Molecular insights into inclusion complex formation between β- and γ-cyclodextrins and rosmarinic acid

Article abstract of DOI:10.1016/j.molliq.2020.113802

In this study, we have investigated the stable inclusion complexes (IC) formation between β- and γ-cyclodextrin (γ-CyD) with rosmarinic acid (RA), a substance known to have antioxidant and anti-inflammatory effects. The β- and γ-CyD: RA ICs were prepared

Full text of DOI:10.1016/j.molliq.2020.113802

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Molecular insights into inclusion complex formation between β-
and γ-cyclodextrins and rosmarinic acid
F. Fateminasab, A.K. Bordbar, S. Shityakov, A.A. Saboury
PII:
S0167-7322(20)32906-8
DOI:
https://doi.org/10.1016/j.molliq.2020.113802
MOLLIQ 113802
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
6 May 2020
29 June 2020
10 July 2020
Please cite this article as: F. Fateminasab, A.K. Bordbar, S. Shityakov, et al., Molecular
insights into inclusion complex formation between β- and γ-cyclodextrins and rosmarinic
acid, Journal of Molecular Liquids (2018), https://doi.org/10.1016/j.molliq.2020.113802
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2018 Published by Elsevier.

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Molecular insights into inclusion complex formation between β- and γ-
cyclodextrins and rosmarinic acid
a
a,b*
c
d
F. Fateminasab , A. K. Bordbar . S. Shityakov , A. A. Saboury
a
Department of Chemistry, University of Isfahan, Isfahan, 8174673441, Iran
b
California Institute for Quantitative Biosciences (QB3), University of California, Berkeley,
California 94720, USA
c
Department of Bioinformatics, University of Würzburg, 97074, Würzburg, Germany
d
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
To whom correspondence should be addressed
b
Emails: akbordbar@berkeley.edu and akbordbar@gmail.com
Tel: +1 (408) 594 6952
d
Email: saboury@ut.ac.ir
Tel: +98-21-66956984
1

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Abstract
In this study, we have investigated the stable inclusion complexes (IC) formation between β- and
-cyclodextrin (γ-CyD) with rosmarinic acid (RA), a substance known to have antioxidant and

anti-inflammatory effects. The β- and γ-CyD: RA ICs were prepared and characterized using
1
experimental ( H-NMR, FTIR, ICD and XRD, SEM) and computational techniques, such as
molecular dynamics simulations and molecular docking. The thermodynamic parameter analysis
showed that the formation of these ICs with the 1:1 molar ratio (A type) and also, the apparent
L
stability constant (K ) for β- and γ-CyD: RA ICs at 25 °C, were 79.07 ± 13.84 and 63.62 ± 9.31
C
-1
L mol respectively. Moreover, these results indicated that the encapsulation RA into the cavity
0
of β- and γ-CyD: RA are exothermic (ΔH <0) driven by the hydrophobic force and H-bond
formations. Additionally, the experimental and theoretical methods confirmed that the β-CyD:
RA IC is more stable than γ-CyD: RA IC. The computational results also confirm the formation
of two stable IC. These results indicated that β- and γ-CyDs are promising excipients to
formulate different substances, including RA to potentiate its antioxidant and anti-inflammatory
activity bioavailability, solubility, and stability.
Keywords: β-cyclodextrin; γ-cyclodextrin; molecular dynamics simulations; rosmarinic acid;
antioxidant; photo-stability.
2

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Abbreviations
Antioxidant activity
AC
2
,2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid
ABTS
β-CyD
RA
Beta-cyclodextrin
Rosmarinic acid
scanning electron microscopy
SEM
Fourier-transform infrared spectroscopy
Gamma-cyclodextrin
FT-IR
γ-CyD
Trolox
MD
6
-hydroxy-2, 5, 7, 8-tetramethychroman- 2-carboxylic acid
Molecular dynamics
Molecular mechanics generalized Born surface area
Molecular mechanics Poisson–Boltzmann surface area
The powder X-ray diffraction
MM-GBSA
MM-PBSA
PXRD
RMSD
RDF
Root mean square displacement
Radial distribution function
1
Hydrogen nuclear magnetic resonance spectroscopy
solvent accessible surface area
Buried surface area
HNMR
SASA
BSA
Center of mass
CoM
3

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Introduction
Phenolic acids are the group of low molecular weight polyphenols, also known as plant
secondary metabolites that can be divided into benzoic acid and cinnamic acid derivatives [1].
These compounds are believed to be quite effective at neutralizing free radicals [2]. They also
include rosmarinic acid (RA), which is a caffeic acid ester of 3-(3,4-dihydroxyphenyl)lactic acid
found in some plants, such as rosemary, basil, oregano and sage [3–5]. It has a variety of
activities biological, containing antioxidant [6], anti-inflammatory, anticancer, antiviral,
antimicrobial, properties, and neuroprotective effect [5,7–9]. The improvements in RA solubility
and stability can be achieved via its efficient complexation with hydrophilic cyclodextrins
(CyDs), which are broadly used in the pharmaceutical industry [10].
CyDs are composed of the units of α-1, 4-linked cyclic oligosaccharides of D-glucopyranose
forming a doughnut-like molecule. CyDs can be applied in the pharmaceutical, food, cosmetic,
and textile industries [11–15]. In particular, β-CyD is most widely used for pharmaceutical
applications due to its low price, safety, biocompatibility, suitable cavity size, and efficient drug
loading [16]. On the other hand, γ-CyD has a larger central cavity, which is more flexible and
non-coplanar, increasing its solubility [16,17].
The native, unmodified CyDs are named α-CyD (6 units), β-CyD (7 units), and γ-CyD (8 units)
[
11,18] and forming the hydrophobic central cavity and the hydrophilic outer surface (Del Valle,
004). As the host molecules, CyDs can form inclusion complexes (IC) with several
2
hydrophobic guest compounds [11,18–20]. The insertion of "guest" molecules, such as drugs,
vitamins, or food ingredients into the CyD cavity of CyDs, is mediated by the release of water
molecules from this cavity [11,21].
Some CyDs has already been investigated to form ICs with antioxidant molecules, such as RA
[
19,21]. Some investigations have studied the formation of ICs of modified and unmodified
CyDs with RA by CUPRAC (CUPric Reducing Antioxidant Capacity) methodology at the
presence of modified and unmodified CyDs in the aqueous environment [3]. Moreover, the
formation of β-CyD/RA IC has been explored by Medronho and et al. (2014) to determine its
1
stoichiometry by H-NMR titration [8]. Additionally, the RA antioxidant effects were measured
in the presence of β-CyD to showing an increase in scavenging free radicals from the cells [8].
Therefore, this study aims to confirm the previous results and to provide new molecular insights
into inclusion complex formation between native cyclodextrins and rosmarinic acid
4

