Irreversible UV-induced quercetin and rutin degradation in solution studied by UV spectrophotometry and HPLC chromatography

Article abstract of DOI:10.2298/JSC110618180Z

The irreversible degradation of quercetin and rutin, dissolved in methanol and water, induced by continuous UV-irradiation from two different sub-ranges (UV-B and UV-C) were studied in this work. The degradation of both flavonoids is related to the formation of UV-induced degradation products: both processes follow first-order kinetics. The degradation and rate constants of the formation of the products are both dependent on the involved UV-photons energy input in both solvents.

Full text of DOI:10.2298/JSC110618180Z

J. Serb. Chem. Soc. 77 (3) 297–312 (2012)
JSCS–4269
UDC 547.918+547.972.3+542.92:544.032.6–
31+543.544.5.068.7:544.351:544.43
Original scientific paper
Irreversible UV-induced quercetin and rutin degradation in
solution studied by UV spectrophotometry and
HPLC chromatography
JELENA B. ZVEZDANOVIĆ, JELENA S. STANOJEVIĆ, DEJAN Z. MARKOVIĆ*
and DRAGAN J. CVETKOVIĆ
University of Niš, Faculty of Technology, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia
(Received 18 June, revised 8 August 2011)
Abstract: The irreversible degradation of quercetin and rutin, dissolved in me-
thanol and water, induced by continuous UV-irradiation from two different
sub-ranges (UV-B and UV-C) were studied in this work. The degradation of
both flavonoids is related to the formation of UV-induced degradation pro-
ducts: both processes follow first-order kinetics. The degradation and rate con-
stants of the formation of the products are both dependent on the involved UV-
photons energy input in both solvents.
Keywords: quercetin; rutin; UV-irradiation; degradation; products; kinetics.
INTRODUCTION
Ultraviolet (UV) radiation is part of the natural sunlight spectrum that reaches
the Earth’s surface. Depending on the source, the reported UV-portion of the to-
tal sunlight does not exceed 5–6 %, of which, under normal circumstances, >95 %
1
is UV-A (320–400 nm) radiation, the rest being UV-B (290–320 nm) light; UV-C
radiation (200–280 nm) is completely absorbed by the atmosphere. Besides being
the obligatory driving force of photosynthesis, light may also be damaging factor
2
in the biosphere. The recent depletion of the stratospheric ozone layer has led to
an increase on the biologically most damaging UV-B portion of light at ambient
levels. Plants generally respond to an increase in UV-B radiation through very
different mechanisms, including the synthesis of UV-absorbing, protective pig-
3,4
ments, such as flavonoids.
Flavonoids are a large class of natural polyphenol compounds of low mole-
cular mass, widely distributed in plant world, where they perform several very
5–7
important functions, including anti-oxidation.
They are benzo-γ-pyrane deri-
vatives consisting of pyrane and phenolic rings. The most important structural
*Corresponding author. E-mail: dejan_markovic57@yahoo.com
doi: 10.2298/JSC110618180Z
297
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ZVEZDANOVIĆ et al.
elements related to their functions are: an o-dihydroxy group in the B ring (ca-
techol structure) as a potential radical target, a double bond between positions 2
and 3 of the C-ring conjugated with keto group in position 4 (because of its
capacity to delocalize the uncoupled electron of the flavonoid radical) and C-3,
C-5 and C-7 hydroxyl groups (of the C and A rings) as potential free radical sca-
8–10
vengers (Fig. 1).
Quercetin (flavonol) and rutin (flavon) are members of the
flavonoid family, with the structures shown in Figs. 1A and 1B, respectively. The
main difference in their structures lies in position C-3, where instead of an OH
group (quercetin), a glycoside residue has been inserted (rutin). Updated studies
connect the antioxidant activity of flavonoids with presence (or absence) of an
OH group at position 3 of the C-ring, in combination with a catechol B-ring
5–11
structure.
The importance of this position may be estimated – among other
ways – by comparing the stabilities of quercetin and rutin toward an externally
induced stress, such as UV-irradiation.
Fig. 1. Structure of A – quercetin and B – rutin.
