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Contribution of DFT computed molecular descriptors in the study of radical scavenging activity trend of natural hydroxybenzaldehydes and corresponding acids
Food Research International 48 (2012) 538–543
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Contribution of DFT computed molecular descriptors in the study of radical scavenging activity trend of natural hydroxybenzaldehydes and corresponding acids Nikolaos Nenadis ⁎, Maria Z. Tsimidou Laboratory of Food Chemistry and Technology, Aristotle University of Thessaloniki, Department of Chemistry, 541 24, Thessaloniki, Greece
a r t i c l e
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Article history: Received 1 February 2012 Accepted 9 May 2012 Keywords: Radical scavenging activity assays Bond dissociation enthalpy (BDE) Ionization potential (IP) Hydrogen atom transfer (HAT) Single electron transfer (SET) Hydroxybenzaldehydes
a b s t r a c t The present study focuses on the examination of the radical scavenging activity trend of some natural hydroxybenzaldehydes and their corresponding acids with the aid of DFT/B3LYP. Values of computed molecular descriptors (bond dissociation enthalpy, BDE; ionization potential, IP; proton affinity, PA; electron transfer energy, ETE) characterizing the hydrogen atom or electron donating efficiency of parent compounds and ions were discussed considering published experimental findings for the radical scavenging activity of the same compounds using various methods (Rancimat, crocin bleaching, trolox equivalent antioxidant capacity and 2,2-diphenyl-1picrylhydrazyl assays). Calculations in the gas and/or liquid phase (benzene, methanol, water) were performed to approximate the activity in a lipid substrate or in solution. BDE values could predict the activity of the compounds when hydrogen atom transfer prevailed. Thus, protocatechuic (1) aldehyde, syringaldehyde (2) and their respective acids (1′, 2′) were predicted to be the most efficient hydrogen atom donors, in line with Rancimat data. In polar media, where electron donation should be favored for hydroxybenzaldehydes, experimental findings could not always be supported by molecular descriptors. Combined information from various descriptors, as shown for DPPH• data, could justify the higher efficiency of 1 over that of 1′ and 2′. The results document further the use of computed molecular descriptors as a green tool that can facilitate antioxidant activity studies and selection of efficient antioxidants. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Evaluation of antioxidant activity of natural antioxidants has been thoroughly investigated in the last thirty years using in vitro and in vivo experiments (Nenadis & Tsimidou, 2010). The recommendation to use more than one assay to study the activity of antioxidants (Frankel & Meyer, 2000) often leads to conflicting results. This is due to “the complex chemistry behind antioxidant activity assays” (Huang, Ou, & Prior, 2005), which influence the order and the magnitude of efficiency of test compounds (e.g. Balk, Bast, & Haenen, 2009; Niki, 2010). Continuing the work on the usefulness of theoretical calculations in the field of antioxidant activity assessment, the present study focuses on the examination of the radical scavenging activity trend of some natural hydroxybenzaldehydes and their corresponding acids. Hydroxybenzaldehydes are known mainly for their flavoring and antimicrobial properties (Friedman, Henika, & Mandrell, 2003; Mangas, Rodriguez, Moreno, Suarez, & Blanco, 1996). Less is known for their antioxidant activity (e.g. Bors, Michel, & Saran, 1984; Bortolomeazzi, Sebastianutto, Toniolo, & Pizzariello, 2007; Setzer, 2011) in contrast to their acid counterparts (Tsimidou, Nenadis, &
⁎ Corresponding author. Tel.: + 30 2310 997731; fax: + 30 2310 997779. E-mail address: [email protected] (N. Nenadis). 0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2012.05.014
Zhang, 2006). The selected compounds (Fig. 1) were recently examined in our laboratory (Bountagkidou, Ordoudi, & Tsimidou, 2010) for their efficiency to scavenge lipid peroxyl (Rancimat test) or synthetic radicals via assays [crocin bleaching (CB), trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assays] that still raise controversial viewpoints regarding the prevailing mechanism. Suggested chemical mechanisms for each assay, experimental findings that varied among assays (Table 1) and points raised by Bountagkidou et al. (2010) are confronted using quantum chemical calculations. Molecular descriptors (Justino & Vieira, 2010) characterizing the potency of the test compounds to scavenge free radicals by the different mechanisms proposed for phenolic (AH) antioxidants (Scheme 1) namely hydrogen atom transfer, HAT [R.1], single electron transfer, SET [R.2] or by sequential proton loss followed by electron transfer, SPLET [R.3] were computed. Density functional theory (DFT) employing the Becke's 3 and Lee Yang Parr (B3LYP) hybrid functional was used as the most common approach in similar studies (Leopoldini, Russo, & Toscano, 2011). Calculations for the parent compounds were first carried out in the gas phase to obtain primary information on their activity (Leopoldini, Marino, Russo, & Toscano, 2004) in short time. Computations in the liquid phase (benzene, methanol, water) were then accomplished to approximate the activity of test compounds in a lipid substrate or in solution (e.g. Nenadis & Sigalas, 2011). The present work is expected to add to the usefulness of theoretical calculations in the antioxidant activity research area and in particular to
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Fig. 1. Structure of the test compounds.
highlight differences between antioxidant activity of closely related substances such as the hydroxybenzaldehydes and corresponding acids. Such an approach can be seen as a “green tool” that may assist selection of efficient antioxidants, reducing, thus, the cost of an experimental study and providing insight to the chemistry behind antioxidant activity assays (Nenadis & Tsimidou, 2010).
2. Theoretical calculations All calculations for compounds 1–5, 1′–5′ and phenol (used as reference) were performed by the Gaussian 03W ver. 6.0 set of programs (Frisch et al., 2003). The B3LYP functional was used for geometry optimization and computation of harmonic vibrational frequencies using the 6-31G basis set (unrestricted B3LYP for the resulting radicals). Single-point electronic energies were then obtained using the 6-311++G (2d,2p) basis set. Employing the electronic energies (298 K) at 6-311++G (2d,2p) and thermal contributions to enthalpy obtained at 6-31G, the bond dissociation enthalpy (BDE) values that characterize HAT activity were determined according to the equation: BDE = Hr + Hh − Hp [1], where Hr is the enthalpy of the radical generated by H-atom abstraction, Hh is the enthalpy of the H-atom (−0.499897 hartree at this level of theory), and Hp is the enthalpy of the parent molecule. The ionization potential (IP) values that characterize SET efficiency were determined according to the equation: IP = Ecr − Ep [2], where p and cr stand for the parent molecule and the corresponding cation radical generated after electron transfer. Proton affinity (PA) values that define the first step of SPLET mechanism were calculated according to the formula: PA = Ha + Hpr − Hp [3], where Ha is the enthalpy of the anion generated after deprotonation, Hpr is the enthalpy of the proton (0.00236 hartree), and Hp is the enthalpy of the parent
molecule. Then, electron transfer energy (ETE) or otherwise ionization potential values of the anions were obtained via the equation: ETE = Er − Ea [4], where r and a are referred to the phenoxy radical and the anion, respectively. No spin contamination was found for radicals; the bS 2> values being about 0.750 in all cases. Implicit solvent effects (benzene, methanol, and water) were taken into account with the aid of integral equation formalism of the polarized continuum model (IEF-PCM) and the united atom for Hartree Fock (UAHF) solvation radii (Barone, Cossi, & Tomasi, 1997; Cances, Mennucci, & Tomasi, 1997). Explicit solvent effects were not considered to avoid higher deviation from experimental findings (Guerra, Amorati, & Pedulli, 2004) and concomitantly a substantial increase in computational time (Kozlowski et al., 2007). All values of molecular descriptors were expressed in kcal/mol (1 hartree = 627.509 kcal/mol).
