432 FARMACIA, 2009, Vol.

57, 4

THE EVALUATION OF ANTIOXIDANT

POTENTIAL OF VERONICA OFFICINALIS AND
ROSMARINUS OFFICINALIS EXTRACTS BY
MONITORING MALONDIALDEHIDE AND
GLUTATHIONE LEVELS IN RATS
 
BÉLA KISS1,*, DANIELA-SAVETA POPA1, GIANINA CRIŞAN2,
MARIUS BOJIŢĂ3, FELICIA LOGHIN1
1
Department of Toxicology, University of Medicine and Pharmacy “Iuliu
Haţieganu”, Faculty of Pharmacy, no. 6 Pasteur, 400349, Cluj-Napoca,
Romania
2
Department of Pharmaceutical Botany, University of Medicine and
Pharmacy “Iuliu Hatieganu”, Faculty of Pharmacy, no. 13 Emil Isac,
400023, Cluj-Napoca, Romania
3
Department of Drug Analysis, University of Medicine and Pharmacy
“Iuliu Haţieganu”, Faculty of Pharmacy, no. 6 Pasteur, 400349, Cluj-
Napoca, Romania
*corresponding author: [email protected]

Abstract
Malondialdehide and glutathione are two of the main biomarkers of oxidative
stress. These biomarkers were used to evaluate the antioxidant potential of two plant
extracts, obtained from Rosmarinus officinalis and Veronica officinalis, in Wistar rats. In
the case of malondialdehide the assay was based on derivatization with
dinitrophenylhydrazine, followed by chromatographic analysis with UV detection at
307nm. The reduced and total glutathione were quantified from rat plasma, after
derivatization with o-phtalaldehyde, using a HPLC method with fluorescence detection.
The results indicated a potential protective effect of these plants against oxidative stress.
Rezumat
Malondialdehida şi glutationul reprezintă doi biomarkeri importanţi ai stresului
oxidativ. Aceşti biomarkeri au fost utilizaţi pentru a evalua potenţialul antioxidant al
extractelor obţinute din Rosmarinus officinalis şi Veronica officinalis, la şobolani Wistar. În
cazul malondialdehidei, metoda de dozare s-a bazat pe derivatizarea cu
dinitrofenilhidrazină, urmată de analiza cromatografică cu detecţie în UV la 307nm.
Cuantificarea glutationului redus şi total din plasma de şobolan s-a realizat printr-o metodă
HPLC cu detecţie în fluorescenţă, după o prealabilă derivatizare cu o-ftalaldehida.
Rezultatele obţinute au indicat un potenţial efect protector al plantelor testate faţă de stresul
oxidativ.
Keywords: Rosmarinus officinalis, Veronica officinalis, malondialdehide,
glutathione, oxidative stress, chromatography

 
FARMACIA, 2009, Vol. 57, 4 433

Introduction
Oxidative stress is due to some very reactive species, mainly
oxygen radical derivatives and peroxides. This process becomes obvious
when the antioxidant defense mechanisms of the cell are overwhelmed by
the levels of free radicals. Reactive free radicals can oxidize biomolecules,
such as proteins, lipids and nucleic acids. It is well known the involvement
of the oxidative stress in the pathogenesis of multiple diseases (e.g.
neurodegenerative, cardiovascular, inflammatory diseases) [3-5,7].
There are many cases when a disease is treated through the
modulation of the antioxidants level or by using drugs with antioxidant
activity. For this reason, there is a great need to identify also plant materials
which may develop antioxidant potential.
The plants chosen to be tested in this work were Veronica officinalis
(Lamiaceae) and Rosmarinus officinalis (Scrophulariaceae). These species
were selected based on their content in some compounds with antioxidant
potential, such as flavonoids and rosmarinic acid [1]. Flavonoids are phenolic
components, present in many plant species, which have beneficial effects in
preventing some cardiovascular and inflammatory diseases [6, 10-12]. In
order to perform the tests on animals, alcoholic extracts were prepared from
Veronica officinalis leaves and Rosmarinus officinalis herba.
The influence of these extracts on oxidative stress can be evaluated by
monitoring the specific biomarkers of radical induced damage and toxicity [2].
In previous papers we presented two chromatographic methods for
the quantification in rat microsomial preparations of malondialdehyde and
glutathione as markers of oxidative stress [8, 9].
The aim of this work was to evaluate the antioxidant effect of these
two plant extracts, through the quantification of malondialdehyde (MDA, a
lipid peroxidation end-product) and glutathione (GSH, a very potent
endogenous protective agent against oxidative stress) in rat plasma samples
exposed to carbon tetrachloride, an agent capable of inducing oxidative
stress. In order to perform these tests, we made some changes in the assays
for MDA and GSH from microsomes, in order to quantify the two
biomarkers, from plasma samples [8, 9].

