Exploring the oxidation and iron binding profile of a cyclodextrin encapsulated quercetin complex unveiled a controlled complex dissociation through a chemical stimulus

Article abstract of DOI:10.1016/j.bbagen.2018.06.006

Background: Flavonoids possess a rich polypharmacological profile and their biological role is linked to their oxidation state protecting DNA from oxidative stress damage. However, their bioavailability is hampered due to their poor aqueous solubility. This can be surpassed through encapsulation to supramolecular carriers as cyclodextrin (CD). A quercetin- 2HP-β-CD complex has been formerly reported by us. However, once the flavonoid is in its 2HP-β-CD encapsulated state its oxidation potential, its decomplexation mechanism, its potential to protect DNA damage from oxidative stress remained elusive. To unveil this, an array of biophysical techniques was used. Methods: The quercetin-2HP-β-CD complex was evaluated through solubility and dissolution experiments, electrochemical and spectroelectrochemical studies (Cyclic Voltammetry), UV–Vis spectroscopy, HPLC-ESI-MS/MS and HPLC-DAD, fluorescence spectroscopy, NMR Spectroscopy, theoretical calculations (density functional theory (DFT)) and biological evaluation of the protection offered against H₂O₂-induced DNA damage. Results: Encapsulation of quercetin inside the supramolecule's cavity enhanced its solubility and retained its oxidation profile. Although the protective ability of the quercetin-2HP-β-CD complex against H₂O₂ was diminished, iron serves as a chemical stimulus to dissociate the complex and release quercetin. Conclusions: We found that in a quercetin-2HP-β-CD inclusion complex quercetin retains its oxidation profile similarly to its native state, while iron can operate as a chemical stimulus to release quercetin from its host cavity. General significance: The oxidation profile of a natural product once it is encapsulated in a supramolecular carrier was unveiled as also it was discovered that decomplexation can be triggered by a chemical stimilus.

Full text of DOI:10.1016/j.bbagen.2018.06.006

Accepted Manuscript
Exploring the oxidation and iron binding profile of a cyclodextrin
encapsulated quercetin complex unveiled a controlled complex
dissociation through a chemical stimulus
Dimitrios A. Diamantis, Sarka Ramesova, Christos M.
Chatzigiannis, Ilaria Degano, Paraskevi S. Gerogianni,
Constantina Karadima, Sonia Perikleous, Dimitrios Rekkas,
Ioannis P. Gerothanassis, Dimitrios Galaris, Thomas
Mavromoustakos, Georgia Valsami, Romana Sokolova, Andreas
G. Tzakos
PII:
DOI:
Reference:
S0304-4165(18)30168-5
doi:10.1016/j.bbagen.2018.06.006
BBAGEN 29136
To appear in:
BBA - General Subjects
Received date:
Revised date:
Accepted date:
25 April 2018
4 June 2018
6 June 2018
Please cite this article as: Dimitrios A. Diamantis, Sarka Ramesova, Christos M.
Chatzigiannis, Ilaria Degano, Paraskevi S. Gerogianni, Constantina Karadima, Sonia
Perikleous, Dimitrios Rekkas, Ioannis P. Gerothanassis, Dimitrios Galaris, Thomas
Mavromoustakos, Georgia Valsami, Romana Sokolova, Andreas G. Tzakos , Exploring
the oxidation and iron binding profile of a cyclodextrin encapsulated quercetin complex
unveiled a controlled complex dissociation through a chemical stimulus. Bbagen (2018),
doi:10.1016/j.bbagen.2018.06.006
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ACCEPTED MANUSCRIPT
Exploring the oxidation and iron binding profile of a cyclodextrin encapsu-
lated quercetin complex unveiled a controlled complex dissociationthrough
a chemical stimulus
Dimitrios A. Diamantis1, Sarka Ramesova2, Christos M. Chatzigiannis1, Ilaria Degano3, Par-
askevi S. Gerogianni4, Constantina Karadima6, Sonia Perikleous6, Dimitrios Rekkas6, Ioannis
P. Gerothanassis1, Dimitrios Galaris4, Thomas Mavromoustakos5, Georgia Valsami6, Romana
Sokolova2*, Andreas G. Tzakos1*
1Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of
Ioannina, Ioannina 45110, Greece, agtzakos@gmail.com
2J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova
3, 18223 Prague, Czech Republic, romana.sokolova@jh-inst.cas.cz
3Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13,
56124 Pisa, Italy
4Laboratory of Biological Chemistry, University of Ioannina, School of Health Sciences,
Faculty of Medicine, 451 10 Ioannina, Greece
5Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopo-
lis Zografou 15771, Greece
6Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopo-
lis Zografou 15771, Greece

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Abstract
Background
Flavonoids possess a rich polypharmacological profile and their biological role is linked to
their oxidation state protecting DNA from oxidative stress damage. However, their bioavaila-
bility is hampered due to their poor aqueous solubility. This can be surpassed through encap-
sulation to supramolecular carriers as cyclodextrin (CD). A quercetin- 2HP-β-CD complex
has been formerly reported by us. However, once the flavonoid is in its 2HP-β-CD encapsu-
lated state its oxidation potential, its decomplexation mechanism, its potential to protect DNA
damage from oxidative stress remained elusive. To unveil this, an array of biophysical tech-
niques was used.
Methods
The quercetin-2HP-β-CD complex was evaluated through solubility and dissolution experi-
ments, electrochemical and spectroelectrochemical studies (Cyclic Voltammetry) UV-Vis
spectroscopy, HPLC-ESI-MS/MS and HPLC-DAD, fluorescence spectroscopy, NMR Spec-
troscopy, theoretical calculations (density functional theory (DFT)) and biological evaluation
of the protection offered against H2O2 -induced DNA damage.
Results
Encapsulation of quercetin inside the supramolecule’s cavity enhanced its solubility and oxi-
dation profile is retained in its encapsulated state. Although the protective ability of the quer-
cetin-2HP-β-CD complex against H2O2 was diminished, iron serves as a chemical stimulus to
dissociate the complex and release quercetin.
Conclusions
We found that in a quercetin-2HP-β-CD inclusion complex quercetin retains its oxidation
profile similarly to its native state, while iron can operate as a chemical stimulus to release
quercetin from its host cavity.
General Significance
The oxidation profile of a natural product once it is encapsulated in a supramolecular cy-
clodextrin carrier as also it was discovered that decomplexation can be triggered by a chemi-
cal stimulus.
Keywords: Antioxidants, Metal complexes, Oxidation mechanism, Spectroelectrochemistry,
quercetin, DNA protection

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Abbreviations
Que: Quercetin, 2HP-β-CD: 2-hydroxypropyl-β-cyclodextrin, NMR: Nuclear Magnetic Res-
onance, UV-Vis: Ultraviolet visible. CD: Cyclodextrin.

