Food Research International 44 (2011) 297–303

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Food Research International

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

a r t i c l e i n f o a b s t r a c t

Article history: Different solvent extracts of endemic Sideritis (Labiatae) species, Sideritis congesta Davis et Huber-Morath and
Received 20 July 2010 Sideritis arguta Boiss et Heldr, were analyzed for free flavonoids (quercetin, apigenin, myricetin and
Accepted 8 October 2010 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
Keywords: rosmarinic acid, chlorogenic acid and myricetin which were quantified in raw samples. Antioxidant activities
Antioxidant activity
of extracts of these two plants and many of their components in pure form were evaluated based on DPPH. and
Cinnamic acid derivatives
Flavonoids
ABTS.+ assays. In general, S. arguta extracts displayed higher antioxidant activity than S. congesta extracts
Sideritis arguta possibly due to their richness in antioxidant components of strong activity. Acetone extract of S. arguta, with
Sideritis congesta 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 phenolic structure and are widely distributed in photosynthesizing cells

(Havsteen, 1983). Flavonoids can be subdivided into several classes:
Extensive number of evidences has pointed out free radicals as flavones, flavonols, flavanones, isoflavones, flavans, flavanols, and
major contributors to aging and degenerative diseases such as cancer, anthocyanins. Tea and herbal preparations provide the major source
cardiovascular disease, cataracts and immune system decline (Ames, of these two groups of phenolic compounds in human diet (Shahidi,
Shigenaga, & Hagen, 1990; Young & Woodside, 2001). However, free 2000). Investigation of the presence and activity of flavonoid and
radical formation is controlled naturally by compounds known as cinnamic acid derivatives as antioxidants in tea and herbs has been
antioxidants. The damage in biological systems can be cumulative performed in various studies (Zaveri, 2006; Erkan, Ayranci, & Ayranci,
when the concentration of radical species and antioxidants are not in 2008; Atoui, Mansouri, Boskou, & Kefalas, 2005). It is known that
balance (Swanson, 1998). Antioxidants are capable of neutralizing, or extracts obtained with organic solvents from dried leaves of such plants
scavenging free radicals by hydrogen donation before the latter attack are of much interest due to the high capacity of these solvents in
cells and other biological components. Thus, they are vital for well- extracting antioxidant compounds.
being and protecting optimal health (Percival, 1998). Synthetic The aerial parts of plants from the genus Sideritis, commonly
antioxidants, such as butylated hydroxytoluene (BHT), butylated known as ‘mountain tea’ are widely used as a popular folk medicine in
hydroxyanisole (BHA) and tertiary butyl hydroquinone (TBHQ) have Mediterranean countries such as Greece, Turkey and Spain. The genus
been widely used as antioxidants in the food industry until recently. Sideritis is represented in the Turkish flora by 46 species, 31 of which
However, their uses have been limited for the fact that they may be are endemic (Davis, Mill, & Tan, 1988). Different biological activities of
responsible for liver damage and carcinogenesis (Grice, 1988; Wichi, Sideritis species, including anti-inflammatory (Hernandez-Perez &
1986). This problem has been overcome by supplementing diets with Rabanal, 2002), anti-ulcer (Aboutabl et al., 2002), antioxidant
antioxidants from natural sources, most of which are plants, fruits and (Armata, Gabrieli, Termentzi, Zervou, & Kokkalou, 2008) and
vegetables (Knekt, Jarvinen, Reunanen, & Maatela, 1996). antimicrobial (Aligiannis, Kalpoutzakis, Chinou, & Mitakou, 2001)
Flavonoids and cinnamic acid derivatives, the groups of compounds activity have been reported up to now. Many of these activities have
that most of the antioxidant activity of plants comes from, contain 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).
⁎ Corresponding author.Tel.: + 90 242 310 2315; fax: + 90 242 227 8911. In this article, we report for the first time a detailed study on
E-mail address: [email protected] (E. Ayranci). investigating the antioxidant activities of two endemic Sideritis species,

0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.10.016
298 N. Erkan et al. / Food Research International 44 (2011) 297–303

Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et 7 μL, respectively. Among the phenolic compounds analyzed, ros-
Heldr, and analyzing the extracts of these plants for free flavonoids and marinic acid, chlorogenic acid and myricetin were quantified in raw
cinnamic acid derivatives. extracts, while ferulic acid, caffeic acid, p-coumaric acid, quercetin,
apigenin and kaempferol were quantified in acid-hydrolyzed extracts.
2. Materials and methods All standard compounds were diluted in methanol before analysis.
Peak identification in HPLC analysis was performed by comparison
2.1. Materials of retention time with respective reference standards. Quantification
of individual flavonoids was done using the peak area of identified
All solvents used were HPLC grade and purchased from Merck. 1,1- compounds. Peak purity values, which were provided by the
diphenyl-2-picryl-hydrazyl (DPPH) radical, quercetin, myricetin, instrument facility, were checked in each component analysis. They
apigenin, caffeic acid and p-coumaric acid were obtained from were found to be within the threshold limit in each case.
Sigma. Trolox, gallic acid, chlorogenic acid, ferulic acid and rosmarinic
acid were from Aldrich. 2,2-azino-bis(3-ethylbenzothiazoline-6-sul- 2.3. DPPH radical scavenging assay
fonic acid) diammonium salt (ABTS) and kaempferol were purchased
from Fluka. The standard compounds were in their highest purity and This assay was carried out as described by Blois (1958) with some
all other reagents were of analytical grade. Deionized water was used modifications. 1 mL of various dilutions of the test materials (pure
to prepare aqueous solutions. antioxidants or plant extracts) was mixed with 2 mL of a 0.2 mM
S. congesta and S. arguta were collected from Akseki county of methanolic DPPH. solution. After an incubation period of 30 min at
Antalya, Turkey in July, 2009. Taxonomic identification was per- 25 °C, the absorbances at 515 nm were recorded as Asample using a
formed by botanists in the Biology Department, Faculty of Arts and Cary 100 Bio UV/VIS spectrophotometer. A blank experiment was also
Sciences, Akdeniz University. The aerial parts of plants were washed carried out applying the same procedure to a solution without the test
with deionized water and dried at room temperature for 15 days. material and the absorbance was recorded as Ablank.
Dried samples were kept in a deep freezer at − 20 °C until use. The free radical scavenging activity of each solution was then
calculated as percent inhibition according to the following equation:
2.2. Preparation, acid hydrolysis and analysis of plant extracts
 
