Dissertations / Theses on the topic 'Phytochemical evaluation'

Cancer and malaria are among the most life-threatening diseases globally. Cancer is responsible for about 125,000 annual deaths globally. In 2015, the World Health Organization report estimated that 236000-635000 people died of malaria. These diseases are complicated by the development of resistance to available chemotherapeutic agents. Natural products have been recognized for their major applications in the identification of drug leads in drug discovery. Viola philippica Car, Viola yedoensis Makino (Violaceae), Triclisia subcordata Oliv (Menispermeaceae) and Cyclicodiscus gabunensis Harms (Fabaceae) are medicinal plants traditionally used for the treatment of various diseases including malaria or cancer in China and West Africa. However, the bioactive compounds are unknown. Therefore, this study evaluated the in vitro anticancer and antimalarial activities of the four medicinal plants and searched their bioactive compounds. The in vitro anti-ovarian cancer and antimalarial assays were demonstrated respectively using sulforhodamine B dye and Syber green 1 fluorescence assay methods. Bioassay-guided fractionation and purification were performed. Structural elucidation was performed by nuclear magnetic resonance spectroscopy and mass spectrometry analysis. Results revealed the anticancer and antimalarial activities of T. subcordata; V. philippica, and V. yedoensis to be bisbenzylisoquinoline alkaloids (cycleanine, isochondodendrine and 2′-norcocsuline) and/or cyclotides. The cycleanine analogues were synthesized and found to be more potent than cycleanine. Induction of apoptosis by these alkaloids has also been determined. This study could serve as basis for the support of use of these plants in cancer and/or malaria treatment. The BBIQ alkaloids and analogues could serve as lead compounds in drug discovery. Future in vivo studies need to be carried out on these alkaloids to get drug approval.

The development and advancement of prostate cancer is supported by a plethora of genetic and proteomic abnormalities, including events of post-translational modifications. The protein arginine methyltransferase 5 (PRMT5) enzyme regulates epigenetic events of histone modifications and protein post-translational modifications within protein signaling pathways. PRMT5 functions by catalyzing the symmetric dimethylation of terminal arginine residues on target protein substrates. Under abnormal conditions of overexpression and upregulation, PRMT5 methyltransferase activity constitutively drives the growth and proliferation of dysregulated cells. Overexpression or upregulation of PRMT5 correlates with disease progression as observed among numerous cancer types, including breast, colorectal, leukemia, lung, melanoma and prostate cancers. We demonstrated previously that PRMT5 knockdowns attenuated both growth and proliferation of lung and prostatic tumors, in vitro and in vivo. Plants naturally produce chemical toxins as mechanisms of defense against microbial and other biological threats. Human exploitation, consumption and application of agents isolated from plants for therapeutic intervention dates back throughout the millennia. In this study, we extracted, purified and evaluated natural, small, chemical compounds from plant products that antagonize PRMT5 activity in prostate cancer cells. We found that crude and purified extracts of Dendrobium aurantiacum var. denneanum (D. denneanum) plants attenuated prostate tumor growth and proliferation by selective inhibition of PRMT5 methyltransferase activity. These findings establish the first set of natural PRMT5-specific inhibitors reported.

There is an increasing need of automation for routine tasks like sorting agricultural produce in large scale post-harvest processing. Among different kinds of sensors used for such automation tasks, near-infrared (NIR) technology provides a rapid and effective solution for quantitative analysis of quality indices in food products. As industries and farms are adopting modern data-driven technologies, there is a need for evaluation of the modelling tools to find the optimal solutions for problem solving. This study aims to understand the process of evaluation of the modelling tools, in view of near-infrared data obtained from green leafy vegetables. The first part of this study deals with prediction of the type of leafy green vegetable from the near-infrared reflectance spectra non-destructively taken from the leaf surface. Supervised classification methods used for the classification task were k-nearest neighbors (KNN), support vector machines (SVM), linear discriminant analysis (LDA) classifier, regularized discriminant analysis (RDA) classifier, naïve Bayes classifier, bagged trees, random forests, and ensemble discriminant subspace classifier. The second part of this study deals with prediction of total glucosinolate and total polyphenol contents in leaves using Partial Least Squares Regression (PLSR) and Principal Component Regression (PCR). Optimal combination of predictors were chosen by using recursive feature elimination. NIR spectra taken from 283 different samples were used for classification task. Accuracy rates of tuned classifiers were compared for a standard test set. The ensemble discriminant subspace classifier was found to yield the highest accuracy rates (89.41%) for the standard test set. Classifiers were also compared in terms of accuracy rates and F1 scores. Learning rates of classifiers were compared with cross-validation accuracy rates for different proportions of dataset. Ensemble subspace discriminants, SVM, LDA and KNN were found to be similar in their cross-validation accuracy rates for different proportions of data. NIR spectra as well as reference values for total polyphenol content and total glucosinolate contents were taken from 40 samples for each analyses. PLSR model for total glucosinolate prediction built with spectra treated with Savitzky-Golay second derivative yielded a RMSECV of 0.67 μmol/g of fresh weight and cross-validation R2 value of 0.63. Similarly, PLSR model built with spectra treated with Savitzky-Golay first derivative yielded a RMSECV of 6.56 Gallic Acid Equivalent (GAE) mg/100g of fresh weight and cross-validation R-squared value of 0.74. Feature selection for total polyphenol prediction suggested that the region of NIR between 1300 - 1600 nm might contain important information about total polyphenol content in the green leaves.

Magister Pharmaceuticae - MPharm
Evaluation of the antinociceptive, anti-inflammatory and antipyretic activities of Ruta graveolens L. in mice and rats FIRDOWS LOONAT M. Pharm. Pharmaceutical Sciences thesis: School of Pharmacy, University of the Western Cape Ruta graveolens (Rutaceae) L. is a medicinal plant that is commonly used to manage and treat essential events such as pain, inflammation and fever. Despite its popularity, particularly as a medicinal plant in the Calvinia district and Bredasdorp region of South Africa, scientific data to substantiate its widespread traditional use and the possible mechanisms of action for this plant species is lacking. Therefore, the objectives of this study were: to scientifically evaluate and validate the antinociceptive, anti-inflammatory and antipyretic activities of Ruta graveolens using the acetic-acid writhing test and hot-plate test, the carrageenan rat paw oedema test, and the E. coli-induced pyrexia test, respectively; to investigate the possible mechanisms of the antinociceptive, anti-inflammatory and antipyretic activities of the plant using interaction studies; to determine some secondary metabolites present in the plant species using standard phytochemical analytical procedures; to characterise the plant species using HPLC techniques; and to determine the safety profile of the plant species using an acute toxicity study.Three percent (3 %) acetic acid (0.25 ml, i.p.) produced a substantial number of writhes in mice. The leaf methanol extract of Ruta graveolens (100 mg/kg, i.p.) significantly reduced the number of writhes induced by 3 % acetic acid (0.25 ml, i.p.). R. graveolens (100 mg/kg,i.p.) produced 54 % inhibition of 3 % acetic acid-induced writhes. Indomethacin (20 mg/kg,i.p.) and paracetamol (500 mg/kg, i.p.) significantly reduced the number of 3 % acetic acidinduced writhes. Indomethacin (20 mg/kg, i.p.) and paracetamol (500 mg/kg, i.p.) produced 57 % and 80 % inhibition of 3 % acetic acid-induced writhes, respectively. R. graveolens (25– 50 mg/kg, i.p. and 200 – 400 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) did not significantly reduce the number of writhes induced by 3 % acetic acid. However, combined therapy of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) significantly reduced the number of 3 % acetic acid-induced writhes. The combined therapy of the lowest and sub-effective doses of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) produced 59 % inhibition of the writhes elicited by 3 % acetic acid. The leaf methanol extract of R. graveolens (50 – 400 mg/kg, i.p.) greatly delayed the reaction time in mice to thermal stimulation produced with hot-plate. 50 – 400 mg/kg (i.p.) of the leaf methanol extract of R. graveolens significantly antagonised rat paw oedema induced by 1 % carrageenan (0.1 ml, subplantar) over the 4 h period of testing. In addition, indomethacin (10 mg/kg, i.p.) significantly antagonised 1 % carrageenan-induced rat paw oedema. R. graveolens (25 mg/kg, i.p.) and indomethacin (2mg/kg, i.p.) given separately did not significantly alter rat paw oedema induced by 1 % carrageenan. However, combined therapy of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.) and indomethacin (2 mg/kg, i.p.) significantly reduced 1 % carrageenan-induced rat paw oedema. The leaf methanol extract of R. graveolens (400 mg/kg, i.p.) significantly reduced the mean rectal temperature of normothermic rats. Ruta graveolens (100 – 400 mg/kg, i.p.) significantly reduced pyrexia induced by E. coli (50 μg/kg, i.m.) over the 5 h period of testing. In addition, pentoxifylline (50 mg/kg, i.p.) significantly reduced E. coliinduced pyrexia. Ruta graveolens (25 – 50 mg/kg, i.p.), paracetamol (500 mg/kg, i.p.) and pentoxifylline (10 mg/kg, i.p.) did not significantly reduce pyrexia induced by E. coli.However, combined therapy of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.)and pentoxifylline (10 mg/kg, i.p.) significantly reduced E. coli (50 μg/kg, i.m.)-induced pyrexia.The phytochemical studies of the powdered leaves of Ruta graveolens indicated the presence of alkaloids, cardiac glycosides, flavonoids, saponins, tannins and triterpene steroids. The HPLC fingerprint indicated characteristic peaks at the following retention times; 1.654 min,2.271 min, 2.403 min, 4.705 min and 7.691 min. The LD50 obtained for Ruta graveolens after oral administration was probably greater than 4000 mg/kg which shows that the plant extract is non-toxic to mice.In conclusion, the data obtained indicate that Ruta graveolens possesses antinociceptive, antiinflammatory and antipyretic activities. Since prostaglandins have been shown to mediate acetic acid-induced writhes, prostaglandins, histamine, serotonin, capsaicin and bradykinin implicated in carrageenan-induced rat paw oedema, and tumor necrosis factor-α (TNF-α) implicated in E.coli-induced pyrexia, it is possible that R. graveolens may be producing its antinociceptive, anti-inflammatory and antipyretic activities by affecting these chemical mediators. The data obtained also justify the use of the plant species by traditional medicine practitioners for the treatment of painful and inflammatory conditions, and pyrexia.

Infectious diseases are responsible for an overwhelming number of deaths and morbidity worldwide. In tropical regions of the world, in particular, developing countries like Zambia, poor health is prevalent and diseases such as malaria, meningitis, pneumonia, tuberculosis and gastrointestinal infections strongly persist. Folkloric medicines are still actively used against some of these infections as primary care before seeking conventional treatment at hospitals. Members of the genus Ficus (Moraceae) are traditionally used in Zambia against many diseases caused by bacterial, fungal and protozoal infections. Thus according to the plant parts used traditionally for herbal preparations, aerial and root parts of eight Ficus species namely; F. ingens, F. lutea, F. natalensis, F. ovata, F. sansibarica subsp. macrosperma, F. sycomorus subsp. gnaphalocarpa, F. sycomorus subsp. sycomorus and F. wakefieldii were collected from different parts of Zambia. The main aim of this thesis was to evaluate the medicinal potential of members of the genus Ficus. This was achieved by three objectives, which involved the phytochemical profiling of the crude extracts and subextracts of the Ficus for their constituents using chromatographic methods such as thin layer chromatography (TLC), proton Nuclear Magnetic Resonance (1H NMR) and high performance liquid chromatography (HPLC). Secondly, the extracts were screened for various biological activities after which they were evaluated against recombinant FAS-II elongation enzymes, FabG, FabI and FabZ as potential targets in liver stage malaria parasites. In this case, finely ground dried plant material was extracted with methanol (MeOH) to yield the crude methanol extracts (CR-MeOH) which, were further partitioned to provide a coarse separation of the crude extracts according to polarity. The three subextracts obtained included n-hexane, chloroform (CHCl3) and aqueous methanol (aq-MeOH). The obtained extracts and subextracts were screened for biological activities such as: antifungal and antibacterial activities using the broth dilution and agar disc diffusion assays, antitubercular activity using the MTT assay, antischistosomal activity using the microscopic in vitro assay. In addition, antiprotozoal activitites which included antileshmanial activity using an assay against amastigotes of L. donovani strain MHOM/ET/67/L82, trypanocidal activity against T. cruzi and T. brucei rhodesiense STIB 900 strain, and antiplasmodial activity by modified [3H]-hypoxanthine incorporation assay, using the chloroquine/pyrimethamine resistant K1 strain were performed. Cytotoxity activity was also performed using rat skeletal myoblasts L6-cells. The chemical profiling was done by TLC, NMR and RP-HPLC. Meanwhile the chemical compound isolation for F. sansibarica was attempted by different chromatographic techniques and characterization by spectroscopic methods. The phytochemical profiling revealed the presence of closely related polyphenolic compounds to which some of the biological activities were attributed to. For instance, the antibacterial and the FAS-II enzyme inhibition activities were mostly retained in the aq-MeOH subextracts, which were composed of very polar metabolites including flavonoids. Antiplasmodial activity was observed mostly in the less polar metabolites which were retained in the hexane and CHCl3 subextracts of the stem barks. This pattern was similar with antitrypanosomal and antileishmanial activities, though with lesser sensitivity. The same subextracts including those of the root barks showed the most activity against M. tuberculosis with MIC values of 256 and 128 μg/ml, and against Schistosoma, for both larval and adult worms. The extracts did not exert any antifungal activity by the agar disc diffusion method we used. Detailed phytochemical investigation of the leaves of F. sansibarica was performed, and led to the isolation of two compounds; epicatechin and apigenin-6-C-glucoside from the chloroform and aq. MeOH subextracts respectively. The predominant constituent of the CR-MeOH extract of F. sansibarica was identified as having a molecular weight of 432 g/mol by LC-MS analysis which could be set as an identification chemical marker for F. sansibarica. The results highlight the potential that Ficus species could have as a valuable source for potent compounds which can be identified as scaffolds for the development of novel liver stage antimalarial drugs. Our results support previous research on the antimicrobial activity of Ficus species and they also provide an in vitro scientific basis supporting the use of Ficus species in traditional herbal preparations against some bacterial and parasitic infections.

