INTRODUCTIONLipid oxidation is one of the major chemicalchanges that occur during food processing, storage, andpreparation. Lipid molecules are rapidly oxidized in thepresence of oxygen, especially in the case of unsaturatedfatty acids [1]. Antioxidants are widely used today toreduce the rate of oxidation reaction of fats in foods.Antioxidants are molecules or compounds thatact against free radicals which damage to molecules,resulting in the loss of their function. Antioxidantsprovide a primary defense against such oxidativedegradations [2]. In industrial processes, syntheticantioxidants – such as butyl hydroxy toluene and butylhydroxy anisol – are mainly used to increase the food’sshelf life. In this regard, nutritionists have found that thesecompounds can have adverse effects on the body [3].Therefore, it is necessary to use strong antioxidantswith lower toxicity and greater efficacy. In recentdecades, natural antioxidants have drawn the attention offood researchers due to their safety in food formulation.These are extracts and essential oils of various plantsthat produce positive effects on nutrient oxidationreactions.Pre-extraction seed treatment is one of the mostessential steps to ensure high quality extraction. Oneof the treatment methods is the use of a pulsed electricfield. It is an important non-thermal method of treatingfoodstuffs by placing them in a chamber between two187Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195electrodes and subjecting to high-voltage pulses fora short time. A pulsed electric field focuses mainly onthe microscopic scale so that pores are created in thecell membrane, accelerating the exit of intercellularcompounds. This process preserves qualitative,nutritional, and energy consumption properties, as wellas increases productivity in food production [4].Most importantly, a pulsed electric field destroysthe cell wall and its membrane and increases the masstransfer rate. Indeed, when a living cell is affected bysuch a field, the cell wall and its membrane are naturallydamaged. The inside material is easily removed andthe surrounding material enters the cell, resulting in itsdestruction. With increased permeability of plant andanimal cells, their intracellular material is extractedmore easily and quickly. Therefore, this treatment canbe used as a pre-processing step in the extraction ofvaluable cellular materials [5, 6].Salvia leriifolia L. is one of the plants that containantioxidant compounds. It is a native species ofLamiaceae family to Khorasan and Semnan provinces,Iran [7]. It grows in cold and semi-arid or arid regions ataltitudes between 900 and 1650 meters, with an averagerainfall of 80 mm. A special shape of its leathery leaves,especially white villi on both sides, and a wide growthon the surface of the soil make this plant resistant toharsh winter winds or severe heat [8].Various studies have reported therapeutic propertiesof Salvia leriifolia. For example, its aqueous andalcoholic root extracts have neuroprotective propertiesagainst topical anemia in the rat brain [9]. The analgesicand sedative activity of Salvia leriifolia leaf extractin the amount of 500 mg/kg is comparable to that ofdiazepam in the amount of 5 mg/kg [8]. In treatingchronic inflammation, the plant’s extract is similar todiclofenac [10]. Its aqueous and alcoholic leaf extractswere found to prevent gastric ulcers in rats similarly toSucralfate [11].In addition, the plant’s root and leaf extractsshowed considerable antimicrobial activity [9]. Theyalso have strong antioxidant properties that preventthe oxidation of oils. This property is competitivewith that of antioxidants commonly used in the foodindustry, such as butylated hydroxy toluene and alphatocopherol.It is due to the presence of a secondarymetabolite of chalcones, called butin, in this plant.Finally, Salvia leriifolia is of industrial importance. Inthis regard, researchers have found that its seeds contain26% yellow oil, with a very low peroxide index and ahigh antioxidant index, which increases its shelf lifecompared to other oils [12].Another plant with antioxidant properties is Linumusitalissmum L. It is a one-year-old plant of Linaceaefamily that grows in bushes. This plant has over 200species but only Linum usitalissmum has economicimportance. In addition, its seeds have several powerfulantioxidants, including lignans. 