University of Szeged Faculty of Pharmacy Department of Pharmacognosy PHYTOCHEMICAL, PHARMACOLOGICAL AND TOXICOLOGICAL STUDIES OF ALKALOID- AND SESQUITERPENE LACTONE- CONTAINING MEDICINAL PLANTS Ph.D. Thesis Tivadar Kiss Supervisors: Prof. Judit Hohmann DSc. Dezső Csupor PhD. Szeged, Hungary 2017 LIST OF PUBLICATIONS RELATED TO THE THESIS I. Kiss T; Orvos P; Bánsághi Sz; Forgó P; Jedlinszki N; Tálosi L; Hohmann J; Csupor D. Identification of diterpene alkaloids from Aconitum napellus subsp. firmum and GIRK channel activities of some Aconitum alkaloids FITOTERAPIA 2013; 90: 85-93. doi: 10.1016/j.fitote.2013.07.010 II. Kiss T; Szabó A; Oszánci G; Lukács A; Tímár Z; Tiszlavicz L; Csupor D. Repeated-dose toxicity of common ragweed on rats PLOS ONE 2017; 12: e0176818 (18p) doi: 10.1371/journal.pone.0176818 III. Kiss T; Borcsa B; Orvos P; Tálosi L; Hohmann J; Csupor D. Diterpene lipo-alkaloids with selective activities on cardiac K+ channels PLANTA MEDICA 2017; accepted doi: 10.1055/s-0043-109556 IV. Kiss T; Mácsai L; Csupor D; Datki Zs. In vivo screening of diterpene alkaloids using bdelloid rotifer assays Acta Biologica Hungarica 2017; 68 (4) accepted V. Kiss T; Cank K; Orbán-Gyapai O; Liktor-Busa E; Rutkovska S; Zomborszki Z; Pučka I; Németh A; Csupor D. Phytochemical and pharmacological investigation of Spiraea chamaedryfolia – A contribution to the chemotaxonomy of Spiraea genus BMC Research Notes 2017; submitted TABLE OF CONTENTS 1. Introduction.................................................................................................................................... 2 2. Aims of the study ........................................................................................................................... 4 3. Literature overview ........................................................................................................................ 5 3.1. Botany of the investigated species ......................................................................................... 5 3.1.1. Botany of Aconitum species ............................................................................................. 5 3.1.2. Botany of Spiraea genus ................................................................................................... 5 3.1.3. Botany of Ambrosia artemisiifolia L. ................................................................................ 6 3.2. Chemistry and pharmacology ................................................................................................. 7 3.2.1. Recent advances in phytochemistry and pharmacology of Aconitum species ................ 7 3.2.2. Aconite lipo-alkaloids ....................................................................................................... 9 3.2.3. Activity of aconite diterpene alkaloids on Na+ and K+ channels .................................... 10 3.2.4. Phytochemistry and pharmacology of Spiraea genus .................................................... 12 3.2.5. Phytochemistry and pharmacology of Ambrosia artemisiifolia ..................................... 14 4. Materials and methods ................................................................................................................ 19 4.1. Plant material ........................................................................................................................ 19 4.2. Ragweed puree ..................................................................................................................... 19 4.3. Diterpene alkaloids, lipo-alkaloids and fatty acids ................................................................ 19 4.4. Purification and isolation of the compounds ........................................................................ 20 4.5. Extraction of plant compounds ............................................................................................. 21 4.5.1. Preparation and phytochemical screening of A. napellus subsp. firmum extracts ........ 21 4.5.2. Preparation of crude alkaloid extract from Aconitum napellus subsp. firmum ............. 22 4.5.3. Preparation of Spiraea extracts for alkaloid-content screening .................................... 22 4.5.3. Preparation of Spiraea chamaedryfolia fractions .......................................................... 22 4.6. Identification and structure elucidation of compounds ....................................................... 23 4.6.1. Identification of diterpene alkaloids by LC-MS .............................................................. 23 4.6.2. Sesquiterpene lactone content identification in common ragweed puree ................... 23 4.6.3. Structure elucidation ...................................................................................................... 23 4.7. Pharmacological tests ........................................................................................................... 24 4.7.1. GIRK channel inhibitory assay ........................................................................................ 