Kaempferol, one of the most bioactive plant flavonoids was theoretically (at M05-2X/6-311G(d,p) level of theory) investigated for its ability scavange potentially highly damaging hydroxyl and superoxide anion radicals. Theoretical calculations point to HAT and SPLET mechanisms as operative for kaempferol in all solvents under investigations.Teoretski (na M05-2X/6-311G(d,p) nivou teorije) je ispitivana sposobnost kempferola, važnog bioaktivnog biljnog jedinjenja flavonoida, da reaguje sa potencijalno veoma štetnim hidroksi radikalom i superoksid radikal anjonom. Proračuni su pokazali da su HAT i SPLET mogući mehanizmi u svim rastvaračima

Dimitrić-Marković, Jasmina

Djorović, J

Jeremić, S

Marković, Zoran

Milenković, D

English

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“XIX  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 19.(21), 2014.  287 INVESTIGATION OF ANTIOXIDATIVE MECHANISAMS OF KAEMPFEROL WITH HYDROXYL RADICAL AND SUPEROXIDE RADICAL ANION  D. Milenković1, Z. Marković2, J.Dimitrić Marković3, S. Jeremić2, J. Đorović1  Abstract: Kaempferol, one of the most bioactive plant flavonoids was theoretically (at M05-2X/6-311G(d,p) level of theory) investigated for its ability scavange potentially highly damaging hydroxyl and superoxide anion radicals.  Theoretical calculations point to HAT and SPLET mechanisms as operative for kaempferol in all solvents under investigations.   Key words: kaempferol, hyrdoxyl radical, superoxide anion radical, HAT and SPLET mechanisms  Introduction  Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one) (Fig.1) is a natural flavonoid that can be found mainly in broccoli, tea, kale, ginko, cabbage, endive, leek, beans, tomato, strawberries, grapes and many medical herbs used in traditional medicine (Calderón-Montaño et al., 2011).  The present paper aims to provide quantitative tools to thoroughly and comprehensively determine antiradical mechanisms of kaempferol by calculating the energy requirements of the reactions of kaempferol and selected radical species in different media. Calculated energy requirements may indicate which radical scavenging mechanism is thermodynamically preferred and point out active sites for radical inactivation.  Material and methods  The conformations of different kaempferol forms (neutral, radical, radical-cation, and anion) are fully optimized by the new local density functional method (M05-2X), developed by the Truhlar group (Zhao and Truhlar, 2008) and 6-311G(d,p) basis set (Shakila et al, 2011; Taşal and Kumalar, 2013) implemented in the Gaussian 09 package (Frisch et al., 2009). Potential energy surfaces are obtained in relation to the torsion angle τ between the rings B and C, defined by the O1–C2–C1'–C2' atoms (Figure 1). The torsion angle τ was scanned in steps of 10° without constrains on all other geometrical parameters.                                                   1 Bioengineering Research and Development Center , Prvoslava Stojanovića 6, 34000 Kragujevac, Republic of Serbia (deki82@kg.ac.rs) 2 Department of Chemical-Technological Sciences, State  University of  Novi Pazar, Vuka Karadžića bb, 36300 Novi Pazar, Republic of Serbia (zmarkovic@np.ac.rs) 3 Faculty of Physical Chemistry, University of  Beograd, Studentski trg 12-16, 11 000 Beograd, Republic of Serbia (markovich@ffh.bg.ac.rs) “XIX  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 19.(21), 2014.  288  Slika 1. Hemijska struktura kempferola Figure 1. Chemical structure of kaempferol  In order to examine the influence of different radicals to an antiradical mechanism of the most stable kaempferol conformer (conformer I) (Fig. 2), the reactive particle RO• was  introduced. In the present paper this particle represents superoxide anion and hydroxyl radicals. In HAT mechanism, the hydrogen atom is transferred from phenolic compound to the free radical RO: K−OH + RO → K−O + ROH                         (1) ΔHBDE for the HAT mechanism can be calculated using the following equation: ΔHBDE=H( KO) + H(ROH) - H( K−OH) - H(RO)                       (2) where the H( KO), H(ROH), H( K−OH), and H(RO) are the enthalpies of the phenolic radical, molecule obtained after hydrogen atom abstraction from the phenolic compound, starting phenolic compound, and reactive radical species, respectively.  