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J. Sep. Sci.

2011, 34, 12611267

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Mantas Stankevicius1 Ieva Akuneca2 Ida Jakobsone2 Audrius Maruska1


1

Research Article

Department of Biochemistry and Biotechnologies, Faculty of Natural Sciences, Vytautas Magnus University, Lithuania 2 Department of Food Chemistry, Faculty of Chemistry, University of Latvia, Latvia

Comparative analysis of radical scavenging and antioxidant activity of phenolic compounds present in everyday use spice plants by means of spectrophotometric and chromatographic methods
Comparative analysis of radical scavenging and antioxidant activities of phenolic compounds present in everyday use spice plants was carried out by means of spectrophotometric and chromatographic methods. Six spice plant samples, namely onion (Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves and leaves of dill (Anethum graveolens) were analyzed. Total amount of phenolic compounds and radical scavenging activity (RSA) was the highest in celery leaves and dill extracts and was the lowest in celery roots. Comparing commonly used spectrophotometric analysis of 2,2-diphenyl-1picrylhydrazyl (DPPH) RSA of extracts with the results obtained using reversed-phase chromatographic separation with on-line post-column radical scavenging reaction detection, good correlation was obtained (R2 5 0.848). Studies using HPLC system with electrochemical detector showed that bioactive phytochemicals can be separated and antioxidant activities of individual compounds evaluated without the need of a complex HPLC system with reaction detector. The results obtained using electrochemical detection correlate with the RSA assayed using spectrophotometric method (R2 5 0.893). Keywords: 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity postcolumn reaction detection / Electrochemical detection / HPLC / Phenolic compounds / Spice plants DOI 10.1002/jssc.201000915

Received December 20, 2010 Revised February 22, 2011 Accepted March 8, 2011

1 Introduction
Culinary herbs have a long history of use as important food ingredients reducing food spoilage and controlling against the growth of food-borne pathogens. Many herbs also contribute to the enhancement of avor in both food and beverages. Parsley, celery, dill and onion are spices of everyday use in local cuisines and may offer many health benets. They have been proved to counteract oxidative stress [1]. In addition, culinary herbal extracts and essential oils have become increasingly popular as alternative sources of natural preservative agents, mainly because herbs are widely cultivated, effective and safe for consumption. Many culinary herbs (e.g. rosemary, sage and thyme) function as
ka, Vytautas Magnus Correspondence: Professor Audrius Marus University, Faculty of Natural Sciences, Department of Biochemistry and Biotechnologies, Vileikos 8, Kaunas, LT-44404, Lithuania E-mail: a.maruska@gmf.vdu.lt Fax: 137-03-73-27-908

Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; ED, electrochemical detection; GAE, gallic acid equivalents; HPLC-RD, HPLC with DPPH reaction detector; TPC, total content of phenolic compounds

natural antioxidants [2]. Components of fresh parsley leaf scavenge superoxide anion in vitro [3]. It was reported that supplementation of diets with fresh parsley leaf can increase antioxidant capacity of rat plasma and decrease oxidative stress in humans [4, 5]. Many culinary spices (e.g. garlic, onion, cinnamon, clove and mustard) have also been effectively used to inhibit microbial spoilage in foods. Fresh and dried parsley inhibits the growth of Listeria monocytogenes, L. innocua and Escherichia coli [6]. Biological properties of phenolic compounds depend on their bioavailability. Indirect evidence of their absorption through the gut barrier is the increase in the antioxidant capacity of plasma after the consumption of polyphenol-rich foods. This has been observed for a wide range of foodstuffs [7]. The average total intake of phenolic compounds is ca. 1 g/day, as it was suggested 25 years ago [8]. However, large uncertainties in the intake of phenolic compounds and in variations of the intake remain. Comprehensive surveys on the content of some important phenolic compounds classes (e.g. anthocyanins, proanthocyanins, phenolic acids) are still lacking [7]. Different methods are used to evaluate phenolic compounds and radical scavenging activity (RSA) of plant

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extracts. The most widely used methods based on photometric reactions or complexation and determinations are carried out spectrophotometrically or chromatographically [9]. Some applications of the off-line [10] and on-line [11] capillary electrophoresis for evaluation of RSA of various plants extracts were reported as well. Electrochemical detectors with electrode arrays were demonstrated as potential means for selective determination of antioxidant compounds in different plant extracts and food matrices in combination with HPLC [12, 13]. The main task of this study was to carry out a comparative evaluation of the content of phenolic compounds, RSA and antioxidant activity in different spice plants, traditionally used in the local cuisine, by means of various methods of instrumental analysis.

