original papers - Advances in Clinical and Experimental Medicine

ORIGINAL PAPERS
Adv Clin Exp Med 2008, 17, 5, 503–512
ISSN 1230−025X
© Copyright by Silesian Piasts
University of Medicine in Wrocław
BOŻENA REGULSKA−ILOW, RAFAŁ ILOW
Assessment of the Influence of Quercetin on
the Activities of Selected Enzymes in Experimental Rats
Under Oxidative Stress Due to Oxidized Dietary Fats
Ocena wpływu kwercetyny na aktywność wybranych enzymów
u szczurów doświadczalnych w stresie oksydacyjnym
wywołanym tłuszczami pokarmowymi
Department of Food Science and Dietetics, Silesian Piasts University of Medicine in Wrocław, Poland
Abstract
Background. The kind and quality of dietary fat may influence the bioactivity of quercetin manifested as its effect
on the activities of selected enzymes.
Objectives. The aim of the study was to evaluate the effect of quercetin on the oxidative potential of blood and
liver functions in rats under oxidative stress due to dietary fats.
Material and Methods. The rats were fed diets with 8% content of fat and 0.5% addition of cholesterol.
Subsequent evaluations involved the effect of quercetin on the activities of superoxide dismutase (SOD) in ery−
throcytes and glutathione peroxidase (GPx) in full blood as well as those of alanine and asparginate aminotrans−
ferases (ALT and AST, respectively) and alkaline phosphatase in plasma and liver extracts. The dietary source of
prooxidants included oxidized sunflower oil and lard. The four−week experiment was performed on 80 male
Buffalo rats, of which 40 were given a 0.075% addition of quercetin as a dietary supplement (60 quercetin per
mg/kg of rat body mass).
Results. Quercetin as well as the quality and kind of fat in the diet were found to exert a significant effect on the
investigated parameters. The addition of quercetin resulted in a significant activation of SOD in the rats on a diet
with fresh oil and decreased SOD activity in the rats fed oxidized lard. It decreased the activity of GPx in rats fed
oxidized lard and oil. The activity of plasma alanine and asparginate aminotransferases was in the physiological
range and did not indicate damage to the liver cells. Quercetin decreased significantly the activity of ALT in the
plasma of rats on a diet containing oxidized oil. Quercetin reduced significantly plasma AP activity in rats fed fresh
lard and increased its activity in rats on a diet containing oxidized oil. The changes in AP activity in plasma and in
liver extracts followed the same directions. Quercetin exerted an unfavorable effect in the form of increased accu−
mulation of fat in the liver cells, which was indicated by high levels of the de Ritis index. The degree of fatty degen−
eration of the liver was differentiated and it depended on the kind and quality of the dietary fat.
Conclusions. The direction of quercetin activity was determined by the kind of fat in the diet. Beneficial effects
were observed only in selected groups of animals, i.e. decreased ALT activity was observed only in animals fed
a diet with oxidized oil and increased SOD activity only in rats on a diet with fresh oil (Adv Clin Exp Med 2008,
17, 5, 503–512).
Key words: aminotransferases, SOD, GPx, oxidized fat, quercetin, rats.
Streszczenie
Wprowadzenie. Rodzaj i jakość tłuszczu w diecie mogą warunkować aktywność biologiczną kwercetyny, przeja−
wiającą się jej wpływem na aktywność wybranych enzymów.
Cel pracy. Ocena wpływu kwercetyny na potencjał oksydacyjny krwi i czynność wątroby u szczurów w stresie
oksydacyjnym wywołanym tłuszczami pokarmowymi.
Materiał i metody. Zastosowano u szczurów diety z 8% zawartością tłuszczu i 0,5% dodatkiem cholesterolu,
i oceniano wpływ kwercetyny na aktywność dysmutazy ponadtlenkowej (SOD) w krwinkach czerwonych i pero−
ksydazy glutationowej (GPx) we krwi pełnej oraz aktywności aminotransferaz: alaninowej (ALT) i asparaginiano−
wej (AST) i fosfatazy zasadowej (AP) w osoczu i ekstraktach wątrobowych. Źródłem prooksydantów w diecie
504
B. REGULSKA−ILOW, R. ILOW
szczurów były utlenione: olej słonecznikowy i smalec. Czterotygodniowe doświadczenie żywieniowe przeprowa−
dzono u 80 szczurów, samców, rasy Buffalo, 40 z nich otrzymywało 0,075% dodatek kwercetyny jako suplement
diety (60 mg kwercetyny/kg masy ciała).