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1. Introduction
Natural products, also known as secondary metabolites, have served as a rich reservoir for the
generation of potential drugs and their polypharmacological activity has been extensively ex-
plored [1]. Almost half of the FDA approved drugs over the last three decades originated
from natural products [2]. Among them, flavonoids present an intriguing class of chemotypes
bearing multifunctional biological activities.
Quercetin is a widely exploited flavonoid due to its diverse benefits in human health [3-5]. Its
biological role is linked to its oxidation state protecting DNA from oxidative stress damage.
Along these lines, the electrochemical oxidation of quercetin and other flavonols was inten-
sively studied [6-10]. The first oxidation site is located at hydroxyl groups at the ring B (Fig.
1) and two-electron and two-proton oxidation is described by ECEC reaction mechanism
(Electrochemical reaction – Chemical reaction - Electrochemical reaction – Chemical
reaction). As the result of this complicated oxidation process is the formation of a
benzofuranone derivative. The phenolic hydroxyl groups of ring A are oxidized in the second
oxidation wave [7-10].
Although the diverse polypharmacological potential of quercetin, its use is limited due to its
low aqueous solubility and thus, its low bioavailability [11]. To enhance its bioavailability, it
is necessary to develop formulations with hosts like calixarenes, cyclodextrins, nanogels etc.
Cyclodextrins (CDs) are supramolecules that can host in their cavities a variety of guest-
molecules. Due to their toroidal shape, there is an internal hydrophobic side that traps the
guest molecules while their external side is hydrophilic, thus enhancing water solubility and
bioavailability [12, 13]. CDs and their water-soluble derivatives such as sulfobutylether of β-
CD (SBE-β-CD), the hydroxypropyl derivative of β-CD (HP-β-CD), and the randomly meth-
ylated β-CD (RM-β-CD) have been utilized for a wide range of applications to surmount lim-
itations such as poor solubility, bioavailability, plasma stability, metabolic inactivation etc
[14]. Among CDs derivatives, 2HP-β-CD is the most versatile excipient and it is used in for-
mulations for oral, rectal, dermal, ocular, and parenteral administration [15, 16]. Α mono-
graph for 2HP-β-CD is available in both the Ph. Eur. and the United States Pharmacopoeia
(USP), while it is also cited in the FDAs list of Inactive Pharmaceutical Ingredients. CDs and
their water-soluble derivatives are also used Another application is their use as Drug Delivery
Systems (DDS) to avoid ectopic delivery and undesirable side effects to normal tissue. Such

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complexes were developed to enhance the bioavailability and aqueous solubility of the anti-
inflammatory drug Naproxen for colon targeted delivery [17]. In another study, the complex
formation between β-CD and 2-aminodiphenylamine (ADPA) was reported to confirm the
expediency of its further application for the design of supramolecular nanovalves on the ex-
ternal surface of mesoporous silica particles [18]. Recently, a complex of silibinin with 2HP-
β-CD that led to the enhancement of the aqueous solubility and a 10 fold increase of oral bio-
availability compared to the parent compound was reported [19, 20]. Selective release of the
guest molecule from the host can be triggered through different stimulus, though this is not
trivial to be identified [14, 21-23]. Concerning quercetin several inclusion complexes have
appeared in the recent literature using different cyclodextrin derivatives (βCD, 2HP-β-CD
and SBE-CD respectively) with the highest solubility enhancement being observed for SBE-
CD [24]. More recently, a quercetin mono-6-deoxy-6aminoethylamino-β-cyclodextrin inclu-
sion complex was reported demonstrating 2.3 fold higher solubility compared to the
QUE/SBE-β-CD [24, 25]. Although intense research is devoted for CD mediated inclusion
complexes several critical points remain elusive. For instance, what are the chemical stimulus
that can trigger the decomplexation of such complexes? In the case of flavonoids that their
activity is linked to their oxidation potential it is not clear if their oxidation profile or metal
ion binding profile is altered once they are encapsulated in a supramolecular carrier. Also,
questions exist on the capability of such inclusion complexes to protect DNA from oxidative
damage. To resolve these issues an array of biophysical techniques was applied on the inclu-
sion complex of quercetin with 2HP-β-CD [26]. Herein is described an intensive exploration
of the oxidative profile of quercetin once is in the cavity of the CD host as also its metal ion
binding profile, its DNA protection behavior against oxidative stress conditions and a chemi-
cal stimulus was revealed that can lead to a controlled release of the flavonoid from the host
core. Such information could allow exploiting the untapped potential that can be offered for
natural products implicated in host-guest complexes.
Figure 1: (A) Structure of 2HP-β-CD and (B) Structure of quercetin and the 3 possible bind-
ing sites (Ι-ΙΙΙ) for metal ion chelation.

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2. Materials and methods
2.1 Complex preparation
The lyophilized complex of quercetin with 2HP-β-CD (Que-2HP-β-CD) was prepared as pre-
viously described using a freeze-drying procedure [26]. Briefly, Que-2HP-β-CD aqueous so-
lutions for freeze-drying were prepared in a molar ratio of 1:2, using the neutralization meth-
od. More specifically, 0.030 g of Que and 0.306 g of 2HP-β-CD were weighed accurately,
transferred in a 50 mL beaker and suspended with 20 mL of water. Small amounts of ammo-
nium hydroxide were then added under continuous stirring and pH monitoring until complete
dissolution and pH adjustment to a value between 9 and 10 has been obtained. The resulting
o
solution at molar ratio of 1:2 was thereafter frozen at -80 C and freeze-dried using a Kryo-
dos-50 model Telstar lyophilizer.
2.2 Solubility studies and Dissolution experiments
Solubility studies for Que and the Que-2HP-β-CD lyophilized product were performed using
a thermostated shaking bath (Unitronic orbital J.P. Selecta) with adapted thermostating unit,
while a Shimadzu UV-1700 Pharma Spec UV/VIS spectrometer was used for spectrophoto-
metric measurements. The aqueous solubilities of pure Que and Que obtained from the lyoph-
o
ilized Que-2HP-β-CD product were measured at pH 1.2, 4.5 and 6.8 at 37 C as described in
the relevant guidelines of both the European Medicines Agency (EMA) [15] and the Ameri-
can Food and Drug Administration (FDA) [27]. Specifically, an excess amount of Que or
Que-2HP-β-CD lyophilized product was added to 10 mL of either HCl solution pH 1.2 or ac-
etate buffer pH 4.5 or phosphate buffer 6.8. After equilibration for 0, 3, 6, 15, 24 and 48 h at
o
the thermostated shaking bath (37 ± 0.5 C, 50 rpm) suspensions were filtered through regen-
erated cellulose syringe filters of 0.45 μm pore size and the Que concentration was deter-
mined spectrophotometrically after dilution with mixed MeOH-aqueous medium solution
(50:50 v/v).

