ANTIOXIDANT ACTIVITIES OF ENDEMIC SIDERITIS LEPTOCLADA AND MENTHA DUMETORUM AQUEOUS EXTRACTS USED IN TURKEY FOLK MEDICINE HULYA AYAR-KAYALİ1, RAZİYE OZTURK UREK1, MAHMURE NAKİBOGLU2 and LEMAN TARHAN1,3 1 Education Faculty Chemistry 2 Biology Department Dokuz Eylul University 35150 Buca-Izmir, Turkey Accepted for Publication March 27, 2008 ABSTRACT Total phenolic contents, hydroxyl radicals and one 1-diphenyl-2picrylhydrazyl (DPPH) scavenging activities, antioxidant capacities and reducing powers of Sideritis leptoclada, which is an endemic plant to Turkey, and Mentha ¥ dumetorum were investigated in water extracts. Total phenolic contents were detected to be 0.32 mg gallic acid/mg dry biomass in the presence of 1.33 and 9.82 mg/mL, respectively. Fifty percent scavenging activities of hydroxyl and DPPH radical were determined with 0.03 and 0.16 mg/mL extracts of S. leptoclada and 0.21 and 1.6 mg/mL extracts of Mxdumetorum. In addition, total antioxidant capacities of S. leptoplada were also higher than Mxdumetorum and there was a significant difference between the controls. The result of this study suggests that extracts of S. leptoclada and Mxdumetorum can be used as potential source of natural antioxidants and as a possible food supplement or in the pharmaceutical industry. PRACTICAL APPLICATIONS The research is focused on the determination of new potential plant sources of antioxidants that can be used as food. Antioxidants are vital substances that possess the ability to protect the body from damage caused by reactive oxygen species induced oxidative stress. In this study, infusions in boiled water of Sideritis leptoclada, which is a plant endemic to Turkey, and Mentha x 3 Corresponding author. TEL: leman.tarhan@deu.edu.tr 90-232-420-48-82-1317; FAX: Journal of Food Processing and Preservation 33 (2009) 285–295. © 2009 The Author(s) Journal compilation © 2009 Wiley Periodicals, Inc. 90-232-420-48-95; EMAIL: 285 286 H. AYAR-KAYALİ ET AL. dumetorum have high levels of antioxidant capacity. The results of the study show that extracts of S. leptoclada and Mxdumetorum can be used as a supplement agent to increase in quality of food and for the pharmaceutical industry. INTRODUCTION In living systems, oxygen and nitrogen radicals are produced by some aerobic metabolic processes and oxidative stress conditions (Halliwell and Gutteridge 1990). The oxygen radicals such as superoxide anion (O2- ·), hydroxyl (·OH) radical and hydrogen peroxide (H2O2) are implicated in damage to membrane lipids, proteins and DNA, and their toxicity is the result of the degree of oxidative stress exceeding the capacity of the cell defense system (Ester-Bauer 1985; Halliwell and Gutteridge 1990; Öztürk-Ürek and Tarhan 2001; Ayar Kayali and Tarhan 2005). Both artificial and naturally occurring antioxidants have been reported to play major roles in inhibiting free radicals induced oxidative damage to membranes and tissues (Yen and Duh 1994). Most living organisms have antioxidant systems against the damaging effects of oxidative stress. However, different external factors and aging decrease the efficiency of such protecting systems, resulting in disturbances of the redox equilibrium established under healthy conditions. Thus, antioxidants that scavenge reactive oxygen species may be of great value in preventing the onset and propagation of oxidative diseases (Shahidi and Wanasundara 1992). In recent years, there has been increasing interest in finding natural antioxidants, since they can protect the human body from free radicals and retard the progress of many chronic diseases carcinogenicity, cardiovascular, and neurodegenerative changes associated with aging (Croft 1999; Shon et al. 2003). As another antioxidant group, natural phenolic compounds, in pure forms or in their extracts from different plant sources (vegetables, fruits and medicinal plants), were also studied in vitro using different model systems (Pratt 1992). Epidemiological studies show that the consumption of vegetables and fruits could protect humans against oxidative damage by inhibiting or quenching free radicals and reactive oxygen species (Klipstein-Grobusch et al. 2000; Slattery et al. 2000). Therefore, the search for newer natural antioxidants, especially of plant origin, is important. Chemistry of natural products is a research field with endless potential, and is especially important in countries possessing great biodiversity, such as Turkey. In the last years, intensive research has been carried out on the extraction, characterization and utilization of natural antioxidants from plants that may serve as potent candidates in combating carcinogenesis and aging processes. In the study, Sideritis leptoclada and Mentha ¥ dumetorum were investigated. One of them, S. leptoclada is an endemic plant of Mediterranean ANTIOXIDANT CAPACITIES OF ENDEMIC S. LEPTOCLADA AND MXDUMETORUM 287 element that distributed in South West Anatolia of Turkey. In folk medicine, they are used as a nervous system stimulant and as an anti-inflammatory, antispasmodic, carminative, analgesic, sedative, antitussive, stomachic and anticonvulsant, in the treatment of coughs due to colds and for curing gastrointestinal disorders (Ezer and Abbasoǧlu 1996; Kırımer et al. 1999; Bondi et al. 2000). Other species, Mentha, is strongly aromatic like other plants that have essential oils. Mentha species have analgesic, antispasmodic, tonic stomach leafs, antirheumatism, antiarthritis, carminative, anthelmintic, cardiac neurosis, eupeptic, antidiarrhea and poisonous protector leafs. In this study, we investigated the antioxidant capacity variations of S. leptoclada and Mxdumetorum by employing various in vitro assay systems, such as hydroxyl and 1-diphenyl-2-picrylhydrazyl (DPPH) radicals’ scavenging capacities, reducing power and total antioxidant capacities and total phenolic contents with respect to water infusion conditions. MATERIALS AND METHODS Materials All solvents or chemicals used were of analytical grade and obtained from Sigma Chemical Co. (St. Louis, MO). Plants The genus Sideritis numbers more than 150 species occurring mainly in the Mediterranean area (Obon de Castro and Nunez 1994). Sideritis is represented in Turkey by 52 taxa belonging to 44 species of which 34 are endemic to Turkey (Davis 1982). S. leptoclada is perennial and an endemic plant. Generally known under the names “Kızlan” or “dağçayı” in Turkey. The genus Mentha is represented in Turkey by nine species and 13 taxa (Davis 1982; Tarimcilar and Kaynak 1997). Mxdumetorum is a perennial and pungent plant and generally known under the names “Kara nane” in Turkey. It is widely used as food industry and folk medicine. Taxonomical description of the species has been made according to Davis (1982). The plants were identified by Associate Professor Dr. M. Nakipoglu and voucher specimens of S. leptoplada (EGE 27308) and Mxdumetorum (EGE 38194) were deposited in the Herbarium of Aegean Agricultural Research. Infusion S. leptoclada and Mxdumetorum were collected from Turkey. The species were dried under darkness at room temperature and stored in the freezer 288 H. AYAR-KAYALİ ET AL. (-20C) until analyzed. Dried plant samples were crushed in a coffee grinder for 2 min, but at 15 s intervals the process was stopped for 15 s to avoid heating of the sample. Powdered plant samples (0.26 g) were infused in boiled water (20 mL) by agitating in magnetic stirrer for 5 min. Then, the obtained extracts were filtered and centrifuged at 5000 rpm for 10 min. Determination of Total Phenolics Total phenolic contents were measured by using the Prussian Blue Assay (Graham 1992). Gallic acid was used as the standard. This method is based on oxidation and reduction of iron. 0.10 mL sample, 50.0 mL distilled water and 3.0 mL 0.10 M FeNH4(SO4)2 (in 0.10 M HCI) were added to Erlenmeyer flask and mixed. Exactly 20 min after the addition of the ferric ammonium sulphate, additions of 3.0 mL 0.008 M K3Fe(CN)6 were achieved and mixed. Definitely 20 min after the addition of the ferricyanide, absorbance at 720 nm was read against to blank. Determination of OH · - Radical Scavenging Activity Deoxyribose has often been used to measure the formation of ·OH radical in biochemical systems (Halliwell and Gutteridge 1990). Reaction mixture contained in a final volume of 1.0 mL, following reagents at the final concentrations stated: deoxyribose (2.8 mM), FeCl3 (100 mM), ethylenediaminetetraacetic acid (EDTA) (104 mm), H2O2 (1 mM) and ascorbate (100 mm). If a Fe2+–EDTA chelate is incubated with deoxyribose in phosphate buffer at pH 7.4, ·OH radicals are formed. Deoxyribose degradation is increased by including ascorbic acid and H2O2. Reaction mixture was incubated at 37C for 1 h, and color developed with TBA. Then absorbance at 532 nm was measured as a pink MDA-thiobarbituric acid (TBA) chromagen. Butylated hydroxyanisole (BHA) was used as a positive control. Determination of DPPH Radical Scavenging Activity The hydrogen atom or electron donation abilities of the S. sipylea and O. sipyleum extracts were measured from the bleaching of the purple-colored methanol solution of the 2,2′-DPPH (Shimada et al. 1992). A 1 mL of DPPH· solution (1 mM) in methanol included 3 mL of extracts solution was incubated at 25C for 30 min. Then, absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. Vitamin C was used as a positive control. Determination of Reducing Power The reducing power of the