Quercetin and its glycosides (such as rutin) are believed to be responsible for
prevention of extended UV radiation-induced damage to a variety of plants.
Quercetin absorbs UV radiation with absorbance maxima in the UV-A (λ
=
max
–1
–1
–1
= 365 nm, ε = 28400 M cm ) and UV-C range (λ
cm ), suggesting that one plausible photoprotective mechanism could be direct
absorption of UV radiation, thereby preventing the formation of reactive oxygen
= 256 nm, ε = 28300 M
max
–1
12
species (ROS) and consequent direct DNA damage. The UV energy absorbed
13
by quercetin may be dissipated as heat or converted into decomposition pro-
11
ducts. UV-irradiation of flavonoids in solution and in liposome systems (in vit-
ro), as well as in vivo, results in their irreversible breakdown, accompanied by
14,15
the appearance of a number of decomposition products.
16
In the recently accepted report, the stability of quercetin and rutin in so-
lution toward UV-irradiation from the three ranges (UV-A, UV-B and UV-C)
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UV-INDUCED CHANGES OF QUERCETIN AND RUTIN IN SOLUTION
299
were compared, as were their antioxidant activities in the presence of a lipoidal
mixture as a protection target. It was concluded that: 1) rutin expressed higher
stability than quercetin toward UV-irradiation, while the stability of both flavo-
noids was found to be affected by the energy of incident photons; 2) however, the
higher stability of rutin had only a partial effect on its antioxidant ability to pro-
tect a lipid mixture from peroxidation, i.e., its dominance in stability over quer-
cetin (towards UV-irradiation) was not supported by its antioxidant ability com-
pared to that of quercetin under the same experimental conditions.
16
This study is a continuation of the previous investigation. Thus, quercetin
and rutin were again irradiated in solution but this time reversed phase HPLC
techniques in combination with absorption spectrophotometry were employed to
analyze the formation of the degradation products quercetin and rutin induced by
continuous UV-irradiation to provide the data for a kinetic analysis to follow.
EXPERIMENTAL
Samples preparations
Quercetin and rutin standards were purchased from Merck. Quercetin was dissolved in
methanol and rutin in both methanol and water to give a concentration of 0.1 mM.
UV-Irradiation treatment
The irradiation of the pigments in solution was performed in a cylindrical photochemical
reactor “Rayonnet”, with 7 symmetrically placed lamps, with emission maxima in two dif-
ferent UV sub-ranges, 300 (UV-B) and 254 nm (UV-C). The total emitted energy flux (mea-
sured using a UV-meter, Solarmeter, model 8.0 UV meter, Solartech) was about 10.5 W m-2
for 300 nm and 12.5 W m-2 for 254 nm at a 10 cm distance from the lamps. The samples (2
ml) were irradiated in quartz cuvettes placed on a rotating circular holder.
UV Spectroscopy
The UV spectra of rutin and quercetin in different solvents were recorded on a Varian
Cary-100 spectrophotometer. All spectra, before and after irradiation with UV-light, were
recorded in the range from 200 to 500 nm.
High performance liquid chromatography – photodiode array detector (HPLC–DAD)
The HPLC analyses were performed at 15 °C on an Agilent 1100 Series system, Wald-
bron, Germany, using a diode array detector set at 257, 295 and 372 nm. Aliquots of 20 µl
were injected into a 4.6×250 mm RPC-18 column (Zorbax Eclipse XDB-C18) with 5 µm
particle size. Elution was realized in the gradient mode proposed by Veit et al. using two
mobile phase components: A = 0.15 % phosphoric acid in water–methanol (77:23 v/v) mix-
ture, B = methanol.17 The gradient mode was set as follows: 0 to 3.6 min, isocratic 100 % A;
3.6–24.0 min, linear gradient from 100 to 80.5 % A; 24–30 min, isocratic 80.5 % A; 30–60
min, linear gradient from 80.5 to 51.8 % A; 60–67.2 min, linear gradient to 100 % B; 67.2–
–90.0 min, isocratic 100 % B. The flow rate was 1.0 cm3 min-1. The HPLC peaks (areas)
recorded at 372 nm were used for the determination of quercetin and rutin degradation
(bleaching) induced by UV-irradiation, i.e., kinetic analysis. New peaks were detected at 295
and 257 nm because of quercetin and rutin UV-irradiation in solution; their areas were plotted
against irradiation time, providing possibilities for kinetic analysis.
