Antioxidant activities of Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr: Identification of free flavonoids and cinnamic acid derivatives

Food Research International 44 (2011) 297–303

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Antioxidant activities of Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr: Identification of free flavonoids and cinnamic acid derivatives Naciye Erkan a, Huseyin Cetin b, Erol Ayranci a,⁎ a b

Department of Chemistry, Faculty of Arts and Sciences, Akdeniz University, Antalya, 07058, Turkey Department of Biology, Faculty of Arts and Sciences, Akdeniz University, Antalya, 07058, Turkey

a r t i c l e

i n f o

Article history: Received 20 July 2010 Accepted 8 October 2010 Keywords: Antioxidant activity Cinnamic acid derivatives Flavonoids Sideritis arguta Sideritis congesta

a b s t r a c t Different solvent extracts of endemic Sideritis (Labiatae) species, Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr, were analyzed for free flavonoids (quercetin, apigenin, myricetin and kaempferol) and cinnamic acid derivatives (rosmarinic acid, ferulic acid, caffeic acid, p-coumaric acid and chlorogenic acid) using HPLC-DAD. All the phenolics were quantified in acid-hydrolyzed extracts, except rosmarinic acid, chlorogenic acid and myricetin which were quantified in raw samples. Antioxidant activities of extracts of these two plants and many of their components in pure form were evaluated based on DPPH. and ABTS.+ assays. In general, S. arguta extracts displayed higher antioxidant activity than S. congesta extracts possibly due to their richness in antioxidant components of strong activity. Acetone extract of S. arguta, with its strikingly high TEAC value of 3.2 mM trolox and low IC50 value of 38.3 μg/mL showed the highest antioxidant potency among all extracts. α-tocopherol, the positive control, displayed IC50 and TEAC values of 33.8 μg/mL and 2.9 mM trolox, respectively. No direct correlation was found between antioxidant activities and total phenolic contents of the plant extracts studied. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Extensive number of evidences has pointed out free radicals as major contributors to aging and degenerative diseases such as cancer, cardiovascular disease, cataracts and immune system decline (Ames, Shigenaga, & Hagen, 1990; Young & Woodside, 2001). However, free radical formation is controlled naturally by compounds known as antioxidants. The damage in biological systems can be cumulative when the concentration of radical species and antioxidants are not in balance (Swanson, 1998). Antioxidants are capable of neutralizing, or scavenging free radicals by hydrogen donation before the latter attack cells and other biological components. Thus, they are vital for wellbeing and protecting optimal health (Percival, 1998). Synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertiary butyl hydroquinone (TBHQ) have been widely used as antioxidants in the food industry until recently. However, their uses have been limited for the fact that they may be responsible for liver damage and carcinogenesis (Grice, 1988; Wichi, 1986). This problem has been overcome by supplementing diets with antioxidants from natural sources, most of which are plants, fruits and vegetables (Knekt, Jarvinen, Reunanen, & Maatela, 1996). Flavonoids and cinnamic acid derivatives, the groups of compounds that most of the antioxidant activity of plants comes from, contain

⁎ Corresponding author.Tel.: + 90 242 310 2315; fax: + 90 242 227 8911. E-mail address: [email protected] (E. Ayranci). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.10.016

phenolic structure and are widely distributed in photosynthesizing cells (Havsteen, 1983). Flavonoids can be subdivided into several classes: flavones, flavonols, flavanones, isoflavones, flavans, flavanols, and anthocyanins. Tea and herbal preparations provide the major source of these two groups of phenolic compounds in human diet (Shahidi, 2000). Investigation of the presence and activity of flavonoid and cinnamic acid derivatives as antioxidants in tea and herbs has been performed in various studies (Zaveri, 2006; Erkan, Ayranci, & Ayranci, 2008; Atoui, Mansouri, Boskou, & Kefalas, 2005). It is known that extracts obtained with organic solvents from dried leaves of such plants are of much interest due to the high capacity of these solvents in extracting antioxidant compounds. The aerial parts of plants from the genus Sideritis, commonly known as ‘mountain tea’ are widely used as a popular folk medicine in Mediterranean countries such as Greece, Turkey and Spain. The genus Sideritis is represented in the Turkish flora by 46 species, 31 of which are endemic (Davis, Mill, & Tan, 1988). Different biological activities of Sideritis species, including anti-inflammatory (Hernandez-Perez & Rabanal, 2002), anti-ulcer (Aboutabl et al., 2002), antioxidant (Armata, Gabrieli, Termentzi, Zervou, & Kokkalou, 2008) and antimicrobial (Aligiannis, Kalpoutzakis, Chinou, & Mitakou, 2001) activity have been reported up to now. Many of these activities have been attributed to various phytochemicals of the plant among which flavonoids, phenolic acids and diterpenoids have been identified (Janeska, Stefova, & Alipieva, 2007; Gómez-Serranillos et al., 1998). In this article, we report for the first time a detailed study on investigating the antioxidant activities of two endemic Sideritis species,