Cancer is a growing health problem around the world and according to estimates from the International Agency for Research on Cancer (IARC), 14.1 million new cancer cases and 8.2 million cancer deaths worldwide have been reported in 2012 (Ferlay et al., 2015). By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths simply due to the growth and aging of the population. Indicaxanthin ((2S)-2,3-dihydro-4-[2-[(2S)-2a-carboxypyrrolidin-1- yl]ethenyl]pyridine-2a,6-dicarboxylic acid), a betalain pigment from cactus pear fruit, has been the object of sound experimental work over the latest years. As many phytochemicals, indicaxanthin is a redox-active compound and has been shown to act as antioxidant in a number of in vitro studies (Allegra et al., 2005; Turco Liveri et al., 2009). Interestingly, thanks to its charged portions, ionizable groups and lipophilic moieties, it is amphiphilic at physiological pH (Turco Liveri et al., 2009) and has been demonstrated to interact with cell membranes (Tesoriere et al., 2006; Turco Liveri et al., 2009). This feature is critical to allow bioactive compounds to interact with cells and to initiate signaling events. In this regard, indicaxanthin has been showed to modulate specific redox-dependent signaling pathways involved in macrophage activation and apoptosis, epithelial and endothelial dysfunction in vitro (Allegra et al., 2014; Tesoriere et al., 2015). Remarkably, and in contrast with the majority of dietary phytochemicals, indicaxanthin is highly bioavailable (Tesoriere et al., 2004). The molecule has been shown to cross unaltered intestinal epithelial cell in vitro being absorbed through paracellular junctions (Tesoriere et al., 2013). In line with that, indicaxanthin has been found in human plasma at a 7 μM peak concentration 3 h after the ingestion of four cactus pear fruits containing 28 mg of the pigment (Tesoriere et al., 2004). Moreover, its amphiphilicity allows it to cross the blood-brain-barrier and localize within the CNS (Allegra et al., 2015). Finally, thanks to its bioavailability and redox-modulating properties, indicaxanthin exerts significant pharmacological effects in vivo. Indeed, oral administration of the PhC at nutritionally-relevant doses (2 μmol/kg) generates, in rats, a plasma peak concentration of 0.2 μM able to exert strong anti-inflammatory effects in an in vivo model of acute inflammation (Allegra et al., 2014). The causative link between inflammation and melanoma has accurately been explored in the recent years (Bald et al., 2014; Meyer et al., 2011; Reinhardt et al., 2017; Soudja et al., 2010). Experiments in mice revealed that UV-induced skin inflammatory responses can cause the reactive proliferation and migration of melanocytes (Zaidi et al., 2011). More recently, it has been shown that reciprocal interactions between melanoma and immune cells in a pro-inflammatory microenvironment provide a source of phenotypic heterogeneity that drives therapy resistance and metastasis (Bald et al., 2014; Landsberg et al., 2012). In keeping this perspective, we decided to investigate the effects of Indicaxanthin against human melanoma cell proliferation and in a model of cutaneous melanoma. We here demonstrate that indicaxanthin induces apoptosis of human melanoma cells through the inhibition of the NF-κB pathway and the downstream anti-apoptotic signaling events in vitro and these effects were paralleled in vivo in a murine model of melanoma. Finally, preliminary data on six healthy volunteers, showed that indicaxanthin is able to modulate TNF- and Il-6 production in a whole blood ex vivo model. Furthermore, the phytochemical induces an increase in the phagocytosis of 5 different Gram-negative pathogens on whole blood assay, without exerting antimicrobial effects on them. Interestingly, preliminary data on 4 of the 6 volunteers showed that the observed effects maybe attributed to the modulation of LTB4 levels, strictly correlated to the activation of immune cells.

Syzygium cordatum is a medicinal plant indigenous to South Africa and Mozambique, commonly used to treat stomach aches, diabetes, respiratory problems and tuberculosis. In spite of the folklore use, adequate scientific data to credit its widespread traditional use is lacking. The objectives of this study were: to evaluate and validate scientifically the successful therapeutic claims by traditional medicine practitioners that Syzygium cordatum is effective in treating diarrhoea and diabetes
to determine the effects of the plant extract on gastrointestinal transit of a charcoal meal in mice
to determine the effects on castor oilinduced intestinal fluid accumulation
to determine the safety profile of the plant by carrying out acute toxicology study and to carry out preliminary screening of the active compounds present in the plant using standard phytochemical analytical procedures. The aqueous leaf extract of Syzygium cordatum (3.125 -50mg/kg, p.o) significantly reduced the faecal output caused by castor oil (0.7ml). All the doses used, reduced faecal output from 100% produced by castor oil to between 40 and 61%. S.cordatum (6.25 &ndash
50mg/kg, p.o) significantly and in a dose dependent manner, delayed the onset of castor oil-induced diarrhoea.

Despite decades of intense research, malaria remains a deadly worldwide disease. Resistance of Plasmodium falciparum to chemical treatment still remains important. Efforts are now being directed towards the discovery and development of new chemically diverse anti-malarial agents. In the course of the search for new antimalarial compounds, a study of plants traditionally used against malaria by the people inhabiting the forests located near Bhubaneswar, Orissa, India was made, which permitted the identification of 34 plants currently used. Among these, 27 plants were selected for testing for antiplasmodial activity aimed at identifying the most effective plants for further research. Also, their activities were compared with 27 randomly collected plant species in order to asess the value of an ethno-medical approach.

The phytochemical and biological study of the species S. lentiscifolius, S. terebinthifolius, S. molle and S. polygamus known as ―aroeiras‖ (Anacardeaceae) distributed in Rio Grande do Sul, was carried out to establish the chemical composition of volatile oils obtained by hydrodistillation of aerial parts of leaves, flowers and fruits collected in different seasons. In addition, leaves and fruits were analyzed and compared to determine seasonal variations in the oils constitution. The oils chemical composition was determined by Gas Chromatography coupled with a Mass Spectrometry (GC-MS). The yields of the studied species oils essential aerial parts ranged from 0.14-2-13%, depending on the stage of development and part collected. One hundred and thirty-five compounds were identified from the oils essential, representing approximately 90% of the oils from leaves, flowers and fruits collected between spring and summer in 2005 to 2011. The volatile oils of the analyzed species showed a constant qualitative standard in the compounds: α-pinene, β-pinene, δ-2- carene, limonene for majority monoterpenes; germacrene-D, bicyclogermacrene, δ-cadinene, spatulenol, caryophyllene oxide and -cadinol for majority sesquiterpenes in the leaves oil; α- pinene, mircene epi-cubenol, cubenol and spatulenol for majority sesquiterpenes in the flowers oil; α-pinene, β-pinene mircene, epi-cubenol, cubenol, spatulenol, caryophyllene oxide and -cadinol for majority sesquiterpenes in the oil fruits. The quantitative variations among the different constituents in oils of different species were quite relevant. Additionally, considering the therapeutic potential and the compounds described, the phytochemical study and antioxidant and antimicrobial activity were evaluated in the oils, crude extracts, aqueousextracts, isolated and derivative compounds. The aqueous extracts obtained by 9 hydrodestillation, in part were dried by lyophilization and in part were subjected to a liquidliquid partition using hexane solvent, ethyl acetate and n-butanol. Besides these, the crude extract was prepared from the leaves of S. lentiscifolius. The chromatographic fraction of crude extract and ethyl acetate led to the isolation and identification of nonadecanol (1) and moronic acid (2). From moronic acid (2) by employing NaBH4 reduction reaction were obtained the derivatives morolic acid (3) and 3-epi-morolic acid (4). The analysis of S. lentiscifolius ethyl acetate aqueous extract led to the isolation of the compound FLA-1 (5) and galic acid (6), from this was obtained the derivative methyl gallate (7). The S. molle nbutanol aqueous extract led to the isolation of the flavonoid quercetin (8) and rutin (9). The substances were identified by physical data and spectroscopic techniques (1H NMR, 13C, COSY, HMQC, HMBC), mass spectrometry and X-rays. The aqueous extracts analysis by High-Performance Liquid Chromatography (HPLC) led to the characterization of galic acid (6), quercetin (8) and rutin (9) comparing to the existing laboratory standards. All extracts present galic acid (6), except to S. molle. The oils antimicrobial activity of the species S. lentiscifolius, S. terebinthifolius, S. molle and S. polygamus were evaluated by microdilution method using a collection of pathogenic microorganisms composed of bacteria and fungi. The results showed that the obtained oils from different plants were active using the most of the microorganisms tested. Best results were found in the fruits oils of S. terebinthifolius. The extracts and fractions of S. lentiscifolius, S. terebinthifolius and S. molle have also been tested as antibacterial and antifungal activity. Best results were found for the extract of S. lentiscifolius with a Minimum Inhibitory Concentration (MIC) from 31.2 to 12.5 mg/mL. In addition, isolated compounds extracts were evaluated as antioxidant activity using 2,2-diphenyl-picrylhydrazyl (DPPH). All extracts and compounds tested showed inhibition results for free radical DPPH.
O estudo fitoquímico e biológico das espécies S. lentiscifolius, S. terebinthifolius, S. molle e S. polygamus conhecidas como ―aroeiras‖ (Anacardiaceae), distribuídas no Rio Grande do Sul, foi realizado visando o estabelecimento da composição química de óleos voláteis obtidos por hidrodestilação das partes aéreas (folhas, flores e frutos) coletadas em diferentes estações do ano. Além disso, folhas e frutos foram analisados e comparados para determinar as variações sazonais na constituição dos óleos. A composição química dos óleos foi determinada através de Cromatografia Gasosa acoplada à Espectrometria de Massas (CGEM). Os rendimentos dos óleos essenciais das partes aéreas das espécies estudadas variaram de 0,14-2,13%, dependendo do estágio de desenvolvimento e da parte coletada. Aproximadamente cento e trinta e cinco compostos foram identificados a partir dos óleos essenciais, representando aproximadamente 90% dos óleos das folhas, flores e frutos, coletados entre a primavera e o verão no período 2005 a 2011. Os óleos volatéis das espécies analisadas apresentaram um padrão qualitativo constante dos compostos: α-pineno, β-pineno, δ-2-careno, limoneno como monoterpênicos majoritários; germacreno-D, biciclogermacreno, δ-cadineno, espatulenol, óxido de cariofileno -cadinol com os esquiterpenos majoritários no óleo das folhas; α-pineno, mirceno epi-cubenol, cubenol, espatulenol como sesquiterpenos majoritários no óleo das flores; α-pineno, β-pineno mirceno como monoterpenos e epicubenol, cubenol, espatulenol, óxido de cariofileno e -cadinol como sesquiterpenos majoritários no óleo dos frutos. As variações quantitativas entre os diferentes constituintes 7 nos óleos das diferentes espécies foram marcantes. Adicionalmente, considerando-se o potencial terapêutico e os compostos descritos para o gênero, foi realizado o estudo fitoquímico e avaliadas as atividades antimicrobianas e antioxidantes dos óleos, dos extratos brutos, dos aquosos, dos compostos isolados e dos derivados. Os extratos aquosos obtidos do processo de hidrodestilação, parte foi seco por liofilização parte foi submetido à partição líquido-líquido com os solventes hexano, acetato de etila e n-butanol. Além desses, foi preparado o extrato bruto das folhas de S. lentiscifolius. O fracionamento cromatográfico do extrato bruto de acetato de etila de S. lentiscifolius levou ao isolamento do álcool graxo (119), ácido morônico (120). Também foram obtidos dois derivados do ácido morônico (120), por reação de redução empregando NaBH4, os quais foram caracterizados como ácido morólico (121) eácido 3-epi- morólico (122). A análise do extrato aquoso de acetato de etila de S. lentiscifolius levou ao isolamento do composto FLA-1 (115) e do ácido gálico (116), do qual foi obtido o derivado galato de metila (118). Do extrato aquoso n-butanólico de S. molle foi isolado o flavonóide quercetina (90) e rutina (117). As substâncias foram identificadas por dados físicos e técnicas espectroscópicas (RMN de 1H, 13C, COSY, HMQC e HMBC) espectrometria de massas e raios-X. A análise dos extratos aquosos por Cromatografia Líquida de Alta Eficiência (CLAE) levou à caracterização do ácido gálico (116), quercetina (90) e rutina (117) por comparação com padrões existentes no laboratório. Todos os extratos apresentaramo ácido gálico (116), exceto nos extratos de S. molle. A atividade antimicrobiana dos óleos das espécies S. lentiscifolius, S.terebinthifolius, S.molle e S. polygamus foram avaliadas pelo método de microdiluição frente uma coleção de microorganismos patogênicos, compostos por bactérias e fungos. Os resultados demonstraram que os óleos obtidos das diferentes plantas foram ativos frente à maioria dos microorganismos testados. Os melhores resultados foram encontrados para os óleos dos frutos de S. terebinthifolius. Os extratos brutos e frações de S. lentiscifolius, S. terebinthifolius e S. molle foram testados também quanto à sua atividade antibacteriana e antifúngica. Os melhores resultados foram encontrados para o extrato de S. lentiscifolius com uma Concentração Inibitória Mínima (CIM) 31,2- 12,5μg/mL. Além disso, os extratos e os compostos isolados foram avaliados quanto à atividade antioxidante, utilizando-se 2,2-difenil-picrilhidrazil (DPPH). Todos os extratos e compostos testados apresentaram resultados de inibição ao radical livre DPPH.

