Effect of abiotic elicitation on main health-promoting compounds, antioxidant activity and commercial quality of butter lettuce (Lactuca sativa L.)

Food Chemistry 148 (2014) 253–260

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Effect of abiotic elicitation on main health-promoting compounds, antioxidant activity and commercial quality of butter lettuce (Lactuca sativa L.) Urszula Złotek ⇑, Michał S´wieca, Anna Jakubczyk Department of Biochemistry and Food Chemistry, University of Life Sciences, Ul. Skromna 8, 20-704 Lublin, Poland

a r t i c l e

i n f o

Article history: Received 1 June 2013 Received in revised form 3 October 2013 Accepted 7 October 2013 Available online 19 October 2013 Keywords: Elicitation Lettuce Health-promoting compounds Quality of lettuce

a b s t r a c t The study presents changes in the phytochemical levels, antiradical activity and quality of lettuce caused by different chemical elicitors: arachidonic acid (AA), jasmonic acid (JA), and abscisic acid (ABA). The application of 1 lM and 100 lM JA induced an increase in the concentration of phenolic compounds, including flavonoids and phenolic acids. Flavonoid levels were also increased after treatment with 100 lM AA and ABA. Some of the elicitor concentrations used also caused an increase in the levels of other phytochemicals, such as chlorophyll a (1 lM and 100 lM AA, 50 lM ABA); chlorophyll b (100 lM AA); carotenoids (100 lM AA, 1 lM JA and 100 lM ABA) and vitamin C (100 lM AA, 100 lM JA). The highest antiradical activity was noted after treatment with 100 lM AA, 100 lM JA. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging ability was positively and significantly correlated with flavonoid, chlorophyll and carotenoid levels. These results may suggest that the antiradical activity of lettuce was determined not only by phenolics, but also by other bioactive compounds. Elicitation did not change the sensory quality of lettuce. Therefore, treatment with elicitors could be a useful tool for improving the health-promoting qualities of lettuce without the loss of sensory quality. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Diets rich in fresh fruit and vegetables are associated with a lower risk of many chronic diseases (Llorach, Martínez-Sánchez, Tomás-Barberán, Gil, & Ferreres, 2008). Lettuce (Lactuca sativa L.) is one of the most popular leaf vegetables, which is predominantly consumed fresh (Mulabagal et al., 2010). The pro-health properties of lettuce are attributed to its low calorific value, and its large supply of vitamins, micro- and macronutrients and others phytochemicals, including polyphenols and fibre (Llorach et al., 2008). There are basically five different types of lettuce: butterhead, crisphead (iceberg), leaf, romaine and stem, but the most popular are butterhead and iceberg. However, butterhead and iceberg lettuces are characterized by a lower nutritional quality compared to the other varieties of lettuce and, based on the fact that consumer interest in the quality and safety of food is steadily increasing, research aimed at improving the quality of lettuce is fully justified (Mulabagal et al., 2010). The value of fresh vegetables depends on visual characteristics as well as on nutritional quality (Kim et al., 2008). A number of fac-

⇑ Corresponding author. Tel.: + 48 81 4623328; fax: +48 81 4623324. E-mail address: [email protected] (U. Złotek). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.10.031

tors, including plant genotype and growing conditions, can have a large impact on the quality of vegetable crops, especially on phytochemical levels. There are three main ways to improve the quality of fresh vegetables: genetic modification, agronomical manipulation (especially fertilization) and elicitation (Ramakrishna & Ravishankar, 2011; Schreiner, 2005). Due to the lack of public acceptance of transgenic food, elicitation has recently become a popular way not only to enhance the resistance of plants to biotic and abiotic stresses, but also to improve the quality of edible plants, especially in relation to their health-promoting phytochemicals (Martínez-Ballesta et al., 2008). To date, elicitation has been widely used to increase production or to induce de novo synthesis of secondary metabolites in in vitro plant cell cultures (Ramakrishna & Ravishankar, 2011), but recently there has been growing interest in using elicitation as a method of improving the quality of whole edible plants. There are some studies regarding increasing the resistance of lettuce to pathogens and the effect of chemical elicitors on some phytochemicals but little has been reported about changes in the consumer quality of butter lettuce (Kim, Fonseca, Choi, & Kubota, 2007). JA, AA and ABA, which are endogenous phytohormones, are potent elicitors and/or signaling agents and play a key role in plant growth and development as well as being involved in stress responses in plants. ABA is a natural compound synthesized by

