Comparison of some antioxidant properties of plant extractsfrom Origanum vulgare, Salvia officinalis, Eleutherococcussenticosus and Stevia rebaudiana

Ladislav Vaško & Janka Vašková & Andrea Fejerčáková &

Gabriela Mojžišová & Janka Poráčová

Received: 15 October 2013 /Accepted: 17 March 2014 / Editor: T. Okamoto# The Society for In Vitro Biology 2014

Abstract Phenolic compounds from plants are known fortheir antioxidant properties and have been proposed as thera-peutic agents to counteract oxidative stress. However, undernormal circumstances, the body only receives a very smallamount of these substances in the diet. We have investigatedthe effect of extracts from known and frequently used plants aspart of diet, food seasoning, medicinal tea, and sweetener atdifferent concentrations on the ability to scavenge free radi-cals, to affect antioxidant enzymes, and finally in the survivalof cancer cell lines. We found extract concentrations of about100 μg.ml−1 more indicative in the assessment of all param-eters investigated. Ginseng possessed a very good ability toscavenge superoxide and hydroxyl radicals, while stevia alsomanifested significant effects against hydroxyl radicals. Bothextracts also showed NO decomposition ability. The antioxi-dant defense system against the excessive production of rad-icals in mitochondria was sufficient. In contrast, the range ofoperating concentrations for sage and oregano mainly present-ed no significant effects against reactive oxygen and nitrogenspecies. Taken together with the significantly reduced activityof glutathione peroxidase, this led to the depletion of glutathi-one. The demonstrated modulation of redox state capabilitywas sufficient to affect the viability of all tested cancer cell

lines, but especially A-549, CEM and HeLa by oreganoextract. Results support the promising role of the tested ex-tracts as a source of compounds for further in vivo studies withthe ability to powerfully interfere with or modify the redoxstate of cells according to the type of disease, which is expect-ed to be associated with oxidative stress.

Keywords Oregano . Radicals . Sage . Siberian ginseng .

Stevia

Introduction

Essential oils from aromatic and medicinal plants have beenknown to possess biological activity, such as antimicrobialand antioxidant properties (Bakkali et al. 2008), and efficacyin retarding the process of lipid oxidation (Silva et al. 2010).Oregano vulgare (L.) is an important aromatic plant widelyused in many countries for seasoning foods (Quiroga et al.2013). Oregano is used in folk medicine to treat respiratorydisorders, dyspepsia, painful menstruation, rheumatoid arthri-tis, scrofula, and urinary tract disorders (Gruenwald et al.2000). Salvia officinalis (L.), known as sage, comes fromEurope. Apart from biological activities such as antioxidantproperties (Bandonienė et al. 2002), sage is known for itssoothing and carminative effects (Schhultz et al. 1998). Inaddition, it has been reported that sage exhibits CNS acetyl-choline receptor activity, with both nicotinic and muscarinicbinding properties (Wake et al. 2000). Eleutherococcussenticosus (Rupr. and Maxim.) Maxim. (Siberian ginseng),native to the eastern areas of the Russian taiga and the northernregions of Korea, Japan, and China (Boon and Smith 1999), isprimarily known as an adaptogen. This term suggests thatsuch a plant possesses four general properties: it is harmlessto the host; it has a general, rather nonspecific effect; itincreases the resistance of the recipient to a variety of physical,

L. Vaško : J. Vašková (*) :A. FejerčákováDepartment of Medical and Clinical Biochemistry, Faculty ofMedicine, Pavol Jozef Šafárik University, Tr. SNP 1, 040 66 Košice,Slovak Republice-mail: janka.vaskova@upjs.sk

G. MojžišováDepartment of Experimental Medicine, Faculty of Medicine, PavolJozef Šafárik University, Tr. SNP 1, 040 66 Košice, Slovak Republic

J. PoráčováDepartment of Biology, Faculty of Humanities and Natural Science,University of Prešov, 17th November Street 1, 081 16 Prešov,Slovak Republic

