Thèses sur le sujet « Triterpenoilds »

This thesis describes the investigation of the bioactivity of triterpenoids, limonoids and spiro-triterpenoids, isolated from the order of the Rutales. The focus of this research has been towards the development of botanical insecticides with non-neurotoxic modes of action in insects. It follows the recent development of Neem (Azadirachta indica, Meliaceae) as a botanical insecticide containing a limonoid azadirachtin as its main active compound. Because so few Meliaceae members have been examined for insecticidal activity the project was initiated by the screening of over sixty ethanol extracts of various parts of twenty species (collected from Costa Rica and the Tropical Museum in Miami, Florida) for bioactivity against the European corn borer (Ostrinia nubilalis) and the Variegated cutworm (Peridroma saucia). The data suggest that a majority of the extracts studied inhibited growth significantly; some are more active than neem leaf extracts. Swietenia mahogani bark, Trichilia glabra bark, T. hirta leaves, T. americana bark, T. trifolia wood, T. pleana and Azadirachta indica wood showed potent activity against the cutworm. Ruptiliocarpon caracolito bark, Cedrela odorata leaves, Aphanamixis polystachys wood, T. glabra wood and T. pleana bark showed good activity against the corn borer larvae. Bioassay guided fractionation of the most active crude extract, the bark of Ruptiliocarpon caracolito, has resulted in the isolation of six very novel spiro-CD-triterpenoids, the spirocaracolitones. The spirocaracolitones, at 100 ppm, were screened for antifungal activity against Fusarium using a hyphal growth bioassay. After 48 h a high degree of inhibition of growth (59-79%) was observed. These novel triterpenoids exhibited no antimalarial activity when screened against Plasmodium falciparum. A study of the effects of these compounds, incorporated into artificial diet, on the neonate life cycle of the European corn borer was conducted. All the spirocaracolitones effected the growth and the development of the insects but spirocaracolitone B and spirocaracolitone C were substancially more active than the other spirocaracolitones. Two postulates dealing with the biosynthesis of these unique spiro-compounds are also presented. Both commence with a friedelin derivative because canophyllol was isolated in large quantities from the hexane extract. They envisage generation of a carbocation at C12 followed by migration of the C23 methyl group this results in the formation of a carbocation at C13. The spirosystem results from migration of the C8-C14 bond. An investigation of the structure/activity relationships of gedunin was conducted to determine the moieties responsible for its antimalarial and/or antifeedant properties. Ten derivatives of gedunin were prepared (1,2-dihydrogedunin, 1,2-epoxygedunin, 1,2-dihydro-3$\beta$-gedunol, 3$\beta$-acetoxy-1,2-dihydrogedunin, 7-deacetylgedunin, 7-ketogdunin, hexahydrogedunin, tetrahydrogedunin, 21-acetylgedunin, 23-acetylgedunin) along with five other limonoids (limonin, epilimonol, nomilin, obacunone, hirtin) closely related to gedunin were evaluated for antifeedant activity (Ostrinia nubilalis) and antimalarial activity (Plasmodium falciparum). Evaluation of these compounds for antimalarial activity presented no increase in activity however it was clearly determined that alterations to the enone in ring A and at C7 of gedunin resulted in large losses of activity. Evaluation of the effects of these limonoids on the neonate life cycle of the European corn borer at 5 and 50 ppm incorporation into the artificial diet did lot produced dramatic effects on the growth and development of the larvae. At these concentrations structure/activity relationships could not be proposed.$\sp*$ ftn$\sp*$Please refer to the dissertation for diagrams.

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The Meliaceae family comprises has about 50 genus and approximately 700 species, generally arboreus, occurring in regions Pantropical. The predominant metabolites in the family are triterpenoids with cycloartane, dammarane, tirucallane and apotirucallane skeleta, further degraded triterpenoids (limonoids and steroids). The genus Guarea possesses about 50 species however chemical studies are limited to ten species, whose reports are sesquiterpenes, diterpenes and triterpenes (cycloartane, tirucallane lanostane and apotirucallane), further limonoids. Although the occurrence of approximately 30 species of Guarea in Amazon have few relates on chemical studies, this acted as stimulus for our investigation about Guarea convergens T.D. occurrent in Amazon. Thus, leaves and branches collected in the Adolpho Ducke Forest Reserve were submitted to maceration to obtain the organic extracts. The chromatographic fractionning of the CH2Cl2 and MeOH leaves extracts led to the isolation of apotirucallane triterpenes: the 24R-acetoxy,25-hydroxy-3,7-dioxo-apotirucalla-14-en-21,23- olide (1) and the 7a,24R,25-trihydroxy-3-oxo-apotirucalla-14-en-21,23-olide (4), steroids: ergosta-5,24-diene-3 ,4 ,22S-triol (2) and ergosta-5,24-diene-3 ,7 -21-triol (3). The compounds 1, 3 and 4 are being reported for first time. The CH2Cl2 branches extract yielded a mixture of steroids (b-sitosterol and stigmasterol, 5) and the tirucallane triterpenes namely melianone (6) and melianodiol (7). This is the first chemical study effected with species G. convergens whose isolated and identified compound provide contributions to knowledge of the chemosystematic of the Rutales order.
A família Meliaceae possui cerca 50 gêneros e aproximadamente 700 espécies, geralmente arbóreas de ocorrência nas regiões Pantropicais. Os metabólitos predominantes na família são os triterpenos com esqueletos cicloartano, damarano, tirucalano e apotirucalano além dos triterpenos degradados (limonóides e esteróides). O gênero Guarea apresenta cerca de 50 espécies, no entanto os estudos químicos são limitados a dez espécies cujos relatos são de sesquiterpenos, diterpenos e triterpenos (cicloartano, tirucalano, lanostano e apotirucalano), além dos limonóides. Apesar da ocorrência de aproximadamente 30 espécies de Guarea no Amazonas, existem poucos relatos sobre os estudos químicos, o que nos estimulou a seleção de G. convergens ocorrente no Amazonas. Assim, folhas e galhos coletados na Reserva Florestal Adolfo Ducke foram submetidos à maceração para obtenção dos extratos orgânicos. O fracionamento cromatográfico dos extratos CH2Cl2 e MeOH de folhas resultou no isolamento dos triterpenos com esqueleto apotirucalano, 24R-acetoxi-25-hidroxi-3,7-oxoapotirucala- 14-en-21,23-olideo (1) e 7a,24R,25-trihidroxi-3-oxo-apotirucala-14-en-21,23- olideo (4) e dos esteróides do tipo ergostano, ergosta-5,24-dieno-3 ,4 ,22S-triol (2) e ergosta-5,24-dieno-3 ,7a,21-triol (3). As substâncias 1, 3 e 4 estão sendo relatadas pela primeira vez. Nos extratos dos galhos, além de -sitosterol (5) foram isolados os triterpenos do tipo tirucalano, melianona (6) e melianodiol (7). Esse é o primeiro estudo químico da espécie G. convergens cujos compostos isolados e identificados fornecem contribuições para o conhecimento da quimiossistemática da ordem Rutales.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Earth, Atmospheric, and Planetary Sciences, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 22-24).
Sedimentary rocks of all ages abound with geostable lipids of microbial origin, but many biomarkers lack known organismal sources and clear environmental contexts. Here we used Rhodospirillum rubrum, a metabolically versatile, genetically tractable c-Proteobacterium, to explore the diversity of its non-polar terpenoids as a function of growth condition and growth phase. We analyzed the nonpolar fraction of lipids extracted from R. rubrum grown under aerobic, anaerobic, heterotrophic and phototrophic conditions and detected a variety of bicyclic, tricyclic, tetracyclic and pentacyclic triterpenoids, derived from the enzymatic cyclization of squalene and produced in amounts comparable to diploptene. Identified compounds included bicyclic polypodatetraenes, malabaricatriene, euphadiene, adianane, and fernene. Prior to this work, malabaricatriene was an "orphan" biomarker suspected to have a microbial origin, yet it lacked a proven source. We observed similar patterns of polycyclic terpenoids in other hopanoid-producing c-proteobacteria, including Zymomonas mobilis, Rhodopseudomonas palustris, and Rhodomicrobium vannielii. The presence and relative abundance of polycyclic triterpenoids in R. rubrum varied with the growth stage (exponential versus early stationary phase) and growth condition (photoheterotrophic versus photoautotrophic growth). Since R. rubrum's genome contains a single squalene-hopene cyclase gene, the array of triterpenoids produced by it and other c-proteobacteria likely evolves from this enzyme performing low-fidelity cyclization. The observed diversity of sedimentary triterpenoids might therefore result from a select few squalene-hopene cyclase enzymes operating with varying specificity under a range of physiological and environmental conditions, rather than reflecting a great diversity of squalene-hopene cyclases.
by Katherine Harris.
S.M.

An extract of the sponge Rhabdastrella globostellifera was active in an assay measuring stabilization of the binding of DNA with DNA polymerase β. From this extract, four isomalabaricane triterpenoids were isolated and characterized, three of which were active in the binding assay. All compounds were active in the A2780 ovarian cancer cell line assay. Bioassay-guided fractionation of an extract of a sponge of species Dysidea using the A2780 bioassay yielded the known scalarane sesterterpenoid heteronemin in good yield. Four derivatives of heteronemin were prepared semisynthetically from the natural product, tested for their bioactivity, and their structure-activity dependence was observed. Bioassay guided-fractionation of an extract of a Tuemoya sp. green alga, using an assay for inhibitors of the enzyme Tie2 kinase, afforded a two sulfated cycloartanol triterpenoids. Both the major and minor compounds were identified by spectroscopic methods. Bioassay-guided fractionation of an extract of Petalonyx parryi yielded three known oleanane triterpenoids which inhibited the lyase domain of DNA polymerase β. The structures were confirmed by NMR spectroscopic techniques. This is the first reported study of the chemical components of Petalonyx parryi. As part of our antitumor natural product drug discovery efforts, several extracts were selected for bioassay-guided fractionation based on their activity in initial in vitro screens. A new dereplication method using aminopropyl SPE cartridges was applied to six of these extracts, and four of the extracts were dropped due to the presence of long-chain fatty acids (LCFAs). We present results for the testing and application of this SPE-based method for LCFA dereplication. The cell cycle kinase Chk1 is an interesting target for the development of agents which might potentiate DNA damaging agents. Typical assays for Chk1 involve the use of expensive or radioactive reagents. To facilitate the development of new assays using shorter peptide substrates, small libraries of peptides have been synthesized and tested for their activity as Chk1 substrates. Several of the substrates synthesized displayed activity in the Chk1 assay.
Ph. D.

It has been identified that triterpenoids saponins extracted from plants can have cardiovascular, antitumor, and anti-inflammatory activities. The Australian Acacia plant has a broad range of triterpenoids saponins. Current methods for separation and isolation of triterpenoids saponins involve using a large amount of organic solvents, which can be a drawback of using the extract as a functional food and nutraceutical.

The triterpenes are one of the largest and most structurally diverse classes of plant specialised metabolites, with important commercial, agronomical and medical potential. However the structural complexity and low abundance of these compounds in nature presents significant challenges for exploiting this diversity. In recent years, advances in our understanding of triterpene biosynthesis in various plant species have greatly facilitated the ability to produce these compounds in other hosts. Plant-based host systems hold great promise because they may provide substrates, cofactors and subcellular compartmentalisation mechanisms facilitating engineering production of complex triterpenoids. Agroinfiltration is a rapid and scalable process that enables transient expression of biosynthetic genes in amenable plants such as Nicotiana benthamiana. This technique holds great promise for selective production and modification of triterpenes, but studies in this area have been limited. In this PhD thesis, transient expression is used to produce and oxidise triterpenes in N. benthamiana. Triterpene yields are improved through expression of rate-limiting genes in the mevalonate pathway (Chapter 3). An enzymatic ‘toolkit’ is established from a collection of previously characterised triterpene biosynthetic genes, allowing production of milligram quantities of a diverse array of simple and oxidised triterpenes in N. benthamiana. Furthermore, combinatorial biosynthesis is utilised to engineer production of novel oxidised triterpenes in planta (Chapter 4). A set of oxidised derivatives of β-amyrin are purified and evaluated for antiproliferative and anti-inflammatory activity in human cell lines, yielding insights into structural features underlying these activities in humans (Chapter 5). Finally, N. benthamiana is used for functional screening of cytochrome P450s implicated in the biosynthesis of agronomically important antifungal triterpene glycosides (avenacins) in oat, leading to identification of new P450s required for disease resistance (Chapter 6). This work highlights the utility of N. benthamiana as triterpene engineering platform and provides a basis for future studies exploring triterpene structure-activity relationships.

