Antioxidant activity and chemical composition of red and white wines of south of brazil

This article was downloaded by: [Gaziosmanpasa Universitesi]On: 28 September 2011, At: 04:16Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Natural Product ResearchPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gnpl20

Antioxidant activity and chemicalcomposition of Sideritis libanoticaLabill. ssp. linearis (Bentham) Borm.(Lamiaceae)Ibrahim Demirtas a , Bulent Ayhan a , Ayse Sahin a , Hüüseyin Aksita , Mahfuz Elmastas a & Isa Telci ba Laboratory of Plant Research, Department of Chemistry, Facultyof Science and Art, Gaziosmanpasa University, TaççlıçiftlikCampus, 60240 Tokat, Turkeyb Department of Field Crops, Faculty of Agriculture,Gaziosmanpasa University, Taççlıçiftlik Campus, 60240 Tokat,Turkey

Available online: 08 Jul 2011

To cite this article: Ibrahim Demirtas, Bulent Ayhan, Ayse Sahin, Hüüseyin Aksit, Mahfuz Elmastas& Isa Telci (2011): Antioxidant activity and chemical composition of Sideritis libanotica Labill. ssp.linearis (Bentham) Borm. (Lamiaceae), Natural Product Research, 25:16, 1512-1523

To link to this article: http://dx.doi.org/10.1080/14786410903293191

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching and private study purposes. Anysubstantial or systematic reproduction, re-distribution, re-selling, loan, sub-licensing,systematic supply or distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,

demand or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

Natural Product ResearchVol. 25, No. 16, September 2011, 1512–1523

Antioxidant activity and chemical composition of Sideritis libanoticaLabill. ssp. linearis (Bentham) Borm. (Lamiaceae)

Ibrahim Demirtasa*, Bulent Ayhana, Ayse Sahina, Huseyin Aksita,Mahfuz Elmastasa and Isa Telcib

aLaboratory of Plant Research, Department of Chemistry, Faculty of Science and Art,Gaziosmanpasa University, Tacl{ciftlik Campus, 60240 Tokat, Turkey; bDepartment of FieldCrops, Faculty of Agriculture, Gaziosmanpasa University, Tacl{ciftlik Campus, 60240 Tokat,Turkey

(Received 9 May 2009; final version received 18 September 2009)

Sideritis libanotica ssp. linearis was screened for the isolation of new naturalantioxidant compounds. The antioxidant activity of flavones obtained fromthe methanol extract of the plant was evaluated in vitro using totalantioxidant, reduction power and free radical scavenging activity. Resultswere compared with the positive controls of antioxidant standards(�-tocopherol and butylated hydroxytoluene). The results indicate that theflavones possess a higher antioxidant activity when compared to the othercomponents in the plant. The lowest antioxidant activity was observed infatty acids (FAs) and hydrocarbons. The FAs were methylated with MeOHand KOH and analysed by GC–MS. The structures of the isolatedcompounds were established based on spectroscopic evidence (NMR,GC–MS, HPLC, IR and UV). In this work, the isolated pure flavone(30-O-methylhypolaetin 7-O-[600 0-O-acetyl-�-D-allopyranosyl-(1! 2)]-600-O-acetyl-�-D-glucopyranoside) was found to possess the highest antioxidantactivity.

Keywords: antioxidant activity; methanolic extract; Sideritis libanoticalinearis; flavonoid; FAs

1. Introduction

The aerial parts of plants from the genus Sideritis, generally known as ‘mountaintea’, are widely used as a popular folk medicine in Spain, Greece and Turkey. Thegenus Sideritis is represented in the Turkish flora by 46 species, 31 of which areendemic (Aytac & Aksoy, 2000; Duman, 2000), including Sideritis libanotica Labill.ssp. linearis (Bentham) Borm. Some species are used in the treatment of gastroin-testinal ailments and common colds as well as a herbal tea in Turkish folk medicine(Baytop, 1999; Yesilada et al., 1995). Sideritis species grown in Turkey are known tobe rich in essential oils (Baser, 2002; Ezer, Vila, Canigueral, & Adzet, 1996),diterpenes (Sahin, Ezer, & Cal|s� , 2004; Sahin, Tasdemir, Ruedi, Ezer, & Cal|s� , 2005;Topcu, Goren, K|l|c, Y|ld|z, & Tumen, 2002), flavonoids and phenylethanoidglycosides (Ezher, Sakar, Rodriguez, & De la Torre, 1992; Sahin et al., 2004, 2005).