Wyniki. Kwercetyna oraz jakość i rodzaj tłuszczu w diecie miały istotny wpływ na badane wskaźniki. Kwercety−
na istotnie aktywizowała SOD w grupie szczurów otrzymujących dietę ze świeżym olejem oraz zmniejszała ak−
tywność SOD w grupie otrzymującej smalec utleniony. Obniżała aktywność GPx u szczurów karmionych utlenio−
nymi: smalcem i olejem. Aktywność aminotransferaz, alaninowej i asparaginianowej w osoczu, z zakresu warto−
ści fizjologicznych nie wskazywała na uszkodzenie komórek wątroby. Kwercetyna istotnie obniżała aktywność
ALT w osoczu szczurów na diecie z utlenionym olejem. Kwercetyna istotnie obniżała aktywność AP w osoczu
szczurów na diecie ze świeżym smalcem i podwyższała aktywność na diecie z olejem utlenionym. Kierunek zmian
aktywności AP w osoczu i ekstraktach wątrobowych pokrywał się. Kwercetyna miała niekorzystne działanie
uboczne związane ze wzmożonym gromadzeniem tłuszczu w komórkach wątrobowych, na co wskazywała wyso−
ka wartość współczynnika de Ritisa. Stłuszczenie wątroby było zróżnicowane i zależne od rodzaju i jakości tłusz−
czu w diecie.
Wnioski. O kierunku działania kwercetyny decydował rodzaj tłuszczu w diecie. Korzystny wpływ, polegający na
obniżeniu aktywności ALT, dotyczył tylko grup zwierząt na diecie z utlenionym olejem oraz polegający na akty−
wizacji SOD tylko grupy szczurów na diecie ze świeżym olejem (Adv Clin Exp Med 2008, 17, 5, 503–512).
Słowa kluczowe: aminotransferazy, SOD, GPx, tłuszcz utleniony, kwercetyna, szczury.
Fats which have undergone oxidative changes
are dangerous to the health of people who consume
foods containing such products. This is associated
with the introduction of free radicals, primary and
secondary products of oxygenation, into the organ−
ism with the diet as well as with the depletion of
the bioactivity of unsaturated fatty acids. Free rad−
icals and lipid peroxides damage biological mem−
branes and cause their permeability [1]. Aldehydes,
formed from peroxides, damage internal organs,
among others the liver [2]. Polymers of fatty acids
decrease the digestibility of fat.
Quercetin, a common bioflavonoid most abun−
dantly occurring in foodstuff [3], has a multidirec−
tional activity [4, 5], exerting, among others a chol−
agogic antioxidative effect and affects the activities
of many enzymes. Due to its antioxidative proper−
ties, it may alleviate the harmful effects of oxidized
dietary fats. However, the fact that the activity is so
multidirectional hampers the evaluation of its
potential effects on metabolic parameters in oxida−
tive stress. The literature contains numerous
reports on the hepatoprotective and antioxidative
effects of quercetin [6, 7], although its hepatopro−
tective activity under conditions of oxidative stress
caused by the consumption of oxidized fats has not
been fully investigated. The aim of the study was to
evaluate the effect of quercetin on the condition
and excretory function of the liver (through estima−
tion of the activity of index and excretive enzymes)
and the oxidative status of blood (through mea−
surement of SOD and GPx activity).
Material and Methods
Experimental Design
The study was carried out on eighty male
Buffalo rats, with an initial mean body mass of 147
± 28.3 g and a final body mass of 226.0 ± 28.4 g.
For the four weeks of the experiment the rats were
kept in appropriate conditions (room temperature,
12 hour light−dark rhythm). All the procedures for
the animal experiments were approved by the local
authorities.
The animals were divided into eight groups of
10 animals. Half of the rats (n = 40) were given
a diet with an 8% sunflower oil content and the
other half (n = 40) were given a diet with an 8%
pork lard content. Within these two subgroups
receiving sunflower oil or lard, half (n = 20) had
a diet with oxidized sunflower oil or lard and the
other half fresh fat. Half of the rats eating a diet with
fresh fat (n = 10) and half of the rats eating a diet
with oxidized fat also had a bioflavonoid quercetin.
The supplement amounted to 0.75 g per kilo of diet
(60 mg of quercetin per kg of rat body mass). The
experiment was performed using Quercetin dihy−
drate reagent (Fluka, cat. no. 83370).
The rats had unlimited access to the fodder
and water. Their consumption of fodder and water
was checked every two days and their body mass
measured once a week. The diet was prepared
according to the method described in [8] (Table 1).
Preparation of Oxidized Fat
for Use as an Ingredient
of the Diet
Two equal portions of pork lard and two of
refined sunflower oil were prepared. One portion
of each was kept fresh, while the other was sub−
jected to thermal oxidation. The oxidation process
was carried out by heating the fat under a quartz
lamp for 65 hours. A 2.5−cm thick layer of fat was
placed in a porcelain dish, 21 × 29 cm. The quartz
lamp was kept 14 cm from the surface of the fat.
