Accepted Manuscript
Biological potentials and cytotoxicity of various extracts from endemic Origa‐
num minutiflorum O. Schwarz & P.H. Davis
Feyza Oke, Belma Aslim
PII:S0278-6915(10)00213-9
DOI:10.1016/j.fct.2010.03.053
Reference:FCT 5348
To appear in:Food and Chemical Toxicology
Received Date:12 January 2010
Accepted Date:31 March 2010
Please cite this article as: Oke, F., Aslim, B., Biological potentials and cytotoxicity of various extracts from endemic Origanum minutiflorum O. S chwarz & P.H. Davis, Food and Chemical Toxicology (2010), doi: 10.1016/j.fct. 2010.03.053
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minutiflorum O. Schwarz & P.H. Davis
Feyza Oke1*and Belma Aslim1
1Department of Biology, Faculty of Art and Science, Gazi University, Ankara 06500, Turkey
*Corresponding author. Feyza Oke,Tel.: +90 312 2021201, Fax: +90 312 2122279, E-mail: feyzaoke@https://www.doczj.com/doc/6b1911611.html,.tr
Running title: Biological potentials of Origanum minutiflorum
minutiflorum O. Schwarz & P.H. Davis
Feyza Oke1*and Belma Aslim1
1Department of Biology, Faculty of Art and Science, Gazi University, Ankara 06500, Turkey
*Corresponding author. Feyza Oke,Tel.: +90 312 2021201, Fax: +90 312 2122279, E-mail: feyzaoke@https://www.doczj.com/doc/6b1911611.html,.tr
Running title: Biological potentials of Origanum minutiflorum
Abstract
The aim of the present study was to investigate antioxidative, antimicrobial and cytotoxic effects of endemic Origanum minutiflorum(O. Schwarz & P.H. Davis).Antioxidant activities of the extracts from O. minutiflorum were evaluated by using DPPH radical scavenging, -carotene bleaching and metal chelating activity assays. In addition, the amounts of total phenol components in the plant extracts were determined. In the -carotene bleaching test, the extracts exhibited in the range of 58.1 ± 0.2% - 98.2 ± 0.3% inhibition against linoleic acid oxidation. The antimicrobial efficiency of the plant was evaluated according to agar well diffusion and microdilution broth methods. The n-hexane extract of O. minutiflorum having an inhibition zone of 20.2 ± 0.2mm, had the maximum antibacterial efficiency against S. sonnei. Cytotoxic effects of the extracts were determined by MTT assay. O. minutiflorum extracts (at concentration of 10-100 μg/ml) did not show any cytotoxic
effect on baby hamster kidney fibroblast cell line. The results showed that O. minutiflorum could be used as a natural source in food industry.
Keywords:Origanum minutiflorum; Antioxidant activity; Antimicrobial activity; Cytotoxicity
1. Introduction
The oxidative deterioration of fats and oils in foods is responsible for rancid odours and flavours, with a consequent decrease in nutritional quality and safety caused by the formation of secondary, potentially toxic, compounds. Antioxidants have been widely used as food additives to protect food quality mainly by the prevention of oxidative deterioration of constituents of lipids. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used as potential inhibitors of lipid peroxidation and thereby stabilizing fat-containing food-stuffs (Moure et al., 2001; Sherwin, 1990). However, these antioxidants have been restricted for use in foods as they are suspected to be carcinogenic (Stone et al., 2003; Wichi, 1988;Fukushima and Tsuda, 1985). The use of spices and herbs as antioxidants in processed foods is a promising alternative to the use of synthetic antioxidants (Madsen and Bertelsen, 1995).
Microbiological food safety has been great concern in every nation irrespective of developed or undeveloped. From the earliest times man has devised ways for preserving foods. In general foods were preserved by use of heat, cold, drying, salting and fermentation. In recent years chemicals such as food additives are extensively used (Kokubo, 1994). Due to consumer concerns about the safety of food containing synthetic chemicals as preservatives, there is a growing interest in the use of natural antibacterial compounds, like extracts of herbs and spices, for the preservation of foods, as these possess characteristic flavors and sometimes show antioxidant activity as well as antimicrobial activity (Smid and Gorris, 1999).
