Effect of inclusion complex on nitrous acid reaction with flavonoids

Article abstract of DOI:10.1016/j.saa.2011.06.070

The kinetic of the nitrous acid reactions with quercetin and catechin has been studied using spectrophotometric method in aqueous solution. The results show that these antioxidants participate in oxidation reactions with nitrous acid which is derived from

Full text of DOI:10.1016/j.saa.2011.06.070

Spectrochimica Acta Part A 81 (2011) 661–665
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Effect of inclusion complex on nitrous acid reaction with flavonoids
Lida Khalafia,∗, Mohammad Rafieeb, Sajjad Sedaghata
a Department of Chemistry, Faculty of Science, Islamic Azad University, Shahr-e-Qods Branch, Tehran, Iran
b Department of Chemistry, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
The kinetic of the nitrous acid reactions with quercetin and catechin has been studied using spectropho-
tometric method in aqueous solution. The results show that these antioxidants participate in oxidation
reactions with nitrous acid which is derived from protonation of nitrite ion in mild acidic conditions. Cor-
responding o-quinones as relatively stable products were detected by spectrophotometric techniques.
pH dependence of the reactions has been examined and the rate constants of reactions were obtained by
non-linear fitting of kinetic profiles. The effect of ␤-cyclodextrin on the oxidation pathway was another
object of this study. It is shown that ␤-cyclodextrin has an inhibitory effect on the oxidation reaction. The
rate constants of oxidation reactions for complexed forms and their stability constants were obtained
based on changes in the reaction rates as a function of ␤-cyclodextrin concentration.
Received 27 March 2011
Received in revised form 18 June 2011
Accepted 26 June 2011
Keywords:
Catechin
Quercetin
Nitrous acid
␤-Cyclodextrin
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
are significant components in environmental and biological chem-
istry [11–13]. Several studies have revealed that nitrous acid can
An antioxidant is defined as a molecule capable of slowing
or preventing the oxidation of other molecules [1]. Among them
flavonoids are the most widely used group which are isolated
from a wide range of vascular plants [2]. They are found in
substantial levels in commonly consumed fruits, vegetables and
beverages. Flavonoids have aroused considerable interest because
of their potential beneficial biochemical and antioxidant effects on
human health. They have several biological activities including rad-
ical scavenging, anti-inflammatory, antimutagenic and anti-cancer
activities [3]. It also has been shown that they enhance product sta-
bility, quality, and shelf life in food technology [4]. In seeds, they
act in protection against predators and pathogens [5], increase seed
coat (testa)-imposed dormancy [6,7] and protect against UV radia-
tions [8]. Quercetin and catechin (Scheme 1) as natural flavonoids
are selected for this study; quercetin is a frequent component of
major dietary constituents, such as onions and apples. Catechin is
extracted from plants and present in natural food and drinks, such
as green tea.
react with flavonoids [14–16]. We have also studied the reaction of
nitrous acid and nitrite ion with catechols and catecholamines in
some kinetic details [13,17].
Finally cyclodextrins (CDs) are cyclic oligosaccharides com-
posed of d(+)glucopyranose units with the shape of a torus with
a hydrophilic exterior and a hydrophobic interior. They are well
known in host guest chemistry due to their unique ability to form
inclusion complexes with numerous compounds. The interaction
of guest molecules with CD leads to apparent changes in their
chemical properties, such as kinetic and thermodynamic parame-
ters [18–20]. The actual or potential uses of CDs in pharmaceuticals,
foods and cosmetics are well-known [21–24]. This paper focuses
on the reactions of nitrous acid with quercetin and catechin in the
presence of ␤-cyclodextrin (␤-CD) and effect of inclusion complex
on their reactions.
2. Materials and methods
They have been described extensively due to their broad biolog-
ical properties, which are related to their antioxidant activity [9].
They are often referred as antioxidants on account of their ability of
protecting against damages caused by reactive oxygen or nitrogen
species, ROS/RNS [10].
2.1. Materials and solutions
All experiments were performed with analytical reagent grade
chemicals purchased from E. Merck. These chemicals were used
without further purification. The stock solutions of, catechin,
sodium nitrite and ␤-CD were prepared fresh, daily by dissolv-
ing them in distilled water. The stock solution of quercetin was
prepared daily by dissolving it in distilled water/ethanol (60/40)
solution. The buffered solutions were prepared based on Kolthoff
tables, and the concentration of the prepared buffers was 0.15 M
[17]. Nitrogen gas with a purity of 99.999% was used to remove
On the other hand nitrous acid and nitrite ion are conjugate
pair of species found at ambient levels in the atmosphere. They
∗
Corresponding author. Tel.: +98 2146842936; fax: +98 2146842936.
E-mail address: l khalafi@yahoo.com (L. Khalafi).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.06.070