In Vitro Cell.Dev.Biol.—AnimalDOI 10.1007/s11626-014-9751-4

chemical, or biological stressors; and that it acts as a generalstabilizer/normalizer for the user (Davydov and Krikorian2000). Stevia rebaudiana (Bert.) is a plant of the Compositaefamily, native to Paraguay, Brazil, and Central America. It hadbeen used for centuries as both a sweetener and a medicine.The leaf is many times sweeter than refined sugar but containsno carbohydrates or calories (Li et al. 2004). There has beenconsiderable interest in understanding the effects and mecha-nisms of the typical low-dose (submicromolar) concentrationsof phytochemicals as the relatively low amount of phytochem-icals typically consumed is unlikely to achieve direct antiox-idant (micromolar) concentrations in cells. Studies with ex-perimental models of cancer (Soobrattee et al. 2006), cardio-vascular disease (Wu et al. 2004), and neurodegenerativedisorders (Mattson and Cheng 2006) have provided evidencethat at least some phytochemicals exert beneficial effects byactivating adaptive stress response signaling pathways. Thepathways typically involve the activation of kinases and tran-scription factors, resulting in the increased production ofcytoprotective proteins including phase 2 enzymes, antioxi-dant enzymes, heat-shock proteins, growth factors, and pro-teins involved in the regulation of cellular energy metabolism(Mattson and Cheng 2006). In response, cancer cells contin-ually exposed to adverse conditions such as nutrient limita-tion, hypoxia, oxidative stress, and host defense may adapt tothis selection pressure by acquiring robust and elaborate sur-vival mechanisms (Kitano 2004). The emergence of vigorouscancer phenotypes is associated with comprehensive energeticand metabolic reprogramming (DeBerardinis et al. 2008). It isimportant to remember that reactive oxygen species (ROS)may elicit cellular responses ranging from proliferation to celldeath (Halliwell 2007). Consequently, efforts in preventingcancer through use of antioxidants and anticancer treatmentmodalities based on pro-oxidant effects are equally welljustified.

The production of ROS by mammalian mitochondria isimportant because it underlies oxidative damage in manypathologies and contributes to retrograde redox signaling fromthe organelle to the cytosol and nucleus (Murphy 2009).Although many purified mitochondrial proteins can be ma-nipulated so as to produce (O2

·–), the physiological relevanceof this is limited. Therefore, it is important to understand moreabout the production of O2

·– within isolated mitochondriaunder conditions which mimic those that may arise in vivounder physiological or pathological conditions. One constraintof using isolated mitochondria is that the system is complicat-ed and difficult to manipulate (Cochemé and Murphy 2008).The production of O2

·– within the mitochondrial matrix, inter-membrane space, and outer membrane leads to the formationof hydrogen peroxide (H2O2). Some O2

·– can react directlywith nitric oxide (NO·) to form peroxynitrite (ONO2

–)(Murphy 2009). Since mitochondria are the major sites of freeradical generation, they are highly enriched with antioxidants

including GSH and enzymes, such as superoxide dismutase(SOD) and glutathione peroxidase (GPx), which are presenton both sides of their membranes in order to minimize oxida-tive stress in the organelle (Cadenas and Davies 2000).

The aim of this study was to determine the ability ofdifferent concentrations of plant extracts from oregano, sage,ginseng, and stevia to scavenge four types of ROS: O2

·–, NO·,ONO2

–, and hydroxyl radicals (·OH). Subsequently, wefollowed the activities of antioxidant enzymes and reducedglutathione levels on isolated mitochondria as respiratory-metabolic centers of cells but also as appropriate containmentsystems that can modulate their own production of ROS underthese natural extracts. Then, we used the compounds in cancercell line culture to determine whether a change in the redoxconditions is sufficient to affect metabolic aberrations, affect-ing viability of cancer cells in vitro.

Material and Methods

All chemicals used were of analytical grade. Solutions werefreshly prepared using redistilled water. Dried root ethanolicextract from Siberian ginseng (E. senticosus) and extractsfrom leaves of stevia (S. rebaudiana), essential oils fromoregano (O. vulgare), and sage (S. officinalis) were kindlypurchased from Calendula ojsc (Stará Ľubovňa, Slovakia).