*Corresponding author. Email: [email protected]

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2011 Taylor & Francis

DOI: 10.1080/14786410903293191

http://www.informaworld.com

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

Several studies have been conducted on various biological activities of Sideritisspecies (de las Heras, Vivas, & Villar, 1994; Demirtas, Sahin, Ayhan, Tekin, & Telci,2009; Hernandez-Perez & Rabanal, 2002; Hernandez-Perez, Sanches, Montalbetti-Moreno, & Rabanal, 2004). Antioxidant activities of Sideritis raeseri and Sideritisjavalambrensis have been reported elsewhere (Gabrieli, Kefalas, & Kokkalou, 2005;Rios, Manez, Paya, & Alcaraz, 1992). The antioxidant activity of S. libanotica ssp.linearis was reported previously with other members of this genus by Tunalier et al.(2004) and Tepe, Sokmen, Akpulat, Yumrutas, and Sokmen (2006) in the crudemethanolic extract. The literature outlines different approaches for the determina-tion of antioxidant activities of the plant extracts, which are difficult to compare andsometimes conflicting.

To the best of our knowledge, the literature contains no information on theisolation of active compounds from S. libanotica ssp. linearis and its fatty acid (FA)contents. The aim of this study is to investigate the isolation and characterisation ofbiologically active compounds such as flavones, FAs and their antioxidant capacityin the plant.

2. Results and discussion

2.1. Structure elucidation

The flavone (30-O-methylhypolaetin 7-O-[6000-O-acetyl-�-D-allopyranosyl-(1! 2)]-600-O-acetyl-�-D-glucopyranoside, 1, in Figure 1) obtained as the 600,6000-diacetylatedderivative of the diglucose moiety was attached to the oxygen at C-7. The molecularformula of 1 was determined to be C32H36O19. UV absorptions were recordedat 276, 303, 325(sh), and 277, 303, 325(sh), respectively, and spectroscopic data withshifting reagents were indicative of a flavone skeleton. The IR spectra indicatedabsorption bands for a hydroxyl, carbonyl, ester carbonyl and aromatic ring(Section 3). In the 1H NMR spectrum of 1 (Table 1), two singlets at �H6.79 and 6.62were assigned to H-6 and H-3, respectively. The 30, 40-disubstitutions on the B ringwere indicated by a pair of ortho-coupled (J¼ 8.5Hz) doublets at �H 7.60 (H-60)

O

O

OH

OCH3

OH

OH

O

O

O

O

AcO

AcOHO

HO

HO

HO

HO

2

345

6

78

9

10

1'

2'3'

4'

5'

6'1''2''

3''

4''5''

6''

1'''2'''3'''

4'''

5'''

6'''

2

3

4

56

7

8

9

10

11

12

13

17

18

1

19

20

14

15

16

OH

OH

1 2

Figure 1. 30-O-Methylhypolaetin 7-O-[600 0-O-acetyl-�-D-allopyranosyl-(1! 2)]-600-O-acetyl-�-D-glucopyranoside (1) and sideridiol (2).

Natural Product Research 1513

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

and 7.10 (H-50), which appeared as an AA0BB0 system, and meta-coupled (J¼ 2.2Hz)

doublets at �H 7.60 (H-60) and 7.48 (H-20). In addition, two anomeric proton

signals at �H 5.17 (d, J¼ 7.5Hz) and 4.90 (d, J¼ 8.0Hz) indicated a diglycosidic

structure. Assignments of all proton and carbon resonances were achieved by COSY

and HMQC experiments, which showed that the sugars are attached to C7. The

Table 1. 13C and 1H NMR spectroscopic dataa for flavone (30-O-methylhypolaetin 7-O-[600 0-O-acetyl-�-D-allopyranosyl-(1! 2)]-600-O-acetyl-�-D-glucopyranoside, 1, in DMSO-d6,100MHz for 13C and 400MHz for 1H and sideridiol (2) in CDCl3, 100MHz for 13C and400MHz for 1H.

Flavone (1) Sideridiol (2)

C/H �Cppm DEPT �Hppm (J, Hz) C/H �C ppm DEPT �Hppm (J, Hz)

2 164.4 C 1 39.9 CH2 0.68m3 103.8 CH 6.62 s 1.714 182.7 C 2 18.0 CH2 1.445 151.7 C 3 35.1 CH2 1.11 d (11.0)6 99.6 CH 6.79 s 1.52m7 164.4 C 4 37.1 C8 152.7 C 5 38.2 CH 1.72 q9 99.9 C 6 26.2 CH2 1.5210 106.0 C 7 75.4 CH 3.56 bs10 123.5 C 8 53.5 C20 112.5 CH 7.48 d (2.2) 9 44.1 CH 1.3230 151.7 C 10 40.0 C40 147.2 C 11 18.3 CH2 1.4450 113.6 CH 7.10 d (8.5) 12 24.9 CH2 1.4460 119.4 CH 7.60 dd (8.5/2.2) 13 44.7 CH 2.31 bsOCH3 56.2 CH3 3.85 s 14 42.2 CH2 1.32 mGlucose 1.86 d (11.0)100 99.9 CH 5.17 d (7.5) 15 129.8 CH 5.41 s200 82.5 CH 3.59b 16 144.1 C300 75.2 CH 3.53b 17 15.5 CH3 1.67 s400 69.2 CH 3.28b 18 70.9 CH2 2.87 d (11.0)500 73.5 CH 3.76b 3.42 d (11.0)600 71.42 CH2 3.26b 19 17.6 CH3 0.68 s