extract was quantified by the method of Oyaizu (1986). Samples at different concentrations and potassium ferricyanide ANTIOXIDANT CAPACITIES OF ENDEMIC S. LEPTOCLADA AND MXDUMETORUM 289 (1%, w/v) were mixed in phosphate buffer (0.2 M, pH 6.6) and incubated for 20 min at 50C. The reaction was terminated by adding trichloroacetic acid solution (10%, w/v) and the mixture was centrifuged at 5000 rpm for 10 min. The absorbance value was measured at 700 nm after diluted supernatant was mixed with ferric chloride (0.1%, w/v). Increases in absorbance of the reaction mixture indicated the reducing power of the samples. Vitamin C was used as a positive control. Calculation of equivalence was carried out by the calibration graphics of vitamin C. Determination of Total Antioxidant Capacity Total antioxidant capacities of the extracts and BHA as a positive control were determined according to thiocyanate method (Yen and Chen 1995). Linoleic acid emulsion (0.02 M) in phosphate buffer (0.02 M, pH 7.0) was prepared by mixing linoleic acid with an equal amount of 20. Each extract was mixed with linoleic acid emulsion and incubated in darkness at 37C. During the linoleic acid oxidation, peroxides formed. These compounds oxidize Fe2+ to Fe3+ and it forms complex with thiocyanate (SCN-), which had maximum absorbance at 500 nm. Statistical Analysis Tukey test, one of the multiple comparisons, was used for statistical significance analyses. The values are the mean of three separate experiments. RESULTS AND DISCUSSION The antioxidant properties of plant are mainly contributed by the active compounds present in them. In this study, total phenolic compounds, the hydroxyl and DPPH radical scavenging capacities; reducing power and antioxidant capacity of S. leptoclada and Mxdumetorum, which are endemic to Turkey, were determined in water extract. Total Phenolic Contents of S. leptoclada and Mxdumetorum Total phenolics values of S. leptoclada and Mxdumetorum were determined as gallic acid equivalents (GAE). Many published data highlight the potential role of the phenolic components of fruit, vegetables, beverages and grain, which may act as antioxidants. According to the our results, total phenolic contents of S. leptoclada and Mxdumetorum were detected to be 0.32 mg gallic acid/mg dry biomass in the presence of 1.33 and 9.82 mg/mL, respectively. Determination of higher total phenolic amounts in S. leptoclada 290 H. AYAR-KAYALİ ET AL. in comparison to Mxdumetorum had as determined in hydroxyl radical scavenging capacities (P < 0.01) may suggest that phenolics are major components of the antioxidant system in plants because phenolic compounds can play an important role in absorbing and neutralizing of free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. Several studies are focused on the relationship between the antioxidant activity of the phenolics compounds, as hydrogen donating free radical scavengers and their chemical structure. It has been shown that the presence of the ––CH = CH––COOH group in the hydroxylated cinnamates ensures greater H-donating ability and subsequent radical stabilization than the carboxylate group in the hydroxy benzoates (Rice-Evans et al. 1996). Hydroxyl Radical Scavenging Capacities of S. leptoclada and Mxdumetorum OH scavenging, (%) Hydroxyl radical are biologically relevant and extremely reactive oxygen species, which can rapidly react with and degrade susceptible food and biologically relevant substrates, such as polyunsaturated fatty acids, proteins and sugars. The hydroxyl radical scavenging potentials of the obtained S. leptoclada and Mxdumetorum were measured at varying concentrations and the results were depicted in Fig. 1. Both extracts were capable of scavenging hydroxyl radical in an amount-dependent manner (P < 0.01). 0.05 mg/mL of S. leptoclada and Mxdumetorum extracts exhibited 88.84 and 11.25% scavenging activity on hydroxyl radical, respectively. IC50 values for OH radical scavenging were determined as 21 mg/mL for S. leptoclada extract and 2.2 mg/mL for 100 80 60 40 20 10 20 30 40 50 100 200 300 400 500 µg/ml FIG. 1. HYDROXYL RADICAL SCAVENGING ACTIVITIES (%) OF S. LEPTOCLADA (䊉; 10–50 mG/ML) AND MXDUMETORUM (䉱; 100–500 mG/ML) DPPH scavenging, (%) ANTIOXIDANT CAPACITIES OF ENDEMIC S. LEPTOCLADA AND MXDUMETORUM 291 100 80 60 40 20 0.1 0.2 0.3 0.4 0.5 1 2 3 4 5 0.6 6 mg/ml FIG. 2. DPPH RADICAL SCAVENGING ACTIVITIES (%) OF S. LEPTOCLADA (䊉; 0.1–0.6 MG/ML) AND MXDUMETORUM (䉱; 1.0–6.0 MG/ML) BHA, this value was 86, 116, 130, 74 and 129 fold higher than the values determined in Potato peel, Origanum vulgaris, Rosmarinus officinalis, Salvia officinalis, Thymus vulgaris, respectively (Dorman et al. 2003; Singh and Rajini 2004). The result