The antioxidant properties of plant extracts against O2·–

were evaluated by the method of Bauchamp and Fridovich(1971). The reaction mixture contained 8.7 ml of50 mmol l−1 phosphate-buffered solution (pH 6.5 and then7.4) with 0.1 mmol l−1 EDTA, 13 mmol l−1 L-methionine,and the tested extract at final concentrations of 5, 25, 50, 75,and 100 μg ml−1 (PBS). Riboflavin and nitro-blue tetrazoli-um (NBT) were added last. The resultant mixture was thenexposed to UV light for 10 and 20 min. The absorbance ofthe solutions in the presence and the absence of the testedsubstance was determined at 450 and 560 nm before andafter UV illumination. All measurements were taken intriplicate. The percentage inhibition of O2

·– generation wasevaluated by comparing the absorbance values of the controland experimental tubes and was calculated according to thefollowing equation: (%) = [(Acontrol−Asample)/Acontrol] × 100.

OH was generated by the Fenton reaction system, and thescavenging capacity toward ·OH was measured by using thedeoxyribose method (Halliwell et al. 1987). The reactionmixture contained 2-deoxy-D-ribose (9 mmol l−1) dissolvedin 30 mmol l−1 PBS (pH 6.5 and 7.4) containing 40 mmol l−1

NaCl, 30 mmol l−1 ammonium iron(II) sulfate hexahydrate,50 mmol l−1 H2O2, and plant extract sample at final concen-trations of 5, 25, 50, 75, and 100 μg ml−1. The mixture waskept in a water bath at 37°C for 10 min. After incubation, asolution of 0.5 ml thiobarbituric acid reagent (1%), TBA

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dissolved in 50 mmol l−1 NaOH followed by 0.5 ml 5.6%trichloroacetic acid was added to the reaction mixture. Themixture was then heated to 100°C for 8 min and then cooleddown with water. The absorbance of the solution was mea-sured spectrophotometrically at 532 nm. The ·OH scavengingcapacity was evaluated with the inhibition percentage of 2-deoxyribose oxidation on hydroxyl radicals. The scavengingpercentage was calculated according to the following formula:(%) = [A0−(A1−A2)]×100/A0, where A0 is the absorbance ofthe control without a sample. A1 is the absorbance after theaddition of the sample and deoxyribose. A2 is the absorbanceof the sample without deoxyribose.

NO was determined indirectly through the detection of itsproducts, nitrites and nitrates (Miranda et al. 2001). In aque-ous phase, free of biological material, NO exclusivelyautooxidizes to nitrite (Tsikas 2007), so that nitrite only deter-mination was performed without need to reduce nitrates priorto the assay. One milliliter of the test substance was added to2 ml of 10 mmol l−1 sodium nitroprusside solution at differentconcentrations dissolved in PBS (pH 7.4). The mixture wasincubated for 150 min. at 25°C in the dark. Then, 1 ml of themixture was mixed with 1 ml Greiss reagent (1% sulfanilicacid in 5% H3PO4 and 0.1%N-(1-naphthyl) ethylenediaminedihydrochloride). The absorbance of the chromophore formedwas detected at a wavelength of 546 nm.

ONO2–was prepared according toWhiteman and Halliwell

(1996) by mixing H2O2 (1 mol l−1) and KNO2 (1 mol l−1) inthe ratio 1:1 (w/w). Then two volumes of NaOH (1.5 mol l−1)were added. Themixture was freeze-dried at −20°C overnight.A working solution of ONO2

- was obtained by diluting it inPBS (pH 7.4). ONO2

– concentration was determined by spec-trophotometry at 302 nm.

Male Wistar rats weighing 200–250 g were used. Theanimals were sacrificed by cervical decapitation according toprocedures approved by the Animal Care and Use Committeeat Pavol Jozef Šafárik University in Košice. Liver mitochon-dria were isolated using the method of Fernández-Vizzaraet al. (2010). Mitochondrial protein yield was quantified usingthe bicinchoninic acid assay. The measurements were carriedout in a respiratory medium (80 mmol l−1 KCl, 300 mmol l−1

