Effect of jasmonic acid elicitation on the yield, chemical composition, and antioxidant and anti-inflammatory properties of essential oil of lettuce leaf basil (Ocimum basilicum L.)

Food Chemistry 213 (2016) 1–7

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Effect of jasmonic acid elicitation on the yield, chemical composition, and antioxidant and anti-inflammatory properties of essential oil of lettuce leaf basil (Ocimum basilicum L.) Urszula Złotek a,⇑, Monika Michalak-Majewska b, Urszula Szymanowska a a b

Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland Department of Fruits, Vegetables and Mushrooms Technology, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland

a r t i c l e

i n f o

Article history: Received 11 February 2016 Received in revised form 15 June 2016 Accepted 17 June 2016 Available online 18 June 2016 Keywords: Jasmonic acid Elicitation Essential oil Antioxidant activity Anti-inflammatory activity

a b s t r a c t The effect of elicitation with jasmonic acid (JA) on the plant yield, the production and composition of essential oils of lettuce leaf basil was evaluated. JA-elicitation slightly affected the yield of plants and significantly increased the amount of essential oils produced by basil – the highest oil yield (0.78 ± 0.005 mL/100 g dw) was achieved in plants elicited with 100 lM JA. The application of the tested elicitor also influenced the chemical composition of basil essential oils – 100 lM JA increased the linalool, eugenol, and limonene levels, while 1 lM JA caused the highest increase in the methyl eugenol content. Essential oils from JA-elicited basil (especially 1 lM and 100 lM) exhibited more effective antioxidant and anti-inflammatory potential; therefore, this inducer may be a very useful biochemical tool for improving production and composition of herbal essential oils. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Diet rich in vegetables, fruit, and herbs can have a positive effect on health. The health benefits of consumption of this plant food are connected with the content of many secondary metabolites. Herbs and spices are primarily used for flavoring foods, and many of their health promoting effects have also been documented. The prohealth properties of most herbs are associated with anti-allergic, anticancer, antimicrobial, antiseptic, antispasmodic, antifungal, antiviral, anti-inflammatory, analgesic, immuno-stimulating, sedative, and antioxidant activities due to their high content of polyphenols and aromatic compounds (Makri & Kintzios, 2007; Taie, Salama, & Radwan, 2010). One of the major groups of compounds, which have proven health-promoting activity, is essential oils. Essential oils (EOs) are aromatic and volatile oily liquids of an aromatic plant secondary metabolism (Škrinjar & Nemet, 2009). Aroma chemicals present in spices/herbs have had wide application in aromatherapy for a long time, but in recent years attention has been also focused on their health-promoting potential. The main biological activities of EOs are notably antibacterial, antifungal, and antioxidant (Boligon, Feltrin, & Athayde, 2013). Some natural compounds such

⇑ Corresponding author. E-mail address: [email protected] (U. Złotek). http://dx.doi.org/10.1016/j.foodchem.2016.06.052 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

as essential oils from herbs are additionally used for food protection. The general trend toward reducing the use of synthetic food additives has aroused interest in the use of essential oils as food preservatives (Jayasena & Jo, 2013). Thus, many aromatic plants are considered today as the most important sources of aromatic compounds which possess some biological activities. Sweet basil (Ocimum basilicum L.) is a popular aromatic and medicinal plant of the Lamiaceae and is well known for its aromatic leaves, used fresh or dried as a drug in traditional medicine and as a flavoring agent for foods, confectionery products, and beverages (Makri & Kintzios, 2007). There are many cultivars of basil varying in their leaf color (green or purple), flower color (white, red, purple), and aroma. The chemical composition of basil oil has been the subject of a considerable number of studies, which indicated that there is wide diversity in the constituents of basil oils depending on the variety, growing conditions, seasonal variation, etc. (Hussain, Anwar, Hussain Sherazi, & Przybylski, 2008). However, methyl chavicol, linalool, methyl cinnamate, methyl eugenol, eugenol, and geraniol are generally the major components of the oils of different chemotypes of O. basilicum (Sajjadi, 2006). The essential oil biosynthesis may be regulated by many biotic and abiotic stresses, such as wounding, light, as well as water and salinity stress (Bettaieb, Zakhama, AidiWannes, Kchouk, & Marzouk, 2009). Some abiotic elicitors involved in the stress responses in plants may cause similar reactions of plants (Zhao, Davis, & Verpoorte, 2005). Jasmonic

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2. Materials and methods

an Varian Chrompack CP-3800 gas chromatograph equipped with a 4000 GC/MS/MS mass detector and a Flame Ionization Detector (FID) and with a 30 m  0,25-mm VF-5 ms column (Varian). The carrier gas and flow rate was He at 0.5 mL/min, the injector temperature was 250 °C, the split ratio was 1:100, and the inject volume was 1 lL. A temperature gradient was applied (50 °C for 1 min, followed by increments of 4 °C/min up to 250 °C, which was retained for 10 min). The GC system consisted of a Varian 3800 Series instrument (Varian, USA) with a DB-5 column (J&W, USA) operated under the same conditions as the GC/MS (flame ionization detector 250 °C; split ratio 1:100). The identification of the essential oil constituents was based on a comparison of their retention indices relative to (C10–C40) nalkanes, compared to published data and spectra of authentic compounds. The compounds were confirmed by their retention indices taken from the literature (Adams, 2004) and from our own data. The quantitative composition of the volatile oil was determined using GC (FID) and by assuming that the total of the percentages of all oil components was 100%.