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Dissolution experiments for Que and Que-2HP-β-CD lyophilized product were performed us-
ing a USP dissolution apparatus II (Erweka DT6 Dissolution Tester). All experiments were
o
performed in triplicate at pH 1.2, 4.5 and 6.8, at 37 C and 100 rpm. An accurately weighed
amount of Que or an equivalent amount of the lyophilized Que-2HP-β-CD product was
placed into an empty capsule (in triplicate). The filled capsules were placed in the vessels of a
USP dissolution apparatus II (Erweka DT6 Dissolution Tester) and were kept at the bottom
with the help of metal spirals. The dissolution medium was 500 mL of either HCl pH 1.2 or
acetate buffer pH 4.5 or phosphate buffer pH 6.8 and dissolution was performed at 37 ± 0.5
oC and at 100 rpm rotation speed. Samples (2.5 mL) were withdrawn at appropriate time in-
tervals (5, 10, 15, 20, 30, 45, 60, 120 and 180 min) after the beginning of the experiment,
they were filtered through regenerated cellulose syringe filters (pore size 0.45 μm), and were
assayed spectrophotometrically for Que.
2.3 Electrochemical and spectroelectrochemical studies
In electrochemical measurements, potassium chloride, and chemicals for preparation of Brit-
ton-Robinson buffers (0.04 mol·L•1 stock solutions of H3PO4, CH3COOH, H3BO3 and 0.2
mol·L•1 NaOH, Sigma Aldrich), which were used as supporting electrolytes, were of reagent
grade. The solutions were prepared with Millipore ultrapure water. Ethanol was present in so-
lution in the ratio 40% (v/v) to increase quercetin solubility and did not affect the stability of
2HP-βCD complex.
Cyclic voltammetry was measured in a three-electrode system with Ag|AgCl|1M LiCl
reference electrode separated from the examined solution by a salt bridge, glassy carbon
working electrode (diameter 0.7 mm) and platinum net as the auxiliary electrode using a
PGSTAT 12 AUTOLAB potentiostat (Ecochemie, Utrecht, NL). The spectroelectrochemical
measurements were performed in an optically transparent thin-layer electrode (OTTLE) cell
[28] with a three-electrode system (Ag/AgCl reference electrode) mounted in a thin layer
(thickness 0.18 mm) between optical windows (CaF2). A sufficiently optically transparent
platinum gauze (5×5 mm size, 80 mesh) was used as the working electrode. The scan rate
was 5 mV·s-1. Absorption spectra during the electrolysis were registered using an Agilent
8453 diode-array UV-Vis spectrometer. The oxidation products of 2HP-βCD complex

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prepared by the exhaustive oxidative electrolysis at a carbon paste electrode were identified
by HPLC-ESI-MS/MS and HPLC-DAD.
HPLC-DAD analyses were performed on a Jasco International Co., Japan system composed
by a PU-2089 Quaternary Gradient Pump with degasser, with an AS-950 auto-sampler and a
photo-diode array detector MD-2010. Data acquisition and analysis were performed by
ChromNav® software. The separation was performed on an analytical reverse phase Agilent
TC-C18(2) column (150x4.6 mm, 5 μm particle size) connected to a precolumn C-18
(12.5x4.6 mm, 5 μm particle size). The programme was: 85% A (bidistilled water with 0.1%
trifluoroacetic acid) and 15% B (LC grade acetonitrile with 0.1% trifluoroacetic acid) for 5
min, then 25 min gradient from 85% A to 50% A, then 10 min gradient from 50% A to 30%
A, then 1 min gradient from 30% A to 0% A and then 5 min cleaning (100% B) and 10 min
re-equilibrating time (85% A). The flow rate was 1 mL·min-1. The injection volume was 20
μL. DAD spectra were acquired with 4 nm resolution in the range 200-650 nm.
HPLC-ESI-MS/MS analyses were performed on an Agilent Technologies, USA 1200 Infinity
HPLC, coupled to a Quadrupole-Time of Flight tandem mass spectrometer 6530 Infinity Q-
TOF through a Jet Stream ESI interface. The separation took place at 30 °C and was per-
formed on a Zorbax Extend C18 Rapid resolution HT column (50x2.1 mm, 1.8 μm particle
size), with a Zorbax Eclipse Plus C18 Analytical Guard Colum. The programme was: 90% A
(LC-MS grade water with 1% formic acid) and 10% B (LC-MS grade acetonitrile with 1%
formic acid) for 2 min, then to 50% A in 9 min, then linear gradient to 30% A in 3 min, then
to 10% A in 4 min, re-equilibration took 3 min. The flow rate was 0.2 mL·min-1. The injec-
tion volume was 10 μL. The operating conditions of the ESI interface were: drying gas N2
(purity >98%) at 350 °C and 10 L·min-1; sheath gas N2 (purity >98%): 375 °C and 11 L·min-
1, capillary voltage at 4.5 kV; nebulizer gas 35 psig. The fragmentor was operated at 175 V.
High resolution MS and MS/MS spectra were acquired in negative mode in the range 100-
3200 m/z and the voltage applied in the collision cell for the MS/MS experiments ranged be-
tween 20 and 50 V. Collision gas was nitrogen (purity 99.999%). The mass axis was calibrat-
ed daily using the Agilent tuning mix HP0321 prepared in acetonitrile.

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2.4 UV-Vis spectroscopy
The UV-Vis spectra of Que-2HP-β-CD complex were recorded with a Perkin Elmer Lambda
25 spectrometer (slit=1, speed 240 nm/min) at room temperature. The samples were dis-
solved in LC-MS grade H2O, pH was adjusted to 7.25 and 5.81 by the addition of aqueous so-
lution of 10 mmol·L•1 NaOH and 10 mmol·L•1 HCl and incubated under shaking at 37 ± 0.1
oC at 600 rpm using thermostated shaking bath for the proper time period. Then the samples
were centrifuged at 10.000 rpm for 5 minutes and the precipitated Que was filtered off
through regenerated cellulose syringe filters of 0.20 μm pore size. Typical titration experi-
ments were performed by sequential addition/ns of freshly prepared metal ion solutions (30
mmol·L•1 stock solution, freshly prepared in double distilled water) to the same 3 mL sample
solution (17.5 μmol·L•1) in a quartz cuvette.
2.5 Fluorescence spectroscopy
The fluorescence spectra of Que-2HP-β-CD (17.0 μM) were recorded at a JASCO FP-8000
Fluorimeter (slit=5, excitation at 370 nm). Typical titration experiments were performed by
sequential additions of freshly prepared metal ion solutions (30 mmol·L•1 stock solution,
freshly prepared in double distilled water) to the same 3 mL sample solution (17.5 μmol·L•1)
in a quartz cuvette.
2.6 NMR Spectroscopy
1H-NMR spectra of Que-2HP-β-CD complex and 2HP-β-CD were recorded in a Bruker 400
MHz Avance spectrometer using D2O and DMSO-d6 as solvent. Samples were dissolved in
500μL D2O and transferred to 5 mm NMR tubes. Topspin 3.1 was used to control the NMR
system. Typical titration experiments were performed by sequential additions of freshly pre-
pared metal ion solution in D2O (246.25 mmol·L•1 stock solution) to the same sample solu-
tion (3.8 mmol·L•1) in an NMR tube.

