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Research Article
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Rosmarinic Acid Prevents the Oxidation of Low Density Lipoprotein (LDL) In vitro
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Hassan Ahmadvand,
Ali Khosrobeigi,
Leila Nemati,
Maryam Boshtam,
Narges Jafari,
Reza Haji Hosseini,
Yadollah Pournia,
Hassan Ahmadvand,
Ali Khosrobeigi,
Leila Nemati,
Maryam Boshtam,
Narges Jafari,
Reza Haji Hosseini
and
Yadollah Pournia
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ABSTRACT
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The objective of the present study was to assess various antioxidative
activities of Rosmarinic Acid (RA) and its effect on oxidation of Low Density
Lipoprotein (LDL) induced by CuSO4 in vitro. It was demonstrated
that RA was able to inhibit LDL oxidation and decrease the resistance of LDL
against oxidation. Rosmarinic acid showed a decrease the formation of malondialdehyde
(MDA) rate of 31.8, 36.7 and 50.3% at concentrations ranging from 100- 400 μM,
respectively, against oxidation in vitro. The inhibitory effects of
the RA on LDL oxidation were dose-dependent. Total antioxidant capacity of RA
was 0.65±0.03 mmol of ascorbic acid equivalents/mmol RA. The RA showed
remarkable scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC50
0.06±0.004 μM). This study showed that RA is a potent antioxidant
and prevents the oxidation of LDL in vitro and it may be suitable for
use in food and pharmaceutical applications.
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How
to cite this article:
Hassan Ahmadvand, Ali Khosrobeigi, Leila Nemati, Maryam Boshtam, Narges Jafari, Reza Haji Hosseini, Yadollah Pournia, Hassan Ahmadvand, Ali Khosrobeigi, Leila Nemati, Maryam Boshtam, Narges Jafari, Reza Haji Hosseini and Yadollah Pournia, 2012. Rosmarinic Acid Prevents the Oxidation of Low Density Lipoprotein (LDL) In vitro. Journal of Biological Sciences, 12: 301-307. DOI: 10.3923/jbs.2012.301.307 URL: https://scialert.net/abstract/?doi=jbs.2012.301.307
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Received: December 29, 2011;
Accepted: July 03, 2012;
Published: September 06, 2012
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INTRODUCTION
Cardiovascular disease is one of the leading causes of mortality in our society.
The increasing concentration of plasma Low Density Lipoprotein (LDL) is a major
risk factor in this regard, the underlying mechanisms remain unclear and need
more investigations. To date, considerable evidence supports a role for oxidatively
modified LDL in the pathogenesis of atherosclerosis (Holvoet
and Collen, 1998; Steinberg, 1997). The uptake of
oxidized LDL (Ox-LDL) by macrophages results in the formation of foam cells
and cellular cholesterol accumulated in vascular endothelial cells and promotes
the development of the characteristic fatty streaks found in atherosclerotic
lesions (Ani et al., 2007; Yoshida
and Kisugi, 2010). Some diseases, such as cancer, cardiovascular diseases,
diabetes, neurological disorders arthritis and inflammations are related to
the imbalance of oxidants and antioxidants (Momtaz and Abdollahi,
2010). There is increasing interest in research of natural antioxidant products
for use as medicines and food additives (Annegowda et
al., 2010). Vitamin C, vitamin E and carotenoids are some of these widely
used natural antioxidants. Antioxidants played an important role in lowering
oxidative stresses caused by Reactive Oxygen Species (ROS). ROS including hydroxyl
radical, nitroxides, superoxide anion radical and hydrogen peroxide are generated
under physiological and pathological stresses in human body (Afonso
et al., 2007). Rosmarinic acid (α-O-caffeoyl-3, 4-dihydroxyphenyl
lactic acid; RA) is a naturally water-soluble polyphenolic compound. Rosmarinic
acid has antioxidant (Del Bano et al., 2003;
Hossain et al., 2009), antimicrobial activities
(Bernardes et al., 2010), anti-inflammatory (Swarup
et al., 2007), antiangiogenic (Furtado et
al., 2010), antitumor (Osakabe et al., 2004)
and HIV-1-inhibiting properties (Dubois et al., 2008).
Rosmarinic acid is also used for food preservation. There is considerable experimental
evidence to show that several different antioxidant compounds given at high
pharmacological doses are effective in decreasing both LDL oxidation and atherogenesis
in animals (Li et al., 2010). In humans, supplementation
with antioxidants combined at physiological doses is incapable of inhibiting
coronary heart disease in primary prevention (Li et al.,
2007). Antioxidants would have to be given at high pharmacological doses
in humans to inhibit ex vivo Cu2+-induced LDL oxidation (Seo
et al., 2010). Since, the inhibitory effects of RA on LDL oxidation
have not previously been reported, the objectives of the present study were
to assess various antioxidative activities of RA and investigate the effect
of RA on the oxidation of LDL induced by CuSO4 in vitro by
monitoring the formation of conjugated dienes, the formation of Thiobarbituric
Acid Reactive Substances (TBARS).