2.1. Plant materials and growth conditions

2.4. Antioxidant activity

Lettuce leaf basil seeds (O. basilicum L. cv. Crispum) were purchased from Vilmorin Garden Company. Basil seeds were sown in boxes containing universal soil for sowing seeds. Seven days postgermination, the seedlings were transplanted to 600 mL pots containing universal garden soil (four plants per pot and four pots for each variant of treatment). The plants were grown in a growth chamber (SANYO MLR-350H) at 25/18 °C, photoperiod 16/8 h day/night, with PPFD (photosynthetic photon flux density) of 500–700 lmol m
2 s
1 at a plant level, and relative humidity of 70%. The seedlings were fertilized twice (before transplantation and one week after transplantation) at the following levels (in mg L
1): N – 50, P – 50, K – 100, Mg – 60. Twenty-one-day-old plants were sprayed with a water solution of 0.01 lM jasmonic acid (JA1), 1 lM jasmonic acid (JA2), and 100 lM jasmonic acid (JA3) (Sigma) (JA had previously been dissolved in a very small amount of ethanol). The control plants (C) were sprayed with a very small amount of ethanol in deionized water. The concentrations of the elicitors were selected based on literature data (Złotek, Szymanowska, Karas´, & S´wieca, 2016; Złotek et al., 2014) and previous screening experiments (data not published) to avoid negative effects on the health of the plants. Fifteen days after elicitation, the herb was collected.

2.4.1. Free radical scavenging assays The 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid + (ABTS ) radical scavenging method according to Re et al. (1999) and the DPPH (1,1-diphenyl-2-picrylhydrazyl) assay (BrandWilliams, Cuvelier, & Berset, 1995) were used for determination of the antiradical activity of the essential oils. For the ABTS assay, 10 lL of essential oil at a concentration of 0.75, 1.5, 3, and 6 lL/mL were added to 1.9 mL of an ABTS+ solution (ABTS radical cations (ABTS+) were produced by reacting 7 mM stock solution of ABTS with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark for at least 6 h at room temperature prior to use. The ABTS+ solution was diluted to an absorbance of 0.7 ± 0.05 at 734 nm) and the absorbance at 734 nm was measured at the beginning and after 2.5 min of the reaction. The percentage inhibition was calculated using the following equation:

acid (JA), which is an endogenous phytohormone, is a potent elicitor and/or a signaling agent and plays a key role in plant growth and development; it is also involved in stress responses in plants. Some studies have suggested that exogenous JA positively influences the levels of phytochemicals/secondary metabolites in selected crops (Kim, Chen, Wang, & Rajapakse, 2006; Zhao et al., 2005; Złotek, S´wieca, & Jakubczyk, 2014). Jasmonic acid as an inducer of essential oil of two Iranian landraces of basil under reduced irrigation has been studied previously (Malekpoor, Salimi, & Pirbalouti, 2015), but many researches have indicated that the chemical composition of EOs depends also on cultivars of aroma plants and climatic factors (Hussain et al., 2008; Trevisan, Vasconcelos Silva, Pfundstein, Spiegelhalder, & Owen, 2006). Therefore, in the present study, we examined the influence of jasmonic acid elicitation on the yield, chemical composition, and some biological activities of essential oil of lettuce leaf basil.

2.2. Determination of plant yield Before harvest, plant height was determined (20 plants measured from each object). After harvest, the herb was weighed and dried (in a drying house at 35 °C) and air-dry mass was determined. 2.3. Essential oil analysis 2.3.1. Isolation of essential oil Twenty grams of powdered air-dried basil leaves were submitted to water-distillation in Deryng apparatus with 400 mL water for 3 h according to the Polish Pharmacopoeia VI (2002). The essential oil yields were measured and expressed in mL/100 g dw (dry weight). Subsequently, the essential oils obtained were dried over anhydrous sodium sulfate and stored at 4 °C until analysis. 2.3.2. Qualitative and quantitative analysis GC and GC–MS analysis methods were used to determine the composition of the essential oils. GC analysis was performed on

Scavenging % ¼ ½ðAC
AA Þ=AC   100% where: AC – absorbance of the control (solvent instead of essential oil), at 0 min, AA – absorbance of the sample after 2.5 min. The concentration of the sample (lL/mL) necessary to provided 50% of scavenging (IC50) was calculated graphically. For the DPPH assay, 10 lL of essential oil at a concentration of 0.75, 1.5, 3, and 6 lL/mL were added to 0.98 mL of a 1.92 mL 6  10
5 M methanol DPPH solution and the absorbance at 515 nm was measured at the beginning and after 2.5 min of the reaction. The percentage inhibition was calculated using the following equation:

Scavenging % ¼ ½ðAC
AA Þ=AC   100% where: AC – absorbance of the control (solvent instead of essential oil), at 0 min, AA – absorbance of the sample after 2.5 min. The concentration of the sample (lL/mL) necessary to provided 50% of scavenging (IC50) was calculated graphically. 2.4.2. Reducing power For the reducing power of the essential oil, the method of Oyaizu (1986) was used. The reaction mixture containing 100 lL of essential oil (at a concentration of 0.75, 1.5, 3, and 6 lL/mL), phosphate buffer (100 lL, 200 mmol/L, pH 6.6), and 100 lL of a 1 g/100 mL aqueous solution of potassium ferricyanide K3[Fe (CN6)] was shaken and incubated at 50 °C for 20 min. 100 lL of trichloroacetic acid (10 g/100 mL) was added to the mixture, which was then centrifuged at 25g for 10 min. The upper layer of the

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solution (200 lL) was mixed with distilled water (200 lL) and 40 lL of FeCl3 (0.1 g/100 mL). Then, the absorbance was measured at 700 nm. The IC50 value (lL/mL) is the effective concentration of essential oils at which the absorbance was 0.5 for reducing power; it was obtained by interpolation from linear regression analysis. 2.4.3. Inhibition of linoleic acid peroxidation The method of Kuo, Yeh, and Pan (1999) with slight modification was used for determination of the degree of inhibition of peroxidation of linoleic acid by the essential oil. The sample (10 lL) was added to 370 lL of phosphate buffer (5 mmol/L, pH 7) containing 0.05% Tween 20 and 4 mmol/L linoleic acid and then incubated at 37 °C for 3 min. Next, peroxidation of linoleic acid in the above reaction mixture was initiated by adding 20 lL FeCl2 (10 mmol/L) in water, followed by incubation at 37 °C for 10 min. The reaction was stopped by adding 5 mL HCl (0.6%) in ethanol to the reaction mixture. The hydroxyperoxide formed was assayed according to a ferric thiocyanate method with mixing, in sequentially, of 0.1 mL FeCl2 (0.02 mol/L) and 0.1 mL ammonium thiocyanate (30%). The absorbance of the sample was measured at 480 nm. Thus, the antioxidative activity of the sample was calculated as:

% Inhibition ¼ 1
½ðAs
A0 Þ=ðA100
A0 Þ  100% where: As – absorbance of the sample, A0 – absorbance of the base control (without hemoglobin addition), A100 – absorbance of the maximal control (no essential oil addition to the above mixture). 2.5. Anti-inflammatory activity 2.5.1. Lipoxygenase inhibitory activity Lipoxygenase (LOX) inhibitory activity assays were carried out at a temperature of 25 °C as described by Axelroad, Cheesborough, and Laakso (1981). The formation of conjugated diene (HpETEs and HETEs) catalyzed by LOX was measured kinetically by an increase in absorbance at 234 nm during 3 min using a Shimadzu UV/Vis spectrophotometer. The cuvette contained 1/15 M phosphate buffer pH 7.0, 0.02 mL of a lipoxygenase solution (167 U mL
1), and essential oil at concentrations of 0.75, 1.5, 3, and 6 lL/mL. After 3 min, the substrate solution (0.04 mL 2.5 mmol L
1 linoleic acid) was rapidly added, mixed, and the increase in absorbance versus the blank was recorded. A change in absorbance of 0.001/min was adopted as one unit of LOX activity. In parallel, LOX activity without addition of the inhibitor was measured as a control. It was shown that the ability to inhibit the activity of LOX was linearly related to the concentration of the essential oil used in the assay. This allowed determination of the IC50 values, which represent the inhibitor concentration (lL/ mL) required to inhibit the enzyme activity by 50%. 2.5.2. Cyclooxygenase inhibitory activity The cyclooxygenase 2 (COX-2) inhibitory activity of the essential oils was determined fluorometrically with using a Cayman Chemical COX-2 Fluorescent Activity Assay Kit. In this assay, resorufin is formed in the reaction between PGG2 (prostaglandin G – arachidonic acid metabolite) and ADHP (10-acetyl-3,7-dihydroxy phenoxazine). Resorufin fluorescence was measured using Quantech Base Fluorometer Thermo Scientific (excitation wavelength: 530–540 nm, emission wavelength: 585–595 nm). The inhibitory samples contained 100 mM Tris–HCl buffer (pH 8.0), 0.01 mL of heme, 0.01 mL of ADHP solution, 0.01 mL of COX-2, and different concentrations of essential oils (0.75, 1.5, 3 and 6 lL/mL), were incubated for 3 min and the reaction was started by adding 0.01 mL of a substrate (AA) solution. The measurement was performed after one minute fluorescence. The positive control (100% of COX-2 activity) was measured analogously, but without the

inhibitor. The concentration of resorufin formed during the reaction was read from a standard curve (concentration range 0.1– 2 lM). The% inhibition was calculated using the formula:

% Inhibition ¼ ½ðInitial activity
Sample activityÞ=Initial activity  100 The IC50 values represent an inhibitor concentration (lL/mL) required to inhibit the enzyme activity by 50%. 2.6. Statistical analysis The experiments were conducted three times and the whole determination was performed in triplicate. Statistical analysis was performed using STATISTICA 7.0 for mean comparison using Tukey’s test at the significance level P < 0.05. 3. Results 3.1. Effect of JA elicitation on basil plant yield The presented study showed no significant differences in the growth parameters of basil elicited with jasmonic acid, compared to the control plants (Table 1). Only the elicitation with JA3 had a significant effect on plant height, while the elicitation with JA1 increased the dry weight of the basil plants. Additionally, it should be noted that in any case there was no negative effect of the jasmonic acid elicitation on the growth and yield parameters of the studied plants (Table 1). 3.2. Effect of JA elicitation on basil essential oil yield and composition The analysis of basil essential oil yield indicated that the JA elicitation had a significant effect on this parameter (Table 1). All the concentrations of JA used in this study resulted in increased production of essential oils by the tested plants. The highest oil yield (0.78 ± 0.005 mL/100 g dw) was achieved by induction of the basil with 100 lM jasmonic acid, which represented a value greater by 36.8% relative to the control (Table 1). The chemical composition of the control and elicited basil essential oils is presented in Table 2. The GC–MS analysis indicated the presence of 67 compounds representing 95.19–99.92% of the oils. Our study demonstrated that the main components in the essential oil in all the tested samples were methyl eugenol (10.84–40.69%), eugenol (15.29–24.88%), 1,8-cineole (9.05– 20.34%), linalool (4.98–20.88%), and (Z)-caryophyllene (4.53– 9.53%). It should be noted that the JA elicitation induced a change in the content of individual oil components (Table 2). The elicitation with 100 lM jasmonic acid increased the linalool (from 4.98% in the control sample to 20.88% in the JA3 sample), eugenol (from 17.59 to 24.88%), and limonene levels (from 0.64 to 0.88%),

Table 1 Growth parameters and essential oil content of basil plants elicited with jasmonic acid.

C JA1 JA2 JA3

Plant height [cm]

Plant fresh weight [g/plant]

Plant dry weight [g/plant]

Essential oil content [mL/ 100 g dw]

10.5 ± 0.4a 10.67 ± 1.53ab 10.83 ± 1.76ab 11.33 ± 0.38b

4.87 ± 1.16a 4.95 ± 1.87a 4.51 ± 1.43a 4.58 ± 0.37a

0.35 ± 0.06a 0.45 ± 0.08b 0.31 ± 0.1a 0.31 ± 0.06a

0.57 ± 0.01a 0.65 ± 0.005c 0.62 ± 0.01b 0.78 ± 0.005d

Abbreviations: C, control; JA1, 0.01 lM jasmonic acid; JA2, 1 lM jasmonic acid; JA3, 100 lM jasmonic acid. Mean ± standard deviation. Statistically significant differences (P < 0.05) indicated various letters (a,b,c,d).

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Table 2 Effect of jasmonic acid elicitation on basil essential oils composition. RT [min]

Kovats RI

Compounds

6,17 6,24 6,46 6,95 7,06 7,35 7,64 7,80 8,16 8,44 8,79 9,07 9,33 9,49 9,60 9,72 9,84 10,07 10,49 10,91

901 903 910 926 929 939 948 953 965 974 985 994 1001 1003 1005 1007 1008 1012 1018 1024

11,45 11,63 11,95 12,23 12,43

1031 1034 1038 1042 1045

13,65 14,48 14,55 14,84 15,09 15,37 15,48

1063 1074 1075 1079 1083 1087 1089

15,91 17,35 17,36 18,54 18,93

1095 1131 1132 1166 1178

19,82 20,17 20,44 20,72 20,98 21,68 21,84 21,93 22,08 22,15 23,01 23,16 23,71 24,50 24,49 24,62 25,31 25,49 26,14 25,88

1204 1215 1223 1231 1239 1260 1264 1267 1272 1274 1302 1305 1324 1352 1352 1356 1381 1387 1389 1397

26,62 25,94 28,99 26,05 26,63 27,07 27,25 29,23 30,88 32,20 Total

1402 1403 1404 1406 1424 1437 1442 1496 1569 1619

Cumene a-Thujene a-Pinene Camphene Unidentified Unidentified Sabinene b-pinene Myrcene Unidentified d-3-Carene a-Terpinene p-Cymene Limonene 1,8-Cineole (Z)-b-Ocimene Unidentified (E)-b-Ocimene c-Terpinene cis-Sabinene hydrate Terpinolene p-Cymenene Linalool Octen-3-yl acetate cis-p-Mentha-2,8dien-1-ol Camphor d-terpineol Borneol Terpinen-4-ol p-Cymen-8-ol a-Terpineol Methyl chavicol (estragole) Octanol acetate Nerol Geraniol Bornyl acetate trans-Pinocarvyl acetate Methyl nerolate d-Elemene Unidentified a-Terpinyl acetate Eugenol a-Copaene Unidentified Unidentified Methyl Cinnamate b-Elemene Methyl eugenol b-Longipinene (Z)-Caryophyllene (E)-Caryophyllene a-Humulene trans-b-Farnesene Unidentified b-Funebrene a-Guaiene Alloaromadendrene Germacrene D Bicyclogermacrene Spathulenol a-Bulnesene c-Cadinene b-Sesquiphellandrene Eugenol acetate Spathulenol Cubenol a-Cadinol

