Application of spectroscopic methods for identification (FT-IR, Raman spectroscopy) and determination (UV, EPR) of quercetin-3-O-rutinoside. Experimental and DFT based approach

Article abstract of DOI:10.1016/j.saa.2014.12.050

Vibrational (FT-IR, Raman) and electronic (UV, EPR) spectral measurements were performed for an analysis of rutin (quercetin-3-O-rutinoside) obtained from Rutaofficinalis. The identification of rutin was done with the use of FT-IR and Raman spectra. Those experimental spectra were determined with the support of theoretical calculations based on a DFT method with the B3LYP hybrid functional and 6-31G(d,p) basis set. The application of UV and EPR spectra was found to be a suitable analytical approach to the evaluation of changes in rutin exposed to certain physicochemical factors. Differences in absorbance observed in direct UV spectra were used to monitor changes in the concentration of rutin in degraded samples. Spectra of electron paramagnetic resonance allowed studying the process of free-radical quenching in rutin following its exposure to light. The molecular electrostatic potential (MEP) and frontier molecular orbitals (LUMO-HOMO) were also determined in order to predict structural changes and reactive sites in rutin.

Full text of DOI:10.1016/j.saa.2014.12.050

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Application of spectroscopic methods for identification (FT-IR, Raman
spectroscopy) and determination (UV, EPR) of quercetin-3-O-rutinoside.
Experimental and DFT based approach
Magdalena Paczkowska a, Kornelia Lewandowska b, Waldemar Bednarski b, Mikołaj Mizera a,
Agnieszka Podborska c,d, Anna Krause e, Judyta Cielecka-Piontek a,
⇑
a
´
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
b Departament of Molecular Crystals, Institute of Molecular Physics Polish Academy of Sciences, Smoluchowskiego 17, 60-179 Poznan´, Poland
c Department of Physical Chemistry and Metallurgy of Non-Ferrous Metals, AGH University of Science and Technology, al. Adama Mickiewicza 30, 30-059 Kraków, Poland
d Department of Non-Ferrous Metals, AGH University of Science and Technology, al. Adama Mickiewicza 30, 30-059 Kraków, Poland
e PozLab sp.z.o.o, Poznan, Poland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
The FT-IR, Raman, EPR and UV spectra
of quercetin-3-O-rutinoside were
used for its identification and/or
determination.
OH
HO
O
O
OH
OH
HO
O
O
OH
O
OH
ꢀ
ꢀ
Spectroscopic properties of the of the
title compound were calculated and
compared with experiment.
The HOMO–LUMO energies and
related molecular properties were
evaluated.
H3
HO
C
O
HO
OH
2
1
400
600
800
1000
1200
1400
1600
1800
-1
Wavenumber (cm
)
a r t i c l e i n f o
a b s t r a c t
Article history:
Vibrational (FT-IR, Raman) and electronic (UV, EPR) spectral measurements were performed for an anal-
ysis of rutin (quercetin-3-O-rutinoside) obtained from Rutaofficinalis. The identification of rutin was done
with the use of FT-IR and Raman spectra. Those experimental spectra were determined with the support of
theoretical calculations based on a DFT method with the B3LYP hybrid functional and 6-31G(d,p) basis set.
The application of UV and EPR spectra was found to be a suitable analytical approach to the evaluation of
changes in rutin exposed to certain physicochemical factors. Differences in absorbance observed in direct
UV spectra were used to monitor changes in the concentration of rutin in degraded samples. Spectra of
electron paramagnetic resonance allowed studying the process of free-radical quenching in rutin follow-
ing its exposure to light. The molecular electrostatic potential (MEP) and frontier molecular orbitals
(LUMO–HOMO) were also determined in order to predict structural changes and reactive sites in rutin.
Ó 2015 Published by Elsevier B.V.
Received 11 September 2014
Received in revised form 1 December 2014
Accepted 12 December 2014
Available online 25 December 2014
Keywords:
Rutin
UV
FT-IR
Raman
EPR
DFT analysis
Introduction
a naturally occurring flavonoid in the plant Rutaofficinalis [1].
Structurally, rutin is aflavonolquercetin which is connected with
Rutin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[
a
-
L
-rhamno-
disaccharides: rhamnose and glucose (Fig. 1) [2]. It shows antioxi-
dant, anti-inflammatory, anticarcinogenic, antithrombotic, cyto-
protective, vasoprotective and microbial activities. Due to its
activity rutin is used in pharmaceutical dosage forms such as solid
(e.g., tablets) and solutions (e.g., ophthalmic drops) [3–6]. Rutin is
pyranosyl-(1 ? 6)-b-D-glucopyranosyloxy]-4H-chromen-4-one) is
⇑
Corresponding author. Tel.: +48 618546649; fax: +48 618546652.