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RESULTS
The changes in chromatograms of UV-irradiated quercetin (in methanol) and
rutin (in methanol and in water) are shown in Figs. 2 and 3, respectively. The
changes of absorption spectra of quercetin and rutin exposed to UV-irradiation
are shown in the related inserted spectra in Figs. 2 and 3 (right), respectively.
Fig. 2. Quercetin in methanol, irradiated with continuous UV-B (A) and UV-C (B) radiation.
The changes in HPLC-chromatograms recorded at 295 nm (A, B: left); the corresponding
changes in the samples absorption spectra, recorded in the spectral range 200–425 nm (A, B:
right). The exposure times are displayed on the graphs and the arrows show the change in
direction: down (↓) in the case of quercetin, and up (↑) in the case of the degradation products.
Main observed products of UV-induced degradation of quercetin (at different retention times,
t
ret.: 17, 21, 32 and 50 min) were numerated with the numbers 1–4, respectively, in the
chromatograms. mAU – mili (10-3) relative absorbance units.
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UV-INDUCED CHANGES OF QUERCETIN AND RUTIN IN SOLUTION
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Fig. 3. Rutin in methanol (A) and water (B), irradiated with continuous UV-C irradiation. The
chromatograms explanation is the same as for Fig. 2. The only observed products (at different
retention times, tret., 13 and 28 min, respectively) were numerated with numbers 1 and 2,
respectively, in the chromatograms.
Irradiation of quercetin in methanol with either UV-B or UV-C radiation re-
sulted in a decrease in quercetin (t = 55 min) and the appearance of new peaks,
ret.
i.e., four major peaks (corresponding to UV-induced products) at the following
retention times: 1) 17, 2) 21, 3) 32 and 4) 50 min (Fig. 2). Irradiation of rutin in
methanol and in water with UV-C radiation resulted in rutin degradation (t
=
ret.
= 41 min) and the appearance of two new peaks, at 1) 13 and 2) 28 min, respecti-
vely (Fig. 3). The absorption spectra of the four observed UV-induced degra-
dation products of quercetin and the two of rutin are shown in Figs. 4A and 4B,
respectively. The proposed, possible structures of the degradation products of
quercetin are shown in Fig. 4A. These compounds, together with their names,
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ZVEZDANOVIĆ et al.
positions of the absorption maxima (λ ) and related references are given in
max
18–23
Table I.
Fig. 4. Absorption spectra of UV-B and UV-C induced quercetin degradation products (A), as
well as UV-C induced rutin degradation products (B), corresponding to the main observed
peaks in the related HPLC chromatograms (the retention times are marked).
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UV-INDUCED CHANGES OF QUERCETIN AND RUTIN IN SOLUTION
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TABLE I. Possible structures of the products formed as the result of UV-B and UV-C irra-
diation of quercetin in methanol, detected by HPLC. The proposals are based on comparisons
of the absorption spectra of the products with literature spectral data
tret.
min
λmax
nm
Product
Name of product
Structure, obtained by
References cited
1
17 2,4,6-Trihydroxy-
phenylglyoxylic
acid
292
Spectrum taken
from Zhou et al.18
The compound is
also cited in
,
refs.19–21
electrochemical oxidation
2
3
21
2-(3,4-Dihydro-
xybenzoyl)-2,4,6-
-trihydroxy-3(2H)-
-benzofuranone
294, 325 Taken from Zhou
and Sadik.19
The compound is
also cited in ref.19
,
electrochemical, enzymatic,
2,2’-azoisobutyronitrile
oxidation and auto-oxidation
32 1,3,8-Trihydroxy-
9aH,11H-ben-
300
Taken from
Timbola et al.22
zofuro[3,2-b]-
[1]benzopyran-
-7,11-dione cyclic
ether connecting
AC-system with
B-ring
,
electrochemical oxidation
4
50 Methyl 2-{2-[(3,4-
-dihydroxyben-
zoyl)oxy]-4,6-di-
hydroxyphenyl}-
-2-oxoacetate
275, 297
Taken from
Marfak et al.23
,
γ-irradiation-induced
degradation
Decreasing kinetic plots of quercetin and rutin degradation obtained using
the quercetin (t = 55 min) and rutin (t = 41 min) peaks from the chromate-
ret.
