Faschingbauer J.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2019. During the screening of biologically active secondary metabolites of plants carried out at the Department of Pharmaceutical Botany FAF UK, selected taxa of the genus Centaurea (Asteraceae) were investigated. This study is focused on a basic phytohemical research of extracts prepared from Centaurea cyanus, Centaurea jacea, Centaurea scabiosa, Centaurea pseudophrygia, Centuarea stoebe, Centaurea solstitialis a Centaurea benedicta. Extracts were prepared for evidence of the proof reactions of TLC and MS analysis (EI, ESI) to clarify a potential presence of alkaloids. EtOAc and ethanol extracts were evaluated for potential inhibitory activity against human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) and cytotoxicity against selected 9 tumor lines. C. cyanus alkaloid extract had interesting cholinesterase activity which selectively inhibited BChE (IC50 BChE = 22.62 ± 3.62 μg / ml, IC50 AChE = 221.50 ± 44.56 g / ml). Other EtOAc extracts of selected Centaurea species were considered inactive (IC50 > 100 μg/ml)....

Die Alzheimer'sche Erkrankung wird derzeit durch die Gabe von Acetylcholinesterase- Inhibitoren (AChEI) symptomatisch behandelt. Durch die AChE-Hemmung steht mehr Acetylcholin (ACh) für die Neurotransmission zur Verfügung. Bei Progression der Erkran-kung nimmt der Anteil an AChE drastisch ab, so dass die Enzymisoform Butyrylcholin- esterase (BChE) die Hydrolyse des Neurotransmitters ACh übernimmt. In späten Phasen der Alzheimer'schen Erkrankung ist daher der Einsatz selektiver BChE-Hemmer erfolgsver- sprechend. Inhibitoren können verschiedene Bindestellen in der Cholinesterase-Bindetasche adressie-ren und dadurch in dieser stabilisiert werden. Zu den Bindestellen zählen die katalytisch aktive Stelle (CAS) am Ende eines 20 Å langen Bindetaschentunnels, die Oxyanion-Vertie-fung, die Cholinbindestelle, sowie die periphere anionische Bindestelle (PAS), welche am Bindetascheneingang lokalisiert ist. In der vorliegenden Arbeit wurden durch in silico Dockingstudien gezielt Protein-Ligand- Interaktionen untersucht, um Strukturmerkmale hochaffiner Inhibitoren von Cholinesterasen zu identifizieren. Damit soll die zukünftige Entwicklung von Cholinesteraseinhibitoren hinsichtlich der Affinität zum Enzym verbessert werden. Ferner dienten synthetische Untersuchungen eines Naturstoffes dazu, Chinazoliniumverbindungen als Leitstruktur für die Inhibition der Cholinesterasen zu etablieren. Für hochaffine tri- und tetrazyklische aminsubstituierte AChE-selektive Chinazolin- und Chinazolinoninhibitoren sollte die bevorzugte Orientierung der Liganden in der Bindetasche ermittelt werden. Hierfür ist die Lokalisation des Aminsubstituenten in der CAS (invertierter Bindemodus) oder die dortige Bindung des Chinazolin-/Chinazolinongerüstes (klassischer Bindemodus) denkbar. Anhand eines präferierten einheitlichen Bindemodus sollten die Struktur-Aktivitäts-Beziehungen erklärt werden. Dockingstudien zeigten die klare Präferenz für den invertierten Bindemodus, bei dem der Aminsubstituent in der Nähe der CAS platziert wird. Ein strukturelles Merkmal für hochaffine Inhibitoren ist ein unter Assaybedingungen protoniertes Amin, welches eine Kation-π-Wechselwirkung zu dem Indolringsystem des Tryptophans der Cholinbindestelle eingehen kann. Für das Ligandengrundgerüst wurde lediglich für tetrazyklische Verbindungen eine π-π-Interaktion mit der peripheren Bindestelle (PAS) am Bindetascheneingang identifiziert. Der Datensatz umfasste auch chirale Chinazolinon- und Chinazolinderivate mit hydrierter C=N-Doppelbindung, die eine schwächere Affinität zu AChE zeigten. Diese ist vermutlich auf das nicht-planare Ligandengrundgerüst zurückzuführen, da vor allem für tetrazyklische chi-rale Verbindungen die Stabilisierung des Ligandengrundgerüstes durch π-π-Interaktionen am Bindetascheneingang aufgrund der Sterik entweder gar nicht, oder nur für ein Enantio-mer möglich ist. Aufgrund der nanomolaren Affinität der achiralen Chinazolin- und Chinazolinonverbindungen wurden weitere gerichtete Wechselwirkungen in der Bindetasche erwartet. Derartige Wechselwirkungen konnten in Form von Wasserstoffbrücken durch die Verwendung von sieben ausgewählten strukturellen Wassermolekülen im Docking identifiziert werden. Durch diese Wassermoleküle werden Wasserstoffbrücken vom Ligandengrundgerüst zum Protein vermittelt. Diese Wechselwirkungen scheinen essentiell für die Stabilisierung hoch-affiner Chinazolin- und Chinazolinoninhibitoren in der AChE-Bindetasche zu sein. Zwei photochrome Bis-Tacrin-Konstitutionsisomere (Ring-geöffnete und Ring-geschlossene Form) inhibieren die AChE und zeigen einen unterschiedlichen Effekt in der Hemmung der Amyloid-β Fibrillenbildung. Die Fibrillenbildung