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characterizing this IC and analyzing its phase solubility, thermodynamic properties, photo-
stability to improve the antioxidant and other bioactive effects of RA (Scheme 1).
Scheme (1): The molecular structure of a) CyDs (n=7, β-CyD and n=8 γ-CyD) and b) RA
2
2
. Materials and methods
.1. Materials
β- and γ-CyD (>98 %), RA (>96 %), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) (>97%), potassium persulfate, 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS) (>98%,) and methanol were obtained from Sigma-Aldrich (UK). The high-purity
solvent (D O) was purchased from Merck Company (Germany). Ultrapure water (17.0 MΩ/cm
2
resistivity; Nano pure infinity, Barnstead, USA) was applied for preparing phosphate buffer
-1
(
PBS, 10 mmol L , pH 7.4).
2
.2. Preparation of (β- and γ-) CyDs: RA IC
To prepare the (β- and γ-) CyDs: RA ICs formation, according to the values listed in Table 1, (β-
and γ-) CyDs were dissolved in 12 mL deoxygenized ultrapure water (by blowing of N gas for
2
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1
5 minutes) and RA (more than 1 to 1 molar ratio) dissolved in methanol in glass capped vials.
Next, stirred in neutral pH for one day to achieve the thermodynamic equilibrium at ambient
temperature. Ultimately, the mixture of CyDs and RA was centrifuged (10000 rpm, 10 min), and
-1
then the supernatant of the mixture of CyDs and RA (VaCo5, Zirbus, Germany; 1.30
and -50 °C). These powder of IC were stored at -4 °C [21,22].

10 mBar
Table 1. The required values for the preparation of ((β- and γ-) CyDs: RA IC
Host
Weight (g)
0.200
Guest
RA
Weight (g)
0.070
Solvent of RA

-CyD
methanol
methanol
-CyD
0.200
RA
0.060
2
.3. Phase solubility analysis
The studies of phase solubility were performed for (β- and -) CyDs: RA IC by Higuchi and
Connors technique [23]. Firstly, 1900 L of various concentration of CyDs (0, 0.475, 0.95, 1.90,
-1
-1
2
.85, 3.80, and 4.75 mol L ) were provided by the deoxygenized PBS (pH 7.4, 10 mmol L )
-1
and then mixed with 100 L RA dissolved in methanol (10 mmol L ). These solutions were
added to the seal foil coating vials to prevent photo-degradation, evaporation, and oxidation.
Besides, vials were sited in Cooling Shaker Incubator (Jal Tajhiz, JTSDL40, and Iran) for three
days at 15, 18, 21, and 25 C, to equilibrate the complexation. Afterward, the mixtures were
cleared by using the 0.45 μm millipore membrane filter, and the RA concentration was
determined spectrophotometrically at 324 nm in the supernatant by a UV/Vis Spectrophotometer,
equipped with a thermostat (Perkin Elmer, Lambda 265, USA) [24].
The apparent stability constant (K ) for the ICs were evaluated from the phase solubility analysis
C
along with the following equation (1) [11,25]:
slope
K 
C
(1)
S0 (1 slope)
Where S (y-intercept) is the intrinsic solubility of RA in the water in the absence of CyDs.
0
-1
The binding analysis was applied at 15, 18, 21, and 25 C. The ΔG°′ of binding (kJ mol ), was
achieved by the following equation (2):
0′
ꢀ1
훥퐺 = −푅푇푙푛(퐾 ⁄L mol )
퐶
(2)
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The thermodynamic parameters (ΔG°′, ΔH°′, and ΔS°′) were measured by the following equation
(3):
훥퐻0′ 훥푆0′
+
ꢀ
1
ln(퐾 ⁄L mol ) = −
퐶
(3)
푅푇
푅
-1
Where ΔH°′, ΔS°′, ΔG°′, [26] T, and R are the standard enthalpy change (kJ mol ), the standard
-1
-1
entropy change (J mol ), the standard Gibbs free energy change (kJ mol ), absolute temperature
-1
-1
(
K) and gas constant (J mol K ) in that order.
2
.4. The characterization of (β- and γ-) CyDs: RA IC
The Fourier-transform infrared spectra (FT-IR) of pure RA, (β- and γ-) CyDs, and (β- and γ-)
-1
CyDs: RA IC, were recorded at ambient temperature in the 4000–400 cm wavenumber range
-1
(
with 4 cm resolution) with the FT-IR spectrometer (JASCO, FT/IR-6300, Japan). These
compounds were organized on a KBr pellet in vacuum desiccators under a 0.01 torr pressure.
1
Hydrogen nuclear magnetic resonance spectroscopy ( H-NMR) for pure RA, (β- and γ-) CyDs
and the IC of (β- and γ-) CyDs: RA was accomplished in D O at 25 ˚C in 0 to 12 ppm range
2
1
using Spectrometer of H-NMR (Varian-INOVA 500 MHz, USA). The observed sharp signal
around 4.76 ppm is in regards to HDO that produced as a result of the exchange of protons
between (β- and γ-) CyDs and also O-H (phenolic and acidic) in RA with D O. The resonance of
2
HDO, as the lock frequency, set to 4.76 ppm to receive the uniform of values for the chemical
shifts. The change of chemical shift calculation (Δδ) was achieved along with the following
equation (4):
  complex  free
(4)
Where δfree and δcomplex are the chemical shifts of pure compounds (RA and (β- and γ-) CyDs)
and β- and γ-) CyDs: ICs, respectively.
The scanning electron microscope (SEM) demonstrated micrographs of RA, (β- and γ-) CyDs,
and ICs were used (Zeiss, Sigma, VP-500 EDS, and Germany) at an acceleration voltage of 15
kV. A thin gold layer coated these compounds in a vacuum.
Another method for the characterization of IC is X-ray diffraction (XRD) that commonly applied
to probe the hosts (CyDs), guests (drugs), and their ICs crystals structure. The diffraction
patterns of XRD for (β- and γ-) CyDs, RA, and IC of (β- and γ-) CyDs: Cu-X-Ray Tube Anode
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measured RA, Cu, Ni-filtered, Cu Kα radiation wavelength = 1.541874 Å and a 40 kV voltage,
-1
3
5 mA current density and in an angle of diffraction (2θ) range of 5-100 ° at 0.05 ° min rate
achieved with an X-ray diffractometer (Rigaku Ultima IV XRD, Japan). These compound
powders were packed a rectangular aluminum cell and finally exposed to the beam of X-ray.
The spectra of Induced Circular Dichroism (ICD) for RA, (β- and γ-) CyDs, and IC of (β- and γ)
-1
-1
CyDs: RA (0.5 mg mL ) were achieved in a PBS (pH 7.4, 10 mmol L ) on a CD
Spectrophotometer (Aviv, Model-215, USA). A 0.1 cm-thick quartz cell was applied in the
region of near-UV (250 to 350 nm) at ambient temperature.
2
.5. Encapsulation efficiency and loading capacity
In order to obtain the encapsulation efficiency (EE%) and loading capacity (LC%) of the RA in
-1
the cavity of CyDs (β- and γ-), 5 mL of 1 mg mL solution of ICs was stirred at room
temperature for one day. The samples were then centrifuged for 5 min at 10000 rpm, and the
concentrations of the RA were calculated using a spectrophotometer UV-vis during the
wavelength of 324 nm that was the maximum absorbance. All measurements were performed
three times in the same condition. The values of LC% and EE% of the desired complexes and
pure host and guests were determined using the following two equations:
푅퐴exp(mg)
퐿ꢁ(%) =
퐸퐸% =
× ꢂꢃꢃ
(
5)
퐼ꢁ(mg)
푅퐴exp(mg)
× ꢂꢃꢃ
(6)
푅퐴 (mg)
ꢄ
Where, RAexp and RA denote the entrapped amount of RA and the initial quantity of RA,
T
respectively.
2
.6. Photo-stability
-1
-1
The photo-stability studies of the pure RA (50 μmol L ) and complexed RA (1 mg mL ) in
aqueous solution was investigated using two UVC lamps (254 nm, 8 W). At first, the solutions of
RA in free and complexed states with appropriate concentration were prepared and
dehydrogenated. Examining the photo-stability under the UVC lamp, the sample solution was
irradiated in a quartz cuvette and stirred for a specified time. The concentration of the
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decomposed guest compound can be measured by assessing the absorbance at 324 nm by
spectrophotometer of UV/Vis at a given time of 0, 20, 40, 60, 80, 100 and 120 min [27].
2
.7. Antioxidant activity
This antioxidant activity study was accomplished to evaluate and compare the RA
antioxidant activity in pure and complexed states with (β- and γ-) CyDs. These
experiments were performed according to the following steps:

3
1

Preparation of solutions with different concentrations and molar ratios of CyD and drug form
-1
0, 45, 60, 75, and 90 μmol L and different molar ratios of β- and γ-CyDs: RA in 1:1, 1:1.5,
:2, 1:2.5 and 1:3.
•+
-1
Producing of ABTS radical cation (ABTS ), the ABTS stock solution (10 mL, 7 mmol L )
-1
was prepared in water and added potassium persulfate solution (1.1 mL, 24.5 mmol L ) and then
to allow free radical generation, this solution covered with foil and placed in the dark for 14–16
•+
hours. Finally, the ABTS solution was diluted by PBS to reach specific absorbance value of
0


.65-0.75 at 734 nm.
Preparation of 1 mmol L standard solution of Trolox.
To evaluate of scavenging effect of the antioxidant samples, 900 µL of ABTS solution
-
1
•+
added to 100 µL of the sample solutions with different concentrations and molar ratios of (β- and
γ)-CyDs and RA. and then to record the UV/vis absorption at 734 nm after 10 min incubation.
•+

Determination of the samples antioxidant activity as inhibition percent of ABTS via the
following equation:
AS


ABTS Inhibition(%)  (1 )100
(7)
AC
•+
Where A and A are the absorptions of ABTS in the presence and absence of sample, in that
S
C
order.
2
2
.8. Molecular structure calculations
.8.1. Molecular docking study
The molecular docking calculations were achieved by the AutoDock 4.2.6 program [28]. In the
first step, the β- and γ-CyDs initial structures were prepared using the REDDB web server [22].
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The RA structure was minimized by Gaussian 09 using the B3LYP/6-311++G(d,p) basis to
achieve a stable geometry. The 40×40×40 Å and 46×46×46 Å dimensions (x, y, z) were set to for
the β-CyD and γ-CyD grid boxes, respectively. The Lamarckian genetic algorithm (LGA) was
used with energy evaluations, and the number of docking runs set to 25,000,000 and 250, in that
order. The conformational cluster was performed according to the differences in the binding
energy.
2
.8.2. Molecular dynamics simulation
The molecular dynamics (MD) simulations were applied to the molecular docking poses of β-
and γ-CyDs: RA ICs with minimal binding energy by using the AMBER (Assisted Model
Building with Energy Refinement) software with the q4md-CD and GAFF force fields for CyDs
and RA [29,30]. The partial charges to RA were assigned by the restrained electrostatic potential
(RESP) method. The free (β- and γ-) CyDs and these ICs were solvated in a water box of a
truncated octahedron with 10 Å dimensions, implementing the TIP3P model [31]. The energy
minimization of the system was performed with the steepest descent algorithm for at least 10000
steps. The system was heated up to 300K as the equilibration step (NVT) to keep the pressure
and the temperature constant, using the isotropic position scaling and the Berendsen thermostat
method. The Particle-Mesh Ewald (PME) method was applied to preserve the long-range
electrostatic interactions with 10 Å cutoff for van der Waals interactions and PME. Finally, the
for 50 ns trajectory at constant temperature and pressure (NPT) was generated using the
molecular mechanics generalized Born (MM-GBSA) or the Poisson-Boltzmann (MM-PBSA)
surface area explicit solvation models to calculate binding affinity [32].
3
. Results and discussions
3
.1. The analysis of phase solubility
Due to RA with low solubility in aqueous media, the nutritional and therapeutic applications of
this antioxidant are limited. Investigation of the effect of β- and γ-CyDs on the solubility of RA
were performed and also calculated the apparent stability constant, K (eq. 1) [23,24]. As
C
indicated in Fig. 1 and Fig. 2, with increasing concentrations of β-CyD and γ-CyD, the RA
solubility increased linearly at the various temperatures (15, 18, 21, and 25 °C). Accordingly,
solubility graphs represent the A group and the 1 to 1 stoichiometric ratio between guest (RA)
L
1
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and host (CyDs) and. The values of K , ΔHº′, and ΔSº′ (Eqs. 1-3) for -CyD: RA IC and -CyD:
C
RA IC listed in Table 2. In keeping with the van't Hoff diagrams shown in Figs. 1-b and 2-b, the
decrease in K with increasing temperature indicates that the process could be considered as an
C
exothermic chemical reaction (ΔHº′ < 0). This is as a result of the water molecules removal from
the CyDs cavity. Furthermore, the negative entropy values (ΔSº′) indicate that the encapsulation
of RA into the β- γ-CyD cavities ensued a decrease in the degrees of transitional and rotational
freedom. As a result, the negative values of these two parameters and the driving forces of these
IC formations represent the van der Waals interactions and hydrogen bonding as the dominant
interactions. The stability order of the - and -CyD: RA ICs with increasing K is as follows: -
C
CyD: RA IC > -CyD: RA IC. By comparing the stability of these complexes, it can be
concluded that the -CyD has a more suitable cavity size than that of γ-CyD. Therefore, it makes