reduction of FolinCiocalteu reagent by phenols to a mixture of the reaction products, having absorbance maximum at 765 nm. The plant extracts were diluted ten times with aqueous ethanol (1:1, v/v) prior to analysis. One milliliter of each diluted sample was mixed with 5 mL of FCR (10% in distilled water) and 4 mL of sodium carbonate solution (7.5% in distilled water) in test tubes. After vortexing for 30 min at room temperature, the absorbance at 765 nm was measured using spectrophotometer UVIKON 930 (Kontron Instruments, Italy). The linear calibration curve ranged between 0.0075 and 0.06 mg/mL (R2 5 0.9994), which corresponds to the absorbance up to 0.7005 AU. In case of over range, additional dilution of the samples was performed to t the calibration range. The analyses were carried out in triplicate. Results were expressed as gallic acid equivalents (GAE) in mg per 100 g of dry plant material using the following equation: C a g V=m 100 1 where C is the total amount of phenolic compounds (mg GAE/100 g plant), a the dilution number, g the mass concentration obtained from calibration curve (mg/mL), V the volume of aqueous ethanol used for extraction (mL) and m the weight of dry plant sample (g).

2 Materials and methods


2.1 Chemicals Acetonitrile, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and gallic acid were obtained from Sigma-Aldrich (Germany). Chlorogenic acid was obtained from Fluka (Switzerland). Sodium carbonate was from Penta (Poland). FolinCiocalteus phenol reagent was purchased from Scharlau Chemie S.A. (Spain). Ethanol (96%) was obtained from FarmaBalt (Latvia). Citric acid was obtained from Lach-Ner (Czech Republic), sodium hydroxide was from Lachema (Czech Republic), methanol was from Chempur (Poland) and monohydrate of sodium dihydrophosphate was from Merck (Germany). All reagents were of analytical grade.

2.4 DPPH RSA DPPH radical scavenging assay was performed according to the method reported in [16] with some modications. Four hundred microliter of sample or ethanol (blank) was added to 3600 mL of ethanolic solution of 100 mM DPPH and vortexed. After 20 min, absorbance at 517 nm was measured using a spectrophotometer. All measurements were performed in triplicate and the result was an average of three determinations. The DPPH RSA (%) was calculated by the following equation: Radical scavenging activity % Acontrol Asample =Acontrol 100% 2

2.2 Plant materials and preparation of extracts Six spice plant samples, namely onion (Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves and leaves of dill (Anethum graveolens) were obtained from SIA Herbe (Latvia) in 2008. The spice plants were dried using an industrial dryer at 451C. The dried samples were carefully powdered using a coffee grinder for 3 min and kept at room temperature prior to extraction. Dry plant material (500 mg) was soaked in 10 mL of aqueous ethanol (1:1, v/v) and extracted shaking for 1 h at room temperature. Extract was ltered through paper lter. The ltrates were stored at 141C for further analyses. The same extract of every spice plant sample was used to carry out all chromatographic and spectrophotometric determinations.

where Acontrol is the absorbance of blank and Asample is the absorbance of sample.

2.5 RSA screening for separated compounds by means of HPLC with DPPH reaction detector (HPLC-RD) Separation was carried out using a reversed-phase HPLC. The scheme of the chromatographic system with the UV and DPPH reaction detectors (HPLC-RD system) is presented in Fig. 1. Two chromatograms were recorded simultaneously. The upper chromatogram was obtained by registering UV absorbance of efuent at 265 nm prior to the reaction; a mirror chromatogram was obtained recording absorbance at 517 nm after reaction of efuent with DPPH solution in the reaction coil. Mobile phase was supplied to
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2.3 Determination of total content of phenolic compounds (TPC) The TPC in the extracts was assessed according to the method described in [14, 15]. This method is based on the
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Figure 1. Scheme of chromatographic setup with reaction detection (HPLC-RD): 1 gradient pump, 2 sample injector, 3 precolumn, 4 reversed-phase column, 5 UV detector, 6 T connection, 7 solvent delivery system for DPPH solution, 8 reaction coil, 9 photometric detector, 10 data acquisition and handling system. (A and B) Mobile phase components.