Materials and methods

Chemicals
1,1,3,3-tetraetoxipropan (TEP) were purchased from Sigma-
Aldrich (Sigma-Aldrich, Steinheim, Germany). HPLC grade reduced and

 
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oxidized glutathione, were obtained from Fluka (Fluka, Buchs SG,

Switzerland). HPLC grade acetonitrile, methanol, formic acid, acetic acid,
acetone and hexane were purchased from Merck (Merck, Darmstadt,
Germany). All other chemicals were analytical reagent grade and they were
obtained from Merck (sodium hydroxide, hydrochloric acid, sulfuric acid,
perchloric acid, 2,4-dinitrophenylhydrazine (DNPH), sodium tetraborate
decahydrate, o-phtalaldehyde, acetaldehyde, TRIS hydrochloride (Tris
hydroxymethyl aminomethane hydrochloride) and 1,4-dithiothreitol (DTT)).
Deionised water was obtained using a Milli-Q water purification
system (Millipore, Milford, MA, USA).
Animals
Male Wistar rats (mean body weight of 200g) were obtained from
the Animal Breeding Station of University of Medicine and Pharmacy Cluj-
Napoca. During the experiment, the animals were housed in standard
conditions and were allowed free access to standard food and water.
Four groups of animals were used, three groups of 30 rats each
(Rosmarinus, Veronica and positive control group) and a group composed
of six rats (negative control). The plant extracts were standardized in active
compounds. The total flavonoid content of Veronica officinalis extract was
of 2.95 mg/mL, expressed as rutoside and the content of phenyl propane
derivatives was of 170 mg/mL, expressed as caffeic acid. Rosmarinus
officinalis extract contained 77.89 µg/mL rosmarinic acid and 28 mg/mL
phenyl propane derivatives, expressed as caffeic acid.
The plant extracts and the other substances were administered
according to the following protocol:
• For 7 days, alcoholic extract (1part plant material / 2 parts 50º alcohol)
of Rosmarinus officinalis or Veronica officinalis were administered
orally after a 1/4 dilution with water (10mL diluted extract/kg body
weight/day). In case of positive and negative control group, the extracts
were substituted with 50º alcohol. On the 7th day, one hour after the last
dose of extract or 50º alcohol, CCl4 was administered (1200 mg/kg
bodyweight or 120 mg/100 g rat in 0.5 mL sunflower oil). The negative
control did not receive CCl4.
Six animals were sacrificed at 30 minutes after the administration
of CCl4, while at 60, 90 and 120min, 8 animals were sacrificed at each time
point. Blood samples were collected in order to quantify the studied
biomarkers of oxidative stress.