100 g of Linumusitalissmum contains about 9.2 mg of vitamin E, mainlyin the form of gamatocopherol [13].The most common method for extracting compoundsfrom plant tissues uses aqueous and ethanol solvents.Therefore, we aimed to evaluate effects of an electricalpulse pre-treatment and to compare the aqueous andalcoholic extracts of Linum usitalissmum and Salvialeriifolia seeds.STUDY OBJECTS AND METHODSPreparation of raw materials. For this study, Linumusitalissmum L. seeds and Salvia leriifolia L. aeriallimbs, leaves, and stems were obtained from a certifiedapothecary. We also used chemicals produced by Merck(Germany).Extraction of aqueous and alcohol extractsfrom Linum usitalissmum and Salvia leriifolia seedspretreated with a pulsed electric field. Initially,Linum usitalissmum and Salvia leriifolia seeds werecleaned and the external materials and impurities wereseparated and dried in an oven at 45°C. The sampleswere powdered in a household mill (Fama Model Cs,Germany) and passed through a 40-mesh sieve. Finally,they were packed in air- and water-proof packages andkept in a freezer at –18°C until further experimentsto preserve the extract’s antioxidant and functionalproperties.The aqueous and alcoholic extracts were made usingthe Kabiri and Seyyedlangi method [14, 15]. For this,the prepared powders were mixed with a water solvent(aqueous extract) or 80% methanol (alcoholic extract) atthe ratio of 50:1.Subsequently, to apply a pulsed electric fieldpretreatment, each of the extracts was subjectedto an alternating electric field with zero (withoutpretreatment), 3, and 6 kV·cm–1 intensity and a constantpulse number of 30 (Table 1). The linear electric currentin this device is transmitted to a series of capacitors andthe energy stored in the capacitors is discharged to thechamber containing two electrodes with a pulse switch.The discharge chamber is made of Plexiglass 1 and thedistance between the two electrodes is 4 cm. Thesewaves were applied to facilitate the extraction.Evaluation of antioxidant properties of Linumusitalissmum and Salvia leriifolia aqueous andalcoholic extracts. Total phenolic compounds. Theamount of total phenolic compounds was measured bythe Folin-Ciocalteu method according to Oardoz et al.[16]. For this purpose, 10 g of extracts was first extractedwith 200 mL of methanol for 24 h at room temperatureusing a magnetic stirrer. The extract was filtered withWhatman Paper No. 1 and the sediment was extractedagain under the same conditions. The solvent was thenremoved by a vacuum evaporator at less than 40°C andconcentrated as far as possible. Then, 0.5 mL of theextract was mixed with 2.5 mL of 0.2N Folin-Ciocalteureagent and 2 mL of 7.5% sodium carbonate solution.188Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195The mixture was kept at room temperature for 120min. The absorbance rate of the solution was then readby a spectrophotometer at 760 nm. The total content ofphenolic compounds was expressed in mg/g of extractusing the line equation drawn on the basis of gallic acid.The calibration curve was plotted as follows.Different concentrations of gallic acid were firstprepared and 0.5 mL of each was mixed with 2.5 mLof 10% Folin-Ciocalteu reagent (v/v) and 2 mL of 7.5%sodium carbonate for half to 8 min (w/v). The sampleswere stored at room temperature for 30 minutes and thenabsorbed at 760 nm [17]. Distilled water was used as acontrol.Antioxidant activity by DPPH method. To extractantioxidant compounds, 10 g of aqueous and alcoholicextracts with 100 mL methanol was stirred with amagnetic stirrer at a speed of 100 rpm at 25°C for 24 hand finally filtered with Whatman filter paper. Thesolution was then transferred to a freezing dryer formethanol removal and, finally, the dried extract wasstored at –20°C [18]. The antioxidant activity of thesamples was further evaluated by the method of BrandWilliams et al. [19]. 