the column by a model 9012 HPLC gradient pump (Varian, USA) at ow rate 0.75 mL/min. Samples of 20 mL were injected into the HPLC system by means of Cheminert C1 injector (Valco Instruments, USA). Reversed-phase LiChroCart 12.5 0.4 cm column (Merck) and 0.5 0.4 cm precolumn lled with LiChroSpher RP-18e and 5-mm particle size packing material (Merck) were used for separation. The DPPH reagent was prepared dissolving 0.01 M DPPH in 0.1 M sodium citrate buffer, pH 7.6, methanol and acetonitrile (50:25:25 v/v). It was continuously supplied into reaction coil (3 m of 0.25 mm id fused-silica capillary) by a model 2200 HPLC pump (Bischoff, Germany) at ow rate of 0.75 mL/min. Signals were acquired at 265 and 517 nm wavelengths by means of Linear 206 PHD (Linear Instruments, USA) and Linear UVIS 200 UV-VIS (Linear Instruments) detectors, respectively. Solutions A bidistilled water with 0.05% TFA additive and B methanol with 0.05% TFA additive were used as the mobile phase components for gradient elution. Extracts were separated using following gradient: 20% of B at 0 min, 95% of B at 30 min, 95% of B at 34 min and 20% of B at 35 min. Clarity chromatography software (DataApex, Czech Republic) was used for data acquisition.

rate of the mobile phase was 0.75 mL/min. Elution was performed using two components mobile phase. Solvent A was composed of 50 mM sodium dihydrophosphate (pH 3) with 1% (vol) additive of methanol. Solvent B was a mixture of 100 mM sodium dihydrophosphate, pH 3, acetonitrile and methanol (30:60:10 v/v). Phenolic compounds were eluted using the following gradient: 0% of B for 6 min, 100% of B at 45 min, 100% of B at 50 min and 0% of B at 51 min.

3 Results
Table 1 presents the results of two assays of the TPC and RSA in the dried spice plants samples determined spectrophotometrically. Figure 2 shows an exemplary chromatogram of celery leaves extract obtained using the HPLC system with UV and DPPH radical scavenging reaction detection. Peak area in the upper chromatogram is proportional to the concentration of compound in a mixture. In the chromatogram, the negative peaks represent DPPH RSA of the mirrored compounds. As bigger negative peak is recorded, the more powerful a radical scavenger the compound is. If the compound does not have radical scavenging properties, the negative peak is not obtained. Figure 3 represents correlation of total DPPH RSA determined by means of spectrophotometric method and total RSA determined by means of the HPLC-RD method. Figure 4 shows chromatogram of celery leaves extract obtained using four cells electrochemical detector. To test the correlation of antioxidant activity and RSA, the data obtained spectrophotometrically and chromatographically were used for a linear t. In Fig. 5, the spectrophotometrically determined total RSA is plotted against total area of the peaks obtained by chromatography with elctrochemical detection. The peaks were integrated at the potential providing the highest response.

2.6 Chromatography using electrochemical detection (ED) For separation and ED of extracted compounds, gradient HPLC system (ESA, USA) was used. Twenty microliter of sample was injected by means of a model 542 autosampler. High-pressure gradient was formed using two 582 HPLC pumps. Reversed-phase C-18 column 80 4.6 mm, particle size 3 mm, 120 A pore size (ESA) was used for separations. Chromatograms were acquired by means of a model 5600 CoulArray electrochemical detector with the array of cells with the potentials set at 300, 500, 700 and 900 mV. Flow

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Table 1. Results of spectrophotometric analysis obtained for


spice plants

Sample Parsley leaves Parsley roots Celery leaves Celery roots Dill Onion

TPC (mg GAE/100 g) 1247.870.9 555.971.2 1637.170.7 233.170.3 1399.872.4 744.071.1

RSADPPHa) (%) 20.8 15.8 71.6 11.4 51.7 23.7

a) RSD for measurements did not exceed 1.59%.

200 180 160 140 120 100 80 60 40 20 0 -20 -40 -60 -80 0 2 4

chlorogenic acid

UV detection = 265 nm

DPPH reaction detection = 517 nm 6 8 10 12 14 16 18 20 22 24 Retention time, min

Figure 2. Chromatograms of celery leaves obtained by HPLCRD. Injection: 10 mL of ethanolic extract.

80.00%

Spectrophotometric radical scavenging activity, %

70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% 0 2000 4000

y = 0.00011x R = 0.848

6000

8000

10000

HPLC-RD, total area

Figure 3. Correlation of the results of spectrophotometric and HPLC-RD methods of evaluation of DPPH radical scavenging activity obtained for spice plant extracts listed in Table 1.

