The influence of the host-guest interaction on the oxidation of natural flavonoid dyes

Article abstract of DOI:10.1135/cccc2011106

The influence of the molecular cavity protection on degradation processes of bioorganic compounds quercetin and luteolin used as the original dyes in old tapestries was studied. The degradation processes were studied by electrochemical methods in aqueous media. The products of the exhaustive electrolysis were separated and identified by GC-MS analysis. Cyclic voltammetry characteristics indicate that the inclusion complex is formed. The inclusion affects the redox potentials of both oxidation waves related to the different dissociation forms of the flavonoid molecule. It was shown that decomposition products formed by the oxidation of quercetin are stabilized in the cavity of β-cyclodextrin, including the main oxidation product 2(3',4'- dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one. The formation of the 1:1 inclusion complex of luteolin with β-cyclodextrin is supported by the enhancement of fluorescence intensity. In the case of quercetin, a decrease of fluorescence intensity occurs when 1:1 inclusion complex with β-cyclodextrin is formed.

Full text of DOI:10.1135/cccc2011106

Influence of the Host–Guest Interaction on the Oxidation
1651
THE INFLUENCE OF THE HOST–GUEST INTERACTION ON
THE OXIDATION OF NATURAL FLAVONOID DYES
b1
Šárka RAMEŠOVÁa1, Romana SOKOLOVÁa2,*, Ilaria DEGANO
,
Magdaléna HROMADOVÁa3, Miroslav GÁLa4, Viliam KOLIVOŠKA and
a5
b2
Maria Perla COLOMBINI
a
J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i.,
Dolejškova 3, 182 23 Prague, Czech Republic; e-mail: sarka.tce@seznam.cz,
romana.sokolova@jh-inst.cas.cz, magdalena.hromadova@jh-inst.cas.cz,
1
2
4
3
5
miroslav.gal@jh-inst.cas.cz, viliam.kolivoska@jh-inst.cas.cz
b
Department of Chemistry and Industrial Chemistry, University of Pisa, Via Risorgimento 35,
1
2
56100 Pisa, Italy; e-mail: ilariad@dcci.unipi.it, perla@dcci.unipi.it
Received May 20, 2011
Accepted July 18, 2011
Published online January 4, 2012
Paper submitted for a special issue of CCCC devoted to “distinguished followers of the J. Heyrovský
School of Polarography”. This work is dedicated to Dr. L. Pospíšil on the occasion of his 70th birthday.
The influence of the molecular cavity protection on degradation processes of bioorganic
compounds quercetin and luteolin used as the original dyes in old tapestries was studied.
The degradation processes were studied by electrochemical methods in aqueous media. The
products of the exhaustive electrolysis were separated and identified by GC-MS analysis. Cy-
clic voltammetry characteristics indicate that the inclusion complex is formed. The inclu-
sion affects the redox potentials of both oxidation waves related to the different dissociation
forms of the flavonoid molecule. It was shown that decomposition products formed by the
oxidation of quercetin are stabilized in the cavity of β-cyclodextrin, including the main oxi-
dation product 2(3′,4′-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one. The forma-
tion of the 1:1 inclusion complex of luteolin with β-cyclodextrin is supported by the
enhancement of fluorescence intensity. In the case of quercetin, a decrease of fluorescence
intensity occurs when 1:1 inclusion complex with β-cyclodextrin is formed.
Keywords: Oxidation; Inclusion complexes; Protection against oxidation; Flavonoids; Cyclic
voltammetry; Natural products.