3. Results and discussion Selection of 6-31G basis set for structure optimization was based on comparison of optimized bond length and angle values in the gas phase for 4′ with experimental (crystal X-ray analysis of the monohydrate form, Colapietro, Domenicano, & Marciante, 1979) and theoretical data available (Nsangou, Dhaouadi, Jaidane, & Ben Lakhdar, 2008; Vafiadis & Bakalbassis, 2003) at B3LYP/6-31G+(d) and 6-31++G* levels of theory (Table S.1). As evidenced, 6-31G basis set could be seen as a good compromise regarding the computational economy afforded especially in the case of IEF-PCM calculations. Gas phase conformational analysis showed that the most stable conformers for 1–3, 5, 1′–3′ and 5′ possessed an intra-molecular hydrogen bond ranging from 1.762 to 2.133 Å (Fig. 2). A planar structure was evidenced for all compounds except for 2, with the methoxy
Table 1 Radical scavenging trend of test compounds in descending order of activity according to Bountagkidou et al. (2010). Bulk oil oxidationa (Rancimat test) CBAa TEACa DPPHa a
1′>1>>2′~ 2 (3–5, 3′–5′: inactive) 2>>>2′>>1′>1>>3>3′ (4, 5, 4′, 5′: inactive) 1>>2′> 1′≥3′>2>3=5~4′>5′>>4 1>>2′> 1′ (2: negligible activity, 3–5, 3′–5′: inactive)
>>>: 5.6‐fold; >>: 1.8–2.3-fold; >: 1.2–1.6-fold.
Scheme 1. Proposed mechanisms of radical scavenging of phenolic (AH) antioxidants. ROO•: peroxyl radical; HAT: hydrogen atom transfer; SET: single electron transfer; SPLET: sequential proton loss followed by electron transfer.
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group not participating in the intra-molecular hydrogen bond to deviate from the plane by ~ 45°. Investigation of the potential conformers of 2 and 2′ showed that differences in enthalpy between the two most stable ones, a planar and a one with a methoxy group deviating from the plane were marginal (0.1–0.4 kcal/mol). Thus, calculation of molecular descriptors in the gas phase was carried out for both of these conformers for 2 and 2′. In the liquid phase, a negligible difference (0.1 kcal/mol) between the two most stable conformers of 2 was found only in benzene (in methanol and water the planar conformation was more stable by 1.3 and 1.4 kcal/mol, respectively). In the case of 2′ the planar conformation was the most stable in all solvents, as its enthalpy was lower by 2.8–3.2 kcal/mol. Thus, calculations for two conformers using the IEF-PCM model were accomplished only for 2 in benzene. Computed values for molecular descriptors that could characterize HAT, SET or SPLET potency of the test compounds are presented in Tables 2–4 and within the text. Computations in water were carried out for the parent compounds (undissociated in an acidic medium, i.e. pH b3.0) and also for their ions anticipated in a slightly acidic (i.e. 3.0 b pH b 7.0) and a slightly alkaline (pH = 7.4) environment. The latter one simulates the environment in CB and TEAC assays corresponding to physiological conditions. Hydrogen atom transfer is discussed first as phenolic antioxidants retard lipid oxidation via this mechanism (Frankel, 1998). SET and SPLET related to ionization phenomena and occurring mainly in polar media (Litwinienko & Ingold, 2003; Tsimidou et al., 2006) are discussed next. 3.1. Hydrogen atom transfer (HAT) On the grounds of computed BDE values (in the gas and liquid phase) for the parent compounds (Table 2) the most efficient radical scavengers are predicted to be those bearing a catechol and a syringol moiety. The prediction was in line with experimental findings by Bountagkidou et al. (2010) as illustrated in Table 1. The rest ones, having BDE values similar or even higher (2.7–15.3 kcal/mol) than that of phenol, are expected to be inactive. The gas-phase predicted order of activity of hydroxybenzoic acids 1′–4′ was the same with that reported by Vafiadis and Bakalbassis (2003) and in line with general structure–activity relationship (SAR) principles (Tsimidou et al., 2006). The presence of a second methoxy group at C-5 (2, 2′) increased significantly activity (2, 2′ vs 3, 3′), though less efficiently than that of a second –OH group (1, 1′). Intra-molecular hydrogen bond formation between the hydroxyl and the carbonyl group of o-hydroxybenzoic compounds explains the higher BDE values over those of p-counterparts.
Table 2 B3LYP/6-311++G (2d,2p)//B3LYP/6-31G bond dissociation enthalpy (BDE) values at 298 K of test compounds and phenol in the gas phase, benzene, methanol and water (at various pH values). AH
BDE (kcal/mol) Gas-phase
Benzene
Methanol
Water pH b 3.0 3.0 b pH b 7.0 pH = 7.4
1 2 3 4 5 1′ 2′ 3′ 4′ 5′ Phenol a b c
79.9 81.0 (82.4)a 87.0 88.0 99.6 79.6 82.7 (81.0)b 87.0 88.2 99.0 84.3
80.6 80.8 (81.4)a 86.5 88.5 93.7 80.5 81.2 86.4 88.5 94.3 85.2
83.6 81.0 85.9 91.2 94.3 83.0 81.0 85.4 90.8 94.3 85.4
83.9 80.9 86.0 91.4 94.0 83.2 80.7 85.4 90.9 94.3 85.7
83.9 80.9 86.0 91.4 94.0 80.8 79.9 83.7 88.2 94.7 85.7
80.7c – – 91.4 94.0 80.8 79.9 83.7 88.2 94.7 85.7
Conformer with 0.4 kcal/mol higher energy than the global minimum. Conformer with 0.1 kcal/mol higher energy than the global minimum. O–H group at C-3.
Calculations in the liquid phase indicated an increase in the BDE values for catechol (1, 1′) and p-hydroxy (4, 4′) derivatives in methanol and water. BDE values of guaiacol and syringol derivatives were affected only slightly. As a consequence (Table 2) in protic solvents hydrogen atom donation from the –OH group at C-4 of the 1 and 1′ is less favored (BDE >2.8–3.0 kcal/mol) compared to that of 2 and 2′. Such a finding agreed with that reported for caffeic and sinapic acids in ethanol by Lithoxoidou and Bakalbassis (2004). The presence of a –CHO or –COOH group affected hydrogen atom donation of the parent compounds in the same magnitude as the electronic phenomena induced by the two groups were comparable according to Hammet-type values (σp), which are 0.42 and 0.45, respectively (Hansch, Leo, & Taft, 1991). In a slightly acidic environment ionization of the –COOH group having a pKa value ~3–4.5 (Lide, 2002) influences positively the hydrogen atom donating efficiency of hydroxybenzoic acids. An exception was 5′, as its BDE value was unaffected probably due to strong intra-molecular hydrogen bond formation. The lower BDE values predicted for 1′–4′ are related to the electron donating (σp = 0.00) properties of the –COO − group (Hansch et al., 1991). In a slightly alkaline environment (e.g. pH = 7.4) formation of phenoxy anions in aldehydes 1–3 is expected since the corresponding literature pKa values for 4-OH of these
Fig. 2. Optimized structures of the test compounds at the B3LYP/6-31G level in the gas phase.
