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Objective: To evaluate the antibacterial and cytotoxic effect of Myrciaria dubia (Camu camu) (M. dubia) methanol extract, against Streptococcus mutans (ATCC 25175) (S. mutans) and Streptococcus sanguinis (ATCC 10556) (S. sanguinis). Methods: Two methanol extracts of M. dubia were prepared in vitro, from the seeds and pulp. Ten independent tests were prepared for each type of extract, using 0.12% chlorhexidine solution as positive control. Agar diffusion test was used by preparing wells with the experimental solutions cultivated in anaerobic conditions for 48 h at 37 °C. Meanwhile, the minimum inhibitory concentration and the cytotoxic effect over MDCK cell line was found. Results: A higher antibacterial effect was observed with the methanol seed extract with an inhibitory halo of (21.36 ± 6.35) mm and (19.21 ± 5.18) mm against S. mutans and S. sanguinis, respectively. The methanol extract of the pulp had an effect of (16.20 ± 2.08) mm and (19.34 ± 2.90) mm, respectively. The minimum inhibitory concentration of the pulp extract was 62.5 µg/mL for both strains, whereas for the seed antibacterial activity was observed even at low concentrations. The CC50 of the seeds extract was at a higher concentration than 800 µg/mL and 524.37 µg/mL for the pulp extract. / This study was supported by Universidad Peruana de Ciencias Aplicadas (UPC) Lima-Peru with Grant No. MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT UPC-501-2015

Objective To evaluate the cytotoxic and antibacterial effect of Bixa orellana L. (B. orellana) (achiote) methanol extract against Streptococcus mutans (ATCC 25175) (S. mutans) and Streptococcus sanguinis (ATCC 10556) (S. sanguinis). Methods Two methanol extracts of B. orellana were prepared in vitro, from the seeds and leaves. The antibacterial activity of extracts against S. mutans and S. sanguinis was evaluated using the cup-plate agar diffusion method. The minimum inhibitory concentration (MIC) was determined using the microdilution method and the cytotoxic activity was determinated by using the cell line MDCK. Results A stronger antibacterial effect was observed with the leaves methanolic extract with an inhibition zone of (19.97 ± 1.31) mm against S. mutans and (19.97 ± 1.26) mm against S. sanguinis. The methanolic extract of the seeds had an activity of (15.11 ± 1.03) mm and (16.15 ± 2.15) mm against S. mutans and S. sanguinis, respectively. The MIC of the leaf and the seed extracts against S. sanguinis was 62.5 and 125 μg/mL, respectively, and the MIC of the leaf extract against S. mutans was 62.5 μg/mL, and for the seed extract it was 31.25 μg/mL. The 50% cytotoxic concentration was 366.45 and 325.05 μg/mL for the leaves and seeds extracts, respectively. Conclusions The experimental findings demonstrated the antibacterial effect of the methanolic extract of B. orellana (achiote) on S. mutans and S. sanguinis. The extract of this plant is cytotoxic at high concentrations. / Peer review

Orientadores: Patricia da Silva Melo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T21:37:44Z (GMT). No. of bitstreams: 1 Anazetti_MaristellaConte_D.pdf: 5127059 bytes, checksum: e392f2bf0540fd486b7890fd082a7078 (MD5) Previous issue date: 2008 / Resumo: Nos últimos anos, a busca por alternativas mais eficientes e menos tóxicas para a terapia de diversas doenças despertou interesse em pesquisadores de diversas áreas, particularmente as que envolvam a utilização de polímeros biodegradáveis no desenvolvimento de sistemas de liberação controlada de hormônios, fármacos anti-câncer, antibióticos e vacinas. A quimioterapia do câncer é uma importante aplicação de conjugados droga-polímero proporcionando aumento da eficácia terapêutica e estabilidade da droga, assim como, a redução dos efeitos colaterais. Recentes estudos comprovaram a atividade anti-Ieucêmica da desidrocrotonina (DHC), que é uma diterpeno lactona extraída das cascas de Croton cajucara (sacaca), uma planta nativa e endêmica da região Amazônica. Em trabalhos anteriores, mostramos que a DHC induz diferenciação e apoptose em células da leucemia humana, HL60, com envolvimento de estresse oxidativo, ativação de caspases 3 e 9, sugerindo a participação da via mitocondrial. Neste trabalho, avaliou-se comparativamente a a indução de diferenciação celular e de morte celular por apoptose pela DHC, na forma livre e complexada em ?