Sageretia thea (Osbeck.) mediated synthesis of multifunctional nanoparticles and their possible applications
By
Ali Talha Khalil
Department of Biotechnology Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2017
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Sageretia thea (Osbeck.) mediated synthesis of multifunctional nanoparticles and their possible applications
A thesis Submitted to the Department of Biotechnology in the partial fulfillment of the requirement for the degree of Doctor of Philosophy In Biotechnology
By Ali Talha Khalil Department of Biotechnology Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2017
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PRAYER
In the name of ALLAH, The most beneficent, The most merciful.
Praise to Allah, Lord of worlds
The beneficent, The merciful
Master of the Day of Judgment
Thee we worship, Thee we ask for help
Show us the straight path
The path of those whom thou worth favored
Neither of who earn Thins anger
Nor of those who go astray
(Ameen)
Al-Quran Surrah Al-Fatiha
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Dedicated to My parents, Siblings, And Family.
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Table of contents
Contents
Chapter 1: Introduction ...... 1 1.1 Nanoscience in 21st century ...... 2 1.2 Metal oxide nanoparticles ...... 2 1.2.1 Zinc oxide (ZnO) ...... 3
1.2.2 Iron oxide (γ-Fe2O3) ...... 4 1.2.3 Nickel oxide (NiO) ...... 5
1.2.4 Cobalt oxide (Co3O4) ...... 5 1.2.5 Lead oxide (PbO) ...... 5 1.3 Biosynthesis of nanoparticles ...... 6 1.4 Sageretia thea (Osbeck.) ...... 8 1.5 Importance of study ...... 8 1.6 Specific objectives ...... 10 Chapter 2: Sageretia thea (Osbeck.) mediated synthesis of ZnO nanoparticles and its biological applications ...... 11 2.1 Introduction ...... 14 2.2 Materialand methods ...... 15 2.2.1 Plant material processing ...... 15 2.2.2 Biosynthesis of ZnO nanoparticles ...... 17 2.2.3 Characterizations ...... 17 2.2.4 Test sample preparation ...... 18 2.2.5 Antibacterial activity ...... 18 2.2.6 Antifungal activity ...... 19 2.2.7 Protein kinase inhibition ...... 19 2.2.8 Alpha amylase inhibition ...... 20 2.2.9 Antileishmanial activity (amastigotes and promastigotes) ...... 20 2.2.10 Brine shrimp cytotoxicity...... 21 2.2.11 Antioxidant activities ...... 21 2.2.12 Biocompatibility of ZnO nanoparticles with RBC’s ...... 22 2.2.13 Biocompatibility of ZnO nanoparticles with human macrophages ...... 22 2.3 Results and discussion ...... 23 2.3.1 Biosynthesis and characterization of ZnO nanoparticles ...... 23
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2.3.2 Physical characterization...... 25 2.3.3 Antibacterial activity ...... 33 2.3.4 Antifungal activity ...... 38 2.3.5 Antileishmanial potential (promastigotes and amastigotes) ...... 40 2.3.6 Brine shrimp cytotoxicity...... 40 2.3.7 Biocompatibility assessment using Human RBC’s and macrophages ...... 40 2.3.8 Enzyme inhibition activity (protein kinase inhibition and alpha amylase inhibition) .. 42 2.3.9 Antioxidant activities ...... 44
Chapter 3: Biosynthesis of iron oxide (Fe2O3) nanoparticles via aqueous extracts of Sageretia thea (Osbeck.) and their pharmacognostic properties ...... 46 3.1 Introduction ...... 49 3.2 Material and Methods ...... 50 3.2.1 Plant material processing ...... 50 3.2.2 Biosynthesis of IONP’s ...... 51 3.2.3 Physical characterizations ...... 53 3.4 Antimicrobial activities ...... 53 3.4.1 Antibacterial activity ...... 53 3.4.2 Antifungal activity ...... 54 3.5 Cytotoxic activities ...... 54 3.5.1 Antileishmanial activity (Promastigotes and Amastigotes) ...... 54 3.5.2 Brine shrimp cytotoxicity...... 55 3.6 Biocompatibility assays ...... 55 3.6.1 Human RBC’s ...... 55 3.6.2 Human Macrophages ...... 55 3.7 Enzyme inhibition assays ...... 56 3.7.1 Protein kinase inhibition ...... 56 3.7.2 Alpha amylase inhibition ...... 56 3.8 Antioxidant activities ...... 56 3.8.1 DPPH free radical scavenging ...... 56 3.8.2 Total Antioxidant Capacity ...... 57 3.8.3 Total Reducing Power ...... 57 3.9 Results and Discussion ...... 57 3.9.1 Biosynthesis of IONPs ...... 57 3.9.2 Room temperature physical characterizations ...... 58
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3.9.3 Antimicrobial properties of bioinspired IONPs ...... 63 3.9.4 Cytotoxic activities ...... 65 3.9.4.1 MTT cytotoxicity against axenic Leishmania tropica cultures ...... 65 3.9.4.2 Brine shrimp cytotoxicity...... 66 3.9.4.3 Biocompatibility against human macrophages and RBC’s ...... 66 3.9.5 Enzyme inhibition assays ...... 67 3.9.6 Antioxidant activities ...... 68 Chapter 4: Sageretia thea (Osbeck.) modulated biosynthesis of NiO nanoparticles and their in vitro pharmacognostic, antioxidant and cytotoxic potential ...... 73 4.1 Introduction ...... 76 4.2 Materials and methods ...... 79 4.2.1 Plant material processing ...... 79 4.2.2 Biosynthesis of NiO nanoparticles ...... 79 4.2.3 Characterizations of NiO nanoparticles ...... 79 4.3 Antimicrobial assays of NiO nanoparticles ...... 80 4.3.1 Antibacterial activity ...... 80 4.3.2 Antifungal activity ...... 80 4.4 In vitro cytotoxicity assays of of NiO nanoparticles ...... 81 4.4.1 Brine shrimp cytotoxicity...... 81 4.4.2 Antileishmanial assay (Promastigotes and Amastigotes) ...... 81 4.5 Biocompatibility assays of of NiO nanoparticles ...... 81 4.5.1 Hemolytic assay ...... 81 4.5.2 Biocompatibility with Macrophages ...... 82 4.6 Enzyme inhibition assays ...... 82 4.6.1 Protein kinase inhibition ...... 82 4.6.2 alpha amylase inhibition ...... 82 4.7 Antioxidant assays ...... 83 4.7.1 DPPH ...... 83 4.7.2 TAC ...... 83 4.7.3 TRP ...... 83 4.8 Results and Discussion ...... 84 4.8.1 Biosynthesis of NiO nanoparticles ...... 84 4.8.2 Physical characterizations of NiO nanoparticles...... 86 4.8.3 Antimicrobial activities ...... 90
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4.8.4 Antileishmanial activity (promastigotes and amastigotes) ...... 93 4.8.5 Brine shrimp cytotoxicity...... 94 4.8.6 Biocompatibility testing ...... 94 4.8.7 Antioxidant activities ...... 94 4.8.9 Enzyme inhibition assays ...... 96 Chapter 5: Bioinspired synthesis of pure massicot phase lead oxide nanoparticles and assessment of their biocompatibility, cytotoxicity and in-vitro biological properties ...... 100 5.1 Introduction ...... 103 5.2 Materials and methods ...... 104 5.2.1 Plant material processing ...... 104 5.2.2 Biosynthesis of PbO nanoparticles ...... 106 5.2.3 Characterization of bioinspired PbO nanoparticles...... 106 5.2.4 Antibacterial activity of bioinspired PbO nanoparticles ...... 107 5.2.5 Antileishmanial activity (Promastigotes and Amastigotes) ...... 107 5.2.6 Brine shrimp cytotoxicity...... 108 5.2.7 Biocompatibility of PbO nanoparticles ...... 108 5.2.7.1 Biocompatibility with erythrocytes ...... 108 5.2.7.2 Biocompatibility human macrophages ...... 108 5.2.8 Antioxidant activities ...... 109 5.2.8.1 Free radical scavenging ...... 109 5.2.8.2 Total Reducing Power of PbO nanoparticles ...... 109 5.2.8.3 Total Antioxidant Capacity ...... 110 5.2.9 Enzyme inhibition assays ...... 110 5.2.9.1 Alpha amylase inhibition ...... 110 5.2.9.3 Protein kinase inhibition ...... 110 5.3 Results and discussion ...... 110 5.3.1 Biosynthesis ...... 110 5.3.2 Characterizations ...... 112 5.3.3 Antibacterial activities ...... 117 5.3.4 Antileishmanial activities ...... 120 5.3.5 Brine shrimp cytotoxicity...... 120 5.3.6 Bio-compatibility potential ...... 120 5.3.7 Antioxidant assays ...... 122
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5.3.8 Enzyme inhibition assays ...... 123 5.3.9 General comments on the mechanism of cytotoxicity ...... 125 Chapter 6: Physical properties, biological applications and biocompatibility studies on biosynthesized single phase cobalt oxide (Co3O4) nanoparticles via Sageretia thea (Osbeck.) .. 127 6.1 Introduction ...... 130 6.2 Material and methods ...... 131 6.2.1 Plant material processing ...... 131 6.2.2 Biosynthesis procedure ...... 133 6.2.3 Characterization ...... 133 6.2.4 Antibacterial potential of Cobalt oxide nanoparticles ...... 133 6.2.5 Brine shrimp cytotoxicity...... 134 6.2.6 Antileishmanial activity (Promastigotes and Amastigotes) ...... 134 6.2.7 Biocompatibility assessment of biogenic cobalt oxide nanoparticles ...... 134 6.2.7.1 Biocompatibility with macrophages ...... 134 6.2.7.2 Hemolytic assay ...... 135 6.2.8 Antioxidant activities ...... 135 6.2.8.1 DPPH radical scavenging...... 135 6.2.8.2 Reducing power ...... 136 6.2.8.3 Total antioxidant capacity ...... 136 6.2.9 Enzyme inhibition assays ...... 136 6.2.9.1 Protein kinase inhibition ...... 136 6.2.9.2 Alpha amylase inhibition ...... 136 6.3 Results and discussion ...... 137 6.3.1 Biosynthesis of cobalt oxide nanoparticles ...... 137 6.3.2 Physical characterization of cobalt oxide nanoparticles ...... 138 6.3.3 Antibacterial activities ...... 144 6.3.4 Cytotoxic activities of bioinspired cobalt oxide nanoparticles ...... 147 6.3.4.1 Brine shrimp cytotoxicity...... 147 6.3.4.1 Antileishmanial activities ...... 147 6.3.5 Biocompatibility assessment of bioinspired cobalt oxide nanoparticles ...... 148 6.3.6 Mechanism of cytotoxicity...... 150 6.3.7 Antioxidant assays ...... 150 6.3.8 Enzyme inhibition assays ...... 150
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Chapter 7: Biogenic synthesis of zinc oxide nanoplatelets on nickel foam as electrode material for supercapacitor applications ...... 154 7.1 Introduction ...... 156 7.2 Material and methods ...... 157 7.2.1. Collection of plant material...... 157 7.2.2 Biogenic synthesis of zinc oxide nanoplatelets using Sageretia thea ...... 158 7.2.3 Fabrication of NiF/ZnO electrode material ...... 158 7.2.4 Material characterization...... 160 7.3 Results and Discussion ...... 160 7.3.1 Structural analysis of ZnO nanoplatelets and NiF-ZnO electrode material ...... 160 7.3.2. Morphological investigation ...... 161 7.3.3 Electrochemical performance of NiF-ZnO electrode materials ...... 164 Conclusions and Future perspectives ...... 169 Conclusions ...... 170 Future prospects ...... 172 References ...... 173 Annexures ...... 202
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Acknowledgments
ACKNOWLEDGEMENTS All praise to Allah Almighty, The most beneficent, The most merciful, Who gave me strength and enabled me to undertake and execute this research task. Countless salutations upon the Holy Prophet Hazrat Muhammad (SAWW), source of knowledge for enlightening with the essence of faith in Allah and guiding the mankind, the true path of life.
I would like to extend my deepest appreciation to those people, who helped me in one way or other in planning and executing this research work and writing up this thesis manuscript. I am thankful to Prof. Dr. Zabta Khan Shinwari, my PhD supervisor and Dean, Faculty of Biological Sciences for extending the research facilities of the department to accomplish this work. I feel highly privileged in taking opportunity to express my deep sense of gratitude to my supervisor for his scholastic guidance and valuable suggestions throughout the study and presentation of this manuscript. I am thankful to him for his inspiration, reassurance and counseling from time to time. I would like to thank Dr. Muhammad Naeem, Chairman Department of Biotechnology for his kind support. It is inevitable to thank Prof. Malik Maaza, UNSECO-UNISA Africa Chair in nanosciences and nanotechnology for his humble support, professional guidance and continuous encouragement during and after my research visit to Cape town. I also want to say a big thank to Dr. Mlungisi Nkosi, Director Material Research Department, iThemba Labs and Dr. Faical Azaiez Managing Director iThemba labs for providing the necessary facilities during my research visit to Cape town, South Africa. I gratefully acknowledge Prof. Noor Muhammad Butt for his cooperation. I also acknowledge the humble cooperation of Dr. Muhammad Ali.
It is inevitable to thank all of my senior PhD lab fellows Ikram Ullah, Fazal Akbar, and Sohail Ahmad Jan for their encouragement and keen interest. I would also like to say thank to Irum iqrar, Samina bashir, Fouzia tanveer and Haris khan. I am indebted to senior-junior colleagues; Dilawer and Hamza as well as the post-doctoral fellows; Dr. Saleh, Dr. Ahmed, Dr. Sone, Dr. Diallo, Dr. Mayedewa at Material Research Department, iThemba lab for their cooperation. I would also like to thank Xanthene Muller for her selfless cooperation. I wish to express sincere thanks to my junior lab fellows for their continued encouragement, moral support and necessary
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Acknowldgments guidance. Special thanks to my friend Muhammad Manzoor Khan who happily extended his assistance at every instance. Heartiest thanks to my time-tested friends Imran Khan, Shahab Saqib and Jangrez Khan for their enjoyable company, care and concern, persistent support and encouragement in my research and personal problems. I want to say a big thank to my roommate, Muhammad Fayyaz for his cooperation.
Solemn gratitude to my Father, Mother, Brothers and Sisters who deserve special mention for their inseparable support and prayers. They have always been my source of strength and love. It wouldn’t have been this bearable if I didn’t have these in my life. Thank you for your unconditional support with my studies. Thank you for giving me a chance to prove and improve myself through all walks of my life. Finally, I would like to thank everybody who was important to the successful completion of thesis, as well as expressing my apology that I could not mention personally one by one.
Ali Talha Khalil
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List of Figures
List of Figures
Chapter 2 Figure 2. 1: From biosynthesis to biological application a schematic representation...... 16 Figure 2. 2: Bioactive compounds isolated from Sageretia thea as reported in previous studies.. 24 Figure 2. 3: (A) XRD pattern of biogenic ZnO nanoparticles annealed at different temperatures; (B) their average size calculations using Debye-Scherrer approximation ...... 26 Figure 2. 4: HR-TEM image of biogenic ZnO nanoparticles; (A/B) Size distribution at 50 nm; (C) at 10 nm; (D) their shape; (E) HR-TEM image; (E) Typical SAED pattern; (F): HR-SEM ...... 28 Figure 2. 5: (A) FTIR of biogenic ZnO nanoparticles; (B) Their Raman spectra...... 30 Figure 2. 6: ATR-FTIR of bioinspired ZnO nanoparticles using diamond crystal; (A) Dried aqueous plant extracts of Sageretia thea; (B) Biogenic ZnO nanoparticles ...... 31 Figure 2. 7: Elemental composition of bioinspired ZnO nanopowder using Energy Dispersive Spectroscopy ...... 32 Figure 2. 8: (A) UV absorption spectra of biogenic ZnO; (B) Their room temperature excitation emission spectra and band gap ...... 33 Figure 2. 9: (A) Antibacterial activity and MIC of biogenic ZnO nanoparticles; (A): With no UV illumination; (B): Anitbacterial activity after 20 min UV illumination ...... 36 Figure 2. 10:Proposed mechanisms of manifesting cytotoxicity as indicated by numerous earlier studies; ...... 37 Figure 2. 11: (A) Antifungal activity using of biogenic ZnO nanoparticles using linear mycelial growth inhibition; (B) Antifungal activity by well diffusion assay ...... 39 Figure 2. 12: Cytotoxic assays for biogenic ZnO nanoparticles ...... 41 Figure 2. 13: Protein kinase inhibition activity of biogenic nanoparticles. Circular red marks indicate the bald zone of inhibition as a result of protein kinase enzyme inhibition activity of ZnO nanoparticles ...... 43 Figure 2. 14: Antioxidant activities of biogenic ZnO nanoparticles ...... 45 Chapter 3 Figure 3. 1: A schematic representation of the study indicating the various important steps from biosynthesis to characterization and application of IONP’s ...... 52 Figure 3. 2: (A) XRD analysis of bioinspired iron oxide nanoparticles, (B) Average size calculations ...... 59 Figure 3. 3: Morphological investigations of biogenic Iron oxide nanoparticles. (A): HR-TEM at 50 nm; (B): Particle distribution; (C) SAED pattern; (D): HR-TEM image; (E): HR-SEM ...... 60 Figure 3. 4: Room temperature physical characterization of biogenic iron oxide nanoparticles. (A): FTIR of bioinspired iron oxide nanoparticles; (B): their Raman spectra; (C) EDS for elemental composition ...... 62 Figure 3. 5: Antibacterial activities of bioinspired IONP’s...... 64 Figure 3. 6: Antifungal activities of biogenically synthesized IONP’s...... 65 Figure 3. 7: Cytotoxic activities of biogenic IONP’s ...... 67 Figure 3. 8: Protein kinase inhibition by biogenic IONP’s...... 69 Figure 3. 9: Antioxidant activities of biogenically synthesized IONP’s...... 70 Figure 3. 10: Proposed role of biosynthesized iron oxide nanoparticles as protein kinase inhibitor for tumor growth prevention: ...... 71 Figure 3. 11: Different proposed mechanisms* of IONP’s mediated toxicity; ...... 72
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List of Figures
Chapter 4 Figure 4. 1: Schematic representation of biosynthesis, characterization and application of NiO nanoparticles ...... 78 Figure 4. 2: Plausible mechanism for the biosynthesis of NiO nanoparticles via green route...... 85 Figure 4. 3: XRD analysis of biogenic NiO nanoparticles. (A): Typical XRD pattern of NiO annealed at 500°C; (B): Size calculation according to Scherer approximation ...... 87 Figure 4. 4: Morphological investigations of biogenic NiO nanoparticles using HR-TEM/HR- SEM; (A/B/C/D): Size distribution of NiO nanoparticles; (E): HR-TEM image; (F): HR-SEM; (G): SAED pattern ...... 88 Figure 4. 5: (A): Typical ATR-FTIR of NiO nanoparticles; (B): their raman spectra ...... 89 Figure 4. 6: Elemental composition of biogenic NiO nanoparticles using Energy Dispersive Spectroscopy (EDS)...... 90 Figure 4. 7: (A): Antibacterial activities of biogenic NiO nanoparticles without UV-illumination; (B): With UV-illumination...... 92 Figure 4. 8: Antifungal potential of bioinspired NiO nanoparticles...... 93 Figure 4. 9: Assessment of the cytotoxicity of the bioinspired NiO nanoparticles...... 95 Figure 4. 10: Antioxidant activities of bioinspired NiO nanoparticles...... 96 Figure 4. 11: Biological properties of biogenic NiO nanoparticles (A): Protein kinase inhibition potential; (B): Alpha amylase inhibition potential ...... 97 Figure 4. 12: Schematic on the cytotoxic properties of bioinspired NiO as reported in literature; 98 Chapter 5 Figure 5. 1: Scheme for studying biosynthesis, characterization and application biogenic PbO nanoparticles...... 105 Figure 5. 2: Plausible mechanism of biosynthesis lead oxide nanoparticles using Sageretia thea aqueous extracts ...... 111 Figure 5. 3: XRD analysis of biosynthesized lead oxide nanoparticles. (A): XRD spectra; (B): Average size calculation using Scherer size approximation formula...... 113 Figure 5. 4: Various HR-TEM images of biosynthesized lead oxide nanoparticles; (A/B/C/D): size distribution and shape; (E): HR-TEM image; (F): Particle size distribution...... 114 Figure 5. 5: (A/B/C): Various HR SEM images of PbO nanoparticles; (D): their SAED pattern...... 115 Figure 5. 6: Energy Dispersive Spectroscopy of biosynthesized lead oxide nanoparticles...... 115 Figure 5. 7: Raman spectra of biosynthesized lead oxide nanoparticles...... 116 Figure 5. 8: ATR-FTIR spectra of biosynthesized lead oxide nanoparticles...... 116 Figure 5. 9: Antibacterial activities of biogenic PbO nanoparticles; (A): without UV-illumination; (B): with UV- illumination...... 119 Figure 5. 10: Potential cytotoxic effects of PbO biogenic nanoparticles...... 122 Figure 5. 11: Antioxidant assays of biosynthesized lead monoxide nanoparticles...... 123 Figure 5. 12: Enzyme inhibition assays of biogenic PbO nanoparticles; (A): alpha amylase enzyme inhibition; (B): Protein kinase enzyme inhibition assay indicating no visible zones of inhibition...... 124 Figure 5. 13: Schematic of the potential cytotoxic nature of biogenically synthesized lead oxide nanoparticles; ...... 126
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List of Figures
Chapter 6 Figure 6. 1: From biosynthesis to applications of bioinspired cobalt oxide nanoparticles ...... 132 Figure 6. 2: Plausible mechanism of biosynthesis cobalt oxide nanoparticles using Sageretia thea aqueous extracts...... 138 Figure 6. 3: XRD analysis of biosynthesized cobalt oxide nanoparticles; (A): XRD spectra; (B): Average size calculation using Scherer size approximation formula...... 140 Figure 6. 4: Various HR-TEM images of biosynthesized cobalt oxide nanoparticles; (A/B/C/D): size distribution and shape; (E): HR-TEM image; (F): SAED pattern...... 141 Figure 6. 5: (A/B/C): Various HR SEM images; (D): Particle size distribution...... 142 Figure 6. 6: Energy Dispersive Spectroscopy of biosynthesized lead oxide nanoparticles...... 142 Figure 6. 7: Raman spectra of biosynthesized cobalt oxide nanoparticles...... 143 Figure 6. 8: ATR-FTIR spectra of biosynthesized cobalt oxide nanoparticles...... 143 Figure 6. 9: Antibacterial activities of bioinspired cobalt oxide nanoparticles; (A) without UV- illumination; (B): with UV- illumination ...... 146 Figure 6. 10: Cytotoxicity of cobalt oxide nanoparticles; (A): Cytotoxicity against brine shrimps and leishmania; (B): Biocompatibility with RBC’s and macrophages; (C) Median lethal concentrations...... 149 Figure 6. 11: Antioxidant assays of biosynthesized cobalt oxide nanoparticles...... 151 Figure 6. 12: Enzyme inhibition assays of bioinspired cobalt oxide nanoparticles; (A): alpha amylase enzyme inhibition; (B): Protein kinase enzyme inhibition assay...... 152 Figure 6. 13: Schematic of the potential cytotoxic mechanisms of biogenically synthesized cobalt oxide nanoparticles...... 153 Chapter 7 Figure 7. 1: Schematic of deposition, characterization and electrochemical application of biogenic ZnO based electrode material for supercapacitor application ...... 159 Figure 7. 2: XRD patterns of (a) Sageretia thea mediated Zinc oxide nanoparticles, (b) NiF/ZnO nanocomposite electrode ...... 161 Figure 7. 3: SEM micrographs of Bare NiF (a); high magnification SEM of the fabricated NiF/ZnO electrode (inset shows HR-SEM micrographs of the deposited ZnO nano platelets on NiF (b)...... 162 Figure 7. 4: 3d computed tomography of the bare NiF (a) and NiF with deposited ZnO nanomaterial (b)...... 163 Figure 7. 5: Electrochemical measurements ...... 167 Figure 7. 6: Specific capacitance variation of ruthenium oxide electrode at different scan rate. 168 Figure 7. 7: Cycling stability of NiF/ZnO electrode...... 168
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List of Tables
List of Tables Chapter 2 Table 2. 1: Major XRD values and estimated sizes of ZnO nanoparticles biosynthesized via Sageretia thea ...... 27 Table 2. 2: IC50 calculations for biogenic ZnO nanoparticles...... 42
Chapter 3 Table 3. 1: IC50 calculation of IONP’s against selected bacterial strains...... 67
Chapter 4 Table 4. 1: MIC calculations bioinspired for NiO nanoparticles...... 99 Table 4. 2: IC50 calculation for bioinspired NiO nanoparticle...... 99
Chapter 5 Table 5. 1: MIC calculations for lead oxide nanoparticles...... 118 Table 5. 2: IC50 calculation for lead oxide nanoparticles...... 121
Chapter 6 Table 6. 1: MIC calculations of biogenic cobalt oxide nanoparticles...... 145 Table 6. 2: IC50 calculation of bioinspired cobalt oxide nanoparticles...... 148
Chapter 7 Table 7. 1: Physical properties of the NiF and NiF-ZnO achieved through simulation software using 3D nanotomoraphy...... 163
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List of Abbreviations
List of abbreviations eV electron volt g gram µL microliter µg microgram mg milligram mL milliliter nm nanometer MIC Minimum inhibitory concentration MTT Methylethiozoltetrazolium bromide DPPH Diphenyle dipicrylhydrazyl DMSO Dimethyle sulfoxide HBSS Hanks buffer salt solution PBS Phosphate buffer saline
IC50 Median lethal concentration pH Hydrogen ion concentration XRD X-ray diffraction FTIR Fourier transformed infrared spectroscopy EDS Energy dispersive spectroscopy SAED Selected area electron diffraction HR-SEM High resolution scanning electron microscopy HR-TEM High resolution transmission electron microscopy UV Ultraviolet RBC’s Red blood cells OD Optical density FWHM Full width half maximum ATCC American type culture collection FCBP Fungal culture bank of Pakistan ROS Reactive oxygen species
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Summary
Summary We successfully performed green process using the aqueous leaf extracts of medicinal plant Sageretia thea (Osbeck.) to produce multifunctional metal oxide nanoparticles. The biosynthesized Zinc oxide (ZnO), Iron oxide (Fe2O3), Nickel oxide (NiO), Cobalt oxide
(Co3O4) and Lead oxide (PbO) nanoparticles were subjected to intensive physical characterization techniques like XRD, FTIR, Raman, EDS, SAED, HR-SEM and HR- TEM. Debye Scherer approximation was used to determine the size of the biogenic nanoparticles. Average sizes of the nanoparticles were calculated as 12.5 nm (ZnO), 29 nm (Fe2O3), 18 nm (NiO), 27 nm (PbO) and 20.03 nm (Co3O4). As synthesized nanoparticles were investigated for their possible biological applications. Antimicrobial, cytotoxic, enzyme inhibition, antioxidant and biocompatibility assays were performed. Antibacterial properties against 6 pathogenic bacterial strains using disc diffusion assay and their MIC’s were calculated. Significant antibacterial potential was revealed for ZnO nanoparticles. Furthermore, the effect of UV-illumination in the enhancement of antibacterial properties was studied. Agar tube dilution method for linear mycelial growth inhibition was used to determine the antifungal potential of biogenic metal oxide nanoparticles. Significant cytotoxicity was revealed against Artemia salina. Dose dependent cytotoxicity is reported for the biosynthesized nanoparticles against Leishamnia tropica (promastigotes and amastigotes) and HepG2 cell lines using MTT cytotoxic assay, while their biocompatibility was assessed against freshly isolated human macrophages and RBC’s. Median lethal concentration (IC50) values were calculated.
Significant protein kinase inhibition is indicated by Fe2O3 nanoparticles while insignificant alpha amylase inhibition is reported for biosynthesized nanoparticles. Moderate DPPH radical scavenging activity while insignificant total antioxidant and total reducing potential was indicated. In addition, ZnO nanoparticles were deposited on the 3D porous substrate (nickel foam) to fabricate electrode material for supercapacitor applications. The fabricated NiF/ZnO electrode showed high specific capacitance of ~ 453 F g-1 at a relatively high current density of ~ 2 A g-1. The electrode also showed excellent cycling performance with 86% specific capacitance retention after 1000 cycles. Our results suggest that biosynthesized metal oxide nanoparticles can be used in diverse applications.
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Chapter 1 Introduction
Chapter 1: Introduction
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Chapter 1 Introduction
1.1 Nanoscience in 21st century Nanotechnology is considered to be an interdisciplinary field intended to design and fabricate materials for different applications. Nanotechnology is an area of interdisciplinary research in biology, chemistry, physics and material sciences. Nanotechnology is controlling, manipulating and utilizing of matter at the nanometer scale that includes atoms, molecules, and supramolecular structures. It is the apprehension and use of matter at the nanoscale with 1 dimension of 1 to 100 nm. The application of nanotechnology has been mounting swiftly in diverse arenas of physics, catalysis, biology, chemistry, electronics, bioimaging sensing etc. (Saini et al. 2010; Razzaque et al. 2016). Nanoparticulate materials have gained tremendous attention in the last few years due to remarkable chemical, biological, physical, optical and magnetic characteristics at nanoscale which differs from their macroscale counterpart owing to their structural features which vary noticeably from molecules and atoms in nanoscale (Zargar et al. 2014; Alivisatos 1996). Size < 100 nm has a significant influence on the physical, biological and chemical attributes (Biju et al. 2008). Few years back, nanoparticles were studied only for their interesting nanoscale properties while now they are extensively studied for commercial applications (Murray et al. 2000; Paull et al. 2003). Among nanoparticles, nanoscaled metallic oxides have enticed numerous researchers for their fascinating properties as well as numerous applications like biomedicine, optoelectronics, sensors, energy storage and environmental remediation (Oskam 2006). Metallic oxides NPs have emerged as multifunctional materials that intend to revolutionize many fields like biomedicine, drug delivery, energy storage, opto- electronics etc. As of present the synthesis of metal oxide nanoparticles represents captivating area of research because of their diverse applications.
1.2 Metal oxide nanoparticles Metal oxides nanomaterials exhibit a rich compositional chemistry and therefore offers massive potential in applications like catalysis, optics, electronics, energy storage, biomedicine etc. The interplay of various features like morphology, shape, size, surface chemistry & crystal structure contributes to the functional properties (Chadwick and Savin 2010; Jolivet et al. 2010; Rao et al. 2007). Metal oxide nanoparticles possess high
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Chapter 1 Introduction surface area, mechanical stability, unique structure, interesting and unusual catalytic and redox properties and are biocompatible (Fernández‐García and Rodriguez 2011). The development of microbial resistance to antibiotics is increasing at an alarming rate. Existing drugs are becoming ineffective which demands an alternative approaches. Among several possibilities of addressing the issue, nanoscale metallic oxide NPs have arisen as a likely contender due to lower toxicity, high durability, higher stability and selectivity (Stankic et al. 2016). The advantage of using inorganic metal oxides for biomedical applications that they contain mineral elements important to human beings and are effective at very low concentration (Premanathan et al. 2011). Metal oxides nanoparticles are frequently becoming part of the cancer theranostics, neurochemical monitoring and in medical implants. Some of the metal oxides have been successfully used as for cell separation, labeling and as gas sensing nanoprobes. They are also used in “Magnetic Resonance Imaging (MRI)” as contrasting agent and for guided delivery of therputants (Andreescu et al. 2012). In addition to the diverse biomedical applications, metal oxides has the potential for meeting our ever increasing energy demands by offering innovative energy storage applications (Chu and Majumdar 2012). Significant studies are been supported out in the past few decades to fabricate energy smart devices like “Lithium Ion Batteries (LiBs) and supercapacitors. Transition metal oxide have been frequently looked for assembling a supercapacitor (Ismail et al. 2016). Due to their tunable properties metal oxides are potential candidates for high technological applications such as fuel cells, ceramics, sensors, solar cells, solar absorbers, alkaline and lithium battery materials, optical devices etc. A comprehensive overview about the nature and application of certain metal oxides targeted in the present thesis is given below; 1.2.1 Zinc oxide (ZnO) It is a semiconductor metal oxide (n-type) which is explored for various uses because of its tunable and multifunctional spintronic, photonic and morphological features (Özgür et al. 2005; Kaminsky 2012). ZnO is characterized by a direct & wide band gap of 3.37 eV and possess a high excitation energy of 60 meV. ZnO has strong pyroelectric and piezoelectric characteristics (Talam et al. 2012). Owing to its interesting properties, ZnO
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Chapter 1 Introduction has successfully been used in numerous devices such as metal-insulator semiconductor diodes, miniaturized semiconductor lasers, optically transparent electrodes, nanogenerators and ultraviolet photo detectors, surface acoustic devices and gas sensors etc (Kalusniak et al. 2009; Ellmer 2012; Gao et al. 2005). In addition, ZnO is among the 5 compounds of zinc that are listed under the category of “generally recognized as safe” (Sawai 2003). Because of their biocompatible nature, ZnO has been studied for their potential biomedical applications, such as sunscreens, lotions and cosmetics. ZnO has also been used for drug delivery and bioimaging (H.-J. Zhang and Xiong 2013). Extensive research is carried out on ZnO nanoparticles to establish them as an antimicrobial and anticancer agent (Sarwar et al. 2016; Jones et al. 2008; Hassan et al. 2016).
1.2.2 Iron oxide (γ-Fe2O3)
It is a semiconductor (n-type) and possess 2.0 eV band gap (Bakardjieva et al. 2007). Iron oxide represents a perfect material for broad range uses due to being chemical stable, high surface area and supermagnetism (Muthukumar and Matheswaran 2015; Boyer et al. 2010). Among the eight known iron oxides (Cornell and Schwertmann 2003), magnetite
(Fe3O4), hematite (α-Fe2O3) and maghemite (γ-Fe2O3) are prevalent contenders because of polymorphism which results from their temperature mediated phase transitions (Wu et al. 2015). Each of these phases possess unique catalytic, magnetic and biochemical properties which make them suitable for diverse biomedical applications. At 25°C, maghemite possess ferrimagnetism, with up to 92 emu g−1 saturation magnetization
(Yamaura et al. 2004). Size and Shape of γ-Fe2O3 has a significant influence on its various properties. Iron oxide nanoparticles have indicated promising results for in materials science, engineering, cosmetics, biomedicine, bioremediation and other clinical applications (Muthukumar and Matheswaran 2015). Magnetic iron oxide nanoparticles are also known for their applications like tissue repairing, drug delivery, cell separation, detoxification, immunoassay and for MRI contrast enhancement (Laurent et al. 2008). γ-
Fe2O3 has also been looked for potential antimicrobial and anticancer agents (Pal 2014; Prucek et al. 2011).
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Chapter 1 Introduction
1.2.3 Nickel oxide (NiO) NiO represents a p-type semiconducting metal oxide having a wide band gap (3.6-4.0 eV) (Manikandan et al. 2015). It is antiferromagnetic, while exhibits exceptionally high ionization and elevated isoelectric point. It is an attractive material for research because of its electro-catalysis, stability, electron transfer and energy storage features (Thema et al. 2016). The diverse uses include electrochemical storage devices, anodic electrochromic smart windows, gas sensors, battery material etc. (Pejova et al. 2000; Odobel et al. 2010; Bandara and Yasomanee 2006; Park and Cairns 2011; Mu et al. 2011). Being an active materials, its finds uses in the adsorption of inorganic pollutants and hazardous dye (Pandian et al. 2015). As a consequence of its anti- inflammatory nature, nano NiO has been investigated for various biomedical applications. Recent studies have indicated a potential antioxidant, anticancerous and antimicrobial nature (Ezhilarasi et al. 2016; Khashan et al. 2016; Khashan et al. 2017; Saikia et al. 2010).
1.2.4 Cobalt oxide (Co3O4)
It is a semiconductor and multifunctional p-type antiferromagnetic material having an optical bandgap at 1.48 to 2.19 eV (Gulino et al. 2003). Cobalt oxide is an attractive material having diverse applications in energy storage, gas sensing, electrochromic sensing, heterogeneous catalysis, solar energy absorption, ceramic dyes and pigments, anode material in LiBs, magnetic and field emission material (Kandalkar et al. 2008; Chen et al. 2007; Lou et al. 2008; Lian et al. 2007). Cobalt oxide nanoparticles are reported to effectively catalyze the degradation of water pollutants (Liang and Zhao 2012) and combustion of hydrocarbons (Wang et al. 2015). Recent research indicate self- assembly and formation of photonic hyper crystals of cobalt nanoparticles which underline their possible applications in chemical and biological sensing (Smolyaninova et al. 2014; Bossi et al. 2016). Recent research also suggested the potential applications of cobalt oxide nanoparticles in biomedicine (Khan et al. 2015). 1.2.5 Lead oxide (PbO) Lead oxide compounds are available commercially since the refinement of lead for various applications. The earliest applications of lead oxide include its use as an additives to glass and ceramic glaze formulas and pigmentation (Blair 1998). The lead element has
5
Chapter 1 Introduction
many oxide forms like PbO, Pb2O3, Pb3O4, PbO2 (Karami et al. 2008). The electrochemical properties of lead oxide are well known. These NPs possess exceptional characteristics evidenced by their wide spread uses like in photonic crystals, gas sensors, pH sensors, luminescent materials, LiB’s, pigments, paints, superconductors, glasses industries and UV-blockers (Thulasiramudu and Buddhudu 2007; Konstantinov et al. 2006; Barriga et al. 1991; El-Damrawi and Mansour 2005; Blair 1998).