4 Discussion
As shown in Table 1, the TPC, expressed as mg GAE/100 g, varied from 233.1 to 1637.1 and was highest in celery leaves and lowest in celery roots. The TPC value in dill and parsley leaves was signicantly lower than in celery leaves. The DPPH free radical scavenging activities measured after
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20-min incubation were highest in the extracts prepared from celery leaves (71.6%) and dill (51.7%). The results obtained let us conclude that celery leaves contain the highest amount of the phenolic compounds and are stronger radical scavenger than other spices. The advantage of the chromatographic analysis and other separation methods comparing to the spectrophotometric methods of the assessment of different plants and food matrices is that it provides a possibility of evaluation not only for the total amounts of the compounds or total radical scavenging activities but also for the contribution or content of individual compounds present in the extracts. Hyphenation of separation methods with the reaction detection may reveal relative activity or inactivity of any separated compound regarding the selected reagent. Compared to spectrophotometry, chromatographic methods require faster complexation/colorimetric reaction for the reproducible and reliable results. Therefore, reaction conditions and reagent concentration should be optimized in order to obtain linear/dynamic response and as low as possible incubation period of the reaction. This is related with an extra-column chromatographic band broadening of the chromatographic zone occurring in the reaction loop attached to the outlet of the rst on-line (UV) detector and the reagent supply via low volume HPLC T-connection. Minimization of the reaction coil diameter and length reduces the chromatographic band dispersion and increases efciency and sensitivity of the reaction (secondary) detector. It should be kept in mind, however, that the decrease in the reaction coil diameter increases the back pressure in the primary (UV) detector ow cell. Testing of several reaction coil materials revealed that rigid materials, such as stainless steel or fused-silica capillaries, provide more stable back pressure due to the rigid connections in contrast to polytetrauoroethylene or polyether ether ketone (PEEK) tubing. Polymeric material tubing may generate additional back pressure, increasing over time due to exibility and relaxation of the tubing in the connection unions and T-connections. This can cause leakage or damage of the primary detector ow cell. To avoid extra-column chromatographic band broadening, id of the reaction coil should be kept below 300 mm, when analytical columns of 34 mm id and mobile phase ow rates of 0.51.5 mL/min are used. The length and diameter of reaction coil dene its volume and reaction incubation time, when the reagent stays in contact with the analytes present in the efuent. Another factor to consider is the reagent ow rate pulsation, which causes noise of the reaction detector base line and can considerably reduce sensitivity of the method. The pulsation of the HPLC pump is less critical, since it is considerably reduced by the HPLC column. The problem may be overcome using a syringe pump as the reagent supply device. Syringe pumps are completely pulse-free compared to the most common HPLC pumps employing reciprocating piston heads. Since the syringe pumps are of periodic action, the volume of the syringe should exceed the reagent volume needed for one chromatographic run [17]. The pulsation of the continuous
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Response mAU

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Figure 4. Chromatogram of celery leaves extract obtained using HPLC with electrochemical detector; potentials: 300, 500, 700 and 900 mA. Injection: 10 mL of ethanolic extract.

Spectrophotometric radical scavenging activity, %

80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% 0 500 1000 1500 2000 2500 y = 0.00013x R = 0.893

HPLC-ED, total area

Figure 5. Correlation between the results obtained evaluating spice plant extracts (listed in Table 1) by means of spectrophotometric evaluation of total radical scavenging activity and total antioxidant activity evaluation by means of HPLC-ED.

