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Oxidative stress is a condition caused by excess free radicals that leads to the development of various chronic and aging- associated diseases. Phenolic compounds as potent antioxidants have an important role in removing excess free radicals and terminating oxidative stress. These compounds can also inhibit various enzymes and by doing so have an important role in anti-inflammatory and anticancer therapy. In our work, molecular modelling was used to study free radical scavenging mechanisms of selected phenolic compounds (hydrogen atom transfer (HAT), single-electron transfer followed by proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET)), while molecular docking was used to study inhibition of cyclooxygenase-2 and multidrug resistance protein 1. Radical scavenging mechanisms were studied by using DFT as implemented in Gaussian 09 (M06-2X/6-311++G(d, p)/SMD). Our results show HAT and SPLET as thermodynamically probable and competitive processes, while Gibbs free energy change indicates selected compounds as potent scavengers. Docking analysis was performed by using the AutoDock 4.2 program package. Free energy of binding and inhibition constant were estimated. Obtained results suggest that investigated compounds are potential inhibitors of studied proteins, meaning they could be responsible for some health benefits associated with regular intake of (poly)phenol-rich diet.

The thermodynamic preference of different free radical scavenging mechanisms of 3-(4-hydroxy- 3-methoxyphenyl)propanoic acid (HMPPA), a colon catabolite of dietary (poly)phenols, was studied using density functional theory. The role of guaiacyl moiety and carboxyl group of HMPPA in double (2H+/2e-) processes of inactivation of free radicals was theoretically investigated in polar and nonpolar media using Gaussian 09 software package. Obtained results indicate that HMPPA possesses potential for inactivating free radicals of different characteristics (HO•, HOO•, CH3O•, CH3OO•, PhO•, Cl3COO• etc.) via double hydrogen atom transfer (dHAT) and double sequential proton loss electron transfer (dSPLET) mechanisms. Concentration of HMPPA in serum may reach very low micromolar values and may contribute to health benefits associated with regular intake of polyphenol- rich diet by direct scavenging of reactive oxygen species.

Poslednjih godina 1, 2, 4-triazol-3-tioni privlače dosta pažnje zahvaljujući njihovim biološkim osobinama. Petočlani prsten ovih jedinjenja je strukturni deo brojnih farmakološki zanimljivih jedinjenja koja pokazuju antidepresivne, antiinflamatorne, antibakterijske i antikancerogene osobine. U našem prethodnom radu1 kombinovali smo fenolne farmakofore sa 1, 2, 4-triazol-3-tionom u cilju dobijanja novih jedinjenja sa boljim antioksidativnim osobinama. Na osnovu dobijenih rezultata u toj studiji može se reći da je SPLET dominantan mehanizam antioksidativnog delovanja u metanolu. Na dalje je ispitivana mogućnost triazola koji su pokazali najbolje antioksidativne osobine, da neutrališu tri slobodno radikalske vrste (izo- propoksil radikal, vinil peroksil radikal i trihlormetilperoksil radikal). Ravnotežne geometrije svih oblika ispitivanih jedinjenja, kao i slobodno radikalskih vrsta koje učestvuju u reakciji izračunate su na B3LYP-D3/6- 311++G(d, p) nivou teorije, u metanolu i benzenu. Ovi rastvarači korišćeni su da bi se ispitao uticaj polarne i nepolarne sredine. Dobijeni rezultati ukazuju da sva tri slobodna radikala mogu biti neutralisana dejstvom ispitivanih triazola, ali je pitanje koji je mehanizam antioksidativnog delovanja dominantan. SET-PT mehanizam je najmanje verovatan reakcioni put antioksidativnog delovanja. U metanolu, prva dva ispitivana radikala mogu biti neutralisana i HAT i SPLET mehanizmom. U benzenu je dominatan SPLET mehanizam. Rezultati ostvareni za trihlormetilperoksil radikal ukazuju da ovaj radikal može biti inaktiviran reakcijom HAT mehanizma u metanolu, dok su u benzenu HAT i SPLET konkurentni mehanizmi.

Slobodni radikali su atomi ili molekuli s jednim ili više nesparenih elektrona u spoljašnjoj elektronskoj ljusci, što ih čini vrlo nestabilnima. Nastaju kao proizvod raznih fizioloških procesa, te su važna komponenta unutar-ćelijske signalizacije i imunološkog odgovora. Naš je organizam izložen i slobodnim radikalima iz okoline, čiji izvor mogu biti UV i jonizirajuće zračenje, zagađenje, duvanski dim i dr. Iako su slobodni radikali nužni za pravilno odvijanje fizioloških procesa, njihov višak ima negativan učinak na organizam i dovodi do nastanka oksidativnog stresa. Kako je oksidativni stres podloga za nastanak raznih bolesti, naš organizam ima sistem za borbu protiv suvišnih slobodnih radikala koji se zasniva na aktivnosti nekoliko enzima (katalaza, glutation peroksidaza i dr.) i endogenih antioksidansa (npr. mokraćna kiselina). U borbi protiv slobodnih radikala važni su i egzogeni antioksidansi (npr. flavonoidi), koji se mogu naći u hrani i piću. U našem prethodnom radu ispitali smo antioksidacijski potencijal mokraćne kiseline1. Na dalje, ispitali smo antioksidacijski potencijal raznih fenolnih i kumarinskih jedinjenja. Ravnotežne geometrije svih oblika ispitivanih jedinjenja, kao i odabranih slobodno radikalskih vrsta, izračunate su na M06-2X/6-311++G(d, p) nivou teorije, u pentil etanoatu i vodi. Na osnovu dobijenih rezultata može se reći da su HAT i SPLET dominantni i konkurentni mehanizmi antioksidativnog delovanja ispitanih jedinjenja, dok je SET-PT mehanizam najmanje verovatan reakcioni put.

