Non-targeted metabolomics and scavenging activity of reactive oxygen species reveal the potential of Salicornia brachiata as a functional food

journal of functional foods 13 (2015) 21–31

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Non-targeted metabolomics and scavenging activity of reactive oxygen species reveal the potential of Salicornia brachiata as a functional food Avinash Mishra *, Manish Kumar Patel, Bhavanath Jha ** Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar, Gujarat 364002, India

A R T I C L E

I N F O

A B S T R A C T

Article history:

Non-targeted metabolomics implied that Salicornia brachiata contains essential and sulphur-

Received 27 August 2014

rich amino acids, which are recommended by the FAO. Total phenolic content of the plant

Received in revised form 15

extract contains reducing capacity and reactive oxygen species (ROS) scavenging activity.

December 2014

A high content of nutritive indicator polyunsaturated fatty acids (PUFAs; 55–64%), includ-

Accepted 16 December 2014

ing linoleic acid (C18:2; 22–27%) and alpha-linolenic acid (C18:3; 29–41%), was detected under

Available online

both control and stress conditions. About 38% of saturated fatty acids, including 26% palmitic acid (C16:0), was found under the control conditions, which varied under stress. About

Keywords:

19 different metabolites with different bioactivities have, so far, been identified. Metabo-

Antioxidants

lites benzyl sulphate (m/z 93.04) and flavonoid myricatin (m/z 183.14 and 532.98) are known

Bioactivity

for their aroma and nutritive supplements. Bioactive metabolites of pharmaceutical im-

Fatty acids

portance, such as oxomefruside (m/z 131.07; to be used in hypertension), clonidine (m/z 210.19;

Functional food

to treat high blood pressure, anxiety and certain pain), carmustine (m/z 212.19; anti-

Metabolites

neoplastic in nature) and gangliosides (m/z 879.48; anti-inflammatory), were detected in the plant extract. An important dietary supplement selenocystathionine (m/z 269.09) was identified, which is used in hyper-accumulation of anti-cancer agent selenium. Moreover, metabolites with antimicrobial (sodium cefazolin; m/z 459.82), insecticidal or fungicidal activities (dichlorophene, m/z 267.10; oxydisulfoton, m/z 271.10; sulfotep, m/z 303.41 and azothoate, m/z 355.48) were also detected. Non-targeted metabolomics, antioxidants and scavenging activities revealed the nutritional potential of the plant, making it a promising functional food for dietary supplements. © 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar, Gujarat 364002, India. Tel.: +91 278 2567760 Ext. 6260; fax: +91 278 2567562. E-mail address: [email protected] (A. Mishra). ** Corresponding author. Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar, Gujarat 364002, India. Tel.: +91 278 2567352; fax: +91 278 2567562. E-mail address: [email protected] (B. Jha). Abbreviations: 4,11 DCQA, 4,11-dichloro-5,12-dihydroquinolino[2,3-b]acridine-7,14-dione; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline6-sulphonic acid); BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ESI, electrospray ionization; FA, fatty acid; FAME, fatty acid methyl ester; GC, gas chromatography; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectroscopy; MUFA, monounsaturated fatty acid; PITC, phenyl isothiocyanate; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; SFA, saturated fatty acid; TBA, thiobarbituric acid; TCA, trichloroacetic acid; TEA, triethylamine; TOF, time-of-flight; Trolox, 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid http://dx.doi.org/10.1016/j.jff.2014.12.027 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

22

1.

journal of functional foods 13 (2015) 21–31

Introduction

Salinity is emerging as a major constraint worldwide and halophytes provide a unique opportunity to study the mechanistic basis of the adaptation to a diverse saline ecological niche. Halophytes contain potential antioxidant systems and are known for quenching toxic reactive oxygen species (ROS) produced under saline conditions (Bose, Rodrigo-Moreno, & Shabala, 2014). Halophytes frequently encounter various environmental stresses, and metabolites are regarded as the end products of cellular regulatory processes. Metabolites are low-molecularweight osmolytes that play a key role in osmotic adjustment. Metabolic pathways are highly dynamic, and osmotic homeostasis includes cellular compartmentalization of toxic substances during sequestration of non-toxic substances in the cytoplasm (Gagneul et al., 2007; Kruger, Masakapalli, & Ratcliffe, 2012). The metabolome is a set of metabolites synthesized by a biological system in response to environmental changes (Fiehn, 2002). Metabolomics is the understanding of metabolic networks, represented by the identification and quantification of all available metabolites of the corresponding biological system (Dunn & Ellis, 2005). Comprehensive analysis of metabolites under environmental stress exhibits new responses and provides the biochemical status of plants (Král’ová, Jampílek, & Ostrovský, 2012). Metabolites are comprised of heterogeneous groups of solutes and/or polymers that vary in their physicochemical properties, including abundance, size, polarity, solubility, stability and toxicity (Okazaki & Saito, 2012). There is a vast array and a structural diversity of plant metabolites (Nakabayashi & Saito, 2013), and more than 200,000 plant metabolites are reported (Fraire-Velázquez & Balderas-Hernández, 2013; Matsuda et al., 2009), but many are still to be identified and characterized. Several conventional and advanced methods and strategies have been reported for the untargeted metabolomics of unknown plant metabolites under abiotic stress (Dunn et al., 2013; Fraire-Velázquez & Balderas-Hernández, 2013; Matsuda et al., 2009; Nakabayashi & Saito, 2013; Okazaki & Saito, 2012). Synthesis of plant metabolites is influenced by varying stress conditions; therefore, strategies for metabolic profiling with a steady-state level of metabolites are very important for the analysis of complex metabolic networks (Kruger et al., 2012). To date, no single method has been developed for the study of plant metabolomics; therefore, different methodologies must be employed for comprehensive metabolome analysis, according to the complex biological processes (Okazaki & Saito, 2012). Nowadays, different analytical methods, including chromatography and spectroscopy, are developed so that individual and/or integrated applications with comparative homologybased analyses can elucidate comprehensive information about plant metabolites. A succulent halophyte Salicornia brachiata, belonging to the Amaranthaceae, has the capability to grow in salty marshes and requires NaCl (500 mM) along with plant growth regulators for in-vitro micropropagation (Joshi, Mishra, & Jha, 2012). Halophytes are considered to be rich in proteins, oils and fats, which are found to be suitable for human consumption. Salicornia seeds are rich sources of proteins and the shoots contain unique oligosaccharides and antioxidants (Jha, Singh,

