Phenolic Contents and Antioxidant Properties of Echinops ritro L . and E . tournefortii Jaup . Et . Spach Extract

Received: 01 February 2016 Revised: 30 March 2016 Accepted: 26 April 2016 Abstract: Aim of the study was to evaluate antioxidant activity and total phenolic content of Echinops ritro L. and E. tournefortii (Asteraceae). The dried leaves and seeds of E. ritro and E. tournefortii were extracted separately with ethanol, methanol, chloroform and dH2O. Total phenolic content was measured by FolinCiocalteu method. Antioxidant activities of the extracts were determined by two test systems namely, radical scavenging on DPPH and β-carotene bleaching methods. dH2O extracts has the highest phenolic content (92.24 GAE mg/100g). The results were compared to those of BHT as synthetic antioxidant. dH2O extracts were found to be rich as a source of phenolics. According to the results of antioxidant activity, dH2O extracts exhibited higher antioxidant activity than all types of solvent. The strongest antioxidant properties were obtained by dH2O extract. Radical scavenging activities (%) were found to be in the following order: Chloroform<Ethanol<Methanol<dH2O<BHT. These results of the present study demonstrated that the extracts of E. ritro and E. tournefortii may be used as a source of natural antioxidant in the food and pharmaceutical industries.


Introduction
Plants are good source of biologically active secondary metabolites which have many therapeutic potential in many diseases and even in free radical associated disorders [1].Among secondary metabolites synthesized, plant polyphenols are the aromatic hydroxylated compounds which have the most potent and therapeutically useful bioactive substances.Promising radical scavenging ability of the phenolic compounds produced in higher plants is studied extensively [2].Oxidation stress is one of the major concerns of health in modern era and antioxidants have been reported to prevent oxidative damage caused by free radical, via interfering with the oxidation process by reacting with free radicals, by chelating with catalytic metals, and also by acting as oxygen scavengers [3].In the presence of antioxidants, the oxidative rates decrease due to an increased activation energy for reaction, thus increasing the "lifetime" of the substrate, serving as a parameter for the evaluation of the antioxidant activity [4].Although several synthetic antioxidants, such as butylated hydroxyanisole and butylated hydroxytoluene, are available because of their toxicity problems; there is an upsurge of interest in the therapeutic potentials of plants as antioxidants.In addition to the natural antioxidants like vegetables, fruits, spices and tea, scientific evaluation of plant's properties through potent pharmacological activities, toxicity profiling and economic viability are needed for growing recognition for medicinal plants and herbal products as novel antioxidants in recent decades.Therefore, significant consideration has been directed toward the detection of antioxidant properties in plant species.
The genus Echinops L. (Asteraceae) consists of approximately 120 species [5], distributed in Africa and the Mediterranean basin [6].In Turkey, the genus comprises 19 species, including 2 subspecies and 3 varieties [7,8].Echinops plant was reported to possess variety of compounds belonging to various classes like: alkaloids, flavonoids, terpenoids, lipids, steroids and polyacetylenes [9].Echinopsine was quinoline alkaloid isolated in 1900 by M. Greshoff from seeds of the blue globe thistle, E. ritro and its presence was also demonstrated in 14 other species of Echinops like E. latifolius, E. setifer [10].Echinops species have been used as traditional medicine for treatment of migraine, diuretic, heart diseases, urinary infection, as well as worm and hemorrhoid in Ethiopia [11].In the present study, total phenolic of the ethanol, methanol, chloroform and dH 2 O extracts prepared from E. ritro and E. tournefortii the were determined as mg/g GAE.These extracts were tested for their antioxidant activity by using two methods namely βcarotene-linoleic acid test system and DPPH free radical scavenging assay.This study examined the antioxidant activities of these species for the first time.

