Made available in DSpace on 2015-03-26T13:39:43Z (GMT). No. of bitstreams: 1 texto completo.pdf: 466511 bytes, checksum: f3030460e9ee9f5dba51df5d32e2b05b (MD5) Previous issue date: 2006-09-19 / Hahnemann in his book The Organon , on the basis of a long time experimentation, reported that in human organisms similar medicines are antagonist to each other and so they loose effectivity. This statement is supported by experimentation in human body but there is no experimental data in plants. The objective of this research was to test the opposing actions of some homeopathic medicines and to obtain conceptual bases of the antagonism in order to develop homeopathic technologies for plants. The trials were conducted in the greenhouse and the Homeopathy Laboratory of Plant Science Department at Federal University of Viçosa, from august to December of 2005. The experiments follow the split plot model being the treatment in the plots and in the subplot was the determination time (10 minutes) and with 4 replicates. It was evaluated the effect of homeopathic preparations on CO2 assimilations of Ruta graveolens L. plants. Test solutions were the dilution of 4 mL of each homeopathic preparation per 1 L of distilled water being poured 1 mL over the soil at 1 cm around the plant. Along the trial, from 7 to 11 a.m. and after pouring the test solution CO2, assimilation data were taken each minute for 10 consecutive minutes by an Infrared Gas Analyzer (IRGA). Data were submitted to analysis of variance, mean test and regression through SAEG Statistical Analysis System which detected treatment influence on CO2 assimilation. The homeopathic treatment Apis mellifica 6CH did counteract pathogenesy effects caused previously in the same plant by Cantharis 4CH meaning that antagonism action did happen. The energy differences resulted from dynamization process of Ruta graveolens 3CH to 12CH homeopathic preparations were detected by plants as shown by CO2 assimilation rate. / Hahnemann no seu livro O Organon , com base em extensa experimentação, relata que medicamentos semelhantes inseridos no organismo têm comportamento antagônico e perdem efetividade. Essa afirmativa tem base na experimentação em seres humanos, mas não tem base experimental em plantas. O trabalho teve como objetivo gerar dados sobre a atuação conflitante de alguns medicamentos homeopáticos e elaborar a base dos conceitos de antagonismo destinada a futuros procedimentos tecnológicos do uso de homeopatia em plantas. Os experimentos foram conduzidos na casa de vegetação e no Laboratório de Homeopatia do Departamento de Fitotecnia da Universidade Federal de Viçosa-MG, no período de agosto a dezembro de 2005. Utilizou-se o esquema de parcela subdividida, tendo nas parcelas os tratamentos e nas subparcelas os tempos de avaliação (10 minutos) com quatro repetições. Foi avaliada a ação dos preparados homeopáticos sobre a planta Ruta graveolens (L.) quantificada pela assimilação de CO2. Os preparados homeopáticos foram aplicados via água destilada na diluição de 4 mL por litro de água destilada e vertido 1 mL da solução a 1 cm do caule e sobre o solo. Durante o ensaio (7 às 11 horas) após verter as soluções foram coletados os dados de assimilação de CO2 em 10 tempos de minuto a minuto no decorrer de 10 minutos consecutivos por tratamento após cada aplicação. A taxa de assimilação de CO2 foi determinada pelo equipamento IRGA (Analisador de Gás no Infravermelho). Os dados foram interpretados por meio da análise de variância, teste de médias e regressão utilizando o programa SAEG (Sistema para Análises Estatísticas). Os preparados homeopáticos provocaram efeito na assimilação de CO2 da planta Ruta graveolens (L.). O preparado homeopático Apis mellifica 6CH anulou a patogenesia causada por Cantharis 4CH significando antagonismo aos efeitos de Cantharis. As diferenças de energia causadas pelo processo físico de dinamização dos preparados homeopáticos Ruta graveolens 3CH a 12CH foram detectadas pelas plantas Ruta graveolens (L.) e retratadas na taxa de assimilação de CO2.