This thesis describes phytochemical studies on two medically important plants, Buxus natalensis and Drypetes gossweileri. Chemical investigation on the acetylcholinesterase inhibiting chloroform extracts, obtained at pH 7.0 and 9.5 resulted in the isolation of seven natural products: O10-natafuranamine (123), cyclonataminol A (124), 31-demethylbuxaminol A (125), buxaminol A (126), buxaminol C (127), p coumaroylputrescine (128) and methyl syringate (129). Compound 123 is a member of a rarely occurring class of Buxus alkaloids containing a tetrahydrofuran ring incorporated in its structure. Compounds 123-129 were isolated for the first time from this plant. Structures of compounds 123-129 were elucidated with the aid of NMR and MS spectral data. All of these isolates exhibited different levels of AChE inhibitory activities with compound 123 being significantly active in this bioassay with an IC50 of 8.5 µM compared to the rest of the isolates. Compounds 123-129 were inactive in antimicrobial assays. Phytochemical studies on the crude extract of Drypetes gossweileri resulted in the isolation of a new N-linked aromatic glycoside, N-β-glucopyranosyl-p-hydroxy phenyl acetamide (151), along with two known compounds, p-hydroxy phenyl acetic acid (152) and p hydroxyphenyl acetonitrile (153). Compounds 151-153 exhibited moderate to weak AChE inhibitory activities.

This thesis describes phytochemical studies on two medically important plants, Buxus natalensis and Drypetes gossweileri. Chemical investigation on the acetylcholinesterase inhibiting chloroform extracts, obtained at pH 7.0 and 9.5 resulted in the isolation of seven natural products: O10-natafuranamine (123), cyclonataminol A (124), 31-demethylbuxaminol A (125), buxaminol A (126), buxaminol C (127), p coumaroylputrescine (128) and methyl syringate (129). Compound 123 is a member of a rarely occurring class of Buxus alkaloids containing a tetrahydrofuran ring incorporated in its structure. Compounds 123-129 were isolated for the first time from this plant. Structures of compounds 123-129 were elucidated with the aid of NMR and MS spectral data. All of these isolates exhibited different levels of AChE inhibitory activities with compound 123 being significantly active in this bioassay with an IC50 of 8.5 µM compared to the rest of the isolates. Compounds 123-129 were inactive in antimicrobial assays. Phytochemical studies on the crude extract of Drypetes gossweileri resulted in the isolation of a new N-linked aromatic glycoside, N-β-glucopyranosyl-p-hydroxy phenyl acetamide (151), along with two known compounds, p-hydroxy phenyl acetic acid (152) and p hydroxyphenyl acetonitrile (153). Compounds 151-153 exhibited moderate to weak AChE inhibitory activities.

No description available.

<p>Extracts from the root bark, stem bark, and leaves of R. natalensis were screened for antibacterial activity against standard bacterial strains / Staphylococcus aureas, Escherichia coli and Pseudomonas aureginosa, and fungi / Candida albicans, Trichophyton mentagrophytes or Microsporum gyseum. Chromatographic techniques were utilized to isolate pure compounds. This study validates and documents, in a systematic way, the antimicrobial properties of the R. natalensis used for many years by many people of the world. It also provides valuable information for&nbsp / further phytochemical isolation and characterization studies of active compounds, necessary for the development of new drugs. The extractions were carried out using broad spectrum of solvents&nbsp / (hexane, dichloromethane, ethyl acetate, and methanol). Fractionation was done using standard chromatographic techniques. A total of seven (7) compounds were isolated from R. natalensis.