Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2013 , Vol 17 , Num 1
In vitro evaluation of antioxidant and antimicrobial activities of some Centaurea L. species
Ali Şen1, Leyla Bitiş1, Seher Birteksöz-Tan2, Gizem Bulut3
1Marmara University Faculty of Pharmacy, Department of Pharmacognosy, İstanbul, Türkiye
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 Material
Plant 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 yields
The 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.

TABLE 5: The MIC (Minimum Inhibitory Concentration) values (μg/mL) of the methanol extracts obtained from Centaurea species.

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.

Reference

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