Dissertations / Theses on the topic 'Podophyllum'

Lignans are the most important secondary metabolites known in Podophyllum hexandrum and Diphylleia cymosa. Tissue cultures studies were carried out in order to preserve and increase these germplasms. Somatic embryogenesis in both species was obtained and confirmed histologically. In D. cymosa, somatic embryos were induced from leaf and petiole-derived callus. The formation of abnormal embryos, with fused cotyledons, was influenced by the growth regulators used. Embryo maturation was confirmed histochemically through the identification of starch granules in the cotyledons. Regeneration was dependent on the culture media. Somatic embryogenesis in P. hexandrum was achieved through embryogenic cell suspension cultures from root-derived callus. Organogenesis via adventitious bud formation led to plant regeneration in liquid medium. Phenotypically normal plants were recovered. The regenerant showed the somatic chromosome number of 2n=2x=12, although some chromosomes were morphologically abnormal. The recalcitrance of P. hexandrum towards Agrobacterium-mediated transformation was demonstrated with different strains of both A. tumefaciens and A. rhizogenes. The presence of cytotoxic lignans in P. hexandrum may have a role in the inactivation of the bacteria. Aryltetralin lactone lignans isolated from rhizomes and roots of P. hexandrum and characterized by spectroscopic methods were used as standards in phytochemical studies of D. cymosa. Enzymic hydrolysis of the lignan glycosides, followed by reverse-phase HPLC, allowed the screening of lignans in leaf tissues, calli and cell suspension cultures. The antitumour lignin, podophyllotoxin was detected in young leaves of cultivated plants and. for the first time, in vitro petiole-derived calli. Flavonoids were investigated in leaf extracts of Passiflora edulis, P. incarnate and their somatic hybrids. Fractionation of the crude extracts of P. incarnata and the somatic hybrid SHI led to the isolation of compounds with skeletal type of C-glycosylflavones. Isoorientin was identified in P. edulis. whilst vitexin was found in P. incarnata by TLC. All the somatic hybrids showed similar consistant flavonoid banding profiles. Isoorientin and vitexin were detected in the somatic hybrids. HPLC of the parental species revealed a distinct pattern of flavonoids. Isoorientin was clearly detected in P. edulis, whereas isovitexin was present in both species. The fingerprint patterns of the HPLC separations of the flavonoids were similar in all the somatic hybrids, probably due to the clonal nature of the plants analysed. They appeared to be more closely related to P. incarnata than to P. edulis. However, they also exhibited flavonoids intermediate between those of the parental species. This is the first report of the biosynthesis of isoorientin and isovitexin in these novel somatic hybrids and could provide information about their inheritance.

碩士
長庚大學
化工與材料工程研究所
92
The medicinal plant is currently one of the hot research topics. Due to the complicated structures of some medicinal materials, most cancer- resisting substances can not be produced by organic synthesis and must be produced by cell culture . Therefore, the objective of this research is to cultivate the plant tissue cells which can secrete an anticancer composition — podophyllotoxin . The materials used in these experiments include podophyllum pleianthum plants from Yang Ming Shan and the callus and suspension cells of the root , steam and leaf from Sitoul offered by the Development Center of Biotechnology. We disinfect the surface of the explant from Yang Ming Shan, cultivate it on a medium based on B5 basal medium supplemented with 1ppm 2,4-D and 0.1ppm kinetin at dark. Callus can thus be greatly produced. The best induced explant is from leaf and its induced rate can reach 92.5%. HPLC analysis indicates that the podophyllotoxin content of the callus from Yang Ming Shan is higher than that from Sitoul. The callus induced from steam and leaf can produce 0.00367% and 0.00341% (g/g) podophyllotoxin, respectively. Among the suspension cells of the root, steam and leaf from the Development Center of Biotechnology, this study shows that the suspension cells induced from root contain a higher content of podophyllotoxin than those induced from steam and leaf. The content of podophyllotoxin induced from root can reach 0.002% (g/g ).