Herbal medicines are generally perceived as safe products (Kinsel and Straus, 2003). But, scientists have been proving that all the natural things are not good for health. Therefore, study on cytotoxicity could give us an idea of the toxic profile of the extracts.
O. minutiflorum (Labiatae) is an endemic plant of Isparta provice in Turkey. It is used as a spice and as a traditional medicinal herb in Anatolia and it can be added to all kinds of food products as a seasoning. To the best of our knowledge, there are no available reports on antioxidant, antimicrobial and cytotoxic effects of the extracts from O. minutiflorum. Especially, wild oregano is an endemic species in Turkey, so it is of special importance for the study. Therefore, the main objectives of this study were (i) to evaluate the antioxidant activity of the plant extracts by using three complementary in vitro assays (ii) to determine the total phenolic contents of the extracts (iii) to investigate the antimicrobial activity of the extracts of O. minutiflorum against some food pathogen bacteria by broth microdilution and agar well diffusion methods and (iv) to determine the cytotoxic effect of the extracts from O. minutiflorum by MTT assay.
2. Materials and Methods
2.1. Chemicals
Nutrient Broth (NB), Tryptic Soy Broth (TSB), chloroform, anhydrous sodium carbonate, Folin-Ciocalteu’s phenol reagent, acetone, ethanol, methanol, glycerol and n-hexane were purchased from Merck (Darmstadt, Germany). 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazoliumbromide (MTT), Tween 40, dimethylsulphoxide (DMSO), ethyl acetate, ethylenediaminetetraacetic acid (EDTA), -carotene, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulphonic acid)-1,2,4-triazine (ferrozine), iron (II) chloride (FeCl2), gallic acid, butylated hydroxyanisole (BHA),2,6-di-tert-butyl-4-methylphenol (BHT), -tocopherol and linoleic acid were purchased from Sigma-Aldrich
GmbH.(Steinheim, Germany).Dulbecco,s modified Eagle’s medium (DMEM),fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA). All other chemicals were analytical grade and obtained from either Sigma or Merck.
2.2. Plant materials
O. minutiflorum plants were collected during the flowering stage in September, 2004, on Sogut mountain (elevation 1684 m, 370 21’ 14 N – 0300 58’ 38 E) from Sutculer-Isparta in Turkey. The identification of plant materials was confirmed by plant taxonomist, Prof. Dr. Hayri DUMAN, in the Department of Biology, Gazi University, Ankara, Turkey.
2.3. Preparation of the extracts
Collected plant materials were dried in the shade and ground in a grinder with a 2 mm in diameter mesh. Thirty grams of the dried and powdered plant materials were separately extracted with different solvents (HPLC grade) by using Soxhlet apparatus for 6 h. The extracts were filtered and concentrated under vacuum by using a rotary evaporator (Heidolph, Laborota 4000, Schwabach, Germany) and stored in the dark at 4°C until used within a maximum period of one week. Acetone, ethanol, ethyl acetate, methanol, n-hexane and water were used as solvents.
2.4. Antioxidant activity
2.4.1. DPPH radical scavenging assay
Radical scavenging activity was determined by a spectrophotometric method based on the reduction of a methanol solution of DPPH using the method of Blois (1958). The extract solutions were added to 0.004% methanol solution of DPPH. The mixture was shaken vigorously and left to stand at room temperature for 30 min in the dark. Then the absorbance
was measured at 517 nm against a blank by a spectrophotometer (Hitachi, U-1800, Tokyo, Japan). Inhibition of free radical, DPPH, in percent (I%) was calculated according to formula: I% = (A control – A sample) / A control x 100
where A control is the absorbance of the control reaction (containing all reagents except the test compound), and A sample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against extract concentration. BHA, BHT and -tocopherol were used as positive controls.