ret.
grams recorded at 372 nm (not shown), ln S
The plots show a good linear fit, with R values 0.95–0.99. The degradation
= f(t ), are shown in Fig. 5.
372nm
irr
2
kinetics seems to obey a first-order law. The calculated rate constants, k
(min ), for the UV-B and UV-C sub-ranges in the two solvents, are given in
Table II.
degrad
–1
The analogue kinetic ln plots obtained using the peaks areas recorded at 295
nm, ln S
= f(t ) for the four observed major products of UV-irradiated
295nm
irr.
quercetin, 1, 2, 3 and 4, are shown in Fig. 6A–D, respectively. The plots show
2
good linear fits, with R values 0.92–0.99. The calculated rate constants for the
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ZVEZDANOVIĆ et al.
formation of the UV- induced degradation products of quercetin, for UV-B and
UV-C sub-ranges in methanol are given in Table III.
Fig. 5. Kinetic ln plots of UV-B and UV-C induced quercetin (A) and UV-C induced rutin (B)
changes, obtained from the HPLC-chromatograms recorded at 372 nm, as a function of UV-B
and C irradiation time, ln S372nm = f(tirr.). The plots show good linear fits, with R2 values
0.95–0.99. The corresponding rate constants calculated from the slopes
of the linear plots are given in Table II.
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UV-INDUCED CHANGES OF QUERCETIN AND RUTIN IN SOLUTION
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TABLE II. Kinetics of quercetin and rutin degradation in methanol and water (shown in Fig.
5), during increasing UV-irradiation time in two different UV sub-ranges: 300 (UV-B) and
254 nm (UV-C). Peak areas were taken from the corresponding HPLC-chromatograms at 372
nm (analogues to Figs. 2 and 3, not shown). ln S372nm = kdegrad. tirr. + n, where S372nm – peak area
of quercetin and rutin from the corresponding chromatograms recorded at 372 nm, tirr. – UV-
irradiation time, kdegrad. – rate constants for UV-induced degradation of quercetin and rutin in
methanol and water, given as the slopes of the linear plots in Fig. 5, in min-1; c = 1.0×10-4 M
Quercetin, tret.=55 min
In methanol
Rutin, tret.=41 min
In water
UV-irradiation
wavelength, nm
300 (UV-B)
254 (UV-C)
–0.0049
–0.1069
–
–
–0.0206
–0.0043
Fig. 6. Kinetic ln plots of UV-B and C induced quercetin degradation products, obtained from
the HPLC-chromatograms recorded at 295 nm, as a function of UV-B and C irradiation time,
ln S295nm = f(tirr.). The plots show good linear fits, with R2 values 0.95–0.99. The correspond-
ing rate constants calculated from the slopes of the linear plots are are given in Table III.
DISCUSSION
Absorption spectra
The absorption spectra of flavonoids generally consist of two distinctive
24
bands, in a broad range of 240–400 nm. Band I, covering the range 300–380
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ZVEZDANOVIĆ et al.
nm, is attributed to the B-ring (with the A
position around 350–370 nm),
max
while band II, covering the range of 240–280 nm (with the A
position around
max
260–270 nm) is attributed to the A–C benzoyl system; a weak band with an
absorption maximum around 300 nm is also detected, which is attributed to the
C-ring only – shown in Fig. 1 and in the related inserted spectra in Figs. 2 and 3.
