This work describes the phytochemical study of Hippeastrum solandriflorum(Amaryllidaceae)aiming the isolation and structural elucidation of new bioactive compounds, as well as its pharmacological investigation. The chemical investigation realized with the EtOH extract from bulbs, through chromatographic methods, including HPLC (reverse phase), resulting inthe isolation of ten compounds: a furan derivative: 5-(hydroxymethyl)furan-2-carbaldehyde(HS-1), two phenolic derivatives: piscidic acid (HS-2), eucomic acid (HS-3), and seven isoquinoline alkaloids: narciclasin ( HS-4), 2α-hydroxypseudolycorin (HS-5), 10α-hydroxy homolycorin (HS-6), galantamin (HS-7), sanguinin (HS-8),N-oxid galantamin (HS-9) andnarcissidin (HS10). The alkaloids (HS-5) and (HS-6) are being reported for the first time inthe literature, while the other ones have been isolated for the first time in the investigated species. The structures of all isolated compounds were determined based on spectrometricmethods (IR, HRMS, NMR 1H and13Câ1D and 2D), besides comparison with published data. The cytotoxic potential of all alkaloids were evaluated against several tumor cell lines:colon (HCT-116), leukemia (HL-60), ovary (OVCAR-8) and brain (SF-295) showing IC50ranging from 0.01 to 35.7 μM. / Este trabalho descreve o estudo fitoquÃmico de Hippeastrum solandriflorum (Amaryllidaceae) visando o isolamento e elucidaÃÃo estrutural de novos constituintes quÃmicos bioativos, bem como o estudo farmacolÃgico dos compostos obtidos. A investigaÃÃo quÃmica realizada com o extrato etanÃlico dos bulbos, atravÃs de mÃtodos cromatogrÃficos, incluindo CLAE (fase reversa), resultou no isolamento e identificaÃÃo de dez substÃncias, sendo um derivado do furano: 5-(hidroximetil)furan-2-carbaldeido (HS-1), dois derivados fenÃlicos: acido piscidico(HS-2), acido eucÃmico (HS-3) e sete alcaloides isoquinolÃnicos: Narciclasina (HS-4), 2α-hidroxipseudolicorina (HS-5), 10αhidroxi-homolicorina (HS-6), Galantamina (HS-7), Sanguinina (HS-8),N-oxido galantamina (HS-9), Narcissidina (HS-10). Os alcaloides (HS-5)e (HS-6) esta sendo relatado pela primeira vez na literatura e os demais como sendo inÃditos na espÃcie estudada. As substÃncias isoladas tiveram suas estruturas elucidadas por mÃtodos espectromÃtricos (IV, IES-EM e RMN de1H e13C 1D e 2D), alÃm de comparaÃÃo com dados da literatura. O potencial citotÃxicodos alcaloides isolados foi avaliado frente Ãs linhagens decÃlulas tumorais humanas: cÃlon (HCT-116), leucemia (HL-60), ovÃrio (OVCAR-8) e cÃrebro (SF-295) mostrando valores IC50 variando 0,01â35,7 μM.