KH2PO4, 300 mmol l−1 K2HPO4, 15 mmol l−1 TRIS–HCl,6 mmol l−1 MgCl2, 0.78 mmol l−1 EDTA, pH 7.4), where thetested extracts were diluted to final concentrations of 125 and62.5 μg ml−1. Enzyme activities in control samples weremeasured without the addition of extracts. The activity ofglutathione reductase (GR; E.C.1.6.4.2) was measured ac-cording to a modified method described by Carlberg andMannervik (1985); that of glutathione peroxidase (GPx, E.C.1.11.1.9) was measured as described by Flohe and Gunzler(1984) and that of superoxide dismutase (SOD, E.C. 1.15.1.1)by means of the SOD-Assay Kit-WST (Fluka, Japan) follow-ing the user manual provided, and calculated per milligram ofmitochondrial proteins (mg/P). Reduced glutathione (GSH)

content was measured by the modified method of Floreaniet al. (1997) using Ellman’s reagent (R2=0.9984).

The following human cancer cell lines were used for thisstudy: HeLa (cervical carcinoma cells), CEM (acute T-lymphoblastic leukemia), MCF-7 (breast cancer cells),A-549 (carcinomic human alveolar basal epithelial cells),MDA (human breast adenocarcinoma cells), and Caco 2 (ep-ithelial colorectal adenocarcinoma cells). All cell lines usedwere kindly provided by Dr. Hajduch (Olomouc, CzechRepublic, originally purchased from American Type CultureCollection (ATCC)). HeLa and CEM cells were cultured inRPMI 1640 medium (PAA Laboratories, Pasching, Austria).MCF-7, A-549, MDA, and Caco 2 were cultured in growthmedium consisting of high-glucose Dulbecco’s modified Ea-gle medium. Both media were with Glutamax supplementwith 10% fetal calf serum, penicillin (100 IU ml−1), andstreptomycin (100 μg ml−1) (all from Invitrogen, Carlsbad,CA) in an atmosphere of 5% CO2 in humidified air at 37°C.Cell viability, estimated by trypan blue exclusion, was greaterthan 95% before each experiment.

The cytotoxic effects of tested extracts were studied by usingcolorimetric microculture assay with the MTT end point. Themethod is based on the conversion of the tetrazolium salt MTTin cells to insoluble formazan. Briefly, 8×103 cells were platedper well in 96-well polystyrene microplates (SARSTEDT,Nümbrecht, Germany) in the culture medium containing thetested extracts at concentrations of 1,000–3.9 μg ml−1. After72 h of incubation, 10 ml of MTT (5 mg ml−1) was added toeach well. After an additional 4 h, during which insolubleformazanwas produced, 100ml of 10% sodium dodecyl sulfatewas added to each well, and another 12 h was allowed for theformazan to be dissolved. The absorbance was measured at540 nm with the use of the automated MRX microplate reader(Dynatech Laboratories, Billinghurst, West Sussex, UK). Theabsorbance of the control wells was taken as 100%, and theresults were expressed as a percentage of the control.

Results

The ability to scavenge O2·– Fig. 1Awas only 11% in the case

of oregano essential oil at concentrations of 5, 75, and100 μg ml−1 and remained almost unchanged. At 25 and50 mg ml−1, however, the ability to uptake fell below 5%.Sage demonstrated a very weak ability to scavenge O2

·–. Noability was shown at the lowest concentration tested. Theactivity only reached 11% at concentrations of 50 μg ml−1.The ability to scavenge O2

·– showed a slightly increasingtendency with increasing concentrations of ginseng, whilethe highest value reached around 18% at a concentration of75 μg ml−1. A further increase resulted in a decrease in O2

·–

scavenging activity. Stevia, like oregano, showed activityeven at a concentration of 5 μg ml−1 (13.54%). This

ANTIOXIDANT PROPERTIES OF PLANT EXTRACTS

capability, however, was not affected by further increase in thestevia concentration, up to 50 μg ml−1. With further increasesin concentration, however, activity declined.

