1 CHEMICAL COMPOSITION AND BIOLOGICAL (ANTIOXIDANT AND ANTIMICROBIAL) ACTIVITIES OF ESSENTIAL OILS FROM SELECTED PLANTS OF POTHOHAR PLATEAU By MOIN UD DIN M.Phil. (UAF) A THESIS SUBMITTED IN PARTIAL FULFIL L MENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY DEPARTMENT OF CHEMISTRY, FACULTY OF SCIENCES, UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2015 2 To The Controller of Examinations, University of Agriculture, Faisalabad. “We, the Supervisory Committee, certify that the contents and form of thesis submitted by MR. MOIN UD DIN, Regd. No.2003-ag-281, have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner(s) for the award of degree”. Supervisory Committee 1. Supervisor ___________________________ (Dr. Raja Adil Sarfraz) 2. Co-Supervisor ___________________________ (Dr. Abdullah Ijaz Hussain) 3. Member __________________________ (Dr. Ijaz Ahmad Bhatti) 4. Member ___________________________ (Dr. Muhammad Shahid) 3 4 ACKNOWLEDGEMENTS In the name of Almighty Allah, the most beneficent, most merciful, the Lord of the lords, Who guides us in difficulties and blessed me with courage and power to complete this thesis. And all praise for Prophet Hazrat Muhammad (Sallallahhu-Alaih-e-Wasallam), for enlightening our conscious with the essence of faith in Allah and Who is a torch of guidance for the entire humanity, forever. It is a great honor for me to express my profound and cordial gratitude to my kind, respected and worthy supervisor Dr. Raja Adil Sarfraz, Assistant professor, Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad for his valuable and skilled guidance, ever encouraging and constructive criticism, which always helped me to carry in with my research work. I thank with core of my heart of my Co-Supervisor Dr. Abdullah Ijaz Hussain, Assistant professor, Department of Chemistry, Government College University, Faisalabad for his erudite suggestions, sympathetic and inspiring attitude, patronizing supervision and technical guidance during my studies. I express my deep gratitude to Dr. Ijaz Ahmad Bhatti and Dr. Muhammad Shahid Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad for their expert guidance, generous assistance, cooperation and friendly affection during the course of my research and writing of this manuscript. Thanks to Dr Mansoor and Dr Faiz ul Hasan for helping in selection of plant material. I ardently extend my special thanks to Dr. Lyle E. Craker for his warm hospitality, dedicated cooperation and encouragement during the part of my Ph.D. in Medicinal and Aromatic Plants Laboratory, Stockbridge School of Agriculture, University of Massachusetts, Amherst, USA. I’m also thankful to Dr. Masood Sadiq Butt, DG NIFSAT to be very much cooperative and friendly to me during my stay in USA. I am also grateful to my friends Muhammad Yasir and Shazia Nourin to be very much Cooperative and their moral support, meanwhile thanks to other laboratory fellows Nadia, Huma, Aisha and Arfaa for their cooperation. A lot of thanks to my Hitech friends Khalid sb, Fazil Shah sb and other members of lab to be very much caring of me during my whole tenure. I’m thankful to my family for keeping me spare for this study which requires a lot of patience. All credits go to my affectionate and dearest father whose prayers are accompaniment in the journey of my life. I ardently extend thanks to my respectable and dearest Sisters for their help and prayers which has proved courage, goodness and good luck for me. My feelings will not be completed without mention of my dearest brother Nizam for his love, encouragement and good wishes that inspired me to accomplish this humble effort. Thanks to my bhabies and all of my nephews and nieces for their love and mellifluous affections, which hearten me to achieve success in every sphere of life with special thanks to Sara for giving me nice pieces of suggestions and helping me a lot in thesis write up. May Allah give all of them long and happy life (Ameen)! Finally, Thanks to all those who taught me ever one word in my life. Moin ud Din 5 C O N T E N T S Chapters Titles Page No. Chapter 1 Introduction 1-10 Chapter 2 Review of Literature 11-35 2.1. Essential Oils 12 2.2. Sources 12 2.2.1. Parthenium hysterophorus 12 2.2.2. Eucalyptus camaldulensis 13 2.2.3. Eucalyptus citriodora 15 2.2.4. Eucalyptus tereticornis 15 2.2.5. Syzygium aromaticum 16 2.2.6. Myristica fragrans 16 2.2.7. Citrus reticulate 17 2.2.8. Murraya koenigii 18 2.2.9. Piper nigrum 19 2.2.10. Elettaria cardamomum 19 2.2.11. Amomum subulatum 20 2.2.12. Trachyspermum ammi 20 2.2.13. Foeniculum vulgare 21 2.2.14. Cuminum Cyminum 22 2.2.15 Cinnamomum zelenicum 23 2.3. History of Essential Oils 24 2.4. Chemistry of Essential oils 25 2.5. Biological activities of essential oils 30 2.5.1. Antioxidant activities: 30 2.5.2. Antimicrobial activities 32 6 