Investigating of Phytochemicals , Antioxidant , Antimicrobial and Proliferative Properties of Different Extracts of Thymus spathulifolius Hausskn . and Velen . Endemic Medicinal Plant from Sivas , Turkey

Thymus species has been used for antioxidant, antiseptic, antitussive, carminative, anti-inflammatory and antimicrobial activities as well as tonic and herbal teas. The present study was conducted to evaluate antioxidant, antimicrobial and proliferative properties of ethanol crude extract and fractions of Thymus spathulifolius (Hausskn. and Velen.) herbes. The antioxidant properties of ethanol extracts and fractions of Thymus spathulifolius were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, total flavonoid and total phenolic content analysis. Antimicrobial activity of the plant extracts were tested using the microdilution method, while proliferative activity were evaluated by MTT assay. Results showed that IC50 of T. spathulifolius extracts that scavenged 50% of the DPPH radical was found to be ranged from 62.39 to 1000 μg/mL. Hexane extract possessed moderate antimicrobial activity towards grampositive bacteria of S. aureus and fungi of C. albicans. S. aureus was the most sensitive bacteria than other tested microorganisms. The hexane and water extracts exhibited a good proliferative activity with ED50 of 3.28 μg/mL and 2.77 μg/mL, respectively. The antioxidant and antimicrobial activities, together with the ability of proliferation, provide some support for the T.spathulifolius’s traditional use.


INTRODUCTION
Herbal medicine has been used for the effective treatment of various disorders in various forms including decoction, maceration, powdered sample, oleoresins, crud extracts, fixed oil, essential oil etc. [1].It is well recognized that free radicals are critically involved in various pathological conditions such as cancer, cardiovascular disorders, arthritis, inflammation and liver diseases [2].Many studies have shown that natural antioxidants in medicinal plants are closely related the prevention or suppression of aging and many diseases associated with oxidative stress; cancer, cardiovascular diseases, rheumatoid arthritis, autoimmune diseases [3].These beneficial effects have been partly attributed to antioxidants, which may play important roles in inhibition of free radicals and oxidative chain-reactions within tissues and membranes [4].The majority of the active antioxidant compounds are flavonoids, isoflavones, flavones, anthocyanins, coumarins, lignans, catechins, and isocatechins [5].
The genus Thymus L., belonging to Lamiaceae family, comprises approximately 300 evergreen species of herbaceous perennial and subshrubs, native to southern Europe and Asia [6].This genus is represented by 38 species and 64 taxa, 24 of which are endemic in Turkey and the East Aegean Islands [7][8].Various species of Thymus is used all over the world as condiments, ornamentals and sources of essential oil [9].The genus are used in traditional medicine as tonic, antiseptic, antitussive and carminative as well as for treating colds and in pharmaceutical, cosmetic and perfume industry for preservation of several food products or as condiments [10].Members of this genus are locally known as ''kekik'' in Turkish and are used as herbal tea and condiments either in the form of fresh or dry [11].
Thymus spathulifolius Hausskn.and Velen. is a dwarf shrub, up to 10 cm high, endemic in inner Anatolia, especially distributed in Sivas, growing in wild in open steppe on gypsous or poor fertile calcareous soils [12].In terms of Pharmacological and phytochemical studies, many Thymus species have been reported for antioxidant, antimicrobial and other biological activities [13][14][15].In addition, in the search for phytochemicals, plant parts are usually screened for phytochemicals that may be present.The presence of a phytochemical of interest may lead to its further isolation, purification and characterization.Then it can be used as the basis for a new pharmaceutical product.Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction process.This therefore underscore the need to try as much solvent as possible in screening plant parts for phytochemicals [16][17].Nevertheless, to the best of our knowledge, no data on phytochemical studies of the Thymus species are available to now.This study aims to evaluate the antioxidant activity by different methods such as DPPH, metal chelating, FRAP, ferric reducing and ABTS tests, the antimicrobial activity agains five gram-positive and gram-negative bacteria and fungi, and proliferative effect of different extracts prepared from T. spathulifolius .For pharmaceutical application and naturaceutical industry, the present study may supply important information on the phytochemical properties of the Thymus species as well as biological activities.

Plant Materials
Test plants were collected during the flowering period from natural populations in Sivas province of Turkey.Collected locality is: B6 Sivas: Zara-Divriği road, between Bolucan-Sincan.Plant materials were prepared as herbarum vouchers.Thes were registered under collector number as M.Tekin 1744.Voucher specimens are conseved at the Cumhuriyet University, Faculty of Science Herbarium (CUFH), Department of Biology, Sivas, Turkey.