& Mishra, 2012; Mishra, Joshi, & Jha, 2013). Additionally, the plant is rich in vitamins (A and C), sodium, calcium, iodine, iron, magnesium, amino acids, fats (low in calories without cholesterol) and fibers (Stanley, 2008). Salicornia is considered as a potential source of dietary supplementation and is eaten as a salad green. Some species of Salicornia have shown important biological properties, such as antioxidant, anti-inflammatory, hypoglycemic and cytotoxic activities (Isca, Seca, Pinto, & Silva, 2014). Furthermore, Salicornia is being widely explored for abiotic stress-responsive genes and promoters to develop transgenic plants that can withstand adverse environmental conditions (Chaturvedi, Mishra, Tiwari, & Jha, 2012; Chaturvedi, Patel, Mishra, Tiwari, & Jha, 2014; Jha, Sharma, & Mishra, 2011; Joshi, Jha, Mishra, & Jha, 2013; Singh, Mishra, & Jha, 2014a, 2014b; Tiwari, Chaturvedi, Mishra, & Jha, 2014; Udawat, Mishra, & Jha, 2014). No information is available, so far, on the metabolomics of this important succulent halophytic plant. Therefore, the nontargeted metabolomics and ROS scavenging activity of S. brachiata were studied under abiotic stress conditions. The study provides useful insights into the functional food quality and the understanding of the metabolic responses of this halophyte induced by abiotic environmental stress.

2.

Material and methods

2.1.

Plant material and stress treatments

Seeds of Salicornia brachiata were germinated in plastic pots, containing garden soil, under natural conditions. One month old seedlings were transferred to hydroponic condition (Hoagland solution) and grown under laboratory conditions with dark/light cycle of 8/16 h at 25 °C for 10 days. Plants were treated for 24 h with NaCl (500 mM) or kept in incubator at 42 °C for salt and heat stress treatment, respectively.

2.2.

Extraction and analysis of amino acids

Total protein was extracted from Salicornia shoot sample (1 g), using TCA/acetone extraction method (Isaacson et al., 2006) and quantified by Bradford (1976) method. The hydrolysis of total extracted protein was carried out in glass vessel with HCl (6 M, 500 µl). Glass vessels were vacuumed by flushing with N2, sealed and sample was hydrolyzed at 110 °C for 24 h. After hydrolysis, samples were vacuum dried and further used for the derivatization (Kwanyuen & Burton, 2010). Mixture of ethanol– water–TEA (2:2:1, v/v/v; 500 µl) was added to the vessels, containing hydrolyzed protein samples and amino acid standard (AAS18, Sigma, St. Louis, Missouri, USA) for neutralization. Samples were mixed, vacuum dried and subjected to the derivatization by adding a mixture of ethanol–water–TEA– PITC (7:1:1:1, v/v/v/v; 50 µl). The reaction mix was kept at room temperature for 20 min, vacuum dried and dissolved in Na2HPO4 buffer (5 mM, pH 7.4; 400 µl), containing acetonitrile (5%, v/v). The samples were filtered with a 0.2 µm membrane, and amino acid composition was analyzed using HPLC (Waters Alliance model, 2996-seperation module with autosampler), equipped with Luna-C18 reversed-phase (5.0 µm, 4.6 × 150 mm, Phenomenex, Torrance, California, USA) column (Kwanyuen &

journal of functional foods 13 (2015) 21–31

Burton, 2010; Mishra & Jha, 2009). The relative proportion of peak area was calculated to estimate the amino-acid composition.

2.3.

