Synthesis and biological evaluation of olive oil polyphenol metabolites

Universidade do Minho Escola de Ciências Catarina Guimarães Ribeiro Synthesis and biological evaluation of olive oil polyphenol metabolites outubro de 2019 Synthesis and biological evaluation of olive oil polyphenol metabolites Catarina Ribeiro UMinho | 2019 Universidade do Minho Escola de Ciências Catarina Guimarães Ribeiro Synthesis and biological evaluation of olive oil polyphenol metabolites Dissertação de Mestrado em Química Medicinal Trabalho efetuado sob a orientação do Professor Doutor Luís Miguel Oliveira Sieuve Monteiro e da Professora Doutora Maria de Fátima Azevedo Brandão Amaral Paiva Martins outubro de 2019 Despacho RT - 31 /2019 - Anexo 3 Declaração a incluir na Tese de Doutoramento (ou equivalente) ou no trabalho de Mestrado DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ V Synthesis and biological evaluation of olive oil polyphenol metabolites Agradecimentos Queria começar por agradecer ao orientador deste projeto, Professor Doutor Luís Monteiro pela oportunidade que me deu de trabalhar consigo pela segunda vez e por toda ajuda e apoio durante o ano. À Professora Doutora Fátima Paiva-Martins, coorientadora deste projeto, queria agradecer por todo o apoio e disponibilidade total demonstrados ao longo deste último ano. Por toda a dedicação e paciência que apresentou para que o trabalho corresse da melhor forma e fossem obtidos os melhores resultados possíveis, um enorme obrigado. Aos meus colegas de laboratório agradeço pelo apoio e pela força que me deram especialmente nos momentos mais difíceis. Agradeço à Doutora Marlene Costa pela ajuda que me forneceu, pelo apoio dado principalmente nos piores momentos, e por ter um auxílio permanente durante todo o ano. Agradeço também ao meu colega de bancada, Alfredo Esteves pelo companheirismo e por me fazer rir mesmo quando estava chateada, e por ter feito companhia nas horas mais tardias. Ao doutor Ivo Dias agradeço pela paciência e pela ajuda que me deu quando mais precisei durante estes últimos meses. À mestre Sara Fernandes, obrigada pela dedicação para me fornecer resultados a tempo, e pelo stress passado. Aos meus amigos, pela paciência compreensão que tiveram durante este ano. Um especial agradecimento à Ana Cláudia por me ter feito pensar em algo mais além da tese. Também lhe agradeço por me ter ajudado a espairecer. À Joana Ferreira por ter lido e relido a minha tese e por me ter ajudado na escrita. Pelas horas ao telefone, e por continuar a manter a esperança de que tudo é possível. À Ana Filipa agradeço pela paciência que teve por não estar sempre presente nos eventos e por ter tido sempre uma palavra amiga a me dar quando estava mais stressada. Aos meus colegas de mestrado, agradeço por terem sido tão acolhedores durante o ano que estive em Braga, e por me terem feito sentir em casa. À minha família, em especial à minha mãe que sempre me motivou e sempre acreditou que eu ia conseguir o mundo. Por ter me feito companhia nos dias de escrita e me ter apoiado todos os dias. Ao meu querido avô que sempre teve um orgulho enorme por tudo o que nós fazemos ao longo da nossa vida. À minha irmã que mais do que ninguém, teve sempre fé no que VI Synthesis and biological evaluation of olive oil polyphenol metabolites eu fiz e sempre acreditou que este dia chegaria. À minha avó que foi a luz que guiou todo o meu percurso e que era a pessoa que mais queria ver este dia chegar. Obrigada por seres a minha estrelinha. Agradeço a todos os voluntários a colaboração e a disponibilidade para colher um pouco de sangue, pois só assim foi possível realizar o estudo da hemólise. Agradeço à Universidade do Minho e à Faculdade de Ciências da Universidade do Porto, por permitirem que tudo isto se tenha concretizado. O trabalho foi apoiado pela UID / QUI / 50006/2019 com financiamento da FCT / MCTES através de fundos nacionais e pelo PTDC / OCE-ETA / 32492/2017 - POCI-01-0145-FEDER032492 – PHENOLIVA. STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. XIV Synthesis and biological evaluation of olive oil polyphenol metabolites 2.1. Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate (Hydroxytyrosol acetate) 28 2.1.1. Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate using acetyl chloride as acylating agent ........................................................................................................... 29 2.1.2. Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate by transesterification with ethyl acetate catalysed by amberlite IR – 120 ...................................................... 30 2.2. Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate (Tyrosol acetate) ................. 31 2.2.1. Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate using acetyl chloride as acylating agent ........................................................................................................... 31 2.2.2. Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate by transesterification with ethyl acetate catalysed by amberlite IR – 120 ............................................................. 32 2.3. Synthesis of 2-(4-hydroxy-3-methoxyphenyl)ethyl ethanoate (homovanillyl acetate) by transesterification with ethyl acetate catalysed by amberlite IR – 120 ............. 33 2.4. Isolation of secoiridoids, 3,4-DHPEA-EDA and 3,4-DHPEA-EA ........................ 34 2.5. Enzymatic synthesis of 4-HPEA-EDA ............................................................. 34 3. Synthesis of the metabolites of phenolic compounds of olive oil ........................... 35 3.1. Sulfation Reactions ...................................................................................... 35 3.1.1. Synthesis of the sodium salts of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and of 5-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate ....................................... 35 3.1.2. Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-phenyl sulfate ..... 36 3.1.3. Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate ....................................................................................................................... 38 3.1.4. Synthesis of the diethylammonium salt of 4-(2-ethanoyloxyethyl)-2hydroxyphenyl sulfate ................................................................................................. 39 3.1.5. Synthesis of the sodium salts of (Z)-5-(2-((4-formyl-3-(2-oxoethyl)hex-4enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate and of (Z)-4-(2-((4-formyl-3-(2-oxoethyl)hex-4enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate ...................................................................... 40 3.2. Solvolysis Reactions ..................................................................................... 41 XV Synthesis and biological evaluation of olive oil polyphenol metabolites 3.2.1. Synthesis of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and of 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate ........................................................ 41 3.2.2. Synthesis of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate ............ 42 3.2.3. Synthesis of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate ................................................................................................................................. 43 4. Haemolysis Assay ............................................................................................... 44 4.1. Preparation of RBC suspension .................................................................... 44 4.2. AAPH-induced haemolysis ........................................................................... 44 IV. Results and Discussion ...................................................................................................... 47 1. Olive oil Phenolic Compounds ............................................................................. 49 1.1. Acetylation of Phenolic Compounds ............................................................. 49 1.1.1. Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate ............................. 50 1.1.2. Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate ................................... 52 1.1.3. Synthesis of 2-(4-hydroxy-3-methoxyphenyl)ethyl ethanoate ................... 55 1.2. Isolation of secoiridoids from olive tree leaves .............................................. 55 1.2.1. Isolation of 3,4-DHPEA-EDA and 3,4-DHPEA-EA .................................... 55 1.3. Tentative of synthesis of 4-HPEA-EDA ........................................................... 61 2. Synthesis of sulfate metabolites .......................................................................... 62 2.1. Synthesis of the sodium salts of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and of 5-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate ....................................................... 64 2.2. Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)phenyl sulfate .............. 66 2.3. Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate ...................................................................................................................................... 67 2.4. Tentative synthesis of 3-hydroxy-4-(sulfoxy)phenylethyl (Z)-4-formyl-3-(2oxoethyl)hex-4-enoate and 4-hydroxy-3-(sulfoxy)phenylethyl (Z)-4-formyl-3-(2-oxoethyl)hex-4enoate ............................................................................................................................ 68 XVI Synthesis and biological evaluation of olive oil polyphenol metabolites 2.5. Synthesis of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate ....................................................................... 69 2.6. Synthesis of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate .................... 70 2.7. Synthesis of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate ... 71 2.8. Tentative synthesis of diethylammonium 4-(2-ethanoyloxyethyl)-3-hydroxyphenyl sulfate ............................................................................................................................ 72 3. Preliminar in vitro bioactivity evaluation ............................................................... 74 V. Conclusion ......................................................................................................................... 