Inhibition of myeloperoxidase and antioxidative activity of Gentiana lutea extracts

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Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 191– 196

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al h om epage: www.elsev ier .com/ locate / jpba

nhibition of myeloperoxidase and antioxidative activity of Gentiana luteaxtracts

ranislav Nastasijevic a,∗, Tamara Lazarevic-Pasti a, Suzana Dimitrijevic-Brankovic b, Igor Pasti c,na Vujacic a, Gordana Joksic a, Vesna Vasic a

Department of Physical Chemistry, Vinca Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001 Belgrade, SerbiaDepartment of Bioengineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11120 Belgrade, SerbiaFaculty of Physical Chemistry, University of Belgrade, Studentski Trg 12-16, 11158 Belgrade, Serbia

r t i c l e i n f o

rticle history:eceived 27 January 2012eceived in revised form 27 March 2012ccepted 28 March 2012vailable online 6 April 2012

eywords:

a b s t r a c t

The aim of this study was to investigate the inhibitory activity of Gentiana lutea extracts on the enzymemyeloperoxidase (MPO), as well as the antioxidant activity of these extracts and their correlation withthe total polyphenol content. Extracts were prepared using methanol (100%), water and ethanol aqueoussolutions (96, 75, 50 and 25% v/v) as solvents for extraction. Also, isovitexin, amarogentin and gentiopi-croside, pharmacologically active constituents of G. lutea were tested as potential inhibitors of MPO.Antioxidant activity of extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scav-

yclic voltammetryPPH testentiana luteaentiopicosideyeloperoxidase

enging test and also using cyclic voltammetry (CV). Among all extracts, the antioxidant capacity of 50%ethanol aqueous extract was the highest, both when measured using the DPPH test, with IC50 = 20.6 �g/ml,and when using CV. Also, 50% ethanol extract, showed the best inhibition of MPO activity in comparisonwith other extracts. In the group of the selected G. lutea constituents, gentiopicroside has proved to bethe strongest inhibitor of MPO, with IC50 = 0.8 �g/ml. Also, the concentration of G. lutea constituents were

s, usin

PLC determined in all extract

. Introduction

Gentiana lutea belongs to family of Gentianaceae, floweringlants comprising approximately 70–80 genera and 900–1200pecies. It is a perennial herb commonly growing in mountainousreas of central and southern Europe and in western Asia [1,2]. Itrows naturally on uncultivated ground in France, Spain and thealkan mountains [3]. Gentian radix is an official drug in many phar-acopoeias and it is present commercially in the form of dried

ermented rhizomes and roots. The active principles of Gentianaossess different pharmacological activities, ranging from anti-

nflammatory to diuretic [1,4]. These properties can be attributedainly to secoiridoid bitter compounds, such as gentiopicroside,

marogentin, swertiamarin and group of xanthones such as iso-itexin and isogentisin [5–7]. It was shown in animal studies thatoots of G. lutea demonstrate antihelmintic properties, while in rats,ntragastric and intraduodenal administration of gentian extractstimulates bile production [8–10].

In terms of enzyme inhibition G. lutea extracts and some of itsonstituents (isogentisine and isovitexin) showed inhibitory effectsn monoamine oxidase type A and B and xanthine oxidase [6,11,12].

∗ Corresponding author. Tel.: +381 11 3408 872; fax: +381 11 8066 434.E-mail address: [email protected] (B. Nastasijevic).

731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jpba.2012.03.052

g Ultra Performance Liquid Chromatography (UPLC).© 2012 Elsevier B.V. All rights reserved.

However, taking into account the anti-inflammatory properties ofGentiana extracts, it was desirable to explore its inhibitory effectson MPO activity. MPO (EC 1.11.2.2.) is a peroxidase enzyme releasedduring degranulation of neutrophils and monocytes. Through cat-alyzing chloride ion oxidation to hypochlorous acid, MPO enablesmicrobicidal action of neutrophils. However, in contrast to its ben-eficial activity, MPO also contributes to the development of manydisorders, such as cardiovascular, inflammatory, neurodegenera-tive and immune-mediated diseases. Its impact on the processof athrogenesis, as well its role in the pathogenesis of lung can-cer, Alzheimer’s disease and multiple sclerosis has been previouslyreported [13–17].

