Atherosclerosis 240 (2015) 125e130

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Anti-atherosclerotic activity of catechins depends on their

stereoisomerism
_ o
Magdalena Mika a, *, Renata B. Kostogrys b, Magdalena Franczyk-Zar  w b,
Agnieszka Wikiera a, Edyta Maslak b
a
Department of Food Biotechnology, Faculty of Food Technology, Agricultural University, Balicka 122, 30-149 Krakow, Poland
b
Department of Human Nutrition, Faculty of Food Technology, Agricultural University, Balicka 122, 30-149 Krakow, Poland

a r t i c l e i n f o a b s t r a c t

Article history: In terms of stereochemistry, catechins are divided into two groups: (
) epi forms (2R, 3R) and (
) forms
Received 6 August 2014 (2S, 3R). Most of the catechins present in green tea are (
) epi forms (2R, 3R). Under the influence of high
Received in revised form temperatures, in anaerobic conditions, as a result of epimerization the proportion of the (
) form (2S, 3R)
11 February 2015
increases. The data indicate that the presence of thermally modified catechins in the diet more efficiently
Accepted 14 February 2015
Available online 23 February 2015
reduces the development of atherosclerosis in apoE knockout mice than the presence of native catechins.
The addition of the thermally modified formulations to the high-fat diet resulted in a reduction of the
area of atherosclerotic lesions by about 28% (en face method) and 45% (cross-section method) compared
Keywords:
Atherosclerosis
to the group fed the high-fat diet without catechins. Furthermore, the body weight gain and plasma
apoE knockout mice TBARS concentration in mice fed a diet with the addition of catechins depends on the degree of epi-
Catechins merization of catechins and decreases with increasing content of catechins belonging to the (
) form (2S,
Cholesterol 3R). Moreover, plasma HDL cholesterol concentration in mice depends on catechins' stereoisomerism
and increases along with the increasing content of catechins belonging to the (
) form (2S, 3R).
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Catechins are the major group of polyphenols of tea leaves,
constituting 20e30% of the dry weight of the leaf. In terms of ste-
reochemistry, catechins are divided into two groups: (
) epi forms
(2R, 3R) and (
) forms (2S, 3R). (
) Epi forms (2R, 3R) constitute
about 90% of catechins naturally occurring in the tea leaf. Under the
influence of high temperatures, in anaerobic conditions, as a result
of epimerization the proportion of the (
) form (2S, 3R) increases
[1]. The stereoisomerism of catechins affects their properties. It has
been shown that the thermally modified catechin formulations
used as food stabilizers more efficiently prevent oxidative and hy-
drolytic processes occurring in the fats during their storage than
the native catechins formulations [2]. A greater antioxidative po-
tential of the thermally modified catechins formulations than
native catechins formulations is likely to be the result of a syner-
gistic effect of the compounds of both groups of stereoisomers, and
it is not caused by the increased activity of the individual catechins
* Corresponding author.
E-mail address: mmika@ar.krakow.pl (M. Mika).
belonging to the (
) form (2S, 2R). This is indicated by the results
obtained in studies comparing the antioxidant effectiveness of
different pairs of stereoisomers. Xu et al. found that catechins
within individual pairs of stereoisomers have comparable antioxi-
dant potential; only the pairs EGC and GC showed a difference in
favor of epi forms [3].