MATERIALS AND METHODS
Materials: Disodium ethylene diamine tetra acetate (Na2EDTA),
potassium bromide (KBr) , sodium chloride (NaCl), disodium hydrogen phosphate
(Na2HPO4), 1,1-diphenyl-2-picrylhydrazyl (DPPH), trichloroacetic
acid, ferric chloride, sodium acetate, 2,6-di-tert-butyl-4-methyl phenol (BHT),
rosmarinic acid, G, ascorbic acid were purchased from Sigma-Aldrich (St. Louis,
MO, USA). All the solvents used were of analytical grade. The 2-thiobarbituric
acid (TBA) was obtained from Fluka Chemie (Buchs SG, Switzerland).
Blood sampling: Blood samples were taken from ten men. The protocols
for the blood sampling were approved by the Medical University of Lorestan Ethics
Committee and all the informed constants were taken from all the men. Fasting
blood samples after an overnight fasting were collected in EDTA containing tubes
(1.6 mg EDTA mL blood). To obtain fresh plasma, the blood samples were centrifuged
(3000 rpm for 10 min at 4°C) as soon as the samples were collected to avoid
auto-oxidation. To minimize oxidation in vitro, sodium azide (0.06%,
w/v) was added to plasma samples immediately after separation.
Isolation of LDL: The LDL fraction was isolated from fresh plasma by
single vertical discontinuous density gradient ultracentrifugation (Ani
et al., 2007; Crawford et al., 1999).
The density of the plasma was adjusted to 1.21 g mL-1 by the addition
of solid KBr (0.365 g mL-1). Centrifuge tubes were loaded by layering
1.5 mL of density-adjusted plasma under 3.5 mL of 0.154 mol L-1 NaCl
and centrifuged in a Beckman L7-55 ultracentrifuge at 40000 rpm at 10°C
for 2.5 h. The isolated LDL was dialyzed for 48 h at 4°C against three changes
of deoxygenated-PBS (0.01 mol L-1 Na2HPO4,
0.16 mol L-1 NaCl, pH 7.4).
DPPH free radical-scavenging activity: DPPH free radical-scavenging
activity of the test samples was determined according to the method of Blois
(1958). In brief, 4 mL of DPPH radical solution in ethanol (1 mM) was mixed
with 1 mL of RA solution in ethanol containing 0.01-3000 μM of RA and after
30 min, the absorbance was measured at 517 nm. This activity was given as percentage
DPPH scavenging that is calculated as:
The 50% inhibition concentration (IC50), i.e., the concentration
of RA that was required to scavenge 50% of radicals, was calculated. All samples
were analyzed in triplicate.
Total antioxidant activity: Total antioxidant activity of the test samples
was determined (Prieto et al., 1999; Hou
et al., 2011). In brief, 0.3 mL of sample was mixed with 3.0 mL of
reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium
molybdate). Reaction mixture was incubated at 95°C for 90 min under water
bath. Absorbance of the samples was measured at 695 nm. The total antioxidant
activity was expressed as the number of equivalents of ascorbic acid (μmol
g-1).
Oxidation of LDL
Continuous monitoring of formation of conjugated dienes in LDL: After isolation
of total LDL, the protein content of LDL was measured (Bradford,
1976). LDL was adjusted to 150 μg mL-1 of LDL protein with
10 mM PBS, pH 7.4 and then aliquots of RA were added to the solution. The oxidative
modification of LDL was initiated by addition of freshly prepared 10 μM
CuSO4 solution at 37°C in a water bath for 5 h. The kinetics
of LDL oxidation was monitored every 10 min by measuring its absorbance at 234
nm. The lag phase was calculated from the oxidation profile of each LDL preparation
by drawing a tangent to the slope of the propagation phase and extrapolation
into intercept the initial-absorbance axis.
The lag phase represented the length of the antioxidant-protected phase during
LDL oxidation by RA in vitro. The lag time was measured as the time period
until the conjugated dienes began to increase (Navder et
al., 1999). The formation of conjugated dienes was calculated as conjugated
dienes equivalent content (nmol mg-1-protein) at 5 h. The conjugated
dienes concentration was calculated by using the extinction coefficient for
diene conjugates at 234 nm (29500 M-1 cm-1).
Assay of the formation of thiobarbituric acid reactive substances (TBARS):
Lipid peroxidation end products were determined as TBARS according to modified
method of Buege and Aust. After initiating the oxidation process with CuSO4,
the sample mixtures were incubated at 37°C for 5 h in a water bath and the
reaction was terminated by adding EDTA (2 mM). TBARS formation was measured
in a spectrophotometer at 532 nm. The results were recorded as malondialdehyde
(MDA) equivalent content (nmol/mg LDL-protein) (1.56x105 M-1
cm-1) (Sheu et al., 2003).