Quercetin (3,3′,4′,5,7-pentahydroxyflavone) and luteolin (3′,4′,5,7-tetra-
hydroxyflavone) are widely studied because of their pharmacological im-
portance (Chart 1). These flavonoid compounds have antioxidant activity,
antihepatotoxic and anti-inflammatory properties1. They belong to the his-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011106

1652
Ramešová et al.:
torical natural dyes used already in 15th and 16th century. They were ex-
tracted from vegetal and animal sources; yellow dyes were achieved by
extracting flavonoids and their glycosides from a number of widespread
plants (some species of Rhamnus, quercitron bark, Quercus velutina L., Alnus
jorullensis, Salvia sagittata)2,3. The original color of medieval textiles is
transformed by ambient aerobic environment. The oxidative degradation
processes involve the presence of subsequent chemical reactions such as
hydroxylation or dimerization4. The oxidative degradation leads to the for-
mation of low molecular weight compounds such as 3,4-dihydroxybenzoic
acid and gallic acid5,6
.
4'
OH
OH
OH
OH
B
3'
2
HO
O
HO
O
7
A
C
3
OH
5
4
OH
O
OH
O
quercetin
luteolin
CHART 1
In order to establish the complexation properties of flavonoid molecules
in the presence of host molecule it is necessary to have the knowledge of
the dissociation constant of quercetin and luteolin. There are many studies
dealing with estimation of dissociation constant in the literature. The con-
troversy reported on the acid-base properties of both flavonoids was ex-
plained by strong influence of the oxygen on the stability of their
dissociation forms in our recent paper7,8. We reported values of dissociation
constant pK1 = 5.87 0.14 and pK2 = 8.48 0.09 of quercetin and pK1 =
5.99 0.32 and pK2 = 8.40 0.42 of luteolin, which were found under strict
anaerobic conditions. The value of pK1 of quercetin agrees with the lowest
value reported in the literature by Escandar et al.9.
The complexation properties of quercetin were mentioned in the case of
its non-stable Cu(2+)-chelate5. The formation of a flavonoid-glutathione
conjugate was shown by peroxidase-catalysed one electron oxidation of
quercetin and luteolin in the presence of glutathione10. The stability con-
stant of inclusion complex of undissociated quercetin with β-cyclodextrin
–1
(β-CD) determined by phase solubility study at pH 3 is 1028 M according
to Zheng et al.11. Values of stability constant found by fluorescence spec-
troscopy by Jullian et al.12,13 at pH 7.4 correspond to mixture of dissociated
and undissociated forms of quercetin or luteolin.
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Influence of the Host–Guest Interaction on the Oxidation
1653
The present paper proves the protective properties of β-cyclodextrin
against the oxidation process of the flavonoid dyes quercetin and luteolin.
The protection properties of β-CD against the oxidation was found in case
of catechin14, chlorogenic acid15, ferrocene derivatives15,16 and chlor-
promazine17. In addition, cyclodextrins have been successfully immobilized
by physical or chemical bonds on the surface of fibres18,19. Since these
flavonoid dyes undergo decomposition in the alkaline aqueous solution in
the presence of oxygen12, the study of the degradation processes of an an-
ionic molecule of quercetin and luteolin encapsulated by β-CD under inert
atmosphere of argon is proposed. Our preliminary results confirm that hy-
droxyl groups substituted on ring B of the flavonoids are protected against
the oxidation by encapsulating by molecule of β-CD. The protection of
these bioorganic molecules against the oxidation in the environment of
cavity of a cyclodextrin has importance not only for restoration works and
the best conservation of the tapestries and saving of the cultural heritage.
The advantage of the complexation properties of these antioxidants turns
profit in medical and cosmetic applications.
EXPERIMENTAL
Reagents
Quercetin and luteolin were purchased from Fluka. The reagents used as supporting electro-
lyte as tetrabutylammoniumhexafluorophosphate and acetonitrile were of reagent grade.
All the solvents were Carlo Erba (Milan, Italy) HPLC grade except from ethyl acetate
(AcOEt), Anal R, BDH. Hexadecane and 2,4-dihydroxybenzophenone, used as internal stan-
dards, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethyl-
chlorosilane were purchased from Sigma (Milan, Italy). Standard solutions of analytes for
GC-MS analysis were prepared in methanol (Carlo Erba, Milan, Italy).
All reagents and chemicals were used without any further purification.