Preparation of the extract
The dried plant materials were powderd using a grinder.The extraction was done at room temperature.100g of dried and grounded herbs of T. spathulifolius were soaked in 80% ethanol (1000 mL) for 48h with intermittent shaking.Then extracts were filtered through Whatman filter paper No.1.In order to increase the yiled of extract, the procedure was repeated for three times.The filtrates combined together and concentrated under vaccum on a rotary evaporator (Buchi R-100 equipped with Vacuum Pump V-300 and Control unit I-300) at 40°C to dryness and dissolved in distilled water.The aqueous extract was fractioned by successive solvent extraction with hexane, chloroform, n-butanol (pre-water-saturated butanol solution).All of the fraction obtained through solvent extractions were then evaporated to dryness and stored at -20ºC for further use.

DPPH free radical scavenging activity
The antioxidant activity of the plant extracts and the standard was evaluated on the basis of the radical scavenging effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-free radical activity by the method of Blois [18].The stock solution of crude extracts (2 mg/mL) was prepared by dissolving a known amount of dry extract in 10% DMSO of methanol.The working solution (1, 10, 50, 100, 250, 500, 1000 µg/mL) of the extracts were prepared from the stock solution using suitable dilution.Ascorbic acid was used as standard in 1-100 µg/mL solution.0.1mM of DPPH was prepared in methanol and 1 mL of this solution was mixed with 3 mL of sample solution and standard solution in test tubes separately in triplicates.These solution mixtures shanked vigorously, then were allowed to stand at dark for 30 min and optical density was measured at 517 nm using UV-VIS Spectrophotometer.Methanol (3 mL) with DPPH solution (0.1mM, 1 mL) was used as blank.Methanol was used for base line corrections in absorbansce of sample.The effective concentration of sample required to scavenge DPPH radical by 50% (IC50 value) was obtained by linear regression analysis of dose-response curve plotting between % inhibition and concentrations.The optical density was recorded and % inhibition was calculated by the formula given below: Percent (%) inhibition of DPPH activity = (Absorbans of Blank-Absorbans of Test) / Absorbans of Blank × 100

Ferrous ion chelating activity
Ferrozine can form complex by chelates with Fe 2+.This reaction is restricted in the presence of other chelating agents and results in a decrease of the red color of the ferrozine-Fe 2+ complexes.Measurement of the color reduction determines the chelating activity to compete with ferrozine for the ferrous ions .The chelation of ferrous ions is estimated using the method of Dinis et al. [19]. 1 mL of the extract is added to a mixture of 3.7mL methanol and a solution of 0.1 mL ferrous chloride (2 mM).The reaction is started by the addition of 0.2 mL of ferrozine (5 mM) and incubated at room temperature for 10 min and then the absorbance is measured at 562 nm.EDTA was used as a positive control.

ABTS radical cation decolorization assay
In this method, measure the loss of color when an antioxidant is added to the blue-green chromophore ABTS+.The antioxidant reduces ABTS.+ to ABTS and decolorize it.Antioxidant activity can be measured as described by Ree et al. [20].ABTS radical cations are generated with 2.45 mM potassium persulphate and a 7 mM aqueous ABTS stok solutions.The ABTS cation working solution was obtained by mixing the two stok solutions in equal volumes and incubate them to react for 16h at 25°C in the dark.Before using, this solution was dilute with methanol and adjustified the absorbance 0.70 ± 0.2 units at 734nm by spectrophotometer.Frech solvent was prepared for each assay.Trolox, a water-soluble analog of vitamin E, used as an antioxidant standard.A standard calibration curve is constructed for Trolox at 0, 50, 100, 150, 200, 250, and 500 µM concentrations.1mL of diluted samples are mixed with 1 mL of ABTS+ radical cation solution in test tubes, and absorbance is read (at 734 nm) after 7 min.TEAC values can be calculated from the Trolox standard curve and expressed as Trolox equivalents (in µM).