E-mail address: jpiontek@ump.edu.pl (J. Cielecka-Piontek).
http://dx.doi.org/10.1016/j.saa.2014.12.050
1386-1425/Ó 2015 Published by Elsevier B.V.

M. Paczkowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
133
widely used in numerous nutraceuticals and dietary supplements
due to its auxiliary, prophylactic treatment of civilization diseases,
such as hypercholesterolemia, high blood pressure, hemorrhoids,
phlebitis and chronic venous insufficiently [7,8].
(Poland). Hydrochloric acid, sodium hydroxide solution, phospho-
ric buffer and all other chemicals were obtained from P.O.Ch.
(Poland). Acetonitrile of an HPLC grade was supplied by Merck
KGaA (Germany) and triethylamine (99.5%) by J.T. Baker (Nether-
lands). High-quality pure water was prepared using an Exil SA
67120 Millipore purification system (France).
In the case of plant substances, the achievement of biological
activity depends on many factors. A purity and chemical stability
are the most important parameters for efficacy of therapy based
on raw substances [9]. Purity of raw materials depends on its ori-
gin. While stability of raw substance can be changed in effect of
activity of affecting factors on raw substances during its storage.
Development of analytical methods suitable for evaluation of
material purity and monitoring its changes during storage. So it
is important for efficacy and safety of potential phytotherapy. Up
to now, for identification and determination of rutin the following
techniques: gravimetry, polarography, fluorometry, ultraviolet
spectrophotometry and high-performance liquid chromatography
method, were reported [10–14]. A few stability studies of rutin
signed the problem its susceptibility to degradation under the
influence of affecting factors such as hydrogen ions, light and
temperature [15–17]. To our the best knowledge, no complex pro-
cedure based on spectroscopic solvents for identification and
determination of rutin, have been reported.
Spectroscopic measurements
The vibrational infrared spectra of samples of rutin were
recorded in the frequency range between 400 and 1800 cmꢁ1, at
room temperature, with an FT-IR Bruker Equinox 55 spectrometer.
The Raman scattering spectra were obtained with a LabRAM
HR800 spectrometer (HORIBA JobinYvon) with laser excitation
kexc = 633 nm (He–Ne laser). In each case the power of the laser
beam at the sample was less than 1 mW to avoid sample damage.
An UV–VIS Lambda 20 (Perkin Elmer) spectrophotometer equipped
with 1.0 cm-in-width quarts cells and controlled by the UV WinLab
software was utilized. Detection of free radicals and determination
of their concentration were carried out using a Bruker ELEXSYS 500
spectrometer (X-band) at 297 K. EPR spectra were recorded as a
first derivative of the absorption signal. The number of free radicals
was calculated using the integration procedure described else-
where [18].
The study investigated experimentally and theoretically the
application of spectroscopic methods for the identification
(ultraviolet, UV; Fourier transform infrared spectroscopy, FT-IR;
Raman spectroscopy) of rutin and the determination (UV; electron
paramagnetic resonance, EPR) of changes that the compound
undergoes due to degradation.
Photodegradation stability studies of rutin in the solid state
were performed using Suntest CPS+ (AtlasÒ) with Solar ID65 filter.
The cells were exposed to UV radiation (300–400 nm) in an overall
illumination of P210 Wh/m2 in Suntest C+.
All the calculations were made by using the Gaussian 03 pack-
age [19]. In order to interpret the experimental results of IR
absorption and Raman scattering, quantum chemical calculations
were performed, based on a density functional theory (DFT)
method with the B3LYP hybrid functional and 6-31G(d,p) basis set.
Experimental
Materials and chemicals
Rutin (purity >98%) originated from natural sours from Rutaof-
ficinalis and it was supplied by Sigma–Aldrich (Poland). The impu-
rities: quercetin and isoquercetin were obtained by Sigma–Aldrich
Theoretical studies
The optimization of the molecular geometry (MG), spatial
electron distribution of frontier molecular orbitals (FMOs) analysis
and molecular electrostatic potential (MEP) of rutin were obtained
with density functional theory calculations using Becke’s three-
parameter hybrid functional (B3LYP) implemented with the stan-
dard 6-31(d,p) as a basis set. The harmonic vibrational frequencies
of FT-IR, Raman and UV spectra have been calculated using the
same level of theory. All the calculation were made by using Gauss-
ian 03 package and GaussView application was utilized to present
the MG, FMO and MEP [19,20].
