<p>To examine cyclooxygenase-2 (COX-2) inhibitory effects of natural compounds and structurally related compounds, a radiochemical assay for measuring inhibition of COX-2 and COX-1 catalysed prostaglandin biosynthesis was optimised. That process resulted in the development of a reliable assay for finding COX-2 inhibitors in plants, and for investigating plant constituents pertaining to the inhibition of COX-2 and COX-1. </p><p>By means of bioassay-guided isolation of the plant Plantago major L., several inhibitors of COX-2 were found: ursolic, oleanolic, a-linolenic and linoleic acid. Subsequently, structural derivatives of these triterpenoids and fatty acids were investigated. The polyunsaturated and the thioether containing fatty acids were the most potent COX-2 and COX-1 inhibitors, with a-linolenic and all-(Z)-5-thia-8,11,14,17-eicosatetraenoic acid expressing selectivity toward COX-2. </p><p>For rapid evaluation of plant constituents, a COX-2 assay using scintillation proximity assay-technology was used to evaluate 49 ubiquitous plant metabolites of different biosynthetic origin for inhibition of COX-2. Eugenol, cinnamaldehyde, and pyrogallol expressed inhibition. Later, in an attempt to find COX-2 inhibitors in plants, but without prior purification, several approaches to the surface plasmon resonance technique were examined, with the measurement of prostaglandin E2 being most successful.</p><p>In mouse macrophage cells, two fatty acids were analysed for effects on COX-2 and iNOS, mRNA, protein, prostaglandin E2, and nitrite levels. 5-Thia-8,11,14,17-eicosatetraenoic acid decreased COX-2 protein expression.</p><p>This thesis contributes to our growing knowledge of methods for measuring COX-2 inhibition, and also knowledge of COX-2 inhibitory effects of commonly occurring compounds in plants, herbal drugs and functional food. Thus the experimental results can facilitate future searches for new COX-2 inhibitors of natural origin.</p>

<p>In inflammatory events, the cyclooxygenase-2 enzyme (COX-2) catalyses prostaglandin biosynthesis. This is a process, which could possibly be affected by natural compounds including those abundant in food. Two systems have been established to enable investigations of the effects of natural compounds on COX-2. The first method developed was an in vitro method suitable for measuring inhibition of COX-2 catalysed prostaglandin E₂ biosynthesis, based on scintillation proximity assay technology. The second system established, comprises a cell model, suitable for studying the effects of compounds on COX-2 and inducible nitric oxide synthase (iNOS) at different cellular levels, including the effects on mRNA, protein, prostaglandin E₂ and nitrite levels.</p><p>The plants, <i>Plantago major</i> L. and <i>Urtica dioica</i> L., were subjected to bioassay-guided isolation and fatty acids, e.g. α-linolenic acid and linoleic acid, were isolated as COX-2 inhibitory principles. The effects of structurally related fatty acids were also studied and the most potent COX-2 inhibitors were eicosapentaenoic acid and its synthetic derivative all-(<i>Z</i>) -5-thia-8,11,14,17-eicosatetraenoic acid. The latter was also found to decrease COX-2 protein levels assessed by the cell model. The inhibitory effects on COX-2 catalysed prostaglandin biosynthesis of 49 plant metabolites with different biosynthetic origin were studied. The phenolic compounds, eugenol, pyrogallol, but also cinnamaldehyde, were found to inhibit COX-2.</p><p>Recently, COX-2 has attracted attention because of its involvement in cancer. The compounds in our diet are suggested to play an important role in the aetiology of colon cancer. To investigate the influence of diet on COX-2 in relation to colon cancer cells, a number of human faecal water samples were assessed for their effects on COX-2 enzymatic activity and protein synthesis. Of the faecal waters tested, two samples exhibited a weak inhibitory effect on COX-2 enzymatic activity, and one sample decreased COX-2 protein levels in a colon cancer cell line. The chemical content of faecal water was analysed and a variety of phenolic compounds were identified, including flavonoids and phenolic acids. Hitherto, a number of the identified compounds in faecal water have been assessed for the effects on COX-2, but none of the tested compounds were found to be responsible for the observed effects of faecal water on COX-2. Moreover, the cytotoxic effects of natural COX-2 inhibitors, traditional non-steroidal anti-inflammatory drugs, and the selective COX-2 inhibitor rofecoxib were evaluated using a human cancer cell line panel. Of the compounds tested, curcumin was found to be most potent.</p><p>This thesis contributes to increase the general knowledge of the effects on COX-2 of commonly occurring plant constituents, including fatty acids and phenolic compounds. As natural parts of our diet, these compounds, abundant in fruits and vegetables, may affect different targets in our bodies, including COX-2.</p>

