Kinetic model for studying the effect of quercetin on cholesterol oxidation during heating

Article abstract of DOI:10.1021/jf052529r

Inhibition of the heat-induced cholesterol oxidation at 150°C by incorporation of quercetin was kinetically studied. Results showed that without quercetin, the cholesterol oxidation products (COPs) concentration increased with increasing heating time. A low amount (0.002%, w/w) of quercetin was effective in inhibiting the formation of COPs during the initial heating period (≤30 min) at 150°C. However, after prolonged heating (30-120 min), a low antioxidant activity was observed because of the degradation of quercetin. When using nonlinear regression models for kinetic study of cholesterol oxidation in the absence of quercetin, the epoxidation showed the highest rate constant (h^-1 = 683.1), followed by free radical chain reaction (h ^-1 = 453.5), reduction (h^-1 = 290.3), dehydration (h ^-1 = 155.5), triol dehydrogenation (h^-1 = 5.35), dehydrogenation (h^-1 = 0.68), thermal degradation (h^-1 = 0.66), and triol formation (h^-1 = 0.38). However, in the presence of quercetin, the reaction rate constants (h^-1) for epoxidation (551.4), free radical chain reaction (111.7), and thermal degradation (0.28) were reduced greatly. The kinetic model developed in this study can be used to predict the inhibition of COPs by quercetin during the heating of cholesterol.

Full text of DOI:10.1021/jf052529r

1486 J. Agric. Food Chem. 2006, 54, 1486−1492
Kinetic Model for Studying the Effect of Quercetin on
Cholesterol Oxidation during Heating
JOHN-TUNG CHIEN, DA-JUNG HSU, AND BING-HUEI CHEN*
Department of Nutrition and Food Sciences, Fu-Jen University, Taipei 242, Taiwan
Inhibition of the heat-induced cholesterol oxidation at 150 °C by incorporation of quercetin was
kinetically studied. Results showed that without quercetin, the cholesterol oxidation products (COPs)
concentration increased with increasing heating time. A low amount (0.002%, w/w) of quercetin was
effective in inhibiting the formation of COPs during the initial heating period (e30 min) at 150 °C.
However, after prolonged heating (30-120 min), a low antioxidant activity was observed because of
the degradation of quercetin. When using nonlinear regression models for kinetic study of cholesterol
-1
oxidation in the absence of quercetin, the epoxidation showed the highest rate constant (h ) 683.1),
followed by free radical chain reaction (h ) 453.5), reduction (h ) 290.3), dehydration (h-1
-
1
-1
)
55.5), triol dehydrogenation (h ) 5.35), dehydrogenation (h-1 ) 0.68), thermal degradation (h
0.66), and triol formation (h ) 0.38). However, in the presence of quercetin, the reaction rate
-
1
-1
1
)
-
1
-
1
constants (h ) for epoxidation (551.4), free radical chain reaction (111.7), and thermal degradation
(0.28) were reduced greatly. The kinetic model developed in this study can be used to predict the
inhibition of COPs by quercetin during the heating of cholesterol.
KEYWORDS: Cholesterol oxidation; quercetin; heating; kinetics; HPLC
INTRODUCTION
as quercetin possessed 4 times higher antioxidant activity than
vitamins C and E. Thus, it is imperative to learn about the effect
of quercetin on the inhibition of COPs formation during heating.
In this study we tried to use a heating system and develop a
kinetic model for elucidating the inhibition mechanism of
cholesterol oxidation by quercetin.
Cholesterol, an important biological compound that is widely
distributed in egg and meat products (1-3), can undergo
oxidation to form cholesterol oxidation products (COPs) during
heating (4-6) or illumination (7). Many studies have demon-
strated that the consumption of COPs in excess may have
adverse effects on human health (8, 9).
MATERIALS AND METHODS
The formation mechanism of COPs during heating or
illumination of cholesterol has been well illustrated (7, 10). Just
like lipid oxidation, a series of free radical chain reactions can
occur initially during heating and lead to the formation of
various COPs such as 7-ketocholesterol (7-keto), 7R-hydroxy-
cholesterol (7R-OH), 7â-hydroxycholesterol (7â-OH), 5,6R-
epoxycholesterol (5,6R-EP), and 5,6â-epoxycholesterol (5,6â-
EP) (5). In view of the impact of COPs on human health, the
inhibition of COPs formation by the addition of antioxidant is
extremely important. Several studies have shown that the
incorporation of antioxidants was effective in inhibiting cho-
lesterol oxidation during high-temperature processing or storage
Materials. Quercetin, lauryl alcohol, cholesterol, and several COPs
standards, including 5,6R-EP, 5,6â-EP, 7-keto, and 5R-cholestane-
3â,5,6â-triol (triol) were obtained from Sigma Chemical Co. (St. Louis,
MO). Both 7â-OH and 7R-OH standards were from Steraloids Inc.
(Wilton, NH). These standards were used without further purification.
Paraffin oil was from Merck Co. (Darmstadt, Germany). The HPLC-
grade solvents, ethanol, 2-propanol, and n-hexane, were from Mallinck-
rodt Co. (Paris, KY), whereas 1,2-dichloroethane was from Riedel-de
2
H a¨ en Co. (Barcelona, Spain). The NH cartridges were from Unichrom
Scientific Co. (Taipei, Taiwan).
Instrumentation. The HPLC instrument consists of a Jasco
PU-980 pump (Tokyo, Japan), a Jasco 830 refractive index detector,
and an SIC Chromatocoder 12 integrator (System Instruments Co.,
Tokyo, Japan). Two Lichrospher 100 CN columns (244 × 4.0 mm i.d.
each) containing 5-µm particle were from Merck. CHEN-WIN computer
software system (Shuen-Hua Co., Taipei, Taiwan) was used to process
data.
