International Journal of Applied Research in Natural Products

Vol. 5 (2), pp. 19-25.

Directory of Open Access Journals
©2012. IJARNP-HS Publication

Original Research

Evaluation of in vitro antioxidant activity and free

radical scavenging potential of variety of Tagetes erecta
L. flowers growing in Bulgaria
Miglena Valyova1*, Stanimir Stoyanov2, Yuliana Markovska3,
Yordanka Ganeva2
1
Department of Plant Pathology and Chemistry, Faculty of Ecology and Landscape
Architecture, University of Forestry. 10, Kl. Ohridski blvd., 1756 Sofia, Bulgaria.
2
Department of Organic Chemistry, Faculty of Chemistry and Pharmacy, University of Sofia.
1, J. Bourchier blvd., 1164 Sofia, Bulgaria. 3Department of Plant Physiology, Faculty of
Biology, University of Sofia. 8, Dr. Tsankov blvd., 1164 Sofia, Bulgaria.
Summary. Marigold (Tagetes erecta L.) is well known for its antimicrobial, antiseptic, wound and ulcer healing,
antiinflammatory, antioxidant and antiviral properties, and it has a long history of being used as an herbal remedy. T. erecta
L. produces a variety of substances that possess pharmacological effects and antioxidant activity. The present study was
therefore aimed to analyze the antioxidant activity of extracts and fractions of T. erecta L. flowers, cultivated in Bulgaria.
Radical scavenging potential was determined using two different in vitro assays. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and
2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals. The ethyl acetate fraction of T. erecta L. ethanol
extract was found to be the most effective in both assays, DPPH (IC 50 4.3±0.4 μg/ml) and ABTS (0.8±0.2 TEAC). The
greatest total phenolic content was detected in EtOAc fraction from EtOH extract (517.8 mg/g GAE). It was obtained high
quantity of ascorbate and ascorbate/dehydroascorbate ratio more than 1. The quantity of glutathione reduced is low and
glutathione reduced/glutathione oxidized ratio was lower than 1. A correlation between radical scavenging capacities of
samples with total phenolic compound content was observed. The present study revealed that the EtOAc fraction effectively
show the best ability to scavenge the free radicals.
Industrial relevance. Herbal medicines have gained increasing attention worldwide for the treatment of chronic diseases
because of their effectiveness and small side effects as compared to synthetic drugs.Recently, an attention has been directed
toward the antioxidant phytochemicals which have proved as protective against cardiovascular diseases and cancer,
associated with overproduction of reactive oxygen species. The present investigations focus has been to discover plant
extracts and fractions from T. erecta useful to prevent chronic degenerative diseases, especially those mediated by free
radicals damages. The great antioxidant activity indicates the potential of the extracts as a source of natural antioxidants or
nutraceuticals with possible application to reduce oxidative stress with consequent health benefits.
Keywords. Tagetes erecta L.; in vitro antioxidant activity; free radical scavenging potential; phenols; flavonoids; ascorbate,
glutathione.

