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Industrial Crops and Products 36 (2012) 599–606

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l homepage: www.e lsev ier .com/ locate / indcrop

hemical composition of the essential oil and antioxidant activity of methanolicxtracts from fruits and flowers of Hypericum lydium Boiss.

uba Serbetci a,∗, Nurten Özsoyb, Betül Demirci c, Ayse Canb, Sükran Kültürd, K.H. Can Baserc,e

Istanbul University, Faculty of Pharmacy, Department of Pharmacognosy, 34116 Beyazıt, Istanbul, TurkeyIstanbul University, Faculty of Pharmacy, Department of Biochemistry, 34116 Beyazıt, Istanbul, TurkeyAnadolu University, Faculty of Pharmacy, Department of Pharmacognosy, 26470 Eskisehir, TurkeyIstanbul University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 34116 Beyazıt, Istanbul, TurkeyKing Saud University, College of Science, Botany and Microbiology Department, 1145 Riyadh, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 17 June 2011eceived in revised form0 September 2011ccepted 6 November 2011vailable online 14 December 2011

eywords:

a b s t r a c t

This study was conducted to evaluate the essential oil composition and antioxidant activity of methanolicextracts from fruits and flowers of Hypericum lydium Boiss., a wild growing species of the Turkish flora.The antioxidant activities were determined through several biochemical assays. Both extracts showed aninhibitory effect against the formation of TBARS in a phosphatidylcholine liposome model system, moder-ate scavenging effect on DPPH• and superoxide radicals, prominent reducing power and inhibitory effecton deoxyribose degradation in both the nonsite and site-specific assay, but a greater hydroxyl radicalscavenging activity was observed in the non-site specific assay, suggesting that the extracts were better

ypericum lydiumssential oilntioxidantPPHBARSC and GC–MS

at scavenging hydroxyl radicals than at chelating iron. Correlation analysis indicated that there was a lin-ear relationship between antioxidant potency, free-radical scavenging activity, reducing power and thecontent of flavonoids of fruits and flowers extracts. The essential oils obtained by hydrodistillation wereanalyzed by GC and GC–MS. In total forty-three compounds were identified. The most abundant compo-nents were monoterpenes hydrocarbons represented principally by �-pinene. The tested oil showed noantioxidant activity.

. Introduction

Herbs used in the preparation of ethnomedical remedies providettractive templates for the development of new products. Theres an increasing interest in using medicinal and aromatic plantss natural sources in pharmaceutical, food, biotechnology, agricul-ural and cosmetic industries all over the world. Thus, efforts haveeen made to isolate and determinate a wide range of secondaryetabolites produced by higher plants in order to discover novel

ompounds which could be potential bioactive agents (Harvey,008).

Essential oils obtained mainly from plant materials are a mixturef volatile molecules such as terpenoids, phenol-derived aromat-cs and aliphatic components. Different in vitro assays characterize

ost of the essential oils and phenolic compounds as antioxidants

Guimarães et al., 2010). Indeed several medicinal plants have beeneported as sources of safe natural antioxidants (Santos et al., 2010;iccaglia et al., 1993). In living systems antioxidants play a major

∗ Corresponding author.E-mail addresses: serbetci@istanbul.edu.tr, serbetci tuba@hotmail.com

T. Serbetci).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.11.002

© 2011 Elsevier B.V. All rights reserved.

role in the prevention of cancer, arteriosclerosis, malaria, rheuma-toid arthritis, neurodegenerative diseases and aging processes byprotecting the organism against oxidative damage (Moure et al.,2001). Mechanism of the activity can include suppressing reactiveoxygen species formation, either by inhibition of enzymes or bychelating trace elements involved in free-radical production, scav-enging reactive species, and upregulating or protecting antioxidantdefences (Acker et al., 1998).

Reports concerning the antioxidant activities of numerousHypericum species have appeared in the world literature (Confortiet al., 2002; Valentão et al., 2002; Rainha et al., 2011). Phar-macological studies concentrated most particularly on Hypericumperforatum revealing antidepressant, antiviral, antioxidant andantimicrobial properties of the extracts as well as the essen-tial oil demonstrate the great importance of this genus forthe pharmaceutical industry (Josey and Tackett, 1999; Schmittet al., 2001; Radulovic et al., 2007). These activities are mostlyrelated to different groups of components present in the plantsuch as naphtodianthrones, phloroglucinols, flavonoids and phe-

nolic acids being the major ones (Saddiqe et al., 2010). Thegenus Hypericum is represented by about 89 species in Turkeyof which 43 are endemic (Davis, 1988). In Anatolian traditionalmedicines flowered aerial parts of principally H. perforatum,

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ypericum scabrum and Hypericum lydium species find use to treatounds, gastric ulcers, stomach ache, colitis, intestinal disorders

nd hemorrhoids (Sezik et al., 2001; Baytop, 1999).Some Hypericum species growing wild in Turkey has already

een investigated for their antioxidant activities and essentialil content. Ethanolic and water extracts obtained from Hyper-

cum venustum were found to possess strong reducing power,ree radicals and hydrogen peroxide scavenging activity (Spitellert al., 2008). The antioxidant properties of ethanolic extractsbtained from H. scabrum, Hypericum lysimachioides var. lysima-hioides and Hypericum retusum were evaluated using differentests and their antioxidative activities were found to be compa-able with Vitamin E (Barıs et al., 2011). Antioxidant propertiesf Hypericum origanifolium, Hypericum montbretii, H. perforatumnd Hypericum hyssopifolium subsp. elongatum var. elongatumave also been reported (Öztürk et al., 2009; Cakır et al.,003).