Fatty acid extraction and FAME analysis

Total lipid was extracted from Salicornia shoot sample (250 mg), using chloroform–methanol–phosphate buffer (1:2:0.9, v/v/v, pH 7.5; 10 ml) following modified Bligh and Dyer method (Bligh & Dyer, 1959; Kumari, Reddy, & Jha, 2011). The corresponding fatty acid methyl esters (FAMEs) of fatty acids were prepared by transmethylation (Kumari, Bijo, Mantri, Reddy, & Jha, 2013). Transmethylation was carried out by adding NaOH (1%, v/v in methanol; 1 ml) in the vessels, containing extracted lipid followed by heating at 55 °C for 15 min, thereafter methanolic HCl (5%, v/v; 2 ml) was added; vessels were further heated at 55 °C for 15 min and finally milli-Q water–hexane mixture (1:2, v/v; 3 ml) was added. The derivative FAMEs were extracted in hexane three times, vacuumed dried and dissolved in hexane (200 µl). The FAME samples were analyzed on GCMS-QP2010 (Shimadzu, Kyoto, Japan) equipped with an auto-sampler (AOC-5000) using a RTX 5MS capillary column (60 m length, 0.25 mm diameter and 0.50 µm thickness; Restek, Bellefonte, Pennsylvania, USA). Injector temperature was 240 °C, carrier gas helium with flow rate 1.0 ml min−1 and pre-column pressure was 49.7 kPa. The initial column temperature was 40 °C for 3.0 min, thereafter from 40 to 230 °C with 5 ° C min−1 increment and finally 230 °C held for 40 min. Injection volume was 1 µl, ionization mode electron impact at 70 eV, temperature of ion source and quadrupole was 200 °C. FAME peaks were identified by comparing their retention times with standards (FAME Mix C4-C24, Supelco, Bellefonte, Pennsylvania, USA and 7-hexedecenoic acid methyl ester, Cayman Chemicals, Ann Arbor, Michigan, USA) run in GC– MS along with samples.

2.4.

Extraction and identification of metabolites

Plant sample (100 mg) was grounded and total metabolites were extracted from succulent shoots using modified method of De Vos et al. (2007) by adding ice cold aqueous methanol (70%, v/v) followed by vortexing. The sample was kept in an ultrasonic water bath (MRC, Holon, Israel) for 1 h at frequency 40 kHz (25 °C). Supernatant was collected after centrifugation (16,000 g at 25 °C for 10 min) and filtered (0.2 µm membrane). Metabolites were analyzed by LC coupled with TOF MS/MS (Micromass, Waters, Milford, Massachusetts, USA) and identified by comparing LC-TOF MS/MS peaks using on-line METLIN database (Zhu et al., 2013). The LC–MS/MS parameters were as source and desolvation temperatures 110 and 200 °C, respectively, 2.5 kV was applied to the electrospray capillary, cone voltage 25 V and nitrogen used as the collision gas. The samples were directly injected, using syringe pump to the ESI–MS at 50 µl min−1 flow rate, and extracted metabolites were examined in negative-ion ESI/MS–MS mode. The scanning range was 0–1000 m/z with an acquisition rate of 0.25 s and inter-scan delay of 0.1 s. For peak integration, the background of each spectrum was subtracted, the data smoothed, cantered and peaks were integrated, using the Mass Lynx software version 4.1 (Micromass, Waters, Milford, Massachusetts, USA).

2.5.

23

Analyses of ROS activities

Plant shoots (10 g) were harvested, grounded in liquid N2, transferred to aqueous methanol (70%, v/v) and kept overnight for the extraction. The mixture was centrifuged at 7000 g for 10 min and supernatant was collected. The extraction (in aqueous methanol) was repeated twice. Collected supernatants were concentrated in a rotary evaporator (150–100 mbar at 37 °C) and lyophilized. The dried residue was stored at −20 °C until use. To determine different activities (scavenging and antioxidant) and contents (phenolic and flavonoid), absorbance reading of samples (plant extracts) were compared with a standard curve, which was drawn by same method, using known amount of corresponding standard. All tests were performed in triplicate and values were expressed as mean ± SE.

2.6.

Total antioxidant activity

Total antioxidant activity was measured by comparing ABTS·+ scavenging capability of plant extract with standard trolox (Hazra, Biswas, & Mandal, 2008; Re et al., 1999). The ABTS diammonium salt (7 mM) solution was mixed with potassium persulphate (2.45 mM) and mixture were incubated in the dark for 12–16 h at room temperature to generate ABTS·+. The ABTS radical cation solution was diluted with water for an initial absorbance of the solution about 0.70 ± 0.02 at 734 nm. The radical cation scavenging activity was assessed, by using 1 ml of diluted radical cation solution mixed with different concentrations of the plant extracts (10–50 µg ml−1) or standard (1– 5 µg ml−1 trolox). After incubation, the absorbance was read at 734 nm. The percentage inhibition of absorbance was calculated and activity was compared.

2.7.

Total phenolic content

Total phenolic content of plant extract was determined by Folin– Ciocalteu (FC) reagents using gallic acid as standard (Hazra et al., 2008; Singleton & Rossi, 1965). The plant extract was mixed with 2.5 ml of 0.2 M Folin–Ciocalteu reagents (Sigma, St. Louis, Missouri, USA) and incubated for 5 min, followed by addition of 2 ml sodium carbonate (Na2CO3; 75 g l−1). The reaction mixtures were incubated further for 90 min at room temperature. The absorbance was read at 760 nm and total phenolic content was calculated as mg ml−1 gallic acid per 100 mg of extract from standard curve.

2.8.

Total flavonoid content

To determine the total flavonoid content, plant extract was added to 0.3 ml NaNO2 (5%, v/v) and incubated for 5 min at room temperature. Thereafter 0.3 ml AlCl3 (10%, v/v) and 2 ml NaOH (1 M) were added. The reaction mixture was diluted, absorbance was read at 510 nm and the total flavonoid content was calculated as mg ml−1 quercetin per 100 mg of extract from standard curve (Hazra et al., 2008; Zhishen, Mengcheng, & Jianming, 1999).

2.9.

