Food Chemistry 161 (2014) 155–161
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Computational study of the structure–free radical scavenging relationship of procyanidins Ana María Mendoza–Wilson ⇑, Sergio Ivan Castro-Arredondo, René Renato Balandrán-Quintana Coordinación de Tecnología de Alimentos de Origen Vegetal, CIAD, A.C., Carretera a La Victoria km 0.6, Hermosillo, Sonora 83304, Mexico
a r t i c l e
i n f o
Article history: Received 8 January 2014 Received in revised form 9 March 2014 Accepted 24 March 2014 Available online 1 April 2014 Keywords: Procyanidin DFT Computational Free radical Antioxidant
a b s t r a c t Procyanidins (PCs) are effective free radical scavengers, however, their antioxidant ability is variable because they have different degrees of polymerisation, are composed by distinct types of subunits and are very susceptible to changes in conformation. In this work the structure–free radical scavenging relationship of monomers, dimers and trimers of PCs was studied through the hydrogen atom transfer (HAT), sequential proton-loss electron-transfer (SPLET) and single electron transfer followed by proton transfer (SET-PT) mechanisms in aqueous phase, employing the Density Functional Theory (DFT) computational method. The structure–free radical scavenging relationship of PCs showed a very similar behaviour in HAT and SET-PT mechanisms, but very different in the SPLET mechanism. The structural factor that showed more effects on the ability of PCs to scavenge free radicals in aqueous phase was the conformation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The PCs are also known as proanthocyanidins or condensed tannins (Enomoto, Nagasako-Akazome, Kanda, Ikeda, & Dake, 2006) and represent one of the most abundant class of phenolic compounds produced by plant tissues (Andersen & Markham, 2006). The PCs have proven to be 30–50 times more effective free radical scavengers than vitamins C and E (Duda-Chodak, Tarko, Satora, Sroka, & Tusz ynski, 2010; Saint-Cricq, Provost, & Vivas, 1999), so they have beneficial effects on the fruits and vegetable quality, in addition to the health of consumers (Jerez, Touriño, Sineiro, Torres, & Núñez, 2007; Khanal, Howard, & Prior, 2009). However, because PCs have very complex and diverse structures, their antioxidant ability is changeable and depends on the intermediates and oxidation products formed during the antioxidant reactions. The PCs are flavonoids belonging to the flavan-3-ols class and are mainly composed of ()-epicatechin and (+)-catechin subunits, which are linked to form oligomers or polymers (Enomoto et al., 2006). The subunit localised at the end of the chain is called terminal unit and the remaining subunits are called extension units (Fig. 1). Also the PCs are characterised by three chiral centres, C2, C3 and C4, which makes them very susceptible to changes in configuration and conformation. From C4 two conformations are ⇑ Corresponding author. Address: Carretera a La Victoria km 0.6, C.P. 83304, PO Box 1735, Hermosillo, Sonora, Mexico. Tel.: +52 662 2892400x354; fax: +52 662 2800422. E-mail address: [email protected] (A.M. Mendoza–Wilson). http://dx.doi.org/10.1016/j.foodchem.2014.03.111 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
formed: compact and extended (Fig. 2). A way to distinguish these conformations is through the dihedral angles formed between the atoms involved in the interflavanoid linkage (C3–C4–C8–C9). Compact conformation is the one with positive dihedral angle values whereas the extended conformation has negative values. For dimers these values can range from +90° to 90° and for trimers from +100° to 70°, respectively (Khan, Haslam, & Williamson, 1997; Tarascou et al., 2007). There is growing evidence indicating a strong relationship between the antioxidant ability of PCs and structural factors such as degree of polymerisation, type of subunit and conformation, but there are also many controversies about this issue (Leite, Laranjinha, Pereira, & de Freitas, 2003; Santos-Buelga & Scalbert, 2000). On the one hand, it is stated that the greater the degree of polimerization of the PCs the greater their ability to scavenge free radicals (Arteel & Sies, 1999; Vennat, Bos, Pourrat, & Bastide, 1994). On the other hand, it is suggested that the PCs ability to scavenge free radicals is not necessarily greater at a higher degree of polymerisation, as in one study was reported that PCs trimers showed to be more efficient than dimers, but less efficient than monomers to scavenge the hydroxyl radical (Cheynier, Rigaud, & da Silva, 1992). There is a special interest to know the differences in the ability to scavenge free radicals amongst monomers, dimers and trimers, as these represent the most bioavailable forms compared to larger oligomers of PCs. Regarding the type of subunit, most studies have reported higher activity for free radical scavenging in ()-epicatechin against different free radicals (Cheynier et al., 1992; de Freitas,
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Fig. 1. Structure of ()-epicatechin (E), (+)-catechin (C), procyanidin B1 (PB1), procyanidin B2 (PB2) and procyanidin C1 (PC1).
Glories, & Laguerre, 1998a); however, in an experimental-computational study conducted by Mendoza-Wilson et al. (2013), higher radical scavenging activity was found for (+)-catechin against the DPPH. In another computational study performed by MendozaWilson and Glossman-Mitnik (2006) it was determined that (+)catechin and ()-epicatechin have comparable intrinsic reactivity properties, due to their similarity in structure (epimers). Nevertheless they have the ability to form distinct intermediates, which can make the difference in activity against free radicals between these isomers. Additionally, the predominant conformation (compact or extended) in a particular solvent (polar, non polar), is also
dependent on the type of subunit as extension unit (Hatano & Hemingway, 1997). The intermediates and products that can be formed from the PCs during their reaction against free radicals are dependent primarily of the mechanism followed and their reactivity properties. There are three basic mechanisms for free radical scavenging: (1) HAT with a phenoxyl radical as intermediate; (2) SPLET with a phenoxide anion as intermediate, and (3) SET-PT with a radical cation as intermediate (Foti, Daquino, Mackie, DiLabio, & Ingold, 2008; Musialik, Kuzmicz, Pawlowski, & Litwinienko, 2009). The reactivity properties of PCs focus on the oxidation–reduction (redox)
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157
Fig. 2. Compact and extended conformations of the procyanidin B2.