79 VI. References ........................................................................................................................ 84 VII. Annexes............................................................................................................................ 92 XVII Synthesis and biological evaluation of olive oil polyphenol metabolites List of Tables Table 1: Eluent gradient used in ultra-high performance liquid chromatography.................................................... 28 Table 2: Assay conditions in the study of aaph-induced haemolysis in the presence of phenolic compounds and its metabolites. ................................................................................................................................................ 45 Table 3: NMR chemical shifts in deuterated chloroform for secoiridoid 3,4-dhpea-eda. ......................................... 58 Table 4: Chemical shifts in deuterated chloroform for secoiridoid 3,4-dhpea-eda. ................................................. 60 Table 5: NMR chemical shifts in deuterated methanol of the sodium salt of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and sodium 5-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate. ............................................................... 65 Table 6: NMR chemical shifts in deuterated methanol of the sodium salt of 4-(2-ethanoyloxyethyl)-phenyl sulfate. . 67 Table 7: NMR chemical shifts in deuterated methanol of the sodium salt of 4-(2-ethanoyloethyl)-2-methoxyphenyl sulfate. ........................................................................................................................................................ 68 Table 8: NMR chemical shifts in deuterated water of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate. ............................................................................................ 69 Table 9: NMR chemical shifts in deuterated water of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate. .............. 71 Table 10: NMR chemical shifts in deuterated water of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate. ........................................................................................................................................................ 72 Table 11: NMR chemical shifts in deuterated methanol of hydroxytyrosol. ............................................................ 73 XIX Synthesis and biological evaluation of olive oil polyphenol metabolites List of Figures Figure 1: Olive tree ( Olea Europaea L.). ................................................................................................................. 3 Figure 2: Olives, fruit from olive trees. ................................................................................................................... 4 Figure 3: Lipid autoxidation pathways (adapted from Frankel 18). ............................................................................ 7 Figure 4: Mechanisms of photosensitized oxidation (adapted from Frankel 12). ........................................................ 8 Figure 5: α-, βand γ-Tocopherol. ...................................................................................................................... 10 Figure 6: Structures of phenolic alcohols present in olive oil. ............................................................................... 10 Figure 7: Structure of the major secoiridoids found in olive fruit. .......................................................................... 11 Figure 8: Structure of secoiridoids of VOO. .......................................................................................................... 11 Figure 9: Structure of oleuropein and ligstroside aglycones and their derivatives, the major secoiridoids found in olive oil (adapted from Paiva-Martins & Kiritsakis 1). ...................................................................................... 12 Figure 10: Metabolic pathways of HT found in olive oil (adapted from Gomes et al. 47). ......................................... 16 Figure 11: Human red blood cell. ....................................................................................................................... 17 Figure 12: Catalytic reactions in the formation of paps (adapted from Srott 62). .................................................... 19 Figure 13: Reaction scheme for sulfation of hydroxyl groups of phenolic compounds by the sulphur trioxide complex method (adapted by Yan et al. 70). ................................................................................................... 20 Figure 14: Proposal synyhesis of phenolic olive oil metabolites. ........................................................................... 23 Figure 15: Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate using acetyl chloride and dimethyl carbonate. ..... 29 Figure 16: Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate using amberlite ir-120 in ethyl acetate. ................ 30 Figure 17: Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate using acetyl chloride in dry dichloromethane. ............ 31 Figure 18: Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate by transesterification with ethyl acetate catalysed by amberlite IR–120. ....................................................................................................................................... 32 Figure 19: Synthesis of 2-(4-hydroxy-3-methoxyphenyl)ethyl ethanoate by transesterification with ethyl acetate catalysed by amberlite IR–120. .................................................................................................................... 33 Figure 20: Synthesis of 4-HPEA-EDA from 3,4-DHPEA-EDA and tyrosol in THF..................................................... 34 Figure 21: Synthesis of the sodium salts of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and of 5-(2ethanoyloxyethyl)-2-hydroxyphenyl sulfate. .................................................................................................... 36 Figure 22: Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-phenyl sulfate. .................................................. 37 Figure 23: Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate. .................................. 38 Figure 24: Synthesis of diethylammonium 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate. ................................... 39 Figure 25: Synthesis of the sodium salts of (Z)-5-(2-((4-formyl-3-(2-oxoethyl)hex-4-enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate and of ( Z )-4-(2-((4-formyl-3-(2-oxoethyl)hex-4-enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate. ......................... 40 Figure 26: Synthesis of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and of 2-hydroxy-5-(2hydroxyethyl)phenyl sulfate. ......................................................................................................................... 41 Figure 27: Synthesis of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate. ......................................................... 42 Figure 28: Synthesis of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate. ........................................ 43 XX Synthesis and biological evaluation of olive oil polyphenol metabolites Figure 29: Proposed sequence for acetylation of tyrosol using acetyl chloride. ..................................................... 49 Figure 30: Proposed sequence for transesterification with ethyl acetate using amberlite IR-120 as catalyst. ......... 49 Figure 31: NMR in deuterated methanol of the mixture of 2-(4-acetyloxi-3-hydroxyphenyl)ethyl ethanoate and 2-(3,4dihydroxyphenyl)ethyl ethanoate. ................................................................................................................. 50 Figure 32: NMR in deuterated methanol of 2-(3,4-dihydroxyphenyl)ethyl ethanoate. ............................................. 51 Figure 33: NMR in deuterated methanol of the mixture of 2-(4-hydroxyphenyl)ethyl ethanoate and 2-(4acetyloxyphenyl)ethanoate. .......................................................................................................................... 53 Figure 34: Result obtained in liquid-liquid extraction of the reaction mixture using NaOH 1M and HCl 0.74M. ...... 54 Figure 35: NMR in deuterated methanol of 2-(4-hydroxyphenyl)ethyl ethanoate.................................................... 54 Figure 36: Chromatogram of mixture before purification. .................................................................................... 56 Figure 37: Chromatogram of standards (A=3,4-DHPEA-EDA; B=oleuropein; C=3,4-DHPEA-EA). ........................... 56 Figure 38: Chromatogram of sample with 3,4-DHPEA-EDA. ................................................................................. 57 Figure 39: Chromatogram of a sample containing 3,4-DHPEA-EDA (A), Oleocanthal (B) and 3,4-DHPEA-EA (C).... 59 Figure 40: Chromatogram of reaction sample (A=hydroxytyrosol; B=tyrosol; C=3,4-DHPEA-EDA; d=Unknown compound). ................................................................................................................................................. 62 Figure 41: Proposed mechanism of sulfation of tyrosol. ...................................................................................... 62 Figure 42: Decomposition of phenylsulfate. ........................................................................................................ 63 Figure 43: Proposed mechanism of solvation of tyrosol. ...................................................................................... 63 Figure 44: NMR of the sodium salt of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and the sodium salt of 5-(2ethanoyloxyethyl)-2-hydroxyphenyl sulfate. .................................................................................................... 66 Figure 45: NMR of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and 2-hydroxy-5-(2hydroxyethyl)phenyl sulfate. ......................................................................................................................... 70 Figure 46: Proposed mechanism for the demethylation of diethylammonium 4-(2-ethanoyloxyethyl)-3methoxyphenyl sulfate with boron tribromide. ............................................................................................... 