it a better candidate for its complexation with the RA molecule [22,33].
1
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140
130
120
110
100
15 °C
18 °C
21 °C
25 °C
a)
90
80
70
60
0
0.5
1
1.5
[
2
2.5
3
3.5
-
1
-CyD] (mml L )
4
.8
.6
.4
.2
4
b)
4
4
4
3.34
3.36
3.38
3.4
1/T)(K )×10
3.42
3
3.44
3.46
3.48
-
1
(
-1
Fig. 1): a) The study of phase solubility in PBS (pH 7.4, 10 mmol L ) at several
temperatures (15, 18, 21 and 25 C; b) The van’t Hoff plot, for the β-CyD: RA IC
2
formation (R =0.982).
1
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130
120
110
100
a)
15 °C
18 °C
21 °C
25 °C
9
0
0
8
0
0.5
1
1.5
2
2.5
3
3.5
4
-
1
[
γ-CyD] (mmol L )
4
.6
.5
.4
.3
.2
.1
b)
4
4
4
4
4
3.34
3.36
3.38
3.4
3.42
3.44
3.46
3.48
3
-1
(
1/T)×10 (K )
-1
Fig. 2): a) The study of phase solubility in PBS (pH 7.4, 10 mmol L ) at several
temperatures (15, 18, 21 and 25 ᵒC; b) The van’t Hoff plot for the γ-CyD: RA IC
2
formation (R =0.983).
1
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Table 2. a) The constants of phase solubility analysis (K ) and the values of estimated
C
thermodynamic for a) β-CyD: RA IC and b) γ-CyD: RA IC formation at several temperatures.
-
1
2
-1
-1
-1
-1
-1
T (K)
Sₒ (mol.L )
slope
R
K (L mol )
ΔG⁰ ′/kJ mol
ΔH⁰ꢅ/kJ mol
11.25 ± 0.31
ΔS⁰ ′/J K mol
C
-
5
-3
-
2
88 ± 0.10 (7.11 ± 0.49) × 10
(7.77 ± 1.02) × 10
0.982
109.79 ± 7.10
-13.20 ± 0.08
-45.354± 1.49
-
-
-
3
3
3
-
-
-
5
5
5
(
(
(
7.52 ± 0.86) × 10
6.41 ± 0.02) × 10
7.80 ± 1.26) × 10
-11.53 ± 0.17
-10.91 ± 0.12
-10.83 ± 0.88
2
2
2
91 ± 0.10 (7.49 ± 0.26) × 10
94 ± 0.10 (7.59 ± 0.17) × 10
98 ± 0.10 (9.94 ± 0.10) × 10
0.994
0.997
0.986
100.46 ± 4.48
86.70 ± 2.19
79.07 ± 13.84
-12.63 ± 0.72
-12.00 ± 0.92
-11.42 ± 0.14
-.45.22 ± 1.96
-45.59 ± 2.10
-45.25 ± 0.46
-
1
2
-1
-1
-1
-1
-1
T (K)
Sₒ (mol L )
slope
R
K (L mol )
ΔG⁰ ′/kJ mol
ΔH⁰ꢅ/kJ mol
ΔS⁰ ′/J K mol
C
-
5
-3
-24.60 ± 5.09
2
88 ± 0.10 (8.38 ± 0.10) × 10
(7.38 ± 1.02) × 10
0.980
88.70 ± 12.12
-10.74 ± 0.66
-48.13± 15.38
-
-
-
3
3
3
-
-
-
5
5
5
(
(
(
6.98 ± 0.22) × 10
6.22 ± 0.37) × 10
6.05 ± 4.15) × 10
-24.60 ± 5.09
-24.60 ± 5.09
-24.60 ± 5.09
2
2
2
91 ± 0.10 (8.62 ± 0.46) × 10
94 ± 0.10 (8.90 ± 0.13) × 10
98 ± 0.10 (9.56 ± 0.18) × 10
0.974
0.978
0.989
81.55 ± 6.70
70.26 ± 5.31
63.62 ± 9.31
-10.65 ± 0.40
-10.40 ± 0.37
-10.29 ± 0. 86
-.48.94 ± 16.13
-48.28 ± 16.04
-48.02 ± 14.19
3
3
.2. The β- and γ-CyD: RA inclusion complexes characterization
.2.1. Spectroscopy of Fourier-transform infrared
The spectroscopy FT-IR is a valuable technique for characterizing CyD: Drug complexes. This
method, in combination with other techniques, is used to entirely identify solid-state ICs and
compare their structures with the separate host (CyDs) and guest (RA) components. As observed
in Fig. 3, the FT-IR spectra for pure β-CyD, γ-CyD, RA, β- and γ-CyD: RA ICs were
investigated as follows:
The spectra of FT-IR for Pure β-CyD and γ -CyD displayed in Fig. 3a-b, In these spectra,
-1
signified an outstanding absorption band at 3391 and 3396 cm (the stretching vibrations of O-
-1
H, β- and γ-CyD, in that order), 1418 and 1439 cm (the bending vibrations of O-H, β- and γ-
CyD, respectively), the stretching and bending vibrations band for C-H (in CH group) at 2927,
2
-1
2
931 and 1081, 935 cm (β- and γ-CyD, respectively), the stretching vibration band for C-O and
C-O-C in the ether linkage at 1157, 1155 and 1029, 1018 cm-1, (β- and γ-CyD), respectively
34].
The RA FT-IR spectrum (Fig. 3c) indicated an absorption band at 3458 and 1353 cm (the
[
-1
-1
stretching and bending vibrations carboxylic acid O-H), and 3309, 3174 and 3396 cm (the
-1
stretching vibrations phenolic O-H), 1712 and 1678 cm (acidic and esteric C=O stretching
-1
vibrations, respectively), 1610, 1520 and 1464 cm (aromatic ring stretching vibrations) and also
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-1
the peak in 1259, 1074 and 1196 cm was related to stretching vibrations for carboxylic acid,
ester, and phenols, in turns.
-1
The spectra of β-CyD: RA IC (Fig. 3d) demonstrated absorption bands in 3375 cm (for O-H
stretching vibrations) with redshift, and the reduced intensity compared to the spectrum showed
for β-CyD. It is important to note that the –OH spectra of phenolic and carboxylic acid are
-1
covered by –OH spectra of β-CyD. The –CH groups stretching vibrations in 2929 cm were
accompanied by blue shift and increased intensity, indicating the presence of the –CH group in
-1
β-CyD and RA. Also, the characteristic bands of RA in IC included 1709 cm (for C=O
carbonyl groups stretching vibrations) with redshifts and the reduced intensity pure RA and the
-1
ether linkage of O-C-O in complexed β-CyD with 1030 cm along with reduced intensity and C-
-
O esteric, carboxylic acid and phenolic groups stretching vibrations in 1610, 1592 and 1480 cm
1
,
respectively, with reduced intensity. This spectral changes such as intensity and blue or
redshift might be because of the incorporation of RA into β-CyD hydrophobic cavity.
-1