Both the investigated flavonoids expressed clear sensitivity to UV-irradiation,
from both UV-ranges. Continuous UV-irradiation of quercetin and rutin in me-
thanol induced a gradual decrease of the absorbance intensity, i.e., a hypochro-
mic effect, as shown in the related inserted spectra in Figs. 2A, 2B and 3A; simi-
lar spectral behavior of the absorption spectra was also observed for rutin in wa-
ter under UV-C-irradiation (Fig. 3B). Thus, continuous irradiation of quercetin
and rutin in solution resulted in their degradation, which increased with in-
creasing irradiation time. However, rutin underwent a slower degradation in me-
thanol, compared to quercetin (Figs. 3A and 2B, respectively). These results again
confirmed that presence or absence of 3-OH group (quercetin, rutin, respectively)
11,16
plays a major role in the stability of the flavonoids toward UV-irradiation.
TABLE III. Kinetics of the formation of UV-B and UV-C-induced quercetin degradation
products in methanol (the analogues plots to the ones for Table II). The peak areas were taken
from the corresponding HPLC-chromatograms recorded at 295 nm (Fig. 2), i.e. the products
Amax value. kdegrad.prod. – rate constant for the formation of the degradation products in me-
thanol was calculated from Fig. 6, in min-1
Product
UV-irradiation
wavelength, nm
1, tret. = 17 min 2, tret. = 21 min 3, tret. = 32 min 4, tret. = 50 min
300 (UV-B)
–
0.0176
0.0628
0.0295
0.0152
0.0637
254 (UV-C)
0.0405
0.0568a
a
Until 20 min of UV-C irradiation
A clear increase in the absorbance was observed between 270 and 325 nm
for quercetin in methanol, with a maximum at 295 nm (the related spectra are
inserted in Fig. 2) for the two UV-ranges (UV-B and UV-C). For UV-C-irra-
diated rutin in methanol and especially rutin in water, the analogue increase in
the absorbance was not so clear (the related spectra are inserted in Fig. 3). The
existence of A
indicates the formation of product(s) as a result of UV-
max,295nm
-induced quercetin degradation that absorb in the spectral region 270–325 nm
(for this reason, a monitoring wavelength of 295 nm was chosen in the HPLC–
–DAD experiments to estimate the UV-induced changes of quercetin and rutin in
solution). The same spectral behaviors of UV-irradiated quercetin and rutin in
solution were previously observed, but under slightly different conditions of UV-
-irradiation compared to the present experiments, i.e., with about two times larger
16
UV-B and UV-C incident energy.
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UV-INDUCED CHANGES OF QUERCETIN AND RUTIN IN SOLUTION
307
HPLC–DAD Analysis
The results of the HPLC analysis proved quercetin and rutin underwent ire-
versible UV-induced bleaching: a clear decrease of the corresponding main peaks
areas at t = 55 min and 41 min, assigned to quercetin and rutin, respectively, at
ret
all three monitoring wavelengths was observed (at 257 nm, 372 nm – not shown,
and at 295 nm, as can be seen in Figs. 2 and 3). Irradiation of quercetin in
methanol with either UV-C radiation for 30 min or with UV-B light for 120 min
resulted in about 95 and 50 % decreases in the quercetin content, respectively,
and its conversion into four major products (Figs. 2 and 4A). For both UV-B and
UV-C radiation, the peak observed at t = 50 min was dominant on the chro-
ret
matograms (Fig. 2). However, the product corresponding to the peak observed at
t
= 17 min was formed in a better yield as a result of UV-C compared to UV-B
ret.
irradiation (Fig. 2). The UV-spectra corresponding to the four observed products
induced by UV-irradiation (either by UV-B or by UV-C) of quercetin in metha-
nol are shown in Fig. 4A. The proposed, possible structures of the products,
based on a comparison of their absorption spectra with the ones found in the res-
pective literature related to different methods of quercetin oxidation (Table I), are
inserted in Fig. 4A.