N. Nenadis, M.Z. Tsimidou / Food Research International 48 (2012) 538–543 Table 3 B3LYP/6-311++G (2d,2p)//B3LYP/6-31G ionization potential (IP) values at 298 K of test compounds and phenol in gas phase, benzene, methanol and water (at various pH values). AH
IP (kcal/mol) Gas-phase
Benzene
1 2
193.2 178.4 (180.4)a
3 4 5 1′ 2′ 3′ 4′ 5′ Phenol
187.6 200.5 197.7 190.4 176.4 (178.2)b 184.9 198.5 194.2 191.8
a b
168.3 156.8 (159.0)a 164.9 175.6 173.7 166.1 155.8 162.6 174.1 170.8 166.5
Methanol
Water pH b 3.0
3.0b pHb 7.0
pH=7.4
141.8 136.5
140.5 135.6
140.5 135.6
115.3 109.4
140.8 150.0 149.5 139.8 135.5 139.3 149.6 147.7 141.3
140.1 148.9 149.0 138.6 134.7 138.0 147.0 152.7 140.3
140.1 148.9 149.0 131.1 128.3 131.6 138.7 136.1 140.3
114.3 148.9 149.0 131.1 128.3 131.6 138.7 136.1 140.3
Conformer with 0.4 kcal/mol higher energy than the global minimum. Conformer with 0.1 kcal/mol higher energy than the global minimum.
compounds were 7.21, 7.34 and 7.4 (Bountagkidou et al., 2010). Aldehydes 4 and 5 having a pKa value of 7.58 and 8.37 should remain intact. In this respect only 1 could perform as an efficient hydrogen atom donor via the –OH group at C-3. On the contrary, the corresponding hydroxybenzoic acids could act through HAT since their pKa values are higher than 8.67 (Bountagkidou et al., 2010). Bulk oil is a typical medium in which phenolic antioxidants act through donation of hydrogen atoms to the free radicals (Frankel, 1998). The lack of activity observed experimentally for the compounds 3–5 and 3′–5′ (Table 1) was in line with predictions discussed in the previous paragraph. Regarding the trend of activity among 1, 1′, 2 and 2′, theoretical calculations in benzene showed that the four compounds should be of equivalent potency, contrary to experimental data. Nevertheless, 1 and 1′ can donate a second hydrogen atom from the –OH at C-3, forming a quinone as illustrated for 1 in Fig. 3. Considering that the BDE values for quinone formation were 78.2 (1) and 76.5 (1′) kcal/mol, respectively, the trend of activity (1′>1>>2′~2) was in accord with that obtained with Rancimat test. A reverse satisfactory correlation of BDE values in benzene with protection factor (PF) values [PF = induction period of oil containing the antioxidant / induction period of control] was evidenced on a semilogarithmic scale (r = 0.85, p b 0.05, n = 10). An improvement was achieved when BDE values for quinone formation of 1 and 1′ were considered instead of those for the –OH group at C-4 (r = 0.92, p b 0.05, n = 10). Similar was the obtained trend using the gas-phase calculations (BDE1 = 79.0 kcal/mol, BDE1′ = 77.5 kcal/mol for quinone formation). As evident, computation of BDE values for the formation of quinones is important not only to support the superiority of catechol over syringol derivatives as scavengers, but also to explain the higher efficiency of 1′ over that of 1 (Bountagkidou et al., 2010). Such information cannot be obtained by any of the widely used in vitro experimental assays. Table 4 B3LYP/6-311++G (2d,2p)//B3LYP/6-31G proton affinity (PA) values at 298 K of test compounds in methanol. ΑΗ
PA (kcal/mol)
ΑΗ
PA (kcal/mol)
1 2 3 4 5
282.8a/286.4b 286.5 286.3 286.4 292.0
1′ 2′ 3′ 4′ 5′
284.5a/287.2b 288.1 288.2 287.9 295.0
a b
O–H at C-4. O–H at C-3.
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The CB assay, based on competition between crocin and antioxidants for the reaction with peroxyl radicals, is used in the literature to assess HAT efficiency of test compounds (Apak et al., 2007; Huang et al., 2005). In this view, only 1 could scavenge efficiently the peroxyl radicals (Table 2); a prediction that contradicts the lower efficiency of 1 over that of 2 observed experimentally (Table 1). Formation of phenoxy ions at C-4 of 1–3 suggests that scavenging of peroxyl radicals should take place via a different mechanism, questioning that CB assays follow a clear HAT mechanism. Regarding the ABTS•+ assay, reviews on antioxidant activity methods are rather confusing whether scavenging via HAT (Roginsky & Lissi, 2005) or ET (Apak et al., 2007; Huang et al., 2005) mechanism prevails. Reaction environment in the assay is the same as that of CB. Therefore 2 and 3 should be non reactive due to the lack of available –OH. However, all of the test compounds were found to present some activity (Table 1). The superiority of 1 over that of other compounds was not predictable through BDE values. In the case of hydroxybenzoic acids the experimental trend of activity on the basis of TEAC values was in accord with that obtained by BDE ones (r=0.96, pb 0.05, n=5). Such a finding adds to the observation of Tyrakowska et al. (1999) that the efficiency of the monoanionic forms of hydroxybenoic acids (deprotonated carboxylic group) is determined by HAT under the ABTS•+ assay conditions. Greater is the confusion about the mechanism of scavenging behind the DPPH• assay (Apak et al., 2007; Huang et al., 2005; Litwinienko & Ingold, 2003; Nenadis & Tsimidou, 2002; Roginsky & Lissi, 2005). On the basis of BDE values 3–5 and 3′–5′ were predicted to be ineffective in agreement with experimental data. The observed activity for 1, 1′ and 2′ on the basis of EC50 values {where EC50: the [test compound] required to scavenge 50% of [DPPH•]} or the negligible activity of 2, could not be justified in terms of BDE values. Using kinetic data [e.g. antiradical efficiency (ΑΕ) values defined as 1 / EC50 × TEC50; TEC50 = reaction time needed to reach the steady state at EC50 in min], 1′ and 2′ should act via HAT. Syringic acid (AE = 0.42 ± 0.01) was more reactive than protocatechuic acid (AE = 0.28 ± 0.02) in accord with the lower BDE value. Even so, BDE values could not explain the higher reactivity of 1 (AE = 0.78 ± 0.06) and the inferiority of 2. 3.2. Single electron transfer (SET) IP values (Table 3) suggested that 2, 2′, 3 and 3′ bearing methoxy group/s could act as electron donors given that the computed values were lower than that of phenol. The presence of an aldehyde group in the aromatic ring raised IP values by 0.4–3.5 kcal/mol when compared to that of a carboxylic one. Unlike BDE values, those of ΙΡ seem to be more influenced by the solvent polarity since the latter may affect charge separation in a molecule (Tsimidou et al., 2006). The higher ΙΡ values in benzene in comparison to those in the two polar solvents suggest that SET is not favored in the hydrocarbon modeling the bulk oil. Changes in pH are expected to differentiate the electron donation of the test compounds because ionization of the carboxylic and hydroxyl groups at C-4 can take place. The electron donating properties of the carboxylate anion in slightly acidic environment (3.0 b pH b 7.0) result in a decrease of the IP values by 6.4–16.6 kcal/mol. The presence of an aldehyde group does not favor radical scavenging in comparison to that of a carboxylic group at the above range of pH values. In slightly alkaline pH (e.g. 7.4) the formation of phenoxy anions at C4 for aldehydes 1–3 causes a significant decrease in IP values (~25 kcal/mol). This is due to the strong electron donating character of the –O − (σp = −0.81) over that of the –OH (σp = − 0.37). In this case activity of 1–3 could be ascribed to SET mechanism. Still, 1, except of an electron, can also donate a hydrogen atom to the free radicals as discussed earlier. The above findings on the electron donating efficiency of aldehydes 1–3 add to the observations of other investigators (Bortolomeazzi et al., 2007; Ordoudi & Tsimidou, 2006) on the reactivity of some phenols
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Fig. 3. Proposed mechanism of radical scavenging by protocatechuic aldehyde in bulk oil (BDE values are computed in benzene).