wird durch eine unbesetzte periphere Bindestelle (PAS) am Eingang der AChE-Bindetasche katalysiert, weshalb eine unterschiedliche Interaktion der Liganden mit ebendieser Bindestelle vermutet wird. Dockingstudien lieferten für beide Konstitutionsisomere einen ähnlichen Bindemodus, der vor dem Hintergrund der ähnlichen IC50-Werte von 4.3 und 1.8 nM für die Ring-geöffnete und Ring-geschlossene Form plausibel erscheint. Durch die Auswahl einer geeigneten Röntgenstruktur wurden Dockinglösungen erhalten, bei denen ein Tacrinsubstituent in der PAS bindet und dort π-π-Interaktionen mit einem Tryptophan und einem Tyrosin eingeht. Eine solche Lage des PAS-bindenden Tacrinsubstituenten ist energetisch bevorzugt und drückt sich durch bessere Scores gegenüber Dockinglösungen, bei denen dieser auf der Protein-oberfläche lokalisiert ist, aus. Der andere Tacrinsubstituent bindet in der CAS wie dies von bereits kristallisierten Tacrinderivaten bekannt ist. Mittels molekulardynamischer Simulati-onen wurde die Stabilität der Protein-Dockinglösungs-Komplexe beider Konstitutionsiso-mere verglichen. Dabei wurde die bessere Stabilisierung des CAS-bindenden Tacrinsubsti-tuenten für die Ring-geöffnete Form des Liganden ermittelt. Ferner zeigt sich für die Ring-geöffnete Inhibitorform während der Simulation der Einstrom von sechs Wassermolekülen in einen Hohlraum der PAS. Dies hat zur Folge, dass der PAS-bindende Tacrinsubstituent während der Hälfte der Simulationszeit durch Wasserstoffbrücken in der PAS stabilisiert wird. Ein Wasserstoffbrückennetzwerk diesen Ausmaßes kann für die Ring-geschlossene Inhibitorform nicht ermittelt werden. Die bessere Hemmung der Amyloid-β Fibrillenbildung der Ring-geöffneten Inhibitorform wird daher auf die bessere Stabilisierung des Liganden durch Wasserstoffbrücken in der AChE-Bindetasche zurückgeführt. Für carbamatsubstituierte Tetrahydrochinazolinverbindungen sollten die bevorzugten Interaktionen in der BChE-Bindetasche ermittelt werden. Die Carbamatverbindungen sind pseudo-irreversible Inhibitoren und zeigen eine zeitabhängige Hemmung mit diversen Interaktionszuständen zwischen Protein und Ligand. Darüber hinaus stellen Dockingstudien in der BChE bislang eine Herausforderung dar, da es derzeit nur zwei Röntgenstrukturen dieses Enzyms mit reversiblen Liganden gibt, weshalb kaum Studien zur Identifikation einer geeigneten Bewertungsfunktion durchgeführt werden können. Im Docking wurde sich für die Analyse des reversiblen Anlagerungskomplexes entschieden, da das Docking des tetraedrischen Übergangszustandes energetisch entartete Dockinglösungen lieferte. Eine weitere Herausforderung stellte die Größe der BChE-Bindetasche dar, die auch im reversiblen Docking entartete Dockinglösungen lieferte. Aufgrund einer ähnlichen Übertragungsrate aller getesteten Inhibitoren wurde eine konservierte Lage des Carbamates in der Bindetasche angenommen. Deshalb wurde eine repräsentative Dockinglösung einer Referenzverbindung als Ausgangspose für einen Modelling-Ansatz gewählt, die hinsichtlich der Interaktionen in der Bindetasche ausgewählt wurde. Diese Interaktionen sind: 1) Eine Wasserstoffbrückendistanz zwischen der Carbamat-Carbonylgruppe und der Oxyanion-Vertiefung sowie 2) eine Distanz, die den nucleophilen Angriff des Serins auf den Carbamatkohlenstoff erlaubt. Im Modelling-Ansatz wurde die repräsentative Bindepose dazu verwendet die entsprechenden Inhibitoren in der Bindetasche aufzubauen. Die bevorzugte Position der N-Methylgruppe wurde für beide Enantiomere über die berechneten Spannungsenergien der Bindeposen abgeschätzt. Für die S-Enantiomere ergab sich die präferierte Bindung mit quasi-„axialer“ Methlygruppe und für die R-Enantiomere mit quasi-„äquatorialer“ Stellung dieser. Die Carbamatstrukturen liegen somit mit der Heptylkette in der Acyltasche und die Ligandengrundgerüste werden in einer Seitentasche der BChE-Bindetasche platziert, in der hydrophobe Wechselwirkungen dominieren. Zusätzlich zu den hochaffinen Chinazolinonverbindungen sollten artverwandte Chinazolini-umverbindungen als Leitstruktur für Cholinesteraseinhibitoren untersucht werden. Zunächst erfolgten Studien zur chemischen Reaktivität und Stabilität des Naturstoffes Dehydroevodiamin (DHED) sowie seines Benz-Derivates (Benz-DHED). Insbesondere Benz-DHED war unter den bisher verwendeten und in der Literatur beschriebenen Synthesemethoden instabil. Die Untersuchungen erforderten daher zunächst die Einführung einer geeigneten Syntheseroute, in diesem Fall die Oxidation mit KMnO4, einhergehend mit der Verbesserung der Ausbeute und ohne Nebenproduktbildung. Für die zukünftige Synthese von Derivaten wurde die Verwendung einer geeigneten Lewis-Säure-labilen Schutzgruppe herausgearbeitet. Die untersuchten Chinazoliniumverbindungen zeigen die