The initial temperature of the fat was 50ºC and its
final temperature was no higher than 75ºC. The
505
The Influence of Quercetin on the Activities of Selected Enzymes
Table 1. Ingredients of the diets used in the experiments, containing 8% fat
Tabela 1. Skład diety doświadczalnej zawierającej 8% tłuszczu
Diet ingredients
(Składniki diety)
Amount in g/kg diet
Zawartość w g/kg diety
Casein
Fat (oil or lard)
Grain starch
Potato starch
Sucrose
Vitamin supplement (solid)
Vitamin supplement (liquid)
Mineral supplement
Vitamin E
Cholesterol
Distilled water
252
80
100
50
380.7
5
5*
50
0.3*
5
100*
Components of the mineral supplement:
(Skład mieszanki mineralnej:)
NaH2PO4 · 2 H2O
Mg SO4
NaCl
KCl
FeSO4 · 7 H2O
CuSO4 · 5 H2O
MnSO4 · 1 H2O
ZnCO3
Na2MoO4 · 2 H2O
KJO3
CaCO3
25.16
2.95
1.27
3.43
0.62
0.31
0.15
0.50
0.00648
0.0003
17.5
Components of the solid vitamin supplement:
(Skład mieszanki witaminowej stałej:)
inositol
p−aminobenzoic acid
Nicotinic acid (vitamin PP)
Riboflavin (vitamin B2)
Pyridoxal (vitamin B6)
Folic acid
D−calcium pantothenate
Thiamin (vitamin B1)
Vitamin K
Grain starch
amount in g/60 kg diet:
(zawartość w g/60 kg diety:)
6.60
6.60
6.60
1.32
1.32
0.12
4.08
1.32
0.60
complete to 300 g
Components of the liquid vitamin supplement:
(Skład mieszanki witaminowej płynnej:)
Vitamin A
Vitamin D3
Vitamin B12
Distilled water
amount in U/60 kg diet:
(zawartość U/60 kg diety:)
1 200 000
132 000
1.8 mg
to 300 cm3
* Amount in cm3.
* Zawartość w cm3.
oxidation conditions were established based on the
report of Ziemlański et al. [9]. In the fresh and
oxidized fats, the content of the polar fraction was
determined, the fatty−acid component, and the
content of primary and secondary products of oxi−
dation were measured as the peroxide and ani−
sidine values (Table 2).
Preparation of the Biological
Material for Analysis
After completion of the feeding stage of the
experiment, the rats underwent light ether anesthe−
sia and blood was taken directly from their hearts
into test tubes containing heparin. After the rats
had been sacrificed, their livers were prepared. The
organ was rinsed in a physiological salt solution,
blotted dry, and weighed. Institutional approval for
the described animal experiment was obtained.
506
B. REGULSKA−ILOW, R. ILOW
The following parameters were assessed from
the biological material:
– the activity of glutathione peroxidase (GPx,
U/g Hb) via the kinetic method, using a set of
Ransel Glutathione Peroxidase reagents (Randox
UK, cat. no. RS 504). This method is based on that
of Paglia and Valentine [10];
– the activity of superoxide dismutase (SOD,
U/g Hb) via the kinetic method, using a set of
Ransod Superoxid−Dismutase reagents (Randox
UK, cat. no. SD 125). This method is based on that
of Oyanagui [11];
– the activities of alanine [12] and aspartate
[13] aminotransferases using kinetic enzymatic
diagnostic tests from BioSystems (cat. nos. COD
11562 and COD 11561);
– the activity of alkaline phosphatase [14]
using enzymatic diagnostic tests from Biochemtest
(cat. no. 178152149);
– the de Ritis index was calculated as the quo−
tient of asparginate and alanin aminotransferase
activity.
age values for the examined groups was assessed
using one−way analysis of variance (p < 0.05).
Data were tested for homogeneity of variances
with Levene’s test. To assess the influence of dif−
ferences on the analysis of variance (average com−
parison post hoc), Tukey’s Honest Significant
Difference test (HSD) was applied. A Cochrane−
−Cox test was used in case of a normal distribution
of variables but a lack of homogeneity of the vari−
ance. The nonparametric Kruskal−Wallis test was
used for comparisons in cases of lack of a normal
distribution as well as a lack of homogeneity of the
variance of variables. All the statistical calcula−
tions were done with the STATISTICA 6.0 PL
(StatSoft. Inc., USA).
Results
Fat Added to Diet
The changes in the quality of dietary fat are
presented in Table 2. The peroxide number of the
sunflower oil increased 194 times as a result of
oxidation and in lard 114 times. The anisidine
number increased 15 times in oxidized oil and
87 times in oxidized lard. The percentage of
polyunsaturated fatty acids decreased after oxida−
tion of oil by 25%. The composition of fatty acids
in lard changed slightly by oxidation. Oxidation
resulted in an increase in the polar fraction: in lard
from 1.3 to 16.9% and in oil from 2.1 to 36.5%.