bearing ionizable groups using CB assay. Similar comments on SET instead of HAT could be made for the respective aldehydes in the case of the TEAC assay considering data on ionization–efficiency relationship of a series of hydroxyflavones presented by Lemańska et al. (2001). Even so, the higher efficiency of 1 could not be fully justified. In the DPPH• assay, SET is not likely to be favored since 1, being the most efficient in the kinetic approach (Bountagkidou et al., 2010), was predicted to be inactive according to the computed IP value. 3.3. Sequential proton loss followed by electron transfer (SPLET) To further explore activity under the DPPH• assay conditions, the possible contribution of phenolates, which could affect kinetics of the reaction with the radical (Litwinienko & Ingold, 2003) was investigated. Deprotonation of phenolic –OH group/s in methanol was studied by computing proton affinity (PΑ) values (Table 4). Ionization was found to be easier for aldehydes in methanol as their PA values were lower by 1.5–3.0 kcal/mol than those of the respective acids. Partial contribution of phenolates to DPPH• scavenging could be hypothesized, especially for 1. The latter seems to be more prone to the formation of a phenoxy anion than all of the other test compounds (PA1 b1.7– 12.2 kcal/mol). Assuming formation of 1 anion, the corresponding ETE value (115.0 kcal/mol) in methanol was lower by 20–24.8 kcal/mol than the IP values of the unionized acids in the same solvent. This supports the higher reactivity of 1 from that of 1′ and 2′ in terms of kinetics (AE values). Even so, the negligible activity of 2 (~40% radical scavenging on a 3.5-fold higher concentration than that of DPPH•) found by Bountagkidou et al. (2010) was not explained. Recently, Hristea et al. (2009) reported in dichloromethane the formation of a new free radical (42% yield) during the reaction of DPPH• and the phenoxy anion of 2, with an overlapping spectra (λmax = 548 nm) to that of DPPH• (λmax = 515 nm). Phenoxy anion formation for 2 seems more feasible in methanol as computed PA value is 7.0 kcal/mol lower than that in dichloromethane (286.5 instead of 293.5 kcal/mol). To this view, formation of phenolates could possibly account for the negligible activity of 2 with the DPPH• assay (Table 1). However, this cannot be fully justified as ions of 1′ and 3 should also be formed (Table 4) which are more efficient electron donors (ETE values computed in methanol: 113.8 and 113.6 kcal/mol, respectively) than 1 anion. 4. Conclusions On the basis of BDE values it was predicted that 1 and 2 should be of comparable or equal activity to their undissociated acid counterparts when HAT mechanism prevails. Furthermore, the respective compounds should be the most efficient ones from the group studied. Thus, the predicted trend in activity was in line with experimental findings in bulk oil accelerated oxidation. In polar media, where ionization of –OH at C-4 is expected for 1–3, BDE could not be used for activity prediction. Still, it was useful to predict the activity trend of hydroxybenzoic acids. The values of other molecular descriptors characterizing electron donation of parent hydroxybenzaldehydes or their ions could not always support experimental findings. The deviations observed should be related to the complex mechanism involved in the assays. Characteristic was the case of DPPH• data, which could
not be explained by a particular molecular descriptor. Combined information from various descriptors could support the higher efficiency of 1 over that of 1′ and 2′. The negligible activity found for 2 with DPPH• assay was not justified by any molecular descriptor or the data from the other in vitro assays. The present results support further that the radical scavenging efficiency of 1 and 2 should be taken into account for their further exploitation as multi-potent additives by the food industry. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.foodres.2012.05.014. References Apak, R., Güçlü, K., Demirata, B., Özyürek, M., Çelik, S. E., Bektaşoğlu, B., et al. (2007). Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay. Molecules, 12, 1496–1547. Balk, J. M., Bast, A., & Haenen, G. R. M. M. (2009). Evaluation of the accuracy of antioxidant competition assays: Incorrect assumptions with major impact. Free Radical Biology & Medicine, 47, 135–144. Barone, V., Cossi, M., & Tomasi, J. (1997). A new definition of cavities for the computation of solvation free energies by the polarizable continuum model. The Journal of Chemical Physics, 107, 3210–3221. Bors, W., Michel, C., & Saran, M. (1984). Inhibition of the bleaching of the carotenoid crocin. A rapid test for quantifying antioxidant activity. Biochimica et Biophysica Acta, 796, 312–319. Bortolomeazzi, R., Sebastianutto, N., Toniolo, R., & Pizzariello, A. (2007). Comparative evaluation of the antioxidant capacity of smoke flavouring phenols by crocin bleaching inhibition, DPPH radical scavenging and oxidation potential. Food Chemistry, 100, 1481–1489. Bountagkidou, O. G., Ordoudi, S. A., & Tsimidou, M. Z. (2010). Structure–antioxidant activity relationship study of natural hydroxybenzaldehydes using in vitro assays. Food Research International, 43, 2014–2019. Cances, E., Mennucci, B., & Tomasi, J. (1997). A new integral equation formalism for the polarizable continuum model: Theoretical background and applications to isotropic and anisotropic dielectrics. The Journal of Chemical Physics, 107, 3032–3041. Colapietro, M., Domenicano, A., & Marciante, C. (1979). Structural studies of benzene derivatives VI. Refinement of the crystal structure of p-hydroxybenzoic acid monohydrate. Acta Crystallographica. Section B, 35, 2177–2180. Frankel, E. N. (Ed.). (1998). Lipid oxidation. Dundee, Scotland: The Oily Press. Frankel, E. N., & Meyer, A. S. (2000). The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. Journal of the Science of Food and Agriculture, 80, 1925–1941. Friedman, M., Henika, P. R., & Mandrell, R. E. (2003). Antibacterial activities of phenolic benzaldehydes and benzoic acids against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. Journal of Food Protection, 66, 1811–1821. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., et al. (2003). Gaussian 03. Pittsburgh, PA: Gaussian, Inc.. Guerra, M., Amorati, R., & Pedulli, G. F. (2004). Water effect on the O–H dissociation enthalpy of para-substituted phenols: A DFT study. Journal of Organic Chemistry, 69, 5460–5467. Hansch, C., Leo, A., & Taft, R. W. (1991). A survey of Hammett substituent constants and resonance and field parameters. Chemical Reviews, 91, 165–195. Hristea, E. N., Covaci-Cîmpeanu, I. C., Ionit, G., Ionit, P., Draghici, P., Caăproiu, M. T., et al. (2009). Reactions of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with two syringylic phenols or one aroxide derivative. European Journal of Organic Chemistry, 626, 626–634. Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53, 1841–1856. Justino, G. C., & Vieira, A. J. S. C. (2010). Antioxidant mechanisms of quercetin and myricetin in the gas phase and in solution — a comparison and validation of semi-empirical methods. Journal of Molecular Modelling, 16, 863–876. Kozlowski, D., Marsal, P., Steel, M., Mokrini, R., Duroux, J. L., Lazzaroni, R., et al. (2007). Theoretical investigation of the formation of a new series of antioxidant depsides from the radiolysis of flavonoid compounds. Radiation Research, 168, 243–252. Lemańska, K., Szymusiak, H., Tyrakowska, B., Zieliński, R., Soffers, A. E., & Rietjens, I. M. (2001). The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones. Free Radical Biology & Medicine, 31, 869–881.