Heating of Cholesterol. A mixture containing 400 µL of paraffin
oil and 100 µL of lauryl alcohol was poured into a 100-mL round-
bottom flask, and 100 mg of cholesterol standard as well as 0.002 mg
(11, 12). However, there is a paucity of data regarding the kinetic
study of inhibition mechanism.
Flavonoids, a class of polyphenols, are mainly present in fruits
and vegetables and have received considerable attention in recent
years because of their vital roles in the prevention of chronic
diseases such as coronary heart disease (13, 14), which can be
attributed to their high antioxidant activity (15, 16). Rice-Evans
et al. (17) demonstrated that naturally occurring flavonoids such
(0.002% of cholesterol) quercetin (in 100 µL of ethanol) was added.
Lauryl alcohol was used to dissolve quercetin, cholesterol, and COPs.
In addition, lauryl alcohol has been reported to promote triol formation
*
Corresponding author (e-mail nutr1007@mails.fju.edu.tw).
0.1021/jf052529r CCC: $33.50
1
© 2006 American Chemical Society
Published on Web 01/21/2006

Effect of Quercetin on Cholesterol Oxidation during Heating
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1487
through alcoholysis (18). For the control treatment, only 100 mg of
cholesterol standard was added to the mixture of 400 µL of paraffin
oil and 100 µL of lauryl alcohol. The flask was placed in a paraffin oil
bath, which was preheated for 5 min with nitrogen gas flushing into
the flask at the same time, until the internal temperature of the flask
reached 150 ( 1 °C. After that, the oxygen was pumped through, and
heating times of 0, 5, 10, 30, 60, and 90 min were started for the control
treatment. For the quercetin treatment, the heating time was extended
to 120 min. After heating, the flask was inserted into dry ice to terminate
the reaction, and each sample was subjected to HPLC analysis.
Triplicate experiments were performed for each heating treatment, and
the data were subjected to nonlinear regression analysis by SAS (19).
Extraction and Purification of Cholesterol and COPs. A modified
method based on that of Nourooz-Zadeh (20) was used to extract and
purify COPs. The heated mixture was added to 20 mL of hexane/
samples for extraction and HPLC analysis, and a high recovery of 97-
100% was achieved.
Kinetic Analyses of COPs. The concentration changes of cholesterol
and various COPs during heating of cholesterol in the presence or
absence of quercetin were analyzed statistically by using a nonlinear
regression procedure of SAS (19). All of the rate constants for each
reaction were estimated by using the least-squares method with a
nonlinear regression procedure-Marquardt iterative approach until the
convergence of the parameters was best fitted. The precision of the
parameters of the kinetic equations was also evaluated.
Statistical Analyses. Analyses of variance and comparisons among
the residual amounts of cholesterol at different heating time intervals
were conducted using the SAS system (19). After a preliminary F test,
the differences among the residual cholesterol were determined by
Duncan’s multiple-range test at a 5% probability level.
2
-propanol (3:2, v/v) and shaken vigorously for 3 min, after which the
solution was centrifuged at 26000g for 5 min. The upper layer was
collected and poured into a centrifuged tube, and 12 mL of distilled
water was added. The solution was centrifuged again, and the upper
phase was also collected and evaporated to dryness at 35 °C. The residue
was dissolved in 1 mL of hexane/1,2-dichloroethane (1:1, v/v) and
RESULTS AND DISCUSSION
It has been well established that cholesterol is relatively stable
in solid form and is prone to undergo autoxidation in liquid
form (23, 24), especially when the temperature reaches its
melting point (148.5 °C) or higher (5). At a temperature of
2
poured into a NH cartridge. In the beginning, 5 mL of hexane was
added to the cartridge to remove impurities such as hydrocarbon,
cholesterol ester, and triglyceride. Then 25 mL of hexane/1,2-
dichloroethane/2-propanol (50:30:15, v/v/v) was added to elute cho-
lesterol and COPs. The solution was evaporated to dryness at 35 °C,
dissolved in hexane/2-propanol (95:5, v/v), and filtered through a
2
00-300 °C, the thermal degradation of cholesterol has been
reported to dominate, instead of autoxidation (25). After various
trials, the most suitable temperature for studying the inhibition
kinetics of cholesterol oxidation by quercetin was found to be
150 °C. Also, this temperature is similar to that used for low-
temperature frying of foods. Thus, the heating temperature of
0.2-µm membrane filter for HPLC analysis. For recovery study, a fixed
concentration of various COPs standards was added to the sample for
extraction and purification. After HPLC analysis, the recovery of each
COP was obtained on the basis of the ratio of the amount of each COP
standard after and before HPLC.
TLC Analysis of COPs. Preparation of Wurster Dye. The Wurster
dye was prepared according to a method of Smith and Hill (21). One
gram of N,N-dimethyl-p-phenylenediamine dihydrochloride was dis-
solved in 100 mL of 50% methanol solution (in water), which was
shaken, and 1 mL of glacial acetic acid was added. Then the mixture
was poured into a glass vial and stored at -20 °C for use.
1
50 °C was selected in this study to minimize the loss of
cholesterol due to thermal degradation and accelerate the
formation of COPs at the same time.
Due to the poor solubility of the mixture of quercetin,
cholesterol, and COPs in the oil, a solvent system of lauryl
alcohol/paraffin oil (1:4, v/v) was used instead. After several
preliminary studies, it was observed that the incorporation of
0.002 mg (0.002%, w/w) of quercetin was adequate to inhibit
the formation of COPs for 100 mg of cholesterol. No COPs
were detected for the 2-h heating period when a higher amount
of quercetin (>0.01 mg) was used. An average loss of 2.1%
was found for cholesterol when preheated alone from 25 to 150
Separation of COPs by TLC. Initially the plate coated with silica
gel was placed in a glass tank lined with a filter paper (20 × 20 cm)
and equilibrated for 30 min with 200 mL of benzene/ethyl acetate
(60:40, v/v) (22) before separation. A 10-µL volume of sample extract
was spotted on the glass plate with a micropipet, and the solvent front
was allowed to run for a distance of ∼18 cm at room temperature.