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Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr, and analyzing the extracts of these plants for free flavonoids and cinnamic acid derivatives. 2. Materials and methods 2.1. Materials All solvents used were HPLC grade and purchased from Merck. 1,1diphenyl-2-picryl-hydrazyl (DPPH) radical, quercetin, myricetin, apigenin, caffeic acid and p-coumaric acid were obtained from Sigma. Trolox, gallic acid, chlorogenic acid, ferulic acid and rosmarinic acid were from Aldrich. 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and kaempferol were purchased from Fluka. The standard compounds were in their highest purity and all other reagents were of analytical grade. Deionized water was used to prepare aqueous solutions. S. congesta and S. arguta were collected from Akseki county of Antalya, Turkey in July, 2009. Taxonomic identification was performed by botanists in the Biology Department, Faculty of Arts and Sciences, Akdeniz University. The aerial parts of plants were washed with deionized water and dried at room temperature for 15 days. Dried samples were kept in a deep freezer at − 20 °C until use. 2.2. Preparation, acid hydrolysis and analysis of plant extracts Dried samples were blended in a blender and subjected to extraction under reflux for 3 h with 1000 mL of methanol, ethyl acetate or acetone as the extracting solvent for 35 g of aerial parts at the boiling points of the organic solvents; 64.7, 77 or 56.5 °C, respectively. The mixture was filtered through Whatman paper (GF/A, 110 mm) and its solvent was removed by a rotary evaporator (Heidolph) at 40 °C. All the extracts were in dark green color after evaporation of the solvent, due to high chlorophyll content of the plants. In order to remove chlorophyll from the extracts in the highest possible amount, repeated washing steps with hexane were applied to each extract. After filtration, the remaining extracts were kept in a vacuum oven at 35 °C for 1 day for further removal of the solvent residue. Dried samples appeared in varying colors from dark yellow to light green and were kept in the deep freezer at −20 °C until use. An acid hydrolysis step was applied to the extracts to release aglycones of flavonoid glycosides for simplification of peak identification procedure. Acid hydrolysis was performed according to the procedure reported by Huang, Wang, Eaves, Shikany, and Pace (2007) with minor modifications. Briefly, 0.01 g of plant extract was subjected to water bath-incubation at 90 °C for 1.5 h after vortexmixing with 4 mL of 80% methanol and 1 mL of 6 M HCl. At the end of incubation, the mixture was cooled to room temperature, made up to 5 mL with methanol and sonicated for 7 min. Then, the mixture was centrifuged at 3000 ×g for 10 min. The supernatant was separated and its solvent was removed completely. 10 mL of methanol was added to the residue and the solution was filtered through a 0.2 μm syringe filter (Millipore, Bedford, MA) and subjected to analysis (approximate concentration, 1 mg/mL). Untreated extracts were also dissolved in methanol (1 mg/mL) and filtered prior to analysis. HPLC analysis of S. congesta and S. arguta extracts were performed with an Agilent 1100 series HPLC instrument equipped with an autosampler and a diode array detector (DAD). The column was Hypersil ODS C18 type with a 5 μm particle size, 4.6 × 250 mm i.d. used with Hypersil ODS 4.0 × 20 mm i.d. 5 μm guard cartridges. The mobile phase was composed of 5% acetic acid in H2O (solvent A) and methanol (solvent B). It was eluted at a flow rate of 0.9 mL/min. Gradient elution utilized was: 0 min, 5% B; 5 min, 15% B; 25 min, 30% B; 39 min, 42% B; 47 min, 55% B; 50 min, 70% B; 56 min, 75% B and 60 min, 100% B. The chromatograms were acquired at 280 and 330 nm. Column temperature and injection volume were 28 °C and