N. Nenadis, M.Z. Tsimidou / Food Research International 48 (2012) 538–543 Leopoldini, M., Marino, T., Russo, N., & Toscano, M. (2004). Antioxidant properties of phenolic compounds: H-atom versus electron transfer mechanism. Journal of Physical Chemistry A, 108, 4916–4922. Leopoldini, M., Russo, N., & Toscano, M. (2011). The molecular basis of working mechanism of natural polyphenolic antioxidants. Food Chemistry, 125, 288–306. Lide, D. R. (Ed.). (2002). CRC handbook of chemistry and physics (pp. 610). (82nd ed.). London, UK: Chapman & Hall/CRC Press. Lithoxoidou, A. T., & Bakalbassis, E. G. (2004). Liquid-phase theoretical antioxidant activity trend of some cinnamic acid antioxidants. Journal of the American Oil Chemists' Society, 81, 799–802. Litwinienko, G., & Ingold, K. U. (2003). Abnormal solvent effects on hydrogen atom abstraction. 1. The reactions of phenols with 2,2-diphenyl-1-picrylhydrazyl (DPPH•) in alcohols. Journal of Organic Chemistry, 68, 3433–3438. Mangas, J., Rodriguez, R., Moreno, J., Suarez, B., & Blanco, D. (1996). Evolution of aromatic and furanic congeners in the maturation of cider brandy: A contribution to its characterization. Journal of Agricultural and Food Chemistry, 44, 3303–3307. Nenadis, N., & Sigalas, M. P. (2011). A DFT study on the radical scavenging potential of selected natural 3′,4′-dihydroxy aurones. Food Research International, 43, 2014–2019. Nenadis, N., & Tsimidou, M. (2002). Observations on the estimation of scavenging activity of phenolic compounds using rapid 1,1-diphenyl-2-picrylhydrazyl (DPPH•) tests. Journal of the American Oil Chemists' Society, 79, 1191–1194. Nenadis, N., & Tsimidou, M. Z. (2010). Assessing the activity of natural food antioxidants. In E. Decker, R. Elias, & D. J. McClements (Eds.), Oxidation in foods and beverages and antioxidant applications: Understanding mechanisms of oxidation and antioxidant activity (volume 1), part 2 antioxidants in foods and beverages (pp. 332–367). Cambridge, UK: Woodhead Ltd. Ch. 14.
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Contribution of DFT computed molecular descriptors in the study of radical scavenging activity trend of natural hydroxybenzaldehydes and corresponding acids Nikolaos Nenadis ⁎, Maria Z. Tsimidou Laboratory of Food Chemistry and Technology, Aristotle University of Thessaloniki, Department of Chemistry, 541 24, Thessaloniki, Greece
a r t i c l e
i n f o
Article history: Received 1 February 2012 Accepted 9 May 2012 Keywords: Radical scavenging activity assays Bond dissociation enthalpy (BDE) Ionization potential (IP) Hydrogen atom transfer (HAT) Single electron transfer (SET) Hydroxybenzaldehydes
a b s t r a c t The present study focuses on the examination of the radical scavenging activity trend of some natural hydroxybenzaldehydes and their corresponding acids with the aid of DFT/B3LYP. Values of computed molecular descriptors (bond dissociation enthalpy, BDE; ionization potential, IP; proton affinity, PA; electron transfer energy, ETE) characterizing the hydrogen atom or electron donating efficiency of parent compounds and ions were discussed considering published experimental findings for the radical scavenging activity of the same compounds using various methods (Rancimat, crocin bleaching, trolox equivalent antioxidant capacity and 2,2-diphenyl-1picrylhydrazyl assays). Calculations in the gas and/or liquid phase (benzene, methanol, water) were performed to approximate the activity in a lipid substrate or in solution. BDE values could predict the activity of the compounds when hydrogen atom transfer prevailed. Thus, protocatechuic (1) aldehyde, syringaldehyde (2) and their respective acids (1′, 2′) were predicted to be the most efficient hydrogen atom donors, in line with Rancimat data. In polar media, where electron donation should be favored for hydroxybenzaldehydes, experimental findings could not always be supported by molecular descriptors. Combined information from various descriptors, as shown for DPPH• data, could justify the higher efficiency of 1 over that of 1′ and 2′. The results document further the use of computed molecular descriptors as a green tool that can facilitate antioxidant activity studies and selection of efficient antioxidants. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Evaluation of antioxidant activity of natural antioxidants has been thoroughly investigated in the last thirty years using in vitro and in vivo experiments (Nenadis & Tsimidou, 2010). The recommendation to use more than one assay to study the activity of antioxidants (Frankel & Meyer, 2000) often leads to conflicting results. This is due to “the complex chemistry behind antioxidant activity assays” (Huang, Ou, & Prior, 2005), which influence the order and the magnitude of efficiency of test compounds (e.g. Balk, Bast, & Haenen, 2009; Niki, 2010). Continuing the work on the usefulness of theoretical calculations in the field of antioxidant activity assessment, the present study focuses on the examination of the radical scavenging activity trend of some natural hydroxybenzaldehydes and their corresponding acids. Hydroxybenzaldehydes are known mainly for their flavoring and antimicrobial properties (Friedman, Henika, & Mandrell, 2003; Mangas, Rodriguez, Moreno, Suarez, & Blanco, 1996). Less is known for their antioxidant activity (e.g. Bors, Michel, & Saran, 1984; Bortolomeazzi, Sebastianutto, Toniolo, & Pizzariello, 2007; Setzer, 2011) in contrast to their acid counterparts (Tsimidou, Nenadis, &
⁎ Corresponding author. Tel.: + 30 2310 997731; fax: + 30 2310 997779. E-mail address: [email protected] (N. Nenadis). 0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2012.05.014
Zhang, 2006). The selected compounds (Fig. 1) were recently examined in our laboratory (Bountagkidou, Ordoudi, & Tsimidou, 2010) for their efficiency to scavenge lipid peroxyl (Rancimat test) or synthetic radicals via assays [crocin bleaching (CB), trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assays] that still raise controversial viewpoints regarding the prevailing mechanism. Suggested chemical mechanisms for each assay, experimental findings that varied among assays (Table 1) and points raised by Bountagkidou et al. (2010) are confronted using quantum chemical calculations. Molecular descriptors (Justino & Vieira, 2010) characterizing the potency of the test compounds to scavenge free radicals by the different mechanisms proposed for phenolic (AH) antioxidants (Scheme 1) namely hydrogen atom transfer, HAT [R.1], single electron transfer, SET [R.2] or by sequential proton loss followed by electron transfer, SPLET [R.3] were computed. Density functional theory (DFT) employing the Becke's 3 and Lee Yang Parr (B3LYP) hybrid functional was used as the most common approach in similar studies (Leopoldini, Russo, & Toscano, 2011). Calculations for the parent compounds were first carried out in the gas phase to obtain primary information on their activity (Leopoldini, Marino, Russo, & Toscano, 2004) in short time. Computations in the liquid phase (benzene, methanol, water) were then accomplished to approximate the activity of test compounds in a lipid substrate or in solution (e.g. Nenadis & Sigalas, 2011). The present work is expected to add to the usefulness of theoretical calculations in the antioxidant activity research area and in particular to
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Fig. 1. Structure of the test compounds.