2.4.2. -carotene bleaching assay
The antioxidant activities of the plant extracts were evaluated by the spectrophotometric -carotene bleaching test (Miller, 1971). A stock solution of -carotene-linoleic acid mixture was prepared as follows: 0.5 mg -carotene was dissolved in 1 ml of chloroform (HPLC grade); 25 μl linoleic acid and 200 mg Tween 40 were added. Chloroform was completely evaporated by using a vacuum evaporator. Then, 100 ml of distilled water was added with oxygen (30 min at a flow rate of 100 ml min–1) vigorous shaking. Aliquots (2.5 ml) of this reaction mixture were dispensed to test tubes and 350 μl of the extract prepared at 2 mg/ml concentration was added and the emulsion system was incubated for up to 48 h at room temperature. The same procedure was repeated with the synthetic antioxidants; BHA, BHT and -tocopherol as positive controls and solvents as negative controls. After this incubation period, absorbances of the mixtures were measured at 490 nm.
2.4.
3. Metal chelating activity on ferrous ions (Fe2+)
Metal chelating activity was determined according to the method of Decker and Welch (1990), with some modifications (Oke et al., 2009). Briefly, 0.5 ml of the plant extracts was mixed with 0.05 ml 2 mM FeCl2 and 0.1 ml 5 mM ferrozine. Total volume was diluted with
the solvent. Then, the mixture was shaken vigorously and left standing at room temperature for ten minutes. After the mixture had reached equilibrium, the absorbance of the solution was then measured spectrophotometrically at 562 nm. The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the formula given below:
Scavenging effect (%) = [(A control ? A sample) / A control] × 100
where A control is the absorbance of the ferrozine-Fe2+ complex and A sample is the absorbance of the test compound. EDTA was used for comparison.
2.4.4. Determination of total phenolic contents
Total phenolic contents of the extracts were analysed using the Folin-Ciocalteu reagent according to the method of Singleton and Rossi (1965) using gallic acid as standard, with some modifications (Oke et al., 2009). The extract solutions were mixed with 0.2 ml of 50% Folin-Ciocalteu reagent. The mixture was allowed to react for 3 min and 1 ml aqueous solution of 2% Na2CO3 was added. At the end of incubation for 45 min at room temperature, absorbance of each mixture was measured at 760 nm. The same procedure was also applied to the standard solutions of gallic acid. Total phenol contents were expressed as μg gallic acid equivalents per mg of the extracts.
2.5. Antimicrobial activity
2.5.1. Test bacteria
Escherichia coli O157:H7, Shigella sonnei RSKK 878, Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 7644, Pseudomonas aeruginosa ATCC 27853 were used as test bacteria. NB and TSB were used for culturing of test bacteria. All strains were stored at -20°C in the appropriate medium containing 10% glycerol and regenerated twice before use in the manipulations.
2.5.2. Inhibitory effect by the agar well diffusion method
The determination of the inhibitory effect of the extracts on test bacteria was carried out by agar well diffusion method (Kalemba and Kunicka, 2003). Bacterial cultures were grown at 37°C for 24 h in NB. L. monocytogenes ATCC 7644 was cultured in TSB. The culture suspensions were adjusted by comparing against 0.5 McFarland. Petri dishes (90x20 mm) with 20 ml of Nutrient Agar were prepared, previously inoculated with 200 μl of the culture suspension. The wells (7.0 mm in diameter) were made and the extracts were added to wells (100 μl) and the same volumes (100 μl) of solvents were used as controls. The inoculated plates were incubated for 24 h. After incubation, the diameter of the inhibition zone was measured with a calliper. The measurements were done basically from the edge of the zone to the edge of the well.
2.5.
