Food Chemistry 218 (2017) 440–446

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Food Chemistry

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Antiradical activity of delphinidin, and towards hydroxyl and nitric oxide radicals: The energy requirements calculations as a prediction of the possible antiradical mechanisms ⇑ Jasmina M. Dimitric´ Markovic´ a, , Boris Pejin b, Dejan Milenkovic´ c, Dragan Amic´ d, Nebojša Begovic´ e, Miloš Mojovic´ a, Zoran S. Markovic´ c,f a Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia b Department of Life Sciences, Institute for Multidisciplinary Research – IMSI, Kneza Višeslava 1, 11030 Belgrade, Serbia c Bioengineering Research and Development Center, 34000 Kragujevac, Serbia d Faculty of Agriculture, Josip Juraj Strossmayer University of Osijek, Kralja Petra Svacˇic´a 1D, 31000 Osijek, Croatia e Institute of General and Physical Chemistry, Studentski trg 12-16, 11000 Belgrade, Serbia f Department of Chemical-Technological Sciences, State University of Novi Pazar, Vuka Karadzˇic´a bb, 36300 Novi Pazar, Serbia article info abstract

Article history: Naturally occurring flavonoids, delphinidin, pelargonidin and malvin, were investigated experimentally Received 24 August 2015 and theoretically for their ability to scavenge hydroxyl and nitric oxide radicals. Electron spin resonance Received in revised form 28 August 2016 (ESR) spectroscopy was used to determine antiradical activity of the selected compounds and M05-2X/6- Accepted 16 September 2016 311+G(d,p) level of theory for the calculation of reaction enthalpies related to three possible mechanisms Available online 17 September 2016 of free radical scavenging activity, namely HAT, SET-PT and SPLET. The results obtained show that the molecules investigated reacted with hydroxyl radical via both HAT and SPLET in the solvents investi- Chemical compounds studied in this article: gated. These results point to HAT as implausible for the reaction with nitric oxide radical in all the sol- Delphinidin (PubChem CID: 128853) vents investigated. SET-PT also proved to be thermodynamically unfavourable for all three molecules Pelargonidin (PubChem CID: 440832) Malvin (PubChem CID: 5458954) in the solvents considered. Ó 2016 Published by Elsevier Ltd. Keywords: Anthocyanidines and ÅOH and ÅNO radicals ESR spectra Semi-empirical calculations DFT

1. Introduction centage of polyphenolic compounds are associated with a decreased incidence of osteoporosis, cardiovascular diseases, can- Phenolic compounds, plant secondary metabolites, are found cer, diabetes and neurodiseases (Nothlings, Murphy, Wilkens, commonly in herbs and fruits. The term phenolics encompass sev- Henderson, & Kolonel, 2007; Song, Manson, Buring, Sesso, & Liu, eral thousand naturally occurring compounds, which can be subdi- 2005; Tomas-Barberan & Espin, 2001; Ness & Powles, 1996; vided into two major groups: simple phenolics and . Kamei et al., 1995; Block, Patterson, & Subar, 1992; Huang & (flavones, flavanones, flavonols, flavanols, antho- Ferraro, 1992; Weisburger, 1992). These attributes are thought to cyanins, etc.) are a subcategory of polyphenols. Generally, flavo- arise mainly from their antioxidant capacity, which operates at dif- noids are reported to exert a wide range of positive health ferent levels in oxidative processes induced by reactive free radical benefits. This has been confirmed by numerous investigations species (Swain, 1976). where the consumption of food and beverages containing high per- and anthocyanins, hydroxylated and glycosy- lated flavylium compounds, are plant pigments responsible for