properties of their hydroxyl groups and are related to the ease of releasing hydrogen atoms and electrons to scavenge free radicals (Suryan, Kafafi, & Stein, 1989). Amongst these are the thermochemical properties, which describe the energy process of the redox reactions; the polarisability, which describes polar effects related with the deformation of the electron cloud (Foresman & Frisch, 1996), and their water solubility. The PCs, like all flavonoids, have amphipathic properties but they are more soluble in water because have a greater number of hydroxyl groups and can acquire different conformations (Achurra, 2006). The fact that the PCs are water soluble gives them special properties, as this is the medium in which biological reactions are carried out, is a major component in food and as the extraction solvent for potential commercial use of PCs is excellent because is cheap and nontoxic. To establish the relationship of the structure with the free radical scavenging properties of PCs, it is necessary explore together structural factors such as the degree of polymerisation, type of subunit, conformation, intermediates and products formed through the distinct mechanisms for free radical scavenging, as well as their reactivity properties. This is an arduos task since by their polyhydroxylated structure the PCs generate many intermediates with very short half-life and different conformers by their interflavanoid linkage, which do not allow their isolation through experimental methods, so it is necessary to use computational methodologies. Due to the size of the PCs, the computational methods used for their study are those of molecular mechanics and semiempirical, which have been very useful to establish the molecular geometry and conformation but not to calculate properties of reactivity. The DFT currently represents the most suitable computational method to calculate structural and reactivity properties of PCs, due to the high precision, low cost and shorter time performing calculations on medium-sized and large molecules, compared with other electronic structure methods, such as Hartree–Fock and postHartree–Fock. The aim of this work was to study the effect of differences in structure, specially degree of polymerisation, type of subunit, conformation, as well as the reactivity properties on the ability for free radical scavenging of monomers, dimers and trimers of procyanidins through HAT, SPLET and SET-PT mechanisms, employing the DFT computational method. 2. Computational methodology All the calculations reported were performed with the GAUSSIAN 03W code (Frisch et al., 2004). The DFT method implemented in this computational package was used, and the M05-2X hybrid meta exchange–correlation functional (Zhao, Schultz, & Truhlar,
2006) in conjunction with the 6–31G(d,p) and 6–31+G(d,p) basis sets were employed to establish the optimisation of molecular structures, vibrational frequencies and reactivity properties. The reactivity properties were calculated under simulation of an aqueous phase and include thermochemical parameters, polarisability and water solubility. 2.1. Thermochemical properties In order to know the ability of the simulated PCs for free radical scavenging by the HAT mechanism, the bond dissociation energy (BDE) was calculated. In aqueous phase the BDE was determined as the difference of total free solvation energy between parent molecule (ArOH) and its phenoxyl radical (ArO) formed after the hydrogen atom (H) transfer from hydroxyl group, applying the reaction (1) and Eq. (2) (Ochterski, Petersson, & Wiberg, 1995):
ArOH ! ArO þ H X X Dr Hð298 KÞ ¼ ðE0 þ Gcorr Þproducts ðE0 þ Gcorr Þreactants
ð1Þ ð2Þ
The computed total free solvation energy of hydrogen atom (H) in aqueous phase was 0.495216 Hartrees. The ability of the PCs to transfer electrons by the SPLET mechanism was determined in two steps. In the first step the proton affinity (PA) of the phenoxide anion (ArO) formed after the deprotonation (H+) of the PCs (ArOH) was calculated from the Eq. (3) (Markovic et al., 2012):
ArOH ! ArO þ Hþ
ð3Þ
In the second step the electron transfer energy (ETE) of the phenoxide anion was determined by means of Eq. (4) (Markovic et al., 2012):
ArO !ArO þ e
ð4Þ
Both PA and ETE were reported in terms of total free solvation energy in water. The values for H+ = 262.4 kcal/mol and e = 35.5 kcal/mol were employed for calculations (Zhan & Dixon, 2003). To know the ability of the PCs to transfer electrons by the SET-PT mechanism, first the ionisation potential (IP) was calculated from the Eq. (5) (Markovic et al., 2012):
ArOH ! ArOHþ þ e
ð5Þ
Subsequently the proton dissociation energy (PDE) was computed by means of Eq. (6) (Markovic et al., 2012):
ArOHþ ! ArO þ Hþ
ð6Þ
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2.2. Polarisability properties As an intrinsic property of reactivity related with the effects of polarity on the easiness to release hydrogen atoms and electrons, the polarisability (a) was calculated through the following formula (Foresman & Frisch, 1996).
hai ¼ 1=3ðaxx þ ayy þ azz Þ
ð7Þ
2.3. Solubility properties The water solubility was determined by calculating the DG of solvation of the molecules from their neutral energies. The solvent simulation was performed by employing integral equation formalism (IEF-PCM) implemented on the GAUSSIAN 03W code (Cances & Mennucci, 1998). The UAHF set of solvation radii was used (Barone, Cossi, & Tomasi, 1997). 3. Results and discussion 3.1. Optimised molecular structures There are very few reports in the current literature related with the ability of PCs to inhibit free radicals by HAT, SPLET and SET-PT mechanisms, taking into account different degrees of polymerisation, types of subunits and conformation. For this reason these data
were calculated for the ()-epicatechin (E) and (+)-catechin (C) monomers, the procyanidin B1 (PB1) and procyanidin B2 (PB2) dimers, besides the procyanidin C1 (PC1) trimer (Fig. 1); these represent the most common, abundant and bioavailable PCs of the plant tissues, including highly consumed fruits such as apples and grapes (de Freitas, Glories, Bourgeois, & Vitry, 1998b; Tsao, Yang, Young, & Zhu, 2003). The optimised molecular structures of E and C, PB1 and PB2 in their respective compact and extended conformations, as well as of PC1 in all their possible conformations related to interflavanoid linkage, as they are compact–compact (comp–comp), compact– extended (comp–ext), extended–compact (ext–comp) and extended–extended (ext–ext), are showed in the Fig. 3. Dihedral angles values of the interflavanoid linkages (C3–C4–C8–C9) are also included in order to confirm the compact conformation (positive value) and extended conformation (negative value) between extension and terminal units of the PB1, PB2 and PC1 molecules. 3.2. Thermochemical properties The most used pathway for studying the free radical scavenging mechanisms in flavonoids is through thermochemical parameters, focused on the intrinsic properties of the molecules under study and the microenvironmental features such as the reaction medium. Several computational works based on these parameters have proved that the most susceptible site to radical attack in distinct classes of flavonoids, amongst which are included ()-epicatechin and (+)-catechin, is 40 -OH (Bentes, Borges, Monteiro, De Macedo, & Alves, 2011; Kondo et al., 2000; Leopoldini, Marino, Russo, & Toscano, 2004; Mendoza-Wilson, 2006; Mendoza-Wilson et al., Table 1 Thermochemical properties of procyanidins computarized in aqueous phase. Procyanidin
()-Epicatechin (+)-Catechin Procyanidin B1 Compact UE UT Extended UE UT Procyanidin B2 Compact UE UT Extended UE UT
Fig. 3. Optimised molecular structures of ()-epicatechin (E) and (+)-catechin (C); procyanidin B1 (PB1) and procyanidin B2 (PB2) in their compact and extended conformations; procyanidin C1 (PC1) in their compact–compact (comp–comp), compact–extended (comp–ext), extended–compact (ext–comp) and extended– extended (ext–ext) conformations. The dihedral angle values of the interflavanoid linkages are given by C3–C4–C8–C9 atoms.
Procyanidin C1 CompComp UE2 UE1 UT CompExt UE2 UE1 UT ExtComp UE2 UE1 UT ExtExt UE2 UE1 UT
Parameters of free radical scavenging (kcal/mol) HAT
SPLET
BDE
PA
ETE
IP
SET-PT PDE
92 90
34 32
70 70
100 98
4 4
92 89
34 38
70 64
92 91
34 38
70 66
91 88
37 34
66 66
91 91
33 39
71 63
91 85 91
38 38 34
65 60 69
92 86 91
34 32 40
69 66 63
91 91 88
33 35 37
70 68 63
96 91 94
44 45 37
64 58 69
88 15 13 95 8 8 91 12 9 95 9 8 85 19 13 18 89 15 9 14 97 6 6 3 95 13 8 11
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2013; Tyrakowska, Lemanska, Szymusiak, Borkowski, & Rietjens, 2003; Zhang, Skinner, & Keller, 2009). Considering that the first reaction site can make a difference in the antioxidant potential, depending on the mechanism followed, the ability to the HAT, SPLET and SET-PT mechanisms was determined on E, C, PB1, PB2 and PC1 on the basis of the 40 -OH. In order to take in account the effects of the solvent in the redox reaction, the thermochemical parameters were calculated from the total free solvation energy under simulation of an aqueous phase at 298 K. The computarised values of PA < BDE 6 IP showed in the Table 1 indicate that the mechanism which could prevail in an aqueous phase in E, C, PB1, PB2 and PC1 is SPLET, followed by HAT and SET-PT. These results are in accordance with the works previously reported by Markovic et al. (2012) and Mendoza-Wilson et al. (2013). Based on the values of proton affinity (PA), which represent the first step of the SPLET mechanism, is suggested that the most important structural factors of the PCs associated with this mechanism are the conformation, the position and type of subunit, since the degree of polymerisation apparently is not decisive. Under these terms both the C monomer and the PC1 trimer in its compact-extended conformation through its extension unit 1, showed the greatest ability for free radical scavenging because of their low and equal value of PA of 32 kcal/mol (Table 1). In the HAT mechanism is observed that the easiness to transfer hydrogen atoms (BDE values) fluctuated within the same range amongst monomers, dimers and trimers of PCs. However, to analyse the degree of polymerisation, conformation and to compare the reactivity of extension and terminal units of each molecule, the following behaviour was found: the lowest value of BDE (85 kcal/mol) was shown by PC1 in its extension unit 1 of the
Table 2 Polarisabilities calculated in aqueous phase for the procyanidin molecules. Procyanidin
Polarisability (a)
()-Epicatechin (+)-Catechin Procyanidin B1 compact Procyanidin B1 extended Procyanidin B2 compact Procyanidin B2 extended Procyanidin C1 CompComp Procyanidin C1 CompExt Procyanidin C1 ExtComp Procyanidin C1 ExtExt
259.37 260.43 522.74 524.96 523.30 524.25 784.76 786.38 777.72 769.61
The polarisabilities are expressed in atomic units (a.u.).
compact–compact conformation, followed by PB2 (88 kcal/mol) in its terminal unit of the compact conformation, PB1 (89 kcal/ mol) in its terminal unit of the compact conformation, after C (90 kcal/mol) and E (92 kcal/mol) (Table 1). Considering that the smaller the value of BDE the greater the antioxidant potential, it is suggested that in the HAT mechanism the antioxidant ability of the PCs increases with the degree of polymerisation (monomer to trimer), is favored by the compact and compact–compact conformations and is slightly higher in C than in E. In relation to the position of the subunits, it is suggested that the terminal unit is the most reactive in dimers and that in the case of the trimers is the extension unit 1, which corresponds to the intermediate unit. Regarding the SET-PT mechanism, the IP is related to the first step of the free radical scavenging reaction, so it was used as an indicator to establish the differences in antioxidant ability amongst different PCs. However, due to the way the IP was determined computationally, it represents a global property of the molecules.