3.88b

COMe 170.6 C 20 17.7 CH3 1.00 sCH3 20.7 CH3 1.87 sGlucose1000 102.6 CH 4.88 d (8.0)200’ 69.4 CH 3.21b

3000 63.5 CH 3.96b

400’ 66.8 CH 3.41b

5000 70.7 CH 3.88b

6000 71.4 CH2 3.26b

3.88b

COMe 170.7 CCH3 21.0 CH3 2.03 s

Notes: aAll carbon and proton resonances were assigned on the basis of 2D NMR (COSY,HSQC and HMBC) experiments. bSignal patterns are unclear due to overlapping.

1514 I. Demirtas et al.

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

1H NMR spectrum of 1 exhibited acetoxymethyl signals at �H1.87 (s, 3H) and�H 2.03 (s, 3H).

30-O-Methylhypolaetin 7-O-[6000-O-acetyl-�-D-allopyranosyl-(1! 2)]-600-O-acetyl-�-D-glucopyranoside (1): Amorphous yellow powder. UV �max nm (MeOH): 277,303, 325(sh); (NaOMe): 247, 276, 377; (AlCl3): 282, 323, 350; (AlCl3þHCl): 281,322, 347 (sh); (NaOAc): 275, 304, 329 (sh), 386; (NaOAcþH3BO3): 276, 305, 327(sh); IR �max (KBr) cm�1¼ 3414 (OH), 1655 (�-pyrone C¼O), 1727 (ester C¼O),1508, 1600 (aromatic ring); C31H34O18;

13C, 1H NMR (DMSO-d6,13C: 100MHz,

1H: 400MHz) in Table 1.The 1H NMR spectrum of sideridiol (2, Table 1 and Figure 1) showed an AB

system of doublets at �H 2.87 and 3.42 (JAB¼ 11.0Hz), a broad singlet at �H 3.56 andthree singlets at �H 0.68, 1.00 and 1.67. 13C NMR and DEPT-135 spectra revealedsignals at �C 144.1 (C) and 129.8 (CH), which were assigned to the olefinic carbons atC-16 and C-15, respectively (spin system d).

The 13C NMR spectrum showed two oxygenated carbon signals (�C 75.4 and70.9). Of these, the carbon resonance at �C 70.9 (CH2) displayed HSQC correlationswith oxymethylene protons (�H2.87 and 3.42 JAB¼ 11Hz) forming an AB system. Inthe HSQC spectrum, short-range correlations between the carbon resonance at�C 75.4 (CH) and a broad singlet at �H 3.56 was indicative of a secondary hydroxylgroup at C-7. The COSY experiment clarified that the secondary hydroxyl group wasat spin system b. In the 1H NMR spectrum, a downfield methyl signal at �H 1.67 wasindicative of an allylic methyl function. The COSY spectrum also indicatedcorrelations between this methyl group (�H 1.67) and the olefinic proton at �H 5.41(H-15) (spin system d). Long-range couplings between signals observed in 13C and1H NMR spectra were explained by HMBC experiments. HMBC cross-peaksbetween methyl protons and carbons showed that methyl groups (�H 1.67, 1.00, 0.68)are at C-16 (Me-17), C-10 (Me-20) and C-4 (Me-19), respectively. 1D and 2DNMR experiments allowed the structure of the terpenoid to be determined assideridiol (2) and the NMR data are in accord with the literature (Sahin, Ezer, &Calis, 2006).

As shown in Figure 2, the total antioxidant activity of the crude extract anddifferent fractions of S. libanotica ssp. linearis were compared against �-tocopherol.In Figure 2, the fractions 214–282 characterised as flavonoid had the highestantioxidant activity. The high antioxidant activity can be attributed to the presenceof higher content of polyphenols attached to the aryl groups, which were found inhigh percentages in the fractions (Table 2). The antioxidant activity of flavonoid isthe same with �-tocopherol. Figure 2 indicates that the crude extract and otherfractions are not as active as fractions 214–282 and �-tocopherol.

