EFFECT of SALINITY on PHENOLIC COMPOSITION and ANTIOXIDANT ACTIVITY of HALOPHYTES SABA NAZIR Institute of Sustainable Halophyte

EFFECT OF SALINITY ON PHENOLIC COMPOSITION AND ANTIOXIDANT ACTIVITY OF HALOPHYTES

SABA NAZIR

Institute of Sustainable Halophyte Utilization (ISHU)

University of Karachi

Karachi-75270, Pakistan

2018

THESIS

Submitted to the Faculty of Science, University of Karachi

In Fulfillment of the Requirement for the Degree of

Doctor of Philosophy in Botany (ISHU)

SABA NAZIR

Institute of Sustainable Halophyte Utilization (ISHU)

University of Karachi

Karachi-75270, Pakistan

IN THE NAME OF ALLAH THE MOST BENEFICENT THE MOST MERCIFUL

Dedication

To My beloved son Muhammad Omer My parents and family members Without their Love and Care I Would Never Succeed

THESIS APPROVED

Research Supervisor: ______

(Prof. Dr. Bilquees Gul)

Research Co-Supervisor: ______

(Prof. Dr. M. Ajmal Khan)

External Examiner: ______

Table of Contents Page Table of Contents I-II List of Tables III List of Figures IV -VIII Acknowledgments IX Summary in English 1 Summary in Urdu 3 Chapter 1 General introduction 4 Chapter 2 Environmental and phenological variations in 12 phenolic composition and antioxidant activity of medicinal halophytes Abstract 13 Introduction 14 Materials and 17 methods Results 20 Discussion 33 Conclusion 37 Chapter 3 Effect of salinity on growth, ecophysiology and 38 antioxidant status of Calotropis procera Abstract 39 Introduction 40 Materials and 43 methods Results 48 Discussion 70 Conclusion 79 Chapter 4 Effect of salinity on growth, ecophysiology and 80 antioxidant status of Thespesia populnea Abstract 81 Introduction 82 Materials and method I 86

Table of Contents Page Results 89 Discussion 112 Conclusion 121 Chapter 5 General conclusions 122 References 125

S.No. List of Tables Page 1.1. List of radical and non-radical Reactive oxygen species (ROS). 6 1.2. Antioxidant enzymes and their action mechanisms. 8 1.3. Non-enzymatic antioxidant compounds, occurrence at sub- 8 cellular level and their role against reactive oxygen species. 2.1 Table showing habitat selection criteria with coordinate 23 distribution and distance from shore 2.2 List of selected test species with common name, family, 25 flowering period and medicinal uses. 2.3 Correlation coefficient (r) of different antioxidant parameters 32 studied. 2.4 Multivariate analysis (ANOVA) showing effect of species, 32 habitat, season and plant part and their interactions on different antioxidant parameters studied.

3.1 Water potential at full turgor (ѰW0), Water potential at turgor loss 68

point (ѰWTLP) & Bulk elasticity of cell wall (Ɛ Mpa) of C. procera leaves. 3.2 % Contribution to osmolality of organic and inorganic osmolytes 68 of Calotropis procera leaves. 3.3. Phenolic composition (µg g-1 DW) in leaves of C. procera treated 69 with 0, 100 and 300 mM NaCl concentrations.

4.1 Water potential at full turgor (ѰW0), Water potential at turgor loss 109

point (ѰWTLP) & Bulk elasticity of cell wall (Ɛ Mpa) of Thespesia populnea leaves. 4.2 % Contribution to osmolality of organic and inorganic osmolytes 109 of Thespesia populnea leaves. 4.3 Number and area of stomata on upper and lower surface of 109 Thespesia populnea leaves. 4.4 Phenolic composition (µg g-1 DW) in leaves of T.populnea treated 110 with 0, 100 and 300 mM NaCl concentrations III

S.No. List of Figures Page Figure. 1.1 External and internal sources of ROS and their 7 consequences at cellular level and potential health effects. Figure. 2.1 Map of study area showing biogeographic regions of 23 Karachi where plants were collected. Figure 2.2 Mean annual temperatures, rainfall, humidity and wind 24 speed of study area 2014 (Pakistan Meteorological Department). Figure 2.3 Change in total phenol (TPC), total flavnoids (TFC) and 26 proanthocynadin (PC) content of selected medicinal halophytes. Figure 2.4 Change in DPPH, ABTS, FRAP and TAC activities of 27 selected medicinal halophytes. Figure 2.5 Habitat variation in polyphenol (TPC, TFC, PC) and 28 antioxidant activities (DPPH, ABTS, FRAP and TAC) of selected medicinal halophytes. Figure 2.6 Seasonal variation in polyphenol (TPC, TFC, PC) and 29 antioxidant activities (DPPH, ABTS, FRAP and TAC) of selected medicinal halophytes. Figure 2.7 Organ specific variation in polyphenol (TPC, TFC, PC) and 30 antioxidant activities (DPPH, ABTS, FRAP and TAC) of selected medicinal halophytes. Figure 2.8 Stage specific variation in polyphenol (TPC, TFC, PC) and 31 antioxidant activities (DPPH, ABTS, FRAP and TAC) of medicinal halophytes. Figure 3.1 (A) Comparison of Calotropis procera plants grown under 51 different (mM) NaCl treatments for 35 days under green net house. (B) Changes in height of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.2 Changes in plant fresh weight and plant dry weight of 52 (leaf, Stem and root), Root weight ratio (RWR) of IV

C.procera plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.3 Changes in leaf area, number of leaves, leaf moisture%, 53 nodes per plant, Leaf succulence and Girth (cm per plant of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.4 Effect of different NaCl treatments (0, 100, 300mM) on 54 water potential, osmotic potential and turgor potential (MPa) of C procera leaves. Figure 3.5 Effect of different NaCl treatments (0, 100, 300mM) on Pre- 54 dawn and noon water potential (MPa) of C procera leaves. Figure 3.6 Changes in chlorophyll a, chlorophyll b, total chlorophyll 55 and chlorophyll a/b ratio, carotenoid and chlorophyll/ carotenoid ratio of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.7 Changes in anthocyanin, betacyanin, flavnolglycosides, 56 betacarotene and lycopene of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations.

Figure 3.8 Changes in H2O2, MDA and EL% of C.procera plants 57 treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.9 Changes in Proline and soluble sugar of C.procera plants 58 treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.10 Changes in Fv/Fm, qP, ETR, Y (II), Y(NPQ),Y(NO) of C. 59 procera plants leaves treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.11 Changes in Na+ (mol kg-1 DW ) K+ (mole kg-1 DW and Na/K 60 in terms of dry weight of C.procera plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.12 Changes in K+ over Na+ selectivity Selective absorption 61 V

from medium to root ( SA), Selective transport from root to stem (ST1) and selective transport from stem to leaves (ST2), of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.13 Changes in CAT, SOD, APX, GR and GPX of C.procera 62 plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.14 Changes in ascorbic acid content and AsA/DHAsA ratio of 63 C.procera plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.15 Changes in total phenolic content, Total flavonoid content, 64 Proanthocynadin content, and Total tannin content, of C. procera plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.16 Changes in DPPH radical scavenging activity (Inhibition %) 65 , ABTS anti-radical activity (mM Trolox g-1) and Total antioxidant activity of C.procera plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations. Figure 3.17 HPLC chromatogram of (a) standard phenolic compounds 66 (1 Pyrogallol; 2 Gallic acid; 3 Resorcinol; 4 Pyrocatechol; 5 Catechin; 6 Hydroxybenzoic acid; 7 Chlorogenic acid; 8 Vanillic Acid; 9 Caffeic acid; 10 Syringic acid; 11 Coumaric acid; 12 Ferulic acid; 13 Sinapic acid; 14 Rutin; 15 Trans- cinnamic acid; 16 Quercetin; 17 Ellagic acid) and (b) their structures used in this study. Figure 3.18 HPLC chromatograms showing profile of different phenolic 67 acids in hydrolysed leaf extracts of C.procera treated with 0, 100 and 300 mM NaCl concentrations Figure 4.1 Comparison of T. populnea plants grown under different 91 (mM) NaCl treatments for 35 days under green net house (A). Changes in height of T. populnea plants treated with 0, VI

100 and 300 mM NaCl concentrations (B). Figure 4.2 Changes in fresh weight and dry weight of T.populnea 92 plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.3 Changes in Leaf area, number of leaves, leaf moisture%, 93 nodes per plant, Leaf succulence and Girth (cm per plant of T .populnea plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.4 Effect of different NaCl treatments (0, 100, 300mM) on 94 water potential, osmotic potential and turgor potential (MPa) of T.populnea leaves. Figure 4.5 Effect of different NaCl treatments (0, 100, 300mM) on Pre- 95 dawn and noon water potential (MPa) of T.populnea leaves. Figure 4.6 Changes in chlorophyll a, chlorophyll b, total chlorophyll, 96 chlorophyll a/b ratio, carotenoid and chlorophyll/ carotenoid ratio of T.populnea plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.7 Changes in anthocyanin, betacyanin, flavnolglycosides, 97 betacarotene and lycopene content of T.populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

Figure 4.8 Changes in H2O2 and MDA of T.populnea plants treated 98 with 0, 100 and 300 mM NaCl concentrations. Figure 4.9 Changes in Proline and soluble sugar of T.populnea plants 99 treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.10 Changes in Fv/Fm, qP, ETR, Y (II), Y (NPQ), Y(NO) of 100 T.populnea plants leaves treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.11 Changes in Na+ (mol kg-1 DW ) K+ (mol kg-1 DW and 101 Na+/K+ in terms of dry weight of T.populnea plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.12 Changes in K+ over Na+ selectivity Selective absorption 102 VII

from medium to root ( SA), Selective transport from root to stem (ST1) and selective transport from stem to leaves (ST2), of T.populnea plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.13 Changes in CAT, SOD, APX, GR and GPX of T.populnea 103 plants treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.14 Changes in ascorbic acid content of T.populnea plants 104 treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.15 Changes in total phenolic content, Total flavonoid content, 105 Proanthocynadin content and Total tannin content of T.populnea plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.16 Changes in DPPH radical scavenging activity (Inhibition 106 %), ABTS anti-radical activity (mM Trolox g-1) and Total antioxidant activity of T.populnea plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations. Figure 4.17 HPLC chromatogram of (a) standard phenolic compounds 107 (1 Pyrogallol; 2 Gallic acid; 3 Resorcinol; 4 Pyrocatechol; 5 Catechin; 6 Hydroxybenzoic acid; 7 Chlorogenic acid; 8 Vanillic Acid; 9 Caffeic acid; 10 Syringic acid; 11 Coumaric acid; 12 Ferulic acid; 13 Sinapic acid; 14 Rutin; 15 Trans- cinnamic acid; 16 Quercetin; 17 Ellagic acid) and (b) their structures used in this study. Figure 4.18 HPLC chromatograms showing profile of different phenolic 108 acids in hydrolysed leaf extracts of T.populnea treated with 0, 100 and 300 mM NaCl concentrations

VIII

Acknowledgments

I am grateful to Almighty ALLAH for showering his blessings upon me throughout my life. My sincere gratitude is to my supervisor Prof. Dr. Bilquees Gul for her encouragement, patience, guidance and support whenever needed. I would also like to express my gratitude to my Co. supervisor Prof. Dr. M. Ajmal Khan S.I. for his cooperation, motivating discussions and inspiration to complete this thesis.

I would like to extend my warmest thanks to, Dr. M. Qasim for the valuable suggestions, expertise, encouragement and analysis to critical review of my dissertation during the course of my research work. I would like to extend my earnest thanks to Dr. Irfan Aziz, Dr. Zaheer Ahmed and Dr. Zainul Abideen and members of Institute of Sustainable Halophytes Utilization (University of Karachi) for their constant help and cooperation during the course of my research work. I would also like to acknowledge Mr. Irfanuddin for his assistance in office matters. Many thanks to all of my research fellows who contributed a lot during various stages of my research, particularly to Ms. Shahjehan Jilani, Dr. Saman Ehsan, Dr. Sonia Bano, Dr. Sumera Manzoor, and Ms. Rabab Rizvi for their friendship and immense co-operation and moral support especially during field trips. I would like to acknowledge the help and cooperation extended by various Principals of The Intellect School, Karachi, for granting permission to continue my research and my colleagues for their constant support and help to continue my higher education. I am also thankful to the University of Karachi for providing research facilities for this study. Last, but not the least, all the deepest gratitude to my beloved father Nazir Istafa and Mother for their endless love, prayers and motivation to pursue my dream for higher studies. I would like to thank my siblings, my husband Muhammad Sohail for their support, patient, generosity and encouragement during difficult phases of my research.

Saba Nazir

Summary

Halophytes are well adapted to survive in harsh environmental conditions particularly saline lands as they possess distinctive regulatory mechanisms and secondary metabolites. These metabolites have been used in treating various diseases for hundred of years and also have various applications in pharmaceutical and other industries. Comprehensive knowledge about different factors (seasons, habitat, phenology and environment) affecting secondary metabolites and related biological activities of medicinal plants are lacking. To bridge this gap, current study was initiated with the analyses of polyphenols and antioxidant activity of different parts (leaf and stem) of 10 medicinal halophytes (i.e. Aerva javanica, Atriplex stocksii, Cressa cretica, Calotrpois procera, Heliotropium bacciferum, Ipomea pes-caprae, Salsola imbricata, Salvadora persica, Suaeda fruticosa and Thespesia populnea), collected from their coastal and inland habitats during different seasons (summer and winter) and phenological stages (vegetative and reproductive). This study revealed that plants accumulated significantly higher amounts of total polyphenols (TPC), flavonoids (TFC) and proanthocynadins (PC) in their leaves with considerable antioxidant activity, compared to stem. Plants accumulated greater proportion of polyphenols and higher antioxidant activity during drier season (winter) compared to wet season (summer). Similarly, higher polyphenols were found in reproductive stage compared to vegetative stage. The status of metabolites showed slight variation under different habitats compared to seasons. Moreover, all tested species exhibited strong correlations among all antioxidant activity assays, which indicated high radical scavenging and reducing power abilities of these plants. On the basis of polyphenol content plants were categorized into three groups i.e. high, moderate and low. Two plant species were selected for further detailed greenhouse experiments, one from high (T. populnea) and other from moderate (C.procera) category to analyze their salt induced ecophysiological responses as well as secondary metabolites (polyphenols) and antioxidant status. Both T. populnea and C. procera showed salt resistance up to 300 mM NaCl (~60% seawater salinity) and their biomass was not affected at moderate salinity (100 mM NaCl). Both species managed moderate salt stress by adjusting water balance, osmotic potential, selective absorption of K+ and oxidative stress management, while high (300 mM NaCl) salinity appeared damaging for both plants. High salinity resulted in significant decline in actual quantum yield (YII), photochemical quenching (qP) and electron transport rate (ETR). These photosynthetic limitations resulted in oxidative damage by increasing H2O2, MDA and electrolyte leakage ultimately 1 resulted in growth decline. However at moderate salinity, plants effectively managed heat dissipation (YNO), non-photochemical quenching (YNPQ), antioxidative enzyme activity (superoxide dismutase, catalase, glutathione reductase, guaicol peroxidase and ascorbate peroxidase) as well as secondary metabolite content, which in turn improved overall antioxidant status. Salt exposure markedly improved secondary metabolites in C. procera (e.g. natural antioxidants) with profound activities, while T. populnea maintained its antioxidant status at moderate salinity. HPLC analyses showed distinctive composition of phenolic antioxidants including pyrogallol, resorcinol, pyrocatechol, chlorogenic acid, coumaric acid, and rutin, which could be responsible for higher antioxidative performance of these species. This data highlights the importance of both tested species as rich sources of natural antioxidants, which may potentially replace synthetic chemicals. These naturally occurring chemicals are environment friendly which could be used as suitable alternatives in pharmaceutical and cosmetic industries.

CHAPTER 1 GENERAL INTRODUCTION

Halophytes are salt-resistant or salt-tolerant plants that often dominate saline habitats including salt marshes, coastal sand dunes, salt flats, deserts, steppes etc. and can thrive by exploiting salty water up to 200 mM NaCl (Khan and Qaiser, 2006; Flowers and Colmer, 2008). Salinity endurance mechanisms include strategies to minimize accumulation of sodium and other detrimental ions, which can be achieved either by exclusion or inclusion and sometimes both (Koyro and Lieth, 2008). In either case, excessive salt concentration can lead to ion toxicity/imbalance and water deficit (physiological drought), leading to production of harmful free radicals (Koyro, 2003; Geissler et al., 2008, 2009; Koyro et al., 2009). In such conditions, halophytes maintain constant water supply by compartmentalizing toxic inorganic moiety (Na+ and Cl-) in vacuole and accumulating compatible organics (proline, soluble sugars, glycinebetaine, and polyols) in cytosol (Shabala, 2013). Such rearrangements also provide efficient protection to vital enzymes, which otherwise undergo serious damages caused by osmotic, ionic or oxidative imbalances (Flowers and Colmer, 2008). Besides halophytes are also equipped to deal with another challenge of Na+/K+ outbalance, (caused by toxic ions having potential to create serious threats to several metabolic processes) by using it as cheap osmoticum (Munns and Tester, 2008). However, these adaptations requires heavy investment in terms of energy, which can be supplied by compromising in biomass production and consequently plant has to bear a growth penalty to survive in salty mediums.

1 2 - Reactive oxygen species (ROS) such as singlet oxygen ( O2), superoxide anion (O ), * hydrogen peroxide (H2O2) and hyroxyl radical (OH ) are ubiquitous in biological systems (Table 1.1). At lower concentrations, ROS work as signaling molecules, but environmental stresses trigger their excessive generation, which is damaging for biological molecules and membranes (Fig. 1.1). Under such stresses e.g. salinity, plants tend to close stomata to prevent water loss and uptake of excessive salt. Stomatal closure results in over-reduction of green pigment, which accelerates the production of ROS in chloroplasts, consequently reducing photosynthetic efficiency (Hernandez et al., 2000). Increasing non- photochemical quenching Y(NO) and Y (NPQ) and decreasing flow of electrons towards photochemistry (Y (II), Fv/Fm, qP) is one of the strategies which has been adopted by tolerant plants to protect their photosynthetic machinery (Jahns et al., 2012; Abideen et al., 2014; Brestic et al., 2015, 2016). Activation of fluorescence emissions and xanthophyll cycle are also employed to escape from salt induced excessive light damages (Martinez- Penalver et al., 2011). These adaptations can reduce the magnitude of ROS generation, 5 however, ROS production cannot be controlled to safer limits without having a strong antioxidant defence system. Halophytes are supplied with an efficient antioxidant system to quench ROS consisting enzymatic and non-enzymatic components (Table 1.2 and 1.3). Antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR), while, ascorbate and glutathione serves as enzymatic substrates (Noctor and Foyer, 1998; Shalata and Neumann, 2001; Kocsy et al., 2002). Non-enzymatic component includes variety of biologically active secondary compounds i.e. phenols, flavonoids, flavonols, proanthocyanidins, tannins, carotenes, lycopene, anthocyanins, betacyanins etc. which serves to maintain a balance between ROS production and neutralization (El Shaer, 2010; Alhdad et al., 2013). Table.1.1. List of radical and non-radical Reactive oxygen species (ROS).

Radicals Abbreviation Non-radicals Abbreviation

*- Superoxide radicals O2 Hydrogen peroxide H2O2

* Hydroxyl radicals OH Ozone O3

* * 1 Peroxyl radicals LOO /ROO Singlet oxygen O2

* * 3 Alkoxyl radicals LO /RO Dioxygen O2

* -2 Nitric oxide NO Peroxide ion O2 Peroxynitrite ONOO- Lipid peroxide LOOH

Stress (drought, salinity, ultraviolet External sources for ROS light, ionization radiations), drugs, toxins, pathogens, heavy metals

Internal sources for ROS

Peroxisome Chloroplast CYTOSOL

NOO- Peroxidase

O2 ria Enzymatic

Mitochond LOO* H2O2 Antioxidant 1 O2

*RO *OH

DNA damage

Lipid peroxidation Apoptosis

enzymatic antioxidant enzymatic -

Cell toxicity & death, Cancer, Non

Minimize aging, metabolic syndrome, Cardiovascular diseases, Neurodegenerative diseases

Figure. 1.1: External and internal sources of ROS and their consequences at cellular level and potential health effects.

Table.1.2. Antioxidant enzymes and their action mechanisms.

Antioxidant enzymes Reaction Catalysed − + Superoxide dismutase (SOD) O2 + 2H → H2O2 Catalase (CAT) 2 H2O2 → 2 H2O + O2 Ascorbate peroxidase (APX) H2O2 + AsA → 2 H2O + DHA Monodehydroascorbadate reductase ( MDHAR) MDHA + NADPH → AsA + NADP+ Dehydroascorbadate reductase (DHAR) DHA + GSH → AsA+ GSSG Glutathione peroxidase (GPX) 2GSH + H2O2 → GS–SG + 2H2O Glutathione reductase (GR) NADPH + H+ + GSSG → 2GSH + NADP+

Table.1.3. Non-enzymatic antioxidant compounds, occurrence at sub-cellular level and their role against reactive oxygen species. Non-enzymatic Sub cellular location Major role antioxidants Substrates Ascorbic acid Cytosol, mitochondria, Scavenge free radicals, recycles (vitamin C) chloroplast, peroxisome vitamin E Glutathione Cytosol, mitochondria, Multiple roles in cellular chloroplast, peroxisome, antioxidant defense vacuole and apoplast Other compounds Phenolic Vacuole Scavenge free radicals, reduce compounds oxidants, activates antioxidant enzymes, protect against heat, UV and high light Flavonoids Vacuole Scavenge free radicals, reduce oxidants, activates antioxidant enzymes, protect against heat, UV and high light, signaling molecule Tannins Produced in tannosomes and Multiple roles in cellular stored in vacuole antioxidant defense Anthocyanins Vacuole Scavenge free radicals, reduce oxidants, activates antioxidant enzymes, protect against heat, UV and high light Betacyanins Vacuole Multiple roles in cellular antioxidant defense Tocopherol Mostly in membranes Break oxidative chain reaction, (vitamin E) antioxidant in cell membrane Uric acid Scavenger of OH * Carotenoids Chloroplast and other non- Singlet oxygen quencher green plastids Ubiquinones ubiquitous occurrence Reduced forms are efficient antioxidants 1 Proline Mitochondria, cytosol, Scavenger of OH*and O2 prevents chloroplast damage due to LPO

Besides being involved in colour/ sensory characteristics and regulation of several growth/reproductive processes, polyphenols are considered as most powerful natural antioxidants, among secondary metabolites (Balasundram et al., 2006). They protect plants from damaging effects of ROS, in response to high light intensity, UV radiations, heat, pathogen attack, and stimulates antioxidant enzyme system under stress (Chao et al., 2014; Mierziak, 2014; Vicente and Boscaiu, 2018). Several reports suggest that plant based natural compounds such as polyphenols possess a range of biological, pharmacological and health promoting effects (Huang et al., 2009; Cicerale et al., 2010; Cartea et al.,2010; Abbas et al., 2015), which are related to their antioxidant pool. Antioxidant activity of phenolic compounds is mainly attributed to their ability to scavenge free radicals, donate electrons or hydrogen, or chelate metal ions (Tsao and Deng, 2004; Chanwitheesuk et al., 2005). Up till now over 800 structural variants of polyphenol have been discovered. Therefore, interest is now being shifted towards plant phenolic to use them as natural antioxidants for food and pharmaceutical industry (Kahkonen et al., 1999; B alasundram et al., 2006). For instance, Catechin (EGCG), a major component of polyphenols in green tea, has been reported for its potent reductive powers capable of quenching singlet molecular oxygen and peroxyl radicals (Beeche, 2003; Sahin et al., 2010a, b). Tomato and its by-products are reported to be enriched in Lycopene (non-provitamin A), powerful antioxidant that provides protection against damage cellular oxidative damage (Palozza et al., 2012; Ali et al., 2014). p-hydroxybenzoic acid has been reported to intensify the cell wall impermeability, hence improved resistance against pathogen infection (Horvath et al., 2007). Likewise many other polyphenol compounds have extensive applications in industries i.e. medicine, consumer chemicals and food industries (Kweon et al., 2001). Chlorogenic acid found to be effective against bacterial and viral properties (Jiang et al., 2001). Turmeric a source of Curcumin, polyphenol has been shown to overcome oxidative stress and by preserving several antioxidant enzymes activity (He et al., 2012; Tapia et al., 2013). Resveratrol, phytochemical polyphenol (grapes, cranberries and peanuts) prevents alleviate cytotoxic effects of oxidative stress and can specially terminate simultaneous generation of ROS onset of heat stress (Calabrese et al., 2008; Sahin et al., 2012). In the light of the present phytochemical based researches, the animal industry is searching for local, natural and safe materials not only for reducing the feed cost but also for improving the animals’ condition during raising and their final performance traits because of possible relationship between the antioxidant benefits derived from phytochemicals and the growth and productive parameter of animals (Lee et al., 2017). The incorporation of antioxidants

9 in fats and oils/fats and oils based foods is known to alleviate rancidity and associated with improved shelf life. Duh (1999) reported water extracts of Chrysanthemum morifolium (Harng Jyur) varieties and soyabean emulsion tends to show better protection against peroxidation, in the soybean oil, compared to tocopherol (Toc) and butylated hydroxyanisole (BHA), indicating potential role in industrial application. Besides their natural origin, these compounds are reported to have stronger antioxidant activity, less side effects and non-toxic nature than harmful synthetic chemicals (Pappas, 1999; Brown and Rice-Evan, 1998). Variety and intensity of biotic/abiotic stresses tend to modify plant polyphenol synthesis (Naczk and Shahidi, 2004), such as salinity and drought (Gharibi et al., 2016; Caliskan et al., 2017) hence playing potential role in stress tolerance. Plant polyphenolic profile depends on genetic makeup of species (Carvalho et al., 2011; Falleh et al., 2013), varietal difference (Carbone et al., 2011), habitat conditions (Falleh et al., 2012) and organ level distribution (Trabelsi et al., 2012). Edaphic and other environmental factors including temperature, water, nutrients, salinity, light etc. may also effect plant phenolic status (Lisiewska et al., 2006) and associated antioxidant performance. Trabelsi et al (2013) studied the inter- and intra-specific variability in two species of Limoniastrum and observed significant variation in phenolic composition with respect to their habitats. Slight variations were reported in phenolic content and antioxidant capacity of hybrid variety cocoa beans (Jonfia-Essien et al., 2008). Variation in antioxidant activity among plant organs may be attributed to their differences in amount and composition of allocated phenolic compounds (Falleh et al., 2011). Some reports indicate increased levels of phenolic content among different plant tissues when grown under salt (Stefanoudaki, 2004; Weisman et al., 2004) and water stress (Cresti et al., 1994). Erturk et al (2010) observed that the harvest time is also a crucial factor to determine the antioxidant potential and plant phenolic content. Recent studies suggest that scientists are now focussing on halophytes and their isolated constituents, of which a number of species have been reported for their antioxidant potential (Falleh et al., 2011; Trabelsi et al., 2013; Qasim et al., 2017). Under saline drought or flooding synergistic effect of applied stress is alleviated, which in turn increased the synthesis of antioxidant compounds (Alhdad et al., 2013). However, studies on factors affecting secondary metabolites and antioxidant potential of halophytes particularly from subtropical region are scanty. Recently health care system have inducted use of herbal medicines for treatment of several diseases in order to minimize after/side effects of synthetic drugs. In this regards

10 medicinal halophytes are considered better than non-halophytes owing to their superior antioxidant activity and higher TPC (Qasim et al., 2017). According to recent reports surge in global market for medicinal and aromatic plants will reach about 5 trillion USD by 2050. Pakistan meets its requirements by importing 90% medicinal plants from countries like Sri Lanka, China and India at an expense of huge cost roughly 1.5 billion PKR annually (Hussain et al., 2003; Qasim et al., 2011). Poor infrastructure, over-harvesting and lack of knowledge about potential uses of local vegetation/wild plants are the major drawbacks for domestic medicinal usages (Shinwari, 2010). Poor management of herbs is also constantly decreasing the biodiversity of indigenous plant species besides urbanization and global climate change (Baillie et al., 2004). In this scenario, concerte research efforts are required to explore the potentials of indigenous medicinal plants to be domesticated as cash crops. This study is focused on utilization of halophytes as a new source of antioxidant rich secondary metabolites, which could be used in foods, pharmaceuticals, nutraceuticals and other related industries. The work is also attempted to indicate the factors affecting content and composition of secondary metabolites (total phenols, total flavnoids, proanthocynadins, and tannins) and their antioxidant activity. Aim of present study Present study aimed to evaluate halophytes traditionally used as medicines. Selection of medicinal plant was based on their prevalence common to both inland and coastal habitats. The specific objectives were:  Screening of medicinal plants and their different parts (leaves vs stem) for antioxidant potential and quantification of total polyphenolic contents.  Antioxidant potential (contents and activity) of test species was also evaluated in terms of different habitat conditions (inland vs coastal).  Seasonal (summer vs winter) and phenological (vegetative vs reproductive) variations in polyphenolic contents and antioxidant capacity of test species was also determined.  Selection of medicinal plants for detailed greenhouse experiments.  Greenhouse experiments were conducted to study the detailed eco-physiological responses of selected medicinal plants to increasing NaCl salinity and status of their enzymatic and non-enzymatic antioxidants was also determined.  Salt induced accumulation/ de-accumulation of individual phenolic antioxidants in selected plant species was also determined using HPLC.

CHAPTER 2 ENVIRONMENTAL AND PHENOLOGICAL VARIATIONS IN PHENOLIC COMPOSITION AND ANTIOXIDANT ACTIVITY OF MEDICINAL HALOPHYTES

Abstract Halophytes usually complete their life cycle in extreme environmental conditions by up regulating secondary metabolism that facilitates their survival. For this study ten different medicinal halophytes viz. Aerva javanica, Atriplex stocksii, Cressa cretica, Calotrpois procera, Heliotropium bacciferum, Ipomea pes-caprae, Salsola imbricata, Salvadora persica, Suaeda fruticosa and Thespesia populnea were selected from inland and coastal habitats, in order to study the organ specificity, spatiotemporal, and phenological changes in polyphenol and antioxidant activities. Results showed considerable antioxidant activity in terms of DPPH (21.4 to 73.1 I %), ABTS (24.9 to 237.4 µMol TE g-1), FRAP (0.2 to 8.9 mMol Fe+2 g-1) and TAC (12.6 to 71.5 mg AsA g-1). Results indicated that these plants also contained high amount of total phenols (11 to 51 mg GAE g-1), flavonoids (1.4 to 4.6 mg QE g-1) and proanthocynadins (0.2 to 25 mg CE g-1). On the basis of the level of phenols and antioxidant activity plants were categorized into 3 groups i.e. high (T. populnea, I. pes-caprae and S. fruticosa), moderate (S. imbricata, A. javanica, C. cretica and C. procera) and low (S. persica, H. bacciferum and A. stocksii). High correlation among antioxidant activity assays (r = 0.797-0.956) indicated the radical scavenging and reducing power abilities of these plants. Similarly, strong correlations (r = 0.614-0.953) among antioxidant activity measurements and polyphenolic composition suggested that phenolic compounds contributed mainly to the antioxidant activity of these plants. In general, leaf of tested species showed more polyphenols than stem which was higher than most of the other phenol rich species. Little variations were observed in phenolic content and antioxidant activities among plants of contrasting habitat (inland vs coast) however, inland plants had relatively higher values. Season was found to be the dominant factor which altered the polyphenol accumulation and associated antioxidant activity. In general, higher phenolics and antioxidant activities were found during drier period (winter) than wet season (summer). In terms of phenological stage leaf of the medicinal halophytes accumulated greater amount and activity of antioxidants in reproductive stage than vegetative stage. Results of this study provide an insight related to organ specific availability of bioactive compounds in particular season with higher antioxidant activity. These plants could be grown using saline lands, utilizing brackish water in order to provide rich source of natural antioxidants with high industrial and economic value.

Introduction Plants cope various stresses such as higher fluctuations in temperatures, light, salinity and water (Riarh et al., 2010) in saline habitats. These stresses may lead to over production of reactive oxygen species (ROS). Accumulation of ROS may lead to damaged membranes and molecules (lipids, proteins and nucleic acids) which ultimately affect plant growth (Abdi and Ali, 1999; Sharma et al., 2012; Karuppanapandian et al., 2011; You and Chan, 2015). Living system, are well equiped with enzymatic systems and bioactive dietary antioxidants: (ascorbic acid, atocopherol, β-carotene, glutathione, uric acid), hormones (estrogen, angiotensin), and endogenous enzymes (superoxide dismutase, glutathione peroxidase and catalase), to protect cells against oxidative damage (Halliwell and Gutteridge, 1990; Martınez-Cayuela, 1995). Previous studies indicate potential role of oxidative stress in number of disorders (atherosclerosis, cancer, malaria, diabetes, inflammatory joint disease, asthma, cardiovascular diseases, cataracts, immune system decline), and could also play a role in neurodegenerative diseases and ageing process (Florence, 1995; Nakagami et al., 1995; Young and Woodside, 2001; Gandhi et al., 2012; Kim et al., 2015). Halophytes show strong correlation between enzymatic and non-enzymatic antioxidant capacitance and abiotic stress tolerance (Tsugane et al., 1999; Jaleel et al., 2007; Lokhande et al., 2012). These antioxidant compounds are often associated with better plant stress resistance and plant survival under harsh environmental conditions (Ashraf, 2009; Gupta and Huang, 2014). Production and accumulation of antioxidant compounds mostly polyphenols for their defense against ROS has been established under various plant species specially halophytes (Dixon and Paiva, 1995; Sreenivasulu et al., 2000; Naczk and Shahidi, 2004; El Shaer, 2010; Alhdad et al., 2013; Reginato et al., 2014). Stress tolerant species use these compounds as principal protective compound against oxidative damage in stressful habitats (Sanchez-Rodriguez et al., 2011; Ksouri et al., 2012; Reginato et al., 2015). Phenolic compounds are involved in regulation of different metabolic processes, coloring and sensory characteristics, structural component of plant cell wall, and are associated with plant defence against biotic and abiotic stresses (Balasundram et al., 2006; Medini et al., 2015, 2018; Akacha et al., 2017; Qasim et al., 2017). Literature survey indicates potential role of polyphenols as powerful antioxidants at cellular level (Chanwitheesuk et al., 2005; Abideen et al., 2015; Asif, 2015; Stankovic et al., 2015; Qasim et al., 2017). Hydrogen atom or electron donation capacity and metal chelating

14 ability of polyphenols is associated with their strong antioxidant activity (Tsao and Deng, 2004; Chanwitheesuk et al., 2005). Recently, interest is now shifted towards plant phenolics as natural substitute of synthetic antioxidants that are being restricted due to their harmful health effects (Munekata et al., 2017; Soriano et al., 2018; Waheed et al., 2018; Zhang et al., 2018). Phenolic compounds have been associated with various biological properties including antibacterial, anticarcinogenic, anti-inflammatory, antiviral, anti-allergic, estrogenic, and immune-stimulating, anti-cancerous, anti-coagulant and hypo-glycemic activities (Larson, 1988; Meira et al., 2012; Elansary et al., 2017; Abid et al., 2013; Suvarna et al., 2018) and are used in different cosmetic products, food, pharmaceutical industries (Rawlings and Matts, 2005; Maisuthisakul et al., 2007; Galanakis et al., 2018). Although various synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butyl hydroquinone (TBHQ) and propylgallate (PG), are used as food additives used to prevent oxidative damage but, natural compounds are reported to have better antioxidant activity and lesser side effects than synthetic ones (Maisuthisakul et al., 2007; Moure et al., 2001). Industrial polyphenols demand was estimated to be 16, 380 tons in 2015 and this is expected to reach a total of 33,880 tons by 2024 with an estimated monetary value of USD 1.33 billion (Adebooye et al., 2018). This global rising trend in polyphenol extraction and related publication ultimately urged to screen natural vegetation on behalf of their application. Production and accumulation of polyphenols content in plants is often associated antioxidant activity depends upon genotypic (species and/or variety), ontogenic (tissue or organ specific) as well as environmental (season, habitat, type and level of stress) variations (Silva et al., 2007; Carbone et al., 2011; Carvalho et al., 2011; Santos et al., 2011; Trabelsi et al., 2012; Soni et al., 2015). Moreover, edaphic and other abiotic factors (temperature, salinity, water stress and light intensity) may also effect composition and activity of plant antioxidant pool (Lisiewska et al., 2006). Salinity induced polyphenols have been associated with antioxidant activity in different plant organs (Stefanoudaki, 2004; Weisman et al., 2004; Petridis et al.,2012; Rezazadeh et al., 2012) and some reports in drought regimes (Cresti et al., 1994). Habitat level variation in this context was also found in two species of Limoniastrum, where higher values were found in plants of arid areas than humid areas (Trabelsi et al., 2013; Gnanasekaran et al., 2017). Lim et al (2012) reported salt induced accumulation of polyphenols and carotenoid in Fagopyrum esculentum sprouts. Harvest time is another crucial factor which determines the antioxidant potential and phenolic content of plants. Erturk et al (2010) reported seasonal

15 variation in amount and activity of phenolic antioxidants of Camellia sinensis shoots. Similar results were also found in Eugenia uniflora in which hydrolysable tannins were higher in rainy season, whereas flavonoid increased in dry season (Santos et al., 2011). Recently, interest is shifted towards halophytes and studies are now focusing on their phytochemical composition to find out bioactive compounds that can be used as natural antioxidants (Falleh et al., 2011; Trabelsi et al., 2013). Several species have been identified and various approaches have been adopted to enhance the quantity, quality and activity of phytochemical antioxidants and their related health benefits. However, greater variation in this context is linked with spatial and temporal variation of selected plants (Alhdad et al., 2013; Trabelsi et al., 2013). Therefore, analysis of medicinal plants with respect to habitat variation and harvest time could offer better understanding of their antioxidant response and may also provide clues to the likely locations and seasons of species with high antioxidant activity (McCune and Johns, 2007). Literature in this regard is not well documented particularly from subtropical region. Present study was aimed to investigate the effect of environmental (season and habitat), pehnological (vegetative v/s reproductive stages) and organ (leaf v/s stem) level variations on antioxidant activity and polyphenolic content of medicinal halophytes.

Materials and methods Description of study area The study area comprises coastal (Hawks bay/Manora) and inland (University of Karachi) (Fig. 1) which could be classified as arid to semi-arid sub-tropical environment. Fig. 2 shows the environmental data of study area comprises mean annual temperature, rainfall and humidity during the course of study (Pakistan metrological department). Generally, annual monsoon rainfall is low (<20 cm) and unpredictable occurring mostly during late summer. Temperatures vary throughout the year with a long summer and a short winter with variable humidity. The study area was divided into coastal and inland regions. Coastal plants face considerable fluctuations in temperature, soil moisture and salinity due to tidal activity, salt spray, high wind velocity and high irradiance (UV radiations). Whereas, inland vegetation referred to in this study experience higher summer temperatures and lower water availability. Selection of test species In order to study organ specific, seasonal and spatiotemporal variation in phenol and antioxidant activities, ten local medicinal halophytes species were selected, which are distributed in both coastal and inland habitats. Traditional ethnobotanical uses of test species are summarised briefly in (Table 2.2). Chemicals and reagents 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), butylated hydroxytoluene (BHT) and all standards were purchased from Sigma-Aldrich (GmbH, Sternheim, Germany). Iron (III) chloride 6-hydrate, iron (II) sulfate 7-hydrate and acetic acid were obtained from BDH (Poole, UK). Aluminum chloride hexahydrate, ammonium molybdate, butylated hydroxyanysol (BHA), Folin–Ciocalteu’s phenol reagent, hydrochloric acid, methanol, potassium acetate, sodium phosphate, sulfuric acid, 2,4,6- Tri(2-pyridyl)-s-triazine (TPTZ) and vanillin were obtained from Merck (Darmstadt, Germany). Sample collection and preparation Plants selected in this study are of high medicinal importance and are traditionally used in different areas of Pakistan and other parts of the world (Qasim et al., 2010; 2011, 2014). Medicinal plants were collected from their coastal and inland populations during 2014 in winter (January) and summer (June) seasons. Plants were separated into leaves and stem and were dried under shade condition. Plant samples were ground to fine powder using a ball mill (Retsch MM-400) and extracted (1.0 g) with 20 mL of 80% methanol using a

17 shaking water bath (GFL-1092) at 40 oC for 3 h (Li et al., 2008). After cooling, the extracts were centrifuged at 4500 rpm for 15 min. The supernatant was recovered for further analysis. Determination of total phenolic content (TPC) Folin-Ciocalteu colorimetric method was used for estimation of total phenols (Singleton and Rossi, 1965). Gallic acid dilutions were used for polyphenol estimation. Results were expressed as mg gallic acid equivalent per g dry weight (DW). Determination of total flavonoids (TFC) Colorimetric method based on aluminum chloride method was used to quantify total flavonoids (Chang et al., 2002). Quercetin was used to prepare calibration curve and results were expressed as milligram quercetin equivalent per gram dry weight. Determination of proanthocyanidin content (PC) Proanthocyanidin content was determined using (Sun et al., 1998). Catechin was used as standard compound to prepare calibration curve and results were expressed as mg catechin equivalent per gram dry weight (mg CE g-1 DW). Free radical-scavenging ability by the use of a stable DPPH radical Antioxidant activity using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was determined following Brand-Williams et al (1995) assay. Absorbance was recorded at 515 nm and the radical scavenging activity was estimated as the percent inhibition of DPPH (I%) by using the following equation: DPPH I% = [(Acontrol – Asample)/Acontrol] × 100

Where Asample is the absorbance of a sample solution and Acontrol is the absorbance of control solution (including all reagents except test sample). Radical scavenging activity using ABTS radical The antioxidant activity was determined by the ABTS radical scavenging method described by Re et al (1999). The ABTS radical scavenging activity is reported as µMol TE g-1. Total antioxidant capacity using phosphomolybdate complex (TAC) Phosphomolybdate complex method described by Prieto et al (1999) was used to evaluate total antioxidant capacity. Ascorbic acid was used as standard and total antioxidant capacity. Results were presented as mg ascorbic acid equivalent per gram dry weight (mg AsA g-1 DW). Ferric reducing antioxidant power assay (FRAP) The FRAP assay was carried out with the help of the modified method of Benzie and Strain (1996) based on the reduction of Fe3+ to Fe2+ ions. In the presence of TPTZ, the Fe+2

TPTZ complex gives blue color read at 593 nm against FeSO4 standard and the values expressed as mMol Fe+2 g-1 DW. Statistical analyses Multivariate analysis (ANOVA) was used to determine significant differences among experimental factors such as different species, habitat, season and plant part and their interactions on polyphenols (TPC, TFC and PC) and antioxidant activities (DPPH, ABTS, FRAP and TAC) .Values are expressed as means (± standard error) of a minimum of 3 biological replicates with up to 5 technical replicates where necessary. SPSS (version 16) and Sigma plot (version 11) were used for all statistical analyses and graph preparation respectively.