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all plants, which plays important roles throughout plant life and development, affecting seed germination, plant growth, senescence and plant stress responses (Zhao, Davis, & Verpoorte, 2005). AA and JA are lipid-derived plant compounds, which are included in the class of endogenous plant growth regulators. Some studies have suggested that exogenous AA, ABA and JA positively influence the levels of phytochemicals/secondary metabolites in selected crops (Kim et al., 2007; Zhao et al., 2005). The applications of these elicitors in promoting crop quality have been previously studied (Kim, Chen, Wang, & Choi, 2006; Kim et al., 2007; Sun, Yan, Zhang, & Wang, 2012), but further studies are still needed, due to the fact that different horticultural products contain various phytochemicals and react differently to different plant regulators. The objectives of this study were to determine the influence of certain chemical elicitors (JA, AA, ABA) on the quality (i.e., commercial quality, which was estimated by instrumental and sensory methods, health-promoting phytochemicals, antiradical activity) of fresh lettuce. The goal of the research was to develop an alternative to both traditional and genetic modification, which is still not acceptable to consumers.

2.3. Analysis of health-promoting compounds 2.3.1. Extraction and analysis of phenolic compounds Frozen leaf tissue (2.5 g) was ground with a mortar and pestle with 15 mL of 50% (v/v) acidified methanol (0.1 M HCl) and the phenolic compounds were extracted for 20 min at room temperature, then centrifuged at 9000g for 30 min – this procedure was repeated three times and the supernatants were combined – this produced a crude extract of polyphenols. The raw methanolic extract was then evaporated to dryness under a vacuum at a temperature of 40 °C and rinsed with 100% methanol to a final volume of 10 mL. 2.3.1.1. Determination of total phenolic compounds (TPC). The amount of total phenolics was determined using Folin–Ciocalteau reagent (Singleton, Orthofer, & Lamuela-Raventos, 1974). To 0.5 mL of the sample, 0.5 mL H2O, 2 mL Folin–Ciocalteau reagent (1:5 H2O) was added, after 3 min, 10 mL of 10% (w/v) Na2CO3 and the contents were mixed and allowed to stand for 30 min. Absorbance at 725 nm was measured in a UV–Vis spectrophotometer. The amount of total phenolics was calculated as gallic acid equivalent (GAE) in mg per g of fresh weight (FW).

2. Materials and methods 2.1. Chemicals Arachidonic acid (P99% purity), jasmonic acid (P97% purity), abscisic acid (P99% purity), Folin–Ciocalteau reagent, DPPH (1,1diphenyl-2-picrylhydrazyl), standards of phenolic compounds and ascorbic acid (HPLC grade), m-phosphoric acid, DTT (DL-Dithiothreitol), Trizma buffer, Trolox ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid and solvents used for HPLC analysis were purchased from Sigma–Aldrich Company, USA. Any other chemicals were of analytical grade.

2.2. Plant materials and growth conditions The lettuce seeds (Lactuca sativa L. var. capitata) were purchased _ from PNOS Ozarów S.A., Poland. Lettuce seeds were sown into sowing boxes filled with universal soil for sowing seeds. Seven-day-old seedlings were transplanted to 600 mL pots containing universal garden soil with sand (4:1 v/ v) – four plants per pot. Plants were grown in a growth chamber (SANYO MLR-350H) at 25/18 °C, photoperiod 14/10 h day/night, with photosynthetic photon flux density (PPFD) at a plant level of 500–700 lmol m 2s 1 and a relative humidity of 70%. The crop was fertilized as recommended by the Regional Chemical–Agricultural Station in Lublin, Poland at the following levels (in mg l 1): N – 40, P – 445, K – 290, Mg – 100, Ca – 770, Cu – 50. Twenty-oneday-old plants of equal size were selected (eight lettuce plants in each treatment, n = 56) to be sprayed with a solution of the elicitors to be tested (1.5 mL per plant): 1 lM arachidonic acid (AA1), 100 lM arachidonic acid (AA2), 1 lM jasmonic acid (JA1), 100 lM jasmonic acid (JA2), 50 lM abscisic acid (ABA1) and 100 lM abscisic acid (ABA2) prepared in deionized water (elicitors were previously dissolved in very small amounts of ethanol (1 mL) and dispersed in appropriate volumes of water to achieve desirable concentrations). The control plants (C) were sprayed with only ethanol dissolved in deionized water. The concentrations of elicitors were selected based on previous screening experiments (data not published), so as not to evoke negative effects on the health and growth of plants. Fifteen days after elicitation the plants were collected, frozen and used in the biochemical analysis. A proportion of the samples were freeze-dried. The experiments were conducted in triplicate.