Methods
Electrochemical measurements were done using an electrochemical system for cyclic
voltammetry. It consisted of a fast rise-time potentiostat interfaced to a personal computer
via an IEEE-interface card (AdvanTech, model PCL–848) and a data acquisition card
(PCL-818) using 12bit precision. Cyclic voltammetry was also conducted using a PGSTAT 12
AUTOLAB potentiostat. A three-electrode electrochemical cell was used with an Ag|AgCl|
1 M LiCl reference electrode separated from the test solution by a salt bridge. The working
electrodes were platinum electrode (0.8 mm) and glassy carbon electrode (0.7 mm). The aux-
iliary electrode was cylindrical platinum net. Oxygen was removed from the solution by
passing a stream of argon. The oxidation products of quercetin were prepared by exhaustive
electrolysis of its 0.7 × 10–3–1 × 10–3 M solutions on carbon paste electrode.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667

1654
Ramešová et al.:
Gas chromatography. The products were identified using a Trace GC gas chromatograph
(Thermo Electron Corporation, USA) equipped with a PTV injection port and a mass spectro-
metric detector based on an ion trap analyzer (Polaris Q, Thermo Electron Corporation,
USA). The PTV injector was in the CT ′splitless with surge′ mode at 280 °C with a surge pres-
sure of 100 kPa, and the mass spectrometer parameters were: electronic impact ionization
(70 eV), ion source temperature 230 °C, scan range m/z 50–700 and interface temperature
280 °C. Chromatographic separation was performed on a DB-5MS chemically bonded fused
silica capillary column (J & W Scientific, Agilent Technologies) with stationary phase
5% phenyl–95% methylpolysiloxane, and of dimensions 0.25 mm i.d., 0.1 µm film thick-
ness, 25 and 30 m length. The gas chromatographic conditions were as follows: initial tem-
perature 57 °C, 2 min isothermal, then ramped at 10 °C/min up to 200 °C, 3 min
isothermal, then ramped at 20 °C/min up to 300 °C and then isothermal for 20 min. The
carrier gas was He (purity 99.9995%), at a constant flow rate of 1.2 ml/min. The peak assign-
ment was based on comparison with analytical reference compounds and materials with
library mass spectra (NIST 1.7) and with mass spectra reported in the literature.
In order to perform GC-MS analysis, electrolysis products were derivatised with a
silylating agent N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Derivatisation conditions
are: 10 µl of 2,4-dihydroxybenophenone (solution in isopropanol; internal standard IS1) is
added to the sample; the solution is dried and 30 µl of the derivatisation agent BSTFA in
50 µl of AcOEt is added; the reaction takes place in closed glass vials at 60 °C for 30 min.
Just before injection, 10 µl of hexadecane (solution in isooctane; internal standard IS2) and
150 µl of AcOEt is added; 2 µl of the final solution is injected in the GC system.
Fluorescence spectroscopy. Fluorescence spectra were measured on LS50B luminescence spec-
trometer (Perkin–Elmer) in 1 cm2 quartz cuvettes with both excitation and emission slit set
at 10 nm. In the case of both flavonoids, quercetin and luteolin, the emission spectra were
the recorded at the excitation wave length λ = 365 nm, where the highest sensitivity of
emission was obtained. Since the flavonoid compounds decompose in the presence of air
oxygen, all the solutions were prepared under the deaerated conditions. Data were statisti-
cally analyzed according to Eckschlager20
.
