Food Chemistry 151 (2014) 198–206

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Food Chemistry

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Density functional theory study of the structure–antioxidant activityof polyphenolic deoxybenzoins

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.11.064

⇑ Corresponding authors at: School of Pharmacy, Xuzhou Medical College, No.209, Tongshan Road, Xuzhou, Jiangsu 221004, China. Tel.: +86 516 83262137 (Y.Xue), +86 516 83262136 (Y. Liu).

E-mail addresses: xueyunsheng@xzmc.edu.cn, xzmcysxue@sina.com (Y. Xue),njuliuyi2003@gmail.com (Y. Liu).

Yunsheng Xue a,b,⇑, Youguang Zheng a,b, Lin An a,b, Yunyan Dou b, Yi Liu a,b,⇑a Chemical and Biological Pharmaceutical Engineering Research Center, Xuzhou Medical College, No. 209, Tongshan Road, Xuzhou, Jiangsu 221004, Chinab School of Pharmacy, Xuzhou Medical College, No. 209, Tongshan Road, Xuzhou, Jiangsu 221004, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 August 2013Received in revised form 15 October 2013Accepted 13 November 2013Available online 20 November 2013

Keywords:DeoxybenzoinsAntioxidant mechanismDFTHATSET–PTSPLET

Quantum chemical calculations based on the density functional theory (DFT) have been employed tostudy the relationship between the structure and the antioxidant activity of four polyphenolic deoxy-benzoins (DOBs) in solvents and the gas phase. The three main working mechanisms, H-atom transfer(HAT), single electron transfer–proton transfer (SET–PT) and sequential proton loss electron transfer(SPLET) have been investigated. The calculated results closely matched experimental values. The resultsobtained prove that for the HAT mechanism, the most efficient system possessed ortho-dihydroxy func-tionality. The results suggested that HAT would be the most favourable mechanism for explaining theradical-scavenging activity of polyphenolic DOBs in the gas phase, whereas the SPLET mechanism isthe thermodynamically favourable pathway in polar solvents.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Antioxidant therapies have been recognised as potential strate-gies for preventing acute CNS injury, cardiovascular diseases andasthma (Cuzzocrea, Riley, Caputi, & Salvemini, 2001; Gilgun-Sher-ki, Rosenbaum, Melamed, & Offen, 2002; Kirkham & Rahman,2006). In addition, applications of antioxidants as preservative inthe food industry (Winkler, Frick, Schroecksnadelm, Schennach, &Fuchs, 2006) and skin-protective ingredients in cosmetics are alsoreceiving increasing attention and interests (Lupo, 2001). Deoxy-benzoins (DOBs) are intermediates in the synthesis of isoflavoneand are found in several plants, such as Glycyrrhiza sp., Trifoliumsubterraneum and Ononis spinosa, and some marine sources (Kiuchi,Chen, & Tsuda, 1990; Niwa et al., 1999; Sanduja, Weinheimer, &Alam, 1985; Wahala & Hase, 1991). As important derivatives of iso-flavones, deoxybenzoins exhibit a broad variety of biological activ-ities, including antibacterial (Li, Xue, Shi, Gui, & Zhu, 2008), ureaseinhibitory (Xiao et al., 2007) and estrogenic (Fokialakis et al., 2004)activity. The basic skeleton of DOBs possesses similar structuralcharacteristic with dihydrochalcone and stilbene (Fig. 1).

Due to their structural similarities with known natural antioxi-dants, such as resveratrol (stilbene) (Queiroz, Gomes, Moraes, &

Borges, 2009) and phloretin (dihydrochalcone) (Nakamura, Watan-abe, Miyake, Kohno, & Osawa, 2003), DOBs show great potential fordevelopment as antioxidants. Indeed, recently, 2,3,4,40-tetrahydr-oxydeoxybenzoin (THDB) was synthesized and the scavenging ef-fect on hydroxy radical was detected by fluorescentspectrophotometry by Yan, Wu, and Zheng (2005). The experimen-tal results showed that THDB can scavenge hydroxy radical effec-tively. On the other hand, Ng, Ko, and Lu (2009) synthesizedseventeen polyphenolic DOBs and evaluated their antioxidantactivity by various antioxidant assays. Their experimental resultsdisplayed that these DOBs are powerful antioxidants; for example,2,3,4-trihydroxy-30,40-dimethoxydeoxybenzoin possesses an excel-lent anti-lipid peroxidation activity (IC50 = 0.72 ± 0.16 lM), whilst2,4,40,5-tetrahydroxydeoxybenzoin showed the most potent DPPHradical scavenging activity (IC50 = 0.69 ± 0.04 lM), which werebetter than Trolox (IC50 = 9.20 ± 0.44 lM) and vitamin C(IC50 = 73.69 ± 1.76 lM). All DOBs tested showed a weak metalchelating effect suggesting that the anti-lipid peroxidation effectsof these compounds were achieved via radical scavenging ratherthan the metal chelating property.