3.9 mL of DPPH stock was pouredinto the cell and read by a spectrophotometer at 515 nm.Then, 0.1 mL of each extract was added to the DPPHstock solution and after 90 minutes of incubation, theabsorbance of the samples was read at 515 nm. Theinhibition percentage of DPPH radical was calculatedusing Eqs. (1) and (2).I(%)= 100 × (A0−As)/A0 (1)where A0 is control absorption and As is sample absorption.The results were then expressed as IC50 (the amountof antioxidant required to reach 50% of the initial DPPHconcentration). To draw a standard curve, we used aTrolox solution with a concentration of 1000–100 μmol.First, the percentage of radical neutralization activitywas obtained for each sample. Then, we calculated theantioxidant activity of the samples using a standardcurve in μmol of Trolox per gram dry weight (μmol/g).Antioxidant activity by TEACI method. To extractantioxidant compounds, 10 g of the milled sample with100 mL of methanol was mixed with a magnetic stirrerat 100 rpm and 25°C for 24 h and then filtered with aWhatman filter. Then, the methanol was transferred to afreezing dryer and, finally, the dried extract was storedat –20°C [18]. The antioxidant activity of the sampleswas further evaluated by the method of Yu et al. [20].First, we made an aqueous solution of ABTSII ata concentration of 1 mM to prepare the radical ABTS.Potassium persulfate was then added to this solution toreach a final concentration of 2.45 mM. The resultingsolution was incubated at room temperature anddarkness for 2 h. During this time, the ABTS moleculeproduced the ABTS•+ cation radical. Then, 4 μL of thesamples was taken with a Peptide and mixed with 4 mLof the ABTS•+ solution in the cell. Its absorption at a734 nm wavelength was verified at 6 min after mixing(for 30 s). A standard curve was plotted, correspondingto the reaction of 40 μL of Trolox (at concentrations of50, 100, 250, 500, 750, and 1000 μM) to 4 mL of theABTS•+ solution. The inhibition percentage of ABTS•+of the samples was calculated according to Eq. (2). Also,the ABTS•+ radical inhibition activity was expressedbased on the standard Trolox curve as the Troloxsolution equivalent antioxidant capacity (mM TEAC).Statistical design and analysis of results. Theresults of our study were evaluated with SPSS 16software.To extract the essential oil, we used a completelyrandomized design with a three-factor arrangement.In particular, the three factors were a plant source(Salvia leriifolia and Linum usitalissmum), a type ofpretreatment (pulsed electric field at the intensity of zero(no pretreatment), 3 and 6 kV·cm–1), and a type of solvent(aqueous and alcoholic).The samples were obtained in three replicationsand the means were compared by the Duncan test at asignificant level of 5% (P < 0.05). Finally, Excel softwarewas used to plot the diagrams.AA%= [Ablank−Asample/Ablank] × 100 (2)where Ablank is the absorption of a control sample withoutthe active compound and Asample is the absorption of a samplecontaining a distilled extract).RESULTS AND DISCUSSIONTotal phenolic compounds. Fig. 1 presentsindependent effects of the factors, Fig. 2 shows theirbinary effect, while Table 3 indicates the interactionbetween the three factors in their effect on the contentof phenolic compounds in the extracts. We found thata higher intensity of a pulsed electric field and the useof an alcoholic solvent significantly increased totalI Trolox equivalent antioxidant capacityII 2,2’-Azino-Bis(3-ethylbenzothiazoline-6-Sulphonic Acid)Table 1 Treatments investigated in the studyExtractionmethodIntensity of pulsed Herbal sourceelectric field pretreatment,kV·cm–1TreatmentcodeLinum aqueoususitatissimum1 02 alcoholic3 3 aqueous4 alcoholic5 6 aqueous6 alcoholic7 0 Salvia leriifolia aqueous8 alcoholic9 3 aqueous10 alcoholic11 6 aqueous12 alcoholic189Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195ba01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourcecba010203040500 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1ba01020304050aqueous alcoholicTotal phenolic compounds,mg Galic acid/gSolvent typebaba0153045Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourceaqueous alcoholicSolvent typebababa01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal source0 3 6Intensity of electric pulse field, kV·cm1dcdc cba01530450 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1ab0100200300aqueous