 
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Sample preparation
Total MDA
In order to quantify total MDA, 75μL plasma, 75μL 1% H2SO4 (or
TEP working solution in case of calibration standards) and 25μL 6M NaOH
were pipetted in a 1.5mL Eppendorf tube. The sample was maintained at
60ºC, for 30min in a waterbath, in order to hydrolyse the MDA bound to
proteins. After the hydrolysis, 100μL internal standard (IS) (acetaldehyde)
solution was added, followed by a deproteinisation step with 63μL 36%
HClO4. The sample was vortexed thoroughly and centrifuged at 10000rpm
for 10min. 150μL of the supernatant was derivatized with 20μL 5mM
DNPH 5mM in 2M HCl, at room temperature, protected from light. The
obtained hydrazone was extracted in 1.2mL hexane and the organic layer
was evaporated in a centrifugal concentrator at 30°C. The residue was
dissolved in 150μL mobile phase and 50μL of the obtained solution was
injected into the chromatographic column.
Reduced and total glutathione
100μL rat plasma spiked with 50μL GSH working standard was
pipetted into 1.5mL Eppendorf tubes; the sample was deproteinized with
50μL sulfosalicylic acid, vortexed and centrifuged at 10000rpm for 5min.
50μL of the supernatant were treated with 50μL OPA solution (a mixture of
54mg OPA/1mL methanol and 343mg Na2B4O7·10H2O/9mL deionized
water, pH 9.5) by vortexing at room temperature for 1 min. After
derivatization 5μL were injected into the chromatographic column.
In case of total GSH, after deproteinization, the oxidized
glutathione (GSSG) was reduced by incubating 50μL supernatant with
100μL DTT 100mM, for 5 min at room temperature. In the final step the
sample was diluted with 800μL deionized water and 5μL were injected into
the chromatographic column.
Instrumentation and chromatographic conditions
The analysis were performed using a 2695 Waters Alliance HPLC
(Waters, Milford, MA, USA) system composed of a quaternary pump,
autosampler, column heater and solvent degasser. The HPLC unit was
linked to a 996 PDA (photodiode array) detector and a 2475 fluorescence
detector (Waters, Milford, MA, USA).
Total MDA
Chromatographic separation was achieved on a Spherisorb ODS
column (250mmx4mm, 5μm) and a Spherisorb ODS (20mmx4mm, 3µm)
guard column, maintained at 25°C. The mobile phase consisted in a mixture

 
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of 1% formic acid/acetonitrile (62/38, v/v). The flow rate was 1mL/min and
the absorbance of the eluent was monitored at 307nm, corresponding to the
maximum in the UV spectra of the MDA hydrazone obtained after
derivatization.
Reduced and total glutathione
The chromatographic analysis was performed on a Supelcosil LC-
18 analytical column (150mmx4.6mm, 3μm particle size), maintained at
30°C and with a mobile phase consisting in a mixture of 0.25% acetic acid
(pH 6.91, with 6M NaOH) /methanol (85/15, v/v). The flow rate was set at
0.8mL/min. The sample compartment was maintained at 4°C. The
fluorescence detection was performed at λem = 420nm with λexc = 350nm.

Results and discussion

Determination of MDA
The elution times of MDA hydrazone and acetaldehyde hydrazone
in the specified chromatographic conditions were 9.96 min and 20.15 min,
respectively (Fig.1).

Figure 1
The chromatogram of a plasma sample spiked with 5 nmol/mL MDA
and 354nmol/mL acetaldehyde, respectively

A full validation of the method was not performed, but we studied

the selectivity and linearity of the assay over the concentration range of 1-
40ng/mL MDA.
Selectivity was assessed through the analysis of blank rat plasma
samples coming from 6 independent sources in a single analytical run. Since
MDA is an endogenous compound it can be detected and quantified in all
blank samples. Regarding the linearity, the calibration curves were
constructed based on the relation between peak area ratios and concentration
 
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ratios of MDA and internal standard. The method proved to be linear over
the studied concentration range, with a coefficient of correlation of 0.9898.
Determination of glutathione
The elution time of GSH after derivatization with OPA was 3.98
min, with the total analysis time being of 8 min in case of reduced GSH and
23 min for total GSH.
Fig. 2 shows a chromatogram for the reduced GSH from a spiked
plasma sample (2.7nmol/mL).

Figure 2
The chromatograms for the determination of reduced GSH
in spiked plasma samples (2.7 nmol GSH/mL plasma)

The method was linear over the studied concentration range (1-15
nmol/mL for reduced glutathione and 1-30nmol/mL for total glutathione).
Evaluation of the antioxidant properties of the plant extracts
MDA is the most frequently used biomarker of oxidative stress, due
to the fact that it is a lipid peroxidation end-product, a process which is very
intense at cellular level because of the great abundance of membranary lipids.
Another important marker is glutathione, the decrease of its
reduced form indicating an increase of the oxidative stress. Usually, in case
of this biomarker it is possible to work with the reduced glutathione levels
or with the reduced glutathione/total glutathione ratio.
In order to evaluate the antioxidant potential of the selected plants,
Veronica officinalis and Rosmarinus officinalis, total MDA and
reduced/total glutathione were quantified in all plasma samples obtained
from the four groups of rats used during the experiment. The statistical
evaluation of the results was performed using student’s t-test, the results
obtained in case of the two groups treated with the studied plant extracts
being compared to those obtained for the positive control group.
 