Reducing power

Different concentrations of the plant extract (0.2–1 mg ml−1) or ascorbic acid (used as positive standard) were mixed with 1 ml

24

journal of functional foods 13 (2015) 21–31

of phosphate buffer (0.2 M, pH 6.6) and 1 ml of K3Fe(CN)6 (10 mg ml−1) and incubated at 50 °C for 20 min in a water bath (Julabo, Seelbach, Germany). The reaction was terminated by adding 1 ml of TCA (100 mg l−1). The reaction mixture was centrifuged at 7000 g for 10 min at room temperature and supernatant was collected. A measure of 1 ml diluted supernatant was mixed with 0.2 ml freshly prepared FeCl3 (0.1%, w/v) and incubated for 10 min at room temperature. The absorbance was read at 700 nm and reducing power was compared.

2.10.

Hydrogen peroxide scavenging activity

The hydrogen peroxide activity was determined by using different concentrations of plant extracts (0.2–1.0 mg ml−1). Plant extract was mixed with 0.4 ml phosphate buffer (50 mM, pH 7.4) followed by addition of 0.6 ml hydrogen peroxide (43 mM prepared in phosphate buffer). The absorbance of reaction mix was recorded at 230 nm after 10 min against a blank (Rosen & Rauckman, 1984) and scavenging activity was measure as

OD230 of Extract ⎤ ⎡ Scavenging activity (% ) = ⎢1 − × 100 OD230 of Control ⎥⎦ ⎣

2.11.

Hydroxyl radical scavenging assay

Hydroxyl radical scavenging activity was measured by using Fenton reaction, in which different concentrations of plant extract was used for the scavenging of hydroxyl radicals, generated by the Fe3+-ascorbate–EDTA–H2O2 system (Saeed, Khan, & Shabbir, 2012). The reaction mixture containing different concentrations of extract (20–80 µg ml−1), 500 µl of 2-deoxyribose (2.8 mM prepared in 50 mM potassium phosphate buffer, pH 7.4), 200 µl premixed FeCl3–EDTA solution (100 mM each; 1:1, v/v) and 100 µl H2O2 (200 mM). The reaction was triggered by adding 100 µl of ascorbic acid (300 mM) followed by incubation at 37 °C for 1 h. A measure of 1 ml of TCA (2.8%, w/v) and 1 ml of TBA (0.5% TBA in 0.025 M NaOH containing 0.02% BHA) were added to the 0.5 ml of reaction mixture. The mixture was heated at 99 °C for 15 min in a water bath (Julabo, Seelbach, Germany), cooled to room temperature and absorbance was read at 532 nm against a blank. The hydroxyl radical scavenging activity was calculated as

OD532 of Extract ⎤ ⎡ Scavenging activity (% ) = ⎢1 − × 100 OD532 of Control ⎥⎦ ⎣

2.12.

DPPH radical scavenging assay

DPPH is a free radical and its scavenging was determined, using trolox as a standard (Saeed et al., 2012). The DPPH stock solution (0.024%, w/v in methanol) was diluted to make working solution by adding methanol until an absorbance of 0.98 ± 0.02 at 517 nm was obtained. Different concentrations of plant extract (10–80 µg ml−1) was mixed with 3 ml of working stock and incubated overnight at room temperature in the dark. The absorbance was measured at 517 nm and the radical scavenging activity of plant extract was estimated using the following equation

⎡ OD of Control − OD517 of Extract ⎤ Scavenging activity (% ) = ⎢ 517 ⎥ × 100 OD517 of Control ⎣ ⎦

3.

Results

3.1.

Amino-acid composition

In total, 17 amino acids were detected, quantified using HPLC and categorized as non-essential, essential, sulphur-rich and aromatic amino acids (Fig. 1 and Supplementary Fig. S1). Glycine and valine were the most detected (0.3 g per 100 g of crude protein) followed by proline and arginine under controlled conditions. The amount of essential amino acid valine, sulphurrich amino acids cysteine and methionine, and tyrosine increased significantly (p < 0.05) under salt and heat stress. Elevated amounts of almost all amino acids (except some, in which the quantity decreased or was unchanged) was detected under stress compared to the control, but the changes were observed to be statistically insignificant at p < 0.05 (Fig. 1).

3.2.

Fatty acid profiling

The total fatty acid (FA) content of S. brachiata was comprised of 55% polyunsaturated, 38% saturated and 7% monounsaturated fatty acids (Table 1 and Supplementary Fig. S2). A range of FAs (C14–C24) was detected, with dominance of alphalinolenic acid (C18:3, 29%) and 26% of both linoleic (C18:2) and palmitic acids (C16:0). A change in the percent quantity of FAs was observed under abiotic stress. Tridecanoic acid C13:0 (0.6%) was detected under salt stress only, whereas oleic acid C18:1 (6–8%) was found in both control and salt-stress-treated plants. The quantity of linoleic acid C18:2 decreased significantly (p < 0.05) under heat stress compared to the control, whereas an elevated quantity was detected under salt stress. Synthesis of alpha-linolenic acid C18:3 increased significantly (p < 0.05) under salt- (33%) and heat-stress (41%) conditions compared to the control (29%). The amount of palmitic acid C16:0 decreased under salt stress, whereas it was unchanged after heatstress treatment compared to the control. Myristoleic acid C14:1, palmitic acid C16:0 and palmitoleic acid C16:1 quantities decreased under stress, whereas elevated quantities of arachidic acid C20:0, heneicosanoic acid C21:0 and lignoceric acid C24:0 were detected under stress conditions. The amount of heptadecanoic acid C17:0 remained unchanged under stress treatments. Overall, the amount of saturated FAs decreased, whereas the quantity of PUFAs increased under stress. Monounsaturated FAs (MUFAs) also increased under salt stress, but a sudden decrease was found under heat-stress conditions compared to the control plants.

