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Identification and characterization of a novel triyne and cinnamate 4-hydroxylase in Helichrysum aureonitens Sch. Bip.Ziaratnia, Sayed Mahdi 17 October 2009 (has links)
For centuries people have used plants as medicine or food additives with varying success to cure and prevent diseases. Written records about medicinal plants date back at least 5 000 years to the Sumerians. According to World Health Organization (WHO) around 80 % of the population in developing countries is dependent on herbal medicine for basic healthcare needs. Even at the dawn of the twenty-first century, 11 % of the 252 drugs considered as basic and essential by WHO were exclusively of flowering plant origin. The genus Helichrysum, belongs to the family Asteraceae and is represented by approximately 600 species in Africa, of which 244 species are indigenous in South Africa. In Helichrysum aureonitens, galangin is one of the flavonol compounds with good medicinal properties. H. aureonitens was targeted to be enhanced via cell suspension culture to potentially produce valuable natural products. In ethanol extracts of cell suspension cultures, galangin was not detected even though it was present in the leaves of the intact plants. Some other compounds were induced in higher amounts in the cells of H. aureonitens suspension cultures when compared to that produced in the intact plants. To find out the reasons for the absence of galangin in the cells of H. aureonitens suspension cultures, some of the intermediates of the 4’-OH biosynthetic pathway for production of flavonols were analyzed by GC-MS, including cinnamic acid, p-coumaric acid, naringenin and kaempferol. None of these were detected in the H. aureonitens cell suspension cultures. The major compound from H. aureonitens cell suspension cultures was isolated and identified as a new chlorophenol compound named 4-chloro-2-(hepta-1,3,5-triyne-1-yl)-phenol (a triyne). This triyne has previously been proposed as being an intermediate in the acetylene biosynthetic pathway in Helichrysum spp., however only the methyl ether form had previously been isolated from the roots of H. coriaceum. The triyne isolated from the H. aureonitens cell suspension cultures in the present study was detected in intact plant extracts, but at very low concentrations. Results of the anti-tuberculosis assay of the cell suspension culture extracts and the triyne showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the ethanol extract of cell suspension cultures against Mycobacterium tuberculosis H37Rv were found to be 1.0 mg/ml and 2.0 mg/ml respectively and the triyne was not active at 200 µg/ml. The ethanol extract of the cell suspension cultures and the triyne were also evaluated for their cytotoxicity against monkey kidney Vero cells and human prostate epithelial carcinoma (DU145) cell lines. The inhibitory concentrations (IC50) of the crude extract and the triyne was found to be 12.11 and 1.51 µg/ml against the Vero cells respectively. The crude extract and the triyne showed similar activity in the prostate cancer cell lines by exhibiting IC50 values of 3.52 and 2.14 µg/ml respectively. The triyne therefore warrants further investigation for its potential as an anticancer drug. Flavonoids represent the major phenolic compounds which are responsible for the medicinal properties in the Helichrysum genus. Some of flavonols, including kaempferol, quercetin, and galangin are also present in H. aureonitens. In this study both galangin and kaempferol (containing a 4’-OH group) were detected in leaf samples of H. aureonitens. But GC-MS analysis of the leaf samples of H. aureonitens did not show the existence of biosynthetic intermediates such as p-coumaric acid and naringenin (compounds having a 4’-OH) while cinnamic acid and some other compounds with no OH at the 4’ position on the B ring, were detected. The chemical structure analysis of the isolated compounds showed that they are pinocembrin chalcone, pinocembrin, pinobanksin and galangin, all containing no OH group at the 4’ position. This indicates that some part of the 4’-OH biosynthetic pathway for 4’-OH flavonoids is not functional in H. aureonitens. Since the only (yet identified) enzyme responsible for hydroxylation at the 4’ position on the B ring is cinnamate 4-hydroxylase (C4H), it