To examine cyclooxygenase-2 (COX-2) inhibitory effects of natural compounds and structurally related compounds, a radiochemical assay for measuring inhibition of COX-2 and COX-1 catalysed prostaglandin biosynthesis was optimised. That process resulted in the development of a reliable assay for finding COX-2 inhibitors in plants, and for investigating plant constituents pertaining to the inhibition of COX-2 and COX-1. By means of bioassay-guided isolation of the plant Plantago major L., several inhibitors of COX-2 were found: ursolic, oleanolic, a-linolenic and linoleic acid. Subsequently, structural derivatives of these triterpenoids and fatty acids were investigated. The polyunsaturated and the thioether containing fatty acids were the most potent COX-2 and COX-1 inhibitors, with a-linolenic and all-(Z)-5-thia-8,11,14,17-eicosatetraenoic acid expressing selectivity toward COX-2. For rapid evaluation of plant constituents, a COX-2 assay using scintillation proximity assay-technology was used to evaluate 49 ubiquitous plant metabolites of different biosynthetic origin for inhibition of COX-2. Eugenol, cinnamaldehyde, and pyrogallol expressed inhibition. Later, in an attempt to find COX-2 inhibitors in plants, but without prior purification, several approaches to the surface plasmon resonance technique were examined, with the measurement of prostaglandin E2 being most successful. In mouse macrophage cells, two fatty acids were analysed for effects on COX-2 and iNOS, mRNA, protein, prostaglandin E2, and nitrite levels. 5-Thia-8,11,14,17-eicosatetraenoic acid decreased COX-2 protein expression. This thesis contributes to our growing knowledge of methods for measuring COX-2 inhibition, and also knowledge of COX-2 inhibitory effects of commonly occurring compounds in plants, herbal drugs and functional food. Thus the experimental results can facilitate future searches for new COX-2 inhibitors of natural origin.

In inflammatory events, the cyclooxygenase-2 enzyme (COX-2) catalyses prostaglandin biosynthesis. This is a process, which could possibly be affected by natural compounds including those abundant in food. Two systems have been established to enable investigations of the effects of natural compounds on COX-2. The first method developed was an in vitro method suitable for measuring inhibition of COX-2 catalysed prostaglandin E2 biosynthesis, based on scintillation proximity assay technology. The second system established, comprises a cell model, suitable for studying the effects of compounds on COX-2 and inducible nitric oxide synthase (iNOS) at different cellular levels, including the effects on mRNA, protein, prostaglandin E2 and nitrite levels. The plants, Plantago major L. and Urtica dioica L., were subjected to bioassay-guided isolation and fatty acids, e.g. α-linolenic acid and linoleic acid, were isolated as COX-2 inhibitory principles. The effects of structurally related fatty acids were also studied and the most potent COX-2 inhibitors were eicosapentaenoic acid and its synthetic derivative all-(Z) -5-thia-8,11,14,17-eicosatetraenoic acid. The latter was also found to decrease COX-2 protein levels assessed by the cell model. The inhibitory effects on COX-2 catalysed prostaglandin biosynthesis of 49 plant metabolites with different biosynthetic origin were studied. The phenolic compounds, eugenol, pyrogallol, but also cinnamaldehyde, were found to inhibit COX-2. Recently, COX-2 has attracted attention because of its involvement in cancer. The compounds in our diet are suggested to play an important role in the aetiology of colon cancer. To investigate the influence of diet on COX-2 in relation to colon cancer cells, a number of human faecal water samples were assessed for their effects on COX-2 enzymatic activity and protein synthesis. Of the faecal waters tested, two samples exhibited a weak inhibitory effect on COX-2 enzymatic activity, and one sample decreased COX-2 protein levels in a colon cancer cell line. The chemical content of faecal water was analysed and a variety of phenolic compounds were identified, including flavonoids and phenolic acids. Hitherto, a number of the identified compounds in faecal water have been assessed for the effects on COX-2, but none of the tested compounds were found to be responsible for the observed effects of faecal water on COX-2. Moreover, the cytotoxic effects of natural COX-2 inhibitors, traditional non-steroidal anti-inflammatory drugs, and the selective COX-2 inhibitor rofecoxib were evaluated using a human cancer cell line panel. Of the compounds tested, curcumin was found to be most potent. This thesis contributes to increase the general knowledge of the effects on COX-2 of commonly occurring plant constituents, including fatty acids and phenolic compounds. As natural parts of our diet, these compounds, abundant in fruits and vegetables, may affect different targets in our bodies, including COX-2.