&nbsp / Three of the isolates were characterized and their structures were unambiguously established by detailed spectroscopic analysis that involved high resolution mass spectrometry, 1D and&nbsp / 2D-NMR spectral data experiments 1H, 13C, DEPT, COSY, HMBC, and NOESY. These compounds are: 3-(1-(2,4-dihydroxyphenyl)-3,3-bis(4- hydroxyphenyl)-1-oxopropan-2-yl)-7-methoxy- 4H-chromone-4-one (39), Rhuschromone, a&nbsp / novel compound isolated for the first time, 2&rsquo / ,4&rsquo / -dihydroxychalcone-(4-O-5&rsquo / &rsquo / &rsquo / )-4&rsquo / &rsquo / ,2&rsquo / &rsquo / &rsquo / ,4&rsquo / &rsquo / &rsquo / - trihydroxychalcone (40) and 3-((Z)-heptadec-13-enyl) benzene- 1,2-diol (41), an urushiol. Compound 39 recorded the highest activity zone of inhibition (21mm) against S. aureas, which was found to be 50% as active the chloramphenicol standard used. The&nbsp / traditional use of the extracts in infections and inflammatory conditions is rationalized based on the content of theisolated compounds, and it has been proposed that the total crude extract, with its&nbsp / contents of so many bioactive compounds, could be formulated for use in many infections, microbial or fungal. Furthermore, not all of the species studied to date have been fully characterized&nbsp / for potential bioactivities. Thus, there remains a significant research gap spanning the range from lead chemical discovery through process development and optimization in order to better&nbsp / understand the full bioactive potential of many of these plants.</p>

<p>Extracts from the root bark, stem bark, and leaves of R. natalensis were screened for antibacterial activity against standard bacterial strains / Staphylococcus aureas, Escherichia coli and Pseudomonas aureginosa, and fungi / Candida albicans, Trichophyton mentagrophytes or Microsporum gyseum. Chromatographic techniques were utilized to isolate pure compounds. This study validates and documents, in a systematic way, the antimicrobial properties of the R. natalensis used for many years by many people of the world. It also provides valuable information for&nbsp / further phytochemical isolation and characterization studies of active compounds, necessary for the development of new drugs. The extractions were carried out using broad spectrum of solvents&nbsp / (hexane, dichloromethane, ethyl acetate, and methanol). Fractionation was done using standard chromatographic techniques. A total of seven (7) compounds were isolated from R. natalensis.&nbsp / Three of the isolates were characterized and their structures were unambiguously established by detailed spectroscopic analysis that involved high resolution mass spectrometry, 1D and&nbsp / 2D-NMR spectral data experiments 1H, 13C, DEPT, COSY, HMBC, and NOESY. These compounds are: 3-(1-(2,4-dihydroxyphenyl)-3,3-bis(4- hydroxyphenyl)-1-oxopropan-2-yl)-7-methoxy- 4H-chromone-4-one (39), Rhuschromone, a&nbsp / novel compound isolated for the first time, 2&rsquo / ,4&rsquo / -dihydroxychalcone-(4-O-5&rsquo / &rsquo / &rsquo / )-4&rsquo / &rsquo / ,2&rsquo / &rsquo / &rsquo / ,4&rsquo / &rsquo / &rsquo / - trihydroxychalcone (40) and 3-((Z)-heptadec-13-enyl) benzene- 1,2-diol (41), an urushiol. Compound 39 recorded the highest activity zone of inhibition (21mm) against S. aureas, which was found to be 50% as active the chloramphenicol standard used. The&nbsp / traditional use of the extracts in infections and inflammatory conditions is rationalized based on the content of theisolated compounds, and it has been proposed that the total crude extract, with its&nbsp / contents of so many bioactive compounds, could be formulated for use in many infections, microbial or fungal. Furthermore, not all of the species studied to date have been fully characterized&nbsp / for potential bioactivities. Thus, there remains a significant research gap spanning the range from lead chemical discovery through process development and optimization in order to better&nbsp / understand the full bioactive potential of many of these plants.</p>

Condensed tannins or proanthocyanidins (CTs) are polymeric flavonoids and common plant secondary metabolites. They are defined by their protein binding capacity, and anti-oxidant and metal chelating properties. Known functions of CTs include anti-herbivore and anti-microbial defenses. Chemical ecology studies, especially on CTs, have focussed almost exclusively on foliar chemistry and above-ground ecological interactions. However, CT concentrations in roots can match and far exceed those found in leaves, particularly in woody plant species. This suggests that CTs are also likely to have important ecological and physiological roles below-ground. A review of the literature suggests three potential roles of root CTs: 1) defense