3.3.

Metabolite profiling

About 19 different metabolites, including sugar phosphates, lipids and gangliosides, were identified in S. brachiata using liquid chromatography–TOF–mass spectrometry (LC–TOF– MS; Fig. 2 and Supplementary Tables S1 and S2). Plant-origin metabolites, such as an aromatic monocyclic sulphuric acid monoester flavor component benzyl sulphate (m/z 93.04), an

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journal of functional foods 13 (2015) 21–31

1.2

Control Salt 1.0

a

c

Heat

Amount

[g/ 100 g of crude protein]

b

0.8

d

0.6

e

ab

0.4

f cd

0.2

gh gh

ef

Non-Essential

Essential

S-Rich

TYR

PHE

MET

CYS

VAL

THR

LYS

LEU

ILE

HIS

PRO

SER

GLY

GLU

ARG

ASP

ALA

0.0

Aromatic

Amino-acids Fig. 1 – Amino-acid composition of protein extracted from S. brachiata under control and stress conditions. Value represents the mean ± SE followed by similar letters showed significant difference at P < 0.05 according to Tukey HSD.

aromatic heteropolycyclic galloyl flavonol myricatin (m/z 183.14 and 532.98) containing flavan-3-one moiety and a food colorant 4,11-DCQA (m/z 379.48), were identified, which are all used as nutritive supplements in food industries. Bioactive compound oxomefruside (m/z 131.07; a diuretic compound), clonidine (m/z 210.19; an imidazoline derivative) and carmustine (m/z 212.19; an organic derivative of urea that contains semicarbazide functional group in which one amine group is

replaced with a hydrazine group) were detected along with antibacterial antibiotic cefazolin sodium (m/z 459.82; an organic heterocyclic beta lactams that belongs to the cephalosporins group). An alcohol soluble halogenated phenolic compound, dichlorophene (m/z 267.10) and three organophosphorus compounds, oxydisulfoton (m/z 271.10), sulfotep (m/z 303.41) and azothoate (m/z 355.48), known for their insecticidal or fungicidal activities, were also observed in the plant. Intermediated

Table 1 – Fatty acid compositions of S. brachiata under stress determined by GC–MS analysis. Fatty acids

Control

Salt1

Heat2

Tridecanoic acid [C13:0] Myristoleic acid [C14:1] Pentadecanoic acid [C15:0] Palmitic acid [C16:0] Palmitoleic acid [C16:1 (n-7)] Heptadecanoic acid [C17:0] Stearic acid [C18:0] Oleic acid [C18:1 (n-9)] Linoleic acid [C18:2 (n-6)] alpha-Linolenic acid [C18:3 (n-3)] Arachidic acid [C20:0] Heneicosanoic acid [C21:0] Lignoceric acid [C24: 0] SFA [saturated fatty acids] MUFA [monounsaturated fatty acids] PUFA [polyunsaturated fatty acids] n6/n3 n9/n3 n9/n6

nd 0.6950 ± 0.25 0.1432 ± 0.04 25.8166 ± 2.11 0.4108 ± 0.17 0.3883 ± 0.07 7.2054 ± 0.62 6.1294 ± 3.08 25.7036 ± 0.34a 29.0636 ± 0.88b 0.9087 ± 0.12 1.6148 ± 0.17 1.9207 ± 0.23 37.9976 7.2352 54.7672 0.8844 0.2109 0.2385

0.5931 ± 0.14 0.1905 ± 0.08 nd 17.7314 ± 8.35 0.1998 ± 0.06 0.3976 ± 0.07 7.6256 ± 0.70 7.9245 ± 7.08 27.2070 ± 2.34 32.6458 ± 2.61c 1.0198 ± 0.16 1.8702 ± 0.26 2.5948 ± 0.31 31.8324 8.3147 59.8528 0.8334 0.2427 0.2913

nd 0.3427 ± 0.05 0.0989 ± 0.01 24.4117 ± 0.73 0.1781 ± 0.04 0.3859 ± 0.01 5.9682 ± 0.55 nd 22.2759 ± 0.52a 41.3511 ± 1.64bc 1.1790 ± 0.04 1.6386 ± 0.04 2.1701 ± 0.04 35.8522 0.5208 63.6270 0.5387 nd nd

Value (%): Mean ± SE, n = 3. nd: not detected; similar letter represent significant difference at P < 0.05; 1: NaCl [500 mM] for 24 hrs and 2: heat [42 °C] for 24 hrs.

26

1000 900

50

100

150

200

250

300

350

400

450

500

550

600

800

Ganglioside

Lipids: Acidic glycosphingolipids

Clonidine

1100

Carmoisine

1200

Dihydrobiopterin

1300

Myricatine

Cefazolin sodium

1400

Lipids: Neutral glycosphingolipids

journal of functional foods 13 (2015) 21–31

850

900

950

1500 1200 900 800 700 600 500 400 300 200 100 0 1000

100

4,11DCQA

200

Azothoate

300

Myricatine

400

Oxomefruside

Benzyl sulphate

500

Shikimate-3-phosphate

Sugar phosphate Carmustine

600

Sulfotep: organothiophosphate

700

Ribose-di-phosphate

800 Dichlorophene Oxydisulfoton

Intensity

Selenocystathionine

0 50

100

200

250

Mass [m/z]

300

350

400

Fig. 2 – Metabolites detected from S. brachiata extract by LC–MS analysis.