1.3 Biosynthesis of nanoparticles Green synthesis or bioinspired NPs is a rapidly growing space of research that has engrossed substantial attention because it facilitate the eco-friendly synthesis of nanotechnology based products. The interface of medicinal plants and nanotechnology has opened a bright area of research with diverse applications (Ovais et al. 2016). Recent work in therapeutic plants and nanosciences has fascinated numerous scientists for biosynthesis of metal oxide and metal nanoparticles due to their several gains over orthodox synthesis methods (Thovhogi et al. 2015). Medicinal plants have demonstrated as paramount reservoirs of different and bioactive phytochemicals which contribute to the biosynthesis of nanoparticles. Crude extracts of plants possess active phytochemicals such as alkaloids, phenolic, terpenoids, and flavonoids that are the reason behind the reducing of ionic into bulk metallic NPs (Aromal and Philip 2012). Such bioactive metabolites initiate the oxidation-reduction for reaction for biosynthesizing eco-benign nanoparticlulate matter. For biosynthesis of NPs, generally chemical and physical based procedures are applied. Physical approaches are lithographic techniques, ball milling, , plasma arcing, pulsed laser desorption, thermal evaporate, sputter deposition, molecular beam epistaxis, ultra-thin films, layer by layer growth and diffusion flame synthesis and spray pyrolysis (Joerger et al. 2000). Similarly, the chemistry based processes of are sol– gel process chemical solution and vapour deposition, electrodeposition, Langmuir Blodgett method and easy chemical methods like, hydrolysis, catalytic route, wet chemical methods & co-precipitation (Oliveira et al. 2005; Panigrahi et al. 2004). Hitherto, being effective, these chemistry and physics based nanoparticles synthesis possess considerable disadvantages. For example the physical methods has high energy requirements while the chemical processes comprise of using hazardous chemicals as
6
Chapter 1 Introduction agents for reduction and stabilization of NPs which can generate toxic waste streams making both methods hazardous (Thema et al. 2015; Kuppusamy et al. 2016). Furthermore, noxious chemicals can remain attached with the nanoparticles which are synthesized via a chemical route and therefore cannot be applied for the biomedical applications (Zak et al. 2011). To overcome such disadvantages, a one plot, ecofriendly scheme has is suggested for bioinspired synthesis of NPs using biological resources like fungi, bacteria, cyanobacteria, diatoms, seaweed and plants (Kuppusamy et al. 2016). For biosynthesis of nanoparticles the microorganism based methods are not preferred because of the complexity in maintenance of microbial cultures and some hygiene concerns (Raj et al. 2006). Furthermore, relatively longer incubation time is taken by microorganisms for the reduction of the metallic ions (Ghaffari-Moghaddam and Hadi-Dabanlou 2014). Among the various biological resources, medicinal plants provides an ideal platform for the biological synthesis of nanoparticles. Therefore a paradigm shift can be observed towards bio-nano interface using medicinal plants as an effective agent for stabilization and chelation (Ovais et al. 2017). Plant based biosynthesis methods offers several other advantages like easiness in the scaling up, safe in handling and do not require any bio- agents which are used for microorganisms (Baker et al. 2013). Furthermore, biologically synthesis of nanoparticles is simple, rapid, one step, safe and economical and with increased level of biocompatibility (Ovais et al. 2016). Certain reports suggest that green synthesized nanoparticles are more biocompatible and effective. Over the last few years a numerous articles have been published which indicate plants as an resourceful factories in biosynthesis of metal and metal oxide NPs (Parveen et al. 2016) like silver, gold, palladium, titanium oxide, iron oxide, zinc oxide and nickel oxide etc. Various plants like Pelargonium graveolens, Emblica officinalis, Cicer arietinum, Cymbopogon flexuosus, Cinnamommum camphora, Azadirachta indica, Aloe vera, Tamarindus indica, Avena sativa, Agathosma betulina, Aspalathus linearis, Hibiscus Sabdariffa, Callistemon viminalis etc. are already been efficaciously demonstrated for metal and metal oxide nanoparticles biosynthesis (Thovhogi et al. 2015; Thema et al. 2015; Diallo et al. 2015; J. Huang et al. 2007; Shankar et al. 2004; Chandran et al. 2006; Ankamwar et al. 2005a; Ankamwar et al. 2005b; Sundrarajan et al. 2017; Ezhilarasi et al. 2016).
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Chapter 1 Introduction
1.4 Sageretia thea (Osbeck.) M.C. Johnnst. Sageretia thea (Osbeck.) is an evergreen shrub that belong to the Rhamnaceae family. It is locally knowns as “bird plum or Chinese sweet plum” in English, “Sangdong” in Korea, “Gangeer” in Urdu and “Momanra” in Pashto language (Ko et al. 2016; Shah et al. 2013). According to the information retrieved from the Flora of Pakistan, Sageretia thea has various synonyms like Sageretia theezans (Linn.), Rhamnus thea (Osbeck.) and Rhamnus theezans (L.). It is found in East Africa, China, Afghanistan, Pakistan, Nepal, India and Korea. Sageretia thea has been used in the folklore medicine for various ailments such as cardio-vascular and circulatory diseases, fever, jaundice and hepatitis (Hyun et al. 2015; Khan et al. 2014; Murad et al. 2011). In certain parts of Korea and China, it is used in the making of tea. Few studies have been published previously which underline the therapeutic potential of Sageretia thea. It has been known for being rich in antioxidants (Chung et al. 2004; Chung et al. 2009) while also been studied for HIV-1 inhibition (Park et al. 2002). Furthermore, different organic plant extracts showed noteworthy toxicity against breast cancer cells of human in vitro (Ko et al. 2016). Sageretia thea also possess a rich phyto-chemistry. It is investigated to be rich in phenolic and flavonoid compounds. In various fractions tested like methanol, n-hexane, chloroform, n-butanol and water, Sageretia thea revealed 58 different phytochemicals by using GC-MS (Ko et al. 2016). Some articles indicated the presence of Taraxerol Syringic acid, Kaempferol, Daucosterol, Quercetin, Myricetrin, etc. in S. thea (Shen et al. 2009; Chung et al. 2004; Xu et al. 1994). In plant based biosynthesis of nanoparticles, plant with rich phyto-chemistry (specifically phenolic and flavonoids) are preferred which performs the stabilization and chelation of NPs during a biosynthesis reaction (Bala et al. 2015). Owing to its rich phyto-chemistry, the effective role of Sageretia thea for capping and chelating can be pre-concluded.
1.5 Importance of study Considering the exciting potential metal oxide nanoparticles, this current research was designed to demonstrate a simple, rapid, one-step and eco-friendly method for the biosynthesis of metal oxide nanoparticles using aqueous leaf extracts of medicinally important and phyto-chemically rich Sageretia thea. Furthermore, the targeted metal
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Chapter 1 Introduction oxide nanoparticles were studied for their various applications. Various characterization techniques like FTIR and Raman scattering were used to study the vibrational properties of biosynthesized metal oxide nanoparticles. Crystalline nature and elemental composition was studied through x-ray diffraction (XRD) and energy dispersive spectroscopy (EDS). Furthermore, HR-SEM was carried out to study the morphology and shape of the nanoparticles. Size, shape and crystalline nature was also investigated through HR-TEM and SAED. After the detailed characterizations, metal oxide nanoparticles (ZnO, Fe2O3, NiO, Co3O4 and PbO) were extensively and studied for their antimicrobial, antileishmanial, anticancer, antioxidant and enzyme inhibition properties while their biocompatibility was also assessed against freshly isolated RBC’s and macrophages. Furthermore, bioinspired ZnO nanoparticles was successfully deposited on 3D porous substrate i.e. nickel foam and was studied for potential application in supercapacitors as energy storage material. The fabricated electrode material using biogenic ZnO and nickel foam was investigated for its stability and capacitance.
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Chapter 1 Introduction
1.6 Specific objectives Objectives of the current study are: Sageretia thea (Osbeck.) mediated synthesis of ZnO, Fe2O3, NiO, PbO and
Co3O4 nanoparticles. Room temperature characterization of bioinspired nanoparticles using XRD, FTIR, EDS, Raman, SAED, HR-SEM and HR-TEM. Studying the in-vitro antioxidant, cytotoxic, enzyme inhibition and antimicrobial properties of targeted metal oxides. Deposition of ZnO nanoparticles on 3D porous substrate “nickel foam” for supercapacitor applications.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Chapter 2: Sageretia thea (Osbeck.) mediated synthesis of ZnO nanoparticles and its biological applications
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Graphical abstract: Sageretia thea (Osbeck.) mediated synthesis of ZnO nanoparticles and its biological applications
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Abstract Physical and biological properties of bioinspired ZnO NPs by aqueous leaf extracts of Sageretia thea were studied. Nanoparticles of size ~ 12.4 nm were extensively characterized. In vitro antimicrobial, cytotoxic, biocompatibility and enzyme inhibition assays were performed. Significant antimicrobial activities with and without UV illumination are reported. Bioinspired ZnO nanoparticles were found effective against fungal strains. MTT assay was performed to check the leishmaniciadal activity against promastigotes (IC50: 6.2 μg/mL) and amastigotes (IC50: 10.87μg/mL) of Leishmania tropica. Brine shrimp lethality was also indicated by bioinspired ZnO nanoparticles (IC50: 21.29 μg/ml. Hemocompatible nature of bioinspired nanoparticles was revealed. Furthermore, the antioxidant activities were performed. In addition, significant protein kinase while insignificant alpha amylase inhibition was recorded.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.1 Introduction The interface of nanosciences and biology has opened an exciting area of research with numerous applications. While biosynthesis of Au, Ag nanoparticles was of primary focus, the green synthesis using natural extract has been recently extended to various nano-scale functional oxides (Thema et al. 2015; Thema et al. 2016; Thovhogi et al. 2015; Diallo et al. 2015; Sone et al. 2015; Nyangiwe et al. 2015; Ezhilarasi et al. 2016; Fuku et al. 2016). Zinc oxide (ZnO) nanoparticles are among the most exploited metal oxides because of its multifunctional and tunable nature. The multifunctional nature of ZnO NP’s has been demonstrated in numerous applications ranging from electronics devices to cosmetics. ZnO NPs possess a 3.37 eV band gap and 60 meV excitation energy and therefore it is an ideal material to be used in many devices such as transistors, semiconductor diodes, UV- photodetectors etc (Thema et al. 2015; Kaminsky 2012; Kalusniak et al. 2009). In addition, ZnO has a number of biomedical applications such as in drug delivery and bioimaging. It has already been used in cosmetics like sunscreen, lotions and ointments (Mirzaei and Darroudi 2017). Due to their different morphologies, size, shapes and their bio-safe nature, ZnO NPs are considered perfect contender for biomedical uses. To date, mostly chemical and physical means are used as a method of synthesis for ZnO nanoparticles. The chemical methods for synthesis that are mostly employed for nano scaled ZnO synthesis include hydrothermal, spray pyrolysis, sol-gel, sonochemical, solvothermal and electrodeposition. The physical methods that are usually employed for synthesis include thermal evaporation, pulsed and chemical laser deposition, epitaxy etc (Fan et al. 2015; Suntako 2015; Mani and Rayappan 2015; Sonker et al. 2015; M.-H. Wang et al. 2015; Ameen et al. 2015). However, the physical and chemical means of synthesis possess considerable disadvantages. The physical methods of synthesis are accompanied with high energy requirements while the chemical means involve the use of toxic chemicals making both methods environment unfriendly (Diallo et al. 2015). For resolving the glitches of dangerous waste and energy, green approaches are anticipated and successfully employed for the synthesis of ZnO nanoparticles, therefore a paradigm shift can be observed towards bio-nano-interface. Among the various biological resources (plants, bacteria, algae, fungi) plants provides an ideal platform for biosynthesis of nanoparticles (Ovais et al. 2016). The phytosynthesis of MNPs is already been
14
Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications demonstrated however limited data is available on metallic oxide nanoparticles and their biological properties as well as biocompatibility. Considering the limited data available on biosynthesis of zinc oxide nanoparticles, we suggest a ecofriendly and simplistic procedure for the biosynthesis ZnO NPs via complete environmental friendly procedure which involve the use of aqueous extracts of Sageretia thea as efficacious agent for oxidation-reduction and capping. The medicinal uses of Sageretia thea (Bird Plum/English) is well documented and it is mostly used in the treatment of, jaundice, hepatitis, circulatory and cardio-vascular disease (Hyun et al. 2015; Khan et al. 2014; Murad et al. 2011). Diverse techniques have been used to study the single zincite phase of the nanoparticles. The novelty of the manuscript also lies in the first time report on the effectiveness of ZnO nanoparticles against Leishmania tropica. In addition, a comprehensive study has been conducted which includes a wide array of biological applications and biocompatibility of the ZnO nanoparticles. All the experiments were conducted in Biotechnology Department, QAU and Material Research Department of iThemba Labs, Cape Town.
2.2 Materialand methods 2.2.1 Processing of plant sample Sageretia thea was collected from Islamabad and taxonomically verified in the Department of plant sciences while the herbarium specimen (MOSEL-343) was deposited in the herbarium of MoSAEL, Biotechnology Department, QAU. Fresh and healthy leaves were excised, washed with distil water, dried and pulverized by Willy mill. Pulverized test sample was stored and used for aqueous extraction. The general layout of the study has been summarized in Figure (2.1).
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 1: From biosynthesis to biological application a schematic representation
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.2.2 Biosynthesis of ZnO nanoparticles Already established procedure established by Thema et al., 2015 was utilized with negligible modification for the phytosynthesis of ZnO NPs. To obtain the green extracts aqueous solution, 30 g of the grounded plant material was added to 200 mL dH2O and heated at ~ 80°C for 1 hr on a magnetic stirrer hot plate (Snijders). The resultant aqueous solution was filtered thrice with a whattman filter paper to remove the solid residues. To 100 mL of aqueous plant extracts solution (pH 5.7), 6.0 g of the precursor salt zinc acetate dihydrate (Alfa Aesar) was added, followed by stirring and gentle heating (~ 60°C) for 2 hr. Solution pH was noted as 3.94 after the introduction of precursor salt zinc acetate dehydrate (Alfa Aesar) at room temperature. The solution was allowed to cool and centrifuged (10,000 rpm / 10 min; HERMLE Z326K) to collect the precipitate. The precipitate assumed as ZnO/Zn(OH)2 (Mani and Rayappan 2015) was washed 3 times with distill water, dried and annealed at different temperatures in air to obtain highly crystalline ZnO nanoparticles. The synthesized nanoparticles were subjected to extensive characterization techniques to confirm a single phase zincite nanopowder. ZnO nanoparticles annealed at 500°C were used for further experiments. 2.2.3 Characterizations The crystallinity ZnO NPs was confirmed by XRD using X-ray diffractometer (model Bruker AXS D8 Advance) with irradiation line Kα of copper (λ=1.5406 A0). XRD analysis was carried out for all the thermally annealed samples and their corresponding size was calculated using Scherer equation {<Øsize> = K λ / ∆θ1/2 cosθ}. Raman spectroscopy (Ocean Optics QE Pro-Raman) was carried out to study the vibrational properties. A laser line of 473 nm with average excitation power of 2.48 mW was used to record Raman spectrum (200-1000 cm-1). Pressed powdered samples of aqueous plant extracts of S. thea and its corresponding ZnO nanoparticles was carried out using ATR (Thermo scientific Nicolet iS 10 spectrometer equipped with smart iTR ATR accessory with a diamond crystal) and FTIR in the spectral range from 4000-400 cm-1and 4000-400 cm-1. Furthermore, the UV absorption (Agilent 8453 UV-vis deuterium lamp as a UV source) spectra and emission spectra (Ocean Optics Maya2000 Pro) was recorded and their band gap was calculated. Nanoparticles morphology was examined by HR-SEM while EDS (Energy Dispersive X-ray spectroscopy; Oxford instruments X-Max) was
17
Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications utilized to determine the elemental composition. Size distribution and shape was also studied through HR-TEM (EMU Joel JEM). 2.2.4 Test sample preparation To study the in vitro biological properties of biogenic zinc oxide nanoparticles, various concentrations of the test samples were made in DMSO. The stability of the colloidal suspension was investigated by adding 10 mg ZnO nanoparticles to 5 mL DMSO in a test tube, which was sonicated (Elmasonic) for 10 min. A slightly turbid colloidal suspension was obtained which was allowed to stand. UV absorption spectra was checked at every 1 hour, a stable reading at 374 nm was recorded till 24 hours. Hence, it can be inferred that these ZnO colloidal suspension remained stable for at least 24 hours. 2.2.5 Antibacterial activity ZnO nanoparticles were investigated for antibacterial activity using disc diffusion method (Fatima et al. 2015; Thatoi et al. 2016) while their corresponding MIC’s were calculated using broth dilution method (Wiegand et al. 2008). Already available bacterial strains in Department of Biotechnology, QAU, Islamabad were used. The frozen cultures were refreshed by inoculating them on Nutrient Agar media (Oxoid-CM0003). Before the assay, cultures were transferred to the nutrient broth and kept in the shaking incubator (Temp:37°C; RPM:200) for 24 hrs. The microbial cultures were standardized by keeping the optical density at 0.5 which corresponds 1 × 108 CFU/ml. 100 µl of broth cultures were dispensed in the nutrient agar. Filter discs (6 mm) in size were loaded with 10 µL of test dilutions (2000, 1000, 500, 250, 125 and 62.5 µg/ml) air dried, then carefully positioned on the uniform bacterial lawn. Antibiotic discs (10 µg) was utilized as positive control. Bacterial plates were incubated for 24 hr at 37°C. Inhibitory zones were measured in millimeters with vernier caliper. Following the antibacterial screening by disc diffusion assay from 2000-62.5 µg/ml, minimum inhibitory concentration was investigated over the final dilution range (31.25-1.95 µg/ml) using broth dilution method. MIC was defined as the minimum concentration of nanoparticle suspension that inhibit the bacterial growth. Considering the debate of UV-illumination on the enhancement of the bactericidal effect, ZnO nanoparticles test dilutions were illuminated under UV light for 20 min. Germicidal 6 Watt UV Lamp 6GT5 (Sankyo denki- Japan) was used as a source of UV. Same
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications procedure was followed for determining the antibacterial activities for the UV-treated ZnO test dilutions. 2.2.6 Antifungal activity The nanoparticles were investigated for antifungal potential using linear growth mycelial inhibition method with minor modifications as previously described (Ahmad et al. 2016). Sabouraud Dextrose Liquid medium (Oxoid CMO147) supplemented with 1.5% agar technical (Oxoid LP0012) was used to prepare fungal growth media. Briefly, 4 ml of autoclaved media was transferred in autoclaved test tubes, then allowed to cool to ~ 50°C. 66.6 µL from the test dilutions were added and the media and allowed to solidify in a slanting position. Freshly cultured fungal spores were inoculated to the base of SDA slants solidified with nanoparticle dilutions and incubated at 37°C for 48 hrs. 250 µg/mL Positive control was Amphotericin B while negative control was plain media Percent inhibition was calculated as; % inhibition = 100 – [growth in sample (cm) / growth in control (cm)] × 100 To further investigate and confirm the antifungal activity, well diffusion assay was performed with minor modifications (Khan et al. 2015). 30 mL of Sabouraud Dextrose liquid medium (Oxoid:CM0147) was used for preparing the broth cultures of pathogenic fungi, while All the fungal strains were inoculated in a broth and kept in a shaker incubator at 37°C for 24 hours. Sabouraud dextrose agar medium was prepared by supplementing the medium with 1.5% agar technical. The autoclaved medium was poured in the plates and kept in incubator for 48 hours to see if there were any contamination. Assay was performed by adjusting the OD of the broth cultures to 0.5 by adding further sterilized broth. From the already standardized cultures, 50 µL of the broth culture was dispensed on the petri plate and their uniform lawns were prepared. Wells were made using 4 mm borer. 10 µL of molten agar was also added to each well to ensure the samples does not diffuse beneath the media. 20 µL of the test samples were introduced in each well. After 72 hrs incubation ZOI were measured with vernier calipers. 2.2.7 Protein kinase inhibition The experiment was accomplished using the Streptomyces 85E strain (Fatima et al. 2015). Uniform lawn of Streptomyces 85E strain was prepared using ISP4 minimal
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications media. Six millimeter filter discs impregnated with 10 µL of the test nanoparticle dilution were carefully placed on the ISP4 medium after making a uniform lawn. DMSO and surfactin were negative and positive controls respectively. Following 72 hrs of incubation at 30°C, bald and clear ZOI were determined using with Vernier calipers in millimeters. 2.2.8 Alpha amylase inhibition In vitro alpha amylase inhibition was investigated as described previously in a 96 well plate (Ali et al. 2017). To the reaction mixture (15 µl PBS/25 µl α-amylase enzyme), 10 µl of test samples and 40 µl of starch solution was added stepwise. Reaction mixture with all the ingredients was allowed incubation at 50°C for 30 min and then 20 µl (1 M HCL) and 90 µl of iodine solution were added. Blank solution contained deionized water, starch and PBS while positive and negative controls comprised of acarbose and deionized water respectively. Enzyme inhibition was calculated as;
푬풏풛풚풎풆 풊풏풉풊풃풊풕풊풐풏 = [{푶푫푺 − 푶푫푵} ÷ {푶푫푩 − 푶푫푵}] × ퟏퟎퟎ
Whereas, “ODS”, “ODN” and “ODB” corresponds to the optical densities of sample, negative control and blank respectively. 2.2.9 Antileishmanial activity (amastigotes and promastigotes) The cytotoxicity of the biogenic nanoparticles was assessed against the promastigote cultures of Leishmania tropica KMH23 (Khyber Medical Hospital-23) using MTT cell viability assay, as described previously (Ali et al. 2017). M199 media having 10% FBS was used for culturing the parasites. Leishmania culture at a density of 1 × 106 cells/ml were used for the assay. Dilutions of final concentrations (1 to 200 µg/mL) were used while the activity was performed in 96 well microplate. DMSO and Amp B were negative and positive controls. The seeded 96 well microplate with test dilutions was allowed incubation at 24°C for 72 hr and readings were recorded at 540 nm. All the survived promastigotes were counted under the inverted microscope and their LC50 values were found out by table curve software. The activity was performed in 96 well plates. Percent inhibition was calculated as; 푺풂풎풑풍풆 풂풃풔풐풓풃풂풏풄풆 % 풊풏풉풊풃풊풕풊풐풏 = [ퟏ − { }] ×100 푪풐풏풕풓풐풍 풂풃풔풐풓풃풂풏풄풆 Same procedure was applied on the amastigote cultures of Leishmania tropica KMH23. Leishmania exist in amastigote form inside the human body.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.2.10 Brine shrimp cytotoxicity Brine shrimp cytotoxicity experiment was further carried out to confirm the cytotoxicity of bioinspired nanoparticles. Artemia salina (brine shrimp) eggs were purchased from Ocean Star International. The eggs were stored at 28°C. Artificial sea water (34 g/L) was used for hatching of the eggs in a tray near a light source at 37°C. The procedure was carried out 96 as established (Ali et al. 2017; Khan et al. 2015). 10 Fresh hatched nauplii were taken with pasture pipette and moved to the wells. Test samples (1 µg/mL to 200 µg/mL) was introduced in the wells followed by volume adjustment to 300 µL After 24 hrs of exposure, the shrimps were taken out using pasture pipette and counted under magnifying glass. Percentage of dead shrimps was calculated in each well after incubation at 24 hrs. LD50 values were calculated using table curve software. 2.2.11 Antioxidant activities Spectrophotometric method was employed to investigate the radical quenching ability by using 0.0024 molar (9.6 mg/100 ml methanol) DPPH (2, 2-diphenyl 1-picrylhydrazyl) as a stable free radical (Ali et al. 2017; Fatima et al. 2015). Various concentrations of the nanoparticle starting from 200 µg/mL to 0.78 µg/ml were tested for free radical scavenging. DMSO and ascorbic acid were used as negative and positive controls. The 200 µL reaction mixture comprised of 180 µl reagent and 20 µL sample. The reaction mixture was incubated in dark for 20 minutes followed by measuring the optical densities at 517 nm BIOTEK microplate reader. Percent free radical scavenging can be found out using the formula; 푺풂풎풑풍풆 풂풃풔풐풓풃풂풏풄풆 % 푺풄풂풗풆풏품풊풏품 = [ퟏ − { }] ×100 푪풐풏풕풓풐풍 풂풃풔풐풓풃풂풏풄풆 Total antioxidant capacity was investigated using phosphomolybdenum based method as established previously (Jafri et al. 2014). Readings were recorded at 695 nm while results were calculated as number of ascorbic acid equivalents in µg per mg of the sample i.e. µg AAE/mg . Potassium ferricyanide based procedure was utilized to investigate the reducing power of biogenic nanoparticles (Jafri et al. 2014). DMSO was used as negative control while ascorbic acid as positive control. The absorbance intensity was measured using BIOTEK microplate reader at 630 nm. The reducing power was determined as ascorbic acid equivalents per mg.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.2.12 Biocompatibility of ZnO nanoparticles with RBC’s For establishing the bio-safe feature of the biogenic ZnO NPs, percent hemolysis assay was carried out on the newly isolated red blood cells of human (Malagoli 2007). The fresh blood was collected with a sterile syringe from a heathy donor and introduced to the EDTA tube to blood clotting prevent. RBC’s were collected through centrifugation of 1 ml blood for 5 min. 9.8 ml of PBS (pH: 7.2) was introduced to 200 µL of pelleted erythrocyte, and by gently shaked for obtaining a suspension of erythrocytes in PBS buffer. 100 µL from the obtained erythrocyte suspension and test nanoparticle suspension each, were gently introduced to separate eppendorf tubes and incubated at 35°C for 1 hr, then centrifuged at 10,000/10 min. Supernatant was added to 96 well plate and the hemoglobin release was determined at 540 nm through BIOTEK microplate reader. DMSO and Triton were used as negative and positive controls respectively. Results were calculated as percentage hemolysis induced by the nanoparticle dilution calculated through the formula;
% 퐻푒푚표푙푦푠𝑖푠 = [퐴퐵푆 − 퐴퐵푁퐶 ÷ 퐴퐵푃퐶 − 퐴퐵푁퐶] × 100 2.2.13 Biocompatibility of ZnO nanoparticles with human macrophages To further assess the bio-safe nature of ZnO nanoparticles, cytotoxicity was examined against human macrophages isolated from peripheral human blood using Ficoll– Gastrografin (sodium diatrozoate) method for cytotoxicity evaluation (de Almeida et al. 2000). This isolation protocol is based on ficoll-gastrografin density gradient (density=1.070 g/mL). In a general procedure, 5.7 g of ficoll was slowly dissolved in 95 mL deionized water with 5 mL of gastrografin. Hank’s buffer salt solution (HBSS) was used for the dilution of blood layered gradually on ficoll-gastrografin, followed by centrifugation at 400g/30 min and purified with percoll gradient (density 1.064 g/mL) which was adjusted via autoclaved dH2O. Obtained cells were added to RPMI medium supplemented with antibiotics (Streptomycin: 0.1 mg/mL; Penicillin:100 U/mL), FBS (10 %) and Hepes (25 mM). Isolated macrophages were grown to the culture density of 1 × 5 10 cells/well in humidified incubator with 5% CO2. Percentage inhibition was calculated using formula; 푺풂풎풑풍풆 풂풃풔풐풓풃풂풏풄풆 % 풊풏풉풊풃풊풕풊풐풏 = [ퟏ − { }] ×100 푪풐풏풕풓풐풍 풂풃풔풐풓풃풂풏풄풆
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.3 Results and discussion 2.3.1 Biosynthesis and characterization of ZnO nanoparticles Physical means of synthesis of ZnO nanoparticles are either costly, laborious and time consuming while the chemical means often generates toxic waste lines making them environmental unfriendly. Moreover, sometimes noxious chemicals can remain adhered to the surface of nanoparticles which cannot be applied in medical applications (Zak et al. 2011; Darroudi et al. 2014). However, phytosynthesis of ZnO NPs is deprived of such disadvantages and therefore has been considered more acceptable process of synthesis. We have demonstrated for the first time synthesis of nanoscaled ZnO from Sageretia thea, a medicinally important plant also used in making tea in China and Korea. The aqueous extracts of plants are usually rich in phenolic and flavonoid chemical entities that has a pivotal function in reducing and stabilizating of ZnO nanoparticles (Bala et al. 2015). Phytochemicals like Quercetin, Myricetrin, Kaempferol, Syringic acid, Daucosterol, Taraxerol etc. are reported in various studies from S. thea through advanced analytical techniques such as HPLC and NMR (Shen et al. 2009; Chung et al. 2009; Xu et al. 1994). Figure (2.2) shows the various bioactive components isolated from the leaves of S. thea which presumably have played the role of stabilizing and capping the ZnO nanoparticles.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 2: Bioactive compounds isolated from Sageretia thea as reported in previous studies
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.3.2 Physical characterization Figure (2.3) demonstrate the XRD spectra of biogenic nanoparticles annealed at various temperatures (300°C – 500°C). The observed Bragg peaks were found in correspondence to the single phase hexagonal Zincite/Chinese white/ Zinc white pattern (JCP pattern 00-
036-1451). The lattice constants were deduced as
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 3: (A) XRD pattern of biogenic ZnO nanoparticles annealed at different temperatures; (B) their average size calculations using Debye-Scherrer approximation
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Table 2. 1: Major XRD values and estimated sizes of ZnO nanoparticles biosynthesized via Sageretia thea
Bragg reflection (hkl) Bragg angle (θ) Beta (∆θ1/2) Øsize nm rad FWHM rad 400°C 500°C 400°C 500°C 400°C 500°C 100 0.2409 0.2580 0.0092 0.0105 14.8 13.0 002 0.2634 0.2836 0.00456 0.0079 30.1 17.3 101 0.2797 0.2970 0.00645 0.0090 21.1 15.0 102 0.3782 0.3955 0.0106 0.0164 12.9 8.3 110 0.4579 0.4855 0.0133 0.0145 10.2 9.3 103 0.5118 0.5294 0.0181 0.0158 7.5 8.6 112 0.5569 0.5740 0.79306 0.0138 9.8 15.4 Average 15.2 12.4
Note: Crystal size was calculated using Debye Scherer’s equation [<Øsize> = K λ
/ b(∆θ1/2) cosθ] where as;
Øsize: is the crystallite size. K or Kappa: is a dimensionless shape factor which is equal to 0.9. l or Lambda: is the wavelength of the X-radiation which is equal to 1.5406. b (∆θ1/2) or Beta: is the peak’s full width at half maximum (FWHM in radians) θ: is the Bragg angle
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 4: HR-TEM image of biogenic ZnO nanoparticles; (A/B) Size distribution at 50 nm; (C) at 10 nm; (D) their shape; (E) HR-TEM image; (E) Typical SAED pattern; (F): HR-SEM
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Raman spectra of ZnO nanoparticles was recorded using Argon laser green excitation
λexc.= 514 nm. In a hexagonal lattice (P63mc symmetry the group theory predicts 8 sets of phonon normal modes i.e. 2A1, 2E1, 2B1, and 2E2. Figure (2.5 B) shows the Raman vibrational modes and indicate the raman shift at 199 cm-1, 320 cm-1, 425 cm-1. The FTIR spectra was recorded over the spectral range of 400-4000 cm-1 indicated in Figure (2.5 A). The IR absorptions were plotted for further characterize the possible functional groups and nanoparticles itself. The broad peak ~ 3300 cm-1 indicate the O-H stretching mode while a characteristic vibrational mode of Zn-O stretching can be observed ~ 490 cm-1 which is in agreement to some of the earlier studies (Maaza et al. 2015). High crystallinity and purity of ZnO nanoparticles can be pre-concluded because of the intense peak of ZnO as compared to the O-H. FTIR spectrum also shows other infrared absorption peaks at different regions i.e. ~800 cm-1, ~1200 cm-1, ~1500 cm-1 and ~2400 cm-1 which can be attributed to various functional groups like =C-H, -C-O, -C-H and -CN respectively. It can be inferred that the aforementioned vibrations are particular to the rich phytochemistry of the aqueous extracts of S. thea which were used as oxidizing- reducing as well as capping agent in the green synthesis reaction. These associated functional groups remained stable even after high thermal annealing such as at 500°C. Furthermore, to get a clear picture of the synthesis process, an ATR-FTIR equipped with diamond crystal was performed on the aqueous powdered plant extracts and the nanoparticulate material over the spectral range of 4000 cm-1- 400 cm-1 as shown in Figure (2.6). Common peaks are observed centered at ~1200 cm-1 and ~3400 cm-1 attributing to -C-O and O-H (alcoholic) stretching while ATR peak at ~1600 cm-1 attributed to –C=O is diminished. It can be proposed that compounds with –O-H functional group like phenols and -C-O functional group are responsible for the reduction of ZnO nanoparticles, however the mechanism is debatable and further studies needs to be conducted. The FTIR absorption of as synthesized nanoparticles were in correspondence with previous studies (Thema et al. 2015). In general, depending on the synthesis method and plant used, the size and positioning of peaks can vary.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 5: (A) FTIR of biogenic ZnO nanoparticles; (B) their Raman spectra
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 6: ATR-FTIR of bioinspired ZnO nanoparticles using diamond crystal; (A) Dried aqueous plant extracts of Sageretia thea; (B) Biogenic ZnO nanoparticles
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Elemental analysis was performed through Energy Dispersive X-ray spectroscopy (EDS) as shown in Figure (2.7). The appearance of “Carbon” is attributed to the grid support while no other significant element has been found in the EDS spectrum a part from Zinc “Zn” and Oxygen“O” which relates the single phase purity of the nanoparticles. Figure (2.8) shows the UV absorption and excitation emission spectra of biogenic nanoparticles. UV spectrum was recorded. A broad peak is observed at 233 nm, while excitation emission spectra was recorded on optic fiber setup and a broad peak was observed at 614 nm. Band gap was calculated using the Tauc plot (Rusdi et al. 2011). The band gap of the phytosynthesized nanoparticles was calculated as 3.25 eV.
Figure 2. 7: Elemental composition of bioinspired ZnO nanopowder using Energy Dispersive Spectroscopy
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 8: (A) UV absorption spectra of biogenic ZnO; (B) Their room temperature excitation emission spectra and band gap
2.3.3 Antibacterial activity Figure (2.9 A/B) indicate the antibacterial effects of ZnO nanoparticles against two strains of gram positive bacteria i.e. B. subtilis (ATCC: 6633) and S. epidermidis (ATCC: 14990) and three strains of gram negative bacteria i.e. P. aeruginosa (ATCC: 9721), K. pneumonia (ATCC: 4617) and E. coli (ATCC: 15224) using disc diffusion method carried out at different concentrations i.e. from 2000-62.5 µg/ml. It was found that ZnO nanocrystals were most effective against K. pneumonia while comparatively least effective against P. aeruginosa. The bactericidal effects of ZnO NPs has been well documented in earlier reports however the enhancement of the antibacterial effect with and without UV exposure to the nanoparticles has been debatable (Sirelkhatim et al. 2015). To get a comparative picture, the antibacterial assay was carried out without UV light exposure and with 20 min of UV exposure to the test dilutions. In general there is
33
Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications increase in the antibacterial effect after UV exposure, however the enhancement is not significantly high. For instance, the MIC calculated for K. pneumonia was found to be 7.81 µg/ml while with UV exposure the MIC was reduced 3.90 µg/ml. The improved bactericidal effect due to UV illumination can be attributed to the elevated production of ROS due to UV exposure. Elevated production of negative oxygen species due to UV has been previously established (H. Zhang et al. 2011). ROS like H2O2 can penetrate inside the organism to initiate the genotoxic effects. Other researchers have indicated the formation of novel complexes due to UV irradiation that contributes to the increase in antibacterial activity (Zhou et al. 2008). In general, the antibacterial and cytotoxic effects can be manifested through a various other mechanisms Figure (2.10). Surface defects are considered another reason for the impressive antibacterial activity. Being rich in defect chemistry (Padmavathy and Vijayaraghavan 2008), ZnO nanoparticles are accompanied by numerous sharp corners and edges that causes can induce abrasion in the protective membranes after coming in contact (Ramani et al. 2014). By entering inside, ROS generation can take place leading to cytotoxicity. Overall, we report impressive antibacterial activities of biosynthesized ZnO nanoparticles as indicated in Figure (2.9 A/B) which are in correspondence with some of the earlier findings (Liu et al. 2009; Applerot et al. 2009). A reason for such good activities can be bioactive functional groups attached to the nanoparticles. As indicated in the presented results, biogenic ZnO nanostructures produced larger inhibitory zones as compared to the gentamycin 10 µg disc. Added to our results, we further conclude a concentration dependent antimicrobial activity of S. thea mediated ZnO nanoparticles. It is important to mention that disc diffusion assay was used for the final concentrations ranging from 2000-62.5 µg/ml. Following the impressive zones of MIC’s were calculated by micro broth dilution method for final concentrations lower than 62.5 µg/ml. Numerous scholars have tried to investigate the antimicrobial properties of Zinc oxide NPs. Impressive antimicrobial nature against gram negative and positive bacterial strains is reported which is in correspondence with the earlier findings (Jones et al. 2008; Joshi et al. 2009). We report potential antibacterial nature of zinc oxide nanoparticles with and without UV illumination, however the antibacterial activity was found to be enhanced after exposure to UV light. UV illuminated zinc oxide nanoparticles are reported to have increased
34
Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications antibacterial activity which is in correspondence with the present study (Raghupathi et al. 2011).
