UNIVERSITÀ DEGLI STUDI DI NAPOLI “FEDERICO II” DOTTORATO IN SCIENZE VETERINARIE XXIX CICLO PhD Thesis “Isolation and characterization of plant bioactive compounds and assessment of their use in veterinary science” Candidato Tutor Co-Tutor Massimiliano D’Ambola Chiar.ma Prof.ssa Chiar.ma Prof.ssa Lorella Severino Nunziatina De Tommasi DOTTORATO IN SCIENZE VETERINARIE - Segreteria Dott.ssa Maria Teresa Cagiano Coordinamento - Prof. Giuseppe Cringoli Ogni cosa che puoi immaginare, la natura l’ha già creata Albert Einstein Table of contents List of abbreviations 10 Figures index 12 Tables index 14 Abstract 16 Introduction 21 I.I Ethnoveterinary-medicine and animal self-medication 22 I.II Natural compounds in veterinary medicine 24 I.II.I Medicinal Plants Used in Veterinary Practice as Antibacterial 25 I.III Plant molecules for drug discovery 28 AIM OF THE PROJECT 30 References 31 Chapter 1 Psiadia punctulata (DC.) Vatke 35 1.1 Asteraceae family 37 1.2 Psiadia genus 37 1.2.1 Flavonoids 37 1.2.2 Diterpenoids 38 1.3 Psiadia punctulata (DC.) Vatke 39 1.3.1 Previous phytochemical studies on Psiadia punctulata (DC) Vatke 39 1.3.2 P. punctulata biological activities 42 1.3.3 Antimicrobial activity 43 AIM 44 1.4 Experimental 44 1.4.1 Plant material 44 1.4.2 Extraction and isolation 44 1.4.3 In vitro antimicrobial activity 47 1.5 Results and Discussions 49 References 74 Chapter 2 Hypoestes forskaolii (Vahl) Roem. &Schult 79 2.1 Acanthaceae family 81 2.2 Hypoestes genus 81 2.3 Previous phytochemical studies of Hypoestes genus 81 7 Table of contents 2.4 Hypoestes forskaolii (Vahl) Roem. & Schult 83 2.4.1 Previous phytochemical studies on Hypoestes forsskaolii 84 2.4.2 Biological activities 84 2.4.3 Anthelmintic activity of plant extracts. 85 AIM 86 2.5 Experimental 86 2.5.1 Plant material 86 2.5.2 Extraction and isolation 86 2.5.3 Reagents and antibodies 89 2.5.4 Cells and treatment 89 2.5.5 Cell viability and cell cycle 90 2.5.6 Statistical analysis 90 2.5.7 Recovery GIN eggs 90 2.5.8 Faecal cultures 91 2.5.9 Egg hatch assay 91 2.6 Results 92 2.6.1 EHA test 92 2.6.2 Chemical study 93 2.7 Discussion 102 References 105 Chapter 3 Trichilia maynasiana C.DC 111 3.1 Meliaceae family 113 3.2 Trichilia Genus 113 3.2.1 Previous phytochemical studies on Trichilia genus 114 3.3 Trichilia maynasia C. DC. 118 AIM 118 3.4 Experimental 119 3.4.1 Plant material 119 3.4.2 Extraction and isolation 119 3.4.3 Analysis of sulfate group 121 3.4.3.1 Compound 1 122 3.4.3.2 Compound 2 122 3.4.4. Cells and treatment 122 3.4.5. Cell viability and cell cycle 122 3.5 Result and discussion 123 References 128 8 Table of contents Chapter 4 Vernonia nigritiana Oliv. & Hiern 133 4.1 Vernonia genus 135 4.1.1 Vernonia genus: phytochemistry bioactive compounds 135 4.1.2 Vernonia nigritiana Oliv. & Hiern 137 AIM 138 4.2 Experimental 138 4.2.1 Plant materials 138 4.2.2 Extraction and isolation 139 4.3 Results and discussion 142 References 147 Chapter 5 Materials and Methods 151 5.1 Chromatographic techniques 153 5.1.1 Thin Layer Chromatography 153 5.1.2 Chromatographic columns 153 5.2 Chemical-physical techniques 154 5.2.1 Optical rotations 154 5.3 Spectroscopic and spectrometric methods 154 5.2.2 Nuclear Magnetic Resonance 154 5.2.3 Mass spectrometry (ESI-MS) 156 5.3 Solvents 156 9 List of abbreviations List of abbreviations µM micromolar HMBC heteronuclear multiple 1D-2D mono e bidimensional bond correlation ACN acetonitril HPLC high pressure liquid anti-Cdc2 (mouse monoclonal, chromatography sc-8395) HRESIMS high resolution anti-phospho (Thr161)-Cdc2 electrosray mass spectrometry p34 (rabbit polyclonal, sc- HSQC heteronuclear single 101654) quantum correlation AR anthelmintic resistance Hz hertz ATCC American Type Cell IC50 inhibitory concentration Culture half maxima C13-NMR carbon nuclear IPP isopentenyl pyrophpsphate magnetic resonance J NMR coupling costant CA Candida albigans Jurkat T-cell leukemia CD3OD deuterate methanol LC-MS liquid chromatography CH2N2 diazomethane coupled with mass CH3COONa sodium acetate spectrometry CHCl3 chloroform m/z mass/ charge COSY correlation spectroscopy MALDI matrix assisted laser DMEM HeLa desorbtion ionization DMSO dimethyl sulfoxide MeOH methanol DQF double-quantum filtered mg milligram EHA egg hatch assay Mhz mega Hertz ESI-MS electrospray mass MS mass spectrometry specrtometry MSn massspectroscopy tandem FBS fetal bovine serum MTT [3-(4,5-dimethylthiazol- GIBCO Life Technologies, 2-yl)-2,5-diphenyltetrazolium