Then the plate was dried in an oven at 110 °C for 10 min and sprayed
°
C with nitrogen flushing before oxygen pumping, whereas a
slightly higher loss (3.1%) was shown for cholesterol heated
together with quercetin. This result should be due to thermal
degradation of cholesterol under anaerobic condition, and the
presence of quercetin failed to protect cholesterol from thermal
degradation during the preheating period. In other words,
quercetin may be effective only in inhibiting cholesterol
oxidation. Also, there is no significant difference in cholesterol
loss for the presence and absence of quercetin. Therefore, for
kinetic study of each COP formation, initial levels of 96.9 and
with 50% H
UV radiation at 254 nm was recorded. Both 7R-hydroperoxycholesterol
7R-OOH) and 7â-hydroperoxycholesterol (7â-OOH) bands were
2 4
SO , and the color development of each spot (COPs) under
(
identified and quantified on the basis of the Wurster dye method as
shown in a previous study (7).
HPLC Analyses. A mobile phase of hexane/2-propanol (95:5, v/v)
and two LiChrospher 100 CN columns with a flow rate at 1.0 mL/min
-
5
and RI detection (sensitivity ) 16 × 10 RIU) were used to separate
cholesterol and various COPs. Two columns connected in series were
used to enhance the resolution power of COPs. Identification was carried
out by comparing retention times of unknown peaks with standards
and cochromatography with added standards. Because of the absence
of a suitable internal standard, each COP was quantified using an
external calibration method. Seven concentrations of cholesterol (500,
9
7.9 mg of cholesterol were used for studying the heating
treatments with and without quercetin, respectively (Table 1).
Formation of COPs. Figure 1 shows the major reaction
pathways and rate constants of cholesterol oxidation and
degradation based on our previous study (6). In the presence of
lauryl alcohol, 5,6R-EP or 5,6â-EP may undergo alcoholysis
to form triol (6, 18), which can be further dehydrogenated to
cholestan-3â,5R,6-one (26).
Figures 2 and 3 show the HPLC chromatograms of COPs
formed during heating at 150 °C in the absence and presence
of quercetin, respectively. A total of eight major COPs, including
1
000, 2500, 5000, 18000, 25000, and 50000 µg/mL) and each COP
(50, 100, 200, 500, 1000, 2500, and 5000 µg/mL) used to prepare the
standard calibration curves were injected onto the HPLC separately,
and the curve for cholesterol and each COP standard was obtained by
plotting concentration against area. The regression equations and
2
correlation coefficients (r ) were calculated using a CHEN-WIN
2
computer software system, and a high r value (>0.99) was found for
5,6R-EP, 5,6â-EP, 7-keto, 7R-OH, 7â-OH, 7R-OOH, 7â-OOH,
cholesterol and various COPs standards. Each COP was quantified using
and triol were adequately resolved within 40 min, with the elu-
tion order being cholesterol, 5.6R-EP, 5.6â-EP, 7-keto, 7R-OH,
7â-OH, 7R-OOH, 7â-OOH, and triol. No COPs were formed
at 0 min of heating, indicating that cholesterol oxidation did
a method as described by Chien et al. (5). The eluates of 7R-OOH and
7
â-OOH were collected separately and then quantified using the
Wurster dye method (7). As described above, recovery was also
performed by adding standards of cholesterol and various COPs to

1488 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Chien et al.
Table 1. Inhibitory Effect of Quercetin on Percentage Changes of
Cholesterola during Heating at 150
°C
cholesterolb (%)
heating
time (min)
%
%
controlc
remaining
quercetinc
remaining
0
5
97.9
±
±
±
±
±
±
0.6Aa
100.0
95.0
88.4
64.3
49.8
36.2
−
96.9
95.6
93.9
85.9
78.5
69.3
53.0
±
±
±
±
±
±
±
0.3Aa
0.2Ab
0.1Bb
0.1Cb
0.3Db
0.4Eb
2.1F
100.0
98.7
97.0
88.7
81.0
71.6
54.7
93.0
86.6
63.0
48.8
35.5
1.0Ba
0.3Ca
1.0Da
0.6Ea
0.2Fa
10
30
60
90
d
1
20
−
a
Values are expressed as percentage relative to cholesterol content at heating
b
time
)
0 min. Cholesterol (100 mg) was dissolved in 500
µ
L of lauryl alcohol/
paraffin oil (1:4, v/v) solvent in the absence (control) or presence of 0.002 mg of
c
quercetin (quercetin). Mean and standard deviation of triplicate determinations;
entries bearing different letters of A
significantly different (p < 0.05). Not detected.
−
F (a
−
b) in the same column (row) are
d
Figure 2. HPLC chromatograms of COPs formation during heating of
cholesterol alone at 150 °C.
Figure 1. Major autoxidation and degradation pathways of cholesterol.
