Identification of the products of oxidation of quercetin by air oxygen at ambient temperature

Article abstract of DOI:10.3390/12030654

Oxidation of quercetin by air oxygen takes place in water and aqueous ethanol solutions under mild conditions, namely in moderately-basic media (pH ～ 8-10) at ambient temperature and in the absence of any radical initiators, without enzymatic catalysis or irradiation of the reaction media by light. The principal reaction products are typical of other oxidative degradation processes of quercetin, namely 3,4-dihydroxy-benzoic (protocatechuic) and 2,4,6-trihydroxybenzoic (phloroglucinic) acids, as well as the decarboxylation product of the latter - 1,3,5-trihydroxybenzene (phloroglucinol). In accordance with the literature data, this process involves the cleavage of the γ-pyrone fragment (ring C) of the quercetin molecule by oxygen, with primary formation of 4,6-dihydroxy-2-(3,4-dihydroxybenzoyloxy)benzoic acid (depside). However under such mild conditions the accepted mechanism of this reaction (oxidative decarbonylation with formation of carbon monoxide, CO) should be reconsidered as preferably an oxidative decarboxylation with formation of carbon dioxide, CO₂. Direct head-space analysis of the gaseous components formed during quercetin oxidation in aqueous solution at ambient temperature indicates that the ratio of carbon dioxide/carbon monoxide in the gas phase after acidification of the reaction media is ca. 96:4 %. Oxidation under these mild conditions is typical for other flavonols having OH groups at C³ (e.g., kaempferol), but it is completely suppressed if this hydroxyl group is substituted by a glycoside fragment (as in rutin), or a methyl substituent. An alternative oxidation mechanism involving the direct cleavage of the C²-C³ bond in the diketo-tautomer of quercetin is proposed.

Full text of DOI:10.3390/12030654

Molecules, 2007, 12, 654-672
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Identification of the Products of Oxidation of Quercetin by Air
Oxygen at Ambient Temperature
1
,
2
2
Igor G. Zenkevich *, Anna Yu. Eshchenko , Svetlana V. Makarova , Alexander G.
1
1
3
Vitenberg , Yuri G. Dobryakov and Viktor A. Utsal
1
St. Petersburg State University, Research Institute of Chemistry, Universitetsky pr., 26, St.
Petersburg 198504, Russia; E-mails: vitenberg@mail.ru; yura@nonel.pu.ru
Interregional Center “Adaptogen”, Piskarevsky pr., 47/5, St. Petersburg 195067, Russia; E-mails:
anna_eshenko@mail.ru; utsedus@yandex.ru
2
3
Russian Scientific Center “Applied Chemistry”, St. Petersburg 197198, Dobrolubov ave., 14, Russia;
E-mail: utsal@bk.ru
*
Author to whom correspondence should be addressed; E-mails: igor@IZ6246.spb.edu;
izenkevich@mail15.com
Received: 13 January 2007; in revised form: 22 March 2007 / Accepted: 25 March 2007 / Published:
7 March 2007
2
Abstract: Oxidation of quercetin by air oxygen takes place in water and aqueous ethanol
solutions under mild conditions, namely in moderately-basic media (pH ∼ 8-10) at ambient
temperature and in the absence of any radical initiators, without enzymatic catalysis or
irradiation of the reaction media by light. The principal reaction products are typical of
other oxidative degradation processes of quercetin, namely 3,4-dihydroxy-benzoic (proto-
catechuic) and 2,4,6-trihydroxybenzoic (phloroglucinic) acids, as well as the
decarboxylation product of the latter – 1,3,5-trihydroxybenzene (phloroglucinol). In
accordance with the literature data, this process involves the cleavage of the γ-pyrone
fragment (ring C) of the quercetin molecule by oxygen, with primary formation of 4,6-
dihydroxy-2-(3,4-dihydroxybenzoyloxy)benzoic acid (depside). However under such mild
conditions the accepted mechanism of this reaction (oxidative decarbonylation with
formation of carbon monoxide, CO) should be reconsidered as preferably an oxidative
2
decarboxylation with formation of carbon dioxide, CO . Direct head-space analysis of the
gaseous components formed during quercetin oxidation in aqueous solution at ambient
temperature indicates that the ratio of carbon dioxide/carbon monoxide in the gas phase
after acidification of the reaction media is ca. 96:4 %. Oxidation under these mild conditi-

Molecules, 2007, 12
655
3
ons is typical for other flavonols having OH groups at C (e.g., kaempferol), but it is comp-
letely suppressed if this hydroxyl group is substituted by a glycoside fragment (as in rutin),
or a methyl substituent. An alternative oxidation mechanism involving the direct cleavage
2
3
of the C -C bond in the diketo-tautomer of quercetin is proposed.
Keywords: Quercetin; oxidative decarboxylation; air oxygen; mechanism; products;
identification
Introduction
Quercetin [3,5,7,3′,4′-pentahydroxyflavone, 5,7,3′,4′-tetrahydroxyflavonol (1)] and its glycosides
e.g., 3-O-rhamnoside (quercitrin) and especially 3-O-rutinoside (rutin)] are among the most frequently
[
mentioned natural flavonoids isolated from various plant extracts [1-3].
OH
HO
O
O
OH
OH
OH
1
This statement can be easily illustrated by the frequency of the citation of the names of different
flavonoids on the Internet (Google search, November 2006), summarized in Table 1.
Table 1.
Number of
Ratio to quercetin,
Flavonoid
search “hits”
214,000
%
14
1
Kaempferol
Luteolin
Fisetin
17,400
59,600
4
Daidzein
Myricetin
Quercetin
Rutin
349,000
22
6
97,600
1,580,000
3,060,000
100
194
Current interest in the compounds of flavonoid group, including 1, is explained by their varied
biological activity, with antioxidant, antiradical, hepatoprotective, anti-inflammatory and other
interesting properties [4-8].
If 1 is such a widespread compound, its properties, physicochemical constants and analytical
parameters should be described in detail. Its “traditional” characteristics, used in the identification of
this compound, are its melting point (313-315 °C) and the variations of its UV-spectra at the presence