INTRODUCTION its ornamental value, marigold is also well known

Recently interest has increased considerably in
finding natural occurring antioxidants for use in
foods or medicinal purposes. Several studies have
demonstrated high antioxidant activity of extracts
from medicinal plants (Lanchance et al., 2001;
Zakaria et al., 2008; Živcović et al., 2012).
Marigold (Tagetes erecta L.) belongs to the family
Asteraceae. This plant is wild growing and also
cultivated at different parts of the world. It is very
popular as a garden plant and yields a strongly
aromatic essential oil which is mainly used for the
compounding of high-grade perfumes. Apart from
______________________
*Corresponding Author.
 mvalyova@abv.bg
 + +359 2 91907 350
Available online http.//www.ijarnp.org
as an herbal remedy. Different parts of this plant
including flowers are used in folk medicine to cure
various diseases. Different species of Tagetes have
been found to possess antimicrobial,
antiinflammatory, hepatoprotective, wound healing,
insecticidal, analgesic activities (Rhama and
Madhavan, 2011; Kiranmai and Ibrahim, 2012;
Gopi et al., 2012). The pharmacological activity of
T. erecta is related to the content of several
secondary metabolites, and the most important
compounds are terpenes, essential oils, flavonoids,
carotenoids and polyphenols (Marotti et al, 2004;
 Valyova et al.
Xu et al., 2011; Hadden et al., 1999). Recently,
there has been growing interest in antioxidant
activity of Tagetes species (Nirmala et al., 2010;
Kaisoon et al., 2011; Gong et al., 2012). Previous
studies conducted on European marigolds have
found that different species of the plant, as well as
different cultivars of the same species, were
markedly different in their lutein, phenol, and
flavonoid contents and their antioxidant activity
(Cetkovic et al., 2004). Therefore the present study
has been undertaken to investigate the radical
scavenging activity, total phenol and flavonoid
contents of T. erecta cultivated in Bulgaria. The
content of some nonenzymatic and enzymatic
antioxidants in T. erecta flowers also was
determined.
MATERIAL AND METHODS
Chemicals. Folin-Ciocalteu’s phenol reagent,
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS), α-tocopherol, Trolox,
sodium carbonate, aluminium chloride, potassium
chloride, gallic acid, quercetin, (+) catechin,
glutathione oxidized; glutathione reduced; ascorbic
acid; dehydroascorbic acid; nicotinamide adenine
dinucleotide - NADH, nicotinamide adenine
dinucleotide phosphate - NADPH, 5,5’-dithiobis(2-
nitrobenzoic acid) – DTNB; 1-chloro-2,4-
dinitrobenzene – CDNB, cumene hydroxyperoxide
and ascorbate oxidase (1653 U mg-1 prot.),
glutathione reductase (5 U ml-1) were purchased
from Sigma Aldrich-Fluka (Germany).
Plant material. Tagetes erecta L. is not a native
plant of Bulgaria. The flowers of T. erecta were
collected at full flowering stage from a farmland in
Sevlievo region, Bulgaria. The plant was
taxonomically authenticated and a voucher
specimen (SO 105294) has been deposited in the
herbarium of the Faculty of Biology, Sofia
University “St. Kliment Ohridski”.
Preparation of extracts and fractions.The air-
dried flowers of T. erecta (100 g) were extracted
twice with ethanol at 50˚-55˚C for 3 h. The ethanol
solutions were evaporated under vacuum to give
ethanol extract (7.4 g). Methanol extract (5.9 g)
was obtained under the same conditions. The
ethanol extract was dissolved in EtOH-H 2 O (7:3)
and partitioned with petroleum ether, chloroform
and ethyl acetate, respectively. These organic
fractions were concentrated separately on rotary
evaporator and the residues thus obtained were
used to evaluate their in vitro radical scavenging
potential (petroleum ether fraction - 1.2 g;
chloroform fraction - 0.2 g; ethyl acetate fraction -
0.4 g).
DPPH radical scavenging activity. In vitro
DPPH radical scavenging activity of T. erecta
extracts and fractions was carried out by modifying
the methods of Blois (1958) and Schmeda-
Hirschman (2003). To 2.4 ml of each of the
20
different concentrations of the samples, 0.8 ml of
0.1 mM DPPH (methanol) was added, vortexed and
followed by incubation at room temperature for 10
minutes. The control sample was prepared without
any extract or fraction. The decrease in absorbance
in presence of DPPH was measured at 517 nm by
using Unicam UV 500 Spectrophotometer (Thermo
Spectronic, UK). Radical scavenging activity was