The -CyD: RA IC spectra (Fig. 3e) revealed absorption bands in 3381 and 1633 cm (for O-H
stretching and bending vibrations) with redshift and the reduced intensity compared to the
spectrum observed for pure -CyD. It is important to note that the –OH spectra of phenolic and
carboxylic acid are covered by –OH spectra of -CyD. The –CH groups stretching vibrations in
-1
2
927 cm were accompanied by blue shift and increased intensity, indicating the presence of the
-1
–
CH group in γ -CyD and RA. Also, the characteristic bands of RA in IC included 1707 cm (for
C=O carbonyl groups stretching vibrations) with redshifts and the reduced intensity pure RA and
-1
the ether linkage of O-C-O in complexed -CyD with 1028 cm along with reduced intensity and
C-O esteric, carboxylic acid and phenolic groups stretching vibrations in 1604, 1520 and 1448
-1
cm , respectively, with reduced intensity. This spectral changes such as intensity and blue or
redshift might be because of the incorporation of RA into -CyD hydrophobic cavity. In general,
the spectral changes (red or blue shift in wavenumber, sharpening, decreasing, or flattening
spectra intensities) can approve the - and -CyD: RA ICs. This data designates changes in the
drug environment due to the van der Waals interactions and hydrogen bonding formation
between -OH phenolic groups of RA and the -OH groups of CyDs.
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(
C-H) a) CyD
(
O-C)
(O-H)
(
C-O-C)
4
4
4
4
4
000
000
000
000
000
3500
3000
2500
2000
1500
1000
500
500
500
500
500
b) CyD
(C-H)
(
O-C)
(O-H)
(C-O-C)
3500
3000
2500
2000
1500
1000
c) RA
(
C=O)
(O-H)
(
O-C)
3500
3000
2500
2000
1500
1000
(
C=O)
(C-H) d) CyD:RA IC
(
O-C)
(O-H)
(
C-O-C)
3500
3000
2500
2000
1500
1000
(
C=O)
(C-H)
e) CyD:RA IC
(
O-C)
(O-H)
(
C-O-C)
3500
3000
2500
2000
1500
1000
-
1
Wavenumber (cm )
Fig. 3): The spectra of FT-IR of a) β-CyD, b) γ-CyD, c) RA, d) β-CyD: RA IC, b), e) γ-CyD: RA
IC.
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1
3
.2.2. The spectroscopy of H-Nuclear magnetic resonance
1
The spectroscopy of H-NMR is a valuable technique for the analysis of chemical shift changes
in the complexation, and the main difference pure guest and host and IC formed between those
[
35]. The H-5 and H-3 located into the narrower part and broader part of the cavity of β- and -
CyD, respectively. So, these two protons display chemical shifts by the insertion of the guest into
1
the cavities of CyD. As shown in Fig. ESI 1, the H-NMR spectra of the free β-CyD, -CyD, and
RA with the insertion of RA into the cavity of β- and -CyD were compared and assessed
changes of chemical shift between those. Fig. ESI 1a demonstrates the value of 3.93 and 3.83 for
1
H-3 and 5 in pure β-CyD. While the H-NMR spectrum for the β-CyD: RA IC (Fig. ESI 1-d)
displays 3.82 ppm for H-3 (Δδ= -0.09 ppm), 3.66 ppm for H-5 (Δδ= -0.17 ppm) and the most
change of chemical shift for H-8' in RA is Δδ= -0.05 ppm (5.23 ppm in free RA and 5.18 ppm in
complexed RA). The results of chemical shift changes are observed in Table 3a. The most
observable change of chemical shift is indicated for H-5 and H-8' (carboxylic acid). As a result, it
1
could be concluded that the acidic group inserted into the cavity of β-CyD. Therefore, the H-
NMR study approves IC is formed and inserted RA into the β-CyD cavity. In the case of -CyD:
RA IC, Fig. ESI 1-b and e indicate the values of 3.84 and 3.73 ppm (Δδ= -0.11 ppm) and these
values for H-5 in pure and complexed -CyD are 3.58 and 3.64 ppm (Δδ= 0.06 ppm),
respectively. Furthermore, the most chemical shift change for H-7 in RA in free RA and 5.18
ppm in complexed RA are Δδ= -0.30 ppm. According to the data mentioned above, H-3 and H-7
in -CyD and RA display the maximum values for Δδ representing the insertion of RA into the -
CyD cavity and formation of the γ -CyD: RA.
1
Table 3. H-chemical shift (δ [ppm]) for a) free β-CyD, RA and β-CyD: RA IC and b) γ-
CyD, RA and γ-CyD: RA IC in D O at 25 C
2
a) β-CyD
H-1
δ
β-CyD /ppm
5.02
δ
complex /ppm
4.96
Δδ /ppm
-0.06
H-2
3.60
3.55
-0.05
H-3
3.93
3.82
-0.11
H-4
3.55
3.50
-0.05
H-5
3.83
3.66
-0.17
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H-6
RA
3.84
3.73
complex /ppm
7.04
-0.11
Δδ /ppm
-0.01
-0.01
-0.03
+0.03
-0.04
0.03
δ
RA /ppm
7.05
6.77
6.87
6.94
6.74
7.46
6.22
5.23
δ
H-2
H-2'
H-5
6.76
6.84
H-6
6.97
H-6'
H-7
6.70
7.49
H-8
6.23
0.01
H-8'
b) γ-CyD
H-1
5.18
-0.05
Δδ /ppm
-0.05
0.05
δ
γ-CyD /ppm
5.03
δ
complex /ppm
4.98
H-2
3.56
3.61
H-3
3.84
3.73
-0.11
-0.03
0.06
H-4
3.51
3.48
H-5
3.58
3.64
H-6
3.77
3.72
0.05
RA
δ
RA /ppm
7.05
δ
complex /ppm
6.78
Δδ /ppm
-0.27
-0.01
-0.18
-0.23
-0.16
-0.30
0.02
H-2
H-2'
H-5
6.77
6.67
6.87
6.69
H-6
6.94
6.71
H-6'
H-7
6.74
6.58
7.46
7.16
H-8
6.22
6.24
H-8'
5.23
5.49
0.26
3
.2.3. Scanning electron microscope
One of the essential techniques of investigation of ligands, CyD, and their ICs is the SEM
method. The micrographs of pure RA, β- and -CyD, and ICs of β- and γ -CyDs: RA are showed
in Fig. 4. As observed in Fig. 5 a and b, β- and γ -CyD indicated the quadrilateral form, and RA
