Sideritis galatica Bornm.: A source of multifunctional agents for the management of oxidative damage, Alzheimer's's and diabetes mellitus

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Sideritis galatica Bornm.: A source ofmultifunctional agents for the management ofoxidative damage, Alzheimer’s’s and diabetesmellitus

Gokhan Zengin a,*, Cengiz Sarikurkcu b, Abdurrahman Aktumsek a,Ramazan Ceylan a

a Science Faculty, Department of Biology, Selcuk University, Konya, Turkeyb Faculty of Pharmacy, Department of Analytical Chemistry, Suleyman Demirel University, Isparta, Turkey

A R T I C L E I N F O

Article history:

Received 13 May 2014

Received in revised form 19 August

2014

Accepted 25 August 2014

Available online 10 September 2014

A B S T R A C T

The antioxidant and enzyme inhibitory potential of different solvent extracts (petroleum

ether, ethyl acetate, methanol and water) from Sideritis galatica were evaluated. Cholines-

terase, α-amylase and α-glucosidase inhibitory activities of the extracts were tested by

microtiter plate assays. Antioxidant abilities were tested using free radical scavenging (DPPH

(1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid)

and NO), reducing power (FRAP (ferric reducing antioxidant power) and CUPRAC (cupric re-

ducing antioxidant capacity)), total antioxidant capacity and chelating assays. Methanol and

water extracts showed higher phenolic content, DPPH and ABTS scavenging activity and

reducing power activities, while the petroleum ether and ethyl acetate extract had the highest

inhibition abilities on the enzymes. 18 phenolic components in these extracts were de-

tected by using high performance liquid chromatography-diode array detector (HPLC-

DAD). Results obtained in this work indicate that S. galatica may be useful as a source of

natural agents for the management of oxidative process, Alzheimer’s disease and type II

diabetes.

© 2014 Elsevier Ltd. All rights reserved.

Keywords:

Sideritis galatica

Antioxidant

Alzheimer’s

Diabetes

HPLC

1. Introduction

In recent years there has been much interest in phytochemicalswith biological properties, such as antioxidant, antimicrobial,antimutagenic, anti-inflammatory, arthritis and their use for thetreatment of human diseases such as Alzheimer’s disease, car-diovascular disease and diabetes mellitus (Newman & Cragg,2012). Among the phytochemicals, polyphenols have receivedparticular attention due to their antioxidant, antimicrobial,

antiviral, antiallergenic and antithromobotic activities (Ajila, Rao,& Rao, 2010; Del Rio, Costa, Lean, & Crozier, 2010). Theantioxidative properties have great importance in terms of oxi-dative stress which is caused by overproduction of free radicalsthat can damage biological molecules, including lipids, pro-teins and DNA, the damage of which is responsible for chronicand degenerative diseases. Antioxidants are considered impor-tant nutraceuticals, exerting a protective effect in these diseases(Feugang, Konarski, Zou, Stintzing, & Zou, 2006). Because of theimportance of antioxidants, some synthetic substances (BHA,

* Corresponding author. Tel.: +90 332 223 27 81; fax: +90 332 2410106.E-mail address: [email protected] (G. Zengin).

http://dx.doi.org/10.1016/j.jff.2014.08.0111756-4646/© 2014 Elsevier Ltd. All rights reserved.

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butylated hydroxyanisole; BHT, butylated hydroxyltoluene; PG,propyl gallate; and TBHQ, tert-butylhydroquinone) are used asantioxidants in the food industry but they have been found tobe toxic and carcinogenic in animal models at high concentra-tions (Anagnostopoulou, Kefalas, Papageorgiiou, Assimepoulou,& Boskou, 2006; Ito et al., 1986). Consequently it is importantto find new antioxidants especially from natural sources for pre-vention of diseases related to free radicals. In recent years, manyspices, herbs, nuts, cereals, legumes, animal products, and mi-crobial products can serve as sources of natural antioxidants,which are commonly known as phenolics, carotenoids, vita-mins C and E, in the market (Cuevas-Juárez et al., 2014; Pokorný,Yanishlieva, & Gordon, 2001; Romojaro, Botella, Obón, & Pretel,2013).

Alzheimer’s disease (AD) and diabetes mellitus (DM) aremajor global health problems. The most accepted hypothesisin the treatment of these diseases is the inhibition of keyenzyme activity. In the treatment of AD, cholinesterase inhibi-tors are usually used, resulting in the maintenance ofacetylcholine levels and enhancing cholinergic function (Howes& Houghton, 2003). Alpha-amylase and α-glucosidase are knownas key enzymes in starch breakdown and increase of bloodglucose level. Therefore, inhibition of these enzyme activitiesis an important strategy in the management of diabetes mel-litus (Kwon, Apostolidis, & Shetty, 2008). In this direction, somedrugs are developed to act as enzyme inhibitors for the treat-ment of both AD and DM. For example, tacrine, galantamineand rivastigmine are used to treat AD. Likewise, acarbose andmiglitol are used to manage blood glucose level in DM. However,these drugs are reported to have side effects including hepa-totoxicity, gastrointestinal disturbances, and diarrhea(Etxeberria, de la Garza, Campión, Martínez, & Milagro, 2012;van de Laar, 2008). It is for this reason that there is a need fornew natural inhibitors, which have no adverse or undesir-able effects, in the treatment of both AD and DM.

