metal with the 3-hydroxyl group of querce-
tin, resulting in an electronic redistribution between the flavo-
noid molecule and the metal ion, forming an extended π bind-
er system. According to Dehghan and Khoshkam (Cherrak
et al. 2016), the 3-hydroxyl group has a more acidic hydrogen,
so the 3-OH and 4-oxo groups are the first coordination sites
The acute toxicity study was carried out against adult
zebrafish (D. rerio) according to the methods proposed in
the literature (Oecd 1992; Huang et al. 2014). The animals
(n = 8/each) were treated orally with 20 μL of a test sample
(4.0, 12, or 40 mg/kg) or vehicle (control, DMSO 3%, 20 μL)
and allowed to stand to analyze the mortality rate. Ninety-six
hours after treatment, the number of dead fish in each group
was counted and the median lethal dose (LD50, the dose able
to kill 50% of the animals) was determined using the trimmed
Spearman-Karber method with confidence interval of 95%
(Arellano-Aguilar et al. 2015).
Molecular Docking
The structures of quercetin and quercetin-Fe (QFe) ligands
were plotted and optimized using Avogadro’s code (Curtis
et al. 2012), configured to use the classic MMFF94 force field
(Molecular et al. 1996) (Molecular Mechanic Force Field 94),

Neurotox Res
Table 1 Spectroscopy data in the UV-Vis region of the quercetin (Q)
ligand in methanol and the QCo, QCu, QFe, QNi, and QZn complexes in
DMSO
complexation process. The main alteration in the complexes
synthesized is relative to the band assigned to the carbonyl
group (νC=O). The absorption band is shifted to a lower fre-
quency due to a decrease in the absorption of C=O bonding
(1661 cm−1 in free quercetin), while in the complexes, the
bands appear from 1647 to 1606 cm−1. The absorbance for
C–OH ranged from 1361 to 1380 cm−1 in relation to quercetin
at 1375 cm−1. Note also a variation of the order of binding of
the band concerning the deformation of the C–OH phenol
bond (1090 cm−1 in the ligand). The bands observed in the
IV spectra in the range of 1508–1448 cm−1 in the QM com-
plex are attributed to the asymmetric stretching of the C–O–
metal group at the chelating site of the quercetin molecule, and
in accordance with (Birjees Bukhari et al. 2008), the coordi-
nation occurs with the 3-OH group.
The absorption bands related to νM–O appear in the range
of 410–590 cm−1, as also reported by Bukhari (Arellano-
Aguilar et al. 2015). The values of νM–O in the complexes
under study followed Hooke’s law, whereby the larger the
mass of the metal, the lower the vibration frequency of the
O–M bond, where M = FeII, CuII, NiII, ZnII, or CoII.
Figure 2 shows the proposed structure of the synthesized
quercetin complexes with the metals, indicating the coordina-
tion of two quercetin molecules with one metal molecule due
to the proportion used in the reaction.
Compound
Bands (nm)
Band I (nm)
Band II (nm)
Quercetin (Q)
QCo
378, 260, 215
426, 255
378
426
420
442
449
443
260
255
256
256
256
254
QCu
256, 370, 420
256, 379, 442
256, 348, 449
254, 370, 443
QFe
QNi
QZn
involved in the process of complexation and influence the
displacement of the band. The hydroxyl group located in po-
sition 5 (Fig. 1) is not involved in the coordination process due
to its lower acidity and the spatial impediment caused by the
first complexation.
After the formation of the five complexes, band II shifted
by approximately 0–5 nm, while band I was very substantially
shifted to the red region by approximately 100 nm, suggesting
the metal-QC coordination through ring C. According to stud-
ies of the coordination of quercetin with iron ion II (Cherrak
et al. 2016), a band at 438 nm in the electronic spectrum
indicates the coordination of the metal with the 3-OH group
to the metal center of Fe. In this work, the band observed in the
electronic spectrum of QFe appeared at 442 nm, so in com-
parison, it can be stated that the coordination of the iron ion II
also occurred with the 3-OH group. The data obtained in
Table 1 are in agreement with those reported by Zhou et al.
(2001) for the complexes under study. The bands of charge
transfer transitions of the metal-binder type are also in the
region observed for band I, which also corroborates the large
shift.
