Atherosclerosis 192 (2007) 113–122

The effect of ethanol extract of Hypericum lysimachioides on lipid profile

in hypercholesterolemic rabbits and its in vitro antioxidant activity
Fidan Hakimoğlu a , Göksel Kızıl a,∗ , Zeki Kanay b , Murat Kızıl a , Hilmi Isı c
a University of Dicle, Faculty of Science and Art, Chemistry Department, 21280 Diyarbakır, Turkey
bUniversity of Dicle, Faculty of Vetenary Science, Histopathology Department, 21280 Diyarbakır, Turkey
c University of Dicle, Faculty of Medicine, Medicinal Biology and Genetic Department, 21280 Diyarbakır, Turkey

Received 1 February 2006; received in revised form 20 June 2006; accepted 6 July 2006
Available online 9 August 2006

Abstract

Hypercholesterolemia, high cholesterol diet and oxidative stress increase serum total cholesterol and LDL cholesterol levels resulting in
increased risk for development of atherosclerosis. Antioxidants play an important role in inhibiting and scavenging radicals, thus providing
protection to humans against infectious and degenerative diseases. Literature shows that the antioxidant activity is high in medicinal plants.
Realizing the fact that, this study was carried out to determine the effect of ethanol extract of Hypericum lysimachioides Boiss var lysimachioides
(Guttifera) on serum lipid levels and serum lipid peroxidation in hypercholesterolemic rabbits. The rabbits were divided into four groups and
these groups were fed with diets containing standard laboratory diet (Group I), standard laboratory diet and ethanol extracts of H. lysimachioides
(HL) (50 mg/kg body weight) (Group II), standard laboratory diet, ethanol extracts of HL (50 mg/kg body weight) and cholesterol (100 mg/kg
body weight) (Group III), and finally standard laboratory diet and cholesterol (100 mg/kg body weight) (Group IV), for 5 weeks. Feeding
cholesterol increased serum cholesterol and LDL cholesterol levels significantly in Group IV as compared to the other groups. Ethanol extract
of HL with high cholesterol diet significantly lowered LDL cholesterol and total cholesterol levels in the rabbits of Group III as compared to
the Group IV. The level of serum triacylglycerol was found to be similar to all comparison groups. HDL cholesterol levels were also increased
significantly in Groups II and III as compared to Group IV. Statistically significant difference was found in Group IV as compared to all other
groups. The ethanol extract of HL with high cholesterol diet significantly lowered the serum MDA levels in the rabbits of Group III compared to
the Group IV. The histopathological findings confirmed that the ethanol extract of HL restrained the progression of the hydropic degeneration
and fatty changes in the liver and some atherosclerotic lesions in the aorta. The in vitro antioxidant activities of ethanol extract of HL was also
evaluated. The free radical-scavenging properties of HL (IC50 = 28 ␮g/ml) were studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay
system. Since plant phenolic compound is one of the phytochemicals possessing radical scavenging activity, the amount of total phenolic
compound was also determined in ethanol extract of HL and total phenolic content of one-milligram HL ethanol extract was equivalent to
307 ␮g of gallic acid. Total antioxidant activity of ethanol extract of HL was tested by using ferric thiocyanate (FTC) and thiobarbituric acid
(TBA) methods. Antioxidative activities of ethanol extract of HL was found to be comparable with Vitamin E. In conclusion, the use of this
extract could be useful in the management of cardiovascular disease in which atherosclerosis is important.
© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cholesterol fed; Lipid peroxidation; Hypericum lysimachioides; Hypercholesterolemia; Antioxidant activity

1. Introduction

Hypericum is a well-known plant in herbal medicine. Dif-

ferent species of Hypericum have been traditionally used for
∗ Corresponding author. Tel.: +90 412 2488550x3038; the treatment of wounds, eczama and burns. It has also been
fax: +90 412 2488039. used for its sedative, anti-inflammatory and antiseptic effects
E-mail address: gokselk@dicle.edu.tr (G. Kızıl). [1–3]. During the last few years antimicrobial, antifungal and