2.3.1.2. Determination of flavonoid content (TFC). Total flavonoid content was determined according to the method described by Lamaison and Carnet (1990). One millilitre of extract was mixed with 1 mL of 2% (w/v) AlCl3  6H2O solution (in methanol) and incubated at room temperature for 10 min. Thereafter, absorbance at 430 nm was measured. Total flavonoid content was calculated as quercetin equivalent (QE) in lg per g of fresh weight (FW). 2.3.1.3. Determination of phenolic acid content (PAC). Total phenolic acid estimation was carried out according to the Arnov method (Szaufer-Hajdrych, 2004). One millilitre of sample was mixed with 5 mL of distilled water, 1 mL 0.5 M HCl, 1 mL of Arnov reagent (10 g sodium molybdate and 10 g sodium nitrite dissolved in 100 mL of distilled water) and 1 mL 1 M NaOH, and then distilled water was added to a final volume of 10 mL. Absorbance was measured at 490 nm. The total phenolic acid content was expressed as caffeic acid equivalent (CAE) in lg per g of fresh weight (FW). 2.3.1.4. HPLC analysis. Quantitative–qualitative HPLC analysis of the phenolic compounds was performed according to the method described by S´wieca, Gawlik-Dziki, Kowalczyk and Złotek (2012). Samples were analyzed with a Varian ProStar HPLC System separation module (Varian, Palo Alto, USA) equipped with a Varian ChromSpher C18 reverse phase column (250  4.6 mm) and a ProStar DAD detector. The column thermostat was set at 40 °C. The mobile phase consisted of 4.5% acetic acid (solvent A) and 50% acetonitrile (solvent B) at a flow rate of 0.8 ml min 1. At the end of the gradient, the column was washed with 50% acetonitrile and equilibrated to the initial condition for 10 min. Quantitative determinations were conducted with an external standard calculation, using calibration curves of the standards. Gradient elution was used as follows: 0 min 92% A, 30 min 70% A, 45 min 60%, 80 min 61% A, 82 min 0% A, 85 min 0% A, 86 min 92% A, and 90 min 92% A. Detection was performed at 270 and 370 nm. Phenolic compounds present in a sample were identified by comparing retention times and UV–Vis absorption spectra with those of the standard compounds. Phenolics were expressed as lg per g of fresh weight (FW). 2.3.2. Extraction and determination of chlorophylls (chl) and carotenoids (car) levels Chl and car were analyzed according to the method described by Lin et al. (2013). Chl and car were eluted overnight from the freeze-dried leaves samples (0.05 g) with 2 mL 80% (v/v) acetone

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at 4 °C. The sample was then centrifuged at 13,000g for 5 min. The supernatant was applied to determine the absorbances of chl a, chl b and car in acetone, as measured with a spectrophotometer, at the respective wavelengths of 663, 645 and 470 nm. Concentrations of chl a, chl b and car were determined from the following equations: Chl a = 12.72  A663 – 2.59  A645 Chl b = 22.88  A645 – 4.67  A663 Car = (1000  A470 – 3.27  chl a – 104  chl b)/229, and expressed in lg/g DW. 2.3.3. Extraction and determination of vitamin C content Total ascorbic acid content was determined as a sum of ascorbic and dehydroascorbic acid, according to modified versions of the methods described earlier by Campos, Ribeiro, Della Lucia, Pinheiro-Sant’Ana, and Stringheta (2009). Briefly, 100 mg of lyophilized lettuce leaf material was extracted two times with 2 mL of 50% (w/v) m-phosphoric acid (MPA). The mixture was centrifuged at 16,000g, and extracts were combined and used for further determination. 500 lL of extract was mixed with 500 lL DTT 30 mM in 0.5 M Trizma buffer for 5 min. Then, 650 lL of 5% (w/v) MPA was added. Samples were filtered using a 0.45 lm Millipore and analyzed with a Varian ProStar HPLC System separation module (Varian, Palo Alto, CA) equipped with a Varian ChromSpher C18 reverse phase column (250  4.6 mm) column and a ProStar 325 UV–Vis detector. The column thermostat was set at 25 °C. The separation was performed under isocratic elution conditions using a mobile phase consisting of 30 mM KH2PO4 adjusted with 5 M HCl to pH 3, at a flow-rate of 0.8 mL min 1 and detection at 245 nm. Quantitative determination was conducted with an external standard calculation, using calibration curves of the standard. Ascorbic acid content was expressed in mg per 100 g of dry mass (DM). 2.4. Antioxidant activity Antioxidant activity was determined using a free radical scavenging assay. The free radical scavenging activity was measured using DPPH (1,1-diphenyl-2-picrylhydrazyl) – according to Brand-Williams, Cuvelier, and Berset (1995) as the source of the free radicals. For the DPPH assay, the 80 lL of methanolic extracts was mixed with 1.92 mL 6  10 5 M solution of DPPH in methanol. Absorbance at 515 nm was measured immediately and after 2.5 min of incubation. The affinity of the test material to quench DPPH free radicals was evaluated according to the equation: scavenging% = [(AC – AA) / AC)]  100, where: AC – absorbance of control at 0 min., AA – absorbance of sample after 2.5 min. The antiradical activity was related to Trolox (an analogue of vitamin E) and expressed as mM of Trolox per gram of fresh weight (FW) (TEAC, Trolox equivalent antioxidant activity). 2.5. Quality of lettuce 2.5.1. Instrumental analysis Instrumental texture analysis was performed according to Ghorpade, Li, Gennadios, and Hanna, (1995). Firmness was evaluated with a TA.XTplus Texture Analyzer via the puncture test. A sample with a diameter of 50 mm was mounted in a holder in the form of two plates with a hole having a diameter of 30 mm, and then placed in a texture analyzer. Lettuce leaf blades pierced a metal ball with a diameter of 2 mm, acting perpendicular to the sample surface, at a speed of 1 mm/s. The value of the force required to pierce the lettuce leaf was recorded using Exponent version 5.1.2.0 and expressed in Newtons (N). Leaf colour was measured on the leaf surface with an X-RiteColourÒ 8200 colorimeter (X-Rite Inc., USA). The CIE colour values L⁄ (lightness), a⁄ (redness), b⁄ (yellowness) were measured to