Eigenschaft, dass sie in Abhängigkeit der Reaktionsbedingungen in zwei Formen (Ring-geöffnet und Ring-geschlossen = Chi-nazoliniumsalz) isoliert werden können. Mittels UV/Vis-Untersuchungen wurde das Gleich-gewicht dieser Spezies aufgeklärt und in wässrigen alkalischen Lösungen die Anreicherung einer dritten, bislang nicht in diesem Zusammenhang beschriebenen, Spezies beobachtet. Als biologisch aktive Spezies konnte die Chinazoliniumform identifiziert werden. In Dockingstudien der Chinazoliniumform von Benz-DHED, nach dem für Carbamatverbindungen entwickelten Modelling-Ansatz, konnte auch hierfür die Stabilisierung der Docking- lösung über eine Wasserstoffbrücke in der BChE-Bindetasche zu einem strukturellen Wassermolekül identifiziert werden. Dies verdeutlicht erneut, dass die Berücksichtigung von Wassermolekülen in Dockingstudien dazu dienen kann zusätzliche Protein-Ligand-Interaktionen festzustellen. Auf Grundlage der Forschung zu Chinazoliniumverbindungen kann die zukünftige Inhibitorentwicklung von Strukturen basierend auf dieser Substanzklasse erfolgen. Die durchgeführten synthetischen und theoretischen Studien liefern wichtige Beiträge zum Verständnis der Wechselwirkungen zwischen Inhibitoren und Cholinesterasen, die in der zukünftigen Inhibitorentwicklung Anwendung finden können. / Alzheimer's disease is currently treated symptomatically by the administration of acetyl-cholinesterase inhibitors (AChEI). By AChE inhibition more acetylcholine (ACh) is available for neurotransmission. During disease progression the amount of AChE drastically decreases, so that the enzyme butyrylcholinesterase (BChE) takes over hydrolysis of this neurotransmitter. In late stages of Alzheimer's disease, the use of selective BChE in-hibitors may therefore be advantageous. Inhibitors can adress different binding sites in the cholinesterase binding pocket. The bind-ing sites includes the catalytic active site (CAS), which is located at the end of a 20 Å long binding gorge, the oxyanion hole, the choline binding site and the peripheral anionic site (PAS), located at the entrance of the binding gorge. In the present study, specific protein-ligand interactions were investigated by means of in silico docking studies to identify structural features of high-affinity inhibitors of cholinester-ases. Thereby the aim is to improve the affinitiy of newly developed cholinesterase- inhibitors. Furthermore, synthetic studies of a natural product served to establish a new lead compound for cholinesterase inhibitors. For high affinity tri- and tetracyclic amine substituted AChE-selective quinazoline and quinazolinone inhibitors, the aim was to determine the preferred ligand orientation in the binding site. To this end, the localization of the amine substituents in the CAS (inverted binding mode) or the binding of the quinazoline-/quinazolinone moiety there (classical binding mode) is conceivable. The aim was to explain structure-activity-relationships by the identified preferred and consistent binding mode. Docking studies showed a clear preference for the inverted binding mode in which the amine substituent is placed in the vicinity of the CAS. A structural characteristic of high affinity inhibitors is a protonated amine which can form a cation-π-interaction to the tryptophan indole ring system of the choline binding site. Furthermore, a π-π-interaction with the peripheral binding site (PAS) has been identified for the ligand backbone of tetra-cyclic inhibitors. The data set also included chiral quinazolinone- and quinazoline derivatives with hydrogenated C=N-double bond, which showed weaker affinity to AchE compared to non chiral compounds. The weaker affinity is probably due to the non-planar ligand back-bone. Due to steric reasons, especially for tetracyclic chiral compounds, stabilisation of the ligand backbone through π-π interactions is either not possible at all, or possible only for one enantiomeric form. Due to the nanomolar affinity of non chiral quinazoline and quinazolinone compounds, other directional interactions between protein and ligand were expected. Several inter- actions were identified in form of hydrogen bonds through the use of conserved water molecules in docking. These water molecules mediate hydrogen bonds from the ligand backbone to the protein. These interactions seem to be essential for the stabilization of high affinity quinazoline and quinazolinone inhibitors in the AChE binding site. Two photochromic bis-tacrine constitutional isomers (ring-open and ring-closed form) inhibit AChE and show a different effect with regard to the inhibition of amyloid-β fibril formation. Fibril formation is catalyzed by an unoccupied peripheral site (PAS) at the entrance of the AChE-binding pocket, which is why a different