Impact of foliar potassium fertilization on biochemical composition and antioxidant activity of fig (Ficus carica L.)

Scientia Horticulturae 253 (2019) 111–119

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Impact of foliar potassium fertilization on biochemical composition and antioxidant activity of fig (Ficus carica L.)

T

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Badii Gaalichea, , Afef Ladharib, Armando Zarrellic, Mehdi Ben Mimounb a

Laboratory of Horticulture, National Agricultural Research Institute of Tunisia (INRAT), IRESA-University of Carthage, Hédi Karray Street, 1004 El Menzah, Tunis, Tunisia b Université de Carthage, Institut National Agronomique de Tunisie (INAT), Laboratoire GREEN-TEAM (LR17AGR01), 43 avenue Charles Nicolle, 1082, Tunis, Tunisia c Department of Chemical Sciences, Complesso Universitario di Monte S. Angelo, University Federico II, Via Cintia 4, 80126, Naples, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Fig Achenes Fertilization Potassium sulphate Antioxidant activity Phenolic compounds

The metabolic processes involved in fig production are influenced by macro- and micronutrients supply to the trees during the growing season. Potassium is an essential plant nutrient that influences growth and fruit quality. In this study, the effect of foliar potassium sulphate (K2SO4) application on biochemical composition and antioxidant activity of fig was assessed in cv. Bouhouli, a commonly cultivated cultivar in Djebba (Northwest of Tunisia). Foliar potassium sulphate supply at 2% on Bouhouli trees were applied twice during the fruit growth. Results exhibited that potassium fertilization induces a significant change in total phenolic, flavonoid contents and radical scavenging activity in total fruit and achenes compared to the control. A strong correlation was observed between antioxidant activity and different phenolic compounds. The total fruit and achenes methanol extracts possess, respectively, the highest values of polyphenol by 29.3 and 25.1 mg GAE/g DW compared to the control. Similarly, the flavonoids content was increased in methanol extracts, respectively, by 36 and 48%. HPLC analyses revealed the influence of potassium on concentrations of phenolic compounds in fig sprayed with K2SO4. Among the polyphenols, the chlorogenic acid, cyanidin 3-rutinoside and cyanidin 3,5-diglucoside contents increased from 0.87 to 1.70 mg/g DW under potassium spray, whereas those compounds were not detected in fig achenes. The present study provides clear evidence that potassium sulphate can be used to manipulate total phenolic concentrations in fig with strong antioxidant potential that could be benefits to human health. Thus, potassium sulphate application at 2% could improve nutritional and qualitative attributes of fig.

1. Introduction

enzymes activities in plants, by the modulation of photosynthesis rate as well as an increase in the translocation rate from leaves through the phloem to storage tissue, leading to improve the yield and fruit quality (Saykhul et al., 2013). Foliar potassium application is an attractive solution especially in arid zones under low rainfall conditions where the lack of water in summer is drastically low (Ghanem and Ben Mimoun, 2010). Southwick et al. (1996) indicated that uptake of potassium from foliar spray may be more predictable and efficient than uptake from the soil, where soil-cation interactions may delay the process. The efficacy of foliar nutrient fertilization depends not only on the absorption of the nutrients but also on their indirect transfer to young leaves and reproductive tissues (Mengel, 2002). Although, the fruit quality could be influenced by orchard cultural practices, particularly nutrient management. Indeed, the excessive and the insufficient levels of potassium nutrient adversely affect fruit quality of pistachio (Zeng et al., 2001), grapevine (Delgado et al., 2004) and cotton (Pettigrew et al., 2005).

The tremendous demands of global food security are related to the increase of world population and climate change that could pose major challenges for the horticultural industry and researchers with respect to sustainability (Duhamel and Vandenkoornhuyse, 2013). Fertilization of horticultural plants is one of the most promising tools in order to rise the production, but it is an important agrotechnical issues. Plants use more than 60 different elements, but not all of them are designated nutrients (Nurzynska-Wierdak et al., 2012). Mineral nutrition is one of the major tools to optimize fruit yield and quality (Tagliavini and Marangoni, 2002). Foliar nutrition became most popular among fruit tree growers that could contributes to satisfy plant nutrient requirement (Inglese et al., 2002). The potassium is considered an important mineral nutrient for all stages of protein synthesis that contributes for all plant growth processes (Arquero et al., 2006). Potassium controls several

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Corresponding author. E-mail address: [email protected] (B. Gaaliche).

https://doi.org/10.1016/j.scienta.2019.04.024 Received 8 December 2018; Received in revised form 25 March 2019; Accepted 11 April 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

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potassium sulphate improves primary metabolites in plants that could influence the biosynthesis of some antioxidant compounds. Consequently, this treatment will increase the fruit quality for economic and health purposes. Thus, we purposed in this study to understand the effectiveness of potassium in improving fruit quality, by determining the biochemical composition and antioxidant activity of fig cv. Bouhouli sprayed with potassium sulphate. The chemical composition was characterized through the investigation of the phenolic compounds in total fruit and achenes under potassium treatment compared to control.