Antiacetylcholinesterase and Antioxidant In Vitro
Activities
The compounds QCo, QCu, QFe, and QZn presented relevant
acetylcholinesterase inhibition (AChEI) potential at the con-
centrations tested. The best anti-AChE activity observed was
for compound QFe, which showed IC50 values of 18
0.29 μg/mL against the standard substances eserine and gal-
antamine (Table 3).
Infrared Spectrum of Quercetin and Metal Complexes
In relation to the antioxidant activity, we found that
IC50Q = QFe > QCo > QNi = QZn > QCu, being closely relat-
ed to the standard redox potentials of the metals, for Fe (−
0.44), Co (− 0, 28), Ni (− 0.25), Zn (− 0.76), and Cu (0.15)
(Masterton et al. 1990).
Table 2 presents the electronic spectroscopy data in the infra-
red region of quercetin and the complexes under study, in KBr
tablets. The comparative analysis of the infrared spectra of the
quercetin molecule and the complexes under study indicated
the presence of the main bands, with some changes due to the
Copper is an active redox metal that is used to catalyze a
large number of biochemical reactions, such as the conversion
Table 2 Infrared spectroscopy data of quercetin and metal complexes in KBr pellets
Compound
ν(O–H) cm−1
ν(C=O) cm−1
ν(C=C) cm−1
ν(C–OH) cm−1
ν(C–O–C) cm−1
δ(C–OH) cm−1
νM–O
Quercetin
QFe
3250
3417
3403
3403
3421
3407
1661
1635
1610
1639
1647
1642
1602
1595
1560
1606
1599
1611
1375
1360
1380
1367
1361
1367
1239
1273
1263
1268
1268
1273
1090
1036
1093
1089
1114
1088
–
522
412
487
419
590
QCu
QNi
QZn
QCo

Neurotox Res
areas (Magaki et al. 2007; Cassetta et al. 2012), leading to
the hypothesis that this copper deficiency contributes to the
pathogenesis. Studies have shown that this metal, when coor-
dinated with flavonoids, has biological activities such as anti-
tumor (Tan et al. 2009), anti-inflammatory (Pereira et al.
2007), and anticoagulant (Pereira et al. 2007), among others.
In this work, copper presented antioxidant activity correlated
with antiacetylcholinesterase activity and statistically similar
to that of QCo.
According to Raszka et al. (2010), the exposure of snails to
nickel was maintained for 2 weeks, evidencing an increase in
AChE activity. Our results corroborate those, where the QNi
compound caused a slight improvement in AChE inhibition
activity in relation to the free ligand, whereas the antioxidant
activity shown was statistically similar to that of QZn.
Brocardo et al. (2005) reported a zinc inhibitory effect on
AChE activity of the cerebral cortex and hippocampus of rats,
and in the present study, QZn increased inhibition of AChE. It
is known that some metals when in contact with cells induce
oxidative stress leading to neurodegeneration. In previous
studies, it was found that Zn demonstrates neuroprotective
effect to DNA damage by Al-induced oxidative damage in
rat brains, corroborating the results obtained in the present
study (Singla and Dhawan 2013).
Fig. 2 Representation of chemical structure of the complexes with the
quercetin molecule and M = FeII, CuII, NiII, ZnII, or CoII
of superoxide free radical into hydrogen peroxide, facilitating
the export of iron from cells. It also plays a key role in the
electron transport chain in mitochondria, as well as other im-
portant cell functions (Ridge et al. 2008; Robinson and Winge
2010; Horn and Barrientos 2008).
Iron is a very important transition metal in biological sys-
tems. It is present in the Fe2+ and Fe3+ oxidation states and is
used in several essential reactions. The entropy of its distribu-
tion in the brain leads to several pathologies (Madsen and Gitlin
2007). Iron deficiency in patients with Alzheimer’s disease may
be associated with myelin degradation, indicating that the en-
tropy of iron distribution is an early event in Alzheimer’s and its
correlated diseases (Magaki et al. 2007) implying that medici-
nal substances with an abundance of iron may act to prevent
iron deficiency. There are reports of coordination of Fe2+ with
antioxidant activity (Souza and Giovani 2004; Sungur and Uzar
2008). In this study, it was observed that the antioxidant activity
of compound QFe and the ligand quercetin were statistically
similar. As for antiacetylcholinesterase activity, the compound
QFe showed that the coordination of iron with the quercetin
molecule markedly increased the degree of enzymatic inhibi-
tion, and this activity was correlated with the antioxidant activ-
ity, as shown in Table 3.