24 4.7.2. hERG channel inhibitory assay ....................................................................................... 24 4.7.3. Screening for antibacterial activity ................................................................................ 24 4.7.4. Xanthine oxidase assay .................................................................................................. 25 4.7.5. Bdelloid rotifer assays .................................................................................................... 25 4.7.6. Repeated dose toxicity on rats ....................................................................................... 26 4.8. Microscopical analysis ........................................................................................................... 27 4.9. Statistical analysis.................................................................................................................. 27 5. Results .......................................................................................................................................... 28 5.1. Isolation and detection of aconite alkaloids ......................................................................... 28 5.2. Investigation of Spiraea species for alkaloid content ........................................................... 31 5.3. Pharmacological activity of aconite alkaloids ....................................................................... 33 5.3.1. Activity on GIRK and hERG potassium channels ............................................................ 33 5.3.2. Activity of aconite alkaloids in bdelloid viability assays ................................................. 37 5.4. Pharmacological activity of Spiraea chamaedryfolia extracts .............................................. 38 5.5. Toxicology of ragweed puree ................................................................................................ 39 5.5.1. Sesquiterpene lactone content of the product .............................................................. 39 5.5.2. Clinical observation and blood chemistry ...................................................................... 39 5.5.3. General toxicological parameters .................................................................................. 40 6. Discussion ..................................................................................................................................... 42 6.1. Screening and isolation of diterpene alkaloids ..................................................................... 42 6.2. Biological activities ................................................................................................................ 43 6.2.1. Effects of aconite alkaloids on GIRK and hERG channels ............................................... 43 6.2.2. Biological activity of Spiraea chamaedryfolia extracts .................................................. 46 6.2.3. Toxicity of diterpene alkaloids on bdelloid rotifers ....................................................... 46 6.2.4. Toxicological evaluation of common ragweed ............................................................... 47 7. Summary ...................................................................................................................................... 50 8. References .................................................................................................................................... 52 Acknowledgements .......................................................................................................................... 59 Appendix I. ....................................................................................................................................... 60 Appendix II. ...................................................................................................................................... 66 ABBREVIATIONS 1D one-dimensional 2D two-dimensional Ac acetyl AcNi acetonitrile ACON aconitine ALP alkaline phosphatase ALT alanine aminotransferase BAE-11TRI 14-BzA-8-O-eicosa-11Z,14Z,17Z-trienoate BAE-DI 14-BzA-8-O-eicosa-11Z,14Z-dienoate BAE-PENT 14-BzA-8-O-eicosa-5Z,8Z,11Z,14Z,17Z-pentaenoate BAE-TETR 14-BzA-8-O-eicosa-5Z,8Z,11Z,14Z-tetraenoate BAL 14-BzA-8-O-laurate BAP 14-BzA-8-O-palmitate BAPO 14-BzA-8-O-palmitoleate BAS 14-BzA-8-O-stearate BEA-8TRI 14-BzA-8-O-eicosa-8Z,11Z,14Z-trienoate BSI body size index Bz benzoyl BzA benzoyl aconine CC open-column chromatography CH Cl dichloromethane 2 2 CON control COSY correlated spectroscopy COX cyclooxygenase CPC centrifugal planar chromatography CRC cellular reduction capacity DA diterpene alkaloid E-11TRI eicosa-11Z,14Z,17Z-trienoic acid E-8TRI eicosa-8Z,11Z,14Z-trienoic acid ED median effective dose 50 E-DI eicosa-11Z,14Z-dienoic acid E-EN eicosa-11Z-enoic acid E-PENT eicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid E-TETR eicosa-5Z,8Z,11Z,14Z-tetraenoic acid EtOAc ethyl acetate EtOH ethanol FA fatty acid g-BALL 14-BzA-8-O-γ-linolenate GFC gel-filtration chromatography GIRK G protein-coupled inwardly-rectifying potassium channel g-LIN