The antioxidant activity of the phenolic antioxidants is relatedto their structures. It has been reported that the scavenging activityof phenolic antioxidants is related to the phenolic O–H bond disso-ciation enthalpy (BDE), adiabatic ionisation potential (IP), protondissociation enthalpy (PDE), proton affinity (PA) and electrontransfer enthalpy (ETE). Recently, theoretical methods especiallydensity functional theory (DFT) method, have been successfully

(a)

O4

HO3

HO2O1H O5H1

3 5A

B2

4

67 8

910

1112

1314

DOB1

O

HO

OHOH

OH

DOB2

O

HO

OHOH

DOB3

O

HO

OHOCH3

DOB4(b)

O

dihydrochalcone stilbene

Fig. 1. (a) Structure and atomic number of the four studied hydroxydeoxybenzoin(DOB1–DOB4) and (b) the basic representation of two analogues of DOB, dihydr-ochalcone and stilbene.

Y. Xue et al. / Food Chemistry 151 (2014) 198–206 199

used to calculated physiochemical descriptors, such as BDE and IPof polyphenolic compounds and to elucidate the structure–activityrelationship (SAR) for phenolic antioxidants (Kozlowski et al.,2007; Leopoldini, Russo, & Toscano, 2011; Mazzone, Malaj, Russo,& Toscano, 2013; Mikulski, Gorniak, & Molski, 2010; Sadasivama& Kumaresan, 2011; Stepanic, Trošelj, Lucic, Markovic, & Amic,2013; Trouillas, Marsal, Siri, Lazzaroni, & Duroux, 2006; Wright,Johnson, & DiLabio, 2001), However, to the best of our knowledge,no theoretical study regarding the antioxidant activity of DOBs hasbeen reported so far.

Continuing our work on the usefulness of theoretical calcula-tions in the field of antioxidant activity assessment (Xue et al.,2012, 2013a, 2013b), the present study focuses on the examinationof the radical scavenging activity trend of four selected polypheno-lic DOBs (Fig. 1a). In this study, the structural and electronicproperties of the selected polyphenolic DOBs and their radicalswere investigated at DFT level. Our aim is to employ the B3LYP/6–311++G(2d,2p)//B3LYP/6–31G(d,p) level of theory to performthe reliable optimization of the geometrical parameters of thesecompounds and to calculate physiochemical descriptors character-ising their antioxidant ability, including O–H BDE, IP, PDE, PA andETE. Moreover, the highest occupied molecular orbital (HOMO) andthe lowest unoccupied molecular orbital (LUMO) distribution andspin density in free radicals were also calculated. These calcula-tions may illustrate the SAR and radical scavenging mechanismof polyphenolic DOBs, and it will be of benefit to investiagte anddevelop these antioxidants.

2. Materials and methods

2.1. Theoretical and computational methods

In the literature, three main antioxidant mechanisms are pro-posed and widely analysed (Leopoldini et al., 2011; Stepanicet al., 2013; Wright et al., 2001). The first one is referred to ashydrogen atom transfer (HAT, Eq. (1)), in which a free radical R� re-moves a hydrogen atom from the antioxidant ArOH. The secondmechanism is referred to as single electron transfer–proton trans-fer (SET–PT, Eq. (2)), where electron abstraction from ArOH is

followed by proton transfer. The third mechanism is the sequentialproton loss electron transfer (SPLET, Eq. (3)) mechanism; protonloss is followed by electron transfer. All of these mechanismsmay occur in parallel, but with different rates.

R� þ ArOH ! þArO� ðEq:1Þ

R� þ ArOH ! R� þ ArOHþ� ! RHþ ArO� ðEq:2Þ

ArOH ! ArO� þH�

ArO� þ R� ! ArO� þ R� ðEq:3Þ

R� þHþ ! RH

In HAT, the reactivity of an ArOH can be estimated by calculat-ing the O–H bond dissociation enthalpy (BDE), where the lower theBDE value the higher the expected activity. The SET–PT mechanismis described by ionisation potential (IP) and proton dissociation en-thalpy (PDE) from the ArOH�+ radical cation. Molecules with low IPand PDE values are expected to have high activity. The reaction en-thalpy of the first step in the SPLET mechanism corresponds to theproton affinity (PA) of the phenoxide anion, ArO�. The reaction en-thalpy of the following step, abstraction of electron, was denotedas electron transfer enthalpy, ETE. Thus, in the present studyBDE, IP and PA values were used as the main molecular descriptorsin an effort to elucidate the radical scavenging activity of com-pounds under investigation.

From the calculated total enthalpies at 298.15 K, we have deter-mined following quantities:

BDE ¼ HðArO�Þ þHðH�Þ �HðArOHÞ ð1Þ

IP ¼ HðArOH�þÞ þHðe�Þ �HðArOHÞ ð2Þ

PDE ¼ HðArO�Þ þHðHþÞ �HðArOH�þÞ ð3Þ

PA ¼ HðArO�Þ þHðHþÞ �HðArOHÞ ð4Þ

ETE ¼ HðArO�Þ þHðe�Þ �HðArO�Þ: ð5Þ

The molecular enthalpy at 298 K (H) consisted of B3LYP/6–311++G(2d,2p) calculated single-point energy (SPE) values andB3LYP/6–31G(d,p) thermal contributions to enthalpy (TCE, inwhich the vibrational contributions include zero-point vibrationalenergy). To calculate the half reaction enthalpies, the enthalpies ofH-atom, proton, and electron in the gas phase and solvents areneeded. The gas phase enthalpy of hydrogen atom predicted usingthe B3LYP/6–311++G(2d,2p) method is �312.956 kcal/mol (Nena-dis & Tsimidou, 2012). The gas phase enthalpy of proton and elec-tron are 1.483 and 0.752 kcal/mol, respectively (Bartmess, 1994).Proton and electron solvation enthalpies were taken from Rim-arcik, Lukes, Klein, and Ilcin (2010), and hydrogen atom solvationenthalpies from Parker (1992) and Bizarro, Costa Cabral, dos San-tos, and Martinho Simões (1999).