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has a rod-shaped crystal. The β- and -) CyDs: RA ICs appeared as irregularly amorphous
fragments and various dimensions that seem to be due to the relative difference in size and shape
of RA, β-CyD, and γ -CyD. For that reason, these analyses could be proved the existence of a
new solid phase, indicative of the ICs of β-CyD: RA and γ -CyD: RA formation [36,37].
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Fig. 4): The SEM micrographs for a) -CyD, b) -CyD, c) RA, d) -CyD: RA IC and e) -CyD:
RA IC.
3
.2.4. X-ray diffraction
The XRD technique was applied to confirm the formation of ICs between CyDs and RA. The
XRD diffractograms for β-CyD, -CyD, RA, β-CyD: RA IC, and -CyD: RA have been observed
in Fig. 5. Fig. 5-a and b describe the intensity peaks at different diffraction angles (2q) of 10.35,
1
2
4.65, 14.70, 14.75, 22.75, and 40.65° for β -CyD and 12.10, 12.20, 16.24, 16.35, 18.75 and
1.88° for -CyD. Also, the RA diffractogram (Fig. 5-c) shows several peaks with various angles
and intensities in 13.48, 14.94, 14.98, 19.46, 19.48, 23.98, 26.36 and 28.72° that these high-
intensity peaks at different diffraction angles indicate the crystalline nature of this drug
compound. The XRD peak of β-CyD: RA IC and -CyD: RA IC have been observed in Fig. 5-d,
and e. Two diffractograms for these ICs showed the pattern associated with the amorphous
structure after the freeze-drying procedure.
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2
00
80
60
40
20
00
80
0
1
4.7
1
200
1
0
1
6.32
0
0
18.70
b) -CyD
1
1
6.24
1000
0
2
1.68
0
a) -CyD
12.30
1
0
8
00
12.20
1
0
2
2.7
1
6
00
0
0
1
0.34
24.25
0
4
0.65
400
60
0
7
.2
40
200
20
0
0
20
40
60
80
20
40
60
80
2
0
2
( )
3
50
120
100
80
0
17.72
0
2
6.32
300
26.36
0
d) -CyD: RA IC
0
c) RA
0
23.98
1
9.48
250
19.46
0
0
1
4.98
0
0
28.72
200
14.94
60
150
0
1
3.48
100
40
50
20
0
20
40
60
80
0
0
20
40
60
80
2
( )
0
2
( )
80
70
e) -CyD: RA IC
60
50
40
30
20
10
20
40
60
80
0
2
( )
Fig. 5): X-ray diffractograms for a) -CyD, b) -CyD, c) RA, d) -CyD: RA IC and e) -CyD:
RA IC
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3
.2.5. The spectroscopy of induced circular dichroism (ICD)
The ICD spectroscopy is one of the techniques to evaluate the molecular recognition in the
formation of IC between CyDs and optically active molecules of a guest. As a result, this
technique could point to the ICD peak presence for the insertion of optically active guest
molecules into the CyDs cavity [38]. The spectra for -CyD, -CyD, RA, -CyD: RA IC, and -
CyD: RA have been indicated in Fig. 6. The ICD spectrum for RA displays a positive peak in
-1
-1
2
94 nm with Δε= 44.04 L mol cm . As is evident in this Fig. 6, the ICD spectra for β-CyD: RA
IC and -CyD: RA IC seems to be positive ICD at 298 and 304 nm with Δε= 22.01 and 17.10 L
-1
-1
mol cm indicative of the insertion of RA into the cavities of these two CyDs. These positive
peaks indicate the parallel or diagonal insertion of RA into the cavity of these CyDs.
3
.3. Encapsulation efficiency and loading capacity
The EE% and LC% values for ICs of β-CyD: RA and γ-CyD: RA obtained using the freeze-
drying process was evaluated by UV-vis spectroscopic measurements at 324 nm. Concerning
Eqs. 5 and 6, the values of (24.014 ± 4.030) % and (23.505 ± 1.730) % were obtained for LC%
(mg of encapsulated RA/mg of complex powder) ×100) for β-CyD: RA IC and γ-CyD: RA IC,
respectively and also the amounts of (83.220 ± 13.970) % and (76.340 ± 5.620) % were
calculated EE% for (β- and γ-CyD): RA ICs, respectively.
LC % and EE % values for locating of RA into the β-CyD cavity are higher than that for γ-CyD,
which may be because of the size of the cavity in β-CyD better than γ-CyD.
6
5
4
3
2
1
0
0
0
0
0
0
0
β-CyD
β-CyD: RA IC
γ-CyD
γ-CyD: RA IC
RA
2
50
260
270
280
290
300
310
320
330
340
350
-10
Wavelength (nm)
Fig. 6: The ICD spectra for a) -CyD, b) -CyD, c) RA, d) -CyD: RA IC, and e) -CyD: RA IC.
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3
.4. Photo-stability analysis
The photo-stabile investigations of polyphenol compounds are essential in various fields of
science and technology, such as pharmaceutical industries. The hydrogen atoms in
photosensitive phenolic groups in polyphenol compounds such as RA can be radicalized, and
therefore this antioxidant has low stability in several environmental conditions e.g., light,
oxygen, and moisture. As a result, the protection and improvement of this photosensitive
compound versus physical agents are necessary and recommended. [39]. The insertion of RA
could improve the photo-stability of RA into the β- and γ-CyD cavities. So, the photo-
degradation reaction was monitored spectrophotometrically for pure and complexed states of RA
in aqueous media with exposure to UVC lamp.
The apparent rate constant (k) for the RA photo-degradation as a pseudo first-order reaction has
been valued along with the following equation:
(8)