expressed as % inhibition of DPPH radicals. IC 50
values were also calculated. α-Tocopherol was
taken as a referent compound.
ABTS radical scavenging activity. ABTS
radical scavenging activity of T. erecta extracts and
fractions was measured by the ABTS cation
decolorization assay as described by Re et al.
(1999) and Pellegrini et al. (1999) with some
modifications. The ABTS radical cation (ABTS•+)
was produced by reaction of 7 mM stock solution
of ABTS with 2.45 mM potassium persulfate and
allowing the mixture to stand in dark at room
temperature for 12 h before use. The ABTS•+
solution was diluted with methanol to give an
absorbance of 0.7 ± 0.01 at 734 nm. Plant extracts
and fractions (1 ml) were allowed to react with 2
ml of the ABTS•+ solution and the absorbance was
measured at 734 nm after 1 minute. Trolox was
used as a reference compound. The results were
expressed as Trolox equivalent antioxidant capacity
(TEAC) values and calculated as mean value ±
standard deviation (SD) (n = 3).
Determination of total phenolic content. Total
phenolic content in the T.erecta extracts and
fractions was determined using the Folin-Ciocalteu
method as described by Singleton et al. (1999).
Briefly, 0.5 ml of the extracts or fractions was
added to 3 ml of distilled water and 0.25 ml Folin–
Ciocalteu reagent. The mixture was allowed to
stand at room temperature for 2 min and then 0.75
ml of 20% sodium carbonate was added to the
mixture and the volume was made up to 5 ml with
distilled water. The absorbance of thus prepared
solutions was measured at 765 nm after standing
for 2 hours. The content of phenolics was
expressed as gallic acid equivalents (GAE) in mg g-
1
of sample.
Determination of total flavonoid content. The
total flavonoid content of T. erecta was determined
by using of a modified colorimetric method
described previously (Zhishen et al., 1999). An air-
dried plant material (25 mg) was ground in a
mortar with 10 ml 80 % methanol. The
homogenous mixture obtained was allowed to stand
for 20 min. at room temperature, followed by
filtration through filter G4. An aliquot of 0.4 ml of
filtrate was mixed with 0.6 ml distilled water, 5 %
NaNO 2 solution (0.06 ml) and the mixture was
allowed to stand for 5 min at room temperature.
After 6 min 10 % AlCl 3 solution (0.06 ml) was
added to the mixture. Immediately, 1 N NaOH (0.4
ml) and 0.45 ml distilled water were added to the
 Antioxidant activity of Tagetes erecta
mixture and allowed to stand for another 30 min.
Absorbance of the mixture was determined at 510
nm and (+) catechin was used as standard
compound for the quantification of total flavonoid
content. All values were expressed as milligram of
catechin equivalents per 1 gram dry weight. Data
was recorded as mean ± SD for three replicates.
Determination of nonenzymatic antioxidants.
The concentrations of reduced and oxidized
glutathione were determined with an enzyme
recycling assay (Griffith, 1980). The assay was
based on sequential oxidation of glutathione by 5,
5‫׳‬-dithiobis (2-nitrobenzoic acid) (DTNB) and
reduction by NADPH in the presence of
glutathione reductase (GR). To quantify the GSH
plus GSSG and GSSG separately the extract was
processed and subsequently assayed as per method
given earlier (Fadzilla et al., 1997). The assay
mixture in 1 ml contained 0.150 ml 125 mM
potassium phosphate buffer and 6.3 mM EDTA,
pH 6.5, 0.3 mM NADPH, 3 mM DTNB and
processed sample. The reaction was initiated by
addition of 10 µl of GR and the change in
absorbance at 412 nm was recorded. Standard
curves were generated with reduced and oxidized
glutathione. The results were expressed per 1 g
DW.
Ascorbate (AsA) and dehydroascorbate (DAsA)
were assayed according to Foyer et al. (1983). The
reaction mixture for determination of ascorbate
contained 100 mM potassium phosphate buffer, pH
5.6, 0.2 mM ascorbate, 5 ml ascorbate oxidase, 0.1
ml processed sample. The reaction was initiated by
addition the sample and the changes in absorbance
at 265 nm were followed for 1 min. The reaction
mixture for the determination of DAsA contained
100 mM potassium phosphate buffer, pH 8.5, 0.2
mM ascorbate, 10 mM GSH and was incubated for
15 min in water bath at 25оС. The reaction was
initiated by addition the processed sample and the
changes in absorbance at 265 nm were followed for
1 min. Standard curves were generated with AsA
and DAsA. The results were expressed per 1 g DW.