Potassium is also important for proper development and quality of other fruits such as apricot, olive, peach and strawberry (Ben Mimoun and Marchand, 2016; Saykhul et al., 2016; Song et al., 2017; Dbara et al., 2018). Thus, the adequate potassium application improves the ascorbic acid concentrations (Zareei et al., 2018), fruit color and quality (Neilsen et al., 2004; Nava et al., 2007) by increasing fruit weight, sugar and anthocyanin concentrations, firmness and K uptake (Solhjoo et al., 2017). In the other side, the manipulation of fertilizer, especially potassium, influences the levels of primary and secondary compounds in plants (Liaqat et al., 2012). It could lead to different carbon/nitrogen ratios and consequently differences in the production of secondary metabolites (such as phenolics) in plants (Bryant et al., 1983). Several previous studies explained phenolic variation in tomato, sea buckthorn and strawberry grown with different fertilizers, but they were somewhat unconvincing (Wang and Millner, 2009). Thus, regulating phenolic compounds in fruits through mineral nutrition is more considered among health-conscious consumers, because of their higher biological properties (Wang et al., 2009; Zareei et al., 2018). Otherwise, these natural products arouse great interest from scientists for their possible use in agriculture for their allelopathic potential (Ladhari et al., 2013; Gaaliche et al., 2017), antifungal (DellaGreca et al., 2007), antioxidant (Crisosto et al., 2010; Tanwar et al., 2014) and for their use in the prevention of oxidative stress-related pathologies (Di Fabio et al., 2015). Usually, phenolic compounds are naturally occurring in fruits, which act as defense against ultraviolet radiation, and have an important attribute to determine fruit sensory qualities (colour, flavour and taste) (Del Caro and Piga, 2008). Fruits are a huge horticultural group and include a lot of species and numerous cultivars, genotypes, accessions, etc.., occurring in most parts of the world as cultivated, semi-wild and wild. These groups are significant genetic resources of biodiversity, which aid the life system on earth (Korkmaz et al., 2016; Shokouh et al., 2018; Koç et al., 2018; Yildiz and Çolak, 2018; Usanmaz et al., 2018). Fruits in nature are very precious for medicine and chemistry, gaining significance in the avoiding and treatment of illness (Okatan, 2018). Fig (Ficus carica L.) is a common fruit worldwide due to its international trade, while the world fig production is concentrated specially in the Mediterranean countries (Flaishman et al., 2008). It is considered as the most popular food that has been sustaining humanity since the beginning of history (Çalişkan and Polat, 2011) . Figs are considered as a good source of bioactive compounds, mainly flavonoids and phenolic acids (Russo et al., 2014; Veberic, 2016). Both phenolic acids and flavonoids are believed to be responsible for the wide spectrum of pharmacological activities attributed to fig (Mawa et al., 2013). It has been demonstrated that F. carica possesses high nutritional values (Trad et al., 2014), which include dietary fibres, amino acids, vitamins, minerals, sugars, organic acids, carotenoids and antioxidant polyphenols (Lianju et al., 2003; Slatnar et al., 2012). In Tunisia, fig is one of the fruit crop species better adapted to different environmental areas and soils (Gaaliche et al., 2012). Fig plantations are widespread over the country occupying about 30.000 ha and the total annual fig production is estimated by 26.000 metric tons (MARH, 2017). However, fig growers often faced difficulties to reach the sufficient yield and high fruit quality, due to the poor caprification and mineral nutrition (Gaaliche et al., 2011; Ben Mimoun et al., 2017). Therefore, mineral nutrition is considered as an efficient cultural practice to improve fig fruit quality, by enhancing the mineral status and reducing the percentage of fruit ostiole cracking (Irget et al., 2008; Ben Mimoun et al., 2017). In fact, many studies have investigated the effects of potassium fertilization on the vegetative and yield aspects of other species, but relatively few studies have investigated the response of plant secondary metabolites. However, there is no information about the phytochemical responses of F. carica to potassium nutriment. The phytochemical characterization of fig under potassium fertilization could be the suitable tool for their valorisation. According to the obtained previous results in literature, the