Chemical space is basically infinite, and comprises all molecules that could possibly exist. Intelligent ways to efficiently navigate through chemical space and to select promising compounds in drug discovery are important tasks, and the focus of this thesis. In this work a new model for chemical space navigation, ChemGPS-NP, was developed. This model is based on a methodology where a global chemical space map is defined through principal component analysis of physico-chemical properties of a reference set of compounds. Through interpolation from the reference set, positions of novel compounds can be defined on this map and interpreted as chemical properties. ChemGPS-NP was demonstrated to be able to chart the entire biologically relevant chemical space, including both drug-like and natural compounds. This is an important improvement considering the present interest in natural products (NPs) in the pharmaceutical industry, as well as the track record of NPs to serve as basis for more than 50% of all marketed drugs. ChemGPS-NP proved able to handle and process large data sets, to aid in efficient selection of test objects, and to extract useful information from the results of high-throughput screening campaigns. Using ChemGPS-NP, it was shown that NPs occupy unique regions of chemical property space in comparison to drug-like compounds, and a number of features distinguishing NPs from medicinal chemistry compounds were identified. ChemGPS-NP was also shown to be able to reliably predict mode of action of anticancer agents based on chemical structure, a finding that has potential to improve cancer research efficiency. Applying a property based similarity search based on calculated eight dimensional Euclidean distances from ChemGPS-NP rendered a tool to identify NP inspired potential leads for drug discovery. Furthermore, ChemGPS-NPWeb, an online version of ChemGPS-NP, was developed, which provides scientists with open access to the tool via http://chemgps.bmc.uu.se/.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 6 uppsatser.

<p>The development of resistance by tumour cells to chemotherapeutic agents is a major problem in cancer treatments. One way to counter this is to find compounds with cytotoxic mechanisms other than those of drugs in clinical use today. The biological and chemical diversity encountered in Nature provide opportunities to discover completely new chemical classes of compounds. Some of these may represent previously unknown anticancer agents, and in some cases, novel, potentially relevant cytotoxic mechanisms. </p><p>The selection of plants for the cytotoxic investigation in this project was designed to cover large parts of the angiosperm system, providing a broad representation of species. Extracts of the plants were subjected to a polypeptide fractionation protocol, followed by bioassay-guided isolation, yielding series of fractions with increasing purity and cytotoxicity. The cytotoxicity assay included tumour cells from patients and a cell-line panel including ten different cell lines representing several types of resistant and non-resistant tumours. This screening strategy allowed fractions and compounds acting with novel mechanisms to be detected at an early stage. </p><p>The compounds isolated represent substantial chemical diversity and originate from diverse parts of the phylogenetic spectrum examined. They include the highly potent cytotoxic alkaloid, thiobinupharidine, the structure of which was determined by NMR techniques. Furthermore, two types of compound were shown to have previously unreported cytoxic activity: cyclotides (small macrocyclic polypeptides, in this case from violets) and polypeptides, possibly of thionine type, of loranthaceaeous mistletoes (collected in Panama). The well known cardiac glycosides from the foxglove, Digitalis, were identified as being responsible for the anti-tumour activity of this species.</p><p>In conclusion, the results obtained in this project show that selection based on phylogenetic information, together with a robust and reliable method to detect cytotoxicity, can be a useful approach for exploring the plant kingdom for cytotoxic substances.</p>