All computations were performed using the GAUSSIAN 09 soft-ware package (Frisch et al., 2009). The B3LYP functional was usedfor geometry optimization and computation of harmonic vibra-tional frequencies using the 6–31G(d,p) basis set (unrestrictedB3LYP for the resulting radicals). Single-point electronic energieswere then obtained using the 6–311++G(2d,2p) basis set. For alloptimised structures, we calculated the harmonic vibrational fre-quencies of the corresponding bonds to determine stationarypoints on the potential energy surface.

The HOMO and LUMO orbital distribution were determined byusing the B3LYP/6–31G(d,p) level of theory for the fully optimisedstructure of the compounds. In addition, the spin density for each

Table 1B3LYP/6–311++G(2d,2p)//B3LYP/6–31G(d,p) bond dissociation enthalpy (BDE) valuesof test compounds and reference compounds in gas phase and solvents.

Moleculea BDE (kcal/mol) IC50 (lM)b

Gas Benzene Methanol Water

200 Y. Xue et al. / Food Chemistry 151 (2014) 198–206

atom of the radicals studied was computed using the unrestrictedB3LYP/6–311++G(2d,2p) level of theory. Implicit solvent effects(benzene, methanol and water) were taken into account with theaid of integral equation formalism of the polarised continuummodel (IEF-PCM) (Tomasi, Mennucci, & Cammi, 2005).

DOB1 5.69 ± 0.692-OH 91.0 90.8 88.2 85.83-OH 77.4 78.0 76.4 74.14-OH 81.7 82.5 81.4 79.140-OH 82.8 83.7 82.7 80.5DOB2 0.69 ± 0.042-OH 94.0 93.2 89.5 87.34-OH 76.2 78.1 78.5 76.35-OH 71.2 73.3 74.1 71.940-OH 82.9 83.8 82.7 80.4DOB3 25.20 ± 2.212-OH 103.1 103.8 102.5 100.24-OH 88.1 89.4 88.9 86.740-OH 82.8 83.7 82.7 80.5DOB4 57.40 ± 5.082-OH 103.1 103.8 102.5 100.24-OH 88.0 89.3 88.9 86.7Phenol 83.5 84.5 83.7 81.4Ascorbic acid 73.9 75.5 75.2 73.0 73.69 ± 1.76Trolox 73.0 73.9 72.9 70.7 9.20 ± 0.44

a DOB1, 2,3,4,40-tetrahydroxydeoxybenzoin; DOB2, 2,4,5,40-tetrahydroxydeoxy-benzoin; DOB3, 2,4,40-trihydroxydeoxybenzoin; DOB4, 2,4-dihydroxy-40-methoxydeoxybenzoin.

b 50% Inhibition concentration of DPPH assay, see Ng, Ko and Lu (2009).

3. Results and discussion

3.1. The optimised geometries of DOBs and their radicals

Conformational analysis of DOBs using molecular mechanicsand DFT calculations revealed that each molecule can adopt threelow energy conformations stabilized by the formation of a hydro-gen bond between 2-OH and the carbonyl oxygen (Fig. S1). Con-formers I (E1 = �916.8644241 hartree) and II (E2 = �916.8642772hartree) are bended and further stabilized through the hydropho-bic interaction of the phenyl rings, while conformer III(E3 = �916.8631379 hartree), with a relative energy of about0.8 kcal/mol compared to the global minimum conformer I, exhib-its an extended shape with respect to other two conformers. In thefollowing study, we only consider the most stable conformer I.

The computational method (B3LYP/6–31G(d,p)) selected forstructure optimization was evaluated by comparing calculatedoptimised bond length and angle values with experimental ones(crystal X-ray) for 2,3,4,40-tetrahydroxydeoxybenzoin (DOB1)(Yan et al., 2005). Some selected calculated and experimentalstructural parameters are reported in Table S1 in the SupportingInformation. In general, and taking into account that the experi-mental data were obtained by crystal X-ray analysis and not inthe gas phase, one can observe quite a satisfactory agreement.Bond lengths agree within 0.063 Å, while the largest deviation ofbond angles appears to be about 3�. Thus, the selected methodwas considered accurate and it was applied in the structure opti-mization. The optimised structure and the geometrical parametersfor neutral molecules and their radicals are given in Fig. S2 andTable S2. An intramolecular hydrogen bond (IHB) between the2-hydroxylic hydrogen and the carbonylic oxygen was found ineach molecule (Fig. S2). The resulting hydrogen bond lengths forDOB1–DOB4 are about 1.63 Å. From the bond distances and bondangles data, it can be seen that no significant geometrical changewas observed when going from the neutral molecule to the phen-oxy (ArO�) and cation (ArOH+�) radicals and the anion forms (ArO�).