3. Determination of minimum inhibitory concentrations (MIC)
Antimicrobial activity of the extracts was evaluated through the determination of the MIC by the microdilution method in culture broth (Koneman et al., 1997). Stock solution of the extracts was prepared in DMSO and then serial dilutions of the extracts were made in a concentration range from 125–2000 μg/ml. The 96-well plates were prepared by dispensing into each well 95 μl of NB, 100 μl of the extract (dissolved in DMSO) and 5 μl of the inoculum. The inoculum of microorganisms was prepared using 24 h cultures and suspensions were adjusted to 0.5McFarland standard turbidity. The final volume in each well was 200 μl.
A positive control (containing 5 μl inoculum and 195 μl NB) and negative control (containing 100 μl of extract dissolved in DMSO, 100 μl N
B without inoculum) were included on each microplate. The contents of the wells were mixed by pipetting and the microplates and the microplates covered with its own sterile bag were incubated at 37°
C for 24 h. The MIC was
defined as the lowest concentration of the compounds to inhibit the growth of microorganisms.
2.6.Cytotoxicity analysis
2.6.1. Cell culture
Baby hamster kidney fibroblast (BHK 21) cells were obtained from Sap Institute (Ankara, Turkey). The cells were grown at 37oC in humidified 5% CO2, 95% air mixture in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100U/ml of penicillin and 100 μg/ml streptomycin. The medium was replaced 3 times in a week until the cells were near confluency.
2.6.2. Cytotoxic effect of the extracts
The extracts of the O. minutiflorum were evaluated on baby hamster kidney fibroblast cell line (BHK 21) in order to examine their cytotoxic effects on normal cells. BHK 21 cells were seeded in a 96-well plates (2×104 cells /well) and incubated at 37oC with 5% CO2. After 48 h incubation, the cells were treated with the extracts (10 μg/ml to 100 μg/ml) for 24 h at 37oC. Cytotoxic effect was determined for the aqueous and the methanol extracts which showed highest antioxidant activity. The methanol extract of O. minutiflorum was dissolved in the minimum amount of DMSO, so that the final concentration of DMSO was less than 0.5%, DMSO at this concentration has not been reported toxic for the cells. The aqueous extract was dissolved in DMEM. Following the removal of the extracts from the wells, the cells were washed in phosphate buffered saline. Cell viability was determined by MTT assay.
2.6.
3. MTT bioassay
The MTT assay is a test of metabolic competence based upon assessment of mitochondrial performance. It is a colorimetric assay relying on the conversion of yellow
tetrazolium bromide (MTT) to the purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells (Mosmann, 1983). The cells were incubated in serum-free medium to which MTT (5 mg/ml, 15 μl) was added. Following 4 h incubation, the medium was removed and 100μL of DMSO was added to dissolve the formazan crystals. Absorbance was measured in an ELISA micro plate reader (ELx800, BioTek, US) at 540 nm. Viability was defined as the ratio (expressed as a percentage) of absorbance of treated cells to untreated cells that served as control.
2.7. Statistical analysis
All experiments were done in triplicate, and mean values are presented. The results were expressed as means ± standard deviations (SD). Statistical analysis was performed using the SPSS 11.0 (SPSS, Chicago, IL). Pearson’s correlation analysis was used to determine the differences between the values. The analysis was used for comparison of the values from microdilution broth and agar well diffusion methods and for comparison of the values from total phenol contents and the antioxidant activity assays. The level of statistical significance was taken at p<0.05.
3. Results and Discussion
3.1. The yield of the extracts
Solubility of phenolics is governed by their chemical nature in the plant that may vary from simple to much polymerized substances. Plant materials may contain varying quantities phenolic acids, phenylpropanoids, anthocyanins and tannins, among others. There is a possibility of interaction of phenolics with other plant components such as carbohydrates and proteins. These interactions may lead to the formation of complexes that may be quite insoluble. Solubility of phenolics is also affected by their polarity of solvent(s) used. Solvents
such as methanol, ethanol, acetone, ethyl acetate and their combinations have also been used for the extraction of phenolics (Naczk and Shahidi, 2006). As the results presented in Table 1, the yields obtained from various extracts of O. minutiflorum varied considerably. Differences in the yield of extracts might be attributed to the availability of different extractable components. The highest yield was obtained from water (27.4% w/w) followed by ethanol (20.7% w/w) and methanol (20.3% w/w). The extraction ability of acetone (12.3% w/w) and ethyl acetate (12.2% w/w) were very similar to one another, whereas the extraction yield with n-hexane (4.3% w/w) was only small in comparison with that of the other solvents.