The absorption spectra of the products 1 and 3 (in the mobile phase, acidic
methanol–water solution, see experimental section), obtained from the peaks at
t
ret
= 17 min and 32 min in chromatograms of UV-irradiated quercetin in metha-
nol, are shown in Fig. 4A. These products have absorbance maximums posi-
tioned at 294 nm (product 1) and 300 nm (product 3) Similar absorption spectra
to that of product 1 were found for di- and tri-hydroxy-phenylglyoxylic acid,
products obtained by electrochemical oxidation of quercetin, with maximums at
18
about 288 and 292 nm, respectively (Table I). This proposed, possible structure
requires a C-ring opening (product 1). In the case of product 3, an additional mo-
lecular rearrangement might occur finishing in a closed structure again via a fu-
18,19
ran type of chromophore as a “bridge”.
Absorption spectra of the products 2 and 4 (in the mobile phase) obtained
from the peaks registered at 21 and 50 min are shown in Fig. 4A and the pro-
18–23
posed structures are given in Table I.
Product 2 has two unresolved bands in
at 294
spectral range between 250 and 400 nm (with broad, “shoulder-like” A
max
and 325 nm) and another clear A
has two unresolved bands with A
at 236 nm, as shown in Fig. 4A. Product 4
at 275 and 297 nm, as shown in Fig. 4A. A
max
max
comparison with literature data suggests again C-ring opening for product 4,
while possible structure for product 3 appears to be of a similar type as product 2,
18–23
Table I.
Hence, it may be concluded that although obtained by different
manners of quercetin oxidation (Table I), the formation of the degradation pro-
ducts were very probably initiated by C-ring opening, followed by stabilization
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ZVEZDANOVIĆ et al.
of such structures (products 1 and 4) or further molecular rearrangements (pro-
ducts 2 and 3).
However, to date, none of the four proposed (quercetin-related) structures
(Table I) have been induced by UV-irradiation. In a similar study by Fahlman
12
and Krol (2009), it was shown that UV-A and UV-B irradiated quercetin in
methanol (under energy flux conditions very similar to those used in the present
–2
–2
study, i.e., 7.4 W m at 365 nm, and 13.3 W m at 310 nm, respectively) under-
12
went decomposition as the result of C-ring opening followed by the formation
of the three products (independent of the presence of oxygen in the system), which
were identified as 2,4,6-trihydroxybenzaldehyde, 2-[(3,4-dihydroxybenzoyl)-
oxy]-4,6-dihydroxybenzoic acid and 2(3,4-dihydroxyphenyl)ethanol. Unlike in
the present study, they also used the well-known UV-photosensitizer benzophe-
none to observe its effect on quercetin decomposition*, and found that the photo-
conversion yield was increased in the presence of benzophenone, i.e., in the
absence of benzophenone, only 20 % of the quercetin underwent decomposition
over 11 h, whereas 90 % of the quercetin was decomposed under both UV-A or
UV-B irradiation in less than 1 h when benzophenone was present, by the same
12
C-ring opening mechanism as in the absence of benzophenone. Unfortunately,
the authors did not present the absorption spectra of their proposed degradation
12
products, for their comparison with those presented in the present work.
Irradiation of rutin (0.1 mM) in methanol and water with UV-C radiation for
90 min resulted in about 85 % and 30 % decrease of the initial rutin content and
its conversion to two barely visible products, with retention times of 1 13 min
and 2 28 min, respectively, as determined by the chromatograms recorded at 295
nm (Fig. 3). Absorbance spectrum of product 1 in the mobile phase, which is
related to the peak with t = 13 min in the chromatogram of UV-C-irradiated
ret.
rutin in methanol (Fig. 3A), is shown in Fig. 4B. The spectrum shows one clear
band with absorbance maximum at 287 nm (Fig. 4B). The absorbance spectrum
of product 2 (in the mobile phase), which is related to the peak with t = 28 min
ret.
in the chromatogram of UV-C-irradiated rutin in water (Fig. 3B), is shown in Fig.
4B. The spectrum shows two bands: the first one is intensive with an absorbance
maximum at 253 nm and the second one has a significantly lower A
value at
max
298 nm (Fig. 4B).