Essential oil composition of H. hyssopifolium var. microca-ycinum, H. lysimachioides var. lysimachioides (Toker et al., 2006),. scabrum, H. perforatum (Cakır et al., 1997), Hypericum scabroides

Bagcı and Bekci, 2010), Hypericum apricum, Hypericum davisiiBagcı and Yüce, 2010a), Hypericum pseudolaeve, Hypericum thym-rifolium (Bagcı and Yüce, 2010b), Hypericum capitatum (Bagcınd Yüce, 2011a), Hypericum salsolifolium and H. retusum (Bagcınd Yüce, 2011b) from Turkey have previously been investi-ated.

H. lydium is a perennial herb and can reach a height of up to0 cm with yellow flowers and characteristic translucent glandularots on the sepals margin (Davis, 1988). Previous works on thispecies have revealed the presence of hypericin, pseudohypericin,utin, hyperoside, apigenin-7-o-glucoside, quercitrin and quercetinCırak et al., 2007).

Determination of the essential oil content and antioxidant activ-ty of different extracts is important for the selection of Hypericumpecies with favorable active compounds profile and improvedealth benefit. To our knowledge there is no report on the antiox-

dant activity of extracts and the essential oil obtained from H.ydium. Based on report regarding the presence of polyphenols inerial parts of the plant and on the assumption that their traditionalses might be partially attributed to an antioxidant activity, wevaluated the antioxidant effects of methanolic extracts obtainedrom fruits and flowers of this species through several biochem-cal assays, including inhibition of lipid peroxidation in soybeanhosphatidylcholine liposomes induced with Fe3+/ascorbate, scav-nging effect on DPPH• and superoxide radicals, nonsite andite-specific hydroxyl radical mediated 2-deoxy-d-ribose degra-ation inhibition and reducing power. The antioxidant activitiesf the extracts were compared to that of quercetin as a typicalxample of a naturally occurring flavonol and �-tocopherol, as aypical example of lipid-soluble natural antioxidant. This is the firstime that the antioxidant activity of the extracts obtained fromruits and flowers of H. lydium has been compared. The essentialil composition of the species has also been reported for the firstime.

. Materials and methods

.1. Plant collection and extract preparation

Flowered and fruity aerial parts of H. lydium were col-ected by Dr. Tuba Serbetci from wild plants growing local-

ty in Gümüshane (Northeastern Turkey), Sungurbeyli vil-age, Bozlar area in June 2008. Voucher specimens haveeen deposited in the Herbarium of the Faculty of Phar-acy (ISTE), Istanbul University (ISTE 86015). The plants

d Products 36 (2012) 599–606

were identified by Dr. Tuba Serbetci and Assoc. Prof. SükranKültür, Department of Pharmaceutical Botany, Istanbul Univer-sity.

10 g of dried flowers and fruits of H. lydium were finely pow-dered separately and extracted with 150 ml of boiling methanol ina Soxhlet apparatus for 3 h. After cooling, the extracts were filteredand concentrated by rotary evaporator to give a residue of 1.7 g and1.3 g, respectively. All the extracts were kept at −20 ◦C and weredissolved in water or solvent before use.

2.2. Essential oil isolation and identification

Flowered aerial part of plant material (100 g) was air dried inthe shade at room temperature and the aerial parts were subjectedto hydrodistillation for 3 h. The obtained oils were dried over anhy-drous sodium sulfate and stored at +4 ◦C in the dark until analyzedand tested. GC analysis was carried out using an Agilent 6890NGC system. FID detector temperature was 300 ◦C. To obtain thesame elution order for GC–MS, simultaneous autoinjection wasdone on a duplicate of the same column applying the same oper-ational conditions. Relative percentage amounts of the separatedcompounds were calculated from FID chromatograms. The GC–MSanalysis was carried out with an Agilent 5975 GC-MSD system.An Innowax FSC column (60 m × 0.25 mm, 0.25 �m film thickness)was used with helium as carrier gas (0.8 ml/min). GC oven tem-perature was kept at 60 ◦C for 10 min and programmed to 220 ◦Cat a rate of 4 ◦C/min, and kept constant at 220 ◦C for 10 min andthen programmed to 240 ◦C at a rate of 1 ◦C/min. Split ratio wasadjusted at 40:1. The injector temperature was set at 250 ◦C. Massspectra were recorded at 70 eV. Mass range was from m/z 35 to450. Identification of the essential oil components was carried outby comparison of their relative retention times with either thoseof authentic samples or by comparison of their relative retentionindex (RRI) with a series of n-alkanes. Computer matching againstcommercial (Wiley GC/MS Library, Adams Library, MassFinder 3Library) (Baser and Demirci, 2007) and in-house “Baser Library ofEssential Oil Constituents” built up from genuine compounds andcomponents of known oils, as well as MS literature data (Joulainand KönigKoenig, 1998; Jennings and Shibamoto, 1980) was usedfor the identification.