0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2006.07.013
114 F. Hakimoğlu et al. / Atherosclerosis 192 (2007) 113–122
antioxidant properties have been reported by many experi-
mental studies [4–7].
There has been a growing interest in natural products and
their role in the maintenance and improvement of health and
wellness. The cholesterol-lowering effect of dietary plants
has been well studied and various plants were shown to be
helpful in lowering plasma cholesterol levels and encourag-
ing safety profile [8–11]. Dietary plants therefore are con-
sidered to be useful means to prevent disorders such as
atherosclerosis.
For many years, it has been recognized that excess serum
cholesterol is a major risk factor for atherosclerosis that
results in coranary heart disease (CHD). A definite link is
observed between elevated serum cholesterol and CHD, and
diet modification can lower the risk of CHD. Low density
lipoprotein (LDL), the major cholesterol carrier in plasma,
also appears to be involved in the development of various
degenerative diseases such as atherosclerosis, carcinogene-
sis, aging and diabetes mellitus. Several authors have sug-
gested that peroxidative modification of LDL is an important
factor in atherosclerotic changes [12–15]. Oxidative mod-
ification of LDL alters its structure allowing LDL to be
taken up by scavenger receptors on macrophage, endothe-
lial and smooth muscle cells, leading to the formation of
lipid-laden foam cells, the hallmark of early atherosclerotic
lesions.
Lipid peroxidation is the process that involves the chain
reaction of free radicals with polyunsaturated fatty acids.
These reactions lead to rearrangements of double bonds in
conjugated diens, hydroperoxide generation, lipid breakdown
into lower molecular weight fragments such as ketones, alco-
hol, hydrocarbons, acids and epoxides as well as chemical
modifications in the Apo-B protein. The extent of lipid per-
oxidation can be estimated by measurement of thiobarbituric
reactive substances (TBARS). TBARS can be determined
by reaction of malondialdehyde a secondary breakdown
product of lipid hydroperoxides, with thiobarbituric acid
(TBA).
Many herbal plants contain antioxidants compounds and
these compounds protect cells against the damaging effects
of reactive oxygen species, such as singlet oxygen, super-
oxide, peroxyl radicals, hydroxyl radicals and peroxynitrite
[16–18]. An imbalance between antioxidants and reactive
oxygen species results in oxidative stress, leading to cellu-
lar damage. Several authors demonstrated that antioxidant
intake is inversely related to mortality from coronary heart
disease and to the incidence of heart attacks [19–21]. The
Hearth Protection Study [22] demonstrated that antioxidant
vitamins did not produce any significant reductions in the
5-year mortality from, or incidence of, any type of vascular
disease, cancer, or other major outcome.
Recent studies have demonstrated that, different species
of Hypericum contains compounds such as flavonoids, xan-
thones and phenolic and can be used as antioxidants [23,24].
There are however no reports on the effect of Hypericum
on hypercholesterolemia. This work was therefore aimed to
investigate the effect of ethanol extract of Hypericum lysi-
machioides Boiss var lysimachioides on serum lipid profile
and serum lipid peroxidation in hypercholesterolemic rabbits
and also to determine in vitro the antioxidant capacity of this
extract.
2. Materials and methods
2.1. Collection of plant material
H. lysimachioides var. lysimachioides was collected in
Mardin and Şırnak in the area of South East Turkey in June
2004, by Zuhal Toker. They were identified by Dr. A. Selçuk
Ertekin and a voucher specimen was deposited at the Herbar-
ium of the Department of Biology, Faculty of Science and