662
L. Khalafi et al. / Spectrochimica Acta Part A 81 (2011) 661–665
OH
OH
OH
OH
HO
O
O
HO
O
OH
OH
OH
OH
quercetin
catechin
Scheme 1. Structure of quercetin and catechin.
oxygen from solution. The kinetic experiment was initiated by the
addition 0.5 ml of 5.0 mM stock solution of catechin or quercetin
to 4.5 ml 3.3 mM sodium nitrite solution with desired pH and
␤-CD concentrations in a 5 ml flask. All of the solutions were
thermostated at 25 ◦C before mixing. Measurements were also per-
formed at this temperature. The reaction mixture was shaken and
transferred to the spectrophotometer quartz cell immediately. The
cell placed in cell holder and reaction monitored by full spectral
scan with time.
Fig. 2. Absorption spectra for 0.5 mM quercetin in the presence of 3.0 mM sodium
nitrite with time, time intervals 30 s, pH 4.0.
dized form of quercetin [14]. Two possible oxidation products are
semiquinone or quinine forms, but it has been reported that the
semiquinone form is not stable in aqueous solutions and easily
converts to the more stable quinonic form [25].
2.2. Apparatus
Absorption spectra were obtained with a Scinco UV–vis Spec-
trophotometer S2100. In each experiment, the sample placed in a
1 mm path length quartz cells, and the measurements were per-
formed at 25 ◦C. All of the calculations were performed in MATLAB
7.5 (Math Works, Cochituate Place, MA).
The most probable reactions are oxidation with nitrous acid or
catalytic oxidation by dissolved oxygen. The possibility of oxidation
with dissolved oxygen has been examined and results did not show
any considerable difference in the presence and absence of oxygen,
it was removed by pumping nitrogen. Nitrous acid is a weak and
monobasic acid with dissociation constant (Ka) which is generally
quoted in the literature as 5.1 × 10−4 M, pKa = 3.27 [26]. Considering
this solution composition and pH value nitrous acid act as oxidant in
the reaction. For more details we extended our studies at different
pH values around the pKa of nitrous acid. Fig. 2 shows the absorp-
tion time of quercetin in the presence of sodium nitrite at pH 4.10.
Comparison of Figs. 1 and 2 shows that the reaction rate decreases
with increasing pH. It is due to increase in nitrous acid concentra-
tion at lower pHs which has been discussed in our previous paper
[13].
3. Results and discussion
3.1. Reaction study
Fig. 1 shows the absorption–time of 0.5 mM quercetin in the
presence of 3.0 mM sodium nitrite at pH 3.40. At the initial time
quercetin shows two absorption bands with ꢀmax of 251 and
365 nm respectively. Upon initiation of the reaction, the absorbance
at 251 and 365 nm gradually decrease whereas the absorption band
at 289 nm appears and its height increases with time. At the end of
the reaction, the two peaks with ꢀmax 251 and 365 nm disappear,
and the new band (ꢀmax 289 nm) reaches to its maximum value.
The solution containing sodium nitrite in the same condition and
absence of quercetin did not absorb in this region. These absorbance
changes confirm the reactivity of quercetin toward nitrous acid at
this pH. The absorbance with ꢀmax 289 nm is related to the oxi-
Spectrophotometric study of interaction of catechin has been
performed in the presence of sodium nitrite at various pHs and
shows the same results as quercetin. Fig. 3 shows the spectrum
Fig. 1. Absorption spectra for 0.5 mM quercetin in the presence of 3.0 mM sodium
Fig. 3. Absorption spectra for 0.5 mM catechin in the presence of 3.0 mM sodium
nitrite with time, time intervals 30 s, pH 3.40, solvent 85/15 water/ethanol.
nitrite with time, time intervals 90 s, pH 3.40.