against soil pathogens and root herbivores; 2) facilitating adaptation to toxic soils through heavy metal chelation, and; 3) facilitating or modulating nutrient uptake through the binding of nutrient cations. In this thesis the relationship between root CTs and nutrient uptake will be analysed in P. tremula x alba. Localization of CTs was determined through both quantitative and histochemical techniques. Quantitation of CTs with the 1-butanol:HCl assay clearly showed that CT concentration was higher in the white root zone compared to the brown root zone or cork zone. This is inconsistent with the term ‘condensed tannin zone’ that is commonly used to describe the macroscopic brown zone of the root between the white zone and the cork zone in Pinus. The tissue specific localization of CTs in roots was determined using the CT-specific stain, 4-dimethylaminocinnamaldehyde (4-DMACA), on embedded longitudinal and fresh cross sections. These stained sections showed that CTs were present in cells on the root surface, specifically accumulating in the root cap and epidermal cells. CTs were also sporadically present in the cortical cells of the young, white root zones. CT concentration declined as distance from the root tip increased. The pattern of CT distribution, as indicated by intensity of the staining, corresponded directly to the quantitative assay results. Growing poplars under low nitrogen availability stimulated higher root CT concentrations than when they were grown under high nitrogen availability, suggesting a link between nitrogen and CTs. Since CTs are known to bind cations in vivo, I hypothesized that root CTs may modulate or facilitate nutrient uptake by binding positively charged nutrient ions. The Microelectrode Ion Flux Measurement (MIFE) technique was used to analyse specific fluxes of NH4+, NO3- and Ca2+ at specific root locations in poplar roots, and test for spatial correlation with CT localization. This technique was also used to compare the NH4+, NO3- and Ca2+ fluxes in transgenic high-CT and wild-type lines of Populus tremula x alba to test the impact of CTs on nutrient uptake directly. No correlation was found between fluxes of NH4+, NO3- or Ca2+ and CT distribution. The data indicate that root CTs do not influence nutrient uptake in Populus. This study provides data regarding the pattern of CT distribution as well as fundamental information on nutrient uptake in P. tremula x alba roots. It provides new knowledge that will be used to stimulate investigations on other potential roles of root CTs. / Graduate

Το γένος Hypericum L. περιλαμβάνει πάνω από 450 taxa παγκοσμίως, ενώ στην Ελλάδα έχουν καταγραφεί 50 taxa (35 είδη και 15 υποείδη). Το υπερικό είναι γνωστό από την αρχαιότητα για την επουλωτική και αντικαταθλιπτική του δράση. Το H. perforatum αποτελεί το γνωστότερο είδος από το οποίο προκύπτουν τα εμπορικά διαθέσιμα φυτικά εκχυλίσματα. Η έρευνα της χημικής σύστασης των εκχυλισμάτων του υπερικού έχει αναδείξει την παρουσία ενώσεων μοναδικών ως προς τη δομή και περιορισμένης κατανομής, όπως οι ναφθοδιανθρόνες και οι φλωρογλουκινόλες. Το Hypericum vesiculosum Griseb. ανήκει στο ίδιο γένος με το H. perforatum L., ωστόσο δεν έχει μελετηθεί ως προς την φυτοχημική του σύσταση. Xαρακτηρίζεται υπενδημικό αυτοφυές φυτό και εξαπλώνεται στη βόρεια και ανατολική Ελλάδα, στη βόρεια και κεντρική Ελλάδα και στην Πελοπόννησο. Σκοπός της παρούσας εργασίας ήταν η φυτοχημική ανάλυση του H. vesiculosum και η μελέτη της αγχολυτικής δράσης του μεθανολικού εκχυλίσματος σε γηραιούς θηλυκούς και αρσενικούς μύες. Το φυτό συλλέχθηκε στην Πελοπόννησο από την περιοχή της λίμνης Στυμφαλίας, του νομού Κορινθίας. Τα μεθανολικά εκχυλίσματα των H. vesiculosum και H. perforatum μελετήθηκαν και συγκρίθηκαν ως προς τη φυτοχημική τους σύσταση με τη χρήση υγρής χρωματογραφίας υψηλής απόδοσης με ανιχνευτή συστοιχίας φωτοδιόδων (High Performance Liquid Chromatography – Diode Array, HPLC – DAD), σε στήλη αντίστροφης φάσης Luna C-18. Παρατηρήθηκε ταύτιση των HPLC χρωματογραφημάτων ανάμεσα στα δύο taxa, σε μεγάλο βαθμό, ενώ ταυτοποιήθηκαν και ποσοτικοποιήθηκαν οι ενώσεις χλωρογενικό οξύ, ρουτίνη, υπεροζίτης, ισοκεριστρίνη, κερσιτρίνη, κερσετίνη, αμεντοφλαβόνη, κερσετίνη, υπερικίνη και υπερικίνη στο H. vesiculosum