碩士
國立臺灣大學
生化科技學系
99
Podophyllotoxin is an important precursor of anti-tumor drug. Podophylloum pleianthum Hance, an endemic species in Taiwan, is a nature source of podophyllotoxin. The biosynthesis pathway of podophyllotoxin starts from two molecules of coniferyl alcohol radical coupled directionally forming one molecule of (+)-pinoresinol with the involvement of dirigent protein (DP). The cDNA of dirigent protein was cloned from P. pleianthum Hance total RNA by RT-PCR. The length of the dirigent protein cDNA was 579 base pairs and the predicted molecular weight of the dirigent protein was 22 kDa. The cDNA sequence was aligned with DP of Podophyllum peltatum. The identity was 97%. The identity and similarity of amino acid alignment were 96% and 98% respectively. The dirigent protein could not express functionally in recombinant E. coli system. Nicotiana tabacum hairy root for heterologous expression was taken instead. The fluorescent reporter protein ZsYellow was observed by fluorescent microscope. The dimerization reaction of coniferyl alcohol in vitro was performed. Pinoresinol was first converted and was then transferred to other metabolites.

碩士
國立臺灣大學
農業化學研究所
91
Podophyllum pleianthum Hance has been used for snake bites and healing of wounds in Taiwan. Its secondary metabolite─podophyllotoxin has proven to be an effective anti-tumor agent. Due to slow-growing habit and over-collection, P. pleianthum is listed as an endangered species. The objective of this research is to establish callus and system of suspension cell culture, and to evaluate the content of podophyllotoxin. Leaf, leafstalk, stem (with shoots tip) and root were used as starting material for callus induction. Our results revealed that B5 medium containing 10 mg/L NAA and 0.1 mg/L Kinetin shows good callus induction rate in dark environment. On the other hand, B5 medium containing 15 mg/L NAA and 0.1 mg/L Kinetin shows good callus induction rate in low light condition. Proliferation rate of cells from leaf and stem (with shoots tip) increased 14.2 and 5.4 fold in modified NP medium containing 10 mg/L NAA. The extraction of callus induced from the four different explants was prepared by 3 mL of 80 % methanol under 30 min sonication. It was found that podophyllotoxin can only be detected from the extraction of callus induced from root explant. This metabolite showed a maximum absorption at 205 nm and the retention time at 10.10 min, which was identical to authentic compound of podophyllotoxin. The extraction of cells cultured in a liquid modified NP medium had more complex peaks in HPLC chromatogram than that of callus cultured in a soild B5 medium. These results revealed that some other unknown metabolites were simultaneously produced in the suspension cell culture.

碩士
國立臺灣大學
微生物與生化學研究所
96
Podophyllotoxin possesses strong tumor-specific cytotoxicity, and its chemical derivatives have been employed in clinical cancer treatment. From Podophyllum pleianthum Hance, pinoresinol lariciresinol reductase (PLR) gene was cloned, which can convert pinoresinol to lariciresinol and consequently to secoisolariciresinol, and secoisolariciresinol dehydrogenase (SDH) gene, which can convert secoisolariciresinol to matairesinol. The total RNA of Podophyllum pleianthum Hance was used as the material for sdh cloning by using reverse transcriptase PCR (RT-PCR). The cDNA sequence of sdh which got from Podophyllum pleianthum Hance aligned with sdh cDNA sequence from Podophyllum peltatum. It reached to 98.1% identity. The alignment of amino acid sequence reached to 98.2%. The optimum conditions was used 0.01 mM IPTG inducting 9 hours at 25oC for expression of SDH in E. coli. The result showed a 34 kDa of protein in SDS-PAGE, the same size for sdh with a SDH-Histag design. The conversion of recombinant enzyme reaction was further analyzed by HPLC, the retention time (23.97 min) and the UV absorption spectrum matched with the characters of authentic matairesinol. It indicated that the cDNA sequence of sdh was cloned from Podophyllum pleianthum Hance and expressed in E.coli functionally. Meanwhile, cDNA of plr got from Podophyllum pleianthum Hance by using rapid amplification of cDNA ends PCR (RACE PCR) and RT-PCR, was aligned with plr cDNA sequence from Forsythia intermedia, and it reached to 68.9% identity. Alignment of amino acid sequence also reached to 75.2% identity and 85% similarity. Western blotting proved the expression of target protein PLR in E. coli. The results showed a 39 kDa of recombinant protein expressed in SDS-PAGE, the same size for sdh with a PLR-His tag design.