FRAP Assay
This assay was used to measure the Fe 3+ ion's reducing power according to the method described by Aksu et al. [21] with slight modifications. 1 mL of samples, BHT and Ascorbic acid at different concentrations in appropriate solvent were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1%) and incubated at 50 •C for 20 min.Then, 2.5 mL trichloroacetic acid (10%) was added to the mixture to stop the reaction.2.5 mL of pure water and 0.5 mL of FeCl3 (0.1%) were added to 2.5 mL of the reaction mixture, then allowed to stand for 30 min.The increases in the absorbance were spectrophotometrically measured at 700 nm as an indication of reducing capacity.

Determination of total phenolics contents
The determination of total phenolic content was performed by the Foline-Ciocalteu method [22] with slight modifications.The samples were read at 730 nm in spectrophotometer.The total phenolics content was expressed in milligrams equivalents of gallic acid (GAE) per gram of each fraction.The equation obtained for the calibration curve of gallic acid in the range of 0.1-1 mg.mL -1 was Y =0.7846X + 0.0764 (r = 0.9995).

Determination of total flavonoids contents
The determination of flavonoids was performed according to the method [23] with slight modifications.The absorbance was determined by spectrophotometer at 415 nm.Ethanol was used as a blank.The equation obtained for the calibration curve of quercetin in the range of 0.0625-1.0mg.mL -1 was Y = 3.1965X + 0.2828 (r = 0.9933).The content of flavonoids was established as quercetin mg/g dry extract.The experiments were conducted in triplicate.

Reducing power
Reducing activity was carried out by using the method of Oyaizu [24].Different concentration (1000, 500, 250,100, 10, 1 mg/mL) of extracts and fractions were prepared with methanol and taken in test tube as triplicates.To the test tubes 2.5 mL of sodium phosphate buffer and 2.5 mLof 1% potassium ferric cyanide solution was added.These contents were mixed well and were incubated at 50°C for 20 minutes.After incubation 2.5 mL of 10% TCA was added and were kept for centrifugation at 3000 rpm for 10 minutes.After centrifugation, 5mL of supernatant were taken and to this 5 mL of distilled water was added.To this about 1mL of 1% ferric chloride was added and was incubated at 35 °C for 20 minutes.The absorbans measured at 700 nm and the blank was prepared by adding every other solution but without extract and ferric chloride (0.1%) and the control was prepared by adding all other solution but without extract.

Micro-well dilution assay
In order to determine the minimal inhibitory concentration (MIC) of T. spathulifolius extracts the broth microdilution method was used [25].The antimicrobial activity of the plant extracts were tested against bacterial and fungal strains: Gram positive (Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212)), Gram-negative (Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922)) and fungal strain (Candida albicans (ATCC 10231)).Plant extracts were dissolved in 8% DMSO (20 mg/mL).50 µL sterile distilled water was added in each well of 96-well microtiter plate.50 μL of plant extract was added into the first well and a serial 2-fold dilution was performed by transferring 50 μL of the suspension to the subsequent wells up till the 9th well; the final 50 μL of the suspension was discarded.10th well was added Gentamicin for bacteria and Flucanazole for Candida.11th well was added 50 µL 2% DMSO and 12th well was added 50 µL Mueller Hinton Broth (sterility control).Concentration of plant extract in wells ranged from 5.00 to 0.02 µg/mL.Final inoculum size was 5 ×10 5 CFU/mL at bacteria and 0.5-2.5 ×10 3 CFU/mL at Candida in each well [26][27].Mueller Hinton Broth and Saboraud Dekstroz Broth was used for dilution bacteria and Candida culture's, respectively.Microtiter plates were incubated at 37 °C for bacteria and 35 °C for Candida between 16-24 hours.Afterwards, 50 µL 2 mg/mL 2,3,5-Triphenyltetrazolium chloride (TTC) (Meck, Germany) was added to each well to indicate microbial growth.The microtitre plates were further incubated at 37 °C for 2 h.Reduction in density of formazan's red color after incubation was accepted MIC value.The experiment was performed in duplicate and the standard deviation was zero.

Assessment of In Vitro proliferative activity
The In vitro proliferative activity was evaluated in the colorimetric MTT assay [28].Exponential growing L929 Mouse fibroblast cells were plated in 96-well micropates at a density of 5 × 10 3 cells per well in 100 μL of culture medium and were allowed to adhere for 16 h, cultured in a humidified atmosphere at 37 °C in 5% CO2 before treatment.Increasing concentrations of extract (1-1000 μg/mL) in their respective extraction solvent were then added.The final concentration of ethanol in the culture medium was maintained at 0.5% (volume/volume) in order to avoid solvent toxicity.The cells were incubated for 24 h in the presence or absence of extract or fractions.After incubation, 100 µL of MTT [5 mg/ml in PBS: medium (1:3)] was added per well, and the plate incubated for 4 h to allow reaction of MTT by cellular mitochondrial dehydrogenases.The excess MTT was aspirated and the formazan crystals formed were dissolved with 100 ml of dimethyl sulfoxide (DMSO).The absorbance of purple formazan, proportional to the number of viable cells, was measured at 595 nm using a microplate reader (Epoch, USA).The experiments were carried out in triplicate.The potency of cell proliferation for each extract was expressed as ED50 value, defined as the concentration that caused a 50% of maximum proliferation of cells.

