ORIGINAL PAPERS

ORIGINAL PAPERS
Adv Clin Exp Med 2006, 15, 5, 777–787
ISSN 1230−025X
© Copyright by Silesian Piasts
University of Medicine in Wrocław
BOŻENA REGULSKA−ILOW1, JADWIGA BIERNAT1, RAFAŁ ILOW1, PRZEMYSŁAW KOWALSKI2,
HALINA GRAJETA1, ELIZA LAMER−ZARAWSKA3
The Development of Laboratory Rats
Fed with Fresh and Oxidized Fats and the Influence
of Bioflavinoids from the Radix of Scutellaria baicalensis
on Lipid Metabolism
Rozwój szczurów laboratoryjnych karmionych paszą
ze świeżymi i utlenionymi tłuszczami i wpływ bioflawonoidów
z korzenia tarczycy bajkalskiej na metabolizm tłuszczów
1
Department of Food Science and Nutrition, Silesian Piasts University of Medicine in Wrocław, Poland
Department of Pathological Anatomy, Silesian Piasts University of Medicine in Wrocław, Poland
3
Department of Pharmaceutical Botany, Silesian Piasts University of Medicine in Wrocław, Poland
2
Abstract
Background. The overall daily food intake of humans always includes some oxidized fats, which form during culi−
nary processes. It is possible to prevent the unhealthy effects of oxidation products by introducing anti−oxidants
into the diet, for example bioflavonoids.
Objectives. Assessing the influence of an extract from the root of Scutellaria baicalensis on the development of
rats and on the indicators of fat metabolism under conditions of oxidative stress caused by consumed oxidized fats.
Material and Methods. Using rats fed a diet with an 8% fat content and a 0.5% cholesterol supplement, the influ−
ence of bioflavinoids from Scutellaria baicalensis root on body mass and total lipid (TL), triglyceride (TG), total
cholesterol (TCH), HDL−cholesterol (HDL−CH), and phospholipid concentrations in the serum and liver were
assessed. Histopathological analyses of liver tissue samples were also performed. The source of pro−oxidants in the
rats’ diet was either oxidized sunflower oil or oxidized lard. The four−week experiment involved 80 male Buffalo
rats, of which 40 received a 0.075% root extract as a supplement to their diet.
Results. In the groups of rats on diets containing oxidized fats, the extract had a statistically significant influence
on the atherogenic index, calculated according to the formula: (TCH – HDL−CH)/HDL−CH. In the group fed with
oxidized lard, the extract also influenced the increase in the total cholesterol concentration in the serum. The TCH
and phospholipid concentrations in the serum were reduced by the extract and the rate of body mass increase rose
in the rats fed on a diet containing fresh sunflower oil. Lower concentrations of TCH (1.30 ± 0.2 mmol/l) and TG
(0.85 ± 0.2 mmol/l) were observed in the serum of rats fed a diet containing oxidized oil compared with controls
(1.87 ± 0.4 and 1.41 ± 0.3 mmol/l, respectively). A similar situation was observed for TCH in the serum of rats fed
on a diet containing oxidized lard (1.26 ± 0.2 mmol/l) compared with controls (1.56 ± 0.2 mmol/l).
Conclusions. The bioflavonoids did not display a hepatoprotective activity in rats given fodder containing oxidized
fats; this was evident in the relative increase in liver mass seen in these animals. We also found that fodder con−
taining lard, especially oxidized lard, had a negative influence on the liver in rats. This was confirmed in
histopathological analyses (Adv Clin Exp Med 2006, 15, 5, 777–787).
Key words: Scutellaria baicalensis radix, oxidized fats, rats, lipids, histopathological analyses.
Streszczenie
Wprowadzenie. W całodziennych racjach pokarmowych człowieka stale są obecne tłuszcze utlenione powstające
w wyniku procesów kulinarnych. Niekorzystnym skutkom działania produktów utlenienia w organizmie można za−
pobiegać przez wprowadzenie do diety antyoksydantów, np. bioflawonoidów.
Cel pracy. Ocena wpływu ekstraktu z korzenia tarczycy bajkalskiej na rozwój zwierząt, wskaźniki przemian tłusz−
czowych w warunkach stresu oksydacyjnego wywołanego utlenionymi tłuszczami spożywczymi.
778
B. REGULSKA−ILOW et al.
Materiał i metody. W grupach szczurów karmionych paszami z 8% zawartością tłuszczu i 0,5% dodatkiem chole−
sterolu, oceniono wpływ bioflawonoidów z korzenia tarczycy bajkalskiej na: przyrosty masy ciała, stężenie lipidów
całkowitych (TL), triglicerydów (TG), cholesterolu ogólnego (TCH), HDL−cholesterolu (HDL−CH) i fosfolipidów
w surowicy i wątrobie. Wykonano także histopatologiczne badanie wycinków tkanki wątrobowej. Źródłem prooksy−
dantów w diecie były utlenione: olej słonecznikowy lub smalec. Czterotygodniowe doświadczenie przeprowadzono
na 80 szczurach, samcach, rasy Buffalo, z których połowa otrzymywała 0,075% dodatek ekstraktu do paszy.
