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FARMACIA, 2011, Vol. 59, 5
669
EFFECT OF BETA VULGARIS L. ON
CHOLESTEROL RICH DIET-INDUCED
HYPERCHOLESTEROLEMIA IN RATS
MOHAMMED AL-DOSARI1, SALEH ALQASOUMI1, MAJID
AHMAD2, MOHAMMED AL-YAHYA1, M. NAZAM ANSARI2, SYED
RAFATULLAH1*
1Department of Pharmacognosy and Medicinal, Aromatic & Poisonous
Plants Research Center (MAPPRC), College of Pharmacy, P.O. Box
2457, King Saud University, Riyadh 11451, Saudi Arabia
2Department of Pharmacology, College of Pharmacy, Alkharj
University, Alkharj, Saudi Arabia
∗
corresponding author: srafat@ksu.edu.sa
Abstract
The lyophilized aqueous extract of Beta vulgaris L. (beet root) (BVE) was
investigated for its possible antihypercholesterolemic and antioxidant potential in
cholesterol rich diet-induced hypercholesterolemia in Wistar albino rats. Hyper-
cholesterolemia was induced in rats by feeding 1% cholesterol rich diet for 10 weeks. Lipid
profile and glucose were estimated in serum. Malondialdehyde (MDA) and non-protein
sulfhydryls (NP-SH) levels were measured in liver and heart. Hypercholesterolemic rats
showed a significant increase in total cholesterol and triglycerides and a significant
decrease in high-density lipoprotein-cholesterol (HDL-C) levels. BVE at the doses of 250
and 500 mg/kg body weight for 70 consecutive days showed a significant decrease in total
cholesterol and triglycerides and significant increase in HDL-C. Furthermore,
hypercholesterolemic rats showed free radical generation (lipid peroxidation), evident by a
significant increase in MDA level and a significant reduction in NP-SH content in both
liver and heart homogenates. BVE treatment significantly decreased MDA level and
significantly replenished the reduced NP-SH content in both liver and heart tissue. The
acute toxicity test of BVE showed no mortality or morbidity in rats. The findings indicate
that BVE has a significant antihypercholesterolemic and antioxidant potential and/or free
radical scavenging properties in hypercholesterolemic, rats possibly exerted by the
phytoconstituents present in the beet root.
Rezumat
Studiul experimental evaluează acţiunea antihipercolesterolemiantă şi
antioxidantă a extractului apos liofilizat al rădăcinii plantei Beta vulgaris (Chenopodiaceae).
Studiul a fost realizat pe şobolani albi de laborator, cărora li s-a indus experimental
hipercolesterolemia. A fost evaluat profilul lipidic şi glucidic al animalelor, concentraţia
serică a malonildialdehidei. De asemenea, au fost evaluate (ĭn ţesutul hepatic şi cardiac)
grupările sulfhidril non-proteice. Rezultatele obţinute indică proprietăţile antihiper-
colesterolemiante şi antioxidante, datorate fitoconstituenţilor prezenţi în rădăcina plantei
studiate.
Keywords: Beta vulgaris, beet root, hypercholesterolemia, lipid profile, antioxidant
FARMACIA, 2011, Vol. 59, 5
670
Introduction
Vegetables are edible plants or part of the plants and they may be
aromatic, bitter or tasteless. The nutrients content of different types of
vegetables vary considerably and they do not represent a major source of
carbohydrates compared to starchy foods which form the bulk of food eaten,
but contain vitamins, essential amino acids as well as minerals and
antioxidants.
Recent findings indicated that some of the vegetables and herbs, in
addition to their lipid-lowering ability, can also reduce the production of
reactive oxygen species (ROS) and increase the resistance of plasma
lipoprotein to oxidation that may contribute to their effectiveness in
preventing atherosclerotic disease [18,21,26]. Hypercholesterolemia is a
well known risk factor in the development of atherosclerosis and subsequent
coronary heart disease (CHD). Cardiovascular diseases represent the
primary cause of mortality in the United States, Europe and most parts of
Asia [2,17]. There are strong evidences that hypercholesterolemia increases
the production of ROS [10,24], which may play an important role in the
pathogenesis and/or progression of cardiovascular diseases [8,35].