-ciclodextrinas (?CDs) em diferentes linhagens de células da leucemia mielóide humana: HL60, K562, K562 Lucena e U937. Os resultados mostram-se promissores quanto à atividade antitumoral da DHC complexada à ?CDs, quanto a citotoxícidade e a atividade antitumoral, mantendo a redução da citotoxicidade em células V79 e em hepatócitos, se comparada à forma livre da DHC. / Abstract: In recent years, the search for alternative, more efficient and less toxic to therapy of various diseases awakened interest in researchers from various areas, particularly those involving the use of biodegradable polymers in the development of controlled release systems for hormones, anti-cancer drugs, antibiotics and vaccines. The chemotherapy of cancer is an important application of polymer-drug conjugates providing increased therapeutic efficacy and stability of drugs, as well as the reduction of side effects. Recent studies prove the anti-leukaemic activity of desidrocrotonina (DHC), a diterpeno lactone extracted from the bark Croton cajucara (sacaca), a plant native and endemic of the Amazon region. In previous work, we show that the DHC induces differentiation and apoptosis of leukemia cells in human, HL60, with the involvement of oxidative stress, activation of caspases 3 and 9, suggesting the involvement of the mitochondrial pathway. In this work, it is comparatively assessed aa induction of cell differentiation and cell death by apoptosis by DHC, in the form free and complexada in ?-cicIodextrinas (?CDs) in different cell lines of human myeloid leukemia: HL60, K562, K562 Lucena and U937. The results show is on the promising anti-tumor activity of DHC complexada the ?CDs, on cytotoxicity and antitumor activity while maintaining the reduction of cytotoxicity in cells V79 and in hepatocytes, if compared to how free of DHC. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Os receptores do tipo Toll compreendem a família de receptores de reconhecimento de padrões melhor caracterizados, que podem ativar diferentes respostas imunes, dependendo de quais receptores e conjuntos de adaptadores são utilizados. Os TLRs, como TLR2, TLR4 e TLR9, e sua sinalização foram implicados no reconhecimento de P. brasiliensis e na regulação da resposta imune, no entanto, o papel do TLR3 ainda não está claro. Assim, a compreensão da função endossomal do TLR3 na PCM experimental é crucial. Utilizamos modelos in vitro e in vivo de infecção por P. brasiliensis, camundongos C57Bl/6 e TLR3-/-, para avaliar a contribuição da TLR3 no desenvolvimento da infecção. Mostramos que ausência de TLR3 leva o aumento de óxido nítrico e a capacidade fagocítica por macrófagos nas primeiras 4 horas de interação com leveduras P. brasiliensis. Mostramos ainda que os camundongos TLR3-/- desempenham papel protetor após 30 dias de infecção intratraqueal com P. brasiliensis, mostrando diminuição do aumento de CFU, perfil de resposta Th1 e Th17, bem como aumento de células citotóxicas T CD8+ produtoras de IFN-γ e IL-17. As células citotóxicas T CD8+ mostraram ser essenciais para o controle da infecção nos camundongos TLR3-/-, uma vez que a depleção dessas células levou a progressão da doença. Em estágios iniciais, 3 e 5 dias de infecção, observamos aumento do recrutamento de neutrófilos para o pulmão. Estudos recentes indicam que o TLR3 é um receptor importante para a resposta imune na micose e sua ausência favorece a infecção por fungos. Em contraste, nossos resultados mostram que, no caso do PCM, o TLR3 é prejudicial ao hospedeiro, sugerindo que a ativação do TLR3 pode ser um possível mecanismo de escape de P. brasiliensis. / Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, C57Bl/6 and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show that absence of TLR3 leads to increased nitric oxide and phagocytic capacity by macrophages in the first 4 hours of interaction with yeasts P. brasiliensis. We also showed that TLR3-/- mice play a protective role after 30 days of intratracheal infection with P. brasiliensis, showing a decrease in the CFU increase, Th1 and Th17 response profile, as well as an increase in cytotoxic CD8+ cells producing IFN-γ and IL-17. The cytotoxic T CD8+ cells were shown to be essential for the control of infection in TLR3-/- mice, since the depletion of these cells led to the progression of the disease. In the initial stages, 3 and 5 days of infection, we observed increased recruitment of neutrophils to the lung. Recent studies indicate that TLR3 is an important receptor for the immune response in mycosis and its absence favors fungal infection. In contrast, our results show that in the case of PCM, TLR3 is detrimental to the host, suggesting that TLR3 activation may be a possible escape mechanism of P. brasiliensis.