The genus Sideritis, perennial and annual herbs of theLamiaceae, consists of more than 150 species widely distrib-uted in the Mediterranean area (González-Burgos, Carretero,& Gómez-Serranillos, 2011). The species are commonly knownas “mountain tea”. In Turkey, the genus includes 46 species,31 of which are endemic (Davis, Cullen, & Coode, 1988). Sideritisspecies are known as “dag çayı”, “ada çayı” or “balbası” in Ana-tolia. Plants of this genus are widely used in the treatment ofseveral ailments including gastrointestinal disturbances, coldand cough in different countries and Anatolian traditional medi-cine (Baytop, 1999). Likewise, the important biological activitiesof Sideritis species, which are anti-inflammatory, antifeedant,antimicrobial and antioxidant, are reported in many ethno-botanical and phytochemical papers (Bondi, Bruno, Piozzi, Baser,& Simmonds, 2000; González-Burgos et al., 2011). Sideritis galaticais an endemic species and spreads in Central Anatolia Regionof Turkey (Ankara-Cubuk). No reports are available on the phy-tochemical composition, antioxidant and enzyme inhibitoryactivities of S. galatica in the literature. The objectives of thepresent investigation were to confirm the antioxidant prop-erties of various solvent extracts using different in vitro models(1), to evaluate enzyme inhibitory activities linked to Alzheim-er’s and diabetes mellitus (2), to examine the total antioxidantcomponents (3), and to identify and quantify major phenolicconstituents by HPLC (4).

2. Materials and methods

2.1. Plant material

The aerial parts of S. galatica, which is a densely haired pe-rennial shrub growing to a height of 65 cm, were randomlycollected in July 2013, at the flowering stage, from a wild popu-lation in Ankara: around Çubuk village (Central Anatolia Regionof Turkey, latitude 40°18′30.00″(N), longitude 32°58′39.00″(E), al-titude 1280 m, dry slopes).Taxonomic identification of the plantmaterial was confirmed by the senior taxonomist Dr. OlcayCeylan, at the Department of Biology, Mugla University. Thevoucher specimen has been deposited at the Herbarium of theDepartment of Biology, Mugla University, Mugla-Turkey (VoucherNo: MUH 1466).

2.2. Preparation of the solvent extracts

To produce solvent extracts, the air-dried samples (5 g) of theaerial part of S. galatica were macerated with different sol-vents (100 mL) according to their increasing polarity: petroleumether, ethyl acetate and methanol at room temperature for 24 h.For water extract, the air-dried samples (5 g) were extractedby boiling deionized water (100 mL) for 15 min. Petroleum ether,ethyl acetate and methanol were then removed with a rotaryevaporator.The water extract was freeze-dried. All extracts werestored at +4 °C until analyzed. The yields of different solventextracts from S. galatica are shown in Table 1.

2.3. Quantification of phenolic compounds by RP-HPLC

Phenolic compounds were evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC, ShimadzuScientific Instruments, Kyoto, Japan). Detection and quantifi-cation were carried out with a LC-10ADvp pump, a diode arraydetector, a CTO-10Avp column heater, SCL-10Avp system con-troller, DGU-14A degasser and SIL-10ADvp auto sampler(Shimadzu Scientific Instruments, Columbia, MD, USA). Sepa-rations were conducted at 30 °C on Eclipse XDB C-18 reversed-phase column (250 mm × 4.6 mm length, 5 µm particle size,Agilent, Santa Clara, CA, USA). The eluates were detected at278 nm. The mobile phases were A: 3.0% acetic acid in dis-tilled water and B: methanol. For analysis, the samples weredissolved in methanol, and 20 µL of this solution were in-jected into the column. The elution gradient applied at a flowrate of 0.8 mL/min was: 93% A/7% B for 0.1 min, 72% A/28% Bfor 20 min, 75% A/25% B for 8 min, 70% A/30% B for 7 min andsame gradient for 15 min, 67% A/33% B for 10 min, 58% A/42%B for 2 min, 50% A/50% B for 8 min, 30% A/70% B for 3 min, 20%A/80% B for 2 min, 100%B for 5 min until the end of the run.Phenolic compositions of the extracts were determined by amodified method of Caponio, Alloggio, and Gomes (1999). Gallicacid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid,chlorogenic acid, caffeic acid, (−)-epicatechin, syringic acid, van-illin, p-coumaric acid, ferulic acid, sinapinic acid, benzoic acid,o-coumaric acid, rutin, naringin, hesperidin, rosmarinic acid,eriodictyol, trans-cinnamic acid, quercetin, naringenin, luteolin,kaempferol and apigenin were used as standards. Identifica-tion and quantitative analysis were done by comparison with

539j o u rna l o f f un c t i ona l f o od s 1 1 ( 2 0 1 4 ) 5 3 8 – 5 4 7

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standards. The amount of each phenolic compound was ex-pressed as milligram per gram of extract using externalcalibration curves, which were obtained for each phenolicstandard.

2.4. Determination of total bioactive components

2.4.1. Total phenolic contentThe total phenolic content was determined by employing themethods given in the literature (Slinkard & Singleton, 1977) withslight modification. Sample solution (0.25 mL) was mixed withdiluted Folin–Ciocalteu reagent (1 mL, 1:9, v/v) and shaken vig-orously. After 3 min, Na2CO3 solution (0.75 mL, 1%) was addedand the sample absorbance was read at 760 nm after a 2 h in-cubation at room temperature. The total phenolic content wasexpressed as milligrams of gallic acid equivalents (mg GAE/gextract).

2.4.2. Total flavonoid contentThe total flavonoid content was determined using the Dowd(1959) method as adapted by Arvouet-Grand, Vennat, Pourrat,and Legret (1994). Briefly, sample solution (1 mL) was mixedwith the same volume of aluminum trichloride (2%) in metha-nol. Similarly, a blank was prepared by adding sample solution(1 mL) to methanol (1 mL) without AlCl3. The sample and blankabsorbances were read at 415 nm after a 10 min incubation atroom temperature. The absorbance of the blank was sub-tracted from that of the sample. Rutin was used as a referencestandard and the total flavonoid content was expressed as mil-ligrams of rutin equivalents (mg RE/g extract).