Background. As a result of the AIDS pandemic, many people areimmuno compromised andopportunistic fungal infections (OFIs) such as candidiasis are common. Despite the widespread use of medicinal plants in South Africa, there is a dearth of knowledge regarding the use of such plants in the management of these infections. This study evaluates three South African medicinal plants (Arctotis arctotoides, Pittosporum viridiflorum, and Gasteria bicolor) traditionally used in the treatment of OFIs in HIV/AIDS patients, in the Eastern Cape Province, South Africa. Materials and methods. A six-stage process of documentation, evaluation and analysis of results was conducted: (1) Selection of medicinal plants most frequently used in the treatment of OFIs through ethnomedical studies and the survey of specialised literature; (2) Collection and preparation of the extract of each plant; (3) Antifungal evaluation of the crude plant extracts. (4) Phytochemical and antioxidant evaluation of the active crude plant extracts; (5) Cytotoxicity evaluation of the bioactive extracts using the Chang liver cell line, and (6) Statistical analysis of the results. Ethnobotanical information was obtained through interviews with traditional healers and AIDS patients with the aid of semi-structured questionnaires, direct observations and by reviewing studies reported in the literature. Following the approval from the University of Fort Hare‘s Ethics Committee, 101 HIV/AIDS patients were recruited through convenience sampling into an anonymous cross-sectional questionnaire study. The agar diffusion and micro-dilution methods were used to determine the antifungal activities of the hexane, acetone and aqueous extracts of A. arctotoides, G. bicolor and P. viridiflorum against 10 opportunistic fungi.

The methanolic crude extract of aerial parts of the plant Scrophularia nodosa was shown to have potent DPPH radical scavenging activity (IC50 = 48.75 ± 7.00 μg/ml). Activity-guided fractionation resulted in the isolation of three principal antioxidant compounds; acteoside, angoroside C and angoroside A. Acteoside (yield = 1.21%, IC50 = 15.2 μM) appeared to be the most abundant and most antioxidant-active. The potent antioxidant activity is in support of the traditional use of the plant for wound healing and anti-inflammatory conditions. The methanolic extract of aerial parts of Tanacetum vulgare has potent DPPH radical scavenging activity (IC50 = 37.00 ± 1.20 μg/ml). Activity-guided fractionation on the methanolic extract of T. vulgare resulted in the isolation of 3,5-di-caffeoylquinic acid (3,5-DCQA), axillarin and luteolin. 3,5-DCQA appeared to be the most abundant and most antioxidant-active compound (yield = 7.28%, IC50 = 9.7 μM). The potent antioxidant activity is in support of the traditional use of the herb for fever, rheumatic conditions and anti-inflammatory conditions. The methanolic crude extract of Cassia auriculata and its fractions were shown to have potent scavenging activity on DPPH, hydroxyl and hydroperoxide radicals, moderate superoxide radical scavenging activity and potent ion(III) reducing power. The activity-directed studies resulted in the isolation of kaempferol-3-0-β-D-rutinoside, kaempferol, luteolin, quercetin and unknown antioxidant inactive compound. The previously reported pharmacological aspects of the above flavonoids and flavonoid glycosides (anti-carcinogenic, anti-inflammatory, hyperglycaemic, antidiabetic) along with the shown antioxidant behaviour explain the traditional medicinal values of the plant. Cassia alata L crude extract and its fractions showed potent radical scavenging activity against formation of lipid peroxide radicals. The activity directed isolations resulted in the isolation of kaempferol along with p-hydroxybenzoic acid. These may contribute towards the traditional medicinal values of the plant as an antidiabetic, anti-microbial and for skin diseases.

The aim of this research was to investigate the phytochemistry of two species Sanicula europaea and Teucrium davaeanum which are traditionally used in treatment of wounds. Four compounds were isolated from the 80% methanolic extract of S. europaea; bis-(2-ethylhexyl) phthalate (1), palmitic acid (2), rosmarinic acid (3), saniculoside N (4). Compounds 1 and 2 were isolated for the first time from this species. The structure elucidation of the isolated compounds was on the basis of 1D, 2D NMR spectroscopy and mass spectrometry measurements. Two compounds were isolated from the crude glycosides extract of T.davaeanum; 6 is a phenylethanoid glycoside and 8 is an iridoid glycoside, from the data available these may be new compounds for which the names davaeanuside A and davaeanuside B are proposed respectively." The total polyphenol content of S. europaea L, T. davaeanum leaves-flowers and T. davaeanum stem were found to be 5.0, 1.20 and 0.65 mg per 100 mg dried plant material respectively. A study of the antioxidant activity of the 50 % ethanol extracts of S. europaea and T. davaeanum showed that on a mg/mg basis S. europaea and T. davaeanum have approximately 5%, 8 % antioxidant capacity of Trolox respectively. A study of the cytotoxic activity of davaeanuside A (6), iridoid glycoside (7), davaeanuside B (8) and saponin compound (10) isolated from the crude glycosides extract of T. davaeanum revealed that saponin compound (10) inhibited the growth of Hela cells by 50 % at 50 μg/ml, P< 0.001, but the other compounds did not show activities against the tested cell lines at 100 μg/ml. The results of this work provide some basis for the traditional use of these species in the treatment of wounds.

Dissertation to obtain a Master’s Degree in Chemical and Biochemical Engineering
Using natural ingredients to combat obesity has emerged as a promising alternative to conventional therapies which present many side effects. Satiety induction by the ingestion of certain natural compounds has been proven to be an interesting strategy. In this context, the present study had the following objectives: - Isolation of rucola, watercress and spinach, plum and tomato waste and cladodes of Opuntia ficus-indica extracts, rich in compounds with the potential to induce a prolonged feeling of satiety - Assess the satiety inducing potential of isolated extracts using two enzymatic methods: - Inhibition of pancreatic lipase, an enzyme responsible for the conversion of complex lipids into simple and easily absorbable fat, which when inhibited is associated with a prolongation of satiety and a reduction in fat absorption, being the therapeutic target of Orlistat (the most common anti-obesity drug) - Inhibition of trypsin’s proteolytic activity, which is associated with a prolongation of satiety as it promotes the secretion of cholecystokinin (CCK), a polypeptide which regulates pancreatic enzyme release, which not only promotes satiety but also slows gastric emptying. In this work a method for extracting thylakoids, which are potent inhibitors of pancreatic lipase,was optimized. In addition to spinach, this method was applied for the first time to rucola and watercress. The extracts from these three matrixes exhibited lipase inhibitory activity, with spinach being the most efficient one. Hydroalcoholic extracts were prepared from plum residue, rich in polyphenols and saponins, which also showed efficient inhibitory capacity of pancreatic lipase. A protocol was optimized for the extraction of proteins which applied to the plum residue,tomatoes and cladodes of Opuntia ficus-indica. Only the opuntia and tomato extracts presented effective inhibition of trypsin’s proteolytic activity.

Dissertation to obtain master degree in Biotechnology
Recently there is a growing interest in cancer treatment through the use of natural compounds. In particular, phenolic compounds and monoterpenes found in fruits and vegetables are very attractive in the prevention and chemotherapy of various types of cancer. However, many promising compounds previously tested in in vitro cell models fail to demonstrate activity when evaluated in vivo. Therefore, there is an emerging need to develop more robust and reliable cellular models for pre-clinical evaluation of new chemotherapeutic agents. The main goal of this thesis was the evaluation of the chemotherapeutic potential of natural extracts, rich in bioactive compounds, using 3D models of colon cancer. For this purpose, a 3D model of human colorectal cancer cell line HT29 was developed. By culturing HT29 cells in a stirred culture system, it was possible to obtain 3D cellular spheroids with different size diameter during culture time. It was verified phenotypic changes within the spheroid along culture, such as formation of apoptotic core and altered expression of stem and epithelial markers in different spheroid areas, which are typical features of tumor progression. After an initial screening of the antiproliferative potential of 14 natural extracts performed in a 2D model of HT29 cells, the most promising samples were selected for further analysis in the 3D model. Cherry and orange extracts showed potential anticancer effect in HT29 aggregates through the inhibition of cell proliferation, induction of apoptosis and cell cycle arrest. A decrease on the bioactive effect was verified with the increase of aggregate diameter, probably due to limited diffusion. The anticancer activity was correlated with the phytochemical composition of natural extracts. For cherry extract, perillyl alcohol was the main bioactive compound identified whereas for orange extract, compounds like nobiletin, tangeretin and sinensetin were highlighted. Results of this thesis demonstrated that natural extracts of cherry and orange contain bioactive molecules with promising application on the development of new therapies for colon cancer treatment. The use of 3D cell models is a valuable tool for the study and evaluation of the effect of new chemotherapeutic compounds.

Magister Pharmaceuticae - MPharm
Fast dispersible tablets (FDTs) are solid single-unit dosage forms that are placed in the mouth and allowed to disperse or dissolve in the saliva without the need of water. The basic approach to formulating FDTs consists of adding a superdisintegrant to a tablet formulation. These tablets offer both the advantages of conventional tablets and liquid dosage forms along with distinctive properties which include accurate dosing, ease of administration, quick onset of action, enhanced bioavailability, and increased patient adherence. FDTs have been found to be effective in remedying therapeutic in-adherence caused by dysphagia (swallowing difficulties) particularly in paediatric and geriatric subjects. There is a strong correlation between therapeutic success and patient adherence especially with HIV/AIDS treatment regimens, consequently the dosage form should be patient friendly and devoid of unappealing characteristics. This study aimed at developing a cost effective fast dispersible tablet of lamivudine using alternative excipients and conventional techniques. Only conventional tablets and oral liquid dosage forms of lamivudine are available on the South African market. Two natural polymers reported to have superdisintegrating properties were selected to serve as multipurpose excipients in this study. The polymers were identified, characterised and compared using thermal, spectroscopic and micromeritic analytical tools. The polymer that displayed the best characteristics in terms of micromeritic, tableting and disintegrating properties was retained and used for the optimum formulation. The optimum formulation was composed of 150 mg of lamivudine, 23% w/w unripe banana powder and 2% w/w magnesium stearate. FDTs of lamivudine were obtained using the compression technique with and without wet granulation. The tablets were assessed as per the United States Pharmacopoeia (USP) guidelines and other evaluation procedures pertaining to FDTs. The wet granulated tablets were found to be less friable and thus more resilient than the directly compressed tablets. In-vitro disintegration of the wet granulated tablets occurred within 50±3 sec in deionised water (pH 7) and 35±2 sec in a phosphate buffer solution (pH 6.8). Consequently, the innovative tablets fulfilled the core requirement of FDTs i.e. rapid disintegration. Drug release studies were carried out by analysing dissolution aliquots of the innovative tablets using a validated High Performance Liquid Chromatography (HPLC) method, and comparing them to Aspen Lamivudine®, a conventional tablet of lamivudine presently on the South African market. Complete dissolution in deionised water (pH 7) was attained within 10 minutes and 30 minutes for the innovative tablets and Aspen Lamivudine® respectively.

>Magister Scientiae - MSc
Nanotechnology has emerged as a promising field in the quest to address health conditions. Green nanotechnology is a fairly new branch of nanotechnology, which aims to produce and utilize nanomaterials in a way that is safe for living organisms and their environment. Plant extracts are increasingly used in the green synthesis of gold nanoparticles (AuNPs), which involves the reduction of sodium tetrachloroaurate (III) dehydrate by phytochemicals present in the plant extract. It is probable that the green synthesised AuNPs are more biocompatible than chemically synthesised AuNPs as biomolecules of plant origin are involved in the synthesis process. Therefore, this study aimed to explore various water extracts from indigenous South African plants, which included Perlagonium capitatum, Otholobium bracteolatum, Gerbera linnae, Morrella quercifolia, Searsia lucida, Phylica bubescens, Euclea racemosa, Tetragonia fruticosa, and Searsia glauca for their potential to synthesize AuNPs and to investigate their toxicity towards several microorganisms known to cause skin infections. These organisms play a significant role in delaying the healing of wounds. The antimicrobial properties of nanoparticles are increasing exploited in the production of wound treatments.