Plant materials
Echinops ritro and E. tournefortii was collected from Denizli Kınıklı field during the period of investigation and in July 2015.The voucher specimen of E. ritro was confirmed and deposited in Herbarium at the Department of Biology.The collected plant material was airdried in darkness at room temperature (20 o C).Dried plant parts were cut up and stored in tight-seal dark containers until needed.

Preparation of plant extracts
Echinops ritro and E. tournefortii species cut into small pieces with a blender.Extractions were prepared using different solvents (methanol, ethanol, chloroform and dH 2 O).For extractions 10 g of the plant and 100 mL of solvent (Merck) were used for each sample.The mixture was extracted after being heated in a shaker water bath at 55ºC for 6 h.The extract obtained was filtered through filter paper (Whatman No: 1), and the solvents were evaporated in a rotary evaporator (IKA, RV 10 basic V-C, Germany) at 48 -49°C.The water in each extract was frozen in Freeze-drying (Thermo, savant) machine and then drawn out (stored at -20 o C).

Determination of total phenolic content
The total phenolic content of extracts was determined using to the Folin-Ciocalteu method [12].Briefly, 0.75 mL of Folin-Ciocalteu reagent (1:9; Folin-Ciocalteu reagent: distilled water) and 100 mL of sample (5 mg/mL) were put into a test tube.The mixture was mixed and allowed to stand for 5 min at room temperature.The mixture was allowed to stand at room temperature for 5 min.0.75 mL of 6 % (w/v) Na 2 CO 3 was added to the mixture and then mixed gently.The mixture was homogenized and allowed to stand at room temperature for 90 min.Total polyphenol content was determined using a spectrophotometer at 750 nm.The standard calibration (0.01-0.05 mg/mL) curve was plotted using gallic acid.The total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g plant extract.

Determination of total antioxidant activity
The antioxidant activity of the crude extracts was evaluated using the β-carotenelinoleic acid test system with slight modifications [13].β-Carotene (0.2 mg) in 1mL of chloroform was added to 20 μL of linoleic acid, and 200 mg of Tween-20 emulsifier mixture.The mixture was then evaporated at 40 o C for 10 min by means of a rotary evaporator to remove chloroform.After evaporation of chloroform, 100 mL of distilled water saturated with oxygen, 4.8 mL of this emulsion was placed into test tubes which had 0.2 mg of the sample and 0.2 of the extract in them.For control, 0.2 mL of solvent (methanol, ethanol, chloroform and dH 2 O) was placed in test tubes instead of the extract.As soon as the emulsion was added into the test tubes, initial absorbance was measured with a spectrophotometer (Shimadzu UV-1601, Japanese) to be at 470 nm.The measurement was carried out at 0.5 h intervals for 2 h.All samples were assayed in triplicate.BHT was used as standards.The antioxidant activity was measured in terms of successful bleaching of β-carotene by using the following equation.The measurements were made using the equation below: Where AA is the total antioxidant activity, A0 is the initial absorbance of the sample, At is the initial absorbance of the control, A0o is the sample's absorbance after 120 min, and Ato is the control's absorbance after 120 min.

Determination of DPPH free radical scavenging activity
Free radical scavenging activity of the extracts was determined using the free radical DPPH [14].4 ml of the DPPH's 0.004% metanolic solution was mixed with 1 ml (0.2 -1.0 mg/mL) of the extracts, and their absorbances were measured to be at 517 nm after incubation for 30 min at room temperature the absorbance value of the samples were evaluated against empty control group (where all determinants except the test compound were present).Lower absorbance of the reaction mixture indicates higher free radical scavenging activity.Every test was treated three times and the averages as determined.Free radical scavenging activity was measured using the equation below: where A0 is the absorbance of the control (blank, without extract) and A1 is the absorbance in the presence of the extract.