Molecules, 2007, 12
656
of different reagents [1,2]. The range of additional analytical parameters available includes relative
UV-absorption abundances at various wavelengths [9,10], retention indices (RI) in reversed-phase
HPLC [11] and gas chromatographic RIs of the trimethylsilyl (TMS) derivatives of 1 on standard non-
polar polydimethyl siloxane phases (original data, Tab1e 2).
Table 2.
TMS-derivative of 1
,7,3′,4′-tetra-TMS
,7,3′,4′-tetra-TMS
RI
5
3
3011 ± 3
3092 ± 3
3171 ± 4
penta-TMS
Only as recently as 2003 was the anomalous UV-spectrum of the pentaacetyl derivative of 1
reported and explained; this spectrum is very different from those of the quercetin parent compound
and all other acetyl derivatives (from mono- to tetra-) [12].
One of principal chemical property of flavonols [including quercetin (1)] seems to be their facile
oxidation by air oxygen, which has been the subject of numerous publications to date. In particular the
first report on the oxidation of quercetin was that by Nierenstein in 1912 [13]. This interest can be
verified by the number of corresponding search “hits” on the Internet. However, in most of these
reports the oxidation of quercetin was preferably carried out in strongly basic organic solvents or their
mixtures with water under the influence of various additional factors, namely heating of reaction
media, the presence of radical initiators, under enzymatic catalysis and light or γ-irradiation. The
possible mechanism of photosensitized oxygenation of 3-hydroxyflavones by air [bubbled through
their pyridine solutions under irradiation by light] was proposed first by Matsuura et al. [14,15]. It
includes formation of peroxide intermediates of the 3-hydroxyflavones, followed by their conversion
into the corresponding depsides and the subsequent hydrolysis products (see discussion below),
2
resulting in the cleavage of ring C. Both carbon monoxide (CO) and carbon dioxide (CO ) were
detected in different proportions among the gaseous products of this reaction, depending on the
conditions used. These results suggest that the reaction may actually proceed simultaneously through
two different mechanisms. Only one of these possible mechanisms (involving formation of carbon
1
8
monoxide) was accepted later; it was confirmed with the use of isotope labeled oxygen ( O ) [16].
2
Similar processes of oxidative destruction of 3-hydroxyflavones (including quercetin) by air
oxygen with the formation of the same products were observed under catalysis by the enzymes quer-
cetinase [16] and tyrosinase [17], in the presence of radical initiators like azodiisobutyronitrile [18-20]
2
+
or the CuSO /H O system [21], and in the complexes of flavonols with Cu ions [22,23]. The same
4
2
2
degradation products were detected as a result of γ-irradiation of quercetin solutions [24,25] and even
heating of its solutions at the presence of oxygen [26,27]. Under some conditions the formation of
other products was observed. Namely, electrochemical two-electron oxidation of (1) does not give
depside, but rather 2,4,6-trihydroxy-2-(3,4-dihydroxybenzoyl)-3(2H)-benzofuranone [28]. The same
benzofuranone, together with depside, was detected among the products of reaction of 1 with
azodiisobutyronitrile [19].
The comparison of the various processes of quercetin oxidation by air allows us to conclude that
the features of this reaction under the mildest conditions, i.e. in water and water-ethanol solutions in
moderately basic media (pH ∼ 8-10) at ambient temperature (i.e. without heating) in the absence of

Molecules, 2007, 12
657
radical initiators, any complexing reagents or fermentative catalysis and without γ- or light irradiation
of the reaction media are still not well-characterized at present. The first experiments indicated that
quercetin was quickly destroyed by simply bubbling air through its solutions under such mild
conditions. This allows us to classify 1 as more unstable than the well-known ascorbic acid, which is
inert to contact with air under the same conditions. Of course, there is a high probability that the
products of this reaction should be the same as established previously [14-27], but this should be
confirmed independently. In addition there is the problem is the origin of gaseous products formed in
this reaction (CO or CO ), because the oxidative state of carbon in these oxides determines the
2
possible mechanism of the process.
Hence, the aims of this paper were the identification of the quercetin oxidation products formed
under as mild conditions as possible, including analysis of the resulting the gas phase carbon oxides in
the reaction media. Preliminary data on the instability of quercetin solutions at the contact with air
under such mild conditions have been reported [29].
Results and Discussion
Preliminary experiments
Easy oxidation of quercetin (1) by air oxygen in the moderate-alkaline solutions at ambient
temperature should be considered as one of most important chemical properties of flavonols. A simple
experiment, namely the slow stepwise titration of 1 in 30 % water ethanol solution by sodium
hydroxide within pH range from 2 to 10 (Figure 1), confirms the universal character of this process.
Figure 1. Variations of optical densities of quercetin solution at wavelength 294 ± 2 nm
in result of its slow stepwise titration by NaOH within the pH range 6-10. The
anomaly at pH 7.2-7.1 is caused by the interruption of the process for ca. 30
min; the second one (at pH 8.2-7.7) was obtained after the storage of the
solution during one week.
0,75
0,70
0,65
0,60
0,55
0,50
0,45
6
7
8
9
10
pH of solution

Molecules, 2007, 12
658
After addition of each portion of titrant, the pH of solution was measured and its UV-spectrum
recorded. Surprisingly, any interruption of this uniform procedure leads to a gap in the smooth
dependence of pH vs. volume of titrant curve and disruptions in the UV-abundances at different
wavelengths. These anomalies in the pH-D (294 nm) coordinates are clearly evident in Figure 1.
The first anomalous area at pH ≈ 7.2-7.1 was caused by the storage of the solution under titration
during ca. 30 min; the second one (at pH ≈ 8.2-7.7) was caused by its storage for one week. The
reproducibility of these effects permits us to exclude the influence of experimental errors and conclude
the existence of an actual experimental feature. The complete UV-spectra of 1 during titration within
the pH range 2-10 indicate anomalies as well. Some of them can be explained by the acid-base
-
7
equilibrium ArOH ↔ ArO [рK (1) = 7.3 ± 0.1 (ОН )] [30]. However, the acidification of the basic
а
solution (pH = 10) after slow stepwise titration did not restore its initial UV-spectrum (Figure 2). At
the same time, the quick reversible pH variation (in only two steps) in accordance with the scheme
“
acid → basic → acid” restores a UV-spectrum identical to that of initial solution.
Figure 2. Variations of the UV-spectra of a water-ethanol solution of quercetin during its
slow stepwise titration by NaOH within pH range 2-10, followed by the
acidification up to pH = 4; (a) – UV-spectrum of quercetin in ethanol
solution; (b) – at рН = 2; (c) – at рН = 10; (d) – UV-spectrum of solution after
acidification to рН = 4.
(
b)
(
a)
(
c)
(d)