While UV-induced decomposition of quercetin and rutin seem to be unques-
11
tionable, the possible mechanisms are still much disputed and very rarely pub-
lished. For the three detected products as a result of quercetin irradiation in me-
thanol, Fahlman and Krol (2009) proposed two distinct mechanisms, both requi-
12
ring C-ring opening: one occurred by direct reaction with singlet oxygen (the
creation of which was assured in the used aerated system by energy transfer from
*Benzophenone was recently employed with similar idea to study its effect on bleaching of
carotenoids in a similar in vitro system.25
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UV-INDUCED CHANGES OF QUERCETIN AND RUTIN IN SOLUTION
309
26
the sensitizer, i.e., the benzophenone triplet state, to a normal, triplet oxygen),
while in the case of the other two products, a precedent molecular rearrangement
occurred; in both cases, the quercetin AC–B system (Fig. 1) was decomposed,
and very simple, one-ring benzoyl-type molecules were finally formed. The spec-
tra of the four separated quercetin degradation products in methanol obtained in
this work (Fig. 4A), as well as of the two rutin degradation products in methanol
and water (Fig. 4B), with a clear absence of the flavonoids band I, attributed to
the B-ring of the flavonoids AC–B system (Fig. 1), suggest that the flavonoid
structure was irreversibly decomposed and probably smaller-sized molecules were
formed. The results obtained in the pre-work might support these suggestions,
since the formation of all four quercetin-degradation products were possibly ini-
tiated by C-ring opening.
Flavonoids degradation: kinetic analysis
Both flavonoids undergo substantial degradation expressed through the pro-
portional decrease of peak areas obtained from the related HPLC-chromatograms
recorded at 295 nm (Figs. 2 and 3), and at 257 and 372 nm (data not shown), as a
function of the UV-irradiation time (t ), i.e., ln S
= f(t ). This depen-
irr.
372nm
irr.
dence is expressed as a linear plot (Fig. 5), indicating first order kinetics of the
16,27
degradation of the flavonoids, as already reported.
The values of the calculated quercetin and rutin degradation rate constants,
–1
k
(min ), are comparable but also different in comparison to already pub-
degrad.
16
lished k values, which is in agreement with the somewhat different conditions
of continuous UV-irradiation (such as energy flux and distance from the lamps),
as well as with the different employed techniques (UV-spectrophotometry vs.
HPLC). The ratio of degradation rate constant values for quercetin irradiated with
UV-C and UV-B, k
/ k
, is about 21 (Table II).
Querc.,degrad.UVC
Querc.,degrad.UVB
16
This trend was already observed in a previous study: thus, the degradation of
flavonoids is highly dependent on the UV-energy input. This behavior was ob-
served in another study related to the continuous UV-irradiation of a very similar
28
compound, 3-hydroxyflavone.
However, rutin was generally more stable against UV-irradiation than quer-
cetin (by a factor of 5), as shown in Table II. Obviously, the substituted position
3 in the C-ring (Fig. 1) makes a major difference in the structures of quercetin
and rutin; rutin reportedly has two intra-molecular hydrogen bonds related to the
B-ring and the glycoside residue, i.e., interactions of O-3’ with H-4’, and O-2”’
29
with H-3’ (Fig. 1B), while in the case of quercetin, three hydrogen-bonds were
reported, i.e., the interaction of O-3’ with H-4’, and =O-4 with H-5 and H-3 (Fig.
30
1A). Hence, it is evident that the 3-OH position makes the main difference in
the response of quercetin and rutin to continuous UV-irradiation, making the gly-
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310
ZVEZDANOVIĆ et al.
coside residue in quercetin more susceptible to the action of UV radiation than
11
the glycoside residue in rutin.
In the case of rutin exposed to continuous UV-C radiation, the degradation
–1
rate constant in methanol was higher than in water: 0.0206 vs. 0.0043 min , as
given in Table II. The solubility of rutin in methanol is slight higher than in water
–1
(the corresponding free energies of solvation are –56.35 and –49.42 kcal mol ,
31
respectively). However, the influence of oxygen can not be neglected because
26
of the formation of singlet oxygen that could potentially play a certain role in
the UV-induced degradation of rutin and quercetin. In such circumstances, fla-
vonoids would need to act as sensitizers (for singlet oxygen creation if other sen-
sitizers are not present) while, on the other hand, they could become objects of
degradation themselves. The relevant data to be born in mind when referring to
the susceptibility of rutin vs. quercetin to UV-irradiation are: the solubility of
–3
oxygen in methanol and water at 20 °C are 2.12 and 0.265 g dm , respectively;
the viscosity under the same conditions are 0.597 mPa s for methanol and 1.002
mPa s for water; the lifetimes of singlet oxygen in methanol and water are 10 and
31
4 µs, respectively. Thus, during UV-C irradiation, the higher solubility of oxy-
gen, the longer lifetime of singlet oxygen and the smaller viscosity of methanol
31
than water makes rutin more susceptible to UV-induced degradation in metha-
nol than in water, which is documented by the corresponding k
values gi-
degrad.