% C

JA1

JA2

JA3

0,39% 0,05% 1,45% – – 1,52% 2,36% 0,49% – – 0,08% – 0,64% 16,68% 0,11% 0,22% 2,85% 0,16% 0,31%

0,28% 0,08% 1,75% – – – 1,41% 3,43% 0,80% – 0,05% 0,11% – 0,54% 20,34% 0,07% 0,06% 2,15% 0,19% 0,29%

0,32% 0,07% 1,42% – – 0,09% 1,16% 2,80% 0,67% 0,05% – 0,10% 0,05% 0,44% 17,25% 0,06% – 1,67% 0,17% 0,22%

– – 0,76% 0,31% – – 0,81% 1,49% 0,60% – 0,20% 0,05% 0,07% 0,88% 9,05% – – 0,26% 0,08% 0,42%

0,10% – 4,98% – –

0,10% – 8,64% – –

0,10% – 5,11% – –

0,51% 0,12% 20,88% 0,20% –

– 0,45% – 0,45% – 1,96% 0,23%

– 0,38% – 0,19% – 1,76% 0,11%

– 0,31% – 0,22% – 1,44% 0,09%

2,76% 0,30% 0,30% 0,13% 0,08% 1,96% 0,05%

0,12% 0,06% – – –

0,23% 0,13% – – –

0,13% – 0,07% – –

0,66% – – 5,37% 0,06%

0,14% – – – 17,59% – – – 0,21% 0,35% 25,81% 0,11% 9,53% 1,23% – – 1,48% 0,63% 0,45% 0,87%

0,17% – – – 18,62% – – – 0,22% 0,30% 26,91% 0,11% 8,41% 1,13% – – 0,23% 0,24% 0,07% –

– – – – 15,29% 0,07% – – – 0,17% 40,69% – 6,48% – 1,54% 1,23% – – – 0,20%

– 0,09% 0,46% 0,07% 24,88% 0,29% – 0,28% – 3,41% 10,84% – 4,53% – 0,76% 1,71% – – – 1,83%

0,32% – 0,88% – – – – – – – 95,19%

– – – – – 0,12% – 0,08% – 0,22% 99,92%

– – – – – – – – – – 99,68%

– 0,99% – 0,29% 0,30% – 0,47% 0,15% 0,08% – 99,79%

but decreased the content of some other components, for example methyl eugenol (from 25.81 to 10.84%), 1,8-cineole (from 16.68 to 9.05%), and (Z)-caryophyllene (from 9.53 to 4.53%) – Table 2. However, the elicitation with JA2 caused the highest increase in the methyl eugenol content (from 25.81 to 40.69%) in the basil essential oil, while a slight impact on the contents of linalool, 1,8-cineole, or eugenol was noted. Additionally, the essential oils from the elicited basil exhibited the presence of some compounds that were not detected in the control, for example camphene, camphor, bornyl acetate (in the JA3 sample), a-humulene, and trans-b-farnesene (in the JA2 and JA3 samples) – Table 2. 3.3. Effect of JA elicitation on antioxidant and anti-inflammatory activities of basil essential oils The antioxidant potential of the basil essential oils was evaluated on the basis of four complementary methods. It should be noted that the essential oils from the control and elicited basil exhibited strong antioxidant activity (Table 3). Additionally, the jasmonic acid elicitation influenced some antioxidant activity. A significant increase in the ABTS free radical scavenging activity was observed for the JA2 and JA3 samples (IC50 = 2.35 ± 0.02 lL/ mL and 2.35 ± 0.03 lL/mL, respectively), while the JA1 sample showed lower ability to neutralize ABTS free radicals, compared to the control (Table 3). The essential oil from basil elicited with 100 lM jasmonic acid (JA3) also showed significantly higher activity against DPPH free radicals than the essential oil from the control plants (by approx. 11%). Additionally, the JA2 elicitation resulted in an increase in the reducing power of the basil essential oil (IC50 = 1.73 ± 0.11 lL/mL, while for the essential oil of the control basil IC50 = 1.88 ± 0.05 lL/mL). The other concentrations of the elicitor used did not result in significant changes in the reducing power of the studied essential oils. Similarly, the elicitation applied in the present work did not influence the ability of the essential oil to inhibit lipid peroxidation. As shown in Fig. 1, the compounds contained in the essential oils can also act as LOX and COX-2 inhibitors. Additionally, the elicitation with JA significantly increased this ability, expressed as an IC50 value, in comparison to the control. As regards LOX inhibition, the lowest IC50 value was obtained for the JA3-sample (IC50 = 0.264 ± 0.002 lL/mL), compared to 0.3 ± 0.004 lL/mL for the C-sample (Fig. 1A). A slightly higher value was obtained for the essential oil from the JA1 elicited basil (IC50 = 0.267 ± 0.003 lL/mL), while the ability of the JA2-sample to inhibit LOX did not significantly differ from the control sample (Fig. 1A). The essential oil from the JA3-elicited basil also showed the highest ability to inhibit COX-2 activity (IC50 = 4.09 ± 0.045 lL/ mL), which represented a 12.41% increase in comparison to the control. A somewhat higher value (but positively differing from the control) was observed for the JA2-sample (Fig. 1B).

