The secondary metabolites of citrus such as limonoids and flavonoids constitute an important part of human diet. The present work was undertaken to elucidate the effect of citrus limonoids and flavonoids on the bacterial cell-cell signaling in Vibrio harveyi, Escherichia coli O157:H7 and Salmonella Typhimurium LT2. The first experiment was focused on purification of limonoids from grapefruit and sour orange seeds. The limonoids were extracted using organic solvents and purified by chromatographic techniques. A total of ten limonoids (7 aglycones and 3 glucosides) were purified. Currently, simultaneous measurement of aglycones and glucosides of limonoids is not available. To address this limitation, an analytical method using high performance liquid chromatography was developed with the capability of measuring both aglycones and glucosides in a single run. Furthermore, its applicability in the fruit and juice samples was demonstrated. The third study investigated the V. harveyi cell-cell signaling inhibitory potential of purified limonoids. Isolimonic acid, ichangin, obacunone and nomilin were showed potent inhibitory activity. Furthermore, isolimonic acid and ichangin inhibit the signal transduction pathway by up-regulating the response regulator luxO. Isolimonic acid was also found to be a potent inhibitor of Escherichia coli O157:H7 cell-cell signaling in the fourth study. The results demonstrated that isolimonic acid inhibits the autoinducer/epinephrine mediated cell-cell signaling, biofilm and virulence in QseBC and QseA dependent fashion. Further investigations using limonin analogues, in the fifth study, demonstrated that the analogue limonin-7-methoxime inhibited the E. coli biofilm in type 1 pili and antigen 43 dependent-fashion, by preventing the binding of the adhesins to plastic surfaces. Another limonoid, obacunone was demonstrated to attenuate the Salmonella virulence by repressing Salmonella Pathogenicity Island 1 (SPI-1) in EnvZ/OmpR dependent mecahnism. The seventh study showed that naringenin, among the flavonoids, was the most potent inhibitor of V. harveyi and E. coli O157:H7 cell-cell signaling. Furthermore, naringenin was found to repress the (SPI-1) in PstS-HilD dependent fashion in the eighth study. In conclusion, the current project identified several limonoids and flavonoids with cell-cell signaling inhibitory property in three bacterial species.

Anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert most of their actions by binding to cannabinoid receptors. The effects of the endocannabinoids are short-lived due to rapid cellular accumulation and metabolism, for AEA, primarily by the enzymes fatty acid amide hydrolase (FAAH). This has led to the hypothesis that by inhibition of the cellular processing of AEA, beneficial effects in conditions such as pain and inflammation can be enhanced. The overall aim of the present thesis has been to examine the mechanisms involved in the cellular processing of AEA and how they can be influenced pharmacologically by both synthetic natural compounds. Liposomes, artificial membranes, were used in paper I to study the membrane retention of AEA. The AEA retention mimicked the early properties of AEA accumulation, such as temperature-dependency and saturability. In paper II, FAAH was blocked by a selective inhibitor, URB597, and reduced the accumulation of AEA into RBL2H3 basophilic leukaemia cells by approximately half. Treating intact cells with the tyrosine kinase inhibitor genistein, an isoflavone found in soy plants and known to disrupt caveolae-related endocytosis, reduced the AEA accumulation by half, but in combination with URB597 no further decrease was seen. Further on, the effects of genistein upon uptake were secondary to inhibition of FAAH. The ability to inhibit the accumulation and metabolism of AEA was shared by several flavonoids (shown in paper III). In paper IV, the isoflavone biochanin A and URB597 had effects in vivo, in a model of persistent pain, effects decreased by the cannabinoid receptor 1 antagonist AM251. In paper VI, the cellular processing of the endocannabinoid metabolites following degradation was examined, a mechanism poorly understood. It was found that nitric oxide (NO) donors significantly increased the retention of tritium in cell membranes following incubation with either tritiated AEA or 2-AG. Further experiments revealed that the effect of NO donors mainly involves the arachidonate part of the molecules. Inhibition of FAAH completely reduced the effect of NO donors in cells with a large FAAH component, indicating that the effects were downstream of the enzyme. These results suggest that the cellular processing of endocannabinoids can be affected in a manner of different ways by pharmacological manipulation in vitro and that naturally occurring flavonoid compounds can interact with the endocannabinoid system.

Flavonoids are a large class of dietary polyphenols that have emerged as candidate agents for chemoprevention in prostate cancer. Despite the large number of known flavonoids (over 9000), only a few have been studied in prostate cancer to date. The work presented in this thesis describes the identification of novel anti-proliferative flavonoids, their molecular effects on cell cycle and related proliferation and survival pathways, and their chemopreventive properties in a murine model of prostate carcinogenesis. We identified several novel flavonoids with potent anti-proliferative effects in human prostate cancer cells in vitro. Non-prostate cell lines were generally resistant to the effect of these flavonoids. Two of the most potent flavonoids identified, 2,2-dihydroxychalcone (DHC) and fisetin, induced S and G2 phase cell cycle arrest in LNCaP and PC3 prostate cancer cells. Gene expression studies employing oligonucleotide microarray demonstrated profound down-regulation in gene expression of 75 key cell cycle (predominantly G2 and M phase) genes by DHC and fisetin, and the enhanced expression of 50 stress-response genes with important roles in cell proliferation and survival. DHC and fisetin induced apoptosis, but not accelerated senescence, in prostate cancer cells. The chemopreventive effect of 4 flavonoids identified from the in vitro studies was examined in an autochthonous murine model of prostate cancer (TRAMP). Mice were administered diets supplemented with 1% DHC, 1% fisetin or a combination of flavonoids (0.25% DHC, 0.25% fisetin, 0.25% quercetin, 0.25% luteolin) for 32 weeks. We demonstrated a significant reduction in genitourinary weight, and a reduction in prostate cancer grade in mice administered 1% DHC and combination diets. Flavonoid supplementation was, however, associated with gastrointestinal toxicity in some mice. Liquid chromatography-mass spectrometry demonstrated the accumulation of high levels of flavonoid in the prostates of TRAMP mice. These findings lay the foundation for further studies of flavonoids in clinical chemoprevention trials.