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describe the colour of the lettuce leaf. The measuring aperture diameter was 12.7 mm, and D65/10° was the illuminant/viewing geometry. The colorimeter was calibrated using the standard of white (L⁄ = 95.82; a⁄ = 0.44; b⁄ = 2.5) (McGuire, 1992). 2.5.2. Sensory evaluation The influence of elicitation on the sensory characteristics of lettuce was evaluated according to Murata, Tanaka, Minoura, and Homma (2004). The panel for sensory analysis was composed of 20 members, aged from 21 to 40 years (12 women, 8 men). Appearance, colour, aroma, crispness, flavour and total estimation of lettuce were evaluated on a scale of 1–5 (1 – bad, 5 – good). 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. Data were evaluated using the correlation coefficient (R) at the significance level p < 0.05 to identify relationships between phytochemical content and antiradical activity and the quality parameters of lettuce using STATISTICA 7.0. 3. Results and discussion 3.1. Effect of elicitation on the levels of health-promoting compounds in lettuce leaves Chemicals such as polyamines, salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and arachidonic acid (AA) are involved in stress responses in plants (Ramakrishna & Ravishankar, 2011). Therefore, besides improving the resistance of plants against biotic and abiotic stresses, elicitation may enhance the synthesis of bioactive compounds of interest for human health, thus improving the quality of edible plants. Usually, plants respond rapidly to stress/elicitors by producing secondary metabolites, including bioactive compounds, but the induced levels are commonly not stable for a long time (Kim et al., 2007). Usually, plants respond rapidly to stress/elicitors by producing secondary metabolites, including bioactive compounds, but the induced levels are commonly not stable for a long time. Therefore, from a consumer point of view and for the determination of the real quality of the elicited lettuce, it was important to investigate the content and the activity of health-promoting phytochemicals at a certain time after elicitation (ready to eat leaves). Thus, in the present studies the quality of the lettuce was determined after 15 days of elicitation (36th day of cultivation). In recent years, special attention has been focused on phenolic compounds because of their antioxidant properties and different preventive roles against diseases associated with oxidative stress, such as cancer, and cardiovascular and neurodegenerative diseases (Riedel et al., 2012). It is well known that the content of phenolics in fruit, vegetables and sprouts is generally affected by environmental and genetic factors, such as the cultivar, abiotic factors as illumination and temperature or chemical elicitors (S´wieca et al., 2012). In this research, the content of total phenolics, phenolic acids and flavonoids in the lettuce leaves elicited by JA1 and JA2 increased markedly in comparison to control plants (Fig. 1A–C). Additionally, the amount of phenolic acids in lettuce leaves has also been significantly increased after stimulation of AA2 and ABA2 It should be noted that the overproduction of phenolics caused by jasmonic acid corresponds perfectly with the results obtained by Nafie, Hathout, and Al Mokadem (2011). A similar elicitation effect was also observed for some other plants, such as

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e

Total phenolic compounds content [mg(GAE)/g FW]

1.4

A

1.2

1

d

0.8 0.6

bc

ab

b

ab a

0.4 0.2

0 C

AA1

AA2

JA1

JA2

ABA1

35

B

g

30

Flavonoids content [mg (QE)/g FW]

25

ABA2

f

e

d

20

c

15 b

10

a

5 0 C

AA1

AA2

JA1

JA2

ABA1

ABA2

200

C

e

180 160 Phenolic acids content [mg (CAE)/g FW]

140

d

120 100 80

c

b

60

c

b

a

40 20 0 C

AA1

2.0

AA2

Antiradical activity [mM Trolox/g FW]

JA2

ABA1

ABA2

e

e d

1.5

JA1

D d

c

1.0

b

0.5 a

0.0 C

AA1

AA2

JA1

JA2

ABA1

ABA2

Fig. 1. Effect of elicitation on content of total phenolic compounds (A), flavonoids (B), phenolic acids (C) and antiradical activity (D) of lettuce leaves. C, control;A A1, 1 lM arachidonic acid; AA2, 100 lM arachidonic acid; JA1, 1 lM jasmonic acid; JA2, 100 lM jasmonic acid; ABA1, 50 lM abscisic acid; ABA2, 100 lM abscisic acid. Values designated by the different letters are significantly different (p < 0.05).