Results Interspecific variation in phenolic content and antioxidant activities of medicinal plants This work aims at survey and quantification of plant Secondary Metabolites (phenol, flavnoids and proanthocynadins) of medicinal halophytes distributed both in inland (Karachi University campus) and on the seacoast (Manora) for assessing their potential antioxidant role. A total of 10 plant species from 7 families have been included in this study (Table.2.2). All tested species were perennial shrubs and herbs of either halophytes or xerophytes indicating little or unpredictable rainfall pattern of studied habitats (Fig. 2.2). The antioxidant capacities of selected medicinal plants (Qasim et al., 2014, 2011, 2010) were evaluated by DPPH, ABTS, FRAP and TAC assays. Radical scavenging activity was estimated using DPPH and ABTS assays while reducing ability of plant extracts was determined by FRAP and TAC assays. The antioxidant activity varied greatly among test species and ranged from 21.4 to 73.1% (IC %) for DPPH, 24.9 to 237.4 (µMol TE g-1 DW) for ABTS, 0.2 to 8.9 (mM Fe2+ g-1 DW) for FRAP and 12.6 to 71.5 (mg AsA g-1 DW) for TAC. Thespesia populnea exhibited highest (DPPH I%, TAC and ABTS) antioxidant activity, lowest DPPH%, TAC and ABTS activities were presented by Salvadora persica, Atriplex stocksii and Heliotropium bacciferum respectively (Fig.2.4). Ipomea pes-caprae indicated highest FRAP activity while, lowest values of were obtained for Heliotropium bacciferum. Polyphenolic analysis of all ten species in the present study showed considerable variation in total phenol and its sub classes and ranged from 11 to 51 (mg GAE g-1 DW) for TPC, 1.4 to 4.6 (mg QE g-1 DW) for TFC and 0.2 to 25 (mg CE g-1 DW) for PC. (Fig.2.3). The highest polyphenols and proanthocynadins, were found in Thespesia populnea whereas lowest values were found in Atriplex stocksii, in case of TFC Salvadora persica ranked highest while Suaeda fruticosa indicated lowest value for TFC. The data obtained from all four methods indicate the presence of high antioxidant capacity in most of the species which was in line with their polyphenolic contents (TPC, TFC and PC). Correlation coefficient of TPC, TFC and PC with AC (DPPH, ABTS, FRAP and TAC) is presented in (Table.2.3). All antioxidant activities showed strong positive correlation (≥0.5) among each other. Results indicate variation in correlations of TPC, TFC and PC of 10 medicinal plants with all antioxidant activities. TPC TFC and PC showed strong positive correlation with ABTS (TPC r = 0.61, TFC r = 0.70), FRAP (TPC r = 0.93, PC r = 0.83) TAC (TPC r = 0.95, TFC r = 0.65, PC r = 0.72) and DPPH (TPC r = 0.87, TFC r = 0.73).

Habitat variation in Phenolic content and antioxidant activities of medicinal plant The comparison of two populations (inland vs coast) showed significant variation in TPC and PC content along antioxidant activities. In general, inland populations showed comparatively higher antioxidant compounds (TPC and PC) activity than coastal populations, while TFC remain unchanged overall in both habitats. These results were consistent with antioxidant activity using all antioxidant activity assays (Fig.2.5). Among ten studied species Atriplex stocksii, Aerva javanica, and Salvadora persica did not show significant differences in secondary metabolites and antioxidant activities between inland and coastal habitats. Calotropis procera and Cressa cretica showed higher accumulation of TPC, TFC and PC in coastal a habitats contributing to higher antioxidant activity in coastal habitat while H.baciferum, I.pes-caprae, S.fruticosa, S.imbricata and T.populnea populations showed high polyphenols in inland habitat along high antioxidant activities (data not shown). Seasonal variation in phenolic content and antioxidant activities of medicinal plant Our data indicates season imposed greater fluctuations in plant responses in studied plants. In general, higher polyphenol accumulation and antioxidant activity were observed during winter (dry) season compare to summer post- monsoon (wet) season (Fig.2.6) irrespective to plant species. TPC and TFC increased by 1.13 and 0.6 fold respectively in winters compared to summer season. Similar changes were observed in DPPH and ABTS activities that increased by 0.4 folds in winters while FRAP values were increased by 2.5 fold in winter. Phenolic content and antioxidant activities of medicinal plant organs An organ dependent variation in antioxidant response was observed among all test species. In general, leaf showed comparatively higher antioxidant compounds (TPC, TFC and PC) and activity then stem. In general leaf methanolic extracts were characterized by (60%, 91.3%, 50%) higher TPC, TFC and PC than stem. This can be related to (59%) high FRAP and (45%) TAC values in leaves than stem. Similarly leaves exhibited (41% and36%) high ABTS and DPPH activities than stem extracts (Fig. 2.7). On basis of leaves methanolic extracts TPC accumulation and antioxidant activities, tested plants were divided into three categories (Figure.2.3 and 2.4). Polyphenol rich category include 3 species (≥ 30 mg GAE g-1 DW) i.e.(Thespesia populnea, Suaeda fruticosa and Ipomea pes-caprae), moderate category enlist 4 species (15-30 GAE g-1 DW) including (Aerva javanica,Calotropis procera, Cressa cretica and Salsola imbricate) and low phenolic regime species (< 15 mg GAE g-1 DW) include Atriplex stocksii, Heliotropium bacciferum and Salvadora persica.

Based on polyphenol accumulation of leaves and stem, the screened species can be divided into three categories. Species with equivalent polyphenol in leaves and stem (Cressa cretica and Heliotropium bacciferum), species with significantly higher values in leaves than stem (Atriplex stocksii, Calotropis procera, Ipomea pes-caprae, Suaeda fruticosa, Thespesia populnea and Salsola imbricata), and species with significantly higher values in stem than leaves (Aerva javanica and Salvadora persica). (data not shown). Although some species showed high TPC in stem but higher antioxidant activity was still observed from leaves samples indicating potential presence of bioactive compounds. Progression of Phenol and antioxidant capacities with phenological stage Stage specific variations in antioxidant response were observed among all test species. In general, reproductive stage showed comparatively higher antioxidant compounds (TPC) and activity then vegetative stage. (Fig.2.8). For instance, the total polyphenol contents all together for both leaves and stems was 67 % higher times at the reproductive stage while TFC and PC content remain unchanged in both stages. Similarly FRAP, ABTS, TAC and DPPH activities increased by 36%, 83% and 34% and 25% respectively in reproductive stage.

Inland

Figure. 2.1: Map of study area showing biogeographic regions of Karachi where plants were collected.

Table.2.1. Table showing habitat selection criteria with coordinate distribution and distance from shore.

Location Habitat Coordination Distance from shore

Manora Coast 24°48′00″N, 66°58′00″E 50-100 m

University of Karachi Inland 24°55′50″N, 67°6′55″E 30 Km

Max Temp (oC) Min Temp (oC) Humidity (%) Precipitation (cm) Wind speed (Kmph)

60 50 40 30

Values 20 10 0

Jul

Jun

Oct

Jan

Apr

Mar

Nov

Feb Aug

May Dec

Sep

Months

Figure. 2.2: Mean annual temperatures, rainfall, humidity and wind speed of study area 2014 (Pakistan Meteorological Department).

Table.2.2. List of selected species with common name, family, flowering period and medicinal uses.

Name of Species Common Flowering Medicinal uses References (family) name period Ipomea.pes caprae Beach July-Sep Antidote ,antipruritic, anti- Chopade, 2009; Pongprayoon (Convolvulaceae) Morning inflammatory et al., 1991; Sunthonpalin et Glory al., 1985; Wasuwat, 1970 Atriplex stocksii Phurkival Dec-Jan Fever, jaundice, dropsy, liver Qureshi et al., 2009 (Amaranthaceae) disease Aerva javanica Desert cotton Jan- May antidiarroheal, antioxidant, Nagaratna et al., 2015 (Amaranthaceae) analgesic, antihyperglycemic, and antihelmentic activities Salsola imbricata Lana Aug-Oct Dyspepsia, constipation, Wariss et al., 2014 (Amaranthaceae) digestive problems Salvadora persica Tooth brush Jan-Apr Stem as miswak, Anti Elvin-Lewis, 1980 (Salvadoraceae) tree /pelu inflammatory

Cressa cretica L. Bukkan Year round Antiinflammatory, Antioxidant, Qasim et al., 2011 (Convolvulaceae) Antiviral and for treatment of Sores Heliotropium bacciferum Markondi July- Antihyperlipidemic, antitumor, Ahmad et al., 2014 (Boraginaceae) September antidiabetic, antioxidant, and antimicrobial Suaeda fruticosa (L.) Shrubby sea April-Sept. Antibacterial Rashid et al., 2000; Forssk. blight Qasim et al., 2011 (Chenopodiaceae) Thespesia populnea Indian Tulip June - Sep Antibacterial, antifungal Savithramma et al., 2017 (Roxb.) Kostel tree (Malvaceae) Calotropis procera (Ait.) Rubber bush Year round Tooth and stomach aches Ilahi, 2008; Ait Qasim et al., 2011 (Asclepediaceae)

) High

-1 60

40 Medium Low 20

TPC (mg GAE g GAE (mg TPC 0

)

-1 10 8 6 4 2

TFC (mg QE g QE (mg TFC 0

) 30 -1 25 20 4 3 2 PC (mg CE g CE (mg PC 1 0

S. persica C. cretica A. stocksii C. procera A. javanicaS. imbricata S. fruticosaT. populnea H. bacciferum I. pes-caprae

Figure. 2.3: Change in total phenol (TPC), total flavnoids (TFC) and proanthocynadin (PC) content of selected medicinal halophytes.

)

-1 High 300

200 Medium

Low 100

ABTS (mMol TE g TE (mMol ABTS 0 100 80 60 40

DPPH (I%) DPPH 20

) 0

-1 80

TAC (mg AsA g AsA (mg TAC 20

) 0 -1 12

+2 9

FRAP (mMol Fe (mMol FRAP 0

S. persica C. cretica A. stocksii C. procera A. javanicaS. imbricata S. fruticosaT. populnea H. bacciferum I. pes-caprae

Figure. 2.4: Change in DPPH, ABTS, FRAP and TAC activities of selected medicinal halophytes

ABTS (mMol TE g ***

) *** 150

-1 30 100 20 50

-1

) 10 0

TPC (mg GAE g GAE (mg TPC

*** 80 DPPH (I%) 0 60 4 ) 40

-1 20 3

0 TAC (mg AsA g 2 *** 40

TFC (mg QE g QE (mg TFC 1 30 20 0

-1

10 ) 5 ***

) 0 FRAP (mMol Fe

-1 4 8 *** 3 6

2 4

PC (mg CE g CE (mg PC

1 2 +2

-1

0 0 ) Coast Inland Coast Inland

Figure. 2.5: Habitat variation in polyphenol (TPC, TFC, PC) and antioxidant activities (DPPH, ABTS, FRAP and TAC) of selected medicinal halophytes

ABTS (mMol TE g 40

) *** *** 150

-1 30 100

20 50

-1

) 10 0

TPC (mg GAE g GAE (mg TPC

80 DPPH (I%) *** 0 60 5 ***

) 40

-1 4 20 3

0 TAC (mg AsA g 2 40

TFC (mg QE g QE (mg TFC 1 30 20 0

-1

10 ) 5

) 0 FRAP (mMol Fe

-1 4 *** 8 3 6 2 4

PC (mg CE g CE (mg PC

1 2 +2

-1

0 0 ) Summer Winter Summer Winter

Figure. 2.6: Seasonal variation in polyphenol (TPC, TFC, PC) and antioxidant activities (DPPH, ABTS, FRAP and TAC) of selected medicinal halophytes

ABTS (mMol TE g

30 *** ) *** 150

-1

100 20 50

-1

) 10 0

TPC (mg GAE g GAE (mg TPC

*** 80 DPPH (I%) 0 60 5 ***

) 40

-1 4 20 3

0 TAC (mg AsA g 2 *** 40

TFC (mg QE g QE (mg TFC 1 30 20 0

-1

10 ) 5

) *** 0 FRAP (mMol Fe

-1 4 *** 8 3 6

2 4

PC (mg CE g CE (mg PC

1 2 +2

-1

0 0 ) Stem Leaf Stem Leaf

Figure. 2.7: Organ specific variation in polyphenol (TPC, TFC, PC) and antioxidant activities (DPPH, ABTS, FRAP and TAC) of selected medicinal halophytes

ABTS (mMol TE g *** 200

) ***

-1 30 150 100 20 50

-1

) 10 0

TPC (mg GAE g GAE (mg TPC

*** 80 DPPH (I%) 0 60 5

) 40

-1 4 20

3 0 TAC (mg AsA g *** 50 2 40

TFC (mg QE g QE (mg TFC 1 30 0 20

-1 5 10 )

) 0 FRAP (mMol Fe

-1 4 *** 8 3 6 2 4

PC (mg CE g CE (mg PC

1 2 +2

-1

0 0 )

Vegetative Vegetative Reproductive Reproductive

Figure. 2.8: Phenological variation in polyphenol (TPC, TFC, PC) and antioxidant activities (DPPH, ABTS, FRAP and TAC) of selected medicinal halophytes.

Table.2.3. Correlation coefficient (r) of different antioxidant parameters studied.

TPC TFC PC ABTS FRAP TAC DPPH

TPC 1 TFC 0.646 1 PC 0.696 0.137 1 ABTS 0.614 0.705 0.084 1 FRAP 0.934 0.114 0.836 0.893 1 TAC 0.953 0.652 0.722 0.774 0.956 1 DPPH 0.879 0.731 0.306 0.797 0.749 0.844 1

Table.2.4. Multivariate analysis (ANOVA) showing effect of species, habitat, season and plant part and their interactions on different antioxidant parameters studied.

. TPC TFC PC ABTS FRAP TAC DPPH

Species 1596.398*** 2530.253*** 6887.138*** 668.209*** 8596.762*** 251.642*** 325.185***

Habitat 1258.013*** 0.365 720.943*** 19.871*** 824.259*** 178.339*** 2726.429***

Season 5521.725*** 2275.872*** 362.565*** 312.638*** 13470.37*** 0.662 2399.326***

Part 514.964*** 5960.252*** 492.112*** 159.92*** 1414.834*** 78.197*** 245.987***

Species * Habitat 451.46*** 131.474*** 168.689*** 111.308*** 522.238*** 57.253*** 141.498***

Species * Season 126.146*** 640.944*** 76.435*** 184.986*** 6108.473*** 88.573*** 101.506***

Species * Part 152.34*** 331.976*** 276.605*** 26.919*** 363.426*** 21.163*** 64.305***

Habitat * Season 2.581 272.001*** 1545.447*** 275.634*** 892.184*** 1.39 1201.686***

Habitat * Part 163.62*** 35.296*** 162.302*** 1.792 447.442*** 8.375** 36.904***

Season * Part 251.038*** 53.062*** 398.252*** 134.639*** 629.866*** 11.336*** 112.694***

Species * Habitat * 252.479*** 95.757*** 806.588*** 63.726*** 451.54*** 25.316*** 152.652*** Season Species * Habitat * Part 55.913*** 101.165*** 105.002*** 19.374*** 384.775*** 7.156*** 109.276***

Species * Season * Part 41.045*** 196.254*** 348.529*** 120.981*** 441.66*** 8.867*** 37.851***

Habitat * Season * Part 1.048 238.883*** 200.471*** 63.455*** 645.652*** 3.711* 120.877***

Species * Habitat * 47.431*** 72.532*** 188.623*** 8.689** 414.57*** 5.077*** 46.826*** Season * Part The F values from ANOVA are given and asterisks in superscripts are showing significance level at P<0.05 (*), P<0.001 (**), P<0.0001 (***)

Discussion Medicinal plants are reported active against infection / oxidative stress related damages (Ksouri et al., 2009, 2012). Present study indicates polyphenolic composition (TPC, TFC and PC) and antioxidant activities of 10 medicinal halophytes. (DPPH) and (ABTS) assays were used to determine free radicals scavenging ability while (FRAP) and (TAC) methods estimate the ability of plant extract to reduce iron and molybdenum ions, respectively. All tested species showed high levels of TPC (≥10 mg GAE g-1 DW) even higher than most of the polyphenol rich species like Cuminum cyminum (Dua et al., 2012), Diplotaxis harra and Diplotaxis simplex (Falleh et al., 2013). Irrespective of habitat and season Thespesia populnea, Ipomea pes-caprae and Suaeda fruticosa were categorized TPC rich with considerable high antioxidant activities than other tested species, several previous studies included these species in most potent group due to phenol enrichment and antioxidant richness. (Ksouri et al., 2012; Ehsen et al., 2016; Qasim et al., 2017). It is noteworthy Aerva javanica, Salsola imbricate showed moderate TPC all belonged to Amaranthaceae, with an exception of Cressa cretica (Convolvulaceae) and Calotropis procera (Asclepediaceae). Various studies indicate diversity in secondary metabolites (essential oils, sesquiterpenes, diterpenes, triterpenes, phenolic acids, flavonoids and betalains) in member of Amaranthaceae family, owing to their (antioxidant, positive effects on metabolic, cardiovascular and gastrointestinal health in humans; antimalarial) bio activities (Cai et al., 2005; Ventura et al., 2011; Parida et al., 2013; Mroczek, 2015; Miguel, 2018). Such antioxidant variability between halophytes could be attribute of genetic factor. In general, our result are in accordance with previous studies based on halophytes showing strong positive correlation between phenol and antioxidant activities, relating to their medicinal potential (Li et al., 2008; Abideen et al., 2015; Qasim et al., 2016; Jdey et al., 2017). Higher radical scavenging and reducing power activity of I. pes-caprae demonstrated in this study agrees with the previous reports (Banerjee et al., 2008; Thimnavukkamsu et al., 2010). The presence of catechin, gallic acid, chlorogenic acid, caffeic acid, syringic acid, ferulic acid, coumarin, naringenin, kaempferol, and derivatives of quercetin, isocoumarin, and isochlorogenic acid, supports its high antioxidant potency (Meira et al., 2012; Qasim et al., 2017). Thespesia populnea ever green tree leaves and stem exhibited highest polyphenol and antioxidant activities which can be related to their uses as (astringent, antibacterial, hepatoprotective, haemostatic, anti-diarroheal) herbal medicines. The results of present study are related to previous studies indicating presence of alkaloids, carbohydrates, steroids, proteins, phenols, tannins, flavonoids, glycosides,

33 gums, gaponins and terpenes (Trease and Evans, 1983; Parthasarathy et al., 2009). Edible medicinal halophyte, Suaeda fruticosa potential radical scavenging activity confirm the previous studies (Oueslati et al., 2012 a, b; Qasim et al., 2016). The presence of bioactive compounds such as gallic acid, catechin, chlorogenic acid, caffeic acid, quercetin and kaempferol (Qasim et al., 2017) validates its potential role as medicinal plant (anti- inflammatory, antioxidant, and anticancer activities). Present study indicates both phenolic contents and antioxidant activities of test species were influenced by the harvest site. In general inland populations exhibited greater accumulation of polyphenol and high antioxidant activity than coastal populations. Although coastal populations are believed to have higher antioxidant activity than inland, due to exposure to full sunlight (Masuda et al., 1999), with greater UV radiation in coastal areas, due to reflection of sunlight from sand and sea surfaces. Some studies indicate no specific change in polyphenol and antioxidant activities in inland and coastal habitats of H. tiliaceus (Wong et al., 2009) likewise Hashiba et al (2006) reported erratic variation leaf flavonoid contents of coastal and inland populations of Silene littorea, while contrasting results are reported by others i.e. Nazir et al (2018) reported higher polyphenol and antioxidant activity in coastal population Ipomea pes-caprae and Cressa cretica while Keiko et al (2005) reported higher flavonoid and phenolic acid contents in inland populations of Adenophora triphylla. Seasonal variation in E. uniflora, suggested that environmental factors can strongly affect different phenolic compounds in leaf. In the dry season, the production of flavonoids increased, probably as a response to the combination between higher light incidence and drought stress (Santos et al., 2011). Similar effects were observed in willow and spring wheat species (Turtola et al., 2005; Feng et al., 2007). The higher antioxidant response in inland plants may be associated to multiple environmental factors (light, salinity, water and/or nutrient stress) faced by these species (Spano et al., 2013; Kranner and Seal, 2013). Inland areas showed sporadic rainfall pattern, compared to available saline high under water table in coastal areas. High drought in (inland populations) might have resulted with high oxidative stress than studied coastal habitat. Under stressful conditions the limited CO2 fixation may leads over reduction of photosynthetic machinery resulting in oxidative burst (Selmar and Kleinwächter, 2013). This triggers the role of an antioxidant system to limit ROS to optimum level only sufficient for signaling (Tripathy and Oelmuller, 2012). High PC content in inland population is in accordance with previous study, indicating higher tannin content in Mesembryanthemum edule (Djerba provenance), during dry period suggesting that tannins

34 contributed significantly to water stress tolerance (Ojeda et al., 2002; Attia, 2007; Falleh et al., 2012). In this way, Attia (2007) showed that under water stress conditions, vine plants significantly increased the levels of condensed tannins as an indirect mechanism of drought tolerance. Contrast to our results some reports indicate high antioxidant capacity of coastal plants like Aegiceras corniculatum, Bruguiera parviflora, Salicornia brachiate, Suaeda maritima and Tamarix gallica during summer (Parida et al., 2004; Ksouri et al., 2009; Parida and Jha, 2010; Alhdad et al., 2013). Generally, leaves showed higher accumulation of polyphenols which can be related to high antioxidant activities. Previous reports showed outstanding polyphenol and antioxidant activity of leaves of H. tiliaceus compared to flowers (Wong et al., 2009). Increased production of phenolic metabolites in leaves is believed to protect vital functions by guarding biological molecules and membranes from oxidative damage (Jaleel et al., 2007). Plant accumulated polyphenol in mesophyll and epidermal cells to protect photosynthetic machinery (Tattini et al., 2005) by heat dissipation (Smith and Markham, 1998; Reginato et al., 2014), serve as UV shield (Tattini et al., 2005) and stimulate antioxidant enzymes (SOD, CAT, APX, DHAR and GR (Gill and Tuteja, 2010). Our findings are in accordance with Xu et al (2011), indicating higher synthesis and accumulation of phenolic compounds and antioxidant properties in five grape cultivars in winter than summer. Similar study based on wild fennel showed higher accumulation of leaf polyphenol and profound antioxidant activity with highest pro-inflammatory capability during winter (Pacifico et al., 2018). High DPPH and FRAP activities in winter might be linked with high fluctuation of temperature and high hydric deficit (Stajner et al., 2011). Production of phenols and tannins may be related to (annual, monthly and daily) variations of temperature, which is one of the factors with the highest influence on the plant growth (Evans, 1996). Results of this study are in accordance with previous reports indicating increase production of phenolic metabolites during drier period (winter) than wet season (summer) (Luengas-Caicedo et al., 2007). Anderson et al (1992) reported high concentrations and activity of antioxidant compounds during winter in aerial parts of Pine and suggested their protective role against photooxidative injury, as low temperatures and high light intensities leading to photooxidation, which has been associated with oxidative damage (Anderson et al., 1992). To cope with this situation, plants usually accumulate high quantities of phenolic antioxidants, which were also observed in this study.

Present study indicates that polyphenol accumulation and antioxidant activities were significantly modulated by plant phenological stages. Our results are in accordance with the previous studies reporting the phenological stage variation (Silva et al 2007; Jallali et al., 2012; Achakzai et al., 2017) among different plants. Higher polyphenol content during reproductive stage might be need of transition from simple growth to reproductive functions. Literature indicates a potential role of phenolic compounds (phenolamides) in the flowering stage (Macheix et al., 2005), moreover the periodicity in the production of phenolic compounds in some species with higher quantification has been reported in spring or summer (Plouguerne et al., 2006). Antioxidant activities of flowering stage extracts exhibited significantly higher antioxidant activities, as compared to the vegetative plant extracts, reflecting the highest antioxidant potentialities. Strong positive correlation between phenolic contents and antioxidant activities suggests that potential role of phenolic compounds as major contributors to the antioxidant activities plant extracts. This proposal has been endorsed by many previous research works, which confirmed the role of phenolics as major antioxidants in plants, especially within halophytic species (Maisuthisakul et al., 2007; Ksouri et al., 2008; Jallali et al., 2012).

Conclusions In this study, we assessed the contribution of interfering factors on phenolics composition and antioxidant activity of medicinal halophytes. All tested species exhibited a wide range of different classes of polyphenol contents and antioxidant capacities. These data appeared tightly dependent on a number of biotic (species, organ and phenological stage) and abiotic (seasonal and habitat) factors. In general leaf of tested species showed more polyphenols than stem. Season being dominant factor that altered polyphenol accumulation. Winter plants contained higher phenolic compounds than summer ones, and consequently exhibited higher antioxidant activities. The results of ANOVA showed that all biotic and abiotic factor significantly affected polyphenols (TPC, TFC and PC), antioxidant activities (DPPH, ABTS, FRAP and TAC), and their interactions. Taken together, this information may confirm the interesting potential of halophytes as a valuable source for natural antioxidant molecules. These results are also helpful to select the optimal collection site and time of harvesting for the highest product quality and economical value.

CHAPTER 3 EFFECT OF SALINITY ON GROWTH, ECOPHYSIOLOGY AND ANTIOXIDANT STATUS OF CALOTROPIS PROCERA

Abstract Calotropis procera (Ait.) Ait. F. is a perennial evergreen shrub (family: Asclepiadaceae) and a widely used medicinal plant. It can tolerate adverse climate conditions and poor soils, which explains its efficient adaptation and wide distribution in arid/semiarid and saline conditions. This study was conducted to determine plant growth, photosynthesis, ion homeostasis, water relations, and enzymatic and non-enzymatic antioxidant status of C. procera. Seedlings were grown under 0, 100 and 300 mM NaCl solutions (equivalent to non-saline, 20% and 60% seawater salinity) for 6 months, under semi-controlled greenhouse. Results showed that increasing salinity reduced plant length, stem girth and succulence. However, plant growth in terms of root length, number of leaves and nodes, leaf area, leaf moisture, leaf pigments, and biomass (fresh weight and dry weights) remained unaffected at 100 mM NaCl. However, growth parameters were markedly decreased (up to 50%) at 300 mM NaCl. Plant decreased water and osmotic potentials by accumulating inorganic (Na+ and K+) and organic osmolytes (soluble sugars and proline). Salt treated plants maintained K+ homeostasis by improving selective absorption of K+ (over Na+). Actual quantum yield (YII), photochemical quenching (qP) and electron transport rate (ETR) showed significant decline, while non-photochemical quenching Y(NPQ) and Y(NO) increased upon salt exposure. These photochemical limitations resulted in oxidative damage by increasing H2O2, MDA and electrolyte leakage. To overcome this problem, plant improved heat dissipation Y(NO), activated antioxidative enzymes (superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase and guaiacol peroxidase) and increased secondary metabolite content (polyphenols, anthocyanin, betacyanin, carotenoids, flavonols, betacarotene and dihydroascorbic acid), which in turn improved overall antioxidant status, at moderate salinity. However, these protection were not sufficient at high salinity. Our results indicate that C. procera manages moderate salt stress by adjusting osmotic potentials, water balance, selective absorption of K+ and oxidative stress management, while high (300 mM NaCl) salinity was damaging for C. procera. In addition, moderate salinity induced higher yield of valuable secondary metabolites (e.g. natural antioxidants) with profound activities, which can be utilized at domestic and industrial scale.

Introduction Salinity can adversely affect plant growth by imposing 1) specific ion toxicity 2) osmotic disturbances and 3) nutritional imbalance resulting photosynthesis inhibition and other physiological/ biochemical impairments including oxidative stress (Munns, 2002; Munns and Tester, 2008; Acosta-Motoset al., 2017). Halophytes are naturally equipped with certain mechanisms to withstand high salinity by ion compartmentalization and osmotic adjustments using accumulation of inorganic (Na+, K+, Mg2+, Ca2+, SO-4, Cl-) and organic osmolytes (proline, soluble sugars, glycine betaine, and polyols; Shabala, 2013). Tolerant plants can survive and complete their life cycle by increasing below ground biomass, keeping root/shoot high, maintain K+ homeostasis, restricting Na+ uploading into above ground parts especially leaves, dumping toxic ions into older tissues, retranslocating essential minerals into the metabolically active tissues, synthesizing organic osmolytes, protecting leaf photochemistry, and maintain redox balance (Flowers and Colmer, 2008; Ahmed et al., 2013; Roy and Chakraborty, 2014; Adnan et al., 2016). Differential utilization of essential minerals (like K+, Ca2+and Mg2+) over abundant toxic ions (like Na+ and Cl-) also plays a vital role to ensure salinity tolerance (Benlloch et al., 1994). However, toxic ions can be used effectively for osmotic adjustments because they spare less energy than the biosynthesis of organic molecules (Bell and O’Leary, 2003). However, a sufficient balance of cytosolic K+/Na+ must be ensured while using toxic ions as osmoticum otherwise, it can disturb major biochemical, physiological, and metabolic processes (Munns and Tester, 2008). These adaptations, however, demand ample energy that shifts the carbon stream from biomass production to tolerance mechanisms, which consequently reduces plant growth on salty mediums. High Na+ in the rhizosphere soil disturb ion K+ absorption hence restrict K+ availability in chloroplast leading to photosynthesis inhibition (Sudhir and Murthy, 2004). Furthermore, salt induced degradation of photosynthetic pigments and/or their reduced synthesis, hamper photosystem’s efficiency (Ashraf and Harris, 2013). Salinity induced osmotic stress often leads to stomatal closure, which slowdown transpiration rates, resulting less CO2 intake and decline photosynthesis output (Parida et al., 2005; Souza et al., 2015; Freitas et al., 2017). It also decreases photosynthetic capacity by over-reduction of PSII reaction centers. Salt tolerant plants generally increase their heat dissipation Y(NO) and non-photochemical Y(NPQ) while reducing electron flow towards photochemistry (Y(II), Fv/Fm, qP; Abideen et al., 2014; Ikbal et al., 2014). In addition, activation of xanthophyll cycle and fluorescence emissions also helps to rescue plant from

40 damaging effects of light energy, under salt stress (Martinez-Penalver et al., 2011). In some cases, if chlorophyll fluorescence does not participate enough to dissipate excessive energy, it works significantly to protect leaf photochemistry (Lu et al., 2003). Salt stress when coupled with high light may impose significant damages to photosynthetic machinery by over production of (ROS) reactive oxygen species (Krieger-

Liszkay et al., 2008; Koyro et al., 2013). These damages can be determined through H2O2, MDA and electrolyte leakages (Foyer and Noctor, 2009). To deal with, tolerant plants are well equipped with antioxidant defenses (both enzymatic and non-enzymatic). These plants either show coordinated up regulation (Gueta-Dahan et al., 1997; Hernandez et al., 2001) or constitutively high level of antioxidant system (Gueta-Dahan et al., 1997; Mittova et al., 2003). Antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate per oxidase (APX), guaicol peroxidase (GPX) and glutathione reductase (GR) offset the over-production of ROS. While, non-enzymatic antioxidant substrates provide including ascorbate and glutathione provide further defense by scavenging damaging radicals (Noctor and Foyer, 1998; Shalata and Neumann 2001; Kocsy et al., 2002). In addition to these, a number of phenolic antioxidants play a crucial role to maintain cellular redox status and resist any change in the direction of physiological metabolism imposed by salt stress. For instance, salt induced hyper accumulation of polyphenol and their subsequent antioxidant protection is reported from several halophytes such as Cakile maritima, Cynara cardunculus, Phrigmitis karka, Bruguiera parviflora, Ocimum basilicum L cultivars (Parida et al., 2002; Ksouri et al., 2007; Abideen et al., 2015; Sytar et al., 2018; Scagel et al., 2019). Calotropis procera L. (Asclepiadaceae) is a perennial shrub, distributed in Asian deserts (Mediterranean to the African coast), where rainfall average ranges less than 200 mm (Fu, 1989; Gutterman, 1995). It also known as aak, giant milkweed, sodom apple, rubber tree, and cabbage tree. It is a xerophyte shrub or small tree which grows on waste lands and saline soils. This plant looks evergreen in all stages of life, while its reproductive phase ranges from March to May (Frosi et at., 2013). C. procera is well-known for its property to restore degraded lands. Due to a variety of bioactive medicinal components, different parts of C. procera have been used in traditional systems of a number of Asian and African countries (Kumari et al., 2004). It has a milky latex in its veins, which is enriched with medicinal compounds signify its use for the treatment of tumors, leprosy, piles, ulcers, disorders of liver, spleen, and abdomen muscular pain, toothache, wound healing and to stop bleeding immediately when applied on fresh cuts (Kritikar and Basu,

1999; Kumari, 2004). Its biological and pharmacological effects are antihyperglycaemic, anti-inflammatory, gastroprotective, antinociceptive, cytotoxic, hepatoprotective and antioxidant (Basu et al., 1992; Roy et al., 2005; Teixeira et al., 2011; Tour and Talele, 2011). These effects are mainly due to the presence of biologically active substances in different parts of C. procera such as polyphenols, triterpenoids, flavonoids, alkaloids, cardioactive glycosides, tannins, anthocyanins, saponins resins, and proteolytic enzymes (Shaker et al., 2010). Besides the medicinal potential it is also suggested as a potential bioenergy and biofuel plant (Abideen et al., 2014). Medicinal and pharmacological importance explains the growing demand of C. procera. In addition to these properties this plant can withstand drought and salinity (Al- Sobhi et al., 2006; Ibrahim, 2013; Bairagi et al., 2018) and hence, could be cultivated on saline/marginal lands to produce bioactive raw material for medicinal and chemical usages. However, without knowing the detailed mechanisms involved in salinity tolerance, the propagation and cultivation of this species cannot be done. Therefore, present study was aimed to study the salt tolerance mechanism of this medicinally important xerohalophyte. The main objectives were to determine the effect of NaCl on growth, chlorophyll fluorescence, leaf pigments, ion regulation, water relations, and enzymatic and non-enzymatic antioxidant defense of C. procera.

Material and methods Plant material The growth experiments of (Calotropis procera (Ait.) Ait.,) were carried out in a greenhouse, at Institute of Sustainable Halophytes Utilization, University of Karachi, Karachi, using, the freshly collected seeds from their natural habitats in Karachi University Campus. Growth experiment Wrinkle free fresh intact seeds were selected for growth experiment. Seeds identical in size and colour were selected for growth experiment. Fifty seeds were sown in plastic trays and left to germinate in greenhouse under ambient light (300 µmol m−2s−1), temperature (25/15 ± 2 ºC) and humidity (~70%). Seedlings were transplanted (2 leave stage) to plastic pots (45 cm in diameter and 55 cm height), containing sand and silt in ratio of 1:1. Each pot contained 3 plants. Healthy seedlings of equal size and vigor were selected and grown at Hoagland’s nutrient solution using sub-irrigation method. After 6 weeks of seedling establishment, salt treatment was started using different concentrations of sodium chloride (0, 100, 300 mM). To prevent osmotic shock, salinity treatment was started gradually (at rate of 25 mM daily). Plants were treated with salinity for 6 months, after that non- destructive measurements were taken and plants were then harvested for further morphological, physiological, biochemical and phytochemical analyses. Growth parameters, leaf area, moisture percent and succulence Freshly harvested plants were, briefly rinsed with tap water and dried using blotting paper. Fresh weight (FW) and length (cm) were measured immediately. In addition, vernier calipers measurement were recorded to measure stem girth. Dry weight (DW) were obtained after oven drying at 60°C for 72h until constant weight. ImageJ software (https://imagej.en.softonic.com/download, accessed 23 March 2018) was used to estimate leaf area per plant on scaled photographs of leaves against a white background. All physiological and biochemical analyses in this study were done using fully expanded leaves from the 2nd and 3rd node. Leaf succulence (SUC) was calculated using the formulae described by Pujol et al (2001) Water relations Water potential was measured on 5 mm diameter disc obtained from 2nd node fully expanded leaf, using C-52 sample chamber in a Wescor HR-33 T, dew point micro voltmeter (Wescor International, USA). Results were expressed in mega-pascal (-MPa) units by converting microvolt readings, using a standard curve of NaCl solutions (0-800

43 mM).Sap osmolality of the same sample was determined using a Vapor pressure Osmometer (Vapro-5520, Wescor, Inc.). The osmotic potential was calculated from leaf osmolality by using the Vant-Hoff’s equation (Guerrier, 1996). Diurnal water relations Pre-dawn and noon leaf water potential were observed changes in diurnal water relations of plant after 6 months of salinity treatments. Proline determination Proline was estimated by Bates et al., (1973). For extraction (50 mg) powdered plant material was added in 4 ml (3% sulphosalicylic acid), mixture was sonicated at 30◦C (15 min) and then centrifuged at 3000 × g. For Proline estimation ninhydrin, glacial acetic acid and extracts were taken in ratio of (1:1:1), samples were incubated in boiling water bath (1hour). 2 ml of toluene was added to the reaction mixture after reaction termination using ice bath. Samples were vortexed for 30 s, absorbance of the supernatant was measured at 520 nm and proline concentration was determined against a standard curve using l-proline. Soluble sugar determination Anthrone assay was for estimation of soluble sugars (Ludwig and Goldberg, 1956). 500 ul Anthrone reagent was added with 250 µl hot water extract/standard. Samples were incubated for 11 min in boiling water bath, reaction was terminated using ice bath. The absorbance was measured at 630 nm against distilled water blank, and sugars were estimated by using glucose as a standard. Cation Contents Cation Na+ and K+ were estimated using flame photometer (Intech model I-66, Belgium) A hot-water extract was prepared from finely ground dried plant material in distilled water at 80ºC for 2 h. This filtrate was used to determine cation Na+ and K+ for root, leaves and stem samples adopting (Khan et al., 2000) method. Selective absorption (SA) and selective transport (ST) of K+ over Na+ were also calculated (Wang et al., 2005): SA= (K+ / Na+) root / (K+ /Na+) medium ST1= (K+ / Na+) stem / (K+ /Na+) root ST2= (K+ / Na+) leaf / (K+ /Na+) stem Organic and inorganic osmotica The percent contribution of leaf Na+, K+, soluble sugars (as sucrose equivalents) and proline to leaf osmotic potential was calculated by Van’t Hoff equation as described by Guerrier (1996)

Determination of photosynthetic pigments Pigments extraction was based on (Ritchie, 2006), using 100% ethanol solvent. Pigment estimation (chlorophyll a, b and carotenoid) was by recording absorbance for chlorophyll a, b and carotenoids at 470, 648.6 and 664.2 nm using (Beckman-Coulter DU-730, UV- vis spectrophotometer). Photosynthetic pigments were calculated with equations suggested by Lichtenthaler (1987). Β-carotene and Lycopene contents Extraction was done by using 25mg plant sample with 10ml acetone-hexane mixture (4:6) for 1 minute. Sample was filter (Whatman No.4) filter paper. Reaction mixture absorbance was recorded at 453nm, 505nm, 645, and 663nm. Barros et al (2007). Anthocyanin, β-cyanin and Flavnols Estimation of pigments (anthocyanin, β-cyanin, flavonol glycosides) was carried out by Ganjewala et al (2008). Briefly 25mg plant sample were extracted with 10ml (4:6) acetone- hexane mixture for 30 minute. For estimation of anthocyanin, flavonol glycosides, betacyanin absorbance was recorded at 530, 360, 536 nm respectively. The concentration was estimated by using molar extinction coefficient (ε) of 38,000 L x mol–1 x cm–1 for (anthocyanin), (ε) of 38,000 L x mol–1 x cm–1 for (β-cyanin) and (ε) 65 x 106 cm2 mol-1 for (flavonol glycosides). Chlorophyll Fluorescence Measurements Chlorophyll fluorescence (pulse modulated) meter (Junior PAM, Walz, Germany) was used to determine chlorophyll fluorescence. (Fv/Fm = Fm-Fo/Fm) according to Kitajima and Butler (1975). NPQ was determined according to Bilger and Bjorkman (1990). Effective quantum yield of PS II (YII) by using Genty et al (1989). Photochemical fluorescence quenching coefficient (qP) was measured by using Van-Kooten and Snel (1990) method and Schreiber et al (1986). Non-photochemical quenching (YNPQ) and non-regulated quenching (YNO) by Laisk et al (1997). Linear electron transport rate (ETR) was calculated according of Krall and Edwards (1992). Electrolyte Leakage 0.1g of plant material was added in 10 ml of de-ionized water, samples were vortex and 0 incubate for 2 hours to obtain (EC1). Same samples were then autoclaved at 121 C for 20 minutes to obtain (EC2) after cooling samples. % Electrolyte Leakage was determined by Dionisio-Sese and Tobita (1998), using by following formula:

% EL= EC1/EC2×100

Oxidative stress markers Trichloroacetic acid (TCA) extracts using 5 mL of 3% (w/v) ice-cold along freshly ground plant materials were used for extraction process. KI reagent assay Junglee et al (2014) was used for quantification of Hydrogen peroxide (H2O2) contents. Malondialdehyde (MDA) concentration is marker of lipid peroxidation. Briefly 0.2 g freshly harvested leaves were extracted in 2.0 ml of ice cold TBA solution. Heath and Packer (1968) 2-thiobarbituric acid (TBA) assay was used for determination of MDA. Antioxidant enzyme activities Antioxidant enzymes extraction was carried out using Polle et al (1994) protocol. Enzyme extracts protein content of were determined using Bradford (1976) and enzymes activities were expressed as units per milligrams of the protein (U mg-1 protein). Catalase (CAT) activity was evaluated using (Aeby, 1984). CAT activity was based on

H2O2 decomposition. Calculation of the enzyme activity was determined using extinction coefficient e = 39.1 mM-1 cm-1 Superoxide dismutase (SOD) activity was determined using Beauchamp and Fridovich, (1971). Briefly assay was carried out with two sets of glass tubes one exposed to light other incubated in dark based on of photochemical inhibition of nitroblue tetrazolium (NBT). Guaiacol peroxidase (GPX) activity was measured according to Tatiana et al (1999). Assay was based on increase in absorbance due to tetraguaiacol formation. Calculation of the enzyme activity was determined using extinction coefficient (e =26.6 mM cm-1). Glutathione reductase (GR) activity was quantified by using (Foyer and Halliwell, 1976). Assay was based on oxidation of NADPH. Calculation of the enzyme activity was determined using extinction coefficient (e = 6.2 mM cm-1) Ascorbate peroxidase (APX) activity was based on Nakano and Asada (1981). Activity was based on decrease in absorbance due to the oxidation of ascorbic acid at 290 nm. Calculation of the enzyme activity was determined using extinction coefficient (e =2.8 mM cm-1). Antioxidant substrate Contents of reduced ascorbic acid (AsA) were determined in TCA extracts according to the method of Law et al (1983). AsA and total AsA will be calculated from standard curves, while DAsA is calculated by the difference between total AsA and AsA. Determination of Polyphenol Referred to Chapter 2 materials and methods section.