Determination of enzymatic antioxidants. In order
to prepare crude extracts for determination of
enzymes of ascorbate-glutathione cycle -
glutathione peroxidase (GPX), glutathione S-
transferase (GST) and glutathione reductase (GR),
as well as guaiacol peroxidase (GPO) and catalase
(CAT) the plant material were grinded with 4 ml of
the extraction buffer (100 mM potassium phosphate
buffer, pH 7.8; 5 mM EDTA; 2% PVP) that was
added to 0.3 g of tissue powder. The extraction
buffer for the determination of ascorbate
peroxidase (APX), monodehydroascorbate
reductase (MDHAR) and dehydroascorbate
reductase (DHAR) contained. 50 mM potassium
phosphate buffer, pH 7.0; 1 mM ascorbate; 1 mM
EDTA; 0.2 % PVP and was added to 0.15 g of
tissue powder. The suspensions were centrifuged
21
(16 000g, 15 min, 4oC). All enzymes were assayed
spectrophotometrically by tracing the changes in
absorbance at 27oC using UV-VIS SPECORD or
SPECOL 11.
GPO (EC 1.11.1.7) was assayed according to
Polle et al. (1994) with some modifications in order
to obtained of maximal extractable enzyme
activity. The reaction mixture constituted of 100
mM potassium phosphate buffer, pH 7.0, 20 mМ
quaiacol, 1 mM H 2 O 2 , 0.2 ml enzyme extract. The
reaction was initiated by the addition of H 2 O 2 and
the oxidation of quaiacol was measured by
following the increase in absorbance at 470 nm for
2 min.
CAT (EC 1.11.1.6) was assayed according to
Aebi (1984). The reaction mixture constituted of
100 mM potassium phosphate buffer, pH 7.0, 15
mМ H 2 O 2 , 0.05 m1 enzyme extract. The reaction
was initiated by the addition of H 2 O 2 and its
decomposition was determined by following the
decline in absorbance at 240 nm for 3 min.
GPX (EC 1.11.1.9) was assayed according to
Edwards (1996). The reaction mixture constituted
of 100 mM potassium phosphate buffer, pH 7.0,
0.2% Triton X-100, 0.24 U glutathione reductase, 1
mM GSH and 0.2 ml enzyme extract. After
addition of enzyme extract, cuvettes were
incubated at 30оС for 10 min and 0.15 mM
NADPH was added to measure the basal rate of
GSH oxidation by monitoring the absorbance at
340 nm for 3 min. The reaction was initiated by the
addition of 1 mM cumene hydroperoxide and GPX
activity was expressed as change in absorbance at
340 nm mg protein min-1.
GST (EC 2.5.1.18) was assayed according to Li
et al. (1995). The reaction mixture constituted of
100 mM potassium phosphate buffer, pH 7.4, 1-
chloro-2, 4-dinitrobenzene /CDNB/ and 0.1 ml
enzyme extract. The reaction was initiated by the
addition of 1 mM GSH and formation of S-(2,4-
dinitrophenyl)glutathione(DNP-GS) was monitored
as an increase in absorbance at 334 nm to calculate
the GST specific activity.
GR (EC 1.6.4.2) was assayed according to
Sherwin and Farrant (1998). The reaction mixture
constituted of 300 mM potassium phosphate buffer,
pH 7.5, 3 mM MgCl 2 , 0.1 mM EDTA, 10 mM
GSSG, 0.15 mM NADPH and 0.2 ml enzyme
extract. The reaction was initiated by addition of
NADPH and the decrease in absorbance at 340 nm
for 3 min was recorded.
APX (EC 1.11.1.11) was assayed according to
Nakano and Asada (1981). The reaction mixture
constituted of 50 mM potassium phosphate buffer,
pH 7.0, 0.5 mM ascorbate, 0.1 mM H 2 O 2 and 0.2
ml enzyme extract. The rate of hydrogen peroxide–
dependent oxidation of ascorbate was determined
by monitoring the change in absorbance at 290 nm
for 3 min after adding of NaAsA.
 Valyova et al.
MDHAR (EC 1.6.5.4.) was assayed according
to Miyake and Asada (1992). The reaction mixture
constituted of 50 mM potassium phosphate buffer,
pH 7.6, 2.5 mM ascorbate, 0.1 mM NADH and 0.1
ml enzyme extract. The reaction was initiated by
addition of 4 ml ascorbate oxidase and the decrease
in absorbance at 340 nm for 30 s was recorded.
Correction was made by subtracting values
obtained in the absence of ascorbate oxidase.
DHAR (EC 1.8.5.1.) was assayed according to
Doulis et al. (1997) by the formation of AsA in a
reaction mixture constituted of 50 mM potassium
phosphate buffer, pH 7.0, 2.5 mM GSH, 0.1 mM
EDTA and 0.2 ml enzyme extract. The reaction
was initiated by addition of 0.2 mM
dehydroascorbate and the changes in absorbance at
265 nm were followed for 1 min. Correction was
made by subtracting values obtained in the absence
of enzyme extract.
The protein content was determined after
Lowry et al. (1951).
Determination of Н 2 О 2 levels. Plant material (0.3
g) was ground thoroughly in 3 ml 1% trichloracetic