Molecules, 2007, 12
659
All the mentioned effects can be explained by the instability of quercetin (1) towards the oxidation
by air oxygen during slow titration and all other operations involving its dilute solutions at ambient
temperature. The oxygen ground state is the triplet one. This means that the O molecule exists as a
2
.
.
reactive biradical [ O-O ]. The solubility of oxygen in the water at ambient conditions (760 torr, 20 °C)
is about 8.8-9.4 mg/L [31,32] or about 0.29 mmol/L. This reference value can be accepted without
additional verification and it should remain constant when air is bubbling through aqueous reaction
media. For comparison, the concentration of 1 in the solution used for recording of UV-spectrum was
ca. 20 mg/L, or 0.06 mmol/L. That is, there was at least a 4.5-fold molar excess of oxygen in this
solution.
Figure 3. HPLC chromatograms (regime 1, detection wavelength 254 nm) (a) – initial
solution of quercetin (t 15.2 min); (b) – the same solution after ∼ 1 h of air
R
bubbling at ambient temperature; (c) – the same solution after termination of
the process (air bubbling during more than 3-3.5 h).
(
b)
(c)
(a)
Experimental oxidation of 1 by air was carried out using a simple procedure. Air was bubbled
through water-ethanol solutions (pH ≈ 8-10) of 1 at ambient temperature during up to 3.5 h with
periodical HPLC analyses of the reaction medium at different times. The chromatograms of the initial
R
quercetin solution (individual compound with t 15.2 min, regime 1) after 1 h of air bubbling and that
after termination of the process (3.5 h) are compared in Figure 3. No peak of 1 is observed on the final
chromatogram; only the signal of a hydrophilic compound(s) with t ca. 4.4 min is registered. No
R
additional changes have been detected with the increase of the contact time of this solution with air.
The same transformations after a similar period are observed when the reaction medium was cooled

Molecules, 2007, 12
660
down to 0 °C, which is typical for radical processes. Oxidation of 1 by air oxygen is accompanied by
the appearance of an intense red-brown coloration of the solution. The dry product isolated from this
solution after evaporation of the solvents possesses the same color.
Analysis of oxidation products
HPLC analysis of dry mixture of oxidation products (regime 2) indicate the presence of four
principal products with retention times of 4.15, 4.80, 6.05 and 20.97 min (initial quercetin with t 25.7
R
min is absent in this mixture), as shown in Figure 4. By analogy with literature data it seems
reasonable to believe that oxidation products of 1 possess simpler chemical structures than that of the
initial compound. So far as quercetin itself can be analyzed in the form of its trimethylsilyl (TMS)
derivatives (see their RI values in Table 1), GC-MS analysis of TMS-derivatives of oxidation products
can be used as the method for their identification. Three principal compounds accounting for ca. 96 %
among semi-volatile substances were found in the dry mixture of oxidation products (Table 3).
Figure 4. HPLC chromatogram (regime 2) of dried quercetin oxidation products (initial
compound with t 25.68 min is absent). Component with t 4.15 min – phloro-
R
R
glucinol; 4.890 min – 2,4,6-trihydroxybenzoic acid; 6.05 min – 3,4-
dihydroxybenzoic acid; 12.02 – intermediate depside (2); 20.97 min –
unidentified compounds. Each peak is 100 % pure as there is no co-elution of
compounds.
Table 3.
t ,
Relative
R
MS identification
Reference RI values
min content, %
2
2
2
3
5.48
7.71
8.25
0.72
10
1
Phloroglucinol, tris-TMS (А)
1656
Ethyl-3,4-dihydroxybenzoate, bis-TMS
3,4-Dihydroxybenzoic acid, tris-TMS (B)
2,3,4-Trihydroxybenzoic acid, tetrakis-TMS (C)
-
53
33
1830
1932 (2,3,4-isomer);
2
-
003 (2,4,6-isomer)
3
2.34
2
Unidentified

Molecules, 2007, 12
661
Components A and B can be identified ambiguously with the use of the Wiley 138.L MS database:
they are TMS-derivatives of 1,3,5-trihydroxybenzene (phloroglucinol, A) and 3,4-dihydroxybenzoic
(
protocatechuic) acid (B). The third component C was tentatively identified as the TMS derivative of
,3,4-trihydroxybenzoic acid, but this is not consistent with the absence of a structural fragment with
2
this arrangement of hydroxyl groups in the molecule of 1. A more reliable assignment of the structure
of this oxidation product is 2,4,6-trihydroxybenzoic (phloroglucinic) acid, which is in agreement with
the position of substituents on ring A of the initial molecule. The erroneous GC-MS identification of
this compound is likely caused by the absence of its mass spectrum in the Wiley MS database; as a
result it is recognized as an isomer. The value of GC retention index of this TMS-derivative (1996) is
in the better agreement with reference RI value for TMS derivative of 2,4,6-trihydroxybenzoic acid
(
2003 ± 9) than with that for 2,3,4-trihydroxybenzoic acid (a single reference value 1932 is available).
The comparison of GC-MS results with the data of HPLC analysis (Figure 4) permits us to identify
3
,4-dihydroxybenzoic acid (B) by the coincidence of its retention time (6.05 min) and UV-spectrum
(
λmax 216, 259 and 293 nm) with a reference sample. More hydrophilic components with retention
times 4.15 and 4.80 min are phloroglucinol (A) and 2,4,6-trihydroxybenzoic acid (C), respectively.
The relatively hydrophobic compounds with retention times 12.02 and 20.97 min have no matches in
the GC-MS analysis results and therefore cannot be identified this way. One of them (with t 8.3 min
R
in regime 1 and 12.02 min in regime 2) is the intermediate depside (ester) that is unstable to hydrolysis
in a basic media.
The retention indices of compounds B and C in reversed phase HPLC (with the use of n-alkyl
phenyl ketones as reference compounds) have been determined first time; both of them are in the
accordance with the previously reported RI value of phloroglucinol [11], because the ascending order
of RI values reflects the order of chromatographic elution (Table 4). The same products were isolated
in the result of quercetin oxidation by hydrogen peroxide under the same pH conditions.
Table 4.
Compound
Phloroglucinol (A)
RI
582 [11]
606
2
3
,4,6-Trihydroxybenzoic acid (C)
,4-Dihydroxybenzoic acid (B)
618
The formation of the aforementioned products is in complete agreement with literature data on the
oxidation of quercetin under different conditions [14-27]. To date the oxidative decarbonylation of 1
was considered the likely process which could take place preferably in non-aqueous solvents in the
presence of strong bases and often at above-ambient temperatures (in pyridine during 14 h [14], in
dimethylformamide with additives of potassium tert-butoxide at 37 °C during 3 h [16], etc.). The
2
accepted mechanism of this reaction (Scheme 1) includes the attack of carbon atom C in ring C by
oxygen with formation of a hydroperoxide anion. The cyclization of this anion implies the formation
4
3
of a new O-C bond, followed by elimination of the C carbon atom in the form of carbon monoxide
(
(
CO). The resulting primary product is 4,6-dihydroxy-2-(3,4-dihydroxybenzoyloxy)benzoic acid
depside, 2, CAS # 30048-34-1); its subsequent hydrolysis in basic media gives salts of 3,4-dihydroxy-
and 2,4,6-trihydroxybenzoic acids. Compound 2 has been identified as one of the intermediate