7 μL, respectively. Among the phenolic compounds analyzed, rosmarinic acid, chlorogenic acid and myricetin were quantified in raw extracts, while ferulic acid, caffeic acid, p-coumaric acid, quercetin, apigenin and kaempferol were quantified in acid-hydrolyzed extracts. All standard compounds were diluted in methanol before analysis. Peak identification in HPLC analysis was performed by comparison of retention time with respective reference standards. Quantification of individual flavonoids was done using the peak area of identified compounds. Peak purity values, which were provided by the instrument facility, were checked in each component analysis. They were found to be within the threshold limit in each case. 2.3. DPPH radical scavenging assay This assay was carried out as described by Blois (1958) with some modifications. 1 mL of various dilutions of the test materials (pure antioxidants or plant extracts) was mixed with 2 mL of a 0.2 mM methanolic DPPH. solution. After an incubation period of 30 min at 25 °C, the absorbances at 515 nm were recorded as Asample using a Cary 100 Bio UV/VIS spectrophotometer. A blank experiment was also carried out applying the same procedure to a solution without the test material and the absorbance was recorded as Ablank. The free radical scavenging activity of each solution was then calculated as percent inhibition according to the following equation:   % inhibition = 100 Ablank –Asample = Ablank

ð1Þ

Antioxidant activities of test compounds or extracts were expressed as IC50, defined as the concentration of the test material required to cause a 50% decrease in initial DPPH. concentration. αTocopherol was used as the positive control. 2.4. ABTS.+ radical scavenging assay This assay was carried out according to the procedure described by Re et al. (1999). ABTS.+ radical cation was produced by reacting 7 mM aqueous ABTS with 2.45 mM (final concentration) potassium persulfate and keeping the mixture in the dark at room temperature for 16 h. Bluegreen ABTS.+ was formed at the end of this period. The solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm, wavelength of maximum absorbance in the visible region. Test materials were dissolved in and diluted with ethanol such that, after the introduction of an accurately measured volume of each dilution into the assay, they produced between 10%–90% decrease in the absorbance of the blank solution at 734 nm. After adding 100 μL of the test solution to 3.5 mL of ABTS.+ solution having A734 = 0.70 ± 0.02, absorbance was recorded at 6 min. The results were expressed as trolox equivalent antioxidant capacity (TEAC). TEAC is defined as the mM concentration of a trolox solution whose antioxidant activity is equivalent to the activity of 1.0 mM and 1 mg/mL test solution for pure compounds and plant extracts, respectively. In order to find TEAC values, a separate concentration response curve for standard trolox solutions was prepared. α-Tocopherol was used as the positive control. 2.5. Total phenolic content (TPC) TPCs of Sideritis extracts were determined using Folin-Ciocalteu reagent (FCR) according to the procedure reported by Singleton, Orthofer, and Lamuela-Raventos (1999) with some modifications. The extracts were dissolved in deionized water to provide a concentration of 500 μg/mL. 0.5 mL aliquot of extract or deionized water (control) was mixed with 0.5 mL of FCR by manual shaking for 10–15 s. After 3 min, 0.5 mL of saturated Na2CO3 solution was added and the solution was diluted to 5 mL with deionized water. The reaction mixture was kept in the dark for 2 h and the absorbance was measured at 760 nm. The

