2010 CHEMICAL TRANSFORMATIONS AND PHYTOCHEMICAL STUDIES OF BIOAC- TIVE COMPONENTS FROM EXTRACTS OF ROSMARINUS OFFICINALIS L. By Omobola Oluranti Okoh A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Science and Agriculture at the University of Fort Hare Supervisors Professor A. P. Sadimenko Professor A. J. Afolayan TABLE OF CONTENTS Declaration ............................................................................. 8 Acknowledgements ..................................................................... 9 Dedication ................................................................................. 10 List of tables ............................................................................. 12 List of figures ............................................................................. 13 Abbreviations ............................................................................. 15 Abbreviations for journal titles ..................................................... 17 Abstract ..................................................................................... 24 I. Introduction ......................................................................... 27 II. Review of literature ................................................................. 35 A. Essential oils ..................................................................... 35 B. Secondary metabolites ......................................................... 38 C. Extraction of essential oils ................................................. 38 1. Steam distillation ......................................................... 39 2. Maceration ..................................................................... 39 3. Cold pressing ................................................................. 40 4. Solvent extraction ......................................................... 40 D. Composition of essential oils ................................................. 41 E. Factors affecting composition of essential oils ......................... 42 1. Harvest time .................................................................. 42 2. Method of extraction ...................................................... 43 3. Temperature .................................................................. 43 2 F. Methods of administration of essential oils ............................. 44 G. Properties of essential oils .................................................. 44 1. Antimicrobial properties .................................................. 44 2. Antibacterial properties .................................................. 45 3. Antiviral properties .......................................................... 46 4. Antifungal properties ...................................................... 47 5. Antioxidant properties ...................................................... 48 H. Mechanism of action of essential oils ...................................... 49 1. Mechanism of inhibition by changes in membrane function ... 50 2. Mechanism of inhibition by changes in protein content ......... 51 I. GS-MS analysis of essential oils .............................................. 51 J. Application of essential oils .................................................. 52 1. In cosmetics .................................................................. 52 2. In food .......................................................................... 53 3. In health care .................................................................. 53 K. Rosmarinus officinalis essential oils ..................................... 54 L. Plant products and their transformation ................................. 62 1. Pinenes .......................................................................... 63 2. 1,8-Cineole .................................................................. 67 3. Camphor ...................................................................... 70 4. Verbenone ...................................................................... 72 5. Bornyl acetate .................................................................. 73 M. Chemical transformations in plants ..................................... 73 3 N. Antioxidant activity .............................................................. 77 1. Definition of antioxidant .................................................. 77 2. Natural antioxidants ...................................................... 78 a. Beta carotene and carotenoids ...................................... 78 b. Ascorbic acid (Vitamin C) .............................................. 79 c. Vitamin E .................................................................. 80 d. Selenium .................................................................. 81 3. New oxidants .................................................................. 81 a. Polyphenols .............................................................. 82 b. Flavonoids .................................................................. 84 c. Free radicals .............................................................. 86 4. Diphenylpicrylhydrazyl (DPPH) free radical ......................... 87 5. Some tropical plants with antibacterial and antioxidant properties ...................................................................... 90 O. Structural elucidation and characterization of bioactive compounds isolated from Rosmarinus officinalis L. ................................. 90 III. Materials and methods .......................................................... 93 A. Cultivation .......................................................................... 93 B. Harvesting for essential oil distillation ................................. 93 C. Solvent-free microwave extraction ......................................... 93 D. Hydrodistillation .................................................................. 94 E. GC-MS analyses and identification of components ..................... 95 F. Antimicrobial activity of essential oils ..................................... 96 4 G. Time-kill assay .............................................................. 98 H. Antioxidant activity of essential oils ...................................... 99 1. DPPH radical scavenging activity ...................................... 99 2. b-Carotene bleaching assay .............................................. 99 I. Isolation and characterization of bioactive compounds from leaf extracts of Rosmarinus officinalis L. ......................... 100 1. Introduction .................................................................. 100 2. Plant collection .............................................................. 101 3. Plant preparation .......................................................... 101 4. Extraction and partitioning .............................................. 102 5. Chromatographic analysis .............................................. 102 J. Biological activity methods .................................................. 104 1. Bacterial cultures .......................................................... 104 2. Bioautographic assay of plant extracts ............................. 104 3. Vacuum liquid chromatography (VLC) ................................. 105 4. Purification of VLC fraction A ............................................. 105 K. Phytochemical screening and polyphenolic antioxidant activity of leaf extracts of Rosmarinus officinalis L ............................. 106 1. Preparation of extracts ...................................................... 106 2. Chemicals ...................................................................... 108 3. Phytochemical screening of the plant extract ..................... 108 4. Test for tannins ............................................................. 108 5. Test for alkaloids ............................................................. 109 5 6. Test for flavonoids ......................................................... 109 7. Test for saponins ............................................................. 109 8. Test for steroids ............................................................. 109 9. Determination of total phenolic composition ......................... 110 10. Estimation of total flavonoids ...................................... 110 11. Determination of total proanthocyanidins ..................... 110 12. Determination of reducing power ................................. 111 L. Antioxidant assay of plant extracts ......................................... 111 1. Ferric thiocyanate (FTC) method ...................................... 112 2. Thiobarbituric acid (TBA) method ...................................... 112 3. 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging activity .............................................. 112 4. Scavenging activity of nitric oxide ...................................... 113 5. Hydrogen peroxide scavenging activity .............................. 114 M. Spectroscopic techniques ...................................................... 114 1. Nuclear magnetic resonance .............................................. 116 2. Mass-spectroscopy .......................................................... 117 3. Infrared spectroscopy ...................................................... 117 IV. Results and Discussion .............................................................. 119 A. Essential oil yield .............................................................