MPO catalyzes the oxidation of various substrates with hydro-gen peroxide (H2O2), producing oxidizing and halogenating agents[18,19]. Numerous nonsteroidal and anti-inflammatory drugs, ani-lines, and phenols inhibit MPO reversibly [20]. Therefore, alongsidetesting the extracts, some of G. lutea constituents were alsoinvestigated as potential inhibitors of MPO. For these purposes,gentiopicroside, amarogentin and isovitexin were selected, sincethey possess pharmacological activity.

For determining the antioxidative capacity of extracts, the DPPH

scavenging test and CV were used. Thus, the results obtainedusing different antioxidative tests could be correlated to each otheras well as with the total polyphenol content, with great accu-racy. The content of total polyphenols was also determined, since

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92 B. Nastasijevic et al. / Journal of Pharmaceu

umerous polyphenols, belonging to the class of iridoids, secoiri-oids, xanthones and flavonoids are present in G. lutea root [21].

. Materials and methods

.1. Chemicals

Gentiopicroside (5-Ethenyl-6-(�-d-glucopyranosyloxy)-5,6-ihydro-1H,3H-pyrano[3,4-c] pyran-1-one), amarogentin (2-aS-(4a�,5ß,6�))-3,3′,5-Trihydroxy-(1,1′ - biphenyl)-2- carboxy-

ate) and isovitexin (6-ß-d-Glucopyranosyl-5,7-dyxidroxy-2-(4-ydroxyphenyl)-4H-1-benzopyran-4-one) were purchased fromhromaDex (Irvine, USA). Acetonitrile and methanol were HPLCrade obtained from J.T. Baker (Deventer, Netherlands) and tri-uoroacetic acid (TFA) was spectrophotometric grade (Aldrich).PPH was obtained from Fluka Chemie AG Buchs (St. Louis, USA).olin–Ciocalteu reagent used for total polyphenol content wasought from AppliChem-Biochemica (Darmstadt, Germany) andallic acid from Sigma–Aldrich. MPO from human neutrophilsas obtained from Planta Natural Products (Vienna, Austria).

t was purified to an Rz (A430/A280) 0.84, and its concentrationas calculated using ε430 = 91,000 M−1 cm−1 per hemeHydrogeneroxide working solutions were prepared daily by diluting a0% (w/v) stock solution and concentration was determined using240 = 43.6 M−1 cm−1 [22]. O-dianisidine dihydrochloride wasurchased from Sigma–Aldrich (St. Louis, USA).

.2. Plant material and extracts preparation

G. lutea roots were purchased from the Institute of Medicinallant Research “Dr Josif Pancic”, Belgrade, Serbia. Five grams ofrounded Gentiana roots were extracted with methanol, ethanolqueous (96%, 75%, 50% and 25% v/v) solutions and water, in theatio 1:5 (w/v). Extraction with ethanol aqueous solutions andith methanol was performed at room temperature, for 48 h, with

ccasional shaking. Water extract was prepared by heating in aoiling water bath for ten minutes. After filtering through 0.45 �mlter (Millipore Co. Ltd.) extracts were concentrated using a rotava-or at 35 ◦C and stored in sample tubes until analysis, in a drylace.

.3. MPO inhibition

Peroxidase enzyme activity was monitored by the o-dianisidinessay at 25 ◦C. In the inhibition experiments, MPO (3 nM) wasixed with 50 mM phosphate buffer, pH 6.0. Extracts were incu-

ated for 10 and 15 min with MPO and in both cases five minutesn the presence 0.53 mM o-dianisidine as enzyme substrate. Theeaction was started by adding hydrogen peroxide, to a final con-entration of 0.15 mM in the final volume of 700 �l assay mixture.he temperature in the cell was kept at 25 ± 1 ◦C by means of

thermostated-controlled water bath. Reference measurementsere performed without inhibitors (control value). The spec-

rophotometric measurements were performed on a Perkin Elmerambda 35 UV–vis spectrophotometer, using 1 cm path lengthuvettes. Absorbance at 460 nm was monitored for 1 min. The activ-ty was determined from the linear slope of the progress curvesbtained with an extinction coefficient of ε460 = 11.3 mM−1 cm−1

22].