% inhibition = 100 Ablank –Asample = Ablank ð1Þ
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 Antioxidant activities of test compounds or extracts were
the organic solvents; 64.7, 77 or 56.5 °C, respectively. The mixture was expressed as IC50, defined as the concentration of the test material
filtered through Whatman paper (GF/A, 110 mm) and its solvent was required to cause a 50% decrease in initial DPPH. concentration. α-
removed by a rotary evaporator (Heidolph) at 40 °C. All the extracts Tocopherol was used as the positive control.
were in dark green color after evaporation of the solvent, due to high
chlorophyll content of the plants. In order to remove chlorophyll from 2.4. ABTS.+ radical scavenging assay
the extracts in the highest possible amount, repeated washing steps
with hexane were applied to each extract. After filtration, the remaining This assay was carried out according to the procedure described by
extracts were kept in a vacuum oven at 35 °C for 1 day for further Re et al. (1999). ABTS.+ radical cation was produced by reacting 7 mM
removal of the solvent residue. Dried samples appeared in varying aqueous ABTS with 2.45 mM (final concentration) potassium persulfate
colors from dark yellow to light green and were kept in the deep freezer and keeping the mixture in the dark at room temperature for 16 h. Blue-
at −20 °C until use. green ABTS.+ was formed at the end of this period. The solution was
An acid hydrolysis step was applied to the extracts to release diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm,
aglycones of flavonoid glycosides for simplification of peak identifi- wavelength of maximum absorbance in the visible region. Test materials
cation procedure. Acid hydrolysis was performed according to the were dissolved in and diluted with ethanol such that, after the
procedure reported by Huang, Wang, Eaves, Shikany, and Pace (2007) introduction of an accurately measured volume of each dilution into
with minor modifications. Briefly, 0.01 g of plant extract was the assay, they produced between 10%–90% decrease in the absorbance
subjected to water bath-incubation at 90 °C for 1.5 h after vortex- of the blank solution at 734 nm. After adding 100 μL of the test solution
mixing with 4 mL of 80% methanol and 1 mL of 6 M HCl. At the end of to 3.5 mL of ABTS.+ solution having A734 = 0.70 ± 0.02, absorbance was
incubation, the mixture was cooled to room temperature, made up to recorded at 6 min. The results were expressed as trolox equivalent
5 mL with methanol and sonicated for 7 min. Then, the mixture was antioxidant capacity (TEAC). TEAC is defined as the mM concentration of
centrifuged at 3000 ×g for 10 min. The supernatant was separated and a trolox solution whose antioxidant activity is equivalent to the activity
its solvent was removed completely. 10 mL of methanol was added to of 1.0 mM and 1 mg/mL test solution for pure compounds and plant
the residue and the solution was filtered through a 0.2 μm syringe extracts, respectively. In order to find TEAC values, a separate
filter (Millipore, Bedford, MA) and subjected to analysis (approximate concentration response curve for standard trolox solutions was
concentration, 1 mg/mL). Untreated extracts were also dissolved in prepared. α-Tocopherol was used as the positive control.
methanol (1 mg/mL) and filtered prior to analysis.
HPLC analysis of S. congesta and S. arguta extracts were performed 2.5. Total phenolic content (TPC)
with an Agilent 1100 series HPLC instrument equipped with an
autosampler and a diode array detector (DAD). The column was TPCs of Sideritis extracts were determined using Folin-Ciocalteu
Hypersil ODS C18 type with a 5 μm particle size, 4.6 × 250 mm i.d. used reagent (FCR) according to the procedure reported by Singleton,
with Hypersil ODS 4.0 × 20 mm i.d. 5 μm guard cartridges. The mobile Orthofer, and Lamuela-Raventos (1999) with some modifications. The
phase was composed of 5% acetic acid in H2O (solvent A) and extracts were dissolved in deionized water to provide a concentration of
methanol (solvent B). It was eluted at a flow rate of 0.9 mL/min. 500 μg/mL. 0.5 mL aliquot of extract or deionized water (control) was
Gradient elution utilized was: 0 min, 5% B; 5 min, 15% B; 25 min, 30% mixed with 0.5 mL of FCR by manual shaking for 10–15 s. After 3 min,
B; 39 min, 42% B; 47 min, 55% B; 50 min, 70% B; 56 min, 75% B and 0.5 mL of saturated Na2CO3 solution was added and the solution was
60 min, 100% B. The chromatograms were acquired at 280 and diluted to 5 mL with deionized water. The reaction mixture was kept in