alcoholicDPPH IC50,μg/mL Solvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc100200300DPPH IC50,μgr/mLSolvent typeabcbcdd100200300DPPH IC50,μg/mLSolvent typephenolic compounds at a 95% confidence level. On theother hand, the content of total phenolic compounds washigher in the Salvia leriifolia L. extract, compared toLinum usitalissmum L. (P < 0.05).Various factors, such as plant variety, harvest area,and harvest time, appear to affect the content of phenoliccompounds. Different studies have found differentamounts of total phenolic compounds in the Salvialeriifolia plant. For example, Hamrouni-Sellami et al.,Ahmadi et al., Najafi et al., Abadi et al., and Bahadoriet al. reported total phenolic compounds of 0.399–2.37, 40.47–61.32, 11.28–23.88, 12.68–83.85, and 17.3–294.9 mg of gallic acid per gram of extract, respectively[21–25]. In our study, this value reached 33.24–63.98mg of gallic acid per gram of extract, depending onFigure 1 Independent effects of herbal source (a), pretreatment(b), and solvent type (c) on total phenolic compounds(P < 0.05)ba01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourcecba010203040500 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1ba01020304050aqueous alcoholicTotal phenolic compounds,mg Galic acid/gSolvent typebaba0153045Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourceaqueous alcoholicSolvent typebababa01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal source0 3 6Intensity of electric pulse field, kV·cm1dcdc cba01530450 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1300ab300Intensity of electric pulse field, kV·cm1 maSalvia leriifoliacba010203040500 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aalcoholicbaba0153045Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourceaqueous alcoholicSolvent typeaaaSalvia leriifoliaHerbal source6pulse field, kV·cm1dcdc cba01530450 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typebSalvia leriifoliaabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1adbdc100200300DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1 m(а)(c)(b)Figure 2 Binary effects of herbal source and pre-treatment (a),herbal source and solvent (b), and pre-treatment and solvent (c)on total phenolic compounds (P < 0.05)b01020Linum Usitatissimum Salvia leriifoliaTotal phenolic mg Galic Herbal sourcec010200 3 6Total phenolic mg Galic Intensity of electric pulse field, kV·cm1ba01020304050aqueous alcoholicTotal phenolic compounds,mg Galic acid/gSolvent typebaba0153045Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourceaqueous alcoholicSolvent typebababa01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal source0 3 6Intensity of electric pulse field, kV·cm1dcdc cba01530450 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifolia DPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent type(а)(c)(b)b0102030Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/Herbal sourcec01020300 Total phenolic compounds,mg Galic acid/Intensity of electric ba01020304050aqueous alcoholicTotal phenolic compounds,mg Galic acid/gSolvent typebb0153045Linum Usitatissimum Total phenolic compounds,mg Galic acid/gaqueous Solvent bababa01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal source0 3 6Intensity of electric pulse field, kV·cm10153045Total phenolic compounds,mg Galic acid/gab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceab01002003000 3 DPPH IC50,μg/mLIntensity of electric pulse ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeabc0100200300Linum Usitatissimum DPPH IC50,μg/mL0 Intensity of electric acbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent type0100200300DPPH IC50,μg/mLba01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourcecba010203040500 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1ba01020304050aqueous alcoholicTotal phenolic compounds,mg Galic acid/gSolvent typebaba0153045Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal sourceaqueous alcoholicSolvent typebababa01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal source0 3 6Intensity of electric pulse field, kV·cm1dcdc cba01530450 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeTable 2 