Wyniki. W grupach zwierząt na diecie z utlenionymi tłuszczami dodatek ekstraktu powodował istotny statystycz−
nie wzrost współczynnika aterogenności, obliczonego według wzoru: (TCH – HDL−CH)/HDL−CH. W grupie kar−
mionej smalcem utlenionym powodował także wzrost stężenia cholesterolu całkowitego w osoczu. Ekstrakt obni−
żał w osoczu stężenie TCH i fosfolipidów i zwiększał przyrost masy ciała szczurów karmionych paszą ze świeżym
olejem słonecznikowym. Obserwowano mniejsze stężenie TCH (1,30 ± 0,2 mmol/l) i TG (0,85 ± 0,2 mmol/l)
w osoczu zwierząt karmionych utlenionym olejem w stosunku do kontroli odpowiednio (1,87 ± 0,4; 1,41 ± 0,3
mmol/l), a także TCH w osoczu szczurów na diecie ze smalcem utlenionym (1,26 ± 0,2 mmol/l) w stosunku do
kontroli (1,56 ± 0,2 mmol/l).
Wnioski. Bioflawonoidy nie wykazywały działania hepatoprotekcyjnego u zwierząt karmionych paszą z utlenio−
nymi tłuszczami, co miało swój wyraz w zwiększeniu względnej masy wątroby. Stwierdzono także niekorzystny
wpływ paszy ze smalcem, zwłaszcza utlenionym, na wątrobę zwierząt. Potwierdziły to wyniki badań histopatolo−
gicznych (Adv Clin Exp Med 2006, 15, 5, 777–787).
Słowa kluczowe: korzeń tarczycy bajkalskiej, tłuszcze utlenione, szczury, lipidy, badania histopatologiczne.
The overall daily food intake of humans
always includes some oxidized fats, which form
during culinary processes such as frying, roasting,
and boiling [1–3]. It is possible to prevent the
unhealthy effects of the activity of oxidation prod−
ucts by introducing anti−oxidants to the diet, for
example vitamins A, C, and E or bioflavonoids.
Bioflavonoids are present in small amounts in
commonly consumed vegetables, fruits, teas, and
red wines [4, 5]. Thousand−fold greater quantities
are found in dried culinary herbs such as mint,
thyme, oregano, and cinnamon and in medicinal
herbs such as Scutellaria baicalensis [6]. Existing
information about the influence of bioflavonoids
on the metabolism of lipids, particularly oxidized
lipids, is contradictory.
The aim of this study was to assess the influ−
ence of an extract from the root of Scutellaria
baicalensis on the development of rats and on the
indicators of fat metabolism under conditions of
oxidative stress caused by consumed oxidized fats.
Material and Methods
The study was carried out on eighty male
Buffalo rats with an initial mean body mass of
183.6 ± 21.1 g and a final body mass of 273.5 ±
25.9 g. For the four weeks of the experiment the
rats were kept in appropriate conditions (room
temperature, 12−hour light−dark rhythm).
The animals were divided into eight groups
with 10 animals per group. Forty rats had fodder
that was 8% sunflower oil, with 20 receiving fod−
der with oxidized oil and 20 receiving fodder with
fresh oil. Ten of the former and 10 of the latter had
a supplement to their fodder: an extract from the
root of Scutellaria baicalensis. The other 40 rats
had fodder that was 8% lard, with 20 receiving fod−
der with oxidized lard and 20 receiving fodder with
fresh lard. Ten of the former and 10 of the latter had
a supplement to their fodder: an extract from the
root of Scutellaria baicalensis. The supplement
amounted to 0.75 g per kilogram of fodder, with
the extract having an 80% baicaline content.
Ready−made extract “Oxyd” was obtained from
Wroclaw’s Herbalist Factory “Herbapol”. The
method of its preparation is copyrighted and
patented. The fodder was prepared according to the
method described in [7]. The rats had unlimited
access to the fodder and to water. Their consump−
tion of fodder and water was checked every two
days and their body mass measured once per week.
After the completion of the feeding stage of
the experiment, the rats underwent light ether
anesthesia and blood was taken directly from their
hearts into test tubes containing heparin. After the
rats had been put to sleep, their livers, hearts, kid−
neys, and lungs were prepared. The organs were
rinsed in a physiological salt solution, blotted dry,
and weighed. Liver samples were taken for
histopathological analysis.
Preparation of Oxidized Fat
for Use as an Ingredient
of the Fodder
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.
779
The Influence of Bioflavinoids on Rat Development
The initial temperature of the fat was 50ºC and its
final temperature was not higher than 75ºC. The
oxidation conditions were established based on the
work of Ziemlański et al. [8]. In the fresh and oxi−
dized fats, the content of the polar fraction was
determined and 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 1).
Parameters Determined
from the Biological Material
The parameters determined from analysis of
the biological material were: total cholesterol
(TCH), high−density lipoprotein cholesterol
(HDL−CH), and triglyceride (TG) levels using the
Biosystems enzymatic diagnostic tests (TCH: cat.
not. 11539, HDL−CH: cat. not. 11523, TG: cat. no.
11529), phospholipid concentration by the enzy−
matic method using the bioMerieux Inc. diagnos−
tic test (cat no. 61491), and total lipid (TL) content
by the enzymatic method using the Lachema diag−
nostic tests (cat. no. 1135801).
