Dissertation submitted to the Combined Faculties for Natural Sciences and for Mathematics of Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Farukh Sharopov born in Yovon, Tajikistan Oral examination: 17. 09. 2015 Phytochemistry and bioactivities of selected plant species with volatile secondary metabolites Referees: Prof. Dr. Michael Wink Prof. Dr. Stefan Wölfl Table of contents Zusammenfassung............................................................................................................ IV Summary .......................................................................................................................... V Acknowledgements........................................................................................................... VI Publication and posters .................................................................................................. VII Chapter 1. General introduction........................................................................................ 1 1.1. Medicinal plants.......................................................................................................... 1 1.2. Plant secondary metabolite......................................................................................... 2 1.3. Biological activity and mode of action of secondary metabolite with special reference to essential oil..................................................................................................... 2 1.4. Objective of the present study..................................................................................... 5 Chapter 2. Literature review of the investigated plants............................................... 8 2.1. Anethum graveolens L. - Apiaceae............................................................................ 8 2.2. Ferula clematidifolia Koso-Pol. - Apiaceae............................................................... 9 2.3. Foeniculum vulgare Millar - Apiaceae....................................................................... 9 2.4. Galagania fragrantissima Lypsky - Apiaceae............................................................ 9 2.5. Pastinaca sativa L. - Apiaceae................................................................................... 10 2.6. Achillea filipendulina Lam. - Asteraceae.................................................................... 10 2.7. Artemisia absinthium L. - Asteraceae......................................................................... 11 2.8. Artemisia rutifolia Stephan ex Spreng. - Asteraceae.................................................. 12 2.9. Artemisia scoparia Waldst & Kit. - Asteraceae.......................................................... 13 2.10. Polychrysum tadshikorum (Kudr.) Kovalevsk. - Asteraceae.................................... 14 2.11. Tanacetum parthenium L. Schultz-Bip. - Asteraceae............................................... 14 2.12. Tanacetum vulgare L. - Asteraceae.......................................................................... 15 2.13. Galega x hartlandii - Fabaceae................................................................................ 15 2.14. Geranium macrorrhizum L.- Geraniaceae................................................................ 16 2.15. Philadelphus x purpureomaculatus - Hydrangeaceae............................................. 16 2.16. Hypericum perforatum L. - Hypericaceae................................................................ 17 2.17. Hypericum scabrum L. - Hypericaceae.................................................................... 18 2.18. Hyssopus seravschanicus Pazij - Lamiaceae........................................................... 18 2.19. Melissa officinalis L. - Lamiaceae........................................................................... 19 2.20. Mentha longifolia (L.) Huds. - Lamiaceae............................................................... 20 2.21. Ocimum basilicum L. - Lamiaceae........................................................................... 21 2.22. Origanum tyttanthum Gontsch. - Lamiaceae............................................................ 22 2.23. Salvia discolor Kunth. - Lamiaceae........................................................................ 23 2.24. Salvia officinalis L. - Lamiaceae............................................................................... 23 2.25. Salvia sclarea L. - Lamiaceae................................................................................... 23 2.26. Ziziphora clinopodioides Lam. - Lamiaceae........................................................... 24 2.27. Ficus carica L. - Moraceae....................................................................................... 25 2.28. Papaver somniferum L. - Papaveraceae.................................................................... 26 i Conclusions........................................................................................................................ 26 Chapter 3. Materials and methods................................................................................. 27 3.1. Plant material.............................................................................................................. 27 3.2. Reagents and chemicals.............................................................................................. 27 3.3. Preparation of crude methanol and water extracts...................................................... 30 3.4. Isolation of essential oils............................................................................................. 30 3.5. Separation of alkaloids of the Papaver somniferum................................................... 31 3.6. Gas liquid chromatography/flame ionization detector (GLC/FID)............................ 32 3.7. Gas liquid chromatography/mass spectrometry (GLC/MS) analysis......................... 32 3.8. High performance liquid chromatography analysis.................................................... 33 3.9. Determination of total phenolic contents.................................................................... 33 3.10. Determination of total flavonoid contents................................................................ 34 3.11. DPPH radical scavenging assay................................................................................ 34 3.12. Free radical scavenging capacity using the ABTS assay.......................................... 34 3.13. Antioxidant activity using the linoleic acid peroxidation method............................ 35 3.14. Ferric reducing antioxidant power............................................................................ 35 3.15. Inhibition of 5-lipoxygenase (anti-inflammatory activity)....................................... 36 3.16. Cytotoxicity assay..................................................................................................... 36 3.16.1. Cytotoxicity of combination of doxorubicin with essential oils............................ 36 3.17. TLC bioautography assay......................................................................................... 36 3.18. Antibacterial activity................................................................................................. 37 3.19. The hemolytic activity of essential oils..................................................................... 37 3.20. Microscopy................................................................................................................ 38 3.21. Statistics.................................................................................................................... 38 Chapter 4. Phytochemistry part..................................................................................... 39 4.1. Chemical composition of the essential oil of Ferula clematidifolia........................... 39 4.2. Chemical composition of the essential oil of Foeniculum vulgare............................. 40 4.3. Chemical composition of the essential oil of Galagania fragrantissima................... 43 4.4. Chemical composition of the essential oil of Geranium macrorrhizum..................... 44 4.5. Chemical composition of the essential oil of Pastinaca sativa.................................. 45 4.6. Chemical composition of the essential oil of Philadelphus x purpureomaculatus .... 48 4.7. Chemical composition of the essential oil of Polychrysum tadshikorum .................. 49 4.8. Chemical composition of the essential oil of Salvia dsicolor..................................... 51 4.9. Chemical composition of the essential oil of Salvia officinalis.................................. 53 4.10. Chemical composition of the essential oil of Salvia sclarea.................................... 56 4.11. Chemical composition of the essential oil of Tanacetum parthenium...................... 57 4.12. Chemical composition of the essential oil of Tanacetum vulgare............................ 59 4.13. Alkaloid contents and composition of Papaver somniferum.................................... 61 4.14. Content of polyphenols in methanol extracts of Salvia sclarea, S. officinalis, and S. discolor .......................................................................................................................... 62 Conclusions........................................................................................................................ 64 ii Chapter 5. Antioxidant activity.....................................................................................