interaction of the ligands is expected with this binding site. Docking studies provided a similar binding mode for both constitutional isomers, which appears plausible for the ring-open and ring-closed form in the light of similar IC50-values of 4.3 and 1.8 nM. By selecting a suitable crystal structure, docking solutions were obtained in which one tacrine substituent is placed in the PAS and undergoes π-π-interactions with a tryptophan and a tyrosine. This placement of the PAS-binding tacrine-substituent is energetically favored which is expressed through better scores, compared to docking solutions where the tacrine-substituent is placed on the protein surface. The other tacrine substituent binds in the CAS in a manner which is known from other already crystalized tacrines. Molecular dynamic simulations were subsequently used for stability comparison of the protein-docking solution-complex of both isomers. Here, the better stabilization of the CAS-binding tacrine-substituent for the ring-open form was determined. The complex with the ring-open form shows the influx of six water molecules in a cavity of the PAS at the beginning of the simulation. As a consequence, the PAS-binding tacrine substituent is stabilized by hydrogen bonds to these water molecules during half of the simulation time. A hydrogen bond network of this magnitude was not observed for the ring-closed form of the inhibitor. The better inhibition of amyloid-β fibril formation by the ring-open form may be due to better stabilization of this ligand through hydrogen bonds in the AChE-binding site, which is not observed for the ring-closed form. Preferred interactions could also be identified for carbamate-based quinazoline inhibitors in the BChE-binding site. These carbamate compounds are pseudo-irreversible inhibitors and show a time-dependent inhibition with several interaction possibilities between protein and ligand. Docking studies in the BChE are challenging, as there currently exist only two X-ray structures of this enzyme with reversible ligands. Therefore, it is difficult to identify a suitable scoring function for the data set under investigation. Docking of the tetrahedral transition state delivered energetically degenerate docking solutions wherefore the aim was to investigate the reversible attachment complex in more detail. Thereby the challenge was the size of the BChE-binding pocket, which also supplied degenerate docking solutions in the reversible docking. Due to a similar carbamoylation rate of all the inhibitors tested, a conserved position of the carbamate moiety in the binding pocket was presumed. Therefore, a representative docking solution of a reference compound, which was selected in the binding pocket in terms of interactions, was chosen for a modeling approach. These interactions are 1) A hydrogen bond distance between the carbamate carbonyl and the oxyanion hole and 2) a distance between the carbamate carbon and the serine which allows for a nucleophilic attack and thus the transfer of the carbamate moiety onto the enzyme. For the modeling approach, the representative binding pose was used to construct the appropriate inhibitor structures. The preferred position of the N-methyl group was calculated for both enantiomers via the tension energy of the corresponding binding poses. For S-enantiomers the preferred posi-tion of the methyl group is quasi-"axial" and for R-enantiomers quasi-"equatorial". The conserved binding mode thus is characterized by the heptyl chain being placed in the acyl pocket and the ligand scaffold in a side pocket of the BChE-binding site where hydrophobic interactions dominate. In addition to high affinity quinazoline and quinazolinone inhibitors, it was intended to make quinazolinium compounds accessible as lead compounds for future cholinesterase inhibitors. Synthetic studies initially focused on chemical reactivity and stability of the natural product dehydroevodiamine (DHED) and its benz-derivative (benz-DHED). In particular, benz-DHED was unstable under the synthesis conditions applied so far and described in the literature. The investigations therefore required introduction of a suitable synthetic route, in this case oxidation with KMnO4, accompanied by improvement of yield and no by-product formation. For future syntheses of derivatives, the use of a Lewis-acid-labile protecting group is suggested. Depending on the reaction conditions, the quinazolinium compounds can be isolated in two forms (ring-opened and ring-closed = quinazolinium-salt). By means of UV/Vis-studies, the equilibrium of these two forms was elucidated. In aqueous alkaline solutions the enrichment of a third, hitherto in this context undescribed species, was observed. These studies helped in identification of the quinazolinium-form