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results were expressed in gallic acid equivalent (GAE) as mM GAE/g dry weight (DW) of extract, which was determined utilizing a separately prepared absorbance versus concentration curve for gallic acid. 2.6. Statistical analysis All measurements were made in triplicate and the values were reported as means of the measurements with standard deviations (SD) in tables. The data were analyzed using one-way ANOVA method of the general linear model (GLM) procedure of SAS systems (Windows Release Version 7). Duncan's multiple range test was performed to determine significant differences between mean values at α = 0.05. Principal component analysis (PCA) was conducted on all data points to understand the covariance structure and identify relationships between the variables. Factor analysis was performed to explain the variability of the data and to understand the correlation between variables, as the dimension of the variables (DPPH, ABTS and TPC assays) were small in the current study. Varimax method was used to produce orthogonal transformations to the factors so as to better identify the high and low correlations. Pearson's correlation coefficients were also given to display the correlations between the results obtained from all assays. 3. Results and discussion 3.1. Analysis of plant extracts for free flavonoids and cinnamic acid derivatives Methanol (MET), ethyl acetate (EtAc) and acetone (ACET) extracts of the two Sideritis species, namely S. congesta and S. arguta, that are native to Turkey, have been investigated for their phenolics composition and antioxidant activity. We have focused on rosmarinic acid, ferulic acid, caffeic acid, p-coumaric acid and chlorogenic acid as cinnamic acid derivative antioxidants (Fig. 1a), and quercetin, apigenin, myricetin and kaempferol as flavonoid antioxidants (Fig. 1b) since some of these compounds and their glycosides have already been reported to exist in different Sideritis species (Armata et al., 2008; Pljevljakušić et al., 2011). The phenolics under study usually occur in plants as glycosides and methylated esters, or they may exist in their free form as well (Skerget et al., 2005). Thus, an acid hydrolysis step has been applied to all plant extracts before analysis to simplify peak identification. The acidic hydrolysis conditions used in the present study have previously been proven to be effective in releasing the aglycones from the conjugated phenolic compounds by Huang et al. (2007). They have observed no significant peaks in their work for glycosides by LC/MS which indicated that hydrolysis of most of the glycosides to aglycones have been achieved under acidic hydrolysis conditions. The signals of all pure compounds were observed to be more intense at 330 nm than at 280 nm, except for p-coumaric acid whose signal appeared to be more intense at 280 nm. Thus, the amounts of p-coumaric acid in plant extracts were calculated based on the peak areas obtained at 280 nm. The chromatograms were obtained for extracts of both S. congesta and S. arguta with all three solvents. However since the antioxidant activity results (discussed in the next subsection) showed that S. congestaMET and S. arguta-ACET extracts displayed the highest antioxidant activities among other extracts of S. congesta and S. arguta, respectively, only the chromatograms for these two extracts are presented in Figs. 2 and 3, respectively. Comparison of chromatograms for acid-hydrolyzed extracts and untreated extracts shows that the release of aglycones attached by glycosidic linkage to the majority of the studied flavonoid compounds, or the release of the previously mentioned cinnamic acid derivative compounds from their ester bonds upon hydrolysis improve peak resolution and intensity. However, the peak intensities of rosmarinic and chlorogenic acids were found to be somewhat higher in untreated samples than in acid-

Fig. 1. Chemical structures of (a) cinnamic acid derivatives and (b) free flavonoids.

hydrolyzed samples under the same conditions (Figs 2a–b and 3a–b). This can be explained by the chemical structures (Fig. 1) of these compounds. Since rosmarinic and chlorogenic acids are esters of caffeic acid with 3,4-dihydroxyphenyl lactic acid and quinic acid, respectively, acidic treatment could have diminished the concentrations of these compounds via hydrolysis. Thus, these components were quantified in untreated samples. Table 1 shows the amounts (mg/g) of phenolic compounds found in different solvent extracts of S. congesta and S. arguta. In general, cinnamic acid derivatives were found to be in higher amounts than flavonoid antioxidants, the most abundant ones being ferulic acid and chlorogenic acid in majority of the extracts. Rosmarinic acid was detected to exist in the lowest amounts among acidic antioxidants studied in most of the extracts (p b 0.05), with the exception of S. arguta-EtAc and S. arguta-ACET extracts (p N 0.05) (note each row for comparing the amount of rosmarinic acid with the amount of other acidic antioxidants in each extract in Table 1). S. congesta extracts were found to contain higher amounts of p-coumaric acid compared to S. arguta extracts (p b 0.05). Its amount was found to be the highest in S. congesta-ACET extract (22.8 mg/g, p b 0.05). Flavonoids, on the other hand, were found to exist in higher amounts in S. arguta extracts than S. congesta extracts, with an exception of apigenin and few other cases (Table 1). Although, quercetin could not be detected in S. congesta extracts at all, it was the flavonoid existing in high amounts together with considerable amounts of kaempferol in S. arguta extracts. 3.2. Antioxidant activity of plant extracts and pure components Table 2 shows antioxidant activities of plant extracts and their phenolic components. Antioxidant activities were expressed as IC50 and TEAC values, based on the results obtained from DPPH. and ABTS.+ radical scavenging assays, respectively. It should be recalled that the

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Fig. 2. HPLC-DAD chromatograms of (a) acid-hydrolyzed S. congesta-MET extract recorded at 330 nm, (b) untreated S. congesta-MET extract recorded at 330 nm and (c) acid-hydrolyzed S. congesta-MET extract recorded at 280 nm. Ordinate has the same scale in a, b and c.