Table 3 Computational calculations of solubility in aqueous phase for the procyanidin molecules. Procyanidin
Parent molecule
Phenoxyl radical
Phenoxide anion
Radical cation
()-Epicatechin (+)-Catechin
29 30
25 27
73 71
73 72
35 37
85 81
42 43
92 71
36 40
77 80
43 44
82 71
34 41 38
81 80 72
48 54 50
99 96 64
48 46 54
92 91 92
55 59 56
85 78 101
Procyanidin B1 Compact UE UT Extended UE UT Procyanidin B2 Compact UE UT Extended UE UT Procyanidin C1 CompComp UE2 UE1 UT CompExt UE2 UE1 UT ExtComp UE2 UE1 UT ExtExt UE2 UE1 UT
40
70
46
77
40
69
48
78
39
65
52
78
51
77
66
89
The calculations were performed as DG of solvation and are expressed in kcal/mol.
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Therefore, to specify the preferred site of the reaction was taking into account the value of PDE, which was determined for each subunit of dimers and trimers. As can be seen in Table 1, in the SET-PT mechanism the PCs showed a comparable behaviour to the HAT mechanism. The lowest value of IP (85 kcal/mol) was shown by PC1 in the compact– compact conformation, and according with the lowest value of PDE (13 kcal/mol) the extension unit 1 was the most reactive. For dimers, the lowest IP value corresponded to PB1 (88 kcal/mol) in the compact conformation and based on the value of PDE (13 kcal/mol) the terminal unit was the most reactive, followed by PB2 (91 kcal/mol) in the compact conformation and the terminal unit as the most reactive site (PDE = 9 kcal/mol). In the monomers, C showed the lowest IP (98 kcal/mol). According to these data, it states that in the SET-PT mechanism the ability to scavenge free radicals of the PCs increases with the degree of polymerisation (monomer to trimer), is favored by the compact and compact– compact conformations and is slightly higher in C than E. In relation to the position of the subunits, it is suggested that the terminal unit is the most reactive in dimers and in the case of the trimers is the extension unit 1, which corresponds to the intermediate unit. 3.3. Polarisability properties The polarisability showed a significant rise as the degree of polymerisation of the PCs increased, being observed the following order: PC1 > PB2 P PB1 > C > E (Table 2). This result indicates that PC1 is more reactive than PB2 and PB1, and both are more reactives than C and E. The more reactive monomer was C. The polarisability did not follow a uniform pattern in relation to the conformation, since the dimers in their extended conformation had higher polarisabilities than the dimers in their compact conformation, and the trimer showed an inverse behaviour. A direct relationship amongst the polarisability, the degree of polymerisation and the ability to scavenge free radicals by means of the HAT and SET-PT mechanisms was found. Also, a direct relationship amongst the polarisability, type of monomer, and the ability to scavenge free radicals by means of the HAT, SPLET and SET-PT mechanisms was found.
(Foo & Porter, 1983; Hatano & Hemingway, 1997; Tarascou et al., 2006, 2007). An implicit model (IEF-PCM) was employed to simulate the aqueous phase in this investigation, which is based on the dielectric constant of the solvent, and the applied radii (UAHF) not added individual spheres to the hydrogen atoms of the molecules under study. To determine the effects of adding spheres in the hydrogen atoms of the PCs, some test calculations for ()-epicatechin, PB1 and catechol as reference molecule, were made with the UFF radii. Although the DG values for PCs were not equal to those obtained with the UAHF radii, the same trend and behaviour were observed with both radiis. It is important to note that the UAHF radii is one of the most recommended for the calculations of DG of solvation, because it is considered very accurate even in large molecules such as PCs. 4. Conclusions The ability of PCs to scavenge free radicals by the HAT and SETPT mechanisms tended to increase with the degree of polymerisation (monomer < dimer < trimer), was slightly higher for the monomer C than E, and was totally dependent on the conformation as it was higher in the compact and compact–compact conformations of dimers and trimers, respectively. The terminal unit was the most reactive site in dimers and in the case of the trimers it was the extension unit 1, which corresponds to the intermediate unit. Regarding SPLET mechanism, the most important structural factors were the conformation, the position and type of subunit, since the degree of polymerisation of PCs apparently was not decisive. In the SPLET mechanism the relationship of the structural factors with the ability of PCs to scavenge free radicals did not show a well-defined trend as in the HAT and SET-PT mechanisms. The polarisability and solubility in aqueous phase of the PCs tended to increase with the degree of polymerisation, were higher in C than E, and in relation to the conformation did not follow a uniform pattern. In resume it is concluded that the structure–free radical scavenging relationship of monomers, dimers and trimers of procyanidins is dependent of the mechanism followed; however, the structural factor that showed more effects on the ability of PCS to scavenge free radicals in aqueous phase was the conformation.
3.4. Solubility properties Acknowledgements As can be observed in Table 3, the solubility in an aqueous phase was slightly higher in C than E, and tended to increase with the degree of polymerisation of the PCs. Nevertheless, the conformation, the position of the reaction site (UE or UT) and the intermediates formed from the PCs, showed a more significant effect on this property. Regarding the type of intermediate, the phenoxide anions were more soluble, followed by radical cations, whilst the phenoxyl radicals were less soluble even than parent molecules. In relation to the conformation, the extended form was the most soluble and the compact form the less soluble. This result is consistent, since in the compact conformation of the PCs the hydroxyl groups remain close to each other and the formation of intramolecular hydrogen bonding is favored, whilst in the extended conformation the hydroxyl groups are more exposed to the solvent to form intermolecular hydrogen bonding with it. What seems contradictory is that PB1, PB2 and PC1 in their compact and compact–compact conformations, can more readily donate their hydrogen atoms, electrons and protons, if they are less soluble in aqueous phase. Such behaviour is probably due to that the intramolecular hydrogen bonds give them greater stability. Additionally, some studies report that the predominant conformation in dimers and trimers of PCs in an aqueous medium is the compact, because this conformation allows minimising the effective surface area and consequently the solute–solvent interactions
The authors of this paper are very grateful with the Consejo Nacional de Ciencia y Tecnología (CONACYT) México, which financed the investigation under Grant Number CB2012-183739. Sergio Ivan Castro Arredondo gratefully acknowledges a fellowship from CONACYT for MSc. studies. References Achurra, P. (2006). Investigating protein-polymer interactions on a microfluidic platform (Tesis Doctoral). Stanford University. Andersen, O., & Markham, K. R. (2006). Flavonoids: Chemistry, biochemistry and applications (1st ed.). Boca Raton: CRC Press. Arteel, G. E., & Sies, H. (1999). Protection against peroxynitrite by cocoa polyphenol oligomers. FEBS Letters, 462, 167–170. Barone, V., Cossi, M., & Tomasi, J. (1997). A new definition of cavities for the computation of solvation free energies by the polarizable continuum model. Journal of Chemical Physics, 107, 3210–3221. Bentes, A. L., Borges, R. S., Monteiro, W. R., De Macedo, L. G., & Alves, C. N. (2011). Structure of dihydrochalcones and related derivatives and their scavenging and antioxidant activity against oxygen and nitrogen radical species. Molecules, 16, 1749–1760. Cances, E., & Mennucci, B. (1998). New applications of integral equations methods for solvation continuum models: Ionic solutions and liquid crystals. Journal of Mathematical Chemistry, 23, 309–326. Cheynier, V., Rigaud, J., & da Silva, J. M. R. (1992). Structure of procyanidin oligomers isolated from grape seeds in relation to some of their chemical properties. Plant Polyphenols, 281–294.