3.2. Antioxidant activity of the extracts
3.2.1. DPPH radical scavenging assay
Antioxidants affect the process of lipid oxidation at different stages due to differences in their mode of action. Because of the complexity of the oxidation process itself, the diversity of the substrates and the active species involved, the application of different test methods is necessary in the evaluation of antioxidants (Koleva et al., 2003).The free radical scavenging activity was investigated in DPPH assay. The effect of antioxidant on DPPH radical scavenging was thought to be due to their hydrogen donating ability or radical scavenging activity. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form diphenypicrylhydrazine (non radical) with the loss of this violet colour. DPPH percent scavenging activities of the plant extracts and synthetic antioxidants were measured in different concentrations ranging between 10 and 50 μg/ml. The IC50, meaning the concentration of antioxidant needed to decrease (by 50%) the initial substrate concentration, is a parameter widely used to measure the antiradical efficiency. The lower IC50 values show the higher antioxidant activity. The values for DPPH scavenging activities of the various extracts, BHA, -tocopherol and BHT are compared and
shown in Table 2, as calculated from the percent inhibition versus log concentration of extract curves. With regard to IC50 values of scavenging abilities on DPPH radicals, the effectiveness was in a descending order: BHA > -tocopherol > the aqueous extract > BHT > the methanol extract > the ethanol extract > the acetone extract > the ethyl acetate extract > the n-hexane extract. The highest DPPH radical scavenging effect was detected in the aqueous extract (IC50 = 16.1 ± 0.2μg/ml) followed by the methanol and the ethanol extracts (IC50 = 30.1 ± 0.3μg/ml and IC50 = 40.6 ± 0.3 μg/ml, respectively). Also, the acetone and the ethyl acetate extracts indicated remarkable scavenging activities (IC50 = 42.5 ± 0.2 μg/ml and IC50 = 51.5 ± 0.7μg/ml, respectively). The n-hexane extract exhibited weak antioxidant activity (IC50 = 167.2 ± 0.8 μg/ml). Compared to reference antioxidants, the aqueous extract of O. minutiflorum provided a lower IC50 than BHT (IC50 = 23.1 ± 1.4 μg/ml) and higher IC50 than BHA (IC50 = 3.0 ± 0.1 μg/ml)and -tocopherol (IC50 = 11.3 ± 0.2 μg/ml). These results showed the aqueous extract has more effective scavenging ability on DPPH radicals than synthetic antioxidant BHT.
3.2.2. -carotene-linoleic acid bleaching assay
-carotene bleaching method is based on the loss of the yellow colour of -carotene due to its reaction with radicals formed by linoleic acid oxidation in an emulsion. The rate of -carotene bleaching can be slowed down in the presence of antioxidants (Kulisic et al., 2004). The relative antioxidative activities (RAAs) of the extracts were calculated from the equation, RAA = A sample / A -tocopherol, where A -tocopherol is the absorbance of the control ( -tocopherol) and A sample is the absorbance of the extracts. The calculated RAAs of the extracts are given in Table 2. The extracts exhibited in the range of 58.1 ± 0.2% – 98.2 ± 0.3% inhibition against linoleic acid oxidation. All the oregano extracts showed remerkable inhibition activities. Inhibition of linoleic acid oxidation is an important issue in food
processing and preservation. Therefore, the oregano extracts especially the aqueous extract may be used as food additives to protect food. Also, this result was similar to data from the DPPH radical-scavenging method. In both assays, the aqueous extract showed better antioxidative capacity than the other extracts and synthetic antioxidant BHT.