The antioxidant activity of fractions 143–171 was also noteworthy whencompared to the antioxidant compound used in this study as positive control. Onthe other hand, the non-polar solvent fraction (Fraction 25) exerted the weakestantioxidant activity in this system, as can be seen in Figure 2. GC–MS analysis of thehexane and hexane : chloroform (9 : 1) fractions gave the FAs, alcohols andhydrocarbons (Table 3). Total FA content (%) was calculated from hexane andCHCl3 : hex (1 : 9) fractions as total carbohydrates in 2 kg of the crude extract.

The antioxidant activity of the chemicals obtained from the plant in decreasingorder was flavonoids, terpenoids, alcohols, FAs and hydrocarbons. The most activefraction in all activity test systems, fractions 214–282, contained only pure flavonoid

Natural Product Research 1515

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

glycosides, 30-O-methylhypolaetin 7-O-[6000-O-acetyl-�-D-allopyranosyl-(1! 2)]-600-O-acetyl-�-D-glucopyranoside.

The free radical chain reaction is widely accepted as a common mechanism oflipid peroxidation. Radical scavengers may directly react with and quench peroxideradicals to terminate the peroxidation chain reaction and improve the quality andstability of food products. Assay based upon the use of DPPH. is the most popularspectrophotometric method for determination of the antioxidant capacity of food,beverages and vegetable extracts (Bendini et al., 2006). This chromogen radicalcompound can directly react with antioxidants. Additionally, the DPPH. scavengingmethod has been used to evaluate the antioxidant activity of compounds due itbeing a simple, rapid, sensitive and reproducible procedure (Ozcelik, Lee, & Min,2003).

As shown in Figure 3(a), the reductive capabilities of the CC fractions and crudemethanol extracts of S. libanotica are compared to BHT. For the measurements of

4.5

3.5

2.5

1.5

0.5

4

3

2

1

0

Abs

orba

nce

(500

nm

)

0 5 10 15 20 25 30 35

Incubation time (h)

Negative control

Crude extract

25

40–52

130–142

143–171

Compound 1

a–Tocopherol

Figure 2. Total antioxidant activity of the crude extract, different fractions and �-tocopherolin the linoleic acid emulsion were determined by the thiocyanate method.

Table 2. The fractions obtained from CC using different ratios of MeOH :CHCl3 solventsystems and their applications in the antioxidant activity tests.

Fractions Solvents Isolated compounds Weight (g)

25 Hexane FAs and hydrocarbons 0.2140–52 Hex :CHCl3 (9 : 1) FAs and hydrocarbons 0.51130–142 CHCl3 Sideritiol 0.40143–171 MeOH :CHCl3 (2 : 3) Flavone mixture 3.40214–282 (Compound 1) MeOH :CHCl3 (1 : 1) Flavone (Figure 1) 0.54

1516 I. Demirtas et al.

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

the reductive ability, we investigated the Feþ3–Fe+2 transformation in the presence

of fractions or extract samples using the method of Oyaizu. The reducing capacity of

the compounds may serve as a significant indicator of its potential antioxidant

activity. Figure 3(b) shows the free radical scavenging activity of all fractions and

crude extracts were increased with increasing concentration.Total antioxidant activity determination in linoleic acid emulsion is the most

commonly used method for the evaluation of antioxidant activities of extracts from

the numerous antioxidant methods and modifications (Amarowicz, Naczk, &

Shahidi, 2000; Chang, Yen, Huang, & Duh, 2002; Duh et al., 1999). It was found

that all the fractions and the extract of the doses assayed caused a significant total

antioxidant activity compared with the control, the most effective fractions being

between 214 and 282. The five fractions were obtained from CC using hexane

(fraction 25, which contained FAs), hexane : chloroform (9 : 1, FAs and

hydrocarbons), chloroform (diterpene, sideritiol), methanol : chloroform (2 : 3,

flavone mixtures) and methanol : chloroform (1 : 1, obtained pure flavone). Since

solvent systems and crude extractions have shown a moderate activity in all

performed assays, they were subjected to further analysis in order to identify the

compounds. The phytochemical analysis revealed the presence of FAs in fraction 25,

FAs and hydrocarbons in fractions 40–52, diterpene in fractions 130–142, an

unidentified mixture of flavonoid derivatives in sub-fractions 143–171 as their main

constituents, and the presence of flavonoid derivative in sub-fractions 214–282

(Figure 1). These results indicated that flavones possess as high an antioxidant

activity as �-tocopherol.The seed oil composition of Sideritis species (S. athoa, S. brevidens, S. caesarea,

S. condensata, S. congesta, S. dichotoma, S. erythrantha var. cedretorum,

S. germanicopolitana ssp. germanicopolitana, S. hololeuca, S. lanata, S. libanotica

ssp. violascens, S. lycia, S. niveotomentosa, S. perfoliata, S. phrygia and S. pisidica)

have been reported previously (Ertan, Azcan, Demirci, & Baser, 2001). The dominant

2

1

0.8

0.6

0.4

0.2

00

Abs

orba

nce

(700

nm

)

0 10 20 30 40

Concentration (mg/mL) Concentration (mg/mL)

Crude extract

25

40–52

130–142

143–171

Compound 1

a–TocopherolCrude extract2540–52130–142143–171Compound 1BHT

(a) (b)

1.8

1.6

1.4

1.2

50 100 150 200

120.0

100.0

80.0

60.0

40.0

20.0

0.0

Free

rad

ical

sca

veng

ing

activ

ity (

%)

Figure 3. Reduction power (a) and free radical scavenging activity (b) of the crude extract andfractions of S. libanotica ssp. linearis (BHT).