Qualitative phytochemical screening
Chemical test for the preliminary phytochemical screening and identification of bioactive chemical constituents in the different extracts obtained from T. spathulifolious were performed using the standard procedures as described by Trease and Evans [29], Sofowara [30], and Ugochukwu [31].
Scavenging activity for free radicals of DPPH has been widely used to evaluate the antioxidant activity of natural products from plant and microbial sources.Free radical scavenging activity of ethanolic extract and fractions prepared from the herbs of T .spathulifoliuswas quantitatively determined using DPPH assay.Figure 1 shows the results of DPPH radical scavenging assay.The total phenolic and total flavonoid contents in different extracts of T. spathulifolius are given in Figure 2 and Figure 3.Among studied T. spathulifolius extracts, total phenolic content was highest in the butanol extract and lowest in the hexane extract.Total phenolic content in the extracts ranged from 98.31 to 320.29 mg/g GAE.Total flavonoid content was highest in the butanol extract and lowest in water extract, ranged between 6.55 -160.0 mg quercetin equivalent flavonoid in g dry weight of extracts.Though the bioactivity of flavonoids appears to be mediated through a variety of mechanisms, particular attention has been focused on their direct and indirect antioxidant actions.The antioxidant properties are conferred on flavonoids by the phenolic hydroxyl groups attached to ring structures and they can act as free radical scavenger, reducing agents and metal chelators [32].
In general, the ABTS radical scavenging activity of the extracts is higher than the DPPH scavenging effect.It was reported that, the capacity of extracts in different test system was effected by solubility of the extract and stereoselectivity of radicals [33].In this work, all of the tested extracts of T. spathulifolius in different polarity showed strong scavenging activities against ABTS radicals.Therefore, they may be valuable therapeutical agents in the treatment of some pathological damage due to free radicals.

Antimicrobial activity
MIC values of the different extracts of T. spathulifolius were detected with broth microdilution assay.As can be seen from the Table 1, plant extracts showed different antimicrobial activity against the test microorganisms.MIC values of the extracts in the range between >5-0.31 mg/mL.According to the results, the most sensitive microorganisms against the Hexane extract of T. spathulifolius were S. aureus and C. albicans that have the lowest MIC values 0.31 and 0.62 mg/mL, respectively.

Proliferative assay
The proliferative effect of plant extracts on cell growth was assessed by 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.This colorimetric assay is based on the conversion of the yellow tetrazolium bromide (MTT) to the purple formazan by the action of mitochondrial enzyme succinate dehydrogenase in viable cells.As can be seen from Table 2, the proliferative activities hexane and aqueous fractions with ED50 of 3.28 µg/mL and 2.77 µg/mL, respectively, while ethanolic crude extract showing antiproliferative activity with 12.0 µg/mL.The results in this study suggest that the compounds responsible for proliferative effects may be non-polar or polar compounds.The crude extract has an antiproliferative effect, when fractionated, has the opposite effect, this may be due to the synergistic effect between the compounds in the extract.As can be seen from the study results, it is worth to evaluating of the active extracts in terms of wound healing activity due to the proliferative effect.

Phytochemical screening
The screening of chemical constituents was carried out with different extracts by using standard chemical methods according to the methodology, results were summarized in Table 3.

Figure 4 .
Figure 4. Ferric reducing power of different extracts from herbs of T. spathulifolius.

Figure 5 .
Figure 5. Metal chelating activity of various extracts from herbs of T. spathulifolius

Figure 6 .
Figure 6.ABTS radical scavenging activity of different extracts from herbs of T. Spathulifolius

Table 1 .
Minimum inhibitory concentrations of different extracts of T. spathulifolius herbs

Table 2 .
Proliferative effects of various fractions of Thymus spathulifolius measured using MTT assay and the determined ED50