Determination of Total Tannins Total tannin content was estimated using Pearson (1976). Determination of Antioxidant Assays Referred to Chapter 2 materials and methods section. High performance liquid chromatographic (HPLC) analyses Briefly 0.5 g leaf dried material was extracted in mixture of 40 ml methanol (62.5%) and 10 ml 6 M HCl according to the method described by Proestos et al (2006). Samples were then refluxed in a boiling water bath for 2 h. Filtrate was collected and the final volume was maintained to100 ml with methanol. Before injecting into a HPLC system, mixtures were re-filtered through 0.45 μm membrane filter (Millex-HV). Auto-sampler (SIL-20A), HPLC system (Shimadzu LC-20AT) was equipped with LCSolution software, column oven (CTO-20A), and diode array detector (SPD-M20A). Analytical column, Nucleosil C18, 5 μm 100 A° (250 × 4.60 mm, Phenomenex) coupled with a guard column (Phenomenex) was used. Mobile phase contained (A) sodium phosphate buffer (50 mM; pH 3.3) in 10% methanol and (B) 70% methanol. Flow rate was maintained to 1 ml min−1 using gradient program by Sakakibara et al (2003). Phenolic compounds were identified by comparing retention time and UV–Vis spectra of chromatographic peaks with that of authentic reference standards at 280 nm wavelength. Statistical analyses All statistical analyses including one way ANOVA was performed by using SPSS (version 16) and graphs were plotted using Sigma plot (version 12.5).All results are expressed as means (± standard error) of a minimum of 3 biological replicates with up to 5 technical replicates where necessary. Bonferroni test was carried out to determine if significant (P ≤ 0.05) differences existed among means.

Results Effects of salinity on plant growth Although shoot height was reduced with increasing salinity, however, plant growth in terms of biomass (fresh and dry), number of leaves, number of nodes, leaf moisture, leaf area, leaf succulence and leaf moisture was optimal at both 0 and 100 mM NaCl (Fig 3.1 and 3.2). High salinity (300 mM NaCl) significantly reduced shoot height (-48%), fresh weight, dry weight, number of leaves, number of nodes, leaf area and stem girth (Fig 3.3). Effects of salinity on water relations Leaf water potential (Ψw) and osmotic potential (Ψs) decreased significantly with increasing NaCl concentration (Fig.3.4). However, turgor potential (ΨP) was maintained along all the salinities. Pre-dawn leaf Ψw were substantially more negative than noon values and this difference was consistent with increasing salinity (Fig. 3.5). Both water potentials (ѰW0) at full turgor and (ѰWTLP) turgor loss point decreased linearly with increasing salinity, whereas, bulk elasticity of cell wall (Ɛ Mpa) followed the reciprocal trend (Table 3.1). Effects of salinity on photosynthetic pigments Chlorophyll a, b and total chlorophyll remain unchanged under moderate salinity, however increased significantly by 2.5 (chl a), 4.3 (chl b) and 3.5 (total chl) folds, respectively at 300 mM NaCl. Chlorophyll a/b ratio transiently increased as the salinity of medium increased. Carotenoid followed the similar trend like chlorophylls, it was unchanged at 100 mM NaCl and increased at 300 mM NaCl (4 times) as compared to control. A transient increase in chlorophyll to carotenoid ratio was found with 25% higher values at 100 mM NaCl, as compare to control (Fig.3.6). Other pigments also increased with salinity either at both salinities (like β- cyanin, flavonol glycosides, and β- carotene) or at higher salinity (like anthocyanin and lycopene) (Fig.3.7). Effects of salinity on electrolyte leakage, Malondialdehyde (MDA) and Hydrogen peroxide (H2O2) contents

Moderate salinity did not affect H2O2 content of C. procera, while slight increase in MDA and EL was observed. All damage markers including H2O2, MDA and electrolyte leakage were generally increased with increasing salinity, however their magnitude was 200% or more than 200% higher at 300 mM NaCl (Fig.3.8). Effect of salinity on Soluble Sugar and Proline contents There was a significant increase in proline content with increase in salinity. This enhancement was 3.3 and 5.5 folds at 100 mM and 300 mM NaCl, respectively. Similarly,

48 soluble sugars increased by 36% and 54% under moderate salinity and higher salinity, respectively (Fig.3.9). Chlorophyll fluorescence A significant increase in PSII maximum quantum efficiency of dark-adapted leaves (Fv/Fm) was found at moderate salinity, however decreased and reached to that of control at 300 mM NaCl. Actual yield of (YII) efficiency, photochemical quenching (qP) and electron transport rate (ETR) showed significant reduction in plants irrigated with 100-300 mM NaCl. However, heat dissipation through non-photochemical quenching Y (NPQ) and Y (NO) increased significantly with increasing (Fig.3.10). Cation accumulation Accumulation of cations in C. procera varied with the level of salinity and the organs (Fig.3.11). Despite organs, sodium (Na+) concentration increased linearly with increasing salinity. The highest Na+ concentration was found in leaves, followed by roots, while stem had lowest Na+. Na+ accumulation was not much affected on K+ uptake in all plant parts except root, in which K+ content increased significantly at higher salinity. Increasing salinity significantly (P<0.01) increased Na+/K+ ratio in all plant parts. Selective absorption of K+ (over Na+) from surrounding to medium increased linearly with increasing salinity (Fig.3.12), however, when K+ moved from root to stem its selectivity was unchanged at 100 mM and decreased at higher salinity (Fig.3.11). At the next level, when K+ transported from stem to leaves the selectivity was decreased slightly with increasing salinity (Fig.3.12).

Antioxidant enzyme activities Activity of antioxidant enzymes (CAT, SOD, GR and GPX) was unchanged at moderate salinity, except APX. However, higher salinity significantly increased antioxidant activity of all enzymes, including APX (Fig.3.13). Antioxidant enzyme substrate Ascorbic acid Total ascorbic acid remain unchanged under non-saline and moderate salinity conditions, however its content increased 4 folds at higher salinity (Fig.3.14). Reduced form of ascorbic acid (AsA) significantly increased with increase in salinity and its content was 3 folds higher at 300 mM NaCl as compare to control. The content of oxidized form of ascorbic acid (DHAsA) did not change at moderate salinity but increased by 2 fold at high salinity. AsA/DHAsA ratio remain unchanged at moderate salinity, however it increased at higher salinity (Fig.3.14).

Effect of salinity on secondary metabolites Effect of NaCl on total phenols, flavonoids, proanthocynadin and tannin contents Total phenolic content (TPC) of leaf, stem and root of C. procera increased with increasing salinity (5-8 mg GAE g-1), however the highest value was found at the stem of 300 mM NaCl (Fig.3.15). Total flavonoid content (TFC) of C. procera leaf also increased with increasing salinity (1.4-1.6 mg QE g-1), while root and stem showed no significant effects of salinity on TFC (Fig.3.15). In all plant parts, proanthocyanidin content (PC) did not change with increasing salinity, however total tannin content (TTC) either unchanged (root) or decreased at high salinity (leaf and stem). Effect of NaCl on Antioxidant activity of C. procera The DPPH and ABTS estimates the radical scavenging ability of plant extracts using 1,1- Diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radicals, respectively. The total antioxidant capacity (TAC) methods estimate the ability of plant extract to reduce molybdenum ions. In all test generally, antioxidant activity increased with increasing salinity in all plant parts (Fig.3.16). The highest DPPH and ABTS activity was found in root samples, followed by stem and leaf. While, TAC was highest in leaves compared to root and stem. Phenolic profile using HPLC HPLC analysis was conducted to evaluate the metabolism of phenolic compounds in C. procera treated with increasing NaCl salinity. Phenolic composition of C. procera hydrolysed extracts were determined against reference standards of 1) Pyrogallol; 2) Gallic acid; 3) Resorcinol; 4) Pyrocatechol; 5) Catechin; 6) Hydroxybenzoic acid; 7) Chlorogenic acid; 8) Vanillic acid; 9) Caffeic acid; 10) Syringic acid; 11) Coumaric acid; 12) Ferulic acid; 13) Sinapic acid; 14) Rutin; 15) Trans-cinnamic acid; 16) Quercetin; and 17) Ellagic acid (Fig.3.17). All phenolic compounds were found except gallic, hydroxybenzoic and caffeic acids (Fig. 3.18). Total content of these phenolic compounds amounted to 173.90 μg g-1 DW under non-saline condition, while they were increased significantly with increased under moderate (191.94 μg g-1 DW) and high salinity (209.68 μg g-1 DW). It is noteworthy that pyrocatechol dominated all identified phenolics under non-saline conditions but resorcinol increased by 30% and was the dominant phenolic compound under salinity. Beside this, most of the other polyphenols, increased with salinity like chlorogenic acid, vanillic acid, ferulic acid, sinapic acid, rutin, quercetin, while others were decreased (Table.3.3).

B Shoot a Root 60

40 b c 20

Plant length (cm) Plant

-20 a a a

0 100 300

NaCl (mM) Figure. 3.1: (A) Comparison of Calotropis procera plants grown under different (mM) NaCl treatments for 35 days under green net house. (B) Changes in height of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations.

) Leaf

-1 50 a Stem a Root 40 Ratio

20 b 10

Freshweight (g plant

) 5 a a 0.16

-1

Root / Shoot Root 4 b b 0.14 3 b 2 0.12 a

Dry weightDry (g plant 1 0.10 0 0 100 300

NaCl (mM) Figure. 3.2: Changes in plant fresh weight and plant dry weight of (leaf, Stem and root), Root shoot ratio of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations.

) a -1 a a a 400 (plant Leaves 15

plant 2 300 10 200 b b -1 5 ) 100

Leaf(cm area

0 0

0.25 a a a

8 (plant Nodes b 0.20 6 0.15 c b 4

Girth (cm) Girth 0.10 -1

) 0.05 2

0.00 0 H (g Succulence a 100 10 a a a ab b 80 8

60 6

O g O 40 4

-1 -1

Leaf moisture (%) 20 2 DW)

0 0 0 100 300 0 100 300 NaCl (mM) Figure. 3.3: Changes in leaf area, number of leaves, grith (cm), nodes (plant-1), leaf moisture% and succulence of C.procera plants treated with 0, 100 and 300 mM NaCl concentrations.

a a 1 a

-1 a a a 1 a -2 a 0 -3 -1b a Waterrelations (Mpa) -4 -2 c b a WP c -5 OP -3 TP b

Waterrelations (Mpa) -4 b c 0 100 WP300 c -5 OP TP NaCl (mM)

0 100 300 Figure. 3.4: Effect of different NaCl treatmentsNaCl (0,(mM) 100, 300mM) on water potential, osmotic potential and turgor potential (MPa) of C procera leaves.

-1

a -1 -2 a a b-2 a b -3 c c

Waterpotential (Mpa) -3 c c

Waterpotential (Mpa) c -4 Pre Dawn c Noon -4 Pre Dawn Noon

0 100 300 0 100 300 NaCl (mM) NaCl (mM) Figure. 3.5: Effect of different NaCl treatments (0, 100, 300mM) on Pre-dawn and noon water potential (MPa) of C. procera leaves.

Chlorophyll b ( b Chlorophyll

) -2 10 10

gcm 8 b 8

6 6

4 b 4 cm g

a a -2

2 2 ) Chlorophyll( a a a 0 0

)

Carotenoides ( Carotenoides

-2 b 10 10 b gcm 8 8

6 6

4 4 cm g a a 2 a a 2 -2

)

Totalchlorophylls (

0 0 / Carotenoides Chlorophyll

4 b 3 3 a a 2 2 b a a 1

Chlorophylla/b 1

0 0 0 100 300 0 100 300

NaCl (mM) Figure. 3.6: Changes in chlorophyll a, chlorophyll b, total chlorophyll and chlorophyll a/b ratio, carotenoid and chlorophyll/ carotenoid ratio of C. procera plants treated with 0, 100 and 300 mM NaCl concentrations.

Betacyanins ( Betacyanins

)

-2 50 b c 5

gcm 40 4 a b 30 a 3

g cm g 20 2

-2

10 a 1 )

Anthocyanins( 0 0

)

Betacarotene ( Betacarotene -2 c c 12 1.5 gcm 10 8 1.0 b 6 a b cm g 0.5 4

a -2

2 ) 0.0 0

Flavonolglycosides( b 0 100 300 10

)

-2 NaCl (mM) 8

gcm 6

4 a a 2

Lycopene( 0 0 100 300

NaCl (mM) Figure. 3.7: Changes in anthocyanin, betacyanin, flavnolglycosides, betacarotene and lycopene of C. procera plants treated with 0, 100 and 300 mM NaCl concentrations.

400 b

FW)

-1 300

M g M a ( 200 a

H 100

0 c 12

FW)

-1 9

M g M 6 b a

MDA( 3

0 80 c 60 b 40

EL(%) a 20

0 0 100 300

NaCl (mM)

Figure. 3.8: Changes in H2O2, MDA and EL% of C. procera plants treated with 0, 100 and 300 mM NaCl concentrations.

800 c

FW) -1 600 gg b 400

Proline( 200

0 100

FW) b

-1 b 80 a 60

Solublesugars (mg g 0 0 100 300

NaCl (mM)

Figure. 3.9: Changes in Proline and soluble sugar of C. procera plants treated with 0, 100 and 300 mM NaCl concentrations.

0.81 b a 0.5 a a b Y(II) 0.78 c 0.4

Fv/Fm

0.75 0.3

0.30 a 0.7 b 0.25 a b Y(NPQ) c a 0.6 0.20

0.5 0.15

0.10

120 a 0.54

b c Y(NO) 105 b 0.45 c ETR a 90 0.36 75

100 300

Figure. 3.10: Changes in Fv/Fm, qP, ETR, Y (II), Y (NPQ), Y (NO) of C. procera plants leaves treated with 0, 100 and 300 mM NaCl concentrations.

3.0

Leaf Na g/kg 2.5 Stem Na g/kg Root Na g/kg

2.0

1.5

Y Data

1.0

0.5

0.0 0 100 300

X Data

Root Stem Leaf b 2.5 c

DW)

-1 2.0 1.5 a c 1.0 c b b 0.5 a a

Na (mole Na Kg 0.0 b 1.5

DW) a -1 b 1.0 ab a c a b b 0.5

K(mole Kg 0.0 3 b b

Na/K c a 1 b b b a a 0 0 100 300 0 100 300 0 100 300

NaCl (mM)

Figure. 3.11: Changes in Na (mole kg-1 DW) K (mole kg-1 DW) and Na/K in terms of dry weight of C. procera plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations.

200

Leaf Na g/kg Stem Na g/kg Root Na g/kg 150

100

Y Data

0 0 100 300

X Data

c 200 SA

150

100 b

50 a

0 a ST1 2.5 a 2.0 1.5 1.0 b 0.5

Kselectivity over Na 0.0 ST2 0.3 a c b 0.2

0.1

0.0 0 100 300

NaCl (mM)

Figure. 3.12: Changes in K over Na selectivity, Selective absorption from medium to root (SA), Selective transport from root to stem (ST1) and selective transport from stem to leaves (ST2), of C. procera plants treated with 0, 100 and 300 mM NaCl concentrations.

12 (U mg SOD b b 5 10 4 a a

protein) 8

-1

3 -1 6 a a protein) 4 2

CATmg (U 2 1 0 0 b c 6 (U mg GR 450 b

protein) a a 4

-1

300 a -1

protein)

150 2

APXmg (U

0 0 0 100 300 60 b 50 a a NaCl (mM)

protein) 40

-1 30 20

GPXmg (U 10 0 0 100 300

NaCl (mM) Figure. 3.13: Changes in CAT, SOD, APX, GR and GPX of C. procera plants treated with 0, 100 and 300 mM NaCl concentrations.

Total AsA AsA DHAsA Ratio 6 B 5 c 5 c AsA DHAsA /

-2 4 4 A A 3 3

mMolcm 2 2 b a b 1 a b 1 a a 0 0 0 100 300 NaCl (mM)

Figure. 3.14: Changes in ascorbic acid content, AsA/ DHAsA of C. procera plants treated with 0, 100 and 300 mM NaCl concentrations.

Leaf Stem Root

(mg GAE g GAE (mg ) c

-1 8 c 8 7 b b 7 TPC a a 6 6

TPC c

5 b 5 -1

a ) (mgGAE g 4 4

2.0 2.0 g QE (mg ) b a ab

-1

1.5 1.5 TFC 1.0 a a a 1.0 TFC a a a -1

0.5 0.5 )

(mgQE g 0.0 0.0

(mg CE g CE (mg ) 3 a a a 3 -1 a a a a a a

PC PC 2 2

PC 1 1 -1

)

(mgCE g 0 0

(mg TAE g TAE (mg

) a a 4 4

-1 b TTC 3 a a 3 2 a a a 2 TTC b 1 1 -1

)

(mgTAE g 0 0

100 300

100 300 NaCl (mM)

Figure. 3.15: Changes in total phenolic content, total flavonoid content, proanthocynadin content, and Total tannin content of C. procera plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations.

Leaf Stem Root

(Inhibition %) b

b b DPPH 70 70 a a a 60 b 60 DPPH a ab

(Inhibition %) (Inhibition 50 50

(mMol Trolox g

)

-1 c 40 b 40 b b ABTS 35 a a 35 c 30 30

ABTS ABTS b a 25 25

-1

)

(mMol Trolox g Trolox (mMol 14 14 c

13 13 (

)

g AsA g -1 12 b 12 a TAC 11 a b 11 ab a a TAC 10 a 10

-1 g AsA g AsA g 9 9

)

(

100 300

NaCl (mM)

Figure. 3.16: Changes in DPPH radical scavenging activity (Inhibition %), ABTS anti- radical activity (mM Trolox g-1) and Total antioxidant activity (µg AsA g-1) of C. procera plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations.

05: Catechin 01: Pyrogallol 02: Gallic acid 03:Resorcinol 04: Pyrocatechol

06:Hydroxybenzoic 08: Vanillic 10: Syringic acid 07: Chlorogenic acid 09: Caffeic acid acid acid

11: p- Coumaric 12: Ferulic acid 15: Trans- acid 13: Sinapic 14: Rutin acid cinnamic acid

16: Quercetin 17: Ellagic acid

Figure. 3.17: HPLC chromatogram of (a) standard phenolic compounds (1 Pyrogallol; 2 Gallic acid; 3 Resorcinol; 4 Pyrocatechol; 5 Catechin; 6 Hydroxybenzoic acid; 7 Chlorogenic acid; 8 Vanillic Acid; 9 Caffeic acid; 10 Syringic acid; 11 Coumaric acid; 12 Ferulic acid; 13 Sinapic acid; 14 Rutin; 15 Trans-cinnamic acid; 16 Quercetin; 17 Ellagic acid) and (b) their structures used in this study.

Figure. 3.18: HPLC chromatograms showing profile of different phenolic acids in hydrolysed leaf extracts of C. procera treated with 0, 100 and 300 mM NaCl concentrations.

Table. 3.1. Water potential at full turgor (ѰW0), Water potential at turgor loss point

(ѰWTLP) and Bulk elasticity of cell wall (Ɛ Mpa) of C. procera leaves.

Treatment ѰW0 (Mpa) ѰWTLP (Mpa) Ɛ Mpa (Mpa) Control -1.08 ± 0.09 a -2.5 ± 0.08 a 12.18 ± 2.21a 100 mM -2.9 ± 0.26 b -4.4 ± 0.1 b 17.39 ± 3.59 b 300 mM -3.6 ± 0.09 c -4.9 ± 0.09 b 19.25 ± 2.34 c

Table. 3.2. % Contribution to osmolality of organic and inorganic osmolytes of C. procera leaves. Inorganic contribution Organic contribution Treatment Na% K% CHO % Proline % Control 8.3 ± 0.3 a 7.8 ± 0.4 a 12.1 ± 0.8 a 0.2 ± 0 a 100 mM 16.3 ± 0.3 b 6.4 ± 0.2 ab 13.6 ± 1.3 a 0.3 ± 0 b 300 mM 16.4 ± 1.4 b 5.5 ± 0.4 b 16.4 ± 0.7 b 0.5 ± 0 c

Table.3.3. Phenolic composition (µg g-1 DW) in leaves of C. procera treated with 0, 100 and 300 mM NaCl concentrations.

Retention NaCl (mM) Compounds time 0 mM 100 mM 300 mM

Pyrogallol 9.80 20.68 ± 1.53a 18.44 ± 2.38 a 15.37 ± 2.11 b Resorcinol 14.11 44.51 ± 0.15 a 60.97 ± 3.57b 64.45 ± 2.95b Pyrocatechol 16.58 62.50 ± 2.15a 39.73 ± 1.98b 26.24 ± 2.08b Catechin 22.70 5.48 ± 0.35a 4.22 ± 0.87a 4.13 ± 0.05a Chlorogenic acid 24.22 22.80 ± 1.32a 33.78 ± 2.56b 54.68 ± 3.44c Vanillic acid 26.58 2.07 ± 0.11a 4.68 ± 1.11b 6.62 ± 0.86c Syringic acid 30.35 0.93 ± 0.01a 0.74 ± 0.04a 0.69 ± 0.01a Coumaric acid 36.30 2.87 ± 0.17a 2.47 ± 0.05a 3.85 ± 0.02a Ferulic acid 40.25 1.25 ± 0.09a 2.50 ± 0.02a 4.24 ± 0.08b Sinapic acid 41.09 1.36 ± 0.05a 3.95 ± 0.01b 5.58 ± 0.05b Rutin 49.88 4.60 ± 0.07a 11.07 ± 1.21b 11.25 ± 1.11b Quercetin 59.52 4.85 ± 1.27a 9.39 ± 1.35b 12.57 ± 0.97b 173.90 ± 6.49a 191.94 ± 5.11b 209.68 ± 6.82c

Discussion In the present investigation, C. procera grew well under NaCl concentrations up to 300 mM. Optimal growth was observed in non-saline and moderately (100 mM NaCl) saline conditions. In general, 100 mM NaCl did not affect C. procera growth in terms of FW, DW, number of leaves, nodes, leaf area ,leaf succulence and leaf moisture percent (Fig.3.2 and 3.3), likewise, of the growth of other halophyte species i.e. Phragmites karka, E. angustifolia and Spartina maritima (Naidoo et al., 2012; Abideen et al., 2014, Liu et al., 2018). On the other hand, higher salinity (300mM NaCl) reduced all growth parameters of C. procera (Fig.3.2). The salt induced growth reduction of C. procera could be attributed to extra energy cost used in salt tolerance (Munns and Gilliham, 2015). Generally, plant spend more than half of its photosynthetic energy (60%) for maintaining regular metabolic processes and rest (40%) is invested in growth and biomass production. However, any stress e.g. salinity not only decreases the gross energy supply (through photosynthesis inhibition), but it also disturb the energy allocation from growth to stress maintenance (through ion homeostasis, osmotic adjustment, oxidative stress management etc.) (Munns and Gilliham, 2015). The above ground biomass (stem and leaf) of C. procera was more sensitive to salinity than roots, indicating a non-excreting behavior like in Panicum turgidum, Phragmites karka, Desmostachya bipinnata, Paspalum paspaloides, and Paspalidium geminatum (Koyro et al., 2013; Abideen et al., 2014; Moinuddin et al., 2014; Adnan et al., 2016). Increase in root/shoot ratio and shifting allocation of biomass towards root appears important for salt exclusion, which help plant to uptake water and essential minerals from the saline medium (Sharp et al.,2004; Munns and Tester, 2008). In addition, C. procera did not change its root size under salinity rather increased root to shoot ratio (Fig.3.2). This may help plant to absorb more K+, sequester more Na+ in roots and restrict Na+ entry into the xylem, thus reducing net Na+ influx and its translocation to above ground parts (Munns and Tester, 2008; Gorham et al., 1985). Such a response is also found in other salt resistant species like Cynodon dactylon, Distichlis spicata, Phragmitis karka, Puccinellia ciliate, Sporobolous airoides, and Zoysia japonica (Marcum, 1999; Teakle et al., 2013; Abideen et al., 2014). Leaf fall was also noticed in this experiment, which was maximum at 300 mM NaCl (data not shown). Leaf shedding reduce leaf quantity and ultimately the active evaporative area, which helps plant to overcome water loss and limiting excessive Na+ to enter into main stream, as reported previously from this plant (Ibrahim et al., 2013). Early senescence and leaf curling (Lutts et al., 1996) might be an attempt to minimize

70 transpiration (Hasselquist et al., 2010), and reduce number of leaves to prevent sap salt concentration reaching toxic levels (Munns and Tester, 2008). The unchanged moisture percent of C. procera leaf showing that plant strategically reduced leaf area and number of leaves to control water loss and also minimize light exposure thus, maintaining leaf turgidity under saline condition. One of the prime indicators of salt stress is the alteration in water relations of plant, which can lead to the osmotic stress, ionic imbalance, nutrient deficiency and metabolic changes (Flowers and Colmer, 2008; Yan et al., 2013; Suzuki et al., 2012). Tolerant plants achieve osmotic balance by compartmentalizing toxic ions in vacuoles and their affect is counter balanced in cytosol by the production and accumulation of several organic osmolytes (Flowers and Colmer, 2008; Shabala and Mackay, 2011; Shabala, 2013). C. procera showed a linear decrease in leaf osmotic potential when exposed to increasing NaCl concentrations, indicating an ‘osmoconformor strategy’ (Khan et al., 2000). Reduced osmotic potential with increasing medium salinity enables plant to maintain water potential and decrease cell wall rigidity (Martinez et al., 2004; Verslues et al., 2006) as observed in Zaleya pentandra (Ehsen et al., 2017), Atriplex nummularia (Silveira et al., 2009), and Zygophyllum propinquum (unpublished data). In order to achieve osmotic adjustment, most halophytes prefer to increase leaf osmolality as compare to increase leaf succulence (Koyro, 2006; Gorai et al., 2010; Rozema and Schat, 2013). Water deprivation may include drought avoidance (e.g., changes in leaf area, stomatal conductance and leaf orientation) and/or drought tolerance (maintaining cell turgor through osmotic adjustments or cell wall elasticity) (Touchette et al., 2007). C. procera appeared to possess drought tolerant strategy by maintain turgor as well as increasing cell wall elasticity. Halophytes use Na+ and Cl– as cheap osmoticum to reduce high energy cost for synthesizing organic osmolytes (Wang et al., 2004). For instance, Atriplex halimus, Atriplex canescens, Kochia scoparia, Nitraria tangutorum, Sesuvium portulacastrum, Suaeda salsa, and Zygophyllum xanthoxylum resist physical and physiological water deficit by accumulating inorganic ions as osmolytes (Glenn and Brown, 1998; Martinez et al., 2004; Wang et al., 2004; Yan et al., 2013; Ma et al., 2011, 2017; Slama et al., 2015; Lokhande et al., 2010). Similarly, highly salt tolerant halophytes like Halosarciaper granulata, Sueada fruticosa, Gypsophila oblanceolata and Salicornia dolichostachya also use the similar strategy (Short and Colmer, 1999; Hameed et al., 2012; Katschnig et al., 2013). Lower osmotic and water potentials of C. procera leaf could result from salt or organic solute accumulation under saline conditions (Munns, 2002). Osmotic contribution

71 of inorganic content of C. procera at high salinity was ~22 % (Na+ 16.5% and K+ 5.5%). Whereas, at this salinity, organic osmolytes like soluble sugars and proline contributed 16% and <1% in overall osmotic potential, respectively (Table.3). This study indicated 55% increase in total soluble sugars which appeared to be a reason of reduced growth at high salinity. Accumulation of such a high amount of soluble sugars has been attributed with impaired carbohydrate utilization (Munns and Termaat, 1986) as found in Solanum lycopersicum (Yin et al., 2009). Similarly, proline also increased around one fold and four folds under moderate and higher salinity. Plant either significantly change or little change the diurnal leaf water relations (Turner and Long 1975; Naidoo et al., 2008). These changes could be attributed to decrease osmotic potential and higher tissue rigidity (Touchette, 2006; Rozema and Schat, 2013). Plants having drought avoiding strategy usually have less elastic cell walls and tend to possess greater fluctuations in diurnal water potential hence spend more water. On the other hand, ‘drought tolerant’ plants possess little variations in diurnal water relations hence conserve more water (Salleo, 1983; Gullo et al., 1986; Gullo and Salleo, 1988). C. procera showed significant variations in diurnal water potential (reduced water potential at noon) indicates a drought avoiding strategy by quick uptake of water from the soil (Salleo, 1983). In addition, diurnal variation of water potential was continued till 300 mM NaCl, suggesting a weaker drought tolerance of this plant (Bolanos and Longstreth, 1984; Pardossi et al., 1998; Touchette, 2006). Further research infuture focused on drought and flooding conditions coupled with salinity would be required to confirm this assumption. Adaptation to saline environment depends on plant ability to maintain K+ homeostasis and restrict Na+ uptake and transport to above ground parts (Carden et al., 2003; Wang et al., 2009; Ahmed et al., 2013; Teakle et al., 2013). At high salinity, C. procera allowed less Na+ to enter in leaf as compare to moderate salinity. This may be due to the less transpiration from the leaves as C. procera not only reduce number of leaves but area of each leaf was also shrank to less than half. However, K+ homeostasis was achieved by its better selective absorption from rooting medium. Decreased shoot K+ of high salinity plants may be due to its limited transport from root which may contribute in the reduction of plasma membrane depolarization (Bonales-Alatorre et al., 2013). C. procera maintained KUE to minimize the damages related to K+ deficiency posed under high salinity. Salt stress has been suggested to decrease chlorophyll in many halophytic plants but in some species chlorophyll increased under salinity (Sobrado et al., 2005; Stefanov et

72 al., 2016). C. procera showed unchanged chlorophyll (a, b and total) at moderate salinity but chlorophyll increased sharply at 300 mM NaCl (Figure.3.5). Chlorophyll a/b and chlorophyll/carotenoid ratios showed transient increase. In accordance with our results, chlorophyll a predominated over chlorophyll b in same plant under salinity (Al-Sobhi, 2005). Structural change in light harvesting complex might attribute to reduced chlorophyll content, thereby decreasing photosynthetic efficiency (Kocheva et al., 2004; Geissler et al., 2008). Higher chlorophyll content of C. procera at 300 mM NaCl could be related to both increased number of thylakoids per chloroplast and mesophyll thickness (Pompeiano et al., 2016). In addition, higher chlorophyll content help plant to increase light absorption, which is associated to retain photosynthesis rate similar to control. However, increased carotenoid content possibly prevent formation of singlet oxygen at high salinity (Asrar et al., 2017). Such an increase in chlorophylls and carotenoids was also reported in some other halophytes like Zoysia japonica (Pompeiano et al., 2014); Paspalum vaginatum (Pompeiano et al., 2016); Paspalum scrobiculatum (Shonubi et al., 2007). However, under high salinity high chlorophyll content may lead to overflow of electrons through the photosystems, which multiplies the risk of photoinhibition and oxidative stress (reflected in higher contents of MDA and H2O2). In C. procera, Fv/Fm showed transient increased with increasing salinity. This indicates an improved or maintained PSII efficiency at moderate and high salinity, respectively. Similar response was observed in S. europaea (Fan et al., 2011), Suaeda salsa (Lu et al., 2003) and Arthrocnemum macrostachyum (Redondo-Gomez et al., 2010). Higher photochemical efficiency enhances energy capturing and protects photosystem II from reactive oxygen species (ROS) mediated injuries. Increased Y (NPQ) of C. procera indicates a photo-protective mechanism that protects pigments, lipids and proteins at high salinity. It also safeguard thylakoid membrane from oxidative damage due to over production of ROS by triplet chlorophyll during thermal dissipation of excess energy (Azzabi et al., 2012). Excessive energy has negative impacts on photosystem, energy trapping either by dissipating as heat/fluorescence or non photochemiacal quenching protects damaging D1 protein (Moradi and Ismail, 2007; (Moradi and Ismail, 2007; Kreslavski et al., 2013). Salt stressed plant efficiently regulates Y (NPQ) and non- regulated process Y (NO) to dissipate heat (produced by absorbing excessive light energy) and minimize quantum yield loss. These results are in agreement with the chlorophyll fluorescence responses of of Paspalum paspalodes, Paspalidium geminatum (Moinuddin et al., 2017). Salt stressed plant also dissipate heat energy via xanthophyll cycle by

73 increasing carotenoid content (Moinuddin et al., 2017; Pompeiano et al., 2017). Exposure to high salinity also coupled with the risk of photorespiration- a sink for excessive excitation energy (Hussin et al., 2017). However, coordination between photochemistry and alternate electron sink (xanthophyll cycle, photo-protective compound carotenoids, and photorespiration) is necessary to avoid chronic photo-damage in plant (Lu et al., 2003; Moinuddin et al., 2017; Pompeiano et al., 2017). Anthocyanins, betacyanins, carotenoids, betacarotenes, lycopene and flavonol glycosides increased significantly under salinity. Plants accumulate anthocyanin to induce pigmentation in response to environmental stresses (Chalker-Scott, 1999). Although, little is known about salt-inducible anthocyanin biosynthesis in halophytes, their proposed roles during abiotic stresses include photoprotection, quenching of ROS, xenohormesis and stress signaling. Literature survey reveals that subsequent production and localization of these pigments in different plant parts induce resistance against a range of biotic and abiotic stresses (Ali and Abbas, 2003). Our study showed strong positive correlation of these pigments (betacyanin, flavnolglycosides, betacarotene, lycopene, anthocyanin, betacyanin and flavnolglycosides) with antioxidant activity (DPPH, ABTS, FRAP and TAC) of C. procera (Table.3.3). Production, accumulation and association of these pigments with antioxidant activity of C. procera indicating their role in salt stress management, as found in the salt tolerance of Arabidopsis thaliana and Beta vulgaris and Ligustrum vulgare (Sepulveda-Jimenez et al., 2004; Fini et al., 2011). Anthocyanin and betacyanins are mainly present in vacuole and ROS generated by chloroplast and mitochondria are generally detoxified by anthocyanin-rich vacuole (Kytridis and Manetas, 2006; Tanaka et al., 2008). It was recently reported that salt and UV stress increased the biosynthesis of flavonoid glycosides to a very similar degree to protect plant from salt induced light injuries (Fini et al., 2011). Beside their role in oxidative stress management, higher levels of beta-carotenes and xanthophylls (sub-types of carotenoids) have potential roles in harvesting light energy for photosynthesis (Holt et al., 2005; Bouvier et al., 2005). These compounds have the ability to dissipate excess energy in the form of heat (Demmig- Adams et al., 1996; Nagy et al., 2015).

Leaf H2O2, Malondialdehyde (MDA) and electrolyte leakage (EL) are common indicators of oxidative damage, linked to series of reactions from free radicals generation to lipid peroxidation or membrane damage (Mazliak, 1983; Sairam et al., 2005), which increased significantly with increasing stress. Moderate salinity did not affect H2O2 content of C. procera, while slight increase in MDA and EL was observed. However

74 significantly higher levels of EL, MDA, and H2O2 were found at high salinity as found in some plants specially halophytes (Jithesh et al., 2006; Hameed et al., 2015; Muchate et al.,

2016). H2O2 is the most stable form of ROS which can easily penetrate in the cell, hence behaving as a signaling molecule to provoke stress tolerance mechanisms (Foyer and

Noctor, 2009, Gill and Tuteja, 2010). However, signaling function of H2O2 depends upon its cellular concentration, which relays on plants’ antioxidant defense otherwise, building up of H2O2 can lead to inactivation of Cu/Zn-SOD, Fe-SOD and many calvin cycle enzymes, protein kinases, phosphatases and transcription factors (Halliwell and

Gutteridge, 1999). Elevated level of H2O2 and associated damage markers (MDA and EL) of C. procera, indicate that dissipation mechanisms Y(NPQ), Y(NO) carotenoids and other pigments were insufficient at high salinity, hence leaking of excess energy from over- reduced photosynthetic machinery resulted in the generation of ROS (Turkan and Demiral, 2009). In present study C. procera showed significant increase in activities of all antioxidative enzyme like superoxide dismutase (SOD), catlase (CAT), ascorbate per oxidase (APX), guaicol peroxidase (GPX) and glutathione reductase (GR). SOD is a vital antioxidant enzyme and a first line of defense for salt stressed plants. SOD generates H2O2 by scavenging harmful O2ˉ, and H2O2 is further neutralized into water by different antioxidant enzymes like CAT, APX, and GPX (Jithesh et al., 2006; Hameed and Khan,

2011). In C. procera, Asada–Halliwell pathway enzymes of H2O2 scavenging and AsA regeneration were upregulated in a synchronized manner to save plant from salt induced oxidative injuries (Gomez et al., 1999; Hernandez et al., 2000; Hernandez et al., 2001; Mittova et al., 2003; Rubio et al., 2009). Such a coordinated response was also found in many other halophytes in response to salinity (Amor et al., 2005; Benzarti et al., 2012; Duarte et al., 2013). Apel and Hirt (2004) stated that the expression and activities of most antioxidant enzymes increased linearly by ROS accumulation. A strong positive correlation of H2O2 with all antioxidant enzymes of C. procera validates the results of

Apel and Hirt (2004). Higher contents of H2O2 and MDA in C. procera were in line with increased activities of SOD and CAT, at high salinity. In addition, raised SOD activity also coincided with CAT activity (0.776). These results of C. procera are in agreement with the antioxidative responses of Echinochloa crusgalli, Pennisetum clandestinum, and Spartina alterniflora (Abogadallah et al., 2010; Subudhi and Baisakh, 2011; Muscolo et al., 2013). CAT plays a key role in detoxification of H2O2 from peroxisomes while, APX removes H2O2 from chloroplast (Frederick and Newcomb, 1969; Mittova et al., 2000).