3.4.

Analysis of antioxidant activities

treatments (Fig. 3). Enhanced antioxidant activity was observed under heat stress more so than under salt stress, compared to the control conditions. It was found that the total phenolic content decreased under heat (about 1.4-fold) and salt stresses (about 2.6-fold) compared to the control conditions. In contrast, the total flavonoid content increased 1.3- and 2.4fold under salt and heat stresses, respectively (Fig. 4).

50 45

Control Salt Heat

40 35

% Inhibition

metabolites of different pathways, shikimate-3-phosphate (m/z 235.12), selenocystathionine (m/z 269.09), dihydrobiopterin (m/z 503.92) and gangliosides (m/z 879.48), were found with different sugar phosphates (m/z 353.46) and lipids (m/z 851.41, 955.48 and 975.46). Shikimate-3-phosphate is an intermediate of aromatic amino acid biosynthesis pathway whereas aromatic heteropolycyclic compound dihydrobiopterin is an oxidative product of tetrahydrobiopterin, which is a natural occurring cofactor of aromatic amino acids. Ganglioside is a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids linked on the sugar chain. It was observed that most of metabolites were not synthesized under stress conditions (Supplementary Table S3). Only a few metabolites, benzyl sulphate, myricatin, clonidine, shikimate-3-phosphate and sulfotep, were detected under both salt and heat stress. Metabolites cefazolin sodium and gangliosides were found under heat stress, but they were absent under salt stress. Similarly, sugar phosphates, azothoate and dihydrobiopterin were detected under salt stress, but were absent under heat stress. Some neutral glycosphingolipids and thymidine 5′-triphosphate were only synthesized under salt and heat stresses, respectively (i.e. they were absent in control plants).

30 25 20 15 10 5 0 10

20

30

40

50

Concentration [µg/ml extract]

Total antioxidant activity (expressed in the terms of % inhibition of decolorization of ABTS·+) of the plant extract increased concomitantly with the extract concentration under all stress

Fig. 3 – Total anti-oxidant activity shown by S. brachiata plant extract.

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3.0

16

12 10 8 6

Heat

2.0 1.5 1.0 0.5

4

0.0

0 Heat

Control

TPC

Salt

Heat

TFC

Fig. 4 – Total phenolic and total flavonoid contents extracted from S. brachiata under control and stress conditions. Value represents the mean ± SE.

Analysis of scavenging activities

The reducing capacity and scavenging activity of the plant extract increased concurrently with the extract concentration during all stress treatments (Fig. 5 and Supplementary Fig. S3). The reducing capability of the plant extract was slightly decreased (statistically insignificant at p < 0.05), except for 0.8 mg ml−1 of the extract (salt stress), under both stress conditions compared to the control (Supplementary Fig. S3). The scavenging capacity of hydrogen peroxide was found to be at its maximum under heat stress, whereas reduced activity was detected under salt stress compared to the control plants (Fig. 5a). Approximately, a twofold increase in the H2O2 scavenging activity was observed when the plant extract concentration increased from 0.2 to 0.5 mg ml−1, whereas a 1.4fold increment was detected when the amount of plant extract was further increased. Overall, an approximate threefold increase in activity was observed with a fivefold increase in plant extract concentration (0.2–1.0 mg ml−1). The scavenging of hydroxyl and DPPH radicals was increased under stress conditions. Better hydroxyl-radical-scavenging activity was found under salt stress compared to heat stress (Fig. 5b), but almost consistent scavenging activity was detected for the DPPH radical under both salt and heat stresses (Fig. 5c). There was a 1- to 2.5-fold increase in hydroxyl- and DPPH-radical scavenging activities observed with the increased plant-extract concentration. Overall, a 2.5- to 3.5-fold increase in scavenging of hydroxyl and DPPH radicals was found with a fourfold increase in the concentration of plant extract.

Discussion

Salicornia grows opulently in salt marshes, possessing stress resilience, and it is well known for scavenging toxic ROS produced under stress conditions (Chaturvedi et al., 2014; Singh et al., 2014a). The plant is a rich source of proteins, vitamins, unique oligosaccharides and antioxidants (Jha et al., 2012;

0.2 0.5 Concentration [mg/ml extract]

a 50

Control

Salt

1

Heat

45 40

Scavenging [%]

Salt

35 30 25 20 15 10 5 0 20

b 25

Control

50 70 Concentration [µg/ml extract] Salt

80

Heat

20

Scavenging [%]

Control

4.

Salt

2.5

2

3.5.

Control

14

Scavenging [%]

TPC [mg/ml gallic acid per 100 mg extract] TFC [mg/ml quercetin per 100 mg extract]

18

15 10 5 0

c

10

20 40 Concentration [µg/ml extract]

80

Fig. 5 – Scavenging activities of S. brachiata under stress. Scavenging of (a) H2O2, (b) hydroxyl and (c) DPPH free radicals. Value represents the mean ± SE.