για πρώτη φορά. Όλες οι κορυφές που ταυτοποιήθηκαν ήταν σε μικρότερο ποσοστό στο Η. vesiculosum σε σχέση με το H. perforatum, με εξαίρεση την κερσιτρίνη σε ποσοστό 20,3%. Οι ναφθοδιανθρόνες στο σύνολο τους ήταν λιγότερες σε ποσοστό 85%. Με σκοπό την απομόνωση των ναφθοδιανθρονών από το ξηρό μεθανολικό εκχύλισμα πραγματοποιήθηκαν διαδοχικές κλασματώσεις με εξάνιο και οξικό αιθυλεστέρα, αλλά και κλασική υγρή χρωματογραφία στήλης με Silica 60A και Sephadex LH-20. Ύστερα από καθαρισμό τους με ημιπαρασκευαστικό HPLC και καθαρότητα μεγαλύτερη από 98%, πραγματοποιήθηκε η ταυτοποίηση τους με φασματομετρία μάζας (Mass Spectrometry, MS), όπου διαπιστώθηκε ότι οι απομονωθείσες ενώσεις ήταν η υπερικίνη, με μοριακό βάρος 504 και η ψευδοϋπερικίνη, με μοριακό βάρος 520. H αγχολυτική δράση του μεθανολικού εκχυλίσματος από το H. vesiculosum εξετάσθηκε σε γηραιούς αρσενικούς και θηλυκούς μύες, ηλικίας περίπου 12 μηνών, με τη δοκιμή του ανοικτού πεδίου (Open Field Test). Η χορήγηση του εκχυλίσματος έγινε ενδοπεριτοναϊκά 24 ώρες και 1 ώρα πριν από τη δοκιμασία, σε ποσότητα 250mg/Kg. Η παράμετρος που εξετάστηκε ήταν ο θιγμοτακτισμός μετρώντας τον χρόνο που παρέμειναν οι μύες στην περιφέρεια της συσκευής. Τα αποτελέσματα επιβεβαίωσαν την αγχολυτική δράση του εκχυλίσματος στους γηραιούς μύες και στα δύο φύλα. Συγκεκριμένα ο χρόνος θιγμοτακτισμού μειώθηκε σε ποσοστό 24% στις ομάδες που έλαβαν το εκχύλισμα. Στο H. vesiculosum εδείχθη για πρώτη φορά η παρουσία των ναφθοδιανθρονών και έγινε σύγκριση της φυτοχημικής του σύστασης σε σχέση με το H. perforatum. Η αγχολυτική δράση που επέδειξε το φυτό στους γηραιούς μύες, θα μπορούσε να χρησιμοποιηθεί στην θεραπευτική για μελλοντική εφαρμογή η οποία, ωστόσο, χρειάζεται περαιτέρω διερεύνηση. / The genus Hypericum L. includes over 450 taxa worldwide and in Greece there have been recorded 50 taxa (35 species and 15 subspecies). St. John’s wort is known since ancient times for it’s healing and antidepressant actions. H. perforatum is the best known species from which the commercially available herbal extracts are produced. The investigation of Hypericum extracts’ chemical composition has revealed the presence of unique compounds, in structure and limited distribution, such as naphthodianthrones and phloroglucinols. The taxon Hypericum vesiculosum Griseb. belongs to the same genus with H. perforatum L., however, it has not been studied before for its phytochemical composition. It is a subendemic plant and spreads to the northern and eastern Greece, the northern and central Greece and the Peloponnese. The purpose of this study was the phytochemical analysis of H. vesiculosum and investigation of methanolic extract’s potential anxiolytic activity, in aged female and male mice. The plant was collected from the Peloponnese region of Lake Stymfalia (prefecture of Corinthia). The methanol extract of H. vesiculosum Griseb. and H. perforatum L. were analysed with high performance liquid chromatography, connected with a photodiode array detector (High Performance Liquid Chromatography - Diode Array, HPLC - DAD) and a reversed phase column Luna C-18. The HPLC chromatograms demonstrated great resemblance between the two taxa. In H. vesiculosum, the compounds chlorogenic acid, rutin, hyperoside, isoquercitrin, quercitrin, quercetin, amentoflavone, hypericin and hypericin were identified and quantified for the first time. All peaks were quantified in smaller proportion in H. vesiculosum compared to H. perforatum, with the exception of quercitrin of which the percentage was 20.3 %. The total propotion of naphthodianthrones was less than 85 % in Η. vesiculosum. In order to isolate the naphthodianthrones from the dry methanol extract, sequential fractionations were performed with hexane and ethyl acetate but also classic liquid column chromatography with Silica 60A and Sephadex LH-20. After isolating the two compounds with semipreparative HPLC and purity greater than 98%, their identification was performed with mass spectrometry (Mass Spectrometry, MS). The isolated compounds were hypericin, with molecular weight 504 and pseudohypericin, with molecular weight 520. The potential anxiolytic activity of the methanolic extract of H. vesiculosum was examined in aged male and female mice (12 