Both, CAT and APX along with GR are responsible of maintaining indigenous level of

H2O2 in cytosol (Noctor and Foyer, 1998; Duarte et al., 2013). APX and GR activities of C. procera also increased significantly under high salinity. APX consumes ascorbate

(AsA) to neutralize H2O2 in to H2O and dehydroascorbate (DAsA) (Asada, 1992). Salt stressed plants increased GR activity to increase NADP+/NADPH ratio, which enables the ability of NADP+ to accept electrons leaking from photosynthetic ETC hence, limiting ROS concentration (Muscolo et al., 2003). An increase in APX activity of C. procera showed its strong relation with total ascorbate concentration (r =0.7441), at high salinity (Kumar et al., 2017). High level of oxidized ascorbate (DHAsA) could be explained with increased activity of APX. However, increased levels of reduced ascorbate suggest ascorbate recycling to regulate the redox state of ascorbic acid pool, which is important to improve salt tolerance (Gill and Tuteja, 2010). The regeneration of AsA is an indicative of high activity of dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR). Recent reports suggest that MDHAR and DHAR both are equally important in controlling the level of total AsA and its redox states (Wang et al., 2010; Hasanuzzaman et al., 2011). Decrease in ASA/DHASA ratio mainly controlled by APX activity (as APX use ASA as a reducing agent to detoxify H2O2), meaning that an induction of the ascorbate glutathione cycle for salt acclimatization or salt avoidance (Hasanuzzaman et al., 2011). Effect of salinity on growth of C. procera was also related to polyphenols and antioxidant activity. C. procera not only increased the amount of TPC and TFC but it also hydrolyzed the polymerized from of polyphenols (PC and TTC) to increase the pool of active phenolic metabolites. Strong positive correlations of salinity with TPC (r = 0.903), 2 TFC (R =0.945), DPPH (r = 0.946), ABTS (r = 0.959) and TAC (r = 0.851) indicating their role in plants’ stress tolerance. In addition, their positive relationship with H2O2 (TPC r = 0.655, TFC r = 0.669, DPPH r = 0.956, ABTS r = 0.894 and TAC r = 0.776) and MDA

(TPC r = 0.758, TFC r =0.802, DPPH r = 0.843, ABTS r = 0.835 and TAC r = 0.723) also explained their part in controlling ROS progression and damage. Fixation of damaging ROS using antioxidant compounds like polyphenols appears to be a preferred trait of halophyte species (Zhu et al., 1994) as amount and composition of polyphenols depend on quantum of applied stress (Awika et al., 2005; De Abreu et al., 2005). In this study, TPC and TFC of C. procera increased with increasing salinity, and these results are consistent with all antioxidant activity measurements. In addition, TPC and TFC showed strong positive correlation with DPPH (TPC r = 0.7088, TFC r = 0.802), ABTS (TPC r = 0.803,

TFC r = 0.922) and TAC (TPC r = 0.966, TFC r = 0.976). Chemical structure of phenolic compounds (aromatic rings and free hydroxyl groups) provides ideal configuration to neutralize damaging oxygen radicals (Rice-Evans et al., 1996; Bors and Michael, 2002). TPC of C. procera was comparable with several glycophytes (Chu et al., 2002; Zhou and Yu, 2006) and even higher than some halophytes (Benhammou et al., 2009). Salt induced gradual increase in polyphenols of C. procera indicating an up-regulation of phenylalanine ammonia lyase (PAL) activity (Faller and Fialho, 2010) as reported in strawberry, sugarcane, peppers, Aegiceras corniculatum, Raphanus sativus L, Rosmarinus officinalis, Carpobrotus rossii and Suaeda maritima (Muthukumarasamy et al., 2000; del-Bano et al., 2003; Wahid and Ghazanfer, 2006; Keutgen and Pawelzik, 2008; Parida and Jha, 2010). Our results also validates the previous study about antioxidant potential and bioactivity of different parts of C. procera (Yesmin et al., 2008). The higher accumulation of polyphenolic compounds with profound antioxidant activity in aerial parts of may explain the protective role of plant phenolics against photooxidation of light exposed tissues (Close and McArthur, 2002; Niknam and Ebrahimzadeh, 2002), since light harvesting complex is more prone to ROS production in plants grown under salt stress conditions (Falleh et al., 2011). HPLC profile of C.procera leaf indicated the higher accumulation of several phenolic compounds stimulated by salinity, while some were de-accumulated. Results indicated that pyrocatechol, pyrogallol and syringic acid decreased under saline condition while resorcinol, chlorogenic acid, vanillic acid, ferulic acid, sinapic acid, quercetin and rutin were increased significantly. Phenolic compounds are ubiquitous to higher plants, besides their role in ROS quenching, these compounds have been reported in several physiological roles. Increased concentration of ferulic acid in maize shoots, has been reported to be involved in reducing cell elongation ultimately affecting shoot fresh mass (Uddin et al., 2014). Hura et al (2006) reported the accumulation of ferulic acid under drought stress and hypothesized that it can be involved in adaptation to osmotic stress and resulted in decrease in plant height/biomass by hampering cell elongation. Synthesis and accumulation of chlorogenic acid has been reported to be involved in protection against potentially harmful UV radiation in transgenic tomato plants (Carla Cle et al., 2008). Thus, plant protect their active source of primary metabolism from photooxidation (Niknam and Ebrahimzadeh, 2002). Alkyl derivatives of resorcinols occurs within grains, seed coat and concentrated within the cuticles of leaves (Ross et al., 2003; Ji and Jetter., 2008). Root systems of Oryza spp. exude an alkyl resorcinol mixture, an allelochemical also possessing antifungal activity (Duke., 2003; Suzuki et al., 1998). Suzuki et al (2015) reported

77 accumulation of rutin in various plant parts of buckwheat, thereby deter herbivores. Rutin and associated enzyme (Rutinosidase) are involved in alleviating environmental stress i.e, UV light, low temperature and desiccation. Quercetin derivatives have been associated with protection of chloroplasts from excess visible, light responsible for generation of 2 (1O ) singlet oxygen (Agati et al., 2007). Accumulation of Quercetin 3-O- and luteolin 7- O-glycosides in root zone salinity of Ligustrum vulgare in response to UV-B irradiance have been interpreted as their ability to quench ROS (Agati et al., 2011). Some phenolic compounds (sinapic acid, vanillic acid, gallic acid, salicylic acid, cinnamic acid, p- coumaric acid, ferulic acid, coumarin, chlorogenic acid, rutin, and morin) provoke ABA- induced stomatal closure, indicating potential role of phenolic compounds in ABA signaling under stress (Rai et al., 1986; Purohit et al., 1991; Bi et al., 2017)

Conclusions Results of this study showed that C. procera modulate growth, biochemical and physiochemical characteristics to withstand increasing salinity. Significantly similar biomass production at moderate salinity as compare to control, accompanied by the accumulation of inorganic (Na+) and organic (soluble sugars and proline) osmolytes, which contributed in lower osmotic potential. Chlorophyll fluorescence parameters like Fv/Fm and Y (NPQ) were either increased or maintained at moderate salinity. Similarly, high K+ selectivity and strong antioxidant defense (enzymatic and non-enzymatic) helped to nullify the salt induced ionic and oxidative stresses. However, at high salinity, these protections were not enough to control ROS content (H2O2) and associated damages (MDA and EL) hence, caused significant biomass losses. Therefore, growth maintenance at 100 mM NaCl appeared to be an adaptation, while high (300 mM NaCl) salinity was damaging for C. procera. These results clearly indicate that C. procera can be grown at marginal/ degraded lands affected with moderate salinity and give similar growth comparable to non-saline conditions. Furthermore, it can produce considerable amount of bioactive secondary metabolites (phenols, flavonoids, tannins, anthocyanins, betacyanins, flavonol glycosides, betacarotens etc.) with profound antioxidant activity, hence could be used for medicinal and other industrial purposes.

CHAPTER 4 EFFECT OF SALINITY ON GROWTH, ECOPHYSIOLOGY AND ANTIOXIDANT STATUS OF THESPESIA POPULNEA

Abstract

Thespesia populnea is a widely spread plant, distributed in warm tropical regions of the world and has ability to tolerate up to 300 mM NaCl (≈60% sea water salinity). The information about mechanisms responsible for its survival and tolerance are scarce. Due to high phenolic compounds with profound pharmacological activities such as antioxidant, the leaves of Thespesia populnea have important drug values. In this regard we studied the effects of salinity (0 - 300 mM NaCl) on growth, eco-physiology and antioxidant status of Thespesia populnea, growing under semi-controlled greenhouse conditions. Results showed that increasing salinity reduced plant length, stem girth and leaf area. However, plant growth in terms of root length, number of leaves and nodes, leaf moisture, leaf succulence, leaf pigments, and biomass (fresh weight and dry weights) remained unaffected at 100 mM NaCl. However, growth parameters were markedly decreased (up to 50%) at 300 mM NaCl. Survival under saline conditions resulted by decreasing water and osmotic potentials owing to accumulation of inorganic (Na+ and K+) and organic osmolytes (soluble sugars and proline). Improved selective absorption of K+ (over Na+), helped to maintain ion homeostasis. Salinity stress shifted electron flow from actual quantum yield (YII), photochemical quenching (qP) and electron transport rate (ETR) to non-photochemical quenching Y (NPQ) and Y (NO). To overcome this problem, plant improved heat dissipation Y(NO), activated antioxidative enzymes (superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase and guaiacol peroxidase) and increased secondary metabolite content (anthocyanin, betacyanin, carotenoids, flavonols and betacarotene), and constant amount of (polyphenol)which in turn maintained overall antioxidant status, at moderate salinity. Despite all these protective mechanisms, photochemical limitations led toward increased oxidative damage indicated by increasing

H2O2 and MDA at high salinity. This study indicates T. populnea appeared as potential candidates, which can be cultivated on degraded/saline lands with brackish water under moderately saline conditions without compromising their growth/ biomass and can provide similar (polyphenol; Natural antioxidants) or even higher amount of bioactive natural products (anthocyanin, betacyanin, carotenoids, flavonols and betacarotene), hence could also contribute to minimize desertification and reversing adverse global climatic change.

INTRODUCTION SALINITY AND GROWTH Soil salinity is a major factor of plant distribution due its effects on growth and survival of plant species (Jayatissa et al., 2008; Munns and Tester 2008). Some halophytes are capable of managing highly salinity are termed as “obligate halophytes” (Braun-Blanquet, 1932), while others are “facultative halophytes” which required a certain amount of salt to reach maximum growth. Growth of facultative halophytes compromised when they exposed to supra-optimal salinities (Polunin, 1960). A number of possibilities could be related to growth reduction, including less C fixation, which may alter biomass allocation between root, stem and leaf (Ball, 1988; Lovelock and Ball, 2002), fall in turgor (Clipson et al.,1985; Rozema, 1991; Balnokin et al., 2005), high concentrations toxic ions in apoplast (Harvey et al., 1981; James et al., 2006), and/or changes in cell wall elasticity (Tomos and Wyn Jones, 1982; Touchette, 2006). Halophytes are naturally equipped with certain mechanisms but all these possibilities required substantial energy (Yeo, 1983) i.e. selective ion transport (to regulate net uptake and cellular compartmentation of Na+ and Cl−), synthesis of compatible solutes and antioxidative defense budget. Accumulation of inorganic ions and osmoprotectants (proline, glycine betaine, polyphenols, soluble sugars) is also an orthodox halophyte response to cope environmental stresses (Lokhande and Suprasanna, 2012; Patel et al., 2016). IONIC CONTENT AND WATER RELATIONS Halophytes encounters with osmotic adjustment challenge when exposed to a saline medium with low water potential. High concentration of Na+ in soil disturb K+ absorption, creates K+ imbalance in chloroplast and results in photosynthesis inhibition (Sudhir and Murthy, 2004). Salt stress also affects photosynthesis through stomatal limitation (leading to poor carbon assimilation) and decrease in chlorophyll and carotenoid concentrations (Parida and Das, 2005; Koyro, 2006, Stepien and Johnson 2009, Duarte et al., 2013). Reduced chlorophyll also attributed to pigment-protein complex instability (Levitt, 2015), as well as, interference of toxic ions with the de novo synthesis of chlorophyll proteins (Jaleel et al., 2008). Halophytes achieve salt tolerance either through “osmoregulation”- developing an extremely negative osmotic potential (Karimi, 1984) or “osmoconformor”- progressive decrease of osmotic potential, strategies (Karimi, 1984; Khan et al., 1999; Khan et al., 2000a, b). Some halophytes accumulate Na+ to achieve osmotic balance or compartmentalize it in the vacuole (Lee et al., 2007). Cytoplasmic water potential on the other side of the vacuole is brought to homeostasis through synthesis and/or accumulation

82 of compatible organic solutes, such as proline, glycinebetaine, and sugars. However, high Na+ accumulation sometimes hampered the uptake and transport of K+ (Marcum, 2008; Zhou and Yu, 2009), hence creating K+ imbalance. PHOTOSYNTHETIC PIGMENTS AND CHLOROPHYLL FLUORESCENCE Salt induced photosynthetic limitations can cause over-reduction of PSII reaction centers (Redondo-Gomez et al., 2007). Low photosynthetic rates under salt stress might cause PSII photoinhibition/photoxidative damage (Osmond, 1994). Increase or unchanged chlorophyll contents (Ashraf and Harris, 2013; Acosta-Motos 2015a, b), stomatal closure, thermal energy dissipation are some of the defenses mechanisms halophytes can apply to protect photosynthetic tissues (Logan et al., 2006). Previous reports indicate that salinity tolerance may be associated with decreasing photochemical quenching (Fv/Fm, Y(II), qP) and electron transport rate (ETR), but increasing non-photochemical quenching parameters [qN, NPQ, Y(NPQ)] (Moradi and Ismail, 2007; Lee et al., 2013, Ikbal et al.,2014; Acosta-Motos 2015a,b). However, such manipulations depends on species, and quantum/ duration of applied stress. Non-photochemical quenching ensure safe heat dissipation to minimizing ROS generation which otherwise can lead to damage photosynthetic machinery (Demmig-Adams and Adams, 1992). Xanthophyll cycle is a safe way of dissipating excess excitation energy from PSII, which required conversion of violaxanthin into zeaxanthin (Zhang et al., 2012). OXIDATIVE STRESS TOLERANCE Salt induced over reduction of electron transport chain (ETC) may leads to photooxidation and oxidative damages, which can be determined through H2O2, MDA and electrolyte leakages (Foyer and Noctor, 2005; Takahashi and Murata, 2008; Foyer and Noctor, 2009; Rochaix, 2011; Koyro et al., 2013; Nishiyama and Murata, 2014). Tolerant plants are equipped with special antioxidative defense, either enzymatic/non-enzymatic or both mechanisms to alleviate ROS. Salt-tolerant species have different strategy compared to salt-sensitive species. Salt tolerance species either show coordinated up-regulation of the antioxidant system (Gomez et al., 1999; Rubio et al., 2009) or constitutively high level of antioxidant system (Gueta-Dahan et al., 1997; Lopez-Gomez et al., 2007), while that of salt-sensitive species show an unchanged response or decreased antioxidant enzyme levels than salt-tolerant species (Hernandez et al., 1995, 2003). Salt-tolerant plants overcome oxidative stress by modulating AsA-GSH cycle components (ascorbate per oxidase, guaicol peroxidase and glutathione reductase) or superoxide dismutase (SOD) regulation as enzymatic antioxidant system (Mittova et al., 2003, 2004). Salt sensitive plants vary

83 ascorbate and glutathione levels (non-enzymatic antioxidant) much more than tolerant ones while, substrates provide further defense by scavenging damaging radicals (Hernandez et al., 2000). In addition to these, a number of phenolic antioxidants play a crucial role in quenching ROS and maintaining cellular redox status hence, resist any change in the direction of physiological metabolism imposed by salt stress. For instance, salt induced hyper accumulation of polyphenol and their subsequent antioxidant protection is reported from several halophytes such as Cakile maritima, Cynara cardunculus, Phrigmitis karka, (Ksouri et al., 2007; Jelali et al., 2011; Rezazadeh et al., 2012; Taarit et al., 2012; Valifard et al., 2014; Abideen et al., 2015; Caliskan et al., 2017; Rebey et al., 2017). THESPESIA POPULNEA Thespesia populnea (Linn) Sol. ex Correa (Malvaceae) is a fast growing, large evergreen tree found in regions of West Bengal to Peninsular India and the Andmans. It also known as Indian tulip. It is also grown as a road side tree in tropical and sub-tropical regions and attain height up to 10 m. T. populnea requires full sunlight and can tolerates drought and saline conditions. Due to a variety of bioactive medicinal components, various parts of T. populnea have been used in traditional systems of a number of Asian and African countries (Savithramma et al., 2017). Different parts of this plant are used to treat skin diseases, ringworm, hepatic diseases, relief inflamed, swollen joints and as a tonic. Its biological and pharmacological effects including anti-hyperglycemic, anti-bacterial, antisteroidogenic, anti-diabetic, antiinflammatory, antioxidant, purgative and hepatoprotective activities (Kavimani et al., 1999; Kumar et al., 2009; Suvarna et al., 2018). These activities are related to its bioactive constituents including phenols, flavnoids, sesquiterpenoids, tannin, saponins, alkanes, essential oils, sugars, fatty acids and anti-oxidants and alkaloids (Rajamurugan et al.,20013; Sangeetha and Vedasree, 2012; Belhekar et al., 2013; Suvarna et al., 2018). Beside medicinal and pharmacological importance, T. populnea can withstand drought, direct winds from the ocean, root zone salinity and aerial salinity through coastal salt spray (Bezona et al., 2009; Gohar et al., 2018) and hence, could be cultivated on saline/ marginal lands to produce bioactive raw material for medicinal and chemical usages. However, information is scarce about the mechanisms involved in the salinity tolerance of this plant, which limits its proper propagation and cultivation. Understanding salt tolerance mechanism of mangrove associate T. populnea, can play a major role in stabilizing crop performance under saline conditions. The main objective of this study were to determine the effect of NaCl on

84 growth, chlorophyll fluorescence, leaf pigments, ion regulation, water relations, and enzymatic and non-enzymatic antioxidant defense of T. populnea. Salt induced augmentation in phenolic metabolites and antioxidant activity was also studied.

Material and methods Growth experiment of Thespesia populnea was carried out in a semi-controlled greenhouse, at Institute of Sustainable Halophytes Utilization, University of Karachi, using, the freshly collected seeds from their natural habitats. Preparing and sowing of seed Wrinkle free fresh intact seeds were selected for growth experiment. Seeds of identical size and colour were manually scarified using sand paper. Fifty seeds were sown in plastic trays and left to germinate in greenhouse under ambient light (1133 µmol m−2s−1), temperature (25/15 ± 2 ºC) and humidity (~70%). When seedlings reached to 2 leave stage, they were transplanted to plastic pots (45 cm in diameter and 55 cm height), containing sand and silt in ratio of 1:1. Each pot contained 3 plants. Healthy seedlings of equal size and vigor were selected and grown in Hoagland’s nutrient solution (modified after Epstein, 1972) using sub-irrigation method. After 6 weeks of seedling establishment, salt treatment was started using different concentrations of sodium chloride (0, 100, and 300 mM). To prevent osmotic shock, salinity treatment was started gradually (at rate of 25 mM daily). Plants were treated with salinity for 6 months, after that non-destructive measurements were taken and plants were harvested for further morphological, physiological, biochemical and phytochemical analyses. Growth parameters, leaf area, moisture percent and succulence Referred to Chapter 3 materials and methods section. Water relations Referred to Chapter 3 materials and methods section. Diurnal water relations Referred to Chapter 3 materials and methods section. Proline determination Referred to Chapter 3 materials and methods section. Soluble sugar determination Referred to Chapter 3 materials and methods section. Cation Contents Referred to Chapter 3 materials and methods section. Determination of photosynthetic pigments Referred to Chapter 3 materials and methods section. β-carotene and Lycopene contents Referred to Chapter 3 materials and methods section.

Anthocyanin, β-cyanin and Flavnols Referred to Chapter 3 materials and methods section. Chlorophyll Fluorescence Measurements Referred to Chapter 3 materials and methods section. Electrolyte Leakage Referred to Chapter 3materials and methods section. Oxidative stress markers Referred to Chapter 3 materials and methods section. Antioxidant enzyme activities Referred to Chapter 3 materials and methods section. Antioxidant substrate Referred to Chapter 3 materials and methods section. Determination of Polyphenol Referred to Chapter 2 materials and methods section. Determination of Total Tannins Referred to Chapter 3 materials and methods section. Determination of Antioxidant Assays Referred to Chapter 2 materials and methods section. High performance liquid chromatographic (HPLC) analyses Referred to Chapter 3 materials and methods section. Stomatal morphometry Stomatal morphometry include counting number of stoma, opening, length and area or upper and lower surfaces of fully emerged leaves by peel off method Hilu and Randall (1984). Briefly nail varnish was applied with the brush abaxial and adaxial surfaces of fully expanded leaf from third node avoiding midrib avoided. Varnish was left to dry 5-10 minutes, then peeled off. Placed the peeled off piece on the slide and apply the glycerol. Count the number of the stomata at 100 x under microscope and calculated length, opening and area of stomata by using ImageJ software (https://imagej.en.softonic.com/download, accessed 23 March 2018). Statistical analysis All statistical analyses including One way ANOVA was performed by using SPSS (version 16) and graphs were plotted using Sigma plot (version 12.5). All results are expressed as means (± standard error) of a minimum of 3 biological replicates with up to 5 technical

87 replicates where necessary. Bonferroni test was carried out to determine if significant (P ≤ 0.05) differences existed among means.

Results Plant growth and Morphology T. populnea plant showed less sensitivity to (100mM) moderate salinity (Fig.4.1). Height showed slight decline under moderate salinity followed by 39% decline under high salinity (Fig.4.1B). Whole plant fresh weight (FW) and dry weight (DW) were slightly decreased under moderate salinity, followed by significant decline at high salinity (300 mM; Fig.4.2). Substantial increased in root/shoot ratio (1.7 folds) was found at 300 mM NaCl (Fig.4.2). There was no significant difference in number of leaves and nodes under non-saline and moderate salinity, followed by decrease in number of leaves (54%) and nodes (58%) under higher salinity (Fig.4.3). Leaf area and stem girth decreased linearly with increasing salinity (Fig.4.3). However, leaf moisture and leaf succulence remain unchanged throughout salinity treatment (Fig.4.3). Water relations Leaf water potential (Ψw) and osmotic potential (Ψs) significantly decreased with increasing NaCl concentrations (Fig.4.4). Leaf turgor (Ψp) maintained under moderate salinity, however significantly declined at 300 mM NaCl treatment (Fig.4.3 and 4.4). Water potential became more negative with increase in sunlight and reached their lowest values at noon in 300 mM NaCl treatment (Fig.4.5). Photosynthetic pigments Total chlorophyll, chl a, chl b and carotenoid contents remained unchanged from 0 to 100 mM NaCl, however increased by 1.4, 1, 0.85, and 1.2 folds respectively at 300mM NaCl. Chl a/b and chl/caro increased linearly with increasing salinity, the ratios were increased by 2 and 1.6 folds at higher salinity, respectively (Fig.4.6). Anthocyanins, betacyanins, flavonolglycoides, betacarotenes and lycopenes showed 135, 0.8, 0.5, 1.2 and 2.2 fold increase at 300mM NaCl, respectively (Fig.4.7).

Oxidative Damage (Malondialdehyde (MDA) and Hydrogen peroxide (H2O2) contents

Hydrogen peroxide H2O2 and malondialdehyde (MDA) contents are potent stress indicators at drought and excessive salinity. All stress indicating markers remain unchanged at moderate salinity but increased at high salinity (Fig.4.8). Effect of salinity on soluble sugar and Proline content There was a progressive increase in soluble sugar content with increasing salinity, it increased by 33% and 55% at moderate and high salinity, respectively as compare to non- saline controls. Proline content increased by around one fold and four folds under moderate salinity and higher salinity, respectively (Fig.4.9).

PAM Data PSII maximum quantum efficiency (Fv/Fm) increased significantly at moderate salinity and decreased at higher salinity as compare to control. Actual yield (PS II) showed significant decline at both salinities (Fig.4.10). Non-photochemical quenching (NPQ) increased by 1.7 times at moderate salinity but decreased equivalent to control at high salinity (Fig.4.9). Photochemical quenching qP increased 1.1 times at moderate salinity, while it dropped at higher salinity. Heat dissipation decreased at moderate salinity, but increased by 10 to 20% at high salinity as compare to control. There was a significant decline in ETR with increasing salinity. Morphometric measurements of the stomata showed absence of stomata on adaxial (upper) surface. Moderate salinity did not affect number of stomata on lower surface however, a 2.6 fold decrease was observed at higher salinity. Salinity did not affect on the area of stomata (Table.4.3), however the number of stomata were significantly reduced at higher salinity than control. Cation (Na+ and K+) accumulation Results showed variability in cation accumulation along salinity gradient and the organ of T. populnea (Fig.4.11). Na+ and K+ contents increased in both shoots and roots as NaCl concentration increased from 0 to 300 mM NaCl. Sodium concentration was higher than K+ in all saline treatments (Fig.4.11). Exceptionally, stem showed significant decline in Na+ content under moderate salinity. Moderate salinity had no influence on leaf and root K+, which increased significantly at 300 mM NaCl, while significant decline was observed in root K+ (Fig.4.11). Root Na/K ratio increased significantly with increasing salinity, while in leaves it increased slightly under moderate salinity but decreased at high salinity (Fig.4.11). Selective absorption of K+ over Na+ increased linearly with increasing salinity, while selective transport increased at higher salinity (Fig.4.12) Antioxidant enzyme activities and Antioxidant substrate Salinity linearly increased the activities of antioxidant enzymes SOD, APX, and GPX, while CAT and GR, increased at high salinity (Fig.4.13). Total ascorbate and reduced from of ascorbate (AsA) content decreased with increasing salinity, while, oxidized from of ascorbate (DHAsA) remain unchanged (Fig.4.14). AsA/DHAsA ratio decreased linearly with increasing salinity (Fig.4.14).

Total phenolic, flavonoid Proanthocynadin content and Antioxidant activity of T.populnea In general, moderate salinity did not affect on the contents of secondary metabolites however, all tested metabolites significantly reduced under high salinity (Fig.4.15). Leaf contained the highest contents of total phenols (TPC), flavonoids (TFC), proanthocynadins (PC) and tannins (TTC) compared to stem and root. Roots showed a significant decrease in TFC, and PC at moderate salinity, which remained unchanged at high salinity (Fig.4.15). Whereas, root TPC decreased linearly, while TTC remain unaffected throughout the salinities. Antioxidant activity was tested using DPPH, ABTS and TAC (Fig.4.16). Generally, in all antioxidant measurments, the activity of leaf and stem remain unchanged at moderate salinity and declined significantly at higher salinity. For root, antioxidant activity either did not change (ABTS and TAC) throughout the salinities or decreaed (DPPH) at high salinity. In general, the highest activity in descending order is as follows: leaf > stem > root. Phenolic profile using HPLC HPLC was used to analyze the metabolism of phenolic compounds in salt stressed T. populnea leaves. Phenolic composition of T. populnea hydrolysed extracts were determined after using reference standards of 1 Pyrogallol; 2 Gallic acid; 3 Resorcinol; 4 Pyrocatechol; 5 Catechin; 6 Hydroxybenzoic acid; 7 Chlorogenic acid; 8 Vanillic Acid; 9 Caffeic acid; 10 Syringic acid; 11 Coumaric acid; 12 Ferulic acid; 13 Sinapic acid; 14 Rutin; 15 Trans-cinnamic acid; 16 Quercetin; 17 Ellagic acid (Fig.4.17). Total content of these phenolic acids fractions (determined using HPLC technique) in T. populnea leaves was 107.37 μg g-1 DW under non-saline condition, which decreased to 55.62 μg g-1 DW and 38.11 μg g-1 DW at moderate and high salinity, respectievely. It is noteworthy that Resorcinol dominated among all phenolic compounds at both non-saline and high saline conditions, while Catechin dominated under moderate saline condition. Besides this other polyphenols (Pyrocatechol, Catechin) accumulation remain unchanged under moderate salinity Chlorogenic acid, Vanillic acid, Caffeic acid, Syringic acid, Coumaric acid, Rutin and Cinnamic acid decreased under both moderate and high salinity treatments (Table.4.4).

100 Shoot a Root 80 b

60 c 40

Plant length(cm) Plant 0

-20 a a a -40 0 100 300 NaCl (mM)

Figure. 4.1: (A) Comparison of T. populnea plants grown under different (mM) NaCl treatments for 35 days under green net house. (B) Changes in height of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

200 a

) Leaf -1 a Stem Root 150 Ratio

100 b

Freshweight (g plant 0

a 0.6

) 60

-1 a B 0.5

Root / Shoot Root

40 0.4 A A 0.3 b 20 0.2

weightDry (g plant 0.1

0 0.0 0 100 300

NaCl (mM) Figure. 4.2: Changes in plant fresh weight and plant dry weight of (leaf, Stem and root), Root shoot ratio of T.populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

80 3000 a

) a a

-1

Leaves (plant Leaves 60

plant b

2 2000 40 b

1000 c -1 20 )

Leaf(cm area 0 0 a a

Nodes (plant Nodes ) 0.24 b a 45

-1

c 0.16 30 b

-1

0.08 15 )

Girth (cm Girth plant

0.00 0 H (g Succulence a 90 a 3 a a a a 75

60 2

45 2

O g O

30 1 -1

Leaf moisture (%) 15 DW) 0 0 0 100 300 0 100 300 NaCl (mM) Figure. 4.3: Changes in Leaf area, number of leaves, Girth (cm plant-1), leaf moisture%, and succulence of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

2 WP OP a TP 1 a b 0

-1

a -2 b Waterrelations (Mpa) a -3 b c c

-4 0 100 300

NaCl (mM)

Figure. 4.4: Effect of different NaCl treatments (0, 100, 300mM) on water potential, osmotic potential and turgor potential (MPa) of T. populnea leaves.

-0.5

-1.0 a

-1.5 a

-2.0 b b

Waterpotential (Mpa) -2.5 Pre Dawn Noon c c -3.0 0 100 300

NaCl (mM)

Figure. 4.5: Effect of different NaCl treatments (0, 100, 300mM) on Pre-dawn and noon water potential (MPa) of T. populnea leaves.

1.5 0.8

Chlorophyll b ( b Chlorophyll ) b -2 b 0.6 gcm 1.0

0.4

0.5 cm g a a 0.2

a a -2

)

Chlorophyll( a

0.0 0.0

Carotenoides ( Carotenoides

) -2 b b 2.0 1.5

gcm 1.5 1.0 1.0

g cm g 0.5 0.5 a a a a

-2

)

Chlorophyll / Carotenoides Chlorophyll Totalchlorophylls ( 0.0 0.0 c 4 c 2.0

3 1.5

2 b b 1.0 a a

Chlorophylla/b 1 0.5

0 0.0 0 100 300 0 100 300

NaCl (mM)

Figure. 4.6: Changes in chlorophyll a, chlorophyll b, total chlorophyll, chlorophyll a/b ratio, carotenoid and chlorophyll/carotenoid ratio of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations

225 1.2 c c

)

Betacyanins ( Betacyanins

-2

gcm 150 0.8 b b

75 0.4 cm g a a

-2

)

Anthocyanins( 0 0.0

Betacarotene ( Betacarotene

)

-2 c c 1.5 gcm 0.4 1.0

b cm g 0.2 b a 0.5

-2

a )

0.0 0.0

Flavonolglycosides( c 0 100 300

) 2.7 -2 NaCl (mM)

gcm 1.8 b 0.9 a

Lycopene( 0.0 0 100 300

NaCl (mM)

Figure. 4.7: Changes in anthocyanins, betacyanin, flavnolglycosides, betacarotene and lycopene contents of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

200 b 150 a

FW) -1 a

M g M 100

(

2 2

O 2 50

0 b

FW)

-1 a 60 a

M g M

MDA(

0 0 100 300

Figure. 4.8: Changes in H2O2 and MDA of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

2.0 c

1.5

FW)

-1

1.0 b

0.5 a

Proline(mgg

0.0 b

FW) b

-1 60 a

Solublesugars (mg g 0 0 100 300

NaCl (mM)

Figure. 4.9: Changes in Proline and soluble sugar of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

100

0.28 b 0.78 a a

Y(II) 0.21 0.75 a b Fv/Fm c

0.72 0.14

0.6 0.7 b a 0.6 0.5 b Y(NPQ) a a 0.5 qP c 0.4 0.4

0.3 0.3 c a 0.42 70 a b Y(NO) 0.36 56 b

ETR c 0.30 42

100 300

Figure. 4.10: Changes in Fv/Fm, qP, ETR, Y (II), Y(NPQ), Y(NO) of T. populnea plants leaves treated with 0, 100 and 300 mM NaCl concentrations.

101

Root Stem Leaf b

DW) 1.2 b b ab

-1 0.9 a a a 0.6 a b

(mole Kg

+ 0.3

Na 0.0 0.8 b

DW)

-1 0.6 a a a a 0.4 b b b

(mole Kg 0.2 a

K 0.0 a 6 c 5 4 b b 3 b b a Na+/K+ a c 2 1 0 0 100 300 0 100 300 0 100 300

NaCl (mM) Figure. 4.11: Changes in Na+ (mole kg-1 DW ) K+ (mole kg-1 DW) and Na+/K+ in terms of dry weight of T. populnea plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations.

102

SA c 25 b 20

10 5 a 0 ST1 b b 2.0

1.5

selectivity

+ 1.0 a 0.5

over Na

K 0.0 4 ST2 b

2 a a 1

0 0 100 300

NaCl (mM)

Figure. 4.12: Changes in K+ over Na+ selectivity Selective absorption from medium to root (SA), Selective transport from root to stem (ST1) and selective transport from stem to leaves (ST2), of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations

103

40 7 b c 6 (U mg SOD 30 b 5

protein) a

-1 4

20 a a -1

3 protein) 2 10

CATmg (U 1 0 0 450 b (U mg GR c 10 a b a 8

protein) 300

-1 a 6 -1

protein)

150 4

APXmg (U 2

0 0 0 100 300 60 c NaCl (mM)

protein) 45 -1 b 30 a

GPXmg (U

0 0 100 300

NaCl (mM)

Figure. 4.13: Changes in CAT, SOD, APX, GR and GPX of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

104

Total AsA AsA DHAsA Ratio 2.5 A 2.5 2.0 B

AsA / DHAsA AsA a C 2.0

-2 1.5 ab b a 1.5 1.0 b c

mMolcm 1.0 a a a 0.5 0.5

0.0 0.0 0 100 300 NaCl (mM)

Figure. 4.14: Changes in ascorbic acid content and AsA/DHAsA of T. populnea plants treated with 0, 100 and 300 mM NaCl concentrations.

105

Leaf Stem Root a

(mg GAE g GAE (mg TPC

) a 30 30

-1 a a 20 20 b a b

-1

10 10 ) TPC (mg GAE g b c

0 0 a a a

(mg QE g QE (mg TFC ) 3 3

-1 b a a b 2 2 b b

-1

) TFC (mgQE g 1 1

0 a a 0

a a g CE (mg PC

) 15 15 ab

-1 b 10 10 b

b b -1

) PC (mgCE g 5 5

0 a 0 0.8 0.8

(mg TAE g TAE (mg

)

-1

0.6 0.6 TTC b 0.4 a a 0.4

c a -1

)

TTC (mg TAE g 0.2 b a a 0.2

0.0 0.0 0 100 300 0 100 300 0 100 300

NaCl (mM)

Figure. 4.15: Changes in total phenolic content, Total flavonoid content, Proanthocynadin content and Total tannin content of T. populnea plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations.

106

Leaf Stem Root A B C

80 a a 80 %) ( Inhibition

a a DPPH 60 b a 60 a

DPPH 40 40 b b

( Inhibition ( %) 20 20

0 0 D E F

(mM Trolox g (mM Trolox

) 100 100

-1

a a ABTS 80 a a a a 80 b a a 60 b 60

ABTS

40 40 -1

)

(mM Trolox (mM g 20 20 0 G H I 0 50 40

( ) a a

gAsAg -1 40 30 b TAC a 30 a a a a TAC 20

-1

gAsAg

20 b ) ( 10 10 0 0 100 300 0 100 300 0 100 300 NaCl (mM)

Figure. 4.16: Changes in DPPH radical scavenging activity (Inhibition %), ABTS anti- radical activity (mM Trolox g-1) and Total antioxidant activity (µg AsAg-1) of T. populnea plants leaves, stem and root respectively treated with 0, 100 and 300 mM NaCl concentrations.

107

05: Catechin 01: Pyrogallol 02: Gallic acid 03:Resorcinol 04: Pyrocatechol

06:Hydroxybenzoic 10: Syringic acid 07: Chlorogenic 08: Vanillic 09: Caffeic acid acid acid acid

11: p- Coumaric 15: Trans-cinnamic 12: Ferulic acid 13: Sinapic acid 14: Rutin acid acid

16: Quercetin 17: Ellagic acid Figure. 4.17. HPLC chromatogram of (a) standard phenolic compounds (1 Pyrogallol; 2 Gallic acid; 3 Resorcinol; 4 Pyrocatechol; 5 Catechin; 6 Hydroxybenzoic acid; 7 Chlorogenic acid; 8 Vanillic Acid; 9 Caffeic acid; 10 Syringic acid; 11 Coumaric acid; 12 Ferulic acid; 13 Sinapic acid; 14 Rutin; 15 Trans-cinnamic acid; 16 Quercetin; 17 Ellagic acid) and (b) their structures used in this study.

108

Figure. 4.18. HPLC chromatograms showing profile of different phenolic acids in hydrolysed leaf extracts of T. populnea treated with 0, 100 and 300 mM NaCl concentrations.

109

Table.4.1. Water potential at full turgor (ѰW0), Water potential at turgor loss point

(ѰWTLP) and Bulk elasticity of cell wall (Ɛ Mpa) of Thespesia populnea leaves.