Molecules, 2007, 12
662
products of quercetin oxidation (component with t 8.3 min and 12.02 min on the chromatograms
R
presented in Figures 3 and 4, correspondingly).
Scheme 1. Possible mechanism of quercetin oxidative decarbonylation by oxygen [16].
OH
OH
OH
OH
O
O
O
[
.O2.]
HO
O
O
HO
O
O
OH
OH
O
OH
OH
OH
O
HO
O
HO
O
O
OH
O
O
O
- CO
OH
O
OH
O
2
1
8
This mechanism was partially confirmed by isotope labeling using of O for oxidation [16]. The
2
isotope labels were found distributed in a 1:1 ratio in both acids formed after hydrolysis of depside (2).
Nevertheless, the oxidative decarbonylation of quercetin presented in Scheme 1 seems to not be the
only possible mechanism. First, as reported in the publications of Matsuura et al. [14,15], carbon
monoxide may not be the sole gaseous product of this reaction; the formation of carbon dioxide was
2
detected as well. The CO :CO ratio was about 0.55 if the reaction media (pyridine solution) was irradi-
ated by a tungsten lamp, but it exceeds 1 under UV-irradiation (when a high-pressure mercury lamp
with Pyrex filter was used). In accordance with Scheme 1, the ratio of reacted oxygen and formed
carbon monoxide should be 1:1, i.e., if the oxidation of 1 is carried out in a closed system, the total gas
pressure should remain constant.
Quercetin (like other flavonoids) with a γ-pyrone ring in the molecule (ring C) can exist in two
prototropic forms (Scheme 2); the first of them is well-known enolic structure the γ-pyrone molecular
fragment, while the second one is the tautomeric diketoform 3 (the ratio of tautomers is unknown and
it can depend on the pH of the solution)
Scheme 2. Keto-enol tautomerisation of quercetin.
OH
OH
OH
HO
O
O
HO
O
O
OH
OH
O
OH
OH
3

Molecules, 2007, 12
663
The existence of tautomer 3 was taken into account in the quantum chemical modeling of the
oxidative properties of 1 [33]. It is interesting to note that one of the principal chemical features of α-
diketones is their easy oxidation in basic media by air oxygen (Scheme 3). This reaction goes via
intermediate ketyl anion-radicals [34] and gives two carboxylic acids in result of C-C bond cleavage
[
35,36]. It was characterized in details on the example of 2,2,5,5-tetramethylfuranidin-3,4-dione [35]:
Scheme 3. Example of the oxidative cleavage of α-diketones.
O
O
COO-
O
O , OH-
2
COO-
O
EtOH
A similar scheme for oxidation of another α-diketo compound (dehydroascorbic acid) by
-.
superoxide anion-radicals (O – one of the forms of active oxygen in chemical reactions) with the
2
cleavage of C-C bong and formation of two carboxylic acids was proposed by Frimer and Gilinsky-
Sharon in 1995 [37] (Scheme 4).
-.
Scheme 4. Oxidative cleavage of dehydroascorbic acid by O
2
O O
O
O
O
O
-
.
-.
O2
O2
OH
OH
O
O
- O2
O
O
OH
OH
O O
O
O
O
O
O
O
+
OH HO
OH
H
OH
OH
O
OH2
HO
OH
OH
Keeping in mind these properties of α-diketones and applying them to the quercetin oxidation
permits us to propose the alternative mechanism of the process with the formation of the same depside
2
shown in Scheme 5.
In comparison with Scheme 1 this is not oxidative decarbonylation, but rather what has taken place
2
is a decarboxylation (accompanied by CO formation). Therefore, more than one equivalent of oxygen
is required for the formation of one equivalent of carbon dioxide. As far as this process takes place in
2
-
basic media, the carbon dioxide formed immediately converts into CO
3
ions in the solution; hence no
elimination of gases from the reaction media should be observed. The radical character of this reaction
is confirmed by the absence of changes in its rate as a result of the cooling of the quercetin solution.