All compounds were solubilized in the lauryl alcohol/paraffin (1:4, v/v)
solvent: cholesterol (A), 7-hydroperoxycholesterol (A
terol (B), 7-ketocholesterol (C), 5,6-epoxycholesterol (E), degraded products
D), cholestan-3 ,5 ,6 -triol (T), cholestan-3 ,5 ,6-one (O). k , k
, and k are the corresponding rate constants. The scheme is based
′), 7-hydroxycholes-
(
â
R
â
â
R
−
1 5
3
′,
6
k ′
7
on a study by Chien et al. (6).
not occur with nitrogen flushing prior to heating to 150 °C
(Figures 2 and 3). Without quercetin, the COPs were formed
gradually during the initial heating period (e30 min) and
increased sharply thereafter (Figure 2). Also, the amount of
COPs followed an increased trend for the increase of heating
time. The level of 7-OOH (7R-OOH and 7â-OOH) accounted
for 0.00037% of cholesterol after 10 min of heating and then
reached a plateau (1.2% of cholesterol) in 90 min (Figures 2
and 4). Meanwhile, the degradation of 7-OOH proceeded
quickly as soon as it was formed from cholesterol, which in
turn led to the formation of 7-OH and 7-keto through reduc-
tion and dehydration, respectively. On the basis of the initial
amount of cholesterol, the percentages of 7-OH and 7-keto
formed were 4.2 and 3.8%, respectively, over a 90-min heating
period (Figure 4). On the other hand, with quercetin, an
extremely low amount of COPs was formed during the 30-min
heating period and increased drastically after 60 min (Figure
Figure 3. HPLC chromatograms of COPs formation in the presence of
quercetin (0.002%, w/w) during heating of cholesterol at 150 C.
°
react with peroxy radicals, which are responsible for the process
of radical oxidation. From Figure 4, two important points,
including the autocatalytic nature of the cholesterol oxidation
and the induction period (∼90 min) caused by the addition of
quercetin, appeared to be evident. The former is probably caused
by the thermal decomposition of hydroperoxides with the
formation of free radicals, which accelerate oxidation, whereas
the latter is caused by quercetin to donate a hydrogen atom to
3
), probably because of quercetin degradation and its ability to

Effect of Quercetin on Cholesterol Oxidation during Heating
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1489
Figure 5. Inhibitory effect of quercetin on 5,6-EP and triol formation during
heating of cholesterol at 150
°C: b, mean of experimental data for
cholesterol only; , mean of experimental data for cholesterol in the
O
presence of quercetin.
performed.
s, best fitting line. Triplicate experiments were
formation of 7-OOH. Nevertheless, the amount of 5,6-epoxides
produced after 90 min of heating was 5.3 times lower than for
the control treatment (Figure 5).
The formation of triol was also observed in this study
(
5
(
Figures 2, 3, and 5). Theoretically, triol can be formed from
,6-epoxides in an aqueous solution through acidic hydrolysis
10). In our experiment, triol was probably formed through
alcoholysis because lauryl alcohol was used instead of water
6, 18). In the absence of quercetin, a significant amount of
Figure 4. Inhibitory effect of quercetin on major COPs formed in the C-7
(
oxidation route during heating of cholesterol at 150
experimental data for cholesterol only; , mean of experimental data for
, best fitting line. Triplicate
°C: b, mean of
triol was formed after 30 min of heating, and then a sharp
increase (9.7%) followed after 90 min (Figure 5). In contrast,
with quercetin, an insignificant quantity of triol was found during
the initial heating period, followed by a gradual increase to 9.8%
after extensive heating to 120 min. Despite that, the level of
triol formed was lower than for the control treatment under the
same heating time.
By comparison of the results shown above, it can be
concluded that the total amount of COPs was greatly reduced
in the presence of quercetin. Approximately 0.5% COPs of
cholesterol was formed after 30 min of heating, which was lower
than for the control treatment (5.9%) by 11.8-fold. However,
the level of COPs further increased to 7.7% in 90 min and
attained a peak (24.4%) in 120 min, with the former being lower
than for the control treatment by 4-fold. As explained before,
this is probably because of drastic degradation of quercetin when
it is heated beyond 60 min. It was also shown that the amount
of cholesterol reduced during heating was not equal to that of
COPs formed, which may arise from the phenomenon that, in
addition to oxidation, the thermal degradation of cholesterol may
also proceed simultaneously.
O
cholesterol in the presence of quercetin;
experiments were performed.
s
stabilize peroxy radicals. This phenomenon proved that the
addition of 0.002% (w/w) quercetin should exert a strong
antioxidant effect on cholesterol oxidation during the initial
heating period at 150 °C.
Compared to the control treatment, the levels of 7-OOH were
6.6 and 4.5 times lower for the heating times 0 and 10 min
after quercetin addition, respectively, demonstrating that quer-
cetin may possess a strong antioxidant activity against choles-
terol peroxidation. Similarly, the amounts of 7-OH and 7-keto
were correspondingly decreased by 4.3- and 7.9-fold, respec-
tively, when compared to the control treatment after 90 min of
heating. This phenomenon may be due to the slow formation
of 7-OOH during heating of cholesterol together with quercetin.
However, after prolonged heating for 120 min, the contents of
7-OOH, 7-OH, and 7-keto increased substantially, probably
because of the thermal degradation or free radical scavenging
capacity of quercetin.
The epoxidation of cholesterol also occurred along with the
formation of 7-OOH during heating of cholesterol without
quercetin, and the levels of 5,6-epoxides (5,6R-EP and 5,6â-
EP) showed a tendency to increase (Figures 2 and 5). A small
content of 5,6-epoxides was induced initially, which then rose
to 1.8 and 12.2% of cholesterol, respectively, after 30 and 90
min of heating (Figure 5). The formation curve of 5,6-epoxides
was similar to that of 7-OOH, implying that the cholesterol
epoxidation may proceed quickly as soon as 7-OOH is generated
Thermal Degradation of Cholesterol during Heating.