Negli ultimi decenni, gli antiossidanti alimentari hanno ricevuto crescente attenzione, soprattutto in ambito biologico, medico e nutrizionale, in relazione alla loro capacità di contrastare lo stress ossidativo implicato nella patogenesi di diverse patologie cronico-degenerative. Numerosi sono i metodi attualmente disponibili in letteratura per valutare la capacità antiossidante (CA) in vitro degli alimenti. In tutti i casi si tratta di metodi semplici, rapidi e poco costosi che consentono di misurare la CA sia su composti puri che su matrici complesse, come alimenti e campioni biologici. Tuttavia, i metodi per valutazione della CA in vitro, spesso evidenziano numerose limitazioni sia tecniche che concettuali. La principale limitazione tecnica è legata alla natura chimica sulla quale si fondano che rende difficile il confronto fra i valori di CA ottenuti con le diverse metodiche analitiche. La principale limitazione concettuale, invece, è legata alla difficoltà di poter attribuire alle misure di CA un reale significato fisiologico. Le misure di CA degli alimenti, infatti, non consentono di valutare quella che è la reale biodisponibilità dei composti antiossidanti assunti con la dieta. Esse, infatti non tengono conto né della stabilità in vivo, né della ritenzione nei tessuti né della reattività in situ dei composti antiossidanti. Pertanto, il target di questa ricerca è stato quello di mettere a punto un nuovo approccio che, mediante l’utilizzo di metodologie innovative, consenta di ottenere informazioni sul potenziale valore salutistico degli alimenti che siano realmente indicative della risposta che essi procurano in vivo. Lo studio si è articolato su tre livelli sequenziali di indagine. Il primo livello ha riguardato l’analisi in vitro dell’alimento effettuata mediante misure di CA sui diversi estratti ottenuti dall’alimento. A questo livello, è stato in primo luogo condotto un avanzamento del metodo "QUENCHERABTS" (QUICK, Easy, New, CHEap and Reproducible); il metodo si basa sulla reazione diretta del radicale cationico ABTS·+ con particelle fini di alimenti solidi senza che venga effettuata una preliminare estrazione degli antiossidanti. Mediante una nuova procedura di calcolo, l'applicabilità del metodo QUENCHERABTS è stata estesa allo studio di particelle più grandi (fino a 0,5 mm). Questo può essere interessante nel caso di alcuni prodotti dell’industria molitoria. Oltre all’avanzamento del metodo QUENCHERABTS, la ricerca relativa al primo livello di indagine ha riguardato lo sviluppo del nuovo metodo Lipoxygenase (LOX) -Fluorescein (FL). Il nuovo metodo LOX-FL si basa su un approccio più vicino alle condizioni fisiologiche rispetto ad altri metodi ampiamente utilizzati in letteratura. Esso utilizza l’isoforma 1 della lipossigenasi di soia per produrre specie radicaliche fisiologiche e la fluoresceina come probe per rilevare queste specie in fluorescenza. Il metodo LOX-FL è stato applicato sia alla misura della CA degli estratti ottenuti dall’integratore alimentare ricco in composti bioattivi Lisosan G sia alla misura della CA del siero (di sette soggetti) durante i 240 minuti successivi all’assunzione di 20 g dell’integratore. Per quanto riguarda l’analisi in vitro, il metodo LOX-FL è stato in grado di discriminare in termini di CA tra i quattro diversi estratti del Lisosan G, in modo simile ai metodi ORAC e TEAC, utilizzati come confronto. Al contrario, solo il metodo LOX-FL è stato in grado di evidenziare un aumento generale della CA del siero fino al 40% 30 minuti dopo l'assunzione di Lisosan G. Per la duplice proprietà di poter essere applicato sia a misure in vitro che ex vivo di CA, il metodo LOX-FL è stato utilizzato anche nel secondo livello di indagine, quello relativo alla valutazione dello stato antiossidante del sangue umano dopo l'assunzione dell’alimento. Data la semplicità nelle misure di CA e il facile accesso ai campioni di sangue umano, le misure di CA sul siero si sono diffuse tantissimo negli ultimi anni e hanno generato un elevatissimo numero di pubblicazioni. Purtroppo, i risultati sono abbastanza contrastanti; in alcuni casi, infatti, si è osservato solo un lieve aumento o addirittura un decremento della CA. Ciò suggerisce l'inadeguatezza delle misurazioni della sola CA del sangue e la necessità di considerare anche i cambiamenti dello stato ossidato del siero. Alla luce di ciò, è stato proposto l'uso di un nuovo parametro, definito “Antioxidant/Oxidant Balance” (AOB), che rappresenta il rapporto tra la CA e lo stato ossidato del siero, valutato come "Peroxide Level" (PxL). L'approccio basato sull’uso dell’AOB è stato applicato per valutare gli effetti sullo stato antiossidante del siero nelle prime quattro ore successive all'assunzione di due putative paste funzionali, la pasta Bran Oleoresin (BO) e la pasta Bran Water (BW), arricchite rispettivamente con antiossidanti lipofili e idrofili/ fenolici estratti dalla crusca di frumento duro. Analogamente al Lisosan G altamente bioattivo, l'assunzione di pasta BO ha procurato un significativo incremento dell’AOB del siero, fino al 70% quando calcolato come LOX-FL/PxL. Al contrario, la pasta BW ha un effetto ossidante sul siero, analogamente alla pasta controllo (Reference pasta) e al glucosio, usati come confronto. Complessivamente, questi risultati indicano che il parametro AOB appare uno strumento eccellente per evidenziare l’effetto del consumo di alimenti ricchi di antiossidanti sul sangue; tale effetto, inoltre, non può essere assolutamente previsto dalle misure in vitro della matrice, ne dall'analisi ex vivo della sola CA del siero. Il terzo livello di indagine è quello relativo alla valutazione degli effetti esercitati dai composti bioattivi a livello cellulare e subcellulare. In particolare, gli effetti biologici di alcuni phytochemicals alimentari sono stati valutati sull'attività degli enzimi: i) Gliossalasi I (GloI), implicato nella difesa cellulare contro gli effetti dannosi dello stress da carbonilazione ii) Sirtuine, una famiglia di deacetilasi NAD+-dipendenti coinvolte nella regolazione di importanti processi cellulari quali trascrizione genica, apoptosi, metabolismo e divisione cellulare. Per quanto riguarda l’enzima Glo I, sono stati effettuati due studi. Nel primo studio è stato valutato l’effetto del sulforafano (SR), un isotiocianato abbondante nei broccoli, sull'espressione e l'attività di GloI e sui livelli di glutatione ridotto (GSH) in cellule mononucleate di sangue periferico (Peripheral Blood Mononuclear Cells, PBMCs). L'incubazione per 24/48 ore dei PMBCs con 2,5 μM SR (che simula la quantità assunta con una porzione di broccoli) non ha indotto un aumento sostanziale dell'attività e dell'espressione di GloI, mentre ha causato una riduzione fino al 73% dei livelli di GSH rispetto alle cellule di controllo. Ciò suggerisce la formazione di un addotto GSH-SR che, riducendo significativamente la reale concentrazione di SR all'interno delle cellule durante l'incubazione, spiegherebbe la mancanza di un sostanziale effetto sull'espressione di GloI osservata in questo studio. Nel secondo studio è stato valutato l'effetto di alcuni phytochemicals sull'attività di GloI nella frazione mitocondriale altamente purificata isolata da plantule di frumento duro (Wheat Mitochondria, WM). È stata studiata la GloI mitocondriale poiché i mitocondri rappresentano uno dei principali bersagli della carbonilazione oltre che dello stress ossidativo. In questi organelli è stata dapprima misurata un'elevata attività di GloI; lo studio della cinetica di reazione ha consentito di osservare una dipendenza iperbolica dall’attività enzimatica dalla concentrazione di substrato e di misurare Km e Vmax che sono pari a 0,27 mM e 0,133 UE / mg di proteine, rispettivamente. Per quanto riguarda lo studio della modulazione, tra i phytochemicals testati soltanto curcumina e quercetina sono risultate in grado di inibire l'attività di GloI in WM. Uno studio dettagliato dell’inibizione ha consentito, inoltre, di verificare la natura competitiva dell’inibizione e di calcolare valori di Ki pari a 20 μM e 55 μM per la curcumina e la quercetina, rispettivamente. Infine, è stato condotto uno studio finalizzato a sviluppare un nuovo approccio sperimentale che consenta di misurare in modo affidabile l'attività sirtuina in estratti cellulari e/o in organelli subcellulari. Questo approccio consiste nell’uso combinato di due diversi sistemi di dosaggio basati su un razionale molto diverso, il dosaggio SIRT-Glo™ che lavora in luminescenza e un metodo basato sulla tecnologia HTRF, e confronto con l’enzima ricombinante umano SIRT1 (human SIRT1, hSIRT1). Usando l'approccio appena descritto, è stata determinata un'attività specifica di sirtuine in WM molto alta, pari a 268 ± 10 mU ∙ mg-1 di proteine, con saggio HTRF®, e di 166 ± 12 ng di hSIRT1 eq. ∙ mg- 1 di proteine, con il metodo bioluminescente. Per quanto riguarda la modulazione dell’attività di sirtuina, utilizzando il saggio HTRF®, non è stato osservato nessun significativo effetto da parte di resveratrolo e quercetina sull’attività di sirtuina in WM. Globalmente, i risultati riportati in questa tesi dimostrano l'importanza cruciale dell'approccio metodologico nella valutazione delle potenziali proprietà salutistiche dei phytochemicals alimentari; ciò, infatti, è assolutamente necessario se si vuole evitare un’interpretazione errata e fuorviante dei dati relativi alle proprietà antiossidanti degli alimenti.
During the last few decades, dietary antioxidants have received increasing attention, especially within biological, medical and nutritional fields, owing to their putative protective roles against the deleterious oxidative-induced reactions implicated in the pathogenesis of several human diseases. A considerable number of analytical assays has been developed claiming to ensure a fast, simple, convenient, and reliable in vitro determination of total antioxidant capacity (AC) of pure compounds or complex matrices, such as foods and biological samples. Nevertheless, methods for in vitro AC measurement show many technical and conceptual limitations. Moreover, in vitro AC measurements of foods alone have not been demonstrated to be relevant for the biological effects of specific bioactive compounds since they do not provide information about bioavailability of food antioxidants, as well as their in vivo stability, retention by tissues and in situ reactivity. Therefore, the target of this research was of developing innovative methodologies/approaches able to provide information as much as possible reflecting the in vivo response. The model of study adopted to carry out the research consists of three sequential levels of study. The first level regards AC measurement of plant foodstuffs. At this level, a first methodological advancement of “QUENCHERABTS” (QUick, Easy, New, CHEap and Reproducible) method has been attempted. It is based on the direct reaction of ABTS•+ reagent with fine solid food particles without extraction of antioxidants. In particular, by adopting a new slope calculation procedure, the applicability of QUENCHERABTS method was extended to the study of larger particles (up to 0.5 mm). This may be of interest in the case of some cereal milling products. In addition to the QUENCHERABTS calculation upgrade, research relative to the first level of investigation has regarded the development of the novel Lipoxygenase (LOX)-Fluorescein (FL) method. The new LOX-FL method is based on the LOX-1 isoenzyme reaction to generate physiological reactive species and provides an AC assessment more reliable from a physiological point of view with respect to other widely used assays. This method was applied on both extracts from the antioxidant-rich dietary cereal supplement Lisosan G and serum (seven subjects) during 240 min after intake of 20 g of supplement. Interestingly, LOX-FL method discriminated in vitro AC of four different Lisosan G extracts similarly to ORAC and TEAC methods, used for comparison. Contrarily, only LOX-FL method was able to highlight a general increase of serum AC (up to 40% after 30 min) after Lisosan G intake, thus confirming its physiological effectiveness by ex vivo serum assay. For the property to be applied to both in vitro and ex vivo AC measurements, LOX-FL assay was used also in the second level of investigation, concerning the evaluation of human blood antioxidant status after food intake. Unfortunately, contrasting results have been obtained from blood AC assessment after consumption of antioxidant-enriched foods, suggesting the unsuitability of blood AC measurements alone and the need to consider also changes of serum oxidative status. In the light of this, we propose the use of a novel parameter, the “Antioxidant/Oxidant Balance (AOB)”, representing the ratio between serum AC and serum oxidant status, evaluated as “Peroxide Level” (PxL). AOB approach was applied for the first time to evaluate effects on serum antioxidant status during the first four hours after intake of two antioxidant-supplemented pastas, the Bran Oleoresin (BO) and Bran Water (BW) pastas, enriched respectively with either lipophilic or hydrophilic/phenolic antioxidants extracted from durum wheat bran. Interestingly, similarly to the highly active Lisosan G, intake of BO pasta allowed a significant improvement of serum AOB, until 70% as evaluated as LOX-FL/PxL. Contrarily, BW pasta induced a serum oxidative effect so as a Reference pasta and glucose, used for comparison. Overall, these findings indicate that AOB approach appears an excellent tool in highlighting effects of antioxidant-enriched food consumption, which cannot be predicted by ex vivo analysis of AC alone, as well as by in vitro measurements of cooked foods. The third level of investigation concerns the evaluation of effects exerted by bioactive compounds at the cellular and sub-cellular levels. In particular, biological effects of some phytochemicals were evaluated on activity of: i) Glyoxalase I (Glo I), implicated in enzymatic cell defence against dicarbonyl stress ii) sirtuins, a family of NAD+- dependent deacetylases involved in the regulation of cellular transcription, apoptosis, cell division and metabolism. Concerning Glo I, two studies were carried out. In the first study, the effect of sulforaphane (SR), an isothiocyanate abundant in Brassica vegetables, on the expression and activity of GloI and on the levels of reduced glutathione (GSH) in peripheral blood mononuclear cells (PBMCs) was investigated. Incubation for 24 h and 48 h of PMBCs with 2.5 μM SR (simulating a daily consumption of a broccoli portion) did not induce a substantial increase in GloI activity and expression while caused a reduction until 73% in GSH levels compared to the control cells. This suggests the formation of a GSH-SR adduct able to significantly reduce the actual SR concentration within the cells during incubation, so explaining the lack of a substantial effect on GloI expression observed in this study. In the second study, the effect of some phytochemicals was evaluated on GloI activity in highly purified mitochondrial fraction obtained from durum wheat seedlings (WM). Mitochondrial GloI was chosen since mitochondria represent one of the major targets of MG-mediated carbonylation/oxidative stress. Interestingly, a hight GloI activity was measured in WM, showing a hyperbolic dependence on substrate concentration, with Km and Vmax values equal to 0.27 mM and 0.133 EU/mg of protein, respectively. Concerning the study of modulation by phytochemicals, curcumin and quercetin were found to strongly inhibit WM-GloI activity in a competitive manner. In the last study, a novel experimental approach for reliably measuring sirtuin activity in cell extracts and/or subcellular organelles was proposed. It involves the combined use of very different enzymatic assays, the bioluminescent SIRT-Glo™ and HTRF® technology-based SIRT1 assays, and a comparative determination of activity of a recombinant human sirtuin 1 isoform (hSIRT1). By using the newly proposed approach, a high and nicotinamide-sensitive specific sirtuin activity was determined in WM, equal 268±10 mU∙mg-1 protein, as maeasured by HTRF® assay, and 166±12 ng hSIRT1 eq.∙mg-1 protein, as evaluated by the bioluminescent assay. Concerning sirtuin modulation, no significant effect of resveratrol and quercetin was found on WM-sirtuin and hSIRT1 activities by using HTRF® assay. Taken together, results reported in this PhD thesis strongly demonstrate the crucial importance of the methodological approach in assessment of putative healthful properties of dietary phytochemicals; it is necessary to avoid incorrect and misleading data interpretation about antioxidant properties of foods.

