University of Bath PHD Phytochemistry of norditerpenoid alkaloids from Aconitum and Delphinium Ahmed, Mai Award date: 2016 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 05. Oct. 2021 Phytochemistry of norditerpenoid alkaloids from Aconitum and Delphinium Mai Abdelhadi Mohamed Ahmed A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Pharmacy and Pharmacology 2015 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and they must not copy it or use material from it except as permitted by law or with the consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. Signed on behalf of the Faculty of Science. Signed: ………………………………… Date: ….…………………….. Abstract Aconitum and Delphinium genera are important rich sources of toxic C19- diterpenoid alkaloids. The alkaloidal content of A. napellus and D. elatum seeds have been investigated in detail. After maceration, crude alkaloidal extracts were obtained and then purified by repeated column chromatography (over silica and alumina gels) to sample homogeneity yielding five known C19-diterpenoid alkaloids from A. napellus, aconitine, neoline, 14-O-acetyltalatisamine, 14-O-benzoylaconine, and taurenine, and two others from D. elatum, delpheline (also including its X-ray single crystal analysis) and methyllycaconitine (MLA). These examples showed that mass spectrometry hyphenated with HPLC or other chromatography can be used as a tool for rapid alkaloid content screening of different extracts. NMR spectroscopic (using a variety of techniques and nuclei) data are presented in support of the first report of iminodelsemine A/B as an imino-alkaloid artefact from D. elatum. A detailed chromatographic study across different pH ranges, and over different solid supports, of aconitine and its main degradation product, 14-O-benzoyl-8-O-methylaconine, together with its semi-synthesis and that of its deuterated analogue are reported within studies to minimize artefact formation during the storage or extraction of A. napellus norditerpenoid alkaloids. Likewise, from D. elatum seeds, as a model source of Delphinium alkaloids, we compared the alkaloid yield using different extraction techniques and conventional chromatographic separations. The structures were confirmed by NMR spectroscopy and mass spectrometry. An NMR spectroscopic approach for the pKa determination of some C19-diterpenoid alkaloids has been developed. A modified calculation method for fatty acid composition quantification has also been developed using 1H- NMR spectroscopic methods. 2 This Thesis is dedicated to my parents 3 Acknowledgements My special appreciation goes to my supervisors Dr Ian S. Blagbrough and Dr Michael G. Rowan for their support, patience, valuable constructive suggestions, and encouragement during the planning and development of this research work. I express my thanks to Dr Tim Woodman for assistance with NMR spectroscopic experiments, Mr Chris Rehbein and Dr Anneke Lubben for mass spectrometry, and Dr Gabriele Kociok-Kohn for the X-ray crystallography. A special thanks goes to Don Perry for his valuable technical support, Kevin Smith and all the technical staff in the Department of Pharmacy and Pharmacology, University of Bath, for their help. I am also grateful to all of my colleagues in laboratory 5W3.14 for their help and friendship. I gratefully thank the Egyptian Government for granting me a PhD scholarship. Finally, my greatest gratitude goes to my dear parents for their care, support, and continuous encouragement throughout my studies without which this work would not have been possible. 