Molecules, 2007, 12
664
Scheme 5. Alternative mechanism of quercetin oxidative decarboxylation by oxygen.
OH
OH
OH
OH
HO
O
O
O
O
HO
O
O
HO
[.O2.]
H
O
O
O
[
.O2.]
OH
O
OH
O
[
.O2.]
keto-tautomer
.O2.]
-CO , -H O
2 2
[
OH
OH
OH
OH
HO
O
O
HO
O
O
CO H
O
2
OH
O
OH
O
2
It is important to note that Scheme 5 should be in the agreement with the observed distribution of
O isotope labels among products as Scheme 1 as well. The primary product of this reaction is the
1
8
same depside 2; its subsequent hydrolysis in basic media gives two hydroxybenzoic acids. Besides
that, the hydrolysis of 2 in water-ethanol mixtures gives small amounts of an ethyl ester (ethyl-3,4-
dihydroxybenzoate); this compound was detected with GC-MS analysis of the mixture of reaction
products. The presence of variable amounts of phloroglucinol (A) among the isolated quercetin oxida-
tion products can be explained by the easy decarboxylation of 2,4,6-trihydroxybenzoic acid (B) during
drying of reaction mixtures (at the temperature ca. 100 °C) (Scheme 6).
Scheme 6.
Formation of phloroglucinol in the result of decarboxylation of 2,4,6-
trihydroxybenzoic acid.
O
O
CO H
H
O
2
HO
OH
HO
HO
OH
[
]
-
CO2
OH
OH
OH
Unfortunately, the chemical origin of the compound(s) responsible for the appearance of intensive
color of reaction media and isolated dry products remains unknown. It is reasonable to assume that
these compound(s) should include benzoquinone-like molecular fragments (chromophores), but its
(
their) content may be small. The screening of UV-spectra of all components detected during HPLC
analysis with diode array detector in relation to the absorbance in the visible region gave no results.
Both Schemes 1 and 5 confirm that the principal structural fragment of the quercetin molecule that is
3
responsible for the easy oxidation of this compound by air oxygen is the hydroxyl group at C . The

Molecules, 2007, 12
665
substitution of this group by glycoside fragments completely suppresses the oxidation. For example,
rutin becomes stable, while another flavonol (3,5,7,4′-tetrahydroxyflavon, kaempferol) oxidizes easily
under the same conditions. This fact is in accordance with the literature data, whereby 3-O-methylated
flavonols gave no oxidation products in the presence of radical initiators [18].
Therefore, the selection of the reliable mechanism of quercetin oxidation by air oxygen seems
impossible without direct analysis of gaseous products (CO or CO ) formed in this process in the gas
2
phase contacting with reaction media (so-called head-space analysis, HSA [37]). This experiment was
carried out in closed system containing 1 L of water quercetin solution (2.1 g, 6.2 mmol/L, pH ∼ 11,
basified by NaOH) and 2 L of air and a digital manometer for pressure measurements. The analysis of
gas phase was conducted with the use of gas chromatograph equipped by two packed columns placed
in parallel and thermo conductivity detector (carrier gas was hydrogen). The first column (with non-
polar polymer sorbent Porapak Q) was used for separation of air (no separation for O and N ) and
2
2
carbon dioxide (signals of positive polarity); the second column was used for separation of O , N and
2
2
carbon monoxide (signals of negative polarity). Both sets of signals were summarized on the same
chromatogram.
Figure 5. Gas chromatograms of gas phase under quercetin solution during its oxidation
by air oxygen in a closed system: (a) – under initial quercetin suspension in
the water (pH = 6.56); (b) – under basic solution of quercetin (pH ∼ 11) after 4
days of contacting with air; (c) – after acidification of basic solution up to pH
∼
1.
1
1
1
1
1
1
25
20
15
10
05
00
CO2
air
1
1
1
1
21,0
20,9
20,8
20,7
O2
CO2
CO
9
5
0
1
000 1200
1400
N2
200
a
b
c
9
0
400
600
800
1000
1200
1400
1600
Figure 5a presents the gas phase chromatogram of a quercetin suspension in water at pH = 6.56,
where the ratio of peak areas of O and N are equal to the content of these gases in the air (1:4). The
2
2

Molecules, 2007, 12
666
chromatogram of the gas phase of an alkalinized solution of 1 in the water at pH ∼ 11 after 4 days of
the contact with air in the closed system is shown in Figure 5b; the content of oxygen is visibly less,
but no other gases (CO or CO ) are detected. It is interesting that the total gas pressure in this system
2
during the same time decreased from 1022 up to 930 mbar (- 9 %), which corresponds to the decrease
of oxygen contents in the gas phase without formation of any volatile reaction products. Just this fact is
enough to reject the previously proposed scheme for oxidative decarbonylation of quercetin [16].
Figure 5c presents the chromatogram of the gas phase after acidification of the solution of 1 with
concentrated HCl up to pH ∼ 1; a carbon dioxide peak has appeared that is the direct confirmation of
alternative mechanism of oxidative decarboxylation of quercetin (Scheme 5). However, the variations
of the recorder scale allow us to reveal the presence of small amounts of carbon monoxide in the gas
phase (negative signal with higher retention time than that of CO ). The peak areas of these two carbon
2
oxides can be recalculated from the relative content of these reaction products in the gas phase with the
use of coefficient k = 2.96 (Table 5, see Experimental).
Table 5.
Average
Gas phase
-3
Peak areas, S×10 , mV×ms
content in gas
phase, %
component
CO
2
81.6
1.18
83.9
1.04
84.9
1.06
96.3 ± 0.3
3.7 ± 0.3
CO
Therefore, different mechanisms of quercetin transformation occur during its oxidation by air
oxygen in moderately-basic water and water-ethanol solutions at ambient temperature. Under the
mildest conditions of quercetin oxidation by air oxygen (at ambient temperature in moderately-basic
media) the contribution of oxidative decarboxylation exceeds that of oxidative decarbonylation
approximately by 26 times.
Conclusions
Oxidative destruction of quercetin by air oxygen under mild conditions should be considered as
one of the typical reactions both for this compound and other flavonols. It is observed not only by
heating its solutions in the presence of strong bases, but can take place in water and water-ethanol
solutions in moderately-basic media (pH ∼ 8-10) at ambient temperature without any radical initiators
or irradiation of the reaction media. Revealed anomalies in the experimental results with dilute
quercetin solutions in contact with air (e.g., stepwise titration) indicate that this process takes place
even at pH ∼ 7-8, close to the pH values of physiological media. Therefore, the reaction under
consideration may be one of the general biotransformation paths of quercetin and other flavonols, and
this process may explain the low concentrations of these compounds in blood (pH ∼ 7.4) in the
presence of oxygen.
Direct head-space analysis of the gas phase of acidified quercetin solutions after air oxidation
indicates that the principal gaseous reaction product under these conditions is carbon dioxide. Only
small amounts of carbon monoxide (relative content ca. 4 %) were detected. This means that the