Beta vulgaris L. (Chenopodiaceae), popularly known as Beet root, a
native of the coasts of Mediterranean, is extensively cultivated in Europe,
America and many parts of Asia. It has been used for centuries as a
traditional natural coloring agent in many cuisines. Medicinally, the roots
and leaves of the beet have been employed as a folk remedy to treat a wide
variety of ailments including immune system stimulation, liver and kidney
diseases. It is also employed as a special diet in the treatment of cancer [5].
The seeds are cooling and diaphoretic and the root is a nutrient [6]. In a
preliminary study, aqueous and ethanolic extracts of Beta vulgaris have
been reported to possess free radical-scavenging activity, reducing the
radical cations and phase II enzyme-inducing activities in murine hepatoma
cell in vitro [23]. Beet root extract has also been reported to be one of the
useful means to prevent lung and skin cancers [16]. Furthermore, it was
reported that the phenolic amides isolated from the seeds of Beta vulgaris
produce the inhibitory effect on lipopolysaccharide-induced nitric oxide
production in experimental isolated tissues in a dose dependent manner [32].
The present study was designed to assess whether the BVE could
exert any protective action against cholesterol rich diet-induced
hypercholesterolemia in rats, in order to substantiate the claims of its
folkloric use to reduce cholesterol level.
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Materials and Methods
Plant Material and Preparation of Dosage Form
The fresh roots of Beta vulgaris used in this study were purchased
from the local vegetable market of Riyadh, and identified by an experienced
taxonomist. A voucher (#210309) specimen was deposited in the Medicinal,
Aromatic and Poisonous Plants Research Center of the College of Pharmacy
from King Saud University, for future reference.
The roots of Beta vulgaris were processed in order to obtain juice
using an electric blender. After obtaining the juice, it was lyophilized to get
the dry powder using Freeze Dry System (LABCONCO, England). The
freeze-dried powder was then dissolved in distilled water and used in all
experiments.
Phytochemical Screening
A preliminary phytochemical analysis of the Beet root was
conducted for the detection of alkaloids, cardiac glycosides, flavonoids,
tannins, anthraquinones, saponins, volatile oils, cyanogenic glycosides,
coumarins, sterols and/or triterpenes [9].
Acute Toxicity Test
The acute toxicity of the BVE was evaluated in mice using the up
and down procedure [30]. Six female rats (weight: 200-250g) received BVE
starting at 2g/kg b.w. orally by gavage. The animals were observed for toxic
symptoms continuously for the first 4 h after dosing. Finally, the number of
survivors was noted after 24 h and these animals were further maintained
for 13 days under daily observations.
Animals and diet
Healthy male adult Wistar albino rats, weighing between 150–200 g,
obtained from the Experimental Animal Care Centre, College of Pharmacy,
King Saud University, Riyadh, were used. They were housed in
polyethylene cages in groups of six rats per cage and were kept at a constant
temperature (22±2°C), humidity (55%) and 12 h light-dark conditions for 7
days. The animals were provided with Purina chow rat diet and free access
to drinking water. The experiments and the procedure of sacrifice (using
ether) were approved by the Ethics Committee of the Experimental Animal
Care Society, College of Pharmacy, King Saud University, Riyadh, Saudi
Arabia.
FARMACIA, 2011, Vol. 59, 5
672
Cholesterol supplemented feed
In crushed pellet diet, cholesterol (1%w/w) powder was thoroughly
mixed; the pellets were reconstituted with water and dried properly to avoid
any fungal contamination.
Experimental design
A systematic study was performed on the adult male rats divided in
five groups. Each group comprised 6 animals.
Group 1: Control rats fed with normal pellet diet.
Group 2: Rats fed with cholesterol mixed pellet diet.