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2.7 Theoretical calculations
Molecular orbital calculations were performed using the density functional theory (DFT) em-
ploying B3LYP functional and 6-31G* basis set in the environment of Spartan ’14, v.1.1.0.
(Wavefunction, Inc.) for the Que-2HP-β-CD complex in vacuum.
2.8 Cell cultures
RPMI-1640 growth medium supplemented with L-glutamine and glucose oxidase (from As-
pergillus niger, 18,000 units/g) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fe-
tal bovine calf serum (FBS), Nunc tissue culture plastics, low melting-point agarose and pen-
icillin/ streptomycin antibiotics were obtained from Gibco GRL (Grand Island, NY, USA).
Microscope superfrosted glass-slides were supplied by Menzel-Glaset (Menzel, Germany).
Cell cultures Jurkat cells (ATCC, clone Ε6-1) were grown in RPMI-1640 containing 10%
heat-inactivated FBS, 2mM glutamine, 100 U/ml penicillin and 100ng/ml streptomycin, at
37°C in 95% air and 5% CO2. Jurkat cells in the log phase were harvested by centrifugation
(250g, 10min), resuspended at a density of 1.5 × 106 cells per ml and allowed to stay for 1h
under standard culturing conditions before treatments.
2.9 Evaluation of the protection offered against H2O2 -induced DNA damage
Cells were treated with each compound for the time periods indicated under otherwise stand-
ard culture conditions and then exposed for 10 min to continuously generated H2O2 formed
by the action of the enzyme “glucose oxidase” which was added directly to the growth medi-
um. The amount of the enzyme added was estimated to generate approximately 10μM of
H2O2 per min. Cells were then collected and analyzed for the formation of single strand
breaks in their DNA by comet assay.
The alkaline comet assay was performed essentially as described previously by Singh et
al.[29] with minor modifications [30]. In brief, cells were suspended in 1% (w/v) low-
melting-point agarose in PBS, pH 7.4, and pipetted onto superfrosted glass microscope slides,
precoated with a layer of 1% (w/v) normal melting-point agarose (warmed to 37°C prior to
use). The agarose was allowed to set at 4°C for 10min, and the slides were then immersed for

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1h at 4°C in a lysis solution (2.5M NaCl, 100mM EDTA, 10mM Tris, pH 10, 1% Triton X-
100) in order to dissolve cellular proteins and lipids. Slides were placed in single rows in a
30cm-wide horizontal electrophoresis tank containing 0.3M NaOH and 1mM EDTA, pH ~ 13
(unwinding solution) and kept at 4°C for 40min in order to allow DNA strand separation (al-
kaline unwinding). Electrophoresis was performed for 30min in the unwinding solution at
30V (1V/cm) and 300mA. Finally, the slides were washed for 3 × 5min in 0.4M Tris (pH 7.5,
4°C) and stained with Hoechst 33342 (10mg/ml). Hoechst-stained nucleoids were examined
under a UV-microscope with a 490nm excitation filter at a magnification of ×400. DNA
damage was not homogeneous, and visual scoring was based on the characterization of 100
randomly selected nucleoids, as previously described [30]. In brief, the comet-like DNA for-
mations were categorized into five classes (0, 1, 2, 3 and 4) representing an increasing extent
of DNA damage visualized as a ‘tail’. Each comet was assigned a value according to its class.
Accordingly, the overall score for 100 comets ranged from 0 (100% of comets in class 0) to
400 (100% of comets in class 4). In this way the overall DNA damage of the cell population
can be expressed in arbitrary units.
3. Results and discussion
3.1 Solubility and dissolution studies
For orally administered drugs and especially low aqueous soluble drugs as Que, aqueous sol-
ubility, in conjunction to their rate of dissolution, and intestinal permeability are the main pa-
rameters controlling the rate and extent of gastrointestinal absorption and consequently oral
bioavailability [31]. The aqueous solubility and dissolution characteristics of the prepared
Que-2HP-β-CD lyophilized product in comparison to that of the parent Que were studied, at
the physiological gastrointestinal pH range, recommended in the relevant FDA and EMA
guidelines [15, 27].
The results from the solubility studies for both pure Que and the Que-2HP-β-CD complex are
presented in Fig. 2A and Fig. 2B. In order to probe the amplification offered on quercetin’s
aqueous solubility in its encapsulated form with CD detailed solubility studies were conduct-
ed at pH 1.2, 4.5 and 6.8 according to the relevant FDA and EMA guidelines [15, 27]. These
pH values could be interpreted on the basis of gastrointestinal absorption after oral drug ad-