months old), with the open-field test (Open Field Test). The short-term administration of the extract was intraperitoneally 24 h and 1 h before the test, at the dose of 250mg/Kg body weight. The parameter which was considered was the time the mice stayed in the periphery of the device (thigmotaxis time). The results confirmed the anxiolytic activity of the extract at the aged mice in both sexes. More specifically the thigmotaxis time was decreased in a percentage of 24% for the groups who received the extract. The presence of naphthodianthrones was shown in H. vesiculosum for the first time but also the phytochemical compositions of Η. vesiculosum and H. perforatum were compared. In conclusion, the present results show that H. vesiculosum contains the main bioactive constituents of H. perforatum in almost the same quantities and engenders anxiolytic behavior in both male and female aged mice

Piperine, the major alkaloid derived from black pepper corns, has played an important role in traditional medicine worldwide. Current research has demonstrated piperine to have several anti-inflammatory properties, however, little is known concerning the effect of piperine on B cells. Spleen-derived murine B cells were cultured in the presence or absence of piperine during T-dependent or T-independent activation. Piperine reversibly inhibited B cell proliferation in a dose-dependent manner. This was due to a G0/1-phase cell cycle arrest, and was associated with a reduction in phospho-ERK, phospho-AKT, and Cyclin D1, D2, and D3. Piperine also inhibited antibody and cytokine production. Furthermore, piperine treatment diminished B cell-mediated antigen presentation determined by measuring OT-II transgenic T cell proliferation in response to OVA, which was attributed to the decreased MHC-II ad co-stimulatory molecule expression observed. This in vitro study shows that piperine has potent immuno-suppressive effects on B cell activation and effector function.

Significant morbidity and mortality continues to be associated with breast cancer and its treatments. Fisetin, a phytochemical that is present in many fruits and vegetables, has demonstrated anticancer activity. My research explores fisetin as a possible novel therapeutic modality for breast cancer. Breast cancer cell lines (MDA-MB-468, MDA- MB-231, MCF-7, T47-D, SKBR-3; mitoxantrone-resistant (MITX) and paclitaxel- resistant (Tx400) cell lines) were exposed to fisetin and cell survival was assessed by MTT, crystal violet, acid phosphatase, and colony-forming assays. Normal cells (human mammary epithelial cells, fibroblasts, human umbilical vein endothelial cells) were used as negative controls. The mechanism of action of fisetin was explored using cell cycle analysis and assays for apoptosis/necrosis, including Annexin V-propidium iodide staining and LDH-release. Apoptosis induction pathways were studied using Western blotting, as well as caspase inhibitors and cell viability assays. Flow cytometry was used to assess mitochondrial membrane stability (DiOC6 staining) and reactive oxygen species (ROS) production (dihydroethidium staining). Fisetin had a dose- and time-dependent cytotoxic effect on breast cancer cell lines (e.g., 100 ?M fisetin decreased MDA-MB-468 cell number by 70% at 72h in both crystal violet and acid phosphatase assays). In contrast, the viability of normal cells was not substantially affected by concentrations of fisetin that killed breast cancer cells. Fisetin-treated breast cancer cells showed cell cycle arrest (MDA-MB-468 cells arrested at G2/M phase; MDA-MB-231 cells arrested in S- phase) and death by apoptosis (e.g., MDA-MB-468 cells showed up to 50% apoptosis and 8% late apoptosis/necrosis by Annexin V-staining; cell cycle analysis and LDH- release assays supported these results). Fisetin-induced apoptosis was associated with mitochondrial membrane permeabilization, as well as activation of the caspase cascade since the pro-apoptotic effect of fisetin was reduced in the presence of a pan-caspase inhibitor. In addition, fisetin did not cause ROS production in MDA-MB-468 or 231 cells, ruling out a role for ROS in fisetin-mediated cytotoxicity. My findings suggest that fisetin may be useful in the treatment of breast cancer.