Interaction between herbal source, pre-treatment,and solvent type in their effects on total phenolic compoundsTotal phenoliccompounds (mgof galic acid/g)Intensity of pulsedelectric field pretreatment,kV·cm–1Type ofsolventHerbalsourceLinum aqueous 0 4.28 ± 0.21cusitatissimumalcoholic 8.11 ± 0.54caqueous 3 5.33 ± 0.37calcoholic 8.49 ± 0.181caqueous 6 5.73 ± 0.33calcoholic 10.37 ± 0.81cSalvia aqueous 0 33.24 ± 0.37bleriifolia alcoholic 51.75 ± 1.63abaqueous 3 38.63 ± 0.76balcoholic 54.81 ± 1.30abaqueous 6 44.19 ± 0.63balcoholic 63.98 ± 1.11aP < 0.05Linum usitatissimum Salvia leriifoliaLinum usitatissimum Salvia leriifoliaLinum usitatissimum Salvia leriifolia190Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195Figure 4 Binary effects of herbal source and pre-treatment (a),herbal source and solvent type (b), and pre-treatmentand solvent type (c) on antioxidant activity by DPPH method(P ˂ 0.05)the solvent type and the use of a pulsed electric fieldpretreatment.Total phenolic compounds in Linum usitalissmumoilseed have been reported by Oomah et al., Brodowskaet al., and Russo and Reggiani at 8–10 mg of caffeicacid per gram of extract), 0.988 mg of catechin per gramof extract, and 4.64–9.40 mg caffeic acid per gram ofextract, respectively [26–28]. In our study, their amountranged from 4.28 to 10.37 mg gallic acid per gram ofextract, depending on the solvent type and the use of apulsed electric field pretreatment.The studies showed that the amount of extractedphenolic compounds increased with a higher intensityof a pulsed electric field, reaching their maximum at a6 kV·cm–1 pre-treatment. Schroeder et al. attributed thisb0100Linum Usitatissimum Salvia leriifoliaDPPH μg/Herbal sourcec0 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1a0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeb0100Linum Usitatissimum Salvia leriifoliaDPPH μg/Herbal source01000 3 DPPH IC50,μg/mLIntensity of electric pulse ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeabc0100200300Linum Usitatissimum DPPH IC50,μg/mL0 Intensity of electric acbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent type0100200300DPPH IC50,μg/mL(а)(c)(b)bbb0102030Linum Usitatissimum Salvia leriifoliaTotal phenolic mg Galic Herbal source0 3 6dcdc cb015300 3 6Total phenolic compounds,mg Galic acid/Intensity of electric pulse field, kV·cm1aqueous alcoholicab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typebbb0102030Linum Usitatissimum Salvia leriifoliaTotal phenolic mg Galic Herbal source0 3 6dcdc cb015300 3 6Total phenolic compounds,mg Galic acid/Intensity of electric pulse field, kV·cm1aqueous alcoholicab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeto the electrical degradation of cells and their increasedpermeability due to the use of a pulsed electric field [29].In this regard, Bozinou et al. investigated the extractionof phenolic compounds and antioxidant activity ofdried oak leaves with a 7 kV·cm–1 pulse electric fieldpretreatment [30]. They stated that the highest amount ofphenolic compounds was obtained with a pulse time of20 ms, a pulsing duration of 40 min, and a pulse intervalof 100 ms.Liu et al. examined the enhancement of extractedphenolic compounds in onion pre-treated with apulsed electric field [31]. They stated that the optimumconditions for this purpose were a pulsed electric fieldof 2.5 kV, 90 pulses, and a temperature of 45°C. Inthese conditions, the amounts of extracted phenolicand flavonoid compounds were 86.82 mg of gallicacid per 100 g and 37.58 mg of quencherine per 100 g,respectively. These values were 2.2 times and 2.7 timesas high as those in the control samples, respectively.Figure 3 Independent effects of herbal source (a), pretreatment(b), and solvent type (c) on antioxidant activity byDPPH method (P ˂ 0.05)bbb010Linum Usitatissimum Salvia leriifoliaTotal phenolic mg Herbal source0 3 6d0150 3 6Total phenolic mg Intensity of electric pulse field, kV·cm1aqueous alcoholicab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceab01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mL Intensity of electric pulse field, kV·cm1aqueous alcoholicSolvent type(а)(c)(b)alcoholicbb015Linum Usitatissimum Salvia leriifoliaTotal phenolic mg