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-09-12T17:30:12Z No. of bitstreams: 1 texto completo.pdf: 3868424 bytes, checksum: 30daced885012273e80d7d570fd52b59 (MD5) / Made available in DSpace on 2016-09-12T17:30:12Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3868424 bytes, checksum: 30daced885012273e80d7d570fd52b59 (MD5) Previous issue date: 2014-02-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A crescente demanda por plantas medicinais, como Ruta graveolens L. (Rutaceae), para o isolamento e síntese de compostos de interesse farmacológico levou à superexploração de determinadas culturas, podendo levá-las à extinção e exigindo novas metodologias, como a cultura in vitro, para conservar seu germoplasma e induzir simultaneamente a produção de metabólitos secundários de interesse farmacológico e agroindustrial. No presente estudo avaliou-se o efeito na produção de biomassa e na síntese de metabólitos secundários, como alcaloides e cumarinas em tecidos in vitro de Ruta graveolens, de fitorreguladores como o 2,4-diclorofenoxiacético (2,4-D), ácido naftalenacético (ANA), meta-topolina (m-TP) e cinetina (Kin); e de condições fotoheterotróficas, fotoautotróficas e fotomixotróficas, com ventilação forçada e atmosfera enriquecida com CO2. Estabeleceu-se o efeito de diferentes níveis de trocas gasosas e da irradiância na germinação de sementes, no desenvolvimento, na morfologia e na biossíntese de metabólitos de plântulas de R. graveolens. Diferentes tipos de explantes de R. graveolens induziram alta quantidade de biomassa quando cultivados em meio MS suplementado com ANA, 2,4-D e m-TP. As condições fotoheterotróficas favoreceram a formação de brotos, enquanto que as fotomixotróficas estimularam seu alongamento e a diminuição da hiperidricidade. Tanto a variação dos níveis de trocas gasosas, como da irradiância acarretaram diferenças no desenvolvimento das plântulas, no conteúdo de pigmentos fotossintéticos e mudanças morfológicas nas plantas, principalmente na distribuição das células do mesofilo e na epiderme das folhas. Em todos os tecidos cultivados in vitro foram detectados alcaloides, cumarinas, compostos antracênicos, terpenóides, saponinas e flavonoides. Neste trabalho são reportados pela primeira vez para R. graveolens: o efeito da m-TP na indução de calos e brotos; o crescimento de brotos sob condições fotomixotróficas e fotoautotróficas com ventilação forçada e atmosfera enriquecida com CO2; a influencia dos níveis de trocas gasosas e de luminosidade na morfofisiología de plântulas in vitro de R. graveolens. Contribuindo ao conhecimento sobre o efeito destas variáveis na micropropagação e na produção in vitro de metabólitos secundários de interesse em esta planta medicinal. / The increasing demand for medicinal plants such as Ruta graveolens L. (Rutaceae), for the isolation and synthesis of compounds of pharmacological interest led to overexploitation of certain crops, which can lead them to extinction, requiring new methodologies, such as in vitro culture to conserve their germplasm and simultaneously induce the production of secondary metabolites of pharmacological and agribusiness interest. In the present study was evaluated the effect on biomass production and the synthesis of secondary metabolites, such as alkaloids and coumarins, in in vitro culture of Ruta graveolens, of growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4- D), naphthalene acetic acid (ANA), meta-topolina (m-TP) and kinetin (Kin); and photoheterotrophic, photomixotrophic and photoautotrophic conditions with forced ventilation and CO2-enriched atmosphere. Established the effect of different levels of gas exchange and irradiance on seed germination, development, morphology and biosynthesis of metabolites of seedlings of R. graveolens. Different types of explants of R. graveolens induced high amounts of biomass when grown on MS medium supplemented with NAA, 2,4-D and m- TP. The photoheterotrophic conditions favored the formation of buds, while photomixotrophic stimulated elongation and decreased vitrification. Variations in the levels of gas exchange, as the irradiance led to differences in seedling development in photosynthetic pigments contents and morphological changes in plants, especially in the distribution of cells in the epidermis and mesophyll of leaves. In all R. graveolens tissues cultured, alkaloids, coumarins, antracenic compounds, terpenoids, saponins and flavonoids were detected. This paper first reported for R. graveolens: effect of m-TP on callus induction and shoots; growth of shoots under photomixotrophic and photoautotrophic conditions with forced ventilation and CO2-enriched atmosphere; influences the levels of gas exchange and light in morphophysiology seedling in vitro of R. graveolens. Contributing to knowledge about the effect of these variables in micropropagation and in vitro production of secondary metabolites of interest in this medicinal plant.