Art, Dicle University (voucher no. DUF-2513-b).
2.2. Preparation of crude extract
Aerial parts (stems, leaves and flowers) were dried for
10 days at room temperature The dried aerial parts (240 g)
was ground in an electric blender and then incubated into a
glass flask with 2000 ml of ethanol (70%) for 3 days under
magnetic stirrer. Solvent was evaporated under vacuum and
crude extract (52 g) was obtained as a dark reddish colour
and kept in dark glass bottles at 4 ◦ C until use.
2.3. Animals and treatment
A total of 20 male New Zealand white rabbits with average
body weight of 3000 g were used in this study. Animals were
housed in an air conditioned animal room at 23 ± 2 ◦ C, with
12 h/12 h light/dark photoperiod, and provided with standard
laboratory diet and water ad libitum.
The rabbits were divided into four groups of five rabbits
each and for 5 weeks as follows.
• Group I: Fed with a standard laboratory diet (control
group) (28.00% alfalfa meal, 22.50% ground oat hulls,
13.00% soybean, 14.75% ground barley, 6.00% wheat
middlings, 7.00% wheat bran, 2.00% dried whey, 1.50%
dried molasses, 1.00% Brewer’s dried yeast, 0.50% salt,
0.50% soybean oil, 1.25% dicalcium phosphate, 1.50%
ground limestone, 0.50% (vitamin–mineral premixes).
• Group II: Standard diet + H. lysimachioides ethanol extract
(50 mg/kg body weight per day).
• Group III: Standard diet + H. lysimachioides ethanol
extract (50 mg/kg body weight per day) + cholesterol sus-
pended in vegetable oil (100 mg/kg body weight per day).
• Group IV: Standard diet + cholesterol suspended in veg-
etable oil (100 mg/kg body weight per day).
The H. lysimachioides ethanol extract and cholesterol was
given orally for 5 weeks. The animals were provided from
Health Centre, University of Dicle. Weight-gain was mea-
sured once a week.
 F. Hakimoğlu et al. / Atherosclerosis 192 (2007) 113–122
2.4. Blood collection
Blood samples were collected from marginal ear vein of
overnight fasted rabbits before starting experiments and after
2 and 5 weeks of treatment. The plasma was obtained by
centrifuging the blood samples at 2000 rpm for 15 min. Total
cholesterol and triacylglycerol (TAG) were estimated enzy-
matically using commercial kits (Linear Chemistry, Spain)
and HDL cholesterol was estimated enzymatically using
commercial kits (BioSystems, Spain). LDL cholesterol was
calculated [25]. Blood samples were also analyzed for plasma
total cholesterol by using automatic auto analyzer (Aeroset,
Abbott Lab., Germany), in order to compare the results
obtained from commercial kits.
On the 36th day all the groups of animals, except Group II,
were sacrificed by rapid intracardiac pentobarbital injection
for histopathological analyze.
2.5. MDA determination
Lipid peroxides were measured in serum by TBARS
method [26]. In brief, 0.5 ml each of plasma was mixed with
2.5 ml of 20% trichloroacetic acid (TCA) (Sigma, Germany)
and centrifuged at 3000 rpm for 10 min. The supernatant was
decanted and the precipitate was washed once with 0.05 M
sulphuric acid and then 3 ml of 0.2 g/dl thiobarbituric acid
(TBA) (Sigma, Germany) reagent was added to the precip-
itate. The mixture was heated in a boiling water bath for
30 min. After cooling in cold water, the resulting chromogen
was extracted with 4 ml of n-butyl alcohol. The organic phase
was separated by centrifugation at 3000 rpm for 10 min and
absorbance was recorded at wavelength of 530 nm. A malon-
dialdehyde (MDA) solution made freshly by the hydrolysis
of 1,1,3,3,-tetramethoxypropane (TMP) (Sigma, Germany)
was used as the standard. (MDA was dissolved in 0.05 M
sulphuric acid to prepare 10 mM stock solution. By diluting
the stock solution, different concentrations (1–5 nmol/ml) of
MDA were obtained in order to prepare a standard curve.)