Orientador: Dulce Helena Siqueira Silva / Banca: Marcia Nasser Lopes / Banca: Hosana Maria Debonsi Navickiene / Resumo: A família Sapotaceae é composta de 53 gêneros com aproximadamente 600 espécies. Esta família não é extensivamente estudada e é encontrada nas regiões tropicais e subtropicais como arbustos e árvores, incluindo espécies comestíveis como Manilkara zapote (sapoti), Pouterie caimito (abiu), Chrysophyllum cainito ("star-apple") e Synsepalum dulcificum ("miracle-fruit"). Poucas espécies do gênero Chrysophyllum foram investigadas quimicamente, sendo que algumas apresentaram atividades biológicas importantes. Estudos mais recentes têm focalizado os constituintes de frutos comestíveis, buscando antioxidantes polifenólicos e substâncias com atividade antiinflamatória, como triterpenos derivados do ácido oleanólico e do ácido glicirretínico. Algumas espécies são usadas na medicina popular para desordens gástricas e tratamento de úlcera. Outras apresentaram atividade redutora dos níveis de LDL, colesterol e triglicerídeos séricos. O estudo fitoquímico de Chrysophyllum flexuosum inclui em um projeto de pesquisa que visa conhecer a composição química das espécies vegetais com potencial atividade biológica. Análise preliminar do extrato bruto das folhas e ramos apresentou atividade antioxidante em teste utilizando CCDC nebulizada com solução de ß-caroteno e inibiu a peroxidação lipídica em modelo de lipossomas usando Fe2+ como iniciador de reações radicalares. O extrato etanólico de folhas e ramos da espécie Chrysophyllum flexuosum foi submetido a partição líquido-líquido com hexano, acetato de etila, butanol e metanol/água (9:1). Da fração hexânica foram isolados 4 triterpenos pentacíclicos majoritários identificados como: ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Sapotaceae family is composed of 53 genera with ca. 600 species. This family is not extensively studied and is found in tropical and subtropical regions as shrubs and trees, including edible species as Manilkara zapote (sapoti), Pouterie caimito (abiu), Chrysophyllum cainito (starapple) and Synsepalum dulcificum (miracle-fruit). Few species of Chrysophyllum genus have been investigated although some present important biological properties. Recent studies have focused on the constituents of edible fruits, searching for antioxidant phenolics and anti-inflammatory compounds, as triterpene derivatives of oleanolic acid and glicerretinic acid. Some species have presented reducing activity on LDL, cholesterol and triglyceride blood levels. The phytochemical study on Chrysophyllum flexuosum is included in a research project focusing on the search for bioactiveormolecular compounds from São Paulo flora with potential biological activity. Preliminary analysis of its leaves crude extract by a TLC test nebulized with beta-carotene solution in a liposome model using Fe2+ as radicalar reactions initiator, indicated the presence of antioxidant compounds. The ethanol extract from leaves of Chrysophyllum flexuosum was submitted to liquidliquid partition with organic solvents: hexane, ethyl acetate, butanol and methanol/water (9:1), followed by chromatographic procedures. The hexane fraction afforded four pentacyclic triterpenes, including the new butanolide-[1,3]-a-amyrin (1), 3-O-ß-acetyl-ß-amyrin (2), 3-O-ß-acetyl-lupeol (3); and 3-O-ß-acetyl-a-amyrin (7), comprising ca. one third of its total weight. Lupeol (6), ß-amyrin (5); and a-amyrin (4) were also identified from the hexane fraction. The ethyl acetate, butanol and methanol fractions using several mobile phases and UV wavelength detection disclosed the presence of gallic acid in... (Complete abstract, click electronic access below) / Mestre