Statistical Assessment
of the Results
The normality of the distribution in the exam−
ined groups was assessed with Shapiro−
Wilk’s W test. In case of lack of a normal distrib−
ution, logarithmic transformation was applied. The
significance of the differences between the aver−
Table 2. Changes in quality parameters of fresh and oxidized fats
Tabela 2. Zmiany wskaźników jakości tłuszczów świeżych i utlenionych
Kind of fat/Quality parameter
(Rodzaj tłuszczu/wskaźniki jakości)
Fresh lard
(Świeży smalec)
Oxidized lard
(Utleniony
smalec)
Fresh
sunflower oil
Świeży olej
słonecznikowy)
Oxidized
sunflower oil
(Utleniony olej
słonecznikowy)
Superoxide number:
(Liczba nadtlenkowa:)
[meq O2/kg]
[mg O2/100g]
1.5 ± 0.0
0.05
169.3 ± 0.2
5.3
3.4 ± 0.0
0.11
666.7 ± 0.0
20.8
Anisidine number
(Liczba anizydynowa)
0.5 ± 0.2
39.1 ± 0.4
4.5 ± 0.0
68.8 ± 0.8
Polar fraction
(Frakcja polarna) [%]
1.3
16.9
2.1
36.5
43.3
43.6
8.6
24.5
45.1
45.5
22.3
30.6
10.7
9.8
68.2
42.9
0.9
1.1
0.9
2.0
Fatty acid type [%]:
(Suma kwasów tłuszczowych:)
Saturated
(Nasycone)
Monounsaturated
(Jednonienasycone)
Polyunsaturated
(Wielonienasycone)
Unidentified
(Niezidentyfikowane)
507
The Influence of Quercetin on the Activities of Selected Enzymes
The Effect of Quercetin
on the Activities of Liver
and Antioxidative Enzymes
Decreased plasma ALT activity was observed
in the group fed oxidized oil and quercetin (37.4 ±
± 14.1 U/L) compared with controls (59.5 ± 14.7
U/L). Increased AP activity was observed in the
plasma of rats fed oxidized sunflower oil and
quercetin (196.0 ± 24.3 U/L) compared with the
group fed oxidized oil (109.0 ± 17.2 U/L) and in
the plasma of animals given oxidized oil and
quercetin (196.0 ± 24.3 U/L) compared with the
group fed fresh oil and quercetin (117.9 ± 20.0
U/L). Quercetin significantly reduced the level of
AP in the plasma of rats fed fresh lard (90.0 ± 20.5
U/L) compared with controls (233.4 ± 95.2 U/L).
In the groups on diet containing oxidized oil,
the addition of quercetin resulted in an elevation of
the de Ritis index from 1.5 to 2.5. In the group fed
a diet containing oxidized lard, the addition of
quercetin increased the level of the index from 2.4
to 3.7. High levels of the de Ritis index were
observed in animals receiving quercetin and fresh
(2.5 ± 0.7) or oxidized lard (3.7 ± 0.6). The results
are summarized in Table 3.
The activities of AST and ALT increased in
the livers of the animals on a diet with fresh oil and
quercetin to 58.0 ± 12.6 U/g of liver and (53.7 ±
± 14.1 U/g of liver, respectively, compared with
the animals without quercetin (25.7 ± 4.4 U/g of
liver and 31.7 ± 5.3 U/g of liver, respectively). The
results are summarized in Table 4.
The activity of AP in the liver increased signif−
icantly in the group receiving oxidized oil and quer−
cetin (16.2 ± 2.0 U/g of liver) compared with the
animals on a diet with oxidized oil (3.1 ± 0.7 U/g of
liver) without quercetin.
Quercetin caused a significant increase in the
activity of SOD in the erythrocytes of the rats on
a diet containing fresh oil and a decrease in SOD
activity in the groups on a diet containing oxidized
lard.
Quercetin decreased the activity of GPx in full
blood of the rats on a diet with oxidized oil (41.4 ±
± 2.5 vs. 32.5 ± 2.9 U/g Hb) and decreased the
activity of SOD and GPx in the rats on a diet with
oxidized lard (2954 ± 334 vs. 1894 ± 251 U/g Hb
and 47.4 ± 3.0 vs. 36.6 ± 3.9 U/g Hb, respectively).
Discussion
The polar fraction content in fat should not
exceed 24% and the content of polymers 12%.
Frying fat achieves such parameters after 5–6 days
of frying in the same batch [15, 16]. According to
the accepted criteria, oxidized oil added to the diet
in our experiment achieved the parameters dis−
qualifying it as consumable. The polar fraction has
a liquid consistency and it is not possible to
remove it from fat. It is therefore absorbed by the
fried food and becomes a dietary constituent [17].
The Effect of Dietary
Quercetin on Liver Function
Aminotransferases are index enzymes and an
increase in their activity suggests damage to hepa−
tocyte structure, with discontinuity or increased
permeability of its cell membrane. ALT is charac−
terized by higher organ specificity in relation to the
liver, while AST may also originate from injured
heart, skeletal muscles, lungs, and kidneys. The
de Ritis index has a diagnostic value in diseases of
the liver. This is the quotient of asparginate and
alanin aminotransferase activity. A de Ritis index
of about 1.2 may indicate cirrhosis of the liver,
while 0.7–0.9 suggests acute hepatitis. A de Ritis
index approaching 2 suggests fatty degeneration of
the liver. Increased AP activity is observed in liver
damage of cholestatic origin.
In animals on diets containing fresh fats, sun−
flower oil and lard, the addition of quercetin did
not induce changes in the activity of the plasma
aminotranferases. Quercetin’s beneficial effect of
decreasing effect ALT activity was observed in the
plasma of the animals on a diet with oxidized oil.
This beneficial effect was not observed in the ani−
mals with oxidized lard as the dietary source of fat.
The effect of quercetin depended on the kind of
oxidized dietary fat and the content of polyunsatu−
rated fatty acids, which were 4.4 times more abun−
dant in oxidized oil than in oxidized lard. The
hepatoprotective effect of quercetin has been
observed by numerous authors [18–21]. Positive
effects were obtained by authors using quercetin
and diosmine in a total amount of 60 mg/kg of rat
body mass (in the present experiment: 60 mg of
quercetin per kg of rat body mass) [18].