RESULTS AND DISCUSSION
The Electrochemical Properties
The electrochemical properties of quercetin and luteolin in aqueous media
are related to the presence of their dissociation forms in solution. Similar
behaviour is known in the case of the oxidation of other flavonoid com-
pounds in aqueous solution21. In acidic and neutral solution cyclic voltam-
metry of quercetin on glassy carbon electrode yields three oxidation waves
up to the potential 1.2 V (curve 1, Fig. 1). The first oxidation wave belongs
to two electron oxidation of 3′,4′-dihydroxymoiety on ring B 4,22,23. The
oxidation wave of luteolin (curve 2, Fig. 1) is more reversible in compari-
son with the oxidation wave of quercetin. The quinoid oxidation product
formed by oxidation of quercetin and luteolin undergoes further chemical
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Influence of the Host–Guest Interaction on the Oxidation
1655
0.0
–2.0
–4.0
1.0
0.5
0.0
–0.5
E, V
FIG. 1
Cyclic voltammogram of 2 × 10–4 M quercetin (a) and luteolin (b) in the solution of Britton–
Robinson buffer and ethanol 3:2 (v/v) at pH 6.9 on glassy carbon microelectrode. The scan rate
is 0.25 V s–1
0
b
a
3.0
–2
2.0
1.0
–4
0.0
0.0
0.5
1.0
1.5
ν1/2
1.0
0.5
0.0
E, V
FIG. 2
Cyclic voltammogram of 6.5 × 10–4 M quercetin in 0.1 M KCl and 3.6 × 10–3 M KOH on glassy
carbon microelectrode at different concentration of β-cyclodextrin: 0 (a), 1 × 10–3 M (b). The
scan rate is 0.065 V s–1. The dependence of the anodic peak current on scan rate is shown in
inset
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667

1656
Ramešová et al.:
reaction in the case of quercetin and the oxidation product 2(3′,4′-di-
hydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one is formed24. Its
oxidation occurs at the second oxidation wave28. The complexation study
is performed in alkaline aqueous solution because of poor solubility of both
compounds and to avoid using of ethanol. The molecule of quercetin and
luteolin is under alkaline conditions dissociated (pKque = 5.87 and pKlut
=
5.99)8,9. Cyclic voltammetry of quercetin in alkaline solution on glassy
carbon electrode shows three oxidation waves up to the potential 1.5 V
(curve 1, Fig. 2). The first oxidation wave at 0.05 V corresponds to the one
electron oxidation of dissociated form of quercetin25,26. The consumption
of one electron was confirmed coulometrically. From the linear dependence
of peak current on square root of scan rate was found that this one electron
process is diffusion controlled. At the potential of second oxidation wave
a two electron oxidation of quercetin takes place. The third oxidation
wave belongs to the oxidation of the oxidation product 2(3′,4′-dihydroxy-
benzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one.
When the β-cyclodextrin is present in the solution, the potential of the
first oxidation wave is shifted to the more positive values (curve 2, Fig. 2).
The height of the oxidation wave is significantly lower in the presence
of β-CD compare to the height of a free quercetin. This indicates that
quercetin forms a complex with β-CD. The oxidation of complexed quer-
cetin is more difficult and requires the dissociation of an inclusion com-
plex, i.e. CE mechanism is involved in the oxidation mechanism (Eq. (1))27
Kd
[AH·CD]
AH + CD
KS
electron
transfer
(1)
products
where KS is the constant of stability, Kd is the dissociation constant of a
complex.