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.

Made available in DSpace on 2015-03-26T13:40:06Z (GMT). No. of bitstreams: 1 texto completo.pdf: 812287 bytes, checksum: 02a407733b8a2eb36cdd62e92d1990ff (MD5) Previous issue date: 2007-02-05 / The bioeletrography detects in the eletromagnetic field, diameter and color alterations related with physiological/energetic variations of living organism. With the aim of interpreting bioeletrography of plants treated with homeopatia, an experiment was conducted with subdivided plots in factorial arrangement 2x3: homeopatic medicines (Arnica and Camphora), dinamizations (5, 12 and 30 CH), in subparcels, time of bioeletrographies (5, 10 and 15 minutes after treatment), with 4 replications and one plant per plot. The diluted solution (20 drops of each treatment/100 m L of water) was sprayed in the aerial part. The leaves were photographed by the Kirlian machine (Newton Milhomens standard) with the digitalized photos. The color expansions were quantified by Corel Draw 11 (0 to 100 mm), Yellow (Y), Blue (B), White (W), Magenta (M) and Red (R). Significant differences were detected: colors Red and White (between treatment) color Yellow interation time x dinamization x medicaments and colors Yellow and Magenta interation treatments x time. The colors espansion was bigger in the homeopatizaded plants, Arnica caused greater expansion than Camphora with differences in colors Red and White. The dinamizations were significant in color Yellow, having 5 CH and 12 CH bigger expansion of Yellow color than the 30 CH. Arnica, in 15 minutes, revealed bigger expansion in 5 CH. Camphora caused bigger expansion with 5 CH compared to 30 CH in time 10 minutes (in time 15 min, 12 CH caused the biggest expansion values) the dinamization 30 CH didn t altered the expansion of the color in all times. Compared with the witness there was bigger expansion in Arnica 5 CH and 12 CH in color Yellow, 12 CH in color Red and 30 CH in color White. In color Yellow Arnica caused bigger expansion than Camphora except in dinamizations/times: 5 CH/10 minutes and 12 CH/15 min. The bioeletrography comproved variations in electromagnetic field of plants of Ruta graveolens treated with homeopatic, having the Yellow color bigger possibilities of be the most important indicator of the alterations. / A bioeletrografia detecta, no campo eletromagnético, alterações de diâmetro e cor, relacionadas com variações do estado fisiológico/energético de organismos vivos. Com o objetivo de interpretar padrões de bioeletrografías de plantas tratadas com homeopatia, foi realizado o experimento com parcelas subdivididas em arranjo fatorial 2 x 3: medicamentos (Homeopatias Arnica e Camphora), dinamizações (5, 12 e 30 CH), nas subparcelas tempos de obtenção das bioeletrografias (5, 10 e 15 minutos após tratamento), no delineamento inteiramente casualizado, com quatro repetições e uma planta por parcela. A solução diluída (20 gotas de cada tratamento/ 100 mL de água) foi pulverizada na parte aérea. Os folíolos foram fotografados com a máquina Kirlian (padrão Newton Milhomens). Com as fotos digitalizadas foram quantificadas, pelo Corel Draw11, as expansões das cores (0 a 100 mm): amarela (Am), azul (Az), branca (B), magenta (M) e vermelha (V). Diferenças significativas foram detectadas: cores V e B (entre tratamentos), cor Am (interação tempo x dinamização x medicamento) e cores Am e M (interação tratamentos x tempo). A expansão das cores foi maior nas plantas homeopatizadas. Arnica causou maior expansão que Camphora, com diferenças nas cores V e B. As dinamizações foram significativas na cor Am, tendo 5 CH e 12 CH maior expansão da cor Am que a 30 CH. Arnica, aos 15 minutos, revelou maior expansão em 5 CH. Camphora causou maior expansão com 5 CH, comparada a 30 CH no tempo 10 minutos (no tempo 15 minutos, 12 CH causou os maiores valores de expansão). A dinamização 30 CH não alterou a expansão da cor em todos os tempos. Comparada com a testemunha, houve maior expansão em Arnica: 12 CH, nos tempos 5 e 10 minutos, na cor Am; 12 CH, na cor V; e 30 CH, na cor B. Na cor Am, Arnica causou expansão maior que Camphora. A bioeletrografia comprovou variações no campo eletromagnético de plantas de Ruta graveolens tratadas com homeopatia, tendo a cor amarela maiores possibilidades de ser a indicadora mais importante das alterações.