Forced studies of rutin
For the determination of concentration changes of rutin in the
presence of its impurities and degradation products (quercetin
and isoquercetin) as a result of factors affecting action, changes
in the absorption spectra of rutin were examined. The degradation
of rutin was studied in aqueous solutions: in hydrochloric acid
(pH = 0.3) at 333 K up to 360 min, sodium hydroxide (pH = 13.3)
at 313 K up to 15 min and in hydrogen peroxide (10%) at 353 K
up to 200 min. Samples were prepared by dissolving an accurately
weighed 5.0 mg of rutin in 25.0 mL of the equilibrated solution to
313 K in glass stoppered flasks. At specified times, samples of the
reaction solutions (1.0 mL) were collected and instantly cooled
with a mixture of ice and water, neutralized with 0.8 ml of NaOH
or HCl solutions of suitable concentrations and assayed. The ionic
strength of all solutions was adjusted to 0.5 mol/L with a solution
of sodium chloride (4 mol/L).
Main impurity
OH
HO
O
OH
OH
OH
O
Quercetin
While the degradation of rutin in the solid state was studies at
an increased relative air humidity (RH = 76.4%) at 353 K, in dry air
Fig. 1. Chemical structure of quercetin-3-O-rutinosideand its impurity.

134
M. Paczkowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
2
1
400
600
800
1000
1200
1400
1600
1800
Wavenumber (cm-1)
Fig. 2. Calculation (1) and experimental (2) IR absorption spectra of rutin at room temperature, in polycrystalline powder.
at 383 K, and as the effect of exposition to light (1.2 ꢂ 10ꢁ6 Lux/h).
At specified time intervals, determined by the rate of degradation,
the samples of rutin (5.0 mg) were removed, cooled to room
temperature and their contents were dissolved in 5.0 mL of
methanol. The so-obtained solutions were quantitatively trans-
ferred into measuring flasks and diluted with water to 25.0 mL
and spectroscopic determination was conducted.
1362 cmꢁ1. This band was located at the 1362 cmꢁ1 in experimen-
tal spectra and it had also additional components associated with
the stretching vibration of the CAO bond and bending vibra-
tion of the CACAH bond in dihydoxyphenyl ring, stretching
vibration of the CAC bond in the chromen structure and bending
vibration of the CAOAH bond in benzene ring. In calculated IR
absorption spectra the band associated with the above-listed vibra-
tions was located at the 1332 cmꢁ1. The bands related to the bend-
ing vibration of the CACAH and CAOAH bonds in benzene and
dihydroxyphenyl rings and rocking vibration of the CAH bonds at
mannopyranosyl and glucopyranosyl rings were located at the
1183 and 1207 cmꢁ1 in calculated spectra. In experimental IR spec-
tra these bands were observed at the 1149 and 1166 cmꢁ1 and were
also quit strong intensity and well separated. One of the most
intense bands in experimental IR absorption spectra was located
at the 1295 cmꢁ1 and is related to the bending vibration of the
CACAH bonds and rocking vibration of the CAH bonds in all rings.
In the calculated spectra these band was small and it was located at
the 1307 cmꢁ1. The bands corresponding to the stretching vibration
of the CAC bond in the rings in calculated spectra at 704, 993, 998,
and 1236 cmꢁ1 were observed. In experimental IR spectra the first
band was located at the 729 cmꢁ1, the next two bands in experi-
mental spectra were observed as a single band at 972 cmꢁ1 and at
1205 cmꢁ1. These bands had additional components responsible
to the rocking vibration of the CAH bond in the benzene and dihy-
droxyphenyl rings and bending vibration of the CAOAH in dihy-
droxyphenyl ring, and stretching vibration of the CAO band at the
heterocyclic ring in chromen-4-one. The band corresponding to
the stretching vibration of the CAO bond were also observed at
about 1000 cmꢁ1 and 1500 cmꢁ1. In the calculated and experimen-
tal IR spectra they were respectively located precisely at 1016/
1011, 1063/1043, 1095/1062, 1102/1094, 1495/1494, and 1556/
1553 cmꢁ1. The first band was related to the stretching vibration
of the CAO bond at the dihydroxyphenyl ring and bending vibration
of the CAOAH bond at dihydroxyphenyl ring too. The second and
fourth bonds were related to the stretching vibration of the CAO
bond between the mannopyranosyl and glucopyranosyl ring and
in themannopyranosyl ring. The third band besides vibration of
CAO bond had additional composite related to the stretching vibra-
tion of the CAC bond in mannopyranosyl and glucopyranosyl rings.