2.4.3. Total saponins contentThe total saponin content was determined by the vanillin–sulfuric acid method (Aktumsek, Zengin, Guler, Cakmak, &Duran, 2013). Sample solution (0.25 mL) was mixed with van-illin (0.25 mL, 8%) and sulfuric acid (2 mL, 72%). The mixturewas incubated for 10 min at 60 °C.Then the mixture was cooledfor another 15 min, followed by the sample absorbance mea-surement at 538 nm. The total saponin content was expressedas milligrams of quillaja equivalents (mg QAE/g extract).

2.4.4. Total condensed tannin contentThe total condensed tannin content was determined by thevanillin method (Bekir, Mars, Souchard, & Bouajila, 2013) with

slight modification. Sample solution (0.5 mL) was mixed withvanillin reagent (1.5 mL, 1% in 7 M H2SO4) in an ice bath andthen mixed well. Similarly, a blank was prepared by adding thesample solution (0.5 mL) to 7 M H2SO4 (1.5 mL). The sample andblank absorbances were read at 500 nm after a 15 min incu-bation at room temperature. The absorbance of the blank wassubtracted from that of the sample.The total condensed tannincontent was expressed as milligrams of (+)-catechin equiva-lents (mg CE/g extract).

2.4.5. Total flavanol contentThe total flavanol content was determined by employing themethods given in the literature (Quettier-Deleu et al., 2000) withslight modification. Sample solution (0.25 mL) was added to5 mL of 0.1% (w/v) DMACA (p-dimethylaminocinnamaldehyde)in methanolic/HCl (37%) (3:1 v/v) reagent. The sample absor-bance was read at 640 nm after 10 min incubation at roomtemperature. Catechin was used as a standard and the totalflavanol content was expressed as milligrams of (+)-catechinequivalents (mg CE/g extract).

2.5. Total antioxidant activity

2.5.1. Phosphomolybdenum methodThe total antioxidant activity of the samples was evaluated byphosphomolybdenum method according to Berk, Tepe, Arslan,and Sarikurkcu (2011) with slight modification. Sample solu-tion (0.3 mL) was combined with 3 mL of reagent solution (0.6 Msulfuric acid, 28 mM sodium phosphate and 4 mM ammo-nium molybdate). The sample absorbance was read at 695 nmafter a 90 min incubation at 95 °C. The total antioxidant ca-pacity was expressed as millimoles of trolox equivalents (mmolTE/g extract).

2.5.2. β-Carotene bleaching methodIn this assay antioxidant activity is determined by measur-ing the inhibition of the volatile organic compounds and theconjugated diene hydroperoxides arising from linoleic acid oxi-dation (Sarikurkcu et al., 2012) with slight modification. A stocksolution of β-carotene-linoleic acid mixture was prepared asfollows: 0.5 mg β-carotene was dissolved in chloroform (1 mL,HPLC grade). 25 µL linoleic acid and 200 mg Tween 40 wereadded. Chloroform was completely evaporated using a vacuum

Table 1 – Extraction yield, and total phenolic, flavonoid, condensed tannin, saponin and flavanol contents of the solventextracts from Sideritis galatica (mean ± SD).

Assays Solvent extracts

Petroleum ether Ethyl acetate Methanol Water

Extraction yield (%) 1.19 2.88 17.08 21.04Total phenolic (mg GAE/g extract)b 19.66 ± 1.81aa 22.48 ± 1.85a 63.30 ± 2.70b 70.68 ± 1.34cTotal flavonoid (mg RE/g extract)c 11.93 ± 1.05a 21.27 ± 0.75b 44.33 ± 1.41c 49.56 ± 0.91dTotal condensed tannin (mg CE/g extract)d nd nd 0.36 ± 0.06a 3.60 ± 0.68bTotal saponin (mg QAE/g extract)e 538.19 ± 11.26d 303.11 ± 22.56c 228.75 ± 17.87b 125.27 ± 1.10aTotal flavanol (mg CE/g extract)d 0.96 ± 0.04b 0.90 ± 0.14b 0.78 ± 0.10ab 0.67 ± 0.11a

a In same row marked different letters indicate significant difference (p < 0.05); nd, not determined.b GAE, gallic acid equivalent.c RE, rutin equivalent.d CE, catechin equivalent.e QAE, quillaja equivalent.

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evaporator. Then 100 mL of oxygenated distilled water wasadded with vigorous shaking; 1.5 mL of this reaction mixturewas dispersed to test tubes and the sample solution (0.50 mL,1 mg/mL) was added and the emulsion system was incu-bated for up to 2 h at 50 °C. The same procedure was repeatedwith the standards and a blank. After this incubation period,the sample absorbance was read at 490 nm. Measurement ofabsorbance was continued until the color of β-carotene dis-appeared. The bleaching rate (R) of β-carotene was calculatedaccording to Eq. (1).

Ra bt

= ( )(⎡⎣⎢

⎤⎦⎥

ln, (1)

where ln = natural log, a = absorbance at time 0, and b = ab-sorbance at time t (30, 60, 90, 120 min). The antioxidant activity(AA) was calculated in terms of percent inhibition relative tothe control using Eq. (2).

AA =−( )⎡

⎣⎢⎤⎦⎥

×R R

RControl Sample

Control

100 (2)

2.6. Radical scavenging activity

2.6.1. DPPH radical scavenging activityThe effect of the samples on 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical was estimated according to Sarikurkcu (2011).Sample solution (1 mL) was added to 4 mL of a 0.004% metha-nol solution of DPPH. The sample absorbance was read at517 nm after a 30 min incubation at room temperature in thedark. DPPH radical scavenging activity was expressed as mil-limoles of trolox equivalents (mmol TE/g extract).