2. Materials and methods 2.1. Plant material and experimental design The present study was performed in a commercial fig orchard located in Northwest of Tunisia (Djebba: altitude, 700 m; latitude, 36°40′N; longitude 9°0′E). The climate of this region is sub-humid with mild winter and hot summer. Annual average temperature is around 20 °C. Thermal amplitude is about 16.5 °C in summer and 8 °C in winter. Average annual rainfall is about 600 mm. The experimental orchard has typical alluvial and clay soil with high water retention capacity. Data related to the experimental site for physical and chemical soil properties are displayed in Table 1. Agricultural practices including caprification, pruning and irrigation were done according to standard practices in the area. Djebba region is known by fig culture with many specific fig genotypes that are very appreciated locally and nationally (Gaaliche et al., 2012). ‘Bouhouli’ is the main fig cultivar grown commercially in Djebba and since 2012 it has been designated as protected denomination of origin (AOC Label) “Djebba figs”. According to Trad et al. (2014), this cultivar is characterized by high fruit quality performance, which has the predominance of nutritional compounds compared to other Tunisian fig cultivars. Twenty-year-old fig trees cv. Bouhouli planted at 8 m × 8 m spacing were selected for the current field trial. The foliar potassium treatments were applied twice during the fruit growth on July 20 and August 05, 2016. Ten trees were sprayed by a solution of 2% soluble potassium sulphate (Ben Mimoun et al., 2017). At harvest, all the fruits were picked in their commercial maturity stage, from the 10 treated trees and from 10 controlled trees. The fruit maturity stage was determined on the basis of their softening, development of typical fruit taste and colour. 2.2. Extraction The collected fresh samples were immediately frozen at -20 °C until Table 1 Physiochemical properties of the soil at the experimental site in 0.00.20 m.

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Properties

Values

Clay (%) Loam (%) Sand (%) pH EC (mS/cm at 25 °C) Total calcium (%) Active calcium (%) Organic matter (%) Total N (%) C/N Exchangeable calcium (Ca ppm) Exchangeable magnesium (Mg ppm) Exchangeable sodium (Na ppm) Exchangeable potassium (K2O ppm) Available phosphorus (P2O5 ppm)

39.38 33.28 27.33 8.37 0.16 37.07 14.56 4.98 2.06 14.07 10,111.26 292.44 50.61 364.18 47.11

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thermal block at 95 °C for 90 min. Then the prepared solution was cooled and measured at the absorbance of 765 nm against a blank. The total antioxidant capacity is expressed as equivalents of ascorbic acid or gallic acid. The antioxidant capacity was estimated using following formula: Antioxidant effect (%) = [(control absorbance - sample absorbance)/(control absorbance)] × 100.

extraction. Then, the pulps of fruits were partly thawed (5 h at room temperature) before blending in a food processor. The fig achenes were then separated from the pulp by pressing through a strainer. The total fruits and collected achenes were rinsed several times with running, cold water and left to dry at room temperature. Dried total fruit and achenes were powdered and stored at −20 °C before extraction. Hundred grams of total fruit and achenes from treated and untreated (control) of cv. Bouhouli were extracted successively with petroleum ether, ethyl acetate and methanol in their increasing order of polarity. The extracts were recovered, filtered using 0.45 μm filter and stored at −20 °C until further analysis within a maximum period of one week. The prepared organic extracts were used in this study for phytochemical analysis and antioxidant activities.

2.4. Phytochemical composition 2.4.1. Total phenolics content (TPC) Total phenolics content was determined using the Folin-Ciocalteau method (Sineiro et al., 1996). One hundred microliter of each extract was mixed with 500 μl of Folin-Ciocalteu reagent (previously diluted 10-fold with distilled water) and allowed to stand at 22 °C for 5 min in the dark. Then, 400 μl of sodium bicarbonate (7.5% (v/v)) solution was added to the mixture. After 90 min at 30 °C, absorbance was measured at 765 nm using a spectrophotometer (Lambda 25, UV/vis Spectrometer). Total phenolics content were expressed as mg gallic acid equivalent per g of dry weight using gallic acid calibration curve (R2 = 0.971). Samples of each extraction were analysed in triplicate.

2.3. Evaluation of antioxidant activities The antioxidant activities of petroleum ether, ethyl acetate and methanol extracts of total fruit and achenes of fig at different concentrations were performed with ascorbic acid as standard compound by using three complementary tests, i.e., DPPH radical-scavenging, hydrogen peroxide radical scavenging and phosphomolybdenum assays.

2.4.2. Total flavonoids content (TFC) The total flavonoids in total fruit and achenes extracts was determined according to the described method by Quettier et al. (2000). The aliquots of extracts (500 μl) were added with same volume to 2% aluminium chloride (AlCl3). Absorbance at 430 nm was recorded after 30 min of incubation in the obscurity and flavonoid content was expressed as mg of quercetin equivalent per g of dry weight, using quercetin calibration curve (R2 = 0.966).