The detrimental effect of a diet containing lard
on the liver structure was observed regardless of
the quality of the fat. The high level of the de Ritis
index in the groups fed a diet containing lard sug−
gested fatty degeneration of the liver.
An increased AST/ALT ratio in all the groups
of animals receiving quercetin may also be evi−
dence of fatty degeneration of the liver, and the
changes reached the level of statistical signifi−
cance in the groups fed oxidized fat and quercetin.
Rats on the diet with oxidized lard and
quercetin revealed an unfavorable synergistic
2361 ± 290
45.2 ± 1.6
SOD [U/g Hb]
GP [U/g Hb]
39.4 ± 3.0
a – statystyczna różnica między 1 i 3 lub 2 i 4.
b – statystyczna różnica między 1 i 5 lub 2 i 6.
c – statystyczna różnica między 5 i 7 lub 6 i 8.
d – statystyczna różnica między 3 i 7 lub 4 i 8.
e – statystyczna różnica między 1 i 2 lub 5 i 6.
x ± SD – średnia ± odchylenie standardowe.
42.6 ± 2.0
3161 ± 298
a
3425 ± 504
a – statistically significant difference between 1 and 3 and 2 and 4.
b – statistically significant difference between 1 and 5 and 2 and 6.
c – statistically significant difference between 5 and 7 and 6 and 8.
d – statistically significant difference between 3 and 7 and 4 and 8.
e – statistically significant difference between 1 and 2 and 5 and 6.
X ± SD – mean ± standard deviation.
a e
2.1 ± 0.8
2.2 ± 0.6e
e
117.9 ± 20.0
Ratio de Ritis
233.4 ± 95.2
111.2 ± 14.5
1.5 ± 0.3e
AP [U/L]
95.9 ± 37.7
abe
74.0 ± 16.5
e
73.7 ± 11.2
AST [U/L]
49.0 ± 15.7
35.7 ± 11.3e
51.8 ± 7.6e
ALT [U/L]
d
Sunflower oil
+ quercetin
diet 3
(Olej słoneczniko−
wy + kwercetyna)
(dieta 3)
Control – lard
diet 2
(Grupa kontrol−
na – smalec)
(dieta 2)
Control
– sunflower oil
diet 1
(Grupa kontrol−
na – olej sło−
necznikowy)
(dieta 1)
Variable
(Zmienna)
X ± SD
43.6 ± 3.0
3440 ± 257
2.5 ± 0.7 d
90.0 ± 20.5
85.3 ± 27.8
34.7 ± 7.6
d
a
Lard + quercetin
diet 4
(Smalec +
kwercetyna)
(dieta 4)
c
41.4 ± 2.5c
2991 ± 401
1.5 ± 0.5c
109.0 ± 17.2
80.7 ± 13.9
59.5 ± 14.7c
Oxidized
sunflower oil
diet 5
(Utleniony olej
słonecznikowy)
(dieta 5)
47.4 ± 3.0c
2954 ± 334
2.4 ± 0.4c
93.8 ± 12.6
79.7 ±13.2
33.7 ± 8.1
c
b
Oxidized lard
diet 6
(Utleniony
smalec)
(dieta 6)
32.5 ± 2.9c
3369 ± 232
2.5 ± 0.8c
196.0 ± 24.3
85.0 ± 20.6
37.3 ± 14.1c
cd
Oxidized sunflower
oil + quercetin
diet 7
(Utleniony olej
słonecznikowy
+ kwercetyna)
(dieta 7)
36.6 ± 3.9c
1894 ± 251c
3.7 ± 0.6c d
106.5 ± 17.9
107.0 ± 21.1
29.0 ± 5.2
Oxidized lard
+ quercetin
diet 8
(Utleniony smalec
+ kwercytyna)
(dieta 8)
Tabela 3. Wpływ diet na aktywność: aminotransferaz alaninowej (ALT) i asparaginianowej (AST), fosfatazy zasadowej (AP) w surowicy, dysmutazy ponadtlenkowej (SOD) w krwinkach czer−
wonych oraz peroksydazy glutationowej we krwi szczurów laboratoryjnych
Table 3. The influence of the investigated diets on the activities of alanine (ALT) and aspartate (AST) aminotransferases and those of alkaline phosphatase (AP) in the plasma, dismutase super−
oxide (SOD) in red blood cells, and glutathion peroxide (GP) in the blood of the laboratory rats
508
B. REGULSKA−ILOW, R. ILOW
2.9 ± 0.4e
a – statystyczna różnica między 1 i 3 lub 2 i 4.
b – statystyczna różnica między 1 i 5 lub 2 i 6.
c – statystyczna różnica między 5 i 7 lub 6 i 8.
d – statystyczna różnica między 3 i 7 lub 4 i 8.
e – statystyczna różnica między 1 i 2 lub 5 i 6.
x ± SD – średnia ± odchylenie standardowe.
a – statistically significant difference between 1 and 3 and 2 and 4.
b – statistically significant difference between 1 and 5 and 2 and 6.
c – statistically significant difference between 5 and 7 and 6 and 8.
d – statistically significant difference between 3 and 7 and 4 and 8.
e – statistically significant difference between 1 and 2 and 5 and 6.