The same ability of copper to participate in these biological
pathways also allows it to participate in various activities re-
lated to neurodegenerative mechanisms, such as in Menkes’,
Wilson’s, and Alzheimer’s diseases (Kodama et al. 1999; Bull
et al. 1993; Deibel et al. 1996). In the brains of patients with
Alzheimer’s disease, copper levels decrease in the affected
Table 3 Data from the biological assays of inhibition of
acetylcholinesterase and the antioxidant activity of quercetin and its
derivatives
Compounds
DPPH (μg/mL)
Antiache (μg/mL)
Copper (II) sulfate
Zinc acetate
44.92 0.17f
52.89 0.46g
37.53 0.17e
59.23 0.13h
58.65 0.45h
1.05 0.55a
3.08 0.05b
5.65 0.03d
1.08 0.04a
4.86 0.02c
4.34 0.02c
np
36.65 0.02h
57.52 0.04i
57.30 0.06i
28.18 0.02g
70.80 0.59j
11.53 0.4e
5.99 0.14c
5.94 0.12c
2.18 0.29b
12.36 0.22f
8.92 0.07d
1.15 0.05a
1.07 0.30a
Cobalt chloride
Nickel chloride
Iron (II) sulfate
Quercetin (Q)
Quercetin-cobalt (QCo)
Quercetin-copper (QCu)
Quercetin-iron (QFe)
Quercetin-nickel (QNi)
Quercetin-zinc (QZn)
Eserine
In Vivo Antiacetylcholinesterase Activity Using
Zebrafish
The best anticholinesterasic compound found in vitro experi-
ments was QFe. The cholinergic system is important for the
regulation of various behavioral and cognitive processes, ace-
tylcholine is a signaling molecule of the cholinergic system
that acts on both the CNS and neuromuscular synapses
through nicotinic and muscarinic receptors (Picciotto et al.
2012). AChE cleaves extracellular acetylcholine into choline
Galantamine
np
Similar lower case letters indicate similarities between rows for each
assay (p < 0.0001, ANOVA followed by Tukey’s test)
np not performed

Neurotox Res
locomotor activity of adult zebrafish (ZF) under analgesic
drug action (Batista et al. 2017). In this context, the same
method was used with the samples (quercetin, QZn, QFe,
QNi, QCu, and QCo) to evaluate their actions on the ZF lo-
comotor system. The results are described in Figs. 4 and 5.
Free quercetin did not present an effect on zebrafish loco-
motion at any doses tested, similar to its cobalt derivative, and
only had a slight effect on QFe. In the experiments related to
the other derivatives, the effects on the locomotor activity of
zebrafish were evidenced in the different doses. QZn was
effective at the lowest dose, 4 mg/kg, compared with free
quercetin similarly to other studies (Doboszewska et al.
2014), using zinc hydro aspartate, where it was observed an
anxiolytic effect in rodents, indicating zinc is an agent for
anxiolytic therapy. QCu, among all the quercetin derivatives,
showed strong action on the locomotor system of zebrafish at
doses of 4 mg/kg and 40 mg/kg, demonstrating possible an-
xiolytic activity. There are reports in the literature with studies
with lithium metal, which demonstrate its effects on the mod-
ulation of the hippocampal antioxidant system and reduction
of oxidative stress by normalized conduct with the metal in
animals with high anxiety index. Reduced SO was followed
by changes in protein levels that modulate neurotransmitter
activity in metal-treated chronic stress animals (Popović
et al. 2019). According to Hasan et al. (2016), the action of
coordination compounds derived from naproxen had good
anxiolytic results compared with free naproxen, as well as
antinociceptive, CNS-depressant, and hypoglycemic
properties.
Fig. 3 Effect of QFe on cerebral AChE inhibition in zebrafish (Danio
rerio). Oral treatment; naive—untreated animals; vehicle—DMSO 3%
(2.5 mg/kg, v.o.). Values represent the mean standard error of the mean
(SEM) for μmol SCh/h/mg of protein produced by 6 animals/group;
ANOVA followed by Tukey (**p < 0.001; *p < 0.05 vs. naive or vehicle)
and acetate, potently regulating cholinergic neurotransmission
(Gold et al. 2011). In this study, it was observed that QFe
interacts with the cholinergic system, significantly reducing
acetylcholine-evoked neurosignalizing currents in Danio rerio
CNS cells and directly inhibiting local enzymatic activity. No
data were found in the literature of other metal-coordinated
natural compounds capable of modulating this activity as de-
scribed, which is the first prospective study in this area so far
(Fig. 3).