highlight differences between antioxidant activity of closely related substances such as the hydroxybenzaldehydes and corresponding acids. Such an approach can be seen as a “green tool” that may assist selection of efficient antioxidants, reducing, thus, the cost of an experimental study and providing insight to the chemistry behind antioxidant activity assays (Nenadis & Tsimidou, 2010).
2. Theoretical calculations All calculations for compounds 1–5, 1′–5′ and phenol (used as reference) were performed by the Gaussian 03W ver. 6.0 set of programs (Frisch et al., 2003). The B3LYP functional was used for geometry optimization and computation of harmonic vibrational frequencies using the 6-31G basis set (unrestricted B3LYP for the resulting radicals). Single-point electronic energies were then obtained using the 6-311++G (2d,2p) basis set. Employing the electronic energies (298 K) at 6-311++G (2d,2p) and thermal contributions to enthalpy obtained at 6-31G, the bond dissociation enthalpy (BDE) values that characterize HAT activity were determined according to the equation: BDE = Hr + Hh − Hp [1], where Hr is the enthalpy of the radical generated by H-atom abstraction, Hh is the enthalpy of the H-atom (−0.499897 hartree at this level of theory), and Hp is the enthalpy of the parent molecule. The ionization potential (IP) values that characterize SET efficiency were determined according to the equation: IP = Ecr − Ep [2], where p and cr stand for the parent molecule and the corresponding cation radical generated after electron transfer. Proton affinity (PA) values that define the first step of SPLET mechanism were calculated according to the formula: PA = Ha + Hpr − Hp [3], where Ha is the enthalpy of the anion generated after deprotonation, Hpr is the enthalpy of the proton (0.00236 hartree), and Hp is the enthalpy of the parent
molecule. Then, electron transfer energy (ETE) or otherwise ionization potential values of the anions were obtained via the equation: ETE = Er − Ea [4], where r and a are referred to the phenoxy radical and the anion, respectively. No spin contamination was found for radicals; the bS 2> values being about 0.750 in all cases. Implicit solvent effects (benzene, methanol, and water) were taken into account with the aid of integral equation formalism of the polarized continuum model (IEF-PCM) and the united atom for Hartree Fock (UAHF) solvation radii (Barone, Cossi, & Tomasi, 1997; Cances, Mennucci, & Tomasi, 1997). Explicit solvent effects were not considered to avoid higher deviation from experimental findings (Guerra, Amorati, & Pedulli, 2004) and concomitantly a substantial increase in computational time (Kozlowski et al., 2007). All values of molecular descriptors were expressed in kcal/mol (1 hartree = 627.509 kcal/mol).
3. Results and discussion Selection of 6-31G basis set for structure optimization was based on comparison of optimized bond length and angle values in the gas phase for 4′ with experimental (crystal X-ray analysis of the monohydrate form, Colapietro, Domenicano, & Marciante, 1979) and theoretical data available (Nsangou, Dhaouadi, Jaidane, & Ben Lakhdar, 2008; Vafiadis & Bakalbassis, 2003) at B3LYP/6-31G+(d) and 6-31++G* levels of theory (Table S.1). As evidenced, 6-31G basis set could be seen as a good compromise regarding the computational economy afforded especially in the case of IEF-PCM calculations. Gas phase conformational analysis showed that the most stable conformers for 1–3, 5, 1′–3′ and 5′ possessed an intra-molecular hydrogen bond ranging from 1.762 to 2.133 Å (Fig. 2). A planar structure was evidenced for all compounds except for 2, with the methoxy
Table 1 Radical scavenging trend of test compounds in descending order of activity according to Bountagkidou et al. (2010). Bulk oil oxidationa (Rancimat test) CBAa TEACa DPPHa a
1′>1>>2′~ 2 (3–5, 3′–5′: inactive) 2>>>2′>>1′>1>>3>3′ (4, 5, 4′, 5′: inactive) 1>>2′> 1′≥3′>2>3=5~4′>5′>>4 1>>2′> 1′ (2: negligible activity, 3–5, 3′–5′: inactive)
>>>: 5.6‐fold; >>: 1.8–2.3-fold; >: 1.2–1.6-fold.
Scheme 1. Proposed mechanisms of radical scavenging of phenolic (AH) antioxidants. ROO•: peroxyl radical; HAT: hydrogen atom transfer; SET: single electron transfer; SPLET: sequential proton loss followed by electron transfer.
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group not participating in the intra-molecular hydrogen bond to deviate from the plane by ~ 45°. Investigation of the potential conformers of 2 and 2′ showed that differences in enthalpy between the two most stable ones, a planar and a one with a methoxy group deviating from the plane were marginal (0.1–0.4 kcal/mol). Thus, calculation of molecular descriptors in the gas phase was carried out for both of these conformers for 2 and 2′. In the liquid phase, a negligible difference (0.1 kcal/mol) between the two most stable conformers of 2 was found only in benzene (in methanol and water the planar conformation was more stable by 1.3 and 1.4 kcal/mol, respectively). In the case of 2′ the planar conformation was the most stable in all solvents, as its enthalpy was lower by 2.8–3.2 kcal/mol. Thus, calculations for two conformers using the IEF-PCM model were accomplished only for 2 in benzene. Computed values for molecular descriptors that could characterize HAT, SET or SPLET potency of the test compounds are presented in Tables 2–4 and within the text. Computations in water were carried out for the parent compounds (undissociated in an acidic medium, i.e. pH b3.0) and also for their ions anticipated in a slightly acidic (i.e. 3.0 b pH b 7.0) and a slightly alkaline (pH = 7.4) environment. The latter one simulates the environment in CB and TEAC assays corresponding to physiological conditions. Hydrogen atom transfer is discussed first as phenolic antioxidants retard lipid oxidation via this mechanism (Frankel, 1998). SET and SPLET related to ionization phenomena and occurring mainly in polar media (Litwinienko & Ingold, 2003; Tsimidou et al., 2006) are discussed next. 3.1. Hydrogen atom transfer (HAT) On the grounds of computed BDE values (in the gas and liquid phase) for the parent compounds (Table 2) the most efficient radical scavengers are predicted to be those bearing a catechol and a syringol moiety. The prediction was in line with experimental findings by Bountagkidou et al. (2010) as illustrated in Table 1. The rest ones, having BDE values similar or even higher (2.7–15.3 kcal/mol) than that of phenol, are expected to be inactive. The gas-phase predicted order of activity of hydroxybenzoic acids 1′–4′ was the same with that reported by Vafiadis and Bakalbassis (2003) and in line with general structure–activity relationship (SAR) principles (Tsimidou et al., 2006). The presence of a second methoxy group at C-5 (2, 2′) increased significantly activity (2, 2′ vs 3, 3′), though less efficiently than that of a second –OH group (1, 1′). Intra-molecular hydrogen bond formation between the hydroxyl and the carbonyl group of o-hydroxybenzoic compounds explains the higher BDE values over those of p-counterparts.