Group 3: Rats fed with cholesterol mixed pellet diet plus BVE (250
mg/kg b.w. p.o./day).
Group 4: Rats fed with cholesterol mixed pellet diet plus BVE (500
mg/kg b.w. p.o./day).
Group 5: Rats fed with normal pellet diet along with BVE (500 mg/kg
b.w., p.o./day).
Biochemical determinations
Estimation of the lipid profile and glucose
Blood samples were collected from overnight fasted rats. Serum
total cholesterol, triglycerides and high-density lipoprotein-cholesterol
(HDL-C) levels were determined by commercially available
spectrophotometric assay kits (Crescent Diagnostics, Jeddah, Saudi Arabia).
The serum glucose was estimated using Reflotron® Plus Analyzer and
Roche kits.
Determination of malondialdehyde (MDA)
The method reported by Utley et al [31] was followed. The heart
and liver tissues were removed and each tissue was homogenized in 0.15 M
KCl (at 4°C; Potter-Elvehjem type C homogenizer) to give a 10% w/v
homogenate. Aliquots of homogenate (1 mL) were incubated at 37°C for 3 h
in a metabolic shaker. Then 1 mL of 10% aqueous trichloroacetic acid was
added and mixed. The mixture was then centrifuged at 800 g for 10 min.
One mL of the supernatant was removed and mixed with 1 mL of 0.67%
thiobarbituric acid in water and placed in a boiling water bath for 10 min.
The mixture was cooled and diluted with 1 mL distilled water. The
absorbance of the solution was then read at 535 nm. The content of
malondialdehyde (nmoles/g wet tissue) was then calculated, by reference to
a standard curve of malondialdehyde solution.
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673
Estimation of non-protein sulfhydryls (NP-SH)
Non-protein sulfhydryls were measured according to the method of
Sedlak and Lindsay [29]. The heart and liver tissues were homogenized in
ice-cold 0.02 mmol/L ethylene diaminetetraacetic acid (EDTA). Aliquots of
5 mL of the homogenates were mixed in 15 mL test tubes with 4 mL of
distilled water and 1 mL of 50% trichloroacetic acid (TCA). The tubes were
shaken intermittently for 10 min and centrifuged at 3000 rpm. Two
milliliters of supernatant were mixed with 4 mL of 0.4 mol/L Tris buffer
(pH 8.9). 0.1 mL of 5, 5’-dithio-bis (2-nitrobenzoic acid) (DTNB) was
added and the sample was shaken. The absorbance was measured within 5
min after the addition of DTNB at 412 nm against a reagent blank.
Statistical Analysis
Values are given as arithmetic means ± standard error of the mean
(S.E.M.). Data was statistically analyzed by using One-way analysis of
variance (ANOVA) followed by Dunnett's multiple comparison test.
Results and Discussion
Phytochemical Screening
The preliminary qualitative phytochemical screening of the root of Beta
vulgaris revealed the presence of flavonoids, saponins, sterols and/or
triterpenes.
Acute toxicity test
The extract at the dose of 2 g/kg b.w. was found to be safe, as the
mice did not show any symptoms of toxicity and mortality during a period
of 14 days of observation.
Effect of BVE on serum lipid profile:
Rats fed with cholesterol rich diet developed hypercholesterolemia
and hyperlipidemia significantly by increasing total cholesterol,
triglycerides levels, and a significant decrease in HDL-C levels as compared
with control rats. Treatment with BVE (250 and 500 mg/kg b.w.) along with
the cholesterol rich diet showed a significant decrease in total cholesterol,
triglycerides, and a significant increase in the level of HDL-C compared
with hypercholesterolemic group. The results are presented in table I.
However, rats treated with BVE (500 mg/kg b.w.) alone and maintained on
FARMACIA, 2011, Vol. 59, 5
674
normal diet (per se) did not show any change in serum lipid profile
compared with the control group.
Table I.