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-20T13:17:23Z No. of bitstreams: 1 Marluce de Souza.pdf: 1603093 bytes, checksum: a2ee90b596164ac534147bc4eb9c2e4c (MD5) / Made available in DSpace on 2016-10-20T13:17:23Z (GMT). No. of bitstreams: 1 Marluce de Souza.pdf: 1603093 bytes, checksum: a2ee90b596164ac534147bc4eb9c2e4c (MD5) Previous issue date: 2007-02-27 / The Ruta graveolens is a plant used in the popular medicine. It’s known that it has antiseptic, stimulating, abortive, anti hemorrhagic, anti-inflammatory and cicatrization actions. In it’s composition we can find active substances as cumarins, terpenoids, alkaloids, flavonoids, aliphatic acids and ketones. Based on this information’s, was developed a research to evaluate the efficiency of Ruta graveolens ointment over cicatrization of skin wounds in dogs. Eight dogs with skin wounds attended at the Veterinary Hospital of the UFRPE were used, after a clinical and laboratorial preevaluation. The wounds were submitted to clinical, bacteriological and histopathological evaluations. For the clinical evaluation, was used a descriptive chart in which the hyperemia and edema parameter were observed in the wounds until the 3RD day, the granulation e cicatrization tissues happened since the 6Th day of treatment and the cicatrization occurred in the end lf the 28Th day. The bacteriological test was done in two times (To and T1 respectively) and the beginning of the treatment and after 24 hours. The most frequently isolated bacteria’s in the wounds were Streptococcus sp, Staphylococcus sp, Shigella sp, Proteus sp, Pseudomona sp, Citrobacter freundii, Klebsiella oxytoca and Enterobacter cloacae. The wound’s histological evaluations were done by biopsy on the 7Th, 14Th and 28Th days, showing on the 7Th day a progressive cellular reorganization with no presence of necrotic tissue and crusts with the formation of granulation tissue, with considerable neovascularization. On the 14th day, was observed active fibroblasts, besides the well organized granulation tissue, and over that, the migration of epithelial cells. On the 28Th day, the granulation tissue was more evident and with the presence of epithelization. In all three evaluations there was in common the presence of non reactive neutrophils and macrophages. So it was concluded that the Ruta graveolens ointment helps the cicatrization process when used in dog’s with skin wounds. / A Ruta graveolens é uma planta utilizada na medicina popular com fins fitoterapêuticos e relata-se que ela possui ação anti-séptica, estimulante, abortiva, anti-hemorrágica, antiinflamatória e cicatrizante. Na sua composição encontram-se substâncias ativas como cumatinos, terpenóides, alcalóides, flavonóides, ácidos alifáticos e cetonas. Mediante esses relatos desenvolveu-se um trabalho cujo objetivo foi o de avaliar a eficiência da pomada de Ruta graveolens sobre a cicatrização de feridas cutâneas em cães. Foram utilizados oito cães com feridas cutâneas, mediante prévia avaliação clínica e laboratorial, oriundos do atendimento ambulatorial do Hospital Veterinário da Universidade Federal Rural de Pernambuco. As lesões cutâneas foram submetidas à avaliação clínica, bacteriológica e histopatológicas. Para avaliação clínica utilizou-se uma tabela descritiva onde os parâmetros hiperemia e edema foram observados nas feridas até o 3º dia; os tecidos de granulação e cicatrização ocorreram a partir do 6º dia do tratamento. A cicatrização das feridas ocorreu ao final do 28º dia. O exame bacteriológico foi realizado em dois tempos To e T1 respectivamente antes do inicio tratamento e após 24 horas. Identificou-se Streptococcus sp, Staphylococcus sp, Shigella sp, Proteus sp, Pseudomona sp, Citrobacter freundii, Klebsiella oxytoca e Enterobacter cloacae como sendo as bactérias isoladas e identificadas mais freqüentes nas lesões. As avaliações histopatológicas das feridas foram realizadas por biópsia no 7º, 14º e 28º dias de tratamento, revelando no sétimo dia uma progressiva reorganização celular sem presença de tecido necrótico e crostas com formação insipiente do tecido de granulação, porém com considerável neovascularização. No 14º dia foi possível observar-se fibroblastos ativos além do tecido de granulação bem organizado e sobre este, migração de células epiteliais aos 28º observou-se tecido de granulação evidente e repitelização. Em comum nas três avaliações foi a presença de neutrófilos e macrófagos não reativos. Desta forma conclui-se que a pomada de Ruta graveolens favorece o processo de cicatrização quando utilizada em feridas cutâneas de cães.