γ-linolenic acid HD high dose hERG human ether-à-go-go-related gene HMBC heteronuclear multiple-bond correlation HSQC heteronuclear single-quantum correlation iv. intravenous JMOD J-modulated spin-echo experiment LA lipo-alkaloid LC liquid chromatography LD low dose 1 LD median lethal dose 50 LOX lipoxygenase MCF mastax contraction frequency assay MeOH methanol MRM multiple reaction monitoring MS mass spectrometry NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NOESY nuclear Overhauser effect spectroscopy PALO: palmitoleic acid PLC preparative layer chromatography SD standard deviation SEM standard error of means SiO silica gel 2 SONG songorine SPE solid phase extraction subsp subspecies syn. synonym TCM Traditional Chinese Medicine TLC thin-layer chromatography TSL toxicity and survival lifespan XO xanthine oxidase δ chemical shift 2 1. INTRODUCTION The kingdoms of plants, animals and fungi are rich and important sources of natural products. For centuries, various diseases have been treated with living organisms in raw or processed form. In modern medicine, drugs and natural products are important raw materials for the pharmaceutical industry and serve as lead compounds in the research and development of medicines. Taking into consideration the high ratio of pharmacons of natural origin to synthetic compounds [1], it is indeed the New Golden Age of natural product discovery [2]. Traditionally applied medicinal plants are of great importance. In case of effectiveness and safety, the authorisation often relies on long-term medicinal use. However, the pharmacovigilance data of medicinal herbs reveal a need for continuous re-evaluation [3]. Safety issues might be caused by minor compounds in drugs, which are exerting side effects after long-term, sometimes decades, of application. In many cases, the health risks related to the application of a medicinal plant remains hidden for centuries and only modern chemical and pharmacological studies are able to reveal the real toxicological character of traditionally applied herbs. Till recently, the per os application of Symphytum officinale roots was considered as safe in case of arthritis, thrombophlebitis, gout and in the treatment of diarrhoea. After recognising its hepatotoxic pyrrolizidine alkaloid-content, the application of this drug was limited [4]. Chelidonium majus L. is a popular plant with a wide spectrum of biological activities. Extracts, tincture and other remedies are in use, often applied internally. The per os application of these preparations has to be revised, since there might be a risk of cardiac side effects due to the activity of some extracts on hERG channels [5]. Modern approaches of phytochemistry and pharmacology might lead to new breakthroughs in drug research. The better understanding of traditional application and processing of medicinal herbs resulted in the discovery of novel mechanisms of action and new active constituents of these plants. Aconitum species are good examples how new results can open new ways in the research of traditionally used plants. Several highly toxic Aconitum species have been applied in Traditional Chinese Medicine. The raw herbal substances were cautiously processed in order to reduce their toxicity. Although neither the exact chemistry of these drugs, nor the pharmacological mechanisms have been thoroughly clarified, aconite drugs were among the most popular TCM medicines. Later, the chemical analysis of raw plant materials and processed drugs made it possible to identify their biological active compounds and their change during processing. In the raw plant material, diterpene alkaloids were identified. These compounds, mainly aconitine (18), are highly toxic. Furthermore, the toxicity of these compounds are depending on the number of ester groups present in the molecule, thus the most toxic ones are diester diterpene alkaloids 2 and monoester diterpene alkaloids are less toxic, while the least toxic are those without an ester group. Lipo-alkaloids are diterpene alkaloids esterified with long-chain fatty acids, usually at position C-8. These lipo-alkaloids are minor compounds in unprocessed drugs, and they are proved to be far less toxic than diterpene alkaloids. During processing of the herbal substance, the amount of tri- and diester alkaloids is decreasing, while the amount of unesterified diterpene alkaloids and lipo-alkaloids is increasing [6]. The understanding of these chemical changes [7] and identification of lipo-alkaloids as biologically active constituents in processed aconite drugs triggered experiments to semisynthesize new compounds and investigate their pharmacological effects [8]. Even though the well-known Aconitum species were removed from the Western Pharmacopoeias, natural products of aconite origin are promising as anti-inflammatory or antiarrhythmic pharmacons. The analysis of structure-activity relationships of diterpene alkaloids led to the development of new diterpene alkaloid-based medicines and the discovery of promising pharmacones [9]. In the recent years, medicinal plants are typically marketed as food supplements. Several products contain herbs that have not been extensively used before, and there are no pharmacological, toxicological and phytochemical data available to serve as basis for the assessment of their effectiveness, safety and quality. Plants that have not been consumed to a significant degree by humans in the European Union prior to 1997 are considered as novel food [10]. One example for novel food (though unauthorised) is Ambrosia artemisiifolia, which has become quite popular as a medicinal plant in the recent years in Hungary. However, this plant has not been consumed or used as a medicinal herb before, hence its toxicological profile is unexplored and the risk related to its consumption is unknown. 