La galanthamine est un alcaloïde isoquinoléique, utilisé dans le monde entier pour le traitement palliatif de la maladie d’Alzheimer en raison de son pouvoir inhibiteur de l’acétylcholinestérase. Cet alcaloïde est extrait à partir de bulbes d’Amaryllidaceae, Leucojum aestivum, Galanthus nivalis, et Narcissus sp. ou obtenu par synthèse chimique. La difficulté principale de cette dernière réside dans le respect de la configuration des centres d'asymétrie. La culture de tissus in vitro pourrait constituer une alternative intéressante pour obtenir ce composé à haute valeur ajoutée. Le premier objectif de ce projet, vise à améliorer l’accumulation de cet alcaloïde par les biais des biotechnologies. Le second objectif est de rechercher par criblage phytochimique (HPLC, LCMS, GCMS, et HPTLC-MS) de bulbes in vitro et in vivo d’Amaryllidaceae, de nouveaux alcaloïdes biologiquement actifs. Le troisième objectif porte sur l’étude de la voie de biosynthèse en vue de réaliser une synthèse biomimétique de la galanthamine. Nous avons établi des cultures in vitro de 3 espèces d’Amaryllidaceae. La variation des paramètres exogènes a conduit à une accumulation accrue d’alcaloïdes (0,02 à 0,2 % MS). Le criblage phytochimique a conduit à l’identification d’alcaloïdes nouveaux issus des cultures in vitro, n’existant pas in vivo, et présentant un puissant pouvoir inhibiteur de l’acétylcholinestérase (40 à 80 % Inh). L’ajout, de la 4’-Ométhyl-d3-norbelladine aux cultures in vitro a conduit à sa métabolisation en trois types d’alcaloïdes deutérés. Une stimulation de la synthèse et du relargage de la galanthamine native (0,15 % MS et 0,16 % Milieu) a été observée en présence du précurseur deutéré / Galanthamine is an Amaryllidaceae alkaloid used worldwide for the symptomatic treatment of Alzheimer’s disease because of his capacity to inhibit the acetylcholinesterase enzyme. There are two galanthamine sources for medical applications. One is the total synthesis, a complicated process because galanthamine has three asymmetric carbons, requiring stereochemically controlled synthesis. Galanthamine is also extracted from bulbs of some Amaryllidaceae such as Leucojum aestivum, Galanthus nivalis, and Narcissus sp.. The first aim of this work is to improve the accumulation of this alkaloid using biotechnologies. The second aim consists on the phytochemical screening (HPLC, LCMS, GCMS, et HPTLCMS) of in vivo and in vitro Amaryllidaceae bulbs, in order to identify new alkaloids with important pharmacological activities. Finally, the third aim concerns the study of the biosynthesis pathway in order to establish a biomimetic synthesis of galanthamine. Therefore, we established in vitro cultures of three Amaryllidaceae species. The variation of exogenousparameters led to the obtainment of high galanthamine accumulation (0.02 to 0.2 % DW). The phytochemical screening showed new alkaloids in extracts of in vitro cultures, which did not exist in in vivo extracts, and possessing high acetylcholinesterase activity (40 to 80 % Inh). The 4’-O-methyl-d3-norbelladine is incorporated into three different groups ofAmaryllidaceae alkaloids. The addition of the labelled precursor to shoot cultures stimulated the synthesis of native galanthamine (0.15 % DW and 0.16 % Culture medium)

In this diploma Thesis I dealt with the synthesis of nitrogen compounds that contain quaternary carbon centres and their structure resembles natural substances, specifically alkaloids from the Amaryllidaceae family. Halocarbocyclization or Heck reaction was planned as a key step of the synthesis, which should lead to the formation of quaternary carbon centres. First, starting nitriles and esters with five- and six-membered rings were prepared. Subsequently, the method for the preparation of a stable bicyclic ketone with a five-membered ring from an ester was optimized. The next part of the Thesis is devoted to the synthesis of a substrate for the Heck reaction from the prepared ketone, which was then used for the preparation of the alkaloid skeleton with a quaternary carbon centre, unfortunately without success so far. In the last part of the work, a bicyclic ketone with a six-membered ring, from which it is possible to prepare a substrate for the Heck reaction in several steps already used for five-membered substances, was prepared from the nitrile by carbopalladation. Key words: synthesis, quaternary carbon centres, Heck reaction, halocarbocyclisation, Amaryllidaceae alkaloids