2.3.1. DPPH radical scavenging activity assay The antioxidant activity of the fractions was measured in vitro by 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay through the described method by Bursal and Gülçin (2011). The stock solution was prepared by dissolving 2.4 mg DPPH with 100 ml methanol and stored at 20 °C until required. The obtained solution was diluted with methanol until reaching an absorbance of about 0.98 ± 0.02 at 517 nm using the spectrophotometer. A 3 ml aliquot of this solution was mixed with 100 μl of the sample at various concentrations (100–1000 μg/ml). The reaction mixture was shaken well and incubated in the dark for 30 min at room temperature. The absorbance was measured at 517 nm against blank samples. A decrease in absorbance indicates DPPH free radical scavenging activity. The scavenging activity was estimated through the following equation:

2.4.3. Determination of total tannins content (TTC) Total tannins content was determined spectrophotometrically according to Broadhurst and Jones (1978). The prepared organic extracts (500 μl) contained in a test tube covered with aluminum foil, was mixed with 3 ml of 4% vanillin–methanol solution and then with 1.5 ml of hydrochloric acid. The mixture could stand for 15 min at 20 °C in the dark. The absorbance of the mixture was measured at 500 nm. Different concentrations of tannic acid aqueous solution (30 mg/l) were used for calibration. The results were expressed as mg catechin equivalent/g dry weight (mg CE/g dw) using catechin calibration curve (R2 = 0.996).

Scavenging effect (%) = [(control absorbance- sample absorbance)/ (control absorbance)] × 100 The results were reported as EC50 value, the effective concentration of antioxidant agent (extract) providing inhibition 50% of the initial DPPH radical concentration. The lowest EC50 value indicates the strongest ability of sample to act as an antioxidant. Ascorbic acid was used as an antioxidant standard. Tests were carried out in triplicate.

2.4.4. HPLC analysis The total fruit and achenes methanolic extracts were subjected to HPLC analysis for phenolic compounds identification. The analysis of phenolic compounds was performed using HPLC SHIMADZU mod., equipped with UV–vis detecor array dector (Shimadzu), reversed phase colum (Phenomenex 250 x 10 mm i.d). The solvent system used was a gradient of water–acetic acid (98:2) (A) and methanol (B), starting with 5% (v/v) methanol and installing a gradient to obtain 8% B at 10 min, 15% B at 15 min, 35% B at 20 min, 50% B at 40 min, 60% B at 50 min, and 100% B at 80 min, at a solvent flow rate of 2 ml/min. Triplicate samples were injected at a level of 50 μl. The column effluent was monitored at 280, 320, 350 and 520 nm. Quantification was achieved by injection of standard solutions of known concentrations. The standards used were as following: chlorogenic acid (5-O-caffeoylquinic acid), (+)-catechin, rutin (quercetin-3-rutinoside), quercetin-3-glucoside, quercetin, cyanidin-3-glucoside and cyanidin-3-rutinoside and 2,5 dihydroxybenzoic acid. The compounds in each extract were identified by comparing their retention times and UV–vis spectra with authentic standards and with the library of spectra previously compiled by the authors. Quantification was achieved by the absorbance recorded in the chromatograms relative to external standards.

2.3.2. Hydrogen peroxide scavenging activity Hydrogen peroxide solution (2 mM) was prepared in 50 mM phosphate buffer (pH 7.4). Aliquots (0.1 ml) of the extract was transferred into the test tubes and their volumes were made up to 0.4 ml with 50 mM phosphate buffer (pH 7.4). After addition of 0.6 ml hydrogen peroxide solution (H2O2), tubes were vortexed and absorbance of the hydrogen peroxide at 230 nm was determined after 10 min, against a blank (Ruch et al., 1989). The abilities to scavenge the hydrogen peroxide were calculated using the following equation: Hydrogen peroxide scavenging activity = (1- absorbance of sample/ absorbance of sample) × 100.

2.3.3. Phosphomolybdate assay The total antioxidant capacity assay was determined as described by Moonmun et al. (2017). An aliquot of 0.1 ml of the extract at different concentrations (100–1000 μg/ml) was mixed with 1.0 ml of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were covered and incubated in a 113

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2.5. Statistical analysis

Table 2 Impact of potassium application on radical scavenging activities of total fruit and achenes of fig cv. Bouhouli.

All data were reported as means ± standard deviation (SD) of three replicates and analysed using PC software package SPSS (version 20.0; SPSS Inc., Chicago, IL, USA). Experimental data were subjected to oneway analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test to determine the significance differences among mean values at the probability level of 0.05. Pearson’s correlation coefficients were estimated between the antiradical scavenging activities and biochemical compositions.

Plant extracts

Petroleum ether

3. Results

Ethyl acetate

3.1. Influence of potassium application on antioxidant activities Methanol

3.1.1. DPPH radical scavenging activity The free radical scavenging activity of different extracts from total fruit and achenes were studied by its ability to reduce the DPPH. The DPPH is a stable free radical and any molecule can contribute an electron or hydrogen to DPPH leading to bleach the DPPH absorption. The results showed that the free radical scavenging increased with increasing extracts concentrations (Fig. 1). The petroleum ether and ethyl acetate extracts of total fruit and achenes possess a moderate antioxidant activity, which was increased by 13% after treatment with K2SO4. The highest DPPH radical scavenging activity was detected in