The Euphorbia L. genus has over 2000 species and is regarded as the largest genus of flowering plants. Euphorbia damarana is a small tree belonging to the Euphorbiceae family which grows in the eastern parts of Namibia. It is a slender grey succulent round shrub. It is commonly known as Damara milk-bush or melkbos by the local community. E. damarana is believed to be one of the most poisonous plants in Namibia producing a milky latex that is capable of killing humans and animals. Poisonous plants occur in every garden worldwide thus forming an important part of the indigenous flora of southern African plants. They consist of a mixture of chemical compounds of which either a single or a collective number of compounds can be toxic. There is often variation of toxicity in different parts of the plants. The stem extract of E. damarana yielded a rubbery material and the dried latex extract yielded a sticky yellow material. The hexane and methanol extracts of the stems of E. damarana and the dried latex extract were tested for antibacterial activity against Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, P. mirabilis, Salmonella typhimurium, Achromobacter xyloxidans and unknown bacteria isolated from the soil of Namibia where the plant grows. Good antibacterial activity was obtained with both extracts using the TLC bio-autography method, however with the microtitre dilution method only the stem extract showed good antibacterial activity. The disc diffusion method did not show any inhibition. Toxicity studies of the stem and dried latex extracts on Vero cells did not show significant results. The antibacterial activity of the stem extract was observed with the soil isolate 11 (0.312mg/ml), P. aeruginosa (1.25mg/ml), B. subtilis (1.25mg/ml), isolate 5 (2.5mg/ml) and isolate 10 (2.5mg/ml) bacteria. For dried latex extract antibacterial activity was observed with isolate 11 (5.00mg/ml), P. aeruginosa (5.00mg/ml) and B. subtilis (5.0mg/ml). Bioassay guided fractionation of the stem extract of the plant led to the isolation of five pure compounds. The chemical structures of the isolated pure compounds was elucidated using NMR spectrometry as lupenone, euphol, lupeol, 20-hydroxy-lupan-3-one and 3-oxo-7,24E-tirucalladien-26-oic acid. All the isolated compounds belong to the family of triterpenoids. E. damarana showed promising antibiotic potential in this study. Since this plant has many other antibacterial compounds as seen on TLC plates of the major fractions and has also shown significant activity against some bacteria in bioassays, further fractionation of the major fractions is recommended in order to obtain more pure compounds that could be tested against bacteria. Toxicity studies on the identified compounds and other compounds that are yet to be identified is in progress and will be reported in further studies. There is limited information published on the phytochemical analysis and properties of E. damarana and for this reason it is important to do further research on this plant as it has potential to be used as a natural ethnomedicinal product.
Dissertation (MSc)--University of Pretoria, 2017.
Plant Science
MSc
Unrestricted

Les dysfonctionnements mitochondriaux sont impliqués dans le développement du diabète mais ne sont pas ciblés par les thérapies disponibles. Les mitochondries sont activées par un RCPG activé par les acides biliaires, le TGR5, une cible intéressante à deux niveaux : pour prévenir les maladies métaboliques (mitochondries) et pour traiter ces maladies (sécrétion de GLP-1). Pour étudier TGR5, il fallait identifier des agonistes sélectifs pour contourner les effets pléiotropiques des acides biliaires, les seuls agonistes connus. Après criblage d’une collection de plantes sur un test d’activité TGR5, les feuilles d’olivier Olea Europaea ont montré une activité TGR5 portée par un triterpène, l’acide oléanolique. In vivo, l’acide oléanolique a une activité anti-hyperglycémique, améliore la tolérance au glucose et empêche la prise de poids. L’acide oléanolique stimule les fonctions mitochondriales dans les muscles in vitro et in vivo. Une étude de SAR sur le squelette des triterpènes nous a permis de synthétiser RG239, un puissant agoniste TGR5 in vitro mais inactif in vivo, probablement à cause d’une faible biodisponibilité. RG239 est un outil pharmacologique intéressant pour étudier la biologie du TGR5. Nous avons ainsi montré que RG239 induit les fonctions mitochondriales de façon TGR5-dépendante. Par ailleurs, RG239 stimule la sécrétion de GLP-1 dans les cellules entéroendocrines intestinales, et cet effet est également TGR5-dépendant. Ces résultats confirment TGR5 comme cible thérapeutique et les triterpènes comme agents thérapeutiques potentiels pour prévenir (mitochondries) et traiter (GLP-1) les maladies métaboliques
Mitochondrial dysfunction is implicated in the early stages of diabetes but is not targeted by the available therapies. Mitochondrial functions can be activated by the bile acids-activated GPCR TGR5, in muscle and in adipose tissue. This double effect of TGR5, on mitochondria and GLP-1 secretion, positions this receptor as an interesting target to prevent the early onset of metabolic diseases. To further explore TGR5 potential, our principal challenge was to identify more potent and more selective TGR5 agonists devoid of the pleiotropic effects of bile acids, the only TGR5 agonists known so far. To this end, we used a TGR5 activity assay to screen a plant library. Among the plant extracts tested, Olea Europaea leaves was shown to exhibit an activity supported by the triterpenoid oleanolic acid. In vivo, oleanolic acid showed anti-hyperglycemic activity, improved glucose tolerance and decreased weight gain. Furthermore, oleanolic acid enhanced mitochondria in muscle both in vitro and in vivo. A SAR study based on the triterpenoid scaffold led us to synthetize RG239, a more potent TGR5 agonist in vitro which is inactive in vivo, probably because of poor bioavailability. RG239 is an interesting pharmacological tool to understand TGR5 biology. Indeed, RG239 was shown to induce mitochondria activity and number in muscle and intestinal cell lines in a TGR5 dependant manner. In addition, RG239 stimulated GLP-1 secretion in intestinal enteroendocrine cells in a TGR5 dependant-manner. Altogether, these results confirm TGR5 as therapeutical target and triterpenoids as potential therapeutical agents in the prevention/mitochondria and treatment/GLP-1 of metabolic diseases

Retinal degeneration is a leading cause of impaired vision or even blindness all around the world, unfortunately, most retinal diseases are irreversible, and there is no ideal treatment to cure the diseases, therefore it is important to protect retina from disease progression. In the recent years, natural compounds have shown great pharmacological potentials. Pentacyclic triterpenoids (PTs) are a group of compounds widely existing in nature, they have shown diverse pharmacological effects in vitro and in vivo. In this thesis, we evaluated the antioxidative effect of betulin, betulinic acid and their 18 derivatives (groups were modified at C3 and C28 positions of betulinic acid skeleton) in human retinal pigment epithelium (RPE) and human Müller cells. RPE is a monolayer of cells between the outer neural layer of photoreceptors and choroid, responsible for mediating visual cycle and nourishing photoreceptors. Hypoxia in RPE cells is likely to cause retinal diseases such as the age-related macular degeneration (AMD), which is the leading cause of blindness in old people worldwide. In Chapter 2, we established the hypoxia model in ARPE-19 cells with CoCl2, which induced a dramatic reduced cell viability, increased reactive oxygen species (ROS) level, induced apoptosis and necrosis. H7 pretreatment could significantly reversed the above damage. In addition, H7 could inhibit the activation of Akt, Erk1/2 and JNK pathways stimulated by CoCl2. We also verified the same signaling pathways in human primary RPE cells. Müller cells are predominant neuron-supporting glial cells, they play important roles in maintaining the retinal homeostasis, releasing trophic factors and supporting other retinal cells. One of the key functions is to uptake the neurotransmitter glutamate and convert it into glutamine, therefore high concentrations of glutamate will lead to pathological changes of Müller cells, which may promote progressions of some retinal degenerations like diabetic retinopathy (DR) and glaucoma. In Chapter 3, we tested the antioxidative effect of betulinic derivatives in MIO-M1 cells against glutamate-induced excitotoxicity. The betulinic derivatives H3, H5, H7 and H11 could significantly increase cell survival and reduce necrosis level after glutamate treatment. H3, H5 and H7 could effectively inhibit ROS production. H5 and H7 showed the effects by inhibit the activation of Akt, Erk1/2 and JNK pathways induced by glutamate, while H3 only inhibited Erk1/2 phosphorylation. Studies in Chapter 2 and Chapter 3 indicate that these betulinic derivatives protected RPE and Müller cells by deactivating Akt, Erk1/2 and JNK. However, the effective concentrations of betulinic derivatives in Chapter 2 and Chapter 3 are hard to reach in the retina by systematic administration. Liposome is a widely-used approach to increase drug delivery efficacy and therapeutic effects, therefore we encapsulated the active betulinic derivatives (H3, H5 and H7) into Compritol 888 ATO or Compritol HD5 ATO liposomes and evaluated the cytoprotective effect of each liposome-based formulations in ARPE-19 and MIO-M1 cells. We found that H3 and H5 Compritol 888 ATO liposomes, as well as H5 Compritol HD5 ATO liposomes increased cell viability in glutamate-treated MIO-M1 cells. H5 Compritol 888ATO liposome, and Compritol HD5 ATO liposomes with H3, H5, H7 significantly attenuated ROS production caused by CoCl2. Furthermore, Compritol 888 ATO liposomes particularly enabled encapsulated compounds to release sustainably. Studies in this Chapter indicated that liposomes are a promising drug delivery carrier for betulinic acid derivatives to achieve an improved cellular protective effect against oxidative stress in human RPE and Müller cells.

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.

Les bactériohopanepolyols (BHPs) en C35 sont les principaux hopanoïdes trouvés chez les bactéries. Ces composés présentent une chaîne latérale polyhydroxylée en C5 liée par une liaison carbone/carbone au groupement isopropyle du squelette hopane. Ils présentent de nombreuses variations structurales au niveau de la chaîne latérale qui apportent des informations taxonomiques et physiologiques. Le bactériohopanetétrol (BHT) et l’aminobactériohopanetriol sont les composés majoritaires. En outre, ces deux BHPs seront les parents de la plupart des BHPs complexes. L'élucidation de la biosynthèse de la chaîne latérale en C5 des hopanoïdes est donc très intéressante pour une meilleure compréhension de la distribution phylogénétique et la signification biologique des BHPs.Au cours de ce travail, le ribosylhopane a été isolé pour la premier fois chez une bactérie. Cette découverte est une preuve solide confirmant le rôle du ribosylhopane comme intermédiaire dans la biosynthèse des hopanoïdes. En outre, deux gènes, impliqués dans la formation de la chaîne latérale des BHPs chez Streptomyces coelicolor, ont été caractérisés. L’adénosylhopane serait converti par une phosphorylase en ribosylhopane et une aminotransférase est nécessaire pour la formation de l’aminobactériohopanetriol du ribosylhopane. De plus, nous avons développé des synthèses concises de l’adénosylhopane et d’un isotopomère bisdeutérié. L’analogue marqué a par la suite été incorporé dans le BHT par un système acellulaire de Methylobacterium organophilum et le suivi du marquage nous a permis de démontrer l’implication de l’adénosylhopane dans la biosynthèse des hopanoïdes en C35
C35 Bacteriohopanoids represent the majority of hopanoids produced by bacteria. They bear an additional C5 side chain linked by a carbon/carbon bond to the isopropyl group of the hopane skeleton. The C5 side chains present an impressive structural diversity and carry taxonomic and physiological information. The most common C35 bacteriohopanoids are bacteriohopanetetrol (BHT) and aminobacteriohopanetriol. Moreover, these two compounds are proposed as the parents of most complex bacteriohopanoids. Therefore, elucidation of the biosynthesis of hopanoid side chains is in great interest for a better understanding of the physiological distribution and biological importance of bacteriohopanoids.In this work, ribosylhopane was isolated for the first time from a bacterium. This discovery is a solid proof for the role of ribosylhopane as an intermediate in the biosynthesis of hopanoid side chain. In addition, two genes involved in the hopanoid production in Streptomyces coelicolor have been characterized. Adnosylhopane may be converted into ribosylhopane by a phosphorylase; and an aminotransferase is required for the formation of aminobacteriohopantriol from ribosylhopane. Moreover, we have developed a concise strategy for the hemisynthesis of adenosylhopane and a deuteriated isotopmer. The subsequent incorporation of the deuteriated adenosylhopane into BHT by a cell-free system in Methylobacterium organophilum proved that adenosylhopane is indeed a precursor of C35 bacteriohopanoids

The formation of carbon-carbon bonds in an asymmetric manner is a central goal of modern synthetic chemistry. Many methods have been developed to form a variety of linkages and substitutions, however the asymmetric formation of quaternary carbons of spirocycles is still a formidable challenge. These compounds represent special challenges because of the requirement of double ring formation together with the control of facial selectivity during the formation of a quaternary carbon centre. The present work demonstrates our efforts towards establishing a strategy based on the Ireland-Claisen rearrangement as the key step for the synthesis of the quaternary spiro-fused ring system such as those found in iridal triterpenoids. 28-Deacetylbelamcandal, a spiroiridal-type triterpenoid, is a representative compound found in iridaceous plants and shows important biological activities in tumor promotion. The synthesis of this compound has been planned from the methodology currently in progress in our group.