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iceberg lettuce, soybean, white lupin seedlings, birch leaves, and tomato leaves (Kim et al., 2006). The HPLC results indicated the presence of five hydroxycinnamic and hydroxybenzoic acids and two flavonoids (kaempferol and quercetin) in the control lettuce, whereas in the elicited plants one more flavonoid – luteolin – was identified (Table 1). The dominant phenolic acid fractions in the lettuce leaves were caffeic, chlorogenic, and ferulic acids and derivates of coumaric acid (Table 1). It should be noted that elicitation with JA increased luteolin, ferulic, caffeic, chlorogenic and o-coumaric acid levels, additionally JA1 led to increased quercetin and JA2 kaempferol (Table 1). Moreover, the level of caffeic acid was elevated at a higher degree in plants also elicited with AA and ABA, and chlorogenic acid in plants elicited with AA (Table 1). It has been reported that polyphenols are mainly produced through the phenylpropanoid that can be induced by stresses or elicitors (Zhao et al., 2005). Therefore, it may be suggested that PAL might be related with the induction of the phenolic compounds of the lettuce treated with chemical elicitors (especially JA). Induction of the synthesis of ferulic and chlorogenic acids may be associated with their antifungal function as well as with their role in reducing disease through the formation of defence barriers (especially lignin and suberin) and activation of defence responses (ElKhallal, 2007). Induction of the biosynthesis of ferulic and chlorogenic acids after elicitation with salicylic acid and jasmonic acid was observed by El-Khallal (2007) in the leaves of tomato seedlings. An increase in the level of caffeic and chlorogenic acids in the leaves of lettuce after treatment with methyl jasmonate was also reported by Kim et al. (2007). Similarly, Nafie, Hathout, and Al Mokadem (2011) noted an increase in caffeic and p-coumaric acid biosynthesis in melon cell cultures after stimulation with jasmonic acid. Lettuce, like most green leafy vegetables, is a major source of dietary carotenoids and chlorophylls which also may have specific

dietary activities. The role of this plant’s pigments has been associated with reducing the risk of cancer, heart disease, stroke and cataracts (Caldwell & Britz, 2006). Carotenoids are also secondary metabolites with significant antioxidant activity, whereas some studies have demonstrated that chlorophyll inhibited COX-1 and COX-2 enzymes (Kim et al., 2007; Mulabagal et al., 2010). Vitamin C, present in lettuce leaves, also acts as an effective antioxidant (Mulabagal et al., 2010). Data concerning the chlorophyll, carotenoid and vitamin C contents in lettuce treated with elicitors are shown in Table 2. Chlorophyll a content significantly increased as a result of AA1, AA2 and ABA1 treatments, whereas there was no significant difference in chlorophyll a content between the control and the other elicitors used. No significant change in either chlorophyll b or total chlorophyll contents was observed between abiotic elicitor treatments and the control, except for induction with AA2. Lettuce elicited with AA2, JA1 and ABA2 was characterized by significantly higher levels of carotenoid content compared to controls (43.9%, 48.7% and 24.5%, respectively). Additionally, over a 3-fold increase in vitamin C content was detected in lettuce after elicitation with AA2 and JA2 (Table 2). Previous studies have proved that some abiotic elicitors could increase the levels of those health-promoting compounds studied in this study, including vitamin C, chlorophyll or carotenoids (Sun et al., 2012), although in the study by Kim et al. (2007) the carotenoid content in methyl jasmonate-treated romaine lettuce was not changed. 3.2. DPPH assay The antioxidant activity (determined via DPPH free radical scavenging assay) was significantly increased by both JA and AA treatments; however the highest activity was determined for AA2 and JA2. The capacities reached maximum values of 2 mM of TE/g FW for JA2 and 1.9 mM of TE/g FW for AA2, which constitute

Table 1 Qualitative and quantitative analysis of phenolic compounds in lettuce leaves after treatment with different elicitors. Compounds [lg/g FW]

Elicitor

Kaempferol Quercetin Luteolin Ferulic acid Caffeic acid Chlorogenic acid o-Coumaric acid p-Coumaric acid