Table 3 Effect of jasmonic acid elicitation on antioxidant activity of essential oil of basil. Sample

IC50 [lL/mL] ABTS

C JA1 JA2 JA3

DPPH b

2.46 ± 0.05 2.66 ± 0.10c 2.35 ± 0.02a 2.35 ± 0.03a

RP a

5.74 ± 0.20 6.29 ± 0.16b 5.98 ± 0.24ab 5.08 ± 0.20c

LPO a

1.88 ± 0.05 1.85 ± 0.04a 1.73 ± 0.11b 1.89 ± 0.07a

4.81 ± 0.58a 5.85 ± 1.21a 5.66 ± 1.30a 5.38 ± 0.59a

Abbreviations: C, control; JA1, 0.01 lM jasmonic acid; JA2, 1 lM jasmonic acid; JA3, 100 lM jasmonic acid. Mean ± standard deviation. Statistically significant differences (P < 0.05) indicated various letters (a,b,c).

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Fig. 1. Effect of jasmonic acid elicitation on anti-inflammatory activity determined as LOX inhibition (A) and COX-2 inhibition (B) of essential oils of basil. Abbreviations: C, control; JA1, 0.01 lM jasmonic acid; JA2, 1 lM jasmonic acid; JA3, 100 lM jasmonic acid. Statistically significant differences (P < 0.05) indicated various letters (a,b,c).

4. Discussion Elicitation, being a method of improving plant resistance, can be biotic or abiotic in nature. Abiotic elicitors include common environmental stresses such as temperature, salinity, water, radiation, or mechanical stresses, but also some plant phytohormones can act as elicitors (Zhao et al., 2005). It is known that environmental stress can adversely affect plant yield and growth, but on the other hand, they may stimulate the production of plant bioactive compounds. Elicitation with some chemical compounds like jasmonic acid act as abiotic elicitors may also affect plant growth. Some researchers have reported that elicitation may cause plant growth reduction (Román et al., 2011). On the other hand, there are also reports which demonstrate positive or no effects on the elicitation of plant growth and yield. For example, in the study conducted by Oh, Carey, and Rajashekar (2009), mild heat, chilling, and high light stress treatment of lettuce for a short time had a negligible effect on plant growth. In turn, elicitation with a red algae Kappaphycus alvarezii extract improved the yield of Phaseolus radiata L. (Zodape, Mukhopadhyay, Eswaran, Reddy, & Chikara, 2010) as well as foliar chitosan spray of basil plants caused an increase in the plant height and weight (Kim, Chen, Wang, & Rajapakse, 2005). In the present study, the JA application had no significant effect on the growth parameters of the studied basil. Although a small positive impact of the JA3 elicitation on plant height (by approx. 8%) and the JA1 application on basil dry weight (by 28%) was observed, in other cases there was no significant difference in the yield parameters of the tested plant. Similarly, in the study by

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Alavi-Samani, Kachouei, and Ghasemi Pirbalouti (2015) on thyme, an application of jasmonic acid had no significant effect on plant growth indices. Other researchers (Hamedi, Pirbalouti, & Moradi, 2014) also reported that foliar application of JA had no significant effect on St. John’s Wort (Hypericum perforatum L.) growth parameters e.g. the number of branches, plant height, and dry weight. Therefore, the influence of elicitation on plant growth may depend on plant condition, the elicitor type and dose, etc. As indicated in many studies, elicitation with biotic and abiotic factors can also influence the amount and composition of secondary metabolites produced by plants (Kim et al., 2006; Złotek et al., 2014). Essential oil components are a group of plant secondary metabolites of great importance both for plants and for consumers of herbal plants. The essential oil content in herbal plants definitely depends on the plant species but also on the cultivar as well as environmental conditions and agronomic techniques (Arraiza, Andrés, Arrabal, & López, 2009; Hussain et al., 2008). The yields of aroma chemicals from basil presented in the literature ranged from 0.37% for basil grown in Serbia and Montenegro (Bozˇin, Mimica-Dukic, Simin, & Anackov, 2006) to even 1.9% in the Purple Opal cultivar (Beatovic´ et al., 2015). In our study, the essential oil yields of the control and JA1-, JA2-, and JA3elicited lettuce leaf basil were 0.57 ± 0.01%, 0.65 ± 0.005%, 0.62 ± 0.01%, and 0.78 ± 0.005%, respectively (Table 1). The increase in the essential oil content in the plants obtained in the present study as a result of the elicitation corresponds with investigations conducted by Malekpoor et al. (2015), Gharib (2006), and Fard, Omidbaigi, Sharifi, Sefidkon, and Behmanesh (2012), in which elicitation with jasmonic acid, salicylic acid, and methyl jasmonate, respectively, had a positive effect on the yield of plant essential oils. In our research, the major components in the essential oil of lettuce leaf basil in all the treatments and the control were methyl eugenol, eugenol, 1,8-cineole, linalool, and (Z)-caryophyllene (Table 2). The results obtained in this work partially correspond with other investigations. According to the data available in the literature (Beatovic´ et al., 2015; Lee, Umano, Shibamoto, & Lee, 2005), the major constituents of basil essential oils were linalool, methyl chavicol, eugenol, methyl eugenol and cadinol, neral, 1,8-cineole, geranial, but the percentage of these individual components varies quite significantly in studies reported by various researchers. The results obtained by Beatovic´ et al. (2015) indicated that linalool was the predominant compound in the essential oil from basil cultivars growing in Serbia. In contrast, the major volatiles detected in basil herb by Lee et al. (2005) were linalool, estragole, methyl cinnamate, eugenol, and 1,8-cineole. In the essential oils extracted from basil growing in three provinces (Baghe-Bahadoran, Shahreza and Falavarjan) in Iran, methyl chavicol was present at the highest concentration (Hadipanah, Ghahremani, Khorrami, & Ardalani, 2015). As some researchers suggested, the composition of the essential oil contained in basil varies considerably depending on such factors as basil cultivars, plant origin, and leaf and flower color (Hadipanah et al., 2015; Malekpoor et al., 2015). Additionally, environmental conditions as such as some stresses e.g. wounding, pathogen attack, water, temperature, or osmotic stress can induce production of secondary metabolites in plants. It has been reported that some abiotic elicitors, including plant hormonal chemicals such as jasmonic acid, can act as stresses and can influence production of various groups of plant bioactive compounds (Alavi-Samani et al., 2015; Gharib, 2006; Malekpoor et al., 2015). JA is a plant regulator inducing production a wide variety of plant secondary metabolites including terpenoids, flavonoids, and alkaloids by activation of the JA signaling pathway (Ghasemi, Pirbalouti, Rahimmalek, Elikaei-Nejhah, & Hamedi, 2014). The foliar application of jasmonic acid in the present study also induced biosynthesis of basil essential oils. The different