Treatment ѰW0 (Mpa) ѰWTLP (Mpa) Ɛ Mpa (Mpa)

Control -0.857 ± 0.02 a -2.2 ± 0.05 a 5.96 ± 0.4 a

100 mM -1.12 ± 0.72 b -3.2 ± 0.08 b 6.03 ± 0.3 b

300 mM -2.3 ± 0.05 c -3.8 ± 0.03 b 10.76 ± 0.9 c

Table.4.2. % Contribution to osmolality of organic and inorganic osmolytes of Thespesia populnea leaves. Inorganic contribution Organic contribution Treatment Na% K% CHO % Proline % Control 18.9 ± 0.3 a 10.9 ± 0.2 a 18.3± 2.2 a 0.49 ± 0.01a 100 mM 22.7 ± 0.4 b 9.2 ± 0.6 b 19.8 ± 2.3 b 0.3 ± 0.02 b 300 mM 20.2 ± 1.4 c 15.5 ± 0.3 c 20.7 ± 2.4 c 1.7 ± 0.01 c

Table.4.3. Number and area of stomata on upper and lower surface of Thespesia populnea leaves. Number of stomata per unit area Area of stomata µm2 Treatment Adaxial Abaxial Abaxial Control none 3 ± 4.68111E-16 a 473.50 ± 75.37 a

100 mM none 3 ± 0.580641482 a 483.30 ± 97.83 b

300 mM none 1± 4.60853E-16 b 468.12 ± 55.24 ab

110

Table.4.4. Phenolic composition (µg g-1 DW) in leaves of T. populnea treated with 0, 100 and 300 mM NaCl concentrations

Retention NaCl (mM) Compounds time 0 100 300

Gallic acid 10.84 0.98 ± 0.01a 0.47 ± 0.01b 0.97 ± 0.02a

Resorcinol 14.91 37.75 ± 2.14a 7.58 ± 0.43b 17.74 ± 0.87c

Pyrocatechol 19.06 11.32 ± 1.45a 8.33 ± 0.57a 5.80 ± 0.57b

Catechin 24.60 12.96 ± 1.11b 17.31 ± 0.98a 1.30 ± 0.01c

Chlorogenic acid 25.31 5.46 ± 0.54a 1.71 ± 0.01b 1.76 ± 0.02b

Vanillic acid 28.52 10.54 ± 0.87a 3.64 ± 0.08b 2.38 ± 0.02b

Caffeic acid 29.91 4.95 ± 0.92a 2.50 ± 0.04b 0.99 ± 0.01b

Syringic acid 31.16 2.15 ± 0.24a 1.28 ± 0.08b 0.85 ± 0.02b

Coumaric acid 37.13 7.36 ± 0.31a 4.73 ± 0.14b 2.40 ± 0.01b

Rutin 50.21 7.65 ± 0.22a 3.50 ± 0.19b 1.67 ± 0.01b

Cinnamic acid 57.86 6.25 ± 0.14a 4.56 ± 0.15b 2.26 ± 0.02b

107.37 ± 7.45a 55.62 ± 4.87b 38.11 ± 4.19c

111

Discussion Growth parameters Salinity decreased growth of T. populnea however, at moderate salinity did not affect FW, DW, number of leaves, nodes and leaf moisture (Fig.4.2 and 4.3). Similar results were reported in T. populnea (Qiu et al., 2011; Gohar et al., 2018), Dalbergia sissoo and Eucalyptus camaldulensis (Rawat and Banerjee, 1998), cotton (Meloni et al., 2001), Brassica napus (Qaderi et al., 2006). Acacia nilotica and Dalbergia sissoo (Minhas et al., 1997), Plantago cressifolia (Vicente et al., 2004), Prosopis juliflora (Khan et al., 1987) P. argentina and P. alpataco (Villagra and Cavagnaro, 2005) and Salvadora persica (Alshammary, 2008). In 300 mM NaCl plant managed to survive by reduction in plant height, stem girth, and biomass (fresh and dry) which resulted in over 50% of over-all growth retardation. Plant did not change its number of leaves but reduced leaf size (leaf area), a possible strategy to combat moderate salinity by maintaining photosynthetic area, manage energy expenditure and reduce transpirational water loss. Reduction in transpiration stream might tend to reduce toxic ions to the roots, and keeping away from the photosynthetically active tissues (Munns and Tester, 2008; Acosta-Motos et al., 2015). However, high salinity adevrsly affected plant growth by decreasing both number of leaves and leaf area (Flanagan and Jefferies, 1988; Gohar et al., 2017). Na+ and K+ accumulation and water relations Salinity tolerance in plants is often associated with salt exclusion, extrusion or sequestration in the vacuoles. Besides that salt stress modulates uptake and transport of other essential minerals which is an important aspect of salt tolerance (Taibi et al., 2016). Salt tolerant plants accumulate Na+ in their shoots contrast to sensitive plants. T. populnea accumulated Na+ in roots and leaves, this accumulation of Na+ was found to be associated with an improvement of water content for (moderate salinity) 100 mM, as found for Gossypium hirsutum (Leidi and Saiz, 1997). Under high salinity, T. populnea showed "excluder" behavior as indicated by decreased leaf Na+ content suggesting its re-transfer towards the roots by phloemic tissue. Similar results were obtained in soya bean (Wieneke and Lauchli, 1980), Acacia auriculiformis and Zea mays (Lohaus et al., 2000). Contrary to our results a salt sensitive wheat variety (Blue Silver) exhibited greater Na+ excluder ability at low salinity than high salinity (Kamboh et al., 2002). Higher accumulation of Na+ is generally associated with membrane damage (lipid peroxidation) that leads to the generation of MDA and loss of essential electrolytes (Penella et al., 2016). T. populnea accumulated alomost similar quantity of Na+ in both moderate and high salinities which

112 suggest that membrane damage at high salinity (higher levels of MDA, and EL) were not due to Na+ toxicity. Increasing amount of Na in growth medium affected the levels of root K+ indicating a typical antagonism between Na+ and K+ similar as that of previous field study on T. populnea (Kotmire,1983) Simmondsia chinensis (Hassan and Ali., 2014), and maize (Alberico and Cramer, 1993; Azevedo and Tabosa, 2000). The maintenance of K+ requires support from different organs which can be achieved by a good selectivity. Regulation of Na+ and K+ homeostases helps to maintain a sufficient uptake of K+ in order to maintain a high cytosolic K+/Na+ ratio (Fig.4.11, 4.12), which is an important determinant of salinity tolerance (Abideen et al., 2014). Water relations Mangroves and mangrove associates manage to obtain desalinated water by paying metabolic cost, besides that they possess certain xeromorphic features to conserve water (Saenger, 2013). Presence of thick multilayered upper epidermis with thick waxy cuticle and dense layer of scales on lower epidermis (Heritiera littoralis, Camptostemon spp, Thespesia populnea) to cover salt gland and stoma are considered to curtail water loss (Saenger., 2002). In controlled greenhouse experiment T. populnea showed significant decrease in water potential with increasing salinity. Similar results were observed in T. populnea (Gohar et al., 2018), Spartina maritime, S. densiflora, Arthrocnemum perenne, A. fruticosum (Nieva et al., 1999), Suaeda fructicosa (Khan et al., 2000), Plantago coronopus (Koyro, 2006), Prosopis Argentina and P. alptaco (Villagra and Cavagnaro, 2005), Pistacia lentiscus L. (Cristiano et al., 2016). T. populnea showed significant variations in diurnal water potential (reduced water potential at noon) indicates a drought avoiding strategy by quick uptake of water from the soil (Salleo, 1983). Plants which such a strategy tend to spend more water with have less elastic cell walls and possess greater fluctuations in diurnal water potential. Salinity stress leads to osmotic and ionic imbalance, which ultimately leads to nutrient deficiency and metabolic changes (Flowers and Colmer, 2008; Krasensky et al., 2012). Halophytes compartmentalize toxic ions in vacuoles and their affect is counter balanced in cytosol by the production and accumulation of several organic osmolytes (Zhu, 2007; Flowers and Colmer, 2008; Shabala and Mackay, 2011; Rahman et al., 2013). T. populnea showed a linear decrease in leaf osmotic potential when exposed to increasing NaCl concentrations, indicating an ‘osmoconformor strategy’ (Khan et al., 2000). Reduced osmotic potential with increasing medium salinity enables plant to maintain water potential and decrease cell wall rigidity (Martinez et al., 2004; Verslues et al., 2006) as

113 observed in Haloxylon stocksii (Ehsen et al., 2017), Atriplex nummularia (Lins et al., 2018), and Zygophyllum propinquum (unpublished data). In order to achieve osmotic adjustment, most halophytes prefer to increase leaf osmolality as compare to increase leaf succulence (Koyro, 2006; Gorai et al., 2010; Rozema and Schat, 2012). Similar to our results Gohar et al., 2017 reported water potential of T. populnea decreased with increase in the salinity. T.populnea showed typical mangrove xeromorphic strategy, i.e. increase in leaf succulence was accompanied with increased salt accumulation (Na+ and K+). This may be associated with dilution effect of ions, a common strategy of some halophytes like Rhizophora (Bowman, 1921) and Sonneratia (Walter and Steiner, 1936). Water deprivation may include drought avoidance (e.g. changes in leaf area, stomatal conductance and leaf orientation) and/or drought tolerance (maintaining cell turgor through osmotic adjustments or cell wall elasticity) (Touchette et al., 2006). Salinity affects cell wall properties and leaf turgor thereby affecting total leaf area (Franco et al., 1997; Rodriguez et al., 2005). Bulk modulus elasticity of cell wall increased slightly under moderate salinity but increased by 50% under high salinity leading to rigid cell walls (Table.4.1), however leaf turgor was maintained under moderate salinity but decreased significantly under high salinity, (Fig.4.4), which ultimately decreased leaf area. T. populnea appeared to possess drought tolerant strategy by maintain turgor as well as increasing cell wall elasticity under high salinity. Halophytes use Na+ and Cl– as cheap osmoticum to reduce high energy cost of synthesizing organic osmolytes (Wang et al., 2004). For instance, Atriplex halimus, Atriplex canescens, Kochia scoparia, Nitraria tangutorum, Sesuvium portulacastrum, Suaeda salsa, and Zygophyllum xanthoxylum resist physical and physiological water deficit by accumulating inorganic ions as osmolytes (Glenn and Brown, 1998; Martinez et al., 2004; Wang et al., 2004; Yan et al., 2013). Similarly, highly salt tolerant halophytes like Halosarciaper granulata, Sueada fruticosa, Gypsophila oblanceolata and Salicornia dolichostachya also use the similar strategy (Short and Colmer, 1999; Hameed et al., 2012; Katschnig et al., 2013). Lower osmotic and water potentials of T. populnea leaf could result from salt or organic solute accumulation under saline conditions (Munns, 2002). Osmotic contribution of inorganic content of T. populnea at high salinity was ~35 % (Na+ 20.2% and K+ 15.5%). Whereas, at this salinity, organic osmolytes like soluble sugars and proline contributed 20.7% and <1.7% in overall osmotic potential, respectively (Table.4.2). Accumulation of such a high amount of soluble sugars has been attributed with impaired carbohydrate utilization (Munns and Termaat, 1986). This study indicated 51% increase in total soluble sugars, which appeared to be a

114 reason of reduced growth at high salinity. Similar results were obtained in T. populnea (Gohar et al., 2018), Atriplex halimus (Nedjimi and Daoud, 2006), Sesbania grandiflora (Dhanapackiam and Ilyas, 2010) and as contributor to osmotic adjustment under salinity and water stress (Hassine and Luttus, 2010). Similarly, proline also increased around 4.3 fold and 4 folds under moderate and higher salinity, respectively, which showed consistency with previous study on T. populnea (Qiu et al., 2011). Leaf pigments and chlorophyll fluorescence Salinity stress is often associated with decrease in chlorophyll content in many halophytes, some contrasting reports indicate (Sobrado et al., 2005; Stefanov et al., 2016) increased chlorophyll under salinity. T. populnea showed unchanged chlorophyll (a, b and total) at moderate salinity, but it was increased sharply at 300 mM NaCl (Fig.4.5). Chlorophyll a/b and chlorophyll/carotenoid ratios showed transient increase with salinity. In accordance with our results, chlorophyll a predominated over chlorophyll b in same plant under salinity (Camejo et al., 2005). Increased number of thylakoids per chloroplast /and mesophyll thickness both could contribute in higher chlorophyll content of T. populnea at 300 mM NaCl (Pompeiano et al., 2016). Chlorophyll enrichment helps plant to increase light absorption, in order to retain photosynthesis rate, while increased carotenoid content might prevent formation of singlet oxygen in high salinity plants (Asrar et al., 2017). Salt induced increase in chlorophylls and carotenoids was reported in some other halophytes like Zoysia japonica (Pompeiano et al., 2014); Paspalum vaginatum (Pompeiano et al., 2016); Paspalum scrobiculatum (Shonubi et al., 2007). However high chlorophyll content, under high salinity coupled with high light intensity may lead to overflow of electrons through photosystems, can magnify risk of photoinhibition and oxidative stress (reflected in higher contents of MDA and H2O2). This may lead to provoke antioxidant defence mechanisms depending under stress. In T. populnea, Fv/Fm showed transient increase with increasing salinity. This indicates an improved or maintained PSII efficiency at moderate and high salinity, respectively. Similar results were observed in S. europaea (Fan et al., 2011), Suaeda salsa (Lu et al., 2003) and Arthrocnemum macrostachyum (Redondo-Gomez et al., 2010). Higher photochemical efficiency enhances energy capturing and protects photosystem II from reactive oxygen species (ROS) injury. Increased NPQ of T. populnea indicates a photo-protective streategy under high salinity. It also safeguard thylakoid membrane from oxidative damage due to over production of ROS by triplet chlorophyll during thermal dissipation of excess energy (Azzabi et al., 2012). Excessive energy has negative impacts

115 on energy trapping either by dissipating as heat/fluorescence or damaging D1 protein (Moradi and Ismail, 2007; Kreslavski et al., 2013). Salt stressed plant efficiently regulates Y (NPQ) and non-regulated process Y (NO) to dissipate heat (produced by absorbing excessive light energy) and reduce quantum yield loss. These results are agreement with the chlorophyll fluorescence responses of other halophytes such as Paspalum paspalodes, Paspalidium geminatum (Moinuddin et al., 2017). Xanthophyll cycle and improved carotenoid content are well known heat dissipation mechanisms under salinity (Moinuddin et al., 2017; Pompeiano et al., 2017). Exposure to high salinity also increased the risk of photorespiration as sinks for excessive excitation energy (Voss et al., 2013). However, coordination between photochemistry and alternate electron sink (xanthophyll cycle, photo-protective compound carotenoids, and photorespiration) is necessary to avoid chronic photo-damage in plant (Moinuddin et al., 2017; Pompeiano et al., 2017). Maximum quantum efficiency of photosystem II (Fv/Fm) in dark adapted leaves increased signifiantly in T. populnea seedlings that received moderate salinity treatment. However, the increasing salinity, particularly 300mM NaCl treatment, resulted in reduction of Fv/Fm compared to the non-saline control. High salinity stress affected the Fv/Fm causing a structural and functional deficiency of the photosynthetic apparatus (Morales et al., 2008), which signifies thermal dissipation processes during stress build-up yielding high Y(NO) (Scarascia-Mugnozza et al., 1996; Ranjbarfordoei et al., 2006). The reduction in Fv/Fm under salinity stress indicate susceptibility of PSII reaction center and induced photoinhibition (Satoh et al., 1983; Bjorkman et al., 1988). High light energy reaching the reaction center of PSII due to over production of antenna complex may cause photoinhibition. All pigments including anthocyanins, betacyanin, carotenoids, betacarotene, lycopene and flavonol glycosides increased in T. populnea significantly under salinity. Plants accumulate anthocyanin, to induce pigmentation in response to environmental stresses (Chalker-Scott, 1999). Although, little is known about salt-inducible anthocyanin biosynthesis in halophytes, its proposed roles during abiotic stresses include photoprotection, quenching of ROS, xenohormesis and stress signaling. Previous reports indicate subsequent production and localization of these pigments in different plant parts induce in reponse to biotic and abiotic stresses (Ali and Abbas, 2003). Our study showed strong positive correlation of these pigments (betacyanin, flavnolglycosides, betacarotene, lycopene, anthocyanin, betacyanin and flavnolglycosides) with antioxidant activity (DPPH, ABTS, FRAP and TAC) of T. populnea. The production, accumulation and

116 association of these pigments with antioxidant activity of T. populnea indicating their role in salt stress management. The role of anthocyanin, betacyanin and flavonol glycosides was found in Arabidopsis thaliana, Beta vulgaris and Ligustrum vulgare when exposed salt stress (Fini et al., 2011; Kovinich et al., 2015). Anthocyanin and betacyanins are mainly present in vacuole and ROS generated by chloroplast and mitochondria are generally detoxified by anthocyanin-rich vacuole (Kytridis and Manetas, 2006; Tanaka et al., 2008). It was recently reported that salt and UV stress increased the biosynthesis of flavonoid glycosides to a very similar degree to protect plant from salt induced light injuries (Fini et al., 2011). Beside their role in oxidative stress management, higher levels of betaarotenes and xanthophylls (sub-types of carotenoids) indicate their potential role in harvesting light energy for photosynthesis (Holt et al.,2005; Merzlyak et al., 2002) They also dissipate excess energy in the form of heat (Demmig-Adams et al., 1996; Nagy et al., 2015). Our results indicate that they do not fulfill their protective function by preventing ROS.Thereby showing strong negative correlation with fv/fm and ETR, although they have positive correlation with antioxidant activities but negative correlation with MDA content. Damage markers

H2O2 and Malondialdehyde (MDA) of various plant parts indicate extent of oxidative damage, linked to series of reactions from free radicals generation to lipid peroxidation or membrane damage (Mazliak, 1983; Sairam et al., 2005), which increased significantly with increasing stress. Moderate salinity did not affect H2O2 and MDA contents, indicating better oxidative stress management of T. populnea, while significantly higher levels of

MDA, and H2O2 of T. populnea were found at high salinity (Amor et al., 2005; Jithesh et al., 2006; Hameed et al., 2015). H2O2 is the most stable form of ROS which can easily penetrate in the cell, besides it acts as a signaling molecule provoke stress tolerance mechanisms (Foyer and Noctor, 2009; Gill and Tuteja, 2010). However, signaling function of H2O2 depends upon its cellular concentration which relays on plants’ antioxidant defense Otherwise, building up of H2O2 can lead to inactivation of Cu/Zn-SOD, Fe-SOD and many Calvin cycle enzymes, protein kinases, phosphatases and transcription factors

(Halliwell and Gutteridge, 1999). Elevated level of H2O2 and associated damage markers (MDA) of T. populnea, indicate that dissipation mechanisms Y(NO), Y( NPQ) , carotenoids and other pigments were not ample to alleviate high salinity stress, that leaked excess energy from over-reduced photosynthetic machinery which generate a burst of ROS (Turkan and Demiral, 2009)

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Antioxidant enzymes T. populnea showed significant increase in activities of all antioxidative enzyme like superoxide dismutase (SOD), catalase (CAT), ascorbate per oxidase (APX), guaicol peroxidase (GPX) and glutathione reductase (GR). SOD is a vital antioxidant enzyme and a first line of defense for salt stressed plants. SOD generates H2O2 by scavenging harmful

O2ˉ, and H2O2 is further neutralized into water by different antioxidant enzymes like CAT, APX, and GPX (Jithesh et al., 2006; Hameed and Khan, 2011). In T. populnea, Asada– Halliwell pathway enzymes were upregulated in a harmonized manner to save plant from salt induced oxidative stress (Jithesh et al., 2006; Hameed et al., 2015; Gómez et al., 1999). Such a coordinated response was also found in many other halophytes in response to salinity (Parida et al., 2004b; Amor et al., 2005; Benzarti et al., 2012; Duarte et al., 2013). Apel and Hirt (2004) stated that the expression and activities of most antioxidant enzymes are increase linearly by ROS accumulation. T. populnea strong positive correlation of

H2O2 with all antioxidant enzymes with increasing salinity found similar to that of Apel and Hirt (2004). Besides Asada–Halliwell pathway enzymes high total ascorbate and •− • 1 glutathione are reported to directly quench O2 , OH and O2 (Foyer and Halliwell, 1976; Foyer and Noctor., 2009). The AsA/DHAsA ratio is not only a measure of ascorbic acid availability but also oxidative stress indicator (Koyro et al., 2013). Therefore, it is reasonable to assume decrease of the AsA/DHAsA ratio (Fig.4.14). We also found the higher contents of H2O2 and MDA in T. populnea, which was in line with increased activities of SOD and CAT, at high salinity. These results of T. populnea are in agreement with the antioxidative responseive of Echinochloa crusgalli, Pennisetum clandestinum, and Spartina alterniflora (Abogadallah et al., 2010; Subudhi and Baisakh, 2011). Contrasting results are also found in Lolium perenne and Aeluropus lagopoides (Sobhanian et al., 2010). This imbalanced AsA/DHAsA ratio did not affect MDA and

H2O2 levels under moderate salinity rather contributed in small stomatal aperture (Table.4.3), in response to ABA (Chen and Gallie, 2004). A 50% reduction in total biomass at high salinity might be consequence of limited CO2 availability (Fig.4.9 and 4.2) at higher salinity. CAT plays a key role in detoxification of H2O2 from peroxisomes while, APX removes H2O2 from chloroplast (Frederick and Newcomb, 1969; Cho and Seo, 2005).

Both, CAT and APX along with GR are responsible of maintaining indigenous H2O2 level in cytosol (Noctor and Foyer, 1998; Duarte et al., 2013). APX and GR activities of T. populnea also increased significantly under high salinity. APX consumes ascorbate (AsA) to neutralize H2O2 in to H2O and dehydroascorbate (DAsA) (Asada, 1992). Salt stressed

118 plant increased GR activity to increase NADP+/NADPH ratio, which enables the ability of NADP+ to accept electrons leaking from photosynthetic ETC hence, limiting ROS concentration (Muscolo et al., 2003). An increase in APX activity of T. populnea showed its strong relation with total ascorbate concentration (r = 0.744), at high salinity (AbdElgawad et al., 2016). High level of oxidized ascorbate (DHAsA) could be explained with increased activity of APX. However, increased levels of reduced ascorbate suggest ascorbate recycling to regulate the redox state of ascorbic acid pool, which is important to improve salt tolerance (Gill and Tuteja, 2010). The regeneration of AsA is an indicative of high activity of dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR). Recent reports suggest that MDHAR and DHAR both are equally important in controlling the level of total AsA and its redox states, under stress (Wang et al., 2010; Hasanuzzaman et al., 2011). Salinity effects TPC and antioxidant activity Effect of salinity on growth of T. populnea could be related to polyphenols and antioxidant activity. T. populnea maintained the contents of TPC, TFC and PC under moderate salinity, not only by synthesis but also by the hydrolysis of polymerized from of polyphenols (TTC) to increase the pool of active phenolic metabolites. Strong positive correlations of salinity with TPC (r = 0.903), TFC (r =0.944), DPPH (r = 0.946), ABTS (r = 0.958) and TAC (r = 0.851) indicating their role in plants’ stress tolerance. In addition, their positive relationship with H2O2 (TPC r = 0.669, TFC r = 0.798, DPPH r = 0.894, ABTS r = 0.776 and TAC r = 0.655), MDA (TPC r = 0.700, TFC r =0.713, DPPH r = 0.835, ABTS r = 0.723 and TAC r = 0.696) and EL (TPC r = 0.914, TFC r =0.885, DPPH r = 0.905, ABTS r = 0.933 and TAC r = 0.857) also explained their part in controlling ROS progression and damage. Halophyte tends to reduce deleterious effects of ROS using antioxidant compounds (polyphenols) as, amount and composition of polyphenols depend on quantum of stress (Awika et al., 2005; De Abreu et al., 2005). In this study, polyphenol content ranged TPC (30-14 mg GAEg-1) and TFC (2.5-1.9 mg CE g-1). T. populnea polyphenol remain unchanged under moderate salinity but decreased sharply under high salinity, and these results are consistent with all antioxidant activity measurements as found in Cynara scolymus (Rezazadeh et al., 2012) and Cynara cardunculus (Hanen et al., 2008). High salt concentration could be associated with disturbance of enzymatic activities, resulting in reduced growth, photosynthesis and ultimately production of polyphenols will be decreased (Wong et al., 2006). TPC and TFC showed strong positive

119 correlation with DPPH (TPC r = 0.820, TFC r = 0.922), ABTS (TPC r = 0.967, TFC r = 0.976) and TAC (TPC r = 0.974, TFC r = 0.916). Chemical structure of phenolic compounds (aromatic rings and free hydroxyl groups) provides ideal configuration to neutralize damaging oxygen radicals (Rice-Evans et al., 1996; Bors et al., 2002). TPC of T. populnea was comparable with several glycophytes (Chu et al., 2002; Zhou and Yu, 2006) and even higher than some halophytes (Benhammou et al., 2009). Our results also validates the previous study about antioxidant potential and bioactivity of different parts of T. populnea (Parthasarathy et al., 2009; Chumbhale et al., 2010; Rajamurugan et al., 2013; Qasim et al., 2010; Qasim et al., 2017; Muthukumar and Veerappa, 2018). Higher accumulation of polyphenols in T. populnea leaf with profound antioxidant activity may explain the protective role of plant phenolics against photooxidation of light exposed tissues (Close and McArthur, 2002; Niknam and Ebrahimzadeh, 2002). T. populnea showed a significant variation between plant organs for their antioxidant activity and phenolic composition. However, their accumulation and de-accumulation is often modulated in response to environmental stresses, such as salinity (Dixon and Paiva., 1995). HPLC profile of T. populnea leaf samples indicate the accumulation of phenols (gallic acid and resorcinol) under high salinity, while (pyrocatechol and catechin) remain unchanged under moderate salinity. Qasim et al., 2017 identified polyphenols as potential natural souce of bioactive compounds. Ozfidan-Konakci et al., (2015) reported exogenous application of gallic acid enhanced the tolerance of rice cultivars to osmotic stress. The increased phenolic accumulation in above ground parts of T. populnea could be attributed to their protective effects under high light intensity, since light harvesting complex is more prone to ROS production in plants grown under salt stress conditions (Falleh et al., 2011). On exposure to salinity stress plants induce endogenous plant hormones, including jasmonic acid and its methylated derivate (methyl jasmonic acid), which tend to stimulate phenylpropanoid pathway, including phenylalanine ammonialyase (PAL), thereby resulting in the accumulation of phenolic compounds (Pedranzani et al., 2007).

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Conclusion Present study indicates that T. populnea can easily tolerate salinity up to 300 mM NaCl (≈60% seawater salinity). High salinity did neither disturb nutrient uptake nor disrupt water status of the plant. T. populnea opted growth reduction strategy at high salinity. Sucrose appeared to counter ionic toxicity and Na+ appeared as cheap osmotica under salinity. Stomatal limitation of photosynthesis was a result of decrease in number of stomata in response to high external salinity, leading to low CO2 diffusion, along decrease in the integrity of the PSII system that resulted in shifting energy costing activation of antioxidant enzymes thereby affecting growth. Chlorophyll fluorescence parameters like Fv/Fm and Y (NPQ) were either increased or remained unaffected with increasing salinity.

Unchanged MDA and H2O2 levels when gradually increased values of SOD, CAT, APX, GR, GPX at moderate salinity suggest that the ROS production was either negligible or appropriately managed by antioxidative enzymes, while at higher salinity all these mechanisms proven inadequate to overcome oxidative damage. These results indicate well-coordinated antioxidative system that protects the membrane integrity by ROS damage at moderate salinity but not in higher salinity. Results clearly indicate that T. populnea can be grown at coastal/wastelands with moderate salinity and give optimal growth performance similar to non-saline conditions. Presence of bioactive secondary metabolites (phenols, flavonoids, tannins, anthocyanins, betacyanins, flavonol glycosides, betacarotens etc.) with profound antioxidant activity, emphasise its application in medicinal and other industrial purposes.

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CHAPTER 5 GENERAL CONCLUSIONS

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The search for new sources of polyphenolic compounds as safe and inexpensive natural antioxidants has become a research interest since last millennium. These compounds are essential to plant life, and can also provide safe therapeutics for humans due potent biological activities with minimal or no side effects. This study revealed antioxidant potential of 10 medicinal halophytes, traditionally used in herbal medicines. All tested halophytes are enriched with polyphenols (TPC), flavonoids (TFC) and proanthocynadins (PC) with profound antioxidant activity. Further in depth investigations revealed that harvesting the leaves of these plants during winter (drier period) and/ or during their reproductive stages would yield maximum antioxidant compounds. For this purpose, Thespesia populnea, Ipomoea pes-caprae and Suaeda fruticosa were ranked highest among others for their antioxidant activity and polyphenolic content. However, some other options are also available including Calotropis procera, Salsola imbricate, Aerva javanica, and Cressa cretica from moderate category, while low phenolic species of this study i.e. Salvadora persica, Heliotropium bacciferum and Atriplex stocksii were also not lesser than some of the reported antioxidant rich plants. Strong positive correlations among antioxidant activities and polyphenolic contents indicating the major contribution of polyphenolics to provide halophytes a strong protection against oxidative damages usually mediated by salt stress. This observation has been verified by greenhouse experiment of this study. For this experiment, T. populnea (from high category) and C. procera (from moderate category) were analyzed for salt induced secondary metabolites (polyphenolic antioxidants) production and ecophysiological responses. Moderate salinity (100 mM NaCl) did not affect growth and biomass production of both of the species, however, it either enhanced (C. procera) or maintained (T. populnea) their antioxidant status (both compounds and activity). Both plants successfully managed to tolerate high salinity (300 mM, ~60% sea water salinity), but with the cost of growth. Growth maintenance at moderate salinity was achieved by accumulating inorganic (Na+ and K+) and organic osmolytes (soluble sugars and proline) which helped to maintain sufficient water balance. Plants also improved selective intake of K+ to maintained K+ homeostasis and avoid toxicity. Strong antioxidative protection, either employing enzymatic (superoxide dismutase, catalase, and glutathione reductase, ascorbate peroxidase) way or enzymatic and non-enzymatic (polyphenols, anthocyanin, betacyanin, carotenoids, flavonols, betacarotene and dihydroascorbic acid) both ways, effectively managed salt induced oxidative stress, hence maintained growth and overall plant biomass. Among polyphenols production of strong antioxidant agents including resorcinol, chlorogenic acid, vanillic

123 acid, ferulic acid, sinapic acid, rutin, and quercetin indicating the robust antioxidant defense of these plants. However, at higher salinity, both plants decreased their actual quantum yield (YII), photochemical quenching (qP) and electron transport rate (ETR) and increased non-photochemical quenching Y (NPQ) and Y (NO). These limitations although helped to retain PSII system integrity, but resulted in excessive production of ROS, which resulted in cellular damages (reflected in MDA and electrolyte leakage) and ultimately growth reduction. From the results of this study, both species i.e. C. procera and T. populnea appeared as potential candidates, which can be cultivated on degraded/saline lands with brackish water and produce considerable biomass for the production of natural antioxidants. In addition, both species can grow under moderately saline conditions without compromising their growth/ biomass and can provide similar (T. populnea) or even higher amount of bioactive natural products (C. procera) using brackish water and saline lands. Beside this, they can also bring vast barren/saline land under cultivation, which are considered unproductive, hence could also contribute to halt desertification and climate change.

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References Abbas, Z. K., Saggu, S., Sakeran, M. I., Zidan, N., Rehman, H., and Ansari, A. A. (2015). Phytochemical, antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves. Saudi journal of biological sciences, 22, 322-326. AbdElgawad, H., Zinta, G., Hegab, M. M., Pandey, R., Asard, H., and Abuelsoud, W. (2016). High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. Frontiers in Plant Science, 7, 276. Abdi, S., and Ali, A. (1999). Role of ROS modified human DNA in the pathogenesis and etiology of cancer. Cancer letters, 142, 1-9. Abid, M., Khaled, A. B., Lachiheb, B., Ferchichi, A., Mansour, E., and Bachar, K. (2013). Phenolic Compounds, Antioxidant, and Antibacterial Activities of Peel Extract from Tunisian Pomegranate. Journal of Agriculture Science and technology, 15, 1393-1403 Abideen, Z., Koyro, H. W., Huchzermeyer, B., Ahmed, M. Z., Gul, B., and Khan, M. A. (2014). Moderate salinity stimulates growth and photosynthesis of Phragmites karka by water relations and tissue specific ion regulation. Environmental and Experimental Botany, 105, 70-76. Abideen, Z., Qasim, M., Rasheed, A., Adnan, M. Y., Gul, B., and Khan, M. A. (2015). Antioxidant activity and polyphenolic content of Phragmites karka under saline conditions. Pakistan Journal of Botany, 47, 813-818. Abogadallah, G. M., Serag, M. M., and Quick, W. P. (2010). Fine and coarse regulation of reactive oxygen species in the salt tolerant mutants of barnyard grass and their wild‐type parents under salt stress. Physiologia Plantarum, 138, 60-73. Achakzai, A. K. K., and Masood, A. (2017). Study of Polyphenols at Vegetative and Reproductive Stages of Eight Common Plant Species of Asteraceae Found in Quetta. Journal of the Chemical Society of Pakistan, 39, 1068-1074. Acosta-Motos, J. R., Diaz-Vivancos, P., Álvarez, S., Fernández-García, N., Sánchez- Blanco, M. J., and Hernández, J. A. (2015)a. NaCl-induced physiological and biochemical adaptative mechanisms in the ornamental Myrtus communis L. plants. Journal of plant physiology, 183, 41-51. Acosta-Motos, J. R., Diaz-Vivancos, P., Álvarez, S., Fernández-García, N., Sanchez- Blanco, M. J., and Hernández, J. A. (2015)b. Physiological and biochemical

125

mechanisms of the ornamental Eugenia myrtifolia L. plants for coping with NaCl stress and recovery. Planta, 242, 829-846. Acosta-Motos, J., Ortuño, M., Bernal-Vicente, A., Diaz-Vivancos, P., Sanchez-Blanco, M., and Hernandez, J. (2017). Plant responses to salt stress: adaptive mechanisms. Agronomy, 7, 18. Adebooye, O. C., Alashi, A. M., and Aluko, R. E. (2018). A brief review on emerging trends in global polyphenol research. Journal of Food Biochemistry, e12519. Adnan, M. Y., Hussain, T., Asrar, H., Hameed, A., Gul, B., Nielsen, B. L., and Khan, M. A. (2016). Desmostachya bipinnata manages photosynthesis and oxidative stress at moderate salinity. Flora-Morphology, Distribution, Functional Ecology of Plants, 225, 1-9. Aeby, H. (1984).Catalase in vitro. In Methods in enzymology (Vol. 105, pp. 121- 126).Academic press. Agati, G., Biricolti, S., Guidi, L., Ferrini, F., Fini, A., and Tattini, M. (2011). The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves. Journal of plant physiology, 168, 204-212. Agati, G., Matteini, P., Goti, A., and Tattini, M. (2007). Chloroplast‐located flavonoids can scavenge singlet oxygen. New Phytologist, 174, 77-89. Ahmad, S., Ahmad, S., Bibi, A., Ishaq, M. S., Afridi, M. S., Kanwal, F and Fatima, F. (2014). Phytochemical analysis, antioxidant activity, fatty acids composition, and functional group analysis of Heliotropium bacciferum. The Scientific World Journal, 2014. Ahmed, M. Z., Shimazaki, T., Gulzar, S., Kikuchi, A., Gul, B., Khan, M. A and Watanabe, K. N. (2013). The influence of genes regulating transmembrane transport of Na+ on the salt resistance of Aeluropus lagopoides. Functional Plant Biology, 40, 860-871. Akacha, M., Lahbib, K., and Boughanmi, N. G. (2017). Rhizotoxicity of the invasive species melia azedarach: implication of phenols on its herbicide potential. Journal of Animal and Plant Sciences, 27.1353-1356 Alberico, G. J., and Cramer, G. R. (1993). Is the salt tolerance of maize related to sodium exclusion? I. Preliminary screening of seven cultivars. Journal of Plant Nutrition, 16, 2289-2303.

126

Alhdad, G. M., Seal, C. E., Al-Azzawi, M. J., and Flowers, T. J. (2013). The effect of combined salinity and waterlogging on the halophyte Suaeda maritima: the role of antioxidants. Environmental and Experimental Botany, 87, 120-125. Ali, N. A. L., Mohammed, A. B., and Allow, A. A. (2014). Effect of adding different levels of Lycopene to the ration on some lipid profile traits of the Laying hens ISA- Brown. Journal of Biology, Agriculture and Healthcare, 4, 10-9. Ali, R. M., and Abbas, H. M. (2003). Response of salt stressed barley seedlings to phenylurea. Plant Soil and Environment, 49, 158-162. Alshammary, S. F. (2008). Effect of saline irrigation on growth characteristics and mineral composition of two local halophytes under Saudi environmental conditions. Pak Journal of Biological Science, 11, 216-21. Al-Sobhi, O. A., Al-Zahrani, H. S., and Al-Ahmadi, S. B. (2005). Effect of salinity on chlorophyll andcarbohydrate contents of Calotropis procera seedlings. King Fasil University Journal. (Accepted, in press). Amor, N. B., Hamed, K. B., Debez, A., Grignon, C., and Abdelly, C. (2005). Physiological and antioxidant responses of the perennial halophyte Crithmum maritimum to salinity. Plant Science, 168, 889-899. Anderson, J. V., Chevone, B. I., and Hess, J. L. (1992). Seasonal variation in the antioxidant system of eastern white pine needles: evidence for thermal dependence. Plant physiology, 98(2), 501-508. Dua A., Gupta S.K., Mittal, A.and Mahajan, R. (2012). A Study of Antioxidant Properties and Antioxidant Compounds of Cumin (Cuminum cyminum). International Journal of Pharmaceutical and Biological Archives, 3, 1110-1116 Apel, K., and Hirt, H. (2004). Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology, 55, 373-399. Asada, K. (1992). Ascorbate peroxidase–a hydrogen peroxide‐scavenging enzyme in plants. Physiologia Plantarum, 85(2), 235-241. Ashraf, M. (2009). Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnology advances, 27, 84-93. Ashraf, M. H. P. J. C., and Harris, P. J. C. (2013). Photosynthesis under stressful environments: an overview. Photosynthetica, 51, 163-190. Ashraf, M., and Orooj, A. (2006). Salt stress effects on growth, ion accumulation and seed oil concentration in an arid zone traditional medicinal plant ajwain

127

(Trachyspermum ammi [L.] Sprague). Journal of Arid Environments, 64, 209- 220. Asif, M. (2015). Chemistry and antioxidant activity of plants containing some phenolic compounds. Chemistry international, 1, 35-52. Asrar, H., Hussain, T., Hadi, S. M. S., Gul, B., Nielsen, B. L., and Khan, M. A. (2017). Salinity induced changes in light harvesting and carbon assimilating complexes of Desmostachya bipinnata (L.) Staph. Environmental and Experimental Botany, 135, 86-95. Attia, F. (2007). Effet du stress hydrique sur le comportement écophysiologique et la maturité phénolique de la vigne Vitis vinifera L.: étude de cinq cepages autochtones de midi-Pyrenees (Doctoral dissertation). Awika, J. M., Rooney, L. W., and Waniska, R. D. (2005). Anthocyanins from black sorghum and their antioxidant properties. Food Chemistry, 90, 293-301. Azevedo Neto, A. D. D., and Tabosa, J. N. (2000). Salt stress in maize seedlings: part II distribution of cationic macronutrients and its relation with sodium. Revista Brasileira de Engenharia Agricola e Ambiental, 4, 165-171. Azzabi, G., Pinnola, A., Betterle, N., Bassi, R., and Alboresi, A. (2012). Enhancement of non-photochemical quenching in the Bryophyte Physcomitrella patens during acclimation to salt and osmotic stress. Plant and cell physiology, 53, 1815-1825. Baillie, J., Hilton-Taylor, C., and Stuart, S. N. (Eds.). (2004). 2004 IUCN red list of threatened species: a global species assessment. Iucn. Bairagi, S. M., Ghule, P., and Gilhotra, R. (2018). Pharmacology of Natural Products: An recent approach on Calotropis gigantea and Calotropis procera. Ars pharmaceutica, 59, 37-44. Balasundram, N., Sundram, K., and Samman, S. (2006). Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food chemistry, 99, 191-203. Ball, M. C. (1988). Ecophysiology of mangroves. Trees, 2, 129-142. Balnokin, Y. V., Kotov, A. A., Myasoedov, N. A., Khailova, G. F., Kurkova, E. B., Lun’kov, R. V., and Kotova, L. M. (2005). Involvement of long-distance Na+ transport in maintaining water potential gradient in the medium-root-leaf system of a halophyte Suaeda altissima. Russian Journal of Plant Physiology, 52, 489-496.

128

Banerjee, D., Chakrabarti, S., Hazra, A. K., Banerjee, S., Ray, J., and Mukherjee, B. (2008). Antioxidant activity and total phenolics of some mangroves in Sundarbans. African Journal of Biotechnology, 7. Barros, L., Ferreira, M. J., Queiros, B., Ferreira, I. C., and Baptista, P. (2007). Total phenols, ascorbic acid, β-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food chemistry, 103, 413-419. Basu et al., 1992 A. Basu, T. Sen, R.N. Ray, A.K. Nag Chaudhuri Fitoterapia, 63 (1992), pp. 507-514 Hepatoprotective effects of Calotropis procera root extract on experimental liver damage in animals Bates, L. S., Waldren, R. P., and Teare, I. D. (1973). Rapid determination of free proline for water-stress studies. Plant and soil, 39, 205-207. Beauchamp, C., and Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical biochemistry, 44, 276-287. Beeche GR. (2003) Overview of dietary flavonoids: nomenclature, occurrence and intake. The Journal of Nutrition, 133, 248-54. Belhekar, S. N., Chaudhari, P. D., Saryawanshi, J. S., Mali, K. K., and Pandhare, R. B. (2013). Antidiabetic and antihyperlipidemic effects of Thespesia populnea fruit pulp extracts on alloxan-induced diabetic rats. Indian journal of pharmaceutical sciences, 75, 217. Bell, H. L., and O'Leary, J. W. (2003). Effects of salinity on growth and cation accumulation of Sporobolus virginicus (Poaceae). American Journal of Botany, 90, 1416-1424. Benhammou, N., Bekkara, F. A., and Panovska, T. K. (2009). Antioxidant activity of methanolic extracts and some bioactive compounds of Atriplex halimus. Comptes Rendus Chimie, 12, 1259-1266. Benlloch, M., Ojeda, M. A., Ramos, J., and Rodriguez-Navarro, A. (1994). Salt sensitivity and low discrimination between potassium and sodium in bean plants. Plant and Soil, 166, 117-123. Benzarti, M., Rejeb, K. B., Debez, A., Messedi, D., and Abdelly, C. (2012). Photosynthetic activity and leaf antioxidative responses of Atriplex portulacoides subjected to extreme salinity. Acta Physiologiae Plantarum, 34, 1679-1688. Benzie, I. F., and Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical biochemistry, 239, 70-76.

129

Bezona, N., Hensley, D., Yogi, J., Tavares, J., Rauch, F., Iwata, R., and Clifford, P. (2009). Salt and Wind Tolerance of Landscape Plants for Hawaii. Bi, B., Tang, J., Han, S., Guo, J., and Miao, Y. (2017). Sinapic acid or its derivatives interfere with abscisic acid homeostasis during Arabidopsis thaliana seed germination. BMC Plant Biology, 17, 99. Bilger, W., and Björkman, O. (1990). Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynthesis research, 25, 173-185. Bjorkman, O., Demmig, B., and Andrews, T. J. (1988). Mangrove photosynthesis: response to high-irradiance stress. Functional Plant Biology, 15, 43-61. Bolanos, J. A., and Longstreth, D. J. (1984). Salinity effects on water potential components and bulk elastic modulus of Alternanthera philoxeroides (Mart.) Griseb. Plant Physiology, 75, 281-284. Bonales-Alatorre, E., Shabala, S., Chen, Z. H., and Pottosin, I. (2013). Reduced tonoplast fast-activating and slow-activating channel activity is essential for conferring salinity tolerance in a facultative halophyte, Quinoa. Plant Physiology, 162, 940- 952. Bors, W., and Michel, C. (2002). Chemistry of the antioxidant effect of polyphenols. Annals of the New York Academy of Sciences, 957, 57-69. Bouvier, F., Isner, J. C., Dogbo, O., and Camara, B. (2005). Oxidative tailoring of carotenoids: a prospect towards novel functions in plants. Trends in plant science, 10, 187-194. Bowman, H. H. M. (1921). Histological variations in Rhizophora mangle. Rep. Mich. Acad. Sci, 22, 129-34. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72, 248-254. Brand-Williams, W., Cuvelier, M. E., and Berset, C. L. W. T. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food science and Technology, 28, 25-30. Braun-Blanquet, J. (1932). Plant sociology: the Study of Plant Communities. Transl. by G. D. Fuller and H. S. Conard. New York; NY: McGraw-Hill Book Co.