In the literature there are many reports indicating the health-
related (anti-atherosclerotic, anti-cancer, anti-obesity and even
anti-diabetic) properties of green tea catechins [4]. Anti-
atherosclerotic effects of catechins, among others, are the result
of the impact they have on the metabolism of cholesterol. Green tea
catechins cause a decrease blood plasma total cholesterol level in
experimental animals and in humans [5,6]. Moreover, it has been
revealed that thermally modified catechins added to the diet of rats
reduce total cholesterol [5] and triglycerides [7] in plasma more
effectively than native catechins. Lowering the level of cholesterol
is a complex process and is a result of the effects catechins on: the
decreased absorption of exogenous and endogenous cholesterol
from the digestive tract [8,9], the reduction of bile acid resorption in
the intestine by the inhibitory effect on the apical sodium bile acid
transporter (ASBT) [10], and inhibiting the activity of HMG-CoA
reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase), which is

http://dx.doi.org/10.1016/j.atherosclerosis.2015.02.026
0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
126 M. Mika et al. / Atherosclerosis 240 (2015) 125e130
the key enzyme responsible for the synthesis of cholesterol in the
liver [11]. Reducing the absorption of exogenous and endogenous
cholesterol from the digestive tract is the result of formation of
cholesterol conjugates with catechins [8] and the inhibitory activity
of catechins on phospholipase A2 [9], which allows the catalytic
activity of cholesterol esterase and significantly enhances absorp-
tion of cholesterol by enterocytes.
Anti-atherosclerotic effects of green tea catechins have been
confirmed in a study conducted on apoE knockout mice [12e15].
However, an interesting topic, so far unexplained, is the impact of
catechins' stereoisomerism on their anti-atherosclerotic potency.
The aim of this study was to investigate whether catechins for-
mulations, containing 20% and 35% (
) forms (2S, 3R), added to a
high-fat diet, may inhibit the development of atherosclerosis in
apoE mice more effectively than the native catechins.
2. Materials and methods
2.1. Preparing catechins formulations and their composition
analysis
The green tea Polyphenon 60 from green tea (SigmaeAldrich)
was used. The catechins were used in the native form (A) and
thermally modified (B, C). 5% of aqueous solutions of catechins
were thermally modified. The process was carried out in anaerobic
conditions. The aqueous solutions were bubbled with nitrogen gas
for 2 min and placed in Scott's test tubes. The modification was
performed at the temperature of 140  C for 40 min (B) and 80 min
(C).
The content and the type of catechins in the formulations were
determined by a high-performance liquid chromatography (HPLC)
as described by Lina et al. [16]. A liquid chromatography, spectro-
photometric detector, l ¼ 280 nm and an LUNA C18(2)
(250  4.6 mm) column were used for the above. Samples were
eluted from the column following the programme:
0e10 mine100% of phase A eisocratic elution; 10e25 mine100%e
90% of phase A, 0%e10% of phase B e linear gradient;
25e60 mine90%e70% of phase A, 10%e30% of phase B e linear
gradient. The flow rate was 1 ml/min. Phase A comprised methanol,
formic acid and redistilled water (20:0.3:79.7 v/v/v) and phase B
composed of methanol and formic acid (99.7:0.3 v/v).
2.2. Animal and diets
Homozygous 10-week-old male apolipoprotein E knockout mice
(apoE-knockout) were purchased from Taconic Laboratory (Taconic
Europe A/S, Denmark). The mice were divided into five groups
(n ¼ 7). Each group of mice was kept in a separate cage and housed
in an air-conditioned room (21  C), with 12 h light/dark cycles and
the humidity at 55 ± 10%. Food and water were provided ad libitum.
For the first two weeks the mice were being acclimated to the
experimental conditions. At this time, the animals were fed with
commercial pellets diet for rodents. After the acclimatization
period, the mice were fed AIN-93 modified diets for 12 weeks [17].
A standard diet (DS) containing 52.95% cornstarch, 20% casein, 10%
sucrose, 7% oil, 5% fibre, 3.5% mineral mix (AIN-93G-MX), 1%
vitamin mix (AIN-93-VX), 0.3% L-cystine, 0.25% choline as well as a
high-fat diet (HFA, HFB, HFC) containing 39.95% cornstarch, 20%
casein, 10% sucrose, 19,9% butter, 5% fibre, 3.5% mineral mix (AIN-
93G-MX), 1% vitamin mix (AIN-93-VX), 0.3% L-cystine, 0.25%
choline and 0.09% catechins formulations (A, B, C). Total energy of
the standard diet and high-fat diet was estimated as 3948 kcal/kg
and 4589 kcal/kg. The estimate of caloric content was based on the
standard physiological fuel values for protein, fat, and carbohydrate
of 4, 9, and 4 kcal/g respectively. Animals fed standard diet (DS)
were the reference group, whereas the control group was the mice
fed high-fat diet (HF) without catechins. Diets were divided into the
single daily portions and stored at the temperature of
20  C.
Before feeding the portions were mixed with water at a ratio of 2: 1.