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concentrations of the tested elicitor had slightly different effects on the major compounds of the essential oils. The results presented in Table 2 demonstrate that basil treated with 100 lM jasmonic acid produced the highest levels of linalool (20.88%) and eugenol (24.88%), but basil treated with 1 lM jasmonic acid yielded the highest levels of methyl eugenol (40.69%). In turn, 0.1 lM jasmonic acid resulted in the highest concentration of 1,8-cineole (20.34%) and b-pinene (3.43%). Our results were similar to the previous report that eugenol and linalool could be induced in sweet basil by methyl jasmonate treatment (Kim et al., 2006). In a study conducted by Malekpoor et al. (2015), basil treated with jasmonic acid produced a higher amount of linalool and 1,8-cineole, likewise in our study, but these researchers achieved the highest increase in the content of methyl chavicol in basil elicited with 400 lL of JA. Additionally, in the research conducted by Ghasemi et al. (2014), foliar application of JA at 50 lL caused a significant increase in some summer savoury essential oil components, namely sabinine, a-phellandrene, b-phellandrene, (Z)-b-ocimene, (E)-b-ocimene, linalool, (Z)-verbenol, citronellol, and bornyl acetate and a decrease in the content of two main components – carvacrol and cterpinene. As indicated in other reports, exogenous application of JA also induced biosynthesis of other groups of plant biologically active compounds such as flavonoids, anthocyanins, carotene, or vitamin C (Szymanowska, Złotek, Karas´, & Baraniak, 2015; Złotek et al., 2014, 2016). Therefore, in the literature, jasmonic acid is generally considered an integral signal for the biosynthesis of many plant secondary metabolites (Zhao et al., 2005). As mentioned above, the effect of elicitation with some abiotic inducers on the yield and composition of plant essential oils has already been tested, but still little is known about the influence of plant elicitation on the biological properties of essential oils. Generally, the biological activity of herbal plant essential oils is known and has been widely studied (Boligon et al., 2013). The antioxidant and antimicrobial properties of basil essential oils were previously reported (Beatovic´ et al., 2015; Hussain et al., 2008; Lee et al., 2005). Regarding their biological activities, the composition of the oils is of great importance. As suggested by Beatovic´ et al. (2015), the antioxidant potential of the oils was correlated with the major proportion of volatile compounds and with compounds possessing a phenolic ring with an OH group (for example eugenol). In a study performed by Politeo, Jukic, and Milos (2007), among the compounds present in the essential oil from basil, eugenol was considered the main contributor of the antioxidant capacity. On the other hand, in a study conducted by Trevisan et al. (2006), the essential oil obtained from O. basilicum Var. purpurascens containing linalool as the major component but lacking a phenol ring also showed a relatively strong antioxidant capacity. Similarity, the results obtained in the present study indicated that lettuce leaf basil essential oils act as effective antioxidants (Table 3). Additionally, it should be mentioned that the elicitation with jasmonic acid had a significant impact on some of the antioxidant activities. The highest antiradical potential (against DPPH and ABTS) was exhibited by the essential oil from basil elicited with 100 lM jasmonic acid (JA3 sample). Probably, this could be attributed to the higher levels of linalool and eugenol, in comparison to the control sample. Our results correspond with the study conducted by Alavi-Samani et al. (2015), in which foliar application of 400 lL JA caused an increase in antioxidant activity of thyme essential oil. Trevisan et al. (2006) also indicated a positive correlation between a high proportion of eugenol and antioxidant capacity of essential oils. According to the literature data, some essential oils (depending on the chemical composition) demonstrate anti-inflammatory activities as well (Miguel, 2010; Wei & Shibamoto, 2010). Inflammation and oxidation are closely related: antioxidants quench free radicals that damage cells and lead to inflammation. In the present