Activity of QFe on Zebrafish Enzymes
Evaluation of Locomotor Activity (Open Field Test)
Glutathione (GSH) plays an important role in a multitude of
cellular processes, including differentiation, proliferation, and
apoptosis, and as a result disorders in GSH homeostasis,
which are implicated in the etiology and/or progression of
The open field test in Petri plates, proposed by Ahmad and
Ahmad and Richardson (2013), is employed to evaluate
Fig. 4 Effect of quercetin on the locomotor activity of adult zebrafish
(Danio rerio) in the open field test (0–5 min). Oral treatment; naive—
untreated animals; vehicle—3% DMSO; DZP, diazepam (2.5 mg/kg,
v.o.). Values represent the mean standard error of the mean (SEM) for
6 animals/group; ANOVA followed by Tukey test. Quercetin
(***p < 0.001 vs. naive or vehicle; ###p < 0.001 vs. DZP). QZn
(**p < 0.01; ***p < 0.001 vs. naive or vehicle; ###p < 0.001 vs. DZP).
QFe (***p < 0.001 vs. naive; ###p < 0.001 vs. vehicle; @@@p < 0.001
vs. DZP). The numbers above each column indicate the percentage of
locomotor activity (%LA)

Neurotox Res
Fig. 5 Effect of quercetin derivatives on the locomotor activity of
zebrafish (Danio rerio) adult in the open field test (0–5 min). Oral
treatment; naive—untreated animals; vehicle—DMSO 3%; DZP, diaze-
pam (2.5 mg/kg, v.o.). Values represent the mean standard error of the
mean (SEM) for 6 animals/group; ANOVA followed by Tukey. Q-Ni
(***p < 0.001; *p < 0.05 vs. naive or vehicle; ###p < 0.001 vs. DZP).
QCu (***p < 0.001 vs. naive or vehicle; ###p < 0.001 vs. DZP). QCo
(***p < 0.001; *p < 0.05 vs. naive and vehicle; ###p < 0.001 vs. DZP).
The numbers above each column indicate the percentage of locomotor
activity (%LA)
various human diseases, including cancer, and aging, cystic
fibrosis, and cardiovascular, inflammatory, immune, metabol-
ic, and neurodegenerative diseases (Giustarini et al. 2011).
Aging and many diseases have been linked to oxidative
stress, DNA, and protein damage that result in several negative
effects on cells. As a marker of oxidative stress, lipid peroxida-
tion can be correctly assessed by the malondialdehyde (MDA)
assay (Kil et al. 2014). GSH deficiency or decreased GSH/
glutathione disulfide (GSSG) manifests largely through in-
creased oxidative stress, and the damage is believed to be in-
volved in neurodegenerative disorders such Parkinson’s disease
and Alzheimer’s disease (Ballatori et al. 2009).
active center of catalase, it interacts with two amino acids in
the enzyme polypeptide chain: a histidine and an asparagine
(Li and Schellhorn 2007) (Fig. 6).
Toxicity
The data obtained in the present study revealed that the quer-
cetin molecule and its derivatives did not present ZF toxicity
until 96 h of analysis. However, QZn (4.0 or 12 or 40 mg/kg,
v.o.), QFe (4 mg/kg, v.o.), QNi (4.0 or 12 or 40 mg/kg), QCu
(4.0 or 40 mg/kg), and Qo (12 mg/kg) caused a possible sed-
ative effect, as they decreased ZF locomotor activity.
Central nervous system dysfunction was observed in all
diseases related to errors in MDA and GSH metabolism, sug-
gesting that the brain is particularly susceptible to changes in
these compounds. When stress-induced animals were main-
tained with GSH and MDA levels after treatment, it was pos-
sible to demonstrate the importance for maintaining homeo-
stasis of these compounds. The increase in catalase activity, an
enzyme acting directly on the maintenance of oxidative levels
with the reduction of hydrogen peroxide due to cellular me-
tabolism, was reinforced by its affinity with the iron ions co-
ordinated in the quercetin molecule. When H2O2 enters the
Molecular Docking
After the molecular docking, the following RMSDs (root
mean square deviations) of atomic positions were generated
between the galantamine (redocking), quercetin, QFe, and
protein, with RMSD value of 1932 Å, with affinity energy
of − 7.9 kcal mol−1 and 2 active galantamine twists (GNT);
RMSD of 1824 Å, with affinity energy of − 7.3 kcal mol−1 and
6 kinks active in the quercetin docking; and RMSD of
Fig. 6 Effect of QFe antioxidant activity on zebrafish (Danio rerio). Oral
treatment; naive—untreated animals; vehicle—DMSO 3% (2.5 mg/kg,
v.o.). Values represent the mean standard error of the mean (SEM) for 6
animals/group; ANOVA followed by Tukey (*p < 0.001 vs. naive or
vehicle)

Neurotox Res
Fig. 7 Three-dimensional visualization of acetylcholinesterase complexed with galantamine (a), quercetin (b), QFe (c), and galantamine, quercetin, and
QFe (d)
0.973 Å, with affinity of − 8.8 kcal mol−1 and 10 active twists
in the docking of QFe (Fig. 7).