Molecules, 2007, 12
667
preferable mechanism is not oxidative decarbonylation of quercetin, but its oxidative decarboxylation.
The possibility of facile oxidation of quercetin by air oxygen in solution should be taken into account
in planning of all processes involving its isolation from plant raw materials and subsequent
purification. Unusually large losses of quercetin during its re-crystallization from water solutions can
be explained by this easy oxidation. Besides that, quercetin is widely used as a well known analytical
2
+
4+
reagent in determination of metal cations like Fe , U , in photometric determination of Zr, Hf, Sc,
Mo, Sn, Ge, in extraction-fluorescent analysis of Hf, Th, Nb, Ta, Al, Zr and other elements. In all these
cases the presence of quercetin oxidation products could distort the analytical results and, hence,
should be excluded. The general practical recommendation is to avoid long-term contact of even
weekly-prepared quercetin solutions with air.
Experimental
General
Commercially available quercetin dihydrate (CAS # 6151-25-3) purchased from Sigma was used in
all experiments. Determination of the solubility of (1) in the water-ethanol solutions (30 and 70 % of
ethanol) at ambient temperature (21 ± 1 °C) was carried out by successive stepwise addition of 1 to
known amounts of solvents (60-70 mL). The resulting values were 0.28 mg/mL (30 % ethanol) and 3.5
mg/mL (70 % ethanol).
Oxidation of 1 by air oxygen at ambient temperature
Compound 1 (about 50 mg) was dissolved in 70 % water-ethanol mixture (20 mL) placed in a 100
mL flask, followed by addition of aqueous ammonium hydroxide until the pH was 10.0 ± 0.1. Air was
bubbled through this solution (with a flow rate of ca. 50 mL/min) using a mini-compressor for up to
3
.5 hours. Aliquots of the reaction medium were taken periodically to record the UV-spectra (UV-
spectrometer Shimadzu UV-1700, wavelength range 200-500 nm) and for HPLC analyses. After
termination of the process solvents were evaporated from the reaction mixtures at 100 °C. The yields
of dry products (deep red-brown powder) were ca. 50-70 %. For preparative syntheses the quantities of
1
and solvents were proportionally increased 3-5 fold. Oxidation of 1 at low temperatures (1-2 °C) was
conducted by cooling the reaction flask with ice. Additional purification of hydrophilic oxidation
products from non-reacted residues of 1 included their dissolution in the minimal amount of water,
filtering the less soluble quercetin and evaporation of the solvent.
HPLC analyses of reaction mixtures and oxidation products
HPLC analyses were carried out in two regimes: “quick” (1) and “smooth” (2). Regime (1) was
performed using a Beckman System Gold LC chromatograph equipped with a UV-detector and a Luna
C18 150 × 3.6 mm column (size of sorbent particles 5 µm) with a 20 × 4.6 mm pre-column filled with
the same sorbent. Elution regime: gradient (45 min) of 10 to 100 % acetonitrile with a mixture of 0.03
%
water solution of trifluoroacetic acid (pH ≈ 2.3). Eluent flow 1.0 mL/min, injection size: 20 µL,
detection wavelengths 254 and 360 nm. Regime (2) involved the use of a Shimadzu LC-20

Molecules, 2007, 12
668
chromatograph with a diode array detector and the same column/precolumn combination. Elution
regime: gradient (60 min) of 10 to 70 % CH CN in the mixture with the same aqueous phase. Elution
3
flow 1.0 mL, injection size: 20 µL. UV-control of peak purity indicates the purity of all separated
components.
GC-MS analyses of oxidation products
GC-MS analysis of the quercetin oxidation products was carried out with a HP-5890 gas chroma-
tograph with a HP-5972 mass selective detector and a WCOT column (30 m × 0.25 mm with HP-5,
film thickness 0.25 µm) under the following temperature programming regime: 30 °C (5 min) → ramp
7
°C/min → 300 °C (20 min). Injector temperature was 300 °C, detector temperature – 280 °C. Sample
preparation included addition of methanol (ca. 50 µL) to the dry sample (2 mg), followed by the
treatment of this mixture with bis-(trimethylsilyl)acetamide (BSA, 200 µL) at 80 °C (1 min) until a
homogenous solution was formed. Interpretation of GC-MS data was carried out with Wiley 138.L and
NIST 98.L MS and GC retention indices databases. Mass spectra of principal components of reaction
mixture; m/z (I > 5 %) and their retention times were as follows:
rel
+
tris-TMS-phloroglucinol (А), t
R
25.48 min: 344 (10), 343 (25), 342 (81) М , 341 (13), 328 (21),
3
27 (71), 268 (10), 253 (6), 147 (21), 133 (15), 99 (5), 75 (10), 74 (9), 73 (100), 45 (14).
tris-TMS-3,4-dihydroxybenzoic (protocatechuic) acid (B), t 28.25 min: 371 (7), 370 (21) М , 355
12), 311 (11), 281 (7), 267 (5), 223 (9), 195 (6), 194 (16), 193 (100), 165 (8), 137 (7), 74 (7), 73 (78),
5 (15).
tetrakis-TMS-2,4,6-trihydroxybenzoic acid (C), t
39), 443 (97), 369 (5), 355 (13), 283 (5), 147 (20), 133 (6), 75 (6), 74 (9), 73 (100), 45 (13).
+
R
(
4
+
R
30.72 min: 458 (0.6) М , 446 (6), 445 (22), 444
(
Gas chromatographic analysis of the gas phase of quercetin solutions during their oxidation
The schematics of the device used for head-space analysis of the gas phase during quercetin
oxidation is presented in Figure 6. It is closed system consisting of a glass vessel (3 L volume) filled
with an aqueous quercetin suspension or solution (1 L, volume of gas phase is therefore 2 L) connected
by steel tubes (i.d. 2 mm) with the heated gas sampling valve (temperature 80 °C) for injecting of
samples into the chromatograph and a digital pressure meter. The circulating pump provided
continuous gas flow though the vessel containing the reaction media and the sampling valve with a
flow rate of up to 0.8 L/min. Periodical injections of the gas phase were carried out manually, gas
sample volume was 1 mL, and the duration of the injection was 15 s. Analyses were carried out on a
Tsvett-500 (Tsvett Inc., Dzerzhinsk, Russia) gas chromatograph equipped with two packed columns
placed in parallel and a thermoconductivity detector. Carrier gas was hydrogen with different flow
rates through each column:
1
2
. 2 m × 2 mm with Porapak Q (Supelco, USA); carrier gas flow 17 mL/min;
. 3 m × 2 mm with 5Å molecular sieves (zeolites); carrier gas flow 42 mL/min.
Oven temperature was 40 °C, detector temperature 150 °C, filament current 100 mA. Processing of
GC signals was provided with MultiChrom software (version 1.5; Ampersand Co., Moscow, Russia).
Both columns were connected to the single GC detector, so that components separated on column 1