A.M. Mendoza–Wilson et al. / Food Chemistry 161 (2014) 155–161 de Freitas, V. A., Glories, Y., & Laguerre, M. (1998a). Incidence of molecular structure in oxidation of grape seed procyanidins. Journal of Agricultural and Food Chemistry, 46, 376–382. de Freitas, V. A., Glories, Y., Bourgeois, G., & Vitry, C. (1998b). Characterization of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry. Phytochemistry, 49, 1435–1441. Duda-Chodak, A., Tarko, T., Satora, P., Sroka, P., & Tusz yn´ski, T. (2010). The profile of polyphenols and antioxidant properties of selected Apple cultivars grown in poland. Journal of Fruit and Ornamental Plant Research, 18, 39–50. Enomoto, T., Nagasako-Akazome, Y., Kanda, T., Ikeda, M., & Dake, Y. (2006). Clinical effects of apple polyphenols on persistent allergic rhinitis: A randomized double-blind placebo-controlled parallel arm study. Journal of Investigational Allergology and Clinical Immunology, 16, 283–289. Foo, L. Y., & Porter, L. J. (1983). Synthesis and conformation of procyanidin diastereoisomers. Journal Chemistry and Society, Perkin Transactions, 1, 1535–1543. Foresman, J. B., & Frisch, A. E. (1996). Exploring chemistry with electronic structure methods (2nd ed.). U.S.A.: Gaussian Inc. Foti, M. C., Daquino, C., Mackie, I. D., DiLabio, G. A., & Ingold, K. U. (2008). Reaction of phenols with the 2, 2-diphenyl-1-picrylhydrazyl radical. Kinetics and DFT calculations applied to determine ArO–H bond dissociation enthalpies and reaction mechanism. The Journal of Organic Chemistry, 73, 9270–9282. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., et al. (2004). Gaussian 03, revision C.02. Wallingford, CT: Gaussian Inc. Hatano, T., & Hemingway, R. W. (1997). Conformational isomerism of phenolic procyanidins: Preferred conformations in organic solvents and water. Journal Chemistry and Society, Perkin Transactions, 2, 1035–1044. Jerez, M., Touriño, S., Sineiro, J., Torres, J. L., & Núñez, M. J. (2007). Procyanidins from pine bark: Relationships between structure, composition and antiradical activity. Food Chemistry, 104, 518–527. Khan, M. L., Haslam, E., & Williamson, M. P. (1997). Structure and conformation of the procyanidin B-2 dimer. Magnetic Resonance in Chemistry, 35, 854–858. Khanal, R. C., Howard, L. R., & Prior, R. L. (2009). Procyanidin content of grape seed and pomace, and total anthocyanin content of grape pomace as affected by extrusion processing. Journal of Food Science, 74, 174–182. Kondo, K., Kurihara, M., Fukuhara, K., Tanaka, T., Suzuki, T., Miyata, N., et al. (2000). Conversion of procyanidin B-type (catechin dimer) to A-type: Evidence for abstraction of C-2 hydrogen in catechin during radical oxidation. Tetrahedron Letters, 41, 485–488. Leite, S. P., Laranjinha, J. A., Pereira, N., & de Freitas, V. A. (2003). Antioxidant protection of low density lipoprotein by procyanidins: Structure/activity relationships. Biochemical Pharmacology, 66, 947–954. Leopoldini, M., Marino, T., Russo, N., & Toscano, M. (2004). Antioxidant properties of phenolic compounds: H-atom versus electron transfer mechanism. The Journal of Physical Chemistry A, 108, 4916–4922. Markovic´, Z., Milenkovic´, D., Dorovic´, J., Dimitric´, J. M., Stepanic´, V., Lucˇic´, B., et al. (2012). PM6 and DFT study of free radical scavenging activity of morin. Food Chemistry, 134, 1754–1760. Mendoza-Wilson, A. M. (2006). Estudio computacional del mecanismo antioxidante de los flavonoides quercetina, (+)-catequina y cianidina basado en su secuencia de
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desprotonacion (Tesis Doctoral). Centro de Investigacion en Materiales Avanzados, S.C., Chihuahua, México. Mendoza-Wilson, A. M., & Glossman-Mitnik, D. (2006). Theoretical study of the molecular properties and chemical reactivity of (+)-catechin and ()epicatechin related to their antioxidant ability. Journal of Molecular Structure: Theochem, 761, 97–106. Mendoza-Wilson, A. M., Armenta-Vázquez, M. E., Castro-Arredondo, S. I., EspinosaPlascencia, A., Robles-Burgueño, R., González-Ríos, H., et al. (2013). Potential of polyphenols from an aqueous extract of apple peel as inhibitors of free radicals: An experimental and computational study. Journal of Molecular Structure, 1035, 61–68. Musialik, M., Kuzmicz, R., Pawłowski, T. S., & Litwinienko, G. (2009). Acidity of hydroxyl groups: an overlooked influence on antiradical properties of flavonoids. The Journal of Organic Chemistry, 74, 2699–2709. Ochterski, J. W., Petersson, G. A., & Wiberg, K. (1995). A comparisson model chemistries. Journal of the American Chemical Society, 117, 11299–11308. Saint-Cricq, N., Provost, C., & Vivas, N. (1999). Comparative study of polyphenol scavenging activities assessed by different methods. Journal of Agricultural and Food Chemistry, 47, 425–431. Santos-Buelga, C., & Scalbert, A. (2000). Proanthocyanidins and tannin-like compounds–nature, occurrence, dietary intake and effects on nutrition and health. Journal of the Science of Food and Agriculture, 80, 1094–1117. Suryan, M. M., Kafafi, S. A., & Stein, S. E. (1989). Dissociation of substituted anisoles: substituent effects on bond strengths. Journal of the American Chemical Society, 111, 4594–4600. Tarascou, I., Barathieu, K., Simon, C., Ducasse, M. A., André, Y., Fouquet, E., et al. (2006). A 3D structural and conformational study of procyanidin dimers in water and hydro-alcoholic media as viewed by NMR and molecular modeling. Magnetic Resonance in Chemistry, 44, 868–880. Tarascou, I., Ducasse, M. A., Dufourc, E. J., Moskau, D., Fouquet, E., Laguerre, M., et al. (2007). Structural and conformational analysis of two native procyanidin trimers. Magnetic Resonance in Chemistry, 45, 157–166. Tsao, R., Yang, R., Young, J. C., & Zhu, H. (2003). Polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (HPLC). Journal of Agricultural and Food Chemistry, 51, 6347–6353. Tyrakowska, B., Leman´ska, K., Szymusiak, H., Borkowski, T., & Rietjens, I. (2003). Modified TEAC test for determination of the antioxidant properties of dietary polyphenolic compounds over a wide pH range. Polish Journal of Food and Nutrition Sciences, 12, 141–148. Vennat, B., Bos, M. A., Pourrat, A., & Bastide, P. (1994). Procyanidins from tormentil: Fractionation and study of the anti-radical activity towards superoxide anion. Biological & Pharmaceutical Bulletin, 17, 1613–1615. Zhang, J., Skinner, M. A., & Keller, R. B. (2009). Flavonoids: Byosinthesis biological effects and dietary sources (1st ed.). New York: Nova Science Publisher Inc. (Chapter 2). Zhan, C. G., & Dixon, D. A. (2003). The nature and absolute hydration free energy of the solvated electron in water. The Journal of Physical Chemistry B, 107, 4403–4417. Zhao, Y., Schultz, N. E., & Truhlar, D. G. (2006). Design of density functionals by combining the method of constraint satisfaction with parametrization for thermochemistry, thermochemical kinetics, and noncovalent interactions. Journal of Chemical Theory and Computation, 2, 364–382.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Computational study of the structure–free radical scavenging relationship of procyanidins Ana María Mendoza–Wilson ⇑, Sergio Ivan Castro-Arredondo, René Renato Balandrán-Quintana Coordinación de Tecnología de Alimentos de Origen Vegetal, CIAD, A.C., Carretera a La Victoria km 0.6, Hermosillo, Sonora 83304, Mexico