L. Khalafi et al. / Spectrochimica Acta Part A 81 (2011) 661–665
663
k
1
-
2 +H+
pKa=3.27
NO
(1)
(2)
HNO2
OH
k-1
O
O
OH
+ 2 HNO2
ko
R
R
k-2
k2
k-3
k3
OH
O
O
OH
+ 2 HNO2
k'o
(3)
R
R
O
O
Uncharactrized Products
(4)
(5)
Fig. 4. Absorption spectra for 0.5 mM catechin in the presence of 3.0 mM sodium
nitrite; (a and b) in the absence, (c and d) in the presence of 10.0 mM ␤-CD with time.
(b and d) at 480 s (a and c) at 960 s. Inset: The absorbance–time plots of reactions at
330 nm.
R
O
O
Uncharactrized Products
of 0.5 mM catechin in the presence of 3.0 mM sodium nitrite at pH
3.40. An absorption band with ꢀmax 321 nm belong to quinonic form
of catechin appears and its height increases with time.
R
HO
Catechin:R=
O
HO
Quercetin: R=
O
3.2. The effect of ˇ-cyclodextrin
OH
OH
Fig. 4 shows the absorption spectra for the reaction of catechin
with sodium nitrite; in the absence and presence of ␤-CD at pH 3.40.
In order to compare the reaction rates the absorbance changes at
320 nm were monitored and the kinetic profiles are shown in the
inset of this figure.
OH
OH O
Scheme 2. Proposed mechanism for the reactions of quercetin and catechin with
nitrous acid.
It shows that in the presence of ␤-CD the rate of oxidation
reaction decreases drastically. Addition of ␤-CD to the quercetin
causes to formation of a 1:1 inclusion complex between them. The
decrease in the rate of the reaction by addition of ␤-CD indicates
that the rate constant for the oxidation of the complexed form of
quercetin is smaller than for its free form [19]. The results for the
spectrophotometric study of quercetin in the presence of various
concentrations of ␤-CD are the same as the results that obtained for
catechin. Based on these results the following pathway is presented
for the reaction of these flavonoids in the presence of ␤-CD.
The acid base reaction of nitrous acid and nitrite ion is an equi-
librium. The formation of inclusion complex between flavonoids
and ␤-CD is also reversible and equilibrium reaction. Presence of
equilibrium between the reactants of these parallel reactions fol-
lows a complicated reaction pathway. The observed reaction rates
are roughly proportional to the equilibrium concentration of free
and complex forms of H2Q and nitrous acid. Based on the pro-
posed reaction mechanism consumption of each reactants shifts
the equilibriums to counteract the imposed changes. For example
consumption of H2Q due to its higher rate constants shifts the com-
plexation reaction to produce the free form. Also in the right side
of the reaction the ratio of Q to QCD is the function of their produc-
tion, formation constant of QCD and ␤-CD concentration. Produced
quinones and their complex forms undergo some side reactions
such as nitration, cyclization and degradation. But the results show
that they are relatively stable and the rates of their conversion are
negligible in the time scale of our spectrophotometric study.
ing Eqs. (1)–(3) in Scheme 2 as the main route of reaction we can
write the following equations:
d[HNO2]
= −k1[HNO2] + k [NO−][H+] − 2ko[H2Q][HNO2]
−1
2
dt
−2koꢀ [H2QCD][HNO2]
(6)
d[NO−2 ]
dt
= k1[HNO2] − k−1[NO−][H+]
(7)
(8)
(9)
2
d[H2Q]
dt
= −2ko[H2Q][HNO2] − k2[H2Q][CD] + k−2[H2QCD]
d[Q]
dt
= 2ko[H2Q][HNO2] − k3[Q][CD] + k−3[QCD]
d[H2QCD]
= −2koꢀ [H2QCD][HNO2] + k2[H2Q][CD] − k−2[H2QCD]
dt
(10)
d[QCD]
dt
= 2koꢀ [H2QCD][HNO2] + k3[Q][CD] − k−3[QCD]
(11)
where H2Q and Q stand for uncomplexed forms of flavonoids and
their oxidized products respectively and H2QCD and QCD stand for
their complexed forms. Considering this fact that equilibrium acid
base and complex formation reactions are very fast in comparison
with desired reaction we can write:
[NO−2 ]
[HNO2]
k1
−1[H+]
Ka
[H+]
=
=
(12)
(13)
3.3. Kinetic evaluation
k
[H2QCD]
[H2Q]
k2[CD]
By assuming the formation of a 1:1 inclusion complex between
the antioxidants and their oxidized forms with ␤-CD and consider-
=
= KfH Q[CD]
2
k
−2