The results are expressed as the nmol MDA/ml plasma.
2.6. Histopathological analysis of liver and aorta
The liver and entire aortas were rapidly dissected out and
tissue sections (5 ␮m) of liver and aorta fixed by immersion at
room temperature in 10% formalin solution. For the histolog-
ical examinations, paraffin-embedded tissue sections of aorta
and liver were stained with hematoxylin–eosin (H&E). The
tissue samples were then examined and photographed under
a light microscope (Nikon-Eclipse 400) for observation of
structural abnormality. The severity of aortic atherosclero-
sis as judged by two-independent observers blinded to the
experimental protocol. The following morphological crite-
ria were considered: grade 0; scale 0 (no damage), grade
1; scale + (mild), grade 2; scale ++ (moderate), grade 3;
scale +++ (severe), and grade 4; scale ++++ (highly severe)
[27].
115
2.7. Free radical scavenging activity
Free radical scavenging activity of ethanol extract of
HL was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH)
(Sigma–Aldrich, Germany) using the method of Shimada et
al. [28]. Briefly 0.1 mM solution of DPPH in ethanol was
prepared. Then 1 ml of this solution was incubated with vary-
ing concentrations of ethanol extract of HL (5–500 ␮g/ml).
The reaction mixture was then shaken well and incubated for
30 min in dark at room temperature. The absorbance of the
resulting solution was read at 517 nm against a blank. The free
radical scavenging ability was calculated as follows: scav-
enging ability (%) = (A517 of control − A517 of sample/A517
of control) × 100. Ascorbic acid, butylated hydroxytoluene
(BHT) and ␣-tocopherol (Sigma–Aldrich, Germany) were
used as positive controls. IC50 value was calculated by linear
regression analysis using Prism 2.0 version software.
2.8. Determination of total phenolic compounds
The content of total phenolic compounds in ethanol
extract of HL was determined using Folin–Ciocalteus reagent
according to the method of Singleton et al. [29]. Forty micro-
liters of crude ethanol extract of HL (1 mg/ml) was mixed
with 200 ␮l Folin–Ciocalteus reagent (Sigma–Aldrich, Ger-
many) and 1160 ␮l of distilled water, followed by 600 ␮l 20%
sodium carbonate (Na2 CO3 ) 3 min later. The mixture was
shaken for 2 h at room temperature and absorbance was mea-
sured at 765 nm. All tests were performed in triplicate. Gallic
acid (Sigma–Aldrich, Germany) was used as a standard. The
concentration of total phenolic compounds in HL was deter-
mined as ␮g of gallic acid equivalents per 1 mg of extract
using the following equation obtained from a standard gallic
acid graph (R2 = 0.9877):
absorbance = 0.0012 × gallic acid (μg) − 0.0034
2.9. Antioxidant activity
Ferric thiocyanate (FTC) method: The method of ferric
thiocyanate was followed from Kikuzaki and Nakatani [30]
which was slightly modified by Mitsuda et al. [31] and Osawa
and Namiki [32]. FTC method was used to determine the
amount of peroxide at the initial state of lipid peroxida-
tion. The peroxides reacts with ferrous chloride (FeCl2 ) to
form a reddish ferric chloride (FeCl3 ) dye. In this method,
the concentration of peroxides decreases as the antioxidant
activity increases. A mixture of 4 mg sample was placed in
4 ml of absolute ethanol (Merck), 4.1 mg of 2.52% linoleic
acid (Sigma–Aldrich, Germany) in absolute ethanol, 8 ml of
0.05 M phosphate buffer (pH 7.0) and 3.9 ml of water was
placed in a vial with a screw cap and then placed in an oven
at 40 ◦ C in the dark. To 0.1 ml of this solution, 9.7 ml of 75%
ethanol and 0.1 ml 30% ammonium thiocyanate (Sigma) was
added. Exactly 3 min after the addition of 0.1 ml of 0.02 M
ferrous chloride in 3.5% hydrochloric acid (HCl) to the reac-
116 F. Hakimoğlu et al. / Atherosclerosis 192 (2007) 113–122