a r t i c l e
i n f o
Article history: Received 8 January 2014 Received in revised form 9 March 2014 Accepted 24 March 2014 Available online 1 April 2014 Keywords: Procyanidin DFT Computational Free radical Antioxidant
a b s t r a c t Procyanidins (PCs) are effective free radical scavengers, however, their antioxidant ability is variable because they have different degrees of polymerisation, are composed by distinct types of subunits and are very susceptible to changes in conformation. In this work the structure–free radical scavenging relationship of monomers, dimers and trimers of PCs was studied through the hydrogen atom transfer (HAT), sequential proton-loss electron-transfer (SPLET) and single electron transfer followed by proton transfer (SET-PT) mechanisms in aqueous phase, employing the Density Functional Theory (DFT) computational method. The structure–free radical scavenging relationship of PCs showed a very similar behaviour in HAT and SET-PT mechanisms, but very different in the SPLET mechanism. The structural factor that showed more effects on the ability of PCs to scavenge free radicals in aqueous phase was the conformation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The PCs are also known as proanthocyanidins or condensed tannins (Enomoto, Nagasako-Akazome, Kanda, Ikeda, & Dake, 2006) and represent one of the most abundant class of phenolic compounds produced by plant tissues (Andersen & Markham, 2006). The PCs have proven to be 30–50 times more effective free radical scavengers than vitamins C and E (Duda-Chodak, Tarko, Satora, Sroka, & Tusz ynski, 2010; Saint-Cricq, Provost, & Vivas, 1999), so they have beneficial effects on the fruits and vegetable quality, in addition to the health of consumers (Jerez, Touriño, Sineiro, Torres, & Núñez, 2007; Khanal, Howard, & Prior, 2009). However, because PCs have very complex and diverse structures, their antioxidant ability is changeable and depends on the intermediates and oxidation products formed during the antioxidant reactions. The PCs are flavonoids belonging to the flavan-3-ols class and are mainly composed of ()-epicatechin and (+)-catechin subunits, which are linked to form oligomers or polymers (Enomoto et al., 2006). The subunit localised at the end of the chain is called terminal unit and the remaining subunits are called extension units (Fig. 1). Also the PCs are characterised by three chiral centres, C2, C3 and C4, which makes them very susceptible to changes in configuration and conformation. From C4 two conformations are ⇑ Corresponding author. Address: Carretera a La Victoria km 0.6, C.P. 83304, PO Box 1735, Hermosillo, Sonora, Mexico. Tel.: +52 662 2892400x354; fax: +52 662 2800422. E-mail address: [email protected] (A.M. Mendoza–Wilson). http://dx.doi.org/10.1016/j.foodchem.2014.03.111 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
formed: compact and extended (Fig. 2). A way to distinguish these conformations is through the dihedral angles formed between the atoms involved in the interflavanoid linkage (C3–C4–C8–C9). Compact conformation is the one with positive dihedral angle values whereas the extended conformation has negative values. For dimers these values can range from +90° to 90° and for trimers from +100° to 70°, respectively (Khan, Haslam, & Williamson, 1997; Tarascou et al., 2007). There is growing evidence indicating a strong relationship between the antioxidant ability of PCs and structural factors such as degree of polymerisation, type of subunit and conformation, but there are also many controversies about this issue (Leite, Laranjinha, Pereira, & de Freitas, 2003; Santos-Buelga & Scalbert, 2000). On the one hand, it is stated that the greater the degree of polimerization of the PCs the greater their ability to scavenge free radicals (Arteel & Sies, 1999; Vennat, Bos, Pourrat, & Bastide, 1994). On the other hand, it is suggested that the PCs ability to scavenge free radicals is not necessarily greater at a higher degree of polymerisation, as in one study was reported that PCs trimers showed to be more efficient than dimers, but less efficient than monomers to scavenge the hydroxyl radical (Cheynier, Rigaud, & da Silva, 1992). There is a special interest to know the differences in the ability to scavenge free radicals amongst monomers, dimers and trimers, as these represent the most bioavailable forms compared to larger oligomers of PCs. Regarding the type of subunit, most studies have reported higher activity for free radical scavenging in ()-epicatechin against different free radicals (Cheynier et al., 1992; de Freitas,
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Fig. 1. Structure of ()-epicatechin (E), (+)-catechin (C), procyanidin B1 (PB1), procyanidin B2 (PB2) and procyanidin C1 (PC1).
Glories, & Laguerre, 1998a); however, in an experimental-computational study conducted by Mendoza-Wilson et al. (2013), higher radical scavenging activity was found for (+)-catechin against the DPPH. In another computational study performed by MendozaWilson and Glossman-Mitnik (2006) it was determined that (+)catechin and ()-epicatechin have comparable intrinsic reactivity properties, due to their similarity in structure (epimers). Nevertheless they have the ability to form distinct intermediates, which can make the difference in activity against free radicals between these isomers. Additionally, the predominant conformation (compact or extended) in a particular solvent (polar, non polar), is also
dependent on the type of subunit as extension unit (Hatano & Hemingway, 1997). The intermediates and products that can be formed from the PCs during their reaction against free radicals are dependent primarily of the mechanism followed and their reactivity properties. There are three basic mechanisms for free radical scavenging: (1) HAT with a phenoxyl radical as intermediate; (2) SPLET with a phenoxide anion as intermediate, and (3) SET-PT with a radical cation as intermediate (Foti, Daquino, Mackie, DiLabio, & Ingold, 2008; Musialik, Kuzmicz, Pawlowski, & Litwinienko, 2009). The reactivity properties of PCs focus on the oxidation–reduction (redox)
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157
Fig. 2. Compact and extended conformations of the procyanidin B2.
