DISSERTATION Titel der Dissertation Molecular and phytochemical analyses of the genus Origanum L. (Lamiaceae) angestrebter akademischer Grad Doktorin der Naturwissenschaften (Dr. rer.nat.) Verfasserin / Verfasser: Mag. Brigitte Lukas Dissertationsgebiet (lt. Stu- Botanik dienblatt): Betreuerin / Betreuer: Univ.-Prof. Dr. Rosabelle Samuel Wien, im Juni 2010 CONTENTS INTRODUCTION .................................................................................................................................. 1 PART 1 Tracing evolutionary relationships in section Majorana Benth. ...................................................... 15 Paper 1 .................................................................................................................................................... 17 B. Lukas, R. Samuel, E. Mader, K.H.C. Baser, H. Duman, J. Novak (2010) Marjoram versus Oregano: Complex evolutionary relationships in the section Majorana Benth., genus Origanum L. (Lamiaceae). To be submitted. PART 2 Phytochemical aspects of the genus Origanum .................................................................................. 61 Paper 2 .................................................................................................................................................... 63 B. Lukas, C. Schmiderer, U. Mitteregger, C. Franz, J. Novak (2008) Essential Oil Compounds of Origanum vulgare L (Lamiaceae) from Corsica. Natural Product Communications 3(7): 1127-1131. Paper 3 .................................................................................................................................................... 71 B. Lukas, C. Schmiderer, C. Franz, J. Novak (2009) Composition of Essential Oil Compounds from Different Syrian Populations of Origanum syriacum L. (Lamiaceae). Journal of Agricultural and Food Chemistry 57(4): 1362-1365. Paper 4 .................................................................................................................................................... 77 B. Lukas, C. Schmiderer, U. Mitteregger, J. Novak (2010) Arbutin in marjoram and oregano. Food Chemistry 121: 185-190. PART 3 Tracing factors that influence the qualitative and quantitative essential oil composition in Origanum ............................................................................................................................................... 85 Paper 5 .................................................................................................................................................... 87 B. Lukas, R. Samuel, J. Novak (2009) Oregano or Marjoram? The γ-terpinene synthase affects chemotype formation in the genus Origanum. Israel Journal of Plant Sciences: accepted. ANNEX TO PART 1 .......................................................................................................................... 105 J. Novak, B. Lukas, K. Bolzer, S. Grausgruber-Gröger, J. Degenhardt (2008) Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag. Molecular Ecology Resources 8: 599-601. ................................................................... 107 E. Mader, B. Lukas, J. Novak (2009) A Strategy to Setup Codominant Microsatellite Analysis for High-Resolution-Melting-Curve-Analysis (HRM). BMC Genetics 9:69. .......................................... 113 M. Müller, B. Lukas, J. Novak, T. Simoncini, A. R. Genazzani, A. Jungbauer (2008) Oregano: A Source for Peroxisome Proliferator-Activated Receptor γ Antagonists. Journal of Agricultural and Food Chemistry 56(24): 11621-11630. ................................................................................................ 123 ANNEX TO PART 2 .......................................................................................................................... 135 J. Novak, B. Lukas, C. Franz (2008) The Essential Oil Composition of Wild Growing Sweet Marjoram (Origanum majorana L. Lamiaceae) from Cyprus – Three Chemotypes. Journal of Essential Oil Research 20: 339-341. .................................................................................................................... 137 A. Lamien-Meda, B. Lukas, C. Schmiderer, C. Franz, J. Novak (2009) Validation of a Quantitative Assay of Arbutin using Gas Chromatography in Origanum majorana and Arctostaphylos uva-ursi Extracts. Phytochemical Analysis 20: 416-420. ................................................................................... 143 ANNEX TO PART 3 .......................................................................................................................... 151 J. Novak, B. Lukas, C. Franz (2009) Temperature Influences Thymol and Carvacrol Differentially in Origanum spp. (Lamiaceae). Journal of Essential Oil Research: accepted. ......................................... 