Les furocoumarines sont des métabolites secondaires qui dérivent de la voie de biosynthèse des phénylpropanoïdes. Ces phytoalexines interviennent notamment dans les mécanismes de défense des plantes tels que la résistance aux bioagresseurs. Le déterminisme moléculaire de cette voie de biosynthèse est encore mal connu mais il a néanmoins été démontré depuis les années 1960 que les enzymes catalysant les différentes étapes de la voie la synthèse des furocoumarines appartenaient à différentes familles. Les travaux présentés dans ce document se sont focalisés sur deux familles d'enzymes : les dioxygenases oxoglutarate dépendantes et les cytochromes P450s. La première étape de la voie de biosynthèse des furocoumarines consiste en une ortho-hydroxylation du p-coumarate qui mène à la formation de l'umbelliférone. Basé sur des travaux récents sur Arabidopsis, six gènes codants pour des dioxygénases ont été isolés chez Ruta graveolens, Citrus limetta et Pastinaca sativa. Les protéines correspondantes présentent plus de 58% d'identité avec la Féruloyle 6' Hydroxylase (F6'H) d'A. thaliana. La caractérisation fonctionnelle de ces enzymes a été réalisée dans un système d'expression hétérologue procaryote. Sur les 6 enzymes, trois n'ont pu être exprimée efficacement, et deux présentent une activité F6'H similaire à celle décrite pour A. thaliana. La dernière enzyme dispose de caractéristiques nouvelles non décrites à ce jour. Elle est en mesure de réaliser l'hydroxylation du féruloyle coA et du p-coumaroyle coA. Ces études in vitro ont été complétées par une exploration des fonctions de la protéine dans la plante. Une analyse fine du profil d'expression du gène a permis de mettre en évidence une expression qui est en corrélation avec le niveau de production d'umbelliférone. La fonction de la protéine a également été validée par une analyse des produits formés dans des feuilles de Nicotiana benthamiana transformées transitoirement. Les cytochromes P450 catalysent 60% des réactions de la voie de biosynthèse de furocoumarines. Un travail de criblage fonctionnel de cytochromes P450 identifiés au préalable chez Ammi majus et Thapsia garganica a été entrepris. Les analyses de bio-informatiques et les modifications apportées au niveau du mode opératoire pour l'expression dans la levure ont permis d'émettre des hypothèses concernant le rôle de certains P450 candidats. Ces travaux exploratoires et préliminaires font supposer de nouvelles conjectures relatives à cette voie de biosynthèse / Furanocoumarins are secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These phytoalexins are especially involved in plant defense mechanisms against insects or phytopatogenous fungi and bacteria. The molecular control of this biosynthetic pathway is still poorly understood even though it has been demonstrated since the 1960s that enzymes catalyzing the different steps are belonging to different enzymatic families. The work presented here is focused on two enzyme families: oxoglutarate dependent dioxygenases and cytochrome P450s. The first step in the furanocoumarins biosynthetic pathway is the ortho-hydroxylation of p-coumarate, which leads to the formation of umbelliferone. Based on a recent work done on Arabidopsis, we isolated six genes encoding dioxygenases from Ruta graveolens, Citrus limetta and Pastinaca sativa. The corresponding proteins share more than 58% identity with the A. thaliana féruloyle 6' Hydroxylase (F6'H). The functional characterization of these enzymes was performed in a prokaryotic heterologous expression system. Of the six enzymes, three could not be functionnaly expressed and two exhibited a similar F6'H activity as described for A. thaliana. The last enzyme has new properties not described to date. It is able to achieve both hydroxylation of féruloyle CoA and p-coumaroyle CoA. These in vitro studies were completed by a functional exploration of the protein in planta. A detailed analysis of the