properties of their hydroxyl groups and are related to the ease of releasing hydrogen atoms and electrons to scavenge free radicals (Suryan, Kafafi, & Stein, 1989). Amongst these are the thermochemical properties, which describe the energy process of the redox reactions; the polarisability, which describes polar effects related with the deformation of the electron cloud (Foresman & Frisch, 1996), and their water solubility. The PCs, like all flavonoids, have amphipathic properties but they are more soluble in water because have a greater number of hydroxyl groups and can acquire different conformations (Achurra, 2006). The fact that the PCs are water soluble gives them special properties, as this is the medium in which biological reactions are carried out, is a major component in food and as the extraction solvent for potential commercial use of PCs is excellent because is cheap and nontoxic. To establish the relationship of the structure with the free radical scavenging properties of PCs, it is necessary explore together structural factors such as the degree of polymerisation, type of subunit, conformation, intermediates and products formed through the distinct mechanisms for free radical scavenging, as well as their reactivity properties. This is an arduos task since by their polyhydroxylated structure the PCs generate many intermediates with very short half-life and different conformers by their interflavanoid linkage, which do not allow their isolation through experimental methods, so it is necessary to use computational methodologies. Due to the size of the PCs, the computational methods used for their study are those of molecular mechanics and semiempirical, which have been very useful to establish the molecular geometry and conformation but not to calculate properties of reactivity. The DFT currently represents the most suitable computational method to calculate structural and reactivity properties of PCs, due to the high precision, low cost and shorter time performing calculations on medium-sized and large molecules, compared with other electronic structure methods, such as Hartree–Fock and postHartree–Fock. The aim of this work was to study the effect of differences in structure, specially degree of polymerisation, type of subunit, conformation, as well as the reactivity properties on the ability for free radical scavenging of monomers, dimers and trimers of procyanidins through HAT, SPLET and SET-PT mechanisms, employing the DFT computational method. 2. Computational methodology All the calculations reported were performed with the GAUSSIAN 03W code (Frisch et al., 2004). The DFT method implemented in this computational package was used, and the M05-2X hybrid meta exchange–correlation functional (Zhao, Schultz, & Truhlar,
2006) in conjunction with the 6–31G(d,p) and 6–31+G(d,p) basis sets were employed to establish the optimisation of molecular structures, vibrational frequencies and reactivity properties. The reactivity properties were calculated under simulation of an aqueous phase and include thermochemical parameters, polarisability and water solubility. 2.1. Thermochemical properties In order to know the ability of the simulated PCs for free radical scavenging by the HAT mechanism, the bond dissociation energy (BDE) was calculated. In aqueous phase the BDE was determined as the difference of total free solvation energy between parent molecule (ArOH) and its phenoxyl radical (ArO) formed after the hydrogen atom (H) transfer from hydroxyl group, applying the reaction (1) and Eq. (2) (Ochterski, Petersson, & Wiberg, 1995):
ArOH ! ArO þ H X X Dr Hð298 KÞ ¼ ðE0 þ Gcorr Þproducts ðE0 þ Gcorr Þreactants
ð1Þ ð2Þ
The computed total free solvation energy of hydrogen atom (H) in aqueous phase was 0.495216 Hartrees. The ability of the PCs to transfer electrons by the SPLET mechanism was determined in two steps. In the first step the proton affinity (PA) of the phenoxide anion (ArO) formed after the deprotonation (H+) of the PCs (ArOH) was calculated from the Eq. (3) (Markovic et al., 2012):
ArOH ! ArO þ Hþ
ð3Þ
In the second step the electron transfer energy (ETE) of the phenoxide anion was determined by means of Eq. (4) (Markovic et al., 2012):
ArO !ArO þ e
ð4Þ
Both PA and ETE were reported in terms of total free solvation energy in water. The values for H+ = 262.4 kcal/mol and e = 35.5 kcal/mol were employed for calculations (Zhan & Dixon, 2003). To know the ability of the PCs to transfer electrons by the SET-PT mechanism, first the ionisation potential (IP) was calculated from the Eq. (5) (Markovic et al., 2012):
ArOH ! ArOHþ þ e
ð5Þ
Subsequently the proton dissociation energy (PDE) was computed by means of Eq. (6) (Markovic et al., 2012):
ArOHþ ! ArO þ Hþ
ð6Þ
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2.2. Polarisability properties As an intrinsic property of reactivity related with the effects of polarity on the easiness to release hydrogen atoms and electrons, the polarisability (a) was calculated through the following formula (Foresman & Frisch, 1996).
hai ¼ 1=3ðaxx þ ayy þ azz Þ
ð7Þ
2.3. Solubility properties The water solubility was determined by calculating the DG of solvation of the molecules from their neutral energies. The solvent simulation was performed by employing integral equation formalism (IEF-PCM) implemented on the GAUSSIAN 03W code (Cances & Mennucci, 1998). The UAHF set of solvation radii was used (Barone, Cossi, & Tomasi, 1997). 3. Results and discussion 3.1. Optimised molecular structures There are very few reports in the current literature related with the ability of PCs to inhibit free radicals by HAT, SPLET and SET-PT mechanisms, taking into account different degrees of polymerisation, types of subunits and conformation. For this reason these data
were calculated for the ()-epicatechin (E) and (+)-catechin (C) monomers, the procyanidin B1 (PB1) and procyanidin B2 (PB2) dimers, besides the procyanidin C1 (PC1) trimer (Fig. 1); these represent the most common, abundant and bioavailable PCs of the plant tissues, including highly consumed fruits such as apples and grapes (de Freitas, Glories, Bourgeois, & Vitry, 1998b; Tsao, Yang, Young, & Zhu, 2003). The optimised molecular structures of E and C, PB1 and PB2 in their respective compact and extended conformations, as well as of PC1 in all their possible conformations related to interflavanoid linkage, as they are compact–compact (comp–comp), compact– extended (comp–ext), extended–compact (ext–comp) and extended–extended (ext–ext), are showed in the Fig. 3. Dihedral angles values of the interflavanoid linkages (C3–C4–C8–C9) are also included in order to confirm the compact conformation (positive value) and extended conformation (negative value) between extension and terminal units of the PB1, PB2 and PC1 molecules. 3.2. Thermochemical properties The most used pathway for studying the free radical scavenging mechanisms in flavonoids is through thermochemical parameters, focused on the intrinsic properties of the molecules under study and the microenvironmental features such as the reaction medium. Several computational works based on these parameters have proved that the most susceptible site to radical attack in distinct classes of flavonoids, amongst which are included ()-epicatechin and (+)-catechin, is 40 -OH (Bentes, Borges, Monteiro, De Macedo, & Alves, 2011; Kondo et al., 2000; Leopoldini, Marino, Russo, & Toscano, 2004; Mendoza-Wilson, 2006; Mendoza-Wilson et al., Table 1 Thermochemical properties of procyanidins computarized in aqueous phase. Procyanidin
()-Epicatechin (+)-Catechin Procyanidin B1 Compact UE UT Extended UE UT Procyanidin B2 Compact UE UT Extended UE UT
Fig. 3. Optimised molecular structures of ()-epicatechin (E) and (+)-catechin (C); procyanidin B1 (PB1) and procyanidin B2 (PB2) in their compact and extended conformations; procyanidin C1 (PC1) in their compact–compact (comp–comp), compact–extended (comp–ext), extended–compact (ext–comp) and extended– extended (ext–ext) conformations. The dihedral angle values of the interflavanoid linkages are given by C3–C4–C8–C9 atoms.
Procyanidin C1 CompComp UE2 UE1 UT CompExt UE2 UE1 UT ExtComp UE2 UE1 UT ExtExt UE2 UE1 UT
Parameters of free radical scavenging (kcal/mol) HAT
SPLET
BDE
PA
ETE
IP
SET-PT PDE
92 90
34 32
70 70
100 98
4 4
92 89
34 38
70 64
92 91
34 38
70 66
91 88
37 34
66 66
91 91
33 39
71 63
91 85 91
38 38 34
65 60 69
92 86 91
34 32 40
69 66 63
91 91 88
33 35 37
70 68 63
96 91 94
44 45 37
64 58 69
88 15 13 95 8 8 91 12 9 95 9 8 85 19 13 18 89 15 9 14 97 6 6 3 95 13 8 11
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2013; Tyrakowska, Lemanska, Szymusiak, Borkowski, & Rietjens, 2003; Zhang, Skinner, & Keller, 2009). Considering that the first reaction site can make a difference in the antioxidant potential, depending on the mechanism followed, the ability to the HAT, SPLET and SET-PT mechanisms was determined on E, C, PB1, PB2 and PC1 on the basis of the 40 -OH. In order to take in account the effects of the solvent in the redox reaction, the thermochemical parameters were calculated from the total free solvation energy under simulation of an aqueous phase at 298 K. The computarised values of PA < BDE 6 IP showed in the Table 1 indicate that the mechanism which could prevail in an aqueous phase in E, C, PB1, PB2 and PC1 is SPLET, followed by HAT and SET-PT. These results are in accordance with the works previously reported by Markovic et al. (2012) and Mendoza-Wilson et al. (2013). Based on the values of proton affinity (PA), which represent the first step of the SPLET mechanism, is suggested that the most important structural factors of the PCs associated with this mechanism are the conformation, the position and type of subunit, since the degree of polymerisation apparently is not decisive. Under these terms both the C monomer and the PC1 trimer in its compact-extended conformation through its extension unit 1, showed the greatest ability for free radical scavenging because of their low and equal value of PA of 32 kcal/mol (Table 1). In the HAT mechanism is observed that the easiness to transfer hydrogen atoms (BDE values) fluctuated within the same range amongst monomers, dimers and trimers of PCs. However, to analyse the degree of polymerisation, conformation and to compare the reactivity of extension and terminal units of each molecule, the following behaviour was found: the lowest value of BDE (85 kcal/mol) was shown by PC1 in its extension unit 1 of the
Table 2 Polarisabilities calculated in aqueous phase for the procyanidin molecules. Procyanidin
Polarisability (a)
()-Epicatechin (+)-Catechin Procyanidin B1 compact Procyanidin B1 extended Procyanidin B2 compact Procyanidin B2 extended Procyanidin C1 CompComp Procyanidin C1 CompExt Procyanidin C1 ExtComp Procyanidin C1 ExtExt
259.37 260.43 522.74 524.96 523.30 524.25 784.76 786.38 777.72 769.61
The polarisabilities are expressed in atomic units (a.u.).
compact–compact conformation, followed by PB2 (88 kcal/mol) in its terminal unit of the compact conformation, PB1 (89 kcal/ mol) in its terminal unit of the compact conformation, after C (90 kcal/mol) and E (92 kcal/mol) (Table 1). Considering that the smaller the value of BDE the greater the antioxidant potential, it is suggested that in the HAT mechanism the antioxidant ability of the PCs increases with the degree of polymerisation (monomer to trimer), is favored by the compact and compact–compact conformations and is slightly higher in C than in E. In relation to the position of the subunits, it is suggested that the terminal unit is the most reactive in dimers and that in the case of the trimers is the extension unit 1, which corresponds to the intermediate unit. Regarding the SET-PT mechanism, the IP is related to the first step of the free radical scavenging reaction, so it was used as an indicator to establish the differences in antioxidant ability amongst different PCs. However, due to the way the IP was determined computationally, it represents a global property of the molecules.