The last two strong bands had also additional composite corre-
sponding to the stretching vibration of the C@C bond in the benzene
ring and bending vibration of the CACAH and CAOAH bonds at
benzene and dihydroxyphenyl rings, respectively. Strong band
Results and discussion
The following spectroscopic methods: FT-IR, Raman, UV, EPR,
supporting by theoretical approach, were proposed for identifica-
tion and determination of quercetin-3-O-rutinoside.
FT-IR and Raman spectroscopy
Identification of rutin based on detail analysis of its FT-IR and
Raman scattering spectra where the type, position and intensity
of the bands significant for biological activity of rutin were
discussed. The main characteristic vibrations of quercetin-3-O-ruti-
noside were identified by comparing its experimental spectra with
the theoretical ones based on the density functional theory. Analy-
sis of rutin especially focused on characterization of bands which
monitoring permitted establish to change rutin toward to presence
of its impurities (quercetin and isoquercetin). The most characteris-
tic bands for rutin associated with vibrations of the bonds were
located between 500 and 1500 cmꢁ1.In this range at the 808, 879,
912, and 944 cmꢁ1 vibrations in experimental IR absorption spectra
were observed from the lowest number length, respectively. In cal-
culated spectra in low frequency at 804, 927, 936, and 973 cmꢁ1
these bands were located and related to the wagging vibration of
the CAH bonds, mainly at the dihydroxyphenyl ring. The band at
927 cm-1 had additional components related to the wagging vibra-
tion of the CAH bonds at mannopyranosyl ring and between them
and glucopyranosyl ring. Whereas the band at 973 cmꢁ1 was asso-
ciated with the wagging vibration of the CAH bond at glucopyran-
osyl ring. Strong and quite strong bands corresponding to the
rocking vibration of the CAH bonds at benzene, dihydroxyphenyl
and mannopyranosyl rings in the calculation spectra were located
at the 1351 and 1369 cmꢁ1. The first band was also related to the
bending vibration of the CAOAH bond in benzene, dihydroxy-
phenyl and mannopyranosyl rings. In experimental IR absorption
spectra the two bands were observed as a single signal at the

M. Paczkowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
135
Table 1
Main characteristic vibrational modes of rutin observed in experimental and calculated spectra.
IR
Raman
Band assignment
Wavenumber (cmꢁ1
)
Calc
Exp
630
654
729
808
Calc
Exp
574 Def. all rings
588
647
704
804
588
Def. benzene, heterocyclic and mannopyranosyl rings
CAC s in all rings
793 CAH w at dihydroxyphenyl ring + breathing dihydroxyphenyl ring
850 CAC s in glucopyranosyl ring
899 CAH w at dihydroxyphenyl and mannopyranosyl rings and between mannopyranosyl and glucopyranosyl rings
CAH wop (poza płaszczyzna) atdihydroxyphenyl ring
804
846
927
927
936
973
993
998
1016 1011
1063 1043
1095 1062
879
912
944
CAH w at glucopyranosyl ring
CAC s in mannopyranosyl and glucopyranosyl rings
972
CACAC b in dihydroxyphenyl ring + CAC s in chromen ring structure and dihydroxyphenyl rings
CAO s at dihydroxyphenyl ring + CAOAH b at dihydroxyphenyl ring
CAO s between mannopyranosyl and glucopyranosyl rings
CAO s in heterocyclic and mannopyranosyl rings + CAO s between heterocyclic and mannopyranosyl ring and between mannopyranosyl
and glucopyranosyl rings + CAC s in mannopyranosyl and glucopyranosyl rings + CAC s in 4 and 5 rings
CAO s in and at mannopyranosyl ring
1102 1094
1137 1133 1137 1131 Breathing mannopyranosyl ring + CAO s at mannopyranosyl ring + CAO s between mannopyranosyl and glucopyranosyl rings
1183 1149
1207 1166
1236 1205
CACAH b in benzene and dihydroxyphenyl rings
CAO-H b at benzene ring + CAH r at mannopyranosyl and glucopyranosyl rings
CAC s in heterocyclic ring + CAH r in benzene and dihydroxyphenyl rings + CAO-H b in dihydroxyphenyl ring + CAO s at heterocyclic
ring
1251 1228 CAO-H b at benzene ring + CAO s and CAC s in heterocyclic ring
1302 1299 CACAH b + CAO-H b at benzene, heterocyclic and mannopyranosyl rings
CACAH b in all rings + CAH r at all rings
1307 1295
1332 1362 1332 1365 CAO s at dihydroxyphenyl ring + breathing dihydroxyphenyl ring + CACAH b in dihydroxyphenyl ring + CAC s in benzene and
heterocyclic rings + CAO-H b in benzene ring
1351
1369
1351
1369
CAH r at benzene, dihydroxyphenyl and mannopyranosyl rings + CAO-H b in benzene, dihydroxyphenyl and mannopyranosyl rings