Mishra et al., 2013; Stanley, 2008); therefore, it is considered as a potential source of dietary supplementation. The identification of untargeted metabolites and ROS scavenging activities under control and stress conditions reveal potential of the plant to be used as functional food and provide useful insights into the metabolic responses and metabolite constituents of this halophyte. The composition of amino acids was studied in some halophytes, and a variable pattern was detected under salt-stress condition (Nasir, Batarseh, Abdel-Ghani, & Jiries, 2010). In the present study, a higher content of essential and sulphur-rich amino acids was found in plant (Fig. 1) compared to the reference pattern recommended by the FAO (FAO, 2013). The

28

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results implied that Salicornia contains a large amount of amino acids, and is thus considered as nutritive (Jha et al., 2012). The elevated amounts of the essential amino acid valine, sulphurrich amino acids cysteine and methionine, and tyrosine were detected under salt and heat stresses, along with proline, leucine and isoleucine. Amino acids, especially proline, were synthesized under stress condition to provide osmotic protection to the plant. A high content of sulphur-rich amino acids (cysteine and methionine) was detected, because of the disruption of the sulphur bonds and release of these amino acids under stress. Previously, it was found that the amino acid content increased in Atriplex halimus, Portulaca oleracea and Tamarix aphylla with salinity during spring and autumn seasons (Nasir et al., 2010). It was observed that accumulation of free amino acids not only provides osmotic adjustment, but also involves maintenance of cell-membrane integrity and stability under stress conditions (Tuteja, Gill, & Tuteja, 2011). The reducing capacity and ROS scavenging activities of H2O2, hydroxyl and DPPH increased concomitantly with the concentration of extracts under control and stress conditions (Fig. 5 and Supplementary Fig. S3). Previously, it was observed that high scavenging activity was associated with the accumulation of antioxidants (Alhdad, Seal, Al-Azzawi, & Flowers, 2013; Jallali et al., 2014). Being a halophyte, Salicornia exhibits an advanced antioxidant system in order to combat with salt stress. The plant extract showed enhanced total antioxidant activity and total flavonoid content under stress conditions (Figs. 3 and 4). In contrast, a decrease in total phenolic content was found under stress (Fig. 4). A strong antioxidant activity and phenolic compounds were detected in the ethanol extract of Salicornia herbacea, which was found to be responsible for the radical scavenging properties of the plant (Oh, Kim, Lee, Woo, & Choi, 2007). Its role as an antioxidant was investigated in halophyte Suaeda maritima under salinity and waterlogging conditions (Alhdad et al., 2013). Previously, it was observed that total phenolic contents and its antioxidant activities were increased with roasting temperature in cashew nuts, kernels and testa (Chandrasekara & Shahidi, 2011). Occurrence and activities of total phenolic contents depend on different plant parts and developmental stages. The peel of potatoes contains higher phenolic content and antioxidant activities than their respective flesh (Albishi, John, Al-Khalifa, & Shahidi, 2013). Similarly, higher total phenolic compounds and antioxidant activities were observed in red-fleshed apples compared to white-fleshed apples (Wang et al., 2014). The highest contents of total phenolic compounds were detected in full flowering stage of Opuntia microdasys compared to other vegetative parts and flowering stages (Chahdoura et al., 2014). The influence of abiotic stress on the antioxidant activity and phenolic composition of halophyte Crithmum maritimum was studied (Jallali et al., 2012). It was observed that the antioxidant activity, ROS scavenging activity, phenolic content and flavonoid content depended on several environmental factors. Their synthesis and activity varied under stress to provide stress endurance in the plant. It was depicted that the antioxidant and ROS-scavenging activities exhibited by Salicornia in this study may be attributed to the presence of phenolic and flavonoid compounds analogous to those reported for Salicornia herbacea (Oh et al., 2007). Some fatty acids, called essential FAs, are outsourced by the human body from food, as these cannot be naturally made