Molecules, 2007, 12
669
were registered on chromatogram as peaks of positive polarity, while components eluted from column
– appeared as peaks of opposite (negative) polarity. Average time of the analytical cycle did not
2
exceed 15 min.
Figure 6. Scheme of the device used for head-space analysis of gas phase under quercetin solu-
tion during oxidation by air oxygen: 1 – vessel with solution; 2 – magnetic stirrer; 3 –
gas tubes; 4 – manometer connection; 5 – gas outlet connection; 6 – gas inlet connec-
tion; 7 – digital pressure meter; 8 – circulating pump; 9 – heated gas injection valve;
1
0 – gas sampling loop; 11 – carrier gas supply; 12 – gas chromatograph; 13 – packed
column with molecular sieves (zeolites); 14 – packed column with Porapak Q.
1
1
1
0
1
2
9
8
TCD
7
Р,mbar
1
3
14
6
4
5
3
1
2
The ratio of air peak area (column 1) and the sum peak areas of O and N (column 2) gives the
2
2
coefficient of relative sensitivity of the two channels of the detector (k = 2.96 ± 0.01). To prevent the
concentration of water vapor from gas phase on the sorbents, both columns should be periodically
heated up to 130-150 °C (at least once per working day).

Molecules, 2007, 12
670
References
1
2
3
4
5
6
. Mabry, T.J.; Markham, K.R.; Thomas, M.B. The Systematic Identification of Flavonoids; Springer
Verlag: N.Y. and Heidelberg, 1970.
. Klysheva, L.K.; Bandukova, V.A.; Alunina, L.S. Plants Flavonoids. Spreading, Physicochemical
Properties, Methods of Analysis (in Russian). Nauka Publ. House: Alma-Ata, 1978.
. Andersen, O.M.; Markham, K.R., Eds. Flavonoids. Chemistry, Biochemistry and Applications.
CRR Taylor & Francis: N.Y., 2005.
. Bors, W.; Hellers, W.; Michel, C. Flavonoids as Antioxidants: Determination of Radical
Scavenging Efficiences. Meth. Enzymol. 1990, 186, 343-374.
. Hanasaki, Y.; Ogawa, S.; Fukui, S. The Correlation between Active Oxygen Scavenging and
Antioxidative effects of Flavonoids. Free Rad. Biol. Med. 1992, 16, 605-608.
. Hu, J.P.; Calomme, M.; LAsure, A.; De Bryune, T.; Pieters, L.; Vlietnick A.; Van den Berghe,
D.D.A. Structure-Activity Relationship of Flavonoids with Superoxide Scavenging Activity. Biol.
Trace Elem. Res. 1995, 47, 327-331.
7
8
. Rice-Evans, C.A.; Miller, N.J.; Pananga, G. Structure-Antioxidant Activity Relationships of
Flavonoids and Phenolic Acids. Free Rad. Biol. Med. 1996, 20, 933-956.
. Gulsen, A.; Makris, D.P.; Kefalas, P. Biomimetic Oxidation of Quercetin: Isolation of a Naturally
Occuring Quercetin Heterodimer and Evaluation of its in vitro Antioxidant Properties. Food Res.
Int. 2007, 40, 7-14.
9
1
1
. Zenkevich, I.G.; Kosman, V.M. Relative Absorption at Different Wavelengths – Additional UV-
Spectral Parameter in Identification of Organic Compounds in Reversed-Phase HPLC (in
Russian). Russ. J. Anal. Chem. 1996, 51, 870-875.
0. Kosman, V.M.; Zenkevich, I.G. Informational Maintenance of the Identification of Phenolic
Compounds of Plant Origin in Reversed-Phase HPLC. Flavones, Flavonols. Flavonones and their
Glicosides (in Russian). Plant Resources 1997, 33, 14-26.
1. Zenkevich, I.G.; Kochetova, M.V.; Larionov, O.G.; Revina, A.A.; Kosman, V.M. Retention
Indices as the Most Reproducible Retention Parameters in Reversed Phase HPLC. Calculation for
Hydrophylic Phenolic Compounds Using Reference n-Alkyl Phenyl Ketones. J. Liq. Chromatogr.
Rel. Tech. 2005, 28, 2141-2162.
1
1
1
1
2. Zenkevich, I.G.; Pimenov, A.I. About Reasons of Anomalies in UV-Spectrum of Pentaacetyl-
quercetin (in Russian). Plant Resources 2003, 39, 120-126.
3. Nierenstein, M.; Whedale, M. Anthocyanin. I. Anthocyanin-like Oxidation Products of Quercetin.
Ber. Deut. Chem. Ges. 1912, 44, 3487-3491.
4. Matsuura, T.; Matsushima, H.; Sakamoto, H. Photosensitized Oxygenation of 3-Hydroxyflavones.
Possible Model for Biological Oxygenation. J. Amer. Chem. Soc. 1967, 89, 6370-6371.
5. Matsuura, T.; Matsushima, H.; Nakashima, R. Photoinduced Reactions. XXXVI. Photosensitized
Oxygenation of 3-Hydroxyflavones as a Nonenzymatic Model for Quercetinase. Tetrahedron
1
970, 26, 435-443.
1
1
6. Brown, S.B.; Rajananda, V.; Holroyd, J.A.; Evans, E.G.V. A Study of the Mechanism of
1
8
Quercetin Oxygenation by O Labelling. Biochem. J. 1982, 205, 239-244.
7. Kubo, I.; Nihey, K.; Shimizu, K. Oxidation Products of Quercetin Catalyzed by Mushroom
Tyrosinase. Bioorg. Med. Chem. 2004, 12, 5343-5347.