Fig. 3. HPLC-DAD chromatograms of (a) acid-hydrolyzed S. arguta-ACET extract recorded at 330 nm, (b) untreated S. arguta-ACET extract recorded at 330 nm and (c) acidhydrolyzed S. arguta-ACET extract recorded at 280 nm. Ordinate has the same scale in a, b and c.

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Table 1 Amounts of free flavonoid and cinnamic acid derivative components in acid-hydrolyzed methanol (MET), ethyl acetate (EtAc) and acetone (ACET) extracts of Sideritis congesta and S. arguta. Plant extracts

S. S. S. S. S. S.

congesta-MET extract congesta-EtAc extract congesta-ACET extract arguta-MET extract arguta-EtAc extract arguta-ACET extract

Amounts of phenolic components in Sideritis extracts

a

(mg/g)

Rosmarinic acidb

Ferulic acid

Caffeic acid

p-coumaric acid

Chlorogenic acidb

Quercetin

Apigenin

1.1 ± 0.0 fE 1.6 ± 0.0 cE 1.5 ± 0.0 dG 1.3 ± 0.0 eF 2.7 ± 0.1 aD 2.0 ± 0.0 bF

6.6 ± 0.0 eB 20.5 ± 0.2 cA 22.6 ± 0.1 bA 23.0 ± 1.0 bA 24.4 ± 0.4 aA 18.9 ± 0.7 dB

5.0 ± 0.4 bC 2.5 ± 0.1 cD 5.5 ± 0.7 bD 3.0 ± 0.1 cE 2.1 ± 0.1 cD 6.8 ± 0.9 aD

4.6 ± 0.5 cC 8.4 ± 0.2 bB 22.8 ± 1.0 aA 2.6 ± 0.5 dE 1.1 ± 0.0 eE 1.7 ± 0.1 eF

22.2 ± 0.8 bA 5.4 ± 0.4 eC 14.2 ± 1.0 cB 23.6 ± 1.0 bA 7.8 ± 0.8 dB 30.3 ± 1.3 aA

n.d n.d n.d 7.7 ± 0.6 aC 7.8 ± 0.6 aB 8.2 ± 0.7 aC

3.0 ± 0.1 2.6 ± 0.1 3.0 ± 0.3 1.5 ± 0.1 1.1 ± 0.0 1.9 ± 0.1

aD bD aF dF eE cF

Myricetinb

Kaempferol

4.7 ± 0.2 bC n.d 4.2 ± 0.0 cE 5.6 ± 0.1 aD 5.6 ± 0.1 aC n.d

2.5 ± 0.3 2.8 ± 0.1 7.0 ± 0.9 10.6 ± 1.0 5.9 ± 0.1 5.3 ± 0.1

dD dD bC aB cC cE

a Means followed by the same lowercase letter within each column and the same uppercase letter within each row are not significantly different [Duncan's multiple range test, (p b 0.05)], n.d: not detected. b Amounts in untreated extracts.

antioxidant activity is directly proportional to TEAC value and inversely proportional to IC50 value. Principal component analysis (PCA) was performed to understand how important are the three assays, namely, DPPH. and ABTS.+ radical scavenging ability and TPC in evaluation of the antioxidant activity of the plant extracts. Since IC50 and TEAC values are inversely proportional to each other, IC50 values were converted to 1/IC50 values before PCA and factor analysis to simplify interpretation and to avoid any confusion. Since the TPC assay was not performed on pure compounds, factor analysis was performed on only plant extracts. A factor rotation using the Varimax method was performed for two factor loadings to see the correlations between assays that accounted for the total covariance of the plant extracts. The most significant component (PC1), DPPH. radical scavenging ability, given as 1/IC50 and ABTS.+ radical scavenging ability, given as TEAC in Fig 4, contributed to the largest variation of approximately 73%, while, TPC (PC2) accounted for approximately 27% to the total variation. Total phenol content (TPC) values did not show a significant contribution to the total variation as the other two assays. This is reasonable because TPC shows the content of all of the phenolic type compounds each of which may not necessarily have strong radical scavenging capacity. In Fig. 4, it can be seen that DPPH assay and ABTS assay, shown as 1/IC50 and TEAC, respectively, are highly loaded on Factor 1 (PC1). The close loading of these values to each other indicates the higher contribution of the two assays to the antioxidant activity (Pearson's correlation coefficient, 0.8404). However, TPC seems to be Table 2 Total phenolic contents (TPCs) of Sideritis extracts and antioxidant activities of the extracts and their pure components expressed as IC50 and TEAC values, based on DPPH. and ABTS.+ assays, respectively. Samplec