The human immunodeficiency virus (HIV) is one of the most common and dreaded diseases of the 21th century. Today, the disease is still spreading with increasing incidence. “Since the beginning of the epidemic, almost 75 million people have been infected with the HIV virus and about 36 million people have died of HIV. Globally, 35.3 million [32.2–38.8 million] people were living with HIV at the end of 2012” (http://www.who.int/gho/hiv/en/). Several studies have been conducted on herbs under a multitude of ethnobotanical grounds. The use of medicinal plants for the management of HIV has become a common practice especially, in the Eastern Cape Province of South Africa (Wilfred Otang Mbeng, 2013 PhD thesis, UFH). At the beginning of this programme, an ethnomedicinal survey of plants used for the management of HIV infection was carried out in targeted areas of the Province and information on the names of plants, the parts and the methods of preparation were collected. The survey revealed that 18 species representing 12 families were found to be commonly used for the management of HIV, as well as other opportunistic diseases such as tuberculosis, diabetes mellitus, sores, high blood pressure, etc. Carpobrotus edulis was selected for this research because it was the most frequently used in the Province. The foliar micro morphological contents of the plant, its phytochemical and antioxidant activity, in vitro antimicrobial activity, inhibitory effect against HIV-1 protease and reverse transcriptase, mechanisms of action and cytotoxicity were investigated. In terms of the foliar micro morphological contents in plants, an electron microscopy scanning (SEM) was completed. Investigation revealed that both glandular tricomes and calcium oxalate crystals (CaOX) were observed. Consequently, it is hypothesized that the bioactive therapeutic compounds secreted by C. edulis may be produced in the glandular trichomes. An investigation of phytochemical content of the plant extracts (C. edulis) was completed using four solvent extracts (hexane, acetone, ethanol and water). Results of the phytochemical analysis showed that proanthocyanidins (86.9 ± 0.005%) where highest in the aqueous extract with phenolics at 55.7 ± 0.404% in acetone extract, tannin at 48.9 ± 0.28% in ethanol extract, while the hexane extract had the highest levels of flavonoids (0.12 ± 0.05%) and flavonols (0.12 ± 0.05%). Antioxidant studies of the various extracts revealed that aqueous and ethanol extracts were found to be the best solvents for antioxidant activity in C. edulis leaves. GC-MS analysis of the essential oil from C. edulis leaves revealed that the essential oil contained at least 28 compounds. These included, in order of abundance: Oxygenated monoterpenes (36.61%); fatty acids esters (19.25%); oxygenated diterpenes (19.24%); monoterpenes (10.6%); sesquiterpenes (3.58%); and diterpenes (1.43%). Similarly, a GC-MS analysis of the crude hexane, acetone and ethanol extracts from C. edulis leaves identified a total of 59 compounds. Of the 59 compounds, 12 major phyto-metabolites that are active against infectious diseases were identified. To comfirm the potential use of C. edulis to treat infectious disease, antifungal activity of the crude essential oil extract and the four solvent extracts were tested against Candida albicans, Candida krusei, Candida glabrata, Candida rugosa and Cryptococcus neoformans strains. The essential oil extract was found to be the most active against all the fungal strains tested and performed better than the four extracts used various solvents used to extract (hexane, acetone, ethanol and water) the C. edulis leaves were tested for antibacterial and anti HIV-1 reverse transcriptase (RT) activity. The results indicated that both gram-positive and gram-negative isolates were inhibited by the extracts (hexane, acetone and ethanol) but antimicrobial activity was observed for the water extract. The lowest minimum inhibitory concentration values were obtained for the ethanol extract, followed by acetone and hexane extracts. No inhibition of HIV-1 reverse transcriptase was observed for any of the leaf extracts, even up to concentrations of 16 mg/ml. The potential inhibitory activities of the various solvent extracts against HIV-1 protease were evaluated at four different concentrations (16, 1.6, 0.16 and 0.016 mg/ml). Results indicated that the water extract showed almost 100% inhibition of HIV-1 protease activity, with an IC50 of 0.86 mg/ml leaf extract. Other solvent extracts (hexane, acetone and ethanol) however, did not show any inhibition activity above that observed for the DMSO control. The metabolic components in the water extract were subjected to LC-MS/MS analysis, which identified at least 91 compounds present in the water extract. Further studies involving the molecular modelling need to be carried out to confirm the inhibitory potential of these compounds. The cytotoxicity of the water extract of C. edulis leaves was also screened using human Chang liver cells at concentrations ranging bewteen 0.005 mg/ml and 1 mg/ml. Results indicated that the water extracts were not toxic. In conclusion the results from this study support the use of water extracts of C. edulis leaves by traditional healers to treat HIV infections and have identified possible mechanisms of action of the water extracts of C. edulis.

Malaria affects hundreds of millions of people worldwide and equally claims hundreds of thousands of lives each year. With the current spread of drug resistance to standard antimalarial drugs like chloroquine and the emergence of artemisinin-resistant parasites, new antimalarial drugs and formulations are urgently needed. An ethnobotanical survey was carried out in this study in search of novel compounds with promising antiplasmodial activity. Using the ethnobotanical approach, a total of 61 plant species from 59 genera distributed in 34 plant families were found to be used traditionally for the treatment of malaria in Nigeria. Biological evaluation of the plant¿s methanolic extracts was assessed using the parasite lactate dehydrogenase (pLDH) assay against the chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. A total of five (5) plant species showed more potent antiplasmodial activities against the malaria parasites. These are Acanthospermum hispidum, Cassia occidentalis, Kaempferia aethiopica Prosopis africana and Physalis angulata with MIC values ranging between 7.815µg/ml to 31.25µg/ml (3D7 strain) and 15.63µg/ml to 62.50µg/ml (K1 strain) against the malaria parasites, respectively. Two plants, Prosopis africana (Leguminosae-mimosoideae) and Physalis angulata (Solanaceae) were selected for further study. The phytochemical investigation of the active chloroform extracts of P. africana and P. angulata yielded several compounds with three known alkaloids, namely, prosopinine (I), prosopine (II) and acetamide (III). Their structures were confirmed by MS, 1D and 2D NMR spectroscopy. Compounds I, II and III have moderate in vitro antiplasmodial activity against the malaria parasites. Both chloroquine and artemether were used as standard control.
Association of Commonwealth Universities and the Commonwealth Scholarship Commission in the UK (Commonwealth Scholarship Reference Number: NGCS-2005-259).