EC50 values (μg/ml) of radical scavenging DPPH Radical

Hydrogen peroxide

Phosphomolybdate assay

CF

977.17 ± 5.71a

848.12 ± 10.25a

415.45 ± 16.56b

TF CA TA CF TF CA TA CF TF CA TA

844.01 nd 943.02 726.18 568.91 864.34 646.54 509.47 338.36 371.12 276.03

± 8.18b ± ± ± ± ± ± ± ± ±

11.41a 7.14b 2.34d 3.02a 3.26c 10.92a 8.78c 11.53b 10.62d

853.69 nd nd 791.43 594.26 802.78 752.12 548.44 355.11 461.34 458.07

± 1.66a

± ± ± ± ± ± ± ±

7,45b 12.71c 10.59a 14.09b 10.76a 12.25c 9.34b 8.97b

321.19 648.12 641.37 361.42 297.18 375.39 221.71 232.13 111.08 264.14 188.78

± ± ± ± ± ± ± ± ± ± ±

7.11c 9.71a 12.09a 10.77a 8.14c 10.48a 9.51b 17.86a 9.15c 14.27a 6.12b

Each value in the table is represented as mean ± SD (n = 3). Values in the same column followed by a different letter are significantly different (P < 0.05). ‘nd’: not determined. (CF): control total fruit; (TF): treated total fruit; (CA): control achenes; (TA): treated achenes.

Fig. 1. Antioxidant activities of different extracts at various concentrations from the petroleum ether, ethyl acetate and methanol extracts of total fruit and achenes of control and treated figs with potassium. (A) DPPH radical scavenging activity, (B) Hydrogen peroxide radical scavenging activity and (C) Total antioxidant capacity. Values are performed in triplicate. (Total fruit C and achenes C represent the control; Total fruit T and achenes T represent the treated samples). 114

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0.8 mg GAE/g DW (Fig. 2A). The flavonoids content in total fruit and achenes extracts were highly influenced by K2SO4 treatment (Fig. 2B). The flavonoids content was increased in methanol extracts by 36 and 48% , respectively, in total fruit and achenes. The flavonoids content in total fruit petroleum ether and ethyl acetate extracts were achieved an average level of 0.41 mg QE/g DW compared to control for an average level of 0.11 mg QE/g DW. However, this level was less influenced in achenes petroleum and ethyl acetate extracts, which were respectively increased by 2.7 and 1.36 times under potassium application (Fig. 2B). It was markedly noted that the tannins levels were raised in treated samples with K2SO4 compared to the control (Fig. 2C). The highest tannins content recorded in total fruit methanol extract from treated samples was 7.7 mg CE/g DW, whereas it was 6 mg CE/g DW in the control. The achenes petroleum extract represents the lowest value of 0.53 mg CE/ g DW in control. In fact, the K2SO4 has no significant effect on TTC level in total fruit petroleum extract (Fig. 2C).

the methanol extracts of total fruit and achenes with 72 and 66%, respectively, at 1000 μg/ml, whereas these values were increased to 73.5 and 75.6%, respectively, after potassium treatment. Therefore, the polar and non-polar extracts exhibited considerable DPPH free radical scavenging activity as indicated by their EC50 values and this has been showed in Table 2 and Fig. 1. The potassium treatment enhances the scavenging effect in all extracts compared to the control, but lesser effect than the standard compound. In fact, fig achenes methanol extract showed the lowest EC50 (276.03 μg/ml) after potassium treatment compared to control (371.12 μg/ml), while the highest EC50 was recorded by the petroleum extract of control fruit (977.02 μg/ml) compared to the treated (844.01 μg/ml) (Table 2). 3.1.2. Hydrogen peroxide radical scavenging activity The scavenging effect of different extracts of fig on hydrogen peroxide was concentration-dependent (100–1000 μg/ml) and was comparable to that of the standard, ascorbic acid as shown in Fig. 1. The total fruit and achenes methanol extracts displayed strong H2O2 scavenging activity of 68 and 60.6%, respectively, whereas that of the standard, ascorbic acid exhibited 81% at 1000 μg/ml. These values were slightly increased, respectively, to 69.8 and 66% under potassium application. The lowest scavenging activities was recorded by achenes petroleum ether extract of 30.6% and reached 40.9% after potassium treatment at the highest concentration (Fig. 1). In the present investigation, the EC50 value hydrogen peroxide radical scavenging activity for the total fruit and achenes methanol extracts were 355.11 μg/ ml and 458.07 μg/ml under potassium treatment, respectively, while for the control the values were 548.44 μg/ml and 461.34 μg/ml (Table 2).