ow HPLC pump often is due to the poor function of the check valves at low back pressure. The use of short column or narrow in-line capillary may improve their function and reduce the pulsation. This considerably improves baseline of the reaction detector and reduces noise of the detector signal. A piece of 75-mm fused-silica capillary was installed in-line between the reagent pump and T-connection in our setup. Varying ow rates ratio of efuent and reagent solution revealed that the best sensitivity and the most efcient mixing of efuent with reagent was achieved when this ratio is unity. As shown in Fig. 2, three dominant peaks are observed in the radical scavenging reaction detection chromatogram. The biggest peak eluting at 8.7 min was identied as chlorogenic acid. Integration of the peaks area revealed that chlorogenic acid contributes 47% to the total RSA of celery leaves extract. With the peaks eluting at 14.53 and 16.59, they comprise ca. 85.5% of all RSA of the celery leaves extract. This indicates that the pattern of the radical scavengers in celery leaves extract is less complex compared
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to the chromatographic pattern of UV absorbing compounds observed in the UV chromatogram. Total RSA was determined by HPLC-RD system calculating total area of the peaks that appeared in the radical scavenging reaction monitoring chromatogram. As a scatter plot shows (Fig. 3), these two methods match quite well and the correlation coefcient calculated for a linear regression is R2 5 0.848. This indicates that both independent methods provide fairly adequate results, when comparing RSA of selected spice plants extracts. Both spectrophotometric method and chromatographic method with reaction detection are based on the reaction of the same model compound, namely DPPH as radical, with the radical scavenging compounds present in the extracts of the spice plants. Chromatographic method provides additional information on the contribution of separated compounds to total RSA of the extract. It is well known that the result when assaying individual phenolic compounds is highly dependent on the structural properties of the certain compound [18]. Some deviation from the linear dependence comparing the results obtained by the spectrophotometric and chromatographic methods can be due to the differences in the incubation time of the radical scavenging reaction (20 min using spectrophotometric method and ca. 0.3 min using chromatographic method) and different conditions of reaction (DPPH concentration, composition of reaction medium and pH). Evaluation of kinetics of colorimetric methods used for spectrophotometric evaluation of total amount of phenolic compounds, avonoids or RSA revealed that different samples behave differently [19, 20]. During the rst seconds and minutes, the colorimetric reaction can be very fast and absorbance changes dramatically. After 30 min, the reaction of phenolic compounds with Folin Ciocalteu reagent in the presence of sodium carbonate is nished and later changes of the absorbance are negligible.
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The reaction of avonoids with sodium nitrite and aluminum chloride slows down after 15 min [19]. Depending on the sample qualitative composition, the DPPH radical scavenging reaction can take from 12 min to 16 min and more [20]. The reaction time in the case of chromatographic analysis with on-line DPPH radical scavenging reaction detection is limited by the chromatographic requirements (low volume reaction coil is used for higher efciency). Therefore, differences in the reaction kinetics of different compounds present in various samples affect the result. In spectrophotometric measurements, kinetic differences of radical scavenging by different analytes are not reected, since the reaction is performed off-line (batch-wise) and excessive time period is provided to get all radical scavenges reacted with the DPPH radicals. Therefore, some deviation of the results from linear correlation is predicted using static and dynamic character analytical techniques for RSA evaluation of various qualitative composition extracts. To ensure fast radical scavenging reaction and increase reproducibility of the results carrying out chromatography with the on-line reaction detection, the pH of DPPH solution was adjusted to pH 7.6 [21]. Using gradient elution, the composition of reaction medium is changed with the gradient and it is different for early eluting and late eluting compounds. Comparable and reproducible results are obtained using the same gradient and keeping the ow rates of efuent and reagent solution constant (constant reagent and analytes ratio). RSD of the results obtained for retention time and for reaction detector peak area did not exceed 0.48 and 6.6%, respectively (n 5 3). Linear range of measurement was checked using rutin standard solutions within concentration range of 0.0250.2 mg/mL and linear calibration curve was obtained y 5 15290x (R2 5 0.991), where y peak area was in range of 3522994.7 mAU s. All determined peak areas of RSA were within the linear range of determination (did not exceed 2994.7 mAU s). Detection and determination limits for chromatographic analysis of DPPH RSA expressed in rutin equivalents were 4.75 and 9.5 mg/ mL, respectively (when peaks are three and six times bigger than noise). Due to the high concentrations of phenolic compounds in spice plants extracts, detection limit was not a problem using off-line spectrophotometric determination of total RSA. Linear determination range was easy to extend diluting the extracts. RSD for the spectrophotometric results was lower (up to 1.59%) compared to the chromatographic determinations. Previous studies revealed that extracts of phenolic compounds stored in dark at room temperature or 161C for 3 months do not show signicant differences in their composition [22]. Inuence of daylight exposure at ambient temperature was observed during the rst 23 wk evaluating extracts of single-styled hawthorn (Crataegus monogyna Jacq.). In 3 wk, hyperoside content decreased to 24% of the initial concentration and rutin and vitexin-2-O-rhamnoside content decreased to 8085% of the initial concentration. In the present study, the extracts of spice plants were stored in