.4. UPLC separation of G. lutea extracts

Waters ACQUITY Ultra Performance Liquid ChromatographyUPLC) system coupled with a TUV detector controlled by thempower software was used. Extracts, as well gentiopicro-ide, were separated on an ACQUITY UPLCTM BEH C18 column

nd Biomedical Analysis 66 (2012) 191– 196

(1.7 �m, 100 mm × 2.1 mm) in gradient mode, with the mobilephase consisting of solvent (A) TFA (0.1% v/v in water) and (B)acetonitrile–methanol mixture (85:15 v/v) starting from 5% to 95%of B, within 7 min, at room temperature. Absorbance was measuredat 250 nm. The eluent flow rate was 0.3 ml/min, the injection vol-ume was 10 �l. All extracts were standardized to a concentration of0.1 mg/ml, and filtered through 0.22 �m nylon filter (Phenomenex)before injection.

2.5. DPPH radical-scavenging activity

The free radical scavenging capacity of the extracts, based onthe scavenging activity of the DPPH free radical was determinedby the method described by Brand Williams [23]. DPPH (7.8 mg)was dissolved in methanol (100 ml) to obtain a concentration of78 �g/ml. The following concentrations of extracts were prepared:0.1, 0.5, 1, 2.5, 5 and 10 mg/ml. Then, 50 �l of these extracts werediluted with methanol up to 4 ml, and then mixed with 1 ml ofDPPH (0.2 mM solution). After 30 min, absorbance was measured at� = 517 nm using a control prepared with water instead of extracts,against methanol as a blank. The inhibition of DPPH was calculatedas:

Inhibition(%) = 100 × ADPPH − Asample

ADPPH

where ADPPH is the absorption of the DPPH control solution againstthe blank, and Asample is the absorption of the extract againstthe blank. The extract concentration providing 50% of antioxidantactivity (EC50) was obtained by plotting the DPPH inhibition againstthe plant extract concentration.

2.6. Determination of total polyphenol content usingFolin–Ciocalteu method

The total content of polyphenolics was determined by a col-orimetric method described by Singleton and Rossi [24]. Extractswere diluted to a concentration of 1 mg/ml. Oxidation withFolin–Ciocalteu reagent (500 �l), was performed by mixing with100 �l of each extract solution, followed by the addition of 2 mlof 15% Na2CO3. Reaction mixtures were diluted up to 10 ml withdistilled water. Absorption was measured after 2 h at � = 750 nm,against a blank, prepared with water, using the same proceduresas for samples. Quantification of total polyphenols in the extracts,was made using gallic acid to establish a calibration curve, coveringa range from 2 to 200 �g/ml. The results were expressed as mg ofgallic acid equivalents per 100 g of dry weight of plant extract.

2.7. Cyclic voltammetry

CV measurements were performed at room temperature using aGamry PCI-4/750 system (Gamry, USA) in one-compartment three-electrode electrochemical cell with glass carbon (GC) disk (basesurface area 0.19625 cm2) as a working electrode, and a saturatedcalomel electrode (SCE) as a reference one. Pt foil was used as acounter electrode large area. Prior to each measurement the GCdisk was polished with diamond paste (1–2 �m particles) and thor-oughly washed with ethanol and deionized water. Reproducibilityof electrode preparation was confirmed by recording backgroundcurrents in inert electrolyte, namely 0.1 M K2SO4 solution, prior toeach measurement. Plant extracts (1 ml, concentration 20 mg/ml)were diluted in 0.1 M K2SO4 solution (10 ml) and these solutions

were subjected to electrochemical measurements. Cyclic voltam-mograms were recorded at a scan rate 100 mV s−1, in the potentialwindow between −0.4 and 1.2 V vs. SCE. Prior to and during eachexperiment solutions were purged with high-purity N2.

