Naslov (srp)

Strukturne i antioksidativne osobine hlorogenske kiseline

Autor

Tošović, Jelena, 1990-

Doprinosi

Marković, Svetlana, 1959-
Amić, Dragan.
Petrović, Zorica, 1958-
Dimitrić-Marković, Jasmina, 1965-

Opis (eng)

One part of the enclosed manuscript is devoted to investigation of structural features of chlorogenic acid, whereas the other is associated with examination of the antioxidative potential of chlorogenic acid. Particular attention is dedicated to thermodynamic and kinetic investigation of antioxidative mechanisms of chlorogenic acid. The first part of the investigation was devoted to determination of the most stable conformation of chlorogenic acid in gaseous phase and in solution by comparison of experimental IR, Raman, 1H-NMR, 13C-NMR, and UV spectra with the spectra simulated using density functional theory methods. Detailed conformational analysis has established that chlorogenic acid exists as a mixture of several conformations. A common feature of all conformations is that quinic moeity is characterized with directed hydrogen bonds, with carboxyl hydrogen oriented toward aproximate hydroxyl group, but not toward carboxyl oxygen. The most stable structure in gaseous phase is characterized with O4–H4···O9’, whereas the most stable conformation in solution is characterized with O4–H4···O10’. Within this dissertation, all vibrational modes of the IR spectrum of chlorogenic acid were examined for the first time. Experimentally obtained Raman and NMR spectra of chlorogenic acid are in perfect accord with the literature results. Very good agreement between simulated and experimental spectra indicate correct arrangement of atoms in this compound. The next step was related to the comparison of the antioxidative activity of chlorogenic acid with the antioxidative potentials of its two structural isomers (neochlorogenic and cryptochlorogenic acids) and caffeic acid. For this reason, thermodynamic parameters: Bond Dissociation Enthalpy (BDE), Proton Affinity (PA), Electron Transfer Enthalpy (ETE), Ionisation Potential (IP), and Proton Dissociation Enthalpy (PDE) in solutions of different polarity were examined. To obtain thermodynamic parameters, the enthalpies of the solvated proton and electron in various solutions with different theoretical methods were determined. Detailed examination of radicals, anions, and radical cations derived from caffeic and caffeoylquinic acids was conducted. The properties of the hydroxycinnamic moiety in the corresponding radicals, anions and radical cations are very similar and practically do not depend on the esterification position. Quinic moiety remains the same in the derived reactive species. As a result, all four phenolic acids have similar values of thermodynamic parameters BDE, PA, ETE, IP, and PDE. Taking all these facts into account, it was concluded that caffeic and caffeoylquinic acids exhibit very similar antioxidant activity. The same results have been derived from experiments, however, they have not been supported by chemical theory so far. Subsequently, the behavior of chlorogenic acid in the reactions with free radicals (DPPH•, HO•, and О2 −•) was investigated using ESR spectroscopy. The obtained results showed that chlorogenic acid is selective toward DPPH•, HO•, and HOO•/O2−• radicals. Thermodynamic investigation of all possible antioxidative mechanisms (Hydrogen Atom Transfer (HAT), Radical Adduct Formation (RAF), Sequential Proton Loss Electron Transfer (SPLET), and Single Electron Transfer – Proton Transfer (SET-PT)) of chlorogenic acid in the presence of investigated free radicals was also conducted. The results revealed that no mechanism is suitable for scavenging O2−•, which leads to conclusion that HOO• is responsible for the behavior of HOO•/O2−• mixture. HAT and RAF are plausible antioxidative mechanisms in the case of the reaction of chlorogenic acid with HOO• and HO•, whereas the only possible mechanism in the case of DPPH• radical is HAT. Following the computational methodology, also known as Quantum Mechanics-based test for Overall free Radical Scavenging Activity (QM-ORSA), the antioxidative activity of chlorogenic acid relative to trolox as reference compound was examined. The applied methodology implies thermodynamic and kinetic investigation of all possible mechanisms. This approach also considers various effects that can influence antioxidative activity of a compound. Firstly, investigation in nonpolar solvents was performed. The results showed that both chlorogenic acid and trolox react with HO• via HAT and RAF mechanisms, whereas in the case of CH3OO• HAT is the only favourable reaction pathway. Chlorogenic acid is more reactive toward HO•, but less reactive toward CH3OO• than trolox. The following step was investigation of antioxidative activity of chlorogenic acid relative to trolox in aquous solution at physiological conditions. At pH = 7.4 chlorogenic acid exists in monoanionic (87 %) and dianionic (13 %) forms, whereas dominant form of trolox is monoanionic (> 99 %). The reactions of chlorogenic acid monoanion and dianion, as well as of trolox monoanion, with HO• and CH3OO• were examined. Both anionic forms of chlorogenic acid undergo only HAT raction with CH3OO• radical. Operative mechanistic pathways in the case of HO• are HAT, RAF, SPLET, and SET-PT. Considering that all the reactions with dianion are diffusion-controlled, its contribution to scavenging HO• is almost equivalent to that of more abundant monoanion. The calculated rate constant value for the reaction of chlorogenic acid with HO• is in perfect agreement with corresponding experimentaly obtained value. The order of reactivity toward selected free radicals is the same in polar and nonpolar solution.