130

Brestic, M., Zivcak, M., Kunderlikova, K., and Allakhverdiev, S. I. (2016). High temperature specifically affects the photoprotective responses of chlorophyll b- deficient wheat mutant lines. Photosynthesis research, 130, 251-266. Brestic, M., Zivcak, M., Kunderlikova, K., Sytar, O., Shao, H., Kalaji, H. M., and Allakhverdiev, S. I. (2015). Low PSI content limits the photoprotection of PSI and PSII in early growth stages of chlorophyll b-deficient wheat mutant lines. Photosynthesis research, 125, 151-166. Brown, J. E., and Rice-Evans, C. A. (1998). Luteolin-rich artichoke extract protects low density lipoprotein from oxidation in vitro. Free radical research, 29, 247- 255. Cai, Y. Z., Sun, M., and Corke, H. (2005). Characterization and application of betalain pigments from plants of the Amaranthaceae. Trends in Food Science and Technology, 16, 370-376. Calabrese, V., Cornelius, C., Mancuso, C., Pennisi, G., Calafato, S., Bellia, F., and Rizzarelli, E. (2008). Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity. Neurochemical research, 33, 2444-2471. Caliskan, O., Radusiene, J., Temizel, K. E., Staunis, Z., Cirak, C., Kurt, D., and Odabas, M. S. (2017). The effects of salt and drought stress on phenolic accumulation in greenhouse-grown Hypericum pruinatum. Italian Journal of Agronomy, 12(3). Camejo, D., Rodríguez, P., Morales, M. A., Dell’Amico, J. M., Torrecillas, A., and Alarcón, J. J. (2005). High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility. Journal of plant physiology, 162, 281-289. Carbone, K., Giannini, B., Picchi, V., Scalzo, R. L., and Cecchini, F. (2011). Phenolic composition and free radical scavenging activity of different apple varieties in relation to the cultivar, tissue type and storage. Food Chemistry, 127, 493-500. Carden, D. E., Walker, D. J., Flowers, T. J., and Miller, A. J. (2003). Single-cell measurements of the contributions of cytosolic Na+ and K+ to salt tolerance. Plant physiology, 131, 676-683. Cartea, M. E., Francisco, M., Soengas, P., and Velasco, P. (2010). Phenolic compounds in Brassica vegetables. Molecules, 16, 251-280.

131

Carvalho, I. S., Cavaco, T., and Brodelius, M. (2011). Phenolic composition and antioxidant capacity of six Artemisia species. Industrial crops and products, 33, 382-388. Chalker‐Scott, L. (1999). Environmental significance of anthocyanins in plant stress responses. Photochemistry and photobiology, 70, 1-9. Chang, C. C., Yang, M. H., Wen, H. M., and Chern, J. C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of food and drug analysis, 10, 178-182. Chanwitheesuk, A., Teerawutgulrag, A., and Rakariyatham, N. (2005). Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand. Food chemistry, 92(3), 491-497. Chanwitheesuk, A., Teerawutgulrag, A., and Rakariyatham, N. (2005). Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand. Food chemistry, 92, 491-497. Chao, P. Y., Lin, S. Y., Lin, K. H., Liu, Y. F., Hsu, J. I., Yang, C. M., and Lai, J. Y. (2014). Antioxidant activity in extracts of 27 indigenous Taiwanese vegetables. Nutrients, 6, 2115-2130. Chen, Z., and Gallie, D. R. (2004). The ascorbic acid redox state controls guard cell signaling and stomatal movement. The Plant Cell, 16, 1143-1162. Cho, U. H., and Seo, N. H. (2005). Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to hydrogen peroxide accumulation. Plant Science, 168, 113-120. Chu, Y. F., Sun, J. I. E., Wu, X., and Liu, R. H. (2002). Antioxidant and antiproliferative activities of common vegetables. Journal of agricultural and food chemistry, 50, 6910-6916. Chumbhale, D. S., Pawase, A. A., Chaudhari, S. R., and Upasani, C. D. (2010). Phytochemical, Pharmacological and Phytopharmaceutics Aspects of Thespesia populnea (linn.) Soland.: A Review. Inventi Impact: Ethnopharmacology. Cicerale, S., Lucas, L., and Keast, R. (2010). Biological activities of phenolic compounds present in virgin olive oil. International journal of molecular sciences, 11, 458- 479. Cle, C., Hill, L. M., Niggeweg, R., Martin, C. R., Guisez, Y., Prinsen, E., and Jansen, M. A. (2008). Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; consequences for phenolic accumulation and UV-tolerance. Phytochemistry, 69, 2149-2156.

132

Clipson, N. J. W., Tomos, A. D., Flowers, T. J., and Jones, R. W. (1985). Salt tolerance in the halophyte Suaeda maritima L. Dum. Planta, 165, 392-396. Close, D. C., and McArthur, C. (2002). Rethinking the role of many plant phenolics– protection from photodamage not herbivores?. Oikos, 99, 166-172. Cresti, M., Ciampolini, F., Tattini, M., and Cimato, A. (1994). Effect of salinity on productivity and oil quality of olive (Olea europaea L.) plants. Advances in Horticultural Science, 8,211-214. Cristiano, G., Camposeo, S., Fracchiolla, M., Vivaldi, G. A., De Lucia, B., and Cazzato, E. (2016). Salinity differentially affects growth and ecophysiology of two mastic tree (Pistacia lentiscus L.) accessions. Forests, 7, 156. De Abreu, I. N., and Mazzafera, P. (2005). Effect of water and temperature stress on the content of active constituents of Hypericum brasiliense Choisy. Plant Physiology and Biochemistry, 43, 241-248. De Souza, B. R., Freitas, I. A. S., de Araujo Lopes, V., do Rosario Rosa, V., and Matos, F. S. (2015). Growth of Eucalyptus plants irrigated with saline water. African Journal of Agricultural Research, 10, 1091-1096.

Del Baño, M. J., Lorente, J., Castillo, J., Benavente-García, O., del Río, J. A., Ortuño, A., Quirin, K.W. and Gerard, D. (2003). Phenolic diterpenes, flavones, and rosmarinic acid distribution during the development of leaves, flowers, stems, and roots of Rosmarinus officinalis. Antioxidant activity. Journal of agricultural and food chemistry, 51, 4247-4253. Demmig-Adams, B., and Adams Iii, W. W. (1992). Photoprotection and other responses of plants to high light stress. Annual review of plant biology, 43, 599-626. Demmig-Adams, B., and Adams III, W. W. (1996). The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends in Plant science, 1, 21-26. Dhanapackiam, S., and Ilyas, M. M. (2010). Effect of salinity on chlorophyll and carbohydrate contents of Sesbania grandiflora seedlings. Indian Journal of Science and Technology, 3, 64-66. Dionisio-Sese, M. L., and Tobita, S. (1998). Antioxidant responses of rice seedlings to salinity stress. Plant Science, 135, 1-9. Dixon, R. A., and Paiva, N. L. (1995). Stress-induced phenylpropanoid metabolism. The plant cell, 7, 1085.

133

Dua, A. (2012). A Study of Antioxidant Properties and Antioxidant Compounds of Cumin Cuminum cyminum. International Journal of Pharmaceutical and Biological Archive, 3. Duarte, B., Santos, D., Marques, J. C., and Caçador, I. (2013). Ecophysiological adaptations of two halophytes to salt stress: photosynthesis, PS II photochemistry and anti-oxidant feedback–implications for resilience in climate change. Plant Physiology and Biochemistry, 67, 178-188. Duh, P. D. (1999). Antioxidant activity of water extract of four Harng Jyur (Chrysanthemum morifolium Ramat) varieties in soybean oil emulsion. Food Chemistry, 66, 471-476. Duke SO. (2003). Weeding with transgenes. Trends Biotechnology. 21,192-5. Ehsen, S., Qasim, M., Abideen, Z., Rizvi, R. F., Gul, B., Ansari, R., and Khan, M. A. (2016). Secondary metabolites as anti-nutritional factors in locally used halophytic forage/fodder. Pakistan Journal of Botany 48, 629-636. Ehsen, S., Rizvi, R. F., Abideen, Z., Aziz, I., Gulzar, S., Gul, B., Khan, M.A. and Ansari, R. (2017). Physiochemical responses of zaleya pentandra (l.) Jeffrey to NaCl treatments. Pakistan Journal of Botany, 49, 801-808. El Shaer, H. M. (2010). Halophytes and salt-tolerant plants as potential forage for ruminants in the Near East region. Small Ruminant Research, 91, 3-12. Elansary, H. O., Salem, M. Z., Ashmawy, N. A., Yessoufou, K., and El-Settawy, A. A. (2017). In vitro antibacterial, antifungal and antioxidant activities of Eucalyptus spp. leaf extracts related to phenolic composition. Natural product research, 31, 2927-2930. Elvin-Lewis, M. (1980). Plants used for teeth-cleaning throughout the world. Journal of Preventive Dentistry, 6, 61-70. Erturk, Y., Ercisli, S. E. Z. A. I., Sengul, M. E. M. N. U. N. E., Eser, Z. E. Y. N. E. P., Haznedar, A., and Turan, M. E. T. I. N. (2010). Seasonal variation of total phenolic, antioxidant activity and minerals in fresh tea shoots (Camellia sinensis var. sinensis). Pakistan Journal of Pharmaceutical Science, 23, 69-74. Evans, W.C. (1996) Deterioration of stored Drugs Trease and Evans’ Pharmacognosy. WB Saunders, London, 520-521. Falleh, H., Jalleli, I., Ksouri, R., Boulaaba, M., Guyot, S., Magné, C., and Abdelly, C. (2012a). Effect of salt treatment on phenolic compounds and antioxidant activity

134

of two Mesembryanthemum edule provenances. Plant Physiology and Biochemistry, 52, 1-8. Falleh, H., Ksouri, R., Boulaaba, M., Guyot, S., Abdelly, C., and Magné, C. (2012b). Phenolic nature, occurrence and polymerization degree as marker of environmental adaptation in the edible halophyte Mesembryanthemum edule. South African Journal of Botany, 79, 117-124. Falleh, H., Ksouri, R., Medini, F., Guyot, S., Abdelly, C., and Magné, C. (2011). Antioxidant activity and phenolic composition of the medicinal and edible halophyte Mesembryanthemum edule L. Industrial Crops and Products, 34, 1066- 1071. Falleh, H., Msilini, N., Oueslati, S., Ksouri, R., Magne, C., Lachaâl, M., and Karray- Bouraoui, N. (2013). Diplotaxis harra and Diplotaxis simplex organs: Assessment of phenolics and biological activities before and after fractionation. Industrial crops and products, 45, 141-147. Faller, A. L. K., and Fialho, E. F. N. U. (2010). Polyphenol content and antioxidant capacity in organic and conventional plant foods. Journal of Food Composition and Analysis, 23, 561-568. Fan, P., Feng, J., Jiang, P., Chen, X., Bao, H., Nie, L., Jiang, D., Lv, S., Kuang, T. and Li, Y. (2011). Coordination of carbon fixation and nitrogen metabolism in Salicornia europaea under salinity: comparative proteomic analysis on chloroplast proteins. Proteomics, 11, 4346-4367. Feng, H., Li, S., Xue, L., An, L., and Wang, X. (2007). The interactive effects of enhanced UV-B radiation and soil drought on spring wheat. South African Journal of Botany, 73, 429-434. Fini, A., Brunetti, C., Di Ferdinando, M., Ferrini, F., and Tattini, M. (2011). Stress-induced flavonoid biosynthesis and the antioxidant machinery of plants. Plant Signaling and Behavior, 6, 709-711. Flanagan, L. B., and Jefferies, R. L. (1988). Stomatal limitation of photosynthesis and reduced growth of the halophyte, Plantago maritima L., at high salinity. Plant, Cell and Environment, 11, 239-245. Florence, T. M. (1995). The role of free radicals in disease. Australian and New Zealand journal of ophthalmology, 23, 3-7. Flowers, T. J., and Colmer, T. D. (2008). Salinity tolerance in halophytes. New Phytologist, 179, 945-963.

135

Foyer, C. H., and Halliwell, B. (1976). The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta, 133, 21-25. Foyer, C. H., and Noctor, G. (2009). Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxidants and redox signaling, 11, 861-905. Foyer, C.H., and Noctor, G. (2005). Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17, 1866–1875. Franco, J. A., Fernández, J. A., Bañón, S., and González, A. (1997). Relationship between the effects of salinity on seedling leaf area and fruit yield of six muskmelon cultivars. Horticultural Science, 32, 642-644. Frederick, S. E., and Newcomb, E. H. (1969). Cytochemical localization of catalase in leaf microbodies (peroxisomes). The Journal of cell biology, 43, 343-353 Freitas, I. A. S., Santos, L. V. B. D., Silva, A. C. F. D., Silva, C. R. D., Silveira, P.S. D., and Matos, F. S. (2017). Growth of Tectona grandis seedlings irrigated with saline water. Ciencia Florestal, 27, 961-967. Frosi, G., Oliveira, M. T., Almeida-Cortez, J., and Santos, M. G. (2013). Ecophysiological performance of Calotropis procera: an exotic and evergreen species in Caatinga, Brazilian semi-arid. Acta Physiologiae Plantarum, 35, 335-344. Fu, L. G. (1989). Rare and endangered plants in China. Shanghai, China: Shanghai Education Press (in Chinese). Galanakis, C. M., Tsatalas, P., and Galanakis, I. M. (2018). Phenols from olive mill wastewater and other natural antioxidants as UV filters in sunscreens. Environmental Technology and Innovation, 9, 160-168. Gandhi, S., and Abramov, A.Y. (2012). Mechanism of oxidative stress neuro de generation. Oxidative medicine and cellular longevity, 2012. Ganjewala, D., Boba, S., and Raghavendra, A. S. (2008). Sodium nitroprusside affects the level of anthocyanin and flavonol glycosides in pea (Pisum sativum L. cv. Arkel) leaves. Acta Biologica Szegediensis, 52, 301-305. Geissler, N., Hussin, S., and Koyro, H. W. (2008). Elevated atmospheric CO2 concentration ameliorates effects of NaCl salinity on photosynthesis and leaf structure of Aster tripolium L. Journal of Experimental Botany, 60, 137-151.

136

Geissler, N., Hussin, S., and Koyro, H. W. (2009). Interactive effects of NaCl salinity and

elevated atmospheric CO2 concentration on growth, photosynthesis, water relations and chemical composition of the potential cash crop halophyte Aster tripolium L. Environmental and Experimental Botany, 65, 220-231. Genty, B., Briantais, J. M., and Baker, N. R. (1989). The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta (BBA)-General Subjects, 990, 87-92. Gharibi, S., Tabatabaei, B. E. S., Saeidi, G., and Goli, S. A. H. (2016). Effect of drought stress on total phenolic, lipid peroxidation, and antioxidant activity of Achillea species. Applied biochemistry and biotechnology, 178, 796-809. Gill, S. S., and Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant physiology and biochemistry, 48, 909- 930. Glenn, E. P., and Brown, J. J. (1998). Effects of soil salt levels on the growth and water use efficiency of Atriplex canescens (Chenopodiaceae) varieties in drying soil. American Journal of Botany, 85, 10-16. Gnanasekaran, N., John, J. R., Sakthivel, G., and Kalavathy, S. (2017). The Comparative Studies of the Phytochemical Levels and the in vitro Antioxidant Activity of Tridax procumbens L. from Different Habitats. Free Radicals and Antioxidants, 7, 50-56 Gohar, Z.N., Asghar, U., Perveen, F., Sultana, R. and Ahmad, R. (2018). Effect of saline water irrigation on growth and water potential of Thespesia populnea (l.) Sol.ex correa. International jounal of Biology and Biotechnology. 15, 255-262. Gomez, J. M., Hernandez, J. A., Jimenez, A., Del Rio, L. A., and Sevilla, F. (1999). Differential response of antioxidative enzymes of chloroplasts and mitochondria to long-term NaCl stress of pea plants. Free Radical Research, 31, 11-18. Gorai, M., Ennajeh, M., Khemira, H., and Neffati, M. (2010). Combined effect of NaCl- salinity and hypoxia on growth, photosynthesis, water relations and solute accumulation in Phragmites australis plants. Flora-Morphology, Distribution, Functional Ecology of Plants, 205, 462-470. Gorham, J., Jones, R. W., and McDonnell, E. (1985). Some mechanisms of salt tolerance in crop plants. In Biosalinity in action: Bioproduction with saline water. Springer, Dordrecht. pp. 15-40

137

Gueta-Dahan, Y., Yaniv, Z., Zilinskas, B. A., and Ben-Hayyim, G. (1997). Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in citrus. Planta, 203, 460-469. Gullo, M. L., and Salleo, S. (1988). Different strategies of drought resistance in three Mediterranean sclerophyllous trees growing in the same environmental conditions. New Phytologist, 108, 267-276. Gullo, M. L., Salleo, S., and Rosso, R. (1986). Drought avoidance strategy in Ceratonia siliqua L., a mesomorphic-leaved tree in the xeric Mediterranean area. Annals of Botany, 58, 745-756. Gupta, B., and Huang, B. (2014). Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. International journal of genomics, 2014. Gutterman, Y., Kamenetsky, R., and Van Rooyen, M. (1995). A comparative study of seed germination of two Allium species from different habitats in the Negev Desert highlands. Journal of Arid Environments, 29(3), 305-315. Halliwell B, Gutteridge JMC (1999). The chemistry of free radicals and related 'reactive species'. In Free radicals in biology and medicine. pp 36-104. Oxford University Press, Oxford. Halliwell, B., and Gutteridge, J. M. (1990). Role of free radicals and catalytic metal ions in human disease: an overview. In Methods in enzymology (Vol. 186, pp. 1-85). Academic Press. Hameed, A., and Khan, M. A. (2011). Halophytes: biology and economic potentials. Karachi University Journal of Science, 39, 40-44. Hameed, A., Gulzar, S., Aziz, I., Hussain, T., Gul, B., and Khan, M. A. (2015). Effects of salinity and ascorbic acid on growth, water status and antioxidant system in a perennial halophyte. Annals of Botany Plants, 7. Hameed, A., Hussain, T., Gulzar, S., Aziz, I., Gul, B., and Khan, M. A. (2012). Salt tolerance of a cash crop halophyte Suaeda fruticosa: Biochemical responses to salt and exogenous chemical treatments. Acta Physiologiae Plantarum, 34, 2331-2340. Hanen, F., Ksouri, R., Megdiche, W., Trabelsi, N., Boulaaba, M., and Abdelly, C. (2008). Effect of Salinity on growth, leaf phenolic content and antioxidant scavenging activity in Cynara cardunculus. In: Biosaline Agriculture and High Salinity Tolerance, Abdelli, C., M Ozturk, M Ashraf and Y.C. Grignon, (Eds.). Birkhauser Verlag, Switzerland, pp: 335-343.

138

Harvey, D. M., Hall, J. L., Flowers, T. J., and Kent, B. (1981). Quantitative ion localization within Suaeda maritima leaf mesophyll cells. Planta, 151, 555-560. Hasanuzzaman, M., Hossain, M. A., and Fujita, M. (2011). Selenium-induced up- regulation of the antioxidant defense and methylglyoxal detoxification system reduces salinity-induced damage in rapeseed seedlings. Biological Trace Element Research, 143, 1704-1721. Hashiba, K., Iwashina, T., and Matsumoto, S. (2006). Variation in the quality and quantity of flavonoids in the leaves of coastal and inland Campanula punctata. Biochemical systematics and ecology, 34, 854-861. Hassan, F., and Ali, E. (2014). Effects of salt stress on growth, antioxidant enzyme activity and some other physiological parameters in jojoba ['Simmondsia chinensis'(link) schneider] plant. Australian Journal of Crop Science, 8, 1615. Hasselquist, N. J., Allen, M. F., and Santiago, L. S. (2010). Water relations of evergreen and drought-deciduous trees along a seasonally dry tropical forest chronosequence. Oecologia, 164, 881-890. Hassine, A. B. and S. Luttus (2010). Differential responses of salt bush Atriplex halimus L. exposed to salinity and water stress in relation to senescing hormones abscisic acid and ethylene. Journal of Plant Physiology, 167, 1448-1456. He, H. J., Wang, G. Y., Gao, Y., Ling, W. H., Yu, Z. W., and Jin, T. R. (2012). Curcumin attenuates Nrf2 signaling defect, oxidative stress in muscle and glucose intolerance in high fat diet-fed mice. World journal of diabetes, 3, 94-109 Heath, R. L., and Packer, L. (1968). Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of biochemistry and biophysics, 125, 189-198. Hernández, I., Alegre, L., and Munné-Bosch, S. (2006). Enhanced oxidation of flavan-3- ols and proanthocyanidin accumulation in water-stressed tea plants. Phytochemistry, 67, 1120-1126. Hernández, J. A., Aguilar, A. B., Portillo, B., López-Gómez, E., Beneyto, J. M., and García-Legaz, M. F. (2003). The effect of calcium on the antioxidant enzymes from salt-treated loquat and anger plants. Functional Plant Biology, 30, 1127-1137. Hernández, J. A., Ferrer, M. A., Jiménez, A., Barceló, A. R., and Sevilla, F. (2001). − Antioxidant systems and O2. /H2O2 production in the apoplast of pea leaves. Its relation with salt-induced necrotic lesions in minor veins. Plant Physiology, 127, 817-831.

139

Hernandez, J. A., Jimenez, A., Mullineaux, P., and Sevilia, F. (2000). Tolerance of pea (Pisum sativum L.) to long‐term salt stress is associated with induction of antioxidant defences. Plant, cell and environment, 23, 853-862. Hernandez, J. A., Olmos, E., Corpas, F. J., Sevilla, F., and Del Rio, L. A. (1995). Salt- induced oxidative stress in chloroplasts of pea plants. Plant Science, 105, 151-167. Hilu, K. W. and J. L. Randall (1984). Convenient Method for studying Grass Leaf epidermis. Taxon. 33, 413-415. Holt, N. E., Zigmantas, D., Valkunas, L., Li, X. P., Niyogi, K. K., and Fleming, G. R. (2005). Carotenoid cation formation and the regulation of photosynthetic light harvesting. Science, 307, 433-436. Horváth, E., Szalai, G., and Janda, T. (2007). Induction of abiotic stress tolerance by salicylic acid signaling. Journal of Plant Growth Regulation, 26, 290-300. Huang, W. Y., Cai, Y. Z., and Zhang, Y. (2009). Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. Nutrition and cancer, 62, 1-20. Hura, T., Grzesiak, S., Hura, K., Grzesiak, M., and Rzepka, A. (2006). Differences in the physiological state between triticale and maize plants during drought stress and followed rehydration expressed by the leaf gas exchange and spectro fluorimetric methods. Acta Physiologiae Plantarum, 28, 433-443. Hussain, N., Naseem, A. R., Sarwar, G., Mujeeb, F., and Jamil, M. (2003). Domestication/ cultivation scope of medicinal crops on salt-affected soils. In Proceedings of international workshop (WWF-Pakistan) “Conservation and Sustainable Uses of Medicinal and Aromatic Plants of Pakistan”. December (pp. 2-4). Hussin, S., Geissler, N., El-Far, M. M., and Koyro, H. W. (2017). Effects of salinity and

short-term elevated atmospheric CO2 on the chemical equilibrium between CO2 fixation and photosynthetic electron transport of Stevia rebaudiana Bertoni. Plant Physiology and Biochemistry, 118, 178-186. Ibrahim, A. H. (2013). Tolerance and avoidance responses to salinity and water stresses in Calotropis procera and Suaeda aegyptiaca. Turkish Journal of Agriculture and Forestry, 37, 352-360. Ikbal, F. E., Hernández, J. A., Barba-Espín, G., Koussa, T., Aziz, A., Faize, M., and Diaz- Vivancos, P. (2014). Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants. Journal of plant physiology, 171, 779-788.

140

Jahns, P., and Holzwarth, A. R. (2012). The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochimica et Biophysica Acta (BBA)- Bioenergetics, 1817, 182-193. Jaleel, C. A., Gopi, R., Manivannan, P., and Panneerselvam, R. (2007). Antioxidative potentials as a protective mechanism in Catharanthus roseus (L.) G. Don. Plants under salinity stress. Turkish Journal of Botany, 31, 245-251. Jaleel, C. A., Sankar, B., Sridharan, R., and Panneerselvam, R. (2008). Soil salinity alters growth, chlorophyll content, and secondary metabolite accumulation in Catharanthus roseus. Turkish Journal of Biology, 32, 79-83. Jallali, I., Megdiche, W., M’Handi, B., Oueslati, S., Smaoui, A., Abdelly, C., and Ksouri, R. (2012). Changes in phenolic composition and antioxidant activities of the edible halophyte Crithmum maritimum L. with physiological stage and extraction method. Acta Physiologiae Plantarum, 34, 1451-1459. James, J. J., Alder, N. N., Mühling, K. H., Läuchli, A. E., Shackel, K. A., Donovan, L. A., and Richards, J. H. (2005). High apoplastic solute concentrations in leaves alter water relations of the halophytic shrub, Sarcobatus vermiculatus. Journal of Experimental Botany, 57, 139-147. Jayatissa, L. P., Wickramasinghe, W. A. A. D. L., Dahdouh‐Guebas, F., and Huxham, M. (2008). Interspecific variations in responses of mangrove seedlings to two contrasting salinities. International review of hydrobiology, 93, 700-710. Jdey, A., Falleh, H., Jannet, S. B., Hammi, K. M., Dauvergne, X., Magne, C., and Ksouri, R. (2017). Anti-aging activities of extracts from Tunisian medicinal halophytes and their aromatic constituents. Experimental and Clinical Sciences International online journal for advances in science, 16, 755-769

Jelali, N., Dhifi, W., Chahed, T., and Marzouk, B. (2011). Salinity effects on growth, essential oil yield and composition and phenolic compounds content of marjoram (Origanum majorana l.) leaves. Journal of Food Biochemistry, 35, 1443-1450. Ji, X., and Jetter, R. (2008). Very long chain alkylresorcinols accumulate in the intracuticular wax of rye (Secale cereale L.) leaves near the tissue surface. Phytochemistry, 69, 1197-1207. Jiang, Y., Satoh, K., Watanabe, S., Kusama, K., and Sakagami, H. (2001). Inhibition of

chlorogenic acid-induced cytotoxicity by CoCl2. Anticancer research, 21, 3349- 3353.

141

Jithesh, M. N., Prashanth, S. R., Sivaprakash, K. R., and Parida, A. K. (2006). Antioxidative response mechanisms in halophytes: their role in stress defence. Journal of Genetics, 85, 237. Jonfia-Essien, W. A., West, G., Alderson, P. G., and Tucker, G. (2008). Phenolic content and antioxidant capacity of hybrid variety cocoa beans. Food chemistry, 108, 1155-1159. Junglee, S., Urban, L., Sallanon, H., and Lopez-Lauri, F. (2014). Optimized assay for hydrogen peroxide determination in plant tissue using potassium iodide. American Journal of Analytical Chemistry, 5, 730. Kähkönen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J. P., Pihlaja, K., Kujala, T. S., and Heinonen, M. (1999). Antioxidant activity of plant extracts containing phenolic compounds. Journal of agricultural and food chemistry, 47, 3954-3962. Kamboh, M. A., Oki, Y., and Adachi, T. (2002). Effect of increasing salinity on growth and mineral composition of wheat varieties and role of sodium exclusion capacity in salt tolerance mechanisms. 岡山大学環境理工学部研究報告, 7, 99-106. Karimi, S.H., 1984. Ecophysiological studies of Atriplex triangularis Willd. to environmental stress. Ph.D. Thesis, Ohio University, Athens, USA Karuppanapandian, T., Moon, J. C., Kim, C., Manoharan, K., and Kim, W. (2011). Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Australian Journal of Crop Science, 5, 709. Katschnig, D., Broekman, R., and Rozema, J. (2013). Salt tolerance in the halophyte Salicornia dolichostachya Moss: growth, morphology and physiology. Environmental and Experimental Botany, 92, 32-42. Kavimani, S., Ilango, R., Karpagam, S., Suryaprabha, K., and Jaykar, B. (1999). Antisteroidogenic activity of floral extract of Thespesia populnea Corr. in mouse ovary. Indian Journal of Experimental Biology 37, 1241-42 Keiko, H., Tsukasa, I., and Sadamu, M. (2005). Variation in the quality and quantity of flavonoids in the leaves of coastal and inland populations of Adenophora triphylla var. japonica. Annals of the Tsukuba Botanic Garden, 24, 43-52. Keutgen, A. J., and Pawelzik, E. (2008). Quality and nutritional value of strawberry fruit under long term salt stress. Food Chemistry, 107, 1413-1420. Khan, D., Ahmad, R. A. F. I. Q., and Ismail, S. (1987). Germination, growth and ion regulation in Prosopis juliflora (Swartz) DC under saline conditions. Pakistan Journal of Botany, 19, 131-138.

142

Khan, M. A., and Qaiser, M. (2006). Halophytes of Pakistan: characteristics, distribution and potential economic usages. In Sabkha ecosystems (pp. 129-153). Springer, Dordrecht. Khan, M. A., Ungar, I. A., and Showalter, A. M. (1999). Effects of salinity on growth, ion content, and osmotic relations in Halopyrum mucronatum (L.) Stapf. Journal of Plant Nutrition, 22, 191-204. Khan, M. A., Ungar, I. A., and Showalter, A. M. (2000a). Effects of salinity on growth, water relations and ion accumulation of the subtropical perennial halophyte, Atriplex griffithii var. stocksii. Annals of Botany, 85, 225-232. Khan, M. A., Ungar, I. A., and Showalter, A. M. (2000b). The effect of salinity on the growth, water status, and ion content of a leaf succulent perennial halophyte, Suaeda fruticosa (L.) Forssk. Journal of Arid Environments, 45, 73-84. Kim, G. H., Kim, J. E., Rhie, S. J., and Yoon, S. (2015). The role of oxidative stress in neurodegenerative diseases. Experimental neurobiology, 24, 325-340. Kitajima, M., and Butler, W. L. (1975). Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 376, 105-115. Kocheva, K., Lambrev, P., Georgiev, G., Goltsev, V., and Karabaliev, M. (2004). Evaluation of chlorophyll fluorescence and membrane injury in the leaves of barley cultivars under osmotic stress. Bioelectrochemistry, 63, 121-124. Kocsy, G., Szalai, G., and Galiba, G. (2002). Induction of glutathione synthesis and glutathione reductase activity by abiotic stresses in maize and wheat. The Scientific World Journal, 2, 1699-1705. Kotmire, S. Y. 1983. Ecophysiological studies in the mangroves of western coast of India. Ph.D. Thesis, Shivaji University., Kolhapur (India). 225 p Kovinich, N., Kayanja, G., Chanoca, A., Otegui, M. S., and Grotewold, E. (2015). Abiotic stresses induce different localizations of anthocyanins in Arabidopsis. Plant signaling and behavior, 10, e1027850. Koyro, H. W. (2003). Study of potential cash crop halophytes by a quick check system: determination of the threshold of salinity tolerance and the ecophysiological demands. In Cash crop halophytes: recent studies (pp. 5-17). Springer, Dordrecht. Koyro, H. W. (2006). Effect of salinity on growth, photosynthesis, water relations and solute composition of the potential cash crop halophyte Plantago coronopus (L.). Environmental and Experimental Botany, 56, 136-146.

143

Koyro, H. W. and H. Lieth (2008). Global water crisis: the potential of cash crop halophytes to reduce the dilemma, In: H. Lieth, S. M. Garcia and B. Herzog (Eds.). Pp.7-19. Mangroves and halophytes: restoration and utilization. Tasks for Vegetation Science No. 43. Springer, the Netherlands. Koyro, H. W., and Huchzermeyer, B. (2004). Ecophysiological needs of the potential biomass crop Spartina townsendii Grov. Tropical Ecology, 45, 123-140. Koyro, H. W., Geissler, N., and Hussin, S. (2009). Survival at extreme locations: life strategies of halophytes. In Salinity and Water Stress (pp. 167-177). Springer, Dordrecht. Koyro, H. W., Hussain, T., Huchzermeyer, B., and Khan, M. A. (2013). Photosynthetic and growth responses of a perennial halophytic grass Panicum turgidum to increasing NaCl concentrations. Environmental and Experimental Botany, 91, 22- 29. Krall, J. P., and Edwards, G. E. (1992). Relationship between photosystem II activity and CO2 fixation in leaves. Physiologia Plantarum, 86, 180-187. Kranner, I., and Seal, C. E. (2013). Salt stress, signalling and redox control in seeds. Functional Plant Biology, 40, 848-859. Krasensky, J., and Jonak, C. (2012). Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. Journal of experimental botany, 63, 1593-1608. Kreslavski, V. D., Shirshikova, G. N., Lyubimov, V. Y., Shmarev, A. N., Boutanaev, A. M., Kosobryukhov, A. A., Schmitt, F.J., Friedrich, T. and Allakhverdiev, S. I. (2013). Effect of pre illumination with red light on photosynthetic parameters and oxidant-/antioxidant balance in Arabidopsis thaliana in response to UV-A. Journal of Photochemistry and Photobiology B: Biology, 127, 229-236. Krieger-Liszkay, A., Fufezan, C., and Trebst, A. (2008). Singlet oxygen production in photosystem II and related protection mechanism. Photosynthesis Research, 98, 551-564. Kritikar and Basu, 1999 K.R. Kritikar, B.D. Basu (2nd ed.), Indian Medicinal Plants, vol. 3, International Book Distributors, Dehradun, India (1999) p. 1610 Ksouri, R., Falleh, H., Megdiche, W., Trabelsi, N., Mhandi, B., Chaieb, K., Bakrouf, A., Magné, C. and Abdelly, C. (2009). Antioxidant and antimicrobial activities of the edible medicinal halophyte Tamarix gallica L. and related polyphenolic constituents. Food and Chemical toxicology, 47, 2083-2091.

144

Ksouri, R., Megdiche, W., Debez, A., Falleh, H., Grignon, C., and Abdelly, C. (2007). Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritima. Plant Physiology and Biochemistry, 45, 244-249. Ksouri, R., Megdiche, W., Falleh, H., Trabelsi, N., Boulaaba, M., Smaoui, A., and Abdelly, C. (2008). Influence of biological, environmental and technical factors on phenolic content and antioxidant activities of Tunisian halophytes. Comptes Rendus Biologies, 331, 865-873. Ksouri, R., Smaoui, A., Isoda, H., and Abdelly, C. (2012). Utilization of halophyte species as new sources of bioactive substances. Journal of Arid Land Studies, 22, 41-44. Kumar, A. S., Venkateshwaran, K., Vanitha, J., Saravanan, V. S., Ganesh, M., Vasudevan, M., and Sivakumar, T. (2009). Synergistic activity of methanolic extract of Thespesia populnea (Malvaceae) flowers with oxytetracycline. Bangladesh Journal Pharmacology, 4, 13-16. Kumar, D., Al Hassan, M., Naranjo, M. A., Agrawal, V., Boscaiu, M., and Vicente, O. (2017). Effects of salinity and drought on growth, ionic relations, compatible solutes and activation of antioxidant systems in oleander (Nerium oleander L.). Public Library of Science one, 12, e0185017. Kumar, S., Gupta, A., and Pandey, A. K. (2013). Calotropis procera root extract has the capability to combat free radical mediated damage. International scholarly research notices pharmacology, 2013. Kumari, A., Kumar, A., Kumar, V.R., (2004) Productivity of Calotropis procera in semia- arid regions of Rajasthan and its use as renewable source of energy In: Biomass for Energy, Industry and Climate Protection, (Ed, by L, Sjunnesson, J,E, Carrasco, P, Helm, and A Grassi), ETA-Renewable energies. Florence, Italy, WIP- Munich pp 276-278 Kweon M.H., Hwang, H.J., Sung, H.C., (2001). Identification and antioxidant activity of novel chlorogenic acid derivatives from bamboo (Phyllostachys edulis). Journal of Agricultural and Food Chemistry, 49, 4646-4655. Kytridis VP, Manetas Y. (2006) Mesophyll versus epidermal anthocyanins as potential in vivo antioxidants: evidence linking the putative antioxidant role to the proximity of oxy-radical source. Journal of Experimental Botany 57, 2203-2210. Laisk, A., Oja, V., Rasulov, B., Eichelmann, H., and Sumberg, A. (1997). Quantum yields and rate constants of photochemical and non-photochemical excitation quenching (experiment and model). Plant Physiology, 115, 803-815.

145

Larson, R. A. (1988). The antioxidants of higher plants. Phytochemistry, 27, 969-978. Law, M. Y., Charles, S. A., and Halliwell, B. (1983). Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of paraquat. Biochemical Journal, 210, 899-903. Lee, M. H., Cho, E. J., Wi, S. G., Bae, H., Kim, J. E., Cho, J. Y., Lee, S., Kim, J.H. and Chung, B. Y. (2013). Divergences in morphological changes and antioxidant responses in salt-tolerant and salt-sensitive rice seedlings after salt stress. Plant physiology and biochemistry, 70, 325-335. Lee, M. T., Lin, W. C., Yu, B., and Lee, T. T. (2017). Antioxidant capacity of phytochemicals and their potential effects on oxidative status in animals—A review. Asian-Australasian journal of animal sciences, 30, 299. Leidi, E. O., and Saiz, J. F. (1997). Is salinity tolerance related to Na accumulation in upland cotton (Gossypium hirsutum) seedlings?. Plant and Soil, 190, 67-75. Levitt, J. (1980). Responses of Plants to Environmental Stress, Volume 1: Chilling, Freezing, and High Temperature Stresses. Academic Press. Li, H. B., Wong, C. C., Cheng, K. W., and Chen, F. (2008). Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. LWT-Food Science and Technology, 41, 385-390. Lichtenhaler, H. (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembrane. Methods in Enzymology, 147, 350-82 Lim, J. H., Park, K. J., Kim, B. K., Jeong, J. W., and Kim, H. J. (2012). Effect of salinity stress on phenolic compounds and carotenoids in buckwheat (Fagopyrum esculentum M.) sprout. Food chemistry, 135, 1065-1070. Lins, C. M. T., de Souza, E. R., de Melo, H. F., Paulino, M. K. S. S., Magalhães, P. R. D., de Carvalho Leal, L. Y., and Santos, H. R. B. (2018). Pressure-volume (PV) curves in Atriplex nummularia Lindl. for evaluation of osmotic adjustment and water status under saline conditions. Plant Physiology and Biochemistry, 124, 155-159. Lisiewska, Z., Kmiecik, W., and Korus, A. (2006). Content of vitamin C, carotenoids, chlorophylls and polyphenols in green parts of dill (Anethum graveolens L.) depending on plant height. Journal of Food Composition and Analysis, 19, 134- 140. Liu, Z., Zhu, J., Yang, X., Wu, H., Wei, Q., Wei, H., and Zhang, H. (2018). Growth performance, organ-level ionic relations and organic osmoregulation of Elaeagnus angustifolia in response to salt stress. Public Library of Science one, 13, e0191552.

146

Logan, B. A., Kornyeyev, D., Hardison, J., and Holaday, A. S. (2006). The role of antioxidant enzymes in photoprotection. Photosynthesis Research, 88, 119-132. Lohaus, G., Hussmann, M., Pennewiss, K., Schneider, H., Zhu, J. J., and Sattelmacher, B. (2000). Solute balance of a maize (Zea mays L.) source leaf as affected by salt treatment with special emphasis on phloem retranslocation and ion leaching. Journal of Experimental Botany, 51, 1721-1732. Lokhande, V. H., and Suprasanna, P. (2012). Prospects of halophytes in understanding and managing abiotic stress tolerance. In Environmental adaptations and stress tolerance of plants in the era of climate change (pp. 29-56). Springer, New York, Lokhande, V. H., Nikam, T. D., and Penna, S. (2010). Differential Osmotic Adjustment to Iso-osmotic NaCl and PEG Stress in the in vitro Cultures of Sesuvium portulacastrum (L.) L. Journal of Crop Science and Biotechnology, 13, 251-256. López-Gómez, E., Sanjuán, M. A., Diaz-Vivancos, P., Mataix Beneyto, J., García-Legaz, M. F., and Hernández, J. A. (2007). Effect of salinity and rootstocks on antioxidant systems of loquat plants (Eriobotrya japonica Lindl.): response to supplementary boron addition. Environmental Experimental Botany, 160, 151-158. Lovelock, C. E., and Ball, M. C. (2002). Influence of salinity on photosynthesis of halophytes. In Salinity: environment-plants-molecules (pp. 315-339). Springer, Dordrecht. Lu, C., Qiu, N., Wang, B., and Zhang, J. (2003). Salinity treatment shows no effects on photosystem II photochemistry, but increases the resistance of photosystem II to heat stress in halophyte Suaeda salsa. Journal of Experimental Botany, 54, 851- 860. Ludwig, T. G., and Goldberg, H. J. (1956). The anthrone method for the determination of carbohydrates in foods and in oral rinsing. Journal of dental research, 35, 90-94.

Luengas-Caicedo, P. E., Braga, F. C., Brandão, G. C., and de Oliveira, A. B. (2007). Seasonal and intraspecific variation of flavonoids and proanthocyanidins in Cecropia glaziovi Sneth. Leaves from native and cultivated specimens. Zeitschrift für Naturforschung C, 62, 701-709.

Lutts, S., Kinet, J. M., and Bouharmont, J. (1996). NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Annals of botany, 78, 389-398.