Natural Product Research 1517

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

FA in all species was linoleic acid. Themain components of the oils from all species arelinoleic (45.4–64.0%), oleic (12.3–26.5%), 6-octadecynoic (4.5–26.8%), palmitic(0.3–9.4%) and linolenic (0.8–2.0%) acids. In the present study, the main FA contentin the aerial part of S. libanotica linearis was palmitic acid (Table 3).

The hexane and hexane : chloroform (9 : 1) fractions of MESLL indicated theFAs as well as unsaponifiable matter. GC–MS analysis of FA (Table 1) revealedpalmitic and 15-octadecenoic acids as the major components. GC–MS ofunsaponifiable compounds (Table 3) revealed oleic alcohol as the main component,in addition to long-chain hydrocarbons.

Table 3. FA, alcohols and hydrocarbons of S. libanotica ssp. linearis identified as the methylesters of FA by GC–MS, FA standards and Wiley and NIST databases.

Identified FA(methyl esters)

Retention(Rt)

Hexane(%)

CHCl3 :Hex(1 : 9) (%)

Total FAcontent (%)

FAsC14: 0, Myristic acid 25.62 1.50 0.62 1.43C16: 0, Palmitic acid 33.39 14.34 5.53 13.48C18: 2, Lineloic acid 39.27 6.38 4.99 7.71C18: 1, Oleic acid 39.71 13.14 0.47 9.23C18: 0, Stearic acid 40.60 1.05 0.98 1.37C20: 0, Arachidic acid 53.22 1.40 0.53 1.30C24: 0, Lignoceric acid 77.63 1.70 1.70 2.30Hydrocarbons and alcoholsDodecane 8.16 2.92 6.75 6.56Dodecane, 2,6,10-trimethyl- 10.01 0.38 0.37 0.50Tridecane 10.84 0.67 0.69 0.92Nonane, 2,2,4,4,6,8,8-heptamethyl- 11.43 0.63 0.39 0.691-Hexadecene 13.39 1.52 1.32 1.92Tetradecane 13.62 3.98 3.51 5.081-Octanol, 2-butyl- 15.74 1.35 0.34 1.14Pentadecane 16.27 0.35 0.34 0.46Pentadecane, 3-methyl- 18.72 0.26 0.21 0.31Hexadecen-1-ol, trans-9- 19.82 3.50 2.38 3.99Hexadecane 20.13 3.43 3.09 4.42Pentadecane, 2,6,10-trimethyl- 21.97 0.28 0.17 0.30Nonadecane 24.39 0.51 0.60 0.75Tetradecane, 4-ethyl- 26.41 0.25 0.18 0.29Heptadecane, 3-methyl- 27.25 0.24 0.22 0.311-Nonadecene 28.31 3.79 2.99 4.60Octadecane 28.54 3.12 2.63 3.902-Pentadecanone, 6,10,14-trimethyl- 30.15 1.01 0.22 0.83Tetracosane 32.14 0.35 0.22 0.38Octadecane, 2-methyl- 33.89 0.50 0.21 0.481-Docosene 35.57 3.68 3.22 4.68Eicosane 35.72 1.91 1.70 2.44Oleic alcohol 38.04 12.13 3.55 10.64Phytol 39.83 0.83 0.88 1.16Eicosane, 2-methyl- 41.31 0.96 0.20 0.78Docosane 44.04 1.36 1.11 1.671-Eicosanol 50.20 2.62 0.86 2.36Heptacosane 59.10 1.24 0.90 1.45

1518 I. Demirtas et al.

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

3. Experimental

3.1. Plant material

Sideritis libanotica ssp. linearis was collected from the Ballica district of Tokat,Turkey (5 km from Pazar town to Ballica cave, 942m, 40� 140 85800 N, 36� 170 15500

E), at the flowering stage, on 23 June 2006. The plant was kindly identified by Assist.Prof. Dr H. Askin Akpulat from the Department of Biology of the Faculty ofScience and Letters at Cumhuriyet University. A voucher specimen has beendeposited at the Herbarium of the Department of Biology, Cumhuriyet University,Sivas, Turkey (CUFH – Voucher No: 8413).

