2İstanbul University Faculty of Pharmacy, Department of Pharmaceutical Microbiology, İstanbul, Türkiye
3Marmara University Faculty of Pharmacy, Department of Pharmaceutical Botany, İstanbul, Türkiye DOI : 10.12991/201317391
Summary
Bu çalışmanın amacı, 5 Centaurea türünün (C.stenolepis, C.kilaea, C.cuneifolia, C.iberica, C.solstitialis subsp. solstitialis) kapitulum ve kapitulumsuz toprak üstü kısımlarından maserasyonla hazırlanan toplam 10 metanol ekstresinin antioksidan, antimikrobiyal aktivitelerini ve toplam fenol içeriklerini araştırmaktır. Türlerin serbest radikal süpürücü aktivite tayinleri DPPH metodu ile, toplam fenolik madde miktar tayini ise Folin–Ciocalteu metoduyla yapıldı. Ekstrelerin antimikrobiyal aktivitesi 7 mikroorganizma türüne karşı mikro dilüsyon yöntemiyle test edildi. DPPH radikal süpürücü aktivite tayini deneyinde test edilen 10 ekstrenin İK50 değerlerinin 1.767-4.665 mg, toplam fenolik madde miktarlarının ise g kuru materyalde gallik asite eşdeğer olarak 4.825-12.460 mg aralığında değişkenlik gösterdiği görüldü. Dört ekstre Pseudomonas aeruginosa (MİK: 312 μg/ml)'ya, 7 ekstre Candida albicans (MİK: 312 μg/ml)'a karşı orta derecede, C.cuneifolia'nın toprak üstü kısımlarımlarından hazırlanan metanol ekstresi ise Staphylococcus aureus'a (MİK: 625 μg/ml) karşı zayıf bir antimikrobiyal aktivite göstermiştir.Introduction
Oxidation is vital to most living organisms. It is required to produce the energy which fuels the biological processes. However, oxygen-centred free radicals and other reactive oxygen species (ROS) produced continuously by most living organisms cause to cell death and tissue damage. Therefore, there is an interest in natural antioxidants such as polyphenols which are found in medicinal and dietary plants so as to prevent oxidative damage[1].Some pathogens are resistant against firstly discovered effective antimicrobial drugs. New compounds inhibiting microorganisms such as benzoin and emetine have been isolated from plants. Contrary to presently used antimicrobial drugs, antimicrobial compounds in plants might inhibit bacterial growth by different mechanisms and may be used as antibiotic against resistant microbial strains. Thus there is a need to find new bioactive compounds of plant origin which can be used in the treatment of resistant microbial strains[2].
The genus Centaurea L.(Asteraceae) is represented by 205 taxon in Turkey[3,4,5]. In traditional medicine, they are used for fever, menstrual disorders, vaginal candidiasis ,the treatment of liver, kidney and ulcer diseases, as antidiarrheal, stomachic, tonic, appetitive, antidiabetic, antipyretic, also as a diuretic and expectorant[6,7].
Methods
Plant MaterialPlant samples were collected in the flowering periods from the different regions of Istanbul in 2009 and were identified by Dr.Gizem BULUT. Voucher specimens were deposited in the Herbarium of the Faculty of Pharmacy, Marmara University (MARE) (Table 1).
TABLE 1: List of plants used in this work.
Plant extraction
The dried and powdered capitulums and aerial parts (except
for capitulum) of Centaurea species were extracted by maceration
with MeOH three times (24h×180ml) at room temperature.
All extracts were filtered, dried under vacuum and stored
under refrigeration for further analysis.
Determination of DPPH radical scavenging activity
Free radical scavenging capacity of methanol extracts of Centaurea
species and standart were evaluated according to the
previously reported procedure using the stable DPPH[1].