73 Figure 47: NMR of mixtures of two compounds. Compound that is identified is hydroxytyrosol. ............................ 74 Figure 48: Percentage of inhibition of haemolysis of rbcs at 2 % haematocrit incubated with olive oil phenolic compounds (2.5, 5, 10, 20, 40 and 80 µM) and aaph (60 mM) for 4 hours at 37 ºc. hydroxytyrosol (HT), 2- (3,4-dihydroxyphenyl)ethyl hydrogenosulfate (HT-1-SO4), sodium salt of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and sodium salt of 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate (HT 3+4-SO4), hydroxytyrosol acetate (HTAc), sodium salts of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and 5-(2-ethanoyloxyethyl)-2hydroxyphenyl sulfate (HTAc 3+4-SO4), tyrosol (Ty), sodium salt of 4-(2-hydroxyethyl)phenyl sulfate (Ty 4-SO4), homovanillyc alcohol (HVA), sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate (HVA 4-SO4), homovanillyc acetate (HVAAc), sodium salt of 4-(2-ethanoyloethyl)-2-methoxyphenyl sulfate (HVAAc 4-SO4).2-h ................................................................................................................................................................... 74 XXI Synthesis and biological evaluation of olive oil polyphenol metabolites List of Abbreviations ACT – O -Acetyltransferase AD – Alzheimer’s Disease ADP – Adenosine Diphosphate APPH – 2,2'-Azobis(2-amidinopropane) dihydrochloride APS – Adenosine 5’-phosphosulfate APT – Attached Proton Test ATP – Adenosine triphosphate CHD – Coronary heart disease 13C-NMR – Carbon nuclear magnetic resonance COMT – Catechol methyl transferase COSY – Homonuclear correlation spectroscopy DEA – Diethylamine 3,4-DHPEA-EDA – 2-(3,4-Dihydroxyphenyl)ethyl (4 Z )-4-formyl-3-(2-oxoethyl) hex-4-enoate 3,4-DHPEA-EA – Methyl ( E )-4-(2-(3,4-dihydroxyphenethoxy)-2-oxoethyl)-3-ethylidene-2-hydroxy3,4-dihydro-2 H -pyran-5-carboxylate DMC – Dimethyl carbonate DNA – Deoxyribonucleic acid DOPAC – 3,4-Dihydroxyphenylacetic acid DOPAC-4’ O -sulfate – 3,4-Dihydroxyphenylacetic acid 4’ O -sulfate DOPAC-3’O-sulfate – 3,4-Dihydroxyphenylacetic acid 3’O-sulfate EVOO – Extra virgin olive oil Hb – Haemoglobin HMBC – Heteronuclear multiple bond correlation 1H NMR – Proton nuclear magnetic resonance XXII Synthesis and biological evaluation of olive oil polyphenol metabolites 4-HPEA-EDA – 2-(4-Hydroxyphenethyl)-( Z )-4-formyl-3-(2-oxoethyl)hex-4-enoate HSQC – Heteronuclear single quantum correlation Ht – Haematocrit HVA – 2-(4-Hydroxy-3-methoxyphenyl)ethanol or homovanillyl alcohol HVAAc – 2-(4-Hydroxy-3-methoxyphenyl)ethyl ethanoate or homovanillyl Acetate HVAc – Homovanillyl acid HVA-GlcA – Homovanillyl glucuronide HVA sulfate – Homovanillyl sulfate HVAAc sulfate – Homovanillyl acetate sulfate HVAc sulfate – Homovanillyl acid sulfate HT – 2-(3,4-Dihydroxyphenyl)etanol, 3,4-DHPEA or hydroxytyrosol HTAc – 2-(3,4-diHydroxyphenyl)ethyl ethanoate or hydroxytyrosol acetate HT-3’ OGlcA – Hydroxytyrosol 3’ O -glucuronide HT-4’ O -GlcA – Hydroxytyrosol 4’ O -glucuronide HT-3’ O -sulfate – Hydroxytyrosol 3’ O -sulfate HT-4’ O -sulfate – Hydroxytyrosol 4’ O -sulfate HTAc-3’ O -sulfate – Hydroxytyrosol acetate 3’ O -sulfate HTAc-4’O-sulfate – Hydroxytyrosol acetate 4’O-sulfate LDL – Low-density lipoproteins metHb – Methaemoglobin NMR – Nuclear magnetic resonance PAPS – 3’-Phosphoadenosine 5’-phosphosulfate PBS – Phosphate-buffered saline RBC – Red blood cells ROS – Reactive species of oxygen XXIII Synthesis and biological evaluation of olive oil polyphenol metabolites Sen – Sensitizers 1Sen* -- Excited Singlet State ST – Sulfotransferase TLC – Thin layer chromatography Ty – 2-(4-Hydroxyphenyl)ethanol, 4-HPEA or tyrosol UGT – Glucuronosyltransferase UHPLC – Ultra-high efficiency liquid chromatography UV – Ultra-violet UV-Vis – Visible ultraviolet spectrophotometry VOO – Virgin olive oil 6 Synthesis and biological evaluation of olive oil polyphenol metabolites 2.1. Autoxidation The reaction of oxygen with unsaturated fatty acids constitutes the major means by which fatty substances undergo deterioration. Oxidation is frequently characterized as autoxidation because the rate of oxidation increases as the reaction proceeds.1 Lipid autoxidation occurs via a free radical chain mechanism that proceeds through three distinct stages, namely, initiation, propagation and termination, leading to a series of complex chemical changes (figure 3). Unsaturated lipid molecules (RH), in the presence of initiators such as heat, light/ionizing radiation and metal ions/metalloproteins loose a hydrogen atom and produce free radicals (R•). The lipid radicals, formed at the initiation stage, then react with oxygen to form peroxyl radicals (ROO•). These act as the chain carriers of the rapid processing reaction by attacking a new lipid molecule, to produce the major initial reaction products of oxidation, hydroperoxides (ROOH). In this step, oxidation process is becoming more complicated since the peroxides formed, being unstable, are easily decomposed and form more free radicals that in turn participate in new reactions. This reaction may be repeated for several times during propagation before a hydrogen source is available or the chain is interrupted, for example by antioxidants. Indeed, during propagation, lipid hydroperoxides are produced as primary products of oxidation. They are unstable and break down to form a wide range of secondary oxidation products, including, among others, aldehydes, ketones, alcohols, hydrocarbons, volatile organic acids and epoxy compounds, among others, some of which have undesirable odours with very low threshold values. Therefore, oxidation-related rancidity is generally related to the secondary oxidation products. In the meantime, alkoyl (RO•), peroxyl (ROO•), hydroxyl (HO•) and new lipid radicals (R•) are generated from decomposition of hydroperoxides, and further participate in the chain reaction of free radicals. In the termination stage of oxidation, radicals neutralize each other through radical–radical coupling or radical–radical disproportionation to form stable non-radical products, including a variety of polymer products.1, 15-16 7 Synthesis and biological evaluation of olive oil polyphenol metabolites Figure 3: Lipid autoxidation pathways (adapted from Frankel 18). 2.2. Photooxidation Photooxidation causes serious deterioration of olive oil, due to the presence of chlorophylls and pheophytins. These pigments act as prooxidants under light and as antioxidants in the dark. In addition to the normal or ground state (triplet) oxygen in autoxidation, the excited state (singlet) oxygen can be involved in lipid oxidation by directly reacting with the double bonds of unsaturated fatty acids through non-radical pathways, as commonly encountered in photooxidation. Singlet oxygen (1O2) is an excited stage of oxygen, which can be generated from the triplet state of oxygen by chemical, photochemical and enzymatic means, as well as by decomposition of hydroperoxides. Singlet oxygen is a major reactive species (ROS) that readily participates in the oxidation process (1500 times faster than triplet oxygen).15 Type I photooxidation proceeds via a free radical or free radical ion route, leading to the formation of hydroperoxides, whereas in type II photooxidation, the highly electrophilic singlet oxygen reacts directly with the double bonds of unsaturated fatty acids by addition rather than through free radical intermediates. Hydroperoxides are generated during singlet oxygen attack, accompanied by a shift of the double bonds in the molecule.1, 15 8 Synthesis and biological evaluation of olive oil polyphenol metabolites 2.3. Antioxidants Among the many methods employed for controlling lipid oxidation, the use of antioxidant is the most effective, convenient and economical. Antioxidants are also used in health-related areas for disease risk reduction and health promotion due to their ability to protect the body against oxidative damage. Antioxidants are substances that when present at low concentrations compared to that at an oxidizable substrate, markedly delay or prevent their oxidation. Antioxidants that fit in this definition include, among others, free radical scavengers, singlet oxygen quenchers, inactivators of prooxidative enzymes. Antioxidants are broadly classified by their mechanisms of action as primary or secondary antioxidants. Primary antioxidants inhibit the oxidation chain reaction by acting as hydrogen donors or free radical acceptors and generating more stable radicals. The inhibition reaction is considered to be in competition with the propagation step of lipid oxidation via a chain reaction. They mainly act as chain breakers by donating hydrogen to alkylperoxyl radicals formed during the propagation step of lipid oxidation. Subsequently they form a stable radical (A•) through the well-known reaction: Secondary antioxidants prevent or retard oxidation by suppressing the oxidation promoters, including metal ions, singlet oxygen, pro-oxidative enzymes and other oxidants. Reducing agents 3Sen* 3O2 1O2* + 1Sen 1O2* + RH ROOH hν 1Sen* 3Sen* Sen Type I 3Sen*1 + 3O2 1[Sen - O2] 1[Sen - O2] + RH ROOH + 1Sen Type II Figure 4: Mechanisms of photosensitized oxidation (adapted from Frankel 12). 9 Synthesis and biological evaluation of olive oil polyphenol metabolites can reduce lipid peroxides and related oxidants through redox reactions and are also referred to as oxygen scavengers.15 Antioxidants have received particular attention because of their potential to modulate oxidative stress associated with chronic disease. The lower incidence of coronary heart disease and of some cancers in the Mediterranean area led to the hypothesis that a diet rich in fruit, vegetables and grains has beneficial effects on health. The major fat component of the so-called “Mediterranean diet” is VOO, as has been stated previously. Many studies suggest that components of the minor fraction, such as, phenolic compounds in particular ortho -diphenols, may contribute to the healthy nature of this diet.17, 19-23 The antioxidant properties of ortho -diphenols can be related to hydrogen donation, i.e., their ability to improve radical stability by forming an intramolecular hydrogen bond between the free hydrogen of their hydroxyl group and their phenoxyl radical.6, 22 2.4. Olive oil phenolic compounds Phenolic compounds present in VOO can be classified into a lipophilic group, like tocopherol, or a hydrophilic group, such as, phenolic acids and alcohols, flavonoids, secoiridoids and lignans.24 With respect to tocopherols (figure 5), α-tocopherol is the main tocopherol found in olive oil. βand γ-tocopherol are only present in minor amounts. In virgin olive oils, they contribute together with polyphenols to virgin olive oil stability. Apart from their action as lipid radical scavengers, they also inhibit photooxidation by reacting with singlet oxygen. Thus, they contribute to an increase in the oxidative stability of oils during storage in the presence of light.17 Furthermore, tocopherols, known as vitamin E, are considered one of the most important antioxidant in vivo . 