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ministration, since, under fasting conditions, the pH range in the GI tract vary from 1.4 to 2.1
in the stomach, 4.9 to 6.4 in the duodenum, 4.4 to 6.6 in the jejunum, and 6.5 to 7.4 in the ile-
um [32-34]. UV assay revealed 8% w/w content of Que in the Que-2HP-β-CD complex. The
solubility of pure Que ranged from 0.004±0.002 mg/mL at pH 1.2 and 4.5 to 0.016±0.001
mg/mL at pH 6.8. The solubility of the lyophilized Que-2HP-β-CD complex was reached
maximum at early times at all pH values studied and declined gradually to lower values at the
24th and 48th hour of the experiment probably due to either Que-2HP-β-CD complex decom-
position or initial formation of supersaturated solution and gradual transition to the stable sat-
urated form at later time points. More specifically, the measured Que solubility was found
0.19±0.07 mg/mL at pH 1.2, 0.13±0.2 mg/mL at pH 4.5 and 0.47±0.117 mg/mL at pH 6.8.
These values remained constant for 3h (pH 6.8) to 15h (pH 1.2 and 4.5) and declined to
0.07±0.04 mg/mL at pH 1.2, 0.085±0.004 mg/mL at pH 4.5 and 0.23±0.03 mg/mL at pH 6.8,
after 24h. It is obvious that the interaction of Que with 2HP-β-CD resulted in the increase of
Que’s solubility with a solubilization effect, as reflected on Que-2HP-β-CD to Que solubility
ratio, higher than 10 in all pH studied (Table 1). From the results presented in Table 1 it is ev-
ident that Que inclusion in the cavity of 2HP-β-CD resulted in 47 to 17 times increase of sol-
ubility at pH 1.2, 32 to 21 times increase of solubility at pH 4.5 and 29 to 14 times increase of
solubility at pH 6.8.
Figure 2: Effect of pH on the solubility of (A) pure Que and (B) the Que-2HP-β-CD lyophi-
lized complex and on the dissolution of (C) pure Que and (D) the Que-2HP-β-CD lyophilized
complex.
The results from the dissolution studies are shown in Fig. 2C and 2D for both the pure Que
and the Que-2HP-β-CD lyophilized product at pH 1.2, 4.5 and 6.8. The amount of pure Que
dissolved ranged from 10.3±3.8% w/w at pH 1.2 to 18.7±3.1% w/w at pH 4.5 and 31.1±1.5%
w/w at pH 6.8. These values correspond to the final concentration of Que in the dissolution
medium of 0.007, 0.004 and 0.012 mg/ml at pH 1.2, 4.5 and 6.8, respectively and are con-
sistent with the results from the solubility study for pure Que. Considering the dissolution of
the Que-2HP-β-CD lyophilized product was 3 to 6 times higher than that of pure Que at pH
ranging from 1.2 to 6.8. More specifically, Que was completely dissolved at pH 6.8

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(90.4±8.8% w/w) from its Que-2HP-β-CD lyophilized product, while 70.4±9.8% w/w and
61.1±9.2% w/w dissolution was observed at pH 4.5 and 1.2, respectively, during the three
hours of the dissolution experiments. These results confirm the solubilizing effect of 2HP-β-
CD on Que and are consistent with the observed solubility values for the Que-2HP-β-CD ly-
ophilized product (Table 1, Fig. 2).
Adopted by both FDA and EMA and described in the relevant regulations on waiver of in vi-
vo bioavailability and bioequivalence studies and investigation of bioequivalence, respective-
ly [15, 27], the Biopharmaceutics Classification System (BCS) [34] consists a scientific
framework for classifying drug substances into four classes based on their aqueous solubility
and gastrointestinal permeability, using the highest dose within the physiologically relevant
range of pH 1−6.8, as follows: Class I: High Solubility – High Permeability, Class II: Low
Solubility – High Permeability, Class III: High Solubility – Low Permeability, Class IV: Low
Solubility – Low Permeability.
Recently, Waldaman et al. [36], presented a provisional BCS classification strategy for herbal
markers according to the available information. Quercetin, one of the markers of Gingko bi-
loba, was classified in Class IV, characterized by both low solubility and permeability, in
agreement with the reported low bioavailability of Que [11]. Using the same Classification
Criteria as Waldman et al [36] the dose number (D0=Dose/(Cs*V)), which describes the rela-
tionship between solubility and maximum dose strength, for both Que and the Que-HP-β-CD
lyophilized product was calculated, for V=250 mL and the measured Cs values at pH 1.2, 4.5
and 6.8 (Table 1). Compounds with a D0 lower or equal to 1 are considered highly soluble
[27, 37] and are classified in either Class I or Class III according to their permeability. It is
obvious from the results in Table 1 that the Que-2HP-β-CD lyophilized product is classified
as highly soluble in the entire physiologically relevant range of pH 1−6.8, while for Que
D0>1 was calculated in all cases. This, in conjunction to the rapid and almost complete disso-
lution observed, points out that the Que-2HP-β-CD lyophilized product has an increased bio-
availability after oral administration.

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3.2 Stability evaluation of the Que-2HP-β-CD complex in aqueous solution through UV-
Vis
The UV-Vis spectra of most flavonoids consist two major absorption bands centered approx-
imately at 260 nm and 350 nm originating from the benzoyl and cinnamoyl bands, respective-
ly and can be assigned to HOMO-LUMO energy gaps and π-π* transitions [38].
Initially, the time-dependent oxidation profile of the Que-2HP-β-CD complex (39.5
μmol·L•1) in aqueous solution (pH=7.14) was monitored. As illustrated in Fig.3, the complex
was stable for 3 hours and considerable alterations emerged after this time. The absorbance of
the major bands at 370 nm and 257 nm gradually decreased with parallel shifting to 376 nm
and 254 nm, respectively. On the other hand, the absorbance of the minor band at 288 nm
gradually increased and red-shifted to 294 nm. The band at 294 nm is related to the decompo-
sition of the oxidation product of quercetin (i.e. a benzofuranone derivative) and the for-
mation of the main stable product, which is 3,4 dihydroxybenzoic acid, as demonstrated by
HPLC-DAD and HPLC-ESI-MS analyses [10].
Figure 3: Time-course UV-Vis absorption spectra of an aqueous solution of Que-2HP-β-CD
complex (39.5μM) at pH 7.14.
3.3 Mapping the oxidative state of quercetin in the CD encapsulated form via electro-
chemical and spectroelectrochemical studies
In order to evaluate the oxidation state of quercetin in its encapsulated form and whether this
is altered to its free form electrochemical and spectroelectrochemical studies were utilized.
This is of immense importance since such information, concerning the oxidative profile of a
flavonoid once is in a CD formulation, is currently elusive. Cyclic voltammetry of Que-2HP-
β-CD complex in solution at pH 4.3 and 7.6 showed three oxidation peaks labelled as I., II.,
and III., respectively (Fig. 4 A and B, respectively). The first two oxidation peaks at 0.36 V
and 0.17 V were quasi reversible. The oxidation properties of the complex were in accord-
ance with those of the free molecule of quercetin; a comparison of the free quercetin and its
complexed form is illustrated in Fig. 4C and D for pH 8.8. The oxidation waves of the Que-
2HP-β-CD complex are attributed to the oxidation of the hydroxyl groups located in ring B