CAH r at mannopyranosyl ring
1495 1494 1495 1502 C@C s in benzene ring
1510 CAC s in dihydroxyphenyl ring + CAO-H b at dihydroxyphenyl ring
1556 1553
1646 1556
CACAH b at dihydroxyphenyl ring + CAO-H b at benzene ring
C@C s in benzene and dihydroxyphenyl rings
1664 1573 1664 1608 C@C s in benzene, heterocyclic and dihydroxyphenyl rings
1678 1599 1668 1626 C@C s in benzene and heterocyclic rings
1751 1655 1751 1659 C@O s at heterocyclic ring
Vibrational modes: s – stretching, b – bending, w – wagging, r – rocking.
corresponding to the stretching vibration of the C@O bond at het-
erocyclic in chromen structure in calculated spectra at 1751 cmꢁ1
was observed. In experimental IR spectra it was located at
1655 cmꢁ1. In calculated spectra, the strong three bands associated
with the stretching vibration of the C@C bonds in unsaturated rings
were observed at 1646, 1664, and 1678 cmꢁ1. In the experimental
spectra were observed all three components related to these vibra-
tions at 1556, 1573, and 1599 cm-1, respectively. Above 2900 cmꢁ1
were observed the band corresponding to the stretching vibration
of the CAH bonds and at the range about 3500 cmꢁ1 were located
the bands associated with the stretching vibration of the OAH
bonds. The experimental and calculated IR spectra for rutin were
presented on Fig. 2 [21–22].
bending vibration of the CAOAH and CACAH bonds at the rings
in chromen structure and mannopyranosyl ring, was observed at
1299 cmꢁ1. In calculated spectra these band was observed at the
1302 cmꢁ1. Other band that can be identified in the Raman scatter-
ing, spectra were related mainly to the bending vibration of the
CAOAH bonds, and wagging vibration of the CAH bonds (Table 1).
In the higher frequencies at about 3000 cmꢁ1 the bands corre-
sponding to the stretching vibration of the OAH bonds were also
observed. The experimental and calculated Raman scattering spec-
tra for rutin were presented on Fig. 3.
UV spectroscopy
In the Raman scattering spectra, the strong bands associated
with the stretching vibration of the C@O and C@C bands were
observed and they were located at 1751/1659, 1668/1626, and
1664/1608 cmꢁ1 in calculated/experimental spectra, respectively.
Similarly to the calculated IR spectra, triplet bands were at1332,
The UV spectra of rutin was prepared by dissolving the rutin in
methanol. In the UV spectra of rutin, two inflections of gauss curve
were observed. Those are related to two different excited states of
molecule (Fig. 4). Our studies showed that kind of used solvent
(water, methanol and acetonitrile) did not significantly influence
on shape of direct spectra of rutin. However, differences in solubil-
ity of rutin in solvents (methanol, water, acetonitrile) strongly cor-
relate with intensity of UV spectra of rutin. Spectrophotometric
purity of experimental spectra of rutin in methanol was confirmed
by comparison with theoretical spectra. The application of direct
UV spectroscopy in identification of rutin provides satisfaction
results due to the differences in localization of maxima of direct
UV spectra for rutin (k = 353 nm, calculated k = 357 nm) and its
main impurity – quercetin (k = 371 nm, calculated k = 369 nm)
(Fig. 4). It was proved that during the degradation of rutin during
acidic hydrolysis, quercetin was formed as the main product. While
1351, and 1369 cmꢁ1
, which correspond to single band at
1362 cm-1in the experimental IR spectra. The quite strong band
was observed in the experimental Raman scattering spectra at
1502 cmꢁ1 and it was related to the two bands in calculated spec-
tra located at the 1495 and 1510 cmꢁ1. They were corresponding to
the stretching vibration of the CAO bond at rings in chromen struc-
ture, and to the stretching vibration of the C@C bond in the ben-
zene ring, and had components related to the stretching
vibration of the CAC bond and bending vibration of the CAOAH
bond at dihydroxyphenyl ring. One of the most intensity bands
in the experimental Raman scattering spectra, correspond to the

136
M. Paczkowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
2800
3000
3200
3400
3600
3800
4000
2
1
0
200
400
600
800 1000 1200 1400 1600 1800 2000
Wavenumber (cm-1)
Fig. 3. Calculation (1) and experimental (2) Raman scattering spectra of rutin at room temperature, in polycrystalline powder.