Galic Herbal sourceaqueous alcoholicaaaSalvia leriifoliaHerbal source6pulse field, kV·cm1dcdc cba01530450 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typebSalvia leriifoliasourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1balcoholictypeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 mccSalvia leriifoliaHerbal sourcealcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent type010aqueous alcoholicTotal phenolic mg Solvent typebb015Linum Usitatissimum Salvia leriifoliaTotal phenolic mg Herbal sourceaqueous alcoholicbababa01020304050Linum Usitatissimum Salvia leriifoliaTotal phenolic compounds,mg Galic acid/gHerbal source0 3 6Intensity of electric pulse field, kV·cm1dcdc cb01530450 3 6Total phenolic compounds,mg Galic acid/gIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeab0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal sourceabc01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1ab0100200300aqueous alcoholicDPPH IC50,μg/mLSolvent typeadbdcd0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μg/mLHerbal source0 3 6Intensity of electric pulse field, kV·cm1 macbc0100200300Linum Usitatissimum Salvia leriifoliaDPPH IC50,μgr/mLHerbal sourceaqueous alcoholicSolvent typeabcbcdd01002003000 3 6DPPH IC50,μg/mLIntensity of electric pulse field, kV·cm1aqueous alcoholicSolvent typeLinum usitatissimum Salvia leriifoliaLinum usitatissimum Salvia leriifoliaLinum usitatissimum Salvia leriifoliaIC50,IC50IC , 50,IC50,IC50IC , 50,191Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195Antioxidant activity by DPPH method. Fig. 3presents independent effects of the agents, Fig. 4shows their binary effects, and Table 3 indicates theirinteraction in relation to the antioxidant activity of theextracts derived with the DPPH method. As we can see,their antioxidant activity significantly increased, at a95% confidence level, with a higher intensity of a pulsedelectric field and the use of an alcoholic solvent. At thesame time, we found that the antioxidant activity ofSalvia leriifolia extracts was higher than that of Linumusitalissmum (P < 0.05).The content of phenolic compounds is not anaccurate measure of antioxidant activity. Since theFolin-Ciocalteu reagent nonspecifically reacts withphenolic and other compounds, such as organic acids,sugars are also able to reduce this reagent. Therefore, itis also necessary to measure antioxidant activity in otherways, for example, by the DPPH method, which we usedin our study [32].Phenolic compounds donated hydrogen or electron tothe groups exposed to oxidation [33]. Thus, the contentof phenolic compounds can be used as an importantindicator of antioxidant activity. As noted above, variousfactors, such as plant variety, harvest area, and harvesttime, appear to affect the amount of phenolic compoundsand, subsequently, antioxidant activity.In our study, the antioxidant activity of extracts(IC50) from Salvia leriifolia plant extracted by theDPPH method ranged between 25.28 and 41.38 μg/mL,depending on the type of solvent and the intensity of apulsed electric field.We also investigated various sources of antioxidantactivity (IC50) in Linum usitalissmum. This value wasreported by Brodowska et al. and Alachaher et al. toreach 299.00 and 220.05 μg/mL of extract, respectively[27, 34]. In our study, the amount of total phenoliccompounds extracted from Linum usitalissmum variedfrom 157.37 to 312.51 μg/mL, depending on the solventtype and the use of a pulsed electric field pretreatment.On the other hand, we found that the extracts’antioxidant activity increased with a higher pulsedelectric field intensity. The highest values were observedin the samples with a 6 kV·cm–1 pretreatment. This wasquite predictable from the measurement of total phenoliccompounds, whose content also increased with a higherintensity of the applied electric field. In this regard,Bozinou et al. investigated the extraction of phenoliccompounds and antioxidant activity of dried oak leaveswith a 7 kV·cm–1 pulse electric field pretreatment[30]. They stated that the antioxidant activity wasproportional to the content of total phenolic compounds:the higher the amount of phenolic compounds, thehigher the antioxidant activity. In