3.2. HAT mechanism

The computed BDE values in the gas phase and solvents are re-ported in Table 1. The most active system is able to act through theH-atom transfer mechanism both in the gas phase and in solutionwith the smallest BDE. From Table 1, it can be seen that DOB1 hasthe lowest BDE in 3-OH, while the lowest BDE was found for 5-OHin DOB2. 40-OH and 4-OH has the lowest BDE for DOB3 and DOB4,respectively. The BDEs for DOB1–DOB4 in the gas phase can bearranged in the following order: 3-OH < 4-OH < 40-OH < 2-OH,5-OH < 4-OH < 40-OH < 2-OH, 40-OH < 4-OH < 2-OH and 4-OH <2-OH, respectively. The same sequence was obtained for BDEs inthe three solvents studied. This clearly confirms that H-atomabstraction from 2-OH is more difficult than other OH groups. Thisresult can be attribute to the IHB between 2-OH and the nearbyC4 = O group. In such case, hydrogen atom abstraction requiresalso the IHB disruption.

In order to rationalise the differences in BDE and consequentlythe differences in the reactivity of the OH sites, the assessment ofspin density distribution was undertaken on the free radicals ofDOBs (Fig. 2). The more delocalized the spin density in the radical,the easier the radical is formed and thus the lower the BDE. For

both DOB1 and DOB2, the spin population appears to be slightlymore delocalized for radicals issued from the A-ring (3-OH, 4-OHand 5-OH) than for those located on the B-ring (40-OH). For exam-ple, the spin density is 0.328 on the oxygen atom in the 4-OH rad-ical for DOB1, whereas it is 0.407 for the 40-OH radical. As aconsequence, the BDE is lower for the OH in the A-ring than thatin the B-ring for DOB1 and DOB2. On the contrary, in the case ofDOB3, the spin density appears to be more delocalized for theradicals originating from the B-ring (40-OH) than that located onthe A-ring (4-OH). The spin population is 0.406 on the O-atom inthe 40-OH radical species whereas it is 0.410 in the 4-OH radical.This in turn results in a lower BDE value of the B-ring than thatof the A-ring. For both DOB3 and DOB4, the spin density is highlycondensed on the O-atom (0.588 for DOB3 and 0.586 for DOB4) forthe 2-OH radicals. This could explain why the BDE values for 2-OHin DOB3 and DOB4 are significantly larger than those of otherradicals.

In the present study, compounds DOB1 and DOB2, bearingortho-dihydroxy groups on the aromatic A ring, presented lowerBDE values (71.2–78.0 kcal/mol), in comparison to those of theother two compounds bearing no such groups (80.5–89.3 kcal/mol). For DOB1 and DOB2, if H atom is abstracted from one ofOH groups located at neighbouring carbon atoms, relativelystrong O–H� � �O hydrogen bond is formed that stabilizes thephenoxy radical. Weaker hydrogen bonding is also present in par-ent molecule with ortho-dihydroxy structure. The presence ofthese hydrogen bonds results in an approximate 3 kcal/mol de-crease in O–H BDE.

Table 1 clearly shows that the BDEs declined in the orderDOB4 > DOB3 > DOB1 > DOB2 in all studied environments, so thesequence of hydrogen donating ability is: DOB2 > DOB1 > DOB3> DOB4. This is consistent with the experimental results from theDPPH assay (Ng et al., 2009). Moreover, the sequence indicates thatfrom a thermodynamic point of view, the HAT mechanism is morefavourable for molecules with ortho-dihydroxy moieties (DOB1and DOB2). When the 40-OH group is replaced by the OMe group(DOB3 vs. DOB4), no effect on the remaining OH groups BDEs couldbe observed. As shown in Table 1, the BDEs of 4-OH and 2-OH for

O

HO

OHO

DOB1-R2

HO

-0.122

0.309

-0.035

0.102

0.013

0.329

-0.026 0.002

0.001-0.0010.0280.339

0.095

-0.009

O

HO

OH

OH

O

DOB1-R3

0.200-0.102

0.281

-0.071

0.1740.049 -0.012

-0.019

0.005

0.010

0.003

-0.007-0.0230.095

0.373

0.061

O

O

OH

OH

HO

DOB1-R4

-0.085

0.109

-0.045

0.172

-0.0450.384

-0.024

-0.011

0.013

0.008

0.003

-0.003

-0.0060.069

0.328

0.012

0.125

0.001

O

HO

O

OH

HO

DOB1-R4'

0.001

0.002

0.013

0.011

-0.010 -0.015

-0.019

0.361

-0.144

0.302

-0.051

0.277

-0.1390.0030.001

0.407

0.001

O

HO

OH

O

DOB2-R2

-0.1200.363

-0.130

0.296

-0.070

0.219

-0.029

0.006

0.003

-0.003

-0.001

0.0540.365

-0.012

OH

0.001

-0.001

0.067

0.002 O

O

OH

OH

DOB2-R4

0.013

0.357

-0.085

0.249

0.0120.026

-0.024

-0.019

0.005

0.005

0.001

0.0020.1140.016

0.202

OH

0.002

-0.001 0.001

0.127

O

HO

OH

OH

DOB2-R5

0.123

0.076

0.245

-0.012

0.175

-0.071-0.024

0.005

0.010

0.003

-0.0090.0300.076

0.033

O

-0.001

0.001

0.357

O

HO

O

OH

DOB2-R4'