Fig. 4. UV spectra of rutin (R) and quercetin (Q) and theoretical spectra of rutin (R-T) and quercetin (Q-T).
as the results of degradation of rutin during basic hydrolysis, ther-
molysis, photolysis and oxidation, degradation products did not
containing chromophore structure were formed. To the above,
overlapping effect of degradants were eliminated therefore the lin-
ear concentration changes of rutin during degradation were possi-
ble to record at wavelength 353 nm. The indirect UV spectra for
degraded samples of rutin are presented in Fig. 5.The changes of
absorption of degraded samples of rutin occurs according to the
kinetics of a first-order reaction relative to substrate concentration
because the values of peak amplitudes of the first derivative
action linked to the possibility of pairing reactive oxygen species
(ROS) and that any ingredient of plant origin is prone to change
on exposure to light or increased relative air humidity and
temperature, the work evaluated the influence of those most com-
mon factors affecting the state of solid-phase raw materials during
storage. EPR-based analysis enabled the detection of radicals in
non-degraded samples of rutin and the identification of free
radicals as well as monitoring their concentration in rutin samples
subjected to external factors. The level of radicals in non-degraded
samples of rutin was found to be very low (1.2 ( 0.2) ꢂ 1015 radi-
cals/g) whereas significant changes in the concentration of free
decreased from Amax to A0over a period of time from t0 to t So
1.
far, the application of direct spectroscopy in analysis of rutin pro-
vided satisfaction results for its identification as well as its deter-
mination in presence of degradation products.
radicals
were
observed
as
a
result
of
irradiation
(56.2 ( 3.0) ꢂ 1015 radicals/g) (Fig. 6). The subjection of samples
to visible radiation made it possible to identify radicals I, II and
III with varying spectroscopic coefficients and time-dependent sig-
nals. The values of g–factors (I and II) are very close to those
observed for semiquinone radicals and indicate that an unpaired
electron is localized on the dihydroxyphenyl ring and/or chromen
structure. Unpaired electron for a radical of type III with the
EPR spectroscopy
Considering the facts that the use of rutin in pharmaceutical
preparations and dietary supplements rests on its antioxidative

M. Paczkowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
137
Fig. 5. UV spectra for degraded samples of rutin after irradiation (A), thermolysis in dry air at 383 K (B), thermolysis at an increased relative air humidity (RH = 76.4%) at
353 K (C) and in the presence of oxidizing factor (10% H2O2) at 353 K (D).
Table 2
EPR parameters describing radicals.
Radicals
g-factor
Hyperfine constant A [G]
I
II
III
2.0047 ( 0.0002)
2.0045 ( 0.0002)
2.0023 ( 0.0002)
14 ( 2)
–
–
t
1
t
2
IðtÞ ¼ I1eꢁ þ I2eꢁ þ I0
t
t
where: I(t)
– free radical concentration at any time t after
irradiation, I0 – concentration of stable radicals, I1 – concentration
of radicals disappearing at mean lifetime t1, I2 – concentration of
radicals disappearing at mean lifetime t2. Thus, the time at which
the level of free radicals reached a constant value was determined.
No significant changes in the concentration of radicals were
observed when the solid phase samples were exposed to increased
temperature and relative air humidity.
Theoretical studies
Fig. 6. EPR spectra of nonirradiated and irradiated samples of rutin.
During the theoretical investigation, based on the analysis of
the HOMO structure it was suggested that two radicals (I and
II) formed within the dihydroxyphenyl ring at the O15 and O16
oxygen atoms of its hydroxyl groups in a rutin molecule. Gauss-
ian software was employed to establish the g-factors for the
g-value close to free electron should be probably localized on the
glucopyranosyl or mannopyranosyl ring. The following equation
describes the disappearance of free radicals in the irradiated rutin
samples as a function of time:

138
M. Paczkowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
role in the electronic and optical properties of molecules [23].