as biologically active species. Docking studies of this form, using the modeling-approach which was evolved for carbamates, showed a possible stabilization of this compound via a hydrogen bond mediated by a struc-tural water molecule in the BChE-binding site. The role of water here again shows that consideration of water molecules in docking studies might be able to describe preffered binding modes by means of additional protein-ligand-interactions better, compared to docking studies without explicit water molecule consideration. Based on the basic research carried out for quinazolinium compounds, future inhibitor development based on this substance class can be performed. Finally, the herein conducted synthetic and theoretical studies provide important contributions to the understanding of the interaction between inhibitors and cholinesterases, which can be used for future inhibitor development.

Posttraumatic Stress Disorder (PTSD) is a psychiatric condition that can develop following exposure to a traumatic event involving actual or threatened death or serious injury. Responses include intense fear, helplessness or horror. Symptoms are characterised into clusters, described as re-experiencing, avoidance, and arousal. These symptoms, which are also evident in other conditions, have been associated with dysfunctions in the central acetylcholinergic system. Benefits from administering acetylcholinesterase inhibitors (AChEI) to people suffering these symptoms have been demonstrated. Donepezil hydrochloride, a reversible inhibitor of the enzyme acetylcholinesterase, is used in the treatment of conditions with difficulties in cognitive function, but has not been used in PTSD. The aim of this thesis was to determine (1) whether there was a difference in the ACh system in people with PTSD and (2) whether administration of an AChEI would change the symtomatology. IDEX (I¹ ² ³ iododexetimide) has been useful in imaging muscarinic-ACh receptors using Single Photon Emission Computerised Tomography (SPECT) and was utilised to investigate whether cholinergic activity in PTSD is altered. One hundred and sixty eight potential subjects were screened and eleven PTSD subjects were enrolled in the IDEX SPECT study. Three healthy non-PTSD control subjects also completed the study. Due to technical complications only the data obtained from eight PTSD and two control subjects was available for analysis. Imaging data for 2 further healthy non-PTSD control subjects were obtained from another study. Sixteen subjects were enrolled in the donepezil open label study (assessed at baseline, Week 2, 6 and 10). Nine PTSD subjects completed the 10-week trial and seven withdrew prematurely (at or after Week 2) due to side effects or a worsening of PTSD symptoms. For the IDEX SPECT study, a voxel-by-voxel statistical analysis of the PTSD subject group versus the control group showed both areas of reduced and increased IDEX uptake. Significant clusters in the PTSD group with a reduced IDEX uptake centred around the bilateral hippocampus, left insula and right precuneus, while increased IDEX uptake appeared in the caudate head. For the donepezil study, in the per-protocol analysis (including only the 9 subjects that completed the protocol), all psychological assessments revealed a difference between the totals obtained at the Week 10 visit compared to those at the Baseline visit and the improvement was in the order of 51%. The intention-to-treat analysis (including all 16 subjects), a repeated measures Analysis of Variance (ANOVA) with a mixed models approach showed that all psychological measures demonstrated statistically significant benefits of the treatment. All subjects who completed the protocol recounted considerable improvement in their overall PTSD symptom profile, which covered symptoms in each of the three clusters. The results of the IDEX SPECT study suggest that alterations in ACh binding in PTSD are evident and may begin to explain a part of the altered cognitive symptomatology apparent in this condition. The pilot open label donepezil trial provided some preliminary evidence that treatment with an AChEI can lessen the intrusions and distress associated with traumatic memories in people with PTSD. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374974 / Thesis (M.Med.Sc.) -- University of Adelaide, School of Medicine, 2009