This one electron process is diffusion controlled, what was proved by
linear dependence of peak current on square root of scan rate (see inset in
Fig. 2). The dependence of the peak potential on the scan rate δE/δlog v =
19 mV indicates that a disproportionation reaction takes place. The higher
value of potential in the presence of β-cyclodextrin proves that a cavity of
β-CD serves as a stabilizer of the molecule of quercetin.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667

Influence of the Host–Guest Interaction on the Oxidation
1657
A stability constant of the inclusion complex can be evaluated from the
change of the standard redox potential in the absence and presence of
complexing agent (Eq. (2))28,29
RT
nF
D
RT
RT
nF
(E0 )complex − (E0 )free
=
ln
−
ln KS −
ln[CD]
(2)
D* nF
where D and D* represent the diffusion coefficient of the free and com-
plexed molecule, respectively, KS is a stability constant of the complex. The
values of standard redox potential were determined from cyclic voltam-
mograms of quercetin in the absence and presence of β-CD on platinum
microelectrode (Fig. 3). The inset of Fig. 3 shows the dependence of the
peak current on square root of scan rate and confirmes that the oxidation
process is diffusion controlled. The difference of standard redox potentials
extrapolated from values of the anodic and cathodic potential (Fig. 4) is in-
cluded in Table I. The estimation of standard redox potential for luteolin in
the absence and presence of β-CD is in Fig. 5. The constants stability deter-
mined from the Eq. (2) are summarized in Table I for both flavonoid com-
pounds, quercetin and luteolin. The cyclic voltammetry in the presence
and in the absence of β-cyclodextrin (Fig. 2) shows that both dissociation
forms are affected by the presence of β-CD. The shift of the peak potentials
0.0
–1.0
3.0
b
2.0
a
1.0
–2.0
0.0
0.0
0.5
1.0
1.5
ν1/2
0.0
–0.1
–0.2
–0.3
E, V
FIG. 3
Cyclic voltammogram of 6.5 × 10–4 M quercetin in 0.1 M KCl and 3.6 × 10–3 M KOH on Pt
microelectrode at different concentration of β-cyclodextrin: 0 (a), 1 × 10–3 M (b). The scan rate
is 0.032 V s–1. The dependence of the anodic peak current on scan rate is shown in the inset
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667

1658
Ramešová et al.:
to the higher values confirms that both oxidation places, catechol ring B
and hydroxyl group OH-7 interact with the cavity of β-cyclodextrin as was
calculated by Yan et al.30. Bergonzi et al.31 found that the interaction of
B-ring of quercetin with β-cyclodextrin is the most favored. Jullian et al.13
recently found that in case of the inclusion complex luteolin:β-cyclodextrin
the B-ring of luteolin is oriented toward the primary ring of β-CD, while the
A-ring of luteolin remains exposed to the external surface by the secondary
ring.
a
b
0.0
–0.1
–0.2
a
E
0.0
–0.1
–0.2
a
p
E
p
E
= –0.139 V
0
E
= –0.120 V
0
c
c
E
E
p
p
–1.0 –0.5
0.0
0.5
1/2
1.0
1.5
–2.0 –1.5 –1.0 –0.5 0.0 0.5 1.0 1.5
1/2
ν
ν
FIG. 4
The dependence of the peak potential on the square root of scan rate of 6.5 × 10–4 M quercetin
in 0.1 M KCl and 3.6 × 10–3 M KOH on Pt microelectrode in the absence (a) and in the presence
of 1 × 10–3 M β-cyclodextrin (b)
0.30
a
b
0.25
0.20
0.25
0.20
0.15
E
= 0.154 V
0
0.15
0.10
E
= 0.160 V
0
–1
0
1
2
–2
0
2
1/2
1/2
ν
ν
FIG. 5
The dependence of the peak potential on the square root of scan rate of 1 × 10–3 M luteolin in
0.1 M KCl and 3.6 × 10–3 M KOH on Pt microelectrode in the absence (a) and in the presence of
2 × 10–3 M β-cyclodextrin (b)
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Influence of the Host–Guest Interaction on the Oxidation
1659
TABLE I
The determination of the stability constant of the inclusion complex of dissociated
flavonoid:β-cyclodextrin
Cyclic voltammetry
Fluorescence
–1
–1
(E0)free, V
(E0)complex, V
K, M
K, M
Luteolin
0.1541 0.0049
–0.1392 0.0038
0.1601 0.0058a
–0.1202 0.0041b
520 220
600 200
720 170
515 50
Quercetin
a
b
The ratio of the concentrations luteolin:β-cyclodextrin is 1:2. The ratio of the concentra-
tions quercetin:β-cyclodextrin is 1:2.