Lycorin gilt bei vielen Amaryllidaceae als Hauptalkaloid und die Aufnahme dieser Pflanzen ist eine häufige Vergiftungsursache bei Mensch und Tier. Als Hauptsymptome infolge dieser Pflanzenvergiftungen werden Nausea und Emesis genannt, aber systematische Untersuchungen zu diesen biologischen Effekten, zum Wirkmechanismus und zur Pharmakokinetik von Lycorin, das als auslösendes Agens angenommen wird, existieren bislang nicht. In der vorliegenden Arbeit werden die Zusammenhänge zwischen verabreichter Lycorin-dosis und Lycorin-induzierter Nausea und Emesis, die Beeinflussbarkeit dieser emetischen Effekte durch etablierte Antiemetika und die Pharmakokinetik von Lycorin in einem cross-over und vehikel-kontrollierten Design in vivo untersucht. Die Studie wurde an elf Beagle-Hunden beider Geschlechter durchgeführt. Die Lycorin-induzierten emetischen Effekte wurden quantifiziert und über Videoaufzeichnungen zeitnah dokumentiert. Nausea wird hierbei mittels eines Scoring-Systems quantifiziert, während die Parameter Latenzzeit, Dauer und Anzahl der Brechakte zur Beurteilung der Emesis herangezogen werden. Die subkutane Applikation von Lycorin induziert, beginnend ab einer Dosis von 0,5 mg/kg KGW Nausea und Vomitus. Eine statistische Signifikanz ist allerdings erst ab 1,0 mg/kg und ein maximaler emetischer Effekt bei einer Dosis von 2 mg/kg (ED100) zu verzeichnen. Die Ergebnisse zeigen eine Korrelation zwischen applizierter Lycorin-Dosis und Nausea-Score sowie der Anzahl der Brechakte. Lycorin-induzierte Nausea und Emesis sind in den vorliegenden Untersuchungen selbstlimitierend und dauern maximal 2,5 Stunden an. Lycorin weist in den untersuchten Dosierungen von 0,25 mg/kg bis 2,0 mg/kg eine lineare Plasmakinetik auf. Nach subkutaner Gabe werden maximale Plasmakonzentrationen (Cmax) nach 0,5 h gemessen, die mittlere Plasma-Halbwertszeit beträgt 0,67 h nach subkutaner, respektive 0,51 h nach intravenöser Applikation. Die errechnete orale Bioverfügbarkeit beträgt ca. 40 %. Das Auftreten von Nausea und Emesis, sowie deren Verlauf decken sich weitestgehend mit dem Verlauf der Lycorinkonzentration im Plasma. In keiner der untersuchten Dosisstufen sind blutchemische oder hämatologische Abweichungen aufgetreten. Um Rückschlüsse auf die Zielstrukturen von Lycorin und somit auf den emetischen Wirkungsmechanismus der Lycorin-induzierten Emesis und Nausea zu gewinnen, wurden die Hunde jeweils mit Diphenhydramin, Maropitant, Metoclopramid, Ondansetron oder Scopolamin vorbehandelt. Diese therapeutisch etablierten Antiemetika besitzen eine selektive Rezeptoraffinität und entfalten ihre antiemetische Wirkung über einen Antagonismus an histaminergen H1- (Diphenhydramin), dopaminergen D2- (Metoclopramid), muskarinergen M1-3- (Scopolamin), serotoninergen 5-HT3- (Ondansetron) oder Neurokinin-1-Rezeptoren (NK1) (Maropitant). Durch die Bindung des jeweiligen Antiemetikums an die spezifischen Rezeptoren, soll die anschließende Bindung von Lycorin an den gleichen Rezeptoren verhindert oder reduziert werden, was sich in einer Reduktion oder Abwesenheit von Nausea und Emesis auswirkt. Die Vorbehandlung mit Ondansetron ist mit einer signifikanten Verminderung der Anzahl der Brechakte verbunden und durch die Vorbehandlung mit Maropitant kann Lycorin-induzierte Emesis komplett verhindert werden. Einzig Ondansetron reduziert darüber hinaus den Ausprägungsgrad der Nausea und verlängert die Latenzzeit bis zum Auftreten von Vomitus, was eine Beteiligung von 5-HT3 Rezeptoren bei lycorin-induzierter Nausea nahe legt. Histaminerge (H1), dopaminerge (D2) und muskarinerge (M1-3) Rezeptoren sind vermutlich nicht an Lycorin-induzierter Nausea und Emesis beteiligt. Die Befunde der vorliegenden Arbeit weisen darauf hin, dass Lycorin bei Vergiftungen mit Pflanzen oder Pflanzenteilen, die zu den Amaryllidaceae gehören, eine entscheidende Bedeutung für die klinische Symptomatik und den Verlauf von Intoxikationen hat. Nach den Ergebnissen dieser Arbeit sind eine prädominierende Beteiligung von NK1- und eine etwas geringer ausgeprägte Beteiligung von 5-HT3-Rezeptoren im emetischen Wirkmechanismus wahrscheinlich. Somit erscheint die therapeutische Anwendung von Maropitant beim Hund (und evtl. Apreptitant beim Menschen) und/oder Ondansetron zur symptomatischen Behandlung anhaltender Nausea und Emesis bei Pflanzenvergiftungen mit Amaryllidacaen bei denen die Wirkung von Lycorin dominiert, wissenschaftlich begründet und klinisch von Vorteil gegenüber anderen antiemetischen Prinzipien zu sein.

