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Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 546550 (2009)DOI: 10.1002/ptr

546 D. TAJNER ET AL.

Copyright 2008 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res.23, 546550 (2009)Published online 9 December 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2682

ExploringEquisetum arvenseL.,EquisetumramosissimumL. andEquisetum telmateiaL. as Sources of Natural Antioxidants

Dubravka tajner1*, Boris M. Popovi1, Jasna anadanovi-Brunet2and Goran AnaCkov3

1Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovia 8, 21000 Novi Sad, Serbia and Montenegro2Faculty of Technology, University of Novi Sad, Cara Lazara 1, 21000 Novi Sad, Serbia and Montenegro3Department of Biology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovi a 3, 21000 Novi Sad, Serbia andMontenegro

The antioxidant and scavenging activities of above ground parts of Equisetum arvense L., Equisetum

ramosissimumL. andEquisetum telmateiaL. phosphate buffer (pH 7) extracts were investigated. Activities

of antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase),

quantities of reduced glutathione, malonyldialdehyde, superoxide and hydroxyl radicals and flavonoid, soluble

protein, chlorophyll a, b and carotenoid contents were determined. The total antioxidant capacity was deter-mined by ferric reducing antioxidant power (FRAP) assay. The Equisetum telmateiaextract demonstrated

scavenging and antioxidant properties better than Equisetum ramosissimum and Equisetum arvense. The

ESR signal of DMPO-OH radical adducts in the presence ofEquisetum telmateiaphosphate buffer (pH 7)

extract was reduced by 98.9% indicating that Equisetum telmateiacould be a useful source of antioxidants

with huge scavenging ability. Copyright 2008 John Wiley & Sons, Ltd.

Keywords:Equisetum; antioxidant activity; FRAP; ESR.

Received 3 September 2007

Revised 15 July 2008

Accepted 15 July 2008

* Correspondence to: Dubravka tajner,Faculty of Agriculture, University

of Novi Sad, Trg Dositeja Obradovia 8, 21000 Novi Sad, Serbia andMontenegro.

E-mail: [email protected]/grant sponsor: Ministry of Science and Environmental Protec-tion of the Republic of Serbia; contract/grant number: 142036B.

INTRODUCTION

Oxygen free radicals highly reactive species (O2, OH,

HO2) with one or more unpaired electrons, H2O2and

activated oxygen species (1g, 1g) are formed inalmost every cell of the body at an astonishing rateduring normal oxidative metabolism (Gey, 1994). Envi-ronmental factors such as UV light, ozone, tobacco smoke,different xenobiotics, herbicides, pesticides, ionizingradiation etc. cause their formation to a greater extent(Halliwell and Gutteridge, 1984). They react voraciouslywith almost every cellular component and contributeto many types of pathologies. Antioxidant defensemechanisms counteract free radical formation andreactions. Increasing levels of antioxidants should de-crease pathology. Antioxidants isolated from plants thatcan neutralize free radicals may be of central importancein the prevention of cancer, heart, vascular, diabetesand neurodegenerative diseases (Nagai et al., 2005).Combinations of different natural antioxidants whichcan be found in different medicinal plants work betterthan separate antioxidants alone (Williamson, 2001).

Equisetum arvenseL. (Equisetaceae, genus and sub-genus Equisetum) is a European herb which grows inmoist waste places throughout temperate regions of theworld. The ancient Greeks used E. arvensein the treat-ment of wounds and the Romans used it as a vegetable,an animal feed and a medicine. In some parts of Serbia

it is still used in the diet as a salad. Among severalEquisetumspecies growing wild in Serbia, only E. arvensehas been investigated and used in herbal medicine.

E. arvensehas traditionally been described in Euro-

pean Pharmacopeias constituting Equisetiherba as adiuretic (Graefe and Veit, 1999). The German Com-mission E expert panel has approved E. arvense forthis indication. E. arvense is also used for urinary tractinflammation (Joksiet al., 2003) and occasionally, forosteoporosis and wound healing, but without strongscientific evidence. In addition to a high percentageof silicates, E. arvensecontains a variety of flavonoidssuch as quercetin and kaempferol glycosides as well asflavone glycosides (Veit et al., 1995). Two compoundsisolated from E. arvense, onitin (phenolic petrosin) andluteolin (flavonoid) showed hepatoprotective as well assuperoxide and DPPH scavenging activities (Oh et al.,2004). Some other Equisetumspecies which have beeninvestigated (E. bogotense H.B.K., E. palustre L., E.telmateiaEhrh., E. pretenseEhrh., E. sylvaticumL. andE. fluviatileL.) also contain various flavonoids and theirglycosides (Veit et al., 1995) with possible antioxidantand medicinal properties.