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(labelled as I.) and occurred at all the investigated pH values at the same potential as in the
case of free quercetin. Moreover, oxidation peaks II. (corresponding to the oxidation of a
benzofuranone product formed at the potential of the first wave) and III. (assigned to the oxi-
dation of hydroxyl groups at the ring A) were present in cyclic voltammograms of both com-
plexed and free forms of quercetin. Thus, all the electroactive sites of quercetin that are
bound in the cavity are available. This fact agrees with the chemical structure of the complex,
where ring B and carbon atoms C2 and C3 and keto group C4=O are available from one side
of the cavity, and the hydroxyl groups of ring A are reachable from the other side (Fig.S1.
The position of the molecule of quercetin in the cavity was confirmed by DFT theoretical
calculations (Fig. S1). Resulting data are in the agreement with the electrochemical findings
and show that all three electroactive sites of quercetin are available even if complexed in cav-
ity. Cyclic voltammograms in Fig. 4 C and D show an additional wave Ib, attributed to the
one-electron oxidation of dianion A2- present in solution at this pH, while Ia is the oxidation
wave of the remaining anion AH-. The oxidation properties were systematically investigated
in Britton-Robinson buffers in the range of pH 4.0 – 9.8 (Fig. 5). Oxidation peaks were shift-
ed to less positive values when pH was increased. The inset in Fig. 6 shows a dependence of
peak potential on pH for oxidation waves attributed to the oxidation of the non-dissociated
form of Que-2HP-β-CD complex and its anion. The intersection point of two linear segments
yields the apparent dissociation constant pK’ = 6.7, which is in agreement with the dissocia-
tion constant reported in the literature [10]. The complex was decomposed at pH higher than
8.8 and a decrease of currents was observed. The dependence of the peak current on the
square root of the scan rate is linear and the oxidation process is thus diffusion controlled
(Fig. S2 in Supporting information. Oxidation at the first oxidation wave involved two elec-
trons and two protons, as confirmed by coulometry and from the slope of peak potential vs.
pH dependence (inset in Fig. 5).
Comparison of the oxidation behavior of free quercetin and the Que-2HP-β-CD complex
shows no differences. Quercetin retains its electrochemical properties when it is encapsulated
to the cavity of 2HP-β-CD. It means, that all electroactive sites of quercetin are available in
the cyclodextrin cavity and no significant differences were monitored between cyclic volt-
ammetries of free and complexed quercetin. The potential of peak II. of Que-2HP-β-CD
complex belonging to a product of oxidation is 54 mV lower than in case of free molecule

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(Fig. S2 in Supporting information). This implies that the oxidation product can be more
easily oxidized while protected in cavity against moving from the electrode or its
decomposition.
Figure 4: Cyclic voltammograms A) of 0.2 mM Que-2HP-β-CD complex at pH 4.3, B) of 0.2
mM Que-2HP-β-CD complex at pH 7.6, C) of 0.2 mM Que-2HP-β-CD complex at pH 8.8 and
D) of 0.4 mM quercetin at pH 8.8.
Figure 5: Selected cyclic voltammograms of 0.2 mM Que-2HP-β-CD complex at different pH
values: (a) 4.0, (b) 4.5, (c) 4.7, (d) 4.9, (e) 5.3, (f) 5.6, (g) 5.9, (h) 6.2, (i) 6.8, (j) 7.0, (k) 7.2,
(l) 8.0, (m) 8.2, (n) 8.8. Scan rate was 0.1 V·s•1. Inset: Dependence of peak potential on pH
(Pourbaix plot).
The UV-Vis spectra were recorded also using in situ spectroelectrochemistry in an optically
transparent thin-layer electrode cell. Spectroelectrochemistry at the potential of the first oxi-
dation wave showed a decrease of the bands at 274 and 393 nm and simultaneous increase of
the intensity at 333 nm (Fig. 6A). This process is characterized by a sharp isosbestic point at
365 nm. The resulting spectrum of intermediate changes as the oxidation proceeds, as pre-
sented in Fig. 6B. An increase of the absorption band at 295 nm accompanies the further de-
crease of bands at 393 and 333 nm. These findings support results obtained by cyclic volt-
ammetry mentioned above. No significant difference in oxidation mechanism was found in
the case of quercetin encapsulated in the cavity of 2HP-β-CD compared to free quercetin
[10].
Figure 6: In situ spectroelectrochemistry of 0.2 mM Que-2HP-β-CD complex in 0.1 mol·L•1
KCl (A) at the edge of the first oxidation peak and (B) at the potential behind the first oxida-
tion peak.
The oxidation products of Que-2HP-β-CD complex after electrolysis at the potential of the
first oxidation peak were also identified by HPLC-DAD and HPLC-ESI-MS/MS analysis
(Table 2). The oxidation pathway led to the formation of two species (P1 and P2), both with
molecular ion [M-H]- corresponding to the raw formula C14H8O8 indicative of the decomposi-

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tion products of the semiquinone intermediate. These products were not identified in the
study on the oxidation of free quercetin due to their short lifetime [10]. Two possible isomers
can be formed from the opening of the semiquinone structure, as shown in Scheme 1. The
UV-Vis spectrum acquired by DAD of the specie eluting later is characterised by absorption
maxima at 265 and 360 nm, consistent with the UV-Vis spectroelectrochemistry results. The
tandem mass spectra of both species are characterised by the presence of a fragment ion at
285.00, consistent with the loss of water. Moreover, 2,4,6-trihydroxybenzoic acid (P3) along
with 3,4-dihydroxybenzoic acid (P4) are present, which can be derived from the decomposi-
tion of P1 and P2. Similar open structures were identified as minor oxidation products of fla-
vonols in aqueous solutions, but they were derived from flavonol structure [8]. Compound P3
has been described as the final oxidation product of quercetin in literature [39, 40]. In this
case, the unique protection of the semiquinone intermediate by the cavity of 2HP-β-CD al-
lowed identifying its presence by direct detection, and not through the interpretation of the
oxidation pathway.
Table 2 The oxidation products of Que-2HP-β-CD complex
Scheme 1. Suggested mechanism of oxidative decomposition of quercetin via a semiquinone
intermediate.
3.4 Charting the Fe2+ and Fe3+ binding profile of the encapsulated Quercetin unveiled
chemical stimulus for a controlled dissociation of the 2HP-βCD – Quercetin complex
Although iron has been evolutionary selected as the main catalyst for biological redox reac-
tions, once in its redox-active form, it can catalyze Fenton-type reactions generating extreme-
ly reactive free radicals that are harmful for cell [41]. Along these lines, several natural prod-
ucts can tailor the intracellular labile iron levels and determine the cell fate in conditions of
elevated oxidative stress [42]. As it was recently reported, the potential of natural products to
protect cells against H2O2-induced DNA damage and apoptosis is linked to their capacity to
chelate labile iron [43]. Along these lines it was set out to determine whether quercetin was
able to retain its metal ion binding potential once it is in its encapsulated form to CD. This