Statistical analysis: The data were presented as mean±SD of three
experiments performed in duplicate. The variables used to describe the difference
between the oxidation curves were lag time, conjugated dienes and MDA. These
parameters were obtained using the Mann-Whitney test (using SPSS 13.0 statistical
software) for independent data and the differences were considered significant
when p<0.05.
RESULTS AND DISCUSSION
Antioxidant activity
DPPH scavenging assay: Researchers are recently interested in investigation
and research into extraction of natural antioxidants from medical herbs to replace
synthetic antioxidants. Natural antioxidants are healter and more beneficial
and have fewer side effects than synthetic antioxidants (Rached
et al., 2010; Prasong, 2011; Olajuyigbe
and Afolayan, 2011) Phytochemicals with antioxidant effects include some
cinnamic acids, rosmarinic acid, flavonoids, lignans, monoterpenes, phenylpropanoids,
tannins and triterpenes (Soobrattee et al., 2005;
Shahbudin et al., 2011). Therefore, natural antioxidants
such as rosmarinic acid are taken into consideration in order to inhibit diseases
related to oxidative stress such as coronary heart disease, nephrotoxicity and
diabetes mellitus (Tavafi et al., 2011; Tavafi
and Ahmadvand, 2011; Hasani-Ranjbar et al., 2010).
RA has a number of interesting biological activities, including antiviral, antibacterial,
anti-inflammatory, anti allergic and antioxidant effects. Plant extracts containing
RA also have excellent potential as antioxidants for food preservation (Sanbongi
et al., 2004). Also Rosmarinic acid helps to reduce the risk of cancer
and atherosclerosis. Another study demonstrated that rosmarinic acid is suitable
for the treatment of rheumatoid arthritis (Al-Sereiti et
al., 1999; Hur et al., 2007). Conducting
research on natural antioxidants and evaluating and comparing their antioxidant
effects, as well as newer and more valuable sources of natural antioxidants
can be found and used in special cases.
A stable free radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH) has widely been
used in the assessment of radical scavenging activity of plant extracts, natural
compounds and foods (Tarawneh et al., 2010; Eleazu
et al., 2011; Ramirez-Mares et al., 2010).
The antioxidant activity of RA was evaluated by the DPPH radical scavenging
capacity. Fig. 1 shows the percentage of DPPH radicals scavenging
capacity with BHT as reference. In the DPPH scavenging assay, the IC50
(the concentration required to scavenge 50% of radical) values of RA and BHT
were 0.5±0.03 and 0.06±0.004 μM, respectively. The data obtained
show that RA is a free radical scavenger and may act as a primary antioxidant
which can react with free radicals by donating hydrogen.
Total antioxidant activity: The phosphomolybdenum method has been widely
used in the assessment of total antioxidant activity of plant extracts, natural
compounds and foods. Fig. 2 shows the total antioxidant activity
of ascorbic acid as standard. The total Antioxidant capacity of RA was 0.65±0.03
mmol of ascorbic acid equivalents mmol-1 RA. Our results indicated
that RA is found to posses a good antioxidant activity.
Oxidation of LDL
Continues monitoring of formation of conjugated dienes in LDL and kinetics
of CuSO4-induced LDL oxidation: Recently, it has become widely
accepted that diet may play an important role in health promotion and disease
prevention. Objective data are required by consumers and health care professionals
to improve daily diets and, consequently, reduce the risk of chronic diseases
such as Coronary Heart Disease (CHD) (Noroozi et al.,
2009).
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Fig. 1: |
Free radical scavenging capacities of the RA and BHT measured
in DPPH assay. Each point represents the mean of five experiments |
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Fig. 3: |
The effects of RA on LDL oxidation in 10 mM PBS, pH 7.4 at
37°C for 5 h. (C) n-LDL, (Cu) n-LDL+copper, (RA1) n-LDL+RA (100 μM),
(RA2) n-LDL+RA (200 μM), (RA3) n-LDL+RA (400 μM) and (E1) n-LDL+vitamin
E(100 μM). Each point represents the mean of five experiments |
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Fig. 4: |
The effects of RA on the formation of conjugated dienes of
LDL oxidation. Each value is the mean of five experiments. *,#p<0.005,
as compared with Cu and RA1 by Mann-Whitney test. (C) n-LDL, (Cu) n-LDL+copper,
(RA1) n-LDL+RA (100 μM), (RA2) n-LDL+RA (200 μM), (RA3) n-LDL+RA
(400 μM) and (E1) n-LDL+vitamin E (100 μM) |
Lipid peroxidation is one of principal factors in causing atherosclerosis (Steinberg,
1997). Oxidized LDL is atherogenic, it causes arterial cell death, accumulation
of growth factors and cytokine release. In addition, oxidized LDL contributes
to platelet aggregation, smooth muscle cell proliferation and LDL oxidation
was shown in patients with hypercholesterolemia, hypertension, diabetes mellitus,
chronic renal failure and in smokers (Steinberg, 1997).