The Orchidaceae makes up the largest and most diverse family of flowering plants. Orchids are popular, often expensive ornamentals, with a broad range of ethnobotanical applications. There is very limited documented information on South African medicinal orchid species; no formal pharmacopoeia outlining ethnobotanical uses; and ethnobotanical and distribution records are either scarce or inconsistent and plant populations are becoming gradually smaller. There have been significant developments in medicinal orchid research worldwide with medicinal use and corresponding pharmacological and phytochemical properties being extensively investigated. It is evident from the literature that there is no pharmacological research on South African medicinal orchids; hence the need to explore biological activity and chemical composition of South African medicinal orchid species. The ethnobotanical approach used to select the orchid species for pharmacological and phytochemical research elsewhere, yielded valuable biological compounds. Thus, a similar approach was applied to South African medicinal orchids. There are approximately 20 000 species and 796 genera of orchids distributed across the world. In southern Africa, orchids are widely represented with 55 genera and 494 species. Approximately 75% are endemic to this region. As part of the current investigation a review of available ethnobotanical literature on South African medicinal orchids was prepared. The review revealed that an estimated 49 indigenous orchid species from 20 orchid genera are currently being informally traded and used in South African traditional medicine. They are used primarily for medicinal and cultural purposes, especially by the Zulu community in South Africa. Medicinal uses of orchid species include: treatment of inflammatory, intestinal, neurological and reproductive disorders and emetics are used to cause emesis. Non-medicinal uses of orchid species include: love, fertility, protective and lethal charms. Based on their ethnobotanical uses and endemism, South African orchids were considered to be one of the untapped sources of bioactive compounds that needed to be researched. The current investigation addressed the broader aims of medicinal plant research by determining the efficacy, safety and chemical profile of seven indigenous orchid species used in South African traditional medicine and practices. The biological and toxic effects of orchid plant extracts were assessed using established pharmacological bioassays. The phytochemical evaluation of the seven orchid plant extracts provided insight into the classes of chemical compounds present and their possible role in the observed biological activities. The potential of plant extracts from seven orchid species used in South African traditional medicine, as sources of natural bioactive products, are discussed. The current investigation determined the biological activity and chemical profile of seven orchid species commonly traded in KwaZulu-Natal herbal markets: Ansellia africana Lindl., Bulbophyllum scaberulum (Rolfe) Bolus, Cyrtorchis arcuata (Lindl.) Schltr., Eulophia hereroensis Schltr., Eulophia petersii (Rchb.f.) Rchb.f., Polystachya pubescens (Lindl.) Rchb.f. and Tridactyle tridentata (Harv.) Schltr. Well established in vitro micro-dilution bioassays were used to determine the antibacterial, antifungal, anthelmintic activities of crude orchid extracts. A minimum inhibitory and/or lethal effect of organic and aqueous crude orchid extracts was observed against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans and Caenorhabditis elegans. Tridactyle tridentata aqueous root extract produced the most effective antibacterial activity against S. aureus (0.049 mg/ml). All T. tridentata organic root extracts produced significant inhibitory activities against B. subtilis and S. aureus. Eulophia petersii DCM pseudobulb extracts significantly inhibited all bacterial strains tested (0.39 mg/ml against S. aureus and 0.78 mg/ml against B. subtilis, E. coli, and K. pneumoniae). Eulophia hereroensis 80% EtOH root extract was the only other extract to exhibit significant inhibitory effects against K. pneumoniae (0.65 mg/ml). After 48 h C. albicans was most susceptible to P. pubescens aqueous pseudobulb extract (0.0816 mg/ml). Eulophia petersii DCM pseudobulb extract however, exhibited significant activity against C. albicans (0.65 mg/ml) over 72 h. Cyrtorchis arcuata leaf and root extracts were the most effective anthelmintic extracts with MLCs of 0.041 mg/ml for 80% EtOH leaf and root extracts; 0.049 mg/ml for aqueous leaf extracts and 0.78 mg/ml for aqueous and DCM root extracts. Caenorhabditis elegans was most susceptible to all A. africana and T. tridentata organic root extracts. A similar significant effect was observed for all E. petersii organic pseudobulb extracts, DCM extracts and organic root extracts of B. scaberulum. Only the DCM tuber and root extracts of E. hereroensis exhibited lethal effects on C. elegans. All of the P. pubescens extracts showed poor anthelmintic activity. Similarly, in vitro enzyme based cyclooxygenase (COX) 1 and 2 and acetylcholinesterase (AChE) inhibitory bioassays, revealed significant inhibition of COX-1, COX-2 and AChE enzymes by crude organic and certain aqueous orchid extracts. Out of a total of 53 evaluated extracts, 21 and 13 extracts exhibited significant anti-inflammatory activity in the COX-1 and COX-2 assays respectively. The DCM tuber extract of E. hereroensis was the only extract to significantly inhibit both COX enzymes, 100.02±0.11% and 87.97±8.38% respectively. All B. scaberulum root extracts (DCM, EtOH and water) exhibited COX-2 selective inhibitory activity (100.06±0.01, 93.31±2.33 and 58.09±3.25%). Overall, the DCM root extract of A. africana was found to be the most potent extract (EC50 0.25±0.10 mg/ml). The 80% EtOH root extract of B. scaberulum was the most potent in the COX-2 assay (EC50 0.44±0.32 mg/ml). Generally the root extracts exhibited greater AChE inhibitory activity; where the most active extract was B. scaberulum DCM root extract (EC50 0.02±0.00 mg/ml). All aqueous extracts, except that of A. africana roots and B. scaberulum pseudobulbs, showed poor or no COX-1 and COX-2 inhibition. The antioxidant capacity of crude orchid extracts was determined using: hydrogen atom transfer (HAT) (β-carotene/linoleic acid assay) and single electron transfer (SET) (2,2‟-diphenylpicrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power (FRAP) assay) reaction-based assays. Potent antioxidant effects were observed for certain crude methanolic orchid extracts. Generally, there was a dose-dependent change in radical scavenging activities of crude extracts from which EC50 values were determined. The root extracts of all species, except that of E. petersii, had consistently more effective radical scavenging activity than that of other plant parts within each species. The pseudobulb extract of E. petersii, was the most potent extract (EC50 1.32±0.86 mg/ml). In the β-carotene-linoleic acid assay, based on the oxidation rate ratio (ORR), the leaf extract of T. tridentata and the root extracts of C. arcuata and E. hereroensis exhibited the best antioxidant effects (0.02, 0.023 and -0.15 respectively). Similarly, the average antioxidant activity (%ANT) of these samples was greater than that of BHT (95.88±6.90%) and all other samples. Bulbophyllum scaberulum leaf, pseudobulb and root extracts, E. petersii pseudobulb extract and T. tridentata root extract also exhibited a greater capacity to prevent β-carotene oxidation when compared to BHT. All crude orchid extracts tested demonstrated a general dose-dependent response in the ferric reducing power assay. The reducing power of ascorbic acid (0.08 mM) and BHT (0.05 mM), as measured as absorbance, was 1.12±0.12 and 0.73±0.08 respectively. At 6.25 mg/ml, A. africana root and E. petersii pseudobulb extracts were the most effective in reducing power activity. The short-term bacterial reverse mutation Ames Salmonella/microsome mutagenicity (ASMM) assay, which makes use of mutant histidine-dependent Salmonella typhimurium strains, was used to determine the mutagenicity and toxicity of crude orchid extracts. In the presence of a mutagen S. typhimurium TA98 strain detects frameshift events while the TA100 and TA102 strains detect base-pair substitutions. In the absence of metabolic activation, mutagenic extracts were observed against the TA98 strain only. All A. africana DCM leaf and stem extracts tested, the DCM root extract (0.5, 0.05 mg/ml) and EtOH leaf, stem and root extracts at 5 mg/ml exhibited mutagenic effects. The EtOH root extracts (5, 0.5 mg/ml) of B. scaberulum exhibited mutagenic indices (MI) comparable to that of 4NQO (17.00 and 13.00, respectively). Eulophia petersii PE pseudobulb extract demonstrated mutagenic potential at 5 mg/ml. The ethanolic root extracts of T. tridentata showed mutagenic effects at 5 and 0.5 mg/ml. The mutagenicity index (MI) with metabolic activation (S9) was determined using only the TA98 strain; where no mutagenic effects were observed. In the phytochemical evaluation of crude methanol orchid extracts, the Folin-Ciocalteu assay for total phenolics, butanol-HCl assay for condensed tannins, rhodanine assay for gallotannins and vanillin assay for flavonoids revealed a quantitative chemical profile of the tested samples. The correlation between observed biological effects and chemical compounds present was found to be generally significant. The significant antimicrobial, anthelmintic, anti-inflammatory and antioxidant activity of E. petersii pseudobulb extracts and E. hereroensis tuber and root extracts may be attributed to their high total phenolic content. Alternatively, the significant levels of gallotannin content in E. hereroensis may have contributed to the bioactivity. The flavonoid content of B. scaberulum and T. tridentata may explain the potent activity observed in the anti-inflammatory, antioxidant and acetylcholinesterase inhibitory assays; while the flavonoid content C. arcuata may have contributed to the potent anthelmintic and antioxidant activities. The significantly higher levels of gallotannin content may explain the significant anti-inflammatory and anthelmintic activity of A. africana. A number of biologically active compounds have been isolated from certain Orchidaceae species around the world on the basis of their traditional medicinal uses. The traditional uses of these orchid species were scientifically validated. No pharmacological research has been previously conducted on South African medicinal orchids; therefore the current investigation has produced novel findings on the efficacy and safety of these orchid species and promotes the continued research of medicinal orchids in South Africa.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2011
Coreopsis tinctoria Nutt. is a small aromatic annual and widely distributed plant that belongs to the Asteraceae family. Its use in traditional medicine has been described as early as the XVII century in China, where its decoction has been used for diarrhoea. Other medicinal properties have been added by North American Indians and include treatment of internal pains, bleeding, to strengthen blood and as an emetic. In Portugal, an infusion of the flowering tops has been described to have antidiabetic proprieties, applied to diabetes type 2 treatment. There are some published phytochemical studies on the genus Coreopsis but the information on this species chemical composition, until recently, was scarce. Flavonoids of chalcone, aurone and flavanone structure have been described in C. tinctoria flowering tops although there was no available information on the chemical constitution of bioactive extracts. Flavonoids are polyphenols well known for their antioxidant and cytoprotective activity that have also been shown to act at several levels in the glucose metabolism, which may be of relevance in the prevention and treatment of diabetes and its complications. Thus the present study aimed to: i) search for new antidiabetic lead molecules able to reduce both glycemia and tissue damage; ii) chemically characterize the bioactive extracts, and; iii) study the antidiabetic profile of extracts and metabolites of C. tinctoria as this species lacked any kind of pharmacological and toxicological data. The aqueous extract of C. tinctoria flowering tops was prepared following the traditional recipe. Fractionation by chromatography followed, and three major compounds were isolated and identified by spectroscopic methodologies. Other thirteen phenolic compounds were identified by HPLC-DAD-MS/MS. Through this technique, reproducible methods for quantitative analysis of the main compounds in bioactive extracts were also developed. Further on, to evaluate whether C. tinctoria had a beneficial effect on diabetes some in vivo assays were performed. In order to test for acute antihyhyperglycemic activity, the aqueous extract was administered to normal (euglycemic) rats at different concentrations and blood glucose levels were monitored by an oral glucose tolerance test. None of concentrations tested have shown significant lowering blood glucose effect compared to control group. In order to understand other possible glucose lowering effects, a streptozotocininduced glucose-intolerant rat model was developed. C. tinctoria aqueous extract (500 mg/Kg) was then daily administrated during three weeks which resulted in significant recovery of glucose tolerance. The active extract was quantified in terms of its main compound, the chalcone marein. As means to determine whether flavonoids were the bioactive compounds, a flavonoid-rich AcOEt fraction (125 mg/Kg), with the same marein content as the bioactive aqueous extract, was prepared and administered to the glucose-intolerant model. Results indicated glucose tolerance regain after only two weeks of treatment, an effect that was maintained over the remaining experimental period. Moreover, lipase values normalized indicating re-establishment of pancreatic function. In addition, the oral treatment with either C. tinctoria aqueous extract or AcOEt fraction caused no hepatotoxicity. Regarding cytotoxicity, C. tinctoria extracts were tested in vitro, using a pancreatic mouse insulinoma cell line (MIN6). Cell viability tests were performed and no cytotoxic effects were observed at the concentrations tested (up to 3 mg/mL). Possible insulin-secretagogue action of the bioactive C. tinctoria extracts was evaluated in two ways: i) using MIN6 cells disposed as monolayers through static incubation method or; ii) in a more sensitive model, using MIN6 cells disposed in three dimensional structures (pseudoislets) using a perifusion technique. Both yielded data that seems to indicate that C. tinctoria extracts do not act increasing insulin secretion. C. tinctoria extracts and pure compounds (marein and flavanomarein), known to be good antioxidants, were then tested for their potential protective effects in MIN6 cells challenged with pro-oxidant tert-Butyl-Hydroperoxide (tBHP) or cytokines. Results indicate that C. tinctoria extracts and pure compounds are effective cytoprotectors, which seems to be due to inhibition of the apoptotic pathway, and not through a decrease on superoxide radical production. Overall, these results support the conclusion that phenolic compounds present in C. tinctoria flowering tops extracts promote recovery of pancreatic function and glucose intolerance possibly by an anti-apoptotic mechanism of injured pancreas, suggesting C. tinctoria traditional use in diabetes therapy
Coreopsis tinctoria Nutt. é uma pequena planta aromática anual, amplamente distribuída, pertencente à família das Asteraceas. Trata-se de uma planta cujo primeiro registo de utilização na medicina tradicional, data do século XVII na China, onde a sua decocção tem sido aplicada para a diarreia. Outras aplicações medicinais foram entretanto desenvolvidas por outros povos, nomeadamente, pelos Índios da América do Norte para o tratamento de dores, hemorragias, para fortalecimento do sangue e como um emético. Em Portugal, a infusão dos ápices florais de Coreopsis tinctoria, também conhecida como Estrelas-do-Egipto, foi descrita como tendo propriedades antidiabéticas, passível de ser aplicada no tratamento da diabetes do tipo dois. Existem já alguns estudos publicados na área da fitoquímica, aplicados ao género Coreopsis. No entanto, até muito recentemente, o conhecimento da composição química da espécie Coreopsis tinctoria era escasso. Flavonóides com estrutura do tipo chalcona, aurona e flavanona foram já descritos nos ápices florais desta espécie, apesar de não existir nenhuma informação sobre a constituição química de extractos bioactivos. Os flavonóides são compostos fenólicos bem conhecidos pela sua actividade antioxidante e citoprotectora, tendo também demonstrado efeitos a vários níveis no metabolismo da glucose, o que pode ser relevante na prevenção e tratamento da diabetes e das complicações macro e micro vasculares associadas a esta patologia. Tendo em conta todos estes factores, os principais objectivos deste projecto foram: i) a pesquisa de novas moléculas com potencial antidiabético capazes de reduzir tanto a glicémia como a danificação de tecidos, ii) caracterizar quimicamente os extractos bioactivos, e, iii) desenvolver um estudo do perfil antidiabético dos extractos e metabolitos presentes na Coreopsis tinctoria, na medida em que sobre esta espécie não havia até então, nenhum tipo de dados farmacológicos nem informação relativa à sua possível toxicidade. O extracto aquoso dos ápices florais da Coreopsis tinctoria foi preparado de acordo com a utilização tradicional em Portugal. Seguiu-se o fraccionamento cromatográfico, do qual se obtiveram três compostos maioritários, que foram isolados e identificados recorrendo a metodologias espectroscópicas. Outros treze compostos fenólicos foram identificados por HPLC-DAD-MS/MS. Através desta técnica, foram também desenvolvidos métodos para a análise quantitativa dos principais compostos presentes nos extractos bioactivos. No seguimento dos objectivos deste projecto, de forma a avaliar os potenciais efeitos benéficos da infusão (extracto aquoso) dos ápices florais da Coreopsis tinctoria na diabetes, procedeu-se ao estudo farmacológico, desenvolvendo alguns ensaios in vivo. Para testar a actividade antihiperglicémica aguda, administraram-se diferentes concentrações do extracto aquoso a ratos normais (euglicémicos) e, seguidamente, monitorizaram-se os níveis de glucose no sangue através do teste oral de tolerância à glucose. Nenhuma das concentrações testadas revelou um efeito significativo de diminuição dos valores de glucose no sangue comparando com o grupo controlo. Para compreender a possibilidade de outros efeitos estarem envolvidos na capacidade desta planta afectar os níveis de glucose no sangue, foi desenvolvido, em ratos Wistar, um modelo de intolerância à glucose (40 mg/Kg de estreptozotocina), na medida em que esta é característica numa situação de pré-diabetes e pode constituir por si só um risco no desenvolvimento de doenças cardiovasculares. O extracto aquoso (500 mg/Kg) foi então administrado diariamente e por sonda intragástrica durante três semanas a ratos Wistar intolerantes à glucose, tendo resultado na recuperação dos níveis de glicémia. O composto identificado como maioritário, a chalcona mareina, foi quantificado no extracto activo. Como forma de determinar se os flavonóides eram os compostos bioactivos no extracto aquoso, uma fracção de acetato de etilo, rica em flavonóides mas com igual conteúdo em mareina, foi preparada e administrada (125 mg/Kg) aos ratos Wistar intolerantes à glucose. Os resultados revelaram, logo ao fim de duas semanas de.tratamento, uma normalização das curvas de glicémia, efeito que se manteve até ao final do período experimental. Adicionalmente observou-se uma recuperação da função pancreática medida pelos níveis de lipase plasmática. É de salientar, que os animais tratados com ambos, extracto aquoso e fracção de Acetato de Etilo, não apresentaram sinais de hepatotoxicidade, após três semanas de tratamento. Relativamente à citotoxicidade, os referidos extractos foram testados in vitro usando uma linha celular pancreática derivada de insulinoma de murganho (MIN6). Testes de viabilidade celular foram efectuados não revelando efeitos de toxicidade nas concentrações testadas (até 3 mg/mL). Uma possível acção insulino-secretagoga dos extractos bioactivos da Coreopsis tinctoria foi investigada de duas formas: i) usando células MIN6 dispostas em monocamada, através de um método de incubação estática ou; ii) utilizando um modelo mais sensível, usando células MIN6 dispostas em estruturas tridimensionais, também designadas por “pseudo-ilhéus”, recorrendo a uma técnica de perfusão. Ambas aparentam indicar que os extractos bioactivos de Coreopsis tinctoria não estimulam a secreção insulínica. Os extractos da Coreopsis tinctoria assim como os seus constituintes maioritários, mareina e flavanomareina, conhecidos por apresentarem actividade antioxidante, foram testados com o objectivo de avaliar o seu potencial efeito protector em células MIN6 expostas ao pro-oxidante, terc-Butil-Hidroperóxido (tBHP,) ou a um conjunto de citocinas. Os resultados revelaram, que tanto os extractos da Coreopsis tinctoria, assim como a mareina e a flavanomareina são efectivamente protectores, o que parece ser devido a uma inibição da via apoptótica e não à diminuição da produção do radical superóxido. Em conclusão, estes resultados tendem a indicar, que os compostos fenólicos presentes nos extractos dos ápices florais da Coreopsis tinctoria, promovem a recuperação da função pancreática e consequentemente revertem a situação de intolerância à glucose possivelmente através de um mecanismo anti-apoptótico no tecido pancreático danificado, o que não contraria o uso tradicional da Coreopsis tinctoria na terapia da diabetes.
Fundação para a Ciência e a Tecnologia(FCT)

