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) By 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 II 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.