University of Bradford eThesis This thesis is hosted in Bradford Scholars – The University of Bradford Open Access repository. Visit the repository for full metadata or to contact the repository team © University of Bradford. This work is licenced for reuse under a Creative Commons Licence. PHYTOCHEMICAL INVESTIGATION AND BIOLOGICAL ACTIVITIES OF SANICULA EUROPAEA AND TEUCRIUM DAVAEANUM Isolation and identification of some constituents of Sanicula europaea and Teucrium davaeanum and evaluation of the antioxidant activity of ethanolic extracts of both plants and cytotoxic activity of some isolated compounds. Agela Hussain Mohammed TALAG Submitted for the degree of Doctor of Philosophy Faculty of Life Sciences University of Bradford 2016 Abstract The aim of this research was to investigate the phytochemistry of two species Sanicula europaea and Teucrium davaeanum which are traditionally used in treatment of wounds. Four compounds were isolated from the 80% methanolic extract of S. europaea; bis-(2-ethylhexyl) phthalate (1), palmitic acid (2), rosmarinic acid (3), saniculoside N (4). Compounds 1 and 2 were isolated for the first time from this species. The structure elucidation of the isolated compounds was on the basis of 1D, 2D NMR spectroscopy and mass spectrometry measurements. Two compounds were isolated from the crude glycosides extract of T.davaeanum; 6 is a phenylethanoid glycoside and 8 is an iridoid glycoside, from the data available these may be new compounds for which the names davaeanuside A and davaeanuside B are proposed respectively." The total polyphenol content of S. europaea L, T. davaeanum leaves-flowers and T. davaeanum stem were found to be 5.0, 1.20 and 0.65 mg per 100 mg dried plant material respectively. A study of the antioxidant activity of the 50 % ethanol extracts of S. europaea and T. davaeanum showed that on a mg/mg basis S. europaea and T. davaeanum have approximately 5%, 8 % antioxidant capacity of Trolox respectively. A study of the cytotoxic activity of davaeanuside A (6), iridoid glycoside (7), davaeanuside B (8) and saponin compound (10) isolated from the crude glycosides extract of T. davaeanum revealed that saponin compound (10) inhibited the growth of Hela cells by 50 % at 50 µg/ml, P< 0.001, but the other compounds did not show activities against the tested cell lines at 100 µg/ml. The results of this work provide some basis for the traditional use of these species in the treatment of wounds. Key words Sanicula, Teucrium, saponin glycosides, phenylpropanoids, phenolic acids, antioxidant, cytotoxicity i To my husband who was supporting me through all the four years of my study, To the spirit of my father, To my family. ii CONTENT Abstract i Dedication ii List of Contents iii List of Figures ix List of Tables xvi Abbreviations and symbols xix Acknowledgments xxiii 1 Introduction ....................................................................................... 1 1.1 The use of natural products in drug discovery .................................... 1 1.2 The genus Sanicle.............................................................................. 2 1.2.1 Sanicula europaea L. ........................................................................ 9 1.2.2 Distribution of S. europaea in the world ........................................... 11 1.2.3 Traditional uses of S. europaea . .................................................... 11 1.2.3.1 Use of S. europaea for wound healing ....................................... 11 1.2.4 Other uses of S. europaea .............................................................. 13 1.2.5 Albarello drug jar for Sanicle , Italy , 1601 – 1800 ........................... 14 1.2.6 Phytochemistry of S. europaea ....................................................... 14 1.2.6.1 Volatile oil .................................................................................... 15 1.2.6.2 Saponin glycosides ...................................................................... 15 1.2.6.3 Phenolic acids ............................................................................. 20 1.2.7 Pharmacological actions of S. europaea ......................................... 20 1.3 Teucrium davaeanum species ......................................................... 23 1.4 Botanical description of Teucrium davaeanum L. ............................ 23 1.5 Phytochemistry of Teucrium genus .................................................. 24 1.5.1 Volatile oils ...................................................................................... 24 1.5.2 Diterpenoides compounds ............................................................... 25 1.5.3 Iridoid glycosides from Teucrium species ........................................ 30 iii 1.5.4 Flavonoids ....................................................................................... 31 1.5.5 Biological activities of some Teucrium species .............................. 32 1.6 Aim of the study ............................................................................... 33 1.7 Objectives ........................................................................................ 33 2 Materials and Methods.................................................................... 34 2.1 Plant materials ................................................................................. 34 2.2 Solvents ........................................................................................... 34 2.3 Chemicals ........................................................................................ 34 2.4 Extraction procedures ...................................................................... 34 2.4.1 Preparation of non-polar constituents of S. europaea ..................... 34 2.4.1.1 Preparation of hydrocarbons mixture ........................................... 35 2.4.1.2 Extraction of glycosides from S. europaea .................................. 37 2.4.1.3 Aqueous extraction ...................................................................... 39 2.5 Chromatographic Techniques .......................................................... 39 2.5.1 Normal phase silica gel chromatography ........................................ 39 2.5.2 Reverse phase silica gel C-18 chromatography .............................. 39 2.5.3 Preparative thin layer chromatography............................................ 40 2.5.4 Gel filtration chromatography .......................................................... 40 2.5.5 Thin layer chromatography (TLC) ................................................... 40 2.6 Gas chromatography-Mass spectrometry (GC-MS) ......................... 41 2.7 High performance liquid chromatography (HPLC) ............................ 41 2.7.1 Analytical High performance liquid chromatography (HPLC) .......... 41 2.7.2 Preparative high performance liquid chromatography (PHPLC) ...... 42 2.8 Spray reagents ................................................................................. 42 2.8.1 Anisaldehyde reagent ...................................................................... 42 2.8.2 Vanillin sulphuric acid reagent ......................................................... 42 2.8.3 Ferric chloride reagent .................................................................... 42 iv 2.8.4 Dragendorff’s reagent ...................................................................... 42 2.9 Spectroscopic methods .................................................................... 43 2.9.1 Mass spectrometry .......................................................................... 43 2.9.1.1 Electro spray ES+ and ES- mass spectrometry ........................... 43 2.9.1.2 Accurate mass spectrometry ....................................................... 43 2.9.2 Nuclear Magnetic Resonance spectroscopy (1-D,2-D NMR) .......... 43 3 Results and discussion, phytochemistry of S. europaea ................. 44 3.1 Identification of the lipid fraction of S. europaea ............................... 44 3.2 Preparation and yields of crude glycosides ...................................... 48 3.3 Fractionation of crude glycosides of S. europaea ............................ 48 3.3.1 Normal phase silica gel ................................................................... 49 3.3.2 Sephadex LH20 .............................................................................. 52 3.3.3 Preparative Thin Layer Chromatography (PTLC) ............................ 54 3.3.4 Reverse phase silica gel C-18 ......................................................... 55 3.4 Natural products isolated from S. europaea ..................................... 58 3.4.1 Isolation of bis (2-ethylhexyl) phthalate 1 ....................................... 58 3.4.1.1 Further purification of 1 ................................................................ 59 3.4.1.2 Structure elucidation of 1 ............................................................. 61 3.4.2 Occurrence of 1 in nature ................................................................ 69 3.4.2.1 Occurrence of 1 in foods ............................................................. 69 3.4.2.2 Occurrence of 1 in water treatment plants ................................... 69 3.4.2.3 Occurrence of 1 in marine organisms .......................................... 70 3.4.2.4 Occurrence of 1 in bacteria organisms ........................................ 70 3.4.3 Pharmacological actions of 1 .......................................................... 70 3.4.4 Determination the origin of 1 in