loaded closer on Factor 2 (PC2) on negative side of the graph. This is also supported by the correlation coefficients, −0.5170 and −0.2627, between TPC; and 1/IC50 and TEAC, respectively, obtained for plant extracts. TPC assay, thus, does not seem to be appropriate for interpreting antioxidant activities of the plant extracts for the current study. The order of the antioxidant activity of Sideritis extracts were found to be; S. arguta-ACET extract N S. arguta-MET extract N S. congesta-MET extract ≥ S. arguta-EtAc extract N S. congesta-EtAc extract N S. congesta-ACET extract, based on DPPH. radical scavenging assay. The highest antioxidant activities for S. arguta-ACET and S. arguta-MET extracts were also confirmed by ABTS. assay. Furthermore, the distribution of plant extracts on PCA graph (Fig 4) shows that S. arguta-ACET extract (extract6) and S. arguta-MET extract (Extract 4) are the strongest antioxidant extracts, among all Sideritis extracts (p b 0.05). In order to see the solvent effect on plant extract, Ertas, Ozturk, Boga, and Topcu (2009) studied the antioxidant activity of acetonic, metanolic and etheric extracts of S. arguta and found the acetonic extract to have the highest activity based on DPPH. and superoxideanion scavenging abilities, similar to what has been observed in the present study. Both Sideritis species studied were observed to have stronger antioxidant potency than Sideritis libanotica subsp. linearis (IC50, 109 μg/mL), another Sideritis species, reported by Tepe, Sokmen, Akpulat, Yumrutas, and Sokmen (2006). In general, S. arguta extracts were found to exhibit higher antioxidant activity than S. congesta extracts. This is also consistent with the results obtained by Guvenc, Houghton, Duman, Coskun, and Sahin (2005). They found S. arguta

Antioxidant activity IC50a (μg/mL) TEACa,b (mM trolox) TPC a (mM GAE/g)

S. congesta-MET extract S. congesta-EtAc extract S. congesta-ACET extract S. arguta-MET extract S. arguta-EtAc extract S. arguta-ACET extract Rosmarinic acid Ferulic acid Caffeic acid p-coumaric acid Chlorogenic acid Quercetin Apigenin Myricetin Kaempferol α-Tocopherol

50.9 ± 2.2 ef 54.2 ± 1.7 e 66.2 ± 3.3 d 48.6 ± 2.4 f 52.7 ± 1.9 ef 38.3 ± 1.1 g 12.4 ± 0.8 i 49.6 ± 2.3 ef 12.4 ± 0.7 i 105.3 ± 4.3 c 35.6 ± 2.1 g 12.9 ± 1.0 i 427.7 ± 4.8 b 438.6 ± 6.5 a 26.6 ± 2.5 h 33.8 ± 0.4 g

2.5 ± 0.0 2.4 ± 0.0 2.3 ± 0.0 2.9 ± 0.0 2.5 ± 0.0 3.2 ± 0.1 6.4 ± 0.1 3.5 ± 0.0 3.8 ± 0.1 3.8 ± 0.1 2.4 ± 0.3 6.1 ± 0.1 1.1 ± 0.0 1.5 ± 0.0 3.5 ± 0.3 2.9 ± 0.2

g g g f g e a d c c g b i h d f

1375.6 ± 9.4 e 1722.4 ± 12.2 c 1970.8 ± 10.4 a 1785.2 ± 6.3 b 1363.2 ± 8.6 e 1475.2 ± 7.2 d

a Means followed by the same letter within each column are not significantly different [Duncan's multiple range test, (p b 0.05)]. b For pure compounds, TEAC is defined as the mM concentration of a trolox solution whose antioxidant activity is equivalent to 1.0 mM of test solution. For plant extracts, TEAC is defined as the concentration of trolox solution whose antioxidant activity is equal to 1.0 mg/mL of test solution. c MET: methanol, EtAc: ethyl acetate, ACET: acetone.

