Antioxidant properties of some plants growing wild in Turkey

En este estudio, la actividad antioxidante de extractos metanólicos al 50% en agua de 38 plantas que crecen en la provincia turca de Afyonkarahisar fueron evaluados con algunos ensayos antioxidantes, incluyendo la actividad captadora de radicales libres y de peróxido de oxígeno (H 2 O 2 ) y la actividad quelatante de metales (Fe). Los extractos metanólicos de frutas de las especies Cornus y Morus (actividades captadoras de H 2 O 2 y DPPH y actividad quelatante de Fe) y los extractos metanólicos de hojas de especies de Mentha (actividad captadora de DPPH) son los que mostraron una actividad mayor. Estas propiedades antioxidantes dependieron de la concentración de la muestra.


Antioxidant properties of some plants growing wild in Turkey.
In this study, the antioxidant activity of 50% aqueous methanol extracts of 38 plants growing in the Afyonkarahisar province of Turkey were evaluated by various antioxidant assay, including free radical scavenging, hydrogen peroxide (H 2 O 2 ) scavenging and metal (Fe 2+ ) chelating activities. The methanolic fruit extracts of the Cornus and Morus species (H 2 O 2 and DPPH scavenging activities, Fe 2+ chelating activity) and the methanolic leaf extracts of the Mentha species (DPPH scavenging activities) examined in the assay showed the strongest activities. These antioxidant properties depended on the concentration of samples.

INTRODUCTION
Fruits, vegetables and herbs are recommended at present as optimal sources of chemical constituents with antioxidant activity and supplementing the human diet with plants containing high amounts of compounds capable of deactivating free radicals may have beneficial effects (Madsen andBertelsen, 1995, Velioglu, Mazza, Gao andOomah, 1998;Lutomski, 2001).
Antioxidants are compounds which prevent some toxic materials in the body, especially free radicals. Free radicals which lead to oxidation are basically oxygen sourced metabolites (super oxide anions O 2 , hydrogen peroxide H 2 O 2 , hydroxide radical (OH), hypochloric acid, chloramines, nitrogen dioxide, ozone and lipid peroxides. Antioxidants such as beta, carotene, ascorbic acid, and alfa-tocopherol are proven to prevent the oxidation of free radicals by in vitro and in vivo studies (Cross et al. 1987;Aruoma, 1998;Peter, 1993;Brand-Williams et al. 1995;Stone and Papas, 1997;Zheng and Wang, 2001;Gümrükçüo lu, 2003). Vitamin A takes part in the regulation of protective epitel in the lungs, stomach, urinary tract and other organs in the defense mechanism of the human body. Another antioxidant, tocopherol, protects cells from free radicals, heavy metals, poisonous compounds, medicines and radiation by stabilizing lipid parts of the cell membrane and transporting molecules. Tocopherols prevent the degenerative effects of free radicals in tissue, skin and blood vessels. Another antioxidant, ascorbic acid (Vitamin C) aids in the growth and well being of the body's cells in bones, ligaments and blood vessels. Besides, it helps the body to respond against infections and stress and assists in the proper use of iron (Cross et al. 1987;Aruoma, 1998;Peter, 1993;Brand-Williams et al. 1995;Stone and Papas, 1997;Zheng and Wang, 2001;Gümrükçüo≈lu, 2003).
Afyonkarahisar is rich in a wide variety of flora and vegetation. This richness in flora and vegetation is especially notable in Sultan, Emir, Akda and the Kumalar Mountains. For this reason, many native and foreign botanists collected plant samples from these mountains and other locations in Afyonkarahisar (Akçiçek, 2003).
Afyonkarahisar is in the middle zone of the Mediterranean and the <ran-Turan floristic regions from the point of view of plant geography and there are plants which also represent the Europa-Syberia flouristic region. Around Afyonkarahisar, there are approximately 2500 natural plant species, almost 350 of them endemic, because of its ecological conditions and especially its microclimate (Akçiçek, 2003;Kargıo≈lu, 2003;Köse and Ocak, 2004).
The aim of the present work is to investigate the antioxidant properties of some plants growing wild in the Afyonkarahisar province of Turkey.