438 FARMACIA, 2009, Vol. 57, 4

The mean total MDA levels (nmol total MDA/mL plasma)

obtained for the different animal groups at different time points are
presented in Table I.
Figure 3 indicates the effect of the Veronica officinalis and
Rosmarinus officinalis extracts on the total MDA formation.

Table I
Mean total MDA levels (nmol total MDA/mL plasma)
Total MDA (mean ± SD)
Time Negative Positive control Rosmarinus Veronica
(min) control officinalis test officinalis test
group group
0 2.131(±0.312) - - -
30 - 1.819(±0.409) 1.093(±0.466) 0.745(±0.292)
60 - 4.619(±1.817) 1.938(±1.031) 0.843(±0.547)
90 - 1.942(±0.837) 1.792(±1.095) 1.175(±0.563)
120 - 2.158(±0.664) 0.902(±0.201) 2.426(±0.875)

Figure 3
The influence of Veronica officinalis and Rosmarinus officinalis extracts on the total MDA
levels in rats treated with carbon tetrachloride (*, p < 0.05)

The mean reduced/total glutathione ratio obtained for the different

animal groups at different time points are presented in Table II.
Figure 4 indicates the effect of the Veronica officinalis and
Rosmarinus officinalis extracts on the reduced/total glutathione ratio.

 
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Table II
Reduced/total glutathione ratio in plasma
Reduced GSH/Total GSH (± SD)
Time Negative control Positive control Rosmarinus Veronica
(min) officinalis test officinalis test
group group
0 0.0295(±0.0022) - - -
30 - 0.0250(±0.0035) 0.0454(±0.0070) 0.0259(±0.0027)
60 - 0.0182(±0.0025) 0.0305(±0.0031) 0.0200(±0.0029)
90 - 0.0293(±0.0043) 0.0238(±0.0017) 0.0282(±0.0032)
120 - 0.0311(±0.0018) 0.0163(±0.0020) 0.0276(±0.0013)

Figure 4
The influence of Veronica officinalis and Rosmarinus officinalis extracts on the
reduced/total GSH ratio in rats treated with carbon tetrachloride (*, p < 0.05)

The chromatographic analysis of MDA after derivatization with

DNPH showed that CCl4 induced oxidative stress and lipid peroxidation with
the maximum of this effect at 60 minutes. The results obtained in case of the
Rosmarinus officinalis and Veronica officinalis test groups showed that both
extracts have protective effects against the hepatotoxicity of CCl4, but with
statistically significant results (p< 0.05) only in case of the Veronica extract.
At 60 min (corresponding to the maximum intensity of the effect of CCl4) the
Veronica extract showed superior efficiency regarding the protective effect
against the oxidative stress. It is also noteworthy that, based on the evolution
of MDA levels in time, the Rosmarinus extract showed a less intense
antioxidant effect than Veronica, but with a longer duration.

 
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The determination of reduced/total glutathione ratio confirmed that

the effect of CCl4 presented maximum intensity at 60 minutes after
administration. The results obtained for GSH indicated great differences
regarding the effects of the two tested extracts. While Veronica showed only
a weak protective effect (the mean values were not significantly different
from those obtained for the positive control group) against the toxicity of
CCl4, the Rosmarinus extract induced a significant delay in the onset of the
toxic effect of the same agent (the maximum decrease of the reduced/total
GSH ratio was observed at 120 min, instead of 60 minutes, as it was shown
in case of the positive control and Veronica groups).

Conclusions
The results of this study indicate that it is not possible to use only
one biomarker in order to evaluate oxidative stress or the protective effect of
some agents, because there are different mechanisms of inducing the
oxidative stress or to fight against it. Probably the two tested plant extracts
present different mechanisms of protection due to their different chemical
composition. However, based on these results, both plant materials could
prove efficient in protecting the living organisms against oxidative damage.

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__________________________
Manuscript received: 10.11.2008