2.6.2. ABTS radical cation scavenging activityThe scavenging activity against ABTS radical cation (2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid) was measuredaccording to the method of Re et al. (1999) with slight modi-fication. Briefly, ABTS+ was produced directly by reacting 7 mMABTS solution with 2.45 mM potassium persulfate and allow-ing the mixture to stand for 12–16 in the dark at roomtemperature. Prior to beginning the assay, ABTS solution wasdiluted with methanol to an absorbance of 0.700 ± 0.02 at734 nm. Sample solution (1 mL) was added to ABTS solution(2 mL) and mixed. The sample absorbance was read at 734 nmafter a 30 min incubation at room temperature.The ABTS radicalcation scavenging activity was expressed as millimoles of troloxequivalents (mmol TE/g extract).

2.6.3. Nitric oxide radical scavenging activitySodium nitroprusside in aqueous solution at physiological pHspontaneously generated nitric oxide (NO), which can be mea-sured by the Griess reaction (Srivastava & Shivanandappa, 2011).Sample solution (0.5 mL) was mixed with sodium nitroprus-side (0.5 mL, 5 mM) in phosphate buffer (0.2 M, pH 7.4) andincubated for 150 min at room temperature. Similarly, a blankwas prepared by adding sample solution (0.5 mL) to phos-phate buffer (0.5 mL). Griess reagent diluted with acetic acid(1 mL, 1:1 v/v) was added to the incubated sample and allowedto stand for 30 min. The sample and blank absorbances were

read at 548 nm. The absorbance of the blank was subtractedfrom that of the sample and the nitric oxide radical scaveng-ing activity was expressed as millimoles of trolox equivalents(mmol TE/g extract).

2.7. Reducing power

2.7.1. CUPRAC assayThe cupric ion reducing activity (CUPRAC) was determined ac-cording to the method of Zengin et al. (2014). Sample solution(0.5 mL) was added to premixed reaction mixture containingCuCl2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH4Acbuffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared byadding sample solution (0.5 mL) to premixed reaction mixture(3 mL) without CuCl2. Then, the sample and blank absor-bances were read at 450 nm after a 30 min incubation at roomtemperature. The absorbance of the blank was subtracted fromthat of the sample. CUPRAC activity was expressed as milli-grams of trolox equivalents (mg TE/g extract).

2.7.2. FRAP assayThe FRAP (ferric reducing antioxidant power) assay was carriedout as described by Aktumsek et al. (2013) with a slight modi-fication. Sample solution (0.1 mL) was added to premixed FRAPreagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) (10 mM) in 40 mM HCl and ferricchloride (20 mM) in a ratio of 10:1:1 (v/v/v). Then, the sampleabsorbance was read at 593 nm after a 30 min incubation atroom temperature. FRAP activity was expressed as milli-grams of trolox equivalents (mg TE/g extract).

2.8. Metal chelating activity on ferrous ions

The metal chelating activity on ferrous ions was determinedby the method described by Aktumsek et al. (2013). Briefly,sample solution (2 mL) was added to FeCl2 solution (0.05 mL,2 mM). The reaction was initiated by the addition of 5 mMferrozine (0.2 mL). Similarly, a blank was prepared by addingsample solution (2 mL) to FeCl2 solution (0.05 mL, 2 mM) andwater (0.2 mL) without ferrozine. Then, the sample and blankabsorbances were read at 562 nm after 10 min incubation atroom temperature. The absorbance of the blank was sub-tracted from that of the sample. The metal chelating activitywas expressed as milligrams of EDTA (disodium edetate) equiva-lents (mg EDTAE/g extract).

2.9. Enzyme inhibitory activity

2.9.1. Cholinesterase inhibitionCholinesterase (ChE) inhibitory activity was measured usingEllman’s method, as previously reported (Zengin et al., 2014).Sample solution (50 µL) was mixed with DTNB (5,5-dithio-bis(2-nitrobenzoic) acid) (125 µL) and AChE (acetylcholinesterase(Electric ell acetylcholinesterase, Type-VI-S, EC 3.1.1.7, Sigma)),or BChE (butyrylcholinesterase (horse serum butyrylcho-linesterase, EC 3.1.1.8, Sigma)) solution (25 µL) in Tris–HCl buffer(pH 8.0) in a 96-well microplate and incubated for 15 min at25 °C. The reaction was then initiated with the addition ofacetylthiocholine iodide (ATCI) or butyrylthiocholine chloride

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(BTCl) (25 µL). Similarly, a blank was prepared by adding samplesolution to all reaction reagents without enzyme (AChE or BChE)solution. The sample and blank absorbances were read at405 nm after a 10 min incubation at 25 °C. The absorbance ofthe blank was subtracted from that of the sample and the cho-linesterase inhibitory activity was expressed as milligrams ofgalanthamine equivalents (mg GALAE/g extract).

2.9.2. α-Amylase inhibitionα-Amylase inhibitory activity was performed using the Caraway–Somogyi iodine/potassium iodide (IKI) method (Zengin et al.,2014). Sample solution (25 µL) was mixed with α-amylase so-lution (ex-porcine pancreas, EC 3.2.1.1, Sigma) (50 µL) inphosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 °C. After pre-incubation, the reaction was initiated with the addition of starchsolution (50 µL, 0.05%). Similarly, a blank was prepared by addingsample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated for10 min at 37 °C. The reaction was then stopped with the ad-dition of HCl (25 µL, 1 M). This was followed by the addition ofthe iodine–potassium iodide solution (100 µL). The sample andblank absorbances were read at 630 nm. The absorbance of theblank was subtracted from that of the sample and theα-amylase inhibitory activity was expressed as millimoles ofacarbose equivalents (mmol ACE/g extract).

2.9.3. α-Glucosidase inhibitionα-Glucosidase inhibitory activity was performed by the pre-vious method (Zengin et al., 2014). Sample solution (50 µL) wasmixed with glutathione (50 µL), α-glucosidase solution (fromSaccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50 µL) in phos-phate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside) (50 µL) in a 96-well microplate and incubatedfor 15 min at 37 °C. Similarly, a blank was prepared by addingsample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with theaddition of sodium carbonate (50 µL, 0.2 M). The sample andblank absorbances were read at 400 nm. The absorbance of theblank was subtracted from that of the sample and theα-glucosidase inhibitory activity was expressed as milli-moles of acarbose equivalents (mmol ACE/g extract).