Table 3 Computational calculations of solubility in aqueous phase for the procyanidin molecules. Procyanidin
Parent molecule
Phenoxyl radical
Phenoxide anion
Radical cation
()-Epicatechin (+)-Catechin
29 30
25 27
73 71
73 72
35 37
85 81
42 43
92 71
36 40
77 80
43 44
82 71
34 41 38
81 80 72
48 54 50
99 96 64
48 46 54
92 91 92
55 59 56
85 78 101
Procyanidin B1 Compact UE UT Extended UE UT Procyanidin B2 Compact UE UT Extended UE UT Procyanidin C1 CompComp UE2 UE1 UT CompExt UE2 UE1 UT ExtComp UE2 UE1 UT ExtExt UE2 UE1 UT
40
70
46
77
40
69
48
78
39
65
52
78
51
77
66
89
The calculations were performed as DG of solvation and are expressed in kcal/mol.
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Therefore, to specify the preferred site of the reaction was taking into account the value of PDE, which was determined for each subunit of dimers and trimers. As can be seen in Table 1, in the SET-PT mechanism the PCs showed a comparable behaviour to the HAT mechanism. The lowest value of IP (85 kcal/mol) was shown by PC1 in the compact– compact conformation, and according with the lowest value of PDE (13 kcal/mol) the extension unit 1 was the most reactive. For dimers, the lowest IP value corresponded to PB1 (88 kcal/mol) in the compact conformation and based on the value of PDE (13 kcal/mol) the terminal unit was the most reactive, followed by PB2 (91 kcal/mol) in the compact conformation and the terminal unit as the most reactive site (PDE = 9 kcal/mol). In the monomers, C showed the lowest IP (98 kcal/mol). According to these data, it states that in the SET-PT mechanism the ability to scavenge free radicals of the PCs increases with the degree of polymerisation (monomer to trimer), is favored by the compact and compact– compact conformations and is slightly higher in C than E. In relation to the position of the subunits, it is suggested that the terminal unit is the most reactive in dimers and in the case of the trimers is the extension unit 1, which corresponds to the intermediate unit. 3.3. Polarisability properties The polarisability showed a significant rise as the degree of polymerisation of the PCs increased, being observed the following order: PC1 > PB2 P PB1 > C > E (Table 2). This result indicates that PC1 is more reactive than PB2 and PB1, and both are more reactives than C and E. The more reactive monomer was C. The polarisability did not follow a uniform pattern in relation to the conformation, since the dimers in their extended conformation had higher polarisabilities than the dimers in their compact conformation, and the trimer showed an inverse behaviour. A direct relationship amongst the polarisability, the degree of polymerisation and the ability to scavenge free radicals by means of the HAT and SET-PT mechanisms was found. Also, a direct relationship amongst the polarisability, type of monomer, and the ability to scavenge free radicals by means of the HAT, SPLET and SET-PT mechanisms was found.
(Foo & Porter, 1983; Hatano & Hemingway, 1997; Tarascou et al., 2006, 2007). An implicit model (IEF-PCM) was employed to simulate the aqueous phase in this investigation, which is based on the dielectric constant of the solvent, and the applied radii (UAHF) not added individual spheres to the hydrogen atoms of the molecules under study. To determine the effects of adding spheres in the hydrogen atoms of the PCs, some test calculations for ()-epicatechin, PB1 and catechol as reference molecule, were made with the UFF radii. Although the DG values for PCs were not equal to those obtained with the UAHF radii, the same trend and behaviour were observed with both radiis. It is important to note that the UAHF radii is one of the most recommended for the calculations of DG of solvation, because it is considered very accurate even in large molecules such as PCs. 4. Conclusions The ability of PCs to scavenge free radicals by the HAT and SETPT mechanisms tended to increase with the degree of polymerisation (monomer < dimer < trimer), was slightly higher for the monomer C than E, and was totally dependent on the conformation as it was higher in the compact and compact–compact conformations of dimers and trimers, respectively. The terminal unit was the most reactive site in dimers and in the case of the trimers it was the extension unit 1, which corresponds to the intermediate unit. Regarding SPLET mechanism, the most important structural factors were the conformation, the position and type of subunit, since the degree of polymerisation of PCs apparently was not decisive. In the SPLET mechanism the relationship of the structural factors with the ability of PCs to scavenge free radicals did not show a well-defined trend as in the HAT and SET-PT mechanisms. The polarisability and solubility in aqueous phase of the PCs tended to increase with the degree of polymerisation, were higher in C than E, and in relation to the conformation did not follow a uniform pattern. In resume it is concluded that the structure–free radical scavenging relationship of monomers, dimers and trimers of procyanidins is dependent of the mechanism followed; however, the structural factor that showed more effects on the ability of PCS to scavenge free radicals in aqueous phase was the conformation.
3.4. Solubility properties Acknowledgements As can be observed in Table 3, the solubility in an aqueous phase was slightly higher in C than E, and tended to increase with the degree of polymerisation of the PCs. Nevertheless, the conformation, the position of the reaction site (UE or UT) and the intermediates formed from the PCs, showed a more significant effect on this property. Regarding the type of intermediate, the phenoxide anions were more soluble, followed by radical cations, whilst the phenoxyl radicals were less soluble even than parent molecules. In relation to the conformation, the extended form was the most soluble and the compact form the less soluble. This result is consistent, since in the compact conformation of the PCs the hydroxyl groups remain close to each other and the formation of intramolecular hydrogen bonding is favored, whilst in the extended conformation the hydroxyl groups are more exposed to the solvent to form intermolecular hydrogen bonding with it. What seems contradictory is that PB1, PB2 and PC1 in their compact and compact–compact conformations, can more readily donate their hydrogen atoms, electrons and protons, if they are less soluble in aqueous phase. Such behaviour is probably due to that the intramolecular hydrogen bonds give them greater stability. Additionally, some studies report that the predominant conformation in dimers and trimers of PCs in an aqueous medium is the compact, because this conformation allows minimising the effective surface area and consequently the solute–solvent interactions
The authors of this paper are very grateful with the Consejo Nacional de Ciencia y Tecnología (CONACYT) México, which financed the investigation under Grant Number CB2012-183739. Sergio Ivan Castro Arredondo gratefully acknowledges a fellowship from CONACYT for MSc. studies. References Achurra, P. (2006). Investigating protein-polymer interactions on a microfluidic platform (Tesis Doctoral). Stanford University. Andersen, O., & Markham, K. R. (2006). Flavonoids: Chemistry, biochemistry and applications (1st ed.). Boca Raton: CRC Press. Arteel, G. E., & Sies, H. (1999). Protection against peroxynitrite by cocoa polyphenol oligomers. FEBS Letters, 462, 167–170. Barone, V., Cossi, M., & Tomasi, J. (1997). A new definition of cavities for the computation of solvation free energies by the polarizable continuum model. Journal of Chemical Physics, 107, 3210–3221. Bentes, A. L., Borges, R. S., Monteiro, W. R., De Macedo, L. G., & Alves, C. N. (2011). Structure of dihydrochalcones and related derivatives and their scavenging and antioxidant activity against oxygen and nitrogen radical species. Molecules, 16, 1749–1760. Cances, E., & Mennucci, B. (1998). New applications of integral equations methods for solvation continuum models: Ionic solutions and liquid crystals. Journal of Mathematical Chemistry, 23, 309–326. Cheynier, V., Rigaud, J., & da Silva, J. M. R. (1992). Structure of procyanidin oligomers isolated from grape seeds in relation to some of their chemical properties. Plant Polyphenols, 281–294.