C

AA1

AA2

JA1

JA2

ABA1

ABA2

3.01 ± 0.50c 0.44 ± 0.12b n.d. 10.97 ± 0.76d 1.73 ± 0.32a 2.67 ± 0.40b 0.17 ± 0.05c 0.72 ± 0.16b

n.d. 0.33 ± 0.01a 0.09 ± 0.007a 4.48 ± 0.06b 5.90 ± 0.15b 9.28 ± 0.39f 0.14 ± 0.03bc 0.15 ± 0.04a

n.d. 0.89 ± 0.02c 0.19 ± 0.04a 3.63 ± 0.01a 12.89 ± 0.25d 5.15 ± 0.10e 0.08 ± 0.001a 0.36 ± 0.33a

2.51 ± 0.005b 0.93 ± 0.03c 0.13 ± 0.01a 46.04 ± 0.67f 9.91 ± 0.44c 4.94 ± 0.18d 0.25 ± 0.03d 3.54 ± 0.08c

5.18 ± 0.14d 0.34 ± 0.02ab 0.13 ± 0.008a 21.89 ± 0.42e 6.19 ± 0.16b 3.33 ± 0.20c 0.46 ± 0.04e 0.93 ± 0.05b

n.d. 0.31 ± 0.008a 0.34 ± 0.03b 3.57 ± 0.13a 14.19 ± 1.59d 0.71 ± 0.03a 0.09 ± 0.03a 0.30 ± 0.05a

0.31 ± 0.05a 0.24 ± 0.01a 0.37 ± 0.03b 7.43 ± 0.22c 14.65 ± 0.54d 2.28 ± 0.31b 0.15 ± 0.005bc 0.23 ± 0.02a

Abbreviations: C, control; A A1, 1 lM arachidonic acid; AA2, 100 lM arachidonic acid; JA1, 1 lM jasmonic acid; JA2, 100 lM jasmonic acid; ABA1, 50 lM abscisic acid; ABA2, 100 lM abscisic acid, n.d. – not detected. Values designated by the different letters (a-f) are significantly different (p < 0.05).

Table 2 Influence of chemical elicitors on chlorophyll (chl), chl a, chl b, carotenoids (car) and vitamin C (vit C) contents in lettuce leaves. Elicitor

Compounds (mg/100 g dw) Chl a

C AA1 AA2 JA1 JA2 ABA1 ABA2

Chl b a

247.45 ± 3.8 376.34 ± 17.36c 483.22 ± 104.32d 341.29 ± 25.50ab 208.39 ± 7.82ab 264.79 ± 27.04bc 262.92 ± 19.11a

Chl a + b a

130.64 ± 3.52 173.44 ± 19.07a 311.61 ± 159.02b 157.80 ± 11.02a 100.90 ± 3.52a 129.31 ± 11.13a 122.10 ± 7.60a

Car ab

378.08 ± 7.16 549.78 ± 34.14a 794.83 ± 260.99c 499.10 ± 36.37a 309.29 ± 10.82b 394.10 ± 38.17ab 385.03 ± 26.68ab

Vit C ab

21.85 ± 0.7 24.33 ± 1.27bc 31.45 ± 2.43d 32.50 ± 2.54d 20.98 ± 1.84ab 18.45 ± 3.05a 27.21 ± 2.10c

115.93 ± 1.29d 45.64 ± 5.92a 367.61 ± 11.53b 60.94 ± 1.08a 358.75 ± 28.03b 16.69 ± 0.30c 68.30 ± 0.97a

Abbreviations: C, control; AA1, 1 lM arachidonic acid; AA2, 100 lM arachidonic acid; JA1, 1 lM jasmonic acid; JA2, 100 lM jasmonic acid; ABA1, 50 lM abscisic acid; ABA2, 100 lM abscisic acid. Values designated by the different letters (a-d) are significantly different (p < 0.05).

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Table 3 Sensory evaluation of control and treated with different elicitors lettuce leaves. Elicitor