<p>Organophosphorus pesticides (OPs) are neuroactive compounds that can affect the development of the nervous system and behavior. Using zebrafish, we compared the effectiveness of three different OPs, chlorpyrifos (CPF), diazinon (DZN) and parathion (PA) on survival, AChE inhibition and larval motility. We conclude that at equimolar concentrations CPF is the most effective OP of the three tested. 300nM CPF inhibited AChE when measured at 5 days post fertilization (dpf) by 80%, which correlated to a 35% decrease in larval motility when measured at 6 dpf. </p><p>We isolated the cDNA of <italic>zfkcc2</italic>, which encodes for an important cation-chloride cotransporter responsible for establishing a chloride gradient that allows for the neurotransmitter GABA to become inhibitory for a subset of neurons. By isolating the cDNA of <italic>zfkcc2</italic>, we were able to characterize its expression pattern up to 4 dpf. Using qRT-PCR, we showed that the expression of <italic>zfkcc2</italic> rises from 2 to 96 hours post fertilization (hpf). The expression of <italic>zfkcc2</italic> is found at 1 dpf in the forebrain, midbrain and hindbrain in a distinct pattern. By 3 dpf, zfkcc2 expression is found throughout most of the developing zebrafish brain. We were then able to characterize the expression pattern of zfnkcc1, which encodes for a cation-chloride cotransporter that works opposite to <italic>zfkcc2</italic> and establishes a chloride gradient allowing GABA to be excitatory during development. Expression of <italic>zfnkcc1</italic> is found at 1 dpf throughout most of the developing zebrafish head including in some non-neural tissue, but appears concentrated to the otic vesicle at 3 dpf. Using double in situ hybridization, we investigated the nature of the developmental switch from zfnkcc1 to zfkcc2 by looking at regions where there was an interaction between both genes' expression patterns in the developing zebrafish nervous system. </p><p>We preliminarily examined how affecting nicotinic signaling with exposures to nicotinic agonists/antagonists can affect the expression of <italic>zfnkcc2</italic> and <italic>zfkcc2</italic>. While there were no apparent changes in the expression pattern from any of the exposures at 2 dpf, we found that by 3 dpf nicotine and CPF exposures resulted in an incomplete expression pattern for <italic>zfkcc2</italic> that seemed to be delayed. However, the expression of <italic>zfnkcc1</italic> at the otic vesicle was unaffected by exposure. </p><p>Our results suggest that disrupting nicotinic signaling during development can affect the expression of an important cation-chloride cotransporter, <italic>zfkcc2</italic>, that plays a major role in establishing the necessary chloride gradient for the proper functioning of GABA during development. Future studies should focus on investigating the nature of the developmental switch between <italic>zfnkcc1</italic> and <italic>zfkcc2</italic> to see if the possibility of their regulation being coupled is a target for exposures.</p> / Dissertation

This thesis describes the results obtained from the phytochemical analysis of Matricaria chamomilla, and the microbial transformation of oxymatrine (85) and harmine (87), as summarized below. 1. Chemical investigation of the crude methanolic extract of Matricaria chamomilla resulted in the isolation of a new natural product, matriisobenzofuran (72), along with four known compounds: apigenin (73), apigenin-7-O-β-glucopyranoside (74), scopoletin (75), and fraxidin (76). The structures of compounds 72-76 were elucidated with the aid of extensive NMR and mass spectroscopic studies. All of the aforementioned compounds showed moderate to good inhibitory activities against glutathione S-transferase, an enzyme which has been implicated in the resistance of cancer cells to chemotherapeutic agents. These compounds were also evaluated for antioxidant activity and displayed moderate to good free radical scavenging activity. Additionally, compounds 72-76 were screened for anti-leishmanial activity. Compounds 75 and 76 were significantly active in this assay, while the remaining compounds were weakly active. In the antibacterial and antifungal assays, compounds 72-76 were not active. 2. The second part of this thesis deals with the biotransformation studies on oxymatrine (85) and harmine (87). Oxymatrine (85) was metabolized to the deoxy analogue, matrine (84) by Penicillum chrysogeneum (ATCC 9480), Cunninghamella bainieri (ATCC 9244), Cunninghamella blakesleena (ATCC 9245 and 8688A), Curvularia lunata (ATCC 12017), and Fusarium sp. In the time-based analysis of this transformation, the metabolism of oxymatrine (85) could be detected after 48 hours of incubation. Additionally, incubation of harmine (87) with Mucor plumbeus (ATCC 4740) resulted in the isolation of harmine-N-oxide (94). The biotransformed products (84 and 94) were identified using IR, UV, NMR, and mass spectroscopic techniques. Compound 94 was evaluated for its ability to inhibit the enzyme acetylcholinestrase, whose overexpression has been linked to Alzheimer’s disease, and was found to possess weaker activity than harmine (87).