Tulsi is often regarded as “the incomparable one”, “Elixir of life” and Queen of herbs”. Tulsi is being used for about a thousand years to support a healthy mind, body, and spirit. Tulsi is believed to have originated in northern central India but grows throughout the Indian subcontinent, Nepal, Bangladesh and much of Southeast Asia. Across different cultures, it is a symbol of purity. It is used to treat various types of diseases and disorders from ancient times. It has tremendous medicinal properties and is used to treat heart diseases, digestive issues, respiratory problems, menstrual cramps, skin related disorders like acne, and mouth ulcers and even helps in controlling blood sugar levels. It is significantly used as an immune-modulatory, antihypertensive, antioxidant, antipyretic, antimicrobial and for anticancer activity. Phytochemicals like Eugenol, methyl eugenol, anthocyanins, linalool, ursolic acid are accountable for the pharmacological properties of the Tulsi. In this report we will discuss about the different varieties of Tulsi, its classification, traditional uses, it’s phytochemicals constitution and the pharmacological activities

MSc (Microbiology)
Department of Microbiology
Background: The medicinal value of plants lies in some chemical substances that produce a definite physiological action in the human body. The secondary metabolites help the plants to survive hash conditions and could be used by humans as supplements of their health, as foods additives or for medicinal purposes. This bioactive compounds are not always beneficial to human beings, and some of this plants bioactive compounds can be toxic or genotoxic to human cells. This study used several methods to evaluate of phytochemical constituents and mutagenic properties of Coccinia rehmanni and Jatropha zeyheri plant extracts. Methodology: Methanol was used for extraction of the bioactive compounds from the two selected plants, filtered with Whatman filter paper and evaporated with rotary evaporator. The extracts were fractionated using open column chromatography. Chemical and TLC methods were used to determine phytochemicals of the study plants extracts and fractions. The plants extracts and fractions were tested against Vero cell lines in order to evaluate cytotoxicity and genotoxicity of the plants. NucRed and LTR Hoechst 33342 dyes were used for cytotoxicity and genotoxicity respectively. For the evaluation of cytotoxicity and genotoxicity Quantification of live and dead cells for the screening assay was performed using the ImageXpress Micro XLS Widefield Microscope and acquired images analyses using the MetaXpress software and Multi-Wavelength Cell Scoring Application Module. Antimutagenicity of plants extracts was observed using PARP universal colorimetric assay kit. Acquired data was transferred to an EXCEL spreadsheet and data was analyzed. Results and discussion: C. rehmanni (12.03%) yielded more extract than J. Zeyheri (8.20%). the two plants had different compound composition and were in different stages of maturity. The study revealed the domination of Terpenoids, Cardiac glycosides, Phenolic and tannis. With an exception of two fraction fractions all the fractions was found to be toxic to an extent were genotoxicity of such fraction could not be concluded. The reason for such extreme toxicity could be due to the influence of the retained alcohol during rotary evaporation. xvi | P a g e Conclusion: this study provides and add to existing knowledge on the phytochemicals mutagenicity and anti-mutagenicity of C. rehmanni and J. Zeyheri medicinal plants. The study serves as scientific proof that extensive use of this plant in traditional medicine for treatment of various ailments may lead to some irreversible damages.
NRF

The stem bark of the protected tree species, Curtisia dentata (Burm. f.)C.A.Sm., is one of the most popular plant species harvested and traded at traditional medicine markets in South Africa. The overexploitation of C. dentata trees lead to a “Near Threatened” conservation status and the population trend is portrayed as “declining”. In the KwaZulu-Natal Province of South Africa, C. dentata is completely conservation dependent. This study is not based on drug discovery or toxicological studies, but on the concern that the stem bark of C. dentata trees are harvested, prepared into remedies and consumed as traditional medicine without knowledge regarding the chemical compounds in the stem bark, particularly since the chemical composition of C. dentata stem bark was unknown to date. Phytochemical analyses were firstly conducted to determine the chemical composition of C. dentata stem bark using various solvents and various analytical methods, and secondly, to determine how seasons and regional separation of C. dentata trees affect the chemical profiles of C. dentata stem bark from an environmental and nature conservation perspective. Plants are known to contain numerous chemical compounds. Compounds isolated from a particular plant species are therefore not the only compounds present in that species, and although a plant has proven pharmacological properties, they can still cause harm. Previous studies on C. dentata aimed at validating the plant species as a medicinal plant by examining extracts of the leaves, twigs and stem bark’s potentials against known pathogens and selected cancer cells in vitro and in vivo, and its anti-inflammatory, antioxidant and antiverotoxic properties. Four pentacyclic triterpenoids and one steroidal compound were also previously isolated from C. dentata leaves, however, the leaves are not used in traditional medicines, but were suggested as alternative for stem bark as the harvesting of leaves is less destructive. The efficacy of these compounds as therapeutic agents is, however, compromised by their low solubility in water and thus their potential to penetrate permeating biological membranes. Moreover, in vitro toxicity studies distort the picture of its actual potentials on human health as the whole human metabolome and all its processes, including uptake and phase I and phase II biotransformation are not included. In vivo toxicity studies on mammalian animal species may also not present a true picture of a chemical or extract’s toxic effects on humans as animal metabolisms differ from those of humans. The chemical composition of leaves and stem bark may furthermore also be in contrast to some extent, and therefore chemical compounds were also isolated from C. dentata stem bark in this study. Scientific studies on plant-based medicines generally involve the discovery or identification of compounds that may be beneficial, and which can be exploited in future. Chemical compounds in traditional medicines or other plant-based health products which may cause adverse effects are generally ignored. Moreover, scientific studies that consider that some compounds present in plant extracts may derive from contaminants are equally limited. Traditional plant-based medicines are neither standardized nor regulated in South Africa. Users of traditional plant-based traditional medicines therefore consume uncertain dosages of both beneficial and hazardous substances, as well as contaminants simultaneously. Certain chemical compounds are carcinogens or mutagens or have the ability to accumulate in human tissues. Adverse effects may therefore only manifest after several years of use and will subsequently not be connected to the use of a particular traditional plant-based medicine. The goal of the thesis is therefore to provide a holistic portrayal of the full spectrum of chemical compounds in extracts of C. dentata stem bark and to discuss, where literature is available, the effect(s) each chemical compound may have on human health. Moreover, this thesis investigates variations in chemical composition and concentration in individual trees, seasonal variations and variations in composition and concentrations in the stem bark of C. dentata trees from geographically distinct regions. Most unexpected was that not all C. dentata stem bark samples contained chemical compounds with known beneficial potentials at each sampling date, and that chemical compounds may be region-specific and also tree-specific, which confirms that plants produce secondary metabolites according to the needs of each individual plant. Additional insight into the chemical composition and concentration of C. dentata trees is provided by the distribution profiles of amino acids in C. dentata stem bark. Extreme variations within populations and between geographical areas support the need for the cultivation of C. dentata trees to ensure sustainable production of homogenous material for chemical homogeneity.
Environment Science
PhD. (Environment Science)

MSc (Microbiology)
Department of Microbiology
BACKGROUND: Antibiotic resistance amongst microbial pathogens has become a challenge over past decades, bringing about genuine and frequently deadly contaminations that can't be dealt with by ordinary means. This has led to a search on developing solutions to this problem by searching for new source of antimicrobial agents or chemically altering the existing ones. Traditional medicinal plants and nanoparticles are highly targeted as promising agents to address the challenge. Pyrenacantha grandiflora Baill from Icacenaceae family possess pharmaceutical activities and is used by Vhavenda people to cure gastrointestinal related infections, diarrhea and tooth pain. OBJECTIVES: The present study aimed to synthesize, characterize and evaluate the efficacy of Pyranacantha grandiflora extracts alone and when conjugated with selected nanoparticles against pathogenic microorganisms. Furthermore, this study investigated the efficacy of selected antibiotics when conjugated with nanoparticles against selected pathogenic microbes. METHODS: Pyrenacantha grandiflora Baill (tubers) were collected from Masisi area. Bioactive compounds were extracted using different solvents such as methanol, acetone, hot water, dichloromethane and chloroform. Preliminary phytochemical screening was done to identify different phytochemicals in the extracts and their functional groups were identified by Fourier Transform Infrared spectroscopy (FTIR). Extracts were further assessed for their total phenolic and flavonoids content. Thin layer chromatography was used to separate the compounds from the plant extracts and active compounds/group of compounds were identified by bioautography. The antioxidant ability of the extracts to scavenge free radical DPPH was also determined. Silver and gold nanoparticles were synthesized using chemical and biological methods, characterized by Ultraviolet-Visible Spectrophotometry (UV-VIS), Transmission Electron Microscopy (TEM) and Energy dispersive X-ray analysis (EDX). Plant extracts, nanoparticles and antibiotics were xix conjugated differently, and conjugates were analyzed by FTIR and their antimicrobial activities were evaluated against different bacteria and fungi. The conjugates were tested for antimicrobial activity against extended beta-lactamase producing Escherichia coli (ATCC 35218), Escherichia coli (ATCC 25922), methicillin-resistant Staphylococcus aureus (ATCC 25923), methicillin-susceptible Staphylococcus aureus (ATCC 33591) and beta-lactamase producing Klebsiella pneumonia (ATCC 700603) using agar diffusion assay and the minimum inhibitory concentrations (MIC) were determined using the microdilution method. The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined by sub-culturing from the MIC plates on Mueller-Hinton Agar. RESULTS: Pyrenacantha grandiflora was found to contain phenolics, saponins, alkaloids, tannin, steroids, terpenoids and flavonoids. FTIR spectroscopic studies revealed different characteristic peak values with various functional compounds similar in most extracts but differed with transmittance values. The total phenolic contents in the examined extracts ranged from 14.167 to 19.02 mg GA/g. The total flavonoid content in the examined extracts ranged from 26.603 to 34.621 mg QE/g. Thin-layer chromatography revealed various Rf values and when analyzed with bioautography, well-defined inhibition zones within the Rf value of 0.236 was identified against E. coli and K. pneumonia. The MICs of the extracts were determined, and all the extracts showed some antimicrobial activity against all tested strains ranging from 0.06-7.5 mg ml/g. Some extracts appeared to be fungicidal and hot water extracts were more active against Cryptococcus neoformans with the MFC value of 0.06 mg/ml. The methanol extract was also active against most tested strains including Candida tropicalis with the minimum fungicidal concentration value of 3.75 mg/ml. Pyrenacantha grandiflora tuber extracts conjugated with silver or gold nanoparticles exhibited a good antibacterial activity against all bacterial strain used and very few were able to exhibit bactericidal activity. Penicillin showed improvement of antibacterial activity xx when conjugated with compounds from the acetone extracts and vancomycin was found to be more effective when conjugated with silver nanoparticles and water extracts. CONCLUSION: The present study validated the efficacy of conjugated P. grandiflora tuber extracts which is used in traditional medicine. The results revealed that water extracts which are generally used by the traditional healers are active against most microorganisms tested as well as methanol and acetone extracts and the synergistic effect was observed when they were conjugated to gold and silver nanoparticles. The results of the present investigation clearly indicate that antimicrobial activity of Pyrenacantha grandiflora Baill tuber when conjugated with selected nanoparticles and antibiotics vary with test strain and the type of solvent used during extraction, thus giving hope for future development of drug leads.
NRF