Fig. 4. Principal component analysis results of DPPH, ABTS and TPC assays displayed as 1/IC50, TEAC and TPC, respectively, for plant extracts (S. congesta-MET extract (Extract 1); S. congesta-EtAc extract (Extract 2); S. congesta-ACET extract (Extract 3); S. arguta-MET extract (Extract 4); S. arguta-EtAc extract (Extract 5); (S. arguta-ACET extract (Extract 6)).

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extracts to have higher antioxidant potential than S. congesta extracts, measured by thiobarbituric acid assay (IC50: 1.27 and 0.44 mg/mL, for S. congesta and S. arguta, respectively). On the other hand, pure antioxidants exhibited varying extent of antioxidant activity according to the results of DPPH. and ABTS.+ assays (Table 2). Rosmarinic acid, caffeic acid and quercetin showed distinctively stronger activities than other compounds, based on both assays (p b 0.05). Rosmarinic acid, caffeic acid, quercetin and kaempferol showed strikingly high activities compared to that of α-tocopherol (control) on both assays (p b 0.05). It is interesting to note that ferulic acid displayed contradictory results for the two assays. It shows lower activity than α-tocopherol according to DPPH. assay and higher activity according to ABTS.+ assay. Chlorogenic acid exhibited statistically very close IC50 value and a slightly lower TEAC value (2.4 mM trolox) compared to α-tocopherol, making it a very effective antioxidant component. Apigenin and myricetin exerted the lowest activities among the other components, based on both assays. The highest activity observed for S. arguta-ACET extract can be ascribed to its highest caffeic acid and chlorogenic acid contents among other extracts (p b 0.05 for both compounds). The presence of considerable amounts of ferulic acid, kaempferol and rosmarinic acid in this extract also contributes to its overall activity (Table 1). The activity of S. arguta-MET extract, which is the second highest among the activities of plant extracts, could also be related to its high flavonoid content. Regarding the total phenolic content (TPC) (Table 2) and the amounts of individual compounds in the extracts (Table 1), it is obvious that methanol seems to be the best solvent for S. arguta in extracting phenolic compounds, while acetone seems to be more appropriate for S. congesta. On the other hand, the highest antioxidant activity of S. arguta-ACET and S. congesta-MET is probably originating from the synergistic effects of phenolic compounds present and the presence of other possible phenolics that were not quantified in these extracts. It is to be noted that the effectiveness of a solvent used in extraction procedure may even vary for different species of the same genus (Armata et al., 2008). In general, the higher antioxidant activity of S. arguta extracts than S. congesta extracts can be correlated primarily with their high ferulic acid and caffeic acid contents and partly with the contents of other antioxidant components such as quercetin, kaempferol, rosmarinic acid and caffeic acid. The absence or low content of myricetin and apigenin in some of the extracts studied may not necessarily contribute to the overall antioxidant activity of these extracts in an important way due to their low activity even in pure form (Table 2). Total phenolic contents of all Sideritis extracts ranging from 1363.2 to 1970.8 mM GAE/g (Table 2) were found to be strikingly high. However, no direct correlation was observed between the TPC and antioxidant activity of the extracts, as expressed previously by the correlation coefficients. This indicates that the presence and amount of individual antioxidant components are more important than TPC in determining the overall activity of the extract. Apart from that, the antioxidant activities of Sideritis extracts studied cannot only be based on the phenolic compounds identified in the current study. Although the presence of many other phenolic components, whose identities have not been clarified yet (see unidentified peaks in Figs. 2 and 3), is highly possible, it is interesting to see that antioxidant activities of Sideritis extracts, as determined from their IC50 and TEAC values, are close to the activities of some of their pure antioxidant components. For instance, TEAC values of Sideritis extracts are very close to or even higher from those of chlorogenic acid and the positive control, αtocopherol (Table 2). Similarly, IC50 values of some of the extracts are also very close to those of ferulic acid, chlorogenic acid and αtocopherol. However, it is usually observed in many studies that the aglycones have more antioxidant potency than their glycosylated and methylated forms, which were reported to exist more abundantly in various Sideritis species than their free forms (Kupeli, Sahin, Calis, Yesilada, & Ezer, 2007; Gabrielia, Kefalasb, & Kokkalou, 2005).