The development of resistance by tumour cells to chemotherapeutic agents is a major problem in cancer treatments. One way to counter this is to find compounds with cytotoxic mechanisms other than those of drugs in clinical use today. The biological and chemical diversity encountered in Nature provide opportunities to discover completely new chemical classes of compounds. Some of these may represent previously unknown anticancer agents, and in some cases, novel, potentially relevant cytotoxic mechanisms. The selection of plants for the cytotoxic investigation in this project was designed to cover large parts of the angiosperm system, providing a broad representation of species. Extracts of the plants were subjected to a polypeptide fractionation protocol, followed by bioassay-guided isolation, yielding series of fractions with increasing purity and cytotoxicity. The cytotoxicity assay included tumour cells from patients and a cell-line panel including ten different cell lines representing several types of resistant and non-resistant tumours. This screening strategy allowed fractions and compounds acting with novel mechanisms to be detected at an early stage. The compounds isolated represent substantial chemical diversity and originate from diverse parts of the phylogenetic spectrum examined. They include the highly potent cytotoxic alkaloid, thiobinupharidine, the structure of which was determined by NMR techniques. Furthermore, two types of compound were shown to have previously unreported cytoxic activity: cyclotides (small macrocyclic polypeptides, in this case from violets) and polypeptides, possibly of thionine type, of loranthaceaeous mistletoes (collected in Panama). The well known cardiac glycosides from the foxglove, Digitalis, were identified as being responsible for the anti-tumour activity of this species. In conclusion, the results obtained in this project show that selection based on phylogenetic information, together with a robust and reliable method to detect cytotoxicity, can be a useful approach for exploring the plant kingdom for cytotoxic substances.

<p>The process of drug discovery from natural products starts with the selection of study object. In this project recent knowledge and methods are incorporated to investigate the process of such selection for pharmacognostic investigations. As the model and object of study mistletoes and their content of the small cytotoxic peptides thionins are chosen.</p><p>The thionins are compared in silico to other proposed plant innate defense peptides. Utilizing analysis of amino acid sequences and secondary structures, the thionins are shown to be one of eight distinct groups of cystein-rich plant polypeptides analysed. Common features of thionins are exploited in an investigation of isolation methods, where a simple acidic extraction is equally efficient to isolate thionins as the laborious methods hitherto used. </p><p>An effort to study the relationships of the order Santalales was done. To infer phylogenetic relationships from DNA sequences, we increased the taxon sampling for utilized genes and regions such as <i>rbcL</i>, <i>atpB</i> and ribosomal 18S and 26S rDNA sequences within the Santalales. Analysing these together with published sequences for other tricolpate taxa a position for Santalales as sister to caryophyllids and basal to asterids is implied. This indication is supported by chemical characters such as the presence of cyclopeptide alkaloids of a kind only known from Gentianales.</p><p>To validate the chemosystematic implications from thionin distribution extracts of mistletoes collected in Panama, Taiwan and Madagascar, and the relative <i>Osyris alba</i> (Santalaceae) collected in Spain, were screened with the established fluorescence microculture cytotoxicity assay using the thionin-sensitive human lymphoma cell-line U937GTB. Bioassay guided isolation concludes that the cytotoxic compounds in Loranthaceae may however constitute another group of peptides.</p><p>In conclusion this work shows that the incorporation of informatic techniques may aid prediction and decision making when planning pharmacognostic research.</p>