Dissertations / Theses on the topic 'Oleanic acid'

A thesis submitted to the Cape Peninsula University of Technology in fulfilment of the requirements for the MASTERS DEGREE IN TECHNOLOGY (CHEMISTRY) Department of Chemistry January 2007
An increasing number of natural products possessing the oleanolic acid moiety have been shown to demonstrate a wide spectrum of biological activity. This thesis deals with the extraction and isolation of oleanolic acid from Syzigium aromaticum and the examination of its stereochemistry and crystal structure by X-ray diffraction. The synthetic routes used for converting functional groups on the oleanolic acid molecule to afford derivatives are described in Chapter 5. Oleanolic acid and its derivatives were evaluated for antimicrobial activity. Three different procedures viz. Kirby-Bauer, Broth dilution and Tetrazolium salt chemosensitivity were used. Acceptable results were obtained from the last method and these were used to arrive at conclusions regarding this study.

La diabète de type 2 est associée avec des désordres du métabolisme de glucose dans le foie et les système périphériques, résultant un niveau trop élevé du glucose dans le sang qui est responsable des conséquences graves à long terme. Un agent anti-diabétique idéal doit être capable de réduire le taux de glucose en états- alimentés et à jeun. Le contrôle du métabolisme hépatique du glucose est l'une des principales voies utilisées par l'insuline pour maintenir l'homéostasie du glucose dans le sang. Parmi les différentes possibilités d'influencer la production hépatique du glucose, l'inhibition de la glycogène phosphorylase (GP), enzyme responsable de la dégradation du glycogène, est une approche prometteuse pour le traitement de diabète de type 2. Plusieurs inhibiteurs de se sont montrent efficaces à baisser le taux de glucose dans des animaux et en essai clinique. Récemment, nous avons montré que l'acide oléanolique et d'autres triterpènes pentacycliques représentent une nouvelle classe d'inhibiteurs de glycogène phosphorylase. La structure aux rayons X révèlent que les triterpènes pentacycliques comme l'acide asiatique et l'acide maslinique fixent au site allostérique de OP. Les efforts intensifs ont été réalisés sur la modification structurale des triterpènes naturels afin de trouver des agents préventifs et thérapeutiques. Nous espérions que les dérives de triterpènes pentacycliques puissent être utilisés comme des nouveaux médicaments pour la régulation du métabolisme de glucose. Dans cette étude, nous avons synthétisé plusieurs dérivés de l'acide oléanolique et des conjugués avec des sucres et des nucléosides. Leur propriété inhibitrice vis-à-vis de la glycogène phosphorylase a été évalué avec la GPa du muscle du lapin (RMGPa). Certaines molécules présentent une inhibition de l'ordre de micromolaire
Type 2 diabetes mellitus is associated with disorelers in glucose metabolism by the liver and periphery resulting in elevated blood glucose levels which, in turn, are responsible for fatal long term complications. An ideal anti-diabetic agent should be capable of lowering blood glucose in both fed and fasted states. Control of die hepatic glycogen metabolism is one of the key events through which insulin maintains blood glucose homeostasis. Among other means for influencing glucose production in the liver, inhibition of glycogen phosphorylase (GP), the rate limiting enzyme of glycogen degradation, has been regarded as a promising therapeutic approach ta the treatment of type 2 diabetes. A couple of GP inhibitors have shown efficacy in lowering blood glucose in animal models and clinical trials. We have recently reported that oleanolic acid and related pentacyclic triterpenes represented a new class of inhibitors of glycogen phosphorylases. X-Ray crystallographic studies revealed the molecular basis of their inhibitory effect demonstrating that pentacyclic triterpenes such as asiatic and maslinic acids bind ta OP at the allosteric site. On the other hand, structural modifications based on natural triterpenes have been extensively explored ta find more patent pentacyclic triterpenes as preventive and therapeutic agents. Therefore, we believe that pentacyclic triterpenes will be used as new drugs in regulating glucose and lipid metabolism one day. Thus, in this study, we have synthesized several oleanolic acid derivatives and conjugates with sugar and nucleosides, and evaluated GP inhibitory activity against RMGPa. Some molecules exhibited inhibition in micromolar range. The structural analyses of these cornpounds can be further exploited (by chemical modification) towards the development of better GP inhibitors

Philosophiae Doctor - PhD
Breast cancer is one of the most common cancers among women in South Africa and the second leading cause of cancer death after lung cancer. According to the American Cancer Society 2015, women have a 12% chance of developing invasive breast cancer and a 3% chance of dying from it. Despite the wide variety of breast cancers e.g. lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS), many share the same etiology and target tissue. Estrogen related carcinogenesis with regard to breast cancer typically results from the activation of distinct signalling pathways. These pathways are not mutually exclusive and are often constituted by receptor mediated stimulation of cell proliferation caused by specific transcriptional gene activation, reactive oxygen species (ROS) formation causing DNA damage and consequently mutations. The molecular pathways that cause drug resistance are not fully understood and the search continues to find novel targets for treatment. The effects of non-toxic triterpenes, oleanolic acid and ursolic acid and the role of autophagy and apoptosis as mechanisms to overcome drug resistance in breast cancer were studied in vitro in MCF-7 breast cancer cells and MCF10A breast cells. In this study the first aim was to establish the influence of OA and UA on cell growth and to see if opposing proliferation patterns could observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells. This was followed by morphology studies to establish the possible presence of cytotoxicity and examination of molecular pathways contributing to the anti-cancerous properties of UA and OA and their validity as therapeutic agents. The MCF-7 breast cancer cell line and the immortalized normal mammary cell line, MCF-10A were treated with different concentrations of UA and OA for 6hrs, 12hrs, 24hrs, 48hrs, and 72hrs respectively. Cell morphology was studied in hematoxylin and eosin as well as Hoechst and acridine orange stained cells and viability was measured using crystal violet staining. Molecular techniques employed included the Tali® Apoptosis - and the cellROX assays, flow cytometry and western blotting. Morphological, viability and apoptotic studies have shown that at their lowest concentration, both UA and OA have anti-proliferative and apoptotic effects on MCF-7 and to a lesser extent on MCF-10A. Flow cytometric analysis of treated cells has demonstrated cell arrest in the S- and G2/M phase. The MCF-7 and MCF-10A cells growth inhibition effect may be due to increased autophagy and apoptosis as an alternative to decreased proliferation in MCF-7 cells. This possibility should be evaluated in further studies. The results showed that UA was more effective OA in decreasing cell numbers and it may be applied as treatment for breast cancer. Our observation has shown the treatment with OA and UA increased cell death in MCF-7 cells.The opposing proliferation patterns observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells could possibly be ascribed to ERß forming homodimers that may facilitate proliferation, whereas ERɑ/ß heterodimers (expressed in 59% of breast cancers) are frequently associated with the ERɑ antagonising actions of ERß. The results indicate a trend towards biphasic and anti- proliferative effects of the reactants in breast cancer cells which may contribute towards the development of anti- cancer therapies. However, further work is must be done to identify the OA and UA mechanism(s) responsible for anticancer activity.
Libyan Embassy

Toxoplasma gondii é um protozoário pertencente ao filo Apicomplexa, de distribuição mundial, e que infecta diversas espécies hospedeiros, como mamíferos e aves, possuindo como hospedeiros definitivos o gato e outros felídeos, enquanto o homem e outros animais são os seus hospedeiros intermediários. O tratamento é feito, na maioria das vezes, com uma combinação de sulfadiazina e pirimetamina, agindo na via metabólica do ácido fólico, o qual é necessário para a biossíntese de purinas, pirimidinas e certos aminoácidos. Propusemos estabelecer um protocolo de avaliação sobre esse protozoário, assim como padronizarmos uma metodologia por espectroscopia para uma rápida avaliação ou triagem de substâncias potencialmente ativas contra o parasito. Verificamos a atividade das substâncias ácido ursólico e ácido oleanóico, as quais já demonstraram atividade biológica sobre outras espécies de protozoários, como Plasmodium, Trypanosoma cruzi e Leishmania sp. em sistemas in vitro e in vivo. A metodologia colorimétrica pelo MTT, pelo Alamar Blue®, do kit CyQUANT® NF e a contagem manual em meio líquido das formas taquizoítas do parasito em ensaios biológicos in vitro mostram-se inviáveis, pois há grande dificuldade em manter a integridade do parasita em meio de cultura líquido, o qual se mostra sensível à adição de qualquer outro componente que não aqueles necessários para manter sua viabilidade em ambiente extracelular. O ácido ursólico mostrou-se potencialmente ativo in vitro sobre formas intracelulares de T. gondii. Entretanto, o contato de células infectadas com as substâncias avaliadas por um período de 48 horas não resultou em diminuição mais acentuada na porcentagem de células infectadas do que a ocorrida no tratamento de 24 horas. A comparação dos resultados do tratamento pós infecção celular por 24 horas e do pré-tratamento das formas taquizoítas houve diferenças significativas, indicando principalmente maior ação do pré-tratamento sobre T. gondii. Quando administrado na dose de 7 mg/kg/ dia a camundongos infectados, o ácido ursólico não apresentou atividade sobre o parasita.
Toxoplasma gondii is an Apicomplexa protozoan, of worldwide distribution, that infects several species, from mammalians to birds; its definitive hosts are the cats and other felines, while man and other animals are considered as intermediate hosts. Treatment is, generally, a combination of sulfadiazine and pyrimethamine, triggering the metabolic pathway of folic acid, which is necessary for certain purines, pirimidines and aminoacids biosynthesis. We have proposed an evaluation protocol on this protozoan, and also to establish a methodology by spectroscopy for rapid evaluation of pottentialy bioactive substances against the parasite. The ursolic acid and oleanoic acid bioactivity were tested. These substances have already demonstrated to be effective on another protozoan species, like Plasmodium, Trypanosoma cruzi e Leishmania sp., either in vitro or in vivo. The colorimetric methodology by MTT, Alamar Blue®, CyQUANT® NF kit and manual counting of tachyzoite forms in liquid culture medium showed to be unviable, because there is a great difficult to maintain the parasite viability in liquid culture medium, wich one is sensible to addition of any other component different of that necessary for its survival in extracelular ambient. Ursolic acid was pottentialy active on T. gondii intracellular forms. However, the infected cells in contact with the tested substances for a period of 48 hours did not show a statistical greater reduction as compared to infected cells which underwent treatment for 24 hours. Significant results were observed when comparing pre-treatment of tachyzoite forms and treatment for 24 hours post-cellular infection. Our data pointed in the direction that pre-treatment exerted a higher effectiveness. Any parasiticidal activity was observed when ursolic acid on a concentration of 7 mg/kg/day was administered to infected mice.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
A doenÃa de Chagas à uma doenÃa negligenciada causada pelo parasito Trypanosoma cruzi e constitui um problema de saÃde pÃblica em todo o mundo. O tratamento atual à restrito pelos efeitos colaterias frequentes e pela eficÃcia limitada do benzonidazol. O Ãcido betulÃnico (AB) e Ãcido oleanÃlico (AO), sÃo triterpenos, existentes em diversas plantas medicinais e exibem uma grande variedade de atividades biolÃgicas e farmacolÃgicas, incluindo efeito em tripanossomatÃdeos. O objetivo do presente estudo foi avaliar o efeito tripanocida e mecanismo de aÃÃo dos Ãcidos betulÃnico e oleanÃlico em cepa Y de Trypanosoma cruzi in vitro. O efeito tripanocida do AB e AO (1,56 - 200 μM) foram avaliadas durante 24, 48 e 72 horas, sob formas epimastigotas, tripomastigotas e amastigotas de T. cruzi. A viabilidade da cÃlula LLCMK2, tratada com AB e AO (200 - 1600 μM), foi avaliada durante 24 h atravÃs do teste MTT. Os ensaios de mecanismo de aÃÃo foram realizados nas formas epimastigotas tratadas com CI50 do AB e AO durante 24 h, e marcadas com anexina V/7AAD, Rho123, H2DCFDA, Laranja de Acridina e MDC de acordo com as instruÃÃes do fabricante e analisadas por citometria de fluxo e microscopia confocal. Os dados foram analisados utilizando ANOVA com pÃs-teste de Bonferroni ou teste t Student, * p≤0,05. O AB inibiu o crescimento de formas epimastigotas em 24h (CI50 = 73,43 μM; BZ = 218 μM), 48h (CI50 = 119,8 μM; BZ = 61μM) e 72h (CI50 = 212,2 μM; BZ = 16,5 μM) de incubaÃÃo; inibiu a viabilidade de tripomastigotas (CI50 = 51,88 μM; BZ = 257μM) em 24h. O AO inibiu o crescimento de epimastigotas em 24h (CI50 = 11,66 μM; BZ = 218 μM), 48h (CI50 = 43,15 μM; BZ = 61 μM) e 72h (CI50 = 43,05 μM; BZ = 16,5μM) de incubaÃÃo; inibiu a viabilidade de tripomastigotas (IC50 = 13,97 μM; BZ = 257 μM) em 24h. O AB e AO diminuiram o percentual de cÃlulas infectadas e reduziram o nÃmero de amastigotas por cÃlulas nos tratamentos com 24h e 48 h, respectivamente. Ambos terpenos nÃo apresentaram toxicidade sobre as cÃlulas LLCMK2 nas concentraÃÃes utilizadas. A anÃlise do mecanismo de morte celular do parasito tratado com AB mostrou alteraÃÃes no potencial da membrana mitocondrial, alteraÃÃes na integridade da membrana celular, aumento da formaÃÃo de espÃcies reativas de oxigÃnio e detecÃÃo de compartimentos acÃdicos. O tratamento com AO demonstrou detecÃÃo de compartimentos acÃdicos e de vacuÃlos autofÃgicos. Nossos resultados demonstram que o AB e AO apresentam efeito tripanocida sobre todas as formas evolutivas de cepa Y de T. cruzi, sugerindo que o mecanismo de morte celular do AB à via necrose e do AO via autofagica nas formas epimastigotas.
A doenÃa de Chagas à uma doenÃa negligenciada causada pelo parasito Trypanosoma cruzi e constitui um problema de saÃde pÃblica em todo o mundo. O tratamento atual à restrito pelos efeitos colaterias frequentes e pela eficÃcia limitada do benzonidazol. O Ãcido betulÃnico (AB) e Ãcido oleanÃlico (AO), sÃo triterpenos, existentes em diversas plantas medicinais e exibem uma grande variedade de atividades biolÃgicas e farmacolÃgicas, incluindo efeito em tripanossomatÃdeos. O objetivo do presente estudo foi avaliar o efeito tripanocida e mecanismo de aÃÃo dos Ãcidos betulÃnico e oleanÃlico em cepa Y de Trypanosoma cruzi in vitro. O efeito tripanocida do AB e AO (1,56 - 200 μM) foram avaliadas durante 24, 48 e 72 horas, sob formas epimastigotas, tripomastigotas e amastigotas de T. cruzi. A viabilidade da cÃlula LLCMK2, tratada com AB e AO (200 - 1600 μM), foi avaliada durante 24 h atravÃs do teste MTT. Os ensaios de mecanismo de aÃÃo foram realizados nas formas epimastigotas tratadas com CI50 do AB e AO durante 24 h, e marcadas com anexina V/7AAD, Rho123, H2DCFDA, Laranja de Acridina e MDC de acordo com as instruÃÃes do fabricante e analisadas por citometria de fluxo e microscopia confocal. Os dados foram analisados utilizando ANOVA com pÃs-teste de Bonferroni ou teste t Student, * p≤0,05. O AB inibiu o crescimento de formas epimastigotas em 24h (CI50 = 73,43 μM; BZ = 218 μM), 48h (CI50 = 119,8 μM; BZ = 61μM) e 72h (CI50 = 212,2 μM; BZ = 16,5 μM) de incubaÃÃo; inibiu a viabilidade de tripomastigotas (CI50 = 51,88 μM; BZ = 257μM) em 24h. O AO inibiu o crescimento de epimastigotas em 24h (CI50 = 11,66 μM; BZ = 218 μM), 48h (CI50 = 43,15 μM; BZ = 61 μM) e 72h (CI50 = 43,05 μM; BZ = 16,5μM) de incubaÃÃo; inibiu a viabilidade de tripomastigotas (IC50 = 13,97 μM; BZ = 257 μM) em 24h. O AB e AO diminuiram o percentual de cÃlulas infectadas e reduziram o nÃmero de amastigotas por cÃlulas nos tratamentos com 24h e 48 h, respectivamente. Ambos terpenos nÃo apresentaram toxicidade sobre as cÃlulas LLCMK2 nas concentraÃÃes utilizadas. A anÃlise do mecanismo de morte celular do parasito tratado com AB mostrou alteraÃÃes no potencial da membrana mitocondrial, alteraÃÃes na integridade da membrana celular, aumento da formaÃÃo de espÃcies reativas de oxigÃnio e detecÃÃo de compartimentos acÃdicos. O tratamento com AO demonstrou detecÃÃo de compartimentos acÃdicos e de vacuÃlos autofÃgicos. Nossos resultados demonstram que o AB e AO apresentam efeito tripanocida sobre todas as formas evolutivas de cepa Y de T. cruzi, sugerindo que o mecanismo de morte celular do AB à via necrose e do AO via autofagica nas formas epimastigotas.

Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2007
An increasing number of natural products possessing the oleanolic acid moiety have been shown to demonstrate a wide spectrum of biological activity. This thesis deals with the extraction and isolation of oleanolic acid from Syzigium aromaticum and the examination of its stereochemistry and crystal structure by X-ray diffraction. The synthetic routes used for converting functional groups on the oleanolic acid molecule to afford derivatives are described in Chapter 5. Oleanolic acid and its derivatives were evaluated for antimicrobial activity. Three different procedures viz. Kirby-Bauer, Broth dilution and Tetrazolium salt chemosensitivity were used. Acceptable results were obtained from the last method and these were used to arrive at conclusions regarding this study.

Oleanolic and ursolic acid (OA and UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from S. officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator (PGR) combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the Respiration Activity MOnitoring System® (RAMOS®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The μmax values for these suspension cultures ranged from 0.20 to 0.37°d-1, their OA and UA contents were greater than 1.3 and 1.2 mg g-1, respectively, and they afforded maximum volumetric yields of 21.0 mg l-1 for OA and 32.8 mg l-1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.

Oleanolic and ursolic acid (OA and UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from S. officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator (PGR) combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the Respiration Activity MOnitoring System® (RAMOS®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The μmax values for these suspension cultures ranged from 0.20 to 0.37°d-1, their OA and UA contents were greater than 1.3 and 1.2 mg g-1, respectively, and they afforded maximum volumetric yields of 21.0 mg l-1 for OA and 32.8 mg l-1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.

The discovery that bile acids act as endogenous ligands of the membrane receptor TGR5 and the nuclear receptor FXR increased their significance as regulators of cholesterol, glucose and energy metabolism. Activation of TGR5, expressed on enteroendocrine L cells, by bile acids caused secretion of GLP-1, which stimulates insulin secretion from pancreatic β cells. Expression of TGR5 on pancreatic islet cells and the direct effect of bile acids on the endocrine functions of pancreas, however, are not fully understood. The aim of this study was to identify expression of TGR5 in pancreatic islet cells and determine the effect of bile acids on insulin secretion. Expression of TGR5 was identified by quantitative PCR and western blot in islets from human and mouse, and in α (αTC1-6) and β (MIN6) cells. Release of insulin, glucagon and GLP-1 were measured by ELISA. The signaling pathways coupled to TGR5 activation were identified by direct measurements such as stimulation of G proteins, adenylyl cyclase activity, PI hydrolysis and intracellular Ca2+ in response to bile acids; and confirmed by the use of selective inhibitors that block specific steps in the signaling pathway. Our studies identified expression of TGR5 receptors in β cells and demonstrated that activation of these receptors by both pharmacological ligands (oleanolic acid (OA) and INT-777) and physiological ligand (lithocholic acid, LCA) induced insulin secretion. TGR5 receptors are also expressed in α cells and, activation of TGR5 by OA, INT-777 and LCA at 5 mM glucose induced release of glucagon, which is processed from proglucagon by the selective expression of prohormone convertase 2 (PC2). However, under hyperglycemia, activation of TGR5 in α cells augmented the glucose-induced increase in GLP-1 secretion, which in turn, stimulated insulin secretion. Secretion of GLP-1 from α cells reflected TGR5-mediated increase in PC1 promoter activity and PC1 expression, which selectively converts proglucagon to GLP-1. The signaling pathway activated by TGR5 to mediate insulin and GLP-1 secretion involved Gs/cAMP/Epac/PLC-ε/Ca2+. These results provide insights into the mechanisms involved in the regulation of pancreatic α and β cell function by bile acids and may lead to new therapeutic avenues for the treatment of diabetes.

In part one, betulinic acid (1) was isolated from the American Sycamore (Platanus occidentalis) in 1.6% yield, while ursolic acid (3) was isolated from Fuji and McIntosh apple peels in 1.0% and 0.8% crude yields, respectively. Oleanolic (4) and dehydrocanophyllic (6) acids were previously available, along with several analogs. Additional analogs of 1, 3 and 4 were prepared, including 9 new compounds, for a total of 51 compounds. Compounds were initially screened for cortisol lowering properties in vitro using a fish head kidney cell assay. Platanic acid (43) was selected for in vivo study in rats, along with 1 and a blend of Platanus occidentalis and Souroubea sympetela. No significant cortisol lowering was observed in vivo. In part two, β-ionone-d6 (75) was synthesized in 6.5% yield from ethyl 2-oxo-cyclohexane carboxylate (77). Total deuterium incorporation was 99.85%, with 0.03% d0 analog. 75 was converted to retinoic acid-d6 (93) in 2.2% yield.

A doença de Chagas é um problema de saúde pública, com dados preocupantes referentes ao número de pessoas contaminadas e daquelas que ainda permanecem expostas ao risco de infecção. As doenças tropicais desafiam as pesquisas científicas, pois os medicamentos existentes apresentam sérios efeitos colaterais. As tripanossomíases são doenças de grande importância no Brasil, como é o caso da doença de Chagas. Estudos realizados em diversos países têm relatado que muitas espécies vegetais possuem atividade contra o agente etiológico da doença de Chagas, Trypanosoma cruzi. Ácido ursólico e seu isômero, ácido oleanólico, pertencem à classe de compostos triterpenóides e são amplamente distribuídos no reino vegetal e têm sido frequentemente isolados como mistura isomérica. Nesse sentido, a proposta para o presente estudo foi avaliar a atividade tripanocida, sobre a cepa Y e Bolívia de T. cruzi, dos triterpenos ácido ursólico e ácido oleanólico, sintetizar e avaliar a atividade tripanocida do sal derivado de ácido ursólico. As substâncias foram administradas nas concentrações de 20 e 50 mg/kg por via intraperitoneal e oral, após a avaliação da dose letal média (DL50). As três substâncias empregadas nas concentrações de 20 e 50 mg/kg, administradas por via intraperitoneal não foram capazes de reduzir a parasitemia dos animais experimentalmente infectados com as cepas Y e Bolívia de T. cruzi. Entretanto, as três substâncias exibiram atividade tripanocida significativa, na concentração de 50 mg/kg, administradas por via oral, em animais infectados com a cepa Y de T. cruzi. Para a cepa Bolívia de T. cruzi, as substâncias administradas por via oral exibiram atividade biológica na concentração de 20 mg/kg, sendo significativa apenas para o triterpeno ácido ursólico. A análise histológica realizada não demonstrou uma correlação significativa entre os níveis parasitêmicos e o parasitismo tecidual, para todos os grupos avaliados. A avaliação do perfil hepatobiliar e renal das substâncias foi realizada a fim de se verificar possíveis efeitos tóxicos das mesmas. Entretanto, não foi observada nenhuma diferença estatisticamente significativa entre os grupos tratados e o grupo controle, indicando que o aumento dos níveis parasitêmicos não está associado ao efeito das substâncias no tecido. Através da avaliação do efeito dos triterpenos sobre a resposta imune, verificamos uma diminuição dos níveis plasmáticos de IFN- e um aumento das concentrações de IL-10. Dessa maneira, nós sugerimos que o tratamento com essas substâncias pode direcionar a resposta imune para o padrão Th2 e, em conseqüência desse efeito imunossupressor, uma maior quantidade de formas tripomastigotas poderia ser observada na circulação do hospedeiro.
Chagas disease is a public health problem with worrisome data on the number of infected people, not to mention the population that still remains at risk of infection. Tropical diseases defy scientific research since the existing drugs have serious side effects. Trypanosomiases, such as Chagas disease, are of great importance in Brazil. Studies in several countries have reported that many plant species display activity against Trypanosoma cruzi, the causative agent of Chagas disease. Ursolic acid and its isomer, oleanolic acid, belong to a class of triterpenoid compounds that is widely distributed in the plant kingdom and has frequently been isolated as an isomeric mixture. In the present study, the trypanocidal activity of the triterpenoids ursolic acid and oleanolic acid was evaluated against the Y and Bolivia strains of T. cruzi. In addition, the potassium salt derivative of ursolic acid was synthesized and also tested. The triterpenoids were administered intraperitoneally and orally at concentrations of 20 and 50 mg / kg, after evaluation of the median lethal dose (LD50). At the concentrations of 20 and 50 mg / kg, the intraperitoneal administration of each of the substances was not able to reduce parasitemia of the animals infected with the Y and Bolivia strains of T. cruzi. However, ursolic and oleanolic acid, as well as the potassium salt derivative of ursolic acid exhibited significant trypanocidal activity at a concentration of 50 mg / kg when they were orally administered to animals infected with the Y strain of T. cruzi. On the other hand, oral administration of the tested compounds at a concentration of 20 mg / kg evidenced biological activity, which was significant for ursolic acid only. For the most of studied groups, histological analysis did not demonstrate a significant correlation between the levels of parasitemia and tissue parasitism. Determination and evaluation of biochemical parameters in the serum of experimental animals was performed, to assess the hepatic, biliary, and renal toxic effects of the substances. There were no statistically significant differences between the treated groups and the control group, indicating that the increase in parasitemia is not associated with the effects of the substances in the tissue. We suggest the hypothesis of an immunosuppressive effect, since there was a decreased plasma levels of IFN- after treatment with the triterpenes. Moreover, the treated groups also exhibited increased levels of IL-10. Therefore, we suggest that treatment with the substances could be directing the immune response toward the Th2 type and, as a consequence of this immunosuppressive effect, a greater amount of trypomastigotes could be observed in the circulation of the host.

En dépit des efforts déployés, l'incidence de l'obésité est en augmentation dans le monde entier où la consommation d‘aliments moins coûteux et hyperénergétiquesriches en matières grasses, représente la principale cause. Des études récentes ont révélé l'existence d'un goût de gras et que l'envie de le satisfaire pourrait être responsable de l‘excès des prises alimentaires et de l'attirance pour les aliments riches en gras. Différentes stratégies ont été utilisées pour réduire la teneur engraisse des aliments ou pour la remplacer par des mimétiques et des substituts de graisse. Dans cette thèse, nous avons non seulement exploré des mécanismes de signalisation tels que le rôle des canaux TRPC3 Ca2+ dans les cellules du bourgeon gustatif, mais nous avons également tenté de rechercher de nouvelles classes de molécules artificielles et naturelles, d‘origines végétales, qui pourraient agir comme exhausteurs de goût ou comme activateurs du goût. Sur la base des résultats obtenus par des études biochimiques et comportementales au cours de la thèse actuelle, nous proposons que Zizyphine purifié à partir de Zizyphus lotus, d‘acide oléanolique et de TUG891 (un agoniste chimique de GPR120) puissent être un candidat novateur et prometteur pour le traitement et la prévention de l'obésité et du syndrome métabolique qui lui est associé
Despite efforts, the incidence of obesity is on a rise throughout the world andconsumption of less expensive fat rich and high energy diet remains the major cause.Recent studies have successfully unveiled the existence of a fat taste, and that theurge to satisfy it might be responsible for the overeating behaviour and attractiontowards fat rich foods. Different strategies have been used to reduce fat content offood or replace it with fat mimetics and fat replacers. In this dissertation, we have not only explored signalling mechanisms like role of TRPC3 Ca2+ channels in taste bud cells but also tried to search for newer classes of molecules from botanical and chemical origin that might either act as fat taste enhancers, or fat taste activators. Based on the result of various biochemical and behavioural studies during the current dissertation, we propose that Zizyphin purified from Zizyphus lotus,Oleanolic acid and TUG891 (a chemical agonist of GPR120), might be novel andpromising candidates for the treatment and prevention of obesity and associatedmetabolic syndrome

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model.
AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.