Briefly, extracts and standart solution (0.1 ml) in MeOH at different
concentrations (5-0.3125 mg) were added to 3,9 ml
(6 × 10–5 M) methanol solution of DPPH. The mixture was
shaken vigorously and allowed to stand in the dark at room
temperature for 30 min. Absorbance readings were taken at
517 nm. The percent radical scavenging activity of extracts and
standard against DPPH were calculated according to the following:
DPPH radical-scavenging activity (%) = [(A0–A1)/A0]×100
where A0 is the absorbance of the control (containing all reagents
except the test compounds), and A1 is the absorbance of
the extracts/standard. Extract concentration providing 50%
inhibition (IC50) was calculated from the graph plotting inhibition
percentage against extracts concentration. Tests were carried
out in triplicate. Ascorbic acid (AA) was used as positive
control.
Determination of Total Phenolic Contents (TPC)
Total phenolic contents of methanol extracts of Centaurea species
were measured using Folin–Ciocalteau reagent[8]. 0.1 mL
of extracts in various concentrations (5, 2.5, 1.25 mg/ml) were mixed with 0.2 mL Folin-Ciocalteu reagent (Sigma), 2 mL of
H2O, and 1 mL of 15% Na2CO3, and the absorbance was measured
at 765 nm after 2 h incubation at room temperature. Gallic
acid was used as a standard and the total phenolics were
expressed as mg GAE / g dry plant.
Determination of antimicrobial activity
The antimicrobial activity of the extracts were tested against
six bacteria (Staphylococcus aureus ATCC 6538, Staphylococcus
epidermidis ATCC 12228, Escherichia coli ATCC 25922, Klebsiella
pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 27853,
Proteus mirabilis ATCC 14153) and one yeast (Candida albicans
ATCC 10231) by the microbroth dilutions technique strictly
following the recommendations of Clinical Laboratory Standards
Institute (CLSI)[9,10]. Ciprofloxacin and fluconazole
were used as the reference compounds for bacteria and fungi,
respectively.
Statistical analysis
The data were reported as means±standard deviations and
analysed by one-way analysis of variance (ANOVA) followed
by the Tukey’s multiple comparison tests using GraphPad
Prism 5. Differences between means at p<0.05 level were considered
significant.
Results
Extraction yieldsThe extraction yields of Centaurea species were found to range between 8-13.5 % (Table 2).
TABLE 2: The extraction yields of Centaurea species.
DPPH radical scavenging activities of Centaurea species
A low IC50 value (the concentration of extract, which is required
to scavenge 50% of DPPH free radical) is an indication
of strong antioxidant activity. All extracts showed low antioxidant
activity when compared with standard (p<0,05). Also,
Most of aerial parts of Centaurea species showed stronger antioxidant activity than their capitula. The antioxidant activities
of the plant extracts are in the following order: CSA>CSC>CK
A>CSSC>CSSA>CCA>CKC>CIC>CCC>CIA. Results are presented
as IC50 values in the Table 3.
TABLE 3: IC50 values (mg/ml) of extracts.
Total Phenolic Contents of Centaurea species
The total phenolic contents of extracts were calculated using the
equation obtained from the standard curve of gallic acid graph
(y = 0.0033x - 0.044, R2 = 0.9987). The methanol extract prepared
from the aerials part of C. solstitialis subsp. solstitialis (CSSA) had
the lowest total phenolic content among other Centaurea species
(p<0,05). The total phenolic contents of the plant extracts are in
the following order: CSA> CSC> CKA> CCC> CKC> CIC>
CIA> CSSC> CCA> CSSA. However, there was no correlation
between antioxidant activities and total phenolic contents of the
extracts (R2 = 0.0197). The results are presented in Table 4.
TABLE 4: Total phenolic content of the methanol extracts obtained from Centaurea species.
Antimicrobial activities of Centaurea species
All other extracts except CSC, CCC, CSSC and CSSA extracts, exhibited
moderate activity against Candida albicans. Only CCA extract
showed low activity against Staphylococcus aureus, while
CKC, CKA, CIC and CSSA extracts possessed moderate activity
against Pseudomonas aeruginosa. None of the extracts were active
against Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and
Staphylococcus epidermidis. The results are summarized in Table 5.
The present study clearly shows that five Centaurea species have good free radical scavenging activity but further studies are needed to determine cytotoxic activities of these Centaurea species to be used safely instead of synthetic antioxidants. Moreover, the substantial effect most of the extracts have against Candida albicans confirms traditional uses of Centaurea species on vaginal candidiasis.
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