For the histopathology examination, liver
samples were fixed in buffered 10% formalin and
embedded in paraffin for light microscopy.
Microscopically, all cytoplasmic vesicular lesions
visible in H−E (hematoxylin−eosin) stained liver
slides were considered as fat changes. The intensi−
ty of fat changes in hepatocytes was estimated
semi−quantitative. To confirm the presence of fats,
frozen liver samples were stained with oil red and
Sudan III. The parameters determined were: the
percentage of fat droplets volume inside hepato−
cytes, assessed in ten random HPVs (high−power
views), and the total number of fat−storing hepato−
cytes, calculated as the percentage of total cells.
Statistical Assessment
of the Results
The normality of the distribution in the exam−
ined groups was assessed by the Shapiro−
Wilk’s W test. In the case of a lack of a normal dis−
tribution, logarithmic transformation was applied.
The significance of the differences between the
average values for the examined groups was
assessed using an individual analysis variance (p <
0.05). Levene’s test was used to check individual
variance. The assessment of the influence of two
independent variables on one dependent variable
was done with the ANOVA two−factor analysis of
variance. All the statistical calculations were done
with the STATISTICA 6.0 PL program of StatSoft.
Inc. USA.
Results
Analysis of the Quality Indices
of the Fats Added to the Fodder
The composition of the fatty acids in the lard
underwent slight changes due to oxidation, while
in the oxidized sunflower oil the unsaturated fat
content decreased and the saturated fat content
increased relative to the contents in fresh oil
(Table 1). The anisidine and peroxide values
increased significantly for both types of oxidized
fat compared with the values for the fresh fats; for
the lard this was a 65−fold increase in the anisidine
Table 1. Fatty acid composition, expressed as a percent of the total fatty acid content, and the quality indicators for the
fresh and oxidized fats added to the rats’ fodder
Tabela 1. Skład kwasów tłuszczowych, jako % sumy wszystkich kwasów, i wskaźniki jakości świeżych i utlenionych
tłuszczów dodawanych do paszy szczurów
Analyzed indicator
(Badany wskaźnik)
Fresh lard
(Świeży
smalec)
Oxidized lard
(Utleniony
smalec)
Fresh
sunflower oil
(Świeży olej
słonecznikowy)
Oxidized
sunflower oil
(Utleniony olej
słonecznikowy)
Peroxide value (Liczba nadtlenków)
[mg O2/100 g fat]
0.14
6.94
0.13
22.7
Anisidine value (Liczba anizydynowa)
0.7
46.0
4.8
71.8
Polar fraction (Frakcja polarna) [%]
1.3
23.9
2.1
42.3
Fatty acid type [total]
(Rodzaj kwasów tłuszczowych):
saturated acids (nasycone)
monounsaturated acids (jednonienasycone)
polyunsaturated acids (wielonienasycone)
unidentified acids (niezidentyfikowane)
43.3
45.1
10.7
0.9
43.6
45.5
9.8
1.1
8.6
22.3
68.2
0.9
24.5
30.6
42.9
2.0
780
B. REGULSKA−ILOW et al.
value and a 50−fold increase in the peroxide value,
while for the sunflower oil the respective increas−
es were 15−fold and 175−fold. The peroxide value
was significantly higher for the oxidized oil (22.7
mg O2/100 g fat) than for the oxidized lard (6.9 mg
O2/100 g fat). The digestive system does not
absorb peroxides easily, but their transformation
can give rise to secondary products of oxidation,
such as aldehydes and ketones. Their content is
described by the anisidine value, which was 71.8
for the oxidized sunflower oil and 46.0 for the oxi−
dized lard. Aldehydes and ketones are easily
absorbed from the digestive system. It is notewor−
thy that in oxidized lard, the secondary products of
oxidation dominated in the overall amount of oxi−
dation products, i.e. the polar fraction. The polar
fraction content was 42.3% in the oxidized oil and
23.9% in the oxidized lard.
The Effect of the Different Diets
on the Rats’ Development
In the experiment it was shown that the increase
in the rats’ body mass depended on the type of fat
(oil or lard) in their diet (Table 2). Significantly
larger increases were seen in the group receiving
fodder containing fresh lard than in the group on
fodder containing fresh sunflower oil. Adding
a bioflavonoid supplement to the rats’ diet only
gave rise to a greater body mass increase in the
group on the diet containing fresh oil (group 3)
compared with the rise seen in the control group
(group 1). An influence on the rats’ development in
the form of smaller increases in body mass was seen
in the groups on a diet containing oxidized fats and
bioflavonoids (groups 7 and 8) compared with those
found in the groups given fodder containing fresh
fats and bioflavonoids (groups 3 and 4).
The liver mass to body mass ratio of the rats
was significantly higher in the groups receiving
fodder containing oxidized fats and bioflavonoids
(groups 7 and 8) than in the groups receiving fod−
der with fresh fats and bioflavonoids (groups 3 and
4). There was a similar dependence in the kidneys,
but only in the rats receiving fodder containing
fresh or oxidized sunflower oil and bioflavonoids.