153 CONCLUSIONS AND PERSPECTIVES ........................................................................................ 163 SUMMARY ......................................................................................................................................... 171 ZUSAMMENFASSUNG .................................................................................................................... 173 CURRICULUM VITAE .................................................................................................................... 175 INTRODUCTION Introduction The genus Origanum L. comprises 43 species and 18 hybrids (Carlström, 1984; Danin, 1990; Danin and Künne, 1996; Duman et al., 1995; Duman et al., 1998; Ietswaart, 1980). Origanum species are usually subshrubs or woody perennial herbs that reach heights of up to 80 cm and have ovate leaves and white or purple flowers (Figure 1). The aerial organs of the plant, especially stems, leaves and bracts, are, often densely, covered by glandular and non-glandular hairs. Figure 1: O. cordifulium, an endemic to Cyprus (Troodos Mountains), in its natural habitat. Most of the Origanum species are locally distributed within the Mediterranean region where they grow in mountainous areas on dry, rocky, often calcareous soils. Origanum vulgare L. possesses by far the largest distribution and can be found throughout the Mediterranean region, in most parts of the Euro- Siberian region as well as in the Irano-Turanian region (Figure 2). Figure 2: Distribution of the genus Origanum (dotted line: area of all sections; solid line: all sections except the section Origanum) (Ietwsaart, 1980). 3 About 60 percent of the Origanum taxa are recorded to grow in Turkey indicating this geographical area as the gene centre of the genus. The rate of endemism is high and about 70 percent of the Origanum species occur on an island or mountain (group) only. The glandular hairs covering the aerial parts of Origanum plants secrete an essential oil with a very characteristic odour. Due to these essential oils Origanum species have locally been collected for centuries, for flavouring traditional dishes as well as for numerous purposes in traditional medicine. Today, two aromatic qualities of Origanum plant material, marjoram (O. majorana L.) and oregano (usually from O. vulgare and O. onites L.) are commercially traded and widely used all over the world as popular spices. Beside their sensorial qualities oregano and marjoram possess further valuable properties. Amongst others, antifungal, antibacterial, antioxidant, analgesic, and anticancer activities were observed (reviewed in Baričevič and Bartol, 2002). Apart from the traditional use as kitchen herb and folk remedy, preparations of Origanum plant material can be used in medicine as well as in feed-, food-, pharma- and cosmetic-industry in a wide variety of ways. Despite its economic importance the genus Origanum is often referred to as an underutilized taxon with its potential for utilization not yet fully explored (Padulosi, 1997). This is partly due to a lack of knowledge on chemical and genetical diversity but also to the complex taxonomy of the genus that is complicated by the extent of morphological variation Origanum taxa exhibit in nature. With the intend to contribute to a refined and more complete picture of the genus my PhD studies addressed both, phylogenetic as well as phytochemical analyses of the genus Origanum. The following paragraphs of the introduction provide an overview about the systematical and phytochemical knowledge currently available and introduce the particular aims of my studies. In the subsequent parts of the thesis the results of my investigations as well as the major conclusions are summarized. Taxonomy of the genus Origanum The Lamiaceae family is a difficult plant group and its classification above the genus level has been heavily debated. According to the current status eight subfamilies are recognized (Cantino et al. 1992). The Nepetoiodeae, the largest and economically most important subfamily, is further divided into the four tribes Escholtzieae, Lavanduleae, Mentheae and Ocimeae (Cantino et al., 1992; Wagstaff et al., 1995). The genus Origanum is placed within the Mentheae tribe, with Thymus L., Thymbra L. and Micromeria Benth. as close relatives (Bräuchler et al., 2010; Ietswaart 1980). The generic limits of Origanum were subject of intensive taxonomic discussions and up to recent times two different genus concepts were in use. Some authors followed Bentham’s original genus concept (1834) and treated Amaracus Benth., Majorana Benth. and Origanum Benth. as separate genera. Others followed the Linnaean genus