0.002

0.001

0.009

0.015

-0.007

-0.028

0.362

-0.1460.0010.001

OH

0.406

-0.009

-0.134

0.299

-0.049

0.274

O

HO

OH

O

DOB3-R2

-0.022-0.005

-0.016

0.080

0.040

-0.021

-0.020

0.023 -0.001

-0.003

0.3420.588

0.005

-0.0030.008

0.001

O

O

OH

OH

DOB3-R4

-0.079

0.267

-0.129

0.297

-0.1200.323

-0.036

-0.007

0.007

0.006

0.001

-0.0020.108-0.019

0.410

0.003

-0.003 0.001

O

HO

O

OH

DOB3-R4'

0.001

0.003

0.016

0.006

-0.009

-0.030

0.363

-0.1420.0020.001

0.406

-0.004

-0.141

0.302

-0.050

0.275

0.001

O

HO

OMe

O

DOB4-R2

-0.022

-0.005

-0.014

0.079

0.036

-0.019-0.018

0.021

0.001

-0.004

-0.003

0.3440.586

0.005

-0.0010.011

0.001

O

O

OMe

OH

DOB4-R4

-0.079

0.268

-0.131

0.300

-0.1190.321

-0.037

-0.002

0.003

-0.001

0.004

0.0080.108-0.019

0.408

0.002

-0.006 0.002

Fig. 2. Spin density distribution of phenoxy radicals of DOB1–DOB4 computed at the B3LYP/6–311++G(2d,2p) level of theory in the gas phase.

Y. Xue et al. / Food Chemistry 151 (2014) 198–206 201

DOB4 are almost equal to those of DOB3 in the same medium. Thesubstitution of hydrogen with a methyl group does not signifi-cantly change the possibility to delocalize the unpaired electron.

From Fig. 2, it can be observed that the spin densities on theO-atom of the 2-OH and 4-OH radicals in these two systems differby only 0.002 (DOB3-R2 vs. DOB4-R2 and DOB3-R4 vs. DOB4-R4).

202 Y. Xue et al. / Food Chemistry 151 (2014) 198–206

Solvent affects the values of BDE slightly. From Table 1, it can beobserved that the BDEs are close in all studied environments foreach compound. For instance, the largest deviation between theBDE in the gas phase and in water is only 6.7 kcal/mol. In general,the BDE of 2-OH for DOBs in polar media (water and methanol) arelower than that in the gas phase. A decrease of 2.9–6.7 kcal/molwas obtained for BDE values when computations were carriedout in water. This observation may be attribute to the presenceof strong intramolecular hydrogen bonds between O1H� � �O4,which are weakened more and more when the solvent polarity in-creases. Indeed, polar solvents ease the charge separations. More-over, the higher the solvent polarity, the easier the separation ofcharges (Fifen, Nsangou, Dhaouadi, Motapon, & Jaidane, 2011).

With the exception of the 2-OH in DOB1 and DOB2, the othersshow the highest BDE in benzene; with the increase of the solventpolarity, BDEs tend to decrease slightly, the lowest values reachedin water. In polar media these values are lower than those in thegas phase. The decrease of BDE in polar media may be related togreater stabilization of the corresponding radical in the presenceof the polar media, as shown from the spin density distributionof the 3-OH phenoxy radical of DOB1 in the gas phase and in meth-anol (Fig. S3). The spin density is 0.373 on the oxygen atom in thegas phase whereas it is 0.350 in methanol. This means the stabil-ization of the radical is higher in methanol than in the gas phase.In the case of 4-OH and 5-OH in DOB2, a small increase in BDE val-ues was observed in the polar media with respect to the gas phase.The highest values were found in methanol. In water BDE valuesare almost equal to or slightly higher than those in the gas phase.

As observed from Table 1, the in-gas lowest BDE values of DOBsare lower or slighter larger than that of phenol (83.5 kcal/mol) cal-culated at the same level of theory. This means that the H-atomdonation ability of these compounds should be stronger than orcomparable to that of phenol. In the case of DOB2, the in-gas low-est BDE (71.2 kcal/mol) is lower than those of ascorbic acid(73.9 kcal/mol) and trolox (73.0 kcal/mol). This indicates thatDOB2 has higher H-atom donation ability than ascorbic acid andtrolox. This agrees well with the experimental results, i.e., DOB2has higher DPPH radical scavenge activity than ascorbic acid andtrolox (Ng et al., 2009). As for DOB1, the lowest BDE in each envi-ronment is always slightly larger than those of ascorbic acid and

Table 2B3LYP/6–311++G (2d,2p)//B3LYP/6–31G(d,p) ionisation potential (IP) and proton dissociatand solvents.

Moleculea IP (kcal/mol)

Gas Benzene Methanol W

DOB1 176.4 154.3 120.9 112-OH3-OH4-OH40-OHDOB2 173.7 151.2 116.7 112-OH4-OH5-OH40-OHDOB3 178.4 156.3 122.7 112-OH4-OH40-OHDOB4 174.9 172.1 138.6 112-OH4-OHPhenol 192.3 163.2 125.0 11Ascorbic acid 193.9 166.9 129.3 12Trolox 159.1 136.8 103.3 9

a See the note in Table 1 for the full names of DOB1–DOB4.

trolox, indicating that DOB1 should be of comparable activity toascorbic acid and trolox. This predicted trend is somewhat differ-ent than the experimental results obtained from the DPPH test.Regarding the 50% inhibition (IC50) values of DPPH assay (Table 1),DOB1 should have a higher activity than trolox and ascorbic acid,especially for ascorbic acid. In addition, the superiority of DOB4over that of ascorbic acid was also not predicted through BDE val-ues. These deviations observed could be related to the complexmechanism involved in the DPPH assay (Nenadis & Tsimidou,2012). On the other hand, the disagreement with respect to theexperimental data could also be due to the fact that the evaluationof the scavenging activity is based on the reaction with the DPPHradical, whereas our theoretical results are more general and donot consider the possible differences due to a particular radicalcounterpart.