3 2. AIMS OF THE STUDY In 2000, the research group of the Department of Pharmacognosy (University of Szeged) started a screening programme for isolation and identification of Ranunculaceae alkaloids in order to find new biologically active compounds and the rational explanations of the folk medicinal use of the toxic species. Later, the scope of the experiments was extended to the safety pharmacology, toxicology and chemistry of traditional and newly discovered medicinal plants. According to this comprehensive approach, the aim of the present work was the chemical, pharmacological and toxicological investigation of diterpene alkaloid-containing traditional medicinal plants and one, only recently applied species, to reveal the dangers of their use and to identify their potential role in modern medicine. In order to achieve the aims, the main tasks were:  Review the literature of the Aconitum, Spiraea genera and Ambrosia artemisiifolia, from aspects of the chemistry and pharmacological properties of the plants.  Diterpene alkaloid extraction and identification from Aconitum napellus subsp. firmum.  Investigation of the activity of Ranunculaceae diterpene alkaloids and semisynthetic lipo- alkaloids on GIRK and hERG channels.  Evaluation of antiarrhythmic potential of Ranunculaceae diterpene alkaloids.  In vivo toxicological evaluation of diterpene alkaloids using bdelloid rotifer assays.  Extraction of Spiraea species with various alkaloid extraction methods for alkaloid-content screening, and investigation of the antibacterial and xanthine oxidase inhibitory activity.  In vivo toxicological examination of common ragweed on rats. 4 3. LITERATURE OVERVIEW 3.1. Botany of the investigated species 3.1.1. Botany of Aconitum species The Aconitum genus belongs to Ranunculaceae family, order of Ranunculales, superorder of Ranunculanae, subclass of Ranunculidae, Dicotyledonopsida class, Angiospermatophytina subdivision, Spermatophyta division. The phylogeny of this genus is extremely complex, due to the allopatric and parapatric speciation. The genus, comprised of 300 species, is divided into three subgenera: only one species is rendered to Gymnaconitum (Stapf.) subgenus; Lycoctonum (DC) Peterm. contains around 50 species; the largest subgenus is Aconitum with 250 species [11]. Since molecular phylogenetic studies suggest that the hotspot of Aconitum species speciation was located to Himalaya Mountains, it is reasonable that vast majority of Aconitum species can be found in Asia (cca. 220) [12]. According to Flora Europaea only 7 species are part of the European flora, from which 5 species are native to the Carpathian Basin [13]. Aconitum napellus subsp. firmum Rchb. Gáyer (syn. Aconitum firmum Rchb.) is a perennial herb native to Carpathian Basin, distributed from Czech Republic to East Carpathians in Transylvania. The plant is 50-150 cm tall. The dark-green, alternate leaves are palmate, divided into 5-7 segments. The inflorescence is few-flowered, with 20-35 mm high, blue and hairy flowers. 3.1.2. Botany of Spiraea genus The Spiraea genus, comprising of approximately 100 species, belongs to Rosaceae family, order of Rosales, subclass of Rosidae, Magnoliopsida class, Magnoliphyta subdivision, Spermatophyta division. [14]. Spiraea species are widely distributed all over the world. The centre of the distribution is Asia [15], while in Europe 20 species are part of the Flora [16]. The genus was further divided according to the morphology of the inflorescences into Spiraria, Calospira and Chamaedryon groups. Since the monophyletic origin of these groups is not supported by the latest molecular phylogeny, the classical division has to be revised [17]. Spiraea species are perennial, multi-stemmed, deciduous, spreading and upright featured shrubs with corymb inflorescence. Spiraea creanata L. has reddish-brown branches and grows 1 m tall. The greyish-green leaves are lanceolate to obovate and the 20 mm wide inflorescence comprises of 10 white flowers [18]. S. salicifolia L. is 1.5-2.0 m high shrub with non-spreading branches, and alternate lanceolate leaves. Inflorescences are 5-12 cm high conical panicles with pink or rose flowers. S. nipponica Maxim. is a 1.2-2.5 m tall shrub, with simple leaves with serrated leaf tips. The inflorescence is umbel-like raceme, with white flowers. S. x vanhouttei, 1.8-2.5 m high 5