2.10. Statistical analysis

For all the experiments all the assays were carried out in trip-licate. The results are expressed as mean values and standarddeviation (SD). The differences between the different extractswere analyzed using one-way analysis of variance (ANOVA) fol-lowed by Tukey’s honestly significant difference post hoc testwith α = 0.05. This treatment was carried out using the SPSSv. 14.0 program.

3. Results and discussion

3.1. Total bioactive compounds

It is generally known that the yield of chemical extractiondepends on the type of solvents with varying polarities. The

solubility of phenolics is governed by the polarity of the sol-vents used. Solvents, such as methanol, ethanol, acetone, ethylacetate, and their combinations have been used for the ex-traction of phenolics from plant materials. In particular,methanol has been generally found to be more efficient in ex-traction of lower molecular weight polyphenols while the highermolecular weight flavanols are better extracted with aqueousacetone. Therefore, there is no universal extraction proce-dure suitable for extraction of all plant phenolics (Naczk &Shahidi, 2004).

The contents of total phenolics, flavonoids, flavanols, sa-ponins and tannins in different solvent extracts of S. galatica,as well as the yield of each extracts were given in Table 1. Phe-nolic compounds contained one or more hydroxyl groupsattached to an aromatic phenyl ring and are associated withantioxidant activity. Hence, it is important to quantify pheno-lic content and to assess its contribution to antioxidant activity.Total phenolic content varied widely in the extracts tested,ranging from 19.66 to 70.68 mg GAE/g extract (Table 1). Ac-cording to the results, the total phenolic content depends onthe type of solvent used. Water extracts had significantly higherconcentrations of phenolic compounds than the other ex-tracts that were tested. Petroleum ether extract, a much moreapolar solvent, had low levels of phenolics. This variation wasdue to the polarity of phenolics. Therefore, this case can be ex-plained by the presence of low concentrations of apolarphenolics in S. galatica. Erkan, Cetin, and Ayranci (2011) re-ported total phenolic content for three solvent extracts(methanol, ethyl acetate and acetone) from Sideritis congestaranging from 1375.6 to 1970.8 mM GAE/g. Pljevljakušic et al.(2011) found (from 15.3 to 46.5 mg GAE/g) comparing water ex-tracts of Sideritis raeseri and S. galatica. However, higher phenoliccontent was reported for aqueous and alcoholic extracts ofS. raeseri (Stagos et al., 2012). Many authors reported that dif-ferences were observed in the phenolic content of plant speciesbelonging to the same genus depending on several factors in-cluding temperature, soil content and altitude (Giorgi, Madeo,Speranza, & Cocucci, 2010). Flavonoids are a major class of phe-nolic compounds and exhibited strong antioxidant activities(Robards, Prenzler, Tucker, Swatsitang, & Glover, 1999). Usingthe AlCl3 colorimetric method, the total flavonoid content ofthe extracts varied from 11.93 to 49.56 mg RE/g extract. Similarto the total phenolic content, the water extract showed thehighest flavonoid content as compared to the other extracts.Our data also suggest that flavonoids were a large portion ofthe total phenolics in the extracts. Flavanol or flavan-3-ols areoften called catechins and they are known to be strong anti-oxidants, which are associated with several potential healthbenefits (Tsao, 2010). Total flavanol content of the extracts wasexamined using a colorimetric DMACA method. In contrast tototal phenolic and flavonoid content, the petroleum etherextract showed a higher amount of flavanol content (Table 1).Thus, petroleum ether is an appropriate solvent for flavanolextraction from S. galatica. Total condensed tannin content wasestimated by vanillin–HCl colorimetric assay. Among the studiedextracts, only the methanol and water extract had con-densed tannin content; the contents were found to be 0.36 and3.60 mg CE/g extract, respectively. The saponin content evalu-ated with the vanillin–H2SO4 assay showed differences amongS. galatica extracts, ranging from 125.27 to 538.19 mg QAE/g

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extract. Saponin content was highest in petroleum ether extractand lowest in water extract. Variability in extract saponins isreported in various solvents in several plant species (Aktumseket al., 2013; Zengin et al., 2014).This study reveals that S. galaticaextracts are a good source of antioxidant components.

3.2. Identification of phenolic compounds in the extracts

It is clear that the total phenolic content determined by theFolin–Ciocalteu method does not give a full picture of quali-fication or quantification of the phenolic compounds in theextracts (Sagdic et al., 2011; Wojdylo, Oszmianski, & Czemerys,2007). Therefore, the phenolic compounds in the extracts fromS. galatica were analyzed by HPLC-DAD and the results are pre-sented in Table 2. Eighteen phenolic compounds: gallic acid,protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlo-rogenic acid, caffeic acid, (−)-epicatechin, syringic acid, vanillin,p-coumaric acid, ferulic acid, benzoic acid, naringin, hesperi-din, trans-cinnamic acid, naringenin, kaempferol and apigeninwere identified by comparison with the retention times andUV spectra of authentic standards analyzed under identical con-ditions, while the quantitative data were calculated from theirrespective calibration curves. Analytical characteristics for de-termination of phenolic compounds are given in Table 2.

The most abundant phenolics found in the methanol extractwere benzoic acid, chlorogenic acid and kaempferol. However,(−)-epicatechin was the main component followed by chloro-genic and benzoic acid in the water extract. Knowing thatchlorogenic acid was the most effective antioxidant and radical-scavenger (Silva, Malva, & Dias, 2008; Xiang & Ning, 2008),chlorogenic acid may be responsible for the strong antioxi-dant properties of the methanol extract. This is in accordancewith previous findings that the chlorogenic acid was presentin several Sideritis species (Erkan et al., 2011; Pljevljakušic et al.,2011). According to other research, chlorogenic acid has anti-viral, antiproliferative and antimutagenic roles as well as anantioxidative role (Gordon & Wishart, 2010; Tang & Liu, 2008;Wang et al., 2009). Thus, S. galatica is considered as a naturalresource of chlorogenic acid. In the ethylacetate extract,kaempferol and apigenin were primary phenolics. Not all ofthe active compounds were detected in petroleum ether extract.To the best of our knowledge, this study is the first report onthe phenolic components of S. galatica.