Grand Island, NY, USA bromide] GIN gastrointestinal nematodes n-BuOH normal-buthanol GPP geranil pirophosphate n-HEXANE normal hexane H1-NMR proton nuclear NHR nuclear hormone receptor magnetic resonance NMR nuclear magnetic spectroscopy resonance HCl chloridic acid PI propidium iodide HeLa cervical carcinoma ppm parts per million 10 List of abbreviations Q-TOF quadrupole- time of TLC thin layar flight chromatography ROESY rotating frame TOCSY total correlation Overhause effect spectroscopy spectroscopy RP reverse phase TOF time of flight RPMI 1640 Jurkat U937 tR retention time RU response unit ug microgramm STA Staphylococcus aureus UPLC Ultra Performance TBZ Thiabendazole Liquid Chromatography TFA Trifluoroacetic acid WAAVP Advancement of Veterinary Parasitology 11 Figures index Figures index Introduction 1 Organs of the plant most used in veterinary practices p.24 2 All new approved drugs 1981−2014; n = 1562. (adopted by Newnan et al 2016) p.28 3 The current lead discovery process p.29 Chapter 1 1.1 Flavonoids in Psiadia genus p.38 1.2 Ditrpenoids from Psiadia genus p.39 1.3 Flavonoids from P.punctulata p.41 1.4 Kaurane and trachylobane diterpenes from P. punctulata exudate p.42 1.5 Separation scheme exudate of P. punctulata exudate p.45 1.6 Antibacterial activity of fractions C (Frs C) and H (Frs H) against S. aureus p.51 1.7 NMR spectra of compound 14 p.53 1.8 NMR spectra of compound 28 p.57 1.9 NMR spectra of compound 18 p.59 1.10 NMR spectra of compound 10 p.60 1.11 NMR spectra of compound 19 p.61 1.12 NMR spectra of compound 20 p.62 1.13 NMR spectra of compound 24 p.64 1.14 Antibacterial activity of pure compound 2 (left) and 27 (right) against S. aureus p.65 1.15 Ability of compounds 2, 3, 8 and 27 to inhibit the biofilm formation of C. albicans and S. aureus p.66 1.16 Know compound of P.punctulata p.73 Chapter 2 2.1 Diterpenes of Hypoestes spp p.83 2.2 Diterpenes of H. forskaolii leaves p.84 2.3 Extracts of H. forskaolii leaves p.87 2.4 Separation scheme of H. forskaolii n-hexane extract p.88 2.5 NMR spectra compound 1 p.95 2.6 NMR spectra compound 2 p.96 2.7 NMR spectra compound 10 p.98 2.8 NMR spectra compound 13 p.99 2.9 Known compound of H. forskaolii p.102 12 Figures index Chapter 3 3.1 Monoterpenes Trichilia genus p.114 3.2 Sesquiterpenes of Trichilia genus p.115 3.3 Diterpenes of Trichilia genus p.115 3.4 Triterpenes of Trichilia genus p.117 3.5 Steroids of Trichilia genus p.117 3.6 Limonoids Trichilia genus p.118 3.7 Extracts from T. maynasiana leaves p.119 3.8 Separation scheme n-hexane extract p.120 3.9 Separation scheme chloroformic extract p.121 3.10 Known compound of T.maynasiana p.127 Chapter 4 4.1 Main phenols from Vernonia ssp p.136 4.2 Terpenoids Vernonia genus p.137 4.3 Structures of compounds 1–15 isolated from Vernonia nigritiana p.138 4.4 Percentages and grams of extract obtained from V. nigritiana leaves p.139 4.5 Scheme of silica gel column of chloroformic extract p.140 4.6 Scheme of silica gel column of chloroform:methanol 9:1 extract p.141 4.7 Compounds of V. nigritiana p.146 13 Tables index Tables index Chapter 1 1.1 Fraction (A-I) antimicrobial activity p.50 1.2 MIC values of the compounds 8, 3, 27, 2 p.64 1 13 a 1.3 H and C NMR data of compounds 14–30 (CD3OD, 600 MHz, J in Hz) p.67 1 13 1.4 H and C NMR data of compounds 21–23 (CD3OD, 600 MHz, J in Hz)a p.68 1 13 1.5 H and C NMR data of compounds 25–27–28 (CD3OD, 600 MHz, J in Hz)a p.69 1 13 a 1.6 H and C NMR data of compounds 10-18 (CD3OD, 600 MHz, J in Hz) p.70 1 13 1.7 H and C NMR data of compounds 19–20 (CD3OD, 600 MHz, J in Hz)a p.71 1 13 1.8 H and C NMR data of compounds 22–24 (CD3OD, 600 MHz, J in Hz)a p.72 Chapter 2 2.1 Percentages of GIN eggs and egg hatch after treatment at various concentrations with thiabendazole, leaves n-hexane extract of H. forskaolii, and distillated water p.93 1 13 a 2.2: H and C NMR data of compounds 1–2 (CD3OD, 600 MHz, J in Hz) p.100 1 13 a 2.3 H and C NMR data of compounds 10–13(CD3OD, 600 MHz, J in Hz) p.101 Chapter 3 1 13 3.1 H and C NMR data of compounds 1 and 2 (CD3OD, 600 MHz, J in Hz)a p.126 Chapter 4 1 13 a 4.1 H and C NMR data of compounds 1–2 (CD3OD, 600 MHz, J in Hz) p.145 14 Acknowledgments Firstly, I would like to express my sincere gratitude to my first supervisor Prof.ssa Severino and my second supervisor Prof.ssa De Tommasi for the continuous support of my Ph.D study and related research, for their patience, motivation and immense knowledge.