gene expression pattern highlighted a link with the level of umbelliferone synthesis. The function of the protein was also confirmed by an analysis of the products formed in transiently transformed Nicotiana benthamiana leaves. Cytochrome P450s catalyze 60% of the reactions of the furanocoumarin biosynthetic pathway. Therefore, a functional screening of cytochrome P450 previously identified in Ammi majus and Thapsia garganica was undertaken. The bioinformatic analyses and the changes undertaken in the procedure for expression in yeast allowed drawing hypotheses on the function of some of these P450 candidates. These exploratory and preliminary experiments allowed suggesting new hypotheses about the biosynthetic pathway

Les furanocoumarines (FCs) sont des métabolites secondaires principalement synthétisés chez quatre familles botaniques et dérivent de la voie de biosynthèse des phénylpropanoïdes. Ces phytoalexines interviennent dans les processus de défense de la plante et présentent un fort potentiel thérapeutique. Des travaux réalisés dans les années 1960 sur des cultures cellulaires en parallèle de l’utilisation de précurseurs radiomarqués ont permis de démontrer que de nombreuses enzymes impliquées dans cette voie appartenaient à la famille des cytochromes P450 (P450s). Seules deux d’entre elles avaient pu être identifiées d’un point de vue moléculaire au début de ce travail de thèse. Afin de générer des informations concernant le génome de plantes productrices de FCs, nous avons fait séquencer les ARNm extraits de feuilles de Pastinaca sativa, de Ruta graveolens et de Cullen cinereum. L’analyse in silico de ces trois banques de données a permis d’identifier près de 800 fragments d’ADNc codants pour des P450s. Des travaux antérieurs réalisés au laboratoire et l’analyse comparative des transcriptomes de ces 3 plantes nous ont amenés à nous focaliser sur la sous-famille CYP71AZ au travers d’une étude fine de CYP71AZ3 et CYP71AZ4. La caractérisation fonctionnelle de ces enzymes a été réalisée dans un système d’expression hétérologue eucaryote : Saccharomyces cerevisiae. Les résultats obtenus ont permis de montrer que CYP71AZ4 avait une spécificité de substrat assez large puisqu’elle pouvait métaboliser au moins une FC et 4 coumarines. L’analyse et la comparaison des constantes cinétiques pour chacun de ces substrats indiquent néanmoins que le psoralène est le substrat préférentiel. La caractérisation fonctionnelle de CYP71AZ3 a mis en évidence que cette enzyme pouvait hydroxyler l’esculétine, une coumarine, mais ne jouait aucun rôle dans la synthèse de FCs. Ces travaux mettent en évidence la diversité fonctionnelle au sein d’une même sous-famille enzymatique et permettent d’émettre des hypothèses nouvelles quant à l’apparition de cette voie de biosynthèse chez les Apiacées d’une part, et chez les autres familles botaniques d’autre part / Furanocoumarins (FCs) are secondary metabolites mainly synthetized in four botanical families deriving from the phenylpropanoid biosynthetic pathway. These phytoalexins are involved in plant defense mechanisms and present strong therapeutic potential. Early studies in the 1960s based on cell cultures and the use of radiolabeled precursors have shown that many enzymes involved in this pathway belong to the cytochrome P450 family (P450s). Only two of them had been identified from a molecular point of view at the beginning of this thesis. In order to generate information regarding the genome of plants producing FCs, we sequenced the mRNA extracted from leaves of Pastinaca sativa, Ruta graveolens, and Cullen cinereum. In silico analysis of these three libraries identified nearly 800 cDNA fragments encoding for P450s. Previous studies in the laboratory and comparative transcriptome analysis of these three plants have led us to focus on the subfamily CYP71AZ through a detailed study of CYP71AZ3 and CYP71AZ4. Functional characterization of these enzymes was performed