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 9: Antibacterial activity and MIC of biogenic ZnO nanoparticles; (A) With no UV illumination; (B) Antibacterial activities after 20 min UV illumination
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 10: Proposed mechanisms of manifesting cytotoxicity as indicated by numerous earlier studies; (A) ROS: Proposed pathway of generation; ROS generation is considered as a primary cause of cytotoxicity to cells. Negatively charged ionic species does not have the tendency to enter inside the cell however they are converted to hydrogen peroxide that can easily penetrate inside the cell, subsequently interfering with the cellular machinery leading to cidal effects. (B) Surface defects: Surface defects in ZnO nanoparticles have the ability to rupture the living cell membranes and get inside the organism where they interacts with the cellular machinery as well as facilitate the release of additional ionic species that produce genotoxic and oxidative stress (C) Zn+2 dissolution: Following the internalisation of the ZnO NP's cytosolic dissolution in to Zn+2 occurs. In Eukaryotes the process of internalization and dissolution occurs in endosomes. After the release of Zn+2, these ions can interact with mitochondiral membrance leading to mitochondiral dysfunction.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.3.4 Antifungal activity Antifungal potential of S. thea mediated nanocrystals was measured using linear growth inhibition and well diffusion assay method indicated in the inset Figure (2.11 A/B). ZnO nanoparticles has been extensively studied for antibacterial activities while limited studies have been performed on the antifungal nature of the ZnO nanoparticles which are mostly carried out through disc diffusion method. Present report will be the first one to investigate the antifungal activities of biogenic nanoparticles using linear mycelial growth inhibition in which the percentage inhibition is find out relative to the linear growth of the fungus. Antifungal activity was investigated for the stock concentrations in the range of 2000-500 µg/ml against 5 pathogenic fungal strains Aspergillus fumigatus (FCBP: 66), Aspergillus flavus (FCBP: 0064), Mucor racemosus (FCBP: 0300), Aspergillus niger (FCBP: 0918) and Rhizopus (FCBP 0040). Amp B (250 µg/ml) as positive control. Our results suggest linear growth inhibition at against all tested strains at 2 mg/ml, however the inhibition is not significant as compared to the Amp B. Among the tested strains, M. racemosus and A. fumigatus was inhibited at all concentrations. Low test concentrations were found ineffective against the A. flavus. In addition to the ROS generation (Lipovsky et al. 2011), earlier reports suggest the interference of ZnO nanoparticles with fungal hypea and fungal spores lead to inhibition of the fungal growth (He et al. 2011). Significant and dose dependent fungicidal potential has been indicated in previous research (Lipovsky et al. 2011; Sharma et al. 2010) which corresponds to the current work, however the fungal strains and the method used in the present study has never been reported before. Similar results are also obtained through the well diffusion assay. Mucor racemosus was significantly inhibited at 2 mg/ml while Rhizopus solani was the least inhibited fungal strain. Similarly at low concentrations at 0.5 mg/ml Rhizopus solani was not inhibited as revealed in both methods.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 11: (A) Antifungal activity using of biogenic ZnO nanoparticles using linear mycelial growth inhibition; (B) Antifungal activity by well diffusion assay
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.3.5 Antileishmanial potential (promastigotes and amastigotes) Novel strategies involving nanoparticles such as ZnO has been recently proposed for leishmania treatment. In recent studies, 88% lethality of ZnO nanoparticles (IC50 4.41 and 3.76 µg/ml) are reported (Ali et al. 2017). In addition silver doped ZnO nanoparticles have also yield impressive results (Nadhman et al. 2014). The current paper is a first report on the antileishmanial activity against Leishmania tropica (KMH23) promastigote for ZnO nanoparticles synthesized through green chemistry. IC50 was calculated as 6.2 ± 2.28 µg/ml. Figure (2.12) shows the antileishmanial activity in response to the different concentrations (200-1 µg/ml). Concentration dependent lethality can be concluded against Leishmania tropica. Antileishmanial activity of the bioinspired ZnO nanoparticles were also investigated against the amastigote form of Leishmania. Leishmania transforms to amastigote form (circular shaped) inside the body of human beings. The biogenic ZnO nanoparticles were found equally effective against amastigotes. Median lethal concentration IC50 was calculated as 10.87 µg/ml. Cytotoxicity against the axenic cultures of Leishmania were found in correspondence to earlier reports (Ali et al. 2017; Khan et al. 2015). 2.3.6 Brine shrimp cytotoxicity Percent mortality of shrimps were investigated. Brine shrimps cytotoxicity testing further confirmed the potential cytotoxic behavior of bioinspired zinc oxide nanoparticles as indicated in Figure (2.12). IC50 was calculated as 21.29 µg/ml. 2.3.7 Biocompatibility assessment using Human RBC’s and macrophages In order to confirm the bio-safe nature of biosynthesized nanoparticles, toxicity testing against freshly collected human RBC’s and freshly isolated macrophages was performed. The obtained results are summarized in Figure (2.12). According to the standards of “American Society for Testing and Materials Designation” substances with hemolysis > 5% are hemolytic, 2-5% slightly hemolytic while < 2% non-hemolytic (Aula et al. 2014). The hemolysis is measured as the hemoglobin is released due to RBC rupture after treatment with sample. In the present results, it is indicated that biogenic ZnO nanoparticles at lower concentrations (up to 2 µg/ml), are non-hemolytic, slightly hemolytic under 40-5 µg/ml and hemolytic at > 40 µg/ml. These results were found to be in agreement with earlier reports (Aula et al. 2014). To reassure, safety and
40
Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications biocompatibility of biogenic ZnO nanoparticles, MTT cytotoxicity against freshly isolated human macrophages was also assessed. The macrophage response to the treatment with ZnO nanoparticles was concentration dependent i.e at higher concentrations the cytotoxic effect was manifested while at lower concentrations, there was less cytotoxicity. 34 % inhibition in the growth of macrophages was observed by treating them of 200 µg/ml of biogenic nanoparticles while the survival rate for the human macrophages was found to be ~ 96 % with the treatment of 1 µg/ml. An IC50 values for biogenic ZnO nanoparticles against human RBC’s and macrophages was found to be > 100 µg/ml which generally conclude non cytotoxic behavior of biogenic ZnO nanoparticles against human RBC’s and macrophages at lower concentrations. Human cells, especially the immune cells like macrophages have a built in mechanism to deal with the invading pathogens by ROS generation and these human macrophages have the capacity to deal with the ROS from external sources. Therefore, the ROS generated by the biogenic nanoparticles at low concentrations are not cytotoxic unless the concentration is increased beyond limits (Prach et al. 2013). The IC50 values have been indicated in table 2.2.
Figure 2. 12: Cytotoxic assays for biogenic ZnO nanoparticles.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Table 2. 2: IC50 calculations for biogenic ZnO nanoparticles.
Assay type IC50 Antileishmanial promastigotes 6.27 µg/ml Antileishmanial amastigotes 10.87 µg/ml Brine shrimp cytotoxicity 21.29 µg/ml Human RBC’s > 200 µg/ml Human macrophages > 200 µg/ml Alpha amylase inhibition > 200 µg/ml
2.3.8 Enzyme inhibition activity (protein kinase inhibition and alpha amylase inhibition) Figure (2.13) shows the ability of biogenic ZnO nanoparticles to inhibit protein kinase enzymes. Protein kinases are the enzymes performing the phosphorylation of tyrosine and serine-threonine residues that serves as a pivotal regulatory process for metabolism, cellular differentiation/proliferation and apoptosis. Deregulated phosphorylation can induce genetic abnormalities leading to tumorigenesis. Henceforth, any product having the ability to inhibit protein kinase enzymes can be of significant importance in cancer research (Yao et al. 2011). Protein kinase phosphorylation perform a critical function in development of hyphae in Streptomyces fungal strains, and the same principal is applied to determine the protein kinase inhibition activity. Streptomyces 85E strain has been widely employed to investigate medicinal compounds for investigation of the PK inhibition (Waters et al. 2002). Following the same theme, PK inhibition was investigated using surfactin as a positive control. Zones of inhibition appeared as 10 ± 1.45, 6.10 ± 1.92 and 3.10 ± 1.53 in millimeters were recorded at 1000, 500 and 250 µg/ml concentrations. Therefore a potential signal transduction inhibitor is identified in the form of nanoscaled ZnO that can be further exploited for anti-infective and anticancer properties. Because of its protein kinase inhibition activity, one can pre-conclude that the biogenic ZnO particles can play a role in anticancer therapies. Alpha amylase performs the function of carbohydrates breakdown into glucose, and therefore been related to the postprandial blood glucose excursion in a person having 42
Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications diabetes (Ali et al. 2006). Hence, blocking the breakdown of starch into glucose by inhibition of alpha amylase enzyme represents a key area of diabetes research (Dineshkumar et al. 2010). Bioinspired zinc oxide nanoparticles were investigated for their inhibition potential against alpha amylase enzyme. The results indicate insignificant alpha amylase enzyme inhibition which are in agreement with the previous studies (Ali et al. 2017). Maximum inhibition was reported at the highest concentration 200 µg/ml i.e. 32 %.
Figure 2. 13: Protein kinase inhibition activity of biogenic nanoparticles. Circular red marks indicate the bald zone of inhibition as a result of protein kinase enzyme inhibition activity of ZnO nanoparticles.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
2.3.9 Antioxidant activities Total reducing power (TRP) and DPPH free radical scavenging activity (FRSA) was performed through standardized assay procedures. TAC was based on the conversion of Mo (VI) to Mo (V) and a subsequent formation of greenish Mo (V)-phosphate complex having maximum absorption at 695 nm (Jafri et al. 2014). Results are shown in Figure 2.14. Maximum value for total antioxidants in terms of ascorbic acid equivalents/mg were found as 25.6 ± 1.54 for the tested concentration of 200 µg/ml. TAC indicate the quenching ability of the tested compound towards ROS species. Since in the present study aqueous extracts of S. thea was used as capping as well as reducing/oxidizing agent, it can be inferred that some of the phenolic compounds having the potential to quench the reactive oxygen species are also attached to the ZnO nanoparticles. In order to further assess the presence of the antioxidant species capped to the ZnO nanoparticles, reducing power assay was performed. This method involve the investigation of reductones that are related with anti-oxidation potential by donating H-atom that results in the breakage of free radical chain (Abdel-Hameed 2009). Figure (2.14) also shows the total reducing potential of the biomimetic ZnO nanoparticles at different concentrations. It is clearly indicated that the reducing power becomes lesser with the decreased concentration of test samples. Maximum reducing power was noted at 200 µg/ml (36.3 ± 2.78) and least reducing potential of 7.11 ± 2.6 at 0.78 µg/ml. Later on, DPPH radical scavenging assay was performed to further confirm the presence of antioxidants on biogenic ZnO nanoparticles. DPPH is based on the formation of yellowish diphenyl picrylhydrazine molecule when 2, 2-diphenyl 1-picrylhydrazyl moiety (DPPH) is reduced by accepting a hydrogen or electron from a donor. Moderate DPPH radical scavenging was observed at higher concentration of 200 µg/ml (63.5 ± 2.4) while low radical scavenging is reported at 25 µg/ml. No DPPH radical scavenging was reported for test dilutions below 25 µg/ml. From the results summarized in Figure (2.14), it can be suggested that some of the antioxidant compounds can be involved in the reduction and stabilization during the synthesis process via aqueous extracts of S. thea. Leaves of S. thea are used in making tea in parts of China and Korea, which further support the presence of numerous antioxidants available to reduce and cap the ZnO nanoparticles.
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Chapter 2 Biosynthesis of ZnO nanoparticles and their biological applications
Figure 2. 14: Antioxidant activities of biogenic ZnO nanoparticles.
Variation and disagreement in the present studies to other studies can be attributed to various factors like experimental conditions (location, seasonal variation, humidity) (Khalil et al. 2014) while other factors like method of nanoparticle synthesis and size of the nanoparticles can be significant factors.
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Chapter 3 Biosynthesis of iron oxide nanoparticles
Chapter 3: Biosynthesis of iron oxide (Fe2O3) nanoparticles via aqueous extracts of Sageretia thea (Osbeck.) and their pharmacognostic properties
46
Chapter 3 Biosynthesis of iron oxide nanoparticles
Graphical abstract: Biosynthesis and applications of IONP’s
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Chapter 3 Biosynthesis of iron oxide nanoparticles
Abstract Sageretia thea (Osbeck.) was used as an agent for effective chelation and biosynthesis of Iron Oxide Nanoparticles (IONPs) and extensively characterized through XRD, FTIR, Raman spectroscopy, EDS, HR-SEM/TEM and SAED. Antibacterial assays against five human pathogenic bacterial strains were carried out and minimum inhibitory concentrations were calculated. Pseudomonas aeruginosa (MIC 7.4 µg/mL) was the most susceptible strain to biosynthesized IONPs. All of the fungal strains showed susceptibility to the IONPs. MTT cytotoxic assay was carried out against the promastigote and amastigote cultures of Leishmania tropica and their IC50 values were calculated as 17.2 and 16.75 µg/mL. The cytotoxic potential was further assessed using brine shrimps and the IC50 was calculated as 16.46 µg/mL. Moderate antioxidant activities were reported. Human RBC’s and macrophages were found to be biocompatible with biogenic IONPs
(IC50 > 200 µg/mL).
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Chapter 3 Biosynthesis of iron oxide nanoparticles
3.1 Introduction The interface of green chemistry and nanotechnology presents an exciting horizon for synthesizing green and clean multifunctional metal oxide nanoparticles. Besides silver and gold (Venugopal et al. 2017). Biogenic synthesis of nanoparticles has been extended to various metal oxides that exhibit interesting properties (Diallo et al. 2015; Thovhogi et al. 2015; Sone et al. 2015; Khenfouch et al. 2016; Nyangiwe et al. 2015; Sone et al. 2017; Fuku et al. 2016). Multifunctional Iron oxide nanoparticles (IONPs) have emerged as a promising material for a wide range of applications, such as biomedicine, cosmetics, bioremediation, diagnostics and materials engineering (Höss et al. 2014; Gupta and Gupta 2005; Lee et al. 2008; X.-q. Li et al. 2006). However, applications of IONPs in biomedicine are growing exponentially. The ability of IONPs in the controlled release of drugs for tissue repair, MRI, and treatment of solid tumors has previously been demonstrated (Siddiqi et al. 2016). The wide range of applications can be attributed to its ideal properties like higher surface area, small band gap and stability. Numerous chemical and physical means are used in phytosynthesis of iron nanoparticles. Chemical based methods used in the synthesis of IONPs includes thermal decomposition hydrothermal synthesis, thermolysis reverse micelles, co-precipitation, colloidal chemistry, micro-emulsion technology, sonochemical reactions, sol-gel synthesis and hydrolysis of precursors, electrospray synthesis and flow injection synthesis etc. (Rasheed and Meera 2016). In certain methods like hydrothermal reaction, co- precipitation and sol-gel process, the dispersion and uniformity of size and particle distribution were poor (Nidhin et al. 2008). Apart from these disadvantages, IONPs have been synthesized using solvents like sodium borohydride, hydrazine, sodium dodecyl sulfate etc. (Mahdavi et al. 2013; Shahwan et al. 2011). Such chemical synthesis methods can generate waste lines that are toxic and harmful to the environment and human beings (Smuleac et al. 2011). These chemical routes provide more chances of agglomeration of nanoparticles (Kharissova et al. 2013). Likewise, physical means of synthesis are often costly because of intense energy requirements (Diallo et al. 2015). Hitherto, these physical/chemical methods are effective in synthesis, there is a paradigm shift towards cheap, sustainable, green and ecofriendly methods (Thema et al. 2015) which can be used for the synthesis of IONPs. Numerous biological resources such as algae, bacteria, fungi,
49
Chapter 3 Biosynthesis of iron oxide nanoparticles and plants have emerged as economical and benign processes for the synthesis of nanoparticles. The interface of medicinal plants and biogenic nanoparticles have attracted numerous researchers to fabricate nanomaterials with diverse applications. Medicinal plants have been widely employed and preferred for the synthesis of nanoparticles because of their rich phytochemistry and bioactive components (Ovais et al. 2016). Bioinspired synthesis of IONPs has already been demonstrated in some recent reports. Bioactive components of the plant extracts are used as reducing agents in the synthesis process. Highly crystalline IONPs were reported using the aqueous leaf extracts of Azadirachta indica (Sharma et al. 2015). Camellia sinensis mediated IONPs indicated four times larger surface area relative to the commercial ones (Ahmmad et al. 2013). Lawsonia inermis, Amaranthus dubius, Rosmarinus officinalis, Melaleuca nesophila, Padina pavonica etc. are some of the other medicinal plants being effectively used in the biosynthesis of IONPs (Siddiqi et al. 2016). Furthermore, these green IONPs were found to be nontoxic to human beings as compared to their chemically produced counterparts and also indicated higher biocompatibility and impressive antimicrobial properties (Hoag et al. 2009; Naseem and Farrukh 2015; Huang et al. 2014). The current chapter reports bioinspired synthesis of nano-scaled single phase pure maghemite Fe2O3 nanoparticles, bio-engineered for the first time via complete green process using the aqueous leaf extracts of Sageretia thea as an effective reducing as well as capping agent without addition of any further chemical components (acids/base)
3.2 Material and Methods 3.2.1 Plant material processing Plant sample collected form Islamabad were taxonomically verified as Sageretia thea (Osbeck.) and the herbarium specimen (MOSEL-343) was deposited in the herbarium of Molecular Systematics and Applied Ethnobotany Lab (MoSAEL), Biotechnology Department of QAU. Fresh and healthy leaves were excised, washed with distilled water, dried and pulverized in Willy mill. Grounded test material was stored and used further for aqueous extraction.
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Chapter 3 Biosynthesis of iron oxide nanoparticles
3.2.2 Biosynthesis of IONP’s Standard procedure as established by previous authors for the biosynthesis of metal oxide nanoparticles was used with minor modifications (Thema et al. 2015). For this purpose, 30 g of plant material was added to 200 mL of deionized water and heated at 80°C for 1 h on magnetic stirrer hotplate for the extraction of bioactive components. Solid residues were removed by filtering the resultant solution 3 times using whattman filter paper. 6.0 g of iron sulfate hepta hydrate (Alfa Aesar) was added as a precursor salt to 100 mL of filtered solution (pH:5.7) and heated at 85°C for 2 h. The color of the solution changed from brownish dye to violet color and the pH was recorded as 4.3. Reaction mixture was cooled and centrifuged at 10,000 rpm for 10 min to collect the precipitate assumed as IONPs. The obtained precipitate was washed 3 times with distill water, dried and annealed at 500°C to obtain highly crystalline IONPs. Annealing was performed to ensure full crystallization of the nanoparticles as well as decomposition of any extra compound from the natural extracts, not involved in the biosynthesis reaction. A general study scheme has been summarized in Figure 3.1.
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Chapter 3 Biosynthesis of iron oxide nanoparticles
Figure 3. 1: A schematic representation of the study indicating the various important steps from biosynthesis to characterization and application of IONP’s
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Chapter 3 Biosynthesis of iron oxide nanoparticles
3.2.3 Physical characterizations To confirm the pure phase of maghemite IONPs, the annealed IONPs were subjected to intensive physical characterization techniques such as XRD, EDS, FTIR, Raman spectroscopy, Selected Area Electron Diffraction, High resolution Scanning/Transmission electron microscopy. XRD analysis was carried using X-ray diffractometer (Bruker AXS D8) with irradiation line Kα of copper (λ=1.5406 A°) while their corresponding size was calculated using Debye-Scherer equation {<Øsize> = K λ /
∆θ1/2 cosθ}. Vibrational properties of IONP’s were studied using Raman spectra recorded in the spectral range (0 cm-1 – 2000 cm-1) using laser line of 473 nm with average excitation power of 2.48 mW. To investigate the major functional groups in the aqueous extracts of S. thea, Attenuated Total Reflectance (4000 cm-1 – 600 cm-1) was carried out for the powdered aqueous extracts using Thermo Scientific Nicolet iS10 Spectrometer (Thermo Scientific, Waltham, MA) equipped with a Smart iTR ATR accessory, with a diamond crystal. FTIR analysis for the bioinspired IONP’s was carried out over the spectral range of 400 to 4000 cm-1. Elemental composition was investigated using EDS while for morphological and shape observations HR-SEM and HR-TEM were used.
3.4 Antimicrobial activities 3.4.1 Antibacterial activity Disc diffusion assay (Fatima et al. 2015) was used to investigate the antibacterial activities against 5 pathogenic bacterial strains (Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis, Klebsiella pneumoniae and Pseudomonas aeruginosa) while their minimum inhibitory concentrations were investigated using broth dilution assay. Fresh cultures were standardized to the optical density of 0.5 that corresponds to 1 × 108 CFU/mL. Uniform microbial lawns were prepared using sterilized bent glass rod after dispensing 100 µL of already standardized culture. Filter discs (6 mm) loaded with 10 µL IONPs (1000 µg/mL – 31.25 µg/mL) final concentrations were carefully placed on the microbial lawns while gentamycin disc (10 µg) was used as positive control. Zones of inhibitions were recorded in millimeters on a vernier caliper. MIC’s were calculated using broth dilution assay by applying further lower concentrations of bioinspired IONP’s.
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Chapter 3 Biosynthesis of iron oxide nanoparticles
3.4.2 Antifungal activity Previously described protocol (Ahmad et al. 2016) for the linear mycelial growth inhibition was employed to investigate the antifungal properties of bioinspired IONPs against (Aspergillus niger, Mucor racemosus. Aspergillus flavus, Rhizopus Solanai and Aspergillus fumigatus,). Sabouraud Dextrose Liquid medium (Oxoid CMO147) supplemented with 1.5% agar technical (Oxoid LP0012) was used to prepare fungal growth media. Autoclaved media (4 mL) was poured in sterilized test tubes and allowed to cool to ~ 50°C. 66.6 µL of test IONPs samples (2000 µg/mL – 500 µg/mL) was added and media was solidified in slanting position. Fungal spores from the freshly cultured strains were inoculated to the base of test tubes carefully using sterilized nichrome wire loop. Amphotericin B was used as positive control while slants without any sample was used as negative control. After incubation for 5 days, at 30°C, linear growth inhibition was measured using the below equation; 퐺푟표푤푡ℎ 𝑖푛 푠푎푚푝푙푒 (푐푚) % 퐼푛ℎ𝑖푏𝑖푡𝑖표푛 = 100 − [ ] × 100 퐺푟표푤푡ℎ 𝑖푛 푛푒푔푎푡𝑖푣푒 푐표푛푡푟표푙
3.5 Cytotoxic activities 3.5.1 Antileishmanial activity (Promastigotes and Amastigotes) MTT cytotoxic assay as previously described (Ali et al. 2017) was performed against axenic Leishmania tropica KMH23 promastigotes and amastigotes cultures. Leishmania can exist in 2 forms promastigotes (outside the human body) and amastigotes (inside the human body). Promastigotes are flagellated while amastigotes are ovoid and non- flagellated (Chtita et al. 2016) MI99 medium (GIBCO) added with 10 % FBS heat inactivated, was used for culturing the parasite. Cultures were grown to a density of 1 × 106 cells/mL. IONPs with final concentration of 200 µg/mL – 1 µg/mL were used. Assay was performed in 96 well plate. The seeded plates with IONPs and culture media were incubated at 24°C for 72 h to a density of 1 × 106 cells/well in humified incubator with 5
% CO2, and readings were taken at 540 nm. The lasted promastigotes were counted with hemocytometer under inverted microscope while their IC50 values were calculated using table curve software. Percent inhibition was calculated as; 푺풂풎풑풍풆 풂풃풔풐풓풃풂풏풄풆 % 풊풏풉풊풃풊풕풊풐풏 = [ퟏ − { }] ×100 푪풐풏풕풓풐풍 풂풃풔풐풓풃풂풏풄풆
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3.5.2 Brine shrimp cytotoxicity Artemia salina larvae were used to further investigate the cytotoxic potential of bioinspired IONPs in 96 well plates (Ali et al. 2017; Ahmad et al. 2016). A. salina eggs incubated at 30 °C for 24 h-48 h in light under sea water (38 g/L) added with dried yeast (6 mg/L). 10 mature phototropic nauplii were harvested using Pasteur pipette and shifted to each well of plate. Test concentrations of IONPs were added to each well. DMSO was used as negative control while doxorubicin as positive control. Percent of dead shrimps was calculated in each well after incubation at 24 h. IC50 values were calculated using table curve software.
3.6 Biocompatibility assays 3.6.1 Human RBC’s Hemolytic assay was carried out to study the compatibility of bioinspired IONPs using newly isolated RBC’s from healthy donor (Malagoli 2007). 1 mL of the freshly collected blood was centrifuged at 14,000/5 min for RBC’s isolation followed by adding 200 µL from the isolated erythrocytes to PBS (pH:7.2) forming an erythrocyte suspension. Both the test dilutions and erythrocyte suspension were added in equal amounts (100 µL) in a tube, and left for 1 h at 35°C. After incubation, the tubes were centrifuged at 10,000 rpm/10 min. The percent hemoglobin release was monitored at 530 nm while Triton X- 100 and DMSO were positive and negative controls. Percent hemolysis was calculated as;
% 퐻푒푚표푙푦푠𝑖푠 = [퐴퐵푆 − 퐴퐵푁퐶 ÷ 퐴퐵푃퐶 − 퐴퐵푁퐶] × 100 3.6.2 Human Macrophages Cytotoxicity of bioinspired IONPs was also determined against freshly isolated human macrophages. Macrophages were isolated using the blood of a healthy donor using the previously described standard procedure based In a general procedure, 5.7 g of ficoll was slowly dissolved in 95 mL deionized water with 5 mL of gastrografin. Hank’s buffer salt solution (HBSS) was used for the dilution of blood layered gradually on ficoll- gastrografin, followed by centrifugation at 400g/30 min and purified with percoll gradient
(density 1.064 g/mL) which was adjusted via autoclaved dH2O. Obtained cells were added to RPMI medium supplemented with antibiotics (Streptomycin: 0.1 mg/mL;
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Penicillin:100 U/mL), FBS (10 %) and Hepes (25 mM). Isolated macrophages were 5 grown to the culture density of 1 × 10 cells/well in humidified incubator with 5% CO2. Percent inhibition was calculated using the below formula; 푺풂풎풑풍풆 풂풃풔풐풓풃풂풏풄풆 % 풊풏풉풊풃풊풕풊풐풏 = [ퟏ − { }] ×100 푪풐풏풕풓풐풍 풂풃풔풐풓풃풂풏풄풆
3.7 Enzyme inhibition assays 3.7.1 Protein kinase inhibition Streptomyces 85E strain cultured in ISP4 minimal media was used to investigate the protein kinase inhibition activity of bioinspired IONPs. Filter paper discs loaded with 10 µL of serially diluted IONP’s were placed after on the culture plates after making uniform lawns. Surfactin was used as positive control. Zone of inhibition was measured in millimeters after 72 h incubation at 30°C (Fatima et al. 2015). 3.7.2 Alpha amylase inhibition Microplate method was applied to study the alpha amylase enzyme inhibition potential of the bioinspired IONPs (Ali et al. 2017). Reaction mixture comprised of 15 µL PBS and 25 µL α-amylase enzyme, 10 µL of test samples and 40 µL of starch solution. Positive and negative controls comprised of acarbose and deionized water respectively. Percent enzyme inhibition was calculated;
% 퐸푛푧푦푚푒 𝑖푛ℎ𝑖푏𝑖푡𝑖표푛 = [{푂퐷푆 − 푂퐷푁} ÷ {푂퐷퐵 − 푂퐷푁}] × 100
Whereas, “ODS”, “ODN” and “ODB” corresponds to the optical densities of sample, negative control and blank.
3.8 Antioxidant activities 3.8.1 DPPH free radical scavenging DPPH radical scavenging ability was investigated using different test concentrations (200 µg/mL to 1 µg/mL) by spectrophotometric method (Ali et al. 2017). Reagent solution was prepared by adding 2.4 mg of DPPH (2, 2-diphenyl 1-picrylhydrazyl) to 25 mL of methanol. Ascorbic acid was considered positive control while DMSO was used as negative control. 200 µL of reaction mixture comprised of 180 µL of reagent solution and 20 µL of test sample. After incubation for 1 h, readings were recorded at 517 nm to investigate the percent scavenging of DPPH. Following formula was used;
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푺풂풎풑풍풆 풂풃풔풐풓풃풂풏풄풆 % 푺풄풂풗풆풏품풊풏품 = [ퟏ − { }] ×100 푪풐풏풕풓풐풍 풂풃풔풐풓풃풂풏풄풆
Whereas, “ABS” and “ABC” corresponds to absorbance of sample and absorbance of control. 3.8.2 Total Antioxidant Capacity Total antioxidant capacity was investigated using phosphomolybdenum method (Jafri et al. 2014). In a typical procedure, 20 µL of test samples are added to 180 µL of reagent mixture. Reagent mixture comprises of (0.6 M H2SO4, 28 mM NaH2PO4, 4 mM (NH4) 0 6Mo7O24.4H2O). The reaction mix was incubated at 95 C for 90 min. Readings were recorded at 695 nm. Results are expressed as number of µg equivalents of ascorbic acid per mg of the sample i.e. µg AAE/mg. 3.8.3 Total Reducing Power Potassium ferricyanide based method was used to investigate total reducing power of bioinspired IONPs (Javed et al. 2016). To 50 µL of PBS, 40 µL of test sample was added, followed by incubation at 50°C for 20 min, and then added with 50 µL of (10%) tri- chloro acetic acid. The reaction mix was centrifuged at 3000 rpm for 10 min. To the
166.6 µL of the collected supernatant, 33.3 µL of (0.1%) FeCl3 was added in a 96 well plate. DMSO and ascorbic acid were used as negative and positive controls. Absorbance were recorded at 630 nm and the results were expressed as ascorbic acid equivalents per mg.
3.9 Results and Discussion 3.9.1 Biosynthesis of IONPs Chemical and physical nanoparticle synthesis procedures are either laborious, time consuming or costly. Sometimes even unwanted toxic chemicals can remain adsorbed to the surface of nanoparticles and eventually hinder its widespread therapeutic applications (Zak et al. 2011; Darroudi et al. 2014). A green, cheap, rapid and simple method for the biosynthesis IONPs has been successfully demonstrated for the first time using aqueous leaf extracts of S. thea. Biomimetic synthesis is more acceptable because it has no disadvantages relative to the physical/chemical methods and hence, can be effectively applied for biological applications. Sageretia thea has already been used in traditional medicines and in making tea in parts of Korea and China. Bioactive compounds like
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Taraxerol, Quercetin, Syringic acid, Myricetrin, Kaempferol, Daucosterol have previously been reported from S. thea (Shen et al. 2009; Chung et al. 2004; Xu et al. 1994) which has an intended role in reducing, capping and stabilizing of IONPs. The valency of Fe in sulphate hepta hydrate is 2+ at room temperature which changes to Fe+3 during the reaction process because of electron donating nature of iron at higher temperature. The oxidation process here is governed by the temperature (85°C). At higher temperature, the reduced Fe+2 is changes to Fe+3, which then combines with the oxygen present in the system or released by the phenolic rich components in the plant extracts resulting in the formation of Fe2O3. 3.9.2 Room temperature physical characterizations X-ray diffraction spectra of the biosynthesized IONPs annealed at 500°C in open air furnace was recorded (Figure 3.2 A). The observed Bragg peaks were found consistent with single pure phase (Fe2O3) tetragonal maghemite (JCP pattern: 00-025-1402). Lattice constants were deduced as
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Chapter 3 Biosynthesis of iron oxide nanoparticles simulated the data generated from XRD analysis (Figure 3.3 A/B). Spotty pattern in the SAED analysis also confirms the crystallinity of IONPs (Figure 3.3 C).
Figure 3. 2: (A) XRD analysis of bioinspired iron oxide nanoparticles, (B) Average size calculations
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Figure 3. 3: Morphological investigations of biogenic Iron oxide nanoparticles. (A): HR- TEM at 50 nm; (B): Particle distribution; (C) SAED pattern; (D): HR-TEM image; (E): HR-SEM
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Vibrational features of the biogenic IONPs were studied using FTIR. FTIR spectrum was recorded over the spectral range (4000 cm-1 – 400 cm-1). Characteristics IR absorption were studied (Figure 3.4 A). FTIR absorption peak ~ 500 cm-1 corresponds to the characteristic Fe-O vibration mode as indicated in the earlier studies (Maiti et al. 2013; Khayatian et al. 2013). Broad –OH stretching can be observed at ~ 3400 cm-1, that is usually attributed to the presence of phenolic compounds. Other IR absorption peaks can be observed at ~ 1100 cm-1 – 1200 cm-1, 1600 cm-1, and 2200 cm-1 which can be attributed to the -C-O, -C=O and –CN functional groups respectively. The presence of these IR absorptions are attributed to the phytochemical components that are used in the reduction, capping as well as stabilizing of the IONPs. These bioactive components remained adhered to the biogenically synthesized IONP even after being subjected to high thermal annealing temperature of 500°C. Figure (3.4 B) indicates Raman spectra recorded at using Argon laser green excitation λexc.= 514 nm. Spectrum indicates raman -1 -1 -1 shift at ~ 388 cm , ~ 500 cm and ~ 772 cm which corresponds to T2g, Eg and A1g raman active phonon modes specific to maghemite. Maghemite generally has 3 raman active phonon modes (Jubb and Allen 2010; De Faria et al. 1997). Pronounced peak A1g is enough to assign maghemite phase to the biosynthesized IONPs which is in line with the data obtained from XRD and in agreement with earlier reports. Here, it is noteworthy to mention that positioning of the peaks vary with the method used in synthesis and distribution vacancies within the unit cell (De Faria et al. 1997). To the best of our knowledge, maghemite phase IONPs has never been reported via green chemistry. To further investigate the elemental composition of the bioinspired IONPs, Energy Dispersive Spectroscopy was employed (Figure 3.4 C). No other elements except Carbon has been indicated by the EDS analysis. Peak of Carbon is attributed to the grid support.
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Figure 3. 4: Room temperature physical characterization of biogenic iron oxide nanoparticles. (A): FTIR of bioinspired iron oxide nanoparticles; (B): their Raman spectra; (C) EDS for elemental composition
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3.9.3 Antimicrobial properties of bioinspired IONPs Antibacterial properties of bioinspired IONPs were identified against 5 pathogenic strains (Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Pseudomonas aeruginosa) across different concentrations (1000 µg/mL – 31.25 µg/mL) using disc diffusion assay and there corresponding MIC’s were investigated at concentrations < 31.25 µg/mL. to the bioinspired IONPs. In general, all bacterial strains were susceptible to the biogenic IONPs while Staphylococcus epidermidis and Pseudomonas aeruginosa were found most susceptible with MIC’s 7.8 µg/mL each. It is interesting earlier reports indicated no activity for chemically synthesized IONPs against Pseudomonas aeruginosa at very high concentrations like (50 mg/mL) (Behera et al. 2012). However, as indicated in our results (Figure 3.5), Pseudomonas aeruginosa was significantly inhibited. Similarly, moderate antibacterial activities were reported for IONPs synthesized with Balanites aegyptiaca oil by co-precipitation method (Gasmalla et al. 2016). Medicinal plants with antimicrobial potential have rarely been used for biosynthesis of IONPs. Our results suggest significant antibacterial activities against human pathogenic bacterial strains. Antifungal activities against 5 pathogenic strains were demonstrated (Figure 3.6), using linear growth inhibition. Antifungal activity was performed against M. racemosus, A. niger, A. flavus, A. fumigatus and R. solanai. Linear growth inhibition was recorded at all of the tested concentrations i.e. 2 mg/mL to 0.5 mg/mL. Percent inhibition was found to be highest against R. solanai (79.03 % ± 2.90), A. fumigatus (74.58 % ± 3.15) and M. racemosus (74 % ± 3.20) and at 2 mg/mL. However, none of the tested sample gave percent inhibition greater then positive control Amp B. It is noteworthy to mention that the antifungal activities of the biogenically synthesized IONPs against the selected bacterial strains using linear mycelial growth inhibition assay have never been performed. We further conclude a dose dependent response towards bioinspired IONPs. Several researchers have tried to explain the antimicrobial effects of IONPs. Many researchers have considered generation of reactive oxygen species leading to cellular oxidative damage as a mechanism for the nanomaterial based microbial toxicity. Apart from ROS generations, other non-oxidant factors like membrane damage because of sorption of nanoparticles to the surface can lead to cellular injury. Similarly, surface
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Chapter 3 Biosynthesis of iron oxide nanoparticles defects in nanoparticle symmetry can also cause injury to the cells (Li et al. 2012). In addition, we further propose the role of bioactive functional groups like phenols adhered from the aqueous leaf extracts of S. thea used in capping-stabilizing IONPs have a significant contribution to antimicrobial potency.
Figure 3. 5: Antibacterial activities of bioinspired IONP’s.
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Figure 3. 6: Antifungal activities of biogenically synthesized IONP’s.
3.9.4 Cytotoxic activities 3.9.4.1 MTT cytotoxicity against axenic Leishmania tropica cultures Leishamniasis has an annual incidence rate of ~1.2 million, while the parasite has been endemic to 100 countries. Previous treatment of Leishmania included antimonial compounds which are not preferred anymore because of drug resistance and associated side effects (Jebali and Kazemi 2013). Metal oxide nanoparticle (oxides of zinc, titanium, magnesium) based treatments have gained popularity because of their impressive cytotoxicity against Leishmania (Jebali and Kazemi 2013)however biosynthesized IONPs have never been explored for their toxicity against Leishmania tropica (KMH23). Biogenically synthesized IONPs in the range of (200 µg/mL - 1 µg/mL) were investigated via MTT cytotoxic assay against the promastigote and amastigote axenic cultures of Leishmania tropica (Figure 3.7). Bioinspired IONPs were found to have significant inhibition against Leishmania parasite in promastigote as well as amastigote form. IC50 values were calculated as 17.2 µg/mL and 16.75 µg/mL for promastigote and amastigote concluding the futuristic possibilities of the use of bioinspired IONPs in antileishmanial therapies.
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3.9.4.2 Brine shrimp cytotoxicity To further confirm the cytotoxicity of bioinspired IONPs, they were further screened against brine shrimps to investigate the percent mortality. Brine shrimps (Artemia salina) are used as a standard to screen the cytotoxic compounds that can be further studied for their anticancer and antimicrobial properties. Brine shrimps cytotoxicity testing further confirmed the potential cytotoxic behavior of bioinspired IONPs as indicated in Figure 3.7. Bioinspired IONPs were found to be highly active at 200 µg/mL with 100 % indicated by the 100 % mortality of brine shrimps. At lower concentrations, there is a gradual decrease in the cytotoxic behavior. Median lethal concentration (IC50) was calculated as 16.46 µg/mL. These results confirm the cytotoxic behavior of biosynthesized IONPs. IC50 values from different assays are summarized in table 3.1. 3.9.4.3 Biocompatibility against human macrophages and RBC’s MTT cytotoxic assay was performed against freshly isolated human macrophages through the ficoll method. Macrophages are the important cells responsible for the immunity of a person against infections. Results of the MTT cytotoxicity are indicated in Figure7. Percent inhibition of macrophages at 200 µg/mL was investigated to be 47 ± 2.34 % while at least concentration applied, the percent inhibition was reported as 3.6 ±
1.1 %. IC50 calculated using table curve was found to be significantly higher i.e. > 200 µg/mL. Figure7 also indicate the percent hemolysis induced by bioinspired IONPs. Hemolytic activity is based on the release of hemoglobin which is induced by the test sample. If a given sample is hemolytic, it will cause rupturing of RBC’s, which results in hemoglobin release. This hemoglobin release which has been resulted after incubation of erythrocyte suspension with the test sample (IONPs) can be quantified with the help of spectrophotometer, hence the lysis of RBC’s is quantified. Hemolytic assay was carried out at different concentrations. Median lethal concentration was investigated > 200 µg/mL. As of international standards by “American Society for Testing and Materials Designation” substances with hemolysis > 5% are hemolytic, 2-5% slightly hemolytic while < 2% non-hemolytic (Aula et al. 2014). From our results, it can be inferred that bioinspired IONPs are non-hemolytic at low concentrations. In the results presented in Figure 3.7, it is evident that bioinspired IONPs are more toxic to all cells except human cells as indicated by their biocompatibility. Generally, the
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Chapter 3 Biosynthesis of iron oxide nanoparticles human cells like macrophages have an inherent capacity to deal with the ROS generated from any source. Therefore, the reactive oxygen species generated at low concentrations are not cytotoxic to human cells like RBCs and macrophages unless the concentration is increased beyond limit (Prach et al. 2013). A detailed proposed mechanism for the IONPs based cytotoxicity has been indicated in Figure 3.11.
Figure 3. 7: Cytotoxic activities of biogenic IONP’s
Table 3. 1: IC50 calculation of IONP’s against selected bacterial strains.