2.3. Antioxidant activity

2.3.1. ChemicalsNitroblue tetrazolium (NBT), �-nicotinamide adenine dinu-

cleotide reduced (�-NADH), 2-deoxy-d-ribose, soybean l-�-phosphatidylcholine Type IV-S, butylated hydroxytoluene (BHT),quercetin and catechin were purchased from Fluka ChemicalCo. (Buchs, Switzerland). Phenazine methosulfate (PMS), 2,2-diphenyl-1-picryl-hydrazyl (DPPH•), ethylenediamine tetraaceticacid (EDTA), gallic acid and ascorbic acid were obtained fromSigma Chemical Co. (St. Louis, MO, USA). Potassium ferricyanide,trichloroacetic acid (TCA), thiobarbituric acid (TBA) and ferrouswere obtained from Merck. All other reagents were of analyticalgrade.

2.3.2. Determination of total phenolic compoundsTotal soluble phenolics in the methanolic extracts from fruits

and flowers of H. lydium were determined with Folin–Ciocalteureagent (Slinkard and Singleton, 1977). The amount of total phe-

nolic compounds was calculated as mg of gallic acid equivalents(GAE) from the calibration curve of gallic acid standard solu-tion (covering the concentration range between 0.05 mg/ml and0.4 mg/ml) and expressed as mg gallic acid/g dry weight of the

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lant material. The data were presented as the average of triplicatenalyses.

.3.3. Determination of the total flavonoid contentTotal flavonoid content was determined by using following

ethod (Sakanaka et al., 2005). The results were expressed aseans (±SD) mg of (+)-catechin equivalents of total extractable

ompounds for the triplicate extracts.

.3.4. Antioxidant activity on liposome peroxidationThe antioxidant activities of the extracts from fruits and flowers

f H. lydium were evaluated by quantifying the ability of differ-nt concentrations of extracts to suppress Fe3+-ascorbate inducedipid peroxidation in soybean phosphatidylcholine liposomes. Lipideroxidation assay was based on the method described by Duht al. (1999). The formation of lipid peroxidation products wasssayed by the measurement of MDA levels on the basis of MDAeacted with thiobarbituric acid at 532 nm according to Buegend Aust (1978). The percentage inhibition of lipid peroxida-ion was calculated by comparing the results of the sample withhose of controls not treated with the extract using the followingquation:

nhibition effect (%)=1 − Absorbance of sample at 532 nmAbsorbance of control at 532 nm

× 100.

.3.5. DPPH radical scavenging activityThe DPPH radical scavenging activity of the methanolic extracts

rom fruits and flowers of H. lydium was measured according to therocedure described by Brand-Williams et al. (1995). The ability tocavenge DPPH radical was calculated by the following equation:

PPH radical scavenging activity (%)

= 1 − Absorbance of sample at 517 nmAbsorbance of control at 517 nm

× 100.

.3.6. Superoxide radical scavenging activityThe effects of the methanolic extracts from fruits and flowers

f H. lydium and quercetin on generation of superoxide radicalsere determined by the nitroblue tetrazolium reduction method

Nishikimi et al., 1972). The abilities to scavenge the superoxideadical were calculated using the following equation:

uperoxide radical scavenging activity (%)

= 1 − Absorbance of sample at 560 nmAbsorbance of control at 560 nm

× 100.

.3.7. Nonsite and site-specific hydroxyl radical-mediated-deoxy-d-ribose degradation

The ability of the extracts to inhibit nonsite and site-specificydroxyl radical-mediated 2-deoxy-d-ribose degradation was car-ied out as described by Halliwell et al. (1987). The percentagenhibition of deoxyribose degradation was calculated by compar-ng the results of the sample with those of controls not treated withhe extract using the following equation:

nhibition effect (%)=1−Absorbance of sample at 532 nmAbsorbance of control at 532 nm

×100.

The ability of the extracts to inhibit site-specific hydroxyladical-mediated 2-deoxy-d-ribose degradation was carried out asescribed in the nonsite-specific procedure, except that EDTA waseplaced by buffer.

d Products 36 (2012) 599–606 601

2.3.8. Reducing powerThe reducing powers of the methanolic extracts from fruits

and flowers of H. lydium, quercetin and �-tocopherol weredetermined according to the method described by Chung et al.(2005).

2.3.9. Statistical analysisResults were expressed as mean ± standard deviation. Statisti-

cal comparisons were performed with Student’s t-test. Differenceswere considered significant at p < 0.05. The correlation coefficient(r2) between the parameters tested was established by regressionanalysis.