Colon polyphenolic metabolites can reduce activity of enzymes involved in human carcinogenesis [1], for instance by inhibition of COX-2 [2]. Recent studies have shown the importance of selective inhibition of COX-2 for the anti-inflammatory and anticancer therapy [3], indicating COX-2 as a valid molecular target for cancer prevention and treatment [4]. Theoretical investigations of active site of COX-2 affirmed some natural phenolic antioxidants as potential inhibitors of this enzyme [5]. Radical scavenging mechanisms of selected polyphenolic metabolites were studied in water and pentyl ethanoate as a solvent, by DFT method using Gaussian 09 package [6]. Geometry optimizations and frequency calculations were carried out using the M06- 2X/6-311++G(d, p) level of theory, in conjunction with the SMD continuum solvation model. Inhibitory potency against COX- 2 by colon polyphenolic metabolites and their mono- and di-anionic forms, were theoretically studied. Free energy of binding and inhibition constant for these ligands at the most favourable binding positions were estimated. Hydrogen atom transfer and sequential proton loss electron transfer mechanisms were found to be thermodynamically probable and competitive processes in both media. The Gibbs free energy change for reaction of inactivation of radicals indicate selected metabolites as potent scavengers. Docking analysis with structural forms of selected metabolites indicates dianionic ligands as potent inhibitors of COX- 2. Obtained results indicate that, because polyphenolic metabolites are produced in high mM concentrations and are usually better absorbed than their precursor molecules, they may contribute to health benefits associated with regular intake of polyphenol-rich diet. Acknowledgments: We gratefully acknowledge the financial support to this work from The Foundation of the Croatian Academy of Sciences and Arts, under the project No. 10-102/244-1- 2016.- “Investigations of the antioxidant mechanisms of polyphenols and their metabolites.” [1] C. Miene, A. Weise, M. Glei, Nutr. Cancer, 2011, 63, 653. [2] P.C. Karlsson, U. Huss, A. Jenner, B. Halliwell, L. Bohlin, J.J. Rafter, J. Nutr., 2005, 135, 2343. [3] G. Dannhardt, W. Kiefer, Eur. J. Med. Chem., 2001, 36, 109. [4] R.A. Gupta, R.N. DuBois, Nat. Rev. Cancer, 2001, 1, 11. [5] M. Amaravani, N.K. Prasad, V. Ramakrishna, Springerplus, 2012, 1, 58. [6] M.J. Frisch, G.W. Trucks, H.B. Schlegel et al., GAUSSIAN 09, Wallingford, CT, 2009.

Health benefits of polyphenol-rich diet are related to intestinal metabolites, rather than to polyphenolic compounds originally present in food [1, 2], which usually possess very low bioavailability and low systemic concentration [3, 4]. Particular intestinal metabolites are produced in high M concentrations and may promote health via several possible in vivo mechanisms [5]. 4-hydroxyphenylpropionic acid (4-HPPA) is among the most abundant products of polyphenols degradation in large intestine [5]. Its concentration in colon may reach value of 200 M [5], which could be sufficient to exert biological activity and effective direct radical inactivation. Radical scavenging mechanisms of 4-HPPA, in water and pentyl ethanoate as a solvent, were studied by DFT method using Gaussian 09 package [6]. Geometry optimizations and frequency calculations were carried out using the M06– 2X/6-311++G(d, p) level of theory, in conjunction with the SMD continuum solvation model. Hydrogen atom transfer (HAT) and sequential proton loss electron transfer (SPLET) mechanisms were found to be thermodynamically probable and competitive processes in both media. The Gibbs free energy change for reaction of inactivation of radicals indicates 4-HPPA as a potent scavenger. 4-HPPA possesses potential for inactivating radicals of different characteristics by direct scavenging via HAT and SPLET mechanisms. Because 4-HPPA is produced in high M concentrations and is usually better absorbed than its precursor molecules, it may contribute to health benefits associated with regular intake of polyphenol- rich diet. References [1] Y.S. Chiou, J.C. Wu, Q. Huang, F. Shahidi, Y.J. Wang, C.T. Ho and M.H. Pan, J. Funct. Foods 7 (2014) 3-25. [2] A. Duda-Chodak, T. Tarko, P. Satora and P. Sroka, Eur. J. Nutr. 54 (2015) 325-341. [3] D. Del Rio, A. Rodriguez-Mateos, J.P.E. Spencer, M. Tognolini, G. Borges and A. Crozier, Antioxid. Redox Signal. 18 (2013) 1818-1892. [4] P.C.H. Hollman, Arch. Biochem. Biophys. 559 (2014) 100-105. [5] B. Halliwell, J. Rafter and A. Jenner, Am. J. Clin. Nutr. 81 (2005) 268S−276S. [6] M.J. Frisch, G.W. Trucks, H.B. Schlegel et al., GAUSSIAN 09, Wallingford, CT, 2009. Acknowledgments We gratefully acknowledge the financial support to this work from The Foundation of the Croatian Academy of Sciences and Arts, under the project No. 10-102/244-1- 2016. – "Investigations of the antioxidant mechanisms of polyphenols and their metabolites"