In the group of rats on a diet with oxidized oil and
bioflavonoids, the relative mass of the kidneys
was statistically significantly higher than that of
those rats on a diet with fresh oil and the extract
(groups 7 and 3). The type of fats, oil or lard, also
influenced the mass of the kidneys, which was sta−
tistically significantly higher in those rats receiv−
ing fodder with oil. The relative mass of the lungs
was found to depend on the quality of the sun−
flower oil: it was higher in the group of rats on the
diet with oxidized oil (groups 5 and 1). The extract
containing bioflavonoids was found to contribute
to a significant decrease in lung mass (groups 7
and 5).
The Influence
of the Ingredients of the Diet
on the Lipid Concentration
A difference was found between the triglyc−
eride (TG) and total cholesterol (TCH) concentra−
tions in the serum of rats on diets containing sun−
flower oil (Tables 3 and 4). The TG, TCH, and
phospholipid concentrations decreased in all those
groups relative to the values in the control group.
Any positive influence from the extract from the
radix of Scutellaria baicalensis was found to
depend on the quality of this fat. A statistically sig−
nificant decrease in the values of the aforemen−
tioned indices of fat transformation was only
observed in the rats given fodder containing fresh
sunflower oil. The influence of bioflavonoids in
the fresh lard−containing fodder was only seen in
the reduction of the phospholipid concentration in
the serum of the rats on that diet.
In the groups of rats receiving fodder contain−
ing lard, its quality and the presence or absence of
the Scutellaria baicallensis root extract did not
have any influence on the TG concentration. It was
observed that the TCH concentration was depen−
dent on the quality of the lard used in the fodder.
In the groups of rats receiving fodder containing
oxidized lard, the TCH concentration was found to
be lower than in the controls: 1.26 ± 0.2 compared
with 1.56 ± 0.2 mmol/l. Adding bioflavonoids to
this type of fodder led to a significant increase in
the TCH concentration to 1.97 ± 0.2 mmol/l. It is
very likely that the bioflavonoids, by increasing
the level of bile secretion, caused a more intensive
emulsification of the oxidized lard, thus causing
a greater absorption of fat by the organism.
In order to better interpret these results, the
change in the concentration of the HDL fraction
was analyzed in connection with the changes in
TCH concentration using the atherogenic index.
The effect of fat on the organism is the healthier
the lower the value of this index. Sunflower oil
was found to have a healthier effect on the forma−
tion of the lipid fraction in the serum. In the groups
of rats given fodder containing sunflower oil,
a lower atherogenic index (0.59 ± 0.2) was found
than in the rats on fodder containing lard (0.80 ±
0.3). The addition of the 0.075% Scutellaria
baicalensis root extract had an unhealthy effect on
the atherogenic index, causing it to rise in both the
group of rats given fodder containing fresh lard
16.1 ± 2.6
107 ± 10.9e
3.27 ± 0.16
0.35 ± 0.02
0.68 ± 0.04e
0.73 ± 0.09
15.0 ± 2.6a b e
86.9 ± 4.6e
17.2 ± 2.7b
80.5 ± 18.0e
3.57 ± 0.34
0.32 ± 0.02
0.75 ± 0.04e
0.68 ± 0.08b
4.0 ± 1.8a e
53.5 ±17.3a e
Volume of liver cells
occupied by fat
(Objętość komórek
wątroby zajęta przez
tłuszcz) %
Number of liver cells
accumulating fat
(Liczba komórek
wątroby zajęta przez
tłuszcz) %
Lungs mass
(Masa płuc)
Kidneys mass
(Masa nerek)
Heart mass
(Masa serca)
Liver mass
(Masa wątroby)
Body weight gain
(Przyrost masy ciała)
96.0 ± 3.2a d
44.0 ± 7.7a d
0.73 ± 0.08
0.70 ± 0.04d
0.36 ± 0.01
3.30 ± 0.21d
102.5 ± 9.8d
16.4 ± 2.5
group 3
Sunflower oil
+ extract
(Olej słoneczni−
kowy + ekstrakt)
b
statistically significant difference between groups 1 and 3 and between 2 and 4.
statistically significant difference between groups 1 and 5 and between 2 and 6.
c
statistically significant difference between groups 5 and 7 and between 6 and 8.
d
statistically significant difference between groups 3 and 7 and between 4 and 8.
e
statistically significant difference between groups 1 and 2.
x ± SD – average ± standard deviation.
a
group 2
group 1
Dietary intake
(Spożycie paszy)
Lard – control
(Smalec
– grupa kontrolna)
Sunflower oil
– control
(Olej słonecz−
nikowy – grupa
kontrolna)
Variable (Zmienna)
x ± SD
60.0 ± 14.9
4.5 ± 1.6c
0.91 ± 0.0b c
0.79 ± 0.06
0.35 ± 0.03
3.94 ± 0.36
64.5 ± 20.2
15.3 ± 1.6b c
group 5
Oxidized
sunflower oil
(Utleniony olej
słonecznikowy)
82.5 ± 4.3
33.3 ± 3.1b
0.68 ± 0.08
0.69 ± 0.04
0.34 ± 0.02
3.28 ± 0.17c
98 ± 13.8
16.6 ± 2.3c
group 6
65.0 ± 8.2d
14.7 ± 4.0c d
0.72 ± 0.07c
0.78 ± 0.04d
0.33 ± 0.03
4.25 ± 0.32d
77 ± 17.0d
16.7 ± 1.6c
group 7
Oxidized lard
Oxidized sunflo−
(Utleniony smalec) wer oil + extract
(Utleniony olej
słonecznikowy
+ ekstrakt)
różnice istotne statystycznie między grupami 1 i 3 oraz 2 i 4.