Fluorescence Spectroscopy
In order to support the formation of an inclusion complex of studied
flavonoids with β-cyclodextrin the fluorescence measurements were pro-
vided. The fluorescence spectra of luteolin recorded at different concentra-
tions of β-cyclodextrin are shown in Fig. 6. The increasing concentration of
β-cyclodextrin causes the enhancement of fluorescence intensity of luteo-
lin. Since β-cyclodextrin does not yield emission under the same experi-
8.0
1
7
6.0
6
5
4.0
2.0
0.0
0
4
3
2
1
0
1000
2000
1/c
500
600
700
λ, nm
FIG. 6
Fluorescence emission spectra of 3 × 10–4 M luteolin in 3.6 × 10–3 M KOH at different concen-
tration of β-cyclodextrin: 0 (1), 2.3 × 104 (2), 3.8 × 104 (3), 9.5 × 104 (4), 1.2 × 103 (5), 2.9 × 103
(6), 3.7 × 103
M
(7) at λexc = 365 nm. The Benesi–Hildebrand plot for 1:1 luteolin:
β-cyclodextrin inclusion complex is shown in inset
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667

1660
Ramešová et al.:
mental conditions, this behaviour indicates the formation of an inclusion
complex.
The stability constant (KS) of the inclusion complex can be calculated by
the use of the modified Benesi–Hildebrand equation32,33
1
1
1
=
+
(3)
F − F0
F∞ − F0 (F∞ − F0 )KS [CD]0
where [CD]0 is the initial concentration of cyclodextrin, F0 denotes the
fluorescence intensity of flavonoid in the absence of cyclodextrin, F∞ is the
fluorescence intensity when all of the flavonoids molecules are essentially
complexed with cyclodextrin, F is the observed fluorescence at each tested
concentration of cyclodextrin, and KS is the stability constant of the com-
plex. The inset of the Fig. 6 shows the dependence of 1/(F – F0) on 1/[CD]
and a straight line is obtained (coefficient of determination D = 100R2
=
96.83). This indicates that the ratio luteolin:β-CD is 1:1. The same ratio was
confirmed also in the case of quercetin and agrees with findings in litera-
ture13,34. The continuous variation method confirmed also the ratio 1:1
(Fig. 7). The emission band at 530 nm is characteristic for pyrilium tau-
tomers35. The intensity of fluorescence of luteolin is very low due to the fai-
ble formation of a pyrilium form upon intramolecular proton transfer from
60
50
40
30
20
10
0
0.2
0.3
0.4
0.5
0.6
0.7
r
FIG. 7
Continuous variation plot for quercetin:β-cyclodextrin (᭹) and luteolin:β-cyclodextrin (᭺) in-
clusion complex (Jobb plot)
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667

Influence of the Host–Guest Interaction on the Oxidation
1661
OH-5 to O-4. The binding into the β-cyclodextrin cavity causes the proton-
ation of O-4 and the enhancement of fluorescence is obtained. Dangles et
al.41 underlined the fluorescence behaviour of pyrilium cations formed dur-
ing intramolecular protonation of some flavonoids. The fast intramolecular
proton transfer from OH-7 group to the 4-oxo group was obtained in the
case of 3-hydroxyflavone and 7-hydroxyflavone36,37
.
The change of the fluorescence spectra of quercetin shows a reversed
trend (Fig. 8). The intensity of emission spectra of quercetin decreases with
increasing concentration of β-cyclodextrin. The stability constant of an in-
clusion complex of dissociated quercetin with β-CD was determined from
the linear dependence shown in the inset of Fig. 8 (D = 99.77). The stability
constants are summarized in Table I. The ratio of the guest:β-cyclodextrin
was confirmed also by the continuous variation method (see Fig. 7). The
values found by fluorescence measurement are in agreement with values
obtained from cyclic voltammetry. The strong fluorescence of quercetin is
consistent with a pyrilium-like structure formed by the intramolecular
protonation of O-4 by OH-3 group. The binding into the cavity of β-cyclo-
dextrin probably avoid the intramolecular protonation of O-4 by OH-3
group. Nevertheless, the fluorescence intensity in the presence of β-CD
remains still higher than in the case of luteolin. The protonation of O-4
must occur in the cavity of β-CD.