3.2. Phytochemical study

3.2.1. Phytochemical screening and analytical procedure

Following the collection of the aerial parts of the plant, the plant materials wereair-dried at room temperature for the preparation of extracts. The crude extract wasscreened for carbohydrates, glycosides, flavonoids, saponins, terpenoids andalkaloids using vacuum chromatography (VC), column chromatography (CC),flash chromatography (FC), thin layer chromatography (TC) and chemical tests(CT). All chemicals were analytical reagents, higher grade or distilled solvents.

Air-dried plant material of S. libanotica (2 kg) was successively extracted withMeOH (6L). The MeOH extract was evaporated under reduced pressure at atemperature540�C. The concentrated extract was then partitioned successively withCC using n-hexane, hex : CHCl3 (9 : 1), CHCl3, MeOH :CHCl3 (2 : 3), MeOH :CHCl3(1 : 1) and MeOH. The n-hexane and hexane–CHCl3 extracts yielded FAs andhydrocarbons (0.72 g), the CHCl3 extract yielded pure sideritiol (400mg) and theMeOH–CHCl3 extract yielded a flavonoid mixture (3.94 g) on the removal ofsolvent. The MeOH extract yielded pure glucose.

Official methods were used to determine FAs and hydrocarbons. FA Me esterswere prepared by NaOMe-catalysed trans-esterification and analysed by GC–MS ona Perkin Elmer Clarus 500 equipped with BPX-20 capillary column (0.25 mm ID30m� 250 mm), filled with 5% phenyl polysilphenylene–siloxane, at an ionisationvoltage of 70 eV. Helium was the carrier gas (1mLmin�1). The injector and detectortemperatures were kept at 100�C for 5min and then gradually increased to 250�C ata 5�Cmin�1 rate, and held for 15min. Diluted samples (1/100, v/v, in n-pentane) of1.0mL were injected. The hydrocarbons and FAs were identified using the mixed FAs(Supelco 37) and hydrocarbon standards available in our laboratory.

The high-resolution NMR spectra (1H and 13C) were run on a Brucker AvanceIII spectrometer (400MHz), J values are given in Hertz, and a UV-vis spectro-photometer was used (Jasco V-530 UV-Vis).

3.3. Preparation of methanolic extracts

Air-dried plant samples (1000 g) were cut into small pieces and extracted successivelywith methanol (3.5 L) three times at room temperature. The extracts were filteredthrough Whatman No. 2 filter paper and then concentrated to dryness in vacuo at40�C. The yield of the methyl extract of S. libanotica ssp. linearis (MESLL) was 14%.

Natural Product Research 1519

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

3.4. Antioxidant activity

3.4.1. Chemicals

Ammonium thiocyanate was purchased from E. Merck (Darmstadt, Germany).Ferrous chloride and �-tocopherol were purchased from Sigma (Sigma–AldrichGmbH, Sternheim, Germany). All other chemicals used were of analytical grade andwere obtained from Sigma, E. Merck and Fluka companies.

3.4.2. Determination of total antioxidant activity

In the present study, the total antioxidant activity of the crude extract and fractionsof S. libanotica was measured by the thiocyanate method (Duh, Tu, & Yen, 1999).For stock solutions, 10mg of MESLL were dissolved in 10mL methanol. Then, thesolution which contains different concentration of MESLL (from 25 to 75 mgmL�1)solution in 2.5mL of sodium phosphate buffer (0.04M, pH7.0) was added to 2.5mLof linoleic acid emulsion in sodium phosphate buffer (0.04M, pH7.0). Therefore,5 mL of the linoleic acid emulsion was prepared by mixing and homogenising 15.5mLof linoleic acid, 17.5mg of Tween-20 as emulsifier and 5mL phosphate buffer(pH7.0). On the other hand, 5mL of control was composed of 2.5mL of linoleic acidemulsion and 2.5mL, 0.04M sodium phosphate buffer (pH7.0). The mixed solution(5mL) was incubated at 37�C in glass flask. The peroxide levels were determined byreading the absorbance at 500 nm in a spectrophotometer (Jasco V-530 UV-visspectrophotometer), after the reaction with FeCl2 and thiocyanate at intervals duringincubation. During the linoleic acid oxidation, peroxides were formed and thesecompounds oxidise Feþ2 to Feþ3. The latter Feþ3 ions form a complex with SCN�

and this complex has maximum absorbance at 500 nm. The solutions without addedextract or standard were used as negative controls. All data on total antioxidantactivity are the average of triplicate analyses. In this test, butylated hydroxytoluene(BHT) and �-tocopherol were used as standards. The inhibition of lipid peroxidationin percent was calculated by the following equation:

% Inhibition ¼ 100� ½ðA1=AoÞ � 100�,

where Ao was the absorbance of the control reaction and A1 was the absorbance inthe presence of the sample.