3.3. Radical scavenging activities

To determine free radical scavenging activity of S. galatica ex-tracts, we used three types of radicals, DPPH, ABTS and NO.The radical scavenging assays are performed to estimate thefree radical scavenging activity of a pure compound or plantextract and are based on the reduction of these radicals. An-tioxidants interacting with these radicals can transfer eitheran electron or a hydrogen atom to these radicals, thus neu-tralizing their free radical character. For example DPPH assaysthe color of the reaction changes from purple to yellow, whichcan be quantified by a change in the absorbance at 517 nm.In DPPH and ABTS assays, methanol (0.390 and 0.675 mmol TE/g) and water extracts (0.352 and 0.756 mmol TE/g) displayedsuperior scavenging activity (Table 3). This can be attributedto the higher total phenolic content of these extracts. Similar

Tabl

e2

–Ph

enol

icco

mp

onen

tsin

the

solv

ent

extr

acts

(mg/

gex

trac

t)fr

omSi

deri

tis

gala

tica

(mea

n±

SD

)an

dan

alyt

ical

char

acte

rist

ics

for

det

erm

inat

ion

ofp

hen

olic

s.

No.

Phen

olic

com

pon

ents

Petr

oleu

met

her

Eth

ylac

etat

eM

eth

anol

Wat

erLi

nea

rra

nge

(mg/

L)Li

nea

req

uat

ion

R2

LOD

(mg/

L)LO

Q(m

g/L)

1G

alli

cac

idn

d0.

01±

0.00

1aa

0.07

±0.

01b

nd

0.20

-25.

0y

=64

487x

−15

309

0.99

930.

075

0.22

72

Prot

ocat

ech

uic

acid

nd

0.02

±0.

001a

nd

0.22

±0.

01b

0.20

-25.

0y

=48

107x

−11

153

0.99

910.

086

0.26

03

(+)-

Cat

ech

inn

dn

dn

d0.

50±

0.03

0.90

-113

y=

2034

6x−

2927

50.

9988

0.17

20.

522

4p-

Hyd

roxy

ben

zoic

acid

nd

0.05

±0.

001a

nd

0.65

±0.

02b

0.20

-25.

0y

=62

896x

−11

801

0.99

940.

007

0.02

05

Ch

loro

gen

icac

idn

d0.

66±

0.02

a12

.93

±0.

34c

1.80

±0.

04b

0.35

-45.

0y

=37

172x

−20

503

0.99

880.

080

0.24

16

Caf

feic

acid

nd

nd

0.05

±0.

001a

0.84

±0.

01b

0.16

-21.

0y

=10

1382

x−

1871

20.

9993

0.05

40.

162

7(–

)-Ep

icat

ech

inn

dn

dn

d2.

03±

0.06

0.50

-66.

0y

=30

982x

−22

609

0.99

900.

170

0.51

48

Syri

ngi

cac

idn

dn

dn

d0.

82±

0.02

0.05

-12.

0y

=93

371x

+72

98.5

0.99

950.

030

0.09

09

Van

illi

nn

d0.

03±

0.00

1a0.

13±

0.01

bn

d0.

08-1

0.0

y=

1530

84x

−71

78.3

0.99

950.

020

0.06

010

p-C

oum

aric

acid

nd

0.03

±0.

002a

0.05

±0.

001b

0.87

±0.

05c

0.04

-6.0

y=

1758

72x

−54

64.3

0.99

960.

066

0.19

911

Feru

lic

acid

nd

0.02

±0.

001a

0.30

±0.

01b

0.47

±0.

02c

0.12

-17.

0y

=94

621x

−15

153

0.99

930.

004

0.01

112

Ben

zoic

acid

nd

nd

19.6

6±

0.50

b1.

64±

0.05

a0.

85-5

5.0

y=

9578

.2x

−28

19.6

0.99

980.

111

0.33

513

Nar

ingi

nn

d0.

05±

0.00

1a1.

06±

0.02

c0.

67±

0.03

b0.

24-3

2.0

y=

3743

9x−

1681

00.

9988

0.02

30.

069

14H

esp

erid

inn

dn

d1.

01±

0.02

nd

0.40

-56.

0y

=36

417x

−23

313

0.99

891.

113

3.37

315

tran

s-C

inn

amic

acid

nd

0.01

±0.

001a

0.02

±0.

001b

0.34

±0.

01c

0.02

-7.0

y=

2453

05x

+74

75.1

0.99

980.

148

0.44

716

Nar

inge

nin

nd

nd

0.02

±0.

001a

0.56

±0.

08b

0.12

-17.

0y

=85

149x

−14

606

0.99

930.

017

0.05

317

Kae

mp

fero

ln

d0.

91±

0.02

b2.

90±

0.15

c0.

50±

0.02

a0.

05-1

5.0

y=

7012

0x+

6037

.10.

9996

0.02

10.

062

18A

pig

enin

nd

0.70

±0.

02a

1.75

±0.

07b

0.64

±0.

01a

0.17

-11.

0y

=95

601x

−65

71.1

0.99

970.

034

0.10

4

aIn

sam

ero

wm

arke

dd

iffe

ren

tle

tter

sin

dic

ate

sign

ifica

nt

dif

fere

nce

(p<

0.05

).n

d,n

otd

eter

min

ed;

LOD

,lim

itof

det

ecti

on;

LOQ

,lim

itof

qu

anti

fica

tion

.