Opis (srp)

Priložen rukopis jednim delom obuhvata ispitivanje strukturnih osobina, a drugim delom ispitivanje antioksidativnog potencijala hlorogenske kiseline. Posebna pažnja je usmerena ka termodinamičkom i kinetičkom ispitivanju mehanizama antioksidativnog delovanja ovog jedinjenja. Prvi deo istraživanja se odnosi na utvrđivanje najstabilnije konformacije hlorogenske kiseline u gasovitoj fazi i u rastvoru upoređivanjem eksperimentalnih IR, Raman, 1H-NMR, 13C-NMR i UV spektara sa spektrima simuliranim pomoću teorije funkcionala gustine. Detaljnom konformacionom analizom je ustanovljeno da hlorogenska kiselina postoji u obliku smeše različitih konformera. Zajednička osobina svih ovih konformera je da se na hinskom delu molekula nalaze usmerene vodonične veze, pri čemu je karboksilni kiseonik usmeren ka vodoniku iz susedne hidroksilne grupe, a ne ka karboksilnom kiseoniku. Najstabilnija konformacija u gasovitoj fazi poseduje O4–H4···O9’ vodoničnu vezu, dok najstabilniji konformer u rastvoru poseduje O4–H4···O10’ vodoničnu vezu. U okviru ove doktorske disertacije po prvi put su ispitani svi vibracioni oblici u IR spektru hlorogenske kiseline. Eksperimentalno dobijeni Raman-ski i NMR spektri hlorogenske kiseline su u saglasnosti sa postojećim rezultatima iz literature. Veoma dobro slaganje između simuliranih i eksperimentalnih spektara ukazuje na to da su atomi u hlorogenskoj kiselini pravilno raspoređeni. Sledeći korak se odnosio na upoređivanje antioksidativne aktivnosti hlorogenske kiseline sa antioksidativnom aktivnošću njena dva strukturna izomera (neohlorogenska i kriptohlorogenska kiselina) i kafeinskom kiselinom. Iz tog razloga su ispitivani termodinamički parametari: entalpija disocijacije veze (Bond Dissociation Enthalpy, BDE), afinitet prema protonu (Proton Affinity, PA), entalpija prenosa elektorna (Electron Transfer Enthalpy, ETE), jonizacioni potencijal (Ionisation Potential, IP) i entalpija disocijacije protona (Proton Dissociation Enthalpy, PDE), u rastvaračima različite polarnosti. Za potrebe izračunavanja termodinamičkih parametara određene su entalpije solvatisanog protona i elektrona u različitim rastvaračima pomoću različitih teorijskih metoda. Detaljno su ispitani i radikali, anjoni i radikal katjoni koji nastaju iz kafeinske i kafeoilhinskih kiselina. Osobine hidroksicimetnog dela u odgovarajućim radikalima, anjonima i radikal katjonima su veoma slične i praktično ne zavise od položaja esterifikacije. Hinski deo ostaje nepromenjen u svim novonastalim reaktivnim vrstama. Posledica toga je da sve četiri fenolne kiseline imaju slične vrednosti termodinamičkih parametara: BDE, PA, ETE, IP i PDE. Iz svega navedenog je zaključeno da kafeinska i kafeoilhinske kiseline ispoljavaju veoma sličnu antioksidativnu aktivnost. Isti ovi zaključci izvedeni su i iz eksperimenata, ali do sada nisu imali uporište u hemijskoj teoriji. Zatim je usledilo ispitivanje ponašanja hlorogenske kiseline u reakciji sa slobodno radikalskim vrstama (DPPH•, HO• i O2 −•) pomoću ESR spektroskopije. Izvedeni eksperimenti su pokazali da je hlorogenska