3. Results and discussion

3.1. Essential oil composition and antioxidant activity

The hydrodistillation of the aerial parts of H. lydium yielded0.16% (v/w) of pale yellowish oil. Forty-three compounds wereidentified in the hydrodistilled oil of H. lydium which accounted for99.8% of the total oil composition. The retention indices with thepercentage compositions are given in Table 1. The most abundantcomponents were monoterpene hydrocarbons represented princi-pally by �-pinene (71.2%) followed by oxygenated sesquiterpenes(5.6%) among which caryophyllene oxide (2.9%) and caryophyl-ladienol I (0.6%) prevailed. The oil consists also of oxygenatedmonoterpenes such as trans-pinocarveol (0.5%), trans-verbenol(0.2%) and myrtenal (0.3%) but with a considerably reduced per-centages. The levels of sesquiterpenes hydrocarbons in the oil werealso insignificant.

Among the pre-investigated Hypericum essential oils from wildgrowing species in Turkey, many taxa were characterized bythe high presence of �-pinene except for Hypericum linaroides(Cakır et al., 2005) together with H. lysimachioides var. lysima-chioides, H. hyssopifolium var. microcalycinum (Toker et al., 2006),Hypericum bupleuroides (Demirci and Baser, 2006) and Hyper-icum thymopsis (Özkan et al., 2009). The level of �-pinene wasdramatically higher in essential oil of H. scabrum, our currenttopic H. lydium and H. perforatum (Cakır et al., 1997) comparedto Hypericum adenotrichum (Erken et al., 2001) and Hypericumheterophyllum (Cakır et al., 2004). Besides, Hypericum cerastoides,H. hyssopifolium ssp. elongatum var. elongatum, H. montbretii andHypericum calycinum (Cakır et al., 2004; Erken et al., 2001) con-tains also an important quantity of �-pinene. It is noteworthy that�-pinene was also reported as the main constituent in several H.perforatum essential oils in the world literature (Petrakis et al.,2005).

The essential oil of H. lydium was subjected to screening for thepossible antioxidant activity by two complementary test systemnamely DPPH free radical scavenging and antioxidant activity onliposome peroxidation. The oil has been found ineffective in bothexperiments.

The lack of activity of H. lydium essential oil could be attributedto its high content in �-pinene (71.2%). This inactivity is closelyrelated to this compounds limited hydrogen capacity and watersolubility. Previous results showed that greater antioxidant poten-tial could be ascribed to the oxygenated monoterpenes types ofcompounds, such as thymol, carvacrol (Piccaglia et al., 1993) and/orphenylpropane derivatives such as eugenol (Atsumi et al., 2005). Onthe other hand pinenes are bicyclic monoterpenes hydrocarbonsand of great value as precursors for flavors and fragrances indus-

tries (Yoo and Day, 2002). In consequence numerous Hypericumspecies rich in �-pinene widely distributed in Turkey including H.lydium are of considerable potential interest for application to thefood and cosmetic industries.

602 T. Serbetci et al. / Industrial Crops and Products 36 (2012) 599–606

Table 1Essential oil composition of Hypericum lydium Boiss.

No Constituents RI Percentage(%)