330 nm. Column temperature and injection volume were 28 °C and the dark for 2 h and the absorbance was measured at 760 nm. The
 N. Erkan et al. / Food Research International 44 (2011) 297–303 299

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
Fig. 1. Chemical structures of (a) cinnamic acid derivatives and (b) free flavonoids.
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, hydrolyzed samples under the same conditions (Figs 2a–b and 3a–b).
apigenin, myricetin and kaempferol as flavonoid antioxidants This can be explained by the chemical structures (Fig. 1) of these
(Fig. 1b) since some of these compounds and their glycosides have compounds. Since rosmarinic and chlorogenic acids are esters of
already been reported to exist in different Sideritis species (Armata caffeic acid with 3,4-dihydroxyphenyl lactic acid and quinic acid,
et al., 2008; Pljevljakušić et al., 2011). The phenolics under study respectively, acidic treatment could have diminished the concentra-
usually occur in plants as glycosides and methylated esters, or they tions of these compounds via hydrolysis. Thus, these components
may exist in their free form as well (Skerget et al., 2005). Thus, an acid were quantified in untreated samples.
hydrolysis step has been applied to all plant extracts before analysis to Table 1 shows the amounts (mg/g) of phenolic compounds found
simplify peak identification. The acidic hydrolysis conditions used in in different solvent extracts of S. congesta and S. arguta. In general,
the present study have previously been proven to be effective in cinnamic acid derivatives were found to be in higher amounts than
releasing the aglycones from the conjugated phenolic compounds by flavonoid antioxidants, the most abundant ones being ferulic acid and
Huang et al. (2007). They have observed no significant peaks in their chlorogenic acid in majority of the extracts. Rosmarinic acid was
work for glycosides by LC/MS which indicated that hydrolysis of most detected to exist in the lowest amounts among acidic antioxidants
of the glycosides to aglycones have been achieved under acidic studied in most of the extracts (p b 0.05), with the exception of S.
hydrolysis conditions. The signals of all pure compounds were arguta-EtAc and S. arguta-ACET extracts (p N 0.05) (note each row for
observed to be more intense at 330 nm than at 280 nm, except for comparing the amount of rosmarinic acid with the amount of other
p-coumaric acid whose signal appeared to be more intense at 280 nm. acidic antioxidants in each extract in Table 1). S. congesta extracts
Thus, the amounts of p-coumaric acid in plant extracts were were found to contain higher amounts of p-coumaric acid compared
calculated based on the peak areas obtained at 280 nm. The to S. arguta extracts (p b 0.05). Its amount was found to be the highest
chromatograms were obtained for extracts of both S. congesta and S. in S. congesta-ACET extract (22.8 mg/g, p b 0.05). Flavonoids, on the
arguta with all three solvents. However since the antioxidant activity other hand, were found to exist in higher amounts in S. arguta extracts
results (discussed in the next subsection) showed that S. congesta- than S. congesta extracts, with an exception of apigenin and few other
MET and S. arguta-ACET extracts displayed the highest antioxidant cases (Table 1). Although, quercetin could not be detected in S.
activities among other extracts of S. congesta and S. arguta, congesta extracts at all, it was the flavonoid existing in high amounts
respectively, only the chromatograms for these two extracts are together with considerable amounts of kaempferol in S. arguta
presented in Figs. 2 and 3, respectively. Comparison of chromato- extracts.
grams for acid-hydrolyzed extracts and untreated extracts shows that
the release of aglycones attached by glycosidic linkage to the majority 3.2. Antioxidant activity of plant extracts and pure components
of the studied flavonoid compounds, or the release of the previously
mentioned cinnamic acid derivative compounds from their ester Table 2 shows antioxidant activities of plant extracts and their
bonds upon hydrolysis improve peak resolution and intensity. phenolic components. Antioxidant activities were expressed as IC50 and
However, the peak intensities of rosmarinic and chlorogenic acids TEAC values, based on the results obtained from DPPH. and ABTS.+
were found to be somewhat higher in untreated samples than in acid- radical scavenging assays, respectively. It should be recalled that the
300 N. Erkan et al. / Food Research International 44 (2011) 297–303