Molecules, 2007, 12
671
1
8. Ohashi, H.; Kyogoku, T.; Ishikawa, T.; Kawase, S.I.; Kawai, S. Antioxidative Activity of Tree
Phenolic Constituents. I: Radical-Capturing Reaction of Flavon-3-ols with Radical Initiator. J.
Wood Sci. 1999, 45, 53-63.
1
9. Krishnamachari, V.; Levine, L.H.; Pare, P.W. Flavonoid Oxidation by the Radical Generator
AIBN: A Unified Mechanism for Quercetin Radical Scavenging. J. Agr. Food Chem. 2002, 50,
4
357-4363.
2
2
2
0. Krishnamachari, V.; Levine, L.H.; Zhou, C.; Pare, P.W. In vitro Flavon-3-ol Oxidation Mediated
by a B-Ring Hydroxylation Pattern. Chem. Res. Toxicol. 2004, 17, 795-804.
1. Makris, D.P.; Rossiter, J.T. Hydroxyl-Free Radical-Mediated Oxidative Degradation of Quercetin
and Morin: A Preliminary Investigation. J. Food Composit. Anal. 2002, 15, 103-113.
2. Balogh-Hergovich, E.; Kaiser, J.; Speier, G. Synthesis and characterization of Copper (I) and Cop-
per (II) Flavonolate Complexes with Phthalazine and Their Oxygenation and Relevance to Quer-
cetinase. Inorg. Chim. Acta. 1997, 256, 9-14.
2
3. Balogh-Hergovich, E.; Kaiser, J.; Speier, G.; Argay, G.; Parkanyi, L. Kinetic Studies on the
Copper (II)-mediated Oxygenolysis of the Flavonolate Ligand. Crystal Structure of [Cu(fla) ] (fla
2
=
flavonolate) and [Cu(O-bs) (py) ] (O-bs = O-benzoylsalicylate). J. Chem. Soc. Dalton. Trans.
2 3
1
999, 3847-3854.
2
2
4. Marfak, A.; Trouillas, P.; Allais, D.P.; Champavier, Y.; Calliste, C.A.; Duroux, J.L. Radiolysis of
Quercetin in Methanol Solution: Observation of Depside Formation. J. Agr. Food Chem. 2002, 50,
4
827-4833.
5. Marfak, A.; Trouillas, P.; Allais, D.P.; Calliste, C.A.; Duroux, J.L. Redox Reactions Obtained by
γ-Irradiation of quercetin Methanol Solution are Similar to in vivo Metabolism. Radiat. Res. 2003,
1
59, 218-227.
2
2
6. Buchner, N.; Krumbein, A.; Rohn, S.; Kroh, L.W. Effect of Thermal Processing of the Flavonols
Rutin and Quercetin. Rapid Commun. Mass Spectrom. 2006, 20, 3229-3235.
7. Makris, D.P.; Rossiter, J.T. Quercetin and Ritin (Quercetin 3-O-Rhamnosylglucoside) Thermal
Degradation in Aqueous Media under Alkaline Conditions. Special Publ. – Roy. Soc. Chem. 2000,
2
48, 216-238.
2
2
8. Jorgensen L.V., Cernett C., Justesen U., Skibsted L.H., Dragsted L.O. Two-Electron Electroche-
mical Oxidation of Quercetin and Kaempferol Changes only the Flavonoid C-Ring. Free Rad.
Res. 1998, 29, 339-350.
9. Zenkevich, I.G.; Eshchenko, A.Yu.; Makarova, S.V.; Makarov, V.G. Unusual Chemical
th
Transformations of Natural Flavonoids // Abstr. 54 Annual Congr. on Medicinal Plant Research.
2
9 Aug.-2 Sept. 1006. Helsinki, Finland. № P300. Planta Med. 2006, 72, 1070.
3
3
3
3
0. Kuntic, V.; Pejic, N.; Micic, S.; Malesev, D.; Vujic, Z. Determination of Dissociation Constants of
Quercetin. Pharmazie. 2003, 58, 439-440.
1. http://www.thermidaire.on.ca/do.html; http://cee.citadel.edu;
http://www.usage.army.mil/ publications/eng-manuals/em1110-3-172/a-b.pdf.
2. Vesilind, P.A. Introduction to Environment Engineering; PWS Publishing Company: Boston,
1
996.
3. Trouillas P., Marsal P., Siri D., Lazzaroni R., Duroux J.-L. A DFT Study of the Reactivity of OH
Groups in Quercetin and Taxifolin Antioxidants. The Specificity of the 3-OH Site. Food Chem.
2
006, 97, 679-688.

Molecules, 2007, 12
672
3
3
4. Kutnevich, A.N.; Rudenko, A.P.; Rodina, L.L.; Pragst, F. Anion-Radicals from Cyclic Diketones
with Alkyl Substituents (in Russian). Russ. J. Org. Chem. 1978, 14, 1343.
5. Korobitsina, I.K.; Gurevich, L.S.; Rodina, L.L. Chemistry of Ketones of Furanidine Series. IX.
Alkaline Cleavage of 2,2,5,5-Tetraalkyl-3,4-furanidindiones (in Russian). Russ. J. Org. Chem.
1
969, 5, 567-570.
3
3
3
6. Shimizu M., Orita H., Suzuki K., Hayakawa T., Hamakawa S., Takehira K. Oxidative C-C Bond
Cleavage of vic-Diols with H O Catalyzed by Heteropolyacids. J. Mol. Catal. A: Chem. 1996,
2
2
1
14, 217-220.
7. Frimer, A.A.; Gilinsky-Sharon, P. Reaction of Superoxide with Ascorbic Acid Derivatives:
Insight into the Superoxide-Mediated Oxidation of Dehydroascorbic Acid. J. Org. Chem. 1995,
6
0, 2796-2801.
8. Ioffe, B.V.; Vitenberg, A.G. Head-Space Analysis & Related Methods in Gas Chromatography;
Wiley-Interscience Publishers: N.Y., 1984.
Sample Availability: Not applicable.
2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.
©