147

Ma, Q., Hu, J., Zhou, X., Yuan, H., Kumar, T., Luan, S., and Wang, S. (2017). ZxAKT1 is essential for K+ uptake and K+ /Na+ homeostasis in the succulent xerophyte Zygophyllum xanthoxylum. The Plant Journal, 90, 48–60 Ma, Q., Yue, L. J., Zhang, J. L., Wu, G. Q., Bao, A. K., and Wang, S. M. (2011). Sodium chloride improves photosynthesis and water status in the succulent xerophyte Zygophyllum xanthoxylum. Tree Physiology, 32, 4-13. Macheix, J. J., Fleuriet, A., and Jay-Allemand, C. (2005). Les composés phénoliques des végétaux: UN exemple de métabolites secondaires d'importance économique. PPUR presses polytechniques. Maisuthisakul, P., Suttajit, M., and Pongsawatmanit, R. (2007). Assessment of phenolic content and free radical-scavenging capacity of some Thai indigenous plants. Food chemistry, 100, 1409-1418. Marcum, K. B. (1999). Salinity tolerance mechanisms of grasses in the subfamily Chloridoideae. Crop science, 39, 1153-1160. Martı̀nez, J. P., Lutts, S., Schanck, A., Bajji, M., and Kinet, J. M. (2004). Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L?. Journal of Plant Physiology, 161, 1041-1051. Martinez-Cayuela, M. (1995). Oxygen free radicals and human disease. Biochimie, 77, 147-161. Martínez-Peñalver, A., Reigosa, M. J., and Sánchez-Moreiras, A. M. (2011). Imaging chlorophyll a fluorescence reveals specific spatial distributions under different stress conditions. Flora-Morphology, Distribution, Functional Ecology of Plants, 206, 836-844. Masuda, T., Yonemori, S., Oyama, Y., Takeda, Y., Tanaka, T., Andoh, T., Shinohara, Aand Nakata, M. (1999). Evaluation of the antioxidant activity of environmental plants: activity of the leaf extracts from seashore plants. Journal of Agricultural and Food chemistry, 47, 1749-1754. Mazliak, P. (1983). Plant membrane lipids: changes and alterations during aging and senescence. In Post-harvest physiology and crop preservation (pp. 123-140). Springer, Boston, MA. McCune, L. M., and Johns, T. (2007). Antioxidant activity relates to plant part, life form and growing condition in some diabetes remedies. Journal of Ethnopharmacology, 112, 461-469.

148

Medini, F., and Ksouri, R. (2018). Antimicrobial Capacities of the Medicinal Halophyte Plants. In Natural Antimicrobial Agents (pp. 271-288). Springer, Cham. Medini, F., Bourgou, S., Lalancette, K., Snoussi, M., Mkadmini, K., Coté, I., Abdelly, C., Legault, J. and Ksouri, R. (2015). Phytochemical analysis, antioxidant, anti- inflammatory, and anticancer activities of the halophyte Limonium densiflorum extracts on human cell lines and murine macrophages. South African Journal of Botany, 99, 158-164. Meira, M., E.P.D. Silva, J.M. David and J.P. David (2012). Review of the genus Ipomoea: traditional uses, chemistry and biological activities. Revista Brasileira de Farmacognosi, 22, 682-713. Meloni, D. A., Oliva, M. A., Ruiz, H. A., and Martinez, C. A. (2001). Contribution of proline and inorganic solutes to osmotic adjustment in cotton under salt stress. Journal of Plant Nutrition, 24, 599-612. Merzlyak, M. N., and Solovchenko, A. E. (2002). Photostability of pigments in ripening apple fruit: a possible photoprotective role of carotenoids during plant senescence. Plant Science, 163, 881-888. Mierziak, J., Kostyn, K., and Kulma, A. (2014). Flavonoids as important molecules of plant interactions with the environment. Molecules, 19, 16240-16265. Miguel, M. G. (2018). Betalains in Some Species of the Amaranthaceae Family: A Review. Antioxidants, 7, 53. Minhas, P. S., Singh, Y. P., Tomar, O. S., Gupta, R. K., and Gupta, R. K. (1997). Effect of saline irrigation and its schedules on growth, biomass production and water use by Acacia nilotica and Dalbergia sissoo in a highly calcareous soil. Journal of arid environments, 36, 181-192. Mittova, V., Guy, M., Tal, M., and Volokita, M. (2004). Salinity up‐regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt‐tolerant tomato species Lycopersicon pennellii. Journal of experimental botany, 55, 1105- 1113. Mittova, V., Tal, M., Volokita, M., and Guy, M. (2003). Up‐regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt‐induced oxidative stress in the wild salt‐tolerant tomato species Lycopersicon pennellii. Plant, Cell and Environment, 26, 845-856. Mittova, V., Volokita, M., Guy, M., and Tal, M. (2000). Activities of SOD and the ascorbate‐glutathione cycle enzymes in subcellular compartments in leaves and

149

roots of the cultivated tomato and its wild salt‐tolerant relative Lycopersicon pennellii. Physiologia plantarum, 110, 42-51. Moinuddin, M., Gulzar, S., Ahmed, M. Z., Gul, B., Koyro, H. W., and Khan, M. A. (2014). Excreting and non-excreting grasses exhibit different salt resistance strategies. Annals of Botany Plants, 6. Moinuddin, M., Gulzar, S., Hameed, A., Gul, B., Khan, M. A., and Edwards, G. E. (2017). Differences in photosynthetic syndromes of four halophytic marsh grasses in Pakistan. Photosynthesis research, 131, 51-64. Moradi, F., and Ismail, A. M. (2007). Responses of photosynthesis, chlorophyll fluorescence and ROS-scavenging systems to salt stress during seedling and reproductive stages in rice. Annals of botany, 99, 1161-1173. Morales, F., Abadía, A., and Abadpa, J. (2008). Photoinhibition and photoprotection under nutrient deficiencies, drought and salinity. In Photoprotection, photoinhibition, gene regulation, and environment (pp. 65-85). Springer, Dordrecht. Moure, A., Cruz, J. M., Franco, D., Domı́nguez, J. M., Sineiro, J., Domı́nguez, H., Núñez, M.J. and Parajó, J. C. (2001). Natural antioxidants from residual sources. Food chemistry, 72, 145-171. Mroczek, A. (2015). Phytochemistry and bioactivity of triterpene saponins from Amaranthaceae family. Phytochemistry reviews, 14, 577-605. Muchate, N. S., Nikalje, G. C., Rajurkar, N. S., Suprasanna, P., and Nikam, T. D. (2016). Physiological responses of the halophyte Sesuvium portulacastrum to salt stress and their relevance for saline soil bio-reclamation. Flora, 224, 96-105. Munekata, P. E. S., Domínguez, R., Franco, D., Bermúdez, R., Trindade, M. A., and Lorenzo, J. M. (2017). Effect of natural antioxidants in Spanish salchichón elaborated with encapsulated n-3 long chain fatty acids in konjac glucomannan matrix. Meat science, 124, 54-60. Munns, R. (2002). Comparative physiology of salt and water stress. Plant, cell and environment, 25, 239-250. Munns, R., and Gilliham, M. (2015). Salinity tolerance of crops–what is the cost?. New phytologist, 208, 668-673.

Munns, R., and Termaat, A. (1986). Whole-plant responses to salinity. Functional Plant Biology, 13, 143-160. Munns, R., and Tester, M. (2008). Mechanisms of salinity tolerance. Annu. Rev. Plant Biol., 59, 651-681. 150

Muscolo, A., Sidari, M., and Panuccio, M. R. (2003). Tolerance of kikuyu grass to long term salt stress is associated with induction of antioxidant defences. Plant growth regulation, 41, 57-62. Muthukumar, S., and Veerappa, N. S. (2018). Phytochemical analysis in the root and leaf of Thespesia populnea (Linn) Soland ex correa. Journal of Pharmacognosy and Phytochemistry, 7, 414-417. Muthukumarasamy, M., Gupta, S. D., and Panneerselvam, R. (2000). Enhancement of peroxidase, polyphenol oxidase and superoxide dismutase activities by triadimefon in NaCl stressed Raphanus sativus L. Biologia Plantarum, 43, 317-320. Naczk, M., and Shahidi, F. (2004). Extraction and analysis of phenolics in food. Journal of Chromatography A, 1054, 95-111. Nagaratna, A., Hegde, P. L., and Harini, A. (2015). A Pharmacological review on Gorakha ganja (Aerva lanata (Linn) Juss. Ex. Schult). Journal of pharmacognosy and phytochemistry, 3, 35-39. Nagy, L., Kiss, V., Brumfeld, V., Osvay, K., Börzsönyi, A., Magyar, M., Szabó, T., Dorogi, M. and Malkin, S. (2015). Thermal effects and structural changes of photosynthetic reaction centers characterized by wide frequency band hydrophone: Effects of carotenoids and terbutryn. Photochemistry and photobiology, 91(6), 1368-1375. Naidoo, G., Naidoo, Y., and Achar, P. (2012). Ecophysiological responses of the salt marsh grass Spartina maritima to salinity. African Journal of Aquatic Science, 37, 81-88. Naidoo, G., Somaru, R., and Achar, P. (2008). Morphological and physiological responses of the halophyte, Odyssea paucinervis (Staph) (Poaceae), to salinity. Flora- Morphology, Distribution, Functional Ecology of Plants, 203, 437-447. Nakagami, T., Nanaumi-Tamura, N., Toyomura, K., Nakamura, T., and Shigehisa, T. (1995).Dietary flavonoids as potential natural biological response modifiers affecting the autoimmune system. Journal of food science, 60, 653-656. Nakano, Y., and Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and cell physiology, 22, 867-880.

Nazir, S., Qasim, M., Gul, B., and Khan, M. A. (2018).Antioxidant properties and phenolic composition of coastal halophytes commonly used as medicine. International Journal of Biology and Biotechnology 15, 66-71

151

Nedjimi, B., and Daoud, Y. (2006). Effect of Na 2 SO 4 on the growth, water relations, proline, total soluble sugars and ion content of Atriplex halimus subsp. schweinfurthii through in vitro culture. In Anales de Biologia 28, 35-43. Nieva, F. J. J., Castellanos, E. M., Figueroa, M. E., and Gil, F. (1999). Gas exchange and chlorophyll fluorescence of C3 and C4 saltmarsh species. Photosynthetica, 36, 397-406. Niknam, V., and Ebrahimzadeh, H. (2002). Phenolics content in Astragalus species. Pakistan Journal of Botany, 34, 283-289. Nishiyama, Y., and Murata, N. (2014). Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery. Applied microbiology and biotechnology, 98, 8777- 8796. Niu, C. F., Wei, W. E. I., Zhou, Q. Y., Tian, A. G., Hao, Y. J., Zhang, W. K., Ma, B., Lin, Q., Zhang, Z.B., Zhang, J.S. and Chen, S. Y. (2012). Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell Environment, 35, 1156-1170. Noctor, G., and Foyer, C. H. (1998). Ascorbate and glutathione: keeping active oxygen under control. Annual review of plant biology, 49, 249-279. Ojeda, H., Andary, C., Kraeva, E., Carbonneau, A., and Deloire, A. (2002). Influence of pre-and postveraison water deficit on synthesis and concentration of skin phenolic compounds during berry growth of Vitis vinifera cv. Shiraz. American Journal of Enology and Viticulture, 53, 261-267. Osmond, C. B. (1994). What is photoinhibition? Some insights from comparisons of shade and sun plants. Photoinhibition of photosynthesis-from molecular mechanisms to the field. Environmental Plant Biology, 1-24. Oueslati, S., Karray-Bouraoui, N., Attia, H., Rabhi, M., Ksouri, R., and Lachaal, M. (2010). Physiological and antioxidant responses of Mentha pulegium (Pennyroyal) to salt stress. Acta Physiologiae Plantarum, 32, 289-296. Oueslati, S., Ksouri, R., Falleh, H., Pichette, A., Abdelly, C., and Legault, J. (2012a). Phenolic content, antioxidant, anti-inflammatory and anticancer activities of the edible halophyte Suaeda fruticosa Forssk. Food Chemistry, 132, 943-947.

Oueslati, S., Trabelsi, N., Boulaaba, M., Legault, J., Abdelly, C., and Ksouri, R. (2012b). Evaluation of antioxidant activities of the edible and medicinal Suaeda species and related phenolic compounds. Industrial Crops and Products, 36, 513-518. 152

Ozfidan-Konakci, C., Yildiztugay, E., and Kucukoduk, M. (2015). Protective roles of exogenously applied gallic acid in Oryza sativa subjected to salt and osmotic stresses: effects on the total antioxidant capacity. Plant growth regulation, 75, 219- 234. Pacifico, S., Galasso, S., Piccolella, S., Kretschmer, N., Pan, S. P., Nocera, P., P., Lettieri, A., Bauer, R. and Monaco, P. (2018). Winter wild fennel leaves as a source of anti- inflammatory and antioxidant polyphenols. Arabian journal of chemistry, 11, 513- 524. Palozza, P., Catalano, A., Simone, R., and Cittadini, A. (2012). Lycopene as a guardian of redox signalling. Acta Biochimica Polonica, 59, 21-25 Papas, A. M. (1999). Diet and antioxidant status. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association, 37, 999-1007. Pardossi, A., Malorgio, F., Oriolo, D., Gucci, R., Serra, G., and Tognoni, F. (1998). Water relations and osmotic adjustment in Apium graveolens during long‐term NaCl stress and subsequent relief. Physiologia plantarum, 102, 369-376. Parida, A. K., and Das, A. B. (2005). Salt tolerance and salinity effects on plants: a review. Ecotoxicology and environmental safety, 60, 324-349. Parida, A. K., and Jha, B. (2010). Antioxidative defense potential to salinity in the euhalophyte Salicornia brachiata. Journal of Plant Growth Regulation, 29, 137- 148. Parida, A. K., and Jha, B. (2013). Physiological and biochemical responses reveal the drought tolerance efficacy of the halophyte Salicornia brachiata. Journal of Plant Growth Regulation, 32, 342-352. Parida, A. K., Das, A. B., and Mohanty, P. (2004). Defense potentials to NaCl in a mangrove, Bruguiera parviflora: differential changes of isoforms of some antioxidative enzymes. Journal of plant physiology, 161, 531-542. Parida, A., Das, A. B., and Das, P. (2002). NaCl stress causes changes in photosynthetic pigments, proteins, and other metabolic components in the leaves of a true mangrove, Bruguiera parviflora, in hydroponic cultures. Journal of Plant Biology, 45, 28-36. Parthasarathy, R., Ilavarasan, R., and Karrunakaran, C. M. (2009). Antidiabetic activity of Thespesia populnea bark and leaf extract against streptozotocin induced diabetic rats. International Journal of PharmTech Research, 1, 1069-1072.

153

Patel, M. K., Mishra, A., and Jha, B. (2016). Untargeted metabolomics of halophytes. In Marine OMICS (pp. 329-346). CRC Press. Pearson, D. (1976). Chemical analysis of food. 7th Edition Edinburgh. London and New York: Churchill Livingston. pp. 274-275 Pedranzani, H., Sierra-de-Grado, R., Vigliocco, A., Miersch, O., and Abdala, G. (2007). Cold and water stresses produce changes in endogenous jasmonates in two populations of Pinus pinaster Ait. Plant Growth Regulation, 52, 111-116. Penella, C., Landi, M., Guidi, L., Nebauer, S. G., Pellegrini, E., San Bautista, A., Remorini, D., Nali, C., López-Galarza, S. and Calatayud, A. (2016). Salt-tolerant rootstock increases yield of pepper under salinity through maintenance of photosynthetic performance and sinks strength. Journal of plant physiology, 193, 1-11. Petridis, A., Therios, I., Samouris, G., and Tananaki, C. (2012). Salinity-induced changes in phenolic compounds in leaves and roots of four olive cultivars (Olea europaea L.) and their relationship to antioxidant activity. Environmental and Experimental Botany, 79, 37-43. Plouguerne, E., Le Lann, K., Connan, S., Jechoux, G., Deslandes, E., and Stiger-Pouvreau, V. (2006). Spatial and seasonal variation in density, reproductive status, length and phenolic content of the invasive brown macroalga Sargassum muticum (Yendo) Fensholt along the coast of Western Brittany (France). Aquatic Botany, 85, 337- 344. Polle, A., Otter, T., and Seifert, F. (1994). Apoplastic peroxidases and lignification in needles of Norway spruce (Picea abies L.). Plant Physiology, 106, 53-60. Pompeiano, A., Di Patrizio, E., Volterrani, M., Scartazza, A., and Guglielminetti, L. (2016). Growth responses and physiological traits of seashore paspalum subjected to short-term salinity stress and recovery. Agricultural Water Management, 163, 57-65. Pompeiano, A., Giannini, V., Gaetani, M., Vita, F., Guglielminetti, L., Bonari, E., and Volterrani, M. (2014). Response of warm–season grasses to N fertilization and salinity. Scientia Horticulturae, 177, 92-98. Pompeiano, A., Landi, M., Meloni, G., Vita, F., Guglielminetti, L., and Guidi, L. (2017). Allocation pattern, ion partitioning, and chlorophyll a fluorescence in Arundo donax L. in responses to salinity stress. Plant Biosystems-An International Journal Dealing with all Aspects of Plant Biology, 151, 613-622.

154

Pongprayoon, U., Bohlin, L., and Wasuwat, S. (1991). Neutralization of toxic effects of different crude jellyfish venoms by an extract of Ipomoea pes-caprae (L.) R. Br. Journal of ethnopharmacology, 35, 65-69. Prieto, P., Pineda, M., and Aguilar, M. (1999). Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Analytical biochemistry, 269, 337-341. Proestos, C., Sereli, D., Komaitis, M., 2006. Determination of phenolic compounds in aromatic plants by RP-HPLC and GC–MS. Food Chemistry 95, 44–52. Pujol, J. A., Calvo, J. F., and Ramírez-Díaz, L. (2001). Seed germination, growth, and osmotic adjustment in response to NaCl in a rare succulent halophyte from southeastern Spain. Wetlands, 21, 256-264. Purohit, S., Laloraya, M. M., and Bharti, S. (1991). Effect of phenolic compounds on abscisic acid‐induced stomatal movement: Structure–activity relationship. Physiologia Plantarum, 81, 79-82. Qaderi, M. M., Kurepin, L. V., and Reid, D. M. (2006). Growth and physiological responses of canola (Brassica napus) to three components of global climate change: temperature, carbon dioxide and drought. Physiologia Plantarum, 128, 710-721. Qasim, M., Abideen, Z., Adnan, M. Y., Ansari, R., Gul, B., and Khan, M. A. (2014). Traditional ethnobotanical uses of medicinal plants from coastal areas. Journal Coastal Life Medicine, 2, 22-30. Qasim, M., Abideen, Z., Adnan, M. Y., Gulzar, S., Gul, B., Rasheed, M., and Khan, M. A. (2017). Antioxidant properties, phenolic composition, bioactive compounds and nutritive value of medicinal halophytes commonly used as herbal teas. South African Journal of Botany, 110, 240-250. Qasim, M., Aziz, I., Rasheed, M., Gul, B., and Khan, M. A. (2016). Effect of extraction solvents on polyphenols and antioxidant activity of medicinal halophytes. Pakistan Journal of Botany, 48, 621-7. Qasim, M., Gulzar, S., and Khan, M. A. (2011). Halophytes as medicinal plants. In NAM Meeting in Denizli, Turkey.

Qasim, M., Gulzar, S., Shinwari, Z. K., Aziz, I., and Khan, M. A. (2010). Traditional ethnobotanical uses of halophytes from Hub, Balochistan. Pakistan Journal of Botany, 42, 1543-1551. 155

Qiu, F. Y., Liao, B. W., and Xiao, F. M. (2011). Salt Tolerance of Semi-mangrove Plant Thespesia populnea Seedlings Journal of Forest Research, 1, 011. Qureshi, R., Waheed, A., Arshad, M. U. H. A. M. M. A. D., and Umbreen, T. (2009). Medico-ethnobotanical inventory of tehsil Chakwal, Pakistan. Pakistan Journal of Botany, 41, 529-538. Rahman, M. M., Mostofa, M. G., Rahman, M. A., Miah, M. G., Saha, S. R., Karim, M. A., and Tran, L. S. P. (2018). Insight into salt tolerance mechanisms of the halophyte Achras sapota: an important fruit tree for agriculture in coastal areas. Protoplasma, 1-11. Rai, V. K., and Sharma, S. (1986). Reversal of ABA-induced stomatal closure by phenolic compounds. Journal of Experimental Botany, 37, 129-134. Rajamurugan, R., Shilpa, G.R., Kumaravel, S., Paranthaman, R. (2013). R GC-MS analysis, antioxidant and antibacterial activities of Thespesia populnea Linn. Leaf – in vitro study. Bio Med Rx 1,248-253. Ranjbarfordoei, A., Samson, R., and Van Damme, P. (2006). Chlorophyll fluorescence performance of sweet almond Prunus dulcis (Miller) D. Webb in response to salinity stress induced by NaCl. Photosynthetica, 44, 513-522. Rashid, S., Iftikhar, Q., Arshad, M., and Iqbal, J. (2000). Chemical composition and antibacterial activity of Suaeda fruticosa Forsk. from Cholistan, Pakistan. Pakistan Journal of Biological Sciences (Pakistan), 3, 348-349. Rawat, J. S., and Banerjee, S. P. (1998). The influence of salinity on growth, biomass production and photosynthesis of Eucalyptus camaldulensis Dehnh. and Dalbergia sissoo Roxb. seedlings. Plant and Soil, 205, 163-169. Rawlings, A. V., and Matts, P. J. (2005). Stratum corneum moisturization at the molecular level: an update in relation to the dry skin cycle. J Invest Dermatol, 124, 1099- 1110. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free radical biology and medicine, 26, 1231-1237. Rebey, I. B., Bourgou, S., Rahali, F. Z., Msaada, K., Ksouri, R., and Marzouk, B. (2017). Relation between salt tolerance and biochemical changes in cumin (Cuminum cyminum L.) seeds. Journal of food and drug analysis, 25, 391-402.

156

Redondo‐Gómez, S., Mateos‐Naranjo, E., Figueroa, M. E., and Davy, A. J. (2010). Salt stimulation of growth and photosynthesis in an extreme halophyte, Arthrocnemum macrostachyum. Plant Biology, 12, 79-87. Reginato, M. A., Castagna, A., Furlán, A., Castro, S., Ranieri, A., and Luna, V. (2014).

Physiological responses of a halophytic shrub to salt stress by Na2SO4 and NaCl: oxidative damage and the role of polyphenols in antioxidant protection. Annals of Botany, Plants, 6. Reginato, M., Varela, C., Cenzano, A. M., and Luna, V. (2015). Role of polyphenols as antioxidants in native species from Argentina under drought and salinization. In Reactive Oxygen Species and Oxidative Damage in Plants under Stress (pp. 247-267). Springer, Cham. Rezazadeh, A., Ghasemnezhad, A., Barani, M., and Telmadarrehei, T. (2012). Effect of salinity on phenolic composition and antioxidant activity of artichoke (Cynara scolymus L.) leaves. Research Journal of Medicinal Plant, 6, 245-252. Riadh, K., Wided, M., Hans-Werner, K., and Chedly, A. (2010). Responses of halophytes to environmental stresses with special emphasis to salinity. In Advances in Botanical Research (Vol. 53, pp. 117-145). Academic Press. Rice-Evans, C. A., Miller, N. J., and Paganga, G. (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free radical biology and medicine, 20, 933-956. Ritchie, R. J. (2006). Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. Photosynthesis research, 89, 27-41. Rochaix, J.D. (2011). Regulation of photosynthetic electron transport. Biochimica et Biophysica Acta, 1807, 375-383 Rodrıguez, P., Torrecillas, A., Morales, M. A., Ortuno, M. F., and Sánchez-Blanco, M. J. (2005). Effects of NaCl salinity and water stress on growth and leaf water relations of Asteriscus maritimus plants. Environmental and Experimental Botany, 53(2), 113-123. Ross, A. B., Shepherd, M. J., Schüpphaus, M., Sinclair, V., Alfaro, B., Kamal-Eldin, A., and Åman, P. (2003). Alkylresorcinols in cereals and cereal products. Journal of Agricultural and Food Chemistry, 51, 4111-4118. Roy, S., and Chakraborty, U. (2014). Salt tolerance mechanisms in salt tolerant grasses (STGs) and their prospects in cereal crop improvement. Botanical Studies, 55, 31.

157

Roy, S., Sehgal, R., Padhy, B. M., and Kumar, V. L. (2005). Antioxidant and protective effect of latex of Calotropis procera against alloxan-induced diabetes in rats. Journal of Ethnopharmacology, 102, 470-473. Rozema J, Schat H (2013) Salt tolerance of halophytes, research questions reviewed in the perspective of saline agriculture. Environmental and Experimental Botany 92, 83– 95. Rozema, J. (1991). Growth, water and ion relationships of halophytic monocotyledonae and dicotyledonae; a unified concept. Aquatic Botany, 39, 17-33. Rubio, M. C., Bustos‐Sanmamed, P., Clemente, M. R., and Becana, M. (2009). Effects of salt stress on the expression of antioxidant genes and proteins in the model legume Lotus japonicus. New Phytologist, 181, 851-859. Saenger, P. (2002). Adapting to the ‘Mangrove Environment’. In Mangrove Ecology, Silviculture and Conservation (pp. 49-100). Springer, Dordrecht. Saenger, P. (2013). Mangrove ecology, silviculture and conservation. Springer Science and Business Media. Kluwer, pp. 360 Sahin, K., Orhan, C., Akdemir, F., Tuzcu, M., Iben, C., and Sahin, N. (2012). Resveratrol protects quail hepatocytes against heat stress: modulation of the Nrf2 transcription factor and heat shock proteins. Journal of animal physiology and animal nutrition, 96, 66-74. Sahin, K., Orhan, C., Tuzcu, M., Ali, S., Sahin, N., and Hayirli, A. (2010). Epigallocatechin-3-gallate prevents lipid peroxidation and enhances antioxidant defense system via modulating hepatic nuclear transcription factors in heat- stressed quails. Poultry Science, 89, 2251-2258. Sahin, K., Tuzcu, M., Gencoglu, H., Dogukan, A., Timurkan, M., Sahin, N., Aslan, A.and Kucuk, O. (2010). Epigallocatechin-3-gallate activates Nrf2/HO-1 signaling pathway in cisplatin-induced nephrotoxicity in rats. Life sciences, 87, 240-245. Sairam, R. K., Srivastava, G. C., Agarwal, S., and Meena, R. C. (2005). Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes. Biologia Plantarum, 49, 85. Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H., and Kanazawa, K. (2003). Simultaneous determination of all polyphenols in vegetables, fruits, and teas. Journal of Agricultural and Food Chemistry, 51, 571-581.

158

Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H., and Kanazawa, K. (2003). Simultaneous determination of all polyphenols in vegetables, fruits, and teas. Journal of Agricultural and Food Chemistry, 51, 571-581. Salleo, S. (1983). Water relations parameters of two Sicilian species of Senecio (groundsel) measured by the pressure bomb technique. New Phytologist, 95, 179- 188. Sánchez-Rodríguez, E., Moreno, D. A., Ferreres, F., del Mar Rubio-Wilhelmi, M., and Ruiz, J. M. (2011). Differential responses of five cherry tomato varieties to water stress: changes on phenolic metabolites and related enzymes. Phytochemistry, 72, 723-729. Sangeetha, R., and Vedasree, N. (2012). In Vitro α-amylase inhibitory activity of the leaves of Thespesia populnea. ISRN pharmacology, 2012. Santos, R. M., Oliveira, M. S., Ferri, P. H., and Santos, S. C. (2011). Seasonal variation in the phenol content of Eugenia uniflora L. leaves. Revista Brasileira de Plantas Medicinais, 13, 85-89. Satoh, K., Smith, C. M., and Fork, D. C. (1983). Effects of salinity on primary processes of photosynthesis in the red alga Porphyra perforata. Plant physiology, 73, 643- 647. Savithramma, N., Yugandhar, P., Devi, P. S., Ankanna, S., Suhrulatha, D., Prasad, K. S., and Chetty, K. M. (2017). Documentation of ethnomedicinal information and antimicrobial validation of Thespesia populnea used by Yanadi tribe of Ganugapenta village, Chittoor district, Andhra Pradesh, India. Journal of intercultural ethnopharmacology, 6, 158-196. Scagel, C. F., Lee, J., and Mitchell, J. N. (2019). Salinity from NaCl changes the nutrient and polyphenolic composition of basil leaves. Industrial Crops and Products, 127, 119-128. Scarascia‐Mugnozza, G., De Angelis, P., Matteucci, G., and Valentini, R. (1996). Long‐

term exposure to elevated [CO2] in a natural Quercus ilex L. community: net photosynthesis and photochemical efficiency of PSII at different levels of water stress. Plant, Cell and Environment, 19, 643-654. Schreiber, U., Schliwa, U., and Bilger, W. (1986). Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis research, 10, 51-62.

159

Selmar, D., and Kleinwächter, M. (2013). Stress enhances the synthesis of secondary plant products: the impact of stress-related over-reduction on the accumulation of natural products. Plant and Cell Physiology, 54, 817-826. Sepulveda-Jiménez, G., Rueda-Benítez, P., Porta, H., and Rocha-Sosa, M. (2004). Betacyanin synthesis in red beet (Beta vulgaris) leaves induced by wounding and bacterial infiltration is preceded by an oxidative burst. Physiological and Molecular Plant Pathology, 64, 125-133. Shabala, S. (2013). Learning from halophytes: physiological basis and strategies to improve abiotic stress tolerance in crops. Annals of botany, 112, 1209-1221. Shabala, S., and Mackay, A. (2011). Ion transport in halophytes. In Advances in botanical research (Vol. 57, pp. 151-199). Academic Press. Shaker, K. H., Morsy, N., Zinecker, H., Imhoff, J. F., and Schneider, B. (2010). Secondary metabolites from Calotropis procera (Aiton). Phytochemistry Letters, 3, 212-216. Shalata, A., and Neumann, P. M. (2001). Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation. Journal of Experimental Botany, 52, 2207-2211. Sharma, P., Jha, A. B., Dubey, R. S., and Pessarakli, M. (2012). Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of botany, 2012. Sharp, R. E., Poroyko, V., Hejlek, L. G., Spollen, W. G., Springer, G. K., Bohnert, H. J., and Nguyen, H. T. (2004). Root growth maintenance during water deficits: physiology to functional genomics. Journal of experimental botany, 55, 2343- 2351. Shinwari, Z. K. (2010). Medicinal plants research in Pakistan. Journal of medicinal plants research, 4, 161-176. Shonubi, O. O., and Okusanya, O. T. (2007). The growth and physiological responses of Paspalum vaginatum Sw. and Paspalum scrobiculatum Linn. in relation to salinity. Asian Journal of Plant Sciences, 6, 949-956. Short, D. C., and Colmer, T. D. (1999). Salt tolerance in the halophyte Halosarcia pergranulata subsp. pergranulata. Annals of Botany, 83, 207-213. Silva, F. G., Pinto, J. E. B. P., Nascimento, V. E., Sales, J. F., Souchie, E. L., and Bertolucci, S. K. V. (2007). Seasonal variation in the total phenol contents in cultivated and wild carqueja (Baccharis trimera (Less) DC.). Brazilian Journal of Medicinal Plants, 9, 52-57.

160

Silveira, J. A. G., Araújo, S. A. M., Lima, J. P. M. S., and Viégas, R. A. (2009). Roots and leaves display contrasting osmotic adjustment mechanisms in response to NaCl- salinity in Atriplex nummularia. Environmental and Experimental Botany, 66, 1- 8. Singleton, V. L., and Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American journal of Enology and Viticulture, 16, 144-158. Slama, I., Abdelly, C., Bouchereau, A., Flowers, T., and Savoure, A. (2015). Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Annals of Botany, 115, 433-447. Sobhanian, H., Motamed, N., Jazii, F. R., Nakamura, T., and Komatsu, S. (2010). Salt stress induced differential proteome and metabolome response in the shoots of Aeluropus lagopoides (Poaceae), a halophyte C4 plant. Journal of Proteome Research, 9, 2882-2897. Sobrado, M. A. (2005). Leaf characteristics and gas exchange of the mangrove Laguncularia racemosa as affected by salinity. Photosynthetica, 43, 217-221. Soni, U., Brar, S., and Gauttam, V. K. (2015). Effect of seasonal variation on secondary metabolites of medicinal plants. International journal of Pharmaceutical sciences and research, 6, 3654-3662. Soriano, A., Alañón, M. E., Alarcón, M., García-Ruíz, A., Díaz-Maroto, M. C., and Pérez- Coello, M. S. (2018). Oak wood extracts as natural antioxidants to increase shelf life of raw pork patties packed in map. Food Research International.111, 524-533 Spanò, C., Bruno, M., and Bottega, S. (2013). Calystegia soldanella: dune versus laboratory plants to highlight key adaptive physiological traits. Acta physiologiae plantarum, 35, 1329-1336. Sreenivasulu, N., Grimm, B., Wobus, U., and Weschke, W. (2000). Differential response of antioxidant compounds to salinity stress in salt‐tolerant and salt‐sensitive seedlings of foxtail millet (Setaria italica). Physiologia plantarum, 109, 435-442. Štajner, D., Orlovic, S., Popovic, B. M., Kebert, M., and Galic, Z. (2011). Screening of drought oxidative stress tolerance in Serbian melliferous plant species. African Journal of Biotechnology, 10, 1609-1614. Stanković, M. S., Petrovic, M., Godjevac, D., and Stevanović, Z. D. (2015). Screening inland halophytes from the central Balkan for their antioxidant activity in relation

161

to total phenolic compounds and flavonoids: Are there any prospective medicinal plants?. Journal of Arid Environments, 120, 26-32. Stefanoudaki, E. (2004). Factors affecting olive oil quality (Doctoral dissertation, Ph. D. Thesis, University of Cardiff, UK). Stefanov, M., Yotsova, E., Rashkov, G., Ivanova, K., Markovska, Y., and Apostolova, E. L. (2016). Effects of salinity on the photosynthetic apparatus of two Paulownia lines. Plant Physiology and Biochemistry, 101, 54-59. Stepien, P., and Johnson, G. N. (2009). Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte Thellungiella: role of the plastid terminal oxidase as an alternative electron sink. Plant physiology, 149, 1154-1165. Subudhi, P. K., and Baisakh, N. (2011). Spartina alterniflora Loisel., a halophyte grass model to dissect salt stress tolerance. In Vitro Cellular and Developmental Biology- Plant, 47, 441-457. Sudhir, P., and Murthy, S. D. S. (2004). Effects of salt stress on basic processes of photosynthesis. Photosynthetica, 42, 481-486. Sun, B., Ricardo-da-Silva, J. M., and Spranger, I. (1998). Critical factors of vanillin assay for catechins and proanthocyanidins. Journal of Agricultural and Food Chemistry, 46, 4267-4274. Sunthonpalin, P., and Wasuwat, S. (1985). Jellyfish dermatitis treated by the extract of Ipomea pes-caprac. Siriraj Hospital Gaz, 37, 328-338. Suvarna, C. M., Sriya, P., Arshad, M. D., and Pavan, K. (2018). A review on phytochemical and pharmacological properties of Thespesia populnea. Journal of Drug Delivery and Therapeutics, 8, 1-4. Suvarna, C. M., Sriya, P., Arshad, M. D., and Pavan, K. (2018). A review on phytochemical and pharmacological properties of Thespesia populnea. Journal of Drug Delivery and Therapeutics, 8, 1-4.

Suzuki, N., Koussevitzky, S. H. A. I., Mittler, R. O. N., and Miller, G. A. D. (2012). ROS and redox signalling in the response of plants to abiotic stress. Plant, Cell and Environment, 35, 259-270. Suzuki, T., Mukasa, Y., Morishita, T., Kim, S. J., Woo, S. H., Noda, T., Takigawa, S. and Yamauchi, H. (2015). Possible Roles of Rutin in Buckwheat Plant. The European Journal of Plant science and Biotechnology.6, 37-42

162

Suzuki, Y., Kono, Y., Inoue, T., and Sakurai, A. (1998). A potent antifungal benzoquinone in etiolated sorghum seedlings and its metabolites. Phytochemistry, 47, 997-1001. Sytar, O., Mbarki, S., Zivcak, M., and Brestic, M. (2018). The Involvement of Different Secondary Metabolites in Salinity Tolerance of Crops. In Salinity Responses and Tolerance in Plants, Volume 2 (pp. 21-48). Springer, Cham. Taârit, M. B., Msaada, K., Hosni, K., and Marzouk, B. (2012). Physiological changes, phenolic content and antioxidant activity of Salvia officinalis L. grown under saline conditions. Journal of the Science of Food and Agriculture, 92, 1614-1619. Taïbi, K., Taïbi, F., Abderrahim, L. A., Ennajah, A., Belkhodja, M., and Mulet, J. M. (2016). Effect of salt stress on growth, chlorophyll content, lipid peroxidation and antioxidant defence systems in Phaseolus vulgaris L. South African Journal of Botany, 105, 306-312. Takahashi, S., and Murata, N. (2008). How do environmental stresses accelerate photoinhibition?. Trends in plant science, 13, 178-182. Tanaka, Y., Sasaki, N., and Ohmiya, A. (2008). Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. The Plant Journal, 54, 733-749. Tapia, E., Zatarain-Barrón, Z. L., Hernández-Pando, R., Zarco-Márquez, G., Molina-Jijón, E., Cristóbal-García, M., Santamaría, J. and Pedraza-Chaverri, J. (2013). Curcumin reverses glomerular hemodynamic alterations and oxidant stress in 5/6 nephrectomized rats. Phytomedicine, 20, 359-366. Tatiana, Z., Yamashita, K., Matsumoto, H. (1999) Iron deficiency induced changes in ascorbate content and enzyme activities related to ascorbate metabolism in cucumber roots. Plant Cell Physiology, 40, 273-280. Tattini, M., Guidi, L., Morassi‐Bonzi, L., Pinelli, P., Remorini, D., Degl'Innocenti, E., Giordano, C., Massai, R. and Agati, G. (2005). On the role of flavonoids in the integrated mechanisms of response of Ligustrum vulgare and Phillyrea latifolia to high solar radiation. New Phytologist, 167, 457-470.

Teakle, N. L., Bazihizina, N., Shabala, S., Colmer, T. D., Barrett-Lennard, E. G., Rodrigo- Moreno, A., and Läuchli, A. E. (2013). Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots. Environmental and Experimental Botany, 87, 69-78. Teixeira, F. M., Ramos, M. V., Soares, A. A., Oliveira, R. S., Almeida-Filho, L. C. P., Oliveira, J. S., Marinho-Filho, J.D. and Carvalho, C. P. S. (2011). In vitro tissue 163

culture of the medicinal shrub Calotropis procera to produce pharmacologically active proteins from plant latex. Process biochemistry, 46, 1118-1124. Thimnavukkamsu, P., L. Ramkumar, T. Ramanarhan and G. Silambamsan (2010). Antioxidant activity of selected coastal medicinal plants. World, 2,134-137. Tomos, A.D., Wyn Jones, R.G. (1982). Water relation in the epidermal cells of the halophyte Suaeda maritima. In: Franks F, Mathia S, eds. Biophysics of water. New York, NY, USA: Wiley, 327–331 Touchette B (2006) Salt tolerance in a Juncus roemerianus brackish marsh: spatial variations in plant water relations. Journal of Experimental Marine Biology and Ecology 337, 1–12. Touchette, B. W. (2007). Seagrass-salinity interactions: physiological mechanisms used by submersed marine angiosperms for a life at sea. Journal of Experimental Marine Biology and Ecology, 350, 194-215. Tour, N., and Talele, G. (2011). Anti-inflammatory and gastromucosal protective effects of Calotropis procera (Asclepiadaceae) stem bark. Journal of natural medicines, 65, 598-605. Trabelsi, N., Falleh, H., Jallali, I., Daly, A. B., Hajlaoui, H., Smaoui, A., Abdelly, C. and Ksouri, R. (2012). Variation of phenolic composition and biological activities in Limoniastrum monopetalum L. organs. Acta physiologiae plantarum, 34, 87-96. Trabelsi, N., Waffo-Téguo, P., Snoussi, M., Ksouri, R., Mérillon, J. M., Smaoui, A., and Abdelly, C. (2013). Variability of phenolic composition and biological activities of two Tunisian halophyte species from contrasted regions. Acta physiologiae plantarum, 35, 749-761. Trease, G.E., Evans, I.C. (1983). Pharmacognosy (12 th edn) Bailliere Tindall London. pp. 21-22. Tripathy, B. C., and Oelmüller, R. (2012). Reactive oxygen species generation and signaling in plants. Plant signaling and behavior, 7, 1621-1633.