Results and Discussion
Polyphenols are known for their antioxidant activity as radical scavengers and possible beneficial roles in human health, such as reducing the risk of cancer, cardiovascular disease, other pathologies [15].Plants containing high phenolic compounds can be a good source of antioxidants.For this reason, this information has led to the determination of the total phenolic content of the sample under study.The amounts of total phenolic contents ranged from 31.54-92.24(GAE mg/100g) for extracts respectively (Table 1.).In our investigation, the dH 2 O extract of E. ritro (92.24GAE mg/100g) exhibited the highest total phenol content.According to β-carotene-linoleic acid bleaching assay, β-carotene undergoes rapid discoloration in the absence of an antioxidant, which results in a reduction in absorbance of the test solution with reaction time.This is due to the oxidation of linoleic acid that generates free radicals that attack the highly unsaturated β-carotene molecules in an effort to reacquire a hydrogen atom.When this reaction occurs the β-carotene molecule loses its conjugation and, as a consequence, the characteristic orange color disappears.The presence of antioxidant avoids the destruction of the β-carotene conjugate system and the orange color is maintained Inhibition of linoleic acid was affected by different solvents [16].BHT showed maximum value of inhibition (94.07 %).The antioxidant activity efficiency were also calculated and given in Fig. 1 Fokialakis et al. (2006) reported that the root extracts of E. ritro were evaluated for their antifungal activity using direct-bioautography assays with three Colletotrichum species that cause strawberry anthracnose.Among the bioactive extracts, the dichloromethane extract of the radix of E. ritro was the most potent [19].It was indicated that aerial parts of E. ritro L. and E.spinosissimus from the Greek island of Crete could be extracted, and the extracts obtained have been investigated for in-vitro anti-protozoal activity.The activity against chloroquinesensitive (D6) and resistant (W2) strains of Plasmodium falciparum and Leishmania donovani promastigotes was determined as well as the cytotoxicity on a mammalian kidney fibroblast (Vero) cell line was tested.Dichloromethane of aerial part  The antioxidant activities of methanolic extracts of the E. kotschyi were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and also screened for cytotoxic activity against three human cancerous cell lines (MOLT-4, K562 and MCF7) using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay.The methanolic extract of E. Kotschyi exhibited potent cytotoxic activity against MOLT-4 and K562 cell lines among all extracts tested in this study [22].On the other hand, the ethanolic extract of E. spinosus has efficient action on muscular fibers; anti-inflammatory activity; The ethanolic extract of E. Spinosus (100 mg/kg, intraperitoneal ) exhibited a very good antiinflammatory activity against carrageenan-induced paw edema in mice and rats, and it selectively inhibited prostaglandin E2 (PGE2) -induced inflammation [23].
In the present study, the antioxidant capacities and phenolic content of various extracts (methanol, dH 2 O, ethanol and chloroform) from E. ritro were determined.The dH 2 O extract had higher antioxidant capacity and free radical scavenging activity than other extract at the same concentrations.Results of the study indicated that dH 2 O extract derived from E. ritro possessed remarkable antioxidant activities.Because the synthetic antioxidants (BHA and BHT) have some toxic effects, such as the promotion of carcinogenesis, E. ritro can be considered as a source of both natural antioxidants and lauric acid in the food industry and pharmacological applications.

Conclusion
According present the study, it may be concluded that the extracts of Echinops ritro and E. tournefortii demonstrated in vitro antioxidant activities.Higher levels of total phenolics of plant are probably responsible from the biological activities observed.This finding candidates the plant as a good case for more in-depth studies and we wish our future research lead to the identification of biologically active molecules present in its extracts

Table 1 .
Total phenolic content of extracts extract of E. ritro and E. spinosissimus had moderate activity against L. donovani with no significant anti-malarial activity or cytotoxicity[20].Apigenin-glucopyranoside 1, Apigeninglucoside 2, methoxycarbonylindole 3 and beta-sitositerol 4 were isolated from of E. orientalis dried leaves and seeds.Isolated compounds and extracts were applied to antioxidant activity tests.While seeds and leaves extracts have high DPPH and moderate ABTS radical scavenging activities, the isolated flavones exhibited high cation radical scavenging activities[21].