Doutoramento em Engenharia Química
A exploração de compostos, subprodutos e resíduos naturais é um passo chave para a obtenção de um futuro sustentável. A valorização e comercialização destes materiais dependem da aplicação de técnicas adequadas de conversão e separação/purificação que permitam obter os níveis desejados de pureza e produtividade. Os ácidos triterpénicos, particularmente os ácidos betulínico, oleanólico e ursólico, são compostos de elevado valor que têm atraído interesse devido às suas já reportadas propriedades nutraceuticas e farmacológicas. Estes triterpenoides estão presentes em diversas fontes vegetais mas podem ser encontrados com abundâncias consideráveis na casca do eucalipto (decídua e externa), um resíduo comum da indústria da pasta e papel. O isolamento dos ácidos triterpénicos é uma tarefa difícil devido às suas semelhantes estruturas moleculares, especialmente no caso dos ácidos oleanólico e ursólico, que são isómeros de posição. O leito móvel simulado (simulated moving bed, SMB) é uma técnica cromatográfica de separação contínua e em contracorrente que maximiza a força diretriz de transferência de massa permitindo a separação de moléculas mesmo quando as seletividades se aproximam de um. Por exemplo, é frequentemente utilizada no isolamento de enantiómeros. No SMB clássico a separação de dois componentes é efetuada em quatro zonas, os caudais e concentrações de alimentação são fixos e as portas de entrada/saída são comutadas simultaneamente. Recentemente, novas implementações tais como a modificação da concentração ou caudais da alimentação, a utilização de tempos de comutação variáveis e a introdução de novas zonas para separação multicomponente, têm permitido a obtenção de maior flexibilidade e melhor performance. Uma revisão profunda destes modos de operação foi feita durante esta tese. A modelação e otimização são passos essenciais no dimensionamento e desenvolvimento de qualquer processo e particularmente importantes no SMB. Como ponto de partida para o estudo do fracionamento de ácidos triterpénicos por leito móvel simulado, modelos fenomenológicos conhecidos foram aplicados para desenvolver de um simulador SMB. Durante este trabalho uma estratégia de otimização de unidades de SMB foi desenvolvida combinando as metodologias de desenho de experiências e respostas de superfície com simulações computacionais, com o objetivo de obter condições de operação ótimas com baixa complexidade e um esforço computacional reduzido. Esta técnica de otimização foi aplicada ao estudo da separação dos enantiómeros de óxido de trans-estilbeno, usando informação da literatura, e posteriormente comparada com outros procedimentos de determinação de condições de operação ótimas. Esta técnica permitiu purezas acima de 99.5 % para ambos enantiómeros, necessitando de um baixo número de simulações. A separação de ácidos triterpénicos foi inicialmente estudada à escala analítica, através de uma série de ensaios cromatográficos em diferentes condições para seleção de fase móvel e estacionária apropriada. Os melhores resultados foram obtidos com uma coluna Apollo C18 usando metanol/água 95/5 (%,v/v) e os parâmetros de equilíbrio e transporte foram determinados através de experiências de rutura com os compostos puros. Esta informação foi utilizada com sucesso na simulação da separação de uma mistura ternária, cujos resultados foram validados com experiências de rutura ternárias. A separação em SMB de uma mistura representativa de um extrato natural contendo os ácidos betulínico, oleanólico e ursólico foi desenhada utilizando um processo em dois passos: inicialmente o ácido betulínico foi isolado dos ácidos oleanólico e ursólico e, de seguida, os ácidos oleanólico e ursólico foram fracionados. Esta separação foi otimizada usando a metodologia de desenho de experiências e respostas de superfície combinada com simulações rigorosas e permitiu demonstrar que é possível produzir os ácidos betulínico, oleanólico e ursólico com purezas de 99.4 %, 99.1 %, e 99.4 %. Os polímeros molecularmente impressos (molecularly imprinted polymers, MIPs) são sintetizados para possuírem centros ativos altamente seletivos para moléculas alvo, tornando-os adsorventes muito promissores. Vários MIPs foram sintetizados por polimerização por precipitação usando diferentes formulações. Após a preparação, estes polímeros foram caracterizados por microscopia eletrónica de varrimento e os mais promissores, em termos de características morfológicas, foram diretamente testados através de experiências de adsorção. Os resultados revelam que o material “MIP1b” exibe selectividade infinita para o ácido oleanólico, a molécula alvo usada na sua síntese. Em conjunto com a sua capacidade de adsorção – superior à da previamente estudada fase C18 – estes resultados demonstram o elevado potencial do polímero “MIP1b” para aplicação na separação dos ácidos triterpénicos. Investigação adicional é necessária neste tópico onde diferentes técnicas de separação podem ser antecipadas. Este trabalho culminou com o dimensionamento e montagem de uma unidade laboratorial de leito móvel verdadeiro baseada num sistema de uma-válvula-ST por coluna recentemente patenteado, que permite à unidade operar sobre diferentes estratégias de operação e configurações.
The exploitation of natural compounds, by-products and residues is a key strategy for the pursuit of a sustainable future. The effective valorization and commercialization of these materials depends on the application of adequate conversion and separation/purification techniques that can provide desired levels of purity and productivity. Triterpenic acids, particularly betulinic, oleanolic, and ursolic acids, are high value molecules that have attracted considerable interest due to their reported nutraceutical and pharmacological properties. These triterpenoids are present in diverse vegetable sources but significant abundances are found in Eucalyptus bark (deciduous and external), a common residue from the paper and pulp industry. The isolation of triterpenic acids is a difficult task due to their similar molecular structures, particularly in the case of oleanolic and ursolic acids, which are positional isomers. The simulated moving bed (SMB) technology is a countercurrent continuous chromatographic technique that maximizes the mass transfer driving force, thus allowing the separation of compounds even when selectivities approach one. For instance, it is often used for enantiomers isolation. In a classic SMB, the separation of two components is accomplished across four zones, the flow rates and feed concentrations are fixed, and the inlet/outlet ports are switched synchronously. Recently, new implementations and modifications, such as the modulation of feed concentration or flow rates, variable switch times and additional zones for multicomponent separations, enabled better flexibility and performances to be achieved. A thorough review of these modes of operation was performed in this thesis. Modeling and optimization are necessary steps for the design and development of any process, being particularly important in SMB. As the starting point for the study of the fractionation of triterpenic acids by simulated moving bed, well know phenomenological models were applied to develop a SMB simulator. During this work, an optimization strategy of SMB units was developed combining the design of experiments and response surface methodologies (DoE-RSM) with computer simulations, aimed at providing good operating conditions with low complexity and reduced computational effort. This optimization technique was applied to the separation of trans-stilbene oxide (TSO) enantiomers using data from the literature and compared with other existing procedures for the determination of the best operation conditions. It allowed purities above 99.5 % for both TSO enantiomers while requiring a small number of simulations. The separation of triterpenic acids was initially studied at analytical scale, through a series of chromatographic assays under different conditions, to select appropriate mobile and stationary phases. Best results were obtained using an Apollo C18 column and methanol/water 95/5 (%, v/v), and equilibrium and mass transport parameters were determined through breakthrough experiments with pure compounds. This information was then successfully applied in the simulation of a ternary mixture separation, whose results were validated with ternary breakthrough measurements. The SMB separation of a representative natural extract containing betulinic, oleanolic and ursolic acids was designed using a two-step process: firstly, betulinic acid was isolated from oleanolic and ursolic acids, and secondly, oleanolic and ursolic acids were fractionated. This separation approach was optimized using DoE-RSM combined with rigorous simulations, and it was demonstrated that it is possible to produce betulinic, oleanolic and ursolic acids with purities of at least 99.4 %, 99.1 %, and 99.4 %. Molecularly imprinted polymers (MIPs) are synthesized to possess binding sites highly selective to specific molecules, making them very promising adsorbents. Several MIPs were synthetized by precipitation polymerization using different formulations. After preparation, these polymers were characterized by scanning electron microscopy and the most successful ones, in terms of morphological features, were directly tested carrying out batch adsorption experiments. The results disclosed that “MIP1b” material exhibits infinite selectivity for oleanolic acid, the template molecule used in its synthesis. Together with its adsorption capacity – even higher than that of the previously studied C18 phase – these results demonstrate the high potential of “MIP1b” polymer for application on the separation of triterpenic acids. Additional research is required in this topic, where distinct separation approaches may be anticipated. This PhD work culminated in the design and assembling of a laboratory simulated moving bed unit based on a recently patented system using a one- ST valve per column valve scheme, thus allowing the SMB to run under several operation strategies and configurations.

