Academic literature on the topic 'Podophyllin'

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.

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
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 ).