Effect of BVE on serum lipid profile and glucose
Groups
Glucose
(mg/dL)
Cholesterol
(mg/dL)
Triglycerides
(mg/dL)
HDL-C
(mg/dL)
Group 1: Control
72.53 ± 5.61
86.06 ± 2.92
68.33 ± 2.47
37.77 ± 0.36
Group 2: 1% Cholesterol only
87.67 ± 6.92
174.55 ± 3.87 a
163.33 ± 2.79 a
12.79 ± 0.67 a
Group 3: BVE (250 mg/kg
b.w.) + 1% Cholesterol
76.02 ± 4.67
129.09 ± 5.53*
122.50 ± 2.50*
20.41 ± 0.44*
Group 4: BVE (500 mg/kg b.w.)
+ 1% Cholesterol
70.38 ± 2.98
127.27 ± 11.42*
113.33 ± 5.80*
21.42 ± 0.34*
Group 5: BVE (500 mg/kg b.w.)
69.32 ± 3.63
80.60 ± 4.14
64.17 ± 2.63
38.99 ± 0.53
All values represent mean±SEM.
a p <0.05 (ANOVA, followed by Dunnett's multiple comparison test) as compared with
normal control group.
* p <0.05 (ANOVA, followed by Dunnett's multiple comparison test) as compared with group
fed with 1% cholesterol only.
Effect of BVE on serum glucose:
In table I, rats fed with cholesterol rich diet did not show any
significant change in serum glucose levels as compared to control rats. Also,
no significant changes were recorded in serum glucose levels in treatment
groups (BVE 250 and 500 mg/kg b.w.) and normal diet group (per se)
compared with hypercholesterolemic group.
Effect of BVE on lipid peroxidation in liver and heart tissue:
Rats fed with cholesterol rich diet showed a significant increase
(p<0.05) in liver and heart MDA level compared to control rats.
Treatment with BVE at both doses (250 and 500 mg/kg b.w.) along
with the cholesterol feeding showed a significant (p<0.05) decrease in
malondialdehyde level. However, rats treated with BVE (500 mg/kg b.w.)
and maintained on normal diet (per se) did not show any change in liver and
heart MDA level compared with the control group (Table II).
Effect of BVE on NP-SH levels in liver and heart tissue:
Rats fed with cholesterol rich diet showed a significant decrease
(p<0.05) in liver and heart NP-SH content compared to control rats.
Treatment with BVE (250 and 500 mg/kg b.w.) along with the
cholesterol feeding showed a significant increase in liver and heart NP-SH
content compared with hypercholesterolemic rats (Table III). However, rats
treated with BVE (500 mg/kg b.w.) and maintained on normal diet (per se)
did not show any change in liver and heart NP-SH levels as compared with
the control group.
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Table II
Effect of BVE administration on MDA levels in liver and heart homogenates
Groups
MDA (nmols MDA/g wet tissue)
Liver
Heart
Control
1.87 ± 0.067
1.63 ± 0.043
1% Cholesterol only
5.71 ± 0.48 a
5.41 ± 0.63 a
BVE (250 mg/kg b.w.) +
1% Cholesterol
2.95 ± 0.21*
2.80 ± 0.17*
BVE (500 mg/kg b.w.) +
1% Cholesterol
2.72 ± 0.059*
2.52 ± 0.05*
BVE (500 mg/kg b.w.)
1.88 ± 0.094
1.72 ± 0.05
All values represent mean±SEM.
a p <0.05 (ANOVA, followed by Dunnett's multiple comparison test) as compared with
normal control group.
* p <0.05 (ANOVA, followed by Dunnett's multiple comparison test) as compared with group
fed with 1% cholesterol only.
Table III
Effect of BVE administration on NP-SH levels in liver and heart homogenates
Groups
NP-SH (nmols/g wet tissue)
Liver
Heart
Control
10.86 ± 0.26
9.38 ± 0.33
1% Cholesterol only
3.75 ± 0.18 a
4.44 ± 0.18 a
BVE (250 mg/kg b.w.) +
1% Cholesterol
8.11 ± 0.18*
7.58 ± 0.28*
BVE (500 mg/kg b.w.) +
1% Cholesterol
8.58 ± 0.30*
8.07 ± 0.25*
BVE (500 mg/kg b.w.)