Identificationmethod

1 3-Methyl nonane 965 0.9 a

2 Decane 1000 0.7 a,b

3 �-Pinene 1032 71.2 a,b

4 Undecane 1100 3.5 a,b

5 �-Pinene 1118 3.0 a,b

6 Thuja-2,4(10)-diene 1135 0.7 a

7 Myrcene 1174 0.5 a,b

8 Limonene 1203 0.6 a,b

9 p-Cymene 1280 0.4 a,b

10 Longipinene 1482 0.4 a

11 �-Campholene aldehyde 1499 0.3 a

12 �-Copaene 1497 0.2 a,b

13 �-Caryophyllene 1612 0.4 a,b

14 Myrtenal 1648 0.3 a

15 trans-Pinocarveol 1670 0.5 a,b

16 (Z)-�-Farnesene 1668 0.7 a

17 trans-Verbenol 1683 0.2 a,b

18 �-Muurolene 1704 0.4 a

19 2-Undecanol 1722 0.4 a

20 Valencene 1740 0.3 a

21 �-Selinene 1742 tr a

22 �-Cadinene 1773 0.3 a

23 �-Cadinene 1776 tr a

24 7-epi-�-Selinene 1785 0.2 a

25 Myrtenol 1804 tr a,b

26 Caryophyllene oxide 2008 2.9 a,b

27 Humulene epoxide-II 2071 0.3 a,b

28 Hexahydrofarnesyl acetone 2131 0.1 a

29 Spathulenol 2144 0.1 a,b

30 Copaborneol 2210 0.4 a

31 Clovenol 2211 0.5 a

32 Cadalene 2256 0.3 a

33 Decanoic acid 2298 0.4 a,b

34 Tricosane 2300 0.2 a,b

35 Caryophylla-2(12),6(13)-dien-5�-ol(=Caryophylladienol I)

2316 0.6 a

36 Caryophylla-2(12),6-dien-5�-ol (=CaryophyllenolI)

2389 0.1 a

37 Caryophylla-2(12),6-dien-5�-ol (=CaryophyllenolII)

2392 0.7 a

38 Pentacosane 2500 0.2 a,b

39 Dodecanoic acid 2503 1.0 a,b

40 Tetradecanoic acid 2670 0.8 a,b

41 Heptacosane 2700 0.8 a,b

42 Nonacosane 2900 2.2 a,b

43 Hexadecanoic acid 2931 2.1 a,b

Monoterpene hydrocarbons 76.4Oxygenated monoterpenes 1.7Sesquiterpene hydrocarbons 2.8Oxygenated sesquiterpenes 5.6Fatty acids 4.3Others 9.0

Total 99.8

RRI, relative retention indices calculated against n-alkanes.% calculated from FID data.tr, trace (<0.1%).

a Comparison of mass spectra with the Wiley and Mass Finder libraries and reten-tion times.

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(inh

ibiti

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fruits extractflowers extractquercetinalpha-tocopherol

ers (1.17 ± 0.19 mg/ml) extract was significantly (p < 0.05) better

b Comparison with genuine compounds on the HP Innowax column.

.2. Antioxidant activity of fruit and flower extracts

.2.1. Extract yield (amount of total extractable compounds),xtractable total phenols and flavonoids content and composition

f methanolic extracts from fruits and flowers

No significant difference (p < 0.05) between the amount ofxtractable compounds of the extracts obtained from fruits

Fig. 1. The inhibitory effect of different amounts of the methanolic extracts fromfruits and flowers of H. lydium on TBARS formation. Quercetin and �-tocopherolwere used as reference antioxidants. Values are means ± SD (n = 3).

(193.3 mg/g of dry weight) and flowers (195.0 mg/g of dry weight)of H. lydium was found.

The total phenolic contents of fruits and flowers extracts werefound to be 1.61 ± 0.17 and 1.92 ± 0.22 mg, respectively, in termsof gallic acid equivalents per gram of dry weight of the sample. Thetotal flavonoid contents of fruits and flowers extracts were foundto be 1.19 ± 0.11 and 1.20 ± 0.053 mg, respectively, in terms of cat-echin equivalents/g of dry weight of the sample. This indicated thatthe content of phenolic compounds and flavonoids in flowers andfruits did not varied markedly. According to the results obtained byKızıl et al. (2008), the total phenolic content of Hypericum triquet-rifolium and H. scabroides extracts were 267 and 333 �g gallic acidequivalents per mg of each extract, respectively. Our results on thetotal phenolic compounds of H. lydium extracts appear to be in agood agreement with literature.

It was observed that the amount of flavonoids in the bothextracts showed high correlation with the total amount of phe-nolics (r2 = 0.999).

3.2.2. Antioxidant activity on liposome peroxidationFree radicals are known to attack the highly unsaturated fatty

acids of membrane system to induce lipid peroxidation. An increasein free radicals causes overproduction of thiobarbituric acid-reactive substances (TBARS), which are the end product of lipidperoxidation process and commonly used as a marker of oxidativestress.

The inhibitory effects of both the extracts and reference antiox-idants (quercetin and �-tocopherol) in different concentrations onlipid peroxidation in phosphatidylcholine liposomes, estimated bythe amount of TBARS accumulated 60 min after the incubationof liposomes with Fe3+/ascorbate are shown in Fig. 1. Both theextracts showed a dose-dependent relationship in inhibiting lipidperoxidation. At 5 mg/ml, the fruits (86.40 ± 0.86%) and flowers(88.86 ± 1.43%) extracts showed almost similar inhibitory effectson TBARS formation, which were comparable to that of quercetin(91.60 ± 0.079%) at 0.08 mg/ml and �-tocopherol (89.47 ± 0.28%) at0.625 mg/ml.

Based on the EC50 values (effective concentration at which theantioxidant activity was 50%), the antioxidant activity of flow-

than the fruits (1.76 ± 0.05 mg/ml) extract. It is may be due to theother compounds of the mixture which may potentiate or pre-vent the expected activity. Both of the extracts were significantly

T. Serbetci et al. / Industrial Crops an

0

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1,250 2,5 3,75 5

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DPP

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activ

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fruits extractflowers extractquercetinalpha-tocopherol

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(99.04 ± 0.34%) at 10 mg/ml were nearly equal (p > 0.05) to that ofquercetin (99.33 ± 0.27%) at a concentration of 1.25 mg/ml.