El ácido oleanólico (OA) y su isómero el ácido ursólico (UA), son triterpenos pentacíclicos que existen ampliamente en las plantas naturales, como acido libre o agliconas de saponinas triterpenoides, y son parte integral de la dieta humana. Estos triterpenos son de gran interés como agentes terapéuticos por sus diversas actividades farmacológicas, como antiinflamatoria, anticancerígena, antioxidante, hepatoprotectora, antimicrobiana, entre otras. El objetivo principal de este trabajo fue el diseñar, optimizar y caracterizar sistemas nanoestructurados como nanopartículas poliméricas de PLGA (NPs) y nanoemulsiones (NEm) conteniendo estos compuestos triterpenoides de origen natural (Plumeria obtusa) y sintético, evaluando su acción antiinflamatoria y citotóxica para aplicación tópica (ocular y dérmica). Las NPs fueron desarrolladas por el método de desplazamiento de solvente y optimizadas por un diseño factorial central compuesto, las NPs seleccionadas fue la compuesta por 1.0 mg/mL de OA/UA, 10.0 mg/mL de P188 y pH de 5.5 para la fase acuosa, que obtuvo valores de tamaño de partícula alrededor de los 200 nm, una carga superficial de -27 mV y una encapsulación entre 76-78%. Las NEm fueron desarrolladas por la construcción de diagramas pseudo-ternarios de fases, con una composición final de 20% de aceite de castor, 20% de propilenglicol, 59.80% de Smix con ratio 4:1 (47.84% de labrasol y 11.96% de transcutol-p) junto a las mezclas triterpenoides al 0.2%, presentando apariencia monofásica, tamaño de gota pequeño (~200 nm) e índices de polidispersidad alrededor de 0.2. Ambas formulaciones presentan una viscosidad baja (menor a 60 mPa·s) y siguen un comportamiento Newtoniano. Ambas mezclas de triterpenos tanto en NPs como NEm, no presentaron fenómenos de desestabilización (coalescencia, floculación, sedimentación, cremado, etc.) durante 30 días después de su elaboración, por lo que las formulaciones optimizadas se consideran sistemas estables. El perfil cinético que presentan ambas mezclas encapsuladas en NPs se ajusta a la función de Weibull, que sigue un mecanismo de liberación complejo. Mientras que la liberación de estas mezclas de las NEm, siguen un modelo cinético de la ecuación de Korsmeyer-Peppas, con un mecanismo de liberación de difusión no Fickiana o anómala. La permeación corneal de ambas mezclas en NPs, pone de relieve que hay mayor cantidad retenida en la córnea que cantidad permeada, siendo ligeramente superior en la mezcla natural. La capacidad de permeación en piel de las nanoemulsiones de ambas mezclas, demuestra también que hay mayor cantidad retenida de OA/UA en la piel que la cantidad permeada. El perfil de permeación mostró que a las primeras 4 horas de estudio hay mayor cantidad permeada de la mezcla sintética que de la mezcla natural. Los resultados de los ensayos de tolerancia ocular y dérmica in vitro e in vivo, indican que las formulaciones optimizadas muestran una tolerancia óptima, siendo seguras para aplicación tópica. Ambas mezclas tanto en NPs como en NEm muestran una inhibición eficaz de la inflamación local, siendo este efecto más marcado en las formulaciones de mezcla natural, ya que esta tiende a quedar más retenida en el área de contacto. La actividad citotóxica se llevó a cabo por el ensayo de Alamar blue, donde la nanoemulsión de la mezcla natural muestra actividad anticancerígena en la línea celular de melanoma murino B16, a partir de concentraciones más bajas (5 µM) que el compuesto puro (30 µM). La NEm blanca presenta también actividad anticancerígena desde los 5 µM, lo que podría deberse al aceite de castor, que potencia la acción del compuesto natural puro. Los resultados presentados en este trabajo demuestran claramente la idoneidad de estos sistemas coloidales desarrollados para un efecto anti-inflamatoria local tras la administración tópica.
Oleanolic acid (OA) and its isomer ursolic acid (UA), pentacyclic triterpenes are widely existing in the natural plants and are integral part of the human diet. These triterpenes are of great interest as therapeutic agents for their various pharmacological activities such as anti-inflammatory, anticarcinogenic, antioxidant, hepatoprotective, antimicrobial, among others. The main objective of this work was to design, optimize and characterize nanostructured systems such as PLGA-nanoparticles (NPs) and nanoemulsions (NEm) containing these natural (Plumeria obtusa) an synthetic triterpenoids compounds, evaluating their anti-inflammatory and cytotoxic activities for application topical (ocular and dermal). The NPs were developed by the method of solvent displacement and optimized by central composite factorial design, the selected NPs was composed of 1.0 mg/mL OA/UA, 10.0 mg/mL of P188 and aqueous phase with pH of 5.5, obtained values of particle size around 200 nm, a surface charge of -27 mV and an encapsulation between 76-78%. NEm were developed for the construction of diagrams pseudo-ternary phase, with a final composition of 20% castor oil, 20% propylene glycol, 59.80% of Smix with ratio 4:1 (47.84% of labrasol and 11.96% of transcutol-P) together with 0.2% triterpenoid mixtures presenting monophasic appearance, small droplet size (~200 nm) and polydispersity index around 0.2. The kinetic profile of mixtures encapsulated in NPs fits with Weibull function, which follows a complex release mechanism. While the release of these mixtures from NEm follow a kinetic model of the Korsmeyer-Peppas equation, with release mechanism of non-Fickian or anomalous diffusion. The corneal and dermal permeation indicates that these triterpenoids compounds tend to be retained in greater amount in the contact area than the amount permeated, being retained slightly higher in the natural mixture. Both mixes in NPs and NEm show effective inhibition of local inflammation, this effect being more pronounced in the formulations of natural mixture. The natural mixture of nanoemulsion shows anticancer activity in the murine melanoma cell line B16, since lower concentrations (5 µM) to the pure compound (30 µM). The results presented here clearly demonstrate the suitability of these colloidal systems developed for a local anti-inflammatory effect after topical administration.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
O Ãcido oleanÃlico à um triterpeno pentacÃclico largamente encontrado em vÃrias plantas medicinais. Essa substÃncia demonstrou ter uma variedade de atividades farmacolÃgicas, dentre as quais se destacam: antiinflamatÃria, hepatoprotetora, gastroprotetora e antinociceptiva. Este trabalho objetivou investigar a atividade antinociceptiva do Ãcido oleanÃlico em modelos de nocicepÃÃo aguda induzida por capsaicina (20Âl/ 1,6 μg) e Ãleo de mostarda (0,75%, 50 ÂL/animal) em camundongos, alÃm dos possÃveis mecanismos de aÃÃo envolvidos. Camundongos foram prÃ-tratados com Ãcido oleanÃlico (3, 10, 30, 100 mg/kg, v.o.) ou veÃculo, e os comportamentos de dor foram analisados. As doses de 10, 30 e 100 mg/kg, v.o., foram capazes de reduzir os comportamentos dolorosos expressos pelos animais nos modelos de nocicepÃÃo induzida por capsaicina e Ãleo de mostarda, sendo o maior nÃvel de inibiÃÃo (p<0,001) encontrado na dose de 30 mg/kg. Na tentativa de desvendar os possÃveis mecanismos de aÃÃo do Ãcido oleanÃlico no modelo de nocicepÃÃo induzido por capsaicina, avaliamos a participaÃÃo dos receptores opiÃides, α2, Ãxido nÃtrico e canais de potÃssio. O efeito antinociceptivo do Ãcido oleanÃlico (30 mg/kg, v.o.) foi significantemente revertido pelo prÃ-tratamento com antagonista opiÃide, naloxona (2 mg/kg, i.p.); pelo doador de Ãxido nÃtrico, L-arginina (600 mg/kg, i.p.) e pela glibenclamida (2 mg/kg, i.p.), um antagonista dos canais de potÃssio. Por outro lado, o prÃ-tratamento com um antagonista α2, ioimbina (2 mg/kg, i.p.), nÃo ocasionou a reversÃo da antinocicepÃÃo. Na tentativa de desvendar os possÃveis mecanismos de aÃÃo do Ãcido oleanÃlico no modelo de nocicepÃÃo visceral induzida por Ãleo de mostarda, avaliamos a participaÃÃo dos receptores opiÃides, α2 e TRPV1. O efeito antinociceptivo do Ãcido oleanÃlico (30 mg/kg, v.o.) foi significantemente revertido (p<0,05) pelo prÃ-tratamento com antagonista opiÃide, naloxona (2 mg/kg, i.p.), enquanto que o antagonista α2 , ioimbina (2 mg/kg, i.p.), nÃo teve o mesmo efeito. O prÃ-tratamento com vermelho de rutÃnio (3 mg/kg, s.c.), um antagonista nÃo competitivo do receptor TRPV1 causou inibiÃÃo significativa da nocicepÃÃo (p<0,01) induzida pelo Ãleo de mostarda, entretanto a administraÃÃo conjunta com o Ãcido oleanÃlico nÃo produziu antagonismo nem potenciaÃÃo da antinocicepÃÃo causada pelo Ãcido oleanÃlico. Para avaliar a existÃncia de um impedimento locomotor ou de uma incoordenaÃÃo motora, foram utilizados os testes do campo aberto e o teste do rota rod, respectivamente. Os dados indicaram que o tratamento com o Ãcido oleanÃlico (30 mg/kg, v.o.) nÃo induziu (p>0,05) impedimento locomotor ou incoordenaÃÃo motora nos animais, sendo ainda capaz de reverter (p<0,05) o impedimento locomotor induzido pelo Ãleo de mostarda no teste do campo aberto. Em conjunto os dados revelaram a efetividade do Ãcido oleanÃlico em modelos de nocicepÃÃo possivelmente envolvendo receptores opiÃides, TRPV1, Ãxido nÃtrico e canais de potÃssio.
Oleanolic acid is a triterpene pentacyclic widely distributed in the plant kingdom. Different biologic activities have been reported including: antiinflammatory, hepatoprotective, gastroprotective and antinociceptive. This work was aimed to evaluate the antinociceptive effect of oleanolic acid in acute nociception models induced by capsaicin (20Âl/ 1.6 μg) and mustard oil (0.75%, 50 ÂL/animal) in mice and to establish the likely mechanism(s) of action. Mice were pretreated orally with oleonolic acid (3, 10, 30 and 100 mg/kg) or vehicle, and the pain-related behavioral responses were analysed. The pain behavioral responses were significantly suppressed at doses 10, 30 and 100 mg/kg in acute nociception models induced by capsaicin and mustard oil. The maximal suppression (p<0.001) was observed at the dose of 30 mg/kg. In order to verify the possible mechanisms involved in the antinociceptive action of oleanolic acid in the capsaicin-induced nociception, the involvement of endogenous opioids, α2, nitric oxide and KATP channels were analyzed. The antinociception produced by OA (30 mg/kg, v.o.) was found to be significantly blocked in animals pre-treated with the opioid antagonist, naloxone (2 mg/kg, i.p.); the substrate for oxide nitric synthase, L-arginine (600 mg/kg, i.p.); or a KATP-channel blocker, glibenclamide (2 mg/kg, i.p.) but was unaffected by yohimbine (2 mg/kg, i.p.), an α2 -adrenoceptor antagonist. In order to verify the possible mechanisms involved in the antinociceptive action of oleanolic acid in the mustard oil-induced visceral pain model, opioid, α2 adreno and TRPV1 receptors were analyzed. The antinociceptive effect of oleanolic acid (30 mg/kg, v.o.) was significantly blocked (p<0.05) by pretreatment with the opioid antagonist, naloxone (2 mg/kg, i.p.), but the α2-adrenoceptor antagonist, yohimbine (2 mg/kg, s.c.), had no effect. Pretreatment with ruthenium red (3 mg/kg, s.c.), a non-competitive TRPV1 antagonist alone caused significant inhibition (p<0.01) of mustard oil-induced nociception but its co-administration with oleanolic acid produced neither antagonism nor potentiation of oleonolic acid antinociception. Further, to evaluate a possible motor impairment and motor incoordination effects related to oleanolic acid, open-field and rota-rod tests were performed. The data indicated that the treatment of animals with the oleanolic acid (30 mg/kg, v.o.) was unable to cause motor impairment or motor incoordination effects (p>0.05), being even able to reverse (p<0.05) a mustard oil-induced motor impairment in the open field test. The results taken together strongly suggest the therapeutic potential of oleanolic acid in oblitering nociception through the mechanisms that possibly involve the opioids, TRPV1 receptors, nitric oxide and KATP channels.

Since ancient times, natural products are used as source of medicines to prevent and treat disease. Currently, high cost and time taken for analysis of natural products have been significantly reduced by the use of modern approaches such as chemometric analysis, which gives flexibility to the process of development of new products. This study aimed to chemometric analysis of the ethanol extract, fraction in chloroform and subfractions of the crude extract of leaves Zeyheria tuberculosa (Veil) Bureau (Bignoniaceae), for Nuclear Magnetic Resonance, with the characterization and quantification of the major compounds present in these, as well as construction of a model to select potentially promising extracts, serving as a guide for further isolation of the substances of interest, enabling the discovery of bioactive compounds. The fraction in chloroform which concentrated almost all the ethanol extract metabolites, showed 100% inhibitory activity lymphocyte proliferation at a concentration of 100 gg.m1:1, and is therefore chosen for characterization and quantification of the major compounds present. The NMR spectral analysis of the fraction in chloroform, including 1D experiments (1H, 13C, DEPT 90 °, DEPT135 °) and 2D (J-Resolved, HSQC and HMBC) allowed the detailed structural elucidation of two major compounds, ursolic and oleanolic acids, both identified for the first time in this species. Due to the importance of these acids in the treatment of skin diseases and various cancers, measurements were determined for these triterpenes in ethanol extract (ZTB), fraction in chloroform (ZTP2) and the subfractions, using techniques for this purpose NMR and chemometric tools (Matlab programs, AMIX and SIMCA). All spectra were processed and analyzed using the program topspin (BRUKER). The qualitative and quantitative results of the chemometrics analysis for NMR of the ethanol extract, fraction in chloroform and subfractions of Zeyheria tuberculosa showed that fractions ZTF3 (evaluated in MTT as being non-cytotoxic at concentrations of 10, 50 and 100 pg.mL-1) and ZTF6 (measured in MTT as being non-cytotoxic at the concentration of 10 pg.mL-1 and cytotoxic at concentrations of 50 and 100 pg.mL-1) are the richest of oleanolic and ursolic acids, respectively, and so are more promising for the isolation of these triterpenes. The fraction ZTF4 was second richer fraction both as oleanolic acid and ursolic acid, the most promising for the isolation of these two acids together, also considering that this fraction was evaluated in MTT as non-cytotoxic at concentrations of 10 and 50 g .mL-1, and only the cytotoxic concentration of 100 pg.mL-1. The ursolic and oleanolic acids have a range of therapeutic properties and a high commercial value, being highly relevant the qualitative and quantitative results obtained in this study, referring to these triterpenes found in the leaves of the species Zeyheria tuberculosa. The model proposed in this work was efficient, as indicated by the values of the Q parameter, and suitable for the classification of samples into groups and subgroups according to the presence of ursolic and oleanolic acids (chemical composition) and the concentration of these in the samples, and it may be useful in the identification of potentially promising extracts, serving as a guide for subsequent isolation of the substance of interest.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Desde os tempos antigos, os produtos naturais são utilizados como fonte de medicamentos, para prevenir e tratar doenças. Atualmente, o alto custo e o tempo despendido para as análises de produtos naturais têm sido significativamente reduzidos pelo uso de abordagens modernas, como a análise quimiométrica, que dão agilidade ao processo de desenvolvimento de novos produtos. Este trabalho teve como objetivo a análise quimiométrica do extrato etanólico, da fração em clorofórmio e subfrações do extrato bruto das folhas de Zeyheria tuberculosa (Vell) Bureau (Bignoniaceae), por Ressonância Magnética Nuclear, com a caracterização e quantificação dos compostos majoritários presentes nestes, além da construção de um modelo capaz de selecionar extratos potencialmente promissores, servindo como guia para um posterior isolamento das substâncias de interesse, viabilizando a descoberta de compostos bioativos. A fração em clorofórmio que concentrou a quase totalidade dos metabólitos do extrato bruto, apresentou 100 % de atividade inibitória da linfoproliferação, sendo por isso escolhida para caracterização e quantificação dos compostos majoritários presentes. A análise espectral de RMN da fração em clorofórmio, incluindo experimentos 1D (1H, 13C, DEPT 90°, DEPT 135°) e 2D (J-Resolvido, HSQC e HMBC) permitiu a elucidação estrutural detalhada de dois compostos majoritários, os Ácidos Ursólico e Oleanólico, ambos identificados pela primeira vez nesta espécie. Devido à importância desses ácidos no tratamento de doenças da pele e vários tipos de câncer, foram determinadas as quantificações relativas destes triterpenos no extrato bruto (ZTB), na fração em clorofórmio (ZTP2) e nas subfrações, utilizando-se para este fim técnicas de RMN e ferramentas quimiométricas (programas Matlab, AMIX e SIMCA). Todos os espectros foram processados e analisados utilizando o programa TOPSPIN (BRUKER). Os resultados quantitativos e qualitativos da análise quimiométrica por RMN do extrato bruto, fração clorofórmica e subfrações de Z. tuberculosa mostraram que as frações ZTF3 (avaliada no teste MTT como sendo átoxica nas concentrações de 10, 50 e 100 pg.mL-1) e ZTF6 (avaliada no teste MTT como sendo atóxica na concentração de 10 pg.mL-1 e citotóxica nas concentrações de 50 e 100 pg.mL-1) são as mais ricas dos ácidos oleanólico e ursólico, respectivamente, e por isso são as mais promissoras para o isolamento destes triterpenos. Já a fração ZTF4 foi a segunda fração mais rica tanto em ácido ursólico como em ácido oleanólico, sendo a mais promissora para o isolamento destes dois ácidos juntos, considerando ainda que esta fração foi avaliada no teste MTT como atóxica nas concentrações de 10 e 50 pg.mL-1, e citotóxica apenas na concentração de 100 pg.mL-1. Os ácidos ursólico e oleanólico possuem uma gama de propriedades terapêuticas e um alto valor comercial, sendo de grande relevância os resultados qualitativos e quantitativos, obtidos neste trabalho, referentes a esses triterpenos encontrados nas folhas da espécie Zeyheria tuberculosa. O modelo proposto neste trabalho foi eficiente, conforme indicado pelos valores do parâmetro Q, e satisfatório para a classificação das amostras em grupos e subgrupos, segundo a presença dos ácidos ursólico e oleanólico (composição química) e a concentração destes nas amostras, podendo ser de grande utilidade na identificação de extratos potencialmente promissores, servindo como guia para um posterior isolamento da(s) substância(s) de interesse.