All uneaten food was removed, dried at 60  C and weighed. Mice
were weighed weekly. Two days before ending the experiment, the
blood glucose taken from the tail vein was determined. At the end
of the experiment, the mice were injected with heparin and after
10 min were anesthetized with pentobarbital (40 mg/kg of the body
weight). The blood samples from the abdominal aorta were placed
in tubes. The plasma samples were separated by centrifugation
12 000  g for 2 min and stored at the temperature of
80  C until
the analysis. The organs were washed with PBS by direct injection
in the heart left ventricle. The heart and the aorta were collected
from the animals.
The research involving animals was approved: No. 78/VI/2009
dated 16.06.2009, issued by the Local Ethics Committee for Ex-
periments on Animals in Cracow.
2.3. Determination of plasma antioxidative activity
The concentration of lipid peroxidation products reacting with
2-thiobarbituric acid (TBARS) in animals plasma was determined
spectrophotometrically method using a commercially available kits
(Cell Biolabs, Inc.). TBARS are expressed as mmol of malondialde-
hyde (MDA).
2.4. Determination of plasma triglycerides and cholesterol levels
The concentration of triglycerides (TG), total cholesterol (Chol-
T), cholesterol associated with high density lipoprotein (HDL-C)
and cholesterol associated with low density lipoprotein (LDL-C) in
mice's plasma were determined by an enzymatic method using a
commercially available kits (Cormay).
2.5. Quantification of atherosclerosis in aortas by en face analysis
In anesthetized mice, the thorax was longitudinally opened, the
right atrium was incised, and the heart was perfused with
phosphate-buffered saline (PBS, pH 7.4) through the apex of the left
ventricle. The aorta from arch to bifurcation was dissected out from
surrounding tissues and fixed in 4% paraformaldehyde. Then, it was
opened longitudinally, pinned onto brown silicone plates, and
stained with Sudan IV (SigmaeAldrich, St. Louis, MO, USA). The
aortic lesion area and total aortic area were measured using the
LSM Image Browser.
2.6. Quantification of atherosclerosis in aortic roots by cross-section
analysis
The heart and the ascending aorta were dissected. The excised
heart and ascending aorta were embedded in OCT compound
(CellPath) and snap-frozen. 10 mm-thick cryosections were cut from
the aortic root using a standardized protocol [18,19]. Eight sections
were collected at 100-mm intervals starting at a 100-mm distance
from the appearance of the aortic valves. Sections were thaw-
mounted on poly-L-lysine coated slides and air dried. After fixa-
tion in 4% paraformaldehyde (pH ¼ 7), sections were stained with
Meyer's hematoxylin and oil red-O (SigmaeAldrich). Oil red-O-
stained sections were examined under an Olympus BX50 micro-
scope and used for quantitative evaluation. Images of the aorta
were recorded using an Olympus Camedia 5050 digital camera and
stored as TIFF files of resolution 1024  768 pixels. The total area of
the lesion was measured semiautomatically in each slide using LSM
Image Browser 3 software. For each animal a mean lesion area was
 M. Mika et al. / Atherosclerosis 240 (2015) 125e130
calculated from eight sections, reflecting the cross-section area
covered by atherosclerosis.
2.7. Statistical analysis
Results are presented as mean ± standard deviation (SD). Data
were analyzed using STATISTICA for Windows. Statistical analysis
was performed by ANOVA followed by post hoc Bonferroni multiple
comparison test. Correlations between parameters were assessed
by Pearson's correlation test. P < 0.05 was considered as statistically
significant.