Nitrite production from NO was higher than 25% withoregano essential oil at the lowest concentration used when

compared to the control Fig. 1B. This declined to below 2% asthe concentrations increased. An even higher percentage ofNO conversion (37.8%) was observed at the lowest concen-tration of sage. The activity of essential oil from sageplummeted below 5% even at a concentration of50 μg ml−1, then increased slightly, but not exceeding 11%at 100 μg ml−1. Ginseng had the highest NO conversionability, with a percentage of almost 54% even at the lowestconcentration. The NO conversion capacity of ginseng did notdecrease substantially as the concentration increased, andremained at 40%. Stevia extract also demonstrated a remark-able ability to convert NO. At concentrations of 25, 50, and75μg ml−1, it was as high as 42–45%.With further increase inconcentration, the percentage conversion decreased only to32.31%.

ONO2– concentration in solution only decreased with oreg-

ano extract at the lowest concentration used Fig. 1C. Theconcentration of ONO2

– rose with increasing extract concen-tration and therefore cannot be evaluated as the ability ofinhibition Fig. 2. Sage extract was able to reduce the concen-tration of ONO2

– better at lower concentrations used, alsowith a declining trend from 9.37% to 8.56%. The concentra-tions of sage from 75 to 100 μg ml−1 decreased the ability toconvert ONO2

– from 6.94% to only 2.36%. Ginseng extractshowed a very low ability to transform ONO2

– (around 5%) atconcentrations of 25 and 50 μg ml−1 Fig. 1C. The concentra-tion of ONO2

– in solutions of stevia had upward trend, whichis also shown in Fig. 2.

It was observed (Li 2013) that the organic solvents (espe-cially alcohols) normally used to prepare sample solutionsmay cause strong interference. In the deoxyribose degradationassay, any organic solvent should be completely evaporatedbefore measurement, or the result will be meaningless. There-fore, measurements of hydroxyl radical scavenging ability ofthe extracts form oregano and sage were not provided. Theextract from ginseng exhibited a hydroxyl radical scavengingability of almost 20% at a concentration of 50μgml−1 Fig. 1E.When using higher concentrations, the value reached 30%.

Figure 1. The percentage of inhibition (A) superoxide anion radical, (B)nitric oxide, (C) peroxynitrite, and (D) hydroxyl radical by tested plantextracts.

Figure 2. The percentage increase in the concentration of peroxynitritein treatment with extracts.

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The activity of stevia is very interesting, with an increasedscavenging capacity of 2.35%–9.8%–39.64% rising up to67.04%, the highest measured, with increasing concentrationsof extract.

The addition of the extracts caused no changes in mito-chondrial SOD activity (Table 1). Also, the activity of GPxunder stevia treatment showed no changes in comparison tocontrol. The activity of GPx (Table 1) was significantly de-creased from −75.26% to −88.66% after incubation withoregano extract and increased in sage from −90.72% to−71.13%. Ginseng showed a rather opposite effect, where anincrease in GPx activity (68%) was measured in comparisonto control. A much higher increase in GPx activity was re-corded at a higher concentration of ginseng (226.8%). At aconcentration of 62.5 μg ml−1, no significant effect was mea-sured on GR activity (Table 1). However, a significant reduc-tion in GR activity (61.9%) was measured only at a higherconcentration 125 μg ml−1 of oregano. The addition of oreg-ano at a concentration of 125 μg ml−1 caused a significantdecrease in reduced glutathione levels of 85.47% and, in thecase of sage, of 57.52%. The lower oregano concentrationused caused almost all GSH depletion. This was not the casewith sage, where the measured decrease was only 34.65%when compared to the control.

Survival of different cancer cells (HeLa, CEM, MCF-7,A-549, MDA, Jurkat T) exposed to 72 h incubation with thetested compounds and the IC50 values for each cell line areshown in Table 2. Oregano exhibited cytotoxicity against allcancer cell lines (IC50 29.1–109.7 μg ml−1). There was asignificant effect on the survival of cancer cell lines at con-centrations of 125 and 62.5 μg ml−1 (except on Caco2),achieved by binary dilution. Oregano had a surprising andsignificant impact on the viability of A-549, MCF-7, CEM,and HeLa at a concentration of 125 μg ml−1. The final con-centration of 125 μg ml−1 is also the lowest for sage in whichwe monitored the effect on survival of all cancer cell lines.Survival of A-549, CEM, and MCF-7 cell lines were

significantly affected. Further reduction in sage concentrationhad no effect on the survival of MCF-7 and Caco2 and only avery weak effect on HeLa, MDA, and A-549 (78.03–79.29%and 90.45%). Our data indicate that extracts from ginseng andstevia showed very little to no cytotoxic effect.