Assay type IC50 Antileishmanial promastigotes 17.2 µg/ml Antileishmanial amastigotes 16.75 µg/ml Brine shrimp cytotoxicity 16.46 µg/ml Human RBC’s > 200 µg/ml Human macrophages > 200 µg/ml Alpha amylase inhibition > 200 µg/ml
3.9.5 Enzyme inhibition assays Protein kinase inhibitors represent a fascinating area of research with respect to the treatment therapies for cancer (Yao et al. 2011). These enzymes are responsible for the
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Chapter 3 Biosynthesis of iron oxide nanoparticles phosphorylation of serine-threonine and tyrosine residues that play a pivotal role in cellular division, differentiation, apoptosis and other metabolic processes. Deregulated phosphorylations at serine-threonine and tyrosine residues can lead to the progression of tumor. Hence, chemical entities having capacity to inhibit PK enzymes are extremely valuable. In the Streptomyces 85E strain, protein kinases play a pivotal role in the formation of hyphae. Same strain was employed to investigate the protein kinase inhibition potential (Waters et al. 2002) of bioinspired IONPs. Following disc diffusion assay, significant zones of inhibitions were observed i.e. 39 mm to 17 mm from highly concentrated to the least concentrated samples (2000 µg/mL to 62.5 µg/mL) that relates to the significant PK inhibition activity of the biogenically synthesized IONPs. Therefore, a potent signal transductor inhibitor is identified in the form of bioinspired IONPs. Results are summarized in Figure 3.8 while a proposed mechanism of protein kinase inhibition is indicated in Figure 3.10. In addition, the bioinspired IONPs indicated insignificant alpha amylase inhibition (IC50 > 200 µg/mL). Alpha amylase enzyme is involved in the metabolism of carbohydrates by converting them in to glucose and therefore it is to the postprandial blood glucose excursion in a person suffering from diabetes. Hence, the conversion to glucose from starch can be blocked by inhibition of alpha amylase enzyme and therefore it represents a key area of diabetes research (Ali et al. 2006; Dineshkumar et al. 2010). 3.9.6 Antioxidant activities Figure 3.9 indicates the antioxidant activities performed on the biosynthesized IONPs engineered via the aqueous leaf extracts of Sageretia thea. Free radical scavenging activity was performed using DPPH. Moderate percent radical scavenging was observed at higher concentrations of 200 µg/mL i.e. 54.08 ± 2.3 %. Free radical scavenging was reported for all of the applied concentrations (200 µg/mL – 1 µg/mL). IC50 for the DPPH activity was calculated as 339 µg/mL. DPPH is based on the formation of yellowish diphenyl picrylhydrazine molecule when 2, 2-diphenyl 1-picrylhydrazyl moiety (DPPH) is reduced by accepting a hydrogen or electron from a donor. Total antioxidant capacity (TAC) is based on the conversion of Mo (VI) to Mo (V) and a subsequent formation of greenish Mo (V)-phosphate complex having maximum absorption at 695 nm Jafri and colleagues (Jafri et al. 2014). Maximum antioxidants in terms of ascorbic acid
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Chapter 3 Biosynthesis of iron oxide nanoparticles equivalents/mg were found as 14.3 ± 1.01 for the tested concentration of 200 µg/mL. TAC indicates the ability of a test compound to quench ROS. Reducing power assay was performed and highest reducing power (15.24 ± 1.45) was investigated for the test concentration of 200 µg/mL. Reducing power assay includes the investigation of reductones that are related with anti-oxidation potential by donating H-atom that results in the breakage of free radical chain (Abdel-Hameed 2009). Since aqueous leaf extracts of S. thea were used as a reducing, capping as well stabilizing agent, it can be inferred that some bioactive components which are retained as capping agents, have contributed to the antioxidant activities of the biogenic IONPs.
Figure 3. 8: Protein kinase inhibition by biogenic IONP’s.
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Figure 3. 9: Antioxidant activities of biogenically synthesized IONP’s.
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Figure 3. 10: Proposed role of biosynthesized iron oxide nanoparticles as protein kinase inhibitor for tumor growth prevention: (A) Indicate the activity of protein kinase in response to growth stimuli which is followed by activation of growth factors by phosphorylation activity of protein kinase; (B): By interfering with the protein kinase enzymes, protein kinases could not initiate the signaling cascade responsible for the proliferation of the cell, hence division is inhibited.
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Figure 3. 11: Different proposed mechanisms* of IONP’s mediated toxicity; (A) Extracellular ROS generations which can easily penetrate inside the cell followed by their interference with nuclear material leading to genotoxicity, (B) IONP’s are internalized by receptor mediated endocytosis, followed by entrance in to the lysosomes where dissolution occurs in to Fe++. (D): The generated ions not only interfere with other proteins, but also make their way in to the mitochondria where they disrupts its function by creating further ROS, (C): Surface defects in IONP’s can result in rupturing of cellular membranes.
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Chapter 4 Biosynthesis and applications of NiO nanoparticles
Chapter 4: Sageretia thea (Osbeck.) modulated biosynthesis of NiO nanoparticles and their in vitro pharmacognostic, antioxidant and cytotoxic potential
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Graphical abstract: Biosynthesis and applications of NiO nanoparticles
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Abstract Pure phase nickel oxide (NiO) nanoparticles have been synthesized via an eco-friendly green procedure using Sageretia thea (Osbeck.) aqueous leave extracts, while their biological activities are reported. The NiO nanoparticles (~ 18 nm) were characterized through various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Energy Dispersive X-ray Spectroscopy (EDS), Raman spectroscopy and Selected Area Electron Diffraction (SAED). Morphological investigations and confirmation of the synthesis of nano-micro structures were carried through High Resolution Transmission Electron Microscopy (HR-TEM) and High Resolution Scanning Electron Microscopy (HR-SEM). Antibacterial activity was investigated against six pathogenic bacterial strains (gram positive and gram negative) and their corresponding MIC’s were calculated. UV-exposed nanoparticles were investigated to have reduced MIC’s relative to the NiO nanoparticles not been exposed to UV. Moderate linear fungal growth inhibition was observed while Mucor racemosus (% inhibition 64% ± 2.30) was found to be most susceptible to biogenic NiO. In vitro brine shrimp cytotoxicity was performed that revealed a cytotoxic potential for the biogenic
NiO. Median lethal concentration (IC50) was calculated as 42.60 µg/ml against brine shrimps. MTT cytotoxicity was performed against Leishmania tropica-KWH23 promastigotes and amastigotes revealed significant percent inhibition across the applied concentrations. IC50 values were calculated as 24.13 µg/ml and 26.74 µg/ml for the promastigote and amastigote cultures of Leishmania tropica. In vitro MTT cell viability assay was performed on the freshly isolated human macrophage cells (IC50 > 200 µg/ml) to test the biocompatibility of the as synthesized nano-nickel oxide. To further investigate, hemolytic assay was also performed on freshly isolated RBC’s and IC50 was found to be > 200 µg/ml. In addition, antioxidant assays like DPPH, total reducing power and total antioxidant capacity were investigated. Furthermore, enzyme inhibition assays like protein kinase inhibition and alpha amylase inhibition were also investigated in vitro. The use of natural plant extracts is hereby indicated as cost effective, green and alternative method for synthesis of NiO nanoparticles which can be used in numerous biological applications.
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4.1 Introduction Metallic oxides present a bright area of research because of their interesting physio- chemical, electronic and optical characteristics (Hao et al. 2010; Ezhilarasi et al. 2016). Therefore, metal oxide nanoparticles like zinc oxide, copper oxide, iron oxide, cobalt oxide etc. have been frequently synthesized because of their interesting properties for a wide array of applications in electronics, sensors (Lokesh et al. 2016), storage devices (Manikandan et al. 2015), catalysis (Lokesh et al. 2016), drug delivery (Brigger et al. 2002), magnetic resonance imaging (Ezhilarasi et al. 2016) and as biomedicine (Ezhilarasi et al. 2016). With the ongoing developments on the interface of nanoscience, metal oxide nanoparticles have gained tremendous popularity for the development of new and effective strategies in biomedicine. Nanoscale matter are different from the microscale counter parts in terms many properties like chemical, mechanical, electro- optical, magneto-optical and surface area to volume ratio which signify them as an effective tool in biomedical applications (Whitesides 2005). Because of its super conductance, chemical stability, electro catalysis, and efficient electron transfer ability, nano-NiO has attracted many researchers (Sasi et al. 2002). Nano-scaled nickel oxide, presented by a wide band gap (3.7 – 4.0 eV) is an intrinsic p- type semiconductor (Sone et al. 2016a) used in numerous biological applications. It has been used in adsorbtion of the toxic pollutants and dyes (Pandian et al. 2015). Because of their anti-inflammatory nature, they can be used in biomedicine (Sudhasree et al. 2014). The cytotoxic effects of nano-NiO has been previously established by the release of ROS and Ni++ leading to oxidative damage (Gong et al. 2011). Several chemical and physical methods have been proposed for the synthesis of nickel oxide nanoparticles. Co-precipitation, electro deposition, sol-gel chemistry, hydrothermal synthesis, combustion synthesis, solvothermal route, galvanostatic anodization etc. have been developed to synthesize the NiO nanoparticles (Zhang 2015; Kundu and Liu 2015; Ksapabutr et al. 2015; Soomro et al. 2015; Tao et al. 2015). However, these synthesis methods are accompanied by some disadvantages that limits their wide spread applications (Thema et al. 2016). Chemical synthesis routes often generates toxic chemical waste lines while physical means required massive energy input (Thovhogi et al. 2015; Thema et al. 2015b). To overcome the problem of toxic wastes and energy
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Chapter 4 Biosynthesis and applications of NiO nanoparticles imbalance, greener and ecofriendly methods have been proposed (Thema et al. 2015a). Biological resources such as plants and microorganisms can be used in a rapid, effective, simple and economical way to produce the desired metal oxide nanoparticles. Green procedures involving medicinal plants are becoming a popular approach for nanoparticle synthesis (Ovais et al. 2016). Plant extracts are used as capping and reducing agents in green chemistry approach. These plant extracts can act as a biotemplate which controls the size, shape and morphology of the nanoparticles (Kar and Ray 2014). In the present study/chapter, a complete green procedure for the synthesis of single phase nano-NiO has been reported by using the aqueous leaf extracts of medicinal plant Sageretia thea. The medicinal uses of Sageretia thea (Bird Plum/English) is well documented and it is mostly used in the treatment of jaundice, hepatitis, circulatory and cardio-vascular diseases (Hyun et al. 2015; Khan et al. 2014; Murad et al. 2011). Biosynthesis of NiO nanoparticles has been successfully demonstrated in recent years and therefore there is a growing interest in NiO nanoparticles synthesis via green route. A general study layout is summarized in Figure 4.1. Recently, biosynthesis through fungal biomass has been reported (Salvadori et al. 2015; Salvadori et al. 2014). Plants like Moringa oleifera (Ezhilarasi et al. 2016), Callistemon Viminalis (Sone et al. 2016a), Nephelium lappaceum (Yuvakkumar et al. 2014), Agathosma betulina (Thema et al. 2016), Tamarix serotine (Nasseri et al. 2016) etc. have been successfully used for biosynthesis of nano-nickel oxide. Complementing to the limited literature on biomedical applications of bioinspired NiO nanoparticles, a comprehensive study was designed to investigate their pharmacological properties.
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Figure 4. 1: Schematic representation of biosynthesis, characterization and application of NiO nanoparticles
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4.2 Materials and methods 4.2.1 Plant material processing The collected plant sample was identified as Sageretia thea in the Department of Plant Sciences, QAU, Islamabad, Pakistan. Herbarium specimen (MOSEL-343) was deposited in the herbarium of Molecular Systematics and Applied Ethnobotany Lab (MoSAEL), Department of Biotechnology, QAU. Fresh leaves were rinsed in running distilled water, shade dried and grounded to fine powder in a Willy mill. Grounded plant material was used for aqueous extraction. For extraction of bioactive components from the leaves, 30 g of grounded powder was added to 200 ml deionized water and heated to 80°C for 1 hr on magnetic stirrer hotplate (Thema et al. 2016). The resultant solution was filtered thrice to obtain aqueous extracts. 4.2.2 Biosynthesis of NiO nanoparticles Previously established procedure was applied for the biosynthesis of nickel oxide nanoparticles (Thema et al. 2016). To the obtained aqueous extracts (pH: 5.7), 6.0 g of
NiNO3 was added as a precursor salt for the biosynthesis of NiO nanoparticles. Following the addition of precursor, the solution (pH: 4.3), was heated up to 60°C for ~ 1 hr. Following such heating phase, precipitates were observed. The resultant solution was allowed to cool down to room temperature, followed by centrifugation and washing with distill water for 3 times at 1000 rpm /10 min. Obtained precipitate assumed as NiO or ° Ni(OH)2 was kept for drying at 100 C for ~ 2 hrs. Dried powdered was annealed in a ceramic crucible at 500°C in open air furnace. Following such phase, the dried nanoparticles underwent extensive dehydration, decomposition and evolution of gases leading to highly crystalline nanoparticles with color change from brown to greyish. 4.2.3 Characterizations of NiO nanoparticles Various techniques were used for the characterization of annealed nickel oxide nanoparticles. X-ray diffractometer (model Bruker AXS D8 Advance) with irradiation line Kα of copper (λ=1.5406 A0) was used to investigate the crystalline nature of the biogenically synthesized nanoparticles. XRD analysis was carried out and their corresponding size was calculated using Scherer equation {<Øsize> = K λ / ∆θ1/2 cosθ}. FTIR spectra was recorded in the range of 400 to 4000 cm-1. Raman spectrum was recorded from 0 cm-1 to 2000 cm-1 with a laser line of 473 nm and average excitation
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Chapter 4 Biosynthesis and applications of NiO nanoparticles power of 2.48 mW. Energy Dispersive X-ray spectroscopy was carried out to determine the elemental composition. HR-TEM and HR-SEM were utilized to investigate the particles morphology and size distribution.
4.3 Antimicrobial assays of NiO nanoparticles 4.3.1 Antibacterial activity Previously described disc diffusion assay (Fatima et al. 2015) was used to investigate the antibacterial properties of as synthesized NiO nanoparticles, while their minimum inhibitory concentrations were calculated. Already available gram positive and gram negative strains were used. Microbial cultures were refreshed on a Nutrient Agar media (Oxoid-CM0003), then transferred to the nutrient broth and kept in a shaker incubator (37°C; 200 rpm) for growing to the optical density of 0.5 which corresponds to 1 × 108 CFU/ml. 200 µl of standardized culture were used to make uniform microbial lawns. 6 mm filter discs loaded with 10 µL of test sample were carefully placed on the uniform lawns while gentamycin (10 µg) discs were used as positive control. Bacterial plates were incubated for 24 hr at 370 C and inhibition zones were measured across 1000 µg/ml to 31.25 µg/ml. MIC’s for the bioinspired NiO nanoparticles were further investigated. Antibacterial activities are studied against three gram positive bacterial strains (B. subtilis, S. aureus and S. epidermis) and three gram negative bacterial strains (K. pneumonia, E. coli and P. aeruginosa). Considering the debate of UV-illumination on the enhancement of the bactericidal effect, the NiO nanoparticles were illuminated under UV light for 20 min. Germicidal 6 Watt UV Lamp 6GT5 (Sankyo denki- Japan) was used as a source of UV, while same method was used to determine the antibacterial efficacy. 4.3.2 Antifungal activity Linear mycelial growth inhibition assay was used as described previously (Ahmad et al. 2016). Sabouraud Dextrose Liquid medium (Oxoid CMO147) supplemented with 1.5% agar technical (Oxoid LP0012) was used to prepare fungal growth media. 66.6 µL of the test samples were poured to the autoclaved media and allowed for solidification in a slanting position. Fungal spores were inoculated at the based on the slant with nichrome wireloop, and incubated at 37°C for 48 hrs. Media devoid of any sample was used as negative control while 250 µg/mL amphotericin B was used as positive control.
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Linear mycelial growth inhibition was calculated as; 푮풓풐풘풕풉 풊풏 풔풂풎풑풍풆 (풄풎) % 풍풊풏풆풂풓 품풓풐풘풕풉 풊풏풉풊풃풊풕풊풐풏 = ퟏퟎퟎ − [{ } × ퟏퟎퟎ] 푮풓풐풘풕풉 풊풏 푵풆품풂풕풊풗풆 풄풐풏풕풓풐풍 (풄풎)
4.4 In vitro cytotoxicity assays of of NiO nanoparticles 4.4.1 Brine shrimp cytotoxicity To assess the cytotoxic potential of the bioinspired NiO nanoparticles, brine shrimps lethality was carried out as discussed previously using Artemia salina larvae in a 96 well culture plates (Khan et al. 2015; Ali et al. 2017). Eggs of A. salina (Ocean star, USA) were incubated from 24 hrs to 48 hrs under light at 30°C in sea water (38 g/L supplemented with 6 mg/L dried yeast). Pasteur pipette was used to harvest 10 mature phototropic nauplii. Test concentrations of bioinspired NiO nanoparticles were applied while DMSO and doxorubicin were used as positive and negative control respectively. Number of the dead shrimps was investigated after 24 hrs and percent inhibition was calculated. Median lethal dose (IC50) was calculated using table curve software. 4.4.2 Antileishmanial assay (Promastigotes and Amastigotes) MTT cytotoxic assay (Ali et al. 2017) was performed against promastigote and amastigote cultures of Leishmania tropica KWH23. Leishmania can exist in 2 forms promastigotes (outside the human body) and amastigotes (inside the human body). Promastigotes are flagellated while amastigotes are ovoid and non-flagellated (Chtita et al. 2016). M199 medium added with 10% heat inactivated FBS was used for culturing parasite. Parasites at the density 1 × 106 cells/ml were used against various concentrations ranging from 200 µg/ml – 1 µg/ml were used. The seeded 96 well plate was kept in 5%
CO2 incubator at 24°C for 72 hrs. Readings were taken using spectrophotometer and IC50 values were calculated using table curve software. Percent inhibition was calculated as; 퐴푏푠표푟푏푎푛푐푒 표푓 푠푎푚푝푙푒 % 퐼푛ℎ𝑖푏푡𝑖표푛 = [1 − { }] × 100 퐴푏푠표푟푏푎푛푐푒 표푓 퐶표푛푡푟표푙
4.5 Biocompatibility assays of of NiO nanoparticles 4.5.1 Hemolytic assay To assess the hemo compatible nature of the as synthesized nanoparticles, hemolytic assay was performed as described previously. Freshly isolated erythrocytes were isolated
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Chapter 4 Biosynthesis and applications of NiO nanoparticles by centrifugation (14000 rpm / 5 min.) of 1 ml fresh blood from the apparently healthy human. Erythrocyte suspension was prepared by adding 200 µl erythrocytes to 9.8 ml phosphate buffer saline (pH: 7.2). Erythrocyte suspension (100 µl) was treated with the biogenically synthesized NiO nanoparticles and incubated for 1 hr at 35°C in 96 well plate. Readings were taken at 530 nm to investigate the hemoglobin release. DMSO and Triton X-100 were used as negative and positive controls. Percent hemolysis was investigated as;
% 퐻푒푚표푙푦푠𝑖푠 = [퐴퐵푆 − 퐴퐵푁퐶 ÷ 퐴퐵푃퐶 − 퐴퐵푁퐶] × 100 4.5.2 Biocompatibility with Macrophages MTT cytotoxicity against freshly isolated macrophage cells was carried out as described previously (Ali et al. 2017). Ficoll-gastrografin based method was used for the isolation of macrophages. Blood was diluted with HBSS (Hanks Buffer Salt Solution) which was gently layered on Ficoll-gastrografin (Malagoli 2007). The resultant was centrifuged at 400 g/ 30 min, subsequently purified with percoll gradient (density 1.064 g/ml) pre- adjusted with sterilized deionized water. Isolated macrophages were cultured in RPMI medium with 10% FBS, 25 Mm Hepes, Streptomycin and 0.1 mg/ml; Penicillin:100
U/ml. Culture was incubated in humidified incubator with 5 % CO2 to a density of 1 × 105 cells/well. Percent inhibition was calculated as; % 퐼푛ℎ𝑖푏𝑖푡𝑖표푛 = [1 − {푆푎푚푝푙푒 푎푏푠표푟푏푎푛푐푒 ÷ 퐶표푛푡푟표푙 푎푏푠표푟푏푎푛푐푒}] × 100
4.6 Enzyme inhibition assays 4.6.1 Protein kinase inhibition Pk activity was carried out as discussed previously (Fatima et al. 2015). Uniform lawns of the standardized Streptomyces 85E strain were prepared after pre-adjustment of the optical density to 0.5. Autoclaved 6 mm filter disc were placed carefully on microbial lawn and 10 µl of sample was added. Surfactin and DMSO were used as positive and negative controls respectively. Clear and bald zones were measured. 4.6.2 alpha amylase inhibition Previously established protocol was used to investigate the alpha amylase inhibition (Ali et al. 2017). Reaction mixture contained 15 µl PBS, 25 µl α-amylase enzyme, 10 µl of samples and 40 µl of starch solution which were added stepwise. Reaction mixture with
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Chapter 4 Biosynthesis and applications of NiO nanoparticles all the ingredients was incubated for 30 min at 50°C followed by addition of 20 µl (1 M HCL) and 90 µl of iodine solution. Blank solution contained deionized water, starch and PBS while positive and negative controls comprised of acarbose and deionized water respectively. Enzyme inhibition was calculated as;
% 퐸푛푧푦푚푒 𝑖푛ℎ𝑖푏𝑖푡𝑖표푛 = [{푂퐷푆 − 푂퐷푁} ÷ {푂퐷퐵 − 푂퐷푁}] × 100
Whereas, “ODS”, “ODN” and “ODB” corresponds to the optical densities of sample, negative control and blank.
4.7 Antioxidant assays 4.7.1 DPPH DPPH was carried out using standard procedure (Ali et al. 2017). Various concentrations ranging from 200 µl to 1 µl were used. 25 ml of methanol was added to 2.4 mg of 2, 2- diphenyl 1-picrylhydrazyl (DPPH) to prepare the reagent solution. Ascorbic acid and DMSO were used as positive and negative controls. The electrons in dpph free radicals give maximum absorption at 517 nm. Absorption changes when DPPH becomes paired with a cation and form a reduced DPPH. Color change from deep violet to pale yellow is observed which can be quantified. Readings were recorded at 517 nm. Percent free radical scavenging was calculated as; 퐴퐵 % 푆푐푎푣푒푛푔𝑖푛푔 = [{1 − 푆 }] × 100 퐴퐵퐶 4.7.2 TAC TAC was investigated using previously described phosphomolybdenum method procedure (Jafri et al. 2014). The reagent solution was prepared by adding 0.6 M H2SO4, 0 28 mM NaH2PO4 and 4 mM (NH4) 6Mo7O24.4H2O stepwise followed by incubation at 95 C for 90 min. Readings were recorded at 695 nm and results were expressed as number of µg equivalents of ascorbic acid per mg of the sample i.e. µg AAE/mg 4.7.3 TRP Pottasium ferrcynaide basd method was used to investigate TRP (Javed et al. 2016). 40 µl of test sample was mixed 50 µl of PBS followed by incubation at 50°C for 20 min. 50 µl of (10%) tri-chloro acetic acid was added to the mixture and 3000 rpm for 10 min.
Supernatant (166.6 µl) was added to FeCl3 (33.3 µl) in a 96 well plate. Ascorbic acid and
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DMSO were used as negative and positive controls and absorbance was recorded at 630 nm. Results were expressed as ascorbic acid equivalents per mg.
4.8 Results and Discussion 4.8.1 Biosynthesis of NiO nanoparticles A green route for the biosynthesis of NiO nanoparticles has been revealed using the aqueous leaf extracts of Sageretia thea. Biomimetic synthesis of nanoparticles is considered more acceptable and eco-friendly (Diallo et al. 2015b; Sone et al. 2015; Thovhogi et al. 2016; Diallo et al. 2016; Sone et al. 2016b; Thema et al. 2016; Diallo et al. 2015a; Khenfouch et al. 2016; Venugopal et al. 2017). Sageretia thea has well documented medicinal uses and been used as tea in parts of Korea and China. Various compound like Syringic acid, Taraxerol, Quercetin, Kaempferol, Myricetrin and Daucosterol (Shen et al. 2009; Chung et al. 2004; Xu et al. 1994) etc. have been isolated which can play a role in reduction and stabilization in the biosynthesis of nanoparticles. A plausible mechanism for the biosynthesis is indicated in Figure 4.2. Successful green synthesis of NiO nanoparticles using plants has also been previously reported by various researchers (Ezhilarasi et al. 2016; Sone et al. 2016a; Thema et al. 2016; Nasseri et al. 2016).
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Figure 4. 2: Plausible mechanism for the biosynthesis of NiO nanoparticles via green route.
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4.8.2 Physical characterizations of NiO nanoparticles X-ray diffraction was carried out for the nanomaterial annealed at 500°C in open air furnace. All the obtained Bragg peaks (in Figure 4.3) corresponding to 101, 012, 110, 113 and 202 are the crystallographic reflections of rhombohedral nickel oxide crystallite with standard lattice parameters = 0.29 nm and
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4.6 presents the EDS spectra to investigate the elemental components of the sample. Energy Dispersive Spectroscopy was carried out for the elemental analysis. Peaks from “Ni” and “O” are evident confirming Ni-O in the sample, while peak indicating “C” is attributed to the grid support.
Figure 4. 3: XRD analysis of biogenic NiO nanoparticles. (A): Typical XRD pattern of NiO annealed at 500°C; (B): Size calculation according to Scherer approximation
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Figure 4. 4: Morphological investigations of biogenic NiO nanoparticles using HR- TEM/HR-SEM; (A/B/C/D): Size distribution of NiO nanoparticles; (E): HR-TEM image; (F): HR-SEM; (G): SAED pattern
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Figure 4. 5: (A): Typical ATR-FTIR of NiO nanoparticles; (B): their raman spectra
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Figure 4. 6: Elemental composition of biogenic NiO nanoparticles using Energy Dispersive Spectroscopy (EDS). 4.8.3 Antimicrobial activities Biogenically synthesized nickel oxide nanoparticles were evaluated against 6 pathogenic bacterial strains with and without UV illumination (20 min.). Figure 4.7A presents antibacterial activity across the concentration range of 1000 µg/ml – 31.25 µg/ml. Bacillus subtilis and E. coli were found to be the most susceptible strains with zones of inhibition 15.1 mm and 14.1 mm. Only Bacillus subtilis was inhibited across all the tested concentrations. Minimum inhibitory concentration was found as 15.6 µg/ml using broth dilution method. K. pneumonia and P. aeruginosa were found to be the least susceptible bacterial strains as they were not inhibited at concentrations < 250 µg/ml, which was considered as their MIC. Figure 7B indicate the antibacterial effect of the bioinspired nickel oxide nanoparticles after UV exposure. Increase is observed in the antibacterial effect after been exposed to the UV. All the bacterial strains were inhibited across all of the used concentrations with only exception of K. pneumonia which was not inhibited at concentrations lower than 31.25 µg/ml. E. coli and B. subtilis were found to be the most susceptible strains with MIC’s 15.6 µg/ml each. Here, it is noteworthy to
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Chapter 4 Biosynthesis and applications of NiO nanoparticles mention the use of broth dilution assay to investigate MIC’s for the concentrations found effective up-till 31.25 µg/ml. In general, gram positive bacterial strains were found as more susceptible which is in good agreement with the previous studies (Ezhilarasi et al. 2016; Helen and Rani 2015). Results are summarized in Figure 4.7A/B and table 4.1. Pure gentamycin 10 µg disc was used as positive control. None of the used samples indicated higher zone of inhibition then the positive control. Our results further indicate that UV illumination has contributed to the enhancement of the antibacterial effect. The role of UV illumination in enhancement of the ROS generation has been previously established (H. Zhang et al. 2011). The precise antibacterial mechanism against particular type of bacteria is however debatable. Proposed electrostatic interactions between the negatively charged bacterial cell membrane and positively charged nickel ions (Ni++), released from the nickel oxide nanoparticles may penetrate inside the cell wherein it interferes with cellular physiology leading to their disruption. NiO can also alter the membrane permeability leading to protein leakage (Wong and Liu 2010; Baek and An 2011). Enhancement of the antibacterial activity after UV illumination could be the generation of holes (h+) and electrons (e-) which has higher oxidizing and reducing abilities. Hence, they may react subsequently with water, hydroxyl ions and oxygen to • 2•- yield further reactive oxygen species like H2O2 , O and •OH, that possess a significant tendency to destroy the cells by interfering with proteins, DNA, mitochondria and other cellular components (Burello and Worth 2011; Du and Gebicki 2004). Sageretia thea aqueous leaf extracts were found capable for producing small size nanoparticles ~ 18 nm. Size is a crucial factor in the antimicrobial properties of nanoparticles (Nel et al. 2006; Jiang et al. 2009). Smaller size is associated with enhanced antimicrobial nature. Antifungal activities for bioinspired nickel oxide nanoparticles have been rarely investigated. Antifungal activities were performed against 5 pathogenic fungal strains across different concentrations 2 mg/ml to 0.5 mg/ml with Amphotericin B as positive control. All strains were inhibited across the tested concentration indicated NiO as a potential antifungal agent. M. racemosus and R. solanai were found to be most susceptible indicated by their percent inhibition of 64 % and 63.2 % at 2 mg/ml respectively. A. flavus was indicated as the least susceptible fungal strain. Nanoscale nickel oxide can easily penetrate inside the fungal cells subsequently leading to cellular
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Chapter 4 Biosynthesis and applications of NiO nanoparticles disruption. Antifungal activities of the as synthesized NiO nanoparticles are presented in Figure 4.8.
Figure 4. 7: (A): Antibacterial activities of biogenic NiO nanoparticles without UV- illumination; (B): With UV-illumination.
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Figure 4. 8: Antifungal potential of bioinspired NiO nanoparticles.
4.8.4 Antileishmanial activity (promastigotes and amastigotes) Leishamniasis is a neglected tropical disease and categorized as class 1 disease which is emerging and uncontrolled. It is endemic to 98 countries and to date, have no effective vaccines or treatments. Upto 350 million people are living under the immediate threat of leishamniasis (Abamor 2017; Légaré and Ouellette 2017). Current medications are accompanied by certain disadvantages like cost, side effects, elevated toxicity and long duration of therapy. The gold standard treatment include antimonial drugs however they have lost their efficacy due to drug resistance (Légaré and Ouellette 2017). Leishmania exist in promastigote (motile) form outside the body while transform into amastigote (non-motile) form inside the body. Figure 4.9 reports the antileishmanial activities of the biosynthesized nickel oxide nanoparticles against the axenic promastigote and amastigote cultures of Leishmania tropica using MTT cytotoxic assay. Both cultures were found to be effectively inhibited in a dose dependent manner. Our results indicate median lethal concentration (IC50) of 24.13 µg/ml and 26.74 µg/ml against Leishmania promastigotes and amastigotes respectively. Novel strategies by involving nanoparticles for antileishmanial therapy have been a keen area of research. Recently, ZnO (Ali et al. 2017; Nadhman et al. 2016a) nanoparticles and silver doped zinc oxide nanoparticles (Nadhman et al. 2016b) revealed significant cytotoxic effects against Leishmania. It is noteworthy to mention that we have described the antileishmanial potential of bioinspired
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Chapter 4 Biosynthesis and applications of NiO nanoparticles nickel oxide nanoparticles for the first time. Obtained results also indicated that the NiO nanoparticles were equally effective against the promastigote and amastigote cultures and hence could be used in antileishmanial therapies. 4.8.5 Brine shrimp cytotoxicity In order to further confirm the cytotoxicity of bioinspired nickel oxide nanoparticles, brine shrimp cytotoxic assay was carried out while their percent mortalities were calculated as indicated in Figure 4.9. IC50 value was calculated as 42.60 µg/ml. Artemia salina is considered as an ideal organism for investigating the cytotoxic potential of a compound (Ali et al. 2017). Our results conclude a dose dependent cytotoxic behavior of the bioinspired zinc oxide nanoparticles. A detailed schematic of the cytotoxicity mechanism is indicated in Figure 12. The IC50 values are summarized in table 4.2. 4.8.6 Biocompatibility testing In response to the potential cytotoxic behavior of the as synthesized nickel oxide nanoparticles, their cytotoxicity against the normal human red blood cells and macrophages were assessed. Results are indicated in Figure 4.9. Results were analyzed over the concentration range from 200 µg/ml – 1 µg/ml. The hemolytic assay revealed significantly less cytotoxic nature of nickel oxide nanoparticles to the freshly isolated RBC’s. Highest hemolysis (23.3 %) was observed at the highest dose (200 µg/ml), while no hemolysis was observed at concentrations < 5 µg/ml. As compared to the RBC’s, macrophages were found more effected with percent mortality of 44% at 200 µg/ml. The
IC50 values were investigated to be > 200 µg/ml for the bioinspired nickel oxide nanoparticles against RBC’s and macrophages. In general, the test samples can be categorized as safe at low levels. 4.8.7 Antioxidant activities Biogenically synthesized nickel oxide nanoparticles were found to have DPPH radical scavenging ability 65% to 3.27% across all the tested concentrations ranging from 200 µg/ml to 1 µg/ml. DPPH has been widely used as a gold standard to investigate the free radical scavenging activity of NiO nanoparticles, however to-date bioinspired nickel oxide nanoparticles have not been investigated for free radical scavenging. Previous researchers have considered chemically synthesized nickel oxide nanoparticles as novel antioxidant because of their free radical scavenging potential (Saikia et al. 2010). The
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Chapter 4 Biosynthesis and applications of NiO nanoparticles role of defects in the free radical scavenging ability of chemically synthesized nano nickel oxide is also established (Madhu et al. 2013; Saikia et al. 2010). This study also confirms free radical scavenging potential for the bioinspired nickel oxide nanoparticles. The antioxidant potency of was due to the quenching ability of the DPPH free radical in a concentration dependent manner. DPPH free radical scavenging by nano scale nickel oxide was also supported by the total antioxidant capacity using ferric ions as a chelating agent. Antioxidant capacity was expressed in as total ascorbic acid equivalents (µg) per mg. At 200 µg/ml, 11.3 µg AAE/mg were recorded. Oxidation is a natural process occurring in living cells can lead to the generation of ROS, which can interfere to de-normalize the cellular physiology. Insufficient availability of antioxidants can have dreadful results like lipid peroxidation, enzyme inactivation, protein and DNA damage. Further assessment of the antioxidant potential of the bioinspired NiO nanoparticles was carried out via studying their reducing power potential. Reducing power potential decreased with the lowering of test sample concentration. Overall a good DPPH free radical scavenging and moderate antioxidant capacity and reducing power can be concluded for the bioinspired nano nickel oxide. Results of the antioxidant activities are presented in Figure (4.10).
Figure 4. 9: Assessment of the cytotoxicity of the bioinspired NiO nanoparticles.
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Figure 4. 10: Antioxidant activities of bioinspired NiO nanoparticles.
4.8.9 Enzyme inhibition assays Figure 4.11A presents the protein kinase (Pk) enzyme inhibition potential of the biogenic nickel oxide nanoparticles. Protein kinase enzymes are considered as a crucial area for anticancer research. Pk enzymes are responsible for the phosphorylation of serine- threonine and tyrosine amino acid residues that play role in the cellular differentiation, proliferation and apoptosis. Cancer is associated with the deregulated phosphorylation by Pk enzymes leading to tumor growth. Therefore, particular entities which can inhibit Pk enzymes are of significant interest in anticancer research. Pk phosphorylation is a key factor in the formation of hyphae in Streptomyces and therefore have been extensively used to identify Pk inhibitors. Streptomyces 85E strain was used to screen Pk inhibition potential of the as synthesized NiO nanoparticles. Bald zones were measured in mm. Largest zone (13 mm) was recorded at 1000 µg/ml. All tested concentration produced bald zones except for 31.25 µg/ml which was found ineffective. Our results indicate that biomodulated NiO can be used as a signal transductor inhibitor in inhibition of tumorigenesis. Figure 4.11B indicate the alpha amylase enzyme inhibition potential of the biogenically synthesized NiO nanoparticles. This enzyme catalyze carbohydrate breakdown to glucose and therefore has been related to the postprandial glucose excursion in a patient suffering from diabetes. Hence, alpha amylase enzyme inhibitors have been considered in diabetes
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Chapter 4 Biosynthesis and applications of NiO nanoparticles research. Our result indicate low alpha amylase enzyme inhibition (35 %) at higher concentrations as high as 1000 µg/ml, while the bioinspired nickel oxide were found as ineffective at lower concentrations < 250 µg/ml.
Figure 4. 11: Biological properties of biogenic NiO nanoparticles (A): Protein kinase inhibition potential; (B): Alpha amylase inhibition potential
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Figure 4. 12: Schematic on the cytotoxic properties of bioinspired NiO as reported in literature; (A): ROS generation; (B): Ni++ release from NiO; (C): membrane damage by interference of membrane proteins with ROS or with their interference with surface defected NiO; (D): Interference of NiO nanoparticles/ROS/Ni++ with nuclear material; (E): Their interference with proteins; (F): Entrance to mitochondrial to generate further ROS; (G): Adherence to the membranes and pores.
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Table 4. 1: MIC calculations bioinspired for NiO nanoparticles.
Without UV illumination With UV illumination Gram positive Gram positive Bacterial strain MIC (µg/ml) Bacterial strain MIC (µg/ml) Staphylococcus aureus 62.5 Staphylococcus aureus 15.6 Staphylococcus epidermis 62.5 Staphylococcus 7.8 epidermis Bacillus subtilis 15.6 Bacillus subtilis 7.8 Gram negative Gram negative Klebsiella pneumonia 250 Klebsiella pneumonia 62.5 Pseudomonas aeruginosa 250 Pseudomonas 15.6 aeruginosa Escherichia coli 62.5 Escherichia coli 15.6
Table 4. 2: IC50 calculation for bioinspired NiO nanoparticle.