Conclusion
Figure 7 also shows that after docking simulations, the com-
plex QFe was in the same place as galantamine in relation to the
quercetin molecule, in addition to presenting better affinity value
(8.8 kcal mol−1). Regarding the docking with the active site of
acetylcholinesterase (Cheung et al. 2012), the QFe complex
showed better interaction with respect to the free quercetin co-
ordination. Table 4 shows a smaller distance between them, thus
a better interaction with the residues of amino acids involved in
the active site, explaining the difference in biological activity
between the quercetin molecule and the QFe complex.
The QFe complex presented the best antioxidant and
antiacetylcholinesterase actions, and these results were con-
firmed by molecular docking. In the toxicity and locomotor
evaluation, the quercetin molecules and the synthesized com-
plexes, mainly QCu and QZn derivatives, showed the highest
degree of inhibition of the fish’s locomotor activity, suggest-
ing a possible anxiolytic action. Thus, Fe+2, Cu+2, and Zn+2
are suitable metals for complexation of quercetin to produce
potential agents against Alzheimer’s disease.
Funding Information We thank the governmental agencies Funcap and
CNPq for the financial support of the research project, Ref. Proc. No.
3781047/2017, Program for SUS-PPSUS-Funcap-Decit/ACTIE/MS-
CNPq.
Table 4 Distance between the binders and active site of
acetylcholinesterase
Ligand/residue
Docking
Redocking
References
Quercetin
Quercetin-Fe
Galantamine
Ahmad F, Richardson MK (2013) Exploratory behaviour in the open field
test adapted for larval zebrafish: impact of environmental complex-
ity. Behav Process 92:88–98. https://doi.org/10.1016/j.beproc.2012.
10.014
Arellano-Aguilar O, Solis-Angeles S, Serrano-García L et al (2015) Use
of the zebrafish embryo toxicity test for risk assessment purpose:
case study. J Fisheriessciencescom 9:52–62. https://doi.org/10.1016/
j.jmb.2017.03.014
Ay M, Luo J, Langley M, Jin H, Anantharam V, Kanthasamy A,
Kanthasamy AG (2017) Molecular mechanisms underlying protec-
tive effects of quercetin against mitochondrial dysfunction and pro-
gressive dopaminergic neurodegeneration in cell culture and
Y337
F295
F297
H447
W86
G120
G121
G122
S203
13.6 Å
14.1 Å
19.6 Å
12.6 Å
17.3 Å
20.2 Å
21.3 Å
20.8 Å
15.9 Å
5.2 Å
9.8 Å
6.8 Å
11.6 Å
8.5 Å
11.9 Å
8.8 Å
10.2 Å
12.6 Å
3.3 Å
6.7 Å
5.0 Å
3.8 Å
3.3 Å
3.2 Å
3.3 Å
3.6 Å
4.5 Å

Neurotox Res
MitoPark transgenic mouse models of Parkinson’s disease. J
Neurochem 141:766–782. https://doi.org/10.1111/jnc.14033
Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL
(2009) Glutathione dysregulation and the etiology and progression
of human diseases. Biol Chem 390:191–214. https://doi.org/10.
1515/BC.2009.033
Ageing Dev 132:141–148. https://doi.org/10.1016/j.mad.2011.02.
001
Gold PE, Countryman RA, Dukala D, Chang Q (2011) Acetylcholine
release in the hippocampus and prelimbic cortex during acquisition
of a socially transmitted food preference. Neurobiol Learn Mem 96:
498–503. https://doi.org/10.1016/j.nlm.2011.08.004
Batista FLA, Campos AR, de Sousa CÁPB et al (2017) Adult zebrafish (
Danio rerio ): an alternative behavioral model of formalin-induced
nociception. Zebrafish 14:422–429. https://doi.org/10.1089/zeb.
