Accepted Manuscript The effects of plant growth regulators and L-phenylalanine on phenolic compounds of sweet basil Nülüfer Koca, Şengül Karaman PII: DOI: Reference:

S0308-8146(14)00951-0 http://dx.doi.org/10.1016/j.foodchem.2014.06.065 FOCH 16007

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

3 March 2014 12 June 2014 13 June 2014

Please cite this article as: Koca, N., Karaman, Ş., The effects of plant growth regulators and L-phenylalanine on phenolic compounds of sweet basil, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.06.065

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The effects of plant growth regulators and L-phenylalanine on phenolic compounds of sweet basil

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Nülüfer Kocaa,*, Şengül Karamanb

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a

Department of Medicinal and Aromatic Plants, Turkoglu Vocational High School, Kahramanmaras Sutcu Imam University, 46100 Turkoglu-Kahramanmaras, Turkey b Department of Biology, Faculty of Science, Kahramanmaras Sutcu Imam University, 46100 Avsar CampusKahramanmaras, Turkey * Corresponding author at: Department of Medicinal and Aromatic Plants, Turkoglu Vocational High School, Kahramanmaras Sutcu Imam University, 46100 Turkoglu-Kahramanmaras, Turkey. Tel.: +90 344 280 13 51; fax: +90 344 280 13 52. E-mail addresses: [email protected], [email protected] (N. Koca).

ABSTRACT

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The effects of methyl jasmonate (MeJA), spermine (Spm), epibrassinolide (EBL) and L-phenylalanine on sweet basil (Ocimum basilicum L.) were studied to determine the amount of phenolic compounds and enzymatic activity of phenylalanine ammonia-lyase (PAL). Total phenolic and total flavonoid contents of sweet basils were determined by a spectrophotometer, and individual phenolic compounds and activity of PAL were analyzed by HPLC/UV. The highest total phenolic (6.72 mg GAE/g) and total flavonoid contents (0.92 mg QE/g) obtained from 1.0 mM Spm+MeJA application. Rosmarinic acid (RA) and caffeic acid contents significantly enhanced after the applications but no such differences observed in chicoric acid content or PAL activity. RA was the main phenolic acid in all samples and its concentration varied from 1.04 to 2.70 mg/g FW. As a result the combinations of Spm+MeJA and EBL+MeJA can induce secondary metabolites effectively and those interactions play important role in the production of phytochemicals in plants.

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Chemical compounds studied in this article

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Methyl jasmonate (PubChem CID 5367719); Spermine (PubChem CID 1103); 24Epibrassinolide (PubChem CID 115196); Phenylalanine (PubChem CID 6140); Caffeic acid (PubChem CID 689043); Chicoric acid (PubChem CID 5281764); Rosmarinic acid (PubChem CID 5281792)

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Keywords:

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Ocimum basilicum, phenolic, spermine, precursor, brassinosteroid, MeJA, secondary metabolites.

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1. Introduction

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The health benefits of the foods in humans have been attributed to phytochemicals, particularly secondary metabolites e.g. polyphenolic compounds and flavonoids. Because of their bioactive functional features, there is a high research interest, including investigations on improving their production (Kim, Chen, Wang, & Rajapakse, 2005).

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Jasmonic acid (JA) and MeJA are lipid-based hormones, synthesized from linolenic acid widely occuring in plants. These endogenous phytohormones play important roles in plant 1

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growth and are potent elicitors or signaling agents that change many biochemical and physiological processes (Creelman & Mullet, 1997). Application of MeJA to plants has been shown to stimulate secondary metabolites and increase synthesis of terpenoids, alkoloids and phenolics in Nicotiana species, sweet basil, and radish sprouts (Keinanen, Oldham, & Baldwin, 2001; Kim, Chen, Wang, & Rajapakse, 2006; Kim, Chen, Wang, & Choi, 2006).

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Polyamines are ubiquitous compounds that are important for not only cellular processes and growth, but also in adaptive responses to (a)biotic stresses in plants (Kusano, Yamaguchi, Berberich, & Takahashi, 2007). For instance, the polyamine Spm acts as an intermediary in defending signal system against pathogens, and this pathway is referred to as the “Spm-signal transduction pathway” (Takahashi, Uehara, Berberich, Ito, Saitoh, Miyazaki, Terauchi, & Kusano, 2004). In this report, we supplied Spm exogenously to induced sweet basil seeds to detect their possible effect.

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Brassinosteroids (Brs), a natural occuring plant hormones, provide a resistance to (a)biotic stresses such as salinity, drought, cold, temperature and pathogens besides its primary role in growth and development (Krishna, 2003). It has been shown by Müssig et al. (2000) that expression of OPR3, a gene that upregulates JA synthesis, is increased through treatment with Brs. The authors suggest a possible link between brassinosteroid activity and JA synthesis and the increase in the activity of a gene related to jasmonic acid syntesis with the treatment of Brs showed that there is a correlation between Brs and JA (Mussig, Biesgen, Lisso, Uwer, Weiler, & Altmann, 2000).

