FARMACIA, 2019, Vol.

67, 3
https://doi.org/10.31925/farmacia.2019.3.4 ORIGINAL ARTICLE

ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF

CHRYSANTHEMUM AND TAGETES SELECTIVE EXTRACTS

ANA FLAVIA BURLEC 1#, OANA CIOANCA 1*, CORNELIA MIRCEA 1#, CRISTINA ARSENE 2,
CRISTINA TUCHILUȘ 2#, ANDREIA CORCIOVĂ 1, MONICA HĂNCIANU 1
1
Faculty of Pharmacy, "Grigore T. Popa" University of Medicine and Pharmacy, 16 Universității Street, 700115, Iași, Romania
2
Faculty of Medicine, "Grigore T. Popa" University of Medicine and Pharmacy, 16 Universității Street, 700115, Iași, Romania

*corresponding author: oana.cioanca@gmail.com

#
Authors with equal contribution
Manuscript received: November 2018

Abstract
The present study focuses on two ornamental species of the Asteraceae family and their biological properties, which can be
correlated with the presence of different classes of natural compounds. Given the fact that secondary metabolites found in
such plants can be used as valuable resources for the pharmaceutical industry, we chose to investigate the biological potential
of Chrysanthemum indicum and Tagetes erecta inflorescences. Four types of extracts were obtained for each species, using
organic solvents with different polarities. Bioactive compounds such as polyphenols were quantitatively analysed using a the
Folin-Ciocâlteu method. The investigated compounds were present in all types of extracts in variable quantities, the highest
amounts being found in the methanolic extracts. Positive dose-related values revealed that the investigated extracts exhibit
good inhibitory activity against 15-lipoxygenase and a moderate metal-chelating activity. Moreover, all extracts showed
promising antibacterial and antifungal activities on the tested strains.

Rezumat
Prezentul studiu se axează pe analiza unor specii ornamentale din familia Asteraceae și pe testarea acțiunilor lor biologice ce
pot fi corelate cu prezența diferitelor clase de compuși naturali. Având în vedere faptul că metaboliții ai acestor plante pot fi
utilizați ca resurse valoroase pentru industria farmaceutică, am ales să investigăm inflorescențele speciilor Chrysanthemum
indicum și Tagetes erecta. Pentru fiecare specie au fost obținute patru tipuri de extracte, utilizând solvenți organici cu
polarități diferite. Compușii bioactivi, precum polifenolii, au fost analizați cantitativ utilizând metoda spectrofotometrică.
Compușii investigați s-au regăsit în toate tipurile de extracte în proporții variabile, cele mai mari cantități regăsindu-se în
extractele metanolice. În ceea ce privește testarea acțiunilor biologice, valorile obținute au evidențiat faptul că extractele
analizate prezintă o bună activitate inhibitorie asupra 15-lipooxigenazei și o activitate moderată de chelatare a ionului feros.
Mai mult, toate extractele obținute din speciile investigate au prezentat activități antibacteriene și antifungice promițătoare
asupra tulpinilor testate.

Keywords: Asteraceae, antimicrobial activity, 15-lipoxygenase, polyphenols

Introduction acids, mono-, sesqui- and diterpenoids, steroids,

carotenoids [12].
Various secondary metabolites from plants have been
Several species belonging to the Tagetes genus are
chemically and biologically characterized so far, some
rich in secondary metabolites such as carotenoids,
of which have shown good antioxidant, antimicrobial
flavonoids, thiophenes and several classes of terpenoids
or antiinflammatory actions [4, 8, 28]. The Asteraceae
[22, 24]. Different species of this genus, such as
family is considered to be the vastest family of
Tagetes patula and Tagetes erecta are used for
flowering plants [11]. Its species have been used for
ornamental purposes [2]. However, important active
many centuries for their therapeutic, ornamental and
compounds such as lutein have been isolated from
nutritional properties. However, ornamental species
plants from this genus and are presently used as dietary
have rarely been investigated regarding their potential
supplements that could have a beneficial impact on
therapeutic use [16].
macular degeneration [10, 19]. Therefore, given the
The Chrysanthemum genus includes many species
large variety of secondary metabolites found in these
which are cultivated not only for ornamental purposes,
ornamental plants, we investigated their potential
but also for their use in cosmetic preparations [2, 5].
implications in biochemical processes and inhibition
Chemical investigations have been carried out on
of microorganisms in correlation with their chemical
different species, showing that they contain a variety
profile.
of secondary metabolites such as flavonoids, phenolic