3. Results
3.1. Composition of catechins formulations
High temperature did not cause a reduction in quantity of cat-
echins in the solution and caused only a change in stereoisomerism
of the (
) epi forms (2R, 3R), which changed into the corresponding
(
) forms (2S, 3R). The amounts of individual stereoisomers of
catechins in the diet are shown in Table 1. Total catechins in for-
mulations amounted to 85%. In addition, all formulations contained
a constant amount of gallic acid (3.1%), caffeine (8.1%) and small
amounts of other catechins. Native catechins formulations (A)
contained 8.5% (
) forms (2S, 3R), whereas thermally modified
formulations B and C contained respectively 20% and 35% (
) forms
(2S, 3R).
3.2. Feed intake
The mean food intake did not differ significantly between
groups (Table 2). Quantity of catechins ingested with the HFA, HFB
or HFC diets was approximately 90 mg per kg of mouse body
weight per day. With respect to the human diet this quantity cor-
responds to the intake of approximately 460 mg of catechins within
24 h, which is equivalent to drinking 1 to 2 cups of green tea.
3.3. Body weight gain in mice
The initial body weight of the experimental animals was on
average 25.5 g (Table 2). At the end of the experiment, the animal
body weight gain ranged from 8.0 g to 14.1 g. The highest final body
weight was observed in the mice fed the high-fat diet (HF). The
addition of catechins with a higher degree of thermal modification
to the high-fat diet reduced the body weight gain of mice by 20.6%.
A high correlation coefficient (R ¼
0.99) was demonstrated be-
tween the amount of catechins belonging to the (
) form (2S, 3R)
added into the diet and body weight gain.
3.4. Concentration of cholesterol in the blood plasma of mice
The concentration of total and LDL cholesterol in the blood
Table 1
The contents of (
) epi forms (2R, 3R) and (
) forms (2S, 3R) in the diet.
Diet symbol (
) epi forms (2R, 3R) [mg/kg diet]
EGCG EC ECG
HFA 605c ± 6.2 42c ± 1.5 114c ± 2.7
HFB 515b ± 5.2 34b ± 1.1 103b ± 1.9
HFC 389a ± 4.9 28a ± 1.6 79a ± 1.7
1
Date are means ± SD values of 6 replicates.
2
The mean in the same column not sharing a common superscript are significantly different among groups at p < 0.05 (Test Bonferroni).
3
C e (
) catechin, CG e (
) catechin gallate, EC e (
) epicatechin, ECG e (
) epicatechin gallate, EGCG e (
) epigallocatechin gallate, GCG e (
) gallocatechin gallate, SD e
standard deviation.
127
plasma of the mice fed the high-fat diet was significantly higher
than in the animals fed the standard diet (Table 2). However, we
observed that total cholesterol in the mice fed the high-fat diet with
thermally modified catechins was significantly lower than in the
animals fed the high-fat diet without catechins. The diet that acted
most efficiently was HFB with thermal modification of catechins,
containing 20% (
) form (2S, 3R). This diet resulted in total
cholesterol levels lower by 23% compared to the HFA diet with
native catechins. Plasma LDL-C level in the mice fed the high-fat
diet was almost twice as high as in the animals fed with the stan-
dard diet (Table 2). Moreover, the high-fat diet caused a lower HDL-
C concentration than the standard diet. The addition of catechins to
the high-fat diet had no effect on plasma LDL-C level in the animals.
However, a significant higher HDL-C level in mice fed diet with
thermal modification catechins was observed (Table 2). It was
observed that the concentration of HDL-C increases with the
increasing content of (
) form (2S, 3R) in the catechins formulation
introduced to the diet. The HDL-C level was 33.6% higher in the
presence of catechins with a higher degree of thermal modification.
A correlation (R ¼ 0.89) was demonstrated between the amount of
catechins belonging to the (
) form (2S, 3R) added into the diet and
plasma HDL-C level.
3.5. Concentration of triglycerides in blood plasma of mice
The increased intake of fat with a high-fat diet (HF) relative to a
standard diet (DS) caused a significantly higher level of plasma
triglycerides (TG) in the experimental animals (Table 2). However,
the introduction of catechins to a high-fat diet resulted in the
reduction of this concentration. The smallest concentration of TG
was determined using dietary supplementation of catechins with a
higher degree of thermal modification (HFC group). In these ani-
mals 20% less plasma TG was observed than in the mice fed with
the high-fat diet without the addition of catechins.