Tsao, R., and Deng, Z. (2004). Separation procedures for naturally occurring antioxidant phytochemicals. Journal of chromatography B, 812, 85-99. Tsugane, K., Kobayashi, K., Niwa, Y., Ohba, Y., Wada, K., and Kobayashi, H. (1999). A recessive Arabidopsis mutant that grows photoautotrophically under salt stress shows enhanced active oxygen detoxification. The Plant Cell, 11, 1195-1206. Türkan, I., and Demiral, T. (2009). Recent developments in understanding salinity tolerance. Environmental and Experimental Botany, 67, 2-9. 164

Turner, J., and Long, J. N. (1975). Accumulation of organic matter in a series of Douglas- fir stands. Canadian Journal of Forest Research, 5, 681-690. Turtola, S., Rousi, M., Pusenius, J., Yamaji, K., Heiska, S., Tirkkonen, V., Meier, B. and Julkunen‐Tiitto, R. (2005). Clone‐specific responses in leaf phenolics of willows exposed to enhanced UVB radiation and drought stress. Global Change Biology, 11, 1655-1663. Uddin, M. N., Hanstein, S., Leubner, R., and Schubert, S. (2013). Leaf cell‐wall components as influenced in the first phase of salt stress in three maize (Zea mays L.) hybrids differing in salt resistance. Journal of Agronomy and Crop Science, 199, 405-415. Valifard, M., Mohsenzadeh, S., Kholdebarin, B., and Rowshan, V. (2014). Effects of salt stress on volatile compounds, total phenolic content and antioxidant activities of Salvia mirzayanii. South African Journal of Botany, 93, 92-97. Van Kooten, O., and Snel, J. F. (1990). The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynthesis research, 25, 147-150. Ventura, Y., Wuddineh, W. A., Myrzabayeva, M., Alikulov, Z., Khozin-Goldberg, I., Shpigel, M., Samocha, T.M. and Sagi, M. (2011). Effect of seawater concentration on the productivity and nutritional value of annual Salicornia and perennial Sarcocornia halophytes as leafy vegetable crops. Scientia Horticulturae, 128, 189- 196. Verslues, P. E., Agarwal, M., Katiyar‐Agarwal, S., Zhu, J., and Zhu, J. K. (2006). Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. The Plant Journal, 45, 523-539. Vicente, O., and Boscaiu, M. (2018). Flavonoids: Antioxidant Compounds for Plant Defence... and for a Healthy Human Diet. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 46, 14-21. Vicente, O., Boscaiu, M., Naranjo, M. Á., Estrelles, E., Bellés, J. M., and Soriano, P. (2004). Responses to salt stress in the halophyte Plantago crassifolia (Plantaginaceae). Journal of Arid Environments, 58, 463-481. Villagra, P. E., and Cavagnaro, J. B. (2005). Effects of salinity on the establishment and early growth of Prosopis argentina and Prosopis alpataco seedlings in two contrasting soils: Implications for their ecological success. Austral ecology, 30, 325-335.

165

Voss, I., Sunil, B., Scheibe, R., and Raghavendra, A. S. (2013). Emerging concept for the role of photorespiration as an important part of abiotic stress response. Plant biology, 15(4), 713-722. Waheed, S., Hasnain, A., and Ahmad, A. (2018). Evaluating the potential of botanical extracts and fractions as substitutes of chemical antioxidants in edible oils. Pakistan journal of botany, 50, 1999-2004. Wahid, A., and Ghazanfar, A. (2006). Possible involvement of some secondary metabolites in salt tolerance of sugarcane. Journal of plant physiology, 163(7), 723- 730. Wang, C. M., Zhang, J. L., Liu, X. S., Li, Z., Wu, G. Q., Cai, J. Y., Flowers, T.J. and Wang, S. M. (2009). Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+. Plant, Cell and Environment, 32, 486-496. Wang, S., Wan, C., Wang, Y., Chen, H., Zhou, Z., Fu, H., and Sosebee, R. E. (2004). The characteristics of Na+, K+ and free proline distribution in several drought-resistant plants of the Alxa Desert, China. Journal of Arid Environments, 56, 525-539. Wang, Z., Xiao, Y., Chen, W., Tang, K., and Zhang, L. (2010). Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis. Journal of Integrative Plant Biology, 52, 400-409. Wariss, H. M., Ahmad, S., Anjum, S., and Alam, K. (2014). Ethnobotanical studies of dicotyledonous plants of Lal Suhanra national park, Bahawalpur, Pakistan. International Journal of Science and Research, 3, 2452-60. Wasuwat, S. (1970). Extract of Ipomoea pes-caprae (Convolvulaceae) antagonistic to histamine and jelly-fish poison. Nature, 225, 758-758. Wieneke, J., and Läuchli, A. (1980). Effects of salt stress on distribution of Na+ and some other cations in two soybean varieties differing in salt tolerance. Zeitschrift für Pflanzenernährung und Bodenkunde, 143, 55-67. Wiesman, Z., Itzhak, D., and Dom, N. B. (2004). Optimization of saline water level for sustainable Barnea olive and oil production in desert conditions. Scientia Horticulturae, 100, 257-266. Wong, C. C., Li, H. B., Cheng, K. W., and Chen, F. (2006). A systematic survey of antioxidant activity of 30 Chinese medicinal plants using the ferric reducing antioxidant power assay. Food chemistry, 97, 705-711.

166

Wong, S. K., Lim, Y. Y., and Chan, E. W. C. (2009). Antioxidant properties of Hibiscus: species variation, altitudinal change, coastal influence and floral colour change. Journal of Tropical Forest Science, 21,307-315. Xu, C., Zhang, Y., Zhu, L., Huang, Y., and Lu, J. (2011). Influence of growing season on phenolic compounds and antioxidant properties of grape berries from vines grown in subtropical climate. Journal of agricultural and food chemistry, 59, 1078-1086.

Yan, K., Shao, H., Shao, C., Chen, P., Zhao, S., Brestic, M., and Chen, X. (2013). Physiological adaptive mechanisms of plants grown in saline soil and implications for sustainable saline agriculture in coastal zone. Acta physiologiae plantarum, 35, 2867-2878. Yeo, A. R. (1983). Salinity resistance: physiologies and prices. Physiologia plantarum, 58, 214-222. Yesmin, M. N., Uddin, S. N., Mubassara, S., and Akond, M. A. (2008). Antioxidant and antibacterial activities of Calotropis procera Linn. Am Eurasian. Journal of Agriculture and Environmental Sciences,4, 550-553. Yin, Y. G., Kobayashi, Y., Sanuki, A., Kondo, S., Fukuda, N., Ezura, H., Sugaya, S. and Matsukura, C. (2009). Salinity induces carbohydrate accumulation and sugar- regulated starch biosynthetic genes in tomato (Solanum lycopersicum L. cv. ‘Micro-Tom’) fruits in an ABA-and osmotic stress-independent manner. Journal of Experimental Botany, 61, 563-574. You, J., and Chan, Z. (2015). ROS regulation during abiotic stress responses in crop plants. Frontiers in plant science, 6, 1092. Young, I. S., and Woodside, J. V. (2001). Antioxidants in health and disease. Journal of clinical pathology, 54, 176-186. Zhang, Q. Y., Wang, L. Y., Kong, F. Y., Deng, Y. S., Li, B., and Meng, Q. W. (2012). Constitutive accumulation of zeaxanthin in tomato alleviates salt stress‐induced photoinhibition and photooxidation. Physiologia Plantarum, 146, 363-373.

Zhang, Y. Y., Zhang, F., Thakur, K., Ci, A. T., Wang, H., Zhang, J. G., and Wei, Z. J. (2018). Effect of natural polyphenol on the oxidative stability of pecan oil. Food and chemical toxicology, 119, 489-495. Zhou, K., and Yu, L. (2006). Total phenolic contents and antioxidant properties of commonly consumed vegetables grown in Colorado. LWT-Food Science and Technology, 39, 1155-1162.

167

Zhu, D., and Scandalios, J. G. (1994). Differential accumulation of manganese-superoxide dismutase transcripts in maize in response to abscisic acid and high osmoticum. Plant Physiology, 106, 173-178. Zhu, J. K. (2007). Plant salt stress. Encyclopedia of Life Science,

168

INT. J. BIOL. BIOTECH., 15 (3): 473-482, 2018.

ANTIOXIDANT PROPERTIES AND PHENOLIC COMPOSITION OF COASTAL HALOPHYTES COMMONLY USED AS MEDICINE

Saba Nazir, Muhammad Qasim, Bilquees Gul* and M. Ajmal Khan

Institute of Sustainable Halophyte Utilization (ISHU), University of Karachi, Karachi-75270, Pakistan *Corresponding author’s email: [email protected], phone: +9221-34820253, fax: +9221-34820258

ABSTRACT

Halophytes are well adapted in extreme environmental conditions, regulation of secondary metabolism is one of the keys of their success. In this study five coastal halophytes i.e. Atriplex stocksii, Cressa cretica, Heliotropium bacciferum, Ipomea pes-caprae and Salsola imbricate, which are well known for their therapeutic properties were investigated for their antioxidant activity and polyphenol composition. Medicinal halophytes showed considerable antioxidant activity in terms of DPPH (14.4-64.8 I%), ABTS (22.6-230.6 µMol TE g-1), FRAP (0.5-5.6 mMol Fe+2 g-1) and TAC (17.6-36.1 mg AsA g-1). Results indicated that these plants also contained high amount of total phenols (7.7- 29.6 mg GAE g-1), flavonoids (4.2-17.3 mg QE g-1) and proanthocynadins (0.2-0.6 mg CE g-1). Among these, I. pes- caprae and C. cretica had the highest antioxidant activity and polyphenolic contents. High correlation among antioxidant activity assays (r = 0.877-0.999) indicated the radical scavenging and reducing power abilities of these plants. Similarly, strong correlations (r = 0.654-0.953) among antioxidant activity measurements and polyphenolic composition suggested that phenolic compounds contributed mainly to the antioxidant activity of these plants. Present study reveals coastal halophytes as rich sources of natural antioxidants, which could be used in herbal formulations, pharmaceuticals/ nutraceuticals, food additives and cure for ailments related to oxidative stress. Furthermore, these plants could be grown using saline resources and provide bioactive raw material with high industrial and economic value.

Keywords: Arabian Sea, Karachi coast, Marginal lands, Medicinal plants, Salt tolerant species, Secondary metabolites

INTRODUCTION

The Karachi coast extends over 100 Km on the Arabian Sea, including several islands and beaches (Shameel and Tanaka, 1992). The diversity of halophyte various sub types (Xerohalophytes, hydrohalophytes) have been established in coastal areas of Pakistan (Khan and Qaiser, 2006; Khan and Gul, 2002). These halophytes are well known for multiple economic usages like fuel wood, food, fodder, medicines, oilseed, landscaping, chemicals (Abideen et al., 2011; Khan et al., 2009; Qasim et al., 2011; Weber et al., 2007) and considered as potential candidates to fulfill as secondary source for basic needs of growing population. Plants distributed at coastal regions exposed to various abiotic stress such as fluctuating salinity, temperatures, light, nutrient, and water (Ksouri et al., 2010). These stresses can cause over production of reactive oxygen species (ROS). Although ROS, at low concentration, are essential messengers for vital plant functions (Salganik, 2001), their higher quantities can trigger cell and tissue injuries by damaging membranes and molecules (Abdi and Ali, 1999; Karuppanapandian et al., 2011; Zhu, 2001). However, tolerant plants like halophytes are adapted to harsh environments and can protect deleterious effects of ROS by enzymatic and non-enzymatic antioxidant systems (Gill and Tuteja 2010). For instance, production of antioxidant compounds contribute significantly to plant stress resistance and is often associated with plant survival (Gupta and Huang, 2014). Halophytes can produce large quantities of secondary metabolism including phenols, flavonoids, proanthocyanidins, tannins and other antioxidant compounds (Alhdad et al., 2013; Dixon and Paiva, 1995; El Shaer, 2010). Hydrogen atom or electron donation capacity/ metal chelating ability of polyphenols is associated with their antioxidant activity (Anchana et al., 2005; Tsao and Deng 2004). Besides providing antioxidant defense, these compounds also possess a broad range of biological and Bouayed pharmacological potential and can be used to treat health care issues based herbal formulations (Qasim et al., 2011, 2014). In addition, phenolic compounds are also known for their beneficial health effects for humans (Cicerale et al., 2010; Huang et al., 2009) including anti-cancerous, anti-coagulant and hypoglycemic properties (Meira et al., 2012) and are used in different food, pharmaceutical and cosmetic products (Maisuthisakul et al., 2007). Recently, interest is shifted towards find natural herbal product alternate to synthetic antioxidants known to possess harmful health effects (Hu et al., 2000). Moreover, natural compounds have better antioxidant activity and lesser side effects than synthetic ones (Maisuthisakul et al., 2007). 474 SABA NAZIR ET AL.,

Numerous studies have been conducted related to taxonomy, distribution, and morpho-ecology of coastal plants, but data about their active compounds and secondary metabolite constituents is scanty (Rizvi and Shameel, 2001). Few reports, which incorporated eco-physiological approach in medicinal plant studies, suggested that in search of natural sources of antioxidant compounds, halophytes are the better candidates to focus (Abideen et al., 2015; Qasim et al., 2017). Keeping in mind the above mentioned scenario, this study evaluated the antioxidant activity of five coastal halophytes i.e. Atriplex stocksii, Cressa cretica, Heliotropium bacciferum, Ipomea pes-caprae and Salsola imbricate, that are commonly used in traditional medicines. Composition of polyphenols including total phenols, flavonoids and proanthocynadins and their correlation with antioxidant activity of medicinal halophytes was also determined.

MATERIALS AND METHODS

Collection of plant material Leaves of five medicinal halophytes i.e. Atriplex stocksii, Cressa cretica, Heliotropium bacciferum, Ipomea pes-caprae and Salsola imbricate, were collected from their natural habitats along the coast of Karachi. Taxonomic description of selected halophytes is given in Table 1. Mean monthly temperatures (maximum 28-36 ºC, minimum 9-29 ºC), precipitation (0-9.9 cm) and humidity (25-64%) during 2014 (Fig. 1).

Sample preparation Leaves were dried under shade condition and ground to finely powdered using ball mill (Retsch MM-400). 25 mg of powdered plant material was extracted in 80% methanol (10 mL) at 40 oC for 3 h using a shaking water bath (GFL-1092; Abideen et al., 2015; Qasim et al., 2016). Extracts were centrifuged and supernatant was recovered for further analysis.

Quantification of secondary metabolites Estimation of secondary metabolites was based on calorimetric method, total phenolic content (TPC) was carried out using the Folin–Ciocalteu method (Singleton and Rossi, 1965). Calorimetric methods of Chang et al. (2002) and Sun et al. (1998) were also used for the quantification of total flavonoids (TFC) and proanthocyanidins (PC), respectively.

Determination of Antioxidant capacity Four different tests were employed to determine the antioxidant activity of medicinal halophytes. DPPH (Brand-Williams et al., 1995) and ABTS (Re et al., 1999) assays are based on radical scavenging ability while, ferric reducing antioxidant power (FRAP; Benzie and Strain, 1996) and total antioxidant capacity (TAC; Prieto et al., 1999) methods were used to determine the reducing potential of plant extracts.

Soil analysis Soil samples were collected from the root zone (up to 12 cm deep) of medicinal halophytes and analyzed for moisture content, electrical conductivity (EC) and pH (AOAC, 2005).

Statistical analyses The study was based on a minimum of 5 biological replicates using 5 technical replicates. SPSS (version 20) was used for LSD post-hoc test and Pearson Correlation Coefficient (r). All graphs were plotted using Sigma Plot (version 12.5). Results are presented as means (± standard error).

RESULTS AND DISCUSSION

Antioxidant activity and polyphenolic composition of five medicinal halophytes distributed in coastal areas of Karachi were studied. Selected species were either perennial shrubs or herbs, belongs to 3 botanical families (Table 1). Data revels that the rhizosphere soil was mostly saline (22-27 dSm-1) and dry (0.6-3.5% moisture) with pH ranging 6.8-8.3 (Table 2), which reflects in the xerohalophytic nature of most of the plants (Table 1). Traditionally these plants are used as a folk remedy in nearby communities and some of them are also reported with profound biological activities (Table 2). Antioxidant activity relies not only on the constituents present in the plant extract but also on the testing method. Hence, cannot be completely assessed by single method rather multiple antioxidant assays are used to harness various antioxidant action mechanisms (Wong et al., 2006). Antioxidant activity of medicinal halophytes

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 473-482, 2018. ANTIOXIDANT PROPERTIES AND PHENOLIC COMPOSITION OF COASTAL HALOPHYTES 475 were evaluated using four antioxidant assays i.e. DPPH, ABTS, FRAP and TAC. The DPPH and ABTS estimates the radical scavenging ability of plant extracts using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-Azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) free radicals, respectively. The ferric-reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) methods estimate the ability of plant extract to reduce iron and molybdenum ions, respectively. These are simple, reliable, less expensive and easily reproducible methods which reflect the different rationales of antioxidant activity measurements (Cai et al., 2004; Li et al., 2008). A wide variation among antioxidant activity of five selected species was recorded. It is important to notify that all test species were collected from the same study area to ensure similar edaphic, climatic, and developmental influences on the growth and antioxidant activity. DPPH radical scavenging activity (% inhibition) of curative halophytes ranged between 14.3% (H. bacciferum) to 64.7% (C. cretica) indicating a 3.5 fold variation. The antioxidant activity using FRAP system (mMol Fe+2 g−1) showed a 13 fold variation from 0.48 (H. bacciferum) to 5.6 (I. pescaprae). The ABTS (µMol TE g-1) values ranged from 22.6 (A. stocksii) to 230.6 (C. cretica), while TAC values (mg AsA g-1) ranged from 17.6 (H. bacciferum) to 36.1 (I. pescaprae). In general, medicinal halophytes represent powerful radical scavenging and reducing oxidant activities (Fig. 2), which is related to their bioactive secondary metabolites which make them better than antioxidant rich plants like herbs, medicinal plants, edible plants, and some halophytes (Bourgou et al., 2008). Our results verify previous studies based on equivalent or higher antioxidant activity of coastal medicinal plants (Falleh et al., 2012; Medini et al., 2014; Qasim et al., 2017).

Table 1. Taxonomic detail of medicinal halophytes used in this study. Species Families Habit Plant type Flowering period

Atriplex stocksii Boiss Amaranthaceae Shrub Xerohalophytes December-January

Cressa cretica L. Convolvulaceae Herb Hydrohalophyte Year round Heliotropium bacciferum Boraginaceae Shrub Xerohalophyte July-September Forssk. Ipomoea pes-caprae (L.) R. Convolvulaceae Herb Psammophyte July-September Br. Salsola imbricata Forssk. Amaranthaceae Shrub Xerohalophyte August-October

Table 2. Soil EC, pH and moisture content collected from rhizosphere of medicinal halophytes. Species EC (dS m-1) pH Moisture (%) Atriplex stocksii 27.04 ± 1.341 7.6 ± 0.01 0.93 ± 0.02 Cressa cretica 27.12 ± 3.671 6.8 ± 0.07 3.54 ± 0.42 Heliotropium bacciferum 24.85 ± 2.431 6.9 ± 0.33 2.85 ± 0.65 Ipomoea pes-caprae 25.52 ± 2.511 8.3 ± 0.23 0.56 ± 0.26 Salsola imbricata 22.03 ± 1.351 7.1 ± 0.35 1.36 ± 0.56

Table 3. Medicinal uses of halophytes used in this study. Common Plant Species Preparation Medicinal uses* name part Atriplex stocksii Phurki val Leaf Infusion Fever, jaundice, dropsy, liver disease Cressa cretica Bukkan Whole Decoction, Antiinflammatory, Antioxidant, Antiviral plant paste and for treatment of Sores Heliotropium Markondi Leaf Decoction Antihyperlipidemic, antitumor, antidiabetic, bacciferum antioxidant, and antimicrobial Ipomea.pes caprae Beach Morning Leaf Decoction Diarrhea, pains, vomiting, inflammation of Glory legs, piles Salsola imbricata Lana Fresh Infusion Insecticidal, vascular hypertension twig *Qasim et al., 2011, Qasim et al., 2014, Ahmad et al., 2014

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Table 4. Correlation coefficient (r) of different antioxidant parameters studied.

TPC TFC PC DPPH ABTS FRAP TAC

TPC 1

TFC 0.610 1

PC 0.643 0.865 1

DPPH 0.740 0.839 0.852 1

ABTS 0.654 0.735 0.862 0.930 1

FRAP 0.710 0.950 0.936 0.957 0.888 1

TAC 0.721 0.953 0.942 0.950 0.877 0.999 1 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radicals, ferric- reducing antioxidant power (FRAP), total antioxidant capacity (TAC), total phenolic content (TPC), total flavonoids (TFC) and proanthocyanidins (PC)

Max Temp (oC) Min Temp (oC) Humidity (%) Precipitation (cm) Wind speed (Kmph)

60 50 40 30

Values 20 10 0

Jul

Jun

Oct

Jan

Apr

Mar

Nov

Feb Aug

May Dec

Sep

Months

Fig. 1. Mean annual temperatures, rainfall, humidity and wind speed of study area 2014 (Pakistan Meteorological Department).

Polyphenols are biologically active molecules with high antioxidant activity. Polyphenolic analysis of the studied plants showed considerable variation in total phenol (7.6-26.6 mg GAE g-1), total flavnoids (1.3-17.3 mg QAE g-1) and proanthocynadin (0.2-0.6 CE g-1) contents (Fig. 3). Among all test species, the highest collective polyphenolic content was 42.6 mg g-1, which was found in I. pes-caprae (TPC 24.7 mg GAE g-1 + TFC 17.3 mg QE g-1 + PC 0.6 mg CE g-1) followed by 40.6 mg g-1 in C. cretica (TPC 29.6 mg GAE g-1 + TFC 10.5 mg QE g-1 + PC 0.5 mg CE g-1). A. stocksii (26.7 mg g-1), H. bacciferum (20.1 mg g-1), and S. imbricata (12.2 mg g-1) had relatively lower polyphenols and generally had non-significant differences in TPC, TFC, and PC contents (Fig. 3). Polyphenols of studied medicinal halophytes are comparable to or even higher than antioxidant rich medicinal plants such as Cetraria islandica and halophytes like Mesembryanthemum edule, Cakile maritima, and Limonium delicatulum (Falleh et al., 2012, 2013; Ivanova et al.,2005; Ksouri et al., 2007; Medini et al., 2014).

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 473-482, 2018. ANTIOXIDANT PROPERTIES AND PHENOLIC COMPOSITION OF COASTAL HALOPHYTES 477

) c -1 30 c

b b 20

10 a

TPC(mg GAE g

) 20 c

-1

15 b 10 a a

TFC(mg QAE g 5 a

0.8

) -1 c 0.6 b

a 0.4 a a PC(mg CE g 0.2

0.0

C. cretica A. stocksii S. imbricata H. bacciferumI. pes-caprae

Fig. 2. Analysis of secondary metabolites total phenol (TPC), total flavonoids (TFC) and proanthocynadin content (P.C) in leaves of medicinal halophyte species.

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 473-482, 2018. 478 SABA NAZIR ET AL.,

c Fe (mMol FRAP c b 60 6 b

40 4

b b +2

20 a a 2 g

a -1 ) DPPH%)(Inhibition a

0 0

300 ( ABTS

) 40 b d -1 b d 30

200 g Mol TE a a a 20 c

100 -1

TAC(mg AsA g 10 b ) a 0 0

C. cretica C. cretica A. stocksii S. imbricata A. stocksii S. imbricata H. bacciferumI. pes-caprae H. bacciferumI. pes-caprae

Fig. 3. Variation in antioxidant activity (DPPH, ABTS, FRAP, TAC) of medicinal halophytes.

The correlation (r) of polyphenols (TPC, TFC and PC) and antioxidant activity (DPPH, ABTS, FRAP and TAC) of medicinal halophytes is presented in Table 4. The TPC, TFC and PC showed strong positive correlation with antioxidant activity measurements (DPPH, ABTS, FRAP and TAC). A positive correlation between the antioxidant assays also indicated that extracts of medicinal halophyte contained broad spectrum antioxidant compounds which enables free radicals scavanging and reducing harmful oxidants ability. Our results taken togather with previous studies indicating phenolic compounds are the significant contributors to the antioxidant activity of medicinal plants (Boulanouar et al., 2013; Djeridane et al., 2006, Lizcano et al., 2010, Ouchemoukh et al., 2012,). Antioxidant activity of phenolic compounds relies on number and orientation of OH groups, which either donate hydrogens or electrons (Bouayed et al., 2011a, b; Rice-Evans et al., 1996) to neutralize free radicals or chelation metal ions (Li et al., 2008; Shan et al., 2005). Several studies indicate positive association between TPC and higher antioxidant activity (Kim et al 2003; Skotti et al., 2014). Several phenolic compounds have been reported, either individually or aggregated with profound antioxidant activity / health benefits (Lee and Lee, 2010; Owen et al., 2000). Beside strong antioxidant activity, phenolic compounds are reported as antimicrobial, antiviral, antimalarial, anti-inflammatory, antiplaque-forming, hypotensive, hypoglycemic, hepatoprotective, antitumor, anticancer, and neuro and cardio protective effects (Nagao et al., 2005; Niggeweg et al., 2004).

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Interestingly, studied medicinal halophytes that showed high antioxidant activity are being commonly used among local populations along coastal areas of Karachi and vicinity. For examples, I. pes-caprae is used as herbal tea against several health care issues like common cold and fever (Qasim et al., 2017). Leaf extract is used for treatment of jellyfish sting. This plant also possess antispasmodic, anticancer, antinociceptive, antihistaminic, insulogenic and hypoglycemic activities (Premanathan et al., 1996; Wasuwat, 1970). Higher radical scavenging and reducing power activity of I. pes-caprae demonstrated in this study agrees with the previous reports (Banerjee et al., 2008; Thimnavukkamsu et al., 2010). The presence of catechin, gallic acid, chlorogenic acid, caffeic acid, syringic acid, ferulic acid, coumarin, naringenin, kaempferol, and derivatives of quercetin, isocoumarin, and isochlorogenic acid, supports its high antioxidant potency (Meira et al., 2012; Qasim et al., 2017). Cressa cretica is another important plant used as herbal remedy against several diseases and disorders (Qasim et al., 2017). It is a popular medicinal halophyte, used in folklore medicine for asthma, ulcers, diabetes, stomachic, expectorant, anthelmintic, and aphrodisiac purposes. It has properties to enrich blood, and very helpful in leprosy, constipation, and urinary problems (Priyashree et al., 2010). In-vivo and in-vitro biological testing highlights its efficacy as a potent antimicrobial, antituberculosis, antitussive, antibilous, anti-inflammatory, and anticancer agent (Priyashree et al., 2010; Rizk and El-Ghazaly, 1995). Animal trials proven that C. cretica extracts improves sexuality and testicular functions in rats (Priyashree et al., 2010). Our results reveled the powerful antioxidant nature of this plant are in line with previous studies (Pryianka et al., 2015; Sunita et al., 2011). Presence of scopoletin, syringaresinol, dicaffeoylquinic acid, creticane, cressatetratriacontanoic acid, cressatriacontanone, cressatetracosanoate, cressanaphthacenone, flavonol glycosides, chlorogenic acids, rutin, and derivatives of quercetin and kampferol in C.cretica could be are related to its antioxidant and other biological activities (Abdallah et al., 2017; Priyashree et al., 2010).

Conclusions The antioxidant activity and polyphenolic contents of five coastal halophytes, which are commonly used in traditional herbal remedies, were evaluated. In general, medicinal halophytes showed strong antioxidant activity with considerable amount of phenols, flavonoids, and proanthocyanidins. Among these plants, I. pes-caprae and C. cretica were enriched in natural antioxidants, both in extraction as well as in antioxidant potential. A significant relationship between polyphenols and antioxidant activities indicated that phenolic compounds are the major antioxidants in these plants. A strong correlation between antioxidant activity assays also implied that extracts of medicinal halophytes were capable of reducing oxidants and quenching free radicals. This study highlights the importance of medicinal halophytes as promising sources of natural antioxidants, which can be used for multiple domestic and industrial applications. Moreover, these plants are adapted to saline coast lands and do not require prime agricultural lands and fresh water resources to grow. Sustainable development of saline/marginal lands with these medicinal resources can provide industrial raw material of bioactive natural products, which can be used to replace harmful synthetic derivatives from food, pharmaceutical and cosmetic industries.

Acknowledgements We are thankful to Pakistan Meteorological Department for providing environmental data.

REFERENCES

Abdallah, H. M.I., A.I. Elshamy, A.E.N.G. El Gendy, A.M.A. El-Gawad, E.A. Omer, M. De Leo and L. Pistelli (2017). Anti-inflammatory, antipyretic, and antinociceptive effects of a Cressa cretica aqueous extract. Planta Medica, 83(17): 1313-1320. Abdi, S and A. Ali (1999). Role of ROS modified human DNA in the pathogenesis and ethiology of cancer. Cancer Lett, 142: 1-9. Abideen, Z., M. Qasim, A. Rasheed, M.Y. Adnan, B. Gul and M.A. Khan (2015). Antioxidant activity and polyphenolic content of Phragmites karka under saline condition. Pak J Bot, 47: 813-818. Abideen, Z., R. Ansari and M.A. Khan. (2011). Halophytes: Potential source of ligno-cellulosic biomass for ethanol production. Biomass Bioenerg, 35(5): 1818-1822. Ahmad, S., S. Ahmad, A. Bibi, M.S. Ishaq, M.S. Afridi, F. Kanwal, and F. Fatima (2014). Phytochemical analysis, antioxidant activity, fatty acids composition, and functional group analysis of Heliotropium bacciferum. Sci World J, 2014. Alhdad, G.M., C.E. Seal, M.J. Al-Azzawi and T.J. Flowers (2013). The effect of combined salinity and waterlogging on the halophyte Suaeda maritima the role of antioxidants. Environ Exp Bot, 87: 120-125.

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 473-482, 2018. 480 SABA NAZIR ET AL.,

Anchana, C., T. Aphiwat and R. Nuansri (2005). Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand. Food Chem, 92: 491-497. AOAC (2005). Official Methods of Analysis. 18th ed. Gaitherburg: AOAC International. Banerjee, D., S. Chakrabarti, A.K. Hazra, S. Banerjee, J. Ray and B. Mukherjee (2008). Antioxidant activity and total phenolics of some mangroves in Sundarbans. Afr J Biotechnol, 7(6). Benzie, I.F. and J. Strain (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem, 239: 70–76. Bouayed, J., H. Deuber, L. Hoffmann and T. Bohn (2011a). Bio accessible and dialyzable polyphenols in selected apple varieties following in vitro digestion vs. their native patterns. Food Chem, 13:1466-1472. Bouayed, J., L. Hoffmann and T. Bohn (2011b). Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated gastro-intestinal digestion and dialysis of apple varieties: Bio accessibility and potential uptake. Food Chem, 128: 14-21. Boulanouar, B., G. Abdelaziz, S. Aazza, C. Gago and M.G. Miguel (2013). Antioxidant activities of eight Algerian plant extracts and two essential oils. Ind Crop Prod, 46: 85-96. Bourgou S., R. Ksouri, A. Bellila, I. Skandrani, H. Falleh and B. Marzouk (2008). Phenolic composition and biological activities of Tunisian Nigella sativa L. shoots and roots. CR Biologies, 331(1): 48-55. Brand-Williams, W., M. Cuvelier and C. Berset (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol, 28(1): 25-30. Cai, Y.Z., Q. Luo, M. Sun and H. Corke (2004). Antioxidant activity and phenolic compounds of112 traditional Chinese medicinal plants associated with anticancer. Life Sci, 74: 2157-2184. Chang, C.C., M.H. Yang, H.M. Wen and J.C. Chern (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal, 10(3): 178-182. Cicerale, S., L. Lucas and R. Keast. 2010. Biological activities of phenolic compounds present in virgin olive oil. Int J Mol Sci, 11: 458-479. Dixon, R.A. and N. Paiva (1995). Stress-induced phenylpropanoid metabolism. Plant Cell, 7: 1085-1097. Djeridane, A., M. Yousfi, B. Nadjemi, D. Boutassouna, P. Stocker and N. Vidal (2006). Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds. Food Chem, 97: 654-660. El Shaer, H.M (2010). Halophytes and salt-tolerant plants as potential forage for ruminants in the Near East region. Small Ruminant Res, 91: 3-12. Falleh, H., I. Jalleli, R. Ksouri, M. Boulaaba, S. Guyot, C. Magné and C. Abdelly (2012). Effect of salt treatment on phenolic compounds and antioxidant activity of two Mesembryanthemum edule provenances. Plant Physiol Biochem, 52: 1-8. Falleh, H., N. Msilini, S. Oueslati, R. Ksouri, C. Magne, M. Lachaâl and N. Karray-Bouraoui (2013). Diplotaxis harra and Diplotaxis simplex organs: assessment of phenolics and biological activities before and after fractionation. Ind Crop Prod, 45: 141-147. Gill, S. S. and N. Tuteja (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem, 48(12): 909-930. Gupta, B. and B. Huang (2014). Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. Int J Genomics, 2014: 1-18. Hu, C., Y. Zhang and D.D. Kitts (2000). Evaluation of antioxidant and prooxidant activities of banboo Phyllostachys nigra var. Henon is leaf extract in vitro. J. Agric Food Chem., 48: 3170-3176. Huang, W.Y., Y.Z. Caiand and Y. Zhang (2009). Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. Nutr Cancer, 62: 1-20. Ivanova, D., D. Gerova, T. Chervenkov, and T. Yankova (2005). Polyphenols and antioxidant capacity of Bulgarian medicinal plants. J Ethnopharmacol, 96 (1-2): 145-150. Karuppanapandian, T., H.W. Wang, N. Prabakaran, K. Jeyalakshmi, M. Kwon, K. Manoharan and W. Kim (2011). 2,4-dichlorophenoxyacetic acid-induced leaf senescence in mung bean (Vigna radiata L. Wilczek) and senescence inhibition by co-treatment with silver nanoparticles. Plant Physiol Biochem, 49: 168-177. Khan, M.A., R. Ansari, H. Ali, B. Gul and B.L Nielsen (2009). Panicum turgidum, a potentially sustainable cattle feed alternative to maize for saline areas. Agricult Ecosys Environ., 129: 542-546. Khan, M. A. and B. Gul (2002). Salt tolerant plants of coastal Sabkhas of Pakistan. In: Sabkha Ecosystems: Volume 1: The Arabian Peninsula and Adjacent Countries. (H. Barth. and B. Boer Eds.). Kluwer Academic Press, Netherlands. pp. 123 - 140. Khan, M.A. and M. Qaiser (2006). Halophytes of Pakistan: Distribution, Ecology, and Economic Importance In: Sabkha Ecosystems: Volume II. The South and Central Asian Countries. (M.A. Khan, H. Barth. G.C. Kust and B. Boer Eds.). Springer, Netherlands. pp.129 - 153.

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Kim, D.O., S.W. Jeong and C.Y. Lee (2003). Antioxidant capacity of phenolic phyto chemicals from various cultivars of plums. Food Chem, 81(3): 321-326. Ksouri, R., W. Megdiche, H.W. Koyro and C. Abdelly (2010). Responses of halophytes to environmental stresses with special emphasis to salinity. Adv Bot Res, 53: 117-145. Ksouri, R.,W. Megdiche, A. Debez, H. Falleh, C. Grignon, C. Abdelly (2007). Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritima. Plant Physiol Biochem, 45: 244- 249. Lee, O.H. and B.Y. Lee (2010). Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresour Technol, 101(10): 3751-3754. Li, H.B., C.C. Wong, K.W. Cheng and F. Chen (2008). Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. LWT-Food Sci. Technol, 41: 385-390. Lizcano, L.J, F. Bakkali, B. Ruiz-Larrea and J.I .Ruiz-Sanz (2010). Antioxidant activity and polyphenol content of aqueous extracts from Colombia Amazonian plants with medicinal use. Food Chem, 119: 1566-1570. Maisuthisakul, P., M. Suttajit and R. Pongsawatmanit (2007). Assessment of phenolic content and free radical- scavenging capacity of some Thai indigenous plants. Food Chem, 100: 1409-1418. Medini, F., H. Fellah, R. Ksouri and C. Abdelly (2014). Total phenolic, flavonoid and tannin contents and antioxidant and antimicrobial activities of organic extracts of shoots of the plant Limonium delicatulum. J Taibah Univ Sci, 8: 216-224. Meira, M., E.P.D. Silva, J.M. David and J.P. David (2012). Review of the genus Ipomoea: traditional uses, chemistry and biological activities. Revista Brasileira de Farmacognosi (Brazilian J Pharmacogn), 22: 682- 713. Nagao, T., Y. Komine, S. Soga, S. Meguro, T. Hase, Y. Tanaka and I. Tokimitsu (2005). Ingestion of a tea rich in catechins leads to a reduction in body fat and malondialdehyde modified LDL in men. Am J Clin Nutr, 81: 122- 129. Niggeweg, R., A.J. Michael and C. Martin (2004). Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol, 22: 746-754. Ouchemoukh, S., S. Hachoud, H. Boudraham, A. Mokrani and H. Louaileche, (2012). Antioxidant activities of some dried fruits consumed in Algeria. LWT-Food Sci Technol, 49(2): 329-332. Owen, R. W., A. Giacosa, W.E. Hull, R. Haubner, B., Spiegelhalder and H. Bartsch (2000). The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. Eur J Cancer, 36(10): 1235- 1247. Premanathan, M., H. Nakashima, K. Kathiresan, N. Rajendran and N. Yamamoto (1996). In vitro anti human immuno deficiency virus activity of mangrove plants. Indian J Med Res, 130: 276-279. Prieto, P., M. Pineda and M. Aguilar (1999). Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem, 269: 337-341. Priyashree, S., S. Jha and S.P. Pattanayak (2010). A review on Cressa cretica Linn.: A halophytic plant. Pharmacogn Rev, 4(8): 161-166. Pryianka, S. Partap, M. Verma and K.K. Jha (2015). In vitro antioxidant activity of plant extract of Cressa Cretica. Der Pharmacia Lettre, 7(5): 28-32. Qasim, M., S. Gulzar and M.A. Khan (2011). Halophytes as medicinal plants. In: Urbanisation, Land Use, Land Degradation and Environment. Daya (Ozturk, M., A.R. Mermut and A. Celik Ed.), Publishing House, Dehli, India, pp. 330-343. Qasim, M., Z. Abideen, M.Y. Adnan, R. Ansari, B. Gul and M.A. Khan (2014). Traditional ethnobotanical uses of medicinal plants from coastal areas of Pakistan. J Coast Life Med, 2(1): 22-30. Qasim, M., I. Aziz, M. Rasheed, B. Gul and M.A. Khan (2016). Effect of extraction solvents on polyphenols and antioxidant activity of medicinal halophytes. Pak J Bot, 48(2): 621-627. Qasim, M., Z. Abideen, M.Y. Adnan, S. Gulzar, B. Gul, M. Rasheed and M.A. Khan (2017). Antioxidant properties, phenolic composition, bioactive compounds and nutritive value of medicinal halophytes commonly used as herbal teas. S Afr J Bot, 110: 240-250. Re, R., N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans, (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med, 26: 1231-1237. Rice-Evans, C.A., N.J. Miller and G. Paganga, (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Bio Med, 20(7): 933-956. Rizk, A.M and G.A. El-Ghazaly (1995). Medicinal and poisonous plants of Qatar. In: Scientific and Applied Research Centre, University of Qatar. Doha, Qatar pp. 282-301.

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 473-482, 2018. 482 SABA NAZIR ET AL.,

Rizvi M.A and M .Shameel (2001).Distribution of elements in marine algae of Karachi coast. Pak J Bot 33: 357- 363. Salganik, R.I. (2001). The benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. J Am Coll Nutr, 20: 464-472. Shameel, M. and J. Tanaka (1992). A preliminary check-list of marine algae from the coast and inshore waters of Pakistan. In: Cryptogamic Flora of Pakistan, Vol. 1, (T. Nakaika and S. Malik Eds.) National Science Museum ser. Tokyo, pp.1-64. Shan, B., Y.Z. Cai, M. Sunand and H. Corke (2005). Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. J. Agric. Food Chem., 53: 7749-7759. Singleton, V. and J.A. Rossi (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic, 16(3): 144-158. Skotti, E., E. Anastasaki, G. Kanellou, M. Polissiou and P.A. Tarantilis (2014). Total phenolic content, antioxidant activity and toxicity of aqueous extracts from selected Greek medicinal and aromatic plants. Ind. Crops Prod, 53: 46-54. Sun, B., J.M. Ricardo-da-Silva and I. Spranger (1998). Critical factors of vanillin assay for catechins and proanthocyanidins. J Agr Food Chem, 46: 4267-4274. Sunita, P., S. Jha and S.P. Pattanayak (2011). Anti-inflammatory and in-vivo antioxidant activities of Cressa cretica Linn. a halophytic plant. Middle-East J. Sci. Res, 8(1): 129-140. Thimnavukkamsu, P., L. Ramkumar, T. Ramanarhan and G. Silambamsan (2010). Anti oxidant activity of selected coastal medicinal plants. World, 2(2): 134-137. Tsao, R. and Z. Deng (2004). Separation procedures for naturally occurring antioxidant phytochemicals. J Chromatograph. B, 812(1-2): 85-99. Wasuwat, S. (1970). Extract of Ipomoea pes-caprae (Convolvulaceae) antagonistic to histamine and jellyfish poison. Nature, 225: 758-59. Weber, D.J., R. Ansari, B. Gul, and M.A. Khan (2007). Potential of halophytes as source of edible oil. J. Arid Environ, 68(2): 315-321. Wong, S.P., L.P. Leong and J.H.W. Koh (2006). Antioxidant activities of aqueous extracts of selected plants. Food Chem, 99: 775-783. Zhu, J. K. (2001). Plant salt tolerance. 2001. Trends Plant Sci, 6: 66-71.

(Accepted for publication June 2018)

INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 15 (3): 473-482, 2018.