At 

ln
 kt
A0

Where A and A and are the UV-vis spectroscopy absorption at initial and after exposure to
t
0
UVC lamp in aqueous media at 324 nm, and t is time, respectively. Fig. 7 shows the variation of
ln (A /A ) vs. time for pure RA and β- and γ-CyDs: RA ICs. The values of k and t (half-life
t
0
0.5
time) were listed in Table 4.
Based on the results presented in Table 4, the photo-stability of pure RA and its ICs is as
follows:
RA < -CyD: RA < -CyD: RA
These results show that the insertion of RA into the β-CyD and γ-CyD cavities have mostly
protected this compound against UV light and improved photostability and reduced the photo-
degradation of RA. According to these results, the γ-CyD: RA IC showed less photo-stability
than the β-CyD: RA IC. The reason is that the cavity of γ-CyD is larger than β-CyD cavity and
the rotation of the RA into the γ-CyD cavity.
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0
0.1
0.2
0.3
0.4
0
20
40
60
80
100
120
140
-
-
-
-
RA
β-CyD: RA IC
γ-CyD: RA IC
Time (min)
Fig. 7): The photo-degradation plots for free RA (♦), β-CyD: RA IC (▴) and γ-CyD: RA IC in
the variation of ln (A /A ) vs. time (min) in aqueous media after the exposure to UVC lamp,
t
0
where A and A are the UV–vis absorption spectroscopy at 324 nm at initial and after exposing
0
t
to UVC lamp in aqueous media, respectively.
Table 4: The apparent pseudo first-order rate constant (k) and the half-life time (t ) for photo-
0
.5
degradation reaction of RA, (β- and γ-) CyDs: RA ICs in aqueous media after the exposure to
UVC lamp.
3
-1
Aqueous media (UVC lamp)
k (×10 ) (min )
t (min)
0.5
RA
2.87 ± 0.1
24146 ± 16.87
491.84 ± 17.39
389.76 ± 6.93