3.2.3. Metal chelating activity on ferrous ions (Fe2+)
Among the transition metals, iron is known as the most important lipid oxidation pro-oxidant due to its high reactivity. The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen peroxide and lipid peroxides to reactive free radicals via the Fenton reaction (Fe2+ + H2O2 Fe3+ + ·OH + OH-). Fe3+ ion also produces radicals from peroxides, although the rate is tenfold less than that of Fe2+ ion (Miller et al., 1996). Chelating activities of the extracts were determined by the ferrozine assay. Ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with the result that the purple colour of the complex is decreased. Effects of the extracts on inhibition of ferrylbipyridyl formation are presented in Table 2. Significant differences in chelating activity were observed among the extracts. The aqueous extract showed the highest ferrous iron chelating ability (At 5 mg/ml,74.8 ± 0.2%).Second highest abilities showed in the methanol extract (59.9 ± 0.2%) followed by the ethanol extract (39.4 ± 0.1%) at 5 mg/ml. On the other hand, the acetone, the ethyl acetate and the n-hexane extracts exhibited weak chelating activity (At 5 mg/ml21.2 ± 0.3%, 18.5 ± 0.2%, 7.2 ± 0.2%, respectively). However, EDTA showed an excellent chelating ability of 93.7 ± 0.3% at 5 mg/ml.
3.2.
4. Determination of total phenolic contents
Phenolic compounds, biologically active components, are the main agents that can donate hydrogen to free radicals and thus break the chain reaction of lipid oxidation at the first initiation step. This high potential of phenolic compounds to scavenge radicals may be explained by their phenolic hydroxyl groups (Sawa et al., 1999). The amounts of total phenolics in the extracts were determined spectrometrically according to the Folin-Ciocalteu procedure and calculated as gallic acid equivalents. The standard curve equation is, y (absorbance) = 0.0063 x (μg gallic acid) - 0.0101, R2 = 1. The absorbance value was inserted in the above equation and the total amount of phenolic compound was calculated. The amounts of total phenols found in the plant extracts were very high. Total phenolic content of the extracts were solvent dependent.The content of total phenolics decreased in the order of the methanol extract > the aqueous extract > the ethanol extract > the acetone extract > the ethyl acetate extract > the n-hexane extract (Table 2). The total phenolic contents of the methanolic extract (144.22 ± 0.62 μg/mg) and the aqueous extract (137.40 ± 0.22 μg/mg) higher than those of the other extracts. The aqueous extract exhibited higher -carotene bleaching, DPPH radical scavenging, and metal chelating activities than the methanol extract. But the methanol extract possess higher amounts of phenolics than the aqueous extract. There may be different kinds of total phenolic compounds (hydrophilic and hydrophobic) in different extracts. The n-hexane extract (114.94 ± 0.12 μg/mg) and the ethyl acetate extract (123.19 ± 0.21 μg/mg) have lower total phenolic compounds than the other extracts. Total phenol contents of the extracts showed significant correlation with DPPH IC50 values (80% correlation) and lipid peroxidation inhibition percents (97% correlation) (p<0.05).
3.3. Antimicrobial activity
The antibacterial activities of the herb extracts are presented in Table 3. The n-hexane extract exhibited strong antimicrobial activity against foodborne bacteria. Also, the acetone and the ethyl acetate extracts showed remarkable antibacterial activity. There is no significant difference between the ethanol and the methanol extracts and both of these extracts showed moderate antibacterial activity. The data for the inhibition zones (mm) of various microorganisms indicate that the aqueous extract had no effect against tested bacteria (Table 3). Several researchers have generally reported that the aqueous extracts of plants do not have much antibacterial activity (Paz et al., 1995; Vlietinck et al., 1995). The antibacterial activity of the extracts was in the range of 5.0 ± 0.1 – 20.2 ± 0.2(mm, inhibition zone diameter). The n-hexane extract had a broad spectrum of activity against both the gram-positive and gram negative bacteria. In general, L. monocytogenes ATCC 7644 was the most resistant bacterium, while S. sonnei RSKK 878 was the most sensitive. Among all extracts, the n-hexane extract was the most active against S. sonnei (20.2 ± 0.2mm). The ethanol and the methanol extracts exhibited the least antimicrobial activity as compared to the other three extract.As expected, the control treatment (solvents) had no inhibitory effect on any of the test bacteria.