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) acid-
hydrolyzed S. arguta-ACET extract recorded at 280 nm. Ordinate has the same scale in a, b and c.
 N. Erkan et al. / Food Research International 44 (2011) 297–303 301

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.
a
Plant extracts Amounts of phenolic components in Sideritis extracts (mg/g)

Rosmarinic acidb Ferulic acid Caffeic acid p-coumaric acid Chlorogenic acidb Quercetin Apigenin Myricetinb Kaempferol

S. congesta-MET extract 1.1 ± 0.0 fE 6.6 ± 0.0 eB 5.0 ± 0.4 bC 4.6 ± 0.5 cC 22.2 ± 0.8 bA n.d 3.0 ± 0.1 aD 4.7 ± 0.2 bC 2.5 ± 0.3 dD
S. congesta-EtAc extract 1.6 ± 0.0 cE 20.5 ± 0.2 cA 2.5 ± 0.1 cD 8.4 ± 0.2 bB 5.4 ± 0.4 eC n.d 2.6 ± 0.1 bD n.d 2.8 ± 0.1 dD
S. congesta-ACET extract 1.5 ± 0.0 dG 22.6 ± 0.1 bA 5.5 ± 0.7 bD 22.8 ± 1.0 aA 14.2 ± 1.0 cB n.d 3.0 ± 0.3 aF 4.2 ± 0.0 cE 7.0 ± 0.9 bC
S. arguta-MET extract 1.3 ± 0.0 eF 23.0 ± 1.0 bA 3.0 ± 0.1 cE 2.6 ± 0.5 dE 23.6 ± 1.0 bA 7.7 ± 0.6 aC 1.5 ± 0.1 dF 5.6 ± 0.1 aD 10.6 ± 1.0 aB
S. arguta-EtAc extract 2.7 ± 0.1 aD 24.4 ± 0.4 aA 2.1 ± 0.1 cD 1.1 ± 0.0 eE 7.8 ± 0.8 dB 7.8 ± 0.6 aB 1.1 ± 0.0 eE 5.6 ± 0.1 aC 5.9 ± 0.1 cC
S. arguta-ACET extract 2.0 ± 0.0 bF 18.9 ± 0.7 dB 6.8 ± 0.9 aD 1.7 ± 0.1 eF 30.3 ± 1.3 aA 8.2 ± 0.7 aC 1.9 ± 0.1 cF n.d 5.3 ± 0.1 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 loaded closer on Factor 2 (PC2) on negative side of the graph. This is also
proportional to IC50 value. Principal component analysis (PCA) was supported by the correlation coefficients, −0.5170 and −0.2627,
performed to understand how important are the three assays, namely, between TPC; and 1/IC50 and TEAC, respectively, obtained for plant
DPPH. and ABTS.+ radical scavenging ability and TPC in evaluation of the extracts. TPC assay, thus, does not seem to be appropriate for interpreting
antioxidant activity of the plant extracts. Since IC50 and TEAC values are antioxidant activities of the plant extracts for the current study.
inversely proportional to each other, IC50 values were converted to 1/IC50 The order of the antioxidant activity of Sideritis extracts were
values before PCA and factor analysis to simplify interpretation and to found to be; S. arguta-ACET extract N S. arguta-MET extract N S.
avoid any confusion. Since the TPC assay was not performed on pure congesta-MET extract ≥ S. arguta-EtAc extract N S. congesta-EtAc extra-
compounds, factor analysis was performed on only plant extracts. A factor ct N S. congesta-ACET extract, based on DPPH. radical scavenging assay.
rotation using the Varimax method was performed for two factor loadings The highest antioxidant activities for S. arguta-ACET and S. arguta-MET
to see the correlations between assays that accounted for the total extracts were also confirmed by ABTS. assay. Furthermore, the
covariance of the plant extracts. The most significant component (PC1), distribution of plant extracts on PCA graph (Fig 4) shows that S.
DPPH. radical scavenging ability, given as 1/IC50 and ABTS.+ radical arguta-ACET extract (extract6) and S. arguta-MET extract (Extract 4)
scavenging ability, given as TEAC in Fig 4, contributed to the largest are the strongest antioxidant extracts, among all Sideritis extracts
variation of approximately 73%, while, TPC (PC2) accounted for (p b 0.05).
approximately 27% to the total variation. Total phenol content (TPC) In order to see the solvent effect on plant extract, Ertas, Ozturk,
values did not show a significant contribution to the total variation as the Boga, and Topcu (2009) studied the antioxidant activity of acetonic,
other two assays. This is reasonable because TPC shows the content of all metanolic and etheric extracts of S. arguta and found the acetonic
of the phenolic type compounds each of which may not necessarily have extract to have the highest activity based on DPPH. and superoxide-
strong radical scavenging capacity. In Fig. 4, it can be seen that DPPH assay anion scavenging abilities, similar to what has been observed in the
and ABTS assay, shown as 1/IC50 and TEAC, respectively, are highly loaded present study. Both Sideritis species studied were observed to have
on Factor 1 (PC1). The close loading of these values to each other indicates stronger antioxidant potency than Sideritis libanotica subsp. linearis
the higher contribution of the two assays to the antioxidant activity (IC50, 109 μg/mL), another Sideritis species, reported by Tepe, Sokmen,
(Pearson's correlation coefficient, 0.8404). However, TPC seems to be Akpulat, Yumrutas, and Sokmen (2006). In general, S. arguta extracts
were found to exhibit higher antioxidant activity than S. congesta
Table 2 extracts. This is also consistent with the results obtained by Guvenc,
Total phenolic contents (TPCs) of Sideritis extracts and antioxidant activities of the Houghton, Duman, Coskun, and Sahin (2005). They found S. arguta
extracts and their pure components expressed as IC50 and TEAC values, based on DPPH.
and ABTS.+ assays, respectively.