acid. After centrifugation at 12 000g for 20 min, a
0.5 ml of the supernatant was mixed with 0.5 ml
100 mM potassium phosphate buffer (рН 7.4) and
1 ml 1M KJ. Control samples contained the same
compounds excepting supernatant. All samples
were incubated in the dark at room temperature for
60 min and were shake periodically. The
absorbance was measured at 390 nm. For
quantification, a standard curve was generated by
using graded amount of H 2 O 2 in the reaction
mixture (Dhindsa et al., 1981). The results were
expressed per 1 g DW.
RESULTS AND DISCUSSION
DPPH radical scavenging activity. DPPH assay is
rapid and sensitive way to survey the antioxidant
activity of a specific compounds or plant extracts.
The DPPH radical scavenging capacity in the study
was reported after 10 min reaction time for all
samples investigated. The parameter used to
measure the radical scavenging activity of the
extracts and fractions evaluated is IC 50 value. The
results obtained were shown in Table 1. Ethyl
acetate fraction (IC 50 = 4.3 ± 0.4 μg/ml) of T.
erecta ethanol extract showed the highest
antioxidant potential when compared to other
extracts and fractions. The value is comparable
with that of standard α-tocopherol (IC 50 = 3.5 ± 0.2
μg/ml). The petroleum ether fraction displayed the
lowest radical scavenging capacity (IC 50 = 100.1 ±
12.4).
ABTS radical scavenging activity. The antioxidant
capacity of methanol extract, petroleum ether,
chloroform and ethyl acetate fractions of ethanol
extract were evaluated according to the ABTS
decolorization method. The results of antioxidant
activity of all samples were expressed as TEAC
22
values, as shown in Table 1. The ethyl acetate
fraction of T. erecta displayed the highest radical
scavenging potential (0.8 ± 0.2 TEAC). The
petroleum ether fraction had the lowest antioxidant
capacity of 0.02 ± 0.001 TEAC.
Total phenolic and flavonoid content. Phenolic
compounds are secondary plant metabolites, which
contribute to the overall antioxidant activities of
plants mainly due to their redox properties.
The total phenolic content of T. erecta extracts
and fractions was determined by Folin-Ciocalteu
assay using gallic acid as a standard phenolic
compound. This method can be useful in
characterizing and standardizing plant samples. The
total phenolic contents of T. erecta extracts and
fractions were calculated with a regression equation
based on a standard curve using gallic acid
(y=8.18181x, R2=0.998) and shown in Table 1. The
ethyl acetate fraction had the highest phenolic
content (517.8 ± 23.4 mg GAE g-1), which may
have contributed towards its greatest radical
scavenging activity in both DPPH and ABTS
assays. The lowest values were obtained for
chloroform and petroleum ether fractions (79.5 ±
0.2 mg GAE g-1 and 80.3 ± 3.5 mg GAE g-1,
respectively).
The content of total flavonoids was also
measured spectrophotometrically by using the
aluminium chloride colorimetric assay. The
flavonoid content of T. erecta flowers was
expressed as catechin equivalents in mg/g dry
weight (32.7 ± 1.1 mg/g dry weight) and a
calibration curve of catechin was used. Different
extraction solvents resulted in significant changes
in the total phenolic content, total flavonoids
content and antioxidant activity (Table 1). Organic
reagents (MeOH, EtOH etc.) were often preferred
for the extraction of antioxidant compound from
fruits, vegetables and herbals. According to
Grzegorczyk et al (2007) variations in antioxidant
properties of plant samples with different extraction
solvents may be attributed to the presence of
different secondary metabolites and solvent used
for extraction.
Enzyme assays It is well known that antioxidant
defense system in plants is responsible for
detoxification of harmful reactive oxygen species
(ROS) that are generated due to the stepwise
reduction of molecular oxygen by high-energy
exposure or as a result of electron-transfer chemical
reactions. Dual roles for ROS in plants have been
suggested, as they serve as key regulators of
growth, development, and defense pathways, as
well as at excessive levels they cause oxidative
damage to biomolecules leading to toxicity in the
cell (Mittler et al., 2004). Stressful conditions of
the environment lead to the enhanced generation of
ROS in plants due to the disruption of cellular
homeostasis (Sharma et al., 2010). Scavenging or
detoxification of excess ROS is achieved by an
 Antioxidant activity of Tagetes erecta