ven in Table II.
Kinetic analysis of the formation of the UV-induced products
The kinetics of the formation of the main quercetin degradation products
seem to be first order (Fig. 6). The formation of the products was faster when
quercetin was irradiated with UV-C than with UV-B radiation (Table III). For
example, UV-C-induced formation of quercetin product 2, detected at t = 21
ret.
min in HPLC chromatogram recorded at 295 nm, was about 3.6 times faster than
when UV-B radiation was used (Table III). Similarly, UV-C-induced formation
of the quercetin products 3 and 4, detected at t 32 and 50 min, respectively,
ret.
were about 1.9 and 4 times faster than for their formation induced by UV-B ra-
diation (Table III). The obvious reason again lies in the increased energy input. A
comparison of the rate constants revealed at least: 1) three of the four products,
i.e., products 2–4 (Table III) were formed during continuous irradiation of quer-
cetin with both UV-B and UV-C radiation, in other words changing the energy
input (from UV-B to UV-C) did not lead to a significant qualitative change in the
outcome; 2) however, change in the energy input was reflected in significantly
higher formation rates, by factors of 3.5 (product 2), 1.9 (product 3) and 4.2 (pro-
duct 4) – Table III; 3) this undoubtedly indicates that the formation of the pro-
ducts was directly related to quercetin degradation, which was also energy depen-
dent (Table II).
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UV-INDUCED CHANGES OF QUERCETIN AND RUTIN IN SOLUTION
311
When rutin was exposed to UV-C radiation in methanol and water, forma-
tion of degradation products was also observed over irradiation time (chromato-
grams in Fig. 3), but their formation kinetics was not so clear (not shown). The
reasons for this behavior could be either in already cited higher stability of rutin
16
to UV-irradiation compared to quercetin, or in the insufficiently long irradia-
tion time used in the related experiments. However, extension of the irradiation
times could possibly make comparison of quercetin and rutin responses toward
UV-irradiation more difficult and less relevant.
Acknowledgements. This work was supported by the Ministry of Education and Science
of the Republic of Serbia under Projects No. TR-34012 on the Development of Technology
and OI 172044 on the Basic Investigations.
И З В О Д
ИРЕВЕРЗИБИЛНА UV-ИНДУКОВАНА ДЕГРАДАЦИЈА КВЕРЦЕТИНА И РУТИНА
У РАСТВОРУ, ИСПИТИВАНА UV-СПЕКТРОФОТОМЕТРИЈОМ И
HPLC ХРОМАТОГРАФИЈОМ
ЈЕЛЕНА Б. ЗВЕЗДАНОВИЋ, ЈЕЛЕНА С. СТАНОЈЕВИЋ, ДЕЈАН З. МАРКОВИЋ и ДРАГАН J. ЦВЕТКОВИЋ
Univerzitet u Ni{u, Tehnolo{ki fakultet, Bulevar oslobo|ewa 124, 16000 Leskovac
У овом раду је проучавана иреверзибилна деградација кверцетина и рутина, растворе-
них у метанолу и води, индукована континуалним UV-озрачивањем из две подобласти, UV-А и
UV-B. Деградација оба флавоноида је повезана са UV-индукованим формирањем деграда-
ционих продуката: оба процеса прате кинетику првог реда. Константе брзина и деградације
флавоноида и формирања продуката у оба растварача су зависне од енергије упадних UV-
-фотона.
(Примљено 18. јуна, ревидирано 8. августа 2011)
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