Six new compounds were isolated from the aerial and root parts of S. viridis L. cv. Blue Jeans. Two new triterpenoids, lup-20(29)-ene-2α-acetate-3β-ol, and lup-20(29)-ene-2α-ol-3β-acetate were found in the aerial part together with lup-20(29)-ene-2α-3β-diol, ursolic acid, oleanolic acid, β-sitosterol and β-sitosterol glucoside. Three new diterpenoids, 1-oxomicrostegiol, viroxocane, viridoquinone, together with five known diterpenoids, ferruginol, salvinolonyl 12-methyl ether, microstegiol, 7α-acetoxy-14-hydroxy-8,13-abietadiene-11,12-dione and 7α,14-dihydroxy-8,13-abietadiene-11,12-dione were found in roots. 1-Docosyl ferulate, 2'',3''-di-O-acetyl-martynoside and a mixture of 2-(4'-alkoxy-phenyl) ethyl alkanoates were also isolated from roots. Seven caffeic acid derivatives, five flavonoid glycosides, and salidroside were found in the crude aerial fraction. Four caffeic acid derivatives were known phenylpropanoids, i.e. trans-, cis-verbascoside, leucosceptoside A and martynoside, which are now reported in the genus Salvia for the first time. The others were caffeic acid, rosmarinic acid and 6-O-caffeoyl-glucose. A new flavonoid glycoside, luteolin-7-O-α-rhamnopyranosyl-(1→6)-β-galactopyranoside was also identified in the aerial part with four known flavone glycosides: luteolin-7-O-β-glucopyranoside, luteolin-7-O-β-galactopyranoside, luteolin-7-O-rutinoside and apigenin-7-O-β-glucopyranoside. Verbascoside (acteoside), which is a major component in this plant, showed a significant protective effect against UVA induced damage in a human skin fibroblast model in vitro. It exhibited 1.4 fold protective effect against UVA induced necrosis with 1.4 fold higher in cell survival. 50 μM Verbascoside showed the same protective effect as 100 μM DFO at a high intensity UVA dose (500 kJ/m2). Further determination of organelle specific protection suggested a mechanism of action in mitochondria. Two terpenoids, lup-20(29)-ene-2α-acetate-3β-ol and 7α,14-dihydroxy-8,13-abieta-diene-11,12-dione, exhibited antibacterial activity against Enterococcus faecalis with MIC 50 μM. Microstegiol was also active against Staphylococcus aureus with MIC 50 μM. Ursolic acid, oleanolic acid and ferruginol showed appreciable antibacterial activity against three Gram-positive bacteria, Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus with MIC 12.5-50 μM. The other diterpenoids were active against all three Gram-positive bacteria with MIC 100-200 μM. None of crude fractions was active against three Gram-negative bacteria, Klebsiella pneumoniae, Proteus vulgaris, and Escherichia coli.

A new method has been developed for the rapid separation, purification and identification of triterpenoids from the seeds of the Neem tree (Azadirachta indica) and cell tissue cultures from it. The formation of triterpenoids in tissue culture was studied in collaboration with researchers at ENEA, Rome. Tissue culture samples prepared by them have been analysed for triterpenoids, in particular the natural insecticidal compound azadirachtin. A solid phase extraction method was developed for the partial purification of compounds from small quantities of tissue culture cells. Compounds were quantified using supercritical fluid chromatography. Azadirachtin was isolated from the tissue cultures of the plant for the first time, along with the related triterpenoids 3-acetyl-l-tigloylmeliacarpin, 3-tigloylazadirachtol and 3-acetyl-l-tigloylazadirachtinin. The decomposition of azadirachtin in solution at different conditions of pH and temperature was studied. The compound was most stable in mildly acidic aqueous conditions but readily decomposed in basic solutions. Azadirachtin decomposed more rapidly when heated in aqueous rather than in organic solutions. At room temperature, it was stable in organic solvents over a9 month period. At 90 °C in methanol, azadirachtin was quantitatively converted to 3-acetyl-l-tigloyl-azadirachtinin and 3-tigloylazadirachtol was converted to 11-dehydroxy-3-tigloylazadirachtinin. Methods were found to reduce the reaction time and increase the yield of salanninolide, isosalanninolide, nimbinolide and isonimbinolide, photo-oxidation products from salannin and nimbin. Isosalanninolide, a photo-oxidised product of salannin, was isolated from the seeds for the first time. This and other products were submitted for insecticidal tests. Studies with synthetic model compounds were made, in an attempt to understand the nature of the photo-oxidation reactions.

A new synthetic route to (-)-4-methylcamphor 43 is described which involved initial conversion of (+)-camphor 1 to (-)-2-methylenebornane 41. Acid-catalyzed rearrangement of 41 provided ( +)-4-methylisobornyl acetate 40 which was reduced to (+)-4-methylisoborneol 42. Finally, oxidation of 42. produced (-)-4-methylcamphor 43. The optical purity of compounds 40 and 43 was determined by a chiral lanthanide shift reagent, [Eu[hfc)₃] 45 while the optical purity of 42 was determined by the Anderson-Shapiro reagent 48. Based on a mechanistic rationale, a different approach to the synthesis of optically pure 40 was attempted which involved the synthesis of a C(5)-substituted 2-methylenebornane derivative 58 from ( + ) -endo-3-bromocamphor 5a . The potential of (-)-4-methylcamphor 43 as a useful intermediate in the synthesis of triterpenoids belonging to the lanostane 49 structural sub-group was investigated. Through a series of bromination reactions followed by regioselective C-3 debromination, 43 was converted to 9,10-dibromo-4-(bromomethyl)camphor 75 which was cleaved to give 76, a possible precursor of a potentially useful intermediate 65 in triterpenoid synthesis.[See Thesis for Diagrams]
Science, Faculty of
Chemistry, Department of
Graduate

Dans le cadre de la promotion d'une viticulture durable, le développement d'alternatives écologiques à l’utilisation de pesticides de synthèse pour le contrôle des maladies de la vigne (Vitis vinifera) gagne en importance. Une des méthodes de bio-contrôle proposée est l'induction de l'immunité des plantes par l’application de substances biodégradables, non toxiques pour la santé et l'environnement, appelées éliciteurs ou stimulateurs de défense des plantes (SDP). La résistance conférée contre divers agents pathogènes peut être obtenue grâce à l’emploi de molécules utilisant le plus fréquemment les voies de signalisation de l'acide jasmonique (JA), de l'acide salicylique (SA) et/ou de l'éthylène (ET). De telles voies peuvent déclencher l'induction de gènes liés à la défense tels ceux codant des enzymes responsables de la biosynthèse des stilbènes, des métabolites phénoliques antimicrobiens parmi les plus importants des Vitacées. Au vignoble, pour contrôler les maladies, les éliciteurs peuvent être appliqués en complément des pesticides de synthèse et non en remplacement total car leur efficacité est souvent variable selon les agents pathogènes et les conditions environnementales. Afin de développer leur utilisation, des études supplémentaires qui pourraient notamment élucider leur mécanisme d'action sont nécessaires. L’objectif de cette thèse était d'étudier les réponses de la vigne à des éliciteurs de différents modes d'action, comme le jasmonate de méthyle (MeJA), impliqué dans la voie de signalisation du JA, l’acide S-méthyl ester 2,1,3-benzothiadiazole-7-carbothioïque (BTH), un analogue synthétique du SA, des phosphonates (PHOS), molécules à double action stimulateur-fongicide. Le profil des stéroïdes et des triterpénoïdes pentacycliques a été caractérisé par des analyses en chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS). Dans un premier temps, l'effet éliciteur du MeJA a été évalué dans des suspensions cellulaires (in vitro) de plusieurs cultivars de V. vinifera. Une surproduction de triterpénoïdes pentacycliques a été observée avec des différences selon la variété considérée : induction de l’accumulation de la bétuline et de l'acide oléanolique ou des phytostérols pour le Petit Verdot, le Gamay Teinturier) et le Cabernet Sauvignon, respectivement. Puis les élicitations ont été effectuées au niveau des feuilles de boutures de serre de V. vinifera cv. Cabernet Sauvignon. Un effet stimulant sur les triterpénoïdes pentacycliques liés à la défense a été démontré au détriment de la biosynthèse des stérols, composants structurels essentiels des membranes cellulaires. Par l’utilisation de puces NeoVigen et la chromatographie liquide à ultra haute performance couplée à la spectrométrie de masse (UHPLC-MS), l’induction de l’expression de gènes liés à la défense et l'accumulation de polyphénols (stilbènes, flavanols et flavonols) ont été notées suite aux trois traitements éliciteurs. La protection de la vigne conférée par les éliciteurs a été confirmée par des biotests sur disques foliaires inoculés par l’oomycète biotrophe Plasmopara viticola, l’agent responsable du mildiou. Par ailleurs, il est important d’avoir connaissance de l’impact des éliciteurs sur le métabolisme général afin d'obtenir l’effet optimal entre croissance, rendement et défense. Ainsi, une approche métabolomique utilisant la spectroscopie de résonance magnétique nucléaire du proton (RMN 1H) a été menée. Une reprogrammation similaire et/ou spécifique selon l'éliciteur considéré a été notée en particulier au niveau des glucides, des acides aminés et de certains intermédiaires du cycle de Krebs. Les recherches présentées dans cette thèse, démontrent que la compréhension approfondie de l'interaction entre l'éliciteur, les réponses moléculaires et métaboliques de la plante et le pathogène, est cruciale pour le développement de stratégies de protection efficaces basées sur l'utilisation des SDP pour contrôler les maladies de la vigne
In the frame of promoting sustainable vitiviniculture, the development of eco-friendly alternatives to synthetic chemical products for phytosanitary treatments against grapevine (Vitis vinifera) pests is gaining importance. One of the bio-control methods that can be proposed is the induction of plant immunity by using elicitors, also called plant defense stimulators (PDS), as these substances are biodegradable and, non-toxic to health and environment. A conferred resistance against various pathogens can be obtained with natural molecules acting most frequently through jasmonic acid (JA), salicylic acid (SA), and/or ethylene (ET) signaling pathways. These pathways are involved in the induction of defense-related genes such as those encoding enzymes responsible for the biosynthesis of stilbenes, which are the most important polyphenolic antimicrobial metabolites (phytoalexins) in Vitaceae. For vineyard protection, PDS can be applied as a complement for pesticides and not as a full replacement since their effectiveness is often variable according to pathogens and environmental conditions. In order to develop the strategies based on PDS use, more studies which could elucidate their mechanism of action are needed. The aim of this thesis was to examine the responses of grapevine to elicitors of different mode of action, as methyl jasmonate (MeJA), implicated in JA signaling pathway, 2,1,3-benzothiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic analogue of SA, and phosphonates (PHOS), molecules of a double stimulator-fungicide action. Due to scarce information about steroids and pentacyclic triterpenoids in grapevine, their profile after PDS treatment were characterized in different grapevine experimental models using gas chromatography-mass spectrometry (GC-MS) analyses. Firstly, the effect of elicitation with MeJA was evaluated in cell suspension cultures (in vitro) of V. vinifera. An overproduction of bioactive pentacyclic triterpenoids occurred with differences according to the cultivar studied, i.e., acumulation of betulin and oleanolic acid or phytosterols was noted in respectively Petit Verdot, Gamay Teinturier and Cabernet Sauvignon cell suspension cultures. Then, elicitations were effectuated on the leaves of V. vinifera cv. Cabernet Sauvignon greenhouse cuttings. A stimulatory effect on the potentially defense-related pentacyclic triterpenoids at the expense of the biosynthesis of sterols, which are essential structural components of cell membranes, was shown. By the use of NeoVigen microarrays, and ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS), the accumulation of defense-related transcripts and polyphenols (stilbenes, flavanols and flavonols) were noted after the three elicitors treatments. Grapevine protection conferred by these elicitors was confirmed on foliar discs against the biotrophic oomycete Plasmopara viticola, the causal agent of downy mildew. Furthermore, the impact of PDS on primary metabolism should be evaluated in order to ensure, in the longer term, the best trade-off between growth, yield and defense. Thus, a thorough metabolomic approach using proton nuclear magnetic resonance spectroscopy (1H-NMR) was performed. A reprogramming similar and/or specific to the elicitor applied was noted, particularly within carbohydrates, amino acids, and some of the Krebs cycle intermediates. The research presented in the current dissertation revealed that the thorough comprehension of the interaction between elicitor, plant molecular and metabolic responses and pathogen, is crucial for the development of effective protection strategies based on the use of PDS for grapevine diseases control