⇑ Corresponding author. the red, blue, and purple hues of flowers and fruits in nature E-mail addresses: [email protected] (J.M. Dimitric´ Markovic´), (Harborne, 1967). They are found in an assortment of fruits, veg- [email protected] (B. Pejin), [email protected] (D. Milenkovic´), [email protected] etables (cherries, rapes, plums, apples, radishes, berries, red cab- (D. Amic´), [email protected] (N. Begovic´), [email protected] (M. Mojovic´), bage, etc.) and flowers (tulips, roses, orchids, etc.). Their brilliant [email protected] (Z.S. Markovic´). http://dx.doi.org/10.1016/j.foodchem.2016.09.106 0308-8146/Ó 2016 Published by Elsevier Ltd. J.M. Dimitric´ Markovic´ et al. / Food Chemistry 218 (2017) 440–446 441 colours attract pollinating insects, but they also act as catalysts in antiradical activity and antiradical mechanisms for delphinidin the light phase of photosynthesis and as regulators of iron channels (Dp), pelargonidin (Pg) and malvin (Mv) (Fig. 1) by combining associated with phosphorylation. These compounds also have an experimental measurements and theoretical calculations of the important role in protection of higher plants against short energy requirements for the reactions of these molecules with wavelength-induced damage (especially protection of photosensi- hydroxyl and nitric oxide radicals in different media. Calculated tive biological targets, such as DNA, lipids, proteins and coen- energy requirements might indicate which radical scavenging zymes), against microbial invasion, insects and mammalian mechanisms are thermodynamically preferred and identify active herbivory (Swain, 1976; Harborne, 1967). sites for radical inactivation. During scavenging processes, flavonoids donate hydrogen Hydroxyl and nitric oxide radicals are radical species of partic- atoms from the phenoxyl groups, inactivating a range of radical ular relevance. The hydroxyl radical is considered the most reactive species. This transfer can be visualized through at least three (with the half-life around 109 s) and the most damaging. Uncon- mechanisms characteristic not only of flavonoids but phenolics trolled actions of hydroxyl radicals can have devastating effects generally: hydrogen atom transfer (HAT), sequential proton loss within the body since they react at diffusion rates with virtually electron transfer (SPLET), and single electron transfer followed by any molecule including macromolecules, such as DNA, membrane proton transfer (SET-PT) (Litwinienko & Mulder, 2009; DiLabio & lipids, proteins and carbohydrates. Reactive nitrogen species (RNS) Johnson, 2007; Foti, 2007; Litwinienko & Ingold, 2007; DiLabio & are a family of molecules, derived from nitric oxide (ÅNO) and the Ingold, 2005; Mayer, 2004). activity of inducible nitric oxide synthase 2 (NOS2), which have Generally, there are two types of antioxidant assays in foods antimicrobial activity. Most of the cytotoxicity attributed to ÅNO and biological systems; those measuring free radical scavenging is in fact due to peroxynitrite produced by the diffusion- capacity and those evaluating lipid peroxidation (Miguel, 2010). controlled reaction between ÅNO and the superoxide anion radical. In this paper, we aimed to provide quantitative tools to determine Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or indirect, radical-mediated mechanisms (Halliwell, 2001; Aruoma & Halliwell, 1998; Fridovich, 1978).

2. Experimental

2.1. ESR spectra

ESR spectra were recorded at room temperature using a Bruker Elexsys E540 ESR spectrometer (Bruker, Billerica, MA, USA) operat- ing at X-band (9.51 GHz) with the following settings: modulation amplitude, 2 G; modulation frequency, 100 kHz; microwave power, 2 mW. The spectra were recorded using EXepr software (Bruker BioSpin). Samples of Dp, Pg and Mv (all purchased from Extrasynthese, Genay, France) were drawn into 10 cm long gas- permeable Teflon tubes (Zeus industries, Raritan, USA) (wall thick- ness 0.025 mm and internal diameter 0.6 mm). All measurements were performed under normal conditions, using quartz capillaries into which Teflon tubes were placed. The ESR spin-trapping experiment was carried out as follows: a) selected reactive oxygen species (ROS) were produced using a pure chemical radical generating systems and the amounts determined based on the amplitude of the selected ESR signals, which origi- nated from the spin-adducts formed during a 5-min period by par- ticular trapping radical; b) the same experiment was repeated after addition of Dp, Pg and Mv, which should decrease the intensity of the ESR signal since a proportion of the radicals present will be removed. The capacity of the selected flavonoids to eliminate free radicals was evaluated based on the difference between the relative ampli- tudes of the ESR signals of spin-adduct in radical generating sys- tems, with and without the addition of these compounds. The results are presented as oxidant scavenging (% of radical reduction), which represents the relative decrease in radical pro-

duction: % of radical reduction = 100 (I0 Ia)/I0: where I0 – rela- tive height of the third low-field ESR peak of the spin-adduct of the

control system and Ia – relative height of the same ESR peak in the spectrum of the sample containing Dp, Pg and Mv.