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Fig. 1. Inhibition of MPO activity (as a function of the time (A) and of inhibitor concentration (B)) by the: (A) Gentiana lutea extracts (0.01 mg/ml) in the presence of 53 mMo-dianisidine as substrate in: (a) 96% ethanol-aqueous, (b) water extract, (c) methanol, (d) 25% ethanol-aqueous, (e) 75% ethanol-aqueous, (f) 50% ethanol-aqueous extract;a o-dir ns obt

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three other mixtures. It is evident that the presence of gentiopi-croside in the mixtures leads to an increase in the degree (% fromcontrol) of inhibition.

Table 1Inhibition of MPO activity obtained with the single constituents and their mixtures:(I) Amarogentin and Isovitexin, (II) Amarogentin and Gentiopicroside, (III) Isovi-texin and Gentiopicroside and (IV) Amarogentin, Isovitexin and Gentiopicroside.Concentration of each constituent individually and each of them in the mixtureswas 0.001 mg/ml, within 10 minutes of incubation with enzyme.

Type of inhibitor MPO inhibition (% from control)

Amarogentin 49 ± 2Isovitexin 46 ± 2Gentiopicroside 65 ± 3

nd by (B) gentiopicroside; isovitexin and amarogentin, in the presence of 53 mManging from 1 × 10−5 to 1 × 10−3 mg/ml. IC50 values were calculated from equatiohree experiments performed in duplicate.

. Results and discussion

Investigation of MPO inhibition activity, antiradical activity andotal polyphenol content was directed mainly toward G. luteaxtracts which are traditionally used in folk medicine and as anuxiliary means of healing in alternative medicine. Consequently,xcept methanol extract, all other extracts were prepared withthanol or water as solvents, due their common usage in alcoholinctures, and alcoholic beverages, as well in the form of tea (waterecoctions).

.1. Inhibition of MPO activity

The influence of G. lutea extracts on MPO activity was inves-igated at a concentration of 0.01 mg/ml for each extract as theunction of the incubation time in the intervals of 15 min. Theesults, presented as percentage of inhibition of control are shownn Fig. 1A. They indicate that the percent of enzyme inhibitionncreased with incubation time, reaching a plateau after 15 minncubation. Among all the extracts tested, the most potent MPOnhibitors were 50% ethanol-aqueous extract and 75% ethanolxtract, since they induced almost 100% inhibition of the enzymectivity, respectively, after 15 min of exposure. However, resultsf MPO inhibition obtained for shorter incubation times enablelearer insight of the enzyme inhibition and revealed that 50%thanol extract is the strongest inhibitor of MPO among the extractsxamined. It inhibits 71% of MPO activity after 10 min of exposure,n contrast with 96% ethanol extract which inhibits only 31% of

PO activity for the same incubation time. Previous studies haveemonstrated that some anti-inflammatory drugs may inhibit MPOctivity. Therefore the inhibitory ability of Gentiana extracts towardPO leads to the conclusion that these extracts can be considered

o exert an anti-inflammatory effect [25]. This information may be

seful in the preparation process of products based on Gentianaoot for its traditionally use in folk medicine.

In addition to anti-inflammatory drugs with inhibition abilityoward MPO, numerous phenolic acids derivatives as quercetin,

anisidine. The inhibition curves of constituents were obtained for concentrationstained by sigmoidal curve fitting. The results present the mean values obtained in

curcumin, ferulic, caffeic and gallic have demonstrated a strongMPO inhibition [26]. Accordingly to that, it was desirable to exam-ine whether some Gentiana constituents would exhibit a similareffect. Hence, gentiopicroside, isovitexin and amarogentin wereselected as potential MPO inhibitors, since these pharmacologi-cally active compounds are usually present in G. lutea extracts.IC50 values, which represent the inhibitor concentrations thatinduced 50% inhibition of MPO activity, were obtained from theanalysis of sigmoid shaped inhibition curves. In the group of theselected compounds, gentiopicroside showed the highest levelof inhibition with IC50 = 0.8 ± 0.1 �g/ml, followed by isovitexin(IC50 = 2.2 ± 0.1 �g/ml) and amarogentin (IC50 = 2.4 ± 0.1 �g/ml).