A.M. Mendoza–Wilson et al. / Food Chemistry 161 (2014) 155–161 de Freitas, V. A., Glories, Y., & Laguerre, M. (1998a). Incidence of molecular structure in oxidation of grape seed procyanidins. Journal of Agricultural and Food Chemistry, 46, 376–382. de Freitas, V. A., Glories, Y., Bourgeois, G., & Vitry, C. (1998b). Characterization of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry. Phytochemistry, 49, 1435–1441. Duda-Chodak, A., Tarko, T., Satora, P., Sroka, P., & Tusz yn´ski, T. (2010). The profile of polyphenols and antioxidant properties of selected Apple cultivars grown in poland. Journal of Fruit and Ornamental Plant Research, 18, 39–50. Enomoto, T., Nagasako-Akazome, Y., Kanda, T., Ikeda, M., & Dake, Y. (2006). Clinical effects of apple polyphenols on persistent allergic rhinitis: A randomized double-blind placebo-controlled parallel arm study. Journal of Investigational Allergology and Clinical Immunology, 16, 283–289. Foo, L. Y., & Porter, L. J. (1983). Synthesis and conformation of procyanidin diastereoisomers. Journal Chemistry and Society, Perkin Transactions, 1, 1535–1543. Foresman, J. B., & Frisch, A. E. (1996). Exploring chemistry with electronic structure methods (2nd ed.). U.S.A.: Gaussian Inc. Foti, M. C., Daquino, C., Mackie, I. D., DiLabio, G. A., & Ingold, K. U. (2008). Reaction of phenols with the 2, 2-diphenyl-1-picrylhydrazyl radical. Kinetics and DFT calculations applied to determine ArO–H bond dissociation enthalpies and reaction mechanism. The Journal of Organic Chemistry, 73, 9270–9282. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., et al. (2004). Gaussian 03, revision C.02. Wallingford, CT: Gaussian Inc. Hatano, T., & Hemingway, R. W. (1997). Conformational isomerism of phenolic procyanidins: Preferred conformations in organic solvents and water. Journal Chemistry and Society, Perkin Transactions, 2, 1035–1044. Jerez, M., Touriño, S., Sineiro, J., Torres, J. L., & Núñez, M. J. (2007). Procyanidins from pine bark: Relationships between structure, composition and antiradical activity. Food Chemistry, 104, 518–527. Khan, M. L., Haslam, E., & Williamson, M. P. (1997). Structure and conformation of the procyanidin B-2 dimer. Magnetic Resonance in Chemistry, 35, 854–858. Khanal, R. C., Howard, L. R., & Prior, R. L. (2009). Procyanidin content of grape seed and pomace, and total anthocyanin content of grape pomace as affected by extrusion processing. Journal of Food Science, 74, 174–182. Kondo, K., Kurihara, M., Fukuhara, K., Tanaka, T., Suzuki, T., Miyata, N., et al. (2000). Conversion of procyanidin B-type (catechin dimer) to A-type: Evidence for abstraction of C-2 hydrogen in catechin during radical oxidation. Tetrahedron Letters, 41, 485–488. Leite, S. P., Laranjinha, J. A., Pereira, N., & de Freitas, V. A. (2003). Antioxidant protection of low density lipoprotein by procyanidins: Structure/activity relationships. Biochemical Pharmacology, 66, 947–954. Leopoldini, M., Marino, T., Russo, N., & Toscano, M. (2004). Antioxidant properties of phenolic compounds: H-atom versus electron transfer mechanism. The Journal of Physical Chemistry A, 108, 4916–4922. Markovic´, Z., Milenkovic´, D., Dorovic´, J., Dimitric´, J. M., Stepanic´, V., Lucˇic´, B., et al. (2012). PM6 and DFT study of free radical scavenging activity of morin. Food Chemistry, 134, 1754–1760. Mendoza-Wilson, A. M. (2006). Estudio computacional del mecanismo antioxidante de los flavonoides quercetina, (+)-catequina y cianidina basado en su secuencia de
161
desprotonacion (Tesis Doctoral). Centro de Investigacion en Materiales Avanzados, S.C., Chihuahua, México. Mendoza-Wilson, A. M., & Glossman-Mitnik, D. (2006). Theoretical study of the molecular properties and chemical reactivity of (+)-catechin and ()epicatechin related to their antioxidant ability. Journal of Molecular Structure: Theochem, 761, 97–106. Mendoza-Wilson, A. M., Armenta-Vázquez, M. E., Castro-Arredondo, S. I., EspinosaPlascencia, A., Robles-Burgueño, R., González-Ríos, H., et al. (2013). Potential of polyphenols from an aqueous extract of apple peel as inhibitors of free radicals: An experimental and computational study. Journal of Molecular Structure, 1035, 61–68. Musialik, M., Kuzmicz, R., Pawłowski, T. S., & Litwinienko, G. (2009). Acidity of hydroxyl groups: an overlooked influence on antiradical properties of flavonoids. The Journal of Organic Chemistry, 74, 2699–2709. Ochterski, J. W., Petersson, G. A., & Wiberg, K. (1995). A comparisson model chemistries. Journal of the American Chemical Society, 117, 11299–11308. Saint-Cricq, N., Provost, C., & Vivas, N. (1999). Comparative study of polyphenol scavenging activities assessed by different methods. Journal of Agricultural and Food Chemistry, 47, 425–431. Santos-Buelga, C., & Scalbert, A. (2000). Proanthocyanidins and tannin-like compounds–nature, occurrence, dietary intake and effects on nutrition and health. Journal of the Science of Food and Agriculture, 80, 1094–1117. Suryan, M. M., Kafafi, S. A., & Stein, S. E. (1989). Dissociation of substituted anisoles: substituent effects on bond strengths. Journal of the American Chemical Society, 111, 4594–4600. Tarascou, I., Barathieu, K., Simon, C., Ducasse, M. A., André, Y., Fouquet, E., et al. (2006). A 3D structural and conformational study of procyanidin dimers in water and hydro-alcoholic media as viewed by NMR and molecular modeling. Magnetic Resonance in Chemistry, 44, 868–880. Tarascou, I., Ducasse, M. A., Dufourc, E. J., Moskau, D., Fouquet, E., Laguerre, M., et al. (2007). Structural and conformational analysis of two native procyanidin trimers. Magnetic Resonance in Chemistry, 45, 157–166. Tsao, R., Yang, R., Young, J. C., & Zhu, H. (2003). Polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (HPLC). Journal of Agricultural and Food Chemistry, 51, 6347–6353. Tyrakowska, B., Leman´ska, K., Szymusiak, H., Borkowski, T., & Rietjens, I. (2003). Modified TEAC test for determination of the antioxidant properties of dietary polyphenolic compounds over a wide pH range. Polish Journal of Food and Nutrition Sciences, 12, 141–148. Vennat, B., Bos, M. A., Pourrat, A., & Bastide, P. (1994). Procyanidins from tormentil: Fractionation and study of the anti-radical activity towards superoxide anion. Biological & Pharmaceutical Bulletin, 17, 1613–1615. Zhang, J., Skinner, M. A., & Keller, R. B. (2009). Flavonoids: Byosinthesis biological effects and dietary sources (1st ed.). New York: Nova Science Publisher Inc. (Chapter 2). Zhan, C. G., & Dixon, D. A. (2003). The nature and absolute hydration free energy of the solvated electron in water. The Journal of Physical Chemistry B, 107, 4403–4417. Zhao, Y., Schultz, N. E., & Truhlar, D. G. (2006). Design of density functionals by combining the method of constraint satisfaction with parametrization for thermochemistry, thermochemical kinetics, and noncovalent interactions. Journal of Chemical Theory and Computation, 2, 364–382.