2.2. Generation of ÅOH radical

The ability of Dp, Pg and Mv to scavenge ÅOH radicals was tested using the Fenton reaction as a ‘‘ÅOH producing” system. The Fenton

reaction system contained 0.5 mM H2O2 (Sigma Aldrich, Tauf- Fig. 1. Structures of delphinidin (a), pelargonidin (b) and malvin (c). kirchen Germany) and 0.075 mM FeSO4 (Sigma Aldrich, 442 J.M. Dimitric´ Markovic´ et al. / Food Chemistry 218 (2017) 440–446

Taufkirchen, Germany). The spin-trap DEPMPO (Enzo Life Sciences 3. Results and discussion Inc., Farmingdale, NY, USA) was purified and tested for hydroxy- lamine impurities using an established procedure (Jackson, Liu, 3.1. ESR measurements Liu, & Timmins, 2002). The final concentration of DEPMPO was 50 mM. The final concentration of all samples was 0.01 mM. Sam- The free radical scavenging activities of Dp, Pg and Mv towards ples with no antioxidants served as controls. Deionized 18 MX H2O hydroxyl and nitric oxide radicals were estimated using ESR spec- was used in all experiments. troscopy. It was observed that the molecules investigated quenched ESR signals to different extents, suggesting different rad- Å 2.3. Generation of NO radical ical scavenging activities. The antiradical activity (AA) was calcu- lated with respect to the relative heights of selected hydroxyl Å The spin trapping of NO radicals, generated by the reduction of and nitric oxide ESR peaks. nitrous acid in the form of potassium nitrite, was achieved using a Signals from stable spin-adducts, formed with DEPMPO spin- standard ferro-N-dithiocarboxysarcosine (Fe(DTCS)2) spin- trap in a standard Fenton reaction system (Yoshimoto, Furukawa, trapping system (Kleschyov, Wenzel, & Munzel, 2007). Yamamoto, Horie, & Watanabe-Kohno, 1983)(Fig. 2), were quenched by all three molecules. The results (Table 1) showed that 2.4. Computational methods the efficiency of the different radical species depended on their structure. Both, the number and position of hydroxyl and methoxy PM7 calculations were performed using MOPAC2012TM (Stewart, groups in ring B affect the stability and reactivity of these mole- 2012) and DFT calculations using the Gaussian 09 (Frisch et al., cules and, consequently, their antiradical activity. To some extent 2009). it was noted that, the higher number of hydroxyl groups, the higher the antiradical activity. 2.4.1. MOPAC calculations Data in Table 1 indicate Pg and Dp are stronger antioxidants The different conformations of Dp, Pg and Mv (Fig. S1) were than Mv. Given the structure of Pg (Fig. 1), and the fact that it optimized using the PM7 method included in MOPAC2012TM. The has a somewhat higher activity than Dp, it is possible to deduce eigenvector (EF) optimization procedure was carried out with a that position C4 primarily contributes to the activity, before final gradient norm less than 0.01 kcal mol 1 Å 1. The solvent con- ortho-hydroxy or pyrogallol group in ring B. It is also possible that tribution to the enthalpies of formation for Dp, Pg and Mv was the C5-OH group in Dp and Pg, as demonstrated in structurally determined employing COSMO (Conductor-like Screening Model) related flavone molecules (Dimitric´ Markovic´, Milenkovic´, Amic´, calculations implemented in MOPAC 2012TM (Maia et al., 2012; Mojovic´, et al., 2014), has an important role in selectivity towards Stewart, 2012; Frisch et al., 2009). This approach was used for all hydroxyl radical. The greater activity of Pg against hydroxyl radi- three flavonoids. DOH-1, POH-1 and MOH-1 were found to be cals was in good agreement with the results of Antal, Gârban, the most stable conformers of Dp, Pg and Mv (Figs. S1 and S2). and Gârban (2003) who also confirmed Pg as the most efficient These conformers were also used for the geometry reoptimizations anthocyanidine against hydroxyl radical. The somewhat reduced by DFT methods.