tion mixture, the absorbance was measured at 500 nm every Table 2

24 h until the absorbance of the control reached maximum.
The control and standard were subjected to the same pro-
cedures as the sample, except that for the control, only the
solvent was added, and for the standard, 4 mg sample was
replaced with 4 mg of Vitamin E.
Thiobarbituric acid (TBA) method: The method of
Ottolenghi [33] was used to determine the TBA values of
the samples. The formation of malonaldehyde is the basis for
the well-known TBA method used for evaluating the extent of
lipid peroxidation. At low pH and high temperature (100 ◦ C),
malondialdehyde binds TBA to form a red complex that can
be measured at 532 nm. The increase amount of the red dye
formed correlates with the oxidative rancidity of the lipid.
Two milliliters of 20% trichloroacetic acid (CCl3 COOH) and
2 ml TBA aqueous solution were added to 1 ml of sample
solution prepared as in the FTC procedure, incubated in a
similar manner. The mixture was then placed in a boiling
water bath for 10 min. After cooling, it was centrifuged at
3000 rpm for 20 min and the absorbance of the supernatant
was measured at 532 nm.
Antioxidant activity was determined as follows:
[(absorbance of control on day maximum − absorbance
of sample on the same day)/absorbance of control on day
maximum] × 100. All data about total antioxidant activity
are the average of three replicates analyses.
2.10. Statistical analysis
The parameter values were all expressed as the
mean ± S.E. Significant differences among the groups were
determined by one-way ANOVA using SPSS 12.0 software
package program. The results were considered significant if
the value of P was less than 0.05.
3. Results
Table 1 summarizes the effect of ethanol extract of HL
on plasma lipid profile of rabbits fed on a high cholesterol
diet. Keeping the rabbits on a high cholesterol diet signifi-
Effect of ethanol extract of H. lysimachioides on serum total cholesterol
levels (mg/dl) of rabbits fed a high fat diet by using auto analyzer instrument
Groups 0 week 2 week 5 week
Group I 48.0 ± 2.0 a 37.5 ± 21.9 a 43.5 ± 13.2 a
Group II 35.0 ± 19.8 a 46.5 ± 2.1 a 37.0 ± 11.9 a
Group III 32.0 ± 5.6 a 145.5 ± 6.3 b 309.0 ± 64.1 b
Group IV 43.5 ± 16.2 a 377.0 ± 14.1 c 686.2 ± 37.5 c
Group I: control, Group II: H. lysimachioides ethanol extract, Group III:
H. lysimachioides ethanol extract + cholesterol, Group IV: cholesterol. All
data are mean ± S.E.M. of five rabbits. Means with different letters at a
time differ significantly, P < 0.05. The values sharing common letters are
not significantly different at P > 0.05.
cantly increased total cholesterol and LDL cholesterol levels
in serum of Groups III and IV as compared to Groups I
and II. Similarly, Group IV which was fed with only choles-
terol diet also showed significant increase in total cholesterol
and LDL cholesterol levels as compared to Group III which
received ethanol extract of H. lysimachioides in addition to
cholesterol diet. HDL cholesterol levels increased 26.4 ± 7.3
to 39.4 ± 3.6 and 24.8 ± 2.0 to 54.2 ± 6.0 in Groups II
and III rabbits, respectively. These increases were signifi-
cantly higher (P < 0.05) than values obtained from the others
(Groups I and IV). TAG levels were found to be 124.8 ± 4.7
in Group I, 131.6 ± 6.1 in Group II, 130.4 ± 11.7 in Group
III and 128.4 ± 11.9 in Group IV. No statistically significance
was found between the mean values (P > 0.05). The Group I
(control animals) and Group II (received only ethanol extract
of HL) did not show any significant change in total choles-
terol, LDL cholesterol and triacylglycerol levels.
Serum total cholesterol levels were also analyzed by using
auto analyzer instrument for comparing the results obtained
from the commercial kits. Tables 1 and 2 show clear differ-
ences in the total cholesterol levels between the two methods
used. Although there is the difference observed, it was found
using both methods, that the ethanol extract of HL with high
cholesterol diet significantly lowered total cholesterol levels
in rabbits of Group III as compared to Group IV. Table 2
shows total cholesterol levels on days 0, 15 and 35 for all
groups of rabbits. Groups I and II did not show any signif-
icant changes in total cholesterol levels during 5 weeks of