efficient antioxidative system comprising of the
nonenzymatic as well as enzymatic antioxidants
(Noctor and Foyer, 1998). Ascorbate (AsA),
glutathione (GSH), α–tocopherol, carotenoids and
phenolic compounds serve as potent nonenzymatic
antioxidants within the cell. The enzymatic
antioxidants include superoxide dismutase (SOD),
catalase (CAT), quaiacol peroxidase (GPO),
enzymes of ascorbate-glutathione cycle (AsA-
GSH) such as ascorbate peroxidase (APX),
monodehydroascorbate reductase (MDHAR),
dehydroascorbate reductase (DHAR), glutathione
peroxidase (GPX), glutathione S-transferase (GST)
and glutathione reductase (GR).
The activity of some important antioxidant
enzymes and metabolites in flowers of T. erecta
was measured (Table 2).

Table 1. Radical scavenging activity and total phenolic content of Tagetes erecta L. flowers.

Sample DPPH assay ABTS assay TPC

IC 50 (μg/ml) TEAC (mg GAE g-1)

Methanol extract 7.5±0.1 0.2±0.003 142.2±22.9

Ethanol extract 7.6±0.1 0.2±0.01 154.4±11.02

Petroleum ether fraction 100.1±12.4 0.02±0.001 80.3±3.5

Chloroform fraction 23.1±0.2 0.2±0.01 79.5±0.2

Ethyl acetate fraction 4.3±0.4 0.8±0.2 517.8±23.4

α-Tocopherol 3.5±0.2 - -

Тable 2. Results from the measurement of the activity of some important antioxidant enzymes and metabolites in
flowers of T. erecta (n = 9 ± SD).