The superoxide radical scavenging activities, expressed in theterm of EC50 (the effective concentration at which the superoxide

0

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fruits extractflowers extractquercetin

ig. 2. DPPH radical scavenging activities of the methanolic extracts from fruits andowers of H. lydium. Quercetin and �-tocopherol were used as reference antioxi-ants. Values are means ± SD (n = 3).

ess effective (p < 0.05) than the reference antioxidants, quercetin0.034 ± 0.0015 mg/ml) and �-tocopherol (0.188 ± 0.011 mg/ml).lthough the inhibitory effect of both the extracts on TBARS for-ation was less than that of quercetin or �-tocopherol, H. lydiumay also be expected to protect against damage to cell mem-

ranes, because it can also slow lipid oxidation by inactivating orcavenging free radicals, particularly peroxy radicals, thus inhibit-ng initiation or propagation of oxidative chain reactions. Thentioxidant activity and the total phenolic content showed a goodorrelation in both the fruits (r2 = 0.9541) and flowers (r2 = 0.9070)xtracts. Strong correlation was observed between the antioxi-ant activity and flavonoids (r2 = 0.9624 and 0.9039 for fruits andowers extracts, respectively). In accordance with our results thefficacy of flavonoid-rich extract of H. perforatum to prevent theeroxidation of soybean lecithin liposome was reported (Zou et al.,004). Similarly, the methanol extract of H. triquetrifolium Turaerial part which contains flavonoids was reported to have a strongntioxidant activity against lipid peroxidation (IC50 = 0.062 mg/ml)Conforti et al., 2002). The ethanolic extracts of H. scabroides and. triquetrifolium were reported to significantly inhibit lipid per-xidation in rat brain (Kızıl et al., 2008). Fractions containingavonoids and/or caffeoylquinic acids were found to be responsi-le for both radical-scavenging activity and lipid peroxidation (Silvat al., 2005).

.2.3. DPPH radical-scavenging activityThe effect of antioxidants on DPPH is thought to be due to

heir hydrogen donating abilities. Fig. 2 shows that at 2.5 mg/mlhe methanolic extracts of fruits (90.75 ± 0.87%) and flowers90.77 ± 1.29%) did not differ in their DPPH radical scavenging abili-ies (p > 0.05), which were comparable to quercetin (94.48 ± 2.31%)t 0.16 mg/ml and �-tocopherol (95.88 ± 0.76%) at 0.625 mg/ml.ur results were in agreement with Kızıl et al. (2008) who reported

he similar DPPH radical scavenging activity of 91.3% and 82.6% for. scabroides and H. triquetrifolium, respectively.

The DPPH radical scavenging activities, expressed in termsf EC50 (the effective concentration at which the DPPH radi-als were scavenged by 50%) were similar (p > 0.05) for fruits1.03 ± 0.13 mg/ml) and flowers (0.99 ± 0.25 mg/ml) extracts but

ignificantly different (p < 0.05) from the EC50 values obtained forhe reference antioxidants, quercetin (0.089 ± 0.0017 mg/ml) and-tocopherol (0.369 ± 0.015 mg/ml). Although the DPPH radicalcavenging abilities of the extracts were significantly lower than

d Products 36 (2012) 599–606 603

those of quercetin and �-tocopherol, it was evident that the extractsdid show the proton-donating ability and could serve as freeradical scavengers. Based on EC50 values, Kızıl et al. (2008) demon-strated high DPPH radical scavenging activity of H. triquetrifolium(IC50 = 39.0 �g/ml) and H. scabroides (IC50 = 33.8 �g/ml), which isconsistent with the findings of Zou et al. (2004), who reportedthat H. perforatum extract also showed relatively high DPPH rad-ical scavenging effect with an average IC50 value of 10.63 �g/ml. Itwas evident that H. perforatum, H. triquetrifolium and H. scabroidesextracts had stronger effects on DPPH radicals compared to H.lydium extracts used in this study.

Total phenolics content (r2 = 0.8672 and 0.8332 for fruitsand flowers extracts, respectively) and flavonoids (r2 = 0.8814and 0.8267 for fruits and flowers extracts, respectively) in theextracts correlate with their antiradical activity. This result wasin agreement with previous reports that the phenolic compoundscontribute significantly to the antioxidant activity.

A great number of reports have established that plant phe-nolic compounds including flavonoids are potent antioxidants. Inaddition, it has been reported that free –OH groups in phenolic com-pounds are mainly responsible for the activity (Weng and Wang,2000). Indeed many phenolic compounds such as caffeoylquinicacid, flavonoid and their glycosylated derivatives present in H.perforatum ethanolic extract have been shown to possess strongantioxidant activity (Silva et al., 2005; Zou et al., 2004). In anotherstudy performed with H. hyssopifolium, it was found that antioxi-dant activity was based on flavonoid-type compounds (Cakır et al.,2003). Methanolic extract of H. triquetrifolium with significantantioxidant activity was also considered to be a good source offlavonoids (Conforti et al., 2002; Valentão et al., 2002).

It is worthy of note that both the configuration and total numberof phenolic hydroxyl groups substantially influence the mechanismof the antioxidant activity (Procházková et al., 2011).