study, the ability of the essential oils obtained from the elicited basil to inhibit the activity of two enzymes of the inflammatory process (LOX and COX-2) was studied. According to the literature (Miguel, 2010) as well as our observations, it was possible to conclude that the anti-inflammatory activities of herbal essential oils also depend on their chemical composition. Our results indicate that basil essential oils are a good source of natural antiinflammatory compounds (Fig. 1). Additionally, the elicitation with jasmonic acid increased this capability of the essential oil, especially in the JA-3 sample (Fig. 1). The analysis of the essential oil composition (Table 2) indicated that anti-inflammatory activity was correlated with an increased level of certain individual oil components. Namely, in the essential oil from the JA3 sample, a significant increase in the eugenol, linalool, and limonene content was detected, in comparison to the control sample. Also the literature data indicate that these essential oil components (i.e. eugenol, linalool, and limonene) possess anti-inflammatory activity (Miguel, 2010; Peana et al., 2002; Sumiwi, Sihombing, Subarnas, Abdassah, & Levita, 2015). Additionally, the study performed by Sumiwi et al. (2015) indicated that eugenol and limonone can act as COX-2 inhibitors, which is in agreement with our results, as the JA3 sample was found to have the highest ability to inhibit COX-2 activity in our study. Therefore, it may be assumed that these individual oil components can act as COX-2 inhibitors. As suggested by Miguel (2010), the mechanism of the anti-inflammatory potential of oils may be connected with the ability of some oil components to modulate arachidonic metabolism or cytokine production, or modulation of pro-inflammatory gene expression. In conclusion, our results indicate that elicitation with jasmonic acid may increase the yield of oil in basil and influence the composition of essential oils. Because the essential oils from the JAelicited basil (especially JA2 and JA3) exhibited more efficient antioxidant and anti-inflammatory potential, using these oils in food production can positively influence their health-promoting qualities. Thus, elicitation with jasmonic acid could be a very useful tool for use in herb production, especially in the context of yield and biological activity of essential oil. References Adams, R. P. (2004). Identification of essential oil compounds by gas chromatography/ quadrupole mass spectroscopy. Carol Stream: Allured Business Media. Alavi-Samani, S. M., Kachouei, M. A., & Ghasemi Pirbalouti, A. (2015). Growth, yield, chemical composition, and antioxidant activity of essential oils from two thyme species under foliar application of jasmonic acid and water deficit conditions. Horticulture, Environment, and Biotechnology, 56(4), 411–420. Arraiza, M. P., Andrés, M. P., Arrabal, C., & López, J. V. (2009). Seasonal variation of essential oil yield and composition of thyme (Thymus vulgaris L.) grown in Castilla—La Mancha (Central Spain). Journal of Essential Oil Research, 21(4), 360–362. Axelroad, B., Cheesborough, T. M., & Laakso, S. (1981). Lipoxygenases in soybeans. Methods in Enzymology, 71, 441–451. Beatovic´, D., Krstic´-Miloševic´, D., Trifunovic´, S., Šiljegovic´, J., Glamocˇlija, J., Ristic´, M., & Jelacˇic´, S. (2015). Chemical composition, antioxidant and antimicrobial activities of the essential oils of twelve Ocimum basilicum L. cultivars grown in Serbia. Records of Natural Products, 9 (1), 62–75. Bettaieb, I., Zakhama, N., AidiWannes, W., Kchouk, M. E., & Marzouk, B. (2009). Water deficit effects on Salvia officinalis fatty acids and essential oils composition. Scientia Horticulturae, 120, 271–275. Boligon, A. A., Feltrin, A. C., & Athayde, M. L. (2013). Determination of chemical composition, antioxidant and antimicrobial properties of Guazuma ulmifolia essential oil. American Journal of Essential Oils and Natural Products, 1(1), 23–27. Bozˇin, B., Mimica-Dukic, N., Simin, N., & Anackov, G. (2006). Characterization of the volatile composition of essential oils of some Lamiaceae species and the antimicrobial and antioxidant activities of the entire oils. Journal of Agricultural and Food Chemistry, 54, 1822–1828. Brand-Williams, W., Cuvelier, E., & Berset, C. M. (1995). Use of free radical method to evaluate antioxidant activity. LWT – Food Science and Technology, 28, 25–30. Fard, F. R., Omidbaigi, R., Sharifi, M., Sefidkon, F., & Behmanesh, M. (2012). Effect of methyl jasmonate on essential oil content and composition of Agastache foeniculum. Journal of Medicinal Plant Research, 6(45), 5701–5705. Gharib, F. A. E. (2006). Effect of salicylic acid on the growth, metabolic activities and oil content of basil and marjoram. International Journal of Agriculture & Biology, 8 (4), 485–492.

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