Os receptores do tipo Toll compreendem a família de receptores de reconhecimento de padrões melhor caracterizados, que podem ativar diferentes respostas imunes, dependendo de quais receptores e conjuntos de adaptadores são utilizados. Os TLRs, como TLR2, TLR4 e TLR9, e sua sinalização foram implicados no reconhecimento de P. brasiliensis e na regulação da resposta imune, no entanto, o papel do TLR3 ainda não está claro. Assim, a compreensão da função endossomal do TLR3 na PCM experimental é crucial. Utilizamos modelos in vitro e in vivo de infecção por P. brasiliensis, camundongos C57Bl/6 e TLR3-/-, para avaliar a contribuição da TLR3 no desenvolvimento da infecção. Mostramos que ausência de TLR3 leva o aumento de óxido nítrico e a capacidade fagocítica por macrófagos nas primeiras 4 horas de interação com leveduras P. brasiliensis. Mostramos ainda que os camundongos TLR3-/- desempenham papel protetor após 30 dias de infecção intratraqueal com P. brasiliensis, mostrando diminuição do aumento de CFU, perfil de resposta Th1 e Th17, bem como aumento de células citotóxicas T CD8+ produtoras de IFN-γ e IL-17. As células citotóxicas T CD8+ mostraram ser essenciais para o controle da infecção nos camundongos TLR3-/-, uma vez que a depleção dessas células levou a progressão da doença. Em estágios iniciais, 3 e 5 dias de infecção, observamos aumento do recrutamento de neutrófilos para o pulmão. Estudos recentes indicam que o TLR3 é um receptor importante para a resposta imune na micose e sua ausência favorece a infecção por fungos. Em contraste, nossos resultados mostram que, no caso do PCM, o TLR3 é prejudicial ao hospedeiro, sugerindo que a ativação do TLR3 pode ser um possível mecanismo de escape de P. brasiliensis. / Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, C57Bl/6 and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show that absence of TLR3 leads to increased nitric oxide and phagocytic capacity by macrophages in the first 4 hours of interaction with yeasts P. brasiliensis. We also showed that TLR3-/- mice play a protective role after 30 days of intratracheal infection with P. brasiliensis, showing a decrease in the CFU increase, Th1 and Th17 response profile, as well as an increase in cytotoxic CD8+ cells producing IFN-γ and IL-17. The cytotoxic T CD8+ cells were shown to be essential for the control of infection in TLR3-/- mice, since the depletion of these cells led to the progression of the disease. In the initial stages, 3 and 5 days of infection, we observed increased recruitment of neutrophils to the lung. Recent studies indicate that TLR3 is an important receptor for the immune response in mycosis and its absence favors fungal infection. In contrast, our results show that in the case of PCM, TLR3 is detrimental to the host, suggesting that TLR3 activation may be a possible escape mechanism of P. brasiliensis.