Table 1 shows the percentage changes of cholesterol during
heating at 150 °C for varied lengths of time in the absence or
presence of quercetin. A significant percentage difference
occurred between the control and quercetin treatments for all
heating times. Without quercetin, cholesterol decreased by
35.7% after 30 min of heating, and greater losses by 50.2 and
63.8% were shown after 60 and 90 min of heating, respectively
(Table 1). This level was lower than that in a previous study
by Chien et al. (5), who reported that only ∼33.3% cholesterol
remained after heating a cholesterol thin film at 150 °C for 30
min. Osada et al. (4) also found that the residual percentage
was 40% after 150 °C heating of cholesterol for 24 h. This
difference may be due to the variety of solvents used to dissolve
cholesterol and the pumping rate of oxygen into the heating
system. On the contrary, with quercetin, the degradation of
(22, 27). The amount of 5,6â-EP formed from 7-OOH was
higher than that from 5,6R-EP (Figure 2), demonstrating a
greater stability for the â-form as compared to the R-form (27).
Conversely, with quercetin, the formation of 5,6-epoxides was
greatly reduced by 6.2-fold after 30 min of heating (Figures 3
and 5). However, after heating for 60 min, a large increase was
shown for 5,6-epoxides, which may be attributed to rapid

1490 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Chien et al.
cholesterol proceeded slowly during the initial heating period,
which can be accounted for by 19.0% cholesterol loss after 60
min of heating. However, the loss further increased to 45.3%
thereafter, which was much lower than the for control treatment.
This result demonstrated that quercetin exhibits a strong
antioxidant activity against COPs formation. Several studies
have also proved that flavonoids such as quercetin can inhibit
free radical chain reaction by donating hydrogen atoms to
stabilize free radicals (28, 29).
min of heating time and k4, k5, and k14 are the reaction rate
constants (h ).
-
1
For the control treatment, the concentration ratio of 7-OOH
to cholesterol for the heating time of 30 min was 1/3500.
However, for the quercetin treatment, the concentration of
7-OOH formed after 30 min of heating was much lower than
that of cholesterol (30000[A′] = [A]). Thus, the first term
(k14[A][A′]) on the right of eq 2 is relatively small as compared
to the second term (k5[A]). Furthermore, percentages of 2.8 and
0
3
.3% of 5,6-EP were formed on the basis of cholesterol after
0 min of heating, respectively, with and without quercetin.
Kinetic Studies on Thermal Degradation and Oxidation
of Cholesterol. As both R- and â-forms of COPs possess similar
reactivity toward oxidation, only five major COPs, including
Therefore, cholesterol degradation can be regarded as the main
reaction to proceed during heating of cholesterol. Equation 2
was thus simplified as -d[A]/dt ) k5[A], and the integration
of the above equation gave
7
5
-OOH (7R- and 7â-OOH), 7-OH (7R- and 7â-OH), 7-keto,
,6-EP (5,6R- and 5,6â-EP), and triol, as well as cholesterol
were selected for kinetic study. The initial heating period (e30
min) was considered as a lag period because quercetin showed
a high antioxidant activity. Thus, the concentration changes of
various COPs during heating of cholesterol together with
quercetin for 30 min were subjected to kinetic study by using
the nonlinear regression models. The COPs formation during
the heating period of 30-120 min was not kinetically analyzed
because a low antioxidant activity of quercetin should follow a
reaction rate model similar to that of the control treatment.
-
k5t
[A] ) [A ] e
(3)
0
where [A0] is the percentage concentration of cholesterol at 0
min of heating time.
Equation 1 is in the form of the Bernoulli equation (n ) 2).
By transformation of a variable, [A′], eq 4 was obtained by
integration
According to a paper by Chien et al. (6), the reactions for
the oxidative pathway can be divided into two major routes of
C-7 oxidation and epoxidation (Figure 1). The oxidation
reaction involved the attack of free radicals on cholesterol to
result in the formation of 7-OOH, which belongs to the second-
order, and the rate equation for 7-OOH formation (A f A′)
was listed in the equation, as described in a previous study (5).
It has been established that cholesterol oxidation would be
unlikely to proceed unless there is sufficient 7-OOH ac-
cumulated in the reaction system (5). Besides, 7-OOH can be
further degraded to 7-OH or 7-keto through the first-order
reaction (5, 30, 31). The rate equation for 7-OOH formation
was thus modified as
-
1
k1
k1
1
[A′]0
e-m1t
[A′] )
+
-
(4)
[
(
)
]
m [A′]
m [A′]
1
max
1
max
where [A′0] is the percentage concentration of 7-OOH at 0 min
of heating time and m1 ) k1 - k2 - k3. For the epoxidation
reaction, the formation of 5,6-EP follows second order and is
dependent upon the concentrations of both cholesterol and
7
-OOH (5, 32). Thus, the rate equation for both 5,6-EP
formation and degradation could be given as
d[E]
dt
)
k [A][A′] - k′ [E]
(5)
4
6
d[A′]
dt
[A′]
)
)
k 1 -
[A′] - k [A′] - k [A′]
2 3
where [E] is the percentage concentration of 5,6-EP at t min of
1
(
)
[A′]max
-1
heating time and k4 and k′6 are the reaction rate constants (h ).
As lauryl alcohol is used instead of water in the solvent
system, alcoholysis of 5,6-EP is most likely to occur in our
study for the reaction (E f T) in Figure 1 according to March
k1
2
m [A′] -
[A′]
(1)
1
[
A′]max
(18). Also, triol may be further dehydrogenated to form
where [A′]max is the maximum attainable concentration of
-OOH, [A′] is the percentage concentration of 7-OOH at t min
of heating time, and k1, k2, and k3 are the reaction rate constants
cholestan-3â,5R,6-one (T f O) (26). As a result, the rate
7
equation of triol formation can be written as
-
1
(h ).
d[T]
)
k [L ][E] - k [T] ) k ′[E] - k [T]
(6)
6
0
8
6
7
In addition, the rate equations of C-7 oxidation routes B and
dt
C were written as in a previous study (5). Because both
cholesterol and 7-OOH were involved in the epoxidation
reaction (32), the rate equation for 5,6-EP formation was also
shown as in a previous study (5). Both 7-OOH and 5,6-EP
formation as well as thermal degradation of cholesterol may
lead to a substantial decrease of cholesterol concentration. Thus,
the overall decreasing rate of cholesterol could be regarded as
the sum of the free radical chain reaction, epoxidation, and
thermal degradation and can be written as
where [T] is the percentage concentration of triol at t min of
heating time, [L ] is the percentage concentration of lauryl
0
alcohol at time 0, and k , k , and k are the reaction rate constants
6
7
8
-
1
(h ).
