List of Figures 21 List of Figures Figure 2.1. Worldwide Prevalence

List of Figures List of Figures Figure 2.1. Worldwide prevalence of diabetes in 2000 and estimates for the year 2030 (in millions). 35 Figure 2.2 . Model of the progressive pathogenesis of type 2 diabetes. 38 Figure 2.3. Glucose stimulated insulin secretion. 39 Figur e 2.4. Insulin signal transduction. 40 Figure 2.5. Examples of different drugs used for type 2 diabetes treatment. 46 Figure 2.6. Action sites of main drugs currently used for diabetes treatment. 47 Figure 2.7. Main pathways leading to secondary metabolites 52 Figure 2.8. Flavonoid basic structure 53 Figure 2.9. Structures of the major groups of flavonoids 54 Figure 2.10. The phenylpropanoid biosynthetic pathway 55 Figure 2.11. Mechanism proposed for chalcone formation through enzymes CoA ligase and Chalcone synthase. 56 Figure 2.12. Scavenging of ROS by flavonoids. 58 Figure 2.13. Example of metal ion/flavonoid complexation. 58 Figure 2.14. Scheme representing the pathway of carbohydrate metabolism and targets where polyphenols have been shown to demonstrate multiple activities. 60 Figure 2.15 . General aspect of Coreopsis tinctoria and a detail of the flowering tops. 85 Figure 2.16. Floral phytochemicals from the genus Coreopsis . Butein, okanin, lanceoletin chalcone-aurone derivatives. 87 Figure 2.17. C. tinctoria floral phytochemicals grouped by type of flavonoid structure. 91 Figure 3.1 . Detail of 1H-1H correlations displayed in the COSY spectrum of compound CT-1. 97 2 3 Figure 3.2 . JC-H and JC-H correlations displayed in the HMBC spectrum of compound CT-1. 98 Figure 3.3. 1H NMR spectrum with additional amplified aromatic region, of compound CT-2. 99 2 3 Figure 3.4 . JC-H and JC-H correlations displayed in the HMBC spectrum of compound CT-2. 100 21 List of Figures Figure 3.5 . Chemical structure of the three compounds isolated; flavanokanin ( 88 ), okanin ( 79 ) and marein ( 78 ). 103 Figure 3.6 . HPLC chromatographic profile (280 nm) and total ion chromatogram of C. tinctoria flowering tops aqueous extract. 105 Figure 3.7 . HPLC chromatographic profile (280nm) and total ion chromatogram of C. tinctoria flowering tops AcOEt fraction. 106 Figure 3.8 . Summary of the chemical structures of compounds identified in C. tinctoria’s flowering tops aqueous extract and AcOEt fraction. 108 Figure 3.9. Detail of peaks P10 and P11 present in the one-week old aqueous extract chromatogram at two different wavelengths 420nm and 380 nm. 109 Figure 3.10 . Mechanism of chemical or enzymatic chalcone oxidation to the correspondent aurone. 110 Figure 3.11 . Scheme representing natural isomer 2 S-flavanomarein formation, and isomer 2R. 111 Figure 3.12 . Proposed fragmentation for chalcone 78 (P11 ) and flavanone 85 (P4) pair in negative ion mode . 112 Figure 3.13 . Fragments obtained from marein ( 78 ) in negative ion mode using different collision energies. 114 Figure 3.14. A-Chromatogram of the standard, marein (50 µg/mL), Rt 6.44 min, at 380 nm. B- Chromatogram of aqueous extract (Sample A) of C. tinctoria (1 mg/mL), Rt (main peak) 6.44 min, at 380 nm. 117 Figure 3.1 5. Correlation between peak area and marein concentration in the linearity range 10 µg/mL – 100 µg/mL. 117 Figure 4.1. Effect of phlorizin on blood glucose levels of normal Wistar rats submitted to an oral glucose tolerance test, compared to control. 130 Figure 4.2. C. tinctoria aqueous extract effect on blood glucose levels of normal Wistar rats submitted to an oral glucose tolerance test. A- 25mg/Kg; B- 50 mg/Kg; C- 100 mg/Kg and D- 300mg/Kg. 131 Figure 4.3. Glucose-tolerant and glucose-intolerant Wistar rat behaviour on OGTT throughout the 21 day subchronical study. 132 Figure 4.4. Weight variations of glucose-tolerant and glucose-intolerant controls during 21-day subchronical study. 133 22 List of Figures Figure 4.5. Blood glucose levels in response to the oral glucose challenge in each group at the beginning, week 1, 2 and 3 of the treatment. 134 Figure 4.6 . Hepatotoxicity biomarkers levels, alanine and aspartate transaminases (ALT and AST) comparing with glucose-intolerant control group. 134 Figure 4.7 . Area under the glycemic curve obtained through OGTT at the end of the 1st , 2 nd and 3 rd weeks of treatment. 136 Figure 4.8. Biochemical analysis of hepatotoxicity parameters at the end of the treatment with C. tinctoria AcOEt fraction (125 mg/Kg): Aspartate transaminases and Alanine transaminases (AST and ALT). 136 Figure 4.9. Plasma lipase values at the end of the three-week experimental period. Glucose-intolerant animals treated with C. tinctoria. 137 Figure 5.1. Effect of C. tinctoria (0.1; 0.3 and 1 mg/mL) aqueous extract ( A) and AcOEt fraction ( B) on insulin secretion from MIN6 cells in static incubations at 2 and 20 mM glucose. 146 Figure 5.2. Time course of the effect of C. tinctoria (1mg/mL) aqueous extract and AcOEt fraction on insulin secretion from perifused MIN6 pseudoislets at 2mM and 20mM glucose. 147 Figure 5.3. MIN6 cell viability when pretreated for 24h with Coreopsis tinctoria extracts; (aqueous extract, AcOEt fraction) and pure compounds (marein ( 78 ) and flavanomarein ( 85 )). 150 Figure 5.4. MIN6 cell viability when pretreated for 24h with Coreopsis tinctoria extracts: aqueous extract (Aq. Ext.) and AcOEt fraction (AcOEt Fr.) and challenged with oxidant tBHP (2h). 152 Figure 5.5. MIN6 cell viability when pretreated for 24h with marein and flavanomarein, and challenged with oxidant tBHP (2h). 153 Figure 5.6. Superoxide anion (O 2-) measurements in (A) MIN6 cells challenged with tBHP and tBHP plus SOD. (B) MIN6 cells pretreated with C. tinctoria aqueous extract and AcOEt fraction and challenged with tBHP. 154 Figure 5.7. C. tinctoria extracts pretreatment (24h) effect on untreated and on cytokine-induced MIN6 cells apoptosis. 155 Figure 5.8. C. tinctoria pure compounds, Marein and Flavanomarein pretreatment (24h) effect on untreated and on cytokine-induced MIN6 cells apoptosis. 156 23 List of Figures Figure 8 .1. Scheme representing the extraction procedure for compound isolation from Coeropsis tinctoria (CTD1 ). 182 Figure 8 .2. Scheme representing the extraction procedure for Coeropsis tinctoria extracts used in biological assays, HPLC-DAD-MS/MS characterization and quantification. 183 Figure A1.1. Percentage of DPPH reduction using C. tinctoria samples and appropriate controls, after 30 minutes of exposure. 232 24 .

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