Assay type IC50 Antileishmanial promastigotes 24.13 µg/ml Antileishmanial amastigotes 26.74 µg/ml Brine shrimp cytotoxicity 42.60 µg/ml Human RBC’s > 200 µg/ml Human macrophages > 200 µg/ml Alpha amylase inhibition > 200 µg/ml
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Chapter 5 Biosynthesis and applications of lead oxide nanoparticles
Chapter 5: Bioinspired synthesis of pure massicot phase lead oxide nanoparticles and assessment of their biocompatibility, cytotoxicity and in-vitro biological properties
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Graphical abstract: Bioinspired synthesis of pure massicot phase lead oxide nanoparticles and assessment of their biocompatibility, cytotoxicity and in-vitro biological properties
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Abstract In the current study, green and ecofriendly route for biosynthesis of lead oxide nanoparticles has been successfully demonstrated using aqueous leaf extracts of Sageretia thea (Osbeck.). Biosynthesized PbO (~ 27 nm) nanoparticles were extensively characterized using XRD, FTIR, Raman, EDS etc. Morphology was studied through HR- TEM/SEM. As synthesized nanoparticles were investigated for their iv vitro biological properties. Antibacterial activities revealed enhancement upon modulation by UV in a concentration dependent manner. Pseudomonas aeruginosa was found to be the most resistant bacterial strain (MIC = 250 µg/ml and MICuv = 31.25 µg/ml) against biogenic PbO nanoparticles. MTT cytotoxicity on Leishmania promastigotes and amastigotes revealed significant inhibition as indicated by their IC50 values of 14.7 µg/ml and 11.95 µg/ml respectively. Cytotoxicity of bioinspired PbO nanoparticle was also confirmed using brine shrimp lethality (IC50 = 27.7 µg/ml). Bio-compatibility evaluation indicated cytotoxicity to freshly isolated human macrophages (IC50 = 57.1 µg/ml). Insignificant alpha-amylase inhibition and moderate protein kinase inhibition was revealed. Antioxidant activities indicated free radical scavenging activity (58 ± 2.45) at 200 µg/ml. Moderate total reducing power and total antioxidant activity was also indicated. Overall, we conclude lead oxide as a potential candidate for biological applications, however further studies are recommended on their in vitro and in vivo cytotoxicity.
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5.1 Introduction Biosynthesis of nanoscale metals and metal oxides has gained tremendous importance over the last few years because of their unique and interesting properties (Thema et al. 2015a; Thovhogi et al. 2015; Diallo et al. 2015b; Thema et al. 2015b; Diallo et al. 2015a; Sone et al. 2015; Nyangiwe et al. 2015; Ezhilarasi et al. 2016; Sone et al. 2017; Fuku et al. 2016; Ismail et al. 2016; Thema et al. 2016). The interface of metal oxide nanoparticles and bio-nanotechnology provides exciting avenues for fabricating nano- scaled matter with interesting properties (Ovais et al. 2016; Ovais et al. 2017). Applications of lead oxide has been well documented in lead acid batteries (Shahram Ghasemi et al. 2006). Lead oxide is also used in the transparent conducting films, x-ray imaging detectors and as lead graphite composite electrodes (Perry 2010; Šljukić et al. 2007). Lead oxide is a photosensitive material and therefore can be used in optical sensors. Some of the other applications of lead oxide include their use in solar energy conversion, x-ray sensitive photoconductor and other opto-electronic applications (Perry 2010). Because of their electronic properties and ease in fabrication, there is a great potential for their use in conjunction with other materials for a vast array of applications. Lead oxides basically exist in 2 forms. The alpha-form generally referred as litharge (red- tetragonal) and beta-form which is commonly known as massicot (yellow-orthorhombic). Synthesis of lead oxide has primarily been done through chemical or physical routes. Thermal decomposition of lead precursors, spray pyrolysis, sono-chemical, electrochemical methods have already been applied for synthesis of PbO (Sobanska et al. 1999; Lyons et al. 1992; S Ghasemi et al. 2008; Shahram Ghasemi et al. 2005). However, these processes are accompanied by various disadvantages. Physical synthesis means requires high energy and high vacuum while the chemical means are associated with the generation of toxic and hazardous waste lines making them environmental un-friendly (Diallo et al. 2015b). To reduce the problem of energy consumption and hazardous waste, alternative green and eco-friendly methods have been proposed. The interface of medicinal plants and metal oxide nanoparticles has been considered as a bright area of research for synthesizing multifunctional metal and metal oxide nanoparticles. Although, chemical synthesis of lead oxide has been successfully reported, however there are no reports available in the literature about biosynthesis of lead oxide nanoparticles.
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Complementing the vacuum in literature on the biosynthesis of lead oxide nanoparticles, a comprehensive research was undertaken to biosynthesize lead oxide nanoparticles using aqueous leaf extracts of medicinal plant Sageretia thea (Osbeck.). Sageretia thea is locally referred as “Momanra”/Pashto and “Bird plum”/English has well documented uses in hepatitis, jaundice, circulatory and cardio-vascular diseases. In this manuscript, biogenic synthesis of multifunctional lead oxide has been successfully reported for the first time. Furthermore, in addition to antileishmanial, antimicrobial, antioxidant and enzyme inhibition assays, their cytotoxicity was assessed against normal human RBC’s and macrophages.
5.2 Materials and methods 5.2.1 Plant material processing The collection of Sageretia thea was done from Islamabad, Pakistan followed by it taxonomic identification from Department of Plant Sciences, Quaid-i-Azam University (QAU), Islamabad. The herbarium specimen with voucher number MOSEL-343 was deposited at Molecular Systematics and Applied Ethnobotany Lab (MoSAEL), Department of Biotechnology, QAU. After excision, the fresh and healthy leaves were dried and grounded into fine powder via Willy mill. Aqueous extraction was performed on the plant powder, while remaining was stored for further use. In Figure (5.1) the overall outline of the study has been summarized.
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Figure 5. 1: Scheme for studying biosynthesis, characterization and application biogenic PbO nanoparticles.
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5.2.2 Biosynthesis of PbO nanoparticles The biosynthesis of PbO nanoparticles was performed as described by Thema et al., 2015 with minor modifications (Thema et al. 2015b). The aqueous extract from the plant powder was obtained by mixing 30 g of plant powder into 200 ml of deionized water under continuous heat of ~ 80⁰ C on a magnetic stirring hot plate (Snijders) for 1 hr. Solid residual plant material was removed by filtering the aqueous solution 3 times with whattman filter paper. Biosynthesis of PbO nanoparticles was performed by adding 6.0 g of the precursor salt lead acetate (Alfa Aesar) into 100 ml of aqueous plant extracts solution (pH 5.7), followed by 2 hr of gentle heating and stirring at ~ 600 C. After the addition of lead acetate, the pH of the solution was recorded as 4.6 at room temperature. As the solution cooled down to room temperature, centrifugation was performed at 10,000 rpm / 10 min for the collection of precipitate. Obtained pellet was washed and centrifuged (10,000 rpm / 10 min) with distilled water for 3 times. For obtaining highly crystalline PbO nanoparticles the precipitate was further annealed in open air in furnace 5000 C respectively. The biosynthesized PbO nanoparticles were extensively characterized for confirmation of pure phase massicot PbO nanoparticles. 5.2.3 Characterization of bioinspired PbO nanoparticles XRD analysis using X-ray diffractometer (model Bruker AXS D8 Advance) with irradiation line Kα of copper (λ=1.5406 A0) was performed for all the samples to check the crystalline structure of PbO nanoparticles. The corresponding size of all the thermally annealed PbO nanoparticles was calculated via Scherer equation {<Øsize> = K λ / ∆θ1/2 cosθ}. For the study of vibrational properties Raman spectroscopy was carried out. To record the Raman spectrum (0-500 cm-1) a laser line of 473 nm with average excitation power of 2.48 mW was used. ATR-FTIR for biogenic PbO nanoparticles was carried in the spectral range from 400 to 4000 cm-1. The morphological investigation was carried out through HR-SEM and HR-TEM. Particle size distribution was calculated using image J software after digitizing the various HR-TEM images, while elemental composition was determined by Energy Dispersive X-ray spectroscopy “EDS”. Selected Area Electron Diffraction (SAED) study was also performed.
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5.2.4 Antibacterial activity of bioinspired PbO nanoparticles Agar disk diffusion method (Fatima et al. 2015; Thatoi et al. 2016) was adopted for the determination of antibacterial efficacy of PbO nanoparticles while broth dilution method (Wiegand et al. 2008) was followed for the calculation of corresponding MIC’s. The microbial strains already available at Department of Biotechnology, QAU, Islamabad were refreshed on Nutrient Agar media (Oxoid-CM0003). Prior to assay, the bacterial cultures were shifted to nutrient broth and kept for 24 hrs in shaking incubator (at 370 C;200 rpm). The bacterial broth cultures were standardized to 1 × 108 CFU/ml by adjusting the optical density to 0.5 at with sterilized nutrient broth. On the nutrient agar plate 100 µl of broth cultures were dispensed homogeneously. Filter discs (6mm in diameter) were loaded with 10 µL of sample dilutions, dried and placed accordingly on the bacterial lawn. As a positive control Gentamycin (10 µg) discs were used, while the bacterial plates were incubated for 24 hr at 370 C. For the measurement of zone of inhibitions vernier caliper was used. Antibacterial activity was performed over the test concentrations of 1000-31.25 µg/ml. Minimum inhibitory concentration of test samples were investigated for the samples effective at < 31.25 µg/ml using broth dilution assay. MIC was defined as the minimum concentration of nanoparticle suspension that inhibits the bacterial growth. In addition the effect of UV-illumination was also assessed. Nanoparticles suspensions were kept in a UV-illuminator for 20 min under. The source of UV radiation was, germicidal 6 Watt UV Lamp 6GT5 (Sankyo denki- Japan). 5.2.5 Antileishmanial activity (Promastigotes and Amastigotes) Promastigote cultures of Leishmania tropica KWH23 strain was used for the assessment of biogenic nanoparticles cytotoxicity via MTT cell viability assay, as described previously (Ali et al. 2017). For culturing the strain, M199 media supplemented with 10 % fetal bovine serum (FBS) was used, while the density of 1 × 106 cells/ml were maintained for the assay. The assay was performed in 96 well microplate with dilutions of final concentrations ranging from 200 to 1 µg/ml. As positive and negative controls Amphotericin B and DMSO were used in the assay respectively. After incubation of seeded 96 well microplate for 72 hr at 240 C, readings were taken at 540 nm by BIOTEK microplate reader. Under the inverted microscope survived promastigotes were counted,
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followed by determination of LC50 values using Table curve software. Percent inhibition was calculated via following equation; 퐴푏푠표푟푏푎푛푐푒 표푓 푠푎푚푝푙푒 % 퐼푛ℎ𝑖푏푡𝑖표푛 = [1 − { }] × 100 퐴푏푠표푟푏푎푛푐푒 표푓 퐶표푛푡푟표푙 Similar procedure was applied on the amastigote cultures of Leishmania tropica KWH23. 5.2.6 Brine shrimp cytotoxicity Cytotoxic potential of bioinspired PbO nanoparticles were investigated against Artemia salina larvae following already established protocol in a 96 well plate (Ali et al. 2017; Khan et al. 2015). After incubation for 24 hrs with the test concentrations of biogenic lead oxide nanoparticles and percent inhibition of brine shrimps were calculated by counting the dead shrimps in each well. IC50 values were calculated using table curve software. 5.2.7 Biocompatibility of PbO nanoparticles 5.2.7.1 Biocompatibility with erythrocytes To determine the biocompatible nature of biogenic PbO nanoparticles with red blood cells of human, hemolytic assay was carried out (Malagoli 2007). From a healthy individual fresh blood was collected and dispensed in a sterile EDTA tube. Centrifugation (14000 rpm for 5 min) of 1 ml blood was performed for the separation of RBCs. For preparing an erythrocyte suspension in PBS, 200 µl of the pelleted erythrocyte were added to 9.8 ml of phosphate buffer saline followed by gentle shaking (pH: 7.2). In a test tube both the erythrocyte suspension (100 µl) and test nanoparticle solution (100 µl) was gently mixed, and incubated for one hour at 350 C. The solution was further centrifuged at 10,000 rpm for 10 min and supernatant was collected. The hemoglobin release was monitored using BIOTEK microplate reader at 540 nm after dispensing the supernatant in the 96 well plate. Triton X-100 and DMSO were used as positive and negative controls respectively. Results were calculated as percentage hemolysis induced by the nanoparticle dilution calculated through the formula;
% 푯풂풆풎풐풍풚풔풊풔 = [{푨풃푺 − 푨풃푵푪} ÷ {푨풃푷푪 − 푨풃푵푪}] × ퟏퟎퟎ 5.2.7.2 Biocompatibility human macrophages For further assessment of PbO nanoparticles biocompatibility, Ficoll–Gastrografin (sodium diatrozoate) method was adopted. The cytotoxicity was inspected against human
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Chapter 5 Biosynthesis and applications of lead oxide nanoparticles macrophages isolated from peripheral human blood. This isolation protocol is based on ficoll-gastrografin density gradient (density=1.070 g/ml) (de Almeida et al. 2000). In 95 ml of deionized water combined with 5 ml of gastrografin, 5.7 g of ficoll was slowly dissolved. Hank’s buffer salt solution (HBSS) was used for the dilution of blood which was layered gently on the ficoll-gastrografin. The solution was further centrifuged for 30 min at 400g followed by purification with percoll gradient (density 1.064 g/ml) adjusted with sterilized deionized water. The suspension of isolated cells was made in RPMI medium supplemented with fetal bovine serum (10 %), Hepes (25 mM), and antibiotics
(penicillin:100 U/ml; Streptomycin: 0.1 mg/ml). In humidified incubator with 5% CO2 the isolated macrophages were cultured to the density of 1 × 105 cells/well. Percentage inhibition was calculated using formula; 퐴푏푠표푟푏푎푛푐푒 표푓 푠푎푚푝푙푒 % 퐼푛ℎ𝑖푏푡𝑖표푛 = [1 − { }] × 100 퐴푏푠표푟푏푎푛푐푒 표푓 퐶표푛푡푟표푙 5.2.8 Antioxidant activities 5.2.8.1 Free radical scavenging By using DPPH (2, 2-diphenyl 1-picrylhydrazyl) as a stable free radical, spectrophotometric method (Fatima et al. 2015; Ali et al. 2017) was employed to investigate the radical quenching ability of green synthesized PbO nanoparticles. For determination of free radical scavenging concentrations of PbO nanoparticles starting from 200 µg/ml to 1 µg/ml were tested. As a positive and negative control, ascorbic acid and DMSO were used respectively. After keeping the 200 µl reaction mixture in dark for 20 min optical densities were measured at 517 nm by BIOTEK microplate reader. Percent free radical scavenging can be found using the formula;
% 푫푷푷푯 풓풂풅풊풄풂풍 풔풄풂풗풆풏품풊풏품 = [ퟏ − {푨풃푺} ÷ {푨풃푪] × ퟏퟎퟎ 5.2.8.2 Total Reducing Power of PbO nanoparticles For the determination of reducing power potential of biogenic PbO nanoparticles,
Potassium ferricyanide [K3Fe (Rao et al.)6] based method was used (Javed et al. 2016). DMSO was used as negative control while ascorbic acid as positive control. At 630 nm, the absorbance intensity was measured using BIOTEK microplate reader. Ascorbic acid equivalents per mg was regarded as reducing power.
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5.2.8.3 Total Antioxidant Capacity Previously described phosphomolybdenum based method was used to determine total antioxidant capacity (Jafri et al. 2014). The absorbance was measured at 695 nm while results are expressed as number of ascorbic acid equivalents in µg per mg of the sample i.e. µg AAE/mg. 5.2.9 Enzyme inhibition assays 5.2.9.1 Alpha amylase inhibition In vitro alpha amylase inhibition assay was performed in a 96 well plate as described previously (Javed et al. 2016). Test samples (10 µl) and starch solution (40 µl) were added stepwise in the reaction mixture (15 µl PBS/25 µl α-amylase enzyme), followed by incubation at 50°C for 30 min. After 30 min, 20 µl (1 M HCL) and 90 µl of iodine solution were added into reaction mixture accordingly. Blank solution contained deionized water, starch and PBS, while positive and negative controls comprised of acarbose and deionized water respectively. Enzyme inhibition was calculated by following equation;
푬풏풛풚풎풆 풊풏풉풊풃풊풕풊풐풏 = [{푶푫푺 − 푶푫푵} ÷ {푶푫푩 − 푶푫푵}] × ퟏퟎퟎ
Where, “ODS”, “ODN” and “ODB” corresponds to the optical densities of sample, negative control and blank respectively 5.2.9.3 Protein kinase inhibition Streptomyces 85E strain was used for Protein kinase inhibition assay as described previously (Fatima et al. 2015). On ISP4 minimal media a uniform lawn of the respective strain was prepared, followed by placement of 6mm filter discs steeped with 10 µL of the test PbO nanoparticle dilution. As positive and negative controls surfactin and DMSO were used respectively. Readings were taken after 72 hrs of incubation at 300 C.
5.3 Results and discussion 5.3.1 Biosynthesis Chemical and physical means of synthesis of nanoparticles bears certain disadvantages like their cost and hazardous waste generation. Even sometimes, noxious chemicals can remain adhered to the surface nanoparticles which limit their use especially in biological applications (Zak et al. 2011; Darroudi et al. 2014). On the contrary, biological synthesis
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Chapter 5 Biosynthesis and applications of lead oxide nanoparticles through medicinal plants is overcome such disadvantages. Therefore green synthesis of nanoparticles is considered more acceptable route for synthesis of multifunctional nanomaterials (Ovais et al. 2016). Biosynthesis of lead oxide has never been reported previously through medicinal plants. Herein, aqueous extracts of Sageretia thea was effectively used as chelation agent for the biosynthesis of lead oxide nanoparticles. Sageretia thea has been used in making tea in parts of China and Korea. Certain phenolic and flavonoid compounds (Syringic acid, Quercetin, Myricetrin, Kaempferol, Daucosterol, Taraxerol) are already been reported from Sageretia thea (Shen et al. 2009; Chung et al. 2004; Xu et al. 1994). Such bioactive components can play a critical role in capping and stabilizing of nanoparticles (Park et al. 2011). The possible mechanism is indicated in Figure 5.2.
Figure 5. 2: Plausible mechanism of biosynthesis lead oxide nanoparticles using Sageretia thea aqueous extracts
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5.3.2 Characterizations X-ray diffraction spectra of bioinspired lead oxide nanoparticles has been indicated in Figure 5.3A, while their average size was determined using Debye-Scherer approximation as indicated in Figure 5.3B. The observed Bragg peaks were the crystallographic reflections of orthorhombic pure massicot phase lead monoxide (PbO) having standard lattice parameters of = 0.549 nm, = 0.589 nm,
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Chapter 5 Biosynthesis and applications of lead oxide nanoparticles be pre-concluded because of the intense peak. IR peak centered at ~ 3400 cm-1 represents –OH stretching vibrations. FTIR spectra also show bands at centered at ~ 2200 cm-1 and 1200 cm-1 which indicate the possible attached functional groups.
Figure 5. 3: XRD analysis of biosynthesized lead oxide nanoparticles. (A): XRD spectra; (B): Average size calculation using Scherer size approximation formula.
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Figure 5. 4: Various HR-TEM images of biosynthesized lead oxide nanoparticles; (A/B/C/D): size distribution and shape; (E): HR-TEM image; (F): Particle size distribution.
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Figure 5. 5: (A/B/C): Various HR SEM images of PbO nanoparticles; (D): their SAED pattern.
Figure 5. 6: Energy Dispersive Spectroscopy of biosynthesized lead oxide nanoparticles.
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Figure 5. 7: Raman spectra of biosynthesized lead oxide nanoparticles.
Figure 5. 8: ATR-FTIR spectra of biosynthesized lead oxide nanoparticles.
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5.3.3 Antibacterial activities Bioinspired lead oxide nanoparticles were screened for their antimicrobial potential against 3 gram positive (Staphylococcus aureus, Staphylococcus epidermis and Bacillus subtilis) and 3 gram negative bacterial strains (Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli) across different concentration i.e. 1000 µg/ml – 31.25 µg/ml. In addition, the effect of UV illumination was studied in the enhancement of the antimicrobial potential. Figure 5.9A shows the antibacterial effect of PbO nanoparticles without UV illumination. Klebsiella pneumonia was found as the most susceptible bacterial strain (MIC = 31.25 µg/ml) while Pseudomonas aeruginosa (MIC = 250 µg/ml) was the least susceptible. Upon modulation of UV-illumination, enhancement in the antibacterial potential was observed as indicated in Figure 5.9B. With UV modulation, Pseudomonas aeruginosa was inhibited at lower concentrations then 250 µg/ml, and their MIC was investigated as 31.25 µg/ml. Klebsiella pneumonia was inhibited at lower concentrations then 31.25 µg/ml. The MIC was for the samples effective up to the concentration of 31.25 µg/ml, was investigated by broth dilution assay. The tube dilution assay revealed MIC of 15.6 µg/ml for the lead oxide nanoparticles produced via aqueous root extracts of Sageretia thea. MIC values are summarized in table 5.1. In general we indicate moderate antimicrobial nature for lead oxide nanoparticles relative to the pure gentamycin disc (10 µg) which is used as a positive control. We further conclude the antibacterial response as concentration or dose dependent.
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Table 5. 1: MIC calculations for lead oxide nanoparticles.
Without UV illumination With UV illumination Gram positive Gram positive Bacterial strain MIC (µg/ml) Bacterial strain MIC (µg/ml) Staphylococcus aureus 62.5 Staphylococcus aureus 31.25 Staphylococcus epidermis 62.5 Staphylococcus 31.25 epidermis Bacillus subtilis 125 Bacillus subtilis 31.25 Gram negative Gram negative Klebsiella pneumonia 31.25 Klebsiella pneumonia 15.6 Pseudomonas aeruginosa 250 Pseudomonas 31.25 aeruginosa Escherichia coli 125 Escherichia coli 31.25
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Figure 5. 9: Antibacterial activities of biogenic PbO nanoparticles; (A): without UV- illumination; (B): with UV- illumination.
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5.3.4 Antileishmanial activities Leishamniasis is as a neglected tropical disease which is endemic to 98 countries and with ~ 350 million people living under immediate threat. Until now, there are no effective treatments for its cure while the medications been used for its treatment are not only costly but also accompanied with side effects like prolonged duration for therapy and elevated toxicity (Abamor 2017; Légaré and Ouellette 2017). Antimonials have been widely used for their treatment which however has lost their efficiency due to drug resistance. Leishmania exist in promastigote (motile) form outside the body while transform into amastigote (non-motile) form inside the body. Antileishmanial activities of biogenically synthesized lead oxide are reported against the axenic promastigote and amastigote leishmania as indicated in Figure 5.10A. MTT cytotoxicity suggested potential lethal nature of lead oxide nanoparticles against Leishmania. Percent inhibition was observed across all the tested concentrations while the IC50 values were calculated as 14.7 and 11.95 µg/ml for the promastigote and amastigote cultures of Leishmania. Novel strategies involving nanoparticles are already proposed for the treatment of Leishmania. Recent research has indicated a significant potential of metal oxides to be used as antileishmanial agents (Ali et al. 2017; Nadhman et al. 2016). From our results, we can conclude dose dependent cytotoxicity against Leishmania, while amastigotes were found more susceptible to promastigotes. 5.3.5 Brine shrimp cytotoxicity The cytotoxic potential of the lead oxide nanoparticles was also studied through brine shrimp cytotoxic activity. Brine shrimps (Artemia salina) are widely used to screen chemical entities for their cytotoxicity (Ali et al. 2017). The obtained results are summarized in Figure 5.10A. Percent inhibition was calculated by counting the succumbed shrimps relative to the dead ones after applying the test dilution. Cytotoxicity of the lead oxide nanoparticles was confirmed and the median lethal concentration was calculated as 27.74 µg/ml. The dose dependent response was also confirmed. 5.3.6 Bio-compatibility potential Keeping in view the adverse effects of lead/lead oxide nanoparticles to the human exposure (Lopes et al. 2015), their biocompatibility was assessed. Herein, it is noteworthy to mention that the bio-compatibility of the bioinspired lead oxide has never
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Chapter 5 Biosynthesis and applications of lead oxide nanoparticles been reported. Freshly isolated human RBC’s and macrophages were used to study the bio-compatibility of the as synthesized lead oxide nanoparticles. The results are indicated in Figure 5.10B. Hemolytic activity revealed cytotoxicity of biogenic lead oxide at higher concentrations i.e. 25 % at 200 µg/ml while lowering their concentrations significantly decreases percent hemolysis. On the contrary, the freshly isolated macrophage cells were inhibited significantly as compared to the RBC’s. The MTT cytotoxic assay revealed the median lethal concentration of 57.1 µg/ml against human macrophage cells. These results against human macrophage cells are in good agreement with some of the previous reports where the median lethal concentration against human cells are found to be around 50 µg/ml (Alarifi et al. 2017). These results are indicated in Figure 5.10A/B while the calculated medial lethal concentration (IC50) are summarized in table 5.2. Our results conclude relatively less cytotoxicity to RBC’s as compared to macrophages.
Table 5. 2: IC50 calculation for lead oxide nanoparticles.
Assay type IC50 Antileishmanial promastigotes 14.7 µg/ml Antileishmanial amastigotes 11.95 µg/ml Brine shrimp cytotoxicity 27.74 µg/ml Human RBC’s > 200 µg/ml Human macrophages > 57.1 µg/ml Alpha amylase inhibition > 200 µg/ml
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Figure 5. 10: Potential cytotoxic effects of PbO biogenic nanoparticles. (A): Cytotoxicity against axenic cultures of leishmania promastigotes, amastigotes and brine shrimps; (B): Bio-compatibility with RBC’s and freshly isolated macrophages 5.3.7 Antioxidant assays Antioxidant potential of the bioinspired lead oxide nanoparticles was assessed as indicated in Figure 5.11. Free radical scavenging activity using DPPH suggested radical scavenging potential for the biosynthesized lead oxide nanoparticles. 58% DPPH radical
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Chapter 5 Biosynthesis and applications of lead oxide nanoparticles scavenging was observed at 200 µg/ml. Scavenging potential was directly related to the concentration of the nanoparticles. Radical scavenging activity decreased to 6% at 2 µg/ml while no activity was observed at concentration < 2 µg/ml. Total reducing power and total antioxidant capacity was expressed as micrograms of ascorbic acid/mg of the test samples. Moderate reducing power i.e. 22 µg AAE/mg and moderate antioxidant capacity i.e. 19.6 µg AAE/mg were reported at 200 µg/ml. Results are summarized in Figure 5.11.
Figure 5. 11: Antioxidant assays of biosynthesized lead monoxide nanoparticles.
5.3.8 Enzyme inhibition assays Figure 5.12 A reports the alpha amylase enzyme inhibition potential of the biogenically synthesized lead oxide nanoparticles. Insignificant inhibition of alpha amylase enzyme is investigated. Enzyme inhibition was not recorded at < 10 µg/ml. In addition, no protein kinase inhibition was reported across any of the tested concentration of the biogenically synthesized lead oxide nanoparticles as indicated in Figure 5.12B.
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Figure 5. 12: Enzyme inhibition assays of biogenic PbO nanoparticles; (A): alpha amylase enzyme inhibition; (B): Protein kinase enzyme inhibition assay indicating no visible zones of inhibition.
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5.3.9 General comments on the mechanism of cytotoxicity Taken together, the biogenically synthesized lead oxide nanoparticles showed antimicrobial and cytotoxic potential against bacteria, leishmania and also human macrophages. Generation of reactive oxygen species, mitochondrial dysfunction, DNA fragmentation and interference with the cellular proteins are considered to be the chief cause of lead oxide mediated cytotoxicity (Alarifi et al. 2017; Amiri et al. 2016). Lead oxide nanoparticles can induce cytotoxicity by oxidative stress by the reduction of GSH, increase in lipid peroxides and SOD concentrations in a dose and time dependent manner. A detailed schematic is presented in Figure 5.13 which indicates the lead oxide mediated cytotoxicity.
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Figure 5. 13: Schematic of the potential cytotoxic nature of biogenically synthesized lead oxide nanoparticles; (A): PbO nanoparticles entering the cell; (B): Interference of PbO NP’s and ROS with DNA causing DNA damage and fragmentation; (C): PbO NP’s and ROS interfering with mitochondria causing mitochondrial dysfunction; (D/E): Interference of PbO NP’s and ROS with proteins and enzymes
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Chapter 6: Physical properties, biological applications and biocompatibility studies on biosynthesized single phase cobalt oxide (Co3O4) nanoparticles via Sageretia thea (Osbeck.)
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Graphical abstract: Physical properties, biological applications and biocompatibility studies on biosynthesized single phase cobalt oxide (Co3O4) nanoparticles via Sageretia thea (Osbeck.)
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Abstract Cobalt oxide nanoparticles were successfully biosynthesized by complete green process using aqueous leaf extracts of Sageretia thea as chelating agent. Diverse techniques were applied for characterization. Antibacterial (with and without UV illumination), antileishmanial, antioxidant and enzyme inhibition applications were assessed, while freshly isolated macrophages and red blood cells were used for biocompatibility studies. Good antibacterial nature and enhancement of bactericidal nature upon UV modulation is reported. Staphylococcus aureus and Escherichia coli are indicated as most susceptible bacterial strains. Significant cytotoxic potential is revealed with IC50 calculated as 12.82 µg/ml and 3.16 µg/ml against the axenic leishmanial promastigote and amastigote cultures respectively. Biogenic cobalt oxide nanoparticles indicated DPPH free radical scavenging potential, while moderate antioxidant capacity and reducing power was demonstrated. Bioinspired cobalt oxide also demonstrated alpha amylase and protein kinase inhibition at higher concentrations. Biogenic cobalt oxide was found as more cytotoxic to macrophages (IC50=58.55 µg/ml) then to RBC’s (IC50> 200 µg/ml). Our results indicate green synthesis as an alternative, effective and eco-friendly method for the biosynthesis of cobalt oxide nanoparticles with numerous biological applications.
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6.1 Introduction Bioinspired and multifunctional metal and metallic oxide nanoparticles are considered as a bright area of research because of their exciting physio-chemical and optoelectronics properties (Thema et al. 2016; Thovhogi et al. 2016; Ismail et al. 2016; Diallo et al. 2016; Matinise et al. 2017; Sone et al. 2017; Thema et al. 2015b; Thema et al. 2015a; Thovhogi et al. 2015; Diallo et al. 2015b; Diallo et al. 2015a; Sone et al. 2015; Ezhilarasi et al. 2016). Metal oxide nanoparticles has been frequently synthesized and tested in a wide range of applications. Rapid developments on the nano-biotechnological interface has resulted in a wide array of biomedical applications including drug delivery and vaccine administration (Khan et al. 2015). Nanoparticlulate matter is different from their microscale counter parts in their magneto-optical, electro-optical, mechanical, chemical and surface area to volume ratio which signify them as an effective tool for biomedical applications. Cobalt oxide possess interesting properties and therefore has attracted numerous researchers for studying their possible biomedical applications (Wang et al. 2005). Beside their physiological role as a cofactor of vitamin B12, cobalt can be used in a wide range of applications. Cobalt oxide is a multifunctional, antiferromagnetic p-type semiconductor (with a direct optical bandgap of 1.48 and 2.19 eV) (Raman et al. 2016) has been used in electrochromic sensors, energy storage, heterogeneous catalysis, pigments, dyes, and in lithium ion rechargeable batteries as an anode material (Askarinejad et al. 2010; Li et al. 2005; Shinde et al. 2006; Kaviyarasu et al. 2013; Diallo et al. 2015a). Cobalt based nanostructures have been successfully used in methanol, glucose, nitrites and amino acids (Yang et al. 2006; Shen et al. 2008; Adekunle et al. 2010; Song et al. 2011). Because of their interesting physical properties, cobalt oxide also have spintronic applications (Diallo et al. 2015a). Many physical and chemical techniques have been applied for the synthesis of cobalt oxide nanoparticles. Hydrothermal reaction, thermal decomposition, solution combustion, microwave assisted, micro-emulsion method, chemical spray pyrolysis and vapor deposition method, sono-chemical, co-precipitation and other mechano-chemical processes has been applied for cobalt oxide nanoparticles synthesis (Salavati-Niasari et al. 2009; Dong et al. 2014; Thota et al. 2009; Makhlouf et al. 2013; Zhang et al. 2012;
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Dai et al. 2013; Farhadi et al. 2013). Hitherto, being effective, the afore-mentioned synthesis methods are accompanied by certain disadvantages like being costly, time and energy consuming and being environment unfriendly. To overcome the problem of toxic wastes and energy imbalance, greener and ecofriendly methods have been proposed (Diallo et al. 2015a). Biological resources such as plants and microorganisms can be used in a rapid, effective, simple and economical way to produce the desired metal or metallic oxide nanoparticles (Ovais et al. 2016; Ovais et al. 2017). Plant mediated biosynthesis of cobalt oxide nanoparticles has been successfully demonstrated (Diallo et al. 2015a). With the aim of synthesizing cobalt oxide nanoparticles, a complete green approach was adopted using aqueous leaf extracts of medicinal plant Sageretia thea (Osbeck.), as an effective stabilizing and chelating agent. The fact that there was neither the use of organic/inorganic solvents and nor the use of any surfactants making the process as an ecofriendly and green. The interface of medicinal plants and biosynthesis of nanoparticles provides an exciting opportunities for wide range of biomedical applications. Hence, the as synthesized cobalt oxide nanoparticles were further investigated for their possible biological applications and biocompatibility with human blood cells.
6.2 Material and methods 6.2.1 Plant material processing Sageretia thea was collected from Islamabad, Pakistan and taxonomically verified in Department of Plant Sciences, Quaid-i-Azam University (QAU), Islamabad. The herbarium specimen with voucher number MOSEL-343 was deposited at Molecular Systematics and Applied Ethnobotany Lab (MoSAEL), Department of Biotechnology, QAU. Fresh leaves were excised and grounded to fine powder in a Willy mill. Aqueous extraction was performed on the plant powder, while remaining was stored for further use. In Figure 6.1 the overall outline of the study has been summarized.
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Figure 6. 1: From biosynthesis to applications of bioinspired cobalt oxide nanoparticles
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6.2.2 Biosynthesis procedure Already established procedure for the biosynthesis of metal oxide nanoparticles was used (Thema et al. 2015a). Leaf extracts were obtained by adding 200 ml of deionized water to 30 g of powdered plant material, followed by heating at of ~ 800 C on a magnetic stirring hot plate (Snijders) for 1 hr. The resultant extract solution was filtered thrice with whattman filter paper to remove solid residual waste. To 100 ml of filtered solution 6.0 g of the precursor salt i.e. cobalt acetate was added. pH change was observed from 5.7 to 4.5 after precursor addition. The solution was heated for 2 hrs at ~ 600 C with gentle stirring. The solution was allowed to cool to room temperature and centrifuged (10,000 rpm / 10 min) to collect the pellet that was subsequently washed 3 times with deionized water. Obtained pellet was dried at 100°C for 2 hrs, followed by annealing at 500°C in open air for obtaining highly crystalline pure phase cobalt oxide nanoparticles. A brief biosynthesis mechanism has been indicated in Figure 6.2. 6.2.3 Characterization X-ray diffractometer (model Bruker AXS D8 Advance) equipped with irradiation line Kα of copper (λ=1.5406 A0) was used to record the XRD spectrum and their corresponding size was calculated using Scherer equation {<Øsize> = K λ / ∆θ1/2 cosθ}. Vibrational properties were studied using ATR-FTIR (400 to 4000 cm-1) and raman spectroscopy over the range from 0 to 1000 cm-1. Raman spectrum was recorded using a laser line of 473 nm with average excitation power of 2.48 mW. Morphology and shape was studied via HR-SEM and HR-TEM, while particle distribution was investigated after digitizing the various HR-TEM images using image J software the The morphological investigation was carried out through HR-SEM and HR-TEM. In addition, Selected Area Electron Diffraction (SAED) and Energy Dispersive X-ray spectroscopy (EDS) were also carried out. 6.2.4 Antibacterial potential of Cobalt oxide nanoparticles Previously described disc diffusion method (Fatima et al. 2015) was used for investigating the antibacterial nature of bioinspired cobalt oxide nanoparticles, while broth dilution assay was used to determine their MIC. Bacterial strains already available at the department of biotechnology were refreshed on nutrient agar (Oxoid-CM0003) before the assay. Bacterial cultures were inoculated to nutrient broth and grown in shaker
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Chapter 6 Biosynthesis and applications of cobalt oxide nanoparticles incubator to the optical density of 0.5 at 600 nm which corresponds to 1 × 108 CFU/ml. 100 µl of broth cultures were dispensed in culture plates and uniform microbial lawns were prepared using sterilized cotton swabs. Filter paper discs (6 mm) loaded with 10 µl of the test sample over the concentration range from 1000 µg/ml to 31.25 µg/ml were used to determine the antibacterial activity. Pure gentamycin disc (10 µg) were used as positive control. Zones of inhibition were measured using vernier caliper. Antibacterial potential was also investigated after exposing cobalt oxide nanoparticles to UV for 20 min. For UV-illumination germicidal 6 Watt UV Lamp 6GT5 (Sankyo denki- Japan) was used. 6.2.5 Brine shrimp cytotoxicity The cytotoxicity of biogenic cobalt oxide nanoparticles was assessed using Artemia salina larvae in a 96 well plate as described previously (Ali et al. 2017; Khan et al. 2015). After incubation of the various concentrations of test samples with brine shrimps for 24 hrs, the number of dead shrimps were counted in each well and percent mortality were calculated. IC50 values were calculated using table curve software. 6.2.6 Antileishmanial activity (Promastigotes and Amastigotes) MTT cytotoxic activity (Ali et al. 2017) was carried out against the axenic promastigote and amastigote cultures of Leishmania tropica KWH23 for determining the antileishmanial potential. M199 media supplemented with 10 % fetal bovine serum (FBS) was used for culturing the parasite while their density was maintained at 1 × 106 cells/ml for MTT assay. 96 well microplate was used to test bioinspired cobalt oxide nanoparticles over concentration range from 200 µg/ml to 1 µg/ml. Amphotericin B and DMSO were used as positive and negative control. Median lethal concentration (IC50) was calculated using table curve software. The seeded 96 well microplate was incubated at 24°C for 72 hrs. Readings were taken at 540 nm, while percent inhibition was calculated using; 퐴푏푠표푟푏푎푛푐푒 표푓 푠푎푚푝푙푒 % 퐼푛ℎ𝑖푏푡𝑖표푛 = [1 − { }] × 100 퐴푏푠표푟푏푎푛푐푒 표푓 퐶표푛푡푟표푙 6.2.7 Biocompatibility assessment of biogenic cobalt oxide nanoparticles 6.2.7.1 Biocompatibility with macrophages MTT cytotoxic assay (Ali et al. 2017) was performed on the freshly isolated human macrophages to study the compatibility of biosynthesized cobalt oxide nanoparticles.