MIC values were determined for n-hexane and acetone extracts of O. minutiflorum which showed highest inhibition zones in the agar diffusion method. MIC values for bacterial strains which were sensitive to the n-hexane and the acetone extracts of O. minutiflorum were in the range of 125-2000 μg/ml (Table 3). The highest inhibitory activity for the n-hexane extract was against S. sonnei RSKK 878 which showed the lowest MIC (125 μg/ml) and largest growth inhibition halos for agar well diffusion assay (20.2 ± 0.2 mm). On the other hand, the acetone extract exhibited the highest inhibitory activity against S. aureus ATCC 25923 which showed the lowest MIC (250 μg/ml) and largest growth inhibition halos (14.8 ± 0.2 mm). L. monocytogenes ATCC 7644 was the most resistant bacterium and exhibited
highest MIC (2000 μg/ml) for both of the extracts. The inhibition zones of the extracts on test bacteria showed a significant correlation with MIC values (p<0.05). Control treatment (DMSO) did not show an inhibitor effect on any of the bacteria.
This active extracts for all test bacteria will be helpful devising antimicrobial formulations with which to protect foods against infection by multiple pathogens. This study demonstrates the in vitro antimicrobial activity of the extracts of this endemic remedy against foodborne bacteria, being the first report on the antibacterial properties of O. minutiflorum various extracts. Food safety is an increasingly important public health issue. Many of the cases of acute diarrheal disease in both developing and industrialized countries are due to infection by Shigella species (Alcoba-Florez et al., 2005). The estimated incidence of shigellosis is 164.7 million cases annually, of which 163.2 million occur in developing countries, resulting in 1.1 million deaths. Moreover, 69% of all episodes of Shigella infection and 61% of all Shigella related deaths involve children younger than 5 years old (Ashkenazi, 2004). Shigella dysenteriae and Shigella sonnei are the predominant species in the tropics, while S. sonnei is the predominant species in industrialized countries (Preston, 1994). Controlling the numbers and growth of Shigella sonnei therefore remains an important objective for sectors of the food production industry. O. minutiflorum is a well-known aromatic plant which is frequently used as a spice and as a traditional medicinal herb in Anatolia and may be used as a natural preservative against Shigella sonnei for the food production industry.
3.3. Effect of extracts on cell cytotoxicity
The extracts of O. minutiflorum were evaluated on baby hamster kidney fibroblast cell line (BHK 21) in order to examine their cytotoxic effects on normal cells. Cytotoxic effects were determined for the aqueous and the methanol extracts of O. minutiflorum which showed highest antioxidant activity. Cytotoxicity of the extracts on the growth of the BHK 21 cell line
are summarized in Figure 1. Cell proliferation was analyzed 24 h after BHK 21 cells had been cultured with an extract of 10, 25, 50, 100 g /ml in the final concentration using the MTT assay. The aqueous and the methanol extracts of O. minutiflorum at concentrations of 10–100 μg/ml did not show any cytotoxic effect in BHK 21 cell line.
4. Conclusions
Our results indicated water extraction is a good method to extract antioxidant compounds found in this species. Water is preferred for the extraction of antioxidant compounds mainly because it has not toxicity. The use of extracts of O. minutiflorum as natural preservatives will be suitable for applications on the food industry to prevent the growth of foodborne bacteria and extend the shelf-life of the processed food. These properties are due to many substances, including some vitamins, polyphenols, flavonoids, terpenoids, carotenoids, phytoestrogens, minerals, etc. Further research should be carried out to elucidate the components responsible for biological activity of these extracts.
