Excli 10 230
Excli 10 230
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
Original article:
ABSTRACT
Antioxidant and antimicrobial effects, total phenolic content and flavonoid concentrations of
methanolic, acetone and ethyl acetate extracts from Xeranthemum annuum L. were investi-
gated in this study. The total phenolic content was determined using Folin-Ciocalteu reagent
and ranged between 101.33 to 159.48 mg GA/g. The concentration of flavonoids in various X.
annuum extracts was determined using spectrophotometric method with aluminum chloride
and the results varied from 22.25 to 62.42 mg RU/g. Antioxidant activity was monitored spec-
trophotometrically using DPPH reagent and expressed in terms of IC50 (µg/ml), and it ranged
from 59.25 to 956.81 µg/ml. The highest phenolic content and capacity to neutralize DPPH
radicals were found in the acetone extract. In vitro antimicrobial activity was determined by
microdilution method. Minimum inhibitory concentration (MIC) and minimum microbicidal
concentration (MMC) have been determined. Testing was conducted against 24 microorgan-
isms, including 15 strains of bacteria (standard and clinical strains) and 9 species of fungi.
Statistically significant difference in activity between the extracts of X. annuum L. was ob-
served and the acetone extract was found most active. The activity of acetone extract was in
accordance with total phenol content and flavonoid concentration measured in this extract.
The tested extracts showed significant antibacterial activity against G+ bacteria and weak to
moderate activity against other microorganisms. Based on the obtained results, X. annuum can
be considered as a rich natural source of polyphenolic compounds with very good antioxidant
and antimicrobial activity.
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EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
A large number of known medicinal gated on bacterial and fungal strains by mi-
species belonging to the family Asteraceae crodilution method.
are used in phytomedicine and pharmacy.
For very long time medicinal plants of this MATERIAL AND METHODS
family have been used in the treatment of
Plant material
many diseases of the digestive, respiratory,
In August 2009 aerial parts of X. annu-
and cardiovascular systems and skin dis-
um were collected from natural populations
eases, as well as for the preparation of bev-
on Plackovica hill in the region of Vranje
erages, and as culinary spices. The species
sity in south Serbia: (position:
of the family Asteraceae are very rich in
42°34′51.94′′N, 21°53′53.90′′E, altitude:
phenolic compounds with very strong bio-
1075.00 m, exposition: E, habitat: arid
logical activity and they exhibit strong anti-
thermophilic rocky meadows). Plants
oxidant, antibacterial, antifungal, antiviral
identified were confirmed and voucher
and antiproliferative effects (Özgen et al.,
specimens deposited at the Herbarium of
2004; Boussaada et al., 2008; Jayaraman et
the Department of Biology and Ecology,
al., 2008; Muley et al., 2009; Kasim et al.,
Faculty of Science, University of Kragu-
2011).
jevac. The collected plant material was air-
Literature data indicate that the X.
dried in darkness at ambient temperature
annuum is a medicinal plant in traditional
(20 °C). The dried plant material was cut up
medicine and it is applied as a source of
and stored in tightly sealed dark containers.
active substances (Vogl-Lukasser and Vogl,
2004; Watson and Preedy, 2008). However,
Chemicals
there is very little data on laboratory
Organic solvents and sodium hydrogen
phytochemical studies and biological
carbonate were purchased from „Zorka
activity of extracts and isolated components
pharma“ Šabac, Serbia. Gallic acid, rutin
from X. annuum, which points to the fact
hydrate, chlorogenic acid and 2,2-diphenyl-
that the plant has not been explored
1-picrylhydrazyl (DPPH) were obtained
completely. The existing data often provide
from Sigma Chemicals Co., St Louis, MO,
chemical properties of secondary
USA. Folin-Ciocalteu phenol reagent, and
metabolites from X. annuum and the
aluminium chloride hexahydrate (AlCl3)
description of the most common
were purchased from Fluka Chemie AG,
metabolites (Zemtsova and Molchanova,
Buchs, Switzerland. Nutrient liquid
1979; Skaltsa et al., 2000).
medium, a Mueller–Hinton broth was
Bearing in mind that the family
purchased from Liofilchem, Italy, while a
Asteraceae comprises species that are well
Sabouraud dextrose broth was obtained
known in phytomedicine and pharmaceu-
from Torlak, Belgrade. An antibiotic, doxy-
tical industry, it is obvious that the
cycline, was purchased from Galenika
evaluation of X. annuum as a new source of
A.D., Belgrade, and antimycotic, flucona-
natural medicinal substances may contri-
zole was from Pfizer Inc., USA. All other
bute to the knowledge about the use and
solvents and chemicals were of analytical
importance of the family.
grade.
Therefore, the purpose of this study was
to explore X. annuum as a new potential
natural source of effective antioxidant and Preparation of plant extracts
antimicrobial agents, as well as its total Prepared plant material (10 g) was
phenolic content and flavonoid concentra- transferred to dark-coloured flasks with
tions. Antioxidant activity, phenolic content 200 ml of solvent (methanol, ethyl acetate,
and flavonoid concentrations are deter- acetone) and stored at room temperature.
mined by spectrophotometric methods and After 24 h, infusions were filtered through
in vitro antimicrobial activity was investi- Whatman No. 1 filter paper and residue was
re-extracted with equal volume of solvents.