Este estudo propôs diferentes métodos de incorporação do vanadato de prata nanoestruturado (AgVO3) em uma resina acrílica e avaliou o padrão de incorporação promovido, a capacidade antimicrobiana, o desempenho mecânico, a taxa de liberação de íons prata (Ag) e vanádio (V) e a citotoxicidade. O AgVO3 foi incorporado nas frações de 0-5%, por meio de duas metodologias, (I) Espatulação a Vácuo e (II) Técnica do Filme Polimérico. A caracterização dos espécimes foi realizada por Microscopia eletrônica de varredura e microanálise (MEV/EDS) e Difração de raios X (DRX). A capacidade antimicrobiana foi avaliada por pirosequenciamento genético e o desempenho mecânico por ensaios de resistência à flexão e ao impacto. A taxa de liberação de Ag e V foi analisada por espectrometria de massa com plasma acoplado indutivamente (ICP-MS) e a citotoxicidade, pelo método do MTT. Análise estatística descritiva foi realizada para o teste microbiológico. Os dados das análises mecânicas foram analisados por ANOVA de 2 fatores com múltiplas comparações com ajustes de Bonferroni e os demais por Kruskal-Wallis seguido pelo pós teste de Dunn, e pelo Teste de Mann-Whitney. O nível de significância adotado foi de &alpha;=0,05. Houve diferença no padrão de dispersão promovido pelos dois métodos. O método de incorporação teve pouca influência na microbiota entretanto, o tempo de incubação e a presença de AgVO3 promoveram alterações no perfil taxonômico ao nível de filo e gênero. A adição de AgVO3 reduziu a resistência à flexão e ao impacto das resinas (p<0,001) e a técnica do filme polimérico promoveu melhor resistência à flexão (p<0,001). Maior liberação de Ag e V foi observada no grupo com 5% de AgVO3 (p<0,05). De maneira geral, não houve influência da concentração de AgVO3 e do método de incorporação na viabilidade do fibroblasto gengival humano (p>0,05) porém, o tempo de contato com os materiais foi inversamente proporcional à viabilidade (p<0,05). Conclui-se que foi possível incorporar o AgVO3 na resina acrílica por meio das duas metodologias propostas, havendo diferença no padrão de distribuição. Há diferenças na diversidade do biofilme oral inicial e maduro formado na superfície das resinas e os resultados sugerem que a incorporação de AgVO3 influencia a microbiota colonizadora. A redução das propriedades mecânicas pode ser clinicamente aceitável e os resultados da liberação elementar e da citotoxicidade não inviabilizam o uso dos materiais obtidos, embora outros testes são recomendados / This study proposed different methods of incorporating nanostructured silver vanadate (AgVO3) into an acrylic resin and evaluated the pattern of incorporation promoted, antimicrobial capacity, mechanical performance, the rate of release of silver ions (Ag) and vanadium (V) and cytotoxity. AgVO3 was incorporated in the fractions of 0-5%, by means of two methodologies, (I) Vacuum Spatulation and (II) Polymeric Film Technique. The characterization of the specimens was performed by scanning electron microscopy and microanalysis (SEM / EDS) and X-ray diffraction (XRD). The antimicrobial capacity was evaluated by genetic pyrosequencing and the mechanical performance by flexural strength and impact tests. The release rate of Ag and V was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and cytotoxity by the MTT method. Descriptive statistical analysis was performed for the microbiological test. Data from the mechanical analyzes were analyzed by 2-way ANOVA with multiple comparisons with Bonferroni adjustments and the other by Kruskal-Wallis followed by Dunn\'s post-test and the Mann-Whitney test. The level of significance was &alpha;=0.05. There was a difference in the dispersion pattern promoted by the two methods. The incorporation method had little influence on the microbiota however, the incubation time and the presence of AgVO3 promoted changes in the taxonomic profile at the level of filo and genus. The addition of AgVO3 reduced the flexural and impact strength of the resins (p<0.001) and the polymeric film technique promoted better flexural strength (p<0.001). Greater release of Ag and V was observed in the group with 5% of AgVO3 (p<0.05). In general, there was no influence of AgVO3 concentration and incorporation method on the viability of human gingival fibroblasts (p>0.05), but the time of contact with the materials was inversely proportional to the viability (p<0.05). It was concluded that it was possible to incorporate AgVO3 into the acrylic resin by means of the two methodologies proposed, with sifference in the distribution pattern. There are differences in the diversity of the initial and mature oral biofilm formed on the surface of the resins and the results suggest that the incorporation of AgVO3 influences the colonizing microbiota. The reduction of mechanical properties may be clinically acceptable and the results of elemental release and cytotoxicity do not preclude the use of the materials obtained, although other tests are recommended