2.5.3. Cytotoxicity 34 Chapter 3 Materials and Methods 36 3.1. Materials 36 3.1.1. Reagent 36 3.1.2. Instruments 37 3.1.3. Collection of plant material & Identification 37 3.1.4. Strains of microorganisms 38 3.2. Experimental Protocol 39 3.2.1. Isolation of Essential Oils 39 3.2.2. Analysis of Essential Oil 39 3.3. Biological Activities of Essential oils 40 3.3.1. Evaluation of antioxidant activity of essential oils 40 3.3.2. Evaluation of antimicrobial activities of 42 3.4. Essentialessential oilsoils as food preservatives 44 3.5. Cytotoxicity 45 Chapter 4 Results & Discussion 46-211 4.1.a. Discussion for Parthenium hysterophorus 54 4.2.a. Discussion for Eucalyptus camaldulensis 65 4.3.a. Discussion for Eucalyptus citriodora 75 4.4.a. Discussion for Eucalyptus tereticornis 86 4.5.a. Discussion for Citrus reticulate 96 4.6.a. Discussion for Syzygium aromaticum 106 4.7.a. Discussion for Myristica fragrans (Nutmeg) 116 4.8.a. Discussion for Myristica fragrans (Mace) 127 4.9.a. Discussion for Murraya koenigii 137 4.10.a. Discussion for Piper nigrum 147 4.11.a. Discussion for Elettaria cardamomum 157 4.12.a. Discussion for Amomum subulatum 167 4.13.a. Discussion for Trachyspermum ammi 177 7 4.14.a. Discussion for Foeniculum vulgare 187 4.15.a. Discussion for Cuminum cyminum 198 4.16.a. Discussion for Cinnamomum zelenicum 209 Chapter 5 Summary 212-213 References 214-244 8 LIST OF FIGURES Figure TITLE PAGE 3.1s A typical agar plate showing the inhibition zones exhibited by essential NO. 43 oils 3.2 A typical plate in resazurin microtitre-plate assay showing the color 43 change due to antibacterial effect of essential oils 4.1.1 Antioxidant activity of Parthenium hysterophorus essential oil 47 measured by bleaching of β-carotene-linoleic acid emulsion 4.1.2 GC-MS chromatogram of essential oil from Parthenium hysterophorus 51 4.2.1 Antioxidant activity of Eucalyptus camaldulensis essential oil measured 58 by bleaching of β-carotene-linoleic acid emulsion 4.2.2 GC-MS chromatogram of essential oil from Eucalyptus camaldulensis 62 4.3.1 Antioxidant activity of Eucalyptus citriodora essential oil measured by 69 bleaching of β-carotene-linoleic acid emulsion 4.3.2 GC -MS chromatogram of essential oil from Eucalyptus citriodora 73 4.4.1 Antioxidant activity of Eucalyptus tereticornis essential oil measured 79 by bleaching of β-carotene-linoleic acid emulsion 4.4.2 GC-MS chromatogram of essential oil from Eucalyptus tereticornis 83 4.5.1 Antioxidant activity of Citrus reticulata essential oil measured by 90 bleaching of β-carotene-linoleic acid emulsion 4.5.2 GC-MS chromatogram of essential oil from Citrus reticulata 94 4.6.1 Antioxidant activity of Syzygium aromaticum essential oil measured by 100 bleaching of β-carotene-linoleic acid emulsion 4.6.2 GC-MS chromatogram of essential oil from Syzygium aromaticum 104 4.7.1 Antioxidant activity of Myristica fragrans (N) essential oil measured by 110 bleaching of β-carotene-linoleic acid emulsion 9 4.7.2 GC-MS chromatogram of essential oil from Myristica fragrans (N) 114 4.8.1 Antioxidant activity of Myristica fragrans (M) essential oil measured 120 by bleaching of β-carotene-linoleic acid emulsion 4.8.2 GC-MS chromatogram of essential oil from Myristica fragrans (M) 124 4.9.1 Antioxidant activity of Murraya koenigii essential oil measured by 131 bleaching of β-carotene-linoleic acid emulsion 4.9.2 GC-MS chromatogram of essential oil from Murraya koenigii 135 4.10.1 Antioxidant activity of Piper nigrum essential oil measured by 141 bleaching of β-carotene-linoleic acid emulsion 4.10.2 GC-MS chromatogram of essential oil from Piper nigrum 145 4.11.1 Antioxidant activity of Elettaria cardamomum essential oil measured 151 by bleaching of β-carotene-linoleic acid emulsion 4.11.2 GC-MS chromatogram of essential oil from Elettaria cardamomum 155 4.12.1 Antioxidant activity of Amomum subulatum essential oil measured by 161 bleaching of β-carotene-linoleic acid emulsion 4.12.2 GC-MS chromatogram of essential oil from Amomum subulatum 165 4.13.1 Antioxidant activity of Trachyspermum ammi essential oil measured by 171 bleaching of β-carotene-linoleic acid emulsion 4.13.2 GC-MS chromatogram of essential oil from Trachyspermum ammi 175 4.14.1 Antioxidant activity of Foeniculum vulgare essential oil measured by 181 bleaching of β-carotene-linoleic acid emulsion 4.14.2 GC-MS chromatogram of essential oil from Foeniculum vulgare 185 4.15.1 Antioxidant activity of Cuminum cyminum essential oil measured by 191 bleaching of β-carotene-linoleic acid emulsion 4.15.2 GC-MS chromatogram of essential oil from Cuminum cyminum 195 10 4.16.1 Antioxidant activity of Cinnamomum zeylanicum essential oil 202 measured by bleaching of β-carotene-linoleic acid emulsion 4.16.2 GC-MS chromatogram of essential oil from Cinnamomum zeylanicum 206 11 LIST OF TABLES Tables TITLE PAGE NO.