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EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
After 48 h, the process was repeated. ing the method described by Tekao et al.
Combined supernatants were evaporated to (1994), adopted with suitable modifications
dryness under vacuum at 40 °C using rotary from Kumarasamy et al. (2007). The stock
evaporator. The obtained extracts were kept solution of the plant extract was prepared in
in sterile sample tubes and stored in a methanol to achieve the concentration of
refrigerator at 4 °C. 1 mg/ml. Dilutions were made to obtain
concentrations of 500, 250, 125, 62.5,
Determination of total phenolic contents 31.25, 15.62, 7.81, 3.90, 1.99, 0.97 µg/ml.
in the plant extracts Diluted solutions (1 ml each) were mixed
The total phenolic content was deter- with 1 ml of DPPH methanolic solution
mined using spectrophotometric method (80 µg/ml). After 30 min in darkness at
(Singleton et al., 1999). The reaction mix- room temperature (23 °C) the absorbance
ture was prepared by mixing 0.5 ml of was recorded at 517 nm. The control sam-
methanolic solution (1 mg/ml) of extract, ples contained all the reagents except the
2.5 ml of 10 % Folin-Ciocalteu’s reagent extract. The percentage inhibition was cal-
dissolved in water and 2.5 ml 7.5 % Na- culated using the equation: % inhibition =
HCO3. The samples were incubated at 100 x (A control – A sample)/A control),
45 °C for 15 min. The absorbance was de- whilst IC50 values were estimated from the
termined at λmax = 765 nm. The samples % inhibition versus concentration sigmoidal
were prepared in triplicate and the mean curve, using a non-linear regression analy-
value of absorbance was obtained. Blank sis. The data were presented as mean values
was concomitantly prepared with methanol ± standard deviation (n = 3).
instead of extract solution. The same proce-
dure was repeated for the gallic acid and the Test microorganisms
calibration line was construed. The total Antimicrobial activity of acetone, ethyl
phenolic content was expressed in terms of acetate and methanolic extract was tested
gallic acid equivalent (mg of GA/g of ex- against 24 microorganisms including fifteen
tract). strains of bacteria (standard strains:
Escherichia coli ATCC 25922,
Determination of flavonoid concentrations Staphylococcus aureus ATCC 25923,
in the plant extracts Enterococcus faecalis ATCC 29212,
The concentrations of flavonoids was Pseudomonas aeruginosa ATCC 27853,
determined using spectrophotometric Bacillus subtilis ATCC 6633, Bacillus
method (Quettier et al., 2000). The sample pumilus NCTC 8241 and clinical strains:
contained 1 ml of methanolic solution of Escherichia coli, Staphylococcus aureus,
the extract in the concentration of 1 mg/ml Enterococcus faecalis, Pseudomonas
and 1 ml of 2 % AlCl3 solution dissolved in aeruginosa, Proteus mirabilis, Sarcina
methanol. The samples were incubated for lutea, Salmonella enterica, Bacillus subtilis
an hour at room temperature. The absorb- and Bacillus cereus) and nine species of
ance was determined at λmax = 415 nm. The fungi: Penicillium italicum PMFKG-F29,
samples were prepared in triplicate and the Penicillium digitatum PMFKG-F30,
mean value of absorbance was obtained. Penicillium chrysogenum PMFKG-F31,
The same procedure was repeated for the Trichothecium roseum PMFKG-F32,
rutin and the calibration line was construed. Botrytis cinerea PMFKG-F33; Aspergillus
The concentration of flavonoids in extracts niger ATCC 16404; Candida albicans
was expressed in terms of rutin equivalent (clinical isolate); Rhodotorula sp. PMFKG-
(mg of RU/g of extract). F27 and Saccharomyces boulardii
PMFKG-P34. All clinical isolates were a
Evaluation of DPPH scavenging activity generous gift from the Institute of Public
The ability of the plant extract to scav- Health, Kragujevac. The other microorga-
enge DPPH free radicals was assessed us- nisms were provided from a collection held
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EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
233
EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
234
EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
235
EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
Table 3: Antibacterial activities of acetone, ethyl acetate and methanolic extracts of X. annuum
against tested microorganisms based on microdilution method
Table 4: Antifungal activities of acetone, ethyl acetate and methanolic extracts of X. annuum against
tested microorganisms based on microdilution method
The tested extracts showed high anti- 20 mg/mL. The acetone extract showed a
bacterial activity against G+ bacteria, significant effect against B. pumilus NCTC
especially for species of the genus Bacillus 8241 and B. cereus. Clinical isolate of S.
(clinical isolates and standard strains). aureus also showed a promising sensitivity
MICs values were in range from to acetone extract, MIC was at
0.625 mg/mL to 10 mg/mL, and MMCs 0.625 mg/mL and MMC at 1.25 mg/mL.
values were from 1.25 mg/mL to
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EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
237
EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
238
EXCLI Journal 2011;10:230-239 – ISSN 1611-2156
Received: September 14, 2011, accepted: November 20, 2011, published: November 24, 2011
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