3.6. Antioxidative activity in blood plasma
The addition of catechins to diet effectively reduced lipid per-
oxidation products in blood plasma (Table 2). Plasma TBARS (thi-
obarbituric acid reactive substances) concentration in the mice fed
with the high-fat diet containing catechins with a higher degree of
thermal modification was 22.5% lower than in the mice fed the
high-fat diet without catechins. A correlation (R ¼
0.87) was
demonstrated between the amount of catechins belonging to the
(
) form (2S, 3R) added into the diet and plasma TBARS.
3.7. Quantification of atherosclerosis
The development of atherosclerosis was assessed quantitatively
in aortas using the en face analysis (Fig. 1A) and in aortic roots using
the cross-section analysis (Figs. 1B and 2). In animals fed the
standard diet the area of atherosclerotic lesions was smaller by 45%
(
) forms (2S, 3R) [mg/kg diet] Catechins [mg/kg diet]
GCG C CG
63a ± 1.3 14a ± 1.1 8a ± 0.9 846a ± 13.7
154b ± 1.8 23b ± 1.1 22b ± 1.3 851a ± 12.4
277c ± 3.3 32c ± 1.4 42c ± 2.3 847a ± 15.2
128 M. Mika et al. / Atherosclerosis 240 (2015) 125e130

Table 2
The body weight gain of the mice, plasma cholesterol and plasma TBARS levels in mice after 12 weeks of nutrition.

Group Cholesterol [mmol/l] TG TBARS Initial body weight Final body weight Feed intake Body weight gain

Chol-T LDL-C HDL-C [mmol/l] [mmol MDA/l] [g] [g] [g/mouse/day] [g]
Mi Mf Mf - Mi

DS 37.1a ± 4.0 14.3a ± 2.3 1.82b ± 0.15 0.97a ± 0.11 56.2ab ± 4.6 25.3a ± 1.3 33.3a ± 1.9 3.9a ± 0.13 8.0a ± 1.3
HF 87.9d ± 4.1 27.7b ± 4.1 1.33a ± 0.18 1.32b ± 0.17 61.8b ± 4.1 25.6a ± 1.0 39.7c ± 1.1 3.9a ± 0.14 14.1c ± 0.9
HFA 84.4cd ± 14.4 26.9b ± 2.8 1.59ab ± 0.29 1.09ab ± 0.18 51.9a ± 5.9 25.5a ± 1.6 39.2bc ± 1.7 4.0a ± 0.14 13.7bc ± 2.7
HFB 64.7b ± 15.7 26.4b ± 3.2 1.75ab ± 0.28 1.05ab ± 0.17 50.7a ± 4.2 25.6a ± 2.2 38.4bc ± 2.3 3.9a ± 0.15 12.8bc ± 2.4
HFC 70.0bc ± 12.6 27.1b ± 3.6 1.78b ± 0.21 1.01a ± 0.12 47.9a ± 7.1 25.7a ± 1.2 36.9b ± 1.7 3.8a ± 0.16 11.2b ± 1.8
1
Date are means ± SD values of 7 mice per group.
2
The mean in the same column not sharing a common superscript are significantly different among groups at p < 0.05 (Test Bonferroni).
3
DS e mice fed standard diet, HF e mice fed high-fat diet, HFA e mice fed high-fat diet with native catechins, HFB e mice fed high-fat diet with thermally modified catechins
formulation containing 20% (
) forms (2S, 3R), HFC e mice fed high-fat diet with thermally modified catechins formulation containing 35% (
) forms (2S, 3R).
4
Chol-T (total cholesterol), HDL-C (cholesterol associated with high density lipoprotein), LDL-C (cholesterol associated with low density lipoprotein), MDA (malondialdehyde),
TBARS (thiobarbituric acid reactive substances), TG (triglycerides).