2017.1436
Birjees Bukhari S, Memon S, Mahroof Tahir M, Bhanger MI (2008)
Synthesis, characterization and investigation of antioxidant activity
of cobalt-quercetin complex. J Mol Struct 892:39–46. https://doi.
org/10.1016/j.molstruc.2008.04.050
Hasan MS, Das N, Al Mahmud Z, Abdur Rahman SM (2016)
Pharmacological evaluation of naproxen metal complexes on
antinociceptive, anxiolytic, CNS depressant, and hypoglycemic
properties. Adv Pharmacol Sci 2016:1–7. https://doi.org/10.1155/
2016/3040724
Horn D, Barrientos A (2008) Mitochondrial copper metabolism and de-
livery to cytochrome C oxidase. IUBMB Life 60:421–429. https://
doi.org/10.1002/iub.50
Brocardo PS, Pandolfo P, Takahashi RN, Rodrigues AL, Dafre AL (2005)
Antioxidant defenses and lipid peroxidation in the cerebral cortex
and hippocampus following acute exposure to malathion and/or zinc
chloride. Toxicology 207:283–291. https://doi.org/10.1016/j.tox.
2004.09.012
Bull PC, Thomas GR, Rommens JM, et al (1993) The Wilson disease
gene is a putative copper transporting P-type ATPase similar to the
Menkes gene. naturegenetics 5(4):327–337
Cassetta E, Rossi L, Dal Forno G et al (2012) Elevation of serum copper
levels in Alzheimer’s disease. Neurology 60:1559–1559. https://doi.
org/10.1212/wnl.60.9.1559
Cherrak SA, Mokhtari-Soulimane N, Berroukeche F et al (2016) In vitro
antioxidant versus metal ion chelating properties of flavonoids: a
structure-activity investigation. PLoS ONE 11:1–21. https://doi.
org/10.1371/journal.pone.0165575
Cheung J, Rudolph MJ, Burshteyn F, Cassidy MS, Gary EN, Love J,
Franklin MC, Height JJ (2012) Structures of human acetylcholines-
terase in complex with pharmacologically important ligands. J Med
Chem 55:10282–10286. https://doi.org/10.1021/jm300871x
Collier AD, Kalueff AV, Echevarria DJ (2017) Zebrafish models of
anxiety-like behaviors. https://doi.org/10.1007/978-3-319-33774-6
Curtis DE, Vandermeersch T, Hutchison GR et al (2012) Avogadro: an
advanced semantic chemical editor, visualization, and analysis plat-
form. J Cheminformatics 4. https://doi.org/10.1186/1758-2946-4-17
de Abreu MS, Giacomini ACVV, dos Santos BE et al (2019) Effects of
lidocaine on adult zebrafish behavior and brain acetylcholinesterase
following peripheral and systemic administration. Neurosci Lett
692:181–186. https://doi.org/10.1016/j.neulet.2018.11.004
de Castilho TS, Matias TB, Nicolini KP, Nicolini J (2018) Study of
interaction between metal ions and quercetin. Food Sci Human
Wellness 7:215–219. https://doi.org/10.1016/j.fshw.2018.08.001
Deibel MA, Ehmann WD, Markesbery WR (1996) Copper, iron, and zinc
imbalances in severely degenerated brain regions in Alzheimer’s
disease: Possible relation to oxidative stress. Journal of the
Neurological Sciences 143:137–142. https://doi.org/10.1016/
S0022-510X(96)00203-1
Huang Y, Zhang J, Han X, Huang T (2014) The use of zebrafish (Danio
rerio) behavioral responses in identifying sublethal exposures to
deltamethrin. Int J Environ Res Public Health 11:3650–3660.
https://doi.org/10.3390/ijerph110403650
Kalinowska M, Świderski G, Matejczyk M, Lewandowski W (2016)
Spectroscopic, thermogravimetric and biological studies of Na(I),
Ni(II) and Zn(II) complexes of quercetin. J Therm Anal Calorim
126:141–148. https://doi.org/10.1007/s10973-016-5362-5
Kaur S, Singla N, Dhawan DK (2019) Neuro-protective potential of
quercetin during chlorpyrifos induced neurotoxicity in rats. Drug
Chem Toxicol 42:220–230. https://doi.org/10.1080/01480545.