Materials
Plants (

Preparation of extracts
The aerial parts of plants were dried in the shade at room temperature. About 2.5 g from each dried plant sample were extracted by homogenizing in a mixer (Ultra turrax) with 50 ml solvent (50% water-methanol). The extracts were centrifuged at 4000xg 3 min at 4 °C (Hettich Zentrfügen-Universal 32 R) after draining through coarse filter paper. The filtrate volume was completed to 50 ml and drained through blue band filter paper (No 589). The filtrats were stored at 4 °C until analysis.

Free radical scavenging effect
The radical scavenging activity against the DPPH radical was evaluated according to the method of Brand-Williams et al. (1995) and Lim and Murtijaya (2007) with some minor modifications. The assay mixture contained 1.5 ml of a 0.09 mg/ml of DPPH (Sigma Chem, Co, Str. Lous, USA) in methanol, 1 ml of acetate buffer solution (100 mM, pH 5.5). The dilutions between 0.4 to 4 mg/ml were prepared with methanol. 3.9 ml DPPH solution prepared with 6 ϫ 10 Ϫ5 M (molar) methanol was added to each 0.1 ml of dilutions and shaken well. The mixture was prepared and incubated for 60 min at room temperature in the dark. The absorbance of the remaining DPPH was determined at 517 nm against a blank. The scavenging activity was expressed as IC50 (mg/ml). All analyses were carried out in duplicate. Linear regression equations of absorbance against concentration were determined by measuring the absorbance of seven different concentrations of DPPH (6 ϫ 10 Ϫ5 M) stock solution. A The remained DPPH concentrations against absorbance values of the sample series of different concentrations were calculated and then the remaining DPPH percentage was calculated: The exponential regression equation was determined between the rate of the remaining DPPH percentage and the DDPH amount of sample in vitro and sample concentrations of plants which decreased their initial DPPH concentrations by 50% (efficient concentration [EC 50 ]). Antiradical activity (AE) was calculated by dividing EC 50 values into 1.
Fe ϩ2 chelating activity Modified methods of Lim and Murtijaya (2007) were used for the determination of Fe ϩ2 chelating activities of samples. 1 ml of extract with different concentrations between 6-45 mg/ml and 3.7 ml deionized water were mixed. A 0.1ml 2 mM FeCl 2 solution was added, shaken and kept in the dark at room temperatures for 70 min. Then, 0.2ml 5 mM ferrozin were added and shaken again and the absorbance of obtained Fe ϩ2 -ferrozin complex after 10min was measured at 562 nm. 1 ml water was used instead of the sample for the control. The equation is given below (Yen and Wu, 1999).
Chelating activity (%) ϭ [1 Ϫ (absorbance of sample / absorbance of control)] ϫ 100 The H 2 O 2 inhibition effect of spice and plant extracts can be determined by spectrophotometer (Ruch et al. 1989). 1 ml (2.6 and 10 mg/ml) sample, 3.4 ml 0.1M phosphate buffer (pH 7.4) and 0.6 ml 43mM H 2 O 2 were mixed and after 60 minutes the absorbance of the mixture was measured at 230 nm. Control solutions without H 2 O 2 were prepared for each sample concentration. To determine the H 2 O 2 mM concentration which did not involve the reaction, a linear repression equation was used. 3.4 ml phosphate buffer were added to 0.6 ml 10, 15, 25, 43 and 50 mM H 2 O 2 at 230 nm. Linear regression equations were obtained by the diagram of concentration against absorbance.

Statistical analyses
Results of the research were analyzed for statistical significance by analysis of variance (Püskülcü and <kiz, 1989). This research was performed in three duplicates with a replicate.