In order to determine the contribution of the aforementionedconstituents to MPO inhibition, mixtures of gentiopicroside, iso-vitexin and amarogentin were investigated as inhibitors. Theresults obtained were compared with the results of MPO inhibi-tion obtained with each of these constituents separately and theyare shown in the Table 1. Results show that mixture of isovitexinand amarogentin is significantly less potent MPO inhibitor than the

I 18 ± 1II 44 ± 2III 46 ± 2IV 43 ± 2

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Fig. 2. UPLC chromatograms of Gentiana lutea extracts and gentiopicroside stan-dard: (a) 25% ethanol-aqueous, (b) 50% ethanol-aqueous, (c) 75% ethanol-aqueous,((e

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Table 2Antiradical activity of Gentiana lutea extracts obtained by the DPPH test and totalpolyphenol content of these extracts.a

Type of extract DPPH IC50

inhibition (�g/ml)Total polyphenolcontentb (mg/g)

25% ethanol 36.1 ± 0.6 78.1 ± 0.450% ethanol 20.6 ± 0.4 96.1 ± 1.275% ethanol 25.4 ± 0.9 74.4 ± 0.696% ethanol 82.4 ± 1.1 58.3 ± 0.4Methanol 48.4 ± 0.4 51.1 ± 0.5Water extract 77.2 ± 0.8 99.6 ± 0.9

a Data are represented as means ± standard deviation of three independent exper-

d) 96% ethanol-aqueous, (e) methanol, (f) water extract and (g) Gentiopicroside0.01 mg/ml). A peak with the retention time of 3.25 min was identified in allxtracts, and was ascribed to gentiopicroside.

This is in accordance with the results obtained with the inhi-ition of MPO with an individual constituent (Fig. 1B) which alsohow that gentiopicroside has the strongest inhibitory activitygainst MPO. In addition, the obtained IC50 values for the inves-igated constituents can be compared with the inhibitory activityf the other polyphenolic compounds such as quercetin, whoseC50 is 0.33 �g/ml [20]. This data could be interesting for furthernvestigations since the capability of enzyme inhibition could betructure dependent, as quercetin belongs to a class of flavonoidshile gentipoicroside belongs to secoiridoids, the predominating

lass of compounds in Gentiana root [21].

.2. UPLC analysis

Since gentiopicroside exerted the strongest inhibitory activitygainst MPO, the amount of this compound was determined inll G. lutea extracts. For this purpose UPLC was used and a novelethod for its separation was developed. An additional explana-

ion for elution method is given in Section 2.4. Extracts were runt concentrations of 0.1 mg/ml and their UPLC chromatograms areresented in the Fig. 2a–f. Standard solutions of gentiopicrosideere run in concentrations from 0.005 to 0.03 mg/ml. Quantifi-

ation of gentiopicroside was done by comparing retention timesnd peak areas of samples to peak areas of standards. It is obvi-us that all ethanolic extracts contained higher concentration ofentiopicroside than water and methanol extracts. The highestmount of gentiopicroside was present in 96% and 50% ethanolqueous extracts (18.08 and 13.92%, respectively) while the waternd methanol extracts contained 7.79 and 6.53% of gentiopicroside,espectively. That was also confirmed in other studies where theighest amount of gentiopicroside was extracted with a mixturef ethanol and water (55:45 v/v). At the same time the efficiencyf gentiopicroside extraction was correlated with increasing ofxtraction temperatures [27].

Finally, it is also likely that increased inhibitory activity ofthanolic extracts, toward MPO, could be dependent on higher con-entration of gentiopicroside. This hypothesis is supported by ouresults obtained for MPO inhibition with the mixture of Gentianaonstituents.