Discussion

In this work, we aimed to determine the antioxidant propertiesof known and frequently used extracts as a whole, primarilyfor their extensive use in everyday life, as an integral part ofhuman diet and in animal husbandry. The hypothesis ofMattson (2008) concluded that it is the antioxidant activityof phytochemicals that is responsible for their health benefitsat micromolar concentrations. However, clinical trials andprimary prevention studies of high doses of such antioxidantsin humans have been disappointing at best (Riccioni et al.2007). When testing the efficacy of growth inhibition inbacterial strains, antibiotics are active at concentrations of10 μg ml−1 and plant extracts at concentrations of100 μg ml−1, where they had a good potency level (Rioset al. 1988). To test the ability of extracts to scavenge freeradicals, we have therefore chosen a concentration range from5 to100 μg ml−1.

Those radicals derived from oxygen represent the mostimportant class of such species generated in living systems(Valko et al. 2004). Oregano showed a weak ability to scav-enge O2

·– (under 11%) and only at higher concentrations.Stevia demonstrated a better capacity (over 14%) at a lowerconcentration, while sage only showed this ability at a con-centration of 50 μg ml−1. Otherwise, its ability to scavengeO2

·– is very weak. In summary, from the results obtained,ginseng extract showed the best propensity to scavenge O2

·–

Fig. 1A.Mitochondria generate approximately 2–3 nmol of O2

·– permilligram of protein; the ubiquitous presence of which

Table 1. Mitochondria antioxidant enzymes activities: superoxide dismutase, glutathione peroxidase, glutathione reductase, and reduced glutathionecontent after treatment of tested plant extracts with final concentrations 125 and 62.5 μg ml−1

Dose (μg ml−1) Compound SOD (U/mg/P) GPx (U/mg/P) GR (kat/kg/P) GSH (nmol/mg)

125 Control 4.025±1.277 0.097±0.045 13.43±3.24 17.49±0.11

Oregano 3.307±0.078 0.024±0.013* 5.12±1.36** 2.54±0.76***

Sage 3.344±0.046 0.009±0.003** 11.41±1.34 7.43±1.37***

Ginseng 3.984±1.331 0.317±0.069** 12.57±1.23 37.83±3.83***

Stevia 4.875±1.289 0.096±0.063 17.07±3.52 63.09±1.24***

62.5 Oregano 2.953±0.413 0.011±0.001** 10.35±4.42 1.78±0.52***

Sage 3.224±0.526 0.028±0.003* 9.40±4.78 11.43±0.92***

Ginseng 3.859±0.609 0.163±0.024* 10.99±3.50 87.09±2.07***

Stevia 3.818±0.856 0.097±0.040 15.29±2.00 4.91±0.60***

*P<0.05; **P<0.01; ***P<0.001

ANTIOXIDANT PROPERTIES OF PLANT EXTRACTS

indicates that it is the most important physiological source ofthis radical in living organisms (Inoue et al. 2003). O2

·–

undergoes a dismutation reaction to H2O2 spontaneously andcan be accelerated about four orders in the matrix by Mn-SOD, and in the inter-membrane space by Cu, Zn-SOD (Hanet al. 2003). After the addition of extracts prepared for thetreatment with biological samples (first mitochondria and latercancer cell lines) by binary dilution, we found no significantchanges in the activity of SOD at all. Based on its fast catalyticactivity, the latter may represent the prevalent scavengingpathway for O2

·–. Because of a modest rate constant, thereaction between cytochrome c and O2

·– would representanother O2

·–-scavenging pathway in the inter-membranespace; however, the voltage-dependent anion channel allowsthe movement of O2

·– from the inter-membrane space to the

cytoplasmic surface of the outer membrane of mitochondria(Han et al. 2003).