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point, although of paramount importance also remained unknown to our knowledge, as it has
never been evaluated before in any other antioxidants-CD encapsulated formulations.
Quercetin has the ability to chelate numerous metal ions in different binding sites [38, 44-48].
Metal chelation can take place up to three different sites on the quercetin scaffold (Fig. 1B):
(i) on the 7-phenolic hydroxyl group - 4 keto group on rings A and C respectively, (ii) the 3-
phenolic hydroxyl group - 4 keto group on ring C and (iii) the catechol group of ring B, influ-
encing both the photochemical properties of quercetin and its biological activity. Flavonoids
pro-oxidant effects have been directly correlated with their ability to bind metals like copper
and iron and being affected by their reducing activities. Especially iron is present in various
forms among the biological systems and its reducing properties during interaction with quer-
cetin is of major significance [49].
Upon chelation with metal ions, the UV-Vis spectra of flavonoids change, displaying a bath-
ochromic shift that can be attributed either to the reduced HOMO-LUMO gaps or to Ligand
to Metal Charge Transfer (LMCT) [48, 50]. UV-Vis and fluorescence titrations of Que-2HP-
β-CD inclusion complex (17 μM) with Fe2+ and Fe3+ were performed in aqueous solution at
two different pH values (7.25 and 5.81). Three phenomena could co-exist upon iron titration:
complexation of iron to Que or complexation of iron to cyclodextrin or both. As illustrated in
Fig. 7A and 7B, it was found a decrease in the intensity of the major peak at 370 nm that de-
pends on the amount of titrated Fe2+. The appearance of a clear isosbestic point at 393 nm and
the slight increase in absorbance of the shoulder at approx. 422 nm indicates the formation of
a new complex between Que and Fe2+ (the different molar ratios can be verified by the corre-
sponding calibration curves in Fig.S4) in a similar fashion as previously described for native
quercetin by Maolin Guo [38]. Considering that the major band at 370 nm in the UV-Vis
spectrum of Que-2HP-β-CD complex comes from Que and originates from the cinnamoyl
system between B+C rings, it can be assumed that Fe2+ chelates this particular binding site
[51]. As shown in Fig. 7A and Fig. 7B the formation of the new complex is directly associat-
ed to the pH as hydrogen bonding is favored at physiological pH compared to the slightly
acidic conditions [52, 53]. At physiological pH, one hydroxyl group at ring B is deprotonated
to form anion A- (curve j in Fig. 5 and Panel A in Figure S3), while at alkaline solutions the
second OH group at ring B can be deprotonated [10], thus the formation of metal complexes
is favored at the catechol group.

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Figure 7: Absorption spectra of Que-2HP-β-CD (17 μM) in the presence of different concen-
trations of Fe2 + in H2O at (A) pH 7.25 and (B) 5.81 respectively. Fluorescence spectra of
Que 2HP-β-CD (17 μM) in the presence of different concentrations of Fe2+ in H2O at (C) pH
7.25 and (D) 5.81, upon excitation at 370 nm.
According to Simkovitch et al., the fluorescence spectrum of quercetin in aqueous solutions
is consisted of two emission bands [53]. The first band exhibits a maximum at 440-470 nm
and is assigned to the normal form of quercetin while the second band exhibits a maximum
centered at 535 nm originated from its tautomeric form. The tautomeric form of quercetin is
directly affected by pH and takes place via a proton transfer either from the 5-OH or the 3-
OH position to the carbonyl group forming a six and a five membered ring, respectively [53].
As shown in Fig. 7C and Fig. 7D, the band intensity ratio is clearly highly dependent on the
pH. At physiological pH the fluorescence intensity originated from the tautomeric form is
higher than the slightly acidic pH as the hydrogen bonding is favored [53]. In order to verify
whether the complex between Fe2+ and Que retains the fluorescent intensity of native
quercetin, further experiments were conducted. Upon titrating Fe2+, the fluorescence intensity
of Que-2HP-β-CD complex, upon excitation at 370 nm, was gradualy decreased. Concerning
the turn off fluorescence mechanism it’s possibe that the observed intensity quenching is a
result of the energy transfer from quercetin to the empty d-orbitals of Fe2+ in a similar fashion
as described for native quercetin by K. Kakavand et al. [54].
The binding affinity between Que 2HP-β-CD complex and Fe3+ was studied in a similar fash-
ion. As shown in Fig. 8A and Fig. 8B the complexation between Que and Fe3+ at high pH re-
quires less equivalents of Fe3+ as Que could exist in its deprotonated form favoring the for-
mation of a complex, as indicated by the gradual appearance of a shoulder at 422 nm and the
existence of an isosbestic point at appx. 394 nm. As the UV-Vis spectra changed in the same
manner as in the case of Fe2+ it was assumed that the complexation takes place at the same
binding site. In order to get a deeper understanding, fluorescence experiments were per-
formed, and the results illustrated similar binding as in the case of Fe2+. The gradual additions
of more equivalents of Fe3+ lead to a significant precipitation, resulting a decrease of the ab-
sorbance at 422 nm. Of interest was that the observed dark yellow precipitate (color of quer-
cetin) can potentially assigned to the decomplexation of quercetin.

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Figure 8: Absorption spectra of Que-2HP-β-CD (17 μM) in the presence of different concen-
trations of Fe3+ in H2O at (A) pH 7.25 and (B) 5.81 respectively. Fluorescence spectra of Que
2HP-β-CD (17μM) in the presence of different concentrations of Fe3+ in H2O at (C) pH 7.25
and (D) 5.81 respectively, upon excitation at 370 nm.
In order to verify whether the iron species selectively bind quercetin or interact with cy-
clodextrin, NMR titrations were conducted both for Que 2HP-β-CD complex and native 2HP-
β-CD in D2O (pH=7.17). As shown in Fig.9, Que initially forms a metal complex with the
paramagnetic Fe3+ ions, as it is clear from the extensive line width broadening and change in
the chemical shift [55]. However, after the addition of 1.0 eq of Fe3+ precipitation was clearly
observed. The mixture was centrifuged, and the precipitate was successively washed twice
1
with H2O (2*1 ml). Subsequently, the solid was redisssolved in DMSO-d6 and the H NMR
spectrum revealed that it is native quercetin. Moreover, NMR titration was performed for na-
tive 2HP-β-CD with Fe3+ and it was found that 2HP-β-CD binds Fe3+ (Fig. S6) in a similar
fashion as described by Halaszova et al. [56], so it can be assumed that two phenomena could
co-exist: iron triggers the un-loading/de-complexation of quercetin from 2HP-β-CD and this
originates upon interaction of iron both with Que and 2HP-β-CD.
1
Figure 9: Selected region of the H NMR spectra of: Α) native Que in DMSO-d6; B) Que-
2HP-β-CD complex (3.8mM) in 100% D2O; C) Que-2HP-β-CD complex (3.8mM) in D2O after
the addition of 0.25 eq. of Fe3+. D) Que-2HP-β-CD complex (3.8mM) in D2O after the addi-
tion of 0.50 eq. of Fe3+; E) Que-2HP-β-CD complex (3.8mM) in D2O after the addition of 1.0
eq. of Fe3+; F) the observed precipitate in DMSO-d6 after the addition of 1.0 eq. of Fe3+ in
Que-2HP-β-CD complex (3.8mM).
To conclude, iron species displayed similar alternation motif concerning the photophysical
properties compare to the initial spectra of Que 2HP-β-CD. More specifically, it was observed
that the UV-Vis spectra was red shifted to 422 nm while the fluorescence intensity was
quenched, in a similar fashion as described for native quercetin by Maolin Guo et al and K.
Kakavand et al. for UV-Vis and fluorescence titrations respectively, suggesting metal
complexation with iron species [38] [54]. In the case of ferric iron, the resulting metal com-
plex was insoluble in aqueous solution and precipitation could clearly observed compared to
ferrous iron.