3.2.2. Quantification of individual phenylpropanoids The phenolic compounds present in the methanol extracts of the total fruit and achenes were identified and quantified by HPLC (Fig. 3 and Table 3). The phytochemical profile of fruit and achenes was slightly different. The achenes methanol extract presents ten compounds compared to total fruit (14 compounds). The compounds 1, 2 and 15 were not detected in achenes and the compounds 16 and 8 were not detected in total fruit. The identified compounds in the control and treated figs were as follows: chlorogenic acid, cyanidin 3-rutinoside, cyanidin 3,5-diglucoside, dihydroxybenzoic acid, catechin, caffeic acid, rutin, isoquercetin and quercetin (Table 3). The most abundant polyphenol in the total fruit and achenes methanol extracts was the unknown compounds 14. The HPLC-UV peak areas of other polyphenols were considerably small. The potassium application increased significantly the quercetin from 0.19 to 4.29 mg/g DW in total fruit methanol extract. Among the polyphenols, the chlorogenic acid, cyanidin 3-rutinoside and cyanidin 3,5-diglucoside, contents increased from 0.87 to 1.70 mg/g DW under potassium treatment, whereas those compounds were not detected in achenes methanol extract. However, the potassium reduces significantly the rutin and catechin contents by two times compared to the control. The potassium application affected also the caffeic acid in total fruit and achenes methanol extract, respectively, from 3.13 and 3.23 mg/g DW to 2.51 and 1.44 mg/g DW (Table 3).

3.1.3. Phosphomolybdate assay The antioxidant activity was increased in dose dependent manner at concentration 100 to 1000 μg/ml. The potassium application enhanced the antioxidant capacity for both total fruit and achenes from different extracts and was found to increase in this order: ether petroleum extracts < ethyl acetate extracts < methanol extracts (Fig. 1 and Table 2). The strong antioxidant response of methanol extracts in comparison with ascorbic acid might be helpful in characterizing the significant sources of natural antioxidant reaction. In fact, the total fruit and achenes methanol extracts exhibited an average antioxidant effect of 80% compared to the ascorbic acid (98%), while this effect was increased to 84.46% under potassium treatment. The EC50 value of antioxidant capacity for total fruit methanol extract (111.08 μg/ml) was most pronounced (P < 0.05) than achenes methanol extract (188.78 μg/ml) under potassium treatment, compared to control (Table 2). The strong antioxidant activity of total fruit and achenes methanol extracts could be due to the presence of phenolic compounds that could be influenced by potassium treatment.

3.3. Correlation between antiradical scavenging activities and biochemical compositions Correlation analysis was revealed in order to determine the association between different biochemical constituents and radical scavenging activity. All variables were investigated in the first correlation analyses (Table 4). Significant correlations were also recorded between various methods used to determine the antioxidant potential, especially between TAC and DPPH (r = 0.858, P < 0.01). The lowest correlations were recorded between DPPH, TAC and H2O2 assays. Generally, a positive correlation (r = 0.938, P < 0.01) was detected between TPC and TFC. Similarly, a positive correlation was revealed between DPPH and TFC (r = 0.664, P < 0.05) and TPC (r = 0.822, P < 0.01). The positive correlation between total phenolics, flavonoids with antioxidant capacities indicates that phenolics and flavonoids are the main components for antioxidant capacity in the fruit and achenes extracts. However, the TTC was correlated with TPC (r = 0.756, P < 0.01) and varied with antioxidant essays (Table 4).

3.2. Chemical composition 3.2.1. Effect of potassium application on phenylpropanoids content The phytochemical composition is defined by the total polyphenol (TPC), total flavonoid (TFC) and total tannin (TTC) contents of achenes and total fruit under potassium application in concentration of 2% (Fig. 2). The highest content of polyphenol was significantly represented in total fruit compared to the achenes (Fig. 2A). The polyphenol content was enhanced by the potassium treatment (2%) in fruit and achenes in polar and non-polar extracts. In fact, the methanol extracts represent the highest values of polyphenol respectively, 29.3 and 25.1 mg GAE/g DW under potassium treatment for fruit and achenes compared to the control. This enhancement was significantly observed in achenes ethyl acetate extract, and the level was increased from 6.7 to 9.8 mg GAE/g DW after potassium application. However, the lowest value was observed in achenes petroleum extracts from the control and not exceeded

4. Discussion It has been reported that potassium applications influence vegetative growth, yield and fruit characteristics (Quaggio et al., 2011; Saykhul et al., 2014). Among many plants, potassium (K) display the 115

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Fig. 2. (A) TPC total polyphenol content, (B) TFC total flavonoids content and (C) TTC total tannins content of total fruit and achenes of fig cv. Bouhouli influenced by foliar potassium application at 2%. The bars on each column show standard deviation (n = 3). The same letter indicates no significant differences (P < 0.05) according to Duncan’s multiple range test.