X ± SD – mean ± standard deviation
13.7 ± 1.9a b e
2.8 ± 0.4d
58.0 ± 12.6
40.7 ± 8.9
25.7 ± 4.4
ad
AP [U/L]
e
AST [U/L]
ae
53.7 ± 14.1a
39.5 ± 8.4
31.7 ± 5.3a
ALT [U/L]
Sunflower oil
+ quercetin
diet 3
(Olej słoneczniko−
wy + kwercetyna)
(dieta 3)
Control – lard
diet 2
(Grupa kontrol−
na – smalec)
(dieta 2)
Control
– sunflower oil
diet 1
(Grupa kontrol−
na – olej sło−
necznikowy)
(dieta 1)
Variable
(Zmienna)
X ± SD
1.9 ± 0.2a d
46.4 ± 13.6
48.1 ± 12.0
Lard + quercetin
diet 4
(Smalec +
kwercetyna)
(dieta 4)
3.1 ± 0.7c
33.2 ± 7.2
36.8 ± 7.1
Oxidized
sunflower oil
diet 5
(Utleniony olej
słonecznikowy)
(dieta 5)
3.2 ± 0.6b
45.6 ± 8.6
44.2 ± 15.6
Oxidized lard
diet 6
(Utleniony
smalec)
(dieta 6)
16.2 ± 2.0c d
37.0 ± 4.7
d
45.8 ± 5.2
Oxidized sunflower
oil + quercetin
diet 7
(Utleniony olej
słonecznikowy
+ kwercetyna)
(dieta 7)
3.6 ± 0.4d
44.7 ± 10.9
42.2 ± 13.0
Oxidized lard
+ quercetin
diet 8
(Utleniony smalec
+ kwercytyna)
(dieta 8)
Tabela 4. Wpływ różnych diet na aktywność aminotransferaz alaninowej (ALT) i asparaginianowej (AST), fosfatazy zasadowej (AP) w ekstraktach wątrobowych szczurów laboratoryjnych
Table 4. The influence of the type of diet on the activities of alanine (ALT) and aspartate (AST) aminotransferases and of alkaline phosphatase (AP) in the liver extracts of the laboratory rats
The Influence of Quercetin on the Activities of Selected Enzymes
509
510
B. REGULSKA−ILOW, R. ILOW
effect of the dietary components compared with
the animals on a diet including fresh lard and
quercetin. The observed effect seems to be associ−
ated with the cholagogic activity of quercetin. This
leads to an accumulation of lipids, but also of
harmful products of their oxidation, in the liver.
In this experiment the measurement of plasma
AP activity enabled an unambiguous interpretation
of findings concerning the effect of quercetin on
the excretory function of the liver and the condi−
tion of bile ducts. An unfavorable effect of
quercetin in the form of a significant increase in
the activity of this enzyme in plasma was observed
in the group fed oxidized sunflower oil. Similar
effects were observed by Juśkiewicz and col−
leagues [22]; the addition of grapefruit extract
resulted in increased AP activity.
In the present experiment, the quality of
dietary vegetable fat was an additional factor
affecting the condition of the bile ducts. Quercetin
did not alleviate the detrimental effects of the
products of oil oxidation on the excretory function
of the liver. In the groups of rats on diets contain−
ing quercetin, the activity of AP was significantly
higher in the plasma of the animals given oxidized
oil than in the group on a diet with fresh oil.
Comparison of the findings obtained in the
groups of rats fed oxidized fats with the control
groups did not reveal any effect of the quality of
the fat on the activity of the plasma aminotrans−
ferases. Thus it can be assumed that the structure
of the hepatocyte cell membranes and of their cell
organelles had not been injured. The effect of the
quality of fat on the function and structure of the
liver was observed by Hayam et al. [2]. The oxi−
dized soybean oil which they used in their experi−
ment produced a significant increase in plasma
aminotransferases, which suggested injury to the
hepatic parenchyma.
Oxidized sunflower oil and oxidized lard in
the diet did not result in cholestatic liver injury in
the animals. The high plasma AP activity in the
control group with lard is difficult to explain. No
confirmation that the kind of fresh dietary fat may
significantly affect the activity of alkaline phos−
phatase of hepatic origin has been found in the lit−
erature.
The Effect of Dietary
Compounds on the Activity of
Aminotransferases and Alkaline
Phosphatase in the Livers of
the Experimental Animals
Measurement of hepatic enzymes in homoge−
nates from the liver of experimental animals has
limited diagnostic significance; however, it does
have a certain cognitive value. Under physiologi−
cal conditions, the activity of ALT in the human
liver is about three times higher than in the plasma,
while AST activity is seven times higher. ALT is
found in the cytoplasm, while AST is present
inside the hepatocyte mitochondria. In the course
of changes in ALT activity, a negative correlation
was observed in the liver and plasma of rats on
a diet with oil. The beneficial effect of quercetin
on the condition of the liver, manifested by
decreased ALT activity in plasma, was observed in
the dietary presence of oxidized vegetable fat and
harmful products of its metabolism. No correla−
tions were observed in the course of changes in
ALT activity in the plasma and the liver of animals
on diet containing lard.