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3.5 Evaluation of the protection offered by native quercetin and its encapsulated form
against H2O2-induced DNA damage in living cells
The degree of DNA protection offered by different concentrations of native Que as also of its
formulate with 2HP-β-CD, when added to cells for 20 min, before the exposure to H2O2, was
evaluated in Jurkat cells by using the comet assay methodology, as previously reported for
other phenolic compounds [42, 57]. As shown in Fig. 10Α, preincubation of the cells with Que
offered the highest protection at 80μM. On the contrary, the encapsulated form of Que in 2HP-
β-CD did not offer considerable degree of DNA protection (Fig. 10Β). A much lower protec-
tion is observed also at 80μM that could be potentially attributed to a dissociation of the com-
plex. Based on our previous findings that compounds bearing the ortho-dihydroxyl moiety
were able to chelate labile iron and thereby to prevent H2O2-induced DNA damage [42, 57], it
was hypothesized that the low effectiveness of Que-2HP-β-CD was emanated from its inabil-
ity to penetrate through plasma membrane due to the polar character of 2HP-β-CD. This could
suggests either a) that quercetin, once encapsulated in the CD core, is not able to penetrate the
cell membrane. A small fraction of quercetin is released in a period of time that enters the cells
via passive diffusion offering the observed protective effect, or b) a portion of the encapsulat-
ed complex of quercetin to 2HP-β-CD is uptake from the cell via fluid-phase endocytosis [58]
and then quercetin is released via the available labile iron [43]
Figure 10: Protective effects offered by quercetin and the inclusion complex against H2O2-
induced DNA damage. Jurkat cells in culture (1.5 × 106 cells/ml) were pre-incubated for 20
min with the indicated concentrations (0, 10, 20, 40, and 80 μM) of the quercetin (A) or the in-
clusion complex (B) and then exposed to an amount of glucose oxidase (0.6μg/ml) able to gen-
erate about 10 μM H2O2 per minute. After 10 min of exposure to H2O2, cells were collected
and analyzed for formation of single-strand breaks in their DNA by using the comet assay
methodology. DNA damage was expressed in arbitrary units (a.u.), as described under Mate-
rials and Methods. Each point represents the mean ± SD of duplicate measurements in two
separate experiments.
4. Conclusion

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Herein, are utilized an array of biophysical techniques to unveil the oxidation profile, the
aqueous solubility and rate of dissolution at a wide pH range indicated by the FDA and EMA
regulations, the metal ion chelation properties as also the protection of DNA damage from ox-
idative stress of a flavonoid once it is encapsulated in a supramolecular cyclodextrin carrier.
The enhanced aqueous solubility offered by the Que-2HP-β-CD complex, compared to the na-
tive compound, as well as the amplified dissolution of quercetin originated from the inclusion
complex indicate increased bioavailability of the inclusion complex after oral administration.
In the inclusion complex Que retained the same electrochemical oxidation profile of the native
compound as also the ability to chelate iron species. We also revealed that iron species can op-
erate as a chemical stimulus that can lead to a controlled release of the flavonoid from the host
core. These results could pave the way to unleash the full biological potential and health bene-
fits of a large reservoir of natural products that their bioactivity is largely depreciated due to
their limited solubility.
Conflict of interest:
The authors declare no conflict of interest

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Table 1: Measured solubility (Cs) of parent Que and Que from the Que-2HP-β-CD lyophi-
lized product at pH 1.2, 4.5 and 6.8
Que-2HP-β-CD to Que
pH
1.2
4.5
Cs (mg/ml) ± SD
0.004±0.001a
0.004±0,001b
D0=Dose/(Cs×V)g
solubility ratio
-
32.4
32.4
-
QUE
6.8
0.016±0.004c
-
8.1
1.2 0.19±0.07b – 0.07±0.04d
47.5 – 17.5
32.5 – 21.25
29.4 – 14.4
0.682 – 1.85
0.996 – 1.52
0.276 – 0.563
0.13±0.02b -
QUE-HP-β-CD
4.5
0.085±0.004
0.47±0.117e –
6.8
0.23±0.03f
a mean ± SD from all concentration measurements, b concentrations measured (mean ± SD) at 3, 6 and 15 h of
experiment, c concentration measured (mean ± SD) at 48 h of experiment, d concentration measured (mean ± SD)
at 24 and 48 h of experiment, e concentration measured (mean ± SD) at 3 h of experiment, f concentration meas-
ured (mean ± SD) at 15, 24 and 48 h of experiment; g Dose= 32.4 mg [33]

ACCEPTED MANUSCRIPT
Table 2 The oxidation products of Que-2HP-β-CD complex
Tr
[M-H]-
m/z
product
ions
Species
Assignment
Raw formula
λmax (nm)
(min)
n.d. (eluting
with the
151.004,
125.025,
107.014
P3
1.3
2,4,6-trihydroxybenzoic acid
C7H6O5
169.014
dead vol-
ume)
P4
P1
1.5
3,4-dihydroxybenzoic acid
2-[(3,4-dioxocyclohexa-1,5-
diene-1-carbonyl)oxy] -4,6-
dihydroxybenzoic acid
3,4-dioxocyclohexa-1,5-
dien-1-yl oxo(2,4,6-
C7H6O4
153.019
303.014
109.029
285.001
260, 295
n.d. (below
limit of de-
tection)
12.0
C14H8O8
P2
16.0
C14H8O8
303.016
285.005
265, 360
trihydroxyphenyl)acetate

ACCEPTED MANUSCRIPT
Highlights
 In-depth exploration of quercetin-2HP-β-CD complex
 Electrochemical oxidation profile of quercetin inside the cavity of 2HP-β-CD
 Iron triggers the decomplexation of quercetin from the cavity of 2HP-β-CD
 DNA protection against oxidative stress induced from H2O2 by quercetin -2HP-β-CD