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Materials and Methods Ferrous ion chelating activity
The capacity of the investigated extracts to chelate
Plant material and extraction process.
ferrous ions was determined according to the method
The plant material was represented by the inflorescences
described by Venditti et al. with slight modifications
of two ornamental species belonging to the Asteraceae
[29]. Briefly, the ferrous ion forms with ferrozine a
family: Chrysanthemum indicum and Tagetes erecta.
complex with maximum absorbance at 562 nm. The
The plants were cultivated in ecological conditions
presence of a chelating agent in the reaction medium
in the North-Eastern part of Romania in the year of
decreases the absorbance of the formed complex.
2017. Voucher specimens are deposited at the
Over 0.2 mL sample solution, 0.74 mL of 0.1 M
Department of Pharmacognosy, Faculty of Pharmacy,
acetate buffer (pH 5.25) and 0.02 mL of 2 mM
“Grigore T. Popa” University of Medicine and
ferrous sulphate solution in 0.2 M hydrochloric acid
Pharmacy, Iași, Romania.
were added. After mixing for 10 - 15 seconds, 0.04
Four types of extracts were obtained for each sample,
mL of 5 mM ferrozine solution was also added. The
using as solvents chloroform, dichloromethane, hexane
absorbance of the solution was determined after
and methanol. 5 g of small fragments (0.5 - 1 cm) of
being kept for 10 minutes in the dark, against a
dried inflorescences were extracted using 50 mL of
blank prepared under the same conditions. The
either chloroform, dichloromethane and hexane,
metal chelating activity was determined using the
respectively, while 2.5 g of small fragments were
following formula:
extracted using 100 mL methanol. The first three
extracts were obtained by maceration at room Activity % = 100 x (Ac - Ap)/(Ac),
temperature for 7 days in a mixing chamber, while
where Ac is the absorbance of the control solution
the methanolic extract was obtained through a three
and Ap is the absorbance of the sample solution.
times extraction at 60ºC. Following filtration, the
For each extract, EC50 was determined and expressed
extracts were concentrated to residue.
as mg sample/mL final solution. Ethylenediamine-
Total phenolic content (TPC)
tetraacetic acid (EDTA) was used as positive control.
The quantity of polyphenols found in each extract was
The assay was carried out in triplicate.
established with a well-known spectrophotometric
Antimicrobial susceptibility tests
method using Folin-Ciocâlteu reagent [26], with some
Microorganisms. The antimicrobial activity was studied
modifications [13]. The dry extracts were dissolved
using a Gram-positive bacterial strain (Staphylococcus
in dimethyl sulfoxide for this determination. The
aureus ATCC 25923), a Gram-negative bacterial
absorbance was measured at 765 nm and gallic acid
strain (Escherichia coli ATCC 25922) and pathogenic
was used as standard. The results were expressed as
yeasts (Candida albicans ATCC 90028, Candida
g gallic acid equivalents (GAE)/100 g dry extract.
parapsilosis ATCC 22019).
The determination was carried out in triplicate.
Disc-diffusion method. Antimicrobial tests for the
Inhibition of 15-lipoxygenase (15-LOX)
selected microorganisms were carried out using a
The inhibition of 15-LOX was determined using the
disc-diffusion method [6, 7]. A small amount of each
Malterud method [20] with some modifications [9].
microbial culture was diluted in sterile 0.9% sodium
0.05 mL lipoxygenase buffer solution (pH 9) was
chloride solution until the turbidity was equivalent to
treated with 0.05 mL extract dissolved in dimethyl
McFarland standard no. 0.5. The suspensions were
sulfoxide in various concentrations and kept for 10
further diluted 1:10 in Mueller Hinton agar for bacteria
minutes at room temperature. Afterwards, 2 mL linoleic
(Oxoid) and Mueller-Hinton agar for yeasts (HiMedia)
acid buffer solution 0,16 mM (pH 9) were added.
and then spread on sterile Petri plates. Sterile stainless-
The absorbance was measured at 234 nm for 120
steel cylinders were applied on the agar surface in
seconds. The results were calculated using the
Petri plates. Afterwards, 0.1 mL of each sample was
following formula:
added into cylinders. Commercially available discs
% activity = (AEFI - AECI) x 100/AEFI, containing ciprofloxacin (5 µg/disc), fluconazole
(25 µg/disc) and nystatin (100 µg/disc) were used as
where AEFI is the difference between the absorbance
positive controls. The plates were incubated at 37°C
of the enzyme solution alone after 90 seconds and
for 24 h (bacteria) and at 24°C for 48 h (yeasts). After
the absorbance of the same solution after 30 seconds
incubation, the diameters of the inhibition zones were
and AECI is the difference between the absorbance
measured in mm, including disc size.
of the enzyme solution mixed with the sample after
Broth microdilution method. The samples were tested
90 seconds and the absorbance of the same solution
for the Minimum Inhibitory Concentration (MIC) and
after 30 seconds. For each sample, the half maximal
Minimum Bactericidal Concentration (MBC) against
effective concentration (EC50) was determined and
Staphylococcus aureus ATCC 25923 and Escherichia
expressed as µg sample/mL final solution. Quercetin
coli ATCC 25922. Serial double dilutions of each
was used as positive control.
extract in Mueller Hinton broth were inoculated with
equal volumes of bacterial suspension. MIC represents
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the lowest concentration of extract where complete Results and Discussion
inhibition of visible growth was observed after 24 h
Total phenolic content
incubation at 37°C. MBC values were determined
The comparison between the lipophilic extracts (in
by transferring 0.1 mL sample showing complete
chloroform, dichloromethane and hexane) for Tagetes
inhibition of visible growth on the surface of an
erecta showed a higher concentration of polyphenols
agar plate. The subcultures were incubated 24 h at
in the chloroform extract, while for Chrysanthemum
37°C. MBC is the lowest concentration of extract
indicum the highest concentration was found in the
required to kill more than 99.9% of microorganisms
dichloromethane extract (Table I). For Tagetes erecta,
being tested. MIC of ciprofloxacin towards bacterial
the high content of polyphenols in this type of extracts
strains was also evaluated.
can be explained by the presence of complex
molecules containing some hydroxyl groups [1].
Table I
Total amount of polyphenols found in the analysed extracts
Extract Code Concentration g GAE/100 g dry extract (mean ± SD*)
Chrysanthemum indicum chloroform extract C1 1.654 ± 0.315
Chrysanthemum indicum dichloromethane extract C2 3.144 ± 0.433
Chrysanthemum indicum hexane extract C3 0.902 ± 0.553
Chrysanthemum indicum methanol extract C4 11.856 ± 0.391
Tagetes erecta chloroform extract T1 6.973 ± 0.376
Tagetes erecta dichloromethane extract T2 4.185 ± 0.216
Tagetes erecta hexane extract T3 5.378 ± 0.339
Tagetes erecta methanol extract T4 15.676 ± 1.139
*SD – standard deviation