2.4.2. DFT calculations Most theoretical studies of Dp, Pg and Mv have focused on the rings, where OH groups are located. Geometry optimizations of all species involved in radical scavenging mechanisms have been carried out using M05-2X functional, developed by Truhlar (Zhao & Truhlar, 2008), and 6-311+G(d,p) basis set implemented in the Gaussian 09 (Frisch et al., 2009). This functional has been recom- mended for kinetic and thermochemistry calculations by Zhao, Schultz, and Truhlar (2006) and others (Dimitric´ Markovic´, Milenkovic´, Amic´, Popovic´-Bjelic´, et al., 2014; Markovic´ et al., 2012; Galano, Macias-Ruvalcaba, Medina-Campos, & Pedraza- Chaverri, 2010; Zavala-Oseguera Alvarez-Idaboy, Merino & Galano, 2009). M05-2X is among the best performing functionals for calculat- ing reaction energies involving free radical species (Zhao & Truhlar, 2008). The influence of water, dimethyl sulfoxide (DMSO), ethanol and dimethylformamide (DMF) as solvents was approximated using the SMD solvation model (Marenich, Cramer, & Truhlar, 2009). This model has proven to be better than others in explaining solvation energies for the neutral and ionic species in aqueous sol- vents (Marenich et al., 2009). The geometries were fully optimized without imposing any restrictions. Local minima were confirmed by the absence of the imaginary frequencies. Thermodynamic corrections at 298.15 K were included in the calculation of the relative energies. The vibra- tional frequencies were obtained from diagonalization of the corre- sponding M05-2X Hessian matrices. The nature of the stationary points was determined by analysing the number of the imaginary Fig. 2. ESR spectra of DEPMPO/OH adduct obtained in Fenton reaction in the presence of EtOH solution of: delfinidin (a), malvin (b), pelargonidin (c) and only frequencies: 0 for minimum and 1 for the transition state. There- EtOH as control (d) (The ERS spectra were recorded 5 min after performing Fenton fore, the structures obtained were verified by normal mode analy- reaction. Experimets were performed under normal conditions, room temperature sis. No imaginary frequencies were obtained. and atmosphere). J.M. Dimitric´ Markovic´ et al. / Food Chemistry 218 (2017) 440–446 443