Plus de 300 alcaloïdes d’Amaryllidaceae doués d’activités biologiques ont été isolés à partir des plantes appartenant à cette famille. De nos jours, seule la galanthamine, utilisée pour le traitement palliatif de la maladie d’Alzheimer, est commercialisée. L’accumulation de ces alcaloïdes dans les plantes est limitée. La culture in vitro est une méthode alternative intéressante pour l’obtention plus aisée de ces alcaloïdes à haute valeur ajoutée. Le premier objectif de ce travail vise à développer une méthode de purification efficace, simple et rapide des extraits de plantes préalablement à leur analyse en LCMS et GCMS. Le second objectif est d’étudier l’effet de plusieurs facteurs exogènes, ajoutés au milieu de culture de bulbilles de Leucojum aestivum et de sa variété Gravety Giant en bioréacteurs RITA®, sur les voies de biosynthèse de la galanthamine et de la lycorine. La variation des paramètres exogènes a permis une accumulation accrue en galanthamine et en lycorine (0,814 mg/g et 1,54 mg/g de matière sèche respectivement) dans les bulbilles. Le troisième objectif porte sur l’isolement et l’identification d’endophytes à partir de bulbes in vivo et in vitro de trois espèces d’Amaryllidaceae (L. aestivum, Narcissus pseudonarcissus et Galanthus elwesii). Des bactéries endophytes du genre Bacillus ont été identifiées. Un nouvel alcaloïde a été isolé à partir des cultures bactériennes / Over 300 Amaryllidaceae alkaloids possessing a wide range of biological activities have been isolated from plants belonging to this family. Galanthamine, used for the palliative treatment of Alzheimer’s disease, is the only one commercialized. The biodisponiblity of these alkaloids is low. In vitro culture offers an alternative yet interesting approach for the biotechnological production of these valuable alkaloids. The aim of this work was, first, to develop a fast, efficient and easy purification method of plant extracts prior to their phytochemical analysis both in LCMS and GCMS. Second, the combined effects of bioreactor RITA® culture and feeding with different exogenous factors on the biosynthetic pathway of both galanthamine and lycorine were studied. The experiments were conducted both with Leucojum aestivum and L. aestivum ‘Gravety Giant’ bulblets. The variation of several exogenous parameters resulted in a better accumulation of galanthamine and lycorine (0.814 mg/g and 1.54 mg/g dry weight respectively) in the bulblets. The third aim was to isolate and identify alkaloid producing endophytes from in vivo and in vitro bulbs of three Amaryllidaceae species (L. aestivum, Narcissus pseudonarcissus and Galanthus elwesii). Bacterial endophtes belonging to the Bacillus genus were identified. A new alkaloid was isolated from bacterial liquid cultures

No abstract available. / Thesis (M.Sc.)-University of Natal, Durban, 2001.

Ledebouria ovatifolia (Bak.) lessop, Clivia caulescens R.A.Dyer and Haemanthus pauculifolius Snijman & Van Wyk were the three species investigated in this work. Ledebouria ovatifolia belongs to the family Hyacinthaceae (Liliaceae sensu lato) and, to date, the chemical composition of this species has not been investigated. Members of this family are widely distributed, but are particularly well represented in Southern Africa. The Ledebouria genus was formerly classified as part of the Scilla genus from which a large number of naturally occurring oxygen heterocycles known as homoisoflavonoids have been isolated. In this work the bulbs of L. ovatifolia were investigated and two compounds were isolated. These were the homoisoflavonoid, 5,7-dihydroxy-3-(4'-hydroxybenzyl)-4-chromanone and the chalcone, 6',2'-dimethoxy-4,4'dihydroxychalcone, both of which are known naturally occurring compounds. Clivia caulescens and Haemanthus pauculifolius are both members of the Amaryllidaceae family. The plants of the Amaryllidaceae family form a large group of over sixty genera, which are concentrated mainly in Southern Africa. Plants from this family have been extensively used in traditional medicines and many have pharmacological properties. The compounds responsible for most of these effects are a group of isoquinoline alkaloids, which are found almost exclusively in plants belonging to this family. The alkaloids isolated from plants belonging to this group are known to cause poisoning in low doses and can cause excessive salivation and diarrhoea. Higher doses of the active compounds can cause CNS depression and large enough doses can prove fatal. Although many of these alkaloids are harmful to man, some of the unique Amaryllidaceae alkaloids exhibit anti-tumour and anti-viral activities, and are thus potentially beneficial to man. The Clivia genus is endemic to South Africa and the most common species, C. miniata, is used by traditional healers to facilitate childbirth and as a snake bite remedy. In this chemical investigation both the bulbs and the leaves of C. caulescens were investigated. The ethanol extract of the bulbs yielded four alkaloids, hippeastrine, haemanthamine, lycorine and ll-(S)-hydroxyvittatine. The investigation of the leaf extract also yielded lycorine and hippeastrine as well as an additional alkaloid sternbergine. Haemanthus pauculifilius is a recently described member of the Haemanthus genus, which consists of 27 taxa that are restricted to Southern Africa and Namibia. In this chemical investigation the ethanol extract of the bulbs and leaves yielded the common triterpenoid sitosterol as well as two novel 5,11-methanomorphanthridine type alkaloids, montanine hydrochloride and manthidine. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