in a sufficient quantity (FAO, 2010). Two essential FAs, linoleic acid (C18:2) and alpha-linolenic acid (C18:3), are widely distributed in plants. PUFAs represent an important nutritional indicator, and plants rich in PUFAs, particularly linoleic acid, are considered to have medical importance. In this study, Salicornia contained about 55% PUFAs, which increased under salt- (60%) and heat-stress (64%) conditions (Table 1). High contents of linoleic acid (C18:2) and alpha-linolenic acid (C18:3) were detected at 26 and 29%, respectively. The alpha-linolenic acid content increased significantly under stress conditions. Furthermore, about 38% saturated FAs, including 26% palmitic acid (C16:0), were detected in the plant. Previously, FA profiling was performed to identify potential halophytes as sources of edible oils, and 22–25% PUFAs were detected in some selected halophytes (Weber, Ansari, Gul, & Khan, 2007), which is lower than the S. brachiata plant. Compared to S. brachiata (38%), about 22% SFAs was reported in Salicornia fruticosa seed oil (Weber et al., 2007). About 16–30% palmitic acid was found in seeds of S. brachiata (16.5%) in addition to other halophytes Arthrocnemum macrostachyum, Cressa cretica, Halopyrum mucronatum, Alhagi maurorum and Haloxylon stocksii (Eganathan, Subramanian, Latha, & Rao, 2006; Weber et al., 2007). Seeds of Salicornia europaea contained about 76% linoleic acid, 13% oleic acid, 7% palmitic acid, 3%, linolenic acid and 2% stearic acid (Liu et al., 2005). Salicornia bigelovii seed oil contained high levels of linoleic acid (about 78%), less oleic acid (approximately 15%) and linolenic acid (2%), as well as SFAs palmitic and stearic acids with contents of 8 and 2%, respectively (Anwar, Bhanger, Nasir, & Ismail, 2002). It was also reported that the compositions of S. europaea and S. bigelovii oil were very similar to that of safflower oil, thus having nutritive and medical health values (Anwar et al., 2002; Liu et al., 2005). Furthermore, the seeds of halophytes, particularly Salicornia fruticosa, have been recommended for human consumption (Weber et al., 2007). Succulent halophyte S. brachiata is a potential source of valuable antioxidants, amino acids, phenolic acids, flavonoids and fatty acids. Furthermore, about 19 different metabolites were identified in the present study using LC–TOF–MS/MS (Fig. 2 and Supplementary Tables S1 and S2). This study is the first report on the untargeted metabolomics of halophyte S. brachiata. A flour compound benzyl sulphate and flavonoid Myricatin were identified in the plant. These metabolites are known nutritive supplements and thus provide nutritional value to the plants. Furthermore, flavonoids are also well known to have anti-oxidant and anticancer activities. A strong positive correlation between antioxidant activity and total flavonoid content was reported in Gynura procumbens (Kaewseejan, Sutthikhum, & Siriamornpun, 2015). A flavonoid quercetin (quercetin 3-βD-glucoside) isolated from Satureja montana contained antioxidant activity (López-Cobo, Gómez-Caravaca, Švarc-Gajic´, Segura-Carretero, & Fernández-Gutiérrez, 2014), whereas quercetin (quercetin-3-O-α-L-rhamnopyranoside) obtained from Toona sinensis was reported to be an antioxidant with anticancer activities (Zhang et al., 2014). Additionally, bioactive compounds were identified along with bactericidal, insecticidal and fungicidal activities. Oxomefruside is a diuretic metabolite involved in hypertension treatment. Metabolite clonidine (KEGG C06920) is a bioactive compound used to treat

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high blood pressure, anxiety and certain pain conditions and it was assumed to act as an imidazoline receptor (Zhu et al., 2013). Another bioactive compound was identified as carmustine (KEGG C06873), which is anti-neoplastic in nature and used in the treatment of several types of cancers. Anti-tuberculosis activity was reported in S. brachiata plant extracts (Rathod et al., 2011), whereas S. herbacea is used in the protection of glomerular sclerosis of PAN-induced kidney damage when given as a diet supplement (Son et al., 2007). Some of the Salicornia species are explored as folk medicine for the treatment of bronchitis, hepatitis and diarrhea (Isca et al., 2014). The compound cefazolin sodium (KEGG C08098) was detected in plant extracts, which is a well-known antibiotic that acts on a wide range of bacteria. Previously, aqueous extract of S. brachiata was found to efficiently produce silver nanoparticles containing bactericidal activity against pathogenic bacteria (Seralathan et al., 2014). Additionally, compounds with insecticidal or fungicidal activities were detected in the S. brachiata extract (Supplementary Tables S1 and S2). Intermediate metabolites from different pathways, shikimate-3-phosphate, dihydrobiopterin and ganglioside, were found with different sugar phosphates and lipids. An intermediate metabolite selenocystathionine (KEGG C05699) was detected, which is involved in selenocompound metabolism and formed as a by-product of cystathionine synthesis by the enzyme cystathionine beta-synthase (Çakır, Turgut-Kara, & Arı, 2012; Haug, Graham, Christophersen, & Lyons, 2007). Selenocystathionine is consumed as a functional food supplement, because it is involved in the hyper-accumulation of selenium, which is an anti-cancer agent (Çakır et al., 2012). Shikimate-3-phosphate (KEGG C03175) and dihydrobiopterin (KEGG C06149) are intermediates of aromatic amino acids and folate biosynthesis pathways, respectively. Gangliosides detected in this study are glycosphingolipids, which modulate cell signal transduction and act as specific receptors for certain glycoprotein hormones. It has been reported that dietary gangliosides decrease cholesterol content, thus exerting a potential anti-inflammatory effect (Park et al., 2005). Salicornia does not synthesize most of the metabolites under stress conditions (Supplementary Table S3), as synthesis of metabolites are known to be environmentally dependent and energy diverts to the stress responses that are involved in complex regulatory networks, including metabolism adjustment and gene expression (Fraire-Velázquez & Balderas-Hernández, 2013; Krasensky & Jonak, 2012).

5.

Conclusions

It was observed that S. brachiata exhibited sulphur-rich proteins with unique oligosaccharides (Jha et al., 2012; Mishra et al., 2013), and considered as potential source of nutritive supplement. Furthermore, the present study evidenced that Salicornia contains metabolites with bioactivities, which reveals the medicinal potential of the plant to be used as a functional food. Moreover, nutritional antioxidants, scavenging activities, amino acids, flavonoids, essential FAs and PUFAs make it promising for use as a functional food, a dietary supplement and use in nutraceutical industries.

29

Acknowledgements CSIR-CSMCRI Communication No. PRIS-098/2014. The financial support received from Council of Scientific and Industrial Research (CSIR), Govt. of India, New Delhi [BSC0106-BioprosPR and BSC0109-SIMPLE] is thankfully acknowledged. Analytical Discipline and Centralized Instrument Facility of the institute is duly acknowledged for helping in running samples for the analytical analysis.

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2014.12.027.

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