1
2
0.010
300
3
4
5
6
7
200
0.000
0
2000
4000
1/c
100
0
8
9
500
600
700
λ, nm
FIG. 8
Fluorescence emission spectra of 3 × 10–4 M quercetin in 3.6 × 10–3 M KOH at different concen-
tration of β-cyclodextrin: 0 (1), 6.2 × 105 (2), 2.3 × 104 (3), 3.7 × 104 (4), 5.3 × 104 (5), 8.8 × 104
(6), 1.1 × 103 (7), 1.4 × 103 (8), 1.8 × 103 M (9) at λexc = 365 nm. The Benesi–Hildebrand plot for
1:1 quercetin:β-cyclodextrin inclusion complex is shown in the inset
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1651–1667

1662
Ramešová et al.:
The higher value of the stability constant of luteolin inclusion complex
compare to quercetin reflects the hydrophobicity of a guest molecule. Our
measurements are performed in alkaline solution where both flavonoid
molecules are dissociated. In the case of quercetin the values of the stability
–1
constant of anion:β-cyclodextrin 602 M (found electrochemically) or
–1
515 M (fluorescence spectroscopy) are in agreement with finding in litera-
ture by Zheng et al.12. Zheng et al. demonstrated that the stability constant
of the inclusion complex of undissociated quercetin with hydroxypropyl-
β-cyclodextrin (HPβ-CD) is significantly higher than the stability constants
–1
of its dissociated form (1009 M ). The latter value of stability constant of
dissociated form with HPβ-CD is higher than our value of stability constant
of the complex with β-CD (Table I). This suggests that the anion is bound
more strongly to HPβ-CD than to β-CD. The similar order was found in the
case of undissociated quercetin12 and in the case of a mixture of anion and
undissociated quercetin complexed with β-CD 14. As expected, the value of
stability constant of dissociated quercetin found in this work12 is lower
–1
than the constant for undissociated molecule 1028 M .
The strong influence of air oxygen on emission spectra was found in case
of both flavonoids. This was the reason why all the fluorescence measure-
ments were performed under deaerated conditions as in case of electro-
chemical studies. In the case of luteolin exposed to the air (Fig. 9a) the
fluorescence intensity at 495 nm significantly increases and is accompanied
by blue shift up to 488 nm. The increase of emission band at 488 nm most
30
30
a
b
6
6
20
10
20
10
5
4
5
4
3
3
2
1
1
2
0
0
500
600
500
600
λ, nm
λ, nm
FIG. 9
Fluorescence emission spectra of 3 × 10–4 M luteolin in 3.6 × 10–3 M KOH in the absence (a) and
in the presence of 1.2 × 103 M β-cyclodextrin (b) recorded at the different time (in min) of ex-
posure to the air oxygen: 0 (1), 54 (2), 76 (3), 119 (4), 163 (5), 938 (6). The excitation wave-
length is λexc = 365 nm
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Influence of the Host–Guest Interaction on the Oxidation
1663
likely belongs to an oxidation product of luteolin. When β-CD is present in
the solution of luteolin, the presence of oxygen causes the same effect as in
the absence of β-CD (Fig. 9b). The difference of fluorescence intensities at
488 nm of the curves exposed 15 h and 38 min to the presence of air in the
absence and presence of β-CD is negligible (compare curves 6 in Fig. 9a and
9b). So, the part of molecule of luteolin, which undergoes the oxidation, is
not hidden in the cavity of β-cyclodextrin. This implies that the oxidation
of luteolin takes different reaction pathway than the oxidation process of
quercetin. The cyclic voltammetry of luteolin (curve 2, Fig. 1) shows higher
reversibility of the oxidation process than in the case of quercetin (curve 1,
Fig. 1). This indicates that a presence of a chemical reaction following the
electron transfer plays an important role in the case of quercetin. This is in
agreement with the fast formation of the oxidation product 2(3′,4′-di-
hydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one, which was found
as the oxidation product of quercetin in acetonitrile28.
Identification of Products
The products obtained by an exhaustive electrolysis at 1.02 V were sepa-
rated and GC-MS analysis confirmed the formation of 2(3′,4′-dihydroxy-
benzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one (P5) as the main product.
