UNIVERSITÀ DEGLI STUDI DI SALERNO Dipartimento di Farmacia Dottorato di ricerca in Scienze Farmaceutiche Ciclo XIII NS – 2012-2015 Coordinatore: Chiar.mo Prof. Gianluca Sbardella Characterization and biological properties of Citrus industrial derivatives and waste products for the formulation of nutraceuticals settore scientifico disciplinare di afferenza: CHIM/08 Dottorando Tutore Dott. Francesco Pagano Chiar.mo Prof. Pietro Campiglia Co-tutore Chiar.mo Prof. Gianluca Sbardella INDEX CHAPTER I: Phytochemicals and health: the effect of polyphenols in the inflammation process .......................................................................................................... - 1 – 1. Introduction ............................................................................................. - 2 - 2. Nutraceutical: .......................................................................................... - 2 - 2.1 Definition ................................................................................................ - 2 - 2.2 Nutraceuticals marketing ........................................................................ - 3 - 3. Polyphenols .............................................................................................. - 5 - 4. High Performance Liquid Chromatography (HPLC): ....................... - 8 - 4.1 Definition ................................................................................................ - 8 - 4.2 Fundamental principles .......................................................................... - 9 - 4.3 Chromatographic parameters............................................................... - 12 - 4.3.1 Retention ............................................................................................ - 12 - 4.3.2 Separation .......................................................................................... - 14 - 4.3.3 Column efficiency .............................................................................. - 14 - 5. Optimization of an HPLC separation ................................................. - 18 - 5.1 Stationary phase particle size ............................................................... - 18 - 5.2 Columns with reduced dimensions ....................................................... - 19 - 6. Mass spectrometric detectors .............................................................. - 21 - 6.1 Definition .............................................................................................. - 21 - 6.2 Atmospheric-pressure chemical ionization ........................................... - 22 - 6.3 Electrospray ionization ......................................................................... - 23 - 7. Inflammation ......................................................................................... - 25 - 7.1 Definition .............................................................................................. - 25 - 7.2 Mediators of the inflammatory process ................................................ - 27 - 7.2.1 Nitric oxide synthase (iNOS): ............................................................ - 28 - 7.2.2 Cyclooxygenase ................................................................................. - 30 - 7.2.3 Reactive Oxygen Species (ROS) ........................................................ - 31 - 8. Aim of research ..................................................................................... - 32 – 9. References .............................................................................................. - 33 - CHAPTER II: Development of fast analytical methods for the characterization of flavonoids in Citrus bergamia juice: An UHPLC-IT-TOF approach . - 38 – Abstract...................................................................................................... - 39 - Keywords .................................................................................................... - 39– 1. Introduction ........................................................................................... - 39 - 2. Materials and methods ......................................................................... - 42 - 2.1 Reagents and standards ........................................................................ - 42 - 2.2 Sample preparation............................................................................... - 42 - 2.3 Instrumentation ..................................................................................... - 42 - 2.4 UHPLC conditions ................................................................................ - 43 - 2.5 ESI-IT-TOF-MS parameters ................................................................. - 43 - 2.6 Scavenging of 1, 1 diphenyl-2-pycrylhydrazyl (DPPH) radicals ......... - 44 - 3. Results and discussion .......................................................................... - 45 - 3.1 Optimization of chromatographic parameters ..................................... - 45 - 3.2 Quantitative analysis of lyophilized juices ........................................... - 49 - 3.3 ESI-IT-TOF-MS elucidation of flavonoid profiles ................................ - 50 - 3.4 Antioxidant activity ............................................................................... - 53 - 4. Conclusions ............................................................................................ - 56 - 5. References .............................................................................................. - 56 - CHAPTER III: UHPLC profiling and effects on LPS-stimulated J774A.1 macrophages of flavonoids from Citrus bergamia juice industry, an underestimated waste product with high anti-inflammatory potential ..................................... - 62 - Abstract...................................................................................................... - 63 - Keywords ................................................................................................... - 63 - 1. Introduction ........................................................................................... - 63 - 2. Materials and methods ......................................................................... - 65 - 2.1 Reagents and standards ........................................................................ - 65 - 2.2 Sample preparation............................................................................... - 65 - 2.3 Instrumentation and UHPLC–MS/MS conditions ................................ - 65 - 2.4 Cell culture ........................................................................................... - 66 - 2.5 Antiproliferative activity ....................................................................... - 66 - 2.6 Measurement of intracellular reactive oxygen species (ROS).............. - 67 - 2.7 Nitrite determination and Western blot analysis for iNOS, COX-2 and HO-1 expression ................................................................................................ - 67 - 2.8 Data analysis ........................................................................................ - 68 - 3. Results and discussion .......................................................................... - 68 - 3.1 UHPLC–MS/MS quantitative and qualitative analysis of juice ........... - 68 - 3.2 Effect of bergamot juice on LPS-stimulated macrophages ................... - 73 - 4. Conclusions ............................................................................................ - 79 - 5. References .............................................................................................. - 79 - CHAPTER IV: Flavonoids from Citrus sinensis extract: anti-inflammatory and hypoglycemic evaluation ......................................................................... - 84 - Abstract...................................................................................................... - 85 - Keywords ................................................................................................... - 85 - 1. Introduction ........................................................................................... - 85 - 2. Materials and methods ......................................................................... - 87 - 2.1 Reagents and standards ........................................................................ - 87 - 2.2 Sample preparation............................................................................... - 88 - 2.3 Instrumentation ..................................................................................... - 88 - 2.4 UHPLC-PDA Conditions ...................................................................... - 89 - 2.5 UHPLC-ESI-IT-TOF Conditions .......................................................... - 89 - 2.6 Cells and Reagents for cell culture ....................................................... - 90 - 2.7 Antiproliferative assay .......................................................................... - 90 - 2.8 Nitrite Determination and Western Blot Analysis for iNOS, COX-2 and HO-1 Expression......................................................................................... - 91 - 2.9 TNF-α Determination ........................................................................... - 92 - 2.10 Immunofluorescence Analysis with Confocal Microscopy ................. - 93 - 2.11 Measurement of Intracellular ROS ..................................................... - 93 - 2.12 Glucose uptake ...................................................................................