AST activity was higher in the livers of the
animals fed fodder containing fresh oil and
quercetin than in the control group and it was not
accompanied by changes in AST activity in plas−
ma. In the group on a diet with oxidized oil, the
addition of quercetin resulted in decreased AST
activity in the liver compared with the group on
diet with fresh oil and quercetin. The animals fed
lard did not reveal any statistically significant
changes in AST activity in the liver.
The findings obtained in this experiment sug−
gest that the beneficial effect of quercetin depends
on the kind of dietary fat. Most probably this can
be explained by the fact that the high content of
saturated fatty acids in lard causes decreased flu−
idity and permeability of the cell membranes for
exogenous compounds, including that of hepato−
cytes for quercetin, which may deprive it of the
biogenic activity. In the rats on a diet with veg−
etable oil, the high content of unsaturated fatty
acids in the dietary fat resulted in an increased flu−
idity and permeability of the cell membranes, also
of hepatocytes for quercetin, and thus enabled
sealing activity of the hepatic cell membranes. On
the other hand, the increase in alkaline phos−
phatase in the plasma and liver of the animals fed
fresh lard cannot be explained on the basis of data
from the literature. The increased activity of this
enzyme most probably resulted from extrahepatic
factors.
The Effect of Quercetin
on SOD and GPx Activity
Dietary components affect the activity of
antioxidative enzymes [23, 24]. In the present
experiment, both quercetin as well as the kind of
fat (fresh or oxidized oil or lard) exerted an influ−
ence on the activity of antioxidative enzymes in
the blood of the experimental rats. The administra−
The Influence of Quercetin on the Activities of Selected Enzymes
tion of quercetin affect the activity of superoxide
dismutase, which depended on the quality and
kind of fat. It had an unfavorable, decreasing effect
on SOD activity in the blood cells of rats fed oxi−
dized lard. On the other hand, in the rats on a diet
with fresh sunflower oil, it enhanced the activity of
SOD, which was a beneficial effect. Quercetin
decreased the activity of GPx in the blood of the
rats fed oxidized fats, lard or oil, which was a neg−
ative effect. Such a negative effect of quercetin
was not observed in the rats fed fresh fats.
SOD and GPx destroy the active forms of oxy−
gen before they manage to cause cell injury. The
activity of SOD depends on the amount of free
radicals in the cell. A positive correlation has been
observed between plasma levels of SOD and lipid
peroxides [25]. Other authors observed signifi−
cantly increased activity of certain antioxidative
enzymes in animals on diets containing vegetable
fat (safflower oil, corn oil) compared with animals
fed animal fat (cow, fish) [26, 27]. The results of
the present experiment confirming the synergistic
beneficial effect of quercetin and vegetable fat
suggest that dietary vegetable fats activate the
enzymatic antioxidative defense system in rats and
provide evidence for a mechanism in which
dietary fats affect our health.
511
The authors concluded that the experiment
demonstrated a beneficial effect of quercetin on the
liver manifested by decreased plasma activity of
alanine aminotransferase in rats. The effect depend−
ed on the kind of dietary fat and was observed only
in animals fed oxidized vegetable oil. The addition
of quercetin to the diet did not have any influence
on the plasma activity of asparginate aminotrans−
ferase. This enzyme can be found mainly in hepato−
cyte mitochondria. In the present experiment
quercetin most probably did not interfere with the
structure of these organelles. Moreover, quercetin
revealed a health−promoting activity by the activa−
tion of an antioxidative enzyme, supraoxide dismu−
tase, in rats fed a diet containing fresh vegetable oil.
At the same time, quercetin had a negative effect in
the form of stimulation of fat accumulation in
hepatic cells, which was evidenced by a high level
of the de Ritis index. The degree of fatty degenera−
tion of the liver was differentiated and depended on
the kind and quality of the dietary fat. Moreover, the
experiment demonstrated a negative effect of lard
on hepatic functions regardless of its quality in the
form of fatty degeneration of the liver. At the same
time, the plasma activities of alanine and
asparginate aminotransferases, which were within
the normal range, did not indicate liver cell injury.
References
[1] Lutz M, Bonilla S, Concha J, Alvarado J, Barraza P: Effect of dietary oils, cholesterol and antioxidant vitamin
supplementation on liver microsomal fluidity and xenobiotic−metabolizing enzymes in rats. Nutr Metab 1998, 42,
350–359.
[2] Hayam I, Cogan U, Mokady S: Dietary oxidized oil and the activity of antioxidant enzymes and lipoprotein pe−
roxidation in rats. Nutr Res 1995, 15, 1037–1044.
[3] De Vries JHM, Jansen K, Hollman PCH, van Staveren WA, Katan MB: Consumption of quercetin and ka−
empferol in free−living subjects eating a variety of diets. Cancer Lett 1997, 114, 141–144.
[4] Aherne SA, O’Brien NM: Dietary flavonols: chemistry, food content, and metabolism. Nutrition 2002, 18,
75–81.
[5] Erlund I: Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioava−
ilability, and epidemiology. Nutr Res 2004, 24, 851–874.