The main fragments of the derivatized P5-5TMS correspond to m/z values:
P1
100
a
P2
80
P3
P6
60
40
P4
P5
20
100
b
80
60
40
20
12 14 16 18 20 22 24 26 28 30 32 34 36 38
Time, min
FIG. 10
Gas chromatogram of the solution of quercetin after the exhaustive electrolysis at 1.1 V after
the removing of the precipitate (see the text) in the presence of 1 × 10–2 M β-cyclodextrin (a)
and in the absence of β-cyclodextrin (b)
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1664
Ramešová et al.:
663 ([M – CH3]•+), 397 ([M – ring B]+), 369 ([M – ring B – CO]+), 281 ([M –
ring B – CO – OTMS]). This oxidation product was found also in the litera-
ture as the one final oxidation product in nonaqueous media28,38 and
agrees with reports5,39
.
The difference in the distribution of oxidation products of quercetin in
the presence and in the absence of β-CD is shown in Fig. 10. Oxidation
TABLE II
The products of oxidation of 1 mM quercetin in 0.1 M KCl/3.6 mM KOH at the potential of
the second oxidation wave in the presence of 10 mM β-cyclodextrin
Molecular weight
Tr, min
Peak Structure
Main m/z
Not derivatized Derivatized
COOH
P1
18.1
370, 355, 193 154
370
458
398
HO
OH
OH
OH
O
HO
P2
P3
21.2
22.0
443, 353
383, 281
471, 369
170
182
198
OH
O
OH
O
HO
OH
OH
O
OH
P4
P5
23.4
35.6
468
678
O
HO
OH
OH
OH
OH
O
678, 369, 281 318
OH
O
HO
O
OH
O
OH
OH
P6
37.3
651, 355
306
666
HO
O
O
OH
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Influence of the Host–Guest Interaction on the Oxidation
1665
products labeled as P1–P6 are summarized in Table II. Gas chromatogram
of solution after the electrolysis without presence of β-CD (Fig. 10b) yields
3,4-dihydroxybenzoic acid. The gas chromatogram of solution after elec-
trolysis shows the presence of the main oxidation product P5 and other
compounds. Since the strong influence of the presence of oxygen on the
decomposition of P5 was found8, it is evident that the cavity of β-CD pro-
tects the oxidation of P5 and stabilizes its oxidation products such as oxo-
acetic acid P4.
CONCLUSION
A poor stability of quercetin while kept in the presence of air oxygen was
confirmed by fluorescence spectroscopy. The study proved a protective for-
mation of inclusion complex of quercetin with β-cyclodextrin. It was
shown that decomposition products formed by the oxidation of quercetin
are stabilized in the cavity of β-cyclodextrin, mainly 2(3′,4′-dihydroxy-
benzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one. This oxidation product de-
composes in the presence of oxygen to 3,4-hydroxybenzoic acid and other
hydroxy compounds, when β-cyclodextrin is not present in solution. The
formation of inclusion complex plays a role in the stabilization of oxoacetic
acid found as one of oxidation products in the presence of oxygen.
In the contrary, molecule of luteolin encapsulated in the cavity of β-CD
is oxidised at part, which is not hidden in the cavity. In long time scale, the
molecule of β-cyclodextrin does not protect luteolin against the oxidation
by air oxygen. Nevertheless, the oxidation of luteolin complexed with
β-cyclodextrin is more difficult than the oxidation of its free molecule.
Electrochemical behaviour of quercetin and luteolin confirmed that
both dissociation forms are affected by the interaction with the cavity
of β-cyclodextrin. Nevertheless, values of constants stability 515 50 and
–1
720 170 M for the complex of β-cyclodextrin and dissociated molecule
of quercetin and luteolin, respectively, determined by fluorescence spec-
troscopy are more precise than values found electrochemically. All the mea-
surements were done under inert atmosphere to avoid the influence of
oxygen.
This work is supported by the Czech Science Foundation (grants 203/09/1607 and 203/08/1157).
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