Appearance Colour Aroma Crispness Flavor Total estimation

C

AA1

AA2

JA1

JA2

ABA1

ABA2

4.3 ± 0.61a 4.3 ± 0.44a 4.3 ± 0.74a 4.3 ± 0.24b 4.1 ± 0.67a 4.1 ± 0.67a

4.1 ± 0.67a 4.7 ± 0.44a 4.0 ± 0.60a 4.2 ± 0.18b 3.7 ± 0.74a 4.0 ± 0.43a

4.0 ± 0.60a 4.1 ± 0.79a 4.0 ± 0.74a 3.8 ± 0.21a 3.8 ± 0.71a 4.1 ± 0.51a

4.5 ± 0.48a 4.7 ± 0.44a 4.3 ± 0.44a 3.5 ± 0.15a 4.1 ± 0.67a 4.4 ± 0.47a

4.0 ± 0.60a 4.0 ± 0.60a 3.6 ± 0.87a 4.3 ± 0.61ab 3.7 ± 0.61a 3.9 ± 0.51a

4.2 ± 0.57a 4.1 ± 0.67a 3.9 ± 0.67a 4.1 ± 0.67ab 4.0 ± 0.43a 4.2 ± 0.38a

3.9 ± 0.79a 4.1 ± 0.67a 4.0 ± 0.74a 4.2 ± 0.27b 3.9 ± 0.67a 3.9 ± 0.79a

Abbreviations: C, control; AA1, 1 lM arachidonic acid; AA2, 100 lM arachidonic acid; JA1, 1 lM jasmonic acid; JA2, 100 lM jasmonic acid; ABA1, 50 lM abscisic acid; ABA2, 100 lM abscisic acid. Values designated by the different letters (a-b) are significantly different (p < 0.05).

Table 4 Correlations coefficients (R) between bioactive compounds content and antiradical activity and quality of fresh lettuce. DPPH

Colour parameters L⁄

TPC TFC PAC Kaempferol Quercetin Luteolin Ferulic acid Caffeic acid Chlorogenic acid o-Coumaric acid p-Coumaric acid Car Chl a Chl b Vit C

0.37 0.75⁄ 0.32 0.15 0.71⁄ 0.70⁄ 0.12 0.48⁄ 0.73⁄ 0.09 0.19 0.55⁄ 0.69⁄ 0.55⁄ 0.08

0.20 0.19 0.18 0.46⁄ 0.33 0.47⁄ 0.17 0.17 0.14 0.08 0.12 0.39 0.09 0.17 0.26

a⁄ 0.04 0.11 0.04 0.08 0.13 0.14 0.01 0.04 0.04 0.01 0.03 0.23 0.26 0.31 0.42

Firm b⁄ 0.07 0.05 0.08 0.07 0.11 0.03 0.06 0.00 0.10 0.09 0.01 0.11 0.20 0.27 0.22

Sensory evaluation App

0.37 0.22 0.46⁄ 0.18 0.46⁄ 0.79⁄ 0.12 0.03 0.64⁄ 0.15 0.06 0.81⁄ 0.58⁄ 0.42 0.31

0.13 0.09 0.12 0.16 0.21 0.11 0.08 0.09 0.08 0.11 0.10 0.07 0.17 0.21 0.20

C

Ar 0.08 0.02 0.03 0.01 0.11 0.36 0.12 0.13 0.25 0.01 0.17 0.17 0.22 0.23 0.20

0.27 0.01 0.31 0.15 0.07 0.23 0.28 0.05 0.12 0.36 0.26 0.18 0.09 0.25 0.00

Cri 0.07 0.34 0.03 0.04 0.01 0.07 0.04 0.25 0.42 0.11 0.05 0.01 0.27 0.11 0.08

Fla 0.07 0.34 0.03 0.04 0.01 0.07 0.04 0.25 0.42 0.11 0.05 0.01 0.27 0.11 0.08

Test 0.10 0.29 0.07 0.07 0.06 0.14 0.09 0.13 0.31 0.01 0.04 0.08 0.18 0.09 0.10

Abbreviations: TPC, total phenolic content; TFC, total flavonoids content; PAC, phenolic acids content; Car, carotenoids; Chl a, chlorophyll a; Chl b, chlorophyll b; Vit C, vitamin C; DPPH, radical scavenging ability; Firm, firmness; App, appearance; C, colour; Ar, aroma; Cri, crispness; Fla, Flavor; Test, total estimation. Values designated by ⁄ are statistically significant at p < 0.05.

an increase of 53.8% and 46.1% in relation to the control, respectively (Fig. 1D). This result only partly coincided with the phenolic results. The DPPH scavenging ability of lettuce was highly and positively correlated with flavonoid content (R = 0.74), and positively, but not highly, with total phenolic and phenolic acid contents (R = 0.37, R = 0.32, respectively) – Table 4. Polyphenols are a class of biologically active compounds with widely documented antioxidant properties, but recently attention has been drawn to the many other properties of polyphenols, such as the ability to inhibit enzymes involved in the formation of inflammation, such as lipoxygenase (LOX), xanthine oxidase (XO) or cyclooxygenase (COX) (Gawlik-Dziki, S´wieca, Sugier, & Cichocka, 2011; Mulabagal et al., 2010). Phenolic compounds have been reported to be highly correlated with antioxidant activity in lettuce elicited with methyl jasmonate (Kim et al., 2007). However, an earlier study by Liu et al. (2007) suggested that the antioxidant capacity of a solution containing a mixture of phenolic compounds would depend on the specific phenolic profile, which can be qualitative (i.e., the type of phenolics present) or quantitative (i.e., the relative amounts or proportions of phenolics present). Correlation analysis conducted in the present work has confirmed these findings. In this study, the DPPH scavenging ability of lettuce was significantly and positively correlated with chlorogenic acid and quercetin contents, but significantly and negatively with caffeic acid and luteolin contents (Table 4). Furthermore, besides phenolic compounds, there are various other compounds with significant antioxidant activity in plants, including carotenoids, terpenoids and some vitamins. Numerous studies have reported that vitamin C and carotenoids

participate in the creation of the antioxidant properties of a plant (Sun et al., 2012). In this study, antioxidant activity was significantly and positively correlated with chlorophyll a, b and carotenoid contents (Table 4). Our results suggested that the estimated antioxidant activity might be determited to by phenolics, carotenoids and other unknown antioxidant compounds.