4. Conclusion The differences in radical scavenging activity of pure compounds and complex plant extracts could be explained on the basis of the chemical nature and reactivity of the compounds and the nature of the solvents used in different assays. The presence of glycosidic moieties, the number and position of phenolic hydroxyl and methoxy groups and the synergistic effects of coexisting antioxidant molecules may influence the antioxidant activity (Hidalgo, Sánchez-Moreno, & Pascual-Teresa, 2010). S. congesta and S. arguta, native to Turkey, were found to have strikingly high antioxidant potency. Antioxidant activities of S. argutaACET and S. arguta-MET extracts were found to be the highest among all Sideritis extracts based on DPPH. and ABTS.+ assays, and principal component analysis. Cinnamic acid derivatives were detected to exist in higher amounts than flavonoid antioxidants in plant extracts, the most abundant ones being ferulic acid and chlorogenic acid in majority of the extracts. No direct correlation was observed between the TPC and antioxidant activity of the extracts indicated by Pearson correlation coefficients. These results and chromatographic analyses suggest that other phenolic components may be present in the extracts which need to be investigated. Acknowledgement We would like to thank the Scientific Research Projects unit of Akdeniz University for the support of this work through the Project 2005.01.0200.001. References Aboutabl, E. A., Nassar, M. I., Elsakhawy, F. M., Maklad, Y. A., Osman, A. F., & El-Khrisy, E. A. M. (2002). Phytochemical and pharmacological studies on Sideritis taurica Stephan ex Wild. Journal of Ethnopharmacology, 82, 177−184. Aligiannis, N., Kalpoutzakis, E., Chinou, I. B., & Mitakou, S. (2001). Composition and antimicrobial activity of the essential oils of five taxa of Sideritis from Greece. Journal of Agricultural and Food Chemistry, 49, 811−815. Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1990). Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy of Science, 90, 7915−7922. Armata, M., Gabrieli, C., Termentzi, A., Zervou, M., & Kokkalou, E. (2008). Constituents of Sideritis syriaca. ssp. syriaca (Lamiaceae) and their antioxidant activity. Food Chemistry, 111, 179−186. Atoui, A. K., Mansouri, A., Boskou, G., & Kefalas, P. (2005). Tea and herbal infusions: Their antioxidant activity and phenolic profile. Food Chemistry, 89, 27−36. Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 181, 1199−1200. Davis, P. H., Mill, R. R., & Tan, K. (Eds.). (1988). Flora of Turkey and the East Aegean Islands, Vol. 7. (pp. 947)Edinburgh: Edinburgh University Press. Erkan, N., Ayranci, G., & Ayranci, E. (2008). Antioxidant activities of rosemary (Rosmarinus officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chemistry, 110, 76−82. Ertas, A., Ozturk, M., Boga, M., & Topcu, G. (2009). Antioxidant and anticholinesterase activity evaluation of ent-kaurane diterpenoids from Sideritis arguta. Journal of Natural Products, 72, 500−502. Gabrielia, C. N., Kefalasb, P. G., & Kokkalou, E. L. (2005). Antioxidant activity of flavonoids from Sideritis raeseri. Journal of Ethnopharmacology, 96, 423−428. Gómez-Serranillos, P., Carretero, E., Slowing, K., Palomino, O. M., Villarrubia, A. I., & Villar, A. (1998). HPLC quantitative analysis of diterpenoids in Sideritis (Labiatae) species. Phytotherapy Research, 12, S101−S103. Grice, H. P. (1988). Enhanced tumour development by butylated hydroxyanisole (BHA) from the prospective of effect on forestomach and oesophageal squamous epithelium. Food and Chemical Toxicology, 26, 717−723. Guvenc, A., Houghton, P. J., Duman, H., Coskun, M., & Sahin, P. (2005). Antioxidant activity studies on selected Sideritis species native to Turkey. Pharmaceutical Biology, 43(2), 173−177. Havsteen, B. (1983). Flavonoids, a class of natural products of high pharmacological potency. Biochemical Pharmacology, 32(7), 1141−1148. Hernandez-Perez, M., & Rabanal, R. M. (2002). Evaluation of the antinflammatory and analgesic activity of Sideritis canariensis var. pannosa in mice. Journal of Ethnopharmacology, 81, 43−47. Hidalgo, M., Sánchez-Moreno, C., & Pascual-Teresa, S. (2010). Flavonoid–flavonoid interaction and its effect on their antioxidant activity. Food Chemistry, 121, 691−696. Huang, Z., Wang, B., Eaves, D. H., Shikany, J. M., & Pace, R. D. (2007). Phenolic compound profile of selected vegetables frequently consumed by African Americans in the southeast United States. Food Chemistry, 103, 1395−1402.

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