in an eukaryote heterologous expression system: Saccharomyces cerevisiae. The results showed that CYP71AZ4 had a broad substrate specificity enough as it could metabolize one FC and 4 coumarins. The analysis and comparison of the kinetic constants for each of these substrates indicate, however, that the preferred substrate is psoralen. The functional characterization of CYP71AZ3 showed that this enzyme could hydroxylate esculetin, a coumarin, but played no role in the synthesis of FCs. This study highlights the functional diversity within a single enzyme subfamily and allows to issue new hypotheses about the emergence of this biosynthetic pathway in Apiaceae on one hand, and among other botanical families on the other hand

Submitted by (edna.saturno@ufrpe.br) on 2016-11-08T14:53:32Z No. of bitstreams: 1 Zelia Ferraz Mendes.pdf: 315816 bytes, checksum: 51bdc2df924254a5384aebfb6ff6656d (MD5) / Made available in DSpace on 2016-11-08T14:53:32Z (GMT). No. of bitstreams: 1 Zelia Ferraz Mendes.pdf: 315816 bytes, checksum: 51bdc2df924254a5384aebfb6ff6656d (MD5) Previous issue date: 2007-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / With the objective of evaluating the antimicrobian activity of the Ruta graveolens dye and ointment on isolated bacteria of dogs’ cutaneous wounds, they were provoked by several traumas and considered polluted or infected and proceeding from ambulatorial casuistry of the Veterinary Hospital of DMV of UFRPE. The wounds were submitted to the collecting of bed material of the cruenta area through swab’s for bacterial cultures accomplishment. In the first phase, the isolated bacteria were stocked in Agar Nutrient and submitted to the in vitro evaluation test of the antimicrobian activity by using the Ruta graveolens’ extract with alcoholic content to 65% and their serial dilutions (100, 10-1, 10-2 and 10-3). The isolated bacteria were Staphylococcus sp, Streptococcus sp, Proteus sp, Escherichia coli. It was just observed inhibited effect in the bacterial growth in the concentration 1:1 of Ruta graveolens. In the second phase, the wounds material was collected before the cleaning and 48 hours after the treatment with Ruta graveolens ointment. Before the observed results, it is ended that Ruta graveolens dye presented antimicrobian activity in vitro when it is used in the proportion of 1:1 under the ointment form for the isolated bacteria of dogs’ cutaneous wounds and it could be an option of a phytoterapical to aid in the treatment of polluted or infected cutaneous losses. / Com o objetivo de avaliar a atividade antimicrobiana da tintura e pomada da Ruta graveolens sobre bactérias isoladas de feridas cutâneas em cães, foram utilizadas feridas provocadas por traumas diversos e consideradas contaminadas ou infectadas provenientes da casuística ambulatorial do Hospital Veterinário do DMV da UFRPE. As feridas foram submetidas à coleta de material do leito da área cruenta, através de swab’s, para realização de culturas bacterianas. Na primeira fase, as bactérias isoladas foram estocadas em Ágar Nutriente e submetidas ao teste in vitro para avaliação da atividade antimicrobiana utilizando-se o extrato da Ruta graveolens com teor alcoólico a 65% e suas diluições seriadas (100, 10-1, 10-2 e 10-3). As bactérias isoladas foram Staphylococcus sp, Streptococcus sp, Proteus sp, Escherichia coli, sendo observado efeito inibitório no crescimento bacteriano apenas na concentração 1:1 de Ruta graveolens. Na segunda fase, foi coletado material das feridas antes da limpeza e após 48 horas do tratamento com pomada de Ruta graveolens. Diante dos resultados observados, conclui-se que tintura da Ruta graveolens apresentou atividade antimicrobiana in vitro quando utilizada na proporção de 1:1 e sob a forma de pomada para as bactérias isoladas de feridas cutâneas de cães, podendo ser uma opção de um fitoterápico para auxiliar no tratamento de perdas cutâneas contaminadas ou infectadas.