różnice istotne statystycznie między grupami 1 i 5 oraz 2 i 6.
c
różnice istotne statystycznie między grupami 5 i 7 oraz 6 i 8.
d
różnice istotne statystycznie między grupami 3 i 7 oraz 4 i 8.
e
różnice istotne statystycznie między grupami 1 i 2.
x ± SD – średnia ± odchylenie standardowe.
b
a
89.0 ± 16.0
34.8 ± 8.2a
0.74 ± 0.08
0.65 ± 0.06
0.35 ± 0.03
3.22 ± 0.17d
103 ± 20.3
16.9 ± 3.4
group 4
Lard + extract
(Smalec + ekstrakt)
90.6 ± 4.6
39.4 ± 7.9
0.78 ± 0.06
0.67 ± 0.04
0.35 ± 0.03
3.68 ± 0.36c d
87 ± 16.2
15.3 ± 2.0c
group 8
Oxidized lard
+ extract
(Utleniony smalec
+ ekstrakt)
Tabela 2. Spożycie paszy/dzień/szczura [g], przyrost masy ciała [g], masa wybranych narządów [g/100 g masy ciała] i wyniki badań histopatologicznych wątroby szczurów doświadczalnych
Table 2. The dietary intake of fodder/day/rat [g], increase in body mass [g], mass of selected organs [g/100 g body mass], and histopathological examination of the livers of the rats studied
The Influence of Bioflavinoids on Rat Development
781
abe
0.87 ± 0.1e
0.80 ± 0.3
110.5 ± 10.0a
1.17 ± 0.2a e
0.59 ± 0.2
141.6 ± 29.0a
95.1 ± 11.4a d
0.91 ± 0.3
group 3
17.3 ± 2.3
10.0 ± 1.6
4.5 ± 0.4
3.3 ± 0.6
2.9 ± 0.1
ad
Phospholipids (Fosfolipidy) mg/g liver
a
61.3 ± 11.7 a
45.5 ± 11.6 b
38.5 ± 3.3 a
TL (Lipidy całkowite) mg/g liver
2.2 ± 0.4
2.1 ± 0.2 a b
1.9 ± 0.2
TCH (Cholesterol całkowity) mg/g liver
ad
8.2 ± 1.0
ab
group 2
ab
group 1
Sunflower oil
+ extract
(Olej słonecz−
nikowy
+ ekstrakt)
Lard – control
(Smalec
– grupa
kontrolna)
Sunflower oil
– control
(Olej słonecz−
nikowy – grupa
kontrolna)
TG (Triglicerydy) mg/g liver
Variable (Zmienna)
x ± SD
Tabela 4. Wpływ stosowanych diet na profil lipidowy w wątrobach szczurów
Table 4. The influence of the diets used on the lipid profile in liver samples taken from the rats
* Współczynnik aterogenności = (całkowity cholesterol – cholesterol HDL)/cholesterol HDL.
* Atherogenic index = (total cholesterol – HDL cholesterol)/HDL cholesterol.
Phospholipids (Fosfolipidy) mg/l
Atherogenic index (Współczynnik
aterogenności) *
HDL cholesterol (HDL Cholesterol) mmo/l
a
0.73 ± 0.1a
1.37 ± 0.1
1.56 ± 0.2
1.41 ± 0.3
Triglicerides (Trigliceridy) mmol/l
Total Cholesterol (Cholesterol całkowity) mmol/l 1.87 ± 0.4
1.14 ± 0.1
1.53 ± 0.2
b
be
2.5 ± 0.3
2.6 ± 0.3
group 3
group 2
group 1
Sunflower oil
+ extract
(Olej słonecz−
nikowy
+ ekstrakt)
Lard – control
(Smalec
– grupa
kontrolna)
2.9 ± 0.6
Sunflower oil
– control
(Olej słonecz−
nikowy – grupa
kontrolna)
Total lipids (Lipidy całkowite) g/l
Variable (Zmienna)
x ± SD
Tabela 3. Wpływ stosowanych diet na profil lipidowy w osoczu szczurów
Table 3. The influence of the diets used on the lipid profile in the serum of the rats
d
d
3.6 ± 0.8
54.5 ± 9.9
2.4 ± 0.1 a
13.9 ± 2.5
group 4
ad
Lard + extract
(Smalec
+ ekstrakt)
87.0 ± 4.4a
0.92 ± 0.2
0.77 ± 0.1
1.47 ± 0.2
1.58 ± 0.2
2.5 ± 0.4
group 4
Lard + extract
(Smalec
+ ekstrakt)
3.2 ± 0.3
44.7 ± 6.5
1.7 ± 0.1
10.3 ± 1.8
group 5
b
Oxidized
sunflower oil
(Utleniony
olej słonecz−
nikowy)
165.7 ± 9.8
bc
3.6 ± 0.5
72.3 ± 23.6 b
2.7 ± 0.6 b
13.3 ± 2.7
group 6
Oxidized lard
(Utleniony
smalec)
109.3 ± 14.4
c
0.69 ± 0.4
c
0.26 ± 0.1
1.26 ± 0.2
b c
1.62 ± 0.3
0.76 ± 0.1
b
b
2.8 ± 0.4
group 6
Oxidized lard
(Utleniony
smalec)
1.03 ± 0.1c
1.30 ± 0.2
0.85 ± 0.2
3.0 ± 0.3c
group 5
Oxidized
sunflower oil
(Utleniony
olej słonecz−
nikowy)
3.3 ± 0.3
d
44.5 ± 3.9
1.9 ± 0.2
11.8 ± 1.3
group 7
d
Oxidized sunflo−
wer oil + extract
(Utleniony olej
słonecznikowy
+ ekstrakt)
158.6 ± 16.3d
0.67 ± 0.2
c
0.85 ± 0.1c
1.41 ± 0.2
0.93 ± 0.1
2.3 ± 0.3c
group 7
Oxidized sunflo−
wer oil + extract