Table 1
Effect of ethanol extract of Hypericum lysimachioides on serum lipid profile of rabbits fed with a high fat diet
Groups Duration (week) Total cholesterol (mg/dl) TAG (mg/dl) LDL (mg/dl) HDL (mg/dl)
0 58.6 ± 13.6 a 125.2 ± 4.9 a 26.6 ± 4.7 a 34.4 ± 6.4 a
Group I
5 52.0 ± 9.0 a 124.8 ± 4.7 a 20.6 ± 6.2 a 35.8 ± 8.4 a
0 51.0 ± 6.2 a 128.6 ± 9.7 a 20.4 ± 6.7 a 26.4 ± 7.3 a
Group II
5 49.8 ± 4.1 a 131.6 ± 6.1 a 15.4 ± 1.6 a 39.4 ± 3.6 b
0 56.4 ± 4.7 a 125.6 ± 20.0 a 25.6 ± 4.7 a 24.8 ± 2.0 a
Group III
5 231.2 ± 61.9 b 130.4 ± 11.7 a 140.2 ± 41.6 b 54.2 ± 6.0 b
0 62.2 ± 15.3 a 129.0 ± 12.6 a 30.2 ± 13.2 a 31.8 ± 12.4 a
Group IV
5 516.4 ± 171.8 c 128.4 ± 11.9 a 486.0 ± 168.8 c 33.0 ± 10.2 a
Group I: control, Group II: H. lysimachioides ethanol extract, Group III: H. lysimachioides ethanol extract + cholesterol, Group IV: cholesterol. All data are
mean ± S.E.M. of five rabbits. Means with different letters at a time differ significantly, P < 0.05. The values sharing common letters are not significantly
different at P > 0.05.
 F. Hakimoğlu et al. / Atherosclerosis 192 (2007) 113–122
Fig. 1. Comparison of serum TBARS levels between Groups I, II, III and
IV rabbits. P value < 0.001 Group III compare the Group IV.
treatment. However, total cholesterol levels obtained from
the rabbits of Groups III and IV were increased significantly
(P < 0.05) compared to plasma cholesterol levels obtained
from Groups I and II after following 2 and 5 weeks of treat-
ment. Similarly, during 2 and 5 weeks treatment of rabbits
fed with cholesterol diet (Group IV) also showed approxi-
mately two-fold increases in the levels of total cholesterol as
compared to rabbits (Group III) fed with cholesterol diet in
addition to the ethanol extract of HL. Total cholesterol level
of Group III were significantly (P < 0.05) lower as compared
to Group IV.
The plasma lipid peroxide levels were determined by mea-
suring the TBARS concentration. Fig. 1 shows the compari-
son of serum MDA levels between Group I (1.4 ± 0.1), Group
II (1.6 ± 0.3), Group III (1.7 ± 0.2) and Group IV (2.9 ± 0.5)
rabbits. The ethanol extract of HL with high cholesterol diet
significantly lowered serum MDA levels in the rabbits of
Group III (P < 0.001) compared to Group IV. There was no
statistical significant difference observed between Group I
(control) and Group II which received only ethanol extract of
HL during the treatment.
Fig. 2. Histological analysis of liver (a) and aorta (b) of control group rabbits, stain: hematoxylin and eosin (H&E, 200×). VS: vena central, S: sinozoid, M:
media, L: lumen. (→) Hydropic degenerative changes; (
117
The aorta and liver of Group I rabbit showed normal
histology (Fig. 2). However, aorta and liver of Group III
rabbits (high cholesterol diet in addition to plant extract)
showed some atherosclerotic lesions in the thoracic artery and
fatty and degenerative changes in the liver (Fig. 3) but these
changes were lower than in Group IV rabbits (high choles-
terol diet) (Fig. 4). Increasing atherosclerotic lesions and
degenerative changes were observed in microscopic exam-
ination of Group IV samples as compared to Group III spec-
imen.
The severity of aortic atherosclerosis as judged by gross
grading, Group I did not show any evidence of atherosclerosis
(grade 0; scale 0). Animals given HL extract with cholesterol
diet, Group III, showed a lower degree of atherosclerosis
(grade 2; scale ++). Group IV showed highest degree of
atherosclerosis (grade 4; scale ++++).
Free radical scavenging capacity of crude ethanol extract
of HL was measured by DPPH assays. DPPH is a useful
reagent for investigating free radical scavenging activities of
phenolic compounds. The reduction of DPPH absorbtion is
indicative of the capacity of the extract to scavenge free rad-
icals, independently of any enzymatic activity [34]. DPPH
scavenging potential of HL at varying concentrations was
measured and the results are depicted in Fig. 5. The scaveng-
ing effect increased with increasing HL concentration up to a
certain extent (50 ␮g) and then level off with slight decrease.
The ethanol extract of HL showed high radical scavenging
ability of 90% at 150 ␮g/ml. The scavenging effects for ascor-
bic acid, ␣-tocopherol and BHT were 96%, 91% and 83% at
the concentration of 150 ␮g/ml, respectively. The IC50 value
of HL in the DPPH radical scavenging assay was 28 ␮g/ml.
The amount of total phenolic compounds were investi-
gated in the ethanol extract of HL. Phenols are very impor-
tant plant constituents because of their radical scavenging
ability due to their hydroxyl groups [35]. The total amount
of phenolic compounds in the plant extract determined as
microgram of gallic acid equivalent by using an equation
that was obtained from standard gallic acid graph. One
milligram HL extract was equivalent to 307 ␮g of gallic
acid.
) endothelial cells.
118 F. Hakimoğlu et al. / Atherosclerosis 192 (2007) 113–122