Åþ Å Table 1 DHIP ¼ HðAr OH ÞþHðNO ÞHðAr OHÞHðON Þð8Þ Radical scavenging activity of Dp, Pg and Mv. where the H(Ar-OHÅ+), H(HO) and H(NO) are the enthalpies of the Compound ÅOH ÅNO radical cation of initial phenolic compound and anion generated Delphinidin 83.5% n.o. from the corresponding initial radical. Pelargonidin 85.2% n.o. The second step of this mechanism is deprotonation of Ar-OHÅ+ Malvin 61.2% n.o. by HO and NO: Åþ Å Ar OH þ HO ! Ar O þ HOH ð9Þ activity of Mv (Table 1) could be associated with glycosylation, Åþ Å which decreases the antiradical capacity of anthocyanins by reduc- Ar OH þ NO ! Ar O þ NOH ð10Þ ing the coplanarity to the rings (De Lima, Sussuchi, & De Giovani, D 2007). According to some authors (Antal, Gârban, & Gârban, HPDE can be calculated using the following equations: 2003; Kähkönen & Heinonen, 2003; Wang, Cao, & Proir, 1997) Å Åþ DHPDE ¼ HðAr O ÞþHðHOHÞHðAr OH ÞHðHO Þð11Þ the number and position of sugar residues, especially at C3, can also affect the antiradical activity of anthocyanins. Å Åþ DHPDE ¼ HðAr O ÞþHðHONÞHðAr OH ÞHðNO Þð12Þ Table 1 also shows that none of the molecules showed activity towards nitric oxide radical. The first step in SPLET is deprotonation of the phenolic com- pound by HO,NO or another base. The outcome of this reaction 3.2. Antiradical mechanisms of delphinidin, pelargonidin and malvin is the formation of phenoxide anion Ar-O : Ar OH þ HO ! Ar O þ HOH ð13Þ Scavenging properties of Dp, Pg and Mv were related to their capacity to transfer hydrogen atoms to hydroxyl and nitric oxide Ar OH þ NO ! Ar O þ NOH ð14Þ radicals. Since neighbouring groups and geometry influenced the behaviour of different OH groups in polyphenolic structures, only DHPA can be calculated as follows: the most stable structures were taken into account. DH ¼ HðAr O ÞþHðHOHÞHðAr OHÞHðHO Þð15Þ In the radical scavenging mechanisms reactive radical species PA were inactivated by hydrogen donation from a phenoxy group of DH ¼ HðAr O ÞþHðNOHÞHðAr OHÞHðNO Þð16Þ the flavonoid (Ar-OH). By reducing radicals, flavonoids become PA Å Å phenoxyl radicals, which are less reactive and more stable. In the next step electron transfer from Ar-O to HO and ON The following reactions describe H atom transfer: takes place: Å Å Å Å Ar OH þ HO ! Ar O þ HOH ð1Þ Ar O þ HO ! Ar O þ HO ð17Þ

Å Å Å Å Ar OH þ ON ! Ar O þ ONH ð2Þ Ar O þ ON ! Ar O þ NO ð18Þ

Energy requirements related to the radical scavenging mecha- DHETE can be determined from the equations: nisms, which all have the same net result, are governed by differ- Å Å DH ¼ HðAr O ÞþHðHO ÞHðAr O ÞHðHO Þð19Þ ent molecular properties: bond dissociation enthalpy (BDE) in the ETE HAT; proton affinity (PA) together with electron transfer energy Å Å DH ¼ HðAr O ÞþHðNO ÞHðAr O ÞHðON Þð20Þ (ETE) of ArO in SPLET; ionization potential (IP) and proton disso- ETE Å ciation enthalpy (PDE) of ArOH+ in SET-PT. The reaction enthalpies associated with the three radical scav- In HAT (Eqs. (1) and (2)), hydrogen atoms are transferred from enging mechanisms (HAT, SET-PT and SPLET) were calculated the phenolic OH group to the free radical. DHBDE for HAT can be using M05-2X/6-311+G(d,p) level of theory. Species necessary for calculated using the following equations: these calculations were generated from the most stable conforma- Å Å tions for Dp, Pg and Mv (Fig. S2 Supplementary material). The cal- DHBDE ¼ HðAr O ÞþHðHOHÞHðAr OHÞHðHO Þð3Þ culations were performed for the aqueous phase, DMSO, ethanol Å Å and DMF. The most favourable mechanism was determined based DHBDE ¼ HðAr O ÞþHðONHÞHðAr OHÞHðON Þð4Þ on the lowest values for DH , DH , and DH . The parameters Å Å Å BDE IP PA where the H(Ar-O ), H(HOH), H(Ar-OH), H(HO ), H(ONH) and H(ON ) calculated are presented in Tables 2–4. The corresponding free are the enthalpies of the phenoxyl radical, molecule obtained after energies are listed in Tables S1–S3. hydrogen atom abstraction from the phenolic compound, starting Based on the thermodynamic values (DHBDE, and DHPA) (corre- phenolic compound and reactive radical species, respectively. sponding DG values are presented in Supplementary section, D Å Lower HBDE values, indicating increased stability of Ar-O and bet- Tables S1–S3), presented in Tables 2–4, it was clear that Dp, Pg ter antioxidant capacity of Ar-OH, can be attributed to increased and Mv reacted with hydroxyl radical via both HAT and SPLET in capacity of phenolic compound to donate hydrogen atom to hydro- all the solvents under investigation. Since all DHBDE and DHPA xyl and nitric oxide radicals. values were close both mechanisms are equally probable. The first step in SET-PT is the one-electron transfer from the According to Wang (Wang, Cao, & Prior, 1997), the highest anti- phenolic compound to the free radical which, leads to the forma- radical activity is expected for molecules with two OH groups in B Å+ tion of a phenolic radical cation (Ar-OH ) and the corresponding ring. The additional OH group, as in Dp, or the presence of a single anion: OH group and some other groups, as in Pg and Mv, decreases the Å Åþ 0 Ar OH þ HO ! Ar OH þ HO ð5Þ antiradical activity. As can be seen from Table 2, the C4 –OH group of Dp was the most favoured site for homolytic and heterolytic O–H Å Åþ 0 0 Ar OH þ ON ! Ar OH þ NO ð6Þ breaking in all solvents followed by C5 –OH, C3–OH = C3 –OH, C5– OH and C7–OH. The most reactive position in Dp, via SPLET, was 0 0 0 DHIP for the first step of the SET-PT can be calculated as follows: C4 -OH followed by C5–OH = C7–OH, C3–OH, C3 –OH and C5 –OH. Åþ Å According to literature data (Dimitric´ Markovic´, Milenkovic´, DHIP ¼ HðAr OH ÞþHðHO ÞHðAr OHÞHðHO Þð7Þ Amic´, Mojovic´, et al., 2014), the reactivity of Dp via HAT is similar 444 J.M. Dimitric´ Markovic´ et al. / Food Chemistry 218 (2017) 440–446