Analysis of cuticular wax from Kalanchoe daigremontiana leaves was performed to identify the constituent components within the wax, determine how these changed during leaf ontogenesis, and discover how they were distributed within the cuticle. Analysis of extracted cuticular wax by gas chromatography, mass spectrometry, and comparison with authentic standards led to the identification of triterpenoids including glutinol, friedelin, germanicol, epifriedelanol, glutinol acetate and β-amyrin as well as very long chain fatty acid (VLCFA) derivatives including alkanes, primary alcohols, aldehydes, fatty acids, and alkyl esters. Cuticular wax composition in young K. daigremontiana leaves was dominated by triterpenoids, which made up over 70% of the lipid soluble compounds. During leaf ontogenesis, wax composition changed to include a higher proportion of VLCFA derivatives, which made up approximately 50% of cuticular wax in mature leaves. The most abundant triterpenoids in the wax were glutinol and friedelin, both fairly uncommon pentacyclic triterpenoids with a complex proposed biosynthetic mechanism. Tritriacontane (C33 alkane) was the most abundant compound within the VLCFA derivatives. Cuticular wax accumulation was found to correspond well to leaf growth, with both processes slowing at the same time. Variations in the ratio of friedelin-like compounds to glutinol-like compounds during leaf ontogenesis suggest the presence more than one active triterpenoid synthase enzyme in the leaves of K. daigremontiana. VLCFA compounds were found mainly in the epicuticular wax on both the adaxial and abaxial surfaces, while triterpenoids were relatively more abundant in the intracuticular layer. Two different epicuticular wax crystal forms were observed by scanning electron microscopy (SEM) which can be described as platelets with sinuate margins and twisted ribbons. Based on SEM and chemical data as well as previous reports of crystal composition, it is hypothesized that each crystal type has a unique composition, with the platelets containing one or more triterpenoids and the twisted ribbons containing alkanes and other VLCFA derivatives. Confirmation of this hypothesis will have to await further investigation. This research provides information that will aid in the larger goals of characterizing a glutinol or friedelin synthase and understanding the gradients established within epicuticular and intracuticular wax layers.

Through our continuing search for anticancer agents from Madagascar rainforests as a part of International Cooperative Biodiversity Group (ICBG), we received two extracts which were active against the A2780 human ovarian cancer cell line and hence were selected for further fractionation. Six compounds were isolated from these extracts. The structure elucidation and characterization of these compounds was carried out using mass spectrometry and 1D and 2D NMR techniques. The bioassay-guided fractionation of Roupellina (Strophanthus) boivinii yielded three new and one known cardenolide glycosides. The structure of the known cardenolide glycoside was determined after comparison of NMR data to that found in literature for digitoxigenin 3-O-β-D-glucopyranosyl-(1â 4)-α-L-acofriopyranoside. All four compounds exhibited good antiproliferative activity on the A2780 bioassay. The fractionation of the extract of Grewia sp. led to the isolation of one new and one known triterpenoid. The known triterpenoid was identified as 7β-hydroxy-23-deoxojessic acid and its structure was confirmed by comparison of its 1D and 2D NMR data to that found in literature.
Master of Science

Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Química Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
O cancro continua a ser uma das principais causas de morte a nível mundial. De acordo com os dados estatísticos, em 2012 morreram cerca de 8.2 milhões de pessoas devido a esta doença. Além disso, prevê-se que o número de novos casos diagnosticados de cancro aumente consideravelmente nas próximas décadas. Considerando que os fármacos antineoplásicos usados atualmente na clínica continuam a ter diversas limitações e efeitos secundários graves, é urgente desenvolver novos fármacos que tenham uma maior eficácia terapêutica no tratamento do cancro. Os produtos naturais têm sido uma fonte importante de leads para a descoberta e desenvolvimento de novos fármacos, nomeadamente os anticancerígenos. Na verdade, alguns dos medicamentos anticancerígenos mais usados na prática clínica são derivados de produtos naturais. Os triterpenóides pentacíclicos representam uma vasta classe de produtos naturais que exibe diversas actividades biológicas, incluindo a atividade anticancerígena. Devido a essa atividade, ao seu perfil relativamente seguro e à sua elevada disponibilidade na natureza, os triterpenóides pentacílicos são compostos interessantes para desenhar novos leads visando o desenvolvimento de fármacos inovadores para a prevenção e tratamento do cancro. O ácido asiático é um triterpenóide pentacíclico da série ursano cujas propriedades anticancerígenas têm vindo a ser extensivamente estudadas ao longo dos últimos anos. Este composto demonstrou ter efeitos anticancerígenos promissores tanto em ensaios in vitro como em ensaios in vivo. Além disso, estudos prévios demonstraram também que a modificação estrutural do ácido asiático pode potenciar a sua atividade anticancerígena. Sendo assim, o presente trabalho teve como objetivo a preparação de novos derivados semissintéticos do ácido asiático, visando aumentar a sua actividade anticancerígena e obter compostos que possam vir a ser úteis para o desenvolvimento de novos tratamentos para o cancro. No decorrer desde trabalho foram idealizados e preparados com sucesso três painéis de novos derivados semissintéticos do ácido asiático. Esses painéis incluem derivados fluorados (3.1–3.26), A-nor (4.2, 4.5, 4.8, 4.12–4.30), amidas e ácidos hidroxâmicos (5.6–5.16). As estruturas químicas dos novos compostos foram elucidadas fazendo uso de várias técnicas analíticas incluindo 1H e 13C RMN, espectroscopia de infravermelho, espectrometria de massa e espectrometria de massa de alta resolução. As atividades antiproliferativas dos novos derivados preparados foram avaliadas num painel de linhas celulares cancerígenas (HT-29, HeLa, MCF-7, Jurkat, PC-3, A-375, e MiaPaca-2), e determinaram-se os valores IC50. A maioria dos novos compostos demonstrou maior atividade antiproliferativa que o ácido asiático. Alguns dos derivados exibiram valores de IC50 inferiores aos exibidos pela cisplatina nas células HeLa (2.28 μM), tendo apresentado, inclusive, valores de IC50 inferiores a 1 μM em várias linhas celulares. Foi estabelecida uma relação estrutura-actividade com base nos valores de IC50 determinados nas células HeLa. Os melhores compostos de cada painel foram posteriormente testados numa linha celular não tumoral (BJ) para avaliar a seletividade, tendo exibido menor toxicidade nas células não tumorais do que nas cancerígenas. De seguida, efetuou-se um estudo preliminar do mecanismo de ação responsável pelo efeito anticancerígeno dos compostos mais ativos de cada painel, 3.14, 4.29 e 5.6. Estes compostos foram avaliados relativamente ao seu efeito no ciclo celular e à sua capacidade para induzir apoptose nas células HeLa. Os três compostos induziram a paragem do ciclo celular na fase G0/G1 e morte celular por apoptose. O composto 3.14 induziu apoptose com ativação das caspases 8 e 3, clivagem do PARP, aumento dos níveis de Bax e uma redução dos níveis de Bcl-2, sugerindo o envolvimento das vias intrínseca e extrínseca no processo apoptótico. O derivado 4.29 induziu a morte celular por apoptose nas células HeLa através da ativação das caspases 9, 8 e 3. Este composto conduziu ainda a uma redução dos níveis das proteínas Bcl-2 e Bid e a um aumento dos níveis da proteína Bax. O composto 5.6 induziu apoptose nas células HeLa através de um mecanismo relacionado com activação das caspases. Por fim, verificou-se a existência de sinergismo quando as células HeLa foram tratadas simultaneamente com o composto 5.6 e com a cisplatina. Alguns dos novos derivados semissintéticos do ácido asiático preparados no decorrer deste trabalho, demonstraram efeitos anticancerígenos promissores em ensaios in vitro e justificam estudos pré-clínicos adicionais. Estes derivados podem vir a ser compostos úteis para o desenvolvimento de novos fármacos anticancerígenos.
Cancer is a leading cause of death worldwide, with an estimated 8.2 million cancer-related deaths registered in 2012. Moreover, the number of new cancer cases diagnosed each year is expected to rise significantly over the next decades. Considering that antineoplastic agents available currently in the clinic have several limitations and some serious side effects, it’s urgent the development of new chemotherapeutic agents with increased efficacy. Natural products have been an important source of leads for the development of new drugs, including new anticancer drugs. In fact, some of the most important antineoplastic agents used in the clinic are derived from natural products. Pentacyclic triterpenoids represent a large and structurally diverse class of natural products that display diverse biological activities, including anticancer activity. Because of this activity, their relatively safe profile, and readily availability in nature, pentacyclic triterpenoids are interesting compounds for the design of new leads aimed at the development of new anticancer agents. Asiatic acid is an ursane-type pentacyclic triterpenoid whose anticancer properties have been extensively studied over the last few years. This compound exhibited promising anticancer effects in both in vitro and in vivo studies. Moreover, previous studies reported that structural modification of asiatic acid backbone may improve its anticancer activity. Therefore, the aim of this project was to synthesize new semisynthetic derivatives of asiatic acid in order to improve its anticancer activity and obtain compounds that may be valuable for the development of new anticancer treatments. During this project, three panels of new semisynthetic derivatives of asiatic acid were designed and successfully synthesized. These panels include new fluorinated (3.1–3.26), A-nor (4.2, 4.5, 4.8, 4.12–4.30), amide and hydroxamic acid (5.6–5.16) derivatives of asiatic acid. The chemical structures of the new compounds were elucidated using techniques such as 1H and 13C NMR, IR, MS and HRMS. The antiproliferative activities of the new derivatives were evaluated against several tumor cell lines (HT-29, HeLa, MCF-7, Jurkat, PC-3, A-375, and MiaPaca-2), and the IC50 values were determined. The great majority of the new compounds showed improved growth-inhibitory activity compared with asiatic acid. Some of the new derivatives exhibited a lower IC50 value than that of the reference drug cisplatin (2.28 μM) in the HeLa cell line, and even showed IC50 values that were lower than 1 μM in several cancer cell lines. A structure–activity relationship was established based on the IC50 values determined in the HeLa cell line. The best compounds of each panel were further tested against a nontumor cell line (BJ), to assess selectivity, and exhibited lower toxicity toward nontumor cells than they did toward tumor cells. A preliminary study of the mechanism underlying the anticancer effect of the most active compounds of each panel, 3.14, 4.29 and 5.6, was performed. These compounds were evaluated regarding their effects on the cell cycle and their ability to induce apoptosis in HeLa cells. All three compounds arrested the cell cycle at the G0/G1 phase and induced apoptotic cell death. Compound 3.14-induced apoptosis was driven by caspases, with activation of caspases 8 and 3, cleavage of PARP, upregulation of Bax, and downregulation of Bcl-2, suggesting the involvement of intrinsic and extrinsic pathways in the apoptotic process. The derivative 4.29 induced apoptotic HeLa cell death via the activation of caspases 9, 8, and 3. This compound also induced the downregulation of the Bcl-2 and Bid proteins and the upregulation of the Bax protein. The compound 5.6 induced apoptosis through activation of caspases. Finally, a synergistic effect was observed after simultaneous treatment of HeLa cells with compound 5.6 and cisplatin. Some of the newly synthesized derivatives of asiatic acid showed promising in vitro anticancer effects and clearly warrant further preclinical evaluation. These derivatives may be useful molecules for the development of new anticancer drugs.
Fundação Para a Ciência e Para a Tecnologia (FCT) - SFRH/BD/69193/2010

碩士
高雄醫學大學
藥學研究所
96
Previously the cytotoxic activity of triterpenoids lantanolic acid (1) and lantabetulic acid (2) isolated from the CHCl3 extract of leaves of Rhus javanica L. var. roxburghiana (DC.) Rehd. & Wilson, showed cytotoxic activity in the human lung carcinoma cell line (A549), human breast carcinoma cell line (MCF-7), human rectum carcinoma cell line (HT-29), and human liver carcinoma cell line (Hep3B). Therefore, we isolated lantanolic acid (1) and lantabetulic acid (2), synthesized derivatives of lantabetulic acid (2) and tested the cytotoxic activity aganist two cancer cell lines in vitro. The results indicated that all derivatives did not reveal stronger cytotoxic activity aganist human colorectal carcinoma cell line (HCT116) and non-small cell lung carcinoma cell line (CL1-5) than that of lantabetulic acid (2). It need to further synthesize various derivatives of lantabetulic acid (2) for study the structure-cytotoxicity relationship.