Fig. 3. Histological analysis of liver (a) and aorta (b) of Group III rabbits, stain: hematoxylin and eosin (H&E, 200×). VS: vena central, F: foam cells, fatty
changes, S: sinozoid, M: media, L: lumen, Se: subendothelial. (→) Hydropic degenerative changes; ( ) endothelial cells.

Fig. 4. Histological analysis of liver (a) and aorta (b) of Group IV rabbits, stain: hematoxylin and eosin (H&E, 200×). VS: vena central, F: foam cells, fatty
changes, S: sinozoid, L: lumen, C: cartilage, B: bone. (→) Hydropic degenerative changes; ( ) endothelial cells.

The total antioxidant activity of ethanol extract of HL was
determined by using FTC and TBA methods and compared
with Vitamin E. The effects of ethanol extract of HL and Vita-
min E on peroxidation of linoleic acid emulsion are shown
in Fig. 6. The individual activity of samples by FTC method
showed that ethanol extract of HL and Vitamin E exhibited
quite similar absorbance values. The absorbance value of the
control increased on day 12 and reached maximum levels
on day 14 and finally dropped on day 15. Fig. 7 shows the

Fig. 5. Scavenging effect of ethanolic extract of Hypericum lysimachioides Fig. 6. Absorbance value of ethanol extract of H. lysimachioides and Vitamin
on 1,1-diphenyl-2-picrylhydrazyl radicals. Each value is expressed as E in the linoleic acid emulsion using FTC method. Each value is expressed
mean ± S.D. of three determinations. BHT: butylated hydroxtoluen. as mean ± S.D. of three determinations.
 F. Hakimoğlu et al. / Atherosclerosis 192 (2007) 113–122
Fig. 7. The total antioxidant activity of ethanol extract of H. lysimachioides
and Vitamin E by using FTC and TBA method. Values are mean ± S.D. of
three determinations.
total antioxidant activity of ethanol extract of HL by FTC and
TBA methods. Vitamin E had the highest activity (60%) fol-
lowed by ethanol extract of HL (59%) by using FTC method.
Based on the TBA method, Vitamin E also had the higher
antioxidant activity (25%) followed by ethanol extract of HL
(20%). No significant difference was found between the total
antioxidant activity of ethanol extract of HL compared with
Vitamin E in both methods.
4. Discussion and conclusion
It has been documented that hypercholesterolemia is an
important coronary risk factor [36]. Cardiac morbidity and
mortality are directly related to serum cholesterol levels.
The consumption of a cholesterol enriched diet increases
the degree of lipid peroxidation, which is also one of the
early processes of atherosclerosis. It is generally assumed that
some antioxidants can prevent atherosclerosis by protecting
LDL from oxidation and are also associated with an anti-
hypercholesterolemic effect [37,38]. However, recent study
showed that antioxidant vitamin supplementation (600 mg
Vitamin E, 250 mg Vitamin C, and 20 ␮g ␤-carotene daily)
appeared to be safe. But, although this regimen increased
blood vitamin concentrations substantially, it did not pro-
duce any significant reductions in the 5-year mortality form,
or incidence of, any type of vascular disease, cancer, or other
major outcome [22].
Research in herbal medicine has increased in the world
as a way to rescue ancient traditions as well as an alter-
native solution to health problems in cities. Many plant
extracts have been shown to have hypocholesterolemic activ-
ity in rabbits or rats. Cholesterol lowering property of sev-
eral extracts have been described [9,39–42] and it has been
found that treatment with different plant extracts are use-
119
ful in achieving and maintaining low plasma levels of total
cholesterol.
Hypericum species have been examined and the antimi-
crobial activities of the essential oils or various extracts from
several Hypericum species have been reported previously
[43–47]. Antioxidant properties of Hypericum species were
also demonstrated [7,48]. However, there is no study on the
effect of Hypericum on hypercholesterolemia.
In this study, ethanol extract of HL was investigated for
hypolipidemic and its in vitro antioxidant activity. After 5
weeks treatment of rabbits with ethanol extract of HL with
high cholesterol diet, total cholesterol and LDL cholesterol
levels were found to be decreased compared to rabbits given
a high cholesterol diet. These differences were found to be
statistically significant. Even 2 week oral administration of
this extract to the rabbits showed significant falls in total
cholesterol. These results showed that treatment with this
extract can decrease the rise in total cholesterol and LDL
cholesterol. Phytosterols are thought to be responsible to this
hypocholesterolemic effect. The cholesterol-lowering effect
of dietary plant sterols on blood lipids has been studied and
is well known and various plant sterols were shown to reduce
serum cholesterol levels at low doses [49,50]. Qualitative
analysis of H. hysopifolium methanol extract had shown the
presence of phenols, flavonoids, glycosides, and sterols [22].
It may be possible that these active principles would be inter-
fering in different pathways involved with lipids [51]. The
decrease in cholesterol balance, which indicates the total
change in body pools of cholesterol, may be due to com-
pensatory mechanisms, such as a decrease in resorption of
endogenous cholesterol or an increase in the rate of secretion
into intestinal track or both.
HDL play a key role in the protection against oxidative
damage of membranes [52,53]. The principle role of HDL
in lipid metabolism is the uptake and transport of cholesterol
from peripheral tissues to the liver through a process known as
reverse cholesterol transport. Low HDL cholesterol levels are
strongly associated with an increased risk of coronary heart
disease and coronary artery disease [54,55]. Ethanol extract