543j o u rna l o f f un c t i ona l f o od s 1 1 ( 2 0 1 4 ) 5 3 8 – 5 4 7

Page 7

results for Sideritis or other plant species by DPPH and ABTSassays have been reported (Aktumsek et al., 2013; Erkan et al.,2011). Petroleum ether extract showed the lowest radical scav-enging activity in both assays.

Nitric oxide radical generated from sodium nitroprussideat physiological pH was found to be inhibited by the extracts.In contrast with DPPH and ABTS assays, nitric oxide scaveng-ing activity of the petroleum ether extract was found to be muchgreater than expected. However, other antioxidant com-pounds, including tocopherols, carotenoids and ascorbic acid,may have contributed to the nitric oxide scavenging activity.The decreasing nitric oxide radical scavenging activity orderof the extracts can be ranked as petroleum ether (6.668 mmolTE/g) > methanol (5.569 mmol TE/g) > ethyl acetate (1.779 mmolTE/g) > water (0.773 mmol TE/g) (Table 2).

3.4. FRAP and CUPRAC assays

The reducing capacity of a sample is regarded as a significantindicator of its potential antioxidant activity. For this reason,FRAP and CUPRAC assays were used for the reductive abilityof the extracts.The reducing power values are presented in Table4. In the FRAP assay, the reducing ability of the extracts was inrange of 41.24–142.68 mg TE/g extract. Cupric reducing powersranged from 74.63 to 199.04 mg TE/g extract.The results of thereducing power assays showed a similar tendency to those ofthe total phenolic contents. The water and methanol extractshad the highest reducing potentials followed by the ethyl acetateand methanol extracts for both assays.These results reveal thatthe phenolic compounds in these extracts can act as reductoneswhich are shown to exert antioxidant action by breaking thefree radical chain or by donating a hydrogen atom. Our resultsare in agreement with previous reports that the polyphenolscontribute significantly to the reducing power activities in

medicinal plants (Li, Wu, & Huang, 2009; Osman, Rahim, Isa,& Bakhir, 2009).

3.5. Chelating effect on ferrous ions

Iron is regarded as the most important pro-oxidant and in-creases formation of the hydroxyl radical via Fenton reaction.Therefore, ferrous chelating ability can be an indicator of an-tioxidant activity of the extracts from S. galatica and wasmonitored by measuring the formation of the ferrous ion-ferrozine complex. Table 4 shows the chelating effect of theextracts on ferrous ions. The petroleum ether extract showeda significantly higher chelating effect than those of other ex-tracts. Interestingly, however, water extracts significantly higherpolyphenol levels as well as stronger scavenging activitiesagainst free radicals, and also exhibited lower chelating effects.Similarly, there are contradictory reports in the literature re-garding metal chelating capacities of polyphenols. Rice-Evans,Miller, and Paganga (1996) stated that metal chelation plays aminor role in the overall antioxidant activities of some poly-phenols. Therefore, the highest metal chelating activity ofpetroleum ether extract can be caused by non-phenolic ch-elators, including polysaccharides, peptides and proteins.

3.6. Total antioxidant activities by phosphomolybdenumand β-carotene/linoleic acid assays

Total antioxidant capacities of the extracts from S. galatica wereevaluated by phosphomolybdenum and β-carotene/linoleic acidassays and the results are shown in Table 5. Phospho-molybdenum method is based on the reduction of Mo (IV) toMo (V) by the antioxidants and the subsequent formation ofgreen phosphate/Mo (V) compounds with a maximum absorp-tion at 695 nm. In this assay, total antioxidant abilities of the

Table 3 – Radical scavenging activity of the solvent extracts from Sideritis galatica(mean ± SD).

Samples Radical scavenging activity (mmol TE/g extract)a

DPPH radical ABTS radical cation Nitric oxide radical

Petroleum ether 0.028 ± 0.007ab 0.045 ± 0.012a 6.668 ± 0.225dEthyl acetate 0.129 ± 0.011b 0.163 ± 0.027b 1.799 ± 0.599bMethanol 0.390 ± 0.001d 0.675 ± 0.032c 5.569 ± 0.025cWater 0.352 ± 0.001c 0.756 ± 0.055d 0.773 ± 0.125a

a TE, trolox equivalent.b In same column marked different letters indicate significant difference (p < 0.05).

Table 4 – Reducing power and metal chelating activity of the solvent extracts from Sideritis galatica (mean ± SD).

Assays Petroleum ether Ethyl acetate Methanol Water

Reducing power activityCUPRAC (mg TE/g extract)b 74.63 ± 2.34aa 83.74 ± 2.50b 195.56 ± 4.92c 199.04 ± 3.20cFRAP (mg TE/g extract)b 41.24 ± 0.86a 49.42 ± 1.86b 132.76 ± 3.81c 142.68 ± 2.50d

Metal chelating activityChelating effect (mg EDTAE/g extract)c 52.44 ± 0.38c 16.37 ± 0.85b 7.90 ± 1.42a 6.60 ± 0.77a

a In same row marked different letters indicate significant difference (p < 0.05).b TE, trolox equivalent.c EDTAE, disodium edetate equivalent.

544 j o u rna l o f f un c t i ona l f o od s 1 1 ( 2 0 1 4 ) 5 3 8 – 5 4 7

Page 8

methanol and water extracts were higher than those of otherextracts, probably due to higher phenolics as observed by Johnand Shahidi (2010) in the case of Brazil nut and Chandrasekaraand Shahidi (2010) in millets. Apparently, the study reveals thatthe antioxidant activity of the methanol extract was about 2-foldhigher than that of the petroleum ether extract.