碩士
大仁科技大學
生物科技研究所
105
Antrodia cinnamomea (abbreviated as AC) is a unique type of medical fungus in Taiwan; it grows only inside the rotten trunks or dying wood segments of aged Cinnamomum kanehirae. It will not grow on other kinds of camphor trees, such as C. longepaniculatum, C. micranthum Hayata and Preslvarcamphora etc. The wild fruiting body of AC is rare and expensive; therefore, illegal lumbering of AC has been a serious problem and also causes the decreasing number of wild C. kanehirae trees. To address this issue, solid fermentation of AC could be a good way to avoid the illegal lumbering of AC because the cultivation matrix for this method does not require the trunks of wild C. kanehirae. This study aims to evaluate the triterpenoids content from AC cultured in lab and the commercial ones via HPLC analysis. The comparison indicators include eight kinds of triterpenoids (five ergostanes: antcin A, antcin B, antcin C, antcin H, antcin K；two lanostanes: dehydrosulphurenic acid, dehydroeburicoic acid and one benzenoid compound). Our result showed that total amount of eight triterpenoids can reach 8700.0 ppm from the powder of AC cultured in lab; however, for commercial products, some of them are lack of antcin H, others without antcin H K, the others in shortage of antcin A, B and C. The total amounts for 8 kinds of triterpenoids in commercial products are shown below: 525.7、605.8、3807.9、383.6 ppm. The biochemical analysis showed that the total triterpenoids for AC cultured in lab is 4.4%; while for commercial ones are: 1.4%, 3.4%, 2.8% and 4.2%。In summary, by means of LC/MS/MS and HPLC analysis, all the results indicate the AC cultured in lab contains more triterpenes than any other commercial products. Thus, the AC in our study may have good market potential. Furthermore, among the eight typical triterpenoids for analyzing AC, antcin A, B, C, H and K all have stereo structures of RS forms. In previous conditions, were only able to separate the RS form of antcin K; while there was only one peak for RS forms of other 4 kinds of antcin compounds. To address this issue, we modified the mobile phase gradients and temperature of C18 column and could separate the RS forms of antcin C, H and K; RS forms of antcin B could be slightly isolated; however, antcin A was inseparable. Because antcin A and B are low-polarity ergostanes, the elution step require higher concentration of acetonitrile. Among them, even 100% of acetonitrile was ineffective to elute antcin A. Subsequently, the separation of RS forms of both antcin A and B was successful by using a long carbon chain hydrophobic C30 column.

) Conclusion CONCLUSIONS This doctoral thesis was focused onto the synthesis if new quaternary ammonium salts oÍ. triterpenoids Ířom lupane, oleanane and ursane family for in vilro assessment of ťollowing biological activities: anti-HIV, antimicrobial, antiinflammatory and cýotoxic. Results are summed up in Íbllowing points: 1' Author co-operated pilot project of industrial isolation of betulin (1a) from approximately 400 kg of birch bark. Futhermore, research of side products and optimisation of preparation of bromoethyl-esters triterpenic acids was concluded. 2' Precursors for preparation of quaternary ammonium esters, bromoethyl-esters 2a, 3f, 3i, l1b, I2b, 14a,15a, and chloroethyl-ester 39, were slmthesised. 3' Six series of compounds, each consisting of three quaternary salts and one tertiary amine, were synthesised from betulinic acid (1b), oleanolic acid (1c), ursolic acid (1c), oxoac id, 3e, platanic acid (11a) a dihydrobetulinic acid (12a). Experimetal procedures are suitable for bulk syntheses. other quaternary salts of triterpenoids were also prepared. 4. oxoacid 3e was derivatised to yield group of heterocyclic hydrochlorides 6a, 6b,ód' 6f and group of heterocyclic salts 6c,6e,69. 5. All new cornpound s were assesed in vitro for cýotoxic activity against T-lymfoblatic leukemia CEM in research...

Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Química Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Lung cancer is the most commonly diagnosed cancer and a leading cause of cancer-related deaths worldwide. Lung cancer can be classified as non-small cell lung cancer (NSCLC) or small-cell lung cancer (SCLC), with NSCLC being the most common type (85%). The high mortality rate for patients with lung cancer has been attributed to the difficulty in obtaining an early diagnosis, a high potential for metastasis, and relative insensitivity to chemotherapy. Despite novel therapeutic strategies, the overall 5-year survival rate in many countries remains less than 15%. These statistics highlight the need for the development of new clinical agents. Pentacyclic triterpenoids (PTs) are a large and structurally diverse group of natural products, widely present in several medicinal plants, fruits and vegetables. Medicinal plants containing PTs have been used for centuries in traditional medicine for the treatment of several diseases. Moreover, natural PTs have been extensively reported to possess a wide range of biological activities, including anticancer activity. From a biological point of view, some of the most important and active PTs include betulinic, oleanolic, glycyrrhetinic and ursolic (UA) acids. UA has interesting biological properties that could be explored for the design and synthesis of more effective anticancer derivatives. Additionally, UA can be considered as a potential lead, due to its promising multitarget anticancer activity, low toxicity and commercial availability. The main goal of this PhD thesis was to design, synthesize and evaluate new ursane derivatives as potential anti-NSCLC agents. This work followed three main semisynthetic strategies to modify the ursane scaffold. The first one involved expansion and cleavage of the A- ring, as well as introduction of N-containing groups. The second one aimed at the introduction of oximes and nitrile groups at A-ring. Finally, the third semisynthetic strategy was based on the introduction of esters (saturated or unsaturated), and of an α,β-unsaturated carbonyl system (with an endo- or exocyclic double bond) at A-ring. The novel ursane derivatives synthesized were fully characterized using IR, MS and NMR techniques, and their anticancer activity was evaluated against NSCLC cell lines. Based on the anticancer results obtained, the structure-activity relationships demonstrated that the most active compounds displayed a cleaved A-ring coupled with a secondary amide bearing a small alkyl side chain (3.17), an oxime group conjugated with a carbonyl function at A-ring (4.2), a cleaved A-ring bearing nitrile and imidazole groups (4.16), and an α,β-unsaturated ketone system with an exocyclic double bond conjugated with a heteroaromatic ring at A-ring (5.12). The most active compounds were further tested in a 3D culture model, specifically a multicellular spheroid, which is able to mimic more closely the in vivo tumor microenvironment. Importantly, even in a 3D model, compounds 3.17 and 4.2 were active albeit a slight increase in the IC50 value. The preliminary studies on the anticancer mechanism demonstrated that the newly synthetized derivatives are able to induce cell death via apoptosis, through activation of the extrinsic pathway, and to induce autophagy. Furthermore, these compounds appear to inherit the multitarget potential of UA, by affecting the levels of different proteins involved in the process of cell death. Overall, this work contributed with three new panels of novel ursolic acid derivatives with in vitro anticancer activity in 2D and 3D culture models. These derivatives can be promising candidates for the development of new anti-NSCLC agents.
O cancro do pulmão é dos tipos de cancro mais frequentemente diagnosticados e a principal causa de morte por cancro no mundo. O cancro do pulmão pode ser classificado como carcinoma pulmonar não de pequenas células (CPNPC) ou como carcinoma pulmonar de pequenas células (CPPC), sendo o CPNPC o tipo mais comum (85%). A elevada taxa de mortalidade em doentes com cancro do pulmão tem sido atribuída à dificuldade em obter um diagnóstico precoce, ao elevado potencial de formação de metástases, e à relativa insensibilidade à quimioterapia. Apesar das novas estratégias terapêuticas, a taxa de sobrevivência global aos 5 anos, em muito países, continua a ser inferior a 15%. Estas estatísticas destacam a necessidade para o desenvolvimento de novos agentes clínicos. Os triterpenóides pentacíclicos (TPs) representam um grupo de produtos naturais abundante e estruturalmente diverso, que estão presentes em várias plantas medicinais, frutas e vegetais. As plantas medicinais contendo TPs têm constituído ao longo dos séculos uma base para a medicina tradicional no tratamento de várias doenças. Os TPs naturais têm sido extensivamente estudados pela vasta gama de atividades biológicas, incluindo a atividade anticancerígena. Do ponto de vista biológico, alguns dos TPs mais importantes e ativos incluem os ácidos betulínico, oleanólico, glicirretínico e ursólico (AU). O AU tem propriedades biológicas interessantes que poderão ser exploradas para o design e síntese de novos derivados anticancerígenos mais eficazes. Além disso, o AU pode ser considerado como um potencial lead devido à sua promissora atividade anticancerígena multifuncional, à sua baixa toxicidade e à sua disponibilidade comercial. O principal objetivo desta tese de doutoramento foi o design, a síntese e a avaliação de novos derivados do tipo ursano como potenciais agentes contra o CPNPC. Este trabalho seguiu principalmente três estratégias de semissíntese para modificar a estrutura do ácido ursólico: 1) expansão e clivagem do anel A, assim como, introdução de grupos contendo o elemento azoto; 2) introdução de grupos oxima e nitrilo no anel A; 3) e introdução de ésteres (saturados ou não saturados) e de um sistema carbonilo α,β-insaturado (com dupla ligação endocíclica ou exocíclica) no anel A. Os novos derivados do tipo ursano sintetizados foram totalmente caracterizados através de técnicas de espectroscopia de infravermelho, ressonância magnética nuclear e espectrometria de massa, e a sua atividade anticancerígena foi avaliada em linhas celulares de CPNPC. Com base nos resultados da atividade anticancerígena, as relações estrutura-atividade obtidas permitiram demonstrar que os compostos mais ativos exibem um anel A clivado e acoplado com uma amida secundária com uma pequena cadeia lateral alquílica (3.17), um anel A contendo um grupo oxima acoplado a um grupo carbonilo (4.2), um anel A clivado contendo um grupo nitrilo e um grupo imidazole (4.16), e um anel A com um sistema carbonilo α,β-insaturado, com dupla ligação exocíclica, conjugado com um anel heteroaromático (5.12). Os compostos mais ativos foram testados num modelo de cultura 3D (esferóide multicelular), o qual mimetiza de forma mais próxima o microambiente tumoral in vivo. De facto, mesmo num modelo 3D, os compostos 3.17 e 4.2 foram ativos, apenas com um ligeiro aumento do valor de IC50. Os estudos preliminares sobre o mecanismo de ação anticancerígena demonstraram que os novos derivados do AU induzem morte celular por apoptose, através da ativação da via extrínseca, assim como autofagia. Além disso, estes compostos parecem herdar o potencial multifuncional do AU, afectando os níveis de diferentes proteínas envolvidas nos mecanismos de morte celular. Em suma, este trabalho contribuiu com três novos grupos de derivados do ácido ursólico com atividade anticancerígena in vitro, em modelos de cultura 2D e 3D. Estes novos derivados podem ser considerados como candidatos promissores para o desenvolvimento de novos agentes anti-CPNPC.

Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Química Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Cancer is a major public health problem and, one of the leading causes of morbidity and mortality worldwide. Cancer treatments, including chemotherapy, are crucial to the clinical management of the disease. However, conventional cancer therapies often cause serious side effects and in the majority of advanced cases, offer only modest improvement in survival rates. Therefore, there is an urgent therapeutic need for the development of new active agents against cancer. Plants are a key source of pharmaceuticals and serve as the basis for numerous medicines, including anticancer agents. Pentacyclic triterpenoids are a group of promising secondary plant metabolites that, present a broad range of biological activity, including antitumour activity. They are often used in traditional medicine and are an important source of hits in drug discovery. Celastrol is one of the most active antitumour compounds among the natural triterpenoids. It has been reported to be highly active against a wide variety of tumours and to affect multiple cellular pathways. Considering its biological potential, some semisynthetic derivatives of celastrol have been previously prepared. These studies suggest that structural modification of celastrol may be important to improve its anticancer activity. In the present work, the design and synthesise of new semisynthetic derivatives of celastrol with improved selectivity and enhanced anticancer activity was intended. For that purpose, different synthetic strategies were exploited and new series of celastrol derivatives, including compounds bearing a urea and a carbamate group at C(29), were prepared and their anticancer activity was evaluated. The chemical structures and high purity of the new celastrol derivatives were corroborated by melting point determination, infrared spectroscopy, nuclear magnetic resonance spectroscopy (1H NMR, 13C NMR and DEPT-135), mass spectrometry and elemental analysis. To determine their anticancer potential, the activity of all synthesised analogues over the viability of cancer cells was evaluated against several human tumour cell lines, including lung carcinoma (A549) and pancreatic carcinoma (MIA PaCa-2) cell lines, using the MTT assay. Furthermore, preliminary studies of the mechanism of action of the most promising derivatives were performed in ovarian carcinoma cells (SKOV-3) using techniques, such as flow cytometry, fluorescence microscopy and western blotting. Several of these new celastrol derivatives exhibited an improved growth-inhibition effect on human cancer cells compared with the parent compound, with IC50 values around or below 1 µM. Additionally, to assess selectivity, the best compounds of each series were further tested against a human non-tumour fibroblast cell line (BJ) and showed lower toxicity, which indicated a selective cytotoxic activity for malignant cells. Compound 3.21 — a quinonemethide urea derivative — and compound 4.11 — a dihydrocelastrol diacetate carbamate derivative — were the most promising synthesised compounds. Preliminary studies of the mechanism underlying their anticancer effect showed that both might induce apoptosis through the activation of the extrinsic death receptor pathway. Additionally, a synergistic anticancer effect was evidenced when SKOV-3 cells were simultaneously treated with compound 3.21 and cisplatin and a similar effect was observed when SKOV-3 cells were treated concomitantly with compound 4.11 and carboplatin. Taken together, these results provided a deeper understanding of the structure-activity relationship, chemical reactivity and stability of celastrol derivatives. Moreover, these results demonstrated the remarkable potential of celastrol derivatives, such as compounds 3.21 and 4.11, as promising leads for the development of new cancer therapies.
O cancro representa um importante problema de saúde pública, sendo uma das principais causas de morbilidade e mortalidade a nível mundial. Os tratamentos utilizados atualmente em clínica, incluindo a quimioterapia, são essenciais para o controlo clínico da doença. No entanto, os tratamentos antineoplásicos convencionais causam frequentemente efeitos secundários graves e, nas fases mais avançados do cancro, apenas aumentam ligeiramente a taxa de sobrevivência. Isto indica que há uma necessidade emergente de desenvolvimento terapêutico de novas substâncias ativas anticancerígenas mais eficazes e menos tóxicas. As plantas são uma fonte fundamental de produtos farmacêuticos, sendo a base de inúmeros fármacos, entre eles medicamentos de quimioterapia. Os triterpenóides pentacíclicos são um grupo promissor de metabolitos secundários das plantas e apresentam uma atividade biológica muito diversificada, entre elas a atividade antitumoral. Estes compostos são frequentemente utilizados na medicina tradicional e são uma fonte importante de novas moléculas para a indústria farmacêutica. O celastrol é dos compostos antitumorais mais activos entre os triterpenóides, demonstrando elevada atividade em diferentes tipo de tumores e afetando diversos mecanismos celulares. Considerando o seu elevado potencial biológico, foram realizados alguns estudos com derivados semissintéticos do mesmo, os quais sugeriram que modificações estruturais da molécula do celastrol poderiam ser vantajosas para otimizarem a sua atividade antitumoral. Assim, neste presente trabalho pretendeu-se preparar novos derivados semissintéticos do celastrol com melhor atividade antitumoral e melhor seletividade. Para tal, explorou-se diferentes estratégias sintéticas de forma a preparem-se novos painéis de derivados do celastrol, entre eles ureias e carbamatos na posição C(29). As estruturas químicas e elevada pureza dos novos derivados do celastrol foram confirmadas por determinação do ponto de fusão, espectroscopia de infravermelho, ressonância magnética nuclear (1H RMN and 13C RMN), espectrometria de massa e análise elemental. De forma a determinar-se o potencial anticancerígeno dos novos compostos, avaliou-se a sua atividade na viabilidade celular de várias linhas celulares tumorais humanas, tais como células do carcinoma de pulmão (A549) e do carcinoma de pâncreas (MIA PaCa-2), através de ensaios de MTT. Foram ainda feitos estudos preliminares do mecanismo de ação dos derivados mais promissores em células do carcinoma do ovário (SKOV-3), utilizando técnicas diversas, tais como, citometria de fluxo, microscopia de fluorescência e western blotting. Muitos dos novos derivados do celastrol apresentaram melhores efeitos de inibição do crescimento celular das células tumorais, comparativamente ao celastrol, apresentando valores de IC50 próximos ou inferiores a 1 µM. Os melhores compostos de cada painel foram também avaliados em relação à sua seletividade, utilizando células humanas não tumorais (fibroblastros BJ). Estes compostos revelaram ser menos tóxicos para este tipo de células, o que indica uma seletividade citotóxica para células malignas. O composto 3.21 — um derivado ureia quinonametídeo — e o composto 4.11 — um derivado carbamato diacetato dihidrocelastrol — demonstraram ser os mais promissores entre todos os compostos sintetizados. Os estudos preliminares do mecanismo de ação indicaram que ambos os compostos induzem a morte celular através da ativação da via extrínseca da apoptose. Demonstrou-se ainda uma sinergia antitumoral nas células SKOV-3 quando estas foram tratadas concomitantemente com o composto 3.21 e cisplatina, assim como quando tratadas com o composto 4.11 e carboplatina. Em conclusão, estes resultados permitiram obter uma compreensão mais detalhada da relação estrutura-atividade, reatividade química e estabilidade dos derivados do celastrol. Além disso, demonstrou-se o potencial notável dos derivados do celastrol, tais como os compostos 3.21 e 4.11, como moléculas promissoras para o desenvolvimento de terapias antineoplásicas.

Doctoral Thesis in Pharmaceutical Sciences with specialization in Pharmaceutical Chemistry, presented to the Faculty of Pharmacy of the University of Coimbra.
Over the past few decades, tremendous progress has been made in the understanding, prevention and treatment of cancer. However, despite this progress, the incidence of cancer appears to be increasing. Therefore, there is an urgent need for the development of new chemotherapeutic agents with improved selectivity, efficacy and safety profiles. Numerous studies have highlighted the enormous anticancer potential of triterpenoids. In particular, pentacyclic triterpenoids have been shown to modulate multiple intracellular signaling pathways and exert chemopreventive and antitumor activities in various in vitro and in vivo model systems. Madecassic acid, a pentacyclic triterpenoid of plant origin, has been reported to possess a variety of pharmacological activities. However, only a few studies have attempted to explore the therapeutic potential of this natural compound, particularly regarding to its anticancer activity. In light of this observation, a series of new semi-synthetic derivatives of madecassic acid were designed, synthesized and evaluated for their in vitro cytotoxic activities, to identify promising lead compounds for the development of new anticancer drug therapies. The preparation of new madecassic acid derivatives was designed to follow three main synthetic strategies. The first one focused mainly on the functionalization of the C-2, C-3, C-6 and C-23 hydroxyl groups and the C-28 carboxylic acid. The second one aimed at the conversion of the 6-membered ring into a 5-membered ring with an α,β-unsaturated aldehyde substituent. Finally, the third semi-synthetic strategy was based on the introduction of different substituents at the C-2 position of the pentameric ring. All synthesized compounds were fully characterized using infrared radiation, nuclear magnetic resonance and mass spectrometry techniques, and their anticancer activity was evaluated against the US National Cancer Institute's 60 human cancer cell line (NCI-60) panel using the sulforhodamine blue colorimetric assay. Several analogs exhibited broad-spectrum cytotoxic activities over all nine tumor types represented in the panel, with more potent antiproliferative activities observed against select cancer cell lines, including multidrug-resistant phenotypes. Among them, compound 3.30, a cyclic enol ether derivative bearing a 2-furoyl moiety at C-3, exhibited sub-µM potencies against 26 different tumor cell lines from the NCI-60 panel. The mechanism of action of this compound was predicted by CellMinerTM bioinformatic analysis and confirmed by biochemical and cell-based experiments to involve inhibition of the DNA replication process and disruption of mitochondrial membrane potential. Furthermore, compounds 4.3, 4.7 and 4.10 displayed potent and highly differential antiproliferative activity against 80% of the tumor cells harboring the B-RafV600E mutation within the nanomolar range. Structure-activity analysis revealed that a 5-membered A-ring containing an α,β-unsaturated aldehyde substituted at C-23 with a 2-furoyl group seems to be crucial to produce this particular growth inhibition signature. In silico analysis of the cytotoxicity pattern of these compounds identified two highly correlated clinically approved drugs with known B-RafV600E inhibitory activity. Follow-up analysis revealed inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway through the reduction of rapidly accelerated fibrosarcoma (Raf) protein levels is a key mechanism of action of these compounds. Among these derivatives, compound 4.10 was the most potent compound in suppressing tumor growth of B-RafV600E-mutant cell lines and displayed the highest reduction of Raf protein levels. The 23-methanesulfonyloxy derivatives 5.5 and 5.7 showed different modes of action with broad cytotoxicity seen for compound 5.5 but some selectivity of cellular response seen for compound 5.7. CellMinerTM analysis revealed that compound 5.5 elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a potentially novel mechanism of anticancer action; whereas it identified the tyrosyl-DNA phosphodiesterase 1 (Tdp1) as a potential target of compound 5.7. Taken together, this work contributed to a deeper understanding of structure-activity relationship and chemical reactivity of madecassic acid derivatives. Moreover, it demonstrated the remarkable potential of madecassic acid derivatives, such as compounds 3.30, 4.10, 5.5 and 5.7, as promising leads for the development of new cancer therapies.
Nas últimas décadas tem-se assistido a um tremendo progresso na compreensão, prevenção e tratamento do cancro. No entanto, apesar deste progresso, a incidência do cancro parece estar a aumentar. Há, portanto, uma necessidade premente de desenvolver novos agentes anticancerígenos com melhores perfis de seletividade, eficácia e segurança. Vários estudos têm destacado o enorme potencial anticancerígeno dos triterpenóides. Em particular, os triterpenóides pentacíclicos parecem modular múltiplas vias de sinalização intracelular e exercer atividades quimiopreventivas e antitumorais em vários modelos in vitro e in vivo. O ácido madecássico, um triterpenóide pentacíclico de origem vegetal, demonstrou possuir uma ampla variedade de atividades farmacológicas. No entanto, poucos estudos tentaram explorar o potencial terapêutico deste composto natural, principalmente no que diz respeito à sua atividade anticancerígena. Perante este enquadramento, uma série de novos derivados semi-sintéticos do ácido madecássico foi idealizada e sintetizada e as suas atividades citotóxicas foram avaliadas in vitro, com o objetivo de identificar leads promissores para o desenvolvimento de novas terapias anticancerígenas. A preparação de novos derivados do ácido madecássico foi baseada em três principais estratégias sintéticas. A primeira focou-se principalmente na funcionalização dos grupos hidroxilos a C-2, C-3, C-6 e C-23 e do ácido carboxílico a C-28. A segunda visou a conversão do anel A de seis membros num anel de cinco membros com um substituinte aldeído α,β-insaturado. Finalmente, a terceira estratégia semi-sintética baseou-se na introdução de diferentes substituintes na posição C-2 do anel pentamérico. Todos os compostos sintetizados foram totalmente caracterizados usando técnicas de radiação infravermelha, ressonância magnética nuclear e espectrometria de massa, e as suas atividades anticancerígenas foram avaliadas num painel de 60 linhas celulares tumorais humanas do Instituto Nacional do Cancro dos Estados Unidos (NCI-60) utilizando o ensaio colorimétrico da sulforodamina B. Vários análogos exibiram atividades citotóxicas de amplo espectro nos nove tipos de tumores representados no painel, com atividades antiproliferativas mais promissoras observadas em linhas celulares selecionadas, incluindo fenótipos multirresistentes a fármacos. Entre estes, o composto 3.30, um derivado éter enólico cíclico com um substituinte 2-furoílo em C-3, exibiu actividades sub-µM em 26 linhas de células tumorais do painel NCI-60. O mecanismo de ação deste composto foi determinado utilizando a ferramenta bioinformática CellMinerTM e confirmado por experiências bioquímicas e celulares, envolvendo a inibição do processo de replicação do ADN e a alteração do potencial da membrana mitocondrial. Além disso, os compostos 4.3, 4.7 e 4.10 exibiram atividade antiproliferativa promissora e altamente diferencial dentro da gama nanomolar em 80% das células tumorais avaliadas que possuem a mutação B-RafV600E. Uma análise da relação estrutura-atividade revelou que um anel de cinco membros contendo um aldeído α,β-insaturado substituído em C-23 por um grupo 2-furoílo parece ser crucial para produzir esta assinatura específica na inibição de crescimento. A análise in silico do padrão de citotoxicidade destes compostos identificou dois fármacos clinicamente aprovados altamente correlacionados e com reconhecida atividade inibidora do B-RafV600E. Uma análise posterior revelou que a inibição da via de sinalização da quinase regulada por sinal extracelular (ERK) através da redução dos níveis de proteína do fibrossarcoma rapidamente acelerado (Raf) é um mecanismo de ação chave desses compostos. Entre estes derivados, o composto 4.10 foi o mais potente na supressão do crescimento tumoral de linhas celulares mutantes B-RafV600E e exibiu a maior redução dos níveis de proteína Raf. Os derivados de 23-metanossulfoniloxi 5.5 e 5.7 exibiram diferentes modos de ação, com ampla citotoxicidade observada para o composto 5.5, mas alguma seletividade de resposta celular observada para o composto 5.7. A análise dos resultados obtidos com recurso ao CellMinerTM revelou que o composto 5.5 provocou um perfil único de inibição de crescimento nas linhas celulares tumorais, indicando um mecanismo de ação anticancerígena potencialmente novo, enquanto identificou a tirosil-ADN fosfodiesterase 1 (Tdp1) como um potencial alvo do composto 5.7. Em conclusão, este trabalho contribuiu para uma compreensão mais aprofundada da relação estrutura-atividade e reatividade química dos derivados do ácido madecássico. Além disso, demonstrou o notável potencial dos derivados do ácido madecássico, em particular o dos compostos 3.30, 4.10, 5.5 e 5.7, como leads promissores no desenvolvimento de novas terapias contra o cancro.