The frontier molecular orbitals (FMOs) are named the highest
occupied molecular orbitals (HOMO) and the lowest unoccupied
molecular orbitals (LUMO). The presence of the highest occupied
molecular orbitals (HOMO) and the lowest unoccupied molecular
orbital (LUMO) in molecule is connected with ‘‘filling-empty’’
interactions which can cause the stabilizing all molecule. 3D plots
of the HOMO–LUMO for the rutin was presented on Fig. 8. In the
case of localization of the HOMO and LUMO orbitals of rutin, it
was excluded molecules of mannopyranosyl and glucopyranosyl
rings. The HOMO are localized mainly on dihydroxyphenyl ring
of rutin. While the localization of LUMO was observed generally
on dicyclic ring structure. The energy gap of HOMO–LUMO
explains the charge transfer interaction within molecule and in
the result of characterization the molecular chemical stability,
chemical reactivity of molecule [24]. The value of the energy sep-
aration between the HOMO and LUMO is 3.98 eV. The small dif-
ferences between energy gap of rutin can explain the significant
susceptibility to photodegradation which is connected with trans-
fer electrons between those orbitals. Next important parameter is
molecular electrostatic potential (MEP) which is very useful
descriptor in understanding sites for electrophilic/ nucleophilic
reactions and hydrogen bonding interactions (Fig. 9.). The nega-
tive (red and yellow) regions of MEP which are related to electro-
philic reactivity are associated with carbonyl group (C@O) and
hydroxyl group (C-2) in dicyclic ring structure. The positive
regions (blue) in rutin were localized on hydroxyl group of chro-
men structure which are not neighbors for mannopyranosyl and
glucopyranosyl rings. Such location of MEP for rutin allowed to
suggest the influence of presence of sugars moieties on its reac-
tivity and in the result of its biological properties. The FT-IR
and Raman spectra were predicted for separate molecule of rutin
in vacuum environment. Frequencies were scaled by scale factor
of 0.9945 [25], according to used basis set. Theoretical spectra
were confronted with experimental FT-IR (Fig. 2) and Raman
(Fig. 3) spectra. Significant differences between theory and exper-
iment were observed, which are caused by approximation of cal-
culating FT-IR and Raman spectra for separate molecule. In
experiment, effects of hydrogen bonding and intermolecular
interactions between rutin molecules can incorporate significant
differences to spectra in comparison to theory.
Fig. 7. Optimized geometry of the rutin/2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-
oxo-4H-chromen-3-yl 6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside/(gray
– carbon, white – hydrogen, red – oxygen – theory level B3LYP/6-31G(d)).
obtained radicals (Table 2). The g-factor tensor was calculated
with GIAO G and DFT methods, the latter using the B3LYP
functional and EPR-III basis set optimized for that type of calcu-
lations. It should be noted that since the error for the g-factors
(Dg) was a systematic one, it probably resulted from the calcula-
tion method. The third radical did not form at the AOH groups of
the chromen structure.
As the result of theoretical studies, it was possible to establish
parameters which are important for prediction of reactivity and
stability of rutin. The analysis of optimized geometry of rutin
showed the intra ring stresses in areas of mannopyranosyl and
glucopyranosyl rings. It can suggests those place are the most
susceptible to degradation (Fig. 7) The FMOs play an important
Fig. 8. HOMO (A) and LUMO (B) orbitals of quercetin-3-O-rutinoside.

M. Paczkowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 132–139
139
Acknowledgements
This study was supported by grant from the Ministry of Science
and Higher Education (Diamentowy Grant DI2012 024342).
This research was supported in part by PL-Grid Infrastructure.
References
[1] N. Fabjan, J. Rode, I.J. Kosir, Z.H. Wang, Z. Zhang, A.I. Kreft, J. Agric. Food Chem.
51 (2003) 6452–6455.
[2] European Pharmacopoeia 7.0 vol. 2, monographs: rutosidetrihydrate, 2871–
2873.
[3] N.C. Cook, D. Samman, J. Nutr. Biochem. 7 (1996) 66–76.
[4] J. Yang, J. Guo, J. Yuan, Food Sci. Technol. 41 (2008) 1060–1066.