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Precursor treatment is another experimental approach used in influencing the biosynthetic pathways to increasing the production of secondary metabolites in different plant species (Kovacik, Kron, Repcak, & Backor, 2007). Sivakumar et al. (2004) determined the highest PAL and TAL activity and colchicine production after application of L-phenylalanine (L-Phe) and tyrosine (Tyr) at high concentrations. However, as a response to precursor feeding, phenolic production has so far been investigated in seed application of plants.

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In this study basil was preferred because it is an important culinary herbs in the world and can be easily grown and is commercially produced in greenhouses (Putievsky & Galambos, 1999). The goal of this study was to evaluate the effect of MeJA along with Spm, EBL and LPhe on the production of secondary metabolites of sweet basil plants, and to obtain highly efficient and high quality yield from such culinary and medicinal plants. High performance liquid chromatography (HPLC) was used for the separation and identification of some major phenolic compounds of sweet basil. Total phenolic and total flavonoid contents were also evaluated. Since PAL is the key enzyme for the phenylpropanoids related to the biosynthesis of phenolics, PAL activity was investigated in sweet basil leaves.

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2. Materials and methods 2.1. Plant materials and chemicals

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Sweet basil seeds were obtained from Tezier Company (HM-Clause, France). The basil cultivar (Ocimum basilicum cv. Grand Vert), which has large green leaves, is sold in Europe for culinary purposes. MeJA (95%), Spermine (≥97%), 24-Epibrassinolid (≥85%), L2

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Phenylalanine (≥99%, Bioultra), trans-Cinnamic acid (≥99%), Bovine serum albümin (≥96%), and chemicals for polyphenolic extraction and HPLC analysis were purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Solvents and chemicals for analysis were analytical and HPLC grade. Rosmarinic acid (≥93%) standard was obtained from HWIAnalytik GMBH (Germany), caffeic acid (≥98%), chicoric acid (≥95%), quercetin (≥95%), and gallic acid (97.5-102.5%) standards, Folin & Ciocalteu’s phenol reagent (2 M) and sodium carbonate (ACS reagent, anhydrous, ≥99,5%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

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2.2.

Plant culture and plant growth regulators and precursor treatments

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Sweet basil seeds were sterilized with 1% hypocloride solution for 2-3 min then washed thoroughly with tap water. The seeds were soaked in MeJA (0.5 mM dissolved in 1% ethanol solution) and MeJA combination with 0.5 µM; 2 µM 24-EBL (an active and stable Br, Khripach, Zhabinskii, & De Groot, 2000), 0.05 mM; 0.5 mM L-Phe (Shinde, Malpathak & Fulzele, 2009), 0.1 mM; 1.0 mM Spm (Ozawa et al., 2009) solutions for 24 hours (Karaman, Kirecci & Ilcim, 2008). Two control groups were used, Control A group were soaked in 1% aqueous ethanol solution and Control B was sown without any treatment. All seeds were sown into 77-cell trays containing soil-perlite-peat mixture (1:1:1 ratio, v/v) and watered as needed. The sweet basil was grown in a growth chamber at 27/23 oC (day/night) under cool fluorescence lamps (100 μmol m-2s-1). When the seedlings had four or five leaves, they were transplanted into 1 L plastic pots containing soil-perlite-peat-humus-fertilizer mixture. Fertilizer was a general purpose nutrient from Osmocote® Exact® that contained 15% N, 9% P, 11% P2O5, 11% K2O, 2% MgO and all necessary micronutrients. Seedlings were watered when needed and grown during ten weeks. Before the harvest all applications were watered with 50 ml 0.5 mM MeJA solution twice and all plants were harvested 10 days later. All applications harvested before the flowering period, uniform sized leaves were detached from the same plant parts and immediately stored -80 oC until analysis.

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2.3.

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Leaves were extracted as described in Lee and Scagel (2009) with slide modifications. Leaves were ground on dry ice using a mortar and pestle. Frozen samples (2 g) were mixed with 20 ml acidified methanol (0.1% formic acid, v/v) and were immersed in a boiling water bath for 5 min, then immediately submerged in an ice bath for 10 min. The blanching step was used to avoid the degradation of polyphenolics by native enzymes in basil (Lee & Scagel, 2009). This mixture filtered with Whatman paper and the pellet was re-extracted, twice with 15 ml acidified methanol. The obtained methanolic extract was completed to a final volume of 35 ml. The mixture was centrifuged at 12000 rpm for 15 min, and the extract was stored at 80 oC analyses in HPLC.

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2.4.