10 Synthesis and biological evaluation of olive oil polyphenol metabolites 2-(3,4-diHydroxyphenyl)ethanol (3,4-DHPEA, hydroxytyrosol or HT), and 2-(4hydroxyphenyl)ethanol (4-HPEA, tyrosol or Ty) are the most abundant phenolic alcohols in olive oil (figure 6). The flavonoids include flavonol glycosides such as luteolin-7-glucoside and rutin as well as anthocyanins, cyanidun and delphinidin glycosides.11-12, 25, 26 However, this class of phenols is found usually in very low concentrations in olive oil. The secoiridoids are present in the olive fruit and are released into the oil during the mechanical extraction process. Secoiridoids are present exclusively in plants belonging to the family of Oleaceae that includes Olea europaea L. The olive drupe contains high concentrations of phenolic compounds being oleuropein, demethyloleuropein, ligstroside and nüzhenide the most abundant secoiridoid glucosides in olive fruits (figure 7).27 γ-tocopherol α-tocopherol β-tocopherol Figure 6: Structures of phenolic alcohols present in olive oil. Figure 5: α-, βand γ-Tocopherol. 11 Synthesis and biological evaluation of olive oil polyphenol metabolites Figure 7: Structure of the major secoiridoids found in olive fruit. However, the most abundant secoiridoids in VOO are the dialdehydic form of elenolic acid linked to hydroxytyrosol or tyrosol (3,4-DHPEA-EDA or 4-HPEA-EDA) and an isomer of the oleuropein aglycon (3,4-DHPEA-EA) (figure 8).2, 4, 7-10 Figure 8: Structure of secoiridoids of VOO. During olive oil extraction, specific glycosidases hydrolyse (figure 9) the glycosides, producing aglycones that are more lipophilic and therefore can be retained in the oil phase. After hydrolysis the secoiridoid aglicones undergo two main degradation pathways leading to secoiridoid derivatives (figure 9) or suffer further hydrolysis to HT, Ty and elenolic acid.28-29 12 Synthesis and biological evaluation of olive oil polyphenol metabolites Figure 9: Structure of oleuropein and ligstroside aglycones and their derivatives, the major secoiridoids found in olive oil (adapted from Paiva-Martins & Kiritsakis 1). The amount of phenolic compounds in VOO is an important factor to bear in mind when the quality of virgin olive oils is evaluated. The natural phenols not only improve its resistance to oxidation, but there are also responsible for its sharp bitter taste and pungency.30-31 3. Health effects of olive oil and olive oil polyphenols The Mediterranean diet, in which olive oil is the major fat component, has been associated with a low incidence of coronary heart disease and certain cancers. The beneficial effects of olive oil could be linked to both its monounsaturated fatty acid and its antioxidant content.32-34 The antioxidant activity of Ty and HT has received an increasing attention in the last years since it has been related to the protection against important chronic and degenerative diseases, such as, coronary heart diseases (CHD), ageing neuro-degenerative diseases and tumours at different localizations. They may also have antimicrobial, antidiabetic or anti-inflammatory properties.8, 12, 35-38 Atherosclerosis is a multifactorial disease that represents the primary cause of death worldwide. Elevated levels of circulating low-density lipoproteins (LDL), which are rich in cholesterol 13 Synthesis and biological evaluation of olive oil polyphenol metabolites and cholesterol esters, are a well-established risk factor for developing CHD.39 The uptake of LDL by cells of the arterial wall – monocytes/macrophages – is generally mediated by a self-regulating, receptor, which limits cholesterol deposition when the required intracellular levels are reached. The discovery of a macrophagic “scavenger” receptor, which internalizes oxidatively modified forms of LDL (oxLDL) and is not down-regulated by increasing intracellular cholesterol levels, led to the hypothesis that oxLDLs play a crucial role in the onset of the atherosclerotic lesion. This can be assigned to the fact that their uptake causes infiltration and deposition of cholesterol loaded cells (foam cells) into the arterial wall.22, 40 In recent years, there has been much interest in antioxidants that retard oxidative modification of LDL. The LDL isolated from animals and humans that have been fed with VOO has shown increased stability, when compared with that of animals and humans fed with refined olive oil. This can be attributed to the phenolic compounds found in the oil.21, 40-41 Reactive oxygen species (ROS) responsible for oxidative stress are involved in the all above mentioned diseases through mechanisms that in part have been elucidated. ROS in fact, oxidize lipoproteins deposited on the arteries, leading to arteriosclerosis and, with regard to the carcinogenic process, they are able to produce DNA oxidative damage.12, 40 On the other hand, some studies have shown that olive oil phenolic compounds reduce oxidative damage to both red blood cells and renal cells.40 Recently, p –HPEA–EDA has been demonstrated to have potential neuroprotective properties and contributes to preventing cognitive decline due to neurogenerative diseases. Different mechanisms have been proposed to describe the role that this compound plays in reducing the incidence of Alzheimer’s disease (AD), a neurodegenerative disease that affects about 30 million people worldwide.34 4. Bioavailability of olive oil phenolic compounds The biological properties of phenolic compounds present in olive oil depend on their bioavailability, this is their absorption rate and their metabolism. In fact, the potential health 14 Synthesis and biological evaluation of olive oil polyphenol metabolites benefits of olive polyphenols may be attributed to both parental compounds and their phase I and phase II metabolites. Metabolization usually converts a molecule capable of crossing biological membranes into one more hydrophilic that can be excreted by urine. The metabolism is usually divided in two distinct phases: phase I and phase II, where each of these phases entails different transformations to the molecule that will be metabolized. Throughout the metabolism phases, the polarity of the molecules usually increases (and the lipophilicity decrease), thus determining whether this molecule goes to the kidneys in order to be excreted by the urine or proceeds to a next metabolic phase, which may enable or disable the bioactivity of the molecule. The metabolization products of a given phenol depend on its initial chemical structure.42-45 As stated before, there have been a large number of studies investigating the in vitro antioxidant properties of VOO phenol, as well as their protective effects against cell injury. The biological properties of these phenols in vivo depend on the extent to which they are absorbed and metabolized. Olive oil polyphenols undergo extensive metabolism in the intestines and liver and are thus mainly found in biological fluids as phase II metabolites.33, 46-47 A human study conducted in normal and ileostomy subjects fed with olive oil polyphenols has indicated that the major site for the absorption of olive oil polyphenols is the small intestine.48 Following ingestion of VOO, the levels of HT and Ty increase rapidly achieving a peak concentration after 1 h in plasma and around 2 h in urine. These results support the evidence that the small intestine is the major site of absorption for these compounds. The analysis of human urine has also demonstrated that both HT and Ty are dose-dependently absorbed and are metabolized primarily to O -glucuronidated conjugates in the intestine. HT also undergoes O -methylation by the action of catecholO -methyl-transferase (COMT), and homovanillyl alcohol has been detected in human and animal plasma and urine after oral administration of either VOO or pure HT and Ty.3233, 39, 46, 49-52 The bioavailability of secoiridoids 3,4-DHPEA-EDA and 3,4-DHPEA-EA was studied and showed that both compounds are efficiently absorbed by Caco-2 cells and by rat intestine. The secoiridoids are hydrolyzed with production of hydroxytyrosol and its metabolites, as shown in figure 10. 15 Synthesis and biological evaluation of olive oil polyphenol metabolites In a study with the aim of identifying biomarkers for olive oil consumption after 3 weeks dietary intervention with phenol-enriched olive oils as part of a randomized, double-blind, crossover, and controlled nutrition intervention trial, it was found that hydroxytyrosol sulfate (compounds 3/4) and hydroxytyrosol acetate sulfate (compounds 5/6) and not glucoronides were the metabolites most suitable for monitoring EVOO intake.47 Figure 10 shows the metabolic pathways of secoiridoids and hydroxytyrosol leading to sulfate metabolites. A similar sequence can be drawn for glucuronide metabolites. 