kiselina selektivna prema DPPH•, HO• i HOO•/O2−• radikalima. Usledilo je i termodinamičko ispitivanje mogućih antioksidativnih mehanizama (prenos vodonikovog atoma (Hydrogen Atom Transfer, HAT), obrazovanje radikalskog adukta (Radical Adduct Formation, RAF), gubitak protona praćen prenosom elektrona (Sequential Proton Loss Electron Transfer, SPLET) i prenos elektrona praćen prenosom protona (Single Electron Transfer – Proton Transfer, SET-PT)) ove kiseline u prisustvu ispitivanih radikala. Otkriveno je da u vodenom rastvoru ni jedan mehanizam nije pogodan za reakciju između hlorogenske kiseline i O2−• što vodi ka zaključku da je u kiseloj sredini HOO• odgovoran za ponašanje HOO•/O2−• smeše. HAT i RAF su povoljni antioksidativni mehanizmi u slučaju reakcija hlorogenske kiseline sa HOO• i HO•, dok je u slučaju DPPH• radikala jedini očekivani mehanizam HAT. U narednom koraku je pomoću računarske metodologije koja je poznata pod nazivom: test za procenjivanje ukupne antioksidativne aktivnosti zasnovan na kvantno mehaničkim proračunima (Quantum Mechanics-based test for Overall free Radical Scavenging Activity (QM-ORSA) ispitivana antioksidativna aktivnost hlorogenske kiseline u odnosu na troloks koji je predstavljao referentni sistem. Primenjena metodologija podrazumeva termodinamičko i kinetičko ispitivanje svih mogućih mehanizama i uključuje različite aspekte koji mogu da utiču na antioksidativni kapacitet jedinjenja. Najpre je vršeno ispitivanje u nepolarnim rastvaračima. Dobijeno je da hlorogenka kiselina i troloks sa HO• radikalom reaguju preko HAT i RAF mehanizama, dok je u slučaju CH3OO• radikala HAT jedini mogući reakcioni put. Hlorogenska kiselina je reaktivnija prema HO•, dok je prema CH3OO• manje reaktivna od troloksa. Zatim je vršeno istraživanje antioksidativne aktivnosti hlorogenske kiseline u odnosu na troloks u vodenom rastvoru pri fiziološkim uslovima. Na pH = 7.4 hlorogenska kiselina postoji u obliku monoanjona (87 %) i dianjona (13 %), dok se troloks dominantno nalazi u obliku monoanjona (> 99 %). Ispitivane su reakcije monoanjona i dianjona hlorogenske kiseline i monoanjona troloksa sa HO• i CH3OO• radikalima. Oba anjonska oblika hlorogenske kiseline u prisustvu CH3OO• podležu isključivo HAT mehanizmu. U prisustvu HO• operativni su HAT, RAF, SPLET i SETPT mehanizmi. Kako su sve antioksidativne reakcije dianjona kontrolisane difuzijom, njegov doprinos u „hvatanju“ HO• je gotovo isti kao i doprinos monoanjonskog oblika koji se pri datim uslovima nalazi u mnogo većoj koncentraciji. Izračunata vrednost konstante brzine za reakciju između hlorogenske kiseline i HO• radikala je u savršenoj saglasnosti sa odgovarajućom eksperimentalnom vrednošću. Redosled reaktivnosti hlorogenske kiseline prema izabranim radikalima je isti u polarnoj i nepolarnoj sredini.

Jezik

srpski

Datum

2019

Licenca

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Creative Commons CC BY-NC-ND 2.0 AT - Creative Commons Autorstvo - Nekomercijalno - Bez prerada 2.0 Austria License.

CC BY-NC-ND 2.0 AT

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