This thesis describes the results obtained from the phytochemical analysis of Matricaria chamomilla, and the microbial transformation of oxymatrine (85) and harmine (87), as summarized below. 1. Chemical investigation of the crude methanolic extract of Matricaria chamomilla resulted in the isolation of a new natural product, matriisobenzofuran (72), along with four known compounds: apigenin (73), apigenin-7-O-β-glucopyranoside (74), scopoletin (75), and fraxidin (76). The structures of compounds 72-76 were elucidated with the aid of extensive NMR and mass spectroscopic studies. All of the aforementioned compounds showed moderate to good inhibitory activities against glutathione S-transferase, an enzyme which has been implicated in the resistance of cancer cells to chemotherapeutic agents. These compounds were also evaluated for antioxidant activity and displayed moderate to good free radical scavenging activity. Additionally, compounds 72-76 were screened for anti-leishmanial activity. Compounds 75 and 76 were significantly active in this assay, while the remaining compounds were weakly active. In the antibacterial and antifungal assays, compounds 72-76 were not active. 2. The second part of this thesis deals with the biotransformation studies on oxymatrine (85) and harmine (87). Oxymatrine (85) was metabolized to the deoxy analogue, matrine (84) by Penicillum chrysogeneum (ATCC 9480), Cunninghamella bainieri (ATCC 9244), Cunninghamella blakesleena (ATCC 9245 and 8688A), Curvularia lunata (ATCC 12017), and Fusarium sp. In the time-based analysis of this transformation, the metabolism of oxymatrine (85) could be detected after 48 hours of incubation. Additionally, incubation of harmine (87) with Mucor plumbeus (ATCC 4740) resulted in the isolation of harmine-N-oxide (94). The biotransformed products (84 and 94) were identified using IR, UV, NMR, and mass spectroscopic techniques. Compound 94 was evaluated for its ability to inhibit the enzyme acetylcholinestrase, whose overexpression has been linked to Alzheimer’s disease, and was found to possess weaker activity than harmine (87).

Posttraumatic Stress Disorder (PTSD) is a psychiatric condition that can develop following exposure to a traumatic event involving actual or threatened death or serious injury. Responses include intense fear, helplessness or horror. Symptoms are characterised into clusters, described as re-experiencing, avoidance, and arousal. These symptoms, which are also evident in other conditions, have been associated with dysfunctions in the central acetylcholinergic system. Benefits from administering acetylcholinesterase inhibitors (AChEI) to people suffering these symptoms have been demonstrated. Donepezil hydrochloride, a reversible inhibitor of the enzyme acetylcholinesterase, is used in the treatment of conditions with difficulties in cognitive function, but has not been used in PTSD. The aim of this thesis was to determine (1) whether there was a difference in the ACh system in people with PTSD and (2) whether administration of an AChEI would change the symtomatology. IDEX (I¹ ² ³ iododexetimide) has been useful in imaging muscarinic-ACh receptors using Single Photon Emission Computerised Tomography (SPECT) and was utilised to investigate whether cholinergic activity in PTSD is altered. One hundred and sixty eight potential subjects were screened and eleven PTSD subjects were enrolled in the IDEX SPECT study. Three healthy non-PTSD control subjects also completed the study. Due to technical complications only the data obtained from eight PTSD and two control subjects was available for analysis. Imaging data for 2 further healthy non-PTSD control subjects were obtained from another study. Sixteen subjects were enrolled in the donepezil open label study (assessed at baseline, Week 2, 6 and 10). Nine PTSD subjects completed the 10-week trial and seven withdrew prematurely (at or after Week 2) due to side effects or a worsening of PTSD symptoms. For the IDEX SPECT study, a voxel-by-voxel statistical analysis of the PTSD subject group versus the control group showed both areas of reduced and increased IDEX uptake. Significant clusters in the PTSD group with a reduced IDEX uptake centred around the bilateral hippocampus, left insula and right precuneus, while increased IDEX uptake appeared in the caudate head. For the donepezil study, in the per-protocol analysis (including only the 9 subjects that completed the protocol), all psychological assessments revealed a difference between the totals obtained at the Week 10 visit compared to those at the Baseline visit and the improvement was in the order of 51%. The intention-to-treat analysis (including all 16 subjects), a repeated measures Analysis of Variance (ANOVA) with a mixed models approach showed that all psychological measures demonstrated statistically significant benefits of the treatment. All subjects who completed the protocol recounted considerable improvement in their overall PTSD symptom profile, which covered symptoms in each of the three clusters. The results of the IDEX SPECT study suggest that alterations in ACh binding in PTSD are evident and may begin to explain a part of the altered cognitive symptomatology apparent in this condition. The pilot open label donepezil trial provided some preliminary evidence that treatment with an AChEI can lessen the intrusions and distress associated with traumatic memories in people with PTSD. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374974 / Thesis (M.Med.Sc.) -- University of Adelaide, School of Medicine, 2009

Universiẗat, Diss., 2003--Stuttgart.