The first step in the SET-PT mechanism is transfer of an electron from phenolic compound to free radical species, yielding the phenolic radical cation Ph−OH+ and corresponding anion.    K−OH + RO →  K−OH++ RO−                                                                                                     (3) ΔHIP for the first step of the SET-PT mechanism can be calculated as follows: ΔHIP= H( K−OH+) + H(RO−) - H( K−OH) - H(RO)         (4) where the H( K−OH+) and H(RO−) are the enthalpies of the radical cation of initial phenolic compound and anion generated from the corresponding initial radical. The second step of this mechanism is deprotonation of  K−OH+ by RO−: K−OH++ RO− →  K−O + ROH                                                                     (5) ΔHPDE can be calculated using the following equation:  ΔHPDE= H( K−O) + H(ROH) - H( K−OH+) - H(RO−)         (6) The first step in the SPLET mechanism is deprotonation of phenolic compound by RO− or other base. The outcome of this reaction is the formation of the phenoxide anion  K−O−: K−OH + RO− →  K−O−  + ROH                                                                      (7) ΔHPA can be calculated as follows: ΔHPA = H( K−O−) + H(ROH) - H( K−OH) - H(RO−)                                     (8) “XIX  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 19.(21), 2014.  289 In the next step electron transfer from  K−O− to RO takes place: K−O− + RO →  K−O + RO−                                                                                                                  (9)  ΔHETE can be determined by the equation: ΔHETE = H( K−O) + H(RO−) - H( K−O−) - H(RO)                                     (10)  The potential antiradical activity of kaempferol, for each reactive site (OH group), is simulated in the reactions with hydroxyl (OH) and superoxide anion (OO−) radicals, the radical species among the most important in biological and food chemistry. Reaction enthalpies for the reaction of kaempferol with selected radicals, related to three mechanisms of free radical scavenging activity (HAT, SET-PT and SPLET), are calculated using M05-2X/6-311G (d, p) model.  Results and discussion  To determine the preferred relative positions of the rings B and C, conformational space of kaempferol structure is investigated as a function of torsional angle τ(O1–C2–C1'–C2') between those rings, in gas-phase (Fig. 2). The lowest energy structure (conformer I) is characterized by a torsional C3-C2-C1′-C2′ angle of 7.8° which indicates that rings B and C are almost planar. Another conformer II (dihedral angle of 172.5°) is less stable, for 0.62 kJ/mol, than the conformer I (Fig. 2).   Slika 2. Energetski profil za rotacionu barijeru oko C2–C1' za strukturu kempferola u gasovitoj fazi Figure 2. Energy profile for rotation barrier around the inner bond C2–C1' for structure kaempferol in gas phase  The preferred mechanism of antiradical activity of kaempferol can be estimated from ΔHBDE, ΔHIP, and ΔHPA values. Namely, the lowest of these values indicates which mechanism is favorable.    “XIX  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 19.(21), 2014.  290 Tabela 1. Izračunate reakcione entalpije (kJ/mol) za reakciju kempferola sa hidroksi radikalom i superoksid radikal anjonom Table 1. Calculated reaction enthalpies (kJ/mol) for the reactions of kaempherol with hydroxyl radical and  superoxide radical anion  M05-2X/6-311G(d,p)  Water (ε=78.35) DMSO (ε=46.83) Kaempferol HAT SET-PT SPLET HAT SET-PT SPLET  ΔHBDE ΔHIP ΔHPDE ΔHPA ΔHETE ΔHBDE ΔHIP ΔHPDE ΔHPA ΔHETE   99     242    KOH-3 + .OH -148  -247 -127 -21 -139  -380 -219 80 KOH-4’ + .OH -125  -223 -127 3 -119  -361 -230 111 KOH-5 + .OH -95  -194 -123 28 -76  -317 -207 131 KOH-7 + .OH -95  -194 -141 46 -91  -333 -245 153   373     692    KOH-3 + .OO- 66  -307 19 46 103  -588 -24 128 KOH-4’ + .OO- 89  -284 19 70 123  -569 -36 158 KOH-5 + .OO- 118  -254 23 96 166  -526 -12 179 KOH-7 + .OO- 118  -254 5 114 151  -541 -50 201  Ethanol (ε=24.85) DMF (ε=37.22)   135     244    KOH-3 + .OH -149  -284 -149 -1 -138  -383 -222 83 KOH-4’ + .OH -128  -263 -151 23 -119  -363 -231 112 KOH-5 + .OH -95  -230 -144 49 -76  -320 -207 131 KOH-7 + .OH -97  -232 -165 68 -91  -335 -245 154   454     697    KOH-3 + .OO- 74  -379 12 62 103  -594 -28 