Flavonoids are a large class of dietary polyphenols that have emerged as candidate agents for chemoprevention in prostate cancer. Despite the large number of known flavonoids (over 9000), only a few have been studied in prostate cancer to date. The work presented in this thesis describes the identification of novel anti-proliferative flavonoids, their molecular effects on cell cycle and related proliferation and survival pathways, and their chemopreventive properties in a murine model of prostate carcinogenesis. We identified several novel flavonoids with potent anti-proliferative effects in human prostate cancer cells in vitro. Non-prostate cell lines were generally resistant to the effect of these flavonoids. Two of the most potent flavonoids identified, 2,2-dihydroxychalcone (DHC) and fisetin, induced S and G2 phase cell cycle arrest in LNCaP and PC3 prostate cancer cells. Gene expression studies employing oligonucleotide microarray demonstrated profound down-regulation in gene expression of 75 key cell cycle (predominantly G2 and M phase) genes by DHC and fisetin, and the enhanced expression of 50 stress-response genes with important roles in cell proliferation and survival. DHC and fisetin induced apoptosis, but not accelerated senescence, in prostate cancer cells. The chemopreventive effect of 4 flavonoids identified from the in vitro studies was examined in an autochthonous murine model of prostate cancer (TRAMP). Mice were administered diets supplemented with 1% DHC, 1% fisetin or a combination of flavonoids (0.25% DHC, 0.25% fisetin, 0.25% quercetin, 0.25% luteolin) for 32 weeks. We demonstrated a significant reduction in genitourinary weight, and a reduction in prostate cancer grade in mice administered 1% DHC and combination diets. Flavonoid supplementation was, however, associated with gastrointestinal toxicity in some mice. Liquid chromatography-mass spectrometry demonstrated the accumulation of high levels of flavonoid in the prostates of TRAMP mice. These findings lay the foundation for further studies of flavonoids in clinical chemoprevention trials.

No description available.

This dissertation examines the effects of postharvest treatment and processing on biologically active compounds of orange juice, and ??Rio Red?? grapefruit and their ability to prevent chemically induced colon cancer in rat model. The first study evaluated the differences in flavonoid content of commercial ??made from concentrate?? (MFC) orange juices and ??not from concentrate?? (NFC) orange and grapefruit juices. Total flavonoid content of MFC orange juices (53 mg/100 mL; n = 12) was significantly (P ≤ 0.05) higher than NFC orange juices (36.5 mg/100 mL; n = 14). The second study investigated the ionizing radiation and storage effects on bioactive compounds and quality of ??Rio Red?? grapefruit. Results showed that storage and irradiation significantly (P ≤ 0.05) affected the bioactive compounds in grapefruit, however, the effect of storage was prominent. The third study examined the influence of irradiation and freeze drying on bioactive compounds of grapefruit. Irradiation of grapefruit prior to freeze drying resulted in enhanced (P ≤ 0.05) flavonoid content (naringin and narirutin). Freeze drying markedly reduced (P ≤ 0.05) lycopene content. Freeze drying and irradiation reduced (P ≤ 0.05) volatile compounds (d-limonene and myrcene), with the exception of ethanol. In the fourth study suppression of colon cancer development in Sprague Dawley rats by natural and irradiated grapefruits and their functional compounds, naringin and limonin, were evaluated.The total number of aberrant crypts (AC; P = 0.02), number of high multiplicity AC foci (ACF; P = 0.01), and proliferative index (P = 0.02) were lower and apoptosis (P = 0.02) was higher in azoxymethane (AOM) injected rats on experimental diets. However, only natural grapefruit and limonin only suppressed AOM induced expansion (P = 0.008) of proliferative zone and also enhanced apoptosis more effectively than other experimental diets indicating that natural grapefruit and limonin may serve as better chemopreventive agents compared to IGFPP and naringin. The present study indicates that postharvest quarantine doses of irradiation slightly alter composition of bioactive compounds and in turn marginally reduce the chemopreventive ability of grapefruit against the promotion stage of colon cancer. These results warrant the necessity of testing the impact of post harvest treatments on fruits and vegetables chemopreventive ability.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2009. / Härtill 5 uppsatser.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Flavonoids are a long and well known class of natural products. Their potential health benefits can be attributed to their antioxidant activity, and modulation of cell signaling pathways. Green tea one of the most widely consumed beverages, consist of flavonoids such as catechins and tannins. Epigallocatechin gallate (EGCG) the major catechin of green tea exhibits multiple health benefits due to its antioxidant nature. The radical scavenging activity of EGCG is attributed to its structure. Therefore, a study on molecular features of EGCG would provide valuable information on structural modifications, which may change the physiochemical properties such as bioavailability and solubility. Although flavonoids are abundant and commercially available they are difficult to purify and crystallize. In this respect, crystallizing EGCG was challenging. By exploring different techniques EGCG was crystallized. Here in this study one new form of EGCG and two solvates, acetonitrile and nitrobenzene, have been synthesized and structurally characterized by differential scanning calorimetry (DSC), infrared (IR) and powder X-ray diffraction (PXRD). The crystal structures were solved by single-crystal X-ray diffraction and a detailed description of synthesis and about the supramolecular synthons that exist in these crystal forms are given.

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