.3. DPPH radical-scavenging capacity and total phenolic content

The scavenging activity of the DPPH free radical test revealedhat 50% and 75% ethanol water extracts have the highest

iments.b Total phenolic content was expressed as mg of Gallic acid equivalents per 100 g

dry weight of extracts.

antiradical activity with the IC50 values of 20.6 �g/ml and25.4 �g/ml, respectively. Water and 96% ethanol extracts had thelowest antiradical activity (Table 2). For comparison, other stud-ies have shown the antioxidant capacity of other compounds,determined with DPPH test and expressed as IC50: 3.2 �g/ml forquercetin, 10.62 �g/ml for �-tocopherol and 9.4–40.1 �g/ml forethanolic extracts of Salvia fruticosa IC50 [28,29]. The antioxidantactivity of Gentiana root is, however only moderate in com-parison with some other plant extracts which contain higheramounts of flavonoids. Gentiana in contrast, contains mainly sec-oiridoides, xanthones, and isoflavonoids and dihydroxybenzoicacids [21,30–32]. Similar results were obtained by Kintizos et al. ina comparative study using the DPPH antioxidant test and cell basedbiosensor [33]. The total phenolic content of Gentiana lutea extractswas determined using the Folin–Ciocalteau method which allowsthe estimation of all flavonoids, anthocyanins and nonflavonoidphenolics present in the samples. As shown in the Table 2, waterextract, as well 50% ethanol-aqueous extract had the highest phe-nolic content. Generally, increasing solvent polarity results in animproved extraction of polyphenols from plants [34]. Moreover,in some cases, depending on the plant material and its polyphenolcomposition larger amounts of polyphenols can be extracted with amixture of ethanol and water than those extracted only with water[35].

Regardless of that, it was expected that the higher contentof polyphenols in the extracts entails their greater antioxidantcapacity. However, this was not confirmed by measuring of DPPHinhibition activity in all cases. A weak linear correlation betweenthe total polyphenols content and DPPH inhibition was observed(r = −0.18). Furthermore, strong DPPH inhibition by the waterextract was expected, since it had a high content of total polyphe-nols (Table 2). Therefore, the antioxidant capacity of the extractswas also determined using CV. Using more than one method fordetermination of the antioxidative capacity is also justified becauseof the limitations of each test [33,36]. Namely, even though theDPPH assay is widely used for measuring and comparing of theantioxidant status of phenolic compounds, the results obtained,should be carefully interpreted. The absorbance of DPPH radical at517 nm, after reaction with antioxidants is changeable with light,oxygen, pH, and type of solvent [37]. A difference between theantioxidative capacity when it has been determined by chemicaltest and by biological test, such as cell based biosensor method,has been observed [33]. Therefore, it is essential to determine theantioxidative activity using at least two methods, and then make afinal and more comprehensive evaluation of their activity.

3.4. CV measurement of antioxidant capacity

CV did not revealed any faradaic processes at potentials lowerthan ∼330 mV vs. SCE, for any of the plant extract solutions studiedhere. Furthermore, the electrochemical response was not observed

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Table 3Electrochemical characteristics of anodic oxidation processes of the plant extractsstudied (Eonset – potential at which oxidation processes start, Ep,a – potential of thefirst anodic peak, Q1 – charge associated with oxidation process between Eonset and600 mV vs. SCE, Qtot – total charge under anodic wave).

Sample Eonset/mV vs. SCE Ep,a/mV vs. SCE Q1/�C Qtot/�C

25% EtOH extract 355 486 2.63 45.350% EtOH extract 334 484 4.03 37.075% EtOH extract 359 527 2.18 19.7

ict(ptTna(la

Ceuisaetcspcibttt

F0tr

96% EtOH extract 395 – 0.52 17.4MeOH extract 400 – 0.43 11.7

n the case of the water extract. Hence, that sample will not be dis-ussed further in this section. Oxidation processes were observedo start in the potential window between 330 and 400 mV vs. SCEEonset; Table 3). Some samples revealed a small anodic peak atotentials below 600 mV vs. SCE, while the others displayed mono-onic increase of anodic current with no distinct characteristics.he CV curves obtained here displayed irreversible behavior witho reverse cathodic peaks observable. Where possible, the firstnodic peak was characterized in terms of the peak potential (Ep,a)Table 3). In CV experiments, the antioxidant capacity of a particu-ar compound or a complex mixture is related to the low potentialst which the oxidation commences.