23 Synthesis and biological evaluation of olive oil polyphenol metabolites Many epidemiology studies show that the incidence of cardiovascular disease (CVD) in Mediterranean countries is low, suggesting a crucial protective role of diet in this area where virgin olive oil (VOO) is the principal source of fat. These studies also shown that VOO consumption could reduce oxidative damage in part due to the phenolic compounds, who have an important role against a range of pathologies associate to oxidative stress. Their antioxidant activity has shown a great impact on human health. However, the potential health benefits of these molecules in general may also be attributed to their metabolites. The aim of this study is to understand how the properties of VOO phenols in vivo depends on the extent of their metabolism. A synthetic strategy is used in order to obtain VOO phenol metabolites in considerable yields in order to be possible to in their protective role against AAPHinduced oxidative stress in normal human RBCs. Figure 14: Proposal synyhesis of phenolic olive oil metabolites. III. Materials and Methods 27 Synthesis and biological evaluation of olive oil polyphenol metabolites 1. General methods All reagents and solvents used in the synthetic procedures were at least pure quality. Solvents used in column chromatography were PA quality. When necessary, dry solvents were obtained by treatment with phosphorus pentoxide. All reactions and purifications were monitored by thin layer chromatography (TLC) on precoated silica gel 60 plates F254, and detected by: • UV-Vis, at λ = 254 nm (lamp U.V.: CN-6 from Vilber Lourmal); • Iodine; • Mostain solution [100 mL H2SO4 10%; 5,0 g (NH4)6Mo7O24.H2O; 0,22 g Ce(SO4)2.4H2O]. Products were purified by flash chromatography through silica gel 60 (0,040-0,063 mm, Merck). NMR spectra were recorded on a 400 MHz equipment (Bruker Avance III 400) at room temperature for solutions in CD3OD, CD3COCD3 and D2O. Chemical shifts are referred to the solvent signal and are expressed in ppm. 2D NMR experiments (COSY, APT, HMBC and HMQC) were carried out when necessary to assign the corresponding signals. The ultra-high performance liquid chromatography (UHPLC) system used was a Vanquish (Thermoscientific) chromatograph, with UV-Vis detector of the same model. Chromatography was carried out using a C18 LiChrospher ® column 100 RP-18, 5 μm (Merck) of dimensions 250 mm x 4 mm. The volume of injections was 10 μL and the mobile phase used was a mixture of water:acetic acid (99:1 v/v) (eluent A) and a mixture of methanol:acetonitrile (1:1 v/v) (eluent B) with a total analysis time of 70 min. 28 Synthesis and biological evaluation of olive oil polyphenol metabolites Table 1: Eluent gradient used in ultra-high performance liquid chromatography. 2. Olive oil Phenolic Compounds Hydroxytyrosol was purchased from Seprox Biotech, tyrosol was obtain from Acros Organics and homovanillyl alcohol was purchased from Sigma-Aldrich. Hydroxytyrosol acetate was obtained from hydroxytyrosol, by two different procedures. Tyrosol acetate and homovanillyl alcohol acetate were synthetized from tyrosol and homovanillyl alcohol, respectively by two different procedures. The secoiridoids 3,4-DHPEA-EDA and 3,4-DHPEA-EA, were isolated from olive leaves according to a procedure described by Paiva-Martins.71 An attempt to synthetize 4-HPEA-EDA was made using isolated 3,4-DHPEA-EDA as starting material. 2.1. Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate (Hydroxytyrosol acetate) Two different techniques were used to synthetize 2-(3,4-dihydroxyphenyl)ethyl ethanoate. Hydroxytyrosol was acetylated using acetyl chloride in dimethyl carbonate, following a procedure described by Bernini et al. 72, and by transesterification with ethyl acetate using Amberlite IR-120 as catalyst, based on a procedure from Begines et al. 73 Time / min Eluent A (%) Eluent B (%) Run 0 95 5 25 70 30 35 65 35 40 60 40 50 30 70 55 0 100 70 0 100 29 Synthesis and biological evaluation of olive oil polyphenol metabolites 2.1.1. Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate using acetyl chloride as acylating agent The reaction of hydroxytyrysol with acetyl chloride in dimethyl carbonate (DMC), is shown in figure 15. The synthesis was done in a proportion of hydroxytyrosol to acetyl chloride of 1:1.2. Figure 15: Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate using acetyl chloride and dimethyl carbonate. In a dry Schlenk tube, 0.50 g of hydroxytyrosol (3.24 mmol) was dissolved in 12 mL of DMC. Then, in an ice bath, 280 µL of acetyl chloride (3.93 mmol) was added dropwise. The mixture was left under argon, protected from light, with magnetic stirring for 2 days at room temperature. The reaction was monitored by TLC [ethyl acetate/petroleum ether (2:1)] and plates revealed by iodine. The solvent was evaporated at reduced pressure and the crude mixture dissolved in 20 mL of ethyl acetate. The organic phase was washed with 20 mL of brine, and the aqueous phase was successively washed with ethyl acetate (4 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and the solvent evaporated at reduced pressure to give a yellowish oil (0.38 g). The crude product was purified by flash chromatography [ethyl acetate/petroleum ether (2:1 v/v)] to give 0.33 g of a yellowish oil. 1HNMR indicated that acetylation occurred both at the phenolic and the aliphatic hydroxyl groups, forming two products 2-(3,4-dihydroxyphenyl)ethyl ethanoate and 2-(4-acetoxy-3-hydroxyphenyl)ethyl ethanoate in a 1:8 ratio. Both compounds presented the same retention factor (rf = 0.27 [ethyl acetate/petroleum ether (2:1)]). 1HNMR (400 MHz, CD3OD): δ = 6.91 (d, J = 8.0 Hz, 1H, 19 – H), 6.85 (dd, J = 8.0, J = 2.0 Hz, 20 – H), 6.82 (d, J = 2.0 Hz, 1H, 16 – H), 6.71 (d, J = 8.0 Hz, 1H, 9 – H), 6.69 (d, J = 2.0 Hz, 1H, 6 – H), 6.55 (dd, J = 30 Synthesis and biological evaluation of olive oil polyphenol metabolites 8.0, J = 2.0 Hz, 1H, 10 – H), 4.24 (t, J = 6.8 Hz, 2H, 13 – H), 4.20 (t, J = 7.2 Hz, 2H, 3 – H), 2.85 (t, J = 6.8 Hz, 2H, 14 – H), 2.77 (t, J = 7.2 Hz, 2H, 4 – H), 2.28 (s, 3H, 22 – H), 2.02 (s, 3H, 11 – H), 2.01 (s, 3H, 11 – H) ppm. 2.1.2. Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate by transesterification with ethyl acetate catalysed by amberlite IR – 120 The reaction between hydroxytyrosol and ethyl acetate catalysed by amberlite IR–120, is shown in figure 16. Figure 16: Synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate using amberlite IR-120 in ethyl acetate. In a round bottom flask, 0.50 g of hydroxytyrosol (3.24 mmol) was dissolved in 25 mL of ethyl acetate. Then 1.05 g of amberlite IR-120 (3.37 mmol) was added and the mixture was left under reflux, protected from light and with magnetic stirring for 11 hours. The reaction was monitored by TLC [ethyl acetate/petroleum ether (2:1)] and the plates revealed by iodine. The resin was filtered, and the solvent was evaporated at 30 ºC. The crude extract (yellowish oil, 0.88 g) was purified by flash chromatography on silica gel [ethyl acetate/petroleum ether (2:1 v/v)], to give 0.63 g of a yellow compound corresponding to 98 % of yield. 1HNMR (400 MHz, CD3OD): δ = 6.69 (d, J = 8.0 Hz, 1H, 9 – H), 6.66 (d, J = 2.0 Hz, 1H, 6 – H), 6.54 (dd, J = 8.0, J = 2.0 Hz, 1H, 10 – H), 4.19 (t, J = 7.2 Hz, 2H, 3 – H), 2.76 (t, J = 7.2 Hz, 2H, 4 – H), 2.01 (s, 3H, 1 – H) ppm. 31 Synthesis and biological evaluation of olive oil polyphenol metabolites 2.2. Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate (Tyrosol acetate) As for the synthesis of 2-(3,4-dihydroxyphenyl)ethyl ethanoate, two procedures to synthetize 2-(4-hydroxyphenyl)ethyl ethanoate were attempted. Tyrosol was acetylated using acetyl chloride in dry dichloromethane and by transesterification with ethyl acetate using amberlite IR120 as catalyst. 2.2.1. Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate using acetyl chloride as acylating agent The synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate is shown is figure 17. Tyrosol reacts with acetyl chloride in dry dichloromethane. The synthesis was done in a proportion of tyrosol to acetyl chloride of 1:1.2. Figure 17: Synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate using acetyl chloride in dry dichloromethane. In a Schlenk tube, under argon 1.13 g of tyrosol (8.18 mmol) was dissolved in 15 mL of dry dichloromethane. Then, the mixture was cooled in an ice bath, and 700 µL of acetyl chloride (9.81 mmol) was added dropwise. The mixture was left stirring, under argon, protected from light, for 24 hours at room temperature. The reaction was monitored by TLC [ethyl acetate/petroleum ether (2:1)] and the plates were revealed by iodine. The solvent was evaporated under vacuum, and 20 mL of ethyl acetate was added to the crude mixture. In a separatory funnel the organic layer was washed with 20 mL of brine and the aqueous phase was washed 3 times with 20 mL of ethyl acetate. The organic layer was extracted 38 Synthesis and biological evaluation of olive oil polyphenol metabolites 3.1.3. Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate The synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate was carried out by reacting homovanillyl acetate with sulfur trioxide-pyridine complex is shown in figure 23. Figure 23: Synthesis of the sodium salt of 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate. In a round bottom flask, under argon, 0.57 g of 2-(4-hydroxy-2-methoxyphenyl)ethyl ethanoate (2.71 mmol) was dissolved in 15 mL of dry dioxane and the mixture cooled in an ice bath. Then, 0.86 g of sulfur trioxide-pyridine complex (5.40 mmol) was added to the solution. The mixture was left under magnetic stirring for 15 minutes at 0 ºC under argon and then closed and stored at -20 ºC for 10 days. The reaction was monitored by TLC, [ethyl acetate/methanol (9:1)] and the plates were revealed with a Mostain solution. When TLC showed a decrease of initial reagents and an increase of product, the mixture was defrosted and 3 mL of cooled water was added. Subsequently, ~80 drops of DEA was immediately added to neutralize the mixture (pH ≈ 7). Then the aqueous phase was washed with diethyl ether (3 x 10 mL) and the organic phase was washed 3 times with 2 mL of water. The aqueous layer was evaporated at 30 ºC, giving a yellow oil (1.39 g). The crude extract was purified by flash chromatography on silica gel by elution with ethyl acetate/methanol (9:1 v/v). The mixture of DEA salts was then dissolved in 2 mL of water, applied to a column of cation-exchange resin (Dowex 50WX8, Na+ form, 10 g), and eluted with water. Fractions containing the desired product were lyophilized to obtain sodium salts of 4-(2-ethanoyloxyethyl)-2methoxyphenyl sulfate as a white solid (0.007 g). 