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The Folin-Ciocalteu colorimetric assay (Singleton & Rossi, 1965) was used to determine the TP content. Absorbance of TP content was measured with a UV/visible

Preparation of samples and polyphenolic extraction

Determination of total phenolic (TP) content

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spectrophotometer (Shimadzu UV-1800, Shimadzu Co, Japan) at 760 nm and was expressed as mg gallic acid equivalent (GAE)/g (R2=0,9992) in the sweet basil methanolic extract, as per gram of fresh weight (FW).

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2.5.

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The total flavonoid content was determined in sweet basil plants according to the aluminium chloride colorimetric method. Basil methanolic extract (0,5 ml) was mixed with 2 ml of distilled water and 0.3 ml of (5% w/v) NaNO2 was added to this mixture. After 6 min, 0.3 ml of (10% w/v) AlCl3 and then after another 6 min, 4 ml of (4% w/v) NaOH were added, and the volume was made up to 10 ml with distilled water. The mixture was shaken vigorously and the absorbance at 510 nm was measured. The total flavonoid content was represented as mg quercetin (R2=1) equivalents (QE)/g in methanolic extract of sweet basil as per gram of FW.

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2.6.

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PAL activity was evaluated using HPLC with the following method (Dos Santos, Ferrarese, Finger, Teixeira, & Ferrarese-Filho, 2004). The fresh basil leaves (2 g) were ground at 4 ◦C in sodium borate buffer (0.2 M, pH 8.8). The mixture was centrifuged (12,000×g for 15 min), and the supernatant solution was used as enzyme source (crude extract). The reaction mixture consisting of 500 µl sodium borate buffer (pH 8.7) and 250 µl enzyme extract was pre-incubated at 40 ◦C for 5 min and the reaction was started by adding 300 µl 50 mM L-Phe. The reaction was stopped after 1 h of incubation by adding 50 µl 5 N HCl. The reaction mixture was centrifuged again (12,000×g for 15 min) at room temperature. Samples were filtered through a 0.45 µm syringe filter and analyzed (10 µl) with an Agilent 1100 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with four Ecom pumps (Prague, Czech Republic), and a SGX C-18 column (5 µM, 4.6 mm×150 mm) coupled with UV dedector (Hewlett-Packard 1100 model). The mobile phase was acetonitril: water (57:43, v/v) with a flow rate of 0.5 ml min−1. Absorbance of both samples and the standard were measured at 275 nm. trans-Cinnamic acid (t-Cinnamate), the product of PAL, was identified by comparison of its retention time with that of the standart. PAL activity was expressed in µmol t-cinnamate h−1 mg protein−1 of fresh weight (FW). Protein amount of the extracts was estimated by the Bradford (1976) method using bovine serum albumin as a standard.

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2.7.

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HPLC was used for separation and determination of rosmarinic acid, chicoric acid and caffeic acid in fresh basil samples. The methanolic extract was filtered using a 0.45 µm membrane filter prior to analysis. All samples were analyzed using an Agilent 1100 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with four Ecom pumps (Prague, Czech Republic), and a SGX C-18 column (5 µM, 4.6 mm×150 mm) coupled with a UV dedector (Hewlett-Packard 1100 model). Ten microliters of methanolic extract was injected and eluted with HPLC water containing 0.05% trifluoroacetic acid (TFA) and

Determination of total flavonoid content

PAL activity assay

HPLC analysis of individual phenolic compounds

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acetonitrile at a flow rate of 1.2 ml/min. The absorbance of the eluent was scanned at 330 nm by the UV. Rosmarinic acid, caffeic acid and chicoric acid were detected in basil samples based on their chromatographic retention times and quantified by comparing integrated peak areas to calibration curves prepared with analytical standarts.

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2.8.

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The plant experiments were conducted in the growth room, at USKIM Research Center, Kahramanmaras Sutcu Imam University in 2012. The trials were designed in Randomized Plot Design with three replications and ten sweet basil plants were used in each replication. The data were analyzed by using SPSS Statistics (Version 20.0 SPSS Inc., Chicago, IL). Means were compared using Duncan’s Multiple Range Test. Standard error was also employed to separate the means in the tables.

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3. Results and discussion

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3.1. Effect of treatments on total phenolic content of sweet basil

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The total amount of phenolic compounds of O. basilicum varied from 3.43 to 6.75 mg GAE/g (Table 1). The highest phenolic content was obtained from 1.0 mM Spm+MeJA and 0.1 mM Spm+MeJA applications, which was 40% and 38% higher than that of the control (sown without any treatments), respectively. There was a difference in the total phenolic content between the two control groups, and soaking in water affected total phenolic content of plant negatively. The total phenolic content significantly increased by plant growth regulators and precursor treatments (p

The effects of plant growth regulators and L-phenylalanine on phenolic compounds of sweet basil.

The effects of methyl jasmonate (MeJA), spermine (Spm), epibrassinolide (EBL) and l-phenylalanine on sweet basil (Ocimum basilicum L.) were studied to...
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