-CyD: RA IC
1.41 ± 0.24
1.78 ± 0.03

-CyD: RA IC
3
.5. Antioxidant activity
The presence of phenolic hydroxyl groups causes antioxidant activity (AC) in RA. Because of
the electron-donating hydrogen atoms, these groups act as free radical stabilizers. As shown in
Fig. 8, the results of AC for pure -CyD, -CyD, RA, Trolox, and the -CyD: RA and -CyD:
•+
RA complexes are shown in Fig. 8. ABTS radical scavenging % was measured using Equation
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7
. As a reducing sugar, it would be expected that free β- and -CyDs did not display any AC, and
•+
their ABTS radical scavenging % was in the variety of 9.66 to 16.63 and 15.58 to 24.81 % for
β-CyD and -CyD, respectively. Therefore, the AC observed for these compounds can be due to
interference with other components in the solution, and the interpretations should not be regarded
as an absolute value for them. Also, due to the small changes seen in CyDs, the higher
concentrations of these compounds are not recommended in the industry. The antioxidant
activity of RA increases significantly from ~45.45 to 95.51% (~ 50%) by increasing its
-1
concentration from 30 to 90 μmol L .
The masking effect on the AC was calculated for the encapsulated RA into the CyDs cavity. The
masking effect for the β- and -CyD: RA ICs was 12.17 to 20.04%, and 22.74 to 23.82%,
respectively. The AC masking values are dependently on the binding affinity of RA to β-and -
CyD [35,36]. By incorporation of RA into the CyD cavities, the AC of pure RA has a slight
decrease at all studied concentrations of RA. The AC changes of RA may be due to the
formation of intermolecular H-bonding of the RA with the -OH groups in CyDs in the β- and -
CyD: RA ICs. Therefore, it can be deduced that the formation of β- and -CyD:RA ICs can cause
masking of some hydroxyl groups of RA. When RA is encapsulated with CyDs, ortho-phenolic
groups in this antioxidant are incorporated into the non-polar cavity and thus result in the
oxidation-reduction behavior of the RA compound [40–42]. Also, the insertion of RA into the
hydrophobic and less-polar of CyD cavities not only protects this compound from environmental
damage such as light, oxygen, moisture, and rapid oxidation but also significantly enhances its
solubility in its free state. As a result, the increase in aqueous solubility and protection of this
compound may justify the profound difference in AC of RA compared to the - and -CyD: RA
ICs.
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1
00
80
60
40
20
0
3
4
6
7
9
0 μM
5 μM
0 μM
5 μM
0 μM
Trolox
β-CyD
γ-CyD
RA
β-CyD: RA IC
γ-CyD: RA IC
Concentration (M)
•+
Fig. 8): The antioxidant concentration effects on the ABTS inhibition
3
3
.6. Computational methods
.6.1. Molecular docking analysis
In this study, the interaction of RA into the - and -CyD cavity have been evaluated by
molecular docking. The incorporation of drugs into these CyD cavities, the free binding energies,
and the number of hydrogen bonds in these interactions are also reported. Finally, the molecular
docking results of two CyD types were compared. At the end of each molecular docking
calculation, 250 different configurations of RA are generated. Then, these RA configurations
based on the RMSD of the corresponding atoms are clustered and arranged in energy order. In
the case of RA, the configuration with the lowest energy and the largest population is selected as
the main configuration [43].
In the CyD: RA IC formation, four hydrogen bonds are observed for the RA and -CyD
interaction. These hydrogen bonds include a) The phenolic H (C4) of RA molecule with the O of
-OH (C-6) glycopyranoside unit (No. 1); b) The phenolic H (C3') of RA with O4
glycopyranoside unit (No. 2), c) The H of –OH in phenyl ring (C4') with O of -OH (C-2)
glycopyranoside unit (No. 1) and d) The O of phenolic -OH (C4') with O of -OH (C-3)
glycopyranoside unit (No. 1). In this molecular docking job, 37 clusters were observed for the
orientation of RA into the -CyD cavity and the RA configuration with the largest population
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-1
and lowest energy of -5.97 kcal mol selected as the main orientation of RA into the -CyD
cavity. These results of molecular docking for the orientation of RA into the CyD cavity are in
1
agreement with spectroscopies of H-NMR and ICD. (See in Fig. 9-a).
As observed in Fig. 9-b, the results of molecular docking calculations in the CyD: RA IC
formation indicated three hydrogen bonds for the encapsulation of RA into -CyD cavity. These
hydrogen bonds are formed between the two H-phenolics (C3' and C4') of RA antioxidant with
the O4 glycopyranoside unit (No. 5) and the H-phenolic (C3) with O of –OH (C6)
glycopyranoside unit (No. 8). In this molecular docking analysis, 45 clusters were indicated for
the orientation of RA into the -CyD cavity and the RA configuration with the largest population
-1
and lowest energy of -6.06 kcal mol selected as the primary orientation of RA into the -CyD
cavity. These results of molecular docking for the orientation and encapsulation of RA into the -
1
CyD cavity align with the results of two spectroscopies H-NMR and ICD.
Concerning the results of molecular docking for the interactions of - and -CyDs with RA, it
can be concluded due to the large size of the RA compound, the free binding energy of
interaction of RA with -CyD is higher than that of -CyD. It should be noted, however, that in
the interaction of -CyD with RA, the number of hydrogen bonds is more than these of -CyD:
RA IC, so this interaction is more stable.
b)
a)
Fig. 9): The molecular docking structures for a) -CyD: RA IC and b) -CyD: RA IC.
3
.6.2. Molecular dynamics simulation
The computational results of MD simulations provided evidence that CyDs can form stable ICs
with RA in aqueous media. The MD simulations in water box were accomplished to explore the
conformational change of RA molecule within the of - and -CyD cavities for 50 ns at room
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temperature. For that, we used the root mean square displacement (RMSD), root mean square
fluctuation (RMSF), radial distribution function (RDF), solvent accessible surface area (SASA),
and the distance between the RA center of gravity and β- and -CyDs. Additionally, the MM-
PBSA and MM-GBSA scripts were executed to evaluate the binding affinity together with the
enthalpy-entropy compensation [44]. As shown in Fig. 10, the conformational changes were
observed for β- and -CyDs in a free and bound state during the 50 ns trajectory. The RA
1
molecule was imbedded into the binding cavities of both CyDs supporting the data from the H-
NMR and molecular docking experiments [45].
To compare the stability of β- and γ-CyDs: RA IC after the encapsulation, we have computed
RMSD of the β- and γ-CyDs corresponding to those of the initial structure from docking, in free
and complex state and then the stabilities of all structures during 50 ns were studied [46]. The
RMSD for β- and -CyDs in free and IC states have been observed in Fig. 11. In these Figs., the
RMSD values of β- and γ-CyD in the free state (blue) and complexed state (red) have been
presented during the simulation time. The mean values of RMSD for β-CyD in the pure and
complexed states were 0.848 ± 0.224 Å and 2.157 ± 0.496 Å and for the free and IC states in -
CyD were 1.043 ± 0.336 Å and 1.997 ± 0.314 Å, respectively. The RMSD values for β- and -
CyDs and β- and -CyDs: RA IC agreed with the results of snapshots of configuration changes in
Fig. 10. From Fig. 11-c, the RMSD values for RA in the first IC (β-CyD: RA IC) are lower
(RMSD = 1.392 ± 0.421 Å) than for RA (RMSD = 1.493 ± 0.659 Å) in the second one (-CyD:
RA IC), indicating the restrictive effect of the β-CyD binding cavity. In other words, the more
extended -CyD binding site permits more flexibility for RA than β-CyD. The comparison of the
structural changes (Fig. 10) and RMSD behavior (Fig. 11) lead us to conclude that RA forms a
more stable IC with β-CyD than γ-CyD.
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0
ns
10 ns
25 ns
40 ns
50 ns
a) β-CyD (Free)
β-CyD: RA IC
b) -CyD (Free)

-CyD: RA IC
Fig. 10): Snapshots of in the pure and complexed states of a) β-CyD and b) -CyD with RA at
the several times 0, 10, 25, 40, and 50 ns.
2
9