3.3. SET–PT mechanism

The calculated parameters IP and PDE for the studied moleculesand their radicals are summarized in Table 2. From Table 2, it canbe seen that the lowest value of IP is always found for DOB2 in allstudied environments. This indicates that the electron donatingability of DOB2 is always stronger than other DOBs irrespectiveof the environment. The sequence of IP values in the gas phaseand water is DOB2 < DOB4 < DOB1 < DOB3, whereas the tendencyof IP values in benzene and methanol solutions is quite different:DOB2 < DOB1 < DOB3 < DOB4. By comparison, we found that thetrend for IP values in the gas phase and water is different from thatof the BDE values. This discrepancy can be attributed to the factthat BDE is affected by the local environment induced by the sub-stituents, whereas the IP value is affected by the structure of thewhole molecule, i.e., the extended delocalization and conjugationof the p-electrons (Wright et al., 2001).

Comparatively to solvent effect on BDE, a significant influenceof solvent on IP values was observed (Table 2). It is well known thatcation radicals are charged and quite sensitive to the polarity ofvarious solvents. As expected, the values of IP in solvent aredramatically lower than those in the gas phase. The relative orderof IP in the studied environments is water < methanol < ben-

ion enthalpy (PDE) values of test compounds and reference compounds in gas phase

PDE (kcal/mol)

ater Gas Benzene Methanol Water

5.7230.5 36.2 14.3 18.6216.9 23.6 2.5 6.9221.2 28.1 7.5 11.9222.3 29.3 8.8 13.3

1.0236.3 41.8 20.0 24.5218.4 26.8 8.9 13.5213.4 22.0 4.5 9.2225.2 32.5 13.1 17.6

7.4240.6 47.5 26.9 31.3225.6 33.0 13.4 17.9220.3 27.3 7.1 11.6

4.6244.1 50.3 29.7 34.1229.0 35.8 16.1 20.6

9.5 207.2 21.2 5.8 10.43.8 196.0 8.5 -6.9 -2.38.0 229.8 37.0 16.8 21.2

Y. Xue et al. / Food Chemistry 151 (2014) 198–206 203

zene < gas. This confirms that the solvent, especially polar medium,can facilitate electron donation.

In comparison with the reference compounds, the in-gas IPs ofDOBs 1–4 are about 14�19 kcal/mol lower than that of phenol(192.3 kcal/mol). In addition, these values are smaller than thatof ascorbic acid (193.9 kcal/mol), but, they are higher than thatof trolox (159.1 kcal/mol). This means that the electron donationability of DOBs 1–4 are stronger than that of phenol and ascorbicacid, but weaker than that of trolox. Similar conclusion can bedrawn from the analysis of the data in water.

As shown in Table 2 the PDEs for DOBs in the studied environ-ments can be arranged in the following order: 3-OH < 4-OH < 40-OH < 2-OH, 5-OH < 4-OH < 40-OH < 2-OH, 40-OH < 4-OH < 2-OH,and 4-OH < 2-OH, respectively. These trends observed for PDEsare analogous with those for BDEs, because the second step ofthe SET–PT mechanism results in the phenoxy radical formation,also. Comparison of DOB1 or DOB2 with DOB3 shows that the pres-ence of a 3-OH or 5-OH group causes a decrease in 4-OH PDE. Incomparison to DOB3, 4-OH PDEs in DOB1 and DOB2 are lower byaverage 5.3 kcal/mol in all the studied environments.

The high solvation enthalpies of proton significantly decreasethe PDE value in solutions. As shown in Table 2, PDEs increase inthe order: methanol < water < benzene < gas phase. The averagedifference between methanol and gas-phase reaches �213 kcal/mol. Such a finding agreed with that reported for p-phenylenedi-amine (Rimarcik et al., 2010) and eupatilin (Li et al., 2013).

Data in Table 2 shows that the lowest gas-phase PDEs for thestudied DOBs increase in the order: DOB2 < DOB1 < DOB3 < DOB4.This order is in line with that of BDE. The orders in polar solventsare somewhat different, following the sequence of DOB1< DOB2 < DOB3 < DOB4. Furthermore, the lowest gas-phase PDEs(213.4–229.0 kcal/mol) are larger than those of phenol(207.2 kcal/mol) and ascorbic acid (196.0 kcal/mol), and smallerthan that of trolox (229.8 kcal/mol). This indicates that the protondissociation ability of the studied DOBs is slightly weaker thanphenol and ascorbic acid, but stronger than that of trolox.

By comparison, it can be found that in the four studied environ-ments, BDE values are always lower than those of IP. This indicatesthat the HAT mechanism not the SET–PT seems to represent theprobable process in the studied environments.

Table 3B3LYP/6–311++G (2d,2p)//B3LYP/6–31G (d,p) proton affinity (PA) and electron transfer esolvents.