Regarding the concentrations of the polyphenolic

compounds in the methanolic extracts, the comparison
between the two species shows that Tagetes erecta
contains the highest amount (15.676 ± 1.139 g GAE/
100 g dry extract).
Inhibition of 15-lipoxygenase
Polyphenols as well as other compounds found in
the extract have the capacity of blocking the action
of lipoxygenase, which is responsible for catalysing
the oxidation of linoleic acid, with the reduction of Figure 2.
absorbance measured at 234 nm. The obtained values 15-LOX inhibition of Tagetes erecta extracts and of
reveal that the methanolic extracts of both species show quercetin
good inhibitory activity against 15-LOX (Figure 1,
Figure 2). The EC50 values of the tested extracts can Table II
be found in Table II. EC50 values of tested samples and of quercetin
Sample EC50 (µg/mL final solution)
C1 26.06 ± 0.34
C2 42.56 ± 0.45
C3 92.50 ± 0.61
C4 44.44 ± 0.31
T1 64.23 ± 2.41
T2 49.21 ± 3.24
T3 57.83 ± 3.29
T4 25.85 ± 0.67
Quercetin 17.45 ± 0.33

Figure 1.
15-lipoxygenase is a non-heme iron enzyme. Its action
15-LOX inhibition of Chrysanthemum indicum
can be modified by compounds that block the reversible
extracts and of quercetin
oxidation of Fe2+ to Fe3+. Consequently, the enzyme
will no longer be able to catalyse the transformation of
the substrate through the oxidation-reduction reaction.
The substances that inhibit the enzyme through this
mechanism have reducing capacity, releasing electrons
and protons. Polyphenols and flavonoids found in the
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extracts can reduce or even block the activity of the the EC50 of EDTA was much lower than those of the
enzyme through the mentioned mechanism [17, 20]. tested samples (0.075 ± 0.001 mg/mL). It is generally
The concentration in polyphenols for the investigated believed that polyphenols are some of the most
species can be correlated with the anti-inflammatory important compounds responsible for the metal chelating
and antioxidant activities. Promising inhibition of the activity. While this can be proved by the correlation
enzyme can be observed for the methanolic extracts between the TPC of methanolic extracts and their
of both species. However, this is also true for EC50, we believe that more classes of compounds
Chrysanthemum indicum chloroform and dichloro- can be responsible for the metal-chelating activity
methane extracts, which indicates that lipophilic observed for other types of extracts [30], although
compounds such as carotenoids or terpenoids can different chemical assays are still ongoing.
also be responsible for the inhibitory activity, through Table III
a different mechanism of action [18, 25]. EC50 of tested samples and of EDTA
Ferrous ion chelating activity Sample EC50 (mg/mL final solution)
Ferrous ion chelating capacity of all investigated C1 0.282 ± 0.001
extracts increased with concentration. According to C2 0.382 ± 0.001
the results, the most active extracts were the C3 0.346 ± 0.001
methanolic extracts of both species (C4, T4) and the C4 0.307 ± 0.001
Chrysanthemum indicum chloroform extract (C1), for T1 0.492 ± 0.001