H2O2 diffuses rapidly through membranes (Antunes andCadenas 2000), and the release of H2O2 from mitochondria tocytosol reflects the balance between H2O2 production andconsumption reactions, with the latter mainly involving thereduction of H2O2 to H2O via GPx. GPx activity significantlydecreased after the addition of oregano and sage at selectedconcentrations (Table 1). The activity of GPx is affected by thepresence of another antioxidant enzyme, GR, which continu-ously recycles the oxidized glutathione to the reduced state.However, GR activity was only significantly reduced at thehighest concentration of oregano. GSH levels were signifi-cantly reduced. In the case of oregano, it was also possible toconsider the total depletion. Unlike O2

.-, H2O2 is more stable

Table 2. Effect of selected extracts on viability (%) of different cancer cell lines

Dose (μg ml−1) HeLa CEM MCF-7 A-549 MDA Caco 2

Origanum vulgare 1,000 0.05 0 0 0 0 0

500 0.04 0 0 0 0 0

250 0.66 0 0 0 0 0

125 2.06 1.31 0.60 0 6.22 33.26

62,5 17.41 4.85 17.56 18.39 37.30 100

31.2 42.22 34.78 80.25 6..49 65.45 100

15.6 100 100 96.15 68.22 92.06 100

7.8 100 100 100 78.40 100 100

3.9 100 100 100 82.80 100 100

IC50 29.1 27.56 45.821 42.757 47.83 109.07

Salvia officinalis 1,000 0.48 0.12 0 0 0.10 0.32

500 0.47 0 0 0 0.27 0.69

250 4.97 4.77 2.10 0 1.82 1.95

125 66.31 33.83 41.54 26.85 7.,34 80.04

62,5 78.03 46.21 100 90.45 79.29 100

31.2 87.11 76.27 100 90.54 82.94 100

15.6 100 100 100 100 100 100

IC50 158.24 57.68 115.81 101.89 165.79 173.09

Eleutherococcus senticosus 1,000 29.07 0 23.45 24.89 9.71 9.19

500 64.58 9.42 93.87 78.96 59.55 100

250 89.58 47.68 98.76 91.05 89.43 100

125 91.31 100 100 92.10 94.80 100

62,5 92.56 100 100 100 86.64 100

31.2 95.85 100 100 100 87.39 100

IC50 705.29 244.46 811.49 767.8 595.81 775.3

Stevia rebaudiana 1,000 90.53 4.85 15.57 26.38 13.59 6.34

500 93.30 26.96 100 100 60.89 79.25

250 92.39 100 100 100 80,31 85.69

125 100 100 100 100 89.86 100

62,5 100 100 100 100 93.19 100

31.2 100 100 100 100 95.28 100

IC50 >1,000 421.14 796.1 839.58 615.12 700.59

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and is able to oxidize key thiol residues on proteins and low-mecular-weight thiolating agents such as GSH (Bienert et al.2006). The addition of stevia showed no changes in theactivities of GPx and GR, although the levels of GSH pointedto significant changes. GSH levels were several times higherthan that in controls, and there was an observed decrease asthe treatment concentration decreased. The effect of ginseng atboth concentrations caused a significant increase in the activ-ity of GPx, though glutathione levels were significantly raisedcompared to control, without changes in the activity of GR.These findings seem interesting, because mitochondria do notsynthesize GSH but rather transport and accumulate up toapproximately 15% of the total cellular GSH (Schafer andBuettner 2001). The chemical composition of the biologicalenvironment is of foremost importance, including the concen-tration of NO, or reactive nitrogen oxide species (RNOS)derived from NO (Wink and Mitchell 1998), and cellulartargets. Under conditions of excess NO, nitrosative modifica-tions of biomolecules can occur either through oxidation byNO2 followed by nitrosylation with excess NO or via N2O3

formation. The concentration of NO decomposition products,e.g., oxidative end-product nitrite and nitrate, is reported to bea reasonable approximation of NO levels (Ridnour et al.2004). Oregano and sage extracts alone showed only a weakability to increase the concentration of nitrites, but stevia andespecially ginseng reached more than 40% efficiency at con-centrations in the range of 50–100 μg ml−1. Under conditionsof excess nitrite, it has been suggested that heme proteinsmediate substrate nitrosation through catalytic production ofN2O3 (Ford and Lorkovic 2002). This mechanism may beresponsible for nitrosation of low-molecular-weight thiols andamines. It should be noted that many of the enzyme motifssuch as amines, thiols, and zinc fingers are not inhibited bytrans-nitrosation. Thus, modification of molecular motifs dur-ing nitrosative stress may occur only under conditions ofexcess NO and through the intermediacy of RNOS. This couldalso lead to an explanation of the significant differences inmeasured levels of GSH under the treatment of mitochondriawith different extracts.