Acknowledgements
The authors wish to thanks to Prof. Dr. Hayri DUMAN, Department of Biology, Faculty of Science and Art, University of Gazi, the identification of the plant material collected and Tubitak for fellowship to F. OKE.
References
Alcoba-Flórez, J., Pérez-Roth, E., González-Linares, S., Méndez-álvarez, S., 2005. Outbreak of Shigella sonnei in a rural hotel in La Gomera, Canary Islands, Spain. International Microbiology, 8, 133-136.
Ashkenazi, S., 2004. Shigella infections in children: new insights. Seminars in Pediatric Infectious Diseases. 15, 246-252.
Blois, M. S., 1958. Antioxidant determinations by the use of a stable free radical. Nature, 181, 1199-1200.
Decker, E. A. and Welch, B., 1990. Role of ferritin as a lipid oxidation catalyst in muscle food. Journal of Agricultural and Food Chemistry, 38, 674-677.
Fukushima, N. S. and Tsuda, H., 1985. Carcinogenity and modification of the carcinogenic response by BHA, BHT and other antioxidants. Critical Reviews in Toxicology, 15, 109-150.
Kalemba, D. and Kunicka, A., 2003. Antibacterial and antifungal properties of essential oils. Current Medicinal Chemistry, 10, 813-829.
Kinsel, J.F. and Straus, S.E., 2003. Complementary and alternative therapeutics: rigorous research is needed to support claims. Annual Review of Pharmacology and Toxicology 43, 463–484.
Kokubo, Y., 1994. Hazard analysis critical control point (HACCP) system for the control of food microbiological safety. Japanese journal of food microbiology, 11, 67-74 (In Japanese).
Koleva, I. I., Linssen, J. P. H., van Beek, T. A., Evstatieva, L. N., Kortenska, V., Handjieva, N., 2003.Antioxidant activity screening of extracts from Sideritis species (Labiatae) grown in Bulgaria. Journal of the Science of Food and Agriculture, 83, 809-819.
Koneman, E.W., Allen, S.D., Janda, W.M,, Scherckenberger, P.C., Winn, W.C., 1997. Color Atlas and Textbook of Diagnostic Microbiology. Philadelphia: Lippincott-Raven. Kulisic, T., Radonic, A., Katalinic, V., Milos, M. 2004. Use of different methods for testing antioxidative activity of oregano essential oil. Food Chemistry, 85, 633-640. Madsen, H. L. and Bertelsen, G., 1995. Spices as antioxidants. Trends in Food Science and Technology, 6, 271-277
Miller, H. E., 1971. A simplified method for the evaluation of antioxidants. Journal of American Oil Chemists’ Society, 48, 91.
Miller, N. J., Sampson, J., Candeias, L. P., Bramley, P. M. and Rice-Evans, C. A., 1996. Antioxidant activities of carotenes and xanthophylls. FEBS Letters, 384, 240-242. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival, application to proliferation and cytotoxicity assay. Journal of Immunological Methods, 65, 55- 63. Moure, A., Cruz, J. M., Franco, D., Domínguez, J. M., Sineiro, J., Domínguez, H., Nu?ez, M.J. and Parajó, J. C., 2001. Natural antioxidants from residual sources. Food Chemistry, 72, 145-171.
Naczk, M. and Shahidi, F., 2006. Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41, 1523-1542.
Oke, F., Aslim, B., Ozturk, S., Altundag, S., 2009. Essential oil composition, antimicrobial and antioxidant activities of Satureja cuneifolia Ten. Food Chemistry, 112, 874-879. Paz, E. A., Cerdeiras, M. P., Fernandez, J., Ferreira, F., Moyna, P., Soubes, M., Vázquez, A., Vero, S., Zunino, L., 1995. Screening of Uruguayan medicinal plants for antimicrobial activity. Journal of Ethnopharmacology, 45, 67-70.
Preston, M. A. and Borczyk, A.A., 1994. Genetic variability and molecular typing of Shigella sonnei strains isolated in Canada. Journal of Clinical Microbiology, 32, 1427-1430.