M.Tech. / As computers become more common in the work environment, more people are developing symptoms linked to long-term computer use. These include visual and eye-related symptoms as well as musculo-skeletal symptoms (Sheedy & Shaw-McMinn, 2003; Yan, Hu, Chen & Lu, 2007). This collection of symptoms is known as Computer Vision Syndrome (CVS). There are various allopathic modes of treatment available which are limited in terms of their long term use and side effects. Yan and colleagues (2007) indicate that a correctly designed, ergonomically efficient work place plays a significant role in limiting the development of CVS symptoms. Depending on the reasons for CVS and the symptoms associated with it, alternative treatment options may be appropriate (Yan, et al., 2007). Homoeopathy offers alternate treatment, however no extensive research has been done on the homoeopathic treatment of this condition. The aim of this study was to determine the efficacy of the homoeopathic remedy Ruta graveolens 6cH together with ergonomic interventions in the work environment in the treatment of Computer Vision Syndrome. This was done by means of a selection questionnaire (APPENDIX B) and a symptom index form (APPENDIX C), rating the severity and frequency of symptoms. This research study is paired to a related study which investigated the efficacy of Ruta graveolens 6cH without ergonomic interventions. These two studies can later be compared to determine whether the remedy Ruta graveolens 6cH by itself is sufficient in treating computer vision syndrome. This study was approved by the Higher Degrees Committee (HDC48/2009) and the Academic Ethics Committee (AEC50/09) at the University of Johannesburg on the 19 June 2009. Thirty one people joined the study. They were recruited by means of flyers and advertisements (APPENDIX E) placed at the University of Johannesburg as well as corporate companies. Participation was voluntary and by means of informed consent (APPENDIX A). Participants were required to fill in a selection questionnaire (APPENDIX B) to see if they qualified for the study. It was a double-blind placebo controlled study in which participants were placed into one of 2 groups. Both groups were required to change the layout of their work environment in order to ensure ergonomic efficiency at their work station (APPENDIX D). Of the two groups, one was dispensed vi the medicated prescription and the other an identically presented unmedicated prescription. Participants were requested to take the medication twice a day for 4 weeks. They were also required to complete a symptom index form (APPENDIX C) on a weekly basis. These forms were collected by the researcher at the end of the four weeks. There was no risk associated with taking the medication. Participants’ right to privacy was adhered to and they were free to withdraw from the study at any time. Of the 31 people who joined the study, 29 completed the study.

M. Tech. / The homoeopathic remedy, Ruta graveolens, is used for the treatment of eyestrain and eye related symptoms caused by the over use of the eyes (Kent, 2006). The aim of this study was to determine the efficacy of the homoeopathic remedy Ruta graveolens 6cH in the treatment of Computer Vision Syndrome. This study can be compared to related studies by means of the symptom index form (Appendix C). This double-blind study included thirty participants between the ages of 20 and 35. The participants had to be regular users of a visual display terminal for a minimum of three hours per day. The participants also had to experience at least two or more eye or vision related symptoms due to VDT use for at least one month prior to the start of the study. During the first consultation, each participant was tested for any refractive errors by a qualified optometrist. The participants were divided into two groups of fifteen each. One group received the remedy, Ruta graveolens 6cH, while the other group received the placebo. The participants were required to take four pillules twice a day for the duration of the study. The data was statistically analysed by means of the Mann-Whitney u Test, Friedman Analysis of Variance by Ranks Test and Wilcoxon Signed-Rank Test. Crosstabs were used to check for confounding variables. The two groups, the placebo and the treatment group were tested against each other. The findings from the data illustrated that in the treatment group there was a statistically significant improvement in the severity of symptoms in seven of the fourteen symptoms over the duration of the study, while five of the fourteen symptoms of the placebo group had a statistically significant improvement in severity over the four week period. Overall the severity of the treatment group had an improvement of 42% compared to the 28% improvement of the placebo group. Both groups showed a reduction of the frequency of symptoms over the duration of the study.