Integration after substitution of eqs 3 and 4 into eq 5 gave
eq 7 as follows:
′
[k 6-k5]s
- ′
′
t
e
k 6t
-k 6t
[
E] ) [E ] e
+ k [A ] e
∫
ds
e-m1s
0
4
0
0
m2
1
m2
d[A]
dt
+
-
-
) k [A][A′] + k [A][A′] + k [A] )
m1
[
[A′0] m1
]
1
4
5
(7)
k [A][A′] + k [A] (2)
14
5
Likewise, eq 8 was obtained by integration after substitution
of eq 7 into eq 6
where [A] is the percentage concentration of cholesterol at t

Effect of Quercetin on Cholesterol Oxidation during Heating
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1491
Table 2. Rate Constants of Cholesterol Autoxidation and Degradation
controla
quercetina
rate equationb
reaction
k (h-1)
r 2
k (h-1)
r 2
k1
chol
chol
chol
9
8
7-OOH
free radical chain reaction
epoxidation
453.5
±
±
0.0
58.1
0.02
1.00
0.99
0.99
1.00
1.00
0.99
0.99
0.64
111.7
±
±
0.0
47.4
0.02
1.00
0.88
0.96
1.00
1.00
0.99
1.00
k
4
9
8
5
5,6-EP
683.1
551.4
k
98
degradation
degradation
0.66
±
0.28 ±
k
2
7
7
7
5
-OOH
9
8
3
7-OH
7-keto
7-keto
reduction
290.3
155.5
±
±
0.0
0.0
76.8
31.0
±
±
0.0
0.0
k
-OOH
9
8
dehydration
k3
9
′
-OH
8
dehydrogenation
alcoholysis
0.68
±
0.10
0.11
±
0.02
0.06
k
6
′
,6-EP
9
8
triol
0.38
5.35
±
±
0.07
1.00
1.61
0.0c
±
k
7
triol
98
O
dehydrogenation
a
Control, cholesterol oxidation during heating (0 < t < 90 min). Quercetin, cholesterol oxidation in the presence of quercetin during heating (30 < t < 120 min). b Chol,
cholesterol; 7-OOH, 7-hydroperoxycholesterol; 7-OH, 7-hydroxycholesterol; 7-keto, 7-ketocholesterol; 5,6-EP, 5,6-epoxycholesterol; triol, 5
R
-cholestan-3
â
,5R
,6â
-triol; O,
c
cholesten-3
â
,5R
-diol-6-one; k
1
−k
5
, k
3
′
, k
6
′
, and k
7
are rate constants. No reaction product was detected.
[
T] )
curves fit well with the data points (Figures 4 and 5), except
for both 5,6-EP and triol when heated for 2 h (Figure 5).
[
k′6-k5]s
t
′
ν
e
-
k8t
(k8-k 6)ν
k′ k [A ] e
∫
e
∫
ds dν
6
4
0
For the various oxidation reactions, the correlation coefficients
of the free radical chain reaction and epoxidation were 1.00
and 0.99 in the absence of quercetin and 1.00 and 0.88 in the
presence of quercetin, respectively. This result implied that both
reactions for the formation of 7-OOH and 5,6-epoxide from
cholesterol fit well the second order. A high correlation
coefficient was also observed for the reduction and dehydration
reactions of 7-OOH (Table 2), indicating that the reactions for
the formation of 7-OH and 7-keto fit the first order. Although
the formation of triol by alcoholysis of 5,6-EP for the quercetin
treatment was completely inhibited, the low correlation coef-
ficient (0.64) for the control treatment revealed that the reaction
of T f O (Figure 1) should be reassessed for the first-order
model to better estimate the rate constant of k7. The above results
also suggested that despite the presence or absence of quercetin,
the rate equations could successfully estimate the thermal
degradation of cholesterol and predict the concentration changes
of COPs during the entire heating period.
0
0
m2
1
[A′0]
m2
+
- m1 e-m1s
)
(
m1
[
]
(8)
where [E0] is the percentage concentration of 5,6-EP at 0 min
of heating time and s, t, and υ are time.