2019.1569022
Kazemipour N, Nazifi S, Poor MHH, Esmailnezhad Z, Najafabadi RE,
Esmaeili A (2018) Hepatotoxicity and nephrotoxicity of quercetin,
iron oxide nanoparticles, and quercetin conjugated with nanoparti-
cles in rats. Comp Clin Pathol 27:1621–1628. https://doi.org/10.
1007/s00580-018-2783-5
Kil HN, Eom SY, Park JD, Kawamoto T, Kim YD, Kim H (2014) A rapid
method for estimating the levels of urinary thiobarbituric acid reac-
tive substances for environmental epidemiologic survey.
Toxicological Research 30:7–11. https://doi.org/10.5487/TR.2014.
30.1.007
Kodama H, Murata Y, Kobayashi M (1999) Clinical manifestations and
treatment of Menkes disease and its variants. Pediatrics International
41:423–429. https://doi.org/10.1046/j.1442-200x.1999.01095.x
Li Y, Schellhorn HE (2007) Rapid Kinetic Microassay for Catalase
Activity. Journal of Biomolecular Techniques 18:162–172
Madsen E, Gitlin JD (2007) Copper and iron disorders of the brain. Annu
Rev Neurosci 30:317–337. https://doi.org/10.1146/annurev.neuro.
30.051606.094232
Magaki S, Raghavan R, Mueller C, Oberg KC, Vinters HV, Kirsch WM
(2007) Iron, copper, and iron regulatory protein 2 in Alzheimer’s
disease and related dementias. Neurosci Lett 418:72–76. https://doi.
org/10.1016/j.neulet.2007.02.077
Masterton WL, Slowinski EJ, Stanitski CL (1990) Princípios de Química,
6th edn. Guanabara Koogan, Rio de Janeiro
Doboszewska U, Jastrzębska-Więsek M, Sławińska A et al (2014)
Anxiolytic-like activity of zinc in rodent tests. Pharmacol Rep 63:
1050–1055. https://doi.org/10.1016/s1734-1140(11)70621-1
Egan RJ, Cachat JM, Elkhayat S, Bartels B (2009) NIH public access.
https://doi.org/10.1016/j.bbr.2009.06.022
Mehta M, Adem A, Sabbagh M (2012) Acetylcholinesterase inhibitors
for Alzheimer’s disease. J Alzheimers Dis 3:483–494. https://doi.
org/10.2217/1745509X.3.4.483
Molecular M, Field F, Halgren TA (1996) Merck molecular force field.
17:490–519
Ellman GL, Courtney KD, Andres V, Featherstone RM (2002) A new and
rapid colorimetric determination of acetylcholinesterase activity.
Biochem Pharmacol 7:88–95. https://doi.org/10.1016/0006-
2952(61)90145-9
Müller-Thomsen T, Arlt S, Mann U, Mass R, Ganzer S (2005) Detecting
depression in Alzheimer’s disease: evaluation of four different
scales. Arch Clin Neuropsychol 20:271–276. https://doi.org/10.
1016/j.acn.2004.03.010
Geldmacher DS (2007) Acetylcholinesterase inhibitors for Alzheimer’s
disease. Aging Health 3:483–494. https://doi.org/10.2217/
1745509X.3.4.483
Giustarini D, Dalle-Donne I, Milzani A, Rossi R (2011) Low molecular
mass thiols, disulfides and protein mixed disulfides in rat tissues:
influence of sample manipulation, oxidative stress and ageing. Mech
OECD (1992) OECD 203 fish, acute toxicity test. Guideline for the test-
ing of chemicals 203:1–9
Park HR, Kim BG, Kim SJ et al (2018) Spectroscopic properties of the
quercetin–divalent metal complexes in hydro-organic mixed sol-
vent. Bull Kor Chem Soc 39:951–959. https://doi.org/10.1002/
bkcs.11532

Neurotox Res
Paul R, Borah A (2017) Global loss of acetylcholinesterase activity with
mitochondrial complexes inhibition and inflammation in brain of
hypercholesterolemic mice. Sci Rep 7:1–13. https://doi.org/10.