T2 1.605 ± 0.004
which a 100% chelating activity was observed at a
T3 0.437 ± 0.01
concentration of 5 mg/mL. All Chrysanthemum indicum
T4 0.441 ± 0.002
extracts showed 100% activity at a concentration of
EDTA 0.075 ± 0.001
10 mg/mL, while only the methanolic extract of
Tagetes erecta showed such result at the same
Antimicrobial susceptibility tests
concentration. Chrysanthemum indicum chloroform
The diameters of the inhibition zones corresponding
and methanol extracts (C1, C4) and Tagetes erecta
to the tested samples are shown in Table IV. All
hexane and methanol extracts (T3, T4) have the
assays were carried out in triplicate. Results are
lowest EC50, as it can be seen in Table III. However,
expressed as mean ± SD.
Table IV
Antibacterial and antifungal activities of the tested samples and of positive controls
Diameter of the inhibition zones (mm)
Sample
S. aureus ATCC 25923 E. coli ATCC 25922 C. albicans ATCC 90028 C. parapsilosis ATCC 22019
C1 16.7 ± 0.06 9.0 ± 0.00 15.5 ± 0.50 18.5 ± 0.50
C2 16.0 ± 0.00 11.5 ± 0.50 15.5 ± 0.50 20.5 ± 0.50
C3 15.5 ± 0.50 8.00 ± 0.00 20.5 ± 0.50 19.5 ± 0.50
C4 16.0 ± 0.00 7.0 ± 0.00 19.5 ± 0.50 15.5 ± 0.50
T1 17.0 ± 0.00 10.0 ± 0.00 20.5 ± 0.50 17.0 ± 0.00
T2 18.3 ± 0.57 7.0 ± 0.00 20.5 ± 0.50 15.5 ± 0.50
T3 15.3 ± 0.57 11.3 ± 0.57 14.0 ± 0.00 14.0 ± 0.00
T4 15.3 ± 0.57 11.0 ± 0.00 19.0 ± 0.00 17.0 ± 0.00
Ciprofloxacin 28.7 ± 0.06 36.5 ± 0.50 NT* NT*
Fluconazole NT* NT* 30.5 ± 0.50 21.0 ± 0.00
Nystatin NT* NT* 23.5 ± 0.50 20.0 ± 0.00
*NT-not tested

All extracts have good antibacterial activity against S. MIC and MBC values are shown in Table V. For most
aureus and medium activity against E. coli. Regarding samples, the MBC values were 2 - 4 times greater
the antifungal potential, all samples have shown good than the MIC values. Out of the tested samples, both
activity against the tested strains. Out of these samples, Chrysanthemum indicum and Tagetes erecta extracts
Chrysanthemum indicum hexane extract (C3) and showed good MIC values for S. aureus, while for
Tagetes erecta chloroform and dichloromethane E. coli the values were higher, which indicates that
extracts (T1, T2) had the best antifungal activity the studied extracts have a better activity on Gram-
against C. albicans. The Chrysanthemum indicum positive bacteria than on Gram-negative bacteria.
dichloromethane extract (C2) had a similar activity to
that of nystatin and fluconazole against C. parapsilosis.

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Table V
MIC and MBC values of the tested samples
S. aureus ATCC 25923 E. coli ATCC 25922
Sample
MIC (mg/mL) MBC (mg/mL) MIC (mg/mL) MBC (mg/mL)
C1 0.25 0.5 0.5 > 0.5
C2 0.125 0.5 0.25 0.25
C3 0.125 0.5 0.5 > 0.5
C4 0.125 0.5 0.5 >0.5
T1 0.25 0.5 0.25 0.25
T2 0.125 0.25 0.5 > 0.5
T3 0.125 0.5 0.25 0.5
T4 0.125 0.5 0.25 0.5
Ciprofloxacin 1.0a 1.0a 1.0a 2.0a
a
values are expressed in µg/mL

The results indicate that secondary metabolites found their chemical profile and toxicity still need to be
in these species possess good antimicrobial properties. done.
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