Among the above equations, both eqs 3 and 4 are in the forms
of exact integration. As the exact integrations for eqs 7 and 8
are not possible, we can only get a better approximation to the
integral term in the equation by using a quadratic polynomial
of Taylor’s series. For example, eq 7 can be expressed as
follows:
k′6t
1
2
-
[E] ) e
[E] + k [A] [A′] t + (m - [A′] m +
{
0
4
0
0
(
1
0
2
2
1
6
2
2
0
2
k ′ - k )t + (m - 3[A′] m m + 2[A′] m +
6
5
1
0
1
2
2
2
2
k ′m - 2[A′] k ′m - 2k m + 2[A′] k m + k ′ -
6 1 0 6 2 5 1 0 5 2 6
It was also shown that the k4 value is very large with or
without quercetin, suggesting that the 5,6-EP concentration
increased sharply as soon as 7-OOH was formed during the
free radical chain reaction. It may be postulated that most
2
3
2
k k ′ + k )t
(9)
5
6
5
)}
The experimental data were thus analyzed in sequence and
7-OOH was converted to 5,6-EP in the beginning during heating,
fitted best to the corresponding nonlinear regression models as
shown above. The data for computing k5 in eq 3 should be the
residual cholesterol as the loss of cholesterol can be attributed
mainly to thermal degradation. Therefore, k5 was recalculated
by adding the total amount of COPs to the residual cholesterol,
and thus the 7-OOH concentrations were maintained at a very
low level (0.03% for the control and 0.003% for the quercetin
treatment) over a 30-min heating period. Both are significantly
lower than the initial maximum concentrations of 7-OOH, which
amounted to 2.1 and 1.0% of total cholesterol in the absence
and presence of quercetin, respectively ([A′max] in eqs 1 and
-
1
2
which was found to be 0.66 ( 0.02 h (r ) 0.99) without
-
1
2
quercetin and 0.28 ( 0.02 h (r ) 0.96) with quercetin (Table
). The rate constants in eq 4 and in the quadratic polynomial
series for the eq 7 (i.e., eqs 9 and 8) as well as the others for
-OH and 7-keto were estimated by using the least-squares
4
). However, in the heating system, it is difficult for 7-OOH to
2
maintain this level because it can undergo further degradation
to form 7-OH or 7-keto via the C-7 route and 5,6-EP via the
epoxidation route. Therefore, the actual maximum level of
7-OOH can be increased to only about 1.3% without quercetin
and 0.7% with quercetin after 90 min of heating (Figure 4).
7
method with a nonlinear Marquardt iterative approach until the
convergences of the best-fitted parameters were met. The rate
constants of the reaction pathways of cholesterol degradation
and oxidation during heating at 150 °C with or without quercetin
are shown in Table 2. With the exception of the dehydroge-
nation of triol (r ) 0.64), the r values of all the reactions
were >0.99 in the absence of quercetin and >0.88 in the
presence of quercetin. Each COP was further plotted on the
basis of the corresponding rate equation and the rate constants
which are also shown in Table 2. With quercetin, all of the
-
1
By comparing the rate constants (h ) of the major reaction
pathways of cholesterol oxidation, a similar trend can be found
in the order k4 > k1> k2 > k3 > k7 for both the control and
2
2
-
1
quercetin treatments. All of the rate constants (h ) are much
higher than k5, k6′, and k3′, which are approximately in the same
magnitude. However, the rate constants of COPs formation with
quercetin were 0.2-5.0 times lower than those without quer-

1492 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Chien et al.
cetin, with the exception of k6′ in the alcoholysis reaction (1.61
0.38). Theoretically, quercetin may play an important role
(13) Fuhrman, B.; Lavy, A.; Aviram, M. Consumption of red wine
meals reduces the susceptibility of human plasma and LDL to
lipid peroxidation. Am. J. Clin. Nutr. 1995, 61, 549-554.
14) Arai, Y.; Watanabe, S.; Kimira, M.; Shimoi, K.; Mochizuki, R.;
Kinae, N. Dietary antioxidant flavonols, flavones and isoflavones
by Japanese women and the inverse correlation between quercetin
intake and plasma LDL cholesterol concentration. J. Nutr. 2000,
>
in opening the highly strained three-membered ring of 5,6-EP.
Quercetin contains two ortho-hydroxyl groups and should be
(
+
able to release a proton (H ) in the heating system (33), and
the protonated 5,6-EP may in turn undergo alcoholysis in an
alcoholic solvent through antihydroxylation, which involved SN2
or SN1 attack (18).
1
30, 2243-2250.
(
(
(
(
15) Hopia, A.; Heinonen, M. Antioxidant activity of flavonol
aglycones and their glycosides in methyl linoleate. J. Am. Oil
Chem. Soc. 1999, 76, 139-144.
16) Pekkarinen, S. S.; Heinonen, I. M.; Hopia, A. I. Flavonoids
quercetin, myricetin, kaemferol and (+)-catechin as antioxidants
in methyl linoleate. J. Sci. Food Agric. 2000, 79, 499-506.
17) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Antioxidant
properties of phenolic compounds. Trends Plant Sci. 1997, 2,
Compared to the control treatment, all of the rate constants
with quercetin treatment decreased substantially, especially for
the free radical chain reaction (k1) by 4.1-fold. A similar result
was reported by Nielsen et al. (34), who studied cholesterol
oxidation in a heterogeneous system initiated by water-soluble
radicals. In Figures 4 and 5, the curves fit well with the
experimental data based on the oxidation of cholesterol during
heating. These results suggested that the kinetic models
developed in this study can be used to predict the concentration
changes of cholesterol and COPs during heating of cholesterol
with or without quercetin. In conclusion, the major reaction
pathways during the heating of cholesterol in the presence of
oxygen include the thermal degradation of cholesterol and the
formation of various COPs. With or without quercetin, the
1
52-159.
18) March, J. AdVanced Organic Chemistry, 2nd ed.; McGraw-
Hill: New York, 1977; pp 360-361.
(19) SAS. SAS Procedures and SAS/Graph User’s Guide, version 9.1,
software release 9.1: foundation (TS1M3 DSCS3054) of the SAS
System for Microsoft Windows; SAS Institute: Cary, NC, 2004.
(
20) Nourooz-Zadeh, J. Determination of autoxidation products from
free or total cholesterol: a new multistep enrichment methodol-
ogy including the enzymatic release of esterified cholesterol. J.
Agric. Food Chem. 1990, 38, 1667-1673.
2
correlation coefficients (r ) for all of the reactions ranged from
0
.64 to 1.00 by nonlinear regression analyses. The reactions
(
21) Smith, L. L.; Hill, F. L. Detection of sterol hydroperoxides on
thin layer chromatoplates by means of the Wurster dyes. J.
Chromatogr. 1972, 66, 101-109.
for 7-OOH and 5,6-EP formations fit the second order, whereas
all of the other reactions fit the first order.