[6] Morand Ch, Crespy V, Manach Ch, Besson C, Demigne Ch, Remesy Ch: Plasma metabolites of quercetin and
their antioxidant properties. Am J Physiol 1998, 275, 212–219.
[7] Murota K, Terao J: Antioxidative flavonoid quercetin: implication of its intestinal absorption and metabolism.
Arch Biochem Biophys 2003, 417, 12–17.
[8] Reeves PG, Nielsen FH, Fahey GC: Ain−93 purified diets for laboratory rodents: final report of the American In−
stitute of Nutrition ad hoc writing Committee on the Reformulation of the Ain−76A rodent diet. J Nutr 1993, 123,
1939–1951.
[9] Ziemlański S, Ziemlański Ś, Panczenko−Kresowska B, Budzyńska−Topolewska J, Żelazkiewicz K, Kołakowska A:
Effect of variously oxidized marine fish fat on guinea pig organism. Acta Aliment Pol 1992, 17/31, 159–169.
[10] Paglia ED, Valentine WN: Studies on the quantitative and qualitative characterization of erythrocytes glutathio−
ne peroxidase. J Lab Clin Med 1967, 70, 158–169.
[11] Oyanagui Y: Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal Bio−
chem 1984, 142, 290–296.
[12] IFCC methods for the measurement of catalytic concentration of enzymes. J Clin Chem Clin Biochem 1986, 24,
481–495.
[13] IFCC methods for the measurement of catalytic concentration of enzymes. J Clin Chem Clin Biochem 1986, 24,
497–510.
[14] The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. Recom−
mended method for determination of enzymes in blood. J Clin Lab Invest 1974, 32, 29.
512
B. REGULSKA−ILOW, R. ILOW
[15] Daniewski M, Pawlicka M, Filipek A, Jacorzynski B, Mielniczuk E, Balas J, Domina P: Assessment of qua−
lity control tests for frying fat of french fries. Pol J Hum Nutr Metab 2001, 28, 132–139.
[16] Firestone D: Worldwide regulation of frying fats and oils. INFORM, 1993, 4, 1366–1371.
[17] Andrikopoulos NK, Antonopoulou S, Kaliora AC: Oleuropein inhibits LDL oxidation induced by cooking oil
frying by−products and platelet aggregation induced by platelet−activating factor. Lebensm−Wiss u−Technol 2002,
35, 479–484.
[18] Lahouel M, Boulkour S, Segueni N, Fillastre JP: Protective effect of flavonoides against the toxicity of vinbla−
stine, cyclophosphamide and paracetamol by inhibition of lipid peroxydation and increase of liver glutation.
Haema 2004, 7, 59–67.
[19] Pavanato A, Tunon MJ, Sanchez−Campos S, Marroni CA, Llesuy S, Gonzalez−Gallego J, Marroni N: Effects
of quercetin on liver damage in rats with carbon tetrachloride−induced cirrhosis. Dig Dis Sci 2003, 48, 824–829.
[20] Lee ES, Lee HE, Shin JY, Moon JO: The flavonoid quercetin inhibits dimethylnitrosamine−induced liver dama−
ge in rats. J Pharm Pharmacol 2003, 55, 1169–1174.
[21] Kawada N, Seki S, Inoue M, Kuroki T: Effect of antioxidants, resveratrol, quercetin and N−acetylcysteine, on
the functions of cultured rat hepatic stellate cells and Kupffer cells. Hepatology 1998, 27, 1265–1274.
[22] Juśkiewicz J, Duńczyk Z, Wróblewska M, Oszmiański J, Hernandez T: The response of rats to feeding with
diets containing grapefruit flavonoid extract. Food Res Inter 2002, 35, 201–205.
[23] Ibrahim W, Lee US, Szabo J, Bruckner G, Chow CK: Oxidative stress and antioxidant status in mouse liver:
effects of dietary lipid, vitamin E and iron. J Nutr 1997, 127, 1401–1406.
[24] Lim RM, Toborek M, Watkins BA, Boissonneault GA, Hennie B: Susceptibility to hepatic stress in rabits fed
different animal and plant fats. J Am Coll Nutr 1996, 15, 289–294.
[25] Panczenko−Kresowska B, Ziemlański Ś, Przepiórka M, Wartanowicz M: Peroxidation processes in elderly
people in longitudinal studies. Pol J Hum Nutr Metab 1998, 4, 337–345.
[26] Venkatraman JT, Angkeow P, Satsangi N, Fernandes G: Effects of dietary n−6 and n−3 lipids on antioxidant
defense system in livers of exercised rats. J Am Coll Nutr 1998, 17, 586–594.
[27] Venkatraman JT, Pinnavaia L: Effects of saturated, n−6 and n−3 lipids on activities of enzymes involved in an−
tioxidant defense in normal rats. Nutr Res 1998, 18, 341–350.
Address for correspondence:
Bożena Regulska−Ilow
Department of Food Science and Dietetics
Silesian Piasts University of Medicine
pl. Nankiera 1
50−140 Wrocław
Poland
Tel.: +48 71 784 02 09
E−mail: [email protected]
Conflict of interest: None declared
Received: 28.01.2008
Revised: 6.03.2008
Accepted: 29.05.2008