This thesis investigates the use of plant cell cultures in combination with synthetic chemistry to provide new routes to biologically active compounds. The first chapter consists of approaches to the lignan podophyllotoxin (4), by means of enzyme catalyzed ring closure reactions of dibenzylbutyrolactone intermediates 56 and 57 mediated by Podophyllum peltatum cells. The substrates 56 and 57 studied were synthesized from readily available aromatic aldehydes employing a "one pot", 1,4addition- enolate alkylation sequence, as the key step. Semi-continuous biotransformation experiments performed with 56 illustrate the potential of the technique yielding the ring-closed products 75 and 76. A possible mechanism for the ring closure in vivo is provided, as well as suggestions to alter the stereochemical outcome of the process. The second chapter of this thesis concerns the attempts to oxidize dehydroisoabietanolide (132) with crude enzyme preparations (CFEs) from Tripterygium wilfordii, as part of an ongoing study regarding the biogenesis of the diterpcne triepoxides tripdiolide (5) and triptolide (91). The 12-hydroxy analogue of 132, isotriptophenolide (190), is also studied. Both compounds were prepared in gram quantities from dehydroabietic acid (122). The results from the biotransformation experiments, suggest that triptophenolide (163), an isomer of 190 and bearing a phenolic group at C-14, is a likely candidate for future biotransformation experiments. The present studies also suggest that an "activated" diterpene, that is, a substrate bearing a hydroxyl group in the aromatic ring (190 or an isomer), is a biogenetic precursor for the triepoxide system present in 5 and 91.