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Macrophages were isolated from peripheral human blood through ficoll-gastrografin (density=1.070 g/ml) density gradient method as described previously (de Almeida et al. 2000). Briefly, 5 ml of gastrografin was added to the 95 ml of deionized water, while 5.7 g of ficoll was slowly added. Blood was diluted with Hank’s buffer salt solution (HBSS) followed by gentle layering on ficoll-gastrografin. The solution was further centrifuged for 30 min at 400g followed by purification with percoll gradient (density 1.064 g/ml) adjusted with sterilized deionized water. Macrophages were suspended in RPMI medium supplemented with fetal bovine serum (10 %), Hepes (25 mM), and antibiotics (Streptomycin: 0.1 mg/ml ; Penicillin:100 U/ml). Cells were kept in humified incubator to grow to a density of 1 × 105 cells/well. Percentage inhibition was calculated using formula; 퐴푏푠표푟푏푎푛푐푒 표푓 푠푎푚푝푙푒 % 퐼푛ℎ𝑖푏푡𝑖표푛 = [1 − { }] × 100 퐴푏푠표푟푏푎푛푐푒 표푓 퐶표푛푡푟표푙 6.2.7.2 Hemolytic assay To further assess the biocompatibility against human RBC’s, hemolytic assay was carried out as described previously (Malagoli 2007). Fresh blood was isolated and dispensed in EDTA tube. RBC’s were isolated by centrifugation of blood at 14000 rpm for 5 min. Erythrocytes suspension was prepared by dispensing 200 µl of the isolated pellet to 9.8 ml phosphate buffer saline (pH:7.2). Hemolytic activity was determined by adding 100 µl of test concentration of cobalt oxide nanoparticles with 100 µl of erythrocytes suspension, followed by incubation for 1 hr at 35°C. This was followed by further centrifugation at 10,000 rpm for 10 min and supernatant was dispensed in the 96 well microplate reader to monitor the percent hemoglobin release at 540 nm. Triton X-100 and DMSO were used as positive and negative controls respectively. Percentage hemolysis induced by the nanoparticles was calculated through the formula;
% 푯풂풆풎풐풍풚풔풊풔 = [{푨풃푺 − 푨풃푵푪} ÷ {푨풃푷푪 − 푨풃푵푪}] × ퟏퟎퟎ 6.2.8 Antioxidant activities 6.2.8.1 DPPH radical scavenging Spectrophotometric method (Ali et al. 2017) was employed to investigate the quenching ability of bioinspired cobalt oxide nanoparticles. DPPH is a stable free radical widely used to test the radical scavenging ability of samples. DPPH free radical scavenging was
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Chapter 6 Biosynthesis and applications of cobalt oxide nanoparticles investigated in the concentration range of 200 µg/ml to 1 µg/ml. Ascorbic acid and DMSO were used as positive and negative controls. The reaction mixture (final volume 200 µl) comprised of 20 µl of test sample and 180 µl of reagent. After incubation for 20 min in dark, absorbance was measured at 517 nm and percent radical scavenging was calculated as;
% 푫푷푷푯 풓풂풅풊풄풂풍 풔풄풂풗풆풏품풊풏품 = [ퟏ − {푨푩푺} ÷ {푨푩푪}] × ퟏퟎퟎ 6.2.8.2 Reducing power
Potassium ferricyanide [K3Fe (CN)6] based method (Javed et al. 2016) was used to determine total reducing power of biogenic cobalt oxide nanoparticles. Ascorbic acid and DMSO were used as positive and negative controls respectively. Absorbance was measured at 630 nm and the results were expressed as ascorbic equivalents per mg of sample. 6.2.8.3 Total antioxidant capacity Phosphomolybdenum based method (Jafri et al. 2014) was used to determine total antioxidant capacity. Readings were taken at 695 nm and results were expressed as number of ascorbic acid equivalents in µg per mg of the sample i.e. µg AAE/mg. 6.2.9 Enzyme inhibition assays 6.2.9.1 Protein kinase inhibition PK inhibition was investigated using Streptomyces 85E strain as described previously (Fatima et al. 2015). ISP4 minimal media was used to produce uninform lawns of Streptomyces 85E strain with pre-adjusted optical density of 0.5 at 600 nm. Surfactin and DMSO were used as positive and negative control. 6 mm filter disc loaded with 10 µl of test sample were gently placed on the uniform lawns. Seeded plates were incubated at 30°C for 72 hrs. Zones of inhibition were measured using vernier caliper. 6.2.9.2 Alpha amylase inhibition Alpha amylase inhibition assay was performed in 96 well plat as described previously (Ali et al. 2017). The reaction mix (15 µl PBS/25 µl α-amylase enzyme) was added with test samples (10 µl) and starch solution (40 µl) step wise and incubated at 50°C for 30 min. After incubation, 20 µl of 1M HCL and 90 µl of iodine solution were added to the reaction mix. Blank solution comprised of PBS deionized water and starch, while
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Chapter 6 Biosynthesis and applications of cobalt oxide nanoparticles negative and positive control comprised of DMSO and acarbose respectively. Readings were taken at 540 nm and the enzyme inhibition was calculated by following equation;
푬풏풛풚풎풆 풊풏풉풊풃풊풕풊풐풏 = [{푶푫푺 − 푶푫푵} ÷ {푶푫푩 − 푶푫푵}] × ퟏퟎퟎ
Where, “ODS”, “ODN” and “ODB” corresponds to the optical densities of sample, negative control and blank respectively. 6.3 Results and discussion Although, the biosynthesis of cobalt oxide nanoparticles have rarely been done in previous reports, but their antimicrobial, antileishmanial, antioxidant and enzyme inhibition has never been investigated. Hence it is worthy to mention that this report will be the first of such kind that has successfully produced cobalt oxide nanoparticles via green route but also presented a broad overview on the possible biomedical applications of green cobalt oxide nanoparticles. 6.3.1 Biosynthesis of cobalt oxide nanoparticles Physical or chemical means for synthesis of nanoparticles possess certain disadvantages such as being laborious, hazardous and time consuming. Even it was indicated that certain toxic chemicals used in chemical synthesis can retain on the nanoparticle surface which limits their biomedical applications. On the contrary, green synthesis using medicinal plants is deprived of such disadvantages therefore has been preferred in the present report. A complete green route for the biosynthesis of cobalt oxide nanoparticles was successfully optimized using aqueous extracts of leaf of Sageretia thea. Medicinal uses of Sageretia thea (Bird Plum/English) are well documented. It is used in the treatment of hepatitis, jaundice, circulatory and cardio-vascular diseases. Leaves are used in making tea in parts of Korea and China (Hyun et al. 2015; Khan et al. 2014; Murad et al. 2011). . Bioactive compounds like Taraxerol, Quercetin, Syringic acid, Myricetrin, Kaempferol, Daucosterol have previously been reported from S. thea (C. J. Shen et al. 2009; Chung et al. 2004; Xu et al. 1994). Such phytochemical components has an intended role in stabilizing of nanoparticles (Park et al. 2011). A schematic representation of biosynthesis procedure is indicated in Figure 6.2.
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Figure 6. 2: Plausible mechanism of biosynthesis cobalt oxide nanoparticles using Sageretia thea aqueous extracts.
6.3.2 Physical characterization of cobalt oxide nanoparticles Crystalline, vibrational and morphological properties of biosynthesized cobalt oxide were studied. XRD spectrum confirmed the formation of crystalline and single phase of cobalt oxide nanoparticles. The observed Bragg peaks were the crystallographic reflections of the single phase, face centered cubic cobalt oxide belonging to the space group Fd3m. Brag peaks were found consistent with which are consistent with the JCPDS pattern no.
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00-042-1467. The average lattice parameters were investigated as
Fd3m symmetry, the group theory predicts the following active modes; A1g (R) + Eg (R)
+ F1g (IN) + 3F2g (R) + 2A2U (IN) + 2EU (IN) + 4F1U (IR) + 2F2U (IN) whereas (R), (IR) and (IN) depicts Raman active vibrations, infrared active vibrations and inactive modes. Figure (6.7) indicate the room temperature Raman spectra of the biogenic cobalt oxide nanoparticles. One can single out 6 different Raman active modes centered at ~ 195, ~ 369, ~ 550, ~ 698, ~ 750 and ~ 838 cm-1. Peaks centered at ~ 195 cm-1, 550 cm-1 and 698 cm-1 are in agreement with some of the earlier studies (Diallo et al. 2015a; Jakubek et al. 2015), and therefore confirm cobalt oxide nanostructures. Positioning of Raman peaks, however can change with synthesis methods and distribution of vacancies within the unit cell (De Faria et al. 1997). To further validate the cobalt oxide nature of the biosynthesized nanoparticles, ATR-FTIR analysis were carried out in the spectral range of 400 – 4000 cm-1, as indicated in Figure 6.8. Major IR abortions were singled out by plotting IR optical transmission versus log of wavenumbers. Two sharp IR bands were observed centered at 576 cm-1 and 674 cm-1. These absorption modes are attributed to the
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fingerprint stretching vibrations of Co-O bond in Co3O4 (Ren et al. 2009). The IR absorption observed at 576 cm-1 is the vibration from O-Co, with cobalt depicting the +3 -1 2+ 3+ 2+ Co in octahedral site. Absorption band at 674 cm is attributed to the Co Co O3 (Co is tetrahedral site) vibrations in the spinal lattice (Ai and Jiang 2009; Salavati-Niasari et al. 2009).
Figure 6. 3: XRD analysis of biosynthesized cobalt oxide nanoparticles; (A): XRD spectra; (B): Average size calculation using Scherer size approximation formula.
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Figure 6. 4: Various HR-TEM images of biosynthesized cobalt oxide nanoparticles; (A/B/C/D): size distribution and shape; (E): HR-TEM image; (F): SAED pattern.
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Figure 6. 5: (A/B/C): Various HR SEM images; (D): Particle size distribution.
Figure 6. 6: Energy Dispersive Spectroscopy of biosynthesized lead oxide nanoparticles.
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Figure 6. 7: Raman spectra of biosynthesized cobalt oxide nanoparticles.
Figure 6. 8: ATR-FTIR spectra of biosynthesized cobalt oxide nanoparticles.
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6.3.3 Antibacterial activities As synthesized cobalt oxide nanoparticles were studied for their antibacterial potential against 3 gram negative (Pseudomonas aeruginosa, Klebsiella pneumonia, and Escherichia coli) and 3 gram positive bacterial strains (Staphylococcus epidermis, Staphylococcus aureus and Bacillus subtilis). It was noted that the antibacterial potential increased with increase in the concentration of the nanoparticles. Similarly, upon UV modulation the antibacterial activities showed enhancement. Staphylococcus aureus and Escherichia coli were found as the most susceptible strains with MIC and MICuv as 31.25 and 31.25 µg/ml respectively. Pseudomonas aeruginosa was found to be the least susceptible strain with MIC and MICuv as 250 and 62.5 µg/ml respectively. The antibacterial response was concluded to be dose dependent, while none of the tested samples were found more effective then gentamycin antibiotic disc (10 µg). The control antibiotic indicated significantly higher zones of inhibition as compared to test bioinspired cobalt oxide samples. Previous study (Khan et al. 2015) also indicated cobalt oxide nanoparticles synthesized via chemical route as less effective than the standard drugs which is in agreement to our findings, however the same study reports MIC > 10,000 µg/ml for cobalt oxide nanoparticles which is in disagreement to our findings. Here, it can be suggested that the disagreement in the results are because of the difference in synthesis methods. While using medicinal plants as a stabilizing and capping agents, it can be inferred that some of the bioactive phenolic components are used in capping of the nanoparticles (Gatselou et al. 2016; Durán et al. 2011) and those components remains capped to the nanoparticles. Those phenolic compounds can be the result in the enhancement of the antibacterial potential of biogenic cobalt oxide relative to chemically produced counterparts. The antibacterial activities across different test concentrations are summarized in Figure 6.9 while there minimum inhibitory concentrations are indicated in table 6.1.
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Table 6. 1: MIC calculations of biogenic cobalt oxide nanoparticles.
Without UV illumination With UV illumination Gram positive Gram positive Bacterial strain MIC (µg/ml) Bacterial strain MIC (µg/ml) Staphylococcus aureus 31.25 Staphylococcus aureus 31.25 Staphylococcus epidermis 125 Staphylococcus 31.25 epidermis Bacillus subtilis 125 Bacillus subtilis 31.25 Gram negative Gram negative Klebsiella pneumonia 62.5 Klebsiella pneumonia 31.25 Pseudomonas aeruginosa 250 Pseudomonas 62.5 aeruginosa Escherichia coli 31.25 Escherichia coli 31.25
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Figure 6. 9: Antibacterial activities of bioinspired cobalt oxide nanoparticles; (A) without UV-illumination; (B): with UV- illumination
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6.3.4 Cytotoxic activities of bioinspired cobalt oxide nanoparticles 6.3.4.1 Brine shrimp cytotoxicity Preliminary cytotoxicity of biogenic cobalt oxide nanoparticles was confirmed through the brine shrimp cytotoxicity assay. Brine shrimps are widely used as a reference organism for screening the cytotoxic potential of chemical entities. Figure 6.9 A indicate the cytotoxicity of biosynthesized cobalt oxide nanoparticles towards brine shrimps. Cytotoxicity of the cobalt oxide nanoparticles was confirmed by their dose dependent response while the median lethal concentration was calculated as 19.18 µg/ml. 6.3.4.1 Antileishmanial activities Leishamniasis is deadly disease which comes under the category of neglected tropical disease, is endemic to ~ 100 countries with about ~ 350 million people living under the direct threat. To date, there are not as such effective antileishmanial drugs while the current treatment medications are accompanied by disadvantages like side effects, cost, toxicity and long therapy duration (Abamor 2017). Antimonials were used as a gold standard therapy for leishmania, which however has lost their effectiveness because of drug resistance (Hadighi et al. 2006). Therefore, the treatment for leishmania consequently requires an alternative approach. Metal oxide nanoparticles recently demonstrated their abilities in considerably reducing the leishmania population in invitro studies (Ali et al. 2017; Nadhman et al. 2016). In the present study, the antileishmanial nature of the biogenic nanoparticlulate cobalt oxide has been assessed using MTT cytotoxic assay for the first time. Both of the axenic promastigote and amastigote cultures were found highly susceptible to the tested concentrations. Amastigote leishmania were found more susceptible (IC50= 3.16 µg/ml) relative to promastigote (IC50= 12.82 µg/ml). The life cycle of leishmania is simple digenetic i.e. Leishmania exist in 2 forms (promastigote and amastigote). The amastigotes are present inside the body as circular and non-flagellated forms while the motile promastigotes are found outside the human body (Zilberstein et al. 1991). It was found that the antileishmanial response was dose dependent. Our results further indicate the possible applications of biogenic cobalt oxide nanoparticles in nanomedicine for the treatment of leishmania at any stage of its life cycle. Results are indicated in Figure 6.9A.
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6.3.5 Biocompatibility assessment of bioinspired cobalt oxide nanoparticles In response to the potential inhibitory effects of biogenic cobalt oxide on microorganisms, it was considered imperative to investigate their biocompatibility with other normal human cells. Henceforth the biocompatibility was assessed under invitro conditions, using freshly isolated red blood cells and macrophages. Results about the percent hemolysis are indicated in Figure 6.9B. MTT cytotoxicity was carried out against freshly isolated macrophages and the results indicated their toxicity towards macrophages. Median lethal concentration were investigated as > 200 µg/ml and 58.55 µg/ml for RBC’s and macrophages respectively. Therefore, it can be concluded that biogenic cobalt oxide nanoparticles can be used in therapies at low concentrations. However, these results are preliminary, and further research is recommended on the compatibility of bioinspired cobalt oxide nanoparticles with normal human cells.
Table 6. 2: IC50 calculation of bioinspired cobalt oxide nanoparticles.
Assay type IC50 Antileishmanial promastigotes 12.82 µg/ml Antileishmanial amastigotes 3.16 µg/ml Brine shrimp cytotoxicity 19.18 µg/ml Human macrophages 58.55 µg/ml Human RBC’s > 200 µg/ml Alpha amylase inhibition > 200 µg/ml
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Figure 6. 10: Cytotoxicity of cobalt oxide nanoparticles; (A): Cytotoxicity against brine shrimps and leishmania; (B): Biocompatibility with RBC’s and macrophages; (C) Median lethal concentrations.
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6.3.6 Mechanism of cytotoxicity Taken together, the bioinspired cobalt oxide nanoparticles manifested toxicity. Researchers have proposed different mechanisms for the cobalt oxide mediated cytotoxicity. Recently described Trojan horse is considered one of the mechanism by which cytotoxicity is manifested by cobalt oxide nanoparticles. The endocytosis mechanism for nanoparticles entry to the cells is well established (Contreras et al. 2010; Bregar et al. 2013) however recent research has indicated the uptake of cobalt oxide nanoparticles via non-endocytotic pathway (Li and Malmstadt 2013; Lin and Alexander- Katz 2013). After entering the cells, the cobalt nanoparticles can leach Co++ ions that can lead to impairment in the nearby cellular organelle by a trojan horse like mechanism (Ortega et al. 2014). Similarly, as with case with other metal nanoparticles, the ROS generation in one of the primary cause of cellular disruption by oxidative stress. Active nanoparticle surface of cobalt oxide can readily generate ROS species by interactive with oxygen (Limor et al. 2011). Double strand DNA breaks are also reported for cobalt oxide nanoparticles (Uboldi et al. 2016). A detailed schematic on the cytotoxicity mechanisms and pathways is presented in Figure 6.13. 6.3.7 Antioxidant assays Antioxidant potential of the bioinspired cobalt oxide was studied and results are indicated in Figure 6.11. Free radical scavenging, total antioxidant capacity and total reducing power potential were studied. Highest DPPH radical scavenging (57%) was observed at 200 µg/ml while the scavenging ability decreased at lower concentrations. Similar trends were also observed with total antioxidant activity and total reducing power. Total reducing power and total antioxidant capacity were highest at 200 µg/ml, i.e. 19.8 µg AAE/mg and 23.6 µg AAE/mg of bioinspired cobalt oxide nanoparticles respectively. Overall, good radical scavenging potential, moderate total antioxidant capacity and total reducing power potential is reported for biogenic cobalt oxide nanoparticles. 6.3.8 Enzyme inhibition assays Figure 6.12A indicate the alpha amylase enzyme inhibition potential of the as synthesized cobalt oxide nanoparticles. Alpha amylase enzyme catalyze the breakdown of carbohydrates into glucose and therefore has been associated with the postprandial glucose excursion in a patient suffering from diabetes. Hence, alpha amylase enzyme
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Chapter 6 Biosynthesis and applications of cobalt oxide nanoparticles inhibitors have been considered in diabetes research. Moderate inhibition is reported (34 %) at the highest concentration (200 µg/ml ) while no inhibition is reported at the lowest concentration of (1 µg/ml). Figure (6.12 B) indicate the protein kinase (Pk) enzyme inhibition potential of the biosynthesized cobalt oxide nanoparticles. Pk are considered an important area for anticancer research. Pk enzymes phosphorylates the serine-threonine and tyrosine amino acid which functions in cellular differentiation, proliferation and apoptosis. Deregulated phosphorylations by Pk enzymes at the aforementioned amino acids residues can lead to tumor growth. Such entities capable of Pk inhibition are looked for in cancer therapies. Pk phosphorylations also play a crucial role in hyphae formation in Streptomyces and therefore this microorganism has been readily used in identification of PK inhibitors. Streptomyces 85E strain was used to screen Pk inhibition potential of the as synthesized cobalt oxide nanoparticles. Our results indicate the formation of bald zones up to 50 µg/ml while no zones are reported at concentrations lower than 50 µg/ml. Inhibitory zones were 9.4 mm, 6.1 mm and 4.6 mm at 200, 100 and 50 µg/ml respectively. These results indicate that bioinspired cobalt oxide nanoparticles could be further explored in anticancer therapies via further studies on its protein kinase inhibition mechanism.
Figure 6. 11: Antioxidant assays of biosynthesized cobalt oxide nanoparticles.
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Figure 6. 12: Enzyme inhibition assays of bioinspired cobalt oxide nanoparticles; (A): alpha amylase enzyme inhibition; (B): Protein kinase enzyme inhibition assay.
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Figure 6. 13: Schematic of the potential cytotoxic mechanisms of biogenically synthesized cobalt oxide nanoparticles. (1) indicate the ROS generation by interaction with oxygen; (2) indicate the Trojan horse like process in which Co++ ions are generated in an acidic medium which can interfere with the cellular organelle; (3) Membrane damage; (4/5) Interaction of ROS, Co++ ions and cobalt oxide nanoparticles with cellular proteins and enzymes; (6) indicate interaction of nanoparticles with nuclear material leading to DNA fragments; (7) Trojan horse like process in cellular organelle with acidic media; (8) Mitochondrial disruption by various ROS and cobalt oxide nanoparticles.
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Chapter 7: Biogenic synthesis of zinc oxide nanoplatelets on nickel foam as electrode material for supercapacitor applications
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Abstract Aqueous plant extracts from the leaves of Sageretia thea (Sth) were used as reducing/oxidizing as well as capping agents to synthesis zinc oxide (ZnO) nanoplatelets. Generally, plants extracts provide an ecofriendly, simple and nontoxic system for the biogenic synthesis of nanoscaled matter. A versatile drop coating method was followed to deposit the ZnO nanoplatelets on nickel foam (NiF) to fabricate electrode material (NiF/ZnO) for supercapacitor application. The successful deposition of ZnO nanoplatelets on NiF was confirmed by high resolution scanning electron microscopy and XRD. 3D tomography was used as a complementary technique for the confirmation of the continuous and closely compacted deposition of ZnO nanoplatelets within the micropores of the NiF. The nature of the ZnO NPs was also studied using UV absorption and ATR-FTIR. The fabricated NiF/ZnO electrode showed high specific capacitance of ~ 453 F g-1 at a relatively high current density of ~ 2 A g-1. The cycling performance of the NiF/ZnO electrode material was excellent with 86% specific capacitance retention after 1000 cycles. These results confirmed that the biogenically synthesized ZnO nanoplatelets exhibit high electrochemical performances making them an exciting candidate for the construction of electrodes based energy storage and conversion
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7.1 Introduction The rapid developments in global economy, population explosion, increasing demands of hybrid electrical vehicles and portable electronic devices has led to the increase in the energy consumption at alarming rates (Chu and Majumdar 2012; Arico et al. 2005; Yan et al. 2014). The global energy demands are expected to get doubled by the mid of the current century and tripled by 2100 at the current rate of consumption (Nocera 2009). Considering the depleting resources of energy, massive research has been carried out on not only to sought alternative, sustainable and clean forms of energy but also on smart energy conversion and storage devices (Simon and Gogotsi 2008; Xiao et al. 2012). Lithium ions batteries (LIB’s) and supercapacitors are among the smart devices at the forefront of research for energy storage and conversion, however, due to the low power uptake and delivery, the large scale application of LIB’s are distant (Simon and Gogotsi 2008). Consequently, the burden now overlays on supercapacitors for an efficient energy storage device (Yan et al. 2014). Supercapacitors that are also sometimes referred as ultracapacitors or electrochemical capacitors has attracted significant attention from scientists and industries because of their impressive qualities like long life cycles (>10,000), power density, simple operation, efficient charging and discharging and less maintenance costs (Hall et al. 2010; Zhang and Zhao 2009). Because of the high power density, supercapacitors have widespread applications in consumer portable electronics, power back-ups and hybrid vehicles (Wang et al. 2006) etc. Therefore, a number of number of researchers are attracted to assemble efficient and high energy density supercapacitors (Miller and Simon 2008; Yan et al. 2014). Such devices requires excellent electrode materials that possess features like excellent porosity, electrical conductivity and high surface area (Bello et al. 2014). The systematic reviews of data available on ISI web of science indicates significant upsurge in the research papers on supercapacitors from 1,673 in 2013 to > 3,000 in 2016 (Huang et al. 2016; Wu et al. 2016; Zhang et al. 2017; Naoi et al. 2016; Samsudin et al. 2016; Liu et al. 2016). Supercapacitors can store greater energy than ordinary capacitors and delivers high power bursts than batteries (Yan et al. 2014). Moreover, there are 2 categories of supercapacitors that differ based on the mechanism used for energy storage. They are pseudo-capacitors and electrochemical double layer capacitors. In pseudo-capacitors,
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Chapter 7 Supercapacitor application of biogenic ZnO nanoparticles charge transfer between electrode and electrolyte become the source of energy storage (Khamlich et al. 2017). The second means of storage is the ion adsorption in electrochemical double layer capacitors (EDLC). Pseudo-capacitors offer high energy density and specific capacitance then EDLC (Ismail et al. 2016). Transition metal oxides are widely used in pseudo-capacitors due to their power density and fast redox kinetics (Ismail et al. 2016; Dong et al. 2012). Ruthenium oxide has mostly been studied for electrochemical capacitors because of its prominent electrochemical properties and high specific capacitance however the cost of ruthenium limits the widespread application. Therefore there is a need of constructing supercapacitors using cheap and equally effective metal oxides. One such candidate that can be looked for is zinc oxide which is already used in batteries and has a high energy density (650 Ag-1) (Kalpana et al. 2006). Zinc oxide is a suitable material for supercapacitor applications due to its eco-friendly and low cost production, and having good electrochemical properties in comparison with other metal oxides (Kalpana et al. 2006). In the current article, we for the first time present a complete green chemistry method for the synthesis of ZnO nanoplatelets using S. thea. The synthesized ZnO nanoplatelets were deposited on a highly porous 3d nickel foam substrate for their possible uses in supercapacitor application. Aqueous extracts of the leaves of S. thea were utilized as stabilizing as well as oxidizing/reducing agents for phytosynthesis of zinc oxide nanoparticles. The functionalization of the electrode was achieved through drop coating method using ethanol as a dispersion medium of the ZnO nanoplatelets. The fabricated electrode NiF/ZnO was investigated for specific capacitance, stability and life cycle. The obtained results showed good and promising electrochemical properties.
7.2 Material and methods 7.2.1. Collection of plant material Sageretia thea (Osbeck.) usually refereed as “Bird Plum or Chinese Sweet Plum” is an evergreen shrub belonging from Rhamnaceae. Locally referred to as “Gangeer” and “Momanra” (Shah et al. 2013). Information retrieved from the Flora of Pakistan indicated that the plant has different synonyms like like Sageretia theezans (Linn.), Rhamnus thea
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(Osbeck.) and Rhamnus theezans (L.) Brongn. Traditionally, the plant is used for making of tea in China and Korea, while it has been also used by the indigenous people as a remedy for fevers, jaundice, circulatory, cardio-vascular diseases and hepatitis (Hyun et al. 2015; Khan et al. 2014; Murad et al. 2011). Sageretia thea was collected from Quaid- i-Azam University, Islamabad (33.7462° N, 73.1381° E) and was taxonomically verified in the Department of Plant Sciences, QAU. The plant material was washed with distilled water and kept for shade drying at room temperature for about 2 weeks. Later on, the test material was ground to fine powder for extraction in a Wiley mill. 7.2.2 Biogenic synthesis of zinc oxide nanoplatelets using Sageretia thea Previously described synthesis procedure by Thema et al., (2015), with minor modification was used to synthesis ZnO nanoparticles (Thema et al. 2015). Earlier reports on the use of S.thea indicated the presence of polyphenols and other bioactive compounds that can play a pivotal role in the biosynthesis of nanoscaled particles. Briefly, 30 g of the ground cover plant material was immersed in 100 ml of distilled water and heated at 100 ºC for 1 h on magnetic stirrer with hot plate (Snijders) to extract the green dye. The resultant aqueous solution was filtered twice with a whatman filter paper to remove any solid residuals. pH of the twice filtered solution was recorded at room temperature before and after the addition of zinc acetate dihydrate precursor (Alfa Aesar). To 100 ml of the filtered solution (pH 5.7), 6.0 g of the precursor salt was added and gently heated at 60 ºC on magnetic stirrer with hot plate for 2 h to ensure maximum dissolution of the salt. After cooling the solution to room temperature, the pH of the solution was recorded to be 3.94. The obtained solution was centrifuged at 10,000 rpm for 10 min. The obtained precipitate was washed 3 times with deionized water and kept for drying in oven at 100 ºC for ~ 2 hrs. Dried precipitate of ZnO (black in color) was annealed at 500 ºC for 2 h in open air furnace to obtain highly crystalline nanocrystals of ZnO (white in color) which was later confirmed by structural analysis. 7.2.3 Fabrication of NiF/ZnO electrode material The fabrication of NiF/ZnO electrode was carried out as described by Ismail et al., (2016) (Ismail et al. 2016). Electrode material was prepared using nickel foam (Sigma-Aldrich, thickness 1.6 mm, bulk density 450 g m-2) as a 3d template. NiF was rinsed in distilled water 2-3 times followed by sonication for 15 minutes to remove any particles or
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Chapter 7 Supercapacitor application of biogenic ZnO nanoparticles contamination before the process of deposition. ZnO nanoplatelets were mixed with absolute ethanol and gently dropped on the surface of the NiF using a sterile dropper. The deposited ZnO on NiF was allowed to dry at 60 ºC. A thin layer of the ZnO nanoplatelets on NiF was obtained (Figure 7.1).
Figure 7. 1: Schematic of deposition, characterization and electrochemical application of biogenic ZnO based electrode material for supercapacitor application
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7.2.4 Material characterization The crystalline structure of ZnO nanoplatelets was confirmed using X-ray diffractometer
(Bruker AXS D8) with irradiation line Kα of copper (λ=1.5406 Aº). In order to further investigate the successful deposition, HR-SEM was carried out for the NiF-ZnO electrode material. The HR-SEM microimages were obtained using FE-SEM (Carl Zeiss) operating at 5 keV. Besides the already used characterization techniques (XRD and HR-SEM), nano X-ray computed tomography (CT) was applied as a complementary techniques to build highly interactive 3d images of the electrode material (NiF, NiF-ZnO) to visually and observe the continuous and closely compacted deposition of ZnO nanoplatelets within the micropores of the NiF. A series of radiographs were obtained at various angular positions over 360º and reconstructed to obtain 3D volumetric data by mathematical algorithms. High resolution maps relating to the density of materials were produced by X-ray attenuation in virtual cross sections (du Plessis et al. 2016). As tomographic techniques are nondestructive, it can be readily applied in material characterization (Maire and Withers 2014). In addition, NiF and NiF/ZnO were further analyzed to observe the structural differences summarized in table 7.1. The electrochemical measurements were performed using Autolab workstation (Metrohm Autolab BV) with NOVA 1.11 software. The glassy carbon electrode was used as a counter electrode and biosynthesized electrode (NiF/ZnO) served as a working while the reference electrode was Ag/AgCl (3 M KCl) in 4.0 M KOH electrolyte. Cyclic voltammetry at different scan rates (0.025 to 0.100 V s-1) was performed. The stability of the fabricated electrode was assessed over 1000 cycles while EIS (Electrochemical Impedance Spectroscopy) was performed from 100 KHz to 1 MHz. The specific capacitances was calculated at different current densities
7.3 Results and Discussion 7.3.1 Structural analysis of ZnO nanoplatelets and NiF-ZnO electrode material Room temperature X-ray diffraction of ZnO nanocrystals annealed at 500 ºC indicated a highly crystalline phase that corresponds to hexagonal zincite which is the mineral form of zinc oxide (ZnO) (Figure 7.2A). The average ZnO nanoplatelets crystallite size was found to be ~ 14 nm, as determined by Debye-Scherrer approximation (Thovhogi et al.
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2016). No other peaks related to zinc associated compounds were found, which is attributed to the purity of the biogenically synthesized ZnO nanoplatelets. In addition, the Sth mediated ZnO NPs was verified (Figure 7.2B). The corresponding Miller indexation were indicated on XRD peaks in bracket. All the peaks that occurred were assigned to (001), (002), (101), (102), (110), (103), (112) and (201) crystallographic reflections of the hexagonal zincite-type crystallite with the standard lattice parameters, a = b = 0.32 nm and c= 0.52 nm, according to the JCPDS card no. 36-1451 of the bulk zincite. The peaks denoted with asterisk (*) are attributed to the reticular planes of Nickel structures (H. Xu et al. 2008; Ismail et al. 2016). These analysis indicated high purity and high crystallinity of the synthesized and deposited ZnO nanoplatelets on NiF for supercapacitor application.
Figure 7. 2: XRD patterns of (a) Sageretia thea mediated Zinc oxide nanoparticles, (b) NiF/ZnO nanocomposite electrode
7.3.2. Morphological investigation HR-SEM was used in this study to confirm the successful deposition of the biogenically synthesised ZnO nanoplatelets on the 3d NiF template. Figure 7.3A of the bare NiF showed micro-porous structure with smooth surfaces. From Figure 7.3B, one can observe a dense layered deposition of ZnO nanoplatelets on the surfaces and within the micro- pores of the NiF. Additionally, the rough and textured layers of ZnO was clearly
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Chapter 7 Supercapacitor application of biogenic ZnO nanoparticles deposited on the inner and outer surfaces of NiF which further confirm the successful deposition of ZnO via drop coating method. Moreover, the inset of Figure 7.3B showed nanoplatelet structure of the biogenically synthesized and deposited ZnO on NiF. A systematic review of literature on the biosynthesis of ZnO nanoparticles indicated mostly spherical or quasi-spherical shapes when produced through green chemistry (Thema et al. 2015; Diallo et al. 2015; Matinise et al. 2017) while ZnO nanoplatelets have not been previously reported via a green chemical synthesis. This signifies the effectiveness of Sageretia thea natural extracts for chelation. Numerous phytochemical studies using high throughput techniques such as HPLC and NMR has revealed active phenolic and flavonoid content which can be responsible for chelation and capping phenomena.(Venugopal et al. 2017; Iravani 2011; Sone et al. 2016). It is established that different bioactive compounds such as ascorbate, terpenoids, proteins, poly-phenols are performing capping and reduction process. The natural extracts of S. th has phytochemicals like quercetrin, myricetin, kaemferol and syringic acid that can act as stabilizing agent as well as capping agent for nanoparticle synthesis (Chung et al. 2004; Shen et al. 2009; Xu et al. 1994).
Figure 7. 3: SEM micrographs of Bare NiF (a); high magnification SEM of the fabricated NiF/ZnO electrode (inset shows HR-SEM micrographs of the deposited ZnO nano platelets on NiF (b). To further confirm the successful deposition and stability of the ZnO nanoplatelets on NiF, a complementary X-ray tomography technique was employed. 3d images of the coated electrode were produced by scanning the electrode at a series of angular positions 162
Chapter 7 Supercapacitor application of biogenic ZnO nanoparticles over 360º as indicated in Figure 4. Observable differences were investigated between the bare NiF and NiF coated with ZnO. Figure 4A showed the x-ray tomographic image of bare NiF while Figure 4B showed the deposited ZnO nanoplatelets on NiF. The observed difference in color and surfaces thickness between NiF and NiF/ZnO clearly confirmed a successful deposition of the electrochemically active ZnO nanoplatelets (colored as green while the NiF is indicated as gray in Figure 7.4A/B. Furthermore, in order to deeply investigate the successful deposition of ZnO nanoplatelets on NiF, parameters like absolute permeability (AP) and total flow rate (TFR) were extracted by using simulation software based generated data as summarized in table 7.1. The AP for the deposited NiF- ZnO was found to be 636392 mD which is significantly less than the bared NiF ~ 2864100 mD. Similar pattern can be observed in the total flow rate which is decreased from 1.70345e-009 in NiF to 3.6563e-10 in NiF-ZnO. The observed decrease in the absolute permeability and the total flow rate could be the results of the deposited ZnO on the NiF surfaces which leads to the decrease in the micro-porosity of the NiF.
Figure 7. 4: 3d computed tomography of the bare NiF (a) and NiF with deposited ZnO nanomaterial (b).
Table 7. 1: Physical properties of the NiF and NiF-ZnO achieved through simulation software using 3D nanotomoraphy.
Parameters NiF NiF/ZnO
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Absolute permeability[mD] 2864100 636392 Total flow rate (m3/s) 1.70345e-009 3.6563e-10
7.3.3 Electrochemical performance of NiF-ZnO electrode materials The macroscopic electrochemical surface reactions of the fabricated NiF-ZnO electrode material were first carried out by employing the well-known cyclic voltammetry (CV) electrochemical method in a three-electrode configuration. Figure 5A compares the CV behavior of the bare NiF and NiF/ZnO electrodes in (4.0 M KOH) electrolyte solution measured between 0-0.5 V potential range and a scan rate of 0.1 V s-1. Very low-intensity current peaks in the CV curve of the NiF were observed due to the redox process (Ni+2/Ni+3) of the nickel foam in the KOH electrolyte (Khamlich et al. 2013; Bello et al. 2013). In addition, the fabricated NiF/ZnO electrode possessed a good mirror image in response to the zero current line and a rapid response on voltage reversal at each end potential which demonstrates its pseudocapacitive characteristics. Specially, a pair of well-defined redox peaks could be found for the CV profile of NiF/ZnO electrode, distinguishable from those of electric double-layer capacitors, indicating the presence of a faradic reaction and corresponding to the rapid adsorption and desorption of the electrolyte cations (K+) at the surface sites of ZnO nanoplatelets following the reaction:
discharg e ZnO K e K ZnO (1) surface charg e surface
The mechanisms behind the efficient electrochemical reversibility of ZnO based electrode has been suggested in some earlier reports on electrodes based on ZnO nanofiber@Ni(OH) nanoflake core–shell heterostructures (Niu et al. 2015). The ZnO nanoplatelets coated micro-porous structures of the NiF template contributed to the enhancement of its high surface area (Khamlich et al. 2013). Additionally, the preserved micro-porous structure of NiF after ZnO deposition (as it was confirmed with the HRSEM) allowed an easy flow of the electrolyte ions (i.e. K+) and hence good electrochemical performance of the fabricated NiF/ZnO electrode material (Khamlich et al. 2013). Moreover, the NiF/ZnO demonstrates a single electron reversible transfer process which is consistent with the mentioned reaction (equation 1). Hence, it can be
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Chapter 7 Supercapacitor application of biogenic ZnO nanoparticles concluded that the energy storage of NiF/ZnO electrode is created on the mechanism of charge storage of ZnO in KOH electrolyte because of the quick intercalation of the metal cations (K+) in the electrode as a result of rapid redox processes. Figure 7.5B depicts the CV curves of the NiF/ZnO electrode in 4.0 M KOH electrolyte at different scan rates ranging from 0.025 to 0.100 V s-1. A pair of redox current peaks around 0.25 and 0.35 V (vs. Ag/AgCl) with nearly mirror image in respect to the zero-current line can be clearly observed at all scan rates. These observed peaks confirmed that the capacitance mainly comes from the pseudocapacitance. Furthermore, the position of the observed redox peaks shifted slightly with the increase in scan rates due to the internal resistance of the electrode (Khamlich et al. 2016). With the increase of scan rates, the current response of the fabricated NiF/ZnO electrode was increased and the shapes of the CV curves were retained, indicating excellent rate capability and high reversibility. Further electrochemical measurements were carried out using the galvanostatic charge- discharge measurements within the potential range of 0-0.5 V. The corresponding discharge curves obtained at different current densities are shown in Figure 7.5C. Considering the characteristics shapes of the displayed discharge curves, one can deduce an ideal pseudo-capacitance behavior rather than an electric double layer capacitor (EDLC). Moreover, the obtained discharge curves showed a potential plateau with a sharp response with small internal resistance drop which further confirm the characteristics of a typical pseudo-capacitance behavior previously observed in the CV results. This arising behavior could be caused by the electrochemical redox reaction of the intercalated K+ ions into the deposited ZnO nanoplatelets of the NiF/ZnO electrode (Li et al. 2015) The specific capacitance of the fabricated electrode could be estimated by using the following equation (Khamlich et al. 2016). C= I × ∆t / m × ∆V (2) Where as
C (F g-1), I (mA), ∆t (s), ∆V (V) and m (g) corresponds to the specific capacitance, charge-discharge current, discharging time, voltage drop in the discharge process and mass of the active material (i.e. ZnO).