Free radical scavenging activity
DPPH, as a partially organic radical, is used to determine the antioxidant activities of many plant extracts and compounds (Brand-Williams et al.1995). This method is based on a decrease in alcoholic DPPH solution in the presence of H binding antioxidant (DPPH и ϩ AH → DPPH Ϫ H ϩ ϩ A и ). A DPPH solution is dark violet colored and has a strong absorption range at 517 nm. It loses its color when transformed to DPPH-H and the absorption level decreases. This decrease in absorption shows the cytochiometric decrease in DPPH.
The DPPH radical scavenging effects of plant leaf and fruit extracts are given in Tables 1 and 2, respectively. While the antiradical activity of the leaf extracts of plants varies from 0.258 (Gypsophila pilosa) to 0.693 (Cornus sanguinea L. subsp. australis), the activities of fruit extracts range from 0.503 (Taxus baccata) to 0.928 (Cornus mas). Generally, the antiradical activity of fruit extracts was found higher than those of leaf extracts. This effect is probably due to the high phenolic compound contents of fruit extracts. The antiradical GRASAS Y ACEITES, 60 (2)  fruits (Morus alba L.) from Korea, as between 225.9-537.6 µg (IC 50 ). Effective scavenging concentration (EC 50 ) on DPPH radicals was 0.70 µg/ml in ethyl acetate and tannin fractions and 5.33 µg/ml in the anthocyanin rich fraction of sumac extracts (Koflar et al. 2007). Emami, Asili, Mohaghegbi & Hassanzadeh (2007) reported that the methanol extracts of the leaves of Taxus baccata L. from Armaniolan, Arasbaran and East Azerbayejan, contained high amounts of alkaloids, tannins and flavonoids while the fruit extracts contained high amounts of tannins and these extracts possessed high antioxidant activity (%) as approximately 90, using the TBA method. According to Cao et al. (1996) among the 22 common vegetables studied, garlic had the highest antioxidant activity, with an antioxidant score (automated oxygen radical absorbance capacity assay) of 23.2 based on fresh weight of the vegetable. However, according to Miller et al. (2000), garlic is very high in antioxidants, its activity being about sixfold that of yellow onion (1300 Trolox equivalents/100 g vs. 200 Trolox equivalents /100 g). The difference is probably at least partially due to the different methods used. The radical scavenging and antioxidant results for blackcurrant plants obtained in this study are not in agreement with the earlier literature (Cao et al.,1996;Gazzani et al.,1998). However these contradictory results  activity of Cornus spp. and Morus spp were compared to those of other fruit extracts (Table 2). These plant extracts may be accepted as having a higher H binding capacity against the DPPH radical. The lowest AEs are obtained from Gypsophila eriocalyx Boiss (0.272), Gypsophila pilosa Hudson (0.258) species and Gypsophila parva Barkoudah.