4 Abbreviations ̊C degree Celsius Å Angstrom Ac acetyl Aq aqueous As anisoyl Bz benzoyl COSY correlated spectroscopy CV column volumes 2D two dimensional Da Dalton DAD diode array detector DCM dichloromethane DDA di-ester diterpenoid alkaloid DEPT distortionless enhancement by polarization transfer ED50 median effective dose ent enantiomer ESI electrospray ionisation h hour H2BC heteronuclear 2-bond correlation HMBC heteronuclear multiple-bond correlation HMQC heteronuclear multiple-quantum coherence HSQC heteronuclear single quantum coherence HPLC high performance liquid chromatography HP-TLC high performance thin layer chromatography HRMS high resolution mass spectrometry Hz hertz ibut isobutyl IR infrared J coupling constant kg kilo-gram LC-MS liquid chromatography-mass spectrometry LDA lipo-diterpenoid alkaloid 5 M molar MDA mono-ester diterpenoid alkaloid Me methyl MeCN acetonitrile MeOH methanol mg milli-gram µg micro-gram MH+ protonated molecular ion MHz mega-Hertz min minute mL milli-litre MLA methyllycaconitine mm milli-metre MS mass spectrometry MW relative molecular weight m/z mass over charge nAChR nicotinic acetylcholine receptors NMR nuclear magnetic resonance NOESY nuclear Overhauser effect spectroscopy ppm parts per million Pro propyl TLC thin layer chromatography UPLC ultra performance liquid chromatography UV ultraviolet v/v volume by volume w/v weight by volume 6 Contents Abstract 2 Dedication 3 Acknowledgements 4 Abbreviations 5 Chapter 1. Introduction 1.1. Overview of diterpenoid alkaloids 10 1.2. Classification of diterpenoid alkaloids 1.3. C19-Diterpenoid alkaloid distribution, numbering and ring names 1.4. Suggested biosynthetic pathways of diterpenoid alkaloids 1.5. Structure activity relationship of diterpenoid alkaloids 1.6. Toxicity of Aconitum alkaloids 1.7. Poisoning from Aconitum alkaloids 1.8. Pre-treatment techniques of Aconitum carmichaelii “Fuzi” 1.9. Pharmacological effects of Aconitum and Delphinium alkaloids 1.10. Aims Chapter 2. Review of the literature 2.1. Diterpenoid alkaloids isolated from Aconitum species 29 2.2. Diterpenoid alkaloids isolated from Delphinium species Chapter 3. Experimental 3.1. General techniques and instrumentation, chemicals, reagents, and solvents 59 3.2. Plant materials 3.3. Thin layer chromatography (TLC) 3.4. Column chromatography 3.5. High performance liquid chromatography (HPLC) 3.6. Spectroscopic analysis 3.7. X-ray crystallography 3.8. Extraction of diterpenoid alkaloids from Aconitum napellus seeds using different solvents 3.9. Monitoring aconitine degradation product formation using HPLC/HRMS 7 3.9.1. Semi-synthesis of 14-O-benzoyl-8-O-methylaconine 3.9.2. Semi-synthesis of the deuterated aconitine artefact 3.10. Screening of diterpenoid alkaloids in A. napellus acetone extract 3.11. Extraction and identification of some C19-diterpenoid alkaloids from Aconitum napellus seeds 3.11.1. Isolation of aconitine 3.11.2. Isolation of neoline 3.11.3. Isolation of 14-O-acetyltalatisamine 3.11.4. Isolation of 14-O-benzoylaconine 3.11.5. Isolation of taurenine 3.12. Extraction of diterpenoid alkaloids from Aconitum carmichaelii seeds 3.13. Extraction of diterpenoid alkaloids from Aconitum lycoctonum seeds 3.14. Extraction of diterpenoid alkaloids from Delphinium elatum seeds 3.15. Isolation of some C19-diterpenoid alkaloids from Delphinium elatum seeds 3.15.1. Isolation of delpheline 3.15.2. Isolation of methyllycaconitine 3.15.3. Isolation of iminodelsemine A/B 3.15.4. Effect of different bases on Iminodelsemine A/B 3.16. HPLC method optimisation of aged aconitine sample 3.16.1. Monitoring aged aconitine sample on different stationary phases 3.16.2. Investigating different mobile phase polarities 3.16.3. The influence of the eluent pH on the chromatographic separation of aconitine and 14-O-benzoyl-8-O-methylaconine 3.17. pKa determination by NMR spectroscopy 3.18. Determination of fatty acids composition 3.18.1. Using gas chromatography/mass spectrometry GC/MS 3.18.2. Using NMR spectroscopy Chapter 4. Phytochemical study on norditerpenoid alkaloids 4.1. Extraction 84 4.2. Stability of aconitine in different organic solvents 4.2.1. Monitoring aconitine degradation products using HPLC/HRMS 4.2.2. Possible synchronous fragmentation of aconitine 8 4.2.3. Isolation and identification of aconitine artefact 4.2.4. Semi-synthesis of the deuterated aconitine artefact 4.3. Screening of A. napellus acetone extract by HRMS 4.4. Alkaloids extracted from Aconitum lycoctonum seeds 4.5. Extraction of Delphinium elatum seeds 4.5.1. Methyllycaconitine 4.5.2. Delpheline 4.5.3. Iminodelsemine A/B Chapter 5. Analysis of aged aconitine using different HPLC methods 5.1. Method development 152 5.2. Reversed-phase