Moleculea PA (kcal/mol)

Gas Benzene Methanol W

DOB12-OH 339.9 102.9 48.0 53-OH 336.1 98.4 43.5 44-OH 328.1 91.8 38.5 440-OH 340.1 103.3 49.1 5

DOB22-OH 350.1 111.6 55.7 54-OH 320.4 85.7 35.8 35-OH 328.0 92.8 43.0 440-OH 337.4 104.2 53.1 5

DOB32-OH 348.5 110.0 53.5 54-OH 329.6 93.5 40.8 440-OH 340.9 103.6 48.9 5

DOB42-OH 349.1 110.4 53.7 54-OH 330.1 93.8 40.9 4Phenol 347.8 106.0 49.0 5Ascorbic acid 325.9 87.9 34.2 3Trolox 347.1 109.4 54.0 5

a See the note in Table 1 for the full names of DOB1–DOB4.

3.4. SPLET mechanism

According to the SPLET mechanism, deprotonation of phenolic-OH group(s) and electron transfer tendency in the gas phase andsolvents were studied by calculating proton affinity (PA) and elec-tron transfer enthalpy (ETE) values (Table 3). Among the studiedDOBs, 4-OH PAs are always the lowest in all the studied environ-ments. The PAs of DOBs in the studied environments can bearranged in the following order: 4-OH < 3-OH < 2-OH < 40-OH,4-OH < 5-OH < 40-OH < 2-OH, 4-OH < 40-OH < 2-OH and 4-OH < 2-OH, respectively. This clearly shows that formation of 4-O� anionis easier than that of other anions. From Table 3, it can be seen thatthe lowest PAs are increasing in the order: DOB2 < DOB1< DOB3 < DOB4. It clearly shows that DOB2 and DOB1 always havesmaller PA than other two compounds as a result of the ortho-dihy-droxy structure. Comparison of DOB3 with DOB4 shows thatreplacement of the 40-OH group with a OMe group has a negligibleeffect on 4-OH PA.

Similar to PDE, the PA values decrease significantly from the gasphase to solution, due to the high solvation enthalpies of proton.The average differences between PA in the gas phase and differentsolvents are 243.8 (benzene), 287.5 (methanol) and 281.6 (water)kcal/mol, respectively. This means solvents seem to favour thedeprotonation process. The lowest gas-phase PAs for the four DOBsare in the range of 320.4–330.1 kcal/mol, which are consistent withthe values reported in the literature for polyphenols, which fall in arange of 312.5–327.5 kcal/mol (Leopoldini, Russo, & Toscano,2006). Furthermore, the lowest gas-phase PAs are lower than thoseof phenol and trolox, while these values are smaller or slightly lar-ger than that of ascorbic acid. Thus, deprotonation of phenolic-OHfor the four DOBs is easier than phenol and trolox. In comparison toascorbic acid, only DOB2 in the gas phase and benzene presentsstronger proton donation ability. In methanol and water, all theDOBs have weaker proton donation ability than ascorbic acid.

From the ETE values in Table 3, it can be seen that the lowestETEs in the gas phase and benzene follow the same sequence:DOB1 < DOB3 < DOB2 < DOB4, while in methanol and water thelowest ETEs follow a different order: DOB2 < DOB1 < DOB3 < DOB4.In solution, ETEs are higher than the corresponding gas phasevalues. In benzene, this increase can be considered uniform with

nthalpy (ETE) values of test compounds and reference compounds in gas phase and

ETE (kcal/mol)

ater Gas Benzene Methanol Water

0.8 66.9 87.7 87.3 83.56.4 57.2 79.4 80.0 76.21.4 69.4 90.7 90.0 86.21.9 58.6 80.2 80.8 77.0

8.5 59.9 81.0 81.3 77.88.9 71.7 92.1 90.3 86.44.8 59.1 80.3 79.4 75.66.2 61.4 80.6 77.4 73.4

6.3 70.5 93.7 96.1 92.43.8 74.5 95.8 95.2 91.41.8 57.8 80.0 80.9 77.2

6.4 69.9 93.3 95.9 92.33.9 73.8 95.4 95.1 91.31.8 51.6 78.4 81.8 78.17.2 63.9 87.5 88.2 84.46.8 41.9 64.4 66.0 62.4

204 Y. Xue et al. / Food Chemistry 151 (2014) 198–206

an average value of �22 kcal/mol. The differences between ETEs inthe gas phase and polar solvents lie in 16–26 and 12–22 kcal/molintervals for methanol and water, respectively. In comparison tothe IP values (Table 2) of the neutral form, the corresponding ETEvalues were significantly lower. Accordingly, the single electrontransfer process from the anionic form is more preferable than thatfrom the neutral form. This is in line with the results obtained fromour previous study on chalcones (Xue et al., 2013a, 2013b) andother studies (Francisco-Marquez & Galano, 2012; Li et al., 2013).