39 Synthesis and biological evaluation of olive oil polyphenol metabolites 3.1.4. Synthesis of the diethylammonium salt of 4-(2-ethanoyloxyethyl)-2hydroxyphenyl sulfate The synthesis of the diethylammonium salt of 4-(2-ethanoyloxyehyl)-2-hydroxyphenyl sulfate was carried out following a procedure described by Teixeira, et al.74 Figure 24 shows the reaction of the DEA salt of diethylammonium 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate and boron tribromide in dry dichloromethane. The synthesis was carried out with a proportion of diethylammonium 4-(2-ethanoyloxyethyl)-2-methoxyphenyl sulfate to boron tribromide of 1:2. Figure 24: Synthesis of diethylammonium 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate. In a hydrolysis tube, 0.10 g of the DEA salt of diethylammonium 4-(2-ethanoyloxyethyl)-2methoxyphenyl sulfate (0.275 mmol) was dissolved in 4 mL of dry dichloromethane, under argon at 80 ° C. To this solution, 540 µL of boron tribromide (0.534 mmol) was added, and the reaction was kept at 80 °C for 10 minutes and then allowed to reach room temperature. The reaction was monitored by TLC [dichloromethane/methanol (9:1)] and the plates were revealed by iodine. After 12 hours, the reaction was quenched by cautious addition of 10 mL of methanol and the solvent evaporated. The crude product was purified by flash chromatography on silica gel [dichloromethane/methanol (9:1 v/v)] to give 7.90 mg of the diethylammonium salt of 4-(2ethanoyloxyethyl)-2-hydroxyphenyl sulfate. 40 Synthesis and biological evaluation of olive oil polyphenol metabolites 3.1.5. Synthesis of the sodium salts of ( Z )-5-(2-((4-formyl-3-(2-oxoethyl)hex-4enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate and of ( Z )-4-(2-((4-formyl-3-(2-oxoethyl)hex-4enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate The reaction of 3,4-DHPEA-EDA with sulfur trioxide-pyridine complex in dry dioxane was carried out in order to obtain a mixture of hydrogenosulfate ( Z )-5-(2-((4-formyl-3-(2-oxoethyl)hex-4enoyl)oxy)ethyl)-2-hydroxyphenyl and hydrogenosulfate ( Z )-4-(2-((4-formyl-3-(2-oxoethyl)hex-4enoyl)oxy)ethyl)-2-hydroxyphenyl and subsequently, their corresponding sodium salts, as shown in figure 25. This would be in agreement with the synthesis of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl hydrogenosulfate and 5-(2-ethanoyloxyethyl)-2-hydroxyphenyl hydrogenosulfate and their corresponding sodium salts previously described. Figure 25: Synthesis of the sodium salts of (Z )-5-(2-((4-formyl-3-(2-oxoethyl)hex-4-enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate and of ( Z )-4-(2-((4-formyl-3-(2-oxoethyl)hex-4-enoyl)oxy)ethyl)-2-hydroxyphenyl sulfate. In a round bottom flask, under argon, 0.26 g of 3,4-DHPEA-EDA (0.812 mmol) was dissolved in 5 mL of dry dioxane. Then the mixture was cooled in an ice bath and 0.26 g of sulfur trioxide-pyridine complex (1.63 mmol) was added to the solution. The mixture was left under magnetic stirring for 15 minutes at 0 ºC under argon and then closed and stored at -20 ºC for 5 days. The reaction was monitored by TLC [ethyl acetate/methanol (9:1)] and the plates were revealed on iodine. When TLC showed a decrease of initial reagents and an increase of product, the mixture was defrosted, and 3 mL of cooled water was added. Immediately the mixture was neutralized with 41 Synthesis and biological evaluation of olive oil polyphenol metabolites ~25 drops of DEA (pH ≈ 7). After this step, the aqueous phase was washed with diethyl ether (3 x 10 mL) and the organic phase was washed 2 times with 2 mL of deionized water. The aqueous layer was evaporated, giving a yellow oil (0.33 g). The crude extract was purified by flash chromatography [ethyl acetate/methanol (9:1 v/v)] to give 0.16 g of a yellow oil. As the 1HNMR was inconclusive, it was decided to pass the product through the column of cation-exchange resin (Dowex 50WX8, Na+ form, 10 g), eluting with water. Only 0.0074 g were obtained as purified compound therefore the NMR spectra obtained after this procedure was inconclusive. 3.2. Solvolysis Reactions 3.2.1. Synthesis of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and of 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate The reaction of diethylammonium 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and diethylammonium 5-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate with methanol and potassium carbonate, is shown in figure 26. Solvolysis of the ester group occurred, to give, after elution through cation-exchange resin (Dowex 50WX8, Na+ form), the sodium salts of 2-hydroxy-4-(2hydroxyethyl)phenyl sulfate and of 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate. Figure 26: Synthesis of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and of 2-hydroxy-5-(2hydroxyethyl)phenyl sulfate. In a round bottom flask, 0.23 g of the mixture of the sodium salts of 2-hydroxy-4-(2hydroxyethyl)phenyl sulfate and of 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate were dissolved in 3 42 Synthesis and biological evaluation of olive oil polyphenol metabolites mL of methanol. Then 0.23 g of potassium carbonate was added. The mixture was left at room temperature and protected from light, under stirring for 24 hours. The reaction was monitored by TLC [ethyl acetate/methanol (9:1)] and plates were revealed by iodine. After 24 hours, the solvent was evaporated, and 10 mL of deionized water was added and acidified with amberlite IR-120 resin, until bubbles started to evolve. Then the resin was filtrated under vacuum and the aqueous phase was washed with diethyl ether (6 x 5 mL) and immediately neutralized with ~4 drops of DEA (pH ≈ 7). The aqueous layer was extracted and evaporated at 30 ºC. The DEA salts solution was concentrated to 2 mL of water and applied to a column of cation-exchange resin (Dowex 50WX8, Na+ form, 10 g) and eluted with water, to give a mixture of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and of 2-hydroxy-5-(2hydroxyethyl)phenyl sulfate as a cream-colored solid (0.11 g). 3.2.2. Synthesis of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate The reaction of the diethylammonium salt of 4-(2-ethanoyloxyethyl)-phenyl sulfate with potassium carbonate in methanol is shown in figure 27. The product, sodium 4-(2hydroxyethyl)phenyl sulfate results from solvolysis of the ester group. Figure 27: Synthesis of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate. To a round bottom flask, 0.25 g of the diethylammonium salt of 4-(2-ethanoyloxyethyl)- phenyl sulfate (0.961 mmol) was added and dissolved in 3 mL of methanol. Then, 0.27 g of potassium carbonate (1.95 mmol) was added. The mixture was left at room temperature, protected from light, under magnetic stirring for 24 hours. The reaction was monitored by TLC [ethyl acetate/methanol (9:1)] and the plates revealed by Mostain solution. 43 Synthesis and biological evaluation of olive oil polyphenol metabolites After 24 hours, the solvent was evaporated, and 10 mL of water was added to the crude residue and then acidified with Amberlite IR-120 resin, until bubbles started to evolve. The resin was filtrated, and the aqueous phase was washed with diethyl ether (7 x 5 mL) and immediately neutralized with ~5 drops of DEA (pH ≈ 7). DEA salts were diluted in 2 mL and applied to a column of cation-exchange resin (Dowex 50WX8, Na+ form, 10 g) eluted with water, to give the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate as white a solid (0.09 g). 3.2.3. Synthesis of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate Figure 28 shows the reaction of the diethylammonium salt of 4-(2-ethanoyloxyethyl)-2methoxyphenyl sulfate with methanol and potassium carbonate, to give the sodium salt of 4-(2hydroxyethyl)-2-methoxyphenyl sulfate. Figure 28: Synthesis of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate. In a round bottom flask, 0.10 g (0.320 mmol) of the diethylammonium salt of 4-(2ethanoyloxyethyl)-2-methoxyphenyl sulfate was added and dissolved in 2 mL of methanol. Then 0.10 g (0.724 mmol) of potassium carbonate was added. The mixture was left at room temperature and protected from light, under magnetic stirring for 24 hours. The reaction was monitored by TLC [ethyl acetate/methanol (9:1)] and the plates were revealed by Mostain solution. After 24 hours, the solvent was evaporated, and 5 mL of water was added to the crude residue and then acidified with Amberlite IR-120 resin, until bubbles started to evolve. The resin was then filtrated, and the aqueous phase was washed with diethyl ether (5 x 5 mL) and immediately neutralized with ~3 drops of DEA (pH ≈ 7). DEA salts were diluted in 2 mL and 44 Synthesis and biological evaluation of olive oil polyphenol metabolites applied to a column of cation-exchange resin (Dowex 50WX8, Na+ form, 10 g) eluted with water, to give the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate as a white solid (0.15 g). 4. Haemolysis Assay 4.1. Preparation of RBC suspension Blood was obtained from healthy volunteers by venepuncture. Blood samples (about 9 mL each) were collected into tubes containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant. Blood samples were centrifuged (Gyrozen Multi-Purpose High Speed Centrifuge 1580R) at 4 ºC, 700 g for 10 min; plasma and buffy coat were carefully removed by aspiration and discarded. RBCs were washed three times with phosphate-buffered saline solution (PBS) pH 7.4 and centrifuged at 4 ºC, 700 g for 7 minutes. Supernatant was carefully removed after each wash. RBCs were resuspended in PBS pH 7.4 after the final wash to obtain 2.0% haematocrit. 4.2. AAPH-induced haemolysis In order to evaluate the protection of the different phenolic compounds and its metabolites against the APPH-induced haemolysis of RBCs, in vitro assays (n≥4) were prepared according to table 2 and 3. 