can be postulated that C4H might be able to hydroxylate other substrates in H. aureonitens plants. One copy of C4H was isolated and cloned from H. aureonitens. It has 1518-base pairs (including stop codon, TAA) and an open reading frame encoding a 506-amino-acid polypeptide. It showed the highest homologies to Echinacea angustifolia (Asteraceae) C4H with 83.6 % identity on the nucleotide level but 93 % identity on the amino acid level. The genomic DNA sequence of the isolated C4H from H. aureonitens indicates the presence of three introns with a longer size compared to the Arabidopsis thaliana C4H gene structure. The presence of the first intron has not been reported before in the C4H gene from other plants and it is therefore a new finding from the isolated C4H in H. aureonitens. To check the putative isolated C4H, the full length cDNA of C4H was isolated from H. aureonitens and for the first time integrated in a secreted expression vector, pPICZáC, and transformed into Pichia pastoris. After the 48 hrs induction protein was collected, precipitated by ammonium sulphate and finally column purified. The results of SDS-PAGE electrophoresis and Western blot showed the expression of a protein with a size of 50-60 kDa. The calculated mass of C4H with regarding to a polyhistidine tag is about 60.5 kDa. The secreted expression was found as an effective system for the production of a soluble C4H protein with easy purification. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted
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CLONING OF KNOWN AND NOVEL CYTOCHROME P450S IN SCUTELLARIA BAICALENISBrundage, Meghan Elizabeth 11 October 2001 (has links)
No description available.
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Modification de la synthèse des furocoumarines chez Ruta graveolens L. par une approche de génie métabolique / Functional exploration of the biosynthesis pathway of phenylpropanoids of Ruta graveolens by metabolic engineeringDoerper, Sébastien 12 November 2008 (has links)
La rue officinale (Ruta graveolens L) est une plante connue comme étant particulièrement riche en métabolites secondaires et produisant notamment des molécules d’intérêt pharmaceutique comme les furocoumarines. Nous avons tenté par une approche de génie métabolique d’augmenter la teneur en furocoumarines produites dans les plantes. La mise en place de telles approches nous a également permis de mieux comprendre les mécanismes de régulation de la voie de biosynthèse des phénylpropanoïdes. Pour atteindre ces objectifs nous avons transformé la rue avec différents gènes placés sous le contrôle d’un promoteur constitutif fort, le promoteur 35S du CaMV. Pour chaque série de transformants nous avons étudié la teneur en furocoumarines et analysé les variations de composés phénylpropanoïdes (rutine, umbelliférone, ferulate, scopolétine). Parallèlement à cette analyse métabolique, une corrélation a été réalisée avec le niveau d’expression des transgènes et de certains endogènes par l’utilisation d’approche de PCR quantitative. Les séries de plantes transgéniques surexprimant les gènes codants pour la Coumaroyl ester 3’-Hydroxylase de rue (CYP98A22) et d’A. thaliana (CYP98A3) présentent toutes les deux une augmentation significative d’une facteur 3 de la teneur en furocoumarines. Par contre si les premières sont caractérisées par une diminution de la production en rutine et en umbelliférone, les secondes présentent une augmentation importante de la teneur en Scopolétine et en umbelliférone. Ces résultats suggèrent la coexistence de deux C3’H chez R. graveolens ayant des fonctions différentes, l’une d’entre elles étant impliquée directement ou non dans la synthèse de scopolétine. Si la transformation génétique de rues avec des gènes de la famille CYP98A induit des modifications du métabolisme secondaire, la surexpression d’un gène spécifique à la voie de biosynthèse des furocoumarines (gène cyp71AJ1, codant pour la psoralène synthase d’A. majus) permet d’augmenter uniquement la teneur en furocoumarines (X4). L’ensemble de ces travaux a permis de montrer l’intérêt d’une approche de génie métabolique pour générer des plantes présentant un intérêt potentiel pour la production de molécules d’intérêts pharmaceutiques / Garden Rue (Ruta graveolens L.) is a plant known as being particularly rich in secondary metabolites