E. arvenseis rich in vitamins such as vitamins C, E,K, B1, B2, B6, nicotinic acid, folic acid and pantothenicacid (Science and Technology Agency, 2002). Moreover,E. arvenseis rich in trace elements such as Na, K, Ca,Mg, P, Fe, Zn, Cu, Mn, Si, Sr, Ti (Nagai et al., 2005).Copper and zinc are essential elements, needed forsuperoxide dismutase to act against active oxygen species.

Our previous study concerning the scavenger activities

of E. arvense, E. ramosissimumL. and E. telmateia L.(tajneret al.,2006b) showed that all three Equisetumspecies possess high scavenger abilities against DPPH,NO and HO radicals and also high total antioxidant


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EQUISETUM ARVENSEL AS SOURCE OF NATURAL ANTIOXIDANTS 547


capacity according to the FRAP method. The E.telmateia extract demonstrated the most relevant scav-enger properties. The aim of this study was to exploreother antioxidant characteristics (antioxidant enzymeactivities, nonezymic antioxidants and oxidative stressparameters) of these Equisetums and compare theirantioxidant and scavenger activities in order to find thespecies that could be a source of potential medicinal

agents and nontoxic natural antioxidants.

MATERIALS AND METHODS

Plant material. Equisetum arvenseL., Equisetum ramosis-simumDesf. and Equisetum telmateiaEhrh. (Equisetaceae),aerial parts collected in Fruska gora mountain (smallmountain in Vojvodina, northern province of Serbia)and identified by Vukievi(1970). Voucher specimens(E. arvenseL. No. 2-1969, Fruska gora-Vrdnik, UTM34TDQ09, 05.2004. det.: Pal Boza; E. ramosissimumDesf. No. 2-1970, Fruska gora-Vrdnik, UTM 34TDQ09,05.2004., det.: Pal Boza; E. telmateiaEhrh. No. 2-1970,Fruska gora-Vrdnik, UTM 34TDQ09, 05.2004., det.: PalBoza) were confirmed and deposited at the Herbariumof the Department of Biology and Ecology (BUNSHerbarium) Faculty of Natural Sciences, University ofNovi Sad.

Preparation of the phosphate buffer (pH 7) extracts.One g of fresh plant material was ground with quartzsand in a cold mortar. The ground material was sus-pended in 5 cm30.1 mol/dm3K2HPO4at pH 7.0. After10 min centrifugation at 4 C and 15 000 g, the aliquotsof the supernatant were used for different antioxidant

and scavenger activity determinations.

Determination of antioxidant enzyme activities. All theantioxidant enzyme activities were determined spectro-photometrically at 25 C using phosphate buffer (pH 7)plant extracts. Enzymatic specific activity is expressedas mol of the substrate transformed/min/mg proteinexcept for superoxide dismutase activity.

Superoxide dismutase (SOD) activity was determinedby the method of Misra and Fridovics (1972) based on theinhibition of transformation of adrenaline to adreno-chrome at pH 10.2. One unit SOD can be regarded asthat amount of enzyme which causes a 50% inhibitionin the extinction change in 1 min compared with thecontrol (Matkovics et al., 1977). Measurements weremade at 480 nm.

Guaiacol peroxidase (GPx) activity was determinedusing guaiacol as the substrate at 436 nm (Matkovicset al.1977).

Glutathione peroxidase (GSH-Px) activity was deter-mined using cumene hydroperoxide and reduced gluta-thione (GSH) as substrates at 412 nm (Chiu et al., 1976).

Catalase (CAT) activity was determined at 240 nm.The decomposition of H2O2was followed by a decreasein absorbance (Simon et al., 1974).

All determinations were performed in triplicate. Thevalues are expressed as mean standard error.