664
L. Khalafi et al. / Spectrochimica Acta Part A 81 (2011) 661–665
Table 1
Kinetic and thermodynamic data for the reaction of nitrous acid in the presence of ␤-CD.
ko*(M−1 s−1
)
koꢀ (M−1 s−1
)
Lack of fit %**
*
Compound
KfH (M−1
)
KfQ(M−1
)
Q
2
Quercetin
Catechin
2700
2060
1900
1450
1.9
2.3
0.4
0.5
2.3
1.8
*
Reported rate constants are the average of three measurements and fitting at pHs 3.0, 3.2 and 3.4.
ꢀ
ꢁ
ꢁ
Lack of fit (%) = 100 ×
e2ij/
di2j where eij are residuals and dij are the elements of the experimental data matrix at pH 3.4.
ij
**
ij
Fig. 5. (I) Resolved spectral profiles for (a) free and (b) complex form of reaction product for catechin and (II) concentration profiles for (c) H2Q, (d) Q, (e) H2QCD and (f) QCD
for the reaction of 0.5 mM catechin with 3.0 mM sodium nitrite in the presence of 10.0 mM of ␤-CD.
The kꢀ , KfH and KfQ are obtained by using the same method and
[H2QCD]
[H2Q]
k3[CD]
Q
o
substitutin2g the obtained value for ko in the absence of ␤-CD. The
rate and formation constants of catechin and quercetin are listed in
Table 1. A very good fit of the data is observed (small values for the
lack of fit), which indicates the validity of the underlying model.
Fig. 5 shows the absorption spectra (5I) for Q and QCD and
concentration profiles (5II) related to the analysis of the data for
catechin in presence of 3.0 mM NaNO2 and 10.0 mM ␤-CD. Inter-
estingly, the calculated spectra and concentration profiles are well
consistent with the spectral changes in the data collected.
Estimation of Kf for the investigated flavonoids and their
oxidized forms indicates that they can form relatively stable
complexes with ␤-CD. The rate constants of complex forms are
considerably lower than the free forms and inclusion cause to
decrease in the rate of oxidation reaction. The formation constants
of the oxidized forms are lower than related parent molecules.
The higher complex formation constants of reduced forms and
inhibitory effect of ␤-CD for flavonoid oxidation may be due to the
hydrogen bonding of their hydroxyl groups with hydroxyl groups of
␤-CD [19].
=
= KfQ[CD]
(14)
k
−3
Ka is the acidic constant of nitrous acid. KfH
and KfQ are the
Q
2
inclusion stability constants of H2Q and Q with ␤-CD respectivel−y.
During the consecutive reactions the total concentrations of NO2
,
HNO2, H2QCD and H2Q are varying with time but the actual ratio
of NO2−/HNO2 depends on the pH of solution and Ka value. The
H2QCD/H2Q is also constant and depends on ␤-CD concentration
and Kf value.
A
two-way data matrix Y can be formed by measuring
absorbance under different wavelengths at a series of chosen times
(kinetic spectra). This matrix can be decomposed into the prod-
uct of matrix C, column wise, containing the concentration profiles
of the absorbing species and matrix A, row wise, containing their
molar absorptivities. Matrix E is the residual.
Y = CA + E
(15)
Propose of the fitting consists of finding set of parameters for
which the sum over all the squares, ssq, over all the elements of the
error matrix, E, is minimal.
ꢂ
Ei2j
(16)
4. Conclusion
ssq =
The kinetic profiles were obtained by numerical integration
using MATLAB’s solver, ode45, which is the standard Runge–Kutta
algorithm [27]. Also the values of rate constants and equilibrium
constants as nonlinear parameters which define the matrix C were
calculated by fitting through the data of the concentration pro-
files using Newton–Gauss algorithm. The molar absorptivities of
the H2Q and H2QCD are known and they are used during the fitting
process. The molar absorptivities of the Q and QCD are calculated
as linear parameters based on beer equation ST = C + D, where C+ is
the pseudo-inverse of the matrix C [28].
The above work describes the oxidation of catechol containing
flavonoids exposed to HNO2 in aqueous solution. Reaction mech-
anisms were proposed and related o-quinones were detected as
relatively stable products. The rate and equilibrium constants of
reactions were obtained by chemometrics techniques. Reaction is
pH dependent and grater reaction rates at lower pH values are due
to higher percentage of HNO2 as oxidant. One of the more signif-
icant findings to emerge from this study is the effect of inclusion
complex on the antioxidant properties of selected flavonoids. The
results show that the formation of inclusion complex decreases the
rate of oxidation reaction. On one hand this complexation lead
to more stability of these antioxidants toward the nitrous acid
as reactive nitrogen species. On the other hand more stability of
antioxidants maybe cause to weaker protecting effect in damage
The spectral time data are obtained at pHs lower than 4.0 in
the absence and presence of various concentrations of ␤-CD. The
values of the rate constant for the reaction of H2Q (ko) are obtained
in the absence of ␤-CD considering pKa equal to 3.27 for HNO2.

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665
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