their study, phenoliccompounds were highest with a pulse time of 20 ms,Table 3 Interaction between herbal source, pre-treatment,and solvent type in their effects on antioxidant activity byDPPH methodDPPH IC50,μg/mLIntensity of pulsedelectric field pretreatment,kV·cm–1SolventtypeHerbalsourceLinum aqueous 0 312.51 ± 12.10ausitatissimumalcoholic 220.22 ± 8.06baqueous 3 304.31 ± 10.11aalcoholic 207.63 ± 12.10baqueous 6 267.81 ± 5.81abalcoholic 157.32 ± 4.16bcSalvia aqueous 0 41.38 ± 3.17cleriifolia alcoholic 33.54 ± 2.87caqueous 3 40.14 ± 1.36calcoholic 31.67 ± 2.23caqueous 6 36.91 ± 5.17calcoholic 25.28 ± 1.10cP < 0.05Figure 5 Independent effects of herbal source (a), pretreatment(b), and solvent type (c) on antioxidant activity byTEAC method (P < 0.05)(а)(c)(b)Linum usitatissimum Salvia leriifolia192Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195a pulsing duration of 40 min, and a pulse interval of100 ms. Under these conditions, the sample’s antioxidantactivity was maximum.Liu et al. studied the enhancement of extractedphenolic compounds in onion subjected to a pulsedelectric field [31]. They stated that the optimumconditions for this purpose were a pulsed electric fieldof 2.5 kV, 90 pulses, and a temperature of 45°C. Theresearchers also found that the extract’s antioxidantactivity increased with a higher pulse electric fieldintensity and a larger number of pulses applied.Their finding also proved the correlation betweenthe antioxidant activity and the amount of phenoliccompounds.Lopez Giral et al. also investigated a pulsed electricfield pretreatment to improve the extraction of phenoliccompounds from three different grape varieties(Graciano, Tempranillo, and Grenache) during twoproduction periods [35]. The pretreatment conditionsincluded a pulsed electric field of 7.4 kV·cm–1, a pulsewidth of 20 ms, and a frequency of 400 Hz. They statedthat using a pulsed electric field increased the colorintensity, total phenol index, anthocyanin index, andtotal antioxidant power. These researchers thereforeintroduced a pulsed electric field pretreatment as asuitable technology for extracting phenolic compounds.However, they acknowledged that the ability of themethod depended on the type of grape and the initialamount of phenolic compounds.Similarly, Minussi et al. demonstrated a positiverelationship between antioxidant power and the contentof total polyphenolic compounds in grape juice,particularly compounds such as gallic acid, catechin,and epi-catechin [36].Antioxidant activity by TEAC method.Independent, binary, and combined effects of the agentson the antioxidant activity of the extracts extractedwith the Trolox method are presented in Fig. 5, Fig. 6,and Table 4. As observed, a higher intensity of a pulsedelectric field pretreatment and the use of an alcoholicsolvent significantly increased the TEAC number of theextracts at a 95% confidence level. On the other hand,the number of TEAC extracts of Salvia leriifolia washigher than that of Linum usitalissmum (P < 0.05).As noted earlier, the amount of phenolic compoundsalone is not a precise measure for antioxidant activity.Since the Folin-Ciocalteu reagent nonspecifically reactswith phenolic and other compounds such as organicacids, sugars also can reduce this reagent. Therefore, it isalso necessary to measure antioxidant activity with othermethods [32]. Therefore, we used the Trolox EquivalentAntioxidant Capacity (TEAC) method to measureantioxidant activity.There was a significant correlation between thevalues of antioxidant activity measured by the DPPHFigure 6 Binary effects of herbal source and pre-treatment(a), herbal source and solvent type (b), and pre-treatment andsolvent type (c) on antioxidant activity by TEAC method(P ˂ 0.05)(а)(c)(b)Table 4 Interaction between herbal source, pre-treatment,and solvent type in their effects on antioxidant activity byTEAC methodTEAC (micromoleTrolox per gdry herb weight)Intensity of pulsedelectric field pretreatment,kV·cm–1SolventtypeHerbalsourceLinum aqueous 0 0.89 ± 0.18cusitatissimumalcoholic 12.44 ± 0.21caqueous 3 1.32 ± 0.01calcoholic 13.96 ± 1.45caqueous 6 6.92 ± 0.07calcoholic 21.48 ± 1.23cSalvia aqueous 0 108.54 ± 2.82bcleriifolia alcoholic 