Determination of quantities of reduced glutathione,malonyldialdehyde, superoxide and hydroxyl radicalsand flavonoid, soluble protein, chlorophyll a, b and

carotenoid contents. The amount of reduced glutathione(GSH) was determined with Ellman reagent at 412 nm(Sedlak and Lindsay, 1968).

Lipid peroxidation (LP) was determined by thethiobarbituric acid (TBA) method. Values were givenas equivalent amounts of malonyldialdehyde (MDA).The calibration curve was prepared with malonyl-dialdehyde bis-diacetal (Placer et al., 1968).

Superoxide radical (O2

) was determined by adrena-line autooxidation (Misra and Fridovics, 1972).

Hydroxyl radical (OH) was determined by the in-hibition of deoxyribose degradation (Cheesman et al.,1988).

Total flavonoids were extracted with 70% methanoland estimated according to Marckam (1989) and thesoluble protein content was determined by the methodof Bradford (1976).

Pigments were extracted with acetone and determinedspectrophotometrically using molar extinction coeffi-cients according to Wettstein (1957).

All determinations were performed in triplicate. Thevalues are expressed as mean standard error.

Determination of the radical scavenging activity and totalantioxidant capacity. The influence of the phosphatebuffer (pH 7) Equisetum extract on hydroxyl radical(HO) formation was studied by electron spin resonance(ESR) using a spin trapping method (Hiramoto et al.,1996). The scavenging activity of the extract was estimatedby the percentage decrease of the relative intensity ofthe signal of DMPO-OH radical adduct with referenceto the control without extract. The scavenger effect (SE)was calculated with the formula:

SE = [(h0hx)/ho] 100%

where h0and hxare heights of the second pick in ESRspectra of the control and sample respectively.

The total antioxidant capacity was estimated accord-ing to the FRAP (ferric reducing antioxidant power)assay (Benzie and Strain, 1999). The total reducingpower is expressed as FRAP units. One FRAP unit isequal to 100mol/dm3 Fe2+. FRAP value was calcu-lated with the formula:

FRAP value =Asample/Astandard

where Asampleis the change in absorbance of the sampleand Astandardis the change in absorbance of the standard(100mol/dm3Fe2+) after 4 min incubation at 593 nm.

RESULTS AND DISCUSSION

The results obtained in this study are presented inTables 1 and 2.

The activities of all antioxidant enzymes were detectedin all investigated Equisetumspecies. The E. telmateiaSOD activity (620.2 U/mg protein) was observed inE. telmateia, as well as GPx (334.4 U/mg protein) andGSH-Px (1.322 U/mg protein) activities. However, CATactivity (48.85 U/mg protein) recorded in E. telmateiaphosphate buffer extract was small. The highest CAT

activity was observed in E. ramosissimum(325.6 U/mgprotein). It is obvious that deficit in one antioxidantenzyme activity can be substituted with other anti-oxidant enzymes (tajner et al., 2006a). The antioxidant


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Table 1. Antioxidant enzyme (SOD, CAT, GPx and GSH-Px) activities, reduced glutathione,

MDA, O2and OH quantities and protein, flavonoid, chlorophyll a, b and carotenoid content

in above ground parts of differentEquisetumspecies

Equisetum Equisetum Equisetum

Biochemical parameter arvenseL. ramosissimumL. telmateiaL.

SOD (U/mg protein) 366.1 5.42 118.5 1.74 620.2 11.2

CAT (U/mg protein) 16.28 0.71 325.6 6.56 48.85 2.52

GPx (U/mg protein) 192.9 2.74 231.5 1.23 334.4 3.51

GSH-Px (U/mg protein) 0.775 0.042 0.945 0.030 1.322 0.089

GSH (mol/mg protein) 2.263 0.28 3.516 0.33 8.584 0.14

Total flavonoids (mg/g) 0.905 0.061 1.751 0.092 2.846 0.120

MDA (nmol/mg protein) 139.5 4.73 81.78 1.85 66.16 1.26

O2 (nmol/mg protein) 1679 63.7 420.2 12.2 118.5 8.40

OH (nmol/mg protein) 123.6 2.32 75.66 0.91 60.20 0.13

Protein (mg/g) 3.337 0.31 14.28 3.83 17.13 1.62

Chlorophyll a (mg/g) 1.580 0.040 0.708 0.022 1.501 0.062

Chlorophyll b (mg/g) 0.579 0.021 0.208 0.018 0.574 0.033

Carotenoids (mg/g) 1.011 0.067 0.243 0.010 0.424 0.012

All determinations were made in triplicate. The values are expressed as mean standard error.