(Utleniony olej
słonecznikowy
+ ekstrakt)
d
3.8 ± 0.5
63.9 ± 13.1
2.4 ± 0.6
9.3 ± 2.5 c d
group 8
Oxidized lard
+ extract
(Utleniony
smalec
+ ekstrakt)
92.5 ± 8.3
1.28 ± 0.2c d
0.87 ± 0.1
1.97 ± 0.2c
1.69 ± 0.4
2.7 ± 0.6
group 8
Oxidized lard
+ extract
(Utleniony
smalec
+ ekstrakt)
782
B. REGULSKA−ILOW et al.
The Influence of Bioflavinoids on Rat Development
(0.92 ± 0.2) and the group given fodder containing
fresh sunflower oil (0.91 ± 0.3). A similar
unhealthy increase in this index occurred when the
fodder contained the extract and oxidized lard
(1.28 ± 0.22) or oil (0.67 ± 0.19).
Phospholipids are the most important compo−
nent of the HDL fraction of cholesterol. The ten−
dencies of changes in this component’s content are
in principle concurrent with the changes in the
concentration of HDL cholesterol in the serum of
the studied rats.
In the livers of the rats, bioflavonoids were
found to cause an increase in the TG concentration
relative to the control group regardless of the type
of fat. Using oxidized fats in the diet also
increased the TG concentration relative to the con−
trols, so the quality of the fat was significant. The
Scutellaria baicalensis root extract had the effect
of reducing the TG concentration in the livers of
rats on the diet with oxidized lard compared with
the concentration found in those rats on that fodder
type without the extract: 9.3 ± 2.5 vs. 13.3 ± 2.7
mg/g liver.
In the livers of the rats given fodder with oxi−
dized fats and the extract, the TG concentration
was lower than in the rats receiving fresh fats and
the extract. In the control diet with sunflower oil,
a lower accumulation of TG was found in the liv−
ers of the rats than in the control group receiving
fodder with lard.
The TCH concentration in the livers of the rats
receiving fodder containing lard was higher (2.1 ±
0.2 mg/g liver) in all the groups relative to the con−
trol. The extract caused the TCH concentration to
rise in the livers of the rats on diets with fresh fats.
If there was oxidized oil in the fodder, the choles−
terol level in the liver was lower (1.7 ± 0.1
mg/g liver) than in the controls (1.9 ± 0.2
mg/g liver), probably due to the lower degree of
absorption of fat, while if there was lard in the fod−
der, the cholesterol level in the liver rose.
The addition of Scutellaria baicalensis root
extract to the fodder with oxidized oil led to an
increase in the TCH concentration in the liver (1.9
± 0.2 mg/g liver), probably due to the increased
emulsification of fat compared with the group of
rats fed on a diet with oxidized oil but no extract.
In the groups of rats receiving fodder contain−
ing fresh sunflower oil and the extract, a higher
TCH concentration (2.2 ± 0.4 mg/g liver) was
found in the livers than in the groups of rats receiv−
ing fodder with oxidized oil (1.9 ± 0.2 mg/g liver).
In the groups of rats receiving fodder contain−
ing sunflower oil, the highest TCH concentrations
in the liver and the highest total fat content (TL)
was found in those on fodder with fresh oil and the
extract. In the groups of rats receiving fodder con−
783
taining lard, the highest concentrations of TCH
and total fat in the liver were found in those on
fodder with oxidized lard. The greatest reduction
in TCH concentration relative to controls was
found in the livers of rats receiving oxidized oil;
this may have been due to the organism not
absorbing the fat.
Histopathological Assessments
Steatosis was observed in the histopathologi−
cal sections of the liver, only taking the form of fat
storage in the hepatocytes (Table 2, Figures 1–8).