O2·– generated toward the inter-membrane space may play

an important role in nitration through ONO2– formation when

NO is present (Han et al. 2003). ONO2– can oxidize thiols to

either thyil radicals or to sulfenic acids, leading to proteinglutathionylation (Hurd et al. 2005). The ability of ginsengand sage to scavenge ONO2

– was very low. The presence oforegano or stevia to the contrary led to an increase in ONO2

–

concentration. It is important to note that the reaction of NOwith ONO2

– forms N2O3, which may result in nitrosativestress (Wink and Mitchell 1998). While NO does not interactdirectly with thiols, N2O3, formed by autooxidation or fromacidified nitrite, has a high affinity for thiol-containing pep-tides (Ridnour et al. 2004) and may cause the observed deple-tion of GSH.

The most chemically active species primarily associatedwith mitochondria, microsomes, and peroxisomes (·OH) aregenerated by the Fenton reaction from H2O2 or by thedecomposition of ONO2

– itself (Valko et al. 2005). Due tothe nature of extracts of oregano and sage, it was notpossible to measure the uptake ability of ·OH by theseextracts. The ability of ginseng was very good, around30% and, surprisingly, stevia expressed the best activitytoward this highly reactive radical. GPx catalyzes the con-version of hydrogen peroxide or peroxide to water or thecorresponding alcohols indirectly. As both extracts increasedGPx activity, thereby suggesting a lower probability ofradical formation, we could express the assumption thatthere were no oxidative or nitrosative stress conditions inmitochondria treated with these two extracts.

Since mitochondrial ROS production is modulated in avariety of nontransformed cells, it may also affect cancercells in which ROS levels are characteristically high (Kitano2004). We have found that the viability of all cancer celllines is affected at concentrations above and below100 μg ml−1 using extracts from oregano and, to a lesserextent, sage. Based on our previous findings about theactivities of antioxidant enzymes in mitochondria and prop-erties of extracts against reactive species, we can yet con-sider several options that would result in a reduction ofcancer cell survival under oregano and sage treatment beingdescribed in the range of effective concentrations. All ofthem could lead to GSH depletion and affect the mitochon-drial redox state. Firstly, through possible oxidation by ex-cess peroxides due to lack of GPx activity. Chemical deple-tion of GSH significantly enhanced the cytotoxicity of NOor NO donors (Wink et al. 1994). Secondly, it may actthrough a nitrosative (other nitrosated species) or oxidativemechanism (oxidative nitrosylation). Nitrosative stress canmodulate the dual pathways that lead to pro- and anti-apoptosis, depending on the redox state and transition metalcomplexes within the cells (Kim et al. 2002). Thirdly, wefound very little activity in these extracts in NO decompo-sition and potentiation rather than quenching of ONO2

–.This therefore increases the likelihood of such cyclical ·OHor N2O3 formation, which further suggests conditions ofsignificant RNOS formation.

In summary, the results obtained indicate that the selectedextracts achieve a good potency level at about 100 μg ml−1

but with a different overall effect. While ginseng and steviaexhibit antioxidant properties with increasing concentrations,oregano and sage tend to change the redox state. This mightbe helpful in preventing or slowing the progress of variousoxidative stress-related diseases. It is first necessary to verifytheir effect on in vivo studies, because the effective dosecould still be achievable despite the undeniably strong taste,which under natural conditions, is a natural barrier to in-creased intake.

ANTIOXIDANT PROPERTIES OF PLANT EXTRACTS

Acknowledgments The study was financially supported by the SlovakGrant Agency for Science VEGA no. 1/1236/12, VEGA no. 1/0751/12,and partially supported by the Agency of the Slovak Ministry of Educa-tion for the Structural Funds of the EU, under project ITMS:26220220104 (10%), ITMS: 26220120058 (10%) and ITMS:26220220152 (10%).

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