3.2.4. Superoxide radical scavenging activityIn a phenazine methosulfate/NADH-nitroblue tetrazolium

system, both the extracts exhibited strong superoxide anion scav-enging activity. As is shown in Fig. 3, the scavenging effectsof methanolic extracts from fruits (98.83 ± 0.76%) and flowers

Concentration (mg/ml)

Fig. 3. Superoxide radical scavenging activities of the methanolic extracts fromfruits and flowers of H. lydium. Quercetin was used as reference antioxidant. Valuesare means ± SD (n = 3).

6 ps and Products 36 (2012) 599–606

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04 T. Serbetci et al. / Industrial Cro

adicals were scavenged by 50%) were similar (p > 0.05) for fruits3.08 ± 0.12 mg/ml) and flowers (2.98 ± 0.095 mg/ml) extracts.onetheless, when compared to the EC50 value obtained for theuercetin (0.461 ± 0.014 mg/ml), considered to be a potent super-xide radical scavenger, the scavenging activities of the aboveentioned extracts were found to be significantly lower (p < 0.05).-Tocopherol did not show any detectable superoxide radical scav-nging activity.

The superoxide radical scavenging activity and the flavonoidontent showed a good correlation in both the fruits (r2 = 0.9840)nd flowers (r2 = 0.9788) extracts. This result was in agreementith previous reports that the antioxidant properties of someavonoids are effective mainly via scavenging of superoxide anionadical. H. perforatum has been reported to be a potent inhibitor ofhe superoxide radical in a cell-free and attributed this antioxidantctivity to hypericin (Hunt et al., 2001). Hypericum androsaemumyophilized infusion strongly scavenged the superoxide radicalenerated by the xanthine/xanthine oxidase (IC50 = 2.9 �g/ml) orMS/NADH (25.6–2.9 �g/ml) systems in a concentration depen-ent manner (Valentão et al., 2002). Our results further supporthe view that strong scavenging properties of Hypericum extracts onuperoxide anion possibly renders them as promising antioxidants.

.2.5. Nonsite and site-specific hydroxyl radical-mediated-deoxy-d-ribose degradation

The deoxyribose assay allows assessment of abilities of extractso exert pro-oxidant action, scavenge hydroxyl radicals generatedy Fenton systems, and assessment of abilities to chelate metal ironAruoma, 2003).

When EDTA chelated iron (III) ions were incubated witheoxyribose, reducing agent such as ascorbic acid and H2O2, H.

ydium extracts were capable of inhibiting TBARS formation bycavenging hydroxyl radicals before they reacted with the 2-deoxy--ribose substrate (Fig. 4). At 10 mg/ml the fruit (85.71 ± 1.21%)nd flower (85.24 ± 1.58%) extracts did not differ in their hydroxyladical scavenging abilities. Extracts were less effective (p < 0.05)n scavenging hydroxyl radicals in comparison with quercetin98.18 ± 0.41% at 2.5 mg/ml).

As can be seen from the EC values, hydroxyl radical scav-

50nging activities of the extracts from fruits (1.99 ± 0.30 mg/ml)nd flowers (2.27 ± 0.030 mg/ml) were comparable (p > 0.05).oth the extracts were statistically significantly (p < 0.05) less

0

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ig. 4. Hydroxyl radical scavenging activities of the methanolic extracts from fruitsnd flowers of H. lydium in the nonsite-specific assay. Quercetin was used as refer-nce antioxidant. Values are means ± SD (n = 3).

and flowers of H. lydium in the site-specific assay. Quercetin was used as referenceantioxidant. Values are means ± SD (n = 3).

potent hydroxyl radical scavengers than the reference antioxidantquercetin (0.616 ± 0.043 mg/ml).

Our results were in agreement with Zou et al. (2004) whoreported the similar hydroxyl radical scavenging activity of 92.3% at5 mg/ml for flavonoid-rich extract of H. perforatum. Compared withthe data presented by Kızıl et al. (2008), the antioxidative proper-ties of the extracts obtained from fruits and flowers of H. lydiumon scavenging hydroxyl radicals were more remarkable than thatof H. triquetrifolium (77% at 80 mg/ml) and H. scabroides (83% at100 mg/ml) extracts.

Hydroxyl radical scavenging activity in the nonsite-specificassay and flavonoid content showed a good correlation in both thefruits (r2 = 0.9648) and flowers (r2 = 0.9472) extracts.

Studies using the deoxyribose assay can provide useful infor-mation on the likelihood that molecules could chelate iron ionsin a way that prevents them from catalyzing OH• formation. Thus,when iron was added to the assay mixture as ferric chloride insteadas ferric-EDTA, the extracts were capable of preventing the for-mation of hydroxyl radicals by chelating and deactivating iron. Ascan be seen in Fig. 5 both the extracts and quercetin exhibiteda concentration-dependent increase in their scavenging effect onhydroxyl radical, but a greater hydroxyl radical scavenging activitywas observed in the non-site specific assay. This indicated that theantioxidant activity of the fruits and flowers from H. lydium wasaccounted for the direct interaction of flavonoids with hydroxylradicals than to a metal chelating activity. At 10 mg/ml the fruits(65.55 ± 0.64%) and flowers (65.06 ± 1.73%) extracts did not differin their hydroxyl radical scavenging abilities (p > 0.05), which werecomparable to that of quercetin (67.81 ± 1.62%) at 2.5 mg/ml.