Orientador: Vanderlan da Silva Bolzani / Banca: Jairo Kenupp Bastos / Banca: Patrícia Sartorelli / Resumo: O presente trabalho teve como objetivo o estudo químico da fração diclorometânica das folhas de Senna multijuga, visando o isolamento, purificação e elucidação estrutural de alcalóides piridínicos com potencial farmacológico. Inicialmente foi preparado o extrato hidroalcóolico das folhas, o qual foi submetido à partição líquido/líquido com hexano, diclorometano e acetato de etila. As quatro frações obtidas foram avaliadas por CCD, sendo a fração diclorometânica a de maior concentração alcaloídica. Essa fração foi submetida à extração ácido/base e a fração alcaloídica submetida à CCD preparativa de sílica, que foi desenvolvida com uma mistura dos solventes hexano:CHCl3:AcOEt (1,5:2:6,5), de onde foram isolados cinco alcalóides que apresentaram absorção na região do ultravioleta a 254 e 286 nm. Desta separação obteve-se dois alcalóides puros, 7'-multijuguinona (5), 12'-hidroxi-7'-multijuguinona (2), e os demais foram purificados por cromatografia líquida de alta eficiência (CLAE): 4'-multijuguinato de metila (3), 7'- multijuguinol (4a e 4b) e 12'-hidroxi-7'-multijuguinol (1a e 1b). Estas substâncias tiveram suas estruturas elucidadas pelo uso dos experimentos de Ressonância Magnética Nuclear (RMN) e Espectrometria de Massas com ionização por eletrospray (EM). A partir destas análises foi possível determinar a estrutura de sete substâncias, sendo todas inéditas na literatura. As substâncias apresentaram atividade anticolinesterásica moderada, o que justifica a importância de estudos com metabólitos secundários. Os pares 1a e 1b e 4a e 4b isolados apresentam isomeria que ainda não determinada. Pelas análises do perfil alcaloídico determinado no estudo sobre a espécie, pode-se constatar que os demais órgãos também acumulam alcalóides, porém, em concentrações diferentes, prevalencendo um ou dois deles. / Abstract: This present work deals with the chemical study of the dichloromethane fraction of the Senna multijuga leaves, aiming the isolation, purification and structural elucidation of the new bioactive pyridine alkaloids, which can be useful for further pharmacological evaluation. The leaves ethanol extract was subjected a liquid/liquid partitions with hexane, dichloromethane and ethyl acetate, resulting in four fractions, which were evaluated for TLC, being the dichloromethane the fraction with the highest concentration of alkaloids. This fraction was subjected to acid/basic extraction, and the alkaloidal fraction (1.42 g) was subjected to silica preparative TLC, and eluted with a solvent system: n-hexane:CHCl3:AcOEt (1.5:6.5:2), which were isolated five alkaloids. From this procedures were obtained alkaloids, 7'-multijuguinone (5), 12'-hydroxyl-7'- multijuguinone (2) and the resulting mixture was further purified by HPLC yielding methyl 4'-multijuguinate (3), 7'-multijuguinol (4a e 4b) and 12'-hydroxyl-7'- multijuguinol (1a e 1b). The structure of all alkaloids were determined by RMN and mass spectral data analysis resulting in seven new moderate acetylcholinesterase inhibitor derivatives. The alkaloids pairs, 1a;1b and 4a;4b, are isomeric mixtures, not identified yet. Through the alkaloid profile has been established after several data accumulated from this plant, it was observed that others organs also accumulate the same alkaloids, but in different concentration prevailing one or two alkaloids. / Mestre

The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.