A doença de Chagas é um problema de saúde pública, com dados preocupantes referentes ao número de pessoas contaminadas e daquelas que ainda permanecem expostas ao risco de infecção. A dificuldade do combate a Trypanosoma cruzi, agente etiológico da doença, está intimamente relacionada às interações existentes entre o parasito e o hospedeiro, sendo que até o momento, nenhum medicamento ou substância tem demonstrado real eficácia ao combate ao parasito. Em estudos recentes realizados por nosso grupo de pesquisa, os ácidos ursólico e oleanólico demonstraram um bom potencial tripanocida, além de um efeito imunomodulatório, promovendo a inibição da produção de IFN-? quando de suas utilizações por via intraperitoneal em elevadas concentrações. Considerando essas evidências encontradas em relação a essas substâncias e os efeitos promovidos no processo de avaliação biológica, propusemos como objetivo avaliar o potencial terapêutico dos triterpenos ácido ursólico e ácido oleanólico na fase crônica da infecção chagásica e suas associações ao benzonidazol, fármaco referência indicado ao tratamento da parasitose, verificando a possibilidade de geração de benefícios sobre a patogênese da infecção crônica experimental. A quantificação de linfócitos T CD4+ e T CD8+, e das citocinas IL-2, IL-10, IL-12, IFN-? e TNF-??pela técnica de citometria de fluxo, utilizando o kit BD Cytometric Bead Array®, indicaram que essas substâncias não apresentam efeitos imunomodulatórios significativos sobre a resposta Th1 e Th2 nessa fase da doença, na comparação entre grupos infectados e tratados e aquele que recebeu apenas solvente. O parasitismo tecidual determinado por Real Time PCR e as observações realizadas em cortes histológicos, mostraram, respectivamente, baixo número de cópias de DNA de T. cruzi, e ausência de ninhos amastigotas, porém, com marcante presença de infiltrado inflamatório em todos os grupos. Assim, os dados obtidos levam à conclusão de que apesar da atividade apresentada pelos triterpenos ácidos em estudo na fase aguda da doença de Chagas, o mesmo não ocorre na fase crônica, como seria desejável para favorecer uma melhora do quadro patológico, mesmo não havendo a cura da doença, considerando as conseqüências da infecção de longo prazo.
Chagas disease is a public health problem, with disturbing data about the number of infected people and those who remain at risk of infection. The difficulty of eliminating Trypanosoma cruzi, etiologic agent of the disease is closely related to the interactions between the parasite and the host, and until now, no medicine or substance has demonstrated real effectiveness against the parasite. In recent studies by our research group, the ursolic and oleanolic acids revealed considerable trypanocidal activity, and an immunomodulatory effect, promoting the inhibition of IFN-? with intraperitoneal administration in high concentrations. Considering these evidences and the effects observed during the biological evaluation, our objective was to evaluate the biological potential of the triterpenoids ursolic acid and oleanolic acid in the chronic phase of chagasic infection and their associations to benznidazole, the reference drug indicated for the treatment of this disease, verifying the possibility of benefits on the pathogenesis of experimental chronic infection. Quantification of T CD4+ and T CD8+ cells and of the cytokines IL-2, IL-10, IL-12, IFN-? and TNF-? by flow cytometry using the kit BD cytometric Bead Array®, indicated that these substances do not exhibit significant immunomodulatory effects on Th1 and Th2 responses in this stage of the disease, comparing infected and treated groups and that who received only solvent. The tissue parasitism determined by Real Time PCR and in histological observations showed, respectively, low copy number of T. cruzi DNA, and absence of amastigote nests, however, with marked inflammatory infiltration in all groups. Thus, our data lead to the conclusion that despite the activity presented by the triterpene acids studied in the acute phase of Chagas disease, the same does not occur in the chronic phase, as would be desirable to the reduction of the pathological conditions, even if there is no cure of the disease, considering the consequences of long term infection.

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Depression and anxiety are widely acclaimed as psychiatric disorders of global concern. These disorders are among the leading causes of disability worldwide. Unsatisfactory responses of patients to the available pharmacotherapy make the search for new drugs a necessity. Medicinal plants remain important source of new drugs and new chemical entities. The ethnopharmacological knowledge and previous data have revealed calming and anxiolytic like effects of the organic leaf extract of Pimenta pseudocaryophyllus (Gomes) L.R. Landrum. The present study sought to investigate antidepressive like effect of dichloromethane fraction (DF) of the ethanolic leaf extract of Pimenta pseudocaryophyllus as well as anxiolytic and antidepressive like effects of oleanolic acid (OA), (E) methyl isoeugenol (MIE) and possible mechanisms of action that are involved. Animal models like barbiturate-induced sleep, light dark box test (LDB), elevated plusmaze (EPM), open field (OF), wire hanging test, pentylenetetrazol-induced convulsion test, forced swimming test (FST), tail suspension test (TST) were conducted to evaluate behavioural alterations that were elicited by the administrations of vehicle, DF, OA, MIE or reference drugs. Bioassays (ex vivo and in vitro) of monoamine oxidase (MAO) and quantification of hippocampal level of brain derived neurotrophic factor (BDNF) were conducted in an attempt to elucidate possible mechanisms of action. Oral administration of DF 125, 250 or 500 mg/kg (potentiated the hypnotic effect of sodium pentobarbital). In the TST and FST, DF 125 or 250 mg/kg induced antidepressant-like response. The data obtained in the OF suggest sedative effect of DF at 500 mg/kg. Pretreatment (i.p) with pchlorophenylalanine methyl ester (PCPA) 100 mg/kg (serotonin depletor) or 𝛼-methyl-ptyrosine (AMPT) 100 mg/kg (catecholamine depletor) blocked anti-immobility effect of DF viii in the FST. The enzymatic activity of MAO remained unaltered by DF. Oral administration of OA (5-20 mg/kg) increased the duration of barbiturate - induced sleep and demonstrated anxiolytic like effect in both LDB and EPM. In the FST and TST, OA 5-20 mg/kg elicited antidepressant like effect without altering locomotion activity of the animals. The antidepressant like effect of OA was attenuated by NAN-190 (non-selective antagonist of 5-HT1A), AMPT, PCPA, WAY and PRAZ. Chronic administration of OA increased hippocampal level of BDNF. Oral administration of MIE 250 or 500 mg/kg potentiated hypnotic effect of sodium pentobarbital without protecting mice against PTZ - induced convulsion. The parameters evaluated in the LDB, EPM and OF demonstrated anxiolytic like property of MIE. This effect was blocked by WAY (selective antagonist of 5-HT1A) pretreatment. MIE 125 or 250 mg/kg showed antidepressant like effect in the FST. Locomotion activity of the animal in the OF remained unaltered by MIE administration at 125 or 250 mg/kg. Pretreatment of mice with PCPA attenuated antidepressant like property of MIE. In conclusion, our findings demonstrated anxiolytic and/or antidepressant like effects of dichloromethane fraction, oleanolic acid and (E) methyl isoeugenol, thereby suggesting the involvement of monoaminergic pathway.
Ansiedade e depressão são transtornos psiquiátricos de interesse global. Estes transtornos estão entre as principais causas da incapacidade laboral das pessoas. Apesar de uma gama de farmacoterapias disponíveis, os resultados clínicos mostram que os fármacos não produziram efeitos terapêuticos desejados e se faz necessário a busca de novos fármacos. As plantas medicinais continuam sendo uma das fontes mais importantes para a descoberta de novos fármacos e entidades químicas. Estudos anteriores mostraram efeito calmante e ansiolítico da fração orgânica do extrato das folhas de Pimenta pseudocaryophyllus (Gomes) L. R. Landrum (Myrtaceae). O presente estudo buscou investigar a atividade tipo antidepressiva da fração diclorometano (FD) do extrato etanólico das folhas desta espécie, bem como antidepressiva e ansiolítica do ácido oleanólico (AO), (E)-metilisoeugenol (MIE) e os possíveis mecanismos de ações envolvidos. Modelos experimentais como o sono induzido por barbitúricos, caixa claro escuro (CCE), labirinto em cruz elevado (LCE), campo aberto (CA), teste de arame, teste de convulsão induzida por pentilenotetrazol, teste de natação forçada (TNF) e teste de suspensão pela cauda (TSC) foram realizados para avaliar alterações comportamentais induzidas pela administração do veículo, FD, AO, MIE ou fármacos de referência. Na tentativa de elucidar os possíveis mecanismos de ação, foram realizados bioensaios (ex vivo e in vitro) da monoamina oxidase (MAO) e do fator neurotrófico derivado do cérebro (BDNF do hipocampo). A administração oral da FD 125, 250 ou 500 mg/kg potencializou o efeito hipnótico de pentobarbital sódico. No TNF e TSC, a FD 125 ou 250 mg/kg induziu efeito tipo antidepressivo. Os dados obtidos no campo aberto sugerem efeito sedativo da fração vi diclorometano na dose de 500 mg/kg. O pré-tratamento (i.p) com p - clorofenilalanina metil éster (PCPA) 100 mg/kg (depletor de serotonina) ou α - metil - p - tirosina (AMPT) 100 mg/kg (depletor de catecolamina) bloqueou o efeito tipo antidepressivo da FD no TNF. O bioensaio da atividade enzimática mostrou que a FD não alterou a atividade da MAO. A administração oral do AO (5-20 mg/kg) aumentou a duração do sono induzido por pentobarbital sódico e demonstrou efeito tipo ansiolítico no CCE e LCE. O AO 5-20 mg/kg demonstrou efeito tipo antidepressivo no TNF e TSC sem alterar a atividade locomotora dos animais. O efeito tipo antidepressivo do AO foi atenuado por prétratamento com NAN-190 (antagonista não-seletivo do receptor 5-HT1A), AMPT, PCPA e PRAZ-prazosin (antagonista do receptor α1 adrenérgico). A administração crônica do AO aumentou o nível de BDNF no hipocampo. A administração oral do MIE 250 ou 500 mg/kg potencializou o efeito hipnótico de pentobarbital sódico sem proteger os animais contra a convulsão induzida por PTZ. Os parâmetros avaliados na CCE e LCE sugerem que MIE têm efeito tipo ansiolítico. Este efeito foi bloqueado pelo pré-tratamento com WAY100635 (antagonista seletivo do receptor 5-HT1A). MIE 125 ou 250 mg/kg apresentou efeito tipo antidepressivo no TNF. Não houve alteração na atividade locomotora dos animais no CA após a administração do MIE 125 ou 250 mg/kg. O prétratamento com PCPA atenuou o efeito tipo antidepressivo do MIE no TNF. Os resultados demonstraram efeito tipo ansiolítico e/ou antidepressivo da fração diclorometano, ácido oleanólico e (E)-metilisoeugenol, sugerindo o envolvimento de vias monoaminérgicas nestes efeitos.

碩士
慈濟大學
生命科學系碩士班
99
Hepatocellular carcinoma (HCC) is one of the most malignant tumors with high rate of recurrence and metastasis. Angiogenesis is a very important process that mediates tumor growth and metastasis, and can be used as a therapeutic target among all the strategies for many cancers. Ursolic acid (UA) and its derivative oleanolic acid (OA), are two glycyrrhetinic acid related pentacyclic triterpenoids that we had found anti-tumor effects in HepG2 cells in previous studies. The aim of this study is to further investigate whether UA and OA have the inhibitory effect on angiogenesis in HepG2 cells. Our results indicate that both UA and OA inhibit cell proliferation in a dose-dependent manner by EdU cell proliferation assay and exhibit anti-angiogenesis in a dose-dependent manner are examined by in vitro analysis including wound-healing, transwell and capillary-like tube formation assay. We observed that both UA and OA could potently inhibit angiogenesis by chicken chorioallantoic membrane (CAM) ex vivo assay and matrigel plug in vivo assay. Our investigations showed that UA and OA inhibit angiogenesis directly through phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)-mediated pathways that are important signal molecules among all the pathways associated with cells angiogenesis. This study revealed that both UA and OA, in addition to their anti-tumor effects, can suppress tumor growth through inhibition of tumor angiogenesis in vitro, ex vivo and in vivo via targeting PI3K/AKT and MAPK/ERK signaling pathways. We expect that UA and OA may effectively be used as a therapeutic strategy for HCC in the future.

碩士
國防醫學院
生物化學研究所
94
Oleanolic acid(OA), a naturally occurring triterpenoid, is distributed in the leaves and bark of various plants. Studies indicate that this compound exhibited significant anti- inflammatory and anti-tumor activity. Since OA has a common steroid-like structure, it is expected to display certain steroid-related pharmacological activities. In this study, we investigated its effect on nuclear receptors including estrogen receptor, glucocorticoid receptor, and Liver X receptor. Using cotransfection and reporter gene assays in human breast cancer MCF-7 cells, we found that when used alone, the compound displayed minimal, if any, effect on estrogen receptor. However, when used in combination with estradiol, OA dramatically enhanced the estrogenic activity of estradiol. The effect was specifically inhibited by ER antagonist ICI 182,780, suggest that the effect of OA is ER-dependent. The OA-mediated enhancement in estrogenic activity was also observed in the expression of some estrogen-regulated genes, including the inhibition of estrogen receptor (ER) and induction of progesterone receptor (PR) genes’ expression. On the other hand, OA also enhanced the activity of dexamethasone (Dex) to activate luciferase activity from the glucocorticoid responsive MMTV-luciferase reporter in human HepG2 cells. Western blot analysis indicated that treatment HepG2 cells with OA enhanced nucleus translocation of glucocorticoid receptor (GR) as does dexamethasone (Dex), though in a much lower scale. Moreover, OA inhibited the activity of T0901317 (T1317) to activate luciferase activity from the LXR responsive tk-LXRE×3-luciferase reporter in human HepG2 cells. Taken together, our study indicates that OA exhibited marked xenoestrogenic activity by modulating the estrogenic response of ER. In addition, OA also showed marked activity by modulating the other nuclear receptors. These data are important for understanding the molecular mechanism of actions OA in human cells.