(the en face method) and by 56% (the cross-section method) than in
the mice fed the high-fat diet without catechins. Both methods
showed that the addition of thermally modified catechins inhibited
the progression of atherosclerosis. The addition of the thermally
modified formulations to the high-fat diet resulted in a reduction of
the area of atherosclerotic lesions by about 28% (en face method)
and 45% (cross-section method) compared to the group fed the
high-fat diet without catechins. There were no statistically signifi-
cant differences observed between the effects of the thermally
Fig. 1. A:Quantification (% area) of atherosclerotic plaque in entire aorta (en face) in
apoE knockout mice after 12 weeks of nutrition. B: Quantification (mm2) of athero-
sclerotic plaque area in aortic roots (cross-section) in apoE knockout mice after 12
weeks of nutrition. 1The bars not sharing a common letters are significantly different
among groups at p < 0.05 (Test Bonferroni). 2DS e mice fed standard diet, HF e mice
fed high-fat diet, HFA e mice fed high-fat diet with native catechins, HFB e mice fed
high-fat diet with thermally modified catechins formulation containing 20% (
) forms
(2S, 3R), HFC e mice fed high-fat diet with thermally modified catechins formulation
containing 35% (
) forms (2S, 3R).
modified formulations with varying degrees of epimerization. The
least efficient were native catechins. In that case the area of
atherosclerotic lesions in mice did not differ significantly from le-
sions in the mice fed the high-fat diet without catechins.
4. Discussion
Our results showed that although the mean food intake did not
differ significantly between groups, the final body weight of mice
fed the HFC diet was significantly lower than mice fed the HF diet.
We observed that the weight gain of the mice fed the high-fat diet
with the addition of catechins depended on the degree of epime-
rization of catechins. The weight gain of the mice decreased with
the increasing content of catechins belonging to the (
) form (2S,
3R).
The effect of catechins on the animal body weight gain revealed
by various authors is not clear. Some authors have observed
decreased appetite and reduced weight gain with dietary supple-
mentation of catechins [20], whereas other authors have reported
increasing animal weight gain [21].
The LDL cholesterol level in the mouse blood plasma from the
groups HFB, HFC and HF did not differ. However, our data
demonstrated that in the mice fed the HFC diet there was a lower
total plasma cholesterol level with a simultaneous higher HDL-C
level than in the mice fed the HF diet without catechins. The
lower level of Chol-T in the blood plasma of mice whose diets
contained thermally modified catechins probably resulted from the
lower quantity of cholesterol connected with VLDL and chylomi-
cron remnants. Similarly, Lee et al. observed that thermally modi-
fied catechins more effectively reduced plasma total cholesterol
levels in rats than native catechins [5]. Effects of green tea catechins
on plasma cholesterol levels in animals have also been reported by
other authors [6,12]. On the other hand, Shrestha et al. observed
that supplementation of a high-fructose diet with green tea extract
caused an increase in total cholesterol in the blood plasma of the
experimental animals, with a simultaneous increase in HDL-C [21].
Our results indicate that addition of catechins to the high-fat
diet affects the plasma TG in mice. A lower plasma TG concentra-
tion in the mice fed a high-fat diet with the addition of catechins
probably resulted from the reduced bioavailability of the dietary
fat [22].
The effectiveness of green tea catechins in inhibiting lipid per-
oxidation processes in animals has been observed by many authors
[12,23]. However, our results showed the impact of epimerization
of catechins on this process. Plasma TBARS concentration in mice
fed a diet with the addition of catechins depends on the degree of
epimerization of catechins and decreases with increasing content
of catechins belonging to the (
) form (2S, 3R). A correlation
 M. Mika et al. / Atherosclerosis 240 (2015) 125e130
Fig. 2. Representative images of cross-sections of aortic roots showing aortic plaque in apoE knockout mice after 12-week experimental period. DS e mice fed standard diet, HF e
mice fed high-fat diet, HFA e mice fed high-fat diet with native catechins, HFB e mice fed high-fat diet with thermally modified catechins formulation containing 20% (
) forms (2S,
3R), HFC e mice fed high-fat diet with thermally modified catechins formulation containing 35% (
) forms (2S, 3R).