1038/s41598-017-17911-z
Sharma S, Nemecz SK, Zhu S, Steele VE (1997) Identification of che-
mopreventive agents by screening for induction of glutathione-S-
transferase as a biomarker. Methods Cell Sci 19:49–52. https://doi.
org/10.1023/A:1009750705969
Pereira RMS, Andrades NED, Paulino N, Sawaya AC, Eberlin MN,
Marcucci MC, Favero GM, Novak EM, Bydlowski SP (2007)
Synthesis and characterization of a metal complex containing
naringin and Cu, and its antioxidant, antimicrobial,
antiinflammatory and tumor cell cytotoxicity. Molecules 12:1352–
1366. https://doi.org/10.3390/12071352
Singla N, Dhawan DK (2013) Zinc, a neuroprotective agent against
aluminum-induced oxidative DNA injury. Mol Neurobiol 48:1–12.
https://doi.org/10.1007/s12035-013-8417-7
Souza RFV de, Giovani WF de (2004) Antioxidant properties of com-
plexes of flavonoids with metal ions. Redox Report 9:97–104.
https://doi.org/10.1179/135100004225003897
Pettersen EF, Goddard TD, Huang CC et al (2004) UCSF chimera — a
visualization system for exploratory research and analysis. https://
doi.org/10.1002/jcc.20084
Picciotto MR, Higley MJ, Mineur YS (2012) Acetylcholine as a
neuromodulator: cholinergic signaling shapes nervous system func-
tion and behavior. Neuron 76:116–129. https://doi.org/10.1016/j.
neuron.2012.08.036
Pittman J, Piato A (2017) Developing Zebrafish depression-related
models:33–44. https://doi.org/10.1007/978-3-319-33774-6
Popović N, Stojiljković V, Pejić S et al (2019) Modulation of hippocam-
pal antioxidant defense system in chronically stressed rats by lithi-
um. Oxidative Med Cell Longev:2019. https://doi.org/10.1155/
2019/8745376
Raszka AZ, Ligaszewski M, Dolezych S et al (2010) Effects of nickel
exposure and acute pesticide intoxication on acetylcholinesterase,
catalase and glutathione S-transferase activity and glucose absorp-
tion in the digestive tract of Helix aspersa (Pulmonata, Helicidae).
Int J Environ Pollut 40:380. https://doi.org/10.1504/ijep.2010.
031757
Ridge PG, Zhang Y, Gladyshev VN (2008) Comparative genomic anal-
yses of copper transporters and cuproproteomes reveal evolutionary
dynamics of copper utilization and its link to oxygen. PLoS ONE 3.
https://doi.org/10.1371/journal.pone.0001378
Robinson NJ, Winge DR (2010) Copper metallochaperones. Annu Rev
Biochem 79:537–562. https://doi.org/10.1146/annurev-biochem-
030409-143539
Santabarbara J, Villagrasa B, López-Antón R et al (2019) Clinically rel-
evant anxiety and risk of Alzheimer’s disease in an elderly commu-
nity sample: 4.5 years of follow-up. J Affect Disord:16–20. https://
doi.org/10.1016/j.jad.2019.02.050
Sungur Ş, Uzar A (2008) Investigation of complexes tannic acid and
myricetin with Fe(III). Spectrochimica Acta - Part A: Molecular
and Biomolecular Spectroscopy 69:225–229. https://doi.org/10.
1016/j.saa.2007.03.038
Tan J, Wang B, Zhu L (2009) DNA binding and oxidative DNA damage
induced by a quercetin copper(II) complex: potential mechanism of
its antitumor properties. J Biol Inorg Chem 14:727–739. https://doi.
org/10.1007/s00775-009-0486-8
Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and
accuracy of docking with a new scoring function, efficient optimi-
zation, and multithreading. J Comput Chem 31:455–461. https://doi.
org/10.1002/jcc
Uriarte-Pueyo I, Calvo MI (2011) Flavonoids as acetylcholinesterase in-
hibitors. Curr Med Chem 18:5289–5302. https://doi.org/10.2174/
092986711798184325
Wang L, Cao C, Bai W, Ma S (2017) Extraction , solation and structure
identification of the antioxidant chemicals of the fruits of Cudrania
Tricuspidata. International Journal of Science 4:72–76
Yar M, Arshad M, Farooq A et al (2015) Synthesis and DPPH scavenging
assay of reserpine analogues, computational studies and in silico
docking studies in AChE and BChE responsible for alzheimer’s
disease. Braz J Pharm Sci 51:53–61. https://doi.org/10.1590/
S1984-82502015000100006
Zhou J, Wang L, Wang J, Tang N (2001) Antioxidative and anti-tumour
activities of solid quercetin metal(II) complexes. Transit Met Chem
26:57–63. https://doi.org/10.1023/A:1007152927167
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.