Samplec Antioxidant activity

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

S. congesta-MET extract 50.9 ± 2.2 ef 2.5 ± 0.0 g 1375.6 ± 9.4 e

S. congesta-EtAc extract 54.2 ± 1.7 e 2.4 ± 0.0 g 1722.4 ± 12.2 c
S. congesta-ACET extract 66.2 ± 3.3 d 2.3 ± 0.0 g 1970.8 ± 10.4 a
S. arguta-MET extract 48.6 ± 2.4 f 2.9 ± 0.0 f 1785.2 ± 6.3 b
S. arguta-EtAc extract 52.7 ± 1.9 ef 2.5 ± 0.0 g 1363.2 ± 8.6 e
S. arguta-ACET extract 38.3 ± 1.1 g 3.2 ± 0.1 e 1475.2 ± 7.2 d
Rosmarinic acid 12.4 ± 0.8 i 6.4 ± 0.1 a
Ferulic acid 49.6 ± 2.3 ef 3.5 ± 0.0 d
Caffeic acid 12.4 ± 0.7 i 3.8 ± 0.1 c
p-coumaric acid 105.3 ± 4.3 c 3.8 ± 0.1 c
Chlorogenic acid 35.6 ± 2.1 g 2.4 ± 0.3 g
Quercetin 12.9 ± 1.0 i 6.1 ± 0.1 b
Apigenin 427.7 ± 4.8 b 1.1 ± 0.0 i
Myricetin 438.6 ± 6.5 a 1.5 ± 0.0 h
Kaempferol 26.6 ± 2.5 h 3.5 ± 0.3 d
α-Tocopherol 33.8 ± 0.4 g 2.9 ± 0.2 f
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 Fig. 4. Principal component analysis results of DPPH, ABTS and TPC assays displayed
whose antioxidant activity is equivalent to 1.0 mM of test solution. For plant extracts, as 1/IC50, TEAC and TPC, respectively, for plant extracts (S. congesta-MET extract
TEAC is defined as the concentration of trolox solution whose antioxidant activity is (Extract 1); S. congesta-EtAc extract (Extract 2); S. congesta-ACET extract (Extract 3); S.
equal to 1.0 mg/mL of test solution. arguta-MET extract (Extract 4); S. arguta-EtAc extract (Extract 5); (S. arguta-ACET
c
MET: methanol, EtAc: ethyl acetate, ACET: acetone. extract (Extract 6)).
302 N. Erkan et al. / Food Research International 44 (2011) 297–303

extracts to have higher antioxidant potential than S. congesta extracts, 4. Conclusion

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 The differences in radical scavenging activity of pure compounds
antioxidants exhibited varying extent of antioxidant activity accord- and complex plant extracts could be explained on the basis of the
ing to the results of DPPH. and ABTS.+ assays (Table 2). Rosmarinic chemical nature and reactivity of the compounds and the nature of the
acid, caffeic acid and quercetin showed distinctively stronger solvents used in different assays. The presence of glycosidic moieties,
activities than other compounds, based on both assays (p b 0.05). the number and position of phenolic hydroxyl and methoxy groups
Rosmarinic acid, caffeic acid, quercetin and kaempferol showed and the synergistic effects of coexisting antioxidant molecules may
strikingly high activities compared to that of α-tocopherol (control) influence the antioxidant activity (Hidalgo, Sánchez-Moreno, &
on both assays (p b 0.05). It is interesting to note that ferulic acid Pascual-Teresa, 2010).
displayed contradictory results for the two assays. It shows lower S. congesta and S. arguta, native to Turkey, were found to have
activity than α-tocopherol according to DPPH. assay and higher strikingly high antioxidant potency. Antioxidant activities of S. arguta-
activity according to ABTS.+ assay. Chlorogenic acid exhibited ACET and S. arguta-MET extracts were found to be the highest among
statistically very close IC50 value and a slightly lower TEAC value all Sideritis extracts based on DPPH. and ABTS.+ assays, and principal
(2.4 mM trolox) compared to α-tocopherol, making it a very effective component analysis. Cinnamic acid derivatives were detected to exist
antioxidant component. Apigenin and myricetin exerted the lowest in higher amounts than flavonoid antioxidants in plant extracts, the
activities among the other components, based on both assays. The most abundant ones being ferulic acid and chlorogenic acid in
highest activity observed for S. arguta-ACET extract can be ascribed to majority of the extracts. No direct correlation was observed between
its highest caffeic acid and chlorogenic acid contents among other the TPC and antioxidant activity of the extracts indicated by Pearson
extracts (p b 0.05 for both compounds). The presence of considerable correlation coefficients. These results and chromatographic analyses
amounts of ferulic acid, kaempferol and rosmarinic acid in this extract suggest that other phenolic components may be present in the
also contributes to its overall activity (Table 1). The activity of S. extracts which need to be investigated.
arguta-MET extract, which is the second highest among the activities
of plant extracts, could also be related to its high flavonoid content. Acknowledgement
Regarding the total phenolic content (TPC) (Table 2) and the amounts
of individual compounds in the extracts (Table 1), it is obvious that We would like to thank the Scientific Research Projects unit of
methanol seems to be the best solvent for S. arguta in extracting Akdeniz University for the support of this work through the Project
phenolic compounds, while acetone seems to be more appropriate for 2005.01.0200.001.
S. congesta. On the other hand, the highest antioxidant activity of S.
arguta-ACET and S. congesta-MET is probably originating from the References
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