Table 2 Calculated reaction enthalpies (kJ mol1) for the reactions of Dp with hydroxyl and nitric oxide radicals.

M05-2X/6-311+G(d,p) Water e = 78.35 DMSO e = 46.83 Dp HAT SET-PT SPLET HAT SET-PT SPLET

DHBDE DHIP DHPDE DHPA DHETE DHBDE DHIP DHPDE DHPA DHETE 99 125 DpOH-3+ÅOH 130 228 145 15 136 260 145 10 DpOH-30+ÅOH 130 229 129 1 136 261 121 15 DpOH-40+ÅOH 157 255 163 6 166 290 176 11 DpOH-50+ÅOH 132 231 128 4 138 262 119 19 DpOH-5+ÅOH 117 216 151 34 121 245 152 31 DpOH-7+ÅOH 111 210 151 40 113 237 153 41 386 438 DpOH-3+ÅNO 162 75 8 302 155 119 4 323 DpOH-30+ÅNO 161 76 24 286 155 120 20 298 DpOH-40+ÅNO 135 103 10 293 125 149 35 324 DpOH-50+ÅNO 160 78 25 283 153 121 23 294 DpOH-5+ÅNO 175 63 2 321 170 104 11 344 DpOH-7+ÅNO 181 57 2 327 178 96 12 354 Ethanol e = 24.85 DMF e = 37.22 118 129 DpOH-3+ÅOH 129 247 153 24 135 264 148 12 DpOH-30+ÅOH 129 247 133 4 136 265 123 13 DpOH-40+ÅOH 156 274 171 16 165 294 176 11 DpOH-50+ÅOH 131 249 131 0 137 266 121 17 DpOH-5+ÅOH 115 233 159 44 120 249 154 34 DpOH-7+ÅOH 109 227 159 50 112 241 156 43 408 442 DpOH-3+ÅNO 159 97 4 314 156 123 6 325 DpOH-30+ÅNO 159 98 16 294 155 123 18 300 DpOH-40+ÅNO 133 124 22 305 126 152 35 324 DpOH-50+ÅNO 157 99 18 290 154 125 21 296 DpOH-5+ÅNO 173 84 9 333 171 108 13 347 DpOH-7+ÅNO 179 78 9 339 179 99 14 356

Table 3 Calculated reaction enthalpies (kJ mol1) for the reactions of Pg with hydroxyl and nitric oxide radicals.