[5] P. Iacopini, M. Baldi, P. Storchi, L. Sebastiani, J. Food Compos. Anal. 21 (2008)
589–598.
[6] U. Takahama, M. Tanaka, S. Hirota, R. Yamauchi, Food Chem. 116 (2009) 214–
219.
[7] L.N. Grinberg, E.A. Rachmilewitz, H. Newmark, Biochem. Pharmacol. 48 (1994)
643–649.
[8] K.H. Janbaza, S.A. Saeedb, A.H. Gilani, Fitoterapia 73 (2002) 557–563.
[9] R.J. Nijveldt, E. van Nood, D.E.C. van Hoorn, P.G. Boelens, K. van Norren, P.A.M.
van Leeuwen, Am. J. Clin. Nutr. 74 (2001) 418–425.
[10] Y. Zu, C. Li, Y. Fu, C. Zhao, J. Pharm. Biomed. Anal. 41 (2006) 714–719.
[11] V. Kuntic, N. Peji, B. Ivkovic, Z. Vujic, K. Ilic, S. Mici, V. Vukojevic, J. Pharm.
Biomed. Anal. 43 (2007) 718–721.
[12] F. Fang, J.M. Li, Q.H. Pan, W.D. Huang, Food Chem. 101 (2007) 428–433.
[13] K. Ishii, T. Furuta, Y. Kasuya, J. Chromatogr. B 759 (2001) 161–168.
[14] H. Xu, Y. Li, H.W. Tang, C.M. Liu, Q.S. Wu, Anal. Lett. 43 (2010) 893–904.
[15] J. Wang, L.L. Zhao, G.X. Sun, Y. Liang, F.A. Wu, Z.L. Chen, Afr. J. Biotechnol. 10
(2011) 1460–1466.
[16] G.J. Smith, S.J. Thomsen, K.R. Markhama, C. Andary, D. Cardon, J. Photochem.
Photobiol. A 136 (2000) 87–91.
[17] A.H. Elhamirad, M.H. Zamanipoor, Eur. J. Lipid Sci. Technol. 114 (2012) 602–
606.
[18] V.C. Mai, W. Bednarski, B. Borowiak-Sobkowiak, B. Wilkaniec, S.
Samardakiewicz, I. Morkunas, Phytochemistry 93 (2013) 49–62.
[19] Gaussian 03, Revision D.01, M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E.
Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin,
J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M.
Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H.
Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo,
R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W.
Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G.
Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D.
Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S.
Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I.
Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A.
Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong,
C. Gonzalez, J.A. Pople, GaussianInc, Pittsburgh, PA, 2003.
[20] GaussView, Version 5, R. Dennington, T. Keith, J. Millam, Semichem Inc.,
Shawnee Mission, KS, (2009).
[21] G. Varsanyi, Assignments for Vibrational Spectra of Seven Hundred Benzene
Derivatives, vol. 1, Wiley, New York, 1974.
[22] D.L. Pavia, G.M. Lampman, G.S. Kris, Introduction to Spectroscopy, second ed.,
Harcourt Brace College, 1996. pp. 28–31.
[23] O.E. Piro, G.A. Echeverría, E. Lizarraga, E. Romano, C.A.N. Catalán, S.A. Brandán,
Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 101 (2014) 196–200.
[24] N. Islam, S. Niaz, T. Manzoor, A.H. Pandith, Spectrochim. Acta Part A Mol.
Biomol. Spectrosc. 131 (2014) 461–470.
Fig. 9. MEP of quercetin-3-O-rutinoside.
Conclusions
The spectroscopic methods (FT-IR, Raman, UV) proposed for
the evaluation of rutin allow its identification in the presence of
observed impurities (quercetin and isoquercetin). Especially, FT-
IR spectra (below 1500 cmꢁ1) were useful in identification of
rutin in presence of its impurities. Additionally, the registration
of adsorption spectra of rutin permits selective assays of its and
studying its conversion to degradation products, quercetin and
isoquercetin. Moreover, the indication of spectroscopic methods
as the most appropriate test for monitoring the identity and sta-
bility of glycosidic linkages within the rutin, is a better solution
than the separation methods based on solvents which may induce
the hydrolysis of these bonds. It is also possible to monitor and
classify the radicals formed and to determine their lifespan by
analyzing the electronic spectra (EPR) of rutin samples exposed
to such degradation factors as photolysis and thermolysis in the
solid phase.
[25] A.P. Scott, L. Radom, J. Phys. Chem. 100 (1996) 16502–16513.