As stated above, IP and PA are reaction enthalpies related to thefirst step of the SET–PT and SPLET mechanisms, and therefore BDE,IP and PA determine thermodynamically preferred reaction path-way. Our results demonstrate that the calculated gas phase IPsand PAs of the molecules considered are significantly higher thanO-H BDEs, and hence from the thermodynamic point of view,HAT represents the most probable process in the gas phase. Thevalues of PA in water and methanol are significantly lower thanthe corresponding values of BDE and IP. Therefore, SPLET repre-sents the most thermodynamically probable reaction pathway inpolar media (water and methanol). Available experimental andtheoretical studies for phenolic antioxidants, including flavonoids,also confirmed the important role of the SPLET mechanism in polarsolvents (Musialik, Kuzmicz, Pawlowski, & Litwinienko, 2009;Wright et al., 2001; Zhang & Ji, 2006). Overall, among the threemechanisms, HAT is thermodynamically preferred in the gas phaseand SPLET is favoured more in polar solvents, such as water andmethanol. However, it must be stressed that the relativeimportance of HAT, ET-PT or SPLET is not only determined by

-5.8599 eV DOB1

-5.6899 eV DOB2

-5.9446 eV DOB3

-5.8554 eV DOB4

Fig. 3. The energy and distribution of HOMO

micro-environmental features (lipid phase, aqueous phase) butalso governed by the characteristics of the scavenged radical(Zhang & Ji, 2006).

3.5. Frontier orbitals

Another important molecular parameter correlated with thefree radical scavenging potential is the energy of the HOMO orbital.The molecules with a higher energy HOMO orbital have strongerelectron donating abilities. Besides, the electronic density distribu-tion in these orbitals permits prediction of the most probable sitesin the studied molecules which can be easily attacked by freeradicals and other reactive agents. It can be observed from Fig. 3that DOB2 provided the highest HOMO energy (�5.6899 eV), fol-lowed by DOB4 (�5.8554 eV), DOB1 (�5.8599 eV) and DOB3(�5.9446 eV). This clearly shows that DOB2 has the strongestelectron-donating capability among the compounds studied. More-over, the predicted electron-donating capability order according toHOMO energy was found to be the same as that for IPs (Table 2).Analysis of orbital distribution in Fig. 3 shows that the LUMOcompositions present similar distributions for the four DOBs. Theelectronic density of the LUMO is mainly localised on the A-ringand carbonyl group. In contrast to the LUMO, some differenceswere found in the HOMO compositions of the four DOBs. DOB1and DOB2 present more electron delocalization on the A-ring withrespect to the B-ring, whereas DOB3 and DOB4 show more electrondelocalization on the B-ring. These results show that for DOB1 andDOB2, the A-ring should be the most probable site for radical

-1.5815 eV

-1.5364 eV

-1.5206 eV

-1.4923 eV

(left) and LUMO (right) for DOB1–DOB4.

Y. Xue et al. / Food Chemistry 151 (2014) 198–206 205

attack, whereas the B-ring should be the most probable site forDOB3 and DOB4.

4. Conclusions

In this work, we have studied the thermodynamics of radicalscavenging for four selected polyphenolic deoxybenzoins (DOBs)based on the DFT method. HAT, SET–PT and SPLET mechanismsin the gas phase and solvents were considered, and the BDE, IP,PDE, PA, ETE and other molecular parameters of the target mole-cules were determined.

Conformational analysis indicated that each DOB can adoptthree low energy conformations stabilized by the formation of ahydrogen bond between 2-OH and the carbonyl oxygen. The re-sults of structural optimization showed that no significant geomet-rical change was observed when going from the neutral molecule(ArOH) to the phenoxy (ArO�) and cation (ArOH+�) radicals andthe anion forms (ArO�). In general, the sequence of hydrogendonating ability of the DOBs is: DOB2 > DOB1 > DOB3 > DOB4,which is in accordance with the experimental results from theDPPH assay. The ortho-dihydroxy moiety was found to be the mostimportant structure in the HAT mechanism. Among the studiedDOBs, DOB2 has higher H-atom donation ability in comparison tothe reference antioxidants ascorbic acid and trolox. As expected,the values of IP, PDE and PA in solvents are dramatically lower thanthose in the gas phase. The trends observed for PDEs are analogouswith those for BDEs. DOB2 and DOB1 always have smaller PA thanthe other two compounds in all the studied environments, and for-mation of the 4-O� anion is easier than that of the other anions.

The results demonstrate that the thermodynamically preferredmechanism in the gas phase is HAT followed by SET–PT and SPLET,because IPs are roughly twice as high as BDEs and PAs are higherthan BDEs by one order. In polar media such as methanol andwater, SPLET represents the most probable reaction pathway. Theresults confirmed that the solvent has a large impact on the reac-tion enthalpies in SET–PT and SPLET, as these mechanisms involvecharged species. On the contrary, O–H BDEs related to the HATmechanism are close in all studied environments.

In conclusion, our study will contribute not only to elucidationof the chemical mechanism of radical-scavenging action, but alsoto stimulate further exploitation of these compounds in the fieldof food chemistry and pharmacy.

Acknowledgements

The authors are grateful to Prof. X.D. Gong (Department ofChemistry, Nanjing University of Science and Technology) for pro-viding calculation convenience. This work was supported by theScience and Technology Plan Projects of Xuzhou (XZZD1224), theNational Science Foundation of China (81202490, 81341084), theNatural Science Foundation of Education Ministry of JiangsuProvince (11KJB350005), Zhen Xing Project of XZMC, the PriorityAcademic Program Development of Jiangsu Higher Education Insti-tutions and the Innovative Practice Training Program for Studentsof Jiangsu Higher Education Institutions (2011-1003).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2013.11.064.

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