In relation to the appropriateness of parameters derived fromV experiments for the assessment of antioxidant capacity, Cheviont al. proposed that the total charge under anodic peaks should besed as a measure of the content of low-molecular-weight antiox-

dants, especially when more than a single molecule contributes apecific anodic wave and the identities of the components of a wavere not known [38]. Therefore, Ep,a of the first anodic peak is consid-red as a relevant measure of the antioxidant capacity. In additiono the parameters listed above, we also integrated recorded CVurves up to 600 mV vs. SCE (Q1) and included the results obtained,ummarized in Table 3 and presented in Fig. 3. We consider thisarameter to be appropriate for the assessment of both antioxidantapacity and total polyphenol concentration. The rationale is foundn the theory of CV and linear sweep voltammetry. Results obtainedy CV were correlated to the ones obtained by the DPPH test (in

erms of DPPH IC50 values, Section 3.3) and the ones regardingotal polyphenol content (Section 3.3), which is usually consideredo be responsible for the antioxidant capacity and was found to

1.21.00.80.60.40.2

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ig. 3. Background-corrected cyclic voltammograms of plant extracts in.1 M K2SO4 solution, recorded on GC electrode, sweep rate 100 mV s−1. Onlyhe potential window between 0.2 and 1.2 V vs. SCE is shown as electrochemicalesponse is located in this potential window.

nd Biomedical Analysis 66 (2012) 191– 196 195

correlate well with DPPH radical scavenging activity [39]. It shouldbe noted that the correlation between these parameters was per-formed without taking into account the water extract, since itselectrochemical signal was not detected.

It was clear that Q1 values correlated strongly IC50 DPPH values,with Pearson correlation coefficient r = −0.80, which was some-what better than correlation with the total polyphenol content(r = −0.73). Eonset values were also very well correlated to DPPH IC50values, giving r = 0.81. Qtot values showed much lower correlationwith DPPH IC50 values, with r = −0.49. Excellent correlation of Q1values to total polyphenol content (r = 0.99) was noticed, while Qtot

correlated to the total polyphenol content with r = 0.78. An addi-tional explanation, about correlation between DPPH IC50 valueswith CV parameters (Q1 and Qtot values) as well their correlationbetween total polyphenol content is given in the Supplementarymaterial. The results obtained confirmed that only electrochemicalresponse at low potentials, in terms of charge passed is importantfor estimation of the antioxidant capacity of a complex mixtureand that rather simple CV measurements can be used for rapidevaluation of the antioxidative activity of the plant constituentsstudied.

4. Conclusion

Results demonstrate that 50% ethanol extract has the best inhi-bition activity toward MPO, and the highest antioxidant activitywith the lowest IC50 = 20.6 �g/ml (DPPH determined). Its antiox-idant activity could be attributed to the increased content oftotal polyphenols. This dependence was also confirmed by resultsobtained with CV, where the highest Q1 potential under first anodicpeak corresponds to high phenolic content of 50% ethanol extract.Among the group of selected G. lutea constituents, gentiopicro-side is the most potent inhibitor of MPO, with IC50 = 0.8 �g/ml.Additional studies are needed to demonstrate whether the MPOinhibitory activity of Gentiana extracts depends mainly on gen-tiopicorside or whether it is due to synergistic effects of severalcomponents. Since inhibition of MPO is associated with antiinflam-atory effects, our results obtained could be useful for the futurerapid evaluation of the healing properties of Gentiana root. This isparticularly important since this method of extraction (with alcoholand water) is commonly used in traditional medicine.

Acknowledgments

The authors would like to thank to the Ministry of Education andScience of the Republic of Serbia for their financial support, ProjectNo 172023, as well as to the FUNCFOOD (FP7) project.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi/org10.1016/j.jpba.2012.03.052.

References

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