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a refrigerator at 141C. Chromatographic and spectrophotometric analyses were carried out simultaneously within a 2-month period after the preparation of extracts so that no considerable aging of the extracts and changes in concentrations of phenolic compounds would occur. Summarizing, it should be noted that data obtained by spectrophotometric and chromatographic DPPH RSA determination methods correlate, although the methods differ in static and dynamic character of the measurements and in selectivity. Therefore, they can be used as complimentary methods providing additional data about complex mixtures of radical scavenging compounds. Spectrophotometric analysis of RSA is simple and easy to carry out. It does not require any nonstandard equipment. The results are reproducible, although it is a less time efcient, manual method. It is nonselective method, i.e. the total RSA of the mixture is determined. Chromatographic determination of RSA is time efcient and easy to automate. It requires additional modication of the standard HPLC setup. The results are reproducible, selective and free of synergistic effects, since on-line post-column radical scavenging reaction is performed with separated compounds. Since the RSA of the phenolic compounds is related to their antioxidant properties, it is worth to use chromatography combined with ED (HPLC-ED) for specic determination of the electrochemically active compounds analyzing plant extracts. This method is very attractive due to its high sensitivity and simplicity of detection, since no additional equipment (reagent pump or secondary detector) is needed. Moreover, some modern electrochemical detectors are equipped with the data acquisition software compatible with the mobile phase gradient operation. This offers additional advantage of direct transfer of the developed chromatographic methods for the electrochemical analysis of the samples containing antioxidants. Two analytes, which cannot be separated chromatographically, can be separated and quantied electrochemically. Progressively increasing potential is delivered to the array of electrodes. Analytes move through the array of electrodes; each of them reaches the particular potential of the electrode, which oxidizes or reduces it. In this study, a model CoulArray 5600 electrochemical detector (ESA, USA) with an array of four detection cells equipped with porous glassy carbon electrodes was used in the coulometric detection mode. The potentials providing maximum detection sensitivity for the phenolic compounds depend on pH of eluent and vary from 300 to 900 mV [13]. Parallel analysis of spice plant extracts was made using electrochemical detector. Four detector working electrodes potentials (300, 500, 700 and 900 mV) were set for analysis of the spice plants extracts. As shown in Fig. 4, the chromatographic pattern obtained for celery leaves extract is more complex compared to the on-line post-column DPPH radical scavenging reaction detection, particularly at higher oxidation potentials (700 and 900 mV), when many compounds, which did not
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react with DPPH radical become electrochemically active. Therefore, both methods can be characterized rather as complementary but not identical. As shown in Fig. 5, a satisfactory correlation exists between the results obtained by spectrophotometric analysis and chromatographic analysis, using HPLC-ED system. Correlation coefcient R2 5 0.843 was calculated for a linear dependence of the results obtained by these two independent methods, which indicates that statistically reliable characterization of the RSA of plant extracts can be carried out by means of coulometric detection. Some deviation of the results can be due to the differences between the electrochemical oxidation of the target compounds present in the spice plants extracts and chemical activities of those compounds, when radical scavenging occurs reacting with DPPH radical. It should be noted that DPPH or other synthetic radicals (e.g. 2,20 -azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)) are model radical compounds selected to mimic the action of radicals occurring in living cells. They are used for comparative evaluation of the radical scavenging activities of different food or plant matrices when assay is carried out at identical conditions. In case of experimental results depicted in Fig. 3, some scatter of the results can be caused by different conditions of reaction of radical scavenging, although the same model radical compound was used. It can be concluded that HPLC-EC is a simple and attractive means for evaluation of the antioxidant activities of individual components in the complex matrices, such as spice plants. The results obtained correlate with the RSA assayed by spectrophotometric method.

6 References
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5 Concluding remarks In this study, spice plants of everyday use, namely dill, parsley leaves and roots, celery leaves and roots and onions, have been analyzed using different analytical methods. Total amount of phenolic compounds and RSA of extracts have been evaluated spectrophotometrically. When compared to chromatographic separations with radical scavenging reaction detection data, the correlation coefcient was R2 5 0.848. Studies using the HPLC system with electrochemical detector showed that bioactive phytochemicals can be separated and antioxidant activities of individual compounds evaluated without a need of complex HPLC setup. The results obtained using ED correlate with the RSA assayed using spectrophotometric method (R2 5 0.893). All methods showed that most of radical scavenging compounds occur in the investigated spice plants leaves and less in roots. The work was instrumentally supported in part by the Lithuanian State Science and Studies Foundation, grant No. N-07008. Financial support from European Social Fund project No. 2009/0162/1DP/1.1.2.1.1/09/IPIA/VIAA/004, contract EES 2010/78/MS/T/29 is acknowledged (I. Akuneca).

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