3.3. Effect of elicitation on the quality of lettuce leaves Little information on the sensory evaluation of lettuce, especially elicited lettuce, is available in the literature. Sensory properties are very important for consumer assessment of vegetable quality. Many indicators of lettuce quality, including colour, texture, and flavour, may be influenced by abiotic and biotic factors (Kleinhenz et al., 2003). The loss of quality of fresh fruit and vegetables may be caused by induction of the activity of the main enzymes responsible for the darkening of the plant tissue. Moreover, literature data show that many plant phenolic compounds are characterized by a bitter taste (Lesschaeve & Noble, 2005). Phenolic compounds also play an important role during enzymatic browning of plant tissues, because they are substrates for the browning enzymes (Arnnok et al., 2012). Additionally, the colour of leaf vegetables depends on the level of pigments such as chlorophylls or carotenoids. Nevertheless, research connected with stimulation of the production of phytochemicals in edible plants often does not incorporate the sensory evaluation of this food. Therefore, in the present study, the quality of the elicited lettuce was assessed via instrumental and sensory analysis.

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Firmness [N]

L*

a*

b*

1.8 ab

ab

ab d

Firmness [N]

1.4 1.2

b

68

ab

a

cd

58

bc

bc

48

ab a

38

1.0

a a

a

0.8

a

a

a

a

a

28

L* a* b*

1.6

b

18

0.6

8

0.4 0.2

ab

ab

ab

a

ab

b

-2 b

0.0

-12 C

AA1

AA2

JA1

JA2

ABA1

ABA2

Fig. 2. Effect of elicitation on colour and firmness of lettuce leaves. Colour parameters: L⁄ (lightness), a⁄ (redness), b⁄ (yellowness); C, control; A A1, 1 lM arachidonic acid; AA2, 100 lM arachidonic acid; JA1, 1 lM jasmonic acid; JA2, 100 lM jasmonic acid; ABA1, 50 lM abscisic acid; ABA2, 100 lM abscisic acid. Values designated by the different letters are significantly different (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Examination of the colour of lettuce leaves in the present study shows that the applied elicitors did not cause significant changes in the parameters L ⁄ (brightness), a⁄ (redness), b⁄ (yellowness) – a statistically significant decrease in the brightness occurred only in lettuce treated after elicitation with ABA1 (a reduction in parameter L ⁄ of 3.2%) – Fig. 2. To date, no studies have been conducted to evaluate colour parameter changes as a result of chemical elicitor treatment, but there are some using other abiotic stresses. For example, Kim et al. (2008), studying the influence of salinity stress on lettuce, found that the colour of the lettuce leaves changed with higher doses of NaCl treatments (50 mM, 100 mM, 200 mM), while lower doses (5 mM) did not lead to a change in the colour of the lettuce. Texture analyses performed in the present study demonstrated that elicitation may cause changes in the firmness of lettuce leaves. An increase in this parameter was observed after treatment with AA2 and JA1 (Fig. 2). Appearance, colour, aroma, crispness and flavour are the major marketable properties of fresh lettuce, and therefore were selected for the sensory analysis. The sensory evaluation conducted in this study showed no significant differences in the panelists’ opinion about the quality of either elicited or un-treated lettuce leaves, except for the crispness of lettuce elicited with AA2 and JA1 (Table 3). Lettuce treated with these elicitors had a lower level (respectively 3.8 and 3.5) for this parameter than the control plants (4.3). Additionally, it should be noted that the concentration of the tested elicitors used in this study did not change the yields of lettuce (data not shown). The statistical analysis performed in this study showed that sensory evaluation was not significantly correlated with bioactive compound contents (Table 4). It can suggest that it is probable that the phytochemicals studied in the present work are not involved in the changes in the quality of lettuce. 4. Conclusion In conclusion, this study indicates that lettuce elicitation by tested inducers, especially by JA and AA, may enhance its healthpromoting qualities without a loss in appearance and other sensory quality parameters Follow obtained results using 100 lM arachidonic acid (AA2), 1 lM jasmonic acid (JA1), 100 lM jasmonic acid (JA2) treatment is most effective. These findings reveal that

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