Table 2. Total antioxidant capacity measured by FRAP method and scavenger effect meas-

ured by ESR (data from tajner et al., 2006b.Fitoterapia77: 601604)

Ferric reducing antioxidant

power (FRAP units; ESR

Equisetum species 1 Unit =100MFe2+) (Scavenger effect, %)

Equisetum arvenseL. 2.85 0.45 73.5

Equisetum ramosissimumL. 5.44 0.72 97.8

Equisetum telmateia L. 44.1 2.11 98.9

Determination of ferric reducing antioxidant power was made in triplicate. The values are

expressed as mean standard error.

defense may have been successful partly because ofcorrelated responses in activities of all antioxidantenzymes capable of scavenging reactive oxygen species(Lee et al., 2001). The highest total flavonoid content(2.846 mg/g) and reduced glutathione quantity (8.584mol/mg protein) were also recorded inE. telmateia wherethey act as powerful antioxidants (Wang et al., 2000).

E. telmateia possessed the lowest quantities of O2

(118.5 nmol/mg protein), OH (60.20 nmol/mg protein)and MDA (66.16 nmol/mg protein) where the scaveng-ing system against oxygen free radicals is the mosteffective. The results showed that E. arvense wasexposed to greater oxidative stress than the other twoEquisetumspecies. According to the results, high lipidperoxidation is caused mainly by OH and O2

action.MDA, being the main end-product of lipid peroxidation,can cause the cross-linking of membrane components.It can also react with free amino groups in protein andDNA bases explaining the mutagenic and genotoxicpower of MDA (Halliwell and Gutteridge, 1984). Thevalues obtained by ESR investigation show that E.telmateiacontain potent natural scavengers of hyperac-tive HOradical.

The ESR spectra of DMPO-OH radical adducts inthe presence of Equisetum-sphosphate buffer extracts

are presented in Figs 1, 2 and 3.The scavenger effect of E. telmateiaphosphate buffer

extract of 98.9%, detected by ESR, is higher than inmany other plants already investigated by this method

Figure 1.ESR spectra of DMPO-OH radical adducts in the pres-ence of Equisetum arvense L. phosphate buffer extract (datafrom 7tajner et al., 2006b. Fitoterapia77:601604).

(tajner et al., 2002, 2006a). The total antioxidant capa-city of E. telmateia extract also indicates expressiveantioxidant ability of this plant. These results agree

with high radical scavenging enzyme activities and highflavonoid, protein and pigment contents in E. telmateiaas well as low levels of MDA, O2

and OH (Tables 1and 2).


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EQUISETUM ARVENSEL AS SOURCE OF NATURAL ANTIOXIDANTS 549


Figure 2.ESR spectra of DMPO-OH radical adducts in the pres-ence of Equisetum ramosissimumL. phosphate buffer extract(data from 7tajner et al., 2006b. Fitoterapia77:601604).

Figure 3.ESR spectra of DMPO-OH radical adducts in the pres-ence of Equisetum telmateiaL. phosphate buffer extract (Datafrom 7tajner et al., 2006b. Fitoterapia77:601604).

The results obtained in this study demonstrated thatE. telmateia phosphate buffer (pH 7) extract showedbetter antioxidant properties than the other two inves-tigated Equisetumspecies. E. ramosissimumphosphatebuffer extracts also showed better antioxidant activitythanE. arvense. A search of plant sources may bringnew natural products into food, pharmaceutical andcosmetic industries with safer and better antioxidants(Dillard and German, 2000). Raising interest amongscientists has been directed toward the isolation of natu-ral antioxidants from botanical sources. Crude extractsof herbs rich in flavonoids and polyphenolics are ofthe special interest to the pharmaceutical, cosmetic and

food industries because they have the capacity toincrease oxidative degradation of lipids and proteinsimproving safer and healthy products (Loliger, 1991).It is believed that E. telmateiacontains powerful naturalantioxidants and that the our investigation is a startingpoint for further phytochemical investigations of thisplant.

Acknowledgements

This research is part of the project No. 142036B, which is financiallysupported by the Ministry of Science and Environmental Protectionof the Republic of Serbia.

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