(
22) Maerker, G.; Bunick, F. J. Cholesterol oxides I: isolation and
determination of some cholesterol oxidation products. J. Am. Oil
Chem. Soc. 1986, 63, 767-771.
LITERATURE CITED
(
(
(
23) Kim, S. K.; Nawar, W. W. Oxidation interactions of cholesterol
with triacylglycerols. J. Am. Oil Chem. Soc. 1991, 68, 931-934.
24) Kim, S. K.; Nawar, W. W. Parameters influencing cholesterol
oxidation. Lipid 1993, 28, 917-922.
25) Park, S. W.; Addis, P. B. Further investigation of oxidized
cholesterol derivatives in heated fats. J. Food Sci. 1986, 51,
(
(
(
1) Paniangvait, P.; King, A. J.; Jones, A. D.; German, B. G.
Cholesterol oxides in foods of animal origin. J. Food Sci. 1995,
6
0, 1159-1174.
2) Osada, K.; Kodama, T.; Cui, L.; Yamada, K.; Sugano, M. Levels
and formation of oxidize cholesterol in processed marine food.
J. Agric. Food Chem. 1993, 41, 1, 1893-1898.
1
380-1381.
3) Osada, K.; Hoshina, S.; Nakamura, S.; Sugano, M. Cholesterol
oxidation in meat products and its regulation by supplementation
of sodium nitrite and apple polyphenol before processing. J.
Agric. Food Chem. 2000, 48, 3823-3829.
(
26) Peng, S. K.; Taylor, C. B.; Tham, P.; Werthessen, N. T.;
Mikkelson, B. Cytotoxicity of oxidation derivatives of cholesterol
on cultured aortic smooth muscle cells and their effect on cho-
lesterol biosynthesis. Am. J. Clin. Nutr. 1979, 32, 1033-1042.
27) Smith, L. L. Cholesterol Autoxidation; Plenum Press: New York,
(
(
(
(
(
4) Osada, K.; Kodama, T.; Yamada, K.; Sugano M. Oxidation of
(
(
cholesterol by heating. J. Agric. Food Chem. 1993, 41, 1198-
1
981; pp 1, 52, 59, 126, 242, 342, 494.
1
202.
28) Foti, M.; Piattelli, M.; Baratta, M. T.; Ruberto, G. Flavonoids,
coumarins, and cinnamic acids as antioxidants in a micellar
system. Structure-activity relationship. J. Agric. Food Chem.
5) Chien, J. T.; Wang, H. C.; Chen, B. H. Kinetic model of the
cholesterol oxidation during heating. J. Agric. Food Chem. 1998,
4
6, 2572-2577.
1
996, 44, 497-501.
6) Chien, J. T.; Huang, D.-Y.; Chen, B. H. Kinetic study of the
cholesterol oxidation as inhibited by stearylamine during heating.
J. Agric. Food Chem. 2004, 52, 7132-7138.
7) Chien, J. T.; Lu, Y. F.; Hu, P. C.; Chen, B. H. Cholesterol
photooxidation as affected by combination of riboflavin and fatty
acids methyl esters. Food Chem. 2003, 81, 421-431.
8) Mahfouz, M. M.; Kummerow, F. A. Cholesterol-rich diets have
different effects on lipid peroxidation, cholesterol oxides, and
antioxidant enzymes in rats and rabbits. J. Nutr. Biochem. 2000,
(
(
(
29) Benavente-Garcia, O.; Castillo, J.; Marin, F. R.; Ortuno, A.; Del-
Rio, J. A. Uses and properties of Citrus flavonoids. J. Agric.
Food Chem. 1997, 45, 4505-4515.
30) Yan, P. S.; White, P. J. Cholesterol oxidation in heated lard
enriched with two levels of cholesterol. J. Am. Oil Chem. Soc.
1
990, 67, 927-931.
31) Yan, P. S.; White, P. J. Linalyl acetate and methyl silicone effects
on cholesterol and triglyceride oxidation in heated lard. J. Am.
Oil Chem. Soc. 1991, 68, 763-768.
1
1, 293-302.
(
(
(
32) Smith, L. L. Cholesterol autoxidation 1981-1986. Chem. Phys.
(
9) Leonarduzzi, G.; Sottero, B.; Poli, G. Oxidized products of
cholesterol: dietary and metabolic origin and proatherosclerotic
effects (review). J. Nutr. Biochem. 2002, 13, 700-710.
Lipids 1987, 44, 87-125.
33) Morrison, R. T.; Boyd, R. N. Organic Chemistry, 3rd ed.; Allyn
and Bacon: Boston, MA, 1973; pp 563-566, 797-799.
34) Nielsen, J. H.; Olsen, C. E.; Skibsted, L. H. Cholesterol oxidation
in a heterogeneous system initiated by water-soluble radicals.
Food Chem. 1996, 56, 33-37.
(
10) Maerker, G. Cholesterol autoxidationscurrent status. J. Am. Oil
Chem. Soc. 1987, 64, 388-392.
(
11) Huber, K. C.; Pike, O. A.; Huber, C. S. Antioxidant inhibition
of cholesterol oxidation in a spray-dried food system during
accelerated storage. J. Food Sci. 1995, 60, 909-912, 916.
12) Guardiola, F.; Codony, R.; Rafecas, M.; Grau, A.; Jord a´ n, A.;
Boatella, J. Oxysterol formation in spray-dried egg processed
and stored under various conditions: prevention and relationship
with other quality parameters. J. Agric. Food Chem. 1997, 45,
Received for review October 12, 2005. Revised manuscript received
December 13, 2005. Accepted December 19, 2005. This study was
supported by a grant (NSC-91-2313-B-030-006) from the National
Science Council, Taiwan.
(
2
229-2243.
JF052529R