This work is an account of investigations into the chemistry of one of the members of the Amaryllidacae family, Ammocharis coranica, and one of the members of the Fabaceae family, Sophora velutina. Chapter one is an account of the extractives from the bulbs of Ammocharis coranica. In all, twelve compounds, eight alkaloids and four cycloartane compounds have been isolated of which one alkaloid and one cycloartane compound have not been described previously. Plants belonging to the Amaryllidacae family have been used by traditional healers, especially in Africa, to treat a range of illnesses and diseases. The alkaloids isolated from these plants have been shown to exhibit responses to muscle stimulant, antiviral, antifungal, antiyeast, antimalarial, cytotoxic and antitumoural activities. Ammocharis coranica is used by the Zulu tribe in South Africa to treat any illness believed to be caused by witchcraft. Alkaloids from the three most common types among the isoquinoline group were found in this species. These are lycorine, 1-O-acetyllycorine, hippadine, acetylcaranine, and the novel 1-O- acetyl-9-norpluviine from the lycorine type, 6-α-hydroxypowelline from the crinine type and hamayne and crinamine from the haemanthamine type. Cycloartane compounds have not been reported previously from the Amaryllidaceae family. All four cycloartane compounds had a common side chain, containing an olefinic methylene group at position 24, but differed in their substituents at positions 3 and 4. These compounds were found to be 24-methylenecycloartan-3β-ol, cycloeucalenol, cycloeucalenone and the novel compound 4-methylenepollinastanone. Chapter two is an account of the extractives from the seeds of Sophora velutina. The seeds of other Sophora species have been used in traditional ceremonies by the Indians of the Southwest United States and adjacent Mexico because of their hallucinogenic activity. The seeds of Sophora velutina subsp. zimbabweensis found in Zimbabwe are suspected to have historically been used by the natives for their hallucinogenic properties. These plants have been known to contain several quinolizidine alkaloids, flavonoids and isoflavonoids. One alkaloid, N-methylcytisine and two isoflavones, pseudobaptigenin and calycosin, as well as the common phytosterol, β-sitosterol were isolated from the seeds of this species. N-methylcytisine is a common quinolizidine alkaloid, isolated previously from several Sophora species and pseudobaptigenin and calycosin are well known isoflavones, isolated previously from several species in the Fabaceae. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

Author: Kateřina Breiterová Title: Neurotropic and antioxidative activity of some selected species of monocotyledonous alkaloidal plants in vitro. VIII. Diploma thesis Charles Univerzity in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology 2015, 101 p. More than 50 % cases of dementia are nowadays caused byAlzheimer's disease (AD). AD is a progressive neurodegenerative disease and it causes gradual memory loss, disorientation and behavioral disorders which affect patient's social and occupational life. AD is characteristic by loss of neurons in some regions of brain - for example hippocampus and cortex. Ethiopathogenesis of this disease is not completely known - that is why the treatment is still just symptomatic. Formation of β-amyloid deposits in brain tissue plays an important role - it is a protein which creates extracellular plagues around neurites and causes their degeneration and death. Intracellular tangles are made up of the changed τ-protein. These tangles also cause death of the neuronal cell. The degeneration of neurons is supported by reactive oxygen radicals too. The another problem is a glutamatergic system disorder. This set of excitatory amino acids is important for correct long-term memory formation. Patients with AD suffer from...

4 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Viera Bodoríková Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of Diploma thesis: Synthesis of haemanthamine derivatives and their biological activity Haemanthamine, an isoquinoline Amarillidaceae alkaloid, exhibits a wide and important range of biological activities, including antitumor, antiviral, antioxidant, antimalarial and anticonvulsant. Biological activity of haemanthamine relatives closely with its structure. By modifying the different parts of the molecule, we can identify some structure-activity relationships. With this aim, the thirteen semisynthetic analogues of alkaloid haemathamine were prepared and purified using analytic and preparative TLC methods. The obtained substances were then subjected to structural analysis, specifically, there were used MS, HRMS, 1D and 2D NMR spectroscopic techniques. Prepared compounds were tested on its possibility to inhibit human erythrocytic acetylcholinesterase (HuAChE) and human serum butyrylcholinesterase (HuBuChE). The most promising biological activities have been shown by aromatic esters labelled as LC- 70 (IC50 HuAChE = 0,12 ± 0,01 µM) and LC-73 (IC50 HuAChE =0,17 ± 0,01 µM). The cytotoxic activity of prepared compounds has been...