quality and nutrient uptake over KCl application (Sapkale et al., 2012). The foliar application of K2SO4 at 2% influenced also the primary metabolites by increasing sugar levels in fig (Irget et al., 2008; Ben Mimoun et al., 2017). On the other hand, the primary metabolites were involved in the biosynthesis of the antioxidant phenolic compounds through shikimate pathway in plants (Peled-Zehavi et al., 2015). According to the previous results, the K2SO4 is the focused subject for the present study in order to improve fig fruit quality and the dietary antioxidant contents by enhancing the phenolic compounds levels. In the present work, the antioxidant activity was conducted through DPPH radical scavenging, hydrogen peroxide radical scavenging, phosphomolybdenum assays. Since there is no universal method to assess antioxidant activity quantitatively and accurately, Prior et al. (2005) suggested the measurement of the antioxidant activity through several methods. The result of the antioxidant activity of the total fig fruit and achenes extracts depending on the nature of the polarity of solvent and mainly to the methods of analysis. There was a dose-dependent relationship in radical scavenging activity for the total fruit and achenes extracts within the range of concentrations from 100 to 1000 μg/ml. All samples were proven to have antioxidant activities with significant differences (P < 0.05) between all extracts measured by different antiradical scavenging methods. Similar results were recorded by Soltana et al. (2016) who reported that fig achenes hexane extract presents a strong antioxidant activity with IC50 of 215.86 μg/ml

strongest influence on quality attributes that could determine fruits marketability and consumers choices. In the horticultural production, the most frequently used mineral fertilizers containing potassium that include anions Cl−, SO42- and NO3−, which act differently on the chemical composition of plants. Several studies assessed the different potassium forms in order to find the appropriate application in order to enhance the productivity and fruit quality by enhancing the nutrient absorption in the plant (Fernández et al., 2013). In our pervious study, Ben Mimoun et al. (2017) revealed that the foliar application of K2SO4 at 2% significantly increased the fig fruit weight and total yield per tree compared to the control (data not shown). The fig quality was improved by reducing the ostiole cracking of fruits. Besides that, the treated trees of cv. Bouhouli with 2% K2SO4 increased the potassium accumulation in leaves compared to the control. These results are in accordance with the findings of Restrepo-Diaz et al. (2008) which they suggested that spraying with K₂SO₄ at 2.5% was effective in raising K content of leaves in olive trees. Indeed, Saykhul et al. (2014) evaluated the foliar application of different potassium forms (KCl, KNO3 and K2SO4) with the same amount of K at 1, 2 and 3% on the growth and nutrient status of olive tree. They exhibited that the dry weight was significantly increased only in the treatments with K2SO4 and this increase could be due to the beneficial effect of sulphate in enhancing dry matter percentage. Among different sources of potassium, the foliar application of K2SO4 as a source of K2O showed significant increase in fig yield, 116

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Fig. 3. HPLC separation of phenylpropanoids from total fruit and achenes of cv. Bouhouli under K2SO4 application. Peak detection was carried out at 280 nm. For peak assignment see Table 3. Table 3 Effect of potassium application on identified phenylpropanoid content (mg/g DW) from total fruit and achenes of cv. Bouhouli. Fruit

Achenes

Peaks

Rt (min)

Compounds

Control

1 2 3 4 5 6 10 11 15

17.63 20.96 23.33 25.28 27.20 32.01 41.24 42.25 67.43

Chlorogenic acid Cyanidin 3-rutinoside Cyanidin 3,5-diglucoside Dihydroxybenzoic acid Catechin Caffeic acid Rutin Isoquercetin Quercetin

0.88 0.90 0.84 1.35 4.24 3.13 0.73 0.57 0.19

± ± ± ± ± ± ± ± ±

0.04b 0.06b 0.03b 0.09a 0.04a 0.71a 0.09a 0.01a 0.01b

Treated

Control

Treated

1.69 ± 0.57a 1.90 ± 0.17a 1.23 ± 0.33a 1.14 ± 0.39b 2.2 ± 0.48b 2.51 ± 0.14c 0.35 ± 0.07b 0.50 ± 0.01a 4.25 ± 0.98a

Nd Nd Nd 0.41 ± 0.01c 1.17 ± 0.18d 3.23 ± 0.54b Nd Nd Nd

Nd Nd Nd 0.47 ± .11c 1.8 ± 0.43c 1.44 ± 0.58d Nd Nd Nd

Different letters indicate significant differences between groups (P < 0.05); Nd: not detected. Values are given as mean ± SD of three assays (n = 3).

from 7.04 to 14.6 mg/ml for total fruit and 10.59 mg/ml to 15.45 mg/ ml for pulp extracts through DPPH method. In fact, this activity was enhanced in total fruit and achenes methanol extracts after foliar spraying of fig with K2SO4, compared to petroleum and ethyl acetate extracts. Similar results confirmed that potassium application improved the antioxidant activity in pomegranate (Tehranifar and Tabar, 2009), pineapple (Soares et al., 2005) and black grape (Zareei et al., 2018). The potassium spray could influence the metabolic process of phenolic compounds and could be correlated to the antioxidant activities of fig. Thus, potassium regulates the biosynthesis, conversion and allocation of metabolites that ultimately increases the yield of crops (Abbas et al., 2011; Islam et al., 2015; Ben Mimoun et al., 2017). In our results, the potassium application increased the polyphenol, flavonoid and tannin contents in fig methanol extract compared to the petroleum ether and ethyl acetate extracts. The enhancement of polyphenol and flavonoids contents were estimated by an average of 42% in total fig fruit and achenes methanol extracts, and 29.3 GAE/mg DW was the highest value in fruit after K2SO4 application. However, the levels of polyphenols and flavonoids contents did not exceed, respectively, 26.9 GAE/mg DW and