Table 2 B3LYP/6-311++G (2d,2p)//B3LYP/6-31G bond dissociation enthalpy (BDE) values at 298 K of test compounds and phenol in the gas phase, benzene, methanol and water (at various pH values). AH
BDE (kcal/mol) Gas-phase
Benzene
Methanol
Water pH b 3.0 3.0 b pH b 7.0 pH = 7.4
1 2 3 4 5 1′ 2′ 3′ 4′ 5′ Phenol a b c
79.9 81.0 (82.4)a 87.0 88.0 99.6 79.6 82.7 (81.0)b 87.0 88.2 99.0 84.3
80.6 80.8 (81.4)a 86.5 88.5 93.7 80.5 81.2 86.4 88.5 94.3 85.2
83.6 81.0 85.9 91.2 94.3 83.0 81.0 85.4 90.8 94.3 85.4
83.9 80.9 86.0 91.4 94.0 83.2 80.7 85.4 90.9 94.3 85.7
83.9 80.9 86.0 91.4 94.0 80.8 79.9 83.7 88.2 94.7 85.7
80.7c – – 91.4 94.0 80.8 79.9 83.7 88.2 94.7 85.7
Conformer with 0.4 kcal/mol higher energy than the global minimum. Conformer with 0.1 kcal/mol higher energy than the global minimum. O–H group at C-3.
Calculations in the liquid phase indicated an increase in the BDE values for catechol (1, 1′) and p-hydroxy (4, 4′) derivatives in methanol and water. BDE values of guaiacol and syringol derivatives were affected only slightly. As a consequence (Table 2) in protic solvents hydrogen atom donation from the –OH group at C-4 of the 1 and 1′ is less favored (BDE >2.8–3.0 kcal/mol) compared to that of 2 and 2′. Such a finding agreed with that reported for caffeic and sinapic acids in ethanol by Lithoxoidou and Bakalbassis (2004). The presence of a –CHO or –COOH group affected hydrogen atom donation of the parent compounds in the same magnitude as the electronic phenomena induced by the two groups were comparable according to Hammet-type values (σp), which are 0.42 and 0.45, respectively (Hansch, Leo, & Taft, 1991). In a slightly acidic environment ionization of the –COOH group having a pKa value ~3–4.5 (Lide, 2002) influences positively the hydrogen atom donating efficiency of hydroxybenzoic acids. An exception was 5′, as its BDE value was unaffected probably due to strong intra-molecular hydrogen bond formation. The lower BDE values predicted for 1′–4′ are related to the electron donating (σp = 0.00) properties of the –COO − group (Hansch et al., 1991). In a slightly alkaline environment (e.g. pH = 7.4) formation of phenoxy anions in aldehydes 1–3 is expected since the corresponding literature pKa values for 4-OH of these
Fig. 2. Optimized structures of the test compounds at the B3LYP/6-31G level in the gas phase.
N. Nenadis, M.Z. Tsimidou / Food Research International 48 (2012) 538–543 Table 3 B3LYP/6-311++G (2d,2p)//B3LYP/6-31G ionization potential (IP) values at 298 K of test compounds and phenol in gas phase, benzene, methanol and water (at various pH values). AH
IP (kcal/mol) Gas-phase
Benzene
1 2
193.2 178.4 (180.4)a
3 4 5 1′ 2′ 3′ 4′ 5′ Phenol
187.6 200.5 197.7 190.4 176.4 (178.2)b 184.9 198.5 194.2 191.8
a b
168.3 156.8 (159.0)a 164.9 175.6 173.7 166.1 155.8 162.6 174.1 170.8 166.5
Methanol
Water pH b 3.0
3.0b pHb 7.0
pH=7.4
141.8 136.5
140.5 135.6
140.5 135.6
115.3 109.4
140.8 150.0 149.5 139.8 135.5 139.3 149.6 147.7 141.3
140.1 148.9 149.0 138.6 134.7 138.0 147.0 152.7 140.3
140.1 148.9 149.0 131.1 128.3 131.6 138.7 136.1 140.3
114.3 148.9 149.0 131.1 128.3 131.6 138.7 136.1 140.3
Conformer with 0.4 kcal/mol higher energy than the global minimum. Conformer with 0.1 kcal/mol higher energy than the global minimum.
compounds were 7.21, 7.34 and 7.4 (Bountagkidou et al., 2010). Aldehydes 4 and 5 having a pKa value of 7.58 and 8.37 should remain intact. In this respect only 1 could perform as an efficient hydrogen atom donor via the –OH group at C-3. On the contrary, the corresponding hydroxybenzoic acids could act through HAT since their pKa values are higher than 8.67 (Bountagkidou et al., 2010). Bulk oil is a typical medium in which phenolic antioxidants act through donation of hydrogen atoms to the free radicals (Frankel, 1998). The lack of activity observed experimentally for the compounds 3–5 and 3′–5′ (Table 1) was in line with predictions discussed in the previous paragraph. Regarding the trend of activity among 1, 1′, 2 and 2′, theoretical calculations in benzene showed that the four compounds should be of equivalent potency, contrary to experimental data. Nevertheless, 1 and 1′ can donate a second hydrogen atom from the –OH at C-3, forming a quinone as illustrated for 1 in Fig. 3. Considering that the BDE values for quinone formation were 78.2 (1) and 76.5 (1′) kcal/mol, respectively, the trend of activity (1′>1>>2′~2) was in accord with that obtained with Rancimat test. A reverse satisfactory correlation of BDE values in benzene with protection factor (PF) values [PF = induction period of oil containing the antioxidant / induction period of control] was evidenced on a semilogarithmic scale (r = 0.85, p b 0.05, n = 10). An improvement was achieved when BDE values for quinone formation of 1 and 1′ were considered instead of those for the –OH group at C-4 (r = 0.92, p b 0.05, n = 10). Similar was the obtained trend using the gas-phase calculations (BDE1 = 79.0 kcal/mol, BDE1′ = 77.5 kcal/mol for quinone formation). As evident, computation of BDE values for the formation of quinones is important not only to support the superiority of catechol over syringol derivatives as scavengers, but also to explain the higher efficiency of 1′ over that of 1 (Bountagkidou et al., 2010). Such information cannot be obtained by any of the widely used in vitro experimental assays. Table 4 B3LYP/6-311++G (2d,2p)//B3LYP/6-31G proton affinity (PA) values at 298 K of test compounds in methanol. ΑΗ
PA (kcal/mol)
ΑΗ
PA (kcal/mol)
1 2 3 4 5
282.8a/286.4b 286.5 286.3 286.4 292.0
1′ 2′ 3′ 4′ 5′
284.5a/287.2b 288.1 288.2 287.9 295.0
a b
O–H at C-4. O–H at C-3.