博士
臺北醫學大學
藥學系(博士班)
97
Herbal medicines have been used for more than thousands of years. The market sales increase gradually as the alternative medicine spring up. Herbal medicines are regulated as medicine in Taiwan. Due to the technology development, herbal medicines are taken not only the original type but also extracted powder or tablet prepared from herbal medicines, hence, the intake amounts by human are augmented, the past traditional experience of intake and the concept of safety is not suitable for modern herbal medicine application on human. It is necessary to reevaluate the toxicity from the constituents of herbal medicine commonly used. Among the possible mechanism of intoxication, genotoxicity is the newly developed toxicity field, and it draws human’s attention as well as is capable of conducting on the research till the nineteenth century. It’s a secret worry for lacking of detailed genotoxicty data from herbal medicine but considered as safety from the intake experience. As the understanding on mutagenicity of natural products is confined, the thesis focuses on this field. The aim of the thesis is to establish a low-cost and highly efficient screening mode, and applied to screen huge amounts of samples extensively, following the screening assay, the suspected mutagen is reconfirmed its genotoxic effect and then investigates on the genotoxicity mechanism. Genotoxic screening on thirty-six natural compounds have been finished in the past several years. Some of them are suspected as mutagens, hence, we extend the related researches on parts two and three. Shortly, the thesis consists of three parts, Part I, First screening tests of the natural products, which is short of genotoxicity to support its safety are conducted by in vitro microbial genotoxicity (Ames Test). The chemicals by chemical structures classification include flavonoids, organic acids (phenols), coumarins, terpenoids, alkaloids and glycosides. The tested compound are as follows: allantoin, amygdalin, asarone, asiaticoside, baicalein, baicalin, caffeic acid, cinnamic acid, coumarin, costunolide, daidzein, digitoxin, 6,7-dimethoxycoumarin, ??-escin, ferulic acid, furolic acid, gentiopicroside, 18??-glycyrrhetic acid, hesperidine, imperatorine, isorhamnetin, kaempferol, naringin, neohesperidine, paeonol, podophyllin, podophyllotoxin, puerarin, quercetin, rutin, silymarin, silybin, strychnine, umbelliferone, wogonin and yohimbine. Salmonella typhrium Strains of TA98, TA100 and TA102 were selected to perform the test, with and without the metabolic enzyme, observed the microbial mutagenicity. Quercetin and podophyllin-induced revertants show concentration-response relationship, suggesting quercetin and podophyllin showed strong mutagenic effect potentially in the preliminary test. As podophyllin is easily obtained, the genotoxic activity needs to be cautioned. Hence, podophyllin was selected as the role in the part two. As a mixture of podophyllin, the true contributor for genotoxicity has not been defined; it needs to verify the constituents. The part two consists of two subjects, one describes the chemical analysis of podophyllin (Part II-A) and the other describes the investigation on genotoxic mechanism of podophyllin. An LC/MS/MS method was developed to analyze podophyllin. The identification of constituent was conducted on daughter ion scanning mode, and the determination was conducted on the multiple reacting monitoring mode. The quantitative results from LC/MS/MS show the percentage of podophyllotoxin, kaempferol and quercetin in podophyllin were 31.2, 3.2 and 1.8%. Following the quantitative results, each constituent was used to evaluate the genotoxic potential in order to clarify the podophyllin-induced genotoxicity, whether the mutagenic effect is from known mutagenic effects of quercetin and kaempferol or not (Part II-B). The evaluation method for genotoxicity consists of microbial genotoxicty test and mammal chromosome aberration test in vitro as well as mammal micronucleus formation in vivo. Podophyllin showed positive reaction in Ames test, chromosome test and in vivo micronucleous test, but quercetin and kaempferol related to the contents in podophyllin used only showed genotoxicity in Ames test, especially, in the presence of metabolic activation systems in the strain TA98, but not in chromosome test and in vivo micronucleous test. From the above result, quercetin and kaempferol-induced mutagenic effect were excluded. On the other hand, this finding indicates that another compounds existing in podophyllin induce genotoxicity, and than raises the doubt about the safety of podophyllin application. In order to investigate the genotoxic mechanism of podophyllin, reactive oxygen species (ROS) was also carried out. The ROS production assay showed that podophyllin increases the DCF fluorescenc in CHO cells, implying that ROS production was induced by podophyllin. The ROS production is accepted as related to genotoxicity. Different results are from three constituents by the same ROS assay. Quercetin or podophyllotoxin or kaempferol do not induce ROS production. The results showed that there is no relationship between podophyllin and its three separate components on the induction of ROS production by podophyllin Besides, podophyllotoxin, the major constituent of podophyllin, do not display in vitro genotoxicty, but show the increase on the incidence of micronucleus formation by concentration-dependently. Hence, we continuously investigate the pharmacological and toxicological properties of podophyllin. In the pharmacological study, podophyllin induced apoptosis, and resulted in G2/M arrest by propidium iodide based on DNA analysis (cell cycle). Apoptosis represents DNA damage is from podophyllin, and DNA damage might be result in micronucleus formation or chromosome aberration. Among them, quercetin or kaempferol do not effect on cell cycle expression, but podophyllotoxin shows the same cell cycle modulating effects. We suggest that podophyllotoxin might be as a promutagen and induce gene mutation by the metabolism in vivo Collecting the above results, podophyllin, the commonly used herbal medicine, showed mutagenicty in the mutagenic screening work among thirty-six compounds, and the mutagenic effect is not from quercetin or kaempferol. We are unable to confirm that the podophyllin-induced genotoxicity is from podophyllotoxin even though which shows the increase on micronucleus formation, other constituents contribute the genotoxicity in vitro microbial mutagenicity study. We establish the genotoxic-screening mode for herbal medicine by the accomplishment of the thesis. In addition to verify its mutagenicty, find more safe and alternative substances in advance. We wish the research could expend to other un-testing substances in order to protect the medication safety.