碩士
慈濟醫學院
醫學研究所
88
Peh-Hue-Juwa-Chi-Cao is a popular folk medicine in Taiwan. It is usually used to treat liver disorders according to its antitumor, antitoxic and hepatoprotective effects. This study is focused on the cytotoxic effect of two kinds of anti-tumor components, oleanolic acid and ursolic acid in Peh-hue-juwa-chi-cao by hepatoma cell lines, HepG2 and Hep3B. The possible mechanisms of cell growth inhibition of these components on tumors are further investigated. An optimal concentration of 100mg/mL of both oleanolic acid and ursolic acid was used to treat the HepG2 and Hep3B cells, respectively. Different time after treatment, cells were harvested to observe the structural and morphological changes by light or electron microscope. The results showed that the morphological changes including cell membrane shrinkage, blebbing, the appearance of apoptotic bodies, nuclear and chromatin condensation are similar to what happened during apoptosis. The evaluation of cellular toxicity and tumor growth inhibition by these phytochemicals by MTT assay, showed that ursolic acid is more effective in inhibiting the growth of both cell lines, and Hep3B cells is more sensitive than HepG2 cells under the same condition of drug treatments. DNA fragmentation analyzed by agarose gel revealed that the effect of oleanolic acid and ursolic acid on inhibiting tumor cell growth seems likely by inducing the apoptosis pathway in the cells. In SDS-PAGE analysis, we found that bovine serum albumin will accumulate in cells after drug treatments with a time-dependent manner.

碩士
大同大學
生物工程學系(所)
97
The stem of Hedyotis diffusa willd was used as explant to establish the in vitro culture system. For callus induction, shoot tips from regenerated plantlet were cultured on modified MS medium and the white friable callus proliferated from shoot tips. The green embryogenic callus formed when white callus were subcultured on MS medium supplemented with 0.002 mgl-1 thidiazuron. Plantlets regenerated when embryogenic callus was subcultured on MS medium without plant growth regulator. For cell suspention culture system, the white callus was cultured in modified MS medium and subcultured every month. The content of oleanolic and ursolic acid of four kinds of callus (white callus、green callus、white suspention cell and green suspention cell) were examined by high performance liquid chromatography. The green cell has the highest content of oleanolic and ursolic acid (538±77 μg/g DW) and the growth index of green cell line ( inoculum : 2 g/10 ml ) was 2.2 times within 12 days. In order to enhance the accumulation of oleanolic and ursolic acid in cell, cell was cultured in MS medium with different culture factor and precursor elicitor. The highest content of oleanolic and ursolic acid ( 1591±206 μg/g DW) in cell was shown when cell cultured in MS medium added with 12 mgl-1 methyl jasmonate after 12 days. The production of oleanolic and ursolic acid in cell was also promoted when medium added 50 gl-1 sucrose ( 1017±167 μg/g DW) or 50 mgl-1 pectin supplemented(1356 ±83 μg/g DW).

碩士
國立嘉義大學
農學研究所
94
Studies on the Tissue Culture and Oleanolic acid & Ursolic acid Content of Hedyotis diffusa (Willd.) Abstract Hedyotis diffusa( Wilid) is a kind of herbal medicines usually we used in Taiwan for treating relieveing of poisonously symptoms. The purpose of the study is to establish a micropropagation system of Hedyotis diffusa( Wilid) and to research the possibility of Oleanolic acid & Ursolic acid production using from plantlet and callus regenerated. In the callus induction, leaf blade and stem segment were used as explants and cultured on MS basal medium containing different combinations of NAA 、2,4-D、picloram (0、0.5、1、2 and 4 mg/L) and BA (0、0.5、1 and 2 mg/L). The leaf blade cultured on basal medium with 2,4-D 2 mg/L had the highest percentage of callus formation 100 %. In the shoots induction, leaf blade and stem segment, stem section, stem were used as explants and cultured on MS basal medium containing different combinations of NAA(0、0.1、1 、2 mg/L) and BA (0、0.5、1 、2 and 4 mg/L). Regard there is no blade of vaccining as and cultivate the body, the license is only trained in adding solid culture of MS of 0.1 mg/L NAA and 2 mg/L BA, can get at most the bud body is counted, and train it can get the adventitious bud of hyperplasia with liquid culture of MS of the thicknessing too, utilizing the solid and liquid can promote plants to grow to train alternatively normally. Lead the forming of the adventitious bud root, we can lead adventitious bud root transform with 4 mg/L NAA or the whole quantity MS into and growing best, In vitro rooted plants were transplanted into a mixture soil, vermiculite and peat moss (1:1:1) and successfully grown. The plantlet and callus contains Oleanolic acid & Ursolic acid. analysed the appearance by the HPLC Measure, Not measuring to in originally testing. Keywords：Hedyotis diffusa (Willd.)、Oleanolic acid、Ursolic acid、callus、plantlet、HPLC

Dissertação de mestrado em Genética Molecular
Cancer incidence is increasing worldwide mainly due to changes in diet, life style and increased lifespan. In particular, liver cancer is the fifth most common cancer in the world and the third most common cause of cancer mortality. Plant phytochemicals are a good and promising source of anticancer compounds. In a previous study, we reported the potential of ursolic acid (UA) to induce cell death and to inhibit proliferation in colorectal cancer cells. This natural triterpenoid, UA, was also shown to activate JNK and to modulate molecular markers of autophagy. In the present study, the ability of two isomer triterpenoids, UA and oleanolic acid (OA), to induce cell death and modulate autophagy in the human hepatocellular carcinoma cell line (HepG2 cells) was tested. For that, the effect of these phytochemicals on cell death was evaluated by MTT assay and propidium iodide staining, in complete and starvation medium. Autophagy markers were evaluated by western blot and fluorescence microscopy. Contrary to our previous data with other cell lines, HepG2 cells were less susceptible to UA and, unexpectedly, OA was a more potent inducer of cell death than UA. Interestingly, starvation-induced autophagy sensitized HepG2 cells to cell death caused by OA, but not by UA. The IC50 of OA decreased from about 50 μM in complete medium to 3.5 μM in starvation medium. Although UA and OA increased the levels of autophagy markers LC3 and p62, as well as the number of acidic vacuoles (as assessed by MDC staining), the cell death induced by OA was not prevented by inhibitors of autophagy and of lysosome proteases. Overall, the results seem to indicate that autophagy is not directly involved in cell death induced by OA. Interestingly, methyl- -cyclodextrin (a polymer able to decrease membrane cholesterol content) prevented OA-induced cell death, which indicates that disruption of cholesterol homeostasis, and in particular in lipid rafts, may be involved in OA effects under starvation conditions. The present results suggest the application of OA as a specific drug for cancer treatment in particular cell physiological conditions, such as under metabolic stress.
A incidência do cancro está a aumentar em todo o mundo principalmente devido a alterações da alimentação, do estilo de vida e do aumento da esperança média de vida. Em particular, o cancro do fígado é o quinto cancro mais comum no mundo e a terceira maior causa de morte por cancro. Os fitoquímicos são uma excelente e promissora fonte de compostos anticancerígenos. Num estudo anterior o nosso grupo descreveu o potencial do ácido ursólico (AU) na indução de morte e na inibição da proliferação de células do cancro coloretal. Este triterpenóide de origem natural foi também descrito como ativador da sinalização JNK e modulador de marcadores moleculares de autofagia. No presente trabalho foi testada a capacidade de dois isómeros triterpenóides, o AU e o ácido oleanólico (AO), em induzir morte celular e modular a autofagia numa linha celular do carcinoma hepatocelular humano (células HepG2). Para tal, o efeito destes fitoquímicos na morte celular foi avaliado pelo ensaio de MTT e pela marcação com o iodeto de propídio, tanto em meio completo como em meio com privação de nutrientes. Os marcadores de autofagia foram avaliados por western blot e também por microscopia de fluorescência. Contrariamente a resultados anteriores com outras linhas celulares, as células HepG2 foram menos suscetíveis ao AU, bem como o AO mostrou ser mais potente na indução de morte celular do que o AU. Além disso, a autofagia induzida pela privação de nutrientes suscetibilizou marcadamente as células HepG2 para a morte celular causada pelo AO, e tal já não se verificou com o AU. Nestas condições o IC50 do AO foi de 3,5 μM, enquanto em meio completo era de cerca de 50 μM. Embora, o AU e o AO aumentem os níveis dos marcadores autofágicos LC3 e p62, bem como o número de vacúolos acídicos (avaliado pela marcação com MDC), a morte celular induzida pelo AO não foi prevenida por inibidores de autofagia e nem por inibidores de proteases lisossomais. Em geral, os resultados parecerem indicar que a autofagia não está diretamente envolvida na morte celular induzida pelo AO. No entanto, um polímero capaz de diminuir o conteúdo de colesterol nas membranas celulares, a metil- -ciclodextrina, preveniu a morte celular induzida pelo AO. Este resultado indica-nos que o AO pode induzir uma alteração na homeostasia do colesterol, em particular em domínios lipídicos ricos em colesterol, com efeitos drásticos sob a viabilidade das células quando estas estão sob privação de nutrientes. Estes resultados sugerem-nos que o AO pode ser utilizado no tratamento do cancro em condições fisiológicas específicas, tal como sob stress metabólico.
Fundação para a Ciência e Tecnologia (FCT) – Projeto PTDC/QUI-BIQ/101392/2008

O cancro é uma das causas de morte mais comuns. Nos Estados Unidos, uma em cada quatro mortes é devida ao cancro. A Organização Mundial de Saúde prevê que em 2030, 30 milhões de mortes serão devidas a cancro. Estes dados remetem para a urgência na pesquisa de novas moléculas capazes de melhorar o tratamento e prevenção da doença oncológica. Os triterpenóides representam um grupo de compostos abundante e diversificado, que pode ser encontrado em plantas, frutas e vegetais. As plantas medicinais contendo triterpenóides tem constituído ao longo dos séculos uma base para a medicina tradicional no tratamento de diversas doenças. Triterpenóides como o ácido ursólico, o ácido oleanólico, a betulina, o ácido betulínico, o ácido maslínico e o ácido glicirretínico, foram isolados de extratos de plantas, demonstrando ter várias actividades biológicas, incluindo a anti-tumoral. O potencial interesse em derivados de triterpenóides extraídos de plantas com actividade anti-tumoral está bem patente no crescente número de publicações nesse domínio científico. Uma análise das publicações enquadrando os triterpenóides com actividade anti-tumoral revelou um aumento exponencial no número de artigos e patentes na referida área científica. Estes factos têm impulsionado a comunidade científica para a síntese de novos derivados triterpénicos com melhorada actividade anti-tumoral. Com o objectivo de melhorar a actividade anti-tumoral dos ácidos ursólico e oleanólico, novos derivados semissintéticos heterocíclicos (compostos 2.18-2.58 e 3.15-3.50) e fluorados (compostos 2.59-2.82 e 3.51-3.58) foram sintetizados e caracterizados através de técnicas de espectrometria de massa, espectroscopia de infravermelhos e ressonância magnética nuclear, e por fim avaliados através do uso de MTT para a inhibição do crescimento de linhas celulares tumorais e para os mecanismos de acção relevantes para a actividade anti-tumoral. A actividade antiproliferativa dos compostos estudados na linha tumoral do pâncreas AsPC-1 foi usada para estabelecer a relação estrutura-actividade entre os compostos. II Todos os 61 novos compostos derivados do ácido ursólico, compostos 2.18-2.82, demonstraram uma boa actividade antiproliferativa nas células tumorais pancreáticas AsPC-1, vários dos novos compostos apresentaram melhor actividade antiproliferativa do que o ácido ursólico 2.1, e algums apresentaram mesmo um IC50 inferior a 1 μM. Os compostos com resultados mais promissores foram também avaliados para a sua actividade antiproliferativa em linhas celulares tumorais de mama, fígado, pulmão, e próstata. O derivado heterocíclico 2.57 revelou ser o mais activo neste grupo, tendo sido usado para avaliar o mecanismo de acção responsável pelos efeitos antiproliferativos nas células AsPC-1: este composto, induz o aumento dos níveis de p53 o que leva ao aumento dos níveis de NOXA e p21waf1, conduzindo à activação das caspases 9 e 3 induzindo a apotose nas células AsPC-1 tratadas. O composto fluorado 2.72 revelou ser o mais activo a inibir de forma efectiva o crescimento da linha celular tumoral pancreática através da inibição do ciclo celular na fase G1 a 1 μM e aumento dos níveis de p21waf1 e indução da apoptose a 8 μM, com aumento dos níveis de NOXA e diminuição dos níveis de c-FLIP. Os 42 novos derivados do ácido oleanólico 3.15-3.17 e 3.19-3.58 e o composto 3.18, já descrito, apresentaram uma actividade antiproliferativa superior ao ácido oleanólico 3.1 nas células AsPC-1. Os compostos 3.27, 3.39 e 3.49, que apresentaram os valores de IC50 mais baixos nas células tumorais AsPC-1, viram a sua actividade antiproliferativa estudada em linhas celulares tumorais de mama, próstata, fígado e pulmão. Os estudos revelaram que os compostos têm boa actividade antiproliferativa, com IC50 mais baixos que 5 μM. Os compostos 3.27 e 3.39 induziram apoptose nas células AsPC-1, quando estas foram tratadas durante 24 h a uma concentração de 1.5 μM. Os novos compostos sintetizados derivados dos ácidos ursólico e oleanólico e avaliados na sua actividade antiproliferativa representam um novo grupo de moléculas com potencialidade para o tratamento de tumores sólidos malignos.
Cancer is one of the most common causes of death. In the United States alone, one in four deaths is the consequence of cancer. The World Health Organization (WHO) estimates that 30 million deaths will be related to cancer in the year 2030. These data demonstrate that research aimed at the identification of new molecules that can improve the treatment and prevention of cancer is urgent. Triterpenoids are a large and diverse group of compounds that can be found in medicinal plants, fruits and vegetables. Medicinal plants containing triterpenoids have been used for centuries in traditional medicine for the treatment of various diseases. Triterpenoids, such as ursolic acid, oleanolic acid, betulin, betulinic acid, maslinic acid, and glycyrrhetinic acid purified from plant extracts, have diverse biological activities, including antitumor activity. The potential interest of plant-derived triterpenoids as antineoplastic agents is reflected in the large number of scientific papers appearing in the field. Analysis of the publications concerning antineoplastic triterpenoids abstracted in available the online databases over the past few years revealed an exponential increase in the number of papers and patents in this area of research. These facts prompted the scientific community to pursue the synthesis of new, improved derivatives of the triterpenoids available. With the aim of improving the antitumor activity of ursolic and oleanolic acids, a series of new heterocyclic (compounds 2.18-2.58 and 3.15-3.50) and fluorine (compounds 2.59-2.82 and 3.51-3.58) derivatives were synthesized, fully characterized using MS, IR and NMR techniques and evaluated for their antiproliferative activity via an MTT assay, as well as for their mechanisms of antitumor action. The antiproliferative activity against the AsPC-1 pancreatic cancer cell line was used to establish the structure activity relationship (SAR) between compounds. The new 61 ursolic acid derivatives 2.18-2.82 showed a good antiproliferative activity against AsPC-1 cells, several of the newly synthesized compounds were more IV active than that of ursolic acid 2.1 and some presented IC50s lower than 1 μM. The most promising compounds were evaluated further for their antiproliferative activity in breast, hepatic, lung and prostate cancer cell lines. The best heterocyclic derivative, compound 2.57, was evaluated for its mechanisms of action in AsPC-1 cells: it induced the upregulation of p53, leading to the upregulation of NOXA and p21waf1, these events culminated with the upregulation of caspases 9 and 3, thus inducing apoptosis in AsPC-1 cells. The fluorine derivative 2.72 exhibited the most effective inhibitory activity against pancreatic cancer cell growth, as it arrested the cell cycle at the G1 phase at 1 μM and upregulated p21waf1 and induced apoptosis at 8 M, with the upregulation of NOXA and the downregulation of c-FLIP. The new 42 oleanane derivatives 3.15-3.17 and 3.19-3.58 and the already described derivative 3.18 had better antiproliferative profile than oleanolic acid 3.1 against AsPC-1 cancer cells. Compounds 3.27, 3.39 and 3.49, which had the lowest IC50s in AsPC-1 cancer cells, were studied further for their antiproliferative activity in breast, prostate, liver and lung cancer cell lines. It was found that these compounds also had an excellent antiproliferative activity, with IC50s lower than 5 μM. Compounds 3.27 and 3.39 induced apoptosis in AsPC-1 cells treated for 24 h at a concentration of 1.5 μM. The novel ursane- and oleanane-type triterpenoids synthesized and evaluated for their antiproliferative activity represent a new group of valuable molecules for the treatment of solid cancers.