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Based on the aforementioned eq. (2), and the discharge curves of NiF/ZnO electrode Figure 7.5C, the values of the specific capacitance were calculated and we found relatively higher values: 453, 429, 392 and 371 F g-1 at current densities of 2, 4, 6 and 14 A g-1, respectively. Gradual decrease in capacitance was observed with increasing the current densities as indicated in Figure 6. This decrease could be attributed to the kinetic loss arising from the very slow reaction kinetic of electrolyte ions in the electrode side. And by increasing the current density, the kinetic loss continues taking place which reflect on the decrease of the capacitance of the whole electrode. Also the internal resistance of the electrode could be increased by increasing the current density affecting the electrochemical performance of the electrode (Khamlich et al. 2016). Enhancing the performance of the fabricated electrode at higher current densities could be achieved by increasing the electrical conductivity and porosity of the used active material. This could be done if a suitable nanocomposite material such as ZnO-Graphene could be deposited on NiF as a supercapacitor electrode. However, the current galvanostatic charge- discharge measurements of the NiF/ZnO electrode confirmed that the biosynthesized ZnO nanoplatelets are suitable candidate for the fabrication of electrode materials for energy storage application such as supercapacitors. The electrochemical impedance spectrum was collected to gain a deep insight into the resistive and interfacial properties of the 3d NiF/ZnO electrode (Figure 7.5D). On the whole, the intrinsic ohmic resistance of the internal resistance or equivalent series resistance (ESR) of NiF/ZnO electrode material and the electrolyte was represented by the intercept of the Nyquist plots on the X-axis in the high-frequency region followed by a line that represents the diffusive resistance of the electrolyte in the electrode pores together with electron diffusion in the host material in the low-frequency region (Hao et al. 2015). The ESR value that was obtained from the inset of Figure 7.5D for the NiF/ZnO electrode was 0.5 Ω in 4 M KHO aqueous electrolyte, which indicate a lower charge-transfer resistance and faster ion diffusion of the electrode. Additionally, the nearly vertical slope of the NiF/ZnO electrode indicated that it has almost good capacitive behavior. In order to assess the utility of the fabricated electrode for commercial applications, it was imperative to check the stability over large number of charge-discharge cycles. The
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Chapter 7 Supercapacitor application of biogenic ZnO nanoparticles specific capacitance retention was found to be ~ 86% after 1000 charging discharging cycles as indicated in Figure 7.7. This shows that the fabricated electrode was electrochemically stable, durable and possess a high degree of charge-discharge reversibility. These excellent electrochemical properties and good cycling stability can be attributed to the growth orientation and cross-linked ZnO nanoplatelets structures which efficiently contributed to the agglomeration prevention of the deposited ZnO on NiF. By using nanoplatelets structures, the minimum dimension is represented by the ion diffusion length, and hence, ZnO nanoplatelets can provide a larger electrode–electrolyte contact area which leads to a shorter ion diffuse length (Khamlich et al. 2017).
Figure 7. 5: Electrochemical measurements (a) CV of bare NiF and ZnO coated NiF at scan rate of 0.1 Vs-1; (b) CV cureves of NiF- ZnO electrode at different scan rates; (c) Galvanostatic charge-discharge curves; (d) Nyquist plots of NiF/ZnO.
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Figure 7. 6: Specific capacitance variation of ruthenium oxide electrode at different scan rate.
Figure 7. 7: Cycling stability of NiF/ZnO electrode.
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CONCLUSIONS AND FUTURE PROSPECTS
Conclusions and Future perspectives
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CONCLUSIONS AND FUTURE PROSPECTS
Conclusions
Metal and metal oxide nanoparticles presents a bright area of research for the development of innovative treatment strategies in nanomedicine. However, new methods has been continuously investigated for fast, cheap and effective synthesis of metal and metal oxide nanoparticles. In the current dissertation a complete green and eco-friendly method has been adopted to successfully produce metal
oxide nanoparticles like ZnO, Fe2O3, NiO, Co3O4 and PbO using aquous extracts
of medicinally valuable plant Sageretia thea (Osbeck.).
The focus was to biosynthesize metal oxide nanoparticles and study their potential for various biomedical applications. Beside biomedical potential, the biosynthesized ZnO nanoparticles were also studied for possible application in
supercapacitors.
Significant antibacterial activities are reported with and without UV exposure to the ZnO nanoparticles. However slight increase in the bactericidal effect has been observed post UV exposure. Biogenic ZnO nanoparticles were found to be highly active against Leishmania tropica. We further conclude moderate antifungal potential and antioxidant potential for the biomimetic ZnO nanoparticles. The bioinspired zinc oxide nanoparticles were found to have effectively inhibit protein kinase enzyme while insignificant alpha amylase inhibition is reported. Furthermore, the biogenic ZnO nanoparticles were found to be biocompatible at
lower concentrations.
Our results indicate impressive biological properties of the biosynthesized Fe2O3.
Biosynthesized Fe2O3 have been found as more cytotoxic to the axenic cultures of Leishmania tropica (promastigotes / amastigotes) and brine shrimps while relatively nontoxic to the human RBC’s and macrophages at low concentrations indicating their safe nature and futuristic role in antimicrobial and antileishmanial
therapies.
Bioinspired nickel oxide nanoparticles indicated enhancement in the antibacterial activities after UV-illumination, while they were found to be effective against pathogenic fungal strains. Significant antileishmanial potential and moderate antioxidant potential is revealed by the bioinspired NiO nanoparticles.
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CONCLUSIONS AND FUTURE PROSPECTS
Furthermore, moderate enzyme inhibition activities are also reported. Bioinspired NiO were found to be less toxic to normal human blood cells like RBC and
macrophages.
Bioinspired lead oxide nanoparticles were investigated as relatively less toxic to RBC’s as compared to macrophages. Moderate antioxidant potential while insignificant alpha amylase and protein kinase enzyme inhibition potential is
reported for the bioinspired lead oxide nanoparticles.
Biogenic cobalt oxide nanoparticles indicated a varying degree of antibacterial potential against different pathogenic bacterial strains. Significant antileishmanial potential is reported. In addition, bioinspired cobalt oxide nanoparticles were found as more biocompatible to RBC’s then to macrophages. Significant DPPH radical scavenging properties of the nanoparticles are reported. Moderate
antioxidant, reducing power and enzyme inhibition activities are reported.
The fabricated electrode (ZnO-NiF) showed promising electrochemical properties with high stability and low internal resistance. A specific capacitance of 453 F g-1 at a current densities of 2 A g-1, and a specific capacitance retention of~ 86% after 1000 charging discharging cycles were achieved. These results confirm that the biogenic synthesis of zinc oxide nanoplatelets through Sageretia thea is an effective way to synthesis metal oxide nanomaterials which could be used for
application in energy storage such as supercapacitors.
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CONCLUSIONS AND FUTURE PROSPECTS
Future prospects The current study is the first of its kind and the work should not be restricted to only the targeted metal oxides and targeted plant. Other medicinal plants should be explored to biosynthesize metal oxides with interesting properties. The exact mechanism of biosynthesis is an open area of research. Further research need to be undertaken to highlight the mechanistic aspects of biosynthesis of nanoparticles. We have successfully demonstrated the potential of biogenic metal oxide nanoparticles for biomedical potential. This research should be further extended to in vivo studies. All the targeted metal oxides indicated potential antileishmanial activities. Ointments of these nanoparticles for the topical treatment of leishmaniasis can be developed. Further research on the toxicology and biocompatibility of metal oxide nanoparticles should be carried out. The immune response and especially the release of particular immune chemicals such as interleukins and cytokines should be studied in response to the biogenic nanoparticles. Significant antimicrobial potential of bioinspired metal oxide nanoparticles was demonstrated in the present study. Research should be undertaken to investigate their antimicrobial potential against biofilms. Furthermore, innovative strategies can be proposed using antibiotics and nanoparticles for further increasing their antimicrobial efficiency. Biogenic ZnO nanoparticles have indicated good electrochemical performance and stability for supercapacitor applications. ZnO nanoparticles could be deposited on carbon electrodes to further investigate their potential for super capacitor applications. Other metal oxides should also be studied for their energy storage applications.
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References
173
REFERENCES
Abamor, E. S. (2017). Antileishmanial activities of caffeic acid phenethyl ester loaded PLGA nanoparticles against Leishmania infantum promastigotes and amastigotes in vitro. Asian Pacific Journal of Tropical Medicine, 10(1), 25-34. Abdel-Hameed, E.-S. S. (2009). Total phenolic contents and free radical scavenging activity of certain Egyptian Ficus species leaf samples. Food chemistry, 114(4), 1271- 1277. Adekunle, A. S., Pillay, J., & Ozoemena, K. I. (2010). Probing the electrochemical behaviour of SWCNT–cobalt nanoparticles and their electrocatalytic activities towards the detection of nitrite at acidic and physiological pH conditions. Electrochimica Acta, 55(14), 4319-4327. Ahmad, K., Khalil, AT., & Somayya, R. (2016). Antifungal, phytotoxic and hemagglutination activity of methanolic extracts of Ocimum basilicum. Journal of Traditional Chinese Medicine, 36(6), 794-798. Ahmmad, B., Leonard, K., Islam, M. S., Kurawaki, J., Muruganandham, M., Ohkubo, T., et al. (2013). Green synthesis of mesoporous hematite (α-Fe 2 O 3) nanoparticles and their photocatalytic activity. Advanced Powder Technology, 24(1), 160-167.
Ai, L.-H., & Jiang, J. (2009). Rapid synthesis of nanocrystalline Co3O4 by a microwave- assisted combustion method. Powder Technology, 195(1), 11-14. Alarifi, S., Ali, D., & Alkahtani, S. (2017). Involvement of mitochondrial dysfunction in nanosized lead oxide induced cellular damage in human lung alveolar epithelial cells. Toxicological & Environmental Chemistry, 99(4), 680-690. Ali, A., Ambreen, S., Javed, R., Tabassum, S., ul Haq, I., & Zia, M. (2017). ZnO nanostructure fabrication in different solvents transforms physio-chemical, biological and photodegradable properties. Materials Science and Engineering: C, 74, 137-145. Ali, S., Stone, M., Peters, J., Davies, M., & Khunti, K. (2006). The prevalence of co‐ morbid depression in adults with Type 2 diabetes: a systematic review and meta‐ analysis. Diabetic Medicine, 23(11), 1165-1173. Alivisatos, A. P. (1996). Semiconductor clusters, nanocrystals, and quantum dots. Science, 271(5251), 933. Ameen, S., Akhtar, M. S., & Shin, H. S. (2015). Highly dense ZnO nanowhiskers for the low level detection of p-hydroquinone. Materials Letters, 155, 82-86.
174
REFERENCES
Amiri, A., Mohammadi, M., & Shabani, M. (2016). Synthesis and Toxicity Evaluation of Lead Oxide (PbO) Nanoparticles in Rats. Electronic Journal of Biology. Andreescu, S., Ornatska, M., Erlichman, J. S., Estevez, A., & Leiter, J. (2012). Biomedical applications of metal oxide nanoparticles. In Fine Particles in Medicine and Pharmacy (pp. 57-100): Springer. Ankamwar, B., Chaudhary, M., & Sastry, M. (2005a). Gold nanotriangles biologically synthesized using tamarind leaf extract and potential application in vapor sensing. Synthesis and Reactivity in Inorganic, Metal-Organic and Nano-Metal Chemistry, 35(1), 19-26. Ankamwar, B., Damle, C., Ahmad, A., & Sastry, M. (2005b). Biosynthesis of gold and silver nanoparticles using Emblica officinalis fruit extract, their phase transfer and transmetallation in an organic solution. Journal of nanoscience and nanotechnology, 5(10), 1665-1671. Applerot, G., Lipovsky, A., Dror, R., Perkas, N., Nitzan, Y., Lubart, R., et al. (2009). Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS‐mediated cell injury. Advanced Functional Materials, 19(6), 842-852. Arico, A. S., Bruce, P., Scrosati, B., Tarascon, J.-M., & Van Schalkwijk, W. (2005). Nanostructured materials for advanced energy conversion and storage devices. Nature materials, 4(5), 366-377. Aromal, S. A., & Philip, D. (2012). Green synthesis of gold nanoparticles using Trigonella foenum-graecum and its size-dependent catalytic activity. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 97, 1-5. Arulmozhi, K., & Mythili, N. (2013). Studies on the chemical synthesis and characterization of lead oxide nanoparticles with different organic capping agents. AIP Advances, 3(12), 122122.
Askarinejad, A., Bagherzadeh, M., & Morsali, A. (2010). Catalytic performance of Mn3O4 and Co 3 O 4 nanocrystals prepared by sonochemical method in epoxidation of styrene and cyclooctene. Applied Surface Science, 256(22), 6678-6682. Aula, S., Lakkireddy, S., Swamy, A., Kapley, A., Jamil, K., Tata, N. R., et al. (2014). Biological interactions in vitro of zinc oxide nanoparticles of different characteristics. Materials Research Express, 1(3), 035041.
175
REFERENCES
Baek, Y.-W., & An, Y.-J. (2011). Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb 2 O 3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus. Science of the total environment, 409(8), 1603-1608. Bakardjieva, S., Stengl, V., Subrt, J., Houskova, V., & Kalenda, P. (2007). Photocatalytic efficiency of iron oxides: degradation of 4-chlorophenol. Journal of Physics and Chemistry of solids, 68(5), 721-724. Baker, S., Rakshith, D., Kavitha, K. S., Santosh, P., Kavitha, H. U., Rao, Y., et al. (2013). Plants: emerging as nanofactories towards facile route in synthesis of nanoparticles. BioImpacts, 3(3), 111-117. Bala, N., Saha, S., Chakraborty, M., Maiti, M., Das, S., Basu, R., et al. (2015). Green synthesis of zinc oxide nanoparticles using Hibiscus subdariffa leaf extract: effect of temperature on synthesis, anti-bacterial activity and anti-diabetic activity. RSC Advances, 5(7), 4993-5003. Bandara, J., & Yasomanee, J. (2006). P-type oxide semiconductors as hole collectors in dye-sensitized solid-state solar cells. Semiconductor Science and Technology, 22(2), 20. Barriga, C., Maffi, S., Bicelli, L. P., & Malitesta, C. (1991). Electrochemical lithiation of Pb3O4. Journal of power sources, 34(4), 353-367. Behera, S., Patra, J., Pramanik, K., Panda, N., & Thatoi, H. (2012). Characterization and evaluation of antibacterial activities of chemically synthesized IONPs. World Journal of Nano Science and Engineering, 2(4), 196-200. Bello, A., Fabiane, M., Momodu, D. Y., Khamlich, S., Dangbegnon, J. K., & Manyala, N. (2014). Functionalized graphene foam as electrode for improved electrochemical storage. Journal of Solid State Electrochemistry, 18(9), 2359-2365. Bello, A., Fashedemi, O. O., Fabiane, M., Lekitima, J. N., Ozoemena, K. I., & Manyala, N. (2013). Microwave assisted synthesis of MnO 2 on nickel foam-graphene for electrochemical capacitor. Electrochimica Acta, 114, 48-53. Benelmekki, M., & Martinez, L. M. (2013). Magnetophoresis of iron oxide nanoparticles: a tool for synthesis monitoring and biomagnetic applications. Nanotechnology Vol. 7: Diagnostics and Therapeutics.
176
REFERENCES
Biju, V., Itoh, T., Anas, A., Sujith, A., & Ishikawa, M. (2008). Semiconductor quantum dots and metal nanoparticles: syntheses, optical properties, and biological applications. Analytical and bioanalytical chemistry, 391(7), 2469-2495. Blair, T. (1998). Lead oxide technology—past, present, and future. Journal of power sources, 73(1), 47-55. Bossi, E., Zanella, D., Gornati, R., & Bernardini, G. (2016). Cobalt oxide nanoparticles can enter inside the cells by crossing plasma membranes. Scientific reports, 6. Boyer, C., Whittaker, M. R., Bulmus, V., Liu, J., & Davis, T. P. (2010). The design and utility of polymer-stabilized iron-oxide nanoparticles for nanomedicine applications. NPG Asia Materials, 2(1), 23-30. Bregar, V. B., Lojk, J., Suštar, V., Veranič, P., & Pavlin, M. (2013). Visualization of internalization of functionalized cobalt ferrite nanoparticles and their intracellular fate. International Journal of Nanomedicine, 8, 919-931. Brigger, I., Dubernet, C., & Couvreur, P. (2002). Nanoparticles in cancer therapy and diagnosis. Advanced drug delivery reviews, 54(5), 631-651. Burello, E., & Worth, A. P. (2011). A theoretical framework for predicting the oxidative stress potential of oxide nanoparticles. Nanotoxicology, 5(2), 228-235. Burgio, L., Clark, R. J., & Firth, S. (2001). Raman spectroscopy as a means for the identification of plattnerite (PbO2), of lead pigments and of their degradation products. Analyst, 126(2), 222-227.. Chadwick, A. V., & Savin, S. L. (2010). Metal Oxide Nanoparticles. Low-Dimensional Solids, 1-76. Chandran, S. P., Chaudhary, M., Pasricha, R., Ahmad, A., & Sastry, M. (2006). Synthesis of gold nanotriangles and silver nanoparticles using Aloevera plant extract. Biotechnology progress, 22(2), 577-583. Chen, Y., Zhang, Y., & Fu, S. (2007). Synthesis and characterization of Co 3 O 4 hollow spheres. Materials Letters, 61(3), 701-705. Chtita, S., Ghamali, M., Hmamouchi, R., Elidrissi, B., Bourass, M., Larif, M., et al. (2016). Investigation of Antileishmanial Activities of Acridines Derivatives against Promastigotes and Amastigotes Form of Parasites Using Quantitative Structure Activity Relationship Analysis. Advances in Physical Chemistry, 2016.
177
REFERENCES
Chu, S., & Majumdar, A. (2012). Opportunities and challenges for a sustainable energy future. nature, 488(7411), 294-303. Chung, S.-K., Chen, C.-Y. O., & Blumberg, J. B. (2009). Flavonoid-rich fraction from Sageretia theezans leaves scavenges reactive oxygen radical species and increases the resistance of low-density lipoprotein to oxidation. Journal of medicinal food, 12(6), 1310-1315. Chung, S.-K., Kim, Y.-C., Takaya, Y., Terashima, K., & Niwa, M. (2004). Novel flavonol glycoside, 7-O-methyl mearnsitrin, from Sageretia theezans and its antioxidant effect. Journal of agricultural and food chemistry, 52(15), 4664-4668. Contreras, J., Xie, J., Chen, Y. J., Pei, H., Zhang, G., Fraser, C. L., et al. (2010). Intracellular uptake and trafficking of difluoroboron dibenzoylmethane-poly (lactic acid) nanoparticles in HeLa Cells. ACS nano, 4(5), 2735. Cornell, R. M., & Schwertmann, U. (2003). The iron oxides: structure, properties, reactions, occurrences and uses: John Wiley & Sons. Dai, G., Liu, S., Liang, Y., & Luo, T. (2013). Synthesis and enhanced
photoelectrocatalytic activity of p–n junction Co3O4/TiO2 nanotube arrays. Applied Surface Science, 264, 157-161. Darroudi, M., Sabouri, Z., Oskuee, R. K., Zak, A. K., Kargar, H., & Hamid, M. H. N. A. (2014). Green chemistry approach for the synthesis of ZnO nanopowders and their cytotoxic effects. Ceramics International, 40(3), 4827-4831. de Almeida Lopes, A. C. B., Navas-Acien, A., Zamoiski, R., Silbergeld, E. K., Carvalho, M. d. F. H., Buzzo, M. L., et al. (2015). Risk factors for lead exposure in adult population in southern Brazil. Journal of Toxicology and Environmental Health, Part A, 78(2), 92-108. de Almeida, M. C., Silva, A. C., Barral, A., & Barral Netto, M. (2000). A simple method for human peripheral blood monocyte isolation. Memorias do Instituto Oswaldo Cruz, 95(2), 221-223. De Faria, D., Venâncio Silva, S., & De Oliveira, M. (1997). Raman microspectroscopy of some iron oxides and oxyhydroxides. Journal of Raman spectroscopy, 28(11), 873- 878.
178
REFERENCES
Diallo, A., Beye, A., Doyle, T., Park, E., & Maaza, M. (2015a). Green synthesis of Co3O4 nanoparticles via Aspalathus linearis: physical properties. Green Chemistry Letters and Reviews, 8(3-4), 30-36. Diallo, A., Manikandan, E., Rajendran, V., & Maaza, M. (2016). Physical & enhanced
photocatalytic properties of green synthesized SnO2 nanoparticles via Aspalathus linearis. Journal of Alloys and Compounds, 681, 561-570. Diallo, A., Ngom, B., Park, E., & Maaza, M. (2015). Green synthesis of ZnO nanoparticles by Aspalathus linearis: Structural & optical properties. Journal of Alloys and Compounds, 646, 425-430. Dineshkumar, B., Mitra, A., & Mahadevappa, M. (2010). Antidiabetic and hypolipidemic effects of mahanimbine (carbazole alkaloid) from Murraya koenigii (rutaceae) leaves. International Journal of Phytomedicine, 2(1). Dong, C., Xiao, X., Chen, G., Guan, H., & Wang, Y. (2014). Hydrothermal synthesis of
Co3O4 nanorods on nickel foil. Materials Letters, 123, 187-190. Dong, X., Cao, Y., Wang, J., Chan-Park, M. B., Wang, L., Huang, W., et al. (2012). Hybrid structure of zinc oxide nanorods and three dimensional graphene foam for supercapacitor and electrochemical sensor applications. RSC Advances, 2(10), 4364- 4369. du Plessis, A., le Roux, S. G., & Guelpa, A. (2016). The CT Scanner Facility at Stellenbosch University: An open access X-ray computed tomography laboratory. Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms, 384, 42-49. Du, J., & Gebicki, J. M. (2004). Proteins are major initial cell targets of hydroxyl free radicals. The international journal of biochemistry & cell biology, 36(11), 2334-2343.. Durán, N., Marcato, P. D., Durán, M., Yadav, A., Gade, A., & Rai, M. (2011). Mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides, bacteria, fungi, and plants. Applied Microbiology and Biotechnology, 90(5), 1609-1624. El-Damrawi, G., & Mansour, E. (2005). Electrical properties of lead borosilicate glasses. Physica B: Condensed Matter, 364(1), 190-198.
179
REFERENCES
Ellmer, K. (2012). Past achievements and future challenges in the development of optically transparent electrodes. Nature Photonics, 6(12), 809-817. Ezhilarasi, A. A., Vijaya, J. J., Kaviyarasu, K., Maaza, M., Ayeshamariam, A., & Kennedy, L. J. (2016). Green synthesis of NiO nanoparticles using Moringa oleifera extract and their biomedical applications: Cytotoxicity effect of nanoparticles against HT-29 cancer cells. Journal of Photochemistry and Photobiology B: Biology, 164, 352-360. Fan, F., Feng, Y., Tang, P., & Li, D. (2015). Facile synthesis and photocatalytic performance of ZnO nanoparticles self-assembled spherical aggregates. Materials Letters, 158, 290-294. Farhadi, S., Pourzare, K., & Sadeghinejad, S. (2013). Simple preparation of ferromagnetic Co3O4 nanoparticles by thermal dissociation of the [CoII (NH3) 6](NO3) 2 complex at low temperature. Journal of Nanostructure in Chemistry, 3(1), 16. Fatima, H., Khan, K., Zia, M., Ur-Rehman, T., Mirza, B., & Haq, I.-u. (2015). Extraction optimization of medicinally important metabolites from Datura innoxia Mill.: an in vitro biological and phytochemical investigation. BMC complementary and alternative medicine, 15(1), 376. Fernández‐García, M., & Rodriguez, J. A. (2011). Metal oxide nanoparticles. Encyclopedia of Inorganic and Bioinorganic Chemistry. Fuku, X., Kaviyarasu, K., Matinise, N., & Maaza, M. (2016). Punicalagin Green Functionalized Cu/Cu2O/ZnO/CuO Nanocomposite for Potential Electrochemical Transducer and Catalyst. Nanoscale Research Letters, 11(1), 386. Gao, P. X., Ding, Y., Mai, W., Hughes, W. L., Lao, C., & Wang, Z. L. (2005). Conversion of zinc oxide nanobelts into superlattice-structured nanohelices. Science, 309(5741), 1700-1704. Gasmalla, H. B., Idris, A. M., Shinger, M. I., Qin, D., Shan, D., & Lu, X. (2016). Balanites aegyptiaca Oil Synthesized Iron Oxide Nanoparticles: Characterization and Antibacterial Activity. Journal of Biomaterials and Nanobiotechnology, 7(03), 154. Gatselou, V., Christodouleas, D. C., Kouloumpis, A., Gournis, D., & Giokas, D. L. (2016). Determination of phenolic compounds using spectral and color transitions of rhodium nanoparticles. Analytica chimica acta, 932, 80-87.
180
REFERENCES
Ghaffari-Moghaddam, M., & Hadi-Dabanlou, R. (2014). Plant mediated green synthesis and antibacterial activity of silver nanoparticles using Crataegus douglasii fruit extract. Journal of Industrial and Engineering Chemistry, 20(2), 739-744. Ghasemi, S., Karami, H., Mousavi, M. F., & Shamsipur, M. (2005). Synthesis and morphological investigation of pulsed current formed nano-structured lead dioxide. Electrochemistry communications, 7(12), 1257-1264. Ghasemi, S., Mousavi, M. F., Karami, H., Shamsipur, M., & Kazemi, S. (2006). Energy storage capacity investigation of pulsed current formed nano-structured lead dioxide. Electrochimica acta, 52(4), 1596-1602. Ghasemi, S., Mousavi, M., Shamsipur, M., & Karami, H. (2008). Sonochemical-assisted synthesis of nano-structured lead dioxide. Ultrasonics sonochemistry, 15(4), 448-455. Gong, N., Shao, K., Feng, W., Lin, Z., Liang, C., & Sun, Y. (2011). Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris. Chemosphere, 83(4), 510-516. Gulino, A., Dapporto, P., Rossi, P., & Fragalà, I. (2003). A novel self-generating liquid MOCVD precursor for Co3O4 thin films. Chemistry of materials, 15(20), 3748-3752. Gupta, A. K., & Gupta, M. (2005). Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials, 26(18), 3995-4021. Hadighi, R., Mohebali, M., Boucher, P., Hajjaran, H., Khamesipour, A., & Ouellette, M. (2006). Unresponsiveness to Glucantime treatment in Iranian cutaneous leishmaniasis due to drug-resistant Leishmania tropica parasites. PLoS Med, 3(5), e162. Hall, P. J., Mirzaeian, M., Fletcher, S. I., Sillars, F. B., Rennie, A. J., Shitta-Bey, G. O., et al. (2010). Energy storage in electrochemical capacitors: designing functional materials to improve performance. Energy & Environmental Science, 3(9), 1238-1251. Hao, J., Liao, Y., Zhong, Y., Shu, D., He, C., Guo, S., et al. (2015). Three-dimensional graphene layers prepared by a gas-foaming method for supercapacitor applications. Carbon, 94, 879-887. Hao, R., Xing, R., Xu, Z., Hou, Y., Gao, S., & Sun, S. (2010). Synthesis, functionalization, and biomedical applications of multifunctional magnetic nanoparticles. Advanced Materials, 22(25), 2729-2742.
181
REFERENCES
Hassan, H. F. H., Mansour, A. M., Abo‐Youssef, A. M. H., Elsadek, B. E., & Messiha, B. A. S. (2016). Zinc oxide nanoparticles as a novel anticancer approach; in vitro and in vivo evidences. Clinical and Experimental Pharmacology and Physiology. He, L., Liu, Y., Mustapha, A., & Lin, M. (2011). Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiological research, 166(3), 207-215. Helen, S. M., & Rani, M. H. E. (2015). Characterization and antimicrobial study of nickel nanoparticles synthesized from dioscorea (Elephant Yam) by green route. International Journal of Science and Research, 4(11), 216-219. Hoag, G. E., Collins, J. B., Holcomb, J. L., Hoag, J. R., Nadagouda, M. N., & Varma, R. S. (2009). Degradation of bromothymol blue by ‘greener’nano-scale zero-valent iron synthesized using tea polyphenols. Journal of Materials Chemistry, 19(45), 8671- 8677. Höss, S., Fritzsche, A., Meyer, C., Bosch, J., Meckenstock, R. U., & Totsche, K.U. (2014). Size-and composition-dependent toxicity of synthetic and soil-derived Fe oxide colloids for the nematode Caenorhabditis elegans. Environmental science & technology, 49(1), 544-552. Huang, J., Li, Q., Sun, D., Lu, Y., Su, Y., Yang, X., et al. (2007). Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf. Nanotechnology, 18(10), 105104. Huang, L., Weng, X., Chen, Z., Megharaj, M., & Naidu, R. (2014). Synthesis of iron- based nanoparticles using oolong tea extract for the degradation of malachite green. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 117, 801- 804. Huang, Z., Zhang, Z., Qi, X., Ren, X., Xu, G., Wan, P., et al. (2016). Wall-like hierarchical metal oxide nanosheet arrays grown on carbon cloth for excellent supercapacitor electrodes. Nanoscale, 8(27), 13273-13279. Hyun, T. K., Song, S. C., Song, C.-K., & Kim, J.-S. (2015). Nutritional and nutraceutical characteristics of Sageretia theezans fruit. journal of food and drug analysis, 23(4), 742-749.
182
REFERENCES
Iravani, S. (2011). Green synthesis of metal nanoparticles using plants. Green Chemistry, 13(10), 2638-2650. Islam, M. S., Kusumoto, Y., & Abdulla-Al-Mamun, M. (2012). Novel rose-type magnetic (Fe 3 O 4, γ-Fe 2 O 3 and α-Fe 2 O 3) nanoplates synthesized by simple hydrothermal decomposition. Materials Letters, 66(1), 165-167.
Ismail, E., Diallo, A., Khenfouch, M., Dhlamini, S., & Maaza, M. (2016). RuO2 nanoparticles by a novel green process via Aspalathus linearis natural extract & their water splitting response. Journal of Alloys and Compounds, 662, 283-289.. Ismail, E., Khamlich, S., Dhlamini, M., & Maaza, M. (2016). Green biosynthesis of ruthenium oxide nanoparticles on nickel foam as electrode material for supercapacitor applications. RSC Advances, 6(90), 86843-86850. Jafri, L., Saleem, S., Ullah, N., & Mirza, B. (2014). In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch. Arabian Journal of Chemistry. Jakubek, T., Kaspera, W., Legutko, P., Stelmachowski, P., & Kotarba, A. (2015). Surface versus bulk alkali promotion of cobalt-oxide catalyst in soot oxidation. Catalysis Communications, 71, 37-41. Javed, R., Usman, M., Tabassum, S., & Zia, M. (2016). Effect of capping agents: Structural, optical and biological properties of ZnO nanoparticles. Applied Surface Science, 386, 319-326. Jebali, A., & Kazemi, B. (2013). Nano-based antileishmanial agents: a toxicological study on nanoparticles for future treatment of cutaneous leishmaniasis. Toxicology in Vitro, 27(6), 1896-1904. Jiang, W., Mashayekhi, H., & Xing, B. (2009). Bacterial toxicity comparison between nano-and micro-scaled oxide particles. Environmental pollution, 157(5), 1619-1625. Joerger, R., Klaus, T., & Granqvist, C. (2000). Biologically Produced Silver–Carbon Composite Materials for Optically Functional Thin‐Film Coatings. Advanced Materials, 12(6), 407-409. Jolivet, J.-P., Cassaignon, S., Chanéac, C., Chiche, D., Durupthy, O., & Portehault, D. (2010). Design of metal oxide nanoparticles: Control of size, shape, crystalline
183
REFERENCES
structure and functionalization by aqueous chemistry. Comptes Rendus Chimie, 13(1), 40-51. Jones, N., Ray, B., Ranjit, K. T., & Manna, A. C. (2008). Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS microbiology letters, 279(1), 71-76. Joshi, P., Chakraborti, S., Chakrabarti, P., Haranath, D., Shanker, V., Ansari, Z., et al. (2009). Role of surface adsorbed anionic species in antibacterial activity of ZnO quantum dots against Escherichia coli. Journal of nanoscience and nanotechnology, 9(11), 6427-6433. Jubb, A. M., & Allen, H. C. (2010). Vibrational spectroscopic characterization of hematite, maghemite, and magnetite thin films produced by vapor deposition. ACS Applied Materials & Interfaces, 2(10), 2804-2812. Kalpana, D., Omkumar, K. S., Kumar, S. S., & Renganathan, N. G. (2006). A novel high power symmetric ZnO/carbon aerogel composite electrode for electrochemical supercapacitor. Electrochimica Acta, 52 1309–1315. Kalusniak, S., Sadofev, S., Puls, J., & Henneberger, F. (2009). ZnCdO/ZnO–a new heterosystem for green‐wavelength semiconductor lasing. Laser & Photonics Reviews, 3(3), 233-242. Kaminsky, F. (2012). Mineralogy of the lower mantle: A review of ‘super-deep’mineral inclusions in diamond. Earth-Science Reviews, 110(1), 127-147. Kandalkar, S., Gunjakar, J., & Lokhande, C. (2008). Preparation of cobalt oxide thin films and its use in supercapacitor application. Applied Surface Science, 254(17), 5540- 5544. Kar, A., & Ray, A. K. (2014). Synthesis of nano-spherical nickel by templating hibiscus flower petals. Journal of Nanoscience and Nanotechnology, 2(2), 17-20. Karami, H., Karimi, M. A., Haghdar, S., Sadeghi, A., Mir-Ghasemi, R., & Mahdi-Khani, S. (2008). Synthesis of lead oxide nanoparticles by sonochemical method and its application as cathode and anode of lead-acid batteries. Materials Chemistry and Physics, 108(2), 337-344.
184
REFERENCES
Kaviyarasu, K., Raja, A., & Devarajan, P. A. (2013). Structural elucidation and spectral
characterizations of Co3O4 nanoflakes. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 114, 586-591.. Khalil, A. T., Khan, I., Ahmad, K., Khan, Y. A., Khan, J., & Shinwari, Z. K. (2014). Antibacterial activity of honey in north-west Pakistan against select human pathogens. Journal of Traditional Chinese Medicine, 34(1), 86-89. Khamlich, S., Abdullaeva, Z., Kennedy, J., & Maaza, M. (2017). High performance symmetric supercapacitor based on zinc hydroxychloride nanosheets and 3D graphene-nickel foam composite. Applied Surface Science, 405, 329-336. Khamlich, S., Bello, A., Fabiane, M., Ngom, B., & Manyala, N. (2013). Hydrothermal synthesis of simonkolleite microplatelets on nickel foam-graphene for electrochemical supercapacitors. Journal of Solid State Electrochemistry, 17(11), 2879-2886. Khamlich, S., Mokrani, T., Dhlamini, M., Mothudi, B., & Maaza, M. (2016). Microwave- assisted synthesis of simonkolleite nanoplatelets on nickel foam–graphene with enhanced surface area for high-performance supercapacitors. Journal of colloid and interface science, 461, 154-161. Khan, I., AbdElsalam, N. M., Fouad, H., Tariq, A., Ullah, R., & Adnan, M. (2014). Application of ethnobotanical indices on the use of traditional medicines against common diseases. Evidence-Based Complementary and Alternative Medicine, 2014. Khan, I., Ahmad, K., Khalil, A. T., Khan, J., Khan, Y. A., Saqib, M. S., et al. (2015). Evaluation of antileishmanial, antibacterial and brine shrimp cytotoxic potential of crude methanolic extract of Herb Ocimum basilicum (Lamiacea). Journal of Traditional Chinese Medicine, 35(3), 316-322. Khan, S., Ansari, A. A., Khan, A. A., Ahmad, R., Al-Obaid, O., & Al-Kattan, W. (2015). In vitro evaluation of anticancer and antibacterial activities of cobalt oxide nanoparticles. JBIC Journal of Biological Inorganic Chemistry, 20(8), 1319-1326. Kharissova, O. V., Dias, H. R., Kharisov, B. I., Pérez, B. O., & Pérez, V. M. J. (2013). The greener synthesis of nanoparticles. Trends in biotechnology, 31(4), 240-248. Khashan, K. S., Sulaiman, G. M., Ameer, F. A. K. A., & Napolitano, G. (2016). Synthesis, characterization and antibacterial activity of colloidal NiO nanoparticles. Pakistan journal of pharmaceutical sciences, 1(29), 541-546.
185
REFERENCES
Khashan, K. S., Sulaiman, G. M., Hamad, A. H., Abdulameer, F. A., & Hadi, A. (2017). Generation of NiO nanoparticles via pulsed laser ablation in deionised water and their antibacterial activity. Applied Physics A, 123(3), 190. Khayatian, G., Hassanpoor, S., Azar, A. R., & Mohebbi, S. (2013). Spectrophotometric determination of trace amounts of uranium (VI) using modified magnetic iron oxide nanoparticles in environmental and biological samples. Journal of the Brazilian Chemical Society, 24(11), 1808-1817. Khenfouch, M., Minnis Ndimba, R., Diallo, A., Khamlich, S., Hamzah, M., Dhlamini, M., et al. (2016). Artemisia herba-alba Asso eco-friendly reduced few-layered graphene oxide nanosheets: structural investigations and physical properties. Green Chemistry Letters and Reviews, 9(2), 122-131. Ko, G.-A., Son, M., & Cho, S. K. (2016). Comparative evaluation of free radical scavenging activities and cytotoxicity of various solvent fractions of Sandong Sageretia thea (Osbeck) MC Johnst. branches. Food Science and Biotechnology, 25(6), 1683-1691. Konstantinov, K., Ng, S., Wang, J., Wang, G., Wexler, D., & Liu, H. (2006). Nanostructured PbO materials obtained in situ by spray solution technique for Li-ion batteries. Journal of power sources, 159(1), 241-244. Ksapabutr, B., Nimnuan, P., & Panapoy, M. (2015). Dense and uniform NiO thin films fabricated by one-step electrostatic spray deposition. Materials Letters, 153, 24-28. Kundu, M., & Liu, L. (2015). Binder-free electrodes consisting of porous NiO nanofibers directly electrospun on nickel foam for high-rate supercapacitors. Materials Letters, 144, 114-118. Kuppusamy, P., Yusoff, M. M., Maniam, G. P., & Govindan, N. (2016). Biosynthesis of metallic nanoparticles using plant derivatives and their new avenues in pharmacological applications–An updated report. Saudi Pharmaceutical Journal, 24(4), 473-484. Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Vander Elst, L., et al. (2008). Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chemical reviews, 108(6), 2064-2110.