188.59 ± 2.37abaqueous 3 132.43 ± 1.06balcoholic 196.93 ± 2.24abaqueous 6 156.76 ± 1.14balcoholic 235.87 ± 2.87aP < 0.05Linum usitatissimum Salvia leriifoliaLinum usitatissimum Salvia leriifolia193Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195of the applied electric field. In this regard, Bozinou et al.investigated the extraction of phenolic compounds andantioxidant activity of dried oak leaves by using a pulsedelectric field pretreatment at 7 kV·cm–1 [30]. They statedthat the level of antioxidant activity was proportionalto the amount of total phenolic compounds, so a highercontent of phenolic compounds increased the antioxidantactivity. In their study, the highest amount of phenoliccompounds was associated with a pulse time of 20 ms,pulse duration of 40 min, and pulse interval of 100 ms.Under these conditions, the level of antioxidant activitywas also maximum.CONCLUSIONOur study aimed to compare the antioxidant activityof aqueous and alcoholic extracts derived from Salvialeriifolia L. and Linum usitalissmum L. subjected to apulsed electric field at the intensities of zero (withoutpre-treatment), 3 and 6 kV·cm–1 with a constant pulse of30. We investigated such parameters as total phenoliccompounds and antioxidant activity. According to ourresults, the Salvia leriifolia extract had more phenoliccompounds and higher antioxidant activity than theLinum usitalissmum extract under the same conditions.On the other hand, a pulsed electric fieldpretreatment and the use of an alcoholic solvent(methanol) for extraction increased the content ofphenolic compounds and the extract’s antioxidantactivity. In fact, the solubility of phenolic compoundsdepended on the type of solvent and their interaction.Finally, the extract derived from Salvia leriifolia with analcoholic solvent and a pulsed electric field pretreatment(at 6 kV·cm–1 with 30 pulses) was selected as possessingdesirable antioxidant properties.CONTRIBUTIONThe authors equally participated in the research andpreparation of manuscript.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterests regarding the publication of this article.method and the TEAC method (P < 0.01 and r = 0.932).The Trolox equivalent antioxidant capacity test anddiphenyl picryl hydrazyl are both synthetic free radicalswith similar application. However, the Trolox equivalentantioxidant potential can be used to measure antioxidantactivity of polar and nonpolar compounds [37].The ABTS•+ cation radical is more active thanthe DPPH radical and is therefore widely used inthe measurement of antioxidant activity. In this test,ABTS oxidation first occurred following the reactionwith potassium persulfate. The ABTS•+ cationradical subsequently reacted with antioxidants orother hydrogen donating radicals and transformed ina reduced form [37]. Consequently, the antioxidantinhibition percentage can be measured by determiningthe absorption reduction rate. The radical inhibitionactivity in this test was reported based on the Troloxequivalent antioxidant capacity.As noted above, various factors (plant variety,harvest area and time) appear to affect the amount ofphenolic compounds and, subsequently, antioxidantactivity.We found that the antioxidant activity of Salvialeriifolia extracts measured with the TEAC methodranged between 108.54 and 235.87 μmol of Trolox per gdry plant weight, depending on the type of solvent andthe intensity of pulsed electric field pretreatment.Some studies evaluated the antioxidant activity ofLinum usitalissmum with the TEAC method. Russoand Ragiani and Deng et al. reported the value of 560–860 (for oilseed Linum usitalissmum) and 22 000 μmolTrolox/g dry weight, respectively [28, 28]. In our study,the amount of total phenolic compounds extracted fromLinum usitalissmum ranged from 0.89 to 21.48 μmolTrolox/g dry weight, depending on the solvent type andthe intensity of pulsed electric field pretreatment.On the other hand, the antioxidant activity of theextracts increased with a higher pulsed electric fieldintensity. A pre-treatment of 6 kV·cm–1 providedthe highest amount of compounds. This result waspredictable from the measurement of total phenoliccompounds, which also increased with a higher intensity