碩士
國立臺灣大學
藥學研究所
100
Triterpenoid and steroidal saponins isolated from natural plants have several biological activities, including anti-infection, hemolytic activity, immune-modulation, cytotoxicity, and etc. N-Acetylglucosamine bearing triterpenoid saponins (ATS), such as lotoidoside D, were reported to be a unique type of saponins with potent anti-tumor activity. In order to study the SAR of ATS, we synthesized 27 derivatives of 3-O-N-acetylglucosaminoyl oleanolic acid, with additional 9 sugars glycosylated either at 3'', 4'', and 6'' positions of N-acetylglucosamine. It is highly essential to develop a concise procedure to prepare these derivatives. To circumvent the problem associated with selective glycosylate sugars at specific of hydroxyl groups of D-glucosamine, it was orthogonal protected to 6-O-tert- butyldiphenylsilyl-3-O-levulinoyl-4-O-(2-nitrophenylacetyl)-2-(2,2,2-trichloroethoxylcarbonylamino)-2-deoxy-D-glucopyranosyl trichloroacetimidate (112). Following by glycosylation with benzyl oleanolate at C-3-OH, compound 90 was obtained, which was selected deprotected by using hydrazine to free 3-hydroxyl group from levulinoyl group, or 3% AcCl/MeOH to free 6-hydroxyl group from 6-tert-butyldiphenylsilyl group. The free hydroxyl groups at 3'' or 6'' of glucosamine were further glycosylated with 9 per-benzoylated glycosyl trichloroacetimidates in the presence of TMSOTf at 0 oC to rt to afford 3'' or 6'' glycosylated intermedidates. In an attempt to free 4-hydroxyl group from 2-nitrophenylacetyl group by Zn in ammonia chloride, we suffered from low yield of desired product due to concomitant removal of Troc group in this condition. However, when compound 90 was subjected to TBAF/HOAc condition, we accidently obtained the 4'', 6''-migration of 2-nitrophenylacetyl group in 95% yield to afford compound 149 which was glycosylated with 9 sugar donors to get the desired 4''-glycosylated products. The global deprotection of glycosylated intermediates afforded 27 3-O-N-acetylglucosaminoyl oleanolic acid derivatives. Three N-2-methoxycarbonyl D-glucosaminoyl oleanolic acid derivatives were also obtained as side products in the global deprotecting process. The effect of these derivatives on HL-60 cell line was studied via SRB assay. Compounds 38, 173, and 196 showed more than 80% of cytotoxicity at 30 miuM. The preliminary SAR results indicated that the modification at 3’ position retained most of cytotoxicity of 3-O-N-acetylglucosaminoyl oleanolic acid, modification at 4’ position have moderate cytotoxicity, and modification at 6'' position completely loss the activity. D-Xylose, L-xylose and L-arabinose attached at 3'' or 4'' positions were found to be active among these modifications. Compounds 194 and 196 with N-2-methoxycarbonyl modification at 2''-amino position was found to increase the cytotoxicity.

碩士
國立屏東科技大學
生物科技研究所
98
Abstract The hepatitis B virus X protein (HBx) contains 154 amino acids, encoded a 17 kDa protein. It plays an important role in the development of liver cancer. HBx is a multifunctional regulator that modulates transcriptional activation, signal transduction, cell proliferation, apoptosis, cancer cell migration, and invasion. Many of transcription factors are known to be activated by HBx through the modulation of intracellular signalings. Flavonoids and triterpenes had been confirmed their anti-cancer activities through the inhibition of cancer cell proliferation and apoptosis induction. This research attempts to investigate whether tangeretin、nobiletin、ursolic acid (UA) and oleanolic acid (OA) could inhibit the transactivation function of HBx and to study the involved molecular mechanisms. Utilizing the luciferase reporter assay, HBx could activate Sp-1, Smad3/4 and NF-B ranging from 5 to 20 folds in Huh7 hepatoma cells. In normal hepatocyte FL83B, HBx could activate Sp-1, Smad3/4 ranging from 2 to 4 folds. MTT analysis was used to evaluate the cytotoxicity of tested compounds. Under the non-cytotoxic conditions, UA and OA selectively inhibited the HBx-induced Sp-1 and Smad3/4 transcriptional activation, however, tangeretin and nobiletin did not have this effect. SB203580 and p38 siRNA releases the UA and OA-mediated inhibitory function, however, SP600125 did not affect this phenomenon. These results suggested the important role of p38.Western blotting analysis revealed the UA and OA enhance the phosphorylation level of MKK3/6, p38 and ATF with different kinetics. Ras dominant negative mutant, but not Csk, suppressed the UA and OA-induced p38 activation. Thus, UA and OA activate the p38 signaling through Ras-dependent mechanism. All of UA, OA and a well-known anti-tumor compound silymarin, could effectively prevent HBV-containing hepatoma cells 2.2.15-induced tumor formation in Nu/Nu mice. Key word: UA、OA、HBx、Sp-1、Smad3/4

Diabetes mellitus (DM) has become a global threat in developing and developed countries, where diabetic patients are more prone to cardiovascular complications, a condition called diabetic cardiomyopathy. Studies have shown a direct link between hyperglycaemia and an increase in the production of reactive oxygen species in cardiac cells leading to diabetic cardiomyopathy. This study tests oleanolic acid, a bioactive compound from the plant Syzigium aromaticum as an antioxidant which could have a potential role in management of DM. Aims i) To extract Oleanolic acid (OA) from Syzigium aromaticim, ii) Investigate the antioxidant effects of plant derived OA in an in-vitro model of hyperglycaemia induced oxidative stress. Methods The flower buds of the Syzigium aromaticim [(Linnaeus) Merrill & Perry] (Myrtaceae) plant (commonly called cloves) were used to isolate OA. The ethyl acetate solubles from the cloves were subjected to chromatographic fractionation to yield OA powder. Spectroscopic analysis was done using 1D and 2D 1H and 13C NMR techniques for the identification of the structure of the compound. This compound was then used in vitro to test for its antioxidative properties. H9C2 cardiac myoblasts were employed which were treated with normoglycaemic (5.5 mM) and hyperglycaemic (33 mM) glucose conditions. The cells were then treated with oleanolic acid to test for its antioxidant properties. We looked at a dose-dependent (0, 20, 50 μM) and time-dependent effects of OA treatment (6 and 24 hrs) following 48 hours glucose exposure. ROS levels were measured using H2DCF-DA fluorescence staining using microscopy and flow cytometry techniques for analysis. xviii Results Recrystallisation of the powder with ethanol and inspection of the 1 and 2- dimensional 1H- and 13C-NMR spectra of the compound with comparison to literature data confirmed OA molecular structure and IUPAC numbering similar to that of literature characterized and confirmed the structure of oleanolic acid. In cell specific data high glucose treatments on H9C2 cells showed increased ROS production (22 ± 6 % and 20 ± 7 % n= 3 p< 0.01) for 6 and 24 hrs treatments, respectively, compared to their normoglycaemic control groups. The 6 h OA treated group showed a decrease in ROS production with 26.6 ± 17.4 % for the 20 μM while for 50 μM there was a 37.7 ± 14.3% decrease. A ROS reduction trend was observed in the normoglycaemic group, but this was significant at 24 hrs with 46.8 ± 45.3% and 57.3 ± 9 % for both 20 and 50 μM treatments, respectively. The 24 hrs OA treated group showed a dose-dependent decrease in ROS with 50 μM more pronounced (80.7% ± 4.5 %). The 20 μM OA treatments also showed a 15.7 ± 19 % decrease in ROS. Discussion In the present study, we have evaluated the antioxidant effects of OA in vitro following extraction of the compound from Syzigium aromaticim. The oxidative stress induced by hyperglycaemia was attenuated by oleanolic acid and this also translated into decreased ROS suggesting its use as an antioxidant in alleviating cardiovascular complications associated with diabetes mellitus.

碩士
靜宜大學
應用化學研究所
84
11α-methoxyurs-12-en-28-oic acid was a new triterpenoid compound obta inedfrom the leaves of Alyxia insularis Kanehira &Sasaki (Apocynaceae). This structure was elucidated with the spectroscopic methods. Herein, we attemptto proceed the partical total synthesis of this compound from the startingmateria l ursolic acid.By the way, the reactions of oleanolic acid was also proceeded. The resultswere discussed in this report.