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The highest DPPH radical scavenging effects were determined in the fruit extracts of Cornus and Morus species with values which varied from 1.078-1.212 (EC 50 ). The fruit extracts of Taxus baccata L., Pistacia terebinthus L .subsp. palaestina (Boiss.) Engler and Pistacia terebinthus L. subsp. terebinthus, Sorbus species and Jasminum fruticans L. plants showed the lowest DPPH radical scavenging effects. Termentzi et al. (2006) reported the AE values (DPPH radical scavenging) of the methanol extracts of fruit pulp from ripe Sorbus domestica fruits as 0.682. Tural and Koca (2008) reported that the methanolic extracts of the Cornelian cherry (Cornus mas L.) fruits showed EC 50 (mg/ml) (DPPH reduction) values as 0.52. Topçu et al. (2007) reported that the methanol extracts of Pistacia terebinthus fruits showed activity as high as the standard , at 50µg/ml as 95% for DPPH scavenging effect, %). Bae and Suh (2007) reported the scavenging abilities of DPPH radicals of the ethanolic extracts of five major cultivars of mulberry are most likely due to differences in methodology and experimental conditions used in the different studies. Due to the wide wariety of potential antioxidant compounds, such as vitamins, flavonoids, phenolic acids and sulphur compounds present in plants, differences in the method of sample extraction can results in a wide variation in the antioxidant activity of the extract (Nuutila et al. 2003).
The radical scavenging and antioxidant activity results for these plants show some differences from the earlier reported results above. However these contradictory results are most likely due to differences in methodology and experimental conditions used in the different studies. Due to the wide variety of potential antioxidant compounds, such as vitamins, flavonoids, phenolic acids and sulphur compounds present in plants, differences in the method of sample extraction can results in a wide variation in the antioxidant activity of the extract (Nuutila, Puupponen-Pimia, Aarni & Oksman-Caldentey, 2003). In our study, there was a noticeable correlation between high radical scavenging / antioxidant activity and high amounts of total phenolics. More plants have been used as a source of food, remedy and animal fodder in Turkey (Baytop, 1984). The antioxidant activities of spices and herbs are attributed to their polar phenolic and essential oil contents (Tsimidou and Boskou, 1994;Shahidi, 1997;Özkan and Özcan, 2006).

Fe ϩ2 chelating activity
Chelating agents may have great importance for rancidity of oily foods; even though they are not antioxidant materials. Because iron catalyzes this reaction during lipid peroxidation, Ferrozin forms a complex with Fe ϩ2 . The amounts of complex and red color decrease in the presence of the other chelating agents. A decrease in absorption values can be determined by changes in the color. The decrease in absorption shows the effectiveness of chelating agent added with the exception of ferrozin. Table 3 and 4 show the chelating activities of plant leaf and fruit extracts. The Fe 2ϩ chelating activity of fruit extracts of plants was established as higher than that of leaf extracts. The highest chelating activity was found in Cornus mas fruit extracts. The Chelating activities of Cornus spp. fruit extracts were found higher compared with other fruit extracts (

CONCLUSIONS
The present study demonstrates the antioxidant potential of some herbs and fruits from Turkey which could protect against free radical damage.  and Taxus baccata L. Jasminum fruticans L., Rhus coriaria L., Achillea teretifolia Willd. and Sorbus species also had lower Fe ϩ2 chelating activities than the fruit extracts of other plants used in the assay. Chelating agents may have a great importance for rancidity of oily foods, even if they are not antioxidant materials. Because iron catalyzes this reaction during lipid peroxidation (Yen and Duh, 1994).

H 2 O 2 inhibition activity
This method is used to eliminate O2• Ϫ , even though the superoxide radical anion (O2• Ϫ ) does not initiate lipid oxidation directly. Super reactive hydroxyl radical (.OH) may be formed from the Fenton reaction (Fe ϩ2 ϩ H 2 O 2 → Fe ϩ3 ϩ OH Ϫ ϩ .OH) in the presence of metal ions. For this reason, H 2 O 2 inhibition activity is an important method for the determination of antioxidant characteristics.
The H 2 O 2 inhibition activities of plant leaf and fruit extracts are given in Tables 5 and 6, respectively. The Cornus species showed the highest H 2 O 2 inhibition values (54.32-65.42%) while Taxus baccata L. and Pistacia terebinthus L .subsp. palaestina (Boiss.) Engler had the lowest H 2 O 2 inhibition values at 13.44% and 16.78%, respectively. The Gypsophila species also showed lower H 2 O 2 inhibition values (18.64-25.43%) than the other plant leaf extracts analyzed.
The H 2 O 2 inhibition activity of plant (fruit) extracts was determined higher than those of leaf with values varying from 1.078-1.212 (EC 50 ). The highest chelating activity was observed for the Cornus species ranging from 44.64-45.72%. It is believed that the detection of natural antioxidant sources and proper consumption of them in the daily diet or the use of isolated compounds in clinical practices would be beneficial for a healthy life.