碩士
國立臺灣大學
森林學研究所
87
This research is to study the morphology of spores, gametophytes, and juvenile sporophytes, as well as the mating system of Cyathea podophylla. Spores was tetrahedral with trilete. They began to germinate after 11 days of sowing. The spore germination pattern is of Cyathea type, and the development of gametophytes is the Adiantum or Drynaria type. Antheridia were borne in wings or rhizoid areas by 7-8 weeks and archegonia appeared below the notch by 11-12 weeks. The sequence of ontogeny of the gametangia was from male to hermaphrodite. The adult gametophytes were generally cordate type and with bristle-like hairs below the notch. There were more male gametophytes in dense population than those in sparse one. Juvenile sporophytes with midribless blades appeared after 5-8 months. The diploid sporophyte (2n = 69II) and the high genetic load (96-100%) were found and could indicate that the C. podophylla don't tend to reproduce by intragametophyte selfing. The sporophyte production ratio through intergametophytic selfing and crossing experiments were 8-25% and 42-64% respectly; and suggest that this species mostly reproduces sporophytes through intergametophytic mating, especially by intergametophytic crossing.

Le parc national d'Oka héberge une grande richesse floristique et une grande diversité d'habitats dans le sud du Québec, à proximité du Saint-Laurent. Il s'agit également d'une aire protégée qui accueille de nombreux visiteurs chaque année. C'est ici que se situe toute la problématique, car les espèces menacées et vulnérables du parc sont sujettes à une pression anthropique durant l'été. De plus, peu d'études ou de travaux sont menés afin d'évaluer et de protéger cette biodiversité végétale unique. Pour répondre à ce problème, un projet d'instauration de suivi de plantes menacées ou vulnérables a été proposé. Les étapes préliminaires comme le choix des taxons à suivre et la validation terrain, ont été réalisées en 2010 par les collaborateurs de ce projet. Les plantes sélectionnées étaient Podophyllum peltatum, Toenidia integerrima, Desmodium nudiflorum, Lysimachia hybrida et Agastache nepetoides. Ce mémoire s'est intégré dans ce projet et a pour objectifs d'établir des protocoles de suivi à long terme des populations de ces espèces et de recueillir les données initiales qui serviront de données de référence. Pour ce faire, les plantes sélectionnées ont été étudiées afin de réaliser un protocole spécifique pour chacune d'elles. Le protocole mis en place à l'été 2011 a permis de récolter les premières données. Une seule colonie de Podophyllum peltatum a été retrouvée au parc d'Oka avec un effectif de 100 tiges aériennes. Cette population montrait une faible capacité d'expansion. Pour Toenidia integerrima, deux colonies se situaient autour des oratoires 1 et 3. Des interventions mécaniques ouvrant la canopée et diminuant la compétition interspécifique devraient permettre à ces colonies de se maintenir. Desmodium nudiflorum possédait une population abondante et étendue dans ce parc et montrait un fort potentiel d'expansion. Une corrélation inverse avec le sol nu a été mis en évidence pour la population de Lysimachia hybrida qui est dense, abondante mais peu étendue. Cette espèce est peu connue et une de ses caractéristiques de produire des racines aux nœuds de la tige a été observée. Pour Agastache nepetoides, la comparaison de trois colonies a mis en évidence différentes structures de taille et différentes abondances. Des interventions augmentant la luminosité et diminuant la compétition interspécifique devraient permettre la sauvegarde de ces colonies. Les premiers résultats obtenus seront complétés les années qui suivent afin de caractériser et de pérenniser ces populations sur le long terme et d'enrichir nos connaissances sur ces espèces. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Podophyllum peltatum, Toenidia integerrima, Desmodium nudiflorum, Lysimachia hybrida, Agastache nepetoides, suivi.