(R ¼
0.87) was demonstrated between the amount of catechins
belonging to the (
) form (2S, 3R) added into the diet and plasma
TBARS. Lower TBARS levels in HFA, HFB and HFC groups, compared
with the TBARS levels in the HF group, results most likely from the
lower quantity of substrates for the peroxidation process (Chol-T
and TG) as well as from the antioxidation effect of catechins and
their metabolites present in the blood.
The present study demonstrated that catechins formulations
containing 20% and 35% (
) forms (2S, 3R), introduced into the
high-fat diet, may inhibit the development of atherosclerosis in the
apoE mice more effectively than the native catechins. One of the
important risk factors of atherosclerosis is the elevated concen-
tration of plasma LDL-C. Oxidation of LDL lipids is thought to render
the lipoprotein atherogenic, because oxidized LDL is more readily
taken up by macrophages via scavenger receptors. The addition of
catechins to the high-fat diet did not affect plasma LDL-C level in
mice (Table 2). However, the concentration of lipid peroxidation
products determined in plasma of the mice fed with the high-fat
diet with the addition of catechins was significantly lower than in
mice fed the HF diet (Table 2). Antioxidative effects of catechins and
their metabolites in plasma inhibit the oxidation process of un-
saturated fatty acids present in LDL. However, the antioxidant ac-
tivity is not the only effect of catechins that inhibits the
development of atherosclerosis. The green tea catechins have a
positive effect on the cardiovascular system by inducing the gene
expression of vascular endothelial nitric oxide synthase (ecNOS)
[24], as well as increasing the activity of this enzyme by altering its
phosphorylation [25,26]. Among the green tea native catechins
only EGCG (epigallocatechin gallate) causes an increase in the
synthesis of NO. There is no information on the effect of GCG
(gallocatechin gallate) on endothelial nitric oxide synthesis.
Furthermore, in the studies on venous endothelial cells it was noted
that EGCG inhibits LOX-1 (oxidized LDL receptor-1) gene expression
[27]. Moreover, catechins reduce plasma homocysteine concen-
tration [28]. On the other hand, the high concentration of homo-
cysteine in blood promotes initiation and progression of
atherosclerosis. Green tea catechins also prevent cardiovascular
diseases by their effects on smooth muscle cell proliferation
through inhibition of the PDGF-b receptor [29]. Research conducted
by Chyu et al. showed that the injection of EGCG (10 mg/kg)
significantly reduced the progression of atherosclerosis, but it did
not reverse existing damage, indicating the importance of the
moment at which the treatment is initiated [13]. These observa-
tions indicate the great validity of application for stabilization of
high-fat foods containing antioxidant cholesterol, which will
effectively prevent the initiation of atherosclerosis. The studies
described here suggest that for this purpose one may use catechins
formulations, particularly those subjected to thermal modification.
Unfortunately, in the literature no information was found about
the influence of catechins' epimerization on their bioavailability in
129
mice, although Xu et al. showed that the epimerization of catechins
(EGCG and GCG) has no significant influence on bioavailability in
male rats [3]. However, in the latest studies it was shown that the
catechin with 2,3-trans structure form complexes more easily with
the human protein HSA (taking part in catechin transport in blood)
than the catechin with 2,3-cis structure. This suggests that cate-
chins belonging to the (
) form (2S, 3R) may show greater bio-
logical activity [30].
To conclude, thermally modified catechins reduce atheroscle-
rosis in the apoE knockout mice more effectively than native cat-
echins. Furthermore, the body weight gain and plasma TBARS and
HDL cholesterol concentration in mice fed a diet with the addition
of catechins depends on the degree of epimerization of catechins
and decreases with increasing content of catechins belonging to the
(
) form (2S, 3R). Moreover, plasma HDL cholesterol concentration
in mice depends on catechins' stereoisomerism and increases along
with the increasing content of catechins belonging to the (
) form
(2S, 3R). These results suggest that thermally modified catechins
should be studied further for their possible use in production of
functional food.
Conflicts of interest
None.
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