Changes associated with steatosis degenerativa
were not observed, i.e. there was no degradation of
cytoplasmic structures, lysis of cells in the steatot−
ic tissues, fibrosis around the steatotic cells, or the
formation of fat pseudocysts. The accumulation of
Fig. 1. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – sunflower oil (control)
Ryc. 1. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – olej słonecznikowy
(grupa kontrolna)
Fig. 2. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – lard (control)
Ryc. 2. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – smalec (grupa kon−
trolna)
784
B. REGULSKA−ILOW et al.
Fig. 3. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – sunflower oil + extract
Fig. 6. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – oxidized lard
Ryc. 3. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – olej słonecznikowy
+ ekstrakt
Ryc. 6. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – utleniony smalec
Fig. 4. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – lard + extract
Fig. 7. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – oxidized sunflower oil
+ extract
Ryc. 4. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – smalec + ekstrakt
Ryc. 7. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – utleniony olej sło−
necznikowy + ekstrakt
Fig. 5. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – oxidized sunflower oil
Fig. 8. Storage of fat in the hepatocytes in liver sam−
ples taken from the rats – oxidized lard + extract
Ryc. 5. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – utleniony olej sło−
necznikowy
Ryc. 8. Magazynowanie tłuszczu w hepatocytach wą−
trób pobieranych od szczurów – utleniony smalec
+ ekstrakt
The Influence of Bioflavinoids on Rat Development
fat was mainly of microvesicular type. Depending
on the size of the steatosis, small droplets initially
collected around the circumference of the cell, and
then the size of the droplets increased as the
steatosis progressed and the droplets began to
occupy the area closer to the nucleus, or even
filled the entire cell. However, no coarse−droplet
steatosis was observed and there were no single
large drops forcing the nuclei to the edges of the
cells.
Besides this, there were single cases of very
scant changes in the liver in the form of minimal
proliferations of the membranes of individual bile
canals and very scant fibrosis around the blood
vessels. There was also visible and widespread
inflammatory infiltration. In the majority of the
samples these were made up of single, or at most
a few, scattered cells of a chronic of inflammatory
infiltration. However, they had a nonspecific char−
acter and were found on a few occasions to be
more widespread in the control rats than in the rats
from the other groups.
Discussion
Oxidized fats pose a danger to the health of
people who eat products containing them.
Kołakowska et al. [9] showed that polyunsaturated
fatty acids were lost when fish fat was oxidized;
the rate of loss was directly proportional to the
increase in the peroxide value. However, signifi−
cant loss of polyunsaturated fatty acids only
occurred at very high values of the peroxide value
that are practically unheard of in food products
intended for consumption. Wiliams et al. [1], in
a study on ten healthy men, determined that there
was a disturbance in the functioning of the
endothelium after meals containing fat that had
been repeatedly used for deep−fat frying. The fat
used in that study was of the average quality that
is commonly found in fast−food restaurants. The
observed disturbances can abet arteriosclerotic
changes in the arteries.
The polar fraction content was determined in
the fats added to the fodder in this experiment. The
polar fraction includes oxidized and non−oxidized
mono− and diacylglycerols and cyclic, dimeric,
and trimeric fatty acids. Polymers of fatty acids
reduce the digestibility of fat. Andrikopoulos et al.
[3] showed that the polar fraction contained in fat,
in the amounts commonly occurring during the
frying of French fries in the home or in restaurants,
is significantly enriched in LDL cholesterol. Those
authors claimed that if the oxidative activity of
a solution of copper ions is assumed to be 100%,
then a 20% polar fraction in fat has an oxidative
785
activity of 33%. In some countries, the acceptable
amount of the polar fraction in fat should not be
greater than 24% and the amount of polymers
should not be greater than 12%. Such parameters
are reached by frying fat after 5 to 6 days of use in
the same deep−fat fryer. This indicates that such fat
is no longer suitable for use in the preparation of
food [10, 11]. The polar fraction has a fluid con−
sistency, and in the home or in restaurants it is not
possible to remove it from the fat. Therefore it is
absorbed into the center or into the surface of the
fried food and thus becomes part of the diet of the
consumer.
The Influence
of the Type of Diet
on the Development of Animals
In this study, the differences in the increase in
body mass that were observed between rats from
the two control groups, i.e. those receiving fodder
containing fresh sunflower oil (group 1) or fresh
lard (group 2), could be due to the differences in
the structures of the triglycerols of the two fats and
the differences in the way the organisms could use
them.
That body mass is determined by the type of
fat in the diet was also observed in other studies
[12, 13]. Kawahara et al. [12] observed a greater
increase in body mass in animals receiving fodder
containing vegetable oils (rape seed or sunflower)
than in animals receiving fodder containing beef
fat. In the experiments of Ellisa et al. [13], a dif−
ference was seen between the increase in body
mass of rats depending on whether their diet con−
tained corn oil, rape seed oil, or coconut oil. The
greatest increases were found in those rats on diets
containing corn oil, and the lowest in those on
diets containing coconut oil.
In several studies [14–17], a relationship was
found between the spatial structure and the type of
dietary triacylglycerol fatty acids and their absorp−
tion in the intestines of animals, including humans.