Parameters T. erecta SD

H 2 O 2 [μM gDW-1] 453.6 21.8

CAT [mM mg pr.-1 min] 0.1 0.003

GPX [ΔE mg pr.-1 min] 0.04 0.001

GST [ΔE mg pr.-1 min] 0.2 0.01

GR [mM mg pr.-1 min] 0.02 0.002

GSH [nM gDW-1] 46.7 2.1

GSSG [nM gDW-1] 108.3 2.9

GSH/GSSG 0.4 -

GPO [mM mg pr.-1 min] 0.04 0.002

MDHAR [mM mg pr.-1 min] 0.03 0.002

DHAR [mM mg pr.-1 min] 0.004 0.0002

APX [mM mg pr.-1 min] 0.1 0.03

-1
AsA [nM gDW ] 14.2 1.6

DAsA. [nM gDW-1] 4.2 0.3

AsA/DAsA 3.4 -

23
 Valyova et al.
Hydrogen peroxide, a product of superoxide
dismutase reaction was a strong oxidant and it
initiates localized oxidative damage leading to
disruption of metabolic function and losses of
cellular integrity at sites where it accumulates.
Excessive levels of H 2 O 2 could be minimized
through the activities of CAT and different
peroxidases. Tagetes flowers possessed the highest
APX activity and lower CAT, GPO and GPX
activities. Hydrogen peroxide can be produced by a
number of non-enzymatic and enzymatic processes
in cells. While mitochondria and plastids are the
major sources of H 2 O 2 in the cells, peroxisomes
and glyoxysomes also contain SOD and APX,
which are responsible for its production and
scavenging (Yamaguchi et al., 1995; Jemenez et
al., 1997) CAT and APX, two potential scavengers
of H 2 O 2 , maintain its level and prevented
uncontrolled export of this toxic species from
organelles to cytosol and competed to remove
H 2 O 2 . Measured activities of MDHAR and
especially of DHAR was significantly lower in the
flowers of Tagetes. At the same time the tissue
ascorbate content was greater than DAsA.
Increases in antioxidant enzyme activities may
indicate increased oxidative stress but they give no
indication of changes in overall flux through AGC.
Glutathione reductase, that catalysed the NADPH-
dependent reduction of oxidized glutathione have
not high activity. GSH, that maintains the cellular
redox status and also serves as substrate for
phytochelatin synthesis was lower than GSSG
measured in Tagetes flowers. GR activity
presumably did not allow an abrupt fall in the GSH
level. The decline in the endogenous level of GSH
may be due to its utilization as a reducing substrate
in the synthesis of ascorbate. GSH is also
consumed and degraded in order to protect cellular
membranes from lipid peroxidation. The other
enzyme from glutathione metabolism - glutathione
S-transferase also exhibit alternative activities as
glutathione peroxidase (Edwards, 1996; Bartling et
al., 1993). GPXs catalyse the reduction of H 2 O 2 ,
organic hydroperoxides and lipid hydroperoxides
by GSH. In the present study GST activity was
higher than GPX activity measured.
The phenolics can act as antioxidants by
donating electrons to quiacol-type peroxidases and
they are equivalent to that of ascorbate for
scavenging H 2 O 2 (Sakihama et al., 2002).
CONCLUSION
The present work reveals that the ethyl acetate
fraction of T. erecta ethanol extract exhibit the
highest phenolic content and the greatest
antioxidant activity in both DPPH and ABTS
scavenging assays. We established high
AsA/DAsA (more than 1) and GSH/GSSG (lower
than 1) ratios in the flowers. The total phenolic
24
content and probably high AsA level in
investigated plant samples correlated with
evaluated radical scavenging capacity. The results
obtained showed that T. erecta could be an
interesting source of natural antioxidants with
potential use in food supplements. More work
therefore needs to be carried out on the fractions in
other to isolate, purify and characterize the active
chemical compounds responsible for radical
scavenging activity.
ACKNOWLEDGEMENTS
The authors are grateful to the financial support
of University of Forestry Bulgaria in funding this
project.
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