MSc (Chemistry) / Department of Chemistry / Chromones are well known naturally occurring heterocyclic compounds with oxygen as a heteroatom. Chromones are also one of the major classes of naturally occurring compounds, and the interest in their chemistry is due to their wide range of their biological activity. In this study, three classes of target compounds were synthesized through three different pathways. The first pathway, chromone-3-carbaldehyde analogues were afforded in good to excellent yield followed by the oxidation thereof to 4-oxo-4H-chromene-3-carboxylic acids. A series of chromone-3-carboxamides was obtained from corresponding 4-oxo-4H-chromene-3-carboxylic acid via the in situ generation of the corresponding acid chloride in good yield. The second class of compounds were achieved by reacting corresponding chromone-3-carbaldehyde analogues with thiazolidine-2,4-dione to afford 5-((4-oxo-4H-chromen-3-yl)methylene)thiazolidine-2,4-dione analogues. The third class of compounds followed the same reaction pathway as the second class of compounds from corresponding 8-allyl-chromone-3-carbaldehyde analogues to afford 5-((8-allyl-4-oxo-4H-chromen-3-yl)methylene)thiazolidine-2,4-dione analogues in good yield. Compounds were characterized by 1D NMR (1H, 13C and DEPT), 2D NMR (COSY, HSQC and HMBC), IR and elemental analysis (CHN analysis). Selected synthesized chromone derivatives were evaluated in vitro for two biological assays; namely trypanocidal activity and cytotoxicity. Among all tested compounds, 41A, 55B and 63D displayed promising trypanocidal activity by reducing the percentage parasite viability to 0.61, 0.15 and 0.21 respectively. These results were further substantiated by their IC50 values 4.3, 1.3 and 1.9 μg/mL respectively. Compounds 41B and 59A also showed significant trypanocidal activity, however it was below the positive control. Compounds 41A and 55B displayed cytotoxicity against the HeLa cells whilst compound 63D displayed no toxicity against the HeLa cells. / NRF

MSc (Botany) / Department of Botany / Diabetes and arthritis are the most common chronic diseases. Arthritis is the leading cause of global disability and diabetes has become a major health problem which is increasing rapidly. The purpose of the study was to document medicinal plants that are used to treat and manage diabetes and arthritis by traditional medicinal practitioners around the Vhembe District Municipality as well as to evaluate their in vitro efficacy. Traditional practitioners were interviewed using semi-structured questionnaires. Seventeen plant species belonging to fourteen different families were found to be used in the treatment of diabetes as well as arthritis. Fabaceae family was dominating. Antioxidant, anti-inflammatory, cytotoxicity, alphaamylase and alpha-glucosidase) of five plant species, (Bridellia mollis, Elephantorrihiza burkei, Elaeodendron transvaalense, Senna petersiana and Searsia lancea) used traditionally to manage diabetes were investigated using the standard in vitro procedures. All extracts showed a good nitric oxide inhibition, with highest percentage inhibition found in the highest concentration of 100 μg/ml. They all had good percentage cell viability at lowest concentration which was comparable to quercetin. Only two plant extracts B. mollis (T2) and E. transvaalense (T3) had lower than inhibition of quercetin at 25 μg/ml than at 12.5 μg/ml. In vero cells low toxicity effect was observed at lowest concentration tested, and toxicity increased with the increase in concentration. In bovine dermis cell line all plant extracts had more cell viability at lower concentration than doxorubicin. Ethanol extracts of B. mollis and S. petersiana, and ethyl extract of E. transvaalense had a good alpha-amylase inhibitory activity with IC50 values 58.6, 81.9 and 131.5 mg/ml respectively. Hydro-ethanol, ethyl acetate and ethanol extracts of E. burkei exhibited a significant alpha-glucosidase inhibitory activity with IC50 values 56.9, 52.2 and 129.7 mg/ml respectively. Kinetic analysis revealed non-competitive and un-competitive inhibitions of the plant extracts on alpha-amylase and alpha-glucosidase enzymes respectively. The information obtained showed that people in Vhembe District Municipality still rely on medicinal plants to treat and manage diabetes and arthritis. All plant extracts were toxic to both bovine dermis and vero cell lines. S. lancea (T5) was found to be the most toxic plant extract. The observed good inhibitions of both alpha-amylase and alpha-glucosidase enzymes by plant extracts of B. mollis, S. petersiana, E. transvaalense and E. burkei validate their use in the traditional treatment of diabetes in the region to some extent. Aqueous extracts of these medicinal plants should also be investigated because water is the main solvent which is used by traditional practitioners in the preparation of their herbal medicines.