EC50 values, in ability of both the extracts to interferewith site-specific Fenton chemistry, were similar for fruits(6.29 ± 0.27 mg/ml) and flowers (6.22 ± 0.84 mg/ml) extracts butsignificantly different (p < 0.05) from the EC50 values obtained forquercetin (1.30 ± 0.02 mg/ml).

The extract EC50 values in site-specific assay were higher thanthe corresponding values generated in the nonsite-specific assaysuggesting that the extracts were better at scavenging hydroxylradicals than at chelating iron. Our results were in agreement withKızıl et al. (2008), who showed that ethanol extracts of H. scabroides

and H. triquetrifolium were not good chelating agents.

Most plant polyphenol compounds possess both antioxidantand prooxidant activities. H. lydium extracts did not show any pro-oxidant effect at the concentrations tested. Our results confirm the

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esults of Silva et al. (2005). It has been noted that H. perforatumossess both antioxidant and pro-oxidant properties. Prooxidantffect was also reported by Hunt et al. (2001) on this species.

.2.6. Reducing powerThe reducing capacity of a compound may serve as an indicator

f its potential antioxidant activity. Radical chain reactions could beerminated when substances exhibiting high reducing tendencieso donate electrons which can react with free radicals convert-ng them to more stable products. The data obtained from thexperiment for determining the reducing power of the methanolicxtracts from fruits and flowers of H. lydium in which increasingbsorbance values implied increased conversion of Fe3+ to Fe2+

howed that both the extracts exhibited concentration-dependenterric reducing ability within the range of plant extract concentra-ions used (0.16–5 mg/ml).

At 5 mg/ml fruits and flowers extracts of H. lydium showedtrong reducing powers of 1.676 ± 0.006 and 1.694 ± 0.009 inerms of absorbance values at 700 nm, respectively, which wereomparable to that of quercetin (1.699 ± 0.006) at 0.625 mg/mlnd �-tocopherol (1.698 ± 0.004) at 2.5 mg/ml. EC50 valuesthe effective concentration at which the absorbance was.5) obtained for the fruits (0.480 ± 0.011 mg/ml) and flowers0.449 ± 0.015 mg/ml) extracts were comparable with that of �-ocopherol (0.487 ± 0.016) but significantly different (p < 0.05) fromhe EC50 value obtained for the quercetin (0.124 ± 0.003 mg/ml).

Reducing power correlated well with total phenolic (r2 = 0.9981nd 0. 9990 for fruits and flowers extracts, respectively) andavonoid (r2 = 0.9954 and 0.9992 for fruits and flowers extracts,espectively) contents, an indication that the high reducing powerf the extracts may be attributed to their total phenolic andavonoid content.

The antioxidant activity, measured as inhibition of lipid perox-dation in phosphatidylcholine liposomes (r2 = 0.9475 and 0.8883or fruits and flowers extracts, respectively), DPPH (r2 = 0.8565 and.8092 for fruits and flowers extracts, respectively) and superoxidenion (r2 = 0.9484 and 0.9701 for fruits and flowers extracts, respec-ively) radical scavenging activity, inhibitory effect on deoxyriboseegradation in the nonsite (r2 = and 0.9551 and 0.9391 for fruitsnd flowers extracts, respectively) specific assay and reducingower of both the extracts correlated directly. It may be concludedhat antioxidant activity of the fruits and flowers extracts may bettributed to the combined effects of reducing power, donation ofydrogen atoms and scavenging of active oxygen.

The antioxidant activity of the extracts were significantly cor-elated with their scavenging activity on DPPH (r2 = 0.9617 and.9962 for fruits and flowers extracts, respectively), superoxidenion (r2 = 0.9994 and 0.9390 for fruits and flowers extracts, respec-ively) and hydroxyl radicals, generated in non-site specific Fentonhemistry (r2 = 0.9760 and 0.9910 for fruits and flowers extracts,espectively), indicating that the antioxidant activities measuredn the four methods complemented each other.

. Conclusions

The obtained data show that H. lydium fruits and flowersethanolic extracts possess prominent antioxidant capacities. In

ddition to their potent scavenging properties and reducing power,hey represent strong inhibitory effects on lipid peroxidation thusan be regarded as promising antioxidants and raw material in food

nd/or pharmaceutical industries. In contrast, the essential oil haseen found ineffective in antioxidant test systems. Neverthelesshe considerable percentage of �-pinene can promote the poten-ial interest for H. lydium oils application in flavor and fragrance

d Products 36 (2012) 599–606 605

industries due to a great scope for commercial cultivation of severalaromatic crops in all over the world.

Acknowledgement

This work is supported by the Scientific Research Foundation ofIstanbul University (Project no: 7069).

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