β-Carotene-linoleic acid bleaching inhibition assay is con-sidered to be a good model for membrane based lipidperoxidation. An extract that inhibits β-carotene bleaching canbe described as a free radical scavenger and a primary anti-oxidant (Ferreira, Proença, Serralheiro, & Araújo, 2006).Petroleum ether, ethyl acetate and water extracts, but notmethanol extract, demonstrated an ability to inhibit the bleach-ing of β-carotene by scavenging linoleate-derived free radicals.The greatest activity was caused by the petroleum ether extract(74.49%) followed by the ethyl acetate extract (59.65%) (Table5). When compared to standard antioxidants (BHA, BHT andtrolox), all extracts were found to be less effective than thoseof standard antioxidants. The results obtained by β-carotene-linoleic acid bleaching inhibition method were different fromthose of the radical scavenging and reducing power assays.Thedifferences may be caused from “polar paradox theory”,which states that polar antioxidants are more effective in lesspolar media (such as bulk oils), while nonpolar antioxidantsare more effective in relatively more polar media (such as oil-water emulsions).

3.7. Inhibitory activities on AChE, BChE, α-amylaseand α-glucosidase

All extracts were tested to determine their inhibitory abili-ties as cholinesterase, α-amylase and α-glucosidase and the

results are depicted in Table 6. In contrast to total phenoliccontent, the best inhibitory activity on AChE and BChE was theethyl acetate extract followed by the petroleum ether andmethanol extracts, which were significantly different (p < 0.05).The ethyl acetate extract was also rich in kaempferol and api-genin. However, these compounds were reported as inactiveon AChE and BChE by Orhan, Kartal, Tosun, and Sener (2007).Therefore, the inhibitory activities may be caused by non-phenolics, for example saponins and alkaloids. As in thisresearch, several other studies found no correlation betweenenzyme inhibitory activities (AChE and BChE) and phenolic con-tents. Similar to cholinesterase inhibitory activity, the highestinhibitory activities on both α-amylase and α-glucosidase wereobserved in the petroleum ether and ethyl acetate extracts. Incontrast to both AChE and BChE inhibitory activities, Goncalves,Lajolo, and Genovese (2010) and Kang, Song, and Zhang (2011)reported that kaempferol was an efficient inhibitor of α-amylaseand α-glucosidase. The presence of kaempferol in the ethylacetate extract may be responsible for its antidiabetic activ-ity. However, the level of kaempferol in the methanol extractwas higher than that of ethyl acetate. Therefore, the pres-ence of kaempferol alone cannot explain the antidiabetic effect.These results suggest that the best inhibitory activity ob-tained with the ethyl acetate could be attributed to the possiblesynergistic interaction between phenolic and non-phenolic com-ponents in its chemical composition. Literature is scarce aboutthe inhibition activity of Sideritis species on the enzymes tested(Erdogan-Orhan, Baki, Senol, & Yilmaz, 2010; Loizzo et al., 2008).

4. Conclusion

The present study was undertaken to evaluate antioxidant ca-pacity, cholinesterase, α-amylase and α-glucosidase inhibitoryactivities of different solvent extracts from S. galatica. Bioactivecomponents, antioxidant and enzyme inhibitory abilities varygreatly among the solvent extracts. Water and methanol ex-tracts had the highest phenolic contents and the strongestantioxidant abilities in DPPH, ABTS, phosphomolybdenum andreducing power assays. However, the petroleum ether and ethylacetate extracts, although they contained lower amounts of totalphenolic compounds, were stronger antioxidants in nitric oxidescavenging, metal chelating, β-carotene/linoleic acid andenzyme inhibitory assays. 18 components in the extracts werequantified by HPLC analysis and benzoic acid and chloro-genic acid were the major phenolic components in the waterand methanol extract. These findings suggest that S. galaticais a valuable source of natural agents for human health and

Table 5 – Total antioxidant activity (by β-carotenebleaching and phosphomolybdenum methods) of thesolvent extracts from Sideritis galatica (mean ± SD).

Samples Phosphomolybdenum(mmol TE/g extract)a

β-Carotenebleaching (%)b

Petroleum ether 1.31 ± 0.01ac 74.49 ± 1.11cEthyl acetate 1.50 ± 0.20a 59.65 ± 0.73bMethanol 2.33 ± 0.02b ndWater 2.30 ± 0.01b 30.24 ± 4.68aBHA – 88.28 ± 0.26dBHT – 90.60 ± 0.77dTrolox – 89.39 ± 0.11d

a TE, trolox equivalent; nd, not determined.b At 1 mg/mL concentration.c In the same column marked different letters indicate significant

difference (p < 0.05).

Table 6 – Enzyme inhibitory activity of the solvent extracts from Sideritis galatica (mean ± SD).

Assays Petroleum ether Ethyl acetate Methanol Water

Acetylcholinesterase inhibition (mg GALAE/g extract)b 5.47 ± 0.03ca 5.59 ± 0.04c 3.87 ± 0.13b 0.06 ± 0.01aButyrylcholinesterase inhibition (mg GALAE/g extract)b 24.59 ± 0.51b 27.28 ± 0.38c 18.33 ± 1.26a naα-Amylase inhibition (mmol ACE/g extract)c 0.71 ± 0.02d 0.57 ± 0.02c 0.41 ± 0.01b 0.06 ± 0.01aα-Glucosidase inhibition (mmol ACE/g extract)c 5.66 ± 0.04c 4.19 ± 0.27b 1.68 ± 0.28a na

a In same row marked different letters indicate significant difference (p < 0.05); na, not active.b GALAE, galanthamine equivalent.c ACE, acarbose equivalent.

545j o u rna l o f f un c t i ona l f o od s 1 1 ( 2 0 1 4 ) 5 3 8 – 5 4 7

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could be useful as additives for food, cosmetic or pharmaceu-tical applications. However, further studies are needed forunderstanding in vivo activities of S. galatica extracts.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgement

The authors want to thank Susanna MASTERS (University ofKent, School of Anthropology & Conservation, UK) for proof-reading the present manuscript.

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