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina)
Cancer is a disease characterized by the uncontrolled growth of abnormal cells that beyond the limits of a cell division in normal conditions. Colorectal cancer (CRC) is the third most common tumors and is a major cause of cancer related death worldwide. Cholesterol metabolism has been established as possible source of therapeutic targets in cancer progression, because the cholesterol synthesis pathway provides farnesyl pyrophosphates groups essential to prenylation of proteins involved in cell proliferation and cancer growth. For this reason, targeting cholesterol synthesis with statins (HMG CoA reductase inhibitors) has been explored but with limited results in the clinic due to the toxicity of high dose statin treatment. Additionally, cholesterol is involved in proliferative cell signalling through receptors activation upstream of RAS and PI3K because it is a major constituent of lipid rafts. Thus, the aim of this project is to test if natural compounds with structure similar to cholesterol, such ursolic acid (UA) and oleanolic acid (OA) can alter the lipid composition and influence signalling pathways in order to inhibit the proliferative activity of carcinoma colorectal cells (HCT116). For this we evaluated the effect of the triterpenoids in the amount of cellular cholesterol by cholesterol quantification assay and found that the UA reduces and OA increase cholesterol cellular levels. These effects may be affect AKT signaling pathway induced by insulin because our results indicate that UA, but not OA, causes a decrease in p-AKT. Furthermore, by the method of nuclear condensation we evaluated apoptosis, and both the UA and OA increased tumor necrosis factor alpha (TNF-α) induced apoptosis. These results help to take another step in understanding the mechanisms of action of these natural compounds, helping control the progression of cancer.
O cancro é uma patologia caracterizada pelo crescimento descontrolado de células anormais, que ultrapassam os limites de uma divisão celular em condições normais. O cancro colorrectal representa mundialmente o terceiro cancro mais comum e a maior causa de morte relatada por cancro. O metabolismo do colesterol tem sido proposto como possível alvo terapêutico na progressão desta neoplasia, porque a via do mevalonato responsável pela síntese deste composto proporciona grupos farnesil pirofosfatos essenciais à prenilação de proteínas que ativam vias relacionadas com a proliferação e o crescimento celular. Por esta razão, o combate da síntese de colesterol com estatinas (inibidores da HMG CoA Reductase) tem sido explorado mas com resultados limitados na prática clínica devido a toxicidade causada pelo tratamento com elevadas doses de estatinas. Adicionalmente verifica-se que o colesterol está também envolvido na proliferação através da ação de recetores de membrana que ativam a via da RAS e do PI3K, por ser o maior constituinte dos lipid rafts. Assim este projeto visa testar se compostos naturais com estrutura semelhante à do colesterol, como o ácido ursólico (AU) e ácido oleanólico (AO), conseguem alterar a composição lipídica e influenciar vias de sinalização de maneira a inibir a atividade proliferativa em células do carcinoma colorrectal (HCT116). Para tal avaliamos o efeito dos triterpenoides na quantidade de colesterol celular através do método de quantificação de colesterol, e verificamos que o AU diminui os níveis celulares e o AO provoca o seu aumento. Estes efeitos podem afetar a via de sinalização AKT induzida pela insulina, porque os nossos resultados indicam que o AU, mas não o AO, provoca uma diminuição da proteína p-AKT. Por outro lado, pelo método de condensação nuclear avaliamos a apoptose, e tanto o AU como AO aumentam a apoptose induzida pelo Fator tumoral de necrose alfa (TNF-α). Estes resultados ajudam a dar mais um passo na compreensão dos mecanismos de ação destes compostos naturais, ajudando no controlo da progressão do cancro.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, School of Physiology, fulfilment of the requirements for the degree of Doctor of Philosophy (PhD). Johannesburg, South Africa, 2018.
The neonatal period is a critical window of developmental plasticity. Consumption of fructose-rich diets has been implicated in the increasing global prevalence of metabolic dysfunction (MD) and non-alcoholic liver disease (NAFLD). Interventions during periods of early ontogenic developmental plasticity can induce epigenetic changes which program metabolism for positive health benefits later in life. The phytochemical, oleanolic acid (OA) possesses anti-diabetic, anti-oxidant and anti-obesity effects. I investigated the potential protective effects of neonatal oral administration of OA on the subsequent development of health outcomes associated with fructose-induced MD and NAFLD in male and female rats. The study was divided into two major experiments. In the first short-term experimental study, the potential of neonatal oral administration of OA to acutely protect against the development of fructose-induced oxidative damage, adverse general health outcomes and precocious maturation of the gastrointestinal tract (GIT) in suckling male and female rats was investigated. Male and female suckling rat pups (N=30) were randomly assigned to four groups and gavaged daily with 10 mℓ/kg body mass of: distilled water (DW) with 0.5% (v/v) dimethyl sulphoxide (vehicle control), oleanolic acid (OA; 60 mg/kg), high fructose solution (HF; 20% w/v), or OAHF for 7 days. On day 14, the pups were euthanised. Blood, liver and skeletal muscle samples were collected to determine clinical health profiles, hepatic lipid content and gene expression of anti-oxidant enzymes, superoxide dismutase (SOD2) and glutathione peroxidase (GPx1). Rats in all groups had a significant increase in body mass over the seven day treatment period (ANOVA; P<0.05). There were no significant differences in visceral organ masses, general clinical health profiles, liver lipid content and GIT morphometry across all treatment groups (ANOVA; P>0.05). Neonatal oral administration of fructose lowered the expression of genes for anti-oxidant enzymes (SOD2 and GPx1) which was prevented by OA (ANOVA; P<0.05). Findings from this study provide evidence that short-term neonatal oral administration of OA protects against fructose-induced oxidative damage with seemingly no adverse effects on health or the maturational and developmental changes of the gastrointestinal tract in suckling male and female pups. In the second long-term experimental study, which was further subdivided into two studies, I investigated the potential protective effects of neonatal oral administration of OA on the subsequent development of high fructose diet-induced a) metabolic dysfunction and b) NAFLD in male and female rats. Male and female suckling rats (N=112) were randomly assigned into four groups and gavaged daily with 10 m mℓ/kg body mass of: distilled water (DW) with 0.5% (v/v) dimethyl sulphoxide (vehicle control), oleanolic acid (OA; 60 mg/kg), high-fructose solution (HF; 20% w/v) and OAHF for 7 days. On day 21, the rats were weaned onto normal rat chow and plain drinking water up to day 55. From day 56, half of the rats in each treatment group were continued on plain water whilst the remainder were given a high fructose solution (20 % w/v) as drinking fluid ad libitum for eight weeks. On day 110 the rats were subjected to an oral glucose tolerance test (OGTT) and then euthanised on day 112. Fasting glucose, triglyceride levels and terminal body mass were measured before termination. Blood samples were collected to determine the effects of treatments on fasting levels of cholesterol, insulin, glucose, triglycerides, insulin resistance (HOMA-IR), glucose tolerance (area under the curve for OGTT), a surrogate biomarker of liver function, alanine amino transaminase (ALT) and non-tissue specific alkaline phosphatase (ALP). Body adiposity was determined by measuring visceral and epidydimal fat pad masses. Liver samples were used to measure hepatic lipid accumulation and hepatic histomorphometry. The livers were formalin fixed, paraffin embedded and sectioned at 3μm. The sections were stained with haematoxylin and eosin for assessment of inflammation and Masson’s trichrome for visualisation of connective tissue and steatosis. Male and female rats in all groups of the second experiment had a significant increase in body mass over the study period (ANOVA; P<0.05). In the general metabolic dysfunction study, rats which consumed fructose as neonates and then later as adults (HF+F) and those which consumed fructose only in adulthood (DW+F) had significant increases in terminal body mass (females only), visceral fat mass (males and females), serum triglycerides (females only), epididymal fat (males only), fasting plasma glucose (males and females), impaired glucose metabolism (females only), β-cell dysfunction and insulin resistance (males and females) compared to the other treatment groups (P<0.05). There were no differences in fasting cholesterol levels across all treatment groups in both male and female rats (P>0.05). The sub-study on NAFLD revealed that fructose consumption in adulthood following neonatal fructose intake (HF+F) caused a 47-49% increase in hepatic lipid content of both male and female rats (P˂0.05). However, fructose administered in adulthood only (DW+F), caused a significant increase in liver lipid content in females only (P<0.05). NAFLD activity scores for steatosis were higher in male (HF+F) and female (DW+F and HF+F) rats compared to other treatment groups (P<0.05). Scores for inflammation were higher in female rats that received DW+F and HF+F (P<0.05) but not in male rats (P>0.05). NAFLD area fraction for fibrosis was 3 times higher in male and female rats that received a double hit neonatally and in adulthood (HF+F) and a late hit of fructose (DW+F) compared to the rats in the negative control group (P<0.05). I have shown that administration of a high fructose diet had adverse effects on several health outcomes associated with MD and induced NAFLD. However, it was notable that the timing of the fructose intake in the life stage of rats had an impact on the development of MD and NAFLD phenotype. I also observed sex-specific differences in the metabolic response to dietary fructose, with females appearing to be more vulnerable to the development of MD and NAFLD. It is thus important to note that studies should not just focus on a single sex but should be comparative between the sexes. I have also demonstrated, for the first time, that neonatal oral administration of oleanolic acid protects against the subsequent development of fructose-induced health outcomes associated with metabolic dysfunction and NAFLD by reducing hepatic lipid storage, terminal liver masses and hepatic histomorphological changes associated with NAFLD. I conclude that neonatal interventional treatment with oleanolic acid during the critical window of developmental plasticity protected against the development of fructose diet-induced adverse health outcomes associated with MD and NAFLD in male and female Sprague Dawley rats. Therefore, OA is a phytochemical that exhibits potential in the prevention of neonatal programming of MD and NAFLD later in life. OA should be considered as a natural strategic prophylactic intervention during periods of developmental plasticity with a lot of potential in the fight against the scourge of metabolic disorders that have a significant negative impact on the health systems globally.
LG2018

Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycaemia; this condition is caused by lack of insulin secretion (Type 1) and/or insulin resistance (Type 2). In diabetic patients; carbohydrate, protein and lipid metabolism is disturbed due to the lack of the body’s ability to utilise glucose efficiently. Management of type 1 diabetes involves insulin therapy which may be inconvenient for patients. Therefore alternative methods for management of type 1 diabetes involving medicinal products are being investigated. This study is aimed at investigating the effect of OA on markers of lipid metabolism and on proteins of the insulin signalling pathway in Type 1 diabetic rats as this plant product has anti-hyperglycaemic effects. Male Sprague-Dawley rats were divided into two groups (diabetic and normal). In both groups the rats were further divided into four groups and assigned to treatment as follows: vehicle, insulin, OA and OA plus insulin. Oral glucose tolerance test was performed in fasted and non-fasted diabetic rats for 2 hours. In acute studies the effect OA following treatment of rats was evaluated at 15, 30 and 60 minutes. In sub-chronic studies rats were treated daily for 14 days. OA did not improve glucose tolerance in diabetic rats after 2 hours of administration. However, it enhanced blood glucose lowering effect of insulin and this was statistically significant in fasted rats. In acute studies OA enhanced the effect of insulin in normal and diabetic animals as AKT phosphorylation was increased when insulin was used in combination with OA. OA reduced the expression and activity of HSL in liver tissue after 14 days of treatment in both normal and diabetic rats. In adipose tissue, OA reduced the expression of HSL in diabetic rats. However, OA alone did not reduce the activity of HSL but when it was combined with insulin, a reduction of HSL activity was observed. OA administration had no significant effect on TGA and HDL-c levels but significantly (p < 0.05) reduced total cholesterol and LDL-c in diabetic rats. It had no significant effect on total cholesterol, and increased LDL-c levels in normal rats. Serum AST and ALT levels in diabetic rats were reduced by OA administration but this reduction was not statistically significant. The results of this study suggest that OA enhances the hypoglycaemic effect of insulin, improves lipid profile and possesses hepatoprotective effects. Lastly, OA independently increases AKT phosphorylation and decreases HSL expression and activity.
Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Adverse effects and increasing cost of therapeutic drugs have renewed an interest in the use of medicinal plant products for the treatment of a variety of chronic disorders. One such bioactive plant-derived compound is a pentacyclic triterpenoid, oleanolic acid (3ß-hydroxy-olea-12-en-28- oic acid, OA) present in herbs. OA possesses a variety of pharmaceutical activities and of interest in this study are the anti-diabetic properties. Diabetes is associated with disorders grouped as microvascular (retinopathy and nephropathy) and macrovascular (atherosclerotic) complications. Accordingly, this study further investigated the potential of OA in diabetes management by studying the effects of this triterpene on kidney function as well as proximal tubular Na+ handling in an effort to identify the site of action of OA. Furthermore, the study evaluated the effects of OA in kidney and liver cell lines to establish whether this triterpene exhibits any toxicity in these organs. OA was extracted using a previously validated protocol in our laboratory. Briefly, dried flower buds of Syzygium aromaticum were soaked in dichloromethane overnight, thereafter in ethyl acetate to obtain ethyl acetate solubles which contained a mixture of OA/ursolic and maslinic acid (MA). OA/MA mixture was subjected to column chromatograph and pure OA was obtained through recrystallization in methanol. The absolute stereostructure of OA was elucidated using 1H and 13C NMR spectroscopy and was comparable to previously reported data. In kidney function studies, various doses of OA (30, 60, 120 mg/kg, p.o.) were administered to male Sprague-Dawley rats twice (8h apart) every third day for five weeks. Rats administered deionised water served as controls. Measurements of body weight, food and water intake, blood pressure, Na+, K+, Cl-, urea and creatinine were taken 24 h from dosing. Renal clearance studies investigated the influence of OA on Na+ handling in the proximal tubule of anaesthetized rats using lithium clearance. Animals were given water with lithium (12mmol/l) for 48 hours following which they were anaesthetized and cannulated using a previously validated standard protocol that has been reported from our laboratories. After a 3½ h equilibration, animals were challenged with hypotonic saline for 4 h of 1 h control, 1½ h treatment and 1½ h recovery periods. OA was added to the infusate during the treatment period. In vitro effects of various OA concentrations (5, 10, 20, 40, 80 μmol/l) were investigated in HEK293, MDBK and HepG2cell lines. Cells were exposed to OA for 24, 48 and 72 h, thereafter, 3-4,5 dimethylthiazol-2-yl- 2,5diphenyltetrozolium bromide (MTT) and single cell gel electrophoresis (comet) assays were conducted. All data are presented as means ±SEM. OA significantly (p<0.05) increased urinary Na+ output from week 2 until the end of the experimental period in a dose independent manner. However, this OA-evoked natriuresis was not reflected in plasma collected at the end of the experiment as there was no change in plasma Na+ concentrations compared with control animals at the corresponding time. OA administration had no significant influence on K+ and Cl- excretion rates throughout the experiment. However, OA significantly (p<0.05) reduced plasma creatinine concentration with a concomitant increase in glomerular filtration rate (GFR). Furthermore, OA administration significantly (p<0.05) decreased mean arterial pressure from week 2 until the end of the experimental period. Intravenous infusion of OA at 90 ug/h for 1 ½ h induced a marked increase in urinary excretion rates of Na+. This increase was accompanied by concomitant increase in FENa proximal and FENa distal and FELi which persisted until the end of the experiment without any apparent changes in GFR. The cell viabilities of HepG2, HEK293 and MDBK cell lines were significantly increased after 24 h exposure, however, the viabilities of all the three cell lines dropped after 72 h exposure to values that did not achieve statistical significance in comparison to the respective controls. In addition, all OA-treated cells in the comet assay had intact DNA after exposure for 24, 48 and 72 h. Hence, the decrease in viability that was observed in the MTT assay after 72 h exposure could probably be attributed to the depletion of nutrients in the culture medium. The results of the present study, apart from confirming our previous observations of the natriuretic effects of OA in rats, indicate that this effect is in part mediated via the inhibition of proximal tubular Na+ reabsorption and increased Na+ secretion. We speculate that this increased Na+ secretion could have been due to increased tubular function and not to the toxicity of OA as indicated by MTT and comet assays. These findings suggest that OA does not exhibit toxicity in the kidney and the liver.
Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Treatment of diabetes mellitus is mainly focused on glycaemic control regulated by insulin and takes place in insulin sensitive tissues like skeletal muscle which accounts for 75% of glucose metabolism. Plant derived compounds that have anti-diabetic potential are currently being investigated for diabetes treatment as they are cheap and non-toxic. Oleanolic acid (OA), a triterpene found in a wide variety of plants has been shown to have anti-diabetic effects but its mechanism of action, especially on the insulin signalling cascade has not been fully elucidated. The aim of the present study was to investigate the effects of OA on the PI3K/Akt insulin signalling cascade in skeletal muscle and skin of streptozotocin induced diabetic male Sprague-Dawley rats. Male Sprague-Dawley rats (non-diabetic and diabetic) were treated with insulin (4IU/ kg bw), OA (80 mg/kg bw) and a combination of OA + insulin in an acute and sub-chronic study. The study showed that OA does not reduce blood glucose levels in type 1 diabetic rats but enhances insulin stimulated hypoglycaemic effects. In the acute study OA was shown to activate Akt and dephosphorylate GS in skeletal muscle of streptozotocin induced diabetic rats. In the sub-chronic study OA and OA + insulin increased expression of GS in skeletal muscle of diabetic rats. GP expression was decreased by OA and OA + insulin treatments in skeletal muscle whilst in skin it was increased by both treatments. OA increased both GS and GP in skeletal muscle whilst in skin they were decreased. OA + insulin treatment increased GS and decreased GP activities in skeletal muscle and increased activity of both enzymes in skin of diabetic rats. OA increased the amount of glycogen in both muscle and skin whilst OA + insulin reduced the amount of glycogen. OA and OA + insulin treatment showed some protective effects against liver and muscle damage as there were reductions in serum LDH, ALT and AST levels. In conclusion, oleanolic acid in synergy with insulin can enhance activation of the insulin signalling pathway and there was evidence of OA activation of insulin signaling enzymes independent of insulin.
Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.