Various plant species have been reported traditionally as well as in scientific literature for cytotoxicity against animal species. Isolation of several poisonous compounds from plant species has been reported previously. Elaeodendron croceum is a well-known poisonous plant species of which the poisonous compounds have not yet been isolated. A phytochemical investigation of E. croceum leaves guided by cytotoxicity against Vero cells, led to the isolation of five known compounds; 20-hydroxy-20-epi-tingenone (1), tingenone (2), tingenine B (3), 11α-hydroxy-β-amyrin (4), and naringenin (5). Compounds 1 and 2 showed the highest toxicity against Vero cells (IC50: 2.651 nM and 8.233 μM respectively). Cytotoxicity of the isolated compounds against three human cancer cell lines, HeLa, MCF-7, and SNO was also determined. Compounds 1 and 2 again showed the highest cytotoxicity with IC50 values ranging between 2.478 – 0.427 μM. This is the first report on the isolation, identification, and in vitro evaluation of poisonous compounds from E. croceum. Copyright
Dissertation (MSc)--University of Pretoria, 2010.
Plant Production and Soil Science
unrestricted

碩士
臺灣大學
微生物與生化學研究所
98
Epstein-Barr virus (EBV) is a human herpesvirus, which has two different life cycles: latent and lytic. EBV infects lymphoid and epithelial cells. Infection by this virus causes infectious mononucleosis and is closely related to several malignant diseases, including Burkitt’s lymphoma, T-cell lymphoma, Hodgkin’s disease，gastric cancer and nasopharyngeal carcinoma. At the onset of the EBV lytic cycle, the virus expresses two immediate-early genes, BRLF1 and BZLF1, which encode transcription factors, Rta and Zta, respectively. These two transcription factors are required to activate the EBV early genes and lytic cascade. Clinically effective anti-EBV drugs that target EBV-encoded DNA polymerase, including acyclovir, ganciclovir and indolocarbazole, are commonly used for inhibiting the lytic cycle of EBV. However, viruses are potentially mutagenic for resistance to drugs. In light of this, it is necessary to identify new targets for antivirus chemotherapy and to develop new treatment strategies. Triterpenoids are formed from six isoprene units with 30 carbons. As it is generally known, triterpenoids are major constituents in several herbal remedies or fungi that are reported to have the capacity of anti-inflammatory, anticancer, repressing cell proliferation, inducing cell apotosis program, etc. The purpose of this study is to demonstrate the effect of JYKMA-37, one of the derivatives of triterpenoids, on repressing the EBV lytic cycle. First of all, P3HR1 cells were treated with different concentrations of JYK-MA-37, and then induced the EBV lytic cycle by sodium butyrate (SB) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblot analysis and flow cytometry analysis were performed, the results showed that JYK-MA-37 at 10 μM effectively inhibits the expression of EBV lytic protein, including Rta、Zta and EA-D, after lytic induction in P3HR1 cells (EC50 = 22.083 μM，CC50 = 11.32 μM). Moreover, transient transfection analysis revealed that transactivation of BRLF1 promoter (Rp) and BZLF1 promoter (Zp) were inhibited by JYK-MA-37 in a dose-dependent manner. Finally, real-time PCR showed that JYK-MA-37 substantially reduces the numbers of EBV particles produced by the cells after lytic induction. Taken together, this study demonstrated that JYK-MA-37 poccess the capacity of inhibiting the progression of EBV lytic cycle.

碩士
國立臺南大學
生物科技學系碩士班
107
Ganoderic Acid A is one of the triterpenoids from Ganoderma lucidum, a parasitic fungus with a damp environment and dim light that grows only at the roots of rot trees or trees. Therefore, strains with the ability to convert Ganoderic Acid A were searched, and a total of 283 actinomycetes were screened, and their ability to catalyze Ganoderic Acid A was determined by Ultra high performance liquid chromatography. A positive strain AI 045 was selected for study, and the AI 045 was confirmed to be Streptomyces sp. by the DNA sequence of the 16S rRNA gene. The bioconverted metabolite was purified using a preparative High Performance Liquid Chromatography and the metabolite compound analyzed based on mass and nuclear magnetic resonance spectroscopy data was 3-O-acetyl-ganoic acid A. Ganoderic Acid A’ is one of the triterpenoids from Ganoderma lucidum, a parasitic fungus that survives in the damp environment and dim light. It grows only at the roots of rotten trees. Therefore, observed sample has the ability to convert ‘Ganoderic Acid A’ were searched, and a total of 283 actinomycetes were screened. It also had the ability to catalyze ‘Ganoderic Acid A’, which was determined by Ultra high performance liquid chromatography. A positive strain AI 045 was selected for study, and the AI 045 was confirmed to be Streptomyces sp. by the DNA sequence of the 16S rRNA gene. The bio-converted metabolite was purified using a preparative High Performance Liquid Chromatography and the metabolite compound was analyzed. Based on mass and nuclear magnetic resonance spectroscopy data, it was found to be 3-O-acetyl-ganoic acid A.

碩士
長庚大學
化學工程研究所
86
The fungus Ganoderma lucidum has been used in folk medicine in Chinese culture. Tradientially, the effective methods of extraction biological activity compounds from Ganoderma lucidum are using hot water, methanol and ethanol. The aim of this dissertation is to explore the feasibility of extracting the triterpenoids from Ganoderma lucidum by supercritical fluid, and comparing this method with the traditional one. The techniques of this experiment are using continuous flow type and control the range of temperature 310.5-323.0 K, pressure 2000-4000 psi, and chooses water or ethanol as cosolvent. According to the experimental result, it shows ethanol is a suitable cosolvent. To increase the pressure at constant temperature, the quantity of extraction will increase when the pressrue and density go up, to increase the temperature at constant pressure, the quantity of extraction will either increase or reduce at a certain range of temperature. Moreover, the experiment indicates that in the condition of 323k, 4000 psi and methanol as cosolvent, the maximum yield is about 0.04g essential oil per gram Ganoderma lucidum. What''s more, we also convinced from the research that crossover pressure will exist at 3750 psi. Comparing the essential oil weight and the gradient eluent pattern of HPLC analysis with the traditional extracting mehtod, the result shows that supercritical carbon dioxide with ethanol as cosolvent really shows better extracting effect.

碩士
台北醫學院
藥學研究所
86
To test the possibility of triterpenoids on cornea tissue fluidity as well as to modify enhancement by the alternation of physicochemical propertie s of triterpenoids. Methods. Oleanolic acid (OA), Ursolic acid (UA) and Betuli nic acid (BA) were chosen to perturb the cornea tissue by transcellular marker (14C- estradiol) and paracellular marker (C- mannitol), for evaluation the me mbrane integrity and properties. In addition, cornea was treated with Linoleni c acid (LA), well-known membrane perturbation agent, for comparison. A series of different temperatures (4C, 20C, 37C and 45C), charge density, size, zeta p otential, partition coefficient and structure of these reagents were monitored influentially ability. Results. Transcellular marker had shown that the appar ent permeability coefficients of saturated OA, BA and LA (1mM) at 37C were l arger than control(24.67±1.55*10e-6 cm/sec), 43.70±1.69, 36.19±1.43 and 36. 01±1.39*10e-6 cm/sec, respectively, except UA(27.13±1.18*10e-6 cm/sec). On t he other hand, only LA could affect paracellular marker at 37C condition. Af ter modified the charge density (pH=7.4 to 3) of enhancers, OA and LA enhancem ent were disappered, not BA. Different influence patterns of temperature on es tradiol permeability of cornea by OA, UA, BA and LA were observed and OA was o bserved most sensitivity by thermostat change. Following opposite tansport of OA on cornea was also shown increment of estradiol permeability and it didn't affect on low concentration performance. In the meantime, under SEM performanc e of OA at pH 7.4 and 3, aggregation of OA particle was observed at different pattern. However, the zeta potential and partition coefficient examinations we re not much different among the all tests for these enhancers. Conclusions. OA , UA, BA and LA revealed different perturbation on different temperature and p H condition. We proposed the enhancement mechanism of these perturbation agent s are interference the cornea tissues by inserting OA among the lipid bilayer.