9.68 ± 0.50
9.69 ± 0.61
All values represent mean±SEM.
a p <0.05 (ANOVA, followed by Dunnett's multiple comparison test) as compared with
normal control group.
* p <0.05 (ANOVA, followed by Dunnett's multiple comparison test) as compared with group
fed with 1% cholesterol only.
Conclusions
The present study examined the possible antihypercholesterolemic,
antihyperlipidemic and antioxidant potential of lyophilized aqueous Beta
vulgaris extract (250 and 500 mg/kg b.w., p.o.) in cholesterol rich diet-
induced hypercholesterolemia in rats. Rats fed with the diet rich in
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cholesterol resulted in an increase of total cholesterol and triglycerides in
serum and decreased circulating HDL-C in rats, besides an increase in
malondialdehyde and decreased non-protein sulfhydryl content in liver and
heart. These results are in agreement with earlier studies [1,4], and provide
an experimental model for dietary hyperlipedemia [12].
Higher plasma LDL-C level is related with greater deposition of
cholesterol in artery and aorta thereby increasing risk for coronary artery
disease[25], whereas low HDL-C is the prevalent lipoprotein abnormality
reported [13,14]. In the current investigation BVE treatment decreased the
levels of total cholesterol and triglycerides and increased the levels of HDL-
C suggesting a cardioprotective and lipid lowering potential of Beta
vulgaris. This lipid lowering potential of beet root may be due to flavanoids
and/or saponins which were found to be the main constituents of BVE in our
preliminary phytochemical screening. These findings are in accordance with
the earlier studies demonstrating the effect of flavonoids on cholesterol
metabolism [3, 15]. It has also been reported that saponins from some
medicinal plants reduced the triglycerides and cholesterol levels in rats [15].
Also, flavonoids are considered as active principles in many medicinal
plants [34] and natural products with positive effect on human health [7]
and saponins content has been also suggested to reduce heart diseases [20].
Some of the earlier studies have indicated that hypercholesterolemia
induces oxidative stress by causing a reduction in the enzymatic antioxidant
defense potential of tissues and generation of oxygen free radical like
superoxide anions leading to the development of cardiovascular and
neurodegenerative diseases [10,22,24,28]. High-cholesterol diet provides a
relevant example of endogenous chronic oxidative stress due to the resulting
hypercholesterolemia. In hypercholesterolemic diet, liver, the primary organ
that metabolises the cholesterol ingested in excess, is affected by oxidative
stress. It results from an imbalance between the production of free radicals
and the effectiveness of antioxidant defense system [19]. The present study
confirms the efficiency of cholesterol-enriched diet to produce a state of
oxidative stress with biochemical and biological characteristics of
hypercholesterolemia.
In the current investigation, elevated MDA levels were decreased
and reduced NP-SH levels were replenished significantly in liver and heart
by the treatment with BVE, thereby, enhancing the endogenous hepatic and
myocardial antioxidant levels. These findings are in accordance with the
earlier studies suggesting the antioxidant potential of Beta vulgaris [23,32].
The recorded increment in HDL-C, increased antioxidant activity
and reduced lipid accumulation in hypercholesterolemic animals suggest the
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usefulness of BVE in the treatment of hyperlipidemia. Also, synthetic
hypercholesterolemic drugs lower both the total cholesterol and HDL-C,
simultaneously [33]; thus BVE could prove to be a more effective therapy
due to its ability to significantly increase HDL-C while lowering total
cholesterol. The present study provides a preliminary scientific basis for
hypolipidemic effects of Beta vulgaris, a plant that has been extensively
used ina folkloric medicine. Further studies are however required to reveal
the possible molecular mechanism(s) of action.
Acknowledgements
Authors are thankful to Mr. Malik Sawood Ahmed for his technical
assistance.
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Manuscript received: April 4th 2010