3.4.3. Free radical scavenging activity

The free radical scavenging activity of the crude extract and fractions was measuredby 1,1-diphenyl-2-picryl-hydrazil (DPPH.) using the Blois (1958) method. Briefly,0.1mM solution of DPPH. in ethanol was prepared and 1mL of this solution wasadded to 3mL of the solutions of the crude extract and fractions in ethanol atdifferent concentrations (15, 25, 40 mgmL�1). The mixture was shaken vigorouslyand allowed to stand at room temperature for 30min. Subsequently, the absorbancewas measured at 517 nm in a spectrophotometer. Lower absorbance of the reactionmixture indicated higher free radical scavenging activity. The DPPH� concentration(mM) in the reaction medium was calculated from the following calibration curve,determined by linear regression (R2 : 0.997):

Absorbance ¼ 0:0003� ½DPPH�� � 0:0174:

1520 I. Demirtas et al.

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

The capability to scavenge the DPPH. radical was calculated using the followingequation:

DPPH scavenging effect ð%Þ ¼ ½ðAo � A1=AoÞ � 100�,

where Ao was the absorbance of the control reaction and A1 was the absorbance inthe presence of the crude extract and fractions or standard.

3.4.4. Reducing power

The reducing power of the crude extract and fractions was determined according tothe method of Oyaizu (1986). Different concentrations of the fractions (100, 150,200 mgmL�1) in 1mL of ethanol was mixed with a phosphate buffer (2.5mL, 0.2M,pH6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5mL, 1%). The mixture wasincubated at 50�C for 20min. A portion (2.5mL) of trichloroacetic acid (10%) wasadded to the mixture, which was then centrifuged for 10min. The upper layer ofsolution (2.5mL) was mixed with distilled water (2.5mL) and FeCl3 (0.5mL, 0.1%),and the absorbance was measured at 700 nm in a spectrophotometer. Higherabsorbance of the reaction mixture indicated greater reducing power.

4. Conclusions

In this study, the methanol extract from S. libanotica ssp. linearis demonstratedpronounced antioxidant potential due to the presence of many polyphenols (such asflavonoids) and terpenoids. The antioxidant activity of the crude extract of the planthas been reported by Tunalier et al. (2004) and Tepe et al. (2006). Thus, the resultscould be considered as the first report on the antioxidant activities of the compoundsisolated from the plant and identified. The findings of the present study also supportthe use of S. libanotica as a food additive and a traditional anti-ageing remedy. Themost abundant components of S. libanotica were palmitic acid (13.48%), oleic acid(9.23%) and lineloic acid (7.71%), respectively. Based on the above discussion, thecrude extract of the plant and some fractions can be used for minimising orpreventing lipid oxidation in pharmaceutical products, retarding the formation oftoxic oxidation products, maintaining nutritional quality and prolonging theshelf-life of pharmaceuticals and foods.

Acknowledgements

We thank Dr H.A. Akpulat for characterisation of the plant material, and GaziosmanpasaUniversity (BAP 2006/8) and State Planning Organization, Turkey (DPT 2003K120510) fortheir grants.

References

Amarowicz, R., Naczk, M., & Shahidi, F. (2000). Antioxidant activity of crude tannins ofcanola and rapeseed hulls. Journal of the American Oil Chemists’ Society, 77, 957–961.

Aytac, Z., & Aksoy, A. (2000). A new Sideritis species (Labiatae) from Turkey.

Flora Mediterranean, 10, 181–184.

Natural Product Research 1521

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

Baser, K.H.C. (2002). Aromatic biodiversity among the flowering plant taxa of Turkey.

Pure and Applied Chemistry, 74, 527–545.

Baytop, T. (1999). Therapy with medicinal plants in Turkey (past and present). Istanbul: Nobel

Tip Publications.

Bendini, A., Cerretani, L., Pizzolante, L., Toschi, T.G., Guzzo, F., Ceoldo, S., et al. (2006).

Phenol content related to antioxidant and antimicrobial activities of Passiflora spp.

extracts. European Food Research and Technology, 223, 102–109.Blois, M.S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 44,

307–315.Chang, L.W., Yen, W.J., Huang, S.C., & Duh, P.D. (2002). Antioxidant activity of sesame

coat. Food Chemistry, 78, 347–354.de las Heras, B., Vivas, J.M., & Villar, A. (1994). Anti-inflammatory activity of Sideritis

javalambrensis extracts. Journal of Ethnopharmacology, 41, 15–17.Demirtas, I., Sahin, A., Ayhan, B., Tekin, S., & Telci, I. (2009). Antiproliferative effects of the

methanolic extracts of Sideritis libanotica Labill. subsp. linearis. Records of Natural

Products, 3, 104–109.Duh, P.D., Tu, Y., & Yen, G.C. (1999). Antioxidant activity of water extract of harng jyur

(Chrysanthemum morifolium Ramat). Lebnesmittel-Wissenschaft und Technologie, 32,

269–277.