M05-2X/6-311+G(d,p) Water e = 78.35 DMSO e = 46.83 Pg HAT SET-PT SPLET HAT SET-PT SPLET

DHBDE DHIP DHPDE DHPA DHETE DHBDE DHIP DHPDE DHPA DHETE 105 128 PgOH-3+ÅOH 131 236 144 13 139 267 144 5 PgOH-40+ÅOH 113 218 141 28 116 244 141 25 PgOH-5+ÅOH 118 223 150 32 122 250 149 27 PgOH-7+ÅOH 114 218 150 36 115 244 150 35 392 441 PgOH-3+ÅNO 161 83 9 300 152 126 3 318 PgOH-40+ÅNO 178 65 11 315 175 103 1 338 PgOH-5+ÅNO 173 70 2 319 169 109 8 340 PgOH-7+ÅNO 178 66 3 323 175 102 9 348 Ethanol e = 24.85 DMF e = 37.22 126 133 PgOH-3+ÅOH 130 256 153 23 138 271 144 6 PgOH-40+ÅOH 110 237 148 38 116 249 147 31 PgOH-5+ÅOH 116 242 157 41 121 254 151 30 PgOH-7+ÅOH 111 237 157 46 115 248 152 37 416 446 PgOH-3+ÅNO 159 107 3 313 153 129 2 319 PgOH-40+ÅNO 178 87 2 327 175 107 5 344 PgOH-5+ÅNO 173 92 7 331 170 113 10 343 PgOH-7+ÅNO 177 88 7 336 176 107 11 350

to the reactivity of flavones, baicalein and fisetin. Reactivity of Dp The BDEs of individual hydroxyl groups in Pg differed by up to via SPLET, in all the solvents investigated, was more probable in 20 kJ mol1, suggesting different reactivity of the individual posi- comparison with the same flavones. tions. If the reaction proceeded via HAT, C3–OH group in Pg would J.M. Dimitric´ Markovic´ et al. / Food Chemistry 218 (2017) 440–446 445

Table 4 Calculated reaction enthalpies (kJ mol1) for the reactions of Mv with hydroxyl and nitric oxide radicals.

M05-2X/6-311+G(d,p) Water e = 78.35 DMSO e = 46.83 Mv HAT SET-PT SPLET HAT SET-PT SPLET

DHBDE DHIP DHPDE DHPA DHETE DHBDE DHIP DHPDE DHPA DHETE 91 101 MvOH-40+ÅOH 149 240 150 1 149 250 142 7 MvOH-7+ÅOH 108 199 156 47 113 213 153 41 378 414 MvOH-40+ÅNO 143 87 3 288 142 108 1 307 MvOH-7+ÅNO 183 47 3 334 178 72 12 354 Ethanol e = 24.85 DMF e = 37.22 107 102 MvOH-40+ÅOH 147 254 154 8 148 251 142 -7 MvOH-7+ÅOH 106 213 162 56 114 217 156 41 3977 415 MvOH-40+ÅNO 142 104 5 298 143 109 0 306 MvOH-7+ÅNO 183 63 12 346 177 75 14 354