45 Synthesis and biological evaluation of olive oil polyphenol metabolites Table 2: Assay conditions in the study of AAPH-induced haemolysis in the presence of phenolic compounds and its metabolites. PBS pH 7.4 (µL) DMSO 18.1 % (µL) Compound 800 µM (µL) RBCs suspension Ht=2.6 % (µL) AAPH 600mM (µL) Positive control Without DMSO 85 - - 500 65 DMSO 1.13 % 44.4 40.6 - Negative control Without DMSO 150 - - - DMSO 1.13% 109.4 40.6 - Olive oil phenols and its metabolites 2.5 µM 44.4 38.57 2.03 65 5 µM 36.54 4.06 10 µM 20 µM 40 µM 24.35 16.25 80 µM - 40.6 RBCs suspensions were prepared at 2.0% haematocrit (Ht), and the assays were performed using phenolic compounds at final concentrations of 2.5, 5, 10, 20, 40 and 80 µM, and AAPH (Sigma Aldrich Chemistry) at final concentration of 60 mM (chosen in agreement to previous work41). Sample tests were run in duplicate. Negative controls (RBCs in PBS with and without DMSO 1.13%) and positive controls (RBCs in PBS with AAPH, and with or without DMSO 1.13%) were run in triplicate. RBCs suspensions were incubated at 37 ºC for 10 min with each phenolic compound before addition of AAPH. Subsequently, AAPH was added and the incubations of RBCs suspensions were carried out at 37 ºC for 4 hours, under gentle shaking. Tubes were shaken by inversion at the end of each hour. Haemolysis was determined spectrophotometrically according to the method described by Ko et al.75 After 4 hours of incubation, an aliquot of each tube was diluted with PBS and an aliquot 46 Synthesis and biological evaluation of olive oil polyphenol metabolites with the same volume was diluted with distilled water to yield complete haemolysis. All Eppendorfs were centrifuged (HaraeusTM FrescoTM 21 Centriguge, Thermo Scientific) at room temperature, 700 g for 10 minutes. The supernatant (300 µL) from each Eppendorf was collected and the absorvance was read at 540 nm (ref. 690 nm) (Synergy HTX multi-mode reader, Biotek). The percentage of haemolysis was calculated by the following formula: % 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 = 𝐴 𝐵×100 were A represents the absorption of the supernatant from the aliquot diluted in PBS, while B represents the absorption of the supernatant from the aliquot diluted in distilled water. IV. Results and Discussion 54 Synthesis and biological evaluation of olive oil polyphenol metabolites of them corresponding to tyrosol (starting reagent) and another corresponding to 2-(4hydroxyphenyl)ethyl ethanoate (figure 34). This suggests that by using NaOH 1M hydrolysis of the product occurred. Figure 34: Result obtained in liquid-liquid extraction of the reaction mixture using NaOH 1M and HCl 0.74M. Therefore, the synthesis of 2-(4-hydroxyphenyl)ethyl ethanoate by transterification using tyrosol and ethyl acetate, catalyzed by amberlite IR-120 was attempted. In these conditions the yield in the wanted product was 75%. Figure 35 shows the NMR spectra obtained for 2-(4hydroxyphenyl)ethyl ethanoate. 6,10-H 7, 9-H 3-H 4-H 1-H Figure 35: NMR in deuterated methanol of 2-(4-hydroxyphenyl)ethyl ethanoate. 55 Synthesis and biological evaluation of olive oil polyphenol metabolites 1.1.3. Synthesis of 2-(4-hydroxy-3-methoxyphenyl)ethyl ethanoate 2-(4-Hydroxy-3-methoxyphenyl)ethyl ethanoate was synthesized, using homovanillyl alcohol by transterification with ethyl acetate and amberlite as catalyst. In these conditions the yield in wanted product was 80 %. 1.2. Isolation of secoiridoids from olive tree leaves It has been reported that secoiridoids, 3,4-DHPEA-EDA and 3,4-DHPEA-EA, can be derived from oleuropein aglycone through a process that results from storing the leaves in bags at 37 ºC. This process induces oleuropein to degrade into these two secoiridoids. Thus, fresh olive tree leaves were processed at 40 ºC in a plastic bag for 24 hours and then macerated in ethanol. After solvent evaporation, a crude extract was obtained and after purification of this extract by liquid-liquid extraction and column chromatography, 3,4-DHPEA-EDA and 3,4-DHPEA-EA were obtained and identified by UHPLC, by comparison with standards of 3,4-DHPEA-EDA and 3,4-DHPEA-EA and also by NMR. 1.2.1. Isolation of 3,4-DHPEA-EDA and 3,4-DHPEA-EA After a preliminary purification by liquid-liquid extraction, a crude extract was obtained and further purified by column chromatography. UHPLC analysis of the mixture before purification and comparison with standards, showed the presence of two phenolic compounds in the secoiridoid region of the chromatogram, which were identified as 3,4-DHPEA-EDA and 3,4-DHPEA-EA (figure 36). 56 Synthesis and biological evaluation of olive oil polyphenol metabolites Further purification by column chromatography allowed separation of these two compounds giving two fractions, one containing 3,4-DHPEA-EDA (figure 38) and another containing 3,4-DHPEA-EA (figure 39). A B C Figure 36: Chromatogram of standards (A=3,4-DHPEA-EDA; B=Oleuropein; C=3,4-DHPEA-EA). Figure 37: Chromatogram of mixture before purification. 57 Synthesis and biological evaluation of olive oil polyphenol metabolites After UHPLC analysis, it was found that in the fraction containing 3,4-DHPEA-EDA, only one peak with a retention time of 27.9 min could be observed in the chromatogram, as it shown in figure 36. After solvent evaporation, 0.28 g of compound was obtained as a cream coloured oil. Through analysis of the NMR spectra it was found that the compound did not present any contamination and thus could be considered as pure. It was important the extract be pure, since it was to be used later in synthetic processes. Figure 36: Chromatogram of sample with 3,4-DHPEA-EDA. 58 Synthesis and biological evaluation of olive oil polyphenol metabolites Table 3: NMR chemical shifts in deuterated chloroform for secoiridoid 3,4-DHPEA-EDA. In contrast, the chromatogram obtained from the fraction containing 3,4-DHPEA-EA (figure 39), showed two additional peaks, meaning the extract was not pure and would require further purification. In fact, one peak corresponding to 3,4-DHPEA-EDA at 29.1 min, another at 34.3 min and another peak corresponding to 3,4DHPEA-EA at 42.4 min could be observed. 1HNMR 13CNMR C Number δ (ppm) Signal type J (Hz) δ (ppm) 1 130.11 2 6.59 dd 8.0 2.0 120.82 3 6.78 d 8.0 114.85 4 142.79 5 143.17 6 6.71 d 2.0 115.83 7 2.78 m 33.92 8a 4.22 m 64.86 8b 4.17 m 9 171.58 10a 2.70 dd 15.6 36.61 10b 2.60 dd 15.6 11 3.62 m 26.85 12a 2.76 m 45.88 12b 2.93 dd 18.4 13 9.19 d 2.0 200.61 14 142.50 15 6.64 q 6.8 154.91 16 2.05 d 7.2 14.96 17 9.63 s 195.47 59 Synthesis and biological evaluation of olive oil polyphenol metabolites In view of the retention time of 34.3 min, the peak could be attributed to oleocanthal. After solvent evaporation 0.25 g of impure 3,4-DHPEA-EA (74%) was obtained. The NMR analysis confirmed the presence of 3,4-DHPEA-EA as the major compound in the extract. A B C Figure 37: Chromatogram of a sample containing 3,4-DHPEA-EDA (A), oleocanthal (B) and 3,4-DHPEA-EA (C). 60 Synthesis and biological evaluation of olive oil polyphenol metabolites Table 4: Chemical shifts in deuterated chloroform for secoiridoid 3,4-DHPEA-EDA. 1HNMR 13CNMR C Number δ (ppm) Signal type J (Hz) δ (ppm) 1 130.20 2 6.57 dd 8.0 1.9 120.80 3 6.74 d 8.0 116.70 4 144.90 5 144.60 6 6.73 d 1.9 116.00 7 2.76 t 7.1 34.90 8 4.20 m 65.80 9 172.10 10a 2.84 dd 15.9 37.50 10b 2.35 m 11 3.37 m 27.20 12 107.50 13a 7.55 s 155.40 13b 7.50 d 1.2 14 4.57 qd 6.7 5.7 71.40 15 2.65 m 54.70 16a 9.67 s 200.80 16b 9.54 d 1.4 17a 1.55 d 6.8 19.40 17b 1.38 d 6.7 18 167.50 19 3.66 s 51.40 61 Synthesis and biological evaluation of olive oil polyphenol metabolites 1.3. Tentative of synthesis of 4-HPEA-EDA From 3,4-DHPEA-EDA extracted from olive tree leaves, an attempt to synthetize 4-HPEAEDA by transesterification of tyrosol was undertaken. For this propose, an enzymatic catalyst, novozym 435, was used. The reaction was left for 1 month at 37ºC. Every day, a TLC in dichloromethane/methanol (5:1) and diethyl ether/methanol (35:1) was carried out in order to accompany the evolution of stains and the emergence of new ones. A spot of hydroxytyrosol started to appear, which led to think that transesterification could actually be happening, but the evolution was slow and so, more enzyme was added to accelerate the process. In the last week, the temperature was increased to 50ºC. Subsequently, the hydrotyrosol spot increased and another spot appeared with a very similar retention factor to that of tyrosol. It was decided to stop the reaction when the new stain no longer increased. The enzyme was then filtered and washed with 10 mL of dichloromethane. TLC of the filtrated mixture in dichloromethane/methanol (5:1) and diethyl ether/methanol (35:1) showed the presence of tyrosol, hydroxytyrosol, 3,4-DHPEA-EDA and a further compound over tyrosol spot. Several eluents were tested in order to separate the compound formed from tyrosol. However, no satisfactory solvent was identified. Since we knew where oleocanthal should appears in the UHPLC chromatogram, we decided to determine by UHPLC if the new spot formed was 4-HPEA-EDA before proceeding with separation by column chromatography. Figure 40 shows the chromatogram obtained. Only four peaks appear related to hydroxytyrosol, tyrosol, 3,4-DHPEA-EDA and unknown compound. The chromatogram allowed us to verify that the appearance of hydroxytyrosol had nothing to do with the transesterification because the pick expected for oleocanthal did not appeared in the chromatogram. 62 Synthesis and biological evaluation of olive oil polyphenol metabolites 2. Synthesis of sulfate metabolites The sulfation of olive oil phenolic compounds was done using sulfur trioxide-pyridine complex in dioxane. To avoid reaction of the aliphatic hydroxyl groups with sulfur trioxide, this group needs to be protected prior to sulfation by an acetyl group. The sulfation reaction occurs by the nucleophilic attack of the phenolic hydroxyl group, at the sulfur as shows in figure 41. Figure 39: Proposed mechanism of sulfation of tyrosol. The acidic mixture obtained from the reaction was neutralized with DEA to avoid decomposition of sulfate during evaporation of the solvent. At pH<4, a rapid acid-catalyzed reaction A B C D Figure 38: Chromatogram of reaction sample (A=hydroxytyrosol; B=tyrosol; C=3,4-DHPEA-EDA; D=Unknown compound). 63 Synthesis and biological evaluation of olive oil polyphenol metabolites occurs, in which the sulfate group is protonated, leading to a unimolecular sulfur-oxygen bond fission with elimination of sulfur trioxide (figure 42). This decomposition is avoided with a base stronger than pyridine. Thus, DEA was chosen, since its excess can be easily removed by evaporation. Figure 40: Decomposition of phenylsulfate. After purification by column chromatography, the solvent was evaporated, dissolved in water and applied to a cation-exchange resin column, allowing the exchange between DEA and the physiological cation Na+. When necessary acetyl deprotection was carried out before cation-exchange using the DEA salts (figure 43). The reaction was carried out using methanol and potassium carbonate, to obtain the monosulfated alcohols derivatives. Figure 41: Proposed mechanism of solvation of tyrosol. An acid resin, amberlite IR-120 was added to neutralize the mixture. This process allows removal of the excess of potassium carbonate excess by formation of CO2. After resin filtration, the aqueous layer was washed with diethyl ether to remove the methyl acetate formed and the mixture 70 Synthesis and biological evaluation of olive oil polyphenol metabolites The proton NMR spectrum (figure 45) shows that the solvolysis reaction was successful due to the absence of the ester methyl singlets (2.02, 2.01 ppm). Additionally, the chemical shifts of the triplets of hydrogens 1 and 9 (3.72 and 3.70 ppm) are lower than the chemical shifts of the triplets of hydrogens 1 and 9 (4.23 and 4.21 ppm) of the sodium salt of 4-(2-ethanoyloxyethyl)-2hydroxyphenyl sulfate and sodium 5-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate. This results from an increased shielding effect by the hydroxyl groups formed. 