treatment increased HDL cholesterol levels in Groups II and
III. These differences were found as statistically significant.
In this study plasma MDA level was used to investigate the
effect of this extract on hypercholesterolemic rabbits. MDA
level was measured as TBARS method. This method has been
criticized for its lack of specificity, but it is one of the easiest
and the most frequently used marker of lipid peroxidation
[56]. Several investigators found that high cholesterol diet
had an increasing effect on lipid peroxidation in plasma and
tissues in rabbits [57,58]. The results of this study show sig-
nificant increases in plasma MDA levels in Group IV rabbits
when compared with other groups. This result suggests that
this extract may have an antioxidant effect on hypercholes-
terolemic rabbits.
Histological study demonstrated that ethanol extract of HL
can reduce atherosclerosis lesions in the aorta and some fatty
changes in liver. Fatty changes of macro and microvesicular
120 F. Hakimoğlu et al. / Atherosclerosis 192 (2007) 113–122
type were observed in Groups IV and III rabbit of livers and
along with some atherosclerotic lesions in the aorta. How-
ever, Group III rabbits showed only focal area of fatty droplets
and degenerative changes in liver and lesser atherosclerotic
lesions in aorta as compared to Group IV. Histopathologi-
cal findings confirmed that ethanol extract of HL restrained
the progression of the atherosclerotic lesions in the thoracic
artery and of hydropic degeneration and fatty changes in the
liver. The total cholesterol and LDL cholesterol levels were
not found to be statistically significant between Groups I and
II rabbits. Therefore, the Group II rabbits were not sacrificed
and aortas and livers of group II rabbits were not examined.
Comparative investigations may be useful for further studies.
Several epidemiological studies have shown that increased
dietary intake of natural phenolic antioxidants correlates with
reduced coronary heart disease. Food rich in antioxidants
plays an essential role in the prevention of cardiovascular
diseases, cancer and neurodegenerative disease [59,60]. The
present study was also designed to evaluate the in vitro antiox-
idant capacity and estimate the phenolic content of ethanol
extract of HL. The model DPPH provides a method to evalu-
ate antioxidant activity in a relatively short time compared to
the other methods [61]. The effect of antioxidants on DPPH
radical scavenging was thought to be due to their hydrogen
donating ability. In the present study, HL showed scavenging
of DPPH radical, which may be attributable to its hydrogen-
donating ability.
Phenols are very important plant constituents because of
their scavenging ability due to their hydroxyl groups. There
is a highly positive relationship between total phenols and
antioxidant activity was found in many plant species [62].
It was also reported that phenolic compounds were associ-
ated with antioxidant activity and play an important role in
stabilizing lipid peroxidation [63]. Phenolic compounds are
effective hydrogen donors, which make them good antiox-
idants. Therefore, total phenolic compounds in the ethanol
extract of HL were determined and 1 mg HL ethanolic extract
was found to be equivalent to 307 ␮g of gallic acid.
Antioxidants are known to alleviate oxidative stress which
is generally perceived as one of the major causes for the accu-
mulation of mutations in the genome. Peroxide is gradually
decomposed to lower molecular compounds during the oxi-
dation process and these compounds here measured by FTC
and TBA method. The amount of peroxide at the primary
stage of linoleic acid peroxidation were measured by FTC
method, whereas TBA methods measures at the secondary
stage. Total antioxidant activity of ethanol extract of HL was
determined by using FTC and TBA methods. Total antioxi-
dant activity of the FTC method compared to the TBA method
in which the activity of antioxidant for FTC method is higher
than the TBA method. This may indicate that the amount of
peroxide in the initial state of lipid peroxidation is greater
than the amount of peroxide in the secondary stage. Further-
more, the secondary product such as malondialdehyde is not
stable for a period of time. It will turn into alcohol and acid
which can not be detected by spectrophotometer [33].
Our results demonstrate that ethanol extract of H. lysi-
machoides were effective increasing HDL cholesterol and
reducing plasma total cholesterol, LDL cholesterol and MDA
levels in cholesterol fed rabbits. HL also showed a concen-
tration dependent scavenging of DPPH radical, which may
be attributable to its hydrogen-donating ability. High content
of total phenols in ethanol extract might explain antioxidant
properties in H. lysimachoides. In mouse model of atheroscle-
rosis associated to hyperglycemia and hypercholesterolemia,
the administration of Vitamin E decreases in the arterial wall,
both the fatty deposits and the macrophages accumulation as
well as the mortality rate [64]. It is noteworthy that the total
antioxidant activity of the HL ethanol extract was found to be
comparable with the Vitamin E. Further studies are needed to
isolate the exact active component which are responsible for
the hypolipidaemic and antioxidant activity. In conclusion,
the use of this extract could be useful in the management car-
diovascular diseases in which atherosclerosis plays a major
role.
Acknowledgements
The authors are grateful to Prof. Dr. Aydin Ketani for
histological analysis. Generous financial support by Dicle
University Research Foundation (DUAPK, Project nos. 04-
FF-55 and 03-FF-63) is gratefully appreciated.
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