541
The CB assay, based on competition between crocin and antioxidants for the reaction with peroxyl radicals, is used in the literature to assess HAT efficiency of test compounds (Apak et al., 2007; Huang et al., 2005). In this view, only 1 could scavenge efficiently the peroxyl radicals (Table 2); a prediction that contradicts the lower efficiency of 1 over that of 2 observed experimentally (Table 1). Formation of phenoxy ions at C-4 of 1–3 suggests that scavenging of peroxyl radicals should take place via a different mechanism, questioning that CB assays follow a clear HAT mechanism. Regarding the ABTS•+ assay, reviews on antioxidant activity methods are rather confusing whether scavenging via HAT (Roginsky & Lissi, 2005) or ET (Apak et al., 2007; Huang et al., 2005) mechanism prevails. Reaction environment in the assay is the same as that of CB. Therefore 2 and 3 should be non reactive due to the lack of available –OH. However, all of the test compounds were found to present some activity (Table 1). The superiority of 1 over that of other compounds was not predictable through BDE values. In the case of hydroxybenzoic acids the experimental trend of activity on the basis of TEAC values was in accord with that obtained by BDE ones (r=0.96, pb 0.05, n=5). Such a finding adds to the observation of Tyrakowska et al. (1999) that the efficiency of the monoanionic forms of hydroxybenoic acids (deprotonated carboxylic group) is determined by HAT under the ABTS•+ assay conditions. Greater is the confusion about the mechanism of scavenging behind the DPPH• assay (Apak et al., 2007; Huang et al., 2005; Litwinienko & Ingold, 2003; Nenadis & Tsimidou, 2002; Roginsky & Lissi, 2005). On the basis of BDE values 3–5 and 3′–5′ were predicted to be ineffective in agreement with experimental data. The observed activity for 1, 1′ and 2′ on the basis of EC50 values {where EC50: the [test compound] required to scavenge 50% of [DPPH•]} or the negligible activity of 2, could not be justified in terms of BDE values. Using kinetic data [e.g. antiradical efficiency (ΑΕ) values defined as 1 / EC50 × TEC50; TEC50 = reaction time needed to reach the steady state at EC50 in min], 1′ and 2′ should act via HAT. Syringic acid (AE = 0.42 ± 0.01) was more reactive than protocatechuic acid (AE = 0.28 ± 0.02) in accord with the lower BDE value. Even so, BDE values could not explain the higher reactivity of 1 (AE = 0.78 ± 0.06) and the inferiority of 2. 3.2. Single electron transfer (SET) IP values (Table 3) suggested that 2, 2′, 3 and 3′ bearing methoxy group/s could act as electron donors given that the computed values were lower than that of phenol. The presence of an aldehyde group in the aromatic ring raised IP values by 0.4–3.5 kcal/mol when compared to that of a carboxylic one. Unlike BDE values, those of ΙΡ seem to be more influenced by the solvent polarity since the latter may affect charge separation in a molecule (Tsimidou et al., 2006). The higher ΙΡ values in benzene in comparison to those in the two polar solvents suggest that SET is not favored in the hydrocarbon modeling the bulk oil. Changes in pH are expected to differentiate the electron donation of the test compounds because ionization of the carboxylic and hydroxyl groups at C-4 can take place. The electron donating properties of the carboxylate anion in slightly acidic environment (3.0 b pH b 7.0) result in a decrease of the IP values by 6.4–16.6 kcal/mol. The presence of an aldehyde group does not favor radical scavenging in comparison to that of a carboxylic group at the above range of pH values. In slightly alkaline pH (e.g. 7.4) the formation of phenoxy anions at C4 for aldehydes 1–3 causes a significant decrease in IP values (~25 kcal/mol). This is due to the strong electron donating character of the –O − (σp = −0.81) over that of the –OH (σp = − 0.37). In this case activity of 1–3 could be ascribed to SET mechanism. Still, 1, except of an electron, can also donate a hydrogen atom to the free radicals as discussed earlier. The above findings on the electron donating efficiency of aldehydes 1–3 add to the observations of other investigators (Bortolomeazzi et al., 2007; Ordoudi & Tsimidou, 2006) on the reactivity of some phenols
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Fig. 3. Proposed mechanism of radical scavenging by protocatechuic aldehyde in bulk oil (BDE values are computed in benzene).
bearing ionizable groups using CB assay. Similar comments on SET instead of HAT could be made for the respective aldehydes in the case of the TEAC assay considering data on ionization–efficiency relationship of a series of hydroxyflavones presented by Lemańska et al. (2001). Even so, the higher efficiency of 1 could not be fully justified. In the DPPH• assay, SET is not likely to be favored since 1, being the most efficient in the kinetic approach (Bountagkidou et al., 2010), was predicted to be inactive according to the computed IP value. 3.3. Sequential proton loss followed by electron transfer (SPLET) To further explore activity under the DPPH• assay conditions, the possible contribution of phenolates, which could affect kinetics of the reaction with the radical (Litwinienko & Ingold, 2003) was investigated. Deprotonation of phenolic –OH group/s in methanol was studied by computing proton affinity (PΑ) values (Table 4). Ionization was found to be easier for aldehydes in methanol as their PA values were lower by 1.5–3.0 kcal/mol than those of the respective acids. Partial contribution of phenolates to DPPH• scavenging could be hypothesized, especially for 1. The latter seems to be more prone to the formation of a phenoxy anion than all of the other test compounds (PA1 b1.7– 12.2 kcal/mol). Assuming formation of 1 anion, the corresponding ETE value (115.0 kcal/mol) in methanol was lower by 20–24.8 kcal/mol than the IP values of the unionized acids in the same solvent. This supports the higher reactivity of 1 from that of 1′ and 2′ in terms of kinetics (AE values). Even so, the negligible activity of 2 (~40% radical scavenging on a 3.5-fold higher concentration than that of DPPH•) found by Bountagkidou et al. (2010) was not explained. Recently, Hristea et al. (2009) reported in dichloromethane the formation of a new free radical (42% yield) during the reaction of DPPH• and the phenoxy anion of 2, with an overlapping spectra (λmax = 548 nm) to that of DPPH• (λmax = 515 nm). Phenoxy anion formation for 2 seems more feasible in methanol as computed PA value is 7.0 kcal/mol lower than that in dichloromethane (286.5 instead of 293.5 kcal/mol). To this view, formation of phenolates could possibly account for the negligible activity of 2 with the DPPH• assay (Table 1). However, this cannot be fully justified as ions of 1′ and 3 should also be formed (Table 4) which are more efficient electron donors (ETE values computed in methanol: 113.8 and 113.6 kcal/mol, respectively) than 1 anion. 4. Conclusions On the basis of BDE values it was predicted that 1 and 2 should be of comparable or equal activity to their undissociated acid counterparts when HAT mechanism prevails. Furthermore, the respective compounds should be the most efficient ones from the group studied. Thus, the predicted trend in activity was in line with experimental findings in bulk oil accelerated oxidation. In polar media, where ionization of –OH at C-4 is expected for 1–3, BDE could not be used for activity prediction. Still, it was useful to predict the activity trend of hydroxybenzoic acids. The values of other molecular descriptors characterizing electron donation of parent hydroxybenzaldehydes or their ions could not always support experimental findings. The deviations observed should be related to the complex mechanism involved in the assays. Characteristic was the case of DPPH• data, which could
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