Duman, H. (2000). Sideritis L. In A. Guner, N. Ozhatay, T. Ekim & K.H.C. Bas� er (Eds.),

Flora of Turkey and the East Eagean Islands (Supplement II, Vol. 11, pp. 201–204).

Edinburgh: Edinburgh University Press.Ertan, A., Azcan, A., Demirci, B., & Baser, K.H.C. (2001). Fatty acid composition of Sideritis

species. Chemistry of Natural Compounds, 4(37), 301–303.Ezer, N., Vila, R., Canigueral, S., & Adzet, T. (1996). Essential oil composition of Turkish

species of Sideritis. Phytochemistry, 41, 203–205.Ezher, N., Sakar, M.K., Rodriguez, B., & De la Torre, M.C. (1992). Flavonoid glycosides and

a phenylpropanoid glycoside from Sideritis perfoliata. International Journal of

Pharmacognosy, 30, 61–65.Gabrieli, C.N., Kefalas, P.G., & Kokkalou, E.L. (2005). Antioxidant activity of flavonoids

from Sideritis raeseri. Journal of Ethnopharmacology, 96, 423–428.Hernandez-Perez, M., & Rabanal, R. (2002). Evaluation of the anti-inflammatory and

analgesic activity of Sideritis canariensis var. pannosa in mice. Journal of

Ethnopharmacology, 81, 43–47.Hernandez-Perez, M., Sanches, C.C., Montalbetti-Moreno, Y., & Rabanal, R.M. (2004).

Studies on the analgesic and anti-inflammatory effects of Sideritis candicans Ait. Var.

eriocephala Webb aerial part. Journal of Ethnopharmacology, 93, 279–284.

Oyaizu, M. (1986). Studies on product of browning reaction prepared from glucosamine.

Japan Journal of Nutrition, 44, 307–315.

Ozcelik, B., Lee, J.H., & Min, D.B. (2003). Effects of light, oxygen and pH on the

2,2-diphenyl-1-picrylhydrazyl (DPPH) method to evaluate antioxidants. Journal of Food

Sciences, 68, 487–490.Rios, J.L., Manez, S., Paya, M., & Alcaraz, M.J. (1992). Antioxidant activity of flavonoids

from Sideritis javalambrensis. Phytochemistry, 31, 1947–1950.Sahin, F.P., Ezer, N., & Cal|s� , I. (2004). Three new acylated flavone glycosides from Sideritis

ozturkii Aytac and Aksoy. Phytochemistry, 65, 2095–2099.Sahin, F.P., Ezer, N., & Calis, I. (2006). Terpenic and phenolic compounds from Sideritis

stricta. Turkish Journal of Chemistry, 30, 495–504.Sahin, F.P., Tasdemir, D., Ruedi, P., Ezer, N., & Cal|s� , I. (2005). Erratum to three new

acylated flavone glycosides from Sideritis ozturkii Aytac and Aksoy. Phytochemistry, 66,

125–125.

1522 I. Demirtas et al.

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11

Tepe, B., Sokmen, M., Akpulat, H.A., Yumrutas, O., & Sokmen, A. (2006). Screening ofantioxidative properties of the methanolic extracts of Pelargonium endlicherianumFenzl., Verbascum wiedemannianum Fisch. and Mey., Sideritis libanotica Labill. subsp.linearis (Bentham) Borm., Centaurea mucronifera DC. and Hieracium cappadocicum

Freyn from Turkish flora. Food Chemistry, 98, 9–13.Topcu, G., Goren, A.C., K|l|c, T., Y|ld|z, Y.K., & Tumen, G. (2002). Diterpenes from

Sideritis trojana. Natural Product Letters, 16, 33–37.

Tunalier, Z., Kosar, M., Ozturk, N., Baser, K.H.C., Duman, H., & Kirimer, N. (2004).Antioxidant properties and phenolic composition of Sideritis species. Chemistry ofNatural Compounds, 40, 206–210.

Yesilada, E., Honda, G., Sezik, E., Tabata, M., Fujita, T., Tanaka, T., et al. (1995).Traditional medicine in Turkey V. Folk medicine in inner Taurus mountains. Journal ofEthnopharmacology, 46, 133–152.

Natural Product Research 1523

Dow

nloa

ded

by [

Gaz

iosm

anpa

sa U

nive

rsite

si]

at 0

4:16

28

Sept

embe

r 20

11