碩士
國防醫學院
生物化學研究所
100
Families of cytochrome P450 protein play a crucial role in the metabolism of endogenous compounds and xenobiotics. Among them, CYP3A4 is most important, as it metabolizes at least 50% of all clinically used drugs. Previously studies indicated that the nuclear receptor PXR (Pregnane X receptor) and CAR (Constitutive abdrostane receptor) are essential in transcription activation of CYP3A4 gene expression. PXR is known to be activated by a wide range of endogenous and exogenous compounds, including chemical drugs and natural products form herbal medicines. We have been studying the action mechanism of herbal medicines including ginseng, astragalus, and poria cocos. The main ingredients of these medicines are triterpenoidal compounds, which are generally attributed for most of its pharmacological effects. Since triterpenoids have a common steroid-like structure, they are expected to have certain steroid-related pharmacological activies. In this study, we investigated whether triterpenoids affect the expression of CYP3A4 through PXR and CAR pathways. Transient transfection studies were carried out in HepG2 cells using CYP3A4 reporter plasmid along with PXR or CAR expression vector. We first showed that these compounds induced CYP3A4 transcription through PXR and CAR pathways. Using Q-PCR and Western blot analysis, we then demonstrated that these compounds significantly increase the expression of CYP3A4 both at mRNA and protein level. Using PXR and CAR inhibitors, the role of PXR and CAR in triterpenoid-mediated activation of CYP3A4 gene expression was further confirmed in the HepG2 cells. These compounds activate PXR partly through increase PXR gene expression and stimulate PXR nuclear translocation. Taken together, our results demonstrated that triterpenoid compounds induced the expression of CYP3A4 in hepG2 cells through activation of PXR and CAR pathways.

碩士
國立中央大學
化學工程與材料工程研究所
98
In order to study effectively increase the triterpenoids of Antrodia fungi type and amount of extracellular polysaccharide production and increase production efficiency, this study will explore the influence of light on liquid fermentation results were as follows: (1) of the cell, the red and blue light are conducive to the growth of Antrodia cinnamomea, in terms of red light 400 lux,Yx/s can reach 0.854 g / g. (2) of the extracellular polysaccharide, the Blue light will help the production of extracellular polysaccharide of Antrodia cinnamomea in the blue light 400 lux, Yp/s is 0.033 g / g, than the dark group to enhance 27%. But the fermentation time is more than the dark, so roughly the same yield and dark. On the molecular weight of exopolysaccharide, the irradiation by red light from the dark of the 868 kDa molecular weight decreased slightly to about 779 kDa, while the Blue light is the molecular weight up to 1400 kDa. (3) intracellular production of triterpenoids, the red light illumination increases with positive assistance, Blu-ray irradiation on the triterpenoids, and no impact on production.

碩士
國防醫學院
生物化學研究所
96
Triterpenoids, such as ginsenosides, oleanolic acid (OA), and ursolic acid (UA), are major components of a variety of herbal medicines with multiple pharmacological activities. Despite extensive studies, the mechanism of their action remains to be clarified. In our previous studies, we found that ginsenosides NL-006 and NL-007 elicited potent enhancing and suppressing effects, respectively, on glucose uptake across human intestinal Caco-2 monolayer through modulation of SGLT1 expression. To study the effect of these ginsenosides and the two pentacyclic triterpenoids, OA and UA on glucose uptake in other cell types, differentiated 3T3-L1 adipocytes model system was used. Using non-metabolized [14C]-labeled 2-deoxyglucose as substrate, we showed that both NL-006 and NL-007 enhanced glucose uptake in the 3T3-L1 adipocytes. In contrast, both of the OA and UA suppressed glucose uptake in differentiated adipocytes. To understand the mechanism of the effect on glucose uptake by these compounds, the expression of glucose transporters (GLUT1 and GLUT4) were investigated. NL-006 and NL-007 increased the expression of GLUT4 in 3T3-L1 adipocytes. Fractionation of cellular membrane systems by sucrose density gradient ultracentrifugation, we found that the GLUT4 level in the plasma membrane fraction was enhanced by NL-006 and NL-007. Consistent with immunofluorescent assay, these results indicated that NL-006 and NL-007 enhanced membrane translocation of GLUT4 in 3T3-L1 adipocytes. In the sterol ester-induced 3T3-L1 adipocytes, we found that all of these compounds enhanced glucose uptake. The GLUT4 expression level and translocation to cytosolic membrane were also enhanced by all of these compounds. Taken together, our results demonstrated that natural triterpenoids, despite their similar structures, may affect glucose uptake into adipocytes through different mechanism.

碩士
國立臺灣師範大學
人類發展與家庭學系
104
Ultraviolet B (UVB) radiation plays a vital role in skin photodamage and photoaging. It causes serious inflammation and DNA damage of epidermis, which is the outermost viable layer of the skin and provides skin barrier function. In our previous study, methanolic leaf extract of wild bitter melon (WBM, Momordica charantia L. var abbreviata Seringe) shows anti-tyrosinase activity and significantly reduced UVB-induced reactive oxygen species (ROS) production in vitro. In this study, we investigated the protective effects of bitter melon triterpenoids on UVB-irradiated HaCaT cells. Two cucurbitane triterpenoids, Kuguacin R and 3, 7, 25-trihydroxycucurbita-5, 23-dien-19-al (TCD) were isolated from ethanolic leaf extract of WBM. HaCaT cells were pretreated for 24h with Kuguacin R or TCD prior to UVB irradiation (20-30 mJ/cm2), except for DNA fragmentation test (100 mJ/cm2). Our results showed that Kuguacin R and TCD inhibited UVB-induced cytotoxicity, and also diminished the productions of interleukin (IL)-1β, IL-6, and IL-8. Furthermore, both compounds significantly reduced phosphorylation of MAPKs, NF-κB activation, c-Jun, clyclooxygenase-2 protein levels, and the prostaglandin E2 production. In addition, both compounds inhibited caspase-3 activation and DNA fragmentation. Besides, Kuguacin R and TCD showed potential effects on percutaneous absorption in vitro. Our findings suggested that WBM triterpenoids, Kuguacin R and TCD may be beneficial for UVB-induced damage of keratinocytes and suggested its potential use in skin UV protection.

碩士
朝陽科技大學
應用化學系生化科技碩士班
101
Antrodia camphorata (AC) is a unique mushroom of Taiwan. It parasite on the protected endemic species Cinnamomum kanehirae (Bull camphor tree).The extracted triterpenoids from A. camphorata express excellent bioactivities, like anti-cancer, anti-inflammatory, lowering-blood pressure, lowering-blood lipids and activate nerve cells and other effects. It was widely used in functional (health) foods and medicine development in Asia. Currently, the products of A. camphorata on market come from different sources. Based on liquid culture or solid-state cultured condition, the triterpenoid components are also different. Usually, triterpenoids was analyzed by HPLC, LC-MS/MS was also used to do pharmacokinetic study of triterpenoids in rat blood. No study reports the application of MALDI-TOF MS on analysis of A. camphorata. In this study, a fast, sensitive and enviromental friendly analysis method by MALDI-TOF MS will be applied to determine eight triterpenoid componds of A. camphorata. A octadecyltrichlorosilane (OTS) modified hydrophobic aluminum foil surface was used on target plate to increase the analyte signal intensity. The identified analytes include methyl antcinate B (AC 1), antcin B (AC 5), antcin H (AC 7), antcin A (AC 9), antcin C (AC 10), methyl antcinate A (AC 11), antcin K (AC 12), and methyl antcinate H (AC 13). The results, showing the linear range and r2 value are 2 ppm~20 ppm with r2 = 0.954 ~ 0.996, respective. This method will be able to fast distinguish the different sources of A. camphorata products on market.

T =t a a !r a a a t I a a a - - 5. CONCLUSION 5. CONCLUSION This work was focused on the synthesis of new triterpenoids - named highly oxidized derivatives of 18o-oleanane, their reductive products or products with heteroatoms, for example fluorine, nitrogen, suflur or phosphorus. Part of this thesis was focused on reactions of lupane and I 8cr-oleanane triterpenoids with Lawesson's reagent. l. Highly oxidized l8cr-oleanane derivatives were synthesised, in particular on A-ring. Commercially available reagents were used for these oxidations: MCPBA, SeOr or pero- xyacetic acid (as a persteril). A group of seco derivatives was prepared by reactions with in situ prepared RuOo. A great deal of this section has been already published in scienti- fic journal.2a 2. Agroup of 18ct-oleanane alcohols (e.g. L5,16a,16b, 19, 20,22) was prepared from some oxidized derivatives (e.g.2,3, 13, L8) by reactions with LiAlHo or NaBI{.. 3. Reactions of some ketones were carried out (e.g. 3) with fluorinating agent DAST for preparation of new fluorous derivatives (e.g. 24,25). The same ketones reacted with ethanedithiol to afford new dithiolanes (e.g. 26). Diketone 3 was'also used for synhesis of several nitrogen containing derivatives. Preparation of pyrazine 27 and quinoxaline 28 has been already published in scientific...

碩士
國立臺灣師範大學
生命科學研究所
92
In Taiwan, hepatocellular carcinoma (HCC) is the leading cause of cancer mortality. The development of potential liver protective agents and drugs from herbal medicines for the treatment of HCC deserves great attention. The purpose of this study is to elucidate the biological activities and antitumor pharmacological functions of Ganoderma australe (Fr.) Pat. (subgenus Elfvingia). For years, G. australe has been erroneously identified as G. applanatum in Taiwan. Until 1990 (Yeh, 1990), this species was identified as Ganoderma australe (Fr.) Pat. Comparing to G. lucidum (Fr.) Karst., a famous fungus in traditional Chinese medicine, studies on G. australe are very limited and the pharmacological potential of this fungus remains unknown. The fruiting bodies of G. australe were extracted by methanol (1:20, w/v) to obtain the triterpenoid-enriched crude extracts (designated as GA-M1-1206). Human hepatoma cell line (Hep 3B) was chosen as the in vitro model. Inhibition of hepatoma cell growth was used as a bioassay to guide the isolation of bioactive compounds from G. australe. Cell viability was determined by using the MTT assay. Separation of GA-M1-1206 by silica gel column chromatography gave 25 fractions (GA-M1-C1 to GA-M1-C25). The results of bioassay indicated that the fractions 4, 5, and 6 were the three most effective fractions to inhibit the growth of cultured Hep3B cells. These fractions were pooled and designated as GA-C46. Repeated bioassay was conducted to give the IC50 value (0.078 µg/mL). Male nude mice (BALB/c-nu/nu), inoculated subcutaneously with human hepatoma cells (Hep3B/T2), were used as the animal model to elucidate the antitumor pharmacological function of GA-C46. The results of in vitro assay were used to design the dose range in the animal model. Animals were randomly divided into two groups and treated for three cycles (7 days per cycle). Mice in the CT group (n=13) were fed with a normal diet (Purina 5010) and GA group (n=7) were treated with 20, 40, and 40 mg (pre kg body weight /day) of GA-C46 in cycles 1, 2, and 3, respectively. Tumor size (L×W2×0.52 cm3) was monitored every two days in the entire treatment period. The results showed that the tumor size of GA group (0.83±0.38 cm3), compared with the tumor size (1.28±0.48 cm3) of CT group, was reduced significantly (by 35.2%; p=0.0480) in the end of the first cycle. Reduction of tumor sizes in GA group was continuously observed in the three treatment cycles for 21 days (CT group 3.37±1.53 cm3; GA group 1.81±1.13 cm3) (46.3% reduction, p=0.0317 on day 21) until animals were sacrificed. Serum GOT, GPT, biochemical markers, and tumor weights were recorded and liver and lung metastasis was examined. Results showed that the tumor weight of GA group (0.67±0.37 g) was significantly reduced by 57.1%, compared with that of the CT group (1.56±0.70 g) (p=0.0064). The animal study established the antitumor pharmacological function of GA-C46. Further isolation of active components in GA-C46 was conducted by reversed-phase high performance liquid chromatography (RP-HPLC) (69 fractions were collected totally). By repeatedly using Hep 3B cells as the in vitro model, results showed that the biological activities appeared in seven low-polar fractions (tentatively designated as GA-C46-H43, 54, 57, 58, 60, 62, 63 fractions). These fractions will be further separated by semi-preparative RP-HPLC to obtain pure active compounds for structural elucidation. In conclusion, this study has incorporated in vitro and in vivo models to elucidate the potential of G. australe (GA-C46) to inhibit hepatoma cell growth and reduce implanted tumor. The antitumor potential of GA-C46 from G. australe was comparable or even better than those of G. lucidum and G. tsugae. This study provides valuable information for future evaluation of G. australe as an antitumor agent.