博士
中山醫學大學
營養學系博士班
106
Background：Obstructive sleep apnea（OSA）is an increasing sleeping disorder occurred owing to the repeatedly collapse and obstruction of the upper respiratory tract during the sleep. OSA is considered to be highly associated with the metabolic syndrome. Moreover, life style modification and body weight control should be taken into consideration beforehand while treating OSA patients with obesity. The purpose of this study is to examine how intervention of specific phytochemical-containing medical functional food, fish oil, vitamin C and Coenzyme Q10 affect patient with OSA. Method：Twenty-nine severe OSA patients had received daily multiple nutrient supplements for 12 weeks. At the beginning and the end of the trial, several elements were examined, which are anthropometric measurements, polysomnography（PSG）, Epworth Sleepiness Scale（ESS）, health-related quality of life, blood biochemistry, inflammatory cytokines, antioxidant enzymes, and oxidative stress marker malondialdehyde （MDA）. Results：The average age of test completing participants are 39.17± 7.30 years old. After the intervention, which lasted for 12 weeks, the body weight, BMI, neck circumference, waist circumference, hip circumference, waist to hip ratio and body fat percentage had shown significant decrease. The apnea-hypopnea index （AHI） reduced from 53.43 ± 18.82 to 34.38 ± 24.41 （P＜0.001） and oxygen desaturation index（ODI）also decreased（P＝0.044）. Also, the ESS showed decrease（P＝0.006）.Subjects had experienced reductions in both systolic and diastolic blood pressure at the end of the trial （P＝0.004）. In the blood biochemistry, the total cholesterol, LDL, TG, TG／HDL, GPT, Cr, uric acid increase and HDL were shown to be decreasing significantly. The mean number of metabolic syndrome variables in the beginning is 3.00 ± 1.34 and drop to 2.14 ± 1.03 at the end of trial, with the total reduction of 0.86 ± 1.19（P＝0.001）.The plasma oxidative products MDA and 8-isoprostane had decreased（P＜0.001）. Moreover, the erythrocyte catalase, glutathione peroxidase, and superoxide dismutase(SOD) increased（P＜0.001）. The inflammatory protein hs-CRP(high sensitive C reactive protein), IL-6(interleukin-6), IL-8(interleukin-8), and TNF-α(tumor necrotic factor-α) showed decrease（P＜0.001）. In the SF-36 generic questionnaire, there are 5 dimensions shown to be enhancing significantly, inclusive of the physical functioning, role-physical, general health, social functioning, and vitality. Conclusion：According to the outcome of the trail, the capability of some specific phytochemical-containing medical food and nutritional supplement therapy is shown to be able to improve metabolic variable, oxidant-antioxidant balance, inflammatory status, AHI, ODI, and the quality of life for patients with severe OSA conditions. As for the suggestion for future studies, further large-scale studies are needed to have a better understanding over the influence of nutritional supplement therapy on OSA patients more holistically.

Despite their worldwide abundancy, algae are still considered an underexploited resource with a lot of interest in several industries, namely food and pharmaceuticals. Several reports comparing the diets of different countries, associate seaweed consumption with the oriental welfare and vitality contrarily to western societies. Within all three classes of edible seaweeds, those belonging to Phaeophyta have been receiving a lot of attention particularly due to their abundance in complex polysaccharides, phlorotannins, fucoxanthin and iodine. This work reports the nutritional composition (total fibre content, protein, ash and minerals) and phytochemical content (pigments and phlorotannins) of a pasta fortified with 1% and 5.5% of brown seaweed Fucus vesiculosus, prior and after the cooking process. In parallel the biological activities were evaluated namely antioxidant potential and capacity of inhibiting three metabolic enzymes, α-amylase, α-glucosidase and pancreatic lipase. Lastly, mineral bioaccessibility and bioavailability was accessed. Overall, the gathered results demonstrate that with each increasing seaweed concentration there is a tendency of improving several parameters. On a first approach, both fibres and ash contents increased up to 48% and 60%, respectively, while protein content remained unchanged. Further confirming the higher levels of ash on enriched samples, iodine content was augmented to around 91% of its initial concentration. Furthermore, F. vesiculosus contributed to a phytochemical increase, namely pigments, such as chlorophyll a (84%) and carotenoids (99%), and phlorotannins (87%) on the target pastas. Altogether, these results are most likely related to the improvement of the antioxidant capacity of the samples. Upon cooking, a general decrease on several contents was noted, namely protein, mineral and phytochemicals, which reflected a decline of the antioxidant property. The maintenance of fibre content upon this thermal processing possibly prompted to numerous effects, such as the retention of protein, to an extent, proving itself advantageous, but also the retention of minerals when submitted to simulated gastrointestinal digestion. Amongst all studied minerals, K and Mg fractions presented the highest percentages, 105% and 62% for the bioaccessible ones, and 56% and 24% for the bioavailable ones, respectively. Additionally, iodine stood out with a bioaccessible fraction of 82%. This element also had a bioavailability of 4%; however, it is estimated that the consumption of 100g of pasta could provide approximately 54% of its recommended daily dose. So, despite the positive developments and the good insight on the impact of both the seaweed incorporation and the cooking process, a lot of research is still required to optimize this product and to address the viability beyond in vitro assays.
Apesar da sua abundância natural, as algas ainda são consideradas um recurso pouco explorado, mas com muito interesse em diversos setores, nomeadamente alimentar e farmacêutico. Vários estudos que comparam as dietas de diferentes países, associam o consumo de algas marinhas ao bem-estar e vitalidade oriental contrariamente às sociedades mais ocidentais. Dentro das três classes de algas comestíveis, as pertencentes ao domínio Phaeophyta têm recebido muita atenção devido à sua abundância em polissacarídeos complexos, florotaninos, fucoxantina e iodo. Este trabalho reporta a composição nutricional (teor total de fibras, proteínas, cinzas e minerais) e o conteúdo em fitoquímicos (pigmentos e florotaninos) de uma massa alimentícia fortificada com 1% e 5.5% da alga castanha Fucus vesiculosus, antes e após o processo de cozimento. Paralelamente, foram investigadas algumas atividades biológicas, nomeadamente o potencial antioxidante e a capacidade de inibir três enzimas do metabolismo, α-amilase, αglucosidase e lipase pancreática. Por fim, foi avaliada a bioacessibilidade e biodisponibilidade dos minerais. No geral, os resultados obtidos demonstram que, com cada incremento de alga, houve uma tendência em melhorar vários parâmetros. Para as massas com 5.5% de alga, os teores de fibras e cinzas aumentaram até 48% e 60%, respetivamente, enquanto que o teor de proteínas permaneceu inalterado. Confirmando ainda os níveis mais altos de cinzas em amostras enriquecidas, o teor de iodo aumentou para cerca de 91% da sua concentração inicial. Além disso, a F. vesiculosus contribuiu para um aumento dos fitoquímicos, nomeadamente de pigmentos como clorofila a (84%) e carotenoides (99%), e florotaninos (87%) nas massas. De uma forma geral, esses resultados estarão relacionados com a notória melhoria da atividade antioxidante das amostras. Após o cozimento, observou-se uma diminuição dos vários conteúdos mencionados anteriormente, nomeadamente proteínas, minerais e fitoquímicos, que, por sua vez, refletiram também uma perda da atividade antioxidante. A manutenção do teor de fibras após esse processamento térmico possivelmente contribuiu para inúmeros efeitos, como a retenção de proteínas, até certo ponto, mas também a retenção de minerais quando submetidos a uma digestão gastrointestinal simulada. Entre todos os minerais estudados, destacaram-se as frações de K e Mg, com as percentagens mais elevadas, 105% e 62%, para as bioacessíveis, e 56% e 24%, para as biodisponíveis, respetivamente. Adicionalmente salienta-se o iodo, cuja bioacessibilidade atingiu os 82%. Para este elemento, a biodisponibilidade foi apenas 4%, no entanto, dada a sua elevada quantidade na massa cozida, estima-se que 100 g de massa poderá disponibilizar aproximadamente 54% da sua dose diária recomendada. Assim, apesar dos desenvolvimentos positivos e da boa perceção relativa ao impacto da incorporação de algas e do processo de cozimento, ainda serão necessárias mais pesquisas para otimizar este e outros produtos, e abordar a viabilidade além dos ensaios in vitro.
Mestrado em Bioquímica

Venereal diseases (VDs) are infections that are mainly transmitted through sexual intercourse and amongst these are gonorrhoea, syphilis, chlamydia and trichomoniasis. Gonorrhoea is the most commonly known VD and the widest spread contagious infection in the world. Out of 448 million cases of curable venereal infections, gonorrhoea represents 88 million cases and the rest are syphilis, chlamydia and trichomoniasis. Gonorrhoea has recently been rated as in the emergent multidrug resistance phase. Venereal diseases are amongst the major diseases ravaging many rural communities. People infected with these diseases are considered a disgrace in the community. Indigenous populations, for example the Vha-Venda people tend to use medicinal plants to treat these infectious diseases rather than using western medicines. Vha-Venda people have depended on medicinal plants for their health and survival for millenia. In order to validate and give scientific credence to the use of medicinal plants by the Vha-Venda people for venereal diseases, several pharmacological assays were carried out. The study was aimed at evaluating the; antimicrobial, anti-inflammatory activities, HIV-type 1 reverse transcriptase (RT) inhibition properties and to determine phenolic contents as well as evaluating the mutagenic properties of, 12 medicinal plants used by the Vha-Venda people against venereal and related diseases. An attempt was also made toward isolating and identification of the most active compounds from some extracts that were active against Neisseria gonorrhoeae. Twelve medicinal plants and various plant parts, Adansonia digitata (bark), Acacia karroo (bark), Aloe chabaudii (roots), Bolusanthus speciosus (leaves, bark and stem), Ekebergia capensis (leaves and bark), Elephantorrhiza burkei (roots), Grewia occidentalis (roots), Osyris lanceolata (roots), Pappea capensis (leaves), Peltophorum africanum (bark), Pterocarpus angolensis (leaves and bark) and Ximenia caffra (leaves and roots) were evaluated for their antimicrobial properties against two Gram-positive (Bacillus subtilis and Staphylococcus aureus), three Gram-negative (Neisseria gonorrhoeae, Escherichia coli and Klebsiella pneumonia) bacteria and the fungus Candida albicans. The plant materials were extracted with petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water. Methanol was used for extracting materials for phenolic contents and HIV-1RT assays. The Disc diffusion method was used to determine gonococcal percentage inhibition and a microdilution assay was used to determine minimum inhibition concentration (MIC) and minimum fungicidal concentrations (MFC). Bolusanthus speciosus and X. caffra extracts exhibited the best antigonococcal, antifungal and antibacterial activities whilst A. digitata and A. chabaudii showed poor activities. The medicinal plants were also evaluated for cyclooxygenase (COX-1 and -2) and HIV-1 reverse transcriptase inhibition activity. The DCM and PE extracts of A. digitata bark, B. speciosus bark, P. angolensis bark and P. capensis leaves showed good anti-inflammatory activity against both COX-1 and COX-2. Methanol and water extracts of B. speciosus stems, P. africanum bark, P. angolensis leaves and P. capensis leaves exhibited good anti-HIV-1 RT activity. A. chabaudii roots, E. capensis bark and O. lanceolata roots showed low HIV-1 RT percentage inhibition. Phytochemical analysis using spectrophotometric methods revealed the presence of a variety of phenolic compounds in all the plant extracts including total phenolics, flavonoids, gallotannins and condensed tannins. High levels of total phenolics, flavonoids, gallotannins and condensed tannins were detected in X. caffra. Low amounts of flavonoids, gallotannins and condensed tannins were detected in B. speciosus. The Ames test using Salmonella typhimurium tester strain TA98 with and without S9 metabolic activation revealed that all plant extracts were non-mutagenic toward S. typhimurium strains TA98 without metabolic activation. However, E. burkei roots and E. capensis bark showed mutagenic effects toward TA98 after metabolic activation. Therefore, these two plants need to be used with caution, however more studies are required to confirm this result. Good antimicrobial activity observed in X. caffra leaves prompted an attempt to isolate active compounds. A pure compound from X. caffra leaves exhibited moderate activity (63%) against N. gonorrhoeae. However, the structure of the compound has as yet to be ratified. Pharmacological activity of the twelve medicinal plants used by Vha-Venda people against venereal and related diseases were validated in this study. The results obtained in this study give credence to the use of some of these plants. This study has further confirmed the need for screening these medicinal plants for more pharmacological activities. These plants may offer a new source of chemicals for the effective treatment of venereal and related diseases.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.