would support that S. leptoclada extract has effective hydroxyl radical scavenging potential compared with the other studied plants. DPPH scavenging capacities of S. leptoclada and Mxdumetorum DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction level of DPPH radicals induced by antioxidants was determined by the decrease in its absorbance at 517 nm. Fig. 2 shows that the scavenging capacity of S. leptoclada and Mxdumetorum extracts increased markedly with the concentration of the extract in the assay medium (P < 0.01). Determination of the same scavenging capacity in the presence of 10.3 fold lower extract amounts of S. leptoclada compared with Mxdumetorum may indicate that it has noticeable effect on the scavenging free radicals. The results showed coherence with the reported researches that the high content of phenolics affected also the strong capability of the species examined to deactivate free radical DPPH, especially in S. leptoclada. IC50 value for DPPH radical scavenging was 4.5 mg/mL for ascorbic acid while it was determined as 0.15 mg/mL for S. leptoclada extract. The result indicated that S. leptoclada extract has a noticeable effect on scavenging DPPH free radical. Reducing Power Capacities of S. leptoclada and Mxdumetorum Table 1 shows the reducing power capacities of the extracts using potassium ferricyanide reduction method and reductive capabilities of S. leptoclada 292 H. AYAR-KAYALİ ET AL. TABLE 1. REDUCING POWERS OF S. LEPTOCLADA AND MXDUMETORUM DEPENDING ON THE EXTRACT AMOUNT mg/mL Abs-700 nm Vitamin C equivalence (mg/mL) S. leptoclada 0.47 1.12 1.34 2.76 0.0559 0.1006 0.1252 0.2925 0.354 0.6375 0.792 1.85 Mxdumetorum 2.80 5.42 11.786 15.40 0.1124 0.3010 0.5529 0.7126 0.711 1.905 3.504 4.51 and Mxdumetorum extracts compared with vitamin C. High absorbance levels indicates high reducing power of extracts. According to the results, the reducing power capacities were increased with increasing amount of samples (r = 0.992, 0.994; P < 0.01). Reducing power of S. leptoclada were 44.7, 52.5, 33.1 and 20.9 fold higher than Thymus vulgaris, Tilia argentea, Salvia triloba, Foeniculum vulgare, respectively. (Yıldırım et al. 2000; Dorman et al. 2003; Oktay et al. 2003). The antioxidant activity of putative antioxidants have been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and, converting radicals into more stable and unreactive species (Diplock 1997). In addition, reducing capacity of S. leptoclada was markedly high parallel to the total phenolic compound as well as DPPH radical scavenging capacities. This probably indicates that phenolic compounds present in the extracts are good electron donors – they show the reducing power. Total Antioxidant Capacity Total antioxidant capacities of S. leptoclada and Mxdumetorum were determined by the thiocyanate method. According to the results, total antioxidant capacities of S. leptoclada and Mxdumetorum were significantly higher than control (P < 0.01) and showed significant correlation with the incubation period (r = 0.943, 0.976; P < 0.01) (Fig. 3). In addition, total antioxidant capacity of S. leptoclada was significantly higher than Mxdumetorum as well as other determined antioxidant parameters such as total phenolic contents and hydroxyl, DPPH radicals scavenging capacities. Determination of positive correlation between total phenolic contents and investigated antioxidant scavenging capacities of S. leptoclada and Mxdumetorum indicate that these parameters have synergic effect on the antioxidant properties of these plants. ANTIOXIDANT CAPACITIES OF ENDEMIC S. LEPTOCLADA AND MXDUMETORUM 293 Abs (500 nm) 2.0 1.5 1.0 0.5 0.0 0 20 40 60 80 100 incubation time (h) FIG. 3. TOTAL ANTIOXIDANT CAPACITIES OF CONTROL (䊏), MXDUMETORUM (䊉) AND S. LEPTOCLADA (䉱) IN PRESENCE OF 150 mG/ML EXTRACT CONCENTRATION CONCLUSION The study showed that there are differences in the capacities of the plants commonly used in Turkey. Endemic S. leptoclada and Mxdumetorum extracts showed strong hydroxyl and DPPH radicals scavenging activities, reducing power and total antioxidant capacities when compared with the other studies (Dorman et al. 2003; Singh and Rajini 2004). In addition, the highest antioxidant activity values were detected in S. leptoplada. It was suggested that not only the level of antioxidants but also a synergy occurring between them and the other plant constituents might influence the differences in the antioxidant ability of the plant extracts. The result of this study suggests that extracts of S. leptoplada and Mxdumetorum can be used as potential source of natural antioxidants and as a possible food supplement or in the pharmaceutical industry. 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