131 KOH-4’ + .OO- 96  -358 10 86 122  -575 -37 159 KOH-5 + .OO- 129  -325 17 112 166  -531 -13 179 KOH-7 + .OO- 127  -327 -5 131 150  -547 -51 201  On the basis of the values in Table 1, it is clear that kaempferol reacts with hydroxyl radical via both, HAT and SPLET mechanisms in all solvents. For protic polar solvents (water and ethanol), HAT and SPLET are competitive mechanisms, while in other two aprotic polar solvents (DMSO and DMF), SPLET is the prevailing mechanism. It should be also noted that hydrogen atom is abstracted mainly from 3-OH group on the C ring when reaction takes place via HAT mechanism. The 7-OH group in the ring A is dominant position for reaction via SPLET mechanism in all solvents. In the reaction with superoxide anion radical kaempferol (Table 1) undergoes SPLET mechanism which is dominant in all solvents while the reaction by HAT mechanism is endothermic in all solvents. Moreover, the aprotic polar solvents (DMF and DMSO) are more suitable medium for reactions of kaempferol with superoxide anion radical.    “XIX  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 19.(21), 2014.  291 Conclusion  Reaction enthalpies are calculated using the M052X/6-311G(d,p) level of theory. In protic polar solvents (water and ethanol) kaempferol reacts with hydroxyl radical via competitive HAT and SPLET mechanisms, while in aprotic polar solvents (DMSO and DMF) SPLET prevails.  Preferred sites of action are C3-OH (via HAT) and C7-OH (via  SPLET) functionals. Aprotic polar solvents (DMF and DMSO) are more favorable for the interaction with superoxide anion radical. In those solvents kaempferol undergoes SPLET mechanism.  Acknowledgment  The authors acknowledge financial support of the Ministry of Science of the Republic of Serbia, grants No. 172015 and 174028.        References  Calderón-Montaño J.M., Burgos-Morón E., Pérez-Guerrero C., López-Lázaro M. (2011). A Review on the Dietary Flavonoid Kaempferol. Mini Reviews in Medicinal Chemistry. 11(4): 298-344. Frisch M. J., Trucks G. W., Schlegel H. B., et al. (2009). Gaussian 09, Revision C.01, Gaussian, Inc., 2009. Wallingford, CT, USA. Shakila G., Periandy S., Ramalingam S. (2011). Molecular Structure and Vibrational Analysis of 1-Bromo-2-Chlorobenzene Using ab initio HF and Density Functional Theory (B3LYP) Calculations. Journal Journal of Atomic, Molecular, and Optical Physics. 2011: 1-10. Taşal E., Kumalar M. (2013). Ab initio HF, DFT and experimental (FT-IR) investigation of vibrational spectroscopy of 3-(2-(4-isopropylpiperazin-1-yl)-2-oxoethyl)-6-(4-methoxybenzoyl)benzo[d]thiazol-2(3H)-one. Spectrochimica Acta. Part A: Molecular and Biomolecular Spectroscopy. 101: 204–217 Zhao Y., Truhlar D.G. (2008). The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals. Theoretical Chemistry Accounts. 120 (1-3): 215-241.          “XIX  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 19.(21), 2014.  292 ISPITIVANJE ANTIOKSIDATIVNIH MEHANIZAMA  KEMPFEROLA SA HIDROKSI RADIKALOM I SUPEROKSID RADIKAL ANJONOM  D. Milenković1, Z. Marković2, J.Dimitrić Marković3, S. Jeremić2, J. Đorović1  Izvod: Teoretski (na M05-2X/6-311G(d,p) nivou teorije) je ispitivana sposobnost kempferola, važnog bioaktivnog biljnog jedinjenja flavonoida,  da reaguje sa potencijalno veoma štetnim hidroksi radikalom i superoksid radikal anjonom.  Proračuni su pokazali da su HAT i SPLET mogući mehanizmi u svim rastvaračima.   Ključne reči: kempferol; hidroksi radikal, superoksid radikal anjon, HAT i SPLET mehanizmi                                                                         1 Centar za istraživanje i razvoj bioinženjeringa, Prvoslava Stojanovića 6, 34000 Kragujevac, Republika Srbija (deki82@kg.ac.rs) 2 Departman za hemijsko-tehnološke nauke, Državni Univerzitet u Novom Pazaru, Vuka Karadžića bb, 36300 Novi Pazar, Republika Srbija (zmarkovic@np.ac.rs) 3 Fakultet za fizičku hemiju, Univerzitet u Beogradu, Studentski trg 12-16, 11 000 Beograd, Republika Srbija (markovich@ffh.bg.ac.rs)  

Ispitivanje antioksidativnih mehanizama kempferola sa hidroksi radikalom i superoksid radikal anjonom

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Ispitivanje antioksidativnih mehanizama kempferola sa hidroksi radikalom i superoksid radikal anjonom

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