and in particular producing molecules of pharmaceutical interest like furocoumarines. By the use of a metabolic engineering approach, we tried to increase the content of furocoumarines produced in these plants but also to better understand the regulation mechanisms of the phenylpropanoïd biosynthesis pathway. To achieve these goals we transformed Ruta plants with various genes placed under the control of a strong constitutive promoter, CaMV 35S promoter. The plants we obtained were analyzed for their ability to overproduce furocoumarines but also other phenylpropanoïds like ferulate, umbelliferone, scopoletine or rutin. Using Real Time PCR experiments, a correlation was carried out with the level of expression of each transgene and several endogenous genes. Plants overexpressing either the Ruta or the Arabidopsis Coumaroyl ester 3 '-Hydroxylase (CYP98A22 and CYP98A3 respectively) display both a significant increase (3 time level) of the furocoumarin. However if the S-98A22 plants are characterized by a reduction in the production of rutin and umbelliferone, S-98A3 transgenic plants display a significant increase scopoletine and umbelliferone content. These results suggest the coexistence of two C3'H having different functions in Ruta. One of them might be involved more specifically in the synthesis of scopoletine. If the transformation of Ruta with genes belonging to the CYP98A family generates an enlarged of the secondary metabolism, we also showed that the overexpression of a gene belonging to the furocoumarins biosynthesis pathway (CYP71AJ1, the psoralen synthase) allowed a specific stimulation. Indeed a 4 time increase of the content of furocouramins was noticed in these transgenic plant lines. This work made it possible to make evidence of the interest of a metabolic engineering approach to generate plants of interest for the production of pharmaceutical molecules
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Contribution à l'étude des P450 impliqués dans la biosynthèse des furocoumarines / Study of P450 involved in furocoumarin biosynthesisLarbat, Romain 30 May 2006 (has links)
Les furocoumarines sont des phytoalexines offrant un potentiel thérapeutique important. Les travaux présentés ici portent sur les cytochromes P450 participant à leur voie de biosynthèse. Une étude «structure-fonction» de la cinnamate-4-hydroxylase (C4H) a été réalisée pour identifier les déterminants de la faible sensibilité de la C4H de Ruta graveolens (CYP73A32) au psoralène. Deux régions protéiques semblent impliquées dans l’inactivation différentielle entre CYP73A32 et CYP73A1. L’une, entre les résidus 31 et 58, est responsable de l’affinité pour le psoralène. L’autre, entre les résidus 229 et 379, contrôle la vitesse d’inactivation. La caractérisation de nouveaux P450 de la biosynthèse des furocoumarines a été entreprise. D’une part, plusieurs ADNc partiels ont été clonés chez Ruta graveolens. D’autre part, CYP71AJ1 isolé chez Ammi majus, a été caractérisé comme étant une psoralène synthase. La spécificité de CYP71AJ1 pour la marmésine a été approchée par l’étude d’un modèle 3D. Mots clés : cytochrome P450, psoralène synthase, cinnamate-4-Hydroxylase, C4H, métabolite secondaire, Ruta graveolens, Ammi majus, inactivation autocatalytique, furocoumarines, psoralène, (+)-marmésine, Modélisation 3D, (+)-columbianetine / Furocoumarins are phytoalexins known as efficient therapeutic agents. The work reported here focuses on cytochromes P450 involved in their biosynthesis pathway. A “structure-function” study was realized to understand how C4H from Ruta graveolens (CYP73A32) can resist to psoralen mechanism-based inactivation. Two parts of the protein seems involved in the differential susceptibility of CYP73A32 and CYP73A1 to psoralen. The first, between amino acids 31 and 58, defines differential affinity to psoralen. The second between residues 229 and 379 controls inactivation kinetic. The second part of this work was devoted to cloning and identification of new P450 involved in furocoumarin biosynthesis. On the one hand, several partial cDNA were cloned from Ruta graveolens. On the other hand, CYP71AJ1, cloned from Ammi majus, was identified as a psoralen synthase. The specificity of CYP71AJ1 for marmesin was approached by the study of a 3D model.
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