be the most reactive followed by C30–OH, C5–OH and C7–OH (61%). Regarding the radical scavenging sequence obtained, it is (Table 3). If the reaction proceeded via SPLET, the most reactive possible to deduce that C40-OH is functional and, most probably, (most acidic) positions in Pg, in all solvents under investigation, renders these flavonoids hydroxyl radical scavengers in advance would be C5–OH and C7–OH (Table 3). of ortho-hydroxyl or pyrogallol groups in ring B. This assumption Mv might also react with hydroxyl radical via both HAT and would be supported by Wang et al. (1997) where the highest anti- SPLET. C40–OH group was more reactive via HAT while C7–OH radical activity is expected for molecules with two OH groups in B was more reactive when the reaction proceeded via SPLET (Table 4). ring. Dp and Pg could be taken as cases ‘‘in between”. The increased Malvin has two methoxy groups at the ortho position with respect activity of Pg against hydroxyl radical correlates well with the lit- to 40-OH. This arrangement affects 40-OH BDE and PA values, erature while the somewhat reduced antiradical activity of Mv decreasing the antiradical activity of Mv in comparison with Dp could be considered a consequence of glycosylation, which reduces (Vaganek, Rimarcˇik, Dropkova, Lengyel, & Klein, 2014; Vaganek, the number of free hydroxyl sites. The higher antiradical activity of Rimarcˇik, Lukeš, & Klein, 2012)(Table 4). Comparing BDE values Dp and Pg, in comparison to Mv, could also be explained by the for baicalein and fisetin (Dimitric´ Markovic´, Milenkovic´, Amic´, influence of the 3-OH group that, when not substituted, interacts Mojovic´, et al., 2014) with BDE values for Pg and Mv, it is evident with ring B through a hydrogen bond, thus maintaining its copla- that the flavones were underwent HAT mechanism more readily. narity to rings A and C. The lower activity of Mv against hydroxyl On the other hand, PA values for Pg and Mv were significantly radicals is a consequence of reduced conjugation due to the loss lower in all solvents, indicating their activity via SPLET was of ring B coplanarity with respect to the rest of the molecule. enhanced in comparison with the flavones (Dimitric´ Markovic´, According to the ESR measurements, none of the molecules Milenkovic´, Amic´, Mojovic´, et al., 2014). showed activity towards the nitric oxide radical, which was con- SET-PT proved to be thermodynamically unfavourable for all firmed by our theoretical calculations. three molecules reacting with hydroxyl radical in all the solvents Calculated energy requirements indicate thermodynamically investigated. plausible radical scavenging mechanisms and identify active sites In the case of nitric oxide radicals, HAT reactions were for radical inactivation. Activity of each OH group is determined endothermic in all the solvents (Tables 2–4, and S1–S3), indicating based on the values of the thermodynamic parameters: BDE, PA that HAT is not possible for Dp, Pg and Mv. On the other hand, and ETE. Results indicated the effect of substitution patterns that some DHPA values for Dp, Pg and Mv reacting with nitric oxide rad- altered the activity of the molecules through different mecha- ical were slightly negative or positive indicating SPLET as hardly nisms, specifically inductive, resonance, and steric. Since all DHBDE plausible. The results point to the newly formed radical as less and DHPA values were negative and very close both HAT and SPLET stable implying the polar solvents are not suitable for Dp, Pg and were competitive in all the solvents under investigations. As indi- Mv to react with nitric oxide radicals via SPLET. cated in the literature, the number of hydroxyl groups was not These results are in good agreement with the ESR results essential, albeit important, because the antiradical activity was obtained. SET-PT was thermodynamically unfavourable for all dependent on the BDE of individual hydroxyl groups. This implies three molecules in all the solvents investigated. an efficient antiradical molecule as one having a similar relative BDE value for a certain number of hydroxyl groups. Here, our 4. Conclusions results indicated (Tables 2–4) that the values of thermodynamic parameters followed the same trends for each hydroxyl group. This An experimental and theoretical study regarding antiradical means that each hydroxyl group, acting as hydrogen-bond donor in activity of Dp, Pg and Mv was performed using ESR spectroscopy polar solvents (which is consequently responsible for the increased and M05-2X/6-311+G(d,p) level of theory. The results indicated electron density on the oxygen atom), had nearly the same BDE that different antiradical activities for the molecules investigated and PA values in all the solvents (Tables 2–4). The result are not against hydroxyl and nitric oxide radicals. According to the ESR surprising given the fact that all the solvents considered were measurements, Dp, Pg and Mv were selective towards hydroxyl polar. radical. Pg and Dp both reduced hydroxyl radical to a greater The C40–OH group of Dp was the most favoured site for homo- extent (85% and 83%, respectively) while Mv was less active lytic and heterolytic O–H breaking in protic and aprotic solvents 446 J.M. Dimitric´ Markovic´ et al. / Food Chemistry 218 (2017) 440–446

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