Absorption of the fat was linearly correlated to the
content of palmitic acid in the medial position of
the triacylglycerol [15]. Dietary fats containing
saturated fatty acids in the medial position of tria−
cylglycerol provided more energy per gram of fat
than those that had unsaturated fatty acids in the
medial position [17]. In animal fats, saturated fatty
acids show a tendency to fill the medial position of
triacylglycerol. Triacylglycerols in plant oils have
a different spatial structure; saturated fatty acids
fill the extremes. Because pancreas lipase has the
ability to specifically remove fatty acids solely
from the extreme positions, its activity leads to the
786
B. REGULSKA−ILOW et al.
formation of estrified monoacylglycerides at the
central carbon of glycerol and free fatty acids,
which are different depending on whether the fat
was animal or plant. The absorption of monoacyl−
glyerols in the intestine is higher than that of free
fatty acids. Long−chain saturated free fatty acids
such as stearic acid and palmitic acid are bound in
calcium salts that do not dissolve easily and are
weakly absorbed from the digestive system. The
spatial structure of triacylglycerols can thus have
an influence on the absorption and metabolism of
dietary fats and their activity within the organism
[14, 15].
In this experiment, the addition of bio−
flavonoids to the diet led to a greater increase in
body mass relative to that seen in the control
(group 1) only in the group receiving fresh oil in
their diet (group 3). The increase in body mass in
the rats in that group could be due to the bile−
secretion−promoting activity of the bioflavonoids;
more bile would mean that the fats would be easi−
er to digest and absorb. The ease of uptake of
bioflavonoids in the extract from the radix of
Scutellaria baicalensis to the cell interior and their
influence on the metabolism of the fats in rats
receiving fodder with sunflower oil was probably
a consequence of an increase in the fluidity and
thus porosity of the cell membranes in said rats.
No influence of bioflavonoids on the body
mass of the rats fed with lard was observed
because they probably did not enter the cells due
to the reduction in the fluidity of the cell mem−
branes. The fluidity of biological membranes is
inversely proportional to the length of the fatty
acid chains built into the membranes’ structures.
Saturated fatty acids increase the rigidity of the
membranes and unsaturated fatty acids reduce
rigidity. The fluidity of biological membranes
depends on the qualitative and quantitative com−
position of the fatty acids, on the cholesterol con−
tent, and on the ratio of proteins to lipids in the
membranes. Fatty acids in the medial position of
triacylglycerols are more readily absorbed from
the digestive system than fatty acids bound to the
first or third carbon of the glycerol; the former are
also more readily built into the cell membrane [14,
15]. In lard, 72% of the triacylglycerol has saturat−
ed palmitic acid in the medial position [15]; when
incorporated into the bilayer phospholipid cell
membrane, this acid can reduce the membrane’s
fluidity and limit the uptake of bioflavonoids into
the cell interior.
Juśkiewicz et al. [18] performed experiments
in which animals received fodder with the higher
bioflavonoid contents contained in extracts from
grapefruit (0.1%, 0.2%, or 0.4%). There was no
difference in the animals’ increase in body mass
relative to that in the control groups. Gao et al.
[19] observed smaller increases in body mass in
animals receiving fodder with supplementary
bioflavonoids. However, they added rutin and
baicaline at concentrations over 13 times higher
than those used in this experiment and over twice
those used by Juśkiewicz et al. [18]. The
bioflavonoid content in the diet of the rats could
have influenced the increase in body mass in these
experiments.
The Influence of the
Components of the Diet
on Lipid Concentrations
Reports on the trends of lipid profile changes
of laboratory animals receiving fodder containing
fresh and oxidized fats are contradictory. For
example, in the experiments of Ziemlański et al.
[8], performed on guinea pigs that were given fod−
der containing fresh or oxidized sea−fish fats for
12 weeks, it was shown that the total cholesterol,
LDL cholesterol, and total lipid content in the
serum were higher in those animals on the diet
with oxidized fat. However, in the Lu and Lo stud−
ies [20], rats receiving fodder containing oxidized
oil displayed results similar to those in this exper−
iment, i.e. there was a positive influence of the
oxidized fat on the concentration of the lipid com−
ponents of the serum and on the livers. The results
of this study were probably due to the large polar
fraction content, including fatty acids that were
polymerized, more intensively in the sunflower oil
and less intensively in the lard. This fraction, as it
is not absorbable by the organism, could not be
used as a source of energy. The probable conse−
quence of this was the healthier lipid profile in the
groups receiving fodder with oxidized fats. The
value of the atherogenic index was also lower in
the groups of rats fed diets with oxidized fats.
Reports on the influence of bioflavonoids on
trends of lipid profile changes in the serum of rats
consuming fodder containing fresh fats show that
the activity of the bioflavonoids depends on their
type and dose and on the amount of fat contained
in the fodder [11].
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Address for correspondence:
Bożena Regulska−Ilow
Department of Food Science and Nutrition
Silesian Piasts University of Medicine in Wrocław
pl. Nankiera 1
50−140 Wrocław
Poland
tel.: +48 071 784 02 09
e−mail: [email protected]
Conflict of interest: None declared
Received: 24.04.2005
Revised: 11.08.2006
Accepted: 21.09.2006
Praca wpłynęła do Redakcji: 24.04.2005 r.
Po recenzji: 11.08.2006 r.
Zaakceptowano do druku: 21.09.2006 r.