2.6. Synthesis of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate The deprotection of the ester of diethylammonium 4-(2-ethanoyloxyethyl)-phenyl sulfate was carried out by reaction with methanol. Table 9 shows the NMR chemical shifts of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate obtained after cation-exchange. The yield was 87%. 7-H 12-H 16-H 15-H 4-H 8-H 1-H 9-H 2-H 10-H Figure 43: NMR of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and 2-hydroxy-5-(2hydroxyethyl)phenyl sulfate. 71 Synthesis and biological evaluation of olive oil polyphenol metabolites Table 9: NMR chemical shifts in deuterated water of the sodium salt of 4-(2-hydroxyethyl)phenyl sulfate. 1HNMR 13CNMR Number of C δ (ppm) Signal type J (Hz) δ (ppm) 1 3.77 T 6.4 63.34 2 2.80 T 6.4 38.08 3 ------------ -------------- -------------- 137.97 4 7.26 D 8.8 131.09 5 7.19 D 8.8 122.37 6 ------------- -------------- -------------- 150.46 7 7.19 D 8.8 122.37 8 7.26 D 8.8 131.09 The disappearance of the singlet at 1.90 ppm and the lower chemical shift of the triplet at 3.77 ppm, due to the increased shielding effect caused by the hydroxyl group, shows that solvolysis occurred successfully. 2.7. Synthesis of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate The reaction of diethylammonium 4-(2-ethanoyloethyl)-2-methoxyphenyl sulfate with methanol was carried out to give the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate after cation-exchange. The yield of this reaction was 89%. 72 Synthesis and biological evaluation of olive oil polyphenol metabolites Table 10: NMR chemical shifts in deuterated water of the sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate. 1HNMR 13CNMR Number of C δ (ppm) Signal type J (Hz) δ (ppm) 1 3.76 t 6.4 63.31 2 2.78 t 6.4 38.56 3 ------------ -------------- -------------- 139.19 4 6.79 d 1.6 114.99 5 ------------- -------------- -------------- 151.97 6 ------------- -------------- -------------- 139.38 7 7.23 d 8.0 122.37 8 6.82 dd 8.0 1.6 123.53 9 3.80 s -------------- 56.89 Table 10 shows that the singlet at 2.00 ppm disappeared and the chemical shift of the triplet of hydrogen 1 (3.76 ppm) is lower than chemical shift of the triplet of hydrogen 3 (4.26 ppm) of the sodium salt 4-(2-ethanoyloethyl)-2-methoxyphenyl sulfate. 2.8. Tentative synthesis of diethylammonium 4-(2-ethanoyloxyethyl)-3hydroxyphenyl sulfate Since a mixture of monossulfates are obtained by the direct reaction of the cathecol with SO3, an attemp to obtain only one isomer was performed. For that, diethylammonium 4-(2ethanoyloxyethyl)-3-methoxyphenyl sulfate was used as initial reagent and made to reacted with boron tribromide. A possible mechanism for this reaction is shown in figure 46. 73 Synthesis and biological evaluation of olive oil polyphenol metabolites Figure 44: Proposed mechanism for the demethylation of diethylammonium 4-(2-ethanoyloxyethyl)-3-methoxyphenyl sulfate with boron tribromide. The reaction was left for 24 hours and then 10 mL of methanol were added dropwise to destroy BBr3. After purification, proton NMR gave a spectrum with the peaks and chemical shifts of hydroxytyrosol and not of diethylammonium 4-(2-ethanoyloxyethyl)-3-hydroxyphenyl sulfate. Table 11: NMR chemical shifts in deuterated methanol of hydroxytyrosol. 1HNMR 13CNMR Number of C δ (ppm) Signal type J (Hz) δ (ppm) 1 3.68 t 7.2 63.88 2 2.67 t 7.2 38.91 3 ------------ -------------- -------------- 129.29 4 6.68 d 8.0 116.37 5 ------------- -------------- -------------- 145.36 6 ------------- -------------- -------------- 145.31 7 6.66 d 2.0 115.61 8 6.53 dd 8.0 2.0 120.52 74 Synthesis and biological evaluation of olive oil polyphenol metabolites The chemical shifts of hydrogens 4, 7 and 8 are below those obtained for diethylammonium 4-(2-ethanoyloxyethyl)-3--hydroxyphenyl sulfate, which indicate that there is a greater shielding effect that would not occur if the hydroxyl at position 6 was sulfated. The chemical shifts of carbons 1 and 2 are also lower, which confirms that solvolysis of the ester occurred. In addition to these peaks, there are also two singlets in the aromatic zone and one additional triplet (figure 47). The hydrogen 1 triplet is integrated into 6 protons, leading to the conclusion that another product has been formed. Cosy, APT and HMQC analysis (see annexes) were performed to analyse the correlation of the protons with the carbons. However, it was not possible to determine the chemical structure of the secondary product. 3. Preliminar in vitro bioactivity evaluation The protection against AAPH-induced haemolysis in human RBCs of tyrosol (Ty), hydroxytyrosol (HT), homovanillyc alcohol (HVA), 2-(3,4-dihydroxyphenyl)ethyl hydrogenosulfate (1SO4-HT), the mixture of the sodium salts of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and 2hydroxy-5-(2-hydroxyethyl)phenyl sulfate (3,4-SO4-HT), 4-(2-hydroxyethyl)phenyl sulfate (3-SO4-Ty), hydroxytyrosol acetate (HTAc), homovanillyc acetate (HVAAc), the mixture of the sodium salts of 4- (2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and 5-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate (3,4-SO4-HTAc), and the sodium salt of 4-(2-ethanoyloethyl)-2-methoxyphenyl sulfate (4-SO4-HVAAc) 4-H 7-H 8-H 1-H 2-H Figure 45: MR of mixtures of two compounds. Compound that is identified is hydroxytyrosol. 75 Synthesis and biological evaluation of olive oil polyphenol metabolites was studied. Different concentrations of compounds were used (2.5, 5, 10, 20, 40 and 80 µM) with incubation at 37ºC for 4 hours. A high percentage of inhibition of haemolysis reflects a high protection by the compound in study. The percentage of inhibition of haemolysis was measured by comparing the spectrophotometric absorbance values of aliquots of the supernatant of RBC suspension in PBS (pH=7.4 at λ=540 nm) and other aliquots in water. In this way, it was possible to conclude which compounds, and the respective concentration, confer more protection against haemolysis. Figure 48 shows that in the presence of AAPH, parental compounds protected RBCs from oxidative-induced haemolysis in a concentration dependent manner, but only HT and HTAc showed important activity at 5 µM. In fact, HTAc showed better activity probably because of its higher lipossolubility that may allow a better location of this compound at the RBC membrane. Although with a lower activity, the same rank is observed for HVA and its acetate, with the acetate showing a better protective activity. It is interesting to note that, Ty shows a significantly activity against haemolysis inhibition of RBCs although it is a monophenol. It was able to protect RBC to an extent similar to that obtained by HVA. Most monophenols do not show radical scavenging activity against radicals used for radical scavenging activity evaluation studies such, as DPPH• or ABTS•. However, in vivo, highly reactive radicals can be formed during oxidative injury and they can actually be trapped by these monophenols, and therefore protect cells. This is the case of tyrosine, a monophenolic amino acid present in all membrane proteins. In the case of metabolites, all sulfates showed a much lower protective activity than the parental compound. Only the 1O -sulfate of HT retained much of the parental activity, showing that the two hydroxyl groups at the aromatic ring are important for the bioactivity of these compounds. In fact, the sulfation of one aromatic hydroxyl groups decreased drastically the protective activity of all compounds, in particular in the case of monophenols (Ty and HVA) that did not show any protection at any concentration. Moreover, the increase of hydrophilicity of compounds by the conjugation with the sulfate group also contribute for a lower interaction of these metabolites with RBC membranes, deviating them from the place of action. 76 Synthesis and biological evaluation of olive oil polyphenol metabolites Nevertheless, it is important to point out that the HT and HTAc sulfates still show some protective activity. Although modest, in a nutritional context, this protection can be important since the consumption of olive oil takes place on a regular basis throughout life. It has also been described that conjugated metabolites may behave as carriers of bioactivity compounds in plasma, which may deconjugate in situ in target tissues realising the parental compound, which is the final effector.76-77 77 Synthesis and biological evaluation of olive oil polyphenol metabolites Figure 48: Percentage of inhibition of haemolysis of RBCs at 2% haematocrit incubated with olive oil phenolic compounds (2.5, 5, 10, 20, 40 and 80 µM and AAPH (60 mM) for 4 hours at 37 ºC. Hydroxytyrosol (HT, 2-(3,4-dihydroxyphenyl)ethyl hydrogenosulfate (HT-1-SO4), soium salt of 2-hydroxy-4-(2-hydroxyethyl)phenyl sulfate and odium salt of 2-hydroxy-5-(2-hydroxyethyl)phenyl sulfate (H 3+4 SO4), hydroxytyrosol acetate (HTAc, sodium salts of 4-(2-ethanoyloxyethyl)-2-hydroxyphenyl sulfate and 5-(2-ethanoyloxyethyl)-2-hydroyphenyl sulfate (HTAc 3+4 SO4), tyrosol (Ty), sodium salt of 4-(2-hydroxyethyl)phenyl sulfate (Ty 4-SO4)), homovanillyc alcohol (HVA), sodium salt of 4-(2-hydroxyethyl)-2-methoxyphenyl sulfate (HVA 4-SO4), homovanillyc acetate (HVAAc) and sodium salt of 4-(2-ethanoyloethyl)-2-methoxyphenyl sulfate (HVAAc 4-SO4). V. Conclusion 86 Synthesis and biological evaluation of olive oil polyphenol metabolites 1. Kiritsakis, A.; Shahidi, F., Olives and Olive Oil as Functional Foods: Bioactivity, Chemistry and Processing. John Wiley & Sons: 2017. 2. Özcan, M. M.; Matthäus, B., A review: benefit and bioactive properties of olive (Olea europaea L.) leaves. 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