186
REFERENCES
Lee, J. H., Schneider, B., Jordan, E. K., Liu, W., & Frank, J. A. (2008). Synthesis of complexable fluorescent superparamagnetic iron oxide nanoparticles (FL SPIONs) and cell labeling for clinical application. Advanced Materials, 20(13), 2512-2516. Légaré, D., & Ouellette, M. (2017). Drug Resistance in Leishmania. In Handbook of Antimicrobial Resistance (pp. 313-341): Springer. Li, S., & Malmstadt, N. (2013). Deformation and poration of lipid bilayer membranes by cationic nanoparticles. Soft Matter, 9(20), 4969-4976. Li, W., Xin, L., Xu, X., Liu, Q., Zhang, M., Ding, S., et al. (2015). Facile synthesis of
three-dimensional structured carbon fiber-NiCo2O4-Ni (OH) 2 high-performance electrode for pseudocapacitors. Scientific reports, 5.
Li, W.-Y., Xu, L.-N., & Chen, J. (2005). Co3O4 nanomaterials in lithium‐ion batteries and gas sensors. Advanced Functional Materials, 15(5), 851-857. Li, X.-q., Elliott, D. W., & Zhang, W.-x. (2006). Zero-valent iron nanoparticles for abatement of environmental pollutants: materials and engineering aspects. Critical reviews in solid state and materials sciences, 31(4), 111-122. Li, Y., Zhang, W., Niu, J., & Chen, Y. (2012). Mechanism of photogenerated reactive oxygen species and correlation with the antibacterial properties of engineered metal- oxide nanoparticles. Acs Nano, 6(6), 5164-5173. Lian, S., Wang, E., Gao, L., & Xu, L. (2007). Fabrication of single-crystalline Co 3 O 4 nanorods via a low-temperature solvothermal process. Materials Letters, 61(18), 3893- 3896. Liang, X., & Zhao, L. (2012). Room-temperature synthesis of air-stable cobalt nanoparticles and their highly efficient adsorption ability for Congo red. RSC Advances, 2(13), 5485-5487. Limor, H.-A., James, K. C., Rafi, K., Patrice, N. M., Oded, M., Jessica, P., et al. (2011). Predictive toxicology of cobalt nanoparticles and ions: comparative in-vitro study of different cellular models using methods of knowledge discovery from data. Toxicological Sciences, kfr124. Lin, J., & Alexander-Katz, A. (2013). Cell membranes open “doors” for cationic nanoparticles/biomolecules: insights into uptake kinetics. ACS nano, 7(12), 10799- 10808.
187
REFERENCES
Lipovsky, A., Nitzan, Y., Gedanken, A., & Lubart, R. (2011). Antifungal activity of ZnO nanoparticles—the role of ROS mediated cell injury. Nanotechnology, 22(10), 105101. Liu, J., Aksay, I. A., Kou, R., & Wang, D. (2016). Mesoporous metal oxide graphene nanocomposite materials. Google Patents. Liu, Y., He, L., Mustapha, A., Li, H., Hu, Z., & Lin, M. (2009). Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157: H7. Journal of applied microbiology, 107(4), 1193-1201. Lokesh, K., Kavitha, G., Manikandan, E., Mani, G. K., Kaviyarasu, K., Rayappan, J. B. B., et al. (2016). Effective ammonia detection using n-ZnO/p-NiO heterostructured nanofibers. IEEE Sensors Journal, 16(8), 2477-2483. Lou, X. W., Deng, D., Lee, J. Y., Feng, J., & Archer, L. A. (2008). Self‐supported formation of needlelike Co3O4 nanotubes and their application as lithium‐ion battery electrodes. Advanced Materials, 20(2), 258-262. Lyons, S. W., Xiong, Y., Ward, T. L., Kodas, T. T., & Pratsinis, S. E. (1992). Role of particle evaporation during synthesis of lead oxide by aerosol decomposition. Journal of materials research, 7(12), 3333-3341. Maaza, M., Ngom, B., Achouri, M., & Manikandan, K. (2015). Functional nanostructured oxides. Vacuum, 114, 172-187. Madhu, G., Bose, V. C., Aiswaryaraj, A., Maniammal, K., & Biju, V. (2013). Defect dependent antioxidant activity of nanostructured nickel oxide synthesized through a novel chemical method. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 429, 44-50. Mahdavi, M., Namvar, F., Ahmad, M. B., & Mohamad, R. (2013). Green biosynthesis and characterization of magnetic iron oxide (Fe3O4) nanoparticles using seaweed (Sargassum muticum) aqueous extract. Molecules, 18(5), 5954-5964. Maire, E., & Withers, P. (2014). Quantitative X-ray tomography. International materials reviews, 59(1), 1-43. Maiti, D., Manju, U., Velaga, S., & Devi, P. S. (2013). Phase evolution and growth of iron oxide nanoparticles: Effect of hydrazine addition during sonication. Crystal Growth & Design, 13(8), 3637-3644.
188
REFERENCES
Makhlouf, M. T., Abu-Zied, B., & Mansoure, T. (2013). Effect of calcination temperature
on the H 2 O 2 decomposition activity of nano-crystalline Co3O4 prepared by combustion method. Applied Surface Science, 274, 45-52.. Malagoli, D. (2007). A full-length protocol to test hemolytic activity of palytoxin on human erythrocytes. Invertebrate Survival Journal, 4(2), 92-94. Mani, G. K., & Rayappan, J. B. B. (2015). Facile synthesis of ZnO nanostructures by spray pyrolysis technique and its application as highly selective H 2 S sensor. Materials Letters, 158, 373-376. Manikandan, E., Kennedy, J., Kavitha, G., Kaviyarasu, K., Maaza, M., Panigrahi, B., et al. (2015). Hybrid nanostructured thin-films by PLD for enhanced field emission performance for radiation micro-nano dosimetry applications. Journal of Alloys and Compounds, 647, 141-145. Matinise, N., Fuku, X., Kaviyarasu, K., Mayedwa, N., & Maaza, M. (2017). ZnO nanoparticles via Moringa oleifera green synthesis: Physical properties & mechanism of formation. Applied Surface Science, 406, 339-347. Miller, J. R., & Simon, P. (2008). Electrochemical capacitors for energy management. Science Magazine, 321(5889), 651-652. Mironova-Ulmane, N., Kuzmin, A., Steins, I., Grabis, J., Sildos, I., & Pärs, M. Raman scattering in nanosized nickel oxide NiO. In Journal of Physics: Conference Series, 2007 (Vol. 93, pp. 012039): IOP Publishing. Mirzaei, H., & Darroudi, M. (2017). Zinc oxide nanoparticles: Biological synthesis and biomedical applications. Ceramics International, 43(1), 907-914. Mu, Y., Jia, D., He, Y., Miao, Y., & Wu, H.-L. (2011). Nano nickel oxide modified non- enzymatic glucose sensors with enhanced sensitivity through an electrochemical process strategy at high potential. Biosensors and Bioelectronics, 26(6), 2948-2952. Murad, W., Ahmad, A., Gilani, S. A., & Khan, M. A. (2011). Indigenous knowledge and folk use of medicinal plants by the tribal communities of Hazar Nao Forest, Malakand District, North Pakistan. Journal of Medicinal Plants Research, 5(7), 1072-1086. Murray, C. B., Kagan, C., & Bawendi, M. (2000). Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies. Annual Review of Materials Science, 30(1), 545-610.
189
REFERENCES
Muthukumar, H., & Matheswaran, M. (2015). Amaranthus spinosus leaf extract mediated FeO nanoparticles: physicochemical traits, photocatalytic and antioxidant activity. ACS Sustainable Chemistry & Engineering, 3(12), 3149-3156. Nadhman, A., Khan, M. I., Nazir, S., Khan, M., Shahnaz, G., Raza, A., et al. (2016a). Annihilation of Leishmania by daylight responsive ZnO nanoparticles: a temporal relationship of reactive oxygen species-induced lipid and protein oxidation. International Journal of Nanomedicine, 11, 2451. Nadhman, A., Nazir, S., Khan, M. I., Arooj, S., Bakhtiar, M., Shahnaz, G., et al. (2014). PEGylated silver doped zinc oxide nanoparticles as novel photosensitizers for photodynamic therapy against Leishmania. Free Radical Biology and Medicine, 77, 230-238. Nadhman, A., Sirajuddin, M., Nazir, S., & Yasinzai, M. (2016b). Photo-induced Leishmania DNA degradation by silver-doped zinc oxide nanoparticle: an in-vitro approach. IET nanobiotechnology, 10(3), 129-133. Naoi, K., Kisu, K., Iwama, E., Nakashima, S., Sakai, Y., Orikasa, Y., et al. (2016). Ultrafast charge–discharge characteristics of a nanosized core–shell structured LiFePO 4 material for hybrid supercapacitor applications. Energy & Environmental Science, 9(6), 2143-2151. Naseem, T., & Farrukh, M. A. (2015). Antibacterial activity of green synthesis of iron nanoparticles using Lawsonia inermis and Gardenia jasminoides leaves extract. Journal of Chemistry, 2015. Nasseri, M., Ahrari, F., & Zakerinasab, B. (2016). A green biosynthesis of NiO nanoparticles using aqueous extract of Tamarix serotina and their characterization and application. Applied Organometallic Chemistry, 30(12), 978-984. Nel, A., Xia, T., Mädler, L., & Li, N. (2006). Toxic potential of materials at the nanolevel. science, 311(5761), 622-627. Nidhin, M., Indumathy, R., Sreeram, K., & Nair, B. U. (2008). Synthesis of iron oxide nanoparticles of narrow size distribution on polysaccharide templates. Bulletin of Materials Science, 31(1). Niu, H., Zhou, D., Yang, X., Li, X., Wang, Q., & Qu, F. (2015). Towards three- dimensional hierarchical ZnO nanofiber@ Ni (OH) 2 nanoflake core–shell
190
REFERENCES
heterostructures for high-performance asymmetric supercapacitors. Journal of Materials Chemistry A, 3(36), 18413-18421. Nocera, D. G. (2009). Living healthy on a dying planet. Chemical Society Reviews, 38(1), 13-15. Nyangiwe, N. N., Khenfouch, M., Thema, F. T., Nukwa, K., Kotsedi, L., & Maaza, M. (2015). Free-green synthesis and dynamics of reduced graphene sheets via sun light irradiation. Graphene, 4(03), 54. Odobel, F., Le Pleux, L., Pellegrin, Y., & Blart, E. (2010). New photovoltaic devices based on the sensitization of p-type semiconductors: challenges and opportunities. Accounts of chemical research, 43(8), 1063-1071. Oliveira, M. M., Ugarte, D., Zanchet, D., & Zarbin, A. J. (2005). Influence of synthetic parameters on the size, structure, and stability of dodecanethiol-stabilized silver nanoparticles. Journal of colloid and interface science, 292(2), 429-435. Ortega, R., Bresson, C., Darolles, C., Gautier, C., Roudeau, S., Perrin, L., et al. (2014). Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells. Particle and fibre toxicology, 11(1), 14. Oskam, G. (2006). Metal oxide nanoparticles: synthesis, characterization and application. Journal of Sol-Gel Science and Technology, 37(3), 161-164. Ovais, M., Khalil, A. T., Raza, A., Khan, M. A., Ahmad, I., Islam, N. U., et al. (2016). Green synthesis of silver nanoparticles via plant extracts: beginning a new era in cancer theranostics. Nanomedicine, 12(23), 3157-3177. Ovais, M., Raza, A., Naz, S., Islam, N. U., Khalil, A. T., Ali, S., et al. (2017). Current state and prospects of the phytosynthesized colloidal gold nanoparticles and their applications in cancer theranostics. Applied microbiology and biotechnology, 101(9), 3551-3565. Özgür, Ü., Alivov, Y. I., Liu, C., Teke, A., Reshchikov, M., Doğan, S., et al. (2005). A comprehensive review of ZnO materials and devices. Journal of applied physics, 98(4), 11. Padmavathy, N., & Vijayaraghavan, R. (2008). Enhanced bioactivity of ZnO nanoparticles—an antimicrobial study. Science and technology of advanced materials, 9(3), 035004.
191
REFERENCES
Pal, S. (2014). Antimicrobial activity of iron oxide nanoparticles. Doctoral dissertion Pandian, C. J., Palanivel, R., & Dhananasekaran, S. (2015). Green synthesis of nickel nanoparticles using Ocimum sanctum and their application in dye and pollutant adsorption. Chinese journal of Chemical engineering, 23(8), 1307-1315. Panigrahi, S., Kundu, S., Ghosh, S., Nath, S., & Pal, T. (2004). General method of synthesis for metal nanoparticles. Journal of Nanoparticle Research, 6(4), 411-414. Park, B., & Cairns, E. J. (2011). Electrochemical performance of TiO 2 and NiO as fuel cell electrode additives. Electrochemistry Communications, 13(1), 75-77. Park, J. C., Hur, J. M., Park, J. G., Hatano, T., Yoshida, T., Miyashiro, H., et al. (2002). Inhibitory effects of Korean medicinal plants and camelliatannin H from Camellia japonica on human immunodeficiency virus type 1 protease. Phytotherapy Research, 16(5), 422-426. Park, Y., Hong, Y., Weyers, A., Kim, Y., & Linhardt, R. (2011). Polysaccharides and phytochemicals: a natural reservoir for the green synthesis of gold and silver nanoparticles. IET nanobiotechnology, 5(3), 69-78. Parveen, K., Banse, V., Ledwani, L., Sharma, N. N., Gaol, F. L., & Akhtar, J. Green synthesis of nanoparticles: Their advantages and disadvantages. In AIP Conference Proceedings, 2016 (Vol. 1724, pp. 020048): AIP Publishing Paull, R., Wolfe, J., Peter, H., & Sinkula, M. (2003). Investing in nanotechnology. Nature biotechnology, 21(10), 1144. Pejova, B., Kocareva, T., Najdoski, M., & Grozdanov, I. (2000). A solution growth route to nanocrystalline nickel oxide thin films. Applied Surface Science, 165(4), 271-278. Perry, D. L. (2010). Lead Oxides: Synthesis and Applications. Transparent Electronics: From Synthesis to Applications, 89-101. Prach, M., Stone, V., & Proudfoot, L. (2013). Zinc oxide nanoparticles and monocytes: impact of size, charge and solubility on activation status. Toxicology and applied pharmacology, 266(1), 19-26. Premanathan, M., Karthikeyan, K., Jeyasubramanian, K., & Manivannan, G. (2011). Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation. Nanomedicine: Nanotechnology, Biology and Medicine, 7(2), 184-192.
192
REFERENCES
Prucek, R., Tuček, J., Kilianová, M., Panáček, A., Kvítek, L., Filip, J., et al. (2011). The targeted antibacterial and antifungal properties of magnetic nanocomposite of iron oxide and silver nanoparticles. Biomaterials, 32(21), 4704-4713. Raghupathi, K. R., Koodali, R. T., & Manna, A. C. (2011). Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir, 27(7), 4020-4028. Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y., & Tyagi, S. (2006). Stochastic mRNA synthesis in mammalian cells. PLoS Biol, 4(10), e309. Raman, V., Suresh, S., Savarimuthu, P. A., Raman, T., Tsatsakis, A. M., Golokhvast, K.
S., et al. (2016). Synthesis of Co3O4 nanoparticles with block and sphere morphology, and investigation into the influence of morphology on biological toxicity. Experimental and therapeutic medicine, 11(2), 553-560. Ramani, M., Ponnusamy, S., Muthamizhchelvan, C., & Marsili, E. (2014). Amino acid- mediated synthesis of zinc oxide nanostructures and evaluation of their facet- dependent antimicrobial activity. Colloids and Surfaces B: Biointerfaces, 117, 233- 239. Rao, C., Vivekchand, S., Biswas, K., & Govindaraj, A. (2007). Synthesis of inorganic nanomaterials. Dalton Transactions(34), 3728-3749. Rasheed, R., & Meera, V. (2016). Synthesis of Iron Oxide Nanoparticles Coated Sand by Biological Method and Chemical Method. Procedia Technology, 24, 210-216. Razzaque, S., Hussain, S. Z., Hussain, I., & Tan, B. (2016). Design and utility of metal/metal oxide nanoparticles mediated by thioether end-functionalized polymeric ligands. Polymers, 8(4), 156.
Ren, L., Wang, P., Han, Y., Hu, C., & Wei, B. (2009). Synthesis of CoC2O4· 2H2O
nanorods and their thermal decomposition to Co3O4 nanoparticles. Chemical Physics Letters, 476(1), 78-83. Ruíz-Baltazar, A., Esparza, R., Rosas, G., & Pérez, R. (2015). Effect of the surfactant on the growth and oxidation of iron nanoparticles. Journal of Nanomaterials, 16(1), 202. Rusdi, R., Rahman, A. A., Mohamed, N. S., Kamarudin, N., & Kamarulzaman, N. (2011). Preparation and band gap energies of ZnO nanotubes, nanorods and spherical nanostructures. Powder Technology, 210(1), 18-22.
193
REFERENCES
Saikia, J. P., Paul, S., Konwar, B. K., & Samdarshi, S. K. (2010). Nickel oxide nanoparticles: a novel antioxidant. Colloids and Surfaces B: Biointerfaces, 78(1), 146- 148. Saini, R., Saini, S., & Sharma, S. (2010). Nanotechnology: the future medicine. Journal of cutaneous and aesthetic surgery, 3(1), 32.
Salavati-Niasari, M., Mir, N., & Davar, F. (2009). Synthesis and characterization of Co3O4 nanorods by thermal decomposition of cobalt oxalate. Journal of Physics and Chemistry of Solids, 70(5), 847-852. Salvadori, M. R., Ando, R. A., Nascimento, C. A. O., & Corrêa, B. (2015). Extra and intracellular synthesis of nickel oxide nanoparticles mediated by dead fungal biomass. PloS one, 10(6), e0129799. Salvadori, M. R., Nascimento, C. A. O., & Corrêa, B. (2014). Nickel oxide nanoparticles film produced by dead biomass of filamentous fungus. Scientific reports, 4, 6404. Samsudin, N. A., Zainal, Z., Sook, H.-N. L. Y. S., & Changf, K. (2016). Titania nanotubes synthesised via the electrochemical anodisation method: synthesis and supercapacitor applications. Pertanika Journal of Scholarly Research Reviews, 2(1), 107-128. Sarwar, S., Chakraborti, S., Bera, S., Sheikh, I. A., Hoque, K. M., & Chakrabarti, P. (2016). The antimicrobial activity of ZnO nanoparticles against Vibrio cholerae: Variation in response depends on biotype. Nanomedicine: Nanotechnology, Biology and Medicine, 12(6), 1499-1509. Sasi, B., Gopchandran, K., Manoj, P., Koshy, P., Rao, P. P., & Vaidyan, V. (2002). Preparation of transparent and semiconducting NiO films. Vacuum, 68(2), 149-154. Sawai, J. (2003). Quantitative evaluation of antibacterial activities of metallic oxide powders (ZnO, MgO and CaO) by conductimetric assay. Journal of microbiological methods, 54(2), 177-182. Shah, A., Marwat, S. K., Gohar, F., Khan, A., Bhatti, K. H., Amin, M., et al. (2013). Ethnobotanical study of medicinal plants of semi-tribal area of Makerwal & Gulla Khel (lying between Khyber Pakhtunkhwa and Punjab Provinces), Pakistan. Shahwan, T., Sirriah, S. A., Nairat, M., Boyacı, E., Eroğlu, A. E., Scott, T. B., et al. (2011). Green synthesis of iron nanoparticles and their application as a Fenton-like
194
REFERENCES
catalyst for the degradation of aqueous cationic and anionic dyes. Chemical Engineering Journal, 172(1), 258-266. Shankar, S. S., Rai, A., Ahmad, A., & Sastry, M. (2004). Rapid synthesis of Au, Ag, and bimetallic Au core–Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. Journal of colloid and interface science, 275(2), 496-502. Sharma, D., Rajput, J., Kaith, B., Kaur, M., & Sharma, S. (2010). Synthesis of ZnO nanoparticles and study of their antibacterial and antifungal properties. Thin Solid Films, 519(3), 1224-1229. Sharma, J. K., Srivastava, P., Akhtar, M. S., Singh, G., & Ameen, S. (2015). α-Fe 2 O 3 hexagonal cones synthesized from the leaf extract of Azadirachta indica and its thermal catalytic activity. New Journal of Chemistry, 39(9), 7105-7111. Shen, C. J., Chen, C. K., & Lee, S. S. (2009). Polar Constituents from Sageretia Thea Leaf Characterized by HPLC‐SPE‐NMR Assisted Approaches. Journal of the Chinese Chemical Society, 56(5), 1002-1009. Shen, J., Hu, Y., Li, C., Qin, C., & Ye, M. (2008). Pt–Co supported on single-walled carbon nanotubes as an anode catalyst for direct methanol fuel cells. Electrochimica Acta, 53(24), 7276-7280. Shinde, V., Mahadik, S., Gujar, T., & Lokhande, C. (2006). Supercapacitive cobalt oxide
(Co3O4) thin films by spray pyrolysis. Applied Surface Science, 252(20), 7487-7492. Siddiqi, K. S., ur Rahman, A., & Husen, A. (2016). Biogenic Fabrication of Iron/Iron Oxide Nanoparticles and Their Application. Nanoscale Research Letters, 11(1), 498. Simon, P., & Gogotsi, Y. (2008). Materials for electrochemical capacitors. Nature materials, 7(11), 845-854. Sirelkhatim, A., Mahmud, S., Seeni, A., Kaus, N. H. M., Ann, L. C., Bakhori, S. K. M., et al. (2015). Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. Nano-Micro Letters, 7(3), 219-242. Šljukić, B., Banks, C. E., Crossley, A., & Compton, R. G. (2007). Lead (IV) oxide– graphite composite electrodes: application to sensing of ammonia, nitrite and phenols. Analytica chimica acta, 587(2), 240-246.
195
REFERENCES
Smolyaninova, V. N., Yost, B., Lahneman, D., Narimanov, E. E., & Smolyaninov, I. I. (2014). Self-assembled tunable photonic hyper-crystals. Scientific Reports (Nature Publisher Group), 4, 5706. Smuleac, V., Varma, R., Sikdar, S., & Bhattacharyya, D. (2011). Green synthesis of Fe and Fe/Pd bimetallic nanoparticles in membranes for reductive degradation of chlorinated organics. Journal of membrane science, 379(1), 131-137. Sobanska, S., Wignacourt, J.-P., Conflant, P., Drache, M., Lagrene, M., & Holt, E. M. (1999). Synthesis, thermal analysis and crystal structure of lead (II) diaqua 3, 6- dicarboxylatopyridazine. Evaluation of performance as a synthetic precursor. New Journal of Chemistry, 23(4), 393-396. Sone, B., Diallo, A., Fuku, X., Gurib-Fakim, A., & Maaza, M. (2017). Biosynthesized CuO nano-platelets: Physical properties & enhanced thermal conductivity nanofluidics. Arabian Journal of Chemistry. (in press) Sone, B., Fuku, X., & Maaza, M. (2016a). Physical & Electrochemical Properties of Green Synthesized Bunsenite NiO Nanoparticles via Callistemon Viminalis’ Extracts. Int. J. Electrochem. Sci, 11, 8204-8220.
Sone, B., Manikandan, E., Gurib-Fakim, A., & Maaza, M. (2015). Sm2O3 nanoparticles green synthesis via Callistemon viminalis' extract. Journal of Alloys and Compounds, 650, 357-362.
Sone, B., Manikandan, E., Gurib-Fakim, A., & Maaza, M. (2016b). Single-phase α-Cr2O3 nanoparticles’ green synthesis using Callistemon viminalis’ red flower extract. Green Chemistry Letters and Reviews, 9(2), 85-90. Song, Y., He, Z., Zhu, H., Hou, H., & Wang, L. (2011). Electrochemical and electrocatalytic properties of cobalt nanoparticles deposited on graphene modified glassy carbon electrode: Application to some amino acids detection. Electrochimica Acta, 58, 757-763. Sonker, R. K., Sabhajeet, S., Singh, S., & Yadav, B. (2015). Synthesis of ZnO nanopetals
and its application as NO2 gas sensor. Materials Letters, 152, 189-191. Soomro, R. A., Ibupoto, Z. H., Abro, M. I., & Willander, M. (2015). Electrochemical sensing of glucose based on novel hedgehog-like NiO nanostructures. Sensors and Actuators B: Chemical, 209, 966-974.
196
REFERENCES
Stankic, S., Suman, S., Haque, F., & Vidic, J. (2016). Pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties. Journal of Nanobiotechnology, 14(1), 73. Sudhasree, S., Shakila Banu, A., Brindha, P., & Kurian, G. A. (2014). Synthesis of nickel nanoparticles by chemical and green route and their comparison in respect to biological effect and toxicity. Toxicological & Environmental Chemistry, 96(5), 743- 754. Sundrarajan, M., Bama, K., Bhavani, M., Jegatheeswaran, S., Ambika, S., Sangili, A., et al. (2017). Obtaining titanium dioxide nanoparticles with spherical shape and antimicrobial properties using M. citrifolia leaves extract by hydrothermal method. Journal of Photochemistry and Photobiology B: Biology. Suntako, R. (2015). Effect of synthesized ZnO nanograins using a precipitation method for the enhanced cushion rubber properties. Materials Letters, 158, 399-402. Talam, S., Karumuri, S. R., & Gunnam, N. (2012). Synthesis, characterization, and spectroscopic properties of ZnO nanoparticles. ISRN Nanotechnology, 2012. Tao, K., Li, P., Kang, L., Li, X., Zhou, Q., Dong, L., et al. (2015). Facile and low-cost combustion-synthesized amorphous mesoporous NiO/carbon as high mass-loading pseudocapacitor materials. Journal of Power Sources, 293, 23-32. Thatoi, P., Kerry, R. G., Gouda, S., Das, G., Pramanik, K., Thatoi, H., et al. (2016). Photo- mediated green synthesis of silver and zinc oxide nanoparticles using aqueous extracts of two mangrove plant species, Heritiera fomes and Sonneratia apetala and investigation of their biomedical applications. Journal of Photochemistry and Photobiology B: Biology, 163, 311-318. Thema, F., Beukes, P., Gurib-Fakim, A., & Maaza, M. (2015a). Green synthesis of Monteponite CdO nanoparticles by Agathosma betulina natural extract. Journal of Alloys and Compounds, 646, 1043-1048. Thema, F., Manikandan, E., Dhlamini, M., & Maaza, M. (2015). Green synthesis of ZnO nanoparticles via Agathosma betulina natural extract. Materials Letters, 161, 124-127. Thema, F., Manikandan, E., Gurib-Fakim, A., & Maaza, M. (2016). Single phase Bunsenite NiO nanoparticles green synthesis by Agathosma betulina natural extract. Journal of Alloys and Compounds, 657, 655-661.
197
REFERENCES
Thota, S., Kumar, A., & Kumar, J. (2009). Optical, electrical and magnetic properties of
Co3O4 nanocrystallites obtained by thermal decomposition of sol–gel derived oxalates. Materials Science and Engineering: B, 164(1), 30-37. Thovhogi, N., Diallo, A., Gurib-Fakim, A., & Maaza, M. (2015). Nanoparticles green synthesis by Hibiscus Sabdariffa flower extract: main physical properties. Journal of Alloys and Compounds, 647, 392-396. Thovhogi, N., Park, E., Manikandan, E., Maaza, M., & Gurib-Fakim, A. (2016). Physical properties of CdO nanoparticles synthesized by green chemistry via Hibiscus Sabdariffa flower extract. Journal of Alloys and Compounds, 655, 314-320. Thulasiramudu, A., & Buddhudu, S. (2007). Optical characterization of Eu 3+ and Tb 3+ ions doped zinc lead borate glasses. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 66(2), 323-328. Uboldi, C., Orsière, T., Darolles, C., Aloin, V., Tassistro, V., George, I., et al. (2016). Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways. Particle and fibre toxicology, 13(1), 5. Venugopal, K., Rather, H., Rajagopal, K., Shanthi, M., Sheriff, K., Illiyas, M., et al. (2017). Synthesis of silver nanoparticles (Ag NPs) for anticancer activities (MCF 7 breast and A549 lung cell lines) of the crude extract of Syzygium aromaticum. Journal of Photochemistry and Photobiology B: Biology, 167, 282-289. Wang, H., Chen, C., Zhang, Y., Peng, L., Ma, S., Yang, T., et al. (2015). In situ oxidation of carbon-encapsulated cobalt nanocapsules creates highly active cobalt oxide catalysts for hydrocarbon combustion. Nature communications, 6. Wang, K., Xu, J.-J., & Chen, H.-Y. (2005). A novel glucose biosensor based on the nanoscaled cobalt phthalocyanine–glucose oxidase biocomposite. Biosensors and Bioelectronics, 20(7), 1388-1396. Wang, M.-H., Ma, X.-Y., & Zhou, F. (2015). Synthesis and characterization of monodispersed spherical ZnO nanocrystals in an aqueous solution. Materials Letters, 142, 64-66. Wang, Y.-G., Cheng, L., & Xia, Y.-Y. (2006). Electrochemical profile of nano-particle CoAl double hydroxide/active carbon supercapacitor using KOH electrolyte solution. Journal of power sources, 153(1), 191-196.
198
REFERENCES
Waters, B., Saxena, G., Wanggui, Y., Kau, D., Wrigley, S., Stokes, R., et al. (2002). Identifying protein kinase inhibitors using an assay based on inhibition of aerial hyphae formation in Streptomyces. The Journal of antibiotics, 55(4), 407-416. Whitesides, G. M. (2005). Nanoscience, nanotechnology, and chemistry. Small, 1(2), 172- 179. Wiegand, I., Hilpert, K., & Hancock, R. E. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature protocols, 3(2), 163-175. Wong, K. K., & Liu, X. (2010). Silver nanoparticles—the real “silver bullet” in clinical medicine? MedChemComm, 1(2), 125-131. Wu, W., Wu, Z., Yu, T., Jiang, C., & Kim, W.-S. (2015). Recent progress on magnetic iron oxide nanoparticles: synthesis, surface functional strategies and biomedical applications. Science and technology of advanced materials, 16(2), 023501. Wu, Z., Zhu, Y., Ji, X., & Banks, C. E. (2016). Transition Metal Oxides as Supercapacitor Materials. In Nanomaterials in Advanced Batteries and Supercapacitors (pp. 317- 344): Springer. Xiao, X., Liu, X., Zhao, H., Chen, D., Liu, F., Xiang, J., et al. (2012). Facile shape control of Co3O4 and the effect of the crystal plane on electrochemical performance. Advanced Materials, 24(42), 5762-5766. Xu, H., Mao, X., Zeng, Q., Wang, S., Kawde, A.-N., & Liu, G. (2008). Aptamer- functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for protein analysis. Analytical Chemistry, 81(2), 669-675. Xu, L., Yang, X., & Li, B. (1994). Chemical constituents of Sageretia theezans Brongn. Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi= China journal of Chinese materia medica, 19(11), 675-676, 702. Yamaura, M., Camilo, R., Sampaio, L., Macedo, M., Nakamura, M., & Toma, H. (2004). Preparation and characterization of (3-aminopropyl) triethoxysilane-coated magnetite nanoparticles. Journal of Magnetism and Magnetic Materials, 279(2), 210-217. Yan, J., Wang, Q., Wei, T., & Fan, Z. (2014). Recent advances in design and fabrication of electrochemical supercapacitors with high energy densities. Advanced Energy Materials, 4(4).
199
REFERENCES
Yang, M., Jiang, J., Yang, Y., Chen, X., Shen, G., & Yu, R. (2006). Carbon nanotube/cobalt hexacyanoferrate nanoparticle-biopolymer system for the fabrication of biosensors. Biosensors and Bioelectronics, 21(9), 1791-1797. Yao, G., Sebisubi, F. M., Voo, L. Y. C., Ho, C. C., Tan, G. T., & Chang, L. C. (2011). Citrinin derivatives from the soil filamentous fungus penicillium sp. H9318. Journal of the Brazilian Chemical Society, 22(6), 1125-1129. Yuvakkumar, R., Suresh, J., Nathanael, A. J., Sundrarajan, M., & Hong, S. (2014). Rambutan (Nephelium lappaceum L.) peel extract assisted biomimetic synthesis of nickel oxide nanocrystals. Materials Letters, 128, 170-174. Zak, A. K., Razali, R., Majid, W., & Darroudi, M. (2011). Synthesis and characterization of a narrow size distribution of zinc oxide nanoparticles. International Journal of nanomedicine, 6,1399-1403. Zargar, M., Shameli, K., Najafi, G. R., & Farahani, F. (2014). Plant mediated green biosynthesis of silver nanoparticles using Vitex negundo L. extract. Journal of Industrial and Engineering Chemistry, 20(6), 4169-4175. Zhang, H., Chen, B., Jiang, H., Wang, C., Wang, H., & Wang, X. (2011). A strategy for ZnO nanorod mediated multi-mode cancer treatment. Biomaterials, 32(7), 1906-1914. Zhang, H.-J., & Xiong, H.-M. (2013). Biological applications of ZnO nanoparticles. Current Molecular Imaging, 2(2), 177-192. Zhang, L. L., & Zhao, X. (2009). Carbon-based materials as supercapacitor electrodes. Chemical Society Reviews, 38(9), 2520-2531. Zhang, P., Hu, G.-X., Bao, S.-J., Guo, J., Lei, C., Cai, C.-J., et al. (2012). One step
microwave synthesis and magnetic properties of Co3O4 octahedrons. Materials Letters, 83, 195-197. Zhang, X., Luo, J., Tang, P., Ye, X., Peng, X., Tang, H., et al. (2017). A universal strategy for metal oxide anchored and binder-free carbon matrix electrode: A supercapacitor case with superior rate performance and high mass loading. Nano Energy, 31, 311- 321. Zhang, Y. (2015). Thermal oxidation fabrication of NiO film for optoelectronic devices. Applied Surface Science, 344, 33-37.
200
REFERENCES
Zhao, B., Wang, Y., Guo, H., Wang, J., He, Y., Jiao, Z., et al. (2007). Iron oxide (III) nanoparticles fabricated by electron beam irradiation method. Materials Science Poland, 25(4), 1143-1148. Zhou, G., Li, Y., Xiao, W., Zhang, L., Zuo, Y., Xue, J., et al. (2008). Synthesis, characterization, and antibacterial activities of a novel nanohydroxyapatite/zinc oxide complex. Journal of Biomedical Materials Research Part A, 85(4), 929-937. Zilberstein, D., Blumenfeld, N., Liveanu, V., Gepstein, A., & Jaffe, C. L. (1991). Growth at acidic pH induces an amastigote stage-specific protein in Leishmania promastigotes. Molecular and biochemical parasitology, 45(1), 175-178.
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Annexures
Annexures
202
Annexures
10.1 Un-submitted data
Annexure 10.1: Cytotoxicity of ZnO nanoparticles against HepG2 cancerous cell line
Annexure 10.2: Cytotoxicity of Fe2O3 nanoparticles against HepG2 cancerous cell l
203
Annexures
Annexure 10.3: Cytotoxicity of NiO nanoparticles against HepG2 cancerous cell line
Annexure: 10.4 Cytotoxicity of PbO nanoparticles against HepG2 cancerous cell line
204
Annexures
Annexure 10.5: Cytotoxicity of Co3O4 nanoparticles against HepG2 cancerous cell line
Annexure 10.6: Acetylcholinesterase, butyrylcholinesterase and α-glucocsidase inhibition by Sageretia thea mediated ZnO nanoparticles
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Annexures
Annexure 10.7: Acetylcholinesterase, butyrylcholinesterase and α-glucocsidase inhibition
by Sageretia thea mediated Fe2O3 nanoparticles
Annexure 10.8: Acetylcholinesterase, butyrylcholinesterase and α-glucocsidase inhibition by Sageretia thea mediated NiO nanoparticles
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Annexures
Annexure 10.9: Acetylcholinesterase, butyrylcholinesterase and α-glucocsidase inhibition by Sageretia thea mediated PbO nanoparticles
Annexure 10.10: Acetylcholinesterase, butyrylcholinesterase and α-glucocsidase
inhibition by Sageretia thea mediated Co3O4 nanoparticles
207
List of publications
Title of the manuscript Journal Impact status factor Sageretia thea (Osbeck.) modulated Artificial 5.6 Published biosynthesis of NiO nanoparticles and their cells, invitro pharmacognostic, antioxidant and nanomedicine cytotoxic potential and biotechnology Sageretia thea (Osbeck.) mediated synthesis of Nanomedicine 4.8 Published ZnO nanoparticles and its biological applications Biosynthesis of iron oxide (Fe2O3) Green 1.7 Published nanoparticles via aqueous extracts of Sageretia chemistry thea (Osbeck.) and their pharmacognostic letters and properties reviews Bioinspired synthesis of pure massicot phase Arabian 4.6 Published lead oxide nanoparticles and assessment of Journal of their biocompatibility, cytotoxicity and in-vitro chemistry biological properties Physical properties, biological applications and Arabian 4.6 Published biocompatibility studies on biosynthesized Journal of single phase cobalt oxide (Co3O4) chemistry nanoparticles via Sageretia thea (Osbeck.)
Biogenic synthesis of zinc oxide nanoplatelets RSC advances 3.1 Under on nickel foam as electrode material for review supercapacitor applications Manuscript to be submitted Anticancer, cholinesterase and alpha-glucosidase properties of certain metal oxide nanoparticles biosynthesized through Sageretia thea (Osbeck.)
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Awards and fellowships
Fellowship and membership Fellowship Description Duration UNESCO-UNISA Africa This chair is based in January, 2017 - onwards Chair in Pretoria, South Africa. This Nanosciences/Nanotechnology fellowship is granted to young researchers who has demonstrated active skills and quality research in nanosciences NANOAFNET This membership is January, 2017 - onwards (Nanosciences African awarded to the young Network) researchers committed to excellence in nanotechnology
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