Table 4 Pearson correlations among the different compositions of total fruit and achenes. DPPH DPPH H2O2 TAC TPC TTC TFC

– 0.191 0.858** 0.822** 0.449 0.664*

H2O2 0.191 – 0.289 0.290 0.374 0.208

TAC

TPC **

0.858 0.289 – 0.680* 0.171 0.601*

**

0.822 0.290 0.680* – 0.797** 0.938**

TTC

TFC

0.449 0.374 0.171 0.797** – 0.756**

0.664* 0.208 0.601* 0.938** 0.756** –

TPC: total phenol content; TFC: total flavonoid content; TTC: total tannin content; DPPH: 2,2-diphenyl-1-picrylhydrazyl; TAC: total antioxidant activity; H2O2: hydrogen peroxide activity. ** Correlation is significant at the 0.01 level. * Correlation is significant at the 0.05 level.

under control condition. This strong potential was confirmed also by Harzallah et al. (2016), who found that fig possess a potent antioxidant activity through different methods, whereas the EC50 values ranged 117

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esteemed in Mediterranean area and presents an increasing interest worldwide. Hence, further study will be carried out to identify the unknown major individual phenolic compounds and their antioxidant capacity response to potassium application at different concentrations.

0.98 mg QE/g dw under control condition. In contrast to our results, Soltana et al. (2016) exhibited a very low level of phenolic and flavonoid contents, respectively, by 144.89 mg/kg and 124 mg/kg in fig achenes under control condition. Our result was confirmed by Zareei et al. (2018) who revealed that the antioxidant activity and the polyphenol content were increased in berries after potassium foliar application at 3 g/l. In addition, Yaldiz (2017) showed that the potassium application increased the polyphenol flavonoids contents in thistle milk (Silybum Marianum) and was correlated with the increase of antioxidant activity. Ahanger et al. (2015) explained that potassium exhibited also an increase in phenols content in cereals plants such as oat (Avena sativa), as well as the activities of antioxidant enzymes (SOD, CAT and APX). Several authors had reported correlation between antioxidant activities of plant with the content of phenolic and flavonoids compounds (Kamiloglu and Capanoglu, 2015; Harzallah et al., 2016). The phytochemical profiling of the fig fruit and achenes evaluated in this study revealed a diverse range of bioactive phenolic compounds. In fact, this is the first study comparing the phenolic levels in fig fruit and achenes extracts under potassium treatment. The phytochemical profile of fruit was significantly different to the achenes profile. In fact, fourteen compounds were identified from total fruit, while only three compounds were recorded for achenes methanol extract. Similar result was observed by Russo et al. (2014) in Turkish and Greek dried figs, who confirmed that the chlorgenic acid was not detected in pulp. However, it was detected in higher amount in fruit and were generally higher respect to the values reported in literature (Faleh et al., 2012; Vallejo et al., 2012). A similar result was recorded respect to the finding of Oliveira et al. (2009) who reported that the rutin was not detected in fig pulp except in the Branca traditional cultivar. They mentioned that rutin was the major compound in different fig cultivars and representing 42–87% of total identified phenolics, except for Pingo de Mel pulp in which chlorogenic acid was present at the highest amount (90%). However, in our study the most abundant polyphenol in the total fruit and achenes methanol extracts was the peak 14, which is unknown phenolic compound. On the other hand, the fruit and achenes methanol extracts were significantly influenced by potassium application. Among the polyphenols, the chlorogenic acid, cyanidin 3-rutinoside, cyanidin 3,5-diglucoside and quercetin were increased in total fruit after potassium treatment. However, the potassium reduces significantly the rutin and catechin contents by two times compared to the control. It is well known that high fertilization doses improve plant growth but to some extent reduce the amount of phenolics, often making the plants more susceptible to pests and diseases (Veberic, 2016). The bioactive compound concentration is one of the most important criteria for determining the quality of plant (Li et al., 2008). Many studies indicate that environmental stressors, such as nutrient deficiency, fertilizer overage and water stress affect the level of several secondary metabolites in plants (Kirakosyan et al., 2004; Zhu et al., 2009). Thus, potassium is not only a constituent of the plant structure, but it has also a regulatory function in several biochemical processes related to protein synthesis, carbohydrate metabolism and enzyme activation (Marschner, 1995; Hasanuzzaman et al., 2018).

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