Thus, the consumption of natural antioxidants is beneficial in preventing atherosclerosis.
The effects of vitamin E and dose-gradient concentration of RA on the kinetics
of CuSO4-induced LDL oxidation are shown in Fig. 3.
It shows that CuSO4 dramatically increased oxidation of LDL. The
formation of conjugated dienes, a marker of LDL oxidation, decreased by vitamin
E and RA. Conjugated Diene (CD) formation serves as a marker of the oxidation
process (Chikezie, 2011).
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Fig. 5: |
The effects of RA on lag time of CuSO4-induced
LDL oxidation, Each point represents the mean of five experiments *,#p<0.005,
as compared with Cu and RA1 by Mann-Whitney test. (C) n-LDL, (Cu) n-LDL+copper,
(RA1) n-LDL+RA (100 μM), (RA2) n-LDL+RA (200 μM), (RA3) n-LDL+RA
(400 μM) and (E1) n-LDL+vitamin E(100 μM) |
Figure 4 shows the levels of conjugated dienes at 5 h in
all the experimental groups. CuSO4 increased the level of the conjugated
dienes in LDL about six-fold and was significantly different from the control
LDL. Vitamin E and RA (100, 200, 400 μM) inhibited the final levels of
conjugated dienes in LDL oxidation (p<0.001). RA showed a dose-dependent
inhibition in decreasing of conjugated dienes at 5 h. 100, 200 and 400 μM
concentrations. Figure 5 shows the levels of lag time in all
the experimental groups. In the assay, various concentrations of RA were confirmed
to have a dose-dependent antioxidant activity by increasing lag time. 62.5%
increased lag time by RA at concentration of 100 μM. At 200 and 400 μM,
RA showed an increase rate of 125 and 175%. Vitamin E, as the positive control,
at concentrations of 100 μM, 100% increased lag time, respectively. So
RA has highly strong resistance on peroxidation. RA can provide hydroxyl to
accept electrons and scavenge OH induced by CuSO4. The results are
considered to be noteworthy when compared to the findings of other studies concerning
Antioxidant (Tepe and Sokmen, 2007). Result showed that
antioxidant activity of rosmarinic acid is stronger than vitamin E.
The formation of malondialdehyde (MDA) assay: TBARS is a secondary products
from lipid peroxidation in LDL. TBARS analysis measures the formation of secondary
products of lipid oxidation, mainly malondialdehyde, which may contribute off-flavour
to oxidized oil (Mudron et al., 2007). The antioxidative
effect of RA on LDL was determined and expressed by measurement of MDA equivalent
content. The levels of MDA after 5 h of incubation in all experiment groups
are shown in Fig. 6. Vitamin E significantly inhibited MDA
formation (p<0.005). RA (100, 200, 400 μM) significantly was inhibited
the MDA production in LDL (p<0.005).
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Fig. 6: |
The effects of RA on the formation of TBARS. Each Point is
the means of five experiments |
RA showed a dose-dependent inhibition of MDA formation at 5 h. 100, 200 and
400 μM concentration. This result suggests that RA is good antioxidants
and may be used in suppressing LDL oxidation in vivo. The reduction of LDL oxidation
in vivo may delay the progress of atherosclerosis and reduce the risk
of heart diseases.
The protection of LDL by RA in a copper-induced oxidation system could be due
to both metal-chelating and radical scavenging capacity. However, the mechanism
by which the RA inhibits LDL oxidation in vitro remains unclear. Laranjinha
et al. (1994) suggested possible explanations for the protecting
effects of compounds of extracts on LDL: (i) scavenging of various radical
species in the aqueous phase, (ii) interaction with peroxyl radicals at the
LDL surface, (iii) partitioning into the LDL particle and terminating chain-reactions
of lipid peroxidation by scavenging lipid radicals and (iv) regenerating endogenous
α-tocopherol back to its active antioxidative form. The results showed
that RA is a potent antioxidant and protects LDL in plasma against oxidation.
CONCLUSION
The results of the present study clearly showed that RA is found to posses
a good antioxidant activity and various concentrations of RA have a dose-dependent
antioxidant activity against LDL oxidation by inhibiting the formation of conjugated
dienes and TBARS and increasing lag time. In conclusion, RA is a potent antioxidant
and may be a good alternative to reduce the risk of atherosclerosis and coronary
heart disease and other free radical associated health problems.
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